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N 6 -clyclohexyladenosine (CHA) is an adenosine A 1 receptor agonist that inhibits thermogenesis. Cardiovascular side effects however, limit use of CHA as a therapeutic. We and others have shown that this can be reversed by administering 8-p-(sulfophenyl)theophylline (8-SPT), a nonspecific antagonist that does not cross the BBB. Other evidence shows that CNS actions of CHA may contribute to bradycardia through enhanced vagal tone and other mechanisms. Here we test the hypothesis that 8-SPT pretreatment alone is sufficient to prevent hypotension caused by CHA. To test this hypothesis, we pretreated rats with 8-SPT alone, and in combination with other antagonists to test the hypothesis that direct action of CHA on the heart is the primary mechanism by which CHA induces bradycardia and hypotension. Results show that pretreatment with 8-SPT alone is not sufficient to prevent CHA-induced hypotension. Pretreatment with 8-SPT or atropine alone did not prevent the fall in mean arterial pressure (MAP) and heart rate (HR), however, pretreatment with 8-SPT (25 mg/kg) and atropine (1 mg/kg) 15 min before CHA (1 mg/kg) preserves MAP and HR baseline values after CHA administration. We next asked if blood pressure was managed during the transition into a hypometabolic state, would prolong CHA-mediated inhibition of metabolism after cardiac arrest improve outcome better than anti-shivering medications meperidine and buspirone. We found that CHA-mediated hypotension can be mitigated by pretreatment with atropine and 8-SPT. This combination administered after cardiac arrest facilitated temperature management and metabolic suppression better than meperidine and buspirone, however, did not improve survival., Competing Interests: Declaration of competing interest Kelly Drew has a financial interest in Be Cool Pharmaceutics. Kelly Drew and Bernard Laughlin hold intellectual property for technology related to formulation. For other authors the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024. Published by Elsevier Inc.)

Background: Meperidine is a synthetic opioid that belongs to the phenylpiperidine class and is a weak mu receptor agonist. In horses there are a limited number of published studies describing the analgesic effects of systemically administered meperidine in horses. The objective of this study was to describe the pharmacokinetics, behavioral and physiologic effects and effect on thermal threshold of three doses of intravenously administered meperidine to horses. Eight University owned horses (four mares and four geldings, aged 3-8 years were studied using a randomized balanced 4-way cross-over design. Horses received a single intravenous dose of saline, 0.25, 0.5 and 1.0 mg/kg meperidine. Blood was collected before administration and at various time points until 96 hours post administration. Plasma and urine samples were analyzed for meperidine and normeperidine by liquid chromatography-mass spectrometry and plasma pharmacokinetics determined. Behavioral and physiologic data (continuous heart rate, step counts, packed cell volume, total plasma protein and gastrointestinal sounds) were collected at baseline through 6 hours post administration. The effect of meperidine administration on thermal nociception was determined and thermal excursion calculated., Results: Meperidine was rapidly converted to the metabolite normeperidine. The volume of distribution at steady state and systemic clearance (mean ± SD) ranged from 0.829 ± 0.138-1.58 ± 0.280 L/kg and 18.0 ± 1.4-22.8 ± 3.60 mL/min/kg, respectively for 0.5-1.0 mg/kg doses. Adverse effects included increased dose-dependent central nervous excitation, heart rate and cutaneous reactions. Significant effects on thermal nociception were short lived (up to 45 minutes at 0.5 mg/kg and 15 minutes at 1.0 mg/kg)., Conclusions: Results of the current study do not support routine clinical use of IV meperidine at a dose of 1 mg/kg to horses. Administration of 0.5 mg/kg may provide short-term analgesia, however, the associated inconsistent and/or short-term adverse effects suggest that its use as a sole agent at this dose, at best, must be cautiously considered.

Pethidine is an opiate agonist used orally and parenterally. Pethidine-containing drugs abuse is frequently encountered on health workers and patients. The analysis methods used to determine the abuse of pethidine are important for forensic toxicology. Pethidine is metabolized to norpethidine by the liver. Therefore, the determination of pethidine and norpethidine in urine is one of the methods to determine the abuse of pethidine. In this study, we have developed a precise, simple and rapid ultra-performance liquid chromatography-tandem mass spectrometer method for the determination of pethidine and norpethidine simultaneously. The developed method was validated in terms of selectivity and linearity which was in the range of 9-1800 ng/mL for both pethidine and norpethidine. The intra-assay and inter-assay accuracy and precision were found within acceptable limits of the EMA guideline. Lower limits of quantitation were 9 ng/mL for both pethidine and norpethidine. The developed method was successfully applied for the determination of both analytes in the real samples., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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 ( k cat / K m ) 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.

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.

Bacterial pore-forming toxin aerolysin-like proteins (ALPs) are widely distributed in animals and plants. However, functional studies on these ALPs remain in their infancy. βγ-CAT is the first example of a secreted pore-forming protein that functions to modulate the endolysosome pathway via endocytosis and pore formation on endolysosomes. However, the specific cell surface molecules mediating the action of βγ-CAT remain elusive. Here, the actions of βγ-CAT were largely attenuated by either addition or elimination of acidic glycosphingolipids (AGSLs). Further study revealed that the ALP and trefoil factor (TFF) subunits of βγ-CAT bind to gangliosides and sulfatides, respectively. Additionally, disruption of lipid rafts largely impaired the actions of βγ-CAT. Finally, the ability of βγ-CAT to clear pathogens was attenuated in AGSL-eliminated frogs. These findings revealed a previously unknown double binding pattern of an animal-secreted ALP in complex with TFF that initiates ALP-induced endolysosomal pathway regulation, ultimately leading to effective antimicrobial responses., Competing Interests: The authors declare no competing interests.

Oral fluid assays for quantifying drugs are useful in forensic toxicology and drug monitoring. Compared with blood and urine specimens, oral fluid collection is simple, non-invasive, and more difficult to adulterate. Therefore, we investigated whether meperidine and its metabolites could be detected in oral fluid and whether there was a predictable relationship between oral fluid and plasma concentrations. Male New Zealand white rabbits (n = 10) were administered meperidine hydrochloride (20 mg/kg, intravenous). Then, plasma and oral fluid were collected at various time points up to 10 h after administration. We developed a simple and sensitive gas chromatography-mass spectrometry method for the determination of meperidine and normeperidine in oral fluid and plasma. We estimated the apparent pharmacokinetic parameters for meperidine in oral fluid and plasma and determined the ratio and correlation between oral fluid and plasma concentrations. The results demonstrate that this method has excellent specificity, linearity, precision, and recovery. Meperidine and normeperidine were detected in both body fluids; meperidine was the most abundant analyte in oral fluid. The oral fluid-to-plasma drug concentration ratios did not differ significantly over time (p > 0.05). In addition, oral fluid and plasma levels of meperidine and normeperidine were significantly correlated over time (r = 0.713 and 0.725, respectively; p < 0.05). These results provide context for interpreting meperidine and metabolite concentrations in oral fluid and support the utility of oral fluid as an alternative matrix in clinical and forensic testing.

