Central administration of a cytochrome P450-7B product 7 alpha-hydroxypregnenolone improves spatial memory retention in cognitively impaired aged rats.

Pregnenolone (PREG) and dehydroepiandrosterone (DHEA) have been reported to improve memory in aged rodents. In brain, these neurosteroids are transformed predominantly into 7alpha-hydroxylated metabolites by the cytochrome P450-7B1 (CYP7B). The biological role of steroid B-ring hydroxylation is unclear. It has been proposed to generate bioactive derivatives that enhance cognition, immune, and other physiological processes. In support, 7alpha-hydroxylated DHEA increases the immune response in mice with greater potency than the parent steroid. Whether the memory-enhancing effects of PREG in rats is mediated via its 7alpha-hydroxylated metabolite 7alpha-hydroxyPREG is not known. We investigated this by treating memory-impaired aged rats (identified by their spatial memory performances in the Morris water maze task compared with young controls) with 7alpha-hydroxyPREG or PREG administered intracerebroventricularly using osmotic minipumps and then tested the rats during week 2 of steroid treatment in the eight-arm radial-arm version of the water maze (RAWM) that allows repeated assessment of learning. CYP7B bioactivity in hippocampal tissue (percentage conversion of [14C]DHEA to [14C]7alpha-hydroxyDHEA) was decreased selectively in memory-impaired aged rats compared with both young and memory-intact aged rats. 7alpha-hydroxyPREG (100 ng/h) but not PREG (100 ng/h) administration to memory-impaired aged rats for 11 d enhanced spatial memory retention (after a 30 min delay between an exposure trial 1 and test trial 2) in the RAWM. These data provide evidence for a biologically active enzyme product 7alpha-hydroxyPREG and suggests that reduced CYP7B function in the hippocampus of memory-impaired aged rats may, in part, be overcome by administration of 7alpha-hydroxyPREG.

Bile acid synthesis in man. In vivo activity of the 25-hydroxylation pathway.

During biosynthesis of bile acid, carbons 25-26-27 are removed from the cholesterol side-chain. Side-chain oxidation begins either with hydroxylation at the 26-position, in which case the three-carbon fragment is released as propionic acid, or with hydroxylation at the 25-position, in which case the three-carbon fragment is released as acetone. We have previously shown in the rat that the contribution of the 25-hydroxylation pathway can be quantitated in vivo by measuring production of [14C]acetone from [14C]26-cholesterol. In the present study, we adapted this method to human subjects. 4 d after oral administration of 100 microCi of [14C]26-cholesterol and 1 d after beginning a constant infusion of 16.6 mumol/min unlabeled acetone, three men and two women underwent breath collections. Expired acetone was trapped and purified as the 2,4 dinitrophenylhydrazine derivative. 14CO2 was trapped quantitatively using phenethylamine. Specific activity of breath acetone was multiplied by the acetone infusion rate to calculate production of [14C]acetone. [14C]Acetone production averaged 4.9% of total release of 14C from [14C]26-cholesterol, estimated by 14CO2 output. The method was validated by showing that [14C]acetone production from [14C]isopropanol averaged 86.9% of the [14C]-isopropanol infusion rate. We conclude that in man, as in the rat, the 25-hydroxylation pathway accounts for less than 5% of bile acid synthesis.

Clinical findings with the oral contraceptive combination ethinylestradiol/dienogest in the Czech Republic.

