TERATOLOGY 44:325-334 (1991)
Developmental Toxicity of Pentostatin (2’-Deoxycoformycin) in Rats and Rabbits LORI A. DOSTAL, SANDRA BROWN, JILL BLECK, AND JOHN A. ANDERSON Department of Pathology and Experimental Toxicology, Parke-Dauis Pharmaceutical Research Diuision, Warner-Lambert Company. Ann Arbor. Michigan 48105
ABSTRACT
The developmental toxicity of the potent adenosine deaminase (ADA) inhibitor, pentostatin (2’-deoxycoformycin), was investigated in pregnant rats and rabbits administered daily iv doses during organogenesis. Rats received 0 , 0.01, 0.10, or 0.75 mg/kg on gestation days 6-15 and rabbits received 0 , 0.005, 0.01, or 0.02 mg/kg on gestation days 6-18 and maternal and fetal parameters were evaluated on gestation day 21 (rats) or 30 (rabbits). Live fetuses were examined for external, visceral, and skeletal malformations and variations. In rats, maternal body weight gain and food consumption were significantly suppressed at doses of 0.10 and 0.75 mg/kg during the treatment period but returned to control levels during posttreatment. Increased postimplantation loss and decreased numbers of live fetuses, litter size, and fetal body weight were observed a t 0.75 mglkg. A statistically significant increase in the incidence of vertebral malformations occurred a t 0.75 mg/kg. The incidence of certain skeletal variations (extra presacral vertebrae, extra ribs, hypoplastic vertebrae) was also increased at 0.75 mg/kg. Ossification of cervical centra was reduced a t 0.75 mglkg compared with controls. In rabbits, marked maternal toxicity (death, body weight loss, and decreased food consumption) and reproductive toxicity (abortion and premature delivery) occurred in all pentostatintreated groups. However, there were no significant effects on number of live fetuses, pre- or postimplantation loss, litter size, or fetal body weights in the animals with live litters. There was also no apparent increase in the incidence of malformations or variations in the live fetuses of pentostatin-treated rabbits. Thus, these studies demonstrate developmental toxicity of pentostatin in rats and rabbits, and teratogenicity in rats, at maternally toxic doses.
Pentostatin (2’-deoxycoformycin) is a potent transition-state inhibitor of adenosine deaminase (EC 3.5.4.4) (Woo et al., ’74; Borondy et al., ’77). Adenosine deaminase (ADA), a key enzyme in purine catabolism, converts adenosine or 2’-deoxyadenosine to inosine or 2’-deoxyinosine, respectively. This enzyme is found in all tissues, but its greatest activity is found in lymphoid tissues and, in some species, placenta (Brady and O’Donovan, ’65). The importance of ADA to lymphocyte function is clearly demonstrated by severe combined immunodeficiency disease (SCID) in which the absence of ADA results in a selective deficiency of T and B lymphocytes (Giblett et al., ’72). As a result of its specific action on lymphocytes, 0 1991 WILEY-LISS, INC.
pentostatin was investigated as a treatment for lymphomas and leukemias, and has been shown to be highly effective for the treatment of the rare cancer, hairy cell leukemia. In phase I1 clinical trials, hairy cell leukemia patients who failed to respond to other therapies were treated with pentostatin at 4 mg/m2 intravenously on a weekly to monthly basis and have shown a high degree of response (79%) with minimal side effects (Grem et al., ’89; O’Dwyer et al., ’88;
Received January 24, 1991; accepted April 24, 1991. Address reprint requests to Lori A. Dostal, Ph.D., Pathology and Experimental Toxicology, Parke-Davis Pharmaceutical Research Division, 2800 Plymouth Road, Ann Arbor, MI 48105.
326
DOSTAL ET AL.
