Note: Descriptions are shown in the official language in which they were submitted.
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
ENZYME CATALYZED ANTI-INFECTIVE THERAPEUTIC AGENTS
CROSS-REFERENCE TO RELATED APPLICATIONS
This appliation claims priority under 35 U.S.C. ~ 119(e) to the following U.S.
provisional applications, Serial Nos. 60/153,101, filed September 9, 1999 and
60/145,364, filed July 22, 1999, the contents of which are hereby incorporated
by
reference into the present disclosure.
TECHNICAL FIELD
1 o This invention relates to the field of therapies for infectious diseases,
and in
particular, compositions and methods for the treatment of therapy-resistant
infectious
diseases.
BACKGROUND
15 Throughout and within this disclosure, various publications are referenced
by
first author and date, patent number or publication number. The full
bibliographic
citation for each reference can be found within the specification. The
disclosures of
these publications are hereby incorporated by reference into this disclosure
to more
fully describe the state of the art to which this invention pertains.
2o Resistance to chemotherapeutic and antibiotic treatments for infectious
disease is a major health care problem. In infectious disease, most drug
resistance is
enzyme mediated. Typically, an enzyme expressed by the infectious agent
rapidly
modifies the chemotherapeutic or antibiotic, thereby abolishing its
therapeutic
activity. In infectious disease, amplified expression of beta-lactamases
accounts for
25 more than one-third of all beta-lactam antibiotic resistant isolates
(Felmingham and
Washington (1999) J. Chemother. 11 Suppl 1:5-21), including the majority of
resistant Haemophilis influenza (upper respiratory infections) and Moraxella
catarrhalis (otitis media). In addition, genes confernng resistance to various
alternative types of antibiotics occur in nature and have become increasing
common
3o in populations of infectious organisms. Recently, infectious agents
carrying sets of
genes simultaneously confernng resistance to multiple antibiotic agents have
arisen
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
making treatment by traditional antibiotic therapy difficult. Applicant has
developed
a novel approach to development of therapeutics targeting well characterized
enzymes which are expressed by infectious agents. This technology is distinct
from
prior and historical approaches to therapy for infectious diseases and is
referred to as
"ECTA," for Enzyme Catalyzed Therapeutic Agent.
DISCLOSURE OF THE INVENTION
This invention provides a method for selectively inhibiting the proliferation
of an infectious agent or a cell infected by the infectious agent. Infectious
agents
to suitably treated by the method of this invention express an activating
agent that
selectively activates or converts a prodrug to a toxin. The enzyme is not
inactivated
or inhibited by the substrate prodrug compound. The method requires contacting
the
cell or the agent with an effective amount of the substrate compound thereby
selectively inhibiting the proliferation of the infectious agent, the cell or
the
15 infectious agent within the cell.
This invention also provides a method for screening for prodrugs selectively
converted to a toxin in a cell by an activating enzyme expressed by an
infectious
agent. The screen requires contacting an infectious agent or a cell infected
with the
infectious agent with a candidate prodrug and assaying for activation of the
prodrug
20 into toxic agents by the activating enzyme. Alternatively, activation of
the prodrug is
determined by noting inhibition of the proliferation or growth of the
infectious agent
or the cell infected with the agent.
BRIEF DESCRIPTION OF THE FIGURES
25 Figure 1 schematically shows the mechanism of action of the ECTA prodrugs
of this invention.
Figure 2 is a graph showing fluorescent products from incubation of
bromovinyl 2'-deoxyuridine monophosphate (BVdUMP) with recombinant human
thymidylate synthase (rHUTS). Incubation of BVdUMP with thymidylate synthase
3o results in a time and enzyme dependent generation of fluorescent
product(s).
BVdUMP was incubated with the indicated amounts of rHuTS in the standard
CA 02379834 2002-O1-21
WO 01/07087 PCT/ZJS00/19844
reaction mixture at 30° C (See Materials and Methods below), except
that N5, N10-
methylenetetrahydrofolate was omitted from the reaction. The numbers adjacent
to
each data curve refer to TS enzyme units.
Figure 3 shows that BVdUMP is competitive with deoxyuridine
monophosphate (BUMP) in rHuTS. Thymidylate synthase catalyzed reaction of
converting dUMP into dTMP was run in vitro in the absence (triangles) and in
the
presence of 20 pM BVdUMP (squares). dUMP concentration was varied from 10 to
100 ~M, N5, N10-methylene tetrahydrofolate concentration was 140 ~,M and the
enzyme concentration was 0.1 ~.M. Enzyme activity was determined by measuring
to the increase in A3ao
Figure 4 is the structures of products of in vitro reaction of BVdUMP
catalyzed by rHuTS. Structures I and II are consistent with mass ions
identified in
cell free reaction mixtures.
Figure 5 is a proposed mechanism of NB 1011 activation. NB 1011 must be
able to enter cells and convert to BVdUMP before interacting with TS.
Structures
generated following transformation by TS are proposed to be exocyclic
pyrimidine
nucleotide monophosphates. These compounds may be cytotoxic to cells by a
variety of mechanisms including interference with nucleotide and nucleic acid
metabolism.
2o Figure 6 shows detection of BVdUMP in H630R10 cells treated with
NB 1011. H630 R10 cells were treated with 100 ~M NB 1011 for 5 days, then
analyzed by liquid chromatograph/mass spectroscopy as described in the
Materials
and Methods, below.
Figure 7 demonstrates that NB 1011 does not irreversibly inactivate TS in
vivo. The effect of NB 1 O l 1 on TS activity in intact cells is completely
reversible.
TS activity was measured in intact RKO cells by release of [3H]20 from 5-[
3H]deoxyuridine as described in Materials and Methods. NB1011 was washed out
of
cells by replacing with fresh media, incubating for 60 minutes at 37
°C, then
repeating this procedure. Control and untreated cells were subj ected to the
same
3o washing procedure.
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
Figure 8 shows TS expression level in cells selected with Tomudex or
NB1011, as estimated by SDS PAGE Western blot developed with antibody against
thymidylate synthase and tubilin. Lane 1 shows MCF7 cells, no selection with
drug;
lane 2 shows MCF7 cells selected with 2~,M tomudex; lane 3 shows MCF7 cells as
in lane 2, but after a subsequent selection using NB 1011 as the selective
agent; lane 4
shows MCF7 cells as in lane 2, after a subsequent passaging without tomudex.
MODE(S) FOR CARRYING OUT THE INVENTION
The practice of the present invention will employ, unless otherwise indicated,
to conventional techniques of molecular biology, microbiology, cell biology
and
recombinant DNA, which are within the skill of the art. See, e.g., Sambrook,
Fritsch
and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2"° edition
(1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et
al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.):
15 PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R.
Taylor eds. (1995)) and ANI11~IAL CELL CULTURE (R.I. Freshney, ed. (1987)).
As used in the specification and claims, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates otherwise. For
example,
the term "a cell" includes a plurality of cells, including mixtures thereof.
2o The term "comprising" is intended to mean that the compositions and
methods include the recited elements, but not excluding others. "Consisting
essentially of when used to define compositions and methods, shall mean
excluding
other elements of any essential significance to the combination. Thus, a
composition
consisting essentially of the elements as defined herein would not exclude
trace
25 contaminants from the isolation and purification method and
pharmaceutically
acceptable Garners, such as phosphate buffered saline, preservatives, and the
like.
"Consisting of" shall mean excluding more than trace elements of other
ingredients
and substantial method steps for administering the compositions of this
invention.
Embodiments defined by each of these transition terms are within the scope of
this
30 invention.
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
An "infectious agent" or a "pathogen" is a organism that is pathological to a
cell or organism that it infects. Examples of pathogenic organisms include,
but are
not limited to, bacteria, parasites, viruses or yeast. Examples of viruses
include but
are not limited to Herpes, Varicella zoster, Hepatitis C and Epstein Barr
virus.
Examples of parasites include but are not limited to T. brucei, T. cruzi, and
Plasmodium falcipurum. Examples of bacteria include, but are not limited to,
all
gram positive and gram negative bacteria, especially, Staphylococcus, sp.,
Enterococcus sp., Myoplasma sp., E. coli sp., Psudomonas sp., Nisseria sp..
And,
from among these, preferred pathogens are those which have become resistant to
common antibiotics (see reveiw by Murray, BE "Antibiotic Resistance" (1997)
Adv.
Int. Med. 42:339-367.)
A "composition" is intended to mean a combination of active agent and
another compound or composition, inert (for example, a detectable agent or
label or a
pharmaceutically acceptable Garner) or active, such as an adjuvant.
A "pharmaceutical composition" is intended to include the combination of an
active agent with a Garner, inert or active, making the composition suitable
for
diagnostic or therapeutic use in vitro, in vivo or ex vivo.
As used herein, the term "pharmaceutically acceptable carrier" encompasses
any of the standard pharmaceutical Garners, such as a phosphate buffered
saline
solution, water, and emulsions, such as an oil/water or water/oil emulsion,
and
various types of wetting agents. The compositions also can include stabilizers
and
preservatives. For examples of carriers, stabilizers and adjuvants, see
Martin,
REMINGTON'S PHARM. SCL, 15th Ed. (Mack Publ. Co., Easton (1975)).
An "effective amount" is an amount sufficient to effect beneficial or desired
results. An effective amount can be administered in one or more
administrations,
applications or dosages.
A "subject," "individual" or "patient" is used interchangeably herein, which
refers to a vertebrate, preferably a mammal, more preferably a human. Mammals
include, but are not limited to, murines, simians, humans, farm animals, sport
animals, and pets.
5
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
A "control" is an alternative subject or sample used in an experiment for
comparison purpose. A control can be "positive" or "negative". For example,
where
the purpose of the experiment is to determine a correlation of an altered
expression
level of a gene with a particular type of pathogenic agent, it is generally
preferable to
use a positive control .(a subject or a sample from a subject, carrying such
alteration
and exhibiting syndromes characteristic of that infection), and a negative
control (a
subject or a sample from a subject lacking the altered expression and clinical
syndrome of that disease).
The term "activating enzyme" as used herein means an enzyme that is
to expressed by a pathogen in its native or natural environment. It is
intended to
distinguish enzymes or other agents that are administered to activate a
prodrug.
As used herein, the terms "pathological cells, "target cells", and "test
cells"
encompass cells characterized by the presence of an activating enzyme. The
expression of the activating enzyme occurs as a consequence of infection by a
pathogenic organism, as defined above. Enzymes expressed by the pathogen or
within an infected cell providing targets for this therapy include, but are
not limited
to thymidylate synthase and dihydrofolate reductase. Additional examples are
listed
below.
2o Therapeutic Methods
In one aspect, this invention is directed to methods for inhibiting the
proliferation or growth of an infectious agent or a cell infected with the
agent by
contacting the agent or infected cell with a substrate prodrug that is
selectively
converted to a toxin in the cell by an activating enzyme expressed by the
infectious
agent. The methods and compositions of this invention are useful to
preferentially
inhibit the growth or proliferation of cells that express or contain
activating enzyme,
for example microbial cells, virally infected cells or cells infected with
other
pathogens. Overexpression of the enzyme is not required, as specificity is
related to
the species-specificity of the prodrug to the activating enzyme expressed by
the
3o pathogen. The activating enzyme may or may not be expressed by the host
cell.
However, even if the cell expresses its own version of the enzyme, the prodrug
is
6
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
selective on the basis that it is preferentially activated by the version of
the enzyme
expressed by the infectious agent as compared to the version of the enzyme
expressed by the host cell. The activating enzyme can be the wild-type or a
mutated
version which has developed resistance to prior art therapeutics (Hooker, et
al. (1996)
J. Virol. 70(11):8010-8018).
Examples of activating enzymes that are selective targets for the prodrugs and
methods of this invention include, but are not limited to, thymidylate
synthase (TS),
dihydrofolate reductase (DHFR) and (3-lactamase activating enzymes.
The concepts of this invention are illustrated using the activating enzyme
to thymidylate synthase and its expression in human tumor cells. However, the
use of
TS is merely illustrative and the claims are not to be construed as limited to
systems
which target TS. Thyrnidylate synthase was used herein as the target,
activating
enzyme because of the high degree of characterization of its structure and
function
(Cameras and Santi (1995) Annu. Rev. Biochem. 64:721-726), the fact that it is
encoded by a single gene, not a gene family (compare for example the family of
enzymes noted as glutathione-S-transferase (GST)). In addition, TS
overexpression
is the result of acquired resistance to chemotherapeutics. Similarly, in one
embodiment, the activating enzyme can be expressed as a result of resistance
to prior
therapy.
2o Other target activating enzymes include, but are not limited to viral
reverse
transcriptases and proteases. Examples of viruses that encode these enzymes
include
the retroviruses (eg. HIV-l, both enzymes, see Turner B.G. and Summers M.F.
(1999) J. Mol. Biol. 285:1-32), the picornaviruses (eg., Hepatitis A virus,
Wang
Q.M. (1999) Prog. Drug Res. 52:197-219), and Hepatitis C virus (Kwong A.D. et
al.
(1999) Antiviral Res. 41:67-84). Early clinical success observed with anti-
HIV1
reverse transciptase and protease inhibitors (reviewed by Shafer R.W. and
Vuitton
D.A. (1999) Biomed. Pharamcother. 53:73-86.) has been tempered by the
development of resistance, largely due to mutations in the virally-enoded
enzymes
(Catucci M. et al. (1999) J. Acquir. Immune Defic. Syndr. 21:203-208;
Mahalingam
3o B. et al. (1999) Eur. J. Biochem. 263:238-44; and Palmer, S. et al. (1999)
AIDS
13(6): 661-667. Highly drug-resistant HIV-1 clinical isolates are cross-
resistant to
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
many anti-retroviral compounds in current clinical development. Hooker et al.
(1996) supra. In these cases of resistance, the viral enzymes retain their
catalytic
activity because the mutated version of the enzyme retains the structure of
the wild-
type active site of the enzyme. The prodrugs of this invention are
specifically
designed to interact with the active site and be converted by this interaction
into a
toxin. Accordingly, the drug resistant viral infections are sensitive to
substrate
prodrug, referred to as an ECTA compound, which require the activating enzyme
to
generate toxin in the infected cell. NB 1011 is an example of such a compound,
directed against TS expressed by mammalian and human cells as well as
pathogens.
to In one embodiment, the prodrug is a compound having a structure as defined
in more detail herein. The term prodrug refers to precursors of active
therapeutics.
The perfect prodrug is one that is pharmacologically inert until activated by
the
intended mechanism. Prodrug strategies are meant to target potentially toxic
therapies to the site of disease, thereby avoiding systemic toxicity. A number
of
approaches have been made to this goal. One of the first attempts at a prodrug
for
cancer therapy was reported by Mead et al. (1966) Cancer Res. 26:2374-2379 and
Nichol and Hakala (1966) Biochem. Pharmacol. 15:1621-1623. The guiding
principle of this effort was to target overexpressed dihydrofolate reductase
in
methotrexate-resistant leukemia. Self poisoning of tumor cells was hoped for
as the
2o elevated dihydrofolate reductase in methotrexate-resistant tumor cells was
supposed
to convert homofolate into metabolic poison directed to thymidylate synthase.
It was
later discovered that the modest antitumor effects of homofolate are not due
to
metabolic activation, but more likely to inhibition of folate transport into
cells
(Livingston et al. (1968) Biochem 7(8):2814-2818.) This, and subsequent,
prodrug-
like attempts to leverage tumor selective targets for therapeutics development
are
summarized in Table l, infra. One or more of the following issues has
confounded
these approaches: a) locating the activating enzyme appropriately and/or lack
of
tumor selectivity of the targeted enzyme; b) systemic distribution and
resulting
toxicity of the activated prodrug; and c) achieving the needed substrate
enzyme
3o specificity to prevent activation by enzymes other than the ones targeted.
For
instance, the glutathione-s-transferase (GST) prodrug that has been described
by
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
Morgan et al. (1998) Cancer Res. 58:2568-2575, while "specific" for GST, it
also is
activated by by the tumor overexpressed GST-P1-1 and can be activated by GST-
Al-
1. This leads to inappropriate drug activation and potential toxicity. Many of
these
issues are discussed in more detail in the publications cited in Table 1,
infra. The
methods and compositions disclosed herein avoid the prior prodrug failures by
developing prodrugs that target enzymes that have appropriate properties to
specifically activate the produgs only in the appropriate target cells,
without priming
the cells by prior adminstration of activating enzymes.
