Effects of Midecamycin Acetate(Miocamycin),A New Macrolide Antibiotic,On Reproductive Performances in Rats and Rabbits

1572(128) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 Aug. 1984

EFFECTS OF MIDECAMYCIN ACETATE(MIOCAMYCIN),A NEW MACROLIDE ANTIBIOTIC,ON REPRODUCTIVE PERFORMANCES IN RATS AND RABBITS

MASAHIDEMORIGUCHI,UETO TAKEDA,TOSHIAKI HATA, ATSUKOYAMAMOTO and TAKEMIKOEDA Laboratoryof Pharmacologyand Toxicology,CentralResearch Laboratories, Meiji SeikaKaisha,Ltd., 760,Morooka-cho,Kohoku-ku,Yokohama,Japan ( Receivedfor publicationApril4,1984)

A new macrolide antibiotic miocamycin(MOM),a derivative of midecamycin(MDM)1,2),has superi- or antibacterial activities in vitro and in vivo in most pathogenic microorganisms and its ED50 is equivalent to about one half to one tenth of that of MDM3,4). Clinically,MOM is applied in oral dosage form with much more higher excellent stability quality as well as a higher rate of patient's acceptance due to its bitterlessness. In the present study,we describe the results obtained from studies on the effect of MOM,non-crystalline solid,on reproductive performance in rats and rabbits.

Materials and Methods

1. Substance MOM,non-crystalline solid,is chemically 9,3"-diacetylmidecamycin,as shown in Fig.1.It is easily soluble in chloroform and dichloromethane,rather soluble in methanol,somewhat soluble in ethanol and n-propanol,and almost insoluble in petroleum ether,n-hexane and water.It occurs in odorless and tasteless crystals or crystalline powders.The objective of this study was to determine the reproductive performance in rats and rabbits. 2. Animals Male and female Wistar strain rats were purchased from a commercial breeder(Japan Laboratory Animals,Inc.)at the age of about 5 weeks and quarantined for 2 weeks.Male and female New Zealand white rabbits were purchased from a commercial breeder(Japan Animals,Inc.)at the age of 3 and a half months and quarantined for about 1 month.The animals were housed in experimental rooms under an artificial environment controlled at temperature of 23•}1•Ž,relative humidity of 55•}5% and lighting from 7:00 a.m.to 7:00 p.m.The rats were given animal chow NMF(Oriental Yeast Industry, Co.)and tap water ad libitum.The rabbits were given animal chow GC-4(Oriental Yeast Industry, Co.)once daily by 100g and tap water ad libitum.

Fig.1.Chemical structure of MOM Aug. THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 1573(129) 1984

