U.S. patent application number 09/249790 was filed with the patent office on 2001-09-27 for treatment of chemotherapeutic agent and antiviral agent toxicity with acylated pyrimidine nucleosides.
Invention is credited to BAMAT, MICHAEL K., VON BORSTEL, REID W..
Application Number | 20010025032 09/249790 |
Document ID | / |
Family ID | 23874595 |
Filed Date | 2001-09-27 |
United States Patent
Application |
20010025032 |
Kind Code |
A1 |
VON BORSTEL, REID W. ; et
al. |
September 27, 2001 |
TREATMENT OF CHEMOTHERAPEUTIC AGENT AND ANTIVIRAL AGENT TOXICITY
WITH ACYLATED PYRIMIDINE NUCLEOSIDES
Abstract
The subject invention discloses compounds, compositions and
methods for treatment and prevention of toxicity due to
chemotherapeutic agents and antiviral agents. Disclosed are
acylated derivatives of non-methylated pyrimidine nucleosides.
These compounds are capable of attenuating damage to the
hematopoietic system in animals receiving antiviral or
antineoplastic chemotherapy.
Inventors: |
VON BORSTEL, REID W.;
(POTOMAC, MD) ; BAMAT, MICHAEL K.; (POTOMAC,
MD) |
Correspondence
Address: |
NIXON & VANDERHYE
ATTY LEONARD C MITCHARD
1100 NORTH GLEBE ROAD
8TH FLOOR
ARLINGTON
VA
222014714
|
Family ID: |
23874595 |
Appl. No.: |
09/249790 |
Filed: |
February 16, 1999 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09249790 |
Feb 16, 1999 |
|
|
|
08472210 |
Jun 7, 1995 |
|
|
|
5968914 |
|
|
|
|
08472210 |
Jun 7, 1995 |
|
|
|
08176485 |
Dec 30, 1993 |
|
|
|
5736531 |
|
|
|
|
08176485 |
Dec 30, 1993 |
|
|
|
08061381 |
May 14, 1993 |
|
|
|
08061381 |
May 14, 1993 |
|
|
|
07903107 |
Jun 25, 1992 |
|
|
|
07903107 |
Jun 25, 1992 |
|
|
|
07724340 |
Jul 5, 1991 |
|
|
|
07724340 |
Jul 5, 1991 |
|
|
|
07438493 |
Jun 26, 1990 |
|
|
|
07438493 |
Jun 26, 1990 |
|
|
|
07115929 |
Oct 28, 1987 |
|
|
|
07115929 |
Oct 28, 1987 |
|
|
|
07487984 |
Feb 5, 1990 |
|
|
|
07487984 |
Feb 5, 1990 |
|
|
|
07115923 |
Oct 28, 1987 |
|
|
|
Current U.S.
Class: |
514/50 ;
514/49 |
Current CPC
Class: |
A61K 31/7072 20130101;
A61K 31/515 20130101; A61K 45/06 20130101; A61P 39/02 20180101;
C07H 19/06 20130101; C07H 19/10 20130101; A61K 31/513 20130101;
A61K 31/7068 20130101; A61P 35/00 20180101; A61K 31/70 20130101;
Y02A 50/30 20180101; A61K 31/70 20130101; A61K 31/70 20130101; A61K
31/70 20130101; A61K 2300/00 20130101; A61K 31/515 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
514/50 ;
514/49 |
International
Class: |
A61K 031/70; A01N
043/04 |
Claims
What is claimed is:
1. A method for treating cancer comprising: (a) administering a
pyrimidine nucleoside analog in a dose at least 1.5 fold greater
than the normal maximum tolerated dose, and (b) administering a
pharmaceutically effective amount of an acyl derivative of a
non-methylated pyrimidine nucleoside.
2. A method as in claim 1 wherein said pyrimidine nucleoside analog
is selected from the group consisting of 5-fluorouracil (5-FU),
5-FU prodrugs including tegafur and 5'-deoxyfluorouridine,
fluorouridine, 2'-deoxyfluorouridine, prodrug derivatives of
fluorouridine or 2'-deoxyfluorouridine, fluorocytosine,
trifluoro-methyl-2'-deoxyuridine, arabinosyl cytosine, prodrugs of
arabinosyl cytosine, cyclocytidine, 5-aza-2'-deoxycytidine,
arabinosyl 5-azacytosine, 6-azacytidine,
N-phosphonoacetyl-L-aspartic acid (PALA), pyrazofurin,
6-azauridine, azaribine, thymidine, and 3-deazauridine.
3. A method as in claim 1 wherein said pyrimidine nucleoside analog
is a 5-fluoropyrimidine or 5-fluoropyrimidine nucleoside analog and
said acyl derivative of a non-methylated pyrimidine nucleoside is
an acyl derivative of uridine, cytidine, or deoxyuridine.
4. A method as in claim 3 wherein said 5-fluoropyrimidine or
5-fluoropyrimidine nucleoside analog is selected from the group
consisting of 5-fluorouracil, 5-fluorouracil prodrugs including
tegafur and 5'-deoxyfluorouridine, fluorouridine,
2'-deoxyfluorouridine, prodrug derivatives of fluorouridine,
prodrug derivatives of 2'-deoxyfluorouridine, 5-fluorocytosine,
5-fluorocytidine, or prodrug derivatives of 5-fluorocytidine.
5. A method as in claim 3 wherein said 5-fluoropyrimidine or
5-fluoropyrimidine nucleoside analog is 5-fluorouracil.
6. A method as in claim 5 wherein said administering step (a)
comprises administering a bolus of 900 to 2400 mg/m.sup.2 of
5-fluorouracil, and said administering step (b) comprises
administering 2 to 24 hours after step (a) 1 to 10 grams of an acyl
derivative of a nonmethylated pyrimidine nucleoside, wherein steps
(a) and (b) are repeated 3-6 times.
7. A method as in claim 6 wherein the time interval between each
repetition of step (a) is 4 to 14 days.
8. A method as in claim 5 wherein said administering step (a)
comprises administering a bolus of 600 to 1000 mg/M.sup.2 of
5-fluorouracil daily for 4 to 5 consecutive days, and said
administering step (b) comprises administering 2 to 12 hours after
each step (a) 1 to 10 grams of an acyl derivative of a
nonmethylated pyrimidine nucleoside.
9. A method as in claim 1 wherein said pyrimidine nucleoside analog
is N-phosphonoacetyl-L-aspartic acid (PALA), pyrazofurin,
6-azauridine, azaribine, trifluoromethyl-2'-deoxyuridine, or
3-deazauridine and said acyl derivative of a non-methylated
pyrimidine nucleoside is an acyl derivative of uridine or
cytidine.
10. A method as in claim 1 wherein said acyl derivative of a
non-methylated pyrimidine nucleoside is an acyl derivative of
uridine, cytidine, deoxycytidine or deoxyuridine.
11. A method as in claim 1 wherein said acyl derivative of a
non-methylated pyrimidine nucleoside is triacetyluridine.
12. A method as in claim 1 wherein said acyl derivative of a
non-methylated pyrimidine nucleoside is ethoxycarbonyluridine.
13. A method as in claim 1 wherein said acyl derivative of a
non-methylated pyrimidine nucleoside is triacetylcytidine.
14. A method as in claim 1 wherein said acyl derivative of a
non-methylated pyrimidine nucleoside is diacetyldeoxycytidine.
15. A method as in claim 1 wherein said pyrimidine nucleoside
analog is an antineoplastic analog of cytidine and said acyl
derivative of a non-methylated pyrimidine nucleoside is an acyl
derivative of deoxycytidine.
16. A method as in claim 15 wherein said antineoplastic analog of
cytidine is arabinosyl cytosine or prodrugs thereof, cyclocytidine,
5-aza-2'-deoxycytidine, arabinosyl 5-azacytosine, or
6-azacytidine.
17. A method as in claim 1 wherein said pyrimidine nucleoside
analog is an analog of uridine, said acylated derivative of a
non-methylated pyrimidine nucleoside is an acylated derivative of
uridine, deoxyuridine, or cytidine, and said administering step (b)
also includes administering an inhibitor of uridine
phosphorylase.
18. A method as in claim 17 wherein said inhibitor of uridine
phosphorylase is selected from the group consisting of
benzylacyclouridine, benzyloxybenzylacyclouridine,
aminomethyl-benzylacyclouridine,
aminomethyl-benzyloxybenzylacyclouridine- ,
hydroxymethyl-benzylacyclouridine,
hydroxymethyl-benzyloxybenzylacyclour- idine,
2,2'-anhydro-5-ethyluridine, 5-benzyl barbiturate,
5-benzyloxybenzyl barbiturate,
5-benzyloxybenzyl-1-[(1-hydroxy-2-ethoxy)m- ethyl] barbiturate,
5-benzyloxybenzylacetyl-1-[(1-hydroxy-2-ethoxy)methyl] barbiturate,
and 5-methoxybenzylacetylacyclobarbiturate.
19. A method as in claim 1 wherein said acylated derivative of a
non-methylated pyrimidine nucleoside is an acylated derivative of
cytidine or deoxycytidine, and said administering step (b) also
includes administering an inhibitor of cytidine deaminase.
20. A method as in claim 19 wherein said inhibitor of cytidine
deaminase is selected from the group consisting of
tetrahydrouridine or tetrahydro-2'-deoxyuridine.
21. A method as in claim 1 wherein said acylated derivative of a
non-methylated pyrimidine nucleoside is an acylated derivative of
uridine, cytidine or deoxycytidine, and said administering step (b)
also includes administering an inhibitor of nucleoside
transport.
22. A method as in claim 21 wherein said inhibitor of nucleoside
transport is selected from the group consisting of dilazep,
dipyridamole, probenicid, lidoflazine or
nitrobenzylthioinosine.
23. A method as in claim 1 wherein said administering step (b) also
includes administering an agent which enhances hematopoiesis.
24. A method as in claim 1 wherein said administering step (b) also
includes administering a compound capable of enhancing the uptake
and phosphorylation of nucleosides into cells.
25. A method as in claim 1 wherein said administering step (a) also
includes administering AZT.
26. A method as in claim 1 wherein said fluorinated pyrimidine is
administered in conjunction with a biochemical modulator of
5-fluorouracil efficacy.
27. A method as in claim 26 wherein said modulator is an inhibitor
of purine biosynthesis, an antifolate, an inhibitor of pyrimidine
biosynthesis, or an inhibitor of 5-fluorouracil degradation.
28. A method as in claim 27 wherein said inhibitor of purine
biosynthesis is methylmercaptopurine riboside.
29. A method as in claim 27 wherein said antifolate is methotrexate
or trimetrexate.
30. A method as in claim 27 wherein said inhibitor of pyrimidine
biosynthesis is PALA, brequinar, acivicin, or 6-azauridine.
31. A method as in claim 27 wherein said inhibitor of
5-fluorouracil degradation is an inhibitor of the enzyme
dihydropyrimidine dehydrogenase.
32. A method as in claim 31 wherein said inhibitor of
dihydropyrimidine dehydrogenase is 5-ethynyluracil,
bromovinyluracil, CDHP, uracil, thymine, thymidine or
benzyloxybenzyluracil.
33. A method for treating cancer comprising: (a) administering an
inhibitor of the enzyme dihydropyrimidine dehydrogenase; (b)
administering a 5-fluoropyrimidine or 5-fluoropyrimidine nucleoside
analog; (c) administering a pharmaceutically effective amount of an
acyl derivative of a non-methylated pyrimidine nucleoside.
34. A method as in claim 33 wherein said 5-fluoropyrimidine or
5-fluoropyrimidine nucleoside analog is selected from the group
consisting of 5-fluorouracil, 5-fluorouracil prodrugs including
tegafur and 5'-deoxyfluorouridine, fluorouridine,
2'-deoxyfluorouridine, prodrug derivatives of fluorouridine,
prodrug derivatives of 2'-deoxyfluorouridine, 5-fluorocytosine,
5-fluorocytidine, or prodrug derivatives of 5-fluorocytidine.
35. A method as in claim 33 wherein said inhibitor of
dihydropyrimidine dehydrogenase is 5-ethynyluracil,
bromovinyluracil, cyanodidhydropyridine, uracil, thymine, thymidine
or benzyloxybenzyluracil.
36. A method as in claim 33 wherein said administering step (a)
takes place before or at the same time as said administering step
(b).
Description
[0001] This application is a continuation in part of copending U.S.
application Ser. No. 08/176,485, filed Dec. 30, 1993, which is a
continuation in part of copending U.S. application Ser. No.
08/061,381, filed May 14, 1993, which is a continuation in part of
copending U.S. application Ser. No. 903,107, filed Jun. 25, 1992,
which is a continuation-in-part application of copending U.S.
application Ser. No. 724,340, filed Jul. 5, 1991, which is a
continuation-in-part of U.S. application Ser. No. 438,493, filed
Jun. 27, 1989, which is a continuation-in-part of U.S. application
Ser. No. 115,929, filed Oct. 27, 1987, and Ser. No. 724,340 is a
continuation-in-part application of copending U.S. application Ser.
No. 487,984, filed Feb. 5, 1990, which is a continuation-in-part of
U.S. application Ser. No. 115,923 filed Oct. 27, 1987. All of these
applications are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] This invention relates generally to treatment of
chemotherapeutic agent and antiviral agent toxicity with acylated
derivatives of non-methylated pyrimidine nucleosides. These
compounds are capable of attenuating damage to the hematopoietic
system in animals receiving antiviral or antineoplastic
chemotherapy. This invention also relates to protection of other
tissues affected by antiviral or antineoplastic chemotherapy,
including the gastrointestinal epithelium.
BACKGROUND OF THE INVENTION
[0003] A major complication of cancer chemotherapy and of antiviral
chemotherapy is damage to bone marrow cells or suppression of their
function. Specifically, chemotherapy damages or destroys
hematopoietic precursor cells, primarily found in the bone marrow
and spleen, impairing the production of new blood cells
(granulocytes, lymphocytes, erythrocytes, monocytes, platelets,
etc.). Treatment of cancer patients with 5-fluorouracil, for
example, reduces the number of leukocytes (lymphocytes and/or
granulocytes), and can result in enhanced susceptibility of the
patients to infection. Many cancer patients die of infection or
other consequences of hematopoietic failure subsequent to
chemotherapy. Chemotherapeutic agents can also result in subnormal
formation of platelets which produces a propensity toward
hemorrhage. Inhibition of erythrocyte production can result in
anemia. The risk of damage to the hematopoietic system or other
important tissues can prevent utilization of doses of chemotherapy
agents high enough to provide good antitumor or antiviral
efficacy.
[0004] Many antineoplastic or antiviral chemotherapy agents act by
inhibiting nucleotide biosynthesis, metabolism, or function, or are
in fact nucleoside analogs that substitute for the normal
nucleosides in nucleic acids, producing defective RNA or DNA.
[0005] 5-Fluorouracil is a clinically important cytoreductive
antineoplastic chemotherapy agent that acts in part through
incorporation into RNA, producing defective RNA; inhibition of
thymidylate synthetase by fluorodeoxyuridine monophosphate may also
contribute to the cytotoxicity of 5-FU. The clinical utility of
5-FU is limited by its toxicity (especially to bone marrow).
Specifically, its clinical utility is limited by a low therapeutic
ratio (the ratio of toxic dose to effective dose; a high
therapeutic ratio implies that a drug has efficacy with little
toxicity).
[0006] 5-FU and many other chemotherapy agents also affect other
tissues, especially gastrointestinal mucosa, producing mucositis,
diarrhea and ulceration. Stomatitis (ulceration of mucosa in the
mouth), is particularly troublesome to patients, making eating and
swallowing painful.
[0007] D. S. Martin et al. (Cancer Res. 42:3964-70 [1982]) reported
that a toxic dose of 5-FU (with strong anti-tumor activity) could
be safely administered to mice if followed by administration of a
high dose of uridine beginning several hours later. This "rescue"
strategy has been shown to increase the therapeutic index of 5-FU
in animal tumor models, allowing administration of the high, toxic
doses of 5-FU that are necessary for causing tumor regresssion or
preventing tumor growth while preferentially protecting normal
tissues (especially important is bone marrow) by subsequent
administration of uridine (D. S. Martin et al., Cancer Res.
43:4653-61 [1983]).
[0008] Clinical trials involving the administration of uridine have
been complicated due to the biological properties of uridine
itself. Uridine is poorly absorbed after oral administration;
diarrhea is dose limiting in humans (van Groeningen et al.,
Proceedings of the AACR 28:195 [1987]). Consequently, parenteral
administration of uridine is necessary for clinically significant
reversal of 5-FU toxicity, which requires use of a central venous
catheter, since phlebitis has been a problem in early clinical
trials when uridine was administered via a small intravenous
catheter (van Groeningen et al. Cancer Treat Rep. 70:745-50
[1986]). Prolonged infusion via central venous catheters requires
hospitalization of the patients. Further, there is considerable
discomfort and inconvenience to the patients.
[0009] Orally-active prodrugs of 5FU have been developed which are
enzymatically or spontaneously converted to 5FU, generally after
absorption from the intestine into the bloodstream. This permits
self-administration by patients, without the discomfort of
intravenous administration. Moreover, in some chemotherapy
regimens, sustained exposure, e.g. a constant intravenous infusion
for several days or weeks, of tumors to 5FU is attempted. Oral
administration of 5FU prodrugs can in principle provide such
sustained availability of 5FU to tumors.
[0010] 5-Fluoro-1-(tetrahydro-2-furfuryl)uracil (FT) is an orally
active prodrug of 5-fluorouracil. It is enzymatically converted to
5-fluorouracil primarily in the liver. The liver, however, has
relatively high levels of the enzyme dihydropyrimidine
dehydrogenase, which degrades 5FU, producing metabolites which are
not useful in cancer chemotherapy and which furthermore contribute
to 5-FU toxicity.
[0011] The cytotoxicity of 5FU, the active metabolite of FT, is
believed to be a result of its incorporation into nucleotide pools,
where certain anabolites exert toxic effects, e.g.
5-fluorodeoxyuridine monophosphate inhibits thymidylate synthetase,
thus depriving cells of thymidine for DNA synthesis, and
5-fluorouridine triphosphate incorporation into RNA impairs its
normal functions in translation of genetic information.
[0012] In order to inhibit the catabolism of 5FU derived from FT,
other compounds have been administered with the FT. In particular,
the pyrimidine uracil inhibits the catabolism of 5FU without
inhibiting its cytotoxicity. The most widely used clinical
formulation of FT contains uracil in a 1:4 molar ratio. This
permits a significant reduction in the dose of FT required to
achieve a therapeutic effect. Other pyridimines, including uridine,
thymidine, thymine, and cytosine are either less effective than
uracil or no better in potentiating the antitumor efficacy of FT
without unacceptably potentiating toxicity. Potent synthetic
inhibitors of dihydropyrimidine dehydrogenase (DHPDHase) have also
been utilized with FT or 5FU. 5-chloro-2,6-dihydroxypyridine (CDHP)
is more potent than uracil as an inhibitor of DHPDHase. However,
this compound also enhances the toxicity of 5FU, so that, in its
intended clinical implementation, a third component, oxonic acid,
is co-administered to reduce the intestinal toxicity.
[0013] Several investigators have administered pyrimidines with 5FU
attempting to improve the therapeutic index of this antineoplastic
agent. In vivo, uridine and thymidine when administered at the same
time as 5FU increased both the antitumor efficacy of 5FU and its
toxicity, so that there was no net increase in therapeutic index
(Hartman and Bollag, Med. Oncol. & Tumor Pharmacother.,
3:111-118 [1986]). Burchenal et al. (Cancer Chemother. Rep., 6:1-5
[1960]) summarized comprehensive studies on interactions of 5FU and
5-fluorodeoxyuridine (FUDR) and pyrimidine compounds. They noted
that despite the fact that pyrimidines and pyrimidine nucleosides,
at doses which are inactive alone, markedly potentiate the
antileukemic effects of small doses of FUDR or FU, it has not been
possible with any combination to improve significantly and with any
degree of regularity the results which can be obtained with maximum
tolerated doses of FU or FUDR alone. Similarly, Jato et al. (J.
Pharm Sci., 64:943-945 [1975]), in an investigation of combinations
of deoxyuridine with 5FU and FUDR report that any therapeutic
benefit of the combination therapy could be duplicated with either
5FU or FUDR at a higher dose. Although deoxyuridine, by inhibiting
the catabolism of the fluoropyrimdines permitted adminstration of
lower doses, deoxyuridine there was no improvement in antitumor
activity at equitoxic doses of the combination versus FU or FUDR
alone.
[0014] As in the case of uridine, problems of poor bioavailability
after oral administration limit the clinical utility of
administration of deoxycytidine, cytidine, and deoxyuridine
themselves for modulation of toxicity of chemotherapy agents.
[0015] Arabinosyl cytosine (Ara-C) is an important agent in the
treatment of leukemia, and is also useful as an immunosuppressant.
