Clearance Excretion Research Articles

Induction of open-form bile canaliculus formation by hepatocytes for evaluation of biliary drug excretion

Biliary excretion is a major drug elimination pathway that affects their efficacy and safety. The currently available in vitro sandwich-cultured hepatocyte method is cumbersome because drugs accumulate in the closed bile canalicular lumen formed between hepatocytes and their amounts cannot be mealsured directly. This study proposes a hepatocyte culture model for the rapid evaluation of drug biliary excretion using permeation assays. When hepatocytes are cultured on a permeable support coated with the cell adhesion protein claudins, an open-form bile canalicular lumen is formed at the surface of the permeable support. Upon application to the basolateral (blood) side, drugs appear on the bile canalicular side. The biliary excretion clearance of several drugs, as estimated from the obtained permeabilities, correlates well with the reported in vivo biliary excretion clearance in humans. Thus, the established model is useful for applications in the efficient evaluation of biliary excretion during drug discovery and development.

An exploration on retro-construction of plasma drug concentration-time curves from corresponding urine excretion data and single-point plasma concentrations using a simplified and idealized method.

Despite the availability of various tools of modeling and simulation, clinical pediatric pharmacokinetic (PK) studies remain far less efficient than those on adults due to ethical constraints. One of the optimal solutions is to substitute urine to blood sampling based on explicit mathematic relationships between them. However, this idea is limited by three main knowledge gaps associated with urine data, i.e., complicated excretion equations with excessive parameters, insufficient frequency that is hard to fit, and the mere expression of amounts with no in vivo distribution volume information involved. To overcome these obstacles, we sacrificed the precision from mechanistic PK models with complex excretion equations to expediency of compartmental model in which a constant ke is used to cover all the internal parameters. And the total cumulative amounts of urinary drug excretion were estimated and introduced to the excretion equation so that urine data were likely to be fitted using a semi-log-terminal linear regression method. In addition, urinary excretion clearance (CLr) could be calculated by single point plasma data to anchor the plasma concentration-time (C-t) curve based on the assumption that CLr was kept constant throughout the PK process. After sensitivity analysis of two subjective judgements (the selection of the compartmental model and the selection of plasma time point to calculate CLr), the performance of the optimized models was assessed using desloratadine or busulfan as model drugs in a variety of PK scenarios, from i.v. bolus/infusion to p.o. administration, from a single dose to multiple doses, and from rats to children. The fitting plasma drug concentrations of the optimal model were close to the observed value. Meanwhile, the drawbacks inherent to the simplified and idealized modeling strategy were fully identified. The method proposed by this tentative proof-of-principle study was able to deliver acceptable plasma exposure curves and shed light on the future refinements.

Excretion of Amyloid-β in the Gastrointestinal Tract and Regulation by the Gut Microbiota.

Amyloid-β (Aβ) is important in the etiology of Alzheimer's disease (AD). Removal of Aβ from the brain is a major strategy for the prevention and treatment of AD. To clarify whether Aβ42 can be cleared by intestinal excretion and whether the gut microbiota (GM) can affect the excretory clearance of Aβ42 in the peripheral blood and intestines. Male 8-month-old C57BL6 mice were maintained on either normal chow or received broad-spectrum antibiotics in their drinking water for one week. Sterile saline, fluorescein isothiocyanate (FITC), or FITC-Aβ42 (fluorescein isothiocyanate-labeled amyloid-β42 peptides) was injected 1 h before sampling. Related changes of Aβ42 before and after injection were evaluated. FITC-Aβ42 was injected into mice through the tail vein and could later be detected in feces. Furthermore, the fecal concentrations of FITC-Aβ42 were higher in mice that had been fed antibiotics to alter their GM than in normal mice. However, the FITC-Aβ42 concentrations in blood showed the opposite pattern. Aβ42 can be excreted into the intestinal lumen and is regulated by the GM.

