Title:
COMBINATION THERAPY FOR TREATMENT OF HBV INFECTIONS
Kind Code:
A1


Abstract:
Provided herein is a combination therapy comprising a compound of Formula I and peginterferon alfa-2a, or another interferon analog. The combination therapy is useful for the treatment of HBV infection. Also provided herein are compositions comprising a compound of Formula I and peginterferon alfa-2a, or another interferon analog.



Inventors:
Hartman, George D. (Lansdale, PA, US)
Application Number:
14/615292
Publication Date:
08/06/2015
Filing Date:
02/05/2015
Assignee:
NOVIRA THERAPEUTICS INC.
Primary Class:
Other Classes:
424/85.7, 424/85.6
International Classes:
A61K38/21; A61K31/445
View Patent Images:



Other References:
Marcellin et al., NEJM, 2004, vol. 351, no. 12, pages 1206-1217
Lau et al., NEJM, 2005, vol. 352, no. 26, pages 2682-2695
Primary Examiner:
SPRINGER, STEPHANIE K
Attorney, Agent or Firm:
Lathrop GPM LLP (Boston, MA, US)
Claims:
1. A method of treating an HBV infection in a subject in need thereof, comprising administering to the subject a capsid assembly inhibitor and an interferon.

2. The method of claim 1, wherein the interferon is selected from the group consisting of interferon alpha, interferon alpha-2a, recombinant interferon alpha-2a, peginterferon-alpha 2a, interferon alpha-2b, recombinant interferon alpha-2b, interferon alpha-2b XL, peginterferon alpha-2b, glycosylated interferon alpha-2b, interferon alpha-2c, recombinant interferon alpha-2c, interferon beta, interferon beta-la, peginterferon beta-1a, interferon delta, interferon lambda, peginterferon lambda-1, interferon omega, interferon tau, gamma interferon, interferon alfacon-1, interferon alpha-n1, interferon alpha-n3,albinterferon alpha-2b, BLX-883, DA-3021, PEG-Infergen, and BELEROFON.

3. The method of claim 2, wherein the interferon is selected from the group consisting of peginterferon alpha-2a, peginterferon alpha-2b, glycosylated interferon alpha-2b, peginterferon beta-1a, and peginterferon lambda-1.

4. The method of claim 3, wherein the interferon is peginterferon alpha-2a.

5. The method of claim 1, wherein the capsid assembly inhibitor is a compound of Formula I: embedded image or a pharmaceutically acceptable salt thereof; wherein R4 is H or C1-C6 alkyl; wherein each R5 is independently selected at each occurrence from the group consisting of CH3, C1-C6 alkoxy, halo, —CN, —NO2, —(L)m—SR9, —(L)m—S(═O)R9, —(L)m—S(═O)2R9, —(L)m—NHS(═O)2R9, —(L)m—C(═O)R9, —(L)m—OC(═O)R9, —(L)mCO2R8, —(L)m—OCO2R8, —(L)m—N(R8)2, —(L)—C(═O)N(R8)2, —(L)m—OC(═O)N(R8)2, —(L)—NHC(═O)NH(R8), —(L)m—NHC(═O)R9, —(L)m—NHC(═O) OR9, —(L)m—C(OH)(R8)2, —(L)mC(NF12)(R8)2, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl and —C1-C6 trihaloalkyl; L is independently, at each occurrence, a bivalent radical selected from —(C1-C3 alkylene)-, —(C3-C7 cycloalkylene)-, —(C1-C3 alkylene)m—O—(C1-C3 alkylene)m—, or —(C1-C3 alkylene)m—NH—(C1-C3 alkylene)m—; each R8 is independently, at each occurrence, H, C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, aryl, heteroaryl, —C1-C4 alkyl-(C3-C10 cycloalkyl), —C1-C4 alkyl-(C3-C10 heterocycloalkyl), —C1-C4 alkyl-(aryl), or —C1-C4 alkyl(heteroaryl), and wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl is optionally substituted with 1-5 substituents selected from R2; R9 is C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, a C3-C10 heterocycloalkyl, aryl, heteroaryl, —C1-C4 alkyl-(C3-C10 cycloalkyl), —C1-C4 alkyl-(C3-C10 heterocycloalkyl), —C1-C4 alkyl-(aryl), or —C1-C4 alkyl-(heteroaryl), and wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring is optionally substituted with 0-5 substituents selected from R2; R10 is OH, C1-C6 alkyl, C1-C6 alkyl-OH, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, a C3-C10 heterocycloalkyl, aryl, heteroaryl, —C1-C4 alkyl-(C3-C10 cycloalkyl), —C1-C4 alkyl-(C3-C10 heterocycloalkyl), —C1-C4 alkyl-(aryl), or —C1-C4 alkyl-(heteroaryl), and wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring is optionally substituted with 1-5 substituents selected from R2; R11 is a bond or C1-C3 alkylene, wherein the C1-C3 alkylene is optionally substituted with 1-3 substituents selected from R2; R2 is independently selected at each occurrence from the group consisting of OH, halo, —CN, —NO2, —C1-C6 alkyl, —C1-C6 alkoxy, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, —C1-C6 heteroalkyl, and C(O)—C1-C6 alkyl; w is 0, 1 or 2; each occurrence of x is independently selected from the group consisting of 0, 1, 2, 3 and 4; each occurrence of y is independently selected from the group consisting of 1, 2, and 3; each occurrence of z is independently selected from the group consisting of 0, 1, 2, and 3; each occurrence of m is independently 0, 1 or 2.

6. A method of treating an HBV infection in a subject in need thereof, comprising administering to the subject peginterferon alfa-2a and a compound of Formula I: embedded image or a pharmaceutically acceptable salt thereof; wherein R4 is H or C1-C6 alkyl; wherein each R5 is independently selected at each occurrence from the group consisting of CH3, C1-C6 alkoxy, halo, —CN, —NO2, —(L)m—SR9, —(L)m—S(═O)R9, —(L)m—S(═O)2R9, —(L)m—NHS(═O)2R9, —(L)m—C(═O)R9, —(L)m—OC(═O)R9, —(L)mCO2R8, —(L)m—OCO2R8, —(L)m—N(R8)2, (L)m—C(═O)N(R8)2, —(L)m—OC(═O)N(R8)2, —(L)m—NHC(═O)NH(R8), —(L)m—NHC(═O)R9, —(L)m—NHC (═O)OR9, —(L)m—C(OH)(R8)2, —(L)mC(NH2)(R8)2, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl and —C1-C6 trihaloalkyl; L is independently, at each occurrence, a bivalent radical selected from —(C1-C3 alkylene)-, —(C3-C7 cyclo alkylene)-, —(C1-C3 alkylene)m—O—(C1-C3 alkylene)m-, or —(C1-C3 alkylene)m—NH—(C1-C3 alkylene)m-; each R8 is independently, at each occurrence, H, C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, aryl, heteroaryl, —C1-C4 alkyl-(C3-C10 cycloalkyl), —C1-C4 alkyl-(C3-C10 heterocycloalkyl), —C1-C4 alkyl-(aryl), or —C1-C4 alkyl(heteroaryl), and wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl is optionally substituted with 1-5 substituents selected from R2; R9 is C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, a C3-C10 heterocycloalkyl, aryl, heteroaryl, —C1-C4 alkyl-(C3-C10 cycloalkyl), —C1-C4 alkyl-(C3-C10 heterocycloalkyl), —C1-C4 alkyl-(aryl), or —C1-C4 alkyl- (heteroaryl), and wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring is optionally substituted with 0-5 substituents selected from R2; R1° is OH, C1-C6 alkyl, C1-C6 alkyl-OH, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, a C3-C10 heterocycloalkyl, aryl, heteroaryl, —C1-C4 alkyl-(C3-C10 cycloalkyl), —C1-C4 alkyl-(C3-C10 heterocycloalkyl), —C1-C4 alkyl-(aryl), or —C1-C4 alkyl-(heteroaryl), and wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring is optionally substituted with 1-5 substituents selected from R2; R11 is a bond or Cl-C3 alkylene, wherein the Cl-C3 alkylene is optionally substituted with 1-3 substituents selected from R2; R2 is independently selected at each occurrence from the group consisting of OH, halo, —CN, —NO2, —C1-C6 alkyl, —C1-C6 alkoxy, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, —C1-C6 heteroalkyl, and C(O)—C1-C6 alkyl; w is 0, 1 or 2; each occurrence of x is independently selected from the group consisting of 0, 1, 2, 3 and 4; each occurrence of y is independently selected from the group consisting of 1, 2, and 3; each occurrence of z is independently selected from the group consisting of 0, 1, 2, and 3; each occurrence of m is independently 0, 1 or 2.

