Danny D. Shen - US grants

The overall objective of this research continues to be a study of the effect of uremia on hepatic drug metabolism. In our previous studies, we have shown that in the rat acute renal failure induced by uranyl nitrate can lead to a pronounced inhibition of the hepatic first-pass metabolism of the Beta-adrenergic blocker 1-propranolol. Our data in the acute renal failure rat model are consistent with results of the earlier clinical studies in end stage renal failure patients. In our current application, we proposed to extend our pharmacokinetic investigations to determine if the degree of inhibitory effect of renal failure on the hepatic first-pass metabolism of 1-propranolol is related to the severity and/or duration of uremia. For this purpose, a chronic uremic animal model will be developed. We will also investigate whether chronic pretreatment with 1-propranolol can modulate the effect of renal failure on 1-propranolol metabolism. The second phase of our investigation will focus on identifying which of the primary metabolic pathways of 1-propranolol are affected in renal failure. In vitro metabolism studies will be carried out using the isolated rat hepatocyte. The final phase of proposed work will be directed at elucidating the mechanism(s) responsible for the inhibition of hepatic metabolism of 1-propranolol in uremia. Experiments will be designed to investigate the following postulated mechanisms: (1) presence of "endogenous inhibitor" in uremic blood; (2) alteration in the regulation and intrinsic activity of the hepatic drug metabolizing enzymes; (3) inhibition of 1-propranolol metabolism by metabolites which are retained in renal failure; and (4) regeneration of 1-propranolol by deconjugation of the glucuronide conjugate metabolite.

The antiepileptic drug, valproic acid (VPA) is a simple branched-chain fatty acid with several notable pharmacologic properties. Unlike other antiepileptic drugs, anticonvulsant response to VPA is poorly correlated with plasma drug concentration. It has also been shown in clinical and animal studies that the time course of anticonvulsant activity is dissociated from the plasma kinetics of VPA. Maximal seizure control is not observed until long after the establishment of steady-state serum concentration of VPA. Also, the duration of anticonvulsant activity outlasts the presence of VPA in circulation. These unusual findings suggest that VPA has a distinctly different mechanism of antiepileptic action compared to other anticonvulsants. Despite considerable effort, the antiepileptic mechanism of valproic acid has not been identified. Recent evidence in literature together with preliminary animal studies in our laboratory now suggest that the unusual pharmacodynamics of VPA may be related to the selective accumulation and slow efflux of two unsaturated metabolites of the drug, Delta2-VPA and Delta4-VPA, in brain during chronic drug administration. The objective of this proposed research is to elucidate the role of these unsaturated metabolites in the antiepileptic activity of VPA. The specific aims are: (i) to establish the contribution of Delta2-VPA and Delta4-VPA to the in vivo anticonvulsant activity of VPA; and (ii) to elucidate the pharmacokinetic mechanisms responsible for the slow uptake and efflux of these unsaturated branched-chain fatty acids in the CNS. The proposed pharmacodynamic studies will involve the comparison of anticonvulsant activity between VPA and directly administered synthetic Delta2-VPA and Delta4-VPA during chronic intravenous infusion in rats. A well established pentylenetetrazol rat seizure model will be used to assess anticonvulsant response. Three separate mechanisms, which may explain accumulation and slow clearance of Delta2-VPA and Delta4-VPA in brain, will be explored: (i) rate-limiting transport of unsaturated metabolite across blood-brain-barrier; (ii) in situ generation of metabolites in brain; and (iii) trapping of metabolites by the reversible formation of their CoA esters in brain. The long term goal of this research is to gain an understanding as to how CNS transport processes and brain metabolism modulate the anticonvulsant activity of the branched-chain fatty acids. Such knowledge will be useful in the development of more effective fatty acid like antiepileptic drugs.

