Note: Descriptions are shown in the official language in which they were submitted.
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ANTI-VIRAL GUANOSINE-RICH TETRAD FORMING OLIGONUCLEOTIDES
CROSS REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of 35 U.S.C ~I11(b) provisional
patent application
60/037,374 filed February 4, 1997. This application is also a continuation-in-
part of co-pending U.S.
S Patent App. No. 08/987,574 filed December 9, 1997.
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates generally to the field of oligonucleotide
chemistry and anti-viral
pharmacotherapy. More specifically, the present invention relates to
therapeutically active guanosine
rich intramolecular tetrad fornvng oligonucleotides, to methods of treating
viral diseases using said
oligonucleotides, and to pharmaceutical compositions containing the novel
oligonucleotides.
Description of the Related Art
General In Vitro Studies
Previously, it was believed that "antisense" oligonucleotides inhibit viruses
by interfering with
protein translation via an RNA:DNA duplex structure. More recent research,
however, indicates a variety
of possible mechanisms by which oligo-nucleotides inhibit viral infections.
For example,
oligodeoxycytidine (poly SdC) inhibits HIV-1. Marshall et al., PNAS (1992)
89:6265-6269, discussed
the potential mechanism (competitive inhibition) by which oligodeoxycytidine
directly inhibits viral
reverse transcriptase. Poly SdC also inhibited AMV reverse transcriptase and
Pol I (Klenow fragment)
and pofymerase a, ~i and y. Previously, Matsukura et al., PNAS (1987) 84:7706-
7710, used a similar
phosphorothioate derivative of oligo-deoxycytidine to demonstrate inhibition
of HIV-1 in culture.
Marshall and Caruthers, Science ( 1993) 259:1564-1569, reported the use of
diphosphorothioate oligo-
nucleotides, e.g., antisense-specific, random nucleotide combinations and
oligodeoxycytidine against
HIV-1. In all cases, the mechanism of action was attributed to a direct
inhibition of HIV-1 reverse
transcriptase. Other potential mechanisms of anti-viral action of
oligonucleotides were postulated by
Boiziau et al., PNAS ( 1992) 89:768-772, e.g., promotion of RNAse H activity
and inhibition of reverse
transcriptase initiating cDNA synthesis. In addition, Goa et al., Molecular
Pharmacology ( 1992)
41:223-229 reported that phosphorothioate oligonucleotides inhibit human DNA
polymerases and
ltNAse H, and the adsorption or penetration of the virus into cells. Iyer et
al., Nucleic Acids Research
( 1990) 18:2855-2859 reported that if a base was removed from an anti-sense
polynucleotide forming an
abasic site, the compound did not lose its activity which argues against the
need for the formation of an
~ 1ZNA:DNA antisense mediated hybrid for anti-viral activity. Stein et al.
have characterized the
interaction of poly SdC with the V3 loop of H1V-1 gp120, and postulated that
the specific interaction of
~ poly SdC with the HIV-1 V3 loop may be a mechanism by which an
oligonucleotide could inhibit HIV-1
in vivo.
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It is known that synthetic oligonucleotides may be designed which are capable
of binding to
duplex DNA to form triplex DNA. See U.S. Patent No. 5,176,996 Hogan & Kessler
issued January 5,
1993. That patent discloses a method for making synthetic guanosine-rich
oligonucleotides which are
targeted to specific sequences in duplex DNA and which form collinear
triplexes by binding to the major
S groove of the DNA duplex.
Specific in Vitro Studies/In Vitro HIV Inhibition With T3a177
Infection with the human immunodeficiency virus type I (HIV-I ) and the
subsequent
development of acquired immunodeficiency syndrome (AIDS), has become a threat
to public health on a
global scale. Preventing further spread of this disease is a major health
priority worldwide. Although
HIV-1 was confirmed to be the causative agent of AIDS as early as 1984, few
drugs and no vaccines are
effective at preventing the ultimate onset of A>DS in HIV-1 seropositive
individuals. This is due, in large
part, to the complexity of the causative agent itself, the dynamics of virus
production and the speed at
which drug-resistant mutants can arise. Ho, et al., Nature 373:123-126 (
1995); Wei, et al., Nature
373:117-122 (1995).
Infection of T-cells by HIV-1 results in the insertion of provirai (double-
stranded) DNA into the
host cell genome. Goff, S.P., Annu. Rev. Genet. 26:527-544 ( 1992}. The
integration process involves
both the sequence-specific and sequence independent endonucleolytic and strand
transfer activities of the
virally encoded integrase enzyme. Katz, et al., Ann. Rev. Biochem. 63:133-173
(1994); Vink, et a.,
Trends in Genetics 9:433-43 8 ( 1993). Once the proviral state is established,
the infection may manifest
itself in several ways including a latent infection in which viral replication
is not measurable until the cell
becomes activated or through a chronic infection in which dividing or non-
dividing cells persistently
release virus in the absence of any cytopathic effect. In addition, recent
reports on the kinetics of virus
production (and clearance) indicate a dynamic process in which virtually a
complete replacement of wild-
type virus by drug-resistant virus in plasma can occur after only two to four
weeks of drug therapy. Ho,
2S et al., Nature 373:123-126 (1995); Wei, et al., Nature 373:117-122 {1995).
For this reason it is of utmost
importance to develop new anti-HIV-1 agents which can complement, by additive
or synergistic activity,
current therapies.
One relatively new approach used in the development of antiviral therapeutics
for HIV-1 is the
use of oligonucieotides designed as antisense agents. Letsinger, et al., Proc.
Natl. Acad Sci. USA
86:6553-6556 (1989); Lisziewicz, et al., Proc. Natl. Acad. Sci. USA 90:3860-
3864 (1993); Milligan, et
aL, J. Med. Chem. 36:1923-1937 (1993). While much effort is being spent on
rationally designed
oligonucleotides such as antisense agents there have also been recent findings
of multiple alternative
mechanisms by which oligonucleotides can inhibit viral infections. Gao, et
al., J.B.C. 264:11521-11526
( 1989); Marshall, et al., Proc. Natl. Acad. Sci. USA 89:6265-6269 ( 1992);
Ojwang, et al., J. AIDS 7:560-
570 (1994); Rando, et al, J. Biol. Chem. 270:1754-1760 (1995). For example,
Stein et al. (Stein, et al.,
Antisense Research and Development 3:19-31 ( 1993 )) have characterized the
interaction of
oligodeoxycytidine, containing a phosphorothioate (PT) backbone (poly (SdC)}
with the v3 loop of HIV-
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WO 98!33807 PCT/US98/01974
1 gp 120. It was determined that poly (SdC)2g specifically interacted with the
positively charged V3 loop
with a Kd of approximately 5 x 10-'M. Stein et al. (Antisense Research and
Development 3:19-31
( 1993)) then postulated that the interaction of poly (SdC) with the HIV-1 v3
loop may be a mechanism by
which poly (SdC) could inhibit HIV-1 in vivo. More recently, Wyatt et. al.
(Wyatt, et al., Proc. Natl.
' S Acad. Sci. USA 91:1356-1360 (1994)) have described the interaction of a
short G-rich oIigonucleotide,
synthesized with a total PT backbone, which also interacts with the v3 loop of
HN-1 gp 120. In addition,
' we have previously reported that oligonucleotides containing only
deoxyguanosine (G) and thymidine
(T), synthesized with natural phosphodiester (PD) internucleoside linkages,
were capable of inhibiting
HN-1 in culture. Ojwang, et al., J. AIDS 7:560-570 ( 1994). The most eff
cacious member of this dG
rich class of oiigonucleotides, I100-15, was found capable of folding upon
itself to form a structure
stabilized by the formation of two stacked guanosine-tetrads which yielded a
guanosine-octet. Rando, et
al, J. Biol. Chem. 270:1754-1760 ( 1995). Furthermore, it was observed that
the positions of the
guanosine bases in the I100-15 sequence, found in both the tetrads and
connecting loops in that structure,
were extremely important to the overall anti-HIV-1 activity of the
oligonucleotide. Rando, et al, J. Biol.
Chem. 270:1754-1760. ( 1995).
Site of Activity Studies-Viral Integrase Inhibition
Two events which are characteristic of the life cycle of retroviruses can be
utilized for therapeutic
intervention. One is reverse transcription, whereby the single-stranded RNA
genome of the retrovirus is
reverse transcribed into singled-stranded cDNA and then copied into double-
stranded DNA. The next
event is integration, whereby the double-stranded viral DNA generated by
reverse transcriptase is inserted
into a chromosome of the host cell, establishing the proviral state.
Integration is catalyzed by the
retroviral enzyme integrase which is encoded at the 3'-end of the pol gene.
Varmus, et al. Mobile DNA,
pp. 53-1 O8, Am. Soc. Microbiol, Washington, D.C. ( 1989). Integrase first
catalyzes the excision of the
last two nucleotides from each 3'-end of the linear viral DNA, leaving the
terminal conserved
dinucleotide CA-3'-OH at these recessed 3' ends (Fig. 23A). This activity is
referred to as the 3'-
processing or dinucieotide cleavage. After transport to the nucleus as a
nucleoprotein complex, Varmus,
et al. Mobile DNA, pp. 53-108, Am. Soc. Microbiol, Washington, D.C. ( 1989),
integrase catalyzes a
concerted DNA strand transfer reaction by nucleophilic attack of the two viral
ends onto a host
chromosome. This reaction generates a recombination intermediate resembling an
X structure, analogous
to a Holliday junction intermediate. [For recent reviews see Katz and Skalka,
Katz, et al., Ann. Rev.
Biochem. 63, 133-173 (1994), and Vink and Plasterk, Vink, et al., Trends
Genet. 9, 433-437 (1993)].
Mutation analyses of the viral integrase gene demonstrate that integration is
required for effective
retroviral replication and that it is a legitimate target for the design of
antiretroviral drugs (Engleman, et
ai., J. Yirol. 69, 2729-2736 ( 1995); Englund, et ai, J. Virol. 69, 3216-3218
( 1995)).
It is known that AZT nucleotides can inhibit HIV-1 integrase, Mazumder, et
al., Proc. Natl.
Acad. Sci. 91, 5771-5775 (1994), and that substitution or unsaturation at the
3'-position of the
deoxyribose confers potency against HIV-1 integrase. These results suggested
that the enzyme's
CA 02279488 1999-07-29
WO 98133807 PCTIUS98/01974
nucleotide binding site could serve as a potential drug target. It has been
shown that the potential
stacking interactions gained from the heterocyclic rings can further enhance
potency against HIV-1
integrase.
Recently, oligonucleotides composed of deoxyguanosine and thymidine have been
reported to
inhibit HIV-1 replication. Rando, et al., J. Biol. Chem. 270, 1754-1760
(1995); Wyatt, et al., Proc. Natl.
Acad. Sci. U.S.A. 91, 1356-1360 (1994). OIigonucleotides forming
intramolecular G4s did not block
virus adsorption but rather inhibited viral-specific transcripts. Rando, et
al., J. Biol. Chem. 270, 1754-
1760 (1995}; Ojwang et al. J. Aids 7:560-570 (1994).
Structure-Function Studies
It is known that G-rich nucleic acid sequences can fold, in the presence of
Na+ or K+ ion, to form
orderly structures stabilized by guanosine tetrads. Depending on sequence,
intramolecular folds, Rando et
al. J. Biol. Chem. 270: 1754-1760, 1995), dimers (Smith, F. W., & Feigon, J. (
1992) Nature (London)
34.1, 410-414, Sundquist, W. I. & Klug, A. ( 1989) Nature (London) 334, 364-
366; Kang, et al. ( 1992)
Nature (London) 356, 126131; Balaguumoorthy, P. & Brahmachari, S. K. {1994) J.
Biol. Chem. 269,
21858-21869), tetrameres (Son, D. & Gilbert, W. (1990) Nature (London) 344,
410-414; Jin, et al. (1990)
Science 250, 543-546; Jin, et al. ( 1992) Proc. Natl. Acad. Sci. USA 89, 8832-
8836; Lu et al., ( 1992)
Biochemistry 31, 2455-2459), and higher order associations have been detected.
Such tetrad based
structures have been postulated to serve as the structural basis for telomere
function (Sen, D. & Gilbert,
W. ( 1988) Nature (London) 334, 364-366), and have been hypothesized to play a
role in retroviral
replication (Bock et al. (1992) Nature (London) 355, 564-566), and
transcription regulation (Marshall et
al. ( 1992) Proc. Nalt. Acid Sci. USA 8, 9, 6265-6269; Wyatt et al. ( 1994)
Proc. Natl. Acad. Sci. USA 91,
1356-60).
Recently, several groups have shown that compounds which contain tetrad-based
folds may have
activity as potential drug compounds. Bock and colleagues have shown that an
intramolecular fold,
obtained by a SELEX procedure can bind tightly to thrombin, so as to inhibit
clotting (Bock et al. (1992)
Nature (London) 355, 564-566). Additionally Wyatt et al. (Wyatt et al. (1994)
Proc. Natl. Acad. Sci.
USA 91, 1356-60) has shown that a dimer-wise pairing of phosphorothioate
oligomers with the sequence
T2G4T2 (four stranded intermolecular tetrads) gives rise to anti-HIV activity,
by inhibition of viral
adsorption to the cell surface.
The present inventors have also obtained evidence for sequence-selective
inhibition of HIV-1 by
simple phosphodiester oligonucleotides which form G-tetrad based structures.
The highest activity was
obtained with a I 7mer, referred to as T30177, with composition G 12-T5 (Rando
et al., ( 1994) J. Biol.
Chem. 270, 1754-1760; Ojwang, J. et al. (1995) J. Aids 7, 560-570), with 2
phosphorothioate linkages (I
at each end) to block cellular exonuclease activity (Bishop et al. ( 1996) J.
Biol. Chem. 271 ) 5698-5703 ).
NMR evidence was obtained (Rando et al., ( 1995) J. Biol. Chem. 270, 1754-
1760) to suggest that, by
reference to similar oligomers (Smith, F. W., & Feigon, J. ( 1992) Nature
(London) 344, 410-414),
T30177 forms a stable intramolecular fold which is stabilized by a pair of G-
tetrads, connected by three
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single-stranded loops and a I-2 base long tail to either side of the fold.
Those preliminary studies
suggested that aligomer folding was coupled to K+ ion binding (Rando et al., (
1995) J. Biol. Chem. 270,
1754-1760). Additional studies have suggested that T30177 and related
derivatives are potent inhibitors
of HIV-1 integrase, in vitro (Ojwang et al. ( 1995) Antimicrob. Agent
Chemotherepy 39, 2426-35).
' S Pharmacolduetic Studies-Single Dose
Antisense, triple-helix, duplex decoy, and protein-binding (aptamer)
oligonucieotides have been
' shown to have potential as drugs for the treatment of a variety of human
clinical disorders (Stein and
Cheng, 1993; Marshall and Caruthers, 1993, Science 259: 1564-1570; Chubb and
Hogan, 1992, Trends in
Biotechnology 10: 132-I36; Stall and Szoka, 1995, Pharm. Res. 12: 465-483. A
number of
oligonucleotides have undergone pre-clinical testing, and several are in human
clinical trials. One finding
that has aroused some concern (Black et al., 1994, Antisense Res. Dev. 4: 299-
301 ) is the observation that
total phosphorothioate oligonucleotides cause hemodynamic changes following
rapid intravenous
administration. Severe hypotension, leukopenia, complement activation, and
death have been reported to
occur in primates after rapid infusions of total phosphorothioate
oligonucleotides {Cornish et al., 1993,
Pharmacol. Commun. 3: 239-247; Galbraith et al., 1994, Antisense Res. Deu 4:
201-206). These findings
have raised the question of whether the cardiovascular toxicity is a property
of phosphorothioate
oligonucleotides, or of all oligonucleotides. On the basis of these findings,
an FDA commentary has
recommended that cardiovascular screening be performed for the pre-clinical
safety assessment of
oligonucleotides (Black et al., 1994).
Pharmacokinetic Studies-Repeat Dose
Oligonucieotides have advanced to the stage that they are now considered as
potential
therapeutics for the treatment of a variety of human diseases, and several are
presently in clinical trials.
Pre-clinical studies have generally shown that doses up to approximately 50
mglkg are safe, but that
higher doses can cause kidney and liver damage, and death (Srinivasan and
Iversen, 1995, J. Clin. Lab.
Analysis 9:129-I37} Bolus intravenous administration has posed a particular
concern since it has been
shown to sometimes result in serious hypotensive events in primates (Cornish
et al., 1993; Galbraith et
al., 1994; Black et al., 1995). However, because the number of
oligonucleotides that have been studied
has been small, it is difficult to conclude at the time of making the
invention whether all oligonucieotides
share similar toxicities. In particular, given the various ways of modifying
the backbone of
oligonucleotides (Wu-Pong, 1994, BioPharm 7:20-33) and their ability to fold
into distinct
three-dimensional structures (Stall and Szoka, 1995, Pharm. Res. 12:465-483),
Rando et al. J. Biol.
Chem. 270; 1754-1760, 1995, the safety profile of different oligonucleotides
may be quite distinct.
Human Clinical Trials
In addition to toxicological studies, efficacy studies should be carried out
for oligonucleotide
drugs. In the past, the preferred method of testing drug e~cacy, especially in
HN-1 infected patients,
was to monitor survival of treated patients. However, recent statistical
studies have shown that a good
indicator of anti-HN drug effcacy is the reduction in the numbers of copies of
viral genome per unit of
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patient serum (viral load). Mellors et al. ( 1996) Science 272:1167-1170.
Reductions in viral load of
90%, or more preferably 99% are desired. However, reductions of viral load of
lesser percentages can be
useful, especially where the trend of the overall treatment regime is
consistently downward.
******
Thus, there is a substantial need for antiviral drugs with novel chemistry and
with sites of activity
distinct from drugs presently used. Most highly desired would be antiviral
drugs whose e~cacy in
humans is known.
SIJNIIKARY OF THE INVENTION
In certain embodiments of the present invention, there are provided methods
and compositions
useful in treating pathophysiological states caused by viruses, comprising
administering a
pharmacological dose of an oligonucleotide, the dose being su~cient to inhibit
production of the virus,
wherein the oligonucleotide contains a high percentage of guanosine bases. In
preferred embodiments,
the oligonucleotide has a three dimensional structure and this structure is
stabilized by guanosine tetrads.
In a further embodiment, the oligonucleotide compositions of the invention
have two or more runs of two
contiguous deoxyguanosines. In certain embodiments of the present invention,
the target virus is either
herpes simplex virus, human immunodeficiency virus, human papilloma virus,
human cytomegalovirus,
adenovirus, and hepatitis B virus.
In other embodiments of the present invention, there are provided guanosine-
rich
oligonucleotides having a three dimensional structure, wherein the three
dimensional structure is
stabilized by guanosine tetrads or at least two runs of two contiguous
deoxyguanosines and wherein these
oligonucleotides exhibit anti-viral activity. In a further embodiment, the
oligonucleotides of the present
invention have partially or fully phosphorothioated internucleoside linkages
(backbones) or other
chemical modifications. In a further embodiment, the oligonucleotides of the
present invention have
chemically modified or unnatural (synthetic) bases.
In accordance with the present invention, certain preferred oligonucteotides
that include the
sequence 5'-GTGGTGGGTGGGTGGGT-3' (SEQ ID NO 87) are disclosed. This nucleic
acid sequence
has at least one phosphodiester or phosphorothioate intemucleoside linkage and
is capable of fonming a
stable intramolecuiar stacked tetrad structure. The oligonucleotide may also
have a 5' and/or 3' end that is
modified by a moiety which is capable of increasing cellular uptake, or of
modifying the tissue or
subcellular distribution, or of increasing the biological stability of the
oliganucleotide. Such a modifier
may be propylamine, polyamine, poly-L-lysine, cholesterol, a C2-C24 fatty
acid, or vitamin E or similar
moieties that behave in the same way.
Some oiigonucleotides of the present invention have the sequence 5'-
GNGGNGGGNGGGNGGGN-3' (SEQ m NO 88), where N,~ (the 17th nucleotide located at
the 3' end)
is either omitted entirely (making it a 16 nucleotide long ODN instead of I7),
or is thymidine or another
pyrimidine or modified pyrimidine. In this embodiment, NZ, N5, N9 and N,j are
each, independently,
either has the base missing from the tmcleoside, is thymidine, or is another
pyrimidine or modified
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pyrimidine. Each intemucleoside linkage may be, independently, either
phosphodiester or
phosphorothioate. The oligonucleotide backbone may be either ribophosphate,
deoxyribophosphate or
modified ribo- or deoxyribophosphate backbone. Such a modified backbone might
be, for example, a
2'-O-methyl ribophosphate.
Certain preferred embodiments of the new oligonucleotides have the base
omitted from N2, and
N,~ is either omitted entirely, is thymidine or C-5 propynyl-dU. In these
embodiments N5, N9 and N,3,
independently, either abasic, thymidine or C-5 propynyl-dU and have
internucleoside linkages that are
either phosphodiester or phosphorothioate. In some of these preferred
embodiments NS is C-5 propynyl
dU, and in others N9, N,3 or N,~ is C-5 propynyl-dU. In one preferred
embodiment, N5, N9 and N,3 are all
IO C-5 propynyl-dU.
Certain preferred embodiments of the oligonucleotides of the present invention
have
phosphorothioate linkages between the ultimate and penultimate nucleosides,
i.e., the final nucleotide
linkages at the 5' and 3' ends are phosphorothioate.
Also provided by the present invention is a method of making an antiviral
oligonucleotide.
According to this method, beginning at either a 5' or 3' end, an
oligonucleotide of 16 or 17 nucleotide
length is synthesized following the general sequence 5'-GNGGNGGGNGGGNGGGN-3'
(SEQ ID NO
88). As desired, Nt~ may be omitted entirely or the nucleoside is choosen from
the group consisting of
an abasic riboside, deoxyriboside or modified ribo- or deoxyriboside,
thymidine and another pyrimidine
or modified pyrimidine riboside, deoxyriboside or modified ribo- or
deoxyriboside. Similarly, the N at
positions 2, 5, 9 and 13 is independently selected from the group that
includes an abasic riboside,
deoxyriboside or modified ribo- or deoxyriboside, thymidine and another
pyrimidine or modified
pyrimidine riboside, deoxyriboside or modified ribo- or deoxyriboside. As each
nucleoside is added
sequentially, the type of internucleoside linkage used is selected from the
group consisting of
phosphodiester and phosphorothioate. The resulting oligonucleotide is one that
is capable of
spontaneously folding into a stable four-stranded oligonucleotide structure
containing two stacked G
quartets.
The present invention also provides a pharmaceutical composition that contains
one of the new
oligonucleotides, together with a pharmacologically acceptable carrier.
Still another embodiment of the invention provides a method of inhibiting the
production of a
virus, such as a retrovirus. The method includes contacting a virus-infected
cell or organism with one or
more of the oligonucleotides or pharmaceutical compositions of the invention.
Viruses that are
susceptible to inhibition may include herpes simplex virus, human papiIloma
virus, Epstein Barr virus,
human immunodeficiency virus, adenovirus, respiratory syncytial virus,
hepatitis B virus or human
. cytomegalovirus. In certain preferred embodiments of the invention, the
virus is a human
immunodeficiency virus such as HIV-1.
Another method provided by the present invention is a method of inhibiting the
production of a
virus by contacting the virus itself with a new oligonucleotide or
pharmaceutical composition.
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Yet another method provided by the present invention is a method of inhibiting
the production
of a virus by contacting a protein encoded by the virus with a new
oligonucleotide or pharmaceutical
composition. Such a viral protein may be, for example, an enzyme that is
associated with the
integration of viral nucleic acid into a host genome.
Also comprehended by the present invention is a method of treating a viral
disease in a human
comprising administering a pharmacological dose of one of the new
pharmaceutical compositions to a
person in need of treatment for the disease. The viral disease might be, for
example, a result of infection
by herpes simplex virus, human papiiloma virus, Epstein Barr virus, human
immunodeficiency virus,
adenovirus, respiratory syncytiaI virus, hepatitis B virus or human
cytomegalovirus. In certain preferred
embodiments of the treatment method, a suitable regime for treating a person
infected with human
immunodeficiency virus includes doses of at least about 3.0 mg/kg of body
weight administered
intravenously in seven equal doses over i4 days.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures for Section A
Figure lA shows a 1973 base pair Hind III to Eco Rl sub fragment of the Friend
Marine
Leukemia Virus (FMLV) clone 57 genome. Figure 1 B shows a 172 base pair
(HindllI to StuI) fragment
which is an expanded portion of the 1973 base pair fragment. Within this
fragment is the purine rich
target to which triple helix forming oligonucleotides are directed. Figure 1 C
shows the entire Hind
III/Eco Rl FMLV fragment cloned into the pT7-2 plasmid (United States
Biochemical Corporation)
yielding p275A. In this recombinant the Hind III site is 10 base pairs
downstream of the T7 mRNA start
site. The 5' portion of the triple helix target region is 63 base pairs
downstream of the mRNA start and
the Dde I site is 131 base pairs downstream of the mRNA start site. Figure 1 D
shows the Hind III/Eco
R1 FMLV fragment was cloned into pBS (Stratagene) yielding pBSFMLV. The Hind
III site, triple helix
target site and Dde I site are respectively 50, 103 and 171 base pairs
downstream from the mRNA start
site.
Figure 2 shows that G-Rich phosphorothioated-oligonucleotides induced
reduction in HSV-2
viral titer. VERO cells infected with HSV-2 were treated with various
concentrations of the indicated
drug. The results are plotted as percent virus yield relative to VERO cells
infected with virus but not
treated with drug (titer = 1 ). The filled square (B 106-62) (SEQ. ID. N0. 5)
represents a single
concentration point (20 1M) for this oligonucleotide. B 106-96 is the fully
phosphorothioated version of
B 106-62 (SEQ. )D. NO. 5). B 106-97 is the fully phosphorothioated version of
B 106-71 (SEQ. ID. NO.
6). ACV (4a and 4b) is acyclovir tested against two different stock
concentrations of HSV-2 strain
HG52. In two experiments, after virus infection and before reapplication of
oligonucleotide (BIO-96 or
BIO-97), the cells were rinsed with a pH 3 buffer in order to remove all virus
not yet internalized (96p3
and 97p3).
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Figure 3 shows MT-2 cells infected with 0.01 m.o.i. of HIV-I and then treated
with various
concentrations of oligonucleotide or AZT or ddC. The data represents the
number of viable cells
remaining in the culture dish, i.e., not undergoing virus induced cytopathic
effects (CPE). In this graph,
100% is the level of CPE occurring in cultures infected with virus but not
treated with any drug.
Figure 4 shows the culture media taken from NIH3T3 cells chronically infected
with FMLV was
mixed with various concentrations of I100-51 (SEQ. ID. N0. 29) or I100-12
(SEQ. )D. N0. 27) (fully
phosphorothioate version of I100-00 (SEQ. ID. N0. 20)). The mixtures were then
assayed for the
presence of viral reverse transcriptase. The data is presented as a percent of
measurable reverse
transcriptase in culture medium not treated with oligonucleotide.
Figures SA, SB and SC show the radio-labelled (32P) full-length or truncated
mRNA transcripts
were analyzed by polyacrylamide gel electrophoresis, and then quantitated by
cutting out the specific
transcript and measuring the radioactivity in a scintillation counter. Figure
SA shows that the reduction in
full length transcripts directed by the T7 and T3 promoter when I100-51 (SEQ.
)D. N0. 29) (anti-parallel
triple helix forming oligonucleotide; FMLV2ap) was added. Samples in which no
oligonucleotide was
added were counted and used as 100% transcription reference points. In all
other reactions 4 x 10'~M of
6101-SO (SEQ. ID. N0. 12) (4e-6) was added and where indicated 6101-50 plus
I100-51 at
concentrations ranging from 2 x 10'9 to 2 x 10'~ M (2e-9 to 2e-6). Figure SB
shows the reduction in full
length transcript by I100-O 1 (SEQ. )D. N0. 21 ) (FMLV2p). T7 directed
transcripts were treated as in
Figure A. 6101-50 was added to each reaction except the control (no oligo)
with or without various
concentration of I100-O1 or II00-11 (SEQ. )D. N0. 26) (26% G-ct!). Figure SC
shows the analysis of
truncated (63 base pair) transcript.
Figure 6 shows inhibition of HIV-1 induced syncytia formation four days post-
infection. SUP T1
cells were infected with HIV-ID~ for four hours and then treated with various
concentrations of
oiigonucleotides. Four days post-infection cells were scored for syncytiurn
formation. All assays were
performed in quadruplicate and the average values used to plot this graph. The
legend to the right of the
graph indicates the symbol used far each oligonucleotide tested.
Figure 7 shows continued suppression of HIV-1 p24 production seven days post
removal of
oligonucleotide. Four days post-infection with HIV-1 pv, the media from
infected cells treated with
oligonucleotides (2.5 TM) was removed and replaced with fresh media without
oligonucleotide. The
presence of viral p24 antigen was then assayed 7 and 11-days post infection.
All samples were assayed in
quadruplicate and the average values used to plot this graph. I100-07 (SEQ.
ID. NO. 24): /100-1 S (SEQ.
ID. NO. 33); I100-18 (SEQ. ~. N0. 36); I-10021 (SEQ. m. N0. 39). The legend to
the right of the graph
indicates the symbol used for each oligonucleotide tested.
- Figure 8 shows a Dixon Plot of random oligonucleotide 1232 (SEQ. )D. NO. 41)
obtained from
kinetic analysis of inhibition of HIV-RT with respect to dNTP. The inhibition
constant K; was
determined by simultaneously varying dNTP (without dATP) concentrations at the
same time as inhibitor
(oligonucleotide 1232). The K; determination was performed at 0.125 mM, 0.25
mM and 0.5 mM dNTP
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concentrations with constant Primer-Template concentration of 0.2 pM. HIV-RT
was used at 1 unit in
each reaction. The reported values are the result of simultaneous independent
duplicates determinations.
Figure 9A reveals PBMCs derived from HIV-1 positive patients were mixed with
HIV-1
negative PBMCs in culture medium containing drug I100-I S (SEQ. 1D. NO. 33).
On day 7 the
cocultures were washed and resuspended in fresh medium containing drug. The
p24 levels in medium
collected on day 7 (before medium change) and day i0 were assayed for p24.
Figure 9B HIV-1 negative
PBMCs from two different donors were infected with HIV-1 ov and then incubated
in the presence of
drug for 10 days at which time the culture medium was assayed for the presence
of p24 antigen.
Figures l0A and IOB show inhibition of binding of V3 loop specific Mabs to HIV-
1 gp120 by
phosphorothioate containing oligonucleotides. Matched sequence
oligonucleotides with either
phosphodiester (PD) or phosphorothioate (PT) backbones were assayed for their
ability to inhibit the
interaction of V3 loop specific Mabs with the gp120 molecule: SEQ. ID. NOS. 31
( 1173) and 32 ( I 174);
SEQ )D. NOS. 24 (I100-07) and 39 (I100-21); or SEQ. DJ. NOS. 42 (1229) and 43
(1230). To do this,
immobilized gp120 was preincubated with oligonucleotides before washing and
the addition of Mab
NEA 9284 ( 10 A) or Mab NEA 9301 ( 10 B).
Figure 11 shows a schematic diagram of the HIV-I genome not drawn to scale.
Fig. 11 A shows
DNA primers. Fig. 11B shows RNA primers.
Figure 12 shows analysis of DNA (PCR) and RNA (RT-PCR) extracted from SUP T1
cells three
days post-infection with HIV-1. (Top Panel). PCR analysis of HIV-1 infected
drug treated SUP T1 cell
DNA used 0.1 Tg of total extracted DNA for each reaction. In this experiment
either AZT, at 0.3 TM
which is 10 fold over the ICso value (lane I ) or I100-15 (SEQ. ID. NO. 33) at
5.0 (lane 2) or 0.3 TM (lane
3) were added to SUP T1 cells at the same time as HIV-1. Lanes 4 (AZT), 5 (5.0
TM I100-15 (SEQ. ID.
NO. 33)) and 6(0.3 TM 1100-15) are the results of DNA samples obtained from
cells in which drug was
added 8 hours post-infection. Lanes 8 to 10 contain 10, 100 or 1000 ng of DNA
extracted from HIV-1
infected control SUP T1 cells. The band corresponding to 220 by is the
predicted size of the internal ~
actin control and the 200 by fragment is the predicted size for the amplified
portion of the HIV-1 genome.
The bottom panel contains RT-PCR analysis of extracted RNA ( I Tglreaction)
obtained from cells
treated in an identical fashion as those described in lanes 1-6 of the top
panel. Lanes 7 and 8 are control
HIV-1 infected cell mRNA and lanes 9 and 10 are the results obtained. using
uninfected untreated SUP T1
cell mRNA.
Figure I 3 shows the results of three oligonucleotides ( 10'SM) incubated with
increasing
concentrations (0,7.5,15,30,60 and 120 mM) of KCl (lanes 1-6 for 1100-15 {SEQ.
1D. NO. 33), 7-12 for
1100-18 (SEQ. B7. NO. 36} and 13-18 for 2106-50). The nucleotide markers are
poly dT.
Figure I4 shows a line model for I100-15 (SEQ. ID. NO. 33) folded into an
intramolecular tetrad
of the Oxytricha class is depicted. The 5!-end of the molecule is in the
bottom left hand side. The bases
/D
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(Gs) are stacked on top of each other with the 4 bases in each plane
stabilized through their hydrogen
bonding with each other and their interaction with the K+ ion complex in the
center of the tetrad.
Figure 15 displays a one dimensional NMR analysis of a KCl titration and
thermal melting
parameters for I100-15 (SEQ. ID. NO. 33).
Figures for Section B
Figure 16. Dose responsive profile for T30177, AZT and ddC. CEM-SS cells were
infected
' with HIV-1(0.01 MOI) and treated with various concentrations of each drug
for six days at which time
the degree of HIV-1-induced syncytium formation (cytopathic effect, cpe) was
addressed. The results
shown are the averages of three or more experiments with the standard
deviations indicated.
IO Figure 17. Effect of T30177 on HIV-1 replication in primary macrophages.
Primary
macrophages were obtained from PBMC preparations and infected with HIV-1 p~
for 24 hours in the
presence of the indicated amount of drug. Seven days post-infection the
intracellular levels of p24 were
quantitated using the Coulter p24 antigen capture ELISA kit. The results shown
are the averages of three
or more experiments.
Figure 18. Effect of time of drug addition on the inhibition profile of
T30177, AZT, and
DS5000. MT4 cells, infected with HIV-1,~ at a MOI of 1, were treated at
various times during (time 0)
or post-virus-infection with the test compounds at a concentration 100-fold
higher than their respective
ICSO values. Viral p24 levels in the culture medium were monitored 29 hour
post-infection. The results
shown are the averages of three or more experiments.
Figure 19. HeLa-CD4-(3-galactosidase cell assays. Figure 19A. HeLa-CD4-~i-
galactosidase
cells were incubated in medium containing drug for one hour before virus was
added to the culture
medium. One hour after the addition of virus the cells were washed extensively
to remove unbound virus
and extracellular test material. Forty-eight hours past-infection the cells
were fixed and stained with X-
gal. Blue multinuclear cells were then counted under an inverted microscope
(5). Figure 19B. HeLa-
CD4-(3-galactosidase cells were incubated for 1 hour in the presence of test
compound at which time an
equal number of HL2/3 cells were added to each well. Cells were incubated for
48 hours at which time
they were fixed, stained with X-gal and counted under an inverted microscope.
Figure 20. Long term suppression of HIV-1 ~ after treatment of infected cell
cultures with
T30177. (A) MT4 cells were infected with 0.01 MOI of HIV-1 ~ and then cultured
for 4 days int eh
presence of T30177, AZT, DSS000, JM2763 or JM3100 using a concentration of
drug equivalent to 100
fold over the respective ICSO value. After 4 days the cells were washed
extensively and further incubated
- in drug free medium. The level of viral p24 antigen in the culture medium
was monitored at various
times after removal of drug from the infected cell cultures. The values given
are the averages of three or
more experiments.
Figures 21A-B. Single cycle analysis of viral DNA. CEM-SS cells infected with
HIV-Is~ at an
MOI of 1, were treated with T30177, UC38, CSB or ddC at the indicated time
post-viral infection. Time
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0 indicates the treatment of cell cultures with drug during virus infection.
After 12 hours the DNA was
extracted from the infected cells and used as a template for PCR. The
concentration of drug used in each
assay is equivalent to 10 to 100-fold over their respective IC25" values.
Figure 22. Analysis of replicated viral DNA. CEM-SS cells were infected with
HIV-15~ at an
MOI of 1 and then treated with T30177. Eighteen to 20 hour post-infection the
low molecular weight
Hirt DNA was analyzed using PCR primers which would amplify mitochondria) DNA
{Fig. 22A), early
viral synthesized cDNA (Fig. 22C), viral gag cDNA (Fig. 22D) and viral 2-LTR
circles (Fig. 22B). The
drug concentrations used were 0.0, 0.01, 0.1, 1 and 10 1 M corresponding to
lanes I to 5 respectively.
The unlabeled lane in each panel contains molecular size marker control DNA.
Figures for Section C
Figure 23. Inhibition of HIV-1 integrase 3'-processing and strand transfer and
HIV-1 ~
cytopathicity by guanosine quartets. (Fig. 23A) Schematic diagram showing 3'-
processing (3'P, which
liberates a GT dinucleotide) and strand transfer (S.T., which results in the
insertion of one 3'-processed
oligonucleotide into another target DNA), with 5'-end labeled (asterisk),
blunt-ended oiigonucleotide.
(Fig. 23B) Left panel, concentration-response obtained from a typical
experiment. The DNA substrate
(2lmer), 3'-processing product (l9mer), and strand transfer products (STP) are
shown. Lane 1, DNA
along; lane 2, with integrase; lanes 3-6, with integrase in the presence of
the indicated concentrations of
T30177. Right panel, graph derived from quantitation (see Materials and
Methods) of the dose response
in the left panel showing inhibition of integrase-catalyzed 3'-processing
(open squares) and strand transfer
(filled squares}. (Fig. 23C) Structures of guanosine quartets
oligonucleotides. (Fig. 23D) ICso values for
several G4 oligonucleotides against both activities of HIV integrase and HIV-1
~ in cell culture.
Insertions into the parent compound T30177 are shown by an italicized and
underlined nucleotide while
mutations are designated by a lower case nucleotide. The guanosines involved
in the quartets are shaded
and the loops are designated by the corresponding numbers (see panel C, left).
Figure 24. Inhibition of strand transfer and 3'-processing activities of HIV-1
integrase by the
guanosine quartet T30177. (Fig. 24A) Left, schematic diagam depicting the
strand transfer assay using
the precleaved oligonucleotide ( 19mer substrate). Right Phosphorimager
picture showing inhibition of
strand transfer with T30177. The DNA substrate ( l9mer) and strand transfer
products (STP} are shown.
Lane 1, DNA alone; lane 2, plus integrase; lanes 3-6, plus integrase in the
presence of the indicated
concentrations of T30177. (B) Left, schematic diagram depicting the 3'-
processing assay using the
oligonucleotide labeled at the 3'-end with 32P-cordycepin (*A) (22mer
substrate). Right, phosphorimager
picture showing inhibition of HIV-1 integrase-catalyzed 3'-processing with
T30177. Lane 1, DNA alone;
lane 2, with integrase; lanes 3-6, in the presence of the indicated
concentrations of T30177.
Figure 25. Inhibition of the DNA binding activity of HIV-1 integrase by
guanosine quartets.
DNA binding was measured after LTV crosslinking of reactions in which
integrase was preincubated for
30 minutes at 300C with the guanosine quartet prior to addition of the DNA
substrate. (Fig. 25A)
/~
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Phosphorimager picture showing differential inhibition of DNA binding with
T30177 and T30659. Lane
1, DNA alone (20 nM); lanes 2, 8, and 14, with integrase (200 nM); lanes 3-7,
in the presence of the
indicated concentrations of T30177; lanes 9-13, in the presence of the
indicated concentrations of
T30659. The mitigations of proteins of known molecular weight are shown to the
right of the gel. (Fig.
S 25B) Graph derived from quantitation of the does response in (Fig. 25A)
showing inhibition of integrase
binding by T30177 (open squares) but not by T30659 (filled squares).
Figure 26. Differential activities of T30177 on wild-type and deletion mutants
of HIV integrase.
(Fig. 26A) Schematic diagram showing the three domains of HIV-1 integrase. (B)
Inhibition of wild-
type IN'-z88 (open squares), IN'-z'z (closed squares), and INs°'ziz
(open triangles) in the disintegration
assay. (Fig. 26C} Binding of HIV-1 integrase wild-type (IN'-z88) and deletion
mutants at a final
concentration of 1 1 M to 3zP-end labeled guanosine quartet T30177 at a final
concentration of 250 nM.
The mobility of proteins of known molecular weight (in KDa) are shown to the
right of each figure. Lane
I, T30177 alone; lanes 8-9, binding to wild-type, full-length HIV-1 integrase
(IN'-zg8) in the presence of
the indicted metal; lanes 2-3, binding to IN'-z'z in the presence of the
indicated metal; lanes 6-7, binding
to INso-m in the presence of the indicated metal are lanes 4-5, binding to
INs°-zas
Figure 27. DNA binding activity of the zinc finger domain of HIV-1 integrase.
Binding of IN'-ss
to T30177 or the viral DNA substrate (see Fig. 23A, 2lmer). Lanes I, DNA alone
(50 nM); lanes 2, IN''
ss (2 1 M) with no metal; lanes 3, IN'-ss with manganese (7.5 mM); lanes 4,
IN'-ss with magnesium (7.5
mM); lanes 5, IN'-ss with manganese (7.5 mM) and zinc (4.2 mM); lanes 6,
IN''ss with magnesium (7.5
mM) and zinc (4.2 mM); lanes 7-10, IN'-ss in the presence of the indicated
concentration of zinc alone.
