Formation Clearance Research Articles

Evaluation of 6β-Hydroxycortisol, 6β-Hydroxycortisone, and a Combination of the Two as Endogenous Probes for Inhibition of CYP3A4 In Vivo

An endogenous probe for CYP3A activity would be useful for early identification of in vivo cytochrome P450 (CYP) 3A4 inhibitors. The aim of this study was to determine whether formation clearance (CL(f)) of the sum of 6β-hydroxycortisol and 6β-hydroxycortisone is a useful probe of CYP3A4 inhibition in vivo. In human liver microsomes (HLMs), the formation of 6β-hydroxycortisol and 6β-hydroxycortisone was catalyzed by CYP3A4, and itraconazole inhibited these reactions with half maximal inhibitory concentration (IC(50))(,u) values of 3.1 nmol/l and 3.4 nmol/l, respectively. The in vivo IC(50,u) value of itraconazole for the combined CL(f) of 6β-hydroxycortisone and 6β-hydroxycortisol was 1.6 nmol/l. The greater inhibitory potency in vivo is probably due to circulating inhibitory itraconazole metabolites. The maximum in vivo inhibition was 59%, suggesting that f(m,CYP3A4) for cortisol and cortisone 6β-hydroxylation is ~60%. Given the significant decrease in CL(f) of 6β-hydroxycortisone and 6β-hydroxycortisol after 200-mg and 400-mg single doses of itraconazole, this endogenous probe can be used to detect moderate and potent CYP3A4 inhibition in vivo.

Population pharmacokinetic modeling of tramadol and its O-desmethyl metabolite in plasma and breast milk

The aim of this investigation was to demonstrate that nonlinear mixed-effects population pharmacokinetic (PK) modeling can be used to evaluate data from studies of drug transport/excretion into human milk and hence to estimate infant exposure. A sparse dataset from a previously published study of the use of oral tramadol for post-cesarean pain management in 75 lactating women was used. Milk and plasma samples were collected during days 2-4 of lactation, and tramadol and O-desmethyltramadol (ODT) concentration measurements in these samples were available. Absolute infant dose was obtained from the concentration measurements and estimated milk volume ingested, and expressed in micrograms per kilogram per day. Relative infant dose was calculated as a percentage of the absolute infant dose divided by the maternal dose (μg/kg/day). Nonlinear mixed-effects modeling was used to fit a population PK model to the data. The disposition of tramadol and ODT in plasma and the transition of these substances into milk were characterized by a five-compartment population PK mixture model with first-order absorption. The polymorphic ODT formation clearance in the plasma compartment was able to be characterized in both CYP2D6-poor and -extensive metabolizers. Milk creamatocrit was a significant covariate in ODT transfer between the plasma and milk compartments. The estimated relative infant doses in extensive and poor metabolizers, respectively, were 2.16 ± 0.57 and 2.60 ± 0.57% for tramadol, and 0.93 ± .20 and 0.47 ± 0.10% for ODT. This study demonstrates that a population PK approach with sparse sampling of analytes in milk and plasma can yield quality information about the transfer process and that it also can be used to estimate the extent of infant exposure to maternal drugs via milk.

Tests on Strength of Coped Beams with Web Stiffening

In steel construction, when beams have to be connected to a girder at the same elevation, beam flanges must often be coped to provide enough clearance for practical joint formation. The presence of a cope in a beam will reduce the strength of the beam in the coped region. To improve the strength of coped beams, web reinforcements at the coped region were proposed in previous research studies. This paper reports the results of the first phase of the experimental program. A total of 8 tests were conducted. The test parameters include the length of longitudinal stiffeners, cope depth to beam depth ratio, cope length to beam depth ratio, and doubler plate. The test results show that the strength of the coped beam specimens was significantly increased when stiffeners were used in the cope. The increase in strength due to the presence of the longitudinal stiffeners could be as high as 96%. The longitudinal stiffeners were able to prevent local web buckling from occurring at the cope, however, the final failure mode of the beams was yielding of non-reduced section followed by rigid body movement of stiffener. The doubler plate was able to increase the strength of the beams; however, the final failure mode of the beams was still local web buckling.

