KR20050044587A - Use of phosphonate nucleotide analogue for treating hepatitis b virus infections - Google Patents

Abstract

Provided are pharmaceutically acceptable compositions comprising 2-amino-9-[2-[bis(2,2,2-trifluoroethoxy) phosphonylmethoxy]ethyl]-6-(4-methoxyphenylthio) purine (LY582563) for oral administration to treat hepatitis B virus infections in infected patients. The compositions are in unit dosage form including, per unit dosage, about 2.5 to about 20 mg of the purine. The compositions are adapted for oral administration, preferably in the form of a tablet or capsule, and can be administered in single or multiple doses, provided that the total daily dose of the purine is in the range of from about 2.5 mg to about 20 mg per patient per day. These compositions are particularly advantageous for lowering the plasma HBV DNA levels, or ameliorating symptoms, conditions, or disorders caused by hepatitis B virus, of human patients with chronic HBV infection.

Description

Use of phosphonate nucleotide analogue for treating hepatitis B virus infections

This application discloses US Provisional Patent Application Serial Nos. 60 / 338,242, filed Jan. 2001; And 60 / 381,123, 2002.5.16, the entire contents of which are hereby incorporated by reference in their entirety as claimed.

Field of invention

The present invention relates to 2-amino-9- [2- [bis (2,2,2-trifluoroethoxy) phosphonylmethoxy] ethyl] -6 for the treatment of hepatitis B virus (HBV) infection in human patients. Novel dosages and dosing regimens for the administration of-(4-methoxyphenylthio) purine (LY582563). The present invention is particularly beneficial for lowering plasma HBV DNA levels in patients infected with HBV and ameliorating symptoms, conditions or diseases associated with HBV infection in humans.

Description of the related technology

Hepatitis B is a potentially pathological liver disease caused by infection with the hepatitis B virus, a partial double-helix DNA virus of the Hepadnaviridae family. It is estimated that 350 million individuals worldwide are chronically infected with HBV, and about 1 million die annually as a direct result of HBV-induced sclerosis or liver cancer.

HBV is a liver-friendly virus that replicates in the liver causing acute and chronic liver disease, including liver fibrosis, sclerosis, inflammatory liver disease, and liver cancer, which in some patients can lead to death (WK Joklik, Virology, Third Edition). , Appleton & Lange, Norwalk, Connecticut, 1988). The virus is a blood-mediated pathogen, which is transmitted by exposure to infectious body fluids in a manner similar to human immunodeficiency virus (HIV). However, HBV is more contagious than HIV. Although effective vaccines are available, they are of no therapeutic value for those already infected with the virus. It is estimated that 15-20% of individuals with chronic infection develop from sclerosis or HBV infection to another chronic disease. For patients with subject sclerosis, survival is 84% at 5 years and 68% at 10 years. For carriers with nontarget sclerosis, 5-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 a while. However, it is effective only for less than 40% of patients and has dose limiting side effects such as flu-like symptoms, weight loss, depression, and cytopenia. Lamivudine and adefovir are new compounds currently under development for use in the treatment of HBV. Unfortunately, the emergence of lamivudine-resistant virus occurred up to 70% of patients after 4 years. Therefore, finding an improved and effective anti-HBV anti-hepatitis therapy is essential and highly prioritized (Locamini et. Al. (1996) Antiviral Chemistry & Chemotherapy 7 (2): 53-64).

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

The gist of the invention

Accordingly, the present invention is directed to novel methods of treating HBV infection in humans, including novel pharmaceutical compositions containing LY582563 and optimal dosing regimens for administration of LY582563 for treating HBV infected patients.

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

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

In another aspect, the invention provides 2-amino-9- [2- [bis (2,2,2-trifluoroethoxy) for the manufacture of a medicament for treating a human patient suffering from hepatitis B virus infection. The use of phosphonylmethoxy] ethyl] -6- (4-methoxyphenylthio) purine provides:

Here, the medicament is formulated for oral administration,

The medication is in unit dosage form and contains about 2.5 mg to about 20 mg of such purine per unit dose.

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

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

In this method, the patient may be administered a purine for a time sufficient to lower the plasma level of the patient's HBV DNA as compared to the patient's HBV DNA prior to the administration of the purine. Preferably, the plasma level of HBV DNA of the patient is lowered to at least about 10 ⁴ copies / mL as compared to the plasma level of HBV DNA of the patient prior to administration of purine. Alternatively, purine may be administered to the patient for a time sufficient to treat or ameliorate a symptom, condition, or disease caused by the hepatitis B virus in a patient, such as, for example, liver fibrosis, sclerosis, inflammatory liver disease, or liver cancer. .