The efficacy and safety of the low dose monophasic oral contraceptive (OC) combination containing 30 micrograms of ethinylestradiol (EE) and 2.0 mg of dienogest (DNG) (EE/DNG) was evaluated in a prospective, open-label, multicenter, uncontrolled, phase III trial. The trial was carried out in six hospitals by 36 investigators in the Czech Republic, and included 557 healthy women (aged 18-35 years), over 12 cycles, with a total of 6051 cycles. EE/DNG provided a reliable ovulation inhibition. The contraceptive efficacy study showed an adjusted Pearl index of 0.198 on the basis of three pregnancies occurring during 6051 cycles. EE/DNG provided good cycle control, reduced the incidence of intermenstrual bleedings, the intensity of menstrual bleeding and frequency of dysmenorrhea. Due to the antiandrogenic properties of the progestogen component DNG, EE/DNG improved androgen-related conditions, such as skin blemishes, hair greasiness and acne vulgaris. From 108 women with acne, 62 (57%) improved after the 6th cycle, and 16 (15%) were healed. Similar changes were found after cycle 12. Breast tenderness and headache were the most frequent of the common complaints due to treatment with EE/DNG. The frequency of all complaints decreased steadily over time. Only 7.7% of subjects discontinued due to adverse reactions. No thrombophlebitic events were noticed.

Pharmacokinetics of 1,25(OH)(2)D(3) and 1alpha(OH)D(3) in normal and uraemic men.

BACKGROUND: The therapeutic equivalence of 1,25(OH)(2)D(3) and 1alpha(OH)D(3) on the suppression of PTH synthesis and secretion has not clearly been established. The aim of the present study was to evaluate the pharmacokinetics of 1,25(OH)(2)D(3) and 1alpha(OH)D(3) after oral and i.v. administration in healthy volunteers and uraemic patients. METHODS: Six healthy volunteers and 12 uraemic patients were included in the study. With an interval of 2 weeks, 4 microg of 1,25(OH)(2)D(3) i.v., 4 microg of 1,25(OH)(2)D(3) orally, 4 microg of 1alpha(OH)D(3) i.v. and 4 microg of 1alpha(OH)D(3) orally were administered. Blood samples for analysis of plasma-Ca(2+), plasma-1,25(OH)(2)D(3), and plasma-PTH were drawn at time 0, 0.25, 0.5, 1, 2, 4, 6, 9, 12, 24, 48, and 72 h. The healthy volunteers were studied in all four protocols and the uraemic patients in either the 1alpha(OH)D(3) (n=6) or the 1,25(OH)(2)D(3) (n=6) protocol. RESULTS: After oral administration of 1,25(OH)(2)D(3) the bioavailability of 1,25(OH)(2)D(3) was 70.6+/-5.8/72.2+/-4.8% in healthy volunteers/uraemic patients (n.s.). After i.v. administration the volume of distribution of 1,25(OH)(2)D(3) was similar, 0.49+/-0.14 vs 0.27+/-0.06 l/kg in healthy volunteers vs uraemic patients (n.s.), while the metabolic clearance rate of 1,25(OH)(2)D(3) was 57% lower in the uraemic patients, 23.5+/-4.34 vs 10.1+/-1.35 ml/min in healthy volunteers vs uraemic patients, respectively (P<0.03). The bioavailability of 1,25(OH)(2)D(3) after i.v. administration of 1alpha(OH)D(3) was 42.4+/-11.0/42.0+/-2.0% in healthy volunteers/uraemic patients (n.s.); and after oral administration of 1alpha(OH)D(3) 42.0+/-2.0/29.8+/-3.1% in healthy volunteers/uraemic patients (n.s.). A small, but significant increase in plasma-Ca(2+) was seen after administration of 1,25(OH)(2)D(3) to the uraemic patients, while no increase was seen after administration of 1alpha(OH)D(3). PTH levels were significantly suppressed in the healthy volunteers 24 h after administration of 4 microg of 1,25(OH)(2)D(3) i.v., 4 microg of 1,25(OH)(2)D(3) orally, and 4 microg of 1alpha(OH)D(3) orally by 35+/-7, 30+/-8, and 35+/-4%, respectively (all P<0.03). In the uraemic patients, PTH levels were significantly suppressed after administration of 4 microg of 1,25(OH)(2)D(3) i.v., 4 microg of 1,25(OH)(2)D(3) orally, and 4 microg of 1alpha(OH)D(3) i.v. by 30+/-10, 45+/-7, and 40+/-7%, respectively (all P<0.04). The effect was transitory in the healthy volunteers and lasted for at least 72 h in the uraemic patients. CONCLUSION: The present study found a 57% lower metabolic clearance rate of 1,25(OH)(2)D(3) in uraemic patients, as compared with that of healthy volunteers (P<0.03). The bioavailability of 1,25(OH)(2)D(3) following administration of 1alpha(OH)D(3) i.v. and orally in both healthy volunteers and uraemic patients was markedly lower than after administration of oral 1,25(OH)(2)D(3) (P<0.03). In spite of lower plasma-1,25(OH)(2)D(3) levels after administration of 1alpha(OH)D(3), no significant difference was observed on the suppressive effect of 4 microg i.v. of either 1,25(OH)(2)D(3) or 1alpha(OH)D(3) on the plasma-PTH levels in the uraemic patients. This might suggest the existence of an effect of 1alpha(OH)D(3) on the parathyroid glands which is independent of the plasma-1,25(OH)(2)D(3) levels, that are achieved after oral or i.v. administration of 1alpha(OH)D(3).

