Bio-generation of stable isotope-labeled internal standards for absolute and relative quantitation of phase II drug metabolites in plasma samples using LC-MS/MS.

Quantification of drug metabolites in biological samples has been of great interest in current pharmaceutical research, since metabolite concentrations and pharmacokinetics can contribute to a better understanding of the toxicity of drug candidates. Two major categories of Phase II metabolites, glucuronide conjugates and glutathione conjugates, may cause significant drug toxicity and therefore require close monitoring at early stages of drug development. In order to achieve high precision, accuracy, and robustness, stable isotope-labeled (SIL) internal standards (IS) are widely used in quantitative bioanalytical methods using liquid chromatography and tandem mass spectrometry (LC-MS/MS), due to their capability of compensating for matrix effects, extraction variations and instrument response fluctuations. However, chemical synthesis of SIL analogues of Phase II metabolites can often be very difficult and require extensive exploratory research, leading to higher cost and significant delays in drug research and development. To overcome these challenges, we have developed a generic method which can synthesize SIL analogues of Phase II metabolites from more available SIL parent drugs or SIL conjugation co-factors, using in vitro biotransformation. This methodology was successfully applied to the bio-generation of SIL glucuronide conjugates and glutathione conjugates. The method demonstrated satisfactory performance in both absolute quantitation and assessment of relative exposure coverage across species in safety tests of drug metabolites (MIST). This generic technique can be utilized as an alternative to chemical synthesis and potentially save time and cost for drug research and development.

Combination of electrochemistry and nuclear magnetic resonance spectroscopy for metabolism studies.

During the development of new materials demonstrating biological activity, prediction and identification of reactive intermediates generated in the course of drug metabolism in the human liver is of great importance. We present a rapid and purely instrumental method for the structure elucidation of possible phase I metabolites. With electrochemical (EC) conversion adopting the oxidative function of liver-inherent enzymes and nuclear magnetic resonance (NMR) spectroscopy enabling structure elucidation, comprehensive knowledge on potential metabolites can be gained. Paracetamol (APAP) has been known to induce hepatotoxicity when exceeding therapeutic doses and was therefore selected as the test compound. The reactive metabolite N-acetyl-p-benzoquinone imine has long been proven to be responsible for the toxic side effects of APAP and can easily be generated by EC. EC coupled online to NMR is a straightforward technique for structure elucidation of reactive drug intermediates at an early stage in drug discovery.

Assessment of cryopreserved human hepatocytes as a model system to investigate sulfation and glucuronidation and to evaluate inhibitors of drug conjugation.

Cultured cryopreserved human hepatocytes are extensively used as a model system for studying drug metabolism, although they remain poorly characterized in respect of the major conjugation reactions glucuronidation and sulfation. Using paracetamol (acetaminophen), we assessed eleven samples of cryopreserved human hepatocytes for their suitability to investigate the simultaneous glucuronidation and sulfation of xenobiotics and evaluated inhibitors of conjugation. Kinetic characterization showed broadly similar values for paracetamol conjugation by hepatocytes (as reported in the literature for in vitro systems), with Km values of approximately 6 mM and 0.3 mM for glucuronidation and sulfation, respectively. Substantial interindividual differences were observed. The hepatocytes demonstrated a strong dose-dependent switch from a preponderance of sulfation at low concentrations of paracetamol to glucuronidation at higher doses, consistent with routes of clearance in vivo. A number of drugs, some of which such as probenecid and sulfinpyrazone are known to interact with paracetamol in vivo, were demonstrated to inhibit the sulfation and/or glucuronidation of paracetamol in hepatocytes, demonstrating the potential application of this model system for studying drug-drug interactions involving conjugation.

[Clinical value of estimated half-life in paracetamol poisoning as a complement to Rumack's nomogram]. / Interés clínico de la semivida de eliminación del paracetamol como complemento al nomograma de Rumack en la valoración de la intoxicación por paracetamol.

