Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02230086 1998-02-20
WO 97/08180 PCTIUS96/13721
TITLE OF THE INVENTION
METHOD TO IMPROVE THE BIOLOGICAL
AND ANTIVIRAL ACTIVITY OF PROTEASE INHIBITORS
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates to a method for improving
cellular uptake of therapeutic agents, such as protease
inhibitors and thus increase their activity, especially their
,anti-HIV activity.
Discussion of the Backaround
The disease now known as AIDS was first recognized as
early as 1979. The number of cases reported to the Centers
for Disease Control and Prevention (CDC) increased
dramatically each year since then, and in 1982 the CDC
declared AIDS a new epidemic. Between December 1987 and
November 1988, over 32,000 new cases of AIDS were reported by
the CDC (HIV/AIDS Surveillance Report, 1-16, December 1989).
Over 3,000 new cases were reported in 1984 alone. By early
1995, the World Health Organization (WHO) estimates that at
least 4 million cases of the disease have occurred worldwide.
It has also been estimated that approximately 10 million
people are infected today with HIV.
In the United States, about 441,000 cases of AIDS have
been reported to the CDC to date. As of January, 1995, the
CDC reported that there have been 250,000 deaths due to AIDS
in the United States alone. It is clear that the cost of the
CA 02230086 1998-02-20
WO 97/08180 PC'd'/US96/13721
-2-
AIDS epidemic in terms of human lives is staggering, and the
worst is yet to come.
Retroviruses were proposed as the causative agent of AIDS. Recently, human
immunodeficiency virus type 1 (HIV) has
emerged as a preferred name for the virus responsible for
AIDS. Antibodies to HIV are present in over 80% of patients
diagnosed as having AIDS or pre-AIDS syndrome, and it has also
been found with high frequency in identified risk groups.
There is considerable difficulty in diagnosing the risk
of development of AIDS. AIDS is known to eventually develop
in almost all of the individuals infected with HIV.
A patient is generally diagnosed as having AIDS when a
previously healthy adult with an intact immune system acquires
impaired T-cell immunity. The impaired immunity usually
appears over a period of 18 months to 3 years. As a result of
this impaired immunity, the patient becomes susceptible to
opportunistic infections, various types of cancers such as
Kaposi's sarcoma, and other disorders associated with reduced
functioning of the immune system.
No treatment capable of preventing the disease or
significantly reversing the immunodeficiency of AIDS is
currently available. All patients with opportunistic
infections and approximately half of all patients with Kaposi's sarcoma have
died within two years of diagnosis.
Attempts at reviving the immune system in patients with AIDS
have so far been unsuccessful.
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-3-
While 3'-azido-3'-deoxythymidine (AZT) has been most
often used in treating HIV infection and AIDS, it has
considerable negative side effects such as reversible bone
marrow toxicity, and the development of viral resistance to
AZT by the patient. Thus other methods of treatment are
highly desirable.
Viruses traditionally do not respond to antibiotic
therapy. Therefore, other treatments are used when treating
viral infections. One such recently discovered therapy
revolves around the use of protease inhibitors to disrupt the
viral replication cycle. Protease inhibitor therapy has the
potential to be used in the treatment of a wide range of
diseases, including viral infections, such as those caused by
retroviruses (e.g., HIV), hepadnaviruses (e.g., hepatitis C
virus), herpesviruses (e.g., herpes simplex virus and
cytomegalovirus) and myxoviruses (e.g., influenza virus), as
well as parasitic protozoa (e.g., cryptosporidium and
malaria), in cancer chemotherapy and various pathological
disorders. By way of example, the role of HIV protease on the
HIV replication cycle is discussed below.
HIV Protease and the Renlication Cycle
HIV replicates through a DNA intermediate. Each virus
particle contains two identical, single-stranded RNA molecules
surrounded by the viral nucleocapsid protein. The remaining
core of the virus =is composed of the capsid and matrix
proteins. Enzymes required for replication and integration of
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-4-
the viral genetic materials irto the host cells are also
contained within the capsid. The outer coat of the virus
particle consists of viral envelope glycoproteins and membrane derived from
the host cell.
As with other retroviruses, HIV has the same basic
genetic makeup for the gag, po1, and env genes. The gag and
po1 genes encode the viral capsid proteins and replication
enzymes, respectively. These genes are expressed from one
intermediate form of viral genetic material, called unspliced
messenger RNA, resulting in the synthesis of precursor gag-pol
fusion polyprotein. The polyprotein is then cleaved by the
HIV protease enzyme to yield the mature viral proteins. The
gag precursor is cleaved into p17 (matrix), p24 (capsid), p7
(nucleocapsid), and p6. On the other hand, the pol precursor
is processed into individual protease, reverse transcriptase,
and integrase enzymes. Thus, HIV protease is responsible for
regulating a cascade of cleavage events that lead to the virus
particle's maturing into a virus that is capable of full
infectivity.
HIV protease is a member of the aspartic protease family.
