Contractility of the cell rear drives invasion of breast tumor cells in 3D Matrigel.

Cancer cells use different modes of migration, including integrin-dependent mesenchymal migration of elongated cells along elements of the 3D matrix as opposed to low-adhesion-, contraction-based amoeboid motility of rounded cells. We report that MDA-MB-231 human breast adenocarcinoma cells invade 3D Matrigel with a characteristic rounded morphology and with F-actin and myosin-IIa accumulating at the cell rear in a uropod-like structure. MDA-MB-231 cells display neither lamellipodia nor bleb extensions at the leading edge and do not require Arp2/3 complex activity for 3D invasion in Matrigel. Accumulation of phospho-MLC and blebbing activity were restricted to the uropod as reporters of actomyosin contractility, and velocimetric analysis of fluorescent beads embedded within the 3D matrix showed that pulling forces exerted to the matrix are restricted to the side and rear of cells. Inhibition of actomyosin contractility or ß1 integrin function interferes with uropod formation, matrix deformation, and invasion through Matrigel. These findings support a model whereby actomyosin-based uropod contractility generates traction forces on the ß1 integrin adhesion system to drive cell propulsion within the 3D matrix, with no contribution of lamellipodia extension or blebbing to movement.

Impact of prescriber's handwriting style and nurse's duty duration on the prevalence of transcription errors in public hospitals.

AIMS AND OBJECTIVES: To investigate the prevalence of transcription errors in a main public hospital in Pakistan and to test the impact of medication name and dose writing styles and the nurse duty duration on the occurrence of transcription errors. BACKGROUND: Medication errors occur frequently in public hospitals. Errors occurring at the transcription stage have not been sufficiently investigated. DESIGN: Medications transcripts and dispensed item labels were prospectively reviewed. In the second stage, nurses (n=25) transcribed medication charts in a double-blind randomised cross-over design administered at one, six and 10 hours after the commencement of their duty. METHODS. Inpatient (n=1000), discharge patient (n=1000) medication transcripts and labels of dispensed items for (n=1000) transcripts were reviewed. On medication charts, orthographically similar medications (n=20) were written in lowercase and Tall Man, decimal doses were written covered and uncovered, and metric doses were written with and without trailing zeros. RESULTS: Of the 6583 and 5329 medications transcribed from inpatient and discharge patient charts, error rates were 16·9 and 13·8%, respectively. Labels for 6734 dispensed items were reviewed, and error rate was 6·1%. Tall Man, covered decimal points and avoiding trailing zeros with decimal units significantly reduced transcription errors. CONCLUSION: Errors increased with increasing nurse duty duration. Highlighting orthographically similar medications and the use of proper decimal and metric units reduce errors. RELEVANCE TO CLINICAL PRACTICE: Transcription errors are highly prevalent in Pakistan public hospitals; therefore, elimination of transcription stage is encouraged.

Does modulation of organic cation transporters improve pralidoxime activity in an animal model of organophosphate poisoning?

OBJECTIVES: Pralidoxime is an organic cation used as an antidote in addition to atropine to treat organophosphate poisoning. Pralidoxime is rapidly eliminated by the renal route and thus has limited action. The objectives of this work were as follows. 1) Study the role of organic cation transporters in the renal secretion of pralidoxime using organic cation transporter substrates (tetraethylammonium) and knockout mice (Oct1/2⁻/⁻; Oct3⁻/⁻). 2) Assess whether sustained high plasma concentrations increase pralidoxime antidotal activity toward paraoxon-induced respiratory toxicity. SETTING: INSERM U705, Faculté de Pharmacie, Université Paris Descartes, 4 Avenue de l'Observatoire, 75006 Paris, France. SUBJECTS: Rodents: Knockout mice (Oct1/2⁻/⁻; Oct3⁻/⁻) and Sprague-Dawley rats. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: In rats, the renal clearance of pralidoxime was 3.6-fold higher than the creatinine clearance. Pretreatment with tetraethylammonium (75 mg/kg) in rats or deficiencies in organic cation transporters 1 and 2 in mice (Oct1/2⁻/⁻) resulted in a significant increase in plasma pralidoxime concentrations. Lack of Oct3 did not alter plasma pralidoxime concentrations. The antidotal activity of pralidoxime (50 mg/kg intramuscularly) was longer and with greater effect, resulting in a return to normal values when administered to rats pretreated with tetraethylammonium. CONCLUSIONS: Pralidoxime is secreted in rats and mice by renal Oct1 and/or Oct2 but not by Oct3. Modulation of organic cation transporter activity increased the plasma pralidoxime concentrations and the antidotal effect of pralidoxime with sustained return within the normal range of respiratory variables in paraoxon-poisoned rats. These results suggest a promising approach in an animal model toward the increase in efficiency of pralidoxime. However, further studies are needed before these results are extended to human poisoning.