In an additional aspect of the prodrugs is that they are essentially non-toxic
to
to normal, uninfected cells. This aspect further enhances the selectivity of
the prodrugs
and increases the overall safety of the therapy. The prodrug can selectively
kill the
cell because only infected cells provide an effective amount of the toxic
metabolite of
the prodrug to inhibit proliferation of the pathogen or the cell infected with
the
pathogen. In other words, the ultimate efficacy of the prodrugs of this
invention are
1 5 related to the origin of the activating enzyme. For example, the efficacy
of NB 1011
is unexpectedly more potent as a substrate for human TS than microbial TS, as
would
be expected from published studies. (Barr (1983) J. Biol. Chem. 258(22):13637-
13631) summarized in Table 2, infra.
Various methods are available to determine if the object of the therapeutic
2o method has been met. This include, but are not limited to RT-PCR analysis,
growth
inhibition study (alamar blue assay) and plaque assays. These methods are well
known in the art and described herein.
Applicant also has discovered that cells which have been treated with the
substrate prodrugs may revert to a prior phenotype which is suitably treated
by
25 conventional therapies. Using TS as an example, Applicant has shown that
tumor
cells treated with 5-FU became resistant to the drug. At that time, the cells
were
treated with NB 1011. A subpopulation survived and became resistant to NB 1 Ol
1 but
regained sensitivity to S-FU (see Table 9 and Figure 8). Thus, this invention
provides the methods described above wherein an effective amount of another
anti-
3o infective agent is co-administered with the substrate prodrug of this
invention. In
one aspect, the second or third agent is the drug to which the pathogen had
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
previously developed resistance. The additional agent can be administered
concurrently or subsequenct to administration of the substrate prodrug.
This invention further provides prodrugs that are selectively converted by an
activating enzyme produced or expressed by an infectious agent or pathogen as
compared to the uninfected cell, e.g., an animal cell, a mammalian cell, or a
human
cell. Applicant has discovered several preferential prodrugs for the practice
of this
invention. The structures and synthetic methods for these compounds are
provided in
Materials and Methods, below.
As used herein, the term "contacting" includes in vitro, ex vivo and in vivo
to administration of prodrug. When done in vivo, the prodrug is administered
to a
subject in an effective amount. As used herein, the term "subject" is intended
to
include any appropriate animal model, e.g.; mouse, rat, rabbit, simian. It
also
includes administration to humans patients.
Another aspect of this invention is a method for treating a subject infected
with
a pathogen by administering to the subject a therapeutically effective amount
of a
prodrug that is selectively converted to a toxin in a cell by an activating
enzyme as
defined herein. The enzyme is not necessarily overexpressed. In a father
aspect, an
effective amount of at least one additional therapeutic agent is co-
administered
concurrently, previously or subsequently to administration of the substrate
prodrug.
2o When the prodrug is administered to a subject such as a mouse, a rat or a
human patient, the agent can be added to a pharmaceutically acceptable carrier
and
systemically or topically administered to the subject.
Administration in vivo can be effected in one dose, continuously or
intermittently throughout the course of treatment. Methods of determining the
most
effective means and dosage of administration are well known to those of skill
in the
art and will vary with the composition used for therapy, the purpose of the
therapy,
the target cell being treated, and the subject being treated. Single or
multiple
administrations can be carried out with the dose level and pattern being
selected by
the treating physician. Suitable dosage formulations and methods of
administering
3o the agents can be found below.
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
The pharmaceutical compositions can be administered orally, intranasally,
parenterally or by inhalation therapy, and may take the form of tablets,
lozenges,
granules, capsules, pills, ampoules, suppositories or aerosol form. They may
also
take the form of suspensions, solutions and emulsions of the active ingredient
in
aqueous or nonaqueous diluents, syrups, granulates or powders. In addition to
a
compound of the present invention, the pharmaceutical compositions can also
contain
other pharmaceutically active compounds or a plurality of compounds of the
invention.
More particularly, a compound of the formula of the present invention also
to referred to herein as the active ingredient, may be administered for
therapy by any
suitable route including oral, rectal, nasal, topical (including transdermal,
aerosol,
buccal and sublingual), vaginal, parental (including subcutaneous,
intramuscular,
intravenous and intradermal) and pulmonary. It will also be appreciated that
the
preferred route will vary with the condition and age of the recipient, and the
disease
being treated.
In general, a suitable dose for each of the above-named compounds, is in the
range of about 1 to about 100 mg per kilogram body weight of the recipient per
day,
preferably in the range of about 1 to about 50 mg per kilogram body weight per
day
and most preferably in the range of about 1 to about 25 mg per kilogram body
weight
2o per day. Unless otherwise indicated, all weights of active ingredient are
calculated as
the parent compound of the formula of the present invention for salts or
esters
thereof, the weights would be increased proportionately. The desired dose is
preferably presented as two, three, four, five, six or more sub-doses
administered at
appropriate intervals throughout the day. These sub-doses may be administered
in
unit dosage forms, for example, containing about 1 to about 100 mg, preferably
about
1 to above about 25 mg, and most preferably about 5 to above about 25 mg of
active
ingredient per unit dosage form. It will be appreciated that appropriate
dosages of
the compounds and compositions of the invention may depend on the type and
severity and stage of the disease and can vary from patient to patient.
Determining
3o the optimal dosage will generally involve the balancing of the level of
therapeutic
11
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
benefit against any risk or deleterious side effects of the treatments of the
present
invention.
Ideally, the prodrug should be administered to achieve peak concentrations of
the active compound at sites of disease. This may be achieved, for example, by
the
intravenous injection of the prodrug, optionally in saline, or orally
administered, for
example, as a tablet, capsule or syrup containing the active ingredient.
Desirable
blood levels of the prodrug may be maintained by a continuous infusion to
provide a
therapeutic amount of the active ingredient within disease tissue. The use of
operative combinations is contemplated to provide therapeutic combinations
to requiring a lower total dosage of each drug that may be required when each
individual therapeutic compound or drug is used alone, thereby reducing
adverse
effects.
It is a further aspect of this invention to combine the prodrugs described
herein with additional therapies as described above. For example, the prodrugs
described herein are preferentially combined with drugs that exert their toxic
effect
by a means other that that of the invention prodrugs.
While it is possible for the prodrug ingredient to be administered alone, it
is
preferable to present it as a pharmaceutical formulation comprising at least
one active
ingredient, as defined above, together with one or more pharmaceutically
acceptable
2o Garners therefor and optionally other therapeutic agents. Each carrier must
be
"acceptable" in the sense of being compatible with the other ingredients of
the
formulation and not injurious to the patient.
Formulations include those suitable for oral, recta, nasal, topical (including
transdermal, buccal and sublingual), vaginal, parenteral (including
subcutaneous,
intramuscular, intravenous and intradermal) and pulmonary administration. The
formulations may conveniently be presented in unit dosage form and may be
prepared by any methods well known in the art of pharmacy. Such methods
include
the step of bringing into association the active ingredient with the Garner
which
constitutes one or more accessory ingredients. In general, the formulations
are
prepared by uniformly and intimately bringing into association the active
ingredient
12
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
with liquid carriers or finely divided solid carriers or both, and then if
necessary
shaping the product.
Formulations of the present invention suitable for oral administration may be
presented as discrete units such as capsules, cachets or tablets, each
containing a
predetermined amount of the active ingredient; as a powder or granules; as a
solution
or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water
liquid
emulsion or a water-in-oil liquid emulsion. The active ingredient may also be
presented a bolus, electuary or paste.
A tablet may be made by compression or molding, optionally with one or
more accessory ingredients. Compressed tablets may be prepared by compressing
in
a suitable machine the active ingredient in a free-flowing form such as a
powder or
granules, optionally mixed with a binder (e.g., povidone, gelatin,
hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative,
disintegrant
(e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium
carboxymethyl cellulose) surface-active or dispersing agent. Molded tablets
may be
made by molding in a suitable machine a mixture of the powdered compound
moistened with an inert liquid diluent. The tablets may optionally be coated
or
scored and may be formulated so as to provide slow or controlled release of
the
active ingredient therein using, for example, hydroxypropylmethyl cellulose in
2o varying proportions to provide the desired release profile. Tablets may
optionally be
provided with an enteric coating, to provide release in parts of the gut other
than the
stomach.
Formulations suitable for topical administration in the mouth include
lozenges comprising the active ingredient in a flavored basis, usually sucrose
and
acacia or tragacanth; pastilles comprising the active ingredient in an inert
basis such
as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
Pharmaceutical compositions for topical administration according to the
present invention may be formulated as an ointment, cream, suspension, lotion,
3o powder, solution, past, gel, spray, aerosol or oil. Alternatively, a
formulation may
13
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
comprise a patch or a dressing such as a bandage or adhesive plaster
impregnated
with active ingredients and optionally one or more excipients or diluents.
For diseases of the eye or other external tissues, e.g., mouth and skin, the
formulations are preferably applied as a topical ointment or cream containing
the
active ingredient in an amount of, for example, about 0.075 to about 20% w/w,
preferably about 0.2 to about 25% w/w and most preferably about 0.5 to about
10%
w/w. When formulated in an ointment, the prodrug may be employed with either a
paraffinic or a water-miscible ointment base. Alternatively, the prodrug
ingredients
may be formulated in a cream with an oil-in-water cream base.
to If desired, the aqueous phase of the cream base may include, for example,
at
least about 30% w/w of a polyhydric alcohol, i.e., an alcohol having two or
more
hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol,
glycerol and polyethylene glycol and mixtures thereof. The topical
formulations may
desirably include a compound which enhances absorption or penetration of the
prodrug ingredient through the skin or other affected areas. Examples of such
dermal
penetration enhancers include dimethylsulfoxide and related analogues.
The oily phase of the emulsions of this invention may be constituted from
known ingredients in an known manner. While this phase may comprise merely an
emulsifier (otherwise known as an emulgent), it desirably comprises a mixture
of at
2o lease one emulsifier with a fat or an oil or with both a fat and an oil.
Preferably, a
hydrophilic emulsifier is included together with a lipophilic emulsifier which
acts as
a stabilizer. It is also preferred to include both an oil and a fat. Together,
the
emulsifiers) with or without stabilizers) make up the so-called emulsifying
wax,
and the wax together with the oil and/or fat make up the so-called emulsifying
ointment base which forms the oily dispersed phase of the cream formulations.
Emulgents and emulsion stabilizers suitable for use in the formulation of the
present invention include Tween 60, Span 80, cetostearyl alcohol, myristyl
alcohol,
glyceryl monostearate and sodium lauryl sulphate.
The choice of suitable oils or fats for the formulation is based on achieving
the desired cosmetic properties, since the solubility of the active compound
in most
oils likely to be used in pharmaceutical emulsion formulations is very low.
Thus the
14
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
cream should preferably be a non-greasy, non-staining and washable product
with
suitable consistency to avoid leakage from tubes or other containers. Straight
or
branched chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl
stearate,
propylene glycol diester of coconut fatty acids, isopropyl myristate, decyl
oleate,
isopropyl palinitate, butyl stearate, 2-ethylhexyl palmitate or a blend of
branched
chain esters known as Crodamol CAP may be used, the last three being preferred
esters. These may be used alone or in combination depending on the properties
required. Alternatively, high melting point lipids such as white soft paraffin
and/or
liquid paraffin or other mineral oils can be used.
to Formulations suitable for topical administration to the eye also include
eye
drops wherein the active ingredient is dissolved or suspended in a suitable
carrier,
especially an aqueous solvent for the prodrug ingredient. The prodrug
ingredient is
preferably present in such formulation in a concentration of about 0.5 to
about 20%,
advantageously about 0.5 to about 10% particularly about 1.5% w/w.
Formulations for rectal administration may be presented as a suppository with
a suitable base comprising, for example, cocoa butter or a salicylate.
Formulations suitable for vaginal administration may be presented as
suppositories, tampons, creams, gels, pastes, foams or spray formulations
containing
in addition to the prodrug ingredient, such Garners as are known in the art to
be
2o appropriate.
Formulations suitable for nasal administration, wherein the carrier is a
solid,
include a coarse powder having a particle size, for example, in the range of
about 20
to about 500 microns which is administered in the manner in which snuff is
taken,
i.e., by rapid inhalation through the nasal passage from a container of the
powder
held close up to the nose. Suitable formulations wherein the carrier is a
liquid for
administration as, for example, nasal spray, nasal drops, or by aerosol
administration
by nebulizer, include aqueous or oily solutions of the prodrug ingredient.
Formulations suitable for parenteral administration include aqueous and non
aqueous isotonic sterile injection solutions which may contain anti-oxidants,
buffers,
3o bacteriostats and solutes which render the formulation isotonic with the
blood of the
intended recipient; and aqueous and non-aqueous sterile suspensions which may
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
include suspending agents and thickening agents, and liposomes or other
microparticulate systems which are designed to target the compound to blood
components or one or more organs. The formulations may be presented in unit-
dose
or mufti-dose sealed containers, for example, ampoules and vials, and may be
stored
in a freeze-dried (lyophilized) condition requiring only the addition of the
sterile
liquid Garner, for example water for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared from
sterile
powders, granules and tablets of the kind previously described.
Preferred unit dosage formulations are those containing a daily dose or unit,
to daily subdose, as herein above-recited, or an appropriate fraction thereof,
of a
prodrug ingredient.
It should be understood that in addition to the ingredients particularly
mentioned above, the formulations of this invention may include other agents
conventional in the art having regard to the type of formulation in question,
for
example, those suitable of oral administration may include such further agents
as
sweeteners, thickeners and flavoring agents.
Prodrugs and compositions of the formula of the present invention may also
be presented for the use in the form of veterinary formulations, which may be
prepared, for example, by methods that are conventional in the art.
2o The agents and compositions of the present invention can be used in the
manufacture of medicaments and for the treatment of humans and other animals
by
administration in accordance with conventional procedures, such as an active
ingredient in pharmaceutical compositions.
Screening Assays
This invention further provides a method for screening for prodrugs that are
selectively converted to a toxin by an activating enzyme by providing cells
that express
an activating enzyme and contacting the cells with a candidate prodrug. At
least one
test cell expresses the pathogen's version of the enzyme (wild-type or
mutated) and
3o another test cell is a cell sample from the host organism which may, or may
not express
its own version of the enzyme. One then assays for conversion of the prodrug
into
16
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
toxic agents by the activating enzyme produced by the pathogen. As used
herein, the
test cells can be procaryotic or eucaryotic cells infected with the pathogen
or
alternatively, transformed to express the activating enzyme. For example, a
procaryotic E. coli which does not endogenously express the activating enzyme
TS is
a suitable host cell or target cell. Alternatively, the test cell can be an
infected cell
isolated from the subject, or a cultured cell infected with the pathogen. The
cell can
have a control counterpart (lacking the target enzyme), or in a separate
embodiment,
a counterpart genetically modified to differentially express the target
enzyme, or
enzymes (containing the appropriate species of target enzyme). More than one
to species of enzyme can be used to separately transduce separate host cells,
so that the
effect of the candidate drug on a target enzyme can be simultaneously compared
to
its effect on another enzyme or a corresponding enzyme from another species.
In another embodiment, a third target cell is used as a positive control
because it receives an effective amount of a compound, such as, for example,
the
compounds shown below, which have been shown to be potent prodrugs.
In another embodiment, transformed cell lines, such as ras-transformed NIH
3T3 cells (ATCC, 10801 University Blvd., Manassas, VA 20110-2209, U.S.A.) are
engineered to express variable and increasing quantities of the target enzyme
of
interest from cloned cDNA coding for the enzyme. Transfection is either
transient or
2o permanent using procedures well known in the art and described in Sambrook,
et al.,
supra. Suitable vectors for insertion of the cDNA are commercially available
from
Stratagene, La Jolla, CA and other vendors. The level of expression of enzyne
in
each transfected cell line can be monitored by immunoblot and enzyme assay in
cell
lysates, using monoclonal or polyclonal antibody previously raised against the
enzyme for immuno-detection. The amount of expression can be regulated by the
number of copies of the expression cassette introduced into the cell or by
varying
promoter usage. Enzymatic assays to detect the amount of expressed enzyme also
can be performed as reviewed by Cameras and Santi (1995), supra, or the
methods
described below.
