The distribution in native populations from Mexico and Central America of the C677T variant in the MTHFR gene.

OBJECTIVES: To explore evolutionary hypotheses for the high frequencies of a substitution in the methylenetetrahydrofolate reductase (MTHFR) gene, in Mexican and Central American Indigenous populations. MATERIALS AND METHODS: We obtained allele frequencies for the C677T variant in the MTHFR gene and ecological information for 37 indigenous samples from Mexico and Central America. We calculated Hardy-Weinberg equilibrium and computed Fst statistics. We computed correlations between the samples' allele frequencies and ecological and geochemical variables. RESULTS: Many of the samples have extremely high frequencies of the T allele ( q ¯  = 0.62, median = 0.66). In this region, the frequency of the T allele decreases from Southeast to Northwest and is significantly correlated with longitude, latitude, altitude, and insolation. CONCLUSIONS: The native people of Central America and Mexico evolved high frequencies of an allele which has been shown to produce deleterious clinical effects including neural tube effects, cardiovascular events, and cancer. This allele has a clinal distribution in the region, perhaps associated with solar irradiation. As (Contreras-Cubas et al., 2016) noted, the traditional diet of these populations, which is high in folate, has likely mitigated the negative effect of the allele. It is of primary importance that their rights to their homeland and traditional diets be respected. It is a matter of Public Health to investigate whether this allele is a factor in the current wave of cardiovascular diseases affecting the majority population of this region, since it descends from the Native peoples and the Mediterranean population, which also has high frequencies of the allele.

Impact of P-glycoprotein on intracellular drug concentration in peripheral blood mononuclear cells and K562 cells.

P-glycoprotein (P-gp) expression in lymphocytes is variable and 2-fold higher in rheumatoid arthritis (RA) patients with treatment resistance than in healthy subjects. To date the information on P-gp-mediated drug interaction in lymphocyte is limited. We analyzed the importance on P-gp in lymphocytes using peripheral blood mononuclear cells (PBMCs) together with K562, K562/Adr, and K562/Vin cells, which have various P-gp levels, as cell models, and dexamethasone, nintedanib and apafant as weak to good P-gp substrates. P-gp levels in K562, K562/Adr, and K562/Vin cells were 0.3-, 20-, and 106-fold of healthy PBMCs, respectively. While cell accumulation of apafant and nintedanib decreased in all cells with increasing P-gp levels, dexamethasone accumulation in K562/Adr was comparable to that in healthy PBMCs and K562 cells. Cell accumulations of substrates in cells with low P-gp expression were not significantly changed by the P-gp inhibitors at therapeutic concentrations. However, accumulation increased to 1.4-fold at highest in K562/Adr cells with higher P-gp expression than in PBMCs of the RA patients. These results suggest P-gp controls the cellular concentration of P-gp substrates in PBMCs or K562 cells but cellular concentration of a weak P-gp substrate would not be apparently affected even in cells with a sufficient P-gp expression.

In-Depth Characterization of EpiIntestinal Microtissue as a Model for Intestinal Drug Absorption and Metabolism in Human.

The Caco-2 model is a well-accepted in vitro model for the estimation of fraction absorbed in human intestine. Due to the lack of cytochrome P450 3A4 (CYP3A4) activities, Caco-2 model is not suitable for the investigation of intestinal first-pass metabolism. The purpose of this study is to evaluate a new human intestine model, EpiIntestinal microtissues, as a tool for the prediction of oral absorption and metabolism of drugs in human intestine. The activities of relevant drug transporters and drug metabolizing enzymes, including MDR1 P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), CYP3A4, CYP2J2, UDP-glucuronosyltransferases (UGT), carboxylesterases (CES), etc., were detected in functional assays with selective substrates and inhibitors. Compared to Caco-2, EpiIntestinal microtissues proved to be a more holistic model for the investigation of drug absorption and metabolism in human gastrointestinal tract.

Surveillance of siRNA integrity by FRET imaging.

Techniques for investigation of exogenous small interfering RNA (siRNA) after penetration of the cell are of substantial interest to the development of efficient transfection methods as well as to potential medical formulations of siRNA. A FRET-based visualization method including the commonplace dye labels fluorescein and tetramethylrhodamin (TMR) on opposing strands of siRNA was found compatible with RNA interference (RNAi). Investigation of spectral properties of three labelled siRNAs with differential FRET efficiencies in the cuvette, including pH dependence and FRET efficiency in lipophilic environments, identified the ratio of red and green fluorescence (R/G-ratio) as a sensitive parameter, which reliably identifies samples containing >90% un-degraded siRNA. Spectral imaging of siRNAs microinjected into cells showed emission spectra indistinguishable from those measured in the cuvette. These were used to establish a calibration curve for assessing the degradation state of siRNA in volume elements inside cells. An algorithm, applied to fluorescence images recorded in standard green and red fluorescence channels, produces R/G-ratio images of high spatial resolution, identifying volume elements in the cell with high populations of intact siRNA with high fidelity. To demonstrate the usefulness of this technique, the movement of intact siRNA molecules are observed after introduction into the cytosol by microinjection, standard transfection and lipofection with liposomes.

