Toxicologic Pathology, 43: 354-365, 2015 Copyright # 2014 by The Author(s) ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1177/0192623314544381
Two-year Carcinogenicity Study in Rats with a Nonnucleoside Reverse Transcriptase Inhibitor PRASHANT R. NAMBIAR1, DANIEL MORTON2, LELAND WAYNE DOCHTERMAN3, CHRISTOPHER HOULE1, PETER J. THOMFORD3, GWENDOLYN FATE1, STEVEN A. BAILEY4, AND GREGORY L. FINCH1 1
Pfizer Inc., Groton, Connecticut, USA Pfizer Inc., Cambridge, Massachusetts, USA 3 Covance Laboratories Inc., Madison, Wisconsin, USA 4 Pfizer Inc., Andover, Massachusetts, USA 2
ABSTRACT Administration of lersivirine, a nonnucleotide reverse transcriptase inhibitor, daily by oral gavage to Sprague-Dawley rats for up to 2 yr was associated with decreased survival, decreased body weights, and an increase in neoplasms and related proliferative lesions in the liver, thyroid, kidney, and urinary bladder. Thyroid follicular adenoma and carcinoma, the associated thyroid follicular hypertrophy/hyperplasia, hepatocellular adenoma/adenocarcinoma, altered cell foci, and hepatocellular hypertrophy were consistent with lersivirine-related induction of hepatic microsomal enzymes. Renal tubular adenoma and renal tubular hyperplasia were attributed to the lersivirine-related exacerbation of chronic progressive nephropathy (CPN), while urinary bladder hyperplasia and transitional cell carcinoma in the renal pelvis and urinary bladder were attributed to urinary calculi. Renal tubular neoplasms associated with increased incidence and severity of CPN, neoplasms of transitional epithelium attributed to crystalluria, and thyroid follicular and hepatocellular neoplasms related to hepatic enzyme induction have low relevance for human risk assessment. Keywords:
lersivirine; liver; kidney; neoplasia; rat; thyroid; UK-453061.
next-generation NNRTIs have been developed to overcome disadvantages such as viral resistance mutations associated with first-generation NNRTIs. NNRTIs currently approved in the United States include efavirenz (Sustiva1; Bristol-Myers Squibb Company, Princeton, NJ), nevirapine (Viramune1; Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT), delavirdine (Rescriptor1; Pfizer, New York, NY), etravirine (Intelence1; Janssen Therapeutics, Titusville, NJ), and rilpivirine (Edurant1; Janssen Therapeutics, Titusville, NJ). Lersivirine was developed as a next-generation NNRTI for treating HIV-infected patients. Lersivirine was designed to bind to the reverse transcriptase enzyme in a novel way and was shown to be a highly potent and selective NNRTI, with excellent efficacy against NNRTI-resistant viruses (Corbau et al. 2010). In this report, we describe neoplastic and nonneoplastic findings that were observed in a 2-yr carcinogenicity study in rats administered lersivirine. Lersivirine, like other NNRTIs, is an autoinducer of hepatic microsomal enzymes in preclinical animal species but not in humans (Walker et al. 2009). Short-term and chronic studies have shown evidence of liver and thyroid effects consistent with enzyme induction (Houle et al. 2013).
INTRODUCTION As a key component of antiretroviral therapy, nonnucleoside reverse transcriptase inhibitors (NNRTIs) have been used to treat HIV-infected patients for several decades. The mechanism of action of NNRTI is to inhibit the reverse transcriptase enzyme that is critical for converting the viral RNA into complementary DNA (cDNA), which in turn is essential for hijacking the host normal cellular processes to facilitate viral replication, assembly, and transmission. Current therapeutic regimens for treatment-naive patients include combination therapy with an NNRTI and additional antiretroviral agents. NNRTIs have been inherently more efficacious and safer than protease inhibitors; however, the existing first-generation NNRTI drugs present issues due to their adverse event profiles and low genetic barrier to viral mutations that bestow resistance (Jayaweera and Dilanchian 2012). Accordingly, several The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The author(s) received no financial support for the research, authorship, and/or publication of this article. Address correspondence to: Prashant R. Nambiar, Drug Safety Research and Development, Pfizer Inc., 100 Eastern Point Road, MS 8274-1210, Groton, CT 06340, USA; e-mail: [email protected]. Abbreviations: ANOVA, one-way analysis of variance; AUC, area under the drug concentration curve; CAC, carcinogenicity assessment committee; CPN, chronic progressive nephropathy of rats; CYP, cytochrome p450; EMA, European Medicines Agency; US FDA, Food and Drug Administration; HIV, human immunodeficiency virus; MTD, maximum tolerated dose; NCI, National Cancer Institute; NNRTI, nonnucleoside reverse transcriptase inhibitor; T4, thyroxine; UDP-GT, uridine diphosphate glucuronosyl transferase.
