TERATOLOGY 42:91-100 (1990)
External Genitalia Abnormalities in Male Rats Exposed In Utero to Finasteride, a 5~ReductaseInhibitor ROBERT L. CLARK, JOSEPH M. ANTONELLO, SCOTT J . GROSSMAN, L. DAVID WISE. CHARLES ANDERSON. WALTER J. BAGDON.' SRINIVASA PRAHALADA, JAMES s. MACDONALD, AND RICHARD T. ROBERTSON Merck Sharp & Dohme Research Laboratortes, West Point. Pennsylvania 19486 ~
ABSTRACT A series of studies was conducted to determine the developmental toxicity of the 5a-reductase inhibitor finasteride (MK-0906) in rats. This compound was administered orally once daily to pregnant rats during various extended treatment periods during gestation. F, offspring were evaluated on Day 20 of gestation as well as postnatally through mating to produce an F2 generation. MK-0906 treatment induced dosage-related incidences of hypospadias (penischisis) in male offspring with a threshold dosage level near 0.1 mgl kglday and a 100%effect level of 100 mglkglday (with dosing through Day 20 of gestation). MK-0906 also caused decreased anogenital distance in male offspring. The dosage response for this effect (ranging from a 4.2%decrease at 0.003 mglkglday to a 38% decrease at 100 mglkglday) was more shallow than that for hypospadias. The decreases in anogenital distance were at least partially reversible postnatally with essentially complete recovery at dosages up to 0.1 mglkglday. There was also a dosage-related, temporary induction of nipples in F, males. All of these effects were apparent following treatment on Days 6 through 17 of gestation but were more pronounced when dosing extended to Day 20 of gestation. Slight maternal toxicity consisting of minor decreases in body weight gain occurred only a t dosages of 3 mglkglday and higher, indicating the selective nature of the developmental toxicity. The 5a-reductase enzyme located in the rat fetal genital tubercle was studied in vitro and compared to that in the adult ventral prostate. The values for K,, V,,,, and IC,, for inhibition by MK-0906 were similar in the two tissues, suggesting that the enzymatic proteins in the genital tubercle and ventral prostate may be similar. Androgens are required for the differentiation of the male reproductive system: the penis from the genital tubercle, the penile urethra and prostate from the endodermally derived urogenital sinus, and the vas deferens, epididymis, and seminal vesicles from the mesodermally derived Wolffian (mesonephric) ducts (Wilson et al., '81). In utero exposure of male rat fetuses to antiandrogens (Neumann et al., '70a) or testosterone synthesis inhibitors (Bloch et al., '71; Goldman et al., '76) results in female-like differentiation of the genitalia. Testosterone is the proximate androgen for some but not all of the events of male differentiation. In human male pseudohermaphrodites lacking 5a-reductase, the enzyme which converts tes0 1990 WILEY-LISS, INC.
tosterone to 5a-dihydrotestosterone (DHT), Wolffian differentiation to vas deferens, epididymis, and seminal vesicles is normal but, at birth, the external genitalia are feminized and the prostate is reduced in size (Imperato-McGinley and Peterson, '76; Moore et al., '75). Specifically, the phallus is greatly reduced, the testes are undescended, and the urethra and a blind vaginal pouch lead to a urogenital sinus which opens on the perineum. At puberty, the penis enlarges to near normal size and the testes descend. However, the prostate fails to enlarge normally. Thus, the derivatives of the urogenReceived September 22, 1989; accepted January 2, 1990
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Study No. 1 2 3 4
5 6
Treatment period, gestational days 6-17 6-17 6-17 6-17 6-20 -14-201
TABLE 1 . Outline o f studv Drotocols Evaluation on Day 20 Dosage of gestation Postnatal levels, Litters/ Fetal evaluation, mg/kg/day group examinations litterdgroup 10, 30, 100, 300 10 External 0 0.03. 0.1,0.3, 1. 3 10 External 0 0.006, 0.1, 3;lOO 25 External, visceral. 0.1, 3, 100 0.0003, 0.003, 0.03, 0.3 0.1. 3, 100 ,
,
0 0 20
-
External. visceral, skeletal
15 22 15
'Treatment began 2 weeks prior to cohabitation with untreated males. Those females killed on Day 20 of gestation received their last dose on Day 19 of gestation.
ital sinus, which is endodermal in origin, opment of F, male offspring exposed to MKapparently require (DHT)for differentiation 0906 in utero has been followed starting whereas the derivatives of the Wolffian duct, with external, visceral, and skeletal examwhich is mesodermal in origin, use testoster- inations of fetuses on Day 20 of gestation, continuing with a postnatal assessment of one as the proximate androgen. Recently, highly specific, potent 5a-reduc- F, pups for effects on growth, developmentase inhibitors without affinity for androgen tal signs, and behavior, and terminating receptors have become available (Brooks et with an evaluation of mating performance al., '82; Rasmusson et al., '86) allowing the of the F, generation. In addition, an examdiscrimination between DHT-mediated and ination of the external genitalia including direct testosterone effects in animal models. anogenital distance was performed periodiThese inhibitors are intended for the treat- cally. The effects observed include those ment of benign prostatic hyperplasia (Stoner seen previously with other 5a-reductase inet al., '87, and Tenover et al., '89). It has been hibitors: decreased anogenital distance and found that in utero exposure to these inhib- nipples in male pups. The sequelae of these itors causes decreased anogenital distance in effects have been determined and, in addimale rat pups (Brooks et al., '82; Imperato- tion, a syndrome of malformations of the McGinley et al., '85, '86). The decrease in male external genitalia including hypospaanogenital distance caused by at least one of dias is described. these inhibitors, finasteride (MK-0906: NMATERIALS AND METHODS (2-methyl-2-propyl)-3-oxo-4-aza-5a-androstRats l-ene-17p carboxamide), can be prevented All studies used Sprague Dawley CR1: by the coadministration of DHT (George and Peterson, '88). This inhibitor also impaired CD(SD) BR rats from Charles River Breedthe prenatal growth of the prostate (George ing Laboratories. Animals were individuand Peterson, '88). Another of these inhibi- ally housed in wire-mesh bottom cages (except as specified) with room temperature tors (17~-N,N-diisopropylcarbamoyl-4-aza5a-androstan-3-one)caused the induction of of approximately 23°C and alternating 12 nipples in male rat pups (which normally hour periods of light and dark. Mating was lack them) indicating that DHT is appar- accomplished by cohabiting each female ently required for suppression of nipple dif- with one untreated male of the same strain. ferentiation (Imperato-McGinley et al., '86). Females were considered to be at Day 0 of The expression of these effects is presumably gestation when daily examination revealed dependent on the synthesis of testosterone, a seminal plug or vaginal sperm. Mated fewhich begins in the male rat embryo on Day males designated for natural delivery were transferred to plastic boxes on approxi15 of gestation (Habert and Picon, '84). We have conducted a series of develop- mately Day 16 of gestation where they and mental and reproductive toxicology studies their litters remained until weaning. Study of the inhibitor MK-0906 in rats. The devel- 5 (see Table 1)was conducted at Argus Re-
DEVELOPMENTAL TOXICITY OF MK-0906 IN MALE RATS
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vagina on postnatal Days 33, 35, and 37. The mean day of occurrence was estimated for each developmental sign. One F, pup of each sex from each litter was tested for acDrug administration tivity in an open field during postnatal Suspensions of finasteride (MK-0906, L- Week 6 and for behavior in a water-filled 652,931, Merck) in vehicle (0.5% aqueous T-maze in postnatal Week 8. In the open methylcellulose) were prepared daily. Doses field test, light-beam interruptions were were administered orally by gavage counted during a 3 minute period. In the through a metal catheter once daily in the water maze test, trials to criterion (three morning during the dosing period. Controls consecutive escapes with no wrong turns) received the same dosing volume (5 mlkg) were counted during two test periods with the location of the escape platform switched of vehicle. between test periods. One F, female per litStudy design ter from all groups and two F, males per An outline of the general design of the six litter from the 0.1 and 3 mg/kg/day groups studies reported here is shown in Table 1. (except those with obvious hypospadias) Most of the studies included examination of were mated with untreated colony animals. conceptuses on Day 20 of gestation. Im- F, females reaching an age of 14 weeks plants were removed and classified as live were checked for female hypospadias (urefetuses, dead fetuses, or resorptions. Each thra opening into the vagina). At necropsy fetus was weighed and examined exter- (postnatal Weeks 20 to 211, approximately nally, and the distance between the anterior two F, males per litter were checked again edge of the anus and posterior base of the for nipples, their external genitalia were exgenital tubercle was measured (anogenital amined, and anogenital distance was meadistance). In Studies 3 and 6, the abdominal sured. In Study 5, the parental females were aland thoracic viscera from every third fetus were examined grossly and the heads were lowed to deliver naturally. Anogenital disfixed in Bouin’s fixative for subsequent ex- tance was measured for all pups on postnaamination of freehand coronal sections. All tal Day 0 and for two culled males per litter fetuses from Studies 3 and 6 were processed on postnatal Day 21. The remaining males and stained with alizarin red for examina- were reared to postnatal Week 9 and their external genitalia were examined. tion of their skeletons. Prostates and seminal vesicles were In Studies 4 and 6, 15 dams per group were allowed to deliver their offspring nat- weighed at necropsy in Studies 4, 5, and 6. urally. Litters were culled to 10 pups on The effects of MK-0906 on these organ postnatal Day 3 , s on postnatal Day 7, and 4 weights will be discussed in a separate pubon postnatal Day 21 (balanced by gender lication. when possible). Pups were weighed on post5a-reductase activity in vitro natal Days 0, 7, 14, and 21 and weekly Genital tubercles from male fetal rats a t thereafter. The anogenital distances of 12 pups from each litter were measured on Day 17 of gestation and ventral prostates postnatal Day 0 (the day delivery was com- from 16 week-old adults were homogenized plete) and that of culled males on postnatal in 50 mM phosphate buffer (pH 6.8) containDays 3 and 7 (one per litter) and postnatal ing 0.25 M sucrose, 5 mM magnesium chloDay 21 (two per litter). The sex of all F, ride, 0.5 mM 2-mercaptoethanol, 50 mM sopups was confirmed by postmortem exami- dium chloride, and 0.05 mM disodium nation of gonads. All surviving F, males EDTA by using either a hand-held glass/ were examined for nipples on postnatal Day glass homogenizer (genital tubercles) or a 11. Two male and two female F, pups from Teflon/glass homogenizer (prostates). Veneach litter were examined for the auditory tral prostate homogenates were prepared as startle reflex on postnatal Days 11, 13, and a 25% (w/v)homogenate (protein concentra15 and for the free-fall righting reflex on tion 25 mgiml). Fetal genital tubercles from postnatal Days 12,14, and 16. Surviving F, several litters were collected with the aid of males were examined for descent of testes a dissecting microscope and pooled, and hoon postnatal Days 26,29, and 32. Surviving mogenates were prepared with approxiF, females were examined for patency of the mately 0.025 ml buffer per tubercle (protein
search Laboratories, Perkasie, Pennsylvania. The remaining studies were conducted at MSDRL, West Point, Pennsylvania.