Cannabis expectancies in substance misusers: French validation of the Marijuana Effect Expectancy Questionnaire.

The aim of this study was to evaluate the psychometric properties of the French version of the Marijuana Effect Expectancy Questionnaire (48 items) and study the cannabis expectancies according to the patterns of substance use and psychiatric disorders (DSM-IV). A sample of 263 subjects (average age 33.1 years [SD = 8.7], 56% men) consisting of cannabis users (n = 64), psychiatric inpatients (n = 175, most of whom were hospitalized for withdrawal), and a control group (n = 24) completed the questionnaire. Internal reliability was good (α= .87) and temporal reliability was satisfactory, with 24 of 48 items having a significant κ ≥ .41. Factor analysis showed four main factors that explained 42.1% of the total variance. The women feared Cognitive Impairment and Negative Effects, and Negative Behavioral Effects more than the men. The onset age of cannabis use, onset age of abuse, abuse and dependence were associated with fewer negative expectancies. Cannabis dependents differed from abusers by more Relaxation and Social Facilitation expectancies. Patients with major depressive episodes, panic disorder, social anxiety disorder, or posttraumatic stress disorder feared negative effects the most. Schizophrenic patients expected more Perceptual Enhancement and Craving. The French version of the Marijuana Effect Expectancy Questionnaire has good psychometric properties and is valid to assess cannabis expectancies in adolescents and adults with substance use disorders.

Electronic prescribing reduces prescribing error in public hospitals.

AIMS AND OBJECTIVES: To examine the incidence of prescribing errors in a main public hospital in Pakistan and to assess the impact of introducing electronic prescribing system on the reduction of their incidence. BACKGROUND: Medication errors are persistent in today's healthcare system. The impact of electronic prescribing on reducing errors has not been tested in developing world. DESIGN: Prospective review of medication and discharge medication charts before and after the introduction of an electronic inpatient record and prescribing system. METHODS: Inpatient records (n = 3300) and 1100 discharge medication sheets were reviewed for prescribing errors before and after the installation of electronic prescribing system in 11 wards. RESULTS: Medications (13,328 and 14,064) were prescribed for inpatients, among which 3008 and 1147 prescribing errors were identified, giving an overall error rate of 22·6% and 8·2% throughout paper-based and electronic prescribing, respectively. Medications (2480 and 2790) were prescribed for discharge patients, among which 418 and 123 errors were detected, giving an overall error rate of 16·9% and 4·4% during paper-based and electronic prescribing, respectively. CONCLUSION: Electronic prescribing has a significant effect on the reduction of prescribing errors. RELEVANCE TO CLINICAL PRACTICE: Prescribing errors are commonplace in Pakistan public hospitals. The study evaluated the impact of introducing electronic inpatient records and electronic prescribing in the reduction of prescribing errors in a public hospital in Pakistan.

Sprague-Dawley rats display sex-linked differences in the pharmacokinetics of 3,4-methylenedioxymethamphetamine (MDMA) and its metabolite 3,4-methylenedioxyamphetamine (MDA).

