Fertility and General Reproduction Studies in Rats with the HMG-CoA Reductase Inhibitor, Atorvastatin

FUNDAMENTAL AND APPLIED TOXICOLOGY ARTICLE NO.

32, 285–292 (1996)

0132

Fertility and General Reproduction Studies in Rats with the HMG-CoA Reductase Inhibitor, Atorvastatin LORI A. DOSTAL,* LLOYD R. WHITFIELD,†

AND

JOHN A. ANDERSON*

Departments of *Pathology and Experimental Toxicology and †Pharmacokinetics and Drug Metabolism, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, Ann Arbor, Michigan 48105 Received February 1, 1996; accepted June 4, 1996

Fertility and General Reproduction Studies in Rats with the HMG-CoA Reductase Inhibitor, Atorvastatin. DOSTAL, L. A., WHITFIELD, L. R., AND ANDERSON, J. A. (1996). Fundam. Appl. Toxicol. 32, 285–292. Fertility and reproduction studies were conducted in rats with the 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor, atorvastatin. Male rats received vehicle (0.5% methylcellulose) or atorvastatin at 20, 100, or 175 mg/kg by oral gavage for 11 weeks prior to mating with untreated females; treatment continued throughout mating and until necropsy on Day 115. An untreated control group of males was also included in the same procedures. Dose-related body weight gain suppressions of 17 and 25%, and food consumption suppressions of 7 and 16%, occurred during the 11-week premating treatment period at 100 and 175 mg/kg, respectively, compared with vehicle controls. There were no treatment-related effects on testes, epididymides, or accessory organs weights, testicular or epididymal sperm counts, sperm motility, or sperm morphology during Week 15 of treatment. Plasma drug concentrations during Week 15 increased with dose to a Cmax of 1820 { 1020 ng eq/ml at 175 mg/kg. There were no effects on copulation or fertility indices, number of days to mating, or female reproductive parameters (number of implants, live fetuses, or pre- and postimplantation loss). In the female fertility study, female rats received vehicle (0.5% methylcellulose) or atorvastatin at 20, 100, or 225 mg/kg by oral gavage for 2 weeks prior to mating with untreated males; treatment continued throughout mating and until Gestation Day 7. Sperm-positive females were sacrificed on presumed Gestation Day 13 to 15 for evaluation of reproductive parameters. Body weight gain in atorvastatin groups was comparable to controls during the premating period, but was suppressed by 35% at 225 mg/kg during the treatment period of gestation (Days 0–8), and was significantly increased at 225 mg/ kg during the posttreatment period of gestation (Days 8–13). Plasma drug concentrations on premating treatment Day 14 increased with dose to a Cmax of 7030 { 3680 ng eq/ml at 225 mg/ kg. The mean number of estrous cycles, copulation and fertility indices, number of days to mating, and number of viable litters were comparable between groups. In addition, term sacrifice parameters (number of corpora lutea, implants, live fetuses, preand postimplantation loss) were not significantly different between groups. Thus, these studies demonstrate no adverse effects of atorvastatin on fertility and reproduction in rats at doses up to 175

and 225 mg/kg in males and females, respectively, and 20 mg/kg was a no-effect dose. q 1996 Society of Toxicology

One of the primary aims in the development of therapeutic agents to reduce the risk of atherosclerosis and cardiovascular disease is the regulation and lowering of serum lipids. The inhibition of microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the biosynthesis of cholesterol, has been shown to be an effective mechanism for lowering serum cholesterol in experimental animals and man. Several inhibitors of HMG-CoA reductase have been developed and used clinically for the treatment of hyperlipidemia. Atorvastatin has been shown to be a highly efficacious inhibitor of HMG-CoA reductase that produces greater reductions in LDL-cholesterol in hyperlipidemic patients than other lipid-regulating drugs (Nawrocki et al., 1995). Due to their cholesterol-lowering properties, this class of inhibitors might be expected to have adverse effects on reproduction by reducing the supply of circulating cholesterol which is required for steroidogenesis (Dobs et al., 1993). Most HMG-CoA reductase inhibitors are effective cholesterol-lowering agents in rabbits, guinea pigs, dogs, and humans (Tsujita et al., 1986; Gerson et al., 1989; MacDonald et al., 1988; Alberts et al., 1989; Purvis et al., 1992; Frishman et al., 1989). However, these reductase inhibitors, including atorvastatin, do not significantly lower serum cholesterol levels in chowfed rats (Endo et al., 1979; MacDonald et al., 1988; Tsujita et al., 1986; Krause and Newton, 1995), and several have been shown to have no adverse effects on reproduction in rats (FDA, 1987, 1993; Tanase and Hirose, 1987; Wise et al., 1990). Nevertheless, HMG-CoA reductase inhibitors are considered teratogenic due to studies conducted with lovastatin (Minsker et al., 1983), and sporadic testicular effects have been observed in dogs (MacDonald et al., 1988; Gerson et al., 1989). Therefore, in the course of safety evaluation of atorvastatin for registration as a new drug, fertility studies were conducted according to ICH guidelines (ICH, 1994), and the present results demonstrate the lack of effects on fertility and reproduction in male and female rats.

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0272-0590/96 $18.00 Copyright q 1996 by the Society of Toxicology. All rights of reproduction in any form reserved.

