Dolutegravir and Folic Acid Interaction during Neural System Development in Zebrafish Embryos.

Dolutegravir (DTG) is one of the most prescribed antiretroviral drugs for treating people with HIV infection, including women of child-bearing potential or pregnant. Nonetheless, neuropsychiatric symptoms are frequently reported. Early reports suggested that, probably in relation to folic acid (FA) shortage, DTG may induce neural tube defects in infants born to women taking the drug during pregnancy. Subsequent reports did not definitively confirm these findings. Recent studies in animal models have highlighted the association between DTG exposure in utero and congenital anomalies, and an increased risk of neurologic abnormalities in children exposed during in utero life has been reported. Underlying mechanisms for DTG-related neurologic symptoms and congenital anomalies are not fully understood. We aimed to deepen our knowledge on the neurodevelopmental effects of DTG exposure and further explore the protective role of FA by the use of zebrafish embryos. We treated embryos at 4 and up to 144 h post fertilization (hpf) with a subtherapeutic DTG concentration (1 µM) and observed the disruption of the anterior-posterior axis and several morphological malformations in the developing brain that were both prevented by pre-exposure (2 hpf) and rescued by post-exposure (10 hpf) with FA. By whole-mount in situ hybridization with riboprobes for genes that are crucial during the early phases of neurodevelopment (ntl, pax2a, ngn1, neurod1) and by in vivo visualization of the transgenic Tg(ngn1:EGFP) zebrafish line, we found that DTG induced severe neurodevelopmental defects over time in most regions of the nervous system (notochord, midbrain-hindbrain boundary, eye, forebrain, midbrain, hindbrain, spinal cord) that were mostly but not completely rescued by FA supplementation. Of note, we observed the disruption of ngn1 expression in the dopaminergic regions of the developing forebrain, spinal cord neurons and spinal motor neuron projections, with the depletion of the tyrosine hydroxylase (TH)+ dopaminergic neurons of the dorsal diencephalon and the strong reduction in larvae locomotion. Our study further supports previous evidence that DTG can interfere with FA pathways in the developing brain but also provides new insights regarding the mechanisms involved in the increased risk of DTG-associated fetal neurodevelopmental defects and adverse neurologic outcomes in in utero exposed children, suggesting the impairment of dopaminergic pathways.

Comparison of Efavirenz and Doravirine Developmental Toxicity in an Embryo Animal Model.

In the past, one of the most widely used non-nucleoside reverse transcriptase inhibitors (NNRTI) in first-line antiretroviral therapy (ART) of HIV infection was efavirenz (EFV), which is already used as a cost-effective treatment in developing countries due to its efficacy, tolerability, and availability. However, EFV also demonstrates several adverse effects, like hepatotoxicity, altered lipid profile, neuropsychological symptoms, and behavioral effects in children after in utero exposure. In 2018, another NNRTI, doravirine (DOR), was approved due to its similar efficacy but better safety profile. Preclinical safety studies demonstrated that DOR is not genotoxic and exhibits no developmental toxicity or effects on fertility in rats. Zebrafish (Danio rerio) embryos have been widely accepted as a vertebrate model for pharmacological and developmental studies. We used zebrafish embryos as an in vivo model to investigate the developmental toxicity of DOR compared to EFV. After exposure of the embryos to the drugs from the gastrula stage up to different developmental stages (30 embryos for each arm, in three independent experiments), we assessed their survival, morphology, hatching rate, apoptosis in the developing head, locomotion behavior, vasculature development, and neutral lipid distribution. Overall, DOR showed a better safety profile than EFV in our model. Therapeutic and supra-therapeutic doses of DOR induced very low mortality [survival rates: 92, 90, 88, 88, and 81% at 1, 5, 10, 25, and 50 µM, respectively, at 24 h post fecundation (hpf), and 88, 85, 88, 89, and 75% at the same doses, respectively, at 48 hpf] and mild morphological alterations compared to EFV exposure also in the sub-therapeutic ranges (survival rates: 80, 77, 69, 63, and 44% at 1, 5, 10, 25, and 50 µM, respectively, at 24 hpf and 72, 70, 63, 52, and 0% at the same doses, respectively, at 48 hpf). Further, DOR only slightly affected the hatching rate at supra-therapeutic doses (97, 98, 96, 87, and 83% at 1, 5, 10, 25, and 50 µM, respectively, at 72 hpf), while EFV already strongly reduced hatching at sub-therapeutic doses (83, 49, 11, 0, and 0% at 1, 5, 10, 25, and 50 µM, respectively, at the same time endpoint). Both DOR at therapeutic doses and most severely EFV at sub-therapeutic doses enhanced apoptosis in the developing head during crucial phases of embryo neurodevelopment and perturbed the locomotor behavior. Furthermore, EFV strongly affected angiogenesis and disturbed neutral lipid homeostasis even at sub-therapeutic doses compared to DOR at therapeutic concentrations. Our findings in zebrafish embryos add further data confirming the higher safety of DOR with respect to EFV regarding embryo development, neurogenesis, angiogenesis, and lipid metabolism. Further studies are needed to explore the molecular mechanisms underlying the better pharmacological safety profile of DOR, and further human studies are required to confirm these results in the zebrafish animal model.

Can maternal treatment with metformin during gestation and lactation cause metabolic and cardiovascular disorders in rat offspring?

Objective: The aim was to evaluate if maternal treatment with metformin (MET) during pregnancy and lactation could be safe for metabolic and cardiovascular parameters of adult male and female offspring.Materials and methods: Wistar female rats were treated with MET (293 mg/kg/d) or tap water, by gavage during gestation (METG or CTRG) or gestation and lactation (METGL or CTRGL).Results: At 75 days of life, male and female MET offspring presented similar blood pressure when compared with their CTR. The heart rate of female METGL was higher than in the CTRGL. The insulin sensitivity, basal glycaemia, body weight, Lee index of obesity, plasmatic concentration of triglycerides, total cholesterol and fat acid of male and female MET were similar to CTR groups. Lower fat pad deposition was observed in female METG and METGL.Conclusion: MET exposure during gestational and lactation does not program cardiovascular and metabolic alterations in adult offspring life.

Revision of the ICH guideline on detection of toxicity to reproduction for medicinal products: SWOT analysis.

SWOT analysis was used to gain insights and perspectives into the revision of the ICH S5(R2) guideline on detection of toxicity to reproduction for medicinal products. The current ICH guideline was rapidly adopted worldwide and has an excellent safety record for more than 20 years. The revised guideline should aim to further improve reproductive and developmental (DART) safety testing for new drugs. Alternative methods to animal experiments should be used whenever possible. Modern technology should be used to obtain high quality data from fewer animals. Additions to the guideline should include considerations on the following: limit dose setting, maternal toxicity, biopharmaceuticals, vaccines, testing strategies by indication, developmental immunotoxicity, and male-mediated developmental toxicity. Emerging issues, such as epigenetics and the microbiome, will most likely pose challenges to DART testing in the future. It is hoped that the new guideline will be adopted even outside the ICH regions.