Lack of direct bone effect of tofacitinib, a JAK inhibitor, in juvenile rats and evaluation of the association between offspring growth and femur length.

Bone has recently emerged as a target organ for some Janus kinase (JAK) inhibitors in adult and/or juvenile animal toxicity studies. Oral administration of tofacitinib, a JAK inhibitor, was not associated with clinical or macroscopic effects on bone growth and development in a rat juvenile animal study (JAS) with tofacitinib dosing starting on postnatal day (PND) 21. However, given that previous JAS did not include a targeted evaluation of bone, inclusive of microscopic examination, an additional rat JAS was conducted to further assess this risk. In this subsequent JAS, administration of tofacitinib from PND 7-49 or from PND 21-49 did not result in any direct effects on bone, with no histologic effects on developing bone. The only bone effect in this JAS was nonadverse shorter femur length, which was not considered to be a direct effect of tofacitinib, but rather an indicator of growth delay, as this was associated with lower body weights. There were no effects on femur length or body weight after a 2-month recovery period. To further explore the relationship between body weight and femur length, historical control data were analyzed from control rats in other JAS. This analysis clearly demonstrated that shorter femur length can occur as an indirect effect that is highly associated with lower body weight, consistent with what was observed in the JAS with tofacitinib. These analyses provide a robust and valuable data set to support the interpretation of such data in JAS, and further support the lack of direct effects of tofacitinib on bone growth and development. As with the previously conducted juvenile studies with tofacitinib, the additional JAS did not identify any special JAS-based concerns for use in pediatric patients as young as 2 years of age.

Reproductive and developmental safety of nirmatrelvir (PF-07321332), an oral SARS-CoV-2 Mpro inhibitor in animal models.

Nirmatrelvir (PF-07321332; NMV) the antiviral component of PAXLOVID™ is a potent and selective inhibitor of the SARS-CoV-2 main protease (Mpro), which plays a critical role in viral replication. PAXLOVID, comprised of nirmatrelvir and ritonavir (used as a pharmacokinetic enhancer), is an oral therapy currently in development as a therapeutic option for those infected with SARS-CoV-2 to prevent progression to severe disease, hospitalization, and death. PAXLOVID has been shown to be efficacious against hospitalization and death in two Phase 2/3 clinical studies that evaluated non hospitalized patients both with and without high risk factors for progression to severe illness. Given that males and females of reproductive age are included in the intended patient population, we assessed the potential effects of NMV up to the limit dose of 1000 mg/kg/day in ICH guideline embryo-fetal development studies in rats and rabbits, and a fertility and early embryonic development study in rats. There were no effects on male and female fertility or early embryonic development in rats, and no severe manifestations of developmental toxicity in rats or rabbits. The lack of adverse findings reported here in nonclinical species is consistent with the intended therapeutic target of NMV (a virus specific protein not present in mammalian cells), the favorable off-target selectivity profile, and lack of genetic toxicity. The results of these nonclinical studies with NMV along with existing ritonavir safety information indicate that there are no clinically relevant risks associated with PAXLOVID administration during pregnancy and in males and females of reproductive age.

Reproductive and developmental toxicity assessment of palbociclib, a CDK4/6 inhibitor, in Sprague-Dawley rats and New Zealand White rabbits.

Palbociclib is a selective inhibitor of the cyclin-dependent kinase (CDK) 4/6, approved for the treatment of breast cancer. We assessed the potential effects of oral administration of palbociclib on reproduction and development. There were no effects on female or male fertility indices; however, in the male there was seminiferous tubule degeneration in the testes and secondary findings in the epididymides, lower testicular and epididymal weights, sperm density and motility. Palbociclib was not teratogenic in rats or rabbits; however, in the presence of maternal toxicity (lower maternal body weight gain and food consumption), low fetal body weights were observed in rats and small forepaw phalanges were noted in rabbits. There were, however, no adverse effects on the F1 generation in a pre- and post-natal developmental toxicity study in the rat.