Developmental pharmacology of tramadol during infancy: ontogeny, pharmacogenetics and elimination clearance

To illustrate the complex interaction between ontogeny, i.e., age-dependent maturation, genetic polymorphisms and renal elimination clearance during infancy, based on developmental disposition of intravenous tramadol during infancy. Tramadol (M) is metabolized by O-demethylation (cytochrome P450 [CYP] 2D6) to the pharmacodynamic active metabolite O-demethyl tramadol (M1). This metabolite is subsequently eliminated by renal route while M1 formation will in part depend on ontogeny, i.e., age-dependent activity and CYP2D6 polymorphisms. However, these pathways do not mature simultaneously. A pooled pharmacokinetic analysis of earlier reported time-concentration profiles in neonates and infants was performed with subsequent simulation of the impact of ontogeny, polymorphisms and renal elimination clearance during infancy. Tramadol plasma time-concentration profile changes with postmenstrual age. The highest metabolite concentrations occur in the 52-week infant, where M1 formation clearance (hepatic, CYP2D6) is already mature but metabolite elimination clearance (through glomerular filtration rate) is immature. The phenotypic observations might in part explain unanticipated (side-)effects of tramadol. In addition to the compound-specific clinical implications, it is important to stress that the maturational trends in the elimination processes described can be considered for other compounds (e.g., codeine) that undergo similar elimination routes.

A population pharmacokinetic evaluation of the influence of CYP2D6 genotype on risperidone metabolism in patients with acute episode of schizophrenia

The objective of this prospective study was to characterize the metabolism of risperidone to (+)− and (−)−9-hydroxyrisperidone in vivo and to evaluate the influence of CYP2D6 genotype. A population pharmacokinetic modeling approach was used to estimate the interindividual variability of the pharmacokinetic parameters in 50 hospitalized patients with acute episode of schizophrenia. CYP2D6 genotype remarkably influenced the formation clearances of the risperidone metabolites, while creatinine clearance was related to the plasma clearance of 9-hydroxyrisperidone. CYP2D6 genotype was also associated with the average plasma concentration of risperidone active moiety (a sum of all three active compounds). In comparison to the patients with CYP2D6*1/*1 genotype, average steady-state plasma concentration of risperidone active moiety was 3.3- and 1.6-fold higher in poor metabolizers (both alleles nonfunctional; CYP2D6*3 or *4) and intermediate metabolizers (one nonfunctional allele and one allele for diminished enzyme activity; CYP2D6*10 or *41), respectively. Additionally, average plasma concentration of risperidone active moiety was higher in the patients with dystonia ( p = 0.0066) and parkinsonism ( p = 0.046). The results of this study imply the potential role of CYP2D6 genotyping in personalizing risperidone therapy in patients with schizophrenia to reduce the incidence of adverse extrapyramidal symptoms.

The relative importance of CYP26A1 in hepatic clearance of all-trans retinoic acid

All-trans retinoic acid (RA) is a critical signaling molecule and its concentration is tightly regulated. Several P450 enzymes including CYP26A1, CYP2C8, and CYP3A4 have been proposed to be responsible for RA clearance in the liver but their quantitative importance has not been demonstrated. To determine the contribution of CYP26A1 to hepatic clearance of RA, CYP26A1 protein was quantified in 37 human liver microsomes (HLMs). CYP26A1 expression ranged from not detectable to 2.80 pmol/mg microsomal protein. RA clearance by P450 enzymes abundant in human liver was measured in Supersomes ®. CYP2C8, CYP3A4, CYP3A5 and CYP3A7 metabolized RA with unbound K m values of 3.4–7.2 μM and V max values of 2.3–4.9 pmol/min/pmol P450, but were less efficient than CYP26A1 in clearing RA. Simulations performed for livers with varying P450 expression levels over a range of RA concentrations demonstrated that at both endogenous and therapeutic concentrations of RA, CYP26A1 is the primary enzyme responsible for 4-OH RA formation clearance. HLM incubation data showed that 4-OH RA formation velocity varied from 0.2 to 15.3 pmol/min/mg microsomal protein and velocity in HLMs was significantly correlated ( p < 0.01) to CYP26A1, CYP3A4, and CYP3A5 protein content, but not to CYP2C8. When experimental data were scaled to in vivo clearances, the predicted hepatic clearance of RA (0.07 L/min using combined Supersome ® data) was similar to the published in vivo clearance of RA. These findings suggest that CYP26A1 is the P450 isoform that should be targeted when designing RA metabolism blocking agents.