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3 mg/ml). Tissues were stored a t -70°C until time of assay. The activity of 5a-reductase was measured by a modification of the method of Moore and Wilson ('75). [1,2-3H]Testosterone (47 Ci/mmol, Amersham, Arlington Heights, Illinois) a t 30 nM to 1 pM (final specific activity up to 10 pCi/mmol) was incubated with tissue homogenate (25 pl ventral prostate homogenate or 50 pl genital tubercle homogenate) in a 50 mM phosphate buffer (pH 6.8) containing NADPH with or without MK-0906. Incubations were carried out for 20-30 minutes at 25°C and were quenched by the addition of 1.5 ml of 25% methanol in dichloromethane and 20 pl of cold carriers. Samples were vortexed and centrifuged, and 1 ml of the organic phase was removed and blown to dryness under nitrogen. The samples were dissolved in 250 p1 of 60% methanol in water. The products were chromatographed on a Supelcosil CI8 150 x 4.6 mm column with 60% acetonitrile in water a t 1.0 mlimin. The eluant was sequentially passed through an LC-85 UV detector for estimation of recovery and a Beckman 171 radiochromatography detector for quantitation of radioactivity.
Statistical methods In these studies, the experimental units were considered to be the pregnant F, generation female rats (Haseman and Hogan, '76) and the sampling units were the F, generation fetuses or pups. Therefore, statistical analyses of F, generation parameters were performed on the male or female litter averages. Trend in response with increasing doses of MK-0906 was assessed by using the NOSTASOT sequential trend-testing approach of Tukey et al. ('85). When appropriate, female anogenital distance was included in the analysis as a covariable for male anogenital distance. Tests for trend in F, male anogenital distance and length of 0s penis were one-sided decreasing. Tests for trend in all other parameters were twosided. The statistical significance level was set a t 0.05. When the response variable was discrete, appropriate scores were assigned to the outcome classes. In these cases, trend was assessed by using the extended Mantel-Haenszel test statistic (Mantel, '63). An observed trend score was computed as the product of the dosing level, the response score, and the number of animals giving that response
801 0001
'
001 " 01 '
'
'
'
'
10
'
'
100
1000
rng/kg/doy M K 0906
Fig. 1. Dosage response for MK-0906-induced decrease in fetal weight. Treatment was on Days 6 through 17 of gestation (A,A-Study 1;*,o-Study 2; .,ElStudy 3) or from 2 weeks prior to cohabitation until Day 19 of gestation (X, +-Study 6). Cesarean section was on Day 20 of gestation. Group means are shown separately for males (closed symbols, X) and females (open symbols, +).
score, summed over all dosing levels and response categories. The squared difference between the observed trend score and the expected trend score divided by the variance of the observed trend score follows a ChiSquare distribution with one degree of freedom. This assessment of trend is consistent with the NOSTASOT approach in that the control is included in the analysis as a zero dose. RESULTS
Maternal toxicity High dosages of MK-0906 caused decreased body weight gain during gestation in parental females. In Study 4, there were 13% and 17%decreases in body weight gain between gestational Days 14 and 20 in the 3 and 100 mg/kg/day groups, respectively, compared to control. In Study 6, there were 12% and 20% decreases in body weight gain between gestational Days 12 and 20 at 3 and 100 mglkgiday, respectively. In Study 6, there was a slight (1.8%)but statistically significant (P50.05) increase in the length of gestation in the 100 mg/kg/day group compared to control. There was no other evidence of maternal toxicity in these studies. Slight delays in mating of parental females in Study 6 will be discussed in a separate publication. Decreases in fetal weight In utero exposure to MK-0906 caused dosage-related decreases in the weight of live fetuses on Day 20 of gestation (Fig. 1).The
DEVELOPMENTAL TOXICITY OF MK-0906 IN MALE RATS
95
of approximately 52%) at which point the anogenital distance would be the same as that of females (there was no effect on female anogenital distance). However, there was no evidence of saturation of the effect on males in any study. The dosage response curve was shifted slightly further to the left in Study 5 in which treatment was on Days 6 through 20 of gestation using the lowest dosage levels tested. The extrapolated threshold effect levels for the three dosing periods discussed above range from approximately 0.001 to 0.002 mglkglday. In Study 5, there was a 2.1% decrease at 0.0003 mgl kglday which was statistically significant ( P i 0 . 0 5 )but this is not considered to be biologically significant since the decrease was very slight and the dosage level was below the extrapolated threshold effect levels. The MK-0906-induced decreases in anogenital distance in male offspring occurred at dosages which had no effect on fetal weight and thus could not be explained simply by a decrease in fetal size. decreases occurred approximately equally The decreases in anogenital distance in in male and female fetuses and were more male offspring were a t least partially repronounced when dosing extended to Day 19 versible (Fig. 3). Following cessation of of gestation compared to when dosing was treatment in Studies 4, 5, and 6, the perstopped a t Day 17 of gestation. The observed ineal region of MK-0906-exposed male offdecreases in fetal weight were accompanied spring grew more rapidly than controls. by delays in ossification (Studies 3 and 6; This catch-up growth occurred primarily data not shown). The decreases in fetal during the first 3 postnatal weeks and was weight seen on Day 20 of gestation were ap- more rapid in the higher dosage level parently reversible since there were negli- groups where the effect had been greater. gible effects on F1 pup weights postnatally, The recovery was essentially complete in even a t 100 mglkglday in Studies 4 and 6. the 0.1 mglkglday group in Study 6 in which the anogenital distance of males was 98.0% Decreased anogenital distance in of controls a t postnatal Week 20. male offspring In utero exposure to MK-0906 caused dosNipples in Fl males age-related decreases in anogenital distance in male offspring (Fig. 2). The dosage reIn utero exposure to 3 and 100 mglkglday sponse was very shallow: when measured on MK-0906 in Studies 4 and 6 caused dosageDay 20 of gestation following treatment on related induction of nipples in F, males Days 6 through 17 of gestation (Studies 1,2, which were apparent on postnatal Day 11 and 31, there were partial effects over a (Fig. 4). The incidences were higher in 10,000-fold dosage range-a 5.8% decrease Study 6 (with dosing from before mating unat 0.03 mglkglday and a 29% decrease at til Day 20 of gestation). There was an even 300 mglkglday. The effect on male anogen- more potent induction of nipples in Study 5 ital distance was slightly greater a t each (dosing on Days 6 through 20 of gestation) dosage level in Study 6 in which dosing ex- with a higher incidence at 0.3 mglkglday tended from before mating until Day 19 or than a t 3 mglkglday in Studies 4 and 6. No 20 of gestation. In that study, there was a nipples were seen in males at dosages of 0.1 38% decrease in male anogenital distance mglkglday or less in any study. The preson Day 20 of gestation at 100 mglkglday. ence of the nipples was transient as they This level of effect approaches the presumed were no longer apparent after the animals maximal effect (corresponding to a decrease reached sexual maturity.
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rng/kg/doy MK 0906
Fig. 5. Dosage response for MK-0906-induced incidence of male hypospadias. Treatment was on gestational Days 6 through 17 (A,Study 4), Days 6 through 20 (B, Study 5), or from 2 weeks prior to cohabitation until Day 20 of gestation (0, Study 6). Approximately 30 males per group were examined in Studies 4 and 6 and 110 per group in Study 5. No animal had hypospadias a t 0.0003, 0.003, and 0.03 mgikgiday in Study 5.
