U.S. patent application number 16/115298 was filed with the patent office on 2018-12-27 for modulation of hepatitis b virus cccdna transcription.
The applicant listed for this patent is BARUCH S. BLUMBERG INSTITUTE, DREXEL UNIVERSITY. Invention is credited to HAROLD R. ALMOND, TIMOTHY M. BLOCK, JINHONG CHANG, JU-TAO GUO, WILLIAM A. KINNEY.
Application Number | 20180369323 16/115298 |
Document ID | / |
Family ID | 49674087 |
Filed Date | 2018-12-27 |
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United States Patent
Application |
20180369323 |
Kind Code |
A1 |
GUO; JU-TAO ; et
al. |
December 27, 2018 |
MODULATION OF HEPATITIS B VIRUS CCCDNA TRANSCRIPTION
Abstract
The present invention provides small molecule inhibitors of
hepatitis B virus (HBV) covalently closed circular (ccc) DNA, which
are useful as therapeutics in the management of chronic HBV. The
compounds of the invention achieve epigenetic modification of the
cccDNA, histone modification and histone deacetylase activity
inhibition, thus modulating HBV cccDNA. The present invention
further provides methods for modulating HBV cccDNA, for treating or
preventing HBV in a subject, and for modulating cccDNA
transcription of hepatitis B in a subject.
Inventors: |
GUO; JU-TAO; (LANSDALE,
PA) ; CHANG; JINHONG; (CHALFONT, PA) ; BLOCK;
TIMOTHY M.; (DOYLESTOWN, PA) ; KINNEY; WILLIAM
A.; (NEWTOWN, PA) ; ALMOND; HAROLD R.; (MAPLE
GLEN, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
DREXEL UNIVERSITY
BARUCH S. BLUMBERG INSTITUTE |
PHILADELPHIA
DOYLESTOWN |
PA
PA |
US
US |
|
|
Family ID: |
49674087 |
Appl. No.: |
16/115298 |
Filed: |
August 28, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15483711 |
Apr 10, 2017 |
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16115298 |
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14435675 |
Apr 14, 2015 |
9623071 |
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PCT/US13/43691 |
May 31, 2013 |
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15483711 |
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61654374 |
Jun 1, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/20 20180101;
A61K 31/166 20130101; C07K 5/123 20130101; A61K 31/165 20130101;
A61P 1/16 20180101; A61K 31/16 20130101; A61P 43/00 20180101; A61K
45/06 20130101; C07K 5/126 20130101; A61K 38/12 20130101 |
International
Class: |
A61K 38/12 20060101
A61K038/12; A61K 31/16 20060101 A61K031/16; A61K 31/166 20060101
A61K031/166; A61K 45/06 20060101 A61K045/06; C07K 5/12 20060101
C07K005/12; A61K 31/165 20060101 A61K031/165 |
Claims
1. A method of modulating cccDNA transcription of hepatitis B in a
subject comprising administering to said subject an inhibitor of
histone deacetylase activity.
2. The method according to claim 1 wherein said inhibitor of
histone deacetylase activity is an inhibitor of class I histone
deacetylase activity.
3. The method according to claim 1 wherein said inhibitor of
histone deacetylase activity is Trichostatin A, suberoyl bis
hydroxamic acid, dimethylamino hydroxy-benzamide, Apicidin or an
analog thereof, or a compound according to formula (I) ##STR00017##
wherein R.sub.1 is --(CH.sub.2).sub.n-- or --C(.dbd.O)--; R.sub.2
is --C(.dbd.O)--, 3,5-triazolyl, or --C(Z)N(R.sub.4)--; R.sub.4 is
hydrogen, alkyl, aryl, aralkyl, dialkylaminoalkyl, or carboxyalkyl;
R.sub.3 is --CH(R.sub.5)--, or R.sub.2 is nitrogen and R.sub.3 is
--CH-- and R.sub.2 and R.sub.3 together form piperidinyl; R.sub.5
is hydrogen, --CH.sub.3, or an alpha amino acid R group; R.sub.6 is
--(CH.sub.2).sub.mC(X)Y, --(CH.sub.2).sub.2CH.sub.3, or
--(CH.sub.2).sub.q-phenyl-(CH.sub.2).sub.mC(.dbd.O)NHOH; X is
.dbd.O, H.sub.2, .dbd.N--NH.sub.2, or
.dbd.N--NH--C(.dbd.O)NH.sub.2; Y is NHOH or --CH.sub.2CH.sub.3; Z
is H.sub.2 or O; R.sub.7 is hydrogen or alkoxy; R.sub.8 is alkyl or
carboxyalkyl; n is 0-2; m is 0-6; and, q is 0-3; or a stereoisomer
or pharmaceutically acceptable salt thereof.
4. The method according to claim 1 wherein said inhibitor of
histone deacetylase activity is Apicidin, ##STR00018## wherein
R.sub.1 is --(CH.sub.2)--, and, R.sub.2 is --C(Z)N(R.sub.4)--.
5. The method according to claim 3 wherein said inhibitor of
histone deacetylase activity is ##STR00019##
6. The method according to claim 1 further comprising administering
to said subject a therapeutically effective amount of a further
agent that modulates hepatitis B virus.
7. A method of treating hepatitis B in a subject comprising
administering to said subject an inhibitor of histone deacetylase
activity.
8. The method according to claim 7 wherein said inhibitor of
histone deacetylase activity is an inhibitor of class I histone
deacetylase activity.
9. The method according to claim 7 wherein said inhibitor of
histone deacetylase activity is Trichostatin A, suberoyl bis
hydroxamic acid, dimethylamino hydroxy-benzamide, Apicidin or an
analog thereof, or a compound according to formula (I) ##STR00020##
wherein R.sub.1 is --(CH.sub.2).sub.n-- or --C(.dbd.O)--; R.sub.2
is --C(.dbd.O)--, 3,5-triazolyl, or --C(Z)N(R.sub.4)--; R.sub.4 is
hydrogen, alkyl, aryl, aralkyl, dialkylaminoalkyl, or carboxyalkyl;
R.sub.3 is --CH(R.sub.5)--, or R.sub.2 is nitrogen and R.sub.3 is
--CH-- and R.sub.2 and R.sub.3 together form piperidinyl; R.sub.5
is hydrogen, --CH.sub.3, or an alpha amino acid R group; R.sub.6 is
--(CH.sub.2).sub.mC(X)Y, --(CH.sub.2).sub.2CH.sub.3, or
--(CH.sub.2).sub.q-phenyl-(CH.sub.2).sub.mC(.dbd.O)NHOH; X is
.dbd.O, H.sub.2, .dbd.N--NH.sub.2, or
.dbd.N--NH--C(.dbd.O)NH.sub.2; Y is NHOH or --CH.sub.2CH.sub.3; Z
is H.sub.2 or O; R.sub.7 is hydrogen or alkoxy; R.sub.8 is alkyl or
carboxyalkyl; n is 0-2; m is 0-6; and, q is 0-3; or a stereoisomer
or pharmaceutically acceptable salt thereof.
10. The method according to claim 7 wherein said inhibitor of
histone deacetylase activity is Apicidin, ##STR00021## wherein
R.sub.1 is --(CH.sub.2)--, and, R.sub.2 is --C(Z)N(R.sub.4)--.
11. The method according to claim 10 wherein said inhibitor of
histone deacetylase activity is ##STR00022##
12. The method according to claim 7 further comprising
administering to said subject a therapeutically effective amount of
a further agent that modulates hepatitis B virus.
13. A method of modulating hepatitis B virus covalently closed
circular DNA comprising contacting a hepatitis B virus with an
inhibitor of histone deacetylase activity.
14. The method according to claim 13 wherein said inhibitor of
histone deacetylase activity is an inhibitor of class I histone
deacetylase activity.
15. The method according to claim 13 wherein said inhibitor of
histone deacetylase activity is Trichostatin A, suberoyl bis
hydroxamic acid, dimethylamino hydroxy-benzamide, Apicidin or an
analog thereof, or a compound according to formula (I) ##STR00023##
wherein R.sub.1 is --(CH.sub.2).sub.n-- or --C(.dbd.O)--; R.sub.2
is --C(.dbd.O)--, 3,5-triazolyl, or --C(Z)N(R.sub.4)--; R.sub.4 is
hydrogen, alkyl, aryl, aralkyl, dialkylaminoalkyl, or carboxyalkyl;
R.sub.3 is --CH(R.sub.5)--, or R.sub.2 is nitrogen and R.sub.3 is
--CH-- and R.sub.2 and R.sub.3 together form piperidinyl; R.sub.5
is hydrogen, --CH.sub.3, or an alpha amino acid R group; R.sub.6 is
--(CH.sub.2).sub.mC(X)Y, --(CH.sub.2).sub.2CH.sub.3, or
--(CH.sub.2).sub.q-phenyl-(CH.sub.2).sub.mC(.dbd.O)NHOH; X is
.dbd.O, H.sub.2, .dbd.N--NH.sub.2, or
.dbd.N--NH--C(.dbd.O)NH.sub.2; Y is NHOH or --CH.sub.2CH.sub.3; Z
is H.sub.2 or O; R.sub.7 is hydrogen or alkoxy; R.sub.8 is alkyl or
carboxyalkyl; n is 0-2; m is 0-6; and, q is 0-3; or a stereoisomer
or pharmaceutically acceptable salt thereof.
16. The method according to claim 13 wherein said inhibitor of
histone deacetylase activity is Apicidin, ##STR00024## wherein
R.sub.1 is --(CH.sub.2)--, and, R.sub.2 is --C(Z)N(R.sub.4)--.
17. The method according to claim 13 wherein said inhibitor of
histone deacetylase activity is ##STR00025##
18. The method according to claim 13 further comprising contacting
the hepatitis B virus with a further agent that modulates hepatitis
B virus.
19. A compound according to formula II: ##STR00026## wherein
R.sub.1 is --(CH.sub.2).sub.n-- or --C(.dbd.O)--; R.sub.2 is
--C(.dbd.O)-- or --C(Z)N(R.sub.4)--; R.sub.4 is hydrogen, alkyl,
aryl, aralkyl, dialkylaminoalkyl, or carboxyalkyl; R.sub.3 is
--CH(R.sub.5)--; R.sub.5 is hydrogen, --CH.sub.3, or an alpha amino
acid R group; R.sub.6 is --(CH.sub.2).sub.inC(X)Y,
--(CH.sub.2).sub.2CH.sub.3, or
--(CH.sub.2).sub.q-phenyl-(CH.sub.2).sub.mC(.dbd.O)NHOH; X is
.dbd.O, H.sub.2, .dbd.N--NH.sub.2, or
.dbd.N--NH--C(.dbd.O)NH.sub.2; Y is NHOH or --CH.sub.2CH.sub.3; Z
is H.sub.2 or O; R.sub.7 is hydrogen or alkoxy; R.sub.8 is alkyl or
carboxyalkyl; n is 0-2; m is 0-6; and, q is 0-3; or a stereoisomer
or pharmaceutically acceptable salt thereof.
20. The compound according to claim 19 wherein said compound is
##STR00027## wherein R.sub.1 is --(CH.sub.2)--, and, R.sub.2 is
--C(Z)N(R.sub.4)--.
21. The compound according to claim 19 wherein said compound is
##STR00028##
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of, and claims priority
to, U.S. patent application Ser. No. 15/483,711, filed Apr. 10,
2017, now allowed, which is a continuation of, and claims priority
to, U.S. patent application Ser. No. 14/435,675, filed Apr. 14,
2015, now issued as U.S. Pat. No. 9,623,071, which is the U.S.
National Phase application filed under 35 U.S.C. .sctn. 371
claiming priority to PCT International Application No.
PCT/US2013/043691, filed May 31, 2013, which claims priority to
U.S. Provisional Application No. 61/654,374, filed Jun. 1, 2012,
all of which applications are hereby incorporated herein by
reference in their entireties.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 28, 2018, is named 046528-7059US3_SequenceListing_ST25.txt
and is 1 kilobyte in size.
TECHNICAL FIELD
[0003] The present disclosure pertains to the use of
pharmacological agents, preferably with histone deacetylase
activity, for modulating covalently closed circular DNA of
hepatitis B virus, and for preventing or treating hepatitis B.
BACKGROUND
[0004] There are now seven medications approved by the United
States Food & Drug Administration (FDA) for the management of
chronic hepatitis B, which fall into one of two categories: the
interferons (IFNs) and the polymerase inhibitors (Lok, A. S., and
B. J. McMahon. 2007. Chronic Hepatitis B. Hepatology 45:507-539).
These are recommended for use in approximately 50% or less of the
infected population of more than 350 million. Although this is the
highest risk population, those who fall outside the treatment
guidelines may also benefit from intervention, since they are also
at significantly elevated risk of liver diseases. The IFNs are
limited by significant side effects. The pol inhibitors target the
same viral life cycle step and thus combination therapy, the
bulwark of HIV and curative HCV therapy, is of limited value. They
require lifelong use, and are subject to eventual use limiting
toxicities, as seen with HIV long term medication use, and the
emergence of drug resistant mutants. Thus, alternatives and
complements to the current portfolio of medications are needed.
[0005] There is a growing belief that a "cure", or at least
sustained off-drug control of HBV, will require, or at least
benefit from, drugs that control the viral nuclear genome, the
covalently closed circular DNA (cccDNA). The 2006 NIDDK Liver
Action Plan, reinforced by the 2010 Institute of Medicine report,
all call for cccDNA inhibition as a priority for HBV drug
development.
[0006] However, screening for HBV cccDNA inhibitors has been
difficult, because of technical reasons: HBV cccDNA is made in
amounts to low to be conveniently detected, and most viral gene
products in conventionally transfected cells in culture are derived
from transgenes of the viral genome, not cccDNA. The present
inventors have created cell lines in which HBV gene products such
as the HBeAg are produced only from cccDNA, but not from integrated
viral transgene and in amounts to be robustly detected, making
screening realistic (Cai, D., et al., 2012. Identification of the
Disubstituted Sulfonamide Compounds as Specific Inhibitors of
Hepatitis B Virus Covalently Closed Circular DNA Formation.
