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.

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.

Placental transfer of (125)Iodinated humanized immunoglobulin G2Δa in the Sprague Dawley rat.

Antibody-like biopharmaceuticals cross the placenta by utilizing transport pathways available for transfer of maternal antibodies to the conceptus. To characterize the timing and magnitude of this transfer in the rat, embryo/fetal biodistribution of maternally administered radiolabeled humanized IgG2 was quantified over the course of gestation using gamma counting and whole body autoradiography. The result was humanized IgG2 found in rat embryo/fetal tissues as early as gestation day 11 with a >1000-fold increase in the amount of total IgG2 by day 21. The concentration of IgG2 in rat embryo/fetal tissues generally remained unchanged from gestation day 11 to 17 with a slight increase from day 17 to 21. In addition, fetal-maternal tissue concentration ratios remained stable during organogenesis with a slight increase from gestation day 17 to 21. Based on the empirical amount of antibody present in the embryo/fetus during specific developmental windows, direct antibody binding to biological targets could potentially result in adverse developmental outcomes.

Embryo-fetal distribution of a biopharmaceutical IgG2 during rat organogenesis.

Embryo-fetal biodistribution of a maternally administered humanized IgG2 in rats was evaluated by enzyme-linked immunosorbent assay in dose response and time course studies. Fetal and maternal plasma IgG2 levels increased with dose from 10 to 300mg/kg but fetal:maternal ratio decreased with increasing dose. Plasma IgG2 levels decreased in fetal rat with increasing time post-dose but more slowly than maternal levels. This difference in post-dose kinetics resulted in an increased fetal:maternal ratio with increasing days since last dose. Lastly, IgG2 in embryo-fetal tissue was detected at very low levels on gestation day (GD) 10-12 and levels increased over 100-fold by GD 17. The profile of increasing IgG2 levels as gestation progressed continued in extra-embryonic fluid (GD 12-19) until the end of gestation in fetal plasma (GD 19-21). Based on the current study, there is a potential for direct effects on rat embryo-fetal development following maternal administration of a biopharmaceutical IgG2.

Multiple responses in gene expression in fish treated with estrogen.

During the last decade there has been a significant body of research conducted on environmental estrogens. These include industrial, agricultural and pest-control chemicals that bind to the estrogen receptor and induce biological changes during development or reproduction. Most of these changes are probably due to modified gene expression, since estrogen receptors function at this level. We have mapped qualitative gene expression responses (by differential display reverse transcriptase polymerase chain reaction, DD) in adult male sheepshead minnows (Cyprinidon variegatus) receiving high dose injections (5 mg/kg), or constant flow-through aquatic exposures to environmentally relevant concentrations (100 ng/l) of estradiol-17beta, and found them nearly identical. We have observed both up-regulation and down-regulation of transcripts, which fit into known responses to estradiol. Among the genes up-regulated are vitellogenin and several vitelline envelope proteins indicating that genes for proteins involved in egg development and maturation are susceptible to environmental estrogen exposure. While physiological changes caused by estradiol treatment are not totally explained by changes at the mRNA level, those changes can nevertheless be used as fingerprints to characterize an in vivo estrogenic response.

Effects of p-nonylphenol, methoxychlor, and endosulfan on vitellogenin induction and expression in sheepshead minnow (Cyprinodon variegatus).

Temporal and dose-response relationships of vitellogenin (VTG) mRNA induction and subsequent plasma VTG accumulation were established for sheepshead minnows (Cyprinodon variegatus) treated with p-nonylphenol (an alkylphenol) and the organochlorine pesticides methoxychlor and endosulfan. Thirty-two adult male fish per treatment were continuously exposed to measured concentrations of 0.64, 5.4, 11.8, 23.3, and 42.7 micrograms/L p-nonylphenol; 1.1, 2.5, 5.6, 12.1, and 18.4 micrograms/L methoxychlor; and in two separate tests, 15.9, 36.3, 68.8, 162, 277, 403, 590, and 788 ng/L endosulfan using an intermittent flow-through dosing apparatus. Separate triethylene glycol (50 microliters/L) and 17 beta-estradiol (65.1 ng/L) treatments served as the negative and positive controls, respectively. Four fish were randomly sampled from each test concentration on days 2, 5, 13, 21, 35, and 42 of exposure, and levels of hepatic VTG mRNA induction and serum VTG accumulation were determined for each individual. Overall, fish exposed to p-nonylphenol or methoxychlor demonstrated a rapid, dose-dependent synthesis of VTG mRNA up to day 5 of exposure, followed by a relatively constant dose-dependent expression through day 42. Both chemicals showed a dose-dependent increase in plasma VTG over the entire time course of exposure, with significantly elevated VTG levels by the fifth day of exposure to p-nonylphenol at concentrations of 5.4 micrograms/L or greater and to methoxychlor at concentrations of 2.5 micrograms/L or greater. Exposure to 0.64 microgram/L p-nonylphenol resulted in highly variable plasma VTG levels of less than 6 mg/ml. Exposures with endosulfan failed to induce measurable levels of either hepatic VTG mRNA or serum VTG at the chemical concentrations tested. Our results demonstrate that the sheepshead minnow bioassay is a suitable estuarine/marine teleost model for in vivo screening of potentially estrogenic substances.