In any of the previous methods, purine may be administered to the patient in the form of a pharmaceutically acceptable oral composition, which may be a tablet or capsule. Moreover, the time period during which purine can be administered may be days, weeks, months or years, and purine is 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 The patient may be administered in an amount ranging 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 this range can be extended to about 12 or about 12.5 mg per day. The amount of such purine can be administered in a single daily dose, or in divided doses that add up to a total amount.

Further scope of applicability of the present invention will become apparent from the detailed description provided hereinafter. However, various changes and modifications within the spirit and scope of the present invention may be apparent to those skilled in the art from this detailed description, while presenting the preferred embodiments of the present invention, and thus the detailed description and the specific embodiments are given by way of explanation. Should be understood

The above and other aspects, features, and advantages of the present invention can be better understood from the following detailed description, which is taken in conjunction with all the figures given by way of illustration only, and not by way of limitation.

1 shows an overview of the mean log-change in HBV DNA with standard deviation bars: (BID group).

2 shows an overview of the mean log-change in HBV DNA with standard deviation bars: (QD group).

FIG. 3 is a scatter plot showing log-change in HBV DNA at day 29 with mean and standard deviation bars. Individual open symbols, ie squares, triangles, etc., represent individual patient data points. This is not really necessary as the columns are clearly distinguished, but in that they show some differences between the columns of different daily doses. Within each column, the solid symbol represents the mean. The outer limit of the bar represents the standard deviation from its mean.

4 shows log-change in HBV DNA at 29 days fitted with an Emax model based on total daily dose (TDD).

5 shows the maximum log-change in HBV DNA during the treatment period fitted with the Emax model based on total daily dose (TDD).

Figure 6 shows the area under the curve (AUC) of the maximum log-change in HBV DNA after the first dose fitted with the Emax model based on the total daily dose (TDD).

The following detailed description of the invention is provided to assist those skilled in the art in practicing the invention. Even so, the following detailed description is intended to unduly limit the invention as modifications and variations in the embodiments discussed herein can be made by one skilled in the art without advancement from the spirit or scope of the invention's discoveries. It should not be interpreted.

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

Current treatment options for chronic HBV infection are limited in number and clinical utility due to the release of resistant and resistant viral strains. LY582563, a structure shown below, is a new purine nucleotide analogue prodrug that shows potential activity against HBV in preclinical studies (Ono-Nita et. Al. (2002) Antimicrob. Antigens Chemother. 46 (8): 2602-) 5; Wise et. Al. (2002) J. Gastroenterol. Hepatol. 17 (suppl): A46).

For oral administration of LY582563, US Pat. No. 5,840,716 teaches that clinical dosages may generally be between 0.1 mg and 500 mg / kg, preferably 1 mg and 50 mg / kg, per day for adults. For adults weighing 68 kg (approximately 150 lbs), this range is from about 6.8 mg to about 34 g per day, preferably from about 68 mg to about 3.4 g per day. The '716 patent states that such dosages can be increased or decreased as appropriate depending on age, condition or condition, or the presence or absence of co-administered drugs. In addition, US Pat. No. 5,840,716 teaches that the daily dose may be administered once a day, or twice or several times a day at appropriate intervals. Finally, continuous administration can be performed at intervals of several days.

Determining the safe and effective dose of any new pharmaceutical antigen and the appropriate treatment regimen using the drug is an experimental process. This process generally requires varying the dose of antigen, one or more times per day, varying the time, treating the suffering patient and observing the effects of the accepted diagnostic methods. In the case of HBV, such methods may involve plasma or tissue levels of various antigens, including parts of HBV DNA or viruses, or patient responses such as antibody formation (eg, hepatitis B e antibodies) or alanine aminotransferase (ALT). Biochemical improvements such as levels. One may also measure the effectiveness of treatment by observing an improvement in symptoms, conditions or diseases due to the virus. In the case of HBV, this includes fatigue, anorexia, jaundice, liver fibrosis and inflammation, sclerosis, and liver cancer (hepatocellular carcinoma). However, in addition to demonstrating efficacy, one must also demonstrate acceptable safety, including acceptable toxicity. Toxicity observed with other HBV antiviralities includes mitochondrial toxicity, kidney toxicity, and bone marrow toxicity. The identity of the drug and the relationship between dose and treatment regimen for efficacy and safety can only be determined by clinical trials in humans. Therefore, the determination of dosage and treatment regimen indicators is not predictable or deduced, and the results are essentially new and non-obvious. At the same time, particular attention should be paid to the selection of therapeutic indicators that are effective in treating the disease and do not cause other harm to patients. Given these considerations, the findings disclosed herein relating to the use of LY582563 in treating HBV infection in human patients are surprising and unpredictable before this study was conducted. These results provide a logical basis for safe and effective dosages and therapies using LY582563 in the treatment of HBV.

