Serum bile acid change correlates with improvement in pruritus in patients with primary biliary cholangitis receiving linerixibat.

BACKGROUND & AIMS: Total serum bile acid (TSBA) levels are elevated in patients with primary biliary cholangitis (PBC) and may mediate cholestatic pruritus. Linerixibat, an ileal bile acid transporter inhibitor, improved pruritus in patients with PBC. We explored the relationship between linerixibat dose, TSBA concentration, and pruritus. METHODS: Data from Phase 1/2 trials were used to develop a population kinetic-pharmacodynamic model to characterize the linerixibat dose-TSBA relationship. Individual Bayesian parameter estimates for participants in the GLIMMER study were used to derive the area under the TSBA concentration curve over 24 h (AUC0-24). Time-matched post hoc estimates of AUC0-24 were correlated with pruritus reported on a 0-10 numerical rating scale. Baseline TSBA concentration was correlated with change from baseline (ΔBL) in monthly itch score (MIS). ΔBL in model-estimated TSBA AUC0-24 was correlated with time-matched ΔBL in weekly itch score (WIS) or MIS. RESULTS: Linerixibat dose dependently reduced TSBA AUC0-24, reaching steady state after 5 days. Baseline TSBA levels in GLIMMER did not correlate with ΔBL in MIS. ΔBL in TSBA AUC0-24 correlated with improved WIS over 12 weeks of treatment (r = 0.52, p < 0.0001). Of participants with a ≥30% decrease in TSBA AUC0-24, 60% were pruritus responders (≥2-point improvement in WIS from baseline). CONCLUSIONS: Linerixibat treatment leads to rapid, dose-dependent TSBA reductions. Baseline TSBA levels do not correlate with on-treatment pruritus change, suggesting they do not predict linerixibat response. Change in TSBA AUC0-24 correlates significantly with, and can be predictive of, pruritus improvement in patients with PBC.

Clinical and Economic Outcomes of Erythropoiesis-Stimulating Agent Hyporesponsiveness in the Post-Bundling Era.

RATIONALE & OBJECTIVE: Since the change in erythropoiesis-stimulating agent (ESA) labeling and bundling of dialysis services in the United States, few studies have addressed the clinical importance of ESA hyporesponsiveness and none have considered health care resource use in this population. We aimed to further explore ESA hyporesponsiveness and its consequences. STUDY DESIGN: Retrospective observational cohort study. SETTING & PARTICIPANTS: US Renal Data System Medicare participants receiving dialysis with a minimum 6 months of continuous ESA use from 2012 to 2014. PREDICTORS: Erythropoietin resistance index (≥2.0 U/kg/wk/g/L) and ESA dose were used to identify ESA hyporesponders and hyporesponsive subgroups: isolated, intermittent, and chronic. OUTCOMES: Associations between ESA responsiveness and mortality, cardiovascular hospitalization rates, and health care resource use were evaluated and compared across subgroups. ANALYTICAL APPROACH: Baseline characteristics were compared using Wilcoxon rank sum tests for continuous variables and χ2 tests for categorical variables. Incidence rates of health care resource use were modeled using an unadjusted and adjusted generalized linear model. RESULTS: Of 834,115 dialysis patients in the CROWNWeb database, 38,891 ESA hyporesponders and 59,412 normoresponders met all inclusion criteria. Compared with normoresponders, hyporesponders were younger women, weighed less, and had longer durations of dialysis (all P < 0.001). Hyporesponders received 3.8-fold higher ESA doses (mean, 94,831 U/mo) and erythropoietin resistance index was almost 5 times higher than in normoresponders. Hyporesponders had lower hemoglobin levels and parathyroid hormone levels > 800 pg/mL, and iron deficiency was present in 26.5% versus 10.9% in normoresponders. One-year mortality was higher among hypo- compared with normoresponders (25.3% vs 22.6%). Hyporesponders also had significantly higher rates of hospitalization for cardiovascular events, emergency department visits, inpatient stays, home health agency visits, skilled nursing facility, and hospice days. LIMITATIONS: Only US Medicare patients were included and different hyporesponder definitions may have influenced the results. CONCLUSIONS: This study explored ESA hyporesponsiveness using new definitions and incorporated clinical and economic outcomes. It established that ESA-hyporesponsive dialysis patients had higher mortality, cardiovascular hospitalization rates, and health care costs as compared with ESA-normoresponsive patients.

