- Email: [email protected]
Compounds Interfering with Ovum Implantation and Development
Compounds Interfering with Ovum Implantation and Development I. Alkaloids and Antimetaholites JOHN McLEAN MORRIS, M.D., GERTRUDE vANWAGENEN, PH.D., GILLES D. HURTEAU, M.D., DOUGLAS W. JOHNSTON, M.D., and RAY A. CARLSEN, M.D.
LIE of various compounds which might interfere with implantation or development of the fertilized ovum may be of importance in EVALUATION
contributing to the development of postcoital conception control measures, as well as to knowledge of possible teratogenic effects of drugs taken during pregnancy. Many of the compounds investigated in this study have previously been evaluated in rodents. Species difference in response to such agents, however, is very considerable, as has been noted with thalidomide. This study was directed largely toward the effects in the rabbit and monkey (Macaca mulatta) . There is great likelihood that the response in the macaque parallels that in man. For any such compound to be a useful postcoital antifertility agent it must be: completely nontoxic, without teratogenic manifestation in marginal dosages, and 100% effective. Toxicity studies were carried out with most of the agents employed, and any fetal abnormalities were noted. Structural formulas of some of the compounds investigated are illustrated in Fig. 1. METHOD
New Zealand white rabbits weighing 3.5-4.5 kg. were housed at a constant 70° F., with an automatic lighting cycle of 12-hr. periods of light and dark. Does were isolated for several weeks before mating to reduce the From the laboratories of the Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Conn. Supported by Research Grants M62.62, M63.52, and M65.68 from the Population Council, Inc., New York, N. Y., and by Grant HD-02295 from the National Institute of Child Health and Human Development, U. S. Public Health Service. Presented at the 22nd Annual Meeting of the American Fertility Society, Chicago, Ill., Apr. 29-May 1, 1966.
7
8
MORRIS ET AL.
COLCHICINE
DESACETYLMETHYLCOLCHICINE (COLCEMIDEl
FERTILITY
& STERILITY
DESACETYLAMINOCOLCHICINE
CH30ffD'NH-CH 3 CHp:,..
I
CH 30
"" =0
OCH 3 6-AZAURIDINE
5- FLUOROURACIL
HN~
01N'~ I
CH
/~COH
o
I
\ ~COH CH I
CH 20H
Fig. 1. Chemical structure of some of investigated compounds.
incidences of pseudopregnancy. They were then mated twice within a 4- to 6-hr. interval, and the presence of sperm in the vagina was checked for confirmation. The day of mating was considered to be Day O. On the eighth to twelfth day after mating, laparotomy was performed with intravenous sodium pentobarbital anesthesia to ascertain the number of corpora lutea and implantations. '\Then indicated, certain implantations were removed for microscopic sections. Animals were sacrificed just before term on Days 28-30, to prevent loss of abnormal fetuses. Fetuses and placentas were weighed and measured. Fetuses were autopsied for gross abnormalities. While controls were available throughout the study, a control series in which 1 implantation was removed on Day 10 was carried out to evaluate the possible adverse effect of such a procedure. This did not appear to alter the development of the remaining fetuses: In 10 rabbits, there were 104 corpora lutea and 95 (91.4%) implantations at 8--12 days; of the 85 fetuses left to continue to term, 58 (68.4%) were normal, and 10 (11.8%) were resorbed or their placentas resorbed. When such early implants were removed, the number of remaining fetuses that were left to continue to term were recorded. Resorbing fetuses or placentas are common in control litters; these were recorded as "arrested fetuses." While many hundreds of fetuses are removed at term, no abnormalities were noted in control litters. Although it is known that abnormalities do occur, their incidence is hard to establish, especially as the rabbit mother is apt to devour the abnormal fetus she may deliver. Investigation of agents in the monkey was limited to those that had
VOL.
18, No.1, 1967
INHIBITION OF NIDATION: I
9
proved effective in the rabbit. All monkeys used in this study were Macaca mulatta of known fertility that had recently had viable offspring. In most instances both menstrual cycle and fertility records extended back over many years. Hartman's index of vaginal desquamation was used to obtain approximate estrogen levels. Females were mated on Day 11 of the menstrual cycle with males of known fertility, and the vagina was examined the following morning for the presence of sperm. RESULTS Ergocornine Methanesulfonate
Although reported as an effective anti-implantation agent in the rat,lO this compound showed no demonstrable effect in the rabbit. The dosage schedule employed was similar to that used in the rat (Table 1). Administration was subcutaneous. This compound proved toxic in the rabbit, and at higher doses (4-6 mg. jkg.) caused death. Vinblastine
An alkaloid extracted from the periwinkle, vinblastine (vincaleukoblastine, Velban, * VLB), has been found to inhibit mitosis and has been reported effective in the treatment of choriocarcinoma. 4 Its effect on the nonmalignant trophoblast has not been reported. Toxicity studies, performed in 60 rabbits, resulted in a dosage adjustment of 0.3-0.5 mg.jkg., administered intravenously. In 1 group of rabbits, mating was carried out (with some reluctance on the part of the rabbit) at the time of maximum WBC depression following drug administration. In other groups the drug was administered on Day 1, Day 5, and weekly throughout pregnancy (Table 1). The percentage of normal fetuses at term was reduced to 0-33.3%. However, there were 3 fetal abnormalities produced. While glutamic acid can prevent antileukemic and general toxic effects of VLB in mice,5 the fetal salvage rate in 14 rabbits treated with glutamic acid either before or after vinblastine was very little higher than with VLB alone (a 53.6% implantation rate and 41.2% developing into normal fetuses). Colcemide
A potent antimitotic compound closely related to colchicine is desacetylmethylcolchicine (colcemide, Demecolcint). Its principal effect is on tissues *Eli Lilly & Sons, Indianapolis, Ind. tCiba Pharmaceutical Company, Summit, N. J.