3. Experimental design Reproductive performance was studied according to the following designs: (1) Setup 1) Fertility test(Segment I)in rats 2) Teratogenicity test(Segment II)in rats 3) Peri- and post-natal tests(Segment III)in rats 4) Behavioral test in rat newborns in Segments II and III 5) Teratogenicity test(Segment II)in rabbits (2) Dosage Rats:125,250,500 and 1,000mg/kg.The highest dose level is equivalent to a dose about 100 times higher than that estimated to be a therapeutic daily dose in humans.Exception- ally,a dose of 1,500mg/kg was employed on rat newborns in Segment II in which behavioral tests were undertaken. Rabbits:Maximum 800mg/kg was given because subacute toxicity studies revealed that some animals showed a decrease in body weight in the 800mg/kg and some died in the more than 1,600mg/kg group. (3) Medication MOM,non-crystalline solid suspended in 0.1% carboxymethylcellulose(CMC)solution,was orally given once daily via a stomach tube by 10ml/kg to rats or by 3ml/kg to rabbits.Only 0.1 % CMC solution by the same volume served as control.It was administered as follows: Segment I:For 9 weeks before mating and 2 weeks during mating to males,for 2 weeks before mating and from copulation to day 7 of pregnancy to females which were established to be pregnant. Segment II:From day 7 to 17 of pregnancy to rats or from day 6 to 18 of pregnancy to rabbits. Segment III:From day 17 of pregnancy to day 21 after delivery to dams. Day 0 of pregnancy was considered to be the day on which sperms were found in the vagina by smear test. 4.Observation (1) Segment I Body weight,feed and water intake were recorded everyday for 9 weeks before mating in adult males and for 2 weeks before mating in adult females.They were continuously housed in a mating cage for 2 weeks.The females in which sperms were found by vaginal smear test after overnight housing were separated from the males.The pregnant dams were recorded on body weight everyday until day 7 of pregnancy when the medication was terminated and every 2 days thereafter.They were also recorded everyday on feed and water intake and abnormal signs. On day 20 of pregnancy,all dams were laparotomized to record the status of implantation.The surviving fetuses were recorded on body weight and gross anomalies;one part was fixed in BOUIN'Ssolu- tion for observation on gross anomalies by a modified method according to WILSON,and another part in 70% alcohol solution to observe skeletal malformations by a modified method according to DAWSON. Copulation and pregnancy ratios were calculated based upon the numbers of mated and pregnant animals.All non-mated males and females were sacrificed for autopsy when the duration of mating was terminated;the males were recorded on organ weights. 2) Segment II ( Pregnant dams were recorded on abnormal symptoms and signs and lactation everyday and body weights on days 0,7 to 18 and 20 of pregnancy,3 days after birth and from 7 days to 4 weeks after birth every week.On day 20 of pregnancy,about two-thirds of the dams were laparotomized to observe the effects on their fetuses according to the same procedures as(1).The remaining one-third were subjected to natural delivery to record sex,litter size,body weight,gross anomalies,symptoms and signs,and behavior of their offsprings(F1),at 3 days after birth,and they were lactated until 28 days after birth. At 27 days after birth,every 25 males and 25 females of offspring were randomly selected from each group to examine ophthalmological and auditory functions by light reflex with pen light and pinna reflex with GALTON'Swhistle. In addition,every 10 males and 10 females of offsprings were randomly selected to examine repro- 1574(130) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 Aug. 1984 ductive performances in which F1 animals were subjected to intragroup mating,no brother-sister mating, at 10 weeks after birth to observe the effects on F2.The remaining offsprings were sacrificed 28 days after birth to weigh the main organs. 3) Segment III ( Dams were recorded everyday on abnormal signs,body weight,feed and water intake on days 0, 7,14 and from days 17 to 20 of pregnancy and subjected to natural delivery.The same procedures were employed for recordings on the dams after delivery and their offsprings as(2). 4) Behavioral tests on rat newborns in Segments II and III.Dams ( were recorded on the status of lactation and body weight after delivery.Their newborns(F1)were recorded on the first day of separation of auricula,eruption of incisors,separation of eyelids,appearance of abdominal hair,descent of testes and opening of vagina.Then,they were examined on motor nerve and skeletal muscle functions by pivoting and righting reflex at 7 days after birth and by walking,supporting and audiogenic seizure reflex at 14 days after birth and on ophthalmological and auditory functions by light and pinna reflexes at 27 days after birth.At 7 weeks after birth,they were examined on emotional response and learning ability in performances by open field tests repeated once daily,and in modified shuttle box by MOWRERand MILLERrepeated 10 times daily for 4 days.All newborn F,'s were autopsied to weigh the main organs at 9 weeks after birth. A few dams medicated by 1,500mg/kg,the highest dosage in Segment II,were laparotomized on day 20 of pregnancy to be examined mainly on teratogenesis by the same procedures as(2). 5) Segment II in rabbits ( Dams were recorded everyday on abnormal signs and feed and water intake and their body weight on days 0 and 4 of pregnancy.All the animals were laparotomized to record the status of implantation and fetuses on day 29 of pregnancy. 5. Statistical analyses MOM medicated groups were statistically compared with the control group by STUDENT'St test on the mean values and by FISHER'Sexact probability test on the nonparametric values.

Results

1. Segment I in rats

(1) Male adults(F0) Fig.2 shows body weight,feed and water intake for 9 weeks before mating.