Bone marrow toxicity (myeloid and erythroid) associated with Ara-C
administration can be partially prevented by administration of
deoxycytidine (Belyanchikova et al. Bull. Exp. Biol. Med. 91:83-85
[1981]), while the toxicity of Ara-C to lymphocytes is not as
strongly attenuated by deoxycytidine. In cell cultures, normal
myeloid progenitor cells are protected from Ara-C by deoxycytidine
better than are leukemic cells (K. Bhalla et al. Blood 70:568-571
[1987]). Deoxycytidine also attenuates toxicity of
5-aza-2'-deoxycytidine and arabinosyl 5-azacytosine in cell
cultures (K. Bhalla et al. Leukemia 1:814-819 [1987]). Prolonged (5
day) infusion of high doses of deoxycytidine via a central venous
catheter was proposed as a means for clinical implementation of
modulation of Ara-C toxicity with deoxycytidine (K. Bhalla et al.
Leukemia 2:709-710 [1988]).
[0016] N-phosphonoacetyl-L-aspartic acid (PALA) is an
antineoplastic agent that inhibits the enzyme aspartate
transcarbamoylase, an enzyme indirectly involved in biosynthesis of
pyrimidine nucleotides. Side effects of PALA primarily involve
damage to gastrointestinal toxicity and mucositis. Pyrazofurin (a
carbon linked pyrimidine analog), 6-azauridine, and 6-azacytidine
all interfere with pyrimidine nucleotide synthesis and
metabolism.
[0017] 3'-Azidodeoxythymidine (AZT) is used clinically in patients
infected with Human Immunodeficiency Virus (HIV, the infectious
agent in AIDS). AZT prolongs the lifespan of patients infected with
HIV, but also impairs hematopoiesis, producing leukopenia and
anemia. In cell cultures, uridine ameliorates AZT-induced toxicity
to granulocyte/macrophage progenitor cells without impairing the
antiviral actions of AZT (Sommadossi et al., (1988) Antimicrobial
Agents and Chemotherapy, 32:997-1001); thymidine attenuated both
toxicity and antiviral activity. In mice, parenteral administration
of high doses of uridine provided some amelioration of AZT-induced
anemia, but only at uridine doses which increased mortality during
the study; a low, non-toxic dose of uridine (500 mg/kg/d) did not
reduce AZT-induced hematologic toxicity (A. Falcone, et al. Blood
76:2216-21 [1990]). Sommadossi and el Kouni (U.S. Pat. No.
5,077,280) proposed the administration of uridine by periodic
intravenous injection in order to attenuate AZT toxicity. Bhalla et
al. (Blood 74:1923-1928 [1989]) reported that deoxycytidine
protects normal human bone marrow progenitor cells in vitro against
the cytotoxicity of AZT with preservation of antiretroviral
activity.
[0018] 5-Fluoroorotate, an analog of the pyrimidine nucleotide
precursor orotic acid, has antiproliferative effects on human
cells, but is especially useful for treating infections with
malarial parasites, e.g., Plasmodium yoelii or Plasmodium
falciparum, which are dependent on de novo pyrimidine biosynthesis.
Administration of uridine to mice treated with 5-fluoroorotate
attenuated host toxicity due to the latter without impairing its
antimalarial activity (ZM Gomez and PK Rathod, Antimicrob. Agents
Chemother. 34:1371-1375 (1990).
[0019] Dideoxycytidine (ddC) is also useful against retroviral
infections including HIV; side effects of ddC include peripheral
neuropathy, mouth ulcers, and reduced platelet counts. The toxicity
of ddC on human myeloid progenitor cells in culture can be
ameliorated by deoxycytidine without thereby impairing the
antiretroviral efficacy of ddC (K. Bhalla et al., AIDS 4:427-31
[1990]).
[0020] The methods disclosed in the prior art cited above for
administering these pyrimidine nucleosides to modify chemotherapy
in the clinical setting are neither practical (prolonged infusion
of deoxycytidine or uridine via a central venous catheter requires
hospitalization, risk of infection, and discomfort to the patient)
or satisfactory (orally administered uridine is poorly absorbed;
therapeutically adequate doses of oral uridine produce
diarrhea).
[0021] Commonly owned U.S. patent application Ser. No. 438,493
demonstrates the use of acylated derivatives of cytidine and
uridine to increase blood cytidine or uridine levels.
[0022] Some acyl derivatives of pyrimidine nucleosides have been
synthesized for use as protected intermediates in the synthesis of
oligonucleotides or nucleoside analogs, e.g. 5'-O-benzoyluridine,
triacetylcytidine, and triacetyluridine. See Sigma Chemical Company
1991 catalog, pages 155, 980, and 981 respectively.
OBJECTS OF THE INVENTION
[0023] It is a primary object of this invention to provide a method
for effectively preventing or treating toxic symptoms of antiviral
or anticancer chemotherapy, including but not limited to damage to
the hematopoietic system and to gastrointestinal mucosa.
[0024] A further object of the invention is to provide compounds
and methods to permit administration of higher doses of the
chemotherapy agents.
[0025] A further object of the invention is to provide methods of
increasing blood and tissue levels of uridine and cytidine, and
their corresponding deoxyribonucleosides deoxycytidine and
deoxyuridine through oral administration of a compound or
compounds.
[0026] A further object of the invention is to provide a method for
preventing or ameliorating gastrointestinal epithelium damage due
to cytotoxic chemotherapy agents.
SUMMARY OF THE INVENTION
[0027] These and other objects of the invention are achieved by
oral or parenteral administration of acylated derivatives of
non-methylated pyrimidine nucleosides, e.g. acylated derivatives of
uridine, deoxyuridine, cytidine, or deoxycytidine, which are
administered to animals, including mammals such as humans. The
administration of these compounds alone, or in combination, is
useful in preventing or ameliorating toxic effects of cytoreductive
chemotherapy in animals.
[0028] Thus, the compounds of the invention, alone or in
combinations, are useful in the treatment of disorders of
hematopoiesis induced by chemical agents; are useful as adjuncts to
cancer and antiviral chemotherapy; and are useful for the treatment
of other pathological conditions.
[0029] An important aspect of this invention is the discovery that
acyl derivatives of non-methylated pyrimidine nucleosides have
unexpected therapeutic properties.
COMPOUNDS OF THE INVENTION
[0030] In all cases except where indicated, letters and letters
with subscripts symbolizing variable substituents in the chemical
structures of the compounds of the invention are applicable only to
the structure immediately preceding the description of the
symbol.
[0031] The compounds useful in attenuating toxicity due to
anticancer or antiviral agents have the following general
structures:
[0032] (1) An acyl derivative of uridine having the formula: 1
[0033] wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are the same
or different and each is hydrogen or an acyl radical of a
metabolite, provided that at least one of said R substituents is
not hydrogen, or a pharmaceutically acceptable salt thereof.
[0034] (2) An acyl derivative of cytidine having the formula: 2
[0035] wherein R.sub.1, R.sub.2 R.sub.3 and R.sub.4 are the same or
different and each is hydrogen or an acyl radical of a metabolite,
provided that at least one of said R substituents is not hydrogen,
or a pharmaceutically acceptable salt thereof.
[0036] (3) An acyl derivative of deoxycytidine having the formula:
3
[0037] wherein R.sub.1, R.sub.2, and R.sub.3 are the same or
different and each is hydrogen or an acyl radical of a metabolite,
provided that at least one of said R substituents is not hydrogen,
or a pharmaceutically acceptable salt thereof.
[0038] (4) An acyl derivative of deoxyuridine having the formula:
4
[0039] wherein R.sub.1, R.sub.2, and R.sub.3 are the same or
different and each is hydrogen or an acyl radical of a metabolite,
provided that at least one of said R substituents is not hydrogen,
or a pharmaceutically acceptable salt thereof.
[0040] Compounds of the invention useful in ameliorating toxicity
due to anticancer or antiviral chemotherapy agents include the
following:
[0041] (5) An acyl derivative of uridine having the formula: 5
[0042] wherein R.sub.1, R.sub.2, and R.sub.3 are the same, or
different, and each is hydrogen or an acyl radical of
[0043] a. an unbranched fatty acid with 5 to 22 carbon atoms,
[0044] b. an amino acid selected from the group consisting of
glycine, the L forms of alanine, valine, leucine, isoleucine,
tyrosine, proline, hydroxyproline, serine, threonine, cystine,
cysteine, aspartic acid, glutamic acid, arginine, lysine,
histidine, carnitine and ornithine,
[0045] c. a dicarboxylic acid having 3-22 carbon atoms,
[0046] d. a carboxylic acid selected from one or more of the group
consisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvic
acid, lipoic acid, pantothenic acid, acetoacetic acid,
p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, and
creatine.
[0047] (6) An acyl derivatives of cytidine having the formula:
6
[0048] wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are the same,
or different, and each is hydrogen or an acyl radical of
[0049] a. an unbranched fatty acid with 5 to 22 carbon atoms,
[0050] b. an amino acid selected from the group consisting of
glycine, the L forms of phenylalanine, alanine, valine, leucine,
isoleucine, tyrosine, proline, hydroxyproline, serine, threonine,
cystine, cysteine, aspartic acid, glutamic acid, arginine, lysine,
histidine carnitine and ornithine,
[0051] c. a dicarboxylic acid having 3-22 carbon atoms,
[0052] d. a carboxylic acid selected from one or more of the group
consisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvic
acid, lipoic acid, pantothenic acid, acetoacetic acid,
p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, and
creatine.
[0053] (7) An acyl derivative of deoxycytidine, having the formula
7
[0054] wherein R.sub.1, R.sub.2, and R.sub.3 are the same, or
different, and each is hydrogen or an acyl radical derived from
[0055] a. an unbranched fatty acid with 3 to 22 carbon atoms,
[0056] b. an amino acid selected from the group consisting of
glycine, the L forms of alanine, valine, leucine, isoleucine,
tyrosine, proline, hydroxyproline, serine, threonine, cystine,
cysteine, aspartic acid, glutamic acid, arginine, lysine,
histidine, carnitine and ornithine,
[0057] c. nicotinic acid
[0058] d. a dicarboxylic acid having 3-22 carbon atoms, provided
that not all of R.sub.1, R.sub.2, and R.sub.3 are H, and where
R.sub.3 is not H, then R.sub.1 and/or R.sub.2 may also be acetyl,
or a pharmaceutically acceptable salt thereof.
[0059] (8) An acyl derivative of deoxyuridine, having the formula
8
[0060] wherein R.sub.1, R.sub.2, and R.sub.3 are the same, or
different, and each is hydrogen or an acyl radical derived from
[0061] a. an unbranched fatty acid with 3 to 22 carbon atoms,
[0062] b. an amino acid selected from the group consisting of
glycine, the L forms of alanine, valine, leucine, isoleucine,
tyrosine, proline, hydroxyproline, serine, threonine, cystine,
cysteine, aspartic acid, glutamic acid, arginine, lysine,
histidine, carnitine and ornithine,
[0063] c. nicotinic acid
[0064] d. a dicarboxylic acid having 3-22 carbon atoms, provided
that not all of R.sub.1, R.sub.2, and R.sub.3 are H, and where
R.sub.3 is not H, then R.sub.1 and/or R.sub.2 may also be acetyl,
or a pharmaceutically acceptable salt thereof.
[0065] (9) An acyl derivative of uridine having the formula: 9
[0066] wherein at least one of R.sub.1, R.sub.2, or R.sub.3 is a
hydrocarbyloxycarbonyl moiety containing 2-26 carbon atoms and the
remaining R substituents are independently a hydrocarbyloxycarbonyl
or hydrocarbylcarbonyl moiety or H or phosphate.
[0067] (10) An acyl derivative of cytidine having the formula:
10
[0068] wherein at least one of R.sub.1, R.sub.2, R.sub.3 or R.sub.4
is a hydrocarbyloxycarbonyl moiety containing 2-26 carbon atoms and
the remaining R substituents are independently a
hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety or H or
phosphate.
[0069] (11) An acyl derivative of deoxycytidine having the formula:
11
[0070] wherein at least one of R.sub.1, R.sub.2, or R.sub.3 is a
hydrocarbyloxycarbonyl moiety containing 2-26 carbon atoms and the
remaining R substituents are independently a hydrocarbyloxycarbonyl
or hydrocarbylcarbonyl moiety or H or phosphate.
[0071] (12) An acyl derivative of deoxyuridine having the formula:
12
[0072] wherein at least one of R.sub.1 or R.sub.2 is a
hydrocarbyloxycarbonyl moiety containing 2-26 carbon atoms and the
remaining R substituents are independently a hydrocarbyloxycarbonyl
or hydrocarbylcarbonyl moiety or H or phosphate.
[0073] The invention, as well as other objects, features and
advantages thereof will be understood more clearly and fully from
the following detailed description, when read with reference to the
accompanying results of the experiments discussed in the examples
below.
DETAILED DESCRIPTION OF THE INVENTION
[0074] The subject invention relates to the use of acylated
derivatives of non-methylated pyrimidine nucleosides, i.e. acylated
derivatives of uridine, deoxyuridine, cytidine, or deoxycytidine,
such as triacetyluridine (TAU), to attenuate toxicity of
chemotherapeutic agents and antiviral agents in vivo. The invention
also relates to the administration of these pyrimidine nucleoside
compounds, alone or in combinations, with or without other agents,
to animals.
[0075] In the case of many antineoplastic and antiviral
chemotherapy agents, exposure of affected cells to appropriate
natural nucleosides can prevent or ameliorate damage to those
cells. The compounds and methods of the subject invention make it
possible to reduce toxicity while maintaining therapeutic efficacy
of the antiviral or antineoplastic agent, and conversely, to
increase the dose of the chemotherapeutic agent while maintaining
an acceptable degree of toxicity.
[0076] The present invention provides compounds and methods for
treating or preventing toxic symptoms of antiviral or anticancer
chemotherapy through oral or parenteral administration of acyl
derivatives of non-methylated pyrimidine nucleosides.
[0077] A. Definitions
[0078] The term "non-methylated pyrimidine nucleoside" as used
herein means naturally occurring nucleosides other than thymidine
(5-methyldeoxyuridine) or 5-methylcytidine and other similar
naturally-occurring methylated nucleosides. Examples of
non-methylated pyridimidine nucleosides include uridine, cytidine,
deoxyuridine, and deoxycytidine.
[0079] The term "acyl derivative" as used herein means a derivative
of a non-methylated pyrimidine nucleoside in which a substantially
nontoxic organic acyl substituent derived from a carboxylic acid is
attached to one or more of the free hydroxyl groups of the ribose
moiety of a non-methylated pyrimidine nucleoside with an ester
linkage and/or where such a substituent is attached to the amine
substituent on the pyrimidine ring of cytidine or deoxycytidine,
with an amide linkage. Such acyl substituents are derived from
carboxylic acids which include, but are not limited to, compounds
selected from the group consisting of a fatty acid, an amino acid,
nicotinic acid, dicarboxylic acids, lactic acid, p-aminobenzoic
acid and orotic acid. Advantageous acyl substituents are carboxylic
acids which are normally present in the body, either as dietary
constituents or as intermediary metabolites.
[0080] The term "analog" as used herein means a nucleoside
chemically modified in either the pyrimidine ring or the ribose (or
deoxyribose) moiety by a means other than acylation or attachment
of other biologically labile substituents (e.g. phosphorylation of
hydroxyl groups on the sugar). Specifically, nucleoside analogs, in
the context of this invention, are drugs with structural
similarities to the naturally occurring nucleosides, but with
antiviral, antineoplastic, or cytotoxic properties. Examples of
antineoplastic nucleoside analogs include but are not limited to
the following: 5-fluorouracil (5-FU), 5-FU prodrugs (e.g. ftorafur,
5'-deoxyfluorouridine, carmofur), fluorouridine,
2'-deoxyfluorouridine, prodrug derivatives of fluorouridine or
2'-deoxyfluorouridine, fluorocytosine, arabinosyl cytosine,
prodrugs of arabinosyl cytosine, cyclocytidine,
5-aza-2'-deoxycytidine, arabinosyl 5-azacytosine, 6-azauridine,
azaribine, 6-azacytidine, trifluoro-methyl-2'-deoxyuridine,
thymidine, and 3-deazauridine. Examples of antiviral nucleoside
analogs include but are not limited to the following:
5-ethyl-2'-deoxyuridine, 5-iodo-2'-deoxyuridine,
5-bromo-2'-deoxyuridine, 5-methylamino-2'-deoxyuridine,
arabinosyluracil, dideoxyuridine, dideoxycytidine,
2',3'-dideoxycytidin-2'-ene, 3'-deoxythymidin-2'-ene,
3'-azido-2',3'-dideoxyuridine, and 3'-azidodeoxythymidine (AZT).
Analogs of pyrimidine nucleoside precursors, e.g.
N-phosphonoacetyl-L-aspartic acid (PALA), are encompassed by this
term.
[0081] Some nucleoside analogs are considered to have structural
similarities to particular naturally-occurring nucleosides. In the
context of the compounds of the invention, nucleoside analogs are
divided into cytidine analogs if they have an exocyclic amino group
in the 4 position of the pyrimidine ring (an amino group in that
position signifies the distinction between cytidine and uridine).
Nucleoside analogs that are specifically analogs of cytidine
include but are not limited to: fluorocytosine, arabinosyl
cytosine, prodrugs of arabinosyl cytosine, cyclocytidine,
5-aza-2'-deoxycytidine, arabinosyl 5-azacytosine, 6-azacytidine,
and dideoxycytidine. Nucleoside analogs that are specifically
considered to be analogs of uridine include but are not limited to:
5-fluorouracil (5-FU), 5-FU prodrugs (e.g. ftorafur,
5'-deoxyfluorouridine, carmofur), fluorouridine,
2'-deoxyfluorouridine, prodrug derivatives of fluorouridine,
prodrug derivatives of 2'-deoxyfluorouridine,
trifluoromethyl-2'-deoxyuridine, 6-azauridine, azaribine,
3-deazauridine, 5-ethyl-2'-deoxyuridine, 5-iodo-2'-deoxyuridine,
5-bromo-2'-deoxyuridine, 5-methylamino-2'-deoxyur- idine,
arabinosyluracil, and dideoxyuridine. Some cytotoxic nucleoside
analogs are also specifically analogs of thymidine, e.g. AZT.
[0082] The term "pharmaceutically acceptable salts" as used herein
means salts with pharmaceutically acceptable acid addition salts of
the derivatives, which include, but are not limited to, sulfuric,
hydrochloric, or phosphoric acids.
[0083] The term "coadministered" as used herein means that at least
two of the compounds of the invention are administered during a
time frame wherein the respective periods of pharmacological
activity overlap.
[0084] The term "hydrocarbylcarbonyl" as used herein means an acyl
radical of a carboxylic acid in which the atom adjacent to the
carbonyl carbon atom is another carbon atom. The parent carboxylic
acid may, for example, be a fatty acid, an aromatic acid (e.g.
benzoate, nicotinoate, or their congeners), an amino acid, a
cycloalkylcarboxylic acid, or a dicarboxylic acid.
[0085] The term "hydrocarbyloxycarbonyl" as used herein means an
acyl radical of a carboxylic acid in which the atom adjacent to the
carbonyl carbon atom is oxygen which is furthermore covalently
linked to another carbon atom. This can also be described as a
radical of a carbonate ester of an alcohol, which, when cleaved
from a non-methylated pyrimidine nucleoside following
administration, degrades further into carbon dioxide and an
alcohol. Advantageous alcohols are those which are of low toxicity,
particularly those which enter readily into normal metabolic or
eliminative pathways.
[0086] The term "fatty acids" as used herein means aliphatic
carboxylic acids having 2-22 carbon atoms. Such fatty acids may be
saturated, partially saturated or polyunsaturated.
[0087] The term "amino acids" as used herein includes, but is not
limited to, glycine, the L forms of alanine, valine, leucine,
isoleucine, phenylalanine, tyrosine, proline, hydroxyproline,
serine, threonine, cysteine, cystine, methionine, tryptophan,
aspartic acid, glutamic acid, arginine, lysine, histidine,
ornithine, hydroxylysine, carnitine, and other naturally occurring
amino acids.
[0088] The term "dicarboxylic acids" as used herein means fatty
acids with a second carboxylic acid substituent.
[0089] The term "therapeutically effective amount" as used herein
refers to that amount which provides therapeutic effects for a
given condition and administration regime.
[0090] B. Compounds of the Invention
[0091] In all cases except where indicated, letters and letters
with subscripts symbolizing variable substituents in the chemical
structures of the compounds of the invention are applicable only to
the structure immediately preceding the description of the
symbol.
[0092] The compounds useful in attenuating toxicity due to
anticancer or antiviral agents have the following general
structures:
[0093] (1) An acyl derivative of uridine having the formula: 13
[0094] wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are the same
or different and each is hydrogen or an acyl radical of a
metabolite, provided that at least one of said R substituents is
not hydrogen, or a pharmaceutically acceptable salt thereof.
[0095] (2) An acyl derivative of cytidine having the formula:
14
[0096] wherein R.sub.1, R.sub.2, R.sub.3 and R.sub.4 are the same
or different and each is hydrogen or an acyl radical of a
metabolite, provided that at least one of said R substituents is
not hydrogen, or a pharmaceutically acceptable salt thereof.
[0097] (3) An acyl derivative of deoxycytidine having the formula:
15
[0098] wherein R.sub.1, R.sub.2, and R.sub.3 are the same or
different and each is hydrogen or an acyl radical of a metabolite,
provided that at least one of said R substituents is not hydrogen,
or a pharmaceutically acceptable salt thereof.