Figure 28. Increased binding to and inhibition by guanosine quartets in
magnesium versus
manganese. {Fig. 28A) Phosphorimager picture showing DNA binding of wild-type
integrase to
radiolabeled T30177. Lane 1, DNA alone (27 nM); lanes 2-5; binding of
integrase (200 nM) in
manganese buffer to the indicated concentration of T30177; lanes 6-9, binding
of integrase (200 nM) in
magnesium buffer to the indicated concentration of T30177. The migrations of
proteins of known
molecular weight are shown to the right of the gel. (Fig. 28B) Structures of
T30177 and two analogs in
which the internucleotidic linkages have been changed. (Fig. 28C) graph
derived from quantitation (see
Materials and Methods) of the inhibition of integrase-catalyzed 3'-processing
in the presence of T30177
and analogs in either magnesium or manganese. Inhibition by T30177
{triangles), T30175 (squares), and
T30038 (circles is shown either containing magnesium (filled symbols) or
manganese (open symbols).
(Fig. 28D) Table showing ICsa values for 3'-processing for the guanosine
quartets in buffer containing
manganese and magnesium and the ratio of these values.
Figure 29. Competition of binding to either US viral oligonucleotide (see Fig.
23A, 21 mer) (Fig.
29A) or guanosine quartet T30177. (Fig. 29B) Lanes 1, DAN alone; lanes 2, with
wild-type, full-length
HIV-1 integrase. Lanes 3-6 in panel (Fig. 29A), with integrase in the presence
of the indicated
concentrations of T30177 added after a 5 minute preincubation with the U5
viral DNA oligonucleotide.
/3
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Lanes 3-6 in panel (Fig. 29B), with integrase in the presence of the indicated
concentrations of viral US
DNA oligonucleotide added after a 5 minute preincubation with the guanosine
quartet T30177.
Figure 30. Inhibition of the related retroviral integrases. (Fig. 30A)
Inhibition of 3'-processing
and strand transfer catalyzed by HIV-1 (lanes 2-8), HIV-2 (lanes 9-15), FIV
(lanes 16-22), and SIV (lanes
23-29) integrases in the presence of T30177. Lane 1, DNA alone; lanes 2, 8, 9,
15, 16, 22, 23, and 29,
with integrase; lanes 3-7, 10-14, 17-21, and 24-28, with integrase in the
presence of the indicated
concentrations of T30177. (Fig. 30B) Graph derived from quantitation (see
Materials and Methods) of
the dose responses in {Fig. 30A) showing inhibition of H1V-1 (open
rectangles), HIV-2 (filled
rectangles), FIV (open triangles), or SIV (filled triangles) integrase-
catalyzed 3'-processing.
Figure 31. Three-dimensional drawings of certain guanosine tetrad forming
oligonucieotides
referred to in Tables C-1 and C-2. Halosubstituted, end modified, and
intermolecular guanosine quartets
are shown.
Figure 32. Three-dimensional drawings of certain guanosine tetrad forming
oligonucleotides
referred to in Tables C-l and C-2. Unless otherwise specif ed, all
oIigonucieotides have
I S phosphorothiodiester linkages between the ultimate and penultimate bases
at both the 5' and 3' ends. (*)
denotes the position of the phosphorothiodiester linkages.
Figure 33. Percentage inhibition of 3' processing by certain oiigonucleotides
in Table C-1.
Figure 34. Inhibition of syncytium formation by certain oligonucleotides in
Table C-1.
Figure 35. Mutations in the loops of T30177. Three-dimensional drawings of
certain guanosine
24 tetrad forming oiigonucleotides referred to in Tables C-1 and C-2.
Figure 36. Mutations, deletions and insertions in G quartets. Three-
dimensional drawings of
certain guanosine tetrad forming oiigonucleotides referred to in Tables C-1
and C-2. ICso for 3' proc./str.
tra. is indicated to the right of each tetrad.
Figures for Section D
25 Figure 37. Structure Models. Fig. 37A. The sequence and a structure model
for oligonucieotides
used in this study presented All four oligomers have been modified so as to
include a single
phosphorothioate linkage at the S' and 3' terminus. Proposed sites of G-
quartet fornration have been
identified by dotted lines. The continuity of the phosphodiester backbone is
identified by solid lines. Fig.
37B. A two step kinetic model for ion induced folding of oligomers in this
study. It is proposed that
30 binding a first K+ or Rb+ ion equivalent, marked as a (+), occurs within
the central G-octet, which has
been identified by dotted lines. This first step is relatively fast, and is
associated with higher apparent ion
binding affinity. It is also associated with formation of unstacked loop
domains, and the resultant net loss
of UV hypochromism, as compared to the initial random coil state. The second
step in the process
involves as many as two additional K+ or Rb+ ion equivalents, (+), at the
junction between the core octet
35 and flanking loop regions. This second step requires significant ordering
of the flanking loop domains,
and is therefor associated with an increase of base stacking interaction, and
a generally high activation
energy.
CA 02279488 1999-07-29
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Figure 38. Thermal Stability of Oligomer Folding. Thermal denaturation of
oligomers has been
measured as a function of ion type, ion concentration and strand
concentration. Data have been obtained
at 240 nm, in 20 mM Li3P04, pH 7, as the supporting buffer. Tm values were
calculated from the first
derivative of a plot of absorbance vs. temperature, but similar values were
obtained by using the midpoint
of the overall absorbance change. Fig. 38A. Tm values for T30695 (curve a),
T30177 (curve b), T30376
(curve c), and T30677 (curve d) obtained as a function of added 12 KC 1
concentration. Fig. 38B. The Tm
Of T30695 obtained as a function of KCI, RbCI, NaCI or CsCI concentration.
Fig. 38C. The strand
concentration dependence of Tm has been measured at I mM of added KC 1.
Figure 39. Oligomer Folding Monitored by Circular dichroism (CD). CD data have
been
obtained at 250C in 20 rnM Li3P04 as a function of added ion concentration.
Data have been presented
as molar ellipticity in units of dmole bases. Fig. 39A. The CD spectrum of
T30695 in the presence of 0
mM (curve a), 0.05 mM (curve b), or 10 mM (curve e) of added KC 1. Fig. 39B.
The change in ellipticity
at 264 nm, relative to that measured in the absence of added ion is presented
as a function of added KCl
concentration for T30695 (curve a), T30177 (curve b) and T30676 (curve c). The
overall midpoint of the
measured KCI induced transition has been plotted for each oligomer: 0.02 mM,
0.15 mM and 0.27 mM,
respectively. Fig. 39C. T30695 has been treated with increasing concentration
of several different cations.
The change in ellipticity at 264 nm was then measured as described in part B
as a function of added KC 1
(curve a), RbCI (curve b) or NaC 1 (curve e).
Figure 40. The Kinetics of Ion Induced Folding. Ion was added to oiigomers at
time zero in the
standard 20 mM Li3P04 assay buffer. Data have been presented as absorbance (A)
vs. time after addition
of metal ion. Fig. 40A. Kinetics for T30I 77 were measured at three added KC 1
concentrations: 0.2 mM
(curve a); 1.0 mM (curve b); and 10 mM (curve e). Fig. 40B. Kinetics for
T30695 were measured at three
added RbC 1 concentrations: 1.0 mM (curve a), 5.0 mM (curve b) and 10 mM
(curve e). For both, the data
has been fit to a sum of two exponentials, i.e. A(T) = A, exp(-T/T, ) + AZexp(-
i/T,_).
Figures for Section E
Figures 41 A-D. Mean arterial pressure of cynomolgus monkeys pAor to, during
and following
intravenous administration of AR177 over ten minutes. Blood pressure was
continuously monitored via
an indwelling femoral artery catheter. The values are the mean t s.d. of three
monkeys at each dose.
Figures 42A-D. Neutrophil levels in blood of cynomolgus monkeys prior to,
during and
following intravenous administration of AR177 over ten minutes. Neutrophii
levels were determined
pre-dose (-10 minutes), and at 10, 20, 40, 60,120 and 1440 minutes following
the initiation of the
ten-minute infusion of ARI77 into cynomolgus monkeys. The values are the mean
~ s.d. of three
monkeys at each dose.
Figure 43. aPTT versus time profile following a ten-minute infusion of AR177
to cynomolgus
3$ monkeys. aPTT was determined before and at various time after intravenous
infusion of AR177 as
described in the Methods section. aPTT levels returned to baseline by 24 hours
in all groups. Certain
~ S
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aPTT values in monkeys at the 20 and 50 mg/kg dose time points, denoted by
asterisks, exceeded the
upper limit of the assay.
Figure 44. Complement factor Bb concentration versus time profile following a
ten minute
infusion of AR177 to cynomolgus monkeys. Bb was determined before and at
various times after
intravenous infusion of AR177 as described in the Methods section. Bb levels
returned to baseline by 24
hours in all groups.
Figure 45. CH50 levels in blood of cynomolgus monkeys prior to, during and
following
intravenous administration of AR177 over ten minutes. CH50 levels were
determined pre-dose (-10
minutes), and at 10, 20, 40, 60,120 and 1440 minutes following the initiation
of the ten-minute infusion of
AR177 into cynomolgus monkeys. The values are the mean of two monkeys in the
saline and 50 mg/kg
groups, and three monkeys in the 20 mglkg group. Data for the third monkey in
the saline and SO mg/kg
groups, and for all of the 5 mg/kg group was not available.
Figure 46. Plasma C",~ of AR177 in cynomolgus monkeys administered AR177 as a
ten-minute
intravenous infusion. The plasma concentration of AR177 was determined by
anion-exchange HPLC as
described in the Methods section.
Figure 47. AR177 plasma concentration versus time profiles following a ten-
minute intravenous
infusion to cynomolgus monkeys. The plasma concentration of AR177 was
determined by
anion-exchange HPLC as described in the Methods section. The plasma ARI77
concentration at 24 hours
for the 5, 20 and 50 mglkg groups were <0.020 g/mL for the 5 and 20 mglkg
groups, and 0.24 ~ 0.42
1/mL for the 50 mglkg group.
Figure 48. The relationship between plasma AR177 and aPTT in cynomotgus
monkeys
following a ten-minute intravenous infusion of S mg AR177/kg. The plasma
concentration of ARI77 was
determined by anion-exchange HPLC as described in the Methods section. The
baseline aPTT level (at
10 minutes prior to dosing) was 32.1 ~ 4.4 seconds (mean t s.d.).
Figure 49. The relationship between plasma AR177 and aPTT in cynomolgus
monkeys
following a ten-minute intravenous infusion of 20 mg AR177/kg. The plasma
concentration of AR177
was determined by anion-exchange HPLC as described in the Methods section. The
baseline aPTT level
(at 10 minutes prior to dosing} was 41.6 t 6.7 seconds (mean t s.d.). The aPTT
value in monkeys at the
10 minute time point, denoted by an asterisk, exceeded the upper limit of the
assay.
Figure 50. The relationship between plasma AR177 and aPTT in cynomolgus
monkeys
following a ten-minute intravenous infusion of 50 mg AR177/kg. The plasma
concentration of AR177
was determined by anion-exchange HPLC as described in the Methods section. The
baseline aPU level
(at 10 minutes prior to dosing) was 33.2 t 4.8 seconds (mean t s.d.). Certain
aPTT values in monkeys at
the 10 to 120 time points, denoted by asterisks, exceeded the upper limit of
the assay.
Figures for Section F
Figure 51. AR177 plasma concentration after bolus IV dose 1 or 12 versus dose
amount in
Cynomolgus monkeys. Cynomolgus monkeys were given intravenous doses of 2.5, 10
or 40 mg/kg/day
/h
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every other day for a total of 12 doses. Blood was obtained 5, 30 and 240
minutes following doses 1 and
12. The concentration of AR177 in the plasma of every monkey was determined by
anion-exchange
HPLC as described in the Methods section. There were six monkeys in the 10 and
40 mg/kg groups, and
eight monkeys in the 40 mg/kg group. There was a linear relationship between
each dose and the plasma
concentration that was achieved at each of the sampling times.
Figure 52. AR177 plasma concentration versus time profile following a bolus IV
injection (dose
12) to Cynomolgus monkeys. Cynomolgus monkeys were given intravenous doses of
2.5, 10 or 40
mg/kgtday every other day for a total of 12 doses. This figure shows the
concentration of AR177 in the
plasma 5, 30 and 240 minutes following dose 12. The concentration of AR177 in
the plasma was
determined in every monkey by anion-exchange HPLC as described in the Methods
section. There were
six monkeys in the 2.5 and 10 mglkg groups, and eight monkeys in the 40 mg/kg
group. There were no
apparent difference between the disappearance of AR177 from the plasma
following the 1st (Figure F-3)
and 12th doses.
Figure 53. The relationship between the plasma AR177 concentration and aPTT in
Cynomolgus
monkeys following a bolus IV injection of 2.5 mg AR177/kg. Cynomolgus monkeys
were given
intravenous doses of 2.5 mg/kgJday every other day for a total of 12 doses.
This f gore shows the plasma
AR177 concentration versus aPTT levels 5, 30 and 240 minutes following doses I
and 12. The
concentration of AR177 in the plasma was determined in every monkey by anion-
exchange HPLC as
described in the Methods section. There were six monkeys in the 2.5 mg/kg
group. The baseline aPTT
levels just prior to (pre-dose) doses 1 and 12 were 24.1 t 3.4 seconds and
22.1 ~ 2.2. There was no
change in the aPTT levels at any of the time points after the 1 st or 12th
doses of AR 177 at 2.5 mg/kg.
Figure 54. The relationship between the plasma AR177 concentration and aPTT in
cynomolgus
monkeys following a bolus N injection of 10 mg AR177/kg. Cynomolgus monkeys
were given
intravenous doses of 10 mg/kg/day every other day for a total of 12 doses.
This figure shows the plasma
AR177 concentration versus aPTT levels 5, 30 and 240 minutes following doses 1
and 12. The
concentration of AR177 in the plasma was determined in every monkey by anion-
exchange HPLC as
described in the Methods section. There were six monkeys in the 10 mglkg
group. The baseline aPTT
levels just prior to (pre-dose) doses 1 and 12 were 23.3 t 1.8 seconds and
21.6 t 2.2. There was a close
correlation between the aPTT) levels after the 1st or 12th doses of AR177 at
10 mglkg and the aPTT
levels.
Figure 55. The relationship between the plasma AR177 concentration and aPTT in
cynomolgus
monkeys following a bolus IV injection of 40 mg AR177/kg. Cynomolgus monkeys
were given
intravenous doses of 10 mglkg/day every other day for a total of 12 doses.
This figure shows the plasma
AR177 concentration versus aPTT levels 5, 30 and 240 minutes following doses 1
and 12. The
concentration of AR177 in the plasma was determined in every monkey by anion-
exchange HPLC as
described in the Methods section. There were eight monkeys in the 40 mg/kg
group. The baseline aPTT
levels just prior to (pre-dose) doses 1 and 12 were 24.8 t 3.3 seconds and
22.5 t 2.5. Certain aPTT]
t ~-
CA 02279488 1999-07-29
1472-06223 ~ ~ ~ ~ l ~~ ~ ~ / U 1 7 l '~'
. ~~~;~ 8 MAR 1999'
values in monkeys at the 20 and SO mg/kg dose time points at five minutes
following doses 1 or 12,
denoted by asterisks, exceeded the upper limit of the assay. There was a close
correlation between the
aPTT levels after the 1 st or 12th doses of AR177 at 40 mg/kg and the aPTT
levels.
Figure S6. AR177 pharmacokinetics following a single IV dose of 0.75 mg/kg to
humans. Four
S HIV-positive human patients were administered AR177 at 0.75 mg/kg as a two-
hour intravenous (IV)
infusion. Blood samples were collected in EDTA-coated tubes at various time
points during and
following the IV infusion. Plasma was obtained following low speed
centriguation of the blood. The
concentration of AR177 in the plasma was determined using a validated anion-
exchange HPLC method.
Figure S7. AR177 pharmacokinetics following a single IV dose of 1.S mg/kg to
humans. Four
I-EV-positive human patients were administered AR177 at 1.S mg/kg as a two-
hour intravenous (IV)
r~~
infusion. Blood samples were collected in EDTA-coated tubes at various time
points during and
following the IV infusion. Plasma was obtained following low speed
centriguation of the blood. The
concentration of AR177 in the plasma was determined using a validated anion-
exchange HPLC method.
Figure S8. AR177 phatmacokinetics following a single IV dose o 3.0 mg/kg to
humans. Two
1 S HN-positive human patients were administered AR177 at 3.0 mg/kg as a two-
hour intravenous (IV)
infusion. Blood samples were collected in EDTA-coated tubes at various time
points during and
following the IV infusion. Plasma was obtained following low speed
centriguation of the blood. The
concentration of AR177 in the plasma was determined using a validated anion-
exchange HPLC method.
Figure S9. AR177 pharmacokinetics following a single IV dose of 0.75, 1.S or
3.0 mg/kg to
humans. Ten HIV-positive human patients were administered AR 177 at 0.75, 1.S
or 3.0 mg/kg as a two
y,
hour intravenous (IV) infusion. Blood samples were collected in EDTa-coated
tubes at various time
points during and following the IV infusion. Plasma was obtained following low
speed centriguation of
the blood. The concentration of AR 177 in the plasma was determined using a
validated anion-exchange
HPLC method.
2S Figure 60. AR177 T%z and C,~ following single doses to humans. HIV-positive
human
patients were administered AR177 at 0.75, 1.5 or 3.0 mg/kg as a two-hour
intravenous infusion. The
concentration of AR177 was determined in the plasma using a validated anion-
exchange HPLC method.
The C~ (maximal plasma concentration of AR177) and plasma T'/z (half life of
AR177 in plasma)
were determined using PKAnalyst software (Micro Math, Salt Lake City, UT).
Figure 61. AR177 clearance following single doses to humans. H1V-positive
human patients
were administered AR177 at 0.75, 1.5 or 3.0 mg/kg as a two-hour intravenous
infusion. The
concentration of AR 177 was determined in the plasma using a validated anion-
exchange HPLC method.
The plasma clearance was determined using PICAnalyst software (Micro Math,
Salt Lake City, UT).
Figures for Section H
3S Figure 62. An electrophoretogram showing HIV-1 DNA detection of PCR
amplified samples
for animals 1-42.
Figure 63. Another electrophoretogram showing HIV-1 DNA detection of PCT
amplified
samples for animals 1-42, assayed after 30 days.
18
CA 02279488 1999-07-29 s ,v, f ~"~ , ,", , .
1472-06223
Figure 64A. FACS data plot for mouse # 1 treated with AR177 at 100 mg/kg/day
showing
forward- and side scatter characteristics.
Figure 64B. FACS data plot for mouse #1 treated with AR177 at 100 mg/kg/day
showing
thymocyte depletion.
Figure 64C. FACS data plot for mouse # 1 treated with AR 177 at 100 mg/kg/day
showing
mean channel fluorescence of CD4+CD8+ cells.
Figure 64D. FACS data plot for mouse #2 treated with AR177 at 100 mglkg/day
showing
forward- and side scatter characteristics.
Figure 64E. FACS data plot for mouse #2 treated with AR177 at 100 mg/kg/day
thymocyte
depletion.
~~--..
Figure 64F. FACS data plot for mouse #2 treated with AR177 at 100 mg/kg/day
showing mean
channel fluorescence of CD4+CD8+ cells.
Figure 65 . Bar graph showing implant p24, W632 expression, viral titer and
viral RNA load in
HIV-1 (NLA-3)-Infected SCID-hu Thy/Liv Mice treated intraperitoneally with
AR177 at 10, 30 and 100
mg/kg/day, compared to ddI-treated or mock-infected animals.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Index to Detailed Description of the Preferred Embodiments
Definitions
...............................................................................
..................................................................19
A. General In Vitro
Studies........................................................................
..............................................21
B. Specific In Vitro Studies and In Vitro HIV Inhibition Using
T30177...............................................42
C. Site of Activity Studies-Viral Integrase Inhibition
.............................................................................63
D. Structure-Function Studies
...............................................................................
................................... 76
E. Single-dose hemodynamic toxicity and pharmacokinetics of a partial
phosphorothioate
anti-HIV oligonucleotide (AR177) following intravenous infusion to cynomolgus
monkeys........................................................................
................................................................86
F. Repeat-dose toxicity and pharmacokinetics of a partial phosphorothioate
anti-HIV
oligonucleotide (AR177) following bolus intravenous administration to
cynomolgus
monkeys........................................................................
................................................................ 96
G. Human Clinical Trials
...............................................................................
........................................107
H. Anti-HIV-1 Animal Model Studies of
T30177.........................................................................
........115
I. Synthesis of Oligonucleotides Containing C-5 Propynyl-dU Protected Monomers
.........................131
Definitions
The following terms as defined will be used in the description of the
invention:
OLIGONUCLEOTIDE. The term "oligonucleotide"as used herein is defined as a
molecule comprised
of two or more deoxyribonucleotides or ribonucleotides, preferably more than
ten. Some embodiments
of the inventive oligonucleotides are 10-45 nucleotides in length, and certain
preferred embodiments are
19
CA 02279488 1999-07-29
WO 98/33807 PCT/US98/01974
16-17 nucleotides in length. "Oligonucleotide" includes ribonucleic acids,
deoxyribonucleic acids and
modified ribo- or deoxyribonucleic acids.
BASE. Referrences to "bases" herein include pyrimidines and purines, or
modified or derivatized
versions thereof The following abbreviations are used: "A" refers to adenine,
but depending on the
context, may also refer to its ribose, deoxyribose or modified ribose or
deoxyribose form. Similarly, "T"
refers to thymine or thymidine, "U" refers to uracii or uridine, "G" refers to
guanine or guanosine, and
"C" refers to cytosine or cytidine.
~ITION. The term "inhibition" of viral replication is meant to include partial
and total inhibition
of viral replication as well as decreases in the rate of viral replication.
The inhibitory dose or "therapeutic
dose" of the compounds in the present invention may be determined by assessing
the effects of the
oligonucleotide on viral replication in tissue culture or viral growth in an
animal. The amount of
oligonucleotide administered in a therapeutic dose is dependent upon the age,
weight, kind of concurrent
treatment and nature of the viral condition being treated.
PHARMACOLOGICAL DOSE. The term "pharmacological dose" as used herein refers to
the dose of
an oligonucleotide which causes a pharmacological effect when given to an
animal or human. The
pharmacological dose introduced into the animal or human to be treated, will
provide a sufficient quantity
of oligonucleotide to provide a specific effect, e.g., ( 1 ) inhibition of
viral protein or enzymes, (2)
inhibition of viral-specific replication, (3) preventing the target site from
functioning or (4) damaging the
duplex DNA at the specific site or (5) ablating the DNA at the site or (6)
inhibiting the
transcription/translation of the gene under the regulation of the site being
bound or (7) internal inhibition
of transcription or translation of the gene containing the sequence. One
skilled in the art will readily
recognize that the dose will be dependent upon a variety of parameters,
including the age, sex, height and
weight of the human or animal to be treated, the organism or gene location
which is to be attacked and the
location of the target sequence within the organism. Given any set of
parameters, one skilled in the art
will be able to readily determine the appropriate dose.
PATHOPHYSIOLOGICAL STATE. The term "pathophysiological state" as used herein
refers to any
abnormal, undesirable or life-threatening condition caused directly or
indirectly by a virus.
GTO. The term "GTOs" means an oligonucleotide in which there is a high
percentage of
deoxyguanosine, or contains two or more segments (runs) of two or more
deoxyguanosine residues per
segment.
GUANOSINE TETRAD. As used herein, the term "guanosine tetrads" refers to the
structure that is
foamed of eight hydrogen bonds by coordination of the four O6 atoms of guanine
with alkali cations ,
believed to bind to the center of a quadruplex, and by strong stacking
interactions. Of particular interest
to the I100-1 S class of GTO is the structure of the telomere sequence repeat
T4G4, first detected in
Oxytricha. The oxytricha repeat has been studied in oligonucleotides by NMR
and by crystallographic
methods. See Smith et al., Nature, 1992, 356:164-68, and Kang et al., Nature,
1992 356:126-31. As
predicted from numerous previous physical and biochemical studies, both the
NMR and crystallographic
CA 02279488 1999-07-29
WO 98/33807 PCT/US98/01974
studies suggest that folding is mediated by square planar Hoogsteen H-bonding
among G-residues, with
overall antiparallel orientation of the four strand equivalents comprising the
tetrad fold. As expected, the
crystallography has shown that the structure is selectively stabilized by
tight binding of a small
monovalent cation to the 06 oxygen of guanosine.
The following examples are offered by way of illustration and are not intended
to limit the
invention in any manner.
A. General In Vitro Studies
The present invention provides methods and compositions for treating a
pathophysiological state
caused by a vints, comprising the step of administering a pharmacological dose
of an oligonucleotide, the
dose being suffcient to inhibit the replication of the virus, wherein the
oligonucleotide contains sufficient
contiguous guanosines so that a guanosine tetrad (inter- or infra- molecular)
can form, and the three
dimensional structure of the oligonucleotide is stabilized by guanosine
tetrads formed at strategic
locations. Generally, this method of treating a virus-induced
pathophysiological state may be useful
against any virus. More preferably, the methods of the present invention may
be useful in treating
pathophysiological states caused by viruses such as herpes simplex virus,
human papilloma virus, Epstein
Barr virus, human immunodeficiency virus, adenovirus, respiratory syncytial
virus, hepatitis B virus,
human cytomegalovirus and FTIT.V I and II.
Generally, the oligonucleotides of the present invention contain a percentage
of guanosine bases
high enough to ensure anti-viral e~cacy. The guanosine is important in forming
tetrads which stabilize
the three dimensional structure of the oligonucleotides. Thus, the
oligonucleotides of the present
invention may have any percentage of guanosine bases which will allow for
tetrad formation provided
that the oligonucleotide exhibits anti-viral activity. Preferably, the
oligonucleotides of the present
invention contain two or more segments of two or more guanosine bases, and an
overall high percentage
of G in order to enable the oligonucleotide to form at feast one quanosine
tetrad.
Generally, the oligonucleotides of the present invention may be capped at
either the 3' or the 5'
terminus with a modifier. Preferably, the modifier is selected from the group
consisting of polyamine or
similar compounds that confer a net positive charge to the end of the
molecule, poly-L-lysine or other
similar compounds that enhance uptake of the oligonucleotide, cholesterol or
similar lipophilic
compounds that enhance uptake of the oligonucleotide and propanolamine or
similar amine groups that
enhance stability of the molecule.
The phosphodiester linkage of the oiigonucleotides of the present invention
may be modified to
improve the stability or increase the anti-viral activity. For example, a
phosphodiester linkage of the
oligonucleotide may be modified to a phosphorothioate linkage. Other such
modifications to the
oligonucleotide backbone will be obvious to those having ordinary skill in
this art.
The present invention also provides specific methods of treating viral states.
For example, the
present invention provides a method of treating a pathophysiological state
caused by a virus (in preferred
embodiments, as specific virus such as, herpes simplex virus, human papilloma
virus, Epstein Barn virus,
CA 02279488 1999-07-29
WO 98133807 PCT/US98I01974
human immunodeficiency virus, adenovirus, respiratory syncytial virus,
hepatitis B virus, human
cytomegalovirus and HTLV I and II), comprising the step of administering a
pharmacological dose of an
oligonucleotide, the dose being sufficient to inhibit the replication of the
virus, wherein the three
dimensional structure of the oligonucleotide is stabilized by the formation of
guanosine tetrads.
This invention discloses a novel anti-viral technology. The total number of
antiviral
mechanisms by which oligonucieotides, and especially G-rich oligonucleotides,
work is not completely
known, although the inventors have at least narrowed the sites of action as to
certain oligonucleotide
drugs as will be seen below. However, in the different virus culture systems
listed above, G-rich
oligonucleotides were able to significantly reduce virus production in each.
More importantly, actual
human clinical studies have demonstrated the efficacy of the drug in reducing
viral replicons in AIDS
patients. The present invention is also drawn to oligonucleotides that have
three dimensional structures
stabilized by the formation of guanosine tetrads.
The present invention demonstrates poly and/or oligonucleotides inhibit growth
of HIV-I,
HSV 1, HSV2, FMLV and HCMV and other viruses if the molecule contains a high
percentage of ribo- or
deoxyriboguanosine. The rest of the molecule is composed of thymine, cytosine,
xanthosine or adenine
nucleotides (ribo- or deoxyribo-), their derivatives, or other natura) or
synthetic bases. The 5' and 3'
termini of the oligonucleotide can have any attachment which may enhance
stability, uptake into cells
(and cell nuclei) or anti-viral activity. The backbone which connects the
nucleotides can be the standard
phosphodiester linkage or any modification of this linkage which may improve
stability of the molecule
or anti-viral activity of the molecule {such as a phosphorothioate linkage).
Structural formulas for representative G-rich oligonucleotides disclosed in
the instant invention
are listed below in Table A-1.
TABLE A-1.
SEQ ID NO 5(B 106-62)5'-gtggtggtggtgttggtggtggtttggggggtgggg-3'
SEQ ID NO 6(B 106-71 5'-gtggttggtggtggtgtgtgggtttggggtgggggg-3'
)
SEQ ID NO 21 (I 100-O 5'-tggtgggtgtgtggggggtgttgggggttgttggtggggtggtgg-3'
1 )
SEQ ID NO 24(I100-07) 5'-gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg-3'
SEQ ID NO 28(I100-50) 5'-ggtggtggggtggttgttgggggttg-3'
SEQ ID NO 29(I100-51)5'-ggtggtggggtggttgttgggggttgttgggggtgtgtgggtggt-3'
SEQ ID NO 26(I100-11 5'-gatccatgtcagtgacactgcgtagatccgatgatccagtcgatg-3'
)
SEQ ID NO 12(G101-50) 5'-ggtgggtggtttgtgtggttggtgggtttt-3'
SEQ ID NO 13(G105-50) 5'-ggggggggggtgtgggggggggttgtggtgg-3'
SEQ ID NO 14(G 106-50)5'-ggtgggtgggttggggggtgggtgggg-3'
SEQ ID NO 15(G109-50)5'-tggggritgggtggggggttgggtggttg-3'
SEQ ID NO 16(G110-50) 5'-gggtggtggtgttggtgttgtgtg-3'
SEQ ID NO 17(G113-50) 5'-ggtgggggggttggtgtgtttg-3'
SEQ ID NO 1 (A 100-00)5'-tgggtggggtggggtgggggggtgtggggtgtggggtg-3'
SEQ ID NO 2(A100-50) 3'-tgggtggggtggggtgggggggtgtggggtgtggggtg-5'
SEQ ID NO 4(A101-00)5'-ggtggtgggggggggtggggtggtggtgggggtgttgg-3'
SEQ ID NO 18(HIV26ap) 5'-gtgtgggggggtggggtggggtgggt-3'
a~
CA 02279488 1999-07-29
WO 98/33807 PCT/US98/01974
SEQ m NO 19(HIV26ct1)5'-gggtgggtgggtgggtgggtgggtgg-3'
SEQ ID NO 9(B 107-515'-ggtggggtggtggtggttggggggggggggt-3'
)
SEQ ID NO 10(B133-55)5'-ggtggttggggggtggggggg-3'
SEQ ID NO 11(B133-55)5'-gggtggggtggtgggtggggg-3'
SEQ ID NO 20(I100-00)5'-gttgggggttgttggtggggtggtgg-3'
SEQ m NO 27(I100-12,PT)5'-gttgggggttgttggtggggtggtgg-3'
SEQ B7 NO 22(I100-OS}5'-tggtgggtgtgtggggggtgttgggggttgttggtggggtggtgg-CHOL
SEQ ID NO 23 (I 100-06)5'-gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg-CHOL
SEQ ID NO 25(I100-08)5'-gttgggggttgttggtggggtggtgg-CHOL
SEQ 117 NO 3 5'-gggtgggtgggtgggtgg-3'
SEQ ID NO 30 5'-gggtggttgggtggttgg-3'
SEQ ID NO 31(1173) 5'-gggtgggtgggtgggtgg-3'
SEQ B7 NO 32(1174,PT)5'-gggtgggtgggtgggtgg-3'
SEQ ID NO 33(I100-15)5'-gtggtgggtgggtgggt-3'
I SEQ ID NO 34(I100-16)5'-gtggtgggtgggtgggtggtgggtggt-3'
S
SEQ ID NO 35(I100-17}5'-gtggtgggtgggtgggtggtgggtggtggttgtgggt-3'
SEQ ID NO 36(I100-18)5'-ttgtgggtgggtggtg-3'
SEQ ID NO 37(I100-19)5'-tggtgggtggtggttgtgggtgggtggtg-3'
SEQ ID NO 38(I100-20)5'-gtgggtgggtggtgggtggtggttgtgggtgggtggtg-3'
SEQ ID NO 39(I100-21,PT)S'-gtggtgggtgggtgggtggtgggtggtggttgtgggtgggtggtg-3'
SEQ iD NO 40( 1231 5'-gatccatgtcagtgacac-3'
)
SEQ ID NO 41(1232,PT)S'-gatccatgtcagtgacac-3'
SEQ ID NO 42(1229) 5'-cccccccccccccccccc-3'
SEQ ID NO 43(1230,PT)5'-cccccccccccccccccc-3'
SEQ ID NO 44( 1198) S'-ttcatttgggaaacccttggaacctgactgactggccgtcgttttac-3'
SEQ ID NO 45(1200) 5'-gtaaaacgacggcca-3'
SEQ ID NO 46(I100-25)5'-gtggtgggtgggtgggg-3'
SEQ ID NO 47(I100-26)5'-gtggtgggtgggtggg-3'
SEQ ID NO 48(I100-35)5'-tggtgggtgggtgggt-3'
SEQ ID NO 49(I100-27)5'-gtggtgggtgggt-3'
SEQ ID NO 50(I100-28)5'-gtggtgggt-3'
SEQ ID NO 51 (I100-30)5'-gtgggtgggtgggt-3'
SEQ ID NO 52(I100-29)5'-gtgggtgggt-3'
HSV-2 CULTURE ASSAY
In viral yield reduction assays, Vero cells (4 x 10° cellsltissue
culture well) were incubated with
oligonucleotide(s) for 14 hours before the oiigonucleotide was removed and
virus (HSV-2 strain HG52)
was added to the cells at a multiplicity of infection (m.o.i.) of 0.1 to 1.0
{4 x 103 to 4 x 104 PFU). The
infection was allowed to proceed for 10 minutes after which the cells are
washed and fresh media,
containing the same oligonucleotide was added for an additional 14 hours.
Then, the cells were subjected
to a freezelthaw lysis after which the released virus was titered.
HIV-1 CULTURE ASSAY
The SUP T1 T lymphoma cell line was infected with HIV-1 strain DV at a
multiplicity of
infection (m.o.i.) of 0.1 for one hour at 37°C. After the infection,
free virus was washed, off and the
newly infected cells were plated (5 x 10' cells) in quadruplicate in 96 well
plates that had been prepared
~3
CA 02279488 1999-07-29
WO 98/33807 PCTIUS98/01974
with various dilutions of oligonucleotide. The final concentration of drug
varied between 0.1 and 20
pM. After 3 days of incubation at 37°C, the plates were scored for the
presence of multinucleated giant
cells (syncytia).
In assays designed to inhibit syncytia formation, a number of oligonucleotides
exhibited
anti-HIV-1 activity. The oligonucleotides and their IC50 are listed in Table A-
2. I100-OS is the same as
I100-01 with a cholesterol group attached to the 3' end via a trigiycyl-
linker. I100-08 is the same as
I100-00 with a cholesterol group attached to the 3' end via a triglycyl-
linker. I100-07 was designed as a
sequence isomer to I 100-O I and I100-06 is the cholesterol derivative of I100-
07. A 100-00 is the same
sequence in the opposite orientation to HIB38p (A100-50). I100-07, originally
designed as a control for
I100-O1 to be used in anti-FMLV experiments, was the most efficacious
oligonucleotide tested against
HIV-1.
In other experiments, the HN-1 strain LAV was used to infect MT-2 cells at an
m.o.i of 0.01.
After 7 days, these cells were scored for cytopathic effects (CPE). In anti-
HIV-1 assays in which MT-2
cells were infected at an m.o.i. of 0.01, several G-Rich oligonucieotides were
able to inhibit viral-induced
i S cytopathic effects with effective dose 50's (ICSOs) in the 0.5-1.0 uM
range (Figure 3). The
oligonucleotides shown in Figure 3 were effective in the 0.5 to 1.0 uM range,
including A100-00
(HIV38p) and A100-50 (HIV38ap), A101-00 {HIV38ct1), HIV-26ct1. The oligo-
nucleotide HIV-26ap
exhibited less e~cacy in this assay with an IC50 in the 5 to 3 0 uM range. In
Figure 3, TE represents
buffer alone, i.e., no drug, while AZT and ddC are control drugs.
TABLE A-2
ICso for oligonucleotides in an anti-HIV-1 syncytia formation assay.
G-Rich oli~onucleotide IC50
I 100-00 3 .75 1 M
I100-Ol 4.50 1M
I100-OS 3.25 1 M
I 100-08 3.25 1 M
I100-06 0.70 1 M
I100-07 0.25 1 M
A 100-00 3.25 1 M
FMLV CULTURE ASSAY
Friend Murine Leukemia Virus (FMLV) was grown in a chronically infected murine
fibroblast
cell line (pLRB215) or was propagated in an acute assay system by infection of
NIH3T3 cells. When the
chronically infected cell line was used, pLItB215 cells were split (1 x 105)
into 24 well culture dishes and
incubated 16 to 20 hours at 37°C. The media was then removed and
replaced with media containing
various concentrations of oligonucleotide. After 1, 3 or 5 days, culture media
was assayed for the
presence of the viral reverse transcriptase enzyme.
a~
CA 02279488 1999-07-29
WO 98/33807 PCTIUS98/01974
In acute assays, NIH3T3 cells were split ( 1 x 10°) into 96 well dishes
and allowed to incubate for
16-20 hours. After incubation, culture media was removed and concentrated
virus stock ( 10 pl) was
added to each well in 100 ul of completed media containing 2 pg/ml polybrene.
The virus infection was
allowed to proceed for 18 hours at which time the virus containing media was
removed and complete
media containing various concentrations of oligonucleotide was added. After 4
to 7 days, the culture
media was assayed for the presence of viral reverse transcriptase.
HCMV CULTURE ASSAY
Human cytomegalovirus was cultured in the human diploid lung fibroblast cell
line MRC-5.
These cells were split and placed into 24 well culture dishes and preincubated
for 24 hours with various
concentrations of oligonucleotide (0.5 to 20 pM) in complete media. The
oligonucleotide was then
washed off and virus was added to the cells (approximately 0.1 m.o.i.) for 2
hours at 37°C. The virus
was then removed and complete media containing the same concentration of
oligonucieotide was added.
Cells were then placed at 37°C for 10-12 days at which time virus in
the culture media was titered using a
standard agar overlay procedure.
BACTERIAL T3 AND T7 ASSAYS
In this assay system, a 2 kb fragment (HindIII to EcoRl) of the FMLV virus
(clone 57) was
molecularly cloned between the HindIIIIEcoRl sites 10 by downstream of the
bacterial T7 promoter
(p275A) or 50 by downstream of the bacterial T3 promoter (pBSFMLV2). A
schematic representation
of these two recombinant plasmids can be seen in Figure A-I. Isolated
recombinant DNA was then
digested with DdeI. Oligonucleotides were then incubated with the digested DNA
and the mixture was
subjected to in-vitro transcription using either the T7 or T3 bacterial
enzymes.
REVERSE TRANSCRIPTASE ASSAY
In this assay, reverse transcriptase (either MMLV or FMLV from pLRB215 culture
media) was
incubated with various concentrations of oligonucleotide and then assayed
using the enzyme linked
oligonucleotide sorbent assay (ELOSA), the ELOSA kit which is commercially
available from New
England Nuclear.
EUKARYOTIC IN VITRO TRANSCRIPTION
In this assay, a recombinant plasmid containing the HSV-1 IE175 promoter fused
to the bacterial
chloramphenicol acetyltransferase gene (CAT) was linearized and used as a
template for run off
transcription studies. Commercially available HeLa cell nuclear extracts or
prepared nuclear extracts of
HSV-2 infected VERO cell were used.
INHIBITION OF HSV-2 ACTIVTTY
The oligonucleotide B 106-62 was originally designed to form a triple helix
structure with a
portion of the promoter region of the major immediate early protein of HSV-2
(IEI75). The
phosphorothioate derivative of two oligonucleotides were synthesized and
tested for anti-viral activity
against HSV-2. Figure 2 shows that the B 106-62 oligonucleotide at 20 ~M was
able to reduce viral titers
by approximately 20% whereas the phosphorothioate version (B 106-96) reduced
virus by 50% in the
a~
CA 02279488 1999-07-29
WO 98133807 PCT/US98/01974
submicromolar concentration range. The control oligonucleotide (B 106-97), the
phosphorothioate
backbone derivative of B 106-7 l, was also able to inhibit virus at the same
levels as B 106-96. Even when
an extensive washing procedure at a pH of 3.0 was employed to remove excess
virus not internalized
during the infection, incubation with both B 106-96 and B 106-97 was able to
significantly reduce virus
yield. Thus, the inventors concluded that the mechanism of anti-viral activity
was not merely a blocking
of the adsorption of HSV-2 virions to cells.
Figure 2 also shows the results of acyclovir in the same molar range as the
oligonucleotides.
Acyclovir was tested against two different stocks of HSV-2 strain HG52, as
illustrated in Figure 4.
OLIGONUCLEOTIDE SYNTHESIS
All oligonucleotides used in these examples were synthesized on a DNA
synthesizer (Applied
Biosystems, Inc., model 380B or 394), using standard phosphoramidite methods.