Fig.2.Mean body weight,feed and water intake of male rats treated with MOM(Fertility test) Aug. THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 1575(131) 1984

Table1.Organ weight of male rats treated with MOM(Fertility test)

Organ weight is shown as the mean value and standard deviation. Significantly different from control *:P<0.05

Fig.3.Mean body weight,feed and water intake of female rats treated with MOM(Fertility test)

Body weight:No significant differences.

Feed and water intake:No significant differences. Abnormal signs:Nothing particularly abnormal. Table1 shows organ weight at autopsy.

Gross findings:No abnormality in any animals. Organ weight:No significant differences except in the thymus and spleen with increases 1576(132) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 Aug. 1984

Table2.Laparotomy findings on pregnant rats treated with MOM(Fertility test)

Body weight is shown as the mean value(g) and standard deviation. Significantly different from control *:P<0.05,**:P<0.01

in absolute values(P<0.05) and except in the adrenals with decrease in relative values(P<0.05) in 1,000mg/kg. (2) Female adults(F0) Fig.3 shows body weight,feed and water intake before mating and during pregnancy. Body weight:No significant differences. Feed and water intake:No significant differences with particular tendency. Table2 shows laparotomy findings on pregnant rats. Copulation ratio:No significant differences from the control. Pregnancy ratio:Significant decrease(P<0.05) from the control value of 100% in 1,000 /kg group but no significant differences in less than 500mg/kgmg groups. Corpora lutea:No significant differences. Implantation ratio:Significant decreases(P<0.01 or 0.05) to 81.6,83.6,83.9 and 83.7%, respectively,in 125,250,500,1,000mg/kg groups from the control value of 90.3%. (3) Fetuses(F1) Mortality:No significant differences;22.4% in the control group,16.6% in 125mg/ kg,6.7% in 250mg/kg,5.0% in 500mg/kg,3.0% in 1,000mg/kg. Body weight:No significant differences. Aug. THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 1577(133) 1984

Table3.Skeletal and visceral findings on rat fetuses from dams treated with MOM(Fertility test)

Significantly different from control *:P<0.05,**:P<0.01

Gross findings:No abnormality in any animals. Table3 shows skeletal and visceral findings on rat fetuses. Skeletal findings:Increase in 14th ribs in 125 and 1,000mg/kg groups,increase in cervical ribs in 500mg/kg group. Visceral findings:No significant differences;bleeding of olfactory bulb in 1 fetus(2.8%) in the control and 1 fetus(2.0%)in 250mg/kg group,subarachnoideal bleeding in 1 fetus(2.4%)in 500mg/kg group and 3 fetuses(5.9%)in 1,000mg/kg group,hydronephrosis in 1 fetus(2.8%)in the control, 5 fetuses(10.2%)in 125mg/kg group,4 fetuses(8.2%)in 250mg/kg group,1 fetus(2.4%)in 500mg/kg group and 9 fetuses(17.6%)in 1,000mg/kg group.

2. Segment II in rats (1) Dams Fig.4 shows the mean body weight,feed and water intake of rat dams during pregnancy and lactation. Body weight,feed and water intake:No significant differences except increases in feed intake on days 17 and 20 of pregnancy in the 1,000mg/kg group(P<0.01 or 0.05). (2) Fetuses Table 4 shows laparotomy findings on pregnant rats. Fetal mortality:Increases(P<0.01 or 0.05)in 250 and 500mg/kg groups,17.0% in the control,15.5% in 125mg/kg group,25.3% in 250mg/kg group,30.1% in 500mg/kg group and 19.2% in 1,000mg/kg group. Body weight:Increases(P<0.05)in male and female rats,in 250 and 500mg/kg groups, respectively. Sex ratio:No significant differences. 1578(134) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 Aug. 1984

Fig.4.Mean body weight,feed and water intake of rat dams treated with MOM(Teratogenicity test)

Table4.Laparotomy findings on pregnant rat dams treated with MOM(Teratogenicity test)

Body weight is shown as the mean value(g)and standard deviation. a:General edema with intestinal hernia, Significantly different from control *:P<0.05,**:P<0.01

