U.S. patent application number 10/910197 was filed with the patent office on 2005-12-01 for use of n-substituted-1,5-dideoxy-1,5-imino-d-glucitol compounds for treating hepatitis virus infections.
Invention is credited to Block, Timothy M., Bryant, Martin L., Dwek, Raymond A., Jacob, Gary S., Mueller, Richard A., Partis, Richard A..
Application Number | 20050267153 10/910197 |
Document ID | / |
Family ID | 26697090 |
Filed Date | 2005-12-01 |
United States Patent
Application |
20050267153 |
Kind Code |
A1 |
Mueller, Richard A. ; et
al. |
December 1, 2005 |
Use of N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds for
treating hepatitis virus infections
Abstract
Provided are methods and compositions for treating hepatitis
virus infections in mammals, especially humans. The methods
comprise (1) administering
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds alone or
in combination with nucleoside antiviral agents, nucleotide
antiviral agents, mixtures thereof, or
immunomodulating/immunostimulating agents, or (2) administering
N-substituted-1,5-dideoxy-1,5-imino-D-glucit- ol compounds alone or
in combination with nucleoside antiviral agents, nucleotide
antiviral agents, or mixtures thereof, and
immunomodulating/immuno-stimulating agents.
Inventors: |
Mueller, Richard A.;
(Glencoe, IL) ; Bryant, Martin L.; (Carlisle,
MA) ; Partis, Richard A.; (Evanston, IL) ;
Jacob, Gary S.; (Creve Coeur, MO) ; Block, Timothy
M.; (Jenkintown, PA) ; Dwek, Raymond A.;
(Oxford, GB) |
Correspondence
Address: |
Scott J. Meyer
Pharmacia Corporation
P. O. Box 1027
St. Louis
MO
63006
US
|
Family ID: |
26697090 |
Appl. No.: |
10/910197 |
Filed: |
August 3, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10910197 |
Aug 3, 2004 |
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09249220 |
Feb 12, 1999 |
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6809083 |
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10910197 |
Aug 3, 2004 |
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09023401 |
Feb 12, 1998 |
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60074508 |
Feb 12, 1998 |
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Current U.S.
Class: |
514/328 ;
514/317 |
Current CPC
Class: |
A61K 31/445 20130101;
A61K 45/06 20130101; A61P 31/20 20180101; A61P 31/14 20180101; C07D
211/46 20130101; A61P 1/16 20180101 |
Class at
Publication: |
514/328 ;
514/317 |
International
Class: |
A61K 031/445 |
Claims
1-84. (canceled)
85. A pharmaceutical composition, consisting essentially of an
antiviral effective amount of at least one
N-substituted-1,5-dideoxy-1,5-imino-D-gl- ucitol compound of
Formula I or a pharmaceutically acceptable salt thereof: 4wherein R
is selected from the group consisting of straight chain alkyl
having a chain length of C.sub.7 to C.sub.20, branched chain alkyl
having a chain length of C.sub.3 to C.sub.20 in the main chain,
alkoxyalkyl, arylalkyl, and cycloalkylalkyl, and wherein W, X, Y
and Z are each independently selected from the group consisting of
hydrogen, alkanoyl, aroyl, and trifluoroalkanoyl; and a
pharmaceutically acceptable carrier, diluent, or excipient.
86. The pharmaceutical composition of claim 85, wherein R is
straight chain alkyl having a chain length of C.sub.7 to C.sub.20,
and W, X, Y and Z are each hydrogen.
87. The pharmaceutical composition of claim 86, wherein R is
nonyl.
88. The pharmaceutical composition of claim 85, wherein R is
straight chain alkyl having a chain length of C.sub.7 to C.sub.20,
and W, X, Y, and Z are each alkanoyl.
89. The pharmaceutical composition of claim 88, wherein R is
nonyl.
90. The pharmaceutical composition of claim 89, wherein said
alkanoyl is butanoyl.
91-92. (canceled)
93. A pharmaceutical composition, comprising an antiviral effective
amount of at least one
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of Formula
I or a pharmaceutically acceptable salt thereof: 5substantially
free of a nucleoside, nucleotide, immunomodulator, or
immunostimulant, wherein R is selected from the group consisting of
straight chain alkyl having a chain length of C.sub.7 to C.sub.20,
branched chain alkyl having a chain length of C.sub.3 to C.sub.20
in the main chain, alkoxyalkyl, arylalkyl, and cycloalkylalkyl, and
wherein W, X, Y, and Z are each independently selected from the
group consisting of hydrogen, alkanoyl, aroyl, and
trifluoroalkanoyl; and a pharmaceutically acceptable carrier,
diluent, or excipient.
94. The pharmaceutical composition of claim 93, wherein R is
straight chain alkyl having a chain length of C.sub.7 to C.sub.20,
and W, X, Y, and Z are each hydrogen.
95. The pharmaceutical composition of claim 93, wherein R is
straight chain alkyl having a chain length of C.sub.7 to C.sub.20,
and W, X, Y, and Z are each alkanoyl.
96. The pharmaceutical composition of claim 93, wherein R is
straight chain alkyl having a chain length of C.sub.7 to C.sub.20,
and W, X, Y, and Z are each alkanoyl.
97. The pharmaceutical composition of claim 96, wherein R is
nonyl.
98. The pharmaceutical composition of claim 97, wherein said
alkanoyl is butanoyl.
99-136. (canceled)
137. A salt, comprising an
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of Formula
I: 6wherein R is selected from the group consisting of straight
chain alkyl having a chain length of C.sub.7 to C.sub.20, branched
chain alkyl having a chain length of C.sub.3 to C.sub.20 in the
main chain, alkoxyalkyl, arylalkyl, and cycloalkylalkyl, and
wherein W, X, Y, and Z are each independently selected from the
group consisting of hydrogen, alkanoyl, aroyl, and
trifluoroalkanoyl; and a compound selected from the group
consisting of a nucleoside having an acidic moiety and a
nucleotide.
138. The salt of claim 137, wherein R is straight chain alkyl
having a chain length of C.sub.7 to C.sub.20, and W, X, Y, and Z
are each hydrogen.
139. The salt of claim 138, wherein R is nonyl.
140. The salt of claim 137, wherein R is straight chain alkyl
having a chain length of C.sub.7 to C.sub.20, and W, X, Y, and Z
are each alkanoyl.
141. The salt of claim 140, wherein R is nonyl.
142. The salt of claim 141, wherein said alkanoyl is butanoyl.
143-147. (canceled)
148. A method, comprising reacting
N-(n-nonyl)-1,5-dideoxy-1,5-imino-D-glu- citol and
(-)-2'-deoxy-3'-thiocytidine-5'-triphosphate under salt-forming
conditions.
149. A salt formed by the method of claim 148.
Description
RELATED APPLICATION DATA
[0001] This application claims the benefit of priority of U.S.
provisional application Ser. No. 60/074,508, filed Feb. 12, 1998.
This application is also a continuation in part of U.S. application
Ser. No. 09/023,401, filed Feb. 12, 1998. The contents of each of
these applications is incorporated by reference herein in their
entirety.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to methods and compositions
for treating hepatitis virus infections, especially hepatitis B
virus infections, in mammals, especially humans. The methods
comprise (1) administering
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds alone or
in combination with nucleoside antiviral agents, nucleotide
antiviral agents, mixtures thereof, or
immunomodulating/immunostimulating agents, or (2) administering
N-substituted-1,5-dideoxy-1,5-imino-D-glucit- ol compounds alone or
in combination with nucleoside antiviral agents, nucleotide
antiviral agents, or mixtures thereof, and
immunomodulating/immunostimulating agents. Such combinations of
anti-hepatitis viral agents show unexpected efficacy in inhibiting
replication and secretion of hepatitis viruses in cells of mammals
infected with these viruses.
[0004] 2. Description of Related Art
[0005] Hepatitis Viruses
[0006] Hepatitis B Virus (HBV, HepB) is a causative agent of acute
and chronic liver disease including liver fibrosis, cirrhosis,
inflammatory liver disease, and hepatic cancer that can lead to
death in some patients (Joklik, Wolfgang K., Virology, Third
Edition, Appleton & Lange, Norwalk, Conn., 1988 (ISBN
0-8385-9462-X)). Although effective vaccines are available, there
are still more than 300 million people worldwide, i.e., 5% of the
world's population, chronically infected with the virus (Locarnini,
S. A., et. al., Antiviral Chemistry & Chemotherapy (1996)
7(2):53-64). Such vaccines have no therapeutic value for those
already infected with the virus. In Europe and North America,
between 0.1% to 1% of the population is infected. Estimates are
that 15% to 20% of individuals who acquire the infection develop
cirrhosis or another chronic disability from HBV infection. Once
liver cirrhosis is established, morbidity and mortality are
substantial, with about a 5-year patient survival period (Blume,
H., E., et. al., Advanced Drug Delivery Reviews (1995) 17:321-331).
It is therefore necessary and of high priority to find improved and
effective anti-HBV anti-hepatitis therapies (Locarnini, S. A., et.
al., Antiviral Chemistry & Chemotherapy (1996) 7(2):
53-64).
[0007] Other hepatitis viruses significant as agents of human
disease include Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis
Delta, Hepatitis E, Hepatitis F, and Hepatitis G (Coates, J. A. V.,
et. al., Exp. Opin. Ther. Patents (1995) 5(8):747-756). In
addition, there are animal hepatitis viruses that are
species-specific. These include, for example, those infecting
ducks, woodchucks, and mice.
1,5-dideoxy-1,5-imino-D-glucitol Compounds
[0008] 1,5-dideoxy-1,5-imino-D-glucitol (also known as
1-deoxynojirimycin, DNJ) and its N-alkyl derivatives (together,
"imino sugars") are known inhibitors of the N-linked
oligosaccharide processing enzymes alpha glucosidase I and II
(Saunier et al., J. Biol. Chem. (1982) 257:14155-14161 (1982);
Elbein, Ann. Rev. Biochem. (1987) 56:497-534). As glucose analogs,
they also have potential to inhibit glucose transport,
glucosyl-transferases, and/or glycolipid synthesis (Newbrun et al.,
Arch. Oral Biol. (1983) 28: 516-536; Wang et al., Tetrahedron Lett.
(1993) 34:403-406). Their inhibitory activity against glucosidases
has led to the development of these compounds as anti-hyperglycemic
agents and antiviral agents. See, for example, PCT International
Publication WO 87/03903 and U.S. Pat. Nos. 4,065,562; 4,182,767;
4,533,668; 4,639,436; 4,849,430; 4,957,926; 5,011,829; and
5,030,638.