Rationale: Several opioid analgesics have been related to the prolongation of cardiac repolarization, a condition which can be fatal. In order to establish a correct estimation of the risk/benefit balance of therapeutic doses of meperidine, normeperidine, tramadol, propoxyphene and norpropoxyphene, it was necessary to develop an analytical method to determinate plasma concentrations of these opioids., Methods: Here we describe a method which incorporates strong alkaline treatment to obtain norpropoxyphene amide followed by a one-elution step solid-phase extraction, and without further derivatization. Separation and quantification were achieved by gas chromatography/electron ionization mass spectrometry (GC/EI-MS) in selected-ion monitoring mode. Quantification was performed with 500 μL of plasma by the addition of deuterated analogues as internal standards., Results: The proposed method has been validated in the linearity range of 25-1000 ng/mL for all the analytes, with correlation coefficients higher than 0.990. The lower limit of quantification was 25 ng/mL. The intra- and inter-day precision, calculated in terms of relative standard deviation, were 2.0-12.0% and 6.0-15.0%, respectively. The accuracy, in terms of relative error, was within a ± 10% interval. The absolute recovery and extraction efficiency ranged from 81.0 to 111.0% and 81.0 to 105.0%, respectively., Conclusions: A GC/MS method for the rapid and simultaneous determination of meperidine, normeperidine, tramadol, propoxyphene and norpropoxyphene in human plasma was developed, optimized and validated. This procedure was shown to be sensitive and specific using small specimen amounts, suitable for application in routine analysis for forensic purposes and therapeutic monitoring. To our knowledge, this is the first full validation of the simultaneous determination of these opioids and their metabolites in plasma samples., (Copyright © 2017 John Wiley & Sons, Ltd.)

Taxoids are anti-cancer drugs frequently used to treat solid tumors, but they are sometimes ineffective and tumors may become resistant to their action. Here, we examined the involvement of sphingolipid metabolic enzymes in paclitaxel (PTX) resistance using a human prostate cancer cell line, PC3, and its PTX-resistant subline, PC3-PR. PTX (20 nM) suppressed cell proliferation and increased various ceramide species in PC3, but not PC3-PR, cells. PC3-PR contained higher S1P levels than did PC3, regardless of PTX treatment. Western blotting revealed that PC3-PR cells expressed higher levels of sphingosine kinase 1 (SPHK1) and glucosylceramide synthase (GCS) but lower levels of acid sphingomyelinase (ASMase) and neutral sphingomyelinase 2 than did PC3 cells. Inhibition of SPHK1 using siRNA or a pharmacological inhibitor decreased S1P levels in PC3-PR cells and inhibited proliferation in the presence or absence of PTX, suggesting that SPHK1 is at least partially responsible for PTX resistance. Similarly, GCS inhibitors (PDMP and PPMP) increased cellular ceramides and suppressed the proliferation of PC3-PR. However, inhibition of proteasome function or histone deacetylase activity increased SMase and ceramide levels and suppressed PC3-PR proliferation. These results suggest that modulation of metabolic enzyme expression and alteration of the sphingolipid rheostat protects cancer cells against PTX., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A prolongation of the QTc-interval has been described for several opioids, including pethidine (meperidine)., Objective: To evaluate in the clinical setting the frequency and risk factors associated with the QT-interval prolongation induced by meperidine., Research Design and Methods: We recruited patients requiring meperidine administration and recorded their medical history and comorbidities predisposing to QT-interval prolongation. Ionograms and electrocardiograms (ECGs) were performed at baseline and during treatment; QT was corrected using the Bazzet, Fridericia, Framinghan, and Hogdes formulas. We measured meperidine and normeperidine by gas chromatography. Values are expressed as mean ± SD (range)., Results: 58 patients were studied (43.1% males). All patients received meperidine at a dose of 304 ± 133 (120 - 480) mg/day. Meperidine and normeperidine concentrations were 369 ± 60 (265 - 519) and 49 ± 17 (15 - 78) ng/mL, respectively. Intratreatment control found QTcB 370 ± 30 (305 - 433), QTcFri 353 ± 35 (281 - 429), QTcFra 360 ± 30 (299 - 429), QTcH 359 ± 27 (304 - 427), ΔQTcB +9 ± 42 (-90 to +136), ΔQTcFri +4 ± 45 (-86 to +137), ΔQTcFra +5 ± 40 (-77 to +129), and ΔQTcH +7 ± 40 (-76 to +129) ms. Meperidine concentration correlated with QTc-interval (R > 0.36) and ΔQTc (R > 0.69) but the correlation was even better for normeperidine concentration, QTc (R > 0.52) and ΔQTc (R > 0.81). Depending on the QTc correction formula used, 13 - 15 patients (22.41 - 25.86%) presented ΔQTc values > 30 ms, and 7 - 8 patients (12.07- 13.79%) showed ΔQTc values > 60 ms. Renal failure was associated with risk for ΔQTc > 30 ms of 3.74 (IC95% 1.73 - 8.10) and for ΔQTc > 60 ms of 4.27 (IC 95% 1.26 - 14.48). No patient developed arrhythmias during the study., Conclusions: Meperidine treatment causes ECG changes (QTc-interval prolongation) in high correlation with normeperidine plasma concentration. Renal failure increases the risk.
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Continuing our previous studies analyzing drugs of abuse in municipal wastewater, a method was developed for the analysis of miscellaneous drugs of abuse in wastewater samples using liquid chromatography coupled with tandem mass spectrometry (LC-MS-MS). Eight drugs and metabolites were analyzed including 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrolidine (EDDP), fentanyl, norfentanyl, meperidine, normeperidine, methadone, phencyclidine and tramadol. These drugs were chosen because of their widespread abuse. Wastewater samples were collected at both the Oxford Waste Water Treatment Plant in Oxford, Mississippi (MS) and the University Wastewater Treatment Plant in University, MS. These wastewater samples were collected on weekends in which the University of Mississippi football team (colloquially the "Ole Miss Rebels" football team) held home games (Vaught-Hemingway Stadium, University, MS 38677). The collected samples were analyzed using a validated method and found to contain tramadol in 25 samples at quantifiable levels. EDDP, meperidine, normeperidine and methadone were also detected but were under the limit of quantitation., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Esophageal cancer is one of the least studied and deadliest cancers worldwide with a poor prognosis due to limited options for treatment. Chemotherapy agents such as the microtubule-targeting compounds are the mainstay of palliation for advanced esophageal cancer treatment. However, the toxicity and side effects of tubulin-binding agents (TBAs) have promoted the development of novel, more potent but less toxic TBAs. Herein, we identified 2-[4-(3,4-dimethoxyphenyl)-3-methyl-1H-pyrazol-5-yl]-5-[(2-methylprop-2-en-1-yl)oxy] phenol (PPMP) as a novel TBA for esophageal cancer treatment. PPMP markedly inhibited tubulin polymerization, and decreased viability and anchorage-independent growth of esophageal cancer cell lines, effects that were accompanied by G2/M arrest and apoptosis. Importantly, we produced patient-derived esophageal cancer xenografts to evaluate the therapeutic effect of PPMP in a setting that best mimics the clinical context in patients with esophageal cancer. Overall, we identified PPMP as a novel microtubule-destabilizing compound and as a new therapeutic agent against esophageal carcinoma., Competing Interests: The authors declare no conflicts of interest.