TABLE 1. Results with Various Alkaloids and Antimetabolites in Pregnancy in the Rabbit Fetuses at term Gestation day given
Dose (mg./kg.)
No. of rabbits
Oorpora lutea
Implantations No.
%
Left to continue to term
No.
%
Arrested {No.}
47
29
61.7
1
21
7 3 6 0
33.3 27.2 28.6
3 0 0 0
Normal
Abnormal {No.}
ERGOCORNINE
3-4
1.5-2.5
5
61
57
93.4 VINBLASTINE
-4 1 5 1,7,14,28
0.3 0.5 0.5 0.3 (X 4)
3 5 5 4
30 58 56 30
23 14 44 30
76.9 24.2 78.9 100.0
11
21
1*
It 1:1: 0
COLCEMIDE (EFFECTIVE)
9 12 15 18 21,24 26
2.5 0.5-5.0 0.5-2.5 1.0-5.0 2.5-5.0 1.0--2.5
2 4 3 3 3 4
23 39 33 35 29 34
20 32 34 22 25 33
0 0 0 0 0 0
0 0 0 0 4 33
0 0 0 0
0 0
COLCEMIDE (INEFFECTIVE)
2,5 9 12,15 18 21,24
0.5-5.0 0.1-1.0 0.2 0.5 0.2-1.0
6 3 2 1 7
60 35 25 8 78
50 24 24 8 74
40 15 18 5 59
80.0 62.5 75.0 62.5 79.7
9 3 5 1 8
It 2§ 111 0 0
5-FLUOROURACIL
4-7 10-11 10-13
5 (X 4) 10 (X 2) 5 (X 4)
4 4 2
43 44 18
51
37
28
75.7
0
36 } 18
21
9
42.9
0
BW
4-6
6-8 8-12 12-14 15-20
5.0-20.0 2.5--20.0 5.0-20.0 5.0-20.0 5.0-10.0
2 3 3 4 3
18 29 38 45 28
0 30 37 39 19
57-323 0 0 0 0 10
52.6
0 0 0 24 0
0 0 0 13,,[ 0
All implantations were left to continue to term unless a figure appears under that heading. For comparison of treated animals with controls, see text (second page of article). *Hydrocephalus. tCranioschisis. :j:Gastroschisis. §Spina bifida and hydrocephalus. II Gastroschisis, craniosynostosis, and spina bifida. 'Amelia.
12
MORRIS ET AL.
FERTILITY
&
STERILITY
of high mitotic activity, but its general toxicity is lower than that of colchicine. It has had application in the treatment of chronic myelocytic leukemia. Fig. 1 shows the structural formulas of colchicine and colcemide. In a study of the action of nucleotoxic substances on pregnancy in mice and rabbits, Didock et al. noted that the effect appeared to be directly on the fetus and not the placenta. Colcemide destroyed the fetuses in rabbits 13-16 days pregnant in doses of 2-8 mg.jkg., subcutaneously. The drug was less effective when given orally. Thierschl l found that 1 or 2 doses of 2.5 mg. jkg. of colcemide given intraperitoneally to pregnant rats destroyed all litters when given between the eleventh and nineteenth day of gestation. Fetuses showed marked edema, ascites, and hemorrhagic staining of internal organs and skin 6-12 hr. after administration of the drug. No gross abnormalities were seen, but stunting was observed in a few surviving fetuses of rats treated with lower doses. Tuchmann-Duplessis and MercierParot found that somewhat smaller doses provoked abortion and fetal death in rats, but none of the 53 surviving fetuses showed any malformations. Lutwak-Mann and Hay administered colcemide to pregnant rabbits and found, with the aid of their flat-mount technic, degeneration of blastocysts predominantly in the area of the embryonic disc. Colcemide has been administered in at least 1 human pregnancy with no apparent deleterious effect.7 While the medication was continuous throughout the pregnancy, the daily dosage was between 1 and 4% of the effective dose in animal experimentation. Colcemide* was administered subcutaneously in saline in single doses ranging from 0.1 mg.jkg. to 5.0 mg.jkg. Results obtained in the rabbit (Table 1) indicate that colcemide has relatively little effect until implantation has occurred, but from Day 9 until term it is an extremely toxic agent to the developing fetus. Death and beginning distintegration of the fetus have been observed within 2-4 hr. of administration of the drug to the mother (Fig. 2), and within half an hour or less when administered intraamniotically. It appears to act directly on the fetus, and have little effect on the placenta or implantation site. Areas of hemorrhage were the most common gross finding at fetal autopsy. Normal placentas, weighing only slightly less than those in control animals, were often found at term along with the arrested and decomposing fetuses. There were no significant microscopic changes noted in the placentas. It appears to have its maximum effect on the fetus on Days 12-15 following mating, when single doses of 0.5 mg.jkg. result in complete destruction of the litter. Earlier and later in pregnancy larger doses are *Obtained courtesy of Dr. P. C. Eisman, Ciba Pharmaceutical Company.
