ABSTRACT
Type II transmembrane serine proteases represent pharmacological targets for blocking entry and spread of influenza or coronaviruses. In this study, the depletion rates of the 3-amidinophenylalanine (3-APhA)-derived matriptase/TMPRSS2 inhibitors MI-463, MI-472, MI-485 or MI-1900 were determined by LC-MS/MS measurements over a period of 300 min using suspensions of rat, dog and cynomolgus monkey primary hepatocytes. From these in vitro pharmacokinetic (PK) experiments, intrinsic clearance values (Clint) were evaluated, and in vivo pharmacokinetic parameters (hepatic clearance, hepatic extraction ratio and bioavailability) were predicted. It was found that rat hepatocytes were the most active in the metabolism of 3-APhA derivatives (Clint 31.9-37.8 mL/min/kg), whereas dog and monkey cells displayed somewhat lower clearance of these compounds (Clint 6.6-26.7 mL/min/kg). These data support elucidation of important PK properties of anti-TMPRSS2/anti-matriptase 3-APhAs using mammalian hepatocyte models and thus contribute to the optimization of lead compounds.
ABSTRACT
Although the new coronavirus disease 2019 (COVID-19) outbreak occurred in late 2019, it is still endemic worldwide, and has become a global public health problem. Vaccination against SARS-CoV-2 is considered to be the most effective intervention to prevent the spread of COVID-19. ZF2001 is a recombinant protein vaccine based on SARS-CoV-2 receptor-binding domain (RBD) subunit which contains aluminum adjuvant. In order to advance our research on ZF2001 into clinical trial, we investigated the general toxicity and immunogenicity of ZF2001 in cynomolgus monkeys and assessed the possible target organs for vaccine-induced toxicity. In the present research, we observed no significant systemic toxicities and abnormal cardiovascular and respiratory events following four times injections of intramuscular ZF2001 in cynomolgus monkeys. Histological examination revealed recoverable inflammatory changes in quadricep muscle and adjacent lymph node at the vaccine injection site. As expected, the vaccine can produce a strongly specific binding antibody and neutralizing antibodies in cynomolgus monkeys after inoculation. Taken together, our regulatory toxicology research proves the safety and immunogenicity of the ZF2001 vaccine, supporting its entry into large scale clinical trials.
ABSTRACT
In vitro models of animals vulnerable to SARS-CoV-2 infection can support the characterization of effective antiviral drugs, such as synthetic inhibitors of the transmembrane protease serine 2 (TMPRSS2). Changes in cytochrome P450 (CYP) 1A2 activities in the presence of the potential TMPRSS2/matriptase inhibitors (MI) were measured using fluorometric and luminescent assays. Furthermore, the cytotoxicity of these inhibitors was evaluated using the MTS method. In addition, 60 min-long microsomal stability assays were performed using an UPLC-MS/MS procedure to elucidate depletion rates of the inhibitors. CYP1A2 was influenced significantly by MI-463 and MI-1900 in rat microsomes, by MI-432 and MI-482 in beagle microsomes, and by MI-432, MI-463, MI-482, and MI-1900 in cynomolgus monkey microsomes. The IC50 values in monkey microsomes were 1.30 ± 0.14 µM, 2.4 ± 1.4 µM, 0.21 ± 0.09 µM, and 1.1 ± 0.8 µM for inhibitors MI-432, MI-463, MI-482, and MI-1900, respectively. The depletion rates of the parent compounds were lower than 50%, independently of the investigated animal species. The host cell factor TMPRSS2 is of key importance for the cross-species spread of SARS-CoV-2. Studies of the in vitro biotransformation of TMPRSS2 inhibitors provide additional information for the development of new antiviral drugs.