[0009] Glucosidase inhibitors such as
N-alkyl-1,5-dideoxy-1,5-imino-D-gluc- itol compounds wherein the
alkyl group contains between three and six carbon atoms have been
shown to be effective in the treatment of Hepatitis B infection
(PCT International Publication WO 95/19172). For example,
N-(n-butyl)-deoxynojirimycin (N-butyl-DNJ;
N-(n-butyl)-1-5-dideoxy-1,5-imino-D-glucitol) is effective for this
purpose (Block, T. M., Proc. Natl. Acad. Sci. USA (1994)
91:2235-2239; Ganem, B. Chemtracts: Organic Chemistry (1994) 7(2),
106-107). N-butyl-DNJ has also been tested as an anti-HIV-1 agent
in HIV infected patients, and is known to be well tolerated.
Another alpha glucosidase inhibitor, deoxynojirimycin (DNJ), has
been suggested as an antiviral agent for use in combination with
N-(phosphonoacetyl)-L-aspartic acid (PALA) (WO 93/18763). However,
combinations of N-substituted-imino-D-gluc- itol derivatives and
other antiviral agents for the treatment of hepatitis virus
infections have not been previously disclosed or suggested. From
results obtained in a woodchuck animal model of hepatitis virus
infection, Block et al. ((1998) Nature Medicine 4(5):610-614)
suggested that glucosidase inhibitors such as N-nonyl DNJ, which
interfere with specific steps in the N-linked glycosylation pathway
of hepatitis virus glycoproteins, may be useful in targeting
glycosylation processing as a therapeutic intervention for
hepatitis B virus.
[0010] Nucleoside and Nucleotide Antiviral Agents
[0011] Reverse transcriptase inhibitors, including the class of
nucleoside and nucleotide analogs, were first developed as drugs
for the treatment of retroviruses such as human immunodeficiency
virus (HIV), the causative agent of AIDS. Increasingly, these
compounds have found use against other viruses, including both RNA
and DNA viruses, via viral screening and chemical modification
strategies. Nucleoside and nucleotide analogs exert their antiviral
activities by inhibiting the corresponding DNA and RNA polymerases
responsible for synthesis of viral DNA and RNA, respectively.
Because viruses contain different forms of polymerases, the same
nucleoside/nucleotide compound can have a dramatically different
effect against different viruses. For example, lamivudine (3TC.TM.)
appears to be useful against HBV infection, whereas zidovudine
(AZT.TM.) appears to have little use against the same virus (Gish,
R. G., et al., Exp. Opin. Invest. Drugs (1995) 4(2):95-115).
[0012] Toxicity has been significant with some nucleoside analog
antivirals. For example, clinical tests on the use of the
nucleoside analog fialuridine (FIAU) for treatment of chronic
hepatitis B were suspended recently due to drug-related liver
failure leading to death in some patients. Consequently, there is
still a need for safer drug regimens for the treatment of hepatitis
B infections and hepatitis (Mutchnick, M. G., et. al., Antiviral
Research (1994) 24:245-257).
[0013] Immunomodulators and Immunostimulants
[0014] Immunomodulators/immunostimulators such as interferon alfa
and other cytokines have been used for the treatment of HBV
infection with promising results. Unfortunately, the response rates
are lower than desired. Interferon treatment is currently approved
by the FDA for the treatment of Hepatitis B. Other immune
system-affecting drug candidates are presently being investigated.
These include thymic peptides for use in the treatment of chronic
hepatitis B (CHB), isoprinosine, steroids, Shiff base-forming
salicylaldehyde derivatives such as Tucaresol, levamisol, and the
like (Gish, R. G., et. al., Exp. Opin. Invest. Drugs (1995)
4(2):95-115; Coates, J. A. V., et. al., Exp. Opin. Ther. Patents
(1995) 5(8):747-765).
SUMMARY OF THE INVENTION
[0015] As noted above, the use of the
N-substituted-imino-D-glucitol compounds and derivatives thereof
disclosed herein alone, or in combination with other anti-hepatitis
virus compounds has, to the present inventor's knowledge, neither
been suggested nor disclosed. The use of two or more anti-viral
agents to provide improved therapy for the treatment of hepatitis B
virus infections is desirable due to the morbidity and mortality of
the disease. Combination therapy is also desirable since it should
reduce toxicity in patients as it enables the physician to
administer lower doses of one or more of the drugs being given to a
patient. Combination therapy can also help to prevent the
development of drug resistance in patients (Wiltink, E. H. H.,
Pharmaceutish Weekblads Scientific Edition (1992) 14(4A):268-274).
The result of an improved efficacy configuration combined with a
relative lack of toxicity and development of resistance would
provide a much improved drug treatment profile.
[0016] The present inventors have surprisingly discovered that the
use of the N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds
disclosed herein are effective in treating hepatitis virus
infections. Furthermore, the use of these compounds in combination
with nucleoside or nucleotide antiviral compounds, or combinations
thereof, and/or immunomodulators/immunostimulants, results in
unexpectedly greater anti-hepatitis virus effectiveness of the
compounds compared to the combined antiviral activities expected of
the individual compounds alone. Whether this is due to different
mechanisms of action of the different classes of drugs employed or
some other biological phenomenon is presently unclear.
[0017] Accordingly, in a first aspect, the present invention
provides a method for treating a hepatitis virus infection in a
mammal, comprising administering to said mammal an anti-hepatitis
virus effective amount of at least one
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of Formula
I or a pharmaceutically acceptable salt thereof: 1
[0018] wherein R is selected from the group consisting of straight
chain alkyl having a chain length of C.sub.7 to C.sub.20, branched
chain alkyl having a chain length of C.sub.3 to C.sub.20 in the
main chain, alkoxyalkyl, arylalkyl, and cycloalkylalkyl, and
wherein W, X, Y and Z are each independently selected from the
group consisting of hydrogen, alkanoyl, aroyl, and
trifluoroalkanoyl.
[0019] In a second aspect, the present invention provides a method
for treating a hepatitis virus infection in a mammal, comprising
administering to said mammal an antiviral composition comprising an
antiviral effective amount of at least one
N-substituted-1,5-dideoxy-1,5-- imino-D-glucitol compound of
Formula I or a pharmaceutically acceptable salt thereof, as
above.
[0020] In a third aspect, the present invention provides a method
for treating a hepatitis virus infection in a mammal, comprising
administering to said mammal an antiviral composition consisting
essentially of an antiviral effective amount of at least one
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of Formula
I or a pharmaceutically acceptable salt thereof, as above.
[0021] In a fourth aspect, the present invention provides a method
for treating a hepatitis virus infection in a mammal, consisting
essentially of administering to said mammal an antiviral effective
amount of at least one
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of Formula
I or a pharmaceutically acceptable salt thereof, as above.
[0022] In a fifth aspect, the present invention provides a method
for treating a hepatitis virus infection in a mammal, consisting
essentially of administering to said mammal an antiviral effective
amount of an antiviral compound consisting essentially of at least
one N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of
Formula I or a pharmaceutically acceptable salt thereof, as
above.
[0023] In a sixth aspect, the present invention provides a method
for treating a hepatitis virus infection in a mammal, consisting
essentially of administering to said mammal a first amount of at
least one N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound
of Formula I or a pharmaceutically acceptable salt thereof, as
above, and a second amount of an antiviral compound selected from
the group consisting of a nucleoside antiviral compound, a
nucleotide antiviral compound, an immunomodulator, an
immunostimulant, and mixtures thereof, wherein said first and
second amounts of said compounds together comprise an
anti-hepatitis virus effective amount of said compounds.
[0024] In another aspect, the present invention provides a method
for treating a hepatitis B virus infection in a mammal, comprising
administering to said mammal from about 0.1 mg/kg/day to about 100
mg/kg/day of at least one
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of Formula
I or a pharmaceutically acceptable salt thereof, as above, and from
about 0.1 mg/person/day to about 500 mg/person/day of a compound
selected from the group consisting of a nucleoside antiviral
compound, a nucleotide antiviral compound, and a mixture
thereof.
[0025] In another aspect, the present invention provides a method
for treating a hepatitis B virus infection in a human patient,
comprising administering to said human patient from about 0.1
mg/kg/day to about 100 mg/kg/day of an
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound selected
from the group consisting N-(n-nonyl-)-1,5-dideoxy-1,5-imino-D-g-
lucitol or a pharmaceutically acceptable salt thereof,
N-(n-nonyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate or a
pharmaceutically acceptable salt thereof, and mixtures thereof, and
from about 0.1 mg/person/day to about 500 mg/person/day of
(-)-2'-deoxy-3'-thiocytidine-5'-triphosphate.
[0026] In another aspect, the present invention provides a method
for treating a hepatitis virus infection in a mammal, comprising
administering to said mammal an antiviral effective amount of at
least one N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound
of Formula I or a pharmaceutically acceptable salt thereof, as
above, substantially exclusive of the administration of an
antiviral composition comprising a nucleoside, a nucleotide, an
immunomodulator, or an immunostimulant.
[0027] In another aspect, the present invention provides a method
for treating a hepatitis virus infection in a mammal, comprising
administering to said mammal an antiviral effective amount of at
least one N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound
of Formula I or a pharmaceutically acceptable salt thereof, as
above, substantially exclusive of the administration of antiviral
compounds other than compounds of Formula I.
[0028] In another aspect, the present invention provides a
pharmaceutical composition, comprising an antiviral effective
amount of at least one
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of Formula
I or a pharmaceutically acceptable salt thereof, as above, and a
pharmaceutically acceptable carrier, excipient, or diluent.
[0029] In another aspect, the present invention provides a
pharmaceutical composition, consisting essentially of an antiviral
effective amount of at least one
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of Formula
I or a pharmaceutically acceptable salt thereof, as above, and a
pharmaceutically acceptable carrier, diluent, or excipient.
[0030] In another aspect, the present invention provides a
pharmaceutical composition, comprising an antiviral effective
amount of at least one
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of Formula
I or a pharmaceutically acceptable salt thereof, as above,
substantially free of a nucleoside, nucleotide, immunomodulator, or
immunostimulant, and a pharmaceutically acceptable, carrier,
diluent, or excipient.
[0031] In another aspect, the present invention provides a
pharmaceutical composition, comprising an antiviral effective
amount of at least one
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of Formula
I or a pharmaceutically acceptable salt thereof, as above,
substantially free of antiviral compounds other than compounds of
Formula I, and a pharmaceutically acceptable, carrier, diluent, or
excipient.
[0032] In yet another aspect, the present invention provides a
composition, comprising at least one
N-substituted-1,5-dideoxy-1,5-imino-- D-glucitol compound of
Formula I or a pharmaceutically acceptable salt thereof, as above,
and an antiviral compound selected from the group consisting of a
nucleoside antiviral compound, a nucleotide antiviral compound, an
immunomodulator, an immunostimulant, and mixtures thereof.
[0033] In another aspect, the present invention provides a
pharmaceutical composition, comprising a first amount of at least
one N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of
Formula I or a pharmaceutically acceptable salt thereof, as above;
a second amount of an antiviral compound selected from the group
consisting of a nucleoside antiviral compound, a nucleotide
antiviral compound, an immunomodulator, an immunostimulant, and
mixtures thereof; and a pharmaceutically acceptable carrier,
diluent, or excipient, wherein said first and second amounts of
said compounds together comprise an antiviral effective amount of
said compounds.