3o The test cells can be grown in small mufti-well plates and is used to
detect the
biologic activity of test prodrugs. For the purposes of this invention, the
successful
17
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
candidate drug will block the growth or kill the pathogen but leave the
control cell
type unharmed.
The candidate prodrug can be directly added to the cell culture media or
previously conjugated to a ligand specific to a cell surface receptor and then
added to
the media. Methods of conjugation for cell specific delivery are well known in
the art,
see e.g., U.S. Patent Nos. 5,459,127; 5,264,618; and published patent
specification
WO 91/17424 (published November 14, 1991). The leaving group of the candidate
prodrug can be detectably labeled, e.g., with tritium. The target cell or the
culture
media is then assayed for the amount of label released from the candidate
prodrug.
l0 Alternatively, cellular uptake may be enhanced by packaging the prodrug
into
liposomes using the method described in Lasic, D.D. (1996) Nature 380:561-562
or
combined with cytofectins as described in Lewis, J.G. et al. (1996) Proc.
Natl. Acad.
Sci. USA 93:3176-3181.
It should be understood, although not always explicitly stated, each
embodiment can be fiu-ther modified by providing a separate target cell to act
as a
control by receiving an effective amount of a compound, such as, for example,
the
compounds shown below, which have been shown to be potent prodrugs.
Agents identified by this method are further provided herein.
In one embodiment, the assay of the effect of the prodrug is provided by
2o analysis of intracellular metabolites of the prodrug, as described in the
Materials and
Methods and Experimental Section below; the results of which are shown in
Figure
4. In this embodiment, the prodrug contains a detectable label that is
monitored
during conversion of the prodrug to toxic agent by the activating enzyme. In
an
alternative embodiment, the candidate substrate prodrug is detectably labeled,
e.g., e.g.,
fluorescent marker, or a radioisotope. In a further aspect, the detectable
label
comprises at least two or more variable isotopes of the same atom, e.g.,
bromine. In
this embodiment, one can assay for the modification of the prodrug into toxic
byproducts by mass spectrometry of the reaction products. One means to
accomplish
this assay is by use of mass spectrometry as described in more detail below,
the
3o results of which are shown in Figure 6.
18
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
Using the above screen, one also can pre-screen several prodrugs against
samples taken from a subject such as a human patient. One can use the screen
to
determine the most effective substrate prodrug and therapy for each pathogen
and
subj ect.
I. MATERIALS AND METHODS
A. Synthetic Methodology.
(E)-5-(2-Bromovinyl)-2'-deoxyuridine (BVdU), was prepared by the
method of Dyer et al. (Dyer et al. (1991) Nucleic Acid Chemistry: Improved and
l0 New Synthetic Procedures, Methods and Techniques, Townsend et al. (eds)
John
Wiley & Sons, Inc., New York, pp.79-83). This and commercial (Fisher/Acros) 5-
fluoro-2'-deoxyuridine (SFdU) were each dried in vacuo at 75 °C
adjacent to P2O5
immediately prior to use. Radial chromatography was performed on a
Chromatotron
instrument (Harnson Research, Palo Alto, CA), using Merck silica gel-60 with
fluorescent indicator as adsorbant. (E)-5-(2-Bromovinyl)-2'-deoxyuridine 5'-
monophosphate (BVdUMP), was prepared by standard chemical phosphorylation of
BVdU.
NMR 1H NMR spectra were recorded on a Varian Associates Gemini
spectrometer at 300 MHz, using hexadeuterio-dimethyl sulfoxide (CZH3)ZSO
2o solutions. Chemical shifts are reported relative to internal
tetramethylsilane
reference at d = 0.0 ppm. '3C NMR spectra were recorded at 75 MHz, with
chemical
shifts reported relative to internal pentadeuterio-dimethyl sulfoxide at d =
39.5 ppm.
3'P NMR spectra were recorded at 202 MHz on a Broker spectrometer, with
chemical
shifts reported relative to external 85%H20/15%H3P04, vol/vol, at d = 0.0 ppm.
NB1011 ((E)-5-(2-Bromovinyl)-2'-deoxy-5'-uridyl phenyl L-
alaninylphosphoramidate (BVdU-PA, "NB1011")) was prepared as follows. A
solution of BVdU (420 mg, 1.26 mmol) and imidazole (103 mg, 1.51 mmol) in 2 mL
of anhydrous DMF under argon was treated dropwise with phenyl L-
methoxyalaninyl phosphorochloridate (McGuigan et al. ( 1996) J. Med. Chem.
39:1748-1753(15 drops, 350 mg, 1.26 mmol) and the reaction mixture was stirred
at
23 °C under argon for 24 hours. By TLC on silica gel using
10%MeOH/90%CHZC12,
19
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
vol/vol, as eluent, the generation of the conversion product (Rf = 0.70) from
the
starting material (Rf= 0.53) had occurred but only to a partial extent (ca.
15%), so
additional imidazole (52 mg, 0.75 mmol) and phosphorochloridate reagent (8
drops,
175 mg, 0.63 mmol) was added and the mixture stirred at 23 °C under
argon another
24 hours. By TLC, the conversion had increased to ca. 30% extent. Subsequent
treatment with additional phosphorochloridate and imidazole did little to
promote the
progress of the reaction. The solution was reduced in volume to 0.75 mL by
rotary
evaporation in vacuo at ~ 40 °C, and then an equal volume of CHZC12 was
added and
the solution was applied directly to a dry 4 mm silica gel Chromatotron plate.
At this
to point, the subsequent separation was facilitated if the bulk of the
remaining DMF
was removed by placing the plate in a vacuum desiccator for 30 min. Radial
chromatography using 250 mL of CHzCIz (to elute residual reagents and DMF)
followed by 10%MeOH/90%CHzCl2, vol/vol, (to elute the product and then the
starting material) gave 144 mg (20%) of the conversion product and 294 mg of
the
starting material, for a 67% yield of conversion product based on unrecovered
starting material. If the presence of contaminating imidazole (d = 7.65 and
7.01) or
DMF (d = 7.95, 2.89, and 2.73) was detected by'H NMR, an additional radial
chromatographic purification was performed. In this way, 3 with a purity of
=98%
by TLC and 1H NMR was obtained as a nearly equimolar mixture of phosphorus
2o center-based diastereomers, in oil/gum or foam-powder form: 'H NMR
((CZH3)ZSO)
d = 11.4 (bs, exchanges with ZH20, 1, N3H), 8.28 (pseudo-t, 1, H6), 7.35
(pseudo-t,
2, o-Ph), 7.31 (d, l, vinyl'H), 7.20 (pseudo-t, 3, m- and p-Ph), 6.89 (d, 1,
vinyl 2H),
6.19 (t, l, Hl'), 6.08 (t, exchanges with ZH20, 1, alaninyl NH), 5.45 (bs,
exchanges
with zHzO, 1, 03'H), 4.32 (m, 1, H3'), 4.22 (m, 2, 5'CH2), 3.97 (m, 1, H4'),
3.86 (t, 1,
alaninyl CH), 3.58 (two s, 3, COzMe), 2.15 (m, 2, 2'CH2), 1.23 (pseudo-t, 3,
alaninyl
CH3). Jvinyl CH-vinyl CH = 13.5, JHl'-H2' ~ 6.8, JH2'-H3' ~ 5, JH3'-H4' ~ 0,
Jalaninyl CH-alaninyl NH ~ 6 Hz. Spectral assignments were confirmed by 1H/1H
COSY 2D NMR analysis. '3C NMR ((CzH3)ZSO)) d = 173.7 and 173.6 (alaninyl
COZ), 162.1 and 161.6 (C2), 150.6, 150.5 (ipso-Ph), 149.2 (C4), 139.4 and
139.2
(C6), 129.8 and 129.6 (m-Ph), 124.7 (p-Ph), 120.3, 120.2 (o-Ph), 107.1 (vinyl
Cl),
87.5 (vinyl C2), 84.8 (C4'), 83.8 (C1'), 70.1 (C3'), 66.1 (C5'), 51.9
(alaninyl OMe),
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
49.7 (alaninyl a-H), 29.5 (C2'), 19.6 (alaninyl a-Me). 3JP-C4' = 7.8, 2JP-CS'
= 4.4,
2JP-ipso-Ph = 6.5 Hz. 31P NMR d = 3.99, 3.69. Low-resolution DCI (NH3) mass:
593/591 (MNH4+), 576/574 (MH+).
For convenience only, the generic structures of exemplar prodrugs useful in
the methods of this invention have been classified as Class I and Class II.
General Synthesis of Compounds of Class I
The L and D isomers of the compounds of Class I have the structure:
O OH O
R~ s 4 3NH R~ s \ N R~ s 4 3N
3
6 1 2 °r I 6 1 2 °r I ( 1 2
N O N O N OH
a
Q Q Q
to In the above formulae, R, (at the 5-position) is or contains a leaving
group
which is a chemical entity that has a molecular dimension and electrophilicity
compatible with extraction from the pyrimidine ring by the activating enzyme,
e.g.,
thymidylate synthase, and which upon release from the pyrimidine ring by the
enzyme,
has the ability to inhibit the proliferation of the agent or cell.
15 In the above formulae, Q can be a moiety such as a sugar, carbocylic or
acyclic
compound, a masked phosphate or phosphoramidate derivative containing a
chemical
entity selected from the group consisting of sugar groups, thio-sugar groups,
carbocyclic groups, and derivatives thereof. Examples of sugar groups include,
but are
not limted to, monosaccharide cyclic sugar groups such as those derived from
oxetanes
2o (4-membered ring sugars), furanoses (5-membered ring sugars), and pyranoses
(6-
membered ring sugars). Examples of fizranoses include threo-furanosyl (from
threose,
a four-carbon sugar); erythro-furanosyl (from erythrose, a four-carbon sugar);
ribo-
furanosyl (from ribose, a five-carbon sugar); ara-furanosyl (also often
referred to as
arabino-furanosyl; from arabinose, a five-carbon sugar); xylo-furanosyl (from
xylose,
25 a five-carbon sugar); and lyxo-furanosyl (from lyxose, a five-carbon
sugar).
Examples of sugar group derivatives include "deoxy", "keto", and "dehydro"
derivatives as well as substituted derivatives. Examples of thio sugar groups
include
21
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
the sulfur analogs of the above sugar groups, in which the ring oxygen has
been
replaced with a sulfur atom. Examples of carbocyclic groups include C4
carbocyclic
groups, CS carbocyclic groups, and C6 carbocyclic groups that may further have
one
or more subsituents, such as -OH groups.
In one embodiment, Q is a (3-D-ribofuranosyl group of the formula:
R~ O
R3 R2
wherein R, is attached to the furane at the 5' position and is selected from
the
group consisting of H, a masked phosphate or a phosphoramidate and derivatives
thereof, and wherein RZ and R3 are the same or different and are independently
-H or -
1 o OH.
In some embodiments, R, may contain an alkenyl group, i.e., (-CH=CH)n R4,
wherein n is 0 or is an integer from 1 to 10, and R4 is a halogen such as is I-
or Bf, CN-
or mercury; wherein RZ is H and R3 is -OH; wherein RZ is OH and R3 is H;
wherein RZ
and R3 are H; or wherein Rz and R3 are OH. In another aspect, R4 is or
contains a group
15 selected from the group consisting of H, a halogen, alkyl, alkene, alkyne,
hydroxy,
-O-alkyl, -O-aryl, O-heteroaryl, -S-alkyl, -S-aryl, a cyanide, cyanate and
thiocyanate
halovinyl group, a halomercuric group, -S-heteroaryl, -NH2, -NH-alkyl, -
N(alkyl)2,
-NHCHO, -NHOH, -NHO-alkyl, NHZCONHO-, and NHNH2. In these embodiments,
further aspects include: wherein RZ and R3 are H; wherein Rz is OH and R3 is
H; herein
2o RZ is H and R3 is OH; or wherein Rz and R3 are OH.
A preferred embodiment for the substituent in the R, position is one that
could
undergo an allylic interchange.
22
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
In a still further aspect, the candidate therapeutic agent is a compound of
the
formula:
O
H (CH=CH)"-CH2 O A
~N
O/ N
Q
wherein n is 0 or an integer from 1 to 10; wherein A is a phosphorous
derivative, or a compound of the formula:
O
i -N(CH2CH2C1)2
N H2
and wherein Q is as defined above.
Additionally, in a further aspect, the candidate therapeutic agent is a
compound of the formula:
to
i H2
C=O
N
R-(CH=CH)n (CH2)m O~ ~H
wherein R = 2'-deoxy-5-uridyl, m is 0 or 1, and n is an integer from 0 to 10.
Where appropriate, the compounds can be in any of their enantiomeric,
diasteriomeric, or stereoisomeric forms, including, for example, D- or L-
forms, and
can be in any stereochemical configuration, including, for example, a- or (3-
anomeric
form.
Synthesis of the above noted 5-substituted pyrimidine nucleosides and 5-
substituted pyrimidine nucleoside monophosphates can be accomplished by
methods
23
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
that are well-known in the art. For example, treatment of 5-chloromercuri-2'-
deoxyuridine with haloalkyl compounds, haloacetates or haloalkenes in the
presence
of LiZPdCl4 results in the formation, through an organopalladium intermediate,
of the
5-alkyl, 5-acetyl or 5-alkene derivative, respectively. Another example of C5-
modification of pyrimidine nucleosides and nucleotides is the formation of C5-
trans-
styryl derivatives by treatment of unprotected nucleotide with mercuric
acetate
followed by addition of styrene or ring-substituted styrenes in the presence
of
Li2PdCl4. Bigge et al. (1980) J. Am. Chem. Soc. 102(6):2033-2038. Pyrimidine
deoxyribonucleoside triphosphates were derivatized with mercury at the 5
position of
to the pyrimidine ring by treatment with mercuric acetate in acetate buffer at
50° for 3
hours. Dale et al. (1973) PNAS 70(8):238-2242. Such treatment would also be
expected to be effective for modification of monophosphates; alternatively, a
modified triphosphate could be converted enzymatically to a modified
monophosphate, for example, by controlled treatment with alkaline phosphatase
followed by purification of monophosphate. Other moieties, organic or
nonorganic,
with molecular properties similar to mercury but with preferred
pharmacological
properties could be substituted. For general methods for synthesis of
substituted
pyrimidines, for example, U.S. Patent Nos. 4,247,544; 4,267,171; and
4,948,882; and
Bergstrom et al. (1981) J. Org. Chem. 46(7):1432-1441. The above methods would
2o also be applicable to the synthesis of derivatives of 5-substituted
pyrimidine
nucleosides and nucleotides containing sugars other than ribose or 2'-
deoxyribose,
for example 2'-3'-dideoxyribose, arabinose, furanose, lyxose, pentose, hexose,
heptose, and pyranose. An example of a 5-position substituent is the halovinyl
group, e.g. E-5-(2-bromovinyl)-2'-deoxyuridylate. Barn et al. (1983) J. Biol.
Chem.
258(22):1367-13631 and Biochem. 22:1696-1703.
Alternatively, 5-bromodeoxyuridine, 5-iododeoxyuridine, and their
monophosphate derivatives are available commercially from Glen Research,
Sterling,
VA (LTSA), Sigma-Aldrich Corporation, St. Louis, MO (USA), Moravek
Biochemicals, Inc., Brea, CA (USA), ICN, Costa Mesa, CA (USA) and New England
3o Nuclear, Boston, MA (USA). Commercially-available 5-bromodeoxyuridine and 5-
iododeoxyuridine can be converted to their monophosphates either chemically or
24
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
enzymatically, though the action of a kinase enzyme using commercial available
reagents from Glen Research, Sterling, VA (USA) and ICN, Costa Mesa, CA
(LTSA).
These halogen derivatives could be combined with other substituents to create
novel
and more potent antimetabolites.