Gross findings:Two fetuses(0.9%)with intestinal hernia associated with general edema in the control(Plate 1). Table5 shows skeletal and visceral findings on rat fetuses from dams. Skeletal findings:No malformation except fused ribs in 1 fetus(0.6%)in the control Plate 2),skeletal anomalies such as cervical ribs,incomplete formation( of thoracic vertebral body,14th ribs,delayed ossification of sternebrae, etc.frequently observed in our laboratory. Visceral findings:Hydronephrosis in 3 fetuses(10.0%,Plate 3),subarachnoideal hemorrhage in 1 fetus(3.3%,Plate 4)and anophthalmia in 1 fetus(3.3%,Plate 5)in the control,1 hydronephrosis and 1 retinal hemorrhage(Plate 6)in Aug. THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 1579(135) 1984

Table5. Skeletal and visceral findings on rat fetuses from dams treated with MOM (Teratogenicity test)

Significantly different from control **:P<0.01

Plate 1.A rat fetus with general edema and in- Plate 2.A rat fetus with fused ribs in control testinal hernia in control group(Segment-II) group(Segment-II)

Plate 3.A rat fetus with hydronephrosis in Plate 4.A rat fetus with subarachnoideal control group(Segment-II) hemorrhage in control group(Segment-II) 1580(136) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 Aug. 1984

Plate 5.A rat fetus with anophthalmia on Plate6.A rat fetus with retinal hemorrhage in right eye in control group(Segment-II) MOM 125mg/kg group(Segment-II)

Table 6.Postnatal observation on rat offsprings from dams treated with MOM(Teratogenicity test)

Significantly different from control *:P<0.05,**:P<0.01

125mg/kg group,7(17.1%),8(18.6%)and 5(14.3%)hydronephrosis in 250,500 and 1,000mg/kg groups,respectively(Plate 5). (3) Offsprings Table 6 shows postnatal observation on rat offsprings. Body weight:At 3 days after birth,decreased in 250 and 500mg/kg groups,at 7 days after birth.decreased in 250mg/kg group,thereafter,gradually increased,at 28 days after birth,increased in all medicated groups. Survival ratio:From 14 days after birth,decreased(P<0.01 or 0.05)in more than 250 mg/kg groups. Gross findings:Two fetuses with crooked tail in 125mg/kg group(1.0%)and 1.000mg/ kg group(0.8%). Aug. THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 1581(137) 1984

Table 7.Organ weight of rat offsprings from dams treated with MOM(Teratogenicity test)

Organ weight is shown as the mean value and standard deviation. Significantly different from control *:P<0.05,**:P<0.01

Visceral findings:No abnormality in autopsy of newborns that died during lactation. Ophthalmological and auditory function test:No abnormality at 27 days after birth. Table 7 shows the organ weight of offsprings. Absolute and relative organ weights:The heart and kidneys gained in 125mg/kg group,the thymus gained in 250mg/kg group,the thymus,spleen and uterus & ovaries gained in 1,000mg/kg group,the liver and adrenals lost in 500mg/kg group(P <0.01 or 0.05). Figs.5 and 6 show mean body weight,feed and water intake of F1 male and female offsprings, respectively. These parameters approximate the values of the control up to 10 weeks after birth in every F1 10 male and female newborns selected from each group to examine reproductive performance in the second generation. Table 8 shows reproductive performance of rat offsprings and observations on F2 fetuses. F1:Three males and 2 females in 500mg/kg group had diarrhea from 5 or 6 weeks after birth and aggravated general conditions and were sacrificed for autopsy at 8 weeks after birth. In the results,hydronephrosis,splenic tumor,hepatic swelling and gastrointestinal lymph node swelling etc.were sporadically observed.F1 offsprings were subjected to intergroup mating no brother-sister mating,at 10 weeks after birth resulting in no significant differences in copulation ratio,pregnancy ratio or fetal mortality. 1582(138) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 Aug. 1984

Fig.5.Mean body weight,feed and water intake of F1 male rats from dams treated with MOM (Teratogenicity test)

Fig.6.Mean body weight,feed and water intake of F1 female rats from dams treated with MOM (Teratogenicity test)

F2:Body weight:In male rats,decreased in 125mg/kg group and in female rats,decreased in 125,250 and 1,000mg/kg groups. Gross findings:No anomalies. Skeletal findings:No malformation but sporadic anomalies,as for F1.

