Impact of cytochrome P450 variation on meperidine N-demethylation to the neurotoxic metabolite normeperidine.

1. Meperidine is an opioid analgesic that undergoes N-demethylation to form the neurotoxic metabolite normeperidine. Previous studies indicate that meperidine N-demethylation is catalyzed by cytochrome P450 2B6 (CYP2B6), CYP3A4, and CYP2C19.2. The purpose of this study was to examine the relative P450 contributions to meperidine N-demethylation and to evaluate the effect of CYP2C19 polymorphism on normeperidine generation. Experiments were performed using recombinant P450 enzymes, selective chemical inhibitors, enzyme kinetic assays, and correlation analysis with individual CYP2C19-genotyped human liver microsomes.3. The catalytic efficiency (kcat/Km) for meperidine N-demethylation was similar between recombinant CYP2B6 and CYP2C19, but markedly lower by CYP3A4.4. In CYP2C19-genotyped human liver microsomes, normeperidine formation was significantly correlated with CYP2C19 activity (S-mephenytoin 4´-hydroxylation).5. CYP2C19 inhibitor (+)-N-3-benzylnirvanol and CYP3A inhibitor ketoconazole significantly reduced microsomal normeperidine generation by an individual donor with high CYP2C19 activity, whereas donors with lower CYP2C19 activity were sensitive to inhibition by ketoconazole but not benzylnirvanol.6. These findings demonstrate that the relative CYP3A4, CYP2B6, and CYP2C19 involvement in meperidine N-demethylation depends on the enzyme activities in individual human liver microsomal samples. CYP2C19 is likely an important contributor to normeperidine generation in individuals with high CYP2C19 activity, but additional factors influence inter-individual metabolite accumulation.

Extrapolation of Drug Clearance in Children ≤ 2 Years of Age from Empirical Models Using Data from Children (> 2 Years) and Adults.

BACKGROUND: The application of modeling and simulation approaches in clinical pharmacology studies has gained momentum over the last 20 years. OBJECTIVES: The objective of this study was to develop six empirical models from clearance data obtained from children aged > 2 years and adults to evaluate the suitability of the models to predict drug clearance in children aged ≤ 2 years (preterm, term, and infants). METHODS: Ten drugs were included in this study and administered intravenously: alfentanil, amikacin, busulfan, cefetamet, meperidine, oxycodone, propofol, sufentanil, theophylline, and tobramycin. These drugs were selected according to the availability of individual subjects' weight, age, and clearance data (concentration-time data for these drugs were not available to the author). The chosen drugs are eliminated by extensive metabolism by either the renal route or both the renal and hepatic routes. The six empirical models were (1) age and body weight-dependent sigmoidal maximum possible effect (Emax) maturation model, (2) body weight-dependent sigmoidal Emax model, (3) uridine 5'-diphospho [body weight-dependent allometric exponent model (BDE)], (4) age-dependent allometric exponent model (ADE), (5) a semi-physiological model, and (6) an allometric model developed from children aged > 2 years to adults. The model-predicted clearance values were compared with observed clearance values in an individual child. In this analysis, a prediction error of ≤ 50% for mean or individual clearance values was considered acceptable. RESULTS: Across all age groups and the ten drugs, data for 282 children were compared between observed and model-predicted clearance values. The validation data consisted of 33 observations (sum of different age groups for ten drugs). Only three of the six models (body weight-dependent sigmoidal Emax model, ADE, and semi-physiological model) provided reasonably accurate predictions of clearance (> 80% observation with ≤ 50% prediction error) in children aged ≤ 2 years. In most instances, individual predicted clearance values were erratic (as indicated by % error) and were not in agreement with the observed clearance values. CONCLUSIONS: The study indicated that simple empirical models can provide more accurate results than complex empirical models.

Sphingolipid biosynthetic pathway is crucial for growth, biofilm formation and membrane integrity of Scedosporium boydii.

Aim: Glycosphingolipids are conserved lipids displaying a variety of functions in fungal cells, such as determination of cell polarity and virulence. They have been considered as potent targets for new antifungal drugs. The present work aimed to test two inhibitors, myriocin and DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol, in Scedosporium boydii, a pathogenic fungus which causes a wide range of disease. Materials & methods: Mass spectrometry, microscopy and cell biology approaches showed that treatment with both inhibitors led to defects in fungal growth and membrane integrity, and caused an increased susceptibility to the current antifungal agents. Conclusion: These data demonstrate the antifungal potential of drugs inhibiting sphingolipid biosynthesis, as well as the usefulness of sphingolipids as promising targets for the development of new therapeutic options.