It differs from the mammalian aspartic proteases in that it
functions as a homodimer of two subunits. By contrast, the
mammalian proteases are monomeric polyproteins. However, the two types of
proteases are similar in their overall structure
and function. In 1988, it was observed that mutation or
deletion of the HIV protease gene results in the production of
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-5-
noninfectious, immature virus particles, suggesting that HIV
protease provides an essential function in the replication
cycle of HIV and makes the protease an attractive target for
the design of specific antiviral drugs for AIDS. The vast
body of knowledge accumulated from studies with other aspartic
proteases, most notably human renin, has facilitated the
design and discovery of HIV protease inhibitors. Recent
advances in computer-aided rational drug design have been
widely translated in the pharmaceutical and biotechnology
industries for the development of potent and highly specific
inhibitors of HIV. Protease inhibitors can be rationally
improved in their in vitro activity, if the architecture of
the protease-inhibitor complex is determined by x-ray
analysis.
The late stage of HIV replication requires a virus-
encoded aspartyl protease for maturational processing of
structural proteins and replicative enzyme precursors.
Inhibition of the protease results in immature, non-infectious
virus particles and cessation of virus propagation.
HIV Protease 2nhibltors
To date, numerous compounds that inhibit HIV protease function
have been identified. The following table shows a variety of
HIV protease inhibitors currently in various stages of
clinical testing, and the companies exploring these compounds
in HIV treatment.
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-6-
HIV Protease Inhibitors (I)
Compound Drug Class Company
Ro 31-8959 Hydroxyethylamine Hoffman-LaRoche
MK-639 Hydroxyaminopentane amide Merck, Sharpe &
Dohme
ABT-538 Symmetry-Based Abbott SC-52151 Hydroxyethylurea Searle/Monsanto
XM-323 Cyclic Urea Dupont/Merck
KNI-272 Phenylnorstatine Kyoto Pharm./NCI
U-103,017 Pyranone Upjohn/Pharmacia
AG-1343 Hydroxyethylamine Agouron
VX-478 Hydroxyethylsulfonamide Vertex/Glaxo-
Wellcome
DPM-450 Cyclic Urea Dupont-Merck
BMS-182,193 Aminoalcohol Bristol-Myers
Squibb
CGP-53820 Pseudosymmetric Ciba-Geigy
Inhibitors
CGP-53437 Hydroxyethylene Isosteres Ciba-Geigy
HOE/BAY-793 C2-Symmetric Hoechst/Bayer
Peptidomimetic
RPI-312 Synthetic Peptide Takeda Chemical
Industries
Structures are provided below for a selection of the
above protease inhibitors.
\ / O
H O
H
N N~N 1.7 CH3SO3H
H
O H H
H2N~
O H
Ro 31-8959
suISM s~E~ (RULE 26)
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-7-
=
H O O O ~--N
H
N
I O~ N
N O H H ~S
CH3S
KNI-272
O HNO
N
JC1NX3
MK-639
O H N
S~H
SCH3 H
N NN O S N~ O
ABT-538
SUBST[rUi'E SHEET (RULE 26)
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-8-
/ I
~ s
O O ~t_ NH
\ N N
H VH H
/
CH3 H
H
AG- 1343
SN O O
~O N
~ \
H OH /
CD NHz
O
VX-478
These compounds make up one particularly promising new
class of antiretrovirals because they attack HIV at late
stages of its replication cycle, and thus are potentially
active against viruses harbored in chronically infected cells. 5 By contrast,
currently licensed anti-HIV drugs, such as AZT,
ddI, ddC, and more recently d4T, work as inhibitors of reverse
transcriptase, a viral enzyme acting at early stages of HIV
CA 02230086 1998-02-20
WO 97/08180 PCTIUS96/13721
-9-
replication. While these drugs can block HIV infection and
thus protect cells that are not yet infected, they are
essentially inactive against cells that are already infected,
such as chronically infected cells. Once infection is
established in the cells (i.e., HIV genetic material is
integrated into the host cell genome), reverse transcriptase
is no longer required for viral replication. However,
protease enzyme is essential for virions to produce
infectious, mature virus particles. The function of HIV
protease inhibitors is to render newly produced virus
particles noninfectious. Therefore, drugs active in
chronically infected cells are urgently needed, to be used
either alone or in combination with other anti-HIV drugs, to
improve the chances of success in therapy for HIV infection.
One factor that could affect the relationship between the
in vitro ECso (median effective concentration) of a drug such
as a protease inhibitor and the antiviral concentration of
that drug required under physiological conditions in vivo is
the extent and effect of protein binding. The antiviral
activity of several HIV protease inhibitors has been shown to
decrease in the presence of higher concentrations of human
serum or plasma (Bilello, Abstract #419 ist Intl. Conference
on Human Retroviruses, Dec. 12-16, 1993 Washington, D.C.;
Bilello, Abstract 178, 34th Interscience Conference on
Antimicrobial Agents and Chemotherapy Oct. 4-7, 1994). The
higher binding affinity of HIV protease inhibitors is probably
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-10-
related to their lipophilicity (Kaaavama et al, Antimicro.