The studies disclosed herein have been accepted as having determined the optimal dosage and dosing regimen for treating HBV-infected patients with LY582563 to maximize the safety and therapeutic benefit achievable with this antiviral pharmaceutical antigen. In summary, patients with chronic HBV infection were treated with LY582563 randomly, placebo-controlled doses increased in stages after 28 days.

Doses of LY582563 include 2.5 mg, 5 mg, and 10 mg QD or BID, and 20 mg QD. Placebo was also used. HBV DNA in the treated patients was measured during dosing and up to a 12 week follow-up period. Safety data such as clinical signs (eg blood pressure, pulse rate, respiratory rate, and general physical findings), adverse events (eg epigastric pain, diarrhea, nausea, headache, fatigue, and hair loss), ECG, hematology, in blood Biochemical dimensions (e.g., aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactic acid, blood urea nitrogen (BUN), creatinine, and bilirubin) and renal tubule toxicity (lactate), which is an indicator of high sensitivity Dehydrogenase (LDH), urinary creatinine, and urinary beta-N-acetyl glucosaminidates (beta-NAG) are also measured. The data obtained show favorable safety aspects and effectiveness in patients chronically infected with LY582563. It demonstrates HBV antiviral activity and suggests optimal dosage levels and dosing regimens for long-term safe and effective use of this new HBV antiviral antigen. Based on this result, a suitable dosage of LY582563, which balances both safety and efficacy, is between about 2.5 mg and about 20 mg per patient per day. This dose should be administered to patients over a course of days, weeks or months or months to improve or control unwanted pathogenic consequences of HBV infection.

LY582563 dosage regimen for the treatment of HBV

Oral dosing regimens with LY582563 for the treatment of patients suffering from HBV generally include age, weight, sex, diet and medical condition of the patient, genotype of the infectious virus, the severity of the infection and the activity, efficacy, pharmacokinetics and toxicology of LY582563. It is selected according to a variety of factors such as pharmaceutical considerations such as aspects. Administration of LY582563 generally results in one or more signs, symptoms, conditions, or symptoms present in the patient due to hepatitis B virus infection, until viral titers reach acceptable levels, or indicating that the infection has been controlled or eradicated. It should continue over a period of hours, days, or weeks to months or years until the disease is ameliorated or eliminated completely.

In terms of decreasing plasma HBV DNA levels, the long term goal is a matter of current debate with some practitioners in the field suggesting that the desired level is less than 10 ⁴ copies / mL. The duration for this level of HBV DNA has yet to be defined.

Representative signs of HBV infection other than plasma HBV DNA levels include, but are not limited to, liver fibrosis, sclerosis, inflammatory liver disease, and liver cancer. For a review see Hollinger et al. (2001) in Fields Virology, Fourth Ed., Vol. 2, David M. Knipe et al. , Eds. , "Hepatitis B Virus" Chapter 87, pp. See 2971-3036, Lippincott, Williams, & Wilkins, Philadelphia, PA. In most of the world, liver fibrosis and the like are diagnosed by liver biopsy; In most Asian countries, liver biopsies are not routinely performed and medical practitioners usually rely on clinical measurements to produce a putative diagnosis.

Patients treated with LY582563 may measure HBV DNA levels in their serum, for example, by slot-blot, dot-blot, or PCR techniques, or by HBV in serum and tissues. HBV antigens such as surface antigens (HBsAg) and HBV e antigens (HBeAg) can be commonly observed to determine efficacy of treatment. The method is described in Hoofnagle et al. (1997) New Engl. Jour. Med. 336 (5): 347-356; FD 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 B Virus" Chapter 87, pp. 2971-3036, especially pages 2989-2991, Lippincott, Williams, & Wilkins, Philadelphia, PA, and references cited therein. For chronic hepatitis B, alleviation is achieved by matching experiments that can detect HBeAg despite loss of HBV viral DNA, ie, levels above 10 ⁵ genomes per ml serum, or the presence of HBsAg continues. As measured, it can be characterized by a reduction to an undetectable level. This serological phenomenon is caused by improvements in the biochemical and histological properties of the disease. In the best experiments of antiviral therapy, the stopping point for successful treatment is the loss of HBeAg and viral DNA from serum. In patients with e antigen disappearance, remission usually lasts and becomes an inactive carrier of HBsAg. Many white patients, most of them adults and those receiving interferon therapy, eventually become HBsAg-negative (Hofnagle et al. (1997) New Engl. Jour. Med. 336 (5): 347-356 for review) Reference). However, this does not represent about 90% of the world's HBV population. Ongoing analysis of the data obtained by such methods allows modification of the treatment regimen during treatment, so that the optimal amount of LY582563 can be determined and the duration of treatment can be well determined. Thus, the treatment regimen / dosage plan is reasonably modified during the course of treatment so that the lowest amount of LY582563 can be administered that exhibits satisfactory antiviral efficacy and that administration continues only as long as administration is necessary to successfully treat the infection. Can be.