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MATERIALS AND METHODS Atorvastatin (CI-981, PD 134298-38A) is described chemically as [R(R*,R*)]-2-(4-fluorophenyl)-b,d-dihydroxy-5-(1-methylethyl)-3-phenyl-4[(phenylamino)carbonyl]-1H-pyrrole-1-heptanoic acid calcium salt (2:1). Dosing suspensions of atorvastatin were prepared weekly in 0.5% aqueous methylcellulose, and concentrations were determined to be within 10% of intended values with only minor exceptions. Homogeneity and stability of suspensions were within acceptable limits (within {10%) over the concentration ranges used. Animals. Male and female Sprague–Dawley rats [Crl:CDBR VAF/ Plus] were obtained from Charles River Breeding Laboratories (Portage, MI). Males were 11 weeks of age at the initiation of treatment (male study) or at the time of mating (female study). Females were 12 to 13 weeks of age at the initiation of treatment (female study) or the time of mating (male study). Purina Certified Rodent Chow 5002 and water were provided ad libitum. Animals were housed individually except during mating in stainless steel cages with wire bottoms under controlled environmental conditions of light, temperature, and humidity (NIH, 1985). Animals were acclimated for at least 1 week and were individually identified. Rats were randomly assigned to dose groups by body weight. Insemination was determined by the occurrence of sperm in a vaginal smear and was considered Day 0 of gestation. Male fertility study. Atorvastatin was administered by gavage to groups of 30 male rats at 0 (0.5% methylcellulose), 20, 100, and 175 mg/kg. An additional group of 25 untreated males was included in all procedures for comparison with vehicle controls. The high dose, intended to induce at least some evidence of general toxicity, was selected based on a previous study in which doses of 175 and 225 mg/kg for 13 weeks caused 1 or 2 males to be euthanized in moribund condition, adverse clinical signs, and significant body weight gain suppressions of 9 and 20%, respectively (unpublished experiments). Males were treated for 11 weeks prior to mating, during mating, and until necropsy. Mating was with untreated females in a 1:1 ratio for a maximum of 19 days. Vaginal smears were evaluated in females during the mating period for stage of estrous and the presence of sperm. At the time of insemination, females were separated from the male, body weights were determined during gestation, and females were sacrificed on Gestation Day 13 to 15. At maternal sacrifice, the number of corpora lutea and the location and status of each implant site were recorded (live/ dead embryo, early/late resorption), and a gross necropsy was conducted. Male reproductive parameters. During Treatment Week 15, 10 males per group were sacrificed by carbon dioxide asphyxiation. Immediately after euthanasia, a section (approximately 1 cm) of the left vas deferens was removed for evaluation of sperm motility. The tissue was incubated for 3 min in 10 ml of medium containing 10 mg/ml bovine serum albumin (Fraction V, Sigma) in Dulbecco’s phosphate-buffered saline (D-PBS) (Gibco) in a 10-cm plastic petri dish. The temperature of the medium, microscope, and sample chambers was maintained at 37 { 27C in a custommade Plexiglas box using an ASI 400 airstream incubator (Nicholson Precision Instruments). During the 3-min incubation the vas deferens tissue was removed from the medium when it was visually determined that a sufficient amount of sperm had diffused from the end of the tubule into the medium, and then the sample was gently mixed. After the 3-min incubation, a sample of the sperm suspension was removed by capillary action using a 7-in. Pasteur pipet and loaded into a 20-mm mCell chamber (Spectrum Technologies). Images of sperm movement were made using an Olympus CH-2 microscope with a 41 objective to produce a pseudo-dark-field image. At least 20 nonoverlapping fields were videotaped using a CellSoft camera (Cryo Resources), a Panasonic WJ-810 time–date generator, and a Panasonic Model AG-1970 videotape recorder. Due to technical difficulties, sperm motility was determined manually from the videotapes using transparent plastic sheets and counting total and nonmotile sperm in a minimum of seven fields per animal and a minimum of 200 sperm per animal.

