Defining the Intravital Renal Disposition of Fluorescence-Quenched Exenatide.

Despite the understanding that renal clearance is pivotal for driving the pharmacokinetics of numerous therapeutic proteins and peptides, the specific processes that occur following glomerular filtration remain poorly defined. For instance, sites of catabolism within the proximal tubule can occur at the brush border, within lysosomes following endocytosis, or even within the tubule lumen itself. The objective of the current study was to address these limitations and develop methodology to study the kidney disposition of a model therapeutic protein. Exenatide is a peptide used to treat type 2 diabetes mellitus. Glomerular filtration and ensuing renal catabolism have been shown to be its principal clearance pathway. Here, we designed and validated a Förster resonance energy transfer-quenched exenatide derivative to provide critical information on the renal handling of exenatide. A combination of in vitro techniques was used to confirm substantial fluorescence quenching of intact peptide that was released upon proteolytic cleavage. This evaluation was then followed by an assessment of the in vivo disposition of quenched exenatide directly within kidneys of living rats via intravital two-photon microscopy. Live imaging demonstrated rapid glomerular filtration and identified exenatide metabolism occurred within the subapical regions of the proximal tubule epithelia, with subsequent intracellular trafficking of cleaved fragments. These results provide a novel examination into the real-time, intravital disposition of a protein therapeutic within the kidney and offer a platform to build upon for future work.

Monocarboxylate Transporters (SLC16): Function, Regulation, and Role in Health and Disease.

The solute carrier family 16 (SLC16) is comprised of 14 members of the monocarboxylate transporter (MCT) family that play an essential role in the transport of important cell nutrients and for cellular metabolism and pH regulation. MCTs 1-4 have been extensively studied and are involved in the proton-dependent transport of L-lactate, pyruvate, short-chain fatty acids, and monocarboxylate drugs in a wide variety of tissues. MCTs 1 and 4 are overexpressed in a number of cancers, and current investigations have focused on transporter inhibition as a novel therapeutic strategy in cancers. MCT1 has also been used in strategies aimed at enhancing drug absorption due to its high expression in the intestine. Other MCT isoforms are less well characterized, but ongoing studies indicate that MCT6 transports xenobiotics such as bumetanide, nateglinide, and probenecid, whereas MCT7 has been characterized as a transporter of ketone bodies. MCT8 and MCT10 transport thyroid hormones, and recently, MCT9 has been characterized as a carnitine efflux transporter and MCT12 as a creatine transporter. Expressed at the blood brain barrier, MCT8 mutations have been associated with an X-linked intellectual disability, known as Allan-Herndon-Dudley syndrome. Many MCT isoforms are associated with hormone, lipid, and glucose homeostasis, and recent research has focused on their potential roles in disease, with MCTs representing promising novel therapeutic targets. This review will provide a summary of the current literature focusing on the characterization, function, and regulation of the MCT family isoforms and on their roles in drug disposition and in health and disease. SIGNIFICANCE STATEMENT: The 14-member solute carrier family 16 of monocarboxylate transporters (MCTs) plays a fundamental role in maintaining intracellular concentrations of a broad range of important endogenous molecules in health and disease. MCTs 1, 2, and 4 (L-lactate transporters) are overexpressed in cancers and represent a novel therapeutic target in cancer. Recent studies have highlighted the importance of MCTs in glucose, lipid, and hormone homeostasis, including MCT8 in thyroid hormone brain uptake, MCT12 in carnitine transport, and MCT11 in type 2 diabetes.

Treatment of γ-Hydroxybutyrate (GHB) Overdose with the GABAB Antagonist SGS742.