VOL.
18, No.1, 1967
INHIBITION OF NIDATION:
1
13
required. At 5.0 mg.jkg., 50% of the animals died from toxicity (deaths were excluded from results reported), yet a dose of 2.5 mg. jkg. was necessary to produce litter destruction at certain stages of gestation, which is an extremely narrow margin. One animal died within 18 hr. at the
Fig. 2. Rabbit fetus showing beginning cellular degeneration 6 hr. after maternal administration of colcemide (5 mg./kg.) on Day 9. Note normal implantation site.
2.5-mg. dose level, but in general, doses up to 2.5 mg. appeared to have little effect on the rabbit doe. Transient slight leukocytosis was sometimes noted. The most consistent finding in rabbits that died appeared to be neurological. All displayed loss of coordination, nystagmus, tilting of the head, and inability to stand or eat. In all animals so affected the changes were irreversible and led to immediate or eventual death. Several does receiving colcemide which resulted in interruption of pregnancy (2 receiving 5 mg.jkg.) were remated subsequently. All delivered normal litters. In view of the high degree of effectiveness of colcemide in interrupting pregnancy after implantation in the rabbit, this agent was investigated in the macaque monkey. Results are shown in Table 2. It is apparent even from this small series that a considerable species
14
MORRIS ET AL.
FERTILITY
& STERILITY
TABLE 2. Results with Colcemide in Pregnancy in the Monkey Infant Gestation day injected
mg./kg.
Total
24 45 66 84
2 2 2 1
10.0 9.24 13.3 7.2
Dose
Animal No.
Animal weight (gm.)
Day of delivery
1107 1109 Mm 1059 Mm 953
4930 4620 6740 7200
167 168 166 160
Mm
Mm
Sex F M M M
Weight (gm.)
Sitting height (em.)
345 462 510 450
17.4 19.7 20.0 18.2
All offspring were normal.
difference exists and that this agent does not have the same effectiveness in the primate that it does in the rabbit or rat. At doses employed there was no visible disturbance of the pregnancies, and all fetuses were normal. One monkey reacted unfavorably to the drug by vomiting 2 hr. after injection of 2 mg.jkg. There were no significant or consistent changes in white blood counts. I>esacetylarninocolchicine
This compound, in which the acetylamino group is replaced by a hydrogen atom, has been shown to have considerable more antimitotic activity than either colchicine or colcemide.9 A small amount of this compound was made available for this study.* In a very limited set of experiments in the rabbit it appeared to act in the same manner as colcemide, causing litter destruction at approximately the same dosages. 5-Fluorouracil
Of the incorporated antimetabolites, 5-fluorouracil has been found to be one of the most effective in terms of antineoplastic activity. There has been relatively little work on the antitrophoblastic effect of fluorinated pyrimidines, although some work has been reported on toxicology and teratogenicity in the chick embryo and pregnant rat. 6 The results of a limited series of experiments in the rabbit are shown in Table 1. There appeared in this small series to be surprisingly little effect on the developing fetus. Larger doses (25 mg.jkg. either as a single dose or in 5 daily 5-mg.jkg. doses) resulted in a 50% death rate due to central nervous system toxicity before leukopenia had time to develop. In other *Obtained courtesy of Dr. S. S. Barkulis, Ciba Pharmaceutical Company.
VOL.
18, No.1, 1967
INHIBITION OF NIDATION:
I
15
experiments when 5-fluorouracil was administered at time of ovulation, no effect on ovulation could be noted, whether ovulation had been induced by mating or by administration of human chorionic gonadotrophin. The drug did not appear to affect implantation and had only a limited effect in producing zygote destruction. 6-Azauridine 6-Azauridine, the ribonucleoside of 6-azauracil, is an antimetabolite capable of inhibiting the growth of certain types of leukemic cells without definite toxic effect on normal cells. 15 It is alleged to have antifertility effect in the mouse.14 In a limited series of experiments, dose ranges of 250-500 mg.jkg. for 3-5 days did not appear to affect the fetus prior to implantation. However, when given at 500 mg.jkg. from Days 13-17 to 1 animal there was some reduction in litter size and 1 fetus had an abnormality in development of the nostrils.
Fig. 3. Amelia and other defects in rabbit litter at term, after administration of BW323H (5 mg./kg.) on Days 12, 13, and 14 of pregnancy.
BW 57-323H
One of a group of purines related to 6-mercaptopurine and thioguanine and with similar antitumor activity is BW 57 -323H, 2-amino-6- ( l'-methyl4'-nitro-5'-imidazolyl) mercaptopurine.:!,3 The structural formula is shown in Fig. 1. This agent has been shown by Thiersch12 to be effective in producing litter destruction in rats if given intraperitoneally between Days 4 and 8 of pregnancy.