General Synthesis of Compounds of Class II
In this embodiment, the present invention involves four classes of compounds
activated by enzymes such as TS. Each class is defined by the structure of the
uricil
base, or modified uricil base present. These classes are ECTA compounds
wherein:
to I) the base is a furano-pyrimidinone derivative of uracil; II) the base is
6-fluoro
uracil; and III) the base is 4-hydrazone substituted uracil derivative, or IV)
the base is
uracil. The uracil or modified uracil derived base is used to synthesize
compounds
substituted with toxic leaving groups at the 5 position, attached by an
electron
conduit tether at this 5 position, and including an appropriate spacer moiety
between
is the electron conduit and the toxic leaving group. The ECTA compounds can be
unphosphorylated, 5' monophosphate, 5' phosphodiester, or 5' protected
("masked")
deoxyuridines or comparable derivatives of alternative carbohydrate moieties,
as
described below. Protected 5-substituted deoxyuridine monophosphate
derivatives
are those in which the phosphate moiety has been blocked through the
attachment of
20 suitable chemical protecting groups. Protection of 5-substituted
deoxyuridine
monophosphate derivatives can improve solubility, facilitate cellular
penetration,
facilitate passage across the blood-brain barrier, and prevent action of
cellular or
extracellular phosphatases, which might otherwise result in loss of the
phosphate
group. In another embodiment, 5-substituted uracil or uridine derivatives are
25 administered to cells containing nucleoside kinase activity, wherein the S-
substituted
uracil/uridine derivative is converted to a 5-substituted uridine
monophosphate
derivative. Uridine derivatives may also be modified to increase their
solubility, cell
penetration, and/or ability to cross the blood-brain barrier.
Action of thymidylate synthase upon 5-substituted uridine monophosphate
30 derivatives can release the substituent attached to the 5-position
("leaving group") of
the pyrimidine ring. The released substituent is then capable, either
inherently or
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
following reaction with another cellular component, of acting as a toxin or an
inhibitor of cellular proliferation.
In one embodiment, the L and D isomers of the compounds of this invention
are selected from the group consisting of the compounds having the structures
shown
below:
or II. or III.
HZN
R O NH
O
N - N N -
O ~ O ~ R~ O R,
N ~N -N
O O F '/O
or
IV .
N
R,
N
O
In the above formulae, R' has the formula:
to n ~ m
In the above formulae, RZ is or contains a divalent electron conduit moiety.
In
one embodiment, Rz is or contains a mono- or polyunsaturated electron conduit
acting
to conduct electrons away from the pyrimidine ring and toward the leaving
group R'
15 with the proviso that in compounds of class I, n can be zero. In one
embodiment, RZ
is selected from the group consisting of an unsaturated hydrocarbyl group; an
aromatic
hydrocarbyl group comprising one or more unsaturated hydrocarbyl groups; and,
a
heteroaromatic group comprising one or more unsaturated hydrocarbyl groups.
26
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
In one embodiment, RZ is an unsaturated hydrocarbyl group having a structure
selected from the group consisting of
In one embodiment, RZ and R3, taken together form a structure selected from
the
group consisting of
In one embodiment, RZ is an aromatic hydrocarbyl group having a structure
selected from the group consisting of
and
~o
In an alternative embodiment, RZ is a heteroaromatic group having a structure
selected from the group consisting of:
~~~ I ~ i a~
wherein J is a heteroatom, such as -O-, -S-, or -Se-, or a heteroatom group,
such
as -NH- or -NR"~'-, where R"~'I' is a linear or branched alkyl having 1 to 10
carbon
atoms or a cycloalkyl group having 3 to 10 carbon atoms.
In the above formulae, R3 is a divalent spacer moiety, also referred to as a
spacer unit. In one embodiment, R3 is a divalent spacer moiety having a
structure
selected from the group consisting of
2~
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
R3
CH20 CH2S ~ N=N
O
CH20-C CH2S C ~ CH20 C
S
CH2S C
-CH2 ~ ~ -CHRS- ~ ~ -C(R5)2
-O- ~ ~ -S ~ ~ NH ~ and ~ NR5-
wherein RS is the same or different and is independently a linear or branched
alkyl group having from 1 to 10 carbon atoms, or a cycloalkyl group having
from 3
to 10 carbon atoms or RS is a halogen (F, Cl, Br, I).
In one embodiment, R3 is a divalent spacer moiety having a structure selected
from the group consisting of
-O ~ ~ S ~ ~ NH ~ and ~ NR5-
to
In one embodiment, R3 is a divalent spacer moiety having a structure selected
from the group consisting of:
28
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
R3
CH20- CH2S ~ N=N
O O
CH S ~~ CH20 C
CH20-C 2
S
CH2S C
In the above formula, n is an integer from 0 to 10 and, m is 0 or 1. In one
embodiment, n is 0 or an integer from 0 to 10 and, m is 1. In one embodiment,
n is 0
and m is 0. In one embodiment, when R' is -H, then n is not zero. In one
embodiment, when R' is -H, then m is not zero. In one embodiment, when R' is -
H,
then n is not zero and m is not zero. In one embodiment, when R' is -H, then
R4 is
not a halogen (i.e., -F, -Cl, -Br, -I). In one embodiment, when R' is -H, and
m is
zero, then R4 is not a halogen (i.e., -F, -Cl, -Br, -I). In one embodiment,
when R' is -
H, and m is zero and n is zero, then R4 is not a halogen (i.e., -F, -Cl, -Br, -
I).
In the above formula, R4 is a toxophore moiety. As used herein, the term
"toxophore" shall mean a moiety which is or contains a leaving group which is
a
chemical entity that has a molecular dimension and electrophilicity compatible
with
extraction from the pyrimidine ring by thymidylate synthase, and which upon
release
from the pyrimidine ring by thymidylate synthase, has the ability to inhibit
the
proliferation of the cell or kill the cell.
29
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
In one embodiment, the toxophore is or contains a leaving group that is
activated or released by an intracellular enzyme overexpressed in the cell. In
one
embodiment, R4 is or contains a group having a structure selected from the
group
consisting of
x
s
R O R80 C02R9
N
Rio
X
~O
O O
O Rio
X
Z P N Z F N
N NH2
X
O
-O-NH-C-NH2
CH2
C=O
NH OH
Z-CH2-CH-CH-CH=CH-(CH2)~2CH3
O
Z-CF2-CH2-CHF-C-OH
O
Z-CF2-CHF-CH2-C-OH
Io
O
Z-CF2-CH2-C-OH
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
j H3 O
Z-CF2-CH C-OH
Y
Z-CF2 C-Y
Y
Z-CF2-CH2-CH2-N02
and
Z ~ ~ N02
wherein X is -Cl, -Br, -I, or other potent leaving group (including, but not
limited to, -CN, -OCN, and -SCN); Y is the same or different, and is
independently -
H or -F; and Z is the same or different and is independently -O- or -S-, Rg
and R9 are
lower alkyls, and R'° is H or CH3.
to In one embodiment, R4 is or contains a chemical entity selected from the
group
consisting of -Br, -I, -O-alkyl, -O-aryl, O-heteroaryl, -S-alkyl, -S-aryl, -S-
heteroaryl,
-CN, -OCN, -SCN, -NH2, -NH-alkyl, -N(alkyl)2, -NHCHO, -NHOH, -NHO-alkyl,
NHZCONHO-, NHNH2, -N3, and a derivative of cis-platin, such as:
O=C C=O
O~ ,O
Pt
i ~
H3N ~ H
31
CA 02379834 2002-O1-21
WO 01/07087 PCTlLJS00/19844
In the above formulae, Q is or contains a group which supports functional
binding of the prodrug to the enzyme, e.g., TS or TK. In one embodiment, Q is
selected from the group consisting of
R~ ~ R~ O S ~ R? O
Rs Rs Rs Rs
R~ CH2 O ~ R7 O O
and
Rs Rs Rs
wherein R6 is the same or different and is independently -H, F, -OH, -
OC(=O)CH3, or other protected hydroxyl group (including, but not limited to,
benzoyl,
-COC6H5, and toluoyl, -COC6H4CH3); and, R' , attached at the 5' position of Q,
is
hydrogen , a phosphate group, a phosphodiester group, a phosphoramidate group,
or
other phosphorus containing group.
to In one embodiment, R' is a phosphoramidate group derived from an amino
acid, including, for example, the twenty naturally occurring amino acids. In
one
embodiment, R' is a phosphoramidate group derived from alanine. In one
embodiment, R' is or contains a group having the structure:
O
o-
NH
I
,.CH
CH~3 ~COOCH3
15 The above group, and methods for its preparation, are described in
McGuigan et al. (1993) J. Med. Chem. 36:1048-1052 and McGuigan et al. (1996)
J.
Med. Chem. 39:1748-1753.
32
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
In one embodiment, R' is a phosphoramidate group derived from tryptophan.
In one embodiment, R' is or contains a group having the structure:
O
HO-PI
NH NH
;CH
\COOCH3
The above group, and methods for its preparation, are described in
Abraham et al., (1996) J. Med. Chem. 39:4569-4575.
In one embodiment, R' is a phosphate group. In one embodiment, R' is or
contains a group having a structure selected from the group consisting of
O O
O g~ O
O-i- ~ O-i-
O O and \ O O
J
-O ~s
The first of the two above groups, and methods for its preparation, are
to described in Freed et al. (1989) Biochem. Pharmacol. 38:3193-3198; Sastry
et al.
(1992) Mol. Pharmacol 41:441-445; Farquhar et al. (1994) J. Med. Chem. 37:3902-
3909; and Farquhar et al. (1995) J. Med. Chem. 38:448-495. The second of the
two
above groups, and methods for its preparation, are described in Valette et al.
(1996) J.
Med. Chem. 39:1981; and Benzaria et al. (1996) J. Med. Chem. 39:4958.
15 In one embodiment, R' is or contains a group having a structure selected
from
the group consisting of (where R is an aromatic substituent):
O
II off
P O
and O O-IP
(CH2)~~CH3 OH
33
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
The first of the two above groups, and methods for its preparation, are
described in Meier et al. (1997) Bioorg. Med. Chem. Lett. 7:1577; Meier et al.
(1997)
Bioorg. Med. Chem. Lett 7:99; and Meier et al. (1997) International Antiviral
News.
5:183. The second of the two above groups, and methods for its preparation,
are
described in Hostetler et al. (1997) Biochem. Pharmcol. 53:1815; and Hostetler
et al.,
published International Patent Application No. WO 96/40088 (1996).
In one embodiment, the R' forms a cyclic group within Q. One such
embodiment, and a method for its preparation, is shown below (where DMTr is
4,4'-dimethoxytrityl, Boc is t-butyloxycarbonyl, DCC is 1,3-
dicyclohexylcarbodiimide,
to and 4-DMAP is 4-dimethylaminopyridine):
D
DMTr-O O ~ I
DMTr-CI Boc-L-alanine
DCC, cat. 4-DMAP
OH
O
O
o-P~ -o
1. NCI PhOP(O)CI2
---~ N H
2. Base Imidazole
O
C
In one embodiment, the compound may be in any enantiomeric,
diasteriomeric, or stereoisomeric form, including, D-form, L-form, a-anomeric
form,
and (3-anomeric form.
In one embodiment, the compound may be in a salt form, or in a protected or
prodrug
form, or a combination thereof, for example, as a salt, an ether, or an ester.
34
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
In a separate embodiment, the above structures are further modified to
possess thiophosphodiaziridine instead of phosphodiaziridine groups, using the
methods described below.
Synthesis of the above noted 5-substituted pyrimidine derivatives can be
accomplished by methods that are well-known in the art, and as described
above.
Alternatively, 5-bromodeoxyuridine, 5-iododeoxyuridine, and their
monophosphate derivatives are available commercially from Glen Research,
Sterling, VA (USA), Sigma-Aldrich Corporation, St. Louis, MO (USA), Moravek
Biochemicals, Inc., Brea, CA (LTSA), ICN, Costa Mesa, CA (USA) and New
to England Nuclear, Boston, MA (USA). Commercially-available 5-
bromodeoxyuridine and 5-iododeoxyuridine can be converted to their
monophosphates either chemically or enzymatically, though the action of a
kinase
enzyme using commercial available reagents from Glen Research, Sterling, VA
(USA) and ICN, Costa Mesa, CA (USA). These halogen derivatives could be
combined with other substituents to create novel and more potent
antimetabolites.
The structures at the 5-position of uracil are referred to as the tethers
because
they connect the proposed leaving group (toxophore) to the heterocycle. Upon
activation of the heterocycle by reaction with a Cys residue in the active
site of
human TS, a negative charge is conducted from the 6-position of uracil into
the
tether. This mechanism has been described for the 5'-monophosphorylated
versions
of (L~-5-(bromovinyl)-2'-deoxyuridine (BVDU) by Barr et al. (1983)
Biochemistry
22(7):1696-1703 and of (L~-5-(3,3,3-trifluoro-1-propenyl)-2'-deoxyuridine
(TFPe-
dUrd) by Wataya et al. (1979) J. Med. Chem. 22:339-340; Santi (1980) J. Med.
Chem. 23:103-111; and Bergstrom et al. (1984) J. Med. Chem. 27:279-284.
The tether "spacer" between the toxin and dNMP must be unsaturated so that
it can conduct the toxin-labilizing negative charge supplied by the TS-
Cysteine-
sulfhydryl attack. Of the many unsaturated organic functionalities available
for this
purpose, the vinyl, allyl, and propargyl units are simple, small, and readily
accessible
synthetically. The vinyl and allyl units have the advantage that they can be
prepared
in either of two non-interconvertible geometric isomeric forms. Thus, they can
be
used as "probes" of prodrug accommodation by the TS active site. On the other
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
hand, the propargyl unit has the advantage of being cylindrically symmetrical,
so that
TS-catalyzed toxin release from this type of tether does not depend upon its
orientation with respect to dUMP's uracil ring, as is the case with the vinyl
and allyl
molecules.
Two distinct approaches have been taken to design several of the nucleotide-
based prodrugs of this invention. One is based on the structure of BVDU
monophosphate and features a leaving group/toxin directly attached to the
terminus
of a (poly)vinyl substituent at CS of dUMP. This is the vinyl tether approach.
The
other is based on the structure of TFPe-dUMP and is similar to the first but
has a
1o methylene unit separating the leaving group/toxin and the unsaturated unit
and thus
contains an allyl or propargyl unit. This is the allyl tether approach.
The mechanism of activation of a propargyl version of the allyl tether
approach has a precedent in the interaction of both 5-ethynyl-2'-deoxyuridine
S'-
monophosphate (EdUMP) and 5-(3-hydroxy-1-propynyl)-2'deoxyuridine 5'-
15 monophosphate (HOPdUMP) with TS (Barr et al. (1981) J. Med. Chem. 24:1385-
1388 and Barr et al. (1983) supra.) EdUMP is a potent inhibitor of TS (Ki =
0.1
~M), and likely forms an allene-based species at the active site. HOPdUMP (Ki
=
3.0 p,M) shows unusual inhibition kinetics, which might be due to formation of
a
cumulene-based species at the active site.
20 5-Alkylidenated 5,6-dihydrouracils similar in structure to the intermediate
common to both the vinyl and allyl tether approach mechanisms have been
synthesized recently (Anglada et al. (1996) J. Heterocycl. Chem. 33:1259-
1270).
These were shown to be highly electrophilic. Their ready reaction with ethanol
to
generate 5-(ethoxymethyl)uracils is a precedent for the water addition that
25 regenerates catalytically competent TS. Even more recently, the existence
of the
long-elusive C5 methylene intermediate produced by TS was demonstrated by
trapping studies (Barrett et al. (1998) J. Am. Chem. Soc. 120:449-450).
Synthesis of ECTA compounds with propargyl tethers. The synthesis of
propargylic and allylic alcohol-equipped 2'-deoxyuridines is straightforward.
Many
30 of these and their close derivatives are reported in the literature, and
some have even
been studied in connection with TS. For example, 5-alkynyl-dUMPs including the
5-
36
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
(3-methoxy-1-propynyl) and 5-(3-hydroxy-1-propynyl) ones have been examined as
TS inhibitors (Barn et al. (1981) J. Med. Chem. 24:1385-1388) and some ofthese
have been shown to become incorporated into the DNA of TS-deficient cancer
cells
(Balzarini et al. (1985) FEBS Lett 373(1):41-4).
Both 5-mercuri- (Ruth et al. (1978) J. Org. Chem. 43:2870-2876) and 5-
iodouridines (Robins et al. (1981) Tetrahedron Lett 22:421-424) readily
condense
with alkenes and alkynes in the presence of a palladium catalyst to afford CS
tether-
equipped uridines. The latter route is the more often employed (Robins et al.
(1982)
Can. J. Chem. 60:554-557; Asakura (1988) Tetrahedron Lett. 29:2855-2858; and
1o Asakura (1990) J. Org. Chem. 55:4928-4933). High-yielding condensations of
protected 5-iodo-2'-deoxyuridines with t-butyidimethylsilyl propargyl ether
(Graham
et al. (1998) J. Chem. Soc. Perk. Trans. 1:1131-1138 and De Clercq et al.