[0034] In a further aspect, the present invention provides a
pharmaceutical composition for treating a hepatitis B virus
infection in a mammal, comprising from about 0.1 mg to about 100 mg
of at least one N-substituted-1,5-dideoxy-1,5-imino-D-glucitol
compound of Formula I or a pharmaceutically acceptable salt
thereof, as above; from about 0.1 mg to about 500 mg of a compound
selected from the group consisting of a nucleoside antiviral
compound, a nucleotide antiviral, and mixtures thereof; and a
pharmaceutically acceptable carrier, diluent, or excipient.
[0035] In another aspect, the present invention provides a
pharmaceutical composition for treating a hepatitis B virus
infection in a human patient, comprising from about 0.1 mg to about
100 mg of an N-substituted-1,5-dideoxy-1,5-imino-D-glucitol
compound selected from the group consisting of
N-(n-nonyl)-1,5-dideoxy-1,5-imino-D-glucitol or a pharmaceutically
acceptable salt thereof, N-(n-nonyl)-1,5-dideoxy-1,5-imi-
no-D-glucitol, tetrabutyrate or a pharmaceutically acceptable salt
thereof, and mixtures thereof; from about 0.1 mg to about 500 mg of
(-)-2'-deoxy-3'-thiocytidine-5'-triphosphate; and a
pharmaceutically acceptable carrier, diluent, or excipient.
[0036] In yet another aspect, the present invention provides a
salt, comprising an N-substituted-1,5-dideoxy-1,5-imino-D-glucitol
compound of Formula I, as above, and a compound selected from the
group consisting of a nucleoside having an acidic moiety and a
nucleotide.
[0037] In a further aspect, the present invention provides a
method, comprising reacting
N-(n-nonyl)-1,5-dideoxy-1,5-imino-D-glucitol and
(-)-2'-deoxy-3'-thiocytidine-5'-triphosphate under salt-forming
conditions, as well as a salt produced thereby.
[0038] Further scope of the applicability of the present invention
will become apparent from the detailed description and drawings
provided below. However, it should be understood that the following
detailed description and examples, while indicating preferred
embodiments of the invention, are given by way of illustration only
since various changes and modifications within the spirit and scope
of the invention will become apparent to those skilled in the art
from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] The above and other objects, features, and advantages of the
present invention will be better understood from the following
detailed description taken in conjunction with the accompanying
drawings, all of which are given by way of illustration only, and
are not limitative of the present invention, in which:
[0040] FIG. 1 shows the anti-hepatitis B virus activity of
(-)-2'-deoxy-3'-thiocytidine-5'-triphosphate (3TC) alone and in
combination with N-nonyl-DNJ in vitro.
[0041] FIG. 2 shows the plasma concentration of N-nonyl-DNJ versus
dose of N-nonyl-DNJ for each animal in Example 5, from samples
taken during dosing. Animals are indicated by unique letters, and a
small amount of random noise has been added to the dose value so
that overlapping values can be distinguished.
[0042] FIG. 3 shows the slope of Log(IPDNA+10) to week versus dose.
A distinct letter is used for each animal. The fitted line is from
a four parameter logistic model. The parameters of the fitted curve
and their approximate standard errors are shown on the plot.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The following detailed description is provided to aid those
skilled in the art in practicing the present invention. Even so,
this detailed description should not be construed to unduly limit
the present invention as modifications and variations in the
embodiments discussed herein can be made by those of ordinary skill
in the art without departing from the spirit or scope of the
present inventive discovery.
[0044] The contents of each of the patent documents and other
references cited herein, including the contents of the references
cited within these primary references, are herein incorporated by
reference in their entirety.
[0045] The present inventors have discovered that the use of
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds are
effective when used alone for treating hepatitis virus infections.
They have additionally discovered that combinations of
N-substituted-1,5-dideoxy-1,- 5-imino-D-glucitol compounds with
anti-hepatitis virus nucleosides or nucleotides, and/or
immunomodulators/immunostimulants, are also effective for this
purpose. There is some evidence that certain combinations may be
more effective in inhibiting hepatitis virus replication than would
have been expected via the combined use of the individual
compounds.
[0046] The present invention thus provides pharmaceutical
compositions and methods of treating hepatitis virus infections,
especially hepatitis B virus infections, in humans, other mammals,
and cells using N-substituted-1,5-dideoxy-1,5-imino-D-glucitol
compounds alone or in combination with either an antiviral
nucleoside, an antiviral nucleotide, mixtures thereof, and/or an
immunomodulating or immunostimulating agent. The
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds have basic
nitrogen atoms and may be used in the form of a pharmaceutically
acceptable salt. Nucleosides and nucleotides useful in the present
invention are substituted purine or pyrmidine heterocycles further
substituted with R.sup.1 in Formulas II-VI at the 9 position in the
case of purines or with R.sup.1 at the 1 position in the case of
pyrimidines. The immunomodulating and immunostimulating agents
useful in the present invention include those that stimulate immune
responses effective in controlling or eliminating viruses or other
infectious agents. Non-limiting examples of such immunomodulating
and immunostimulating agents include cytokines, peptide agonists,
steroids, and classic drugs such as levamisol. The drug
combinations of this invention may be provided to a cell or cells,
or to a human or other mammalian patient, either in separate
pharmaceutically acceptable formulations administered
simultaneously or sequentially, formulations containing more than
one therapeutic agent, or by an assortment of single agent and
multiple agent formulations. However administered, these drug
combinations form an anti-hepatitis virus effective amount of
components.
[0047] As used herein, the term "anti-hepatitis-virus effective
amount" refers to an amount of an
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound alone, or a
combined amount of (1) an N-substituted-1,5-dideoxy--
1,5-imino-D-glucitol compound with either an antiviral nucleoside,
an antiviral nucleotide, a mixture of an antiviral nucleoside and
an antiviral nucleotide, or an immunomodulating/-immunostimulating
agent (or mixtures thereof), or (2) a combined amount of an
N-substituted-1,5-dideo- xy-1,5-imino-D-glucitol compound with an
antiviral nucleoside, an antiviral nucleotide, or a mixture
thereof, and an immunomodulating/-immunostimulating agent (or
mixtures thereof) effective in treating hepatitis virus infection.
The antiviral effectiveness of the aforementioned combinations may
involve a variety of different phenomena associated with viral
replication and assembly. These may include, for example, blocking
hepatitis viral DNA synthesis; blocking viral transcription;
blocking virion assembly; blocking virion release or secretion from
infected cells; blocking or altering viral protein function,
including the function of viral envelope protein(s); and/or the
production of immature or otherwise non-functional virions. The
overall effect is an inhibition of viral replication and infection
of additional cells, and therefore inhibition of the progress of
infection in the patient.
N-substituted-1,5-dideoxy-1,5-imino-D-glucose compounds
[0048] N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds
useful in the present invention are represented by structure I
below: 2
[0049] wherein R is selected from straight chain alkyl having a
chain length of C.sub.7 to C.sub.20, more preferably C.sub.8 to
C.sub.20, more preferably C.sub.8 to C.sub.16, more preferably
C.sub.8 to C.sub.12, even more preferably C.sub.8 to C.sub.10, and
most preferably C.sub.9; branched chain alkyl having a chain length
of C.sub.3 to C.sub.20 in the main chain, preferably C.sub.3 to
C.sub.16, more preferably C.sub.3 to C.sub.14, more preferably
C.sub.4 to C.sub.12, more preferably C.sub.6 to C.sub.12, and even
more preferably C.sub.8 to C.sub.10; alkoxyalkyl; arylalkyl; and
cycloalkylalkyl. W, X, Y and Z are independently selected from
hydrogen, alkanoyl, aroyl, and trifluoroalkanoyl.
[0050] The phrase "in the main chain" refers to the longest
contiguous or adjacent chain of carbon atoms starting at the point
of attachment of a branched chain alkyl group to the nitrogen atom
in the compounds of Formula I.
[0051] The terms "alkoxy" and "alkyloxy" embrace linear or branched
oxygen-containing radicals each having alkyl portions of one to
about ten carbon atoms. Alkoxyalkyl groups ("alkylether" or "oxa"
derivatives) useful in the present invention can be C.sub.3 to
C.sub.20, preferably C.sub.4 to C.sub.18, more preferably C.sub.5
to C.sub.16, more preferably C.sub.6 to C.sub.12, and even more
preferably C.sub.8 to C.sub.12, wherein one to five non-terminal
carbon atoms, preferably one to three non-terminal carbon atoms,
more preferably one to two non-terminal carbon atoms, most
preferably one non-terminal carbon atom, can be replaced with
oxygen.
[0052] The term "aryl", alone or in combination with another
radical, means a carbocyclic aromatic system containing one, two or
three rings wherein such rings may be attached together in a
pendent manner or may be fused. The term "aryl" embraces aromatic
radicals such as phenyl, naphthyl, tetrahydronaphthyl, indyl, and
biphenyl.
[0053] The term "arylalkyl" embraces aryl-substituted alkyl
radicals such as benzyl, diphenylmethyl, triphenylmethyl,
phenylethyl, and diphenylethyl.
[0054] The term "cycloalkyl" embraces saturated carbocyclic
radicals having three to about twelve carbon atoms. More preferred
cycloalkyl radicals are "lower cycloalkyl" radicals having three to
about eight carbon atoms. Examples of such radicals include
cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl.
"Cycloalkylalkyl" means an alkyl group susbstituted with a
cycloalkyl group.
[0055] The term "acyl" denotes a radical provided by the residue
after removal of hydroxyl from an organic acid. Examples of such
acyl radicals include alkanoyl and aroyl radicals. "Alkanoyl" means
branched or straight chain alkanecarbonyl having a chain length of
C.sub.1 to C.sub.20, preferably C.sub.1 to C.sub.10, more
preferably C.sub.1 to C.sub.5; "aroyl" means arylcarbonyl; and
"trifluoroalkanoyl" means alkanoyl containing three fluoro
substituents. Examples of such alkanoyl radicals include formyl,
acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl,
pivaloyl, hexanoyl, and radicals formed from succinic, glycolic,
gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic,
maleic, fumaric, pyruvic, mandelic, pantothenic,
.beta.-hydroxybutyric, galactaric and galacturonic acids.
[0056] When used in combination with another radical when referring
to the imino sugars useful in the present invention, the term
"alkyl" means a straight or branched chain hydrocarbon radical
containing from 1 to about 20 carbon atoms, preferably 1 to about
16 carbon atoms, more preferably from about 2 to about 12 carbon
atoms, more preferably from about 3 to about 10 carbon atoms.
[0057] The term "alkenyl" embraces radicals having "cis" and
"trans" orientations, or alternatively, "E" and "Z" orientations.