50
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140
Postnatal Day
Fig. 3. Postnatal recovery from MK-0906-induced decrease in anogenital distance. Panel A Study 4. Panel B Study 6. Treatment was at 0.1 (O),. 3 (A,A), or 100 (0,W mglkgiday. 100
I
/ I
2 1 %
(_j
40
rng/kg/doy MK 0906
Fig. 4. Dosage response for MK-0906-induced incidence of nipples in male pups. Examination for nipples was performed on postnatal Day 11. Treatment was on gestational Days 6 to 17 (0, Study 41, Days 6 through 20 (A, Study 5), or from 2 weeks prior to cohabitation until Day 20 of gestation (0,Study 6). Approximately 50 males per group were examined in Studies 4 and 6 and approximately 150 per group in Study 5.
Malformations of the external genitalia in male ogspring In utero exposure to MK-0906 at 0.3, 3, and 100 mglkglday in Studies 4, 5, and 6
caused dosage-related incidences of hypospadias (penischisis) (Fig. 5). This malformation was most readily apparent after preputial separation when the penis could be fully expressed for examination. In affected animals, the urethral opening extended a variable distance toward the base of the penis on the inferior (ventral) side (Fig. 6). The frenulum often extended to the urethral opening, even in those animals with only slight hypospadias, thus restricting the full extension of the penis. In severely affected animals, the groove on the inferior side of the penis extended to its base. The unattached flaps of penile tissue extended laterally from the expressed penis giving the penis a paddle-like appearance and revealing the 0s penis attached only a t the bottom of the exposed groove. The penis was occasionally reduced in size. In most of the affected animals, there were also anomalies apparent even without expressing the penis. The preputial glands were distended and extended caudally. The tip of the penis sometimes protruded beneath the distended preputial glands through a cleft in the prepuce on its inferior side. Also, some of the affected males, like females, had little hair in the perineal area. One F, male in the 0.1 mg/kg/day group in Study 4 had slight abnormalities of the external genitalia. The urethral opening of this animal was slightly extended proximally on the ventral side of the penis and the preputial skin on the ventral side was slightly shortened. There were no malfor-
DEVELOPMENTAL TOXICITY OF MK-0906 IN MALE RATS
trast, there was no significant (P>0.05) relationship between hypospadias and anogenital distance in study 6 (dosing from before mating until Day 20 of gestation). Decreases in length of 0s penis In utero exposure to 100 mgkglday MK0906 caused slight but statistically significant (P10.05)decreases in the length of the 0s penis of F, males when measured during postnatal Week 20 (Table 2). In Study 6, there was a 9%mean decrease a t 100 rngl kglday compared to control. In Study 4, the group means in the control and 100 mglkgl day groups were the same (9.5 mm) but five of 26 F1 males in the 100 mglkgtday group had a slightly shortened 0s penis (range: 6.8 to 8.2 mm) compared to the 26 control males (range: 8.8 to 11.2 mm). Other developmental endpoints possibly related to MK-0906 exposure in utero In general, there was little evidence that in utero treatment with MK-0906 caused the death of F, offspring either in utero or postnatally or caused structural abnormalities (data not shown) other than the malformations of the external genitalia and decreases in anogenital distance in F, male Fig. 6. Morphology of MK-0906-induced malforma- offspring discussed above. tions of the male external genitalia. The inferior (venTwo F, males from parental females tral) side of the expressed penis of control male rats is treated with 100 mglkglday until Day 20 of shown in panels A and B. The open groove (penischisis) on the inferior side of the penis of MK-0906-exposed gestation (study 6) each had one normal demales is shown in panels C (at arrow)and D. The arrow scended testis and one testis which rein panel D points to the exposed 0s penis in the open mained undescended (and reduced in size) groove (the penis is directed toward the camera). Note until termination during postnatal Week the perineal alopecia in panels C and D. 20. The testes in the remaining animals in that group and in all other MK-0906mations of the external genitalia a t dosages treated groups in Studies 4 and 6 descended of 0.030 mglkglday and lower in Study 5. during the same time period as controls (TaSince there were also no malformations of ble 2). This lack of a dramatic effect on testhe external genitalia in control animals in tes descent is interesting in light of the obany of these studies, the presence of a single servation that the gubernaculum of the affected animal at 0.1 mglkglday may mean gestational Day 17 rat fetus contains high that 0.1 mglkglday is close to a threshold levels of 5a-reductase and that the growth dosage level for the induction of hypospa- of the gubernaculum is inhibited by 50 rngl dias in rats. There was no effect of MK-0906 kglday MK-0906 S.C. (George, '89). In the swimming maze test conducted treatment on the position of the urethral opening in female offspring at any dosage during postnatal Week 8 in study 6, there were statistically significant ( P ~ 0 . 0 5 in) level. In Study 4, with dosing on gestational creases in trials to criterion in both Tests 1 Days 6 through 17, F1males with hypospa- and 2 for F, males in the 100 mglkglday dias in the combined 3 and 100 mglkglday group and in Test 1 for males in the 3 rngl groups had slightly (10%)but statistically kglday group (Table 2). However, there significantly (Pr0.01) shorter anogenital were no effects in the swimming maze test distance on postnatal Day 0 than their in Study 4 conducted at the same dosage groupmates without hypospadiase. In con- levels.
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TABLE 2 . Selected develoamental endaoints o f F , males exaosed in utero to MK-0906' Dosage group, mglkgiday MK-0906 Control 0.1 3 100 Length of 0s penis (mm, mean -t SD) Study 4 9.5 i- 0.6 9.9 i 0.8 9.5 i 1.2s 10.4 +- 0.8NS 9.4 ? 0.5s 10.1 i 0.9NS Study 6 10.3 2 0.9 10.1 -?r 0.7 Testes descent (estimated mean postnatal day of occurrence) 30.3 29.9NS Study 4 29.6 27.6 28.6 28.3NS Study 6 29.2 29.0 Swim maze (trials to criterion, mean i- SD) Study 4' 4.7 1.6 5.9 i 2.7NS 5.4 2 2.1 6.4 -t 2.8 Test 1 7.5 i 2.4 7.9 t 3.7 5.9 i- 2.2NS 6.4 i- 2.5 Test 2 Study 63 4.3 i 1.4 5.6 2 2.7NS 7.1 t 2.8s 6.3 2 2.55 Test 1 6.6 i 2.4 6.4 i- 1.6 8.1 i 2.1s 7.4 -t 2.ONS Test 2
*
INS, trend not statistically significant (P>0.05) through indicated dosage; S, trend statistically significant (Ps0.05) through indicated dosage group. 'Age = 50-52 days. 3Age = 48-50 days.
