BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to novel doses and dosing regimens for the administration of 2-amino-9- [2- [bis (2,2, 2-trifluoroethoxy) phosphonylmethoxy] ethyl]- 6- (4-methoxyphenylthio) purine (LY582563) to treat hepatitis B virus (HBV) infections in human patients. The invention is particularly advantageous for lowering the plasma HBV DNA levels of patients infected with HBV, and ameliorating symptoms, conditions, or disorders associated with HBV infections in humans.

Description of Related Art Hepatitis B is a potentially fatal liver disease caused by infection with hepatitis B virus, a partially double-stranded DNA virus of the Hepadnaviridae family. It is estimated that some 350 million individuals in the world are chronically infected with HBV, and about 1 million individuals die annually as a direct result of HBV-induced cirrhosis or liver cancer.

HBV is a hepatotropic virus that replicates in the liver, resulting in acute and chronic liver disease including liver fibrosis, cirrhosis, inflammatory liver disease, and hepatic cancer that can lead to death in some patients (W. K. Joklik, Virology, Third Edition, Appleton & Lange, Norwalk, Connecticut, 1988). The virus is a blood- borne pathogen, which is transmitted by exposure to infectious body fluids in a fashion similar to human immunodeficiency virus (HIV). However, HBV is much more infectious than HIV. Although effective vaccines are available, they have no therapeutic value for those already infected with the virus. Estimates are that 15 to 20% of individuals who acquire the chronic infection develop cirrhosis or another chronic disability from HBV infection. For patients with compensated cirrhosis, the

survival is 84% at five years and 68% at ten years. In carriers with decompensated cirrhosis, five year survival is only 14% (Lok et al. (2001) Hepatology 34: 1225- 1241).

Alpha-interferon therapy has been available for the treatment of chronic hepatitis B for some time. However, it is effective in fewer than 40% of patients, and has dose-limiting side effects such as flu-like symptoms, weight loss, depression, and cytopenias. Lamivudine and adefovir are new compounds currently in development for us6 in HBV treatment. Unfortunately, the emergence of lamivudine-resistant viruses developing in up to 70% of patients after four years has been observed. It is therefore necessary and of high priority to find improved and effective anti-HBV anti- hepatitis therapies (Locamini et. al. (1996) Antiviral CS1ençistry & Chemotherapy 7 (2): 53-64).

LY582563 (2-amino-9- [2- [bis (2,2, 2-trifluoroethoxy) phosphonyl- methoxy] ethyl]-6- (4-methoxyphenylthio) purine) is a recently discovered phosphonate nucleotide analog (see U. S. Patent No. 5,840, 716) currently under development as an anti-HBV agent. LY582563 is a derivative of 9- [2- (phosphonyl- methoxy) ethyl] adenine (PMEA; adefovir) with improved oral absorption and antiviral activity. In vitro, LY582563 has demonstrated greater activity against HBV than lamivudine or PMEA. Although some of the beneficial effects of LY582563 in the treatment of HBV infections are known, optimization of treatment with this agent would be of significant therapeutic value.

SUMMARY OF THE INVENTION Accordingly, the present invention is directed to novel pharmaceutical compositions comprising LY582563, as well as novel methods of treating HBV infections in humans, including optimal dosing regimens for the administration of LY582563 to treat HBV infected patients.

Thus, in a first aspect, the present invention provides a pharmaceutical composition for oral administration in dosage unit form, comprising: about 2.5 mg to about 20 mg of 2-amino-9- [2- [bis (2,2, 2- trifluoroethoxy) phosphonylmethoxy] ethyl]-6- (4-methoxyphenylthio) purine per dosage unit, and a pharmaceutically acceptable carrier, diluent, or excipient.

The composition can be in the form of a tablet or capsule containing the purine in an amount of about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, or about 20 mg per dosage unit, or in an amount in the range between about 5 mg and about 10 mg, or between about 7.5 mg and about 10 mg, per dosage unit. In each case, the upper limit of these ranges may be extended to about 12 or about 12.5 mg per dosage unit.

In another aspect, the present invention provides the use of 2-amino-9- [2- [bis (2,2, 2-trifluoroethoxy) phosphonylmethoxy] ethyl]-6- (4-methoxyphenylthio) purine for the preparation of a medicament for treating a human patient suffering from a hepatitis B virus infection, wherein: the medicament is formulated for oral administration, and the medicament is in dosage unit form and comprises, per dosage unit, about 2.5 mg to about 20 mg of this purine.

The medicament can be in the form of a tablet or capsule containing the purine in an amount of about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, or about 20 mg per dosage unit, or in an amount in the range between about 5 mg and about 10 mg, or between about 7.5 mg and about 10 mg, per dosage unit. In each case, the upper limit of these ranges may be extended to about 12 or about 12.5 mg per dosage unit.

In another aspect, the present invention provides a method of treating a human patient suffering from a hepatitis B virus infection, comprising administering to said patient a total amount of 2-amino-9- [2- [bis (2,2, 2-trifluoroethoxy) phosphonyl- methoxy] ethyl]-6- (4-methoxyphenylthio) purine in the range of from about 2.5 mg to about 20 mg of the purine per day.

In this method, the purine can be administered to the patient for a period of time sufficient to lower the plasma level of HBV DNA of the patient compared to the plasma level of HBV DNA of the patient prior to administering the purine.

Preferably, the plasma level of HBV DNA of said patient is lowered to at least about 104 copies/mL compared to the plasma level of HBV DNA of the patient prior to administering the purine. Alternatively, the purine can be administered to the patient for a period of time sufficient to treat or ameliorate a symptom, condition, or disorder caused by a hepatitis B virus in the patient, for example liver fibrosis, cirrhosis, inflammatory liver disease, or hepatic cancer.