All oligonucleotides
were synthesized with an amino modified 3'-terminal, which resulted in the
covalent attachment of a
propanoiamine group to the 3'-hydroxyl group or resulted in a cholesterol
moiety attached to the 3'-
tenninal via a triglycyl-linker. Oligonucleotides used in this example were
capped at their 3'-terminal
with either a propanolamine or a cholesterol moiety to reduce degradation by
cellular exonucleases.
Phosphorothioate containing oligonucleotides were prepared using the
sulfurizing agent TETD or
beaucauge reagent. The 3'-cholesterol modified oIigonucleotides were prepared
and purified as described
by Vu et al. (in Second International Symposium on Nucleic Acidr Chemistry,
Sapporo, Japan, 1993).
STABILITY AND TOXICITY
Guanosine-rich oligonucleotides with either full length phosphodiester (PD) or
full length
phosphorothioate (PT) backbones were stable in the culture media for 4 days,
while oligonucleotides
consisting of a more random composition of nucleotides were rapidly degraded.
This indicates that the
3'-modified G-rich oligonucleotides with PD backbones were stable against both
endonuclease and
exonuclease digestion over a defined four day incubation in culture. The
concentration of oligonucieotide
needed to reduce cell proliferation by 50% (TCso) of selected compounds, based
on the dye metabolism
assay was approximately 40 to 50 pM for oligonucleotides with PD backbones and
15 to 40 p.M for those
compounds containing a PT backbone. The TCso for selected oligonucleotides are
presented in Table A-
3. Stability and toxicity tests were replaced as described below
CA 02279488 1999-07-29
WO 98/33807 PCT/US98101974
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CA 02279488 1999-07-29
WO 98/33807 PCTILTS98101974
A. Cytotoxicity and Stability Assays.
The cytotoxicity of selected oligonucleotides was assayed using the CellTiter
96TM Aqueous
Non-Radioactivity Cell Proliferation Assay (Promega). This is a colormetric
method for determining the
number of viable cells in proliferation or chemosensitive assays using a
solution if MTS. Dehydrogenase
enzymes found in metabolically active cells convert MTS into a formazan
product. The SUP T1 cells
used in the cytotoxicity assays were in log phase growth at the time of the
assay. Cytotoxicity profiles for
GTOs with PD backbones such as I100-I S (SEQ. ID. NO. 33) had TCsos (50%
cytotoxic concentration)
in the range of 30 to 50 1iM while GTOs with PT backbones such as I100-15 had
TCsos in the 10 to 30
l.~M range. The TCso for AZT in this assay format was approximately 10 p.M .
Blockage of the hydroxyl terminus of oligonucleotides has been shown by many
investigators to
greatly reduce degradation by cellular exonucleases. Therefore, ail
oligonucleotides used in these studies
were modified at their 3'- end with either a propanolamine group or a
cholesterol group. For stability
studies, 10 wM of GTOs were incubated in MEM (GIBCO) supplemented with 10%
FBS. Aliquots were
taken after 10 min, 1 day, 2 days, 3 days and 4 days. The aliquots at each
time point were immediately
extracted twice with 50:50 phenol-chloroform solution and then precipitated by
the addition of ethanol.
The recovered oligonucieotides were 5'-end-labeled using [y-32P]ATP and
polynucleotide kinase. The
integrity of the oligonucleotides was then analyzed on a 20% polyacrylamide
gel with 7 M urea. The
results indicated that a portion of each GTO with a PD backbone was present in
the culture medium for
three to four days while oligonucleotides composed of a more random assortment
of all four nucleotides
were rapidly degraded. In addition, positions within PD GTOs where there
existed two or more
contiguous pyrimidines were more susceptible to endonuclease digestion than
regions containing purines
or alternating purines and pyrimidines.
INIEIIBITION OF HIV-I PRODUCTION IN CULTURE ASSAYS
B. Long Term Suppression of Acute HIV-1 Infections in SUP Tl cells. The anti-
HIV-1 activity of a
series of guanosine/thymidine oligonucleotides {GTOs), with PD backbones,
containing different
sequences motifs was tested. As seen in Table A-2, one of the sequence motifs
tested (oligonucleotide
I100-07) was 10 fold more active at inhibiting HIV-1 induced syncytium
formation than the other motifs
tested (e.g. I100-00 shown in Table A-1 ). I100-07 and its derivatives (length
and chemical
modifications) were further tested for their ability to inhibit virus in a
dose-dependent fashion by
measurement of syncytium formation and viral p24 production.
Briefly, HIV-Ipv was used to infect the SUP T1 lymphoblastoid cell line at an
m.o.i. of 0.1
TCIDso for one hour at 37°C prior to washing and resuspension in
increasing concentrations of GTOs.
The cells (2 x 104 cells/well) were inoculated in triplicate in 200 ul of RPMI
1640 containing 10% fetal
calf serum. Four days later, the number of syncytia per well or the level of
p24 in the medium was
determined. The results of these assays are presented in Table A-4. which
results indicated that GTOs
CA 02279488 1999-07-29
WO 98133807 PCT/US98101974
with simple PD linkages were capable of inhibiting HIV-1 syncytia formation
and p24 production in
culture.
CA 02279488 1999-07-29
WO 98/33807 PCT/US98/01974
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CA 02279488 1999-07-29
WO 98133807 PCT/US98101974
- In order to determine the effect of backbone modification on GTO anti-viral
activity, the PD
backbone in two oligonucleotides sequences motifs was replaced with a PT
backbone. The
phosphorothioate containing oIigonucleotides (I 100-12 and I100-21 ) where
then tested for their ability
to inhibit HIV-1 induced syncytium formation and production of HIV-1 p24 in
the SUP T1 acute assay
system (Table A-4). The results from these assays indicated that the presence
of sulfur molecules in the
oligonucleotide backbone greatly enhanced the anti-viral activity of I100-00
(I100-12) but had little if
any effect on I100-07 (I100-21 ) (Table A-4).
It was apparent from the studies that the anti-viral activity of I100-07 was
maintained when
steps were taken to reduce the length of the molecule to 17 by deleting
segments from the 3'-end (I100-
15, -16, -17) but not by deletions from the 5'-end (I100-18, -19, -20). To
further determine to optimal
size of the PD oligonucleotide needed for maximal anti-HIV-1 activity, the
I100-I S size variants listed
in Table A-S were synthesized and assayed for antiviral activity.
TABLE A-5.
Inhibition of HIV-1 Induced Syncytia Using Size variants of 1100-15.
oligo Sequence IC50 Syn.(uM)
I100-15* 5' gtggtgggtgggtgggt-3' 0.16
I100-25 S' gtggtgggtgggtgggg-3' 0.25
I100-26* 5' gtggtgggtgggtggg -3' 0.12
I100-35 5' tggtgggtgggtgggt -3' 1.75
I100-27 5' gtggtgggtgggt -3' 4.50
I100-28 5' gtggtgggt -3' 4.50
I100-30 5' gtgggtgggtgggt -3' 4.50
I 100-29 5' gtgggtgggt -3' > 10.00
AZT 0.02
At 5 uM these compounds suppressed virus at least 7 days post-removal of drug.
All other compounds
at 5 uM were the same as AZT 7 days after removal of drug.
The duration of the viral suppression was assayed by changing the medium in
HIV-1 infected
cultures containing 2.5 uM of various oligonucleotides to complete media
without added oligonucleotide
on day 4 post-viral infection. The production of viral p24 antigen was then
assayed on day 7 and day 11
post-infection. The results of this experiment indicated that the shorter
variants of I100-07 (I100-15 and
I100-16) as well as the PT version of this molecule (I 100-21 ), were capable
of totally suppressing HN-1
p24 production for at least 7 days after removal of the drug from the culture
medium (Table A-6). This
substantial level of prolonged inhibition was >99% for II00-15, I100-16 and
I100-21 when compared to
the p24 antigen levels obtained for untreated HN-1 infected cells (Table A-6).
The quantitation of p24
production relative to untreated HIV-I infected SUP T1 cells for all
oligonucieotides tested is presented
in Table A-b. The presence of sulfur molecules in the backbone of
oligonucleotide I100-07 (I100-21)
had a more marked effect on the reduction of virus seven days after removal of
compound from the
culture medium than was observed at the four day post-infection assay point
(Table A-5).
CA 02279488 1999-07-29
WO 98/33807 PCT/US98/01974
TABLE A-6.
Detection of HIV 1 p24 Antigen in the
Culture Media of GTO-Treated SUP Tl Cells.
Percent p24a
Oligonucleotide (2.SuM) Day 46 Day 7 Day
11
Control SUP TI cells 100.0 % 100.0 % 100.0%
I100-07 6.0 /a 15.9 % 8.6
I100-21 (PT)d 0.0 /a 0.0 /a 0.0
I100-15 0.0% 0.0% 0.0%
II00-16 0.0% 0.0/a 0.0%
I100-18 144.5 /a 9.7 % 5.3
I100-19 208.0 % 21.8 % 15.0
1100-12 (PT) 0.0 % 0.0 % 0.0
a Level of detectable p24 in culture medium relative to control (infected but
untreated SUP T1 cells after subtraction of background values.
b Day 4 post-infection culture medium was replaced with fresh medium
without oligonucleotide.
SUP T1 cells infected with HIV-1 but not treated with oligonucleotides or
AZT were used as positive control cells in this experiment.
d 1100-21 and 1 I00-12 contain phosphorothioate backbone linkages (PT).
In control experiments, the culture medium from HIV-1 infected SUP T1 cells
treated with
AZ1" (4 uM) was also replaced on day 4 post-infection with drug free media. In
these experiments, two
days after removal of AZT from the culture medium the presence of syncytium
was observed in the
HIV-1 infected cell cultures and by day 4 ail cells were visibly infected with
HIV-1.
To determine whether the prolonged suppression of HIV-I was due to toxicity of
the
oligonucleotides, SUP T I cells were counted for all treated samples 7 days
after removal of the
oligonucieotides from the infected cell cultures. The results indicated that
for cells treated with 2.5 ~M
of drug there was no difference in the number of cells when compared with
control cultures (uninfected,
untreated) of SUP T 1 cel ls.
C. Inhibition of HIV expression in patient derived peripheral blood
mononuclear cells (PBMCs).
I100-15 was assessed for activity in PBMC cultures derived from AIDS patients.
Briefly, PHA
activated uninfected PBMC's were added to 4PBMC's derived from patients with
HIV infection in the
presence of varying concentrations of oligonucleotide. Anti-H1V activity was
assessed by analyzing
supernatants, collected every three days from these mixed cultures, for the
presence of HIV p24. The
PHA activated PBMC's were grown in the presence of 10 units/ml of IL-1 and
medium was exchanged
every three days for a period of three weeks. HN p24 antigen production was
assayed in drug-treated
as compared to untreated control specimens. It should be noted that the
results in these experiments
CA 02279488 1999-07-29
WO 98/33807 PCT/US98101974
(Figure 9A-B) observed for AZT were obtained when AZT was used at 12 uM which
is roughly 300
fold greater than the ICso for this compound.
D. In-Vitro inhibition of HIV-1 reverse transcriptase (RT). The ability of
oligonucleotides to inhibit
HIV-1 RT in vitro has been well documented. Marshall et al. PNAS 1992 89:6265-
6269 have described
a competitive interaction at the active site as the mechanism by which mono-
or diphosphorothioate
containing oligonucleotides inhibit HIV-1 RT independent of whether the
molecule tested was antisense,
a random sequence or poly SdC.
In order to determine whether 1100-15 or its parent molecule, I100-07 (or the
PT version I100-
21 ), was interacting with HIV-1 RT, the activity of this enzyme was assayed
in the presence of various
concentrations of oligonucleotides. A kinetic analysis of the resultant enzyme
inhibition was conducted
to determine the mechanism of inhibition. The GTOs appeared to be inhibiting
the RNA dependent
DNA polymerase activity of the RT enzyme by competitive inhibition at the
active site of the enzyme.
The K; value for all of the oligonucleotides tested is presented in Table A-7.
The data indicate
that for all oligonucleotides tested the presence of the sulfur group in the
backbone greatly enhanced the
interaction between the oligonucleotides and the enzymes. The median
inhibitory dose ()DSa) for these
oligonucleotides were also calculated (Table A-7). The IDSa results are based
on the ability of these
compounds to inhibit 10 nM of HIV RT.
Short oligonucleotides (18 mers) with PD or PT backbones were assayed to
determine whether
the nature of the nucleotide sequence contributed to inhibition of HIV-1 RT in
this assay system.
Comparison of the effects of the PD versions of a GTO ( 1173 or 1100-15), poly
dC ( 1229} or a random
nucleotide sequence ( 1231 ) suggested that at this length none of the
sequence motifs inhibited RT
(Table A-7). Other 18 mer PD GTO sequence motifs tested yielded similar
results. Enzyme inhibition
monitored by both K, and IDSO was observed for the PT versions of these same I
8 mer oiigonucleotides
(Table A-7). The degree of enhancement of observed enryme inhibition for all
oligonucieotides tested
when the sulfur group was present in the backbone, was between one to three
orders of magnitude
(Table A-7).
TABLE A-7.
In Vitro Inhibition of HIV-1 RT by PD and PT Oligonucleotides.
Oligonucfeotides Length Linkageb Ki (uM) ID50 (uM) _
I 100-00 26 PD 0.37 5.0
I 100-12 26 PT 0.005 0.015
I100-07 45 PD 0.137 2.5
I 100-21 45 PT 0.001 0.004
I 100-15 17 PD >5.0 >5.0
1173 18 PD >5.0 >5.0
33
CA 02279488 1999-07-29
WO 98133807 PCT/US98/01974
1174 18 PT 0.015 0.0154
1229 (poly dC) 18 PD >5.0 >5.0
1230 (poly dC) 18 PT 0.044 0.033
1231 (GATC) 18 PD >5.0 >5.0
1232 (GATC) 18 PT 0.56 0.045
a Each pair of oligonucleotides contain the same sequence and differ only in
the nature of their backbone linkage. Oligonucleotides 1229 and 1230 were
poly dC while the 1231 and 1232 oligonucleotides were a random sequence
of all four bases (GATC).
b The backbone modifications are denoted as PD for phosphodiester and PT
for phosphorothioate.
The results from this set of experiments demonstrated that I100-15 is
minimally inhibitory to
the RNA dependent DNA polymerase activity of HIV-1 RT. The data also indicated
that chemically
modifying GTOs, poly dC or a random sequence oligonucieotide greatly enhanced
the in vitro inhibitory
activity of the molecule. Therefore, chemically modified oligonucleotides such
as the antiviral G-rich
molecule describe by Wyatt et al. ( 1994) has, by nature, a different set of
characteristics from
oligonucleotides with natural PD backbones.
E. Inhibition of the interaction of HIV-1 gp120 with cellular CD4. The outer
envelope glycoprotein
gp120 of HIV-1 mediates viral attachment to the cell surface glycoprotein CD4
in the initial phase of
HIV-1 infection. The effects of both PD and PT modified oiigonucleotides on
this interaction were
examined using a gp120 capture ELISA kit.
The concentration of the gp120 used in these studies (125 nglmi) was
determined to be within
the linear range of the detection assay. The ability of oligonucleotides to
inhibit gp 120/CD4 interactions
by binding to gp 120 was determined by preincubation of the test compounds
with soluble gp 120 before
addition to the immobilized CD4. The results of this experiment (Table A-8)
are presented as the
concentration of oligonucleotide needed to reduce by 50% CD4 bound gp120 (IDso
[gpi20]). The
reciprocal experiment was then performed to measure the ability of the
oligonucleotides to inhibit these
interactions by binding to immobilized CD4. In this set of experiments I100-
00, I100-07 and the PT
versions of these two oligonucleotides were capable of preventing the
interaction of gp i 20 with
immobilized CD4 (IDso [CD4], Table A-8}. For both sequences tested, the PT
version of the
0oligonucleotide had IDSO values which were 50 to 100 fold lower than that of
the PD version.
A fixed length ( I 8 mer) set of oligonucleotides with either PD or PT
backbones were assayed to
determine whether the nature of the nucleotide sequence contributed to
inhibition of gp120/CD4
interactions. As was observed for the inhibition of HIV-1 RT, the PD versions
of these molecules had
little or no measurable effects on the binding of gp 120 with CD4. However,
the PT versions of these
oligonucleotides did yield measurable inhibitory activity. The 18 mer GTO (
1174) interrupted
3~
CA 02279488 1999-07-29
WO 98133807 PCTIUS98101974
. gp 120/CD4 interactions at approximately 10 fold lower concentrations than
poly (SdC)~ 8 ( 1230) while
the random sequence 18 mer ( 1232) had no measurable activity (Table A-7).
TABLE A-8.
In Vitro Inhibition of HIV-1 gp120
Interaction with CD4 by PD and PT Oligonucleotides.
Oliaonucleotide Linkage' 1D50 len 1201(uMl IDSOf CD41(uM)
I 100-00 PD 3.50 18
I 100-12 PT 0.08 0.475
I 100-07 PD 0.80 4.25
I 100-21 PT 0.07 0.048
1173 PD > 100 > 100
1174 PT 0.09 0.45
1229 (poly dC) PD > 100 > 100
1230 (poly dC) PT I .00 3.25
1231 (GATC) PD >100 >50
1232 (GATCI PT > 10 > 10
' Each pair of oligonucleotides contain the same sequence and differ only in
the nature of their backbone linkage.
b The backbone modifications are denoted as PD for phosphodiester and PT for
phosphorothioate.
F. Oiigonucleotide interactions with the v3 loop of HIV-1 gp120. It had been
reported previously
that poly SdC oligonucleotides were able to bind to the third variable loop
domain of HIV-1 gp120 (v3
loop). The degree of interaction was reported to be dependent on the length of
the oligonucleotide
studied, with a rapid decrease in binding affinity observed for compounds
shorter than 18 nucleotides.
It was noted that the detection antibody used to monitor inhibition of gp
120/CD4 interactions in
the capture gp 120 ELISA KIT (HRP-I-GP I 20) as described above (Table A-8)
recognized an epitope in
the gp120 v3 loop (manufacturer's information). For this reason, control
experiments were performed to
determine whether the observed inhibition of gpI20/CD4 interactions was due in
part, or in whole, to
interference with the HRP-I-gp 120 detection antibody. The results indicated
that I 100-07 and I173 (PD
backbones) did not inhibit the detection of immobilized gp I 20. However, the
PT oligonucleotides
tested (I100-21 and 1 I 74) were able to slightly inhibit the detection of gp
120 at oligonucleotide
concentrations above 5 ItM. This level of inhibition was too small to account
for the IDso [gp 120]
values presented for I100-21 and I 174 in Table A-8.
Further analysis of oligonucieotide interactions with the v3 loop was
conducted using a v3 loop
specific murine Mab, NEA-9284 (Figure A-10). PT oligonucleotides were able to
inhibit binding of
NEA-9284 to gp 120. The presence of bound gp 120 specific Mab was determined
using a HRP-labeled
goat-I-mouse antibody. The results of these experiments indicated that PT
oligonucleotides were able to
inhibit binding of NEA -9284 to gp 120. The IDso for the most active
oligonucieotide ( 1100-21 ) was
approximately 4 to 7 pM. This concentration is approximately 10 to 30 fold
higher than the ICso for this
CA 02279488 1999-07-29
WO 98/33807 PCT/US98/01974
oligonucleotide against HIV-1 in culture (Table A-8). The PD oligonucleotides
tested did not inhibit the
binding of any Mab to gp 120. Therefore, it was determined to be unlikely that
this was the mechanism
by which the PD GTOs such as I100-07 (and hence I100-15) were inhibiting HIV-
1.
G. Analysis of HIV-1 RNA and DNA in single cycle assays. Total RNA and DNA
were extracted
from SUP T1 cells 36 hours after infection with 0.1 m.o.i of HIV-IDV. In this
assay, the infected cells
were treated with I100-15 or AZT at various time points before, during or
after infection. Harvesting of
the infected cells at 36 hr post-infection allowed for the analysis of
approximately one round of viral
replication. A schematic diagram of the positions of the PCR primers used in
the DNA and RNA
analysis is shown in Figure 11.
Total extracted DNA was analyzed using a PCR primer set which would amplify a
200 by
portion of the viral genome spanning the repeat element (R) into the gag gene.
The primer set detected
full-length or nearly completely synthesized viral DNA. This is the last
region of the minus strand of
viral DNA that is synthesized. Thus, for DNA to be detected by this primer
set, two template-switching
events have occurred and contiguous 5'LTR to gag seguences must be present on
either the minus or
plus strand of DNA.
In the same reaction mixture, a PCR primer set which would amplify a 220 by
region of the
human ~i-actin gene was used. The results indicated that in cells treated with
AZT there was a marked
decrease in viral DNA synthesis when the drug was added up to 4 hrs post-
infection (data in Figure 12
shows zero hour and 8 hour time of addition studies). The effects of I100-15
on the early rounds of viral
DNA synthesis was minimal.
The results of this experiment indicated that I100-15 did not inhibit virus
entry into the cells
because of the detectable levels of viral DNA even in samples treated with
I100-15 at the same time as
virus infection (zero hour addition). Furthermore, it suggested that I100-1 S
had a different mechanism
of action compared to AZT.
Additional experiments using alternative PCR primers suggested that there may
be alterations
in the viral DNA synthesis caused by I100-15. The observed amplification
products, when primers
clustered in the U3 region of the virus were used, yielded a banding pattern
which was not predicted and
obviously different from the infected cell control (untreated) and the AZT
treated infected cell samples.
RNA extracted from HIV-1 infected cells was analyzed by RT-PCR. In this assay,
the
antisense primer of the PCR primer pairs was used with MMLV RT and extracted
mRNA to synthesize
cDNA strand. The resultant cDNA was then used as a template in PCR reactions.
Two RNA primer
sets were used to analyze unspliced (primers rI and r2) and spliced (primers
rl and r3) HIV-1
transcripts. Predicted sizes of the amplified products were 101 by and 214 by
for the unspliced and
spliced species respectively. The same (3-actin primers used for the analysis
of the DNA samples were
used as controls in this experiment.
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The results obtained using primer pair rl and r3 are depicted in Figure 12.
The results of this
experiment clearly indicated that a reduced level of HIV-I specific transcript
was observed in samples
treated with I100-15 in the samples treated with drug at the same time as
virus infection (zero hours). It
was also clear that while samples treated with AZT had reduced levels of viral
cDNA, viral mltNA was
still being produced. The same decrease in HIV-1 specific transcript was
observed in viral infected cells
treated with I100-15 when the rl and r2 primer pair was used (data not shown).
- H. Structural analysis of I100-15 and I100-26. I100-07, and its derivative
products including I100-15
and I100-26, are composed entirely of deoxyguanosine (G) and deoxythymidine
{T). These G-rich
oligonucleotides were purified using anion exchange reverse phase HPLC. Using
this procedure the
oligonucleotide is purified in the presence of sodium ions. Monovalent cations
are known to encourage
self associated structures for G-rich molecules, all of which involve
formation of G-tetrads. The G-
tetrad formation involves the formation of eight hydrogen bonds by
coordination of the four O6 atoms of
guanine with alkali cations believed to bind to the center of a quadruplex,
and by strong stacking
interactions. The oligonucleotides purified using anion exchange
chromatography then have an
opportunity to form inter- or infra-molecular tetrads. The tetrad structure
can be strengthened by
replacing the sodium ion with potassium.
I. Nondenaturing gel analysis. I100-15 (17 mer, Table A-5) was analyzed using
nondenaturing
polyacrylamide gel electrophoresis. In this experiment, trace concentrations
of radiolabeled
oligonucleotide ( I 0-'M) was incubated with increasing concentrations of cold
oligonucleotide (up to 10'
SM) before gel analysis in the presence of monovaient cation. Under the gel
conditions used, I100-15
migrated as a unique band faster than a random coiled (denatured) 17 mer
oligonucleotide would and it
was shown to do so in a concentration independent fashion (data not shown).
This was in contrast to
I100-18 (16 mer, 10 fold less active than I100-15) which appeared to migrate
as multiple species in a
concentration dependent fashion under the same gel conditions (data not
shown). The same phenomena
was observed when 10-5 oligonucleotide (total cold and radiolabeled
oligonucleatide) was incubated
with increasing concentration of KCl (Figure 13). I100-15 migrated as a unique
species at all
concentrations of KCI while I100-18 and 2106-50 (ggttgggggttggg) migrated as
multiple species.
The results from these assay suggested that I100-15 folds into an
intramolecular structure while
other G-rich oligonucleotides such as I100-18 and 2106-50 aggregate into
higher order intermolecular
structures. It was noted that the total phosphorothioate oligonucleotide G-
rich compound described by
Wyatt et al., P.N.A.S. 1994 91:1356-66, with the sequence TZG4T2, was claimed
to fold into an
intermolecular tetrad. Therefore, I100-15 (PD backbone) is structurally and
chemically different from
the oligonucleotide reported (ISIS PT oligonucleotide).
J. Tetrad Structure. Principally due to its role in telomere formation, the
structure of four stranded
nucleic acid tetrads has been well studied. Most eukaryotes possess a
repeating G-rich sequence of the
form (T/C)nGm where n=1-4 and m=1-8. Of particular interest to the study of
the I100-1 S class of GTO
was the structure of the telomere sequence repeat TzG4, f rst detected in
Oxytricha. The Oxytricha
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repeat has been studied in oligonucleotides by NMR, Smith et al., Nature 1992,
356:164-68, and by
crystallographic methods, Kang et al. Nature, 1992, 356:126-31. As had been
predicted from numerous
previous physical and biochemical studies, both the NMR and crystallographic
studies suggested that
folding is mediated by square planar Hoogsteen H-bonding among G residues,
with overall antiparallel
orientation of the four strand equivalents comprising the tetrad fold. As
expected, the crystallography
has shown that the structure is selectively stabilized by tight binding of a
small monovalent cation to the
O6 oxygen of guanosine. But surprisingly, both NMR and crystallography confirm
that the folded
structure possess alternating synlanti glycosidic bond angles (as opposed to
all anti for most duplex
structures).
Feigon and colleagues have used NMR and modelling to deduce the structure of a
28 base-long
oligonucleotide (G4T4G4T4G4T4GQ,Oxy 3.5) which is capable of forming a well-
defined all-antiparallel
intramoIecular tetrad, Smith et aL, Nature 1992, 356:164-68. The present
inventors postulated that if the
GTO I100-I S were to fold to form a stable intramolecular tetrad, its NMR
properties would be expected
to be similar to those of the Oxy 3.5 molecule.
In the folded state, the salient NMR characteristics of the intramolecular Oxy
3.5 tetrad were as
follows:
I. Narrow linewidths, indicative of monomer formation only.
2. Induction of well-defined guanosine N 1 Hoogsteen imino resonances in the
11.2 to 11.7 ppm range. The chemical exchange rate of these protons is very
slow,
reflective of the high positive cooperativity of tetrad folding and
dissociation.
3. Spectral simplicity, indicative of a single predominant folded structure,
rather
than an equilibrium among different folded structures.
4. Intrabase H8-C I' and interbase H7-C2" NOE connectivity which demands a
pattern of alternating syn-anti glycosidic bond angle throughout the "tetrad
stem" of
the folded structure.
K. One dimensional NMR analysis. Displayed in Figure 14 is a line model for
I100-15, folded to
form an intramolecular tetrad of the Oxytricha class. From a physical
perspective, the possibility that an
intramolecular tetrad structure might form in high KCl or NaCI is not
surprising. What was surprising
was the fact that this model proposed a stem region comprising a single G-
octet and intervening loop
regions which were only two bases long.
In order to test the general feasibility of this model, a detailed 3D
molecular model for a I100-
I S was constructed. In so doing, the inventors assumed that the 8 G's
comprising the octet core of the
structure formed a standard square planar octet, and that giycosidic angles
were as in the crystal and
NMR structures of the antiparallel Oxytricha tetrads, Smith et al., Nature
1992, 356:164-68, and Kang et
al., Nature 1992, 356:126-31. Additionally, a single K+ ion was introduced
into the center of the G-
octet, with octahedral coordination to GO6. Initially, 2 base loop structures
were created so as to
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connect elements of the octet without disruption. Subsequent to this initial
postulation, the structure was
subjected to mechanical refinement with full electrostatics, employing Charmm
parameters in Sybyl.
After refinement, it was observed that coordinates of the octet core were not
significantly
altered and that backbone parameters within the loop domains were within
acceptable energetic limits.
First, the structure was very compact, nearly spherical, with the three loop
regions and the 5'
"GT tail" comprising the surface of the tetrad core. Based upon this
structure, it appeared likely that
interaction with cellular macromolecules would be heavily dominated by the
structures of these surface
loops. In that regard, the inventors believe that it may be inappropriate to
think of such interactions as
"tetrad binding." The inclusion of G-tetrads in such a structure may not be
important as a recognition
element per se, but instead provides a latticework upon which an orderly loop
array is positioned.
Further, although the loop regions did not appear to be under mechanical
stress, they were short
enough so that they possessed very high configurational freedom. Because of
those severe length
constraints, it was found that all feasible loop models display a distinct
"rabbit ears" structure, wherein
the two base planes of the loop region are unstacked, and point outward from
the center of the octet
core. Such rigid, unstacked, single strand loop character was very distinctive
as compared to other
known folded nucleic acid structwes. Therefore, varying the sequence or
chemical structure of these
loops, one at a time, was necessary to determine if bonding interactions
between these loops and cellular
macromolecules are important to the observed anti-HIV activity.
The structures described above possessed a single G-octet core, which was
known to be the
minimum structure required for nucleation of tetrad formation. Therefore, when
paired with the
observed short loop size, the intramolecular tetrad structure proposed for
I100-15 is best described as
meta-stable, relative to other more robust tetrads which have been described
in the literature. An
increase of the core from 2 to 3 stacked tetrads, or an increase in the length
of flexibility of one or more
loops would be expected to increase the thermodynamic and/or kinetic stability
of this structure
significantly. Thus, the observed anti-HIV activity can be improved by
sequence modification which
enhances the stability of the underlying tetrad latticework.
Finally, it was observed that I I00-I S and homologues display profound
resistance to cellular
nucleases. One interesting aspect of the proposed structure was that, even in
the loop domains,
phosphodiester linkages are generally buried from interaction with large
solutes, such as a nuclease.
The structure analysis proposed defined local phosphodiester backbone
structure at low resolution.
When paired with explicit biochemical analysis of phosphodiester cleavage
rate, it is possible to define
sites for selective introduction of backbone modification in I100-15
homologies, for the purpose of
extending the biological half life in vivo.
The gel electrophoresis data described above suggested that I100-15 spends
very little time as a
random coil at 25°C, under native salt conditions. Although the gel
data rules out intermolecular
associations, the data do not constrain the oligomer to any particular folded
monomeric structure.
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Oligonucleotide folding in I100-15 has been studied employing a combination of
high resolution NMR
and methods.
Stable formation of a discrete octet core, mediated by tight binding of a
single monovalent ion
is crucial to the model described above. Given that G-N1 imino protons give
rise to sharp, characteristic
'H NMR signals in such a structure, focus has been on the potassium ion
dependence and temperature
dependence of I100-15 folding, as assessed by'H NMR at SOOmHz.
For these measurements, I100-15 was synthesized at lSuM scale employing fast
deblocking
"Expedite" chemistry on a Milligen synthesizer. Subsequent to purification by
denaturing anion-
exchange chromatography in base ( 1 OmM LiOH, 0.2 to 0.7M NaC 1 ), oligomer
purity was confirmed by
denaturing gel electrophoresis (7M urea, 65°C). For NMR, the oligomer
was desalted and transferred
into 20mM LiC 1 adjusted to pH 6.0, which minimizes folding to form tetrads.
Oligonucleotide strand
concentration was held constant at 2.7 mM. NMR was measured in HBO, employing
a Redfield pulse
sequence to saturate the water resonance, as described previously, Dittrich et
al., Biochemistry, 1994
33:4111-4120.
In Figure 15 a KCl titatation is displayed. At 3000K, in the absence of added
K+, imino proton
signals cannot be resolved in the 10-12 ppm region. Subsequent to addition of
KCI, substantial
narrowing of imino signals was obtained, saturating at an added KCl
concentration of 3mM, which is
very close to one added K+ equivalent per octet. Above 4 mM, it can be seen
that at least two classes of
imino resonance can be detected in the 10-12 ppm range with roughly equal
intensity: a broad envelope
from 10-11 ppm, upon which several sharp resonances are superimposed in the 11-
11.5 ppm region.
By analogy with chemical shifts of other G tetrad structures, the inventors
tentatively ascribed
the sharp imino signals to the 8 Hoogsteen H bonds of the core octet. The
broad envelope was ascribed
to the G and T imino resonances contributed by the loop and 5' terminal
domains. Consistent with
published tetrad NMR data, a broad envelop of signal was detected at 9 ppm,
which most likely results
from unusually slow exchange of guanosine N2 protons engaged in Hoogsteen
pairing.
In order to better distinguish the two classes of imino'H signal and,
additionally, to investigate
the gross stability characteristics of the folded 1 I 00-15 structure, thermal
melting analysis, at 2.7 mM in
strands, 6mM KC 1, 20mM LiCI, pH 6.0 over the range from 3000K to 3450K was
performed.
Substantial line narrowing of "Hoogsteen" imino proton signals was seen at
3100K, which
appears to be accompanied by broadening of the poorly resolved imino envelope
at 10.7 ppm. This
caused a narrowing of the plateau above 3100K, giving rise to 7-8 well-
resolved imino protons at
3200K. By reference to the NMR behavior of the Oxytricia tetrad and other
tetrad structures, the
formation of 7 to 8 narrow, well-resolved imino resonances at elevated
temperatures strongly suggested
that in the presence of one bound K+ ion per octet equivalent, 1100-15 folded
into a discreet tetrad
structure, stabilized by the 8 Hoogsteen H-bonds of the presumed octet.
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In the range from 330 to 3400K, the imino proton spectrum undergoes an abrupt
transition,
which is likely to be representative of cooperative unfolding of the octet.
Stability of this kind,
accompanied by apparently high thermal cooperativity is very striking indeed,
and is generally
indicative of a single, well-defined folded oligonucieotide structure.
The origin of the shallow temperature dependence of the spectral parameters,
leading to
enhanced 'H resolution at 3200K, remains to be determined. It is likely to
have resulted from weak
intermolecular association which occur in the millimolar strand concentration
range. This interpretation
is born out by preliminary analysis of spectral parameters as a function of
strand concentration (not
shown). Independent of interpretation, the data suggested that high quality
NMR data may be obtained
for exchangeable and non-exchangeable I100-I S (SEQ. ID. NO. 33) protons at
35°C, 20 mM, LiCI,
6mM KC1 and 2mM in strand equivalents.
INHIBITION OF HCMV ACTIVITY
Several different oligonucleotides reduced HCMV titers in tissue culture. Each
of the
oligonucleotides contained a different percentage of guanosine residues and a
different number of total
nucleotides in the polymer. The results of this assay are depicted in Table A-
9. All oligonucleotides
were capable of reducing viral titer in culture including G 101-50 which
contained only 53% G residues
( 16 out of 30 total nucleotides). In Table A-9, the length and percent
guanosine nucleotides is indicated
for each oligonucleotide tested.
TABLE A-9
Oligonucleotide Inhibition of HCMV Activity
Viral Yield in plaque forming units (PFi77
oligonucleotide (%G)
Ollgo. 6101-50 (53%) 6105-50 (80%) 6106-50 (78%) 6109-50 (65%) 6113-50 (64%)
Conc. 30mer 3lmer 27mer 29mer 24mer
None 4.5xi0' PFU 4.5x10' PFU 4.5x10' PFU 4.5x10' PFU 4.5x10' PFU
20.0 lM 0 4.5x10' PFU 2.5x10' PFU 8.0x10' PFU 3.5x10' PFU
10.0 IM 2.5x10' PFU 1.8x10' PFU 4.0x10' PFU 4.5x10' PFU 4.0x10' PFU
1.0 IM 7.Ox10~ PFU 1.9x10= PFU 6.0x10' PFU 1.5x102 PFU 5.0x102 PFU
0.5 lM B.OxlOZ PFU 2.7x102 PFU 1.3x102 PFU 3.0x10' PFU 5.4x102' PFU
In NIH3T3 cells chronically infected with FMLV, oligonucleotides (Fig. I }
were capable of
inhibiting virus production. However, oligonucleotide controls in this
experiment were capable of
inhibiting virus production in culture.
IN T'ITRO ENZYMATIC ASSAYS
Culture media containing FMLV reverse transcriptase (RT) was mixed with
various
concentrations of I100-51 or I 100-12, (the phosphodiester backbone of I100-S
1 having been modified to
a phosphorothioate backbone). Reverse transcriptase was measured as described
in the section entitled
"Reverse Transcriptase Assay" above. Figure 4 shows that both oligonucleotides
were capable of
inhibiting the RT enzyme. Inhibitory concentrations for SO% reduction in RT
activity was between 0.5
to 1 1M for I100-51 and less than 0.5 uM for I100-12.
CA 02279488 1999-07-29
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The I100-51 (FMLV2ap), attenuated full length transcription directed by either
the T7 or T3
polymerases (Figure SA). As can be seen in Figure 1, full length transcripts
directed by the T7 promoter
would be 131 bases long while full length transcripts directed by the T3
promoter would be 171 bases
long (position of the Dde I site relative to the mRNA start site). The
sequence isomer of I100-S I
(I100-O1 = FMLV2p), designed parallel to the target strand was also capable of
significantly inhibiting
transcription from the T7 promoter (Figure SB). However, only the anti-
parallel triple helix forming
oligonucleotide FMLV2ap inhibited via attenuation of transcription as can be
seen in the build up of a
truncated transcript in the reaction mix (Figure SC). The truncated transcript
analyzed in Figure SC was
approximately 63 bases long and matched the predicted size fragment when p275A
was used as a
template (T7 promoter). 6101-SO (53% G} inhibited T7, but not T3 directed,
transcription by a
mechanism other than attenuation (Figure SA) since no truncated transcripts
were observed when this
oligonucleotide was used atone. I100-11 (26% G) increased the level of
specific transcripts directed by
the T7 promoter (Figure 4).
In experiments designed to monitor inhibition of transcription initiation of
the HS V-1 IE I 75
promoter, using oligonucleotides, both specific and control G-Rich
oligonucleotides were capable of
inhibiting eukaryotic transcription when a HeLa cell extract system was used.
The oligonucleotides
used were B 133-54; B 133-55 and B 107-51 as specific inhibitors via potential
triple helix mechanism of
action and G 101-50 and I100-I 1 as the low G-content control
oligonucleotides.
The experiments described above clearly demonstrated the anti-viral activity
in tissue culture
assays for several G-Rich oligonucleotides against HSV-2, HN-1, HCMV and FMLV.
In addition,
G-Rich oligonucieotides specifically inhibited the bacterial RNA polymerase
enzymes T7 and T3, the
FMLV and HIV- I reverse transcriptase enzyme and eukaryotic RNA polymerase.
B. Specific In Vitro Studies and In Vitro HIV Inhibition Using T30177
As was demonstrated by the inventors in the studies initially conducted as
described below,
T30177 is an oligonucleotide composed of only deoxyguanosine and thymidine, it
is 17 nucleotides in
length is the same sequence as I100-15 (SEQ. ID. NO. 33), and it contains
single phosphorothioate
internucleoside linkages at its 5' and 3' ends for stability. This
oligonucleotide does not share significant
primary sequence homology with, or possess any complementary (antisense)
sequence motifs to the
HIV-1 genome. As shown below, T30177 inhibited replication of multiple
laboratory strains of HIV-I
in human T-cells lines, peripheral blood lymphocytes, and macrophages. T30177
was also shown to be
capable of inhibiting multiple clinical isolates of HN-I and preventing the
cytopathic effect of HIV-1 in
primary CD4+ T-lymphocytes. In assays using human peripheral blood lymphocytes
there was no
observable toxicity associated with T30177 at the highest concentration tested
( 100 p.M), while the
median inhibitory concentration (ICSO) was determined to be in the 0.1 to 1.0
p.M range for the clinical
isolates tested, resulting in a high therapeutic index for this drug. In
temporal studies, the kinetics of
addition of T30I77 to infected cell cultures indicated that like the known
viral adsorption blocking
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agents dextran sulfate and Chicago sky blue, T30I77 needed to be added to
cells during, or very soon
. after, viral infection. However, analysis of nucleic acids extracted 12 hr-
post infection from cells treated
with T30177, at the time of virus infection, established the presence of
unintegrated viral cDNA,
including circular proviral DNA, in the treated cells. In vitro analysis of
viral enzymes revealed that
T30177 was a potent inhibitor of HIV-I integrase reducing enzymatic activity
by 50% at concentrations
in the range of 0.01 to 0.10 L~M. T30177 was also able to inhibit viral
reverse transcriptase activity,
however, the 50% inhibitory value obtained was in the range of I-10 pM
depending upon the template
used in the enzymatic assay. No observable inhibition of viral protease was
detected at the highest
concentration of T30177 used ( 10 1 M). In experiments in which T30177 was
removed from infected
cell cultures 4 days post-HIV-1 infection, total suppression of virus
production was observed for more
than 27 days. Polymerase chain reaction analysis of DNA extracted from cells
treated in this fashion
was unable to detect the presence of viral DNA 11 days after removal of drug
from the infected cell
cultures. The ability of T30177 to inhibit both laboratory and clinical
isolates of HIV-I and the
experimental data suggested to the inventors that T30177 represented a novel
class of integrase
inhibitors, indicating that this compound was a viable candidate against
evacuation as a therapeutic agent
for HIV-1 in humans.
In the present study the inventors disclose the mechanism by which a variant
of I100-1 S
(T30177) was able to inhibit multiple HIV-1 laboratory strains in acute and
long-term suppression
assays. The data indicated that T30177 is a potent and selective inhibitor of
HIV-1 via at least two
mechanisms. One mechanism involves interfering with CD4- and gp 120-mediated
cell fusion events.