Nonclinical Safety Considerations for the Development of Pediatric-First Drugs: An Industry View.

This paper provides considerations on approaches to the development of medicines initially developed for pediatric use (ie, "pediatric-first" or "pediatric-only" drugs). The most common development approach for these types of medicines involves a first-in-human (FIH) clinical trial with healthy adult volunteers to assess safety and tolerability. This approach generally requires nonclinical repeat-dose studies in adult animals; safety pharmacology and in vivo genetic toxicology studies in adult animals are also performed for small-molecule drugs. Additional studies in juvenile animals may be required prior to clinical trials in pediatric patients, on a case-by-case basis. In this paradigm, the starting dose for pediatric patients is primarily driven by modeling from the adult pharmacokinetic assessment and pharmacology data. A second development approach is where the FIH clinical trial is conducted in pediatric patients. This approach is generally supported by repeat-dose studies in juvenile animals, with the onset of dosing at ages that developmentally correlate to the age of the pediatric patients. Safety pharmacology and in vivo genetic toxicology studies are generally performed in adult animals for small-molecule drugs. To define a safe yet minimally efficacious starting dose for pediatric patients, various complementing approaches can be used, including human equivalent dose, minimal anticipated biological effect level, and physiologically based pharmacokinetic modeling. Case examples for pediatric-first drug candidates show how both drug development approaches (ie, entry into human first in adults or directly in pediatric populations) are used in the pharmaceutical industry.

Toward a comparative retrospective analysis of rat and rabbit developmental toxicity studies for pharmaceutical compounds.

Based on a proposal made at the ICH Workshop in Tallinn, Estonia (2010), the value of the rabbit embryo-fetal development (EFD) versus the rodent EFD was examined by the HESI DART group. A cross-industry data survey provided anonymised EFD and toxicokinetic data from EFD studies on over 400 marketed and unmarketed drugs (over 800 studies) that were entered by experts at RIVM into US EPA's ToxRefDB style database. The nature and severity of findings at the lowest observed adverse effect level (LOAEL) are being reviewed to quantitate the frequency with which lesser signs of embryo-fetal effects (e.g., delays in ossification, minor changes in frequency of variants) are driving the LOAELs. Interpretation was based on exposure rather than administered dose. This paper provides an update of this ongoing project as discussed during a workshop of the European Teratology Society in Ispra, Italy (2013). This was the first presentation of the initial data set, allowing debate on future directions, to provide a better understanding of the implications of either delaying a rabbit EFD or waiving the need in particular circumstances.

Sensitivity of male reproductive endpoints in nonhuman primate toxicity studies: a statistical power analysis.

To determine the sensitivity of male reproductive toxicity endpoints in NHPs we performed a power analysis of routine and triggered endpoints using control data from sexually mature Asian and Mauritian NHPs. The power to detect a 50% change from control was 13-30% for male reproductive organ weights, ∼30% for testicular volume, 6-66% for seminal analyses and 10-78% for male hormones. Overall, male reproductive endpoints have poor power (less than 80%) to detect a 50% change from control with a group size of 3 monkeys. Confidently identifying adverse male reproductive effects with these endpoints would likely require specialized study designs with larger group sizes. Triggering of non-routine endpoints in cases where there is special concern for male reproductive toxicity is unlikely to increase sensitivity to detect adverse effects.

Effects of feed restriction during organogenesis on embryo-fetal development in rabbit.

