Cation-selective Apical Transporters Mediate an Intestinal Cycling Mechanism of Metformin that Enhances its Paracellular Oral Absorption in Mice

Metformin is the most widely prescribed drug for type 2 diabetes mellitus; yet its in vivo mechanism of oral absorption has not been elucidated. A pKa of 12.4 and logDpH6.0 of -6.13 suggest metformin is a hydrophilic cation at all physiologic pHs, limiting its ability to cross biological membranes. However, metformin is well-absorbed with an oral bioavailability ranging from ~40-60% in man. Previous in vitro studies conducted using the Caco-2 Transwell® model of intestinal absorption demonstrated efficient transporter-mediated metformin apical uptake and efflux and poor basolateral egress. Kinetic modeling of these results suggested absorptive transport is predominantly paracellular, and led to the development of a novel mechanism of absorption stating that during oral absorption of metformin, transporter-mediated apical uptake and a lack of basolateral efflux leads to intestinal drug accumulation. Changes in luminal drug concentration as a result of gastrointestinal transit leads to apical efflux of metformin and its enhanced paracellular absorption. Studies presented in this dissertation evaluate this novel metformin absorption mechanism in a mouse model. Gene expression of the mouse orthologs of putative human metformin transporters, namely organic cation transporter 1-3 (mOct1-3), multidrug and toxin extrusion 1 (mMate1), and plasma membrane monoamine transporter (mPmat), was characterized in mouse small intestine. Stable cell lines singly-expressing these transporters were generated, and metformin uptake kinetics for each transporter was determined. Pentamidine, quinidine, and desipramine, were identified as pan transporter inhibitors and were used in subsequent mouse studies. Absorptive transport of metformin in ex vivo experiments using mouse intestinal tissue was similar to results previously reported for Caco-2 cell monolayers, showing high transporter-mediated apical uptake compared to apical-to-basolateral transport. Metformin orally co-administered with pentamidine demonstrated that the intestinal accumulation and absorption of metformin is transporter-mediated. Attenuation of metformin apical efflux in the intestine after oral dosing showed a decreased metformin absorption rate, suggesting an important role for apical efflux of metformin during its oral absorption. Collectively, these studies provide strong circumstantial evidence that metformin is absorbed through the hypothesized mechanism, which can account for the intestinal accumulation and oral pharmacokinetics of metformin observed in human and animal studies.Doctor of Philosoph

Identification of Glycochenodeoxycholate 3-O-glucuronide and Glycodeoxycholate 3-O-glucuronide as Highly Sensitive and Specific OATP1B1 Biomarkers

The aim of this study was to investigate the sensitivity and specificity of endogenous glycochenodeoxycholate and glycodeoxycholate 3-O-glucuronides (GCDCA-3G and GDCA-3G) as substrates for organic anion transporting polypeptide 1B1 (OATP1B1) in humans. We measured fasting levels of plasma GCDCA-3G and GDCA-3G using liquid chromatography-tandem mass spectrometry in 356 healthy volunteers. The mean plasma levels of both compounds were similar to 50% lower in women than in men (P = 2.25 x 10(-18) and P = 4.73 x 10(-9)). In a microarray-based genome-wide association study, theSLCO1B1rs4149056 (c.521T>C, p.Val174Ala) variation showed the strongest association with the plasma GCDCA-3G (P = 3.09 x 10(-30)) and GDCA-3G (P = 1.60 x 10(-17)) concentrations. The mean plasma concentration of GCDCA-3G was 9.2-fold (P = 8.77 x 10(-31)) and that of GDCA-3G was 6.4-fold (P = 2.45x10(-13)) higher in individuals with theSLCO1B1c.521C/C genotype than in those with the c.521T/T genotype. No other variants showed independent genome-wide significant associations with GCDCA-3G or GDCA-3G. GCDCA-3G was highly efficacious in detecting theSLCO1B1c.521C/C genotype with an area under the receiver operating characteristic curve of 0.996 (P <0.0001). The sensitivity (98-99%) and specificity (100%) peaked at a cutoff value of 180 ng/mL for men and 90 ng/mL for women. In a haplotype-based analysis,SLCO1B1*5and*15were associated with reduced, andSLCO1B1*1B, *14, and *35with increased OATP1B1 function.In vitro, both GCDCA-3G and GDCA-3G showed at least 6 times higher uptake by OATP1B1 than OATP1B3 or OATP2B1. These data indicate that the hepatic uptake of GCDCA-3G and GDCA-3G is predominantly mediated by OATP1B1. GCDCA-3G, in particular, is a highly sensitive and specific OATP1B1 biomarker in humans.Peer reviewe