The use of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has increased in recent years; it can lead to life-threatening hyperthermia and serotonin syndrome. Human and rodent males appear to be more sensitive to acute toxicity than are females. MDMA is metabolized to five main metabolites by the enzymes CYP1A2, CYP2D and COMT. Little is presently known about sex-dependent differences in the pharmacokinetics of MDMA and its metabolites. We therefore analyzed MDMA disposition in male and female rats by measuring the plasma and urine concentrations of MDMA and its metabolites using a validated LC-MS method. MDA AUC(last) and C(max) were 1.6- to 1.7-fold higher in males than in females given MDMA (5 mg/kg sc), while HMMA C(max) and AUC(last) were 3.2- and 3.5-fold higher, respectively. MDMA renal clearance was 1.26-fold higher in males, and that of MDA was 2.2-fold higher. MDMA AUC(last) and t(1/2) were 50% higher in females given MDMA (1 mg/kg iv). MDA C(max) and AUC(last) were 75-82% higher in males, with a 2.8-fold higher metabolic index. Finally, the AUC(last) of MDA was 0.73-fold lower in males given 1 mg/kg iv MDA. The volumes of distribution of MDMA and MDA at steady-state were similar in the two sexes. These data strongly suggest that differences in the N-demethylation of MDMA to MDA are major influences on the MDMA and MDA pharmacokinetics in male and female rats. Hence, males are exposed to significantly more toxic MDA, which could explain previously reported sexual dysmorphism in the acute effects and toxicity of MDMA in rats.

Pharmacokinetic-pharmacodynamic modeling of manganese after a single intravenous infusion of mangafodipir in patients with acute alcoholic hepatitis.

To determine the pharmacokinetic (PK) profile of manganese (Mn) after a 2-hour intravenous infusion of mangafodipir at 5 micromol/kg body weight and to correlate Mn concentrations with oxidative stress, early decrease in serum total bilirubin concentration, and prothrombin time (PT) in chronic alcoholic patients with acute alcoholic hepatitis. In 7 patients, a total of 49 serum Mn concentrations were determined on day 1 (before the start of the infusion and 15, 30, and 45 minutes after the end of the infusion) and on days 2, 7, 14, and 21. Fifty-seven PTs, reflecting liver activity, were measured on days 1, 2, 7, 14, and 21 and at months 1, 2, and 3. A population PK-pharmacodynamic model was developed to describe the kinetics of serum Mn concentrations and PT, to estimate interpatient variability, and to test covariate influence. A 2-compartment model with zero-order absorption and first-order elimination best described the data, and a signal transduction model with 2 transit compartments best described PT. Mean PK estimates and the corresponding interindividual variabilities (%) were clearance 23.1 L/h (34%), central and peripheral volume of distribution 35.4 and 1090 L, respectively, intercompartmental clearance 27.3 L (34%), endogenous Mn concentrations 15.8 nmol/L, slope-relating effect to concentration 141 nmol x L(-1) x s(-1) (52%), and mean transit time (tau) 3.8 days (34%). When patients had an early decrease in bilirubin at day 7, tau increased to 28.2 days. Serum Mn concentrations could be related to a decrease in PT; the effect was longer in patients with an early decrease in total bilirubin serum concentrations.

Acute renal failure alters the kinetics of pralidoxime in rats.

There is a trend towards increasing doses of pralidoxime to treat human organophosphate poisonings that may have relevance in subpopulations. Indeed, pralidoxime is eliminated unchanged by the renal route. This study assesses the effect of renal failure on the kinetics of pralidoxime in a rat model of acute renal failure induced by potassium dichromate administration. On the first day, Sprague-Dawley rats received subcutaneously potassium dichromate (study) or saline (control). Forty-eight hours post-injection, animals received pralidoxime methylsulfate (50mg/kg of pralidoxime base) intramuscularly. Blood specimens were sampled during 180min after the injection. Urine was collected daily during the 3 days of the study. Plasma pralidoxime concentrations were measured by liquid chromatography with electrochemical detection. There was a 2-fold increase in mean elimination half-life and a 2.5-fold increase in mean area under the curve in the study compared to the control group. The mean total body clearance was halved in the study compared to the control group. Our study showed acute renal failure does not modify the distribution of pralidoxime but significantly alters its elimination from plasma. These results suggest that dosages of pralidoxime should be adjusted in organophosphate-poisoned humans with renal failure when using high dosage regimen of pralidoxime.