BACKGROUND AND OBJECTIVE: Rumack's nomogram is usually used to indicate the treatment with N-acetilcysteine in the paracetamol poisoning, but it has several limitations. Paracetamol half-life elimination (t1/2) is approximately of 2 h with therapeutic doses and it increases to more than 4 h in patients with hepatotoxicity. The aim of this study was to determine the usefulness of estimated paracetamol t1/2 as greater than or inferior to 4 h by using a simple ratio in relation to the development of hepatotoxicity. PATIENTS AND METHOD: 21 patients with paracetamol overdose were admitted to Son Dureta Hospital (Palma de Mallorca) and Clínic Hospital (Barcelona) over 13 months. The estimated t1/2 is calculated using the quotient between 2 plasma paracetamol concentrations separated by 2 or more hours. RESULTS: We found a significant difference (p < 0.005) between the group with hepatotoxicity (n = 3; t1/2 = 8,5 h; range: 3,6 - 8,7 h); and without hepatotoxicity (n = 18; t1/2 = 2,4 h; (range: 1,6 - 4,3 h). We observed an agreement between positive ratio and a t1/2 > 4 h, and negative ratio with t1/2 < 4 h, bearing in mind that the quotient is obtained through mathematical equations. CONCLUSIONS: Rumack's nomogram should be complemented with t1/2 estimation in all cases of paracetamol poisoning, especially with those patients for whom we are not able to determine the time of ingestion at presentation or if there has been a multiple-timepoint ingestion.

Investigating the human metabolism of acetaminophen using UPLC and exact mass oa-TOF MS.

The ability to rapidly detect and characterize drug metabolites in biological fluids often relies on a combination of a high quality chromatographic separation and sensitive high resolution mass spectrometry. Here, the performance of two high throughput LC/MS approaches, namely monolith columns and sub-2 microm particle Ultra Performance Liquid Chromatography (UPLC) columns is compared for the detection and identification of the human metabolites of acetaminophen in urine. The UPLC system produced approximately three times the sensitivity and detected more metabolites than the monolithic column approach. The sharp peaks produced by UPLC seem to be particularly advantageous when coupled to electrospray mass spectrometry, apparently reducing ion suppression leading to superior sensitivity and hence lower limits of detection.

NCX-701. NicOx.

NicOx is developing NCX-701, a nitric oxide-releasing derivative of paracetamol (acetaminophen), for the potential treatment of inflammation and pain. The compound is currently undergoing phase III clinical trials.

Chronic acetaminophen overdosing in children: risk assessment and management.

Acetaminophen is currently the pediatric analgesic and antipyretic of choice. Although children appear to tolerate single, high-dose ingestions well, the literature is replete with reports of significant morbidity and mortality after repeated supra-therapeutic dosing. Proposed risk factors for injury with chronic use include age, total dose, duration, presence of intercurrent febrile illness, starvation, co-administration of cytochrome P450-inducing drugs, underlying hepatic disease, and unique genetic makeup. Evaluation of these children should include serum acetaminophen concentration, prothrombin time, and serum bilirubin and transaminase concentrations. The Rumack-Mathew nomogram should not be used to estimate the risk of hepatotoxicity in cases of chronic ingestion. Based on history, clinical examination, and laboratory findings, patients may be placed in three categories: those without hepatic injury and with no residual acetaminophen to be metabolized, those without injury but with some acetaminophen to be metabolized, and those with hepatotoxicity. Those without injury and no residual acetaminophen need not be treated or followed. Patients with hepatotoxicity or potential for hepatotoxicity based on residual acetaminophen should be treated with N-acetylcysteine. Most importantly, because so many parents are unaware of the potential risk of inappropriate dosing, education is the key to preventing future cases.

Isolation and characterization of a pseudomonas strain that degrades 4-acetamidophenol and 4-aminophenol.