7. The method of claim 6, wherein the peginterferon alfa-2a and compound of Formula I are in a single formulation or unit dosage form.

8. The method of claim 7, further comprising a pharmaceutically acceptable carrier.

9. The method of claim 6, wherein the peginterferon alfa-2a and compound of Formula I are administered separately.

10. The method of claim 6, wherein the subject is human.

11. The method of claim 6, wherein the treatment comprises administering the peginterferon alfa-2a and compound of Formula I at substantially the same time.

12. The method of claim 6, wherein the treatment comprises administering the peginterferon alfa-2a and compound of Formula I at different times.

13. The method of claim 12, wherein the peginterferon alfa-2a is administered to the subject, followed by administration of a compound of Formula I.

14. The method of claim 12, wherein the compound of Formula I is administered to the subject, followed by administration of the peginterferon alfa-2a.

15. The method of any one of claim 12, wherein the peginterferon alfa-2a and compound of Formula I are in separate formulations or unit dosage forms.

16. The method of claim 6, wherein the peginterferon alfa-2a and compound of Formula I are administered at dosages that would not be effective when one or both of the peginterferon alfa-2a and compound of Formula I are administered alone, but which amounts are effective in combination.

17. A composition comprising peginterferon alfa-2a and a compound of Formula I: embedded image or a pharmaceutically acceptable salt thereof; wherein R4 is H or C1-C6 alkyl; wherein each R5 is independently selected at each occurrence from the group consisting of CH3, C1-C6 alkoxy, halo, —CN, —NO2, —(L)m—SR9, —(L)m—S(═O)R9, —(L)m—S(═O)2R9, —(L)m—NHS(═O)2R9, —(L)m—C(═O)R9, —(L)m—OC(═O)R9, —(L)mCO2R8, —(L)m—OCO2R8, —(L)m—N(R8)2, —(L)m—C(═O)N(R8)2, —(L)m—OC(═O)N(R8)2, —(L)m—NHC(═O)NH(R8), —(L)m—NHC(═O)R9, —(L)m—NHC (═O)OR9, —(L)m—C(OH)(R8)2, —(L)mC(NH2)(R8)2, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl and —C1-C6 trihaloalkyl; L is independently, at each occurrence, a bivalent radical selected from —(C1-C3 alkylene)-, —(C3-C7 cyclo alkylene)-, —(C1-C3 alkylene)m—O—(C1-C3 alkylene)m— or —(C1-C3 alkylene)m—NH—(C1-C3 alkylene)m-; each R8 is independently, at each occurrence, H, C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, aryl, heteroaryl, —C1-C4 alkyl-(C3-C10 cycloalkyl), —C1-C4 alkyl-(C3-C10 heterocycloalkyl), —C1-C4 alkyl-(aryl), or —C1-C4 alkyl(heteroaryl), and wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl is optionally substituted with 1-5 substituents selected from R2; R9 is C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, a C3-C10 heterocycloalkyl, aryl, heteroaryl, —C1-C4 alkyl-(C3-C10 cycloalkyl), —C1-C4 alkyl-(C3-C10 heterocycloalkyl), —C1-C4 alkyl-(aryl), or —C1-C4 alkyl-(heteroaryl), and wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring is optionally substituted with 0-5 substituents selected from R2; R10 is OH, C1-C6 alkyl, C1-C6 alkyl-OH, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, a C3-C10 heterocycloalkyl, aryl, heteroaryl, —C1-C4 alkyl-(C3-C10 cycloalkyl), —C1-C4 alkyl-(C3-C10 heterocycloalkyl), —C1-C4 alkyl-(aryl), or —C1-C4 alkyl-(heteroaryl), and wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring is optionally substituted with 1-5 substituents selected from R2; R11 is a bond or C1-C3 alkylene, wherein the C1-C3 alkylene is optionally substituted with 1-3 substituents selected from R2; R2 is independently selected at each occurrence from the group consisting of OH, halo, —CN, —NO2, —C1-C6 alkyl, —C1-C6 alkoxy, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, —C1-C6 heteroalkyl, and C(O)—C1-C6 alkyl; w is 0, 1 or 2; each occurrence of x is independently selected from the group consisting of 0, 1, 2, 3 and 4; each occurrence of y is independently selected from the group consisting of 1, 2, and 3; each occurrence of z is independently selected from the group consisting of 0, 1, 2, and 3; each occurrence of m is independently 0, 1 or 2.

18. A method of treating an HBV infection in a subject in need thereof comprising administering to the subject an effective amount of the composition of claim 17.

Description:

RELATED APPLICATION

The present application claims priority to U.S. Provisional Application No. 61/936,242, filed Feb. 5, 2014, the entire content of which is incorporated herein by reference.

BACKGROUND

Chronic hepatitis B virus (HBV) infection is a significant global health problem, affecting over 5% of the world population (over 350 million people worldwide and 1.25 million individuals in the U.S.).

Despite the availability of a prophylactic HBV vaccine, the burden of chronic HBV infection continues to be a significant unmet worldwide medical problem, due to suboptimal treatment options and sustained rates of new infections in most parts of the developing world. Current treatments rarely provide a cure and are limited to only two classes of agents (interferon and nucleoside analogues/inhibitors of the viral polymerase); drug resistance, low cure rates, and tolerability issues limit their impact. The low cure rates of HBV can be attributed at least in part to incomplete suppression of HBV replication and to the presence and persistence of covalently closed circular DNA (cccDNA) in the nucleus of infected hepatocytes. However, persistent suppression of HBV DNA slows liver disease progression and helps to prevent hepatocellular carcinoma. Therefore, current therapy goals for HBV-infected patients are directed to reducing serum HBV DNA to low or undetectable levels, and to ultimately reducing or preventing the development of cirrhosis and hepatocellular carcinoma.

Although there is precedent for improved efficacy from combination regimens in other viral diseases such as HIV and HCV, combination of existing HBV drugs have failed to show improved efficacy. Neither the combinations of interferon α (IFN) and nucleos(t)ide polymerase inhibitors nor combinations of nucleos(t)ide polymerase inhibitors have provided improved efficacy in treating HBV compared to monotherapy.

Therefore, there remains a need in the art for improved therapies for treating HBV infection.

SUMMARY OF THE INVENTION

Provided herein is a combination therapy comprising a capsid assembly inhibitor and an interferon. The combination therapy is useful for the treatment of HBV infection. This combination unexpectedly provides additional HBV virus replication suppression efficacy compared to monotherapy with interferon, entecavir, or a compound of Formula I.

Accordingly, in one aspect, provided herein is a method of treating an HBV infection in a subject in need thereof, comprising administering to the subject a capsid assembly inhibitor and an interferon. In one embodiment, the interferon is selected from the group consisting of interferon alpha, interferon alpha-2a, recombinant interferon alpha-2a, peginterferon-alpha 2a, interferon alpha-2b, recombinant interferon alpha-2b, interferon alpha-2b XL, peginterferon alpha-2b, glycosylated interferon alpha-2b, interferon alpha-2c, recombinant interferon alpha-2c, interferon beta, interferon beta-1a, peginterferon beta-la, interferon delta, interferon lambda, peginterferon lambda-1, interferon omega, interferon tau, gamma interferon, interferon alfacon-1, interferon alpha-n1, interferon alpha-n3,albinterferon alpha-2b, BLX-883, DA-3021, PEG-Infergen, and BELEROFON. In another embodiment, the interferon is selected from the group consisting of peginterferon alpha-2a, peginterferon alpha-2b, glycosylated interferon alpha-2b, peginterferon beta-1a, and peginterferon lambda-1. In a particular embodiment, the interferon is peginterferon alpha-2a.

In one embodiment of the method, the capsid assembly inhibitor is a compound of Formula I:

embedded image

or a pharmaceutically acceptable salt thereof.

In another aspect, provided herein is a method of treating an HBV infection in a subject in need thereof, comprising administering to the subject peginterferon alfa-2a and a compound of Formula I:

embedded image

or a pharmaceutically acceptable salt thereof.

In an embodiment, the peginterferon alfa-2a and compound of Formula I are in a single formulation or unit dosage form. In another embodiment, this method further comprises a pharmaceutically acceptable carrier. In yet another embodiment, the peginterferon alfa-2a and compound of Formula I are administered separately. In still another embodiment, the method comprises administering the peginterferon alfa-2a and compound of Formula I at substantially the same time.