High doses of the partial agonist of the GABA B receptor, γ-hydroxybutyric acid (GHB), causes respiratory depression that can lead to death. Previously, it has been shown that GABAB- receptor antagonism is able to prevent respiratory depression and sedation when inhibitors are pre-administered. In order to treat GHB overdoses, safety and efficacy of a treatment strategy at various times after GHB administration is necessary, in order to more closely replicate a true overdose situation. Preliminary studies developed an assay for SGS742 and determined its pharmacokinetics in rats. The effects of SGS742 on GHB-induced respiratory depression were evaluated when SGS742 administration was delayed 1 and 2 hours after intravenous or oral administration of GHB or γ-butyrolactone, a GHB prodrug. SGS742 reversed GHB-induced respiratory depression in a dose-dependent manner at both time points tested, with no effects on its toxicokinetics. However, some of the dosing paradigms resulted in toxicity in the form of tremors, seizures or abnormal movements. The tremors/seizures occurred in a manner that was dependent on both the dose and timing of SGS742 administration, and were not altered with pretreatment with gabazine, a GABAA receptor inhibitor, and only partially reduced with pretreatment with NCS382, a selective GHB receptor antagonist. Additional studies with a second GABAB antagonist SCH50911 demonstrated similar effects, producing reversal of respiratory depression but producing tremors and abnormal movements. Further studies are necessary in order to identify the potential use of GABAB antagonism as a treatment strategy for GHB overdoses. Significance Statement There is no current treatment for overdoses of the drug of abuse γ-hydroxybutyric acid (GHB). Since the toxicodynamic effects of GHB, including respiratory depression and lethality, are mediated through GABAB receptor agonism, GABAB receptor antagonists may represent a therapeutic strategy to treat overdoses. This study demonstrates that while GABAB receptor antagonists are effective as a pretreatment, they are less effective when administered at times after GHB administration and their administration is also associated with time- and dose-associated toxicity.

γ-Hydroxybutyric Acid-Ethanol Drug-Drug Interaction: Reversal of Toxicity with Monocarboxylate Transporter 1 Inhibitors.

The drug of abuse, γ-hydroxybutyric acid (GHB), is commonly co-ingested with ethanol, resulting in a high incidence of toxicity and death. Our laboratory has previously reported that GHB is a substrate for the monocarboxylate transporters (MCTs), necessary for its absorption, renal clearance, and tissue distribution, including across the blood-brain barrier. Our goal was to investigate the drug-drug interaction (DDI) between GHB and ethanol and to evaluate MCT1 inhibition as a strategy to reverse toxicity. The toxicokinetics of this DDI were investigated, including brain-to-plasma concentration ratios, in the presence and absence of ethanol. The toxicodynamic parameters examined were respiratory depression (breathing frequency, tidal volume) and sedation (time of return-of-righting reflex). Ethanol was administered (2 g/kg i.v.) 5 minutes before the intravenous or oral administration of GHB, and MCT1 inhibitors AZD-3965 and AR-C155858 (5 mg/kg i.v.) were administered 60 minutes after GHB administration. Ethanol administration did not alter the toxicokinetics or respiratory depression caused by GHB after intravenous or oral administration; however, it significantly increased the sedation effect, measured by return-to-righting time. AZD-3965 or AR-C155858 significantly decreased the effects of the co-administration of GHB and ethanol on respiratory depression and sedation of this DDI and decreased brain concentrations and the brain-to-plasma concentration ratio of GHB. The results indicate that ethanol co-administered with GHB increases toxicity and that MCT1 inhibition is effective in reversing toxicity by inhibiting GHB brain uptake when given after GHB-ethanol administration. SIGNIFICANCE STATEMENT: These studies investigated the enhanced toxicity observed clinically when γ-hydroxybutyric acid (GHB) is co-ingested with alcohol and evaluated strategies to reverse this toxicity. The effects of the novel monocarboxylate transporter 1 (MCT1) inhibitors AR-C155858 and AZD-3965 on this drug-drug interaction have not been studied before, and these preclinical studies indicate that MCT1 inhibitors can decrease brain concentrations of GHB by inhibiting brain uptake, even when administered at times after GHB-ethanol. AZD-3965 represents a potential treatment strategy for GHB-ethanol overdoses.

Drug-drug interaction between diclofenac and gamma-hydroxybutyric acid.

Gamma hydroxybutyric acid (GHB) has been approved clinically to treat excessive daytime sleepiness and cataplexy in patients with narcolepsy, alcohol and opioid withdrawal, and as an anesthetic. The use of GHB clinically is limited due to its high abuse potential. The absorption, clearance and tissue uptake of GHB is mediated by proton-dependent and sodium-coupled monocarboxylate transporters (MCTs and SMCTs) and inhibition of these transporters may result in a change in GHB pharmacokinetics and pharmacodynamics. Previous studies have reported that non-steroidal anti-inflammatory drugs (NSAIDs) may inhibit these monocarboxylate transporters. Therefore, the purpose of this work was to analyze the interaction between GHB (at a dose of 600 mg/kg i. v.) and the NSAID, diclofenac, by examining the effects of this drug on the in vivo pharmacokinetics and pharmacodynamics in rat studies. The pharmacodynamic effect evaluated was respiratory depression, a measure of toxicity observed by GHB at this dose. There was an improvement in the respiratory rate with diclofenac administration suggesting an effect of diclofenac on GHB toxicity. In vitro studies with rat blood brain endothelial cells (RBE4) that express MCT1 indicated that diclofenac can inhibit GHB transport with an IC50 of 10.6 µM at pH 7.4. In vivo studies found a decrease in brain GHB concentrations and a decrease in the brain-to-plasma concentration ratio following diclofenac treatment. With this study we can conclude that diclofenac and potentially other NSAIDs can inhibit the transport of GHB into the brain, therefore decreasing GHB's pharmacodynamic effects and toxicity.