However, T30177 is 100-fold less effective in inhibiting gp 120-induced cell
fusion events than it is at
inhibiting an early event in the viral life cycle, suggesting a specific point
of interdiction distinct from
that of blocking virus/cell interactions. The data also clearly showed that
T30177 is a potent inhibitor of
the HIV-1 integrase enzyme in vitro and that by blocking these events in the
viral life cycle T30177 is
able to suppress virus production for prolonged periods after an initial short
treatment regimen with the
drug.
Materials Used in In Vitro HIV Inhibition Studies
Oligonucleotides. The deoxyguanosine-rich and other oligodeoxynucleotides used
in this
study were synthesized, purified, and characterized as previously reported.
Ojwang, et al., J. AIDS
7:560-570 (1994); Rando, et al, J. Biol. Chem. 270:1754-1760 (1995). The
sequence and
phosphorothioate (PT) pattern of the oligonucleotides used in antiviral assays
is shown in Table B-7.
Materials. Zidovudine (3'-azido-3'-deoxythymidine, AZT) and the nucleoside
analogs 2',3'-
dideoxyionsine (ddI) and 2',3'-dideoxycytidine (ddC) were obtained from the
AIDS Research and
Reference Reagents Program, National Institute of Allergy and Infectious
Diseases. Dextran sulfate
(DS5000) was purchased from Sigma, and the bicyclam derivatives JM2763 and
JM3100 (De Clereq, et
al., Antimicrob. Agents Chemother. 3 8:668-674 ( 1994)) were obtained from
Johnson Matthey
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(Westchester, Pennsylvania). Chicago sky blue (CSB) was obtained from the Drug
Synthesis and
Chemistry Branch, National Cancer Institute.
Cytotoxicity Analysis. The cytotoxicity of T30177 was assayed as described
above. The
concentration of drug necessary to give one-quarter (TCZS), one-half (TCso) or
95% (TC95) of the
maximum inhibition of growth response was then determined. The degree of cell
proliferation was
determined according to the manufacturer's instructions.
In other experiments the effect of T30177 on the viability of primary human
PBMCs, PBLs and
macrophages was determined using the trypan blue dye exclusion technique.
Griffiths, B., IRL Press, p.
48 ( 1992), or by measuring the degree of [3H]thymidine or [3H]leucine uptake
in these cells (McGrath,
M.S., et al.Proc. Natl. Acad. Sci. USA 86:2844-2848 ( 1989)).
Antiviral assavs
HIV-1 infection assays using cell lines. Laboratory strains of HIV-l, HIV-2,
simian
immunodeficiency virus (SN), or the low passage isolate HIV-1D~ (Ojwang, et
al., J. AIDS 7:560-570
( 1994)), were used to infect established cell lines using the indicated
multiplicity of infection (MOI) of
1 S virus, for one hour at 37°C prior to washing and resuspension in
medium containing increasing
concentrations of drug. The infected cells (2 x 104 cells/well) were
inoculated in triplicate in 200 a of
complete medium which contains RPMI 1640 (Life Technologies) supplemented with
10% FBS,
penicillin (50 U/mL), streptomycin (50 p.g/mL) and L-glutamine, (2 mM). Four
to 6 days post-
infection, drug treated and control wells were analyzed for HIV-1 induced
cytopathic effects, for the
presence of viral reverse transcriptase (RT) or viral p24 antigen in the
culture medium. Buckheit, et al.,
AIDS Research and Human Retroviruses 7:295-302 ( 1991 ); Ojwang, et al., J.
AIDS 7:560-570 ( 1994);
Rando, et aI, J. Biol. Chem. 270:1754-1760 (1995). Cytopathic effects (CPE)
were monitored by either
direct counting of HIV-1 inducted syncytium formation or by staining cells
with the tetrazolium dye XT
or MTT. Buckheit, et ai., AIDS Research and Human Retroviruses 7:295-302 (
1991 ). The AZT
resistant strain of HIV-1 (ADP/141) was kindly provided by Dr. Brendan Larder
and the AIDS Directed
Programme Reagent Project, Medical Research Council, England.
HIV-1 infection of PBMCs. Peripheral blood mononuclear cells (PBMCs) were
isolated from blood of
HIV-1 negative and hepatitis B virus (HBV) negative (healthy) donors by
Ficoll/Hypaque density
gradient centrifugation, cultured as described by Gartner and Popovic (Gartner
et al., In Techniques in
HIV Research, p. 59-63 ( 1990)), then activated with phytohemagglutinin (2
p.g/mL) and cultured in
RPMI 1640 medium supplemented with 15% fetal bovine serum (FBS) and human
recombinant
interleukin 2 (1L-2, 30 units/mL). After 3 days PBMCs (2 x 105 cellslwell)
were infected with various
isolates of HIV-1 at a multiplicity of infection (MOI) of 0.01. After 2 hours
at 37°C cells were washed
and treated with various concentrations of T30177 or AZT, as described by
Buckheit and Swanstrom, id.
( 1991 ). The medium was changed on day 3 or 4 post-infection and fresh drug
was added at these times.
Seven days after infection, HIV-1 replication was analyzed using the Coulter
p24 antigen-capture assay.
CA 02279488 1999-07-29
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Assays were performed in triplicate. Data was obtained by spectrophotometric
analysis at 40 nm using a
Molecular Devices Vmax plate reader.
HIV-1 infection of PBLs. Human peripheral blood lymphocytes (PBLs) were
isolated from blood
drawn from HN-1 and HBV seronegative donors. PBLs were isolated by Ficoll-
Hypaque density
gradient centrifugation. The PBLs were suspended in culture medium (RPMI 1640
medium
supplemented with 2 mM L-glutamine, 20% FBS and 50 ug/mL gentamicin) and the
cells counted using
' the trypan blue exclusion technique. After adjustment of cell density to 1 x
10' cells per mL with
culture medium, the suspension was placed in a T-75 culture flask and
incubated flat at 37°C in a
humidified atmosphere of 5% COz for 2 hours. The non-adherent cell population
was decanted into a
sterile disposable flask. Phytohemagg(utinin (PHA-P) was added to the PBL
suspension at a
concentration of 2 pg/mL and the PBl preparation was then further incubated at
37°C for 48 hours. At
this time an aliquot of the culture was used for virus infectivity studies.
PBLs (5 x 105 cells/well) were
infected with HIV-1 isolates at an MOI of 0.2. This level of infection yielded
a satisfactory virus control
RT activity value result at day 7 post-infection (Buckheit, et ai., id. ( 1991
)). Two hours post-infection,
the cells were separated from the virus by centriguation, washed twice with
culture medium, and
suspended in culture medium containing II,-2 at a concentration 30 units/mL
and at a cell density 2 x 105
PHA-P-stimulated PBL cells/0.1 mL of culture medium. Seven day post-infection,
HIV-1 replication
was analyzed using either the RT or p24 assay systems. Data was obtained in
the p24 assays by
spectrophotometric analysis at 450 nm using a Molecular Devices Vmax plate
reader.
Inhibition of acute infection of primary human macrophages. Human macrophage
cultures were
established as described by Crow et ai. Crowe, et al., AIDS Research and Human
Retroviruses
3(2):135-145 (1987). Briefly, PBMC's isolated from HIV-1 and HBV seronegative
donors was allowed
to adhere to glass at 37°C for two hours in calcium and magnesium free
PBS (pH 7.4). The non-
adherent cells were aspirated and the adherent cells were washed three times
with cold PBS. The
adherent macrophages were scraped free from the plate, counted, and inoculated
into 96 well plates at a
concentration of 105 cells/well in RPMI 1640 medium supplemented with 10%
human serum. The
macrophages were cultivated in RPMI 1640 with 10% human serum. After
incubation overnight at
37°C the macrophages were infected with HN-lpv at a multiplicity of
infection of 0.1 for 24 hours at
37°C in the presence of the indicated amount of drug. Unabsorbed virus
was then washed off and the
cells were further incubated for 7 days at 37°C in complete medium
supplemented with the indicated
amount of drug. On day 7 post-infection the adherent macrophages were washed
extensively with PBS
and lysed with detergent. Cytoplasmic HIV p24 levels were then quantitated and
percent inhibition
were calculated and compared to control infected but untreated cells.
Long term suppression studies. Long term suppression assays were perfotmted in
MT-4 cells infected
with HN-11"~ (MOI of 0.01) using drug concentrations representing I, 10 or 100-
fold over the median
ICsa value for each compound. Four days post-infection, cells were washed
twice with phosphate-
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buffered saline (PBS) and resuspended in complete medium without drug (day 0).
Viral breakthrough
was monitored at several time points by measurement of viral p24 antigen
production in the culture
medium or the presence of intracellular viral DNA as described previously,
(Rando, et al, J. Biol. Chem.
270:1754-1760 (1995)).
Other viral assays. Respiratory syncytial virus (RSV strain A2), and influenza
A (FLUA strain H3N2)
virus assays were performed as described by Wyde et al. {Wyde, et al., Drug.
Dev. Res. 28:467-472
( 1993)) while Herpes Simplex viruses types I and 2 (HSV-1, HSV-2) plaque
reduction assays were
performed as previously described. Lewis, et al, Antimicrob. Agents Chemother.
38:2889-2895 (1994).
Vesicular stomatitis virus (VSV), Vaccinia virus, Sindbis virus, Coxsackie
virus B4, Polio virus-1, and
Semliki forest virus assays were performed as described by De Clercq (De
Clereq, E., Antimicrob.
Agents Chemother. 28:84-89 ( 1985). The arenaviridae assays (Junin and
Tacaribe viruses) were
performed as described by Andrei and De Clercq (Andrei, et al., Arrtiviral
Res. 14:287-299 (1990).
Punts Toro virus (ATCC VR-559) and Yellow fever virus (vaccine strain 17D)
assays were performed
using Vero cells.
Flow cytometric analysis of HIV-1 infected lymphocytes. Seven days post-HIV-I
infection of
PBMCs, the infected cell culture medium was analyzed for HIV-1 production
using the p24 antigen-
capture assay. In addition, cells from both the drug treated and control welts
were analyzed for CD4 and
CD8 antigens by cytofluorometry. Briefly, cells were washed and treated with
fluorochrome-labeled
monoclonal antibodies to CD4 or CD8 (Becton Dickinson). The cells were washed
again and fxed with
2% paraformaldehyde before analysis. Crissman, et al., Flow Cytometry and
Sorting, p. 229-230 ( 1990)
and Crowe et al., AIDS Res. Hum. Retroviruses 3:135-145 ( 1987).
Single cycle analysis of HIV-1 eDNA. CEM-SS cells (2 x 106 ceilslwell) in 0.5
mL of complete
medium were infected with HIV-1 s~ at a MOI of 1.0 for 45 minutes on ice at
which time complete
culture medium ( 10 mL) was added to the cells. The infected cells were then
pel leted ( 1000 RPM for 10
min. at 4°C), washed t<vice and aliquoted into a 24-well flat bottom
plate (2 x 105 cells/well). The
indicated amount of drug was added to the infected cell cultures at various
times during or post-
infection. The cells were harvested 12 hours post-infection at which time cell
pellets were lysed in 100
p. polymerase chain reaction (PCR) Lysis buffer (50 mM KCI, 10 mM Tris-HCI
(pH8.3), 2.5 mM MgClz,
0.1 mg/mL gelatin, 0.45% Nonidet P40, 0.45% Tween 20 and 75 p.g/mL Proteinase
K) at 500C for one
hour followed by 95°C for 10 minutes. The lysate was stored at -
2°C until use.
PCR analysis of viral cDNA was performed using 10 wL of total cell lysate in a
100 pL
reaction buffer as previously described (Rando, et al, J. Biol. Chem. 270:1754-
1760 (1995)). The
primers used were 5'-ATAATCCACCTATCCCAG TAGGAGAAAT-3'
and 5'-TTTGGTCCTTGTCTTATGTCCAGAATCG-3' which will amplify a I15 by segment of
the
HIV-1 genome. The cycle conditions used were 95°C for 10 minutes to
denature the DNA, followed by
30 cycles of 95°C for 75 seconds, 60°C for 75 seconds, and a
final extension step at 60°OC for 10
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minutes. Thirty pL of the amplification reaction were mixed with 10 ul of 2-
3zP-labeled internal probe
(5'-ATCCTGGGATTAAATAAAATAGTAAGAATGTATAGCCCTAC-3'), placed at 95°C for
7.5
minutes and then annealed at 55°C for I S minutes. The resultant
products were separated by
electrophoresis on a 10% polyacrylamide gel.
Analysis of viral replication. CEM-SS cells (2 x 10') were infected with HIV-l
s~ (MOI of I) for 45
minutes at 37°C with gentle mixing. Following virus attachment, the
cells were gently pelleted, washed
twice and resuspended in complete tissue culture medium. The cells were then
divided into aliquots,
treated with various concentrations of drug and placed in T75 culture flasks.
The cells were incubated at
37°C for 18-20 hours and then harvested by centriguation. To extract
nucleic acids for analysis of HIV-
1 integration low- and high-molecular weight DNA were prepared from HN-1
infected cells (untreated
or treated with increasing concentrations of drug) according to the protocol
originally described by flirt
(flirt, B.J., J. Mol. Biol. 26:365-369 ( 1967)) and modified by Gowda et al.
Gowda, et al., J. Immunol.
142:773-780 ( 1989).
DNA (300 ng), obtained from the low-molecular weight flirt fractions, was used
as the
template in PCR analysis undergoing a 30 cycle amplification reaction using
the conditions described by
Steinkasserer et al. (Steinkasserer, et al., J. Virol. 69:814-824 ( 1995)).
PCR primer sets included
control primers for the amplification of mitochondrial DNA (sense,
5'-GAATGTCTGCACAGCCACTTT-3 ; antisense, 5'-ATAGAAAGGCTAGGACCAAAC-3 ;
amplified product, 427 bp); primers for the detection of early viral
transcription events (M667 and
AA55 primers as described by Zack et al. (Zack, et al., Cell 61:213-222 {
1990)), amplified product, 142
bp); primers for the detection of the viral gag gene (sense,
5'-AGTGGGGGGACATCAAGCAGCCATGCAAAT-3 ;
antisense, 5'- TTTGGTCCTTGTCTTATGTCCAGAATG-3', amplified product 300 bp); and
primers
for the detection of circular proviral DNA (sense, 5'-
CCTTTTAGTCAGTGTGGAAAATCTCTAGCA-
3 ; antisense, 5'-CAG TGGGTTCCCTAGTTAGC-3', amplified product, 536 bp). PCR
products were
separated by agarose gel electrophoresis and visualized by ethidium bromide
staining.
Reverse transcriptase enzyme inhibition assays. Purified recombinant RT (HIV-
I BHP o) was obtained
from the University of Alabama, Center for AIDS research. The enzyme assays
utilized three different
template:primer systems, primed ribosomal RNA, gapped duplex DNA, and
poly(rA)p(dT),2_,8 to
evaluate the inhibition of HIV-1 RT as described by White et al. (White, et
al., Arrtiviral Res. 16:257-
266 ( 1991 ), and Parker et al. (Parker, et al., J. Biol. Chem. 266:1754-1762
( 1991 )).
Integrase enzyme assays. Purified recombinant HIV-1 integrase enzyme (wild-
type) was a generous
gift from Dr. R. Craigie, Laboratory of Molecular Biology, National Institute
of Diabetes and Digestive
and Kidney Diseases. The enzyme (0.25 p.M) was preincubated in reaction buffer
at 30°C for 30
minutes. All 3'-processing and strand-transfer reactions were performed as
described previously by
Fresen et al. (Fresen, et al., Proc. Nail. Acad. Sci. USA 90:2399-2403 ( 1993
)) and Mazumder et al.
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(Mazumder, et al., Proc. Natl. Acad. Sci. USA 91:5771-5775 (1994)). Enzyme
reactions were quenched
by the addition of Maxam-Gilbert loading dye, and an aliquot was
electrophoresed on a denaturing 20%
polyacrylamide gel. Gels were then dried and subjected to autoradiography
using Kodax XAR-2 film or
exposed in a Molecular Dynamics PhosphoImager cassette.
Protease assays. HIV-1 protease enzyme (Bachem) was diluted to 166 ug/mL in 50
mM NaOAc, 5
mM DTT, 2 mM EDTA, and 10% glycerol (pH 5.0) and stored as 10 ul aliquots at -
20°C. HIV
protease substrate I (Molecular Probes) was diluted to a working concentration
of 0.32 nmol/TL.
Enzyme (20 uI,), substrate (20 pL) and drug (20 pL) were added to each well of
a microtiter plate.
Positive and negative controls were evaluated in parallel. Fluorescence was
quantitated on a
Labsystems Fluoroskan II using 355 nm for excitation and 460 nm emission
wavelengths at 370C at
time zero and at 30 minute intervals for 2 hours.
HeLa-CD4-8-galactosidase cell assays. Two different assays using genetically
engineered HeLa cells
were performed as described previously. Buckheit, et al., AIDS Research and
Human Reiroviruses
10:1497-1506 ( 1994). These assays utilized the HeLa-CD4-LTR-9-galactosidase
cell line (Kimpton, et
al., J. Virol. 66:2232-2239 ( 1992)), which employ a tat protein-induced
transactivation of the ~3-
galactosidase gene driven by the HIV-1 long terminal repeat (LTR). One assay
involved infecting the
HeLa-CD4-LTR-(3-galactosidase cells with HIV-1 while the second assay
monitored the expression of
~i-galactosidase after incubation of the HeLa-CD4-LTR-(3-galactosidase cell
with HL2/3 cells.
Buckheit, et al., AIDS Research and Human Retroviruses 10:1497-1506 (1994);
Ciminale, et al., AIDS
Research and Human Retroviruses 6:1281-I287 (1990). The HL2/3 cells express
both the HIV-I
envelope glycoprotein and tat gene product so that ca-cultivation of these
cells with the HeLa-CD4-
LTR-(3-galactosidase cells would allow for CD4- and gp 120--mediated cell
fusion. The extent of cell
fusion can then be monitored by the degree of tat transactivation of LTR-
driven (3-galactosidase
expression. Buckheit, et al., AIDS Research and Human Retroviruses 10:1497-
1506 (1994); Ciminale,
et al., AIDS Research and Human Retroviruses 6:1281-1287 ( 1990).
Results of the In Vitro HIV Inhibition Studies
As described above, the anti-HIV-1 activity, in cell culture assays of the
oligonucieotide (I100-
I S) composed entirely of G and T was established by the inventors. See also,
Ojwang, et al., J. AIDS
7:560-570 ( 1994}; Rando, et al, J. Biol. Chem. 270:1754-1760 ( 1995). I 100-
15 was found to inhibit
HIV-IDV in SUP T1 cells with a median inhibitory concentration (ICSO) of 0.125
ulvl. 1100-15 was
synthesized with an unmodified (natural) PD internucloeside linkage and a
propanolamine group
attached to the 3'-terminus to increase the stability of the oligonucleotide.
T30177, a modified variant of
I100-15, has the same sequence as I100-15 but contains an hydroxyl moiety at
its 3'-terminus and a
single PT internucieoside linkage at both the 5'- and 3'-ends.
Cytotoxicity Assays. The cytotoxicity of T30177 was determined using several
different cell lines and
primary human cells as described above. The TC25, TCSO and TC95 values
obtained are shown in Table
,...
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B-1. The cytotoxicity profile obtained for log phase growing cells was
variable depending upon the cell
line used, while the slower growing PBMCs, PBLs, and macrophages all tolerated
the compound at
concentrations exceeding 100 pM as monitored using the trypan blue exclusion,
(3H]thymidine uptake,
or [3H]leucine uptake techniques.
Table B-1. Cytotoxicity of T30177 in established cell lines and primary cells.
CYTOTOXICITY
(TM)a
Cell Type TC25 TCSO TC9s
Cell LinesbCEM-SS 50.8 t 3.2 92.0 t 3.0 >100
MT4 34 t 4.0 70 t 7. > 100
I
CEMxl74 10 t 2.5 50 t 5.2 >100
MT2 27 ~ 3.5 61.2 t 5.5 > 100
AA5 45.66 ~ 2.0 94.2 t 3.1 >100
U937 >100 >100 >100
Vero > 100 > 100 > 100
NIH3T3 >100 >100 >100
Primary PBLs > I 00 > 100 > 100
human
cells
PBMC > I 00 > 100 > 100
Macrophages> 100 > 100 > 100
TCzs, TCso, and TC95 values are the concentrations of T30177 required to
inhibit 25%, 50% and
95% of growth (cell lines) or cell survival (primary human cells).
The cytotoxicity of T30177 in human cell lines was determined using log phase
growing cells.
The cytotoxicity of T30177 in primary human cells was determined using trypan
blue exclusion
technique or by measuring the uptake of [3H]thymidine or [3H]leucine on slow
growing primary
cells.
Inhibition of Viral Replication in Cell Lines. CEM-SS cells were infected with
HIV-1~ at
an MOI of 0.01 and treated with T30I77, AZT or ddC for six days. In this assay
system T30177
inhibited HIV-1,~ replication in a dose-dependent manner with an ICSO value of
0.075 uM while the
control drugs, AZT and ddC, had ICso values of 0.007 and 0.057 N,M
respectively (Figure B-1 ). T30177
was then assayed against additional strains of HIV-1 in a variety of different
cell lines. The results from
these assays showed that the degree of inhibition observed for each strain of
HIV-1 analyzed was greatly
influenced by the cell line used (Table B-2). In addition, as observed for
DS5000, T30177 was
inhibitory for the AZT-resistant strain of HIV-1 tested (ADP/141 ) which has
four mutations in its RT
gene (67N, 70R, 215F and 219Q).
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Table B-2. Inhibitory effects of T30 i 77, AZT, and DS500 on viral
replication.
10
ICso(TM)a
Virus Cell
Line T30177
AZT DSSOOOb
HIV-1
strains'
SKI CEM-SS 0.025 t 0.0060.022 ~ 0.0001-
MT2 0.06 ~ 0.001 0.66 t 0.005-
I RF CEM-SS 0.075 f 0.00020.007 t 0.0002-
MT2 0.270 t 0.04 0.03 f 0.005-
'I MT4 0.037 t 0.03 - 0.018 f 0.02
DV SUP T 0.06 t 0.004 0.03 t 0.005-
1
IIIB CEM-SS 2.83 ~ 0.17 0.002 t 0.0003-
MT2 1.94 t 0.12 0.01 ~ 0.004-
MT4 0.15 f 0.02 - 0.034 f 0.016
i SUP T 0.6 t 0.06 0.03 t 0.006-
1
AA5 < 0.32 < 0.003 -
ADP/141 MT4 0.27 t 0.05 - 0.032 f 0.008
HIV-2/SN strains
~-2ROD MT4 27.5 t 11.6 - 0.082 ~ 0.088
HN-2EHO MT4 5.98 t 1.05 - 0.084 ~ 0.086
SIV"~~5, MT4 1.5 t 1.2 - 0.548 f 0.48
a The IC50 value is the concentration of drug required to inhibit virus
production by 50%. The
results presented are the averages of three or more experiments.
For DS5000 the 1 M units are an approximation based upon the average molecular
weight
(5000) of the material used in these studies.
The MOI used for all HIV-1, HN-2 and SIV strains tested was 0.01.
T30177 was also tested for its ability to inhibit laboratory strains of HN-2
and SIV. The
results (Table B-2) from these assays indicate that T30177 is more active
against the strains of HN-1
and SIV tested than against the two strains of HN-2 tested (ROD and EHO). In
addition, T30177 was
found to be inactive against a variety of enveloped and nonenveloped viruses
tested (Table B-3) with
ICso values found to be greater than the highest concentration of drug tested
(200 ~g~mL or 37 uM).
This is in contrast to DSS000 which was found to be a potent inhibitor of all
of the enveloped viruses
tested except Vaccinia and Semliici forest viruses (Table B-3).
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Table B-3. Inhibition of viral replication in cell lines treated with T30177
or DS5000.
ICso(w~~-)a
T30177 T30177
DSS000 DSSOOOb
nvelo a Vi s:
' HSV-1 KOS >20 2 400 >4 0
SV-2 >200 2 400 >400
HS -1 TK B 0 >200 2 0 >40
HSV-1 T 1837 >2 2 400 >400
Si is vi s > 0 10 > >400
emli ' f re t virus>200 >400 > >400
Vesicular tomat't's>200 20 40 >400
virus
Vaccini viru >2 0 >400 400 >400
Punta ro virus >200 10.9 >200 >400
Yellow Fever vi >200 26 >200 >2 0
s
RSV A2 >200 4 >400 >200
Influen H3N2 > 125 >200 >400
Junin v' >50 13 >50 >200
Tac ' >50 1 . > 0 >200
Non Envelo ed V'
es:
Co a kie virus 4 >200 >40 > >400
Polio virus-1 >200 >400 > >400
a Concentration of drug required to reduce virus-induced cytopathogenicity by
50% (ICso). The
assay results are presented in pg/mL units. For T30177 5.4 pglmL is equal to
pM and for
DSS000 5 pg/mL is approximately equal to 1 ~eM.
a The minimum concentration required to cause microscopically detectable
alterations is normal
cell morphology (MCC). The results presented are the averages of the three or
more
experiments.
Inhibition of HIV-1 Replication in Peripheral Blood Cells. The primary targets
of HIV-I infection in
vivo are CD4+ T lymphocytes and macrophages. Therefore in the following set of
experiments the
inventors tested the efficacy of T30177 on HIV-l replication in PBMCs, PBLs
and macrophages.
Activated PBMCs were infected with laboratory strains of HIV-1 and cultured in
the presence
of T30177, AZT or ddl. Treatment of infected PBMCs with T30177 inhibited the
replication of the all
four HIV-1 isolates tested with ICso values ranging from 0.12 to 1.35 pM
(Table B-4). In this assay
AZT was more efficacious against all HIV-I isolates tested, on a molar scale,
than T30177 while at the
same time T30177 was more potent than ddI against the two HIV-1 strains
tested. It is also interesting
to note that HN-l,nB was more susceptible to T30177 in assays performed using
PBMCs than in assays
using T-cell lines (Tables B-2 and B-4).
S
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Table B-4. HIV-1 replication in primary human cells treated with T30177, ddI
or AZT
Virus Strains
ICSOTM
(Cells) HIV-lIsolate
T30177 ddI
AZT
Laboratory IIIB 0.12 t 0.74 t 0.003 t 0.0002
Isolatesb 0.006 0.05
(PBMCs)
JR~F 0.28 f 2.0 f 0.5 0.0025 t 0.001
0.04
RF 0.75 t ND' 0.272 t 0.003
0.13
MN 1.35 f ND 0.053 ~ 0.001
0.10
Clinical IsolatesdWEJO(SI) 0.30 t 2.18 t 0.017 t 0.0001
(PBLs) 0.01 0.026
j BAKI(SI) 0.23 t 2.61 ~ 0.020 t 0.006
0.005 0.003
WOME(SI) 0.71 t 0.41 t 0.025 t 0.0003
0.002 0.008
ROJO(SI) 3.9 f 0.020.87 t 0.052 t 0.0004
0.001
JOGA(NSI) 0.33 ~ ND >1.0
0.004
BLCH(NSI) 3.08 t ND 0.022 t 0.0008
0.006
VIHU(NSI) 1.3 t 0.021.21 t 0.036 t 0.0007
0.009
S. E. Asia0.58 t ND 0.06 ~ 0.005
0.003
N.Amer. 0.25 f ND 0.01 t 0.004
# 1 0.003
N.Amer. 2.92 t ND I .65 t 0.007
#2 0.005
942716 0.86 f ND 0.002 t 0.003
0.006
942751 0.38 f 2.2 t 0.020.028 f 0.0025
0.003
° Concentration of drug required to inhibit viral production by 50%
(ICso) was determined using the
Coulter p24 antigen capture or RT assays.
b Antiviral assays were performed using laboratory strains of HN-1 in
peripheral blood mononuclear cells
(PBMCs) or using syncytium inducing (SI) or non-syncytium inducing {NSI)
clinical isolates of HIV-1 in
PBLs.
° The value was not determined (ND).
The therapeutic potential of any anti-HIV drug is dependent upon its ability
to inhibit clinical
isolates of the virus obtained from different geographical locations.
Therefore, the inventors evaluated
the ability of T30177 to inhibit the infection of PBLs using a variety of
clinical isolates of HN-1 which
were both syncytium inducing (SI) and non syncytium inducing (NSI) strains of
HN-1. In addition, the
isolates used in this study had their origins in different geographic regions.
After infection with HIV-1
the PBLs were cultured in the presence of T30177, AZT or ddI for seven days.
T30177 inhibited the
viral replication of all the HIV-1 isolates tested with ICSO values ranging
from 0.23 to 3.08 ItM (Table B-
4). In the same assay, AZT and ddI had ICso values ranging from 0.01 to i .65
p.M and 0.41 to 2.61 l.tM,
respectively. It is important to note that T30177 was active against both NSI
and SI isolates and was
very active against he JOGA isolate which was obtained from a pediatric
patient. The JOGA isolate was
also observed to be relatively resistant to AZT treatment (Table B-4).
Another major target cell of HIV-1 infection is the macrophage. Fully
differentiated
macrophages were infected with H1V-lov and treated with T30177 or AZT. T30177
significantly
inhibited HIV-1 replication in macrophages (Figure B-2). However, due to the
long exposure of cells to
5 Z
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virus (24 hours), T30177 and AZT worked best when administered at
concentrations above the ICso
values obtained for these drugs in assays performed in established cell lines.
Variations in Viral MOI. To investigate the effect of variations in the MOI on
the anti-HIV-1 activity
of T30177, CEM-SS or MT4 cells were infected with various MOIs of HIV-1~ or
HIV-1~ (Table B
.' S 5). Unlike AZT, T30177 was much less sensitive to changes in the viral
MOI. For example in these
assays when the MOI of HIV-1~ was changed from 0.01 to 1.28, T30177 only
exhibited a 14-fold
increase in its ICso value while at the same time the ICso value for AZT
increased over 1000-fold (Table
B-5).
Table B-5.
Effect of changes in viral multiplicity of infection (MOI) on the anti-HIV-1
activity of T30177 and AZT.
Multiplicity ICSOIIC90
~ 1 b in 231 Ma AZT
Infection T30177
Isolate/Cell
RF(CEM-SS) 0.01 0.20/0.5010.01/0.030.01/0.1910.001/0.001
0.02 0.41/1.5010.03/0.040.02/0.4710.009/0.046
0.04 0.60/1.5610.01/0.020.07/0.8610.005/0.03
0.08 0.70/1.560.0110.08O.SO/l.Of0.01/0.005
0.16 0.87/1.610.01!0.030.6h10.Ot0.05
0.32 1.2514.70.15/0.278.Sh 10.0
0.64 2.64/4.7510.05/0.16> 1 O.Oh 10.0
1.28 2.81/4.7710.04/0.06>l0.Oh10.0
IIIB(MT4) 0.02 3.1/6.610.23/0.8 0.037/0.220.003
0.01 2.7/9.210.0310.25 0.01 /0.0310.002
0.3 3.38/70.15/0.5 0.15/3.3f0.01/0.05
1 6.8/26f0.53/5.1 0.42/34I2t0.1/10
The concentration of drug needed to limit virus production by 50 (IC50) and 90
((IC90) percent
as measured in the cpe assay
The strain of HN-1 and cell line used for each assay is indicated.
Effect of T30177 on CD4 and CD$ T-cell Subsets. One of the principal
immunological markers
correlated with progression to AIDS is the decline in T lymphocytes which
express the CD4 cell
determining marker (CD4). The change in CD4+ T-lymphocytes is usually
monitored by noting changes
in the ratio of CD4+ to CD8+ lymphocytes in the blood. To determine the effect
of T30177 treatment on
the CD4/CD8 ratio, CD4 and CD8 antigen expression was analyzed on the surface
of cultured PBMCs
seven days post-infection with either laboratory strains or clinical isolates
of HN-1. In these
53
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experiments treatment with either AZT or T30177 increased the number of CD4+ T-
cells in the cell
culture, relative to untreated infected cultures (Table B-6). The observed
increase in CD4+ cells was
dependent on the drug concentration used and was inversely correlated with the
level of virus production
(Figures 16 and Tables B-2 and B-4). These results suggest that the blockage
of HIV-I replication
parallels the suppression of the cytopathic effects of the virus in primary
human lymphocytes.
S '~
f
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WO 98133807 PCTlITS98101974
oo ~3
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CA 02279488 1999-07-29
WO 98/33807 PCTIUS98/01974
In vitro some HIV-1 isolates infect CD4+ lymphocytes, shed infectious virus
into the culture
medium but do not cause destruction of the infected cells (carry, R.F., AIDS
3:683-694 { 1989). This
may explain results obtained when the inventors used the North American
isolate number 1 (N. Amer.
# l, Table B-5). When this virus was used to infect PBMC's, in the absence of
drug, a CD4lCD8 ratio of
0.35 was observed 7 days post-infection. At the same time analysis of the
culture medium from cells
infected with this isolate revealed the presence of viral p24 antigen (Table B-
4) which suggested that a
productive viral infection had occunred.
Time of drug addition studies. T30I77, DS5000 or AZT was added to MT-4 cells
infected with HIV
1 B~ (MOI of 1 ) at various times post-infection. Test compounds were added at
a concentration 100-fold
higher than the determined ICSO value for each drug in the standard assay
performed using MT-4 cells
and the IIIB strain of HIV-1 (Table B-2). Viral p24 antigen levels were
monitored 29 hour post-
infection. The results of this assay indicate that postponing the addition of
T30177 for one hour was
enough to dramatically reduce the inhibitory effects of this compound in a
fashion similar to that of
DS5000 and clearly different from AZT which lost its protective capacity when
added to the cell culture
medium 3 or 4 hours post-infection (Figure 18). A similar result was obtained
when comparing T30177
with CSB, a known inhibitor of both virus binding to cells and fusion related
events (Clanton, et al., J.
Aids 5:771-781 (1992)), in that the antiviral activity of both T30177 and CSB
was greatly reduced if
added to infected cel I cultures one hour post-virus infection (data not
shown).
HeLa-CD4-~i-galactosidase cell studies. To differentiate the effects of T30177
on early events in the
viral life cycle, through integration and subsequent production of the tat
gene product, from the
inhibition of HN-1 gp 120-mediated cell fusion two experimental protocols were
employed. The first
protocol monitored the effects of the drug on the ability of HIV-1,~ to infect
and/or replicate within
HeLa-CD4-LTR-~3-galactosidase cells and was performed as described in Methods.
In this experiment
drug interdiction at any step in the viral life cycle through the production
of the tat gene product would
cause a decrease in expression of the ~3-galactosidase gene, the transcription
of which is regulated by the
HIV-1 LTR. The results show that T30177 is a potent inhibitor of (3-
galactosidase production in this
assay with an ICso value of 0.009 p.M, while the ICSO value obtained for CSB
in the same experiment
was 0.26 1tM (Figure 19A). In control experiments T30177 had no observable
direct effect on (3-
galactosidase enzyme activity at concentrations up to 10 N,M (data not shown).
The second protocol used was a virus-free assay designed to monitor CD4- and
gp120-
mediated cell fusion events. In this assay T30177 was able to interfere with
the fusion process (Figure
19B). However, the observed ICSO value (1 uZvl) was approximately 100-fold
higher than that needed to
interfere with 9~-gaiactosidase production in the virus infection assay
(Figure 19A). In the same assay
system the ICSO value observed for CSB increased approximately 3-fold to 0.8
N.M over the
concentration needed to interrupt (3-galactosidase production in the virus
infection assay (Figure i9).
s~
.T..... ....., .,
CA 02279488 1999-07-29
WO 98133807 PCTIUS98IOI974
The three-dimensional structure of an oligonucleotide with the sequence of
T30177 is stabilized
' by the formation of an intramolecular G-octet, (Rando, et al, J. Biol. Chem.
270:1754-1760 ( I 995 )).
Previously the inventors have reported how the replacement of one of the Gs
involved with tetrad
formation with a deoxyadenosine (A) reduced the anti-HIV-1 activity of the
resultant molecule (Rando,
et al., J. Biol. Chem. 270:1754-1760 (1995). To determine the effects of
intramoiecular tetrad formation
in T30I77 on the observed inhibition of ~3-galactosidase production in the two
assays presented in
Figure B-4, the inventors tested T30526, an oligonucleotide in which a dA has
been substituted for a dG
at a position that would interrupt the formation of one of the two tetrads
involved in the G-octet.
T30526 has the same partial PT patterns as T30177 (Table B-7). T30526 has the
same partial PT pattern
as T30177 (Table B-7). T30526 was found to be approximately 100-fold less
potent that T30177 in
inhibiting HIV-1~ production in culture assays (Table B-7), 10-15-fold less
potent at inhibiting virus-
infected cell (3-galactosidase production (Figure 19A) and did not inhibit
cell fusion at the highest
concentration of drug tested (20 p.M, Figure 19B).
S ~--
CA 02279488 1999-07-29
WO 98/33807 PCT/US98/01974
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CA 02279488 1999-07-29
WO 98133807 PCT/US98101974
Long term suppression of HIV-1. In separate experiments, HIV-1 n,B infected MT-
4 cells were treated
'with T30177, AZT, DS5000, or the bicyclam compounds JM2763 or JM3100 for four
days using drug
concentrations equivalent to 1, 10 or 100-fold over their respective ICso
values (Table B-7). The ICso
values used for JM2763 and JM3100 were from previously reported results, (De
Clereq, et al.,
Antimicrob. Agents Chemother. 38:668-674 (1994}). After four days in culture
the cells were washed
and then further cultured in complete medium without drug. The cells were
monitored daily for the
appearance of viral-induced syncytium formation and every second or third day
for viral p24 antigen in
the culture medium. In cells treated with T30177, at 100-fold over the ICso
value (approximately 10
E.~M), suppression of virus P24 production was observed for at 1st 27 days
after removal of drug from the
infected cell culture (Figure 20). Furthermore, there was no detectable viral
cDNA (by PCR analysis) in
cells examined up to 11 days after the removal of T30177 from the infected
cell culture (data not
shown). Cells treated in the same fashion with AZT, DS5000, JM2763, or JM31000
had measurable
levels of viral p24 antigen in the culture medium within 3 days after removal
of the drug (Figure 20).
The degree of continued suppression was contingent upon the concentration of
T30177 used in the assay
and the duration of the drug treatment regimen (data not shown). The
concentration and duration of
treatment regimen data are consistent with those previously reported for I100-
15, (Rando, et al, J. Biol.
Chem. 270:1754-1760 (1995)).
To determine if exposure of cells to T30177 protects them for subsequent
infection with HIV-1,
cultures of HIV-1 infected MT-4 cells treated for 4 days with T30177 ( 100-
fold over the ICso value)
were washed and then reinfected with HIV-1 "03 before resuspension in fresh
culture medium without
drug. In these assays there was no protection of cells from the second round
of viral infection (data not
shown).
Single cycle analysis of viral cDNA. Total DNA from HIV-ls~ infected CEM-SS
cells was isolated
12 hour post-infection and analyzed for the presence of viral cDNA as
described in Methods. In this
experiment viral cDNA was detected in cells treated with 1 or 10 pM T30177
(approximately 10- to
100-fold over the ICso value) even when the drug was added to the cell culture
at the time of virus
infection (Figure 21 ). This is in contrast to the results obtained when the
adsorption blocking drug CSB
(10 pM), the nucleoside RT inhibitor ddC (10 ~M), or the nonnucleoside RT
inhibitor UC38 (1 l1M)
were used as control drugs. UC38 is an analog of oxathiincarboxanilide. Bader,
et al., Proc. Natl. Acad
Sci. U.S.A. 88:6740-6744 ( 1991 ); McMahon, et al., Proc. Natl. Acad. Sci. USA
( 1995). As expected
there was no detectable viral DNA in cells treated during, or very soon after,
virus infection with any of
the three control drugs when used at concentrations 10- to 100-fold over their
reported ICSO values
(Table B-7, Figure 21 ).
Analysis of replicated viral DNA. The inventors have previously reported on
the presence of viral
cDNA in T30177 treated SUP T1 cells 36 hour post infection with a lower MOI of
HIV-lov. Rando, et
al, J. Biol. Chem. 270:1754-1760 ( 1995). As described above, viral cDNA was
also detected in T30177
CA 02279488 1999-07-29
WO 98/33807 PCTIUS98/01974
treated cells 12 hour post-infection with a high MOI of virus (Figure 2I ). To
determine the extent of
viral replication within these cells PCR primers were used which would
differentiate bet<veen the
different stages of viral replication through the production of circular
proviral DNA (2-LTR circles).
The results of these experiments indicate that viral replication has occurred
in the T30177 treated cells
up to an including the production of 2-LTR circles (Figures 22A-D).
Inhibition of viral enzymes. Oligonucleotides with PT backbones have been
reported to be much more
potent inhibitors of HIV-1 reverse transcriptase (RT) than the same molecules
with PD backbones.
Ojwang, et al., J. AIDS 7:560-570 (1994). T30177 was able to inhibit HIV-1 RT
however, the
concentration needed to inhibit the enzyme by 50% was above 5 LvM when gapped
duplex DNA or
RNA:DNA templates were used (Table B-8). It is interesting to note that when
the primed ribosomal
RNA template was used the ICS° value for T30177 was in the 1 pM range
(Table B-8).
Table B-8.
Inhibition of recombinant HIV-1 Reverse Transcriptase.
IC_soL~
Template T30177 AZT 5'-triphosphate
poly(rA}+p(dT) 12-18 I 1.0 0.59
4.2 0.6
gapped duplex DNA 8.0 0.47
10.0 0.40
ribosomal RNA 1.2 0.019
0.36 0.008
The concentration required to inhibit enzyme activity by SO% (ICsp) is given
for duplicate
experiments in TM units.
