Post-artificial insemination supplementation with calcium salts of soybean oil influences pregnancy establishment factors in beef cows.

The objective of this experiment was to compare hormonal, uterine, and conceptus factors associated with pregnancy establishment in beef cows supplemented or not with Ca salts of soybean oil (CSSO) for 21 d beginning after timed AI. One hundred lactating multiparous Nelore cows were allocated to 20 groups of 5 cows/group and timed inseminated on d 0 of the experiment. After AI, groups were randomly assigned to receive (as-fed basis) 100 g of protein-mineral mix + 100 g of ground corn per cow per day, in addition to 1) 100 g/cow daily of CSSO ( = 10) or 2) 100 g/cow daily of kaolin (CON; rumen-inert indigestible substance; = 10). Groups were maintained in 4 pastures (5 groups from the same treatment within each pasture) with ad libitum access to forage. Groups were segregated daily and individually offered treatments from d 0 to 21. Blood samples were collected and transrectal ultrasonography was performed to verify ovulation and corpus luteum (CL) volume immediately before AI (d 0) and on d 7 and 15. After ultrasonography on d 15, 60 cows (30 cows/treatment and 3 cows/group) diagnosed without the presence of a CL on d 0 but with a CL greater than 0.38 cm3 in volume on d 7 and 15 were assigned to conceptus collection via transcervical flushing with PBS followed by endometrial biopsy in the uterine horn ipsilateral to the CL. Additional blood samples were collected for whole-blood RNA extraction (d 20), and pregnancy status was verified by transrectal ultrasonography (d 30) in cows not assigned to conceptus collection. Cows receiving CSSO had greater ( ≤ 0.04) mean plasma linoleic acid concentration, plasma linoleic:linolenic acid ratio, plasma progesterone (P4) concentration, and CL volume during the experiment compared with CON cows. Moreover, CSSO supplementation increased ( ≤ 0.04) length and mRNA expression of and by the conceptus as well as blood mRNA expression of interferon-stimulated genes on d 20 in gestating cows. No treatment differences were detected ( ≥ 0.30) for endometrial mRNA expression of and . In summary, post-AI CSSO supplementation to B. indicus beef cows increased plasma concentration of linoleic acid and enhanced pregnancy establishment factors, which included CL development and plasma P4 concentration, conceptus growth, and mRNA expression of as well as blood mRNA expression of interferon-stimulated genes.

Effects of different supplemental soya bean oil levels on the performance of prepubertal Saanen goats: Oestrogen and progesterone release.

The aim of this study was to investigate the effects of different levels of soya bean oil in the total diet on the growth rate, metabolic changes, and oestrogen and progesterone release in Saanen goats. After dietary adaptation, 21 prepubertal goats (weight of 29.12 ± 0.91 kg, 230 days old) were randomly distributed among three diets of D2: inclusion of 2% soya bean oil in the total diet; D3: basal diet - inclusion of 3% soya bean oil in the total diet; and D4: inclusion of 4% soya bean oil in the total diet. The basal diet (D3) was formulated to promote a daily gain of 0.140 kg. The goats were weighed, and their blood samples were collected weekly. Glucose, cholesterol, triglycerides, total protein, urea, non-esterified fatty acids, beta-hydroxybutyrate, oestrogen and progesterone in the plasma were measured. Prepubertal goats that were fed D4 exhibited a significantly lower dry matter intake, urea and cholesterol levels compared with the goats that were fed D2 and D3. Indeed, goats that were fed D4 displayed a significantly lower final weight than goats that were fed D2 and D3. In contrast, the inclusion of soya bean oil in the diet increased the progesterone and oestrogen concentrations, and goats that were fed D4 released a significantly higher concentration of progesterone than those that were fed D2 and D3. Furthermore, the percentage of goats with a progesterone level greater than 1 ng/ml (functional Corpus luteum) was significantly higher among the goats that were fed D3 and D4 than among those that were fed D2. In this study, although the inclusion of 4% soya bean oil in the diet decreased dry matter intake and growth rate, it increased progesterone concentration and the percentage of goats with a functional Corpus luteum, suggesting that the inclusion of soya bean oil accelerated puberty in prepubertal goats.

The potential for CYP2D6 inhibition screening using a novel scintillation proximity assay-based approach.

