U.S. patent application number 12/316627 was filed with the patent office on 2009-04-23 for nucleosides with anti-hepatitis b virus activity.
Invention is credited to Gilles Gosselin, Jean-Louis Imbach, Raymond F. Schinazi, Jean-Pierre Sommadossi.
Application Number | 20090105185 12/316627 |
Document ID | / |
Family ID | 23929192 |
Filed Date | 2009-04-23 |
United States Patent
Application |
20090105185 |
Kind Code |
A1 |
Schinazi; Raymond F. ; et
al. |
April 23, 2009 |
Nucleosides with anti-hepatitis B Virus activity
Abstract
A method for the treatment of a host, and in particular, a
human, infected with hepatitis B virus (HBV) is provided that
includes administering an effective amount of a
.beta.-L-nucleotide, optionally in combination therapy with other
drugs for the treatment of HBV or human immuno-deficiency virus
(HIV).
Inventors: |
Schinazi; Raymond F.;
(Decatur, GA) ; Sommadossi; Jean-Pierre;
(Birmingham, AL) ; Gosselin; Gilles; (US) ;
Imbach; Jean-Louis; (US) |
Correspondence
Address: |
KING & SPALDING
1180 PEACHTREE STREET , NE
ATLANTA
GA
30309-3521
US
|
Family ID: |
23929192 |
Appl. No.: |
12/316627 |
Filed: |
December 15, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09879854 |
Jun 12, 2001 |
7468357 |
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12316627 |
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09112878 |
Jul 9, 1998 |
6245749 |
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09879854 |
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08485716 |
Jun 7, 1995 |
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09112878 |
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08320461 |
Oct 7, 1994 |
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08485716 |
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08119470 |
Sep 10, 1993 |
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08320461 |
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Current U.S.
Class: |
514/45 ;
514/49 |
Current CPC
Class: |
A61K 38/21 20130101;
A61K 47/544 20170801; A61K 31/7068 20130101; A61P 31/12 20180101;
A61P 31/18 20180101; A61K 31/70 20130101; A61K 2300/00 20130101;
A61P 31/20 20180101; A61K 38/21 20130101; A61K 31/7076 20130101;
A61P 43/00 20180101; A61K 31/7072 20130101; A61P 1/16 20180101 |
Class at
Publication: |
514/45 ;
514/49 |
International
Class: |
A61K 31/70 20060101
A61K031/70 |
Claims
1. A method for the treatment of HBV infection of ##STR00011##
wherein B is a purine or pyrimidine base; Y.sup.1, Y.sup.2,
Y.sup.3, and Y.sup.4 are independently H, OH, N.sub.3,
NR.sup.1R.sup.2, NO.sub.2, NOR.sup.3, --O-alkyl, --O-aryl, halo
(including F, Cl, Br, or I), --CN, --C(O)NH.sub.2, SH, --S-alkyl,
or --S-aryl, and wherein typically three of Y.sup.1, Y.sup.2,
Y.sup.3, and Y.sup.4 are either H or OH. The --OH substituent, when
present, is typically a Y.sup.1 or Y.sup.3 group. As illustrated in
the structure, Y.sup.2 and Y.sup.4 are in the arabino (erythro)
configuration, and Y.sup.1 and Y.sup.3 are in the threo (ribose)
configuration. R is H, monophosphate, diphosphate, triphosphate,
alkyl, acyl or a phosphate derivative, as described in more detail
below. R.sup.1, R.sup.2, and R.sup.3 are independently alkyl (and
in particular lower alkyl), aryl, aralkyl, alkaryl, acyl, or
hydrogen.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 08/320,461, filed Oct. 7, 1994, which is
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
[0002] This invention is in the area of methods for the treatment
of hepatitis B virus (also referred to as "HBV") that includes
administering an effective amount of one or more of the active
compounds disclosed herein, or a pharmaceutically acceptable
derivative or prodrug of one of these compounds.
[0003] HBV is second only to tobacco as a cause of human cancer.
The mechanism by which HBV induces cancer is unknown, although it
is postulated that it may directly trigger tumor development, or
indirectly trigger tumor development through chronic inflammation,
cirrhosis, and cell regeneration associated with the infection.
[0004] Hepatitis B virus has reached epidemic levels worldwide.
After a two to six month incubation period in which the host is
unaware of the infection, HBV infection can lead to acute hepatitis
and liver damage, that causes abdominal pain, jaundice, and
elevated blood levels of certain enzymes. HBV can cause fulminant
hepatitis, a rapidly progressive, often fatal form of the disease
in which massive sections of the liver are destroyed. Patients
typically recover from acute viral hepatitis. In some patients,
however, high levels of viral antigen persist in the blood for an
extended, or indefinite, period, causing a chronic infection.
Chronic infections can lead to chronic persistent hepatitis.
Patients infected with chronic persistent HBV are most common in
developing countries. By mid-1991, there were approximately 225
million chronic carriers of HBV in Asia alone, and worldwide,
almost 300 million carriers. Chronic persistent hepatitis can cause
fatigue, cirrhosis of the liver, and hepatocellular carcinoma, a
primary liver cancer. In western industrialized countries, high
risk groups for HBV infection include those in contact with HBV
carriers or their blood samples. The epidemiology of HBV is in fact
very similar to that of acquired immunodeficiency syndrome, which
accounts for why HBV infection is common among patients with AIDS
or HIV-associated infections. However, HIV is more contagious than
HIV.
[0005] Daily treatments with .alpha.-interferon, a genetically
engineered protein, has shown promise. A human serum-derived
vaccine has also been developed to immunize patients against HBV.
Vaccines have been produced through genetic engineering. While the
vaccine has been found effective, production of the vaccine is
troublesome because the supply of human serum from chronic carriers
is limited, and the purification procedure is long and expensive.
Further, each batch of vaccine prepared from different serum must
be tested in chimpanzees to ensure safety. In addition, the vaccine
does not help the patients already infected with the virus.
[0006] European Patent Application No. 92304530.6 discloses that a
group of 1,2-oxathiolane nucleosides are useful in the treatment of
hepatitis B infections. It has been reported that the
2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane has anti-hepatitis
B activity. Doong, et al., Proc. of Natl. Acad. Sci, USA, 88,
8495-8499 (1991); Chang, et al., J. of Biological Chem., Vol
267(20), 13938-13942. The anti-hepatitis B activity of the (-) and
(+)-enantiomers of
2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane has been
published by Furman, et al., in Antimicrobial Agents and
Chemotherapy, December 1992, pages 2686-2692.
[0007] PCT/US92/03144 (International Publication No. WO 92/18517)
filed by Yale University discloses a number of .beta.-L-nucleosides
for the treatment of both HBV and HIV. Other drugs exlored for the
treatment of HBV include adenosine arabinoside, thymosin,
acyclovir, phosphonoformate, zidovudine,
(+)-cyanidanol, quinacrine, and
2'-fluoroarabinosyl-5-iodouracil.