16
MORRIS ET AL.
FERTILITY
& STERILITY
This compound* was administered intraperitoneally to pregnant rabbits (Table 1). While completely effective in producing litter destruction when given early in pregnancy, it is relatively ineffective when given late in pregnancy. However, when administered between Days 12 and 14, consistent amelia was noted (Fig. 3) in addition to other deformities. The compound appeared to affect the fetus directly; no placental abnormalities were noted. The drug was moderately toxic, producing agranulocytosis. There were 4 deaths (out of 20 animals) that could be attributed to drug toxicity. A single pregnant macaque monkey (Mm 1110) received 3 daily intraperitoneal injections of BW 57-323H in a dose of 7.5 mg.jkg., on Days 28, 29, and 30 of pregnancy. There was no visible effect on the pregnancy, and a normal female infant weighing 465 gm. was delivered on Day 173. SUMMARY
Alkaloids (ergocornine, vinblastine, colcemide) with reported antifertility effects in the rat, antitrophoblastic effect in man, or other antimitotic activity were investigated in the rabbit. At the dosage schedule employed, ergocornine was ineffective, and vinblastine appeared to be teratogenic. The most effective, colcemide, which produced complete zygote destruction after implantation in the rabbit, was evaluated in the macaque monkey by administration on Days 24, 45, 66, and 84 of pregnancy; there was no detectable effect on the primate fetus. Three antimetabolites, a pyrimidine, a ribonucleoside, and a purine, were also studied. The first, 5-fluorouracil, had surprisingly little effect on the rabbit fetus, as did 6-azauridine, the ribonucleoside of 6-azauracil. The 6-mercaptopurine derivative, BW 57-323H, was effective in producing litter destruction early in pregnancy in the rabbit, markedly teratogenic in the middle of gestation, and ineffective thereafter. This compound did not inhibit pregnancy when administered on Days 28-30 to a macaque. 333 Cedar St. New Haven, Conn.
REFERENCES 1.
K., JACKSON, D., and ROBSON, J. M. The action of some nucleotoxic substances on pregnancy. Brit J Pharmacol 11:437, 1965. 2. ELION, C. B., BIEBER, S., and HITCHINGS, C. H. A summary of investigation with 2-amino-6-(I-methyl-4-nitro-5-imidazolyl) thiopurine (B.W.-323) in animals. Cancer Chemother Rep 8:36, 1960. DIDOCK,
*Obtained courtesy of Dr. G. H. Hitchings, Burroughs Wellcome Research Laboratory,
Tuckllhoe, N. Y.
VOL. 18, No.1, 1967
INHIBITION OF NIDATION: I
17
3. ELION, C. B., CALLAHAN, S. W., HITCIDNGS, C. H., and RUNDLES, R W. The metabolism of 2-amino-6-(I-methyl-4-nitro-5-imidazolyl) thiopurine (B.W. 57-323) in man. Cancer Chemother Rep 8:47, 1960. 4. HERTZ, R, LIpSETT, M. B., and Moy, R H. Effect of vincaleukoblastine on metastatic choriocarcinoma and related trophoblastic tumors in women. Cancer Res 20: 1050, 1960. 5. JOHNSON, 1. S., VLANTIS, J., MATTAS, B., and WRIGHT, H. F. Anti-tumor principles derived from Vinca rosea, Linn. II. Further studies of biological activities of vincaleukoblastine. Canad Cancer Cont 4:339, 1961. 6. KAnNOFSKY, D. A., MURPHY, M. L., and LACON, C. R Comparative toxicologic and teratogenic effects of 5-Huoro-substituted pyrimidines in the chick embryo and pregnant rat. Proc Amer Ass Cancer 2:312, 1958. 7. LESSMANN, K M., and SOKOL, J. K Conception and pregnancy in a patient with chronic myelocytic leukemia under continuous colcemide therapy. Ann Int Med 50: 1512, 1959. 8. LUTWAK-MANN, C., and HAY, M. F. Effect on the early embryo of agents administered to the mother. Brit Med J 11:944, 1962. 9. SCIDNDLER, O. Personal communication. 10. SHELESNYAK, M. C. Fertility control in rats by ergotoxine and by ergocornine treatment during pro gestation. First Int Cong Endocrinol, Copenhagen, Session VII, No. 340, 1960. 11. TIDERSCH, J. B. Effect of N-desacetyl-thio-colchicine (TC) and N-desacetylmethyl-colchicine (MC) on rat fetus and litter in utero. Proc Soc Exp Biol Med 98:479, 1958. 12. TIDERSCH, J. B. Effect of 6-(1'-methyl-4'-nitro-5'-imidazolyl)-mercaptopurine and 2-amino-6-( l'-methyl-4'-nitro-5'-imadazolyl)-mercaptopurine on the rat litter in utero. J Reprod Fertil 4:297, 1962. 13. TUCHMANN-DuPLESSIS, H., and MERCIER-PAROT, L. The teratogenic action of some antimitotic substances in rats. C R Acad Sci (Par) 247:152, 1958. 14. VORHERR, H. Personal communication. 15. WELCH, A. D., HANDSCHUMACHER, R K, FINCH, W. C., JAFFE, J. J., CARDOSO, S. S., and CALABRESI, P. A synopsis of recent investigations of 6-azauridine (NSC32074). Cancer Chemother Rep 9:39, 1960.