T30177 was also tested for its ability to inhibit HIV-1 protease and integrase
enzymes. When
concentrations of T30177 up to I 0 1 M were used in protease inhibition assays
no effect on the viral
enzyme was observed (data not shown). However, when assayed for its effect on
HIV-1 integrase,
T30177 was able to reduce both the 3'-processing and strand transfer
activities of the integrase enzyme
with ICso values of 0.092 and 0.046 p.M, respectively (Table B-7).
To determine if the sequence, three dimensional structure, chemical
composition of the
backbone or a combination of these parameters contributed to the observed anti-
integrase activity of
T30177, the inventors synthesized and tested for enzyme inhibitory activity
the oligonucieotides shown
in Table B-7. T30038, T30175, and T30526 are variations of T30177. T30340,
T30341 and T30659
are variations of the thrombin-binding aptamer sequence reported by Bock et
al. Bock, et al., Nature
355:564-566 (1992). Both the dG-rich sequence of the anti-HIV-I
oligonucleotide T30177 and the
CA 02279488 1999-07-29
WO 98133807 PCT/I1S98/01974
thrombin binding aptamer have been shown to fold upon themselves to form
structures stabilized by
intramolecular G-octets. Rando, et al, J. Biol. Chem. 270:1754-1760 ( 1995).;
Schultze, et al., J. Mol.
Biol. 235:1532-1547 (1994); Wang, et al., Biochem. 32:1899-1904 (1993).
Oligonucleotides T30531,
T30658 and T30662 are variations of the antisense compound GEM91 reported to
be a potent inhibitor
of HIV-1. Agrawal et al., Antisense Research and Development 2:261-266 (
1992}.
The ICso values for each of these oligonucleotides tested in the integrase
assay are shown in
Table B-7. The results of this experiment indicate that any of the sequence
motifs tested were potent
inhibitors of the HN-1 integrase enzyme when the oligonucleotides were
synthesized with a PT
backbone. When the number of PT linkages in the backbone was reduced to one
linkage at each end of
the molecule (pPT) the thrombin binding aptamer (T30559) and the antisense
sequence (T30662) no
longer displayed anti-integrase activity while the level of inhibition
observed using T30177 was
relatively the same as that observed using the total PT version of this
molecule (T30038). For
compounds with total PD backbones only the total PD version of T30177 sequence
motif was able to
inhibit viral integrase with ICso values of 170 and 125 nM for the
3'processing and strand transfer
I 5 enzyme activities, respectively. T30526, the tetrad-disrupted mutant
version of T30177, was still able to
inhibit viral integrase protein in this assay, albeit at a concentration 2-to
3-fold higher than that observed
using T30177.
Conclusions of the In Vitro HIV Inhibition Studies
The inventors expanded upon the earlier observations of their initial studies
(see also, Ojwang,
et al., J. AIDS 7:560-570 ( 1994); ltando, et al, J. Biol. Chem. 270:1754-1760
( 1995)) on the anti-HIV-1
activity of dG-rich oligonucleotides by demonstrating the efficacy of T30177
against multiple laboratory
strains and clinical isolates of HIV-1. Using the cytotoxicity (Table B-1 )
and the efficacy data (Tables
B-2 and B-4), it was found T30177 to have a wide range of therapeutic indices
(TIs) depending upon the
viral strain and cell line used in a given assay. For example, when T30177 was
used to inhibit HN-ls~
in CEM-SS cells a TI of 3680 was obtained. However, when measuring the effect
on HN-1~ in MT2
cells, the TI for T30177 was only 226.
The variability in e~cacy of T30177 in PBMCs and PBLs, which depended upon the
clinical
isolate tested, was very similar to the variation in activity observed for the
nucleoside analogs AZT and
ddI. It is interesting to note that an approximately 20 fold variation in the
ICS value was observed for
T30177 when used to inhibit HIV-I l,~ in CEM-SS cells (2.8 pM) versus PBMSc
(0.12 pM) (Tables B-2
and B-3}. An explanation for this observation may be that when viruses are
propagated continuously in
homogeneous cell fines the "adapt" to those cells and begin to display
phenotypes different from low
passage clinical isolates. Therefore, results obtained using clinical isolates
to infect heterogeneous
populations of primary cells (PBMCs or PBLs) may be more predictive of in vivo
efficacy than data
generated using laboratory strains of HIV-1 in established cell lines. It is
unlikely that HN-1~H is a
resistant strain of HN-1 since T30177 was more effective against this virus in
PBMCs than in cell lines.
However, given the well documented ability of HN-I to mutate and thus develop
resistance to known
6~
CA 02279488 1999-07-29 -
WO 98/33807 PCT/US98/01974
therapies, efforts are underway to determine if resistant mutants can arise
after treatment of HIV-1
infected cells with T30177.
In mechanism of action studies it was found that T30177 displayed some
antiviral which
indicated a mechanism of action similar to the known blockers of virus
adsorption or virus mediated cell
fusion such as dextran sulfate and CSB (Figures i8 and 19). Like CSB and
DS5000, T30I77 needed to
be added to cells at the time of or soon after virus infection. However,
T30177 is 100-fold less effective
in inhibiting gp 120-induced cell fusion events than it is at inhibiting early
events in the viral life cycle,
suggesting a specific point of interdiction with virus distinct at least from
that of CSB. In addition, the
antiviral profile of T30177 also displayed other characteristics which
distinguished T30177 from
DS5000 and CSB. For example, while DS5000 is active against a wide range of
enveloped viruses,
T30I77 appears to be a more selective inhibitor of retroviruses with maximum
efficacy displayed when
used to inhibit strains of HIV-I (Tables B-2 and B-3).
Experimental results presented in Figure B-6 show that unlike control drugs
CSB, AZT, and
UC38, when T30177 was added to cell cultures during virus infection it was
unable to completely block
viral infection even when used at concentrations 100-fold over the ICso value
(~ 10 LtM). Furthermore,
analysis of viral DNA demonstrated that viral replicative intermediates
including circular proviral DNA
were present in infected cells treated with T30177 (Figures 21 and 22). This
data, coupled with the
ability of T30177 to completely suppress virus outbreak (Figure 20), and
possibly clear virus from
infected cell cultures, after removal of drug from infected cells (a profile
not observed for AZT,
DS5000, JM2763 or JM3100), suggests that a second mechanism of action,
distinct from inhibition of
virus binding or inhibition of cell fusion events, is at work. One possible
alternative mechanism is that
T30177 interferes with the viral integration process. A combination of
activities including inhibition of
virus attachment or internalization, virus-mediated cell fusion events and
viral integration could explain
the loss of virus from infected cell cultures. This hypothesis is supported by
the observation that
T30I77 is a potent inhibitor of HIV-1 integrase function in vitro (Table B-7)
and by the observed
accumulation of circularized proviral DNA in the low-molecular weight Hirt DNA
fractions (Figure 22).
It is clear that highly charged molecules such as DS5000 and oligonucleotides
with total PT
backbones are excellent inhibitors of the integrase enzyme in vitro. However,
since T30177, of all the
pPT molecules tested maintained its level of enzyme inhibitory activity (Table
B-7), it is unlikely that
the mechanism of inhibition is totally based upon a polyanion effect as seen
for compounds such as
DS5000 and suramin. Carteau, et al., Arch. Biochem. Biophys. 305:606-610
{1993). It is unclear at this
time whether the G-octet structure, with the two base long dG loops, found in
T30177 is of paramount
importance for inhibition of viral integrase since the G-octet sequence found
in T30659 did not inhibit
integrase activity while T30526 (tetrad disrupting mutant) was able to inhibit
enzyme activity albeit at a
reduced level.
While the time of drug addition studies would suggest interference with virus
internalization as
a key mechanism of action for T30177 it is also clear that readily detectable
viral nucleic acids do enter
CA 02279488 1999-07-29
WO 98/33807 PCT/US98/OI974
the cells. It is quite possible that T30177 inhibits HIV-1 via several
different mechanisms of action.
Another possibility is that T30177 is carried into the cell along with the
infecting virus or is slow to
accumulate within cells (Bishop et al. 1996 J. Biol. Chem. 271:56988-5703)
hence the need to add drug
during virus infection. Experiments designed to address these possibilities
are underway.
The recently reported emergence rate of drug-resistant virus to current
approved therapies for
HIV-1 (T,, of approximately 2 days) suggests that single drug therapy for this
virus cannot succeed (Ho,
et al., Nature 373:123-126 ( 1995); Wei, et ai., Nature 373:117-122 ( 1995),
and therefore, a likely
treatment regimen for any new drug candidate would be in combination with one
or more other drugs
which have differing antiviral mechanisms of action. Further experimentation
might determine that the
actual mechanism of action for T30177 may not be via either inhibition of
virus binding/internalization
or inhibition of viral integration, however, it is unlikely that this
oligonucleotide is acting via the same
mechanism as drugs currently in use for HIV-1. In additional studies the
Applicants have determined
that T30177 is stable in serum and within cells, with a half life measured in
days (Bishop, et aI. J. Biol.
Chem. 1996 271:5698-5703). This information taken together with the ability of
T30177 to suppress
HIV-1 for over four weeks after an initial treatment regimen, in culture,
makes this class of compounds
an attractive candidate for development of oiigonucieotide-based therapeutic
agents for HIV-1.
C. Site of Activity Studies-Viral Integrase Inhibition
Next the inventors undertook studies to demonstrate the potent inhibition of
HIV-1 integrase by
oligonucleotides containing intramolecular guanosine quartets or octets
abbreviated (G4s) and to
provide better understanding of the structure-activity results from a series
of these structures and the site
of molecular interactions with HIV-1 integrase. The relevance of these
findings with respect to HN-1
integrase binding to its DNA substrate and to dimerization of the retroviral
genome was also reviewed.
Materials Used in Site of Activity Studies
Preparation of oligonucleotide substrates and inhibitors. The following HPLC
purified
oligonucleotides were purchased from Midland Certified Reagent Company
(Midland, TX):
AE1 I7, 5'-ACTGCTAGAGATTTTCCACAC-3 ;
AE 118, 5'-GTGTGGAAAATCTCTAGCAGT-3 ;
AE157, 5'-GAAAGCGACCGCGCC-3 ;
AE146, 5'-GGACGCCATAGCCCCGGCGCGGTCGCTTTC-3 ;
AE156, 5'-GTGTGGAAAATCTCTAGCAGGGGCTATGGCGTCC-3 ;
AE118S, 5'-GTGTGGAAAATCTCTAGCA-3 ;
RM22M, 5'-TACTGCTAGAGATTTTCCACAC-3'.
The AE i 17, AE 118, and the first 19 nucleotides of AE 156, correspond to the
US end of the HIV-1 long
terminal repeat (LTR).
To analyze the extents of 3'-processing and strand transfer using 5'-end
labeled substrates,
AE118 was 5'-end labeled using T4 polynucleotide kinase (Gibco BRL) and y-
[32P]-ATP (Dupont-
NEN). The kinase was heat-inactivated and AE 117 was added to the same final
concentration. The
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CA 02279488 1999-07-29
WO 98133807 PCTIUS98/01974
mixture was heated at 95°C, allowed to cool slowly to room temperature,
and run on a G-25 Sephadex
quick spin column {Boehringer Mannheim) to separate annealed double-stranded
oligonucleotide firom
unincorporated label.
To analyze the extent of strand transfer using the "precleaved" substrate,
AE118S was 5'-end
labeled, annealed to AE117, and column purified as above.
To analyze the choice of nucleophile for the 3'-processing reaction, AE118 was
3'-end labeled
using a-[3zP]-cordycepin triphosphate (Dupont-NEN) and terminal transferase
(Boehringer Manheim).
Engleman, et al, Ce1167, 1211-1221 (1991); Vink, et al., Nucleic Acids Res.
19, 6691-6698 {1991). The
transferase was heat-inactivated and RM22M was added to the same final
concentration. The mixture
was heated at 950C, allowed to cool slowly to room temperature, and run on a G-
25 spin column as
before.
To determine the extent of 30mer target strand generation during
disintegration, Chow, et al.,
Science 25 S, 723-726 ( 1992), AE I 5 7 was 5'-end labeled, annealed to AE
156, AE I46, and AE 1 I 7,
annealed, and column purified as above.
Oligonucleotides composed of deoxyguanosine and thymidine were synthesized,
purified, and
incubated with potassium ion to generate the G4s. The guanosine quartet (G4)
forming structures were
then purified as previously described. Rando, et al., J. Biol. Chem. 270, 1754-
1760 (1995).
Integrase proteins and assays. Purified recombinant wild-type HIV-1 integrase,
deletion mutants IN'-
ziz~ INso.zaa~ ~so-zm~ Bushman, et al., Proc. Natl. Acad Sci. U.S.A. 90, 3428-
3432 (1993), and IN~'ss and
site-directed mutants INF~ssxiczsos and INF'asx~czaosnnzr~nt~brr ',,ere
generous gifts of Drs. T. Jenkins and R.
Craigie, Laboratory of Molecular Biology, NIDDK, NIH, Bethesda, MD. Dr.
Craigie also provided the
expression system for the wild-type HIV-1 integrase. A piasmid encoding the
HIV-2 integrase was
generously provided by Dr. R.H.A. Plasterk (Netherlands Cancer Institutes).
Purified recombinant wild-
type FiV and SIV integrases were generous gifts of Drs. S. Chow (UCLA) and R.
Craigie (NIDDK),
respectively.
Integrase was preincubated at a final concentration of 200 (for HIV-1 and HIV-
2) or 600 nM
(for F1V and SIV) with inhibitor in reaction buffer (50 mM NaCI, 1 mM HEPES,
pH 7.5, 50 N,M
dithiothreitol, 10% glycerol (wt/vol), 7.5 mM MnClz or MgClz (when specified),
0.1 mgJmL bovine
serum albumin, 20 mM 2-mercaptoethanol, 10% dimethyl suIfoxide, and 25 mM
MOPS, pH 7.2) at
30°C for 30 minutes. When magnesium was used as the divalent metal ion,
polyethylene glycol was
added at a final concentration of 5% to increase activity as previously
described (Engelman & Craigie,
1995). Preincubation for 30 minutes of the enzyme with inhibitor was performed
to optimize increases
the inhibitory activity in the 3'-processing reaction (Fesen et al., 1994).
Then, 30 nM of the 5'-end 3zr-
labeled linear oligonucleotide substrate was added, and incubation was
continued for an additional 1 hr.
The final reaction volume was 16 wL.
w
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Disintegration reactions, Chow, et al., Science 255, 723-726 ( 1992), were
performed as above
. with a Y oligonucleotide (i.e., the branched substrate in which the US end
was "integrated" into target
DNA) was used.
Electrophoresis and quantitation. Reactions were quenched by the addition of
an equal volume ( 18
N.L) of loading dye (98% deionized formamide, 10 mM EDTA, 0.025% xylene
cyanol, 0.025%
bromophenol blue). An aliquot (5 uL) was electrophoresed on a denaturing 20%
polyacrylamide gel
(0.09 M Tris-borate pH 8.3, 2mM EDTA, 20% acrylamide, 8M urea). Gels were
dried, exposed in a
Molecular Dynamics Phosphorimager cassette, and analyzed using a Molecular
Dynamics
phosphorimager (Sunnyvale, CA). Percent inhibition was calculated using the
following equation:
I 0 100 X[ 1 - (D - C)I(N - C)],
where C, N, and D are the fractions of 21 mer substrate converted to 19mer (3'-
processing product) or
strand transfer products for DNA alone, DNA plus integrase, and integrase plus
drug, respectively. ICso
was determined by plotting the drug concentration versus percent inhibition
and determining the
concentration which produced 50% inhibition.
UV crosslinking experiments. The method used has been described by Engleman et
al. Engelman, et
al., J. Virol. 68, 5911-5917 (1994). Briefly, integrase (at the indicated
concentration) was incubated
with substrate in reaction buffer as above for 5 minutes at 300C. Reactions
were then irradiated with a
W transilluminator (254 nm wavelength) from 3 cm above (2.4 mW/cmz) at room
temperature for 10
minutes. An equal volume (16 p.L) of 2X SDS-PAGE buffer (100 mM Tris, pH 6.8,
4% 2-
mercaptoethanol, 4% SDS, 0.2% bromophenol blue, 20% glycerol) was added to
each reaction. Twenty
~L aliquots were heated at 95°C for 3 minutes prior to loading on a 12%
or 18% SDS-polyacrylamide
gel. The gel was run at 120 V for 1.5 hours, dried, and exposed in a
Phosphorlmager cassette. For
inhibition of DNA binding experiments (Fig. C-3), integrase (200 nM) was
preincubated with the
guanosine quartet (at the indicated concentration) for 30 minutes at
30°C prior to the subsequent
addition of the radiolabeled viral DNA substrate (20 nM). For the competition
experiments (Fig. 29},
integrase (200 nM) was preincubated with either the radiolabeled viral DNA
substrate (20 nM) or
T30177 (20 nM) for 5 minutes at 30°C prior to the addition of
competitor DNA at the indicated
concentration.
Results of the Site of Activity Studies
Guanosine quartet ofigonucleotides inhibit HIV-1 integrase. The inhibition of
HIV-1 integrase by a
series of oligonucieotides which can form G4s is shown in Figure 23.
Oligonucleotides T30177 and
' T30659 (Ojwang, et al., Antimicrob. Agents Chemother. 39, 2426-2435 (1995))
fold upon themselves
into structures stabilized by two G4s stacked upon each other to form a
guanosine octet (Rando, et al., J.
Biol. Chem. 270, 1754-1760 (1995); Schultze, et al., J. Mol. Biol. 235, 1532-
1547 (1994)).
Interestingly, T30177 is active against H1V-1 in cell culture and against
purified HIV-1 integrase in vitro
(Ojwang, et al., Antimicrob. Agents Chemother. 39, 2426-2435 (1995)) while
T30659 is not. For
~P~
CA 02279488 1999-07-29
WO 98133807 PCT/US98/01974
example, inhibition of both the 3'-processing and strand transfer activities
of HIV-1 integrase (Fig. 23A)
by T30177 was observed in the nanomolar range (see Fig. 23B).
In order to ascertain why T30177 was effective and T30659 was not, the
inventors made a
series of compounds to incrementally change one compound into the other. The
structures of these
compounds are shown in panels C and D of Figure 23. The differences between
T30177 and T30659
(i.e., the presence of additional bases at both ends, different sequences in
all three loops, and extension
of loop 2) manifest themselves in dramatic increases in the IC50 values (Fig.
C-1D). To distinguish the
contributions of each of these changes, the inventors first added the same 5'-
and 3'-nucleotides to
T30659 as are present on T30177, yielding T30674 (Fig. 23C). These changes did
not confer potency
(Fig. 23D). Then it was undertaken to change either loop 1 to obtain T30675
{Fig. 23C) or the three
bases in loop 2 into those found in T30177, yielding compound T30677 (Fig.
23C). Neither change by
itself conferred potency (Fig. 23D). However, when the change was accomplished
in two of the loops to
resemble T30177, yielding T30676 or T30678 (Fig. 23C), the inventors were able
to significantly
improve the activity over that of T30659. Interestingly, a two- to three-fold
decrease in potency was
also observed when a second quartet was unable to form, yielding T30526 (Fig.
23D). These data
suggest not only that the octet structure is critical but also that the loops
are important for interaction
with HIV-1 integrase.
The activities of the oligonucleotides in the cellular assays did not strictly
correlate with the in
vitro anti-integrase activity (Fig. 23D). The correlation is complicated by
the differential stabilities and
susceptibilities to nuclease digestion of the oligonucleotides in vivo (Joshua
O. Ojwang and Robert F.
Rando, unpublished).
In Figure 23, G4 oligonucleotides were initially tested in a dual assay which
measures both 3'-
processing and strand transfer. Craigie, et al., Cell 62, 829-837 ( 1990);
Katz, et al., Cell 63, 87-95
( 1990). A strand transfer assay using "preprocessed" (3'-recessed) substrate
( l9mer in Fig. 24A, left
panel) was also performed to determine whether the strand transfer reaction
was truly being inhibited or
whether the inhibition of the 3'-processing reaction caused the decrease in
the subsequent strand transfer
products. Inhibition of strand transfer using this substrate was observed in
the same concentration range
(Fig. 24A, right panel) as that seen with the blunt-ended, duplex
oligonucleotide substrate (Fig. 23A,
top). Therefore, G4 oligonucleotides inhibit both steps of the integrase
reactions: 3'-processing and
strand transfer.
Inhibition of 3'-processing was confirmed using DNA substrates labeled at the
3'-end,
(Engleman, et at, Cell 67, 1211-1221 ( 1991 ); V ink, et al., Nucleic Acids
Res. 19, 6691-6698 ( 1991 ))
(Fig. 24B, left panel), showed that all of the G4s tested inhibited
glycerolysis, hydrolysis, and circular
nucleotide formation to the same extent (Fig. 24B, right panel). Thus, G4
oligonucleotides exert a
global inhibition of the three nucleophiles in the 3'-processing reaction
(glycerol, water, or the hydroxyl
group of the viral DNA terminus).
CA 02279488 1999-07-29
WO 98/33807 PG"TIUS98/01974
Having demonstrated that the catalytic activities of integrase could be
inhibited by G4
oligonucleotides, the inventors next examined whether DNA binding was also
affected. They
performed UV crosslinking of integrase-DNA reactions to address this question.
Crosslinking of
substrate DNA to integrase followed by electrophoresis results in a product
having a molecular weight
of approximately 39 kDa (Engleman et al., 1994, Yoshinaga et al., 1994). As
seen in Figs. C-3A and C-
3B, binding of HIV-1 integrase to radiolabeled US DNA substrate was inhibited
by preincubation of the
enzyme with T30177 in the same concentration range as its ICS° value
for strand transfer (lanes 3-7). In
contrast, preincubation of the enzyme with T30659, which was poorly active in
the 3'-processing/strand
transfer assay (Fig. 23D), resulted in only modest inhibition of DNA binding
even at a T30659
concentration of 500 nM (Fig. 25A, lanes 9-13).
Importance of the HIV-1 integrase zinc finger region for guanosine quartet
oligonucleotide
interactions. Integrase can catalyze in vitro an apparent reversal of the DNA
strand transfer reaction
called disintegration. Chow, et al., Science 255, 723-726 ( 1992). In contrast
to the 3'-processing and
strand transfer reactions, disintegration requires neither the N-terminal zinc-
finger region nor the C-
1 S terminal DNS-binding domain of integrase. Bushman, et al., Proc. Nat1
Acad. Sci. U.S.A. 90, 3428-
3432 (1993). For this reason, the HIV-1 integrase catalytic core domain,
Ins°-Zm (Fig. C-4A), can be use
din the intramolecular disintegration assay and for testing the site of action
of inhibitors. Mazumder, et
al., Proc. Natl. Acad. Sci. 9I, 5771-5775 (1994); Mazumder, et al., AIDS Res.
Hum. Retrov. 11, 115-125
( 1995 ).
In the disintegration assay, only the In'-Z88 and IN'-2'2 proteins (Fig. 26B)
were inhibited by
T30177 (with ICS°s of 270 and 600 nM, respectively) while neither
INS°~2'Z (Fig. 26B} nor INS°-2$$ (data
not shown) showed more than 30% inhibition at a 3 1 M concentration of T30177.
The concentration of
T30177 required for inhibition of disintegration was higher than that required
for inhibition of either 3'-
processing or strand transfer. These results are consistent with those
observed with other molecules
(Fesen et al., 1994, Mazumder et al, 1994). This observation suggests that the
active site of HIV-1
integrase may tolerate drug-induced protein or DNA distortion during the
disintegration reaction,
consistent with the relative tolerance of integrase to mutagenesis of either
substrate features (Chow &
Brown, 1994) or protein structural domains (Bushman et al., 1993) in this
reaction. This is the first
example of an HIV-I integrase inhibitor requiring the enzyme zinc-finger
region for inhibitory activity.
These results suggest that the zinc-finger may assist in stabilizing binding
to T30177.
This hypothesis was investigated further by monitoring binding of wild-type,
full length
integrase (IN''2g8) and of the deletion mutants to radiolabeled T30177. The
concentration of T30177
required for DNA-protein complex formation was the same as that required for
complex formation using
the viral US DNA substrate (i.e., in the 20 nM range). UV crosslinking assays,
Engelman, et al., J.
Yirol. 68, 5911-5917 (1994), showed that IN'-28g formed a DNA-protein complex
of the expected
molecular weight in the absence or presence of added manganese (Fig. 26C,
lanes 8 and 9). The IN'-2'z
protein, which has previously been shown to bind to linear viral DNA only at
high concentrations
~O ~
CA 02279488 1999-07-29
WO 98133807 PCT/US98/01974
(approximately 2.56 p.M) and only in the presence of divalent metal ion,
(Engelman, et al., J. Virol. 68,
5911-5917 (I994)), was able to crosslink to T30177 with the same efficiency as
wild-type integrase in
the absence or presence of added manganese (lanes 2 and 3). The INso-zss
protein. which contains a
nonspecific DNA-binding domain, was also able to crosslink to T30177 with the
same efficiency as
wild-type integrase in the absence or presence of added manganese (lanes 4 and
5), consistent with its
ability to bind to viral US DNA (Engelman et al., 1994). The extent of
crosslinking was significantly
diminished in the case of the core mutant INso-z~z compared to INl-n2 in the
absence or presence of
manganese (compare lanes 2 and 3 with 6 and 7, faster migrating complex). The
higher molecular
weight species in lane 6, having the expected molecular weight of a dimer, has
been reproducibly
observed, but its density has not been confirmed. These data support the
notion that the N-terminus of
HIV-1 integrase assist in the formation or stabilization of an HIV-1 integrase-
T30177 complex, perhaps
by binding the oligonucleotide.
DNA-binding activities of the HIV-1 integrase zinc finger domain. To further
analyze the binding of
the N-terminal zinc finger region to T30177 and compare these results to the
viral US substrate, UV
crosslinking was performed with an In''S5 deletion mutant (Fig. 26A)
containing only this domain. As
seen in Figure 27A-B, this mutant could not bind either the T30177
oligonucleotide or the viral DNA
substrate when only manganese or magnesium was present left and right panels,
lanes 3 and 4).
However, the FN'-SS protein could bind to both DNAs in the presence of zinc
and either manganese or
magnesium (left and right panels, lanes 5 and 6). Significantly, the In''S5
protein was able to bind to the
T30177 G4 oligonucleotide, but not the viral DNA substrate, in the presence of
zinc atone (left and right
panels, lanes 9 and 10). These results are in accord with the known zinc-
binding ability of this domain.
Bushman, et al., Proc. Natl. Acad Sci. U.S:A. 90, 3428-3432 (1993); Burke, et
al., J. Biol. Chem. 267,
9639-9644 ( 1992). But they also suggest that the N-terminal domain of
integrase has DNA binding
capabilities on its own. Finally, these experiments demonstrate comparable
binding of the HIV-I
integrase zinc finger domain to an oligonucleotide containing in G4s than to a
double-stranded, linear,
viral DNA oligonucleotide when both manganese (or magnesium) and zinc are
present but more
efficient binding to the G4 oligonucleotide under non physiological conditions
(zinc alone). The
inventors also found that the nucleocapsid protein of HIV-1, a nucleic acid
annealing protein which
contains two CCHC zinc fingers and which is essential for dimerization of the
retroviral RNA genome,
Tsuchihashi, et al., J. Yirol. 68, 5863-5870 (1994}, was able to bind
efficiently to T30177 (data not
shown). The ability of zinc to confer DNA binding ability on the IN'-55
protein was examined by
replacement of this ion with other transition metals. Consistent with
spectroscopic data (Burke et al.,
1992), only zinc was able to induce detectable DNA binding to the G4
oligonucleotide (data not shown).
Increased potency of guanosine quartets in magnesium. In contrast to IN''S5,
the extent of
crosslinking (and presumably binding) of wild-type integrase to radiolabeled
guanosine quartet was
increased in the presence of magnesium relative to manganese at several
concentrations of the guanosine
quarter (Fig. 28A). This observation led us to examine whether the inhibitory
activity of T30177 and
6g
CA 02279488 1999-07-29
WO 98/33807 PCT/US98/01974
analogs could also be enhanced by buffer containing magnesium. In order to
address this question, the
inventors tested three versions of T30177 as shown in Fig. 28B. T30175 has the
same base sequence as
T30177 but is composed entirely of phosphorothiodiester internucleotidic
linkages. The inhibition of 3'-
processing catalyzed by HIV-I integrase by these guanosine quartets is shown
in Fig. 28C. Both
S T30175 and T30177 showed four to five-fold increases in potency when
magnesium was used as the
divalent metal instead of manganese. In contrast, T30038 showed no significant
increase in potency
when magnesium was used as the ion (Fig. 28D). These data are in accord with
the increased stability
constants for magnesium-nucleotide complexes when oxygen replaces sulfur
(Pecoraro et al., 1984).
The opposite is true for manganese. Therefore, the greater inhibitory potency
of T30177 in buffer
containing magnesium versus manganese may reflect a requirement for magnesium
ion coordination
along the phosphodiester backbone of T30177 in order to confer inhibitory
activity and optimum
interaction of T30177 with HIV-1 integrase. This coordination can occur with
more stability when
either T30177 or T30175 are assayed in buffer containing magnesium rather than
manganese and is
manifested in a greater potency against 3'-processing.
I5 DNA competition experiments. The relative affinity for the G4
oligonucleotide was probed by
attempting to compete off the integrase bound to radiolabeled HIV-1 viral US
DNA with increasing
concentrations of unlabeled T30177 (Fig. 29A). The converse experiment, where
binding of integrase to
radiolabeled G4 oligonucieotide was carried out prior to the addition of
increasing concentrations of
unlabeled HIV-I viral US DNA, was also performed (Figure 29B). In each case, a
band having the
apparent mobility of an integrase-DNA complex was evident. In Fig. 29A, the
viral DNA-integrase
complex has a molecular weight of 38,500 while in Fig. 29B, the T30177-
integrase complex has a
molecular weight of 37,000. Neither complex could not be competed off by
either competitor DNA
even at concentrations where the competitor was in 500-fold excess (Fig. 29A,
lane 6). Similar results
were seen when the Ins-z~z and INso-z~z proteins were used in competition
experiments (data not shown).
Therefore, the stability of the G4 oligonucleotide DNA-integrase complex is
comparable to that of the
viral DNA-integrase complex is comparable to that of the viral DNA-integrase
complex. Ellison, et al,
Proc. Natl. Acad. Sci. U.S.A. 91, 7316-7320 (1994); Vink, et al., Nuc. Acids
Res. 22, 4103-4110 (1994).
Inhibition of related lentiviral integrases. T30177 was tested for inhibition
of the related retroviral
integrases from HIV-2 (van Gent et al., 1992), simian immunodeficiency virus
(SIV) and feline
immunodeficiency virus (FIV) (Vink et al., 1994b). As seen in Figs. 30A and
30B, T30177 inhibited 3'-
processing catalyzed by HIV-1 integrase in the expected concentration range
(Fig. 30A, lanes 2-8; ICso
= 55 nM). Inhibition of HIV-2 integrase (using HIV-1 DNA) was also observed in
the same range
(lanes 9-I S; ICso = 90 nM). However, F1V integrase was inhibited at three-
fold higher concentrations of
T30177 (lanes 16-22; ICSO = 175 nM), and SIV integrase was inhibited at seven-
fold higher
concentrations to T30177 (lanes 23-29; ICso = 420 nM). Therefore, the T30177
G4 oligonucleotide
displayed some selectivity among the lentiviral integrases.
Conclusions Regarding the Site of Activity Studies
~9
CA 02279488 1999-07-29
WO 98133807 PCTlUS98/01974
The present study demonstrates for the first time the binding of DNA guanosine
quartet
structures to HIV-1 integrase, and that some oligonucleotides recently shown
to exhibit antiviral activity
are potent H1V-1 integrase inhibitors.
Guanosine Quartet Oligonucleotides are Novel and Potent Inhibitors of HIV-1
integrase.
Oligonucleotides composed of deoxyguanosine and thymidine and forming
guanosine-tetrads
(G4) structures have previously been shown in inhibit HIV replication. Rando,
et al., J. Biol. Chem.
270, 1754-1760 ( 1995}; Wyatt, et al., Proc. Natl. Acad Sci. U.S.A. 91, 1356-
1360 ( 1994). Two
mechanisms have been invoked. First, some oIigonucleotides have been shown to
bind to the V3 loop
of the envelope protein gp 120 and subsequently inhibit virus adsorption and
cell fusion. Wyatt, et al.,
Proc. Natl. Acad. Sci. U.S.A. 91, 1356-1360 (1994). Secondly, oligonucieotides
such as those described
in the present study also inhibit viral-specific transcripts, Rando, et al.,
J. Biol. Chem. 270, 1754-1760
( 1995), presumably by inhibiting viral integration. Ojwang, et al.,
Antimicrob. Agents Chemother. 39,
2426-2435 (1995). The present finding that inhibition of the HIV-1~ strain in
cell culture parallels that
of purified integrase in vitro in the series of G4 oligonucleotides tested
(Fig. C-1D) further demonstrates
the possibility that HIV-1 integrase can be targeted by some G4
oligonucleotides.
G4 oligonucleotides differ from previously published HIV-1 integrase
inhibitors in several
ways. (Table C-1) First, they are among the most potent inhibitors to date
with IC50's in the nanomolar
range. Their potency range is comparable to flavone, Fesen, et al., Biochem.
Pharmacol. 48, 595-608
( 1994), and tyrophostin derivatives, Mazumder, et al, Biochemistry 34, in
press ( 1995}, which, however,
generally fail to show antivirai activity. Secondly, the zinc finger domain of
HIV-1 integrase
contributes to the inhibition by G4 oligonucleotides, as truncation mutant
enzymes lacking this domain
are resistant to the G4 oligonucleotides. This property is unique, as all the
other inhibitors to date are
active against the HIV integrase catalytic core domain. (Table C-2) Mazumder,
et al., Proc. Natl. Acad
Sci. 91, 5771-5775 (1994); Mazurnder, et al., Mol. Pham. submitted (1995);
Fesen, et al., Biochem.
Pharmacol. 48, 595-608 (1994); Mazumder, et al, Biochemistry 34, in press
(1995). Finally, G4
oligonucleotides form stable enzyme complexes that cannot be displaced by
excess viral DNA
oligonucleotide.
CA 02279488 1999-07-29
WO 98/33807 PCT/US98/01974
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CA 02279488 1999-07-29
WO 98133807 PCTIUS98101974
Role of the HIV-1 Integrase Zinc Finger Region. Mutation and deletion analyses
show that the zinc
finger motif (H-H-C-C) of retroviral integrases is required for integration
(3'-processing and strand
transfer) activity. Engelman, et al., J. Virol. 66, 6361-6369 ( 1992).
However, the structural role of this
region has not been elucidated. It has been postulated too provide DNA
sequence-specificity, Bushman,
' S et al., Proc. Natl. Acad Sci. U.S.A. 90, 3428-3432 (1993), stabilize DNA-
enzyme, Vink, et al., Nuc.
Acids Res. 22, 4103-4110 (1994), and enzyme multimer complexes. Ellison, et
al, J. Biol. Chem. 270,
3320-3326 ( 1995). These data provide the first direct evidence that the HIV-1
integrase N-terminus
region (amino acids I-55 [Fig. 26A]) can interact directly with viral DNA in
the presence of both zinc
and magnesium (or manganese). The fact that the IN~-55 protein binds to the G4
oiigonucieotides in the
presence but not in the absence of zinc is consistent with the formation of a
zinc finger in this region.
Burke, et al., J. Biol. Chem. 267, 9639-9644 ( 1992). Hence, it is possible
that the zinc finger region can
selectively bind to non-B DNA structures. It is noteworthy that the recently
solved structure of HIV-1
integrase, Dyda, et al., Science 266, 1981-1984 (1994), resembles that of the
Rev C Hoiliday junction-
resolving enzyme, Ariyoshi, et al, Cell 78, 1063-1072 ( 1994), and of the
bacteriophage Mu transposes
core. Rice, et al., Cell 82, 209-220 ( 1995). These structurally related
proteins also bind multiple double
helices, generating X structure intermediates. (For review See Katz et al.,
Ann. Rev. Biochem 63:133-
173 (1994) and Vink et al; Trends in Genetics 9:433-438 (1993).
Although the zinc finger region of integrase is required for inhibition by the
G4s, the INS°'nz
protein, which contains only the central catalytic domain, was capable of
binding to T30177 (Fig. 26C).
The inventors also found that an HIV-1 integrase mutant with the two zinc
finger histidines mutated to
asparagines was able to bind to G4 oligonucleotides (data not shown). These
data suggest that HIV-1
integrase may have two separate binding sites, one for viral DNA and one for
target or "host" DNA.
This scenarios would be expected if integrase were to bind both the viral and
host DNA at sites which
were instinct but in close proximity in vivo. Vincent, et al., J. Virol. 67,
425-437 (1993). It should be
noted that the existence of a single binding site on HIV integrase for both
viral and target DNA has been
proposed by others. Vink, et al., Nucleic Acids Res. 21, 1419-1425 (1993).
Biological relevance of G4 structures. Several similarities exist between
retroviral genomes and
telomeric regions of eukaryotic chromosomes. The two RNA strands comprising
the HIV-I genome
can potentially dimerize and form intermolecular G4s in vitro, Sundquist, et
al, Proc. Natl. Acad Sci.
U.S.A. 90, 3393-3397 (1993); Awang, et al., Biochemistry 32, 11453-11457
(1993), as does telomeric
DNA. Sundquist, et al., Nature 342, 825-829 ( 1989). In addition, the [3
subunit of the Oxytrichia
telomere binding protein has bene proposed as a molecular chaperone for the
formation of G4s at the
end of chromosomes by enhancing the rate of a thermodynamically favored
transition. Fang, et al., Cell
74, 875-885 (1993}. In retroviruses, the nucleocapsid protein may also act as
a molecular chaperone to
enhance dimer formation. Sundquist, et al, Proc. Natl. Acad. Sci. U.S.A. 90,
3393-3397 (1993). In this
manner, it may facilitate the formation of and bind to the G4. The inventors
also found that G4s can
bind to purified nucleocapsid protein (data not shown). Thus, G4s may be
structurally important as
CA 02279488 1999-07-29
WO 98133807 PCT/US98101974
molecular scaffolds in both retroviral preintegration complexes and telomeres,
and these structures may
.have associated chaperones in both cases. Finally, a G4 containing structure
may act as a negative
regulator of telomere elongation in vivo due to its ability to inhibit
telomerase in vitro. Zahler, et al.,
Nature 350, 718-720 ( 1991 ). Analogously, G4 structures may act to inhibit
integrase (Fig. 23A-D) and
thereby act as a block to auto integration or digestion of the viral DNA prior
to insertion into the host
chromosome.
The existence of G4s in vivo has not been demonstrated. However, they have
been shown to
form in vitro in telomeric sequences, Sundquist, et al., Nature 342, 825-829
(1989); Smith, et al., Nature
356, 164-168 (1992); Kang, et al., Nature 356, 126-131 (1992), HIV-1 RNA
sequences, Sundquist, et al,
Proc. Natl. Acad. Sci. U.S.A. 90, 3393-3397 (1993); Awang, et al.,
Biochemistry 32, 11453-11457
(1993), fragile X syndrome nucleotide repeats, Fry, et al., Proc. Natl. Acad.
Sci. U.S.A. 91, 4950-4954
( 1994), the retinoblastoma susceptibility gene, Murchie, et al., Nuc. Acids
Res. 20, 49-53 { 1992),
immunoglobulin switch region sequences, Sen, D., et al., Nature 334, 364-366 (
1988), and possibly
during meiotic recombination. Liu, et al., Cell 77, 1083-1092 ( 1994). Given
these results, it is not
surprising that proteins such as thrombin, Bock, et al., Nature 355, 564-566 (
1992), (not normally
known to bind nucleic acids), chick topoisomerase II, Chung, et al., Nucleic
Acids Res. 20, 1973-1977
( 1992), MyoD (a transcription factor that regulates myogenesis), Walsh, et
al., J. Biol. Chem. 267,
13714-13718 ( 1992), an hepatocyte chromatin protein, Weisman-Shomer, et al.,
J. Biol. Chem. 268,
3306-3312 (1993), macrophage scavenger receptors, Pearson, et al., J. Biol.
Chem. 268, 3546-2554
( 1993), and a protein from Tetrahymena thermophila, Schierer, et al.,
Biochemistry 3 3, 2240-2246
(1994), have been found to bind G4 containing nucleic acids. Another G4
binding protein, KEM1, has
been isolated and implicated in recombination-type reactions in vivo. Liu, et
al., Cell 77, 1083-1092
( 1994). The catalytic activities of this protein and of the integrase protein
are DNA endonucieolytic
cleavage and strand transfer. However, unlike KEMI, integrase does not
catalyze endonucleolytic
cleavage reactions on G4s (data not shown). Thus, G4s may be mechanistically
relevant in a diverse set
of biological processes involving enzymes with similar activities.
In summary, the inventors demonstrated that oligonucleotides containing
intramoiecular G4s
are potent inhibitors of HIV-1 integrase. Inhibition is dependent on the zinc
finger region of integrase
and on the structure and sequence of the G4s. These findings also suggest that
novel AIDS therapies
could be based upon G4s as inhibitors of HIV-1 integrase.
D. Structure-Function Studies
As shown previously, the inventors obtained evidence for inhibition of HIV-1
infection by
treatment with phosphodiester oligonucleotides containing only G and T bases.
Additional studies noted
above suggested to the inventors that such oligomers were potent inhibitors of
HIV-1 integrase, in vitro.
The highest activity was obtained using the l7mer, referred to as T30177, with
composition GI2-T5.
NMR evidence suggested to the inventors that T30177 forms an intramolecular
fold which is stabilized
.._ ,
CA 02279488 1999-07-29
WO 98133807 PCTIUS98/01974
by a pair of G-tetrads, connected by three single stranded loops, with a 1-2
base tail to either side of the
fold.