In another embodiment, the treatment comprises administering the peginterferon alfa-2a and compound of Formula I at different times. In one embodiment, the peginterferon alfa-2a is administered to the subject, followed by administration of a compound of Formula I. In another embodiment, the compound of Formula I is administered to the subject, followed by administration of the peginterferon alfa-2a. In still another embodiment, the peginterferon alfa-2a and compound of Formula I are in separate formulations or unit dosage forms.

In an embodiment of any of the above methods, the subject is human.

In an aspect, provided herein is a composition comprising peginterferon alfa-2a and a compound of Formula I:

embedded image

or a pharmaceutically acceptable salt thereof.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a line graph of viral load reduction from baseline (Log10; ordinate) as a function of time (days; abscissa) in a uPa-SCID humanized mouse model of HBV infection. Murine subjects were administered amounts of either: capsid inhibitor only; Entecavir (ETV) only; pegylated interferon α (IFN) (PEGASYS) only; a mixture of a capsid inhibitor and Entecavir (capsid inhibitor+ETV); or a mixture of a capsid inhibitor and interferon (capsid inhibitor+PEG-IFNα). Control subjects were administered dimethyl sulfoxide (DMSO) only. N=6

FIG. 2 is a line graph of HBV DNA (Log10 copies/mL; ordinate) as a function of time (days; abscissa) in a murine model for HBV genotype C infection of human chimeric liver. Murine subjects were administered amounts of either: capsid inhibitor only; pegylated interferon α (PEG-IFNα) (PEGASYS); or a mixture of a capsid inhibitor and pegylated interferon α (capsid inhibitor+PEG-IFNα).

DETAILED DESCRIPTION

It has been discovered that administering a combination of a compound of Formula I, or a pharmaceutically acceptable salt thereof, and peginterferon alfa-2a (PEGASYS), or another interferon analog, provides surprising, improved effects for treating HBV infection in a subject. Such an approach—combination or co-administration of the two types of agents—can be useful for treating individuals suffering from an HBV infection who do not respond to or are resistant to currently-available therapies. The combination therapy comprising a compound of Formula I and peginterferon alfa-2a, or another interferon analog, provided herein is also useful for improving the efficacy and/or reducing the side effects of currently-available HBV therapies for individuals who do respond to such therapies.

Certain terms used herein are described below. Compounds of the present invention are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

Combination Therapy

Provided herein is a combination of therapeutic agents and administration methods for the combination of agents to treat HBV infection. As used herein, a “combination of agents” and similar terms refer to a combination of two types of agents: (1) a compound of Formula I, or a pharmaceutically acceptable salt thereof, and (2) and peginterferon alfa-2a or another interferon analog.

Pegylated interferon alpha 2a or peginterferon alfa-2a is a conjugate of poly(ethylene glycol) (PEG) and interferon alpha 2a One brand name for pegylated interferon alpha 2a is PEGASYS. Pegylated interferon alpha 2a compositions and/or methods of making pegylated interferon alpha-2a are disclosed, e.g. in U.S. Pat. No. 5,382,657, U.S. Pat. No. 5,762,923 and WO 08/145323, all of which are incorporated herein by reference. Pegylated interferon alpha 2a may be prepared using the procedures described in these references.

Compounds of Formula I are useful in the treatment and prevention of HBV in man. In one aspect, the compounds of the invention are useful in HBV treatment by binding to the HBV core protein and thereby disabling all or a subset of the functions HBV core protein plays in HBV replication and persistence such as disrupting, accelerating, reducing delaying and/or inhibiting normal viral capsid assembly and/or disassembly of immature or mature particles, thereby inducing aberrant capsid morphology and leading to antiviral effects such as disruption of virion assembly and/or disassembly and/or virion maturation, and/or virus egress, and/or cccDNA production, maintenance or transcription, and/or modulation of the host innate immune response.

Capsid assembly plays a central role in HBV genome replication. HBV polymerase binds pre-genomic HBV RNA (pgRNA), and pgRNA encapsidation must occur prior to HBV DNA synthesis. Moreover, it is well established that nuclear accumulation of the cccDNA replication intermediate, which is responsible for maintenance of chronic HBV replication in the presence of nucleoside suppressive therapy, requires the capsid for shuttling HBV DNA to the nuclei. Therefore, the HBV core inhibitors or capsid assembly disruptors of the invention have the potential to increase HBV functional cure rates through improved suppression of viral genome replication and through suppression of cccDNA when used alone or in combination with existing HBV drugs such as interferons and nucleos(t)ide inhibitors. The core inhibitors or capsid assembly disruptors of the present invention may also alter normal core protein degradation, potentially leading to altered MHC-1 antigen presentation, which may in turn increase seroconversion/eradication rates through immuno-stimulatory activity, more effectively clearing infected cells. Thus, the compounds of the present invention may have the potential to bind to HBV core protein and alter the function of that protein by interfering with, accelerating, decelerating, disrupting or otherwise modifying the functions associated with HBV core protein.

The compounds useful within the invention may be synthesized using techniques well-known in the art of organic synthesis. The starting materials and intermediates required for the synthesis may be obtained from commercial sources or synthesized according to methods known to those skilled in the art.

In one aspect, the combination therapy comprises a compound of Formula I:

embedded image

or a pharmaceutically acceptable salt thereof;

wherein

R4 is H or C1-C6 alkyl;

wherein each R5 is independently selected at each occurrence from the group consisting of CH3, C1-C6 alkoxy, halo, —CN, —NO2, —(L)m—SR9, —(L)m—S(═O)R9, -(1-)m-S(═O)2R9, —(L)m—NHS(═O)2R9, —(L)m—C(═O)R9, —(L)m-OC(═O)R9, —(L)mCO2R8, 4L)m-OCO2R8, —(L)m—N(R8)2, —(L)m—C(═O)N(R8)2, —(L)m-OC(═O)N(R8)2, —(L)m-NHC(=O)NH(R8), —(L)m—NHC(═O)R9, —(L)m—NHC(═O)OR9, —(L)m—C(OH)(R8)2, —(L)mC(NH2)(R8)2, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl and —C1-C6 trihaloalkyl;

L is independently, at each occurrence, a bivalent radical selected from —(C1-C3 alkylene)-, —(C3-C7 cycloalkylene)-, —(C1-C3 alkylene)m—O—(C1-C3 alkylene)m-, or -(C1-C3 alkylene)m—NH—(C1-C3 alkylene)m—;

each R8 is independently, at each occurrence, H, C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, aryl, heteroaryl, —C1-C4 alkyl-(C3-C10 cycloalkyl), —C1-C4 alkyl-(C3-Cto heterocycloalkyl), —C1-C4 alkyl-(aryl), or —C1-C4 alkyl(heteroaryl), and wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl and heteroaryl is optionally substituted with 1-5 substituents selected from R2;

R9 is C1-C6 alkyl, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, C1-C6 heteroalkyl, C3-C1( )cycloalkyl, a C3-C10 heterocycloalkyl, aryl, heteroaryl, —C1-C4 alkyl-(C3-C10 cycloalkyl), —C1-C4 alkyl-(C3-C10 heterocycloalkyl), —C1-C4 alkyl-(aryl), or —C1-C4 alkyl-(heteroaryl), and wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring is optionally substituted with 0-5 substituents selected from R2;

R10 is OH, C1-C6 alkyl, C1-C6 alkyl-OH, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, a C3-C10 heterocycloalkyl, aryl, heteroaryl, —C1-C4 alkyl-(C3-C10 cycloalkyl), —C1-C4 alkyl-(C3-C10 heterocycloalkyl), —C1-C4 alkyl-(aryl), or —C1-C4 alkyl-(heteroaryl), and wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring is optionally substituted with 1-5 substituents selected from R2;

R11 is a bond or C1-C3 alkylene, wherein the C1-C3 alkylene is optionally substituted with 1-3 substituents selected from R2;

R2 is independently selected at each occurrence from the group consisting of OH, halo, —CN, —NO2, —C1-C6 alkyl, —C1-C6 alkoxy, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, —C1-C6 heteroalkyl, and C(O)—C1-C6 alkyl;

w is 0, 1 or 2;

each occurrence of x is independently selected from the group consisting of 0, 1, 2, 3 and 4;

each occurrence of y is independently selected from the group consisting of 1, 2, and 3;

each occurrence of z is independently selected from the group consisting of 0, 1, 2, and 3;

each occurrence of m is independently 0, 1 or 2.