MATERIALS
AND
METHODS
Animals and Husbandry Male and female Sprague-Dawley (Crl:CD[SD]) rats 6 to 7 weeks old were obtained from Charles River Laboratories (Portage, Michigan). Seventy animals/sex were randomly assigned to the control and high-dose groups to enable a longer dosing 354
Downloaded from tpx.sagepub.com at University of Otago Library on April 22, 2015
Vol. 43, No. 3, 2015
LERSIVIRINE RAT NEOPLASTIC FINDINGS
TABLE 1.—Design for 2-yr carcinogenicity study of lersivirine in Sprague-Dawley rats. No. of animals Groupa
Male
Carcinogenicity animals Vehicle controls 70d Low dose 60d Middose 60e High dose 70f Toxicokinetic animals Vehicle controls 6 Low dose 6 Middose 6 High dose 6
Female
Dose levelb (mg/kg/day)
Dose concentrationc (mg/mL)
70g 60g 60g 70g
0 50 150 500
0 5 15 50
6 6 6 6
0 50 150 500
0 5 15 50
a Controls received vehicle control article [0.5% (w/v) methylcellulose and 0.1% (v/ v) Tween 80 prepared in reverse osmosis water] only. bAnimals were dosed at a volume of 10 mL/kg. cConcentrations were corrected for lot specific potency. dDosing ceased on day 680 for control males and males given 50 mg/kg/day. Remaining surviving males were necropsied at the end of the scheduled dosing period during week 104 (day 728). e Dosing of males given 150 mg/kg/day ceased on day 616 followed by euthanasia on day 653. fDosing of males given 500 mg/kg/day ceased on day 586 followed by euthanasia on day 597. gBecause of low survival, dosing ceased on days 652, 635, 623, and 654 for females given 0, 50, 150, or 500 mg/kg/day respectively, and all surviving females in all dose groups (including controls) were euthanized on day 656.
period if high mortality was observed in either group (Table 1). Sixty animals per sex were assigned to the low- and intermediate-dose groups. Additional animals (6/sex/group) were assigned to separate groups for toxicokinetic analysis. Animals were identified with implantable microchips and cage cards. Male and female rats were housed individually in polycarbonate cages with paper bedding and offered Certified Rodent Diet #2016C (Harlan Laboratories, Inc., Madison, WI) ad libitum (except when fasted prior to the scheduled necropsy) and water ad libitum. Environmental controls were set to maintain the following animal room conditions: temperature range of 18 C to 26 C, relative humidity range of 30 to 70%, 10 or greater air changes/hr, and a 12-hr light/12-hr dark cycle. All procedures were approved by the institutional animal care and use committee and performed in compliance with the US Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and federal regulations. Dose Selection and Administration Dosages of 100, 300, and 1,000 mg/kg/day administered by oral gavage were suggested to the US Food and Drug Administration (FDA) Carcinogenicity Assessment Committee (CAC). This was based on observed mortality in animals given 1,500 mg/kg/day and the exposure plateau achieved in males given 500 mg/kg/day by gavage in a 13-week range finding study. The dose of 1,000 mg/kg/day was near the maximum tolerated dose (MTD) in the 13-week study; this was based on the unexplained death of 1 rat at 1,000 mg/kg/day and findings such as decreased body weight (males only), hepatocellular degeneration/necrosis and inflammation, increased kidney weights, regeneration of renal tubules (males), renal tubular
355
basophilia (females), and granular material in the bladder with hypertrophy/hyperplasia of transitional epithelium at 500 mg/kg/day. A 6-mo study was conducted in mice instead of rats because mice achieved higher exposures of lersivirine than rats; therefore, 6-mo data from rats were not available to contribute to dose selection. Dose selection could not be performed based on multiples of clinical exposure due to enzyme induction and resulting extensive metabolism of lersivirine in the rat with resulting low human exposure multiples. The CAC recommended dosage levels of 0, 50, 150, and 500 mg/kg given once daily by oral gavage, presumably due to concerns that a dosage of 1,000 mg/kg/day would not be tolerated, and these were accepted for use in the carcinogenicity study. The dosage levels of 50 and 150 mg/kg/day were determined on the basis of approximately half-log decreases from the high-dosage level. The vehicle given to control animals was 0.5% (w/v) methylcellulose and 0.1% (v/v) Tween 80 prepared in reverse osmosis water. This study was designed and conducted in compliance with all relevant regulatory guidance. Pre-term Euthanasia Criteria High mortality was observed in males at 150 and 500 mg/ kg/day and in female control rats. Prespecified criteria were developed in consultation with the CAC to halt dosing when survival for each group and sex (except controls) reached
