Enantioselective drug-protein interaction between mexiletine and plasma protein

Objectives This study examined the interaction of mexiletine enantiomers with human plasma, human serum albumin (HSA), and human α1‐acid glycoprotein (hAGP), and characterized the binding modes of mexiletine enantiomers with hAGP in the molecular level. Methods Enantiomer separation of mexiletine was performed using precolumn derivatization chiral HPLC. The ultrafiltration technique was used to separate the free mexiletine in plasma matrix. Molecular dynamics simulations and free energy calculations were assessed using molecular mechanics and the generalized Born surface area method. Key findings Significant differences in enantioselective binding to human plasma were observed (R > S). The hAGP–mexiletine binding profile exhibited similar enantioselectivity (R > S) to that in human plasma, whereas HSA–mexiletine interaction was S > R at pH 7.4. Moreover, the results of comparative studies indicated that mexiletine had the highest binding affinity for F1‐S, a variant of hAGP. Based on the computational studies, residues such as Arg90, Leu79, Ser89 and Phe89 showed an energy difference of more than ?0.35 kcal/mol between the enantiomers. Conclusions hAGP may be one of the key proteins leading to the enantioselective protein bindings of mexiletine in human plasma (R > S). The residues Arg90, Leu79, Ser89 and Phe89 of hAGP may have important roles in the observed enantioselectivity.     

Stereoselective binding of the glucuronide conjugates of carprofen enantiomers to human serum albumin

The stereoselective binding of carprofen enantiomers and carprofen glucuronide diastereomers to human serum albumin (HSA) was studied using an ultrafiltration method. Carprofen glucuronides exhibit a considerable and stereoselective affinity to HSA, although less than that seen for the parent enantiomers. The (S)-glucuronide showed a higher binding affinity to HSA than the (R)-glucuronide. The (S)-enantiomer of unmetabolized carprofen was bound to fatty acid free HSA to a much greater extent than the (R)-enantiomer. Warfarin reduced the binding of the glucuronides to a greater extent than did diazepam, but diazepam displaced the unconjugated enantiomers to a greater extent than did warfarin. These results suggest differences in binding region between the carprofen enantiomers and their glucuronides on the albumin molecule.     

Irreversible Binding of Tolmetin Glucuronic Acid Esters to Albumin in Vitro

Tolmetin glucuronide (TG), extracted and purified from human urine, was incubated with albumin in vitro. The degradation profile and irreversible binding to protein were investigated and kinetic parameters calculated. Standard conditions were as follows: TG, 30 µg/ml; human serum albumin (HSA), 3%; pH 7.45; 37°C. Lower pH enhanced TG stability and reduced both the extent and the rate of irreversible binding. HSA also increased TG stability, compared to protein-free buffer, but the opposite was observed with bovine serum albumin (BSA). With BSA, irreversible binding was much less, but the rate of adduct formation was the same as with HSA. Essentially fatty acid free HSA behaved similarly to HSA. Preincubation of HSA with warfarin, or diazepam, or an excess of tolmetin, did not influence irreversible binding significantly. In buffer, acyl migration led predominantly to one isomer. This isomer bound irreversibly to HSA, although more slowly and to a lesser extent than the 1-isomer. Incubation of TG with poly-L-lysine also resulted in irreversible binding but to a lesser extent than with HSA. Our results suggest that there is more than one binding mechanism, with the preferential pathway a function of the isomers present and the experimental conditions.     

Stereoselective drug distribution and anticoagulant potency of the enantiomers of phenprocoumon in rats

The elimination, distribution and anticoagulant activity of S(—)-, R(+)-, and R,S(±)-phenprocoumon were determined in male Wistar-Lewis rats after intravenous injection of a single dose of 0·6 mg kg^?1. From the plasma concentrations which elicited the same anticoagulant effect, S(—)-phenprocoumon was 4 to 5 times more potent than R(+)-phenprocoumon. The potency of the racemate was between those of the enantiomers. The mean biologic half-life of the S(—)-enantiomer was shorter (12·5 h) than that of R(+)-phenprocoumon (17·8 h). No differences were observed in the apparent volume of distribution. However, the mean liver: plasma concentration ratio was higher for the S(—)-(6·9) than for the R(+)-enantiomer (5·2). At a total concentration of 16·8 μg ml^?1 the percentage of unbound drug in rat serum was significantly higher for the S(—)- (1·13%) than that for the R(+)-enantiomer (0·76%). Values obtained for the racemate were always between those of the enantiomers. It is concluded that stereoselective differences in the distribution between plasma and liver, and consequently in the rate of elimination are most likely due to stereoselective differences in serum protein binding. The greater anticoagulant potency of the S(—)- over the R(+)-enantiomer, cannot be explained primarily by the observed pharmacokinetic differences.     

