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Rencofilstat (RCF) demonstrated antifibrotic effects in preclinical models and was safe and well tolerated in Phase 1 studies. The aim of this Phase 2a study was safety, tolerability, pharmacokinetics, and exploration of efficacy biomarkers in subjects with nonalcoholic steatohepatitis (NASH). This Phase 2a, multicenter, single-blind, placebo-controlled study randomized 49 presumed F2/F3 subjects to RCF 75 mg once daily (QD), RCF 225 mg QD, or placebo for 28 days. Primary safety and tolerability endpoints were explored using descriptive statistics with post hoc analyses comparing active to placebo groups. Pharmacokinetics were evaluated using population pharmacokinetics methods. Efficacy was explored using biomarkers, transcriptomics, and lipidomics. RCF was safe and well tolerated, with no safety signals identified. The most frequently reported treatment-emergent adverse events were constipation, diarrhea, back pain, dizziness, and headache. No clinically significant changes in laboratory parameters were observed, and RCF pharmacokinetics were unchanged in subjects with NASH. Alanine transaminase (ALT) reduction was greater in active subjects than in placebo groups. Nonparametric analysis suggested that ALT reductions were statistically different in the 225-mg cohort compared with matching placebo: -16.3 ± 25.5% versus -0.7 ± 13.4%, respectively. ProC3 and C6M reduction was statistically significant in groups having baseline ProC3 15.0 ng/ml. RCF was safe and well tolerated after 28 days in subjects with presumed F2/F3 NASH. Presence of NASH did not alter its pharmacokinetics. Reductions in ALT, ProC3, and C6M suggest direct antifibrotic effects with longer treatment duration. Reductions in key collagen genes support a mechanism of action via suppression and/or regression of collagen deposition. Conclusion: These results support advancement of rencofilstat into a larger and longer Phase 2b study.

Rencofilstat (RCF) is a novel cyclophilin inhibitor under development for the treatment of nonalcoholic steatohepatitis and hepatocellular carcinoma. This phase 1, randomized, open-label study in healthy participants assessed the relative bioavailability of a single dose of RCF 225-mg soft gelatin capsules in both fasted and high-fat conditions. Forty-four participants were enrolled to either the fasted (n = 24) or the high-fat fed (n = 20) arm. Noncompartmental pharmacokinetics were evaluated following a single 225-mg oral dose. Administration of RCF with a high-fat meal led to increases in maximum concentration, area under the concentration-time curve (AUC) from time 0 to 24 hours, and AUC from time 0 to infinity fed-to-fasted geometric mean ratios of 102.2%, 114.5%, and 132.9%, respectively. All AUC geometric mean ratios were outside of the 80% to 125% range, suggesting that a high-fat meal can increase the extent of RCF exposure. Time to maximum concentration increased from 1.5 to 1.8 hours in the fasted and high-fat groups, respectively, suggesting slightly delayed absorption. High fat intake may delay gastric emptying while increasing the absorption and bioavailability of RCF. No treatment-emergent adverse events were observed in the fasted group, and 1 treatment-emergent adverse event occurred in the high-fat meal group. The differences in observed whole-blood concentrations are unlikely to have clinically relevant effects given the wide therapeutic index of RCF demonstrated in previous phase 1 studies.

The amount of bisulfite (HSO3−, hydrated SO2) needed in beer to extend flavor stability is controversial with results that show elevated HSO3− levels in beer improves beer shelf-life analytically, ...

We previously reported that the non-immunosuppressive cyclophilin inhibitors (CypIs)-cyclosporin A analog CRV431 and sanglifehrin analog NV556-efficiently inhibit HCV replication in vitro. In this study, we asked whether they can also reduce HCV replication in vivo. We found that a single oral administration of CRV431 and NV556 to HCV-infected humanized-liver mice drastically reduced HCV blood levels. The antiviral effect was observed when CRV431 or NV556 were each individually administered with HCV, 3, 6 weeks or even 3 months post-infection when viral replication is robust. These results were confirmed in chimeric mice implanted with human hepatocytes isolated from three distinct donors. Remarkably, no viral rebound was observed 5 months after a single dose administration of 50 mg/kg of CRV431 or NV556 four weeks post-HCV infection, indicating the possibility of suppression of an established viral infection. Since we recently demonstrated that both CRV431 and NV556 also inhibit the development of liver fibrosis and hepatocellular carcinoma in nonviral-induced non-alcoholic steatohepatitis mouse models, our present data suggest that the two entirely structurally different CypIs-CRV431 and NV556-derived from unrelated natural products, represent attractive partners to current direct-acting agent (DAA) regimens for the treatment of hepatitis C and liver diseases.

PURPOSE: The objective of this study was to develop a self-microemulsifying drug delivery system (SMEDDS) formulation for the oral delivery of CRV431, a non-immunosuppressive analogue of cyclosporine A. Relative to cyclosporine A, CRV431 is poorly soluble in lipid solvents and thusly presents a challenge for the development of a formulation of sufficient oral bioavailability for clinical use. METHODS: The solubility of CRV431, a cyclosporine derivative, was determined in a range of commonly used surfactants, oils and co-solvents. A pseudo-ternary phase diagram was constructed from the most soluble excipients and prototype formulations, SERIES 1 and SERIES 2 were developed. The pharmacokinetics, following single oral doses of 1 and 3 mg/kg of CRV431 SMEDDS, was studied in healthy human volunteers using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). RESULTS: The maximum drug load for the SERIES 1 formulations was less than 40 mg/ml. Manipulation of the excipient ratios allowed for the development of SERIES 2 formulations, which had higher drug loading capacity and stability for CRV431 compared to SERIES 1. Further improvements allowed for the development of an optimized SMEDDS formulation containing up to 90 mg/ml CRV431 and which generated a microemulsion mean particle size of 25 nm when dispersed into aqueous media. The pharmacokinetics of the optimized CRV431 SMEDDS displayed excellent total body exposure and dose-proportional effects in humans, and high drug levels in the liver of rats. CONCLUSIONS: The developed SMEDDS formulation should allow for effective clinical development of CRV431, targeted to the treatment of liver diseases including hepatitis B (HBV), fibrosis, and hepatocellular carcinoma.

