Severe acute organophosphate poisoning managed with 2-month prolonged atropine therapy: a case report.

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.

Spontaneous bacterial peritonitis complicating extensive splanchnic vein thrombosis, a rare manifestation of essential thrombocythemia: A case report.

Key Clinical Message: Clinicians should be mindful of the rare occurrence of spontaneous bacterial peritonitis in essential thrombocythemia with extensive splanchnic vein thrombosis, especially when patients with ascites exhibit fever and abdominal pain. Abstract: Spontaneous bacterial peritonitis (SBP) complicating extensive splanchnic vein thrombosis (SVT) is a rare manifestation of essential thrombocythemia (ET). In the absence of any hypercoagulable state, JAK2 mutation can be an important risk factor for extensive SVT. Evaluation for SBP is crucial when non-cirrhotic patient exhibits fever, abdominal pain and tenderness in the background of ascites after ruling out common pathologies such as tubercular peritonitis, acute pancreatitis, Budd-Chiari syndrome and ovarian malignancy. We present a case of SBP complicating pre-hepatic portal hypertension with ascites in a 44-years-old female. On further evaluation, extensive SVT with portal cavernoma in the setting of ET was identified. She was managed with cytoreductive therapy and anticoagulation, resulting in symptom resolution.

City-Scale Agent-Based Simulators for the Study of Non-pharmaceutical Interventions in the Context of the COVID-19 Epidemic: IISc-TIFR COVID-19 City-Scale Simulation Team.

We highlight the usefulness of city-scale agent-based simulators in studying various non-pharmaceutical interventions to manage an evolving pandemic. We ground our studies in the context of the COVID-19 pandemic and demonstrate the power of the simulator via several exploratory case studies in two metropolises, Bengaluru and Mumbai. Such tools may in time become a common-place item in the tool kit of the administrative authorities of large cities.

Synthesis and Preclinical Evaluation of 11C-UCB-J as a PET Tracer for Imaging the Synaptic Vesicle Glycoprotein 2A in the Brain.

UNLABELLED: The synaptic vesicle glycoprotein 2A (SV2A) is found in secretory vesicles in neurons and endocrine cells. PET with a selective SV2A radiotracer will allow characterization of drugs that modulate SV2A (e.g., antiepileptic drugs) and potentially could be a biomarker of synaptic density (e.g., in neurodegenerative disorders). Here we describe the synthesis and characterization of the SV2A PET radiotracer (11)C-UCB-J ((R)-1-((3-((11)C-methyl-(11)C)pyridin-4-yl)methyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-2-one) in nonhuman primates, including whole-body biodistribution. METHODS: (11)C-UCB-J was prepared by C-(11)C-methylation of the 3-pyridyl trifluoroborate precursor with (11)C-methyl iodide via the Suzuki-Miyaura cross-coupling method. Rhesus macaques underwent multiple scans including coinjection with unlabeled UCB-J (17, 50, and 150 µg/kg) or preblocking with the antiepileptic drug levetiracetam at 10 and 30 mg/kg. Scans were acquired for 2 h with arterial sampling and metabolite analysis to measure the input function. Regional volume of distribution (VT) was estimated using the 1-tissue-compartment model. Target occupancy was assessed using the occupancy plot; the dissociation constant (Kd) was determined by fitting self-blocking occupancies to a 1-site model, and the maximum number of receptor binding sites (Bmax) values were derived from baseline VT and from the estimated Kd and the nondisplaceable distribution volume (VND). RESULTS: (11)C-UCB-J was synthesized with greater than 98% purity. (11)C-UCB-J exhibited high free fraction (0.46 ± 0.02) and metabolized at a moderate rate (39% ± 5% and 24% ± 3% parent remaining at 30 and 90 min) in plasma. In the monkey brain, (11)C-UCB-J displayed high uptake and fast kinetics. VT was high (∼25-55 mL/cm(3)) in all gray matter regions, consistent with the ubiquitous expression of SV2A. Preblocking with 10 and 30 mg/kg of levetiracetam resulted in approximately 60% and 90% occupancy, respectively. Analysis of the self-blocking scans yielded a Kd estimate of 3.4 nM and Bmax of 125-350 nM, in good agreement with the in vitro inhibition constant (Ki) of 6.3 nM and regional Bmax in humans. Whole-body biodistribution revealed that the liver and the brain are the dose-limiting organs for males and females, respectively. CONCLUSION: (11)C-UCB-J exhibited excellent characteristics as an SV2A PET radiotracer in nonhuman primates. The radiotracer is currently undergoing first-in-human evaluation.