In any of the foregoing methods, the purine can be administered to the patient in the form of a pharmaceutically acceptable oral composition, which can be a tablet or capsule. Furthermore, the period of time over which the purine is administered can

be several days, several weeks,, several months, or several years, and the purine can be administered to the patient in a total amount of about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, or about 20 mg per day, or in an amount in the range between about 5 mg and about 10 mg per day, or between about 7.5 mg and about 10 mg per day. In each case, the upper limit of these ranges may be extended to about 12 or about 12.5 mg per day. These amounts of purine can be administered in a single dose, or in divided subdoses totaling the total amount per day.

'Further scope of the applicability of the present invention will become apparent from the detailed description provided below. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the present invention, are given by way of illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS The above and other aspects, features, and advantages of the present invention will be better understood from the following detailed description taken in conjunction with the accompanying drawings, all of which are given by way of illustration only, and are not limitative of the present invention, in which: Figure 1 shows the Profile of Mean Log-change in HBV DNA with standard deviation bars: (BID groups).

Figure 2 shows the Profile of Mean Log-change in HBV DNA with standard deviation bars: (QD groups).

Figure 3 is a scatter plot showing the Log-Change in HBV DNA on Day 29 with mean and standard deviation bars. The individual open symbols, i. e. , squares, triangles, etc. , represent individual patient data points. These differ to show some differences between the columns of data at different daily doses, although this is not really necessary as the columns are clearly differentiated. The solid symbol in each column represents the mean. The outer limits of the bars represent the standard deviation from that mean.

Figure 4 shows the Log-Change in HBV DNA on Day 29 with Fitted Emax model based on Total Daily Dose (TDD).

Figure 5 shows the Maximum Log-Change in HBV DNA during treatment period with fitted Emax model based on Total Daily Dose (TDD).

Figure 6 shows the area under the curve (AUC) of Log-Change in HBV DNA after first dose with fitted Emax model based on Total Daily Dose (TDD).

DETAILED DESCRIPTION OF THE INVENTION The following detailed description of the invention is provided to aid those skilled in the in practicing the present invention. Even so, the following detailed description should not be construed to unduly limit the present invention as modifications and variations in the embodiments discussed herein can be made by those of ordinary skill in the art without departing from the spirit or scope of the present inventive discovery.

The contents of each of the references cited herein are herein incorporated by reference in their entirety.

Current treatment choices for chronic HBV infection are limited in number and clinical utility due to issues of tolerability and the emergence of resistant viral variants. LY582563, the structure of which is shown below, is a new purine nucleotide analogue prodrug that has shown potent activity against HBV in preclinical studies (Ono-Nita et. al. (2002) Antimicrob. Agents Chemother. 46 (8): 2602-5; Wise et. al. (2002) J. Gastroenterol. Hepatol. 17 (suppl): A46).

LY582563

For oral administration of LY582563, U. S. Patent 5,840, 716 teaches that the clinical dose may generally be 0.1 mg to 500 mg/kg per day, preferably 1 mg to 50 mg/kg per day, of the compound for an adult. For an adult weighing 68 kg (approximately 150 lbs), these ranges are approximately 6.8 mg to approximately 34 g per day, preferably approximately 68 mg to approximately 3.4 g per day. The '716 patent notes that these doses may be appropriately increased or decreased depending on age, conditions or symptoms, or the presence or absence of a co-administered drug.

In addition, U. S. Patent 5,840, 716 teaches that the above daily doses may be administered once a day, or dividedly administered twice or several times a day with appropriate intervals. Finally, continual administration may be carried out at intervals of several days.

Determination of a safe and effective dose of any new pharmaceutical agent, and an appropriate treatment regimen employing the drug, is an empirical process.

This process generally requires treating afflicted patients with varying doses of the agent, one or more times daily, over varying time periods, and monitoring effectiveness by accepted diagnostic methods. In the case of HBV, such methods can include measurement of plasma or tissue levels of HBV DNA or various antigens that comprise part of the virus, or patient response such as antibody formation (for example hepatitis B e antibody) or biochemical improvement such as decreased alanine aminotransferase (ALT) levels. In addition, one can also measure the effectiveness of treatment by monitoring the amelioration of symptoms, conditions, or disorders caused by the virus. In the case of HBV, these include fatigue, anorexia, jaundice, liver fibrosis and inflammation, cirrhosis, and hepatic cancer (hepatocellular carcinoma). In addition to demonstrating efficacy, however, one must also demonstrate acceptable safety, which includes acceptable toxicity. Toxicities that have been observed with other HBV antivirals include mitochondrial toxicity, renal toxicity, and bone marrow toxicity. The relationship between dose and treatment regimen for a new drug entity, and effectiveness and safety, can only be determined by clinical testing in humans. Therefore, determination of dose and treatment regimen parameters cannot be predicted or deduced, and the results are necessarily novel and unobvious. Particular attention must be paid to selecting treatment parameters that, while being effective to treat a disease, do not cause other harm to the patient. Given these considerations, the findings disclosed herein relating to the use of LY582563 in treating HBV infections in human patients are surprising, and could

not have been predicted before the present studies were conducted. These results provide a rational basis for safe and effective dosing and treatment regimens employing LY582563 in the treatment of HBV.

, The studies disclosed herein were undertaken to determine optimal doses and dosing regimens for treating HBV-infected patients with LY582563, thereby maximizing the safety and therapeutic benefits obtainable with this antiviral pharmaceutical agent. Briefly, patients with chronic HBV infection were treated with ( LY582563 in a randomized, placebo controlled, dose escalating study over 28 days.