Transient, Tunable Expression of NTCP and BSEP in MDCKII Cells for Kinetic Delineation of the Rate-Determining Process and Inhibitory Effects of Rifampicin in Hepatobiliary Transport of Taurocholate

In predicting the hepatic elimination of compounds, the extended clearance concept has proven useful. Yet, its experimental proof was scarce partly due to the lack of models with the controlled expression of transporters. Here, the uptake and efflux transporters [NTCP (SLC10A1) and BSEP (ABCB11), respectively] were doubly and transiently expressed in MDCKII cells by electroporation-based transfection (with the BSEP plasmid amount varied and with the NTCP plasmid fixed), achieving the activity levels of NTCP and BSEP comparable to those of sandwich cultured human hepatocytes. The biliary excretion clearance for taurocholate increased proportionally to the BSEP expression level. Under the same conditions, the basal-to-apical transcellular clearance of taurocholate displayed an initial increase, and a subsequent plateau, indicating that the basolateral uptake of taurocholate became rate-limiting. The doubly transfected MDCKII cells were also used to kinetically analyze the inhibitory effects of rifampicin on BSEP and NTCP. The obtained results showed a bell-shaped profile for cell-to-medium concentration ratios over a range of rifampicin concentrations, which were quantitatively captured by kinetic modeling based on the extended clearance concept. The present study highlights the utility of the transient, tunable transporter expression system in delineating the rate-determining process and providing mechanistic insights into intracellular substrate accumulation.

SO052EFFECT OF SEVERE RENAL IMPAIRMENT OR END-STAGE RENAL DISEASE ON THE PHARMACOKINETICS AND PHARMACODYNAMICS OF ROXADUSTAT, AN ORAL HYPOXIA-INDUCIBLE FACTOR PROLYL HYDROXYLASE INHIBITOR

Abstract Background and Aims Roxadustat is an orally active hypoxia-inducible factor prolyl hydroxylase inhibitor used at titrated doses to treat anaemia in CKD patients. This study investigated the pharmacokinetics (PK), metabolic profile, pharmacodynamics, and safety of roxadustat in subjects with different degrees of renal function (RF). Method This was a phase 1, open-label study conducted in Germany and the United Kingdom (EudraCT Number: 2015-002565-28). Subjects were enrolled in four RF groups: normal RF (eGFR ≥90 mL/min/1.73 m2); severely impaired RF (not on dialysis; eGFR <30 mL/min/1.73 m2); end-stage renal disease (ESRD) on continuous ambulatory or automated peritoneal dialysis (CAPD/APD); or ESRD on 4-hour haemodialysis or haemodiafiltration (HD/HDF). Subjects in the normal RF, severely impaired RF, and ESRD on CAPD/APD groups received a single dose of 100-mg oral roxadustat on Day 1. A single-sequence design with two treatment periods (P1/P2) separated by a washout period was used for subjects with ESRD on HD/HDF; a single administration of 100-mg oral roxadustat occurred 2 hours after completion of a HD/HDF session (P1) and 2 hours before initiation of a HD/HDF session, 2 days after the previous session (P2). Plasma and urine PK of roxadustat and its circulating metabolites (O-glucuronide-, O-glucoside-, and sulphate of hydroxy-roxadustat) and plasma erythropoietin (EPO) were evaluated. Continuous heart rate monitoring was conducted. Results Of 73 screened subjects, 34 (46.6%) were enrolled and received roxadustat (normal RF, n=12; severely impaired RF, n=9; ESRD on CAPD, n=1; ESRD on HD/HDF, n=12). The geometric least-square mean ratio of area under the plasma concentration-time curve (AUC) from administration to infinity (AUCinf) relative to subjects with normal RF was 223% (90% CI: 185, 268) and 195% (90% CI: 165, 229) in subjects with severely impaired RF and in those with ESRD on HD/HDF, respectively. Roxadustat’s maximum concentration (Cmax) and terminal elimination half-life (t1/2) were comparable between groups. The PK of roxadustat and its metabolites were not affected by HD/HDF. In subjects with normal RF, the amount of roxadustat excreted unchanged in urine was <1%; urinary excretion and renal clearance of roxadustat and its metabolites decreased with lower baseline RF. Mean AUCinf and t1/2 for roxadustat’s circulating metabolites were higher in subjects with severely impaired RF and with ESRD on HD/HDF than in those with normal RF. Metabolite to parent ratio of AUCinf was <1% for O-glucuronide- and O-glucoside-roxadustat and <10% for sulphate of hydroxy-roxadustat. EPO baseline-corrected AUC from administration to last measurable concentration and EPO maximum effect were higher in subjects with severely impaired RF or with ESRD on HD/HDF than in those with normal RF. Conclusion The AUC for roxadustat and its metabolites was higher in patients with severely impaired RF or with ESRD on HD/HDF than in those with normal RF. Roxadustat’s Cmax and t1/2 were comparable among all groups. Roxadustat and its metabolites were not significantly cleared by HD/HDF.