Bouroncle et al., '87; Spiers e t al., '87; Johnston et al., '86; Foon et al., '86; Kraut et al., '89). In these studies, complete remission rates of 60-90% have been achieved. The present studies were conducted to determine the developmental toxicity, including the teratogenic potential, of pentostatin administered throughout organogenesis to rats and rabbits.
nously to groups of 20 rats (222-325 g body weight) on days 6 through 15 of gestation at doses of 0 (0.9%sodium chloride), 0.01, 0.10, or 0.75 mg/kg (0.06, 0.6, or 4.5 mg/m2). Groups of 20 inseminated rabbits (3.5-4.9 kg body weight) were dosed on days 6 through 18 of gestation at doses of 0 (0.9% sodium chloride), 0.005, 0.01, or 0.02 mg/kg (0.055, 0.11, or 0.22 mglm'). A dose volume of 1.0 mllkg (rats)or 0.5 ml/kg (rabbits) was MATERIALS AND METHODS injected a t a n approximate rate of 0.1 m1/5 Chemicals sec. An untreated control group was also inPentostatin (2'-deoxycoformycin) is de- cluded for each species. Body weight and scribed chemically as (R)-3-(2-deoxy-P-~- food consumption were measured every 3 erythro-pentofuranosyl) -3,6,7,8- tetrahydro- days and clinical signs were monitored imidazo[4,5-d][l,3]diazepin-8-01, and was daily. Necropsies were performed on all anconsidered 100% active. Dosing solutions of imals. On gestation day 21 (rats)or 30 (rabpentostatin were prepared daily in 0.9% so- bits), the animals were sacrificed by carbon dium chloride injection, USP and filtered dioxide asphyxiation (rats) or by overdose through 0.2-p.m filters prior to intravenous injection with Surital (Parke-Davis) (rabinjection. bits) and the number of corpora lutea was determined. The uterus was examined for Animals a n d treatment the numbers of implantation sites, resorpFemale Sprague-Dawley rats [Crl: tions, and live and dead fetuses. Uteri of CD@BR]were obtained from Charles River animals which appeared nonpregnant were Breeding Laboratories, Portage, MI and stained with 10% ammonium sulfide to dewere approximately 13 weeks of age on the termine pregnancy status (Kopf et al., '64). day of mating with sexually mature male All live fetuses were sexed, weighed, and rats of the same strain and source. Female examined externally, including the palate, New Zealand White rabbits were obtained for malformations and variations. Rat fefrom Hazleton Research Animals, Inc. (Den- tuses were fixed in formalin and examined ver, PA) and were 7-9 months of age on the for visceral malformations and variations day of artificial insemination. Rats were using a modification of the Staples ('74) given Purina Certified Rodent Chow@5002 technique. Approximately one-third of the and water ad libitum. Rabbits were given rat fetuses were examined for head abnorPurina Certified High Fiber Chow@ 5325 malities by slicing, while the remaining rat and water ad libitum. Animals were housed fetuses were eviscerated, cleared, stained individually in stainless steel cages with with alizarin red S, and subsequently examwire bottoms under controlled environmen- ined for skeletal malformations and variatal conditions of light, temperature, and hu- tions. All rabbit fetuses were examined for midity (NIH, '85). Animals were acclimated visceral abnormalities by fresh dissection for 2 weeks (rats) or 3-5 months (rabbits) using a modification of the Staples ('74) and were individually identified by tail tat- technique, and subsequently examined for too (rats) or ear tag (rabbits). Rats were as- skeletal malformations and variations. Rabsigned to dose groups a t the time of success- bit litters which were delivered early on ful mating by consecutive placement into gestation day 30 were included in the evalrandomly selected groups. Rabbits were uation of external, visceral, and skeletal randomly assigned to dose groups based on malformations and variations but were not body weight. Each doe was artificially in- included in most calculations of reproducseminated with semen from a proven male tive and litter parameters or skeletal ossifirabbit of the same strain and source. Fol- cation determinations. lowing insemination, ovulation was induced Doses for the definitive studies were seby iv injection of 100 IU of human chorionic lected based on dose-range finding studies gonadotropin (Organon Inc). The day of in- in which doses of 0,0.05,0.1,0.5,0.75,or 1.0 semination or occurrence of sperm in a vag- mg/kg were given to rats on gestation days inal smear was considered gestation day 0. 6 through 15, and doses of 0, 0.01,0.02,0.05, Pentostatin was administered intrave- 0.2, or 1.0 mg/kg were given to rabbits on