Thus, the inventors undertook studies to determine sequence dependence of the
intramolecular
folding mechanism, in a set of four closely related 16-17 base oligonucleotide
homologues, with
sequences in the range G 10-12-T4-7. The original T30177 compound was
included, along mth three
derivatives which were designed so as to alter the structure of loop domains,
while keeping the pair of
G-tetrads intact. Based on thermal denaturation, CD and kinetic analysis, the
inventors were able to
show that a single base alteration within the loop or tail domains can produce
a very large change in
folding stability. The K+ ion dependence of these data suggested a preliminary
model wherein the loop
and tail domains interact to form stable metal ion-binding sites. A l6mer
derivative (T30695) was
designed within the context of that model, with the intent of enhancing the
interaction between K+ and
the 5' terminus of the oiigomer. The inventors showed that T30695 folding is
indeed more stable than
other members of the group and is highly specific for K+, as assessed from the
ion dependence of
thermal denaturation, CD spectra and W detected folding kinetics.
To assess the relationship between biological activity and formation of the
ion-selective
oligomer fold, the inventors compared tertiary structure stability at three K+
concentrations with the
capacity of the folded oligomers to inhibit the HIV-1 integrase enzyme in
vitro, or HIV-I infection in
cell culture. The stability and activity data are found to be highly
correlated, as a function of sequence
alteration, suggesting that formation of the stable intramolecular fold may be
a prerequisite for both
integrase inhibition and anti-HIV-1 activity. Although the structure of the
folded state has not yet been
confirmed at high resolution, the data presented here suggested that the
structure of the T30695 complex
with K+ ion may be of pharmaceutical significance and could serve as the basis
for additional
improvement of the observed HIV-1 activity.
Materials and Methods for the Structure/Function Studies.
Oligonucleotide Synthesis. All oligonucleotides used in this study were
synthesized on an Applied
Biosystems Inc. DNA synthesizer, model 380B or 394, using standard
phosphoramidite chemistry, or
fast deblocking Expedite chemistry on a Milligen synthesizer. All oligomers
possessed 2
phosphorothioate linkages (one on each terminus) which were introduced by the
H-phosphonate
method. Oligonucleotides were purified by preparative anion exchange HPLC, on
Q-Sepharose. Chain
purity was confirmed by analytical Q-sepharose chromatography, and by
denaturing electrophoresis of
32P labeled oligomers on a 20% poiyaerylamide ( I 9:1 ), 7M urea gel matrix
(Rando et al., ( 1994) J. Biol.
Chem. 270. 1754-1760, 17). In all instances, greater than 90% of the purified
oligomer was determined
to be full length. Oligomer folding was monitored by native gel
electrophoresis on I S% acrylamide
( 19:1 ) matrix in TBE. Folded, 32P labeled samples were loaded subsequent to
annealing in 20 mM
Li3P04, pH 7.0, 10 mM KCl at 7 uM in strands, as described below.
Annealing. Prior to W, CD or kinetic analysis, oligonucleotides were annealed
at 20 mM Li3P04, pH 7
at 3-I S uM in strands. Samples were heated to 900C for 5 min and then
incubated for I hour at 37°G.
CA 02279488 1999-07-29
WO 98133807 PCT/US98I01974
Metal ion could be added as the chloride either before or after the
37°C incubation, with no measurable
difference in final state, as assessed by LJV, CD or gel analysis. As assessed
by native gel
electrophoresis (not shown), this annealing method was found to produce a
single product with mobility
consistent with a folded monomer over the strand concentration range from 3-15
uM, at all ion
concentrations described.
Ultraviolet Spectroscopy. L1V measurements were obtained on a HP 8452A diode
array spectrometer,
using a HP 89090A temperature regulator. Except where noted, thermal
denaturation profiles were
obtained at a rate of 1.25°C/min over the range from 20°C-
80°C, on samples at 20 mM Li3P04, pH 7, at
7 uM in strands. Absorbance was monitored at 240 . nm, which was determined to
be the point of
maximal temperature induced change. For melting analysis, metal ion was added
to the desired
concentration, followed by a one hour pre-incubation at 37°C, to ensure
compete annealing. Folding
kinetics were obtained by manual addition of metal ion at t=O, followed by
absorption measurement at
264 nm. Mixing dead time was determined to be 10 sec. Kinetics were monitored
over the range from
10 sec to 15 min at 25°C.
Circular Dic6roism. CD spectra were obtained at 250C in 20 mM Li3P04, pH 7, at
15 uM in strands, on
a Jasco J-500A spectropolarimeter. Metal ion was added to the desired
concentration, followed by one
hour of pre-incubation at 37°C. Each spectrum in the text represents 5
averaged scans. To conform to
traditional standards, data are presented in molar ellipticity (deg-cm2-dmolE-
1) as measured in base,
rather than strand equivalents.
Antiviral assay. The RF laboratory strain of HIV- 1 was used to infect
established cell lines for one
hour at 37°C prior to washing and resuspension in medium containing
increasing concentrations of drug.
Four to six days post-infection, drug treated and control wells were analyzed
for HIV- 1 induced
cytopathic effects, for the presence of viral reverse transcriptase (RF) or
viral p24 antigen in the culture
medium as previously described by Ojwang et al. (Bishop et al., ( 1996) J.
Biol. Chem. 271, 5698-5703).
Purified recombinant HN-1 integrase enzyme (wild-type) was a generous gift
from Dr. Craigie,
Laboratory of Molecular Biology, NIDDK. All 3'-processing and strand-transfers
were performed as
described previously by Fresen et al. (Fresen et al. (1993} Proc. Natl. Acad.
Sci. hSA 90, 2399-2403)
and Mazurnder et al. (Mazumder et al. (1994) Proc. Natl. Acad. Sci. USA 91,
5771-75).
Results of Structure/Function Studies
The structure of the oligonucleotides in this study are presented in Figure
37A. For the purposes of
clarity, they have been represented in the context of a particular folding
model which places eight of the
guanosincs as a central octet and the remainder of the oligomer in either a
loop region, or as part of a 1-2
base long tail region at the 5' or 3' terminus. Previous electrophoresis and
B7 NMR data (Rando et al.,
( 1994) J. Biol. Chem. 270, 1754-1760) have strongly suggested that T30177
folds so as to form an
intramolecular G-tetrad based structure which is stabilized by a single
central G-octet. Therefore, for
T30177, the simple model presented in Figure 37A is adequately substantiated
by structural data. The
T.... ....... .,..
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validity of a similar structural model for the other members of the series is
legitimately assumed based
upon their sequence similarity, to be tested in terms of the data presented
below.
Thermal Denaturation Analysis. Based upon previous NMR data, and the general
literature, the
inventors postulated that folding of T30177 should be strongly dependent upon
K+ binding. To quantify
this, they measured the thermal stability of T30177, as a function of added KC
1 concentration. Coupled
equilibrium theory predicts that, in the instance that K+ binding stabilizes
formation of an intramolecular
fold, measured TM values should increase linearly with the Log of the KCl
concentration. Such data are
shown in Figure 38A, line b. It is seen that in the presence of 20 mM Li3P04,
measured Tm values for
T30177 increase from 38°C to 65°C in the range from 0.1 to 10 mM
of added KC1. This very large
increase in Tm below 10 mM of added KCI, in the presence of 20 mM of Li3P04 as
buffer, argues
strongly that the effect of K+ binding is not a simple ionic strength effect.
The inventors have noticed that the measured Tm values for T30177 are
consistently higher, by
1030°C, than has been seen for other small intramolecular folds (Smith,
F. W., & Feigon, J. ( 1992)
Nature (London) 344, 410-414; Schultze, et al., J. (1994) J. Mol. Biol. 235,
1532-1547). Since T30177
differs from these other homologues only in terms of the proposed loop
domains, the inventors have
synthesized homologues of T30177 where the central G-octet remains constant,
but where the loop
domains to either side have been modified by addition or replacement of a
single base. In the context of
the simple folding model (Figure 37A) the T30676 homologue is identical to
T30177, but has been
modified so as to add an additional G into the topmost coop of the structure.
As seen in Figure 38A, line
c, this one base addition produces a 20°C decrease in Tm over the
entire range of K+ ion tested.
Similarly, the T30677 homologue was prepared (Figure 37A), which is identical
to T30177, but has
been modified so as to convert a pair of Gs in the bottommost loop domain. As
seen in Figure 38A, line
d, this two base loop substitution produces a 30°C decrease in Tm over
the range of K+ ion tested.
In the context of these substantial stability changes, the inventors sought to
confirm that the
general mechanism of folding had not been altered by base substitution. Thus,
Tm analysis was repeated
at 1 mM KCI as a function of strand concentration in the range from 3 to 10
p.M (Figure 38C). As seen,
a measurable strand concentration dependence could not be detected over this
three fold range of
variation, for any of the derivatives, thus verifying that the folding
equilibrium remains intramolecular
throughout. This was confirmed by native gel electrophoresis, which continued
to display a single
folded oligomer state (not shown), similarly, it was observed that the thermal
difference spectrum for all
three homologues was very similar (not shown).
Oligomer Design Improvement. Based upon the unusually high thermal stability
of T30177, relative to
intramolecular folds in the literature, and upon the 20-40°C decrease
in Tm observed as a function of
what should have been a simple loop modification (Figure 37A), the inventors
concluded that
interactions within the loop domains may contribute to stability.
Specifically, it is proposed that K+ ions
may engage in stable binding to the loop domains of T30177. Simple docking
calculations, performed
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.with BIOSYM software (not shown) suggested that the TGTG loop configuration
at the lower face of
these folded homologues could, in cooperation with the proximal G-tetrad, give
rise to tight K+
coordination which is similar to that seen when K+ (or Na+) ion coordinates
between G-tetrads (Bishop
et al., (1996) J. Biol., Chem. 271, 5698-5703). In the context of that
proposal, the inventors noticed that,
if the penultimate T were removed from the 5' terminus of T30177, the
distribution of nucleotide bases
in the uppermost face of the fold would be similar to that of the lower face,
but with one less
internucleotidyl phosphate linkage.
Those considerations served as the basis for the design of the l6mer
oligonucleotide, T30695 (Figure
37A). As seen in Figure 38A, line a, even though T30695 is one base shorter
than the T30177
homologue, it was found to melt at approximately 10°C higher
temperature, over the entire K+ range
tested. As for the other homologues, Tm values for T30695 were found to be
strand concentration
independent, confining the general similarity of the folding process (Figure
38C).
For T30695, the K+ ion dependence of thermal stability was very striking. In
the presence of 20
mM of Li3P04 as buffer, measured Tm values increase from 40°C to
65°C over the added KC 1 range
from 50 EM to 1 mM. Again, this ion dependence argues that the observed
stabilization is likely to
result from site-specific ion binding, rather than simple ion-screening
effects.
In order to explore the selectivity of ion binding by T30695, Tm values have
been measured for
alkaline metal ions with differing radius: Na+ (0.99A), K+ ( 1.38A), Rb~ (
1.49A), and Cs+ ( 1.69A). As
seen in Figure 38B, significant K+ ion selectivity is detected. Although Rb+
is very similar to K+ in
general chemical properties, and differs by only +O.11A in ion radius, it is
seen that the Rb+ complex
with T30695 melts at approximately 20-30°C lower temperature over the
entire concentration range
studied. Na+ ion and Cs+ ion, which differ from K' in ion radius by -0.37A and
+0.29A, respectively,
are seen to be even more destabilizing. Similar ion binding selectivity were
obtained by this method for
the T30177 homologue (not shown).
Circular Dichroism. In order to explore the nature of these ion binding
effects, the inventors monitored
the folding of T30695 by circular dichroism (CD) methods. It is known that G-
quartet based folding,
both infra and intermolecular, gives rise to large induced ellipticity values
(Balagurumoorthy, P. &
Brahmachari, S. K. ( 1994) J. Biol. Chem. 269, 21858-21869; Jin, et aI. ( I
992) Proc. Natl. Acad. Sci.
USA 89, 8832-8836; Lu et al. ( 1992) Biochemistry 31, 2455-2459; Gray et al. (
1992) Methods in
Enrymology 211, 389406). Stable tetrad folds are characterized by
nonconservative spectra, with
maxima at 264 nm (~ 1 x 10+$ deg-cm2/dmol) and 210 rim (~5x 10+4 deg-cm2/dmol)
and a minima at
240 rim (-4x1044 deg-cm2/dmol).
In Figure 39A, the inventors monitored the CD spectrum of T30695 at 0, 0.05
and I 0rnM KC 1.
As seen, at the highest added KC 1 concentration, the induced CD spectrum is
very similar to that
predicted for an orderly G-tetrad based fold. Interestingly, the spectrum
obtained in the absence of added
KCl is indicative of significant folding in the absence of added K+ ion, at
25°C in the supporting 20 mM
CA 02279488 1999-07-29
WO 98133807 PCTIUS98l01974
Li3P04 buffer. Tm data for T30695 in Figure 38A suggests an extrapolated Tm
near to 20°C in the limit
of very low added K+ ion. That extrapolated value is consistent with the
structure evidenced at 0 mM
KC 1 in Figure 39A.
Detailed K' titration data of that kind have been presented in Figure 39B, for
T30695, T30177,
and T30676. As seen, all three oligomers displayed a generally similar
increase in elliptic as a function
of added K+ ion concentration, which is consistent with the hypothesis that
they fold ire a fashion similar
' to the simple model of Figure D-lA. However, the ion concentration
dependence of the folding process
is quantitatively different for the three. As would have been predicted from
the Tm data of Figure D-2A,
it was found that the coupling between K+ ion binding and folding is stronger
for T30695 (transition
midpoint near to 0.02 mM), than is the case for T30177 (0.15 mM) or T30676
(0.27 mM). The T30677
oligomer, which was the least stable of the oligomers tested by Tm analysis,
showed very little ellipticity
change over the 0- 100 mM KC 1 range, and therefore has not been presented in
Figure 39B.
Closer inspection of the data in Figures 39A and 39B suggested that the CD
titration for T30695
is biphasic, with a first step completed by 0.1 rnM, and a second step which
is complete in the 1-2 mM
1 S range. In order to confirm that the K' induced folding process involves
two steps, the inventors have
performed CD titrations with different alkaline metal ions (Figure 39C). The
inventors Rb+ induced
folding of T30695 is associated with an overall ellipticity increase which is
very similar to that induced
by K+. This argues that the Rb+ and K+ complexes are folded in a similar
fashion. However, as expected
from the Tm differences seen in Figure 38B, it is observed that Rb+ ion
induced folding is quantitatively
different, occurring only at relatively high added ion concentration (0.5 mM
midpoint as compared to
0.02 mM for K+). This confirms that Rb+ is a much poorer effector of the
folding process. A very similar
K+ vs. Rb+ differential was seen for T30177 (not shown), which suggests that
the two oiigomers display
similar overall ion binding selectivity.
The biphasic character of the T30695 folding process now easily detected upon
addition of Rb+
(Figure 39C). The magnitude of the CD change associated with the first and
second ion induced steps
are similar for both K+ and Rb+, confirming that the folding process has not
been significantly altered in
a qualitative fashion by Rb+. For comparison, it was observed that folding of
T30695 as a function of
Na+ ion binding is not biphasic, and is associated with a total ellipticity
increase which is no larger than
that of the first transition seen in the presence of K+ and Rb+ ion. One
interpretation of this difference is
that Na+ ion is capable of driving the first, but not the second step in the
folding process. This proposal
will be discussed below.
Folding Kinetics. In order to investigate the two step folding process in more
detail, the inventors have
measured the kinetics of oligomer folding, for T30695 and T30177 at
25°C in the standard 20 mM
Li3P04 buffer. Data were obtained by manual addition of K+ or Rb+ ion at time
zero, followed by
measurement of LTV absorbance change at 264 nm, in the 0 to 300 second time
range. In Figure 40A, K+
ion has been added to T30I77 at 0.2 uM (curve a), 1.0 mM (curve b) or 10 uM
(curve c). In Figure 40B,
CA 02279488 1999-07-29
WO 98133807 PCTIUS98/01974
Rb+ ion has been added to T30695 at I uM (curve a), 5 uM (curve b) or 10 uM
(curve c). These three
values are approximately those required to obtain the midpoint, endpoint and
ten times the endpoint of
the K+ induced (Figure 39B, curve b) or Rb+ induced folding process (Figure
39C). Although not shown,
the kinetic data described below were found to be nucleic acid concentration
independent over the range
from 3-10 uM in strands, confirming that the folding process is
intramolecular.
As seen in Figure 40A, upon addition of K+ to T30177 to 0.2 mM (curve a), a
single slow
kinetic process is detected with a time constant near 18 sec. Interestingly,
this component is
hyperchromic, indicating a net loss of base stacking interaction during this
first step of the folding
process. Upon addition of sufficient K+ to drive the folding transition to
completion ( 1 uM, curve b), a
second kinetic component is detected (~=-IS sec, t2=1x104 sec). The second
component is hypochromic,
indicative of a net increase in base stacking, and is very slow. Upon an
additional increase of K+ ion to
10 1 M (curve c), the first kinetic component becomes nearly too fast to be
detected in the current
apparatus, while the time constant for the second step has decreased to about
50 sec. Very similar
kinetics, but approximately 20-fold slower, have been obtained upon addition
of Rb+ ion to T30I 77 (not
shown).
In Figure 40B, the inventors have performed a similar folding analysis on
T30695, but with Rb+
instead of K+ ion. This was done because, for T30695, the kinetics of K+
induced folding were too fast to
be detected in the simple optical apparatus employed. As seen in Figure D-4B,
upon addition of Rb+ to
1 mM (curve a), a single slow kinetic process is detected, similar to that
obtained at low K+ ion
concentration with T30695 (Taul=48 sec). Again, this component is
hyperchromic, indicating a net loss
of base stacking interaction. Upon addition of sufficient Rb+ to drive the
T30695 folding transition to
completion (5 uM, curve b), a second kinetic component is detected. Again, the
second component is
hypochromic, indicative of a net increase in base stacking. Upon additional
increase of Rb+ ion
concentration to 10 pM (curve c), the first kinetic component becomes nearly
too fast to be detected,
while the time constant for the second step has decreased from about 60 sec
(curve b) to about 16 sec.
Although these initial kinetic data are not sufficient to solve for rate
constants, the
absorbance-detected kinetic data for both T30I77 and T30695 are consistent
with the equilibrium
binding data obtained by CD (Figure 39B and C). Both techniques suggest that
for K+ and Rb+, the ion
induced oligomer folding process is aphasic. Kinetic data obtained with Na+
ion (not shown), suggest
that only the first, hyperchromic transition is obtained at any concentration
in the 0-200 mM range. That
observation is also generally consistent with Na+ titration data (Figure 39C).
A structural model is
proposed below to rationalize those observations.
A Relationship Between Structure and Function. The inventors' interest in
T30I77 and its derivatives
has arisen because this class of oligonucleotide is a potent inhibitor of HIV
infection in culture (Rando et
ai., ( 1994) J. Biol. Chem. 270) 1754-1760; Ojwang et al. ( 1994) J. AIDS 7,
560-570; Bishop et al.,
(1996) J. Biol. Chem. 271, 5698-5703; Ojwang et al. (1995) Antimicrob. Agent
Chemotherepy 39,
g~2
CA 02279488 1999-07-29
WO 98133807 PCTIUS98101974
2426-35), and in vitro, has been shown herein to be the most potent inhibitor
of HIV-1 integrase to have
'been identified thus far (see also Ojwang et al. {1995) Antimicrob. Agent
Chemotherepy 39, 2426-35). In
Table D-1, there is provided a catalog of the melting temperatures of the
closely related set of
derivatives used in this study, as an index of their stability as an
intramolecular tetrad-based fold.
Stability has been presented at three different added K+ ion concentrations,
spanning a range of Tm
values which differ by 50°C. This was done to ensure that stability-
activity correlations would not be
limited to any particular K+ ion concentration.
Three kinds of activity data have been presented. Integrase inhibition by
these oligonucleotides
has been monitored for both the 3' exonuclease and strand transfer activities
of the purified HIV-1
integrase (Ojwang et al. (1995) Antimicrob. Agent Chemotherepy 39, 2426-35).
Data are presented in
Table D-1 as the IC50, in nM of added oligonucieotide. Antiviral activity has
been obtained as described
herein and elsewhere (Ojwang et al. (1995) Antimicrob. Agent Chemotherepy 39,
2426-35), and is
presented as the 1C50, in nM, of added oligonucleotide.
Inspection of Table D-1 suggests that, relative to any added K+ ion
concentration, there is a
correlation between thermal stability of the folded state and the capacity to
inhibit the exonuclease or
strand transfer activity of purified HIV-1 integrase. A qualitative
correlation is also obtained when
comparing thermal stability with measured anti-HIV activity in cell culture. A
relationship between
thermal stability and function can only be meaningful for folded structures
which are very similar.
However, given the sequence similarity among these four homologues in Table D-
1, and the similarity
of their ion-induced folding process, the correlations are likely to be
meaningful.
Conclusions Regarding the StructurelFunction Studies
Data were obtained suggesting that the anti-HIV oligonucleotide drug T30177
and its homologue
T30695, fold via intramoiecular G-tetrad formation, to yield a structure which
is stabilized by K'' ion
binding. It is well known from the literature that alkaline metal ions can
stabilize G-tetrad formation
(Williamson, J. R. ( 1994) Annul Rev. Biophys. Biomal. Struct. 27, 703-730).
What distinguishes the
behavior of these two oligomers is the unusually high stability of the folded
state (Figure 38A), the high
selectivity shown for K' ion (Figures 38B and 39C) and the possibility that K+
coordination may be
strongly coupled to loop structure within the oligonucleotide fold (Figures
38A and 39B). Consistent
with the idea that ion binding may occur with G-tetrads and with loops, the
inventors have observed that
the folding of T30695 and T30177 appears to occur as a two step process, as
detected by equilibrium
(Figure 39) and kinetic methods (Figure 40).
In order to relate these various observations, the inventors have found it
useful to propose a simple,
two step folding model (Figure 37B). They suggest that the first, higher
affinity ion binding step occurs
by coordination of metal ion with the central-most pair of G-tetrads, thereby
generating a core octet
which is similar to that seen in related intramolecular folds (Williamson, et
al. (1989) Cell 59, 871-880;
Panyutin, et al. ( 1990) Proc. Natl. Acad Sci. USA 87, 867-870; Smith, F. W.,
& Feigon, J. ( 1992)
Nature (London) 34~f. 410-414; Schuitze, et al. ( 1994) J. Mol. Biol. 235,
1532-1547). It is proposed that,
~3
CA 02279488 1999-07-29
WO 98133807 PCTlUS98/01974
by analogy to those other, better understood G-tetrad based structures, this
first ion binding step has
rather modest selectivity among the alkaline metal ions (Williamson, J. R.
(1994) Annul Rev. Biophys.
Biomal. Struct. 27, 703-730). The inventors propose that the second step in
the folding process involves
binding of additional ion equivalents to the loop regions of the structure. It
is also proposed that this
second process, which occurs at higher added ion concentration (Figure 39) and
which is associated with
the slow kinetic step of Figure 40, is coupled to a rearrangement of the Loop
domains to yield two
additional sites for metal ion coordination.
In preliminary modeling studies (not shown), the inventors have confirmed that
orderly structures of
the proposed kind can be obtained in which carbonyl oxygens from T and G base
plains are organized in
the loops so as to complement the end of the G-octet, resulting in octahedral
coordination of one K+
equivalent at each of the two junctions between loop and core octet domains.
It is proposed that this
capacity for additional K+ ion binding is the origin for the remarkable
stability of T30695, the corollary
being that other homologues described in this work are less stable because
they have lost one or the
other of the proposed K+ coordination sites. A second corollary of the model
is that the high ion
selectivity seen for these oligomer folds is dominated by the structural
requirements for ion binding to
the loops, rather than from ion binding within the core octet. Preliminary NMR
data (Ding & Hogan,
unpublished data) suggests that the additional binding step involves 2
equivalents of K+, yielding 3 K+
equivalents per oligomer fold, at saturation.
Confirmation of this model awaits detailed structure analysis. However, the
data at hand (Table
D-1 ) suggest that formation of the ion-selective oligomer fold described
herein may be a necessary
pre-condition for anti-integrase and the overall anti-HN activity of these
compounds. As such,
refinement of the present folding model could prove useful as the basis for
pharmaceutical
improvement.
is
CA 02279488 1999-07-29
WO 98/33807 PCTIUS98101974
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CA 02279488 1999-07-29
WO 98/33807 PCT/US98101974
Pharmacokinetic Studies
'E. Side-dose hemodynamic toxicity and pharmacokinetics of a partial
nhosnhorothioate anti-HIV
oligonucleotide (AR1771 following intravenous infusion to c nv omolgus monkeys
As part of the pre-clinical assessment of AR177, a toxicity study of AR177
(T30177) was
conducted with the objective of establishing the dose-response relationship
between intravenous
infusion of AR177 and hemodynamic parameters in cynomolgus monkeys.
Intravenous infusion is the
proposed route of administration of AR177 to humans. The present study was
conducted using the short
term infusion protocol recommended by the Food and Drug Administration (Black
et al., 1994), with
measurement of central blood pressure, serum chemistry, hematology,
coagulation factors, complement
factors, and plasma ARI77 concentrations.
Materials and Methods for Pre-Clinical Toxicology Screens
Materials. AR17? was synthesized at Aronex on a Milligen 8800 oligonucleotide
synthesizer, and
made into a stock solution at 25 mg~mL in sterile phosphate-buffered saline.
AR177 has a molecular
weight of 5793 daltons, and is a fully neutralized sodium salt. The structure
of AR177 was characterized
by phosphorus and proton NMR, sequencing, base composition, laser Resorption
mass spectrometry,
anion exchange HPLC and polyacrylamide gel electrophoresis. The AR177 was
approximately 94%
pure according to HPLC and electrophoretic analysis. All analyses are
consistent with the proposed
structure.
For HPLC analysis of plasma ARI77, tris was obtained from Fisher, NaBr and
NaCI were
obtained from Sigma, and methanol was purchased from J. T. Baker. The Gen-Pak
Fax anion-exchange
HPLC column (4.6 x 100 mm; cat. no. #15490) was purchased from Waters.
Dosing. Twelve experimentally naive cynomolgus monkeys were assigned to four
groups of three
animals each. Prior to dosing, each animal was lightly sedated with a
combination of ketamine ( 1 0 mg /
kg) and diazepam (0.5 mg l kg), and a catheter was introduced into the femoral
artery for recording
central arterial pressure. Monkeys were given a single intravenous infusion of
5, 20, or 50 mg AR177Ikg
or saline over ten minutes through a cephalic vein catheter using a Harvard
infusion pump. Arterial
blood samples were drawn at -10, +10, +20, +40, +60 and +120 minutes relative
to the initiation of
infusion into EDTA-containing tubes for hematology, complement factors,
coagulation assay, serum
chemistry, and plasma AR177 determination. At 24 hours post-infusion, blood
was drawn via the
femoral vein into EDTA-containing tubes for these same parameters. The
concentration of AR177 in
dosing solutions was confirmed post experiment by absorbance at 280 nm on a
spectrophotometer. For
the determination of AR177 by HPLC, the plasma fraction was obtained by low
speed centrifugation of
blood, and stored at -20°C until used. Electrocardiograms (ECGs),
central pressure, and heart rate were
recorded continuously for 120 minutes following the initiation of dosing.
Table E-1 summarizes the
study design. The animals were observed twice daily for pharmacotoxic signs
and general health
beginning two days before dosing and for seven days following dosing. The
monkeys were not
necropsied at the end of the study.
CA 02279488 1999-07-29
WO 98/33807 PCT/~TS98/01974
Serum chemistry parameters. The following were determined: sodium, potassium,
chloride, carbon
dioxide, total bilirubin, direct bilirubin, indirect bilirubin alkaline
phosphatase, lactate dehydrogenase,
aspartate aminotransferase, aianine aminotransferase, gamma-
glutamyltransferase, calcium, phosphorus,
glucose, urea nitrogen, creatinine, uric acid total protein, albumin,
globulin, cholesterol and
S triglycerides. The samples were analyzed at Sierra Nevada Laboratories
(Reno, NV).
Hematology and coagulation parameters. The following were determined: red
blood cell count and
morphology, total and differential white blood cells, hemoglobin, hematocrit,
prothrombin time,
fibrinogen, mean cell hemoglobin, mean corpuscular volume, mean corpuscular
hemoglobin
concentration, platelet count, and activated partial thromboplastin time.
Hematology parameters were
I O determined at Sierra Nevada Laboratories (Reno, NV).
Complement factors. The complement split product Bb and total hemolytic
complement CH50 were
determined. The choice of measuring the Bb split product, as opposed to other
complement factors, was
based on a published study showing the involvement of the alternative pathway
in complement
activation induced by oligonucieotides (Galbraith et al, 1994). Complement
determinations were
15 performed in the laboratory of Dr. Patricia Giclas at the Complement
Laboratory, National Jewish
Center for Immunology (Denver, CO).
HPLC analysis of AR177 plasma concentrations. AR177 was assayed in the plasma
using an
anion-exchange HPLC method on a Waters HPLC system with a 626 pump, 996
photodiode array
detector, 717 autosampler and Millennium system software controlled by an NEC
Image 466 computer.
20 Buffer A consisted of 0.1 M Tris base, 20% methanol, pH 12, and Buffer B
consisted of 0.1 M Tris
base, 1.0 M NaBr, pH 12. The anion-exchange column (Gen-Pak Fax column) was
equilibrated at 80%
buffer A/20% buffer B for 30 minutes before each HPLC run. Fifty microliters
of 0.2 % filtered, neat
plasma were analyzed per run. The elusion conditions were: a) five-minute
isoaatic run at 80% A/20%
B. during which the majority of the plasma proteins eluted, b) 25-minute
linear gradient to 30% A/70%
25 B during which AR177 elutes, c) five-minute Socratic run at 30% AI70% B. d}
one-minute linear
gradient to I00% B. e) two- minute run at 100% B for column clean-up, and fl
two-minute linear
gradient to 70% AI30% B for the step in the HPLC clean-up. The high pH ( 12)
of the elusion buffers
was necessary to dissociate AR177 from tissue constituents, which bind AR177
tightly around
physiological pH. AR177 is completely stable at pH 12. This method can clearly
distinguish between the
30 full length ARI77 and n-1, n-2, etc. species, which are potential metabolic
products. The ultraviolet
detection wavelength was 260 nm. The flow rate was 0.5 mLlminute in all steps.
Column clean up
between runs was performed by a 500 pL bolus injection of 0.1 M Tris base, 2 M
NaCI, pH 10.5,
followed by: a) ten-minute linear gradient to 60% A/40% B. b) one-minute
linear gradient to 100% B. c)
a three-minute isocratic run at i 00% B and d) one-minute linear gradient to
80% A/20% B.
35 A standard curve was generated by spiking AR177 into cynomolgus monkey
plasma in order to
achieve concentrations of 0.04 to 128 li/mL. The plasma standards and unspiked
plasma (control) were
g ~-
CA 02279488 1999-07-29
WO 98/33807 PCT/ITS98/01974
run on the anion-exchange HPLC column using the above conditions. The Waters
Millennium software
was used to determine the area under the peak for each AR177 standard at 260
nm. The HPLC peak area
versus AR177 concentration was plotted using Cricket Graph III 1.5.1 software.
There were one to three
HPLC replicate runs per AR177 standard. The limit of quantitation was 25 ng/mL
{SO pL injection),
whereas the limit of detection was 5 nglmL (50 pL injection). The overall
correlation coeffcient of the
fitted lines on the standard curve plots was greater than 0.999 on two
different standard curves used in
this study. The standard curve was Linear over an approximate 3,200-fold
range. The variability of the
replicates was 1-2% at all concentrations. There was one HPLC run per monkey
plasma sample. This
method was validated (Wallace, T.L., Bazemore, S.A., Kornbrust, D.J. and
Cossum, P.A. (199ba) J.
Pharmacol. Exp. T7zer. 278: 1306-12).
Pharmacolcinetic parameters. The volume of distribution (Vd)was calculated by
dividing the total
dose administered by the concentration at the end of the infusion (Rowland and
Tozer, 1995). The Cue,
(maximum concentration) was taken from the plasma concentration at the
conclusion of the ten-minute
intravenous infusion.
I5 Results/CIinical Observations and Hemodynamic Parameters. Aside from an
anticoagulant effect
described below, there were no indications of significant toxicity. No clearly
treatment-related changes
in blood pressure (Figure 41 ), heart rate (data not shown) or
electrocardiographic activity (data not
shown) were observed, no animals died following AR177 infusion. One high-dose
(50 mglkg) monkey
exhibited a rise in arterial pressure during the infusion followed by a
decline to approximately 20-30 mm
Hg below the pre-infusion blood pressure. These changes are qualitatively
similar to, but less
pronounced, than those seen in monkeys given total phosphorothioate
oligonucleotides (GaIbraith et al.,
1994). Although suggestive of a treatment effect, the alterations in blood
pressure in the subject animal
could not be clearly distinguished from normal fluctuations that occurred in
other animals, including one
control monkey. The only treatment related clinical sign was emesis during the
infusion, which occurred
in two of the three animals in the 50 mg/kg group and all of the monkeys that
received the 20 mg/kg
dose.
Serum Chemistry. There were no changes in any of the serum chemistry
parameters that could be
attributed to AR177.
Hematology. There were no changes in hematology values attributable to AR177.
Neutrophil counts
were increased to a similar extent in all groups, including the saline control
group, probably as a result
of the stress associated with the experimental procedure (Figure 42). The
characteristic neutropenia and
rebound neutrophilia that has been reported with other oiigonucleotides did
not occur in the
AR177-treated monkeys, which is consistent with the reiativeiy small changes
in complement Bb split
product (Figure 44) and CH50 (Figure 45) levels.
3S Coagulation parameters. The most salient effect of AR177 observed in this
study was a pronounced,
albeit transient, dose-dependent, reversible prolongation of aPTT in the 20
and 50 mg/kg groups, which
reflected inhibition of the intrinsic coagulation pathway. There was at least
a four-fold prolongation of
CA 02279488 1999-07-29
WO 98/33807 PCT/US98101974
aPTT in the 20 and 50 mg/kg dose groups at the conclusion of the infusion of
AR177. Determination of
' the upper aPTT value was limited by the range of the assay. (See Figure 43).
This change was reversible
in both dose groups. The aPTT was increased beyond the upper limit of the
assay in the 50 mg/kg group
for all or most of the two-hour monitoring period, but had returned to normal
by the following day.
Baseline aPTT values were reestablished by two hours after termination of
dosing with the 20 mg/kg
dose. In the 5 mg/kg group, only a small and transient rise in aPPT was
observed, and there was no
change in prothrombin time (PT). Similar changes have been observed with other
oligonucleotides, and
are believed to be, at least in part, attributable to direct and reversible
binding of the oligonucleotide to
thrombin (Henry et al., 1994, Pharmaceutical Res. 11: S353, 1994). PT was
affected to a much lesser
extent than aPTT in the 20 and 50 mg AR1771kg groups (data not shown),
indicating little or no effect
on the extrinsic pathway.
Complement activation. Plasma levels of the complement split product Bb, a
marker for activation of
the alternative pathway, were increased 60-85% over baseline in the 5 mg/kg
group, approximately
2-fold over baseline in the 20 mglkg group, and approximately 2- to 4-fold in
the 50 mg/kg group at the
end of infusion. (See Figure 44). The elevation in Bb persisted through the
duration of the 2-hour
monitoring period, but the values had returned to normal by the following day.
These increases in Bb
were small in magnitude. There were also small and transient decreases in the
CH50 levels (Figure 45)
in the 20 and 50 mglkg doses, but there was no dose-CH50 level relationship.
In confirmation of this
minimal change in CH50, AR177 had no effect on complement CH50 at doses up to
236 l.~/mL when it
was tested in vitro in human or cynomolgus monkey plasma. (See below). Thus, a
large increase in
complement activation, and resulting characteristic neutropenia and rebound
neutrophilia, that has been
reported with other oligonucleotides (Galbraith et al., 1994) did not occur in
the AR177-treated
monkeys (Figure 42).
Plasma AR177 concentration. Plasma concentrations of AR177 were maximal at the
end of the
infusion and declined thereafter with an approximate initial half life of 20-
30 minutes (Figure 46).
Another more complete study in cynomolgus monkeys has shown the terminal half
life to be
approximately 24 hours (See below). These half lives are much longer than that
reported by Lee et al.
(( 1995) Pharmaceut. Res. 12:1943-1947} in cynomolgus monkeys for GS-522, a 1
Smer oligonucieotide
that has a tetrad structure similar to AR177. No metabolites of AR177 could be
observed in the plasma
at any time point or dose. This contrasts with the results of Lee et al. (
1995), who found significant
amounts of shorter species of GS522 in monkey plasma following intravenous
infusion. The results with
AR177 suggest that AR177 does not undergo metabolism. There was a direct
relationship between the
AR177 plasma Cmax and the dose that was administered as a ten-minute
intravenous infusion to the
monkeys (Figure 45). Plasma Cmaxs of 83.2 +/- 7.2, 397.8 +/- 30.8, and 804.7
+/- 226.3 p.g/mL were
achieved for the 5, 20, or 50 mg/kg doses, respectively, at the end of the
infusion (+10 minute time
point) (Table E-2; Figure 47).
$%
CA 02279488 1999-07-29
WO 98/33807 PCT/US98/01974
The initial volume of distribution (Vd) of the three doses ranged from 200-248
mL (mean +
s.d.) (Table E-2) at the conclusion of the intravenous infusion. The mean body
weight of the monkeys in
the AR 177 dose groups was 3.67 kg. Assuming that plasma volume is 4% of body
weight (Davies and
Morris, 1993, Pharmaceutical Res. ID: 1093-1095), the plasma volume would be
147 mL. Thus, the
initial Vd was slightly greater than the plasma volume.
In general, there was a direct relationship between the plasma concentration
of ARI77 and
aPTT for the 5 (Figure 48) and 20 (Figure 49) mg/kg doses. For the 50 mg/kg
dose (Figure 50), the
aPTT values were off scale during the two-hour sampling period so it was not
possible to determine the
relationship between the plasma concentration and aPPT. There was a no effect
plasma AR177
concentration versus aPPT of approximately 60-100 ug AR177lmL, above which
there was prolongation
of aPTT. Doubling of aPTT was observed at plasma AR177 concentrations of
approximately 100-250
ug ARI77/mL. Tripling of aPTT was observed at plasma AR177 concentrations of
approximately 250-
300 fig AR1771mL, after which no correlation was possible because the aPTT
values were beyond the
aPTT assay range. The disappearance of AR177 from plasma was roughly
correlated with the return of
the aPTT to baseline, which is consistent with direct and reversible binding
of the oligonucleotide to one
or more clotting factors. By contrast, there did not appear to be a
correlation between the plasma
concentration of AR177 and complement split product Bb (data not shown).
In addition to the in vivo study in cynomoigus monkeys, an in vitro study was
performed which
investigated the effect of AR177 on the coagulation- cascade and complement
activity in cynomolgus
monkey and human plasma (coagulation) and serum (complement), respectively.
AR177 caused a
two-fold increase in aPTT at a concentration between 30 and 59 leg/mL of human
plasma, whereas the
compound caused a two-fold increase in aPTT at a concentration between 118 and
236 p.g/mL of
cynomolgus monkey plasma in vitro. AR177 had no effect on thrombin time in
human plasma, but
caused approximately a 2.5-fold increase in thrombin time in cynomolgus monkey
plasma at 236
p.g/mL. AR177 had no effect on either fibrinogen or complement CH50 at doses
up to 236 ug/mL in
human or cynomolgus monkey plasma. AR177 caused a 30% increase in prothrombin
time in human
plasma and approximately a I S% increase in prothrombin time in cynomolgus
monkey plasma at 236
p,g/mL.
Discussion. Using an identical dosing regimen to that used in previous
experiments that resulted in
profound hemodynamic effects, the present study showed that AR177 was very
safe. Although limited
conclusions can be drawn from the present study because only one partial
phosphorothioate (ARI77)
was examined, it is possible that the lack of the cardiovascular toxicity is
due to the limited number of
phosphorothioate linkages (two) in AR177. It is also speculated that the lack
of toxicity could be due to
the three-dimensional (i.e. tetrad) shape of AR177 (Rando et al., 1995, J.
Biol. Chem. 270: 1754-1760).
In confirmation of the lack of toxicity of AR177 found in the present study,
AR177 does not cause
Po
r
CA 02279488 1999-07-29
WO 98133807 PCT/US98/01974
toxicity when it is administered as a bolus intravenous injection to
cynomolgus monkeys every other day
'at 40 mg/kg for a total of 12 doses (see below).
There was minimal activation of the complement system following administration
of AR177.
Small increases (2-4 fold) in plasma Bb levels occurred at plasma ARI77
concentrations as high as 750
- 5 1/mL after a dose of 50 rngJkg given as a ten-minute intravenous infusion.
Minimal changes (-25%) in
the CHX levels occurred at plasma AR177 concentrations as high as 750 pg/mL
after a dose of 50
mglkg given as a ten-minute intravenous infusion. The complement activation
that was seen with
AR177 at these high doses did not even result in hypotension. By contrast,
Galbraith et al. (1994) have
reported that GEM91, a 25-mer phosphorothioate oligonucieotide, caused an 80%
decrease in
complement CH50, a 700% increase in the level of complement CSa, and death in
two out of four
monkeys following the intravenous infusion of 20 mg/kg over ten minutes. The
mechanism by which
oligonucleotides activate the complement system is unknown. However, this
phenomenon bears
resemblance to a similar phenomenon in human patients during dialysis in which
contact between blood
and dialyser membrane induces complement activation and profound neutropenia
(Heierli et al., 1988,
Nephrol. Dial. Transplant 3: 773-783; Jacobs et al., 1989, Nephron 52: 119-
124). The present work
indicates that although AR177 induces some minimal complement activation, this
is not translated into
the hemodynamic toxicity that has been seen with other oligonucleotides.