Pharmacokinetic and pharmacodynamic basis for developing modified-release formulations of furosemide

Furosemide has a narrow absorption window restricted to the upper part of the gastrointestinal tract due to its ionization in the gut. Therefore, dissolution of the dose entirely in the stomach was the main aim in the development of gastroretentive extended-release dosage forms in order to sustain its delivery without losing bioavailability. Just as pharmacokinetics (PK) is a prerequisite for observing a pharmacodynamic response (PD) in the individual, the intensity of such a response could also modulate the PK of the drug whenever some physiological mechanism triggered an increase or decrease in the renal fraction of cardiac output. The urine volume increase caused by furosemide triggers the arrival of a supplementary number of molecules at the site of action, and therefore its urinary excretion rate experiences an increase in line with this new cardiac output distribution. It was found that the diuretic effect of furosemide conditioned its urinary excretion clearance. Therefore, its urinary recovery would not reflect the systemic bioavailability of formulations, but both their actual bioavailability at the biophase and their diuretic responses instead. Extended drug input yielded a more efficient diuretic response than the whole dose given rapidly, provided that drug levels at the action site were above the minimum effective concentration. A deep knowledge in the pharmacodynamics-pharmacokinetics (PD-PK) relationships is fundamental to achieve successful furosemide dosage forms. The relation between: drug excretion rates and the intensity of urine output; diuretic efficiency and drug delivery rates; and the benefit of sustained drug delivery are here analyzed. All this together leads us to conclude that the best strategy for developing gastroretentive dosage forms of furosemide would be by including two moieties within the formulation: an accelerated-release loading dose and an extended-release maintenance dose.

Mechanistic Modeling of the Hepatic Disposition of Estradiol-17β-Glucuronide in Sandwich-Cultured Human Hepatocytes.

Estradiol-17β-glucuronide (E217G) is an estrogen metabolite that has cholestatic properties. In humans, circulating E217G is transported into hepatocytes by organic anion transporting polypeptides (OATPs) and is excreted into bile by multidrug-resistance associated protein 2 (MRP2). E217G is also a substrate of the basolateral efflux transporters MRP3 and MRP4, which translocate E217G from hepatocytes to blood. However, the contribution of basolateral efflux to hepatocyte disposition of E217G has not been evaluated previously. To address this question, E217G disposition was studied in sandwich-cultured human hepatocytes and mechanistic modeling was applied to calculate clearance values (mean ± S.D.) for uptake, intrinsic biliary excretion (CLint,bile) and intrinsic basolateral efflux (CLint,BL). The biliary excretion index of E217G was 45% ± 6%. The CLint,BL of E217G [0.18 ± 0.03 (ml/min)/g liver) was 1.6-fold higher than CLint,bile [0.11 ± 0.06 (ml/min)/g liver]. Simulations were performed to study the effects of increased CLint,BL and a concomitant decrease in CLint,bile on hepatic E217G exposure. Results demonstrated that increased CLint,BL can effectively reduce hepatocellular and biliary exposure to this potent cholestatic agent. Simulations also revealed that basolateral efflux can compensate for impaired biliary excretion and, vice versa, to avoid accumulation of E217G in hepatocytes. However, when both clearance processes are impaired by 90%, hepatocyte E217G exposure increases up to 10-fold. These data highlight the contribution of basolateral efflux transport, in addition to MRP2-mediated biliary excretion, to E217G disposition in human hepatocytes. This elimination route could be important, especially in cases where basolateral efflux is induced, such as cholestasis. SIGNIFICANCE STATEMENT: The disposition of the cholestatic estrogen metabolite estradiol-17β-glucuronide (E217G) was characterized in sandwich-cultured human hepatocytes. The intrinsic basolateral efflux clearance was estimated to be 1.6-fold higher than the intrinsic biliary excretion clearance, emphasizing the contribution of basolateral elimination in addition to biliary excretion. Simulations highlight how hepatocytes can effectively cope with increased E217G through the regulation of both basolateral and biliary transporters.

Combined Supplementation with Glycine and Tryptophan Reduces Purine-Induced Serum Uric Acid Elevation by Accelerating Urinary Uric Acid Excretion: A Randomized, Single-Blind, Placebo-Controlled, Crossover Study.