[0008] An essential step in the mode of action of purine and
pyrimidine nucleosides against viral diseases, and in particular,
HBV and HIV, is their metabolic activation by cellular and viral
kinases, to yield the mono-, di-, and triphosphate derivatives. The
biologically active species of many nucleosides is the triphospahte
form, which inhibits DNA polymerase or reverse transcriptase, or
causes chain termination. The nucleoside derivatives that have been
developed for the treatment of HBV and HIV to date have been
presented for administration to the host in unphosphorylated form,
notwithstanding the fact that the nucleoside must be phosphorylated
in the cell prior to exhibiting its antiviral effect, because the
triphosphate form has typically either been dephosphorylated prior
to reaching the cell or is poorly absorbed by the cell. Nucleotides
in general cross cell membranes very inefficiently and are
generally not very not very potent in vitro. Attempts at modifying
nucleotides to increase the absorption and potency of nucleotides
have been described by R. Jones and N. Bischofberger, Antiviral
Research, 27 (1995) 1-17, the contents of which are incorporated
herein by reference.
[0009] In light of the fact that hepatitis B virus has reached
epidemic levels worldwide, and has severe and often tragic effects
on the infected patient, there remains a strong need to provide new
effective pharmaceutical agents to treat humans infected with the
virus that have low toxicity to the host.
[0010] Therefore, it is another object of the present invention to
provide a method and composition for the treatment of human
patients or other hosts infected with HBV.
SUMMARY OF THE INVENTION
[0011] A method for the treatment of a host, and in particular, a
human, infected with HBV is provided that includes administering an
HBV-treatment amount of a nucleoside of the formula:
##STR00001##
wherein:
[0012] R.sup.1 is hydrogen, fluoro, bromo, chloro, iodo, methyl or
ethyl; and R.sup.2 is OH, Cl, NH.sub.2, or H; or a pharmaceutically
acceptable salt of the compound, optionally in a pharmaceutically
acceptable carrier or diluent.
[0013] In an alternative embodiment, the .beta.-L-enantiomer of a
compound of the formula:
##STR00002##
wherein R.sup.5 is adenine, xanthine, hypoxanthine, or other
purine, including an alkylated or halogenated purine is
administered to a host in an HBV-treatment amount as described more
fully herein.
[0014] In another alternative embodiment, the nucleoside is of the
formula:
##STR00003##
[0015] wherein B is a purine or pyrimidine base;
[0016] Y.sup.1, Y.sup.2, Y.sup.3, and Y.sup.4 are independently H,
OH, N.sub.3, NR.sup.1R.sup.2, NO.sub.2, NOR.sup.3, --O-alkyl,
--O-aryl, halo (including F, Cl, Br, or I), --CN, --C(O)NH.sub.2,
SH, --S-alkyl, or --S-aryl, and wherein typically three of Y.sup.1,
Y.sup.2, Y.sup.3, and Y.sup.4 are either H or OH. The --OH
substituent, when present, is typically a Y.sup.1 or Y.sup.3 group.
As illustrated in the structure, Y.sup.2 and Y.sup.4 are in the
arabino (erythro) configuration, and Y.sup.1 and Y.sup.3 are in the
threo (ribose) configuration. R is H, monophosphate, diphosphate,
triphosphate, alkyl, acyl or a phosphate derivative, as described
in more detail below. R.sup.1, R.sup.2, and R.sup.3 are
independently alkyl (and in particular lower alkyl), aryl, aralkyl,
alkaryl, acyl, or hydrogen.
[0017] In a preferred embodiment, the nucleoside is provided as the
indicated enantiomer and substantially in the absence of its
corresponding enantiomer (i.e., in enantiomerically enriched
form).
[0018] In another embodiment, the invention includes a method for
the treatment of humans infected with HBV that includes
administering an HBV treatment amount of a prodrug of the
specifically disclosed nucleosides. A prodrug, as used herein,
refers to a pharmaceutically acceptable derivative of the
specifically disclosed nucleoside, that is converted into the
nucleoside on administration in vivo, or that has activity in
itself. Nonlimiting examples are the 5' and N.sup.4-pyrimidine or
N.sup.6-purine acylated or alkylated derivatives of the active
compound.
[0019] In a preferred embodiment of the invention, the nucleoside
is provided as the monophosphate, diphosphate or triphosphate in a
formulation that protects the compound from dephosphorylation.
Formulations include liposomes, lipospheres, microspheres or
nanospheres (of which the latter three can be targeted to infected
cells). In an alternative preferred embodiment, the nucleoside is
provided as a monophosphate, diphosphate or triphosphate derivative
(i.e., a nucleotide prodrug), for example an ester, that stabilizes
the phosphate in vivo.
[0020] The disclosed nucleosides, or their pharmaceutically
acceptable prodrugs or salts or pharmaceutically acceptable
formulations containing these compounds are useful in the
prevention and treatment of HBV infections and other related
conditions such as anti-HBV antibody positive and HBV-positive
conditions, chronic liver inflammation caused by HBV, cirrhosis,
acute hepatitis, fulminant hepatitis, chronic persistent hepatitis,
and fatigue. These compounds or formulations can also be used
prophylactically to prevent or retard the progression of clinical
illness in individuals who are anti-HBV antibody or HBV-antigen
positive or who have been exposed to HBV.
[0021] In one embodiment of the invention, one or more of the
active compounds is administered in alternation or combination with
one or more other anti-HBV agents, to provide effective anti-HBV
treatment. Examples of anti-HBV agents that can be used in
alternation or combination therapy include but are not limited to
the (-)-enantiomer or racemic mixture of
2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane ("FTC",
see WO 92/14743), its physiologically acceptable derivative, or
physiologically acceptable salt; the (-)-enantiomer or racemic
mixture of 2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane, its
physiologically acceptable derivative, or physiologically
acceptable salt; an enantiomer or racemic mixture of
2'-fluoro-5-iodo-arabinosyluracil (FIAU); an enantiomer or racemic
mixture of 2'-fluoro-5-ethyl-arabinosyluracil (FEAU); carbovir, or
interferon.
[0022] Any method of alternation can be used that provides
treatment to the patient. Nonlimiting examples of alternation
patterns include 1-6 weeks of administration of an effective amount
of one agent followed by 1-6 weeks of administration of an
effective amount of a second anti-HBV agent. The alternation
schedule can include periods of no treatment. Combination therapy
generally includes the simultaneous administration of an effective
ratio of dosages of two or more anti-HBV agents.
[0023] In light of the fact that HBV is often found in patients who
are also anti-HIV antibody or HIV-antigen positive or who have been
exposed to HIV, the active anti-HBV compounds disclosed herein or
their derivatives or prodrugs can be administered in the
appropriate circumstance in combination or alternation with
anti-HIV medications, including but not limited to
3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (DDI),
2',3'-dideoxycytidine (DDC), 2',3'-dideoxy-2',3'-didehydrothymidine
(D4T), 2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane
(FTC), or 2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane
(BCH-189), in racemic or enantiomeric form. Non-nucleoside
RT-inhibitors such as the Tibo class of compounds, nevirapine, or
pyrimidinone can also be administered in combination with the
claimed compounds.
[0024] The active anti-HBV agents can also be administered in
combination with antibiotics, other antiviral compounds, antifungal
agents, or other pharmaceutical agents administered for the
treatment of secondary infections.
BRIEF DESCRIPTION OF THE FIGURES
[0025] FIG. 1 is an illustration of the chemical structures of
13-L-2',3'-dideoxycytidine (.beta.-L-FddC),
.beta.-D-2',3'-dideoxycytidine (.beta.-D-ddC),
13-L-2',3'-dideoxy-5-fluorocytidine (.beta.-L-ddC),
(-)-.beta.-L-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane
((-)-.beta.-L-FTC),
(+)-.beta.-D-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-dioxolane
((+)-.beta.-D-FDOC), and
.beta.-L-2-amino-6-(R.sup.4)-9-[(4-hydroxymethyl)-tetrahydrofuran-1-yl]pu-
rine.