The term "alkynyl" embraces linear or branched radicals having at
least one carbon-carbon triple bond of two to about twenty carbon
atoms or, preferably, two to about twelve carbon atoms. More
preferred alkynyl radicals are "lower alkynyl" radicals having two
to about six carbon atoms. Examples of alkynyl radicals include
propargyl, 1-propynyl, 2-propynyl, 1-butyne, 2-butenyl and
1-pentynyl.
[0058] The term "cycloalkylalkyl" embraces alkyl radicals
substituted with a cycloalkyl radical. Preferred cycloalkylalkyl
radicals are C.sub.4 to C.sub.20; more preferred cycloalkylalkyl
radicals are C.sub.8 to C.sub.14. "lower cycloalkylalkyl" which
embrace lower alkyl radicals substituted with a lower cycloalkyl
radical as defined above. Examples of such radicals include
cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl and
cyclohexylmethyl.
[0059] The present invention comprises any tautomeric forms of
compounds of Formula I. The present invention also comprises
compounds of Formula I having one or more asymmetric carbons. It is
known to those skilled in the art that those imino sugars of the
present invention having asymmetric carbon atoms may exist in
diastereomeric, racemic, or optically active forms. All of these
forms are contemplated within the scope of this invention. More
specifically, the present invention includes enantiomers,
diastereomers, racemic mixtures, and other mixtures thereof.
[0060] Representative N-substituted-imino-D-glucitol compounds
useful in the present invention include, but are not limited
to:
[0061] N-(n-heptyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0062] N-(n-octyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0063] N-(n-nonyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0064] N-(n-decyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0065] N-(n-undecyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0066] N-(n-dodecyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0067] N-(n-tridecyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0068] N-(n-tetradecyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0069] N-(n-pentadecyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0070] N-(n-hexadecyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0071] N-(n-heptadecyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0072] N-(n-octadecyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0073] N-(n-nonadecyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0074] N-(n-eicosyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0075] N-(n-heptyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate
[0076] N-(n-octyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0077] N-(n-nonyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0078] N-(n-decyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0079] N-(n-undecyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0080] N-(n-dodecyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0081] N-(n-tridecyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0082] N-(n-tetradecyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0083] N-(n-pentadecyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0084] N-(n-hexadecyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0085] N-(n-heptadecyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0086] N-(n-octadecyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0087] N-(n-nonadecyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0088] N-(n-eicosyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0089] N-(2-ethylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0090] N-(4-ethylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0091] N-(5-methylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0092] N-(3-propylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0093]
N-(1-pentylpentylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0094] N-(1-butylbutyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0095] N-(7-methyloctyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0096] N-(8-methylnonyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0097] N-(9-methyldecyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0098] N-(10-methylundecyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0099] N-(6-cyclohexylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0100] N-(4-cyclohexylbutyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0101] N-(2-cyclohexylethyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0102] N-(1-cyclohexylmethyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0103] N-(1'-phenylmethyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0104] N-(3-phenylpropyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0105]
N-(3-(4-methyl)-phenylpropyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0106] N-(6-phenylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0107] N-(2-ethylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0108] N-(4-ethylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0109] N-(5-methylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0110] N-(3-propylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0111] N-(1-pentylpentylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0112] N-(1-butylbutyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0113] N-(7-methyloctyl-)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0114] N-(8-methylnonyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0115] N-(9-methyldecyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0116] N-(10-methylundecyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0117] N-(6-cyclohexylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0118] N-(4-cyclohexylbutyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0119] N-(2-cyclohexylethyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0120] N-(1-cyclohexylmethyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0121] N-(1-phenylmethyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0122] N-(3-phenylpropyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0123]
N-(3-(4-methyl)-phenylpropyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0124] N-(6-phenylhexyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0125] N-(7-oxa-n-decyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0126] N-(7-oxa-n-decyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetrabutyrate;
[0127] N-(7-oxa-n-decyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetraacetate;
[0128] N-(3-oxa-n-decyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0129] N-(9-oxa-n-decyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0130] N-(7-oxa-n-nonyl)-1,5-dideoxy-1,5-imino-D-glucitol;
[0131] N-(3-oxa-n-nonyl)-1,5-dideoxy-1,5-imino-D-glucitol,
tetraacetate;
[0132] N-(3-oxa-n-nonyl)-1,5-dideoxy-1,5-imino-D-glucitol; and
[0133]
N-(7,10,13-trioxa-n-tetradecyl)-1,5-dideoxy-1,5-imino-D-glucitol.
[0134] Preferred compounds are
N-(n-nonyl-)-1,5-dideoxy-1,5-imino-D-glucit- ol and
N-(n-nonyl-)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate.
[0135] The N-substituted-imino-D-glucitol compounds, including
pro-drugs, useful in the present invention, can be prepared by
methods well known in the art. For example, Example 13 of U.S. Pat.
No. 5,144,037 discloses a method for the preparation of N-nonyl
DNJ. At column 4, line 62, U.S. Pat. No. 4,806,650 discloses the
preparation of various alkoxy compounds, i.e., with alkyl chains
substituted with alkoxy groups. U.S. Pat. No. 4,260,622 discloses
the preparation of numerous compounds. Additional documents
relevant to the preparation of N-substituted-imino-D-glucitol
compounds and pro-drugs include U.S. Pat. Nos. 4,182,767,
4,260,622, 4,611,058, 4,639,436, and 5,003,072, 5,411,970, and
5,151,519; PCT International Publication WO 95/19172; and Tan et
al. (1991) Journal of Biological Chemistry 266(22):14504-14510; and
the references cited therein. Methods for introducing oxygen into
alkyl side chains are disclosed in Tan et al., (1994) Glycobiology
4(2):141-149, and van den Broek et al. (1994) Recl. Trav. Chim.
Pays-Bas 113:107-116 discloses the preparation of ether
oxygen-containing DNJ compounds.
[0136] Non-limiting illustrative preparation procedures are
presented below in Examples 1 and 2.
[0137] In treating hepatitis virus infections, one can use the
present N-substitututed-1,5-dideoxy-1,5-imino-D-glucitol compounds
alone or in combination in the form of salts derived from inorganic
or organic acids. These salts include but are not limited to the
following: acetate, adipate, alginate, citrate, aspartate,
benzoate, benzenesulfonate, bisulfate, butyrate, camphorate,
camphorsulfonate, digluconate, cyclopentanepropionate,
dodecylsulfate, ethanesulfonate, glucoheptanoate, glycerophosphate,
hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride,
hydrobromide, hydroiodide, 2-hydroxy-ethanesulfonate, lactate,
maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate,
oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate,
picrate, pivalate, propionate, succinate, tartrate, thiocyanate,
tosylate, mesylate, and undecanoate.
[0138] The basic nitrogen-containing groups can be quaternized with
agents such as lower alkyl halides, such as methyl, ethyl, propyl,
and butyl chloride, bromides, and iodides; dialkyl sulfates such as
dimethyl, diethyl, dibuytl, and diamyl sulfates; long chain halides
such as decyl, lauryl, myristyl, and stearyl chlorides, bromides,
and iodides; aralkyl halides such as benzyl and phenethyl bromides,
and others. Water- or oil-soluble or dispersible products are
thereby obtained as desired. The salts are formed by combining the
basic compounds with the desired acid.
[0139] Nucleosides and Nucleotides
[0140] Nucleosides and nucleotides useful in the present invention
are purine (II) base compounds or pyrimidine (III) base compounds,
or analogs such as compounds IV or V. 3
[0141] Position numbering for purines and pyrmidines is as shown in
structures II and III. R.sup.1 can be selected from hydroxyalkyl,
hydroxyalkenyl, carboxyalkyl, carboxyalkenyl, thiolalkyl,
alkylthioalkyl, alkoxyalkyl, alkoxyalkenyl, heterocycle,
heterocyclo-alkyl, hydroxyalkylalkoxyalkyl, alkoxyalkylalkoxyalkyl,
and cycloalkylalkyl. The purine compounds can be further
substituted at positions 1, 2, 3, 6, 7, or 8 of the purine
heterocycle, and the pyrimidine compounds can be substituted at
positions 2, 3, 4, 5, or 6 of the pyrimidine heterocycle. Such
substituents can be selected from hydroxy, alkoxy, halo, thiol,
amino, carboxyl, mono-substituted amino, di-substituted amino, and
alkyl.
[0142] The following definitions are applicable only to the
structures of Formulas II, III, IV, V and VI of this invention.
When used in combination with another radical when referring to the
purines and pyrimidines useful in the present invention, the term
"alkyl" means a straight or branched chain hydrocarbon radical
containing from 1 to 8 carbon atoms, preferably 1 to 4 carbon
atoms. When used in combination with another radical, the term
"alkenyl" means a straight or branched chain hydrocarbon radical
having I or more double bonds, containing from 2 to 8 carbon atoms,
preferably 1 to 4 carbon atoms. When used alone when referring to
purines and pyrimidines useful in the present invention, the term
"alkyl" means a straight or branched chain alkyl radical containing
from six to 14 carbon atoms, preferably seven to 12 carbon atoms,
and most preferably eight to 11 carbon atoms. The term "aryl" alone
or in combination with another radical means a phenyl, naphthyl, or
indenyl ring, optionally substituted with one or more substituents
selected from alkyl, alkoxy, halogen, hydroxy, or nitro. "Alkanoyl"
means branched or straight chain alkanecarbonyl having a chain
length of C.sub.1 to C.sub.20, preferably C.sub.2 to C.sub.14, more
preferably C.sub.4 to C.sub.10; "aroyl" means arylcarbonyl; and
"trifluoroalkanoyl" means alkyl containing three fluoro
substituents. "Halogen" means fluorine, chlorine, bromine, or
iodine. "Thiol" means sulfur substituted with hydrogen (--SH).
"Amino" means nitrogen with two hydrogen atoms; "monosubstituted
amino" and "disubstituted amino" mean amino groups further
independently substituted with one or more alkyl or arylalkyl
groups. "Hydroxyalkyl" means an alkyl group substituted with one or
more hydroxyl groups; "hydroxy-alkenyl" means an alkenyl group
substituted with one or more hydroxyl groups; "thioalkyl" means an
alkyl substituted with one or more thiol (SH) groups; "alkoxyalkyl"
means an alkyl substituted with one or more alkyl ether groups;
"alkoxyalkenyl" means an alkenyl group substituted with one or more
alkyl ether groups; "hydroxyalkylalkoxyalkyl- " means an
alkoxyalkyl group substituted with a hydroxyalkyl group;
"alkoxyalkyl-alkoxyalkyl" means an alkoxyalkyl group substituted
with an alkoxyalkyl group; "cycloalkylalkyl" means an alkyl group
substituted with a cycloalkyl group. The term "heterocycle," alone
or in combination, means a saturated or partially unsaturated 5 or
6-membered ring containing one or more oxygen, nitrogen, and/or
sulfur heteroatoms. Said heterocycle can further be substituted
with one to four substituents, which can be independently, hydroxy,
hydroxyalkyl, thiol, alkoxy, azido, nitro, a halogen atom, amino,
mono-substituted amino, or disubstituted amino. Heterocycloalkyl
means an alkyl group wherein one or more hydrogen atoms are
replaced by a substituted or unsubstituted heterocyclic ring.