Previous studies show that cyclophilins contribute to many pathologic processes, and cyclophilin inhibitors demonstrate therapeutic activities in many experimental models. However, no drug with cyclophilin inhibition as the primary mode of action has advanced completely through clinical development to market. In this study, we present findings on the cyclophilin inhibitor, CRV431, that highlight its potential as a drug candidate for chronic liver diseases. CRV431 was found to potently inhibit all cyclophilin isoforms tested-A, B, D, and G. Inhibitory constant or IC50 values ranged from 1 to 7 nM, which was up to 13 times more potent than the parent compound, cyclosporine A (CsA), from which CRV431 was derived. Other CRV431 advantages over CsA as a nontransplant drug candidate were significantly diminished immunosuppressive activity, less drug transporter inhibition, and reduced cytotoxicity potential. Oral dosing to mice and rats led to good blood exposures and a 5- to 15-fold accumulation of CRV431 in liver compared with blood concentrations across a wide range of CRV431 dosing levels. Most importantly, CRV431 decreased liver fibrosis in a 6-week carbon tetrachloride model and in a mouse model of nonalcoholic steatohepatitis (NASH). Additionally, CRV431 administration during a late, oncogenic stage of the NASH disease model resulted in a 50% reduction in the number and size of liver tumors. These findings are consistent with CRV431 targeting fibrosis and cancer through multiple, cyclophilin-mediated mechanisms and support the development of CRV431 as a safe and effective drug candidate for liver diseases. SIGNIFICANCE STATEMENT: Cyclophilin inhibitors have demonstrated therapeutic activities in many disease models, but no drug candidates have yet advanced completely through development to market. In this study, CRV431 is shown to potently inhibit multiple cyclophilin isoforms, possess several optimized pharmacological properties, and decrease liver fibrosis and tumors in mouse models of chronic liver disease, which highlights its potential to be the first approved drug primarily targeting cyclophilin isomerases.

Hepatitis B virus (HBV) infection is a major health burden worldwide with 240 million chronically infected individuals. Nucleos(t)ide analogs and interferons are the current standards of care due to their suppression of HBV replication, but the treatments rarely eradicate HBV from individuals. Similar to current treatments for human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV) patients, improved HBV therapies will require the combination of multiple drugs which target distinct steps of the HBV life cycle. In this study, we tested the potential of a cyclophilin inhibitor, CRV431, to affect HBV replication in transgenic mice. We found that oral treatment with CRV431 (50 mg/kg/day) for a period of 16 days significantly reduced liver HBV DNA levels and moderately decreased serum HBsAg levels. We observed an additive inhibitory effect on liver HBV DNA levels in mice treated with a combination of low doses of CRV431 (10 mg/kg/day) and the nucleotide prodrug, tenofovir exalidex (TXL), (5 mg/kg/day). No toxicity was observed in CRV431-treated mice. Although it is well known that CRV431 neutralizes the peptidyl-prolyl isomerase activity of cyclophilins, its anti-HBV mechanism(s) of action remains unknown. Nevertheless, this study provides the first demonstration of a beneficial effect of a cyclophilin inhibitor in vivo in an HBV transgenic mouse model. Altogether our data reveal the potential of CRV431 to be part of improved new therapies for HBV patients.

We and others previously reported that the direct-acting agents (DAA) NS5A inhibitors (NS5Ai) and the host-targeting agents cyclophilin inhibitors (CypIs) inhibit HCV replication in vitro. In this study, we investigated whether the combination of NS5Ai and CypI offers a potent anti-HCV effect in vivo. A single administration of NS5Ai or CypI alone to HCV-infected humanized-mice inhibits HCV replication. The combination of NS5Ai with CypI suppresses HCV (GT1a, GT2a, GT3a and GT4a) replication in an additive manner. NS5Ai/CypI combinations provide a statistically more profound anti-HCV inhibition for GT2a and GT3a than GT1a and GT4a, leading to a fastest and deepest inhibition of GT2a and GT3a replications. Combining CypI with NS5Ai prevents the viral rebound normally observed in mice treated with NS5Ai alone. Results were confirmed in mice implanted with human hepatocytes from different donors. Therefore, the combination of NS5Ai with CypI may serve as a regimen for the treatment of HCV patients with specific genotypes and disorder conditions, which diminish sustain viral response levels to DAA, such as GT3a infection, cirrhosis, and DAA resistance associated with the selection of resistance-associated substitutions present at baseline or are acquired during treatment.

The cytochrome P450-mediated Phase I in vitro metabolism of CRV431 was studied using selective chemical inhibition and recombinant human enzymes. Additionally, the metabolic profile of CRV431 in human, rat, and monkey liver microsomes was investigated. Liver microsomes were incubated for 0–80 min with CRV431, and the metabolite profile was assessed by electrospray ionization liquid chromatography mass spectrometry (ESI-LCMS). CRV431 was extensively metabolized through oxidation to produce various hydroxylated and demethylated species. Species identified included monohydroxylated CRV431 (two distinct products), dihydroxylated CRV431, demethylated CRV431 (two distinct products), demethylated and hydroxylated CRV431 (two distinct products), didemethylated and hydroxylated CRV431, and didemethylated and dihydroxylated CRV431. The magnitude of metabolism was greatest in monkey, followed by human, followed by rat. Importantly, all of the species identified in human microsomes were correspondingly identified in monkey and/or rat microsomes. Human liver microsome studies using selective chemical inhibition, as well as studies using recombinant human cytochrome P450 enzymes, revealed that the major enzymes involved are cytochromes P450 3A4 and 3A5. Enzymes 1A2, 2B6, 2C8, 2C9, 2C19, and 2D6 are not involved in the in vitro metabolism of CRV431. This information will be useful for the further development of CRV431 both preclinically and clinically.

Aims Voclosporin is a novel calcineurin inhibitor intended for prevention of organ graft rejection and treatment of lupus nephritis. Pharmacokinetic drug interactions between voclosporin and a CYP3A inhibitor, inducer and substrate and a P-glycoprotein inhibitor and substrate were evaluated. Methods Voclosporin 0.4 mg kg−1 was administered to 24 subjects in each of five studies, as follows: every 12 h (Q12H) alone and concomitantly with ketoconazole 400 mg once daily (QD); single dose before and single dose after rifampin 600 mg QD; Q12H where midazolam 7.5 mg was administered as a single dose alone before voclosporin and with last the dose of voclosporin; Q12H alone and concomitantly with verapamil 80 mg every 8 h; and Q12H with digoxin 0.25 mg QD. The noncompartmental pharmacokinetic parameters maximal concentration (Cmax) and area under the concentration–time curve (AUC) were obtained, and geometric least squares mean ratios and 90% confidence intervals were evaluated. Results Ketoconazole increased voclosporin Cmax (6.4-fold) and AUC (18-fold); rifampin reduced voclosporin AUC (0.9-fold); voclosporin did not change exposure of midazolam or α-hydroxy-midazolam; verapamil increased voclosporin Cmax (2.1-fold) and AUC (2.7-fold); and voclosporin increased digoxin Cmax (0.5-fold), AUC (0.25-fold) and urinary excretion (0.2-fold). Conclusions Administration of voclosporin concomitantly with strong inhibitors and inducers of CYP3A resulted in increased and decreased exposures, respectively, and should be considered contraindicated. Drug–drug interactions involving voclosporin and CYP3A substrates are not expected. Administration of voclosporin concomitantly with inhibitors and substrates of P-glycoprotein resulted in increased voclosporin and substrate exposures, respectively. Appropriate concentration and safety monitoring is recommended with co-administration of voclosporin and P-glycoprotein substrates and inhibitors.

