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Introduction and Importance: Organophosphate (OP) poisoning is a common and potentially fatal condition that requires prompt and aggressive treatment with atropine, oximes, and supportive care. We report a rare case of OP poisoning that needed high doses of atropine and intensive care for 60 days. Case Presentation: A 39-year-old male ingested 200 ml of chlorpyrifos, an OP compound, and presented with vomiting and epigastric pain. He received an initial dose of atropine of 60 ml (36 mg, 1 ml=0.6 mg), followed by an infusion of 16 ml/h (9.6 mg/h). He developed hypoxia, cardiac arrest, delirium, fever, and persistent bronchorrhea. He was intubated, resuscitated, and transferred to ICU, where he continued showing signs of OP excess and therefore, he received up to 170 ml/h (102 mg/h) of atropine infusion, along with triple inotropes and sedation. He underwent tracheostomy and gradual weaning of atropine. He recovered completely and was discharged in stable condition. Clinical Discussion: This case demonstrates the need for prolonged monitoring of patients with OP poisoning wherein the patient can develop signs of OP excess even after initial atropinization, the effectiveness of multiple doses of atropine in OP poisoning, and the importance of monitoring for complications associated with a prolonged hospital stay. It also shows the potential need for prolonged atropine therapy and intensive care in OP poisoning. Conclusion: OP poisoning can be life-threatening and requires early and aggressive treatment with atropine, oximes, and supportive care. Clinicians should be aware of the potential need for prolonged atropine therapy in OP poisoning cases to improve the chances of survival.
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Permeability-glycoprotein (P-glycoprotein, P-gp), an efflux transporter at the human blood-brain barrier (BBB), is a significant obstacle to central nervous system (CNS) delivery of P-gp substrate drugs. Using positron emission tomography imaging, we investigated P-gp modulation at the human BBB by an approved P-gp inhibitor, quinidine, or the P-gp inducer, rifampin. Cerebral blood flow (CBF) and BBB P-gp activity were respectively measured by administration of (15)O-water followed by (11)C-verapamil. In a crossover design, healthy volunteers received quinidine and 11-29 days of rifampin treatment during different study periods. CBF and P-gp activity was measured in the absence (control; prior to quinidine treatment) and presence of P-gp modulation. At clinically relevant quinidine plasma concentrations, P-gp inhibition resulted in a 60% increase in (11)C-radioactivity distribution across the human BBB as measured by the brain extraction ratio (ER) of (11)C-radioactivity. Furthermore, the magnitude of BBB P-gp inhibition by quinidine was successfully predicted by a combination of in vitro and macaque data, but not by rat data. Although our findings demonstrated that quinidine did not completely inhibit P-gp at the human BBB, it has the potential to produce clinically significant CNS drug interactions with P-gp substrate drugs that exhibit a narrow therapeutic window and are significantly excluded from the brain by P-gp. Rifampin treatment induced systemic CYP3A metabolism of (11)C-verapamil; however, it reduced the ER by 6%. Therefore, we conclude that rifampin, at its usual clinical dose, cannot be used to induce P-gp at the human BBB to a clinically meaningful extent and is unlikely to cause inadvertent BBB-inductive drug interactions.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/fisiología , Barrera Hematoencefálica/diagnóstico por imagen , Barrera Hematoencefálica/metabolismo , Tomografía de Emisión de Positrones , Quinidina/sangre , Rifampin/sangre , Adulto , Animales , Barrera Hematoencefálica/efectos de los fármacos , Circulación Cerebrovascular/efectos de los fármacos , Circulación Cerebrovascular/fisiología , Estudios Cruzados , Femenino , Humanos , Macaca , Masculino , Tomografía de Emisión de Positrones/métodos , Quinidina/farmacología , Ratas , Rifampin/farmacología , Adulto JovenRESUMEN
Hepatic microsomal cytochrome P450 (CYP) enzymes have broad and overlapping substrate specificity and catalyze a variety of monooxygenase reactions, including aliphatic and aromatic hydroxylations, N-hydroxylations, oxygenations of heteroatoms (N, S, P and I), alkene and arene epoxidations, dehalogenations, dehydrogenations and N-, O- and S-dealkylations. Individual CYP enzymes typically catalyze the oxidative metabolism of a common substrate in a regioselective and stereoselective manner. In addition, different CYP enzymes often utilize different monooxygenase reactions when oxidizing a common substrate. This review examines various oxidative reactions catalyzed by a CYP enzyme acting on a single substrate. In the first example, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a halogenated aromatic environmental contaminant, was oxidatively biotransformed by human CYP2B6. Nine different metabolites of BDE-47 were produced by CYP2B6 via monooxygenase reactions that included aromatic hydroxylation, with and without an NIH-shift, dealkylation and debromination. In the second example, lithocholic acid (3α-hydroxy-5ß-cholan-24-oic acid), an endogenous bile acid, served as a substrate for human CYP3A4 and yielded five different metabolites via aliphatic hydroxylation and dehydrogenation reactions.