Within the context of the present invention, the terms "ameliorate", "treat", "treatment", "therapeutic use" or "treatment regimen" as used herein ) "Includes prophylactic, alleviative and therapeutic aspects of the administration of LY582563 and includes signs, symptoms, conditions for treating signs, disease stages, conditions, symptoms or diseases caused by hepatitis B virus or associated with HBV infection. Or any use of this purine to prevent, arrest, retard, or reverse disease progression. Thus, any prevention, improvement, alleviation, retrograde, or complete elimination of unwanted signs, disease stages, symptoms, conditions or diseases associated with HBV infection is encompassed by the present invention. As used herein, the term “dosage unit” refers to a separate means of transport, eg, but not limited to, tablets or capsules for the administration of a dose of active LY582563 purine.

The following examples are provided to illustrate various aspects of the invention and are not to be construed as limiting in any way.

Example 1

Multiple Dose Escalation Study in Healthy Subjects and Chronic Hepatitis B Patients

A randomized, single-term, multiple dose-increasing, simple blind study of LY582563 was performed in two parts. Part 1 was performed in healthy subjects, and Part 2 was performed in subjects with chronic HBV infection (hereinafter referred to as “patients”). The study was designed to assess the safety and pharmacodynamics of multiple doses of LY582563. This is the first multi dose study in humans.

Dose escalation was performed with a careful increase in healthy subjects who received BID therapy for the 2-week period. Safety data were reviewed prior to each dose increase.

Part 1 (Healthy Subjects)

In Part 1 there were 4 groups with 8 subjects in each group (6 subjects on study medication and 2 subjects on placebo). Table 1 shows the characteristics of the subjects in this study. 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, LY582563 multiple BIDs were administered for approximately 14 days except for the last day of QD administration. There was a dose increase for each group of subjects. Safety and other data, as appropriate, were reviewed prior to each dose escalation step. Plasma levels of LY582563 and its metabolism (602074, 602075 and 602076) were measured after a single dose of LY582563 and up to 24 hours after the last dose by the identified LC / MS / MS method (Advion BioSciences, Inc., Ithaca, NY). Subjects were followed at regular intervals for approximately 4 weeks after the last dose of LY582563.

Table 1. Characteristics of healthy subjects

Part 2 (Patient)

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. LY582563 doses are: 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 (1 day) after a single dose of LY582563, LY582563 was administered in multiple BIDs for 28 days except for the last day (29 days) administered with QD (ie, 2 to 29 days). Patients are only studied following the review of safety data from the same dosage level in Part 1. Patients were followed at regular intervals for approximately 12 weeks after the last dose of LY582563. Plasma levels of LY582563 and its metabolism (602074,602075 and 602076) are samples up to 24 hours after a single dose and the last dose of LY582563. Subjects were followed at regular intervals for approximately 12 weeks after the last dose of LY582563.

Table 2. Patient Characteristics

*: HBV DNA baseline = geometric mean, -28 days, -14 days and predose of HBV DNA at screening.

cure

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

LY582563 and placebo preparations for oral administration are as follows:

Table 3.2.5 mg LY582563 tablets

ingredient Amount (mg / tablet) function Active ingredient LY582563 2.5 Active ingredient Other Ingredients D-Mannitol Corn Starch Hydroxypropyl Cellulose Low Substituted Hydroxypropyl Cellulose Magnesium Stearate Hydroxypropylmethyl-Cellulose 2910 Propylene Glycol Titanium Dioxide Talc Curing Oil 92.523.13.11.0 2.53.35 0.770.330.550.0625 Diluent Diluent Binder Disintegrant Lubricant Coating Coating Agent Coating Agent Coating Agent

Table 4. 10 mg LY582563 Tablets

ingredient Amount (mg / tablet) function Active ingredient LY582563 10 Active ingredient Other Ingredients D-Mannitol Corn Starch Hydroxypropyl Cellulose Low Substituted Hydroxypropyl Cellulose Magnesium Stearate Hydroxypropylmethyl-Cellulose 2910 Propylene Glycol Titanium Dioxide Talc Curing Oil 84.521.13.13.8 2.53.35 0.770.330.550.0625 Diluent Diluent Binder Disintegrant Lubricant Coating Coating Agent Coating Agent Coating Agent

Table 5.20 mg LY582563 Tablets

ingredient Amount (mg / tablet) function Active ingredient LY582563 20 Active ingredient Other Ingredients D-Mannitol Corn Starch Hydroxypropyl Cellulose Low Substituted Hydroxypropyl Cellulose Magnesium Stearate Hydroxypropylmethyl-Cellulose 2910 Propylene Glycol Titanium Dioxide Talc Curing Oil 74.518.63.16.3 2.53.35 0.770.330.550.0625 Diluent Diluent Binder Disintegrant Lubricant Coating Coating Agent Coating Agent Coating Agent