Developmental endpoints not directly affected by treatment with MK-0906 There were no direct effects of in utero exposure to MK-0906 on the development of the auditory startle and free-fall righting reflexes, the timing of vaginal canalization, or activity in an open field (data not shown). Mating performance and fertility of F1 males with normal external genitalia in groups dosed with 0.1 and 3 mg/kg/day MK0906 were comparable to control (data not shown). In addition, four F, males with slight hypospadias and a slightly shortened prepuce were cohabited with females in Study 4-one in the 0.1 mgtkgtday group and three in the 3 mg/kg/day group. All four malformed males impregnated females within the first 4 days of cohabitation thus demonstrating the lack of effect of their abnormal genitalia on mating performance. Similarly, there was no effect on mating performance or fertility of F, females (data not shown). 5a-reductase kinetics in vitro and inhibition by MK-0906 The affinity of 5a-reductase for testosterone was similar in the fetal genital tubercle and the adult ventral prostate. Under the conditions used, the K, values were 360 and 500 nM for fetal genital tubercle and ventral prostate, respectively. The maximal velocity of 5a-reductase was slightly higher in tubercle (288 pmol/hour/mg protein) than in ventral prostate homogenate (132 pmoll hour/mg protein). The rate of formation of 5a-reduced metabolites was inhibited by
MK-0906 in a concentration-dependent manner (Table 3). The dose response curves from both tissues were similar, with an IC,, of approximately 25 nM. DISCUSSION
The effects of MK-0906 indicate that dihydrotestosterone (DHT) synthesized by the action of 5a-reductase is apparently required for the differentiation of the penile urethra in the rat. DHT is also apparently involved in the formation (and/or separation) of the prepuce, particularly on the inferior (ventral) side of the penis. The generally normal or nearly normal size of the adult penis indicates either that the growth of the penis is not very dependent on DHT synthesized in utero or that the androgen produced postnatally (after the end of the period of dosing with MK-0906) was sufficient to allow normal or catch-up growth. This point highlights the differences between this 5a-reductase inhibitor and antiandrogens which cause a much more complete feminization of male rat offspring (Neumann et al., '70b). The observed decreases in anogenital distance induced by MK-0906 likely indicate simply decreased growth of the perineal region including the epithelium, mesenchyme, and urethra and thereby suggest the role of DHT in that growth process. The mesenchymal cells of the genital tubercle contain androgen receptors (Murakami '87), and it may be that the mesenchymal cells in the neighboring perineal region also contain androgen receptors which make them
DEVELOPMENTAL TOXICITY OF MK-0906 IN MALE RATS
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at non-maternotoxic dosages with a threshold effect level near 0.1 mglkgtday. Anogenin tiitro ital distance in male offspring was de% of control activity creased at much lower dosages (at least as MK-0906 (nM) Genital tubercle Ventral prostate low as 0.003 mglkglday). The relationship between decreased anogenital distance and 2 95.8 f 3.0 91.3 f 8.4 47.9 t 3.9 25 53.8 f 1.6 the occurrence of hypospadias is unclear. 250 3.6 ? 2.6 1.9 t 3.8 Some of the data in our studies suggest an association between decreased anogenital Mean 2 S.E.M., N = 3. Testosterone concentration = 1 +M. distance and hypospadias and some do not. One possibility is that the growth of the pervery sensitive to the ambient levels of DHT. ineal region up to Day 17 of gestation is The period of responsiveness to androgens related to the formation of hypospadias and may correspond t o the period of the most the growth of that region after Day 17, alrapid growth (normal and catch-up) of the though still potentially affected by MKperineal region in males (from Day 17 of 0906 treatment, is no longer correlated with gestation to approximately postnatal Day the occurrence of hypospadias. Additional 21). The extreme potency of MK-0906 as studies to clarify this point are in progress. In summary, most of the effects seen in measured by its ability to cause decreased anogenital distance in male rat fetuses male rats exposed to MK-0906 in utero are (which occurred a t dosages at least as low as expected pharmacological effects. These 3 Fglkglday) is not very different from its findings are important for assessing the popotency for suppression of serum DHT in tential hazards of inadvertent exposure of adult men. MK-0906 caused a 63% decrease women to MK-0906. The risk assessment in serum DHT levels following treatment in based on the induction of hypospadias indiman for 14 days a t only 0.04 mglday (ap- cates that the exposure of women of childproximately 0.7 Fglkglday; Gormley et al., bearing potential to even relatively low levels of MK-0906 should be avoided. The '89). The similar K,'s and IC5Gs for 5a-reduc- significance of the slight MK-0906-induced tase from fetal genital tubercle and adult decreases in anogenital distance a t dosages ventral prostate suggest that the enzyme in below the threshold dosage level for producthese two tissues may be the same. The MK- ing hypospadias is unclear and is currently 0906 concentration dependence for the inhi- under investigation. Since this agent is inbition of 5a-reductase in vitro does not to- tended for treatment of benign prostatic hytally account for the very shallow dosage perplasia, our findings have no direct impliresponse for the MK-0906-induced decrease cations for the intended patient population. in anogenital distance in vivo (slope = apACKNOWLEDGMENTS proximately 6 to 10% decrease per log unit of dosage) since there was little or no inhiWe gratefully acknowledge the expert bition of 5a-reductase at 2 nM in vitro and technical assistance of Gabriel A. Battisti, nearly complete inhibition at 250 nM, a con- Kristie Deyerle-Brooks, Kristin Collevecentration only 125-fold higher. This differ- chio, Wendy A. Cusick, Warren D. Ditzler, ence in dose response slope may be at least Donald M. Duchai, Donna K. Parcell, Janice partially explained by the ability of testos- L. Sina, Judith A. Trojnar, Maureen G. Urterone to bind to the androgen receptor (al- baniak, and Christine M. Vetter. We also beit with a lower affinity than DHT) and thank Dror M. Rom, who performed some of thereby partially counteract the decrease in the statistical analyses reported, and anogenital distance due to decreased DHT. Thelma M. Demetrius, who provided excelTestosterone levels may be increased in the lent secretarial assistance. Study 5 was conpresence of MK-0906 [as is the case in men ducted at Argus Research Laboratories un(Tenover et al., '89) and young male rats der the direction of Drs. Alan M. Hoberman (George et al., '89) treated with MK-09061, and Elizabeth A. Lochry. thus further compensating for the lack of LITERATURE CITED DHT. These studies demonstrate that MK-0906 Bloch, E., M. Lew, and M. Klein (1971) Studies on the is a potent selective teratogen in rats proinhibition of fetal androgen formation. Inhibition of ducing frank malformations (hypospadias) testosterone synthesis in rat and rabbit fetal testes TABLE 3. Inhibition of fetal genital tubercle and adult ventral prostate 5a-reductase by MK-0906
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with observations on reproductive tract development. Endocrinology, 89: 16-31. Brooks, J.R., C. Berman, M. Hichens, R.L. Primka, G.F. Reynolds, and G.H. Rasmusson (1982) Biological activities of a new steroidal inhibitor of A4-5a-reductase. Proc. SOC. Exp. Biol. Med., 169.57-73. George, F. (1989) Developmental pattern of 5a-reductase activity in the rat gubernaculum. Endocrinology, 124:727-732. George, F.W., L. Johnson, and J.D. Wilson (1989) The effect of a 5a-reductase inhibitor on androgen physiology in the immature male rat. Endocrinology, 125: 2434-2438. George, F.W., and K.G. Peterson (1988)5a-dihydrotestosterone formation is necessary for embryogenesis of the rat prostate. Endocrinology, 122:1159-1164. Goldman, A.S., R.D. Eavey, and M.K. Baker (1976) Production of male pseudohermaphroditism in rats by two new inhibitors of steroid l7a-hydroxylase and C 17-20 lyase. J . Endocrinol., 71:289-297. Gormley, G., R. Rittmaster, H. Gregg, K. Lasseter, D. Ferguson, and E. Stoner (1989) Dose response effect of an orally active 5-alpha-reductase inhibitor (MK0906) in man. Endocrinology, in press. Habert, R., and R. Picon (1984)Testosterone, dihydrotestosterone and estradiol-17P levels in maternal and fetal plasma and in fetal testes in the rat. J . Steroid Biochem., 21t183-198. Haseman, J.K., and M.D. Hogan (1976) Selection of the experimental unit in teratology studies. Teratology, I2:165-171. Imperato-McGinley,J., Z. Binienda, A. Arthur, D. Mininberg, E.D. Vaughan, and F. Quimby (1985) The development of a male pseudohermaphrodite rat using an inhibitor of the enzyme 5a-reductase. Endocrinology, 116t807-812. Imperato-McGinley, J., Z. Binienda, J. Gedney, and E.D. Vaughan (1986) Nipple differentiation in fetal male rats treated with an inhibitor of the enzyme 5areductase: Definition of a selective role for dihydrotestosterone. Endocrinology, 118:132-137. Imperato-McGinley, J., and R. Peterson (1976) Male pseudohermaphroditism: The complexities of male phenotypic development. Am. J . Med., 61r251-272.
Mantel, N. (1963) Chi-square tests with one degree of freedom; extensions of the Mantel-Haenszel procedure. J . Am. Stat. Assoc., 58:690-700. Moore, R., J. Griffin, and J . Wilson (1975) Diminished 5a-reductase activity in extracts of fibroblasts cultured from patients with familial incomplete male pseudohermaphroditism, Type 2. J . Biol. Chem., 250: 7168-7172. Moore, R.J., and J.D. Wilson (1975) In: Methods in Enzymology. B.W. OMalley and J.G. Hardman, eds. Academic Press, New York, Vol. 36, pp. 466-474. Murakami, R. (1987) Autoradiographic studies of the localisation of androgen-binding cells in the genital tubercles of fetal rats. J. Anat., 151:209-219. Neumann, F., R. von Berswordt-Wallrabe, W. Elger, H. Steinbeck, J.D. Hahn, and M. Kramer (1970a) Aspects of androgen-dependent events as studied by antiandrogens. Recent Prog. Horm. Res., 26:337-410. Neumann, F., W. Elger, and H. Steinbeck (1970b) Antiandrogens and reproductive development. Philos. Trans. R. SOC.Lond. [Biol.], 259:179-184. Rasmusson, G., G. Reynolds, N. Steinberg, E. Walton, G. Patel, T. Liang, M. Cascieri, A. Cheung, J . Brooks, and C. Berman (1986) Azasteroids: Structure-activity relationships for inhibition of 5a-reductase and of androgen receptor binding. J. Med. Chem., 29: 2298-23 15. Stoner, E., H. Gregg, J . Otterbein, E. Stein, G. Lamkin, P. Vlasses, J . Danzeisen, and J . Tobert (1987) Administration of MK- 0906, a 4-azasteroid compound results in marked suppression of serum dihydrotestosterone in healthy men. Clin. Res., 35t402A. Tenover, J., M. Zeitner, and S. Plymate (1989) Effects of 24-week administration of a 5a-reductase inhibitor (MK-906) on serum levels of testosterone (T), free T, and gonadotropins in men. Endocrine Society Program and Abstracts Book. Williams and Wilkins, Baltimore, MD. p. 168. Tukey, J.W., J.L. Ciminera, and J.F. Heyse (1985) Testing the statistical certainty of a response to increasing doses of a drug. Biometrics, 41:295-301. Wilson, J.D., F.W. George, and J.E. Griffin (1981) The hormonal control of sexual development. Science, 21I: 1278-1284.