[0143] Also included are the tautomers of the substituents on the
compounds of the invention. Non-limiting examples of tautomers are
ketone/enol tautomers, imino/amino tautomers, N-substituted
imino/N-substituted amino tautomers, thiol/thiacarbonyl tautomers,
and ring-chain tautomers such as the five and six membered ring
oxygen, nitrogen, sulfur, or oxygen- and sulfur-containing
heterocycles also containing substituents alpha to the heteroatoms.
Also specifically included in the present invention are enantiomers
and diastereomers, as well as racemates and isomeric mixtures of
the compounds discussed herein.
[0144] Representative nucleoside and nucleotide compounds useful in
the present invention include, but are not limited to:
[0145]
(+)-cis-5-fluoro-1-[2-(hydroxy-methyl)-[1,3-oxathiolan-5-yl]cytosin-
e;
[0146] (-)-2'-deoxy-3'-thiocytidine-5'-triphosphate (3TC);
[0147]
(-)-cis-5-fluoro-1-[2-(hydroxy-methyl)-[1,3-oxathiolan-5-yl]cytosin-
e (FTC);
[0148] (-)2',3', dideoxy-3'-thiacytidine [(-)-SddC];
[0149]
1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine
(FIAC);
[0150]
1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-iodocytosine
triphosphate (FIACTP);
[0151]
1-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-5-methyluracil
(FMAU);
[0152] 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide;
[0153] 2',3'-dideoxy-3'-fluoro-5-methyl-dexocytidine
(FddMeCyt);
[0154] 2',3'-dideoxy-3'-chloro-5-methyl-dexocytidine
(ClddMeCyt);
[0155] 2',3'-dideoxy-3'-amino-5-methyl-dexocytidine (AddMeCyt);
[0156] 2',3'-dideoxy-3'-fluoro-5-methyl-cytidine (FddMeCyt);
[0157] 2',3'-dideoxy-3'-chloro-5-methyl-cytidine (ClddMeCyt);
[0158] 2',3'-dideoxy-3'-amino-5-methyl-cytidine (AddMeCyt);
[0159] 2',3'-dideoxy-3'-fluorothymidine (FddThd);
[0160] 2',3'-dideoxy-beta-L-5-fluorocytidine (beta-L-FddC);
[0161] 2',3'-dideoxy-beta-L-5-thiacytidine;
[0162] 2',3'-dideoxy-beta-L-5-cytidine (beta-L-ddC);
[0163] 9-(1,3-dihydroxy-2-propoxymethyl)guanine;
[0164] 2'-deoxy-3'-thia-5-fluorocytosine;
[0165] 3'-amino-5-methyl-dexocytidine (AddMeCyt);
[0166]
2-amino-1,9-[(2-hydroxymethyl-1-(hydroxymethyl)ethoxy]methyl]-6H-pu-
rin-6-one (gancyclovir);
[0167] 2-[2-(2-amino-9H-purin-9y)ethyl]-1,3-propandil diacetate
(famciclovir);
[0168]
2-amino-1,9-dihydro-9-[(2-hydroxy-ethoxy)methyl]6H-purin-6-one
(acyclovir);
[0169] 9-(4-hydroxy-3-hydroxymethyl-but-1-yl)guanine
(penciclovir);
[0170] 9-(4-hydroxy-3-hydroxymethyl-but-1-yl)-6-deoxy-guanine,
diacetate (famciclovir);
[0171] 3'-azido-3'-deoxythymidine (AZT);
[0172] 3'-chloro-5-methyl-dexocytidine (ClddMeCyt);
[0173]
9-(2-phosphonyl-methoxyethyl)-2',6'-diaminopurine-2',3'-dideoxyribo-
side;
[0174] 9-(2-phosphonylmethoxyethyl)adenine (PMEA);
[0175] acyclovir triphosphate (ACVTP);
[0176] D-carbocyclic-2'-deoxyguanosine (CdG);
[0177] dideoxy-cytidine;
[0178] dideoxy-cytosine (ddC);
[0179] dideoxy-guanine (ddG);
[0180] dideoxy-inosine (ddI);
[0181] E-5-(2-bromovinyl)-2'-deoxyuridine triphosphate;
[0182] fluoro-arabinofuranosyl-iodouracil;
[0183]
1-(2'-deoxy-2'-fluoro-1-beta-D-arabinofuranosyl)-5-iodo-uracil
(FIAU);
[0184] stavudine;
[0185] 9-beta-D-arabinofuranosyl-9H-purine-6-amine monohydrate
(Ara-A);
[0186] 9-beta-D-arabinofuranosyl-9H-purine-6-amine-5'-monophosphate
monohydrate (Ara-AMP);
[0187] 2-deoxy-3'-thia-5-fluorocytidine;
[0188] 2',3'-dideoxy-guanine; and
[0189] 2',3'-dideoxy-guanosine.
[0190] A preferred compound is
(-)-2'-deoxy-3'-thiocytidine-5'-triphosphat- e (3TC).
[0191] Synthetic methods for the preparation of nucleosides and
nucleotides useful in the present invention are likewise well known
in the art as disclosed in Acta Biochim. Pol., 43, 25-36 (1996);
Swed. Nucleosides Nucleotides 15, 361-378 (1996), Synthesis 12,
1465-1479 (1995), Carbohyd. Chem. 27, 242-276 (1995), Chem.
Nucleosides Nucleotides 3, 421-535 (1994), Ann. Reports in Med.
Chem., Academic Press; and Exp. Opin. Invest. Drugs 4, 95-115
(1995).
[0192] The chemical reactions described in the references cited
above are generally disclosed in terms of their broadest
application to the preparation of the compounds of this invention.
Occasionally, the reactions may not be applicable as described to
each compound included within the scope of compounds disclosed
herein. The compounds for which this occurs will be readily
recognized by those skilled in the art. In all such cases, either
the reactions can be successfully performed by conventional
modifications known to those skilled in the art, e.g., by
appropriate protection of interfering groups, by changing to
alternative conventional reagents, by routine modification of
reaction conditions, and the like, or other reactions disclosed
herein or otherwise conventional will be applicable to the
preparation of the corresponding compounds of this invention. In
all preparative methods, all starting materials are known or
readily preparable from known starting materials.
[0193] While nucleoside analogs are generally employed as antiviral
agents as is, nucleotides (nucleoside phosphates) must sometimes
have to be converted to nucleosides in order to facilitate their
transport across cell membranes. An example of a chemically
modified nucleotide capable of entering cells is
S-1-3-hydroxy-2-phosphonylmethoxypropyl cytosine (HPMPC, Gilead
Sciences).
[0194] Nucleoside and nucleotide compounds of this invention that
are acids can form salts. Examples include salts with alkali metals
or alkaline earth metals, such as sodium, potassium, calcium, or
magnesium, or with organic bases or basic quaternary ammonium
salts.
[0195] Immunomodulators and Immunostimulants
[0196] A large number of immunomodulators and immuno-stimulants
that can be used in the methods of the present invention are
currently available. A list of these compounds is provided in Table
1, below.
1 TABLE 1 AA-2G adamantylamide dipeptide adenosine deaminase, Enzon
adjuvant, Alliance adjuvants, Ribi adjuvants, Vaxcel Adjuvax
agelasphin-11 AIDS therapy, Chiron algal glucan, SRI algammulin,
Anutech Anginlyc anticellular factors, Yeda Anticort antigastrin-17
immunogen, Ap antigen delivery system, Vac antigen formulation,
IDBC antiGnRH immunogen, Aphton Antiherpin Arbidol Aviron azarole
Bay-q-8939 Bay-r-1005 BCH-1393 Betafectin Biostim BL-001 BL-009
Broncostat Cantastim CDRI-84-246 cefodizime chemokine inhibitors,
ICOS CMV peptides, City of Hope CN-5888 cytokine-releasing agent,
St DHEAS, Paradigm DISC TA-HSV J07B I01A I01Z ditiocarb sodium
ECA-10-142 ELS-1 endotoxin, Novartis FCE-20696 FCE-24089 FCE-24578
FLT-3 ligand, Immunex FR-900483 FR-900494 FR-901235 FTS-Zn
G-proteins, Cadus gludapcin glutaurine glycophosphopeptical GM-2
GM-53 GMDP growth factor vaccine, EntreM H-BIG, NABI H-CIG, NABI
HAB-439 Helicobacter pylori vaccine, herpes-specific immune factor
HIV therapy, United Biomed HyperGAM+CF ImmuMax Immun BCG immune
therapy, Connective immunomodulator, Evans immunomodulators,
Novacell imreg-1 imreg-2 Indomune inosine pranobex interferon,
Dong-A (alpha2) interferon, Genentech (gamma) interferon, Novartis
(alpha) interleukin-12, Genetics Ins interleukin-15, Immunex
interleukin-16, Research Cor ISCAR-1 J005X L-644257 licomarasminic
acid LipoTher LK-409 LK-410 LP-2307 LT(R1926) LW-50020 MAF,
Shionogi MDP derivatives, Merck met-enkephalin, TNI
methylfurylbutyrolactones MIMP mirimostim mixed bacterial vaccine,
Tem MM-1 moniliastat MPLA, Ribi MS-705 murabutide murabutide,
Vacsyn muramyl dipeptide derivative muramyl peptide derivatives
myelopid N-563 NACOS-6 NH-765 NISV, Proteus NPT-16416 NT-002 PA-485
PEFA-814 peptides, Scios peptidoglycan, Pliva Perthon, Advanced
Plant PGM derivative, Pliva Pharmaprojects No. 1099 Pharmaprojects
No. 1426 Pharmaprojects No. 1549 Pharmaprojects No. 1585
Pharmaprojects No. 1607 Pharmaprojects No. 1710 Pharmaprojects No.
1779 Pharmaprojects No. 2002 Pharmaprojects No. 2060 Pharmaprojects
No. 2795 Pharmaprojects No. 3088 Pharmaprojects No. 3111
Pharmaprojects No. 3345 Pharmaprojects No. 3467 Pharmaprojects No.
3668 Pharmaprojects No. 3998 Pharmaprojects No. 3999 Pharmaprojects
No. 4089 Pharmaprojects No. 4188 Pharmaprojects No. 4451
Pharmaprojects No. 4500 Pharmaprojects No. 4689 Pharmaprojects No.
