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Leflunomide is an immunosuppressant drug displaying teratogenicity in mice, rats, and rabbits. Its immunosuppressive effect occurs via inhibition of dihydroorotate dehydrogenase (DHODH) and tyrosine kinases. In this study, we coadministered Leflunomide and uridine, a precursor substance of pyrimidine nucleotides, to pregnant CD-1 mice, and examined whether or not a decreased level of intracellular pyrimidine nucleotides with inhibition of DHODH is related to the teratogenicity of Leflunomide. Then we examined the alteration of the nucleotide level in fetal tissue by Leflunomide and the effect of coadministered uridine. We administered Leflunomide with or without uridine to pregnant mice on gestation day 10, and used the vehicle of Leflunomide as a control. Leflunomide caused multiple malformations in all fetuses, but coadministration with uridine inhibited most of its teratogenicity. Leflunomide decreased the concentration of pyrimidine nucleotides, not purine nucleotides, whereas uridine coadministered with Leflunomide partially restored the level of pyrimidine nucleotides. These results indicate that the inhibitory effect of DHODH activity is related to the teratogenicity of Leflunomide.

Leflunomide has inhibitory effects on dihydroorotate-dehydrogenase activity and protein tyrosine kinase activity. In the present study, a single dose of 50 mg/kg Leflunomide was administered to pregnant mice on one of gestation days (GD)6-11. Characteristic external malformations were craniofacial defects following dosing on GD7, cleft palate on GD9, cleft palate and limb and tail deformities on GD10, and limb deformities on GD11. Skeletal examination revealed cervical to caudal vertebral malformations after treatment on GD7, GD8, GD9 or GD10. In the viscera, cardiovascular deformities were observed in the GD7 and GD9 Leflunomide-treated groups. These results demonstrate that multiple malformations were seen in various organs and most of the malformations observed appeared to be developmental stage-specific responses to Leflunomide treatment.

Cefmatilen hydrochloride hydrate (S-1090) was administered daily by gavage to rats at doses of 100, 300 or 1000 mg potency/kg/day prior to and in the early stage of pregnancy to assess its adverse effects on parental reproductive ability and embryo-fetal development. Loose and/or reddish brown feces were observed in both males and females of all the S-1090 dosing groups, and abdominal distention was also observed in males throughout the dosing period. No drug-related deaths occurred in either males or females. In males, body weight and food consumption were increased at a dose of 1000 mg potency/kg/day throughout the dosing period. In females, body weight gain was restrained during late pregnancy, and food consumption was decreased transiently following the initiation of dosing, and then remained high on the day before parturition in all the S-1090 dosing groups. Necropsy of male and female rats revealed an increase in the cecum weight. The reproductive ability of males and females was normal in all the S-1090 dosing groups. No effects of S-1090 were observed in the implantation ratio, embryo-fetal viability, fetal body weight, and incidence of external, skeletal and visceral anomalies. Based on these results, the no observed adverse effect levels of S-1090 are estimated to be less than 100 mg potency/kg/day for parental general toxicity, 1000 mg potency/kg/day for reproductive toxicity, and 1000 mg potency/kg/day for developmental toxicity in embryo-fetuses under the conditions of the present study.

Cefmatilen hydrochloride hydrate (S-1090) was administered daily by gavage to female rats at doses of 100, 300 or 1000 mg potency/kg/day from Days 7 to 17 of pregnancy to assess its effects on dams and on development of the embryo-fetuses and offspring. Loose or reddish-brown feces were observed in dams of all the S-1090 dosing groups. Body weight gain was increased from the early stage of administration to the end of pregnancy, food consumption was transiently decreased at the early stage of administration, and water consumption was increased from the middle to the end of pregnancy in all the S-1090 dosing groups. However, no effects on pregnancy, parturition and lactation were observed. Necrospy revealed an increased cecum weight in pregnant and lactating dams of all the S-1090 dosing groups. No effects of S-1090 were observed in viability, growth, incidences of external, skeletal and visceral anomalies, and degree of ossification in F1 fetuses. No effects of S-1090 were observed in such parameters as viability, incidence of external and skeletal anomalies, physical development, sensory functions/reflexes, behavior and reproductive function in F1 offspring. No adverse effects were observed in F2 offspring. On the basis of these results, the no observed adverse effect levels of S-1090 are estimated to be less than 100 mg potency/kg/day for maternal general toxicity, 1000 mg potency/kg/day for maternal reproductive toxicity and the developmental toxicity in the embryo-fetuses and offspring under the conditions of the present study.

Cefmatilen hydrochloride hydrate (S-1090) was administered daily by gavage to female rats at doses of 100, 300 or 1000 mg potency/kg/day from Day 17 of pregnancy to Day 20 of lactation to assess its effects on pregnant/lactating females and on development of the offspring. In dams, loose feces/reddish brown feces, increased cecum weight, decreased weights of the heart, spleen and submaxillary gland in all the S-1090 dosing groups and a decreased weight of the thymus in the 1000 mg potency/kg dosing group were observed. However, no effects on parturition and lactation were observed in any of the dosing groups. In F1 offspring, although increased cecum weight was found at weaning in all the S-1090 dosing groups, no abnormalities in viability, physical development, sensory functions/reflexes, behavior and reproductive function were observed. No adverse effects were observed in F2 fetuses and offspring. On the basis of these results, the no observed adverse effect levels of S-1090 are estimated to be less than 100 mg potency/kg/day for maternal general toxicity, and 1000 mg potency/kg/day for maternal reproductive toxicity and for developmental and reproductive toxicity in offspring under the conditions of the present study.