Muscarinic acetylcholine receptor binding affinities of pethidine analogs.

A series of pethidine analogs were synthesized and their affinities for the [(3)H]N-methyl-scopolamine (NMS) binding site on muscarinic acetylcholine receptors (mAChRs) were determined using M1, M3 or M5 human mAChRs expressed by Chinese hamster ovary (CHO) cell membranes. Compound 6b showed the highest binding affinities at M1, M3 and M5 mAChRs (Ki=0.67, 0.37, and 0.38 µM, respectively).

Determination of pethidine hydrochloride using potentiometric coated graphite and carbon paste electrodes.

A new approach for lowering the detection limit of a pethidine ion-selective electrode is presented. A coated graphite (CGE) and carbon paste (CPE) electrodes for pethidine ions based on pethidine-phosphotungstate (PD-PT) as ion-pair complex are described. The sensors exhibit a Nernstian slope of 58.1 and 54.2 mVdecade(-1) for pethidine ion over a wide concentration range from 2.6 × 10(-7) to 1.0 × 10(-2) M and 2.1 × 10(-6) to 1.0 × 10(-2) M with a detection limit of 1.8 × 10(-7) M and 7.3 × 10(-7) M for pethidine coated graphite (PD-CGE) and pethidine carbon paste electrode (PD-CPE), respectively. These sensors exhibited a fast response time (about 5-8 s) and good stability. The standard electrode potentials, E(o) , were determined at different temperatures and used to calculate the isothermal temperature coefficient (dE(o) /dT) of the PD-CGE and PD-CPE, which was 0.0062 and 0.0071 V/ °C, respectively. Selectivity coefficients, determined by matched potential method (MPM) and separate solution method (SSM), showed high selectivity for pethidine hydrochloride (PDCl) over a large number of inorganic cations, organic cations, sugars, urine components, and some common drug excipients. The sensors were applied for determination of PDCl in ampoule and in spiked urine samples using potentiometric determination, standard addition and the calibration curve methods. The results obtained were satisfactory with excellent percentage recovery comparable and sometimes better than those obtained by other routine methods for the assay.

In vivo investigation of brain and systemic ketobemidone metabolism.

Ketobemidone metabolites have previously been identified in urine and plasma; here we show, for the first time, that norketobemidone and ketobemidone N-oxide are present in in vivo microdialysate from rat brain (striatum) after reverse microdialysis, suggesting striatal metabolism of ketobemidone. Ketobemidone metabolites were also identified in in vivo microdialysate samples from brain and blood, as well as in urine from rats, after subcutaneous administration of ketobemidone. Three Phase I metabolites (norketobemidone, ketobemidone N-oxide and hydroxymethoxyketobemidone) and three Phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in the microdialysates after subcutaneous administration. Coupled capillary liquid chromatography tandem mass spectrometry (LC-MS/MS) and SPE (boronate)-MS/MS were utilized for the analysis of the biological samples. The Phase I metabolites were identified by comparing the retention times and tandem mass spectra of the microdialysates with synthetic standards. The Phase II metabolites were identified by determination of exact masses and by comparing the tandem mass spectra of the microdialysates with those of synthetic standards for the aglycones. Hydroxyketobemidone, a catechol-type Phase I metabolite, was selectively isolated by solid-phase boronate-complexation but identified in urine alone. This work demonstrated that the in vivo microdialysis technique in combination with LC-MS/MS can be used to study the local metabolism of a drug in the brain.

The effect of oral and subcutaneous meperidine on the maximal electroshock seizure (MES) in mice.

The likely effect of oral and subcutaneous meperidine on maximal electroshock seizure (MES) in mice was studied. Convulsive current fifty (CC50) was assessed to be 46m A, an electrical pulse causing seizure in 50% of test animals. Doses of 15, 30, 60, or 120 mg/kg meperidine given orally or subcutaneously increased the convulsion threshold of MES as evidenced by a significant dose-dependent reduction of MES below control value (p < .05). An initial hyperactivity reaction that was worsened by noisy and tactile stimuli and tail erection followed by sedation was observed after s.c. injection of 60 or 120 mg/kg meperidine. No significant difference was found between meperidine-induced reductions of control MES values obtained one and two hours after oral doses; the depressed MES values obtained one hour after oral administration of meperidine were significantly different and more powerful than those obtained two hours after s.c. drug administrations (p < .05). Combining previous literature information with the present results, we conclude that such an effect of meperidine can be attributed to cerebellar stimulation.