Two-year Carcinogenicity Study in Rats with a Nonnucleoside Reverse Transcriptase Inhibitor PRASHANT R. NAMBIAR1, DANIEL MORTON2, LELAND WAYNE DOCHTERMAN3, CHRISTOPHER HOULE1, PETER J. THOMFORD3, GWENDOLYN FATE1, STEVEN A. BAILEY4, AND GREGORY L. FINCH1 1
Pfizer Inc., Groton, Connecticut, USA Pfizer Inc., Cambridge, Massachusetts, USA 3 Covance Laboratories Inc., Madison, Wisconsin, USA 4 Pfizer Inc., Andover, Massachusetts, USA 2
ABSTRACT Administration of lersivirine, a nonnucleotide reverse transcriptase inhibitor, daily by oral gavage to Sprague-Dawley rats for up to 2 yr was associated with decreased survival, decreased body weights, and an increase in neoplasms and related proliferative lesions in the liver, thyroid, kidney, and urinary bladder. Thyroid follicular adenoma and carcinoma, the associated thyroid follicular hypertrophy/hyperplasia, hepatocellular adenoma/adenocarcinoma, altered cell foci, and hepatocellular hypertrophy were consistent with lersivirine-related induction of hepatic microsomal enzymes. Renal tubular adenoma and renal tubular hyperplasia were attributed to the lersivirine-related exacerbation of chronic progressive nephropathy (CPN), while urinary bladder hyperplasia and transitional cell carcinoma in the renal pelvis and urinary bladder were attributed to urinary calculi. Renal tubular neoplasms associated with increased incidence and severity of CPN, neoplasms of transitional epithelium attributed to crystalluria, and thyroid follicular and hepatocellular neoplasms related to hepatic enzyme induction have low relevance for human risk assessment. Keywords:
lersivirine; liver; kidney; neoplasia; rat; thyroid; UK-453061.
next-generation NNRTIs have been developed to overcome disadvantages such as viral resistance mutations associated with first-generation NNRTIs. NNRTIs currently approved in the United States include efavirenz (Sustiva1; Bristol-Myers Squibb Company, Princeton, NJ), nevirapine (Viramune1; Boehringer Ingelheim Pharmaceuticals, Inc., Ridgefield, CT), delavirdine (Rescriptor1; Pfizer, New York, NY), etravirine (Intelence1; Janssen Therapeutics, Titusville, NJ), and rilpivirine (Edurant1; Janssen Therapeutics, Titusville, NJ). Lersivirine was developed as a next-generation NNRTI for treating HIV-infected patients. Lersivirine was designed to bind to the reverse transcriptase enzyme in a novel way and was shown to be a highly potent and selective NNRTI, with excellent efficacy against NNRTI-resistant viruses (Corbau et al. 2010). In this report, we describe neoplastic and nonneoplastic findings that were observed in a 2-yr carcinogenicity study in rats administered lersivirine. Lersivirine, like other NNRTIs, is an autoinducer of hepatic microsomal enzymes in preclinical animal species but not in humans (Walker et al. 2009). Short-term and chronic studies have shown evidence of liver and thyroid effects consistent with enzyme induction (Houle et al. 2013).
INTRODUCTION As a key component of antiretroviral therapy, nonnucleoside reverse transcriptase inhibitors (NNRTIs) have been used to treat HIV-infected patients for several decades. The mechanism of action of NNRTI is to inhibit the reverse transcriptase enzyme that is critical for converting the viral RNA into complementary DNA (cDNA), which in turn is essential for hijacking the host normal cellular processes to facilitate viral replication, assembly, and transmission. Current therapeutic regimens for treatment-naive patients include combination therapy with an NNRTI and additional antiretroviral agents. NNRTIs have been inherently more efficacious and safer than protease inhibitors; however, the existing first-generation NNRTI drugs present issues due to their adverse event profiles and low genetic barrier to viral mutations that bestow resistance (Jayaweera and Dilanchian 2012). Accordingly, several The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The author(s) received no financial support for the research, authorship, and/or publication of this article. Address correspondence to: Prashant R. Nambiar, Drug Safety Research and Development, Pfizer Inc., 100 Eastern Point Road, MS 8274-1210, Groton, CT 06340, USA; e-mail: [email protected]. Abbreviations: ANOVA, one-way analysis of variance; AUC, area under the drug concentration curve; CAC, carcinogenicity assessment committee; CPN, chronic progressive nephropathy of rats; CYP, cytochrome p450; EMA, European Medicines Agency; US FDA, Food and Drug Administration; HIV, human immunodeficiency virus; MTD, maximum tolerated dose; NCI, National Cancer Institute; NNRTI, nonnucleoside reverse transcriptase inhibitor; T4, thyroxine; UDP-GT, uridine diphosphate glucuronosyl transferase.
