Summary Basis of Decision for Baraclude

3.2.1 Pharmacodynamics

The pharmacology program consisted of in vitro and in vivo studies to examine the pharmacodynamics of entecavir.

In vitro studies demonstrated that entecavir is a potent, selective inhibitor of HBV. Entecavir (ETV) is efficiently phosphorylated to entecavir-triphosphate (ETV-TP) by cellular nucleoside kinases. By competing directly with the natural deoxyguanosine triphosphate (dGTP), ETV-TP potently inhibits all three functional activities of the HBV polymerase (priming, reverse transcription and DNA-dependent DNA synthesis). ETV-TP had little effect on the DNA polymerases; activity against cellular polymerases is unlikely at clinically relevant concentrations.

In vivo studies showed entecavir to be effective and well tolerated in the woodchuck model for chronic HBV infection. A long-term study with entecavir in woodchuck hepatitis virus (WHV) infected animals indicated that after an initial daily dosing period of 8 weeks, viral replication was suppressed for up to 3 years with only a weekly maintenance dosing regimen. Viral DNA levels were decreased more than 7 logs and significant reductions in levels of covalently closed circular DNA (a key replicative intermediate) were observed. Survival rates for animals treated for 14 or 36 months were 40% and 80% at the end of the prolonged monitoring period, respectively, compared to only 4% for historical controls. Compared to historical data, the incidence of hepatocellular carcinoma in these animals was reduced markedly and no emergence of WHV resistant substitutions was observed during the prolonged treatment period.

Entecavir was also tested in vivo and in vitro for anti-hepadnaviral activity in ducklings and in duck hepatitis B virus (DHBV) infected hepatocytes. Entecavir was shown to be >1000 fold more potent than lamivudine in DHBV infected hepatocytes, had superior potency, and was well tolerated in the duckling DHBV model.

Effects of entecavir on vital functions including the cardiovascular, respiratory, and central nervous system were studied in vitro and in vivo. The in vitro studies and the in vivo study performed in anesthetized dogs were in accordance to the ICH Guidelines 7B (May 2005) for assessing the potential of entecavir to delay ventricular repolarization. Based on the results of the battery of in vivo and in vitro safety pharmacology studies, there is minimal potential for unwanted pharmacologic effects including unwanted cardiac electrophysiological effects in humans receiving entecavir. Combination studies of HIV nucleoside reverse transcriptase inhibitors with entecavir showed no antagonistic effects on HBV or HIV antiviral activities in vitro.

3.2.2 Pharmacokinetics

Absorption

Entecavir was rapidly absorbed in many species including rats, dog, mice, woodchucks, rabbits, and ducks following oral administration. The bioavailability of entecavir was high in rats and dogs but was low in monkeys. The rate of absorption was also slower in monkeys relative to rats and dogs. The low bioavailability in monkeys was due to poor absorption and not related to first-pass metabolism.

Dose-related increases in systemic exposure to entecavir were observed in all species. In rats, drug exposure was higher in males than in females. This difference between the sexes was species-specific and was attributable to the greater production of the major metabolite m² in female rats compared to male rats.

The terminal phase plasma half-life of entecavir following IV administration ranged from approximately 2-9 hours in selected species. Systemic accumulation of entecavir was not observed after repeated administration in rats and monkeys. Low to moderate accumulation was observed in dogs (1.1-2.5 fold increase). In humans the systemic accumulation ratio of entecavir was similar and ranged from 1.6-2.7 in the dose range of 0.1-1.0 mg/day.

Distribution

In vitro , the serum protein binding levels of entecavir were low in mouse, rat, dog, monkey and human (ranging from 7.9%-24%) and entecavir was uniformly distributed between plasma and the red blood cells in human blood. Values for the steady state volume of distribution in rats, dogs, and monkeys were greater than the volume of total body water in these species suggesting extravascular distribution of the drug and/or preferential binding in tissues. Tissue distribution studies in mice and rats indicated that entecavir was rapidly and extensively distributed throughout the body, including the liver which is the intended site of action. Drug-related radioactivity was widely distributed in maternal and foetal tissues of pregnant rats as well, and was secreted in the milk of lactating rats. Low concentrations of entecavir were found in the cerebrospinal fluid of mice, rats, dogs and monkeys indicating that entecavir can cross the blood brain barrier.

Metabolism

In vitro entecavir is not a substrate, inhibitor, or inducer of the human cytochrome P450 isoenzymes at concentrations up to 10 μM (approximately 340 times higher than the maximum concentration observed in humans following multiple daily doses of 1.0 mg). Consistent with this finding, no oxidative metabolism was detected in any of the in vivo studies. Only Phase II (conjugation reactions) metabolites (glucuronide and sulfate) of entecavir were found in rats, dogs, monkeys and humans. All of the circulating metabolites identified in humans were also present in rats, dogs and monkeys. The total amount of metabolites as a percentage of the total radioactivity in urine or faeces was low in all species indicating that metabolic clearance does not play a major role in entecavir elimination.