External Genitalia Abnormalities in Male Rats Exposed In Utero to Finasteride, a 5~ReductaseInhibitor ROBERT L. CLARK, JOSEPH M. ANTONELLO, SCOTT J . GROSSMAN, L. DAVID WISE. CHARLES ANDERSON. WALTER J. BAGDON.' SRINIVASA PRAHALADA, JAMES s. MACDONALD, AND RICHARD T. ROBERTSON Merck Sharp & Dohme Research Laboratortes, West Point. Pennsylvania 19486 ~
ABSTRACT A series of studies was conducted to determine the developmental toxicity of the 5a-reductase inhibitor finasteride (MK-0906) in rats. This compound was administered orally once daily to pregnant rats during various extended treatment periods during gestation. F, offspring were evaluated on Day 20 of gestation as well as postnatally through mating to produce an F2 generation. MK-0906 treatment induced dosage-related incidences of hypospadias (penischisis) in male offspring with a threshold dosage level near 0.1 mgl kglday and a 100%effect level of 100 mglkglday (with dosing through Day 20 of gestation). MK-0906 also caused decreased anogenital distance in male offspring. The dosage response for this effect (ranging from a 4.2%decrease at 0.003 mglkglday to a 38% decrease at 100 mglkglday) was more shallow than that for hypospadias. The decreases in anogenital distance were at least partially reversible postnatally with essentially complete recovery at dosages up to 0.1 mglkglday. There was also a dosage-related, temporary induction of nipples in F, males. All of these effects were apparent following treatment on Days 6 through 17 of gestation but were more pronounced when dosing extended to Day 20 of gestation. Slight maternal toxicity consisting of minor decreases in body weight gain occurred only a t dosages of 3 mglkglday and higher, indicating the selective nature of the developmental toxicity. The 5a-reductase enzyme located in the rat fetal genital tubercle was studied in vitro and compared to that in the adult ventral prostate. The values for K,, V,,,, and IC,, for inhibition by MK-0906 were similar in the two tissues, suggesting that the enzymatic proteins in the genital tubercle and ventral prostate may be similar. Androgens are required for the differentiation of the male reproductive system: the penis from the genital tubercle, the penile urethra and prostate from the endodermally derived urogenital sinus, and the vas deferens, epididymis, and seminal vesicles from the mesodermally derived Wolffian (mesonephric) ducts (Wilson et al., '81). In utero exposure of male rat fetuses to antiandrogens (Neumann et al., '70a) or testosterone synthesis inhibitors (Bloch et al., '71; Goldman et al., '76) results in female-like differentiation of the genitalia. Testosterone is the proximate androgen for some but not all of the events of male differentiation. In human male pseudohermaphrodites lacking 5a-reductase, the enzyme which converts tes0 1990 WILEY-LISS, INC.
tosterone to 5a-dihydrotestosterone (DHT), Wolffian differentiation to vas deferens, epididymis, and seminal vesicles is normal but, at birth, the external genitalia are feminized and the prostate is reduced in size (Imperato-McGinley and Peterson, '76; Moore et al., '75). Specifically, the phallus is greatly reduced, the testes are undescended, and the urethra and a blind vaginal pouch lead to a urogenital sinus which opens on the perineum. At puberty, the penis enlarges to near normal size and the testes descend. However, the prostate fails to enlarge normally. Thus, the derivatives of the urogenReceived September 22, 1989; accepted January 2, 1990
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Study No. 1 2 3 4
5 6
Treatment period, gestational days 6-17 6-17 6-17 6-17 6-20 -14-201
TABLE 1 . Outline o f studv Drotocols Evaluation on Day 20 Dosage of gestation Postnatal levels, Litters/ Fetal evaluation, mg/kg/day group examinations litterdgroup 10, 30, 100, 300 10 External 0 0.03. 0.1,0.3, 1. 3 10 External 0 0.006, 0.1, 3;lOO 25 External, visceral. 0.1, 3, 100 0.0003, 0.003, 0.03, 0.3 0.1. 3, 100 ,
,
0 0 20
-
External. visceral, skeletal
15 22 15
'Treatment began 2 weeks prior to cohabitation with untreated males. Those females killed on Day 20 of gestation received their last dose on Day 19 of gestation.
ital sinus, which is endodermal in origin, opment of F, male offspring exposed to MKapparently require (DHT)for differentiation 0906 in utero has been followed starting whereas the derivatives of the Wolffian duct, with external, visceral, and skeletal examwhich is mesodermal in origin, use testoster- inations of fetuses on Day 20 of gestation, continuing with a postnatal assessment of one as the proximate androgen. Recently, highly specific, potent 5a-reduc- F, pups for effects on growth, developmentase inhibitors without affinity for androgen tal signs, and behavior, and terminating receptors have become available (Brooks et with an evaluation of mating performance al., '82; Rasmusson et al., '86) allowing the of the F, generation. In addition, an examdiscrimination between DHT-mediated and ination of the external genitalia including direct testosterone effects in animal models. anogenital distance was performed periodiThese inhibitors are intended for the treat- cally. The effects observed include those ment of benign prostatic hyperplasia (Stoner seen previously with other 5a-reductase inet al., '87, and Tenover et al., '89). It has been hibitors: decreased anogenital distance and found that in utero exposure to these inhib- nipples in male pups. The sequelae of these itors causes decreased anogenital distance in effects have been determined and, in addimale rat pups (Brooks et al., '82; Imperato- tion, a syndrome of malformations of the McGinley et al., '85, '86). The decrease in male external genitalia including hypospaanogenital distance caused by at least one of dias is described. these inhibitors, finasteride (MK-0906: NMATERIALS AND METHODS (2-methyl-2-propyl)-3-oxo-4-aza-5a-androstRats l-ene-17p carboxamide), can be prevented All studies used Sprague Dawley CR1: by the coadministration of DHT (George and Peterson, '88). This inhibitor also impaired CD(SD) BR rats from Charles River Breedthe prenatal growth of the prostate (George ing Laboratories. Animals were individuand Peterson, '88). Another of these inhibi- ally housed in wire-mesh bottom cages (except as specified) with room temperature tors (17~-N,N-diisopropylcarbamoyl-4-aza5a-androstan-3-one)caused the induction of of approximately 23°C and alternating 12 nipples in male rat pups (which normally hour periods of light and dark. Mating was lack them) indicating that DHT is appar- accomplished by cohabiting each female ently required for suppression of nipple dif- with one untreated male of the same strain. ferentiation (Imperato-McGinley et al., '86). Females were considered to be at Day 0 of The expression of these effects is presumably gestation when daily examination revealed dependent on the synthesis of testosterone, a seminal plug or vaginal sperm. Mated fewhich begins in the male rat embryo on Day males designated for natural delivery were transferred to plastic boxes on approxi15 of gestation (Habert and Picon, '84). We have conducted a series of develop- mately Day 16 of gestation where they and mental and reproductive toxicology studies their litters remained until weaning. Study of the inhibitor MK-0906 in rats. The devel- 5 (see Table 1)was conducted at Argus Re-
DEVELOPMENTAL TOXICITY OF MK-0906 IN MALE RATS
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vagina on postnatal Days 33, 35, and 37. The mean day of occurrence was estimated for each developmental sign. One F, pup of each sex from each litter was tested for acDrug administration tivity in an open field during postnatal Suspensions of finasteride (MK-0906, L- Week 6 and for behavior in a water-filled 652,931, Merck) in vehicle (0.5% aqueous T-maze in postnatal Week 8. In the open methylcellulose) were prepared daily. Doses field test, light-beam interruptions were were administered orally by gavage counted during a 3 minute period. In the through a metal catheter once daily in the water maze test, trials to criterion (three morning during the dosing period. Controls consecutive escapes with no wrong turns) received the same dosing volume (5 mlkg) were counted during two test periods with the location of the escape platform switched of vehicle. between test periods. One F, female per litStudy design ter from all groups and two F, males per An outline of the general design of the six litter from the 0.1 and 3 mg/kg/day groups studies reported here is shown in Table 1. (except those with obvious hypospadias) Most of the studies included examination of were mated with untreated colony animals. conceptuses on Day 20 of gestation. Im- F, females reaching an age of 14 weeks plants were removed and classified as live were checked for female hypospadias (urefetuses, dead fetuses, or resorptions. Each thra opening into the vagina). At necropsy fetus was weighed and examined exter- (postnatal Weeks 20 to 211, approximately nally, and the distance between the anterior two F, males per litter were checked again edge of the anus and posterior base of the for nipples, their external genitalia were exgenital tubercle was measured (anogenital amined, and anogenital distance was meadistance). In Studies 3 and 6, the abdominal sured. In Study 5, the parental females were aland thoracic viscera from every third fetus were examined grossly and the heads were lowed to deliver naturally. Anogenital disfixed in Bouin’s fixative for subsequent ex- tance was measured for all pups on postnaamination of freehand coronal sections. All tal Day 0 and for two culled males per litter fetuses from Studies 3 and 6 were processed on postnatal Day 21. The remaining males and stained with alizarin red for examina- were reared to postnatal Week 9 and their external genitalia were examined. tion of their skeletons. Prostates and seminal vesicles were In Studies 4 and 6, 15 dams per group were allowed to deliver their offspring nat- weighed at necropsy in Studies 4, 5, and 6. urally. Litters were culled to 10 pups on The effects of MK-0906 on these organ postnatal Day 3 , s on postnatal Day 7, and 4 weights will be discussed in a separate pubon postnatal Day 21 (balanced by gender lication. when possible). Pups were weighed on post5a-reductase activity in vitro natal Days 0, 7, 14, and 21 and weekly Genital tubercles from male fetal rats a t thereafter. The anogenital distances of 12 pups from each litter were measured on Day 17 of gestation and ventral prostates postnatal Day 0 (the day delivery was com- from 16 week-old adults were homogenized plete) and that of culled males on postnatal in 50 mM phosphate buffer (pH 6.8) containDays 3 and 7 (one per litter) and postnatal ing 0.25 M sucrose, 5 mM magnesium chloDay 21 (two per litter). The sex of all F, ride, 0.5 mM 2-mercaptoethanol, 50 mM sopups was confirmed by postmortem exami- dium chloride, and 0.05 mM disodium nation of gonads. All surviving F, males EDTA by using either a hand-held glass/ were examined for nipples on postnatal Day glass homogenizer (genital tubercles) or a 11. Two male and two female F, pups from Teflon/glass homogenizer (prostates). Veneach litter were examined for the auditory tral prostate homogenates were prepared as startle reflex on postnatal Days 11, 13, and a 25% (w/v)homogenate (protein concentra15 and for the free-fall righting reflex on tion 25 mgiml). Fetal genital tubercles from postnatal Days 12,14, and 16. Surviving F, several litters were collected with the aid of males were examined for descent of testes a dissecting microscope and pooled, and hoon postnatal Days 26,29, and 32. Surviving mogenates were prepared with approxiF, females were examined for patency of the mately 0.025 ml buffer per tubercle (protein
search Laboratories, Perkasie, Pennsylvania. The remaining studies were conducted at MSDRL, West Point, Pennsylvania.