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Paired testes, paired epididymides, and accessory sex organs were weighed. Testicular spermatid head counts were determined as follows. The left testis parenchyma was weighed after removal of the tunica albuginea, placed in approximately 75 ml of dilute Triton X-100 (one drop Triton X100 in 1000 ml) in 0.9% saline, and minced with scissors. The tissue was homogenized for 1 min and sonicated for 3 min, and 10 ml of 1% Eosin Y stain was added. Spermatid heads were counted on a hemocytometer in four replicate samples. Epididymal sperm counts were determined in the left cauda epididymis as follows. The cauda was finely minced in a few drops of cold D-PBS with two razor blades on a glass dish. The minced tissue was rinsed into a cylinder and brought to a total volume of 100 ml with cold D-PBS. Sperm were counted on a hemocytometer in four replicate samples. Sperm morphology was determined in a smear made from the cauda epididymal mince (above). The dried smears were stained with Eosin Y and 200 sperm were evaluated for head and tail abnormalities and classified as normal or abnormal based on criteria similar to that described for mice by Wyrobek and Bruce (1975). The right testis and epididymis were fixed in Bouin’s, embedded in paraffin, stained with hematoxylin and eosin, and examined microscopically for abnormalities. Female fertility study. Atorvastatin was administered by gavage to groups of 30 female rats (5 rats for plasma drug analysis) at 0, 20, 100, and 225 mg/kg. The high dose was chosen based on a 13-week study in which 225 mg/kg caused adverse clinical signs and 5 females were euthanized in moribund condition whereas a dose of 175 mg/kg resulted in only one moribund sacrifice and fewer clinical signs. Current ICH guidelines recommend a study of fertility and early embryonic development to implantation in females which should detect effects on estrous cycle, embryo transport, implantation, and development of preimplantation stages of the embryo (ICH, 1994). Therefore, females were treated for 2 weeks prior to mating, during mating, and until implantation (Gestation Day 7). Mating was with untreated males in a 1:1 ratio for a maximum of 19 days. Vaginal smears were evaluated daily for stage of estrus for 15 days prior to treatment, during treatment, and during the mating period. At the time of insemination, females were separated from the male, body weights were determined every other day during gestation, and females were sacrificed between Gestation Days 13 and 15. At maternal sacrifice, the number of corpora lutea and the location and status of each implant site were recorded (live/dead embryo, early/late resorption), and a gross necropsy was conducted. Plasma sample collection and drug concentration analysis. During Treatment Week 15 of the male fertility study, and Treatment Day 14 during the premating treatment period of the female fertility study, five rats from each atorvastatin group were bled before (0 hr) and 1, 2, 8, 12, and 24 hr postdose for the determination of plasma atorvastatin-equivalent concentrations. Blood was collected by orbital puncture under anesthesia with isoflurane at the 0-, 1-, 2-, 8-, and 12-hr time points and by cardiac puncture under carbon dioxide anesthesia at 24 hr postdose. Five vehicle controls for each of the male and female studies were bled by cardiac puncture at 2 hr postdose. Plasma samples were stored at 0207C until assayed. Plasma samples were analyzed for atorvastatin-equivalent concentrations utilizing a HMG-CoA reductase enzyme inhibition assay validated over a 0.36 to 16 ng/ml concentration range. Two hundred and fifty microliters of calibration standards, quality control samples, plasma blanks, and unknown rat plasma samples was transferred to screw-capped vials. One milliliter of acetonitrile:acetone (95:5) was added to precipitate plasma proteins. Tubes were shaken in a mechanical shaker for 15 min and centrifuged at 2600g for 15 min. A 700-ml aliquot of the supernatant was transferred to a glass tube and evaporated to dryness at 377C under nitrogen. The residue was reconstituted in 50 ml of distilled water and then 50 ml of a buffer prepared by dissolving 52 mg NADPH and 26 mg D,L-dithiothreitol in 0.7 ml of 2 M phosphate buffer (pH 7.4), 1.4 ml of 0.1 M EDTA, and 5 ml of water and 100 ml of rat liver microsomes (0.75 to 1.0 mg protein/ml) were added

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FERTILITY STUDIES WITH ATORVASTATIN as the source of HMG-CoA reductase. The reaction mixture was incubated for 30 min in a 377C shaking water bath. The tubes were removed from the water bath and 50 ml of a 1 M phosphate buffer (pH 7.4) containing 0.2 mg/ml of [14C]HMG-CoA (approximately 100,000 dpm/ml) was added. The mixture was vortexed and incubated for 35 min at 377C in a shaking water bath. Then 25 ml of concentrated HCl was added, the tubes were vortexed, and tubes were incubated 25 min at 377C to lactonize [14C]mevalonic acid to [14C]mevalonolactone. The tubes were removed from the shaking water bath, cooled to room temperature, and vortexed, and a 200ml aliquot of the mixture was transferred to a wet AG 1X8 anion-exchange column. Each column was eluted with five 1-ml volumes of water and all five eluates were collected in a scintillation vial. Ten milliliters of scintillation cocktail was added to each vial, the vial was vortexed, and [14C] radioactivity was measured in a Packard Tricarb 2500TR counter, using a Transformed Spectral Index (tSIE) standard method of quench correction. The relationship between percentage inhibition of HMG-CoA reductase and log atorvastatin concentration in calibration standards was used to construct a calibration curve. The assay measures atorvastatin and atorvastatin metabolites in plasma capable of inhibiting HMG-CoA reductase. Therefore, atorvastatin concentration is expressed in terms of atorvastatin equivalents. Mean total recovery of [14C]atorvastatin from rat plasma by protein precipitation was 95.1%. The lower limit of quantitation was 0.36 ng eq/ml. For purposes of statistical analysis, one concentration which was below this limit (a 20 mg/kg female at Time 0) was assigned a value of zero. The relative standard deviation (%RSD) of calibration standards (range 0.36 to 16 ng/ml) ranged from 1.19 to 3.47% and relative error (%RE) of backcalculated values was within {1.67%. Assay precision and accuracy, based on quality control sample %RSD and %RE values, ranged from 2.68 to 8.62% and were within {5%, respectively. Pharmacokinetic and statistical methods. Statistical comparisons between the treated groups and the vehicle control used p £ 0.05 as the level of significance. Pairwise comparisons of the untreated control group with the vehicle control group in the male study were conducted using the Wilcoxon rank sum test for continuous data and Fisher’s exact test for dichotomous data. To control for the multiplicity of statistical comparisons (i.e., to reduce the likelihood of false-positive conclusions), the parameters were divided into distinct classes of related parameters (e.g., body weights in one class, food consumption in another). The classwise significance level was then allocated to each parameter proportionally by the inverse of the square root of the number of parameters in a class (Tukey et al., 1985). Continuous data were analyzed by Tukey’s sequential trend test using the rank-dose scale and rank-transformed data (Tukey et al., 1985; Park, 1985) at the 5% classwise significance level. A trend reversal test was performed at the 1% classwise significance level. Dichotomous data were analyzed by sequential application of a weighted Cochran–Armitage test for linear trend in proportions at the 5% classwise significance level. To ensure that the linear dose–response relationship was realistic, a nonlinearity test was performed at the 1% classwise significance level. Atorvastatin-equivalent pharmacokinetic parameters were obtained by noncompartmental analysis of plasma concentration–time data (Gibaldi and Perrier, 1982). Maximum plasma atorvastatin-equivalent concentrations (Cmax) and their corresponding times (tmax) were recorded as observed. Area under the plasma concentration–time curve from Time 0 to 24 hr [AUC(0-24)] was estimated using the linear trapezoidal rule. The apparent elimination rate constant (lz) was estimated as the absolute value of the slope of a least-squares linear regression of natural logarithm of plasma atorvastatin-equivalent concentration versus time during the terminal phase of the plasma concentration–time profile. Typically the 12- and 24-hr plasma concentration–time data were used to estimate lz . Apparent elimination half-life values (t1/2) were calculated as 0.693/lz . Comparison of atorvastatin-equivalent Cmax, tmax, AUC(0-24), and apparent half-life values between male and female rats were made using a two-way analysis of variance. Cmax and AUC(0-24) values for all dose groups were normalized for dose before analysis of variance was performed.