The authors previously confirmed the serum uric acid-lowering effects of the combination of glycine and tryptophan in subjects with mild hyperuricemia. This study examined whether combined supplementation with glycine and tryptophan suppressed the elevation in serum uric acid levels caused by purine ingestion and accelerated urinary uric acid excretion in subjects with lower urate excretion using a randomized, single-blind, placebo-controlled, crossover clinical trial design. Healthy Japanese adult males with lower urate excretion ingested water containing purines in addition to dextrin (placebo), tryptophan, glycine, or a glycine and tryptophan mixture. The combined supplementation with glycine and tryptophan significantly reduced the elevated serum uric acid levels after purine ingestion. Glycine alone and in combination with tryptophan significantly increased urinary uric acid excretion and urate clearance compared with the effects of the placebo. Urinary pH increased by the ingestion of the mixture. These results suggested that the improved water solubility of uric acid due to increased urinary pH contributed to the increase of urinary uric acid excretion.

Towards Further Verification of Physiologically-Based Kidney Models: Predictability of the Effects of Urine-Flow and Urine-pH on Renal Clearance.

In vitro-in vivo extrapolation (IVIVE) of renal excretory clearance (CLR) using the physiologically based kidney models can provide mechanistic insight into the interplay of multiple processes occurring in the renal tubule; however, the ability of these models to capture quantitatively the impact of perturbed conditions (e.g., urine flow, urine pH changes) on CLR has not been fully evaluated. In this work, we aimed to assess the predictability of the effect of urine flow and urine pH on CLR and tubular drug concentrations (selected examples). Passive diffusion clearance across the nephron tubule membrane was scaled from in vitro human epithelial cell line Caco-2 permeability data by nephron tubular surface area to predict the fraction reabsorbed and the CLR of caffeine, chloramphenicol, creatinine, dextroamphetamine, nicotine, sulfamethoxazole, and theophylline. CLR values predicted using mechanistic kidney model at a urinary pH of 6.2 and 7.4 resulted in prediction bias of 2.87- and 3.62-fold, respectively. Model simulations captured urine flow-dependent CLR, albeit with minor underprediction of the observed magnitude of change. The relationship between drug solubility, urine flow, and urine pH, illustrated in simulated intratubular concentrations of acyclovir and sulfamethoxazole, agreed with clinical data on tubular precipitation and crystal-induced acute kidney injury. This study represents the first systematic evaluation of the ability of the mechanistic kidney model to capture the impact of urine flow and urine pH on CLR and drug tubular concentrations with the aim of facilitating refinement of IVIVE-based mechanistic prediction of renal excretion.

Ursodeoxycholate Restores Biliary Excretion of Methotrexate in Rats with Ethinyl Estradiol Induced-Cholestasis by Restoring Canalicular Mrp2 Expression.

The in vivo relevance of ursodeoxycholate (UDCA) treatment (100 mg/kg/day, per oral tid for 5 days before cholestasis induction followed by the same dosing for 5 days) on hepatic function was investigated in rats with 17α-ethinylestradiol (EE, 10 mg/kg, subcutaneous for 5 days)-induced experimental cholestasis. The bile flow rate and the expression level of hepatic multidrug resistance-associated protein 2 (Mrp 2) that were decreased in cholestasis were restored after UDCA treatment. Consistent with this, the biliary excretion clearance (CLexc,bile) of a representative Mrp2 substrate—methotrexate (MTX)—was decreased in cholestatic rats but was restored after UDCA treatment. Consequently, the plasma concentrations of MTX, which were increased by cholestasis, were decreased to control levels by UDCA treatment. Thus, the restoration of CLexc,bile appears to be associated with the increase in Mrp2 expression on the canalicular membrane by UDCA treatment followed by Mrp2-mediated biliary excretion of MTX. On the other hand, the hepatic uptake clearance (CLup,liver) of MTX was unchanged by cholestasis or UDCA treatment, suggestive of the absence of any association between the uptake process and the overall biliary excretion of MTX. Since UDCA has been known to induce the expression of canalicular MRP2 in humans, UDCA treatment might be effective in humans to maintain or accelerate the hepatobiliary elimination of xenobiotics or metabolic conjugates that are MRP2 substrates.

Modulated function of multidrug resistance-associated proteins in cisplatin-induced acute renal failure rats.

