The sheep as a model of preclinical safety and pharmacokinetic evaluations of candidate microbicides.

When developing novel microbicide products for the prevention of HIV infection, the preclinical safety program must evaluate not only the active pharmaceutical ingredient but also the product itself. To that end, we applied several relatively standard toxicology study methodologies to female sheep, incorporating an assessment of the pharmacokinetics, safety, tolerability, and local toxicity of a dapivirine-containing human vaginal ring formulation (Dapivirine Vaginal Ring-004). We performed a 3-month general toxicology study, a preliminary pharmacokinetic study using drug-loaded vaginal gel, and a detailed assessment of the kinetics of dapivirine delivery to plasma, vaginal, and rectal fluid and rectal, vaginal, and cervical tissue over 28 days of exposure and 3 and 7 days after removal of the ring. The findings of the general toxicology study supported the existing data from both preclinical and clinical studies in that there were no signs of toxicity related to dapivirine. In addition, the presence of the physical dapivirine ring did not alter local or systemic toxicity or the pharmacokinetics of dapivirine. Pharmacokinetic studies indicated that the dapivirine ring produced significant vaginal tissue levels of dapivirine. However, no dapivirine was detected in cervical tissue samples using the methods described here. Plasma and vaginal fluid levels were lower than those in previous clinical studies, while there were detectable dapivirine levels in the rectal tissue and fluid. All tissue and fluid levels tailed off rapidly to undetectable levels following removal of the ring. The sheep represents a very useful model for the assessment of the safety and pharmacokinetics of microbicide drug delivery devices, such as the vaginal ring.

7alpha-Methyl-ethinyl estradiol is not a metabolite of tibolone but a chemical stress artifact.

OBJECTIVE: This study was conducted to establish whether 7alpha-methyl-ethinyl estradiol (7alpha-MEE) in plasma from postmenopausal women treated with tibolone is a metabolite or an artifact. DESIGN: Clinical samples with known levels of tibolone metabolites, plus plasma samples spiked with tibolone and metabolites, were analyzed for levels of 7alpha-MEE using liquid chromatography-mass spectometry (LC-MS/MS) with and without derivatization. RESULTS: Approximately 20 to 40 pg/mL 7alpha-MEE was detected using LC-MS/MS with derivatization in plasma samples from postmenopausal women treated with tibolone. In plasma samples spiked with 200 ng/mL tibolone or Delta-tibolone, LC-MS/MS with derivatization revealed the generation of around 200 and 36 pg/mL 7alpha-MEE, respectively, whereas LC-MS/MS without derivatization showed no detectable chemical conversion of tibolone to 7alpha-MEE. Generation of 7alpha-MEE is increased by the "stress conditions" used in the derivatization procedure; simply drying the sample also shows this artifactual conversion. The major active and sulfated 3-hydroxy metabolites of tibolone are not converted to 7alpha-MEE. Without derivatization, and avoiding stress conditions, no detectable levels (<20 pg/mL) of 7alpha-MEE were found in plasma samples from postmenopausal women treated with single (eight participants at 13 time points) or multiple (seven participants at 18 time points) doses of tibolone. CONCLUSIONS: 7alpha-MEE is not a metabolite of tibolone but is a chemical artifact generated during analytical procedures with derivatization. Using LC-MS/MS without derivatization, 7alpha-MEE cannot be demonstrated in plasma from postmenopausal women after single or multiple doses of tibolone.