Agents and Chelaothsr., 38:1107-1111, 1994). Sommadossi et al
(Abstract "A Human Serum Glycoprotein Profoundly Affects
Antiviral Activity of the Protease Inhibitor SC-52151 by
Decreasing Its Cellular Uptake" The Second Nat'l Conference on
Human Retroviruses and Related Infections, Washington, DC,
January 30, 1995) and Bilello, et al (1993 supra) have
recently shown that human alpha-l-acid glycoprotein (AAG) but
not albumin, both major components of human plasma, can
markedly reduce the antiviral activity of the protease
inhibitors A77003 and SC-52151 and their analogs. AAG is an
acute-phase protein with normal physiological levels of 0.5 to
1.5 mg/mi, which can increase after disturbance of homeostatis
by infections, cancer, inflammation and injuries. The average
value of AAG has been reported to be 50-100% higher in AIDS
and cancer patients, as compared to healthy persons (Oei et al
J. AIDS, 6:25-27, 1993).
The antiviral activity of certain experimental anti-HIV
agents, including dextran sulfates, oligonucleotides and
peptidomimetic-based protease inhibitors, and to a lesser
extent, nucleoside analogs, (Kagavama et al (supra); Hartman
et al AIDS Res. Hum. Retroviruses, 6:805-812, 1990) are
markedly affected by high concentrations of human serum or
components of human plasma. Although the structure of a
particular drug candidate cannot be used to predict the in
vitro plasma protein-binding affinity (Schmid in "The Plasma
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-11-
Proteins, Structure, Function and Genetic Control" Ed. by F.W.
Putnam, Academic Press, Inc. New York, pp 184-228, 1975), the
reduction in antiviral potency in the presence of
physiologically relevant concentrations of plasma, or plasma
components such as albumin or AAG, could be clinically
important.
Another factor which affects the ECso and the antiviral
concentration of protease inhibitors is the development of
viral strains which are resistant to the protease inhibitors.
Nearly every protease inhibitor and reverse transcriptase
inhibitor used has the potential to result in resistant viral
strains. This is especially the case in treatment of HIV
infection, due to the ability of HIV to readily mutate and
develop resistance. Mellors et al, (International Antiviral
News, 3(1), pp 8-13, 1995) have recently reported on a variety
of nucleoside RT inhibitors and protease inhibitors to which
HIV has developed resistant mutations. (See also Condra et al
Nature 374, pp. 569-571, 1995).
Thus, if therapy can be performed using less protease
inhibitor while maintaining the same or even higher levels of
antiviral activity, the tendency of the infectious agent being
treated to mutate into a strain which is resistant to the
protease inhibitor should be decreased.
In conclusion, binding of protease inhibitor to AAG leads
to a major decrease in drug cellular uptake which results in
alteration of anti-HIV activity. Therefore, sustained
CA 02230086 1998-02-20
WO 97/08180 PC'E'/US96/13721
-12-
cellular uptake of protease inhibitors is critical for their
in vivo anti-HIV activity, and for such cellular
incorporation, the effect of AAG must be countered.
SUMMARY OF THE INVENTION
Accordingly one object of the present invention is to
provide a method for increasing the cellular uptake and
cellular concentration of protease inhibitors.
A further object of the present invention is to provide a
method for increasing the cellular uptake and cellular
concentration of one or more protease inhibitors in the
presence of one or more additional therapeutic agents selected
from protease reverse transcriptase inhibitors,
antifusion/binding agents, anti-integrase agents and antiviral
oligonucleotides.
A further object of the present invention is to provide a
method for increasing the cellular uptake and cellular
concentration of protease inhibitors for various pathogenic
and infectious diseases including viral, fungal, antirenin,
parasitic protozoan, cancer and antimicrobial diseases.
A further object of the present invention is to provide a
method for improving the anti-HIV activity of HIV protease
inhibitors.
A further object of the present invention is to provide a
method to competitively bind AAG in the presence of a protease
inhibitor.
CA 02230086 2007-05-10
-13-
A-further object of the present invention is to provide a
method to decrease the amount of protease inhibitor
administered in protease inhibitor based therapy by increasing
the availability of the protease inhibitor for cellular
uptake.
These and other objects of the present invention have
been satisfied by the discovery of potent AAG binders, such as
antibiotics of the Marcrolide and Lincosamide families, some
of which bind AAG with a binding constant greater than the AAG
protease inhibitor binding constant, thus increasing the
cellular uptake of protease inhibitor, and the use of this
discovery in a method for improving the cellular uptake and
antiviral activity of therapeutic agents, such as protease
inhibitors, especially the anti-HIV activity of protease
inhibitors.
According to one aspect of the present invention, there
is provided use of an effective amount of one or more AAG-
binding compounds for improving cellular uptake of a protease
inhibitor during protease inhibitor-based therapy, in a
subject, wherein said one or more AAG-binding compounds bind
to AAG in the presence of said protease inhibitor.
According to another aspect of the present invention,
there is provided use of an effective amount of one or more
AAG-binding compounds for the manufacture of a medicament
improving cellular uptake of a protease inhibitor during
protease inhibitor-based therapy in a subject, wherein said
one or more AAG-binding compounds bind to AAG in the presence
of said protease inhibitor.
According to another aspect of the present invention,
there is provided use of an HIV protease inhibitor and one or
more AAG-binding compounds for the treatment of an HIV
infection in a subject, wherein said one or more AAG-binding
CA 02230086 2007-05-10
-13a-
compounds is suitable for administration in an amount
effective to increase cellular uptake of said HIV protease
inhibitor when compared to cellular uptake of said HIV
protease inhibitor in the absence of said one or more AAG-
binding compounds and wherein said one or more AAG-binding
compounds bind to AAG in the presence of said HIV protease
inhibitor.