Induction of gene expression in sheepshead minnows (Cyprinodon variegatus) treated with 17beta-estradiol, diethylstilbestrol, or ethinylestradiol: the use of mRNA fingerprints as an indicator of gene regulation.

The recent interest in hormonally active environmental contaminants has sparked a drive to find sensitive methods to measure their effects on wildlife. A molecular-based assay has been developed to measure the induction of gene expression in sheepshead minnows (Cyprinodon variegatus) exposed in vivo to the natural and pharmaceutical estrogens 17beta-estradiol, ethinylestradiol, and diethylstilbestrol. This method used differential display reverse transcriptase polymerase chain reaction assays to compare the expression of individual mRNAs from control and estrogen-exposed fish. Forty-eight differentially expressed cDNAs were isolated by this method, including cDNAs for vitelline envelope proteins and vitellogenin. The mRNA expression patterns for fish injected with a pharmacological dose of estradiol (5 mg/kg) were identical to those obtained in fish receiving constant aqueous exposure to 212 ng estradiol/liter. Further, the cDNA "fingerprint" pattern observed in the estradiol-treated fish also matched that obtained in fish receiving continuous-flow aqueous exposures to 192 ng ethinyl estradiol/liter and a nominal concentration of 200 ng diethylstilbestrol/liter. The results demonstrate a characteristic expression pattern for genes upregulated by exposure to a variety of natural and anthropogenic estrogens and suggest this approach may be valuable to examine the potential effects of environmental contaminants on other endocrine-mediated pathways of reproduction, growth, and development.

Estrogen-induced vitellogenin mRNA and protein in sheepshead minnow (Cyprinodon variegatus).

Many environmentally persistent xenobiotic chemicals appear to disrupt normal endocrine function by acting as ligands for endogenous steroid receptors, including the estrogen receptor. Xenobiotics that bind to the estrogen receptor may elicit several effects, one of which is activating estrogen-responsive genes, such as vitellogenin (Vtg). Primers to vitellogenin mRNA have been used to amplify a portion of the coding sequence in sheepshead minnow (SHM) (Cyprinodon variegatus). Two Vtg cDNA fragments from SHM were isolated exhibiting 72% sequence homology and corresponding to the two Vtg genes identified in the mummichog, Fundulus heteroclitus. Using these Vtg cDNA fragments as sensitive genetic probes, we evaluated the initial estrogenic response of fish exposed to natural or anthropogenic chemicals. These probes were used to study in vivo gene induction in SHM exposed to 17beta-estradiol (E(2)) and ethinylestradiol (EE(2)) under controlled laboratory conditions. Hepatic Vtg mRNA was upregulated and plasma Vtg synthesis in estrogen-induced SHM was assessed. Two in vivo time-course experiments were conducted; a single injection of E(2) followed over 72 h and a double E(2) injection examined for 12 days. These two protocols provided evidence for differential hepatic Vtg mRNA regulation resulting from a single or a double injection. In a separate experiment using an aqueous flowthrough system, constant exposures to low doses of E(2) (200 ng/L) and EE(2) (100 ng/L) induced hepatic Vtg mRNA and plasma Vtg to levels comparable with the E(2) injections. Larger aqueous exposure doses (2000 ng/L E(2) or 1000 ng/L EE(2)) in the flowthrough experiment resulted in greater responses of hepatic Vtg mRNA and plasma Vtg at 7 days. Constant aqueous exposure to E(2) (2000 ng/L) or EE(2) (1000 ng/L) may thus be more effective than a single large-dose injection (5 mg/kg) to stimulate Vtg gene activation and synthesis.

Contrasting effects of estrogen on transthyretin and vitellogenin expression in males of the marine fish, Sparus aurata.

A partial cDNA encoding for the C-terminus of vitellogenin (VTG) was cloned from liver of Sparus aurata male treated with 17beta-estradiol (E(2)). E(2) treatment of S. aurata males resulted in increased synthesis and secretion of VTG protein into the plasma, determined by a specific enzyme-linked immunosorbent assay (ELISA) in a time-dependent manner. While VTG mRNA was induced by E(2) treatment, transthyretin (TTR) mRNA levels were reduced. These data provide the first demonstration that estrogen exhibits contrasting effect on VTG and on TTR gene expression in teleosts.