In one embodiment of Formula I, R2 is independently selected at each occurrence from the group consisting of halo, —CN, —NO2, —C1-C6 alkyl, —C1-C6 alkoxy, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 trihaloalkyl, —C1-C6 heteroalkyl, and C(O)—C1-C6 alkyl;

In one embodiment, compounds of Formula I are of the Formula IVa:

embedded image

or a pharmaceutically acceptable salt thereof.

In embodiments of Formulae I or IVa,

each R5 is independently selected at each occurrence from the group consisting of CH3, C1-C6 alkoxy, halo, —CN, —NO2, —C1-C6 haloalkyl, —C1-C6 dihaloalkyl, —C1-C6 and trihaloalkyl;

RI° is OH, halo, C1-C6 alkyl, C1-C6 alkyl-OH, —C1-C6 chloroalkyl, —C1-C6 dichloroalkyl, —C1-C6 trichloroalkyl, —C1-C6 fluoroalkyl, —C1-C6 difluoroalkyl, —C1-C6 trifluoroalkyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, a C3-C10 heterocycloalkyl, aryl, heteroaryl, —C1-C4 alkyl-(C3-C10 cycloalkyl), —C1-C4 alkyl-(C3-C10 heterocycloalkyl), —C1-C4 alkyl-(aryl), or —C1-C4 alkyl-(heteroaryl), and wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring is optionally substituted with 1-5 substituents selected from R2;

R11 is a bond or C1-C3 alkylene, wherein the C1-C3 alkylene is optionally substituted with 1-3 substituents selected from R2;

R2 is independently selected at each occurrence from the group consisting of halo, —CN, —NO2, -C1-C6 alkyl, —C1-C6 alkoxy, —C1-C6 fluoroalkyl, —C1-C6 heteroalkyl, C(O)—C1-C6 alkyl, and C(O)—C1-C6 alkoxy.

In other embodiments of Formulae I or IVa, each R5 is independently selected at each occurrence from the group consisting of CH3, C1-C6 alkoxy, halo, fluoromethyl, difluoromethyl, trifluoromethyl, chloromethyl, dichloromethyl, and trichloromethyl;

R10 is OH, halo, C1-C6 alkyl, C1-C6 alkyl-OH, C1-C6 fluoroalkyl, C1-C6 difluoroalkyl, C1-C6 trifluoroalkyl, C1-C6 heteroalkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, aryl, heteroaryl, —C1-C4 alkyl-(C3-C10 cycloalkyl), —C1-C4 alkyl-(C3-C10 heterocycloalkyl), —C1-C4 alkyl-(aryl), or —C1-C4 alkyl-(heteroaryl), and wherein the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl ring is optionally substituted with 1-5 substituents selected from R2;

R11 is a bond or C1-C3 alkylene;

R2 is independently selected at each occurrence from the group consisting of halo, —CN, —NO2, -C1-C6 alkyl, —C1-C6 alkoxy, —C1-C6 fluoroalkyl, —C1-C6 heteroalkyl, and C(O)—C1-C6 alkyl, and C(O)—C1-C6 alkoxy.

In other embodiments of Formulae I and IVa, R5 (i.e., (R5)y) is 3-F, 3—Cl, 3—CH3, 3—CH2F, 3—CHF2, 4-F, 3—CH3-4-F, 3-C1-4-F, 3-Br-4-F, 3,4,5-trifluoro, 3,4,5-trichloro, or 3-chloro-4,5-difluoro. In another embodiment, w is 1 or 2.

In yet other embodiments of Formulae I and IVa,

R11 is a bond or C1-C3 alkylene;

R2 is OH, halo, C1-C6 alkyl, C1-C6 alkyl-OH, —C1-C6 chloroalkyl, —C1-C6 dichloroalkyl, —C1-C6 trichloroalkyl, —C1-C6 fluoroalkyl, —C1-C6 difluoroalkyl, —C1-C6 trifluoroalkyl, C3-C10 cycloalkyl, C3-C10 heterocycloalkyl, or phenyl, wherein the C3-C10 cycloalkyl, a C3-C10 heterocycloalkyl, or phenyl groups are optionally substituted with 1-5 substituents selected from halo, —C1-C6 alkyl, and —C1-C6 alkoxy; and

z is 0 or 1.

In another embodiment, compounds of Formula I are of the Formula IVb:

embedded image

or pharmaceutically acceptable salts thereof;

wherein G1 is independently selected at each occurrence from CH3, OCH3, halo, CF3, CCl3, CH2C1, CCl2H, CF2H, CH2F, and CF3;

G2 is H, C1-C4 alkyl, or halo;

G3 is OH, CH2OH, or CH2CH2OH;

G4 is H, OH, halo, C1-C6 alkyl, C1-C6 -C1-C6 chloroalkyl, —C1-C6 dichloroalkyl, —C1-C6 trichloroalkyl, —C1-C6 fluoroalkyl, —C1-C6 difluoroalkyl, —C1-C6 trifluoroalkyl, or phenyl, wherein the phenyl group is optionally independently substituted with 1-5 substituents selected from halo, —C1-C6 alkyl, and —C1-C6 alkoxy; and

y is 1, 2, or 3.

In an embodiment of Formula IVb, G1 is independently selected at each occurrence from halo, CF3, CCl3, CH2Cl, CCl2H, CF2H, CH2F, and CF3.

In another embodiment, compounds of Formula I are of the Formula IVc:

embedded image

or pharmaceutically acceptable salts thereof;

wherein X is halo;

G1 is hydrogen or halo;

G2 is H, C1-C4 alkyl, or halo; and

G4 is H, halo, C1-C4 alkyl, or OH.

In one embodiment of Formula IVc, G2 is C1-C4 alkyl or halo, and wherein G2 is in the 2, 3, or 4 position of the phenyl ring.

In a particular embodiment, the compound of Formula I is a compound provided in the following table, or a pharmaceutically acceptable salt thereof:

Structure
MS (M + H)+Cmp. ID
embedded image  960_D1
embedded image  960_D2
embedded image  890
embedded image  893
embedded image  946_D1
embedded image  946_D2
embedded image  925
embedded image 1080
embedded image 1084_D1
embedded image 1084_D2
embedded image 1085
embedded image 1088
embedded image 1100
embedded image 1161
embedded image  916
embedded image 1057
embedded image 1060
embedded image 1081_D1
embedded image 1081_D2
embedded image 1130
embedded image 1135_D1
embedded image 1135_D2
embedded image 1073
embedded image 1077_D1
embedded image 1077_D2
embedded image 1076

Examples of compounds of Formula I include the compounds described in U.S. Pat. No. 8,629,274, which is incorporated herein by reference in its entirety. Methods of making compounds of Formula I, including the compounds of the above table, can be found in U.S. Pat. No. 8,629,274.

Compounds of Formula I may be prepared by the reaction sequence that is illustrated in Scheme 1.

embedded image

The compound of Formula (IV) from Scheme 1 may be reacted with chlorosulfonic acid to yield the sulfonyl chloride of formula (V). The compound of Formula (V) may be reacted with a secondary or primary amine of formula HNR6R6, in a solvent such as but not limited to tetrahydrofuran, dichloromethane, ethyl ether or a mixture thereof, preferably in the presence of a tertiary base such as but not limited to triethylamine, diisopropylethylamine or pyridine, to yield the compound of Formula (VI), which may be coupled to an amine via an amide bond, yielding the compound of Formula (II). The amide coupling may be performed in the presence of a coupling agent, such as but not limited to DCC (N,N′-dicyclohexyl carbodiimide), DIC (N,N′-diisopropylcarbodiimide), EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide), HBTU (O-benzotriazole-N,N,N′,N′-tetramethyl-uronium-hexafluoro-phosphate), HATU (2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate methanaminium), HCTU ((2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate), TBTU (O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate), or PyBOP (benzotriazol-1-yl-oxytripyrrolidino-phosphonium hexafluorophosphate), in a solvent such as but not limited to tetrahydrofuran, dichloromethane, or a mixture thereof, and in the optional presence of a tertiary base, such as but not limited to triethylamine, diisopropylethylamine or pyridine. Alternatively, the sulfonyl chloride of Formula (V) may be reacted with a chlorinating reagent, such as but not limited to thionyl chloride, phosgene, diphosgene or triphosgene, to yield the acyl chloride of Formula (VII). The compound of Formula (VII) may then be reacted with an amine in a solvent such as but not limited to tetrahydrofuran, dichloromethane, ethyl ether or a mixture thereof, under conditions that do not promote the reaction of the sulfonyl chloride group with the amine, to yield the compound of Formula (VIII), which may then be reacted with the amine HNR6R6 in a solvent such as but not limited to tetrahydrofuran, toluene, dichloromethane, or a mixture thereof, and in the presence of a tertiary base, such as but not limited to triethylamine, diisopropylethylamine or pyridine, to yield the compound of Formula (II).