According to another aspect of the present invention,
there is provided use of an HIV protease inhibitor and one or
more AAG-binding compounds in the manufacture of a medicament
for the treatment of an HIV infection in a subject, wherein
said one or more AAG-binding compounds is suitable for
administration in an amount effective to increase cellular
uptake of said HIV protease inhibitor when compared to
cellular uptake of said HIV protease inhibitor in the absence
of said one or more AAG-binding compounds and wherein said one
or more AAG-binding compounds bind to AAG in the presence of
said HIV protease inhibitor.
According to another aspect of the present invention,
there is provided use of an effective amount of one or more
AAG-binding compound and one or more protease inhibitors and
one or more first therapeutic agents selected from the group
consisting of protease reverse transcriptase inhibitors,
antifusion/binding agents, anti-integrase agents and antiviral
oligonucleotides for improving cellular uptake of a second
therapeutic agent, wherein said one or more AAG-binding
compound bind to AAG in the presence of said one or more
protease inhibitors.
According to another aspect of the present invention,
there is provided use of an effective amount of one or more
AAG-binding compound and one or more protease inhibitors and
one or more first therapeutic agents selected from the group
CA 02230086 2007-05-10
-13b-
consisting of protease reverse transcriptase inhibitors,
antifusion/binding agents, anti-integrase agents and antiviral
oligonucleotides for the manufacture of a medicament for
improving cellular uptake of a second therapeutic agent,
wherein said one or more AAG-binding compound bind to AAG in
the presence of said one or more protease inhibitors.
According to another aspect of the present invention,
there is provided a composition for treatment of a pathogenic
or infectious disease of viral, fungal, antirenin, parasitic
protozoan, cancer or antimicrobial origin, comprising an
effective amount of one or more protease inhibitors admixed
with one or more AAG-binding compounds, wherein said one or
more AAG-binding compounds bind AAG in the presence of said
one or more protease inhibitors.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention relates to a method for improving
the cellular uptake and antiviral activity of protease
inhibitors, alone or in combination with one or more
additional therapeutic agents, comprising administering, to a
subject in need thereof, an effective amount of an AAG-binding
compound having an affinity of AAG which is stronger than the
affinity of AAG for the protease inhibitor.
In the present method, not all AAG binding compounds work
experimentally. In fact, the AAG-binding compounds of
CA 02230086 1998-02-20
WO 97/08180 PCTIUS96/13721
-14-
the present invention must have a strong enough affinity for
binding AAG to competitively bir:d the AAG in the presence of
protease inhibitor.
Preferred AAG-binding compounds for use in the present
invention include the Macrolide antibiotics and the
Lincosamide antibiotics. More preferred Macrolide antibiotics
include erythromycin, troleandomycin, clarithromycin and
roxithromycin. Most preferred Macrolide antibiotics are
clarithromycin and roxithromycin. A most preferred
Lincosamide antibiotic is lincomycin.
In performing the method of the present invention, the
AAG binding compound can be administered in any of the
conventional methods for administering drug compositions.
Such methods include, but are not limited to, intravenously,
intraperitoneally, and orally. The compositions can be
administered in the form of injectable solutions, ingestible
solutions, tablets, capsules, lozenges, powders, etc. The
preferred methods of administration are intravenously or
orally. In the methods of the present invention, the AAG
binding compounds can be given alone or as mixtures of two or
more AAG binding compounds. Additionally, the AAG binding
compound can be administered just prior to administration of a
protease inhibitor, simultaneously with the administration of
a protease inhibitor or after administration of the protease
inhibitor, preferably prior to or simulaneously with
administration of the protease inhibitor.
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-15-
Any protease inhibitor used in protease inhibitor therapy
may be used in the methods of the present invention. Protease
inhibitors may be administered singly or as a mixture of two
or more protease inhibitors. Preferred protease inhibitors
include those listed in the Table on page 6 above, with SC-
52151 being most preferred.
If the one or more protease inhibitors and one or more
AAG binding compounds are being administered simultaneously
they may be admixed immediately prior to administration or can
be administered in any of the above mentioned forms for
administration of the AAG binding compounds.
In a further embodiment of the present invention, one or
more AAG binding compounds of the present invention may be
administered in conjunction with administration of one or more
protease inhibitors combined with one or more additional
therapeutic agents selected from protease reverse
transcriptase inhibitors, antifusion/binding agents, anti-
integrase agents and antiviral oligonucleotides.
The AAG binding compounds of the present invention are
used in a dosage range of from 0.1 times their normal dosage
range in non-protease inhibitor based theranv un to their
maximum tolerated dose (based on toxicity of the compounds).
Preferably, the A.AG binding compounds of the present invention
are used in a dosage range of from 1 to 10 times their normal
dosage in non-protease inhibitor based therapy. For example,
roxithromycin is conventionally used in treatment of bacterial
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-16-
infections in an amount of 150 mg two times a day. However,
in the method of the present invention, roxithromycin is
administered in an amount ranging from 15 mg to 3000 mg twice
a day.
