R.D. Simmons

Prenatal diazepam exposure in rats: long-lasting, receptor-mediated effects on hypothalamic norepinephrine-containing neurons

The concentration and turnover of catecholamines (CAs) were measured in the cortex, hippocampus, and hypothalamus of rats exposed in utero to diazepam (DZ, 1.0, 2.5, or 10.0 mg/kg/day) over gestational days 13-20. Prenatal DZ induced a regionally specific, dose-related decrease in the level of norepinephrine (NE) (maximum decrease, 65%) and turnover rate of NE (maximum decrease, 85%) in the hypothalamus of 90-day-old adult rats. Dopamine levels were not altered in this region and neither of the CAs were altered in the other regions. Dividing the prenatal exposure period into two shorter periods revealed that late gestation (days 17-20) was the period when factors influencing NE function in the hypothalamus were most sensitive to DZ. Analysis of the development of the CA in the hypothalamus demonstrated that the effect of the prenatal exposure on NE levels did not become apparent until after 28 days of age. However, at 28 days, there was a dose-related increase in turnover rate of NE (maximum increase, 52%). Concurrent administration of the specific benzodiazepine (BZ) antagonist RO15-1788 with DZ (2.5 mg/kg) to pregnant rats effectively reversed the effects of DZ in the hypothalamus of the adult offspring, suggesting that the effects were mediated via the BZ receptor. These data have provided insight into the intricate processes of neuronal development; in particular the importance of target cell-nerve terminal interactions and the role of early developing receptors.

Prenatal exposure to diazepam alters central and peripheral responses to stress in adult rat offspring

Central and peripheral responses to restraint stress were evaluated in 90-day-old rats exposed prenatally to diazepam (1.0, 2.5, or 10.0 mg/kg/day) over gestational days 13-20. As a measure of a central response to stress, the utilization of norepinephrine (NE) by hypothalamic NE neurons was assessed by determining the effect of stress on the loss of NE after synthesis inhibition. The stress-induced changes in plasma corticosterone and prolactin levels were evaluated as a physiologic index of stress. While stress increased the loss of NE after synthesis inhibition in the non-exposed control animals, it totally prevented any loss of NE after synthesis inhibition in offspring prenatally exposed to DZ. Additionally, the stress-induced change in plasma corticosterone was attenuated in a dose-related manner by prenatal exposure to DZ. The stress-induced change in plasma prolactin was also altered in a dose-related manner by the prenatal exposure. Both the altered response to stress within hypothalamic NE neurons and the attenuated change in plasma corticosterone induced by prenatal exposure to DZ (2.5 mg/kg) were prevented by concurrent administration of the centrally acting benzodiazepine antagonist Ro15-1788 to the pregnant dam, indicating that the effects of DZ were mediated via binding of the drug to central sites during gestation. These results indicate that activation of specific binding sites during early development can induce neural alterations in the adult offspring which can be reflected in functional changes which may compromise the organism.

Efficacy of AR-R15896AR in the Rat Monofilament Model of Transient Middle Cerebral Artery Occlusion

The monofilament technique of transient middle cerebral artery occlusion (MCAO) was used in 3 separate studies to evaluate the efficacy of the low-affinity, use-dependent N-methyl-d-aspartate receptor antagonist, AR-R15896AR. First, a dose-response curve was attempted. Wister Kyoto rats received 2 hours of MCAO. Five minutes later, a 30-minute intravenous infusion of AR-R15896AR was given, followed by subcutaneous implantation of Alzet minipumps that were calibrated to maintain specified plasma levels (approximately 682, 1885, or 2682 ng/mL) of AR-R15896 (free base) for 1 week. The highest plasma level attained significantly decreased the percentage of damage to the subcortex, cortex, and total brain. Second, the high-dose, 1-week treatment regimen was repeated to determine if neuroprotection would extend to 8 weeks after MCAO. Indeed, in separate groups of animals, significant reduction in the percentage of damage, which was generally confined to the cortex and subcortex, was observed at 1, 2, 4, and 8 weeks. Third, verification was achieved in another laboratory. Lister Hooded rats received 60 minutes of transient MCAO. At 70 minutes, an acute dose of AR-R15896AR (20.3 mg/kg) was injected intraperitoneally and the rats were killed 23 hours later. This treatment group also exhibited significant reduction in the volume of infarction in the subcortex, cortex, and total brain. The outcome of these investigations supports the ongoing Phase II clinical trials in patients with acute stroke.