(1983) J.
Med. Chem. 26:661-666), methyl propargyl ether (Tolstikov et al. (1997)
Nucleosides Nucleotides 16:215-225) and even propargyl alcohol itself
(Chaudhuri et
15 al. (1995) J. Am. Chem. Soc. 117:10434-10442 and Goodwin et al. (1993)
Tetrahedron Lett. 34:5549-5552) have been achieved. The 3-hydroxy-I-propynyl
substituent introduced by the latter reaction can also be accessed by DIBAL-H
reduction of a methacrylate group (Cho et al. (1994) Tetrahedron Lett. 25:1149-
1152), itself arising from the same Heck reaction used in the synthesis of
BVDU.
2o These palladium-catalyzed reactions are so versatile that they can used to
condense
very long and elaborately-functionalized propargyl-based tethers to 5-iodo-2'-
deoxyuridines (Livak et al. (1992) Nucleic Acids Res. 20:4831-4837 and Hobbs
(1989) J. Org. Chem. 54:3420-3422). (Z)-Allyl-based tethers are generated by
the
partial hydrogenation of a propargylic precursor over Undiar catalyst (Robins
(1983)
25 J. Org. Chem 5(11):3546-3548 and Barr (1983) J. Biol. Chem. 258(22):13627-
13631
and Biochem. 22:1696-1703) whereas the (E)-allyl-based ones are best prepared
by
Heck coupling of an (E)-tributylstannylated ethylene (Crisp (1989) Synth.
Commun.
19:2117-2123).
Closely following the literature procedures, a t-butyldimethylsilyl propargyl
30 ether-equipped 3', 5'-di-O-protected 2'-deoxyuridine (Graham et al. (1998)
supra;
De Clercq et al. (1983) J. Med. Chem. 26:661-666) is prepared and a portion of
it,
37
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
converted to the corresponding (Z)-allyl ether, (Barr et al. (1983) supra) is
reduced.
Because the TBAF-mediated removal of a TBDMS group generates an oxyanion that
can be functionalized in situ, these TBDMS-protected propargyl- and (Z)-
allytic-
tethered nucleosides will serve as convenient precursors to some of the
toxophore-
equipped targets. For the (E)-allyl alcohol equipped nucleoside, the known O-
tetrahydropyranyl ether derivative is prepared by the literature Heck coupling
of an
(E)-tributylstannylated ethylene (Crisp (1989) supra).
=C-CH2-OH
~ropargylic,
E)-allylic, or
Z)-a11y1ic
Using a two step literature protocol (Phelps et al. (1980) J. Med. Chem.
23:1229-1232; and Hsiao and Bardos (1981) J. Med. Chem. 24:887-889), the
propargylic and (E) and (Z)-allylic alcohols are converted to their
corresponding bis-
aziridinyl phosphoramidates or thiophosphoramidates so that TS processing of
the
5'-mononucleotide versions will release an active metabolite of the cytostatic
drugs
TEPA or ThioTEPA (Dirven et al. (1995) Cancer Res. 55:1701-1706),
respectively.
38
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
H O
N
O~ C= =C-C h~-O H
PG- propargylic,
(E)-allylic, or
(Z)-allylic
H O
D(S)
O C= =C-C I~-O-P-N
PC' ~ propargylic,
_ (E)-allylic, or
(Z)-allylic
PC
Bis-aziridin-1-yl-phosphinic acid 3-[2-deoxyuridin-5-yl]-prop-2-ynyl ester
(TEPA) was synthesized and analyzed by'H NMR to yield the following result:
'H NMR ((CD3)ZSO) complicated due to noise. Salient features: 8 8.28 (d, 1,
H6),
6.10 (pseudo-t, 1, H1'), 5.26 (m, exchanges with D20, l, 3'-OH), 5.13 (m,
exchanges
with D20, 1, 5'-OH), 4.81 (q or dd, 2, propargyl-CHz), 4.24 (m, 1, H3'), 3.57
(m, 2,
5'-CHZ), 2.15-2.0 (m, 8, aziridine-CHZ).
1o Bis-aziridin-1-yl-phosphinothioic acid 3-[2-deoxyuridin-5-yl]-prop-2-ynyl
ester (Thio TEPA) was also synthesized and analyzed by'H NMR to yield the
following result:
'H NMR ((CD3)ZSO) complicated due to noise. Salient features: 8 8.29 (d, 1,
H6),
6.10 (pseudo-t, 1, Hl'), 5.22 (m, exchanges with D20, l, 3'-OH), 5.10 (m,
exchanges
with D20, l, 5'-OH), 4.88 (q or dd, 2, propargyl-CHz), 4.31 (m, 1, H3'), 3.52
(m, 2,
5'-CHZ), 2.15-2.0 (m, 8, aziridine-CHZ).
Synthesis of furano-pyrimidinones. Synthesis of furano-pyrimidinones
begins with synthesis of a CS propargylic -alcohol-equipped 2'-deoxyuridine.
Furano-pyrmidinone compounds are then be formed from the O-tetrahydropyranyl
39
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
ether derivative described above. Synthesis proceeds by reaction of the second
carbon of the propargyl bond with the oxygen attached to the C4 position of
the
pyrimidine ring to yield a fluorescent furano-pyrimidinone which can be
readily
separated from the reaction mix. Such compounds provide an additional basis
for
synthesis of ECTA compounds through various combinations of specific electron
conduits, spacers and toxic leaving groups.
R~
O=
to The faro[2,3-d]pyrimidinone nucleosides were prepared by condensing 2',3'-
di-O-p-toluoyl or 2',3'-di-O-acetyl-5-iodo-2'-deoxyuridine with 1-
(tetrahydropyranyloxy)-2-propyne (Jones and Mann (1953) J. Am. Chem. Soc.
75:4048-4052) under conditions known to promote the formation of these
fluorescent
compounds (Barn et al. (1983) supra). Base-catalyzed removal of the
carbohydrate
15 protecting groups gave the 6-(tetrahydropyran-2-yloxymethyl)-substituted
bicyclic
nucleoside which was either subjected to standard acidic THP group hydrolysis
(TFA
in CHZC12) or was regioselectively S'-phosphoramidated by the same procedure
used
to prepare BVDU-PA and SFUdR-PA. After the phosphoramidation, the THP group
could be removed by acidic hydrolysis.
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
H O 0 O O 0
N N N
~c ca~amP O
RO O I ~ RO N ~ O ~ HO ~N ~ O
N (Ph3P}zPdCl=, '4
CuI, Et3N
RO
RO HO
R = 4-CH3C6FIaC(~) or CH3C(=O)
OOH O
~N- N-
Ph0-pP v \ ~ IOI O N- ~ OH
I !a'~ ~ Ph0-P N ~ O O
M~,.~N\H M~ I HO N /
~H
MeO2C HO M~ ~ HO
HO
3-(2-Deoxy-(3-D-ribofuranosyl)-6-(tetrahydropyran-2-
yloxymethyl)faro[2,3-d]pyrimidin-2(3H)-one. 'H NMR ((CD3)ZSO) 8 8.80 (s, 1,
H4), 6.74 (s, 1, HS), 6.16 (pseudo-t, l, H1'), 5.27 (d, exchanges with DzO, 1,
3'-OH),
5.12 (t, exchanges with D20, 1, 5'-OH), 4.72 (m, 1, THP-H2), 4.56 (q, 2,
CHZOTHP),
3.92 (m, 1, H4'), 3.64 (m, 2, 5'-CHZ), 2.40 (m, 1, H2'a), 2.03 (m, 1, H2'b),
1.68 and
1.50 (m, 8, THP). Low-resolution mass spectrum (DCI-NH3) on bis-TMS
derivative,
mlz 323 (B+TMS+H+), 511 (MH+), 583 (M+TMS+).
l0 3-(2-Deoxy-[i-D-ribofuranosyl)-6-(hydroxymethyl)faro[2,3-d]pyrimidin-
2(3H)-one. 'H NMR ((CD3)ZSO) 8 12.0 (bs, 1, OH), 8.24 (s, 1, H4), 6.53 (s, l,
HS),
5.51 (pseudo-t, 1, Hl'), 4.42 (m, 2, CHZOH). Low-resolution mass spectrum (DCI-
NH3), m/z 167 (B+2H+), 184 (B+NH4+).
1-[6-(Tetrahydropyran-2-yloxymethyl)faro[2,3-d]pyrimidin-2(3H)-on-3-yl]-
2-deoxy-(3-D-ribofuranos-S-yl phenyl methoxy-L-alaninylphosphoramidate. 1H
NMR ((CD3)2S0) complicated due to presence of diastereomers. Salient features:
8
8.62 and 8.59 (each s, each 1, H4), 7.4-7.1 (m, 5, Ph0), 6.61 and 6.60 (each
s, each l,
HS), 6.25 (m, 1, H1'), 4.56 (q, 2, propargyl-CH2), 3.56 and 3.54 (each s, each
3,
C02Me), 2.0 (m, 1, H2'b), 1.22 (m, 3, alaninyl-a-Me). Low-resolution mass
2o spectrum (DCI-NH3), m/z 167 (B+2H+), 184 (B+H++NH4+-THP).
1- [6-(Hydroxymethyl)furo [2,3-d] pyrimidin-2 (3H)-on-3-yl]-2-deoxy-(3-D-
ribofuranos-5-yl phenyl methoxy-L-alaninylphosphoramidate. 'H NMR (CDC13)
complicated due to presence of diastereomers. Salient features: 8 8.5 (s, 1,
H4), 7.4-
41
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
7.1 (m, 5, Ph0), 6.36 and 6.30 (each s, each 1, HS), 6.23 (m, 1, H1'), 3.67
and 3.65
(each s, each 3, COZMe), 2.69 (m, 1, H2'a), 2.10 (m, 1, H2'b), 1.35 (m, 3,
alaninyl-a-
Me). Low-resolution mass spectrum (DCI-NH3), m/z 525 (MPI+), 595 (MNH4+).
The 4-nitrophenyl ether derivative of 5-(3-hydroxy-1-propynyl)-2'
deoxyuridine was prepared according to a standard ether synthesis as shown
below.
02N ~ ~ OH
oZiPr
N=N
PriOzC
Ph P O2N
5-[3-(4-Nitrophenoxy)-1-propynyl]-2'-deoxyuridine. A solution of pre-
dried 5-(3-hydroxy-1-propynyl)-2'-deoxyuridine ("Nucleic Acid Compounds. 39.
Efficient Conversion of 5-Iodo to S-Alkynyl and Derived 5-Substituted Uracil
Bases
to and Nucleosides" (Barr et a1.(1983) supra) (565 mg, 2 mmol) in 40 mL of
anhydrous
THF under argon was treated with 4-nitrophenol (696 mg, 5 mmol),
triphenylphosphine (787 mg, 3 mmol), and diisopropyl azodicarboxylate (590 L,
3
mmol), and the reaction mixture heated at 60 °C until the solution was
clear, and then
1 h longer. The mixture was allowed to cool to 23 °C and then it was
evaporated
15 onto Si02 and purified by chromatography using MeOH/CHZC12 as eluent to
afford
107 mg (13%) of the desired ether product: mp 112-118 °C. 'H NMR
((CD3)ZSO) 8
11.65 (s, exchanges with DzO, 1, NH), 8.29 (s, 1, H6), 8.24 (d, J= 9.3 Hz, 2,
m-
ArH), 7.23 (d, J= 9.3 Hz, 2, o-ArH), 6.09 (pseudo-t, 1, H1'), 5.17 (s, 2,
propargyl-
CHZ), 4.22 (m, 1, H3'), 3.80 (m, 1, H4'), 3.59 (m, 2, 5'-CHZ), 2.13 (pseudo-t,
2, 2'-
20 CHZ). Low-resolution mass spectrum (DCI-NH3) on per-trimethylsilyated
material,
m/z 547 [M(TMS)ZH+], 565 [M(TMS)ZNHQ+], 620 [M(TMS)3H+].
TS ECTA compounds based on furano-pyrimidinones. Toxic R4 leaving
groups can be attached to the furan-2 methyl alcohol using methods similar to
those
employed to attach toxic leaving groups to the hydroxyl on the CS propargyl
uridine
25 compound, as explained with the synthesis of the TEPA and ThioTEPA
derivatives
described above. A variety of alternative toxic leaving groups, apparent to
one
42
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
skilled in the art, are envisioned. In addition, modifications to the length
and
composition of the RZ electron conduit component and of the composition of the
R3
spacer element are also envisioned.
TS ECTA compounds based on fi~rano-pyrimidinones can also consist of
variously modified "Q" moeities. Many 5-substituted 2'-deoxyuridines are not
substrates for human TK, but interestingly 5-(4-hydroxy-1-butynyl)-2'-
deoxyuridine
was found to be an exception (Barn et al. (1981) supra). Thus, it is expected
that
some of the toxophore equipped nucleosides will also possess propitious TK
substrate activity. Thus, the ECTA compounds can have a free 5' hydroxyl, a 5'
monophosphate, or a 5' phosphoramidate group attached to alternative
carbohydrate
groups. A novel method for synthesis of such phosphoramidate compounds is
accomplished by reacting a 2-deoxy 3'-hydroxy, 5'-hydroxy unprotected
nucleotide
with a phosphochloridate in the presence of an HCl scavenger. In a preferred
embodiment, the phosphochloridate comprises a phosphorus substituent which is
derived from an amino acid such as alanine. For example, the phosphochloridate
can
be phenyl-L-methoxyalanine phosphorochloridate.
C6 Fluoro uridine and C4 hydozone based compounds. The neutral thiol
addition to the pyrimidine CS-C6 double bond proceeds as an exothermic
reaction (3-
9 kcal per mol; see review by (Les et al (1998) Biomolecular Structure and
Dynamics
15(4):703-71 S) in the normal TS reaction with dUMP. Alternative substituents
to the
TS reactive hydrogen at the 6 position that can facilitate the formation of
the
sulfydryl bond with the enzyme, via an active human TS cysteine (homologous
with
cys-198 of L. casei), include fluorine. Such substituents at other positions
in the
pyrimidine ring can also facilitate the reaction between the substrate and TS.
For
instance, a 4-hydrazone substitution on the uracil (as described by Les et al.
(1998),
supra) facilitates formation of the thiol with TS. It is important that the
resulting
nucleotide-thiol (TS) intermediate rearranges in such a way as to release the
altered
nucleotide which can be accomplished passively via hydrolysis.
43
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
O=
The introduction of fluorine at the C6 position has not been previously
reported, but it can be synthesized by following the synthetic descriptions of
Krajewskas and Shugar (1982) Biochem. Pharmacol. 31(6):1097-102, who describe
the synthesis of a number of 6 substituted uracil and uridine analogues.
Chemistry facilitating substitutions at the C4 position of the pyrimidine base
are well known by those skilled in the art. Examples of literature
descriptions
include Wallis et al. (1999) Pharmaco. 54(1-2):83-89; Negishi et al. (1996)
Nucleic
Acids Symp. Ser. 35 (Twentythird Symposium on Nucleic Acids Chemistry) 137-
l0 138; Barbato et al. (1991) Nucleosides Nucleotides 10(4):853-66; Barbato et
al.
(1989) Nucleosides Nucleotides 8(4):515-528; and Holy et al. (1999) J. Med.
Chem.
42(12):2064-2086. These synthetic techniques also enable combinations of
substitutions, for instance at the C4 and C5 positions of the pyrimidine ring
(Pluta et
al. (1999) Boll. Chim. Farm. 138(1):30-33) or the C2 and C4 positions of the
15 pyrimidine ring (Zeid et al. (1999) Nucleosides Nucleotides 18(1):95-111).
H2N
NH
N
O
N
Q
44
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
In another embodiment of the invention, ECTA compounds are synthesized
by addition of alternative electron conduits, spacer moieties and toxic
leaving groups
to either the C6 fluoro-uridine base or the C4 hydrazone modified pyrimidine.
Methods described above for synthesis of 2'-deoxyuridine based ECTA compounds
can again be employed for synthesis of such molecules.