The antiepileptic drug valproic acid (VPA) is a branched-chain fatty acid with unique pharmacodynamic properties. Unlike other commonly used antiepileptics, the anticonvulsant activity of VPA is poorly correlated with plasma drug concentration. Recent studies in neurosurgical patients showed that, during chronic VPA therapy, the concentrations of VPA in human brain were much lower than either the total or unbound serum drug concentrations. More significant is the fact that the steady-state brain- to-serum concentration ratios varied widely between individuals. It appears that the failure to demonstrate a clearly definable relationship between clinical effect and circulating concentration of VPA in patients with epilepsy may be related to the unusually large interindividual variation in blood-brain partitioning of the drug. The overall objective of this project is to investigate the cause of this low and variable distribution of VPA into brain. We hypothesize that (1) the translocation of VPA across blood-brain and blood-cerebrospinal fluid (CSF) barriers is mediated by membrane transport carriers for endogenous carboxylic acids, (2) individual differences in the physiologic regulation of these transport processes accounts for the observed variation in CNS distribution of VPA between patients, and (3) the low brain-to-blood concentration gradient of VPA is due to an asymmetry in drug transport between brain and blood. Thus, a series of in vivo and in vitro studies in animal models are proposed to identify and characterize the putative transport carriers for VPA and its 2-unsaturated analog (E)-delta2-VPA. The latter compound is currently under development as a second generation alkanoate anticonvulsant. We will test the specific hypothesis that VPA and (E)- delta2-VPA are shuttled across the brain capillary endothelium by the monocarboxylic acid (MCA) transporter, which mediates the exchange of ketone bodies and lactate between blood and brain. Cerebrovascular transport studies will be performed using the in situ brain perfusion technique in rats and the in vitro bovine brain microvessel endothelial cell culture model. Evidence that the branched-chain fatty acids and endogenous MCAs share the same endothelial transport system will be sought. The contribution of an efflux pathway at the choroidal epithelium will be investigated by in vitro uptake studies with isolated rabbit choroid plexus and by transepithelial transport studies with an in situ lateral ventricle choroid plexus preparation in the rabbit. Specifically, we will test if the medium, branched-chain fatty acids are transported by the organic anion exchanger at the brush-border membrane of the choroid plexus. Information on the CNS transport of branched-chain fatty acids may be useful in developing mechanism-based strategies for improving the brain delivery of alkanoate antiepileptics.

The long-term objective of this research is to improve clinical management of persistent pian states. The goal of the proposed work is to evaluate the ability for a norepinephrine re-uptake inhibitor and a serotonin releaser (desipramine and fenfluramine) to enhance human opioid analgesia. Analgesia effect measures will be derived from a human testing model based on painful tooth pulp stimulation: Subjective pain report, dental evoked potentials, and EEG measures will be obtained. The first stage in this work will address the hypothesis that desipramine and fenfluramins exert an analgesic effect when administered alone. The second stage of the work will evaluate the ability of these drugs to enhance opioid analgesia. To accomplish the latter we will administer the opioid alfentanil at three different plasma concentrations to normal volunteers using pharmacokinetically tailored infusions. These infusions will permit the evaluation of the effects of the candidates enhancers (also infuse at steady-state concentrations) while the subject is experiencing opioid analgesia at steady- state, We predict that neither of the candidate enhancers used above will demonstrated analgesic effects, but each of the enhancers when used in combination with the alfentanil infusion is predicted to increase opioid analgesia and alter the relationship of opioid side effects to plasma concentration. Finally, we will evaluate the effects of proglumide, a cholecystokinin antagonist, alone and in combination with alfentanil, on measures of analgesia. A model is proposed for the modulation of opioid analgesia in which norepinephrine and serotonin systems are balanced against the cholecystokinin system in an opponent process relationship.

