Melanie A. Felmlee

Overview of the Proton-coupled MCT (SLC16A) Family of Transporters: Characterization, Function and Role in the Transport of the Drug of Abuse γ-Hydroxybutyric Acid

The transport of monocarboxylates, such as lactate and pyruvate, is mediated by the SLC16A family of proton-linked membrane transport proteins known as monocarboxylate transporters (MCTs). Fourteen MCT-related genes have been identified in mammals and of these seven MCTs have been functionally characterized. Despite their sequence homology, only MCT1–4 have been demonstrated to be proton-dependent transporters of monocarboxylic acids. MCT6, MCT8 and MCT10 have been demonstrated to transport diuretics, thyroid hormones and aromatic amino acids, respectively. MCT1–4 vary in their regulation, tissue distribution and substrate/inhibitor specificity with MCT1 being the most extensively characterized isoform. Emerging evidence suggests that in addition to endogenous substrates, MCTs are involved in the transport of pharmaceutical agents, including γ-hydroxybuytrate (GHB), 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibitors (statins), salicylic acid, and bumetanide. MCTs are expressed in a wide range of tissues including the liver, intestine, kidney and brain, and as such they have the potential to impact a number of processes contributing to the disposition of xenobiotic substrates. GHB has been extensively studied as a pharmaceutical substrate of MCTs; the renal clearance of GHB is dose-dependent with saturation of MCT-mediated reabsorption at high doses. Concomitant administration of GHB and l-lactate to rats results in an approximately two-fold increase in GHB renal clearance suggesting that inhibition of MCT1-mediated reabsorption of GHB may be an effective strategy for increasing renal and total GHB elimination in overdose situations. Further studies are required to more clearly define the role of MCTs on drug disposition and the potential for MCT-mediated detoxification strategies in GHB overdose.

Cytochrome P450 Expression and Regulation in CYP3A4/CYP2D6 Double Transgenic Humanized Mice

Analysis of the developmental and sexual expression of cytochrome P450 drug-metabolizing enzymes is impeded by multiple and varied external factors that influence its regulation. In the present study, a CYP2D6/CYP3A4-double transgenic (Tg-CYP2D6/CYP3A4) mouse model was employed to investigate hepatic CYP2D6 and CYP3A4 ontogeny and sexual dimorphism. Both age and sex have considerable effects on hepatic CYP3A4 protein expression in 3- to 8-week-old transgenic mice, whereas neither factor alters CYP2D6 content. Constitutive CYP2D6 expression resulted in 2- to 3-fold higher dextromethorphan O-demethylase activity in Tg-CYP2D6/CYP3A4 mouse liver microsomes compared with wild-type mice. In contrast, expression of CYP3A4 in transgenic mouse livers did not increase dextromethorphan N-demethylase and midazolam 1′-hydroxylase activities. Pretreatment with pregnenolone 16α-carbonitrile (PCN) and 1,4-bis-2-(3, 5-dichloropyridyloxy)-benzene (TCPOBOP) elevated CYP3A4 expression in double transgenic mice. Interestingly, induction of hepatic CYP3A4 was greater in females than age- and treatment-matched males. Consequently, the increase in midazolam 1′-hydroxylase activity was markedly higher in 8-week-old female mice than in corresponding males (8-fold versus 6-fold for PCN treatment and 6-fold versus 5-fold for TCPOBOP). Furthermore, increases in testosterone 6β-hydroxylase activity after CYP3A induction were relatively lower compared with those in midazolam 1′-hydroxylation for age-, sex-, and treatment-matched mice. The difference in CYP3A4 expression and induction between male and female mice suggests that women may be more susceptible to CYP3A4-mediated drug-drug interactions, and the extent of drug-drug interactions could be substrate dependent.

Concentration-Effect Relationships for the Drug of Abuse γ-Hydroxybutyric Acid

γ-Hydroxybutyric acid (GHB) is an endogenous neurotransmitter that is abused because of its sedative/hypnotic and euphoric effects. The objectives of this study were to evaluate the concentration-effect relationships of GHB in plasma, cerebrospinal fluid (CSF), brain (whole and discrete brain regions), and brain frontal cortex extracellular fluid. This information is crucial for future studies to evaluate effects of therapeutic interventions on the toxicodynamics of GHB. GHB (200–1000 mg/kg) was administered intravenously to rats, and plasma and frontal cortex microdialysate samples were collected for up to 6 h after the dose, or plasma, CSF, and brain (whole, frontal cortex, striatum, and hippocampus) concentrations were determined at the offset of its sedative/hypnotic effect [return to righting reflex (RRR)]. GHB-induced changes in the brain neurotransmitters γ-aminobutyric acid (GABA) and glutamate were also determined. GHB, GABA, and glutamate concentrations were measured by liquid chromatography/tandem mass spectrometry. GHB-induced sleep time significantly increased in a dose-dependent manner (20-fold increase from 200 to 1000 mg/kg). GHB concentrations in plasma (300–400 μg/ml), whole brain (70 μg/g), discrete brain regions (80–100 μg/g), and brain microdialysate (29–39 μg/ml) correlated with RRR. In contrast, CSF GHB and GABA and glutamate concentrations in discrete brain regions exhibited no relationship with RRR. Our results suggest that GHB-induced sedative/hypnotic effects are mediated directly by GHB and that at high GHB doses, GABA formation from GHB may not contribute to the observed sedative/hypnotic effect. These results support the use of a clinical GHB detoxification strategy aimed at decreasing plasma and brain GHB concentrations after GHB overdoses.