Behavioral Effects of AR-R 15849, a Highly Selective CCK-A Agonist

The behavioral effects of AR-R 15849, a novel cholecystokinin agonist with high affinity and selectivity for the CCK-A receptor subtype, were examined. Initially, using an operant feeding paradigm to test for anorectic activity and specificity, acute administration of AR-R 15849 was found to alter the intake and pattern of feeding in a manner similar to prefeeding. Further, AR-R 15849 did not induce compensatory feeding as did CCK-8, and did not affect performance on running rates of responding, or motor activity on a running wheel, as did fenfluramine. In tests for subchronic anorectic activity, daily intraperitoneal injections of AR-R 15849 significantly reduced food intake in fasted rats over a 9-day test period with greater efficacy compared to its nonselective predecessor AR-R 14294 (formerly FPL 14294). The sustained decrease in food intake with AR-R 15849 treatment resulted in a significant reduction in body weight gain over 9 days. Finally, an experiment designed to determine the effect of caloric deprivation and subchronic drug exposure on the overall efficacy of AR-R 15849 indicated that pharmacological tolerance does not develop following subchronic treatment.

ARL 15849: A Selective CCK-A Agonist With Anorectic Activity in the Rat and Dog

Cholecystokinin octapeptide (CCK-8) and the peptide analog ARL 14294, formerly FPL 14294, [Hpa(SO3H)-Met-Gly-Trp-Met-Asp-N(Me)Phe-NH2], have been reported to induce satiety by interaction with the CCK-A receptor subtype. ARL 15849 [Hpa(SO3H)-Nle-Gly-Trp-Nle-N(Me)-Asp-Phe-NH2] is an improved ARL 14294 analog with enhanced CCK-A receptor selectivity, greater stability, and a longer duration of action. The affinity of ARL 15849 for the CCK-A receptor (Ki = 0.034 nM) is 6,600 fold greater than for the CCK-B receptor (Ki = 224 nM), whereas CCK-8 and ARL 14294 are nonselective. Although comparable in potency to contract isolated gallbladder and induce pancreatic phosphatidylinositol hydrolysis, ARL 15849 is 3- and 100-fold more potent than ARL 14294 and CCK-8, respectively, to inhibit 3-h feeding in rats. The duration of feeding inhibition was significantly longer for ARL 15849 (>5 h), compared to equipotent doses of ARL 14294 (3 h), and CCK-8 (1 h). Intranasal administration of ARL 15849 inhibits feeding in beagle dogs with a greater separation between doses that induce emesis and those that inhibit feeding. Therefore, ARL 15849 is a potent, selective, intranasally active anorectic agent which may be useful in the treatment of eating disorders.

Selection of orally active antifungal agents from 3,5-substituted isoxazolidine derivatives based on acute efficacy-safety profiles.

Routine in vitro screening of a new synthetic series of 3,5-substituted 2-methylisoxazolidines revealed that three imidazole analogs (PR 967-248, PR 967-234, and PR 969-566) and, to a lesser extent, a triazole analog (PR 988-399) exerted rather potent antifungal activity against three systemic and four dermatophytic classes of fungi. When tested in vivo for ability to eradicate Candida vaginitis in the rat, the triazole derivative, PR 988-399, was effective after oral administration. In this in vivo test for efficacy, PR 967-234 and PR 969-566 reduced but did not eradicate the infection, while PR 967-248 was inactive. PR 988-399 was, moreover, 4- to 13-fold less potent than the three imidazoles in inhibiting testosterone synthesis in isolated rat Leydig cells. After oral or intravenous administration, PR 988-399 and PR 969-566 elicited the fewest cardiovascular and behavioural side effects in conscious dogs. The rat safety study consisted of oral dosing followed by evaluation of the exploratory motor activity of the naive animals in a novel environment. Motor activity was suppressed least by PR 988-399 and most by PR 969-566. In a battery of mouse behavioural-neuromuscular-drug interaction tests, PR 988-399 and PR 969-566 produced the fewest central-behavioural-neuromuscular signs. These efficacy-safety evaluations were performed with ketoconazole as a positive reference standard. The sequence of drug testing with respect to efficacy-safety considerations appears to be a suitable approach for early detection of orally active antifungal agents such as PR 988-399 for more advanced development.