The aims of this population-pharmacokinetic/pharmacodynamic (POP-PKPD) analysis of voclosporin in renal allograft patients were to build a POP-PKPD model for voclosporin and calcineurin activity (CNa) and identify clinically relevant covariates that could assist dosing of the drug. POP-PKPD modeling was performed using a stochastic approximation of the standard expectation maximization (SAEM) algorithm for nonlinear mixed-effects as implemented in Monolix™ 3.2. Voclosporin whole blood concentrations were obtained from de novo renal allograft patients and assayed using a validated LC/MS/MS assay. CNa was measured using a (32)P-radiolabeled assay. A two-compartment model with simultaneous sigmoid inhibitory Emax model was used to describe the PKPD relationship between voclosporin concentration and CNa. The POP-PKPD model was then utilized to simulate an optimal initial dosing strategy. Eighty-seven patients were included in the POP-PKPD study. Population mean estimates (relative standard error, rse) for oral clearance (CL/F) and first compartment volume of distribution (V1), were 717 mL min(-1) (35%) and 2010 mL (17%), respectively. Maximum CNa Inhibition (Imax), effective concentration (C50), and baseline immunosuppression (S0) were 0.87 pmol/min/mg (8.0%), 123 ng/mL (10%), and 1.15 pmol/min/mg (4.0%), respectively. Covariate analyses demonstrated that age and body surface area significantly influenced CL/F: CLi=717(Agei/48.8)-0.57(BSAi/1.99)1.1, while serum triglycerides significantly altered S0: S0i=1.15(TRIGi/1.97)0.15.

Voclosporin is a novel calcineurin inhibitor intended for prevention of organ graft rejection and treatment of lupus nephritis. These studies evaluated the effect of renal or hepatic impairment on pharmacokinetics of voclosporin. Thirty-three subjects were enrolled into 1 of 4 groups based on renal function as defined by creatinine clearance and 18 subjects were enrolled into 1 of 3 groups based on hepatic function defined by Child-Pugh classes. Voclosporin 0.4 mg/kg was administered orally. Geometric mean ratios (renal/hepatic impairment-to-normal) and 90% confidence intervals for Cmax and AUC were calculated. A default no-effect interval of 80-125% was set. Although 90% confidence intervals exceeded the no-effect intervals for both parameters, individual Cmax and AUC plots indicate almost complete overlapping range of values for mild and moderate renal impairment and normal subjects. Severe renal impairment resulted in a 1.5-fold increase in AUC without an increase in Cmax . Mild to moderate hepatic impairment resulted in a 1.5- to 2-fold increase in voclosporin exposure. Voclosporin can be administered safely to patients with mild to moderate renal impairment without dose modification. Appropriate safety monitoring with concentration-based adjustments in transplantation are recommended for patients with severe renal impairment, and for patients with hepatic impairment.

Voclosporin (VCS) is a novel calcineurin (CN) inhibitor intended for prevention of organ graft rejection and treatment of lupus nephritis. These studies evaluated the single ascending dose pharmacokinetics (PK) and pharmacodynamics (PD, CN activity) of VCS and the effect of food. VCS was administered orally in single doses of 0.25 through 4.5 mg/kg in 62 subjects in the single ascending dose study and as a single oral 1.5 mg/kg dose to 18 subjects after fasting, consumption of a low-fat and high-fat meal. Non-compartmental PK, PD, and PKPD correlation were evaluated. Following single oral doses, systemic exposure increased in a linear manner and demonstrated 1:1 dose-proportional, first-order linear PK above 1.5 mg/kg. VCS inhibited CN activity in a dose-related fashion with maximal inhibition peaking at 3.0 mg/kg. PKPD correlation indicated an EC50 of 78.3 ± 6.8 ng/mL. Administration of VCS with a low-fat and high-fat meal decreased C(max) by 29% and 53%, respectively, and AUC(inf) by 15% and 25%, respectively. Following ascending single doses of VCS, exposure increased in a linear fashion. A food effect on exposure was demonstrated, with a more pronounced effect following a high-fat meal. VCS concentrations were also found to correlate with CN activity.

Dimethyl sulfide (DMS) is an important flavor compound in American and European lagers, so brewers strive to control its level in their products. Although the main approach to controlling DMS involves judicious selection of malting and wort production regimes to control one of its precursors, S-methyl methionine (SMM), DMS formation during fermentation by yeast caused by an enzyme coined "dimethyl sulfoxide (DMSO) reductase" cannot be overlooked. During the process of altering the wort carbohydrate spectrum from one predominantly made up of maltose to one much richer in glucose through adjunct changes, a significant reduction in beer DMS levels was noted. An investigation into malt SMM levels demonstrated no significant reduction in this main precursor, suggesting other mechanisms could be involved. Since it is known that glucose inhibits the development of both mitochondria and their unique lipid, cardiolipin, we speculated that this organelle could be involved. Through a series of replicated incubation experiments aimed at altering cardiolipin content in yeast, it was found that beer DMS levels at the end of fermentation were moderately higher (approx. 10 μg/L) under incubation conditions that promote cardiolipin formation, i.e., incubation in glycerol and 1-thyroxine. In comparison, conditions that limit cardiolipin formation (control and inositol incubation) resulted in lower DMS levels (P < 0.05). Assuming equal losses due to CO 2 stripping during fermentation, these results suggest a possible role involving yeast mitochondria. Two suggestions are provided, of which we believe the latter is most likely based on DMSO- and methionine sulfoxide (MetSO)-addition experiments conducted within this study. First, if a true bacterial-like DMSO reductase protein were responsible, then defects in iron-sulfur cluster (ISC) formation or protein maturation would be likely, because ISCs are catalytic sites for electron transfer of Fe-S proteins. However, due to complete MetSO inhibition of DMSO reduction to DMS in the yeast strain studied, it seems more plausible that this was the result of decreased MetSO reductase activity, specifically an MsrB protein, which is speculated to be localized to the mitochondria, and that somehow the cardiolipin composition of the mitochondrial membrane is involved.

