Development Of A Therapeutic Approach Of Chronic Toxoplasmosis In Mouse Model

Abstract

Background: Toxoplasma gondii is an obligate intracellular parasite. Its infective tachyzoites can evade the healthy host’s immune system by forming cysts in the host’s tissues, particularly the brain. The bradyzoites in the cysts remain dormant in brain, but may re-activate as tachyzoites if the host’s immunity is compromised. Re- activated cysts lead to acute toxoplasmic encephalitis, a deadly complication in these patients. The current treatment regime of toxoplasmosis still fail to eliminate T. gondii from the infected host, mainly due to low drug concentration attained in the brain. Spiramycin is a macrolide antibiotic [P-glycoprotein substrate, P-gp], tested effective against acute and chronic murine toxoplasmosis. Metronidazole is a nitro-imidazole antibiotic which inhibits the P450 [CYP] 3A4 & P-glycoprotein.

Objectives: The aim of this study is to see the effectiveness of spiramycin in combination with a P-gp inhibitor (metronidazole) to enhance the drug delivery and uptake in brain of an animal model and to determine its efficacy in an animal model with chronic toxoplasmosis.

Methods: A HPLC method was developed to determine and quantify spiramycin and metronidazole simultaneously. A Phenomenex C-18 [150 x 3.8 mm, 5 μm] column and acetonitrile-phosphate buffer [pH 2.5] with a gradient elution [20/ 80 to 30/ 70 in 3 min] at 1 mL/ min flow, 29°C and 232 nm was used for the study. In order to study the in vivo interactions between both the drugs and to establish the pharmacokinetic profile of spiramycin in plasma and brain, three groups of male Balb/c mice were dosed with only metronidazole 500 mg/ kg, only spiramycin 400 mg/ kg and spiramycin 400 mg/ kg co-administered with metronidazole 500 mg/ kg respectively. All the drugs were given orally. The animals were euthanized at pre-determined time points; 0.5, 1.0, 2.0, 2.5, 4.0, 6.0, 8.0, 10.0 and 12.0 hours. Brain and blood samples were obtained, processed and subjected to HPLC analysis. The pharmacokinetic parameters were calculated using non-compartmental techniques. In order to determine the onset and development of chronic toxoplasmosis, the male Balb/c mice were challenged with tachyzoites of T. gondii [ME 49 Strain]. The survival and disease state conversion were analyzed. At selected time-point based on the survival study, the mice were treated either with spiramycin 400 mg/ kg or metronidazole 500 mg/ kg or spiramycin 400 mg/ kg co-administered with metronidazole 500 mg/ kg, including infected untreated and positive control groups. The treatment efficacy was determined by microscopic counting of brain cysts and cysts cultured in vitro.

Results: The HPLC method was linear [0.25– 50.0 μg/ mL], the LLOQ was 0.25 μg/ mL, intra- and inter-day 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. In plasma, metronidazole did not affect the pharmacokinetics of spiramycin. In brain, metronidazole had more profound effects on the bio-distribution of spiramycin to the brain with a 1.7-fold increase in CMAX [p<0.05] and the AUC0–, was 64% greater [p<0.001]. Brain cysts developed two weeks post-acute infection [865 ± 86 cysts] and reached plateau phase at four weeks [1448 ± 81 cysts]. Reactivation of cysts in vitro was seen after one week of incubation and the bradyzoites-tachyzoites conversion was not influenced by the age of the brain cysts. In the study on treatment efficacy, the co- administration of spiramycin with metronidazole, showed a 15-fold and 10-fold reduction of brain cysts [79  23] with reference to the control group and spiramycin treated group respectively. The enhanced brain spiramycin concentration is probably due to the inhibition of P-glycoprotein transporter at the blood brain barrier by metronidazole, which facilitates the uptake of spiramycin into the brain.

Conclusion: The combination of spiramycin and metronidazole was more effective than spiramycin alone in reducing brain cysts and cysts in vitro. This synergistic effect of drug interaction may have clinical translatability in the treatment and prevention of chronic toxoplasmosis.