B. Derivatives of the Compounds of Class I and II
Salts, esters, and ethers of the above compounds disclosed herein are also
within the scope of this invention. Salts of the prodrugs of the present
invention may
be derived from inorganic or organic acids and bases. Examples of acids
include
hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, malefic,
phosphoric,
glycollic, lactic, salicyclic, succinic, toluene-p-sulfonic, tartaric, acetic,
citric,
methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-
sulfonic
and benzenesulfonic acids. Other acids, such as oxalic, while not in
themselves
pharmaceutically acceptable, can be employed in the preparation of salts
useful as
intermediates in obtaining the compounds of the invention and their
pharmaceutically
acceptable acid addition salts. Examples of bases include alkali metal (e.g.,
sodium)
hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and
compounds of formula NW4+, wherein W is C,~ alkyl.
2o Examples of salts include: acetate, adipate, alginate, aspartate, benzoate,
benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,
fumarate,
flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate,
hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate,
maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,
palmoate,
pectinate, persulfate, phenylproprionate, picrate, pivalate, propionate,
succinate,
tartrate, thiocyanate, tosylate and undecanoate. Other examples of salts
include
anions of the compounds of the present invention compounded with a suitable
cation
such as Na+, NH4+, and NW4+ (wherein W is a C,~ alkyl group).
For therapeutic use, salts of the compounds of the present invention will be
pharmaceutically acceptable. However, salts of acids and bases which are non-
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
pharmaceutically acceptable may also find use, for example, in the preparation
or
purification of a pharmaceutically acceptable compound.
Esters of the prodrugs or compounds identified by the method of this
invention include carboxylic acid esters (i.e., -O-C(=O)R) obtained by
esterification
of the 2'-, 3'- and/or 5'-hydroxy groups, in which R is selected from (1)
straight or
branched chain alkyl (for example, n-propyl, t-butyl, or n-butyl), alkoxyalkyl
(for
example, methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (for
example,
phenoxymethyl), aryl (for example, phenyl optionally substituted by, for
example,
halogen, C,~alkyl, or C,~alkoxy or amino); (2) sulfonate esters, such as
alkylsulfonyl
(for example, methanesulfonyl) or aralkylsulfonyl; (3) amino acid esters (for
example, L-valyl or L-isoleucyl); (4) phosphonate esters and (5) mono-, di- or
triphosphate esters. The phosphate esters may be further esterified by, for
example, a
C,-ZO alcohol or reactive derivative thereof, or by a 2,3-di-(C~24)acyl
glycerol. In such
esters, unless otherwise specified, any alkyl moiety present advantageously-
contains
from 1 to 18 carbon atoms, particularly from 1 to 6 carbon atoms, more
particularly
from 1 to 4 carbon atoms. Any cycloalkyl moiety present in such esters
advantageously contains from 3 to 6 carbon atoms. Any aryl moiety present in
such
esters advantageously comprises a phenyl group. Examples of lyxo-furanosyl
prodrug derivatives of the present invention include, for example, those with
2o chemically protected hydroxyl groups (e.g., with O-acetyl groups), such as
2'-O-
acetyl-lyxo-furanosyl; 3'-D-acetyl-lyxo-furanosyl; 5'-O-acetyl-lyxo-furanosyl;
2',3'-
di-O-acetyl-lyxo-furanosyl and 2',3',5'-tri-O-acetyl-lyxo-furanosyl.
Ethers of the compounds of the present invention include methyl, ethyl,
propyl, butyl, isobutyl, and sec-butyl ethers.
In a further embodiment, the substrate may not be chemically related to
pyrimidines or folates, but rather synthesized based upon known parameters of
rational
drug design. (See Dunn et al. (1996) J. Med. Chem. 39:4825).
Chemical assays for products, for example, where a reaction product is an
anti-metabolite of the bromovinyl-derivatives of dUMP, are described in the
3o Examples provided below or by (Barr et al. (1983) supra).
46
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
C. RT-PCR analysis of matched normal and tumor tissues. Transcript
levels of human thymidylate synthase in colon cancer tissues and matched
normal
colon tissues were quantified by using Reverse RT-PCR amplification.
Oligonucleotide primers for amplification of the human thymidylate synthase
and (3-
actin were designed as following: thymidylate synthase sense primer 5'-
GGGCAGATCCAACACATCC-3' (corresponding to bases 208-226 of thymidylate
synthase cDNA sequence, Genbank Accession No. X02308), antisense primer 5'-
GGTCAACTCCCTGTCCTGAA-3' (corresponding to bases 564-583), (3-actin sense
primer S'-GCCAACACAGTGCTGTCTG-3' (corresponding to bases 2643-2661 of
~3-actin gene sequence, Genbank Accession No. M10277) and antisense primer 5'-
CTCCTGCTTGCTGATCCAC-3' (corresponding to bases 2937-2955).
Human colon tumor tissues and matched normal tissues were obtained from
Cooperative Human Tissue Network (CHTN, Western Division, Cleveland, OH).
Total RNAs were isolated using Tri pure isolation reagent (obtained from
Boehringer
Mannheim Corp., Indianapolis, ll~, followed manufactureis protocol. To monitor
for possible DNA contamination, the primers for amplification of (3-actin were
designed to span the exon4/intron5/exon5 junction. Genomic DNA template leads
to
a 313 by (3-actin fragment, and cDNA template generates a 210 by product.
Reverse transcriptions were performed, using Superscript preamplification
system (Gibco/BRL, Gaithersburg, MD). 3 ~.g total RNA was applied in a volume
of
20 ~1 buffer to conduct reverse transcription reaction, followed manufacture's
protocol.
PCR reactions were performed in a volume of 96 ~1, containing 5~,1 of cDNA
mixture from reverse transcription reaction, 3 mM MgCl2 , 50 mM KCI, 20 mM
Tris
Cl, pH 8.4, 0.2 mM of each dNTP, 0.3 ~,M of thymidylate synthase sense and
antisense primers and 5 units of Tag DNA polymerase (obtained from Promega,
Madison, WI). The reaction mixtures were incubated at 94°C for 3 min,
followed by
9 cycles of 1 min incubation at 94°C , 1 min incubation at 58°C
, and then 1 min
incubation at 72°C. After 9 cycles, human (3-actin primers in 4 ~l were
added to
3o achieve a final concentration of 0.2 ~M, bringing the final reaction volume
to 100 ~1.
47
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
PCR reaction was continued to a total of 30, 32 or 34 cycles, followed by a 7
min
incubation at 72 °C.
~L of PCR products were resolved by electrophoresis in 2% agarose gel,
followed by staining with SYBR Gold nucleic acid gel stain (obtained from
5 Molecular probes, Eugene, OR). Result of quantification indicated that
amplification
of thymidylate synthase and ~3-actin was linear between cycles 30 and 34. The
DNA
bands corresponding to thymidylate synthase were quantified and normalized to
that
of ~i-actin by Molecular Dynamics Storm. The quantified expression levels were
expressed as values of ratio between TS and ~i-actin. This assay is also
useful for
to detecting pathogens in mammalian cells, as described in Nagata et al.
(1999) J.
Hepatol-30:965-969.
D. Cell lines and transfection. HT1080 cells were grown in PRMI1640
medium supplemented with 10% fetal calf serum, and transfected with GFP-TS
expression vector. 48 hours after, transfection cells were tripsinized and
replated in
culture medium containing 750 ~g/ml 6418. After selection with 6418 for two
weeks, surviving cells were sorted based upon fluorescence expression. One
clone
with higher fluorescence expression (named as TSH/HT1080) and one clone with
lower fluorescence expression (named as TSL/HT1080) were selected and expanded
2o into cells lines. The stable HT1080 cells transfected with pEGFP-C3 were
used as
control.
E. Construction of GFP-TS expression vector. A cDNA fragment
encoding conserved region of human thymidylate synthase (amino acids 23 to
313)
was obtained by PCR amplification using following primers: Sense primer, 5'-
CGGAAGCTTGAGCCGCGTCCGCCGCA-3' and antisense primer, 5'-
GAAGGTACCCTAAACAGCCATTTCCA-3'. The cDNA was cloned into HindIII
and KpnI sites of mammalian expression vector pEGFP-C3 ( Clontech
Laboratories.
Inc., Palo Alto, CA), in-frame with GFP sequence. The cDNA insert was
confirmed
3o by DNA sequencing.
48
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
F. Western Blot analysis. Human normal and cancer cells were grown in
RPMI 1640 medium supplemented with 10% fetal bovine serum. Cells were grown
until confluent in 100 mm culture dish and lysed in 0.5 ml of RIPA buffer ( 50
mM
Tris-HCI, pH 7.5, 150 mM NaCI, 0.5% Triton X-100, 0.1% SDS, 0.5% Deoxycholic
acid, sodium salt and protease inhibitors ). Protein concentrations were
determined
by using BCA-200 protein assay kit (obtained from Pierce, Rockford, IL). 1 S
pg of
total protein from each cell strain/line was resolved by 12% SDS-PAGE. The
separated proteins were transferred onto PVDF membrane, followed by immunoblot
with human thymidylate synthase monoclonal primary antibody (manufactured by
to NeoMarkers, Fremont, CA) and horseradish peroxidase linked sheep anti-mouse
Ig
secondary antibody (obtained from Amersham, England). The ECL plus kit
(Amersham) was used for detection of immunoreactivity. The bands corresponding
to thymidylate synthase were quantified and normalized to that of tubulin by
Molecular Dynamics Storm. The quantified expression levels were expressed as
values relative to that of cell strain CCD 18co.
G. TS Activity Assay by Tritium Release from dUMP 3H. Cells were
plated in 24 well plates to a density of 30,000 cells/plate and incubated for
16 hours
to allow adhesion to the plastic surface of the plate.
2o Immediately prior to the thymidylate synthase assay, the media was replaced
with RPMI+10% dialyzed fetal calf serum. 0.5 pCi of S-[ 3H]deoxyuridine was
added to each well, and plates were incubated for 60 minutes at 37 °C
without
additional CO2. [3H] release was measured by adsorbing 5-[3H]deoxyuridine to
activated charcoal (10% in 1 x PBS) for 5 minutes at room temperature. After
centrifugation for 5 minutes at 13,000 RPM, the amount of [3H] in the
supernatant
was determined by liquid scintillation counting.
H. Growth Inhibition Studies. Cells growing exponentially were
transferred to 384-well flat bottom tissue culture plates. All cell types were
plated at
3o a density of S00 cells per well in 25 ~L of complete medium (RPMI 1640 +
10%
fetal bovine serum + antibiotics/antimycotics). After 24 hours (day 0), 25~L
of
49
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
complete medium containing the experimental compounds over the dose range of
10
fi3 to 10 ~'° M were added in triplicate. Drug exposure time was 120
hours (day 5),
after which growth inhibition was assayed. Sp,L of the redox indicator,
alamarBlue,
was added to each well (10% v/v). After 4 hours incubation at 37 °C,
fluorescence
was monitored at 535 nm excitation and 595 nm emission.
Concentration vs. relative fluorescence units (RFL~ were plotted, and sigmoid
curves were fit using the Hill equation. ICS° , indicated by the
inflection point of the
curve, is the concentration at which growth is inhibited by 50%.
The same growth inhibition/cytotoxicity assays can be used to measure
to cytotoxicity of toxins released from ECTA compounds by activating enzymes
encoded by infectious agents as described in the patent. As noted above,
infectious
agents of this class include, but are not limited to, Mycobacterium sp.,
Chlamydia
sp., Rickettsia sp. And Pheumocystis sp. pathogenic Enterococcus sp.,
Moraxella sp.,
Haemophilis sp., and Staphylococcus sp. Colony formation assays can be used to
measure cytotoxicty of metabolized ECTA compounds on extracellular pathogenic
bacteria or other pathogens on plates or in liquid media (Miller, J.H. A Short
Course
in Bacterial Genetics: A Laboratory Manual and Hardbook for E. Coli and
Related
Bacteria, Cold Spring Harbor Press (1992)).
I. Tomudex Inhibition of NB1011 Cytotoxicity. MCF7-TDX were
transferred to a 384 well assay plate at S00 cells in 25 pL complete medium
per well.
After 24 hours (day 0), 25 pL complete medium containing a combination of
NB1011 in doubling serial dilutions from 1mM and tomudex at discrete
concentrations (0,1,10,100,1000 nM) were added in duplicate. Drug exposure
time
was 120 hours (day 5) after which growth inhibition was measured with
alamarBlue
as described above in Growth Inhibition Studies.
J. Enzyme Preparation. Cloned human thymidylate synthase plasmid
pBCHTS was subcloned into E. coli. BL21 (DE3)/pET-28a(+) (Novagen) using the
3o NdeI nSacI insertion site, in order to add an amino terminal His tag.
Enzyme was
expressed in E. coli. by induction with IPTG, and purified by affinity
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
chromatography on a Ni2+ His Bind metal chelation resin (Novagen). The column
Niz+ His Bind metal chelation column was washed with 20 mM Tris pH 7.9, 5 mM
imidazole, 0.5 M NaCI; thymidylate synthase activity was eluted with 20 mM
Tris
pH 7.9, 60 mM imidazole, 0.5 M NaCI.
K. Enzyme Assays and Kinetic Measurements. Thymidylate synthase
assays were done in 96 well Costar UV transparent plates in a reaction volume
of 200
~1, consisting of 40 mM Tris pH 7.5, 25 mM MgClz, 1 mM EDTA, 25 mM-
mercaptoethanol, 125 M dUMP, and 65 pM N5, N10-methylene tetrahydrofolate
to indicated. Tetrahydrofolate stock solutions were prepared by dissolving
tetrahydrofolic acid (Sigma) directly into 0.2 M Tris pH 7.5, 0.5 M-
mercaptoethanol;
stock solutions were stored at -80 °C. N5, N10-methylene
tetrahydrofolate was
prepared by adding 12 ~1 of 3.8% formaldehyde to 1 ml of a 0.65 mM solution of
tetrahydrofolate and incubating for 5 minutes at 37 °C. N5, N10-
methylene
15 tetrahydrofolate was kept on ice and used within 2 hours of preparation.
Conversion of BVdUMP to fluorescent products) by thymidylate synthase
was measured in 200 thymidylate synthase reactions containing 125 M BVdLJMP in
96 well Dynex Microfluor Black "U" bottom microtiter plates using an
excitation
wavelength of 340 nm and emission wavelength of 595 nm. Fluorescence was
2o measured with a Tecan Spectrafluor Plus fluorimeter.
Enzyme kinetic constants (Kn, and V",~X) were determined for the human
thymidylate synthase substrates dUMP and BVdUMP using the enzyme assay
conditions described above. The initial rates of the enzyme reactions was
determined
by measuring the increase in A3ao for the reaction with dUMP, and decrease in
A29a
25 for the reaction with BVdUMP. The catalytic efficiency of the enzyme (K~a~
was
calculated from the kinetic constants K", and V",~.
L. Liquid Chromatography/Mass Spectroscopy. Cells were washed three
times with PBS at room temperature, then subjected to freeze/thaw lysis in 5
ml PBS.
3o Cell extracts were centrifuged for 10 minutes at lOKRPM, then adsorbed to
Sep-Pak
C,8 and washed with 10 ml PBS. BVdLIMP was eluted with 1 ml distilled water.
51
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
LC/MS samples were analyzed by reverse phase chromatography on a C,8 column
using a linear gradient of 0.1 % formic acid-0.1 % formic acid/95%
acetonitrile. Mass
spectroscopy was done with a Micromass Quattro II triple quadropole
spectrometer.
M. Reversal of Resistance. The origin and characteristics of the human
breast cancer MCF7 TDX cell line have been previously described (Drake et al.
(1996) Biochem. Pharmcol. 51(10):1349-1355). Briefly, MCF-7 breast cancer
cells
were selected in vitro for resistance to Tomudex by continuous exposure to
stepwise
increases in TDX concentrations up to 2.0 ~M. A resistant subline was selected
for
resistance to NB 1011 by continuous exposure of the parental MCF7 TDX cell
line to
medium supplemented without TDX but with 50 pM NB1011, a concentration
approximately 16 times higher than the ICso for NB 1011 in the parental MCF7
TDX
cell line. After a dramatic initial cell killing effect, resistant colonies
emerged, and
vigorously growing monolayers were formed. TS protein level and ICSO for 5-FU,
TDX, and NB1011 were determined for the resultant MCF7 TDX/1011 cell line as
described in "Materials and Methods" by western blot and the alamarBlue
cytotoxicity assay, respectively.
II. EXPERIMENTAL
2o A. In vitro Reaction of BVdUMP with Human Thymidylate Synthase
1. The cell-free processing of BVdUMP by rHuTS generates fluorescent
product(s).