Ventilatory effects of low-dose paraoxon result from central muscarinic effects.

Paraoxon induces respiratory toxicity. Atropine completely reversed parathion- and paraoxon-induced respiratory toxicity. The aim of this study was to assess the peripheral or central origin of ventilatory effects of low-dose paraoxon. Male Sprague-Dawley rats were given paraoxon 0.215 mg/kg subcutaneously and treated with either atropine (10 mg/kg sc) or ascending doses of methylatropine of 5.42 (equimolar to that of atropine), 54.2, and 542 mg/kg administered subcutaneously 30 min after paraoxon. Ventilation at rest was assessed using whole-body plethysmography and rat temperature using infra-red telemetry. Results are expressed as mean+/-SE. Statistical analysis used two-way ANOVA for repeated measurements. Paraoxon induced a significant decrease in temperature 30 min after injection lasting the 90 min of the study period. This effect was partially corrected by atropine, but not by methylatropine whatever the dose. Paraoxon induced a decrease in respiratory rate resulting from an increase in expiratory time associated with an increase in tidal volume. Atropine completely reversed the ventilatory effects of low-dose paraoxon while the equimolar dose of methylatropine had no significant effects. The 54.2 and 542 mg/kg doses of methylatropine had no significant effects. Atropine crosses the blood-brain barrier and reverses peripheral and central muscarinic effects. In contrast, methylatropine does not cross the blood-brain barrier. Atropine completely reversed the ventilatory effects of low-dose paraoxon, while methylatropine had no significant effects at doses up to 100-fold the equimolar dose of atropine. We conclude that the ventilatory effects of low-dose paraoxon are mediated by disrupted muscarinic signaling in the central nervous system.

Pharmacokinetic studies of scorpion venom before and after antivenom immunotherapy.

The improvement of the immunotherapeutic treatment of envenomations requires a better knowledge of the pharmacological actions of the scorpion venom and of the mechanism of its in vivo neutralization by antivenom. In the present work, we determined the toxicokinetic parameters of the toxic fraction of Androctonus australis garzonii venom in the absence and after antivenom immunotherapy, in experimentally envenomed rabbits. After subcutaneous injection of the scorpion venom, toxins showed a fast and complete resorption from the site of injection associated with a simultaneous distribution in a large extracellular compartment and with an important body clearance. The precocious intravenous injection of an appropriate antivenom dose was shown to induce an immediate, complete and durable neutralization of toxins, as well as their rapid redistribution from the peripheric compartment to the vascular one. On the contrary, the intramuscular injection of the same antivenom dose produced a slower and partial redistribution of toxins, leading to a delayed neutralization of the venom. The intravenous injection of smaller antivenom doses induced transient decreases of circulating toxins, indicating that a minimal antivenom dose has to be administered to allow an efficient and durable neutralization of the venom. We concluded also that this minimal effective dose of antivenom has to be injected precociously, by intravenous route, to achieve an efficient immunotherapy.

Buprenorphine and midazolam act in combination to depress respiration in rats.

High dose buprenorphine is used as substitution treatment in human heroin addiction. Deaths have been reported in addicts using buprenorphine, frequently in association with benzodiazepines. In the current study, we observed the effects of buprenorphine and midazolam alone and in combination on arterial blood gases. Four groups of 10 male Sprague-Dawley rats received a parenteral injection of aqueous solvent, buprenorphine (30 mg/kg, iv), midazolam (160 mg/kg, ip), or buprenorphine (30 mg/kg, iv) plus midazolam (160 mg/kg, ip). Serial blood gases were obtained over 3 hours. There was a mild but significant effect of buprenorphine alone in comparison with the aqueous solvent on PaCO2 at 60 min (6.24 vs. 5.65 kPa, p< 0.01). There was also a mild but significant effect of midazolam alone in comparison with aqueous solvent on arterial pH at 90 min (7.33 vs. 7.41,p< 0.001) and PaCO2 at 60 min (6.52 vs. 5.65 kPa,p< 0.01). The combination of midazolam and buprenorphine produces a rapid, profound, and prolonged respiratory depression, as demonstrated by an increase in PaCO2 at 7.65 +/- 0.12 kPa at 20 min and a decrease in arterial pH at 7.25 +/- 0.02 at 20 min, with appearance of delayed hypoxia with a decrease in PaO2 at 8.74 +/- 0.20 kPa at 120 min. These data show that high doses of midazolam and buprenorphine alone have limited effects on arterial blood gases in rats while midazolam and buprenorphine appear to act in an additive or synergistic fashion to depress ventilation in rats.