Monocarboxylate Transporter-Mediated Transport of γ-Hydroxybutyric Acid in Human Intestinal Caco-2 Cells

The objectives of this study were to determine mRNA expression of monocarboxylate transporters (MCT) and to evaluate intestinal transport of the MCT substrates γ-hydroxybutyrate (GHB) and d-lactate in human intestinal Caco-2 cells. The presence of mRNA for MCT1, 2, 3, and 4 was observed in Caco-2 cells. The uptake of both GHB and d-lactate in Caco-2 cells was demonstrated to be pH- and concentration-dependent and sodium-independent. The uptake of GHB and d-lactate was best described by a Michaelis-Menten equation with passive diffusion (GHB: Km = 17.6 ± 10.5 mM, Vmax = 17.3 ± 11.7 nmol/min/mg, and P = 0.38 ± 0.15 μl/min/mg; and d-lactate: Km = 6.0 ± 2.9 mM, Vmax = 35.0 ± 18.4 nmol/min/mg, and P = 1.3 ± 0.6 μl/min/mg). The uptake of GHB and d-lactate was significantly decreased by the known MCT inhibitor α-cyano-4-hydroxycinnamate and the MCT substrates GHB and d-lactate but not by the organic cation tetraethylammonium chloride. Directional flux studies with both GHB and d-lactate suggested the involvement of carrier-mediated transport with the permeability in the apical to basolateral direction higher than that in the basolateral to apical direction. These findings confirm the presence of MCT1–4 in Caco-2 cells and demonstrate GHB and d-lactate transport characteristics consistent with proton-dependent MCT-mediated transport.

Mechanism-Based Pharmacodynamic Modeling

Pharmacodynamic modeling is based on a quantitative integration of pharmacokinetics, pharmacological systems, and (patho-) physiological processes for understanding the intensity and time-course of drug effects on the body. Application of such models to the analysis of meaningful experimental data allows for the quantification and prediction of drug-system interactions for both therapeutic and adverse drug responses. In this chapter, commonly used mechanistic pharmacodynamic models are presented with respect to their important features, operable equations, and signature profiles. In addition, literature examples showcasing the utility of these models to adverse drug events are highlighted. Common model types that are covered include simple direct effects, biophase distribution, indirect effects, signal transduction, and irreversible effects.

Brain Uptake of the Drug of Abuse γ-Hydroxybutyric Acid in Rats

γ-Hydroxybutyric acid (GHB) is an endogenous compound and a substrate for the ubiquitous monocarboxylate transporter (MCT) family. GHB is also a drug of abuse due to its sedative/hypnotic and euphoric effects, with overdoses resulting in toxicity and death. The goal of this study was to characterize the distribution of GHB into the brain using in vivo microdialysis and in vitro uptake studies and to determine concentration-effect relationships for GHB in a rat animal model. GHB was administered to rats (400, 600, and 800 mg/kg i.v.), and blood, dialysate, and urine were collected for 6 h post-GHB administration. The GHB plasma and extracellular fluid (ECF) concentration-time profiles revealed that GHB concentrations in ECF closely followed plasma GHB concentrations. Sleep time increased in a dose-dependent manner (91 ± 18, 134 ± 11, and 168 ± 13 min, for GHB 400, 600, and 800 mg/kg, respectively). GHB partitioning into brain ECF was not significantly different at 400, 600, and 800 mg/kg. GHB uptake in rat and human brain endothelial cells exhibited concentration dependence. The concentration-dependent uptake of GHB at pH 7.4 was best-fit to a single-transporter model [Km = 18.1 mM (human), 23.3 mM (rat), Vmax = 248 and 258 pmol · mg−1 · min−1 for human and rat, respectively]. These findings indicate that although GHB distribution into the brain is mediated via MCT transporters, it is not capacity-limited over the range of doses studied in this investigation.

γ-Hydroxybutyrate blood/plasma partitioning: effect of physiologic pH on transport by monocarboxylate transporters.