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Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP3A/química , Éteres Difenilos Halogenados/química , Ácido Litocólico/química , Animales , Catálisis , Citocromo P-450 CYP2B6/metabolismo , Citocromo P-450 CYP3A/metabolismo , Éteres Difenilos Halogenados/metabolismo , Humanos , Hidroxilación , Ácido Litocólico/metabolismo , Microsomas Hepáticos/enzimología , Especificidad por SustratoRESUMEN
UNLABELLED: Studies in animals and postmortem human brain tissue support a role for P-glycoprotein in clearance of cerebral ß-amyloid across the blood-brain barrier (BBB). We tested the hypothesis that BBB P-glycoprotein activity is diminished in Alzheimer disease (AD) by accounting for an AD-related reduction in regional cerebral blood flow (rCBF). METHODS: We compared P-glycoprotein activity in mild-AD patients (n = 9) and cognitively normal, age-matched controls (n = 9) using PET with a labeled P-glycoprotein substrate, (11)C-verapamil, and (15)O-water to measure rCBF. BBB P-glycoprotein activity was expressed as the (11)C-verapamil radioactivity extraction ratio ((11)C-verapamil brain distributional clearance, K1/rCBF). RESULTS: Compared with controls, BBB P-glycoprotein activity was significantly lower in the parietotemporal, frontal, and posterior cingulate cortices and hippocampus of mild AD subjects. CONCLUSION: BBB P-glycoprotein activity in brain regions affected by AD is reduced and is independent of rCBF. This study improves on prior work by eliminating the confounding effect that reduced rCBF has on assessment of BBB P-glycoprotein activity and suggests that impaired P-glycoprotein activity may contribute to cerebral ß-amyloid accumulation in AD. P-glycoprotein induction or activation to increase cerebral ß-amyloid clearance could constitute a novel preventive or therapeutic strategy for AD.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Barrera Hematoencefálica/metabolismo , Adulto , Anciano , Enfermedad de Alzheimer/diagnóstico por imagen , Enfermedad de Alzheimer/fisiopatología , Barrera Hematoencefálica/diagnóstico por imagen , Radioisótopos de Carbono , Estudios de Casos y Controles , Circulación Cerebrovascular , Femenino , Humanos , Masculino , Persona de Mediana Edad , Radioisótopos de Oxígeno , Tomografía de Emisión de Positrones , Verapamilo , Agua , Adulto JovenRESUMEN
Drug delivery across the brain-blood interfaces is a complex process involving physicochemical drug properties, transporters, enzymes, and barrier dysfunction in diseased conditions. Intact blood-brain barrier (BBB) limits the entry of potentially harmful compounds into the brain but may also reduce the CNS permeability of therapeutic agents. BBB permeability is typically assessed by measuring brain-to-plasma ratio in rodents (referred to as B/P ratio, BB, or Kp, often calculated as logBB), an approach that suffers significant limitations as discussed in the present review. Kp is not a permeability measurement but a partition coefficient mainly driven by the relative binding to plasma and brain tissue components including lipids, phospholipids, and proteins. Compounds with high Kp are often lipophilic with low free fraction available to mediate CNS activities. Efforts should be more concentrated on measuring pharmacologically relevant free drug concentrations at the target site. Using healthy rodents to predict brain penetration in patients might be biased due to species differences in BBB-related parameters such as transporter expression and functional activities. In addition, pathophysiological conditions such as aging, multiple sclerosis, and Alzheimer's and Parkinson's diseases have been described to affect BBB permeability, with barrier leakage and altered transporter activity. The impact of these species differences and disease states on drug delivery to the brain is largely overlooked. More data are needed to better understand their clinical implication in order to design more appropriate screening strategies and ultimately better mitigate the risk for failure in late stage development.