A rapid, accurate, and reproducible liquid chromatography electrospray tandem mass spectrometry (LC/ESI-MS/MS) method was developed and validated for the therapeutic drug monitoring of voclosporin in human whole blood. Sample aliquots of 100muL were processed utilizing a protein precipitation procedure that contained a mixture of methanol, 0.2M ZnSO(4), and deuterated voclosporin internal standard. Supernatant was injected onto a Zorbax SB-C8, 2.1x12.5mm column (at 60 degrees C), and washed with water-acetonitrile, supplemented with 0.02% glacial acetic acid and 0.02mM sodium acetate, to remove poorly retained components. After washing, water-MeOH (with 0.02% glacial acetic acid and 0.02mM sodium acetate) was used to elute the voclosporin and internal standard to the Applied Biosystems/MDS-Sciex API3000 mass spectrometer for detection in multiple reaction monitoring. Analytical performance was assessed in the range of 1-200ng/ml in whole blood. This method has been used to quantify concentrations of voclosporin in whole blood from healthy volunteers participating in a pharmacokinetic study.

HCV-related liver disease is the main cause of morbidity and mortality of HCV/HIV-1 co-infected patients. Despite the recent advent of anti-HCV direct acting antivirals (DAAs), the treatment of HCV/HIV-1 co-infected patients remains a challenge, as these patients are refractory to most therapies and develop liver fibrosis, cirrhosis and liver cancer more often than HCV mono-infected patients. Until the present study, there was no suitable in vitro assay to test the inhibitory activity of drugs on HCV/HIV-1 co-infection. Here we developed a novel in vitro "co-infection" model where HCV and HIV-1 concurrently replicate in their respective main host target cells--human hepatocytes and CD4+ T-lymphocytes. Using this co-culture model, we demonstrate that cyclophilin inhibitors (CypI), including a novel cyclosporin A (CsA) analog, CPI-431-32, simultaneously inhibits replication of both HCV and HIV-1 when added pre- and post-infection. In contrast, the HIV-1 protease inhibitor nelfinavir or the HCV NS5A inhibitor daclatasvir only blocks the replication of a single virus in the "co-infection" system. CPI-431-32 efficiently inhibits HCV and HIV-1 variants, which are normally resistant to DAAs. CPI-431-32 is slightly, but consistently more efficacious than the most advanced clinically tested CypI--alisporivir (ALV)--at interrupting an established HCV/HIV-1 co-infection. The superior antiviral efficacy of CPI-431-32 over ALV correlates with its higher potency inhibition of cyclophilin A (CypA) isomerase activity and at preventing HCV NS5A-CypA and HIV-1 capsid-CypA interactions known to be vital for replication of the respective viruses. Moreover, we obtained evidence that CPI-431-32 prevents the cloaking of both the HIV-1 and HCV genomes from cellular sensors. Based on these results, CPI-431-32 has the potential, as a single agent or in combination with DAAs, to inhibit both HCV and HIV-1 infections.

Acebutolol (AC) is a chiral beta-adrenergic receptor-blocking agent, which has been shown to be clinically effective in hypertension. The plasma concentration-time profiles of AC exhibit two peaks following oral administration of racemate for both R- and S-enantiomers. In the present study, the absorption of AC after a single dose was studied as a function of gastric pH in male Sprague-Dawley rats. Furthermore, the effect of cimetidine (CIM) on pharmacokinetic parameters of AC and its metabolite diacetolol (DC) was evaluated. CIM (50 mg kg(-1)) was administered via jugular vein 30 min prior to AC administration to elevate the intragastric pH. AC (50 mg kg(-1)) was administered orally by gavage and serial blood samples were collected before and for 8h after AC administration. Plasma samples were assayed for AC and DC, pharmacokinetic parameters were estimated and compared with those of control. The concentration-time profiles and the pharmacokinetics of AC were unchanged after co-administration of CIM. The oral absorption of AC, as assessed by the area under the plasma concentration-time curve (AUC) and the amount of unchanged drug recovered in the urine were not affected by CIM. The amount of metabolite recovered in the urine and the rate of absorption, however, were significantly altered. These are unlikely to be of clinically importance as we have found that the extent of absorption was not changed. We, therefore, concluded that intragastric elevation of pH has no effect either on generation of multiple peaking or on pharmacokinetic parameters of AC.

Beta-adrenoceptor blocking drugs are widely used as effective antihypertensive and antianginal agents. We have determined the effect of beta-blockade in the rat to ascertain whether there are differences between metoprolol (MET) and acebutolol (AC) with respect to regional blood flow (RBF). Both AC and MET were administered as a single or multiple intravenous (iv) doses in Sprague-Dawley rats. Microspheres labelled with (85)Sr and (141)Ce were used to measure cardiac output (CO) and RBF before and after drug administration. CO and RBF were measured 1 and 10 min after the i.v. administration of AC (30 mg/kg) and MET (10 mg/kg). After acute administration of MET, CO decreased by 65% and 31% after 1 and 10 min measurements, respectively. These values were 54% and 28% for AC as compared with baseline values. After chronic administration of either AC or MET, however, there were no significant reductions in CO as compared with saline. Both MET and AC significantly reduced RBF in most organs either after 1 or 10 min measurements when compared with the baseline values. It is concluded that both AC and MET reduced CO and RBF after acute administration. The CO and RBF however, returned to normal after chronic administration.

Purpose. The effect of hyperlipidemia on nifedipine pharmacokinetics was studied. The mechanisms by which hyperlipidemia affects pharmacokinetics of drugs are mainly undetermined. Hyperlipidemia may decrease the fraction of unbound drug in plasma and/or decrease intrinsic ability of the cytochrome P-450 systems due to excess membrane cholesterol. Hyperlipidemia is a primary risk factor for coronary artery disease leading to hypertension and ischemic heart disease, for which nifedipine, a calcium channel blocker, is used.

Acebutolol (AC), is a chiral, beta-adrenergic blocking agent which possesses partial agonist activity and is metabolized to an equipotent chiral metabolite, diacetolol (DC). The enantiomeric disposition of AC is reported following racemic administration as a single oral (p.o., 50 mg kg(-1)) or as a multiple thrice daily intravenous (i.v.) or p.o. dosing for four days in male Sprague-Dawley rats (n = 6). Enantiomeric concentrations of AC and DC in plasma and urine were determined using a stereospecific HPLC assay. The bioavailabilities of R- and S-enantiomer were 0.40 and 0.39 after single dose administration of AC respectively. These values were increased to 0.51 and 0.53 after multiple dosing. Although no significant differences were found in AUC0-infinity after single i.v. as compared with AUC0-tau after multiple i.v. dosing of AC, the 39 and 45% increase in mean AUC0-tau were found after multiple p.o. dosing over the corresponding AUC0-infinity, for the single p.o. dose of AC for R- and S-enantiomer, respectively. The disposition of DC as well as the urinary excretion of metabolite was stereoselective in favor of R-enantiomer after oral administration of AC. These results indicate that AC enantiomers have low availability and moderate extraction through the first-pass metabolism in a rat model. The higher AUC values after p.o. multiple dosing may suggest a saturable first-pass metabolism of AC.