Table 6. Placebo Tablets

ingredient Amount (mg / tablet) function Active ingredient LY582563 0 Active ingredient Other Ingredients D-Mannitol Corn Starch Hydroxypropyl Cellulose Low Substituted Hydroxypropyl Cellulose Magnesium Stearate Hydroxypropylmethyl-Cellulose 2910 Propylene Glycol Titanium Dioxide Talc Curing Oil 90.522.63.16.3 2.53.35 0.770.330.550.0625 Diluent Diluent Binder Disintegrant Lubricant Coating Coating Agent Coating Agent Coating Agent

Method of manufacture

LY582563, D-mannitol, and cornstarch are mixed. Hydroxypropylcellulose dissolved in purified water is added for granulation by a liquid-bed granulator. After drying, the granules are sifted by a tornado mill. The filtered granules are mixed with a magnesium stearate and a low substituted hydroxypropylcellulose by a V-shaped mixer.

The mixed granules are compressed into tablets. Tablets are coated with hydroxypropylmethyl-cellulose 2910, propylene glycol, titanium dioxide, and talc by conventional methods and covered with a thin film. Finally, the tablets are coated with some hardened oil.

dosage

 The 2.5 mg dose was administered as one tablet of 2.5 mg LY582563 or one corresponding placebo. The 5 mg dose was administered as two 2.5 mg LY582563 tablets or two corresponding placebos. The 10 mg dose was administered as one tablet of 10 mg LY582563 or as one corresponding placebo. The 15 mg dose was administered as the corresponding placebo corresponding to two tablets of 2.5 mg LY582563 and one tablet of 10 mg LY582563 or tablets of 2.5 and 10 mg LY582563. The 20 mg dose was administered as one tablet of 20 mg LY582563 or as one corresponding placebo.

Each participant was randomly distributed to each dose group and received either LY582563 or placebo. Healthy Subjects in Part 1 Patients in Parts 2 and 2 were assigned according to 4 (3 study medications versus 1 placebo) non-displacement block. Study medication was administered in BID for healthy subjects in Part 1 (Table 7) and in BID or QD for patients in Part 2 (Table 8).

Table 7. Dosage Groups for Part 1 (Healthy Subjects)

* There was a dose increase for each successive group. Safety and other data, as appropriate, were reviewed prior to each dose escalation step.

Table 8. Dose Groups for Part 2 (Patients)

* Grouping order based on time the group started study medication. Patients are only studied according to a review of safety data from healthy subjects at the same or equivalent dose levels in Part 1. For patients starting with 2.5 mg (3A and 3B) in this study, QD dosing is followed by 10 mg (3B and 4B) and 20 mg (4A). A review of safety data in healthy subjects at a 15 mg BID dose determined that no QD dose above 20 mg was studied. Therefore, a 5 mg QD dose was studied in the 5A group.

** One subject in the 5A dose group that was randomized to receive LY582563 dropped out of the study on Day 1 prior to receiving study medication.

Pharmacodynamic Evaluation

In Part 2 of the study, blood samples were taken from hepatitis B infected patients to determine the concentration of HBV DNA levels. Levels of plasma HBV DNA were determined using confirmed HBV polymerase chain reaction (PCR) analysis (NGI HBV SuperQuant ^™ Quantitative PCR Assay; National Genetics Institute, Los Angeles, CA). All HBV DNA levels were converted to a log ₁₀ scale. Baseline measurements were defined as geometric mean of non-log transformed data obtained prior to dosing. The maximum log-change in the subject at the HBV DNA value during the treatment period can be derived by subtracting the minimum HBV DNA value during the treatment period from the baseline value. For two patients with HBV DNA values greater than baseline during treatment (2108 and 3308, both under placebo treatment), the maximum log-change was assigned a value of zero.

Considering the drop and regression rates of HBV DNA values over the study period, the area under the curve (AUC) for the logarithmic change between the horizontal lines passing through each HBV DNA time and baseline score was calculated. HBV DNA is first converted to a baseline score when they are higher than the baseline score, and the area between the curve and the horizontal baseline is calculated using a trapezoidal rule to adjust for the difference in time interval between measurements.

In the assay, three HBV DNA values in the specialized assay range (5x10 ⁹ genomic copy / mL) without performing any dilution assay were designated 5x10 ⁹ copies / mL and the following specialized assay range (100 genome copy / One HBV DNA value in mL) was assigned a value of 100 copies / mL.