Megalin-mediated albumin endocytosis in cultured murine mesangial cells.

Endocytosis by podocytes is gaining increased attention as a biologic means of removing large proteins such as serum albumin from the glomerular barrier. Some of this function has been attributed to the megalin/cubilin (Lrp2/Cubn) receptor complex and the albumin recycling protein FcRn (Fcgrt). However, whether other glomerular cells possess the potential to perform this same phenomenon or express these proteins remains uncharacterized. Mesangial cells are uniquely positioned in glomeruli and represent a cell type capable of performing several diverse functions. Here, the expression of megalin and FcRn in murine mesangial cells along with the megalin adaptor protein Dab-2 (Dab2) was shown for the first time. Cubilin mRNA expression was detected, but the absence of the cubilin partner amnionless (Amn) suggested that cubilin is minimally functional, if at all, in these cells. Mesangial cell endocytosis of albumin was characterized and shown to involve a receptor-mediated process. Albumin endocytosis was significantly impaired (p < 0.01) under inducible megalin knockdown conditions in stably transduced mesangial cells. The current work provides both the novel identification of megalin and FcRn in mesangial cells and the functional demonstration of megalin-mediated albumin endocytosis.

Contribution of Monocarboxylate Transporter 6 to the Pharmacokinetics and Pharmacodynamics of Bumetanide in Mice.

Bumetanide, a sulfamyl loop diuretic, is used for the treatment of edema in association with congestive heart failure. Being a polar, anionic compound at physiologic pH, bumetanide uptake and efflux into different tissues is largely transporter-mediated. Of note, organic anion transporters (SLC22A) have been extensively studied in terms of their importance in transporting bumetanide to its primary site of action in the kidney. The contribution of one of the less-studied bumetanide transporters, monocarboxylate transporter 6 (MCT6; SLC16A5), to bumetanide pharmacokinetics (PK) and pharmacodynamics (PD) has yet to be characterized. The affinity of bumetanide for murine Mct6 was evaluated using Mct6-transfected Xenopus laevis oocytes. Furthermore, bumetanide was intravenously and orally administered to wild-type mice (Mct6+/+) and homozygous Mct6 knockout mice (Mct6-/-) to elucidate the contribution of Mct6 to bumetanide PK/PD in vivo. We demonstrated that murine Mct6 transports bumetanide at a similar affinity compared with human MCT6 (78 and 84 µM, respectively, at pH 7.4). After bumetanide administration, there were no significant differences in plasma PK. Additionally, diuresis was significantly decreased by ∼55% after intravenous bumetanide administration in Mct6-/- mice. Kidney cortex concentrations of bumetanide were decreased, suggesting decreased Mct6-mediated bumetanide transport to its site of action in the kidney. Overall, these results suggest that Mct6 does not play a major role in the plasma PK of bumetanide in mice; however, it significantly contributes to bumetanide's pharmacodynamics due to changes in kidney concentrations. SIGNIFICANCE STATEMENT: Previous evidence suggested that MCT6 transports bumetanide in vitro; however, no studies to date have evaluated the in vivo contribution of this transporter. In vitro studies indicated that mouse and human MCT6 transport bumetanide with similar affinities. Using Mct6 knockout mice, we demonstrated that murine Mct6 does not play a major role in the plasma pharmacokinetics of bumetanide; however, the pharmacodynamic effect of diuresis was attenuated in the knockout mice, likely because of the decreased bumetanide concentrations in the kidney.

Characterization and Proteomic-Transcriptomic Investigation of Monocarboxylate Transporter 6 Knockout Mice: Evidence of a Potential Role in Glucose and Lipid Metabolism.