Ketobemidone is a substrate for cytochrome P4502C9 and 3A4, but not for P-glycoprotein.

The role of the major drug-metabolizing cytochrome P450 (CYP) enzymes as well as P-glycoprotein (PGP) was investigated in the disposition of ketobemidone in vitro. Formation of norketobemidone from ketobemidone was studied and compared with the activities of 11 major CYP enzymes in human liver microsomes. The formation of norketobemidone from ketobemidone (1 microM) correlated best with CYP2C9 activity, measured as losartan oxidation (rs = 0.82, n = 19, p < 0.001), but there was also a strong correlation with CYP3A4 activity. Additionally, a good correlation was observed with CYP2C19, CYP2C8 and CYP2B6 at a ketobemidone concentration of 50 microM. Inhibition studies confirmed the involvement of CYP2C9 and CTP3A4 in the formation of norketobemidone. The formation rate of norketobemidone was three times higher in the CYP2C9*1*1 genotype group compared with the CYP2C9*1*2, CYP2C9*1*3 and CYP2C9*3*3 genotypes (p < 0.01). Treatment with verapamil as a PGP inhibitor did not affect the transport of ketobemidone in Caco-2 cells, indicating that PGP is not involved. The data suggest that CYP2C9 and CYP3A4 play a major role in the formation of norketobemidone at clinically relevant concentrations.

CYP2B6, CYP3A4, and CYP2C19 are responsible for the in vitro N-demethylation of meperidine in human liver microsomes.

Meperidine is an opioid analgesic metabolized in the liver by N-demethylation to normeperidine, a potent stimulant of the central nervous system. The purpose of this study was to identify the human cytochrome P450 (P450) enzymes involved in normeperidine formation. Our in vitro studies included 1) screening 16 expressed P450s for normeperidine formation, 2) kinetic experiments on human liver microsomes and candidate P450s, and 3) correlation and inhibition experiments using human hepatic microsomes. After normalization by its relative abundance in human liver microsomes, CYP2B6, CYP3A4, and CYP2C19 accounted for 57, 28, and 15% of the total intrinsic clearance of meperidine. CYP3A5 and CYP2D6 contributed to < 1%. Formation of normeperidine significantly correlated with CYP2B6-selective S-mephenytoin N-demethylation (r = 0.88, p < 0.0001 at 75 > microM meperidine, and r = 0.89, p < 0.0001 at 350 microM meperidine, n = 21) and CYP3A4-selective midazolam 1'-hydroxylation (r = 0.59, p < 0.01 at 75 microM meperidine, and r = 0.55, p < 0.01 at 350 microM meperidine, n = 23). No significant correlation was observed with CYP2C19-selective S-mephenytoin 4'-hydroxylation (r = 0.36, p = 0.2 at 75 microM meperidine, and r = 0.02, p = 0.9 at 350 microM meperidine, n = 13). An anti-CYP2B6 antibody inhibited normeperidine formation by 46%. In contrast, antibodies inhibitory to CYP3A4 and CYP2C8/9/18/19 had little effect (<14% inhibition). Experiments with thiotepa and ketoconazole suggested inhibition of microsomal CYP2B6 and CYP3A4 activity, whereas studies with fluvoxamine (a substrate of CYP2C19) were inconclusive due to lack of specificity. We conclude that normeperidine formation in human liver microsomes is mainly catalyzed by CYP2B6 and CYP3A4, with a minor contribution from CYP2C19.

Sensitive determination of pethidine in body fluids by gas chromatography-tandem mass spectrometry.

We have presented a simple and sensitive method for determining pethidine, a narcotic analgesic drug in body fluids by gas chromatography-tandem mass spectrometry (GC-MS/MS). Pethidine and 4'-piperidinoacetophenone (internal standard) were extracted from body fluids with Bond Elut C(18) columns; the recoveries were above 85% for both compounds. The calibration curves for blood and urine showed good linearities in the range of 1.25-40 ng/ml. Its detection limits (signal-to-noise ratio=3) were estimated to be approximately 0.5 ng/ml of whole blood and urine.

Procainamide and quinidine inhibition of the human hepatic degradation of meperidine in vitro.