BACKGROUND: Appropriate maternal nutrition and body weight gain during pregnancy is well established as a major factor in healthy prenatal development in humans. Given the role of nutrition and body weight gain in normal development, pharmaceuticals intended to reduce appetite and promote weight loss will generate developmental toxicity data that may be challenging to interpret. To aid with this, the effects of feed restriction, and subsequent reduction in maternal body weight gain, on embryo-fetal development was investigated in the rabbit. METHODS: Groups of 15 pregnant New Zealand White rabbits were offered 150 (control), 110, 75, 55, 35, and 15 g feed/day from gestation day (GD) 7-19. Cesarean sections were carried out on GD 29 and fetuses were examined for external, visceral, and skeletal development. RESULTS: Maternal body weights at the end of the feed restriction period (GD 20) were 0.97, 0.98, 0.93, 0.94, and 0.86 x control for the 110, 75, 55, 35, and 15 g feed/day groups, respectively. Only at 15 g feed/day was there a net maternal body weight loss (the GD 20 body weight was 0.93 x the GD 6 body weight) at the end of the feed restriction period. Six does aborted in the 15 g feed/day group; there were no other abortions associated with feed restriction. Fetal body weight was significantly reduced at 75, 55, 35, and 15 g feed/day (0.95, 0.90, 0.86, and 0.84 x control, respectively). There were no external or visceral malformations or variations, and no skeletal malformations associated with feed restriction. The incidence of fetuses with sternebrae 5 or 6 unossified was increased at feed levels < or = 75 g/day. At a feed level of 35 g/day there was an increase in unossified metatarsals and metacarpals, and an increase in the number of fetuses with a reduced number of caudal vertebrae ossified. Although these findings were not increased at a feed level of 15 g/day, the lack of dose response was likely due to increased abortion and subsequent decrease in fetuses available for evaluation at 15 g feed/day. CONCLUSION: These data demonstrate that feed restriction to feed levels that produce substantial reductions in maternal body weight gain can result in developmental toxicity expressed by abortion, reduced fetal weight, and alterations in ossification. Abortion only occurred when feed was restricted to an amount that produced maternal body weight loss (15 g feed/day) whereas reduced fetal weight and increased incidence of fetuses with unossified sternebrae, metatarsals, metacarpals, or caudal vertebrae were noted at feed levels of < or = 75 g/day. There were no fetal malformations associated with feed restriction.

The effects of feed restriction during organogenesis on embryo-fetal development in the rat.

BACKGROUND: Given the role of nutrition and body weight gain in normal development, pharmaceuticals intended to reduce appetite and promote weight loss will generate safety data that may be challenging to interpret. To aid with this, the effects of feed restriction and subsequent body weight reductions on embryo-fetal development were investigated in the rat. METHODS: Groups of 20 timed pregnant female Sprague-Dawley rats were offered Certified Rodent Diet 5002 either ad libitum or in restricted amounts of 20, 15, 10, and 7.5 g/day from Gestation Day (GD) 6-17. Clinical signs, body weights, and food consumption were recorded. Cesarean sections were performed on GD 21 and fetuses were sexed, weighed, and examined for external, visceral, and skeletal development. RESULTS: Mean maternal body weights at the end of the feed restriction period, GD 18, were reduced 0.87 x, 0.80 x, 0.69 x, and 0.63 x control mean in the 20, 15, 10, and 7.5 g/day groups, respectively. Mean body weight gains for the restriction period inclusive, GD 6-18, were 0.49 x and 0.24 x control at 10 and 7.5 g/day, respectively, and a mean body weight loss occurred at 10 and 7.5 g/day (0.95 x and 0.85 x mean GD 6 body weight, respectively). Fetal body weights were reduced 0.95 x, 0.93 x, 0.90 x, and 0.76 x control at 20, 15, 10, and 7.5 g/day, respectively. This resulted in a reduction in gravid uterine weight at 10 and 7.5 g/day. There were no external, visceral, or skeletal malformations attributed to feed restriction. There was an increase in the skeletal variation of wavy ribs and a decrease in ossification at 7.5 g/day. CONCLUSIONS: These data demonstrate that feed restriction-induced reductions in maternal gestational body weight gain of approximately 50% compared to ab lib fed rats only caused a reduction in fetal body weight. Even up to a 15% maternal gestational body weight loss had no effect on embryo viability in rats, but retarded fetal growth significantly enough to induce minor changes in skeletal development. There were no external, visceral, or skeletal malformations associated with any of the levels of maternal body weight reduction or loss.