Monocarboxylate transporter 6 [(MCT6), SLC16A5] is an orphan transporter with no known endogenous substrates or physiological role. Previous in vitro and in vivo experiments investigated MCT6 substrate/inhibitor specificity in Xenopus laevis oocytes; however, these data remain limited. Transcriptomic changes in the livers of mice undergoing different dieting schemes have suggested that Mct6 plays a role in glucose and lipid metabolism. The objectives of this study were 1) to develop a novel knockout (KO) mouse model (Mct6-/-) using CRISPR/Cas9 technology, 2) to characterize the KO animal model by examining physiological and biochemical parameters, and 3) to understand the physiological role of MCT6 in vivo through global proteomic and liver transcriptomic profiling. mRNA tissue analysis demonstrated knockout of Mct6, which showed greater than 90% knockdown of Mct6 (Slc16a5) gene expression in all major tissues analyzed when normalized to Mct6+/+ mice. Proteomic analyses identified greater than 4000 unique proteins in kidney, liver, and colon tissues, among which 51, 38, and 241 proteins were significantly altered, respectively (for each tissue), between Mct6+/+ and Mct6-/- mice. Additionally, Mct6-/- mice demonstrated significant changes in 199 genes in the liver compared with Mct6+/+ mice. In silico biological pathway analyses revealed significant changes in proteins and genes involved in glucose and lipid metabolism-associated pathways. This study is the first to provide evidence for an association of Mct6 in the regulation of glucose and lipid metabolism. SIGNIFICANCE STATEMENT: This paper focuses on elucidating the innate biological role of an orphan transporter in vivo, which has not been investigated thus far. Using efficient and high-throughput technologies, such as CRISPR/Cas9 gene editing, liquid chromatography-tandem mass spectrometry-based proteomic and RNA-sequencing transcriptomic analyses, our laboratory provides the first existence and characterization of a Mct6 knockout mouse model. The evidence gathered in this paper, as well as other laboratories, support the importance of MCT6 in regulating a variety of glucose and lipid metabolic pathways, which may indicate its significance in metabolic diseases.

Treatment of γ-Hydroxybutyric Acid and γ-Butyrolactone Overdose with Two Potent Monocarboxylate Transporter 1 Inhibitors, AZD3965 and AR-C155858.

The illicit use of γ-hydroxybutyric acid (GHB), and its prodrug, γ-butyrolactone (GBL), results in severe adverse effects including sedation, coma, respiratory depression, and death. Current treatment of GHB/GBL overdose is limited to supportive care. Recent reports indicate that GHB-related deaths are on the rise; a specific treatment may reduce lethality associated with GHB/GBL. Pretreatment with inhibitors of monocarboxylate transporter 1 (MCT1), a transporter that mediates many of the processes involved in the absorption, distribution (including brain uptake), and elimination of GHB/GBL, has been shown to prevent GHB-induced respiratory depression by increasing the renal clearance of GHB. To identify whether MCT1 inhibition is an effective treatment of GHB overdose, the impact of two MCT1 inhibitors, (S)-5-(4-hydroxy-4-methylisoxazolidine-2-carbonyl)-1-isopropyl-3-methyl-6-((3-methyl-5-(trifluoromethyl)-1H-pyrazol-4-yl)methyl)thieno[2,3-day]pyrimidine-2,4(1H,3H)-dione (AZD3965) and 6-[(3,5-dimethyl-1H-pyrazol-4-yl)methyl]-5-[[(4S)-4-hydroxy-2-isoxazolidinyl]carbonyl]-3-methyl-1-(2-methylpropyl)thieno[2,3-day]pyrimidine2,4(1H,3H)-dione (AR-C155858), on the toxicokinetics and toxicodynamics of GHB/GBL was assessed when the administration of the inhibitor was delayed 60 and 120 minutes (post-treatment) after administration of GHB/GBL. AR-C155858 and AZD3965 reduced the toxicodynamic effects of GHB when GHB was administered intravenously, orally, or orally as the prodrug GBL. The impact of these inhibitors on GHB toxicokinetics was dependent on the route of GHB administration and the delay between GHB/GBL administration and administration of the MCT1 inhibitor. The reduction in GHB plasma exposure did not explain the observed effect of MCT1 inhibition on GHB-induced respiratory depression. The efficacy of MCT1 inhibition on GHB toxicodynamics is likely driven by the pronounced reduction in GHB brain concentrations. Overall, this study indicates that inhibition of MCT1 is an effective treatment of GHB/GBL overdose.

Pharmacokinetics of the Monocarboxylate Transporter 1 Inhibitor AZD3965 in Mice: Potential Enterohepatic Circulation and Target-Mediated Disposition.