3. Segment III in rats

(1) Dams Fig.7 shows the mean body weight,feed and water intake of dams. Aug. THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 1583(139) 1984

Table 8.Reproductive performance of rat offsprings and observation on F2 fetuses(Teratogenicity test)

Body weight is shown as the mean value(g) and standard deviation. Significantly different from control *:P<0.05,**:P<0.01

Fig.7.Mean body weight,feed and water intake of material rats treated with MOM(Peri-and post-natal tests)

Body weight:No significant differences in the less than 500mg/kg group during pregnancy and lactation,slightly lower weight gains from day 17 of pregnancy but return to the control level during lactation in the 1,000mg/ kg group. Food and water intake:No significant decreases. Delivery ratio:No significant differences. Autopsy:Nothing abnormal at the end of lactation. 1584(140) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 Aug. 1984

Table 9.Postnatal observation on rat offsprings from dams treated with MOM(Peri- and post- natal tests)

Significantly different from control *:P<0.05,**:P<0.01

(2) Offsprings Table 9 shows the postnatal observation on rat offsprings from dams. Body weight:Significant increases from control levels in medicated groups. Survival ratio:No significant differences. Gross findings:No particular anomalies. Ophthalmological and auditory function tests:No particular abnormality at 27 days after birth. Autopsy:No particular abnormality at 28 days after birth. Table 10 shows the organ weight of rat new bores from dams. Organ weight:The thymus and testes increased in medicated groups,the adrenals increased in the 125,250mg/kg groups. Figs.8 and 9 show body weight,feed and water intake of F1 male and female rats from dams. There were no significant decreases in body weight,feed and water intake up to 10 weeks in F1 for examinations on the reproductive performances in the second generation. Table 11 shows reproductive performance of rat offsprings and observation on F2 fetuses. Body weight:No significant decrease except decrease in the 1,000mg/kg group of male. Gross findings:No anomalies. Skeletal findings:Skeletal anomalies such as cervical ribs,14th ribs,retarded ossification of sternebrae and incomplete formation of thoracic vertebral body were observed frequently. Aug. THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 1585(141) 1984

Table 10.Organ weight of rat offsprings from dams treated with MOM (Peri- and post-natal tests)

Organ weight is shown as the mean value and standard deviation. Significantly different from control *:P<0.05,**:P<0.01

Fig.8.Mean body weight,feed and water intake of F1 male rats from dams treated with MOM (Peri- and post-natal tests)

4. Behavioral tests on rat offsprings

(1) Segment II 1) Dams Body weight:Lower gains during the later half of medication in the 1,500mg/kg 1586(142) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 Aug. 1984

Fig.9.Mean body weight,feed and water intake of F1 female rats from dams treated with MOM Peri- and post-natal tests) (

Table 11.Reproductive performance of rat offsprings and observations on F2 fetuses(Peri- and post-natal tests)

Body weight is shown as the mean value(g)and standard deviation. Significantly different from control *:P< 0.05,**:P<0.01

group.Other parameters resulted in approximately the same results as the Segment II in above. 2) Newborns Body weight:In the less than 500mg/kg dosed groups,decreased at 3 days after birth but not significantly different from the control in the 1,500mg/kg group. Physical development:Retardation of all parameters except separation of auricula in the 125 /kg group,retardation of eruption of incisors and opening of vaginamg in the 250mg/kg group,retardation of separation of auricula and eruption of incisors in the 500mg/kg group and retardation of eruption of incisors in the 1,500mg/kg group were observed(Table 12). Aug. THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 1587(143) 1984

Table 12. Physical and behavioral development of rat offsprings from dams treated with MOM (Teratogenicity test)

Significantly different from control *:P<0.05,**:P<0.01

Table 13.Behavioral test of rat offsprings from dams treated with MOM(Teratogenicity test)

No significant difference from control at P<0.01 level,Unit:frequency/3min. 1588(144) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 Aug. 1984

Table 14-1.Organ weight of 9-week-old rat offsprings(male) from dams treated with MOM (Teratogenicity test)