LIE of various compounds which might interfere with implantation or development of the fertilized ovum may be of importance in EVALUATION
contributing to the development of postcoital conception control measures, as well as to knowledge of possible teratogenic effects of drugs taken during pregnancy. Many of the compounds investigated in this study have previously been evaluated in rodents. Species difference in response to such agents, however, is very considerable, as has been noted with thalidomide. This study was directed largely toward the effects in the rabbit and monkey (Macaca mulatta) . There is great likelihood that the response in the macaque parallels that in man. For any such compound to be a useful postcoital antifertility agent it must be: completely nontoxic, without teratogenic manifestation in marginal dosages, and 100% effective. Toxicity studies were carried out with most of the agents employed, and any fetal abnormalities were noted. Structural formulas of some of the compounds investigated are illustrated in Fig. 1. METHOD
New Zealand white rabbits weighing 3.5-4.5 kg. were housed at a constant 70° F., with an automatic lighting cycle of 12-hr. periods of light and dark. Does were isolated for several weeks before mating to reduce the From the laboratories of the Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Conn. Supported by Research Grants M62.62, M63.52, and M65.68 from the Population Council, Inc., New York, N. Y., and by Grant HD-02295 from the National Institute of Child Health and Human Development, U. S. Public Health Service. Presented at the 22nd Annual Meeting of the American Fertility Society, Chicago, Ill., Apr. 29-May 1, 1966.
7
8
MORRIS ET AL.
COLCHICINE
DESACETYLMETHYLCOLCHICINE (COLCEMIDEl
FERTILITY
& STERILITY
DESACETYLAMINOCOLCHICINE
CH30ffD'NH-CH 3 CHp:,..
I
CH 30
"" =0
OCH 3 6-AZAURIDINE
5- FLUOROURACIL
HN~
01N'~ I
CH
/~COH
o
I
\ ~COH CH I
CH 20H
Fig. 1. Chemical structure of some of investigated compounds.
incidences of pseudopregnancy. They were then mated twice within a 4- to 6-hr. interval, and the presence of sperm in the vagina was checked for confirmation. The day of mating was considered to be Day O. On the eighth to twelfth day after mating, laparotomy was performed with intravenous sodium pentobarbital anesthesia to ascertain the number of corpora lutea and implantations. '\Then indicated, certain implantations were removed for microscopic sections. Animals were sacrificed just before term on Days 28-30, to prevent loss of abnormal fetuses. Fetuses and placentas were weighed and measured. Fetuses were autopsied for gross abnormalities. While controls were available throughout the study, a control series in which 1 implantation was removed on Day 10 was carried out to evaluate the possible adverse effect of such a procedure. This did not appear to alter the development of the remaining fetuses: In 10 rabbits, there were 104 corpora lutea and 95 (91.4%) implantations at 8--12 days; of the 85 fetuses left to continue to term, 58 (68.4%) were normal, and 10 (11.8%) were resorbed or their placentas resorbed. When such early implants were removed, the number of remaining fetuses that were left to continue to term were recorded. Resorbing fetuses or placentas are common in control litters; these were recorded as "arrested fetuses." While many hundreds of fetuses are removed at term, no abnormalities were noted in control litters. Although it is known that abnormalities do occur, their incidence is hard to establish, especially as the rabbit mother is apt to devour the abnormal fetus she may deliver. Investigation of agents in the monkey was limited to those that had
VOL.
18, No.1, 1967
INHIBITION OF NIDATION: I
9
proved effective in the rabbit. All monkeys used in this study were Macaca mulatta of known fertility that had recently had viable offspring. In most instances both menstrual cycle and fertility records extended back over many years. Hartman's index of vaginal desquamation was used to obtain approximate estrogen levels. Females were mated on Day 11 of the menstrual cycle with males of known fertility, and the vagina was examined the following morning for the presence of sperm. RESULTS Ergocornine Methanesulfonate
Although reported as an effective anti-implantation agent in the rat,lO this compound showed no demonstrable effect in the rabbit. The dosage schedule employed was similar to that used in the rat (Table 1). Administration was subcutaneous. This compound proved toxic in the rabbit, and at higher doses (4-6 mg. jkg.) caused death. Vinblastine
An alkaloid extracted from the periwinkle, vinblastine (vincaleukoblastine, Velban, * VLB), has been found to inhibit mitosis and has been reported effective in the treatment of choriocarcinoma. 4 Its effect on the nonmalignant trophoblast has not been reported. Toxicity studies, performed in 60 rabbits, resulted in a dosage adjustment of 0.3-0.5 mg.jkg., administered intravenously. In 1 group of rabbits, mating was carried out (with some reluctance on the part of the rabbit) at the time of maximum WBC depression following drug administration. In other groups the drug was administered on Day 1, Day 5, and weekly throughout pregnancy (Table 1). The percentage of normal fetuses at term was reduced to 0-33.3%. However, there were 3 fetal abnormalities produced. While glutamic acid can prevent antileukemic and general toxic effects of VLB in mice,5 the fetal salvage rate in 14 rabbits treated with glutamic acid either before or after vinblastine was very little higher than with VLB alone (a 53.6% implantation rate and 41.2% developing into normal fetuses). Colcemide
A potent antimitotic compound closely related to colchicine is desacetylmethylcolchicine (colcemide, Demecolcint). Its principal effect is on tissues *Eli Lilly & Sons, Indianapolis, Ind. tCiba Pharmaceutical Company, Summit, N. J.
