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.

Clinically relevant anterograde amnesia and its relationship with blood levels of benzodiazepines in suicide attempters who took an overdose.

The relationship between anterograde amnesia, sedation and plasma levels of benzodiazepines was studied prospectively in a group of 24 patients who took an overdose of benzodiazepines. Patients were tested on two sequential days after having taken an overdose. Anterograde amnesia was tested by using a verbal recall test and a photo recognition test. Sedation was scored on a visual analogue scale (VAS) by the patient and the interviewer. The concentration of benzodiazepines in plasma was measured by using a radioreceptor assay that adds benzodiazepines and their active metabolites. The cumulative amount of benzodiazepines was expressed as diazepam equivalents (DZE). Diazepam equivalents determined by this radioreceptor assay were significantly higher on the first day than on the second day. Ratings on the verbal recall test were significantly lower on the first day than on the second day. There was a significant relation between decrease of diazepam equivalents and increase of verbal recall: more than 30% of increase of verbal recall was explained by decrease of diazepam equivalents. There was not a strong relation between decrease of diazepam equivalents and reduction of level of sedation as scored by the patients. There was almost no relation between decrease of diazepam equivalents and reduction of level of sedation as scored by the interviewer. No relation was found between verbal recall, sedation and diazepam equivalents. There was no relation between diazepam equivalents and photo recognition. It was concluded that anterograde amnesia was strongly associated with benzodiazepines in patients who take benzodiazepines in an overdose. Sedation does not predict the degree of anterograde amnesia.

Development and validation of fluorescent receptor assays based on the human recombinant estrogen receptor subtypes alpha and beta.

This article describes the development and validation of two fluorescent receptor assays for the hRec-estrogen receptor subtypes alpha and beta. As a labelled ligand an autofluorescent phyto-estrogen (coumestrol) has been used. The estrogen receptor (ER) belongs to the nuclear receptor family, a class of soluble DNA binding proteins, mainly present in the cytoplasm of the cell, that act as ligand-activated enhancer factors. It consists of two different forms, expressed as ER-alpha (66 kDa) and ER-beta (59 kDa). The ER-alpha is mainly located in the uterus and the ER-beta can be found in vascular tissue. Detection and identification of compounds having estrogenic effects is of importance in drug discovery programmes within the pharmaceutical industry for their search for ER-subtype selective (ant)agonists which may prove to be of therapeutic value in treating a variety of estrogen-linked pathologies (breast cancer, osteoporosis, cardiovascular disease, type II diabetes and Alzheimer disease). Furthermore, interactions of (xeno-)estrogens with the endogenous hormonal system of the exposed organism can affect embryos, gonads, and reproductive behaviour. The latter can eventually lead to reduced reproduction and deterioration of a population. For that reason, monitoring of (xeno-)estrogens in food products and in the environment, attracts considerable attention by health councils throughout the world. The following characteristics were obtained for the human recombinant (hRec) estrogen receptor-beta assay, which is suitable for ER subtype selective drug-discovery purposes (IC50 values for 17-beta-estradiol and genistein were 5.1 nM and 25 nM, respectively): goodness of fit (R2) was always > 0.98 (x = 0.9933, n = 10). LLOQ of the assay is typically > or = 500 picomolar, whereas the ULOQ of the assay is < or = 20.0 nanomolar. For the hRec-estrogen receptor-alpha assay, which is suitable for monitoring of (xeno-)estrogens (IC50 values for 17-beta-estradiol and genistein were 0.68 nM and 65 nM, respectively) the following characteristics were obtained: goodness of fit (R2) was always > 0.96 (x = 0.9838, n = 10). LLOQ of the assay is typically > or = 200 picomolar, whereas the ULOQ of the assay is < or = 5.0 nanomolar.

Determination of the benzodiazepine plasma concentrations in suicidal patients using a radioreceptor assay.

Impairments in memory and psychomotor function appear to be induced by benzodiazepines not only after long-term use, but also after administration of a single dose. Because it is known on which neurotransmitter system the benzodiazepines exert their action, the use of a quantitative radioreceptor assay (RRA) can be a useful tool in studying the interrelationship between the neurochemical and memory processes. The RRA measures the sum of the main compound(s) and all active metabolites present, where it relates the biological activity to the pharmacodynamic effect instead of relating it to the plasma levels of the individual compounds. To correlate the loss of memory with the benzodiazepine concentration, plasma concentrations were determined in suicidal patients. From suicidal patients (n = 84), the benzodiazepines in plasma were measured with a direct radioreceptor assay using tritiated flunitrazepam as the labelled ligand. The receptor material was a lyophilized preparation from calf cortex. Furthermore, the samples were subjected to high-performance liquid chromatographic (HPLC) analysis, and the HPLC data were converted to diazepam equivalents using cross-reactivities of the individual compounds. Patients who had ethanol residues in their plasma were excluded from this correlation experiment. The data (n = 40) obtained with the two analytical techniques were compared and correlated to assess the validity of the radioreceptor assay in establishing the relationship between the loss of memory and the total amount of benzodiazepines present. The cumulative amount of diazepam determined with the RRA and the sum of compounds determined with the HPLC method, after correction using the cross-reactivities, were plotted and correlated using regression analysis. Regression analysis showed an x variable of 0.75 and a correlation coefficient of 0.67. The intercept was not significantly different from zero (P = 0.49, t-test), whereas the slope was significantly different from zero (P < 0.01). Benzodiazepines can be directly determined in plasma using this radioreceptor assay. The data obtained from HPLC analysis were easily converted to diazepam equivalents using the cross-reactivities. A discrepancy between the data obtained from the two analytical techniques, however, indicates that certain metabolites are present, which were not quantitated in the HPLC analysis, but were measured in the radioreceptor assay. Therefore, the radioreceptor assay proved to be a valuable tool for the assessment of clinical effects, such as the demonstration of the loss of memory in suicidal patients after a benzodiazepine overdose.