The effect of cisplatin-induced acute renal failure (ARF) on the function and expression of multidrug resistanceassociated proteins (MRPs) was evaluated in rats. Rats received an intraperitoneal injection of cisplatin (9 mg/kg), and the induction of ARF state with high plasma concentrations of indoxyl sulfate and creatinine was observed 72 h after cisplatin treatment. The function of MRPs in the liver, kidney and brain was evaluated by measuring the tissue accumulation and biliary excretion of 2,4-dintrophenyl-S-glutathione (DNP-SG), a substrate for MRPs, after administration of 1-chloro-2,4-dintrobenzene (CDNB), a precursor of DNP-SG, in rats. The levels of MRP1-4 expression were evaluated by Western blot analysis. Effect of ARF plasma components on MRP function was also examined by using calcein acetoxymethyl ester (calcein-AM) in HepG2 cells. In ARF rats (72 h after cisplatin treatment), the accumulation of DNP-SG in the liver, kidney and brain was significantly higher than those in control and cisplatin-treated rats (1 h after treatment). In ARF rats, intrinsic biliary excretion clearance of DNP-SG, estimated by dividing the biliary excretion rate of DNP-SG with the liver concentration, was also significantly reduced, though the expression levels of MRP1-4 in the liver remained unchanged. ARF rat plasma (5%) significantly increased the accumulation of calcein, a MRP substrate, in HepG2 cells after application of calcein-AM. In conclusion, MRP function was found to be suppressed not only in the kidney but also in the liver and brain in cisplatin-induced ARF rats, possibly due to the accumulation of some MRP substrates/inhibitors in plasma.

Delineating the Role of Various Factors in Renal Disposition of Digoxin through Application of Physiologically Based Kidney Model to Renal Impairment Populations.

Development of submodels of organs within physiologically-based pharmacokinetic (PBPK) principles and beyond simple perfusion limitations may be challenging because of underdeveloped in vitro-in vivo extrapolation approaches or lack of suitable clinical data for model refinement. However, advantage of such models in predicting clinical observations in divergent patient groups is now commonly acknowledged. Mechanistic understanding of altered renal secretion in renal impairment is one area that may benefit from such models, despite knowledge gaps in renal pathophysiology. In the current study, a PBPK kidney model was developed for digoxin, accounting for the roles of organic anion transporting peptide 4C1 (OATP4C1) and P-glycoprotein (P-gp) in its tubular secretion, with the aim to investigate the impact of age and renal impairment (moderate to severe) on renal drug disposition. Initial PBPK simulations based on changes in glomerular filtration rate (GFR) underestimated the observed reduction in digoxin renal excretion clearance (CLR) in subjects with moderately impaired renal function relative to healthy. Reduction in either proximal tubule cell number or the OATP4C1 abundance in the mechanistic kidney model successfully predicted 59% decrease in digoxin CLR, in particular when these changes were proportional to reduction in GFR. In contrast, predicted proximal tubule concentration of digoxin was only sensitive to changes in the transporter expression/ million proximal tubule cells. Based on the mechanistic modeling, reduced proximal tubule cellularity and OATP4C1 abundance, and inhibition of OATP4C1-mediated transport, are proposed as possible causes of reduced digoxin renal secretion in renally impaired patients.

Effects of Adjuvant-Induced Inflammation on Disposition of Diclofenac and Its Metabolites in Perfused Rat Liver

The reactive metabolites of diclofenac (DF) such as 1-O-acyl glucuronide (DF-Glu) are hypothesized to result in idiosyncratic hepatotoxicity. However, it is unclear whether inflammation affects the hepatic disposition of DF and its metabolites. To clarify the alterations in the disposition of DF and its metabolites in inflammatory conditions, we performed in situ perfused rat liver experiments. Using adjuvant arthritis rats as a model of inflammation, the elimination of DF, 4’-hydroxydiclofenac, and DF-Glu from the perfusate was observed to be delayed in comparison with the control. Parameter sensitivity analysis for hepatic DF disposition revealed that the area under the plasma concentration-time curve (AUC) and the maximum concentration (Cmax) of DF-Glu in the liver markedly increased along with a decrease in intrinsic excretion clearance of DF-Glu (CLint,bile,Glu) and an increase in intrinsic glucuronidation clearance (CLint,Glu) of DF-Glu. It is possible that the elimination of DF-Glu from the perfusate in adjuvant arthritis rats was delayed via reduction of biliary excretion of DF-Glu.