Doses of LY582563 included 2.5 mg, 5 mg, and 10 mg QD or BID, and 20 mg QD.

A placebo was also employed. HBV DNA in treated patients was measured during dosing and for up to twelve weeks of follow-up. Safety data, e. g. , clinical signs (e. g., blood pressure, pulse, respiratory rate, and general physical findings), adverse events (e. g. + upper abdominal pain, diarrhea, nausea, headache, fatigue, and alopecia), ECGs, hematology, biochemistry measures in blood (e. g. , aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactic acid, blood urea nitrogen (BUN), creatinine, and bilirubin), as well as highly sensitive urinary markers of renal tubular toxicity (lactate dehydrogenase (LDH), urinary creatinine, and urinary beta-N-acetyl glucosaminidase (beta-NAG) ), were also measured. The data obtained demonstrate that LY582563 exhibits a favorable safety profile and effective HBV antiviral activity in chronically infected patients, and suggest optimal dose levels and dosing regimes for long term safe and effective use of this new HBV antiviral agent. Based on the present results, an appropriate dose of-LY582563 balancing both safety and efficacy is between about 2.5 mg and about 20 mg per patient per day. These doses may have to be administered to patients over the course of several days, several weeks, or several months or years in order to ameliorate or control undesirable pathogenic consequences of HBV infection.

Dosing Regimen for the Treatment of HBV with LY582563 The oral dosing regimen for treating a patient suffering from HBV with LY582563 is generally selected in accordance with a variety of factors, including the age, weight, sex, diet, and medical condition of the patient, genotype of the infecting virus, the severity of the infection, and pharmacological considerations such as the activity, efficacy, pharmacokinetic, and toxicology profiles of LY582563.

Administration of LY582563 should generally be continued over a period of several

hours, days, or weeks to several months or years until virus titers reach acceptable levels, or until one or more indicia, symptoms, conditions, or disorders present in a patient due to hepatitis B virus infection has been ameliorated or completely eliminated, indicating that infection has been controlled or eradicated.

With respect to reduction in plasma HBV DNA levels, the long term goal is currently a matter of debate, with some workers in the field proposing that a desirable level is below 104 copies/mL. The duration for this level of HBV DNA has yet to be defined.

Representative indicia of HBV infection other than plasma HBV DNA levels include, but are not limited to, liver fibrosis, cirrhosis, inflammatory liver disease, and hepatic cancer. See Hollinger et al. (2001) in Fields Virology, Fourth Ed. , Vol. 2, David M. Knipe et al. , Eds. ,"Hepatitis B Virus, "Chapter 87, pp. 2971-3036, Lippincott, Williams, & Wilkins, Philadelphia, PA for a review. In most parts of the world, liver fibrosis, etc. , is diagnosed via liver biopsy ; in most Asian countries, liver biopsies are not routinely performed, and medical practitioners usually rely on clinical measures to make presumptive diagnoses.

Patients undergoing treatment with LY582563 can be routinely monitored by measuring HBV DNA levels in their serum by, for example, slot-blot, dot-blot, or PCR techniques, or by measurement of HBV antigens, such as HBV surface antigen (HBsAg) and HBV e antigen (HBeAg), in serum and tissue to determine the effectiveness of therapy. Methods therefor are discussed in Hoofnagle et al. (1997) New Engl. Jour. Med. 336 (5): 347-356 ; F. D. Hollinger (1996) in Fields Virology, Third Ed. , Vol. 2, Bernard N. Fields et al. , Eds. ,"Hepatitis B Virus, "Chapter 86, pp.

2738-2807, Lippincott-Raven, Philadelphia, PA; and Hollinger et al. (2001) in Fields Virology, Fourth Ed. , Vol. 2, David M. Knipe et al. , Eds. ,"Hepatitis BVirus,"Chapter 87, pp. 2971-3036, especially pages 2989-2991, Lippincott, Williams, & Wilkins, Philadelphia, PA, and the references cited therein. In chronic hepatitis B, remissions are characterized by the disappearance of HBV viral DNA, i. e. , reduction to undetectable levels as measured by, for example, hybridization tests capable of detecting levels 2105 genomes per ml of serum, or HBeAg from serum despite the continued presence of HBsAg. These serologic events are followed by improvement in the biochemical and histologic features of the disease. The end point of successful treatment in most trials of antiviral therapy is the disappearance of HBeAg and viral DNA from serum. In patients in whom the e antigen disappears, remission is usually

sustained, and results in an inactive HBsAg carrier state. Many caucasian patients, most of whom acquire the disease in adulthood and receive interferon therapy, eventually become HBsAg-negative (see Hoofnagle et al. (1997) New Engl. Jour.

Med. 336 (5): 347-356 for a review). However, this is not representative of about 90% of the world's HBV population. Continuous analysis of the data obtained by such methods permits modification of the treatment regimen during therapy so that an optimal amount of LY582563 is administered, and so that the duration of treatment can be determined as well. Thus, the treatment regimen/dosing schedule can be rationally modified over the course of therapy so that the lowest amount of LY582563 which exhibits satisfactory antiviral effectiveness is administered, and so that administration is continued only so long as is necessary to successfully treat the infection.