Modulation of P-glycoprotein at the Human Blood-Brain Barrier by Quinidine or Rifampin Treatment: A Positron Emission Tomography Imaging Study.

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.

Regioselective Versatility of Monooxygenase Reactions Catalyzed by CYP2B6 and CYP3A4: Examples with Single Substrates.

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.

Activity of P-Glycoprotein, a ß-Amyloid Transporter at the Blood-Brain Barrier, Is Compromised in Patients with Mild Alzheimer Disease.

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.

Hepatic bile acid metabolism and expression of cytochrome P450 and related enzymes are altered in Bsep (-/-) mice.

The bile salt export pump (BSEP/Bsep; gene symbol ABCB11/Abcb11) translocates bile salts across the hepatocyte canalicular membrane into bile in humans and mice. In humans, mutations in the ABCB11 gene cause a severe childhood liver disease known as progressive familial intrahepatic cholestasis type 2. Targeted inactivation of mouse Bsep produces milder persistent cholestasis due to detoxification of bile acids through hydroxylation and alternative transport pathways. The purpose of the present study was to determine whether functional expression of hepatic cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) is altered by Bsep inactivation in mice and whether bile acids regulate CYP and mEH expression in Bsep (-/-) mice. CYP expression was determined by measuring protein levels of Cyp2b, Cyp2c and Cyp3a enzymes and CYP-mediated activities including lithocholic acid hydroxylation, testosterone hydroxylation and alkoxyresorufin O-dealkylation in hepatic microsomes prepared from female and male Bsep (-/-) mice fed a normal or cholic acid (CA)-enriched diet. The results indicated that hepatic lithocholic acid hydroxylation was catalyzed by Cyp3a/Cyp3a11 enzymes in Bsep (-/-) mice and that 3-ketocholanoic acid and murideoxycholic acid were major metabolites. CA feeding of Bsep (-/-) mice increased hepatic Cyp3a11 protein levels and Cyp3a11-mediated testosterone 2ß-, 6ß-, and 15ß-hydroxylation activities, increased Cyp2b10 protein levels and Cyp2b10-mediated benzyloxyresorufin O-debenzylation activity, and elevated Cyp2c29 and mEH protein levels. We propose that bile acids upregulate expression of hepatic Cyp3a11, Cyp2b10, Cyp2c29 and mEH in Bsep (-/-) mice and that Cyp3a11 and multidrug resistance-1 P-glycoproteins (Mdr1a/1b) are vital components of two distinct pathways utilized by mouse hepatocytes to expel bile acids.

Confounding parameters in preclinical assessment of blood-brain barrier permeation: an overview with emphasis on species differences and effect of disease states.

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.

Interindividual variability in hepatic expression of the multidrug resistance-associated protein 2 (MRP2/ABCC2): quantification by liquid chromatography/tandem mass spectrometry.

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.

3-ketocholanoic acid is the major in vitro human hepatic microsomal metabolite of lithocholic acid.

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.

Identification of human hepatic cytochrome p450 enzymes involved in the biotransformation of cholic and chenodeoxycholic acid.

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.

Biotransformation of lithocholic acid by rat hepatic microsomes: metabolite analysis by liquid chromatography/mass spectrometry.

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.