Liposomal 1,25 (OH)2 vitamin D3 compounds block proliferation and induce differentiation in myelomonocytic leukaemia cells.

The vitamin D3 derived hormone 1.25 (OH)2 vitamin D3 (1,25 D3) is able to induce growth arrest and differentiation in myelomonocytic leukaemia cells. In order to allow for specific delivery to leukaemic cells the lipophilic compound was incorporated into the lipid membranes of liposomes. Liposomal 1.25 D3 reduced proliferation as measured by 3H-thymidine incorporation in HL60 leukaemia cells by up to 60%. When liposomes were prepared at different concentrations of 1,25 D3 65% inhibition was achieved at 48 nM. The MC 1288 stereoisomer of 1,25 D3 was more potent and had the same activity at 4.8 nM. The effect of the liposomal compounds was specific to myeloid cells as they reduced proliferation in myelomonocytic HL60, monoblastic U937 and monocytic Mono Mac 6 cells but not in the T-cell lines Jurkat and Molt 4. The antiproliferative effect of liposomal 1,25 D3 was associated with an induction of differentiation since treated HL60 cells showed a monocytic morphology, increased expression of CD14 and decreased expression of CD33. When peripheral blood leukaemic cells from M4 and M5 acute myeloid leukaemia (AML) patients were admixed with liposomal compounds an antiproliferative effect was seen in all five cases, including the two cases where free compounds led to enhanced growth. Liposomal delivery of 1,25 (OH)2 vitamin D3 may offer a novel approach to treatment of myelomonocytic leukaemia.

Is cytochrome P450 2C9 genotype associated with NSAID gastric ulceration?

AIMS: The aim of this study was to explore whether genetic variation of cytochrome P450 2C9 (CYP2C9) contributes to NSAID-associated gastric ulceration. The hypothesis tested was that CYP2C9 poor metabolizer genotype would predict higher risk of gastric ulceration in patients on NSAIDs that are metabolized by CYP2C9, due to higher plasma NSAID concentrations. METHODS: Peripheral blood DNA samples from 23 people with a history of gastric ulceration attributed to NSAIDs metabolized by CYP2C9, and from 32 people on NSAIDs without gastropathy, were analysed to determine CYP2C9 genotype. RESULTS: The following genotypes were found: *1/*1 (wild type) in 70% of cases and 58% of controls, *1/*2 in 17% of cases and 29% of controls, *1/*3 in 13% of cases and 13% of controls. The difference between case and control nonwild-type genotype frequency was 11.5% (95% CI -14,37%), with the direction of the difference being against the hypothesis. No individuals with homozygote poor metaboliser genotype were identified. The differences in genotype frequencies between the two groups were not significant and the frequencies were similar to those in a large published population study. Ninety-five percent binomial confidence interval analysis confirms that there is no apparent clinically significant relationship between CYP2C9 genotype and risk of gastric ulceration although a small difference in risk in poor metabolizers cannot be excluded. CONCLUSIONS: These results do not support the hypothesis that gastric ulceration resulting from NSAID usage is linked to the poor metabolizing genotypes of CYP2C9.