Carisoprodol pharmacokinetics and distribution in the nucleus accumbens correlates with behavioral effects in rats independent from its metabolism to meprobamate.

Carisoprodol (Soma®) is a centrally-acting skeletal-muscle relaxant frequently prescribed for treatment of acute musculoskeletal conditions. Carisoprodol's mechanism of action is unclear and is often ascribed to that of its active metabolite, meprobamate. The purpose of this study was to ascertain whether carisoprodol directly produces behavioral effects, or whether metabolism to meprobamate via cytochrome P450 (CYP450) enzymatic reaction is necessary. Rats were trained to discriminate carisoprodol (100 mg/kg) to assess time course and whether a CYP450 inhibitor (cimetidine) administered for 4 days would alter the discriminative effects of carisoprodol. Additionally, pharmacokinetics of carisoprodol and meprobamate with and without co-administration of cimetidine were assessed via in vivo microdialysis combined with liquid-chromatography-tandem mass spectrometry from blood and nucleus accumbens (NAc). The time course of the discriminative-stimulus effects of carisoprodol closely matched the time course of the levels of carisoprodol in blood and NAc, but did not match the time course of meprobamate. Administration of cimetidine increased levels of carisoprodol and decreased levels of meprobamate consistent with its interfering with metabolism of carisoprodol to meprobamate. However, cimetidine failed to alter the discriminative-stimulus effects of carisoprodol. Carisoprodol penetrated into brain tissue and directly produced behavioral effects without being metabolized to meprobamate. These findings indicate that understanding the mechanism of action of carisoprodol independently of meprobamate will be necessary to determine the validity of its clinical uses.

Automatic Dissolution Testing with High-Temporal Resolution for Both Immediate-Release and Fixed-Combination Drug Tablets.

Dissolution testing plays many important roles throughout the pharmaceutical industry, from the research and development of drug products to the control and evaluation of drug quality. However, it is a challenging task to perform both high-efficient separation and high-temporal detection to achieve accurate dissolution profile of each active ingredient dissolved from a drug tablet. In our study, we report a novel non-manual-operation method for performing the automatic dissolution testing of drug tablets, by combining a program-controlled sequential analysis and high-speed capillary electrophoresis for efficient separation of active ingredients. The feasibility of the method for dissolution testing of real drug tablets as well as the performance of the proposed system has been demonstrated. The accuracy of drug dissolution testing is ensured by the excellent repeatability of the sequential analysis, as well as the similarity of the evaluation of dissolution testing. Our study show that the proposed method is capable to achieve simultaneous dissolution testing of multiple ingredients, and the matrix interferences can be avoided. Therefore it is of potential valuable applications in various fields of pharmaceutical research and drug regulation.

Peri-operative Medication Dosing in Adult Obese Elective Surgical Patients: A Systematic Review of Clinical Studies.

BACKGROUND: Despite the increasing numbers of obese patients undergoing elective surgery, there is a lack of evidence-based dosing guidelines for peri-operative medications in obesity. OBJECTIVE: The objective was to systematically review the dosing and outcomes of peri-operative medications used in obese elective surgical patients. METHODS: Medical subject headings and general keywords were used to systematically search multiple databases (PubMed, EMBASE, Cochrane Library and CINAHL). Studies of medications in obese surgical patients were included if they had a non-obese control or comparative dosing scalar group. The National Health and Medical Research Council GRADE tool was used to assess quality of evidence for each drug. RESULTS: Thirty-three studies of six drug classes were identified: anaesthetics (n = 6), muscle relaxants (n = 10), neuromuscular reversal agents (n = 3), analgesics (n = 2), antibiotics (n = 5) and anticoagulants (n = 7). A variety of dose scalars and/or recommendations was observed for various medications. Lean body weight was proposed as a suitable weight scalar for induction of anaesthesia with propofol whereas total body weight for maintenance of anaesthesia with propofol and depolarizing muscle relaxants. Ideal body weight was reported as an appropriate dosing scalar for non-depolarizing muscle relaxants and neuromuscular reversal agents. Both corrected body weight 40% and ideal body weight were reported as suitable weight scalars for post-operative analgesia with morphine. The standard 2-g dose of cefazolin appeared effective in the prevention of surgical site infection. Body mass index stratified dosing of enoxaparin was effective for venous thromboembolism prevention. CONCLUSION: No drug recommendation achieved an "Excellent" quality of evidence. Limited data suggest that clinicians should consider each individual class of medication when selecting a dose for obese surgical patients. Routine use of fixed-dosing regimens is likely to under- or overdose obese patients thus predisposing them to adverse drug events or treatment failure leading to patient harm.