AR177 administration resulted in the dose-dependent inhibition of the
intrinsic coagulation
pathway, reflected by prolongation of aPTT. The effect was maximal at the end
of infusion and was
reversed in parallel with clearance of AR177 from plasma. The inhibition of
coagulation was significant
at the highest dose level, but marginal and not considered clinically
significant at the 5 mgJkg dose level.
An anticoagulant effect has been reported to be a class effect of
oligonucleotides (Henry et al., 1994).
The anticoagulam results with AR177 thus agree with results that have been
seen with other
oligonucleotides, although AR177 is 40-fold less potent than the thrombin-
binding aptamer
oligonucleotide that has been reported by Grin et al. ( 1993).
In conclusion, AR177 does not cause mortality, cardiovascular toxicity, or
alterations in clinical
chemistry in cynomolgus monkeys receiving doses up to 50 mgJkg as a ten-minute
intravenous infusion.
However, there was a reversible prolongation of coagulation time at doses of
20 and 50 mg/kg. Taken
together, the data suggest that AR177 does not have the hemodynamic toxicities
that are associated with
total phosphorothioate oligonucleotides, and can be administered safely as an
intravenous infusion over
ten minutes.
CA 02279488 1999-07-29
WO 98/33807 PCT/I1S98/01974
TABLE E-1. Monkey Dosing Information
Dose Monkey
Group TreatmentDose Dose volume MaleFemale weight
level Conc. (mL/kg) (kg)
(mg/kg) (mglmL) mean t
s.d.
I Placebo0 0 4.0 1 2 3.67 t
0.38
2 AR177 5 L.25 4.0 2 ! 4.12 f
0.83
3 AR177 20 5.0 4.0 2 1 3.67 f
0.29
4 AR177 SO 4.0 4.0 1 2 3.23 f
0.76
Table E-1 - Monkey dosing information. Cynomolgus monkeys were given ten-
minute, intravenous
infusions of 5, 20 or 50 mg AR177/kg at a volume of 4 mL/kg.
TABLE E-2. Plasma AR177, Vd, aPTT and complement Bb values at C,",,~
Dose AR177 plasmaVd (mL) aPTT Bb
(mglkg) (ug/mL) (seconds) (uglmL)
5 83.2 f 7.2 247.6121.4 45.91 11.4 0.5910.07
20 397.8 f f 84.5 f > 166.8 1.48 t 0.44
30.8 14.3 t 23.8
50 804.7 t 200.7 t > 170.0 1.28 t 0.46
226.3 56.4 t 34.6
Table E-2 - Plasma AR177 Cm,x, aPTT and complement Bb levels. The ARI77 plasma
Cue,
aPTT, and Bb values are the means t standard deviations of data at the +10
minute tune point (end of
the infusion). The baseline ( 10 minutes prior to dosing) aPTT levels were
32.1 t 4.4, 41.6, 6.7 and
33.2 t 4.8 seconds for the 5, 20, and 50 mg/kg doses, respectively (mean t
s.d.). The baseline ( 10
minutes prior to dosing) Bb levels were 0.44 ~ 0.14, 0.78 f 0.46 and 0.49 ~
0.21 E.~/mL for Me 5, Z0,
and 50 mg/kg doses. Volume of distribution (Vd) = dose/plasma C,,~,~, where
the dose is the total mg
of AR177.
r
CA 02279488 1999-07-29
WO 98/33807 PCTIUS98/01974
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CA 02279488 1999-07-29
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CA 02279488 1999-07-29
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CA 02279488 1999-07-29
WO 98/33807 PCTJUS98/01974
F Repeat-dose toxicity and pharmacokinetics of martial nhosphorothioate anti
HIV oliaonucleotide
(AR1771 following bolus intravenous administration to cynomoigus monks
AR177 is a 17-mer partial phosphorothioate oiigonucleotide with the sequence
S'GTGGTGGGTGGGTGGGT-3', with sulfurs at the terminal internucleoside linkages
at the 3' and 5'
ends. It is a potent inhibitor of HIV integrase and HIV production in vitro
(Rando et al., 1995; Ojwang et
al., 1995), and has a long tissue half life in rodents (unpublished data).
AR177 does not have an
antisense- or triplex-based mechanism of action. A previous study has shown
that AR177 does not cause
the characteristic hypotension or neutropenia of other oligonucleotides
(Cornish et al., 1993; GaIbraith et
aL, 1994) following a ten-minute intravenous infusion, at doses up to SO mg/kg
(Wallace, T.L.,
Bazemore, S.A., Kornbrust, D.J., Cossum, P.A ( 1996b) J. Pharmacol. Exp. Ther.
278: 13 I3-7). As part
of the pre-clinical assessment of AR177, an intravenous toxicity study of
AR177 was conducted in
cynomolgus monkeys with the objective of establishing the clinical and
histopathological changes that
occur following repeated doses.
I 5 Materials and Methods for Repeat Dose Studies
Materials. For HPLC analysis of plasma AR177 concentrations, tris was obtained
from Fisher, NaBr
and NaCI were obtained from Sigma, and methanol was purchased from J. T.
Baker. The Gen-Pak Fax
anion-exchange HPLC column {4.6 x 100 mm, cat. no. #15490) was purchased from
Waters.
AR177 was synthesized at Biosearch, a division of PerSeptive Biosystems, on a
Milligen 8800
oligonucleotide synthesizer, and vialed at 25 mg/mL in phosphatebuffered
saline. AR177 has a
molecular weight of 5793, and is a fully neutralized sodium salt. The
structure of AR177 was
characterized by phosphorus and proton NMR, sequencing, base composition,
laser Resorption mass
spectrometry, anion exchange HPLC and polyacrylamide gel electrophoresis. Ail
analyses were
consistent with the proposed structure. The AR177 was approximately 94% pure
according to HPLC
and electrophoresis analysis.
The monkeys used in this study were laboratory bred (C.V. Primates, Indonesia
or Yunnan
National Laboratory, China) and were experimentally naive prior to the study.
The age of the monkeys
was 3 to 61 /2.
Dosing. AR177 was administered intravenously over 1-2 minutes into unsedated
monkeys every other
day for 23 days (12 doses) by injection into the femoral vein. (See Table E-
I). The monkeys were not
sedated, but were restrained during dosing. The highest dose level (40
mg/kg/injection) was selected
based on observations in a previous single-dose study of pronounced
anticoagulant activity of AR177 at
a dose of 50 mg/kg infused over 10 minutes (Wallace et al., 1996b). A
comparable or greater degree of
anticoagulation was expected to occur with fast ( I-2 minute) infusion of 40
mg/kg, and was confirmed
by the results of this study. The dosing schedule (every other day) was chosen
in order to avoid
excessive accumulation of the test material, which, based on pharmacokinetic
data obtained in rats
(Waliace et al., 1996b), would be expected to occur with daily administration.
t.
CA 02279488 1999-07-29
WO 98133807 PCTIL1S98/01974
The monkeys were observed twice daily for general health, changes in appetite
and clinical
signs of adverse events. Body weights were measured within a few days prior to
the first dose (Day 1 )
and approximately weekly thereafter. Electrocardiographic (ECG) recordings
were obtained from all
animals prior to the study and on Day 22, and from recovery animals on Day 35.
Blood samples were
collected for evaluation of serum chemistry, hematology and coagulation
parameters from all animals
prior to the initiation of the study, on the first day of dosing (Dose 1; Day
1 ), and on the last day of
dosing (Dose 12; Day 23). The sample collection on Days 1 and 23 was timed
relative to dose
administration in order to characterize possible acute effects on hematology
parameters. An additional
clinical pathology evaluation was conducted for all animals on Day 24, as well
as for recovery animals
an Day 37. Blood was collected from ail animals at 5 minutes, 30 minutes and 4
hours post-dosing on
Days I and 23 for analysis of the plasma AR177 concentration.
On Day 25 (two days after the last dose), three males and three females from
each group were
humanely euthanized and necropsied, while the remaining two animals each in
the high-dose and control
groups were continued on study for an additional two-week treatment-free
"recovery" period, and were
euthanized on Day 38. Complete gross necropsies were performed on all animals
at their scheduled
termination. Urine was collected from each animal during necropsy by bladder
puncture and submitted
for routine urinalysis. Weights of 13 major organs were recorded, and numerous
tissues were collected,
preserved and processed for histology.
Serum chemistry parameters. Serum chemistry was determined pre-study, on day
24 (one day after
the 12th dose), and on day 37 in the recovery monkeys. The following were
determined: sodium,
potassium, chloride, carbon dioxide, total bilirubin, direct bilirubin,
indirect bilirubin alkaline
phosphatase, lactate dehydrogenase, aspartate aminotransferase, alanine
aminotransferase, gamma
glutamyltransferase, calcium, phosphorus, glucose, urea nitrogen, creatinine,
uric acid total protein,
albumin, globulin, cholesterol and triglycerides. Serum chemistry was
determined at Sierra Nevada
Laboratories (Reno, NV).
Hematology and coagulation parameters. Hematology and coagulation parameters
were determined
9-11 days prior to the start of the study, just prior to administering doses I
(day 1 ) and 12 (day 23), five
minutes after dosing (coagulation only), 30 minutes and 4 hours following
dosing, one day after the 12th
dose (day 24), and in recovery monkeys at sacrifice (day 37). The following
were determined: red blood
cell count and morphology, total and differential white blood cells,
hemoglobin, hematocrit,
prothrombin time, fibrinogen, mean cell hemoglobin, mean corpuscular volume,
mean corpuscular
. hemoglobin concentration, platelet count, activate partial thromboplastin
time, and D-dimer.
Hematology was determined at Sierra Nevada Laboratories (Reno, NV).
AR177 plasma HPLC analysis. Blood was taken for plasma analysis of AR177 just
prior to, and at 5,
30 and 240 minutes following administration of doses 1 and I2. The plasma
fraction was obtained by
low speed centrifugation of blood, and stored at -20°C until analyzed
for the AR177 concentration.
Plasma AR177 concentrations were assayed using an anion-exchange HPLC method
on a Waters HPLC
CA 02279488 1999-07-29
WO 98/33807 PCTIUS98I01974
system with a 626 pump, 996 photodiode array detector, 7I7 autosampler and
Millennium system
software controlled by an NEC Image 466 computer. Buffer A was 0.1 M Tris
base, 20% methanol, pH
12, and Buffer B was 0.1 M Tris base, 1.0 M NaBr, pH 12. anion-exchange column
teen-Pak Fax
column) was equilibrated at 80% buffer A120% buffer B for 30 minutes before
each HPLC run. Fifty
microliters of plasma were analyzed per run. The elusion conditions were: a)
five-minute isocratic run at
80% A/20% B. during which the majority of the plasma proteins eluted, b) 30-
minute linear gradient to
30% A/70% B during which the AR177 eluted, c) five-minute isocratic run at 30%
A/70% B. d) one -
minute linear gradient to 100% B. e) two- minute run at 100% B for initial
column cleanup, and fl
two-minute linear gradient to 70% A/30% B for the initial step in the clean-up
method for HPLC
column clean-up. The high pH (12) of the elusion buffers was necessary to
dissociate AR177 from tissue
constituents, which bind AR177 tightly around physiological pH. AR177 is
completely stable at pH 12.
This method can clearly distinguish between the full length AR177 and n-1, n-
2, etc. species, which are
potential metabolic products. The W detection wavelength was 260 nm. The flow
rate was 0.5
mL/minute in all steps. All runs were performed at room temperature. Column
clean up between runs
1 S was performed by a 500 uL bolus injection of 0.1 M Tris base, 2 M NaCI, pH
10.5, followed by: a)
ten-minute linear gradient to 60% A/40% B. b) one-minute linear gradient to
100% B, c) a three-minute
isocratic run at 100% B and d) one-minute linear gradient to 80% A120% B.
AR177 was spiked into cynomolgus monkey plasma in order to achieve
concentrations of
0.0635 to 125 p.g/mL for the standard curve. The plasma standards and unspiked
plasma (control) were
run on the anion-exchange HPLC column using the above conditions. The Waters
Millennium software
was used to determine the area under the peak for each AR177 standard at 260
nm. The HPLC area
versus AR177 concentration was plotted using Cricket Graph m 1.5.1 software.
There were two HPLC
replicate runs per AR177 standard. The areas which represented the lowest
concentration were at least
two times the background area at 260 nm. The overall correlation coefficient
of the fitted lines on the
standard curve plots was greater than 0.999. There was a linear concentration
versus A260 relationship
over a minimum 6,SOOfold range. The variability of the replicates was 1-2%.
This method was validated.
Necropsy and histopathology. A complete necropsy was conducted on all monkeys,
and included
examination of the external surface of body (body orifices; dosing site;
cranial, nasal, paranasal,
thoracic, abdominal and pelvic cavities), and the external surface of the
brain and spinal cord. The organ
weights of the adrenals, epididymies, liver, pituitary, spleen, thyroids,
parathyroids, brain, heart, lungs,
prostate, testes, uterus, cervix, kidney, ovaries, seminal vesicles, and
thymus were recorded.
A histopathological assessment was made of 46 hematoxylin and eosin-stained
tissues by a
veterinary pathologist. These included tissues from the cardiovascular,
digestive, respiratory, urogenital,
lymphoid/hematopoietic, skin/musculoskeletal and nervous systems, and all
major organs.
Results
CA 02279488 1999-07-29
WO 98133807 PCT/US98/01974
Clinical observations. No animals died during the course of the study, and
there were no effects on
body weight. The only treatment-related clinical sign was an incidence of
discoloration around the eyes
in three high-dose animals, which occurred on only one occasion (Day 16 or 18)
for two of the animals,
and on four consecutive days (Days 18-21 ) in the third animal. The latter
monkey also had swelling
.- 5 around the eyes on Day I 8. The reaction was transient and was limited to
the high-dose group.
ECG, clinical chemistry, urinalysis and hematology. No abnormalities in the
ECG recordings were
noted, and there were no treatment-related changes in serum chemistry or
urinalysis parameters. The
only changes in hematology parameters considered possibly treatment-related
were an acute and
transient increase in lymphocytes in the high-dose group, and an acute
decrease in eosinophils which
was seen in ail groups, but appeared to be more pronounced in the AR177-
treated groups. Both of these
changes were observed shortly following dosing on Days 1 and 23 (i.e., those
days when clinical
pathology was evaluated at several time points post-dosing), but were largely
absent on Day 24 (one day
after the last dose). The values generally remained within the normal range
and were not considered
indicative of significant toxicity.
Necropsy and Histopathology. No clearly treatment-related histopathologic
changes were seen in any
organs or tissues, and no effects on organ weight were evident. Eosinophilic
material was seen in a few
tubules in the medullary area of the kidneys of three monkeys in the high dose
group on day 25, but was
not seen in the controls or in the recovery animals. Although this may be
treatment-related, eosinophilic
material can sometimes be observed in the renal tubules of healthy, untreated
monkeys.
Plasma AR177 concentration. Figure 51 shows that there was no difference
between the AR177
plasma concentrations that were achieved after the first and twelfth (last)
doses at either 2.5, 10 or 40
mg/kg. The plasma concentration versus time profile of AR177 is shown in
Figure 52. At the earliest
sampling time point (five minutes after initiation of dosing), maximal plasma
levels of 35.79 t 5.99,
135.43 t 16.19 and 416.54 t 54.55 Li/mL were achieved for dose #1 at 2.5, 10
and 40 mglkg (Figure
52). At the earliest sampling time point (five minutes after initiation of
dosing), maximal plasma levels
of 33.98 + 9.98, 113.71 t 26.55 and 386.39 t 70.29 I.JmL were achieved for
dose #12 at 2.5, 10 and 40
mg/kg. The decay kinetics of the 2.5 mg/kg dose appeared to be different than
the decay kinetics of the
10 and 40 mg/kg doses after either dose 1 (Figure 52), although no definite
conclusions can be drawn
because of the limited number of time points. No metabolites (i.e. n-I, n-2,
etc.) could be observed in the
plasma at any time or any dose. This confirms results in rats showing no
metabolism of AR177 (Wallace
et al., 1996b).
Coagulation parameters. Dose-dependent anticoagulant activity was manifested
at the 10 (Figure 54)
and 40 (Figure 55) mg/kg doses, whereas there was no anticoagulant activity
following the 2.5 mg/kg
dose (Figure 53}. This activity was evident from the prolongation of activated
partial thromboplastin
time (aPTT), which reflects a primary effect on the intrinsic coagulation
pathway. Following both the
1 st and I 2th doses, mean aPTT in the 10 mg/kg group was increased to
approximately twice the
CA 02279488 1999-07-29
WO 98133807 PCTIUS98101974
. pre-dose value by 5 minutes post-dosing, but had returned to baseline levels
by four hours. Following
both the 1st and 12th doses, mean aPTT in the 40 mg/kg group exceeded the
upper limit of the assay
five minutes after dosing. By 30 minutes post-dosing, aPTT values in the 40
mg/kg group had declined
to approximately 2 to 4-fold above the pre-dose level. By four hours, the aPTT
had returned to the
pre-dose levels in all but one monkey.
The relationship between the AR177 plasma concentration and aPTT is also shown
in Figures
53, 54, and 55 for doses 2.5, 10, and 40 mg/kg, respectively. There was a no
effect plasma AR177
concentration versus aPTT of approximately 60-100 ~g AR177/mL, above which
there was
prolongation of aPTT. Doubling of aPTT was observed at plasma AR 177
concentrations of
approximately 100-220 p,g AR177/mL. Tripling of aPTT was observed at plasma
AR177 concentrations
of approximately 220-300 pg AR177/mL, after which no correlation was possible
because the aPTT
values were off scale. There was no change in aPTT after the f rst or twelfth
doses of 2.5 mg/kg (Figure
53), since the AR177 plasma concentration did not reach the threshold of
approximately 60-100 lg/mL.
There was a maximal two-fold increase in aPTT after the first or twelfth 10
mgJkg doses (Figure 54).
The elimination kinetics of AR177 and the return of aPTT to baseline levels
were similar after the first
or twelfth doses.
Discussion
These results indicate that AR177, administered as bolus intravenous
injections up to 40 mg/kg
every other day for 12 doses, did not cause mortality, histopathological or
cardiovascular events that
have been described for other oligonucleotides (Galbraith et al., 1994;
Srinivasan and Iversen, 1995).
The only significant change that was observed was a prolongation of aPTT,
which was reversible. To
our knowledge, this is the first oligonucleotide that has not been observed to
cause liver and kidney
toxicity following intravenous administration.
The structure of ARI77 may contribute to its lack of general toxicity. AR177
contains only two
phosphorothioate bonds at the 3' and 5' termini. These phosphorothioate bonds
were designed to help
prevent endonuclease-induced cleavage of AR177. We speculate that the small
number of sulfurs may
have reduced the propensity to bind to proteins, a phenomenon that has been
observed for full
phosphorothioates, which has been speculated to cause toxicity (Srinivasan and
Iversen, 1995). ARl7Ts
three-dimensional shape may also contribute to its lack of toxicity. AR177 has
been shown to form a
structure in which hydrogen bonds form between deoxyguanosine residues to
create a "G-tetrad" (Rando
et al., 1995). This tetrad structure imparts a compact shape which makes it
resistant to degradation
(Bishop et al., 1996) and may make it relatively non-toxic by minimizing
reactive sites. The resistance
to degradation has been noted in single and repeat dose pharmacokinetics
studies in rodents (Wallace et
al., 1996a;1996b), and in a more complete pharmacokinetic study in cynomoigus
monkeys which
showed a terminal plasma half life of greater than 24 hours (data not shown).
CA 02279488 1999-07-29
WO 98/33807 PCT/US98101974
The results of the AR177 plasma analysis demonstrated that there was no
difference between
the AR177 plasma concentrations that were achieved after the first or twelfth
(last) doses of 2.5, 10 or
40 mg/kg. These results can be interpreted to mean that AR177 does not induce
metabolic enzymes that
would, if they were induced, reduce the concentration of AR177 by increasing
its metabolism. This has
the important implication that repeat doses of AR177, at least when given
every other day for 23 days,
will not result in pharmacokinetic tolerance.
The results of the AR177 plasma analysis demonstrated that there was a close
relationship
between the ARI?7 plasma concentration and aPTT. There was a no erect plasma
ARi 77 concentration
versus aPTT of approximately 60-100 pg AR177/mL, above which there was
prolongation of aPTT.
The ability to prolong coagulation has been noted to be a feature of other
oligonucleotides (Bock et al.,
1992; Henry et al., 1994}. An oligonucleotide composed of deoxyguanosines and
thymidines has been
described that binds to thrombin (Paborsky et al., 1993), demonstrates
sequence dependent inhibition of
coagulation in vitro (Bock et al. 1992), has a G-tetrad structure (Wang et
al., 1993), and is active as a
short acting anticoagulant in vivo (Griffin et al., 1993; DeAnda et al.,
1994). The structure of the
oligonucleotide, (GGTTGGTGTGGTTGG) bears some resemblance to AR177
(GTGGTGGGTGGGTGGGT). Both oligonucleotides form G-tetrad structures. A
comparison of the
anticoagulant properties of these oligonucleotides indicates that the
oligonucleotide is approximately
10-100 times more potent than AR177. An examination of the anti-HIV properties
of the
oligonucleotide showed that it had little or no anti-HIV activity (unpublished
data). Thus, although both
oligonucleotides are composed of deoxyguanosines and thymidine, and form G-
tetrads, they have
distinct biological properties.
In conclusion, administration of up to 40 mg/kg of AR177 to cynomolgus monkeys
by bolus
intravenous injection every other day for 23 days was well tolerated. No
mortality or clinical signs of
signifccant toxicity occurred. The most salient alteration in clinical
pathology parameters was the
prolongation of aPTT in the 10 and 40 mglkg groups, which reflects inhibition
of the intrinsic
coagulation pathway. The approximate doubling of aPTT observed in the middle-
dose group ( 10
mg/kg) is considered to be marginally clinically significant following bolus
intravenous injection. The
severe inhibition of coagulation in the 40 mg/kg group may not be dose
limiting since aPTT values had
returned to baseline levels fours hours following dosing. It is probable that
prolongation of aPTT at
these doses could be circumvented by administering AR177 as a slow infusion
over the course of
several hours in order to stay below the threshold for anticoagulation, which
was established to be
60-100 p,g/mL of AR177. The absence of clinical pathology abnormalities or
tissue histopathology at
even the highest dose (40 mg/kg) after repeated intravenous administration
suggests that there is little
potential for cumulative toxicity with T31077 with any type of administration.
CA 02279488 1999-07-29
WO 98133807 PCT/US98/01974
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CA 02279488 1999-07-29
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CA 02279488 1999-07-29
WO 98/33807 PCT/US98/01974
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CA 02279488 1999-07-29
WO 98/33807 PCT/ITS98/01974
G. Human Clinical Trials
Four HIV-infected patients/group were dosed with AR177 at 0.75 mg/kg and 1/5
mg/kg, and
two HN-infected patients were dosed so far with AR 177 at 3.0 mg/kg by
intravenous infusion over two
hours.
,. 5 Methods. Blood was collected in EDTAp coated tubes at 0.25, 0.5, 1, 2,
2.05, 2.5, 3, 3.5, 4, 6, 8, 11, 14,
26, 48, 98, and 122 hours following initiation of drug administration. Plasma
was obtained by low
speed centrifugation of the blood, and was stored frozen until analyzed by
HPLC for AR177
concentration. The concentration of AR177 was determined in patient plasma
using a validated anion-
exchange HPLC method at the Division of Clinical Pharmacy of the University of
California, San
Francisco. This method has a limit of quantitation of 15 ng/mL in human
plasma.
Pharmacoitinetic analysis. Pharmacokinetic parameters were calculated using
PKAnalyst software
(MicroMath, Salt Lake City, UT). The pharmacokinetic data best fit a two
compartment model for all of
the patients. The alpha and beta half lives were almost identical in each of
the patients, based on the
software interpretation of the AR177 plasma concentration versus time plot
(Figures 56-59). For this
I S reason, only one half life is reported. (Note that in monkeys, a third
half life of approximately 24 hours
was observed at a dose of 5 mg/kg given as an intravenous infusion over two
hours. A third half life
was not evident in human data, except perhaps for patient # 10.) For each
pharmacokinetic parameter,
the mean ts.d. of n=4 was calculated for the 0.75 and 1.5 mg/kg groups and the
mean ts.d. of n=2 was
calculated for the 3.0 mglkg group.
Results. The plasma concentrations of AR I 77 fol lowing intravenous infusion
are shown in Figure 56
(0.75 mg/kg), Figure 57 ( 1.5 mg/kg), Figure 58 (3.0 mg/kg) and Figure 59 (all
doses). Analysis of this
data indicate that the plasma pharmacokinetics of AR177 are not directly
proportional to the dose (Table
G-1 ). The increase in the Cm~ and AUC were proportionally much greater than
the increase in the dose
from 0.75 to 3.0 mg/kg. The increase in the Cm~ and AUC were much greater than
the increase in the
dose. The C",~ value in the 0.75 mglkg group was 5. i t 1.4 Tg/mL and the
C",aX value in the 3.0 mg/kg
group was 37.5 t 0.1 ug/mL, approximately a seven-fold increase (Figure 60).
The AUC value in the
0.75 mg/kg group was 703.6 t 154.7 ug-min/mL and the AUC value in the 3.0
mg/kg group was
8,277.8 ~ 2.937.4 ug-min/mL, approximately a 12-fold increase (Table 1 ).
The plasma clearance and Vd values reflected the Cm~ and AUC data. ' The
plasma clearance in
the 0.75 mg/kg group was 1.1 t 0.2 mL/min/kg and the clearance in the 3.0
mg/kg group was 0.4 f 0.2
mL/min/kg, approximately a 65% decrease (Figure 61 ). The initial and steady-
state volumes of
. distribution in the 0.75 mg/kg group were 0.16 ~ 0.05 L/kg and 0.14 t 0.05
L/kg, respectively, whereas
the initial and steady-state volumes of distribution (Vd) in the 3.0 mg/kg
group were 0.08 t 0.00 L/kg
and 0.05 t 0.03 Llkg, respectively (Table 1).
~6 ~-
CA 02279488 1999-07-29
WO 98133807 PCT/US98101974
In agreement with the above data, the plasma half life in the 0.75 mg/kg group
was 28.0 t 12.7
minutes, and the half life in the 3.0 mg/kg group was 120.1 f 60.7 minutes,
approximately a four fold
increase (Figure 60).
Conclusions. These results indicate that the plasma pharmacokinetics of AR177
are non-linear and
suggest that there is a saturable mechanism for the elimination of the drug.
~e~
CA 02279488 1999-07-29
WO 98133807 PCT/US98/01974
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CA 02279488 1999-07-29
WO 98133807 PCT/US98101974
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CA 02279488 1999-07-29
WO 98/33807 PCT/LTS98101974
h
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CA 02279488 1999-07-29
WO 98/33807 PCTIUS98/01974
Multi-Dose Trials. Zintevir~ (AR177; T30177) was next used in multiple dosing
experiments with
AIDS patients. Supporting rationale include:
~ anti-HIV-1 activity at sub-micromolar concentrations in lymphocytes infected
with clinical isolates
of HIV-1;
S ~ prevention of cytopathic effects if HIV-I in primary CD4+T-cell
lymphocytes;
~ activity at high multiplicities of infection (MOI); and
~ a novel mechanism of action that does not involve inhibition of either
reverse transcriptase (RT) or
protease activity.
Study Design. This was an open-label, single-center, study to evaluate the
safety, pharmacokinetic
profile and virologic/immunologic activity of ARI77 in HIV patients. Patients
that met the screening
criteria received multiple doses of AR177 infused every other day for 14 days
(seven doses). Patients
were allowed to participate in the study at ONLY one dose level. Patients were
confined to the
Research Unit from Day 0 through Day 18. Patient activities outside the unit
had to be acceptable to,
and agreed upon prior to study initiation.
Drugs. AR177 was provided by Aronex Pharmaceuticals, Inc. AR177 was obtained
from multiple lots
during the course of the study. The study drug was available in two vial
sizes. These clear glass vials
contained 2.2 cc or 15.9 cc of product. Each ml of active drug wilt deliver a
25 mg dose; thus, the
expected total mg dose per vial is 55.0 milligrams and 397.50 milligrams,
respectively.
Dosages. Patients meeting all entry criteria were given a two-hour continuous
infusion of AR177 every
other day for a total of seven infusions. The dosing schedule utilized is
shown in the following table (G-
2).
DOSAGE SCHEDULE
Group Study MedicationDose Level Number of Patients
I AR177 1.5 mg/kg 3
*2 AR177 3.0 mg/kg 8
*Escalation
will occur
at a I 00%
increment
from I .5
mg/kg (Group
1 ) to 3.0
mg/kg
(Group 2),
if no >_
Grade III
toxicity(ies)
occurs.
Dose escalation occurred at a 100% increment from the starting does of 1.50
mg/kg (Group 1 ) to 3.0
mg/kg (Group 2), if no ? Grade III toxicities are observed. Details regarding
does escalation and/or de-
escalation, and the number of patients to be enrolled at each dose level if
toxicity is observed, was
determined.
CA 02279488 1999-07-29
WO 98/33807 PCTIUS98101974
DOSE AD~STRATION
The intravenous infusion of study medication will be administered continuously
via an
indwelling LV. catheter at a rate of 2 mLlmin for two hours.
RESULTS
' S The plasma levels of HIV-1 RNA are an accepted measure of the plasma viral
titer and are
directly related to the progression of HIV infection to acquired immunodef
ciency disease syndrome
~ (A)DS) and death in humans. Mellors et al. ( 1996) Science 272:1167-1170.
Striking results were
obtained over the course of a 14-day treatment. In each of the three patients
given 3.0 mg/kg dosages,
viral load was significantly reduced.
~~ 3
CA 02279488 1999-07-29
WO 98133807 PCT/US98I01974
0 0 0
N
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y
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CA 02279488 1999-07-29
WO 98/33807 PCT/CTS98/01974
H. Anti-HIV-1 Animal Model Studies of T30177
As disclosed hereinabove, and as reported by Ojwang et al. (Antimicrobial
Agents and
' Chemotherapy, 39: 2426-2435 (1995)) the guanosine-rich oligonucleotide
T30I77, which is stabilized
by an intramolecular guanosine octet, is a potent inhibitor of laboratory
strains and clinical isolates of
human immunodeficiency virus type 1. Rabin et al. (Rabin, L., et al.
Antimicrobial Agents and
Chemotherapy, 40: 755-762 (March 1996) have disclosed a standardized procedure
for infection of
SCID-hu thy/liv mice with human immunodeficiency virus type 1. This
publication is incorporated
herein by reference to the extent that it provides materials and methods not
specifically set forth
herein.
in vivo Anti-HIV-1 AcNvity of the T30177 ODN
A representative ODN, T30177, was tested for its anti-human immunodeficiency
virus type 1
activity in vivo using the procedures and materials described below. The
experimental results are
shown in Tables H1-H6 and Fig. 65. Shown in Fig. 65 are the implant p24, W632
expression, viral
titer, and viral RNA load in an HIV-1 infected animal model treated
intraperitoneally with T30177
(AR177) at doses of 10, 30 and 100 mg/kg/day. Corresponding data for
untreated, mock-infected,
and ddI treated SC1D-hu mice are also shown in Fig. 65.
Table H-1 gives a summary of the results of these studies, which are described
in more detail
below. In brief, untreated SCID-hu Thy/Liv mice supported HIV-I replication
after direct inoculation
of their human ThyILiv implants with 630-1300 TCID50 of HIV-1 (NL4-3). Viral
replication in the
implants was apparent 12 days after inoculation by the presence of p24 antigen
(620 pg per I06 cells),
infectious virus, HN-1 proviral DNA, HIV-I RNA ( 1 O6'' copies per 1 O6
cells), and a 5-fold increase
in HLA class I (W632) expression by implant thymocytes. No depletion of CD4+
CDS+ immature
cortical thymocytes and a small reduction in the CD4/CD8 ratio were observed.
Intraperitoneal administration of AR177 resulted in potent antiviral activity
in this model
when treatment was initiated 24 h before inoculation. Statistically
significant, dose-dependent
reductions in implant p24 level, viral titer, viral RNA, and W632 expression
were observed. Implants
from 4 of 6 mice treated with AR177 at 100 mg/kg/day had no detectable p24
antigen, infectious
virus, HIV-1 RNA, or proviral DNA after 13 days of treatment. Treatment with
AR177 also prevented
virus-induced reduction of the CD4/CD8 ratio in the Thy/Liv implants.
CA 02279488 1999-07-29
WO 98133807 PCT/US9810I974
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CA 02279488 1999-07-29
WO 98133807 PCT/US98101974
The SCID-hu mouse model (McCune, J.M. et ai. ( 1988) Science 241:1632-1639)
was
developed to study mechanisms of HIV-1 pathogenesis in vivo and to serve as a
model for preclinicai
evaluation and prioritization of compounds possessing anti-HIV-I activity in
vitro. This model is
constructed by transplantation of interactive human lymphoid organs into
immunodeficient C.B 17
scidlscid mice. The SCID-hu model has been optimized by use of conjoint
implants of human fetal
thymus and liver to create SC)D-hu Thy/Liv mice. The human fetal tissue
becomes vascularized and
' grows when implanted beneath the kidney capsule, eventually reaching a total
mass of 10'-108 human
cells in 80-90% of recipients (Namikawa, R., et al. (1988) J. Exp. Med
172:1055-1063). Importantly,
the Thy/Liv implants sustain multiiineage human hematopoiesis and provide for
a continuous source
of human CD4+ T cells for up to 12 months (Krowka, J., et ai. (1991) J.
Immunol. 145:3751-3756;
Namikawa, R., et al. ( 1988) J. Exp. Med. 172:1055-1063; Vandekerckhov,
B.A.E., et al. ( 1991) J.
Immunol. 46:4173-4179; V andekerckhove, B.A.E., et al. ( 1992) J. Exp. Med
176:16 i 9-1624). The
implants support viral replication after direct inoculation of HIV-1
(Namikawa, R., et al. (1988)
Science 242:1684-1687), and thymocyte depletion occurs with some viral
isolates within 3-5 weeks
(Aldrovandi, G., et ai. (1993) Nature 363:732-736; Bonyhadi, M.L., et al.
(1993) Nature 363:728-
732; Bonyhadi, M.L., et al. ( 1993) Nature 363:728-732; Kaneshima, H., et al.
( 1994) J. Yirol.
68:8188-8192; Stanley, S.K., et al. 1993) J. Exp. Med 178:1151-1163). This
depletion includes loss
of CD4+ CD8+ immature cortical thymocytes and a decrease in the CD4/CD8 ratio
in the thymic
medulla and in peripheral blood. Several mechanisms have been associated with
these events,
including indirect, apoptotic destruction of uninfected thymocytes and direct
infection and destruction
of CD3- CD4+ CDB- intrathymic T-progenitor (ITTP) cells (Su., L., et al. (
1995) Immunity 2:25-36).
After administration of antiviral nucleoside analogs, such as zidovudine (AZT)
and didanosine (ddl),
and anti-HIV bicyclams to SC)17-hu Thy/Liv mice, HN-1 replication within the
implants is inhibited
(Datema, R., et a1. (1996) Antibicrob. Agents Chemother. 40:750-754; McCune,
J.M., et al. (1990)
Science 247:564-566; Rabin, L., et al. (1996)Antimicrob. Agents Chemoiher.
40:755-762).
The 17-mer oligonucleotide AR177 (5'-gtggtgggtgggtgggt-3') inhibits
replication of multiple
laboratory strains and clinical isolates of HIV-1 in human T cell lines,
peripheral blood lymphocytes,
and macrophages (Bishop, J.S., et al. (1996) J. Biol. Chem. 271:5698-5703;
Ojwang, J.O., et al.
(1995} Antimicrob. Agents Chemother. 39:2426-2435). The molecule contains
single
phosphorothioate internucleoside linkages at both the 5' and 3' ends for
stability and under
physiological conditions, the 17-mer folds upon itself to form an
intramolecular guanosine octet. This
a oligonucleotide does not possess a sequence complementary (antisense) to the
HIV-1 genome, but it is
a potent inhibitor of HIV-1 integrase (Ojwang, J.O., et al. (1995). This study
was designed to
evaluate the antiviral activity of AR177 against HIV-1 (NL4-3) infection in
SCID-hu Thy/Liv mice
treated by intraperitoneal administration 24 h before virus inoculation. As
assessed by p24 ELISA for
viral antigen, quantitative microculture, PCR-based detection of HIV-1
proviral DNA, branched DNA
assay for HIV-1 RNA, and by flow cytometry for perturbations in HLA class I
expression and T cell
CA 02279488 1999-07-29
WO 98/33807 PCTIUS98/01974
subsets, treatment with AR177 at 30 and 100 mg/kg/day had potent antiviral
activity in the model.
Preparation of phytohemaggtutinin (PHA)-activated peripheral blood mononuclear
cells.
Peripheral blood mononuclear cells (PBMC) were isolated from leukocyte-
enriched fractions of
human blood (Stanford Blood Bank). Equal volumes of cells and phosphate-
buffered saline (PBS)
containing 8 U of heparin per ml were mixed and underlayered with 15 ml
Histopaque 1077 (Sigma)
in 50-ml conical centrifuge tubes and centrifuged at 450 x g for 30 min. The
interface cells were
collected, washed twice with PBS, counted, and adjusted to 2 x 106 cells/ml in
Dulbecco's modified
Eagle medium (Mediatech) supplemented with 10% fetal bovine serum {PBS), 2 mM
L-glutamine,
100 U penicillin per ml, 100 pg streptomycin per ml (complete medium), and 1
p.g PHA-P (Sigma)
per ml. Cells from individual blood donors were incubated separately in 150-
cm2 flasks at 37°C with
5% C02 for three days and treated with 50 U IL-2 (human lymphocyte, Boehringer-
Mannhiem,
Indianapolis, Ind.) per ml during the final 24 h of incubation. The cells were
then pooled, divided into
1-ml aliquots of 3 x 10' cells per vial in 90% FBS-10% dimethysulfoxide, and
frozen in liquid N2 for
future use.
1 S Stock virus preparation. All procedures using infectious HIV-I were
carried out in a Biosafety
Level 3 (BL3) facility or in a restricted animal barrier facility under BL3
guidelines. A plasmid
containing the molecularly cloned virus pNL4-3 (Adachi, A., et al. ( 1986) J.
Virol. 59:284-291 ) was
obtained from the NIH A)DS Research and Reference Reagent Program, National
Inst: Lute of Allergy
and Infectious Diseases. Seed virus was prepared by electroporation of 25 wg
of HIV-1 DNA per 5 x
106 fresh PHA-activated PBMC {Bio-Rad Gene Pulser) at 960 OFD and 280 volts.
Working stocks
were prepared by inoculating 1 O8 fresh PHA-activated PBMC with 2 x 10$ TCID50
of virus in 5 ml of
complete medium containing 5 ug polybrene (Sigma) per ml. After 2 h at
37°C, the cells were diluted
to a density of 2-3 x 106 per ml in complete medium containing 50 U IL-2 per
ml {virus culture
medium). On day 2 the cells were pelleted, fresh medium was added, and the
supernatant was
collected 24 hours later. Supernatant collection was repeated daily on days 4-
8, with the addition of
fresh virus culture medium after each collection. Supernatants were aliquoted
and frozen and stored in
liquid N2. Aliquots were analyzed for p24 content by enzyme-linked
immunosorbent assay (ELISA)
and for infectious virus titer by limiting dilution (TCIDso) assay in PHA-
activated PBMC.
TCIDgO assay for HIV-1. Thawed PHA-activated PBMC were cultured overnight in
virus culture
medium in 25-cmz flasks and seeded into 96-well plates ( 10$ cells in 25 pI
per well). Serial 10-fold
dilutions of virus were prepared in medium containing 10 Pg polybrene per ml,
and 25 pl of each
dilution was added to quadruplicate wells of PBMC. After 2 h at 37°C,
200 pl of virus culture
medium was added to each well, and the plates were incubated at 37°C in
a humidified 5% C02
atmosphere. After 7 days, the plates were centrifuged at 400 x g for 5 min,
supernatants were
removed, and cell pellets were assayed for p24 antigen. The TCIDso is the
reciprocal of the dilution at
!t~
CA 02279488 1999-07-29
WO 98133807 PCTIUS98/01974
which 50% of the wells contained detectable p24 (>_30 pg/ml) and specifies the
number of infectious
doses per 25 pl.
p24 ELISA assay. For quantitation of HIV-1 p24 within cells, samples
containing I x 105 to 5 x 106
cells were lysed overnight at 4°C in p24 lysing buffer { 1 % Triton X-
100, 0.5% sodium deoxycholate,
' S 5 mM EDTA, 25 mM Tris CI, 250 mM NaCI and 1% aprotinin). Pellets were
lysed in 100 p.l lysing
buffer for the TC)Dso assay and in 400 ui for implant p24 determination. The
lysed samples were then
transferred into HIV p24 antibody-coated microplates (Dupont) for quantitative
ELISA. A standard
curve was generated with HXB2-infected H9 cells, and results were calculated
as pg p24 per 106 cells
or per ml.
I O Construction of SCID-hu Thy/Liv mice. Homozygous C.B-17 scidlscid mice
(SCID) were bred at
SyStemix (Palo Alto, Cali~) and treated prophylactically with trimethoprim-
sulfamethoxazole
(Septra) pellets in the food bin to prevent opportunistic infection with
Pneumocystis carinii (McCune,
J.M. et al. ( 1988). For surgical procedures, 8-week-old male mice were
anesthetized with 100 mg/kg
ketamine and 8 mg/kg xylazine, given intraperitoneally. Implantation of
fragments of human fetal
15 liver and human fetal thymus to create SCID-hu Thy/Liv mice was carried out
as described
(Namikawa, R., et al. ( 1990)), and a cohort of 47 mice was produced with
tissue from the same donor.