Closing the gaps: a full scan of the intestinal expression of p-glycoprotein, breast cancer resistance protein, and multidrug resistance-associated protein 2 in male and female rats.

Intestinal ATP binding cassette (ABC) transporters may affect the bioavailability and effectiveness of orally administered drugs. Available studies on regional expression of intestinal efflux transporters were done with selected intestinal segments only and inconsistent with regard to the variability of transporter expression and the course of expression along the intestine. For an evaluation of the consistency between mRNA and protein expression, relative expression levels of P-glycoprotein (Pgp; ABCB1), breast cancer resistance protein (Bcrp; ABCG2), and multidrug resistance-associated protein (Mrp) 2 (ABCC2) were determined using quantitative real-time-polymerase chain reaction and Western blot in rat intestinal segments from duodenum, jejunum, ileum, and colon. In addition, the protein expression of Pgp, Bcrp, and Mrp2 from the entire rat intestine was studied by a complete 3-cm segmentation to evaluate the predictive power of expression analyses from selected intestinal segments. Pgp showed an increase from proximal to distal regions, Bcrp showed an arcuate pattern with highest expression toward the end of small intestine, and Mrp2 decreased along the intestinal axis from proximal to distal parts. No gender specific differences could be observed. Regarding the concordance of mRNA and protein expression, Pgp and Bcrp mRNA samples allow good estimations about the corresponding protein expression (for Pgp limited to the mdr1a isoform), but for Mrp2, pronounced deviation could be observed. All transporters showed considerable intra- and interindividual variability, especially at the protein level, making it problematic to take transporter expressions of small sections exemplary for general assumptions on intestinal abundances.

Concept: The Use of Targeted Immunoaffinity Proteomics for Routine Assessment of In Vitro Enzyme Induction.

In vitro investigations on enzyme induction are indispensable for assessing drug-drug interactions of drug candidates. Regulatory bodies require measurement of changes of mRNA in cultured human hepatocytes. However, such data provide only indirect assessments of effects of enzyme induction in vivo. We describe the quantification of cytochrome P450 (CYP) enzyme protein levels by liquid chromatography-mass spectrometry for the routine assessment of enzyme induction. Protein concentration of CYP1A2, 2B6, 3A4, and 2C8 were measured in human hepatocytes after incubation with prototypical enzyme inducers and drug candidate BI-X using an antibody-based capturing method. In addition, CYP mRNA levels and CYP enzyme activities were determined. Except for CYP2B6, mRNA levels consistently showed more pronounced induction effects than CYP activity or CYP protein concentration. Induction of CYP activities was better reflected on the level of CYP protein. The described method requires small sample amounts and can be integrated in routine in vitro enzyme induction studies using tissue culture in 48- and 96-well plates. Assessment of changes of enzyme protein levels adds valuable information to conventional measurements of enzyme induction and can improve the use of in vitro data for the prediction of clinical outcomes.

Regional absorption of fexofenadine in rat intestine.

PURPOSE: Expression of Pgp along the GI-tract of rats increases towards distal segments. We assessed the in vivo relevance by determining the absorption of fexofenadine from proximal and distal parts of rat intestine. Since it has been reported that fexofenadine is also actively taken up by OATP/oatps, we quantified rat oatp1a5 and oatp2b1 mRNA in gut mucosa to elucidate a possible contribution of an oatp-mediated active uptake of fexofenadine. METHODS: Absorption was determined after drug administration into ligated segments of rat duodenum/upper jejunum and terminal ileum, respectively, in the absence and the presence of the Pgp blocker PSC-833s. Portal vein blood was sampled. Expression of Pgp in the mucosa of proximal and distal intestinal segments was analyzed by Western Blot. Oatp1a5 and oatp2b1 mRNA in intestinal mucosa was quantified by real-time qRT-PCR. RESULTS: Portal vein AUC(0-90min) of fexofenadine was significantly higher after absorption from proximal segments compared to distal segments. Accordingly, Pgp expression was significantly lower in proximal compared to distal segments. Inhibition of Pgp by PSC-833 affected fexofenadine absorption only in distal segments resulting in AUC values comparable to the proximal data. Both oatp1a5 and oatp2b1 mRNA expression increased along the small intestine. CONCLUSION: The study demonstrates that Pgp is responsible for a limitation of fexofenadine absorption from distal small intestine. These findings are supported by the found pattern of expression for oatp1a5 and oatp2b1, showing that an active oatp-mediated uptake plays no role for fexofenadine absorption in rats.