PURPOSE: To evaluate the pharmacokinetics (PK) of the monocarboxylate transporter 1 (MCT1) inhibitor AZD3965 in mice after IV and oral administration and to develop mechanistic PK models to assess the potential enterohepatic circulation (EHC) and target-mediated drug disposition (TMDD) of AZD3965. METHODS: Female BALB/c mice were administered AZD3965 by IV injection (10, 50 and 100 mg/kg) or oral gavage (100 mg/kg). Plasma samples were analyzed using LC/MS/MS, and PK parameters determined by compartmental and non-compartmental analyses. RESULTS: AZD3965 exhibited a large volume of distribution and rapid oral absorption, with a high oral bioavailability. Prominent reentry peaks were observed after both oral and IV administration, suggesting potential EHC of AZD3965 or of a potential glucuronide conjugate. The dose-dependent studies indicated greater than proportional increases in exposure, an increase in the terminal half-life, and decrease in clearance and volume of distribution with increasing IV doses, indicating nonlinear pharmacokinetics and potential TMDD of AZD3965. Mechanistic compartmental models were developed to characterize the complex pharmacokinetics of AZD3965. CONCLUSIONS: The current study represents the first comprehensive report of the pharmacokinetics of AZD3965 in mice, indicating the potential contribution of EHC and TMDD in the disposition of AZD3965.

Increased megalin expression in early type 2 diabetes: role of insulin-signaling pathways.

The megalin/cubilin complex is responsible for the majority of serum protein reclamation in the proximal tubules. The current study examined if decreases in their renal expression, along with the albumin recycling protein neonatal Fc receptor (FcRn) could account for proteinuria/albuminuria in the Zucker diabetic fatty rat model of type 2 diabetes. Immunoblots of renal cortex samples obtained at worsening disease stages demonstrated no loss in megalin, cubilin, or FcRn, even when proteinuria was measured. Additionally, early diabetic rats exhibited significantly increased renal megalin expression when compared with controls (adjusted P < 0.01). Based on these results, the ability of insulin to increase megalin was examined in a clonal subpopulation of the opossum kidney proximal tubule cell line. Insulin treatments (24 h, 100 nM) under high glucose conditions significantly increased megalin protein ( P < 0.0001), mRNA ( P < 0.0001), and albumin endocytosis. The effect on megalin expression was prevented with inhibitors against key effectors of insulin intracellular signaling, phosphatidylinositide 3-kinase and Akt. Studies using rapamycin to inhibit the mechanistic target of rapamycin complex 1 (mTORC1) resulted in a loss of insulin-induced megalin expression. However, subsequent evaluation demonstrated these effects were independent of initial mTORC1 suppression. The presented results provide insight into the expression of megalin, cubilin, and FcRn in type 2 diabetes, which may be impacted by elevated insulin and glucose. Furthermore, proximal tubule endocytic activity in early diabetics may be enhanced, a process that could have a significant role in proteinuria-induced renal damage.

Prediction of the Effects of Renal Impairment on Clearance for Organic Cation Drugs that Undergo Renal Secretion: A Simulation-Based Study.

Renal impairment (RI) is a major health concern with a growing prevalence. RI leads to various physiologic changes, in addition to a decrease in glomerular filtration rate, that impact the pharmacokinetics (PK) and, specifically, the renal clearance (CLR) of compounds, including alterations of drug transporter (DT)/drug-metabolizing enzyme expression and activity, as well as protein binding. The objectives of this study were to use a physiologically based pharmacokinetic modeling platform to 1) assess the impact of alterations in DT expression, toxin-drug interactions (TDIs), and free fraction (fu) on PK predictions for the organic cation transporter 2/multidrug and toxin extrusion protein 1 substrate metformin in RI populations; and 2) use available in vitro data to improve predictions of CLR for two actively secreted substrates, metformin and ranitidine. The goal was to identify changes in parameters other than glomerular filtration rate-namely, fu and DT expression/activity-that are consistent with in vitro and clinical data in RI, and predict the importance of these parameters in the PK of metformin and ranitidine in RI patients. Our results demonstrated that including alterations in DT expression and fu, and including TDIs affecting DT activity, as indicated by in vitro data, improved the simulated predictions of CLR and other PK parameters for both metformin and ranitidine in RI. Our simulations suggest that modifications of DT expression/activity and fu are necessary for improved predictions of CLR in RI for compounds that are actively secreted, and that improvement of PK predictions in RI populations for metformin and ranitidine can be obtained by incorporating in vitro data.

Effects of renal impairment on transporter-mediated renal reabsorption of drugs and renal drug-drug interactions: A simulation-based study.