As used herein, the expression “Cx-Cy-alkyl”, wherein x is 1-5 and y is 2-10 indicates a particular alkyl group (straight- or branched-chain) of a particular range of carbons. For example, the expression C1-C4-alkyl includes, but is not limited to, methyl, ethyl, propyl, butyl, isopropyl, tert-butyl and isobutyl.

As used herein, the term “C3-6 cycloalkyl” refers to saturated or unsaturated monocyclic or bicyclic hydrocarbon groups of 3-6 carbon atoms, preferably 5 carbon atoms. Exemplary monocyclic hydrocarbon groups include, but are not limited to, cyclopropyl, cyclobutyl, and cyclopentyl.

The term “halogen” or “halo” refers to chloro, bromo, fluoro, and iodo groups.

Agents may contain one or more asymmetric elements such as stereogenic centers or stereogenic axes, e.g., asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms. These compounds can be, for example, racemates or optically active forms. For compounds with two or more asymmetric elements, these compounds can additionally be mixtures of diastereomers. For compounds having asymmetric centers, it should be understood that all of the optical isomers and mixtures thereof are encompassed. In addition, compounds with carbon-carbon double bonds may occur in Z- and E-forms; all isomeric forms of the compounds are included in the present invention. In these situations, the single enantiomers (optically active forms) can be obtained by asymmetric synthesis, synthesis from optically pure precursors, or by resolution of the racemates. Resolution of the racemates can also be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example a chiral HPLC column.

Unless otherwise specified, or clearly indicated by the text, reference to compounds useful in the combination therapy of the invention includes both the free base of the compounds, and all pharmaceutically acceptable salts of the compounds.

As used herein, the term “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. 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 of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

Provided herein is a combination therapy comprising a compound of Formula I, or a pharmaceutically acceptable salt thereof, and PEGASYS. Administration of the combination includes administration of the combination in a single formulation or unit dosage form, administration of the individual agents of the combination concurrently but separately, or administration of the individual agents of the combination sequentially by any suitable route. The dosage of the individual agents of the combination may require more frequent administration of one of the agent(s) as compared to the other agent(s) in the combination. Therefore, to permit appropriate dosing, packaged pharmaceutical products may contain one or more dosage forms that contain the combination of agents, and one or more dosage forms that contain one of the combination of agents, but not the other agent(s) of the combination.

The term “single formulation” as used herein refers to a single carrier or vehicle formulated to deliver effective amounts of both therapeutic agents to a patient. The single vehicle is designed to deliver an effective amount of each of the agents, along with any pharmaceutically acceptable carriers or excipients. In some embodiments, the vehicle is a tablet, capsule, pill, or a patch. In other embodiments, the vehicle is a solution or a suspension.

The term “unit dose” is used herein to mean simultaneous administration of both agents together, in one dosage form, to the patient being treated. In some embodiments, the unit dose is a single formulation. In certain embodiments, the unit dose includes one or more vehicles such that each vehicle includes an effective amount of at least one of the agents along with pharmaceutically acceptable carriers and excipients. In some embodiments, the unit dose is one or more tablets, capsules, pills, or patches administered to the patient at the same time.

The term “treat” is used herein to mean to relieve, reduce or alleviate, at least one symptom of a disease in a subject. Within the meaning of the present invention, the term “treat” also denotes, to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease or symptom of a disease) and/or reduce the risk of developing or worsening a symptom of a disease.

The term “subject” is intended to include animals. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from an HBV infection.

The term “about” or “approximately” usually means within 20%, more preferably within 10%, and most preferably still within 5% of a given value or range. Alternatively, especially in biological systems, the term “about” means within about a log (i.e., an order of magnitude) preferably within a factor of two of a given value.

The terms “capsid assembly inhibitor,” “capsid inhibitor,” “capsid assembly disruptor,” and “core inhibitor” refer to the same mode of action. Without being limited by any theoretical explanation, this mode of action may be initiated by binding of compounds of the invention to HBV core protein and altering the function of that protein by interfering with, accelerating, decelerating, disrupting or otherwise modifying the functions associated with HBV core protein.

The term “combination therapy” refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present disclosure. Such administration encompasses co-administration of these therapeutic agents in a substantially simultaneous manner, such as in a single capsule having a fixed ratio of active ingredients or in multiple, or in separate containers (e.g., capsules) for each active ingredient. In addition, such administration also encompasses use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. In either case, the treatment regimen will provide beneficial effects of the drug combination in treating the conditions or disorders described herein.

The combination of agents described herein provide improved HBV suppression or HBV cure efficacy compared to the respective monotherapies. In certain embodiments, the combination of agents described herein display a synergistic effect. The term “synergistic effect” as used herein, refers to action of two agents such as, for example, a compound of Formula I, or a pharmaceutically acceptable salt thereof, and Pegasys, producing an effect, for example, slowing the symptomatic progression of cancer or symptoms thereof, which is greater than the simple addition of the effects of each drug administered by themselves. A synergistic effect can be calculated, for example, using suitable methods such as the Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), the equation of Loewe additivity (Loewe, S. and Muischnek, H., Arch. Exp. Pathol Pharmacol. 114: 313-326 (1926)) and the median-effect equation (Chou, T. C. and Talalay, P., Adv. Enzyme Regul. 22: 27-55 (1984)). Each equation referred to above can be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the drug combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively.

In an embodiment, provided herein is a combination therapy comprising an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, and PEGASYS. An “effective amount” of a combination of agents is an amount sufficient to provide an observable improvement over the baseline clinically observable signs and symptoms of the disorders treated with the combination.

An “oral dosage form” includes a unit dosage form prescribed or intended for oral administration.

Methods of Treatment

In one aspect of the invention, provided herein is a method of treating an HBV infection in a subject in need thereof, comprising administering to the subject a capsid assembly inhibitor and an interferon.

In one embodiment, the interferon is selected from the group consisting of interferon alpha, interferon alpha-2a, recombinant interferon alpha-2a, peginterferon-alpha 2a, interferon alpha-2b, recombinant interferon alpha-2b, interferon alpha-2b XL, peginterferon alpha-2b, glycosylated interferon alpha-2b, interferon alpha-2c, recombinant interferon alpha-2c, interferon beta, interferon beta-1a, peginterferon beta-1a, interferon delta, interferon lambda, peginterferon lambda-1, interferon omega, interferon tau, gamma interferon, interferon alfacon-1, interferon alpha-n1, interferon alpha-n3,albinterferon alpha-2b, BLX-883, DA-3021, PEG-Infergen, and BELEROFON. In a particular embodiment, the interferon is selected from the group consisting of peginterferon alpha-2a, peginterferon alpha-2b, glycosylated interferon alpha-2b, peginterferon beta-1a, and peginterferon lambda-1. In a specific embodiment, the interferon is peginterferon alpha-2a.

In still another embodiment, the capsid assembly inhibitor is a compound of Formula (I).

The invention includes a method of treatment of an HBV infection in an individual in need thereof, comprising administering to the individual the combination therapy of the invention (i.e., a compound of Formula I in combination with peginterferon alfa-2a).

The invention also includes a method of reducing viral load associated with an HBV infection in an individual in need thereof, comprising administering to the individual the combination therapy of the invention.

The invention further includes a method of reducing reoccurrence of an HBV infection in an individual in need thereof, comprising administering to the individual the combination therapy of the invention.

The invention also includes a method of reducing the physiological impact of an HBV infection in an individual in need thereof, comprising administering to the individual the combination therapy of the invention.

The invention further includes a method of reducing, slowing, or inhibiting an HBV infection in an individual in need thereof, comprising administering to the individual the combination therapy of the invention.

The invention also includes a method of inducing remission of hepatic injury from an HBV infection in an individual in need thereof, comprising administering to the individual the combination therapy of the invention.

The invention further includes a method of reducing the physiological impact of long-term antiviral therapy for HBV infection in an individual in need thereof, comprising administering to the individual the combination therapy of the invention.

The invention also includes a method of eradicating an HBV infection in an individual in need thereof, comprising administering to the individual the combination therapy of the invention.

