HPLC-MS/MS-based quantification of human monoclonal antibodies targeting SARS-CoV-2 in the presence of endogenous SARS-CoV-2 antibodies in human serum.

Antibodies for treatment and prophylaxis against SARS-CoV-2 are needed particularly for immunocompromised individuals, who cannot adequately benefit from vaccination. To address this need, Aerium Therapeutics is developing antibodies targeting the SARS-CoV-2 spike protein. A bioanalytical method to quantify fully human monoclonal antibodies in a population with widely varying anti-spike antibody titers is required to investigate the pharmacokinetics of these antibodies in clinical trials. To eliminate interference from endogenous anti-spike protein antibodies, an HPLC-MS/MS assay was developed to quantify the investigational monoclonal antibodies (AER001 and AER002) by targeting signature peptides spanning the monoclonal antibodies' CDR regions. By optimizing and comparing affinity capture and ammonium sulphate precipitation, it was demonstrated that both procedures allowed accurate and precise quantification of AER001 and AER002 in human serum with comparable sensitivity. Ammonium sulphate precipitation outperformed immunocapture due to its simplicity and speed at lower cost and a full bioanalytical method validation was performed in human serum. The assay was also validated for human nasal lining fluid extract with a 50-fold lower limit of quantification and was shown to deliver similar sensitivity to previously published affinity capture HPLC-MS/MS assays. Finally, the CDR-derived signature peptides were also generated by tryptic digestion of blank serum in some individuals, an important caveat for HPLC-MS/MS strategies targeting human monoclonal antibodies. In summary, the presented results show that ammonium sulphate precipitation and HPLC-MS/MS allow accurate and precise quantification of monoclonals in clinical studies. The developed methods demonstrate that HPLC-MS/MS can reliably quantify human monoclonal antibodies even when endogenous antibodies with overlapping specificities are present and are crucial for the clinical testing of two investigational COVID-19 monoclonals.

Preclinical 28-day inhalation toxicity assessment of s-nitrosoglutathione in beagle dogs and Wistar rats.

To support clinical development of S-nitrosoglutathione (GSNO) as a therapeutic agent, 28-day toxicology studies in rats and dogs were conducted. Rats (21-25/sex) and dogs (3-5/sex) were exposed for 4 hours or 1 hour, respectively, to inhaled GSNO (0, 3, 9.3, 19, and 28 mg/kg per d in rats and 0, 4.6, 9.0, and 16.2 mg/kg per d in dogs) or vehicle daily via a nebulizer. Animals were monitored throughout the 28-day dosing period and during a postexposure recovery period. Complete necropsy and tissue examinations were performed. Experimental end points included clinical pathology, toxicokinetics, and immunotoxicology. No biologically significant adverse findings were noted in either species, and the no observed adverse effect levels (NOAELs) under these conditions were the highest achieved doses (28 and 16.2 mg/kg per d in rats and dogs, respectively). These data demonstrate that GSNO is well tolerated in rodents and dogs and predict a favorable toxicity profile in humans, thus supporting future clinical development of GSNO or closely related compounds.

Genotoxicity assessment of the antiepileptic drug AMP397, an Ames-positive aromatic nitro compound.

AMP397 is a novel antiepileptic agent and the first competitive AMPA antagonist with high receptor affinity, good in vivo potency, and oral activity. AMP397 has a structural alert (aromatic nitro group) and was mutagenic in Salmonella typhimurium strains TA97a, TA98 and TA100 without S9, but negative in the nitroreductase-deficient strains TA98NR and TA100NR. The amino derivative of AMP397 was negative in wild-type strains TA98 and TA100. AMP397 was negative in a mouse lymphoma tk assay, which included a 24h treatment without S9. A weak micronucleus induction in vitro was found at the highest concentrations tested in V79 cells with S9. AMP397 was negative in the following in vivo studies, which included the maximum tolerated doses of 320mg/kg in mice and 2000mg/kg in rats: MutaMouse assay in colon and liver (5x320mg/kg) at three sampling times (3, 7 and 31 days after the last administration); DNA binding study in the liver of mice and rats after a single treatment with [14C]-AMP397; comet assay (1x2000mg/kg) in jejunum and liver of rats, sampling times 3 and 24h after administration; micronucleus test (2x320mg/kg) in the bone marrow of mice, sampling 24h after the second administration. Based on these results, it was concluded that AMP397 has no genotoxic potential in vivo. In particular, no genotoxic metabolite is formed in mammalian cells, and, if formed by intestinal bacteria, is unable to exert any genotoxic activity in the adjacent intestinal tissue. These data were considered to provide sufficient safety to initiate clinical development of the compound.

First Steps Towards an Understanding of a Mode ofCarcinogenic Action for Vanadium Pentoxide.

Inhalation of vanadium pentoxide clearly increases the incidence of alveolar/bronchiolar neoplasms in male and female B6C3F1 mice at all concentrations tested (1, 2 or 4 mg/m(3)), whereas responses in F344/N rats was, at most, ambiguous. While vanadium pentoxide is mutagenic in vitro and possibly in vivo in mice, this does not explain the species or site specificity of the neoplastic response. A nose-only inhalation study was conducted in female B6C3F1 mice (0, 0.25, 1 and 4 mg/m(3), 6 h/day for 16 days) to explore histopathological, biochemical (α-tocopherol, glutathione and F2-isoprostane) and genetic (comet assays and 9 specific DNA-oxo-adducts) changes in the lungs. No treatment related histopathology was observed at 0.25 mg/m(3). At 1 and 4 mg/m(3), exposure-dependent increases were observed in lung weight, alveolar histiocytosis, sub-acute alveolitis and/or granulocytic infiltration and a generally time-dependent increased cell proliferation rate of histiocytes. Glutathione was slightly increased, whereas there were no consistent changes in α-tocopherol or 8-isoprostane F2α. There was no evidence for DNA strand breakage in lung or BAL cells, but there was an increase in 8-oxodGuo DNA lesions that could have been due to vanadium pentoxide induction of the lesions or inhibition of repair of spontaneous lesions. Thus, earlier reports of histopathological changes in the lungs after inhalation of vanadium pentoxide were confirmed, but no evidence has yet emerged for a genotoxic mode of action. Evidence is weak for oxidative stress playing any role in lung carcinogenesis at the lowest effective concentrations of vanadium pentoxide.