327
DEVELOPMENTAL TOXICITY OF PENTOSTATIN
TABLE 1 . Summary
dose range finding studies in pregnant rats and rabbits with pentostatin Dose imnike) 0.1 0.5 0.75 0 0.05 Rat study 5 4 5 5 4 No. pregnant animals at term 0 1 0 0 0 No. deaths 0 0 0 0 0 No. with resorptions only 19.0 16.3 -8.0 46.0 17.0 Weight change during treatment (g) 118 183 129 219 176 Food consumption during treatment (g) 15.3 10.5 13.0 15.6 14.0 No. live fetusesidam 3.3 35.4 4.0 9.7 9.8 Postimplantation loss (%) Fetal body weight (g) 5.3 5.1 4.4 5.3 5.1 Males 5.0 5.0 4.5 5.0 4.8 Females External malformations (no. fetusesino. litters) 0 111 0 0 211 of
Rabbit studv No. pregnant animals at term No. deathsimoribund sacrifice No. abortions No. with resorptions only Weight change during treatment (g) Food consumption during treatment (g) No. viable litters a t term No. live fetusesidam Postimplantation loss (%I Fetal body weight ( g ) Males Females External malformations (no. fetuses)
0 5 0 0 0 269 2020 5 7.2 10.8
0 0 0 149 2218 4 6.0 4.2
0 0 0 -149 1200 5 3.4 17.5
0 1 2 -204 75 1 2 5.5 71.7
1 -589 551
53.8 52.5 0
55.7 56.8 0
48.6 45.3 1
43.4 39.7 1
-
gestation days 6 through 18 (Table 1). In addition to maternal and fetal reproductive parameters, fetuses were examined for external malformations and variations only.
Statistics Statistical comparisons between the treated groups and the vehicle control were made using P
Developmental Toxicity of Pentostatin (2’-Deoxycoformycin) in Rats and Rabbits LORI A. DOSTAL, SANDRA BROWN, JILL BLECK, AND JOHN A. ANDERSON Department of Pathology and Experimental Toxicology, Parke-Dauis Pharmaceutical Research Diuision, Warner-Lambert Company. Ann Arbor. Michigan 48105
ABSTRACT
The developmental toxicity of the potent adenosine deaminase (ADA) inhibitor, pentostatin (2’-deoxycoformycin), was investigated in pregnant rats and rabbits administered daily iv doses during organogenesis. Rats received 0 , 0.01, 0.10, or 0.75 mg/kg on gestation days 6-15 and rabbits received 0 , 0.005, 0.01, or 0.02 mg/kg on gestation days 6-18 and maternal and fetal parameters were evaluated on gestation day 21 (rats) or 30 (rabbits). Live fetuses were examined for external, visceral, and skeletal malformations and variations. In rats, maternal body weight gain and food consumption were significantly suppressed at doses of 0.10 and 0.75 mg/kg during the treatment period but returned to control levels during posttreatment. Increased postimplantation loss and decreased numbers of live fetuses, litter size, and fetal body weight were observed a t 0.75 mglkg. A statistically significant increase in the incidence of vertebral malformations occurred a t 0.75 mg/kg. The incidence of certain skeletal variations (extra presacral vertebrae, extra ribs, hypoplastic vertebrae) was also increased at 0.75 mg/kg. Ossification of cervical centra was reduced a t 0.75 mglkg compared with controls. In rabbits, marked maternal toxicity (death, body weight loss, and decreased food consumption) and reproductive toxicity (abortion and premature delivery) occurred in all pentostatintreated groups. However, there were no significant effects on number of live fetuses, pre- or postimplantation loss, litter size, or fetal body weights in the animals with live litters. There was also no apparent increase in the incidence of malformations or variations in the live fetuses of pentostatin-treated rabbits. Thus, these studies demonstrate developmental toxicity of pentostatin in rats and rabbits, and teratogenicity in rats, at maternally toxic doses.