Coexistence of passive and proton antiporter-mediated processes in nicotine transport at the mouse blood-brain barrier.

Nicotine, the main tobacco alkaloid leading to smoking dependence, rapidly crosses the blood-brain barrier (BBB) to become concentrated in the brain. Recently, it has been shown that nicotine interacts with some organic cation transporters (OCT), but their influence at the BBB has not yet been assessed in vivo. In this study, we characterized the transport of nicotine at the mouse luminal BBB by in situ brain perfusion. Its influx was saturable and followed the Michaelis-Menten kinetics (K(m)=2.60 mM, V(max)=37.60 nmol/s/g at pH 7.40). At its usual micromolar concentrations in the plasma, most (79%) of the net transport of nicotine at the BBB was carrier-mediated, while passive diffusion accounted for 21%. Studies on knockout mice showed that the OCT Oct1-3, P-gp, and Bcrp did not alter [(3)H]-nicotine transport at the BBB. Neither did inhibiting the transporters Mate1, Octn, or Pmat. The in vivo manipulation of intracellular and/or extracellular pH, the chemical inhibition profile, and the trans-stimulation experiments demonstrated that the nicotine transporter at the BBB shared the properties of the clonidine/proton antiporter. The molecular features of this proton-coupled antiporter have not yet been identified, but it also transports diphenhydramine and tramadol and helps nicotine cross the BBB at a faster rate and to a greater extent. The pharmacological inhibition of this nicotine/proton antiporter could represent a new strategy to reduce nicotine uptake by the brain and thus help curb addiction to smoking.

Blood concentrations are better predictors of chioroquine poisoning severity than plasma concentrations: a prospective study with modeling of the concentration/effect relationships.

INTRODUCTION: Chloroquine causes rare but life-threatening toxicity. The prognostic value of plasma chloroquine concentrations in acute poisonings remains poorly investigated. We investigated the hypothesis that blood chloroquine concentrations better predicted chloroquine poisoning severity than plasma concentrations. METHODS: A prospective study of consecutive chloroquine poisonings admitted to an intensive care unit from 2003 to 2007 was performed with simultaneous measurements of blood and plasma chloroquine (chloroquine and desethylchloroquine) concentrations. A population pharmacokinetic-pharmacodynamic model described epinephrine infusion rate, our surrogate marker of cardiovascular toxicity, as function of blood or plasma chloroquine concentrations. RESULTS: Forty-four patients [29F/15M, 33 years (25-41), median (25-75th percentile), 34% with cardiac arrest] were included. Management included mechanical ventilation (80%), 8.4% sodium bicarbonate (66%), epinephrine [73%, maximal rate: 2.8 mg/h (0.8-5.0)], and extracorporeal life support (16%). Seven patients died. Blood [6.7 mg/L (4.0-13.0)] and plasma [1.5 mg/L (1.2-2.9)] chloroquine concentrations were weakly, although significantly correlated (r = 0.66, p < 0.0001, Spearman test). Admission chloroquine concentrations correlated with the reported ingested dose (r = 0.70 for blood vs. 0.48 for plasma), QRS duration (r = 0.82 vs. 0.64), lactate concentrations (r = 0.63 vs. 0.47), and epinephrine infusion rates (r = 0.70 vs. 0.62). Chloroquine concentrations differed significantly between patients with and without cardiac arrest (p = 0.0002 for blood vs. 0.02 for plasma). A one-compartment pharmacokinetic (PK) model adequately described blood chloroquine concentrations. An effect compartment linked to the blood compartment adequately described plasma chloroquine concentrations. Using a sigmoidal E(max) pharmacodynamic (PD) model, epinephrine infusion rate was better predicted with blood than plasma concentrations (p < 0.01), suggesting that time-course of blood concentrations is a better prognostic value than plasma concentrations. CONCLUSION: Immediate and serial measurements of blood chloroquine concentrations are better than plasma for predicting cardiovascular severity of chloroquine poisonings.