[0026] FIG. 2 is an illustration of the numbering scheme used in
the chemical nomenclature for nucleosides in this text.
DETAILED DESCRIPTION OF THE INVENTION
[0027] As used herein, the term "enantiomerically pure" refers to a
nucleoside composition that includes at least approximately 95%,
and preferably approximately 97%, 98%, 99%, or 100% of a single
enantiomer of that nucleoside.
[0028] The term alkyl, as used herein, unless otherwise specified,
refers to a saturated straight, branched, or cyclic, primary,
secondary, or tertiary hydrocarbon of C.sub.1 to C.sub.10, and
specifically includes methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, t-butyl, pentyl, cyclopentyl, isopentyl, neopentyl,
hexyl, isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl,
2,2-dimethylbutyl, and 2,3-dimethylbutyl. The alkyl group can be
optionally substituted with one or more moieties selected from the
group consisting of hydroxyl, amino, alkylamino, arylamino, alkoxy,
aryloxy, nitro, cyano, sulfonic acid, sulfate, phosphonic acid,
phosphate, or phosphonate, either unprotected, or protected as
necessary, as known to those skilled in the art, for example, as
taught in Greene, et al., "Protective Groups in Organic Synthesis,"
John Wiley and Sons, Second Edition, 1991. The term lower alkyl, as
used herein, and unless otherwise specified, refers to a C.sub.1 to
C.sub.4 ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl,
sec-butyl, or t-butyl group.
[0029] As used herein, the term acyl specifically includes but is
not limited to acetyl, propionyl, butyryl, pentanoyl,
3-methylbutyryl, hydrogen succinate, 3-chlorobenzoate, benzoyl,
acetyl, pivaloyl, mesylate, propionyl, valeryl, caproic, caprylic,
capric, lauric, myristic, palmitic, stearic, and oleic.
[0030] The term aryl, as used herein, and unless otherwise
specified, refers to phenyl, biphenyl, or naphthyl, and preferably
phenyl. The aryl group can be optionally substituted with one or
more moieties selected from the group consisting of hydroxyl,
amino, alkylamino, arylamino, alkoxy, aryloxy, nitro, cyano,
sulfonic acid, sulfate, phosphonic acid, phosphate, or phosphonate,
either unprotected, or protected as necessary, as known to those
skilled in the art, for example, as taught in Greene, et al.,
"Protective Groups in Organic Synthesis," John Wiley and Sons,
Second Edition, 1991.
[0031] The term purine or pyrimidine base includes, but is not
limited to, adenine, N.sup.6-alkylpurines, N.sup.6-acylpurines
(wherein acyl is C(O)(alkyl, aryl, alkylaryl, or arylalkyl),
N.sup.6-benzylpurine, N.sup.6-halopurine, N.sup.6-vinylpurine,
N.sup.6-acetylenic purine, N.sup.6-acyl purine,
N.sup.6-hydroxyalkyl purine, N.sup.6-thioalkyl purine,
N.sup.2-alkylpurines, N.sup.2-alkyl-6-thiopurines, thymine,
cytosine, 6-azapyrimidine, 2- and/or 4-mercaptopyrmidine, uracil,
C.sup.5-alkylpyrimidines, C.sup.5-benzylpyrimidines,
C.sup.5-halopyrimidines, C.sup.5-vinylpyrimidine,
C.sup.5-acetylenic pyrimidine, C.sup.5-acyl pyrimidine,
C.sup.5-hydroxyalkyl purine, C.sup.5-amidopyrimidine,
C.sup.5-cyanopyrimidine, C.sup.5-nitropyrimidine,
C.sup.5-aminopyrimidine, N.sup.2-alkylpurines,
N.sup.2-alkyl-6-thiopurines, 5-azacytidinyl, 5-azauracilyl,
triazolopyridinyl, imidazolopyridinyl, pyrrolopyrimidinyl,
pyrazolopyrimidinyl. Functional oxygen and nitrogen groups on the
base can be protected as necessary or desired. Suitable protecting
groups are well known to those skilled in the art, and include
trimethylsilyl, dimethylhexylsilyl, t-butyldimethylsilyl, and
t-butyldiphenylsilyl, trityl, alkyl groups, acyl groups such as
acetyl and propionyl, methylsulfonyl, and p-toluoylsulfonyl.
[0032] As used herein, the term natural amino acid includes but is
not limited to alanyl, valinyl, leucinyl, isoleucinyl, prolinyl,
phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl,
threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl,
aspartoyl, glutaoyl, lysinyl, argininyl, and histidinyl.
[0033] The invention as disclosed herein is a method and
composition for the treatment of HBV infection and other viruses
replicating in a like manner, in humans or other host animals, that
includes administering an effective amount of one or more of the
above-identified compounds, or a physiologically acceptable
derivative, or a physiologically acceptable salt thereof,
optionally in a pharmaceutically acceptable carrier. The compounds
of this invention either possess anti-HBV activity, or are
metabolized to a compound or compounds that exhibit anti-HBV
activity.
I. Structure and Preparation of Active Nucleosides
Stereochemistry
[0034] The compounds used in the methods disclosed herein are
enantiomers of 2',3'-dideoxycytidine, 2',3'-dideoxy-5-(halo or
methyl)cytidine,
2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-dioxolane, or
2-amino-6-(OH, Cl, NH.sub.2, or
H)-9-[(4-hydroxymethyl)-tetrahydrofuran-1-yl]purine.
[0035] Since the 1' and 4' carbons of the sugar or dioxolanyl
moiety (referred to below generically as the sugar moiety) of the
nucleosides are chiral, their nonhydrogen substituents (CH.sub.2OR
and the pyrimidine or purine base, respectively) can be either cis
(on the same side) or trans (on opposite sides) with respect to the
sugar ring system. The four optical isomers therefore are
represented by the following configurations (when orienting the
sugar moiety in a horizontal plane such that the "primary" oxygen
(that between the C1' and C4'-atoms; see FIG. 2) is in back): cis
(with both groups "up", which corresponds to the configuration of
naturally occurring nucleosides), cis (with both groups "down",
which is a nonnaturally occurring configuration), trans (with the
C2 substituent "up" and the C5 substituent "down"), and trans (with
the C2 substituent "down" and the C5 substituent "up"). As
indicated schematically in FIG. 1, the "D-nucleosides" are cis
nucleosides in a natural configuration and the "L-nucleosides" are
cis nucleosides in the nonnaturally occurring configuration.
[0036] The nucleosides useful in the disclosed method to treat HBV
infection are .beta.-L-enantiomers, with the exception of FDOC,
which is used in its .beta.-D-enantiomeric form, because it has
been discovered that the .beta.-D-enantiomer of FDOC is
surprisingly less toxic than the .beta.-L-enantiomer of FDOC.