Correction to Identification of Quinoline-Based RIP2 Kinase Inhibitors with an Improved Therapeutic Index to the hERG Ion Channel.

[This corrects the article DOI: 10.1021/acsmedchemlett.8b00344.].

Understanding Pharmacokinetic Disconnect in Preclinical Species for 4-Aminoquinolines: Consequences of Low Permeability and High P-glycoprotein Efflux Ratio on Rat and Dog Oral Pharmacokinetics.

Receptor Interacting Protein 2 (RIP2) kinase inhibitors have been reported for therapeutic opportunities in inflammatory bowel diseases such as Ulcerative Colitis and Crohn's disease. During lead optimization, team identified 4-aminoquinoline series and several compounds from this series were investigated in rat and dog pharmacokinetic studies. While compounds such as GSKA and GSKB demonstrated acceptable pharmacokinetics in rat and dog, further progression of these compounds was halted due to adverse findings in advanced safety studies. Structurally similar analogues incorporating polarity at C-7 position of 4-aminoquinoline resulted in identification of GSKC - GSKF. Interestingly, following oral administration to rat at similar low dose, GSKC - GSKF demonstrated significantly low systemic drug exposure compared to GSKA and GSKB (3-17-fold difference). However, in dog, dose normalized oral systemic exposure for GSKC - GSKF was comparable to GSKA and GSKB (within 2-fold). A series of studies were conducted to understand the disconnect which highlighted that an intrinsic reduction in permeability and high P-glycoprotein (P-gp) efflux ratio for C-7 substituted analogues were driving pharmacokinetic disconnect between rat and dog. Oral absorption was minimally impacted in dog by P-gp mediated efflux compared to rat because the leakier gastrointestinal tract in dog likely overcomes this effect.

Elicitation of disease concepts in patients with diabetic foot ulcers: a qualitative study.

OBJECTIVE: To identify in-depth information directly from patients with diabetic foot ulcers (DFU) on DFU symptoms, impacts on functioning and effects on health-related quality of life (HRQoL). METHOD: Semi-structured, qualitative concept elicitation interviews were conducted with patients with DFUs (Wagner grade 1 or 2) until saturation was reached. Qualitative analysis (using MAXQDA, VERBI GmbH, Germany) of interview transcripts was conducted to identify concepts relevant to patients with DFUs, based on the frequency of mentions, and elucidate themes regarding impacts on HRQoL. RESULTS: Of the 18 participants, most were male (n=14; 78%) and 10 (56%) presented with a Wagner grade of 1. Frequently reported symptoms were pain/discomfort (n=15; 83%), weeping/discharge (n=10; 56%), bleeding (n=10; 56%) and swelling (n=8; 44%). Overall, patients reported more impacts than symptoms-wound care/treatment burden (n=14; 78%), limitations on exercise/physical activity (n=13; 72%), mobility limitations (n=12; 67%), and offloading (n=12; 67%) were the most frequently mentioned. Based on findings from the patient interviews, a draft conceptual model was developed outlining interrelationships between DFU symptoms, impacts, and HRQoL from the patient perspective. CONCLUSION: Qualitative interviews captured the breadth of disease-related concepts of direct importance to patients. The draft conceptual model developed from the analysis can help identify measures or instruments for use in assessing patient-reported symptoms or HRQoL in clinical practice and may have wider research applicability, including evaluation of treatment benefits in patients with DFUs.

Discovery of Pyrazolocarboxamides as Potent and Selective Receptor Interacting Protein 2 (RIP2) Kinase Inhibitors.

Herein we report the discovery of pyrazolocarboxamides as novel, potent, and kinase selective inhibitors of receptor interacting protein 2 kinase (RIP2). Fragment based screening and design principles led to the identification of the inhibitor series, and X-ray crystallography was used to inform key structural changes. Through key substitutions about the N1 and C5 N positions on the pyrazole ring significant kinase selectivity and potency were achieved. Bridged bicyclic pyrazolocarboxamide 11 represents a selective and potent inhibitor of RIP2 and will allow for a more detailed investigation of RIP2 inhibition as a therapeutic target for autoinflammatory disorders.