Antimicrobial Agents and Chemotherapy: In Press; Zhou, T, et al.,
2006. Hepatitis B virus e antigen production is dependent upon
covalently closed circular (ccc) DNA in HepAD38 cell cultures and
may serve as a cccDNA surrogate in antiviral screening assays.
Antiviral Research 72:116-124).
[0007] Given such challenges, it is unsurprising that there are no
HBV therapeutics in use that target HBV cccDNA and, there have been
few, if any, programs to screen and develop cccDNA inhibitors. This
is largely due to technical difficulties (see Block, T M, et al.
2003. Molecular viral oncology of hepatocellular carcinoma.
Oncogene 22:5093-5107; Locarnini, S. 2005. Therapies for hepatitis
B: where to from here? Gastroenterology 128:789-792; Lok, A. S.
2011. Does antiviral therapy for hepatitis B and C prevent
hepatocellular carcinoma? J Gastroenterol Hepatol 26:221-227). In
addition, the role of host functions in regulating HBV cccDNA
transcription and stability is poorly understood further
frustrating development of therapeutics. Thus, any work in this
area would be innovative, and would address the outstanding and
long-felt need for drugs that control the viral nuclear genome of
hepatitis B and otherwise provide treatment for HBV infection.
SUMMARY
[0008] Provided are methods of modulating cccDNA transcription of
hepatitis B in a subject comprising administering to the subject an
agent that provides epigenetic modification of the cccDNA, a
histone modifying agent, or an inhibitor of histone deacetylase
activity. For example, the epigenetic modifying agent, histone
modifying agent, or inhibitor of histone deacetylase activity may
be pharmacological, such as a small molecule.
[0009] Also provided are methods of treating hepatitis B in a
subject comprising administering to the subject an inhibitor of
histone deacetylase activity.
[0010] The present disclosure also pertains to method of modulating
hepatitis B virus covalently closed circular DNA comprising
contacting a hepatitis B virus with an inhibitor of histone
deacetylase activity.
[0011] Also disclosed are compounds according to formula II:
##STR00001##
wherein R.sub.1-R.sub.8 are defined as provided herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 provides data demonstrating that HBV cccDNA is
efficiently formed and transcriptionally active in dstet5
cells.
[0013] FIG. 2 relates to experiments demonstrating that cccDNA can
be inhibited by IFN-.alpha..
[0014] FIG. 3 pertains to the present finding that Apicidin and TSA
repress cccDNA transcription.
[0015] FIG. 4 relates to the discovery that HDAC inhibitors
dose-dependently stimulate DHBV pgRNA synthesis from transgene
integrated in a host cellular chromosome.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0016] The present invention may be understood more readily by
reference to the following detailed description taken in connection
with the accompanying figures and examples, which form a part this
disclosure. It is to be understood that this invention is not
limited to the specific products, methods, conditions or parameters
described and/or shown herein, and that the terminology used herein
is for the purpose of describing particular embodiments by way of
example only and is not intended to be limiting of the claimed
invention.
[0017] The disclosures of each patent, patent application, and
publication cited or described in this document are hereby
incorporated herein by reference, in their entirety.
[0018] As employed above and throughout the disclosure, the
following terms and abbreviations, unless otherwise indicated,
shall be understood to have the following meanings.
[0019] In the present disclosure the singular forms "a," "an," and
"the" include the plural reference, and reference to a particular
numerical value includes at least that particular value, unless the
context clearly indicates otherwise. Thus, for example, a reference
to "a compound" is a reference to one or more of such compounds and
equivalents thereof known to those skilled in the art, and so
forth. Furthermore, when indicating that a certain chemical moiety
"may be" X, Y, or Z, it is not intended by such usage to exclude in
all instances other choices for the moiety; for example, a
statement to the effect that R.sub.1 "may be alkyl, aryl, or amino"
does not necessarily exclude other choices for R.sub.1, such as
halo, aralkyl, and the like.
[0020] When values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment. As used herein, "about X" (where X is a
numerical value) preferably refers to .+-.10% of the recited value,
inclusive. For example, the phrase "about 8" refers to a value of
7.2 to 8.8, inclusive; as another example, the phrase "about 8%"
refers to a value of 7.2% to 8.8%, inclusive. Where present, all
ranges are inclusive and combinable. For example, when a range of
"1 to 5" is recited, the recited range should be construed as
including ranges "1 to 4", "1 to 3", "1-2", "1-2 & 4-5", "1-3
& 5", and the like. In addition, when a list of alternatives is
positively provided, such listing can be interpreted to mean that
any of the alternatives may be excluded, e.g., by a negative
limitation in the claims. For example, when a range of "1 to 5" is
recited, the recited range may be construed as including situations
whereby any of 1, 2, 3, 4, or 5 are negatively excluded; thus, a
recitation of "1 to 5" may be construed as "1 and 3-5, but not 2",
or simply "wherein 2 is not included." In another example, when a
listing of possible substituents including "hydrogen, alkyl, and
aryl" or "hydrogen, alkyl, or aryl" is provided, the recited
listing may be construed as including situations whereby any of
"hydrogen, alkyl, and aryl" or "hydrogen, alkyl, or aryl" is
negatively excluded; thus, a recitation of "hydrogen, alkyl, and
aryl" or "hydrogen, alkyl, and aryl" may be construed as "hydrogen
and/or aryl, but not alkyl", or simply "wherein the substituent is
not alkyl".
[0021] As used herein, the terms "component," "composition of
compounds," "compound," "drug," "pharmacologically active agent,"
"active agent," "therapeutic," "therapy," "treatment," or
"medicament" are used interchangeably herein to refer to a compound
or compounds or composition of matter which, when administered to a
subject (human or animal) induces a desired pharmacological and/or
physiologic effect by local and/or systemic action.
[0022] The abbreviations in the specification correspond to units
of measure, techniques, properties, or compounds as follows: "min"
means minute(s), "g" means gram(s), "mg" means milligram(s),
".mu.g" means microgram(s), "eq" means equivalent(s), "h" means
hour(s), "pt" means microliter(s), "mL" means milliliter(s), "mM"
means millimolar, "M" means molar, "mmol" or "mmole" means
millimole(s), "cm" means centimeters, "SEM" means standard error of
the mean, and "IU" means International Units. "IC.sub.50 value" or
"IC.sub.50" means dose of the compound which results in 50%
alleviation or inhibition of the observed condition or effect.
[0023] "Apicidin" is a compound derived from a Fusarium species
fungal metabolite. It has the structure
cyclo(N--O-methyl-L-tryptophanyl-L-isoleucinyl-D-pipecolinyl-L-2-amino-8--
oxodecanoyl).
[0024] "Natural analogs of Apicidin" refers to analogs of Apicidin
that are produced in fermentations of Fusarium pallidoroseum
species ATCC74322 and ATCC47289 (Apicidins A, B, C, D1, D2, D3,
which are described in JOC 67, 815 (2002) and Tet Lett, 37, 8077
(1996), and in WO 1996/9603428.
[0025] As used herein, "alkyl" refers to an optionally substituted,
saturated straight, or branched, hydrocarbon radical having from
about 1 to about 20 carbon atoms (and all combinations and
subcombinations of ranges and specific numbers of carbon atoms
therein). Where appropriate, "alkyl" can mean "alkylene"; for
example, if X is --R.sub.1R.sub.2, and R.sub.1 is said to be
"alkyl", then "alkyl" may correctly be interpreted to mean
"alkylene".
[0026] "Amino" refers to --NH.sub.2 and may include one or more
substituents that replace hydrogen. "Amino" is used interchangeably
with amine and is also intended to include any pharmaceutically
acceptable amine salts. For example, amino may refer to
--NH.sup.+(X)(Y)Cl.sup.-, wherein X and Y are preferably and
independently hydrogen or alkyl, wherein alkyl may include one or
more halo substitutions.
[0027] As used herein, "aryl", "arene", and "aromatic" each refer
to an optionally substituted, saturated or unsaturated, monocyclic,
polycyclic, or other homo-, carbo- or heterocyclic aromatic ring
system having from about 3 to about 50 ring members (and all
combinations and subcombinations of ranges and specific numbers of
carbon atoms therein), with from about 5 to about 10 ring atom
members being preferred. Such moieties encompass (include)
"heteroaryl" and "heteroarene" as defined infra. Where appropriate,
"aryl" can mean "arene"; for example, if X is --R.sub.1R.sub.2, and
R.sub.1 is said to be "aryl", then "aryl" may correctly be
interpreted to mean "arene".
[0028] As used herein, "alkenyl" refers to an alkyl radical having
from about 2 to about 20 carbon atoms and one or more double bonds
(and all combinations and subcombinations of ranges and specific
numbers of carbon atoms therein), wherein alkyl is as previously
defined. In some embodiments, it is preferred that the alkenyl
groups have from about 2 to about 6 carbon atoms. Alkenyl groups
may be optionally substituted.
[0029] As used herein, "aralkyl" refers to alkyl radicals bearing
one or more aryl substituents and having from about 4 to about 50
carbon atoms (and all combinations and subcombinations of ranges
and specific numbers of carbon atoms therein), wherein aryl and
alkyl are as previously defined. In some preferred embodiments, the
alkyl moieties of the aralkyl groups have from about 1 to about 4
carbon atoms. In other preferred embodiments, the alkyl moieties
have from about 1 to about 3 carbon atoms. Aralkyl groups may be
optionally substituted.
[0030] "Alkylamino" signifies alkyl-(NH)--, wherein alkyl is as
previously described and NH is defined in accordance with the
provided definition of amino. "Arylamino" represents aryl-(NH)--,
wherein aryl is as defined herein and NH is defined in accordance
with the provided definition of amino. Likewise, "aralkylamino" is
used to denote aralkyl-(NH)--, wherein aralkyl is as previously
defined and NH is defined in accordance with the provided
definition of amino. "Alkylamido" refers to alkyl-CH(.dbd.O)NH--,
wherein alkyl is as previously described. "Alkoxy" as used herein
refers to the group R--O-- where R is an alkyl group, and alkyl is
as previously described. "Aralkoxy" stands for R--O--, wherein R is
an aralkyl group as previously defined. "Alkylsulfonyl" means
alkyl-SO.sub.2--, wherein alkyl is as previously defined.
"Aminooxy" as used herein refers to the group amino-(O)--, wherein
amino is defined as above. "Aralkylaminooxy" as used herein is used
to denote aryl-alkyl-aminooxy-, wherein aryl, alkyl, and aminooxy
are respectively defined as provided previously.
[0031] As used herein, "alkylene" refers to an optionally branched
or substituted bivalent alkyl radical having the general formula
--(CH.sub.2).sub.n--, where n is 1 to 10. Non-limiting examples
include methylene, trimethylene, pentamethylene, and
hexamethylene.
[0032] "Alkyleneamino" refers to --(CH.sub.2).sub.n--NH--, where n
is 1 to 10 and wherein the bivalent alkyl radical may be optionally
branched or substituted, and the amino group may include one or
more substituents that replace hydrogen.
[0033] As used herein, "heteroaryl" or "heteroarene" refers to an
aryl radical wherein in at least one of the rings, one or more of
the carbon atom ring members is independently replaced by a
heteroatom group selected from the group consisting of S, O, N, and
NH, wherein aryl is as previously defined. Heteroaryl/heteroarene
groups having a total of from about 3 to about 14 carbon atom ring
members and heteroatom ring members are preferred. Likewise, a
"heterocyclic ring" is an aryl radical wherein one or more of the
carbon atom ring members may be (but are not necessarily)
independently replaced by a heteroatom group selected from the
group consisting of S, O, N, and NH. Heterocyclic rings having a
total from about 3 to 14 ring members and heteroatom ring members
are preferred, but not necessarily present; for example,
"heterocyclohexyl" may be a six-membered aryl radical with or
without a heteroatom group.
[0034] "Halo" and "halogen" each refers to a fluoro, chloro, bromo,
or iodo moiety, with fluoro, chloro, or bromo being preferred.
[0035] "Haloalkyl" signifies halo-alkyl- wherein alkyl and halo,
respectively, are as previously described.
[0036] The phrase reading "[moiety] is absent" may mean that the
substituents to which the moiety is attached are directly attached
to each other.
[0037] Typically, substituted chemical moieties include one or more
substituents that replace hydrogen. Exemplary substituents include,
for example, halo (e.g., F, Cl, Br, I), alkyl, cycloalkyl,
alkylcycloalkyl, cycloalkylalkyl, alkenyl, alkynyl, aralkyl, aryl,
heteroaryl, heteroaralkyl, spiroalkyl, heterocycloalkyl, hydroxyl
(--OH), nitro (--NO.sub.2), cyano (--CN), amino (--NH.sub.2),
--N-substituted amino (--NHR''), --N,N-disubstituted amino
(--N(R'')R''), oxo (.dbd.O), carboxy (--COOH), --O--C(.dbd.O)R'',
--C(.dbd.O)R'', --OR'', --C(.dbd.O)OR'',
-(alkylene)-C(.dbd.O)--OR'', --NHC(.dbd.O)R'', aminocarbonyl
(--C(.dbd.O)NH.sub.2), --N-substituted aminocarbonyl
(--C(.dbd.O)NHR''), --N,N-disubstituted aminocarbonyl
(--C(.dbd.O)N(R'')R''), thiol, thiolato (--SR''), sulfonic acid
(--SO.sub.3H), phosphonic acid (--PO.sub.3H),
--P(.dbd.O)(OR'')OR'', --S(.dbd.O)R'', --S(.dbd.O).sub.2R'',
--S(.dbd.O).sub.2NH.sub.2, --S(.dbd.O).sub.2NHR'',
--S(.dbd.O).sub.2NR''R'', --NHS(.dbd.O).sub.2R'',
--NR''S(.dbd.O).sub.2R'', --CF.sub.3, --CF.sub.2CF.sub.3,
--NHC(.dbd.O)NHR'', --NHC(.dbd.O)NR''R'', --NR''C(.dbd.O)NHR'',
--NR''C(.dbd.O)NR''R'', --NR''C(.dbd.O)R'' and the like. In
relation to the aforementioned substituents, each moiety R'' can
be, independently, any of H, alkyl, cycloalkyl, alkenyl, aryl,
aralkyl, heteroaryl, or heterocycloalkyl, for example.