TABLE 1. Results with Various Alkaloids and Antimetabolites in Pregnancy in the Rabbit Fetuses at term Gestation day given
Dose (mg./kg.)
No. of rabbits
Oorpora lutea
Implantations No.
%
Left to continue to term
No.
%
Arrested {No.}
47
29
61.7
1
21
7 3 6 0
33.3 27.2 28.6
3 0 0 0
Normal
Abnormal {No.}
ERGOCORNINE
3-4
1.5-2.5
5
61
57
93.4 VINBLASTINE
-4 1 5 1,7,14,28
0.3 0.5 0.5 0.3 (X 4)
3 5 5 4
30 58 56 30
23 14 44 30
76.9 24.2 78.9 100.0
11
21
1*
It 1:1: 0
COLCEMIDE (EFFECTIVE)
9 12 15 18 21,24 26
2.5 0.5-5.0 0.5-2.5 1.0-5.0 2.5-5.0 1.0--2.5
2 4 3 3 3 4
23 39 33 35 29 34
20 32 34 22 25 33
0 0 0 0 0 0
0 0 0 0 4 33
0 0 0 0
0 0
COLCEMIDE (INEFFECTIVE)
2,5 9 12,15 18 21,24
0.5-5.0 0.1-1.0 0.2 0.5 0.2-1.0
6 3 2 1 7
60 35 25 8 78
50 24 24 8 74
40 15 18 5 59
80.0 62.5 75.0 62.5 79.7
9 3 5 1 8
It 2§ 111 0 0
5-FLUOROURACIL
4-7 10-11 10-13
5 (X 4) 10 (X 2) 5 (X 4)
4 4 2
43 44 18
51
37
28
75.7
0
36 } 18
21
9
42.9
0
BW
4-6
6-8 8-12 12-14 15-20
5.0-20.0 2.5--20.0 5.0-20.0 5.0-20.0 5.0-10.0
2 3 3 4 3
18 29 38 45 28
0 30 37 39 19
57-323 0 0 0 0 10
52.6
0 0 0 24 0
0 0 0 13,,[ 0
All implantations were left to continue to term unless a figure appears under that heading. For comparison of treated animals with controls, see text (second page of article). *Hydrocephalus. tCranioschisis. :j:Gastroschisis. §Spina bifida and hydrocephalus. II Gastroschisis, craniosynostosis, and spina bifida. 'Amelia.
12
MORRIS ET AL.
FERTILITY
&
STERILITY
of high mitotic activity, but its general toxicity is lower than that of colchicine. It has had application in the treatment of chronic myelocytic leukemia. Fig. 1 shows the structural formulas of colchicine and colcemide. In a study of the action of nucleotoxic substances on pregnancy in mice and rabbits, Didock et al. noted that the effect appeared to be directly on the fetus and not the placenta. Colcemide destroyed the fetuses in rabbits 13-16 days pregnant in doses of 2-8 mg.jkg., subcutaneously. The drug was less effective when given orally. Thierschl l found that 1 or 2 doses of 2.5 mg. jkg. of colcemide given intraperitoneally to pregnant rats destroyed all litters when given between the eleventh and nineteenth day of gestation. Fetuses showed marked edema, ascites, and hemorrhagic staining of internal organs and skin 6-12 hr. after administration of the drug. No gross abnormalities were seen, but stunting was observed in a few surviving fetuses of rats treated with lower doses. Tuchmann-Duplessis and MercierParot found that somewhat smaller doses provoked abortion and fetal death in rats, but none of the 53 surviving fetuses showed any malformations. Lutwak-Mann and Hay administered colcemide to pregnant rabbits and found, with the aid of their flat-mount technic, degeneration of blastocysts predominantly in the area of the embryonic disc. Colcemide has been administered in at least 1 human pregnancy with no apparent deleterious effect.7 While the medication was continuous throughout the pregnancy, the daily dosage was between 1 and 4% of the effective dose in animal experimentation. Colcemide* was administered subcutaneously in saline in single doses ranging from 0.1 mg.jkg. to 5.0 mg.jkg. Results obtained in the rabbit (Table 1) indicate that colcemide has relatively little effect until implantation has occurred, but from Day 9 until term it is an extremely toxic agent to the developing fetus. Death and beginning distintegration of the fetus have been observed within 2-4 hr. of administration of the drug to the mother (Fig. 2), and within half an hour or less when administered intraamniotically. It appears to act directly on the fetus, and have little effect on the placenta or implantation site. Areas of hemorrhage were the most common gross finding at fetal autopsy. Normal placentas, weighing only slightly less than those in control animals, were often found at term along with the arrested and decomposing fetuses. There were no significant microscopic changes noted in the placentas. It appears to have its maximum effect on the fetus on Days 12-15 following mating, when single doses of 0.5 mg.jkg. result in complete destruction of the litter. Earlier and later in pregnancy larger doses are *Obtained courtesy of Dr. P. C. Eisman, Ciba Pharmaceutical Company.