Forecasting gastrointestinal precipitation and oral pharmacokinetics of dantrolene in dogs using an in vitro precipitation testing coupled with in silico modeling and simulation.

The aim of the current research was to determine the precipitation kinetics of dantrolene sodium using canine biorelevant in vitro testing and to model the precipitation kinetics by appropriately coupling the data with an in silico tool adapted for dogs. The precipitation profiles of dantrolene sodium solutions were obtained with the in vitro paddle apparatus at a revolution rate of 50rpm. The in silico prediction tool was designed using STELLA software and the predicted plasma concentration profiles of dantrolene using the in vitro precipitation data were compared with the observed in vivo pharmacokinetics in beagle dogs. The plasma profiles of dantrolene, which served as a model weakly acidic drug which precipitates in the upper gastrointestinal tract, was successfully predicted using the in vitro precipitation testing coupled with the in silico modeling and simulation approach. The approach was subsequently used to forecast the effect of pharmaceutical excipients (HPMC/PG) on the ability of the drug to supersaturate in the gut and the resulting pharmacokinetics. The agreement of the simulated pharmacokinetics with the observed values confirms the ability of canine biorelevant media to predict oral performance of enhanced dosage forms in dogs.

Synthesis, Computational and Pharmacological Evaluation of N-(2-benzoyl- 4-chlorophenyl)-2-(4-(Substituted Phenyl) Piperazin-1-yl) Acetamides as CNS Agents.

BACKGROUND: A series of new N-(2-benzoyl-4-chlorophenyl)-2-(4-(substituted phenyl) piperazin-1-yl) acetamides (3a-j) have been synthesized by the chloroacetylation of 2-amino-5- chlorbenzophenone which was further reacted with substituted phenylpiperazine. MATERIAL: The chemical structures of the compounds were confirmed on the basis of their TLC, IR, 1HNMR, 13CNMR and by elemental analysis. The physicochemical similarity of the target compounds with respect to standard drug diazepam was assessed by calculating from a set of physicochemical properties using software programs. CONCLUSION: Molecular docking studies revealed that the target compounds correctly dock into the binding pocket of the GABAA receptor, while their bioavailability/drug-likeness was predicted to be acceptable but requires future optimization. The anxiolytic and skeletal muscle relaxant activity of the target compounds (3a-j) were evaluated in albino mice. Among them, compound 3h showed potent anxiolytic and skeletal muscle relaxant activity.

Liquid chromatography high resolution mass spectrometry for the determination of baclofen and its metabolites in plasma: Application to therapeutic drug monitoring.

Baclofen is used to manage alcohol dependence. This study describes a simple method using liquid chromatography coupled to high-resolution mass spectrometry (LC-HR-MS) developed in plasma samples. This method was optimized to allow quantification of baclofen and determination of metabolic ratio of its metabolites, an oxidative deaminated metabolite of baclofen (M1) and its glucuronide form (M2). The LC-HR-MS method on Exactive® apparatus is a newly developed method with all the advantages of high resolution in full-scan mode for the quantification of baclofen and detection of its metabolites in plasma. The present assay provides a protein precipitation method starting with 100 µL plasma giving a wide polynomial dynamic range (R2 > 0.999) between 10 and 2000 ng/mL and a lower limit of quantitation of 3 ng/mL for baclofen. Intra- and inter-day precisions were <8.1% and accuracies were between 91.2 and 103.3% for baclofen. No matrix effect was observed. The assay was successfully applied to 36 patients following baclofen administration. Plasma concentrations of baclofen were determined between 12.2 and 1399.9 ng/mL and metabolic ratios were estimated between 0.4 and 81.8% for M1 metabolite and on the order of 0.3% for M2 in two samples.

Effect of piperine on CYP2E1 enzyme activity of chlorzoxazone in healthy volunteers.