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Bonferoni’s method was used for all pairwise multiple comparisons. Statistical comparisons were made using the p õ 0.05 level of significance. Dose proportionality was assessed by visual inspection of pharmacokinetic parameter values. These studies were conducted in accordance with U.S. FDA Good Laboratory Practice regulations and current guidelines for animal welfare (NIH, 1985).

RESULTS

Male Fertility Study No treatment-related deaths, clinical signs, or gross pathological changes were observed. There were no differences between untreated and vehicle control groups, with the exception of accessory sex organ weights which were significantly greater in untreated controls. Statistically significant dose-related body weight gain suppressions of 17 and 25% occurred during the 11-week premating treatment period at 100 and 175 mg/kg, respectively, compared with vehicle controls (Table 1). Weight gain was most affected during the first 4 weeks of treatment at those doses. Food consumption was statistically significantly decreased by 7 to 16% at 100 and 175 mg/kg during the first 2 to 3 weeks of treatment and was decreased by 5% at 175 mg/kg during the 11-week premating period compared with vehicle controls. There were no treatment-related differences in absolute weights of the testes, epididymides, or accessory sex organs (Table 1). Relative testes weights were statistically significantly increased at 175 mg/kg compared with vehicle controls, which was primarily due to the decrease in body weight. There were no effects on epididymal sperm or testicular spermatid counts and no effects on sperm motility or morphology. No histopathological abnormalities were observed in testes or epididymides. There were no significant differences between groups in body weight gain during gestation for pregnant females. There were also no biologically significant differences between groups in copulation or fertility indices or in number of days to mating or number of viable litters. The group mean numbers of corpora lutea, implants, live fetuses, and pre- and postimplantation losses were comparable in all groups (Table 2). Female Fertility Study One pregnant female at 225 mg/kg had clinical signs of dehydration, emaciation, salivation, red material around mouth, urinary stain, and diarrhea on Gestation Days 7 and 8 and was found dead on Gestation Day 9 with additional necropsy findings of decreased body fat, watery cecal contents, gas-filled small intestine, and small thymus. The relationship of the death to treatment was considered equivocal since no other animals died at this dose and due to the nonspecific nature of the clinical signs and pathological findings. An increased incidence of alopecia and sporadic salivation occurred at 225 mg/kg. No other treatment-related

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TABLE 1 Body Weight, Food Consumption, and Reproductive Parameters in Male Rats Given Atorvastatin (Male Fertility Study) Male dose (mg/kg)

Untreated

Body weight gain (g) Days 0–77 Food consumption (g) Days 0–77 Week 15 body weight (g) Testes weight (g) Epididymides weight (g) Accessory organs weight (g) Sperm (106) Per gram cauda epididymis Per cauda epididymis Spermatids (106) Per gram testis Per testis Sperm motility (% motile) Sperm morphology (% normal sperm)

185.3 { 8.73 1958 568 3.54 1.52 3.62

{ { { { {

34.5 16.2 0.072 0.031 0.114**

0

20

180.7 { 6.76 1955 565 3.59 1.50 3.18

{ { { { {

194.4 { 7.92

32.2 17.5 0.076 0.026 0.102

1997 581 3.72 1.56 3.15

{ { { { {

28.9 11.3 0.071 0.030 0.124

100

175

150.2 { 5.91*

134.9 { 7.30*

1885 557 3.59 1.50 3.09

{ { { { {

26.9 13.6 0.060 0.028 0.151

1861 533 3.81 1.55 3.20

{ { { { {

30.0 13.2* 0.059 0.031 0.073

1506 { 38.3 496 { 17.4

1738 { 111.5 502 { 38.7

1529 { 67.2 480 { 22.7

1541 { 77.9 457 { 22.5

1418 { 79.0 464 { 26.1

156 { 5.1 251 { 10.0 93.8 { 0.94

160 { 9.9 264 { 16.4 94.1 { 1.01

157 { 4.6 263 { 11.0 97.5 { 0.55

160 { 5.5 255 { 7.6 95.3 { 1.07

164 { 4.6 285 { 9.7 94.0 { 1.65

96.3 { 1.75

98.9 { 0.49

97.2 { 0.85

97.0 { 1.11

98.3 { 0.63

Note. Male rats (mean body weight 378–381 g) were given daily oral doses of atorvastatin or 0.5% methylcellulose vehicle for 11 weeks prior to mating, during mating, and until necropsy. Ten males per group were sacrificed during Week 15 and male reproductive parameters were determined. Values represent the mean { SE for N Å 14–30 (body weight and food consumption) and N Å 10 for Week 15 reproductive organ weights and sperm parameters. * Significantly different from vehicle control at p õ 0.0500. ** Significantly different from vehicle control at p õ 0.0204.