Ten randomly-chosen mice were anesthetized and examined 14 weeks after
implantation to evaluate
growth of the Thy/Liv implants. Because 9 of the 10 examined implants were
>_30 mm3 (3 mm x 3
mm x 3 mm), the entire cohort was entered into the antiviral efficacy
experiment. Four mice from a
20 different cohort were used for group G.
Drug preparation. ARI77 (lot #IM127-04, 68.5% oligonucleotide) was provided by
Aronex
Pharmaceuticals, Inc., The Woodlands, Texas and stored in the dark at -
4°C. The test agent was
dissolved in sterile PBS without Ca++ or Mg++ (Digene Diagnostics, Inc.,
Beltsville, Md.) at 1.5, 4.4,
and 15 mg/mi (for dosing at 10, 30, and 100 mg/kg/day, respectively), and the
solutions were not
25 filtered. These concentrations were based on oligonucleotide weight alone,
excluding the weight
contributed by salt. A positive antiviral control, ddI (NSC-612049, lot #5
PC2793), was obtained from
the NIH AIDS Research and Reference Reagent Program and stored at -
20°C. The ddI was dissolved
in pH 9 sterile water at I S mg/ml, adjusted to pH 7 with 8% sodium
bicarbonate, and sterilized by
filtration. All dosing solutions were prepared the day before treatment
initiation and were stored in the
30 dark at 4°C until dosing.
~/ S
CA 02279488 1999-07-29
WO 98133807 PCTlUS98101974
Drag dosing. Groups of 6 or 7 mice were treated with AR177 at 100 mg/kg/day
(group A), 30
mg/kg/day (group B), and 10 mglkg/day (group C) by intraperitoneal injection
(200 p l ) with a 26-
gauge x 1/2-inch needle. Mice in group D were treated intraperitoneally with
ddI at 100 mglkg day,
and mice in group E were not treated. Mice were rnoculated 24 h after the
first dose (2 h after the
second dose), and dosing was performed once daily at 7:00-10:00 AM for 13
days. The untreated
mice in group F were mock-infected with medium alone. Four mice (group G) were
not inoculated
and were treated with the AR177 at 100 mglkg/day for 13 days as described
above. Their implants
were removed 2 h after the last dose, snap-frozen in liquid N~, and stored at -
70°C for shipment to
Aronex and determination of implant AR177 concentrations.
IO HIV-1 infection of SC1D-hu Thy/Liv mice. Inoculations were performed on
anesthetized mice in a
restricted animal barrier facility under BL3 guidelines (i.e., with mask, eye-
covering, gown, etc.). To
maximize visual and manual access, teams of three operators worked side-by-
side on a bench top.
One operator shaved the left flank of the mouse and exposed the left kidney
carrying the ThyILiv
implant through a 1.0- to 1.5-cm incision. The second operator then gently
immobilized the kidney
with forceps and marked an opening in the kidney capsule over the implant with
India ink injected
with a 1-cc tuberculin syringe and 30-gauge x 1/2-inch sharp needle. Using the
ink mark as a guide,
the Thy/Liv implant was injected with 25-50 ~l of undiluted stock virus (630-
1300 TC1175o) in 1 to 3
places with a 250-~tl Hamilton glass syringe and 30-gauge x 1/2-inch blunt
needle. A third operator
closed the incision with one stitch in the peritoneal lining and one skin
staple. For this study, implants
were inoculated 17 weeks after tissue implantation.
Thy/Liv implant tissue processing. Mice were euthanized by C02 inhalation
followed by cervical
dislocation, and the human Thy/Liv implants were surgically excised and
transferred into 6-well
tissue culture plates containing sterile PBS/2% FBS at 4°C. A single
cell suspension was made by
placing the implant into a sterile nytex bag, submerging the bag in PBSI2% FBS
in a 60-mm tissue
culture dish, and gently grinding the tissue between the nytex layers with
forceps. The cells were
counted with a Coulter counter, and appropriate numbers of cells were
aliquoted for each assay. For
p24 ELISA, pellets of 2.5 x 106 cells were resuspended in 400 pl of p24 lysis
buffer, rotated overnight
at 4°C, and stored at -20°C. For DNA PCR, pellets containing 1 x
103 cells were processed
immediately or stored at -80°C. For bDNA assay, dry pellets of 2 x 10'
to 5 x 106 cells were frozen
and stored at -80°C. For FACS analysis, 106 cells per well were placed
in a 96-well plate, stained, and
analyzed on the same day. Results are shown in Table H-4.
Quantitative microculture assay. For quantitation of infectious HIV-1 in
implants, implant
thymocytes were serially diluted in S-fold increments in virus culture medium,
added to 96-well plates
containing 105 PHA-activated PBMC per well, and incubated at 37°C. A
range of 32 to 100,000
thymocytes per well were cocultivated in duplicate with the PBMC. After 5
days, cell pellets were
lysed and assayed for p24, and wells containing detectable p24 (>30 pg/mi)
were scored positive for
CA 02279488 1999-07-29
WO 98/33807 PCT/US98/01974
HIV-1 infection. Implant HIV-1 titers are expressed as TCIDS° per
106
implant thymocytes, and the log,° values were used for calculation of
geometric means. The limit of
detection was 10~'° TC1D5° per 106 cells.
DNA PCR. Infection of human cells in the Thy/Liv implant was assessed by PCR
amplification with
primers specific for the conserved US-gag region of the HIV-1 genome (SK145
and SK431, Perkin-
Eimer Cetus). Amplification of human /3-globin DNA was performed as a control
for human DNA.
Pellets ( 1 x 103 cells) were lysed in 100 pl of 1 % NP-40, 1 % Tween-20, 2.5
mM MgCl2, 5 mM KCl
and IO pg proteinase K. Samples were vortexed, microfuged, incubated at
60°C for I h, heated at
95°C for 10 min to inactivate the proteinase K, vortexed, microfuged,
and assayed by a standard PCR
assay as previously described (cCune, J.M., et al. ( 1988). Amplification of
10 pl lysate (corresponding
to 100 cells) was continued for 40 cycles (cycle 1: 6 min at 95°C, 2
min at 60°C, and 1.5 min at 72°C;
cycles 2-5: 2 min at 95°C, 2 min at 60°C, and 1.5 min at
72°C; cycles 6-40: 1 min at 95°C, 1 min at
60°C, and 1.5 min at 72°C; final extension: i0 min at
72°C and 8 h at 27°C). Amplified products
were subjected to electrophoresis in a 3% agarose gel with molecular weight
standards (BstE II-
digested lambda DNA). Gels contained 0.5 ~g ethidium bromide per ml, and
images were acquired on
an Eagle Eye II gel imager (Stratagene). Control samples A, B, and C were
prepared by mixing HIV-
1-positive ACH2 cells with HIV-I-negative implant thymocytes at concentrations
of I0%, 2%, and
0%, respectively. Control A should yield a dark HIV-I DNA band (scored +),
control B a light HIV-1
band (+/-), and conirol C no HIV-1 band (-). The limit of HIV-1 DNA detection
is therefore one to
two HIV-1-positive cells per 100 input cells. The H20 control sample (-)
should yield neither human
(3-globin nor HIV-1 DNA bands. (Table 4 and Figs. 63 and 64)
Viral RNA quantitation by bDNA assay. Cells were disrupted with sterile
disposable pestles and a
cordless motor grinder (Kontes, Vineland, N.J.) in 8 M guanidine HC1 with 0.5%
sodium N-
lauroylsarcosine. The RNA was extracted by adding 5 ml 100% EtOH containing 20
pg polyadenylic
acid (Sigma) per ml, and each sample was vortexed and pelleted at 12,000 x g
for 20 mm at 4°C.
Supernatants were aspirated to remove DNA, and RNA pellets were washed with 5
ml 70% EtOH,
placed on dry ice, and digested with reagents supplied by the manufacturer
(Quantiplex HIV-1 RNA
assay 2.0, Chiron Corporation, Emeryville, Cali~). Implant HIV-1 RNA load is
expressed as copies
per 106 implant thymocytes, and the logo values were used for calculation of
geometric means. The
limit of detection was 1023 RNA copies per 106 cells. Results are shown in
Table H-4.
FRCS analysis for thymocyte depletion and HLA class I (W632) expression.
Pellets containing
106 cells were resuspended in 50 ul of a monoclonal antibody cocktail
containing phycoerythrin (PE)-
conjugated anti-CD4, (Becton Dickinson), tricolor (TC)-conjugated anti-CD8
(Caltag), and
fluorescein isothiocyanate (FITC)-conjugated anti-W632 (SyStemix). Cells from
one uninfected
implant were stained with conjugated, isotype-matched antibodies to control
for nonspecific antibody
/02-~
CA 02279488 1999-07-29
WO 98133807 PCT/US98/01974
binding. Cells were incubated for 30 min in the dark, washed 3 times with
PBS/2% PBS, resuspended
in 200 ~1 of PBS/2% PBS containing 1 % paraformaldehyde in 1.5-ml FACS tubes,
and analyzed on a
FACScan (Becton Dickinson).
After collecting 10,000 events, percentages of marker-positive (CD4+, CD8+
(CD4+ CD8+)
thymocytes in the impiant samples were determined by gating on a lymphoid cell
population
identified by forward- and side-scatter characteristics. In addition, W632-
positive mean channel
fluorescence of CD4+ CD8+ cells was determined for each sample. Examples of
the FACS analyses
are shown in Figs. 62A-F (for mouse #1 and #2, for AR177 at 100 mglkglday).
Data analysis. The p24 results (in pg per 106 cells) were converted to % of
control by dividing values
for treated mice by the value for untreated mice. All results are expressed as
the mean ~ SEM.
Nonparametric statistical analyses were performed by use of the Mann-Whitney U
test (StatView 4.1,
Abacus Concepts, Berkley, Calif.). Data for mice in each dosage group were
compared to those for
untreated infected mice, and the resulting p values appear in Table H-7 (p
values _< 0.05 were
considered statistically significant).
I 5 The data obtained in these investigations is shown in Tables H i-6 and
Figs. 62-65.
Size and quality of Thy/Liv implants. At the time of inoculation, implants
were mostly small or
medium in size and were of good quality. As shown in Table H-3, two mice (#4
and #26 were
rejected because of lack of implant or insu~cient implant size. At
termination, implants were mostly
small or medium in size and were of good quality.
CA 02279488 1999-07-29
WO 98133807 PCT/US98/01974
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CA 02279488 1999-07-29
WO 98133807 PCTILTS98101974
Adverse affects in treated mice. No significant weight loss occurred in any of
the treated groups
(Table H-4). The apparent reduction in implant cell yield in the high-dose
AR177-treated mice (mean
of 85 x 106 versus 140 x 106 for untreated infected mice) is not statistically
significant. Three mice in
group A and two mice in group G, ali of which received 100 mg/kglday AR 177,
had minor
subcutaneous bruising at the site of intraperitoneal injection.
HIV-1 replication, induction of HLA class I expression, and thymocyte subsets
in Thy/Liv
implants of untreated mice. Implants from all 8 untreated infected mice (group
E) had detectable p24
{mean of 620 pg per 106 cells), infectious virus (IO26 TC)DS° per 106
cells), HIV-I DNA, HIV-1 RNA
(106'1 per 106 cells), and a mean 5.2-fold increase in HLA class I (W632)
expression by implant
thymocytes compared to untreated mock-infected mice (Table H-1 and Figure 65).
These results are
consistent with previous results obtained in the model (Aldrovandi, G., et al.
(1993); Bonyhadi, M.L.,
et al. ( 1993 ); Datema, R., et al. ( 1996); Krowka, J., et al. ( 1991 ) J.
Immunol. 145:3 75 I -3756;
Namikawa, R., et al. (1988); Rabin, L., et al. (1996) Antimicrob. Agents
Chemother. 40:755-762;
Stanley, S.K., et al. (1993) J. Exp. Med. 178:1151-1163; Su, L., et al. (1995)
Immunity 2:25-36). No
I S depletion of CD4+ CD8+ immature cortical thymocytes {85% for infected
versus 88% for mock-
infected mice) and a small reduction in the CD4/CD8 ratio ( 1.7 for infected
versus 2.6 for mock-
infected mice) were observed.
Effect of AR177 on IiIV-1 replication and induction of HLA class I expression
in Thy/Liv
implants. Intraperitoneal administration of AR177 resulted in potent antiviral
activity when
treatment was initiated 24 h before inoculation. Statistically significant
reductions in implant viral
load occurred at all three doses of AR177, and the reductions in cell-
associated p24, infectious virus,
viral RNA, and W632 expression were ali dose dependent. Implants from 4 of 6
mice treated with
AR177 at 100 mg/kg/day had no detectable p24 antigen, infectious virus, HIV-1
DNA, or HIV-1
RNA after 13 days of treatment. Treatment with 30 mg/kg/day caused a 17-fold
reduction in p24, a 9-
fold reduction in viral titer, and a 29-fold reduction in viral RNA load
(Table H-2).
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CA 02279488 1999-07-29
WO 98!33807 PCTlUS98/01974
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CA 02279488 1999-07-29
WO 98133807 PCTIUS98101974
Effect of ARI77 on thymocyte subsets in HIV-1 infected ThyILiv implants. No
depletion
of CD4+ CD8+ thymocytes was apparent in untreated mice by the 12-day
termination time point, so
the effect of drug treatment on depletion could not be determined in this
study. Treatment of mice
with 30 and 100 mg/kg/day AR177 prevented virus-induced reduction of the
CD4/CD8 ratio in the
Thy/Liv implants.
Effect of ddI on HIV-1 infection in Thy/Liv implants. Intraperitoneal
administration of ddI at 100
mg/kglday caused substantial reductions in viral load and HIV-1-induced class
I expression, as
expected for this positive antiviral control drug. At termination, implants
from ddI-treated mice had a
mean p24 level of 46 pg per 106 cells versus 620 pg per 106 cells in untreated
mice, a mean viral RNA
load of 105'' copies per 106 cells versus lObv per 106 cells, and W632 mean
channel fluoresence of 250
versus 620 in untreated mice. Implants from all four ddI-treated mice were
positive for HIV-1 DNA
by PCR. (Table H-2).
~~v
CA 02279488 1999-07-29
WO 98/33807 PCT/US98/01974
a
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p N N N l~ pf N N N N N N c0 P7 t~J C7 In N N N N !h P7 N ~7 P1 N N N
m tp t0 b m m 10 t0 f0 m b m m m m (0 m m m b b b b m m f0 t0
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\ 1 \ \ \ \ \ \ \ 1 ~. ' \ \ \ \ \ \ \ \ 1 \ \ \ \ \ \ \
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N N N P7 N N N N N l9 (n (O Pf N N N N N N M nJ N i~l c~ N N N
b m b b b m b m m m ~o b m m b m m m m m m m m m m m eo b m m ~u eo
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s m H CI O m A ID A N C1 0~ A ~ O O O O m ID m m IH N 01 N b b N
p v N P! N N N N N N N c! !9 t0 (9 N N N N ~ N t9 t9 N c9 M N N N
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t N N N N N N N N N N N N N N N N N N N N N N N N N N N N N = N
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~ N N N N N N N N N O7 l9 W !~ Ff M
CA 02279488 1999-07-29
WO 98/33807 PCT/US98101974
i
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CA 02279488 1999-07-29
WO 98133807 PCT/US98101974
TABLE H-5
o..a,w..l~w..
..w.Ti O~ID.DMf
n.. foul
r:.. Mtn. to
r:.. Mm. too
na. Mln. ae
n.. Lei. too
ns. NC. NOIE
n~. rc. Nolrt
xn.. twt
xna. Mnr. to
xrsl. Mm. loo
xm. ARtn. ao
xr>N. su. loo
xnl, NC. NONE
lGPtl. PC. NONE
04, tWV
ou. Mm. to
011. Ml7f. 140
OAI. MtTI. ~0
G4 Iit. 100
ON.IC.NONE
~~»
RNA Taut
RNA. Mm. to
RNA Mln, too
RNA. ARtTf. 70
RNA. 1m. 100
RNA NQ IME
RNA, lC'" IidE
01. TWI
01. ARtTT. td
x. Mtn. too
w. Mtn, m
Dr. a:, loo
n.NGNaiE
a.re,ItoItE
CON. TeIY
Cl7h. MITt. t0
COIv. MtT7. 100
coo.. ARIn. m
coo.. aa. too
Cp~.NGIbNF
4~h, tG NONE
CDh. TWI
CDH. M17T. 10
Cow. Mln, too
CD1.. Mlff. io
COH. IIL 100
COE.. NG NOIE
COIw 1G NOlE
RATIO, talal
RATIO, M171. 10
RATIO. MITT. t00
MTIO. MITT. 70
RATIO. 11l 100
MfIQ NG NONi
RAnO. ~C. NONE
WE1~. T1111
WE~f. M177. td
wm. Mln. too
WIil. MiT7. 70
WE7l, ddl, 100
WNi 1C. NONE
Call Ya111. T1111
G1 YaW, Mltl, t0
GI YW. M1T7. 100
Gll rial4 MtTI. 70
Ga YfW. 111. t00
G1 Ylak. NG NONE
Gn rrtt PG NONE
CA 02279488 1999-07-29
WO 98/33807 PCT/US98/01974
a o
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CA 02279488 1999-07-29
WO 98/33807 PCT/US98/01974
I. Oligonucleotides Containing C-5 Propvnyl-dU Protected Monomers
Additional novel guanosine-rich oligonucleotides were prepared in the present
investigations
and have been examined for in vitro anti-viral activity and for inhibition of
integrase activity. These
new anti-viral oligonucleotides, which also form intramolecular stacked
guanosine quartet structures
under physiological conditions, are based on the T30695 motif (SEQ ID NO 87),
and are exemplified
by T30925, T30926, T30927, T30928, and T30929 (SEQ ID NO 87). These ODNs
contain a C-5
propynyl-deoxyuridine, variously positioned at 2, 5, 9, 13 and 17, from the 5'
end. These new
oligonucleotides are even more effective than T30177 in in vitro tests of anti-
HIV activity (as shown
in Table I-1 ) and are expected to demonstrate similar or even greater
therapeutic efficacy than T30177
against human immunodeficiency virus type 1 (HIV-1) in a SCID-hu mouse model.
ODN Synthesis. C-5 propynyl-dU protected monomers were obtained from Glen
Research or
synthesized at Aronex Pharmaceuticals, Inc. and other 5'-protected nucleoside
phosphoramidite
monomers were obtained from either PerSeptive Biosystems (Framingham, MA) or
Glen Research
(Sterling, VA). Oligodeoxynucleotides (ODNs) were synthesized using a standard
protocol employing
amidite monomers synthesized at Aronex, or obtained from Glen Research or
PerSeptive Biosystems,
on a PerSeptive Biosysterns DNA synthesizer Expedite model 8909.
Phosphorothiolated (PT) ODN
linkages were prepared using the Beaucage reagent as described previously
(Ojwang et al. 1995).
ODNs with phosphodiester and partial PT backbones were cleaved and deprotected
in ammonium
hydroxide at 56°C for 16 hrs.
Propynyl modified pyrimidine ODNs were synthesized using the standard
phosphoramidite
methods with the following modifications. The coupling time was extended to
300 seconds and the
resulting ODN was cleaved from the solid support and deprotected in ammonium
hydroxide at room
temperature for 48 hours as opposed to 56°C for 16 hrs.
Crude ODNs were purified by anion-exchange chromatography on a Q-Sepharose
column
( I .5 x 10 cm) using a Waters high performance liquid chromatography (HPLC)
system. Standard
sodium chloride (0.5-3 M)/sodium hydroxide ( 10-15 mM) mobile phases were used
depending on the
backbone. The purified ODNs were desalted on SepPak Plus C 1 g cartridges
purchased from Waters.
The purity and integrity of the ODNs was confirmed using one or more of the
following procedures
on ail ODNs synthesized: analytical HPLC, gel electrophoresis in a 20%
polyacrylamide gel
containing 7M urea, or mass spectroscopy.
~3~
CA 02279488 1999-07-29
WO 98/33807 PCTlUS98/01974
Integrase Assay. Integrase assays were performed essentially as described by
Mazumder et al.
( 1996). In these assays integrase (HIV-1) was preincubated at a final
concentration of 200 nM with
inhibitor in reaction buffer [50 mM NaCI, 1 mM HEPES, pH 7.5, 50 pM EDTA, 50
pM DTT, 10%
glycerol (w/v), 7.5 mM MnCl2 or MgCl2, 0.1 mg/ml BSA, 10 mM 2-mercaptoethanol,
10% DMSO,
and 25 mM MOPS, pH 7.2] at 30°C for 30 mm. When magnesium was used as
the divalent metal
ion, polyethylene glycol) was added at a final concentration of 5%.
Preincubation for 30 mm of the
enzyme with inhibitor was performed to optimize the inhibitory activity in the
3'-processing reaction.
Then, 20 nM of the 5'-end 3zP-labeled linear oligonucleotide substrate was
added, and the incubation
was continued for an additional 1 hr. The final reaction volume was 16 ~1.
The disclosures of the following technical papers attached hereto are
incorporated herein to
the extent that they provide materials and methods not specifically set forth
herein.
Ojwang, J.O., Buckheit, R.W., Pommier, Y., et al. "T30177, an Oligonucleotide
Stabilized by
an Intramolecuiar Guanosine Octet, Is a Potent Inhibitor of Laboratory Strains
and Clinical Isolates of
Human Immunodeficiency Virus Type 1" Antimicrobial Agents and Chemotherapy.
39:2426-2435
1 S ( 1995).
Mazumder, A., Neamati, N., Ojwang, J.O., Sunder, S., Rando, R.F. and Pommier,
Y.
"Inhibition of the Human Immunodeficiency Virus Type 1 Integrase by Guanosine
Quartet
Structures" Biochemistry 35:13762-13771 (1996).
In Vitro Activity of C-5 Propynyl dU Modified ODNs. The exemplary
oligonucleotides
listed in Table I-1 were assayed for inhibition of the integrase enzyme and
for acute HIV-1
infection in tissue culture cells using essentially the procedures described
hereinabove. The
spaces or gaps in these sequences represent a ribonucleotide or
deoxyribonucleotide from
which the base has been removed ("abasic") without causing a break in the
oligonucleotide
backbone. As shown in Table I-1, the T30695, T30925, T30926, T30927, T30928,
T30929
oligonucleotides were found to be 10-20 fold more active than T30177. In vivo
activity of
these new ODNs is expected to be similar or superior to that of T30177.
CA 02279488 1999-07-29
WO 98/33807 PCTIUS98/01974
TABLE I-1
SAR studies using
variants of
T30695
IC50 (nlV1}
Enzyme Assay Culture b
Assay
- 5 ODN Sequence' 3'-processing
1118
RF Strain
Strain
130177 5'- gtggtgggtgggtgggt-3' 92 250 150
130695 5'- g ggtgggtgggtgggt-3' 43 230 -
130925 5'- g ggXgggtgggtgggt-3' 17 - 10
130926 5'- g ggtgggXgggtgggt-3' 33 - 9
130927 5'- g ggtgggtgggXgggt-3' 52 30 7
130928 5'- g ggtgggtgggtgggX-3' 29 - 10
130929 5'- g ggXgggXgggXgggt-3' 24 - 12
a X = C-5 propynyl-dU
b Acute infection assays for HIV-1 strains RF and IIIB were assayed in MT-4
cells as
described by Ojwang et al. (1995).
/ 33
CA 02279488 1999-07-29
WO 98/33807 PCT/US98101974
(1) GENERAL INFORMATION:
(i) APPLICANT: Rando, Robert F.
Ojwang, Joshua O.
Hogan, Michael E.
Wallace, Thomas L.
Cossum, Paul A.
(ii) TITLE OF INVENTION: Anti-Viral Guanosine-Rich
Tetrad Forming Oligonucleotides
(iii) NUMBER OF SEQUENCES: 88
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Conley, Rose & Tayon, P.C.
(B) STREET: 600 Travis, Suite 1800
(C) CITY: Houston
(D) STATE: Texas
(E) COUNTRY: U.S.A.
(F) ZIP: 77002-2912
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: MS Word 97 (saved as .txt file)
(vi.) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/037,374
(B) FILING DATE: 04-FEB-97
(C) APPLICATION NUMBER:
(D) FILING DATE: 09-DEC-97
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: McDaniel, C. Steven
(B} REGISTRATION NUMBER: 33,962
(C) REFERENCE/DOCKET NUMBER: 1472-06223
(ix} TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 713/238-8010
(B) TELEFAX: 713/238-8008
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 38
~ 3~
CA 02279488 1999-07-29
WO 98133807 PCT/US98/01974
(D) OTHER INFORMATION: /note= "Amine moiety
- attached to 3' end"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:1:
TGGGTGGGGT GGGGTGGGGG GGTGTGGGGT GTGGGGTG 38
(2} INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii} MOLECULE TYPE: DNA (genomic)
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
GTGGGGTGTG GGGTGTGGGG GGGTGGGGTG GGGTGGGT 38
{2) INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3:
GGGTGGGTGG GTGGGTGG lg
(2} INFORMATION FOR SEQ ID N0:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3B base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:4:
GGTGGTGGGG GGGGGTGGGG TGGTGGTGGG GGTGTTGG 38
(2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
,
(A} LENGTH: 36 base pairs
_
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
3.1~
CA 02279488 1999-07-29
WO 98133807 PCT/LTS98/01974
GTGGTGGTGG TGTTGGTGGT GGTTTGGGGG GTGGGG 36
(2} INFORMATION FOR SEQ ID N0:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
GTGGTTGGTG GTGGTGTGTG GGTTTGGGGT GGGGGG 36
(2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix} FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 36
(D) OTHER INFORMATION: /note= "phosphorothioate
backbone"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
GTGGTGGTGG TGTTGGTGGT GGTTTGGGGG GTGGGG 36
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 36 base pairs
{B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 36
(D) OTHER INFORMATION: /note= "phosphorothioate
backbone"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:8:
GTGGTTGGTG GTGGTGTGTG GGTTTGGGGT GGGGGG 36
(2) INFORMATION FOR SEQ ID N0:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
~3~
CA 02279488 1999-07-29
WO 98/33807 PCTIUS98101974
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:9:
GGTGGGGTGG TGGTGGTTGG GGGGGGGGGG T 31
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GGTGGTTGGG GGGTGGGGGG G 21
(2} INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GGGTGGGGTG GTGGGTGGGG G 21
(2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
GGTGGGTGGT TTGTGTGGTT GGTGGGTTTT 30
(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
~ 3~
CA 02279488 1999-07-29
WO 98133807 PCTIUS98/01974
{xi) SEQUENCE DESCRIPTION: SEQ N0:13:
ID
GGGGGGGGGG TGTGGGGGGG GGTTGTGGTG 31
G
(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ N0:14:
ID
GGTGGGTGGG TTGGGGGGTG GGTGGGG 27
(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ N0:15:
ID
TGGGGTTTGG GTGGGGGGTT GGGTGGTTG 29
(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
{ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ N0:16:
ID
GGGTGGTGGT GTTGGTGTTG TGTG 24
(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ N0:17:
ID
GGTGGGGGGG TTGGTGTGTT TG 22
(2) INFORMATION FOR SEQ ID N0:18:
(3 ~
CA 02279488 1999-07-29
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- (i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:18:
GTGTGGGGGG GTGGGGTGGG GTGGGT 26
(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
GGGTGGGTGG GTGGGTGGGT GGGTGG 26
(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 26
(D) OTHER INFORMATION: /note= "Amine moiety
attached to 3' end"
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:20:
GTTGGGGGTT GTTGGTGGGG TGGTGG 26
(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 45 base pairs
(B} TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix} FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 45
(D) OTHER INFORMATION: /note= "Amine moiety
~3p
CA 02279488 1999-07-29
WO 98133807 PCT/US98101974
attached to 3' end"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
TGGTGGGTGT GTGGGGGGTG TTGGGGGTTG TTGGTGGGGT GGTGG 45
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
{ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 45
(D) OTHER INFORMATION: /note= "cholesterol moiety
attached to 3' end"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
TGGTGGGTGT GTGGGGGGTG TTGGGGGTTG TTGGTGGGGT GGTGG 45
(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 45
(D) OTHER INFORMATION: /note= "cholesterol moiety
attached to 3' end"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
GTGGTGGGTG GGTGGGTGGT GGGTGGTGGT TGTGGGTGGG TGGTG 45
(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 45
(D) OTHER INFORMATION: /note= "Amine moiety
attached to 3' end"
t
r
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
~ GTGGTGGGTG GGTGGGTGGT GGGTGGTGGT TGTGGGTGGG TGGTG 45
(2} INFORMATION FOR 5EQ ID N0:25:
' (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
{D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 26
{D) OTHER INFORMATION: /note= "cholesterol moiety
attached to 3' end"
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
GTTGGGGGTT GTTGGTGGGG TGGTGG 26
(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA {genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 45
(D) OTHER INFORMATION: /note= "Amine moiety
attached to 3' end"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
GATCCATGTC AGTGACACTG CGTAGATCCG ATGATCCAGT CGATG 45
(2} INFORMATION FOR SEQ ID N0:27:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
- (A) NAME/KEY: misc_feature
(B) LOCATION: 26
{D) OTHER INFORMATION: /note= "phosphorothioate
backbone and amine moiety attached to
backbone"
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
GTTGGGGGTT GTTGGTGGGG TGGTGG 26
(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
GGTGGTGGGG TGGTTGTTGG GGGTTG 26
(2) INFORMATION FOR SEQ ID N0:29:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic}
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
GGTGGTGGGG TGGTTGTTGG GGGTTGTTGG GGGTGTGTGG GTGGT 45
(2) INFORMATION FOR SEQ ID N0:30:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
GGGTGGTTGG GTGGTTGG 18
(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 18
(D) OTHER INFORMATION: /note= "Amine moiety
CA 02279488 1999-07-29
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attached to 3' end"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
GGGTGGGTGG GTGGGTGG lg
(2} INFORMATION
FOR SEQ ID N0:32:
(i} SEQUENCE CHARACTERISTICS:
(A) LENGTH: lg base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc
feature
_
(B) LOCATION: 18
{D) OTHER INFORMATION: /note= moiety
"Amine
attached to 3' end and phosphothioate
backbone"
(xi) SEQUENCE DESCRIPTION: SEQ ID
N0:32:
GGGTGGGTGG GTGGGTGG lg
(2) INFORMATION
FOR SEQ ID N0:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA {genomic)
(ix) FEATURE:
(A) NAME/KEY: misc
feature
_
(B} LOCATION: 17
(D) OTHER INFORMATION: /note= moiety
"Amine
attached to 3' end"
(xi) SEQUENCE DESCRIPTION: SEQ ID
N0:33:
GTGGTGGGTG GGTGGGT 17
(2) INFORMATION
FOR SEQ ID N0:34:
- (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc
feature
_
(B} LOCATION: 27
(D) OTHER INFORMATION: /note= moiety
"Amine
izf3
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attached to 3' end"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:34:
GTGGTGGGTG GGTGGGTGGT GGGTGGT 27
(2) INFORMATION FOR SEQ ID N0:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 37
(D) OTHER INFORMATION: /note= "Amine moiety
attached to 3' end"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:35:
GTGGTGGGTG GGTGGGTGGT GGGTGGTGGT TGTGGGT 37
(2) INFORMATION FOR SEQ ID N0:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 16
(D) OTHER INFORMATION: /note= "Amine moiety
attached to 3' end"
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
TTGTGGGTGG GTGGTG 16
(2) INFORMATION FOR SEQ ID N0:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 29
(D) OTHER INFORMATION: /note= "Amine moiety
attached to 3' end"
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:37:
TGGTGGGTGG TGGTTGTGGG TGGGTGGTG 29
(2) INFORMATION FOR SEQ ID N0:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
{ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 38
(D) OTHER INFORMATION: /note= "Amine moiety
attached to 3' end"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:38:
GTGGGTGGGT GGTGGGTGGT GGTTGTGGGT GGGTGGTG 38
(2) INFORMATION FOR SEQ ID N0:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 45
(D) OTHER INFORMATION: /note= "phosphorothioate
backbone and amine moiety attached to 3' end"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:39:
GTGGTGGGTG GGTGGGTGGT GGGTGGTGGT TGTGGGTGGG TGGTG 45
(2) INFORMATION FOR SEQ ID N0:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix} FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 18
(D) OTHER INFORMATION: /note= "Amine moiety
attached to 3' end"
CA 02279488 1999-07-29
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:40:
GATCCATGTC AGTGACAC 18
(2) INFORMATION FOR SEQ ID N0:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 18
(D) OTHER INFORMATION: /note= "Amine moiety
attached to 3' end and phosphorothioate
backbone"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:41:
GATCCATGTC AGTGACAC 18
(2) INFORMATION FOR SEQ ID N0:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 18
(D) OTHER INFORMATION: /note= "Amine moiety
attached to 3' end"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:42:
CCCCCCCCCC CCCCCCCC 18
(2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc-feature
(B) LOCATION: 18
(D) OTHER INFORMATION: /note= "Amine moiety
attached to 3' end and phosphorothioate
backbone"
r
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
CCCCCCCCCC CCCCCCCC lg
(2) INFORMATION FOR SEQ ID N0:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 47 base pairs
(B) TYPE: nucleic acid
- (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:44:
TTCATTTGGG AAACCCTTGG AACCTGACTG ACTGGCCGTC
GTTTTAC 47
(2) INFORMATION FOR SEQ ID N0:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:45:
GTAAAACGAC GGCCA 15
(2} INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:
GTGGTGGGTG GGTGGGG 17
(2) INFORMATION FOR SEQ ID N0:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA igenomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:47:
GTGGTGGGTG GGTGGG 16
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(2) INFORMATION FOR SEQ ID N0:48:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 16 base pairs
(B} TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:48:
TGGTGGGTGG GTGGGT 16
(2) INFORMATION FOR SEQ ID N0:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:49:
GTGGTGGGTG GGT 13
(2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:50:
GTGGTGGGT
(2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
GTGGGTGGGT GGGT 14
(2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 base pairs
(B) TYPE: nucleic acid
CA 02279488 1999-07-29
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(C) STRANDEDNESS: single
~ (D) TOPOLOGY: linear
(ii} MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:52:
~ GTGGGTGGGT lp
17
(2) INFORMATION FOR SEQ ID N0:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D} TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
GGTTGGTGTG GTTGG 15
{2) INFORMATION FOR SEQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
{ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:
GTGGTTGGTG TGGTTGG 17
(2) INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
GTGGTTGGTG TGGTTGGT lg
(2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 1B base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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- (ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:
GTGGTGGGTG TGGTTGGT 18
(2) INFORMATION FOR SEQ ID N0:57:
(i) SEQUENCE CHARACTERISTICS:
tA) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:57:
GTGGTGGGTG TGGTGGGT 18
(2) INFORMATION FOR SEQ ID N0:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
{B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA {genomic)
(ix) FEATURE:
{A) NAME/KEY: misc_feature
(B) LOCATION: 11
(D) OTHER INFORMATION: /note= "the base is
removed from this nucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:58:
GUGGUGGGUG GGUGGGU 17
(2) INFORMATION FOR SEQ ID N0:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: i7 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: RNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:59:
GUGGUGGGUG GGUGGGU 17
(2) INFORMATION FOR SEQ ID N0:60:
(i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
l.5 (J
r
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 2
(D) OTHER INFORMATION: /note= "the base is
removed from this nucleotide"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:60:
GNGGTGGGTG GGTGGGT 17
(2) INFORMATION FOR SEQ ID N0:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:61:
GTGGGTGGTG GGTGGGT 17
(2) INFORMATION FOR SEQ ID N0:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:62:
GTGGTGGGGT GGTGGGT 17
(2) INFORMATION FOR SEQ ID N0:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:63:
GTGGTGGGTGG GGTGGT 17
(2) INFORMATION FOR SEQ ID N0:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
~S
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(D) OTHER INFORMATION: /note= "C-5 propynl dU"
{xi) SEQUENCE DESCRIPTION: SEQ ID N0:67:
GTGGNGGGTG GGNGGGT 17
(2) INFORMATION FOR SEQ ID N0:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 2
(D) OTHER INFORMATION: /note= "the base is
removed from this nucleotide"
{ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 5,13
(D) OTHER INFORMATION: /note= "C-5 propynl dU"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:68:
GUGGUGGGUG GGUGGGU 17
(2) INFORMATION FOR SEQ ID N0:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
{ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 2
(D) OTHER INFORMATION: /note= "the base is
removed from this nucleotide"
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 6,13
(D) OTHER INFORMATION: /note= "C-5 propynl dU"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:69:
GNGGGTGGTG GGTGGGT 17
(2) INFORMATION FOR SEQ ID N0:70
(i) SEQUENCE CHARACTERISTICS:
r
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(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:64:
GTGGGTGGTGG GGTGGT 17
~ (2) INFORMATION FOR SEQ ID N0:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii} MOLECULE TYPE: DNA (genomic}
(ix} FEATURE:
(A) NAME/KEY: misc_feature
CBI LOCATION: 5
(D) OTHER INFORMATION: /note= "C-5 propynl dU"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:65:
GTGGNGGGGT GGTGGGT 17
(2) INFORMATION FOR SEQ ID N0:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
{B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii} MOLECULE TYPE: DNA {genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
{B) LOCATION: 13
(D) OTHER INFORMATION: /note= "C-5 propynl dU"
(xi) SEQUENCE DESCRIPTION: 5EQ ID N0:66:
GTGGTGGGTG GGNGGGT 17
(2) INFORMATION FOR SEQ ID N0:67:
{i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
{D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
{A) NAME/KEY: misc_feature
{B) LOCATION: 5,13
/s3
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(A) LENGTH: 17 base pairs
- (B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc-feature
(B) LOCATION: 2
(D) OTHER INFORMATION: /note= "the base is
removed from this nucleotide"
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 1,5,6,9,10,13,14,17
(D) OTHER INFORMATION: /note= "deoxyinosine"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:70:
NNGGNNGGNN GGNNGGN 17
(2) INFORMATION FOR SEQ ID N0:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc-feature
(B) LOCATION: 6,13
(D) OTHER INFORMATION: /note= "C-S propynl dU"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:71:
GNGGGTGGTG GGTGGGT 17
(2) INFORMATION FOR SEQ ID N0:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc-feature
(B} LOCATION: i3
(D) OTHER INFORMATION: /note= "3' cholesterol via
triglycyl linker"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:72:
GTGGTGGGTG GGTGGGT 17
S
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WO 98133807 PCT/US98J01974
'(2) INFORMATION FOR SEQ ID N0:73:
~ (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 13
(D) OTHER INFORMATION: /note= "5-bromo dU"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:73:
GTGGTGGGTG GGNGGGT 17
(2) INFORMATION FOR SEQ ID N0:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(BI LOCATION: 5,9,13
(D) OTHER INFORMATION: /note= "5-bromo dU"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:74:
GTGGNGGGNG GGNGGGT 17
(2) INFORMATION FOR SEQ ID N0:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 5
(D) OTHER INFORMATION: /note= "5-iodo dU"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:75:
GTGGNGGGTG GGTGGGT 17
(2) INFORMATION FOR SEQ ID N0:76:
(i) SEQUENCE CHARACTERISTICS:
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WO 98133807 PCTIUS98/01974
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 9
(D) OTHER INFORMATION: /note= "5-iodo dU"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:76:
GTGGTGGGNG GGTGGGT 17
(2) INFORMATION FOR SEQ ID N0:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 13
(D) OTHER INFORMATION: /note= "5-iodo dU"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:77:
GTGGTGGGTG GGNGGGT 17
(2) INFORMATION FOR SEQ ID N0:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 5,9,13
(D) OTHER INFORMATION: /note= "5-iodo dU"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:78:
GTGGNGGGNG GGNGGGT 17
(2) INFORMATION FOR SEQ ID N0:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
is6
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- (ii) MOLECULE TYPE: DNA (genomic}
(xi) SEQUENCE DESCRIPTION: N0:79:
SEQ ID
GTGGCGGG TG GGTGGGT 17
(2) INFO RMATION FOR SEQ ID N0:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: N0:80:
SEQ ID
GTGGTGGG CG GGTGGGT 17
(2) INFORMATION
FOR SEQ ID
N0:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
{D) TOPOLOGY: linear
{ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: N0:81:
SEQ ID
GTGGTGGGTG GGCGGGT 17
{2) INFORMATION
FOR SEQ ID
NO: B2:
(i) SEQUENCE CHARACTERISTICS:
(A} LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA {genomic)
(xi) SEQUENCE DESCRIPTION: N0:82:
SEQ ID
GTGGCGGGCG GGCGGGT 17
(2) INFORMATION
FOR SEQ ID
N0:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: N0:83:
SEQ ID
/5~ ~
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TGGGAGGTGG GTCTG 15
(2) INFORMATION FOR SEQ ID N0:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
{D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:84:
TGGGAGGTGG GTCTG 15
(2) INFORMATION FOR SEQ ID N0:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
{D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:85:
TGGGAGGTGG GTCTG 15
(2) INFORMATION FOR SEQ ID N0:86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:86:
GCGGGGCTCC ATGGGGGTCG 20
(2} INFORMATION FOR SEQ ID N0:87:
{i) SEQUENCE CHARACTERISTICS:
{A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(xi} SEQUENCE DESCRIPTION: SEQ ID N0:87:
GTGGTGGGTG GGTGGGT 17
(2) INFORMATION FOR SEQ ID N0:88:
~S
r
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
_ (C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic)
(ix) FEATURE:
(A) NAME/KEY: misc_feature
(B) LOCATION: 2,5,9,13
(D) OTHER INFORMATION: /note= "N is abasic,
thymidine or another pyrimidine or
modified pyrimidine"
(ix) FEATURE:
(AI NAME/KEY: misc_feature
(B) LOCATION: 17
(D) OTHER INFORMATION: /note= "N is absent,
thymidine or another pyrimidine or
modified pyrimidine"
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:88:
GNGGNGGGNG GGNGGGN 17