In the context of the present invention, the terms"ameliorate,""treat," "treatment, ""therapeutic use,"or"treatment regimen"as used herein are meant to encompass prophylactic, palliative, and therapeutic modalities of administration of LY582563, and include any and all uses of this purine that remedy an indicium, a disease state, a condition, a symptom, or a disorder caused by a hepatitis B virus, or which prevent, hinder, retard, or reverse the progression of indicia, symptoms, conditions, or disorders associated with HBV infection. Thus, any prevention, improvement, alleviation, reversal, or complete elimination of an undesirable indicium, disease state, symptom, condition, or disorder associated with HBV infection is encompassed by the present invention. The term"dosage unit"as used herein refers to an individual delivery vehicle, for example, but not limited to, a tablet or capsule, for administration of a dose of the active LY582563 purine.

The following example is provided to illustrate various aspects of the present invention, and should not be construed to be limiting thereof in any way.

Example 1 Multiple Dose-Escalation Study in Healthy Subjects and Patients with Chronic Hepatitis B Infection A randomized, single period, multiple dose-escalation, single-blind study of LY582563 was carried out in 2 parts. Part 1 was conducted in healthy subjects, and Part 2 was conducted in patients with compensated chronic HBV infection (hereafter

multiple doses of LY582563. This was the first multiple dose study in humans.

Dose escalation was carried out in conservative increments in healthy subjects who were given BID regimen for a 2-week period. Safety data were reviewed prior to each dose escalation.

Part 1 (Healthy Subiects) 'In part 1, there were 4 groups with 8 subjects in each group (6 subjects on study drug and 2 on placebo). Table 1 shows the characteristics of the subjects in this study. The LY582563 doses were: 2.5 mg BID, 5 mg BID, 10 mg BID and 15 mg BID. Approximately 24 hours after a single dose of LY582563, multiple BID dosing for approximately 14 days of LY582563 was administered, except for the last day of dosing which was administered QD. There was a dose escalation for each consecutive group of subjects. Safety and other data, as appropriate, were reviewed prior to each dose escalation step. Plasma concentrations of LY582563 and its metabolites (602074,602075 and 602076) were measured up to 24 hours after a single dose of LY582563 and after the last dose by a validated LC/MS/MS method (Advion BioSciences, Inc. , Ithaca, NY). Subjects were followed up at regular intervals for approximately 4 weeks after the last dose of LY582563.

Table 1. Characteristics of Healthy Subjects LY582563 Dose 2.5 mg BID 5.0 mg BID 10 mg BID 15 mg BID Placebo No. of Patients, n 6 6 6 6 8 Age (years) 23. 5+1. 8 23. 5+2. 6 23. 0+1. 1 22. 8+1. 0 23. 4+2. 3 Male : Female 6 : 0 6 : 0 6 : 0 6 : 0 8 : 0 Ethnicity, n (% Chinese) 4 (67%) 4 (67%) 6 (100%) 5 (83%) 7 (87%) BMI, kg/M2 21. 7+1. 0 20. 9+1. 6 25, 8+1. 1 23. 72. 5 24. 6+2. 8 Part 2 (Patients) In part 2, there were 7 groups with 8-12 patients in each group. Table 2 shows the characteristics of the subjects in this study. The LY582563 doses were: 2.5 mg BID (n=8), 5 mg BID (n = 8), 10 mg BID (n = 8), 2.5 mg QD (n = 8), 5 mg QD (n = 11), 10 mg QD (n = 12) and 20 mg QD (n = 11). Approximately 24 hours after a

single dose of LY582563 (day 1), multiple BID dosing for approximately 28 days (i. e. , day 2 to day 29) of LY582563 was administered, except for the last day (day 29) of dosing, which was QD. Patients were only studied following review of safety data from healthy subjects at the same dose level in Part 1. Patients were followed up at regular intervals for approximately 12 weeks after the last dose of LY582563. Plasma concentrations of LY582563 and its metabolites (602074,602075 and 602076) were sampled up to 24 hours after a single dose of LY582563 and after the last dose.

Subjects were followed up at regular intervals for approximately 12 weeks after the last dose of LY582563.

Table 2. Patient Characteristics LY582563 Dose 2.5 mg 5.0 mg 10 mg 2.5 mg 5.0 mg 10 mg QD 20 mg QD Placebo BID BID BID QD QD No. of 6 6 6 6 7 9 9 16 Patients, n Age (years) 32.5~7.7 35.7~6.0 38.8~9.0 28.0~4.6 28.4~7.6 34.9~11.5 38.4~10.2 34.2~8.8 Male:Female 6:0 6:0 5:1 6:0 7:0 9:0 9:0 14:2 ethnicity, 5 (83%) 5 (83%) 6 (100%) 6 (100%) 6 (86%) 9 (100%) 8 (89%) 16 (100%) n (% Chinese) BMI, kg/m2 23.4~2.3 25.4~3.2 25.1~3.1 23.8~2.4 24.6~3.1 24.0~3.3 23.8~3.4 22.7~3.0 HBe Ag 6 (100%) 6 (100%) 3 (50%) 5 (83%) 6 (86%) 5 (56%) 6 (67%) 12 (75%) positive, n (%) HBVDNA*(pg /mL) - Geometric 1.19x109 2.71x109 5.03x108 2.92x108 6.24x108 3.73x108 7.39x108 6.78x108 Mean - Range 3.17x108 7.99x107 4.64x105 8.02x106 2.85x105 2.46x106 1.58x105 2.95x105 - - - - - - - - 3.69x109 5.69x109 2.20x109 7.50x108 1.85x109 8.22x108 2.80x109 3.29x109 *: HBV DNA Baseline = Geometric mean of HBV DNA at screening, -28 day, -14 day and predose.