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Barrera Hematoencefálica/metabolismo , Animales , Permeabilidad Capilar , Fármacos del Sistema Nervioso Central/administración & dosificación , Fármacos del Sistema Nervioso Central/sangre , Fármacos del Sistema Nervioso Central/farmacocinética , Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Enfermedades del Sistema Nervioso Central/metabolismo , Sistemas de Liberación de Medicamentos , Humanos , Especificidad de la EspecieRESUMEN
Multidrug-associated protein 2 (MRP2) is an efflux transporter that is expressed at the bile canalicular membrane. To allow in vitro to in vivo extrapolation of the contribution of MRP2 toward hepatic disposition of its substrates, data on the interindividual variability of hepatic MRP2 protein expression are required. Therefore, we quantified the expression of MRP2 in the University of Washington (UW) human liver bank (n = 51) using a modified version of a previously validated liquid chromatography/tandem mass spectrometry assay. An unlabeled (LTIIPQDPILFSGSLR) and stable isotope-labeled (LTIIPQDPILFSGSL[(13)C(6)(15)N(1)]R) surrogate peptide for MRP2 were used as the calibrator and internal standard, respectively. After isolation of the membrane fraction from the liver tissue, in-solution tryptic digestion was conducted. Quality control samples created by spiking human serum albumin or pooled human liver (n = 51) matrix with three different MRP2 synthetic peptide concentrations generated error and precision values of less than 15%. As determined by the surrogate peptide, the average MRP2 expression in the UW liver bank samples was 1.54 ± 0.64 fmol/µg liver membrane protein and was found to be independent of age (7-63 years) or sex. A single nucleotide polymorphism in the promoter region (rs717620), previously thought to affect MRP2 expression, did not influence hepatic expression of MRP2. In contrast, the single nucleotide polymorphism 21214G>A (V417I; rs2273697) was associated with significantly higher hepatic MRP2 expression.
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Expresión Génica , Hígado/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Polimorfismo de Nucleótido Simple , Adolescente , Adulto , Factores de Edad , Membrana Celular/metabolismo , Niño , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Control de Calidad , Estándares de Referencia , Reproducibilidad de los Resultados , Factores Sexuales , Espectrometría de Masas en Tándem , Adulto JovenRESUMEN
3alpha-Hydroxy-5 beta-cholan-24-oic (lithocholic) acid is a relatively minor component of hepatic bile acids in humans but is highly cytotoxic. Hepatic microsomal oxidation offers a potential mechanism for effective detoxification and elimination of bile acids. The aim of the present study was to investigate the biotransformation of lithocholic acid by human hepatic microsomes and to assess the contribution of cytochrome P450 (P450) enzymes in human hepatic microsomes using human recombinant P450 enzymes and chemical inhibitors. Metabolites were identified, and metabolite formation was quantified using a liquid chromatography/mass spectrometry-based assay. Incubation of lithocholic acid with human liver microsomes resulted in the formation of five metabolites, which are listed in order of their rates of formation: 3-oxo-5 beta-cholan-24-oic (3-ketocholanoic) acid, 3 alpha,6 alpha-dihydroxy-5 beta-cholan-24-oic (hyodeoxycholic) acid, 3 alpha,7 beta-dihydroxy-5 beta-cholan-24-oic (ursodeoxycholic) acid, 3 alpha,6 beta-dihydroxy-5 beta-cholan-24-oic (murideoxycholic) acid, and 3 alpha-hydroxy-6-oxo-5 beta-cholan-24-oic (6-ketolithocholic) acid. 3-Ketocholanoic acid was the major metabolite, exhibiting apparent K(m) and V(max) values of 22 muM and 336 pmol/min/mg protein, respectively. Incubation of lithocholic acid with a of human recombinant P450 enzymes revealed that all five metabolites were formed by recombinant CYP3A4. Chemical inhibition studies with human liver microsomes and recombinant P450 enzymes confirmed that CYP3A4 was the predominant enzyme involved in hepatic microsomal biotransformation of lithocholic acid. In summary, the results indicate that oxidation of the third carbon of the cholestane ring is the preferred position of oxidation by P450 enzymes for lithocholic acid biotransformation in humans and suggest that formation of lithocholic acid metabolites leads to enhanced hepatic detoxification and elimination.
