Application of monoclonal antibodies to measure metabolism of an anti-trypanosomal compound in vitro and in vivo.

Human African trypanosomiasis (HAT), also called African sleeping sickness, is a neglected tropical parasitic disease indigenous to sub-Saharan Africa. Diamidine compounds, including pentamidine and CPD-0801, are potent anti-trypanosomal molecules. The latter is a potential drug in the development at the UNC based Consortium for Parasitic Drug Development. An orally bioavailable prodrug of CPD-0801, DB868, is metabolized primarily in the liver to the active form. A monoclonal antibody developed against a pentamidine derivative has shown significant reactivity with CPD-0801 (EC(50) 65.1 nM), but not with the prodrug (EC(50)>18,000 nM). An inhibitory enzyme-linked immunosorbent assay (IELISA) has been used to quantitatively monitor prodrug metabolism by detecting the production of the active compound over time in a sandwich culture rat hepatocyte system and in rats. These results were compared with the results of the standard LC/MS/MS assay. Spearman coefficients of 0.96 and 0.933 (in vitro and in vivo, respectively) indicate a high correlation between these two measurement methods. This novel IELISA provides a facile, inexpensive, and accurate method for drug detection that may aide in elucidating the mechanisms of action and toxicity of existing and future diamidine compounds.

Identification of a cranberry juice product that inhibits enteric CYP3A-mediated first-pass metabolism in humans.

An in vivo study in rats showed a cranberry juice product to inhibit the intestinal first-pass metabolism of the CYP3A substrate nifedipine. However, a clinical study involving the CYP3A probe substrate midazolam and a different cranberry juice product showed no interaction. Because the composition of bioactive components in natural products can vary substantially, a systematic in vitro-in vivo approach was taken to identify a cranberry juice capable of inhibiting enteric CYP3A in humans. First, the effects of five cranberry juices, coded A through E, were evaluated on midazolam 1'-hydroxylation activity in human intestinal microsomes. Juice E was the most potent, ablating activity at 0.5% juice (v/v) relative to control. Second, juice E was fractionated to generate hexane-, chloroform-, butanol-, and aqueous-soluble fractions. The hexane- and chloroform-soluble fractions at 50 microg/ml were the most potent, inhibiting by 77 and 63%, respectively, suggesting that the CYP3A inhibitors reside largely in these more lipophilic fractions. Finally, juice E was evaluated on the oral pharmacokinetics of midazolam in 16 healthy volunteers. Relative to water, juice E significantly increased the geometric mean area under the curve (AUC)(0-infinity) of midazolam by approximately 30% (p=0.001), decreased the geometric mean 1'-hydroxymidazolam/midazolam AUC(0-infinity) ratio by approximately 40% (p<0.001), and had no effect on geometric mean terminal half-life, indicating inhibition of enteric, but not hepatic, CYP3A-mediated first-pass metabolism of midazolam. This approach both showed a potential drug interaction liability with cranberry juice and substantiated that rigorous in vitro characterization of dietary substances is required before initiation of clinical drug-diet interaction studies.

Validation of a high-throughput absorption, distribution, metabolism, and excretion (ADME) system and results for 60 literature compounds.

A high-throughput analytical system for the support of absorption, distribution, metabolism, and excretion (ADME) was constructed in collaboration with Gubbs Inc. to commercialize corresponding software. We sought to quickly build and validate this system for microsomal clearance assessment using 60 commercially available non-proprietary compounds that are non-DEA-restricted in addition to 36 proprietary Millennium compounds that had already been assessed using a low-throughput infrastructure. The system was constructed such that a approximately 45 second total cycle time was achieved injection-to-injection. Software was successfully coded to enable the analyst to submit multiple batches, and modify multiple methods very quickly for use with Applied Biosystems Analyst 1.3. After acquisition the software was used to simultaneously integrate multiple injection chromatograms, regress the data, and calculate clearance such that all of the data could be easily and immediately reviewed by both bioanalytical and enzymology personnel. Unfortunately, despite an exhaustive search of the literature, we were unable to find a large number of non-proprietary compound data for validation, so we provide such a source of data here. Results are presented for the 60 literature compounds that were assessed. A good correlation was observed between literature results for 16 compounds that we were able to find and the results obtained using the system. The Millennium proprietary compounds that we assessed using both low- and high-throughput approaches also correlated well. We present here a system for the support of high-throughput in vitro ADME analysis and also present the results of 60 non-proprietary, non-DEA-scheduled compounds to facilitate the validation efforts of others. Finally, we present commercially available software to facilitate high-throughput ADME systems in the community.