Telbivudine was manufactured at Microbiologica Quimica e Farmaceutica (Rio de Janeiro, Brazil) in compliance with concurrent good manufacture practice. The purity was ≥99.1%. Telbivudine was formulated in 0.5% (wt/vol) carboxymethylcellulose and administered to mice, rats, rabbits, or monkeys at a dose volume of 5 or 10 ml/kg.
CD-1 and CB6F1 mice were obtained from Charles River Laboratories (Raleigh, NC). CB6F1-Tg rasH2 mice were obtained from the Central Institute for Experimental Animals (Kanagawa, Japan). Sprague-Dawley rats [Crl:CD(SD)IGS BR] were obtained from Charles River Laboratories (Raleigh, NC). Female New Zealand White rabbits were obtained from Covance Research Products, Inc. (Denver, PA). Cynomolgus monkeys (Macaca fascicularis) were obtained from Charles River Primates (Houston, TX), Covance Research Products, Inc. (Alice, TX), Primate Products, Inc. (Miami, FL), or Guangdong Scientific Instruments and Materials Import and Export Corporation (Guangdong, China). Animal studies were conducted at SNBL USA Ltd. (Everett, WA), Charles River Laboratories (Horsham, PA, and Worcester, MA), or Covance Laboratories Inc. (Vienna, VA) according to good laboratory practice or accepted scientific and industrial standards.
Safety pharmacology studies.
Safety pharmacology studies were performed to assess the potential for telbivudine to produce any adverse pharmacological effects. Telbivudine was administered to conscious telemeterized male cynomolgus monkeys (4 males/group) on four separate occasions (4-by-4 Latin Square design) at doses of 0, 250, 750, or 2,000 mg/kg by oral gavage to evaluate the effects on the function of the respiratory and cardiovascular systems. In the central nervous system safety study, single doses of 0, 150, 500 and 1,000 mg/kg of telbivudine were orally administered to 8 rats/sex/group.
The cardiac hERG (human ether-à-go-go-related gene) channel is a potassium channel responsible for rapid delayed rectifier outward currents (IKr). Inhibition of IKr is the most common cause of cardiac action potential prolongation by noncardiac drugs, leading to prolongation of the QT interval and an associated ventricular arrhythmia disorder, torsade de pointes. The effect of telbivudine on the hERG current was determined at concentrations of 10, 100, 1,000, and 10,000 μM in human embryonic kidney cells (HEK293) stably transfected with a human ERG gene (3 to 4 cells/group).
Acute toxicity studies.
Four groups of 5 rats/sex/group were orally administered with a single dose of vehicle (0 mg/kg) or telbivudine at 500, 1,000, or 2,000 mg/kg. One group of 2 monkeys/sex received a starting dose of 20 mg/kg of telbivudine. As no overt toxicity was observed, the monkeys received escalating doses of 100, 500, 1,000, and finally 2,000 mg/kg with a washout period of 3 to 4 days between doses. Rats were observed for 14 days, whereas monkeys were observed for 3 days following the last dose administration. Acute toxicity was evaluated based on clinical observations, body weights, clinical pathology, and macroscopic pathology.
Repeat-dose toxicity studies.
The multiple-dose nonclinical safety studies conducted in support of the telbivudine program are listed in Table . Telbivudine was administered by oral gavage to mice for 28 days or 13 weeks at doses up to 3,000 mg/kg/day and to rats and monkeys for 28 days or 3, 6 (rats only), or 9 (monkeys only) months at doses up to 2,000 mg/kg/day. Repeat-dose toxicity was evaluated based on mortality; clinical observations; body weight; food consumption; ophthalmology; clinical pathology, including hematology, coagulation, and chemistry; and/or macroscopic and microscopic pathology. Electrocardiogram and vital signs such as blood pressure, heart rate, and body temperature were also collected in the monkey studies for toxicity assessment.
Repeat-dose oral toxicity studies with telbivudine in mice, rats, and monkeys
Reproductive and developmental toxicity studies.
Five studies were conducted to evaluate the effects of telbivudine on fertility in male and female rats, on embryo-fetal development in female rats and New Zealand White rabbits, and on multiple generation development in male and female rats (Table ).
Reproductive and developmental toxicity studies with telbivudine in rats and rabbits
In a combined rat fertility and embryo-fetal development study (combined segments I and II), male rats (25 animals/group) were orally administered telbivudine at doses of 0, 100, 500, or 1,000 mg/kg/day, starting 28 days prior to cohabitation and continuing through the day before the completion of cohabitation. Female rats (25 animals/group) were dosed starting 15 days prior to cohabitation and continuing through gestation day 17. Male rats were sacrificed at the completion of cohabitation, whereas females were sacrificed on gestation day 21.
In a follow-up male fertility study (segment I), male rats (25 to 26 animals/group) were dosed with telbivudine at doses of 0, 1,000, or 2,000 mg/kg/day, starting 28 days prior to cohabitation and continuing through the day before the completion of cohabitation. Male rats were sacrificed at the completion of cohabitation, whereas untreated cohabitated females were sacrificed on gestation day 13. In another follow-up female fertility study (segment I), female rats (25 animals/group) were dosed with telbivudine at doses of 0 or 2,000 mg/kg/day, starting 15 days prior to cohabitation and continuing through gestation day 7. Females were sacrificed on gestation day 13, whereas untreated cohabitated male rats were sacrificed at the completion of cohabitation.