The drug of abuse γ-hydroxybutyrate (GHB) displays nonlinear renal clearance, which has been attributed to saturable renal reabsorption by monocarboxylate transporters (MCTs) present in the kidney. MCT1 is also present in red blood cells (RBCs); however, the significance of this transporter on the blood/plasma partitioning of GHB is unknown. The purpose of this research was to characterize the transport of GHB across the RBC membrane and assess GHB blood/plasma partitioning in vivo in the presence and absence of a competitive MCT inhibitor, l-lactate. In vitro experiments were performed using freshly isolated rat erythrocytes at pH values of 6.5 and 7.4. Inhibition with p-chloromercuribenzene sulfonate and 4,4′-diisothiocyanostilbene-2,2′-disulfonate were used to determine the contribution of MCT1 and band 3, respectively, on GHB uptake. For in vivo experiments, rats were administered GHB (400–1500 mg/kg) with and without l-lactate. In vitro experiments demonstrated that GHB is transported across the RBC membrane primarily by MCT1 at relevant in vivo concentrations. The Km for MCT1 was lower at pH 6.5 than that at pH 7.4, 2.2 versus 17.0 mM, respectively. The in vivo blood/plasma partitioning of GHB displayed linearity across all concentrations. l-Lactate coadministration increased GHB renal clearance but had no effect on the blood/plasma ratio. Unlike its MCT-mediated transport in the intestine and kidneys, GHB blood/plasma partitioning appears to be linear and is unaffected by l-lactate. These findings can be attributed, at least in part, to differences in physiologic pH at different sites of MCT-mediated transport.

Quantitation of human cytochrome P450 2D6 protein with immunoblot and mass spectrometry analysis

Accurate quantification of cytochrome P450 (P450) protein contents is essential for reliable assessment of drug safety, including the prediction of in vivo clearance from in vitro metabolism data, which may be hampered by the use of uncharacterized standards and existence of unknown allelic isozymes. Therefore, this study aimed to delineate the variability in absolute quantification of polymorphic CYP2D6 drug-metabolizing enzyme and compare immunoblot and nano liquid chromatography coupled to mass spectrometry (nano-LC/MS) methods in identification and relative quantification of CYP2D6.1 and CYP2D6.2 allelic isozymes. Holoprotein content of in-house purified CYP2D6 isozymes was determined according to carbon monoxide difference spectrum, and total protein was quantified with bicinchoninic acid protein assay. Holoprotein/total CYP2D6 protein ratio was markedly higher for purified CYP2D6.1 (71.0%) than that calculated for CYP2D6.1 Supersomes (35.5%), resulting in distinct linear calibration range (0.05–0.50 versus 0.025–0.25 pmol) that was determined by densitometric analysis of immunoblot bands. Likewise, purified CYP2D6.2 and CYP2D6.10 and the CYP2D6.10 Supersomes all showed different holoprotein/total CYP2D6 protein ratios and distinct immunoblot linear calibration ranges. In contrast to immunoblot, nano-LC/MS readily distinguished CYP2D6.2 (R296C and S486T) from CYP2D6.1 by isoform-specific proteolytic peptides that contain the altered amino acid residues. In addition, relative quantitation of the two allelic isozymes was successfully achieved with label-free protein quantification, consistent with the nominated ratio. Because immunoblot and nano-LC/MS analyses measure total P450 protein (holoprotein and apoprotein) in a sample, complete understanding of holoprotein and apoprotein contents in P450 standards is desired toward reliable quantification. Our data also suggest that nano-LC/MS not only facilitates P450 quantitation but also provides genotypic information.

SLC and ABC Transporters: Expression, Localization, and Species Differences at the Blood-Brain and the Blood-Cerebrospinal Fluid Barriers

The blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) separate the brain and cerebrospinal fluid (CSF) from the systemic circulation and represent a barrier to the uptake of both endogenous compounds and xenobiotics into the brain. For compounds whose passive diffusion is limited due to their ionization or hydrophilicity, membrane transporters can facilitate their uptake across the BBB or BCSFB. Members of the solute carrier (SLC) and ATP-binding case (ABC) families are present on these barriers. Differences exist in the localization and expression of transport proteins between the BBB and BCSFB, resulting in functional differences in transport properties. This review focuses on the expression, membrane localization, and different isoforms present at each barrier. Diseases that affect the central nervous system including brain tumors, HIV, Alzheimer’s disease, Parkinson’s disease, and stroke affect the integrity and expression of transporters at the BBB and BCSFB and will be briefly reviewed.

A novel mutant Na+/HCO3− cotransporter NBCe1 in a case of compound-heterozygous inheritance of proximal renal tubular acidosis

The inheritance of two defective alleles of SLC4A4, the gene that encodes the widely‐expressed electrogenic sodium bicarbonate cotransporter NBCe1, results in the bicarbonate‐wasting disease proximal renal tubular acidosis (pRTA). In the present study, we report the first case of compound‐heterozygous inheritance of pRTA (p.Arg510His/p.Gln913Arg) in an individual with low blood pH, blindness and neurological signs that resemble transient ischaemic attacks. We employ fluorescence microscopy on non‐polarized (human embryonic kidney) and polarized (Madin–Darby canine kidney) renal cell lines and electrophysiology on Xenopus oocytes to characterize the mutant transporters (R510H and Q913R). Both mutant transporters exhibit enhanced intracellular retention in renal cells, an observation that probably explains the HCO3− transport deficit in the individual. Both mutants retain a close‐to‐normal per molecule Na+/HCO3− cotransport activity in Xenopus oocytes, suggesting that they are suitable candidates for folding‐correction therapy. However, Q913R expression is uniquely associated with a depolarizing, HCO3− independent, Cl−‐conductance in oocytes that could have pathological consequences if expressed in the cells of patients.