4833 Pharmaprojects No. 494 Pharmaprojects No. 5217 Pharmaprojects
No. 530 pidotimod pimelautide pinafide PMD-589 podophyllotoxin,
Conpharm POL-509 poly-ICLC poly-ICLC, Yamasa Shoyu PolyA-PolyU
Polysaccharide A protein A, Berlox Bioscience PS34WO pseudomonas
MAbs, Teijin Psomaglobin PTL-78419 Pyrexol pyriferone Retrogen
Retropep RG-003 Rhinostat rifamaxil RM-06 Rollin romurtide RU-40555
RU-41821 rubella antibodies, ResCo S-27609 SB-73 SDZ-280-636
SDZ-MRL-953 SK&F-107647 SL04 SL05 SM-4333 Solutein SRI-62-834
SRL-172 ST-570 ST-789 staphage lysate Stimulon suppressin T-150R1
T-LCEF tabilautide temurtide Theradigm-HBV Theradigm-HPV
Theradigm-HSV THF, Pharm & Upjohn THF, Yeda thymalfasin thymic
hormone fractions thymocartin thymolymphotropin thymopentin
thymopentin analogues thymopentin, Peptech thymosin fraction 5,
Alpha thymostimulin thymotrinan TMD-232 TO-115 transfer factor,
Viragen tuftsin, Selavo ubenimex Ulsastat ANGG- CD-4+ Collag+
COLSF+ COM+ DA-A+ GAST- GF-TH+ GP-120- IF+ IF-A+ IF-A-2+ IF-B+
IF-G+ IF-G-1B+ IL-2+ IL-12+ IL-15+ IM+ LHRH- LIPCOR+ LYM-B+ LYM-NK+
LYM-T+ OPI+ PEP+ PHG-MA+ RNA-SYN- SY-CW- TH-A-1+ TH-5+ TNF+ UN
[0197] Dosages
[0198] The N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds
useful in the present invention can be administered to humans in an
amount in the range of from about 0.1 mg/kg/day to about 100
mg/kg/day, more preferably from about 1 mg/kg/day to about 75
mg/kg/day, and most preferably from about 5 mg/kg/day to about 50
mg/kg/day.
[0199] The nucleoside or nucleotide antiviral compound, or mixtures
thereof, can be administered to humans in an amount in the range of
from about 0.1 mg/person/day to about 500 mg/person/day, preferably
from about 10 mg/person/day to about 300 mg/person/day, more
preferably from about 25 mg/person/day to about 200 mg/person/day,
even more preferably from about 50 mg/person/day to about 150
mg/person/day, and most preferably in the range of from about 1
mg/person/day to about 50 mg/person/day.
[0200] Immunomodulators and immunostimulants useful in the present
invention can be administered in amounts lower than those
conventional in the art. For example, thymosin alpha 1 and thymosin
fraction 5 are typically administered to humans for the treatment
of HepB infections in an amount of about 900 .mu.g/m.sup.2, two
times per week (Hepatology (1988) 8:1270; Hepatology (1989) 10:575;
Hepatology (1991) 14:409; Gastroenterology (1995) 108:A1127). In
the methods and compositions of the present invention, this dose
can be in the range of from about 10 .mu.g/m.sup.2, two times per
week to about 750 .mu.g/m.sup.2, two times per week, more
preferably from about 100 .mu.g/m.sup.2, two times per week to
about 600 .mu.g/m.sup.2, two times per week, most preferably from
about 200 .mu.g/m.sup.2, two times per week to about 400
.mu.g/m.sup.2, two times per week. Interferon alfa is typically
administered to humans for the treatment of HepC infections in an
amount of from about 1.times.10.sup.6 units/person, three times per
week to about 10.times.10.sup.6 units/person, three times per week
(Simon et al., (1997) Hepatology 25:445-448). In the methods and
compositions of the present invention, this dose can be in the
range of from about 0.1.times.10.sup.6 units/person, three times
per week to about 7.5.times.10.sup.6 units/person, three times per
week, more preferably from about 0.5.times.10.sup.6 units/person,
three times per week to about 5.times.10.sup.6 units/person, three
times per week, most preferably from about 1.times.10.sup.6
units/person, three times per week to about 3.times.10.sup.6
units/person, three times per week.
[0201] Due to the enhanced hepatitis virus antiviral effectiveness
of these immunomodulators and immunostimulants in the presence of
the N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds useful
in the present invention, reduced amounts of other
immunomodulators/immunostimul- ants can be employed in the methods
and compositions disclosed herein. Such reduced amounts can be
determined by routine monitoring of hepatitis virus in infected
patients undergoing therapy. This can be carried out by, for
example, monitoring hepatitis viral DNA in patients' serum by
slot-blot, dot-blot, or PCR techniques, or by measurement of
hepatitis surface or other antigens, such as the e antigen, in
serum. Methods therefor are discussed in Hoofnagle et al., (1997)
New Engl. Jour. Med. 336(5):347-356, and F. B. Hollinger in Fields
Virology, Third Ed., Vol. 2 (1996), Bernard N. Fields et al., Eds.,
Chapter 86, "Hepatitis B Virus," pp. 2738-2807, Lippincott-Raven,
Philadelphia, Pa., and the references cited therein.
[0202] Patients can be similarly monitored during combination
therapy employing N-substituted-1,5-dideoxy-1,5-imino-D-glucitol
compounds and nucleoside and/or nucleotide antiviral agents to
determine the lowest effective doses of each.
[0203] The doses described above can be administered to a patient
in a single dose or in proportionate multiple subdoses. In the
latter case, dosage unit compositions can contain such amounts of
submultiples thereof to make up the daily dose. Multiple doses per
day can also increase the total daily dose should this be desired
by the person prescribing the drug.
[0204] Pharmaceutical Compositions
[0205] The compounds of the present invention can be formulated as
pharmaceutical compositions. Such compositions can be administered
orally, parenterally, by inhalation spray, rectally, intradermally,
transdermally, or topically in dosage unit formulations containing
conventional nontoxic pharmaceutically acceptable carriers,
adjuvants, and vehicles as desired. Topical administration may also
involve the use of transdermal administration such as transdermal
patches or iontophoresis devices. The term parenteral as used
herein includes subcutaneous, intravenous, intramuscular, or
intrasternal injection, or infusion techniques. Formulation of
drugs is discussed in, for example, Hoover, John E., Remington's
Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (1975),
and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage
Forms, Marcel Decker, New York, N.Y. (1980).
[0206] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions, can be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, and
isotonic sodium chloride solution. In addition, sterile, fixed oils
are conventionally employed as a solvent or suspending medium. For
this purpose, any bland fixed oil may be employed, including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are useful in the preparation of injectables. Dimethyl
acetamide, surfactants including ionic and non-ionic detergents,
and polyethylene glycols can be used. Mixtures of solvents and
wetting agents such as those discussed above are also useful.
[0207] Suppositories for rectal administration of the compounds
discussed herein can be prepared by mixing the active agent with a
suitable non-irritating excipient such as cocoa butter, synthetic
mono-, di-, or triglycerides, fatty acids, or polyethylene glycols
which are solid at ordinary temperatures but liquid at the rectal
temperature, and which will therefore melt in the rectum and
release the drug.
[0208] Solid dosage forms for oral administration may include
capsules, tablets, pills, powders, and granules. In such solid
dosage forms, the compounds of this invention are ordinarily
combined with one or more adjuvants appropriate to the indicated
route of administration. If administered per os, the compounds can
be admixed with lactose, sucrose, starch powder, cellulose esters
of alkanoic acids, cellulose alkyl esters, talc, stearic acid,
magnesium stearate, magnesium oxide, sodium and calcium salts of
phosphoric and sulfuric acids, gelatin, acacia gum, sodium
alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then
tableted or encapsulated for convenient administration. Such
capsules or tablets can contain a controlled-release formulation as
can be provided in a dispersion of active compound in
hydroxypropylmethyl cellulose. In the case of capsules, tablets,
and pills, the dosage forms can also comprise buffering agents such
as sodium citrate, or magnesium or calcium carbonate or
bicarbonate. Tablets and pills can additionally be prepared with
enteric coatings.
[0209] For therapeutic purposes, formulations for parenteral
administration can be in the form of aqueous or non-aqueous
isotonic sterile injection solutions or suspensions. These
solutions and suspensions can be prepared from sterile powders or
granules having one or more of the carriers or diluents mentioned
for use in the formulations for oral administration. The compounds
can be dissolved in water, polyethylene glycol, propylene glycol,
ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl
alcohol, sodium chloride, and/or various buffers. Other adjuvants
and modes of administration are well and widely known in the
pharmaceutical art.
[0210] Liquid dosage forms for oral administration can include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups, and elixirs containing inert diluents commonly used in the
art, such as water. Such compositions can also comprise adjuvants,
such as wetting agents, emulsifying and suspending agents, and
sweetening, flavoring, and perfuming agents.
[0211] The amount of active ingredient that can be combined with
the carrier materials to produce a single dosage form will vary
depending upon the patient and the particular mode of
administration.
[0212] Certain of the pharmaceutical compounds of this invention
which are administered in accordance with the methods of the
invention can serve as prodrugs to other compounds of this
invention. Prodrugs are drugs that can be chemically converted in
vivo or in vitro by biological systems into an active derivative or
derivatives. Prodrugs are administered in essentially the same
fashion as the other pharmaceutical compounds of the invention.
Non-limiting examples are the esters of the
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds of this
invention.
[0213] Compounds of the combinations of this invention, for example
N-(n-nonyl)-1,5-dideoxy-1,5-imino-D-glucitol and various
nucleosides or nucleotides, may be acids or bases. As such, they
may be used to form salts with one another. Nucleosides are purine
or pyrimidine compounds lacking a phosphate ester. Compounds of
Formulas II, III, IV, V, or VI herein without a phosphate ester but
containing a carboxylic acid moiety could form a salt with an
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound of the
present invention. Nucleotides are purine or pyrimidine compounds
that are mono-, di-, or triphosphate esters. These phosphate esters
contain free --OH groups that are acidic, and that can form salts
with inorganic bases or organic bases. Salt formation with organic
bases depends on the pKa of the acid and base. The
N-substituted-1,5-dideoxy-1,- 5-imino-D-glucitol compounds
disclosed herein are basic, and form pharmaceutically acceptable
salts. In the present case, useful salts can be formed not only
with pharmaceutically acceptable acids, but also with biologically
active acids such as the nucelosides and nucleotides disclosed
herein. These salts can be prepared in the conventional manner for
preparing salts, as is well known in the art. For example, one can
treat the free base of an
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compound with a
nucleotide analog of Formula II, III, IV, V, or VI to form a salt.
This can be performed as a separate chemical reaction, or as part
of the formulation process. The limiting reagent in the salt
forming reaction is either the acid or base, as selected by the
artisan to obtain a suitable biological result. The formulation can
contain mixtures of different salts, acids, or free bases as
desired. For example, the phosphoric acid form of
(-)-2'-deoxy-3'-thiocytidine-5'-triphosphate will form a salt with
the base form of N-(n-nonyl)-1,5-dideoxy-1,5-imino-D-glu- citol or
N-(n-nonyl)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate. This
type of salt can then be provided to the patient in a
pharmaceutically acceptable formulation, as a pure single salt, or
as part of a mixture.