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3 mg/ml). Tissues were stored a t -70°C until time of assay. The activity of 5a-reductase was measured by a modification of the method of Moore and Wilson ('75). [1,2-3H]Testosterone (47 Ci/mmol, Amersham, Arlington Heights, Illinois) a t 30 nM to 1 pM (final specific activity up to 10 pCi/mmol) was incubated with tissue homogenate (25 pl ventral prostate homogenate or 50 pl genital tubercle homogenate) in a 50 mM phosphate buffer (pH 6.8) containing NADPH with or without MK-0906. Incubations were carried out for 20-30 minutes at 25°C and were quenched by the addition of 1.5 ml of 25% methanol in dichloromethane and 20 pl of cold carriers. Samples were vortexed and centrifuged, and 1 ml of the organic phase was removed and blown to dryness under nitrogen. The samples were dissolved in 250 p1 of 60% methanol in water. The products were chromatographed on a Supelcosil CI8 150 x 4.6 mm column with 60% acetonitrile in water a t 1.0 mlimin. The eluant was sequentially passed through an LC-85 UV detector for estimation of recovery and a Beckman 171 radiochromatography detector for quantitation of radioactivity.
Statistical methods In these studies, the experimental units were considered to be the pregnant F, generation female rats (Haseman and Hogan, '76) and the sampling units were the F, generation fetuses or pups. Therefore, statistical analyses of F, generation parameters were performed on the male or female litter averages. Trend in response with increasing doses of MK-0906 was assessed by using the NOSTASOT sequential trend-testing approach of Tukey et al. ('85). When appropriate, female anogenital distance was included in the analysis as a covariable for male anogenital distance. Tests for trend in F, male anogenital distance and length of 0s penis were one-sided decreasing. Tests for trend in all other parameters were twosided. The statistical significance level was set a t 0.05. When the response variable was discrete, appropriate scores were assigned to the outcome classes. In these cases, trend was assessed by using the extended Mantel-Haenszel test statistic (Mantel, '63). An observed trend score was computed as the product of the dosing level, the response score, and the number of animals giving that response
801 0001
'
001 " 01 '
'
'
'
'
10
'
'
100
1000
rng/kg/doy M K 0906
Fig. 1. Dosage response for MK-0906-induced decrease in fetal weight. Treatment was on Days 6 through 17 of gestation (A,A-Study 1;*,o-Study 2; .,ElStudy 3) or from 2 weeks prior to cohabitation until Day 19 of gestation (X, +-Study 6). Cesarean section was on Day 20 of gestation. Group means are shown separately for males (closed symbols, X) and females (open symbols, +).
score, summed over all dosing levels and response categories. The squared difference between the observed trend score and the expected trend score divided by the variance of the observed trend score follows a ChiSquare distribution with one degree of freedom. This assessment of trend is consistent with the NOSTASOT approach in that the control is included in the analysis as a zero dose. RESULTS
Maternal toxicity High dosages of MK-0906 caused decreased body weight gain during gestation in parental females. In Study 4, there were 13% and 17%decreases in body weight gain between gestational Days 14 and 20 in the 3 and 100 mg/kg/day groups, respectively, compared to control. In Study 6, there were 12% and 20% decreases in body weight gain between gestational Days 12 and 20 at 3 and 100 mglkgiday, respectively. In Study 6, there was a slight (1.8%)but statistically significant (P50.05) increase in the length of gestation in the 100 mg/kg/day group compared to control. There was no other evidence of maternal toxicity in these studies. Slight delays in mating of parental females in Study 6 will be discussed in a separate publication. Decreases in fetal weight In utero exposure to MK-0906 caused dosage-related decreases in the weight of live fetuses on Day 20 of gestation (Fig. 1).The
DEVELOPMENTAL TOXICITY OF MK-0906 IN MALE RATS
95
of approximately 52%) at which point the anogenital distance would be the same as that of females (there was no effect on female anogenital distance). However, there was no evidence of saturation of the effect on males in any study. The dosage response curve was shifted slightly further to the left in Study 5 in which treatment was on Days 6 through 20 of gestation using the lowest dosage levels tested. The extrapolated threshold effect levels for the three dosing periods discussed above range from approximately 0.001 to 0.002 mglkglday. In Study 5, there was a 2.1% decrease at 0.0003 mgl kglday which was statistically significant ( P i 0 . 0 5 )but this is not considered to be biologically significant since the decrease was very slight and the dosage level was below the extrapolated threshold effect levels. The MK-0906-induced decreases in anogenital distance in male offspring occurred at dosages which had no effect on fetal weight and thus could not be explained simply by a decrease in fetal size. decreases occurred approximately equally The decreases in anogenital distance in in male and female fetuses and were more male offspring were a t least partially repronounced when dosing extended to Day 19 versible (Fig. 3). Following cessation of of gestation compared to when dosing was treatment in Studies 4, 5, and 6, the perstopped a t Day 17 of gestation. The observed ineal region of MK-0906-exposed male offdecreases in fetal weight were accompanied spring grew more rapidly than controls. by delays in ossification (Studies 3 and 6; This catch-up growth occurred primarily data not shown). The decreases in fetal during the first 3 postnatal weeks and was weight seen on Day 20 of gestation were ap- more rapid in the higher dosage level parently reversible since there were negli- groups where the effect had been greater. gible effects on F1 pup weights postnatally, The recovery was essentially complete in even a t 100 mglkglday in Studies 4 and 6. the 0.1 mglkglday group in Study 6 in which the anogenital distance of males was 98.0% Decreased anogenital distance in of controls a t postnatal Week 20. male offspring In utero exposure to MK-0906 caused dosNipples in Fl males age-related decreases in anogenital distance in male offspring (Fig. 2). The dosage reIn utero exposure to 3 and 100 mglkglday sponse was very shallow: when measured on MK-0906 in Studies 4 and 6 caused dosageDay 20 of gestation following treatment on related induction of nipples in F, males Days 6 through 17 of gestation (Studies 1,2, which were apparent on postnatal Day 11 and 31, there were partial effects over a (Fig. 4). The incidences were higher in 10,000-fold dosage range-a 5.8% decrease Study 6 (with dosing from before mating unat 0.03 mglkglday and a 29% decrease at til Day 20 of gestation). There was an even 300 mglkglday. The effect on male anogen- more potent induction of nipples in Study 5 ital distance was slightly greater a t each (dosing on Days 6 through 20 of gestation) dosage level in Study 6 in which dosing ex- with a higher incidence at 0.3 mglkglday tended from before mating until Day 19 or than a t 3 mglkglday in Studies 4 and 6. No 20 of gestation. In that study, there was a nipples were seen in males at dosages of 0.1 38% decrease in male anogenital distance mglkglday or less in any study. The preson Day 20 of gestation at 100 mglkglday. ence of the nipples was transient as they This level of effect approaches the presumed were no longer apparent after the animals maximal effect (corresponding to a decrease reached sexual maturity.
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rng/kg/doy MK 0906
Fig. 5. Dosage response for MK-0906-induced incidence of male hypospadias. Treatment was on gestational Days 6 through 17 (A,Study 4), Days 6 through 20 (B, Study 5), or from 2 weeks prior to cohabitation until Day 20 of gestation (0, Study 6). Approximately 30 males per group were examined in Studies 4 and 6 and 110 per group in Study 5. No animal had hypospadias a t 0.0003, 0.003, and 0.03 mgikgiday in Study 5.