[0099] (4) An acyl derivative of deoxyuridine having the formula:
16
[0100] wherein R.sub.1, R.sub.2, and R.sub.3 are the same or
different and each is hydrogen or an acyl radical of a metabolite,
provided that at least one of said R substituents is not hydrogen,
or a pharmaceutically acceptable salt thereof.
[0101] Compounds of the invention useful in ameliorating toxicity
due to anticancer or antiviral chemotherapy agents include the
following:
[0102] (5) An acyl derivative of uridine having the formula: 17
[0103] wherein R.sub.1, R.sub.2, and R.sub.3 are the same, or
different, and each is hydrogen or an acyl radical of
[0104] a. an unbranched fatty acid with 5 to 22 carbon atoms,
[0105] b. an amino acid selected from the group consisting of
glycine, the L forms of alanine, valine, leucine, isoleucine,
tyrosine, proline, hydroxyproline, serine, threonine, cystine,
cysteine, aspartic acid, glutamic acid, arginine, lysine,
histidine, carnitine and ornithine,
[0106] c. a dicarboxylic acid having 3-22 carbon atoms,
[0107] d. a carboxylic acid selected from one or more of the group
consisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvic
acid, lipoic acid, pantothenic acid, acetoacetic acid,
p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, and
creatine.
[0108] (6) An acyl derivatives of cytidine having the formula:
18
[0109] wherein R.sub.1, R.sub.2, R.sub.3, and R.sub.4 are the same,
or different, and each is hydrogen or an acyl radical of
[0110] a. an unbranched fatty acid with 5 to 22 carbon atoms,
[0111] b. an amino acid selected from the group consisting of
glycine, the L forms of phenylalanine, alanine, valine, leucine,
isoleucine, tyrosine, proline, hydroxyproline, serine, threonine,
cystine, cysteine, aspartic acid, glutamic acid, arginine, lysine,
histidine carnitine and ornithine,
[0112] c. a dicarboxylic acid having 3-22 carbon atoms,
[0113] d. a carboxylic acid selected from one or more of the group
consisting of glycolic acid, pyruvic acid, lactic acid, enolpyruvic
acid, lipoic acid, pantothenic acid, acetoacetic acid,
p-aminobenzoic acid, betahydroxybutyric acid, orotic acid, and
creatine.
[0114] (7) An acyl derivative of deoxycytidine, having the formula
19
[0115] wherein R.sub.1, R.sub.2, and R.sub.3 are the same, or
different, and each is hydrogen or an acyl radical derived from
[0116] a. an unbranched fatty acid with 3 to 22 carbon atoms,
[0117] b. an amino acid selected from the group consisting of
glycine, the L forms of alanine, valine, leucine, isoleucine,
tyrosine, proline, hydroxyproline, serine, threonine, cystine,
cysteine, aspartic acid, glutamic acid, arginine, lysine,
histidine, carnitine and ornithine,
[0118] c. nicotinic acid
[0119] d. a dicarboxylic acid having 3-22 carbon atoms, provided
that not all of R.sub.1, R.sub.2, and R.sub.3 are H, and where
R.sub.3 is not H, then R.sub.1 and/or R.sub.2 may also be acetyl,
or a pharmaceutically acceptable salt thereof.
[0120] (8) An acyl derivative of deoxyuridine, having the formula
20
[0121] wherein R.sub.1, R.sub.2, and R.sub.3 are the same, or
different, and each is hydrogen or an acyl radical derived from
[0122] a. an unbranched fatty acid with 3 to 22 carbon atoms,
[0123] b. an amino acid selected from the group consisting of
glycine, the L forms of alanine, valine, leucine, isoleucine,
tyrosine, proline, hydroxyproline, serine, threonine, cystine,
cysteine, aspartic acid, glutamic acid, arginine, lysine,
histidine, carnitine and ornithine,
[0124] c. nicotinic acid
[0125] d. a dicarboxylic acid having 3-22 carbon atoms, provided
that not all of R.sub.1, R.sub.2, and R.sub.3 are H, and where
R.sub.3 is not H, then R.sub.1 and/or R.sub.2 may also be acetyl,
or a pharmaceutically acceptable salt thereof.
[0126] (9) An acyl derivative of uridine having the formula: 21
[0127] wherein at least one of R.sub.1, R.sub.2, or R.sub.3 is a
hydrocarbyloxycarbonyl moiety containing 2-26 carbon atoms and the
remaining R substituents are independently a hydrocarbyloxycarbonyl
or hydrocarbylcarbonyl moiety or H or phosphate.
[0128] (10) An acyl derivative of cytidine having the formula:
22
[0129] wherein at least one of R.sub.1, R.sub.2, R.sub.3 or R.sub.4
is a hydrocarbyloxycarbonyl moiety containing 2-26 carbon atoms and
the remaining R substituents are independently a
hydrocarbyloxycarbonyl or hydrocarbylcarbonyl moiety or H or
phosphate.
[0130] (11) An acyl derivative of deoxycytidine having the formula:
23
[0131] wherein at least one of R.sub.1, R.sub.2, or R.sub.3 is a
hydrocarbyloxycarbonyl moiety containing 2-26 carbon atoms and the
remaining R substituents are independently a hydrocarbyloxycarbonyl
or hydrocarbylcarbonyl moiety or H or phosphate.
[0132] (12) An acyl derivative of deoxyuridine having the formula:
24
[0133] wherein at least one of R.sub.1 or R.sub.2 is a
hydrocarbyloxycarbonyl moiety containing 2-26 carbon atoms and the
remaining R substituents are independently a hydrocarbyloxycarbonyl
or hydrocarbylcarbonyl moiety or H or phosphate.
[0134] Also encompassed by the invention are the pharmaceutically
acceptable salts of the above-noted compounds.
[0135] Advantageous compounds of the invention are fatty acid
esters of uridine and deoxycytidine, especially those with 4 or
fewer carbon atoms in the acyl substituent.
Particularlyadvantageous compounds are fatty acid esters of uridine
or deoxycytidine with 2 or 3 carbon atoms in the acyl
substituent.
[0136] Other advantageous compounds of the invention are
hydrocarbyloxycarbonyl derivatives of uridine and deoxycytidine,
particularly those with 3 to 6 carbon atoms in the
hydrocarbyloxycarbonyl moiety.
[0137] In one embodiment of the invention, prodrugs of the
compounds of the invention with enhanced water solubility are
prepared by attaching phosphate to a free hydroxyl group on the
aldose moiety of the acylated non-methylated pyrimidine
nucleoside.
[0138] C. Compositions of the Invention
[0139] Compositions of the invention include one or more of the
above-noted compounds along with a pharmaceutically acceptable
carrier.
[0140] In another embodiment, the compositions of the invention
include in addition to one or more compounds of the invention and
at least one of the following agents which enhance hematopoiesis:
oxypurine nucleosides, congeners of oxypurine nucleosides, and acyl
derivatives of oxypurine nucleosides and their congeners, e.g.
fatty acid esters of guanosine or deoxyguanosine (see U.S. Ser. No.
653,882, filed, Feb. 8, 1991, hereby incorporated by reference), a
nonionic surfactant, an interleukin such as
IL-1,-2,-3,-4,-5,-6,-7,-8 (advantageously IL-1, 3, or 6), a
colony-stimulating factor, for example granulocyte
colony-stimulating factor (G-CSF), granulocyte/macrophage
colony-stimulating factor (GM-CSF), stem cell factor (SCF),
erythropoietin (EPO), glucan, polyinosine-polycytidine, or any
other agent having beneficial effects on hematopoiesis.
[0141] Acyl derivatives of oxypurine nucleosides which enhance
hematopoiesis and which are optionally administered in conjunction
with the compounds of the invention have the following general
structure:
[0142] R.sub.A.dbd.H or an acyl radical of a carboxylic acid with 2
to 30 carbon atoms, and
[0143] R.sub.B.dbd.H or an acyl radical of a carboxylic acid with 2
to 30 carbon atoms, and
[0144] Z.dbd.H, OH, .dbd.O, or NHR.sub.C where R.sub.C.dbd.H or an
acyl radical of a carboxylic acid with 2 to 30 carbon atoms,
and
[0145] L.dbd.H or OR.sub.D, where R.sub.D.dbd.H or an acyl radical
of a carboxylic acid with 2 to 30 carbon atoms, and
[0146] M.dbd.H or OR.sub.E, where R.sub.E.dbd.H or an acyl radical
of a carboxylic acid with 2 to 30 carbon atoms, with the proviso
that at least one of L and M is H, and
[0147] Q.dbd.H, a halogen, NHR.sub.F where R.sub.F is H or an acyl
or alkyl radical containing 1 to 10 carbon atoms, S divalently
bound to the carbon in which case the adjacent carbon-nitrogen
double bond is a single bond and an H is then attached to that
nitrogen, SR.sub.G where R.sub.G is H or an acyl or alkyl radical
containing 1 to 10 carbon atoms, O divalently bound to the carbon,
in which case the adjacent carbon-nitrogen double bond is a single
bond and an H is then attached to that nitrogen, or OR.sub.H where
R.sub.H is H or an acyl or alkyl radical containing 1 to 10 carbon
atoms, and
[0148] the C--C bond between the 2' and 3' positions of the aldose
moiety is optionally present.
[0149] In another embodiment of the invention, an acylated
non-methylated pyrimidine nucleoside is formulated with a compound
capable of enhancing the uptake and phosphorylation of nucleosides
into cells such as insulin or an insulinogenic carbohydrate.
[0150] In another embodiment of the invention, the
compositioncomprises at least one compound of the invention and an
antiviral or antineoplastic agent (see detailed discussion of these
agents in the section below entitled Therapeutic Uses of the
Compounds and Compositions of the Invention).
[0151] In another embodiment, the compositions of the invention
comprise an acyl derivative of uridine or deoxyuridine and a
compound capable of inhibiting uridine phosphorylase. Uridine
phosphorylase is the primary enzyme involved in the catabolism of
uridine, forming uracil and ribose phosphate. Administration of a
compound which inhibits uridine phosphorylase will modify the
pharmacokinetics and biological activity of uridine or deoxyuridine
produced by deacylation of acylated derivatives of these two
non-methylated pyrimidine nucleosides. Examples of suitable
inhibitors of uridine phosphorylase include but are not limited to
5-benzyl barbiturate or 5-benzylidene barbiturate derivatives
including 5-benzyl barbiturate, 5-benzyloxybenzyl barbiturate,
5-benzyloxybenzyl-1-[(1-hydroxy-2-ethoxy)methyl] barbiturate,
5-benzyloxybenzylacetyl-1-[(1-hydroxy-2-ethoxy)methyl] barbiturate,
and 5-methoxybenzylacetylacyclobarbiturate,
2,2'-anhydro-5-ethyluridine, and acyclouridine compounds,
particularly 5-benzyl substituted acyclouridine congeners including
but not limited to benzylacyclouridine,
benzyloxybenzylacyclouridine, aminomethyl-benzylacyclouridine,
aminomethyl-benzyloxybenzylacyclouridine,
hydroxymethylbenzylacyclouridin- e, and
hydroxymethyl-benzyloxybenzylacyclouridine. See also WO 89/09603
and WO 91/16315, hereby incorporated by reference.
[0152] In another embodiment of the invention, the composition
comprises an acyl derivative of a non-methylated pyrimidine
nucleoside and a compound which inhibits cellular uptake or
excretion of non-methylated pyrimidine nucleosides, and thereby
promotes maintenance of blood nucleoside levels after enzymatic
deacylation of administered doses of acylated derivatives of
non-methylated pyrimidine nucleosides. Such modulators of uridine
transport or excretion include but are not limited to dipyridamole,
dilazep, probenicid, lidoflazine or nitrobenzylthioinosine.
[0153] In another embodiment of the invention, the composition
comprises an acyl derivative of cytidine and a compound capable of
inhibiting the enzyme uridine phosphorylase. Inhibition of this
enzyme is useful in conjunction with cytidine since cytidine is in
part deaminated in the bloodstream after deacylation of its acyl
derivatives, providing uridine to the tissues.
[0154] In another embodiment of the invention, the composition
comprises an acyl derivative of cytidine or deoxycytidine and a
compound capable of inhibiting deoxycytidine deaminase. By
inhibiting the deamination of deoxycytidine or cytidine, inhibitors
of cytidine deaminase or deoxycytidine deaminase such as
tetrahydrouridine or tetrahydro-2'-deoxyuridine modify the efficacy
of acyl derivatives of cytidine or deoxycytidine. In another
embodiment of the invention, an inhibitor of cytidine deaminase or
deoxycytidine deaminase is used to modify the toxicity of an
antiviral or anticancer nucleoside analog (see Example 11).
[0155] In another embodiment of the invention, especially for
prevention or treatment of damage to the gastrointesinal mucosa,
the composition comprises an acyl derivative of a non-methylated
pyrimidine nucleoside and an agent or agents with utility in
promoting mucosal healing or in reducing discomfort. Examples of
such agents include but are not limited to sucralfate, mixtures of
two or more deoxyribonucleosides as disclosed in U.S. patent
application Ser. No. 341,925, filed Apr. 21, 1989 (hereby
incorporated by reference), allopurinol, antibiotics like
chlorhexidine gluconate or local anesthetics like benzocaine.
[0156] In another embodiment of the invention, the composition
comprises a combination of an acyl derivative of a non-methylated
pyrimidine nucleoside and an orally-active antineoplastic
nucleoside analog. An advantageous combination is an acyl
derivative of uridine with an orally active fluorinated
pyrimidines, especially prodrugs of 5-fluorouracil. In such
compositions, the acyl derivative of a non-methylated pyrimidine
nucleoside is mixed with (or otherwise adminstered with) the
antineoplastic nucleoside analog in molar ratios, ranging from 1:1
to 12:1. Molar ratios ranging from 2:1 to 8:1 are generally
advantageous. Suitable orally-active fluorinated pyrimidines
include tegafur, 5'-deoxyfluorouridine, 5-fluorouracil,
5-fluorouridine, 2'-deoxy-5-fluorouridine,
N.sup.4-trimethoxybenzoyl-5'-deoxy-5-fluorocyti- dine, or acyl
derivatives thereof.
[0157] The compositions, depending on the intended use, are
manufactured in the form of a liquid, a suspension, a tablet, a
capsule, a dragee, an injectable solution, a topical solution, or a
suppository (see discussion of formulation below).
[0158] As an alternative to formulation of compositions containing
a compound of the invention and another active agent (as discussed
above), in another embodiment, the compounds of the invention are
coadministered with the other active agents.
[0159] D. Therapeutic Uses of the Compounds and Compositions of the
Invention
[0160] The compounds of the invention are useful to prevent or
treat damage to the process of hematopoiesis and immune system
function in animals. The compounds reduce damage to the process of
hematopoiesis by minimizing loss in blood cell counts after bone
marrow damage or suppression caused by antiviral or antineoplastic
agents which affect nucleotide biosynthesis, metabolism, or
utilization. The compounds of the invention are useful in treating
humans; however, the invention is not intended to be so limited, it
being within the contemplation of the invention to treat all
animals that experience a beneficial effect from the administration
of the active compounds of the invention.
[0161] The invention is furthermore embodied in the administration
of a pharmaceutical compound or composition of the invention, or in
combinations, for the purpose of preventing, attenuating, or
ameliorating toxicity associated with administration of antiviral
or antineoplastic agents which affect nucleotide biosynthesis,
metabolism, or utilization.
[0162] Specific conditions where advantages are achieved using the
compounds, compositions, and methods of the invention include
situations where the hematopoietic system has suffered or is likely
to suffer damage from chemotherapy, particularly chemotherapy that
affects nucleotide biosynthesis, metabolism, or utilization. Such
conditions include treating animals, e.g. human patients, subjected
to cytoreductive cancer chemotherapy or antiviral chemotherapy.
Specifically included are veterinary applications requiring
maintenance of blood cell counts.
[0163] The compounds and compositions are also useful for
preventing or treating damage caused by anticancer or antiviral
chemotherapy agents to other tissues, including but not limited to
gastrointestinal epithelium. For this purpose, the compounds and
compositions are optionally administered orally, as a suppository,
or parenterally.
[0164] By attenuating damage to the hematopoietic and immune
systems caused by anticancer or antiviral chemotherapy, the
compounds and methods of the invention reduce the risk of
susceptibility to opportunistic or secondary infections (bacterial,
viral, or fungal).
[0165] The efficacy of the compounds of the invention is enhanced
by coadministration of agents which stimulate the uptake and
phosphorylation of pyrimidine nucleosides by cells. Such agents
include hematopoietic growth factors (e.g. G-CSF, GM-CSF, SCF,
acylated oxypurine nucleosides and their congeners, erythropoietin,
and interleukins), insulin, and insulinogenic carbohydrates such as
glucose or glucose polymers.
[0166] Treatment of Complications Associated with Cancer
Chemotherapy
[0167] The white blood cell counts, and particularly the neutrophil
counts, of patients treated with standard anti-neoplastic
chemotherapy agents (e.g., 5-fluorouracil, fluorodeoxyuridine,
vinca alkaloids, cyclophosphamide and other nitrogen mustard
alkylating agents, daunorubicin, doxorubicin, methotrexate,
cytosine arabinoside, 6-mercaptopurine, thioguanosine,
podophyllotoxins, cisplatin or combinations of such cytoreductive
agents) are often greatly diminished. In the case of cytotoxic
agents which act by affecting nucleotide biosynthesis, metabolism,
or utilization, daily administration (oral or parenteral) of an
effective dose, (for example, 0.1-10.0 grams) of a compound of the
invention such as triacetyluridine (or other acyl derivatives of
uridine, cytidine, deoxycytidine, or deoxyuridine) for several days
reduces the severity of the neutropenia which typically occurs
several days to several weeks after chemotherapy is initiated. This
reduces the likelihood of infection throughout the course of
treatment, and makes it possible for the patient to receive larger
doses of the chemotherapeutic agents and/or to receive repeated
doses sooner than comparable patients not treated with the uridine
derivative(s). Similarly, chemotherapy-induced alterations in
counts of other blood cell types (lymphocytes, platelets,
erythrocytes, etc.) are ameliorated by administration of the
compounds and compositions of the invention.
[0168] Antineoplastic agents with which the compounds and methods
of the invention are particularly useful include: 5-fluorouracil
(5-FU), 5-FU prodrugs (e.g. ftorafur, 5'-deoxyfluorouridine,
carmofur), fluorouridine, 2'-deoxyfluorouridine, prodrug
derivatives of fluorouridine or 2'-deoxyfluorouridine,
fluorocytosine (which also has antifungal activity), arabinosyl
cytosine, prodrugs of arabinosyl cytosine, cyclocytidine,
5-aza-2'-deoxycytidine, arabinosyl 5-azacytosine,
N-phosphonoacetyl-L-aspartic acid (PALA), pyrazofurin,
6-azauridine, azaribine, 6-azacytidine,
trifluoro-methyl-2'-deoxyuridine, thymidine, and 3-deazauridine.
Such antineoplastic agents and various other therapeutic nucleoside
analogs act by affecting nucleoside or nucleotide biosynthesis,
utilization, or metabolism; hence, amelioration of their toxic
effects is accomplished by administration of the pyrimidine
compounds of the invention.
[0169] In addition to reduction of toxicity of antineoplastic
nucleoside analogs, acyl derivatives of nonmethylated pyrimidine
nucleosides are also useful for reduction of toxicity of
antineoplastic agents that are cleared from the body via
glucuronidation. Antineoplastic agents that are eliminated by
glucuronidation include but are not limited to epirubicin and
camptothecins like irinotecan and topotecan. In this process,
glucuronic acid is attached to toxic compounds to facilitate their
elimination. Uridine diphosphoglucuronic acid (UDPGA) is necessary
for attachment of glucuronic acid to other molecules.
Administration of an acyl derivative of either uridine or cytidine
increases cellular UDPGA levels and thereby enhances
glucuronidation of toxic compounds. In this situation, the acyl
derivative of cytidine or uridine is administered before or at the
same time as the antineoplastic agent. In a typical clinical
situation, 1 to 10 grams of an acyl derivative of uridine is
administered once to four times prior to or during administration
of the antineoplastic compound.
[0170] The compounds of the invention are administered before,
during, and/or after administration of the anti-neoplastic or
antiviral agents. Typically, the compounds of the invention are
administered after a dose of a cancer chemotherapy agent, as a
means of "rescuing" normal tissues after administration of an
effective antineoplastic dose of the agent.
[0171] Gastrointestinal epithelium is sensitive to cancer
chemotherapy agents like fluorouracil. Mucositis, stomatitis, or
ulceration of the gastrointestinal mucosa are common side effects
of cancer chemotherapy, resulting in discomfort, diarrhea,
electrolyte imbalances and weight loss. The compounds of and
compositions of the invention are useful in preventing or treating
damage to the gastrointestinal tract (including the mouth) caused
by cancer chemotherapy agents. The compounds and compositions of
the invention are optionally administered for this purpose as a
solution or suspension in liquid form (as a mouthwash, as a
composition to be swallowed, or as an enema), as a capsule, dragee,
or tablet, as an injectable solution, or as a suppository. Systemic
administration of the compounds and compositions of the invention
also reduces damage to gastrointestinal mucosa caused by anticancer
or antiviral nucleoside analogs.