Reproductive toxicity assessment of lasofoxifene, a selective estrogen receptor modulator (SERM), in female rats.

BACKGROUND: Lasofoxifene is a nonsteroidal selective estrogen receptor modulator (SERM). With high affinity to the alpha and beta human estrogen receptors and greater potency than other SERMs, lasofoxifene is potentially a superior treatment for postmenopausal osteoporosis. In light of the known effects of estrogen-modulating compounds on female reproductive indices, two studies were conducted to evaluate the effects of lasofoxifene on female rat cyclicity, reproduction, and parturition. METHODS: One study evaluated effects of lasofoxifene on estrous cyclicity, and the second study assessed effects on implantation and parturition. In the cyclicity study, lasofoxifene was administered to female rats at doses of 0.1, 0.3, and 1.0 mg/kg/day for 14 consecutive days. After treatment, there was a 3-week reversibility phase followed by a mating phase. In the implantation study, lasofoxifene was administered to pregnant female rats at doses of 0.01, 0.03, and 0.1 mg/kg/day for 7 consecutive days (gestation day [GD] 0-6). Some animals were euthanized on GD 21, and the remainder of the group was allowed to deliver the F1 generation. Several developmental indices were evaluated in the F1 pups through post-natal day (PND) 21. RESULTS: In the cyclicity study, all lasofoxifene-treated females were anestrous by Study Day 7 (1.0 mg/kg) or 9 (0.3 and 0.1 mg/kg). The reversibility phase resulted in restoration of normal estrous cycles by the end of 1 (0.1 mg/kg) or 2 weeks (0.3 and 1.0 mg/kg). During the mating phase, no adverse effects occurred in pregnancy success or reproductive parameters. In the implantation study, all doses of lasofoxifene increased pre- and post-implantation losses, increased gestation length, and reduced litter size. None of the developmental parameters measured on the F1 generation was adversely affected. CONCLUSION: Lasofoxifene reversibly altered the estrous cycle and inhibited implantation, consistent with what would be expected from a member of the SERM class.

Comparison of the developmental toxicity of aspirin in rabbits when administered throughout organogenesis or during sensitive windows of development.

BACKGROUND: A review of the nonsteroidal anti-inflammatory drug (NSAID) literature suggested occurrences of low-level incidences of cardiovascular and midline defects in rabbit fetuses exposed in utero. Aspirin (acetylsalicylic acid, ASA) is a widely used NSAID that irreversibly inhibits cyclooxygenases (COXs) 1 and 2. ASA has been studied extensively in rats and has consistently increased low-incidence cardiovascular malformations and defects in midline closure. The objectives of the current study were to comprehensively define the developmental toxicology profile of ASA in rabbits by using a dosing paradigm encompassing the period of organogenesis and to test the hypothesis that maternal gastrointestinal toxicity after repeated dose administrations hampers the detection of low-incidence malformations with ASA in rabbits by limiting ASA administration to sensitive windows for cardiovascular development and midline closure. METHODS: ASA was administered to pregnant New Zealand White rabbits from gestation days (GDs) 7 to 19 at dose levels of 125, 250, and 350 mg/kg per day and as single doses of 500, 750, or 1000 mg/kg on GD 9, 10, or 11. Cesarean sections were performed on GD 29, and the fetuses were examined for external, visceral and skeletal development. RESULTS: In the repeated dose study, maternal toxicity was exhibited in the 250- and 350-mg/kg per day groups by mortality and decreased food consumption and body weight gain. In the single dose studies, maternal toxicity was exhibited at all doses by reductions in body weight gain and food consumption for 3 days after treatment. Fetal body weight was significantly reduced in the repeated dose study at 350 mg/kg per day. Fetal weights were not affected by single doses of ASA on GD 9, 10, or 11. There were no treatment-related external, visceral or skeletal malformations associated with ASA administration throughout organogenesis or with single doses administered during critical developmental windows. CONCLUSION: These findings supported previous work demonstrating that ASA is not teratogenic in rabbits, as opposed to rats, even when large doses are administered on single days during specific windows of development.