Prodrug Formulations
[0037] The nucleosides disclosed herein can be administered as any
derivative that upon administration to the recipient, is capable of
providing directly or indirectly, the parent active compound, or
that exhibits activity in itself. In one embodiment, the hydrogen
of the 5'-OH group is replaced by a C.sub.1-C.sub.20 alkyl; acyl in
which the non-carbonyl moiety of the ester group is selected from
straight, branched, or cyclic C.sub.1-C.sub.20 alkyl, phenyl, or
benzyl; a naturally occurring or nonnaturally occurring amino acid;
alkoxyalkyl including methoxymethyl; aralkyl including benzyl;
aryloxyalkyl such as phenoxymethyl; aryl including phenyl
optionally substituted with halogen, C.sub.1 to C.sub.4 alkyl or
C.sub.1 to C.sub.4 alkoxy; a dicarboxylic acid such as succinic
acid; sulfonate esters such as alkyl or aralkyl sulphonyl including
methanesulfonyl; or a mono, di or triphosphate ester.
[0038] One or both hydrogens of the amino groups on the purine or
pyrimidine base can be replaced by a C.sub.1-C.sub.20 alkyl; acyl
in which the non-carbonyl moiety of the ester group is selected
from straight, branched, or cyclic C.sub.1-C.sub.20 alkyl, phenyl,
or benzyl; alkoxyalkyl including methoxymethyl; aralkyl including
benzyl; aryloxyalkyl such as phenoxymethyl; aryl including phenyl
optionally substituted with halogen, C.sub.1 to C.sub.4 alkyl or
C.sub.1 to C.sub.4 alkoxy.
[0039] The active nucleoside can also be provided as a 5'-ether
lipid, as disclosed in the following references, which are
incorporated by reference herein: Kucera, L. S., N. Iyer, E. Leake,
A. Raben, Modest E. J., D. L. W., and C. Piantadosi. 1990. Novel
membrane-interactive ether lipid analogs that inhibit infectious
HIV-1 production and induce defective virus formation. AIDS Res Hum
Retroviruses. 6:491-501; Piantadosi, C., J. Marasco C. J., S. L.
Morris-Natschke, K. L. Meyer, F. Gumus, J. R. Surles, K. S. Ishaq,
L. S. Kucera, N. Iyer, C. A. Wallen, S. Piantadosi, and E. J.
Modest. 1991. Synthesis and evaluation of novel ether lipid
nucleoside conjugates for anti-HIV activity. J Med. Chem.
34:1408.1414; Hostetler, K. Y., D. D. Richman, D. A. Carson, L. M.
Stuhmiller, G. M. T. van Wijk, and H. van den Bosch. 1992. Greatly
enhanced inhibition of human immunodeficiency virus type 1
replication in CEM and HT4-6C cells by 3'-deoxythymidine
diphosphate dimyristoylglycerol, a lipid prodrug of
3,-deoxythymidine. Antimicrob Agents Chemother. 36:2025.2029;
Hostetler, K. Y., L. M. Stuhmiller, H. B. Lenting, H. van den
Bosch, and D. D. Richman, 1990. Synthesis and antiretroviral
activity of phospholipid analogs of azidothymidine and other
antiviral nucleosides. J. Biol. Chem. 265:6112.7.
Nucleotide Prodrugs
[0040] Any of the nucleosides described herein can be administered
as a nucleotide prodrug to increase the activity, bioavailability,
stability or otherwise alter the properties of the nucleoside. A
number of nucleotide prodrug ligands are known. In general,
alkylation, acylation or other lipophilic modification of the mono,
di or triphosphoate of the nucleoside will increase the stability
of the nucleotide. Examples of substituent groups that can replace
one or more hydrogens on the phosphate moiety are alkyl, aryl,
steroids, carbohydrates, including sugars, 1,2-diacylglycerol and
alcohols. Many are described in R. Jones and N. Bischofberger,
Antiviral Research, 27 (1995) 1-17. Any of these can be used in
combination with the disclosed nucleosides to achieve a desired
effect. Nonlimiting examples of nucleotide prodrugs are described
in the following references.
[0041] Ho, D. H. W. (1973) Distribution of Kinase and deaminase of
1.beta.-D-arabinofuranosylcytosine in tissues of man and muse.
Cancer Res. 33, 2816-2820; Holy, A. (1993) Isopolar
phosphorous-modified nucleotide analogues. In: De Clercq (Ed.),
Advances in Antiviral Drug Design, Vol. I, JAI Press, pp. 179-231;
Hong, C. I., Nechaev, A., and West, C. R. (1979a) Synthesis and
antitumor activity of 1.beta.-D-arabinofuranosylcytosine conjugates
of cortisol and cortisone. Biochem. Biophys. Rs. Commun. 88,
1223-1229; Hong, C. I., Nechaev, A., Kirisits, A. J. Buchheit, D.
J. and West, C. R. (1980) Nucleoside conjugates as potential
antitumor agents. 3. Synthesis and antitumor activity of
1-(.beta.-D-arabinofuranosyl)cytosine conjugates of corticosteriods
and selected lipophilic alcohols. J. Med. Chem. 28, 171-177;
Hostetler, K. Y., Stuhmiller, L. M., Lenting, H. B. M. van den
Bosch, H. and Richman, D. D. (1990) Synthesis and antiretrioviral
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Preparation of the Active Compounds
[0043] The nucleosides used in the disclosed method to treat HBV
infections in a host organism can be prepared according to
published methods. .beta.-L-Nucleosides can be prepared from
methods disclosed in, or standard modifications of methods
disclosed in, for example, the following publications: Jeong, et
al., J. of Med. Chem., 36, 182-195, 1993; European Patent
Application Publication No. 0 285 884; Genu-Dellac, C., G.
Gosselin, A.-M. Aubertin, G. Obert, A. Kirn, and J.-L. Imbach,
3-Substituted thymine .alpha.-L-nucleoside derivatives as potential
antiviral agents; synthesis and biological evaluation, Antiviral
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Brankovan, and J. C. Martin, Preparation of the geometric isomers
of DDC, DDA, D4C and D4T as potential anti-HIV agents, Bioorg. Med.
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[0044] 2',3'-Dideoxycytidine (DDC) is a known compound. The
D-enantiomer of DDC is currently being marketed by Hoffman-LaRoche
under the name Zalcitabine for use in the treatment of persons
infected with HIV. See U.S. Pat. Nos. 4,879,277 and 4,900,828.
[0045] Enantiomerically pure .beta.-D-dioxolane-nucleosides such as
.beta.-D-FDOC can be prepared as disclosed in detail in
PCT/US91/09124. The process involves the initial preparation of
(2R,4R)- and (2R,4S)-4-acetoxy-2-(protected-oxymethyl)-dioxolane
from 1,6-anhydromannose, a sugar that contains all of the necessary
stereochemistry for the enantiomerically pure final product,
including the correct diastereomeric configuration about the 1
position of the sugar (that becomes the 4'-position in the later
formed nucleoside). The (2R,4R)- and
(2R,4S)-4-acetoxy-2-(protected-oxymethyl)-dioxolane is condensed
with a desired heterocyclic base in the presence of SnCl.sub.4,
other Lewis acid, or trimethylsilyl triflate in an organic solvent
such as dichloroethane, acetonitrile, or methylene chloride, to
provide the stereochemically pure dioxolane-nucleoside.
[0046] Enzymatic methods for the separation of D and L enantiomers
of cis-nucleosides are disclosed in, for example, Nucleosides and
Nucleotides, 12(2), 225-236 (1993); European Patent Application
Nos. 92304551.2 and 92304552.0 filed by Biochem Pharma, Inc.; and
PCT Publication Nos. WO 91/11186, WO 92/14729, and WO 92/14743
filed by Emory University.