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)., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Acquired resistance to cisplatin treatment is a caveat when treating patients with non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM). Ceramide increases in response to chemotherapy, leading to proliferation arrest and apoptosis. However, a tumour stress activation of glucosylceramide synthase (GCS) follows to eliminate ceramide by formation of glycosphingolipids (GSLs) such as globotriaosylceramide (Gb3), the functional receptor of verotoxin-1. Ceramide elimination enhances cell proliferation and apoptosis blockade, thus stimulating tumor progression. GSLs transactivate multidrug resistance 1/P-glycoprotein (MDR1) and multidrug resistance-associated protein 1 (MRP1) expression which further prevents ceramide accumulation and stimulates drug efflux. We investigated the expression of Gb3, MDR1 and MRP1 in NSCLC and MPM cells with acquired cisplatin resistance, and if GCS activity or MDR1 pump inhibitors would reduce their expression and reverse cisplatin-resistance., Methods: Cell surface expression of Gb3, MDR1 and MRP1 and intracellular expression of MDR1 and MRP1 was analyzed by flow cytometry and confocal microscopy on P31 MPM and H1299 NSCLC cells and subline cells with acquired cisplatin resistance. The effect of GCS inhibitor PPMP and MDR1 pump inhibitor cyclosporin A for 72h on expression and cisplatin cytotoxicity was tested., Results: The cisplatin-resistant cells expressed increased cell surface Gb3. Cell surface Gb3 expression of resistant cells was annihilated by PPMP whereas cyclosporin A decreased Gb3 and MDR1 expression in H1299 cells. No decrease of MDR1 by PPMP was noted in using flow cytometry, whereas a decrease of MDR1 in H1299 and H1299res was indicated with confocal microscopy. No certain co-localization of Gb3 and MDR1 was noted. PPMP, but not cyclosporin A, potentiated cisplatin cytotoxicity in all cells., Conclusions: Cell surface Gb3 expression is a likely tumour biomarker for acquired cisplatin resistance of NSCLC and MPM cells. Tumour cell resistance to MDR1 inhibitors of cell surface MDR1 and Gb3 could explain the aggressiveness of NSCLC and MPM. Therapy with GCS activity inhibitors or toxin targeting of the Gb3 receptor may substantially reduce acquired cisplatin drug resistance of NSCLC and MPM cells., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Calcinosis cutis is a rare disease entity characterized by deposits of calcium in the skin and subcutaneous tissue causing hard-to-heal ulcers. This is a case report on a patient with femoral ulcers in connection with densely mineralized skin caused by ketobemidon injections. Next to surgical excision of calcified tissue the patient received extracorporeal shockwave therapy (ESWT). On the basis of excellent healing, partial skin transplant was feasible. We advocate for randomized trials on ESWT as an adjunctive therapy for complex non-healing wounds.

Glucosylceramide synthase (GCS) catalyzes the first committed step in the biosynthesis of glucosylceramide (GlcCer)-related glycosphingolipids (GSLs). Although inhibitors of GCS, PPMP and PDMP have been widely used to elucidate their biological function and relevance, our comprehensive literature review revealed that the available data are ambiguous. We therefore investigated whether and to what extent GCS inhibitors affect the expression of lactosylceramide (LacCer), neolacto (nLc4 and P1), ganglio (GM1 and GD3) and globo (Gb3 and SSEA3) series GSLs in a panel of human cancer cell lines using flow cytometry, a commonly applied method investigating cell-surface GSLs after GCS inhibition. Their cell-surface GSL expression considerably varied among cell lines and more importantly, sublethal concentrations (IC10) of both inhibitors preferentially and significantly reduced the expression of Gb3 in the cancer cell lines IGROV1, BG1, HT29 and T47D, whereas SSEA3 was only reduced in BG1. Unexpectedly, the neolacto and ganglio series was not affected. LacCer, the precursor of all GlcCer-related GSL, was significantly reduced only in BG1 cells treated with PPMP. Future research questions addressing particular GSLs require careful consideration; our results indicate that the extent to which there is a decrease in the expression of one or more particular GSLs is dependent on the cell line under investigation, the type of GCS inhibitor and exposure duration., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Aggregate-prone mutant proteins, such as α-synuclein and huntingtin, play a prominent role in the pathogenesis of various neurodegenerative disorders; thus, it has been hypothesized that reducing the aggregate-prone proteins may be a beneficial therapeutic strategy for these neurodegenerative disorders. Here, we identified two previously described glucosylceramide (GlcCer) synthase inhibitors, DL-threo-1-Phenyl-2-palmitoylamino-3-morpholino-1-propanol and Genz-123346(Genz), as enhancers of autophagy flux. We also demonstrate that GlcCer synthase inhibitors exert their effects on autophagy by inhibiting AKT-mammalian target of rapamycin (mTOR) signaling. More importantly, siRNA knock down of GlcCer synthase had the similar effect as pharmacological inhibition, confirming the on-target effect. In addition, we discovered that inhibition of GlcCer synthase increased the number and size of lysosomal/late endosomal structures. Although inhibition of GlcCer synthase decreases levels of mutant α-synuclein in neurons, it does so, according to our data, through autophagy-independent mechanisms. Our findings demonstrate a direct link between glycosphingolipid biosynthesis and autophagy in primary neurons, which may represent a novel pathway with potential therapeutic value for the treatment of Parkinson's disease. Inhibition of GlcCer synthase enhances autophagy by inhibiting AKT-mTOR signaling, and increases the number and size of lysosomal/late endosomal structures. Furthermore, inhibition of GlcCer synthase decreased levels of mutant α-synuclein in neurons, which may represent a potential therapeutic target for Parkinson's disease., (© 2014 International Society for Neurochemistry.)

Context: Normeperidine accumulates in patients with impaired renal function and may cause central neurotoxicity. However, some uremic patients still undergo meperidine treatment for chronic pain., Objectives: To prevent normeperidine side effects and complications, we investigated the clearance rate and extraction ratio of meperidine and normeperidine in hemodialysis patients with chronic pain., Methods: Three hemodialysis patients, with diagnoses of chronic pancreatitis, chronic back pain, and intractable intra-abdominal pain, received long-term (more than six months) administration of meperidine for chronic noncancer pain. During regular hemodialysis, 72 blood samples in total were collected from the afferent port, efferent port, and ultradiafiltrate port at eight time points. The plasma concentrations of meperidine and normeperidine were determined by high-performance liquid chromatography., Results: The prehemodialysis plasma concentrations of meperidine and normeperidine were 2963 ± 315 and 2369 ± 1974 ng/mL, which declined to 591 ± 109 and 853 ± 765 ng/mL, with 80% and 65% reduction, respectively. The plasma clearance and extraction ratios of meperidine were 22.7 ± 9.8 mL/minute and 10.1 ± 5.6% and for normeperidine 26.0 ± 11.4 mL/minute and 10.8 ± 2.5%, respectively., Conclusion: Hemodialysis can efficiently remove meperidine and its active metabolite, normeperidine, in uremic patients receiving long-term meperidine therapy for chronic noncancer pain., (Copyright © 2014 U.S. Cancer Pain Relief Committee. Published by Elsevier Inc. All rights reserved.)