Previous studies with rats indicate that nifedipine undergoes both hepatic and extrahepatic presystemic metabolism after peroral (po) administration, and that its bioavailability is increased and absorption delayed by concomitant administration of grapefruit juice concentrate (GJC). Hence, the effects of GJC could be to delay stomach emptying and inhibit nifedipine metabolism in the small-intestinal wall and liver or, alternatively, to impede nifedipine absorption until reaching the large intestine where gut wall presystemic metabolism is not a factor. The mechanism(s) of action of GJC might be partially resolved by comparison with orange juice concentrate (OJC), which has a similar consistency but lacks inhibitory effects on nifedipine presystemic metabolism, and also by giving regular-strength solutions of the two juices, both on which should not significantly affect stomach emptying. This study compared the po bioavailability of nifedipine (6 mg kg-1) in male Sprague-Dawley rats coadministered GJC, OJC, grapefruit juice regular strength (GJRS), orange juice regular strength (OJRS), or (tap) water. Nifedipine plasma concentration-time profiles in the GJRS, OJRS, and (tap) water groups displayed a single peak. Both GJC and OJC groups have double-peak profiles (indicating delayed gastric emptying); however, the majority of the nifedipine dose in both cases was absorbed during the interval of the second peak, which occurred several hours postdosing. GJC significantly increased nifedipine bioavailability (relative bioavailability 2.02, compared with (tap) water), indicating that GJC may affect both extrahepatic and hepatic first-pass metabolism, although a reduction in systemic nifedipine clearance cannot be ruled out. Surprisingly, GJRS had no significant effect on nifedipine bioavailability. OJC did not increase nifedipine bioavailability, further suggesting that the delay in nifedipine absorption by GJC or OJC results from delayed gastric emptying.

The nifedipine gastrointestinal therapeutic system (GITS) produces relatively linear fractional absorption-time plots between 6 and 24 h and about 70% of the available dose is absorbed within 24 h in human beings. However, conventional single-phase in vitro dissolution tests with the GITS demonstrate pseudo zero-order release of nifedipine suspension between about 2 to 20 h and ≥90% of the labeled dose is released within 24 h. Using the two-phase dissolution test developed in part I, mean in vitro data obtained from 2 strengths of the nifedipine GITS were compared with mean in vivo data from a human bioequivalency study in anticipation of improved in vitro-in vivo correlations. For the in vivo data, deconvolution was performed using the Wagner-Nelson method (model-dependent, WN) and DeMonS which is a new method of numerical deconvolution (model-independent, DS). Calculated zero-order rates of drug absorption (0.95(WN)/1.03(DS) mg h −1 and 1.88(WN)/1.94(DS) mg h −1 ; 30 mg and 60 mg strengths, respectively) compared reasonably well with the calculated zero-order rates of drug transfer (0.96 and 2.02 mg h −1 ; 30 mg and 60 mg, respectively). These values, however, are considerably less than zero-order drug release rates obtained using single-phase dissolution methods or reported as the manufacturer's design specifications (1.7 and 3.4 mg h −1 ; 30 mg and 60 mg strengths, respectively). Hence, improved in vitro-in vivo correlations were demonstrated with the two-phase dissolution test, yielding 1:1 (Level A) correlations ( r 2 >0.99 in all cases).

The nifedipine gastrointestinal therapeutic system (GITS) incorporates a push–pull osmotic pump to release–in zero-order fashion–a finely-divided suspension of nifedipine, which must then undergo dissolution in the GI tract before the drug can be absorbed. Classical, differential (ALZA) and flow-through type dissolution methods adequately characterize the in vitro nifedipine suspension release rate from the nifedipine GITS: however, these methods fail to measure the in vitro dissolution rate of the suspended particles–a potentially significant shortcoming considering that nifedipine is poorly water-soluble (≤10 μg ml−1). Therefore, an in vitro two-phase dissolution system was developed. This system measured the rate of nifedipine `transfer' from an aqueous phase (simulated intestinal fluid, USP, without pancreatin containing a nifedipine GITS tablet) into an organic phase (n-octanol), a process dependent on release of the drug suspension from the tablet, dissolution in the aqueous phase and partitioning in the organic phase. For the 30 mg and 60 mg nifedipine GITS formulations tested the two-phase method indicated that about 90% of the drug was `transferred' within 30 h. This is in contrast to the results from single-phase dissolution methods showing that about 90% of drug is `released' within 24 h. Results obtained from the two-phase dissolution method appear to be in better agreement with published in vivo studies of the nifedipine GITS with regard to the rate and duration of nifedipine absorption from the GI tract. This emphasizes the importance of differentiating between drug release and drug dissolution for this type of formulation: but, the two-phase dissolution method may also be useful for other pharmaceutical formulations and poorly water-soluble drugs.

The peroral (p.o.) bioavailability of nifedipine is reported to range from about 45 to 58% in the rat; this compares favourably to human beings. The metabolism of nifedipine is similar in rats and humans (oxidation of the dihydropyridine ring), with the liver believed to be solely responsible for the systemic clearance of the drug and the observed first-pass effect after p.o. dosing. The purpose of this study was to determine whether intestinal metabolism also contributes to the first-pass elimination of nifedipine in the rat. The systemic availabilities of nifedipine doses given by po, intracolonic (i.c.), and intraperitoneal (i.p.) routes of administration were compared to that for an intravenous (i.v.) dose (in each case a dose of 6 mg kg-1 was given) using adult male Sprague-Dawley rats (249-311 g, n = 6 or 7/group). The geometric mean of systemic nifedipine plasma clearance after i.v. dosing was 10.3 mL min-1 kg-1. The nifedipine blood-to-plasma ratio was found to be about 0.59. Therefore, the systemic blood clearance of nifedipine was about 17.5 mL min-1 kg-1; which, compared to the hepatic blood flow of rats (55 to 80 mL min-1 kg-1) showed that nifedipine is poorly extracted by the liver (0.22 < or = EH < = 0.32). The mean absolute bioavailabilities of the p.o., i.p., and i.c. doses were 61, 90, and 100%, respectively. Assuming complete absorption of the extravascular nifedipine doses these results indicate that, in addition to hepatic extraction, substantial first-pass elimination of nifedipine occurs within the wall of the small intestine but not the colon of the rat.