Relationship between cyclooxygenase 1 and 2 selective inhibitors and fetal development when administered to rats and rabbits during the sensitive periods for heart development and midline closure.

BACKGROUND: A review of the scientific literature suggested the occurrence of low-level incidences of ventricular septal defect (VSD) and midline defect (MD) in rat fetuses and diaphragmatic hernia (DH), VSD, and MD in rabbit fetuses after maternal exposure to nonsteroidal anti-inflammatory drugs (NSAIDs). Aspirin, an NSAID that irreversibly inhibits cyclooxygenase 1 (COX-1) and COX-2, induces DH, VSD, and MD when administered as one dose during the sensitive periods of development in rats. Unlike aspirin, other NSAIDs, including selective COX-2 inhibitors, reversibly inhibit COX activity. To evaluate whether the dysmorphogenesis observed after maternal NSAID exposure correlates with COX-1 or COX-2 inhibition, a series of compounds with different capacities to inhibit COX-1 and COX-2 were administered to pregnant rats and rabbits during the sensitive period for heart development and midline closure. METHODS: The compounds selected, ranked from the most COX-2 selective to the most COX-1 selective based onCOX inhibition in a human whole blood assay, were CJ-19,209, meloxicam, diclofenac, diflunisal, ibuprofen, and ketorolac. Rat dams were treated on gestation days (GDs) 9 and 10, and rabbit does were treated on GDs 9, 10, and 11. The doses selected for evaluation represented the maximum tolerable dose for the compound, with the exception of CJ-19,209, which was dosed at 1000 mg/kg. Fetuses were collected by cesarean section on GDs 21 and 29 for rats and rabbits, respectively, and all fetuses were examined for external and visceral developmental anomalies. RESULTS: In rabbits, diflunisal induced DH, VSD, and MD (omphalocele) and single incidences of VSD and MD (gastroschisis) were noted in the ibuprofen group; no other developmental findings were associated with treatment. In rats, ibuprofen, diflunisal, and ketorolac induced increases in the incidence of VSD. In general the induction of developmental defects was associated with compounds that selectively inhibit COX-1 or have a high ratio of COX-1 to COX-2 inhibition. CONCLUSIONS: Inhibition of COX-1 may be involved in the disruption of heart development, whereas the selective inhibition of COX-2 (as assessed with CJ-19,209) appears to have no effect on heart development and midline closure in rats and rabbits.

Proposal for a tiered approach to developmental toxicity testing for veterinary pharmaceutical products for food-producing animals.

In order to establish the safety of veterinary drug residues in human food, a number of toxicological evaluations are required, including the assessment of potential risks to development. In the light of the use of developmental toxicity testing for risk characterization, we evaluated whether conducting these tests in more than one species was redundant. Review of the published Summary Reports of recommendations of the EU Committee for Veterinary Medicinal Products supplemented with data from the Joint FAO/WHO Expert Committee on Food Additives (JECFA) reports on veterinary drug residues in food identified 120 compounds, of which 105 had teratogenicity information in more than one species. The analysis of these compounds, representing a variety of chemical and pharmacological classes, provided justification for consideration of the use of a tiered approach for developmental toxicity evaluation of veterinary drugs for food-producing animals. The tiered approach begins with developmental toxicity testing in a rodent species, preferably the rat. If teratogenicity is observed, no testing in a second species would be required, except under specific circumstances where the ADI is determined based on the NOEL from this study. If a negative or an equivocal result for teratogenicity were observed in the rodent, then a developmental test in a second species, preferably the rabbit, would be conducted. The tiered approach provides thorough hazard identification, based on the use of a second species for compounds negative for teratogenicity in the rodent, and maintains maximum public protection based on the extremely low potential for human exposure to these compounds, while making a genuine attempt to limit unnecessary animal testing.

Effects of HCFC-123 exposure to maternal and infant rhesus monkeys on hepatic biochemistry, lactational parameters and postnatal growth.