50
1
0
20
1
40
60
80
100
1
120
140
Postnatal Day
Fig. 3. Postnatal recovery from MK-0906-induced decrease in anogenital distance. Panel A Study 4. Panel B Study 6. Treatment was at 0.1 (O),. 3 (A,A), or 100 (0,W mglkgiday. 100
I
/ I
2 1 %
(_j
40
rng/kg/doy MK 0906
Fig. 4. Dosage response for MK-0906-induced incidence of nipples in male pups. Examination for nipples was performed on postnatal Day 11. Treatment was on gestational Days 6 to 17 (0, Study 41, Days 6 through 20 (A, Study 5), or from 2 weeks prior to cohabitation until Day 20 of gestation (0,Study 6). Approximately 50 males per group were examined in Studies 4 and 6 and approximately 150 per group in Study 5.
Malformations of the external genitalia in male ogspring In utero exposure to MK-0906 at 0.3, 3, and 100 mglkglday in Studies 4, 5, and 6
caused dosage-related incidences of hypospadias (penischisis) (Fig. 5). This malformation was most readily apparent after preputial separation when the penis could be fully expressed for examination. In affected animals, the urethral opening extended a variable distance toward the base of the penis on the inferior (ventral) side (Fig. 6). The frenulum often extended to the urethral opening, even in those animals with only slight hypospadias, thus restricting the full extension of the penis. In severely affected animals, the groove on the inferior side of the penis extended to its base. The unattached flaps of penile tissue extended laterally from the expressed penis giving the penis a paddle-like appearance and revealing the 0s penis attached only a t the bottom of the exposed groove. The penis was occasionally reduced in size. In most of the affected animals, there were also anomalies apparent even without expressing the penis. The preputial glands were distended and extended caudally. The tip of the penis sometimes protruded beneath the distended preputial glands through a cleft in the prepuce on its inferior side. Also, some of the affected males, like females, had little hair in the perineal area. One F, male in the 0.1 mg/kg/day group in Study 4 had slight abnormalities of the external genitalia. The urethral opening of this animal was slightly extended proximally on the ventral side of the penis and the preputial skin on the ventral side was slightly shortened. There were no malfor-
DEVELOPMENTAL TOXICITY OF MK-0906 IN MALE RATS
trast, there was no significant (P>0.05) relationship between hypospadias and anogenital distance in study 6 (dosing from before mating until Day 20 of gestation). Decreases in length of 0s penis In utero exposure to 100 mgkglday MK0906 caused slight but statistically significant (P10.05)decreases in the length of the 0s penis of F, males when measured during postnatal Week 20 (Table 2). In Study 6, there was a 9%mean decrease a t 100 rngl kglday compared to control. In Study 4, the group means in the control and 100 mglkgl day groups were the same (9.5 mm) but five of 26 F1 males in the 100 mglkgtday group had a slightly shortened 0s penis (range: 6.8 to 8.2 mm) compared to the 26 control males (range: 8.8 to 11.2 mm). Other developmental endpoints possibly related to MK-0906 exposure in utero In general, there was little evidence that in utero treatment with MK-0906 caused the death of F, offspring either in utero or postnatally or caused structural abnormalities (data not shown) other than the malformations of the external genitalia and decreases in anogenital distance in F, male Fig. 6. Morphology of MK-0906-induced malforma- offspring discussed above. tions of the male external genitalia. The inferior (venTwo F, males from parental females tral) side of the expressed penis of control male rats is treated with 100 mglkglday until Day 20 of shown in panels A and B. The open groove (penischisis) on the inferior side of the penis of MK-0906-exposed gestation (study 6) each had one normal demales is shown in panels C (at arrow)and D. The arrow scended testis and one testis which rein panel D points to the exposed 0s penis in the open mained undescended (and reduced in size) groove (the penis is directed toward the camera). Note until termination during postnatal Week the perineal alopecia in panels C and D. 20. The testes in the remaining animals in that group and in all other MK-0906mations of the external genitalia a t dosages treated groups in Studies 4 and 6 descended of 0.030 mglkglday and lower in Study 5. during the same time period as controls (TaSince there were also no malformations of ble 2). This lack of a dramatic effect on testhe external genitalia in control animals in tes descent is interesting in light of the obany of these studies, the presence of a single servation that the gubernaculum of the affected animal at 0.1 mglkglday may mean gestational Day 17 rat fetus contains high that 0.1 mglkglday is close to a threshold levels of 5a-reductase and that the growth dosage level for the induction of hypospa- of the gubernaculum is inhibited by 50 rngl dias in rats. There was no effect of MK-0906 kglday MK-0906 S.C. (George, '89). In the swimming maze test conducted treatment on the position of the urethral opening in female offspring at any dosage during postnatal Week 8 in study 6, there were statistically significant ( P ~ 0 . 0 5 in) level. In Study 4, with dosing on gestational creases in trials to criterion in both Tests 1 Days 6 through 17, F1males with hypospa- and 2 for F, males in the 100 mglkglday dias in the combined 3 and 100 mglkglday group and in Test 1 for males in the 3 rngl groups had slightly (10%)but statistically kglday group (Table 2). However, there significantly (Pr0.01) shorter anogenital were no effects in the swimming maze test distance on postnatal Day 0 than their in Study 4 conducted at the same dosage groupmates without hypospadiase. In con- levels.
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TABLE 2 . Selected develoamental endaoints o f F , males exaosed in utero to MK-0906' Dosage group, mglkgiday MK-0906 Control 0.1 3 100 Length of 0s penis (mm, mean -t SD) Study 4 9.5 i- 0.6 9.9 i 0.8 9.5 i 1.2s 10.4 +- 0.8NS 9.4 ? 0.5s 10.1 i 0.9NS Study 6 10.3 2 0.9 10.1 -?r 0.7 Testes descent (estimated mean postnatal day of occurrence) 30.3 29.9NS Study 4 29.6 27.6 28.6 28.3NS Study 6 29.2 29.0 Swim maze (trials to criterion, mean i- SD) Study 4' 4.7 1.6 5.9 i 2.7NS 5.4 2 2.1 6.4 -t 2.8 Test 1 7.5 i 2.4 7.9 t 3.7 5.9 i- 2.2NS 6.4 i- 2.5 Test 2 Study 63 4.3 i 1.4 5.6 2 2.7NS 7.1 t 2.8s 6.3 2 2.55 Test 1 6.6 i 2.4 6.4 i- 1.6 8.1 i 2.1s 7.4 -t 2.ONS Test 2
*
INS, trend not statistically significant (P>0.05) through indicated dosage; S, trend statistically significant (Ps0.05) through indicated dosage group. 'Age = 50-52 days. 3Age = 48-50 days.
