Andrew G. Hendrickx

Fetal surgery in the primate I. Anesthetic, surgical, and tocolytic management to maximize fetal—Neontal survival

There are compelling physiologic arguments for correcting certain malformations before birth. Although fetal surgery has been successful in sheep and lower animals, it has proven difficult in primates because the gravid uterus is exquisitely sensitive to induction of preterm labor and abortion. Because the feasibility of fetal intervention can be determined only in a primate model, we have investigated the variables affecting fetal-neonatal survival after fetal surgery in 25 monkeys. As we improved our anesthetic and surgical techniques and refined our tocolytic therapy, mortality fell from 73.3% (11/15) to 20% (2/10). Since spontaneous perinatal loss in 56 controls was 21.4% we can now operate on the late second and early third trimester fetal monkey without increasing maternal or fetal-neonatal mortality. Success in this rigorous model is a requisite for attempted correction of human malformations in utero.

Evaluation of Valproic Acid (VPA) Developmental Toxicity and Pharmacokinetics in Sprague—Dawley Rats

This study was undertaken to assess the pharmacokinetics and developmental toxicity of the anticonvulsant, valproic acid (VPA), a human teratogen, in Sprague-Dawley rats. Oral administration of 200-800 mg/kg VPA (5-20x human therapeutic dose) from Gestational Days (GD) 8 to 17 resulted in increasing maternal toxicity at the higher doses with 100% maternal lethality at 800 mg/kg. Although there was an increased incidence of resorptions at 600 mg/kg (48 +/- 43%) compared to controls (18 +/- 24%), it was not statistically significant. Fetal examination on GD 20 revealed dose-dependent fetal growth retardation (p less than or equal to 0.05) as evidenced by decreased fetal weight and length in addition to underossification of both the axial and appendicular skeleton. The incidence of skeletal defects, including abnormal vertebrae, ribs, and craniofacial dysmorphia, also increased with higher doses of VPA. Cardiac anomalies observed in the two highest treatment groups consisted of great vessel malformations with or without associated ventricular septal defects (VSDs). Urogenital defects were also noted in the 600 mg/kg group. The plasma elimination half-life on GD 8 was 1.0 +/- 0.3 hr at 200 mg/kg and 2.3 +/- 0.7 hr at 600 mg/kg. Maximal concentrations of total and free drug were 341 +/- 18 micrograms/ml and 181 +/- 11 micrograms/ml, respectively, in the low-dose group and 911 +/- 379 micrograms/ml and 542 +/- 224 micrograms/ml in the high-dose group. No significant changes in any pharmacokinetic parameters (t1/2, AUC, Cmax, tmax) were observed over the 10-day treatment period at either dose level.

Periconceptional vitamin a use: How much is teratogenic?

The objective of the review is to determine whether preformed vitamin A (retinol and retinyl esters) is teratogenic at dosages commonly used by women living in industrialized countries. Published human and animal data and research developed by the authors are reviewed. It is well known that vitamin A is essential for normal reproduction and development. Although doses of 10,000 IU/d or less of preformed vitamin A (retinyl esters and retinol) are considered safe, doses > 10,000 IU/d as supplements have been reported to cause malformations in a single epidemiologic study. Nonhuman primate data show no teratogenicity at doses of 30,000 IU/d. Daily periconceptional exposures greater than 25,000 IU/d of preformed vitamin A have not been sufficiently studied to establish specific risk. Because no study reports adverse effects of 10,000 IU/d preformed vitamin A supplements and this dose is more than the Recommended Dietary Allowance for pregnant women (2670 IU or 800 RE/d), we recommend that women living in industrialized countries or who otherwise have nutritionally adequate diets may not need to ingest more than the Recommended Dietary Allowance of preformed vitamin A as supplements. If periconceptional vitamin A exposures to levels up to 30,000 IU/d (9,000 micrograms RE/d) do occur unintentionally, multiple animal studies do support only very low risk. Human epidemiologic studies do not establish at what level vitamin A becomes teratogenic; however, pharmacokinetic data presented in this paper indicate that blood levels of retinoids from women taking 30,000 IU/d of preformed vitamin A are not greater than retinoid blood levels in pregnant women during the first trimester who delivered healthy babies. Interestingly, neither teratogenicity nor vitamin A toxicity has been observed in multiple species exposed to high doses of beta-carotene.

Titration of an SIVmac251 stock by vaginal inoculation of Indian and Chinese origin rhesus macaques: Transmission efficiency, viral loads, and antibody responses

The purpose of this study was to determine whether rhesus monkeys of Chinese origin are suitable for studies of mucosal lentivirus transmission by comparing the relative ability of these animals and rhesus macaques of Indian origin to become infected by vaginal (IVAG) inoculation with SIVmac251. In addition, we sought to test the hypothesis that differences in viral load during the first few weeks after inoculation were due to the relative strength of the anti-SIV immune responses in the two populations of rhesus macaques. Significant difference was not observed between the number of Indian and Chinese origin monkeys that were infected after IVAG SIV inoculation in this study. For 8-9 weeks after infection there was considerable overlap in the range of viral loads among the Indian and Chinese animals and the variation among the Indian origin animals was greater than the variation among the Chinese origin monkeys. By 6 weeks postinfection, viral loads in SIV-infected Chinese origin monkeys tended to be at the lower end of the range of viral loads observed in SIV-infected Indian origin monkeys. The strength of the anti-SIV antibody response was also more variable in the Indian origin rhesus macaques, but at 6-8 weeks postinfection, Chinese and Indian origin rhesus macaques had similar titers of anti-SIV antibodies. Microsatellite allele frequencies differed between Chinese and Indian rhesus macaques; however, the majority of alleles present in Indian-origin animals were also found in Chinese macaques. Together these results show that host factors, other than geographic origin, determine the ability of a rhesus macaque to be infected after IVAG SIV exposure and that geographic origin does not predict the viral load of SIV-infected animals during the first 8-9 weeks after IVAG inoculation.