MATERIALS
AND
METHODS
Animals and Husbandry Male and female Sprague-Dawley (Crl:CD[SD]) rats 6 to 7 weeks old were obtained from Charles River Laboratories (Portage, Michigan). Seventy animals/sex were randomly assigned to the control and high-dose groups to enable a longer dosing 354
Downloaded from tpx.sagepub.com at University of Otago Library on April 22, 2015
Vol. 43, No. 3, 2015
LERSIVIRINE RAT NEOPLASTIC FINDINGS
TABLE 1.—Design for 2-yr carcinogenicity study of lersivirine in Sprague-Dawley rats. No. of animals Groupa
Male
Carcinogenicity animals Vehicle controls 70d Low dose 60d Middose 60e High dose 70f Toxicokinetic animals Vehicle controls 6 Low dose 6 Middose 6 High dose 6
Female
Dose levelb (mg/kg/day)
Dose concentrationc (mg/mL)
70g 60g 60g 70g
0 50 150 500
0 5 15 50
6 6 6 6
0 50 150 500
0 5 15 50
a Controls received vehicle control article [0.5% (w/v) methylcellulose and 0.1% (v/ v) Tween 80 prepared in reverse osmosis water] only. bAnimals were dosed at a volume of 10 mL/kg. cConcentrations were corrected for lot specific potency. dDosing ceased on day 680 for control males and males given 50 mg/kg/day. Remaining surviving males were necropsied at the end of the scheduled dosing period during week 104 (day 728). e Dosing of males given 150 mg/kg/day ceased on day 616 followed by euthanasia on day 653. fDosing of males given 500 mg/kg/day ceased on day 586 followed by euthanasia on day 597. gBecause of low survival, dosing ceased on days 652, 635, 623, and 654 for females given 0, 50, 150, or 500 mg/kg/day respectively, and all surviving females in all dose groups (including controls) were euthanized on day 656.
period if high mortality was observed in either group (Table 1). Sixty animals per sex were assigned to the low- and intermediate-dose groups. Additional animals (6/sex/group) were assigned to separate groups for toxicokinetic analysis. Animals were identified with implantable microchips and cage cards. Male and female rats were housed individually in polycarbonate cages with paper bedding and offered Certified Rodent Diet #2016C (Harlan Laboratories, Inc., Madison, WI) ad libitum (except when fasted prior to the scheduled necropsy) and water ad libitum. Environmental controls were set to maintain the following animal room conditions: temperature range of 18 C to 26 C, relative humidity range of 30 to 70%, 10 or greater air changes/hr, and a 12-hr light/12-hr dark cycle. All procedures were approved by the institutional animal care and use committee and performed in compliance with the US Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, and federal regulations. Dose Selection and Administration Dosages of 100, 300, and 1,000 mg/kg/day administered by oral gavage were suggested to the US Food and Drug Administration (FDA) Carcinogenicity Assessment Committee (CAC). This was based on observed mortality in animals given 1,500 mg/kg/day and the exposure plateau achieved in males given 500 mg/kg/day by gavage in a 13-week range finding study. The dose of 1,000 mg/kg/day was near the maximum tolerated dose (MTD) in the 13-week study; this was based on the unexplained death of 1 rat at 1,000 mg/kg/day and findings such as decreased body weight (males only), hepatocellular degeneration/necrosis and inflammation, increased kidney weights, regeneration of renal tubules (males), renal tubular
355
basophilia (females), and granular material in the bladder with hypertrophy/hyperplasia of transitional epithelium at 500 mg/kg/day. A 6-mo study was conducted in mice instead of rats because mice achieved higher exposures of lersivirine than rats; therefore, 6-mo data from rats were not available to contribute to dose selection. Dose selection could not be performed based on multiples of clinical exposure due to enzyme induction and resulting extensive metabolism of lersivirine in the rat with resulting low human exposure multiples. The CAC recommended dosage levels of 0, 50, 150, and 500 mg/kg given once daily by oral gavage, presumably due to concerns that a dosage of 1,000 mg/kg/day would not be tolerated, and these were accepted for use in the carcinogenicity study. The dosage levels of 50 and 150 mg/kg/day were determined on the basis of approximately half-log decreases from the high-dosage level. The vehicle given to control animals was 0.5% (w/v) methylcellulose and 0.1% (v/v) Tween 80 prepared in reverse osmosis water. This study was designed and conducted in compliance with all relevant regulatory guidance. Pre-term Euthanasia Criteria High mortality was observed in males at 150 and 500 mg/ kg/day and in female control rats. Prespecified criteria were developed in consultation with the CAC to halt dosing when survival for each group and sex (except controls) reached