Procainamide and quinidine inhibition of the degradation of meperidine in human liver was investigated by incubation of two concentrations of either drug with meperidine in homogenates of human liver over 24 and 36 h. Meperidine concentrations declined by 26% after incubation for 24 h and by 42% after incubation for 36 h. In the presence of procainamide, however, they decreased by only 15% to 18% at 24 h and by only 26% to 28% at 36 h. In the presence of quinidine, they declined by only 18% to 19% at 24 h and by only 27% to 28% at 36 h. Procainamide and quinidine may inhibit human hepatic carboxylesterase hCE-1, which is responsible for catalyzing the hydrolysis of meperidine. This inhibition may prolong the biological half-life of meperidine in patients receiving the drug together with either procainamide or quinidine.

Meperidine misuse in a patient with sphincter of Oddi dysfunction.

OBJECTIVE: To report a seizure occurring secondary to meperidine treatment despite normal renal and central nervous system (CNS) function, and to provide a review of meperidine's role in pain management, including its use in pancreatitis and sphincter of Oddi dysfunction. CASE SUMMARY: A 55-year-old white woman with a history of sphincter of Oddi dysfunction presented to the emergency department with severe abdominal pain. On admission to the hospital, the serum creatinine level was 0.6 mg/dL with slightly elevated aspartate aminotransferase of 56 U/L (normal range 0-31) and alanine aminotransferase of 34 U/L (0-31). The patient received repeated and escalating doses of intravenous meperidine, resulting in a generalized seizure on day 4 of hospitalization. The accumulated meperidine dose was 2125 mg. Buprenorphine was substituted in place of meperidine, and the patient had no further reported complications. She was then transferred to a tertiary-care facility for sphincter of Oddi reevaluation. An objective causality assessment revealed the adverse drug event as probable. DISCUSSION: Despite alternative opioids, meperidine continues to be used in pain management. Meperidine is different from other opioids because its active metabolite, normeperidine, is neurotoxic. Patients with renal insufficiency, liver failure, or CNS dysfunction are at increased risk for adverse drug reactions related to normeperidine accumulation. Due to normeperidine's extended half-life, however, accumulation of normeperidine can occur in any patient receiving repeated doses of meperidine. CONCLUSIONS: This case demonstrates the potential hazards that exist when using meperidine in any patient. Meperidine's inherent risks of both undertreating pain and causing adverse drug reactions should prompt clinicians and health organizations to restrict its use in pain management. This restriction should not make exceptions to meperidine's traditional use in pancreatitis or sphincter of Oddi dysfunction.

Synthesis, dopamine and serotonin transporter binding affinities of novel analogues of meperidine.

A series of meperidine analogues was synthesized and the binding affinities for the dopamine and serotonin transporters were determined. The substituents on the phenyl ring greatly influenced the potency and selectivity of these compounds for the transporter binding sites. In general, meperidine (3) and its analogues were more selective for serotonin transporter binding sites and the esters 9 were more potent than the corresponding nitriles 8. The 3,4-dichloro derivative 9e was the most potent ligand of the series for dopamine transporter binding sites while the 2-naphthyl derivative 9g exhibited the most potent binding affinity and was highly selective for serotonin transporter binding sites.

Norpethidine accumulation and generalized seizure during pethidine patient-controlled analgesia.

A 35-year-old, 47 kg female presented for elective laparatomy, adhesiolysis and ileostomy formation. Pre-existing neurological problems precluded placement of an epidural and IV PCA was used for postoperative analgesia. A patient request for pethidine was allowed. Twenty-three hours postoperatively, a brief generalized seizure occurred without adverse sequelae. This had been immediately preceded by myoclonic-type jerking. The cumulative pethidine dose was 3,000 mg and the norpethidine level was 1.8 micrograms.ml-1. Avoidance of pethidine for IV PCA where large cumulative doses are anticipated is advised. Seizures associated with pethidine/norpethidine toxicity can occur early during pethidine usage, and there is considerable variation in measured norpethidine levels.

Binding and hydrolysis of meperidine by human liver carboxylesterase hCE-1.