Plasma samples for HBV DNA were quantified using the NGI HBVSuperQuant ^™ quantitative PCR assay identified by the National Genetics Institute (Los Angeles, CA) with a specialized assay range of 100 genome copies / mL to 5 × 10 ⁹ genome copies / mL. Approximately 5 mL of blood samples were collected for screening prior to dosing as well as approximately 4 and 2 weeks prior to dosing; Measurements were made during the dosing period (approximately 3 days after the first dose, then 1, 2, 3 and 4 weeks) and the follow up period (5, 6, 8, 12 and 16 weeks after the first dose). 8 weeks after dosing Depending on the level of hepatitis B viral inhibition, subjects may have additional samples obtained approximately 10 and 14 weeks after the first dose.

At the day of administration, blood samples were taken prior to morning administration. Sampling times can be adjusted, added, or deleted based on substantial considerations, clinical needs, or needs for pharmacodynamic data.

Mean and standard deviation of HBV DNA values can be obtained at each scheduled timeline for each dose group.

For log-drop at day 29, the maximum log-change and the AUC for log-change, retrograde model were used to compare mean parameter values in the dosing group. The dose level placed within the dosing regimen was used as a fixed effect and the baseline was used as a control. Differences in mean values were obtained with 95% confidence intervals. Preliminary Emax models were also applied to these parameters to obtain a suitable retrograde curve with the total daily dose as a variable.

Pharmacodynamic Evaluation

An overview of the mean log-change in HBV DNA is shown in FIGS. 1 and 2.

The placebo group shows a constant mean value of 0 on each measurement day (ie, the baseline is not significantly different). For BID administration, 2.5, 5, and 10 mg doses of LY582563 produced a linear drop in log-scale of HBV DNA during the treatment period (FIG. 1). The same was observed in the QD dose group, but the 2.5 mg dose group showed a lower drop rate and the 5 mg group showed a lower drop rate after 7 days. In general, HBV DNA values gradually returned to baseline after the last dose of LY582563, and the time to return to baseline or 1/2 log change of baseline appeared to increase at higher doses. The mean HBV DNA for the 10 mg BID, 10 mg QD, and 20 mg QD groups was lower compared to baseline at 112 days (FIGS. 1 and 2), indicating that viral load did not return to baseline after the 3 month period Indicates.

The degree of dispersion of the log-change from the baseline of HBV DNA is shown in FIG. 3. Pair-wise comparisons of the mean log-changes at 29 days in each dosing regimen are listed in FIG. 9. Dual differences are tested at the 5% level of significance without any diversity control.

Table 9. Average log-change from baseline of HBV DNA values with 95% confidence intervals at 29 days

Note: Confidence intervals that do not contain 0 show a statistically significant difference between the two treatments at the 5% level.

One subject did not have HBV DNA data at 29 days.

For BID administration, mean log-change at 29 days was observed at 2.02, 1.98, and 2.53 for the 2.5, 5, and 10 mg dose groups, respectively. All mean log-changes are statistically significant when compared to those of the placebo group. There was no statistically significant difference between the three BID dose levels. For QD administration, the mean log-changes observed at 29 days 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 are statistically significant when compared to those of the placebo group. The mean log-change for the 10 and 20 mg dose groups was similar and significantly different from that of the 2.5 mg group.

The E _max model fits the log-change with the total daily dose as an explanatory variable (FIG. 4) and makes it easy to understand the relationship between pharmacodynamic effects and total daily dose. The fitted regression curves suggest that congestion performed the HBV DNA drop at approximately 10 mg total daily dose.

Pairwise comparisons of mean log-changes in HBV DNA values over the treatment period within each dosing regimen are listed in FIG. 9. Dual differences are tested at the 5% level of significance without any diversity control.

Table 10. Average maximum log-change from baseline of HBV DNA values with 95% confidence intervals during the treatment period (1 day to 29 days)

Note: Confidence intervals that do not contain 0 show a statistically significant difference between the two treatments at the 5% level.

For BID administration, mean maximum log-change during the treatment period was observed at 2.39, 1.82, and 2.93 for the 2.5, 5, and 10 mg dose groups, respectively. All mean log-changes are statistically significant when compared to those of the placebo group. Although there was a significant difference in mean log-change between the 10 and 5 mg dose groups, there was no significant difference between the 10 mg and 2.5 mg dose groups. For QD administration, the mean maximum log-changes observed 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 are statistically significant when compared to that of the placebo group. The mean log-change for the 5, 10 and 20 mg dose groups was found to be similar and significantly different from that of the 2.5 mg group.

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

The E _max model fits the maximum log-change with the total daily dose as an explanatory variable (FIG. 5). The fitted regression curves reached the plateau for maximum HBV DNA drop during treatment at approximately 10 mg total daily dose.

