Development of a Robust Manufacturing Route for Molnupiravir, an Antiviral for the Treatment of COVID-19.

Herein is described the development of a large-scale manufacturing process for molnupiravir, an orally dosed antiviral that was recently demonstrated to be efficacious for the treatment of patients with COVID-19. The yield, robustness, and efficiency of each of the five steps were improved, ultimately culminating in a 1.6-fold improvement in overall yield and a dramatic increase in the overall throughput compared to the baseline process.

Expression of Phox2b by brainstem neurons involved in chemosensory integration in the adult rat.

Central congenital hypoventilation syndrome is caused by mutations of the gene that encodes the transcription factor Phox2b. The syndrome is characterized by a severe form of sleep apnea attributed to greatly compromised central and peripheral chemoreflexes. In this study, we analyze whether Phox2b expression in the brainstem respiratory network is preferentially associated with neurons involved in chemosensory integration in rats. At the very rostral end of the ventral respiratory column (VRC), Phox2b was present in many VGlut2 (vesicular glutamate transporter 2) mRNA-containing neurons. These neurons were functionally identified as the respiratory chemoreceptors of the retrotrapezoid nucleus (RTN). More caudally in the VRC, many fewer neurons expressed Phox2b. These cells were not part of the central respiratory pattern generator (CPG), because they were typically cholinergic visceral motor neurons or catecholaminergic neurons (presumed C1 neurons). Phox2b was not detected in serotonergic neurons, in the A5, A6, and A7 noradrenergic cell groups nor within the main cardiorespiratory centers of the dorsolateral pons. Phox2b was expressed by many solitary tract nucleus (NTS) neurons including those that relay peripheral chemoreceptor information to the RTN. These and previous observations by others suggest that Phox2b is expressed by an uninterrupted chain of neurons involved in the integration of peripheral and central chemoreception (carotid bodies, chemoreceptor afferents, chemoresponsive NTS neurons projecting to VRC, RTN chemoreceptors). The presence of Phox2b in this circuit and its apparent absence from the respiratory CPG could explain why Phox2b mutations disrupt breathing automaticity during sleep without causing major impairment of respiration during waking.

Afferent and efferent connections of the rat retrotrapezoid nucleus.

The rat retrotrapezoid nucleus (RTN) contains candidate central chemoreceptors that have extensive dendrites within the marginal layer (ML). This study describes the axonal projections of RTN neurons and their probable synaptic inputs. The ML showed a dense plexus of nerve terminals immunoreactive (ir) for markers of glutamatergic (vesicular glutamate transporters VGLUT1-3), gamma-aminobutyric acid (GABA)-ergic, adrenergic, serotonergic, cholinergic, and peptidergic transmission. The density of VGLUT3-ir terminals tracked the location of RTN chemoreceptors. The efferent and afferent projections of RTN were studied by placing small iontophoretic injections of anterograde (biotinylated dextran amine; BDA) and retrograde (cholera toxin B) tracers where RTN chemoreceptors have been previously recorded. BDA did not label the nearby C1 cells. BDA-ir varicosities were found in the solitary tract nucleus (NTS), all ventral respiratory column (VRC) subdivisions, A5 noradrenergic area, parabrachial complex, and spinal cord. In each target region, a large percentage of the BDA-ir varicosities was VGLUT2-ir (41-83%). Putative afferent input to RTN originated from spinal cord, caudal NTS, area postrema, VRC, dorsolateral pons, raphe nuclei, lateral hypothalamus, central amygdala, and insular cortex. The results suggest that 1) whether or not the ML is specialized for CO(2) sensing, its complex neuropil likely regulates the activity of RTN chemosensitive neurons; 2) the catecholaminergic, cholinergic, and serotonergic innervation of RTN represents a possible substrate for the known state-dependent control of RTN chemoreceptors; 3) VGLUT3-ir terminals are a probable marker of RTN; and 4) the chemosensitive neurons of RTN may provide a chemical drive to multiple respiratory outflows, insofar as RTN innervates the entire VRC.