DESCRIPTION (provided by applicant):Extract of SJW; Hypericum perforatum, has gained widespread popularity as an over-the-counter, natural antidepressant. Until recently, SJW was thought to be well tolerated and relatively safe. Within the past year, adverse metabolic interactions have been reported between SJW and several narrow therapeutic index drugs, notably cyclosporine indinavir, and digoxin. The interactions are now recognized to involve induction of two drug disposition mechanisms: cytochrome P450 3A4 enzyme and the active efflux pump, P-glycoprotein, both leading to profound reductions in blood or plasma drug concentration that compromises the therapeutic efficacy of the affected drug. Natural and synthetic opioids are the first-line agents for the palliative treatment of severe pain that results from cancer and cancer treatment. It is well recognized that depression is a co-morbid condition of severe and poorly controlled cancer-related pain. Given the widespread recognition of St. Johns wort as a "mood enhancer" and natural antidepressant, cancer pain patients receiving opioid analgesics may well turn to this herbal preparation for relief of depressive symptoms. The overall objective of this research proposal is to investigate if significant interactions occur between two widely used opioid analgesics - oxycodone and fentanyl and St. John wort extract through laboratory-based studies in healthy volunteers. The studies will assess the potential clinical significance of the interactions with respect to opioid analgesia efficacy and side effects, and provide scientific insights into the pharmacokinetic mechanisms underlying any observed interactions. The oxycodone arm of the study is designed to 1) investigate the induction of CYP3A4-mediated N-demethylation which is the major detoxification pathway for oxycodone, and 2) resolve the inductive effects of SJW on intestinal and hepatic CYP3A4 through intravenous and oral administrations of a CYP3A-specific, in vivo catalytic probe -midazolam. The fentanyl arm of the study will 1) assess the effects of SJW on the brain uptake and efflux kinetics of fentanyl through pharmacokinetic-pharmacodynamic (PK-PD) modeling of miotic response over time during and following intravenous infusion of the opioid, and 2) to evaluate the changes in analgesia and side effects of fentanyl upon pretreatment with SJW that may have resulted from induction of Pgp at the BBB. A third arm of the study will assess whether SJW has analgesic properties of its own, or is capable of promoting opioid analgesia. Overall, the proposed research will provide a definitive assessment of the potential and clinical significance of adverse interactions between SJW and opioids in the context of cancer pain therapy.

St Johns wort; antidepressants; fentanyl; analgesics; pharmacokinetics; drug interactions; P glycoprotein; analgesia; intravenous administration; blood brain barrier; drug adverse effect; medicinal plants; plant extracts; patient oriented research; alternative medicine; human subject; clinical research;