[0172] Topical application of the compounds (e.g. to the scalp) of
the invention is useful for preventing chemotherapy-induced
alopecia.
[0173] Acyl derivatives of uridine are advantageous in preventing
or treating toxicity due to fluorouracil or related fluorinated
analogs of uridine (e.g. fluorouridine or prodrugs thereof,
fluorodeoxyuridine or prodrugs thereof, ftorafur,
5'-deoxyfluorouridine). For oral administration, advantageous acyl
derivatives of uridine are those substituted with short-chain fatty
acids, (especially acetate) or with short chain carbyloxycarbonates
(e.g. ethoxycarbonate). Acyl derivatives of cytidine or
deoxyuridine are also useful in treating toxicity due to
fluorouracil or related fluorinated pyrimidine analogs.
[0174] In a typical therapeutic situation, a patient receives a
dose of fluorouracil, either as a single treatment agent or as part
of a regimen also involving administration of other antineoplastic
drugs like methotrexate, leucovorin, PALA, or cyclophosphamide.
Several hours to one day (i.e. 2 to 24 hours) after administration
of the 5-FU, the patient receives an oral dose of 1 to 10 grams of
triacetyluridine. The patient receives additional doses of TAU of
similar size every 6 to 8 hours over the course of the next 1 to 4
days. The patient may receive additional courses of 5-FU plus TAU
on a weekly basis or less frequently.
[0175] For treatment of cancer in humans, 5FU is often administered
as a weekly bolus injection, for a period of six weeks. The normal
maximum tolerated dose of 5FU in such a regimen is 500 to 600
mg/m.sup.2. Delayed administration (2 to 24 hours after 5FU) of an
acyl derivative of a nonmethylated pyrimidine nucleoside permits
escalation of the weekly 5FU dose to levels greater than 1.5 times
the normal maximum tolerated dose (i.e. greater than 900
mg/m.sup.2), advantageously 900 to 2000 mg/m.sup.2, more
advantageously 1000 to 1600 mg/m.sup.2. An alternative method for
use of 5FU in the clinic involves injection of doses from 400 to
500 mg/m.sup.2 daily for four or five days in a row. This
administration regimen is repeated every three to four weeks.
Delayed administration of an acyl derivative of a nonmethylated
pyrimidine nucleoside in this case permits similar 5FU dose
escalation to levels at least 1.5 fold greater than normal
tolerated doses, advantageously 600 to 1000 mg/m.sup.2, more
advantageously 700 to 900 mg/m.sup.2. In the case of daily 5FU
administration, delayed administration of an acyl derivative of a
nonmethylated pyrimidine nucleoside occurs 2 to 12 hours after each
5FU dose. Another dosing regimen for 5FU involves infusion of a
total dose of 1800 to 2600 mg/m.sup.2 over 24 hours repeated once
every week to once every three to four weeks. When 5FU is
administered as an infusion (compared to a bolus) high cumulative
doses are administered because of extensive degradation of 5FU. In
the case of a 24 hour 5FU infusion, an acyl derivative of a
nonmethylated pyrimidine nucleoside is administered 2 to 24 hours
after the termination of the 5FU infusion, permitting escalation of
the 5FU dose by at least 1.5 fold over normal tolerated doses,
advantageously 2800 to 4000 mg/m.sup.2/24 hours.
[0176] An alternative regimen for use of high-dose 5FU with delayed
administration of an acyl derivative of a nonmethylated pyrimidine
nucleoside is weekly bolus administration of 5FU for 3 weeks,
followed by one or more weeks of rest. This regimen permits
administration of higher 5FU doses per week than the common weekly
.times.6 regimen described above. In a weekly .times.3 regimen
utilizing an acyl derivative of a nonmethylated pyrimidine
nucleoside for reducing 5FU toxicity, bolus 5FU doses in the range
of 1000 to 2400 mg/m.sup.2 are advantageous.
[0177] In all regimens involving escalation of the dose of an
antineolastic nucleoside analog with delayed administration of an
acyl derivative of a nonmethylated pyrimidine nucleoside, the
maximum dose of the antineoplastic nucleoside analog is the dose
which produces the maximum clinically acceptable degree of toxicity
to the recipient.
[0178] Clinical response to 5FU is a function of weekly dose
intensity (Hryniuk et al. Seminars in Oncology, 14 (No. 4; Suppl
4): 3-11 [1987]). 5FU dose escalation in conjunction with delayed
administration of an acyl derivative of a nonmethylated pyrimidine
nucleoside increases the antitumor efficacy of 5FU. Other
modulators of 5FU efficacy including but not limited to
methotrexate, trimetrexate, methylmercaptopurine riboside, PALA,
leucovorin, inhibitors of 5FU catabolism, levamisole, interferon,
or cisplatin are optionally administered in conjunction with 5FU
and an acyl derivative of a nonmethylated pyrimidine nucleoside.
When used with 5FU in conjunction with an acyl derivative of a
nonmethylated pyrimidine nucleoside, such modulators are
administered in doses and schedules similar or identical to thoses
determined suitable for their use with 5FU in the absence of
delayed administration of a an acyl derivative of a nonmethylated
pyrimidine nucleoside. Some such modulators also increase 5FU
toxicity and thereby reduce the maximum tolerated dose that may be
administered to patients. However, in these cases, delayed
administration of an acyl derivative of a nonmethylated pyrimidine
nucleoside permits escalation of the 5FU dose to levels more than
1.5 fold greater than could be tolerated in the absence of an acyl
derivative of a nonmethylated pyrimidine nucleoside. Similarly,
delayed administration of an acyl derivative of a nonmethylated
pyrimidine nucleoside permits escalation of the normal maximum
tolerated dose of other fluorinated pyrimidines, 5FU prodrugs, or
other cytotoxic nucleoside analogues.
[0179] There are individual differences in the sensitivity of
patients to 5FU. However, in any individual patient, use of an acyl
derivative of a nonmethylated pyrimidine nucleoside in conjunction
with 5FU permits dose escalation of the fluorinated pyrimidine to
at least 1.5 fold greater than the normal maximum tolerated dose.
The normal maximum tolerated dose of 5FU for any individual patient
is determined either by 5FU challenge, by judgment based on a
physician's experience, or by estimation guided by measurements of
enzymes involved in 5FU degradation or anabolism.
[0180] 5FU itself can be administered in conjunction with a
compound which inhibits 5FU degradation by the enzyme
dihydropyrimidine dehydrogenase (DPD). DPD is the initial enzyme in
5FU degradation and is found in high concentrations in the liver.
Inhibition of this enzyme reduces the rate of 5FU clearance from
the bloodstream by the liver and other tissues and thereby permits
oral administration of 5FU. DPD inhibition also reduces the
formation of 5FU catabolites which might contribute to its toxicity
or which might reduce its antitumor efficacy. Examples of
inhibitors of DPD include but are not limited to 5-ethynyluracil,
bromovinyluracil, uracil, thymine, or benzyloxybenzyluracil
(BBU).
[0181] In one embodiment of the invention, 5FU or a 5FU prodrug is
administered in conjunction with an inhibitor of the enzyme DPD. In
this embodiment, an inhibitor of DPD is administered prior to, or
at the same time as, a 5FU or another cytotoxic fluorinated
pyrimidine. An acyl derivative of uridine or cytidine is
administered 2 to 24 hours after the 5FU. Delayed administration of
an acyl derivative of uridine or cytidine permits safe
administration of higher doses of 5FU than are normally tolerated
in conjunction, with a consequent improvement in antitumor
efficacy.
[0182] Acyl derivatives of uridine and, secondarily, cytidine, are
also advantageous for treatment or prevention of toxicity due to
N-phosphonoacetyl-L-aspartic acid (PALA), pyrazofurin,
6-azauridine, azaribine, trifluoro-methyl-2'-deoxyuridine, and
3-deazauridine.
[0183] For modulating toxicity and efficacy of orally-active
antineoplastic drugs, particularly orally-active fluorinated
pyrimidine or prodrugs of fluorinated pyrimidines (such as 5'
deoxyfluorouridine derivatives like tegafur
(5-fluoro-1-(tetrahydro-2-furfuryl)uracil), 5'-deoxyfluorouridine,
or related derivatives), acyl derivatives of non-methylated
pyrimidine nucleosides may be used in several ways. In one
embodiment of the invention, the acyl derivative of a
non-methylated pyrimidine nucleoside is administered several hours
to one day after a dose of a fluorouracil prodrug such as tegafur,
similar to the situation with parenteral administration of
fluorouracil described above. In this context, the delayed
administration of the acyl derivative of a non-methylated
pyrimidine nucleoside results in reduced toxicity of the
fluorinated pyrimidine toward normal tissues. In another embodiment
of the invention, the acyl derivative of a non-methylated
pyrimidine nucleoside is administered at the same time as, or
within about an hour of, the orally-active antineoplastic
agent.
[0184] Tegafur, an orally active 5-fluorouracil prodrug, is
currently administered clinically in a formulation containing
uracil in a molar ratio of four parts uracil to one part tegafur.
In this context, uracil potentiates the antitumor efficacy of
5-fluorouracil produced by degradation of tegafur (during and after
absorption from the intestinal tract into the bloodstream) by
competing with 5-fluorouracil for the enzyme which breaks down both
pyrimidine molecules, dihydropyrimidine dehydrogenase. However,
uracil also potentiates the toxicity of 5-fluorouracil toward
normal tissues, particularly the intestine. Gastrointestinal damage
is the primary dose-limiting toxicity of a mixture of tegafur and
uracil. Co-administration of tegafur (or other orally active
antineoplastic pyrimidine analogs) with an acyl derivative of a
non-methylated pyrimidine nucleoside results in the desirable
potentiation of the systemic toxicity of, for example,
5-fluorouracil derived from tegafur, without potentiating its
toxicity toward the intestinal mucosa as drastically as does uracil
itself. The acyl derivative of a non-methylated pyrimidine
nucleoside and the orally active antineoplastic nucleoside analog
are administered in ways and at dosages and molar ratios typically
used for administration of uracil and an orally active
antineoplastic agent. See, for example, U.S. Pat. No. 4,328,229,
hereby incorporated by reference. Higher doses of the orally active
antineoplastic agent are also possible with the use of the acyl
derivatives of the subject invention. This embodiment of the
invention is experimentally demonstrated in Examples 13 and 14
below.
[0185] Acyl derivatives of deoxycytidine are advantageous in
treating or preventing toxicity due to antineoplastic nucleoside
analogs that are specifcally analogs of cytidine, e.g. arabinosyl
cytosine or prodrugs thereof, cyclocytidine,
5-aza-2'-deoxycytidine, arabinosyl 5-azacytosine, or 6-azacytidine.
For oral administration, advantageous acyl derivatives of
deoxycytidine are those substituted with short-chain fatty acids,
especially acetate.
[0186] In a typical clinical situation involving the use of
arabinosyl cytosine or related antineoplastic analogs of cytidine,
which are primarily utilized for treatment of leukemias, the acyl
derivative(s) of deoxycytidine are administered orally, in a dose
of 0.5 to 10 grams, either before or after administration of a dose
of Ara-C is completed, or concurrently with the dose of Ara-C.
Further doses of the acyl derivative of deoxycytidine are
administered every six to eight hours for 1 to 4 days. Repetitions
of this treatment regimen are initiated once per week or less
frequently, depending on the clinical response.
[0187] It is intended that the antineoplastic agents be used for
treating the types of tumors for which they are normally utilized,
e.g. Ara-C and its related cytidine analogs are effective in
leukemias, fluorouracil and related fluorinated uridine analogs are
useful in treating tumors of the colon, stomach, pancreas, breast,
and head-and-neck. In one embodiment, the antineoplastic agents are
administered in their normal doses, in which case the compounds of
the invention primarily reduce the severity of toxic side effects.
In another embodiment, the antineoplastic agents are administered
in doses higher than normal, in which case acyl derivatives of
nonmethylated pyrimidine nucleosides permit safer administration of
such higher, therapeutically aggressive doses of the anticancer
drugs. Furthermore, the increases in therapeutic index of
anticancer agents resulting from use of the compounds and
compositions of the invention permit the use of particular
antineoplastic agents for treating tumors for which they are not
currently standard therapy, including but not limited to melanoma,
prostate, renal carcinoma, ovarian carcinoma, or lung cancer.
[0188] A method for identifying tumor types or individual cancer
patients with a high probability of responding to high-dose
fluorinated pyrimidines in conjunction with an acyl derivative of
nonmethylated pyrimidine nucleosides involves measurement of the
enzyme fatty acid synthetase (FAS) in serum or in tumor biopsies.
FAS is highly expressed in many cancers, where it may have a role
in providing lipids necessary for rapid cell division (Kuhajda et
al., Proc. Nat. Acad. Sci. 91:6379-6383 [1994]). FAS requires a
supply of NADPH as a cofactor. A substantial fraction of NADPH
utilized for fatty acid synthesis is regenerated from NADP through
the hexose monophosphate shunt (HMP shunt) pathway of glucose
metabolism. The HMP shunt is activated by depletion of cellular
NADPH. A product of NADPH generation through the HMP shunt is
ribose and various ribose phosphates. In order for cells to rapidly
anabolize high concentrations of 5FU, a supply of either
phosphoribosyl pyrophosphate (PRPP) or ribose-1-phosphate (R-1-P)
is necessary, depending on whether the 5FU is converted to
intracellular fluorouridine monophosphate via the enzyme orotate
phosphoribosyl transferase or uridine phosphorylase respectively.
High levels of FAS in serum or tumor biopsies is a marker for
tumors which are actively producing ribose necessary for efficient
anabolism of 5FU, and which are particularly responsive to high
doses of 5FU.
[0189] Treatment of Complications Associated with Viral
Infection
[0190] HIV-infected patients, especially those whose infection has
progressed to "acquired immunodeficiency syndrome" (AIDS), suffer
from a variety of symptoms and diseases which result from and, in
some cases, further exacerbate a severely compromised immune
system. Many of these patients are given antiviral chemotherapeutic
agents, such as AZT, which also have detrimental effects on the
body's immune function and upon hematopoiesis, further lowering
resistance to infections of all kinds. Administration of the
compounds of the invention--orally, intravenously, or by parenteral
injection--raises the low blood cell counts due to antiviral
chemotherapy agents, particularly those that modify nucleotide
synthesis, metabolism, or utilization, such as AZT or
dideoxycytidine. Because anemia and greater susceptibility to
infections are dose- and rate-limiting factors in chemotherapeutic
treatment of AIDS patients, treatment of the patients with these
compounds reduces chemotherapeutic side effects (and thus improves
the quality of life) and, if appropriate, permits a more intensive
chemotherapeutic regimen to be employed. AZT and dideoxycytidine
produce deleterious side effects in tissues other than bone marrow,
including muscle and the peripheral nervous system. The compounds
and compositions of the invention are also useful for treating or
preventing such side effects.
[0191] Various antiviral nucleoside analogs other than AZT and
dideoxycytidine are used to treat viral infections, including but
not limited to HIV, herpes, or hepatitis. Examples of such agents
include 5-ethyl-2'-deoxyuridine, 5-iodo-2'-deoxyuridine,
5-bromo-2'-deoxyuridine, 5-methylamino-2'-deoxyuridine,
2',3'-dideoxycytidin-2'-ene, 3'-deoxythymidin-2'-ene,
3'-azido-2',3'-dideoxyuridine, arabinosyluracil, dideoxyuridine,
2',3'-dideoxy-3'-fluorothymidine and
(S)-1-(3-hydroxy-2-phosphonyl-methoxypropyl) cytosine (HPMPC); see
also WO 89/09603, hereby incorporated by reference. The compounds
of the invention are used to treat or prevent deleterious side
effects of these and related other antiviral nucleoside
analogs.
[0192] In treatment or prevention of toxicity due to antiviral
chemotherapy, the compounds and compositions are administered prior
to, during and/or after administration of the antiviral agents.
Typical antiviral chemotherapy regimens, especially for chronic
viral infections such as HIV infection, involve daily (often
multiple daily) administration of the antiviral agent or agents.
The compounds of the invention are administered, several times
daily, daily, or less frequently, depending on the clinical effect
observed. In all cases, the antiviral drugs are typically
administered in their normal regimens for the types of viral
infections-for which they are clinically useful. Treatment of
patients receiving antiviral nucleoside analogs is undertaken
either to reduce side effects of a standard dose or to permit
administration of doses of antiviral agents higher than are
normally tolerated or utilized.
[0193] For treatment of toxicity due to AZT, acyl derivatives of
either or both uridine, cytidine, or deoxycytidine are useful.
Particularly advantageous are acyl derivatives of deoxycytidine.
For oral administration, acyl derivatives of deoxycytidine,
uridine, and cytidine substituted with short chain fatty acids
(particularly acetate) or with short chain carbyloxycarbonates
(e.g. ethoxycarbonate), are advantageous.
[0194] In a typical clinical situation, a patient receives AZT two
to four times daily, and must generally do so indefinitely. Doses
of 1 to 10 grams of acyl derivatives of uridine, cytidine, or
deoxycytidine (or mixtures of two or all three) are administered
orally once per week up to about four times per day, depending on
the clinical response.
[0195] For treatment or prevention of toxicity due to
dideoxycytidine, acyl derivatives of deoxycytidine are
advantageous.
[0196] Treatment of Complications Associated with Malarial
Infection
[0197] Malarial parasites, e.g. Plasmodium yoelii or Plasmodium
falciparum, are dependent upon de novo synthesis pathways for
pyrimidine nucleotide biosynthesis; mammalian cells in general can
utilize either de novo pathways or "salvage" pathways, through
which advanced nucleotide precursors such as uridine or cytidine
are incorporated into intracellular nucleotide pools.
[0198] 5-Fluoroorotate, an analog of the pyrimidine nucleotide
precursor orotic acid, is toxic toward malarial parasites which are
dependent on de novo pyrimidine biosynthesis. Other inhibitors of
de novo pyrimidine biosynthesis, such as PALA, pyrazofurin or
6-azauridine are also similarly toxic toward malaria parasites.
Inhibitors of pyrimidine biosynthesis, including especially
fluoroorotate, are also toxic toward mammals. However,
administration of uridine to mammals treated with 5-fluoroorotate
(or other inhibitors of pyrimidine biosynthesis) attenuates host
toxicity due to the latter without impairing its antimalarial
activity. Orally active agents which elevate blood uridine levels
are advantageous sources of uridine in this context. Such agents
include the acyl derivatives of uridine or cytidine of the
invention. In treatment of malaria, an effective anti-malarial dose
of fluoroorotate is administered. Before, after, or at the same
time as fluoroorotate administration, an acyl derivative of uridine
or cytidine (triacetyluridine is particularly advantageous) is
administered, in a dose sufficient to attenuate fluoroorotate
toxicity. Typical doses of an acylated uridine or cytidine
derivative such as triacetyl uridine range from 1 to 10 grams,
administered as often as needed to minimized fluoroorotate
toxicity, e.g. one to four times per day. Doses of fluoroorotate or
uridine are optionally repeated as necessary to overcome the
malarial infection and to reduce host toxicity respectively.
[0199] E. Administration and Formulation of Compounds and
Compositions of the Invention
[0200] The compounds and compositions of the invention are
administered orally, by parenteral injection, intravenously,
topically, or by other means, depending on the condition being
treated.
[0201] The optimal doses and dose schedules for triacetyluridine
(or other acyl derivatives of uridine, cytidine, deoxycytidine or
deoxyuridine) are readily determined by one skilled in the art, by
monitoring the therapeutic effect.
[0202] The compounds and compositions of the invention are
administered chronically or intermittently. The compounds and
compositions are administered prior to, during, or after an
exposure to cytoreductive or antiviral chemotherapy agents,
depending on the characteristics of the toxicity of the
chemotherapy agents.
[0203] Advantageous acyl derivatives of uridine, cytidine,
deoxycytidine, or deoxyuridine for oral administration are those
substituted with short chain (2-6 carbon) fatty acids on the
hydroxyl groups of their ribose or deoxyribose rings. Also
advantageous for oral administration are pyrimidine nucleosides
substituted on their hydroxyl groups with hydrocarbyloxycarbonyl
radicals containing 3-7 carbon atoms.
[0204] Dosages for orally adminstered acyl derivatives of uridine,
cytidine, deoxycytidine or deoxyuridine typically range from 0.5 to
20 grams per day, most commonly 2 to 10 grams per day.
[0205] Powdered acyl derivatives of uridine, cytidine,
deoxycytidine or deoxyuridine are administered orally in capsule or
tablet form, although solutions, emulsions, or suspensions are also
useful for oral administration.
[0206] The compounds of the invention are optionally formulated in
biodegradable, bioerodible, or other gradual-release matrices for
sustained release of the compounds after oral administration or
subcutaneous implantation. In the case of intravenous or
intramuscular injection, the compounds are optionally formulated in
liposomes.
[0207] The pharmacologically active compounds optionally are
combined with suitable pharmaceutically acceptable carriers
comprising excipients and auxiliaries which facilitate processing
of the active compounds. These are administered as tablets,
dragees, capsules, and suppositories. The compositions are
administered, for example, orally, rectally, vaginally, or released
through the buccal pouch of the mouth, and are optionally applied
in solution form by injection, orally or by topical administration.