Organ weight is shown as the mean value and standard deviation. Significantly different from control *:P<0.05,**:P<0.01

Table 14-2.Organ weight of 9-week-old rat offsprings(female) from dams treated with MOM (Teratogenicity test)

Organ weight is shown as the mean value and standard deviation . Significantly different from control *:P<0.05,**:P<0.01 Aug. THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 1589(145) 1984

Table 15.Physical and behavioral development of rat offsprings from dams treated with MOM (Peri- and post-natal tests)

Significantly different from control *:P<0.05,**:P<0.01

Table 16.Behavioral test of rat offsprings from dams treated with MOM(Peri- and post-natal tests)

No significant difference from control at P<0.01 level,Unit:frequency/3min. 1590(146) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 Aug. 1984

Table 17-1.Organ weight of 9-week-old rat offsprings(male) from dams treated with MOM Peri- and post-natal tests) (

Organ weight is shown as the mean value and standard deviation. Significantly different from control *:P<0.05,**:P<0 .01

Table 17-2.Organ weight of 9-week-old rat offsprings(female) from dams treated with MOM Peri- and post-natal tests) (

Organ weight is shown as the mean value and standard deviation . Significantly different from control *:P<0.05 ,**:P<0.01 Aug. THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 1984 1591(147)

Behavioral development:No significant differences. Table 13 shows behavioral tests on rat offsprings from dams. Open-field test:No significant differences. Shuttle box test:No significant differences in conditioned avoidance responses in both males and females. No significant differences or malformation were found in the implantations,surviving fetuses,dead fetuses,fetal body weight or skeletal and visceral findings in 1,500mg/kg in teratogenicity test. Tables 14-1,-2 show the organ weight of 9-week-old rat offsprings from dams. Gross findings:No abnormality. Organ weight:Sporadic increases or decreases in medicated groups but without dose- dependency. (2) Segment III in rats 1) Dams:The results were approximately the same as for above Segment II. 2) Offsprings Body weight:Decrease at 3 days after birth but not significantly different thereafter and returned to the control level at weaning in the more than 500mg/ kg groups. Table 15 shows the physical and behavioral development of rat offsprings from dams. Physical development:Retardation of opening of vagina in the 125mg/kg group and separation of auricula in the 500mg/kg group. Behavioral development:Decreases in walking and supporting in the 250mg/kg group, pivoting in the 500mg/kg group and pivoting,walking and supporting in the 1,000mg/kg group but without a definite tendency. Table 16 shows behavioral tests of rat offsprings from dams. Open-field test:No significant differences in either sex. Shuttle box test:No significant differences in conditioned avoidance responses in either sex. Tables 17-1,-2 show the organ weight of 9-week-old rat offsprings from dams. Visceral findings:No morphological abnormality. Organ weight:Sporadic significant differences in medicated groups.

5. Segment II in rabbits

(1) Dams Fig.10 shows the mean body weight and water intake of rabbit dams. Body weight:No significant differences in the less than 400mg/kg groups but decrease P<0.05) during the experiment in the highest dosage level,800mg/kg.( Feed intake:Approximately the same changes in the less than 400mg/kg groups but decreases in many dams in the 800mg/kg group. Water intake:Infrequent increase in the 200mg/kg group and sporadic decrease in the 800mg/kg group,but without a definite tendency because of greater deviations. Table 18 shows laparotomy findings on pregnant rabbits. Dams having spontaneous abortion were 1(10.0%) in the control group,2(20.0%) in the Aua. 1592(148) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 1984

Fig.10.Mean body weight and water intake of maternal rabbits treated with MOM(Teratogenicity test)

Table 18.Laparotomy findings on pregnant rabbits treated with MOM(Teratogenicity test)

Body weight is shown as the mean value(g) and standard deviation. Significantly different from control *:P<0.05,**:P<0.01

200mg/kg group,1(10.0%) in the 400mg/kg group,and 4(36.4%) in the 800mg/kg group.There were no significant differences between medicated groups or the control,nor were there any deaths. Visceral findings:No abnormalities at laparotomy. Corpora lutea and implantations:No significant differences. Aug. THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII 8 1593(149) 1984