Population pharmacokinetics of 3,4-methylenedioxymethamphetamine and main metabolites in rats.

The pharmacokinetics of the recreational drug 3,4-methylenedioxymethamphetamine (MDMA) and its mains metabolites have never been modeled together. We therefore designed a model with which to analyze the pharmacokinetics of MDMA, 3,4-methylenedioxyamphetamine (MDA), 4-hydroxy-3-methoxymethamphetamine (HMMA), and 4-hydroxy-3-methoxyamphetamine (HMA) and to test the effect of covariates like gender and body weight on the pharmacokinetics. Rats (18 males and 18 females) were given 1 mg/kg MDMA iv, and the concentrations of MDMA, MDA, and HMMA were measured by high-performance liquid chromatography-mass spectrometry. Another 30 rats (15 males) were given 1 mg/kg MDA, and MDA and HMA were measured. A population pharmacokinetic model was developed to describe the changes in MDMA, HMMA, MDA, and HMA concentrations over time and to estimate interanimal variability. The influence of gender was tested using a likelihood ratio test. Estimated exposures of males and females to MDMA and its metabolites were compared using the Wilcoxon nonparametric test. An integrated six-compartment model adequately described the data. MDMA (two compartments) was transformed irreversible to HMMA (one compartment) and MDA (two compartments), which then produced HMA (one compartment). All rate constants were first order. Females given MDMA had significantly smaller MDMA distribution volumes than males, and they converted less MDMA to MDA than did males. Our MDMA, MDA, HMA, and HMMA model is suitable for examining the relationship between drug concentrations and its pharmacological/toxicological effects. Male rats were exposed to significantly more MDA and HMA than were females, which could explain why males are more sensitive to MDMA toxic effects than females.

Acute renal failure enhances the antidotal activity of pralidoxime towards paraoxon-induced respiratory toxicity.

We recently showed in a rat model of dichromate-induced acute renal failure (ARF) that the elimination but not the distribution of pralidoxime was altered resulting in sustained plasma pralidoxime concentrations. The aim of this study was to compare the efficiency of pralidoxime in normal and acute renal failure rats against paraoxon-induced respiratory toxicity. Ventilation at rest was assessed using whole-body plethysmography after subcutaneous administration of either saline or paraoxon (50% of the LD(50)), in the control and ARF rats. Thirty minutes after administration of paraoxon, either saline or 50mg/kg of pralidoxime was administered intramuscularly. ARF had no significant effects on the ventilation at rest. The effects of paraoxon on respiration were not significantly different in the control and ARF group. Paraoxon increased the total time (T(TOT)), expiratory time (T(E)) and tidal volume (V(T)), and decreased the respiratory frequency (f). In paraoxon-poisoned rats with normal renal function, pralidoxime had a significant but transient effect regarding the T(TOT) and V(T) (p<0.05). In the ARF group, the same dose of pralidoxime significantly decreased the T(TOT), T(E), and V(T) and increased f during 90 min (p<0.01). In conclusion, pralidoxime had partial and transient effects towards paraoxon-induced respiratory toxicity in control rats; and a complete and sustained correction in ARF rats.

Clonidine transport at the mouse blood-brain barrier by a new H+ antiporter that interacts with addictive drugs.