[0038] As used herein, the terms "treatment" or "therapy" (as well
as different word forms thereof) includes preventative (e.g.,
prophylactic), curative or palliative treatment.
[0039] As employed above and throughout the disclosure the term
"effective amount" refers to an amount effective, at dosages, and
for periods of time necessary, to achieve the desired result with
respect to the treatment of the relevant disorder, condition, or
side effect. It will be appreciated that the effective amount of
components of the present invention will vary from patient to
patient not only with the particular compound, component or
composition selected, the route of administration, and the ability
of the components to elicit a desired response in the individual,
but also with factors such as the disease state or severity of the
condition to be alleviated, hormone levels, age, sex, weight of the
individual, the state of being of the patient, and the severity of
the pathological condition being treated, concurrent medication or
special diets then being followed by the particular patient, and
other factors which those skilled in the art will recognize, with
the appropriate dosage ultimately being at the discretion of the
attendant physician. Dosage regimens may be adjusted to provide the
improved therapeutic response. An effective amount is also one in
which any toxic or detrimental effects of the components are
outweighed by the therapeutically beneficial effects. As an
example, the compounds useful in the methods of the present
invention are administered at a dosage and for a time such that the
level of activation and adhesion activity of platelets is reduced
as compared to the level of activity before the start of
treatment.
[0040] "Pharmaceutically acceptable" refers to those compounds,
materials, compositions, and/or dosage forms which are, within the
scope of sound medical judgment, suitable for contact with the
tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem complications
commensurate with a reasonable benefit/risk ratio.
[0041] Within the present invention, the disclosed compounds may be
prepared in the form of pharmaceutically acceptable salts.
"Pharmaceutically acceptable salts" refer to derivatives of the
disclosed compounds wherein the parent compound is modified by
making acid or base salts thereof. Examples of pharmaceutically
acceptable salts include, but are not limited to, mineral or
organic acid salts of basic residues such as amines; alkali or
organic salts of acidic residues such as carboxylic acids; and the
like. The pharmaceutically acceptable salts include the
conventional non-toxic salts or the quaternary ammonium salts of
the parent compound formed, for example, from non-toxic inorganic
or organic acids. For example, such conventional non-toxic salts
include those derived from inorganic acids such as hydrochloric,
hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like;
and the salts prepared from organic acids such as acetic,
propionic, succinic, glycolic, stearic, lactic, malic, tartaric,
citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic,
glutamic, benzoic, salicylic, sulfanilic, 2-acetoxybenzoic,
fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic,
oxalic, isethionic, and the like. These physiologically acceptable
salts are prepared by methods known in the art, e.g., by dissolving
the free amine bases with an excess of the acid in aqueous alcohol,
or neutralizing a free carboxylic acid with an alkali metal base
such as a hydroxide, or with an amine.
[0042] Compounds described herein throughout, can be used or
prepared in alternate forms. For example, many amino-containing
compounds can be used or prepared as an acid addition salt. Often
such salts improve isolation and handling properties of the
compound. For example, depending on the reagents, reaction
conditions and the like, compounds as described herein can be used
or prepared, for example, as their hydrochloride or tosylate salts.
Isomorphic crystalline forms, all chiral and racemic forms,
N-oxide, hydrates, solvates, and acid salt hydrates, are also
contemplated to be within the scope of the present invention.
[0043] Certain acidic or basic compounds of the present invention
may exist as zwitterions. All forms of the compounds, including
free acid, free base and zwitterions, are contemplated to be within
the scope of the present invention. It is well known in art that
compounds containing both amino and carboxy groups often exist in
equilibrium with their zwitterionic forms. Thus, any of the
compounds described herein throughout that contain, for example,
both amino and carboxy groups, also include reference to their
corresponding zwitterions.
[0044] "Hydrate" refers to a compound of the present invention
which is associated with water in the molecular form, i.e., in
which the H--OH bond is not split, and may be represented, for
example, by the formula R.H.sub.2O, where R is a compound of the
invention. A given compound may form more than one hydrate
including, for example, monohydrates (R.H.sub.2O) or polyhydrates
(R.nH.sub.2O wherein n is an integer >1) including, for example,
dihydrates (R.2H.sub.2O), trihydrates (R.3H.sub.2O), and the like,
or hemihydrates, such as, for example, R.n.sub./2H.sub.2O,
R.n.sub./3H.sub.2O, R.n.sub./4H.sub.2O and the like wherein n is an
integer.
[0045] "Solvate" refers to a compound of the present invention
which is associated with solvent in the molecular form, i.e., in
which the solvent is coordinatively bound, and may be represented,
for example, by the formula R. (solvent), where R is a compound of
the invention. A given compound may form more than one solvate
including, for example, monosolvates (R.(solvent)) or polysolvates
(R.n(solvent)) wherein n is an integer >1) including, for
example, disolvates (R.2(solvent)), trisolvates (R.3(solvent)), and
the like, or hemisolvates, such as, for example,
R.n.sub./2(solvent), R.n.sub./3(solvent), R.n.sub./4(solvent) and
the like wherein n is an integer. Solvents herein include mixed
solvents, for example, methanol/water, and as such, the solvates
may incorporate one or more solvents within the solvate.
[0046] "Acid hydrate" refers to a complex that may be formed
through association of a compound having one or more base moieties
with at least one compound having one or more acid moieties or
through association of a compound having one or more acid moieties
with at least one compound having one or more base moieties, said
complex being further associated with water molecules so as to form
a hydrate, wherein said hydrate is as previously defined and R
represents the complex herein described above.
[0047] The term "stereoisomers" refers to compounds that have
identical chemical constitution, but differ as regards the
arrangement of the atoms or groups in space.
[0048] "Racemic" means having the capacity for resolution into
forms of opposed optical activity.
[0049] As used herein, the term "partial stereoisomer" refers to
stereoisomers having two or more chiral centers wherein at least
one of the chiral centers has defined stereochemistry (i.e., R or
S) and at least one has undefined stereochemistry (i.e., R or 5).
When the term "partial stereoisomers thereof" is used herein, it
refers to any compound within the described genus whose
configuration at chiral centers with defined stereochemistry
centers is maintained and the configuration of each undefined
chiral center is independently selected from R or S. For example,
if a stereoisomer has three chiral centers and the stereochemical
configuration of the first center is defined as having "S"
stereochemistry, the term "or partial stereoisomer thereof" refers
to stereoisomers having SRR, SRS, SSR, or SSS configurations at the
three chiral centers, and mixtures thereof.
[0050] An "isotopically substituted analogue" is a compound of the
present disclosure in which one or more atoms have been replaced
with an isotope of that atom. For example, hydrogen (protium) may
be substituted with deuterium or tritium. Other atoms that may be
replaced with an isotope thereof in order to form an isotopically
substituted analogue thereof include, for example, carbon (replaced
with C.sup.13), nitrogen (replaced with N.sup.15), iodine (replaced
with I.sup.131), fluorine (replaced with F.sup.18), or sulfur
(replaced with S.sup.31). Any available isotope may be used to form
an isotopically substituted analogue thereof, and those of ordinary
skill in the art will recognize available techniques for forming
such analogues from a given compound.
[0051] "Prodrug" refers to compounds which are themselves inactive
or minimally active for the activity desired, but through
biotransformation can be converted into biologically active
metabolites. For example, a prodrug of the present invention would
include, inter alio, any compound which is convertible in vivo by
metabolic means to a compound claimed or described in the present
disclosure.
[0052] "N-oxide" refers to compounds wherein the basic nitrogen
atom of either a heteroaromatic ring or tertiary amine is oxidized
to give a quaternary nitrogen bearing a positive formal charge and
an attached oxygen atom bearing a negative formal charge.
[0053] When any variable occurs more than one time in any
constituent or in any formula, its definition in each occurrence is
independent of its definition at every other occurrence.
Combinations of substituents and/or variables are permissible only
if such combinations result in stable compounds.
[0054] The term "administering" means either directly administering
a compound or composition of the present invention, or
administering a prodrug, derivative or analog which will form an
equivalent amount of the active compound or substance within the
body.
[0055] "Dosage unit" refers to physically discrete units suited as
unitary dosages for the particular individual to be treated. Each
unit may contain a predetermined quantity of active compound(s)
calculated to produce the desired therapeutic effect(s) in
association with the required pharmaceutical carrier. The
specification for the dosage unit forms of the invention may be
dictated by (a) the unique characteristics of the active
compound(s) and the particular therapeutic effect(s) to be
achieved, and (b) the limitations inherent in the art of
compounding such active compound(s).
[0056] "Subject" or "patient" refers to an embryonic, immature, or
adult animal, including the human species, that is treatable with
the compositions, and/or methods of the present invention.
[0057] It has presently been discovered that hepatitis B virus
covalently closed circular DNA (cccDNA), existing and being
expressed as an "episome" in the nucleus of an infected cell, is
regulated differently than HBV DNA integrated in to the host
chromosome, and that RNA expression from the HBV cccDNA can be
pharmacologically suppressed, selectively, as compared to other
genes (as described more fully herein). Indeed, the present
inventors have identified numerous compounds that repress DHBV
cccDNA transcription in a reproducible and robust manner, and that
occurs at low concentrations and under conditions of no apparent
toxicity. These results represent the first time that selective
pharmacological suppression has been achieved, by design, with
small molecules. The result that gene expression from HBV cccDNA is
regulated differently that the same or similar DNA integrated in to
the host chromosomes is surprising and a highly useful observation,
in that it enables therapies that selectively repress cccDNA DNA
(for example, as compared with integrated HBV DNA) without
suppressing or otherwise affecting host chromosomal DNA. The
present finding that HBV cccDNA can be suppressed pharmacologically
was heretofore unknown, and offers the proof of useful concept of
the prior statement, and demonstrates that such pharmacological
suppression is possible.
[0058] Accordingly, the present disclosure provides, inter alia,
methods of modulating cccDNA transcription of hepatitis B in a
subject comprising administering to the subject an agent that
provides epigenetic modification of the cccDNA, a histone modifying
agent, or an inhibitor of histone deacetylase activity. For
example, the epigenetic modifying agent, histone modifying agent,
or inhibitor of histone deacetylase activity may be
pharmacological, such as a small molecule. The epigenetic modifying
agent, histone modifying agent, or inhibitor of histone deacetylase
activity may be selective for the inhibition of cccDNA, as compared
with integrated HBV DNA, i.e., does not inhibit integrated HBV DNA,
and/or as compared with cellular host DNA, i.e., does not inhibit
cellular host DNA. The inhibitor of histone deacetylase activity
may be an inhibitor of multiple classes of histone deacetylase, or
may be selective for a particular class of histone deacetylase. For
example, the inhibitor may be an inhibitor of class I histone
deacetylase activity, class II histone deacetylase activity, or
both. Preferably, the inhibitor of histone deacetylase activity is
an inhibitor of class I histone deacetylase activity. Numerous
inhibitors of histone deacetylase activity are known, and any such
HDAC inhibitor may be used pursuant to the present methods.
[0059] The present methods of modulating cccDNA transcription of
hepatitis B may also include--in addition to the administration to
the subject an agent that provides epigenetic modification of the
cccDNA, a histone modifying agent, or an inhibitor of histone
deacetylase activity--administering to the subject a
therapeutically effective amount of a further agent that modulates
hepatitis B virus. The further agent may be administered
simultaneously with, or simply as a part of the same general
therapy regimen as the agent that provides epigenetic modification
of the cccDNA, histone modifying agent, or inhibitor of histone
deacetylase activity. The further agent may be any substance that
is presently used for modulation of HBV, of which numerous types
are known among those skill in the art. For example, existing drugs
for the modulation of HBV include interferons (e.g., interferon
alpha, pegylated interferon), nucleoside analogues (e.g.,
lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir,
clevudine, amdoxovir), non-nucleoside antivirals (e.g., BAM 205,
ANA380, myrcludex B, HAP Compound Bay 41-4109, REP 9AC,
nitazoxanide, dd-RNAi compound, ARC-520, NVR-1221), non-interferon
immune enhancers (e.g., thymosin alpha-1, interleukin-7, DV-601,
HBV core antigen vaccine, GS-9620, GI13000), and post-exposure
and/or post-liver transplant treatment drugs (e.g., hyperHEP S/D,
Nabi-GB, Hepa Gam B).
[0060] In particular, the further agent may be any other Direct
Acting Antiviral anti hepatitis B agent (such as the polymerase
inhibitors Barraclude, Tenofovir, lamivudine, telbivudine, and
adefovir) and/or any other directing acting antiviral agents that
work at a step in the virus life cycle other than suppression of
cccDNA transcription, such as capsid inhibitors, secretion
inhibitors, or entry inhibitors. The further agent may also be any
other non-direct acting antiviral agent, such as an interferon or
other immunomodulatory agent.