Pentostatin (2’-deoxycoformycin) is a potent transition-state inhibitor of adenosine deaminase (EC 3.5.4.4) (Woo et al., ’74; Borondy et al., ’77). Adenosine deaminase (ADA), a key enzyme in purine catabolism, converts adenosine or 2’-deoxyadenosine to inosine or 2’-deoxyinosine, respectively. This enzyme is found in all tissues, but its greatest activity is found in lymphoid tissues and, in some species, placenta (Brady and O’Donovan, ’65). The importance of ADA to lymphocyte function is clearly demonstrated by severe combined immunodeficiency disease (SCID) in which the absence of ADA results in a selective deficiency of T and B lymphocytes (Giblett et al., ’72). As a result of its specific action on lymphocytes, 0 1991 WILEY-LISS, INC.
pentostatin was investigated as a treatment for lymphomas and leukemias, and has been shown to be highly effective for the treatment of the rare cancer, hairy cell leukemia. In phase I1 clinical trials, hairy cell leukemia patients who failed to respond to other therapies were treated with pentostatin at 4 mg/m2 intravenously on a weekly to monthly basis and have shown a high degree of response (79%) with minimal side effects (Grem et al., ’89; O’Dwyer et al., ’88;
Received January 24, 1991; accepted April 24, 1991. Address reprint requests to Lori A. Dostal, Ph.D., Pathology and Experimental Toxicology, Parke-Davis Pharmaceutical Research Division, 2800 Plymouth Road, Ann Arbor, MI 48105.
326
DOSTAL ET AL.
Bouroncle et al., '87; Spiers e t al., '87; Johnston et al., '86; Foon et al., '86; Kraut et al., '89). In these studies, complete remission rates of 60-90% have been achieved. The present studies were conducted to determine the developmental toxicity, including the teratogenic potential, of pentostatin administered throughout organogenesis to rats and rabbits.
nously to groups of 20 rats (222-325 g body weight) on days 6 through 15 of gestation at doses of 0 (0.9%sodium chloride), 0.01, 0.10, or 0.75 mg/kg (0.06, 0.6, or 4.5 mg/m2). Groups of 20 inseminated rabbits (3.5-4.9 kg body weight) were dosed on days 6 through 18 of gestation at doses of 0 (0.9% sodium chloride), 0.005, 0.01, or 0.02 mg/kg (0.055, 0.11, or 0.22 mglm'). A dose volume of 1.0 mllkg (rats)or 0.5 ml/kg (rabbits) was MATERIALS AND METHODS injected a t a n approximate rate of 0.1 m1/5 Chemicals sec. An untreated control group was also inPentostatin (2'-deoxycoformycin) is de- cluded for each species. Body weight and scribed chemically as (R)-3-(2-deoxy-P-~- food consumption were measured every 3 erythro-pentofuranosyl) -3,6,7,8- tetrahydro- days and clinical signs were monitored imidazo[4,5-d][l,3]diazepin-8-01, and was daily. Necropsies were performed on all anconsidered 100% active. Dosing solutions of imals. On gestation day 21 (rats)or 30 (rabpentostatin were prepared daily in 0.9% so- bits), the animals were sacrificed by carbon dium chloride injection, USP and filtered dioxide asphyxiation (rats) or by overdose through 0.2-p.m filters prior to intravenous injection with Surital (Parke-Davis) (rabinjection. bits) and the number of corpora lutea was determined. The uterus was examined for Animals a n d treatment the numbers of implantation sites, resorpFemale Sprague-Dawley rats [Crl: tions, and live and dead fetuses. Uteri of CD@BR]were obtained from Charles River animals which appeared nonpregnant were Breeding Laboratories, Portage, MI and stained with 10% ammonium sulfide to dewere approximately 13 weeks of age on the termine pregnancy status (Kopf et al., '64). day of mating with sexually mature male All live fetuses were sexed, weighed, and rats of the same strain and source. Female examined externally, including the palate, New Zealand White rabbits