Acebutolol (AC) is a chiral beta-adrenergic blocking drug, possessing intrinsic sympathomimetic activity (ISA), and is useful clinically as the racemate in treating hypertension. Utilizing a stereospecific high-performance liquid chromatographic (HPLC) assay, the enantiomeric disposition of AC and its major metabolite diacetolol (DC) are reported after intravenous administration of single 5, 15, 30, and 50 mg kg-1 doses of racemate to male Sprague-Dawley rats. The mean area under the plasma concentration versus time curve (AUC) values display a linear relationship with respect to the administered dose. No statistical differences are observed in apparent volume of distribution (Vd), terminal elimination half-life (t1/2), total body clearance (Clt), or renal clearance (Clr) with respect to dose administered. Generally, R-S ratios for AUC following AC administration are statistically different from unity (p < 0.05). However, for the 50 mg kg-1 doses the R-S ratio for AUC is not statistically different from one. For DC, the plasma disposition is nonstereoselective in plasma. The amount of R-DC recovered in urine, however, was greater than that of the antipode (R:S = 1.92 +/- 0.29). This study suggests that the enantiomeric disposition of intravenous AC is linear within the investigated range of 5-50 mg kg-1 racemate in rats.

Verapamil is a chiral calcium channel blocking drug which is useful clinically as the racemate in treating hypertension and arrhythmia. The published pharmacokinetic data for verapamil enantiomers in the rat model are limited. Utilizing a stereospecific high-performance liquid chromatographic (HPLC) assay, the enantiomeric disposition of verapamil is reported after intravenous (1·0 mg kg−1) and oral (10 mg kg−1) administration of racemic verapamil to the rat model. After intravenous administration the systemic clearance of R-verapamil was significantly greater than that of S-verapamil; 34·9 ± 7 against 2·7 ± 3·7 mL min−1 kg−1 (mean ± SD), respectively. After oral administration, the clearance of R-verapamil was significantly greater than that of S-verapamil, 889 ± 294 against 351 ± 109 mL min−1 kg−1, respectively. The apparent oral bioavailability of S-verapamil was greater than that of R-verapamil, 0·074 ± 0·031 against 0·041 ± 0·011, respectively. These data suggest that the disposition of verapamil in the rat is stereoselective; verapamil undergoes extensive stereoselective first-pass clearance after oral administration and the direction of stereoselectivity in plasma is opposite to that observed in the human. © 1997 John Wiley & Sons, Ltd.

Significant daily variability occurs in the bioavailability and peak plasma concentrations of nifedipine given as an immediate-release (IR) oral dosage form, which is attributed to increased absorption or reduced presystemic metabolism in the morning. The nifedipine gastrointestinal therapeutic system (GITS) is an oral osmotic delivery device that exhibits controlled-release properties within the gastrointestinal tract, and is designed to provide zero-order nifedipine release for nearly a full day. Thus, nifedipine's chronopharmacokinetic properties might be observed following single-dose administration of a GITS formulation by comparison of evening plasma concentrations with those of the following morning (e.g., 12 versus 24 h postdosing, respectively). Time-dependent and variable subject kinetics were assessed in a bioequivalency study conducted with 4 commercially available nifedipine GITS formulations (2 lots of both 30 and 60 mg strengths obtained from the same manufacturer) given in single-dose fashion to 12 healthy subjects. As expected, mean C max and AUC(0-t) values of the 60 mg doses were approximately double those of the 30 mg doses suggesting a linear dose versus concentration relationship; however, typical standards of bioequivalence were not met because of large inter- and intrasubject variability that is only partially explained by the relatively small number of study participants. This latter finding may have important ramifications for others attempting to develop a bioequivalent product. Interestingly, mean plasma nifedipine concentrations increased up to 24 h post-dosing with each of the 4 lots tested. In individual kinetic profiles, t max was measured as 24 h (corresponding to about 8 AM) more than half the time (56%), consistent with previous chronopharmacokinetic findings. Alternatively, the results suggest that the location of the GITS tablet in the gastrointestinal tract influences nifedipine bioavailability.

Biliary clearance (Clb) of sotalol (STL) enantiomers was assessed in anaesthetized Sprague-Dawley rats (419 +/- 9 g, mean +/- SEM, n = 4) following administration of a 10 mg kg-1 i.v. dose of the racemate. Clb for S- and R-STL (0.0675 +/- 0.0090 and 0.0662 +/- 0.0089 mL min-1 kg-1, respectively) represented approximately 0.3% of systemic clearance (Cls) values for S- and R-STL (20.4 +/- 2.2 and 20.7 +/- 2.0 mL min-1 kg-1, respectively). Bile:plasma concentration ratios at 1, 2, and 3 h post-dose were approximately 1.4, 1.3, and 1.2 for both STL enantiomers. Renal clearance (Clr) and intestinal clearance (Cli) of STL enantiomers were assessed in conscious Sprague-Dawley rats (325 g, n = 4) following administration of a 10 mg kg-1 i.v. dose of the racemate. STL enantiomers were predominantly eliminated intact in the urine: Clr for S- and R-STL (26.3 +/- 3.2 and 28.7 +/- 4.2 mL min-1 kg-1 respectively) accounted for approximately 96% of Cls for S- and R-STL (27.5 +/- 3.3 and 29.9 +/- 4.2 mL min-1 kg-1, respectively). Approximately 4% of the dose was recovered in the faeces, corresponding to Cli values of 1.16 +/- 0.17 and 1.26 +/- 0.19 mL min-1 kg-1 for S- and R-STL, respectively. Total recovery of the administered dose in urine and faeces was 99.7 +/- 0.2 and 99.8 +/- 0.5% for S- and R-STL, respectively. It is concluded from these results in the rat model that (i) STL enantiomers are predominantly eliminated intact in urine; (ii) STL enantiomers are excreted intact in bile, and to a much larger extent in the faeces, thus suggesting the presence of intestinal exsorption of STL; (iii) STL does not appear to be metabolized; and (iv) Cls, Clr, Clb, and Cli are negligibly stereoselective.

Sotalol (STL) is an amphoteric, chiral β-adrenergic blocking drug useful in the treatment of both hypertension and ventricular arrhythmias. In the human and rat, STL enantiomers are predominantly cleared from the body by the kidney as intact drug. The renal clearance (Cl r ) of STL enantiomers substantially exceeds the glomerular filtration rate (GFR) in the human and rat. In this report, the hypothesis that STL enantiomers are excreted by an active renal transport system was investigated in the rat by coadministering racemic STL (10 mg kg -1 ) with cimetidine, an inhibitor of renal tubular secretion of organic cations. To compare the effects of short-term and sustained cimetidine exposure on STL enantiomer disposition, cimetidine was administered either as a single bolus (30 mg kg -1 , n=7) immediately prior to the STL dose, or as a 30 mg kg -1 bolus plus a 50 mg kg -1 infusion over the 6 h study period (n = 7). Blood and urine samples were collected over 6 h, during which time anaesthesia was maintained via intraperitoneal administration of pentobarbital. Cimetidine bolus and cimetidine infusion reduced STL enantiomer Cl r by 43 and 59%, respectively, compared with respective saline controls. Significant stereoselectivity was observed in the cimetidine infusion group : systemic clearance, Cl r (R>S), and AUC (S>R), although the magnitude of stereoselectivity was less than 5%. This study supports the hypothesis that STL enantiomers are predominantly cleared from the rat via a renal cationic transport mechanism, and that this system can be competitively inhibited by the presence of cimetidine.