Discovery of a First-in-Class Receptor Interacting Protein 2 (RIP2) Kinase Specific Clinical Candidate, 2-((4-(Benzo[d]thiazol-5-ylamino)-6-(tert-butylsulfonyl)quinazolin-7-yl)oxy)ethyl Dihydrogen Phosphate, for the Treatment of Inflammatory Diseases.

RIP2 kinase has been identified as a key signal transduction partner in the NOD2 pathway contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP2 kinase or its signaling partners on the NOD2 pathway that are suitable for advancement into the clinic have yet to be described. Herein, we report our discovery and profile of the prodrug clinical compound, inhibitor 3, currently in phase 1 clinical studies. Compound 3 potently binds to RIP2 kinase with good kinase specificity and has excellent activity in blocking many proinflammatory cytokine responses in vivo and in human IBD explant samples. The highly favorable physicochemical and ADMET properties of 3 combined with high potency led to a predicted low oral dose in humans.

Identification of Quinoline-Based RIP2 Kinase Inhibitors with an Improved Therapeutic Index to the hERG Ion Channel.

RIP2 kinase was recently identified as a therapeutic target for a variety of autoimmune diseases. We have reported previously a selective 4-aminoquinoline-based RIP2 inhibitor GSK583 and demonstrated its effectiveness in blocking downstream NOD2 signaling in cellular models, rodent in vivo models, and human ex vivo disease models. While this tool compound was valuable in validating the biological pathway, it suffered from activity at the hERG ion channel and a poor PK/PD profile thereby limiting progression of this analog. Herein, we detail our efforts to improve both this off-target liability as well as the PK/PD profile of this series of inhibitors through modulation of lipophilicity and strengthening hinge binding ability. These efforts have led to inhibitor 7, which possesses high binding affinity for the ATP pocket of RIP2 (IC50 = 1 nM) and inhibition of downstream cytokine production in human whole blood (IC50 = 10 nM) with reduced hERG activity (14 µM).

Effects of Low- and High-Mineral Content Water on the Relative Bioavailability of a Coformulated Abacavir/Dolutegravir/Lamivudine Dispersible Tablet in Healthy Adults.

BACKGROUND: The fixed-dose combination (FDC) tablet formulation of abacavir/dolutegravir/lamivudine is indicated for the treatment of HIV-1 infection in adults and pediatric patients weighing ≥40 kg. Alternative formulations with acceptable palatability and convenient dosing are needed for children who require smaller doses and have difficulty swallowing tablets. SETTING: A phase 1, open-label, randomized study was conducted in healthy adults to evaluate the relative bioavailability of a novel dispersible FDC tablet of abacavir 150 mg/dolutegravir 10 mg/lamivudine 75 mg administered under 4 different dosing conditions compared with dolutegravir plus abacavir/lamivudine nondispersible, film-coated tablets. METHODS: The test treatments were 4 dispersible FDC tablets reconstituted in water with high- or zero-mineral content and administered either immediately or after a 30-minute delay. The reference treatment was 4 nondispersible dolutegravir 10-mg tablets plus 1 nondispersible abacavir 600-mg/lamivudine 300-mg tablet administered with zero-mineral content water. The primary endpoints were area under the concentration-time curve from time 0 extrapolated to infinity and the maximum observed plasma concentration. RESULTS: Following administration of dispersible abacavir/dolutegravir/lamivudine, the relative bioavailability of dolutegravir was approximately 50% higher. Abacavir and lamivudine demonstrated bioequivalence when administered as the dispersible FDC tablet compared with coadministration of dolutegravir plus abacavir/lamivudine nondispersible, film-coated tablets. Neither the mineral content of the water nor dosing times affected the pharmacokinetics of individual components. The dispersible tablet was safe and well tolerated, and the palatability was acceptable. CONCLUSIONS: These pharmacokinetic results support further development of a dispersible FDC tablet of abacavir/dolutegravir/lamivudine for future use in pediatric patients.

Application of the Stable Isotope Label Approach in Clinical Development-Supporting Dissolution Specifications for a Commercial Tablet Product with Tafenoquine, a Long Half-life Compound.