By employing the present AAG binding compounds during
protease inhibitor therapy, the present method provides for
increased cellular uptake of the protease inhibitor. While
the present inventors do not wish to be bound by any
particular theory on the mode of action of the AAG binding
compounds of the present invention, it is believed that the
AAG binding compounds competitively bind the AAG in the
presence of the protease inhibitor thus giving more free
(unbound) protease inhibitor to be unbound to AAG. This is
believed to provide more available protease inhibitor for
cellular uptake.
The present method is useful in a variety of protease
inhibitor based therapies, including, but not limited to, the
treatment of various viral infections, such as those caused by
retroviruses (e.g., HIV), hepadnoviruses (e.g., hepatitis C
virus), herpesviruses (e.g., herpes simplex virus and
cytomegalovirus) and myxoviruses (e.g., influenza virus), as
well as parasitic protozoa (e.g., cryptosporidium and
malaria), in cancer chemotherapy and pathological disorders
which are treated using protease inhibitors.
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-17-
By way of example, thet effects of the present method on
cellular uptake of an HIV protease inhibitor are described
below.
SC-52151 is a member of a potent class of HIV protease
inhibitors incorporating the (hydroxyethyl)urea isostere that
shows a strong preference for the (R)-hydroxyl isomer in
contrast to, for example, renin inhibitors where preference is
for the (S)-hydroxyl configuration. The structure of SC-52151
is shown below as Formula I.
Q
H O
O
~ N N N
C~N
O OH
H2N
SC-52151 has similar in vitro antiviral potency, and
selectivity as compared to other HIV protease inhibitors
(Chang, J. Physicians Assoc. Aids Res., July pp. 8-18, 1994).
The HIV protease inhibitor SC-52151 is a tight-binding
transition state analog containing a hydroxyethylurea
isostere. In recent clinical trials, SC-52151 produced no
measurable effect on markers of anti-HIV activity, including
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-18-
PCR RNA, P24 antigen or CD4+ counts, despite an oral
absorption which leads to plasma levels five-to-eighty-fold
above the in vitro EC50. Human serum AAG has been shown to
interfere with the virologic effects of various protease
inhibitors. The following table shows the effect of AAG on
the in vitro antiviral activity of protease inhibitors in HIV-
infected CEM cells.
Effect of human serum alpha-i acid glycoprotein (AAG) on in
vitro antiviral activity of protease inhibitors in HIV-
infected CEM cells.
EC90a (ng/ml)
Fold Increase in EC90
AAG AAG Relative to Compound
Compound (0 mg/ml) (2 mg/ml) Alone
AZT 9 10 0
SC-52151 80 1970 24.6
VX-478 40 1200 30
Ro 31-8959 15 96 6.4
MK-639 20 100 5.0
'EC90 value was estimated from curve fit related to inhibition
of reverse transcriptase activity associated with the
clarified supernatants of HIV-infected cells.
Human serum AAG at physiological concentrations is seen
to interfere with the in vitro anti-HIV activity of protease
inhibitors with a 5 to 6-fold enhancement of the EC90 value of
Ro 31-8959 and MK-639 and a 25 fold increase in the EC90 value
of SC 52151. VX-478 and KNI-272 antiviral activity are also
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-19-
mc;stly affected by the presence of AAG. Protein binding
st.udies revealed that SC-52151, VX-478 and KNI-272 were bound
to AAG and to human plasma protein, respectively. Exposure of
human HIV-infected PHA-activated peripheral blood mononuclear
cells (PBMC) to 1 M SC-52151 resulted in intracellular
steady-state levels of 1.5 to 4.0 pmole/106 cells within 30
min, a 2-3 fold increase over uninfected cells. No difference
in cellular content of SC-52151 was detected when cells were
infected with either a low or high multiplicity of infection
(MOI). Physiological concentrations of AAG, but not albumin,
substantially affect the antiviral potency of SC-52151.
The AAG-binding compounds of the present invention
provide increased activity of protease inhibitors, such as SC-
52151, by binding the AAG present, freeing up the protease
inhibitors, or by preventing binding of protease inhibitor to
AAG.
Having generally described this invention, a further
understanding can be obtained by reference to certain specific
examples which are provided herein for purposes of
illustration only and are not intended to be limiting unless
otherwise specified.
CA 02230086 1998-02-20
WO 97/08180 PCTIUS96/13721
-20-
EXAMPLES
Prenaration of SC-52151
Protease inhibitor SC-52151 was prepared using the method
of Getman et al (J. Med. Chem., 36:288-291 (1993)).
Effect of AAG-bindincr comipounds on cellular uptake of SC-52151
The cellular accumulation of HIV protease inhibitor SC-
52151 in phytohemagglutanin (PHA)-stimulated human peripheral
blood mononuclear cells (PBMC) was measured after exposure to
1 M of SC-52151 in the presence of 1 mg/ml of AAG and a
modulating agent including AAG-binding compounds of the
present invention at various concentrations. Each of the
modulating agents was added 15 minutes before the addition of
AAG and SC-52151 and the experiments were performed for 2
hours prior to measurement. All of the following experiments
used human AAG. Similar results can be obtained using bovine
AAG, although the activity and effect on cellular uptake of
the present AAG binding compounds is attenuated using bovine
AAG. The results are provided in the tables below.