Organic Cation Transporter 1 (OCT1/mOct1) Is Localized in the Apical Membrane of Caco-2 Cell Monolayers and Enterocytes

ABSTRACT Organic cation transporters (OCTs) are members of the solute carrier 22 family of transporter proteins that are involved in absorption, distribution, and excretion of organic cations. OCT3 is localized in the apical (AP) membrane of enterocytes, but the literature is ambiguous about OCT1 (mOct1) localization, with some evidence suggesting a basolateral (BL) localization in human and mouse enterocytes. This is contrary to our preliminary findings showing AP localization of OCT1 in Caco-2 cell monolayers, an established model of human intestinal epithelium. Therefore, this study aims at determining the localization of OCT1 (mOct1) in Caco-2 cells, and human and mouse enterocytes. Functional studies using OCT1-specific substrate pentamidine showed transporter-mediated AP but not BL uptake in Caco-2 cells and human and mouse intestinal tissues. OCT1 inhibition decreased AP uptake of pentamidine by ∼50% in all three systems with no effect on BL uptake. A short hairpin RNA-mediated OCT1 knockdown in Caco-2 cells decreased AP uptake of pentamidine by ∼50% but did not alter BL uptake. Immunostaining and confocal microscopy in all three systems confirmed AP localization of OCT1 (mOct1). Our studies unequivocally show AP membrane localization of OCT1 (mOct1) in Caco-2 cells and human and mouse intestine. These results are highly significant as they will require reinterpretation of previous drug disposition and drug-drug interaction studies where conclusions were drawn assuming BL localization of OCT1 in enterocytes. Most importantly, these results will require revision of the regulatory guidance for industry in the United States and elsewhere because it has stated that OCT1 is basolaterally localized in enterocytes

Metformin modulates glucose uptake and transport in Caco-2 cell monolayers

Metformin is an anti-diabetic drug widely used to treat type 2 diabetes mellitus. Several studies have demonstrated that in vivo, metformin decreases both hepatic glucose production and intestinal glucose transport, and increases intestinal glucose utilization, all of which contribute to the blood glucose-lowering effects of metformin in diabetics. Increased intestinal glucose utilization is thought to contribute to metformin-induced intestinal lactic acidosis. Studies show that the highest accumulation of metformin occurs in the intestine following oral administration, which raises the question whether high metformin intestinal concentrations modulate cell function. Since intestinal glucose absorption occurs from the apical (AP) and basolateral (BL) membranes, it warrants investigating whether high metformin concentrations affect intestinal glucose absorption and glucose transporter (GLUT) translocation to the membranes of intestinal cells. There is little information about how metformin mediates GLUT transporters, although some evidence shows that metformin promotes translocation of GLUT transporters to the AP membrane. This study aims at elucidating the effect of metformin on glucose uptake and transport as a result of the GLUT transporters being translocated to AP and BL membranes of Caco-2 cell monolayers, a well-established model of human intestinal epithelia. Real-time polymerase chain reaction to determine relative expression of candidate glucose transporters GLUT1-3 in Caco-2 cells, and time- and concentration-dependent transport and uptake studies of the non-metabolizable radiolabeled glucose surrogate 2-deoxy-D-glucose (2DG) will provide information on the effect of metformin on intestinal glucose uptake and transport and contribute to knowledge about the risk of developing lactic acidosis