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Ácido Litocólico/análogos & derivados , Ácido Litocólico/metabolismo , Microsomas Hepáticos/metabolismo , Biotransformación , Colestanos/metabolismo , Cromatografía Líquida de Alta Presión , Inhibidores Enzimáticos del Citocromo P-450 , Sistema Enzimático del Citocromo P-450/metabolismo , Interpretación Estadística de Datos , Inhibidores Enzimáticos/farmacología , Humanos , Indicadores y Reactivos , Cinética , Espectrometría de Masas , Proteínas Recombinantes/metabolismoRESUMEN
3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic (cholic) and 3alpha,7alpha-dihydroxy-5beta-cholan-24-oic (chenodeoxycholic) acids are the predominant hepatic and biliary bile acids of most mammalian species including humans. Cholic and chenodeoxycholic acids are synthesized from cholesterol and accumulate in the liver during cholestasis. Biotransformation by hepatic cytochrome P450 (P450) enzymes represents a potentially effective pathway for elimination of these lipid-soluble bile acids. We developed a liquid chromatography/mass spectrometry-based assay to identify and quantify the human hepatic microsomal metabolites of cholic acid and chenodeoxycholic acid, and using a panel of human recombinant P450 enzymes, we determined the P450 enzymes involved. Incubation of cholic acid with human hepatic microsomes and NADPH produced a single metabolite, 7alpha,12alpha-dihydroxy-3-oxo-5beta-cholan-24-oic (3-dehydrocholic) acid. Of the recombinant P450 enzymes tested, only CYP3A4 catalyzed 3-dehydrocholic acid formation. Similar experiments with chenodeoxycholic acid revealed the formation of 7alpha-hydroxy-3-oxo-5beta-cholan-24-oic acid and 3alpha,6alpha,7alpha-trihydroxy-5beta-cholan-24-oic (gamma-muricholic) acid as major metabolites and 3alpha-hydroxy-7-oxo-5beta-cholan-24-oic (7-ketolithocholic) acid and cholic acid as minor metabolites. Among the human recombinant P450 enzymes examined, CYP3A4 exhibited the highest rates of formation for 7alpha-hydroxy-3-oxo-5beta-cholan-24-oic acid and gamma-muricholic acid from chenodeoxycholic acid. Formation of 7-ketolithocholic acid and cholic acid from chenodeoxycholic acid has not been reported previously and could not be attributed to any of the recombinant P450 enzymes tested. In conclusion, the predominant pathway for the biotransformation of both cholic and chenodeoxycholic acids in human hepatic microsomes was oxidation at the third carbon of the cholestane ring. This study highlights a major role for CYP3A4 and suggests a possible route for the elimination of these two bile acids.
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Ácido Quenodesoxicólico/metabolismo , Ácido Cólico/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Microsomas Hepáticos/enzimología , Biotransformación , Cromatografía Liquida , Humanos , Cinética , Espectrometría de Masas , Proteínas Recombinantes/metabolismoRESUMEN
Lithocholic acid is a lipid-soluble hepatotoxic bile acid that accumulates in the liver during cholestasis. A potential detoxification pathway for lithocholic acid involves hydroxylation by hepatic cytochrome P450 (P450) enzymes. The purpose of the present study was to identify the hepatic microsomal metabolites of lithocholic acid by liquid chromatography/mass spectrometry and to determine the P450 enzymes involved. Incubation of lithocholic acid with rat hepatic microsomes and NADPH produced murideoxycholic acid (MDCA), isolithocholic acid (ILCA), and 3-keto-5beta-cholanic acid (3KCA) as major metabolites and 6-ketolithocholic acid and ursodeoxycholic acid as minor metabolites. Experiments with hepatic microsomes prepared from rats pretreated with P450 inducers and with inhibitory antibodies indicated that CYP2C and CYP3A enzymes contribute to microsomal MDCA formation. Results obtained with a panel of recombinant P450 enzymes and CYP2D6 antiserum showed that CYP2D1 can also catalyze MDCA formation. Similar experimental evidence revealed that formation of 3KCA was mediated primarily by CYP3A enzymes. ILCA formation appeared to be catalyzed by a distinct pathway mediated largely by microsomal non-P450 enzymes. Based on the results obtained using lithocholic acid and 3KCA as substrates, a mechanism for the formation of ILCA involving a geminal diol intermediate is outlined. In conclusion, lithocholic acid was extensively metabolized by multiple P450 enzymes with the predominant biotransformation pathway being hydroxylation at the 6beta-position. This study provides an insight into possible routes of detoxification of lithocholic acid.