1. The purpose of the present study was to investigate the effect of piperine (PIP) on CYP2E1 enzyme activity and pharmacokinetics of chlorzoxazone (CHZ) in healthy volunteers. 2. An open-label, two period, sequential study was conducted in 12 healthy volunteers. A single dose of PIP 20 mg was administered daily for 10 days during treatment phase. A single dose of CHZ 250 mg was administered during control and after treatment phases under fasting conditions. The blood samples were collected at predetermined time intervals after CHZ dosing and analyzed by HPLC. 3. Treatment with PIP significantly enhanced maximum plasma concentration (Cmax) (3.14-4.96 µg/mL), area under the curve (AUC) (10.46-17.78 µg h/mL), half life (T1/2) (1.26-1.82 h) and significantly decreased elimination rate constant (Kel) (0.57-0.41 h - 1), apparent oral clearance (CL/F) (24.76-13.65 L/h) of CHZ when compared to control. In addition, treatment with PIP significantly decreased Cmax (0.22-0.15 µg/mL), AUC (0.94-0.68 µg h/mL), T1/2 (2.54-1.68 h) and significantly increased Kel (0.32-0.43 h - 1) of 6-hydroxychlorzoxazone (6-OHCHZ) as compared to control. Furthermore, treatment with PIP significantly decreased metabolite to parent (6-OHCHZ/CHZ) ratios of Cmax, AUC, T1/2 and significantly increased Kel ratio of 6-OHCHZ/CHZ, which indicate the decreased formation of CHZ to 6-OHCHZ. 4. The results suggest that altered pharmacokinetics of CHZ might be attributed to PIP mediated inhibition of CYP2E1 enzyme, which indicate significant pharmacokinetic interaction present between PIP and CHZ. The inhibition of CYP2E1 by PIP may represent a novel therapeutic benefit for minimizing ethanol induced CYP2E1 enzyme activity and results in reduced hepatotoxicity of ethanol.

Evaluation on activity of cytochrome p450 enzymes in turbot via a probe drug cocktail.

Cytochrome P450s (CYPs) are the main catalytic enzymes for metabolism by a variety of endogenous and exogenous substrates in mammals, fish, insects, etc. We evaluated the application of a multidrug cocktail on changes in CYP1, CYP2, and CYP3 activity in Turbot Scophthalmus maximus. The probe drugs were a combination of caffeine (5 mg/kg body weight), dapsone (5 mg/kg), and chlorzoxazone (10 mg/kg). After a single intraperitoneal injection of the cocktail, the concentration of all three probe drugs in the plasma increased quickly to a peak and then decreased gradually over 24 h. Pharmacokinetic profiles of the three probe drugs were determined using a noncompartmental analysis, and the typical parameters were calculated. In the assay for CYP induction, pretreatment with rifampicin significantly reduced the typical pharmacokinetic metrics for caffeine and chlorzoxazone, but not dapsone, indicating that the activity of CYP1 and CYP2 in turbot were induced by rifampicin.

The action of cytochrome b(5) on CYP2E1 and CYP2C19 activities requires anionic residues D58 and D65.

The capacity of cytochrome b(5) (b(5)) to influence cytochrome P450 activities has been extensively studied and physiologically validated. Apo-b(5) enhances the activities of CYP3A4, CYP2A6, CYP2C19, and CYP17A1 but not that of CYP2E1 or CYP2D6, suggesting that the b(5) interaction varies among P450s. We previously showed that b(5) residues E48 and E49 are required to stimulate the 17,20-lyase activity of CYP17A1, but these same residues might not mediate b(5) activation of other P450 reactions, such as CYP2E1-catalyzed oxygenations, which are insensitive to apo-b(5). Using purified P450, b(5), and reductase (POR) in reconstituted assays, the D58G/D65G double mutation, of residues located in a hydrophilic α-helix of b(5), totally abolished the ability to stimulate CYP2E1-catalyzed chlorzoxazone 6-hydroxylation. In sharp contrast, the D58G/D65G double mutation retained the full ability to stimulate the 17,20-lyase activity of CYP17A1. The D58G/D65G double mutation competes poorly with wild-type b(5) for binding to the CYP2E1·POR complex yet accepts electrons from POR at a similar rate. Furthermore, the phospholipid composition markedly influences P450 turnover and b(5) stimulation and specificity, particularly for CYP17A1, in the following order: phosphatidylserine > phosphatidylethanolamine > phosphatidylcholine. The D58G/D65G double mutation also failed to stimulate CYP2C19-catalyzed (S)-mephenytoin 4-hydroxylation, whereas the E48G/E49G double mutation stimulated these activities of CYP2C19 and CYP2E1 equivalent to wild-type b(5). We conclude that b(5) residues D58 and D65 are essential for the stimulation of CYP2E1 and CYP2C19 activities and that the phospholipid composition significantly influences the b(5)-P450 interaction. At least two surfaces of b(5) differentially influence P450 activities, and the critical residues for individual P450 reactions cannot be predicted from sensitivity to apo-b(5) alone.