[0061] In accordance with the present methods of modulating cccDNA
transcription of hepatitis B, the inhibitor of histone deacetylase
activity may be, for example, Trichostatin A, suberoyl bis
hydroxamic acid,
4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl]benzamide,
Apicidin, an Apicidin analog (for example, a natural analog of
Apicidin or an analog that is synthesized de novo), or a compound
according to formula (I)
##STR00002##
wherein
[0062] R.sub.1 is --(CH.sub.2).sub.n-- or --C(.dbd.O)--;
[0063] R.sub.2 is --C(.dbd.O)--, 3,5-triazolyl, or
--C(Z)N(R.sub.4)--;
[0064] R.sub.4 is hydrogen, alkyl, aryl, aralkyl,
dialkylaminoalkyl, or carboxyalkyl;
[0065] R.sub.3 is --CH(R.sub.5)--, or R.sub.2 is nitrogen and
R.sub.3 is --CH-- and R.sub.2 and R.sub.3 together form
piperidinyl;
[0066] R.sub.5 is hydrogen, --CH.sub.3, or an alpha amino acid R
group;
[0067] R.sub.6 is --(CH.sub.2).sub.mC(X)Y,
--(CH.sub.2).sub.2CH.sub.3, or
--(CH.sub.2).sub.q-phenyl-(CH.sub.2).sub.mC(.dbd.O)NHOH;
[0068] X is .dbd.O, H.sub.2, .dbd.N--NH.sub.2, or
.dbd.N--NH--C(.dbd.O)NH.sub.2;
[0069] Y is NHOH or --CH.sub.2CH.sub.3;
[0070] Z is H.sub.2 or O;
[0071] R.sub.7 is hydrogen or alkoxy;
[0072] R.sub.8 is alkyl or carboxyalkyl;
[0073] n is 0-2;
[0074] m is 0-6; and,
[0075] q is 0-3;
or a stereoisomer or pharmaceutically acceptable salt thereof.
[0076] As used herein, the phrase "alpha amino acid R group" refers
to a side chain group from a a natural or unnatural amino acid.
[0077] In certain embodiments, the inhibitor of histone deacetylase
activity is Apicidin,
##STR00003## [0078] wherein
[0079] R.sub.1 is --(CH.sub.2)--,
[0080] and,
[0081] R.sub.2 is --C(Z)N(R.sub.4)--
or a stereoisomer or pharmaceutically acceptable salt thereof.
[0082] In other embodiments, the inhibitor of histone deacetylase
activity is
##STR00004##
or a stereoisomer or pharmaceutically acceptable salt thereof.
[0083] The present disclosure also pertains to methods of treating
hepatitis B in a subject comprising administering to the subject an
agent that provides epigenetic modification of the cccDNA, a
histone modifying agent, or an inhibitor of histone deacetylase
activity. For example, the epigenetic modifying agent, histone
modifying agent, or inhibitor of histone deacetylase activity may
be pharmacological, such as a small molecule. The epigenetic
modifying agent, histone modifying agent, or inhibitor of histone
deacetylase activity may be selective for the inhibition of cccDNA,
as compared with integrated HBV DNA, i.e., does not inhibit
integrated HBV DNA, and/or as compared with cellular host DNA,
i.e., does not inhibit cellular host DNA. The inhibitor of histone
deacetylase activity may be an inhibitor of multiple classes of
histone deacetylase, or may be selective for a particular class of
histone deacetylase. For example, the inhibitor may be an inhibitor
of class I histone deacetylase activity, class II histone
deacetylase activity, or both. Preferably, the inhibitor of histone
deacetylase activity is an inhibitor of class I histone deacetylase
activity. Numerous inhibitors of histone deacetylase activity are
known, and any such HDAC inhibitor may be used pursuant to the
present methods.
[0084] In accordance with the present methods of treating hepatitis
B in a subject, the inhibitor of histone deacetylase activity may
be, for example, Trichostatin A, suberoyl bis hydroxamic acid,
4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl] benzamide,
Apicidin, an Apicidin analog (for example, a natural analog of
Apicidin or an analog that is synthesized de novo), or a compound
according to formula (I)
##STR00005##
wherein
[0085] R.sub.1 is --(CH.sub.2).sub.n-- or --C(.dbd.O)--;
[0086] R.sub.2 is --C(.dbd.O)--, 3,5-triazolyl, or
--C(Z)N(R.sub.4)--;
[0087] R.sub.4 is hydrogen, alkyl, aryl, aralkyl,
dialkylaminoalkyl, or carboxyalkyl;
[0088] R.sub.3 is --CH(R.sub.5)--, or R.sub.2 is nitrogen and
R.sub.3 is --CH-- and R.sub.2 and R.sub.3 together form
piperidinyl;
[0089] R.sub.5 is hydrogen, --CH.sub.3, or an alpha amino acid R
group;
[0090] R.sub.6 is --(CH.sub.2).sub.mC(X)Y,
--(CH.sub.2).sub.2CH.sub.3, or
--(CH.sub.2).sub.q-phenyl-(CH.sub.2).sub.mC(.dbd.O)NHOH;
[0091] X is .dbd.O, H.sub.2, .dbd.N--NH.sub.2, or
.dbd.N--NH--C(.dbd.O)NH.sub.2;
[0092] Y is NHOH or --CH.sub.2CH.sub.3;
[0093] Z is H.sub.2 or O;
[0094] R.sub.7 is hydrogen or alkoxy;
[0095] R.sub.8 is alkyl or carboxyalkyl;
[0096] n is 0-2;
[0097] m is 0-6; and,
[0098] q is 0-3;
or a stereoisomer or pharmaceutically acceptable salt thereof.
[0099] In certain embodiments, the inhibitor of histone deacetylase
activity is Apicidin,
##STR00006## [0100] wherein
[0101] R.sub.1 is --(CH.sub.2)--,
[0102] and,
[0103] R.sub.2 is --C(Z)N(R.sub.4)--
or a stereoisomer or pharmaceutically acceptable salt thereof.
[0104] In other embodiments, the inhibitor of histone deacetylase
activity is
##STR00007##
or a stereoisomer or pharmaceutically acceptable salt thereof.
[0105] The present methods of treating hepatitis B in a subject may
also include--in addition to the administration to the subject an
agent that provides epigenetic modification of the cccDNA, a
histone modifying agent, or an inhibitor of histone deacetylase
activity--administering to the subject a therapeutically effective
amount of a further agent that modulates hepatitis B virus. The
further agent may be administered simultaneously with, or simply as
a part of the same general therapy regimen as the agent that
provides epigenetic modification of the cccDNA, histone modifying
agent, or inhibitor of histone deacetylase activity. The further
agent may be any substance that is presently used for modulation of
HBV, of which numerous types are known among those skill in the
art. For example, existing drugs for the modulation of HBV include
interferons (e.g., interferon alpha, pegylated interferon),
nucleoside analogues (e.g., lamivudine, adefovir dipivoxil,
entecavir, telbivudine, tenofovir, clevudine, amdoxovir),
non-nucleoside antivirals (e.g., BAM 205, ANA380, myrcludex B, HAP
Compound Bay 41-4109, REP 9AC, nitazoxanide, dd-RNAi compound,
ARC-520, NVR-1221), non-interferon immune enhancers (e.g., thymosin
alpha-1, interleukin-7, DV-601, HBV core antigen vaccine, GS-9620,
GI13000), and post-exposure and/or post-liver transplant treatment
drugs (e.g., hyperHEP S/D, Nabi-GB, Hepa Gam B).
[0106] In particular, the further agent may be any other Direct
Acting Antiviral anti hepatitis B agent (such as the polymerase
inhibitors Barraclude, Tenofovir, lamivudine, telbivudine, and
adefovir) and/or any other directing acting antiviral agents that
work at a step in the virus life cycle other than suppression of
cccDNA transcription, such as capsid inhibitors, secretion
inhibitors, or entry inhibitors. The further agent may also be any
other non-direct acting antiviral agent, such as an interferon or
other immunomodulatory agent.
[0107] Also disclosed are methods of modulating hepatitis B virus
covalently closed circular DNA comprising contacting a hepatitis B
virus with an agent that provides epigenetic modification of the
cccDNA, a histone modifying agent, or an inhibitor of histone
deacetylase activity. For example, the epigenetic modifying agent,
histone modifying agent, or inhibitor of histone deacetylase
activity may be pharmacological, such as a small molecule. The
epigenetic modifying agent, histone modifying agent, or inhibitor
of histone deacetylase activity may be selective for the inhibition
of cccDNA, as compared with integrated HBV DNA, i.e., does not
inhibit integrated HBV DNA, and/or as compared with cellular host
DNA, i.e., does not inhibit cellular host DNA. The inhibitor of
histone deacetylase activity may be an inhibitor of multiple
classes of histone deacetylase, or may be selective for a
particular class of histone deacetylase. For example, the inhibitor
may be an inhibitor of class I histone deacetylase activity, class
II histone deacetylase activity, or both. Preferably, the inhibitor
of histone deacetylase activity is an inhibitor of class I histone
deacetylase activity. Numerous inhibitors of histone deacetylase
activity are known, and any such HDAC inhibitor may be used
pursuant to the present methods.
[0108] In accordance with the present methods of modulating
hepatitis B virus covalently closed circular DNA, the inhibitor of
histone deacetylase activity may be, for example, Trichostatin A,
suberoyl bis hydroxamic acid,
4-(dimethylamino)-N-[7-(hydroxyamino)-7-oxoheptyl] benzamide,
Apicidin, an Apicidin analog (for example, a natural analog of
Apicidin or an analog that is synthesized de novo), or a compound
according to formula (I)
##STR00008##
wherein
[0109] R.sub.1 is --(CH.sub.2).sub.n-- or --C(.dbd.O)--;
[0110] R.sub.2 is --C(.dbd.O)--, 3,5-triazolyl, or
--C(Z)N(R.sub.4)--;
[0111] R.sub.4 is hydrogen, alkyl, aryl, aralkyl,
dialkylaminoalkyl, or carboxyalkyl;
[0112] R.sub.3 is --CH(R.sub.5)--, or R.sub.2 is nitrogen and
R.sub.3 is --CH-- and R.sub.2 and R.sub.3 together form
piperidinyl;
[0113] R.sub.5 is hydrogen, --CH.sub.3, or an alpha amino acid R
group;
[0114] R.sub.6 is --(CH.sub.2).sub.mC(X)Y,
--(CH.sub.2).sub.2CH.sub.3, or
--(CH.sub.2).sub.q-phenyl-(CH.sub.2).sub.mC(.dbd.O)NHOH;
[0115] X is .dbd.O, H.sub.2, .dbd.N--NH.sub.2, or
.dbd.N--NH--C(.dbd.O)NH.sub.2;
[0116] Y is NHOH or --CH.sub.2CH.sub.3;
[0117] Z is H.sub.2 or O;
[0118] R.sub.7 is hydrogen or alkoxy;
[0119] R.sub.8 is alkyl or carboxyalkyl;
[0120] n is 0-2;
[0121] m is 0-6; and,
[0122] q is 0-3;
or a stereoisomer or pharmaceutically acceptable salt thereof.
[0123] In certain embodiments, the inhibitor of histone deacetylase
activity is Apicidin,
##STR00009## [0124] wherein
[0125] R.sub.1 is --(CH.sub.2)--,
[0126] and,
[0127] R.sub.2 is --C(Z)N(R.sub.4)--
or a stereoisomer or pharmaceutically acceptable salt thereof.
[0128] In other embodiments, the inhibitor of histone deacetylase
activity is
##STR00010##
or a stereoisomer or pharmaceutically acceptable salt thereof.
[0129] The present methods of modulating hepatitis B virus
covalently closed circular DNA may also include--in addition to the
contacting of a hepatitis B virus with an agent that provides
epigenetic modification of the cccDNA, a histone modifying agent,
or an inhibitor of histone deacetylase activity--contacting the
hepatitis B virus with a therapeutically effective amount of a
further agent that modulates hepatitis B virus. The contacting of
the further agent with the HBV may occur simultaneously with, or
simply as a part of the same procedure that involves contacting the
HBV with the agent that provides epigenetic modification of the
cccDNA, histone modifying agent, or inhibitor of histone
deacetylase activity. The further agent may be any substance that
is presently used for modulation of HBV, of which numerous types
are known among those skill in the art. For example, existing drugs
for the modulation of HBV include interferons (e.g., interferon
alpha, pegylated interferon), nucleoside analogues (e.g.,
lamivudine, adefovir dipivoxil, entecavir, telbivudine, tenofovir,
clevudine, amdoxovir), non-nucleoside antivirals (e.g., BAM 205,
ANA380, myrcludex B, HAP Compound Bay 41-4109, REP 9AC,
nitazoxanide, dd-RNAi compound, ARC-520, NVR-1221), non-interferon
immune enhancers (e.g., thymosin alpha-1, interleukin-7, DV-601,
HBV core antigen vaccine, GS-9620, GI13000), and post-exposure
and/or post-liver transplant treatment drugs (e.g., hyperHEP S/D,
Nabi-GB, Hepa Gam B).
[0130] In particular, the further agent may be any other Direct
Acting Antiviral anti hepatitis B agent (such as the polymerase
inhibitors Barraclude, Tenofovir, lamivudine, telbivudine, and
adefovir) and/or any other directing acting antiviral agents that
work at a step in the virus life cycle other than suppression of
cccDNA transcription, such as capsid inhibitors, secretion
inhibitors, or entry inhibitors. The further agent may also be any
other non-direct acting antiviral agent, such as an interferon or
other immunomodulatory agent.
[0131] The present disclosure also pertains to compound according
to formula II:
##STR00011##
wherein
[0132] R.sub.1 is --(CH.sub.2).sub.n-- or --C(.dbd.O)--;
[0133] R.sub.2 is --C(.dbd.O)-- or --C(Z)N(R.sub.4)--;
[0134] R.sub.4 is hydrogen, alkyl, aryl, aralkyl,
dialkylaminoalkyl, or carboxyalkyl;
[0135] R.sub.3 is --CH(R.sub.5)--;
[0136] R.sub.5 is hydrogen, --CH.sub.3, or an alpha amino acid R
group;
[0137] R.sub.6 is --(CH.sub.2).sub.mC(X)Y,
--(CH.sub.2).sub.2CH.sub.3, or
--(CH.sub.2).sub.q-phenyl-(CH.sub.2).sub.mC(.dbd.O)NHOH;
[0138] X is .dbd.O, H.sub.2, .dbd.N--NH.sub.2, or
.dbd.N--NH--C(.dbd.O)NH.sub.2;
[0139] Y is NHOH or --CH.sub.2CH.sub.3;
[0140] Z is H.sub.2 or O;
[0141] R.sub.7 is hydrogen or alkoxy;
[0142] R.sub.8 is alkyl or carboxyalkyl;
[0143] n is 0-2;
[0144] m is 0-6; and,
[0145] q is 0-3;
or a stereoisomer or pharmaceutically acceptable salt thereof,
[0146] For example, the compound may be
##STR00012##
wherein
[0147] R.sub.1 is --(CH.sub.2)--,
[0148] and,
[0149] R.sub.2 is --C(Z)N(R.sub.4)--.