[0214] In some cases, the salts can also be used as an aid in the
isolation, purification, or resolution of the compounds of this
invention.
[0215] Treatment Regimen
[0216] The regimen for treating a patient suffering from a
hepatitis virus infection with the compounds and/or compositions of
the present invention is selected in accordance with a variety of
factors, including the age, weight, sex, diet, and medical
condition of the patient, the severity of the infection, the route
of administration, pharmacological considerations such as the
activity, efficacy, pharmacokinetic, and toxicology profiles of the
particular compounds employed, and whether a drug delivery system
is utilized.
[0217] Administration of the drug combinations disclosed herein
should generally be continued over a period of several weeks to
several months or years until virus titers reach acceptable levels,
indicating that infection has been controlled or eradicated. As
noted above, patients undergoing treatment with the drug
combinations disclosed herein can be routinely monitored by
measuring hepatitis viral DNA in patients' serum by slot-blot,
dot-blot, or PCR techniques, or by measurement of hepatitis
antigens, such as hepatitis B surface antigen (HBsAg) and hepatitis
B e antigen (HBeAg), in serum to determine the effectiveness of
therapy. In chronic hepatitis B, for example, remissions are
characterized by the disappearance of hepatitis B viral DNA, i.e.,
reduction to undetectable levels as measured by hybridization tests
capable of detecting levels .gtoreq.10.sup.5 genomes per ml of
serum, and HBeAg from serum despite the continued presence of
HBsAg. These serologic events are followed by improvement in the
biochemical and histologic features of the disease. The end point
of successful treatment in most trials of antiviral therapy is the
disappearance of HBeAg and viral DNA from serum. In patients in
whom the e antigen disappears, remission is usually sustained, and
results in an inactive HBsAg carrier state. Many patients
eventually become HBsAg-negative (see Hoofnagle et al., (1997) New
Engl. Jour. Med. 336(5):347-356 for a review).
[0218] Continuous analysis of the data obtained by these methods
permits modification of the treatment regimen during therapy so
that optimal amounts of each component in the combination are
administered, and so that the duration of treatment can be
determined as well. Thus, the treatment regimen/dosing schedule can
be rationally modified over the course of therapy so that the
lowest amounts of each of the antiviral compounds used in
combination which together exhibit satisfactory anti-hepatitis
virus effectiveness are administered, and so that administration of
such antiviral compounds in combination is continued only so long
as is necessary to successfully treat the infection.
[0219] The following non-limiting examples serve to illustrate
various aspects of the present invention.
EXAMPLE 1
Preparation of 1,5-(butylimino)-1,5-dideoxy-D-glucitol
[0220] A solution of 1,5-dideoxy-1,5-imino-D-glucitol (5.14 g,
0.0315 mole), butyraldehyde (3.35 ml, 0.0380 mole) and Pd black (1
g) in 200 ml methanol was hydrogenated (60 psi/29.degree. C./21
hrs.). After filtering the resulting mixture, the filtrate was
concentrated in vacuo to an oil. The title compound was
crystallized from acetone, and recrystallized from
methanol/acetone, m.p. ca. 132.degree. C. The structure assignment
was supported by NMR, infrared spectra and elemental analysis.
[0221] Analysis calcd. for C.sub.10H.sub.21NO.sub.4: C, 54.78; H,
9.65; N, 6.39. Found: C, 54.46; H, 9.33; N, 6.46.
EXAMPLE 2
Preparation of 1,5-(butylimino)-1,5-dideoxy-D-glucitol,
tetraacetate
[0222] Acetic anhydride (1.08 g, 0.0106 mole) was added to the
title compound of Example 1 (0.50 g, 0.0023 mole) in 5 ml pyridine
and stirred for 17 days at room temperature. The product was
evaporated under nitrogen gas. The resulting title compound was
purified by silica gel chromatography. Structure assignment was
supported by NMR, infrared spectra and elemental analysis.
[0223] Analysis calcd. for C.sub.18H.sub.29NO.sub.8: C, 55.80; H,
7.54; N, 3.62. Found: C, 55.42; H, 7.50; N, 3.72.
EXAMPLE 3
Anti-Hepatitis B Virus Activity of Various
N-Substituted-1,5-Dideoxy-1,5-I- mino-D-Glucitol Compounds In
Vitro
[0224] The anti-hepatitis B virus activity and effect on cell
viability of a number of different
N-substituted-1,5-dideoxy-1,5-imino-D-glucitol compounds were
assessed using an in vitro assay employing chronically hepatitis B
virus secreting HepG2.2.15 cells. The method employed was
essentially that described in Block et al. (1994) Proc. Natl. Acad.
Sci. USA 91:2235-2239. The results are shown in Tables 2 and 3.
TABLE 2
Effect of N-Substituted-1,5-Dideoxy-1,5-Imino-D-Glucitol Compounds
on Hepatitis B Virus Secretion and Viability of HepG2.2.15
Cells
[0225]
2TABLE 2 Effect of N-Substituted-1,5-Dideoxy-1,5-Im- ino-D-Glucitol
Compounds on Hepatities B Virus Secreation and Viability of
HepG2.2.15 Cells Relative amount of Compound and HBV secreted, as
[Concentration].sup.1 % Viable +/- 1 S.D..sup.2 a % of
control.sup.3 Control 90 +/- 7 (n = 4) 100 NBDNJ.sup.4 [200] 94 +/-
6 (n = 10) 37.0 +/- 13 (n = 15) NBDNJ.sup.4 [1000] 88 +/- 8 (n =
10) 3.2 +/- 5 (n = 15) 1 [200] 90 +/- 2 (n = 4) 85.0 +/- 5 (n = 8)
1 [1000] 87 +/- 3 (n = 4) 35.0 +/- 6 (n = 8) 2 [200] 90 +/- 6 (n =
4) 107.0 +/- 12 (n = 3) 2 [1000] 89 +/- 4 (n = 4) 38.0 +/- 15 (n =
3) 3 [200] n.d..sup.5 45.0 +/- 30 (n = 3) 3 [1000] n.d..sup.5 5.0
+/- 20 (n = 3) .sup.1Chronically HBV secreting 2.2.15 cells
(approximately 500,000 per well) were incubated in the presence of
indicated compound for three days. .sup.2After 3 days of culture in
the absence or presence of compound, cells were removed by trypsin
treatment, incubated with trypan blue, and visually examined for
dye exclusion by microscopy. Values are the percentage, relative to
the total number of cells examined, of cells excluding trypan blue
(trypan blue exclusion was considered equivalent to viability).
.sup.3After 3 days of incubation in the absence or presence of
compound, secreted virions were immunoprecipitated from the culture
medium with monoclonal antibody specific for preS1 antigen (Meisel
et al. (1995) Intervirology 37: 330-339; Lu et al. (1995) Virology
213: 660-665). Viral DNA present in the immunoprecipitates was
detected by densitrometric quantification # of the DNA fragment of
the correct size resulting from a polymerase chain reaction. The
amount of DNA amplified from control (cells receiving no compound)
is assumed to be 100%. .sup.4NBDNJ:
N-(n-butyl-)-1,5-dideoxy-1,5-imino-D-glucitol; N-butyl DNJ.
.sup.5Although trypan blue viability staining was not performed,
cells appeared unremarkable (healthy) by gross microscopic
examination. S.D.: standard deviation. Compounds: 1:
N-(3-phenylpropyl)-1,5-dideoxy-1,5-imino-D-glucitol 2:
N-(n-butyl)-1,5-dideoxy-1,5-imino-D-glucitol, tetrabutyrate 3:
N-(2-ethylbutyl)-1,5-dideoxy-1,5-imino-D-glucitol
[0226]
3TABLE 3 Effect of N-Substituted-1,5-Dideoxy-1,5-Im- ino-D-Glucitol
Compounds on Hepatities B Virus Secreation and Viability of
HepG2.2.15 Cells Concentration Resulting Concentration Resulting In
90% HBV In 50% Compound Secretion Inhibition.sup.1 Reduction in
MTT.sup.2 1 0.5-1.0* 100-200 2 >200** n.d..sup.3 .sup.1in .mu.gs
per ml. and based upon duplicate PCR results. .sup.2in .mu.gs per
ml.; MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide. The MTT-based colorimetric assay is a measurement of cell
viability (Heo et al. (1990) Cancer Research 50: 3681-3690).
.sup.3Not determined. *lowest concentration tested. **there was no
inhibition seen at the highest concentration used (200 .mu.gs/ml).
Compounds: 1: N-(n-nonyl)-1,5-dideoxy-1,5-imi- no-D-glucitol 2:
N-(n-butyl)-1,5-dideoxy-1,5-imino-D-glucitol, 3,4-diacetate
[0227] Compounds:
[0228] 1: N-(n-nonyl)-1,5-dideoxy-1,5-imino-D-glucitol
[0229] 2: N-(n-butyl)-1,5-dideoxy-1,5-imino-D-glucitol,
3,4-diacetate
EXAMPLE 4
Anti-Hepatitis B Virus Activity of
(-)-2'-deoxy-3'-thiocytidine-5'-triphos- phate (3TC) Alone and in
Combination with N-nonyl-DNJ
[0230] The anti-hepatitis B virus effect of
(-)-2'-deoxy-3'-thiocytidine-5- '-triphosphate (3TC) alone and in
combination with N-nonyl-DNJ was determined according to Korba
((1996) Antiviral Research 29(1):49-51), using the "combostat"
strategy (Comstat Program, Combostat Corp., Duluth, Minn.). The
combostat method involves serially diluting the IC-90 of each
compound. The IC-90 of N-nonyl-DNJ has been determined to be
between 4 and 10 .mu.g/ml (T. Block and G. Jacob, unpublished
observation). The accepted IC-90 for 3TC in HepG 2.2.15 (2.2.15)
cells is 300 nM to 500 nM (Doong et al. (1991) Proc. Natl. Acad.
Sci. USA 88:8495-8499).
[0231] 2.2.15 cells, described in Sells et al. (1987) Proc. Natl.
Acad. Sci. USA 84:1005-1009, were maintained in RPMI 1640 medium
(Gibco BRL, #31800-022) supplemented with 10% fetal bovine serum,
200 .mu.g/ml G418 (Gibco BRL 066-1811). Cells were seeded into 25
cm.sup.2 flasks at 80% confluency. Five days later, flasks in
triplicate received either no compound, serial dilutions of 3TC
alone, or serial dilutions of 3TC plus N-nonyl-DNJ. At 2, 4, and 6
days after addition of compound (with medium replacement on those
days), the amount of hepatitis B virus (HBV) DNA in the culture
medium was determined by PCR analysis of
polyethyleneglycol-sedimented particles. Thus, in these
experiments, enveloped particles were not distinguished from
nucleocapsids. PCR-amplified products were resolved by agarose gel
electrophoresis (1,5% agarose), and the 538 nucleotide fragment was
quantified by band scanning (HP Jet Imager). The amount of HBV
recovered from untreated cells is assumed to be 100%. Data from the
6-day time point are presented in FIG. 1 as the average values from
at least three separate flasks, and the standard error was never
greater than 20%, with an average error of 12%.