VOL.
18, No.1, 1967
INHIBITION OF NIDATION:
1
13
required. At 5.0 mg.jkg., 50% of the animals died from toxicity (deaths were excluded from results reported), yet a dose of 2.5 mg. jkg. was necessary to produce litter destruction at certain stages of gestation, which is an extremely narrow margin. One animal died within 18 hr. at the
Fig. 2. Rabbit fetus showing beginning cellular degeneration 6 hr. after maternal administration of colcemide (5 mg./kg.) on Day 9. Note normal implantation site.
2.5-mg. dose level, but in general, doses up to 2.5 mg. appeared to have little effect on the rabbit doe. Transient slight leukocytosis was sometimes noted. The most consistent finding in rabbits that died appeared to be neurological. All displayed loss of coordination, nystagmus, tilting of the head, and inability to stand or eat. In all animals so affected the changes were irreversible and led to immediate or eventual death. Several does receiving colcemide which resulted in interruption of pregnancy (2 receiving 5 mg.jkg.) were remated subsequently. All delivered normal litters. In view of the high degree of effectiveness of colcemide in interrupting pregnancy after implantation in the rabbit, this agent was investigated in the macaque monkey. Results are shown in Table 2. It is apparent even from this small series that a considerable species
14
MORRIS ET AL.
FERTILITY
& STERILITY
TABLE 2. Results with Colcemide in Pregnancy in the Monkey Infant Gestation day injected
mg./kg.
Total
24 45 66 84
2 2 2 1
10.0 9.24 13.3 7.2
Dose
Animal No.
Animal weight (gm.)
Day of delivery
1107 1109 Mm 1059 Mm 953
4930 4620 6740 7200
167 168 166 160
Mm
Mm
Sex F M M M
Weight (gm.)
Sitting height (em.)
345 462 510 450
17.4 19.7 20.0 18.2
All offspring were normal.
difference exists and that this agent does not have the same effectiveness in the primate that it does in the rabbit or rat. At doses employed there was no visible disturbance of the pregnancies, and all fetuses were normal. One monkey reacted unfavorably to the drug by vomiting 2 hr. after injection of 2 mg.jkg. There were no significant or consistent changes in white blood counts. I>esacetylarninocolchicine
This compound, in which the acetylamino group is replaced by a hydrogen atom, has been shown to have considerable more antimitotic activity than either colchicine or colcemide.9 A small amount of this compound was made available for this study.* In a very limited set of experiments in the rabbit it appeared to act in the same manner as colcemide, causing litter destruction at approximately the same dosages. 5-Fluorouracil
Of the incorporated antimetabolites, 5-fluorouracil has been found to be one of the most effective in terms of antineoplastic activity. There has been relatively little work on the antitrophoblastic effect of fluorinated pyrimidines, although some work has been reported on toxicology and teratogenicity in the chick embryo and pregnant rat. 6 The results of a limited series of experiments in the rabbit are shown in Table 1. There appeared in this small series to be surprisingly little effect on the developing fetus. Larger doses (25 mg.jkg. either as a single dose or in 5 daily 5-mg.jkg. doses) resulted in a 50% death rate due to central nervous system toxicity before leukopenia had time to develop. In other *Obtained courtesy of Dr. S. S. Barkulis, Ciba Pharmaceutical Company.
VOL.
18, No.1, 1967
INHIBITION OF NIDATION:
I
15
experiments when 5-fluorouracil was administered at time of ovulation, no effect on ovulation could be noted, whether ovulation had been induced by mating or by administration of human chorionic gonadotrophin. The drug did not appear to affect implantation and had only a limited effect in producing zygote destruction. 6-Azauridine 6-Azauridine, the ribonucleoside of 6-azauracil, is an antimetabolite capable of inhibiting the growth of certain types of leukemic cells without definite toxic effect on normal cells. 15 It is alleged to have antifertility effect in the mouse.14 In a limited series of experiments, dose ranges of 250-500 mg.jkg. for 3-5 days did not appear to affect the fetus prior to implantation. However, when given at 500 mg.jkg. from Days 13-17 to 1 animal there was some reduction in litter size and 1 fetus had an abnormality in development of the nostrils.
Fig. 3. Amelia and other defects in rabbit litter at term, after administration of BW323H (5 mg./kg.) on Days 12, 13, and 14 of pregnancy.
BW 57-323H
One of a group of purines related to 6-mercaptopurine and thioguanine and with similar antitumor activity is BW 57 -323H, 2-amino-6- ( l'-methyl4'-nitro-5'-imidazolyl) mercaptopurine.:!,3 The structural formula is shown in Fig. 1. This agent has been shown by Thiersch12 to be effective in producing litter destruction in rats if given intraperitoneally between Days 4 and 8 of pregnancy.
16
MORRIS ET AL.