Peroxisome proliferators are a class of nongenotoxic rodent hepatocarcinogens that cause peroxisome proliferation and liver tumors when administered to rats and mice; but other species, including guinea pigs, dogs, and primates are less sensitive or refractory to the induction of peroxisome proliferation. Therefore, rodent peroxisome proliferators are not believed to pose a hepatocarcinogenic hazard to humans. Some peroxisome proliferators produce developmental toxicity in rats that is expressed as suppressed postnatal growth. To evaluate the relevance of the rat developmental effect to primates, groups of 4 lactating female Rhesus monkeys and their infants were exposed for 6 h/day, 7 days/week for 3 weeks to air or 1000 ppm HCFC-123. Animals were evaluated for clinical signs, body weights, clinical pathology parameters, and biochemical and pathological evaluations of liver biopsy samples. The effect of HCFC-123 exposure on milk quality (protein and fat concentration) was evaluated. The concentrations of HCFC-123 and the major metabolite, trifluoroacetic acid (TFA), were measured in the blood of the mothers and infants and in the milk. Exposure of monkeys to 1000 ppm HCFC-123 did not result in exposure-related clinical observations, or changes in body weight, appetence and behavior. There were no exposure-related effects on serum triglycerides, cholesterol, or glucose levels. HCFC-123 and TFA were present in milk, although maternal HCFC-123 exposure did not affect milk protein and fat content. In general, HCFC-123 was not detected in maternal or infant blood. TFA was detected in the majority of the mothers and TFA levels in infants ranged from 2 to 6 times higher than levels in the corresponding maternal blood. A pharmacokinetic analysis in a maternal animal indicated a peak concentration of TFA at approximately 1 h post-exposure, with a half-life of approximately 20 h. Liver microsomal P450 and peroxisome oxidase activities showed exposure-related decreases in CYP4A1 and CYP2E1 and acyl-CoA oxidase for animals exposed to HCFC-123. Microscopic evaluation of maternal liver from HCFC-123 exposed animals revealed mild to moderate centrilobular hepatocyte vacuolation, trace to mild centrilobular necrosis, and trace to mild subacute inflammation. The histopathological damage and altered hepatic biochemical activities produced by HCFC-123 in monkeys are not consistent with the HCFC-123 peroxisome proliferation response observed in rat livers. These findings demonstrate that HCFC-123 is not a peroxisome proliferator in adult Rhesus monkeys and postnatal exposure to HCFC-123 does not affect body weight of nursing infant monkeys.

Effects of the rat hepatic peroxisome proliferator, Wyeth 14,643, on the lactating goat.

Some peroxisome proliferators have been reported to reduce body weight gain in suckling rats, possibly through a lactational effect. Decreases in milk production or nutritional quality, either as a result of peroxisome proliferator-induced reductions in lipid content or alterations in the hormonal milieu necessary for milk production, could result in pup growth retardation. Wyeth-14,643 (WY) is hypolipidemic agent and a potent inducer of hepatic peroxisome proliferation in rats and mice. As is commonly seen with rodent hepatic peroxisome proliferators, WY produces minimal or no peroxisome induction in guinea pigs or non-human primates. Goats are an excellent model for studying lactation, however, their sensitivity to peroxisome proliferating chemicals is not known. The present study was performed to assess the sensitivity of goats to the hypolipidemic and peroxisome proliferator properties of WY and to determine the effects of WY on milk quantity and quality. Six lactating adult female goats were assigned to either control or treated groups. Goats in the treated group were administered WY (40 mg/kg/day) for 14 consecutive days. The goats were milked twice daily in order to maintain lactation and the quantity of milk collected was recorded. Milk quality was evaluated by determining the content of total fat, protein, and carbohydrate in milk samples collected following 7 and 14 days of treatment. WY administration had no effects on final body weight, liver weight or, gross and histopathological findings. Milk quantity and quality were unaffected by treatment. Serum cholesterol and triglyceride levels were reduced by 25% compared to controls, although only the difference in cholesterol was statistically significant. Hepatic beta-oxidation (3 x control) and aromatase (1.5 x control) activities were significantly greater in the treatment group; however, there was no treatment-related effect in the total content of hepatic cytochrome P450. There was no difference in aromatase activity in a pooled ovarian microsome sample. Milk estradiol and prolactin concentrations were not affected by treatment. These findings indicate that goats are weak responders to the hepatic peroxisome proliferator effects of WY. Additionally, the slight serum hypolipidemic effect does not impact milk production or nutritional value.