[0232] For each of the three time point series tested, the
combination of 3TC plus N-nonyl-DNJ was significantly more
effective in inhibiting HBV secretion than either compound alone.
Conclusions based upon PCR analysis alone make it difficult to
assign precise IC-50 values. The extreme sensitivity and delicate
nature of PCR, for example, may account for the inability to
achieve greater than 90% inhibition of HBV by 3TC alone, even at
300 nM. Every experiment included controls to assure that PCR was
performed in a range of concentrations of DNA in which the reaction
yields results proportional to the amount of DNA in the sample.
Resolution is approximately 3-fold, i.e., 3-fold differences in DNA
concentrations can be detected. The inability to consistently
detect less than 3-fold differences probably explains the failure
of 3TC alone to achieve 90% inhibition. This suggests that a very
high standard of inhibition must be met for the PCR to detect
inhibition. Consequently, the trend, over three separate time
points, is clear: the combined effect of 3TC plus N-nonyl-DNJ is
greater than that of either compound alone, or the additive
individual effects of each compound. These data suggest that the
IC-50 of 3TC has been moved from about 60 nM to about 0.48 nM when
0.016 .mu.g/ml N-nonyl-DNJ is present.
EXAMPLE 5
Anti-Hepatitis B Virus Effect of N-nonyl-DNJ Alone in a Woodchuck
Model
[0233] In order to evaluate the efficacy of N-nonyl-DNJ in
combination with 3TC (or other nucleoside or nucleotide analogs)
against Hepatitis B virus in a woodchuck animal model, an
monotherapy experiment using N-nonyl-DNJ alone was first conducted.
This was necessary to determine if N-nonyl-DNJ has any anti-HBV
effect in the woodchuck and, if N-nonyl-DNJ has a beneficial
effect, to design a combination study based on the dose-response
relationship of this drug alone.
[0234] Therefore, five groups of four animals each (all groups had
both sexes, all but the control had two of each sex) were assigned
to 0, 12.5, 25, 50, and 100 mg/kg/day with BID oral dosing. These
were lab-reared wild animals. All animals were infected with
woodchuck hepatitis virus (WHV) as neonates, and had been tested
positive on serological tests for WHV surface antigen. Blood
samples were drawn one week prior to dosing (-1 week), immediately
before dosing (0 weeks), weekly during dosing (1, 2, 3, and 4
weeks), and after the end of dosing (5, 6, 8, and 10 weeks).
[0235] There are two measures of drug efficacy: reduction in total
HBV DNA (measured by quantitative PCR), and reduction in HBV DNA
from capsids with intact surface glycoproteins, which is the active
form of the virus (measured by an ELISA-like immune precipitation
assay followed by quantitative PCR). Cell culture experiments with
N-nonyl-DNJ demonstrated little or no effect of this compound on
total HBV DNA, but a marked effect on the immune precipitated DNA
(IPDNA). Not surprisingly, the IPDNA assay is quite variable; as a
partial compensation for this, four assay runs were conducted, each
containing samples from all animals, but different subsets of the
study weeks.
[0236] To summarize the results, N-nonyl-DNJ had no effect on total
HBV DNA measurements, which were essentially constant for all dose
levels over the pre-dose and dosed portions of the study. On the
other hand, IPDNA levels were not constant over the study period.
The low dose animals tended to have increasing levels of IPDNA over
the dosing period (weeks 0-4), while high dose animals tended to
have decreasing levels of IPDNA over the same period. Fitting a
straight line to each animal's weekly responses gave a significant
difference in the slope of these lines due to either dose or plasma
level of drug. The plasma levels of drug were also quite variable:
animals with the lowest plasma levels in their dose group had lower
plasma levels than the animals with the highest plasma levels from
the next lower dose group. There were no differences between
responses of males and females on any of the measures.
[0237] Plasma Levels
[0238] There were no clear patterns in the changes in plasma levels
of N-nonyl-DNJ which could be related to week of dosing or time
since previous dose. Because the plasma levels within an animal
seemed reasonably consistent during dosing, the median plasma level
for each animal was used for subsequent modeling. The plasma levels
for each week of the dosing period are plotted for each animal vs.
dose (a small amount of random noise is added to the dose level so
points which would lie on top of each other on the plot can be
distinguished) (FIG. 2).
[0239] HBV DNA
[0240] The total HBV DNA levels were essentially constant over time
within each animal (data not shown). There was a faint hint of a
dose-response relationship with decreasing levels of virus with
increasing levels of drug, except that three animals at the highest
dose had very high virus levels. It is not possible to conclude
that there is any relationship between dose of N-nonyl-DNJ and
total HBV DNA. It is possible that there are two populations of
animals, responders (such as animal r) and non-responders (animals
i, m, and d), but more data would be required to permit a firm
conclusion on this point.
[0241] Immune Precipitated HBV DNA
[0242] Substantial variation existed in the IPDNA assay, both
between assay runs and within assay runs (data not shown). Even so,
it was possible to observe and model a slope over weeks 0-4 which
is generally increasing for low dose animals and decreasing for
high dose animals. This change in slope was statistically
significant (p<0.005).
[0243] Before models are fitted to the data, a log transform was
applied because: 1) the variation in IPDNA increases with
increasing IPDNA values; the log transformation gives values with a
nearly constant variation, and 2) it is expected that drug effects
will appear as a constant multiplier of the IPDNA level. Because
there are zero values of IPDNA, a small value (about 1/2 of the
smallest non-zero value) was added to all values before the log
transform.
[0244] Two approaches were used to model the changes in slope to
week with dose of N-nonyl-DNJ: a linear modeling approach and a
nonlinear model. Both approaches assume that the (linear) rate of
change of the Log(IPDNA) measure over the dosing period is the
"right" measure to reflect the effect of the drug on the virus.
Both approaches are fit in stages, and the first stage is common to
both approaches. First, a simple straight line regression model is
fit using weeks 0-4 to predict log(IPDNA+10) separately for each
animal by run combination. In the second stage, the response
variable is the slope fitted in the first stage.
[0245] For the linear approach, a model is fit with slope to week
as the response where run is considered a block, dose has a
significant effect (almost all of this effect is due to a slope to
dose), and the relevant error for testing the effect of dose is the
variation among animals treated alike (after the adjustment for the
runs as blocks). This is similar to using the calibration data
within each run to first adjust each run's data to a common virus
DNA concentration; the difference is that here the data from the
woodchucks are used for the run adjustment rather than only the
calibration data.
[0246] For the nonlinear approach, a four parameter logistic model
is fit with the slope to week as the response and the dose as the
predictor. Again, run is considered a block, but because no run has
all weeks, it is not possible to fully reflect the blocking in the
nonlinear approach. Even so, the nonlinear model yields an EC50 of
7.88 mg/kg/BID dose. The average maximum slope observed was 2.71
additional Log(IPDNA .mu.g/mL)/week, or an increase of about
150%/week, the average minimum slope observed with N-nonyl-DNJ is
0.31 fewer Log(IPDNA .mu.g/mL)/week), or about a decrease of about
25%/week. The slopes, the fitted model, the parameter estimates
from the model, and the approximate standard errors for these
parameters are all shown in FIG. 3. The data indicate an
approximate effective monotherapy dose of N-nonyl-DNJ in woodchucks
of about 16 mg/kg/day. Whether in woodchucks or humans, the
effective dose of both the N-alkyl-DNJ and nucleoside or nucleotide
antiviral agent administered in combination therewith can be
administered in two equal daily subdoses (i.e., B.I.D.).
[0247] FIGS. 2 and 3 show letters to indicate animals. Table 4
shows two of the animal codes, the sex, and the dose.
4TABLE 4 Animal Codes, Sex, and Dose Animal Number Letter Code Sex
Dose F95343 b F 0 M96364 n M 0 F96304 k F 0 F96301 j F 0 M96285 h M
6.25 F96283 g F 6.25 F96391 o F 6.25 M96305 l M 6.25 F96271 f F
12.5 M96256 e M 12.5 M96404 s M 12.5 F96392 p F 12.5 F96163 c F 25
M96414 t M 25 F96393 q F 25 M95322 a M 25 M96286 i M 50 F96231 d F
50 F96402 r F 50 M96363 m M 50
EXAMPLE 6
Antiviral Study to Test the Activity of N-nonyl-DNJ in Combination
with 3TC in a Woodchuck Model of Hepatitis B Virus Infection
[0248] The combined activity of N-nonyl-DNJ and the nucleoside
analog 3TC can be assessed using the woodchuck model of hepatitis B
virus infection. Twenty-eight woodchucks with persistent woodchuck
hepatitis virus (WHV) infection can be utilized. Groups of
woodchucks can be treated orally with 3TC alone (s.i.d.), with
N-nonyl-DNJ alone (b.i.d.), or with combinations of the two drugs.
The antiviral activity of the individual drugs and combinations can
be assessed by measuring serum WHV DNA during treatment, and
comparing the results of treated groups to placebo treated
controls.
[0249] Twenty-eight woodchucks with established persistent WHV
infection can be used, all of which were experimentally infected
with WHV during the first week of life. All can be WHsAg positive
at the time the study is initiated.
[0250] A total of eight experimental groups can be used. Woodchucks
in each group can be stratified on the basis of gender, body
weight, and age. 3TC can be administered orally as an aqueous
suspension of Epivir (Glaxo-Wellcome) tablets one time per day.
N-nonyl-DNJ can also be administered orally in aqueous solution, in
two divided doses. Treatment with both drugs can be followed by the
administration of 4 to 5 mls of semisynthetic liquid woodchuck diet
to insure complete ingestion of the drugs.
[0251] The experimental groups can be as follows:
5 Group 3TC N-nonyl-DNJ ID No. (mg/kg/day) (mg/kg/day) 1 4 0.0 0.0
2 3 3.0 0.0 3 3 9.0 0.0 4 3 0.0 4.0 5 3 0.0 12.0 6 4 1.5 2.0 7 4
4.5 6.0 8 4 9.0 12.0
[0252] Woodchucks can be anesthetized (50 mg/kg ketamine, 5 mg/kg
zylazine), weighed, and blood samples obtained prior to initial
treatment, at weekly intervals during the six week period of
treatment, and at 1, 2, and 4 weeks following treatment. Serum can
be harvested and divided into aliquots. One aliquot can be used for
analysis of WHV DNA by dot blot hybridization and for WHsAg by
ELISA. CBCs and clinical biochemical profiles can be obtained prior
to treatment and at the end of treatment. A second aliquot can be
maintained as an archive sample. Other aliquots of serum can be
used for drug analysis and special WHV DNA analyses.
[0253] The invention being thus described, it will be obvious that
the same can be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of the present
invention, and all such modifications and equivalents as would be
obvious to one skilled in the art are intended to be included
within the scope of the following claims.
* * * * *