The cell-free processing of BVdUMP by L. casei TS has been shown to
create potentially reactive intermediates (Barn et al. (1983) supra). For this
reason it
has been thought that processing of BVdUMP by TS leads to irreversible
inactivation
of human TS (Balzarini (1987) Mol. Pharmacol. 32(3):410-6). The cell-based
experiments by DeClercq, Balzarini and colleagues (Balzarini (1987) supra;
Balzarini (1993) J. Biol. Chem. 268(a):6332-7; Balzarini (1995) FEBS Lett
373(1):41-4) support the concept that, once BVDU is converted to the
3o monophosphate in cells (e.g. via herpes virus thymidine kinase), then it
binds to and
inactivates the HUTS enzyme during processing. However, the actual reaction of
52
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
human TS with BVdUMP has never been adequately characterized. Santi and
colleagues (Ban et al. (1983) supra) utilized a bacterial TS for their work to
show
generation of product from the BVdUMP + TS reaction, and DeClercq and
colleagues utilized cells and cell lysates, not purified human TS (Balzarini
(1987)
supra); Balzarini (1993) supra; Balzarini (1995) supra).
Because of Applicant's interest in generating therapeutic substrates that can
be specifically activated by TS, the interaction of BVdUMP with purified
recombinant human TS (rHuTS) was revisited. When BVdUMP was incubated with
rHuTS in the standard reaction mixture, the reaction results in the formation
of
fluorescent products) (Figure 2). The time dependent increase in fluorescence
is
accompanied by a decrease in the initial BVdUMP concentration. The products)
produced have been partially characterized, and appear to be exocyclic
pyrimidine
nucleotide derivatives (see below).
This result is surprising because previous results supported the idea that TS
reaction with BVdLTMP should inactivate the human TS enzyme (Balzarini et al.
(1987), (1993) supra and Balzarini et al. (1995) supra). Because the reactions
described above were done in a cell-free system with purified components, it
remained possible that the intracellular milieu could provide components that
would
result in TS inactivation following conversion of NB 1 O 11 to the free
nucleotide
monophosphate inside the cell. This issue is addressed in more detail below.
2. Comparative reaction kinetics of dLTMP and BVdLTMP with rHuTS.
Previously reported work by Barr et al., utilizing the L. casei TS (Balzarini
(1995) supra; Balzarini (1987) supra; and Balzarini (1993) supra) using cells
and
cell lysates, leaves unclear whether the reaction of BVdUMP with human TS will
result in irreversible inactivation of the enzyme. To address this question,
the
kinetics of interaction of BVdUMP with rHuTS, in the presence or absence of
dUMP, were determined.
Competitive inhibition is most consistent with a reaction in which BVdUMP
does not inactivate the TS enzyme. To help further clarify this situation, an
extended
incubation of rHuTS with BVdUMP was done in order to measure any inactivation
53
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00119844
that may occur over a period of time longer than that in which the kinetics
were
performed (Figure 4).
These data show that even after a 20 hour incubation of rHuTS with
BVdLJMP, little or no enzymatic inactivation is apparent as measured by rate
of
conversion of THF DHP dUMP as substrate. This result is consistent with the
hope
for ability of overexpressed TS to convert BVdUMP into cytotoxic metabolites
in
cells, preferentially in cells which overexpress TS, and finally, without
inactivating
the enzyme.
l0 3. Characterization of BVdUMP reaction with TS: Cofactors and
Inhibitors
The best characterized reaction of TS is the conversion of dLTMP to dTMP.
This reaction involves the transfer of a methylene group from NS,N10-methylene
tetrahydrofolate (THF) to the C-5 position of dLTMP (Carreras CW (1995)
supra).
15 This reaction is dependent upon the cofactor (THF), and is inhibited by the
uridylate
mimic, SF-dUN>P, which, upon reaction with the enzyme, results in the
formation of
a stable complex and loss of enzymatic activity. A second well characterized
inhibitor of TS activity is Tomudex, which occupies the folate binding site of
the TS
homodimer, prevents the binding of THF, and blocks TS activity in the cell
(Drake et
20 al. (1996) Biochem. Pharmacol. 51(10):1349-1355; Touroutoglou and Pazdur
(1996)
Clin. Cancer Res. 2(2):227-243). As part of a preliminary effort to
characterize the
reaction of rHuTS with BVdUMP, the effects of SF-dUMP, Tomudex and cofactor
were compared on the reaction of the enzyme with dUMP and BVdUMP. These
experiments (Table 1) have shown that, similarly to the case of dITMP, SF-dUMP
can
25 prevent conversion of BVdUMP to fluorescent product(s). In addition,
Tomudex can
also prevent product formation from both dLTMP and BVdLTMP. However,
consistent with earlier reported results with L. casei TS (Barr et al. (1983)
supra),
THF is not required for the conversion of BVdUMP to fluorescent product(s). In
addition, the data shown in Table 1 also demonstrate that THF stimulates the
3o production of fluorescent products) in the BVdUMP reaction with rHuTS. This
result is not expected from the earlier data reporting that THF has no effect
on this
54
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
reaction (Barr et al. (1983) supra), and illustrates a potentially important
possibility
that cofactors, or cofactor agonists, like leucovorin , could modulate the
reaction of
BVdLTMP with human TS.
Analysis of the Michaelis-Menton kinetics of this reaction showed that
inhibition of BVdITMP by dUMP fits the expected form for competitive
inhibition,
consistent with both nucleotides behaving as substrates for rHuTS.
As shown in Table 2, infra, previously reported data with the L. casei TS
indicated that BVdUMP is 385 times less efficient a substrate as dUMP (Barr et
al.
(1983) supra and Santi (1980) supra). The experiments reported herein have
shown
to that this situation is quite different with the human enzyme. For rHuTS the
relative
catalytic efficiency of dUMP compared with BVdITMP is 60x. This represents a >
6.4 fold increase in catalytic efficiency as compared to endogenous substrate.
The
previous result with L. casei TS leads to the prediction that the efficiency
of
enzymatic reaction within the cell would be too low for NB 1011 to be an
effective
therapeutic substrate, since it would have to compete with large amounts of
endogenous dUMI'. The discovery reported herein, ie. that the human enzyme has
a
greater than 6.4-fold improved efficiency of conversion of BVdUMP, is an
important
factor enabling utility of NB 1011 as a selective anti-infective. The
increased
efficiency of BVdUNJP utilization by the human enzyme as compared to the L.
casei
2o enzyme also establishes that species specific substrates are possible and
can be
designed. These substrates are applied in the treatment of infections (either
viral or
cell-mediated) in which the infectious agent expresses a TS enzyme distinct
from that
encoded by the host. Examples of viral infections that can be treated using
this
approach include hepatitis virus, herpesviruses, or other viruses that express
their
own TS enzyme; and bacterial infections, especially drug resistant bacteria
like
multiply resistant staphyloccoccus aureus, and other infectious agents for
example
Pneumocystis carnii and Plasmodium falciparum. The ability to specifically
inhibit
heterologous enzymes via binding to species specific regions on the surface of
L.
casei vs. human TS has recently been reported (Stout (1999) Biochemistry
38(5):1607-17 and Costi et al. (1999) J. Med. Chem. 42(12):2112-2124).
Differences in specificity relating to the active site of TS, which is
composed of the
CA 02379834 2002-O1-21
WO 01/07087 PCT/LJS00/19844
most highly conserved regions of the protein (Cameras (1995) supra) is
surprising
and has not been reported previously.
Products of the cell-free enzymatic reaction of rHuTS with BVdUMP were
analyzed by mass spectroscopy. The molecular structures I and II shown in
Figure 5
have masses that are consistent with the mass of molecular ions detected in TS
reaction mixtures following incubation of BVdUMP with purified rHuTS.
Knowledge of the products of this reaction may be used to understand the final
mechanism of action of NB 1 O 11. In addition, this information could be used
to
design novel chemotherapeutics, since the products of the TS-BVdUMP reaction
1o could, themselves, be potentially used as chemotherapeutics.
4. NB 1011 is converted to the monophosphate in tumor cells
NB 1 Ol 1 is converted from the phosphoramidate to the monophosphate form
in cells, as a prerequisite for binding to TS. The proposed pathway for
unmasking the
i5 phosphate ofNB1011, its binding to TS and conversion to toxic metabolites
is shown
in Figure 5.
To detemnine whether this crucial step in conversion was taking place
advantage was taken of an unusual property of the bromine atom, ie. that it
exists in
nature in two equally abundant isotopic fomns. This situation allows detection
of the
2o bromine containing monophosphate by focusing the mass spectrometry analysis
on
the predicted mass ions of BVdUMP (411 and 413 daltons). H630 R10 tumor cells
(which express high levels of TS) were incubated with 1 OOuM NB 1011. Extracts
of
treated cell lysates were prepared as described in Materials and Methods,
above.
Detection using mass spectroscopy, following an initial purification with
liquid
25 chromatography relied upon formation of the unprotected nucleotide mass
ions of
BVdUMP which have identical retention times on reverse phase chromatography.
These results (Figure 6) are consistent with NB 1 Ol 1 following the first
step in
the activation pathway.
3o B. Characterization of the cytotoxic activity of NB1011.
1. The tumor/normal cell screen.
56
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
As an initial step in characterizing the biological activity of NB 1011, a
large
series of normal and tumor cell types were tested in the alamar blue assay for
sensitivity to both NB 1011 and 5-fluorouracil.
Assays were carried out as described in Methods, above. Therapeutic index is
calculated as the ratio of the average ICso for normal cells to the average
ICso for
tumor cells. All assays were done at least three times.
These data show that NB 1011 has met the primary design goal for TS ECTA
compounds, i.e. increased potency on tumor cells vs. normal cell types.
Overall,
NB 1 O11 is about 2-fold more cytotoxic to tumor cells vs. normal cells, while
SFU is
l0 3-fold more toxic to normal cells than it is to tumor cells. The total
benefit of
NB 1011 is therefore (2) x (3) = 6-fold improvement in therapeutic index for
NB 1 O 11
as compared with SFU. A critical tactic that allows for selection of
chemotheraputics
with a positive therapeutic index is screening of activity on both normal and
tumor
cell types. This approach has not been consistently employed in the field of
new
cancer drug discovery. For instance, screening of new candidate compounds on
normal cell types is not part of the National Cancer Institute's screening
procedure
(Curt (1996) Oncologist 1 (3):II-III).
2. NB 1 O 11 does not inactivate TS in vivo
The results described above indicate that BVdUMP, generated intracellularly
from NB101 l, is unlikely to inactivate TS during its transformation to
product(s).
However, the cell free system is different from the intracellular milieu. In
order to
further explore this question, cell-based assays for TS activity were
performed. In
these experiments exogenous 5-(3H) deoxyuridine is added to cell culture
medium
and the release of tritiated water is monitored (Carreras et al. (1995) supra,
and
Roberts (1966) Biochem. 5(11) 3546-3548). Figure 7 shows that the presence of
NB 1011 in cell culture media reduces the rate at which [3H]ZO is released
from 5-[
3H]BUMP. In order to determine whether this is the result of irreversible
inhibition
of TS, NB1011-treated cells were allowed to briefly recover in fresh culture
media,
then assayed for TS activity. Cells that have been allowed to recover in
culture
media lacking NB 1011 have the same level of TS activity as untreated cells.
This
57
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
result supports the proposal that NB 1 Ol 1 does not irreversibly inactivate
the TS
enzyme following intracellular processing.
An additional approach was taken to understanding whether NB 1 O 11 might
interfere with cell growth primarily by inactivating TS. This approach is
based upon
thymidine rescue of TS-blocked cells. Cells which are blocked by Tomudex or by
SFdUMP (following treatment by SFdUrd) do not make dTMP by de novo synthesis.
For this reason, they survive only by scavenger mechanisms. One of the
important
scavenger mechanisms is utilization of extracellular thymidine. Thymidine
incorporated by target cells can be converted to dTMP, usually by thymidine
kinase,
1o and thus continue DNA synthesis. Other pathways for use of exogenous
thymidine
have also been described If an important mechanism for NB 1011 activity is via
inhibition of endogenous TS, then the cytotoxicity should be relieved when
thymidine is added to the cell culture media. For this experiment, a number of
tumor
cell lines were screened for their sensitivity to Tomudex and SFdUrd, and
ability to
be rescued from these agents via thymidine supplementation. The normal colon
epthelial cell, CCDl8co, was used because of its measurable sensitivity to
NB1011,
SFUdR and Tomudex. Experiments were carned out as described by (Patterson et
al.
(1998) Cancer Res. 58:2737-2740) with or without lOuM thymidine, except that
the
alamar blue assay (see Materials and Methods) was employed to determine
2o cytotoxicity. The results showed a 15-fold rescue from Tomudex (ICSO change
from
6.SnM to 95 nM), a greater than 590-fold rescue from SFudR (from an ICso of
less
than 0.01 pM to greater than 5.9 pM), and a slight decrease in the absence of
thymidine to 223 ~M in the presence of 10 ~M thymidine.
3. Relationship between TS level and NB1011-mediated cytotoxicity on
tumor cell lines.
Confirmation that TS participates in NB1011-mediated cytotoxicity was
established using several approaches: 1 ). the activity of NB 1 O 11 was
examined on
normal colon cells vs. high TS expressing, SFU-resistant, tumor cells; 2).
transfection
of TS into a tumor cell background, and generating clonal derivatives which
differ
58
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
primarily by TS expression level, but are otherwise very similar; and 3). use
of a
specific inhibitor of TS, Tomudex, to decrease intracellular TS activity.
In the initial analysis, of NB 1011 and SFUdR-mediated cytotoxicity were
compared on the CCDl8co normal colon epithelial cell type and H630R10, SFU-
resistant colon tumor cell line (Copur S. et al. (1995) Biochem Pharm.
49(10):1419-
1426). This allows a determination of cytotoxicity vs. normal cells (CCDl8co)
as
well as a measure of cytotoxicity vs. drug-resistant tumor cells (H630R10),
which
overexpress TS. This is important because a significant limitation to current
chemotherapeutics is their toxicity to normal tissues. The results are
presented in
to Table 4.
This experiment shows that SFUdR is about 18-fold more toxic to normal
colon cells (CCDl8co) than to SFU-resistant H630R10 tumor cells. This ne. ae;
five
therapeutic index characterizes the major limitation of current chemotherapy,
ie. its'
toxicity to normal tissue. Such a negative therapeutic index has also been
reported
for doxorubicin (Smith et al. (1985) J. Natl. Cancer Inst. 74(2):341-7 and
Smith et al.
(1990) Cancer Res. 50(10):2943-2948). In contrast to SFUdR, however, NB1011
has
more than an 11-fold improved activity on drug-resistant H630R10 cells (ICSO =
216.7 pM) vs. normal colon epithelial cells (ICso greater than 2500 ~M). This
result
suggests that: 1). activity of NB 1011 is more pronounced on high TS
expressing
2o tumor cells; and 2). a total improvement in therapeutic index of (18) x
(11) = 198-
fold is achievable using TS ECTA compounds vs. SFUdR.
4. Overexpression of TS in HT1080 tumor cells enhances their
sensitivity to NB 1 O 11.
Activation of NB1011 requires several steps. These include cell penetration
conversion to the nucleotide monophosphate, binding to TS, and subsequent
toxic
metabolism. The precise mechanisms of cell penetration and conversion are not
fully
defined. Cell entry may depend in part on nucleoside transport mechanisms
(Cass et
al. (1998) Biochem. Cell Biol. 76(5):761-70). Similarly, processing from the
3o phosphoramidat+e to the monophosphate employs poorly defined mechanisms
(Abraham et al. (1996) J. Med. Chem. 8:39(23):4589-4575.
59
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
These results are particularly significant because they demonstrate, in a
fairly
uniform genetic background, that increasing TS levels predicts enhanced
sensitivity
to NB 1011. In addition, the data also show that increasing TS levels predicts
resistance to fluoropyrimidines, a result consistent with reports in the
literature
(Copur et al. (1995) Bioche. Pharm. 49(10):1419-1426 and Banerjee et al.
(1998)
Cancer Res. 58:4292-4296).