Bioavailability/bioequivalence studies supporting clinical drug development or commercial supply of drug formulations are often time, cost, and resource intensive. The drug's pharmacokinetic (PK) variability, systemic half-life, and safety issues may pose additional challenges. The stable isotope label (SIL) approach provides a useful tool to significantly reduce the study size in clinical PK studies. Tafenoquine (TQ) is an 8-aminoquinoline under development for preventing Plasmodium vivax malaria relapse. This SIL study assessed the impact of differences in the in vitro dissolution profiles on in vivo exposure of TQ tablets. Fourteen healthy volunteers received a single dose of 300 mg TQ Intermediate Aged or 300 mg TQ Control formulations in this single-center, two-arm, randomized, open-label, parallel-group study. Endpoints included the geometric means ratio of the area under the concentration-time curve (AUC(0-t) and AUC(0-∞); primary endpoint) and maximum plasma concentration (Cmax) for Intermediate Aged versus Control TQ; correlation of PK parameters for venous versus peripheral (via microsample) blood samples; and safety and tolerability endpoints. Geometric mean ratios for PK parameters (AUC and Cmax) and their 90% confidence intervals fell well within standard bioequivalence limits (0.80-1.25). Only one mild adverse event (skin abrasion) was reported. In summary, this SIL methodology-based study demonstrates that the observed differences in the in vitro dissolution profiles between the Control and Intermediate Aged TQ tablets have no clinically relevant effect on systemic TQ exposure. The SIL approach was successfully implemented to enable the setting of a clinically relevant dissolution specification. CLINICAL TRIAL: This study (GSK study number 201780) is registered at clinicaltrials.gov with identifier NCT02751294.

The Identification and Pharmacological Characterization of 6-(tert-Butylsulfonyl)-N-(5-fluoro-1H-indazol-3-yl)quinolin-4-amine (GSK583), a Highly Potent and Selective Inhibitor of RIP2 Kinase.

RIP2 kinase is a central component of the innate immune system and enables downstream signaling following activation of the pattern recognition receptors NOD1 and NOD2, leading to the production of inflammatory cytokines. Recently, several inhibitors of RIP2 kinase have been disclosed that have contributed to the fundamental understanding of the role of RIP2 in this pathway. However, because they lack either broad kinase selectivity or strong affinity for RIP2, these tools have only limited utility to assess the role of RIP2 in complex environments. We present, herein, the discovery and pharmacological characterization of GSK583, a next-generation RIP2 inhibitor possessing exquisite selectivity and potency. Having demonstrated the pharmacological precision of this tool compound, we report its use in elucidating the role of RIP2 kinase in a variety of in vitro, in vivo, and ex vivo experiments, further clarifying our understanding of the role of RIP2 in NOD1 and NOD2 mediated disease pathogenesis.

Crystal structures of human RIP2 kinase catalytic domain complexed with ATP-competitive inhibitors: Foundations for understanding inhibitor selectivity.

Receptor interacting protein 2 (RIP2) is an intracellular kinase and key signaling partner for the pattern recognition receptors NOD1 and NOD2 (nucleotide-binding oligomerization domain-containing proteins 1 and 2). As such, RIP2 represents an attractive target to probe the role of these pathways in disease. In an effort to design potent and selective inhibitors of RIP2 we established a crystallographic system and determined the structure of the RIP2 kinase domain in an apo form and also in complex with multiple inhibitors including AMP-PCP (ß,γ-Methyleneadenosine 5'-triphosphate, a non-hydrolysable adenosine triphosphate mimic) and structurally diverse ATP competitive chemotypes identified via a high-throughput screening campaign. These structures represent the first set of diverse RIP2-inhibitor co-crystal structures and demonstrate that the protein possesses the ability to adopt multiple DFG-in as well as DFG-out and C-helix out conformations. These structures reveal key protein-inhibitor structural insights and serve as the foundation for establishing a robust structure-based drug design effort to identify both potent and highly selective inhibitors of RIP2 kinase.

Catalytic in vivo protein knockdown by small-molecule PROTACs.