Verapamil is a racemic calcium channel-blocking drug that undergoes extensive hepatic first-pass metabolism to an active metabolite, norverapamil. The enantiomers of verapamil and norverapamil have differing negative inotropic, chronotropic, and dromotropic activities and differing effects on vascular smooth muscles; the S-enantiomers having greater activity. It is hypothesized that the R/S concentration ratio of verapamil enantiomers may be input-rate dependent. The pharmacokinetics of verapamil and norverapamil enantiomers were studied in 11 young, healthy male and female volunteers after oral administration of 80 mg immediate-release (IR) verapamil every 8 hours, and a 240 mg dose once daily of controlled-release (CR) formulation on two separate occasions. Both dosage regimens were continued for 1 week with a minimum 1-week period between the two drug treatments. After the last dose of each regimen, plasma samples were collected over the period corresponding to the dosing interval. Enantiomer concentrations were determined using a microwave-facilitated precolumn derivatization with high performance liquid chromatographic quantification. Stereospecific assay revealed that: (1) stereoselective R- and S-enantiomer disposition occurred regardless of formulation administered; (2) a trend of R:S concentration ratios of verapamil differed between the two formulations; and (3) fluctuations between Cmax and Cmin values of the two formulations were statistically different over respective dosing intervals (greater fluctuation after CR administration). Using nonstereospecific data analyses, however, the pharmacokinetic parameters for verapamil and norverapamil were similar for both formulations over a 24-hour period. We suggest that kinetic differences can be attributed to differences in release rates of drug from the tablet matrices. The relative bioavailabilities of verapamil and norverapamil from the two products may, therefore, be subject to input rate-dependent processes.

The protein binding of sotalol (STL) enantiomers was evaluated using an ultrafiltration technique with serum from young (32 +/- 2 years, n = 5) and elderly (73 +/- 6 years, n = 5) male and female humans, and young (8 weeks, n = 4) and elderly (60 weeks, n = 3) male Sprague-Dawley rats. Serum samples were collected and immediately frozen at -20 degrees C. Within 1 week, the serum samples were thawed at room temperature, and adjusted to pH 7.4 using 0.05 M phosphate buffer, pH 5.0. Aliquots were spiked with 250 ng mL-1 and 500 ng mL-1 of each STL enantiomer, placed in ultrafiltration sets (Microsep, 30K molecular weight cut-off), capped, equilibrated to 37 degrees C, and centrifuged at 1850g for 1.5 h at 37 degrees C. Aliquots of ultrafiltrate and unspun serum were analysed for STL enantiomer concentration using a stereospecific HPLC assay. In all groups, bound fraction was less than 7% for both STL enantiomers. There were no significant differences in bound fraction between groups, or between enantiomers. Adsorption of STL enantiomers to the ultrafiltration device and membrane, evaporative loss of serum samples during centrifugation, and protein concentration in each ultrafiltrate sample were all negligible. It is concluded that the binding of STL in human and rat serum at therapeutic concentrations and physiological temperature and pH is negligible and non-stereoselective.

This report describes an HPLC assay developed for the quantification of the enantiomers of lomefloxacin (LFLX), a quinolone antibiotic, in plasma. Following addition of racemic acebutolol (internal standard, IS), plasma samples were extracted at pH 7 with a mixture of chloroform-isopentyl alcohol-diethyl ether (71.25:3.75:25, v/v/v). The organic layer was evaporated, and LFLX and IS enantiomers in the resulting residue were derivatized with chloroform solutions of 1% triethylamine and 1% ( S )-(+)-(1-naphthyl)ethyl isocyanate, followed by 2% ethyl chloroformate (ECF) 1 min later. Ethanolamine was added 30 s after the addition of ECF. The enantiomers were separated as diastereomers on an 8 × 100 mm Radial Pak normal phase column using a mobile phase of hexane-chloroform-methanol (64.5:33:2.5, v/v/v) pumped at 2.0 ml min −1 . The IS was detected by fluorescence at 245 and 420 nm (excitation and emission, respectively) during the first 12 min, after which time the wavelengths were 280 and 470 nm for detection of LFLX. The method: (1) was sensitive and showed excellent linearity (10–1000 ng ml −1 , r 2 > 0.99) between added enantiomer concentrations and peak-area-ratio (LFLX/IS); and (2) separated LFLX and IS enantiomers within 25 min. The assay is suitable for the quantification of LFLX enantiomers in plasma samples.

Nifedipine (NIF), a 1,4-dihydropyridine calcium channel antagonist, undergoes photodegradation to dehydronifedipine (DNIF) upon exposure to ultraviolet (UV) light and to the nitroso analogue of dehydronifedipine (NDNIF) when exposed to sunlight. NIF photodegradation products do not contribute to clinical activity, thus the content of NIF must remain uniform between equipotent formulations. Large differences in light stability between bioequivalent NIF products could potentially result in the therapeutic failure of unstable preparations. Consequently, if large photostability differences do exist between NIF preparations, product substitution may not be warranted. The light stability of 10 intact immediate- or controlled-release oral NIF formulations, obtained from several European and North American manufacturers, was studied using direct continuous artificial sunlight exposure extending over a 12-week period. The content of both NIF and NDNIF for each product was measured to determine the extent of photodecomposition using a specific and sensitive reversed-phase high pressure liquid chromatographic (HPLC) method. In addition, NIF photodegradation was measured using both pure NIF powder and methanolic NIF solution to determine the effectiveness of the artificial sunlight source used in this study. After 12 weeks of artificial sunlight exposure, less than 3% of NDNIF (w/w initial NIF content) was present in each of the 10 tested dosage forms. Photodegradation was greater than 10% (w/w initial NIF content) in ∼5–10 min (mean t 1 2 = 31 min), and in ∼24 h (mean t 1 2 = 7.7 days) of artificial sunlight exposure for methanolic NIF solution and pure NIF powder samples, respectively. Therefore, the tested NIF formulations all appear to be photostable up to at least 12 weeks of continuous artificial sunlight exposure, compared with pure NIF powder and methanolic NIF solution. It is concluded that if therapeutic failures or pharmacodynamic differences between the tested NIF formulations were observed, photoinstability as a major contributory factor would be unlikely.