The purpose of this study was to develop a primary culture system using serum-free medium for rat placental trophoblast cells and to investigate the factors that control rat placental lactogen-II (rPL-II) secretion in vitro. The placentae of day 13 pregnant rats were dissociated in Medium 199 containing 0.1% collagenase and 0.002% DNAase. Dissociated cells were fractionated into five segments by centrifugation through a 40% Percoll density gradient and incubated on rat tail collagen bed in medium SFM-101 for up to 7 days. Fraction B at the Percoll gradient density of 1.05 g ml-1 was enriched with rPL-II-producing cells and the time course of rPL-II secretion was characterized by a rapid increase in the first 2 days, remaining at high values (mean: 14-16 ng micrograms-1 DNA) for the following 2-3 days and decreasing thereafter. The rPL-II-producing cells from faction B identified by immunocytochemical examination accounted for approximately 69% of total cultured cells and consisted of a few giant cells and polygonal cells. Growth factors (bovine insulin, 0.1-20 micrograms ml-1; recombinant human insulin-like growth factor (IGF)-I, IGF-II, 0.1-1.0 micrograms ml-1; murine epidermal growth factor (EGF), 0.001-10 micrograms ml-1), rat pituitary hormones (rat growth hormone, rat prolactin, 0.1-10 micrograms ml-1) and hypothalamic hormones (human growth hormone-releasing hormone (GHRH), corticotrophin-releasing hormone (CRH), LHRH, 0.1-10 micrograms ml-1) were individually added to the culture medium to investigate the putative factors that directly control rPL-II secretion by the trophoblast cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study was designed to develop a procedure for purifying rPL-II and a homologous radioimmunoassay (RIA) for rPL-II. Molecular profiles of rPL-II were also investigated in tissue and plasma. rPL-II was purified 3,780-fold, based on its radioreceptor assay (RRA) activity compared to ovine prolactin (0PRL), from the placenta of day 18 pregnant rats using ammonium sulfate precipitation and chromatography on phenyl-Sepharose, DEAE-TOYOPEARL 650S, AF-chelate TOYOPEARL 650M and Sephadex G-100. Electrophoretic analysis on SDS gel revealed molecular weight heterogeneity of purified rPL-II, which consisted of three proteins; a major form with a molecular weight of 20.0 K and two minor forms with molecular weights of 20.6 K and 21.0 K under non-reducing conditions. One of the minor forms of rPL-II observed under non-reducing conditions disappeared with 2-mercaptoethanol treatment and the rest of the hormones migrated as 24.5 K and 25.0 K molecular weight species, suggesting that it is a cleaved form of rPL-II. Purified rPL-II displaced 125I-labelled oPRL from binding sites on rabbit mammary gland membranes in a dose-dependent manner. rPL-II and rPRL were, respectively, 21 and 2 per cent as effective as oPRL in the displacement. Antibody to purified rPL-II was raised in rabbits and a homologous RIA for rPL-II was developed. No displacement was observed with rPRL, rGH, oPRL, and other pituitary hormones up to 1,000 ng/ml. Molecular profiles of rPL-II in the placental tissue and plasma from day 18 pregnant rats were examined by gel chromatography on Sepharcryl S-300 HR and by Western blotting. Chromatography of the placental extracts revealed a single peak, which accounted for 86 per cent of the total RIA activity. Anti-rPL-II antiserum detected proteins of at least three molecular sizes as monomeric forms with molecular weights of 20.0, 20.6, and 21.0 K in the non-reducing placental extracts. One of them disappeared with 2-mercaptoethanol treatment and other two proteins had molecular weights of 24.5 and 25.0 K, indicating monomeric heterogeneity of rPL-II in the tissue. The elution profile of day 18 plasma in RIA activity gave two major peaks; the first, eluting just after the void volume (approximate molecular weight of 530 K) accounted for 35 per cent of the total RIA activity, and the second coinciding with the same elution volume as the monomeric form in the placental extract constituted about 26 per cent of the total RIA activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Rat placental lactogen-II (rPL-II) and growth hormone (rGH) in maternal and fetal serum, amniotic fluid, and placental tissue were measured by a homologous radioimmunoassay during the last half of pregnancy. rPL-II appeared first in maternal circulation and the placental tissue on day 11 of pregnancy. The maternal serum rPL-II concentration increased progressively and reached the peak value (684 +/- 76 ng/ml) on day 19, and declined thereafter up to term. rPL-II content in the tissue had a similar pattern to the maternal serum profile of rPL-II, while its concentration in the tissue increased dramatically on day 12 and remained high until day 19. Fetal serum rPL-II was detected on days 17 and 18, though its concentration was much lower (ranged between 3-10 ng/ml) than that of maternal serum. rPL-II in amniotic fluid was also detectable only on days 12-14 of pregnancy, and the peak value on day 13 was 22% of the maternal serum rPL-II concentration. The rGH concentration increased gradually as pregnancy advanced with a decline on the day before parturition. Although rGH in fetal serum increased on day 20 with a decline on the following day, it was slightly detectable in amniotic fluid on the last two days of pregnancy. The molecular profile of rPL-II in amniotic fluid and maternal serum of day 13 pregnant rats were examined by Western blotting. Anti-rPL-II serum detected two proteins with molecular weights (mol wt) of 19.5K and 20.5K in amniotic fluid and one protein with a mol wt of 20.5K in maternal serum under nonreducing conditions.(ABSTRACT TRUNCATED AT 250 WORDS)