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-21-
Table 1
Modulating Human AAG Cellular Uptake Percent of
Agent (MM) (1 mg/ml) (pmole/106 cells') Control
(~)
None - 1.44 100
None + 0.12 8.3
Erythromycin (50) + 0.55 38.2
Erythromycin (100) + 0.85 59.0
Erythromycin (500) + 1.50 104.2
Troleandomycin (50) + 0.42 29.2
Troleandomycin .(100) + 0.64 44.4
Troleandomycin (500) + 1.29 89.6
1 pmole/10' cells = approx. 1 M
Table 1 shows the effect of erythromycin and
troleandomycin on cellular uptake of HIV protease inhibitor
SC-52151 in the presence of AAG, compared to administration of
SC-52151 in the absence of AAG and administration of HIV
protease inhibitor SC-52151 in the presence of 1 mg/ml of AAG.
As shown in the table, throughout the range of 50 M to 500 M
the cellular concentration of the protease inhibitor markedly
increases to essentially 100% of the level obtained in the
absence of AAG. Even at low levels of erythromycin and
troleandomycin of 50 M the cellular concentration is at 30-
40% of the control with no AAG, compared to the control
experiment performed in the presence of AAG which gave reduced
cellular concentration to only 8.3% of that obtained in the
absence of AAG. Thus, it is clearly shown that macrolide
antibiotics such as erythromycin and troleandomycin can
significantly increase the cellular concentration of protease
inhibitors.
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-22-
Clarithromycin provides an even stronger effect on
protease inhibitor cellular concentration than found with
erythromycin or troleandomycin, as shown by the increase in
cellular concentration of the protease inhibitor at lower
doses of clarithromycin as compared to erythromycin or
troleandomycin. This is shown in the results in Table 2.
Table 2
Modulating Human AAG Cellular Uptake Percent of
Agent (MM) (1 mg/ml) (pmole/10` cells") Control
M
None - 1.02 100
None + 0.09 8.8
Clarithromycin (50) + 0.54 52.9
None - 1.25 100
None + 0.13 10.4
Clarithromycin (100) + 1.02 82.0
1 pmole/106 cells = approx. 1 f.cM
Not all AAG binding compounds are likely to be useful in
the present invention at low concentrations. Further, not all
macrolide antibiotics are useful, as shown below in Table 3
which provides the results obtained using midecamycin,
oleandomycin and spiramycin. While some improvement in
cellular concentration of the HIV protease inhibitor is
obtained using these macrolide antibiotics, the effects are
clearly inferior to the macrolide antibiotics having stronger
binding affinities for AAG.
CA 02230086 1998-02-20
WO 97/08180 PCTIUS96/13721
-23-
Table 3
Modulating Human AAG Cellular Uptake Percent of
Agent (pM) (1 mg/ml) (pmole/106 cells') Control
($)
Midecamycin (100) + 0.24 19.3
Oleandomycin (100) + 0.29 23.3
Spiramycin (100) + 0.23 18.5
Azithromycin (100) + 0.18 17.7
Josamycin (100) + 0.37 26.4
Rokitamycin (100) + 0.34 24.3
1 pmole/10' cells = approx. 1 pM
One of the strongest macrolide antibiotics for use in the
present invention is roxithromycin. Tables 4 and 5 show the
results obtained from two independent experiments using
roxithromycin. It is interesting to note that the cellular
concentration of the HIV protease inhibitor SC-52151 is not
only improved compared to experiments performed in the
presence of AAG and absence of roxithromycin, but the cellular
concentration of SC-52151 is increased compared to controls
performed in the absence of AAG, with the improvement in
cellular concentration being as high as a 72.4% increase over
the control. Accordingly, roxithromycin enhances the cellular
concentration of protease inhibitors above and beyond the
extent previously attainable when AAG is absent. As is
evident from these results, the AAG binding ability of the
macrolide antibiotic is not the only factor affecting the
cellular concentration of the protease inhibitor since
compounds having known potent AAG binding capability, such as
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-24-
midecamycin, even at 100 uM, did not markedly influence the
cellular uptake of the protease in:iibitor.
Table 4
Modulating Human AAG Cellular Uptake Percent of
Agent ( M) (1 mg/ml) (pmole/106 cells') Control
M
None - 1.27 100
None + 0.11 8.7
Roxithromycin (10) + 0.27 21.6
Roxithromycin (50) + 2.08 163.8
Roxithromycin (100) + 2.19 172.4
' 1 pmole/10' cells = approx. 1MM
Table 5
Modulating Human AAG Cellular Uptake Percent of
Agent ( M) (1 mg/ml) (pmole/10' cells') Control
($)
None - 1.47 100
None + 0.10 6.8
Roxithromycin (10) + 0.30 20.4
Roxithromycin (20) + 0.97 66.0
Roxithromycin (30) + 1.33 90.5
Roxithromycin (40) + 1.96 133.3
Roxithromycin (50) + 2.16 147.0
` 1 pmole/10' cells = approx. 1pM
Table 6 shows the improvements in cellular concentration
obtained using the Lincosamide antibiotic of lincomycin,
compared to another Lincosamide antibiotic, clindamycin, which
is known to bind AAG. The results again show that AAG binding
is not the only factor at play in increasing cellular uptake
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-25-
of the protease inhibitor, since clindamycin shows neagligible
increase in protease inhibitor cellular uptake.