The current predominant therapeutic paradigm is based on maximizing drug-receptor occupancy to achieve clinical benefit. This strategy, however, generally requires excessive drug concentrations to ensure sufficient occupancy, often leading to adverse side effects. Here, we describe major improvements to the proteolysis targeting chimeras (PROTACs) method, a chemical knockdown strategy in which a heterobifunctional molecule recruits a specific protein target to an E3 ubiquitin ligase, resulting in the target's ubiquitination and degradation. These compounds behave catalytically in their ability to induce the ubiquitination of super-stoichiometric quantities of proteins, providing efficacy that is not limited by equilibrium occupancy. We present two PROTACs that are capable of specifically reducing protein levels by >90% at nanomolar concentrations. In addition, mouse studies indicate that they provide broad tissue distribution and knockdown of the targeted protein in tumor xenografts. Together, these data demonstrate a protein knockdown system combining many of the favorable properties of small-molecule agents with the potent protein knockdown of RNAi and CRISPR.

RIP3 induces apoptosis independent of pronecrotic kinase activity.

Receptor-interacting protein kinase 3 (RIP3 or RIPK3) has emerged as a central player in necroptosis and a potential target to control inflammatory disease. Here, three selective small-molecule compounds are shown to inhibit RIP3 kinase-dependent necroptosis, although their therapeutic value is undermined by a surprising, concentration-dependent induction of apoptosis. These compounds interact with RIP3 to activate caspase 8 (Casp8) via RHIM-driven recruitment of RIP1 (RIPK1) to assemble a Casp8-FADD-cFLIP complex completely independent of pronecrotic kinase activities and MLKL. RIP3 kinase-dead D161N mutant induces spontaneous apoptosis independent of compound, whereas D161G, D143N, and K51A mutants, like wild-type, only trigger apoptosis when compound is present. Accordingly, RIP3-K51A mutant mice (Rip3(K51A/K51A)) are viable and fertile, in stark contrast to the perinatal lethality of Rip3(D161N/D161N) mice. RIP3 therefore holds both necroptosis and apoptosis in balance through a Ripoptosome-like platform. This work highlights a common mechanism unveiling RHIM-driven apoptosis by therapeutic or genetic perturbation of RIP3.

The immune receptor NOD1 and kinase RIP2 interact with bacterial peptidoglycan on early endosomes to promote autophagy and inflammatory signaling.

The intracellular innate immune receptor NOD1 detects Gram-negative bacterial peptidoglycan (PG) to induce autophagy and inflammatory responses in host cells. To date, the intracellular compartment in which PG is detected by NOD1 and whether NOD1 directly interacts with PG are two questions that remain to be resolved. To address this, we used outer membrane vesicles (OMVs) from pathogenic bacteria as a physiological mechanism to deliver PG into the host cell cytosol. We report that OMVs induced autophagosome formation and inflammatory IL-8 responses in epithelial cells in a NOD1- and RIP2-dependent manner. PG contained within OMVs colocalized with both NOD1 and RIP2 in EEA1-positive early endosomes. Further, we provide evidence for direct interactions between NOD1 and PG. Collectively, these findings demonstrate that NOD1 detects PG within early endosomes, thereby promoting RIP2-dependent autophagy and inflammatory signaling in response to bacterial infection.

An orally active TRPV4 channel blocker prevents and resolves pulmonary edema induced by heart failure.

Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca(2+) influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.

N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), a novel and potent transient receptor potential vanilloid 4 channel agonist induces urinary bladder contraction and hyperactivity: Part I.

The transient receptor potential (TRP) vanilloid 4 (TRPV4) member of the TRP superfamily has recently been implicated in numerous physiological processes. In this study, we describe a small molecule TRPV4 channel activator, (N-((1S)-1-{[4-((2S)-2-{[(2,4-dichlorophenyl)sulfonyl]amino}-3-hydroxypropanoyl)-1-piperazinyl]carbonyl}-3-methylbutyl)-1-benzothiophene-2-carboxamide (GSK1016790A), which we have used as a valuable tool in investigating the role of TRPV4 in the urinary bladder. GSK1016790A elicited Ca2+ influx in mouse and human TRPV4-expressing human embryonic kidney (HEK) cells (EC50 values of 18 and 2.1 nM, respectively), and it evoked a dose-dependent activation of TRPV4 whole-cell currents at concentrations above 1 nM. In contrast, the TRPV4 activator 4alpha-phorbol 12,13-didecanoate (4alpha-PDD) was 300-fold less potent than GSK1016790A in activating TRPV4 currents. TRPV4 mRNA was detected in urinary bladder smooth muscle (UBSM) and urothelium of TRPV4+/+ mouse bladders. Western blotting and immunohistochemistry demonstrated protein expression in both the UBSM and urothelium that was absent in TRPV4-/- bladders. TRPV4 activation with GSK1016790A contracted TRPV4+/+ mouse bladders in vitro, both in the presence and absence of the urothelium, an effect that was undetected in TRPV4-/- bladders. Consistent with the effects on TRPV4 HEK whole-cell currents, 4alpha-PDD demonstrated a weak ability to contract bladder strips compared with GSK1016790A. In vivo, urodynamics in TRPV4+/+ and TRPV4-/- mice revealed an enhanced bladder capacity in the TRPV4-/- mice. Infusion of GSK1016790A into the bladders of TRPV4+/+ mice induced bladder overactivity with no effect in TRPV4-/- mice. Overall TRPV4 plays an important role in urinary bladder function that includes an ability to contract the bladder as a result of the expression of TRPV4 in the UBSM.

Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: Part 2.

The transient receptor potential (TRP) vanilloid subtype 4 (V4) is a nonselective cation channel that exhibits polymodal activation and is expressed in the endothelium, where it contributes to intracellular Ca2+ homeostasis and regulation of cell volume. The purpose of the present study was to evaluate the systemic cardiovascular effects of GSK1016790A, a novel TRPV4 activator, and to examine its mechanism of action. In three species (mouse, rat, and dog), the i.v. administration of GSK1016790A induced a dose-dependent reduction in blood pressure, followed by profound circulatory collapse. In contrast, GSK1016790A had no acute cardiovascular effects in the TRPV4-/- null mouse. Hemodynamic analyses in the dog and rat demonstrate a profound reduction in cardiac output. However, GSK1016790A had no effect on rate or contractility in the isolated, buffer-perfused rat heart, and it produced potent endothelial-dependent relaxation of rodent-isolated vascular ring segments that were abolished by nitric-oxide synthase (NOS) inhibition (N-nitro-L-arginine methyl ester; L-NAME), ruthenium red, and endothelial NOS (eNOS) gene deletion. However, the in vivo circulatory collapse was not altered by NOS inhibition (L-NAME) or eNOS gene deletion but was associated with (concentration and time appropriate) profound vascular leakage and tissue hemorrhage in the lung, intestine, and kidney. TRPV4 immunoreactivity was localized in the endothelium and epithelium in the affected organs. GSK1016790A potently induced rapid electrophysiological and morphological changes (retraction/condensation) in cultured endothelial cells. In summary, inappropriate activation of TRPV4 produces acute circulatory collapse associated with endothelial activation/injury and failure of the pulmonary microvascular permeability barrier. It will be important to determine the role of TRPV4 in disorders associated with edema and microvascular congestion.

Synthesis of 11-hydroxyl O-methylsterigmatocystin and the role of a cytochrome P-450 in the final step of aflatoxin biosynthesis.

The major skeletal rearrangements (anthraquinone --> xanthone --> coumarin) that occur in the complex biosynthesis of aflatoxin B(1) are mediated by cytochromes P-450. Previous experiments have suggested that two successive monooxygenase reactions are required to convert the xanthone O-methylsterigmatocystin (OMST) to aflatoxin, a process we demonstrate is mediated by a single P-450, OrdA, in Aspergillus parasiticus in accord with findings in A. flavus. The first oxidative cycle is proposed to result in the formation of 11-hydroxy O-methylsterigmatocystin (HOMST), while the second entails aryl ring cleavage, demethylation, dehydration, decarboxylation, and rearrangement to give aflatoxin - a remarkable sequence of transformations. To test this hypothesis, HOMST has been synthesized by an alkylnitrilium variant of the Houben-Hoesch reaction. The troublesome xanthone carbonyl was protected as a butylene to allow further elaboration of the molecule, and then the product xanthone was restored in a uniquely facile peracid deprotection. Methods were devised to construct the sensitive dihydrobisfuran and to maintain the oxidation state of the partially methylated hydroquinone. Expression of ordA in a yeast membrane preparation enabled the intermediacy of HOMST both to be detected in the conversion of OMST to aflatoxin and to be established directly in the biosynthesis of the mycotoxin. Having secured the role of HOMST in aflatoxin formation, the mechanism of the second oxidative cycle of this P-450 is considered.