Male fertility toxicity was evaluated based on mating and impregnating rate; sperm count, mobility, and morphology; and microscopic examinations of testes and epididymides. Female fertility was evaluated based on estrous cycling, mating and pregnancy rate, number of corpora lutea, number and distribution of implantation sites, early resorption, and microscopic examination of ovaries. Fetotoxicity and teratologic potential were evaluated based on mid and late resorptions; the numbers of live and dead fetuses; external sex; body weight; and gross external alterations and microscopic internal alterations, such as major malformations, minor external visceral and skeletal anomalies, and common skeletal variants.
In an embryo-fetal development study (segment II) in female New Zealand White rabbits, four groups of 20 artificially inseminated New Zealand hybrid rabbits were orally administered telbivudine at doses of 0, 50, 250, or 1,000 mg/kg/day on day 6 through day 18 postinsemination. The does were caesarean sectioned on day 29 postinsemination. The numbers of corpora lutea, implantation sites, early and late resorptions, and live and dead fetuses were recorded. Fetuses were examined for external sex, body weight, gross external alterations, and microscopic internal alterations. Pregnancy rates and pre- and postimplantation losses were calculated.
In a multiple generation development study (segment III) in male and female rats, telbivudine was administered by oral gavage at doses of 0, 100, 250, or 1,000 mg/kg/day to female Crl:CD(SD)IGS BR rats (25 animals/group) on gestation day 7 through postpartum day (PD) 20. Females were allowed to deliver and maintain their progeny until PD 21. On PD 21, dams were sacrificed and implantation sites were counted. Pups not scheduled for continued evaluation were sacrificed on PD 21 and examined for gross lesions and microscopic changes on selected tissues. From the surviving pups, one rat/sex/group of the first offspring (F1) was evaluated for passive avoidance tests for learning and short-term and long-term retention starting from PD 24 ± 1. Females were examined for the age of vaginal patency starting from PD 28. Males were examined for the age of preputial separation starting from PD 39. On approximately PD 70, one rat/sex/litter/group was evaluated in a water-filled M-maze for overt coordination, swimming ability, learning, and memory. At the age of 90 days, one rat/sex/group was cohabitated for a maximum of 21 days. Once females were observed to have spermatozoa in their vaginal smears or a copulatory plug, they were housed independently. F1 males were sacrificed at the end of cohabitation, and the testes and epididymides were weighed. F1 females were sacrificed on gestation day 21 and evaluated in like manner to the F0 females.
Genetic toxicology studies.
Three in vitro assays and one in vivo test were performed to assess the genotoxic potential for telbivudine. Reverse mutation potential was assayed with Salmonella enterica serovar Typhimurium strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2uvrA. The mutagenic potential was tested with in vitro chromosomal aberration assays in Chinese hamster ovary cells and in human peripheral blood lymphocytes. The in vitro assays were performed at concentrations up to 5,000 μg/plate (bacterial mutation), 2,442 μg/ml (human peripheral blood lymphocytes), or 5,000 μg/ml (Chinese hamster ovary cells) with or without metabolic activation (S9). An in vivo micronucleus test was performed in CD-1 mice given single oral doses of 0, 500, 1,000, or 2,000 mg/kg. The numbers of micronuclei in bone marrow preparations obtained at 24 and 48 h postdose were determined. For all in vitro assays and in vivo tests, telbivudine was prepared as a solution in dimethyl sulfoxide and appropriate negative and positive controls were used.
The carcinogenic potential of telbivudine was tested in two bioassays. In a 6-month carcinogenicity study in CB6F1-Tg rasH2 mice, telbivudine was administered once daily by oral gavage to groups of 25 mice/sex/group at doses of 0, 500, 1,000, or 2,000 mg/kg/day. In addition, an additional group of 25 mice/sex received a known carcinogen, N-methyl-N-nitrosourea (MNU), intraperitoneally at a dose of 75 mg/kg on day 1 as a positive control. In a traditional 2-year carcinogenicity study in Sprague-Dawley rats [Crl:CD (SD)IGS BR], telbivudine was administered to groups of 65 rats/sex/group by oral gavage at doses of 0, 500, 1,000, or 2,000 mg/kg/day. As mortality reached 38% (25/65) in both male and female rats dosed at 2,000 mg/kg/day at week 85, dosing of this group was discontinued after the completion of 85 weeks. The remaining animals continued to receive dosing until the completion of 95 weeks of dosing. Animals were sacrificed at weeks 95 (group 2 males), 96 (groups 1, 3, and 4 males), and 97 (all females). Toxicity and carcinogenicity were evaluated based on survival; clinical observations, including grossly visible and/or palpable masses; body weight; food consumption; clinical pathology; and macroscopic and microscopic pathology.
For continuous data such as body weight, food consumption, clinical pathology, and organ weight, Bartlett's test or Levene's test was used to test for variance homogeneity. In the case of heterogeneity of variance at P ≤ 0.05, rank transformation was used to stabilize the variance. One-way analysis of variance was used to analyze data. When the result was significant (P ≤ 0.05), Dunnett's test was used for comparisons between dosed and control groups. Group comparisons (dosed groups versus control groups) were evaluated at the 5.0% two-tailed probability level.
For data that exhibited heterogeneity after transformations, a Kruskal-Wallis test was applied. If the result was significant, a nonparametric Dunn's test was employed for comparisons between dosed and control groups.
Histopathology data were analyzed for incidence of neoplastic and nonneoplastic lesions using Fisher's exact test to compare dosed groups and positive control groups versus the vehicle control groups. The incidence of common background findings was compared with controls and published values from different laboratories (26