Renal impairment (RI) significantly impacts the clearance of drugs through changes in the glomerular filtration rate, protein binding and alterations in the expression of renal drug transport proteins and hepatic metabolizing enzymes. The objectives of this study were to evaluate quantitatively the effects of renal impairment on the pharmacokinetics of drugs undergoing renal transporter-mediated reabsorption. A previously published semi-mechanistic kidney model incorporating physiologically relevant fluid reabsorption and transporter-mediated active renal reabsorption (PMID: 26341876) was utilized in this study. The probe drug/transporter pair utilized was γ-hydroxybutyric acid (GHB) and monocarboxylate transporter 1 (SCL16A1, MCT1). γ-Hydroxybutyric acid concentrations in the blood and amount excreted into urine were simulated using ADAPT 5 for the i.v. dose range of 200-1500 mg/kg in rats and the impact of renal impairment on CLR and AUC was evaluated. A 90% decrease in GFR resulted in a > 100-fold decrease in GHB CLR . When expression of reabsorptive transporters was decreased and fu was increased, CLR approached GFR. The effect of renal impairment on CLR was reduced when the expression of drug metabolizing enzymes (DME) was increased as a result of increased metabolic clearance; the converse held true when the DME expression was decreased. In conclusion, this study quantitatively demonstrated that the effects of renal insufficiency on the clearance of drugs is modulated by transporter expression, contribution of renal clearance to overall clearance, expression of drug metabolizing enzymes, fraction unbound and drug-drug interactions with inhibitors of renal transporters that may be increased in the presence of renal impairment.

Quercetin, Morin, Luteolin, and Phloretin Are Dietary Flavonoid Inhibitors of Monocarboxylate Transporter 6.

Monocarboxylate transporter 6 (MCT6; SLC16A5) has been recognized for its role as a xenobiotic transporter, with characterized substrates probenecid, bumetanide, and nateglinide. To date, the impact of commonly ingested dietary compounds on MCT6 function has not been investigated, and therefore, the objective of this study was to evaluate a variety of flavonoids for their potential MCT6-specific interactions. Flavonoids are a large group of polyphenolic phytochemicals found in commonly consumed plant-based products that have been recognized for their dietary health benefits. The uptake of bumetanide in human MCT6 gene-transfected Xenopus laevis oocytes was significantly decreased in the presence of a variety of flavonoids (e.g., quercetin, luteolin, phloretin, and morin), but was not significantly affected by flavonoid glycosides (e.g., naringin, rutin, phlorizin). The IC50 values of quercetin, phloretin, and morin were determined to be 25.3 ± 3.36, 17.3 ± 2.37, and 33.1 ± 3.29 µM, respectively. The mechanism of inhibition of phloretin was reversible and competitive, with a Ki value of 22.8 µM. Furthermore, typical MCT substrates were also investigated for their potential interactions with MCT6. Substrates of MCTs 1, 2, 4, 8, and 10 did not cause any significant decrease in MCT6-mediated bumetanide uptake, suggesting that MCT6 has distinct compound selectivity. In summary, these results suggest that dietary aglycon flavonoids may significantly alter the pharmacokinetics and pharmacodynamics of bumetanide and other MCT6-specific substrates, and may represent potential substrates for MCT6.

Effect of chronic γ-hydroxybutyrate (GHB) administration on GHB toxicokinetics and GHB-induced respiratory depression.

BACKGROUND: γ-hydroxybutyrate (GHB) has a high potential for illicit use; overdose of this compound results in sedation, respiratory depression and death. Tolerance to the hypnotic/sedative and electroencephalogram effects of GHB occurs with chronic GHB administration; however, tolerance to respiratory depression has not been evaluated. GHB toxicodynamic effects are mediated predominantly by GABAB receptors. Chronic treatment may affect monocarboxylate transporters (MCTs) and alter the absorption, renal clearance and brain uptake of GHB. OBJECTIVES: To determine effects of chronic GHB dosing on GHB toxicokinetics, GHB-induced respiratory depression, and MCT expression. METHODS: Rats were administered GHB 600 mg/kg intravenously daily for 5 days. Plasma, urine and tissue samples and respiratory measurements were obtained on days 1 and 5. Plasma and urine were analyzed for GHB by LC/MS/MS and tissue samples for expression of MCT1, 2 and 4 and their accessory proteins by QRT-PCR. RESULTS: No differences in GHB pharmacokinetics or respiratory depression were observed between days 1 and 5. Opposing changes in MCT1 and MCT4 mRNA expression were observed in kidney samples on day 5 compared to GHB-naïve animals, and MCT4 expression was increased in the intestine. CONCLUSIONS: The lack of tolerance observed with GHB-induced respiratory depression, in contrast to the tolerance reported for the sedative/hypnotic and electroencephalogram effects, suggests that different GABAB receptor subtypes may be involved in different GABAB-mediated toxicodynamic effects of GHB. Chronic or binge users of GHB may be at no less risk for fatality from respiratory arrest with a GHB overdose than with a single dose of GHB.