Enantioselective disposition of clenbuterol in rats

Clenbuterol is a long‐acting β2‐adrenoceptor agonist and bronchodilator that is used for the treatment of asthma, but the desired activities reside almost exclusively in the (‐)‐R‐enantiomer. This study examined enantioselectivity in the disposition of clenbuterol following administration of clenbuterol racemate to rats. Concentrations of clenbuterol enantiomers in plasma, urine and bile were determined by LC‐MS/MS assay with a Chirobiotic T column. This method was confirmed to show high sensitivity, specificity and precision, and clenbuterol enantiomers in 0.1 ml volumes of plasma were precisely quantified at concentrations as low as 0.25 ng/ml. The pharmacokinetic profiles of clenbuterol enantiomers following intravenous and intraduodenal administration of clenbuterol racemate (2 mg/kg) in rats were significantly different. The distribution volume of (‐)‐R‐clenbuterol (9.17 l/kg) was significantly higher than that of (+)‐S‐clenbuterol (4.14 l/kg). The total body clearance of (‐)‐R‐clenbuterol (13.5 ml/min/kg) was significantly higher than that of the (+)‐S‐enantiomer (11.5 ml/min/kg). An in situ absorption study in jejunal loops showed no difference in the residual amount between the (‐)‐R‐ and (+)‐S‐enantiomers. Urinary clearance was the same for the two enantiomers, but biliary excretion of (‐)‐R‐clenbuterol was higher than that of the (+)‐S‐enantiomer. The fractions of free (non‐protein‐bound) (‐)‐R‐ and (+)‐S‐clenbuterol in rat plasma were 48.8% and 33.1%, respectively. These results indicated that there are differences in the distribution and excretion of the clenbuterol enantiomers, and these may be predominantly due to enantioselective protein binding. Copyright © 2013 John Wiley & Sons, Ltd.     

Stereoselective binding of chiral anti-diabetic drug nateglinide to plasma proteins

The binding of nateglinide (NA) enantiomers with human plasma (HP), human serum albumin (HSA) and bovine serum albumin (BSA) was investigated. The protein binding was studied over a drug concentration range of 5-100 μM at a protein concentration of 600 μM. Unbound drug concentrations were determined by direct chiral liquid chromatography using chiralcel OJ-RH column. At therapeutic drug concentrations, the protein binding of each enantiomer was >98%. The results showed that the binding of NA enantiomers was stereoselective, mutually competitive and non-linear. The binding characteristics were, however, opposite for the two most important plasma binding proteins. Opposite stereo-selectivity was observed between BSA and HSA while stereo-selectivity was identical between HSA and HP. Scatchard analysis was used to illustrate the different binding affinities of NA enantiomers to BSA, HSA and HP. The interaction between enantiomers observed in HP and serum albumins was confirmed as a competitive type interaction at the high affinity site. Scatchard analysis was used to illustrate the different binding affinities of NA enantiomers to BSA, HSA and HP.     

Uptake and Stereoselective Binding of the Enantiomers of MK-927, a Potent Carbonic Anhydrase Inhibitor,by Human Erythrocytes in Vitro

MK-927 [5,6-dihydro-4H-4(isobutylamino)thieno(2,3-B)thiopyran-2-sulfonamide-7.7 dioxide], a potent carbonic anhydrase inhibitor, contains a chiral center and exists as a racemate. In order to understand the kinetic behavior of the enantiomers of MK-927 in the body, the uptake and binding of these compounds were studied in human erythrocytes in vitro. Since no degradation or metabolism of the enantiomers occurred during incubation in blood, one can describe the equilibration of the drugs between plasma and erythrocytes by a closed two-compartment system. Erythrocytes were considered as a compartment composed of two parts: one in which free drug is exchangeable to plasma and the other in which drug is tightly bound to carbonic anhydrase in a Michaelis–Menten type binding. After the addition of the enantiomers individually to fresh blood, they were taken up by erythrocytes rapidly in a concentration-dependent manner. The time to achieve equilibrium decreased as the concentration increased, suggesting saturation of binding sites. With the assumption of simple diffusion, the binding and transfer kinetics were determined simultaneously by computer fitting. There were no Stereoselective differences in the transfer process of the enantiomers across the erythrocyte membrane, while binding of the enantiomers exhibited stereoselectivity. The penetration of the unbound enantiomer across the erythrocyte cell membrane was rapid, with a mean transit time of about 3 sec. The S-( + )-enantiomer was bound to the high-affinity carbonic anhydrase isoenzyme more strongly than the R-( – )-enantiomer by approximately 10-fold. For the low-affinity isoenzyme, the R-( – )-enantiomer was bound more strongly than the S-( + )-enantiomer.     

