CYP1A1 and CYP1B1-mediated biotransformation of the antitrypanosomal methamidoxime prodrug DB844 forms novel metabolites through intramolecular rearrangement.

DB844 (CPD-594-12), N-methoxy-6-{5-[4-(N-methoxyamidino)phenyl]-furan-2-yl}-nicotinamidine, is an oral prodrug that has shown promising efficacy in both mouse and monkey models of second stage human African trypanosomiasis. However, gastrointestinal (GI) toxicity was observed with high doses in a vervet monkey safety study. In the current study, we compared the metabolism of DB844 by hepatic and extrahepatic cytochrome P450s to determine whether differences in metabolite formation underlie the observed GI toxicity. DB844 undergoes sequential O-demethylation and N-dehydroxylation in the liver to form the active compound DB820 (CPD-593-12). However, extrahepatic CYP1A1 and CYP1B1 produced two new metabolites, MX and MY. Accurate mass and collision-induced dissociation mass spectrometry analyses of the metabolites supported proposed structures of MX and MY. In addition, MY was confirmed with a synthetic standard and detection of nitric oxide (NO) release when DB844 was incubated with CYP1A1. Taken altogether, we propose that MX is formed by insertion of oxygen into the amidine CN to form an oxaziridine, which is followed by intramolecular rearrangement of the adjacent O-methyl group and subsequent release of NO. The resulting imine ester, MX, is further hydrolyzed to form MY. These findings may contribute to furthering the understanding of toxicities associated with benzamidoxime- and benzmethamidoxime-containing molecules.

Airway drug pharmacokinetics via analysis of exhaled breath condensate.

Although the airway surface is the anatomic target for many lung disease therapies, measuring drug concentrations and activities on these surfaces poses considerable challenges. We tested whether mass spectrometric analysis of exhaled breath condensate (EBC) could be utilized to non-invasively measure airway drug pharmacokinetics and predicted pharmacological activities. Mass spectrometric methods were developed to detect a novel epithelial sodium channel blocker (GS-9411/P-680), two metabolites, a chemically related internal standard, plus naturally occurring solutes including urea as a dilution marker. These methods were then applied to EBC and serum collected from four (Floridian) sheep before, during and after inhalation of nebulized GS-9411/P-680. Electrolyte content of EBC and serum was also assessed as a potential pharmacodynamic marker of drug activity. Airway surface concentrations of drug, metabolites, and electrolytes were calculated from EBC measures using EBC:serum urea based dilution factors. GS-9411/P-680 and its metabolites were quantifiable in the sheep EBC, with peak airway concentrations between 1.9 and 3.4 µM measured 1 h after inhalation. In serum, only Metabolite #1 was quantifiable, with peak concentrations ∼60-fold lower than those in the airway (45 nM at 1 h). EBC electrolyte concentrations suggested a pharmacological effect; but this effect was not statistical significant. Analysis of EBC collected during an inhalation drug study provided a method for quantification of airway drug and metabolites via mass spectrometry. Application of this methodology could provide an important tool in development and testing of drugs for airways diseases.

Acute hyperkalemia associated with inhalation of a potent ENaC antagonist: Phase 1 trial of GS-9411.