Use of Enantiomeric Bupropion and Hydroxybupropion to Assess CYP2B6 Activity in Glomerular Kidney Diseases

Chronic kidney disease affects more than 50 million people in the U.S.1. The top three etiologies for end-stage kidney disease are diabetes mellitus (45% of patients), hypertension (29% of patients), and glomerulonephritis (19% of patients).2 While it is well established that chronic kidney diseases can result in decreased elimination of drugs via the kidneys, the effects of kidney disease on non-renal clearance processes, especially specific metabolic routes, are less well described. Experimental models of chronic kidney disease have reported reductions in hepatic cytochrome P450 (CYP) enzymes including 3A1, 3A2, 2C11, and N-acetyltransferases.3–5 Reductions in nonrenal clearance ranging from 30% to 67% have been reported for substrates of the CYP3A4, CYP2D6, CYP2B6, and CYP2C9 enzymes in patients with kidney disease.6 In order to fully understand the clinical significance of altered metabolic routes associated with kidney disease, it will be necessary to evaluate the effects that specific forms of kidney diseases have on these pathways and whether these processes effect drug disposition. Numerous studies have reported various probe drugs or cocktail approaches to evaluate the in vivo function of various drug metabolizing enzymes and transporters in patients.7, 8 However, only a few studies9–12 were actually conducted in chronic kidney disease patients, and hence applicability of most of the published studies beyond the healthy control population remains to be established. As chronic kidney disease patients are commonly prescribed ~10–12 different daily medications13, studies that evaluate metabolic pathway alterations should address requirements for modifications in drug regimens as well as drug interaction potential. While studies designed to assess and report the pharmacokinetics of bupropion exist14, 15, studies designed specifically to assess the influence of chronic kidney diseases on CYP2B6 activity are currently nonexistent. CYP2B6 is responsible for the metabolism of 3–8% of the currently marketed drugs16, 17 including bupropion, cyclophosphamide, efavirenz, selegiline, methadone, and sertraline18–22 and several drugs, including clopidrogrel, ticlopidine, clotrimazole, itraconazole, sertraline, and raloxifene have been purported to inhibit CYP2B6.23 Data from healthy subjects demonstrate that both the R and S enantiomers of bupropion and its hydroxy-metabolite are present in plasma, but only stereoselective S bupropion and (S,S) hydroxybupropion formation clearance have been shown to be a phenotypic probe for CYP2B624, complicating the assessment of in vivo activity. The purpose of the current study was to evaluate the pharmacokinetics of enantiomeric bupropion and its hydroxybupropion metabolite in patients with kidney diseases affecting the glomerulus in order to provide an assessment of CYP2B6 activity in this disease state.

What is the learning curve for Paediatric Percutaneous Nephrolithotomy?

Purpose Paediatric urinary tract stones are infrequent in the U.K. In our unit. PCNL is performed jointly by the paediatric urologist and paediatric radiologist only. We reviewed our experience to determine what constituted the ‘learning curve’ for paediatric PCNL. Material and Methods We reviewed the notes of 26 children who underwent PCNL in 2 years. Measures of outcome to achieve competence was defined as time taken (number of cases) to attain the plateau phase of the learning curve. Defined outcome measures were success of first pass puncture and track formation, complete stone clearance by preoperative fluoroscopy and postoperative ultrasonography, overall time required for the procedure and complications. Results There were 28 episodes of PCNL. The first 4 cases performed under supervision had a successful puncture and track formation and complete stone clearance without complications. The overall time required was 97 minutes (46-128). Of the next 4, two had a failed puncture. The remaining two had successful track formation and complete stone clearance without complications. The overall operating time was 183 minutes (150-237). The subsequent 20 (including the two with failed puncture) had a successful puncture and complete stone clearance in all but the last case with abnormal calyceal anatomy in a scarred upper pole. Of these a first pass puncture was successful in all cases after case 12. There were no complications. The overall operating time was 146 minutes (90-220). All children were discharged within 48 hours of surgery. Conclusions Numerous factors are responsible for determining the learning curve for paediatric PCNL including the stone burden, dilatation of the collecting system and renal scarring. In our review a consistent steady state was achieved after 12 cases. Failure may still occur in difficult cases despite overcoming the learning curve.