Background: Local anaesthetics (LA) injected intraperitoneally have been found to decrease postoperative pain. This double-blind randomized study was performed comparing continuous infusion or patient-controlled intraperitoneal (i.p.) bolus injection of LA. The primary endpoint was supplemental opioid consumption during the first 24 postoperative hours., Methods: Two multi-hole catheters were placed intraperitoneally at the end of the surgery in 40 patients undergoing elective abdominal hysterectomy. The patients were randomized into two groups: Group P: patients self-injected 10 ml of levobupivacaine 1.25 mg ml(-1) via the i.p. catheter as needed, maximum once per hour, and had continuous saline infusion 10 ml h(-1) into the second catheter. Group C: patients received a continuous infusion of 10 ml h(-1) of levobupivacaine 1.25 mg ml(-1) intraperitoneally through one catheter and 10 ml saline as bolus as needed via the other. Ketobemidone was administered intravenously as rescue medication., Results: Total ketobemidone consumption during 0-24 h was lower in Group P compared with Group C (mean 23.1 vs 35.7 mg, P=0.04). No differences in the median pain scores were found between the groups. Earlier return of gastrointestinal (GI) function was found in Group P vs Group C (mean 1.5 vs 2.2 days, P<0.01), which also resulted in earlier home-readiness (mean 1.9 vs 2.7 days, P=0.04)., Conclusions: A statistically significant opioid-sparing effect was found when patient-controlled levobupivacaine was administered intraperitoneally as needed compared with continuous infusion. This was associated with a faster return of GI function and home-readiness. There was, however, a wide confidence interval in the primary endpoint, opioid consumption.

Meperidine (Demerol(®)) is a mu- and kappa-opiate receptor agonist used for moderate to severe pain. Overdose can result in respiratory depression, hypotension and coma, while accumulation of its toxic metabolite, normeperidine, can cause delirium and seizures. Little data exist examining the inter- and intrasubject variability of the normeperidine-to-meperidine metabolic ratio (MR) in urine. This retrospective data analysis examined meperidine and normeperidine urine concentrations collected from chronic pain patients. In 98 subjects with multiple visits, the geometric mean urinary MR = 6.1 (coefficient of variation, %CV = 68%). From single specimens obtained from 799 subjects, the geometric mean urinary MR = 6.2 (%CV = 212%). The urinary MR increased in young subjects compared with elderly (P = 0.004) and middle-aged subjects (P = 0.01). A 27% difference was found between the male and female urinary MR (male geometric mean MR = 5.1, female geometric mean MR = 7.0, P = 0.02). Intersubject variability in meperidine metabolism was 3-fold greater than intrasubject variability. A significant difference in the urinary MR was found between males and females. The substantial variability in meperidine metabolism and the serious side effects of its metabolite normeperidine require greater vigilance in patient medication monitoring.

Lactosylceramide (LacCer) is a member of the glycosphingolipid family and is known to be a bioactive lipid in various cell physiological processes. However, the direct targets of LacCer and cellular events mediated by LacCer are largely unknown. In this study, we examined the effect of LacCer on the release of arachidonic acid (AA) and the activity of cytosolic phospholipase A2α (cPLA2α). In CHO-W11A cells, treatment with 1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), an inhibitor of glucosylceramide synthase, reduced the glycosphingolipid level, and the release of AA induced by A23187 or platelet-activating factor was inhibited. The addition of LacCer reversed the PPMP effect on the stimulus-induced AA release. Exogenous LacCer stimulated the release of AA, which was decreased by treatment with an inhibitor of cPLA2α or silencing of the enzyme. Treatment of CHO-W11A cells with LacCer induced the translocation of full-length cPLA2α and its C2 domain from the cytosol to the Golgi apparatus. LacCer also induced the translocation of the D43N mutant of cPLA2α. Treatment of L929 cells with TNF-α induced LacCer generation and mediated the translocation of cPLA2α and AA release, which was attenuated by treatment with PPMP. In vitro studies were then conducted to test whether LacCer interacts directly with cPLA2α. Phosphatidylcholine vesicles containing LacCer increased cPLA2α activity. LacCer bound to cPLA2α and its C2 domain in a Ca(2+)-independent manner. Thus, we propose that LacCer is a direct activator of cPLA2α.

This study aims to develop an analytical method for the simultaneous determination of pethidine and norpethidine in mouse plasma using the liquid chromatography-electrospray ionization source-tandem mass spectrometry (LC-ESI-MS-MS) method. After being alkalified, the plasma specimens were extracted by liquid-liquid extraction with ethyl acetate. Chromatographic separation was performed on a C18 column (150 × 2.1 mm internal diameter, 5 μm) using a mobile phase consisting of acetonitrile/water (5 mM ammonium acetate in water, pH 6.2, 40:60, v/v) during a total run time of 4.0 min. The mass spectrometer was operated under the positive ionization mode. The multiple reaction monitoring with the transitions of m/z 248.1 → 174.2, 234.2 → 160.0 and 389.2 → 201.1 was used to quantify pethidine, norpethidine and cetirizine (internal standard), respectively. Linear calibration curves were obtained over concentration ranges of 8.24-8,440.00 ng/mL for pethidine, and 6.15-6,300.00 ng/mL for norpethidine. The intra- and interday precisions and accuracies were <15% (relative standard deviations, RSDs), and the accuracies were within the interval of 94-103% for both. The lower limits of quantification in mouse plasma were 8.24 ng/mL for pethidine and 6.15 ng/mL for norpethidine. Subsequently, the validated method was successfully applied to a pharmacokinetics study of pethidine and its metabolite, norpethidine, in mice.

Most patients with advanced cancer experience severe pain and are often treated with opiates. Cancer patients are especially susceptible to opportunistic infections due to treatment with immunosuppressive and cytostatic drugs. Since opiates have been demonstrated to have immunomodulatory effects, it is of clinical importance to evaluate potential differences between commonly used opiates with regard to their effect on the immune system. The aim of this study was to evaluate the effect of morphine, tramadol, fentanyl and ketobemidone on the functioning of the immune system with special reference to TNF and IL-8 release. Method. U-937 cells were preincubated with different concentrations of opioids followed by stimulation with LPS 100 μg/ml for three hours. The effect of opioids on the levels of cytokine mRNA was studied using RT-PCR. Erk and Akt phosphorylation was also measured by Western blot. Results. All opioids with the exception of fentanyl were capable of inhibiting TNF release from U-937 cells. Morphine had no effect on IL-8 release but the effect of other opiates was almost the same as the effect on TNF. All opioids with the exception of fentanyl were capable of inhibiting production of mRNA for TNF and IL-8. The observed effects of opiates were not always reversible by naloxone, suggesting that the effects might be mediated by other receptors or through a non-receptor mediated direct effect. Although preliminary evidence suggests the involvement of Erk and Akt pathways, further studies are needed to unravel the intracellular pathways involved in mediating the effects of opiates. Our data suggests that the order of potency with regard to inhibition of cytokine release is as follows: tramadol > ketobemidone > morphine > fentanyl. Conclusion. Further studies are needed to understand the clinical implications of the observed immunosuppressive effects of tramadol and ketobemidone and to improve opioid treatment strategies in patients with cancer.