Identifying drug transporters and their in vivo significance will help to explain why some central nervous system (CNS) drugs cross the blood-brain barrier (BBB) and reach the brain parenchyma. We characterized the transport of the drug clonidine at the luminal BBB by in situ mouse brain perfusion. Clonidine influx was saturable, followed by Michaelis-Menten kinetics (K(m)=0.62 mmol/L, V(max)=1.76 nmol/sec per g at pH 7.40), and was insensitive to both sodium and trans-membrane potential. In vivo manipulation of intracellular and/or extracellular pH and trans-stimulation showed that clonidine was transported by an H+-coupled antiporter regulated by both proton and clonidine gradients, and that diphenhydramine was also a substrate. Organic cation transporters (Oct1-3), P-gp, and Bcrp did not alter clonidine transport at the BBB in knockout mice. Secondary or tertiary amine CNS compounds such as oxycodone, morphine, diacetylmorphine, methylenedioxyamphetamine (MDMA), cocaine, and nicotine inhibited clonidine transport. However, cationic compounds that interact with choline, Mate, Octn, and Pmat transporters did not. This suggests that clonidine is transported at the luminal mouse BBB by a new H+-coupled reversible antiporter.

Sprague-Dawley rats display metabolism-mediated sex differences in the acute toxicity of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy).

The use of the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) has been associated with unexplained deaths. Male humans and rodents are more sensitive to acute toxicity than are females, including a potentially lethal hyperthermia. MDMA is highly metabolized to five main metabolites, by the enzymes CYP1A2 and CYP2D. The major metabolite in rats, 3,4-methylenedioxyamphetamine (MDA), also causes hyperthermia. We postulated that the reported sex difference in rats is due to a sexual dimorphism(s). We therefore determined (1) the LD50 of MDMA and MDA, (2) their hyperthermic effects, (3) the activities of liver CYP1A2 and CYP2D, (4) the liver microsomal metabolism of MDMA and MDA, (5) and the plasma concentrations of MDMA and its metabolites 3 h after giving male and female Sprague-Dawley (SD) rats MDMA (5 mg.kg(-1) sc). The LD50 of MDMA was 2.4-times lower in males than in females. MDMA induced greater hyperthermia (0.9 degrees C) in males. The plasma MDA concentration was 1.3-fold higher in males, as were CYP1A2 activity (twice) and N-demethylation to MDA (3.3-fold), but the plasma MDMA concentration (1.4-fold) and CYP2D activity (1.3-fold) were higher in females. These results suggest that male SD rats are more sensitive to MDMA acute toxicity than are females, probably because their CYP1A2 is more active, leading to higher N-demethylation and plasma MDA concentration. This metabolic pathway could be responsible for the lethality of MDMA, as the LD50 of MDA is the same in both sexes. These data strongly suggest that the toxicity of amphetamine-related drugs largely depends on metabolic differences.

ABC transporters and the accumulation of imatinib and its active metabolite CGP74588 in rat C6 glioma cells.

Imatinib (Glivec, Gleevec, STI571) is a small tyrosine kinase inhibitor that is currently in phase II clinical trials in patients with recurrent glioblastoma. Its therapeutic benefit is minimal, although it is greater in some patients when combined with hydroxyurea. Imatinib is transported by human and rodent ATP-binding cassette (ABC) transporters like P-glycoprotein (Pgp) and the breast cancer resistance protein (BCRP). We have investigated whether ABC transporters determine the pharmacokinetics of imatinib and its pharmacological active metabolite CGP74588 in rat C6 glioma cells. ABC transporter expressions were measured by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). C6 cells express high concentrations of the Pgp-encoding gene Mdr1b and a 10-fold smaller amount of the Pgp-encoding gene Mdr1a. The relative expression of ABC transporter genes are: Mdr1b>Mrp4>Mrp1>Mrp5>Mdr1a>Mrp3>Mrp2>Bcrp. The accumulation of imatinib into C6 cells increased linearly with the extracellular concentration of imatinib (0.5-50microM) and was not increased by zosuquidar (selective Pgp inhibitor) or elacridar (inhibitor of both Pgp and Bcrp). In contrast, there was less CGP74588 than imatinib in C6 cells and its concentration increased with the extracellular concentration in a sigmoid fashion. Lastly, 10microM valspodar (selective Pgp inhibitor), elacridar and zosuquidar all increased the accumulation of CGP74588 by 2.5-fold. Thus CGP74588 is readily transported by the Pgp in rat C6 gliomas cells, which raises the question of the role of Pgp in the resistance of recurrent glioblastomas to imatinib.