Neuropathogenesis induced by rhesus cytomegalovirus in fetal rhesus monkeys (Macaca mulatta)

Rhesus cytomegalovirus (RhCMV) infection of rhesus macaques offers opportunities to analyze mechanisms of CMV pathogenesis in a primate species. Four fetal rhesus monkeys were inoculated intraperitoneally with RhCMV early in the second trimester, and pregnancies were terminated by hysterotomy during the third trimester. Three fetuses had evidence of severe CMV disease, including intrauterine growth restriction, ventriculomegaly, microcephaly, lissencephaly, and extensive degenerative changes of the cerebral parenchyma. Histopathologic examination revealed polymicrogyria, gliosis, leptomeningitis, periventricular calcifications, and inclusion-bearing cells. These results demonstrate that the developing macaque brain is susceptible to infection with RhCMV early in the second trimester and that intrauterine infection results in neuropathologic outcomes similar to those observed in humans congenitally infected with CMV.

The effect of contraceptives containing nonoxynol-9 on the genital transmission of simian immunodeficiency virus in rhesus macaques *

The ability of two nonoxynol-9 spermicide preparations to prevent the genital transmission of SIV in rhesus macaques was compared. Administration of one mL of contraceptive foam before the intravaginal inoculation of cell-free SIV prevented the genital transmission of SIV to three of six animals, and using one mL of contraceptive gel prevented the genital transmission of SIV to two of six animals. Thus, both contraceptive foams and gels containing nonoxynol-9 provided protection against the genital transmission of SIV.

Frequency of prenatal loss in a macaque breeding colony

An accurate knowledge of the historical incidence of prenatal loss is essential for management of breeding colonies and for performing developmental toxicity studies in nonhuman primates. Data from the California Regional Primate Research Center indoor (timed‐mated) and outdoor (random‐mated) colonies of rhesus, cynomolgus, and bonnet macaques (Macaca mulatta, M. fascicularis, and M. radiata) were evaluated for a 10 year breeding period from 1984 to 1993.

Reproductive toxicity testing of therapeutic biotechnology agents

Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan 48105 (J.WH.); Miles, Inc., Elkhart, Indiana 46515 (K.G.H.); USFDA, Rockville, Maryland 20852 (M.A.S., J.A.C.); California Regional Primate Research Center, Davis, California 95616 (A.G.H.); Hoffman-LaRoche, Inc., Nutley, New Jersey 07110 (N.D.A.); Skyline Technology Consulting Group, Znc., San Francisco, California 94133 (A.H.C.K.); and Genentech, Znc., South San Francisco, California 94080 (J.M.)

Predicting the human teratogenic potential of the anticonvulsant, valproic acid, from a non-human primate model☆

The anticonvulsant, valproic acid (VPA) is a suspected human teratogen. This study, employing the rhesus monkey as an animal model, demonstrates that VPA has a significant teratogenic potential in the monkey. Timed pregnant monkeys were exposed orally to VPA at approx. 1X, 10X, and 30X (20, 200, and 600 mg/kg/day, respectively) the human therapeutic dose, daily, during organogenesis (gestation days 21-50). All fetuses of mothers exposed to greater than 1X exhibited some form of embryotoxicity. The highest dose, 30X, was 100% embryolethal, while offspring of the 10X dose group exhibited craniofacial and skeletal defects, and low body weights. Maternal pharmacokinetic parameters and plasma metabolites were determined for VPA on the first and last day of dosing for the 10X dose group. Comparison of the kinetic and metabolite data with that obtained for man indicates that the rhesus monkey is a good model for predicting the teratogenic potential of VPA in the human.

Early implantation and embryonic development of the baboon: stages 5, 6 and 7

SummaryImplantation stages of the olive baboon, Papio cynocephalus anubis, showing embryonic development equivalent to Carnegie stages 5, 6 and 7 of development, were collected by hysterotomy and examined histologically. The younger specimens (stage 5) consisted of a thick trophoblastic plate composed of cytotrophoblast and syncytiotrophoblast with multiple small clefts, and a bilaminar disk embryo with a small slit-like amniotic cavity. An epithelial plaque response was present in the uterine epithelium immediately peripheral to the implantation site, within an area of pronounced uterine edema. The bilaminar embryonic disk consisted of columnar epiblast cells underlying the amniotic cavity, and thickened visceral endodermal cells that form part of the yolk sac. The slightly further developed placenta (stage 6) consisted predominantly of cytotrophoblast including primary villi and syncytotrophoblast lining large spaces containing maternal blood. Secondary placental villi were present in the oldest group (stage 7), and there was modest decidualization of the uterine stroma. An epithelial plaque response persisted, but varied in extent. The sequence of events in early development in the baboon is similar to that in the rhesus monkey insofar as blood space formation and endometrial responses are concerned. However, the plaque response is not so great as in the rhesus; there is no secondary placenta, and the decidual response is slightly more extensive.