Background/aims: Resistance to genotoxic therapy is a characteristic feature of glioma cells. Acid sphingomyelinase (ASM) hydrolyzes sphingomyelin to ceramide and glucosylceramide synthase (GCS) catalyzes ceramide metabolism. Increased ceramide levels have been suggested to enhance chemotherapy-induced death of cancer cells., Methods: Microarray and clinical data for ASM and GCS in astrocytomas WHO grade II-IV were acquired from the Rembrandt database. Moreover, the glioblastoma database of the Cancer Genome Atlas network (TCGA) was used for survival data of glioblastoma patients. For in vitro studies, increases in ceramide levels were achieved either by ASM overexpression or by the GCS inhibitor DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) in human glioma cell lines. Combinations of alkylating chemotherapy or irradiation and ASM overexpression, PPMP or exogenous ceramide were applied in parental cells. The anti-glioma effects were investigated by assessing proliferation, metabolic activity, viability and clonogenicity. Finally, viability and clonogenicity were assessed in temozolomide (TMZ)-resistant cells upon treatment with PPMP, exogenous ceramide, alkylating chemotherapy, irradiation or their combinations., Results: Interrogations from the Rembrandt and TCGA database showed a better survival of glioblastoma patients with low expression of ASM or GCS. ASM overexpression or PPMP treatment alone led to ceramide accumulation but did not enhance the anti-glioma activity of alkylating chemotherapy or irradiation. PPMP or exogenous ceramide induced acute cytotoxicity in glioblastoma cells. Combined treatments with chemotherapy or irradiation led to additive, but not synergistic effects. Finally, no synergy was found when TMZ-resistant cells were treated with exogenous ceramide or PPMP alone or in combination with TMZ or irradiation., Conclusion: Modulation of intrinsic glioma cell ceramide levels by ASM overexpression or GCS inhibition does not enhance the anti-glioma activity of alkylating chemotherapy or irradiation.

Pethidine (meperidine), a synthetic opiate, formally used as an analgesic in surgery and obstetrics, has been an abused drug of choice for some doctors. A case is presented in which a doctor, who previously admitted to using pethidine, was suspected of re-using, following a second positive urine test. A laboratory had reported the presence of pethidine in the doctor's urine; however, the doctor denied re-use. The norpethidine (normeperidine) metabolite, normally found in urine, had not been detected, raising concern over the laboratory's conclusion and necessitating an independent investigation. Because the major metabolite of pethidine is pethidinic acid (meperidinic acid), accounting for approximately 40% of the excreted dose, its presence or absence were deemed to be important criteria in interpreting the laboratory result. Pethidinic acid was synthesized by alkaline hydrolysis of pethidine and used as a control. Urine samples from a patient receiving pethidine for pain, from the previous pethidine use of the doctor, and the urine under question plus the control were analyzed for the presence of pethidinic acid using electrospray mass spectrometry. Pethidinic acid was found in all samples except the one under dispute. The absence of pethidinic acid appeared to corroborate the doctor's denial of re-use.

Background: Ketobemidone is often used as an alternative to morphine in children in the Scandinavian countries. In an earlier study, we have examined the pharmacokinetic properties in children in different age groups but have not focused on neonates. The aim of this clinical trial was to explore the pharmacokinetics of ketobemidone in neonates., Methods: Fifteen full-term neonates (eight females) from 37 gestational weeks at birth and scheduled for elective surgery were included in the trial. Their median age was 3 days (range 1-18 days). Ketobemidone hydrochloride was administered as a single intravenous bolus dose, and ketobemidone concentrations were measured by liquid chromatography-mass spectrometry over 10 h. Pharmacokinetic parameters were calculated with standard compartmental methods., Results: The median (range) values for ketobemidone clearance, apparent volume of distribution, volume of central compartment, distribution half-life and elimination half-life were 0.46 (0.23-0.84) l/h/kg, 4.64 (3.50-7.31) l/kg, 1.71 (0.16-3.47) l/kg, 2.85 (1.04-10.78) min and 7.26 (3.5-11.3) h., Conclusion: Compared with our previous study in children older than 1 year of age, the elimination of ketobemidone appeared to be slower in full-term neonates. Despite a low pharmacokinetic variability of ketobemidone as observed in the present neonatal patient population, we recommend individualizing the dose of ketobemidone based on observations of analgesic efficacy., (© 2012 The Authors. Acta Anaesthesiologica Scandinavica © 2012 The Acta Anaesthesiologica Scandinavica Foundation.)

Background: There is no information about the distribution of pethidine into breast milk and/or exposure of the breastfed infant during pethidine patient-controlled epidural analgesia after caesarean delivery., Methods: We conducted an observational study among 20 women. The mean (95% confidence interval) pethidine dose administered was 670 (346-818) mg over 41 (35-46) h. Maternal plasma and milk and neonatal plasma were collected near the time of pethidine cessation and 6h later. Absolute and relative infant doses via milk and infant exposure were calculated. Infant behaviour was assessed using the Neurologic and Adaptive Capacity Score., Results: At first and second sampling times, mean absolute infant doses for pethidine were 20 (14-27) μg/kg/day and 10 (7-13) μg/kg/day, while mean relative infant doses were 0.7 (0.1-1.4)% and 0.3 (0.1-0.5)% respectively. Similar values for norpethidine (expressed as pethidine equivalents) were 21 (16-26) μg/kg/day and 22 (12-32) μg/kg/day; and 0.7 (0.3-1)% and 0.6 (0.2-1)% respectively. Mean pethidine and norpethidine concentrations in neonatal plasma were 3 (0-6.1) μg/L and 0.6 (0.2-1) μg/L. Compared with a time-matched maternal sample, the infant's exposure was 1.4 (0.2-2.8)% for pethidine and 0.4 (0.2-0.6)% for norpethidine. The mean (95% confidence interval) neurologic and adaptive capacity score was 33.6 (32.2-34.9)., Conclusion: The combined absolute infant dose of pethidine and norpethidine received via milk was 1.8% of the neonatal therapeutic dose and the combined relative infant dose was below the 10% recommended safety level. Breastfed infants are at low risk of drug exposure when mothers self-administer epidural pethidine after caesarean delivery., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