Plasma and brain methamphetamine concentrations in neonatal rats.

D-Methamphetamine (D-MA) treatment during the neonatal period has been shown to induce acoustic startle hyperreactivity and Morris maze spatial learning deficits, but not to significantly affect Cincinnati maze sequential learning. In order to characterize the internal dose in these experiments, MA was measured in plasma and brain of neonatal rats at one of two ages, and using one of three dose schedules, two of which were selected to be representative of those used in previously published neurobehavioral studies. Plasma parameters showed few age and dose-frequency effects; however, brain concentrations showed more consistent age-dependent effects. Brain area under the concentration (AUC) values were consistently higher, regardless of dosing schedule, in offspring treated on postnatal day (P) 1 compared to those treated on P11. Previous results with the multiple-dose schedules have shown that Morris maze spatial learning deficits only occur in those exposed beginning on P11, whereas acoustic startle hyperreactivity is associated with exposure beginning on either P1 or P11. The pharmacokinetic parameters did not predict the long-term spatial learning and memory effects of neonatal MA administration, nor are they well correlated to the acoustic startle effects. The plasma concentrations obtained in rats are within the range for human MA abusers based on extrapolations from human low-dose values to those expected for heavy users.

Evaluation of neonatal exposure to cocaine on learning, activity, startle, scent marking, immobility, and plasma cocaine concentrations.

Prenatal cocaine treatment produces equivocal effects on spatial learning and memory; however, no data are available on neonatal treatment as a model of human third-trimester exposure. Sprague-Dawley rats were treated on postnatal days (P) 1-10 or 11-20 with cocaine (15 mg/kg x 4 per day at 2-h intervals) or saline (P1-P20) and evaluated as adults in the Morris water maze and on tests of activity, startle, scent marking, swimming immobility, and sequential learning. Neonatal cocaine had no effect on mortality; however, early treatment reduced body weight, whereas later treatment did not. Neonatal cocaine had no effects on exploratory activity, swimming ability, sequential learning, multiday activity rhythms, scent marking, or swimming immobility, but augmented acoustic startle amplitude in the early-treated group. Neonatal cocaine also produced an interaction on spatial learning in which the cocaine early-treated males performed slightly more efficiently than controls. Plasma cocaine concentrations were significantly higher in the early-treated group than the later-treated group despite receiving the same weight-adjusted doses. It was concluded that neonatal cocaine, when administered during a stage of brain development analogous to human third trimester, induces few behavioral effects based on the assessments used in this study.

Time-course of methamphetamine-induced neurotoxicity in rat caudate-putamen after single-dose treatment.

The time-course of monoamine and tyrosine hydroxylase depletion after single-dose administration of D-methamphetamine (40 mg/kg s.c.) was investigated in caudate-putamen of male Sprague-Dawley rats. Times evaluated were 6, 12, 48, 72 and 240 h following treatment. Tyrosine hydroxylase was significantly reduced by 29, 60, 66, 76 and 76% of control at each of the respective post-treatment time intervals. Dopamine was not reduced 6 h following treatment. Dopamine was significantly reduced by 53, 57, 68 and 74% 12, 48, 72 and 240 h post-treatment, respectively. Reductions in caudate-putamen serotonin began earlier and were ultimately larger than for dopamine, with significant reductions of 28, 33 55, 74 and 81% at each of the respective post-treatment intervals. Confirmation of neurotoxicity was provided by measurement of glial fibrillary acidic protein (GFAP) 240 h post-treatment. GFAP was increased at this time interval by 150% above control. Methamphetamine-induced hyperthermia during the 6 h immediately after treatment was comparable among the groups of animals used for analyses at each time interval. The results demonstrate that methamphetamine-induced monoamine reductions in the caudate-putamen occur rapidly, peak at 75-80% below controls, and last for at least 10 days after a single dose. These effects are as large or larger than those reported after the commonly used 10 mg/kgx4 dose treatment regimen administered at 2-h intervals and provides an alternate model for the investigation of methamphetamine-induced neurotoxicity.