Antioxidant and antihyperglycaemic effects of naringenin arrest the progression of diabetic nephropathy in diabetic rats

Background Long-standing diabetes declines the kidney function and is responsible for diabetic nephropathy (DN). The plant and the phytoconstituents have a promising therapeutic potential in the management of diabetes and diabetes complications. Aim This study aimed to evaluate the protective effect of naringenin in diabetic-induced nephropathy in experimental rats. Materials and methods Diabetes was induced in Sprague-Dawley rats fed a high-fat diet and injected with streptozotocin (STZ) (35 mg/kg body weight, intraperitoneal). At 48 h after injection, hyperglycaemia was confirmed by estimating the blood glucose levels and the rats were left untreated for 4 weeks. The diabetic rats were orally treated with different doses of naringenin 25 and 50 mg/kg body weight, for the next 4 weeks. At the end of the treatment body weight and kidney weight were recorded, serum and urine were used for various biochemical estimations. Oxidative stress levels and histopathological studies were performed on isolated kidneys. The efficacy of treatment was statistically analysed with diabetic control rats. Results Increased blood glucose, lipid levels and abnormal kidney functions were noticed in diabetic rats. Moreover, increased levels of oxidative stress markers and altered histological structure were noted in the kidney of diabetic rats, which mimic DN. Naringenin treatments (25 and 50 mg/kg) in diabetic rats significantly restored their kidney functions and reversed hyperglycaemia and lipids level. Hyperfiltration and increased microalbuminuria, urinary albumin excretion and creatinine clearance were effectively attenuated within 4 weeks of naringenin treatments. Increased levels of oxidative stress and histological alteration were restored towards normal. Conclusion Naringenin treatments in diabetic rats arrest the progression of DN due to its multivariate actions such as antihyperglycaemic and antioxidant.

Recent Advances in Understanding of Kinetic Interplay Between Phase II Metabolism and Efflux Transport.

Mechanistic understanding of the metabolism-transport interplay assumes great importance in pharmaceutical fields because the knowledge can help to interpret drug/xenobiotic metabolism and disposition studies as well as the drug-drug interactions in vivo. About 10 years ago, it started to recognize that cellular phase II metabolism is strongly influenced by the excretion (efflux transport) of generated metabolites, a kinetic phenomenon termed "phase II metabolism-transport interplay". This interplay is believed to have significant effects on the pharmacokinetics (bioavailability) of drugs/chemicals undergoing phase II metabolism. In this article, we review the studies investigating the phase II metabolism-transport interplay using cell models, perfused rat intestine, and intact rats. The potential confounding factors in exploring such interplay is also summarized. Moreover, the mechanism underlying the phase II metabolism-transport interplay is discussed. Various studies with engineered cells and rodents have demonstrated that there is an interaction (interplay) between phase II enzymes and efflux transporters. This type of interplay mainly refers to the dependence of phase II (conjugative) metabolism on the activities of efflux transporters. In general, inhibiting efflux transporters or decreasing their expression causes the reductions in metabolite excretion, apparent excretion clearance (CLapp) and total metabolism (fmet), as well as an increase in the intracellular level of metabolite (Ci). The deconjugation mediated by hydrolase (acting as a "bridge") is essential for the interplay to play out based on pharmacokinetic modeling/simulations, cell and animal studies. The hydrolases bridge the two processes (i.e., metabolite formation and excretion) and enable the interplay thereof (a bridging effect). Without the bridge, metabolite formation is independent on its downstream process excretion, thus impact of metabolite excretion on its formation is impossible. Deconjugation (mediated by hydrolases) plays an essential role in the conjugation-transport interplay.

Effects of exenatide on glycemia and renal water and ion excretion differ in frogs and rats

The aim of this study was to compare the effects of exenatide, a glucagon-like peptide-1 (GLP-1) mimetic, on glucose and water–salt homeostasis in animals with different levels of renal proximal tubular reabsorption, rats (Rattus norvegicus) and frogs (Rana temporaria). Following the glucose tolerance test, in rats exenatide promoted fast recovery of normoglycemia, whereas in frogs it delayed this process. In water-loaded rats, exenatide augmented solute-free water clearance and enhanced natriuresis in furosemide-treated animals. In frogs, exenatide did not alter the urinary flow rate, urinary sodium excretion and solute-free water clearance under water diuresis and furosemide treatment. It is suggested that the involvement of GLP-1 in regulation of water–salt homeostasis in mammals was preceded by a key evolutionary transformation, the increase in the glomerular filtration rate and proximal tubular reabsorption.