Treatments LY582563 and matching placebos were supplied as either 2.5 mg, 10 mg, or 20 mg tablets. LY582563 and matching placebo tablets were supplied by Mitsubishi Pharma Corporation, Tokyo, Japan.

LY582563 and placebo formulations for oral administration were as follows: Table 3.2. 5 mg LY582563 Tablet Quantity Ingredient (mg/tablet) Function Active Ingredient LY582563 2.5 Active ingredient Other Ingredients D-mannitol 92.5 Diluent Corn starch 23.1 Diluent Hydroxypropylcellulose 3.1 Binder Low substituted 1. 3 Disintegrant hydroxypropylcellulose Magnesium stearate 2. 5 Lubricant Hydroxypropylmethyl-3. 35 Coating agent cellulose 2910 Propylene Glycol 0.77 Coating agent Titanium Dioxide 0.33 Coating agent Ta] c 0.55 Coating agent Hydrogenated Oil 0.0625 Coating agent

Table 4.10 mg LY582563 Tablet Quantity Ingredient (mg/tablet) Function Active Ingredient LY582563 10 Active ingredient Other Ingredients D-mannitol 84.5 Diluent Corn starch 21. 1 Diluent Hydroxypropylcellulose 3. 1 Binder Low substituted 3.8 Disintegrant hydroxypropylcellulose Magnesium stearate 2.5 Lubricant Hydroxypropylmethyl-3. 35 Coating agent cellulose 2910 Propylene Glycol 0.77 Coating agent Titanium Dioxide 0.33 Coating agent Talc 0.55 Coating agent Hydrogenated Oil 0.0625 Coating agent Table 5.20 mg LY582563 Tablet

Quantity Ingredient (mg/tablet) Function Active Ingredienti LY582563 20 Active ingredient Other Ingredients D-mannitol 74.5 Diluent Corn starch 18.6 Diluent Hydroxypropylcellulose 3. 1 Binder Low substituted 6.3 Disintegrant hydroxypropylcellulose Magnesium stearate 2.5 Lubricant Hydroxypropylmethyl-3. 35 Coating agent cellulose 2910 Propylene Glycol 0.77 Coating agent Titanium Dioxide 0.33 Coating agent Talc 0.55 Coating agent Hydrogenated Oil 0.0625 Coating agent

Table 6. Placebo Tablet Quantity Ingredient (mg/tablet) Function Active Ingredient' LY582563 0 Active ingredient Other Ingredients D-mannitol 90.5 Diluent Corn starch 22.6 Diluent Hydroxypropylcellulose 3.1 Binder Low substituted 6.3 Disintegrant hydroxypropylcellulose Magnesium stearate 2.5 Lubricant Hydroxypropylmethyl-3. 35 Coating agent cellulose 2910 Propylene Glycol 0.77 Coating agent Titanium Dioxide 0.33 Coating agent Talc 0.55 Coating agent Hydrogenated Oil 0.0625 Coating agent

Method of Manufacture LY582563, D-mannitol, and corn starch are mixed. Hydroxypropylcellulose dissolved in purified water is added for granulation by fluid-bed granulator. After drying, granules are screened by tornado mill. Screened granules are mixed with magnesium stearate and low substituted hydroxypropylcellulose by V-shaped mixer.

Mixed granules are compressed into tablets. Tablets are filmed coated with hydroxypropylmethyl-cellulose 2910, propylene glycol, titanium dioxide, and talc by conventional methods. Finally, tablets are coated with a little hydrogenated oil.

Dosing The 2.5 mg dose was administered as 1 tablet of 2.5 mg LY582563 or 1 matching placebo. The 5 mg dose was administered as 2 tablets of 2.5 mg LY582563 or 2 matching placebos. The 10 mg dose was administered as 1 tablet of 10 mg LY582563 or 1 matching placebo. The 15 mg dose was administered as 2 tablets of 2.5 mg LY582563 and 1 tablet of 10 mg LY582563 or matching placebos corresponding to the 2.5 and 10 mg LY582563 tablets. The 20 mg dose was administered as 1 tablet of 20 mg LY582563 or 1 matching placebo.

Each participant was randomly assigned to each dose group and to receive either LY582563 or placebo. Healthy subjects in Part 1 and patients in Part 2 were allocated according to a randomization block of 4 (3 study drug to one placebo).

Study drug was administered BID for healthy subjects in Part 1 (Table 7) and BID or QD for patients in Part 2 (Table 8).

TABLE 7. Dosing Groups for Part 1 (Healthy Subjects) Dose Group* Dose of LY582563 (mg) and n (on study n (on Dosing Schedule drug) placebo) 1 2. 5 BID 6 2 2 5 BID 6 2 3 10 BID 6 2 4 15 BID 6 2 *There was a dose escalation for each consecutive group. Safety and other data, as appropriate, were reviewed prior to each dose escalation step.

TABLE 8. Dosing Groups for Part 2 (Patients) Dose Group* Dose of LY582563 (mg) & n (on study n (on Dosing Schedule drug) placebo) 1 2. 5 BID 6 2 , 2 5 BID 6 2 3A 2. 5 QD3 1 10 BID 6 2 3B 2.5 QD 3 1 10 QD 6 2 4A 20 QD 9 2 4B 10 QD 3 1 5A 5 QD 8** 3 *The order of grouping was based on the time when the group started study drug therapy. Patients were only studied following review of safety data from healthy subjects at the same or equivalent dose level in Part 1. In this study, QD dosing for patients started with 2.5 mg (3A and 3B), followed by 10 mg (3B and 4B) ) and 20 mg (4A). It was decided that QD doses higher than 20 mg would not be studied following the review of safety data in healthy subjects of 15 mg BID dose. Therefore, 5 mg QD dose was studied in 5A group.