clinical signs or gross necropsy findings were observed in any treatment group. No biologically significant differences in body weight gain occurred during the 2-week premating treatment period (Table 3). However, group mean body weight gain was statistically significantly reduced by 35%

at 225 mg/kg during the treatment period of gestation (Days 0–8) compared with vehicle controls. During the posttreatment period of gestation (Days 8–13), body weight gain at 225 mg/kg was increased by 25% and food consumption was increased by 19% compared with vehicle controls.

TABLE 2 Female Body Weights and Reproductive Parameters (Male Fertility Study) Male dose (mg/kg) Body weight gain (g) Gestation Days 0–7 Gestation Days 7–13 Reproduction data Copulation index (%) Fertility index (%) No. days to mating No. with viable litters Term sacrifice parameters No. implant sites No. live fetuses No. resorptions Preimplantation loss (%) Postimplantation loss (%)

Untreated

0

20

100

175

40.1 { 2.19 35.0 { 2.48

43.6 { 3.05 30.2 { 5.42

39.8 { 1.43 36.3 { 1.63

38.0 { 1.50 35.7 { 1.06

36.9 { 1.68 39.6 { 2.08

100 100 3.4 { 0.48 24

100 96.7 3.6 { 0.72 29

100 93.3 3.5 { 0.50 28

100 93.3 3.0 { 0.39 28

100 96.7 3.2 { 0.43 29

16.9 15.9 1.0 6.7 5.8

{ { { { {

0.30 0.37 0.18 1.67 1.07

16.3 15.2 1.0 7.8 6.8

{ { { { {

0.62 0.66 0.23 2.01 1.65

16.4 15.2 1.2 8.2 7.1

{ { { { {

0.59 0.55 0.18 2.71 1.05

16.1 15.0 1.1 5.9 7.2

{ { { { {

0.44 0.48 0.23 2.10 1.64

16.3 14.9 1.4 6.2 9.0

{ { { { {

0.54 0.57 0.23 2.46 1.65

Note. Untreated female rats (mean body weight 275–277 g) were mated with male rats treated with atorvastatin for 11 weeks prior to mating and during mating. Females were sacrificed on Gestation Days 13–15 and reproductive parameters were determined. Copulation index Å [No. sperm positive/ No. cohabitated] 1 100%; Fertility index Å [No. pregnant/No. sperm postive] 1 100%. Values are the mean { SE for N Å 18–29, where appropriate.

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FERTILITY STUDIES WITH ATORVASTATIN

TABLE 3 Body Weights and Reproductive Parameters of Female Rats Treated with Atorvastatin (Female Fertility Study) Female dose (mg/kg) Body weight gain (g) Premating Days 0–15 Gestation Days 0–8 Gestation Days 8–13 Food consumption (g) Premating Days 0–6 Premating Days 6–15 Gestation Days 0–8 Gestation Days 8–13 Reproduction data Copulation index (%) Fertility index (%) No. days to mating No. with viable litters Term sacrifice parameters No. corpora lutea No. implant sites No. live fetuses No. resorptions Preimplantation loss (%) Postimplantation loss (%)

0

20

100

225

21.5 { 1.81 48.3 { 2.17 29.4 { 1.37

21.6 { 1.64 45.6 { 2.28 30.3 { 1.90

28.9 { 2.12 47.2 { 1.66 30.8 { 1.75

23.6 { 2.14 31.4 { 4.52* 36.8 { 2.11*

115 182 212 133

{ { { {

1.7 2.8 4.0 6.3

100 96.0 2.9 { 0.48 24 18.0 15.9 15.0 1.0 12.6 7.0

{ { { { { {

0.55 0.97 1.00 0.19 4.91 1.83

114 181 216 141

{ { { {

1.8 3.8 5.1 5.1

100 100 3.4 { 0.68 25 19.2 17.3 15.8 1.5 9.0 7.9

{ { { { { {

0.71 0.65 0.55 0.32 2.99 1.51

114 198 218 150

{ { { {

2.2 3.4* 4.0 3.3*

100 92.0 2.4 { 0.20 23 19.2 17.3 16.0 1.3 9.1 7.2

{ { { { { {

0.50 0.62 0.52 0.27 2.85 1.36

104 192 198 158

{ { { {

2.3* 2.8* 9.4 3.3*

100 96.0 3.1 { 0.65 23 19.5 16.8 14.7 2.0 14.9 12.9

{ { { { { {

0.70 1.00 1.15 0.64 4.38 4.14

Note. Groups of 30 female rats (mean body weight 275–279 g) were treated with atorvastatin for 2 weeks prior to mating; 25 continued treatment during mating with untreated males and until Gestation Day 7. Females were sacrificed on Gestation Days 13–15 and reproductive parameters were determined. Copulation index Å [No. sperm positive/No. cohabitated] 1 100%; Fertility index Å [No. pregnant/No. sperm positive] 1 100%. Values are the mean { SE for N Å 23–30, where appropriate. * Significantly different from vehicle control at p õ 0.0224.

