Pretreatment of rhesus monkeys with transdermal patches containing physostigmine and procyclidine: implications of the delivery system for the potential application against VX nerve agent intoxication in humans.

Physostigmine (Phs) is a reversible inhibitor of acetylcholinesterase (AChE) that penetrates the blood-brain barrier (BBB) and could be used to protect the central nervous system (CNS) against the effects of nerve agents. For prophylactic effectiveness, long, steady, and adequate inhibition of AChE activity by Phs is needed to broadly protect against the CNS effects of nerve agents. Here, we evaluated the efficacy of transdermal patches containing Phs and procyclidine (PC) as prophylactic agents. Patches (25 cm2) containing 4.4 mg Phs and 17.8 mg PC had a protective ratio of approximately 78.6-fold in rhesus monkeys challenged with VX nerve agent and given an antidote. Physiologically based pharmacokinetic model in conjunction with an indirect pharmacodynamic (PBPK/PD) was developed for Phs and scaled to rhesus monkeys. The model was able to reproduce the concentration profile and inhibitory effect on AChE of Phs in monkeys, as evidenced by correlation coefficients of 0.994 and 0.992 for 25 cm2 and 49 cm2 patches, respectively (i.e., kinetic data), and 0.989 and 0.968 for 25 cm2 and 49 cm2 patches, respectively (i.e., dynamic data). By extending the monkey PBPK/ PD model to humans, the effective human dose was predicted to be five applications of a 25 cm2 patch (i.e., 22 mg Phs), and two applications of a 49 cm2 patch (i.e., 17.4 mg Phs). Therefore, given that patch application of Phs in rhesus monkeys has a prolonged effect (namely, AChE inhibition of 19.6% for the 25 cm2 patch and 23.0% for the 49 cm2 patch) for up to 216 h, patch formulation of Phs may provide similar protection against nerve agent intoxication in humans.

Visualization of a novel human monoclonal antibody against Claudin-3 for targeting ovarian cancer.

INTRODUCTION: Claudin-3 (CLDN3), a tight junction protein, regulates cell-to-cell interactions in epithelial or endothelial cell sheets. During tumorigenesis, epithelial cells are transformed, and tumor cells proliferate through out-of-plane division, resulting in external exposure of CLDN3. Since alterations of CLDN3 expression are associated with cancer progression and higher CLDN3 expression is observed in most ovarian cancers, we tested the feasibility of using a CLDN3-specific antibody as a novel imaging tracer. MATERIALS AND METHODS: After reducing the CLDN3-specific antibodies to expose the -SH groups, click chemistry was used to conjugate the radioactive isotope 111In or the fluorescent protein FNR648. Human ovarian cancer OVCAR-3 and glioblastoma U87MG cells were used as CLDN3-positive and -negative cells. Flow cytometry was used to determine the CLDN3 IgG1 monoclonal antibody binding to both cell lines. OVCAR-3 cells were injected subcutaneously into mice to establish a xenograft model. 111In-labeled CLDN3 antibodies (370 kBq/50 µL) were administered intravenously into mice. After 24 h, organs, including tumors, were excised and measured with a γ-counter. Images were acquired with the IVIS optical imaging system and SPECT/CT. RESULTS: The labeling efficiency of NOTA-111In and antibody-NOTA-111In was 98.52% and 100%, respectively. FNR648-labeled CLDN3 antibody bound to the cell surface of OVCAR-3 and U87MG with 83.4% and 5.7% specificity, respectively. In OVCAR-3 tumor xenografted mice, CLDN3 IgG1 antibody showed a 2.5-fold higher tumor uptake (20.4 ± 7.4% ID/g) than human IgG1 (8.8 ± 2.6% ID/g) at 24 h post injection. The CLDN3 antibody fluorescence signal in the tumor peaked at 24 h post injection. CONCLUSION: We have successfully conjugated a radioisotope and a fluorescent protein with CLDN3-specific antibodies and verified the specific binding of labeled antibodies to OVCAR-3 tumors in a mouse model. Our data suggested that CLDN3-specific human monoclonal antibodies could be used as a useful theranostic tracer.