5. Inhibitors of NB 1 O11-mediated cytotoxicity.
Tomudex is a chemotherapeutic that acts primarily via inhibition of TS. If
NB 1 Ol 1 exerts cytotoxicity via the TS enzyme, then inhibition of TS with
Tomudex
should decrease NB1011-mediated cytotoxicity. To test this hypothesis
directly,
Tomudex-resistant MCF7 cells, which overexpress TS 11-fold compared to the
parental MCF7 cell line, were exposed to NB 1011 in the presence of increasing
concentrations of TDX.
Cells were plated and exposed to indicated concentrations of compounds) as
described in the Materials and Methods, above.
The data show that blockade of TS using the specific inhibitor Tomudex,
results in up to about 25-fold inhibition of NB 1011-mediated cytotoxicity.
These
results support the concept that activity of NB 1011 results from its
metabolism by
2o TS.
To further characterize the intracellular metabolism of NB 1011, combination
experiments with leucovorin (LV; 5-formyltetrahydrofolate) were performed.
This
experiment was initiated because we had observed that THF stimulates
production of
fluorescent products) in the cell-free reaction of BVdUMP and rHuTS. It was
hypothesized that if the fluorescent products are related to the cytotoxic
effects of
NB101 l, then enhancing intracellular levels of THF by providing LV in the
culture
media would also enhance NB1011-mediated cytotoxic effects. Surprisingly, in
the
presence of 3pM LV, NB1011 activity on the H630R10 cell line was diminished by
more than 90%, compared to NB 1011 alone, as determined in the alamar blue
assay.
3o The fact that NB 1011 activity is abolished by LV, which supplements
intracellular
reduced folate pools, suggests that NB 1 O11 may work in part by diminishing
these
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
pools. Alternatively, LV (or a metabolite) could directly impact the
metabolism of
BVdUNlP by interfering with its interaction with TS.
To explore whether LV could directly impact the reaction of BVdUMP with
TS, reactions were carned out +/- THF with BVdUMP, or with THF + dUMP , and
+/_
Methotrexate (MTX), LV or Tomudex (TDX).
The results (Table 6) are surprising in two respects: 1 ). Although an
increase
in fluorescent product was noted from BVdUMP in the presence of THF, a
decreased
rate of substrate consumption (BVdUMP) utilization occurs in the presence of
the
to cofactor; and 2). In the presence of cofactor, all three compounds tested
(MTX, TDX
and LV) dramatically inhibited the BVdUMP + rHuTS reaction. In each case, the
inhibition was more pronounced than that seen in the dUMP + rHuTS reaction, or
the
reactions with BVdUMP in the absence of THF.
The results described above, demonstrating inhibition of the BVdIIMP + TS
reaction by LV, MTX and TDX, and further, that this effect is more pronounced
in
the presence of cofactor (THF), suggests that NB 1011 activity may be
modulated by
other chemotherapeutics. Importantly, rescue of NB 1011-treated cells is
feasible by
providing LV, similar to the LV rescue from MTX. In the case of MLX, LV rescue
occurs via supplementation of intracellular folate pools, which are diminished
via
2o MTX inhibition of dihydrofolate reductase and TS. If reduced folates are
diminished
within the cell during BVdUMP reaction with TS, then other compounds that
diminish intracellular thymidine or purine nucleotide pools by distinct
mechanisms
may give additive or synergistic anti-cellular effects when used together with
NB1011. Examples of such compounds (Dory and Von Hoff (1994) supra),include
6-mercaptopurine, thioguanine and 2'-deoxycoformycin, all of which interfere
with
purine metabolism. Azacytidine-mediated inhibition of orotidylate
decarboxylase
blocks pyrimidine biosynthesis, and so could lower intracellular thymidine
levels in a
cell by a mechanism distinct from that of NB 1011.
3o C. Pharmacogenomics of TS ECTA
Comparison of TS and HER2.
61
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
An important aspect of the current approach to discovery and development of
novel therapeutics is the ability to identify patients who are most likely to
respond to
treatment (a positive pharmacogenomics selection). One of the pioneering drugs
in
this area is Herceptin, now used to treat breast cancers which overexpress the
HERZ
protooncogene. Early data with anti-HER2 antibodies showed that activity on
randomly selected tumor cells and normal cells was minimal. However, if tumor
cell
lines were selected that had at least a 4-fold increased expression of HER2,
then a
significant activity and anti-HER2 antibody could be demonstrated, as compared
to
normal cells or tumor cells expressing lower amounts of the HER2 gene product
l0 (Shepard et al. (1991) J. Nat. Cancer Inst. 74(2):341-347 and Lewis (1993)
Cancer
Immoral. Immunother. 37(4):255-63).
The cell line results shown in Figure 2 may suggest an additional similarity
between the TS and HERZ/NEU systems. The similarity is that each has a similar
overexpression requirement (about 4-fold) which predicts more aggressive
disease
for both TS and HER2/NEU overexpressing patients (Johnston et al. (1994) J.
Clin.
Oncol. 12:2640-2647).
2. NB 1011 is active against SFU and Tomudex-resistant colon and
breast tumor cell lines.
2o Because NB1011 has promising anticancer activity, it is important to
compare it with other chemotherapeutics with respect to safety. The utility of
NB 1011 in the treatment of cancer is further strengthened when it is compared
with
Tomudex, a chemotherapeutic which, like SFU, is often used to treat colon and
breast
cancer, among other malignancies.
The results show that while NB 1 O l 1 is more than 10-fold less toxic than
TDX vs. normal cells (CCDl8co), it is more than 30-fold more potent than TDX
on
MCF7-TDX resistant tumor cells. Similar results have been obtained for other
TDX-
resistant tumor cell lines. The low level of toxicity vs. normal cells and the
high
activity vs. TDXR tumor cells supports the application of NB 1011 to drug
resistant
3o cancers that overexpress TS.
62
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
3. NB 1011 is more dependent upon TS protein levels than TS activity as
measured by tritium release from dLTMP 3H.
Four types of assays have been used to characterize TS levels in cells and
tissues. Most commonly used is the antibody-based technique (Johnston (1994)
J.
Clin. Oncol. 12:2640-2647; Johnston and Allegra (1995) Cancer Res. 55:1407-
1412)
but RT-PCR, SFdUMP-binding and tritium release (van Laar (1996) Clin Cancer
Res
2(8):1327-33; van Triest (1999) Clin. Cancer. Res. 5(3):643-54; Jackman (1995)
Ann. Oncol. 6(9):871-81; Larsson (1996) Acta. Oncol. 35(4):469-72; Komaki
(1995)
Breast Cancer Res. Treat. 35(2):157-62; and Mulder (1994) Anticancer Res.
1o 14(6B):2677-80) have also been measured in various studies. For
characterization of
cell lines Applicants have focused on western blotting and tritium release
from 3H-
dUMP. These assays were chosen because antibody-detection is commonly used for
clinical samples and tritium release from labeled deoxyuridine is a direct
measure of
TS catalytic activity in cells.
Cells were grown and characterized as described in Methods. TS expression
level is relative to CCDl8co, a normal colon epithelial cell line. Tritium
release is
background substracted as described in Methods. ND = Not detectable above
background.
Analysis of the data presented in Table 7 indicates that there is a closer
2o relationship between TS protein level and sensitivity to NB 1011 than
between TS
activity (tritium release from 3H-dUMP) and NB 1 O 11 sensitivity. In each set
of
matched parental and drug-resistant tumor cell types, the drug-resistant
derivatives,
each with more TS protein than the parent, also have an increased sensitivity
to
NB 1011. However, when the same comparison is done with respect to TS
activity,
the parental cell lines often have comparable, or greater, TS activity and are
less
sensitive to NB1011-mediated cytotoxicity.
While these results could occur via a number of different mechanisms, or
combinations of mechanisms, it is likely that 3H-dUMP conversion to dTMP (and
accompanying tritium release) may be subject to limitation by some component,
3o perhaps cofactor availability. However, since conversion of BVdUMP is not
dependent upon cofactor, then its reaction with TS can continue even in a
cellular
63
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
milieu in which cofactor is limiting. This observation is important because TS
substrates as therapeutics would not be attempted based upon the results of
typical
tritium release assays for TS activity in which the most aggressive, and drug-
resistant, tumor cells are observed to have a lower TS activity than their
precursors.
These results lend additional support to the proposal of selecting patients
for TS
ECTA therapy based simply on the level of TS detected by antibody staining.
64
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
Table 1. Comparison of Prodrug Strategies
Technolo~y Acronym Description Key References
Metabolic activationNone Conversion of folateMead et al.
analogs to
toxins via 'lethal ( 1966) supra.
synthesis.'
Antibody directedADEPT Antibody-enzyme complexSyrigos and
prodrug therapy binds to tumor selectiveEpenetos
antigen. (1999)
Prodrug is administeredAnticancer
and Res.
activated when it 19(1A):605-13
encounters the
antibody bound enzyme.
Gene directed ADEPT Gene encoding activatingConnors and
prodrug
therapy enzyme is transducedKnox (1995)
into large t
cells Stem Cells
13:501-511
Enzyme directed EDEPT Prodrugs are administeredBreistol
prodrug which et al.
therapy are activated by (1998)
extracellular
enzymes present a Eur. J. Cancer
high levels
only at tumor site. 34(19):1602-
1606 and
Bosslet
et al. (1998)
Cancer Res.
58:1195-1201.
Tumor Activated 'TAC' Prodrugs activated Morgan et
by al.
Cytotoxin glutathione-s-transferase(1998)
supra
Enzyme catalyzedECTA Prodrugs are activatedAs disclosed
by
therapeutic agents enzymes overexpressedherein
as a
result of tumor suppressor
gene
loss and in vivo
selection by
chemotherapy
Table 2. Comparison of Kenetic Parameters of Bacterial and rHuTS
Kinetic ConstantsLactobacillusrHuTS
casei
DUMP Km 3.O~M 7.7~M
K~ 6.4s' 0.2s'
K~,,Km 2.1 x 106M-'s2.6 x 104M-'s
' '
K; (of BVdUMP)0.6~M 4.S~M
BvdUMP Km 3.3 ~M 16~M_
K~a, 0.018s' 0.0067sv'
K~a,,Km 5.6 x 103M-'5'4.2 x 103M-'5'
K; (of dUMP) 2.O~M 17.S~M
Relative catalytic 385-fold 60-fold
efficiency
(BUMP vs BVdUMP)
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
Enzyme kinetics were done as described in Methods. Data for Lactobacillus
casei
are derived from Barr et al. (1983) supra. The rHuTS was prepared as described
in
Methods, above.
Table 3. Inhibition of rHuTS reactions by Tomudex and 5-FdUMP
Substrate + CofactorNo InhibitorTomusdex (500 5-FdUMP (500
nM) nM)
10916 673 442
BvdUMP + THF RFU/min (61%) (40%)
( 100%)
7511 343 9313
BvdUMP - THF ( 100%) (45%) ( 129%)
1500 _+ 690 _+40 290 _+ 70
20
dUMP + THF nmoles/min (46%)
( 100%)
( 19%)
to Inhibition of rHuTS reactions by Tomudex and 5-FdUMP. Thymidylate synthase
reactions containing enzyme inhibitors or cofactor were incubated at
30°C as
described in Materials and Methods, and the initial rates of the enzyme
reaction were
determined by measuring the increase in relative fluorescence units at 340 nm
excitation, 595 nm emission for the BVdUMP reactions, or increase in A3ao for
the
1 5 dUNII' reaction.
66
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
Table 4 Cytotoxicity of NB1011 vs. SFi1 on Normal and Tumor Cell Strains
Normal IC50 Tumor 1C50 (~.M)
Cells (~M) Cells
NB 101.1SFU NB 1 SFU
O 1.1
CCD1800(Colon)562 2.0 H630R10 (Colon) 65 41.6
DET551 (Skin) 262 0.8 HT1080 (Colon) 449 0.8
NHDF (Skin) 359 0.8 COL0320 (Colon) 401 1.5
H527 (Skin) 273 1.6 COL0205 (Colon) 105 1.3
W138 (Lung) 335 1.0 SW620 (Colon) 374 4.6
MRC9 (Lung) 303 1.1 SKCOl (Colon) 184 1.4
NHLF (Lung) 139 0.9 HCTC (Colon) 280 2.8
NHA (Brain)839 0.9 MCF7 (Breast) 141 1.0
NHOST (Bone) 642 4.7 MDAMB (Breast 365 S.0
361
NPRSC (Prostat369 1.7 MDAMB (Breast) 172 4.4
e) 468
NHEPF (Liver)2085 1.7 SW527 (Breast) 431 4.3
NCI H520(Lung) 135 0.6
Average561 1.6 SKLU1 (Lung)' 270 7.9
SOAS2 (Bone) 232 1.4
PANC1 (Pancreas)492 1.9
SKOV3 (Ovary) 484 3.0
PC3 (Prostate)184 0.9
HEPG2 (Liver) 704 22.8
SKHEP (Liver) 247 1.7
1
A431 (Skin) 266 0.2
MCIxc (Brain) 61. 1.2
Average 288 5.3
NB101.1 SFU
Therapeutic index (N/T) 1.95 0.30
Cells were analyzed for response to either NB1011 or SFU in the alamar blue
assay
(Methods). All assays were performed at least three times. The standard
deviation is less
than 20%. Therapeutic index was calculated as the ratio of ICSO (mean of all
cell types) to ICso
(mean of all tumor cell lines).
67
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
Table 5 NB1011 cvtotoxicity on cell lines engineered to express HUTS
Cell Line TS Level ICSo
(%)* NB 1011 FUDR 5-FU TDX
(~M) (~M) (~M) (~.M)
C/HT1080 100 320 <0.1 1.0 3.6
TSL/HT1080409 196 2.2 1.7 24
TSL/HT1080702 0.8 3.1 3.5 153
A cDNA encoding rHuTS was subcloned into ventor pEGFP-C3, in-frame with GFP.
The
construct was transfected into HT1080 cells and selected with 6418 (750 ug/ml)
in order to
obtain clones that stably express fusion rHuTS. Individual cells were cloned
based upon high
or low fluorescence expression as described in Methods. *TS levels were
determined by
using Western blot analysis, the quantified expression levels were expressed
as values relative
1o to that of cell strain CCDl8co.
Table 6 Tomudex Inhibits NB1011 Mediated Cytotoxicity
[Tomudex] OnM 1nM IOnM 100nM 1000nM
M)
NB 101 l 5.7 25.5 87.7 140.3 103.0
ICso
(~M)
Fold Protection1 4.5x 15.4x 24.6x 18.1x
The Tomudex rescue assay (alamar blue) was done with TDX-resistant MCF7 breast
tumor
cells as described in Methods. "Fold Protection" was calculated as the ratio
of ICso with and
without added TDX.
68
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
Table 7 Impact of Folate Inhibitors
InhibitorBVdUMP, with BVdUMP, w/o THF dUMP, with
THF THF
None 100% 138% 100%
MTX 10% 24% 31
LV 17% 97% 77%
TDX 0% 25% 18%
Cell-free assays using rHuTS, the appropriate substrate and other components
were combined
as described in Methods. MTX (140~.M), LV (140~.M) or TDX (5~M) were added to
evaluate their inhibitory activity. Utilization of substrate (either BVdUMP or
THF) was
employed as a measure of reaction rate. The numbers indicate remaining
activity.
to
Table 8 NB1011 activity is more associated with TS protein than with tritium
release
Cell Line Drug TS ProteinTritium NB1011-
Selection Release ICso
H630 None 288 3206 414
Colon cancer 5FU 2350 1840 65
TDX 671 3980 2.3
RKO None 142 4920 136
Colon cancer TDX 279 1625 28
MCF7 None 178 5185 327
Breast cancerTDX 1980 875 2.8
N1 S 1 None 197 12,565 494
SFU 1241 ND 204
69
CA 02379834 2002-O1-21
WO 01/07087 PCT/US00/19844
Table 9 MDF7 TDX cells selected for resistance to
NB1011 are more sensitive to 5-Fluorouracil and Tomudex
ICSO (micromolar)' Relative TS
5- FU Tomudex NB 1011 Protein Level
MCF7 10- .026- 291- 1X-
MCF7 TDX 32 >10 2 11X
MCF7 TDX/1011 2 .041 240 4X
* = as determined by the alamar blue assay described in Materials and Methods
TDX = Tomudex; 1011 = NB 1011
1o The preceding discussion and examples are intended merely to illustrate the
invention
of the claims. As is apparent to one of skill in the art, various
modifications can be made to
the examples and claimswithout departing from the spirit and scope of this
invention.