A quantitative threshold for high/low extent of urinary excretion of compounds in humans.

In this study, a quantitative threshold was determined for the high/low extent of urinary excretion (UE) of compounds in humans, using a straightforward but robust statistical method known as receiver operating characteristic curve (ROC) analysis, and also 18 potential physicochemical determinants of UE were evaluated. Data on the percent of drug excreted unchanged into the urine, %Ae , were used to determine the threshold for high/low UE. Compounds can be divided into high/low UE groups using the threshold value of Ae  = 16.8%, namely those with Ae  > 16.8% are classified as high UE and those with Ae  ≤ 16.8% as low UE. The %Ae negatively correlated with cLogP (r = -0.56); however, cLogP could not quantitatively predict the value of %Ae (R(2) adj. = 0.32). Several determinants of the extent of UE, including cLogP, ACD labs cLogP and ACD labs cLogD(pH=7.4) , were successfully evaluated as a priori indicators of the extent of UE using two cut-off values for each parameter. Moreover, 87% of the 90 compounds in the external validation set were correctly classified using this approach. Analysis of the physicochemical spaces of compounds in these two groups showed significant overlap, which hinders the a priori determination of the extent of UE of compounds using a single threshold/cut-off value of simple physicochemical parameters. In conclusion, 16.8% is a quantitative threshold value to distinguish between high and low UE and new molecular entities with cLogP and ACD labs cLogP values of ≤0.7 and ≥1.0 and ACD labs cLogD(pH=7.4) values of ≤0.0 and ≥0.5 could be identified as exhibiting high and low UE, respectively. Copyright © 2016 John Wiley & Sons, Ltd.

Quantitative structure-pharmacokinetic relationships for the prediction of renal clearance in humans.

Renal clearance (CLR), a major route of elimination for many drugs and drug metabolites, represents the net result of glomerular filtration, active secretion and reabsorption, and passive reabsorption. The aim of this study was to develop quantitative structure-pharmacokinetic relationships (QSPKR) to predict CLR of drugs or drug-like compounds in humans. Human CLR data for 382 compounds were obtained from the literature. Step-wise multiple linear regression was used to construct QSPKR models for training sets and their predictive performance was evaluated using internal validation (leave-one-out method). All qualified models were validated externally using test sets. QSPKR models were also constructed for compounds in accordance with their 1) net elimination pathways (net secretion, extensive net secretion, net reabsorption, and extensive net reabsorption), 2) net elimination clearances (net secretion clearance, CLSEC; or net reabsorption clearance, CLREAB), 3) ion status, and 4) substrate/inhibitor specificity for renal transporters. We were able to predict 1) CLREAB (Q(2) = 0.77) of all compounds undergoing net reabsorption; 2) CLREAB (Q(2) = 0.81) of all compounds undergoing extensive net reabsorption; and 3) CLR for substrates and/or inhibitors of OAT1/3 (Q(2) = 0.81), OCT2 (Q(2) = 0.85), MRP2/4 (Q(2) = 0.78), P-gp (Q(2) = 0.71), and MATE1/2K (Q(2) = 0.81). Moreover, compounds undergoing net reabsorption/extensive net reabsorption predominantly belonged to Biopharmaceutics Drug Disposition Classification System classes 1 and 2. In conclusion, constructed parsimonious QSPKR models can be used to predict CLR of compounds that 1) undergo net reabsorption/extensive net reabsorption and 2) are substrates and/or inhibitors of human renal transporters.

A Novel Monocarboxylate Transporter Inhibitor as a Potential Treatment Strategy for γ-Hydroxybutyric Acid Overdose.