Plate 7.A rabbit fetus with hematoma of outer myocardial wall in MOM 800mg/kg group Plate 8.A rabbit fetus with fused ribs in control Segment-II) ( group(Segment-II)

Plate 9.A rabbit fetus with fused costicartilage in MOM 400mg/kg group(Segment-II)

(2) Fetuses Mortality:Increases in the 200mg/kg group,especially frequent in late resorption and dead fetuses in the 800mg/kg group. Body weight:No significant differences except for decreases in males in the 100mg/kg group. Gross findings:No anomalies. Visceral findings:Two fetuses with hydronephrosis and I with hematoma of outer myocardial wall(Plate 7) in the 800mg/kg group. Skeletal findings:One fetus with fused ribs(Plate 8) in the control and I fetus with fused costicartilage(Plate 9) in 400mg/kg group. In addition,anomalies such as retarded ossification of sternebrae and delayed formation of the 13th ribs were sporadically observed but without significant differences.

Discussion and Conclusion

1. Fertility tests on rats receiving MOM,non-crystalline solid,before and during organogenesis No particular influences on male and female adults were observed and general conditions and autopsy revealed no abnormality. There were no significant differences in the copulation ratio.Pregnancy ratio resulted in a decrease (72.7%) in only the 1,000mg/kg group. Implantations decreased to 81.6 to 83.9% in all medicated groups,which might have no significance, as the control values were as high as 90.3% in the present study in comparison to our background data (83.5%) in Wistar strain rats. 1594(150) THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 Aug. 1984

It can also be estimated that MOM had no influence on embryonic development after implantation, with approximately the same occurrence of dead fetuses in each group and no significant differences in fetal body weights. No fetal gross anomalies nor skeletal malformations occurred.Skeletal anomalies were limited to those which had been frequently observed in our laboratory.The frequencies were not heightened by MOM medication.No visceral abnormality was found. 2. Segment II in rats receiving MOM,non-crystalline solid,during fetal organogenesis Toxic effects on dams were considered extremely few from the viewpoints of progressive gains in maternal body weight,no abnormal visceral organs by laparotomy and favorable course in feed and water intake and general conditions. Higher fetal mortality in the MOM medicated groups might be due to 2 or 3 dams with an extremely larger percentage per litter. If this case could be omitted,there were no significant differences in overall fetal mortality. Fetuses were little influenced:No loss of fetal body weight and no skeletal or visceral malformations. Decreases in survival ratio of newborns from 14 days after birth in the more than 250mg/kg groups were negligible,because the values were within the normal range of our background data. Gross anomalies such as crooked tail in 2 fetuses from the 125 and 1,000mg/kg groups are known to occur spontaneously,which might not be attributable to MOM medication. Diarrhea with aggravated general conditions found in the course of growth after weaning in the 500 mg/kg group might not be attributable to MOM medication either because there were no symptoms or signs in other medicated groups and no common abnormalities at autopsy. Behavioral tests on newborns revealed no definite tendency,in spite of infrequent retardation of development. Emotional responses and learning ability showed no significant differences between MOM medicated groups and the control in both sexes. MOM might have no influence on reproductive performance in the F1 group because of no significant differences in copulation ratio,pregnancy ratio,maternal body weight or fetal mortality. Influences on the F2 group might also not be attributable to MOM medication,although the fetal body weight decreased,except in the 500mg/kg group. 3. Segment III in rats receiving MOM,non-crystalline solid,during peri- and post-natal periods Dams lost body weight during the perinatal period in the 1,000mg/kg group but recovered during the postnatal period,with approximately the same changes as in the control.Feed and water intake and general conditions were not affected.In general,MOM might have had a little influence on the dams. Newborns progressed favorably after birth,without body weight changes,nor did they show gross anomalies or visceral malformations at autopsy. Their physical development,emotional responses,learning ability and reproductive performance were almost the same as in 2. 4. Segment II in rabbits receiving MOM,non-crystalline solid,during organogenesis Dams lost body weight in the 800mg/kg group.Some of them rather frequently showed a tendency toward spontaneous abortion in the 800mg/kg group,together with more frequent later resorption and dead fetuses.MOM,non-crystalline solid,might have had some influence on the dams or their fetuses. Surviving male and female fetuses lost body weight in the 100mg/kg group,which might not be attributable to MOM,non-crystalline solid,medication because of larger litter size. There were no abnormalities in gross,skeletal or visceral findings attributable to MOM,non-crystalline solid,medication.HIGAKI et al.5) reported in experimental studies on the placental passage of antibiotics into mice fetuses that the placental passage of an antibiotic is greatly regulated by its binding ability and placental functions,and that macrolide antibiotics such as erythromycin or oleandomycin with little solubility in water have a higher percentage of passage into fetuses and concentration of drugs in fetuses than those of other antibiotics such as benzylpenicillin with much solubility in water. NAKATSUKAet al.') confirmed in investigations on the placental passage of macrolide antibiotics into fetuses that they easily reach fetuses. Aug. THE JAPANESE JOURNAL OF ANTIBIOTICS XXXVII-8 1595(151) 1984