Enantioselective carbonyl reduction of eperisone in human liver microsomes.

Eperisone, 4-ethyl-2-methyl-3-piperidinopropiophenone, is a centrally acting muscle relaxant widely used to relieve muscle stiffness and back pain. In this study, enantioselectivity for carbonyl reduction of eperisone was investigated in human liver microsomes, and the enzymes involved in the carbonyl reduction were characterised. Carbonyl reduction of eperisone predominantly occurred in microsomal fractions and 11ß-hydroxysteroid dehydrogenase type 1(11ß-HSD 1) played a major role in this reaction as judged by selective inhibition of the activity by BVT-14225 and KR-66344. The kinetic study with (+)-S- and (-)-R-eperisone showed that the formation of the carbonyl reduced metabolite (M5) from the (-)-R-isomer was more efficient than that from the (-)-S-isomer. As eperisone is a racemic compound with one chiral centre, the carbonyl reduced metabolite of eperisone (M5) may have four possible diastereoisomeric structures. Chiral separation of incubation mixtures of racemic eperisone with human liver microsome revealed that (1S, 2S)-M5 and (1R, 2R)-M5 were generated specifically from (+)-S- and (-)-R-eperisone, respectively. Selective formation of anti-diastereomers was further confirmed by incubation of individual enantiomer with microsomes. Carbonyl reduction of eperisone by microsomal 11ß-HSD 1 may significantly contribute to the metabolic disposition of eperisone in human and (-)-R-isomer is preferentially reduced by this enzyme.

Pharmacokinetics and metabolism of dantrolene in horses.

Dantrolene is a skeletal muscle relaxant used commonly in performance horses to prevent exertional rhabdomyolysis. The goal of the study reported here was to begin to characterize cytochrome P450-mediated metabolism of dantrolene in the horse and describe the pharmacokinetics of the compound, formulated as a capsule or a compounded paste formulation, following oral administration. Dantrolene is rapidly metabolized to 5-hydroxydantrolene both in vivo and in vitro. Preliminary work with equine liver microsomes suggest that two enzymes are responsible for the metabolism of dantrolene, as evidenced by two distinct K(m) values, one at high and one at low substrate concentrations. For the pharmacokinetic portion of the study, a randomized, balanced 2-way crossover design was employed wherein eight healthy horses received a single oral dose of either capsules or paste followed by a 4 week washout period prior to administration of the second formulation to the same horse. Blood samples were collected at time 0 (prior to drug administration) and at various times up to 96 h postdrug administration. Plasma samples were analyzed using liquid chromatography-mass spectrometry and data analyzed using both noncompartmental and compartmental analysis. Peak plasma concentrations were 28.9 ± 21.6 and 37.8 ± 12.8 ng/mL for capsules and paste, respectively and occurred at 3.8 h for both formulations. Dantrolene and its major metabolite were both below the limit of detection in both plasma and urine by 168 h postadministration.

Bioconcentration of two pharmaceuticals (benzodiazepines) and two personal care products (UV filters) in marine mussels (Mytilus galloprovincialis) under controlled laboratory conditions.

Bioaccumulation is essential for gaining insight into the impact of exposure to organic micropollutants in aquatic fauna. Data are currently available on the bioaccumulation of persistent organic pollutants, but there is very little documentation on the bioaccumulation of pharmaceuticals and personal care products (PPCPs). The bioconcentration of selected PPCPs was studied in marine mussels (Mytilus galloprovincialis). The selected PPCPs were two organic UV filters, i.e., 2-ethylhexyl-4-trimethoxycinnamate (EHMC) and octocrylene (OC), and two benzodiazepines (BZP), i.e., diazepam (DZP) and tetrazepam (TZP). Laboratory experiments were performed in which M. galloprovincialis was exposed to these compounds either directly from water, for the less lipophilic substances (BZP) or via spiked food for lipophilic UV filters. M. galloprovincialis uptook and eliminated BZP following first-order kinetics. The biological half-life (t (1/2)) of TZP was 1.4 days, resulting in a bioconcentration factor of 64 and 99 mL g(-1) dry weight (dw), respectively, for 2.3 and 14.5 µg L(-1) of exposure, while the biological half-life (t (1/2)) of DZP was 0.4 days, resulting in a bioconcentration factor of 51 mL g(-1) dw for 13.2 µg L(-1) of exposure. The uptake of UV filter was rapid in mussels, followed by elimination within 24 h. EHMC increased from 15 to 138 ng g(-1) dw in 1 h and decreased to 25 ng g(-1) after 24 h for 11.9 µg L(-1) exposure. OC reached 839 ng g(-1) dw after 1 h and decreased to 33 ng g(-1) after 24 h for 11.6 µg L(-1) exposure. However, EHMC and OC were slightly accumulated in 48 h, i.e., 38 and 60 ng g(-1) dw, respectively.