[0150] In other embodiments, the compound may be
##STR00013##
[0151] As will be readily understood, functional groups present may
contain protecting groups during the course of synthesis.
Protecting groups are known per se as chemical functional groups
that can be selectively appended to and removed from
functionalities, such as hydroxyl groups and carboxyl groups. These
groups are present in a chemical compound to render such
functionality in room temperature chemical reaction conditions to
which the compound is exposed. Any of a variety of protecting
groups may be employed with the present invention. Protecting
groups that may be employed in accordance with the present
invention may be described in Greene, T W. and Wuts, P. G. M.,
Protective Groups in Organic Synthesis 2d. Ed., Wiley & Sons,
1991.
[0152] In a further aspect, the present disclosure relates to
pharmaceutical compositions comprising a compound according to
formula (I) or (II), or a pharmaceutically acceptable salt,
isotopically substituted analogue, or stereoisomer thereof and a
pharmaceutically acceptable carrier, diluent, or excipient. The
applicable carrier, diluent, or excipient may be selected on the
basis of the chosen route of administration and standard
pharmaceutical practice as described, for example, in Remington's
Pharmaceutical Sciences (Mack Pub. Co., Easton, Pa., 1985), the
disclosure of which is hereby incorporated by reference in its
entirety. The pharmaceutical compositions may further comprise a
therapeutically effective amount of a further agent that modulates
hepatitis B virus. For example, the further agent that modulates
virus may be a known anti-viral agents. In certain embodiments, the
present compositions comprise a therapeutically effective amount of
a compound according to formula (I) or (II) which is administered
in combination with immunizations or vaccines that are effective in
preventing or lessening the symptoms of HBV. Examples include
antibodies, immune suppressants, anti-inflammatory agents, and the
like.
[0153] As used herein, the term "contacting" refers to the bringing
together into physical or chemical communication of indicated
moieties in an in vitro system or an in vivo system. For example,
"contacting" an HBV virus with a compound in the invention may
include the administration of a compound in the present invention
to an individual or patient, such as a human, having an HBV
infection, as well as, for example, introducing a compound of the
invention into a sample containing a cellular or purified
preparation containing cccDNA.
[0154] As used herein, the term "individual" or "patient," used
interchangeably, refers to any animal, including mammals, such as
mice, rats, other rodents, rabbits, dogs, cats, swine, cattle,
sheep, horses, or primates, such as humans.
[0155] As used herein, the phrase "therapeutically effective
amount" refers to the amount of active compound or pharmaceutical
agent that elicits the biological or medicinal response that is
being sought in a tissue, system, animal, individual or human by a
researcher, veterinarian, medical doctor or other clinician, which
includes one or more of the following:
[0156] (1) preventing the disease; for example, preventing a
disease, condition or disorder in an individual who may be
predisposed to the disease, condition or disorder but does not yet
experience or display the pathology or symptomatology of the
disease;
[0157] (2) inhibiting the disease; for example, inhibiting a
disease, condition or disorder in an individual who is experiencing
or displaying the pathology or symptomatology of the disease,
condition or disorder (i.e., including arresting further
development of the pathology and/or symptomatology); and
[0158] (3) ameliorating the disease; for example, ameliorating a
disease, condition or disorder in an individual who is experiencing
or displaying the pathology or symptomatology of the disease,
condition or disorder (i.e., including reversing the pathology
and/or symptomatology).
[0159] A subject or patient in whom administration of the
therapeutic compound is an effective therapeutic regimen for a
disease or disorder is preferably a human, but can be any animal,
including a laboratory animal in the context of a clinical trial or
screening or activity experiment. Thus, as can be readily
appreciated by one of ordinary skill in the art, the methods,
compounds and compositions of the present invention are
particularly suited to administration to any animal, particularly a
mammal, and including, but by no means limited to, humans, domestic
animals, such as feline or canine subjects, farm animals, such as
but not limited to bovine, equine, caprine, ovine, and porcine
subjects, wild animals (whether in the wild or in a zoological
garden), research animals, such as mice, rats, rabbits, goats,
sheep, pigs, dogs, cats, and the like, avian species, such as
chickens, turkeys, songbirds, and the like, i.e., for veterinary
medical use.
[0160] The compounds of this invention may be administered orally
or parenterally, neat or in combination with conventional
pharmaceutical carriers, diluents, or excipients, which may be
liquid or solid. The applicable solid carrier, diluent, or
excipient may function as, among other things, a binder,
disintegrant, filler, lubricant, glidant, compression aid,
processing aid, color, sweetener, preservative,
suspensing/dispersing agent, tablet-disintegrating agent,
encapsulating material, film former or coating, flavors, or
printing ink. Of course, any material used in preparing any dosage
unit form is preferably pharmaceutically pure and substantially
non-toxic in the amounts employed. In addition, the active compound
may be incorporated into sustained-release preparations and
formulations. Parenteral administration in this respect includes
administration by, inter alia, the following routes: intravenous,
intramuscular, subcutaneous, intraocular, intrasynovial,
transepithelial including transdermal, ophthalmic, sublingual and
buccal; topically including ophthalmic, dermal, ocular, rectal and
nasal inhalation via insufflation, aerosol, and rectal
systemic.
[0161] In powders, the carrier, diluent, or excipient may be a
finely divided solid that is in admixture with the finely divided
active ingredient. In tablets, the active ingredient is mixed with
a carrier, diluent or excipient having the necessary compression
properties in suitable proportions and compacted in the shape and
size desired. For oral therapeutic administration, the active
compound may be incorporated with the carrier, diluent, or
excipient and used in the form of ingestible tablets, buccal
tablets, troches, capsules, elixirs, suspensions, syrups, wafers,
and the like. The amount of active compound(s) in such
therapeutically useful compositions is preferably such that a
suitable dosage will be obtained. The therapeutic compositions
preferably contain up to about 99% of the active ingredient.
[0162] Liquid carriers, diluents, or excipients may be used in
preparing solutions, suspensions, emulsions, syrups, elixirs, and
the like. The active ingredient of this invention can be dissolved
or suspended in a pharmaceutically acceptable liquid such as water,
an organic solvent, a mixture of both, or pharmaceutically
acceptable oils or fat. The liquid carrier, excipient, or diluent
can contain other suitable pharmaceutical additives such as
solubilizers, emulsifiers, buffers, preservatives, sweeteners,
flavoring agents, suspending agents, thickening agents, colors,
viscosity regulators, stabilizers, or osmo-regulators.
[0163] Suitable solid carriers, diluents, and excipients may
include, for example, calcium phosphate, silicon dioxide, magnesium
stearate, talc, sugars, lactose, dextrin, starch, gelatin,
cellulose, methyl cellulose, ethylcellulose, sodium carboxymethyl
cellulose, microcrystalline cellulose, polyvinylpyrrolidine, low
melting waxes, ion exchange resins, croscarmellose carbon, acacia,
pregelatinized starch, crospovidone, HPMC, povidone, titanium
dioxide, polycrystalline cellulose, aluminum methahydroxide,
agar-agar, tragacanth, or mixtures thereof.
[0164] Suitable examples of liquid carriers, diluents and
excipients for oral and parenteral administration include water
(particularly containing additives as above, e.g. cellulose
derivatives, preferably sodium carboxymethyl cellulose solution),
alcohols (including monohydric alcohols and polyhydric alcohols,
e.g. glycols) and their derivatives, and oils (e.g. fractionated
coconut oil and arachis oil), or mixtures thereof.
[0165] For parenteral administration, the carrier, diluent, or
excipient can also be an oily ester such as ethyl oleate and
isopropyl myristate. Also contemplated are sterile liquid carriers,
diluents, or excipients, which are used in sterile liquid form
compositions for parenteral administration. Solutions of the active
compounds as free bases or pharmacologically acceptable salts can
be prepared in water suitably mixed with a surfactant, such as
hydroxypropylcellulose. A dispersion can also be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations may contain a preservative to prevent the growth of
microorganisms.
[0166] The pharmaceutical forms suitable for injectable use
include, for example, sterile aqueous solutions or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. In all cases, the form is
preferably sterile and fluid to provide easy syringability. It is
preferably stable under the conditions of manufacture and storage
and is preferably preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier, diluent, or
excipient may be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol, propylene
glycol, liquid polyethylene glycol and the like), suitable mixtures
thereof, and vegetable oils. The proper fluidity can be maintained,
for example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of a
dispersion, and by the use of surfactants. The prevention of the
action of microorganisms may be achieved by various antibacterial
and antifungal agents, for example, parabens, chlorobutanol,
phenol, sorbic acid, thimerosal and the like. In many cases, it
will be preferable to include isotonic agents, for example, sugars
or sodium chloride. Prolonged absorption of the injectable
compositions may be achieved by the use of agents delaying
absorption, for example, aluminum monostearate and gelatin.
[0167] Sterile injectable solutions may be prepared by
incorporating the active compounds in the required amounts, in the
appropriate solvent, with various of the other ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions may be prepared by incorporating the
sterilized active ingredient into a sterile vehicle which contains
the basic dispersion medium and the required other ingredients from
those enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation may include vacuum drying and the freeze drying
technique that yields a powder of the active ingredient or
ingredients, plus any additional desired ingredient from the
previously sterile-filtered solution thereof.
[0168] The compounds of the invention may be administered in an
effective amount by any of the conventional techniques
well-established in the medical field. The compounds employed in
the methods of the present invention including the compounds of
formulas (I) or (II) may be administered by any means that results
in the contact of the active agents with the agents' site or sites
of action in the body of a patient. The compounds may be
administered by any conventional means available.
[0169] Preferably the pharmaceutical composition is in unit dosage
form, e.g. as tablets, buccal tablets, troches, capsules, elixirs,
powders, solutions, suspensions, emulsions, syrups, wafers,
granules, suppositories, or the like. In such form, the composition
is sub-divided in unit dose containing appropriate quantities of
the active ingredient; the unit dosage forms can be packaged
compositions, for example packeted powders, vials, ampoules,
prefilled syringes or sachets containing liquids. The unit dosage
form can be, for example, a capsule or tablet itself, or it can be
the appropriate number of any such compositions in package form. In
addition, dosage forms of the present invention can be in the form
of capsules wherein one active ingredient is compressed into a
tablet or in the form of a plurality of microtablets, particles,
granules or non-perils. These microtablets, particles, granules or
non-perils are then placed into a capsule or compressed into a
capsule, possibly along with a granulation of the another active
ingredient.
[0170] The dosage of the compounds of the present invention that
will be most suitable for prophylaxis or treatment will vary with
the form of administration, the particular compound chosen and the
physiological characteristics of the particular patient under
treatment. Generally, small dosages may be used initially and, if
necessary, increased by small increments until the desired effect
under the circumstances is reached. Generally speaking, oral
administration may require higher dosages.
[0171] The desired dose may conveniently be presented in a single
dose or as divided doses administered at appropriate intervals, for
example, as two, three, four or more sub-doses per day. The
sub-dose itself may be further divided, e.g., into a number of
discrete loosely spaced administrations. The dose may also be
provided by controlled release of the compound, by techniques well
known to those in the art.
[0172] Additional information regarding the preparation of the
present compounds for administration and the formulation of
compositions according to the present invention is provided
infra.
[0173] The compounds useful in the methods of the present invention
may be prepared in a number of ways well known to those skilled in
the art. The compounds can be synthesized, for example, by the
methods as described below, or variations thereon as appreciated by
the skilled artisan. The reagents used in the preparation of the
compounds of this invention can be either commercially obtained or
can be prepared by standard procedures described in the literature.
All processes disclosed in association with the present invention
are contemplated to be practiced on any scale, including milligram,
gram, multigram, kilogram, multikilogram or commercial industrial
scale.
[0174] For compounds herein in which a variable appears more than
once, each variable can be a different moiety selected from the
Markush group defining the variable. For example, where a structure
is described having two R groups that are simultaneously present on
the same compound, the two R groups can represent different
moieties selected from the Markush group defined for R.
[0175] It is further appreciated that certain features of the
invention, which are, for clarity, described in the context of
separate embodiments, can also be provided in combination in a
single embodiment. Conversely, various features of the invention
which are, for brevity, described in the context of a single
embodiment, can also be provided separately or in any suitable
subcombination.
[0176] The present invention is further described in the following
Examples. It should be understood that these examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only, and should not be construed as limiting the
appended claims. From the above discussion and these examples, one
skilled in the art can ascertain the essential characteristics of
this invention, and without departing from the spirit and scope
thereof, can make various changes and modifications of the
invention to adapt it to various usages and conditions.
EXAMPLES
[0177] Modulation of HBV cccDNA
[0178] DHBV cccDNA in LMH derived dstet5 cells is efficiently
produced and transcriptionally active. Most HBV producing cells
lines produce HBV gene products from an HBV transgene integrated
into the host chromosome, and thus cccDNA is not the major source
of viral product. This makes screening for drugs that target cccDNA
difficult. Cell lines were produced in which viral gene products
are dependent upon cccDNA. It was established human Hep G2 and
chicken hepatoma (LMH)-stable cell lines for this purpose with
tetracycline (tet) regulated HBV/DHBV. As shown in FIG. 1, after
culture in the absence of tet and presence of 2 mM of foscarnet
(PFA) to block viral reverse transcription, DHBV RNAs accumulate,
but DHBV replication is arrested at the stage of pgRNA-containing
nucleocapsids (lane 0). Upon addition of tet back to media to block
transgene transcription, and removal of PFA to allow the viral DNA
synthesis in the pgRNA-containing capsid to proceed, there is a
rapid decline of viral RNA (day 1 and 2), with an eventual increase
to a higher level when cccDNA is made after day 3.