[0047] Separation of the acylated or alkylated racemic mixture of D
and L enantiomers of cis-nucleosides can be accomplished by high
performance liquid chromatography with chiral stationary phases, as
disclosed in PCT Publication No. WO 92/14729.
[0048] Mono, di, and triphosphate derivative of the active
nucleosides can be prepared as described according to published
methods. The monophosphate can be prepared according to the
procedure of Imai et al., J. Org. Chem., 34(6), 1547-1550 (June
1969). The diphosphate can be prepared according to the procedure
of Davisson et al., J. Org. Chem., 52(9), 1794-1801 (1987). The
triphosphate can be prepared according to the procedure of Hoard et
al., J. Am. Chem. Soc., 87(8), 1785-1788 (1965).
General Procedures for the Preparation of Bis (S-acyl-2-thioethyl)
Phosphoester of .beta.-L-Dideoxynucleosides [Bis (SATE) .beta.-L
ddx MP]
##STR00004##
[0049] Bis (SATE) .beta.-L-ddxMP
Y.sub.1, Y.sub.2, Y.sub.3, Y.sub.4=H, F, N.sub.3, NR.sub.1R.sub.2,
NO.sub.2, NOR, O-alkyl, O-aryl
R.sup.1=CH.sub.3, (CH.sub.3).sub.2 CH, (CH.sub.3).sub.3C,
C.sub.6H.sub.5.
[0050] (i) ICH.sub.2CH.sub.2OH, DBU/C.sub.6H.sub.5CH.sub.3; (ii)
Cl.sub.2PN(iPr).sub.2, NEt.sub.3/THF; (iii)
.beta.-L-dideoxynucleoside, 1H-tetrazole/THF, then
ClC.sub.6H.sub.4CO.sub.3H/CH.sub.2Cl.sub.2
[0051] 1H-Tetrazole (0.21 g, 3.0 mmol) was added to a stirred
solution of .beta.-L-dideoxynucleoside (1.0 mmol) and the
appropriate phosphoramidite C (1.2 mmol) in tetrahydrofuran (2 mL)
at room temperature. After 30 minutes, the reaction mixture was
cooled to -40.degree. C. and a solution of 3-chloroperoxybenzoic
acid (0.23 g, 1.3 mmol) in dichloromethane (2.5 mL) was added; the
mixture was then allowed to warm to room temperature over 1 h.
Sodium sulfite (10% solution, 1.3 mL) was added to the mixture to
destroy the excess 3-chloroperoxybenzoic acid, after which the
organic layer was separated and the aqueous layer washed with
dichloromethane (2.times.10 mL). The combined organic layers were
washed with saturated aqueous sodium hydrogen carbonate (5 mL),
then water (3.times.5 mL), dried over sodium sulfate, filtered and
evaporated to dryness under reduced pressure. Column chromatography
of the residue on silica gel afforded the title Bis(SATE)
.beta.-L-ddxmP.
EXAMPLE=.beta.-L-2',3'-Dideoxyadenosin-5'-yl
bis(2-pivaloylthioethyl) phosphate [Bis (SATE) .beta.-L-ddAMP].
##STR00005##
[0052] Following the above general procedure, pure
Bis(SATE).beta.-L-ddAMP was obtained as a colorless oil in 72%
yield after silica gel column chromatography [eluent: stepwise
gradient of methanol (0-3%) in dichloromethane]; .sup.1NMR
(DMSO-d.sub.6) .delta. ppm: 8.26 and 8.13 (2s, 2H each, H-2 and
H-8), 7.20 (br s, 2H, NH.sub.2), 6.24 (t, 1H, H-1'; J=6.0 Hz),
4.35-4.25 (m, 1H, H-4'), 4.25-4.00 (m, 2H, H-5', 5''), 3.96 (m, 4H,
2 SCH.sub.2CH.sub.2O), 3.04 (t, 4H, 2 SCH.sub.2CH.sub.2O; J=6.3
Hz), 2.5-2.4 (m, 2H, H-2',2'') 2.2-2.0 (m, 2H, H-3',3''), 1.15 [s,
18H, 2 (CH.sub.3).sub.3C]; .sup.31PNMR (DMSO-d.sub.6) .delta.
ppm=-0.76 (s); UV (EtOH), .lamda..sub.max=259 nm (.epsilon. 15400);
mass spectrum (performed in: glycerol, thioglycerol, 1:1,
.nu./.nu.), FAB>O 604 (M+H).sup.+, 136 (BH.sub.2).sup.+.
General Scheme for the Sterospecific Synthesis of 3'-Substituted
.beta.-L-Dideoxynucleosides
##STR00006##
[0053] EXAMPLE
1-(3-Azido-2-3-dideoxy-.beta.-L-erythro-pentofuranosyl) thymine
[.beta.-L-AZT]
##STR00007##
[0054] A mixture of diethyl azodicarboxylate (0.46 mL; 2.9 mmol)
and diphenyl phosphorazidate (0.62 ml; 2.9 mmol) in THF (2.9 ml)
was added dropwise over 30 min. to a solution of
1-(2-deoxy-5-O-monomethoxytrityl-.beta.-L-threo-pentofuranosyl)
thymine 8 [0.5 g, 0.97 mmol] and triphenylphosphine (0.76 g, 2.9
mmol) in THF 11.6 ml) at 0.degree. C. The mixture was stirred for
3.5 h at room temperature, and ethanol was added. After
concentration to dryness in vacuo, the residue was dissolved in a
mixture of acidic acid (240 ml) and water (60 ml) in order to
remove the mMTr protecting group. The mixture was stirred for 5
hours at room temperature and was diluted with toluene. The
separated aqueous phase was concentrated to dryness in vacuo. The
residue was purified over a silica gel column eluted with ethyl
acetate to afford .beta.-L-AZT (105 mg, 40%, crystallized from
ethyl acetate). The physicochemical data of .beta.-L-AZT were in
accordance with literature data [J. Wengel, J-Lau, E. B. Ledersen,
C. N. Nielsen, J. Org. Chem. 56 (11), 3591-3594 (1991)].
General Scheme for the Stereospecific Synthesis of 2'-Substituted
.beta.-L-Dideoxynucleosides
##STR00008##
[0055]
EXAMPLE=1-(2-Fluoro-2,3-dideoxy-.beta.-L-threo-pentofuranosyl)-5-fl-
uorocytosine [2'-F-.beta.-L .beta.-L-FddC]
##STR00009##
[0056] Hitherto unknown 2'-F-.beta.-L-FddC was synthesized in five
steps from
1-(5-O-benzoyl-3-deoxy-.beta.-L-erythro-pentofuranosyl)-5-fluorourac-
il 17 with an overall yield of 28%. m.p. 209-210.degree. C.
(crystallized from absolute ethanol); UV (Et OH) .lamda..sub.max
276 .sub.nm (.epsilon., 9000), .lamda..sub.min 226 nm (E, 4000);
.sup.19F-NMR (DMSO-d.sub.6) .delta. ppm: -179.7 (m, F.sub.2),
-167.2 (dd, F.sub.5; J.sub.F,6=7.3 Hz, J.sub.F,1=1.5 Hz);
.sup.1H-NMR (DMSO-d.sub.6) .delta. ppm: 8.30 (d, 1H, H-6;
J.sub.6,F=7.3 Hz), 7.8-7.5 (br s, 2H, NH.sub.2), 5.80 (d, 1H, H-1'
J.sub.1',F=17.4 Hz), 5.34 (t, 1H, OH-5'; J=4.8 Hz), 5.10 (dd, 1H,
H-2'; J.sub.2',F=51.2 Hz; J.sub.2,3=3.4 Hz), 4.3 (m, 1H, H-4'),
3.8-3.6 (m, 2H, H-5',5''), 2.2-2.0 (m, 2H, H-3', H-3''); mass
spectra (performed in: glycerol-thioglycerol, 1:1 .nu./.nu.),
FAB>0:248 (M+H).sup.+, 130 (BH.sub.2).sup.+; FAB<0:246
(M-H).sup.-; [.alpha.].sup.20.sub.D=-16.5 (-c 0.85, DMSO). Anal.