For further explanation, FIG. 4 shows log-change in HBV DNA at 29 days fitting the E _max model based on total daily dose (TDD); Figure 5 shows the maximum log-change in HBV DNA during the treatment period that fits the E _max model based on TDD. Both figures are needed to be considered as the maximum log-change for some patients occurring at times other than 29 days. Taken together, these figures allow for a better understanding of aspects of pharmacodynamic effects, ie HBV DNA change with dose.

Paired comparisons of mean changes in AUC from the first dose to the end of the study period (Day 112) after each dosing regimen are listed in Table 11. The twin differences are tested at 5% level of significance without any diversity control. .

Table 11.Mean AUC of log-change from baseline of HBV DNA values with 95% confidence interval after first dose (day 1 to last visit)

Note: Confidence intervals that do not contain 0 show a statistically significant difference between the two treatments at the 5% level.

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

The E _max model was fitted to the AUC for log-change with the total daily dose as an explanatory variable, similar to the E _max model for log-change in HBV DNA (FIG. 6). The fitted regression curves suggest that a 10 mg total daily dose can perform 80% of the effect of a 20 mg total daily dose on AUC log-change in HBV DNA.

Conclusion on the efficacy of LY582563

As evidenced by the data presented above, all doses of LY582563 tested in this study are related to a decrease in plasma HBV DNA and a dose-dependent decrease during treatment. The 10 mg QD, 10 mg BID, and 20 mg QD dose groups had an average drop in HBV DNA (FIG. 3), an average maximum log-viral drop (FIG. 10), and a log-viral drop after the first dose at 29 days. The largest viral inhibition was seen as seen in mean AUC change (FIG. 11). This level of reduction performed with antigens such as interferon alpha, lamivudine, and adefovir proved to continue to progress to further inhibition of HBV, DNA with cumulative increase in seroconversion with the duration of treatment. The data suggest the identity of the effect before the 10 mg total daily dose. Dosages between about 5 mg daily and about 10 mg daily, or between about 7.5 mg daily and about 10 mg daily will be optimal. In each case, the upper limit of this range can be extended to about 12 or about 12.5 mg per day. Such administration may be in the form of tablets or capsules.

 Viral load gradually returns to baseline after discontinuation of LY582563 treatment, and higher dose groups demonstrate slower recovery of viral load (FIGS. 1 and 2). Slower recovery of viral replication in the higher dose group may be due to a greater range of viral inhibition during treatment, leading to smaller remaining pools of infected hepatocytes. The viral load at 29 days was different in each dose group as the higher dose group had lower viral load compared to the lower dose group with high viral load (FIGS. 1 and 2), and baseline after discontinuation of LY582563 treatment. There was no analysis performed to determine viral regression towards. This study is not designed to detect mean log-change from baseline of HBV DNA values at 29 days between dose groups.

LY582563 safety

 The most widely reported adverse events in healthy subjects were drowsiness, epigastric pain and diarrhea. The most widely reported adverse events in patients with chronic hepatitis B infection are sleepiness, headache, and intravenous intubation-related symptoms. The maximum dose level (10 mg BID, 20 mg QD) is comparable to or lower than that of placebo. There was no incidence of adverse events, and no dose relationship was observed for adverse events. No subjects withdrew from the study because of adverse events.

It was shown that the maximum dose group had the highest prevalence of ALT increase from baseline, both in healthy subjects (approximately 30%) and patients with targeted chronic HBV infection (approximately 50-70%), with a dose relationship for ALT improvement. The maximum extent of ALT improvement attributable to LY582563 was usually three times the upper limit.

In both healthy subjects and patients, clinical biochemical markers (serum phosphate, urea, and creatinine) and clinical urine markers (LDH / creatinine and beta-NAG / creatinine ratios) did not reveal any dose-related renal tubule toxicity. Clinical trials of plasma lactate and serum creatine kinase also suggested no dose-related mitochondrial or musculoskeletal toxicity as repeated doses of LY582563 were tested. There was no clinically significant abnormality of vital sign, electrocardiogram (ECG), QT _c smoke greater than 30 msec in either healthy subject or patient given LY582563.

The results shown above indicate that LY582563 is administered in multiple doses of 2.5 mg to 30 mg per day for up to 14 days in safe and healthy subjects, and multiples of 2.5 mg to 20 mg per day for up to 28 days in patients with targeted chronic HBV infection. Demonstrate well toleration when administered in a dosage. It was shown that the maximum dose group had the highest prevalence of ALT increases from baseline in both healthy subjects and patients with subject chronic HBV infection, indicating that there was a dose relationship for ALT enhancement. The maximum extent of ALT improvement attributable to LY582563 was usually three times the upper limit.

There was no other biochemical marker indicating any relationship to dose. There were no significant abnormalities in vital signs, ECG (including evaluation of QTc smoke), among healthy subjects or patients with chronic chronic HBV infection. All doses of LY582563 were associated with a reduction in viral load (plasma HBV DNA) during the treatment periods that appeared to be dose-related.