There were no differences between groups in the mean number of estrous cycles completed or the number of animals with abnormal estrous cycles (not shown). Copulation and fertility indices, number of days to mating, and number of viable litters were comparable between groups (Table 3). In addition, term sacrifice parameters (corpora lutea, implants, live fetuses, and pre- and postimplantation loss) were not significantly different between groups. Pharmacokinetics For male rats, Cmax and AUC(0-24) values for atorvastatin equivalents during Treatment Week 15 generally increased with increasing dose (Table 4; Figs. 1 and 2). Mean Cmax value ({SE) was 1820 { 456 ng eq/ml at 175 mg/ kg and the corresponding AUC(0-24) value was 14,500 { 4338 ng eqrhr/ml. Mean tmax ranged from 2.4 to 4.6 hr in all groups, and mean apparent half-life values ranged from 5.53 to 9.95 hr. For female rats, increases in Cmax and AUC(0-24) values on premating Treatment Day 14 were generally greater than proportional to increasing dose (Table 4; Figs. 1 and 2). The mean Cmax value was 7030 { 1646 ng eq/ml at 225 mg/ kg. The corresponding mean AUC(0-24) value was 56,200 { 20,751 ng eqrhr/ml. Mean tmax and apparent half-life

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values ranged from 1.00 to 5.20 hr and 2.42 to 4.85 hr, respectively. The mean Cmax for females was statistically greater than for males at 175/225 mg/kg based on dosenormalized values. Atorvastatin-equivalent concentrations were below the detection limit in controls. DISCUSSION

Atorvastatin, administered to rats prior to and during mating at doses which induced toxicity in the parental animals (175 mg/kg in males, 225 mg/kg in females), had no adverse effects on fertility and reproduction. In the male fertility study, significant body weight gain suppression and decreased food consumption occurred at 100 and/or 175 mg/kg, doses which are at least 60-fold greater than the maximum therapeutic dose of 80 mg per day in humans (Nawrocki et al., 1995). Despite the toxicity, there were no effects on copulation, fertility, number of days to mating, number of offspring or live litters, male reproductive organ weights, sperm counts, sperm motility, or sperm morphology. In the female fertility study, significant body weight gain suppression occurred at 225 mg/kg with no effects on estrous cycles, copulation, fertility, number of days to mating, or number of offspring or live litters. A dose of 20 mg/kg was a noeffect dose in males and females.

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TABLE 4 Atorvastatin Pharmacokinetic Parameters in Male and Female Rats 20 mg/kg Male Cmax (ng eq/ml) tmax (hr) AUC (0–24) (ng eqrhr/ml) t1/2 (hr)

110 4.60 926 5.53

{ { { {

13.3 2.29 155 1.29

175/225 mg/kga

100 mg/kg Female

80.7 1.00 480 3.62

{ { { {

12.5 0.0 42.6 0.53

Male 1,320 2.40 7,240 6.33

{ { { {

Female

279 1.40 1851 1.92

2,030 2.40 9,760 4.85

{ { { {

470 1.40 2,462 1.23

Male 1,820 3.80 14,500 9.95

{ { { {

456 1.71 4,338 5.59

Female 7,030 5.20 56,200 2.42

{ { { {

1,646* 1.71 20,751 0.44

Note. Values are means { SE, n Å 5. Cmax , maximum plasma concentration; tmax , time of maximum plasma concentration; AUC(0–24), area under the plasma concentration–time curve from Time 0 to 24 hr; t1/2, apparent half-life. a Male and female rats received 175 and 225 mg/kg doses, respectively. All Cmax and AUC(0–24) comparisons were based on dose-normalized values. * Male versus female values significantly different at p õ 0.05.

The results of this study show that atorvastatin-equivalent pharmacokinetics are highly variable and differ between male and female rats. Regarding variability, a coefficient of

FIG. 1. Mean { SE (n Å 5) atorvastatin plasma concentrations in male (open circles) and female (solid circles) rats after receiving 20, 100, and 175 or 225 mg/kg daily oral doses for 15 weeks for males and 14 days for females. Male and female rats received the same doses with the exception that high-dose male rats received 175 mg/kg and female rats received 225 mg/kg.

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variation in excess of 50% was common for many mean pharmacokinetic parameter values (data not shown). Variability appeared to increase with increasing dose. Mean atorvastatin Cmax and AUC(0-24) values for male rats generally increased with increasing dose. In female rats, mean Cmax and AUC(0-24) values generally increased in a greater than proportional manner with increasing dose. Apparent halflife and tmax values did not appear to change as a function of dose for either male or female rats. Because of the high pharmacokinetic variability and discrepant results between male and female rats, it is not clear whether atorvastatin pharmacokinetics are dose proportional in rats. Gender differences in the pharmacokinetics of atorvastatin equivalents in rats are supported by AUC(0-24) values generally being greater in female rats than male rats. The increase in AUC(0-

FIG. 2. Relationship between atorvastatin dose and AUC(0-24) in male and female rats. Mean { SE for males (open circles) and females (solid circles) after receiving 20, 100, and 175 or 225 mg/kg daily oral doses for 15 weeks for males and 14 days for females.