Calc. for C.sub.9H.sub.11N.sub.3O.sub.3F.sub.2: C, 43.73; H, 9.49;
N. 17.00; F. 15.37. Found: C, 43.56; H,
[0057] 4.78; N, 16.75; F, 14.96.
##STR00010##
II. Anti-HBV Activity of Nucleosides
[0058] The ability of the active compounds to inhibit HBV can be
measured by various experimental techniques. The assay used herein
to evaluate the ability of the disclosed compounds to inhibit the
replication of HBV is described in detail in Korba and Gerin,
Antiviral Res. 19: 55-70 (1992). For purposes of illustration only,
and without limiting the invention, the results of the evaluation
of toxicity and anti-HBV activity are provided below for
.beta.-L-2',3'-dideoxycytidine (.beta.-L-FddC),
.beta.-L-2',3'-dideoxy-5-fluorocytidine (.beta.-L-ddC), and
(+)-.beta.-D-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-dioxolane
((+)-.beta.-D-FDOC). The toxicity and anti-HBV activity of
(-)-.beta.-L-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane
((-)-.beta.-L-FTC) and .beta.-D-2',3'-dideoxycytidine
(.beta.-D-ddC) are included as controls. The other compounds
disclosed herein can be evaluated similarly.
[0059] The samples of .beta.-L-ddC and .beta.-L-5-FddC used in the
anti-HBV assays were characterized as follows.
[0060] 2',3'-Dideoxy-.beta.-L-cytidine (.beta.-L-DDC).
m.p.=220-220.degree. C.; UV (EtOH 95) max 273 nm, .lamda.min 252
nm; NMR-.sup.1H (DMSO-d.sub.6) .delta. ppm=7.89 (d. 1H. H-6; J=7.4
Hz). 7.15-6.95 (d large, 2H, NH.sub.2), 5.91 (dd. .sup.1H, H-1';
J=3.0 et 6.5 Hz), 5.66 (d, 1H, H-5; J 7.4 Hz), 4.99 [t. 1H, OH-5';
J-5.2 Hz]. 4.05-3.95 (m, 1H, H-4'), 3.60-3.70 (m, 1H, H-5'; after
D.sub.2O exchange: dd, 3.64 ppm, J=3.6 et 12.O Hz). 3.60-3.50 (m.
1H, H-5''; after D.sub.2O exchange: dd, 3.50 ppm, J=4,1 et 12.0
Hz), 2.30-2.15 (m. 1H, H-2'), 1.9-1.65 (m. 3H, H-2'', -3' et 3'');
[.alpha.].sub.D.sup.20-103.6 (c 0.8 MeOH); mass spectrum (performed
in: glycerol-thioglycerol, 50:50. v/v); FAB>0 423 [2M+H].sup.+,
304 [M+glycerol+H].sup.+. 212 [M+H].sup.+, 112 [BH.sub.2].sup.+,
101 [s].sup.+; FAB<O 210 [M-H].sup.-. Anal. Calc. for
C.sub.9H.sub.13N.sub.3O.sub.3 (M=211.21); C 51.18; H, 6.20; N 19.89
found; C 51.34; H 6.25; N 20.12.
[0061] 2',3'-Dideoxy-.beta.-L-5-fluorocytidine (.beta.-L-5-FDDC).
m.p.=158-160.degree. C.; UV (EtOH 95) .lamda.max 281 nm (.epsilon.,
8100) et 237 nm (.epsilon., 8500); min 260 nm (.epsilon., 5700) et
225 nm (.epsilon., 7800); NMR-.sup.1H (DMSO-d.sub.6) .delta. ppm
8.28 (d. 1H, H-6; J-7.4 Hz), 7.7-7.4 (d large, 2H, NH.sub.2), 5.83
(dd poorly resolved, 1H, H-1'), 5.16 (t. 1H, OH-5'; J=5.1 Hz),
4.05-3.95 (m, 1H, H-4'), 3.8-3.70 [m, 1H, H 5'; after D20 exchange:
dd, 3.71 ppm. J=2.7 et 12.3 Hz], 3.60-3.50 [m. 1H, H-5''; after
D.sub.20 exchange: dd, 3.52 ppm; J=3.3 et 12.3 Hz], 2.35-2.15 (m,
1H, H-2'). 1.95-1.75 (m, 3H, H-2'', 3' et 3''):
[.alpha.].sub.D.sup.20-80.0 (-c 1.0, DMSO); Mass spectrum
[performed in: 3-nitrobenzyl alcohol] FAB>0 230 [M+H].sup.+ et
101 [s].sup.+; FAB<O 228 [M-II].sup.-. Anal. Calculated for
C.sub.9H.sub.12N.sub.3FO.sub.3(M=229.21); C 47.16; II 5.28; N
18.33, F 8.29, Found. C 16.90; H 5.28; N 18.07; F 8.17.
[0062] The antiviral evaluations were performed on two separate
passages of cells, two cultures per passage (4 cultures total). All
wells, in all plates, were seeded at the same density and at the
same time.
[0063] Due to the inherent variations in the levels of both
intracellular and extracellular HBV DNA, only depressions greater
than 3.0-fold (for HBV virion DNA) or 2.5-fold (for HBV DNA
replication intermediates) from the average levels for these HBV
DNA forms in untreated cells are generally considered to be
statistically significant [P<0.05] (Korba and Gerin, Antiviral
Res. 19: 55-70, 1992). The levels of integrated HBV DNA in each
cellular DNA preparation (which remain constant on a per cell basis
in these experiments) were used to calculate the levels of
intracellular HBV DNA forms, thereby eliminating technical
variations inherent in the blot hybridization assays.
[0064] Typical values for extracellular HBV virion DNA in untreated
cells range from 50 to 150 pg/ml culture medium (average of
approximately 76 pg/ml). Intracellular HBV DNA replication
intermediates in untreated cells range from 50 to 100 pg/ug cell
DNA (average approximately 74 pg/ug cell DNA). In general,
depressions in the levels of intracellular HBV DNA due to treatment
with antiviral compounds are less pronounced, and occur more
slowly, than depressions in the levels of HBV virion DNA.
[0065] For reference, the manner in which the hybridization
analyses were performed for these experiments results in an
equivalence of approximately 1.0 pg intracellular HBV DNA/ug
cellular DNA to 2-3 genomic copies per cell and 1.0 pg of
extracellular HBV DNA/ml culture medium to 3.times.10.sup.5 viral
particles/ml.
[0066] Toxicity analyses were performed in order to assess whether
any observed antiviral effects were due to a general effect on cell
viability. The method used was based on the uptake of neutral red
dye, a standard and widely used assay for cell viability in a
variety of virus-host systems, including HSV (herpes simplex virus)
and HIV.