Comparisons of ketoconazole, PR 969-566, PR 967-234, and PR 967-248 as antifungals in vitro and in the rat model of candidal vaginitis based on efficacy/safety profiles.

Critical factors in the rational development of new antifungal drugs are standardized, quantitative, and coordinated approaches involving chemical discovery, preclinical evaluations, and comparisons of novel antifungal candidates with positive reference compounds. The reference compound ketoconazole has been found to be effective clinically against vaginal candidiasis as well as in the rat model of candidal vaginitis. A new chemical series of isoxazolidine compounds was targeted for preclinical evaluations involving 1) in vitro testing; 2 ) in vivo analysis in the rat model of candidal vaginitis; and 3 ) safety considerations involving hormonal, central nervous system (CNS), and cardiovascular (CV) effects. The following antifungal candidates were first compared to ketoconazole for in vitro activity: 1) PR 969-566 or cis-3-(4-chlorophenyl) 3 [ ( 1Himidazol1 yl) methyl] 2 -methyl 5 { [ ( 4 methylphenyl) thiolmethyl) isoxazolidine; 2) PR 967-234 or cis-3,5-bis(4-chlorophenyl)-3-[ ( ltl-imidazoll-yl)methyl]-2-methylisoxazolidine; and 3) PR 967-248 or cis-5-[ (4-chlorophenoxy ) methy1]-3-(4-~hlorophenyl)-3-[( lH-imidazol1 -yl)methyl]-2-methylisoxazolidine. In the in vitro evaluations, minimum inhibitory concentration (MIC) values were used for comparing antifungal activity of all four compounds against two strains of Candida

PRENATAL DIAZEPAM EXPOSURE IN RATS: LONG-LASTING FUNCTIONAL CHANGES IN THE OFFSPRING

Adult (90-100 day-old) rats exposed prenatally (13-20 days of gestation) to diazepam (DZ) at 1.0, 2.5, or 10 mg/kg/day, were examined for functional deficits. Tests were designed to (1) correlate with known neural alterations induced by the prenatal exposure, or (2) to address the problem of developing tests of functional teratology in animals which have relevance to complex human functions. The careful selection of functional tests has disclosed several effects of prenatal DZ exposure on adult rat progeny. The dose-effect relationship was found to vary with the different tests, indicating that a thorough evaluation of the consequences of prenatal drug exposure necessitates utilization of several functional approaches. The results of these studies on the effects of prenatal exposure to DZ support the hypothesis that in utero exposure to drugs targeted for action on the central nervous system can induce long-lasting alterations on the neural substrates of behavior on the offspring with resulting functional consequences.

prenatal diazepam exposure induces alterations in central and peripheral responses to restraint stress in adult rat progeny

The adenylate cyclase system, which is the membranous relay between substances of the ex t race l l u la r mi l ieu and cAMP, the i n t r a c e l l u l a r messenger, play a s i gn i f i can t role in cel l migration and d i f f e r e n t i a t i o n during ear ly development. In recent studies we investigated the developmental p ro f i l e of the enzyme in the chick brain. We found that adenylate cyclase was present in the chick embryo gastrula and was markedly high from the 3rd through the 8th day of incubat ion. This embryonic period coincides with active neurogenesis and gl iogenesis. To explore fur ther the role of adenylate cyclase in neural growth and d i f f e r e n t i a t i o n , we have used neuronal-enriched cultures derived from 6-day-old chick embryo brain in DMEM (Dulbeccos Modified Eagle Medium) plus 10% FCS ( feta l ca l f serum), plated on polylysine-coated petr i dishes; and g l ia l -enr iched cultures from 15-day-old chick embryo cerebral hemispheres in DMEM plus 20% FCS, p la t ed on p las t ic pet r ic dishes. Both basal and NaF-stimulated adenylate cyclase a c t i v i t ies were present in both neuronaland g l ia l -enr iched cul tures as ear ly as 3 days in cu l ture. Prel iminary data show that cel l density has a dramatic e f fect on adenylate cyclase a c t i v i t y leve ls . Neural t issue cul ture is proving to be a valuable tool to study the role of adenylate cyclase in embryonic development. (Supported by research grants AA 03527 from NIAAA and DA 02131 from NIDA.)