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24) values was accompanied by higher Cmax values in female rats than male rats as well. The greater-than-proportional increase in AUC in female rats may be due to saturation of an enzyme system responsible for the metabolism of atorvastatin and/or atorvastatin metabolite(s). There was no apparent gender difference in rate of absorption as reflected by similar tmax values for male and female rats. In general, apparent half-life values were based on the 12- and 24-hr plasma concentration–time data because the apparent terminal elimination phase did not begin until about 12 hr postdose. Since half-life values generally were not characterized over a four or five half-life time period, they may underestimate the true half-life values. The results of these two studies are consistent with results obtained with other HMG-CoA reductase inhibitors. Lovastatin, pravastatin, simvastatin, and fluvastatin were each shown to have no adverse effects on fertility and reproduction in rats (FDA, 1987, 1993; Tanase and Hirose, 1987; Wise et al., 1990). In addition to the traditional evaluations of copulation and fertility indices and production of live offspring, the present male fertility study included evaluations of testis histopathology and various sperm parameters. Although mating and fertility indices represent the integrated functions of the entire male reproductive process, fertility is generally considered to be an insensitive index of spermatogenesis in the rat since numerous studies have shown that significant decreases in sperm number can be associated with normal fertility in rodents (Meistrich, 1982; Robaire et al., 1984; Blazak et al., 1985). Evaluations of multiple sperm parameters such as epididymal and testicular count, motility, and morphology, and testicular histopathology, can be more sensitive indicators of effects on male reproduction in rodents than fertility (Morrissey et al., 1988; Toth et al., 1992; reviewed by Blazak et al., 1993). Therefore, the inclusion of multiple sperm parameters in the present study further substantiates the lack of adverse effects of atorvastatin on male reproduction. Varying degrees of testicular degeneration were observed sporadically in dogs treated with simvastatin, but not in rats, mice, or monkeys at comparable doses (Gerson et al., 1989). Similarly a low incidence of testicular degeneration was observed in dogs treated with lovastatin (MacDonald et al., 1988). In contrast, a recent study with atorvastatin treatment for 2 years in dogs demonstrated no effects on testicular histopathology, reproductive organ weights, or semen sperm count, motility, or morphology (Dostal et al., 1995). Sperm motility was slightly but significantly reduced in men with Type II hyperlipoproteinemia by lovastatin treatment, while all other indications of testicular function (testis size, sperm count, morphology, and serum androgen concentration) were unaffected (Farnsworth et al., 1987). Short-term treatment of familial hypercholesterolemic patients with simvastatin had no effects on sperm quality, seminal plasma concentra-

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tions of various sex gland products, or the serum concentrations of cortisol, testosterone, LH, FSH, or prolactin (Purvis et al., 1992). No indications of adverse effects on steroidogenesis in females have been reported. Despite these sporadic reports of rather minor changes in dogs and humans, there has not been any conclusive evidence of adverse reproductive effects in the large numbers of patients who have taken HMG-CoA reductase inhibitors. In addition to the lack of effects of atorvastatin on fertility and reproduction in the present studies, previous studies have shown atorvastatin to be nonteratogenic in rats and rabbits (Dostal et al., 1994), whereas other HMG-CoA reductase inhibitors, lovastatin (Minsker et al., 1983) and fluvastatin (FDA, 1993), were shown to produce malformations in rats. Thus, these previous studies and the present studies demonstrate that atorvastatin, a potent HMG-CoA reductase inhibitor, is not teratogenic in rats or rabbits and has no effects on fertility and reproduction in rats. ACKNOWLEDGMENTS The authors are grateful for the excellent technical assistance of Mr. William Craft, Ms. Sheri Gonyea-Polski, Ms. Ann Marie Morse, and Ms. Michelle Cole and the plasma drug concentration analyses performed by Ms. Helen Huang. We also thank Ms. Jamie Colgin for the statistical analyses, Dr. Robert Sigler for histopathological evaluation of testes, Mr. Y. Y. Shum for analysis of the plasma drug concentration data, and Ms. Carol Faber and Mr. Michael Oswalt for sperm evaluations.

REFERENCES Alberts, A. W., MacDonald, J. S., Till, A. E., and Tobert, J. A. (1989). Lovastatin. Cardiovasc. Drug Rev. 7, 89–109. Blazak, W. F., Rushbrook, C. J., Ernst, T. L., Stewart, B. E., Spak, D., DiBiasio-Erwin, D., and Black, V. (1985). Relationship between breeding performance and testicular/epididymal functioning in male Sprague– Dawley rats exposed to nitrobenzene. Toxicologist 5, 121. Blazak, W. F., Treinen, K. A., and Juniewicz, P. E. (1993). Application of testicular sperm head counts in the assessment of male reproductive toxicity. In Methods in Toxicology: Male Reproductive Toxicology (R. E. Chapin and J. J. Heindel, Eds.), Vol. 3, Part A, pp. 86–94. Academic Press, San Diego. Dobs, A. S., Sarma, P. S., and Schteingart, D. (1993). Long-term endocrine function in hypercholesterolemic patients treated with pravastatin, a new 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor. Metabolism 42, 1146–1152. Dostal, L. A., Schardein, J. L., and Anderson, J. A. (1994). Developmental toxicity of the HMG-CoA reductase inhibitor, atorvastatin, in rats and rabbits. Teratology 50, 387–394. Dostal, L. A., Faber, C. K., Rothwell, C. E., and Anderson, J. A. (1995). Semen analysis and sperm counts in beagle dogs treated with the HMGCoA reductase inhibitor, atorvastatin. Toxicologist 15, 248. Endo, A., Tsujita, Y., Kuroda, M., and Tanzawa, K. (1979). Effects of ML236B on cholesterol metabolism in mice and rats: Lack of hypocholesterolemic activity in normal animals. Biochim. Biophys. Acta 575, 266– 276. Farnsworth, W. H., Hoeg, J. M., Maher, M., Brittain, E. H., Sherins, R. J., and Brewer, H. B., Jr. (1987). Testicular function in Type II hyperlipopro-