Direct high pressure liquid chromatographic analysis and preliminary pharmacokinetics of enantiomers of oxazepam and temazepam with their corresponding glucuronide conjugates

Three high pressure liquid chromatographic systems for the separation of oxazepam, temazepam and their glucuronides (system A), the separation of theirR,S glucuronide diastereomers (system B) and the chiral separation of the parent drugs (system C) are described. Preliminary pharmacokinetics ofR,S-oxazepam andR,S-temazepam in a human volunteer reveal that the protein binding of the glucuronides is lower than that of the parent drugs, but that there is no difference in protein binding between theR-oxazepam/temazepam andS-oxazepam/temazepam and their corresponding glucuronides. TheS-glucuronide is the main metabolite formed and excreted by man. The plasma ratioR/S-glucuronide is 11 for both oxazepam and temazepam. The renal clearances ofR-temazepam, andS-temazepam are similar, and those ofR-oxazepam andS-oxazepam tend to be different.     

Effect of naproxen co-administration on valproate disposition

The effects of co-administration of the antiepileptic agent valproic acid (VPA) and the non-steroidal anti-inflammatory drug naproxen (NAP) on their relative dispositions (particularly with respect to glucuronidation) were investigated in human volunteers. Seven healthy males received each drug alone and then in combination (orally twice daily for seven days, 500 mg sodium VPA, 500 mg NAP). On day 7 of each dosing phase, serial plasma and 24 h urine samples were collected for analysis. Co-administration of NAP resulted in significant increases (about 20%, p<0.05) in the apparent plasma clearance of total VPA and in the unbound fraction of VPA in plasma, with the apparent plasma clearance of unbound VPA being unchanged. There were associated increases in the formation clearances to urinary VPA-glucuronide and 3-oxo-VPA, though these were relatively greater for the glucuronidation pathway (and remained significant when formation clearances were calculated using the unbound fraction of drug in plasma). The data thus point to a shift towards glucuronidation as a result of the NAP-induced increase in the unbound fraction of VPA in plasma. By contrast, VPA co-administration caused a decrease (of about 10%, p<0.05) in the apparent plasma clearance of total NAP. Taken in hand with in vitro results showing a VPA-induced displacement (of about 40%) of NAP from plasma protein binding sites, the data strongly support a role for diminished glucuronidation of NAP and its desmethyl metabolite in the presence of co-administered VPA.     

ENANTIOSELECTIVE DISTRIBUTION OF VERAPAMIL AND NORVERAPAMIL INTO HUMAN AND RAT ERYTHROCYTES: THE ROLE OF PLASMA PROTEIN BINDING

In this in vitro study, the distribution of the enantiomers of verapamil (VER) and its active metabolite, norverapamil (NOR), into the red blood cells (RBCs) of humans and rats was investigated using a chiral liquid chromatographic assay. When plasma was replaced with buffer, the distribution of VER and NOR enantiomers into both human and rat RBCs was substantial (RBC:blood concentration ratios, 1·39–1·79), non-stereoselective, concentration (125–1000 ng mL⁻¹) linear, and species independent. However, in the presence of plasma, the RBC distribution of VER and NOR was stereoselective, with opposite stereoselectivity for human (S>R) and rat (R>S) blood. Additionally, the presence of plasma caused a reduction in the extent of RBC distribution for both VER and NOR enantiomers and in some cases resulted in nonlinearity in the RBC distribution of the enantiomers. Plasma protein binding studies revealed opposite stereoselectivity in the free fractions in human (S>R) and rat (R>S) plasma for both VER and NOR. These data suggest that the stereoselective protein binding is responsible for the apparent stereoselectivity in the RBC distribution of VER and NOR. The data are also in agreement with the opposite stereoselectivity in the plasma concentrations of VER observed in vivo in rats and humans.     