A series of benzyl esters of meperidine and normeperidine were synthesized and evaluated for binding affinity at serotonin, dopamine and norepinephrine transporters. The 4-methoxybenzyl ester 8b and 4-nitrobenzyl ester 8c in the meperidine series and 4-methoxybenzyl ester 14a in the normeperidine series exhibited low nanomolar binding affinities at the SERT (K(i) values <2nM) and high SERT selectivity (DAT/SERT >1500 and NET/SERT >1500)., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Synthesis of glucosylceramide via glucosylceramide synthase (GCS) is a crucial event in higher eukaryotes, both for the production of complex glycosphingolipids and for regulating cellular levels of ceramide, a potent antiproliferative second messenger. In this study, we explored the dependence of the early branching eukaryote Giardia lamblia on GCS activity. Biochemical analyses revealed that the parasite has a GCS located in endoplasmic reticulum (ER) membranes that is active in proliferating and encysting trophozoites. Pharmacological inhibition of GCS induced aberrant cell division, characterized by arrest of cytokinesis, incomplete cleavage furrow formation, and consequent block of replication. Importantly, we showed that increased ceramide levels were responsible for the cytokinesis arrest. In addition, GCS inhibition resulted in prominent ultrastructural abnormalities, including accumulation of cytosolic vesicles, enlarged lysosomes, and clathrin disorganization. Moreover, anterograde trafficking of the encystations-specific protein CWP1 was severely compromised and resulted in inhibition of stage differentiation. Our results reveal novel aspects of lipid metabolism in G. lamblia and specifically highlight the vital role of GCS in regulating cell cycle progression, membrane trafficking events, and stage differentiation in this parasite. In addition, we identified ceramide as a potent bioactive molecule, underscoring the universal conservation of ceramide signaling in eukaryotes.

Previous studies suggest that membrane lipids may regulate proteolytic processing of the amyloid precursor protein (APP) to generate amyloid-beta peptide (Abeta). In the present study, we have assessed the capacity for a series of structurally related synthetic ceramide analogues to modulate APP processing in vitro. The compounds tested are established glucosylceramide synthase (GS) inhibitors based on the d-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) structure. PDMP and related compounds PPMP and EtDO-P4 inhibited Abeta secretion from Chinese hamster ovary cells expressing human APP (CHO-APP) with approximate IC(50) values of 15, 5, and 1 microM, respectively. A trend for reduced secretion of the APP alpha-secretase product, sAPPalpha, was also observed in PDMP-treated cells but not in PPMP- or ETDO-P4-treated cells, whereas levels of the cellular beta-secretase product APP C-terminal fragment, CTFbeta, were increased by both PDMP and PPMP but unaltered with EtDO-P4 treatment. Our data also revealed that EtDO-P4 inhibits endogenous Abeta production by human neurons. In conclusion, this study provides novel information regarding the regulation of APP processing by synthetic ceramide analogues and reveals that the most potent of these compounds is EtDO-P4., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: Ketobemidone is often used as an alternative to morphine in children in the Scandinavian countries. The aim of this clinical trial was to explore the pharmacokinetics of ketobemidone in children because these properties have not been reported previously., Methods: Thirty children, newborn to 10 years, scheduled for elective surgery were included in the trial. Ketobemidone hydrochloride was administered as a single intravenous bolus dose and ketobemidone and norketobemidone concentrations were measured by LC-MS over 8 h. Pharmacokinetic parameters were determined using compartmental methods., Results: Six children were excluded from pharmacokinetic analysis because of incomplete blood sampling. The values of ketobemidone clearance (l/h/kg) given as median (range) were 0.84 (0.29-3.0) in Group A (0-90 days), 0.89 (0.55-1.35) in Group B (1-2.5 years) and 0.74 (0.50-0.99) in Group C (7-10 years). The corresponding values for apparent volume of distribution (l/kg) were 4.4 (3.7-6.9) (Group A), 2.6 (2.0-5.6) (Group B) and 3.9 (2.7-5.0 (Group C), and for elimination half-life (h) 3.0 (1.4-8.9) (Group A), 2.0 (1.2-4.7) (Group B) and 3.7 (2.4-6.9) (Group C), respectively. In the two neonates the elimination half-life was almost 9 h. The metabolite norketobemidone did not reach levels above the limit of quantification (0.07 ng/ml) in any of the patients., Conclusion: The pharmacokinetic parameters of ketobemidone in children older than 1 month appear to be similar to those in adults. Because of the large interindividual variability of the pharmacokinetics in neonates, further studies especially in this age group are warranted.

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 GM1/caveolin-1 lipid raft mediated endocytosis mechanism was explored for generation 5 and 7 poly(amidoamine) dendrimer polyplexes employing the Cos-7, 293A, C6, HeLa, KB, and HepG2 cell lines. Expression levels of GM1 and caveolin-1 were measured using dot blot and Western blot, respectively. The level of GM1 in the cell plasma membrane was adjusted by incubation with exogenous GM1 or ganglioside inhibitor PPMP, and the level of CAV-1 was adjusted by upregulation with the adenovirus vector expressed caveolin-1 (AdCav-1). Cholera toxin B subunit was employed as a positive control for uptake in all cases. No evidence was found for a GM1/caveolin-1 lipid raft mediated endocytosis mechanism for the generation 5 and 7 poly(amidoamine) dendrimer polyplexes.

One of the major shortcomings of many commonly used opioids is the fact that they are P-gp substrates, which represents a major obstacle towards effective pain management. P-gp can affect opioids' oral absorption, CNS accumulation, systemic clearance, antinociceptive activity, and tolerance development to their analgesic effects. Moreover, P-gp can be the locus of drug-drug interactions between opioids and other concomitantly administered drugs that are P-gp substrates/inhibitors. The objective of this study was to identify opioids that are non-P-gp substrates to overcome some of the mentioned shortcomings. We evaluated the P-gp affinity status (substrate, non-substrate, or inhibitor) of a series of morphine analogs (10 opioid agonist and 2 opioid antagonists) and compared them to previously reported meperidine analogs. The fold stimulation of the morphine analogs ranged from 1.01 to 1.54 while for the meperidine analogs the fold stimulation ranged from 1.10 to 3.66. From each series (morphine and meperidine analogs) we selected potential candidate opioids that are non-P-gp substrates and conducted in vivo assessments of their antinociceptive effects using P-gp knockout and P-gp competent mice. 6-Desoxymorphine, meperidine and N-phenylbutyl normeperidine did not significantly (p>0.05) stimulate the basal P-gp ATPase activity, where, the fold stimulations of the basal P-gp ATPase activity were 1.01+/-0.11, 1.51+/-0.29 and 1.10+/-0.23, respectively. Evaluation of the influence of P-gp ablation on their antinociceptive effects indicated that P-gp did not significantly (p>0.05) affect their antinociceptive effects. Among the evaluated opioids in vivo, 6-desoxymorphine showed high potency and induced no apparent toxicity upon low- and high-dose administration. 6-Desoxymorphine is therefore an ideal lead compound to create a library of opioids that have negligible P-gp affinity for better management of pain.

