Developmental safety of nirmatrelvir in zebrafish (Danio rerio) embryos

Abstract

Background Nirmatrelvir, in combination with ritonavir, is one of the first orally available antiviral treatment for coronavirus disease 2019 (COVID‐19). Symptomatic pregnant women are at increased risk for severe illness and complications that can affect the developing baby. No malformations or lower embryo‐fetal survival have been observed when nirmatrelvir were administered to pregnant rats and rabbits. Safety evaluation of drugs used for treating COVID‐19 also in pregnancy is urgent for public health, then in this study we further investigated nirmatrelvir developmental toxicity using zebrafish as in vivo model. Material and Methods Using the standardized Fish Embryo Toxicity (FET) test, we first determined the lethal concentration 50 (LC50), exposing embryos from gastrula stage up to 120 hr post fertilization (hpf) and daily recording lethality. Then, we exposed embryos to five doses comprising the human therapeutic one and up to the LC50 (25 μM). Morphology was evaluated at 72 and 120 hpf. Results Nirmatrelvir did not affect survival rate and did not induce morphological defects up to the human therapeutic dose. Exposure at higher doses (2.4× and 3× the human Cmax) however resulted in decreased hatching rate, reduced growth, slower heartbeat with pericardial edema, reduction of eye dimension, absence of the swim bladder and disruption of the anterior–posterior axis, with lack of tail detachment, spinal curvature and straight and smaller head. Conclusions Our findings in zebrafish embryos add further information about developmental nirmatrelvir safety. Further studies are needed for pharmacological safety assessment of nirmatrelvir exposure during pregnancy.

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

“…ngn1 is expressed in neuronal precursors at the very onset of neurogenesis in the zebrafish neural plate prior to neural lineage determination, and it is crucial for cellular differentiation in primary neurons that control embryo motility, dopaminergic neuron progenitors of the forebrain and progenitors of sensory neurons of the dorsal root ganglia [52]. Fish were bred in a recirculating aquaculture system (Techniplast ZebTEC, Buguggiate, Italy) in fish water (0.1 g/L Instant Ocean Sea Salts, 0.1 g/L sodium bicarbonate, 0.19 g/L calcium sulfate) at 28.5 • C in a 14 h light and 10 h dark daily cycle, essentially as previously described [75]. The fish were fed three times per day with dry granular feed (GEMMA Micro Skretting, Tooele, UT, USA) and one time per day with fresh Artemia.…”

“…As developmental indicators, we considered gross morphological changes like alterations in the head regions (particularly main brain areas like forebrain, midbrain, hindbrain, ventricle regions, midbrain-hindbrain boundary length and eye diameter), tail morphology, maintenance of a correct anterior-posterior (AP) axis or its deformation and normal growth or growth delay (body length). Body length, eye diameter and midbrain-hindbrain boundary length measurements for control and 1 µM DTG-exposed embryos were performed on digital images using the ImageJ Fiji software (https://imagej.nih.gov/ij/, accessed on 13 February 2024), as previously described [75] and considering 10 embryos for each experimental condition and for each of three experiments (a total of 30 embryos). The phenotype of the embryos after drug exposure at the blastula stage was then classified as normal, mild or severe, based on the abnormality of the developmental morphological endpoints that are described in Table 1.…”

Adverse outcomes in SARS-CoV-2–infected pregnant mice are gestational age–dependent and resolve with antiviral treatment

“…To date, published animal models of SARS-CoV-2 infection during pregnancy have been limited ( 25 , 41 ), which has hindered investigation into both host and viral factors that may underlie the severe outcomes observed in humans. Animal models have only been used to study the potential reproductive toxicity of nirmatrelvir in rats, rabbits, and zebrafish, with no evidence of embryonic toxicity, fetal abnormalities, maternal toxicity, or other adverse outcomes ( 42 , 43 ). Whether equivalent dosing of nirmatrelvir administered during pregnancy is equally efficacious against SARS-CoV-2 infection in pregnant as in nonpregnant animals has not been reported.…”

Developmental toxicity of the emerging contaminant cyclophosphamide and the integrated biomarker response (IBRv2) in zebrafish

“…23,24 Thus, zebrash embryos represent an excellent non-invasive 3Rs (reduction, replacement and renement) model to assess the eco-toxicity of pharmaceutical drugs. 25 Hatching, malformation, and the heart rate of zebrash embryos are routinely used physiological endpoints in ecotoxicity assessments. 26 Acetylcholinesterase (AChE) is a neurotransmitter which plays an important role in neural system-related activities in zebrash.…”

“…23,24 Thus, zebrafish embryos represent an excellent non-invasive 3Rs (reduction, replacement and refinement) model to assess the eco-toxicity of pharmaceutical drugs. 25…”