[0179] These results imply that cccDNA is efficiently formed and
transcriptionally functional in dstet5 cells. These results are
more thoroughly demonstrated in FIG. 2, where, under the conditions
specified in which the transgene transcription is blocked with tet,
appearance of new HBV RNA is closely associated with appearance of
cccDNA (FIG. 2 Group II, core DNA shown), whereas, viral
transcripts are rapidly degraded (1/2 life .about.3 hrs) in cells
in which both cccDNA synthesis and new transgene transcription is
blocked (FIG. 2, Group I).
[0180] Identification of Compounds that Potently Repress DHBV
cccDNA Transcription.
[0181] With a system and conditions under which viral transcripts
are produced in a cccDNA dependent manner (FIGS. 1, 2B),
approximately 100 compounds were screened, including those from the
inventors' in-house small compound library, those present in the
inventors' Natural Products collection, and selected compounds
including inhibitors of cellular epigenetic modification enzymes,
including HDACs, HATs, Sirtuins, histone methyltransferases,
histone demethylases and DNA methyltransferases. Numerous
compounds, including the four compounds shown in Table 1,
significantly reduced the amounts of cccDNA-derived DHBV pgRNA. All
possess HDAC class I inhibitory activity.
TABLE-US-00001 TABLE 1 Compounds that repress HBV cccDNA function
and their activity against HDACs.sup.1 4-(dimethylamino)- N-[7-
(hydroxyamino)-7- Suberoyl bis oxo- Trichostatin hydroxamic
heptyl]benzamide Hit Apicin A acid (SBHA) (M344) HBV 0.183 0.480
2.50 6.25 cccDNA (EC.sub.50, uM) Toxicity >20.00 >20.00
>40.00 >100.00 (CC.sub.50, uM).sup.2 Selectivity >100
>40 >16 >16 Index (SI).sup.3 HDAC-I YES.sup.4,5
YES.sup.5,6,9 YES.sup.4,6,7,10 YES.sup.4,7 inhibitor? HDAC-II
NO.sup.3,7 YES.sup.4,5,6,9 YES & YES & inhibitor? HDAC
III.sup.11 HDAC III.sup.7,11 .sup.1Compounds found to suppress HBV
cccDNA function in the dstet5 system, described in Prelim Evid., as
illustrated in FIG. 2. .sup.2Toxicity from our assays on dstet5
cells, as in text; Selectivity Index (SI) is the toxicity CC.sub.50
divided by the Effectiveness EC50, see text. .sup.3Selectivity
Index (SI) is concentration that reduces 50% of cell viability
(CC.sub.50) divided by the concentration that reduces 50% of the
HBV specific signal (RNA and/or HBeAg) (EC.sub.50). .sup.4(7);
.sup.5 (15)(26); .sup.6(45); .sup.7Reaction Biology Monograph;
.sup.8(33); .sup.9(13); .sup.10(14); .sup.11(38)
Structures
##STR00014##
[0182] As shown in FIG. 3, since Apicidin potently inhibited cccDNA
(EC50.about.180 nM), with no toxicity at up to 20 uM for five days,
and has nanomolar activity against class I but not class II HDACs,
it appears that HDAC II inhibition is not necessary to suppress HBV
in this system.
[0183] Apicidin and TSA Repress HBV cccDNA Transcription.
[0184] FIG. 3 shows that Apicidin and TSA repress cccDNA
transcription in Dstet5 cells. Evidence was also obtained
demonstrating that these compounds also repress HBV cccDNA
transcription in the HepG2 cells. In marked contrast, it was
observed that Apicidin and TSA dose-dependently stimulate DHBV
pgRNA transcription from transgene integrated in host cellular
chromosome (FIG. 4). This is more typical of cellular gene
responses to HDAC inhibitions and suggests that unlike chromosomal
DNA, transcription from cccDNA "minichromosomes" are regulated
differently. Moreover, there is even evidence that cccDNA levels
were reduced, indicating, as seen in the Duck system, that
transcriptional repression is followed by destabilization.
Effect of Compounds Upon HBV cccDNA Transcription in Human Hepatoma
Cells.
[0185] HepDE19 cells are seeded into 6-well plates, cultured in the
presence of tetracycline until confluence. Tet is removed from the
culture media to allow pgRNA transcription, DNA synthesis and
cccDNA formation to occur. Tet is added back to culture media to
shut off transgene transcription. After day 3, the cells, in
different wells, are left untreated or treated varying
concentrations (i.e., 0.1 to 10.0 uM) of each of the "Test"
compounds (four "hits" from Table 1 and .about.20 analogues) for 2
days. Intracellular HBV cccDNA, viral RNA and core DNA are
quantified by Southern/Northern blot hybridization assays as
described above and in known procedures. Intracellular full-length
HBeAg precursor and secreted HBeAg are quantified with Western blot
and ELISA assays respectively. HepG2.2.15 cells are used as a
control, because all HBV expression is primarily from the HBV
transgene in these cells. Interferon alpha, which has been shown to
inhibit cccDNA transcription, and disubstituted-sulfonamides (DSS)
CCC-0975, which inhibits cccDNA formation (from our screen, Guo
2012) will be included as positive drug controls. In some
experiments, cultures are maintained for varying times (days) after
removal of "Test" drug from the culture medium, to determine the
durability of any drug induced repression of HBV cccDNA. In order
to determine the selectivity of the testing compounds on cccDNA
transcription, effects of the testing compounds on the expression
of a panel of cellular genes, including, but not limited to, alpha1
antitrypsin, albumin, are also measured by quantitative RT-PCR or
Northern blot hybridization. The cytotoxicity of the compounds are
determined by MTT assay in parallel cultures.
[0186] The amount (0-100%) of reduction of HBeAg and HBV
transcripts is taken as a measure of HBV cccDNA transcriptional
repression. The amount (0-100%) of HBV cccDNA reduction is taken as
a measure of destabilization and degradation of HBV cccDNA. The
amount (0-100%) of repression of A1AT and/or albumin mRNA reduction
is taken as a measure of cellular function inhibition in
specificity determination. The amount of MTT (0-100%) activity is
taken as a measure of cell viability and the basis of cell
cytoxicity (CC). The Selectivity Index (SI) is as in the Table 1
legend.
[0187] Effect of Compounds Upon WHV cccDNA Transcription in Primary
Woodchuck Hepatocytes.
[0188] Woodchuck Hepatitis Virus (WHV) has been a useful model for
evaluating therapeutics for HBV. Therefore, it is useful to know,
for planning, if the lead compounds are active against WHV. Primary
woodchuck hepatocytes cultures (PWHCs) are prepared by plating
collagenase treated tissues, derived from small section biopsies
obtained from chronically infected woodchucks, under conditions
where 50-90% of the hepatocytes harbor WHV, and the cultures (90%
or greater) are hepatocytes and can be maintained for at least two
months in culture, as done previously (see Fletcher S P, et al.
2012. Transcriptomic analysis of the woodchuck model of chronic
hepatitis B. Hepatology: In press). Within 7 days of seeding,
cultures are incubated in the absence or presence of test
compounds, and the amount of WHV gene product in the culture medium
(WHV virion associated DNA; WHs) and intracellularly (WHV DNA, WHV
RNA transcripts) are determined, using the similar methods as
previously used and published (Guo, H., et al. 2010. Production and
function of the cytoplasmic deproteinized relaxed circular DNA of
hepadnaviruses. J Virol 84: 387-396; Guo, et al. 2011. Alkylated
porphyrins have broad antiviral activity against hepadnaviruses,
Flaviviruses, filoviruses, and arenaviruses. Antimicrob Agents
Chemother 55:478-486)
[0189] Where WHV is sensitive (as suspected) to Apicidin and other
candidate cccDNA inhibitors, at SI's similar to that in the avian
and human systems, then chronically infected woodchucks are used
for an in vivo proof of efficacy study.
[0190] The inhibitors are ranked (i) by their selectivity index
(SI), with the most selective in inhibiting HBV cccDNA
transcription versus cellular viability, and then cellular
function, being the most attractive; (ii) by their potency of
inhibiting cccDNA transcription (lowest EC50) and finally, (iii) by
"critical chemistry" (scalability type/formulation) issues. The
compounds with the lowest values of EC.sub.50 (concentration that
inhibits 50% of the cccDNA-transcribed RNA) and greatest SIs, are
the most attractive.
Identification of HDAC Isoform
[0191] Each of the compounds identified in the primary screen share
the property of having HDAC inhibitory activity (see, e.g., Table
1). It is likely that HDAC inhibition is either part of, or central
to, the mechanism of the HBV antiviral action of these compounds.
Although it is not necessary to precisely know the compound's
mechanism, this information would be helpful in selecting or
designing modified compounds, as well as in forecasting and
reducing possible in vivo toxicities, and designing clinical
studies. Also, since crystal structures are available for many
HDACs, future drug design can be assisted. Taken together, the
growing experience with HDAC inhibitors in research and in people,
can could provide direction for the present clinical designs and
future plans.
[0192] HDACs deacetylate polypeptides (i.e., histones) and are
classified into four categories, based on function and DNA sequence
homology. Class I and II HDACs are inhibited by trichostatin A
(TSA). Apicidin efficiently inhibits class I, but not class II
HDACs. Class III HDACs, called sirtuins, are a family of
NAD+-dependent proteins, not affected by TSA. Class IV is
considered an atypical category, based on DNA sequence. Because
Apicidin and TSA potently inhibited cccDNA transcription, HDAC
isozymes in classes I and II are most relevant. However, since
Apicidin inhibits only Class I, the initial focus is only on this
class.
[0193] Experimental Details: Silencing of HDAC isozyme transcripts
with shRNA and the effect upon HBV cccDNA function. Short hairpin
RNAs (shRNAs) expressed from lentivirus transduction vectors are
now standard tools to repress translation of the transcripts to
which the shRNA is homologous. A focus is placed on the class I
HDAC isozymes. Therefore, confluent monolayers of HepDE19 cells
expressing HBV gene products in a cccDNA dependent manner (as
described supra, after tet repression) are transduced with 100 ul
of lentivirus (.about.5.times.10.sup.7/ml) in transduction mixture,
expressing shRNA selective for class I-1,2,3 or 8 isozymes under
conditions where at least 95% of the cells receive and express the
shRNA. This is determined by expression of reporter from the
retroviral transgene. The shRNA lentiviral expression vectors are
provided by the vendor as transduction ready, and each vector
targets different HDACs of sub-class I. They are purchased from
vendors (e.g., Santa Cruz Bio, OpenBio), and contain and express
short hairpins with 19-25 nts homologous to each HDAC isozyme
transcript to be targeted. For example, one HDAC 1 specific shRNA
contains 5'-GAT CCC CGC AGA (SEQ ID NO: 1) . . . ATC TGC TTT TTG
GAA A (SEQ ID NO: 2)-3', and others are similarly designed but
specific for the other shRNAs, as provided by vendor and from
previous work. There is 4- and 5-fold coverage for each HDAC
isozyme. Control vectors contain scrambled sequences, and are used
as negative controls.
[0194] After 5 days of shRNA lentiviral transduction, repression of
the specific HDAC is quantified by RNA analysis and western blot
(with HDAC specific probes and monoclonal antibodies provided by
vendor), and the amount of HBV cccDNA and cccDNA dependent
transcript, and HBeAg, is measured as was performed in preceding
description. Where transient transduction approaches are
unsatisfactory, although a bit more involved, stable transductions
are used, since shRNA constructs, with selectable markers are used.
The amount of cellular gene expression (A1AT and albumin mRNA) are
also quantified, as described in above, as a specificity control.
Positive controls include incubation of the HepDE19 cells with
Apicidin at 1000 nM, a concentration which represses HBV cccDNA
(and will have been validated on HepDE19). Each of the different
HDACs has been associated with specific cellular functions (i.e. 1,
up regulation of p53, 2 & 3 p21, 8, deacetylation of H4) which
are quantified as evidence of successful HDAC sub class inhibition,
should that be desired.
[0195] Given the potency of Apicidin, it is expected that silencing
at least one of the Class I HDACs will result in significant
repression of HBV cccDNA function. It is recognized that the HDAC
inhibitors, which act on multiple HDACs, may have a greater effect
than can be achieved by a single HDAC transcript knock down.
However, we knock down experiments are also performed using
lentivirus combinations covering all class I enzymes, since this
should repress HBV cccDNA if class I enzymes are involved (as
Apicidin suggests), but multiple enzymes must be repressed to
detect the HBV cccDNA inhibition.
[0196] Where silencing a specific or group of HDAC transcripts
results in repression of HBV cccDNA function, this is validated as
a target for HBV antiviral action, and corroborated the finding
that a mechanism of anti-HBV cccDNA action of the identified
compounds involves HDAC inhibition. The compounds could, of course,
use other mechanisms for HBV cccDNA suppression, but it will at
least be known that HDAC inhibition does repress HBV cccDNA, and
the door is now open for this new class of HBV therapeutic
strategies.
[0197] Determining which of the Identified Compounds have the
Greatest Inhibitory Effect Upon the HDACs Isozymes Responsible for
HBV cccDNA Repression.
[0198] Having identified specific HDAC isozymes that are
responsible for regulating HBV cccDNA, it is useful to identify the
compounds that have the greatest selectivity for inhibiting the HBV
cccDNA regulating isozyme. This allows for advancing the compounds
with the greatest selectivity and help avoid off target effects
resulting from needlessly inhibiting HDAC isozymes that are not
involved in regulating HBV cccDNA. We note that of the four
compounds denoted above in Table 1, Apicidin has the greatest
selectivity index, and is also the one with the narrowest HDAC
inhibitory profile (selective for HDAC class I). Therefore, it is
possible to achieve even greater selectivity by avoiding broad HDAC
inhibitors and zooming in on the specific HDAC sub-isozyme that is
sufficient to repress HBV cccDNA.