**lsubject of 5A dose group who was randomized to receive LY582563 was withdrawn from the study on day 1 prior to receiving study drug.

Pharmacodvnamic Assessment Blood samples were taken from hepatitis B infected patients for the measurement of plasma concentrations of HBV DNA levels in Part 2 of the study.

The level of plasma HBV DNA was determined using a validated HBV polymerase chain reaction (PCR) assay (NGI HBV SuperQuant Quantitative PCR Assay; National Genetics Institute, Los Angeles, CA). All HBV DNA levels were transformed to the log, o scale. Baseline measurements were defined as the geometric mean of the non log-transformed data obtained prior to dosing. Within-subject maximum log-changes in HBV DNA values from baseline during the treatment period were derived by substracting the minimum HBV DNA value during treatment period from the baseline value. For two patients (2108 and 3308, both on placebo treatment) where the HBV DNA values during treatment were all greater than the baseline, the maximum log-change was assigned a value of zero.

To take into consideration the rate of decline and rate of return of HBV DNA values during the study period, the area under the curve (AUC) for log change between each HBV DNA time curve and a horizontal line passing through the baseline score were computed. The HBV DNA values were first converted to baseline score when they were higher than baseline score, and area between the curve and the

horizontal baseline was computed using the trapezoidal rule to adjust for the difference in time interval between measurements.

In the analysis, three HBV DNA values that were above the specified assay range (5x109 genome copies/mL) without any dilution assays performed were assigned a value of 5x109 copies/mL, and one HBV DNA value that was below the specified assay range (100 genome copies/mL) was assigned a value of 100 copies/mL Plasma samples for HBV DNA were quantitated using the validated NGI HBV SuperQuantTM Quantitative PCR assay with a specified assay range of 100 genome copies/mL to 5x109 genome copies/mL at National Genetics Institute (Los Angeles, CA). Blood samples of approximately 5 mL were collected at screening, at approximately 4 and 2 weeks before dosing, as well as just prior to dosing; measurements were then made during the dosing period (approximately 3 days, then 1,2, 3, and 4 weeks after the first dosing) and the follow up period (5,6, 8,12 and 16 weeks after the first dosing). Depending on the level of hepatitis B viral suppression 8 weeks after dosing, subjects would have additional samples taken approximately 10 and 14 weeks after the first dosing.

On dosing days, the blood samples were taken prior to the morning dose.

Sampling times were adjusted, added, or deleted based on practical considerations, clinical needs, or need for additional pharmacodynamic data.

The means and standard deviations of HBV DNA values were obtained at each scheduled time point for each dosing group.

For the log-drop on day 29, maximum log-change and AUC for log-change, a regression model was used to compare the mean parameter values of the dosing groups. The dose level nested within dosing regimen was used as fixed effect, and baseline value used as covariate. Differences in the mean values were obtained with 95% confidence intervals. An exploratory Emax model (J. Gabrielsson and D.

Weiner (1998), Pharnmacolinetic and Pharmacodynaryic Data Analvsis : Concepts and Applications, Second Edition, Swedish Pharmaceutical Press) was also applied to the above parameters to obtained a fitted regression curve with total daily dose as explanatory variable.

Pharmacodynamic Evaluation The profiles of mean log-change in HBV DNA are shown in Figures 1 and 2.

The placebo group exhibited a consistent mean value of zero (i. e. , not significantly different from baseline) at each measurement day. For BID dosing, the 2.5, 5, and 10 mg doses of LY582563 resulted in a linear decline in log-scale of HBV DNA during the treatment period (Figure 1). The same was also observed for QD dosing groups, although the 2.5 mg dose group appeared to have a slower rate of decline, and the 5 mg group exhibited a slower rate of decline after Day 7 (Figure 2). Generally, the HBV DNA values gradually returned to baseline after the last dose of LY582563, and the time of return to the baseline or 1/2 log change of baseline appeared to increase with higher doses. The mean HBV DNA for 10 mg BID, 10 mg QD, and 20 mg QD groups was lower compared to the baseline at day 112 (Figures 1 and 2), indicating that the viral load did not return to baseline after a 3-month period.

A scatter plot of the log-change from baseline of HBV DNA is presented in Figure 3. The pair-wise comparisons of mean log-change on day 29 within each dosing regimen are listed in Table 9. Pair-wise differences were tested at 5% level of significance without any multiplicity adjustments.

Table 9. Mean Log-Change from Baseline of HBV DNA Value with 95% Confidence Interval on Day 29 Dose (mg) n Mean Difference vs. Difference vs. Difference vs. Difference Placebo 2. 5mg 5mg vs. 10mg Placebo 15 0.0639 2. 5mg BID 6 2.02 1.95 (1. 35, . 2. 56) 5. Omg BID 5 1. 98* 1.92 (1. 11, -0.04 (-1.01, 2.72) 0.94) 10mg BID 6 2.53 2.47 (2.02, 0.51 (-0.22, 0.55 (-0.36, 2.92) 1.25) 1.46) 2. 5mg QD 6 1.52 1.46 (1. 09, 1.83) 5. 0mg QD 7 1.96 1.89 (1. 13,0. 44 (-0.39, 2.66) 1.26) 1 Omg QD 9 2.49 2.43 (1. 83,0. 97 (0.30, 0.54 (-0.42, 3.03) 1.64) 1.49) 20mg QD 9 2.50 2.44 (1. 88,0. 98 (0.34, 0.54 (-0.39, 0.01 (-0.79, 3. 00) 1.62) 1.48) 0.80) Note: Confidence interval (shown in parenthesis) that does not contain zero indicates a statistically significant difference between two treatments at the 5% level.

