Abstract

Treatments fail to eliminate Toxoplasma gondii due to low drug brain penetration. Spiramycin is an Mrp2, Pglycoprotein substrate, active in acute and chronic murine toxoplasmosis. Metronidazole is a CYP3A4 inhibitor and Pglycoprotein substrate. We developed a simple HPLC method to analyze spiramycin and metronidazole simultaneously. Male Balb/c mice were randomly selected in three groups and were dosed orally either 500 mg/kg metronidazole (group 1, n=4) or 400 mg/kg spiramycin (group 2, n=4) or coadministered 500 mg/kg metronidazole 30 min prior to 400 mg/kg spiramycin (group 3, n=4). Mice were euthanized 2 hours after spiramycin administration. Metronidazole and spiramycin brain and plasma concentrations were measured by HPLC using a Phenomenex® C-18 (150x3.8 mm, 5 μm) column and acetonitrile-phosphate buffer (pH 2.5) gradient elution (20/80 to 30/70 in 3 min) at 1 mL/min flow, 29°C and 232 nm. The method was linear (0.25-50.0 μg/mL), the LLOQ was 0.25 μg/mL, intra- and interday variability, precision and accuracy were within 15%. Recoveries were above 75% and there was no matrix interference. Metronidazole eluted at 3 min and spiramycin at 5 min. Spiramycin did not affect plasma metronidazole concentration (6.93±0.48 μg/mL in combination vs. alone 7.65±0.55 μg/mL) or brain (2.96±0.60 μg/g after coadministration vs. control, 4.02±0.78 μg/g). Metronidazole did not change spiramycin plasma concentration (coadministration: 3.94±1.30 μg/mL, control: 3.71±0.94 μg/mL). However, spiramycin brain concentration increased 2-fold after metronidazole coadministration from 2.44±0.33 μg/g to 4.83±1.25 μg/g (P < 0.05). Metronidazole increased spiramycin brain uptake, probably due to P-glycoprotein inhibition, which may improve toxoplasmosis encephalitis treatments.

Keywords: Brain uptake, Drug-drug interaction, Metronidazole, Spiramycin, Toxoplasma gondii, CYP3A4 inhibitor, HPLC, LLOQ, Plasma, Acute murine toxoplasmosis