[0199] Enzyme assays for each of the HDAC class I (1,2,3,8)
isozymes are available as commercial kits, with positive and
negative competitive inhibitor controls. Kits are purchased
corresponding to the relevant isozyme as identified above, from
BioTeK, BPS Bioscience, or other available sources. Briefly, with
the BioTek system, sub class specific purified HDAC enzyme
(recombinant, at .about.10-50 ng/vessel) is provided, with a
fluorogenic substrate, detected following deacetylation, with
developer in a premixed reaction. The enzymes that were shown by
silencing to be involved in HBV cccDNA repression are purchased.
Varying amounts of control or each of the experimental compounds
are incubated with the enzyme reaction mix
[0200] The assay read-out is optimized for linearity both as a
function of time and enzyme concentration. Kits from the
concentrations of the testing compounds required to inhibit 50% of
the deacetylase activity of an HDAC isoform (i.e. IC50) are
calculated by regression analysis using SigmaPlot software (Systat
Software, Inc., San Jose, Calif.).
[0201] Ideally, and most logically, compounds found to be active
according to the procedures described above are active against
HDACs found to be most involved in HBV cccDNA regulation, and these
represent the favored compounds. Compounds that are active but
broadly inhibit HDACs, some of which are found to be irrelevant to
HBV cccDNA regulation, are somewhat less favored, since they may
bring unnecessary side effects. Where, on the other hand, there is
a disconnect, and the compounds active in the preceding assays do
not inhibit the HDACs found to be most important to HBV cccDNA
regulation, the compounds are advanced based on HBV cccDNA
suppressive activity, and not HDAC inhibitory ranking.
Evaluation of Lead Compounds for their In Vitro Absorption,
Distribution, Metabolism and Toxicity (ADMET) Properties HBV
Producing Cells and Non Producing Cells
[0202] Introduction and Rationale
[0203] In vivo experiments are expensive and ethically constrained.
Before testing in animals, it is therefore prudent to initially
profile compounds for potential toxicity and other
cell-serum--interactive properties that are, to the extent
possible, predictive of in vivo performance. These studies have
become standards in the field. Toxicity in replicating cells has
also been found to be a good way to rank compounds with respect to
toxicity. Finally, differing formulations are also usually
necessary, before moving on to in vivo work, because solvents used
in the tissue culture setting are not always compatible with in
vivo administration. These are used, as below. An innovation in in
vitro "ADMET" is presently proposed, in which the profiling is
carried out with HBV producing cells in the presence of a currently
approved antiviral therapies, in addition to the routine ADMET.
[0204] It is likely that new anti-HBV drugs, will be used in
combination with the other HBV antiviral drugs, in current use.
Combination therapy is standard for HIV and HCV and other
infectious diseases. It is important to know if a new drug to treat
HBV has toxicities or other altered profiles in the presence of the
current standards of care, since there is evidence that many
otherwise well tolerated medications have selective toxicities in
chronically infected individuals. HBV producing cells may be more
sensitive to some emdications than are non producing cells (Block,
in progress). Therefore, the toxicity experiments, below, are
carried out in the absence as well as the presence of HBV
polymerase inhibitors and, in some cases, interferon alpha
(IFNa).
[0205] Some of the present lead compounds may have already been
used in animals (by others), there may be considerable information
available. On the other hand, some of the leads may be new
compounds for which there is no animal data. Compound profiles are
also examined in the context of HBV infection, for the reason
stated above.
[0206] Finally, compounds that suppress wild type HBV cccDNA
function and are well tolerated in vitro are tested for their
ability to suppress cccDNA from HBV that is resistant to HBV
polymerase inhibitors. Depending on the results of the preceding
studies, human and/or duck HBV transfection (and for the duck,
infection) systems are used.
[0207] For every experiment described below, controls with known
toxicity, metabolism, protein permeability, membrane transport and
defined formulation properties are included. For example,
Barraclude and FIAU are included as controls for compounds that
have no detectable toxicity in HBV producing cells, and those that
do, respectively, and have reported PK and TK properties for which
comparisons can be made.
[0208] Experimental Detail: In Vitro "Administration, Distribution,
Metabolism, "Elimination" and Toxicity" (ADMET) Studies.
[0209] Some of these experiments are carried out under contract by
a Vendor (i.e. Absorption Systems) and others, particularly where
HBV producing cells and material are used, are carried out by the
present inventors, as indicated, below.
[0210] Standard Cytoxicity Assays:
[0211] Human hepatoma (HepG2, Huh7, HepRG) and HepG2-derived cell
lines supporting constitutive (HepG2.2.15) and
tetracycline-inducible HBV replication (HepDE19 and HepDES19) are
seeded into 96-well plates at a density of 2.times.10.sup.4 cells
per well. Cells are treated with a serial dilution of testing
compounds. The culture media is changed every other day. MTT assays
are performed at day 2, 4, 6, 8 and 10 day since treatment.
[0212] Toxicity to Multiplying Cells:
[0213] Varying concentrations of lead compound(s) are incubated
with HepRG cells seeded at low density (100 cells per well of 32 mm
dish) under HBV producing and non producing conditions, and
cultured for 10 days, with media changes every 3 days.
[0214] Metabolic Stability in Human and Mouse Liver Microsomes:
[0215] The compounds are incubated with human and mouse liver
microsomes from HBV producing and non producing cells (tissue
culture source as above) in the presence of NADPH. In addition, the
stability of compounds are evaluated in the presence of human
simulated gastric fluid and simulated intestinal fluid. The purpose
of this set of experiments is also to determine if the compounds
are metabolized by the digestive enzymes. Since orally available
compounds are pursued, it is important to find out what
metabolites, if any, might be produced in the GI tract.
[0216] The toxicity and metabolic stability studies are carried out
in the absence and presence of concentrations of lamivudine,
barraclude, telbivudine, tenofovir and/or adefovir that are equal
to and multiples (.about.0.1 ug/ml, for barraclude, -10 ug/ml for
lamivudine) or interferon alpha (IFNa) of the serum levels
typically achieved in people. The cccDNA suppressive test compounds
are used at 10 times their IC50, as determined in assays described
above. Control compounds (with established toxicities and
established metabolic profiles) are also included with each panel
of tests (i.e. FIAU, statins, etc).
[0217] Plasma Protein Binding:
[0218] Equilibrium dialysis is used in this assay to determine the
percentage of compound that binds to human plasma proteins (by
Vendor).
[0219] Bidirectional Permeability:
[0220] This assay is used to determine the permeability of
compounds through Caco-2 cell monolayers in the
apical-to-basolateral and basolateral-to-apical direction.
(Contractor)
[0221] Antiviral Activity of Lead Compounds in the Presence of
Interferons (IFNs).
[0222] The experiments above explore the in vitro ADMET of the lead
compounds when used in combination with polymerase inhibitors or
interferons in uninfected cells. It is also important to determine
if the lead compounds have an impact upon an established antiviral
agent's antiviral properties. Compared with pol inhibitors, IFN
alpha (a) is less frequently used to manage HBV. When used, it is
only for a period of months, unlike pol inhibitors, which are used
for years and more likely to be co-administered with a cccDNA
inhibitor. However, given the fact that IFNa mechanisms of
antiviral action and toxicities may involve HDACs, it does make
sense to evaluate the presently disclosed cccDNA inhibitors for
their interaction profiles with IFNa, to the extent this can be
evaluated in vitro. Therefore, the dSTET cells and AD38 cells
programmed to produce transcripts from HBV cccDNA (as in prelim
evidence and Cai 2012) seeded at cloning densities (for growth
studies) and semi confluence (for antiviral/cccDNA transcription
studies) are incubated in the absence and presence of varying
concentrations of candidate cccDNA inhibitor and the absence and
presence of amounts of either avian IFN or human IFNa known to
suppress HBV in vitro. Cell viability and the amount of HBV cccDNA
derived gene products (transcripts) produced are determined as in
previously described procedures and those known in the
literature.
[0223] The compounds are also tested for in vitro activity in the
presence of the currently used polymerase inhibitors. The emergence
of mutant viruses resistant to the nucleoside/tide inhibitors of
the HBV polymerase is a problem in the management of chronic
infection, although the problem varies with the polymerase
inhibitor used. Thus, compounds that suppress wild type HBV cccDNA
function are tested for their ability to suppress cccDNA from HBV
that is resistant to HBV polymerase inhibitors. All of the mutant
viruses (DHBV and WHV) needed are available. Human and/or Duck HBV
transfection (and for the Duck, infection) systems are used. Given
the distinct mechanism of action, the present compounds retain
antiviral activity.
[0224] Formulation Optimization:
[0225] For selected compounds, dosing vehicle development suitable
for oral gavage are evaluated. The test vehicles include 1) pH
manipulation, 2) co-solvents (such as glycin, polyethylene glycol
propylene glycol, ethanol etc), 3) surfactants (such as
polysorbates, polozamer, polyoxyl castor oil, glyceryl and PEG
esters), 4) Non-aqueous systems (such as sesame oil, medium chain
triglycerides, soybean oil, oleic acid), 5) complexing agents (such
as cyclodextrins).
[0226] From an ADMET perspective, preferred are compounds that have
properties similar to Barraclude, with respect to tolerability.
Also preferred are compounds that have the same toxicity and
metabolic stability profiles in the absence of HBV polymerase
inhibitors (lamivudine, barraclude, interferon etc) as in their
presence. Compounds with selective toxicity to HBV producing cells
are disfavored, disqualified, or advanced with extra caution.
Compounds that have enhanced, or enhance, the toxicity of current
HBV antivirals, or antagonize the antiviral, activity those
compounds, are still advanced, but with caution and tested in in
vivo experiments for the possibility of enhanced toxicity in
combination. It is possible to propose that the cccDNA active
compounds not be used (or only used cautiously) in combination.
Lead Compounds with Favorable In Vitro Properties are Scaled Up and
Tested for In Vivo Toxicity, Pharmaco Kinetics (PK) and
Efficacy
[0227] Pharmacokinetic, Toxico-Kinetic (TK), and Dose Range Finding
Studies.
[0228] Prior to conducting in vivo efficacy studies, which are
expensive, ethically constrained, and consume great amounts of
compound, it is necessary to determine the maximum tolerated doses
(MTDs) and pharmacokinetic properties (PK) of the candidate drugs,
in vivo, in uninfected animals. This permits the identification of
compounds worthy of advancement and establish proper dosing and
routes of administration. Compounds are tested for efficacy in
either (or both) duck and/or woodchuck models of chronic
hepadnavirus infection, since these are the established and
predictive animal models. The rationale for duck versus woodchuck
is described below. Regarding Apicidin itself, a great deal will
already be known about its PK/TK in animals, since it has already
been used in mice. However, even for Apicidin, and certainly for
any other of the present compounds, new PK, TK for the Duck and
woodchuck study are needed. Therefore, a series of murine and rat
PK and TK studies are conducted as follows.
[0229] Experimental Detail--
[0230] Single Dose Pharmacokinetic Study in Mice, Ducks and, if
Indicated, Woodchucks.
[0231] The objective of this study is to obtain volume of
distribution, systemic clearance, half-life (T1/2), maximal plasma
concentration (Cmax) and bioavailability. These parameters are used
to evaluate the clearance and bioavailability of each imino sugars
so that the compounds can be ranked by their ability to maintain
plasma concentration. In general, greater than 50% bioavailability
is preferred for compounds to be advanced.
[0232] As described above, candidates are administered via i.v.
injection (5 mg/kg) or given orally (25 mg/kg) to mice (6 week old
Balb/c; 6 mice/group); Peking Ducks (6 week old) or woodchucks (3
per group). Clinical observations are recorded at several intervals
after dosing. Blood and urine samples for pharmacokinetics are
collected predose, and at 5, 15, and 30 min, 1, 2, 4, 6, 8, 16 and
24 h post-dose. Samples are analyzed for the presence and amounts
of administered drug (drug or prodrug) and in the case of
administered prodrug, for the presence and amount of "drug"
metabolite" as well. The samples are analyzed by Absorption
Systems, who has established mouse plasma assays for our other
compounds.
[0233] Tissue Distribution (Murine).
[0234] Tissue is taken from mice (3 per dose group) receiving a
single oral or iv administration of compound at various times after
administration. Knowledge of the tissue distribution of a compound
can significantly aid in evaluating potential as successful drug
candidate. Although other in vitro parameters, such as plasma
protein binding and volume of distribution have prediction values
for rate and extent of distribution to extravascular tissues, the
liver tissue concentration of drug is probably most relevant to
efficacy. A focus is maintained on liver, in comparison to serum,
kidney and abdominal fat tissue/lymph nodes, for tissue
concentration of candidates, using endpoint samples, following the
single administration of the compound by an i.p. and oral route in
mice. One point of interest is if active compound builds up in key
tissue, which provides insights regarding its effective half life,
in tissue. That is, although the serum half life of a drug might be
.about.2 hours, it could have a tissue half life in liver several
fold times that, explaining a greater than expected efficacy (for a
given dosing regimen), or greater than expected toxicity.
[0235] Dose-Finding Maximum Tolerated Dose (NTD) Study.
[0236] Since the compounds are evaluated for antiviral activity in
murine models, it is important to know the tolerability of the
compounds in mice. Balb/c mice (6 week old, 6 per group); Ducks (6
week old, 3 per group) will be dosed by oral gavage (since we are
pursuing orally available compounds) either "vehicle" alone, or
vehicle in which compound has been dissolved. From previous
experience, the range of compound administered is likely between
100 mg/kg to 500 mg/kg, 5 mice per dose group. Animals will be
observed for up to 14 days, with daily readings of weight and an
endpoint of survivability. Routine histology and clinical chemistry
studies are be performed. The highest dose of compound that does
not result in any mortality/toxicity is considered to be the MTD.