*One subject did not have HBV DNA data on day 29.

For BID dosing, the mean log-change on day 29 was observed to be 2.02, 1.98, and 2. 53 for the 2.5, 5, and 10 mg dose groups, respectively. All mean log- changes were statistically significant when compared to those of the placebo group.

No statistically significant difference was detected between the three BID dose levels.

For QD dosing, the observed mean log-changes on day 29 were 1.52, 1.96, 2.49, and 2.50 for the 2.5, 5,10, and 20 mg dose groups, respectively. All mean log-changes were statistically significant when compared to those of the placebo group. The mean log-changes for the 10 and 20 mg dose groups were similar, and were significantly different from that of 2.5 mg group.

An Emax model was fitted to the log-change with total daily dose as the explanatory variable (Figure 4), facilitating understanding the relationship between pharmacodynamic effects and total daily dose. The fitted regression curve suggests that a plateau was achieved for HBV DNA drop at approximately 10 mg total daily dose.

The pair-wise comparisons of mean maximum log-change in HBV DNA values during the treatment period within each dosing regimen are listed in Table 10.

Pair-wise differences were tested at 5% level of significance without any multiplicity adjustments.

Table 0. Mean Maximum Log-Change from Baseline of HBV DNA Value with 95% Confidence Interval During Treatment Period (Day 1 to Day 29) Dose (mg) n Mean Difference vs. Difference vs. Difference vs. Difference vs. Placebo | 2. 5mg 5mg 10mg Placebo 16 0. 34 2. 5mg BID 6 2.39 2.05 (1. 50, 2.60) 5. Omg B1D 6 1.82 1.48 (0. 75,-0. 57 (-1. 45, 2.21) 0.30) 10mg BID 6 2.93 2.59 (2.03, 0.54 (-0.21, 1. 11 (0.22, 3. 14) 1. 29) 2.00) 2. 5mg QD 6 1.53 1. 19 (0.82, 1.57) 5. Omg QD 7 2.19 1.85 (1. 33,0. 66 (0.06, 2.37) 1.25) 10mg QD 9 2.58 2.24 (1. 66, 1. 04 (0.39, 0.39 (-0. 35, 2.81) 1.69) 1.13) 20mg QD 9 2.68 2.34 (1. 79,1. 14 (0.52, 0.49 (-0.23, 0. 10 (-0.66, 2. 88) 1.77) 1. 21) 0.86) Note: Confidence interval (shown in parenthesis) that does not contain zero indicates a statistically significant difference between two treatments at the 5% level.

For BID dosing, the mean maximum log-changes during the treatment period were observed to be 2.39, 1.82, and 2.93 for the 2.5, 5, and 10 mg dose groups, respectively. All mean maximum log-drops were statistically significant when compared to that of the placebo group. Although there appeared to be a significant difference in mean log-change between the 10 mg and 5 mg dose groups, there was no significant difference between the 10 mg and 2.5 mg dose groups. For QD dosing, the observed mean maximum log-changes during the treatment period were 1.53, 2.19, 2. 58, and 2.68 for the 2.5, 5,10, and 20 mg dose groups, respectively. All mean log- drops were statistically significant when compared to that of the placebo group. The mean log-change for the 5,10, and 20 mg dose groups appeared to be similar, and were significantly different from that of 2.5 mg group.

At 10 mg BID, 10 mg QD, and 20 mg QD, all patients demonstrated a maximum log change in HBV DNA greater than 1.5 during the treatment period.

An Emax model was fitted to the maximum log-change with total daily dose as the explanatory variable (Figure 5). The fitted regression curve suggests achievement of a plateau for maximum HBV DNA drop during treatment at approximately 10 mg total daily dose.

For further clarification, Figure 4 shows the log-change in HBV DNA on day 29 with fitted EmaX model based on total daily dose (TDD); Figure 5 shows the maximum log-change in HBV DNA over the treatment period with fitted Emax model based on TDD. Both figures need to be taken into consideration as the maximum log- change for some patients occurred at times other than at day 29. Taken together, these figures permit a better understanding of the profile of pharmacodynamic effect, i. e. , HBV DNA change, with dose.

The pair-wise comparisons of mean change in AUC after the first dose until the end of the study period (day 112) within each dosing regimen are listed in Table 11. Pair-wise differences were tested at the 5% level of significance without any multiplicity adjustments.

Table 11. Mean AUC of Log-Change from Baseline of HBV DNA Value with 95% Confidence Interval After First Dose (Day 1 to Last Visit) Dose (mg) n Mean Difference vs. Difference vs. Difference vs. Difference vs. Placebo 2. 5mg 5mg 10mg Placebo 15 18.4 2. 5mg 6 76.2 57.7 (31.9, 83.6) BID 5. 0mg 5 91. 6 73.1 (34.9, 111) 15.4 (-24. 0, BID 54.7) lOmg BID 6 135 117 (71.7, 162) 59.1 (11.2, 43.7 (-12.0, 107) 99.4) 2. 5mg QD 6 53.4 34.9 (18.0, 51.9) 5. 0mg QD 7 75.2 56.7 (26.4, 87.0) 21.8 (-5.33, 48.9) lOmg QD'9 142 123 (58.7, 188) 88.5 (25.1, 66.7 (-1.43, 152) 135) 20mg QD 9 125 106 (35.5, 177) 71.1 (1. 84,' 49.4 (-24. 4,-17. 4 (-111, 140) 123) 76.0) Note: Confidence interval (shown in parenthesis) that does not contain zero indicates a statistically significant difference between two treatments at the 5% level.