The invention further includes a method of prophylactically treating an HBV infection in an individual in need thereof, wherein the individual is afflicted with a latent HBV infection, comprising administering to the individual the combination therapy of the invention.

In one embodiment, the individual is refractory or non-responsive to other therapeutic classes of HBV drugs (e.g., HBV polymerase inhibitors, interferons, viral entry inhibitors, viral maturation inhibitors, literature-described capsid assembly modulators, antiviral compounds of distinct or unknown mechanism, and the like, or combinations thereof). In another embodiment, the method of the invention reduces viral load in an individual suffering from an HBV infection to a greater extent compared to the extent that other therapeutic classes of HBV drugs reduce viral load in the individual.

In one embodiment, the method of the invention reduces viral load in an individual suffering from an HBV infection, thus allowing lower doses or varying regimens of combination therapies to be used.

In one embodiment, the method of the invention causes a lower incidence of viral mutation and/or viral resistance compared to other classes of HBV drugs, thereby allowing for long term therapy and minimizing the need for changes in treatment regimens.

In one embodiment, the method of the invention increases the seroconversion rate beyond that of current treatment regimens.

In one embodiment, the method of the invention increases and/or normalizes and/or restores normal health, elicits full recovery of normal health, restores life expectancy, and/or resolves the viral infection in the individual in need thereof.

In one embodiment, the method of the invention eradicates HBV from an individual infected with HBV, thereby obviating the need for long term and/or life-long treatment, or shortening the duration of treatment, and/or allowing for reduction in dosing of other antiviral agents.

Accordingly, in one embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a compound of Formula IVa, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a compound of Formula IVb, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a compound of Formula IVc, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 960, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 890, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 893, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 946, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 925, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 1080, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 1084, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 1085, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 1088, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 1100, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 1161, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 916, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 1057, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 1060, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 1081, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 1130, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 1135, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 1073, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 1077, or a pharmaceutically acceptable salt thereof, and PEGASYS.

In another embodiment, provided herein is a method of treating an HBV infection in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of compound 1076, or a pharmaceutically acceptable salt thereof, and PEGASYS.

Dosages

The optimal dose of the combination of agents for treatment of disease can be determined empirically for each individual using known methods and will depend upon a variety of factors, including, though not limited to, the degree of advancement of the disease; the age, body weight, general health, gender and diet of the individual; the time and route of administration; and other medications the individual is taking. Optimal dosages may be established using routine testing and procedures that are well known in the art.

The amount of combination of agents that may be combined with the carrier materials to produce a single dosage form will vary depending upon the individual treated and the particular mode of administration. In some embodiments the unit dosage forms containing the combination of agents as described herein will contain the amounts of each agent of the combination that are typically administered when the agents are administered alone.

In an embodiment of the combination provided herein, each agent is administered at dosages that would not be effective when one or both of the agents are administered alone, but which amounts are effective in combination. For example, in an embodiment, peginterferon alfa-2a and a compound of Formula I are administered at dosages that would not be effective when one or both of the peginterferon alfa-2a and compound of Formula I are administered alone, but which amounts are effective in combination.

Frequency of dosage may vary depending on the compound used and the particular condition to be treated or prevented. In general, the use of the minimum dosage that is sufficient to provide effective therapy is preferred. Patients may generally be monitored for therapeutic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those of ordinary skill in the art.

In an embodiment of the combination provided herein, one or more agents are administered for a duration that is shorter compared to the duration when either of the agents are administered alone. For example, current treatment guidelines recommend interferon treatment for 12 months. In an embodiment of the combination provided herein (e.g., a compound of Formula I and interferon), the duration of interferon treatment is 12 months or less, e.g., 11 months or less, e.g., 10 months or less, e.g., 9 months or less, e.g., 8 months or less, e.g., 7 months or less, e.g., 6 months or less, e.g., 5 months or less, e.g., 4 months or less, e.g., 3 months or less, e.g., 2 months or less, e.g., 1 month or less. In another embodiment, a treatment of peginterferon alfa-2a and a compound of Formula I are administered for 12 months or less, e.g., 11 months or less, e.g., 10 months or less, e.g., 9 months or less, e.g., 8 months or less, e.g., 7 months or less, e.g., 6 months or less, e.g., 5 months or less, e.g., 4 months or less, e.g., 3 months or less, e.g., 2 months or less, e.g., 1 month or less.

The dosage form can be prepared by various conventional mixing, comminution and fabrication techniques readily apparent to those skilled in the chemistry of drug formulations.

The oral dosage form containing the combination of agents or individual agents of the combination of agents may be in the form of micro-tablets enclosed inside a capsule, e.g., a gelatin capsule. For this, a gelatin capsule as is employed in pharmaceutical formulations can be used, such as the hard gelatin capsule known as CAPSUGEL, available from Pfizer.

Many of the oral dosage forms useful herein contain the combination of agents or individual agents of the combination of agents in the form of particles. Such particles may be compressed into a tablet, present in a core element of a coated dosage form, such as a taste-masked dosage form, a press coated dosage form, or an enteric coated dosage form, or may be contained in a capsule, osmotic pump dosage form, or other dosage form.

The drug compounds of the present invention are present in the combinations, dosage forms, pharmaceutical compositions and pharmaceutical formulations disclosed herein in a ratio in the range of 100:1 to 1:100. For example, the ratio of a compound of Formula I : peginterferon alfa-2a (or another interferon analog) can be in the range of 1:100 to 1:1, for example, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:5, 1:2, or 1:1 of Formula I : peginterferon alfa-2a. In another example, the ratio of peginterferon alfa-2a : a compound of Formula I can be in the range of 1:100 to 1:1, for example, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:5, 1:2, or 1:1 of peginterferon alfa-2a: a compound of Formula I.

The optimum ratios, individual and combined dosages, and concentrations of the drug compounds that yield efficacy without toxicity are based on the kinetics of the active ingredients' availability to target sites, and are determined using methods known to those of skill in the art.

The pharmaceutical compositions or combinations provided herein can be tested in clinical studies. Suitable clinical studies may be, for example, open label, dose escalation studies in patients with proliferative diseases. Such studies prove in particular the improvement of efficacy of the active ingredients of the combination of the invention. The beneficial effects on proliferative diseases may be determined directly through the results of these studies which are known as such to a person skilled in the art. Such studies may be, in particular, suitable to compare the effects of a monotherapy using the active ingredients and a combination of the invention.

The administration of a combination therapy of the invention may result not only in a beneficial effect, e.g. an improved therapeutic effect, e.g. with regard to alleviating, delaying progression of or inhibiting the symptoms, but also in further surprising beneficial effects, e.g. fewer side-effects, an improved quality of life or a decreased morbidity, compared with a monotherapy applying only one of the pharmaceutically active ingredients used in the combination of the invention.

A further benefit may be that lower doses of the active ingredients of the combination of the invention may be used, for example, that the dosages need not only often be smaller but may also be applied less frequently, which may diminish the incidence or severity of side-effects. This is in accordance with the desires and requirements of the patients to be treated.

It is one objective of this invention to provide a pharmaceutical composition comprising a quantity, which may be jointly therapeutically effective at targeting or preventing HBV infection. In this composition, a compound of Formula I and peginterferon alfa-2a (or another interferon analog) may be administered together, one after the other or separately in one combined unit dosage form or in two separate unit dosage forms. The unit dosage form may also be a fixed combination.

The pharmaceutical compositions for separate administration of both compounds, or for the administration in a fixed combination, i.e. a single galenical composition comprising both compounds according to the invention may be prepared in a manner known per se and are those suitable for enteral, such as oral or rectal, and parenteral administration to mammals (warm-blooded animals), including humans, comprising a therapeutically effective amount of at least one pharmacologically active combination partner alone, e.g. as indicated above, or in combination with one or more pharmaceutically acceptable carriers or diluents, especially suitable for enteral or parenteral application.

Formulations

The drug combinations provided herein may be formulated by a variety of methods apparent to those of skill in the art of pharmaceutical formulation. The various release properties described above may be achieved in a variety of different ways. Suitable formulations include, for example, tablets, capsules, press coat formulations, and other easily administered formulations.