Developmental endpoints not directly affected by treatment with MK-0906 There were no direct effects of in utero exposure to MK-0906 on the development of the auditory startle and free-fall righting reflexes, the timing of vaginal canalization, or activity in an open field (data not shown). Mating performance and fertility of F1 males with normal external genitalia in groups dosed with 0.1 and 3 mg/kg/day MK0906 were comparable to control (data not shown). In addition, four F, males with slight hypospadias and a slightly shortened prepuce were cohabited with females in Study 4-one in the 0.1 mgtkgtday group and three in the 3 mg/kg/day group. All four malformed males impregnated females within the first 4 days of cohabitation thus demonstrating the lack of effect of their abnormal genitalia on mating performance. Similarly, there was no effect on mating performance or fertility of F, females (data not shown). 5a-reductase kinetics in vitro and inhibition by MK-0906 The affinity of 5a-reductase for testosterone was similar in the fetal genital tubercle and the adult ventral prostate. Under the conditions used, the K, values were 360 and 500 nM for fetal genital tubercle and ventral prostate, respectively. The maximal velocity of 5a-reductase was slightly higher in tubercle (288 pmol/hour/mg protein) than in ventral prostate homogenate (132 pmoll hour/mg protein). The rate of formation of 5a-reduced metabolites was inhibited by
MK-0906 in a concentration-dependent manner (Table 3). The dose response curves from both tissues were similar, with an IC,, of approximately 25 nM. DISCUSSION
The effects of MK-0906 indicate that dihydrotestosterone (DHT) synthesized by the action of 5a-reductase is apparently required for the differentiation of the penile urethra in the rat. DHT is also apparently involved in the formation (and/or separation) of the prepuce, particularly on the inferior (ventral) side of the penis. The generally normal or nearly normal size of the adult penis indicates either that the growth of the penis is not very dependent on DHT synthesized in utero or that the androgen produced postnatally (after the end of the period of dosing with MK-0906) was sufficient to allow normal or catch-up growth. This point highlights the differences between this 5a-reductase inhibitor and antiandrogens which cause a much more complete feminization of male rat offspring (Neumann et al., '70b). The observed decreases in anogenital distance induced by MK-0906 likely indicate simply decreased growth of the perineal region including the epithelium, mesenchyme, and urethra and thereby suggest the role of DHT in that growth process. The mesenchymal cells of the genital tubercle contain androgen receptors (Murakami '87), and it may be that the mesenchymal cells in the neighboring perineal region also contain androgen receptors which make them
DEVELOPMENTAL TOXICITY OF MK-0906 IN MALE RATS
99
at non-maternotoxic dosages with a threshold effect level near 0.1 mglkgtday. Anogenin tiitro ital distance in male offspring was de% of control activity creased at much lower dosages (at least as MK-0906 (nM) Genital tubercle Ventral prostate low as 0.003 mglkglday). The relationship between decreased anogenital distance and 2 95.8 f 3.0 91.3 f 8.4 47.9 t 3.9 25 53.8 f 1.6 the occurrence of hypospadias is unclear. 250 3.6 ? 2.6 1.9 t 3.8 Some of the data in our studies suggest an association between decreased anogenital Mean 2 S.E.M., N = 3. Testosterone concentration = 1 +M. distance and hypospadias and some do not. One possibility is that the growth of the pervery sensitive to the ambient levels of DHT. ineal region up to Day 17 of gestation is The period of responsiveness to androgens related to the formation of hypospadias and may correspond t o the period of the most the growth of that region after Day 17, alrapid growth (normal and catch-up) of the though still potentially affected by MKperineal region in males (from Day 17 of 0906 treatment, is no longer correlated with gestation to approximately postnatal Day the occurrence of hypospadias. Additional 21). The extreme potency of MK-0906 as studies to clarify this point are in progress. In summary, most of the effects seen in measured by its ability to cause decreased anogenital distance in male rat fetuses male rats exposed to MK-0906 in utero are (which occurred a t dosages at least as low as expected pharmacological effects. These 3 Fglkglday) is not very different from its findings are important for assessing the popotency for suppression of serum DHT in tential hazards of inadvertent exposure of adult men. MK-0906 caused a 63% decrease women to MK-0906. The risk assessment in serum DHT levels following treatment in based on the induction of hypospadias indiman for 14 days a t only 0.04 mglday (ap- cates that the exposure of women of childproximately 0.7 Fglkglday; Gormley et al., bearing potential to even relatively low levels of MK-0906 should be avoided. The '89). The similar K,'s and IC5Gs for 5a-reduc- significance of the slight MK-0906-induced tase from fetal genital tubercle and adult decreases in anogenital distance a t dosages ventral prostate suggest that the enzyme in below the threshold dosage level for producthese two tissues may be the same. The MK- ing hypospadias is unclear and is currently 0906 concentration dependence for the inhi- under investigation. Since this agent is inbition of 5a-reductase in vitro does not to- tended for treatment of benign prostatic hytally account for the very shallow dosage perplasia, our findings have no direct impliresponse for the MK-0906-induced decrease cations for the intended patient population. in anogenital distance in vivo (slope = apACKNOWLEDGMENTS proximately 6 to 10% decrease per log unit of dosage) since there was little or no inhiWe gratefully acknowledge the expert bition of 5a-reductase at 2 nM in vitro and technical assistance of Gabriel A. Battisti, nearly complete inhibition at 250 nM, a con- Kristie Deyerle-Brooks, Kristin Collevecentration only 125-fold higher. This differ- chio, Wendy A. Cusick, Warren D. Ditzler, ence in dose response slope may be at least Donald M. Duchai, Donna K. Parcell, Janice partially explained by the ability of testos- L. Sina, Judith A. Trojnar, Maureen G. Urterone to bind to the androgen receptor (al- baniak, and Christine M. Vetter. We also beit with a lower affinity than DHT) and thank Dror M. Rom, who performed some of thereby partially counteract the decrease in the statistical analyses reported, and anogenital distance due to decreased DHT. Thelma M. Demetrius, who provided excelTestosterone levels may be increased in the lent secretarial assistance. Study 5 was conpresence of MK-0906 [as is the case in men ducted at Argus Research Laboratories un(Tenover et al., '89) and young male rats der the direction of Drs. Alan M. Hoberman (George et al., '89) treated with MK-09061, and Elizabeth A. Lochry. thus further compensating for the lack of LITERATURE CITED DHT. These studies demonstrate that MK-0906 Bloch, E., M. Lew, and M. Klein (1971) Studies on the is a potent selective teratogen in rats proinhibition of fetal androgen formation. Inhibition of ducing frank malformations (hypospadias) testosterone synthesis in rat and rabbit fetal testes TABLE 3. Inhibition of fetal genital tubercle and adult ventral prostate 5a-reductase by MK-0906
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with observations on reproductive tract development. Endocrinology, 89: 16-31. Brooks, J.R., C. Berman, M. Hichens, R.L. Primka, G.F. Reynolds, and G.H. Rasmusson (1982) Biological activities of a new steroidal inhibitor of A4-5a-reductase. Proc. SOC. Exp. Biol. Med., 169.57-73. George, F. (1989) Developmental pattern of 5a-reductase activity in the rat gubernaculum. Endocrinology, 124:727-732. George, F.W., L. Johnson, and J.D. Wilson (1989) The effect of a 5a-reductase inhibitor on androgen physiology in the immature male rat. Endocrinology, 125: 2434-2438. George, F.W., and K.G. Peterson (1988)5a-dihydrotestosterone formation is necessary for embryogenesis of the rat prostate. Endocrinology, 122:1159-1164. Goldman, A.S., R.D. Eavey, and M.K. Baker (1976) Production of male pseudohermaphroditism in rats by two new inhibitors of steroid l7a-hydroxylase and C 17-20 lyase. J . Endocrinol., 71:289-297. Gormley, G., R. Rittmaster, H. Gregg, K. Lasseter, D. Ferguson, and E. Stoner (1989) Dose response effect of an orally active 5-alpha-reductase inhibitor (MK0906) in man. Endocrinology, in press. Habert, R., and R. Picon (1984)Testosterone, dihydrotestosterone and estradiol-17P levels in maternal and fetal plasma and in fetal testes in the rat. J . Steroid Biochem., 21t183-198. Haseman, J.K., and M.D. Hogan (1976) Selection of the experimental unit in teratology studies. Teratology, I2:165-171. Imperato-McGinley,J., Z. Binienda, A. Arthur, D. Mininberg, E.D. Vaughan, and F. Quimby (1985) The development of a male pseudohermaphrodite rat using an inhibitor of the enzyme 5a-reductase. Endocrinology, 116t807-812. Imperato-McGinley, J., Z. Binienda, J. Gedney, and E.D. Vaughan (1986) Nipple differentiation in fetal male rats treated with an inhibitor of the enzyme 5areductase: Definition of a selective role for dihydrotestosterone. Endocrinology, 118:132-137. Imperato-McGinley, J., and R. Peterson (1976) Male pseudohermaphroditism: The complexities of male phenotypic development. Am. J . Med., 61r251-272.
Mantel, N. (1963) Chi-square tests with one degree of freedom; extensions of the Mantel-Haenszel procedure. J . Am. Stat. Assoc., 58:690-700. Moore, R., J. Griffin, and J . Wilson (1975) Diminished 5a-reductase activity in extracts of fibroblasts cultured from patients with familial incomplete male pseudohermaphroditism, Type 2. J . Biol. Chem., 250: 7168-7172. Moore, R.J., and J.D. Wilson (1975) In: Methods in Enzymology. B.W. OMalley and J.G. Hardman, eds. Academic Press, New York, Vol. 36, pp. 466-474. Murakami, R. (1987) Autoradiographic studies of the localisation of androgen-binding cells in the genital tubercles of fetal rats. J. Anat., 151:209-219. Neumann, F., R. von Berswordt-Wallrabe, W. Elger, H. Steinbeck, J.D. Hahn, and M. Kramer (1970a) Aspects of androgen-dependent events as studied by antiandrogens. Recent Prog. Horm. Res., 26:337-410. Neumann, F., W. Elger, and H. Steinbeck (1970b) Antiandrogens and reproductive development. Philos. Trans. R. SOC.Lond. [Biol.], 259:179-184. Rasmusson, G., G. Reynolds, N. Steinberg, E. Walton, G. Patel, T. Liang, M. Cascieri, A. Cheung, J . Brooks, and C. Berman (1986) Azasteroids: Structure-activity relationships for inhibition of 5a-reductase and of androgen receptor binding. J. Med. Chem., 29: 2298-23 15. Stoner, E., H. Gregg, J . Otterbein, E. Stein, G. Lamkin, P. Vlasses, J . Danzeisen, and J . Tobert (1987) Administration of MK- 0906, a 4-azasteroid compound results in marked suppression of serum dihydrotestosterone in healthy men. Clin. Res., 35t402A. Tenover, J., M. Zeitner, and S. Plymate (1989) Effects of 24-week administration of a 5a-reductase inhibitor (MK-906) on serum levels of testosterone (T), free T, and gonadotropins in men. Endocrine Society Program and Abstracts Book. Williams and Wilkins, Baltimore, MD. p. 168. Tukey, J.W., J.L. Ciminera, and J.F. Heyse (1985) Testing the statistical certainty of a response to increasing doses of a drug. Biometrics, 41:295-301. Wilson, J.D., F.W. George, and J.E. Griffin (1981) The hormonal control of sexual development. Science, 21I: 1278-1284.