The compositions may contain from about 0.1 to 99 percent,
preferably from about 50 to 90 percent, of the active compound(s),
together with the excipient(s).
[0208] For parenteral administration by injection or intravenous
infusion, the active compounds are suspended or dissolved in
aqueous medium such as sterile water or saline solution. Injectable
solutions or suspensions optionally contain a surfactant agent such
as polyoxyethylenesorbitan esters, sorbitan esters, polyoxyethylene
ethers, or solubilizing agents like propylene glycol or ethanol.
The solution typically contains 0.01 to 5% of the active compounds.
The active compounds optionally are dissolved in pharmaceutical
grade vegetable oil for intramuscular injection. Such preparations
contain about 1% to 50% of the active compound(s) in oil.
[0209] Suitable excipients include fillers such as sugars, for
example lactose, sucrose, mannitol or sorbitol, cellulose
preparations and/or calcium phosphates, for example tricalcium
phosphate or calcium hydrogen phosphate, as well as binders such as
starch paste, using, for example, maize starch, wheat starch, rice
starch or potato starch, gelatin, tragacanth, methyl cellulose,
hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose
and/or polyvinyl pyrrolidone.
[0210] Auxiliaries include flow-regulating agents and lubricants,
for example, silica, talc, stearic acid or salts thereof, such as
magnesium stearate or calcium stearate and/or polyethylene glycol.
Dragee cores are provided with suitable coatings which, if desired,
are resistant to gastric juices. For this purpose, concentrated
sugar solutions are used, which optionally contain gum arabic,
talc, polyvinyl pyrrolidone, polyethylene glycol and/or titanium
dioxide, lacquer solutions and suitable organic solvents or solvent
mixtures. In order to produce coatings resistant to gastric juices,
solutions of suitable cellulose preparations such as
acetylcellulose phthalate or hydroxypropylmethylcell- ulose
phthalate are used. Dyestuffs or pigments are optionally added to
the tablets or dragee coatings, for example, for identification or
in order to characterize different compound doses.
[0211] The pharmaceutical preparations of the present invention are
manufactured in a manner which is itself known, for example, by
means of conventional mixing, granulating, dragee-making,
dissolving, or lyophilizing processes. Thus, pharmaceutical
preparations for oral use are obtained by combining the active
compound(s) with solid excipients, optionally grinding the
resulting mixture and processing the mixture of granules, after
adding suitable auxiliaries, if desired or necessary, to obtain
tablets or dragee cores.
[0212] Other pharmaceutical preparations which are useful for oral
delivery include push-fit capsules made of gelatin, as well as
soft-sealed capsules made of gelatin and a plasticizer such as
glycerol or sorbitol. The push-fit capsules contain the active
compound(s) in the form of granules which optionally are mixed with
fillers such as lactose, binders such as starches and/or lubricants
such as talc or magnesium stearate, and, optionally stabilizers. In
soft capsules, the active compounds are preferably dissolved or
suspended in suitable liquids such as fatty oils, liquid paraffin,
or polyethylene glycols. In addition, stabilizers optionally are
added.
[0213] Suitable formulations for parenteral administration include
aqueous solutions of the active compounds in water soluble form,
for example, water soluble salts. In addition, suspensions of the
active compounds as appropriate in oily injection suspensions are
administered. Suitable lipophilic solvents or vehicles include
fatty oils, for example, sesame oil, or synthetic fatty acid
esters, for example, ethyl oleate or tri-glycerides. Aqueous
injection suspensions optionally include substances which increase
the viscosity of the suspension which include, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension
optionally contains stabilizers.
[0214] In another embodiment, the active compounds are formulated
as part of a skin lotion for topical administration. Suitable
lipophilic solvents or vehicles include fatty oils, for example
sesame oil or coconut oil, or synthetic fatty acid esters, for
example ethyl oleate or triglycerides.
[0215] In another embodiment, the active compounds are formulated
in vehicles suitable for direct treatment of gastrointestinal
mucosa. Examples include mouthwashes, liquids (solutions or
suspensions) to be swallowed, or viscous fluids (e.g. solutions of
methylcellulose, carboxymethylcellulose, xanthan gum, etc.) which
are administereded orally or rectally.
[0216] Other pharmaceutical preparations which are used rectally,
especially for treatment of the colon and rectum, include, for
example, suppositories which consist of a combination of active
compounds with a suppository base. Suitable suppository bases are,
for example, natural or synthetic triglycerides, paraffin
hydrocarbons, polyethylene glycols or higher alkanols. In addition,
gelatin rectal capsules which consist of a combination of the
active compounds with a base are useful. Base materials include,
for example, liquid triglycerides, polyethylene glycols, or
paraffin hydrocarbons.
[0217] F. Synthesis of the Compounds of the Invention
[0218] Acylated derivatives of non-methylated pyrimidine
nucleosides are synthesized by reacting a pyrimidine nucleoside
with an activated carboxylic acid. An activated carboxylic acid is
one that has been treated with appropriate reagents to render its
carboxylate carbon more susceptible to nucleophilic attack than is
the case in the original carboxylic acid. Examples of useful
activated carboxylic acids for synthesis of the compounds of the
invention are acid chlorides, acid anhydrides, n-hydroxysuccinimide
esters, or carboxylic acids activated with BOP-DC. Carboxylic acids
are alternatively linked to pyrimidine nucleosides with coupling
reagents like dicyclohexylcarbodiimide (DCC).
[0219] During preparation of the acyl compounds of the invention,
when the acid source of the desired acyl derivative has groups
which interfere with the acylation reactions, e.g., hydroxyl or
amino groups, these groups are blocked with protecting groups,
e.g., t-butyldimethylsilyl ethers or t-BOC groups, respectively,
before preparation of the anhydride. For example, lactic acid is
converted to 2-t-butyldimethylsiloxypropionic acid with
t-butyl-dimethylchlorosilane, followed by hydrolysis of the
resulting silyl ester with aqueous base. The anhydride is formed by
reacting the protected acid with DCC. With amino acids, the N-t-BOC
or N-CBZ derivative is prepared, using standard techniques, which
is then converted to the anhydride with DCC. With acids containing
more than one carboxylate group (e.g., succinic, fumaric, or adipic
acid) the acid anhydride of the desired dicarboxylic acid is
reacted with a pyrimidine nucleoside in pyridine or pyridine plus
dimethylformamide or dimethylacetamide.
[0220] Amino acids are coupled to the exocyclic amino groups of
cytosine and deoxycytosine, and to hydroxyl groups on the aldose
moiety of pyrimidine nucleosides, by standard methods using DCC in
a suitable solvent, particularly a mixture of (i) methylene
chloride and (ii) dimethylacetamide or dimethylformamide.
[0221] Carbyloxycarbonyl derivatives of non-methylated pyrimidine
nucleosides are prepared by reacting the nucleoside with the
appropriate carbylchloroformate in a solvent such as pyridine or
pyridine plus dimethylformamide under anhydrous conditions. The
solvent is removed under vacuum, and the residue is purified by
column chromatography.
[0222] It will be obvious to the person skilled in the art that
other methods of synthesis may be used to prepare the compounds of
the invention.
[0223] The following examples are illustrative, but not limiting of
the methods and compositions of the present invention. Other
suitable modifications and adaptations of a variety of conditions
and parameters normally encountered in clinical therapy which are
obvious to those skilled in the art are within the spirit and scope
of this invention.
EXAMPLES
Example 1
[0224] Oral Administration of Triacetyluridine Ameliorates
Hematologic Toxicity of 5-fluorouracil
[0225] Purpose
[0226] This study was undertaken in order to determine if oral
administration of TAU could rescue mice from 5-FU toxicity more
effectively than oral administration of uridine itself. Bone marrow
cellularity and peripheral blood cell counts were used as an index
of 5-FU toxicity.
[0227] Methods
[0228] Forty-five female Balb/C mice (20 grams each) were given
5-fluorouracil (150 mg/kg, i.p.) at 12:00 noon on the initial day
of the experiment. These animals were then divided into 5 groups:
control (water, p.o.), oral uridine at 400 mg/kg/dose, oral uridine
at 800 mg/kg/dose, parenteral (i.p.) uridine at 400 mg/kg/dose, and
oral TAU at 500 mg/kg/dose.
[0229] Two hours after administration of 5-FU, the rescue
treatments with uridine or triacetyluridine were begun. Groups
received their designated treatment at 2:00 p.m., 4:00 p.m., and
6:00 p.m. on the day of 5-FU administration, and at 9 a.m., 11:00
a.m., 1:00 p.m., 3:00 p.m. and 5:00 p.m. on the following day. Each
dose of uridine or TAU was administered in 0.2 ml of water by
gavage or in 0.2 ml of saline by i.p. injection, as
appropriate.
[0230] Seven days after administration of 5-FU, blood (0.2-0.3 ml)
from five mice in each group was collected from the suborbital
sinus into EDTA for subsequent differential blood cell counting.
Mice were sacrificed by cervical dislocation; femurs were removed,
and their cell contents were expelled for counting; spleens were
also removed and weighed. Thirteen days after administration of
5-FU, the remaining four mice in each group were bled, sacrificed,
and their spleens removed.
[0231] Results
[0232] Day Seven
[0233] 5-FU administration resulted in declines in counts of all
blood cell types examined. Seven days after administration of 5-FU,
neutrophil, lymphocyte, and platelet counts in animals treated with
oral TAU were significantly higher than in control animals (Table
1). It is particularly noteworthy that leukocyte counts in the mice
that received TAU were higher than in mice that received an
equimolar dose of uridine by intraperitoneal injection. Cell counts
in the mice that received oral TAU were also higher than in mice
that received uridine orally at either equimolar (400 mg/kg/dose)
or twice equimolar doses (800 mg/kg/dose).
[0234] Platelet counts were within the normal range (700-800
K/.mu.l) in the mice that received oral TAU and i.p. uridine. They
were subnormal in the other groups, and lowest in the control mice
(Table 1).
[0235] Bone marrow cell counts were significantly greater in the
mice treated with TAU orally and with uridine parenterally than any
of the other groups (Table 2)
[0236] Day Eleven
[0237] Importantly, neutrophil and RBC levels were higher in the
group that received TAU than in the other treatment groups,
including mice that received an equimolar dose of uridine by
intraperitoneal injection (Table 3).
[0238] Spleen weight is an index of hematopoietic activity in mice
recovering from bone marrow damage. Eleven days after 5-FU, spleen
weight was significantly higher in mice given oral TAU compared to
the other treatment groups; spleens were smallest in the control
group (Table 4).
[0239] Conclusion
[0240] The results of this study show that oral administration of
TAU rescues mice from 5-FU toxicity more effectively than oral
administration of equimolar and twice equimolar uridine itself, and
more effectively than an equimolar dose of uridine given by
intraperitoneal injection.
1TABLE 1 Blood cell counts 7 days after 5-FU Lympho- WBC
Neutrophils cytes Platelets RBC Control 3.4 .+-. .1 0.24 .+-. .05
3.18 .+-. 387 .+-. 8.05 .+-. .11 .07 72 K/.mu.l Urd 400 oral 4.4
.+-. .5* 0.48 .+-. .23 3.01 .+-. 434 .+-. 7.66 .+-. .12 .77 114 Urd
800 oral 4.6 .+-. .4* 0.88 .+-. .22* 3.76 .+-. 646 .+-. 8.24 .+-.
.26 .27 108 Urd 400 i.p. 4.6 .+-. .4* 0.83 .+-. .24* 3.80 .+-. 773
.+-. 8.06 .+-. .05 .30 54** TAU 500 5.9 .+-. .4* 1.20 .+-. 23**
4.72 .+-. 723 .+-. 8.25 .+-. .16 oral .36** 57** All blood cell
count units are K/.mu.l except RBC's, which are M/.mu.l *= greater
than control value, P < .05 **= greater than control value, P
< .01
[0241]
2TABLE 2 Marrow cell counts and spleen weight 7 days after 5-FU
Marrow cell counts Spleen weight Control 1.5 .+-. .2 .times.
10.sup.6/femur 71 .+-. 2.6 mg Urd 400 oral 1.6 .+-. .2 .times.
10.sup.6 74 .+-. 2.4 Urd 800 oral 2.9 .+-. .9 .times. 10.sup.6 74
.+-. 6.4 Urd 400 i.p. 5.0 .+-. .7 .times. 10.sup.6** 78 .+-. 5.5
TAU 500 oral 3.0 .+-. .5 .times. 10.sup.6* 78 .+-. 3.5 *= greater
than control value, P < .05 **= greater than control value, P
< .01
[0242]
3TABLE 3 Blood cell counts 11 days after 5-FU Lympho- WBC
Neutrophils cytes Platelets RBC Control 5.2 .+-. .3 0.48 .+-. .12
4.68 .+-. .11 2280 .+-. 193 7.31 .+-. .05 Urd 400 4.6 .+-. .5 0.84
.+-. .19 3.65 .+-. .27 1940 .+-. 177 7.44 .+-. .14 oral Urd 800 4.9
.+-. .4 0.82 .+-. .19 4.07 .+-. .38 2127 .+-. 143 7.33 .+-. .16
oral Urd 400 5.1 .+-. .5 1.20 .+-. .21* 3.84 .+-. .38 1706 .+-. 88
7.78 .+-. .22 i.p. TAU 5.4 .+-. .5 1.89 .+-. .12** 3.29 .+-. .36
1446 .+-. 160 8.05 .+-. .16** 500 oral All blood cell count units
are K/.mu.l except RBC's, which are M/.mu.l *= greater than control
value, P < .05 **= greater than control value, P < .01
[0243]
4TABLE 4 Spleen weight 11 days after 5-FU Spleen weight Control 76
.+-. 7 mg Urd 400 oral 103 .+-. 15 Urd 800 oral 99 .+-. 6* Urd 400
i.p. 104 .+-. 6* TAU 500 oral 143 .+-. 11** *= greater than
control, P < .05 **= greater than control value, P < .01
Example 2
[0244] TAU Accelerates Hematopoietic Recovery in 5-FU-treated
Animals in a Dose Dependent Manner
[0245] Purpose
[0246] The purpose of this experiment was to confirm and extend the
previous findings that orally administered TAU accelerates
hematopoietic recovery in mice treated with 5-fluoruracil (5-FU),
and to observe the relationship between increasing doses of TAU and
the responses of the hematopoietic system of these 5-FU-treated
mice.
[0247] Methods
[0248] Seventy female Balb/C mice weighing approximately twenty
grams were each given an i.p. injection of 5-FU (150 mg/kg) at 1:00
p.m. on the initial day of the study. These animals were then
divided into five different treatment groups: control (water), and
oral TAU at doses of 100, 250, 500, and 1,000 mg/kg/treatment. The
test compounds were then given at 3:00, 5:00, 7:30, and 10:00 p.m.
on the day of 5-FU administration; at 9:00 a.m., 11:00 a.m., and
1:00, 3:00, 6:00, and 10:00 p.m. the following day; and a final
administration at 11:00 a.m. two days following the single 5-FU
injection. Each treatment was given orally in a volume of 0.2 ml
water (by gavage), except the highest dose of TAU which was
administered in 0.4 ml of water.
[0249] On days seven and eleven following 5-FU administration,
blood (0.2-0.3 ml) was collected from seven mice in each group by
retro-orbital bleeding into EDTA for subsequent differential blood
cell counting. Mice were sacrificed by cervical dislocation; their
right femurs removed and the contents expelled for cell counting;
and their spleens removed and weighed.
[0250] Results
[0251] Day Seven.
[0252] With increasing doses of TAU there were increasing numbers
of nucleated cells in the bone marrow. While the number of such
cells in the group receiving TAU 100 was approximately equal to
that in the control group, differences in bone marrow cellularity
were significantly greater in the TAU 500 and TAU 1,000 groups
compared that of controls (Table 5).
[0253] Treatment with TAU at doses of 500 and 1,000 mg/kg/treatment
resulted in white blood cell counts significantly greater than
those in the control group.
[0254] Total neutrophil counts also appeared to increase in a
dose-dependent manner, reaching approximately three-fold higher
levels in the TAU 1,000 group compared to controls.
[0255] In both the TAU 500 and TAU 1,000 groups lymphocyte counts
were significantly elevated compared to those in the control
group.
[0256] Platelet counts were significantly elevated in the TAU 250,
500, and 1,000 groups. The dose-response curve appeared to plateau
at about 500 mg/kg/treatment (Table 6).
5TABLE 5 Effect of increasing doses of TAU on hematopoiesis in mice
seven days after 5-FU administration. All blood cell counts are
K/.mu.l. Marrow WBC Neut Lym Control 1.70 .+-. .35 2.46 .+-. .13
.026 .+-. .011 2.43 .+-. .13 TAU 100 1.77 .+-. .18 2.47 .+-. .04
.014 .+-. .008 2.46 .+-. .04 TAU 250 2.91 .+-. .48 2.76 .+-. .20
.027 .+-. .012 2.71 .+-. .20 TAU 500 3.93 .+-. .39* 4.06 .+-. .14*
.037 .+-. .020 3.99 .+-. .15* TAU 1000 4.42 .+-. .34* 3.57 .+-.
.25* .071 .+-. .027 3.44 .+-. .23* *indicates different from
control, p < .01
[0257]
6TABLE 6 Effect of increasing doses of TAU on red blood cells (RBC)
and platelets (PLT) in mice seven days after 5-FU administration.
RBC PLT (M/.mu.l) (K/.mu.l) Control 8.38 .+-. .18 308 .+-. 52 TAU
100 8.20 .+-. .07 440 .+-. 46 TAU 250 8.51 .+-. .06 601 .+-. 46**
TAU 500 8.43 .+-. .16 712 .+-. 42** TAU1000 8.65 .+-. .15 715 .+-.
35** **indicates different from control, p < .01
[0258] Day Eleven.
[0259] Spleen weight was somewhat elevated at every dose of TAU
compared to controls, but reached statistical significance only in
the TAU 250 group (104.2.+-.3.5 mg versus 79.7.+-.3.1;
p<0.05)
[0260] Total neutrophil counts were significantly greater than
control values in the TAU 250, 500, and 1,000 groups.
[0261] Red blood cell counts were significantly increased in the
TAU 500 group (8.72.+-.0.18.times.10.sup.6 per microliter) and the
TAU 1000 group (8.63.+-.0.16.times.10.sup.6 per microliter)
compared to controls (7.90.+-.0.09.times.10.sup.6 per microliter).
The hematocrit followed a similar pattern (Table 7).
7TABLE 7 Effect of increasing doses of TAU on hematocrit (HCT) in
mice seven (7d) and eleven (11d) days after 5-FU administration.
Control T100 T250 T500 T1000 HCT 38.50 .+-. 37.77 .+-. 40.04 .+-.
39.50 .+-. 40.84 .+-. .88 .37 .38 .61 .68 (7d) HCT 36.94 .+-. 36.80
.+-. 37.47 .+-. 41.16 .+-. 41.55 .+-. 0.51 .88 .83 .88 .77 (11d) p
< .01 p < .001
[0262] Conclusions
[0263] The results of this experiment confirm and extend the
previous findings that treatment of animals receiving the
chemotherapeutic agent 5-FU with TAU dramatically reverses the
detrimental effects of 5-FU on the hematopoietic system, and that
it does so in a dose-dependent manner.
Example 3
[0264] Acyl Derivatives of Uridine Ameliorate Bone Marrow Toxicity
of 5-fluorouracil
[0265] Purpose
[0266] The purpose of this experiment was to test and compare the
efficacy of uridine and derivatives of uridine in attenuating
damage to the hematopoietic system of mice caused by the
chemotherapeutic agent 5-flourouracil (5-FU).
[0267] Methods
[0268] Ninety-eight female Balb/C mice weighing approximately 20
grams each were given a one-time 150 mg/kg injection (i.p.) of 5-FU
at 1 p.m. on the initial day of the study. These animals were then
divided into seven groups: control (saline), uridine (300
mg/kg/treatment), triacetyluridine (TAU; 455 mg/kg/treatment),
benzoyluridine (BU; 428 mg/kg/treatment), ethoxycarbonyl (ECU; 389
mg/kg/treatment), octanoyluridine (OU; 455 mg/kg/treatment), and
valeryluridine (VU; 403 mg/kg/treatment). All of these doses are
equimolar, and were administered in a volume of 0.4 ml by i.p.
injection. The groups were treated at 3:30, 6:00, and 8:30 p.m. on
the initial day with their respective agents. On the following day
these compounds were administered at 9:30 a.m., 12:00 noon, 2:30,
and 5:00 p.m. One final treatment was given on the next day at
10:00 a.m.
[0269] On days seven and eleven following 5-FU administration,
blood (0.2-0.3 ml) was collected from seven mice in each group by
retro-orbital bleeding into EDTA for subsequent differential blood
cell counting. Mice were sacrificed by cervical dislocation; their
right femurs removed and the contents expelled for cell counting;
and their spleens removed and weighed.
[0270] Results
[0271] Day Seven.