SHOMURA7) also confirmed that MOM,non-crystalline solid,orally given to rats during the later stage of pregnancy has its placental passage into fetuses but in the lower concentration than maternal

blood levels.

However,there have been few reports on teratogenesis induced by macrolide antibiotics.We already confirmed that MDM causes to teratogenesis in rats receiving MDM,even at the dose levels

greater than its LD50.. In reviewing the results obtained in the present study,we conclude that MOM,non-crystalline

solid,might have no teratogenesis and little influence on dams and their fetuses and offsprings.

Abstract

Miocamycin(MOM) is a derivative of midecamycin,a macrolide antibiotic isolated from a culture

broth of Streptoinyces mycarofaciens.The objective of this study was to determine the effect of MOM on

reproductive performance in rats and rabbits.MOM,non-crystalline solid,was suspended in 0.1%

CMC solution immediately before use.

In this study,reproductive performance was studied according to the following designs:

1. Fertility test(Segment I) in Wistar rats

2. Teratogenicity test(Segment II) in Wistar rats

3. Peri- and post-natal tests(Segment III) in Wistar rats

4. Behavioral test in rat newborns in Segments II and III

5. Teratogenicity test(Segment II) in New Zealand white rabbits

In conclusion,MOM,non-crystalline solid,might have no teratogenesis and little influence on dams

and their fetuses and offsprings.

References

1) YOSHIDA, T.; T. WATANABE, T. SHOMURA, S. SOMEYA, R. OKAMOTO, S. ISHIHARA, K. MIYAUCHI & Y.KAZU-

NO: Bacteriological evaluation of midecamycin acetate and its metabolites. Jap. J. Antibiotics 35: 1462•`1474, 1982 2) OMOTO, S.; K. IWAMATSU, S. INOUYE & T. NIIDA: Modifications of a macrolide antibiotic midecamycin

SF-837). I. Synthesis and structure of 9, 3"-diacetylmidecamycin. J. Antibiotics 29: 536•`548,( 1976 3) KAWAHARAJO, K.; Y. SEKIZAWA & M. INOUE: In vitro and in vivo antibacterial activity of 9, 3"-di-O-

acetyl midecamycin(MOM), a new macrolide antibiotic. J. Antibiotics 34: 436•`442, 1981 4) NAKAMURA, T.; S. FUKATSU, S. SEKI & T. NIIDA: A convenient method for the preparation of the acylated

macrolide antibiotic midecamycin using molecular sieves and acylchloride. Chemistry Letters -1978: 1293•`1296,1978

5) HIGAKI, Y.; T. HIROZANE, I. OBONARI & H.ARATANI: Transfer of antibiotics in fetus. Jap. J. Pharma- col. 62: 194, 1966

6) NAKATSUKA, M.; Y. HIGAKI, T. NAKAGAWA & T. HASHIMOTO: Transfer of antibiotics in fetus. Jap. J. Pharmacol. 63: 154, 1967

7) SHOMURA, T.: Transfer of MOM in fetus.(in house report)