Carisoprodol withdrawal after internet purchase.

INTRODUCTION: Carisoprodol is a centrally acting muscle relaxant used in the treatment of various musculoskeletal disorders whose main metabolite, meprobamate, is a controlled substance in the United States due to its sedative properties and potential for abuse. CASE DESCRIPTION: We report a case of a 51-year-old man with cognitive impairment and tremor who developed worsening tremor, anxiety, myoclonus, ataxia, and psychosis on abrupt cessation of carisoprodol. At hospital discharge, his cognitive function significantly improved compared with when he was on carisoprodol. CONCLUSION: Carisoprodol withdrawal is an important and under-recognized syndrome that should be considered in patients presenting with neurologic symptoms who are taking the medication. Carisoprodol withdrawal can be successfully treated with the use of benzodiazepines, although further studies are needed to identify the most appropriate treatment protocol.

Anesthesia in adult cardiac surgery without maintenance of muscle relaxants: a randomized clinical trial.

There may be no need for muscle paralysis during cardiac surgery when adequate anesthesia is provided. We studied intra- and post-operative conditions during cardiac surgery without maintenance muscle relaxant therapy. Eighty adult patients who were candidates for elective coronary artery bypass graft surgery were randomly allocated into two groups. In the noMR or study group (noMR group; n = 40) only an intubation dose of cisatracurium (0.15 mg kg(-1)) was administrated, as opposed to the control group (MR group; n = 40), who had a continuous infusion added to the intubation dose. The anesthesia level was maintained at a Bispectral score of 40-50 using a propofol infusion. A remifentanil infusion was titrated to control patient hemodynamic response. During surgery, any minor (fine body or respiratory muscle movements) or major (coarse body movements or bucking/caught) movements were recorded. Postoperatively, analgesia was provided by remifentanil. The surgical condition was classified into three states: good (no movement), acceptable (minor movements), or poor (major movements). Anesthesia, surgery and postoperative characteristics were compared between the two groups. Statistical analysis was performed in only 78 patients (noMR = 38, MR = 40). The demographic and preoperative characteristics of the two groups were comparable. Intra-operative propofol consumption was the same, but significantly more remifentanil was used in the noMR group (p = 0.001). Post-operative characteristics and complication rates did not differ between the two groups. There were no movements in the MR group patients, while in the noMR group one patient had major movement and three had minor movements. We concluded that omitting maintenance muscle relaxants in adult cardiac surgery or eliminating residual muscle paralysis at the end of the surgery without improving early outcome can increase patient intra-operative movement risk.

Metabolic studies of tetrazepam based on electrochemical simulation in comparison to in vivo and in vitro methods.

During the last 2 years, the knowledge on the metabolic pathway of tetrazepam, a muscle relaxant drug, was expanded by the fact that diazepam was identified as a degradation product of tetrazepam. The present study demonstrates that this metabolic conversion, recently discovered by in vivo studies, can also be predicted on the basis of a purely instrumental method, consisting of an electrochemical cell (EC) coupled to online liquid chromatography (LC) and mass spectrometry (MS). By implementing a new electrochemical cell type into the EC-LC-MS set-up and by an enhanced oxidation potential range up to 2V, one limitation of the electrochemical metabolism simulation, the hydroxylation of alkanes and alkenes, has been overcome. Instead of commonly used flow-through cell with a porous glassy carbon working electrode, a wall-jet cell with exchangeable electrode material was used for this study. Thereby, the entire metabolic pathway of tetrazepam, in particular including the hydroxylation of the tetrazepam cyclohexenyl moiety, was simulated. The electrochemical results were not only compared to microsomal incubations, but also to in vivo experiments, by analysing urine samples from a patient after tetrazepam delivery. For structure elucidation of the detected metabolites, MS/MS experiments were performed. The comparison of electrochemistry to in vitro as well as to in vivo experiments underlines the high potential of electrochemistry as a fast screening tool in the prediction of metabolic transformations in drug development.