[0272] Even at this first time point each of the uridine
derivatives accelerated one or more aspects of hematopoietic
recovery following 5-FU damage. Thus, spleen weight was elevated in
all of the groups compared to saline controls. These differences
reached statistical significance (p<0.05) in the uridine
(80.4.+-.7.8 mg), ECU (75.7.+-.5.0 mg), and VU (69.1.+-.1.7 mg)
groups when compared to controls (62.7.+-.1.8).
[0273] TAU increased bone marrow cellularity by 40% over control
values (4.50.+-.0.77.times.10.sup.3 per microliter versus
2.78.+-.0.45.times.10.sup.3 per microliter, respectively).
[0274] White blood cell counts were significantly elevated in both
the ECU-treated (7.26.+-.0.31.times.10.sup.3 per microliter;
p<0.01) and VU-treated (6.57.+-.0.49.times.10.sup.3 per
microliter; p<0.05) groups compared to saline controls
(4.60.+-.0.70.times.10.sup.3 per microliter).
[0275] Platelet counts were significantly greater in the groups
treated with uridine (785.3.+-.57.5.times.10.sup.3 per microliter;
p <0.02), BU (829.6.+-.x10.sup.3 per microliter; p<0.01), and
VU (825.7.+-.26.7.times.10.sup.3 per microliter; p<0.002) than
those in the saline-treated controls (523.2.+-.71.4.times.10.sup.3
per microliter).
[0276] There was a trend toward higher total neutrophil counts in
nearly all of the treatment groups, but only VU
(0.141.+-.0.027.times.10.sup.3 per microliter) was actually
statistically significantly greater (p<0.002) than saline
controls (0.013.+-.0.009.times.10.sup.3 per microliter).
[0277] Lymphocyte counts were significantly greater in the groups
treated with ECU (7.19.+-.0.32.times.10.sup.3 per microliter;
p<0.01) and VU (6.42+0.49.times.10.sup.3 per microliter) than in
the saline-treated controls (4.59.+-.0.70.times.10.sup.3 per
microliter).
[0278] At the doses used in this particular experiment,
octanoyluridine, having the longest carbon chain of any of the
other derivatives, proved to be somewhat detrimental. There were
not enough animals from this group to provide day 11 data. However,
in dose optimization studies (see Example 3A), octanoyluridine
administered at a lower dose showed very beneficial effects on
hematopoietic recovery following 5-FU.
[0279] Day Eleven.
[0280] Virtually every index of hematopoietic function, including
spleen weight, white blood cell counts, red blood cell counts,
hematocrit, neutrophil counts, and lymphocyte count, was
significantly improved by treatment with the uridine derivatives
used in this experiment (Table 8). Spleen weights were elevated
above those of controls (94.7.+-.7.4) in each treatment group,
reaching statistical significance (p<0.05) in the uridine
(121.6.+-.9.7), BU (126.9.+-.12.3), and VU (139.4.+-.8.0) groups.
ConclusionsA wide variety of the uridine derivatives of
theinvention are effective in ameliorating damage caused by
administration of the chemotherapeutic agent 5-FU.
8TABLE 8 Effect of uridine and derivatives of uridine on
hematopoiesis in mice eleven days after 5-FU administration. White
blood cell counts (WBC), neutrophil counts (Neut), and lymphocyte
counts (lym) are all expressed in K/.mu.l; red blood cell counts
(RBC) in M/.mu.l. WBC RBC HCT % Neut Lym Con 3.37 .+-. .68 7.11
.+-. .1 32.97 .+-. 0.4 .09 .+-. 3.3 .+-. .7 .047 Urd 5.20 .+-. .47*
7.32 .+-. .2 34.67 .+-. 0.9 .60 .+-. 4.6 .+-. .4 .180** TAU 6.23
.+-. .55** 7.35 .+-. .2 35.01 .+-. 1.2 .55 .+-. 5.6 .+-. 4* .164**
BU 6.20 .+-. .49** 7.62 .+-. .1** 36.87 .+-. 0.6** 1.04 .+-. 5.1
.+-. .4* .295** ECU 5.51 .+-. .64* 7.65 .+-. .2* 37.54 .+-. 1.0**
.66 .+-. 4.8 .+-. .5* .166** VU 6.31 .+-. .42** 7.83 .+-. .1**
38.31 .+-. 0.8** .97 .+-. 5.2 .+-. .4* .136** *indicates different
from Control, p < .05 **indicates different form Control, p <
.01 Con = control; Urd = uridine; TAU = triacetyluridine; BU =
benzoyluridine; ECU = ethoxycarbonyluridine; VU =
valeryluridine
Example 3A
[0281] Octanoyl Uridine Attenuates Hematological Toxicity of
5-FU
[0282] Purpose
[0283] The purpose of this experiment was to test the efficacy of
octanoyluridine (Oct-U) in ameliorating the toxic effects of
5-flourouracil (5-FU) on hematopoiesis.
[0284] Methods
[0285] Fourteen Balb/C female mice weighing approximately 20 grams
each were given a one-time 75 mg/kg i.p. injection of 5-FU at 11:00
a.m. on the initial day of the study. Half of these animals were
subsequently treated with Oct-U (100 mg/kg/treatment, i.p.), while
the other half (controls) were injected with physiological saline.
Administration of Oct-U and saline occurred at 2:30, 4:30, and 7:00
p.m. on the initial day, and at 9:30 a.m., 12:00 noon, 2:30, and
5:00 p.m the following day. An additional group of seven mice
(basals) received no 5-FU and no treatments.
[0286] Results
[0287] Administration of 5-FU resulted in statistically significant
damage to the hematopoietic system as measured by each and every
index employed in this study, including spleen weight, bone marrow
cellularity, WBC count, total neutrophil count, lymphocyte counts,
RBC count, hematocrit percent, and platelet count (Tables 9 and
10). Subsequent treatment of mice receiving 5-FU with Oct-U
resulted in substantial improvement in each and all of these
parameters of hematopoietic function (Table 9 and 10).
[0288] Conclusion
[0289] Treatment of mice receiving 5-FU with octanoyl uridine
ameliorates the toxic effects of 5-FU on hematopoiesis.
9TABLE 9 Effect of octanoyluridine on bone marrow cellularity and
myelopoiesis in mice seven days after 5-PU administration. All
counts are in thousands per microliter. Marrow WBC Neut Lym Basal
8.96 .+-. .32 6.89 .+-. .25 1.85 .+-. .24 4.78 .+-. .23 Control
4.16 .+-. .40* 4.04 .+-. 19* 0.26 .+-. .03* 3.74 .+-. .19* Oct-U
7.73 .+-. .68** 5.96 .+-. .47** 1.19 .+-. .22** 4.62 .+-. .29**
*indicates p < .01 versus basals **indicates p < .01 versus
controls
[0290]
10TABLE 10 Effect of octanoyluridine on red blood cells (RBC),
platelets (PLT), and spleen weight in mice seven days after 5-FU
administration. RBC PLT Spleen weight (M/.mu.l) (K/.mu.l) (mg)
Basal 8.56 .+-. .17 897 .+-. 21 83.6 .+-. 1.7 Control 7.98 .+-.
.13* 963 .+-. 13* 75.4 .+-. 2.4* Oct-U 8.13 .+-. .14 1243 .+-. 59**
87.2 .+-. 3.7** *indicates p < .05 versus basal **indicates p
< .05 versus control
Example 4
[0291] Plasma Uridine Levels after Administration of Acyl
Derivatives of Uridine
[0292] Methods
[0293] Plasma uridine levels were determined in mice at various
times (15 minutes, 30 minutes, 1 hour, and 2 hours) after
administration of the acyl derivatives of uridine utilized in
Example 3 for attenuation of toxicity caused by 5-FU. Groups of
mice (n=3 per compound per time point) received intraperitoneal
injections of uridine (300 mg/kg), triacetyluridine (TAU; 455
mg/kg/treatment), benzoyluridine (BU; 428 mg/kg/treatment),
ethoxycarbonyluridine (ECU; 389 mg/kg/treatment), octanoyluridine
(OU; 455 mg/kg/treatment), and valeryluridine (VU; 403
mg/kg/treatment). The doses of the acyl derivatives of uridine are
the molar equivalent of 300 mg/kg uridine. At the appropriate time
points, blood samples (200 .mu.l) were taken from mice via the
retro-orbital sinus and immediately centrifuged. 75 .mu.l of the
resulting plasma was deproteinized with 2 volumes of methanol
followed by centrifugation. The supernatant was lyophilized and
reconstituted with 50 mM potassium phosphate buffer, pH 6.0, and
analyzed for uridine content by HPLC on a reverse phase (C.sub.18)
column. Uridine was separated from other plasma components in 50 mM
potassium phosphate buffer, pH 6.0, with a methanol gradient (2% to
35% over 15 minutes). Uridine was detected and quantified by UV
absorbance at 260 nM.
[0294] Results
[0295] Administration of all of the acyl derivatives of uridine
tested resulted in increased plasma uridine levels, as shown in
Table 11. Plasma uridine levels in control animals (mice that
received no exogenous uridine or cytidine derivatives) were
1.1.+-.0.1 .mu.M.
11TABLE 11 Plasma uridine concentration in mice after
administration of acyl derivatives of uridine Time: 15 min 30 min 1
hr 2 hr Compound: Plasma Uridine Concentration (.mu.M) Uridine 911
.+-. 35 415 .+-. 86 185 .+-. 21 5.3 .+-. 1.5 Triacetyluridine 666
.+-. 70 348 .+-. 28 24.5 .+-. 7.5 15.1 .+-. 1.6 Benzoyluridine 1160
.+-. 45 353 .+-. 87 178 .+-. 54 8.6 .+-. 1.7 Valeryluridine 1001
.+-. 113 347 .+-. 117 266 .+-. 41 7.4 .+-. 0.4 Octanoyluridine 56
.+-. 22 14 .+-. 7.4 17 .+-. 2.9 2.2 .+-. 0.04 Ethoxycarbonyl- 250
.+-. 18 83 .+-. 0.1 44 .+-. 12 2.0 .+-. 1.6 uridine
[0296] Administration of all of the acyl derivatives of uridine
results in elevated plasma uridine levels. The acyl derivatives
provide sustained formation of uridine via gradual deacylation.
This may not be reflected in plasma uridine levels, since cellular
uptake of uridine can remove uridine from the circulation as it is
formed by deacylation of the acylated uridine derivatives. It is
important to note that the acylated uridine derivatives are
generally superior to an equimolar quantity of uridine in
attenuating toxicity due to 5-FU (Table 8 in Example 3)
Example 5
[0297] Improved Therapeutic Index of 5-FU (I): TAU Rescue with 5-FU
Alone in tumor-bearing Mice
[0298] Purpose
[0299] The purpose of this experiment was to assess and compare the
ability of uridine and TAU to increase the therapeutic index of
5-FU in a tumor-bearing mouse model.
[0300] Methods
[0301] Sixty CD8F1 (BALB/C.times.DBA/8) female mice with first
generation transplants of CD8F1 spontaneous mammary adenocarcinoma
were treated with a weekly chemotherapy regimen which included a
single dose of 5-FU (150 mg/kg) followed by various rescue
strategies. The average tumor size was 157 mg at the start of the
chemotherapy. The weekly chemotherapy course was completed three
times.
[0302] In order to evaluate the various rescue therapies, the
animals were divided into six groups of ten animals each as
follows:
12 1. Saline: Saline (no 5-FU) 2. 5-FU alone: 5-FU (150 mg/kg i.p.)
3. 5-FU + vehicle: 5-FU (150 mg/kg i.p.) + vehicle.sup.1 4. 5-FU +
i.p. uridine: 5-FU (150 mg/kg i.p.) + Uridine (3,500 mg/kg i.p.) 5.
5-FU + oral uridine: 5-FU (150 mg/kg i.p.) + Uridine (5,000 mg/kg
p.o.).sup.2 6. 5-FU + oral TAU: 5-FU (150 mg/kg i.p.) + TAU (7,582
mg/kg p.o.).sup.2 .sup.11:1 oil-water emulsion + 2.5% Tween-80
.sup.27,582 mg TAU and 5,000 mg uridine are molar equivalent
doses.
[0303] Results
[0304] At the conclusion of three weeks of chemotherapy, mortality
in each group was compared and, where a sufficient number of
animals survived, body weight and tumor size were compared. The
results are summarized as follows in Table 12:
13TABLE 12 Effect of combined administration of 5-FU and TAU or
uridine Average Final Group Mortality Tumor Weight (mg) 1. Saline
3/10 7,391 2. 5-FU alone 9/10 * 3. 5-FU + vehicle 10/10 * 4. 5-FU +
i.p. uridine 1/10 1,604 5. 5-FU + oral uridine 0/10 896 6. 5-FU +
oral TAU 1/10 1,013 *Not meaningful due to high mortality.
[0305] Mortality in the group receiving only saline was due to the
progress of the disease, whereas mortality among the groups
receiving 5-FU but no rescue was due to the toxicity of 5-FU
itself.
[0306] Conclusion
[0307] TAU and uridine are effective in rescuing tumor-bearing mice
from the toxic effects of 5-FU. Both agents increase the
therapeutic index of 5-FU in tumor-bearing mice, and allow higher
doses of the drug to be tolerated, with a commensurate increase in
anti-cancer effect.
Example 6
[0308] Improved Therapeutic Index of 5-FU (II): TAU Rescue with
Combination Chemotherapy in Tumor-bearing Mice
[0309] Purpose
[0310] The purpose of this experiment was to assess and compare the
ability of TAU and uridine to increase the therapeutic index of
5-FU when used in combination with Phosphonacetyl-L-aspartate
(PALA), methotrexate (MTX), and leucovorin (LV), in a drug dosing
regimen which increases the cytotoxic potential of 5-FU.
[0311] Methods
[0312] Forty male CD8F1 mice with transplanted CD8F1 spontaneous
breast tumors (initial tumor weight of 155 mg) were treated weekly
with the following regimen: 25
[0313] In order to evaluate and compare the efficacy of TAU and
uridine, the mice were divided into four groups-of ten animals
each.
14 1. Control: Saline 2. Uridine i.p.: Uridine (3,500 mg/kg) 3.
Uridine (oral): Uridine (4,000 mg/kg) 4. TAU (oral): TAU (6,066
mg/kg.sup.1) .sup.1Molar equivalent of 4000 mg/kg uridine.
[0314] Saline, uridine, or TAU was administered every eight hours
for a total of 5 treatments starting two hours after each weekly
dose of 5-FU. This weekly regimen was repeated for three successive
weeks. One week after the third course of chemotherapy regimen,
mortality in each group was assessed and, where a sufficient number
of animals survived, body weight and tumor weight were also
measured.
[0315] Results
15TABLE 13 Effect of TAU on mortality of tumor-bearing mice
receiving combined chemotherapy Average Final Group Mortality Tumor
Weight (mg) Control 10/10 ** Uridine i.p. 0/10 110 Uridine p.o.
5/10 ** TAU p.o. 1/10 162 ** Not meaningful due to high
mortality.
[0316] Mortality in the control group was due to toxicity of 5-FU.
The tumor weight in untreated mice at this time point averages
approximately 3000 milligrams.
[0317] Conclusion
[0318] TAU and uridine improve the therapeutic index of 5-FU used
in this clinically relevant combination of agents. Oral TAU was as
effective as intraperitoneally-administered uridine and more
effective than an equimolar dose of orally-administered
uridine.
Example 7
[0319] Oral Administration of Diacetyldeoxycytidine Attenuates
Hematopoietic Toxicity of Arabinosyl Cytosine
[0320] Purpose
[0321] The purpose of this experiment was to test the efficacy of
diacetyldeoxycytidine (DAdC) and palmitoyldeoxycytidine (PdC) in
ameliorating the toxic effects of the chemotherapeutic agent
arabinosylcytosine (Ara-C) on the hematopoietic system.
[0322] Methods
[0323] Twenty-one Balb/C female mice weighing approximately 20
grams each received a daily intraperitoneal injection of Ara-C (100
mg/kg) for five days. These mice were divided into three treatment
groups: oral administration of water (controls); oral
administration of DAdC (411 mg/kg/treatment); and intraperitoneal
administration of PdC (200 mg/kg/treatment in 0.2% Tween 80). Mice
were treated with either water, DAdC, or PdC twice daily, at 9 a.m.
and 6 p.m. Treatment volume in each case was 0.2 ml. An additional
seven mice received no Ara-C and no treatment at all (basals).
[0324] On days seven and eleven following 5-FU administration,
blood (0.2-0.3 ml) was collected from the seven mice in each group
by retro-orbital bleeding into EDTA for subsequent differential
blood cell counting. Mice were sacrificed by cervical dislocation,
and their spleens removed and weighed.
[0325] Results
[0326] Ara-C administration resulted in significantly depressed
spleen weights, WBC counts, total neutrophil counts, lymphocyte
counts, and platelet counts in the control mice compared to the
basal mice (Table 14). No toxic effects of Ara-C were observed on
erythropoiesis per se (RBC counts, hemoglobin, and hematocrit).
[0327] Treatment of mice with DAdC orally and treatment with PdC
intraperitoneally significantly reversed the detrimental effects of
Ara-C on hematopoiesis. Spleen weight, WBC counts, total neutrophil
counts, and platelet counts were all significantly greater than
those in control mice (Table 14).
[0328] Conclusions
[0329] Administration of DAdC or PdC to mice receiving Ara-C
ameliorates the toxic effects of Ara-C on the hematopoietic
system.
16TABLE 14 The effect of diacetyldeoxycytidine (DAdC) or
palmitoyldeoxycytidine (Pdc) treatment on hematopoiesis in mice
receiving arabinosylcytosine (Ara-C) for five days. Spleen weight
is in milligrams, WBC, neutrophil, and platelet counts are
expressed as K/.mu.l. Spleen WBC Neut PLT Basal 93.0 .+-. 2.5 8.66
.+-. .53 1.84 .+-. .12 854 .+-. 30 Control 37.1 .+-. 1.3 3.60 .+-.
.55 0.04 .+-. .02 230 .+-. 21 DAdC 87.8 .+-. 3.5** 5.00 .+-. .42
0.42 .+-. .05** 728 .+-. 64** PdC 126.0 .+-. 5.3** 5.20 .+-. .35*
1.22 .+-. .27** 327 .+-. 30* *indicates p < .05 versus control
**indicates p < .001 versus control
Example 8
[0330] Orally Administered TAU Ameliorates the Detrimental Effects
on the Hematopoietic System of the Antiviral Chemotherapeutic Agent
Azidothymidine (AZT) in Drinking Water
[0331] Purpose
[0332] The purpose of this experiment was to test the efficacy of
orally administered triacetyluridine (TAU) in attenuating the
hematopoietic damage caused by the antiviral chemotherapeutic agent
azidothymidine (AZT).
[0333] Methods
[0334] Forty-two Balb/C female mice, each weighing approximately 19
grams, were divided into three different groups of 14 animals each.
The three groups were: basal (no AZT, no treatment), control (AZT,
water), and TAU (AZT, TAU at 460 mg/kg/treatment). AZT was
administered ad libitum in the drinking water at a concentration of
1.5 mg/ml throughout the course of the experiment. The volume of
AZT solution consumed in all treatment groups was similar and
averaged 2.25 ml per day per mouse, resulting in a daily AZT dose
of about 170 mg/kg per day. Water and TAU were administered in a
volume of 0.2 mL three times per day for the first 24 days of the
study, and twice each day thereafter.
[0335] All of the animals were weighed on the day the experiment
began, once each week, and immediately prior to sacrifice. After
weighing the mice on days 24 and 35, blood (0.2-0.3 ml) was
collected from seven mice in each group by retro-orbital bleeding
into EDTA for subsequent differential blood cell counting,
including reticulocytes. Mice were then sacrificed by cervical
dislocation; their right femurs removed and the contents expelled
for cell counting; and their spleens removed and weighed.
[0336] Results
[0337] Body weights of the mice receiving AZT alone were
significantly reduced (17.55.+-.0.47 grams, p<0.002) compared to
those of basal animals (19.78.+-.0.38) at day 7, while the body
weights of mice treated with TAU (19.51.+-.0.38) were nearly
identical to those of basals and significantly greater (p<0.005)
than those of controls at this same time point. This trend was also
observed on day 13, although there were no statistically
significant differences between groups. Again on day 24 of this
experiment, the body weights of the AZT controls were statistically
depressed compared to both basals (p<0.001) and TAU-treated mice
(p<0.05). At this time point the body weights of the TAU-treated
animals were also less than those of basal animals.
[0338] Day 24
[0339] The RBC counts of the mice receiving AZT only (8.49.+-.0.16)
were significantly reduced (p<0.01) compared to basals
(9.07.+-.0.11). The RBC counts of those animals treated with TAU
(9.01.+-.0.09) were not significantly different from basals and
were significantly greater (p<0.02) than those of controls.
[0340] Total neutrophil counts were also significantly reduced
(p<0.02) in the mice receiving only AZT (0.67.+-.0.09) compared
to basals (1.02.+-.0.08). Neutrophil levels in those animals
treated with TAU (0.91.+-.0.06) were not significantly different
from basals, and were significantly greater (p<0.05) than those
in the AZT-only group.