Background and Objective: Local anaesthetics administered intraabdominally have been found to reduce analgesic requirements postoperatively after hysterectomy. This study was designed to assess the optimal dose of local anaesthetics for best pain relief., Methods: Sixty patients undergoing abdominal hysterectomy were randomly divided into three groups to receive 10 ml h infusion of levobupivacaine intraabdominally postoperatively for 48 h in a double-blind manner: group L, 7.5 mg h; group M, 12.5 mg h and group H, 17.5 mg h. Pain intensity was measured using the numeric rating scale, ketobemidone consumption over 48 h was measured with a patient-controlled analgesia pump, recovery parameters, expiratory muscle strength, time to home readiness, plasma concentration of levobupivacaine and health-related quality of life were all measured at defined time points postoperatively., Results: No differences were found between the active groups in pain intensity, recovery parameters or health-related quality of life. Pain intensity was maximal during 0-4 h and during coughing. Expiratory muscle strength decreased significantly during 0-4 h in all active groups, with no differences between the groups. Plasma concentration of levobupivacaine was below known toxic concentrations in humans, and no patient had symptoms of local anaesthetic toxicity. Health-related quality of life showed improved scores at 3 months after the operation compared with preoperative values, but no differences between the groups were found in any of the parameters., Conclusion: Satisfactory analgesia can be achieved with low doses of levobupivacaine administered intraabdominally, except during the early postoperative period. No advantages were seen in this study when higher doses of levobupivacaine were administered as a continuous infusion for postoperative pain relief.

The stimulation of programmed cell death can either enhance or inhibit antigen presentation by classic major histocompatibility complex molecules. In the current study, we report that the induction of apoptosis by topoisomerase I inhibition or elevation of intracellular ceramide levels substantially impairs CD1d-mediated antigen presentation. In the former case, such a reduction occurred via the regulation of both the p38 mitogen-activated protein kinases and protein kinase C delta signal transduction pathways as well as the caspase cascade, whereas the latter was p38-(but not caspase)-dependent. Confocal microscopic analysis showed an altered intracellular distribution of CD1d following the inhibition topoisomerase I or by an increase in intracellular ceramide levels, that was prevented by p38 and caspase inhibitors. Thus, the induction of apoptosis in antigen presenting cells severely compromises CD1d-mediated antigen presentation by multiple mechanisms.

Numerous studies have shown that many clinically employed opioid analgesics are substrates for P-glycoprotein (P-gp), suggesting that up-regulation of P-gp may contribute to the development of central tolerance to opioids. The studies herein focus on the development of SAR for P-gp substrate activity in the meperidine series of opioids. Addition of a 3-OH to meperidine and the ketone analog of meperidine yielding bemidone and ketobemidone, respectively, significantly increased P-gp substrate affinity. The results of this study have implications in the development of novel analgesics to be utilized as tools to study the contribution of P-gp on the development of central tolerance to opioids.

Methodologies for identification of ketobemidone metabolites in microdialysate samples utilizing coupled-column capillary liquid chromatography-electrospray quadrupole time-of-flight tandem mass spectrometry are presented. Two different methods were developed to efficiently analyze the metabolites norketobemidone, ketobemidone N-oxide and hydroxyketobemidone, respectively. Both methods include on-line desalting and trapping of the analytes on micro-solid-phase extraction columns with different retention mechanisms. Norketobemidone and ketobemidone N-oxide were trapped on a C18 column and then eluted by back-flush followed by isocratic separation on a fluorinated reversed-phase type silica gel column (Fluofix). Retention equations are proposed for the chromatographic observations made on the Fluofix column. Hydroxyketobemidone was trapped on a phenylboronic acid column by complex formation at basic pH and then eluted at acidic pH directly into to the mass spectrometer. Oxidation of hydroxyketobemidone to its corresponding quinone was also observed. The methods were successfully used to analyze synthetic ketobemidone metabolites in dilute low-volume microdialysis samples.

Direct analysis, with minimal sample pretreatment, of antidepressant drugs, fluoxetine, imipramine, desipramine, amitriptyline, and nortriptyline in biofluids was developed with a total run time of 8 min. The setup consists of two HPLC pumps, injection valve, capillary RAM-ADS-C18 pre-column and a capillary analytical C18 column connected by means of a six-port valve in backflush mode. Detection was performed with ESI-MS/MS and only 1 microm of sample was injected. Validation was adequately carried out using FLU-d(5) as internal standard. Calibration curves were constructed under a linear range of 1-250 ng mL(-1) in plasma, being the limit of quantification (LOQ), determined as 1 ng mL(-1), for all the analytes. With the described approach it was possible to reach a quantified mass sensitivity of 0.3 pg for each analyte (equivalent to 1.1-1.3 fmol), translating to a lower sample consumption (in the order of 10(3) less sample than using conventional methods).

A variety of observations suggest that decreasing glycolysis and increasing levels of reduced glutathione, generated by metabolism of glucose through the pentose phosphate pathway, would have an anticonvulsant effect. Because fructose-1,6-bisphosphate (F1,6BP) shifts the metabolism of glucose from glycolysis to the pentose phosphate pathway, it was hypothesized to have anticonvulsant activity. The anticonvulsant activity of F1,6BP was determined in rat models of acute seizures induced by pilocarpine, kainic acid, or pentylenetetrazole. The efficacy of F1,6BP was compared with that of 2-deoxyglucose (2-DG; an inhibitor of glucose uptake and glycolysis), valproic acid (VPA), and the ketogenic diet. One hour before each convulsant, Sprague Dawley rats received either saline (as seizure controls), F1,6BP (0.25, 0.5 or 1 g/kg), 2-DG (0.25 or 0.5 g/kg), or VPA (0.3 g/kg). Additional animals received the ketogenic diet (starting at 20 or 60 d old). Time to seizure onset, seizure duration, and seizure score were measured in each group. F1,6BP had dose-dependent anticonvulsant activity in all three models, whereas VPA had partial efficacy. 2-DG was only effective in the pilocarpine model. The ketogenic diet had no effect in these models. F1,6BP was also partially effective when given at the first behavioral seizure after pilocarpine. Administration of sodium lactate, which bypasses the block in the glycolytic pathway, abolished the anticonvulsant activity of 2-DG in the pilocarpine model, but only decreased the efficacy of F1,6BP. These data demonstrate that F1,6BP has significant anticonvulsant efficacy.

P-Glycoprotein (P-gp) is an efflux transporter which is up-regulated at the blood-brain barrier in both morphine- and oxycodone-tolerant rats. Numerous studies have shown that many clinically employed opioid analgesics are substrates for P-gp, suggesting that up-regulation of P-gp may contribute to the development of central tolerance to opioids. The studies herein focus on the development of SAR for P-gp substrate activity in the meperidine series of compounds, and show that a meperidine analog of greater potency, N-phenylbutyl-N-normeperidine, has low activity as a P-gp substrate and has the potential to be utilized as a tool to study the contribution of P-gp to the development of central tolerance to opioids.