A high-performance liquid chromatographic (HPLC) assay suitable for the analysis of the enantiomers of the non-steroidal anti-inflammatory drug ibuprofen (IB) in plasma was developed. Following the addition of racemic fenoprofen as internal standard (I.S.), samples are acidified and extracted with a mixture of isooctane-isopropanol (95:5, v/v). After evaporation of the organic layer, the drug and I.S. are derivatized with S-(-)-1(1-naphthyl)ethylamine (S-NEA) after addition of ethyl chloroformate as the coupling reagent. Ethanolamine is added 3 min after the addition of S-NEA to react with the excessive ethyl chloroformate. The resultant diastereomers corresponding to IB and I.S. were chromatographed at ambient temperature on a 100 mm x 4.6 mm I.D. C18 reversed-phase column using acetonitrile-water-acetic acid-triethylamine (60:40:0.1:0.02) as the mobile phase pumped at a flow-rate of 1.2 ml/min. Detection of the fluorescent chromophore was at 280 and 320 nm for excitation and emission, respectively. The suitability of the assay for clinical pharmacokinetic studies of IB was determined by the analysis of plasma samples obtained from a healthy volunteer, following administration of a single 400-mg oral dose of racemic IB.

The determination of the in vitro dissolution profiles of three different modified-release formulations of (±)-verapamil were determined and compared to a reference (Calan SR). The determination of (±)-verapamil utilized a microwave-facilitated derivatization reaction with an enantiomerically pure reagent followed by measurement of drug by reversed-phase high-performance liquid chromatography (HPLC). The dissolution profile of each of the four modified-release formulations was followed for the first hour in simulated gastric fluid (pH 1.2) and then in simulated intestinal fluid (pH 7.5) for up to a total of 12 h. The resulting dissolution profiles of each modified-release formulation suggested that only one of the three test formulations had a similar dissolution to the Calan SR reference. Interestingly, the (S:R)-enantiomer ratios of two of the test products were significantly different from unity, and the third test product was also significantly different from unity if 4 out of 5 outliers were omitted. It is suggested that dissolution testing of modified-release formulations containing chiral active ingredients must be stereospecific in order to discern whether a drug-excipient interaction occurs. Additionally, it may be suggested that dissolution techniques relying more upon diffusion of medium through the tablet matrix, rather than erosion of the tablet, may accentuate enantiomeric differences in release rates. © 1993 Wiley-Liss, Inc.

The chiral beta-blocker, sotalol (STL), is marketed as a racemic mixture. Although both STL enantiomers have equal Class III antiarrhythmic activity, beta-blocking activity has been ascribed mainly to the R-enantiomer. The pharmacokinetics of STL enantiomers were studied in young (mean age 32 +/- 3 years), healthy male volunteers after oral administration of 160 mg. Subsequent plasma and urine samples were collected over 24 hours, and STL enantiomer concentrations were determined using a stereospecific high-performance liquid chromatography assay. There were no significant differences between pharmacokinetic parameters of enantiomers. The area under the time-concentration curves (mean +/- standard deviation [SD]) were 6.95 +/- 0.85 and 6.76 +/- 1.2 (mg/L)hour for S- and R-STL, respectively. Maximal plasma concentrations of S- and R-STL were 615 +/- 167 and 619 +/- 164 ng/mL, respectively, which were obtained on average, 3.13 +/- 0.60 hours after dosing. The mean residence time (mean +/- SD) was 13.2 +/- 1.2 and 12.9 +/- 1.8 hours for S- and R-STL, respectively. Respective renal clearance values for S- and R-STL were 8.98 +/- 1.5 and 9.46 +/- 2.3 L/hour, and were approximately 1.5 times greater than creatinine clearance. Renal clearance constituted approximately 76% of the oral clearance. Although stereoselective disposition of STL was absent after racemate administration, these results should not be extrapolated to patients with significantly altered physiology, or to the pharmacokinetics of S-STL after administration of pure-S-STL.

A valid, sensitive high-performance liquid chromatographic technique is reported for the separation of the two enantiomers of metoprolol in human plasma. The procedure involves pre-column derivatization with the homochiral reagent S-(+)-1-(1-naphthyl)-ethyl isocyanate. Once formed, the diastereomers are separated using normal-phase high-performance liquid chromatography. Fluorescence detection (220 nm excitation; no emission filter) was utilized, resulting in baseline resolution (Rs > 1.5). The peaks corresponding to metoprolol enantiomers were free from interference throughout the examined range of 5–500 ng/ml; accuracy and precision were within approximately 10%. Analysis of a plasma sample collected from a healthy volunteer demonstrated that the assay is applicable to clinical studies.

A sensitive high-performance liquid chromatographic assay was developed for the determination of tocainide enantiomers in plasma. Following extraction of tocainide from plasma, the enantiomers were derivatized with S-(+)-1-(1-naphthyl)ethylisocyanate. The resulting diastereomers were separated and quantified using normal-phase chromatography with fluorescence detection set at 220/345 nm (excitation/emission). The peaks, resolved with a resolution factor greater than 1.5, were free from interference. Linearity was established over the concentration range 0.25-10.0 mg/l for each enantiomer in plasma (r2 greater than 0.998). The inter-assay variability was less than 10% at all concentrations examined. The method can be used to determine the pharmacokinetics of tocainide enantiomers in man.

A convenient high-performance liquid chromatographic (HPLC) assay was developed for determination of sotalol (STL) enantiomers in plasma. Following addition of the internal standard (IS; racemic atenolol), enantiomers of STL and IS were extracted using ethyl acetate. After evaporation of the organic layer, samples were derivatized with a solution of S-( + )-l-(l-naphthyl)ethyl isocyanate (NEIC). The resulting diastereomers were chromatographed with normal-phase HPLC with chloroform:hexane:methanol [65:33:2 (v/ v)] as the mobile phase at a flow rate of 2 ml/min. The fluorescence detection wavelength was set at 220 nm for excitation with no emission filter. The suitability of the assay for pharmacokinetic studies was determined by measuring STL enantiomers in the plasma of a healthy subject after administration of a single 160-mg oral, racemic dose of STL.

A sensitive high-performance liquid chromatographic technique is described for the separation of R- and S-acebutolol in human plasma and urine. The procedure involves derivatization with the chiral reagent S-(+)-1-(1-naphthyl)ethyl isocyanate. The resulting diastereoisomers are quantified using normal-phase high-performance liquid chromatography with fluorescence detection (220/389 nm). Virtual baseline separation, free from interference, with achieved (resolution factor = 1.45). Excellent linearity (r greater than 0.998) was observed throughout the range 10-500 ng/l and 2-100 mg/l in plasma and urine, respectively. Inter-assay variability was less than 5% for each enantiomer at concentrations of 10 ng/ml. This method is applicable for the determination of the pharmacokinetics, in man, of acebutolol enantiomers in plasma and urine.