Mild Hypothermia Alters Midazolam Pharmacokinetics in Normal Healthy Volunteers

The clinical use of therapeutic hypothermia has been rapidly expanding due to evidence of neuroprotection. However, the effect of hypothermia on specific pathways of drug elimination in humans is relatively unknown. To gain insight into the potential effects of hypothermia on drug metabolism and disposition, we evaluated the pharmacokinetics of midazolam as a probe for CYP3A4/5 activity during mild hypothermia in human volunteers. A second objective of this work was to determine whether benzodiazepines and magnesium administered intravenously would facilitate the induction of hypothermia. Subjects were enrolled in a randomized crossover study, which included two mild hypothermia groups (4 degrees C saline infusions and 4 degrees C saline + magnesium) and two normothermia groups (37 degrees C saline infusions and 37 degrees C saline + magnesium). The lowest temperatures achieved in the 4 degrees C saline + magnesium and 4 degrees C saline infusions were 35.4 +/- 0.4 and 35.8 +/- 0.3 degrees C, respectively. A significant decrease in the formation clearance of the major metabolite 1'-hydroxymidazolam was observed during the 4 degrees C saline + magnesium compared with that in the 37 degrees C saline group (p < 0.05). Population pharmacokinetic modeling identified a significant relationship between temperature and clearance and intercompartmental clearance for midazolam. This model predicted that midazolam clearance decreases 11.1% for each degree Celsius reduction in core temperature from 36.5 degrees C. Midazolam with magnesium facilitated the induction of hypothermia, but shivering was minimally suppressed. These data provided proof of concept that even mild and short-duration changes in body temperature significantly affect midazolam metabolism. Future studies in patients who receive lower levels and a longer duration of hypothermia are warranted.

Inhibition of Morphine Metabolism by Ketamine

Clinical observation of a synergistic effect of ketamine on morphine analgesia remains controversial. Although a pharmacodynamic basis for an interaction has been explored in animal and clinical studies, the possibility of a pharmacokinetic mechanism has not been investigated. Whereas both morphine and morphine-6-glucuronide are effective analgesics, morphine-3-glucuronide (M3G) lacks activity. Thus, changes in the metabolism and disposition of morphine may result in an altered response. First, we investigated the interaction between morphine and ketamine in the isolated perfused rat liver preparation. The clearance of morphine was decreased from 16.8 +/- 4.6 ml/min in the control period to 7.7 +/- 2.8 ml/min in the ketamine-treatment period, with the formation clearance of M3G decreasing from 8.0 +/- 4.1 ml/min to 2.1 +/- 1.1 ml/min. Fractional conversion of morphine to M3G was significantly decreased from 0.46 +/- 0.17 in the control period to 0.28 +/- 0.14 upon the addition of ketamine. The possible mechanism of the interaction was further investigated in vitro with rat liver microsomes as the enzyme source. The formation of M3G followed single-enzyme Michaelis-Menten kinetics, with a mean apparent K(m) of 2.18 +/- 0.45 mM and V(max) of 8.67 +/- 0.59 nmol/min/mg. Ketamine inhibited morphine 3-glucuronidation noncompetitively, with a mean K(i) value of 33.3 +/- 7.9 microM. The results demonstrate that ketamine inhibits the glucuronidation of morphine in a rat model.