[0067] The test compounds were used in the form of 40 mM stock
solutions in DMSO (frozen on dry ice). Daily aliquots of the test
samples were made and frozen at -20.degree. C. so that each
individual aliquot would be subjected to a single freeze-thaw
cycle. The daily test aliquots were thawed, suspended into culture
medium at room temperature and immediately added to the cell
cultures. The compounds were tested at 0.01 to 10 .mu.M for
antiviral activity. The compounds were tested for toxicity at
concentrations from 1 to 300 .mu.M. The results are provided in
Table 1.
TABLE-US-00001 TABLE 1 EFFECT OF D-DDC, L-DDC, L-FDDC, FDOC and
(-)-FTC AGAINST HEPATITIS B VIRUS IN TRANSFECTED HEPG-2 (2.2.15)
CELLS Selectivity Index HBV viron.sup.a HBV RI.sup.b Cytotoxicity
IC.sub.50/EC.sub.90 Compound EC.sub.50 .+-. SD EC.sub.90 .+-. SD
EC.sub.50 .+-. SD EC.sub.90 .+-. SD IC.sub.50 .+-. SD Virion RI
.beta.-D-DDC .sup. 1.3 .+-. 0.2.sup.c 2.1 .+-. 0.3 8.1 .+-. 1.7
12.0 .+-. 2.4 .sup. 219 .+-. 28.sup.C 104 18 1.5 .+-. 0.7 9.4 .+-.
2.5 3.2 .+-. 0.6 11.0 .+-. 2.0 216 .+-. 22 23 20 .beta.-L-DDC 0.033
.+-. 0.003 1.1 .+-. 0.2 0.107 .+-. 0.012 1.8 .+-. 0.2 493 .+-. 64
448 274 .beta.-L-FDDC 0.12 .+-. 0.01 0.30 .+-. 0.03 2.8 .+-. 0.4
4.8 .+-. 0.6 438 .+-. 57 1,460 91 (+)-.beta.-D-FDOC 0.020 .+-.
0.003 0.195 .+-. 0.027 0.062 .+-. 0.012 0.23 .+-. 0.02 251 .+-. 23
1,287 1,091 (-)-.beta.-L-FTC 0.017 .+-. 0.005 0.15 .+-. 0.02 0.049
.+-. 0.008 0.18 .+-. 0.03 292 .+-. 13 1,947 1,622
.sup.aExtracellular DNA .sup.bReplicative intermediates
(Intracellular DNA) .sup.c.mu.M
EXAMPLE 2
Toxicity of Compounds
[0068] The ability of the active compounds to inhibit the growth of
virus in 2.2.15 cell cultures (HepG2 cells transformed with
hepatitis virion) was evaluated. As illustrated in Table 1, no
significant toxicity (greater than 50% depression of the dye uptake
levels observed in untreated cells) was observed for any of the
test compounds at the concentrations 100 .mu.M. The compounds were
moderately toxic at 300 .mu.M, however, all three compounds
exhibited less toxicity at this concentration than .beta.-D-ddC. It
appears that the IC.sub.50 of .beta.-L-ddC and .beta.-L-FddC is
approximately twice that of .beta.-D-ddC.
[0069] Toxicity analyses were performed in 96-well flat bottomed
tissue culture plates. Cells for the toxicity analyses were
cultured and treated with test compounds with the same schedule as
used for the antiviral evaluations. Each compound was tested at 4
concentrations, each in triplicate cultures. Uptake of neutral red
dye was used to determine the relative level of toxicity. The
absorbance of internalized dye at 510 nM (A.sub.510) was used for
the quantitative analysis. Values are presented as a percentage of
the average A.sub.510 values (.+-.standard deviations) in 9
separate cultures of untreated cells maintained on the same 96-well
plate as the test compounds. The percentage of dye uptake in the 9
control cultures on plate 40 was 100.+-.3. At 150-190 .mu.M
.beta.-D-ddC, a 2-fold reduction in dye uptake (versus the levels
observed in untreated cultures) is typically observed in these
assays (Korba and Gerin, Antiviral Res. 19: 55-70, 1992).
EXAMPLE 3
Anti-Hepatitis B Virus Activity
[0070] The positive treatment control,
.beta.-D-2',3'-dideoxycytosine [.beta.-D-ddC], induced significant
depressions of HBV DNA replication at the concentration used.
Previous studies have indicated that at 9-12 .mu.M of .beta.-D-ddC,
a 90% depression of HBV RI (relative to average levels in untreated
cells) is typically observed in this assay system (Korba and Gerin,
Antiviral Res. 19: 55-70, 1992). This is consistent with the data
presented in Table 1.
[0071] The data presented in Table 1 indicates that all three test
compounds ((.beta.-L-FddC), (.beta.-L-ddC), and .beta.-D-FDOC)),
were potent inhibitors of HBV replication, causing depression of
HBV virion DNA and HBV RI to a degree comparable to, or greater
than, that observed following treatment with .beta.-D-ddC.
EXAMPLE 4
[0072] The effect of selected .beta.-L-derivatives against
Hepatitis B virus replication in transfected Hep G-2 cells is
described in Table 4.
TABLE-US-00002 TABLE 1 Effect of L-derivatives against Hepatitis B
virus replicaTion in transfected Hep G-2 (2.2.15) cells.
Selectivity HBV HBV Cyto- Index Virion.sup.a RI.sup.b toxicity
IC.sub.50/EC.sub.50 Compound EC.sub.50 EC.sub.50 IC.sub.50 Virion
RI .beta.-L-ddA 5.0.sup.C 5.0 250 50 50 Bis (Sate) .beta.-L-ddAMP
0.45 0.35 200 445 571 .beta.-L-AZT >10 >10 1000 NA NA Bis
(Sate) .beta.-L-AZTMP 7.5 8 200 27 25 2'-F-.beta.-L-5FDDC 1.7 5.0
210 124 42 .sup.aExtracellular DNA .sup.bReplicative intermediates
(Intracellular DNA) .sup.C.mu.M
EXAMPLE 5
[0073] The Comparative inhibitory effect of selected triphospahtes
on woodchuck hepatitis virus DNA polymerase is set out in Table
5.
TABLE-US-00003 TABLE 2 Comparative inhibitory activities of
L-nucleoside triphosphates on woochuck hepatitis virus DNA
polymerase and human DNA polymerase .alpha. and .beta.. WHB DNA Pol
DNA Pol .alpha. DNA Pol .beta. Inhibitor IC.sub.50 (.mu.M) Ki
(.mu.M) Ki (.mu.M) .beta.-L-AZTPP 0.2 >100 >100
.beta.-L-ddATP 2.1 >100 >100 3-TC-TP 1.0 >100 >100
.beta.-L-5FDDCTP 2.0 >100 >100
III. Preparation of Pharmaceutical Compositions
[0074] The compounds disclosed herein and their pharmaceutically
acceptable salts, prodrugs, and derivatives, are useful in the
prevention and treatment of HBV infections and other related
conditions such as anti-HBV antibody positive and HBV-positive
conditions, chronic liver inflammation caused by HBV, cirrhosis,
acute hepatitis, fulminant hepatitis, chronic persistent hepatitis,
and fatigue. These compounds or formulations can also be used
prophylactically to prevent or retard the progression of clinical
illness in individuals who are anti-HBV antibody or HBV-antigen
positive or who have been exposed to HBV.