Genetic differences in spatial learning between Dark Agouti and Sprague-Dawley strains: possible correlation with the CYP2D2 polymorphism in rats treated neonatally with methamphetamine.

Following neonatal exposure to d-methamphetamine, adult rats have previously been shown to exhibit augmented acoustic startle and spatial learning deficits. d-Methamphetamine is structurally similar to several phenylethylamines that are metabolized by CYP2D6. In humans, allelic differences in the CYP2D6 confer the extensive or poor metabolizer phenotype for the more than three dozen drugs that are members of the CYP2D6-mediated 'debrisoquine/sparteine panel.' An analogous genotype exists with the CYP2D2 gene in rats. Female Dark Agouti rats show the poor metabolizer phenotype, whereas Sprague-Dawley rats show the extensive metabolizer phenotype; male Dark Agouti rats are intermediate. We sought to test the possibility that these strains might exhibit altered d-methamphetamine-induced developmental neurotoxicity. Dark Agouti and Sprague-Dawley litters (11-20 days of age) were given d-methamphetamine or vehicle alone subcutaneously twice daily (15 mg/kg). Offspring were assessed as adults (beginning at 50 days of age) on acoustic startle, straight-channel swimming, and spatial learning and memory in a Morris hidden platform maze. Increases in d-methamphetamine-induced acoustic startle were found in both male and female Dark Agouti rats, but not Sprague-Dawley rats. In the Morris maze, d-methamphetamine-induced spatial navigation deficits were found in both strains among males, suggesting some mechanism other than the CYP2D2 polymorphism. In contrast, among females only the d-methamphetamine-treated Dark Agouti rats showed deficits in spatial navigation. The maze deficits in Dark Agouti females, and enhanced acoustic startle in Dark Agouti females and males, support the hypothesis that the CYP2D2 poor metabolizer phenotype confers increased vulnerability to d-methamphetamine-induced developmental neurotoxicity, indicating that the parent drug rather than a CYP2D2-mediated metabolite is responsible for this behavioural defect--which occurs in adults who had been exposed to d-methamphetamine during the neonatal period.

Enhancement of cocaine-induced hyperthermia fails to elicit neurotoxicity.

The neurotoxic potential of cocaine when administered under conditions conducive to the initiation of hyperthermia was investigated. Rats were administered cocaine at ambient temperatures of 22 degrees C or 30 degrees C. To determine the thermal response, body temperatures were measured every 30 min and the total thermal response (TTR), representing the area under the temperature vs. time curve, was calculated. Saline administered at 22 degrees C or 30 degrees C resulted in a normal thermal response (TTR = 9.8+/-0.9 and 11.2+/-0.9, respectively). Cocaine administration resulted in ambient temperature-dependent hyperthermia. Cocaine (4 x 25 mg/kg) administered at 22 degrees C resulted in a TTR of 15.1+/-0.9 whereas cocaine (4 x 15 or 25 mg/kg) administered at 30 degrees C resulted in TTRs of 22.2+/-0.9 and 21.9+/-0.8, respectively. Regardless of the dose or thermal response, cocaine administration did not result in depletion of dopamine (DA) or serotonin (5-HT) in the caudate-putamen. Cocaine administration also failed to induce an increase in the concentration of glial fibrillary acidic protein (GFAP), a marker for neurotoxicity. These results demonstrate that hyperthermia does not promote cocaine-induced neurotoxicity in the rat caudate-putamen.