BACKGROUND: Inhaled epithelial sodium channel (ENaC) blockers are designed to increase airway surface liquid volume, thereby benefiting cystic fibrosis patients. This study evaluated the safety, tolerability, and pharmacokinetics of multiple doses of ENaC blocker GS-9411, in healthy participants. METHODS: This randomized, double-blind, placebo-controlled, parallel-group, residential, Phase 1 study evaluated inhaled GS-9411 (2.4, 4.8, and 9.6 mg) or placebo, dosed twice daily for 14 days. RESULTS AND CONCLUSIONS: GS-9411 was well tolerated; 86.1% of treated participants completed dosing (n=31/36). Cough and dizziness (27.8% participants each; most of mild severity) were the most commonly reported adverse events and occurred in both placebo and GS-9411 treatment groups. Arrhythmias were not observed for GS-9411-treated participants, and electrocardiographic changes were not considered clinically significant. Serum potassium levels exceeded the upper limit of normal (>5 mmol/L), 4 hr after the morning dose in GS-9411 (n=16/24) and placebo (n=4/12) treatment groups (38 incidences total). Retesting revealed levels had returned to normal within 2-3 hr. In urine electrolyte analyses, obtained 0-6 hr after the Day 1 morning dose, mean sodium/potassium ratios significantly increased from values 0-6 hr before dosing. Increased urine sodium/potassium ratios corresponded with high urine concentrations of active GS-9411 metabolites, which inhibited sodium reabsorption in the kidney, leading to the observed transient hyperkalemia in these participants. Inhaled GS-9411 was well tolerated except for the emergence of transient clinically significant hyperkalemia; this finding resulted in termination of further clinical development of this drug and will necessitate development of a new generation of ENaC blockers, which provide a sustained improvement in mucociliary clearance, while reducing renal exposure to ENaC blockade. Transient increases in mean urine sodium/potassium ratios appeared to be the first signal of electrolyte imbalances resulting from drug-induced block of ENaC in the kidney. The results of this study strongly suggest that clinical trials of novel ENaC blockers will require intensive measurement of plasma and urine electrolyte levels.

An in vitro assay to assess transporter-based cholestatic hepatotoxicity using sandwich-cultured rat hepatocytes.

Drug-induced cholestasis can result from the inhibition of biliary efflux of bile acids in the liver. Drugs may inhibit the hepatic uptake and/or the biliary efflux of bile acids resulting in an increase in serum concentrations. However, it is the intracellular concentration of bile acids that results in hepatotoxicity, and thus serum concentrations may not necessarily be an appropriate indicator of hepatotoxicity. In this study, sandwich-cultured rat hepatocytes were used as an in vitro model to assess the cholestatic potential of drugs using deuterium-labeled sodium taurocholate (d(8)-TCA) as a probe for bile acid transport. Eight drugs were tested as putative inhibitors of d(8)-TCA uptake and efflux. The hepatobiliary disposition of d(8)-TCA in the absence and presence of drugs was measured by using liquid chromatography/tandem mass spectrometry, and the accumulation (hepatocytes and hepatocytes plus bile), biliary excretion index (BEI), and in vitro biliary clearance (Cl(biliary)) were reported. Compounds were classified based on inhibition of uptake, efflux, or a combination of both processes. Cyclosporine A and glyburide showed a decrease in total (hepatocytes plus bile) accumulation, an increase in intracellular (hepatocytes only) accumulation, and a decrease in BEI and Cl(biliary) of d(8)-TCA, suggesting that efflux was primarily affected. Erythromycin estolate, troglitazone, and bosentan resulted in a decrease in accumulation (total and intracellular), BEI, and Cl(biliary) of d(8)-TCA, suggesting that uptake was primarily affected. Determination of a compound's relative effect on bile acid uptake, efflux, and direct determination of alterations in intracellular amounts of bile acids may provide useful mechanistic information on compounds that cause increases in serum bile acids.

Characterization of sandwich-cultured hepatocytes as an in vitro model to assess the hepatobiliary disposition of copper.

