Evaluation of novel biomarkers of nephrotoxicity in Cynomolgus monkeys treated with gentamicin.

Most studies to evaluate kidney safety biomarkers have been performed in rats. This study was conducted in Cynomolgus monkeys in order to evaluate the potential usefulness of novel biomarkers of nephrotoxicity in this species. Groups of 3 males were given daily intramuscular injections of gentamicin, a nephrotoxic agent known to produce lesions in proximal tubules, at dose-levels of 10, 25, or 50mg/kg/day for 10days. Blood and 16-h urine samples were collected on Days -7, -3, 2, 4, 7, and at the end of the dosing period. Several novel kidney safety biomarkers were evaluated, with single- and multiplex immunoassays and in immunoprecipitation-LC/MS assays, in parallel to histopathology and conventional clinical pathology parameters. Treatment with gentamicin induced a dose-dependent increase in kidney tubular cell degeneration/necrosis, ranging from minimal to mild severity at 10mg/kg/day, moderate at 25mg/kg/day, and to severe at 50mg/kg/day. The results showed that the novel urinary biomarkers, microalbumin, α1-microglobulin, clusterin, and osteopontin, together with the more traditional clinical pathology parameters, urinary total protein and N-acetyl-ß-D-glucosaminidase (NAG), were more sensitive than blood urea nitrogen (BUN) and serum creatinine (sCr) to detect kidney injury in the monkeys given 10mg/kg/day gentamicin for 10days, a dose leading to an exposure which is slightly higher than the desired therapeutic exposure in clinics. Therefore, these urinary biomarkers represent non-invasive biomarkers of proximal tubule injury in Cynomolgus monkeys which may be potentially useful in humans.

Dried Blood Spot Methodology in Combination With Liquid Chromatography/Tandem Mass Spectrometry Facilitates the Monitoring of Teriflunomide.

BACKGROUND: Teriflunomide, a once-daily oral immunomodulator approved for treatment of relapsing-remitting multiple sclerosis, is eliminated slowly from plasma. If necessary to rapidly lower plasma concentrations of teriflunomide, an accelerated elimination procedure using cholestyramine or activated charcoal may be used. The current bioanalytical assay for determination of plasma teriflunomide concentration requires laboratory facilities for blood centrifugation and plasma storage. An alternative method, with potential for greater convenience, is dried blood spot (DBS) methodology. Analytical and clinical validations are required to switch from plasma to DBS (finger-prick sampling) methodology. METHODS: Using blood samples from healthy subjects, an LC-MS/MS assay method for quantification of teriflunomide in DBS over a range of 0.01-10 mcg/mL was developed and validated for specificity, selectivity, accuracy, precision, reproducibility, and stability. Results were compared with those from the current plasma assay for determination of plasma teriflunomide concentration. RESULTS: Method was specific and selective relative to endogenous compounds, with process efficiency ∼88%, and no matrix effect. Inaccuracy and imprecision for intraday and interday analyses were <15% at all concentrations tested. Quantification of teriflunomide in DBS assay was not affected by blood deposit volume and punch position within spot, and hematocrit level had a limited but acceptable effect on measurement accuracy. Teriflunomide was stable for at least 4 months at room temperature, and for at least 24 hours at 37°C with and without 95% relative humidity, to cover sampling, drying, and shipment conditions in the field. The correlation between DBS and plasma concentrations (R = 0.97), with an average blood to plasma ratio of 0.59, was concentration independent and constant over time. CONCLUSIONS: DBS sampling is a simple and practical method for monitoring teriflunomide concentrations.

Pharmacokinetics of ferroquine, a novel 4-aminoquinoline, in asymptomatic carriers of Plasmodium falciparum infections.

Ferroquine (SSR97193), a ferrocene-quinoline conjugate, is a promising novel antimalarial currently undergoing clinical evaluation. This study characterizes its pharmacokinetic properties. Young male African volunteers with asymptomatic Plasmodium falciparum infection were administered a single oral dose (n = 40) or a repeated oral dose (n = 26) given over 3 days of ferroquine in two dose-escalation, double-blind, randomized, placebo-controlled clinical trials. In addition, a food interaction study was performed in a subsample of participants (n = 16). The studies were carried out in Lambaréné, Gabon. After single-dose administration of ferroquine, dose linearity was demonstrated in a dose range of 400 to 1,200 mg for maximum mean blood concentrations ([C(max)] 82 to 270 ng/ml) and in a dose range of 400 to 1,600 mg for overall exposure to ferroquine (area under the concentration-time curve [AUC], 13,100 to 49,200 ng · h/ml). Overall mean estimate for blood apparent terminal half-life of ferroquine was 16 days and 31 days for its active and major metabolite desmethylferroquine (SSR97213). In the 3-day repeated-dose study, C(max) and overall cumulated exposure to ferroquine (AUC(cum)) increased in proportion with the dose from day 1 to day 3 between 400 and 800 mg. No major food effect on ferroquine pharmacokinetics was observed after single administration of 100 mg of ferroquine except for a slight delay of time to maximum blood concentration (t(max)) by approximately 3 h. The pharmacokinetics of ferroquine and its active main metabolite are characterized by sustained levels in blood, and the properties of ferroquine as a partner drug in antimalarial combination therapy should be evaluated.