Human liver carboxylesterases catalyze the hydrolysis of apolar drug or xenobiotic esters into more soluble acid and alcohol products for elimination. Two carboxylesterases, hCE-1 and hCE-2, have been purified and characterized with respect to their role in cocaine and heroin hydrolysis. The binding of meperidine (Demerol) and propoxyphene (Darvon) was examined in a competitive binding, spectrophotometric assay. The hCE-1 and hCE-2 bound both drugs, with Ki values in the 0.4- to 1.3-mM range. Meperidine was hydrolyzed to meperidinic acid and ethanol by hCE-1 but not hCE-2. The Km of hCE-1 for meperidine was 1.9 mM and the kcat (catalytic rate constant) was 0.67 min-1. Hydrolysis of meperidine by hCE-1 was consistent with its specificity for hydrolysis of esters containing simple aliphatic alcohol substituents. Hence, hCE-1 in human liver microsomes may play an important role in meperidine elimination. Propoxyphene was not hydrolyzed by hCE-1 or hCE-2. This observation is consistent with the absence of a major hydrolytic pathway for propoxyphene metabolism in humans.

A comparison of 50, 100 and 200 mg of intra-articular pethidine during knee joint surgery, a controlled study with evidence for local demethylation to norpethidine.

Pethidine (meperidine) is a compound with both local anaesthetic and opioid agonist properties. We have in a recent study demonstrated that pethidine could be an interesting alternative to prilocaine in arthroscopy with local anaesthetic technique. Therefore, we investigated, in a controlled randomized double-blind study, the effect of three doses of pethidine compared with a standard local anaesthetic, in patients subjected to arthroscopic knee joint surgery. Ten patients in each group received 50 mg (P50), 100 mg (P100), 200 mg (P200) of pethidine or prilocaine (5 mg/ml) + adrenaline (4 mg/ml) (PC), injected intra-articularly (i.a.) before surgery. We measured pain intensity and discomfort during arthroscopy and pain intensity at rest and at movement, nausea and tiredness for 3 days post-operatively at regular intervals using the VAS-technique. We also measured the concentration of pethidine and its demethylated metabolite, norpethidine, in plasma by collecting blood samples at 20, 40, 60, 80, 140 and 200 min following injection, and in synovial fluid which was collected through the arthroscope at the start and the end of the surgery. It was found that significantly more patients in the P50 group (n = 6) needed general anaesthesia due to intense pain than those in the P100 group (n = 1), P200 group (n = 0) or the PC group (n = 1). The PC group required significantly more analgesics and had a significantly higher calculated total sum of pain scores at movement post-operatively, than the other three groups. The P200 group more often reported tiredness post-operatively than the other three groups. We conclude that 100 or 200 mg pethidine i.a. produces satisfactory anaesthesia for surgery. There was a rapid transfer of pethidine from synovial fluid to plasma, resulting in plasma levels earlier reported to produce centrally mediated effects, such as analgesia and tiredness. We found much higher concentrations of norpethidine in the synovial fluid than in plasma, suggesting a local demethylation in the knee joint tissues. This site of drug oxidation has not earlier been demonstrated neither in vitro nor in vivo. The results suggest that pethidine given i.a. in the dose range of 50 to 200 mg results in analgesia due to both peripheral and central mechanisms. The significant systemic uptake of pethidine can cause unwanted side-effects.

Ketobemidone plus (RS)-3-dimethylamino-1,1-diphenylbut-1-ene (A29) is more potent at NMDA receptors than ketobemidone alone: evidence for A29 as a non-competitive NMDA receptor antagonist.

The opioid, ketobemidone, has previously been shown to be a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist. In Denmark, ketobemidone is available in a formulation which contains ketobemidone and a spasmolytic compound, (RS)-3-dimethylamino-1,1-diphenylbut-1-ene, hydrochloride (A29), in a one to five ratio. Using in vitro receptor binding techniques and an in vitro electrophysiological preparation consisting of rat cerebral cortex, we have characterized the interaction between A29 and the different glutamate receptor subtypes. A29 selectively inhibited binding of the non-competitive NMDA receptor antagonist 3H-MK-801 with a Ki value 16 +/- 4.5 microM, but was inactive in assays measuring affinities for other glutamate receptors. In agreement with the binding studies, A29 was found to selectively inhibit responses to NMDA in the rat cortical wedge preparation, whereas responses to kainate and AMPA were unaffected. Analysis of dose response curves showed A29 to be a NMDA receptor antagonist with an IC50 value of 100 microM versus responses to 10 microM NMDA. The inhibitory effects of ketobemidone and A29 on responses to 10 microM NMDA were additive. These data show that the combination of A29 and ketobemidone exert more potent antagonism at the NMDA receptor than does ketobemidone alone.