For BID dosing, the mean AUC for log-change after the first dose for the 2.5, 5, and 10 mg dose groups was observed to be statistically significant when compared to that of the placebo group. A significant difference in mean AUC for log-change between the 10 mg and 2.5 mg dose groups was also observed. For QD dosing, the observed mean AUC for log-change for the 2.5, 5,10, and 20 mg dose groups was statistically significant compared to that of the placebo group. The mean AUC for log-change for the 10 and 20 mg dose groups appeared to be similar, and were significantly different from that of the 2.5 mg group. A trend of significant difference between the 10 mg and 5 mg doses was also observed.

An Emax model was fitted to the AUC for log-change with total daily dose as the explanatory variable, similar to the E,,, x model for log-change in HBV DNA (Figure 6). The fitted regression curve suggests that a 10 mg total daily dose would achieve 80% of the 20 mg total daily dose's effect on AUC log-change in HBV DNA.

Conclusions Regarding Efficacy of LY582563 As demonstrated by the data presented above, all doses of LY582563 tested in this study were associated with a reduction of plasma HBV DNA during the treatment period, and the reduction appeared to be dose-dependent. The 10 mg QD, 10 mg BID, and 20 mg QD dose groups showed the greatest viral suppression, as seen in the mean

drop of HBV DNA on day 29 (Figure 3), the mean maximum log-viral decline (Table 10), and the mean AUC change in log-viral decline after first dosing (Table 11). This level of reduction achieved with agents such as interferon alpha, lamivudine, and adefovir has been demonstrated to continue to progress to further depression of HBV, DNA, with a cumulative increase in seroconversion with duration of therapy. The data suggest a plateau of effect before 10 mg total daily dose. A dose in the range between about 5 mg per day and about 10 mg per day, or between about 7.5 mg per day add about 10 mg per day, may be optimal. In each case, the upper limit of these ranges may be extended to about 12 or about 12.5 mg per day. These doses can be administered in tablet or capsule form.

The viral, load gradually returned to baseline after cessation of LY582563 treatment, with the higher dose groups demonstrating a slower recovery of viral load (Figures 1 and 2). Slower return of viral replication in the higher dose groups could be due to a greater extent of viral suppression during treatment, thus leading to a smaller remnant pool of infected hepatocytes. As the viral load at day 29 was different in each dose group, with higher dose groups having a lower viral load compared to the lower dose groups that had a higher viral load (Figures 1 and 2), no analysis was performed to determine the return of viral load toward baseline after the cessation of LY582563 treatment. This study was not designed to detect the mean log-change from baseline of HBV DNA value on day 29 between dose groups.

Safety of LY582563 The most commonly reported adverse events in healthy subjects were somnolence, upper abdominal pain, and diarrhea. In patients with chronic hepatitis B infection, the most commonly reported adverse events were somnolence, headache, and intravenous cannula related events. There did not appear to be a dose relationship to adverse events, with the highest dose levels (10 mg BID, 20 mg QD) having an adverse event incidence comparable to or less than that of placebo. No subject was withdrawn from the study because of adverse events.

There appeared to be a dose relationship to ALT elevations, with higher dose groups having the highest prevalence of ALT increases from baseline, in both healthy subjects (approximately 30%) and patients with compensated chronic HBV infection (approximately 50-70%). The maximum extent of ALT elevations attributable to LY582563 was approximately three times the upper limit of normal.

Evaluations of clinical biochemical markers (serum phosphate, urea, and creatinine) as well as of clinical urinary markers (LDH/creatinine and beta- NAG/creatinine ratios) in both healthy subjects and patients did not reveal any dose- related, renal tubular toxicity. Clinical laboratory results of plasma lactate and serum creatine kinase also suggested that there was no dose-related mitochondrial or musculoskeletal toxicity following repeated dosing with the doses of LY582563 examined. There were no clinically significant abnormalities of vital signs, electrocardiogram (ECG), or QTc prolongations of greater than 30 msec in either healthy subjects or patients given LY582563.

The results presented above demonstrate that LY582563 was safe and well tolerated when administered as multiple doses of 2.5 mg to 30 mg daily for up to 14 days in healthy subjects, and as multiple doses of 2.5 mg to 20 mg daily for up to 28 days in patients with compensated chronic HBV infection. There appeared to be a dose relationship to ALT elevations, with higher dose groups having the highest prevalence of ALT increases from baseline, in both healthy subjects and patients with compensated chronic HBV infection. The maximum extent of ALT elevations attributable to LY582563 was approximately three times the upper limit of normal.

No other biochemical markers exhibited any relationship to dose. There were no significant abnormalities of vital signs or ECGs (including assessment for QTc prolongations) in healthy subjects and patients with compensated chronic HBV infection. All doses of LY582563 were associated with a reduction in viral load (plasma HBV DNA) during the treatment period that appeared to be dose-related.

The greatest decline was observed in the 10 mg QD, 10 mg BID, and 20 mg QD dose groups, resulting in an approximately 2.5 mean log, decline after four weeks of therapy. The viral load gradually returned to baseline after cessation of LY582563 treatment. This appeared to be delayed in the higher doses. Based on these results, an appropriate dose of LY582563 that balances safety with efficacy is between about 2.5 mg and about 20 mg per patient per day. These doses may have to be administered to patients over the course of several days, several weeks, or several months or years in order to ameliorate or control undesirable pathogenic consequences of HBV infection.

The invention being thus described, it is obvious that the same can be varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the present invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.