Suitable pharmaceutical formulations may contain, for example, from about 0.1% to about 99.9%, preferably from about 1% to about 60%, of the active ingredient(s). Pharmaceutical formulations for the combination therapy for enteral or parenteral administration are, for example, those in unit dosage forms, such as sugar-coated tablets, tablets, capsules or suppositories, or ampoules. If not indicated otherwise, these are prepared in a manner known per se, for example by means of conventional mixing, granulating, sugar-coating, dissolving or lyophilizing processes. It will be appreciated that the unit content of a combination partner contained in an individual dose of each dosage form need not in itself constitute an effective amount since the necessary effective amount may be reached by administration of a plurality of dosage units.

In particular, a therapeutically effective amount of each of the combination partner of the combination of the invention may be administered simultaneously or sequentially and in any order, and the components may be administered separately or as a fixed combination. For example, the method of treating a disease according to the invention may comprise (i) administration of the first agent in free or pharmaceutically acceptable salt form and (ii) administration of the second agent in free or pharmaceutically acceptable salt form, simultaneously or sequentially in any order, in jointly therapeutically effective amounts, preferably in improved therapeutically effective amounts, e.g. in daily or intermittently dosages corresponding to the amounts described herein. The individual combination partners of the combination of the invention may be administered separately at different times during the course of therapy or concurrently in divided or single combination forms. Furthermore, the term administering also encompasses the use of a pro-drug of a combination partner that convert in vivo to the combination partner as such. The instant invention is therefore to be understood as embracing all such regimens of simultaneous or alternating treatment and the term “administering” is to be interpreted accordingly.

The effective dosage of each of the combination partners employed in the combination of the invention may vary depending on the particular compound or pharmaceutical composition employed, the mode of administration, the condition being treated, the severity of the condition being treated. Thus, the dosage regimen of the combination of the invention is selected in accordance with a variety of factors including the route of administration and the renal and hepatic function of the patient. A clinician or physician of ordinary skill can readily determine and prescribe the effective amount of the single active ingredients required to alleviate, counter or arrest the progress of the condition.

Preferred suitable dosages for the compounds used in the treatment described herein are on the order of about 1 mg to about 600 mg, preferably about 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580 to about 600 mgs total.

Accordingly, in one embodiment, provided herein is a composition comprising an interferon and a compound of Formula I. In another embodiment, provided herein is a composition comprising peginterferon alfa-2a and a compound of Formula I. In an embodiment, the compound of Formula I is compound 960, compound 890, compound 893, compound 946, compound 925, compound 1080, compound 1084, compound 1085, compound 1088, compound 1100, compound 1161, compound 916, compound 1057, compound 1060, compound 1081, compound 1130, compound 1135, compound 1073, compound 1077, or compound 1076. In still another embodiment, the composition further comprises a pharmaceutically acceptable carrier.

Experimental

FIG. 1 is a line graph of viral load reduction from baseline (Log10 ordinate) as a function of time (days; abscissa) in an uPa-SCID humanized mouse model of HBV infection. Murine subjects were administered amounts of either: capsid inhibitor only; Entecavir (ETV) only; interferon α (IFN) (PEGASYS) only; a mixture of a capsid inhibitor and Entecavir (capsid inhibitor+ETV); or a mixture of a capsid inhibitor and interferon (capsid inhibitor+IFN). Control subjects were administered dimethyl sulfoxide (DMSO) only. N=6. Surprisingly, the combination of PEGASYS with a capsid inhibitor showed improved efficacy compared to treatment with either of PEGASYS, a capsid inhibitor, ETV, or ETV in combination with PEGASYS.

FIG. 2 is a line graph of HBV DNA (log10 copies/ml; ordinate) as a function of time (days; abscissa) in a murine model for HBV genotype C infection of human chimeric liver. Murine subjects were administered amounts of either: capsid inhibitor only; pegylated interferon α (PEG-IFN) (PEGASYS); or a mixture of a capsid inhibitor and pegylated interferon α (capsid inhibitor+PEG-IFN).

Mouse Study Protocol

Study TitlePK/Tolerability study of a capsid inhibitor in non-PXB grade mice and
PXB-mice (4-week)
Expected StudyPre-dose blood sampling: June 21 (Day-7)
ScheduleGroup assignment: June 27 (Day-1)
Administration period: from June 28 (pm) to(from Day 0 to
July 26 (am)Day 27) (Day
Necropsy: July 26 (pm) 28)
Study end: August 30
ObjectivesThe objective of this study is to evaluate the tolerability and liver toxicity
of a capsid inhibitor in non-PXB grade mice and PXB-mice.
TestIdentification: Capsid Inhibitor
CompoundsLot: PCV-CRA1.113-6
Nature: solid
Provided amount: 35 g
Storage conditions: store at <25 degrees C.
Source: Study sponsor
AnimalsSpecies: Mouse
Strain:
PXB-mouse [Genotype: eDNA-uPA+/+/SCID, uPA+/+: B6; 129SvEv-Plau, SCID:
C.B-1711cr-scid lscid Jcl, Mouse containing human hepatocytes with an
estimated replacement index of 70% or more, which is calculated based on
the blood concentration of human albumin (h-Alb)]
non-PXB grade mouse [Genotype: eDNAuPA+/+/SC1D, uPA+/+: B6; 129SvEv-
Plau, SCID: C.B-17/1cr-scid/scid Jcl, Mouse containing human hepatocytes
with an estimated replacement index of less than 70%, which is calculated
based on the blood concentration of h-Alb]
Number: 16 (PXB-mouse: 2, non-PXB grade mouse: 14)
Identification: Ear punching
AcclimationAll the candidate animals will be weighed and individual health conditions
will be checked. After this the mice will be acclimated to the study room for
at least 7 days prior to the start of the administration. During the acclimation
period, health condition observations and body weight measurements will be
conducted once a day for all the candidate animals.
GroupOn Day-7, all the candidate animals will have pre-dose blood sampling for
Assignmentthe measurements of blood h-Alb concentration and serum ALT/AST
and Criteriaactivities. These analyses will be performed using the procedures
for Animaldescribed in the “Observations, Measurement, Sampling and Other
SelectionMethods” section. The remaining serum will be stored at −80° C. until being
shipped to the Sponsor.
On Day-1, the animals with a healthy appearance and which meet all of the
criteria specified below will be assigned to the groups. To minimize
variance between the groups, the group composition will be randomized
based on the arithmetic mean values for body weight and geometric mean
values for blood h-Alb concentration.

Age: 12 to 16-weeks on Day 0
Weight: 15.6 g or more on Day −1
Blood h-Alb level: 7.0 mg/int, or more on Day −7 (for PX13-mouse) less than 7.0 mg/mL on Day −7 (for non-PXB grade mouse) Donor of hepatocytes:

Dosing

1. Group Composition

No. ofDose
MiceTestLevelConc.Volume
GroupStrain(ID)compound(1118/k8)(mg/mL)(mL/k8)RouteFrequency
1non-PXB4Capsid Inhibitor454.510p.o.BID, 28 clays
(101-Days 0 to 27
104)
2non-PXB4Capsid Inhibitor13513.510p.o.BID, 28-days
(201-Days 0 to 27
204)
3PXB2Capsid Inhibitor40540.510p.o.BID. 28-days
(301-Days 0 to 27
302)
non-PXB2Capsid Inhibitor40540.510p.o.BID, 28-days
(303-Days 0 to 27
304)
4non-PXB4Vehicle0010p.o.BID, 28-days
(401-Days 0 to 27
404)

2. Preparation of 0.5% w/v Methocel E50 dispersion
    • 1) 1 g of Tween 80 will he weighed into a beaker (Vessel 1), and 40 mL of purified water, pre-heated to range 70° C.±5° C., will be added to Vessel 1 and the vessel will be hold at this temperature.
    • 2) The Tween 80 in Vessel 1 will be dissolved at range 70° C. ±5° C. in 3 minutes to obtain a clear solution.
    • 3) 0.5 g of Methocel E50 will be weighed and added over a period of minute to the Tween 80 solution in Vessel 1, whilst mixing to create a vortex. The contents in Vessel 1 will be mixed for 5 minutes at range 70° C.±5° C. to form a consistent dispersion of Methocel E50.
    • 4) 50 mL of purified water at ambient temperature will be added to Vessel 1. The contents will be mixed with avoidance of excessive frothing to obtain a clear Methocel E50 dispersion, After that the Methocel E50 dispersion will be cooled to range 20° C.±3° C. whilst stirring. A cold water bath may be used to speed up the cooling rate.
    • 5) The contents of Vessel 1 will be transferred into graduated measuring cylinder and adjusted with water to 100 mL. The cylinder will be sealed and the contents mixed for 1 minute by repeated inversion of the measuring cylinder.
    • 6) 0.5% w/v Methocel E50 dispersion will be stored at 4° C. for up to 1 week.