Development and validation of an LC‐MS/MS method after chiral derivatization for the simultaneous stereoselective determination of methylenedioxy‐methamphetamine (MDMA) and its phase I and II metabolites in human blood plasma

3,4‐Methylenedioxymethamphetamine (MDMA, ecstasy) is a racemic drug of abuse and its two enantiomers are known to differ in their dose‐response curves. The S‐enantiomer was shown to be eliminated at a higher rate than the R‐enantiomer. The most likely explanation for this is a stereoselective metabolism also claimed in in vitro studies. Urinary excretion studies showed that the main metabolites in humans are 4‐hydroxy 3‐methoxymethamphetamine (HMMA) 4‐sulfate, HMMA 4‐glucuronide and 3,4‐dihydroxymethamphetamine (DHMA) 3‐sulfate. For stereoselective pharmacokinetic analysis of phase I and phase II metabolites in human blood plasma useful analytical methods are needed. Therefore the aim of the presented study was the development and validation of a stereoselective liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method for the simultaneous quantification of MDMA, 3,4‐methylenedioxyamphetamine, DHMA, DHMA 3‐sulfate, HMMA, HMMA 4‐glucuronide, HMMA 4‐sulfate, and 4‐hydroxy 3‐methoxyamphetamine in blood plasma for evaluation of the stereoselective pharmacokinetics in humans. Blood plasma samples were prepared by simple protein precipitation and afterwards all analytes were derivatized using N‐(2,4‐dinitro‐5‐fluorophenyl) L‐valinamide resulting in the formation of diastereomers which were easily separable on standard reverse phase stationary phases. This simple and fast method was validated according to international guidelines including specificity, recovery, matrix effects, accuracy and precision, stabilities, and limits of quantification. The method proved to be selective, sensitive, accurate and precise for all tested analytes except for DHMA. Copyright © 2014 John Wiley & Sons, Ltd.     

Binding of 3-alkyl-1,4-benzodiazepin-2-one stereoisomers to human serum albumin

The binding of both enantiomers of 3-alkyl-1,4-benzodiazepin-2-ones to human serum albumin (HSA) strengthens with increasing size of the 3-alkyl substituent. 3,3-Dimethylderivatives show diminished binding affinities although they bind in the favourable conformation assumed by the (S)-enantiomers. N(1)-Methyl substitution increases the enantioselectivity of binding by selectively enhancing the binding strength of (S)-antipodes.     

Stereoselective Interactions of Organic Cations with the Organic Cation Transporter in OK Cells

Recent studies have suggested that certain organic cations, such as pindolol and the diastereomers, quinine and quinidine, may be stereoselectively secreted by the kidney in humans. The goal of this study was to determine if the enantiomers of pindolol, verapamil, and disopyramide and the diastereomers, quinine and quinidine, interact stereoselectively with the organic cation transporter in the brush border membrane of the opossum kidney cell line. All organic cations tested inhibited the uptake of tetraethylammonium (TEA). The IC₅₀ values (mean ± SD) were as follows: quinine (17 ± 2 µM), quinidine (51 ± 13 µM), S-(–)-pindolol (23 ± 4 µM), R-( + )-pindolol (30 ± 4 µM), S-(–)-verapamil (0.4 ± 0.04 µM), R-( + )-verapamil (7 ± 2 µM), R-(–)-disopyramide (27 ± 4 µM), and S-( + )-disopyramide (66 ± 12 µM). Each individual organic cation pair showed significant stereoselective differences in their IC₅₀ values, with quinine, S-(–)-pindolol, S-(–)-verapamil, and R-(–)-disopyramide being the more potent species. Both enantiomers of pindolol, quinine, and quinidine appear to exhibit simple competitive inhibition of TEA uptake based upon a derived slope similar to 1.0, using a sigmoidal inhibition model. The enantiomers of verapamil and disopyramide exhibited a slope of much less than 1.0, suggesting a more complex interaction of these organic cations with the TEA transporter. Our results suggest that organic cations stereoselectively interact with the organic cation transporter in the brush border membrane of OK cells. Stereoselective interactions with the organic cation transporter may be responsible for the stereoselective renal clearance of basic drugs known to occur in humans.     