Characterization of human cytochrome P450 enzymes involved in the biotransformation of eperisone.

Eperisone is a centrally acting muscle relaxant widely used for the therapeutic treatment of spastic patients to relieve muscle stiffness and back pain. The objective of this study was to characterize the metabolic pathway involved in the biotransformation of eperisone mediated by human cytochrome P450 (CYP) enzymes. Eperisone was metabolized to seven metabolites via oxidation and carbonyl reduction in human liver microsome. Among them, M3 and M4 were found to be primary major metabolites which were generated by CYPs. The kinetics study with (-)-R- and (+)-S-eperisones revealed that CYPs-mediated hydroxylation did not have significant stereoselectivity for metabolic clearance of eperisone. Incubation with recombinant CYP isozyme, chemical inhibition assay, and immuno-inhibition assay showed that multiple CYPs were involved in M4 formation, but mainly CYP2J2 in M3 formation. In addition, intestinal microsomes metabolized eperisone to M3 and M4 via CYP2J2- and CYP3A4-mediated reactions, which are supposed to contribute to presystemic metabolism of eperisone.

Tolperisone: a typical representative of a class of centrally acting muscle relaxants with less sedative side effects.

Tolperisone, a piperidine derivative, is assigned to the group of centrally acting muscle relaxants and has been in clinical use now for decades. The review summarizes the known pharmacokinetics, pharmacodynamics, toxicology and side effects in humans and the clinical use of tolperisone. A future perspective for further exploration of this drug is given.

Is the frequency of carisoprodol withdrawal syndrome increasing?

Carisoprodol is a commonly used centrally acting muscle relaxant. A number of case reports have suggested that the drug may have abuse potential, presumably because it is metabolized to the anxiolytic drug, meprobamate, which is a controlled substance at the federal level. Two recent case reports described symptoms of withdrawal after the cessation of carisoprodol. We present two additional cases that support the concept of a withdrawal syndrome with this drug. Symptoms of carisoprodol withdrawal include anxiety, tremulousness, insomnia, jitteriness, muscle twitching, and hallucinations. These symptoms are most likely caused by withdrawal from the meprobamate that accumulates after large amounts of carisoprodol are ingested. Although carisoprodol is not a controlled substance at the federal level, clinicians should be aware of its significant potential for abuse.

Tolfenamic acid is a potent CYP1A2 inhibitor in vitro but does not interact in vivo: correction for protein binding is needed for data interpretation.

OBJECTIVE: Our aim was to correlate the in vitro and in vivo CYP1A2 inhibition potential of tolfenamic acid, an NSAID highly (99.7%) bound to plasma proteins, to study the significance of protein binding of inhibitor in metabolic drug interactions. METHODS: The effect of tolfenamic acid on CYP1A2 (phenacetin O-deethylation) was studied using human liver microsomes, with and without albumin (0-10 mg/ml). In a randomized, crossover study, 10 volunteers took 200 mg tolfenamic acid or placebo t.i.d. for 3 days. On day 2, a caffeine test was performed. On day 3, each ingested 4 mg of the CYP1A2 substrate tizanidine. Plasma tizanidine, its metabolites (M) and tolfenamic acid, and pharmacodynamic variables were measured. RESULTS: Tolfenamic acid strongly inhibited phenacetin-O-deethylation in vitro (IC(50) 1.8 microM without albumin). Albumin decreased its inhibitory effect in a concentration-dependent manner; the IC(50) exceeded 100 microM with 10 mg/ml of albumin. Tolfenamic acid had no effect on the area under the concentration-time curve (AUC(0-oo)), peak concentration, time of peak concentration or half-life of tizanidine or M-3; only the AUC(0-oo) of secondary metabolite M-4 was slightly decreased (13%, P = 0.004). The caffeine test and the pharmacodynamic effects of tizanidine were unchanged. CONCLUSIONS: Tolfenamic acid potently inhibits CYP1A2 in vitro when studied without albumin, but not in vivo. This apparent discrepancy is due to the high protein binding of tolfenamic acid. To avoid overestimation of the interaction potential, the inhibitory effect of highly albumin-bound compounds should also be studied in vitro with albumin, or their exact unbound plasma concentration should be used in predictions.