Excretion

Entecavir is eliminated mainly as unchanged drug in the urine in animals (rats, dogs and monkeys) and humans. Biliary excretion was not a major route of elimination.

Overall, the ADME (absorption, distribution, metabolism, excretion) profiles of entecavir in mice, rats, dogs and monkeys compared to humans suggest that these species were appropriate for the safety assessment of entecavir and its metabolites.

3.2.3 Toxicology

The pivotal toxicology studies supporting the safety of entecavir were appropriately designed and conducted in compliance with Good Laboratory Practices. The monohydrate of entecavir was used in all studies; therefore, all doses or concentrations used are expressed in terms of entecavir free base.

Acute Toxicity

In single-dose studies in mice and rats, oral doses up to 200 mg/kg were well tolerated and doses ≥1000 mg/kg were overtly toxic.

Long-Term Toxicity

Long-term toxicology studies were ≥6 months in duration (3 months for dogs). The long-term studies are more relevant for entecavir as this drug is anticipated to be administered daily for ≥1 year. The principal target organs/tissues in these studies were the central nervous system (CNS), kidney, liver, lung, skeletal muscle, and testes.

CNS

CNS inflammation was observed at doses ≥0.3 mg/kg in a 3-month dog study. This was not associated with overt signs of CNS toxicity. In a second 3-month study in dogs, a dose of 0.1 mg/kg was demonstrated to be a no-effect dose for all drug-related changes (exposure 23 and 13 times higher than in humans at doses of 0.5 mg and 1.0 mg dose, respectively). At the highest dose tested in the second study (15 mg/kg), the CNS changes were found to be reversible following a 3-month post-dose period. CNS inflammation was not observed in the other species evaluated in the toxicology studies. Based on this and the fact that the CNS changes occurred at exposures >23 times that in humans suggest that the CNS findings in dogs are not relevant to human safety.

Kidney

Kidney changes (spontaneous nephropathy in mice, and renal tubular degeneration in dogs) were observed only at overtly toxic doses in the 6- and 3- month studies, respectively. At the no-effect doses for kidney changes in mice (5 mg/kg) and dogs (3 mg/kg), entecavir exposure was ≥46 and ≥458 times higher than that in humans, respectively. Nephrotoxicity was not reported in clinical trials with entecavir. The lack of nephrotoxicity in the clinical trials along with the fact that the kidney changes in animals occurred only at overtly toxic doses in 2 out of the 4 species tested suggest that the kidney is not a target organ in humans.

Liver

In mice and rats, centrilobular degeneration in the liver (without associated increases in serum transaminases) was observed at doses ≥0.2 mg/kg and 0.02 mg/kg, respectively. Mitochondrial enlargement was associated with this finding in rats at doses ≥0.6 mg/kg; however it was considered to be a non-specific reaction to the liver degeneration as it lacked the characteristics of hepatocellular megamitochondria observed with fialuridine, and entecavir was not shown to have any direct effects on mitochondria. Liver degeneration was not observed in dogs (even at overtly toxic doses) or monkeys at exposures to entecavir that were higher than those associated with liver changes in rodents. Therefore, entecavir -induced liver degeneration appears to be a rodent-specific finding.

Lung

In mice, alveolar histiocytosis was observed in the lungs at doses ≥ 1.0 mg/kg and bronchioloalveolar hyperplasia and benign lung adenomas were observed at doses ≥10 mg/kg. In mice, the no-effect dose for lung changes (0.2 mg/kg) was 22 times the exposure in humans at the 0.5 mg dose (12 times at the 1.0 mg dose). The lung changes appeared to be species-specific as no similar changes were observed in rats, dogs or monkeys.

Skeletal Muscle

Skeletal muscle myopathy occurred in mice at doses ≥1.0 mg/kg and in rats at doses ≥0.6 mg/kg in the 6-month studies. Exposure to entecavir at the no-effect doses for these findings were ≥4 times the exposure in humans at the 1.0 mg dose. The skeletal muscle myopathy appears to be specific to rodents and is unlikely to be relevant to human safety.