Table 6
Modulating Human AAG Cellular Uptake Percent
Agent ( M) (1 mg/ml) (pmole/106 cells*) of
Control
(~)
None - 1.44 100
None + 0.12 8.3
Lincomycin (50) + 0.43 29.9
Lincomycin (100) + 0.55 38.2
Lincomycin (500) + 1.28 88.8
Clindamycin (100) + 0.15 10.4
1 pmole/106 cells = approx. 1 M
The present invention requires the use of AAG binding
compounds which have sufficient binding affinity with AAG to
disrupt or prevent the AAG-protease inhibitor binding and
increase the cellular uptake of the protease inhibitor. Table
7 shows that not all AAG binding compounds are useful in the
present invention. The compounds of Table 7 are known to bind
AAG (Kremer et al, Pharm. Rev. 40(1), 1-47, 1988 and
references cited therein). However, even though these
compounds are known to bind AAG, they do not appear to have a
significant effect on protease inhibitor concentration at
clinically relevant concentration, with the exception of
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-26-
verapamil, which shows a moderate improvement in SC-52151
cellular uptake.
Ta.ble 7
Modulating Human AAG Cellular Uptake Percent of
Agent (uM) (1 mg/ml) (pmole/106 cells') Control
($)
None - 1.44 100
None + 0.12 8.3
Thioridazine (5) + 0.17 11.8
None - 0.65 100
None + 0.06 9.2
Verapamil (10) + 0.23 35.4
None - 0.58 100
None + 0.074 12.6
Prazocin (5) + 0.089 15.2
Disopyramide (5) + 0.11 19.3
Dipyridamole (1) + 0.084 14.3
Indomethacin (5) + 0.083 14.2
Oxprenolol (5) + 0.10 17.7
1 pmole/10' cells = approx. 1 M
The increased cellular uptake of protease inhibitor
exhibited in the above studies prompted further investigation
into whether the antiviral activity of protease inhibitors
would be restored in the presence of macrolide antibiotics
which were shown to increase cellular uptake of the antiviral
compound.
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-27-
Antiviral studies
Cells. Human PBMC from healthy HIV-1 seronegative and
hepatitis B virus seronegative donors were isolated by Ficoll-
Hypaque discontinuous gradient centrifugation at 1,000 x g for
30 minutes, washed twice in phosphate-buffered saline (pH 7.2;
PBS), and pelleted at 300 x g for 10 minutes. Before
infection, the cells were stimulated by phytohemagglutinin
(PHA) at a concentration of 8 g/ml for three days in RPMI
1640 medium supplemented with 15% heat-inactivated fetal calf
serum, 1.5 mM L-glutamine, penicillin (100 U/mi), streptomycin
(100 g/ml), and 4 mM sodium bicarbonate buffer.
Viruses. HIV-1 (strain LAV-1) was obtained from the CDC,
Atlanta, GA. The virus was propagated in human PBMC using
RPMI 1640 medium, as described previously (McDouaal et al, J.
immun. Meth. 76:171-183, 1985) without PHA or fungizone and
supplemented with 100 U/ml recombinant interleukin-2 (Cetus)
and 7/ig/ml DEAE-dextran (Pharmacia, Uppsala, Sweden). Virus
obtained from cell-free culture supernatant was titrated and
stored in aliquots at -70 C until use.
Inhibition of virus replication in human PBMC.
Uninfected PHA-stimulated human PBMC were uniformly
distributed among 25 cm2 flasks to give a 5 ml suspension
containing about 2 x 106 cells/ml. Suitable dilutions of virus
were added to infect the cultures. The mean reverse
transcriptase (RT) activity of the inocula was 60,000 dpm RT
activity/10 cells (MOI = 0.01). The drugs at twice their
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-28-
final concentrations in 5 ml of RPMI 1640 mediu:a, supplemented
as described above, were added to the cultures. Uninfected
and untreated PBMC at equivalent cell densities were grown in
parallel as controls. The cultures were maintained in a
humidified 5% C02-95% air incubator at 37 C for six days after
infection at which point all cultures were sampled for
supernatant RT activity. Previous studies had indicated that
maximum RT levels were obtained at that time. Concentrations
which provided a 90% decrease in RT activity associated with
cell supernatent as compared to untreated controls are
reported below as EC90 values.
RT activity assay. Six ml supernatant from each culture
was clarified from cells at 300 x g for 10 minutes. Virus
particles were pelleted from 5 ml samples at 40,000 rpm for 30
minutes using a Beckman 70.1 Ti rotor and suspended in 200 l
of virus disrupting buffer (50 mM Tris-HC1, pH 7.8, 800 mM
NaCl, 20% glycerol, 0.5 mM phenylmethyl sulfonyl fluoride, and
0.5% Triton X-100).