Effects of folic acid supplementation on the pharmacokinetics and anticoagulant effect of warfarin: An open-label, prospective study of long-term administration in adults

Background: Folic acid supplementation in patients with folic acid deficiency has been associated with increased clearance of phenytoin to its cytochrome P450 (CYP) 2C9-mediated metabolite, 5-(4′-hydroxyphenyl)-5-phenylhydantoin. Objective: The aim of this study was to determine whether folic acid supplementation increases the dosage requirement of the CYP2C9 substrate warfarin, and the formation clearance of the CYP2C9-mediated product, ( S)-7-hydroxywarfarin. Methods: Patients aged ≥18 years with folic acid deficiency who were receiving long-term treatment with a stable dosage of warfarin were studied prospectively, before and 30 to 60 days after the initiation of supplementation with folic acid. Warfarin dosage and international normalized ratio (INR) were documented, and the formation clearance of ( S)- and ( R)-7-hydroxywarfarin and the oral clearance of ( S)- and ( R)-warfarin were determined. Results: Twenty-four white patients (14 males; mean (SD) age, 55.0 [19.7] years; body mass index, 30.64 [6.8] kg/m 2) were enrolled. Treatment with folic acid was associated with a significantly increased mean (SD) formation clearance of ( S)-7-hydroxywarfarin (1.096 [0.816] vs 1.608 [1.302] mL/min; P = 0.048). Before folic acid supplementation, the mean (SD) warfarin dosage was 5.98 (2.12) mg/d, and the INR was 2.51 (0.55). During supplementation, the warfarin dosage was 6.17 (2.31) mg/d and the INR was 2.63 (0.65) (both, P = NS vs before supplementation). Conclusions: Folic acid supplementation was associated with significantly increased formation clearance of ( S)-7-hydroxywarfarin. Changes in warfarin dosage requirements and INR were nonsignificant.

Disposition and Metabolism of Semagacestat, a γ-Secretase Inhibitor, in Humans

Semagacestat is a functional gamma-secretase inhibitor that has been shown to reduce the rate of formation of amyloid-beta in vitro and in vivo. This study was conducted to characterize the disposition of semagacestat in humans. After a single 140-mg dose of [(14)C]semagacestat administered as an oral solution to six healthy male subjects, semagacestat was rapidly absorbed (T(max) approximately 0.5 h) and eliminated from the systemic circulation (terminal t(1/2) approximately 2.4 h). The major circulating metabolites of semagacestat, M2 (hydrolysis of the amide bond proximal to the benzazepine ring) and M3 (benzylic hydroxylation of the benzazepine ring), accounted for approximately 27 and 10% of total radioactivity exposure, respectively, as calculated from relative area under the plasma concentration versus time curve from 0 to 24 h derived from the plasma radiochromatograms. The radioactive dose was almost completely recovered after 7 days postdose, with 87% of the dose in urine and 8% in feces. Unchanged [(14)C]semagacestat in urine accounted for approximately 44% of the dose, which indicates that renal excretion played an important role in elimination. Metabolites M2 and M3, with their related secondary metabolites, each accounted for approximately 20% of the dose in excreta. In vitro data indicate the formation of M3 is primarily mediated by CYP3A, with cDNA-expressed CYP3A5 approximately 2 times more efficient than CYP3A4 in forming M3. Thus, the relative content of CYP3A4 and CYP3A5 in humans will likely determine the formation clearance of M3 after exposure to semagacestat.

Morphine Glucuronidation in Preterm Neonates, Infants and Children Younger than 3 Years

A considerable amount of drug use in children is still unlicensed or off-label. In order to derive rational dosing schemes, the influence of aging on glucuronidation capacity in newborns, including preterms, infants and children under the age of 3 years was studied using morphine and its major metabolites as a model drug. A population pharmacokinetic model was developed with the nonlinear mixed-effects modelling software NONMEM V, on the basis of 2159 concentrations of morphine and its glucuronides from 248 infants receiving intravenous morphine ranging in bodyweight from 500 g to 18 kg (median 2.8 kg). The model was internally validated using normalized prediction distribution errors. Formation clearances of morphine to its glucuronides and elimination clearances of the glucuronides were found to be primarily influenced by bodyweight, which was parameterized using an allometric equation with an estimated exponential scaling factor of 1.44. Additionally, a postnatal age of less than 10 days was identified as a covariate for formation clearance to the glucuronides, independent of birthweight or postmenstrual age. Distribution volumes scaled linearly with bodyweight. Model-based simulations show that in newborns, including preterms, infants and children under the age of 3 years, a loading dose in microg/kg and a maintenance dose expressed in microg/kg1.5/h, with a 50% reduction of the maintenance dose in newborns younger than 10 days, results in a narrow range of morphine and metabolite serum concentrations throughout the studied age range. Future pharmacodynamic investigations are needed to reveal target concentrations in this population, after which final dosing recommendations can be made.