High throughput inhibition screens for human cytochrome P450s (CYPs) are being used in preclinical drug metabolism to support drug discovery programs. The versatility of scintillation proximity assay (SPA) technology has enabled the development of a homogeneous high throughput assay for cytochrome P450 2D6 (CYP2D6) inhibition screen using [O-methyl-(14)C]dextromethorphan as substrate. The basis of the assay was the trapping of the O-demethylation product, [(14)C]HCHO, on SPA beads. Enzyme kinetics parameters V(max) and apparent K(m), determined using pooled human liver microsomes and microsomes from baculovirus cells coexpressing human CYP2D6 and NADPH-cytochrome P450 reductase, were 245 pmol [(14)C]HCHO/min/mg protein and 11 microM, and 27 pmol [(14)C]HCHO/min/pmol and 1.6 microM, respectively. In incubations containing either pooled microsomes or recombinant CYP2D6, [(14)C]dextromethorphan O-demethylase activity was inhibited in the presence of quinidine (IC(50) = 1.0 microM and 20 nM, respectively). By comparison, inhibitors selective for other CYP isoforms were relatively weak (IC(50) > 25 microM). In agreement, a selective CYP2D6 inhibitory monoclonal antibody caused greater than 90% inhibition of [(14)C]dextromethorphan O-demethylase activity in human liver microsomes, whereas CYP2C9/19- and CYP3A4/5-selective antibodies elicited a minimal inhibitory effect. SPA-based [(14)C]dextromethorphan O-demethylase activity was also shown to correlate (r(2) = 0.6) with dextromethorphan O-demethylase measured by high-performance liquid chromatography in a bank of human liver microsomes (N = 15 different organ donors). In a series of known CYP2D6 inhibitors/substrates, the SPA-based assay resolved potent inhibitors (IC(50) < 2 microM) from weak inhibitors (IC(50) >or= 20 microM). It is concluded that the SPA-based assay described herein is suitable for CYP2D6 inhibition screening using either native human liver microsomes or cDNA-expressed CYP2D6.

Screening of drug candidates for their drug--drug interaction potential.

Within the past year, additional papers have been published that focus on higher-throughput drug-interaction screening. Some papers have described enzyme assays that can be used to evaluate inhibition or induction of the human cytochrome P450s. At the same time, numerous investigators have developed computational (in silico) methods to predict interactions and have validated the approach using in vitro (assay-derived) data. These so called 'in silico--in vitro' correlations have great potential and may complement existing 'in vitro--in vivo' correlations.

Role of human liver cytochrome P4503A in the metabolism of etoricoxib, a novel cyclooxygenase-2 selective inhibitor.

Etoricoxib, a potent and selective cyclooxygenase-2 inhibitor, was shown to be metabolized via 6'-methylhydroxylation (M2 formation) when incubated with NADPH-fortified human liver microsomes. In agreement with in vivo data, 1'-N'-oxidation was a relatively minor pathway. Over the etoricoxib concentration range studied (1-1300 microM), the rate of hydroxylation conformed to saturable Michaelis-Menten kinetics (apparent K(m) = 186 +/- 84.3 microM; V(max) = 0.76 +/- 0.45 nmol/min/mg of protein; mean +/- S.D., n = 3 livers) and yielded a V(max)/K(m) ratio of 2.4 to 7.3 microl/min/mg. This in vitro V(max)/K(m) ratio was scaled, with respect to yield of liver microsomal protein and liver weight, to obtain estimates of M2 formation clearance (3.1-9.7 ml/min/kg of b.wt.) that agreed favorably with in vivo results (8.3 ml/min/kg of b.wt.) following i.v. administration of [(14)C]etoricoxib to healthy male subjects. Cytochrome P450 (P450) reaction phenotyping studies-using P450 form selective chemical inhibitors, immunoinhibitory antibodies, recombinant P450s, and correlation analysis with microsomes prepared from a bank of human livers-revealed that the 6'-methyl hydroxylation of etoricoxib was catalyzed largely (approximately 60%) by member(s) of the CYP3A subfamily. By comparison, CYP2C9 (approximately 10%), CYP2D6 (approximately 10%), CYP1A2 (approximately 10%), and possibly CYP2C19 played an ancillary role. Moreover, etoricoxib (0.1-100 microM) was found to be a relatively weak inhibitor (IC(50) > 100 microM) of multiple P450s (CYP1A2, CYP2D6, CYP3A, CYP2E1, CYP2C9, and CYP2C19) in human liver microsomes.

Preclinical drug metabolism in the age of high-throughput screening: an industrial perspective.

With the advent of genomics, combinatorial paradigms and high-throughput screen (HTS)-based pharmacological testing, the number of compounds flowing through the discovery pipeline is likely to escalate. At the same time, with increased knowledge of the human drug-metabolizing enzymes and the availability of in vitro absorption-metabolism (AM) models, Preclinical Drug Metabolism is poised to meet the challenges of HTS. In order to be successful, however, a rational HTS strategy (vs. serendipitous HTS) has to be employed. Such a strategy is based on automation, validation and integration of in vitro AM models and database management (AVID). A generalized strategy for rational (AVID-based) HTS in Preclinical Drug Metabolism is described briefly.