A DBS method for quantitation of the new oral trypanocidal drug fexinidazole and its active metabolites.

AIM: Fexinidazole (FEX) is a nitroimidazole being developed as a new trypanocide treatment for human African trypanosomiasis/sleeping sickness. Its main metabolites, fexinidazole sulfoxide (M1) and fexinidazole sulfone (M2), show the same in vitro pharmacological activity as FEX. METHODS & RESULTS: An LC-MS/MS assay was developed for quantitation of FEX in DBS, collected via finger-prick from healthy subjects. The DBS assay was specific, accurate and reproducible for FEX, M1 and M2 when validated against the current plasma assay. DBS samples were stable for 24 h at 37°C with 95% relative humidity, and 58 weeks desiccated at room temperature. CONCLUSION: DBS finger-prick sampling offers a simple, practical method for determining FEX, M1 and M2 concentrations in clinical studies in Africa.

Heparin-like glycosaminoglycan/amine salt-bridge interactions: a new potential tool for HLGAGs analysis using mass spectrometry.

Characterization of glycosaminoglycans poses a challenge for current analytical techniques, as they are highly acidic, polydisperse and heterogeneous compounds. The purpose of this study is the separation and analysis of a partially depolymerized heparin-like glycosaminoglycan by on-line ion-pairing reversed-phase high-performance liquid chromatography/electrospray mass spectrometry. The gas-phase behavior of two synthesized glycosaminoglycans has been investigated. Dibutylamine was found to be the best suited ion-pairing reagents for mass spectrometry analysis. The optimized ion-pairing conditions provide reproducible and easily interpretable electrospray mass spectra in both negative and positive ESI modes. The glycosaminoglycans are detected as a non-covalent complex with amines. In fact, the observed ionic species and their gas-phase dissociation under CID conditions revealed the presence of salt bridge interactions in the gas phase.

Bioavailability of a co-formulated combination of amodiaquine and artesunate under fed and fasted conditions. A randomised, open-label crossover study.

OBJECTIVES: To assess the effect of food on the pharmacokinetics of the anti-malarial amodiaquine (AQ, CAS 6398-98-7) and artesunate (AS, CAS 182824-33-5) and their active metabolites [desethylamodiaquine (DSA, CAS 81496-81-3) and dihydroartemisinin (DHA, CAS79352-78-6), respectively] in healthy volunteers. METHODS: In an open, two-way crossover study, 22 healthy male volunteers fasted overnight and were randomised to receive a single oral administration of 4 tablets of a fixed-dose combination containing 135 mg AQ and 50 mg AS in the absence or presence of a standardised high-fat breakfast, administered 30 min before drug administration. Blood samples were collected up to Day 10 and AQ, DSA, AS and DHA were assayed by an LC/MS/MS method. RESULTS: Relative to the fasting state, the administration of the fixed-dose combination after a high-fat breakfast resulted in delayed median Tmax values for AQ (15 min and for DSA (2.3 h). The geometric mean ratios (GMR) of fed to fasting conditions indicated increased Cmax values for AQ (GMR 1.22) (90% CI: 1.07-1.39) and DSA (GMR 1.21) (90% CI: 1.05-1.39) and increased AUC0-t values for AQ (GMR 1.59) (90% CI: 1.39-1.83) and for DSA (GMR 1.13) (90% CI: 1.04-1.24). The median Tmax values were not delayed for AS as opposed to DHA (approximately 1 h delay). The Cmax values were decreased for AS (GMR 0.36) (90% CI: 0.30-0.47) and for DHA (GMR 0.51) (90% CI: 0.44-0.60). The AUC0-t values were slightly decreased for AS (GMR 0.89) (90% CI: 0.74-1.06) and for DHA (GMR 0.93) (90% CI: 0.84-1.02). CONCLUSION: Intake of AQ and AS with a high fat meal resulted in (1) a statistically significant increase in blood levels of AQ and DSA which may affect the safety and tolerability of the study drugs and (2) a decrease in AS and DHA blood levels which may affect efficacy. These results suggest that the fixed-dose combination should not be administered with a high-fat meal.