3. Preparation of the Dose Formulations

    • 1) CMP drug substance will be weighed and transferred into a mortar.
    • 2) 1 mL of 0.5% Methocel E50 dispersion will be added drop-wise and the capsid inhibitor powder will be mixed with a pestle to make a capsid inhibitor paste.
    • 3) A further 4 mL of 0.5% Methocel E50 dispersion will be added drop-wise whilst mixing with the pestle to make a pourable capsid inhibitor slurry. The slurry will be transferred into a tared glass vial.
    • 4) The mortar and pestle will be rinsed with 3.0 mL volumes of 0.5% Methocel E50 dispersion and the rinses will be added to the capsid inhibitor slurry.
    • 5) The final weight will he adjusted with 0.5% Methocel E50 dispersion to 9.33 g in the tared glass vial.
    • 6) Using a homogenizer (MH-1000, As One Corporation, Osaka, Japan), the white capsid inhibitor suspension will be mixed for 2 minutes at 8000 rpm.
    • 7) The dose formulations will be stored at room temperature for 24 hours and stirred during the dosing to ensure the homogeneity of the suspension.

4. Dose Administration

    • All doses will be calculated based on the individual body weights of the mice which are taken prior to the 1st (first) administration on the days of dosing. The dose volume factor will be 10 ml/kg. All the subject mice will receive an oral dose of the dose formulation via gavage twice a day (approx. 8 pm and 8 am; dosing times will be recorded) for 28 days from Days 0 to 27 using disposable plastic sondes (Fuchigami Kikai Co., Kyoto, Japan) and disposable 1.0 mL plastic syringes (Terumo Corporation, Tokyo, Japan).

5. Storage Conditions for the Remaining Dose Formulation

    • Dose formulations will be prepared daily. After dosing, a -100 RL sample of the remaining dose formulation will be stored at <25 degrees Celsius until the completion of all data analysis from the study, to enable the quantification of a capsid inhibitor dosed, if necessary. Any additional unused dose formulation will be disposed of according to the chemical waste disposal regulations at PhoenixBio.

Observations, Measurement, Sampling and Other Methods

The first day of administration will be set as Day 0. The following observations, measurements and samplings will be conducted:

1. General Condition Observation Detailed observations of general condition will be conducted once a day prior to Pre-1st dose blood sampling, 1st administration on days of dosing and terminal blood sampling.
2. Body Weight Measurement Individual body weights will be taken once a day prior to Pre-1° dose blood sampling. 1° administration on days of dosing and terminal blood sampling.

3. In-Life Phase Sample Collections

A detailed blood collection schedule is as follows:

Blood Volume
( μL)
Serum Volume
h-Alb(μL)
DayTime pointSubject animal(μL)ALT/ASTPK
0Pre-1st doseAll animals100240
7Pre-1st dose#1 and #3 animals15022040
3 hours post-#2 and #4 animals15022040
1st dose
14Pre-1st dose#1 and #3 animals15022040
3 hours post-#2 and #4 animals15022040
I″ dose
21Pre-1st dose#1 and #3 animals100240
3 hours post-#2 and #4 animals100240
1st dose
1 hour Post#1 animal≧400220≧140
2nd dose
273 hours Post-#2 animal≧400220≧140
2nd dose
6 hours Post#3 animal≧400220≧140
2nd dose
2812 hours #4 animal≧400220≧140
Post-2nd dose

At each time point on Days 0, 7, 14 and 21, target volume of blood will be collected under isotlurane (Escain, Mylan, Osaka, Japan) anesthesia from all animals via the retro-orbital plexus/sinus using calibrated pipettes (Drummond Scientific Company. PA. USA). Two microliters (2 μL) from the collected blood will be used for these measurements. The remaining blood will be centrifuged to separate serum.

At 1 hour, 3 hours and 6 hours post-2′d dose on Day 27 and at 12 hours post 2nd dose on Day 27 (Day 28), all the subject animals will he anesthetized with isoflurane and a minimum of 400 μL of blood will be collected from each animal via the heart into syringes after which the animals will be sacrificed by cardiac puncture and exsanguination. Two microliters (2 μL) from each collected blood sample will he centrifuged to separate serum.

Necropsy will be performed after the whole blood has been collected at sacrifice. Individual whole livers will be harvested, blot-dried, divided into 6 approximately equal sized pieces, weighed, then transferred into a tube and flashed frozen in liquid nitrogen. The frozen liver samples will be stored at −80° C. until being shipped to the Sponsor.

4. Serum Separation

The individual blood samples of the animals will be transferred to labeled blood collection tubes and left to coagulate at room temperature for at least 5 minutes and then centrifuged at 13200×g, 4° C. for 3 minutes to obtain scrum.

Target volume of serum from each separated serum sample will be transferred into a separate, labeled microtube.

These serum samples will he stored at −80° C. until use and being shipped to the Sponsor.

5. Laboratory Investigations

The blood h-Alb concentration will be measured by PhoenixBio using latex agglutination immuronephelometry (LX Reagent “Hiken” Alb II, Eiken Chemical Co.. Ltd., Tokyo, Japan). Serum ALT/AST activities will be determined using Drichem 7000 (Fujifilm, Tokyo, Japan).

6. Adverse Events

If unexpected abnormalities such as weight loss of more than of the initial body weight, moribundity or death are observed during the in-life phase. PhoenixBio will report the details of such an incident to the text missing or illegible when filed

APPENDIX 1
Study Schedule
Blood Volume (μL)
SerumSample List
Subjecth-AlbVolume (μL)(tubes)
DayTime PointScheduleanimal(μL)ALT/ASTPKSerumLiver
−7Pre-dose bloodAll the150260204016
samplingcandidate
−1Group assignmentAll the
candidate
Pre-1st doseSerial blood All animals1002404016
sampling
01st AdministrationAll animals
2nd AdministrationAll animals
1st AdministrationAll animals
2nd AdministrationAll animals
21st AdministrationAll animals
2nd AdministrationAll animals
31st AdministrationAll animals
2nd AdministrationAll animals
41st AdministrationAll animals
2nd AdministrationAll animals
51st AdministrationAll animals
2nd AdministrationAll animals
1st AdministrationAll animals
2nd AdministrationAll animals
Pre-1st doseSerial blood #1 and #31502602040
samplinganimals
1st AdministrationAll animals
3 hours Post-Serial blood #2 and #415026020408
1st dosesamplinganimals
2nd AdministrationAll animals
1st AdministrationAll animals
2nd AdministrationAll animals
91st.AdministrationAll animals
2nd AdministrationAll animals
101st AdministrationAll animals
2nd AdministrationAll animals
111st AdministrationAll animals
2nd AdministrationAll animals
121st AdministrationAll animals
2nd AdministrationAll animals
131st AdministrationAll animals
2nd AdministrationAll animals
Pre-1stSerial blood#1 and #315026020408
dsamplinganimals
1st AdministrationAll animals
143 hoursSerial blood #2 and #41502602040
Post-1stsamplinganimals
2nd AdministrationAll animals
151st AdministrationonAll animals
2nd AdministrationAll animals
161st AdministrationAll animals
2nd AdministrationAll animals
171st AdministrationAll animals
2nd AdministrationAll animals
181st AdministrationAll animals
2nd AdministrationAll animals
191st AdministrationAll animals
2nd AdministrationAll animals
201st AdministrationAll animals
2nd AdministrationAll animals
Pre-1stSerial blood #1 and 4310024040
dosesamplinganimals
211st AdministrationAll animals
ThreeSerial blood #2 and #4100240408
hourssamplinganimals
2nd AdministrationAll animals
221st AdministrationAll animals
2nd AdministrationAll animals
231st AdministrationAll animals
2nd AdministrationAll animals
241st AdministrationAll animals
2nd AdministrationAll animals
251st AdministrationAll animals
2nd AdministrationAll animals
261st AdministrationAll animals
2nd AdministrationAll animals
1st AdministrationAll animals
2nd AdministrationAll animals
271 hourTerminal blood#1 animal≧4002≧16020≧140
Post-2ndNecropsy4 I animal24
dose
3 hoursTerminal blood#2 animal≧4002≧16020≧140
Post-2ndNecropsy#2 animal24
dose
6 hoursTerminal blood#3 animal≧4002≧16020≧140
Post-2ndNecropsy#3 animal24
dose
2812 hoursTerminal blood#4 animal≧4002≧16020≧140
Post-2ndNecropsy#4 animal24
dose