Novel minimal physiologically-based model for the prediction of passive tubular reabsorption and renal excretion clearance

PurposeDevelop a minimal mechanistic model based on in vitro–in vivo extrapolation (IVIVE) principles to predict extent of passive tubular reabsorption. Assess the ability of the model developed to predict extent of passive tubular reabsorption (Freab) and renal excretion clearance (CLR) from in vitro permeability data and tubular physiological parameters. MethodsModel system parameters were informed by physiological data collated following extensive literature analysis. A database of clinical CLR was collated for 157 drugs. A subset of 45 drugs was selected for model validation; for those, Caco-2 permeability (Papp) data were measured under pH6.5–7.4 gradient conditions and used to predict Freab and subsequently CLR. An empirical calibration approach was proposed to account for the effect of inter-assay/laboratory variation in Papp on the IVIVE of Freab. ResultsThe 5-compartmental model accounted for regional differences in tubular surface area and flow rates and successfully predicted the extent of tubular reabsorption of 45 drugs for which filtration and reabsorption were contributing to renal excretion. Subsequently, predicted CLR was within 3-fold of the observed values for 87% of drugs in this dataset, with an overall gmfe of 1.96. Consideration of the empirical calibration method improved overall prediction of CLR (gmfe=1.73 for 34 drugs in the internal validation dataset), in particular for basic drugs and drugs with low extent of tubular reabsorption. ConclusionsThe novel 5-compartment model represents an important addition to the IVIVE toolbox for physiologically-based prediction of renal tubular reabsorption and CLR. Physiological basis of the model proposed allows its application in future mechanistic kidney models in preclinical species and human.

Breast Cancer Resistance Protein and P-Glycoprotein Influence In Vivo Disposition of 11C-Erlotinib.

(11)C-erlotinib is a PET tracer to distinguish responders from nonresponders to epidermal growth factor receptor-targeted tyrosine kinase inhibitors and may also be of interest to predict distribution of erlotinib to tissues targeted for treatment. The aim of this study was to investigate if the known interaction of erlotinib with the multidrug efflux transporters breast cancer resistance protein (humans, ABCG2; rodents, Abcg2) and P-glycoprotein (humans, ABCB1; rodents, Abcb1a/b) affects tissue distribution and excretion of (11)C-erlotinib and has an influence on the ability of (11)C-erlotinib PET to predict erlotinib tissue distribution at therapeutic doses. Wild-type and Abcb1a/b or Abcg2 knockout mice underwent (11)C-erlotinib PET/MR scans, with or without the coinjection of a pharmacologic dose of erlotinib (10 mg/kg) or after pretreatment with the ABCB1/ABCG2 inhibitor elacridar (10 mg/kg). Integration plot analysis was used to determine organ uptake (CLuptake) and biliary excretion (CLbile) clearances of radioactivity. (11)C-erlotinib distribution to the brain was restricted by Abcb1a/b and Abcg2, and CLuptake into the brain was only significantly increased when both Abcb1a/b and Abcg2 were absent (wild-type mice, 0.017 ± 0.004 mL/min/g of tissue; Abcb1a/b((-/-))Abcg2((-/-)) mice, 0.079 ± 0.013 mL/min/g of tissue; P < 0.001). The pretreatment of wild-type mice with elacridar increased CLuptake into the brain to levels comparable to Abcb1a/b((-/-))Abcg2((-/-)) mice (0.090 ± 0.007 mL/min/g of tissue, P < 0.001). The absence of Abcb1a/b and Abcg2 led to a 2.6-fold decrease in CLbile (wild-type mice, 0.025 ± 0.005 mL/min/g of tissue; Abcb1a/b((-/-))Abcg2((-/-)) mice, 0.0095 ± 0.001 mL/min/g of tissue; P < 0.001). There were pronounced differences in distribution of (11)C-erlotinib to the brain, liver, kidney, and lung and hepatobiliary excretion into intestine between animals injected with a microdose and pharmacologic dose of erlotinib. ABCG2, ABCB1, and possibly other transporters influence in vivo disposition of (11)C-erlotinib and thereby affect its distribution to normal and potentially also tumor tissue. Saturable transport of erlotinib leads to nonlinear pharmacokinetics, possibly compromising the prediction of erlotinib tissue distribution at therapeutic doses from PET with a microdose of (11)C-erlotinib. The inhibition of ABCB1 and ABCG2 is a promising approach to enhance brain distribution of erlotinib to increase its efficacy in the treatment of brain tumors.