[0341] Day 35
[0342] Significant detrimental effects of AZT were seen with
virtually every parameter employed at this time point. Bone marrow
cellularity, WBC counts, and lymphocyte counts were significantly
depressed as were RBC counts, hemoglobin, hematocrit compared to
those from basal mice (Table 15). Mean cell volume, mean cell
hematocrit, and platelet counts of the control mice were
significantly elevated compared to those of the basal mice (Table
14). All of these parameters indicate AZT-induced hematopoietic
damage.
[0343] Treatment with oral TAU resulted in significantly higher RBC
counts, hemoglobin, and hematocrit compared to AZT controls (Table
15), as well as significantly lower mean cell volume, mean cell
hematocrit, and platelet counts compared to the control values
(Table 16).
[0344] While reticulocyte number was significantly elevated in the
control group (0.256.times.10.sup.6 per microliter; p<0.001)
versus basal counts (0.129.times.10.sup.6 per microliter), those
mice treated with TAU had reticulocyte levels significantly higher
(0.371.times.10.sup.6 per microliter) than both basals (p<0.001)
and controls (p<0.01).
[0345] Conclusions
[0346] From these data it is clear that orally administered TAU has
beneficial effects in animals with AZT-inducedhematopoietic
damage.
17TABLE 15 TAU ameliorates the detrimental effects of AZT on
erythropoiesis in mice. Red blood cell (RBC) counts are in M/.mu.l,
hematocrit (HCT) is expressed as a percentage, and hemoglobin (HGB)
is gm/dl. RBC HCT HGB Basal 9.04 + .08 42.52 .+-. .34 15.57 .+-.
.09 Control 7.53 + .13 39.29 .+-. .59 14.57 .+-. .26 TAU 8.20 .+-.
.20** 41.26 .+-. .62* 15.14 .+-. .12* *indicates p < .05 versus
control **indicates p < .01 versus control
[0347]
18TABLE 16 TAU attenuates AZT-induced cell damage. Platelet counts
(PLT) are in K/.mu.l, mean cell volume (MCV) is in f1, and mean
cell hematocrit (MCH) is measured in picograms. PLT MCV MCH Basal
736 .+-. 24 47.03 .+-. .16 17.23 .+-. .13 Control 903 .+-. 18 52.16
.+-. .37 19.37 .+-. .20 TAU 809 .+-. 06** 50.41 .+-. .48* 18.49
.+-. .28* *indicates p < .05 versus control **indicates p <
.01 versus control
Example 9
[0348] Orally administered TAU Ameliorates the Detrimental Effects
on the Hematopoietic System of Intraperitoneally Administered
AZT
[0349] Purpose
[0350] The purpose of this experiment was to test the efficacy of
orally administered TAU in reversing the hematopoietic damage
caused by parenteral administration of azidothymidine (AZT).
[0351] Methods
[0352] AZT (100 mg/kg i.p.) was given three times daily at 9 a.m.,
4 p.m., and at 10 p.m. to fifty-six female Balb/C miceweighing
approximately 19 grams each. Three times each day, at 9 a.m., 4
p.m., and 10 p.m., animals were treated with either water (control)
or TAU at doses of 230, 460, or 920 mg/kg/treatment by oral
intubation. Treatment volume was 0.2 ml for the control and TAU 460
groups, 0.1 ml for the TAU 230 group, and 0.4 ml for the TAU 920
group. One additional group of fourteen mice was not given AZT or
any treatments (basal).
[0353] All of the animals were weighed on the day the experiment
began, on day six and on day 13. After weighing the mice on days
six and thirteen blood (0.2-0.3 ml) was collected from seven mice
in each group by retro-orbital bleeding into EDTA for subsequent
differential blood cell counting, including reticulocytes. Mice
were then sacrificed by cervical dislocation; their right femurs
removed and the contents expelled for cell counting; and their
spleens removed and weighed.
[0354] Results
[0355] Day Six.
[0356] Bone marrow cellularity was significantly greater
(p<0.05) in the group treated with the 230 mg/kg TAU
(8.89.+-.0.46) than in the group receiving AZT alone
(7.54.+-.0.23).
[0357] White blood cell counts were 8.23.+-.0.38.times.10.sup.3 per
microliter in the basal group. AZT reduced the WBC count to
6.8.+-.0.66.times.10.sup.3 per microliter, but when mice were
treated with TAU (920 mg/kg) in addition to receiving AZT, WBC
counts were restored to basal levels (8.46.+-.0.63.times.10.sup.3
per microliter).
[0358] AZT administration resulted in a drop in RBC levels from
9.14.+-.0.10.times.10.sup.6 per microliter (basals) to
8.80.+-.0.31.times.10.sup.6 per microliter (controls). No such
decrement was seen in mice receiving TAU (460 mg/kg/treatment) and
AZT (9.15.+-.0.07.times.10.sup.6 per microliter).
[0359] Day Thirteen.
[0360] For virtually every parameter employed in this study those
mice given AZT alone for 13 days showed statistically significant
evidence of hematopoietic damage. Thus, WBC counts, RBC counts,
hemoglobin, hematocrit, reticulocyte counts, total neutrophil
counts, and lymphocyte counts were all significantly depressed,
while mean cell hematocrit and platelet counts were significantly
elevated. Concomitant treatment of mice receiving AZT with TAU (460
mg/kg/treatment) resulted in statistically significant improvement
in each and all of these measures (Table 17 and 18).
[0361] Conclusions
[0362] Concomitant treatment of mice with AZT and TAU significantly
improves hematopoietic function. This is true for both the white
blood cell and red blood cell indices.
19TABLE 17 Oral TAU attenuates AZT-induced damage to the
myelopoietic system in mice. These data were obtained on day 13 of
the study. WBC Neutro. Lymph. Platelets K/.mu.l K/.mu.l K/.mu.l
K/.mu.l Basal 6.8 .+-. 1.27 .+-. 5.14 .+-. 680 .+-. 0.6 0.11 0.33
30 Control 5.1 .+-. 0.49 .+-. 4.02 .+-. 1025 .+-. 0.28 * 0.07 *
0.27 * 44 * TAU 7.0 .+-. 0.62 .+-. 6.06 .+-. 863 .+-. 0.37 ** 0.03
0.38 ** 26 ** * = different from Basal group, P < .05 ** =
different from Control group, P < .05
[0363]
20TABLE 18 Oral TAU attenuates AZT-induced damage to the
erythropoietic system in mice. These data were obtained on day 13
of the study. RBC HGB HCT Retic M/.mu.l G/dL % M/.mu.l Basal 9.16
.+-. 15.32 .+-. 43.18 .+-. 0.298 .+-. 0.06 0.18 0.54 0.028 Control
8.15 .+-. 14.08 .+-. 38.90 .+-. 0.086 .+-. 0.07 * 0.13 * 0.43 *
0.007 * TAU 8.58 .+-. 14.54 .+-. 40.83 .+-. 0.162 .+-. 0.08 ** 0.13
** 0.37 ** 0.014 ** * = different from Basal group, P < .05 ** =
different from Control group, P < .05
Example 10
[0364] Oral Administration of Diacetyldeoxycytidine (DAdC)
Ameliorates Hematopoietic Toxicity Produced by Intraperitoneally
Administered AZT in DBA Mice
[0365] Purpose
[0366] The purpose of this experiment was to test the efficacy of
orally administered DAdC in reversing the hematopoietic damage
caused by parenteral administration of the antiviral
chemotherapeutic agent azidothymidine (AZT).
[0367] Methods
[0368] AZT (100 mg/kg i.p.) was given three times daily at 9 a.m.,
4 p.m., and at 10 p.m. to twenty-eight female DBA mice weighing
approximately 20 grams each. Three times each day,at 9 a.m., 4
p.m., and 10 p.m., animals were treated with either water (control)
or DAdC (411 mg/kg/treatment) by oral intubation. Treatment volume
was 0.2 ml. One additional group of fourteen mice was not given AZT
or any treatment (basal).
[0369] During the first few days of treatment a total of eight mice
died from accidents occurring during oral administration of the
water and DAdC. Therefore, the number of animals in the control
group and the DAdC group were reduced on the days of sacrifice as
follows: On day 6 there were four mice in the control group and
five in the DAdC group; on day 13 there were seven in the control
group and three in the DAdC group. The number of basal animals was
seven at both time points.
[0370] On days six and thirteen blood (0.2-0.3 ml) was collected
from mice in each group by retro-orbital bleeding into EDTA for
subsequent differential blood cell counting, including
reticulocytes. The animals were then sacrificed by cervical
dislocation; their right femurs removed and the contents expelled
for cell counting; and their spleens removed and weighed.
[0371] Results
[0372] Day Six
[0373] By day 6 AZT administration resulted in statistically
significant hematopoietic damage, especially in those mice not
receiving DAdC (controls). Thus, WBC and lymphocyte counts were
significantly depressed in the controls compared to the basals, as
were RBC counts, hemoglobin (HGB), hematocrit (HCT), and
reticulocyte counts (Table 17). Platelet counts were significantly
elevated in these control mice. Bone marrow cell counts were also
reduced 23% in the control group compared to the basals.
[0374] In contrast, those mice receiving AZT but also treated with
DAdC had only a slight (2.5%) reduction in bone marrow cellularity
and were not statistically different from basals. RBC counts, HGB,
HCT, and reticulocyte counts in the DAdC group were not different
from those in the basal group, but were significantly greater than
those in the control group (Table 19).
[0375] Day Thirteen
[0376] Mice given AZT alone for 13 days showed statistically
significant evidence of hematopoietic damage in nearly every
category compared to basal animals. Concomitant treatment of mice
with DAdC markedly attenuated or reversed the AZT-induced
erythropoietic damage (Table 20) as was seen on day 6. Platelet
counts were also significantly improved in micetreated with DAdC
(769.+-.32; p<0.02) compared to control animals (950.+-.34).
[0377] Conclusions
[0378] Treatment of mice receiving AZT with DAdC
significantlyimproves hematopoietic function, especially
erythropoiesis.
21TABLE 19 The effect of diacetyldeoxycytidine (DAdC) treatment on
erythropoiesis in mice receiving AZT for six days. RBC HGB HCT
Reticulocytes Basal 9.47 .+-. .18 13.93 .+-. .20 40.83 .+-. 0.83
.326 .+-. .039 Control 8.53 .+-. .33* 12.60 .+-. .40* 36.32 .+-.
1.54* .017 .+-. .004* DAdC 9.49 .+-. .32** 13.96 .+-. .40** 40.84
.+-. 1.35** .205 .+-. .075** *indicates different from Basal, p
< .05 **indicates different from Control p < .05
[0379]
22TABLE 20 The effect of diacetyldeoxycytidine (DAdC) treatment on
erythropoiesis in mice receiving AZT for thirteen days. RBC HGB HCT
Reticulocytes Basal 9.95 .+-. .13 14.83 .+-. .19 42.83 .+-. 0.41
.637 .+-. .044 Control 8.51 .+-. .05* 13.06 .+-. .07* 37.21 .+-.
0.13* .365 .+-. .022* DAdC 9.16 .+-. .32** 13.97 .+-. .39** 41.53
.+-. 1.17** .814 .+-. .069** *indicates different from Basal, p
< .05 **indicates different from Control, p < .05
Example 11
[0380] Orally Administered Diacetyldeoxycytidine (DAdC) or
Tetrahydrouridine (THU) Ameliorate the Detrimental Effects on the
Hematopoietic System of Intraperitoneally Administered AZT in
Balb/C Mice
[0381] Purpose
[0382] The purpose of this experiment was to test the efficacy of
orally administered DAdC or parenterally administered THU in
reversing the hematopoietic damage caused by parenteral
administration of the antiviral chemotherapeutic agent
azidothymidine (AZT).
[0383] Methods
[0384] AZT (100 mg/kg i.p.) was given three times daily at 9 a.m.,
4 p.m., and 10 p.m. to twenty-one female Balb/C mice weighing
approximately 20 grams each. Three times each day, at 9 a.m., 4
p.m., and 10 p.m., seven animals were treated with either water
(control, p.o.) or DAdC (300 mg/kg/treatment, p.o.), or THU (12.5
mg/kg/treatment in 0.2%Tween 80, i.p.). Treatment volume was 0.2
ml. One group of seven mice was not given AZT or any treatment
(basal).
[0385] On day thirteen blood (0.2-0.3 ml) was collected from mice
in each group by retro-orbital bleeding into EDTA for subsequent
differential blood cell counting, including reticulocytes. The
animals were then sacrificed by cervical dislocation; their right
femurs removed and the contents expelled for cell counting; and
their spleens removed and weighed.
[0386] Results
[0387] Concomitant treatment of mice with DAdC markedly attenuated
or reversed AZT-induced erythropoietic damage (Table 21). Total
neutrophil counts were also significantly improved in mice treated
with DAdC (1.39.+-.0.14; p<0.01) compared to control animals
(0.74.+-.0.10).
[0388] Mice given AZT and receiving THU treatment showed
significant improvements in myelopoiesis compared to controls as
well as erythropoiesis. Total white blood cell counts were
significantly greater in the THU group (6.06.+-.0.35; p<0.05)
than in the control group (4.73.+-.0.36). Significant differences
(p<0.05) were also observed comparing the total neutrophils
counts in the THU-treated group (1.16.+-.0.15) with those of the
controls (0.74.+-.0.10). Lymphocyte counts were improved, but the
differences did not reach statistical significance in this
experiment. In addition, reticulocyte indices (percent and M/.mu.l)
were significantly greater (p<0.05) in the THU-treated group
compared to those in the control group.
[0389] Conclusions
[0390] Treatment of Balb/C mice receiving AZT with DAdC or THU
significantly improves hematopoietic function.
23TABLE 21 The effect of diacetyldeoxycytidine (DAdC) treatment on
erythropoiesis in Balb/C mice receiving AZT for thirteen days. RBC
HGB HCT Retic Basal 8.63 .+-. .11 15.37 .+-. .08 41.01 .+-. 0.52
.089 .+-. .009 Control 7.55 .+-. .11* 13.71 .+-. .22* 35.96 .+-.
0.46* .039 .+-. .004* DAdC 8.26 .+-. .13** 14.97 .+-. .15** 39.51
.+-. 0.61** .059 .+-. .006** *indicates different from Basal, p
< .05 **indicates different from Control, p < .05
Example 12
[0391] Plasma Uridine Levels after Oral Administration of Uridine
or Triacetyluridine (TAU), with or without Dipyridamole
[0392] Purpose
[0393] The purpose of this experiment was to demonstrate that TAU
is a more effective orally-active agent for elevating plasma
uridine levels than is uridine itself, and furthermore to
demonstrate the effect of dipyridamole (DPM), a nucleoside
uptake-blocker which also has antiviral activity, on plasma uridine
levels after administration of TAU or uridine.
[0394] Methods
[0395] Female Balb/C mice with a body weight of 20 grams were
divided into four groups of 8 animals each:
24 1. Uridine (1000 mg/kg) p.o. 2. TAU (1500 mg/kg) p.o. 3. Uridine
(1000 mg/kg) p.o. + DPM (25 mg/kg i.p.) 4. TAU (1500 mg/kg) p.o. +
DPM (25 mg/kg i.p.) (1500 mg/kg TAU is the molar equivalent of 1000
mg/kg uridine)
[0396] Dipyridamole was administered by intraperitoneal injection
30 minutes prior to uridine or TAU.
[0397] Uridine was administered orally by gavage as an aqueous
solution in a volume of 0.4 ml.
[0398] TAU was administered by gavage in an emulsion vehicle (1:1
corn oil/water with 2.5% Tween 80).
[0399] Two mice from each group were bled from the suborbital
plexus at each time point: 0 (Basal uridine levels before TAU or
uridine administration), 0.5, 1, 2, and 4 hours after
administration of TAU or uridine.
[0400] Plasma samples (0.1 ml) were deproteinized by addition of
0.2 ml methanol followed by centrifugation. Samples were
lyophilized and then reconstituted with HPLC buffer (100 mM
ammonium acetate, pH 6.5, for subsequent assay of uridine by
reverse phase HPLC with UV absorbance detection (254 nm).
[0401] Data points are the mean of two samples for each time
point.
[0402] Results
[0403] After oral administration of uridine, plasma uridine reached
peak concentrations of 6 micromolar. In contrast, oral
administration of an equimolar dose of TAU resulted in peak plasma
uridine levels of 260 micromolar, thus demonstrating the marked
advantage of TAU over uridine as an orally active means of
elevating plasma uridine levels.
[0404] Dipyridamole further enhanced (approximately two-fold) the
amplitude (peak uridine levels of 460 micromolar) and duration of
blood uridine levels after oral administration of TAU.
[0405] Dipyridamole similarly improved blood uridine level
maintenance after oral uridine, although levels were much lower
than in the corresponding mice that received TAU (peak plasma
uridine levels of 20 micromolar).
[0406] These results are summarized in Table 22.
25TABLE 22 Plasma uridine concentrations after oral administration
of uridine or TAU, with or without dipyridamole. Plasma uridine
levels (.mu.M) after TAU or uridine administration Treatment 0 hr
0.5 hr 1 hr 2 hr 4 hr Uridine 2 4 6 4 2 TAU 2 240 260 110 12
Uridine + DPM 2 10 20 10 4 TAU + DPM 2 280 460 440 32
[0407] Conclusions
[0408] Plasma uridine levels after oral administration of TAU were
much higher than were observed after oral administration of
uridine. TAU is thus a much better source of plasma uridine after
oral administration than is uridine itself. Dipyridamole inhibits
uridine uptake into some cell types, and thereby enhances the
amplitude and duration of plasma uridine concentrations after
administration of TAU or uridine.
Example 13
[0409] Modulation of Toxicity of Oral Tegafur with TAU and
Uracil
[0410] Purpose:
[0411] Tegafur (5-fluoro-1-(tetrahydro-2-furfuryl)uracil) is an
orally active prodrug of 5-fluorouracil; it is enzymatically
converted to 5-FU during and after absorption from the intestinal
tract into the bloodstream. For treatment of cancer, tegafur is
typically administered orally in a formulation also containing
uracil, in a 1:4 molar ratio of tegafur to uracil; tegafur
formulated with uracil is currently used clinically in humans.
Uracil potentiates the activity of the 5-FU formed from tegafur by
competitively inhibiting dihydrouracil dehydrogenase, an enzyme
which degrades 5-FU.
[0412] After administration of high doses of tegafur+uracil, mice
lose a substantial amount of body weight, indicating
gastrointestinal toxicity. After oral administration, it is
believed that uracil potentiates the local toxicity of 5-FU formed
in intestinal cells during passage of Tegafur into the bloodstream.
It is therefore desirable to utilize with tegafur, or other orally
active prodrugs of 5-FU, an agent which inhibits breakdown of 5-FU
(or otherwise potentiates its activity) primarily in the
circulation (after absorption) rather than locally in the gut.
[0413] Triacetyluridine (TAU), like other acyl derivatives of
uridine and cytidine of the invention, is converted to uridine and
uracil during and after absorption into the bloodstream; when
present at the same time as 5-FU, both uridine and uracil are
capable of potentiating 5-FU cytotoxicity. Therefore, the
cytotoxicity of oral tegafur+uracil versus tegafur+TAU was
assessed. Blood cell counts were utilized as an index of
cytotoxicity of 5-FU in the circulation (and by extension, of
antitumor potency). Body weight loss was used as an index of
gastrointestinal toxicity.
[0414] Methods:
[0415] Three groups of mice received tegafur by oral intubation in
a dose of 400 mg/kg per mouse. The initial body weight of mice in
each group was 19.0.+-.0.6 grams. One of these groups also received
uracil in a molar ratio of 4:1 to the tegafur dose, and another
group received TAU, also in a 4:1 molar ratio to tegafur. Body
weights were monitored. Six days after tegafur administration,
blood samples were taken for differential cell counts.
[0416] Results:
[0417]
[0418] Tegafur alone at a dose of 400 mg/kg produced a significant
drop in neutrophil numbers, but did not significantly affect
platelet counts; body weight was reduced only slightly (7%)
compared to untreated (basal) animals. Tegafur plus uracil produced
a more severe drop in neutrophil counts than did tegafur alone, and
also reduced platelet counts and caused a substantial (29%) loss of
body weight. Tegafur plus TAU produced blood cell count changes
similar to those found after tegafur plus uracil, but did not cause
a change in body weight. The blood cell counts found after oral
administration of either tegafur+TAU or tegafur+uracil are similar
to those observed after a therapeutically effective dose of
5-fluorouracil (e.g. 150 mg/kg) administered systemically. However,
the weight loss caused by tegafur+uracil is unacceptable in the
context of cancer chemotherapy. The excellent systemic cytotoxicity
of oral tegafur+TAU (better than tegafur alone and at least
equivalent to tegafur+uracil) and the absence of a loss in body
weight (especially in contrast to the marked weight loss in animals
receiving tegafur+TAU) indicate that TAU is useful in potentiating
systemic cytotoxicity (and therefore antitumor potency) of
orally-active fluorouracil prodrugs without a proportional increase
in gastrointestinal toxicity. Combination of an orally active
fluorouracil prodrug with an acylated non-methylated pyrimidine
nucleoside derivative therefore permits better oral delivery of
therapeutically effective amounts of the important antineoplastic
drug fluorouracil than is obtained with current methods and
compositions.