吡喹酮对映异构体与血浆蛋白结合的立体选择性

本实验用平衡透析法研究吡喹酮(POT)对映异构体与血浆蛋白结合的立体选择性。当透析液药物初始浓度在1～32μmol/L范围内时,(±)-POT及其对映异构体与血浆蛋白的结合属非饱和类型。无论牛血清白蛋白(BSA)然度为1.47或5.88×10^-4mol/L,(+)-PQT结合能力(nK)均大于(-)-POT,表现较明显的立体选择性。(-)-和(+)-PQT与兔血浆蛋白结合率分别为73.7±4.4和58.3±10.1%(n=8,p<0.05),其立体选择性与BSA结合的选择方向相反。(-)-和(+)-POT与人血浆蛋白结合率分别为81.9±4.2和83.2±6.9%(n=10,P>0.05),无明显立体选择性。POT对映异构体和血浆蛋白结合立体选择性表现种属差异。     

Effects of turpentine oil pretreatment on β-blocker pharmacokinetic parameters in rats

Abstract— Turpentine oil treatment (0·2 mL kg^?1, s.c.) was used to increase the plasma concentration of α₁-acid glycoprotein (0·13 mg mL^?1 in control rats) to 1·72 mg mL^?1 after 2 days, and allow assessment of its effects on the pharmacokinetics and stereoselective binding of three β-blockers. Racemates (5 mg kg^?1) were administered intravenously to control and turpentine oil-pretreated rats and the plasma concentrations were determined up to 90 min. Stereoselective analysis showed the apparent distribution volume and the area under plasma concentration-time curves (AUC) of R-(+)-propranolol to be, respectively, one-quarter and twice those of the S-(–)-enantiomer and differences in pharmacokinetic parameters between the two were magnified by turpentine oil pretreatment. Pharmacokinetic parameters of oxprenolol enantiomers were essentially similar for the controls but after turpentine oil pretreatment, a higher affinity of the R-(+)-enantiomer for plasma was observed. Acebutolol enantiomers behaved non-stereospecifically throughout. These results were consistent with predictions from the in-vitro stereospecific binding properties of these agents to purified rat α₁-acid glycoprotein.     

Plasma protein binding of the enantiomers of hydroxychloroquine and metabolites

Summary The in vitro binding of the enantiomers of hydroxychloroquine and its three major metabolites in pooled plasma obtained from four healthy volunteers and the binding of the enantiomers of hydroxychloroquine to purified plasma proteins has been investigated.The plasma protein binding of hydroxychloroquine was found to be stereoselective. The (S)-enantiomer of hydroxychloroquine was 64% bound in plasma, while (R)-hydroxychloroquine was 37% bound. Fifty % of (S)-hydroxychloroquine was bound to a 40 g·l^–1 solution of human serum albumin, while only 29% of the (R)-enantiomer was bound. The enantioselectivity of hydroxychloroquine binding was reversed in a 0.7 g·l^–1 solution of ₁-acid glycoprotein with (R)-hydroxychloroquine being bound to a greater extent than its optical antipode (41% versus 29%). The enantiomers of the metabolites of hydroxychloroquine were bound to a similar extent to plasma and purified plasma proteins.Binding of hydroxychloroquine to plasma and purified proteins was found to be linear over the racemic concentration range of 50 to 1000 ng·ml^–1 and hydroxychloroquine metabolite binding to plasma was linear over the range 25 to 500 ng·ml^–1.     

Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins

Abstract

1.?This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR).

2.?Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (f_u?>?0.8) to extensive (f_u?<?0.1). Of the compounds screened, the highest binding to both HSA and LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for β-estradiol (a polar, neutral compound).

3.?The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP.

4.?Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP.     

Stereoselective disposition of carvedilol in man after intravenous and oral administration of the racemic compound