[unreadable] DESCRIPTION (provided by applicant): Garlic supplements are widely used for the treatment of high blood cholesterol and lipids. Until recently, garlic supplements were thought to be well tolerated and relatively safe. Adverse interactions between garlic supplements and several narrow therapeutic drugs, including anticoagulants (warfarin, fuindione) and HIV protease inhibitors (saquinavir, ritonavir), have been reported within the past 2 years. Animal and in vitro studies with known organosulfur constituents of garlic, as well as garlic extracts, are known to simultaneously inhibit and induce several CYP enzymes (1A, 2B, 2C, 2E and 3A subfamilies) in the liver, as well as the efflux transporter P-gylcoproptein (Pgp) in the intestinal mucosa. However, very recent studies in human subjects have produced conflicting data as to the effects of garlic supplements on cytochrome P450 function. Our specific hypotheses are: 1) the effects of garlic supplements on CYP and Pgp function vary with the type of garlic preparation (viz. garlic powder, steamed-distilled garlic oil, and aged garlic); and 2) garlic's effect is time- and regimen-dependent (e.g. it acutely inhibits and chronically induces CYP3A4 and Pgp activities in human subjects). Two separate "cocktails" of CYP and Pgp probe substrates, including caffeine (1A2), buproprion (2B6), chlorzoxazone (2E1), tolbutamide (2C9), omeprazole (2C19), metoprolol (2D6), midazolam (3A4/5), and digoxin (Pgp), will be administered to a panel of healthy human volunteers at various times during and shortly after discontinuation of daily intake of garlic supplement. Pharmacokinetics of known bioactive garlic constituents and their biotransformation products will be simultaneously studied through collection of blood, breath and urine samples. Garlic metabolite data may offer clues as to which garlic-derived compounds may be responsible for the putative metabolic effects. Specifically, we will measure metabolites deriving from allicin and allylic sulfides, which are the main constituents of garlic powder and garlic oil. We will also measure metabolites of S-allylcysteine, which is derived from hydrolysis of allylic cysteinyl peptides in garlic powder and aged garlic. These metabolic studies in human subjects will be conducted at the University of Washington General Clinical Research Center. The results will provide critical information for the design of further clinical studies to characterize and elucidate potentially important adverse garlic-drug interactions. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Garlic supplements are widely used by cancer patients for their purported immune enhancement and anticancer effects. Epidemiological evidence and laboratory studies suggest that dietary garlic or garlic extracts may prevent or halt cancer through multiple biochemical and cellular mechanisms, including inhibition of tumor promotion and proliferation, as well as suppression of carcinogen activation through inhibition of bioactivating enzymes and induction of detoxification enzymes. Garlic is particularly known for its induction of cytochrome P450 enzymes in the intestine and liver, which constitute a major detoxification system for drugs. In addition, recent reports suggest that garlic can also modulate the activity of the efflux membrane transporter P-gylcoproptein (Pgp), which plays an important gatekeeper's role in intestinal drug absorption. Hence, there is a high potential for undesirable interactions between garlic supplements and drugs used in the treatment of cancer and related medical complications. The main objective of this proposal is to assess the likelihood of a clinically significant interaction between garlic supplements and oxycodone-a commonly used oral opioid for the treatment of cancer pain. We hypothesize that garlic extract induces intestinal and hepatic CYP3A4 isoenzyme and Pgp, lowers opioid bioavailability, and attenuates the effectiveness of opioid analgesia. We propose a placebo-controlled, crossover trial in healthy human subjects to investigate whether acute (2 days) and chronic (4-weeks) treatment with 2 select commercial garlic supplements (dried garlic powder and garlic oil) would affect the pharmacokinetics as well as the analgesic response and side effects of a clinically relevant dose of oxycodone. Modulation of CYP3A4 and Pgp activities will be demonstrated by phenotyping tests with midazolam and digoxin. Pharmacokinetics of oxycodone and its metabolites derived from CYP3A4 pathways will be characterized. Oxycodone analgesia will be measured by subjective responses in an experimental pain paradigm-cold pressor test. A battery of biobehavioral instruments that have been specifically designed for opioid effects will be applied to obtain: 1) subjective measures of somatic and cognitive-affective side effects and mood disturbance, 2) measures of cognitive function (visual-motor coordination, memory, verbal reasoning), and 3) physiological responses (pupil constriction, respiration). The results will guide further studies on the liability of adverse herb-drug interactions with garlic in chemotherapy and palliative treatment of cancer. [unreadable] [unreadable]