Though many microorganisms that are capable of using phenol as sole source of carbon have been isolated and characterized, only a few organisms degrading substituted phenols have been described to date. In this study, one strain of microorganism that is capable of using phenol (3,000 ppm), 4-aminophenol (4,000 ppm) and 4-acetamidophenol (4,000 ppm) as sole source of carbon and energy was isolated and characterized. This strain was obtained by enrichment culture from a site contaminated with compounds like 4-acetamidophenol, 4-aminophenol and phenol in Pakistan at Bhai Pheru. The contaminated site is able to support large bacterial community as indicated by the viable cell counts (2 x 10(4) - 5 x 10(8)) per gram of soil. Detailed taxonomic studies identified the organisms as Pseudomonas species designated as strain STI. The isolate also showed growth on other organic compounds like aniline, benzene, benzyl alcohol, benzyl bromide, toluene, p-cresol, trichloroethylene and o-xylene. Optimum growth temperature and pH were found to be 30 degrees C and 7, respectively, while growth at 4, 25 and 35 degrees C and at pH 8 and 9 was also observed. Non growing suspended cells of strain ST1 degraded 68, 96 and 76.8% of 4-aminophenol (1,000 ppm), phenol (500 ppm) and 4-acetamidophenol (1,000 ppm), respectively, in 72 hrs. The isolation and characterization of Pseudomonas species strain STI, may contribute to efforts on phenolic bioremediation, particularly in an environment with very high levels of 4-acetamidophenol and 4-aminophenol.

Quantitative assessment of the binding of acetaminophen metabolites to mouse liver microsomal phospholipid.

Phospholipids were quantitatively extracted from microsomes and separated by an h.p.l.c. gradient system with a solvent mixture of n-hexane/n-propanol/water/acetic acid. In a model reaction using horseradish peroxidase/H2O2 in order to activate acetaminophen and inactivated microsomes as target, a covalent binding of 10 nmol drug metabolite per mg microsomal lipid was found. In isolated intact microsomes from methylcholanthrene-pretreated male albino mice, a binding of 0.1 nmol acetaminophen metabolite per mg phospholipid was determined while the binding of metabolites to protein amounted to 3 nmol/mg. The results demonstrate that in mouse liver microsomes metabolizing acetaminophen, about one out of 10(4) phospholipid molecules is modified.

In vitro assessment of pharmacogenetic susceptibility to toxic drug metabolites in humans.

An experimental approach to the pharmacogenetics of human idiosyncratic drug reactions requires an assay for determining individual differences in susceptibility that does not expose patients to further drug-related risk. We have developed an in vitro drug toxicity assay designed to test the hypothesis that differences in susceptibility may be based on genetic abnormalities in the detoxification of electrophilic drug metabolites. Lymphocytes are challenged with metabolites generated by a murine hepatic microsomal system. By using cells from patients deficient in glutathione synthetase, we found that cells with decreased glutathione defenses are more sensitive to toxicity from metabolites of drugs such as acetaminophen, nitrofurantoin, and metronidazole. The assay was then applied to studying the pharmacogenetics of phenytoin hepatotoxicity. We found an inherited defect in the detoxification of phenytoin arene oxide metabolites in cells from patients and their relatives. The studies have led to an elucidation of a genetically heterogeneous group of detoxification defects for arene oxide metabolites of various aromatic drugs. Such experimental approaches may be useful in diagnosing idiosyncratic drug reactions, in establishing their pharmacogenetic basis, and perhaps in predicting toxicity potential of drugs for selected patients and families.

Assessment of biotransformation during transfer of propoxyphene and acetaminophen across the isolated perfused human placenta.

The purpose of this investigation was to assess the extent of biotransformation of drugs by the human placenta during their transfer from the maternal to the fetal circulation. Propoxyphene was used to determine N-demethylation, and acetaminophen served as a substrate for glucuronide and sulfate conjugation. Human full-term placentae were dually perfused in vitro, with one or the other drug being added to the maternal circulation. Propoxyphene and acetaminophen concentrations reached an essentially constant fetal/maternal ratio within 1 hour, with a half-time of about 20 minutes. The concentrations of both drugs in the placental tissues were higher than in the perfusion fluids; this accumulation was particularly pronounced in the case of propoxyphene. No metabolites of either drug were found in the maternal or fetal circulations, but norporpoxyphene, the N-demethylated metabolite of propoxyphene, was detected in placental tissue.