FERTILITY
& STERILITY
This compound* was administered intraperitoneally to pregnant rabbits (Table 1). While completely effective in producing litter destruction when given early in pregnancy, it is relatively ineffective when given late in pregnancy. However, when administered between Days 12 and 14, consistent amelia was noted (Fig. 3) in addition to other deformities. The compound appeared to affect the fetus directly; no placental abnormalities were noted. The drug was moderately toxic, producing agranulocytosis. There were 4 deaths (out of 20 animals) that could be attributed to drug toxicity. A single pregnant macaque monkey (Mm 1110) received 3 daily intraperitoneal injections of BW 57-323H in a dose of 7.5 mg.jkg., on Days 28, 29, and 30 of pregnancy. There was no visible effect on the pregnancy, and a normal female infant weighing 465 gm. was delivered on Day 173. SUMMARY
Alkaloids (ergocornine, vinblastine, colcemide) with reported antifertility effects in the rat, antitrophoblastic effect in man, or other antimitotic activity were investigated in the rabbit. At the dosage schedule employed, ergocornine was ineffective, and vinblastine appeared to be teratogenic. The most effective, colcemide, which produced complete zygote destruction after implantation in the rabbit, was evaluated in the macaque monkey by administration on Days 24, 45, 66, and 84 of pregnancy; there was no detectable effect on the primate fetus. Three antimetabolites, a pyrimidine, a ribonucleoside, and a purine, were also studied. The first, 5-fluorouracil, had surprisingly little effect on the rabbit fetus, as did 6-azauridine, the ribonucleoside of 6-azauracil. The 6-mercaptopurine derivative, BW 57-323H, was effective in producing litter destruction early in pregnancy in the rabbit, markedly teratogenic in the middle of gestation, and ineffective thereafter. This compound did not inhibit pregnancy when administered on Days 28-30 to a macaque. 333 Cedar St. New Haven, Conn.
REFERENCES 1.
K., JACKSON, D., and ROBSON, J. M. The action of some nucleotoxic substances on pregnancy. Brit J Pharmacol 11:437, 1965. 2. ELION, C. B., BIEBER, S., and HITCHINGS, C. H. A summary of investigation with 2-amino-6-(I-methyl-4-nitro-5-imidazolyl) thiopurine (B.W.-323) in animals. Cancer Chemother Rep 8:36, 1960. DIDOCK,
*Obtained courtesy of Dr. G. H. Hitchings, Burroughs Wellcome Research Laboratory,
Tuckllhoe, N. Y.
VOL. 18, No.1, 1967
INHIBITION OF NIDATION: I
17
3. ELION, C. B., CALLAHAN, S. W., HITCIDNGS, C. H., and RUNDLES, R W. The metabolism of 2-amino-6-(I-methyl-4-nitro-5-imidazolyl) thiopurine (B.W. 57-323) in man. Cancer Chemother Rep 8:47, 1960. 4. HERTZ, R, LIpSETT, M. B., and Moy, R H. Effect of vincaleukoblastine on metastatic choriocarcinoma and related trophoblastic tumors in women. Cancer Res 20: 1050, 1960. 5. JOHNSON, 1. S., VLANTIS, J., MATTAS, B., and WRIGHT, H. F. Anti-tumor principles derived from Vinca rosea, Linn. II. Further studies of biological activities of vincaleukoblastine. Canad Cancer Cont 4:339, 1961. 6. KAnNOFSKY, D. A., MURPHY, M. L., and LACON, C. R Comparative toxicologic and teratogenic effects of 5-Huoro-substituted pyrimidines in the chick embryo and pregnant rat. Proc Amer Ass Cancer 2:312, 1958. 7. LESSMANN, K M., and SOKOL, J. K Conception and pregnancy in a patient with chronic myelocytic leukemia under continuous colcemide therapy. Ann Int Med 50: 1512, 1959. 8. LUTWAK-MANN, C., and HAY, M. F. Effect on the early embryo of agents administered to the mother. Brit Med J 11:944, 1962. 9. SCIDNDLER, O. Personal communication. 10. SHELESNYAK, M. C. Fertility control in rats by ergotoxine and by ergocornine treatment during pro gestation. First Int Cong Endocrinol, Copenhagen, Session VII, No. 340, 1960. 11. TIDERSCH, J. B. Effect of N-desacetyl-thio-colchicine (TC) and N-desacetylmethyl-colchicine (MC) on rat fetus and litter in utero. Proc Soc Exp Biol Med 98:479, 1958. 12. TIDERSCH, J. B. Effect of 6-(1'-methyl-4'-nitro-5'-imidazolyl)-mercaptopurine and 2-amino-6-( l'-methyl-4'-nitro-5'-imadazolyl)-mercaptopurine on the rat litter in utero. J Reprod Fertil 4:297, 1962. 13. TUCHMANN-DuPLESSIS, H., and MERCIER-PAROT, L. The teratogenic action of some antimitotic substances in rats. C R Acad Sci (Par) 247:152, 1958. 14. VORHERR, H. Personal communication. 15. WELCH, A. D., HANDSCHUMACHER, R K, FINCH, W. C., JAFFE, J. J., CARDOSO, S. S., and CALABRESI, P. A synopsis of recent investigations of 6-azauridine (NSC32074). Cancer Chemother Rep 9:39, 1960.