PURPOSE: Monocarboxylate transporter (MCT) inhibition represents a potential treatment strategy for γ-hydroxybutyric acid (GHB) overdose by blocking its renal reabsorption in the kidney. This study further evaluated the effects of a novel, highly potent MCT inhibitor, AR-C155858, on GHB toxicokinetics/toxicodynamics (TK/TD). METHODS: Rats were administered GHB (200, 600 or 1500 mg/kg i.v. or 1500 mg/kg po) with and without AR-C155858. Breathing frequency was continuously monitored using whole-body plethysmography. Plasma and urine samples were collected up to 8 h. The effect of AR-C155858 on GHB brain/plasma partitioning was also assessed. RESULTS: AR-C155858 treatment significantly increased GHB renal and total clearance after intravenous GHB administration at all the GHB doses used in this study. GHB-induced respiratory depression was significantly improved by AR-C155858 as demonstrated by an improvement in the respiratory rate. AR-C155858 treatment also resulted in a significant reduction in brain/plasma partitioning of GHB (0.1 ± 0.03) when compared to GHB alone (0.25 ± 0.02). GHB CLR and CLoral (CL/F) following oral administration were also significantly increased following AR-C155858 treatment (from 1.82 ± 0.63 to 5.74 ± 0.86 and 6.52 ± 0.88 to 10.2 ± 0.75 ml/min/kg, respectively). CONCLUSION: The novel and highly potent MCT inhibitor represents a potential treatment option for GHB overdose.

Semi-mechanistic kidney model incorporating physiologically-relevant fluid reabsorption and transporter-mediated renal reabsorption: pharmacokinetics of γ-hydroxybutyric acid and L-lactate in rats.

This study developed a semi-mechanistic kidney model incorporating physiologically-relevant fluid reabsorption and transporter-mediated active reabsorption. The model was applied to data for the drug of abuse γ-hydroxybutyric acid (GHB), which exhibits monocarboxylate transporter (MCT1/SMCT1)-mediated renal reabsorption. The kidney model consists of various nephron segments--proximal tubules, Loop-of-Henle, distal tubules, and collecting ducts--where the segmental fluid flow rates, volumes, and sequential reabsorption were incorporated as functions of the glomerular filtration rate. The active renal reabsorption was modeled as vectorial transport across proximal tubule cells. In addition, the model included physiological blood, liver, and remainder compartments. The population pharmacokinetic modeling was performed using ADAPT5 for GHB blood concentration-time data and cumulative amount excreted unchanged into urine data (200-1000 mg/kg IV bolus doses) from rats [Felmlee et al (PMID: 20461486)]. Simulations assessed the effects of inhibition (R = [I]/KI = 0-100) of renal reabsorption on systemic exposure (AUC) and renal clearance of GHB. Visual predictive checks and other model diagnostic plots indicated that the model reasonably captured GHB concentrations. Simulations demonstrated that the inhibition of renal reabsorption significantly increased GHB renal clearance and decreased AUC. Model validation was performed using a separate dataset. Furthermore, our model successfully evaluated the pharmacokinetics of L-lactate using data obtained from Morse et al (PMID: 24854892). In conclusion, we developed a semi-mechanistic kidney model that can be used to evaluate transporter-mediated active renal reabsorption of drugs by the kidney.

Flavonoids are inhibitors of human organic anion transporter 1 (OAT1)-mediated transport.

Organic anion transporter 1 (OAT1) has been reported to be involved in the nephrotoxicity of many anionic xenobiotics. As current clinically used OAT1 inhibitors are often associated with safety issues, identifying potent OAT1 inhibitors with little toxicity is of great value in reducing OAT1-mediated drug nephrotoxicity. Flavonoids are a class of polyphenolic compounds with exceptional safety records. Our objective was to evaluate the effects of 18 naturally occurring flavonoids, and some of their glycosides, on the uptake of para-aminohippuric acid (PAH) in both OAT1-expressing and OAT1-negative LLC-PK1 cells. Most flavonoid aglycones produced substantial decreases in PAH uptake in OAT1-expressing cells. Among the flavonoids screened, fisetin, luteolin, morin, and quercetin exhibited the strongest effect and produced complete inhibition of OAT1-mediated PAH uptake at a concentration of 50 µM. Further concentration-dependent studies revealed that both morin and luteolin are potent OAT1 inhibitors, with IC50 values of <0.3 and 0.47 µM, respectively. In contrast to the tested flavonoid aglycones, all flavonoid glycosides had negligible or small effects on OAT1. In addition, the role of OAT1 in the uptake of fisetin, luteolin, morin, and quercetin was investigated and fisetin was found to be a substrate of OAT1. Taken together, our results indicate that flavonoids are a novel class of OAT1 modulators. Considering the high consumption of flavonoids in the diet and in herbal products, OAT1-mediated flavonoid-drug interactions may be clinically relevant. Further investigation is warranted to evaluate the nephroprotective role of flavonoids in relation to drug-induced nephrotoxicity mediated by the OAT1 pathway.