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 equi- valent 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-crystal- line 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 be- havioral 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 re- flex 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 separa- tion 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.