Quantitative Evaluation of mMate1 Function Based on Minimally Invasive Measurement of Tissue Concentration Using PET with [(11)C]Metformin in Mouse.

To evaluate the function of multidrug and toxin extrusion proteins (MATEs) using (11)C-labeled metformin ([(11)C]metformin) by positron emission tomography (PET). PET was performed by intravenous bolus injection of [(11)C]metformin. Pyrimethamine at 0.5 and 5mg/kg was intravenously administered to mice 30min prior to the scan. Integration plot analysis was conducted for calculating liver (CLuptake,liver), kidney (CLuptake,kidney) tissue uptake, intrinsic biliary (CLint,bile) and urinary (CLint,urine) excretion clearances of [(11)C]metformin. Visualization by PET showed that pyrimethamine increased concentrations of [(11)C]metformin in the liver and kidneys, and decreased the concentrations in the urinary bladder without changing the blood profiles. Pyrimethamine had no effect on the CLuptake,liver and CLuptake,kidney, which were similar to the blood-flow rate. CLint,bile with regard to the liver concentration was unable to be determined, but administration of 0.5 and 5mg/kg of pyrimethamine increased the liver-to-blood ratio to 1.6 and 2.3-fold, respectively, indicating that pyrimethamine inhibited the efflux of [(11)C]metformin from the liver. CLint,urine with regard to the corticomedullary region concentrations was decreased 37 and 68% of the control by administration of 0.5 and 5mg/kg of pyrimethamine, respectively (P < 0.05). Tissue concentration based investigations using [(11)C]metformin by PET enables the functional analysis of MATEs in the liver and kidneys.

Molecular mechanisms governing different pharmacokinetics of ginsenosides and potential for ginsenoside-perpetrated herb-drug interactions on OATP1B3.

Ginsenosides are bioactive saponins derived from Panax notoginseng roots (Sanqi) and ginseng. Here, the molecular mechanisms governing differential pharmacokinetics of 20(S)-protopanaxatriol-type ginsenoside Rg1 , ginsenoside Re and notoginsenoside R1 and 20(S)-protopanaxadiol-type ginsenosides Rb1, Rc and Rd were elucidated. Interactions of ginsenosides with human and rat hepatobiliary transporters were characterized at the cellular and vesicular levels. A rifampin-based inhibition study in rats evaluated the in vivo role of organic anion-transporting polypeptide (Oatp)1b2. Plasma protein binding was assessed by equilibrium dialysis. Drug-drug interaction indices were calculated to estimate potential for clinically relevant ginsenoside-mediated interactions due to inhibition of human OATP1Bs. All the ginsenosides were bound to human OATP1B3 and rat Oatp1b2 but only the 20(S)-protopanaxatriol-type ginsenosides were transported. Human multidrug resistance-associated protein (MRP)2/breast cancer resistance protein (BCRP)/bile salt export pump (BSEP)/multidrug resistance protein-1 and rat Mrp2/Bcrp/Bsep also mediated the transport of the 20(S)-protopanaxatriol-type ginsenosides. Glomerular-filtration-based renal excretion of the 20(S)-protopanaxatriol-type ginsenosides was greater than that of the 20(S)-protopanaxadiol-type counterparts due to differences in plasma protein binding. Rifampin-impaired hepatobiliary excretion of the 20(S)-protopanaxatriol-type ginsenosides was effectively compensated by the renal excretion in rats. The 20(S)-protopanaxadiol-type ginsenosides were potent inhibitors of OATP1B3. Differences in hepatobiliary and in renal excretory clearances caused markedly different systemic exposure and different elimination kinetics between the two types of ginsenosides. Caution should be exercised with the long-circulating 20(S)-protopanaxadiol-type ginsenosides as they could induce hepatobiliary herb-drug interactions, particularly when patients receive long-term therapies with high-dose i.v. Sanqi or ginseng extracts.