26TABLE 23 Uracil vs TAU for enhancing cytotoxicity of Tegafur Body
weight Neutrophils Platelets (grams) (K/.mu.l) (K/.mu.l) Basal 19.6
.+-. 0.5 2.60 .+-. 0.510 984 .+-. 25 Tegafur-400 18.2 .+-. 0.7 0.40
.+-. 0.085 * 956 .+-. 30 Tegafur-400 + Uracil 14.0 .+-. 0.3 * 0.02
.+-. 0.002 * 448 .+-. 31 * FT-400 + TAU 19.8 .+-. 0.4 0.02 .+-.
0.003 * 334 .+-. 33 * * Different from Basal values, P < .01
[0419] All drugs were administered orally in a single dose. Blood
samples were taken for cell counts 6 days after drug
administration; body weights were also recorded at this time.
Example 14
[0420] Antitumor efficacy of tegafur and TAU vs tegafur and
Uracil
[0421] Purpose
[0422] Tegafur (FT) is an orally active prodrug of fluorouracil.
Uracil, at an optimum molar ratio of 4:1, potentiates the antitumor
efficacy of FT. The dose limiting toxicity of the FT-uracil
combination is damage to the intestinal mucosa. Increasing doses of
FT-uracil also result in hematopoietic damage.
[0423] The purpose of the present study was to compare the
antitumor efficacy of FT in combination with either uracil or
triacetyluridine (TAU) in rats bearing the Walker 256
carcinosarcoma. The essential question addressed in this experiment
was whether or not FT+TAU inhibits tumor growth as well as
FT+uracil, while causing less intestinal toxicity (body weight
changes). Blood cell damage was also assessed and compared.
[0424] Methods
[0425] Animals
[0426] Male Sprague-Dawley rats with an initial body weight of
approximately 120 grams.
[0427] Tumors
[0428] Pooled ascites fluid containing Walker 256 cells at a
density of 2.47.times.10.sup.8 cells/milliliter was collected from
three donor rats. An aliquot containing 4.94.times.10.sup.7 cells
was injected subcutaneously in the right flank of each rat in the
anti-tumor efficacy study. This resulted in formation of solid
subcutaneous tumors.
[0429] Treatment
[0430] FT was administered at doses of 40, 60, and 80 mg/kg/day in
a 1:4 molar ratio with either uracil or TAU. The vehicle used was
1% hydroxypropylmethylcellulose. Treatment was initiated (day 1)
five days after tumor implantation. Vehicle and vehicle plus drugs
were administered orally by gavage each day for seven days (day 1
through 7) in a volume of 1.2 milliliters per 100 grams of body
weight. Tumor size was measured in situ on day 8. On day 10 blood
samples were obtained and, after sacrifice of the animals, tumor
size and weight were determined.
[0431] Treatment Groups
[0432] 1. Vehicle (control)
[0433] 2. FT 40 mg/kg+uracil
[0434] 3. FT 60 mg/kg+uracil
[0435] 4. FT 80 mg/kg+uracil
[0436] 5. FT 40 mg/kg+TAU
[0437] 6. FT 60 mg/kg+TAU
[0438] 7. FT 80 mg/kg+TAU
[0439] n =7 animals/group
[0440] Measurements
[0441] Body weights were determined prior to treatment on days 1,
3, 5, and 7. On day 10 the animals were weighed prior to sampling
and sacrifice. Body weights are expressed as the percent of body
weight change over the course of the experiment.
[0442] Tumor size was measured in situ on day 8 and the tumor
volume was then calculated using the following formula: 1 length
.times. width 2 2
[0443] Following sacrifice on day 10 the tumor was exposed, the
size measured in situ, and the tumor volume calculated using the
formula above. The tumor was then removed and the tumor weight
determined. Tumor data are also expressed as T/C %. T/C % is: 2
mean tumor size in drug-treated rats .times. 100 mean tumor size in
control rats
[0444] Complete blood cell counts with differential were determined
using blood samples obtained by cardiac puncture immediately prior
to sacrifice on day 10.
[0445] Results
[0446] The effects of FT+uracil and FT+TAU on tumors and on body
weights as assessed on day 8 are summarized in Table 24. The data
show that FT+TAU has greater anti-tumor efficacy and preserves body
weight better than equivalent doses of FT+uracil at each dose of
FT. For example, at the FT 60 dose the tumor volume and T/C percent
for the FT-TAU treated group areless than half those of the
uracil-treated group. At that same dose level (FT 60) the body
weight change is 52.1% in the FT-TAU group compared to 7.8% in the
FT-uracil group. Higher doses of FT are more effective in
preventing tumor growth than lower doses.
[0447] Tumor and body weight data obtained on day 10 are presented
in Table 25. Tumor values--T/C percent, tumor volume and tumor
weight--are significantly lower and body weight gains significantly
greater in the rats treated with FT-TAU than in those treated with
FT-uracil.
[0448] The hematopoietic effects of FT treatment are recorded in
Table 26. Platelet counts are preserved at all FT dose levels in
the FT-TAU treated animals, while dropping precipitously at
increasing doses of FT in the FT-uracil groups. Total white blood
cell counts and lymphocytes are also maintained better in the
FT-TAU groups at the higher, more effective doses of FT. At the FT
40 dose, neutrophil counts are less severely attenuated in the
FT-TAU group than in the equivalent FT-uracil group. In fact, at
each FT-TAU dose level, neutrophil counts are approximately twice
those observed in the corresponding FT-uracil group.
[0449] The results of this experiment indicate that the use of oral
TAU in combination with FT has significant advantages over the
FT-uracil combination. FT-TAU has greater anti-tumor activity than
equivalent doses of FT-uracil while causing less intestinal and
hematopoietic damage.
27TABLE 24 Antitumor effects of FT with uracil or TAU in rats
bearing Walker 256 Carcinosarcoma: Day 8 Group Tumor volume T/C
Body wt. change (mm.sup.3 .+-. SE) (%) (%) Control 3098 .+-. 372
100.0 100.0 FT 40 + Uracil 1651 .+-. 135 53.3 51.0 FT 60 + Uracil
1051 .+-. 123 30.8 -1.7 FT 80 + Uracil 848 .+-. 124 27.4 -26.0 FT
40 + TAU 1244 .+-. 209 40.2 78.0 FT 60 + TAU 1006 .+-. 114 32.5
52.0 FT 80 + TAU 557 .+-. 49 18.0 23.7 FT dose = mg/kg/day for 7
days FT/Uracil = 1:4 molar ratio FT/TAU = 1:4 molar ratio
[0450]
28TABLE 25 Antitumor effects of FT with uracil or TAU in rats
bearing Walker 256 Carcinosarcoma: Day 10 Group Tumor weight T/C
Body wt. change (g .+-. SE) (%) (%) Control 4.16 .+-. 0.55 100.0
100.0 FT 40 + Uracil 3.62 .+-. 0.35 87.0 62.2 FT 60 + Uracil 2.80
.+-. 0.28 67.3 39.0 FT 80 + Uracil 1.50 .+-. 0.58 36.1 -7.5 FT 40 +
TAU 2.14 .+-. 0.31 51.4 93.2 FT 60 + TAU 2.18 .+-. 0.42 52.4 58.9
FT 80 + TAU 1.77 .+-. 0.25 42.5 30.3 FT dose = mg/kg/day for 7 days
FT/Uracil = 1:4 molar ratio FT/TAU = 1:4 molar ratio
[0451]
29TABLE 26 Blood cell counts after FT + uracil and FT + TAU: Day 10
WBC Neutrophils Lymphocytes Platelets Control 11.8 .+-. 0.8 1.84
.+-. .24 9.9 .+-. 0.8 844 .+-. 44 FT 40 + Uracil 9.7 .+-. 1.8 0.68
.+-. .18 8.5 .+-. 1.5 866 .+-. 117 FT 60 + Uracil 5.9 .+-. 1.1 0.27
.+-. .11 5.6 .+-. 1.0 602 .+-. 147 FT 80 + Uracil 3.6 .+-. 1.0 0.14
.+-. .12 4.3 .+-. 1.1 171 .+-. 49 FT 40 + TAU 8.2 .+-. 0.9 0.91
.+-. .21 7.0 .+-. 0.8 913 .+-. 117 FT 60 + TAU 5.3 .+-. 0.9 0.27
.+-. .05 5.0 .+-. 0.9 830 .+-. 83 FT 80 + TAU 5.6 .+-. 0.8 0.12
.+-. .07 5.4 .+-. 0.7 534 .+-. 136 All blood cell count units are
K/.mu.l FT dose = mg/kg/day for 7 days FT/Uracil = 1:4 molar ratio
FT/PN401 = 1:4 molar ratio
Example 15
[0452] 5FU Dose Escalation with Delayed Triacetyluridine Improves
Efficacy in Murine Colon Cancer
[0453] The murine tumor Colon 26 is a colon carcinoma that is
relatively resistant to 5FU. Maximum tolerated doses of 5FU alone
or 5FU plus leucovorin slow down the growth of Colon 26 but do not
produce regressions of established tumors and do not ultimately
prevent tumor-related death of the host animal.
[0454] Mice:
[0455] Female Balb/C mice approximately 8 weeks old and weighing
about 20 grams were used in all experiments. The maximum tolerated
dose of 5FU in these mice on a weekly bolus .times.3 schedule is
100 mg/kg.
[0456] Tumors:
[0457] Colon 26 was obtained from the Frederick Cancer Research and
Development Center and maintained by serial transplantation as a
subcutaneous solid tumor. For studies on antitumor efficacy, 0.3 ml
of a 5% brei was injected subcutaneously on the left flank. Tumor
length and width were determined with a vernier caliper and
tumor-volume was estimated according to the formula:
Volume=L(mm).times.W.sup.2(mm.sup.2)/2.
[0458] Tumors were allowed to grow for 10 days to a size of 100 to
200 mm.sup.2 and mice were then sorted into groups of 10 mice each
containing approximately equal distributions of tumor size. Tumor
volumes were measured at least weekly during treatments; body
weights were recorded at the same times. Mice received three weekly
bolus doses of 5FU (with or without TAU), and the final evaluation
of tumor size and body weight were recorded seven days after the
last dose of 5FU.
[0459] Drugs:
[0460] 5-Fluorouracil was dissolved in saline and administered by
intraperitoneal injection in a volume of 0.2 ml.
[0461] TAU was suspended in 0.75% hydroxypropylmethylcellulose with
a tissue homogenizer at a concentration of 200 mg/ml. TAU was
administered in 5 doses administered every 8 hours, beginning two
hours after 5FU administration.
[0462] 5FU dose-response: Effect of 5FU Dose Escalation with TAU
rescue in Advanced Colorectal Carcinoma in Mice.
30 Tumor Volume Body wt Regressions * Group (mm.sup.3) (% change)
Dead PR CR A. Control (no 5FU) 3406 .+-. 263 -5% 3/10 -- -- B. 5FU
100 mg/kg 1503 .+-. 267 -7% 4/10 -- -- C. 5FU 150 mg/kg + TAU 691
.+-. 256 -1% 0/10 1/10 -- D. 5FU 175 mg/kg + TAU 19 .+-. 15 +2%
0/10 9/10 4/10 E. 5FU 200 mg/kg + TAU 12 .+-. 10 -1% 0/10 10/10
8/10 * PR = Partial Response; > 50% decrease in tumor size
(includes CR) * CR = Complete Response; no tumor detectable Tumor:
Colon 26 carcinoma, initial size 198 .+-. 14 mm.sup.3 5FU: i.p.
injection, weekly .times. 3 TAU: 4000 mg/kg p.o. .times. 5 at 8 hr
intervals, beginning 2 hours after 5FU Evaluation: 7 days after
last 5FU dose Discussion:
[0463] There is a strong improvement in antitumor efficacy when the
5FU dose is elevated to 175 mg/kg/week. Further elevation to 200
mg/kg in creases the incidence of complete responses. It is
important to note that a high incidence of regressions was obtained
without significant toxicity as measured by body weight loss. 5FU
at the normal maximum tolerated dose (100 mg/kg) failed to produce
regressions, and only inhibited tumor growth by about 50%.
[0464] The lifespan of the animals treated with 5FU at 175 or 200
mg/kg was substantially prolonged. All of the animals in groups A
and B died of tumor progression within 40 days after tumor
transplantation. The animals in groups D and E were alive and
maintaining normal body weights even after 50 days. The complete
regressions as determined by absence of palpable tumor tissue were
durable; tumors did not regrow in these animals during a subsequent
observation period of more than 90 days.
Example 16
[0465] Treatment of B16 Melanoma with High-dose 5-fluorouracil and
Triacetyluridine
[0466] Mice:
[0467] Female C57BL/6 mice approximately 8 weeks old and weighing
about 20 grams were used in this experiment. The maximum tolerated
dose of 5FU in these mice on a weekly bolus .times.3 schedule is
100 mg/kg.
[0468] Tumors:
[0469] Murine B16 melanoma was obtained from the Frederick Cancer
Research Foundation and maintained by serial transplantation as a
subcutaneous solid tumor. For studies on antitumor efficacy, 0.3 ml
of a 5% brei was injected subcutaneously on the left flank. Tumor
length and width were determined with a vernier caliper and tumor
volume was estimated according to the formula:
Volume=L(mm).times.W.sup.2(mm.sup.2)/2.
[0470] Tumors were allowed to grow for 10 days to a size
ofapproximately 300 mm.sup.2 and mice were then sorted into groups
of eight mice each containing approximately equal distributions of
tumor size. Tumor volumes were measured at least weekly during
treatments; body weights were recorded at the same times. Mice
received three weekly bolus doses of 5FU (with or without TAU), and
the final evaluation of tumor size and body weight were recorded
seven days after the last dose of 5FU.
[0471] Drugs:
[0472] 5-Fluorouracil was dissolved in saline and administered by
intraperitoneal injection in a volume of 0.4 ml.
[0473] TAU was suspended in 0.75% hydroxypropylmethylcellulose with
a tissue homogenizer at a concentration of 200 mg/ml. TAU was
administered in 5 doses given once every 8 hours, beginning two
hours after 5FU administration.
[0474] Effect of 5FU Dose escalation with TAU rescue on Growth and
Metastasis of B16 Melanoma
31 Tumor wt Mice with Metastases Groups (g) metastases per lung
Control (no 5FU) 5.8 .+-. 1.1 100% 7.9 5FU 100 mg/kg (MTD) 4.7 .+-.
1.4 86% 5.6 5FU 225 mg/kg + TAU 0.9 .+-. 0.3 12% 0.38 Tumor: s.c.
B16 melanoma, initial size 344 .+-. 58 mg
[0475] Discussion:
[0476] B16 melanoma, like human melanomas in general, is relatively
resistant to chemotherapy. 5FU at the normal maximum tolerated dose
of 100 mg/kg has little effect on tumor growth and spontaneous
metastases to the lung. Escalation of the weekly 5FU dose to 225
mg/kg with delayed administration of TAU to reduce 5FU toxicity
resulted in a significant inhibition of tumor growth and a strong
reduction in spontaneous metastases to the lungs. Since melanomas
are not ordinarily responsive to fluorouracil, the observation that
significant activity was obtained with 5FU dose escalation and TAU
suggests that this approach can be effective in tumor types for
which 5FU is not normally a drug of choice.
Example 17
[0477] Triacetyluridine Improves the Therapeutic Activity of Oral
5FU Administered with an Inhibitor of 5FU Degradation
[0478] The bioavailability of orally administered 5-fluorouracil
(5FU) is low and variable because of degradation of 5FU by the
enzyme dihydropyrimidine dehydrogenase (DPD). Administration of an
inhibitor of this enzyme permits oral administration of 5FU
(Baccanari et al. Proc. Nat. Acad. Sci. 90:11064-11068, 1994).
Since the acyl derivatives of nonmethylated pyrimidine nucleosides
are active after oral administration, their use with oral 5FU
results in an improvement in efficacy and therapeutic index. A
completely oral treatment for some types of cancer presents
advantages in terms of hospital expenses and convenience.
[0479] One goal of oral administration of 5FU in conjunction with
an inhibitor of DPD is to provide 5FU exposure similar to a
continuous intravenous infusion. This involves daily (or more
frequent) administration of 5FU plus a DPD inhibitor.
Alternatively, higher doses of 5FU are given less frequently, e.g.
weekly. The following experiment was designed to determine the
effect of weekly oral high dose 5FU with delayed oral
administration of TAU upon antitumor efficacy. 5-ethynyluracil, a
potent, irreversible inhibitor of DPD, was used to permit oral
administration of 5FU.
[0480] Mice:
[0481] Female Balb/C mice approximately 8 weeks old and weighing
about 20 grams.
[0482] Tumors:
[0483] Colon 26 was obtained from the Frederick Cancer Research
Foundation and maintained by serial transplantation as a
subcutaneous solid tumor. For studies on antitumor efficacy, 0.3 ml
of a 5% brei was injected subcutaneously on the left flank. Tumor
length and width were determined with a vernier caliper and tumor
volume was estimated according to the formula:
Volume=L(mm).times.W.sup.2(mm.sup.2)/2.
[0484] Tumors were allowed to grow for 10 days to a size of 100 to
200 mm.sup.2 and mice were then sorted into groups of 10 mice each
containing approximately equal distributions of tumor size. Tumor
volumes were measured at least weekly during treatments; body
weights were recorded at the same times. Mice received three weekly
bolus doses of 5FU (with or without TAU), and the final evaluation
of tumor size and body weight were recorded seven days after the
last dose of 5FU.
[0485] Drugs:
[0486] 5-ethynyluracil was dissolved in saline and administered
orally to mice in a volume of 0.2 ml one hour before 5FU.
[0487] 5-Fluorouracil was dissolved in saline and administered
orally by gavage in a volume of 0.2 ml.
[0488] TAU was suspended in 0.75% hydroxypropylmethylcellulose with
a tissue homogenizer at a concentration of 200 mg/ml.
[0489] TAU was administered in 5 doses administered every 8 hours,
beginning two hours after 5FU administration.
[0490] 5FU Dose Escalation with delayed TAU Rescue: Oral SFU with
5-ethynyluracil
32 Tumor Vol. Body wt Regressions Group (mm.sup.3) (% change) CR PR
Control 1230 .+-. 154 -14 0/10 0/10 EU + 5FU 20 mg/kg 319 .+-. 79
-6 0/10 2/10 Weekly x2 EU + 5FU 50 mg/kg + TAU 15 .+-. 11 -3 4/10
5/10 Weekly x2 EU + 5FU 2 mg/kg 924 .+-. 98 -7 0/10 0/10 Daily x9
Tumor: Colon 26 carcinoma, initial size 82 .+-. 9 mm.sup.3 EU +
5FU: 5-ethynyluracil (EU) 2 mg/kg p.o. 1 hr before 5FU TAU: 4000
mg/kg p.o. x5 at 8 hr intervals, beginning 2 hours after 5FU
Evaluations: 14 days after first treatment dose (24 days after
tumor implantation) Regressions: CR = Complete regression; PR =
partial (>50%) regression
[0491] Delayed administration of oral TAU permitted 5FU dose
escalation in conjunction with an inhibitor of 5FU degradation. The
maximum tolerated dose of 5FU is reduced in the presence of a DPD
inhibitor, since the clearance of 5FU is inhibited. Normally, more
than 90% of an administered dose of 5FU is degraded by DPD rather
than incorporated into cellular nucleotide pools. Therefore,
inhibition of 5FU degradation has the potential to strongly
increase the toxicity of 5FU. The approximate maximum tolerated
dose of oral 5FU in mice pretreated with 5-ethynyluracil is 20
mg/kg on a weekly .times.3 schedule. Delayed administration of TAU
permitted 5FU doseescalation to 50 mg/kg/week with a consequent
improvement in antitumor efficacy. Daily administration of a lower
dose of 5FU (2 mg/kg daily for 9 days), which approximates a
continuous infusion of 5FU, was less effective in inhibiting tumor
growth than was a weekly schedule of 5FU administration.
Example 18
[0492] Synthesis of Ethoxycarbonyluridine
[0493] To an ice cold solution of 0.5 grams (1.76 millimoles) grams
of 2',3'-isopropylidene uridine in 10 ml pyridine, 2.64 mmoles (1.5
equivalents) of ethylchloroformate was addeddropwise while
stirring. The reaction was allowed to warm up to room temperature
(25.degree. C.) and stirred overnight (18 hours), at which point
TLC (9:1 chloroform/methanol) showed complete conversion of
starting material to a single product. The solvent was removed by
rotary evaporation under high vacuum, giving a light beige syrup
which was carried over into the subsequent deprotection step.
[0494] The syrup was dissolved in 15 ml of 50% formic acid and
heated at 60-70.degree. C. for two hours, at which point TLC showed
quantitative removal of the isopropylidene group. Water and formic
acid were removed by evaporation under high vacuum, giving a light
beige-pink syrup which was applied to a silica gel column and
eluted with 95:5 chloroform/methanol. Fractions containing the
product were collected, pooled, and evaporated, giving a faintly
pink glassy product.
[0495] The foregoing is intended as illustrative of the present
invention but not limiting. Numerous variations and modifications
may be effected without departing from the true spirit and scope of
the invention.
* * * * *