Effect of rutin on warfarin anticoagulation and pharmacokinetics of warfarin enantiomers in rats

The effects of the flavonoid rutin on the anticoagulant activity of oral warfarin and the protein binding and pharmacokinetics of its enantiomers were investigated in rats. A single dose of racemic warfarin, 1.5 mg/kg, was administered orally to rats either alone or on day 5 of an 8-day oral regimen of rutin, 1 g/kg daily. Rutin reduced the anticoagulant effect of racemic warfarin, evident as a 31% reduction in the area under the prothrombin complex activty-time curve (P < 0.05). Rutin had no apparent effect on pre-treatment baseline blood coagulation. It enhanced the in-vitro serum protein binding of S- and R-warfarin (reflected by 40% and 26% reductions in unbound fraction, respectively), and thus restricted distribution by 33 and 21%, respectively. Treatment with rutin significantly decreased the elimination half-life of S-warfarin by 37% as a result of the 69% increase in unbound clearance of the S-enantiomer. This effect was attributed to a significant 77% increase in the unbound formation clearance of the overall oxidative and reductive metabolites, and an increase in the unbound renal clearance of the more potent S-enantiomer of warfarin. Concurrent rutin administration is likely to reduce the anticoagulant effect of racemic warfarin, reflecting a significant decrease in the elimination half-life of the more potent S-enantiomer.

Effect of rutin on warfarin anticoagulation and pharmacokinetics of warfarin enantiomers in rats

Abstract Objectives The effects of the flavonoid rutin on the anticoagulant activity of oral warfarin and the protein binding and pharmacokinetics of its enantiomers were investigated in rats. Methods A single dose of racemic warfarin, 1.5 mg/kg, was administered orally to rats either alone or on day 5 of an 8-day oral regimen of rutin, 1 g/kg daily. Results Rutin reduced the anticoagulant effect of racemic warfarin, evident as a 31% reduction in the area under the prothrombin complex activty–time curve (P &amp;lt; 0.05). Key findings Rutin had no apparent effect on pre-treatment baseline blood coagulation. It enhanced the in-vitro serum protein binding of S- and R-warfarin (reflected by 40% and 26% reductions in unbound fraction, respectively), and thus restricted distribution by 33 and 21%, respectively. Treatment with rutin significantly decreased the elimination half-life of S-warfarin by 37% as a result of the 69% increase in unbound clearance of the S-enantiomer. This effect was attributed to a significant 77% increase in the unbound formation clearance of the overall oxidative and reductive metabolites, and an increase in the unbound renal clearance of the more potent S-enantiomer of warfarin. Conclusions Concurrent rutin administration is likely to reduce the anticoagulant effect of racemic warfarin, reflecting a significant decrease in the elimination half-life of the more potent S-enantiomer.

Methadone metabolism and clearance are induced by nelfinavir despite inhibition of cytochrome P4503A (CYP3A) activity