[0075] Humans suffering from any of these conditions can be treated
by administering to the patient an effective HBV-treatment amount
of one or a mixture of the active compounds described herein or a
pharmaceutically acceptable derivative or salt thereof, optionally
in a pharmaceutically acceptable carrier or diluent. The active
materials can be administered by any appropriate route, for
example, orally, parenterally, intravenously, intradermally,
subcutaneously, or topically, in liquid or solid form.
[0076] The active compound is included in the pharmaceutically
acceptable carrier or diluent in an amount sufficient to deliver to
a patient a therapeutically effective amount without causing
serious toxic effects in the patient treated.
[0077] A preferred dose of the active compound for all of the
above-mentioned conditions will be in the range from about 1 to 60
mg/kg, preferably 1 to 20 mg/kg, of body weight per day, more
generally 0.1 to about 100 mg per kilogram body weight of the
recipient per day. The effective dosage range of the
pharmaceutically acceptable derivatives can be calculated based on
the weight of the parent nucleoside to be delivered. If the
derivative exhibits activity in itself, the effective dosage can be
estimated as above using the weight of the derivative, or by other
means known to those skilled in the art.
[0078] In one embodiment, the active compound is administered as
described in the product insert or Physician's Desk Reference for
3'-azido-3'-deoxythymidine (AZT), 2',3'-dideoxyinosine (DDI),
2',3'-dideoxycytidine (DDC), or
2',3'-dideoxy-2',3'-didehydrothymidine (D4T) for HIV
indication.
[0079] The compound is conveniently administered in unit any
suitable dosage form, including but not limited to one containing 7
to 3000 mg, preferably 70 to 1400 mg of active ingredient per unit
dosage form. A oral dosage of 50-1000 mg is usually convenient.
[0080] Ideally the active ingredient should be administered to
achieve peak plasma concentrations of the active compound of from
about 0.2 to 70 .mu.M, preferably about 1.0 to 10 .mu.M. This may
be achieved, for example, by the intravenous injection of a 0.1 to
5% solution of the active ingredient, optionally in saline, or
administered as a bolus of the active ingredient.
[0081] The active compound can be provided in the form of
pharmaceutically acceptable salts. As used herein, the term
pharmaceutically acceptable salts or complexes refers to salts or
complexes of the nucleosides that retain the desired biological
activity of the parent compound and exhibit minimal, if any,
undesired toxicological effects. Nonlimiting examples of such salts
are (a) acid addition salts formed with inorganic acids (for
example, hydrochloric acid, hydrobromic acid, sulfuric acid,
phosphoric acid, nitric acid, and the like), and salts formed with
organic acids such as acetic acid, oxalic acid, tartaric acid,
succinic acid, malic acid, ascorbic acid, benzoic acid, tannic
acid, pamoic acid, alginic acid, polyglutamic acid,
naphthalenesulfonic acids, naphthalenedisulfonic acids, and
polygalacturonic acid; (b) base addition salts formed with cations
such as sodium, potassium, zinc, calcium, bismuth, barium,
magnesium, aluminum, copper, cobalt, nickel, cadmium, sodium,
potassium, and the like, or with an organic cation formed from
N,N-dibenzylethylene-diamine, ammonium, or ethylenediamine; or (c)
combinations of (a) and (b); e.g., a zinc tannate salt or the
like.
[0082] Modifications of the active compound, specifically at the
N.sup.6 or N.sup.4 and 5'-O positions, can affect the
bioavailability and rate of metabolism of the active species, thus
providing control over the delivery of the active species.
[0083] The concentration of active compound in the drug composition
will depend on absorption, inactivation, and excretion rates of the
drug as well as other factors known to those of skill in the art.
It is to be noted that dosage values will also vary with the
severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that the
concentration ranges set forth herein are exemplary only and are
not intended to limit the scope or practice of the claimed
composition. The active ingredient may be administered at once, or
may be divided into a number of smaller doses to be administered at
varying intervals of time:
[0084] A preferred mode of administration of the active compound is
oral. Oral compositions will generally include an inert diluent or
an edible carrier. They may be enclosed in gelatin capsules or
compressed into tablets. For the purpose of oral therapeutic
administration, the active compound can be incorporated with
excipients and used in the form of tablets, troches, or capsules.
Pharmaceutically compatible binding agents, and/or adjuvant
materials can be included as part of the composition.
[0085] The tablets, pills, capsules, troches and the like can
contain any of the following ingredients, or compounds of a similar
nature: a binder such as microcrystalline cellulose, gum tragacanth
or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring. When the dosage unit form
is a capsule, it can contain, in addition to material of the above
type, a liquid carrier such as a fatty oil. In addition, dosage
unit forms can contain various other materials which modify the
physical form of the dosage unit, for example, coatings of sugar,
shellac, or other enteric agents.
[0086] The active compound or pharmaceutically acceptable salt or
derivative thereof can be administered as a component of an elixir,
suspension, syrup, wafer, chewing gum or the like. A syrup may
contain, in addition to the active compounds, sucrose as a
sweetening agent and certain preservatives, dyes and colorings and
flavors.
[0087] The active compound, or pharmaceutically acceptable
derivative or salt thereof can also be mixed with other active
materials that do not impair the desired action, or with materials
that supplement the desired action, such as antibiotics,
antifungals, antiinflammatories, or other antivirals, including
anti-HBV, anti-cytomegalovirus, or anti-HIV agents.
[0088] Solutions or suspensions used for parenteral, intradermal,
subcutaneous, or topical application can include the following
components: a sterile diluent such as water for injection, saline
solution, fixed oils, polyethylene glycols, glycerine, propylene
glycol or other synthetic solvents; antibacterial agents such as
benzyl alcohol or methyl parabens; antioxidants such as ascorbic
acid or sodium bisulfite; chelating agents such as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates
or phosphates and agents for the adjustment of tonicity such as
sodium chloride or dextrose. The parental preparation can be
enclosed in ampoules, disposable syringes or multiple dose vials
made of glass or plastic.
[0089] If administered intravenously, preferred carriers are
physiological saline or phosphate buffered saline (PBS). In a
preferred embodiment, the active compounds are prepared with
carriers that will protect the compound against rapid elimination
from the body, such as a controlled release formulation, including
implants and microencapsulated delivery systems. Biodegradable,
biocompatible polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Methods for preparation of such formulations will
be apparent to those skilled in the art. The materials can also be
obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc.
[0090] Liposomal suspensions (including liposomes targeted to
infected cells with monoclonal antibodies to viral antigens) are
also preferred as pharmaceutically acceptable carriers. These may
be prepared according to methods known to those skilled in the art,
for example, as described in U.S. Pat. No. 4,522,811 (which is
incorporated herein by reference in its entirety). For example,
liposome formulations may be prepared by dissolving appropriate
lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl
phosphatidyl choline, arachadoyl phosphatidyl choline, and
cholesterol) in an inorganic solvent that is then evaporated,
leaving behind a thin film of dried lipid on the surface of the
container. An aqueous solution of the active compound or its
monophosphate, diphosphate, and/or triphosphate derivatives are
then introduced into the container. The container is then swirled
by hand to free lipid material from the sides of the container and
to disperse lipid aggregates, thereby forming the liposomal
suspension.
[0091] This invention has been described with reference to its
preferred embodiments. Variations and modifications of the
invention, will be obvious to those skilled in the art from the
foregoing detailed description of the invention. It is intended
that all of these variations and modifications be included within
the scope of the appended claims.
* * * * *