Woodchucks can not be used for this MTD study; extrapolations from
the murine study, combined with the PK woodchuck study will be
necessary.
[0237] The compounds are ranked for their oral bioavialibility,
tolerability, and half lives. The ideal compound is able to reach
and sustain serum or liver tissue levels at least 10 times the
tissue culture IC50 concentration, with soluble, oral, single day
dosing, and have MTDs more than 100 times that of the tissue
culture IC50. Compounds are ranked with respect to these qualities,
and the best and second best will be advanced.
[0238] Is the Lead Compound Efficacious in Chronically Infected
Animal Models, in Vivo?
[0239] Having demonstrated in vitro efficacy, and determined safe
and rationale dosing for in vivo work, it is be important to know
if the lead compounds can control viral levels in validated animal
models of chronic HBV. This represents the first time a small
molecule drug that targets cccDNA will have been tested in animals.
Outcomes consistent with a safe, selective and cccDNA targeting
agent are of interest. Efficacy end points include: rapid and
coordinated reduction in viremia, antigenemia as well as amount of
intra-liver cccDNA and replicative forms which would be indicative
of cccDNA suppression. These goals dictate the animal models that
are used, and length of treatment that is studied.
[0240] Several animal models of chronic HBV infection exist, and
each has virtues as well as disadvantages. Ducks and woodchucks can
be experimentally chronically infected with duck and woodchuck
hepadnavirus, respectively. There are now several murine models,
but since transgenic mice bearing HBV transgenes do not produce HBV
from cccDNA templates, to test a cccDNA targeting compound, a
chrimeric mouse with human hepatocytes would be necessary, such as
the uPA mice. Practical considerations require making a choice.
Experiments are designed for evaluation in the Duck model of
chronic HBV, since the compounds are active against the Duck virus
in avian cells in culture are already known. Studies in the
chronically infected woodchuck are also prepared, since this is an
established model for testing HBV therapeutics and is a natural
infection. The uPA mice are very expensive but will are if
woodchucks are not sensitive to the drugs, but human HBV is.
[0241] Therefore, preferred compounds are scaled up to the amount
necessary and tested for efficacy, as defined below, in the
following Duck, and if appropriate, woodchucks.
[0242] Experimental Detail--Scale Up Production of Preferred
Compounds.
[0243] Apicidins are produced in fermentations by Fusarium (i.e.
sp. ATCC 74322). The strain is inoculated into a nutrient medium
called MED5, shaken at 220 rpm, for 12-16 days in a controlled
humidity atmosphere. At harvest, whole broth is extracted with
methylethylketone and the extract is fractionated by gel filtration
on Sephadex followed by final purification by RP-HPLC. Yields are
on the order of 250 mg/L so scale up to gram amounts are
routine.
[0244] Duck Hepadnavirus Efficacy Study.
[0245] Since it is known that Apicidin is highly active against the
DHBV, in culture, it is tested in a chronically infected duck. The
goal of this study is to determine the antiviral potential of
preferred compounds. Serology and histology are secondary.
[0246] Six-week-old Peking Ducks, chronically infected with DHBV
type 16 (Alberta Strain), are used. At 6 weeks, viremia and liver
mass in ducks tends to have stabilized. Ducks are given, by either
i.m. or oral gavage (depending on Aim 4 PK/TK results), test
compound (3 dose groups, with dosing amount and frequency to depend
on PK results, but aiming to achieve stable serum levels of at
least 10 times the tissue culture IC50). There are three dose
groups with 5-6 animals per dose group. Control dose groups (6
animals each group) include placebo treated animals and animals
treated with either barraclude (1 mg/kg) or lamivudine (40 mg/kg)
per day. At least three animals from all dose groups contribute at
least one pre treatment and one post treatment liver biopsy.
Treatment is for 10 weeks, since this exceeds the time for
lamivudine to suppress viremia to beneath detectable levels and the
reported 1/2 life of cccDNA in the duck. Ducks are followed with
weekly serum collections for an additional 4 weeks after withdrawal
of drug. Serum will be collected weekly.
[0247] Weekly serum is tested for standard "lab values"
(hematology, albumin, AST, ALTs, The amount of DHBV viral DNA, sAg,
sAb in the circulation is determined. Liver tissue derived from
biopsies (some pre treatment and end of treatment from the same
animals) is examined for DHBV DNA (cccDNA, replicative forms) and
DHBV core (immunostained).
[0248] WHV-Infected Woodchuck Study.
[0249] The study uses 10 groups, with 5 animals per group, with
drug treatment for 10 weeks followed by 10 weeks off drug (to test
durability of affect). Due to variability in the levels of viremia
and antigenemia, animals are stratified to groups by WHV viremia
and antigenemia levels as determined seven days prior to study
start, so that the average levels of both viral markers are evenly
distributed among all groups of animals. Animals with abnormally
low WHVsAg levels are not used in this study. Compound is
administered daily, by a route and frequency to be finalized after
bioavailability studies in rodents. The first day of dosing on the
study is Study Day 1. Study Day 1 dose levels are calculated on a
pretest body weight, and body weights are taken weekly for dose
administration. Dosing range is as for the mouse study over four
doses, with Group 10 treated with Barraclude as a reference
compound (Tennant).
[0250] The primary endpoint is a dose dependent reduction in
viremia and antigenemia on and off drug achieving durable off drug
reductions.
[0251] Viability and Animal Health.
[0252] Clinical observations are performed and recorded once daily
for morbidity and mortality. Further toxicology is addressed via
hematology, serum chemistry, and histology examination. It is also
important to consider all biochemical and immunological endpoints
in the context of general animal health to insure that decreases in
viremia or antigenemia or other putative beneficial outcomes are
not a secondary consequence protocol (compound) toxicity. Gross
physical characteristics (weight, stool and urine output and
characterization, are determined on a weekly basis. In addition,
liver function tests (performed on samples collected monthly),
hematology and chemistry (performed on pre, mid and end of
treatment samples (as described in the table) and, for selected
animals (at predose, mid dose and end of treatment times),
histology on punch biopsy derived liver sections are also performed
for assessment of toxicity as well as efficacy
[0253] Liver function test are determined by commercial service in
the monthly samples as a marker of liver viability
[0254] Evidence of Humoral Responsiveness.
[0255] The presence of antibodies that recognize WHsAg are
determined by an ELISA. This assay is such that even WHs Abs
complexed with antigen are detected.
[0256] Toxicology.
[0257] Careful toxicology is carried out via hematology and serum
chemistry as described for the mouse studies. In addition,
histological examination of the punch biopsies of the livers is
undertaken, including inflammation, bile duct proliferation, and
portal and lobular hepatitis.
[0258] WHV Virus Levels in the Serum.
[0259] An assessment is performed on weekly (as slot blot
hybridization and PCR or bi-monthly (southern blot).
[0260] Biopsies.
[0261] Liver biopsies are collected before the start, middle, end
of treatment, and end of study and used for histology and
intracellular WHV DNA examination. Levels of replicative form and
intrahepatic covalently-closed circular WHV DNA (WHV cccDNA) are
quantitatively determined based on Hirt extraction. For
immunostaining, separate tissue is used and accumulation of core
and WHsAg in treated versus untreated animals will be
determined.
[0262] For both the Duck and woodchuck studies, no technical
difficulties are expected, since these studies are fairly routine,
with all methods and reagents needed for evaluation being in hand.
One possible problem with Ducks is the variations in
viremia/antigenemia that occur without drug. This is mitigated by
using Ducks after 6 weeks of age, in which virology as usually
stabilized.
[0263] The benchmark of positive activity is LFMAU treated animals.
These animals are expected to have uniformly lost HBV viremia and
even antigenemia, by 3 and 10 weeks of treatment, in the Duck and
Woodchuck, respectively, with numbers of HBV infected hepatocytes
greatly reduced, relative to pretreatment and untreated groups.
[0264] Inhibition of cccDNA transcription (and stability) should
reduce the intracellular and extracellular amounts of all viral
gene products (at a rate influenced by their serum half lives),
even before there are reductions in the numbers of HBV infected
cells (and possibly, out of proportion to the number of HBV
infected cell loss). Realistically, the clearest evidence of
efficacy of our new compounds is time and dose dependent statically
significant reductions in HBV DNA viremia and sAg antigenemia.
Given the efficacy of the present compounds, in vitro, an at least
a ten-fold reduction of serum surface antigen in either or both
models is expected.
[0265] DHBsAgWHsAb levels are also measured. Control, chronically
infected animals are expected to have no detectable (or very little
detectable) Ag. There is a growing body of evidence that
chronically infected people (and woodchucks) are capable, and do
make, sAb, but it is suppressed or bound with circulating sAg. It
is therefore possible that if and as Ag declines, sAb will declare
itself.
[0266] Biopsy analysis is performed on immunostained for HBV core,
sAg, using mounted liver tissue, and with extracts to examine the
amounts of HBV nucleic acid, before and after treatment. Ideally,
the numbers of infected cells will decline as a function of drug
treatment. Useful information includes whether this occurs in a
setting of increased hepatitis (cell infiltration).
[0267] Serum from animals for 10 & 4 weeks (Woodchuck and Duck,
respectively) is also evaluated after drug treatment has been
stopped. Stable, off drug, repression of antigenemia, viremia, with
appearance of sAbs is considered the obtaining all major
objectives. On drug suppression of viremia and antigenemia by
amounts exceeding placebo, in the absence of any adverse reactions
or events, is considered proof of a drug specific affect.
[0268] The animal studies outlined above permit definitive
conclusions as to whether the compounds are effective at reducing
antigenemia in an in vivo context.
[0269] Where inhibition of an HDAC is determined to repress HBV
cccDNA transcription, the results are as surprising as they are
useful, since HDAC inhibition has generally been associated with
gene activation, including HBV DNA integrated into host
chromosomes. The results may represent an example of how different
is the regulation of HBV cccDNA from most cellular genes and, even
if the inhibitors identified herein are not ultimately used in
human systems, it is demonstrated that it is possible to
non-catalytically inhibit cccDNA with small, pharmacologically,
active compounds.
[0270] Taken together, this work delivers two very critical
answers. First, it indicates the selective suppression of HBV
cccDNA function in human and woodchuck cultures. Second, it
determines which HDAC (the target of Apicidin) regulate HBV cccDNA.
We understand that HDAC inhibition in HBV infected people must
proceed with caution, and this work represents direction regarding
how to go proceed with a revolutionary new therapeutic
strategy.
General Synthesis
[0271] The compounds of this invention can be prepared from readily
available starting materials using the following general methods
and procedures. It will be appreciated that where typical or
suitable process conditions (i.e., reaction temperatures, times,
mole ratios of reactants, solvents, pressures, etc.) are given,
other process conditions can also be used unless otherwise stated.
Optimum reaction conditions may vary with the particular reactants
or solvent used, but such conditions can be determined by one
skilled in the art by routine optimization procedures.
[0272] The processes described herein can be monitored according to
any suitable method known in the art. For example, product
formation can be monitored by spectroscopic means, such as nuclear
magnetic resonance spectroscopy (e.g., .sup.1H or .sup.13C NMR),
infrared spectroscopy (IR), spectrophotometry (e.g., UV-visible),
or mass spectrometry, or by chromatography such as high performance
liquid chromatography (HPLC) or thin layer chromatography.
[0273] Preparation of compounds can involve the protection and
deprotection of various chemical groups. The need for protection
and deprotection, and the selection of appropriate protecting
groups can be readily determined by one skilled in the art. The
chemistry of protecting groups can be found, for example, in P. G.
M. Wuts and T. Greene, Greene's Protective Groups in Organic
Synthesis, 4th. Ed., Wiley & Sons, 2006, which is incorporated
herein by reference in its entirety.
[0274] The reactions of the processes described herein can be
carried out in suitable solvents which can be readily selected by
one of skill in the art of organic synthesis. Suitable solvents can
be substantially nonreactive with the starting materials
(reactants), the intermediates, or products at the temperatures at
which the reactions are carried out, i.e., temperatures which can
range from the solvent's freezing temperature to the solvent's
boiling temperature. A given reaction can be carried out in one
solvent or a mixture of more than one solvent. Depending on the
particular reaction step, suitable solvents for a particular
reaction step can be selected. The compounds of the invention can
be prepared, for example, using the reaction pathways and
techniques as described below.
Compound Synthesis
[0275] Apicidins have been derivitized and recent analogs 1 and
2
##STR00015##
(see Horne, W. S., C. A. Olsen, J. M Beierle, A. Montero, and M. R.
Ghadiri. 2009. Probing the bioactive conformation of an archetypal
natural product HDAC inhibitor with conformationally homogeneous
triazole-modified cyclic tetrapeptides. Angew Chem Int Ed Engl
48:4718-4724; Vickers, C. J., C. A. Olsen, L. J. Leman, and M. R.
Ghadiri. 2012. Discovery of HDAC Inhibitors That Lack an Active
Site Zn2+-Binding Functional Group. ACS Medicinal Chemistry
Letters) demonstrate that the Apicidin structure can be modified
without loss of anti HDAC potency.
[0276] Further analogs were prepared with a focus on improving
pharmaceutical properties relative to Apicidin, which has very poor
aqueous solubility, oral bioavailability, and half life in vivo.
Apicidin derivatives were prepared, inter alia, by standard solid
and solution phase methods. In certain embodiments, the reduced
beta-isoleucine amino acid derived fragments 4
##STR00016##
were prepared in a suitably protected form (PG=suitable protecting
group, such as Fmoc or Boc) by solution phase methods and
introduced into the amino acid sequence by solution or solid phase
means, followed by cyclization using established methods.
Sequence CWU 1
1
2112DNAArtificial SequenceChemically synthesized 1gatccccgca ga
12216DNAArtificial SequenceChemically synthesized 2atctgctttt
tggaaa 16
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