The RT assay was performed in 96-well microtiter plates,
as described by Spira et al (J. Clin. Microbiol. 25:97-99,
1987). The reaction mixture, which contained 50 mM Tris-HC1
pH 7.8, 9 mM MgCla, 5 mM dithiothreitol, 4.7 g/ml (rA);,=(dT)Z2_
18, 140 M dATP, and 0.22 M [3H]TTP (specific activity 78.0
Ci/mmol, equivalent to 17,300 cpm/pmol; NEN Research Products,
Boston, MA.), was added to each well. The sample (20 l) was
added to the reaction mixture which was then incubated at 37 C
CA 02230086 1998-02-20
WO 97/08180 PCTIUS96/13721
-29-
for hours. The reaction was terminated by the addition of
100 ~1 10% trichloroacetic acid (TCA) containing 0.45 mM
sodium pyrophosphate. The acid-insoluble nucleic acids which
precipitated were collected on glass filters using a Skatron
semi-automatic harvester (setting 9). The filters were washed
with 5% TCA and 70% ethanol, dried, and placed in
scintillation vials. Four ml of scintillation fluid (Ecolite,
ICNm, Irvine, CA) were added and the amount of radioactivity
in each sample was determined using a Packard Tri-Carb liquid
scintillation analyzer (model 2,000CA). The results were
expressed in dpm/ml of original clarified supernatant. The
procedures for the anti-HIV-1 assays in PBMC described above
have been published (see Schinazi, et al. in Antimicrob.
Agents Chemother. 32:1784-1789, 1988 and Schinazi, et al. in
Antimicrob. Agents Chemother. 34:1061-1067 1990). The CEM
studies were performed as described by Schinazi, et al. in
Antimicrob. Agents Chemother. 36:2423-2431, 1992.
The following Table 8 provides the results of the
antiviral studies on restoration of the anti-HIV activity of
SC-52151 by Macrolide antibiotics in acutely infected PBM and
CEM cells.
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-30-
v
V1 -1
U lim >t4
UU+i~n~ m
o .-r
'O m m 41
v v mu a =r
+J C W C
y, O rp A
~r 0 G p~ Fs,
M..,~
y .
U 1
a.r L) e
j y
.rEi y~ ~ r=1 N ~ ~ m Of O
=o osr,
~ eia G7 MA p O
w L) W 0 V
OI
a
U ~r > L
L V 0% 4)
C4 .-r r ~ In m
+1 G. C R! N "a ~ r==1 N m ~ r-1 N
A N N
L .a +i .-f tt1
RC. C ,.y d U
r6 0 11
U
ro s
r+ o
rn
o a,
~'+u
m a~i vm c
~ 4J M 1-I 'i +' =-i rl
c 0 o N e ~ ~ ~ E a f f f s f s a a a x x
_ tp a rn a s s a a z a z a a a a
.==~ .-~'i al G
rn d +~ o in p
ufeti U
U U m
y C
w V 'y 7
O a+ i, s~ ~ Oo
U G 'O "'r y
~ u ~ 0 tNl1 1y0 ~ Ul o 0 o N t~t tD N O m ~T t~ f+f ~ tfl
+i C M O C C C O O N ~O N = U1 QV r..y A 14 . . 'i "' 01 tt1 m O
= a=/ . . . O
o O V O o O o O
.,> ,r ~ V W m p1 .Q O A A A O O O
O o O
aJ .1. C. ` 1r OE
V W ~ O U
9 7
+~ C
-4
O ZL E _ E E S T S E
O in a p~ ~ a z a
t3l cn
'q ' m 0 0 s
O O O
m S 7
1J O
o r.r r=r r+f I[I
Rl V ` O O O ...
0 ~ U C C ~ .... .... .~
-.+ +y ` "' .-. C C C C
CWl ri U O O O
a .~ O LOi E E U U "'r E
m L a.r t E E E S.~ l~ Sa l~
L ='y L M LI 1-I ~ JJ L 1J iJ
c C M H y~ s .c C = i -r -.a
(y7 M C ,~,r 3.1 Ip iJ JJ y X X X X
a ~ ,n U V u u ~ W a cK a z
ED O O + + + ~, + + + + +
r^ ~a r +
r
E EQ)
cn m x E
U w C N E E E E E E E E E
N
.a .~ .i
an
+ Q st + + + +
+ '~ + + +
~ m m .~ .-r ~ vi tn tn tn
~"'r N N N .-1 rl N N N N N
N lIi T T N N N tll VI t!l Ifl
tn
6 ~ cUi] tUi] U ~ rn vJ V1
0
SUB STITUTE SHEET (RULE 26)
CA 02230086 1998-02-20
WO 97/08180 PCT/US96/13721
-31-
From the above data, it is important to note that
clarithromycin, erythromycin and roxithromycin are all
essentially inactive with respect to antiviral activity
against HIV-1 up to 600 N. When only SC-52151 is present in
the PBM or CEM cells, the EC90 of SC-52151 is 0.14 M. When
AAG is added, the EC90 of SC-52151 in PBM cells increases an
order of magnitude to 1.22 M (1.18 M in CEM cells).
However, when the AAG-binding compound of the present
invention, namely clarithromycin, erythromycin and
roxithromycin, are added there is a dose related decrease in
EC90, indicating greater antiviral potency. Specifically, the
addition of the AAG-binding compounds of the present invention
provides increased cellular uptake of SC-52151, which
translates to increased antiviral activity. In fact, the AAG-
binding compounds in the above table provide increases in
antiviral activity beyond that achieved with SC-52151 alone.
This enhanced activity is not cell line dependent, as
confirmed by the above results in both PBM cells and CEM
cells.
Obviously, additional modifications and variations of the
present invention are possible in light of the above
teachings. It is therefore to be understood that within the
scope of the appended claims, the invention may be practiced
otherwise than as specifically described herein.