PROJECT SUMMARY OF THE ADMINISTRATIVE CORE The Administrative Core is responsible for two main tasks: 1) provide overall administrative support for the U54 Center and 2) develop and disseminate Best Practices on natural product (NP)-drug interaction (NPDI) research. The U54 Center will be coordinately managed by the Executive Committee and the Center Leadership Committee. The Executive Committee, consisting of the two Co-PIs (Dr. Shen and Dr. Paine) and the Co-Leader of the Administrative Core (Dr. McCune), will manage the administrative operation of the U54 Center. The Center Leadership Committee, consisting of members of the Executive Committee and Leaders of the Pharmacology, Analytical, and Informatics Core, will oversee the programmatic aspects of the Center. Regarding the first task, the Administrative Core will 1) support the operations of the Pharmacology, Analytical, and Informatics Cores in accomplishing the assigned milestones; 2) coordinate the activities of the joint Center-NCCAM Steering Committee; 3) participate in the Interaction Project Teams and monitor their progress; 4) manage the flow of samples and information from the Interaction Projects to the Pharmacology and Analytical Cores, and in turn the transfer of resulting data to the repository and public portal of the Informatics Core; and 5) solicit input from the multidisciplinary consultants on the Internal Advisory Panel. The Administrative Core will establish and maintain a system of communication that fosters a close working relationship between the U54 Center members located in three different institutions. Specifically, this Core will support the communication link between the Steering Committee, the External Advisory Panel, and the U54 Center investigators. Within the U54 Center, the Administrative Core will coordinate regularly scheduled teleconferences and face-to-face meetings for the Core units, Center Leadership Committee, and the Executive Committee. There will be an annual meeting of the Internal Advisory Panel. The Steering Committee will hold separate annual face-to-face meetings to review progress of the NPDI Interaction Projects and plan for the subsequent year. Regarding the second task, the Administrative Core will lead the U54 investigators and NCCAM officials in developing the Best Practice guidelines to communicate clearly the U54 Center?s experience during the planning, preparation, implementation, and analysis of the Interaction Projects. A dissemination plan has been developed to ensure effective communication with the primary target audience, specifically researchers in clinical pharmacology, NP chemistry and pharmacognosy, and naturopathic and herbal medicine. With support from the communication experts in the Informatics Core, multi-modal outreach will be organized that includes publication of commentaries and white papers in scholarly journals, sponsoring of symposia or forums at professional society meetings, making Best Practice documents available via the U54 Center web-based public portal, and creating a blog site that will allow the tracking of thoughts and opinions of the researchers.

? DESCRIPTION (provided by applicant): As natural products (NP) sales increase, the risk of adverse NP-drug interactions (NPDIs) increases, yet the pharmacokinetic (PK) elucidation and clinical relevance of putative NPDIs remain elusive. Assessing the risk of NPDIs is more challenging than that of drug-drug interactions (DDIs), often due to relatively scant PK knowledge of individual NP constituents that perpetrate these interactions. The proposed U54 Center will develop a roadmap for NPDI research through a series of well-designed human in vitro and in vivo studies (termed Interaction Projects) on 4-6 priority NPs that pose a potential risk for clinically relevant NPDIs. A repository will be developed for the data generated from the Interaction Projects, and results will be communicated to various target audiences through a public portal. This U54 Center is composed of an accomplished team of investigators, including pharmacokineticists with expertise in NPDIs, complemented by expertise in NP chemistry, DDIs, and health information communication. In collaboration with the Steering Committee, an innovative strategy that combines a mechanistic approach with practical considerations (e.g., popularity of the NPs) will be used to select and prioritize 4-6 NPs for further investigation in te Interaction Projects. Mechanistic aspects include curated clinical NDPI data from the widely used Drug Interaction Database (DIDB) of the Informatics Core, structural alerts, and compelling preliminary clinical and in silico NPDI data. Once selected, the NPs will be entered into a Decision Tree for assessment of NPDI liability and probable significance of interactions with victim drugs. In parallel, the Pharmacology Core will develop detailed Statements of Work for the human in vitro and in vivo studies - while the Analytical Core will source, acquire, and characterize the selected NPs - for the Interaction Projects. Upon completion of each project, the Analytical Core will analyze the PK samples, and the Pharmacology Core will develop physiologically-based PK models for further assessment of the clinical relevance of the Interaction Project results. Throughout these projects, the Informatics Core will create a data repository embedded within a public portal web-based application named, NaPDI app, for Natural Product-Drug Interaction application. The repository, built using the DIDB framework, will allow researchers to access both raw data and summarized results. The U54 Center will also develop and share Best Practices recommended for the conduct of NPDI studies, based on the experience with and results from the Interaction Projects, with the research communities via the public portal. Effective dissemination of the Interaction Project results will be ensured through user experience and brand content studies with target audiences - researchers/practitioners and lay public - to refine the public portal content. The Informatics Core will ensure that the U54 Center's results are archived, organized, analyzed, and well-publicized, allowing for improved design of future NPDI research and ultimately, improved decisions on the optimal management of clinically relevant NPDIs.