 The maximum drop was observed in the 10 mg QD, 10 mg BID, and 20 mg QD dose groups, occurred at approximately 2.5 mean logs, and dropped after 4 weeks of treatment. Viral loads gradually returned to baseline after discontinuation of LY582563 treatment. This has been shown to be delayed at higher doses. Based on these results, the doses of LY582563 that are efficacious and modulate stability are about 2.5 mg and about 20 mg per patient per day. This dosage may be administered to a patient over a course of days, weeks, or months or years to ameliorate or control unwanted pathogenic consequences of HBV infection.

It is obvious that according to the invention described, the same can be varied in many ways. Such variations are not to be regarded as having evolved from the spirit and scope of the invention, and all such modifications apparent to those skilled in the art are deemed to be within the scope of the following claims.

Claims (16)

About 2.5 mg to about 20 mg of 2-amino-9- [2- [bis (2,2,2-trifluoroethoxy) phosphonylmethoxy] ethyl] -6- (4-methoxy per unit dose Phenylthio) Purine and a pharmaceutical composition for oral administration in unit dosage form containing a pharmaceutically acceptable carrier, diluent, or excipient. The composition of claim 1 in the form of a tablet or capsule. The compound of claim 1 or 2, wherein the 2-amino-9- [2- [bis (2,2,2-trifluoroethoxy) phosphonylmethoxy] ethyl] -6- (4-methoxy Phenylthio) purine is present in an amount of about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, or about 20 mg per unit dose. 2-amino-9- [2- [bis (2,2,2-trifluoroethoxy) phosphonylmethoxy] ethyl for the manufacture of a medicament for treating a human patient suffering from hepatitis B virus infection. Use of -6- (4-methoxyphenylthio) purine, wherein the medicament is formulated for oral administration, wherein the medicament is in unit dosage form, and from about 2.5 mg to about 20 mg of the purine per unit dose Containing). 5. Use according to claim 4, wherein the medicament is in the form of a tablet or capsule. The compound of claim 4 or 5, wherein the 2-amino-9- [2- [bis (2,2,2-trifluoroethoxy) phosphonylmethoxy] ethyl] -6- (4-methoxy Phenylthio) purine is present in an amount of about 2.5 mg, about 5 mg, about 10 mg, about 15 mg, or about 20 mg per unit dose. About 2.5 mg per day of the total amount of 2-amino-9- [2- [bis (2,2,2-trifluoroethoxy) phosphonylmethoxy] ethyl] -6- (4-methoxyphenylthio) purine A method of treating a human patient suffering from hepatitis B virus infection comprising administering to the patient within the range of said purine in the range of from about 20 mg. The 2-amino-9- [2- [bis (2,2,2-trifluoroethoxy) phosphonylmethoxy] ethyl] -6- (4-methoxyphenylthio) purine according to claim 7, A method of administering to a patient for a time sufficient to lower the plasma level of the patient's HBV DNA as compared to the plasma level of the patient's HBV DNA prior to the administration of purine. The method of claim 8, wherein the plasma level of HBV DNA of the patient is lowered to at least about 10 ⁴ copies / mL compared to the plasma level of HBV DNA of the patient prior to administration of purine. The method of claim 7, wherein the patient is treated with 2-amino-9- [2- [bis (2,2,2-tree for a time sufficient to ameliorate signs, symptoms, conditions or diseases caused by the hepatitis B virus. Fluoroethoxy) phosphonylmethoxy] ethyl] -6- (4-methoxyphenylthio) purine. The method of claim 10, wherein the signs, symptoms, conditions, or diseases are selected from the group consisting of liver fibrosis, sclerosis, inflammatory liver disease, and liver cancer. The compound of any one of claims 7-11, wherein 2-amino-9- [2- [bis (2,2,2-trifluoroethoxy) phosphonylmethoxy] ethyl] -6- (4 -Methoxyphenylthio) purine is administered to the patient in the form of a pharmaceutically acceptable oral composition. The method of any one of claims 7-12, wherein the pharmaceutically acceptable oral composition is in the form of a tablet or capsule. The method of claim 8, wherein the time is days, weeks, months or years. The method according to any one of claims 7 to 14, wherein 2-amino-9- [2- [bis (2,2,2-trifluoroethoxy) phosphonylmethoxy] ethyl] -6- (4 -Methoxyphenylthio) purine is administered to a 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. The compound of claim 7, wherein 2-amino-9- [2- [bis (2,2,2-trifluoroethoxy) phosphonylmethoxy] ethyl] -6- (4 Administering the total amount of methoxyphenylthio) purine in a single daily dose, or in divided doses that add up to the total amount.