Background Methadone plasma concentrations are decreased by nelfinavir. Methadone clearance and the drug interactions have been attributed to CYP3A4, but actual mechanisms of methadone clearance and the nelfinavir interaction are unknown. We assessed nelfinavir effects on methadone pharmacokinetics and pharmacodynamics, intestinal and hepatic CYP3A4/5 activity, and intestinal P-glycoprotein transport activity. CYP3A4/5 and transporters were assessed using alfentanil and fexofenadine, respectively. Methods Twelve healthy HIV-negative volunteers underwent a sequential crossover. On three consecutive days they received oral alfentanil plus fexofenadine, intravenous alfentanil, and intravenous plus oral methadone. This was repeated after nelfinavir. Plasma and urine analytes were measured by mass spectrometry. Opioid effects were measured by pupil diameter change (miosis). Results Nelfinavir decreased intravenous and oral methadone plasma concentrations 40–50%. Systemic clearance, hepatic clearance, and hepatic extraction all increased 1.6- and 2-fold, respectively, for R- and S-methadone; apparent oral clearance increased 1.7- and 1.9-fold. Nelfinavir stereoselectively increased (S > R) methadone metabolism and metabolite formation clearance, and methadone renal clearance. Methadone bioavailability and P-glycoprotein activity were minimally affected. Nelfinavir decreased alfentanil systemic and apparent oral clearances 50 and 76%, respectively. Nelfinavir appeared to shift the methadone plasma concentration–effect (miosis) curve leftward and upward. Conclusions Nelfinavir induced methadone clearance by increasing renal clearance, and more so by stereoselectively increasing hepatic metabolism, extraction and clearance. Induction occurred despite 50% inhibition of hepatic CYP3A4/5 activity and more than 75% inhibition of first-pass CYP3A4/5 activity, suggesting little or no role for CYP3A in clinical methadone disposition. Nelfinavir may alter methadone pharmacodynamics, increasing clinical effects.

P4-406: Tarenflurbil (MPC-7869), a selective Aβ42-lowering agent, has no significant effect on drugs metabolized by CYP2C9

Alzheimer's disease is the most common type of dementia among the elderly. Its histopathologic features include phosphorylated Tau neurofibrillary tangles and amyloid beta senile plaques. In randomized, double-blind Phase II studies, the Selective Aβ42-Lowering Agent (SALA) tarenflurbil (MPC-7869) has demonstrated efficacy and tolerability. In earlier studies, CYP2C9 inhibition was demonstrated in vitro. Therefore, tarenflurbil has the potential to inhibit CYP2C9 in vivo. Sixteen healthy subjects participated in this third-party blind, placebo-controlled, randomized, crossover, Phase I study. Participants received either placebo or two 400-mg oral tablets of tarenflurbil every 12 hours for 9 consecutive days. On Day 6, each subject received one 500-mg oral tablet of tolbutamide, a CYP2C9 probe substrate. After the 14-day washout period, the treatments were switched (ie, subjects received the other treatment). Blood samples were collected throughout the study. Blood and urine samples were obtained on Day 6, prior to the morning dose of tarenflurbil or placebo and tolbutamide, and throughout the 96 hours after tolbutamide administration. The 90% confidence intervals for tolbutamide AUC(0–∞) (110%–120%) and Cmax (88%–112%) were within the standard bioequivalence criteria (80%–125%). Five days after administration of tarenflurbil 800 mg BID, the difference between mean tolbutamide exposure AUC(0–∞) with and without tarenflurbil was less than 15%. Furthermore, the formation clearance of 4-hydroxytolbutamide was similar for the treated and untreated states. Tarenflurbil has low potential for inhibition-based metabolic drug-drug interactions. Because of its amyloid-lowering properties, tarenflurbil holds promise as a disease-modifying agent for Alzheimer's disease.

Identification of the Human Enzymes Involved in the Oxidative Metabolism of Dasatinib: An Effective Approach for Determining Metabolite Formation Kinetics