Sandwich-cultured hepatocytes (SCH) from rats (SCRH), dogs (SCDH), and humans (SCHH) were used as an in vitro model to assess the hepatobiliary disposition of copper (Cu). The expression of Cu transporters, ceruloplasmin synthesis, Cu uptake, and biliary excretion and species differences in drug-induced alterations in Cu disposition were determined in SCH from all species. Western blot analysis verified basolateral Cu uptake transporter 1 (CTR1) and canalicular Cu efflux transporter (ATP7B) expression: enzyme-linked immunosorbent assay verified synthesis/secretion of ceruloplasmin (major Cu binding protein found in blood). Endogenous Cu in SCRH, SCDH, and SCHH were 17.2 +/- 7.00, 490 +/- 44.8, and 43.5 +/- 15.8 ng/well, respectively. The hepatobiliary disposition of Cu as measured by uptake (increase in intracellular Cu in comparison to endogenous levels) and biliary excretion (increase in Cu in wash solutions obtained from hepatocytes exposed to calcium-free versus standard buffer) was determined as a function of Cu concentration and incubation time. In general, an increase in Cu concentration or incubation time resulted in an increase in Cu uptake and/or biliary excretion; however, the extent to which they affected Cu disposition was species dependent. 5-(1,1-Dioxido-1,2-thiazinan-2-yl)-N-(4-fluorobenzyl)-8-hydroxy-1,6-naphthyridine-7-carboxamide (L-000870810) (an anti-HIV compound, the development of which was halted due to an observed Cu-specific toxicity in the liver and kidneys of dogs after long-term exposure) showed no effect on Cu disposition in SCRH; however, it increased the biliary excretion of Cu in SCDH and SCHH. This is the first report to demonstrate the utility of SCH as a model to assess hepatobiliary disposition of Cu in an in vitro system.

O-alkoxyamidine prodrugs of furamidine: in vitro transport and microsomal metabolism as indicators of in vivo efficacy in a mouse model of Trypanosoma brucei rhodesiense infection.

Five O-alkoxyamidine analogues of the prodrug 2,5-bis[4-methoxyamidinophenyl]furan were synthesized and evaluated against Trypanosoma brucei rhodesiense in the STIB900 mouse model by oral administration. The observed in vivo activity of these prodrugs demonstrates that compounds with an O-methoxyamidine or O-ethoxyamidine group effectively cured all trypanosome-infected mice, whereas prodrugs with larger side-chains did not completely cure the mice. Permeability across Caco-2 cell monolayers and microsomal metabolism were used to identify the underlying mechanisms of prodrug efficacy.

Identification of Mycobacterium marinum virulence genes using signature-tagged mutagenesis and the goldfish model of mycobacterial pathogenesis.

Mycobacterium marinum, a causative agent of fish tuberculosis, is one of the most closely related Mycobacterium species (outside the M. tuberculosis complex) to M. tuberculosis, the etiologic agent of human tuberculosis. Signature-tagged mutagenesis was used to identify genes of M. marinum required for in vivo survival in a goldfish model of mycobacterial pathogenesis. Screening the first 1008 M. marinum mutants led to the identification of 40 putative virulence mutants. DNA sequence analysis of these 40 mutants identified transposon insertions in 35 unique loci. Twenty-eight out of 33 (85%) loci encoding putative virulence genes have homologous genes in M. tuberculosis.

High-throughput screening for stability and inhibitory activity of compounds toward cytochrome P450-mediated metabolism.

With the advent of combinatorial chemistry and high-throughput screening technology, thousands of molecules can now be rapidly synthesized and screened for biological activity against large numbers of protein targets, greatly increasing the speed with which lead compounds are identified during the early stages of drug discovery. However, rapid optimization of parameters that determine whether a high-affinity ligand or a potent inhibitor will become a successful drug remains a challenge in improving the efficiency of the drug discovery process. Parameters that define absorption, distribution, metabolism, and excretion properties of drug candidates are important determinants of therapeutic efficacy, and thus should be optimized during early stages of drug discovery. Although the speed with which drugs are screened for properties such as absorption, cytochrome P450 (CYP) inhibition, and metabolic stability has increased over the past several years, the screening rate/capacity is still several orders of magnitude lower than those for high-throughput methods used in lead identification, resulting in a bottleneck in the drug discovery process. This review discusses current methods used in the in vitro screening of drugs for their stability toward CYP-mediated oxidative metabolism. This is a critical screen in the drug discovery process because metabolism by CYP represents an important clearance mechanism for the vast majority of compounds, thus affecting their oral bioavailability and/or duration of action.