The racemic compound carvedilol is a multiple-action oral antihypertensive drug that exhibits both vasodilator and non-selective beta-adrenergic blocking activities. The effects of the levorotatoryS-enantiomer [S( – )-CARV] are vasodilatation and beta-blockade. TheR (+)-enantiomer [R (+)-CARV] is a pure vasodilating agent. Quantitative determination of the enantiomers in human plasma by HPLC was carried out after formation of diastereoisomers with the chiral reagent 2,3,4,6-tetraO-acetyl--d-glucopyranosyl isothiocyanate (GITC). The pharmacokinetics of the enantiomers were studied following i. v. (12.5 mg in 1 h) and p. o. (50 mg) administration of racemic carvedilol in ten healthy male subjects according to a randomized crossover design. The AUCs ofS (–)-CARV were significantly lower than those ofR (+)-CARV after both i. v. and p. o. administration. The systemic clearance of the two enantiomers was significantly different, whereas half-lives and apparent distribution volumes were comparable. Following p. o. administration, the absolute bioavailability (31.1% and 15.1%, respectively) and maximal plasma concentrations ofR (+ )-CARV were twice those ofS (–)-CARV A similar difference was found in the half-lives. A close correlation existed between enantiomeric ratios after i.v. and after p. o. administration, demonstrating slight intraindividual variability. The preferential systemic clearance of theS ( – )-enantiomer suggests stereoselective hepatic metabolism of carvedilol, becoming especially apparent after p. o. administration. The small intrasubject variability in enantiomer ratios indicates a relatively constant relation of beta-blockade to vasodilation during chronic treatment.     

Apparent stereoselectivity in propafenone uptake by human and rat erythrocytes

The distribution of propafenone (PPF) enantiomers between the plasma and red blood cells (RBCs) was investigated using human and rat blood. In separate experiments, effects of incubation time (15-60 min), blood concentration (100-5000 ng ml-1), and plasma proteins on the RBC uptake of the enantiomers were studied. In both humans and rats, the distribution of propafenone enantiomers into RBCs was rapid, extensive, and stereoselective. However, the extent of RBC uptake and the direction of stereoselectivity were different in these two species. In humans, preferential distribution of (-)-PPF into RBCs resulted in lower plasma concentrations for this enantiomer, whereas in rat plasma, (-)-PPF was the dominant enantiomer. When the plasma was replaced with buffer, the stereoselectivity in the RBC uptake of the enantiomers was abolished. This suggested that stereoselective protein binding may be responsible for this phenomenon. A direct measurement of the extent of binding of PPF enantiomers to rat and human plasma proteins further confirmed this. Moreover, the distribution of the enantiomers in RBCs was not affected by low temperatures or addition of ouabain, suggesting passive diffusion as the underlying mechanism. These results suggest that stereoselective red blood cell uptake may be responsible, at least in part, for the differences in the plasma pharmacokinetics of PPF enantiomers observed after the drug administration to humans and rats.     

Carvedilol Stereopharmacokinetics in Rats: Affinities to Blood Constituents and Tissues

Carvedilol, a lipophilic β-adrenoceptor antagonist with vasodilating activities, is characterized by a high as well as stereoselective metabolic clearance and distribution volume. Tissue distribution of carvedilol enantiomers and their conjugates were determined under steady-state conditions in rats (p.o., 10 mg/kg, repetitive dosage; n = 5) and after single i.v. administration in control rats and rats with surgical portacaval shunt (pcs) (10 mg/kg; n = 3 each group). In addition, in vitro plasma protein binding was evaluated. - The plasma protein binding of carvedilol in rats is > 98% for total plasma (tp) and > 96% for rat serum albumin (rsa) solution (4%), with enantioselectivity ratios of 1.53 (tp) and 1.27 (rsa). Significantly higher unbound fractions were observed in pcs rats, in part due to reduced protein concentrations. - In contrast to plasma, where a preponderance of the R-enantiomer with an S/R ratio of 0.6 was found, S-carvedilol was predominant in all tissues (heart, liver, kidneys, lung, spleen, muscle, and adipose tissue), with S/R ratios of 1.3-1.4 in most of these tissues and 2.3 in liver. This preferential tissue partitioning of S-carvedilol was in accordance with its higher unbound fraction in plasma. Carvedilol accumulated predominantly in the highly perfused and/or eliminating organs liver, kidneys, and lung (tissue/plasma ratios; lung: S 76, R 34; liver: S 21, R 5; kidney: S 8, R 3). A similarly enantioselective distribution into the heart of control as well as pcs rats was observed, where the S-enantiomer concentrations exceeded the plasma concentrations 7-fold. Probably because of the impaired liver function in pcs rats with increased importance of the renal route, kidney concentrations were higher in these rats. The kidney/plasma ratio was elevated approximately 2-fold for the parent compound (control: S 7, R 2; pcs: S 14, R 4) and 4-fold for the R-carvedilol conjugate (control: S 2, R 1; pcs: S 3, R 4). The conjugates of carvedilol were detectable in all organs, with significantly smaller concentrations than those of the aglycones and with varying stereoselectivities.