Buprenorphine alters desmethylflunitrazepam disposition and flunitrazepam toxicity in rats.

High-dosage buprenorphine (BUP) consumed concomitantly with benzodiazepines (BZDs) including flunitrazepam (FZ) may cause life-threatening respiratory depression despite a BUP ceiling effect and BZDs' limited effects on ventilation. However, the mechanism of BUP/FZ interaction remains unknown. We hypothesized that BUP may alter the disposition of FZ active metabolites in vivo, contributing to respiratory toxicity. Plasma FZ, desmethylflunitrazepam (DMFZ), and 7-aminoflunitrazepam (7-AFZ) concentrations were measured using gas chromatography-mass spectrometry. Intravenous BUP 30 mg/kg pretreatment did not alter plasma FZ and 7-AFZ kinetics in Sprague-Dawley rats infused with 40 mg/kg FZ over 30 min, whereas resulting in a three-fold increase in the area under the curve (AUC) of DMFZ concentrations compared with control (p < 0.01). In contrast, BUP did not significantly modify plasma DMFZ concentrations after intravenous infusion of 7 mg/kg DMFZ, whereas resulting in a similar peak concentration to that generated from 40 mg/kg FZ administration. Regarding the effects on ventilation, BUP (30 mg/kg) as well as its combination with FZ (0.3 mg/kg) significantly increased PaCO(2), whereas only BUP/FZ combination decreased PaO(2) (p < 0.001). Interestingly, FZ (40 mg/kg) but not DMFZ (40 mg/kg) significantly increased PaCO(2) (p < 0.05), whereas DMFZ but not FZ decreased PaO(2) (p < 0.05). Thus, decrease in PaO(2) appears related to BUP-mediated effects on DMFZ disposition, although increases in PaCO(2) relate to direct BUP/FZ additive or synergistic dynamic interactions. We conclude that combined high-dosage BUP and FZ is responsible for increased respiratory toxicity in which BUP-mediated alteration in DMFZ disposition may play a significant role.

Is P-glycoprotein (ABCB1) a phase 0 or a phase 3 colchicine transporter depending on colchicine exposure conditions?

This study investigates the P-glycoprotein (Pgp)-mediated transport of its substrates in accumulation or efflux modes under steady-state conditions. The kinetics of colchicine uptake and efflux, a substrate of both Pgp and intracellular tubulin, were studied in HL60 and HL60/DNR cells; HL60/DNR cells contain 25 times more Pgp than do HL60 cells. HL60/DNR cells in a medium containing 6.25 nM colchicine, which mimics therapeutic conditions, reached steady-state twice as rapidly as did HL60 cells, and accumulated 24-times less colchicine than did HL60 cells. The Pgp inhibitor GF120918, increased colchicine uptake by HL60 cells 1.2-fold and that of HL60/DNR cells 17-fold, while it had no effect on colchicine efflux from either cell line that had been incubated with colchicine for 24 h. Colchicine kinetics fitted well a two closed-compartment model, showing that the low intracellular accumulation of colchicine in HL60/DNR cells resulted from a 11-fold decrease in colchicine uptake and a 2.3-fold increase in colchicine efflux, that could be attributed to Pgp-mediated efflux activity in HL60/DNR cells. Intracellular colchicine was mainly and similarly distributed in the cytosol in both cell lines. These data demonstrate that the kinetics of the intracellular colchicine accumulation depend on the density of Pgp and that Pgp is more a phase 0 (preventing cellular uptake) than a phase 3 (effluxing intracellular substrate) transporter under steady-state conditions, although the situation is reversed after a short incubation time (30 min), when intracellular free colchicine concentration is probably high enough for it to be removed from the cell by Pgp.