Induction of tyrosine hydroxylase by forskolin: modulation with age.

With aging, circulating catecholamines are elevated in both humans and animals. This may be related to the increased basal levels of tyrosine hydroxylase messenger RNA (mRNA) levels and tyrosine hydroxylase enzyme activity in the adrenal medulla of senescent compared with younger animals. In addition, tyrosine hydroxylase gene expression in the senescent rat is resistant to further stimulation by cold exposure as compared with younger animals. Collectively, these observations suggest either that tyrosine hydroxylase expression is already maximally stimulated in senescent rats or that tyrosine hydroxylase gene induction pathways are impaired with senescence. To help distinguish between these possibilities, we examined the induction of tyrosine hydroxylase mRNA, tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase enzyme activity in the adrenal medulla following forskolin administration to young and old F-344 rats. Forskolin at doses of 1.8 and 3.5 mg/kg increased tyrosine hydroxylase mRNA levels 2.5-fold in adrenal medulla from young rats but did not increase either tyrosine hydroxylase immunoreactivity or tyrosine hydroxylase enzyme activity 5 h after administration. Prolonged treatment with forskolin (3 doses, 12 h apart) increased tyrosine hydroxylase mRNA levels and tyrosine hydroxylase immunoreactivity and tyrosine hydroxylase enzyme activity. In senescent rats, the baseline level of tyrosine hydroxylase mRNA was more than 2-fold higher compared with young rats. A single injection of the lower dose of forskolin increased tyrosine hydroxylase mRNA levels by the same increment in senescent as compared with young rats. These data indicate that the tyrosine hydroxylase gene in the adrenal medulla from senescent rats is still capable of further stimulation.

Cooling augments human saphenous vein reactivity to electrical stimulation.

Human saphenous veins were obtained at operation and assayed immediately (n = 10). The veins were cut into rings, suspended in organ chambers, and connected to force transducers for recording of isometric tension. One ring served as control; others were treated with either the alpha 1-adrenoceptor antagonist prazosin (Pz, 3 x 10(-7) M) or the alpha 2-adrenoceptor antagonist rauwolscine (Rw1, 10(-7) M). Cooling from 37 degrees to 24 degrees C had no significant effect on the resting tone of quiescent rings. Electrical stimulation (0.2-16 Hz) caused frequency-dependent contractions in control vessels. The contractions were inhibited by Pz (p < 0.001) and by Rw (p < 0.001). In control rings, cooling potentiated contractions evoked at all frequencies. Similar augmentations were induced by cooling in rings treated with the alpha 1-antagonist Pz. In contrast, rings treated with Rw before being electrically stimulated showed no significant change in contractile force when cooled. The data indicate that in the human saphenous vein, both alpha 1- and alpha 2-adrenoceptors are innervated, contributing to contractile response evoked by neuronal excitation. Cold augments saphenous vein reactivity to endogenously released norepinephrine (NE) by an apparent increase in the responsiveness of alpha 2-adrenoceptors to agonists. This relationship between temperature and adrenoceptor responsiveness is consistent with the hypothesized role of alpha 2-adrenoceptors in cold-induced vasospasm.

Cooling augments alpha 2-adrenoceptor-mediated contractions in rat tail artery.

Experiments were performed to assess the effects of acute moderate cooling on postjunctional alpha 1- and alpha 2-adrenoceptors in isolated rings of tail arteries from male Sprague-Dawley rats. Rings were contracted with norepinephrine (NE; 10(-9) to 10(-4) M) alone or in the presence of prazosin (Pz; 3 x 10(-7) M) or rauwolscine (Rw; 10(-7) M). NE concentration-response curves were inhibited by alpha 1-blockade (Pz) but not significantly affected by alpha 2-blockade (Rw). In all rings, cooling caused an increase in the slope of the dose-response curve and a significant increase in the concentration of agonist required to evoke contractions, as assessed by that concentration of NE required to evoke a contraction equal to 10% of maximal (EC10). Cooling inhibited contractions evoked by the selective alpha 1-adrenergic agonist phenylephrine (PE) as assessed by EC10 but had no significant effect on the weak contractions elicited by the selective alpha 2-adrenergic agonist B-HT 920. Prior elevation of tone with either KCl or prostaglandin F2 alpha enhanced alpha 2-mediated contractions. These contractions were augmented by cooling, whereas those caused by either KCl or prostaglandin F2 alpha alone were not significantly affected. Our results suggest that alpha 2-adrenoceptor-mediated responses in this blood vessel are dependent on the level of preexisting tone and are potentiated by cooling.