N-(2-Chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl]-2-methyl-4-pyrimidinyl]amino]-5-thiazolecarboxamide (dasatinib, Sprycel, BMS-354825; Bristol-Myers Squibb, Princeton, NJ) is a potent protein kinase inhibitor to treat chronic myeloid leukemia. In vivo studies have shown that the primary oxidative metabolites of dasatinib are M4 (N-dealkylation), M5 (N-oxidation), M6 (carboxylic acid formation), M20, and M24 (hydroxylation). To identify the enzymes responsible for the formation of these metabolites, [(14)C]-dasatinib and nonradiolabeled dasatinib were incubated with human cDNA-expressed enzymes [cytochromes P450 (P450s) and flavin-containing monooxygenase (FMO) 3] or human liver microsome (HLM) in the presence of selective P450 inhibitors (antibodies and chemical inhibitors). The results of these experiments showed that metabolites M4, M20, and M24 were mainly generated by CYP3A4; M5 was primarily formed by FMO3; and M6 was formed by a cytosolic oxidoreductase. The enzyme kinetic analysis showed that the formation of M4 and M5 in HLM followed the Michaelis-Menten kinetics, and the formation data of M20 and M24 fitted well to a partial substrate inhibition kinetic model. The K(m) values were determined by the kinetic analysis of the substrate-dependent metabolite formation plots from a large number of incubations with the nonlabeled dasatinib; the V(max) values were calculated with the predetermined K(m) values and the metabolite formation rates from a limited number of incubations with [(14)C]dasatinib. The intrinsic formation clearance values (V(max)/K(m)) of 52, 14, 274, and 20 microl/mg protein/min for the formation of M4, M5, M20, and M24, respectively, suggested that the formation of M20 was more efficient than other metabolites. Collectively, multiple in vitro experiments showed that dasatinib was predominately metabolized by CYP3A4.

Possible Differential Induction of Phase 2 Enzyme and Antioxidant Pathways by American Ginseng, Panax quinquefolius

Human immunodeficiency virus (HIV)-infected patients often take herbal medicines, which may interact with antiretrovirals. American ginseng induces phase 2 and antioxidant enzymes in vitro and might increase the clearance of zidovudine and/or enhance antioxidant activity. Ten healthy volunteers received 300 mg of zidovudine orally before and after 2 weeks of treatment with a ginsenoside-enriched American ginseng extract 200 mg twice daily. This ginseng extract induced the phase 2 enzyme quinone reductase with an average concentration of doubling of enzyme activity of 190 microg/mL. Total ginsenoside content was 8.5 +/- 0.5%. Pharmacokinetic profiles of zidovudine and oxidative stress marker concentrations were measured post-zidovudine dose. American ginseng does not significantly affect the formation clearance of zidovudine to its glucuronide (ratio post- to pre-American ginseng = 1.17; 90% confidence interval: 0.95-1.45; P = .21), total clearance (ratio = 0.97; 0.82-1.14; P = .70), or plasma zidovudine AUC0-8 (ratio = 1.03; 0.87-1.21; P = .77). Oxidative stress biomarkers are reduced post-American ginseng (F2-isoprostane ratio = 0.79; 0.72-0.86; P < .001; 8-hydroxy-deoxyguanosine ratio = 0.74; 0.59-0.92; P = .02). Two weeks of American ginseng does not alter zidovudine pharmacokinetics but reduces oxidative stress markers.

The metabolism of amiodarone by various CYP isoenzymes of human and rat, and the inhibitory influence of ketoconazole

To evaluate the metabolism of amiodarone (AM) to desethylamiodarone (DEA) by selected human and rat cytochrome P450, and the inhibitory effect of ketoconazole (KTZ). Some important CYP isoenzymes (rat CYP1A1, 1A2, 2C6, 2C11, 2D1, 2D2, and 3A1 and human CYP1A1, 1A2, 2D6 and 3A4) were spiked with various concentrations of AM to determine the relative kinetic parameters for formation of DEA in the presence and absence of various concentrations of KTZ. The formation of DEA was observed when AM was exposed to each of the CYP tested, although the rates were varied. Human CYP1A1 followed by 3A4 had the highest intrinsic clearance (CLint) for DEA formation whereas in rat, CYP2D1 followed by CYP2C11 had the highest CLint. Human and rat CYP1A2 seemed to have the lowest CLint. At high concentrations of AM and KTZ, near those expected in vivo, significant inhibition of all isoforms except for rat CYP1A2 was observed. At lower concentration ranges of both drugs, the inhibitory constant was determined. At these levels, KTZ was found to potently inhibit human CYP1A1 and 3A4 and rat 2D2 and 1A1. Human CYP1A1 and 3A4 and rat CYP2D1 and 2C11 were most efficient in converting AM to DEA. For DEA formation, the in vivo administration of KTZ could inhibit other CYP isoforms besides CYP3A in human and rat.