Objective: To compare clinical outcome 6 months after treatment with bilateral laparoscopic occlusion of the uterine artery versus uterine leiomyoma embolization., Methods: Sixty-six premenopausal women with symptomatic uterine leiomyomata were randomized to treatment with either laparoscopic occlusion of uterine arteries or uterine leiomyoma embolization. The primary outcome was reduction of blood loss from pretreatment to 6 months postoperatively, measured by a Pictorial Bleeding Assessment Chart. Secondary outcomes included patients' own assessment of symptom reduction, postoperative pain assessed using visual analog scales, ketobemidone used postoperatively, complications, secondary interventions, and failures., Results: Fifty-eight women were included; 6-month follow-up data were available for 28 participants in each group. The percentage reduction in Pictorial Bleeding Assessment Chart scores did not differ between the treatment groups (52% after uterine leiomyoma embolization and 53% after laparoscopy, P=.96). The study had 52% power to detect a 20% difference on the Pictorial Bleeding Assessment Chart. Fewer participants in the group treated with uterine leiomyoma embolization complained of heavy bleeding after 6 months (4% compared with 21%, P=.044). The postoperative use of ketobemidone was higher after uterine leiomyoma embolization (46 mg compared with 12 mg, P<.001)., Conclusion: Both laparoscopic occlusion of uterine vessels and embolizaton of uterine leiomyoma improved clinical symptoms in the majority of patients. Participants with the laparoscopic procedure had less postoperative pain but heavier menstrual bleeding 6 months after treatment. A larger study and longer follow-up is necessary before a definite conclusion can be made regarding the most effective treatment., Clinical Trial Registration: (www.ClinicalTrials.gov), NCT00277680, Level of Evidence: I.

Background: The aim of the present study was to investigate whether a combination of rofecoxib and gabapentin could improve pain relief and reduce opioid requirements, compared with rofecoxib alone, during the first 5 days after tonsillectomy., Methods: In a randomized, double-blind, placebo-controlled study, 49 patients received gabapentin 1200 mg pre-operatively, followed by gabapentin 2 x 600 mg on the day of operation and gabapentin 3 x 600 mg for the next 5 days, or placebo. Both groups were given rofecoxib 50 mg daily. In the post-operative care unit, intravenous morphine was administered in doses of 2.5 mg on request. From 4 h to 5 days post-operatively, ketobemidone was offered as escape drug. Pain at rest and during swallowing, and side-effects, were assessed using a four-point verbal rating scale., Results: As a result of the global withdrawal of rofecoxib, the study had to be terminated prematurely. This report comprises the results from 22 patients in the gabapentin group and 27 patients in the placebo group. Gabapentin reduced ketobemidone requirements during the first 24 h post-operatively [4.5 mg (standard deviation, 3.0 mg) in the placebo group vs. 2.0 mg (standard deviation, 2.0 mg) in the gabapentin group; P < 0.003]. Gabapentin induced more dizziness (P < 0.002), gait disturbance (P < 0.02) and vomiting (P < 0.05) during days 0-5 than placebo. No other statistically significant differences were observed., Conclusion: Gabapentin reduced opioid requirements in the first 24 h after tonsillectomy. The benefits of the reduced opioid intake may be overshadowed by the drawbacks of side-effects.

The structure-activity relationships of 3',4'-dichloro-meperidine were investigated at dopamine (DAT) and serotonin transporters (SERT). Large ester substituents and lipophilic groups at the 4-position favored molecular recognition at the SERT. The benzyl ester of 3',4'-dichloro-meperidine exhibited high potency and high selectivity for the SERT (DAT/SERT=760). Chemical modification of the ester group and N-substitution generally led to compounds with decreased DAT affinity. Only small esters and alkyl groups were tolerated at the 4-position of the meperidine ring system by the DAT. Overall, the meperidine analogues were generally more selective for the SERT than for the DAT.

Ketogenic diet (KD) is used to manage intractable epilepsy; however, the mechanisms underlying its therapeutic effects are not known. Steroid hormones, such as progesterone and testosterone, are derived from cholesterol, and are readily 5alpha-reduced to dihydroprogesterone and dihydrotestosterone, which are subsequently converted to 5alpha-pregnan-3alpha-ol-20-one (3alpha,5alpha-THP) and 3alpha-androstanediol, neuroactive steroids that can influence seizures. The present study examined the effects of the KD on circulating concentrations of these neuroactive steroids, and their precursors, in intact female rats. Thirty-six, 22-day-old female Sprague-Dawley rats (weaned at 21 days) were fasted for 8 hours prior to placement on one of three dietary regimens for 6 weeks: ad libitum chow, calorie-restricted chow, or KD. After 6 weeks of the diet, when six rats in each dietary condition were in diestrus and six were in behavioral estrus, all rats were administered pentylenetetrazole (PTZ, 70 mg/kg, i.p.). The latency and incidence of seizures were recorded by an observer who was uninformed of the estrous cycle and dietary treatment conditions of the rats. Immediately after each test, trunk blood was obtained for later measurement of pregnane (progesterone, dihydroprogesterone, 3alpha,5alpha-THP) and androstane (testosterone, dihydrotestosterone, 3alpha-androstanediol) neuroactive steroid concentrations in plasma by radioimmunoassay. KD tended to lengthen the latency to, and significantly reduced the number of, PTZ-induced barrel roll seizures. KD also significantly reduced plasma levels of the pregnane (dihydroprogesterone, 3alpha,5alpha-THP) and androstane (dihydrotestosterone, 3alpha-androstanediol) 5alpha-reduced metabolites. These data suggest that levels of pregnane and androstane neuroactive steroids, or their precursors, may underlie some of the antiseizure effects of KD.

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.

Approximately one third of children with epilepsy have persistent seizures despite trials of multiple antiepileptic medications. For some of these patients, epilepsy surgery may provide freedom from seizures. However, in many cases, epilepsy surgery is not a viable treatment option. Nonpharmacological approaches are a useful adjunct to help manage seizures in these children. This review examines the role of vagus nerve stimulation, the ketogenic diet, and various forms of EEG biofeedback therapy in children with intractable epilepsy. Although the mechanism of action is not known precisely for any of these adjunctive therapies, they add an important and evolving dimension to the management of difficult to control epilepsy in children. In addition, pyridoxine-dependent seizures are discussed as an example of an etiology of refractory seizures that responds well to replacement therapy.

The hexosamine pathway (HP) is a biochemical hypothesis recently proposed explaining cellular alterations occurring during diabetic microvascular complications. Diabetic retinopathy is a common microvascular complication of diabetes, and it is known that cell proliferation is severely affected during the development of the disease. Particularly, early stages are characterized by death of the retinal microvascular cells, pericytes. Gangliosides have often been described to regulate cell growth; however, very few studies focused on the potential role of gangliosides in diabetic microvascular alterations. The aim of this article was to investigate the effect of the HP activation on pericyte proliferation and determine the potential implication of gangliosides in this process. Results indicate first that HP activation, mimicked by glucosamine treatment, decreased pericyte proliferation. Second, glucosamine treatment induced a modification of gangliosides pattern, particularly GM1 and GD3 were significantly increased. Next, results showed that exogenous addition of a-series gangliosides (GM3, GM2, GM1, GD1a) and b-series ganglioside (GD3) caused a decrease of pericyte proliferation, whereas nonsialylated precursors glucosylceramide and lactosylceramide were without effect. Furthermore, when ganglioside biosynthesis was blocked using PPMP, a glucosylceramide synthase inhibitor, the effects of glucosamine on pericyte proliferation were partially reversed. Our results suggest that in retinal pericytes, gangliosides and particularly GM1 and GD3 that are increased in response to glucosamine, are involved in the antiproliferative effect of glucosamine. These observations also underlie the potential involvement of gangliosides in a pathological context, such as diabetic microvascular complications.