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teinemic patients treated with lovastatin (mevinolin) or neomycin. J. Clin. Endocrinol. Metab. 65, 546–550. FDA (1987). Summary basis for approval of NDA 19-643 (lovastatin), p. 13. FDA (1993). Summary basis for approval of NDA 20-261 (fluvastatin), Pharmacology Review of Amendment Conducted August, 1988. Frishman, W. H., Zimetbaum, P., and Nadelmann, J. (1989). Lovastatin and other HMG-CoA reductase inhibitors. J. Clin. Pharmacol. 29, 975– 982. Gerson, R. J., MacDonald, J. S., Alberts, A. W., Kornbrust, D. J., Majka, J. A., Stubbs, R. J., and Bokelman, D. L. (1989). Animal safety and toxicology of simvastatin and related hydroxy-methylglutaryl-coenzyme A reductase inhibitors. Am. J. Med. 87(Suppl. 4A), 28S–38S. Gibaldi, M., and Perrier, D. (1982). Pharmacokinetics, 2nd ed., Dekker, New York. ICH (1994). International conference on harmonisation: Guideline on detection of toxicity to reproduction for medicinal products. Fed. Reg. 59(183), 22, 48746–48752. Krause, B. R., and Newton, R. S. (1995). Lipid-lowering activity of atorvastatin and lovastatin in rodent species: Triglyceride-lowering in rats correlates with efficacy in LDL animal models. Atherosclerosis 117, 237– 244. MacDonald, J. S. , Gerson, R. J., Kornbrust, D. J., Kloss, M. W., Prahalada, S., Berry, P. H., Alberts, A. W., and Bokelman, D. L. (1988). Preclinical evaluation of lovastatin. Am. J. Cardiol. 62, 16J–27J. Meistrich, M. L. (1982). Quantitative correlation between testicular stem cell survival, sperm production and fertilization in mouse after treatment with different cytotoxic agents. J. Androl. 3, 58–68. Minsker, D. H., MacDonald, J. S., Robertson, R. T., and Bokelman, D. L. (1983). Mevalonate supplementation in pregnant rats suppresses the teratogenicity of mevinolinic acid, an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase. Teratology 28, 449–456. Morrissey, R. E., Lamb, J. C., Schwetz, B. A., Teague, J. L., and Morris, R. W. (1988). Association of sperm, vaginal cytology, and reproductive organ weight data with results of continuous breeding reproduction studies in Swiss (CD-1) mice. Fundam. Appl. Toxicol. 11, 359–371.

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Nawrocki, J. W., Weiss, S. R., Davidson, M. H., Sprecher, D. L., Schwartz, S. L., Lupien, P.-J., Jones, P. H., Haber, H. E., and Black, D. M. (1995). Reductions of LDL cholesterol by 25% to 60% in patients with primary hypercholesterolemia by atorvastatin, a new HMG-CoA reductase inhibitor. Arterioscler. Thromb. Vasc. Biol. 15, 678–682. NIH (1985). Guide for the care and use of laboratory animals. NIH Publication 86-23. Park, Y. C. (1985). Nonparametric sequential trend test. Proc. Tenth Annu. SAS Users Group Intl. Conference, 809–813. Purvis, K., Tollefsrud, A., Rui, H., Haug, E., Norseth, J., Viksmoen, L., Ose, L., and Lund, H. (1992). Short-term effects of treatment with simvastatin on testicular function in patients with heterozygous familial hypercholesterolaemia. Eur. J. Clin. Pharmacol. 42, 61–64. Robaire, B., Smith, S., and Hales, B. F. (1984). Suppression of spermatogenesis by testosterone in adult male rats: Effect on fertility, pregnancy outcome and progeny. Biol. Reprod. 31, 221–230. Tanase, H., and Hirose, K. (1987). Reproduction study of pravastatin sodium administered prior to and in the early stages of pregnancy in rats. Jpn. Pharmacol. Ther. 15, 4975–4982. Toth, G. P., Kelty, K. C., George, E. L., Read, E. J., and Smith, M. K. (1992). Adverse male reproductive effects following subchronic exposure of rats to sodium dichloroacetate. Fundam. Appl. Toxicol. 19, 57–63. Tukey, J. W., Ciminera, J. L., and Heyse, J. F. (1985). Testing the statistical certainty of a response to increasing doses of a drug. Biometrics 42, 295– 301. Tsujita, Y., Kuroda, M., Shimada, Y., Tanzawa, K., Arai, M., Kaneko, I., Tanaka, M., Masuda, H., Tarumi, C., Watanabe, Y., and Fujii, S. (1986). CS-514, a competitive inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase: Tissue-selective inhibition of sterol synthesis and hypolipidemic effect on various animal species. Biochim. Biophys. Acta 887, 50–60. Wise, L. D., Minsker, D. H., Robertson, R. T., Bokelman, D. L., Akutsu, S., and Fujii, T. (1990). Simvastatin (MK-0733): Oral fertility study in rats. Oyo Yakuri/Pharmacometrics 39, 127–141. Wyrobek, A. J., and Bruce, W. R. (1975). Chemical induction of sperm abnormalities in mice. Proc. Natl. Acad. Sci. USA 72, 4423–4429.

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