were obtained for malformations and variations. Rat fefrom Hazleton Research Animals, Inc. (Den- tuses were fixed in formalin and examined ver, PA) and were 7-9 months of age on the for visceral malformations and variations day of artificial insemination. Rats were using a modification of the Staples ('74) given Purina Certified Rodent Chow@5002 technique. Approximately one-third of the and water ad libitum. Rabbits were given rat fetuses were examined for head abnorPurina Certified High Fiber Chow@ 5325 malities by slicing, while the remaining rat and water ad libitum. Animals were housed fetuses were eviscerated, cleared, stained individually in stainless steel cages with with alizarin red S, and subsequently examwire bottoms under controlled environmen- ined for skeletal malformations and variatal conditions of light, temperature, and hu- tions. All rabbit fetuses were examined for midity (NIH, '85). Animals were acclimated visceral abnormalities by fresh dissection for 2 weeks (rats) or 3-5 months (rabbits) using a modification of the Staples ('74) and were individually identified by tail tat- technique, and subsequently examined for too (rats) or ear tag (rabbits). Rats were as- skeletal malformations and variations. Rabsigned to dose groups a t the time of success- bit litters which were delivered early on ful mating by consecutive placement into gestation day 30 were included in the evalrandomly selected groups. Rabbits were uation of external, visceral, and skeletal randomly assigned to dose groups based on malformations and variations but were not body weight. Each doe was artificially in- included in most calculations of reproducseminated with semen from a proven male tive and litter parameters or skeletal ossifirabbit of the same strain and source. Fol- cation determinations. lowing insemination, ovulation was induced Doses for the definitive studies were seby iv injection of 100 IU of human chorionic lected based on dose-range finding studies gonadotropin (Organon Inc). The day of in- in which doses of 0,0.05,0.1,0.5,0.75,or 1.0 semination or occurrence of sperm in a vag- mg/kg were given to rats on gestation days inal smear was considered gestation day 0. 6 through 15, and doses of 0, 0.01,0.02,0.05, Pentostatin was administered intrave- 0.2, or 1.0 mg/kg were given to rabbits on
327
DEVELOPMENTAL TOXICITY OF PENTOSTATIN
TABLE 1 . Summary
dose range finding studies in pregnant rats and rabbits with pentostatin Dose imnike) 0.1 0.5 0.75 0 0.05 Rat study 5 4 5 5 4 No. pregnant animals at term 0 1 0 0 0 No. deaths 0 0 0 0 0 No. with resorptions only 19.0 16.3 -8.0 46.0 17.0 Weight change during treatment (g) 118 183 129 219 176 Food consumption during treatment (g) 15.3 10.5 13.0 15.6 14.0 No. live fetusesidam 3.3 35.4 4.0 9.7 9.8 Postimplantation loss (%) Fetal body weight (g) 5.3 5.1 4.4 5.3 5.1 Males 5.0 5.0 4.5 5.0 4.8 Females External malformations (no. fetusesino. litters) 0 111 0 0 211 of
Rabbit studv No. pregnant animals at term No. deathsimoribund sacrifice No. abortions No. with resorptions only Weight change during treatment (g) Food consumption during treatment (g) No. viable litters a t term No. live fetusesidam Postimplantation loss (%I Fetal body weight ( g ) Males Females External malformations (no. fetuses)
0 5 0 0 0 269 2020 5 7.2 10.8
0 0 0 149 2218 4 6.0 4.2
0 0 0 -149 1200 5 3.4 17.5
0 1 2 -204 75 1 2 5.5 71.7
1 -589 551
53.8 52.5 0
55.7 56.8 0
48.6 45.3 1
43.4 39.7 1
-
gestation days 6 through 18 (Table 1). In addition to maternal and fetal reproductive parameters, fetuses were examined for external malformations and variations only.
Statistics Statistical comparisons between the treated groups and the vehicle control were made using P