Testes

In both rats and mice, decreased weight of the testes was observed at doses ≥5.0 mg/kg and 15 mg/kg, respectively. In mice, seminiferous tubular degeneration was observed only at the overtly toxic doses of 10 mg/kg and 20 mg/kg. An increased incidence of seminiferous tubular degeneration was noted at the 1.4 mg/kg dose in the carcinogenicity study in rats. These changes did not affect the reproductive function in male rats as demonstrated in the reproductive study. Exposure to entecavir at this dose was ≥35 times the exposure in humans at the 1.0 mg dose. In dogs, decreased weight of the testes was observed at the 0.3 mg/kg dose and seminiferous-tubular degeneration was observed at doses ≥3 mg/kg, both of which were non-overtly toxic doses. At 3 mg/kg, the threshold dose for microscopic changes in the testes of dogs, exposure to entacavir was 813 times higher than that in humans at the 0.5 mg dose and 458 times higher at the 1 mg dose. In addition, these changes showed evidence of reversibility at 15 mg/kg following a 3-month post-dose period. Therefore, the testis is not a likely target organ in humans administered entecavir.

Reproductive and Developmental Toxicity

In rats, e ntecavir demonstrated no selective developmental toxicity, no effects on reproductive function or fertility, and no adverse findings in a perinatal/postnatal study at exposures ≥28 times that in humans at the1.0 mg dose daily. In rabbits, entecavir caused embryo-foetal toxicity at 16 mg/kg without producing maternal toxicity; therefore entecavir caused selective developmental toxicity in rabbits. The exposure to entecavir at this dose was ≥883 times the exposure to humans taking the 1.0 mg dose. Statistically significant increases in the average number of ossified ribs (13^th rib) occurred at all doses. The sponsor states that this is not drug-related at the lower doses (1.0  mg/kg and 4.0 mg/kg), because there is considerable variability within control populations and this was the only foetal finding at the lower doses. At the no-effect dose, exposure to entecavir was approximately 210 times higher than that in humans at the 1.0 mg dose. Overall, adequate safety margins relative to the exposures in humans were evident for entecavir.

Genetic Toxicity

Entecavir was not mutagenic in the Ames assay or in a mammalian-cell forward gene mutation assay. Entecavir was not clastogenic in an oral micronucleus study in rats. In addition, entecavir was not mutagenic in an oral DNA repair study in rats and it did not increase the frequency of morphologically transformed colonies with Syrian hamster embryo cells. However, in an in vitro chromosome aberration assay in primary human lymphocytes, entecavir was clastogenic at ≥10 μg/mL (36 μM). This was not unexpected as all marketed antiviral nucleoside analogues show clastogenic activity in in vitro and/or in vivo assays. It was postulated that deoxynucleotide-triphosphate (dNTP) pool imbalances were a potential mechanism for clastogenicity. The concentrations required for dNTP imbalances and clastogenicity are in the micromolar range whereas therapeutic concentrations in humans are in the low nanomolar range, suggesting an unlikely risk to humans.

Carcinogenicity

Positive carcinogenic results were observed in the two-year carcinogenicity studies conducted in mice and rats. E ntecavir was associated with an increased incidence of benign and malignant neoplasms involving a variety of organ sites. Of particular concern, because of the low equivalence to human exposure, was the development of lung adenomas in male mice at exposures three times that in humans at the 1.0 mg dose. It is postulated that the lung tumours are species-specific, since a key event in tumour development, pneumocyte proliferation, was not observed in rats, dogs or monkeys. In addition, the chemotactic effect of entecavir on the recruitment of macrophages, which leads to the sustained proliferation of Type II pneumocytes, was not observed in a human monocytic cell line. The Product Monograph identifies this potential hazard and includes detailed information on the results of the carcinogenicity studies.

3.2.4 Summary and Conclusion

Based on the pre-clinical data, entecavir is a potent and selective inhibitor of chronic HBV infection. E ntecavir was shown to be very effective in two common animal models for chronic hepatitis B virus: woodchucks chronically infected with woodchuck hepatitis virus and ducks infected with duck hepatitis B virus.

The non-clinical toxicity profile of entecavir , characterized in an extensive battery of invitro and in vivo studies including carcinogenicity studies in rats and mice, supports the clinical use of entecavir. The pivotal toxicology studies supporting the safety of entecavir were appropriately designed and conducted in compliance with Good Laboratory Practices.

The positive results of the rodent carcinogenicity testing were not judged to warrant a black box warning considering their uncertain clinical significance and the levels of entecavir exposure involved. However, the results are prominently displayed in the product label immediately following the black box warnings.

The sponsor has submitted a Pharmacovigilance Plan which outlines additional measures to further characterize and monitor the theoretical cancer risk in humans. This includes a postmarketing commitment to the US FDA to conduct a large simple safety study of entecavir in order to assess the major outcomes of death, progression of liver disease, and cancer in a large population of chronic HBV patients. The proposed study protocol describes an observational study with a target enrolment of 12,500 subjects, randomized 1:1 to receive entecavir or another nucleoside/tide analogue with the subjects followed for 5-8 years.

Based on a risk/benefit assessment, it was found that there are no non-clinical pharmacology and toxicology issues which would preclude the market authorization of Baraclude.