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1.  Effects of chronic kidney disease and uremia on hepatic drug metabolism and transport 
Kidney international  2013;85(3):522-528.
The pharmacokinetics of non-renally cleared drugs in patients with chronic kidney disease is often unpredictable. Some of this variability may be due to alterations in the expression and activity of extra-renal drug metabolizing enzymes and transporters, primarily localized in the liver and intestine. Studies conducted in rodent models of renal failure have shown decreased mRNA and protein expression of many members of the cytochrome P450 enzyme (CYP) gene family and the ATP-Binding Cassette (ABC) and Solute Carrier (SLC) gene families of drug transporters. Uremic toxins interfere with transcriptional activation, cause down-regulation of gene expression mediated by proinflammatory cytokines, and directly inhibit the activity of the cytochrome P450s and drug transporters. While much has been learned about the effects of kidney disease on non-renal drug disposition, important questions remain regarding the mechanisms of these effects, as well as the interplay between drug metabolizing enzymes and drug transporters in the uremic milieu. In this review, we have highlighted the existing gaps in our knowledge and understanding of the impact of chronic kidney disease on non-renal drug clearance, and identified areas of opportunity for future research.
PMCID: PMC4276411  PMID: 24132209
2.  Specialty supplement use and biologic measures of oxidative stress and DNA damage 
Oxidative stress and resulting cellular damage have been suggested to play a role in the etiology of several chronic diseases, including cancer and cardiovascular disease. Identifying factors associated with reduced oxidative stress and resulting damage may guide future disease-prevention strategies.
In the VITamins And Lifestyle (VITAL) biomarker-study of 209 persons living in the Seattle area, we examined the association between current use of several specialty supplements and oxidative stress, DNA damage, and DNA repair capacity. Use of glucosamine, chondroitin, fish oil, methylsulfonylmethane (MSM), co-enzyme Q10 (CoQ10), ginseng, ginkgo, and saw palmetto was ascertained by a supplement inventory/interview, while use of fiber supplements was ascertained by questionnaire. Supplements used by more than 30 persons (glucosamine and chondroitin) were evaluated as the trend across number of pills/week (non-use, <14 pills/week, 14+ pills/week), while less-commonly used supplements were evaluated as use/non-use. Oxidative stress was measured by urinary 8-isoprostane and PGF2α concentrations using enzyme immunoassays (EIA), while lymphocyte DNA damage and DNA repair capacity were measured using the Comet assay. Multivariate-adjusted linear regression was used to model the associations between supplement use and oxidative stress/DNA damage.
Use of glucosamine (p-trend:0.01), chondroitin (p-trend:0.003), and fiber supplements (p:0.01) was associated with reduced PGF2α concentrations, while CoQ10 supplementation was associated with reduced baseline DNA damage (p:0.003).
Use of certain specialty supplements may be associated with reduced oxidative stress and DNA damage.
Further research is needed to evaluate the association between specialty supplement use and markers of oxidative stress and DNA damage.
PMCID: PMC3901246  PMID: 23917455
DNA damage; DNA repair; epidemiologic studies; oxidative stress; dietary supplements
3.  CYP3A5 Gene Variation Influences both Systemic and Intrarenal Tacrolimus Disposition 
We evaluated the hypothesis that CYP3A5 expression can affect intrarenal tacrolimus accumulation. An oral dose of tacrolimus was administered to 24 healthy volunteers who were selected based on their CYP3A5 genotype. Compared to CYP3A5 nonexpressors, expressors had a 1.6-fold higher oral tacrolimus clearance and 2.0- to 2.7-fold higher metabolite/parent AUC ratios for 31-DMT, 12-HT and 13-DMT. In addition, the apparent urinary tacrolimus clearance was 36% lower in CYP3A5 expressors, compared to nonexpressors. To explore the mechanism behind this observation, we developed a semi-physiological model of renal tacrolimus disposition and predicted that tacrolimus exposure in the renal epithelium of CYP3A5 expressors is 53% of that for CYP3A5 nonexpressors, when normalized to blood AUC. These data suggest that at steady state, intrarenal accumulation of tacrolimus, and its primary metabolites, will depend on the CYP3A5 genotype of the liver and kidneys. This may contribute to inter-patient differences in the risk of tacrolimus-induced nephrotoxicity.
PMCID: PMC4038024  PMID: 23073208
4.  Enhancement of hepatic 4-hydroxylation of 25-hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: Implications for drug-induced osteomalacia 
Long-term therapy with certain drugs, especially P450 inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4 and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25OHD3 were evaluated following exposure to P450 inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine and rifampin significantly increased the levels of CYP3A4 but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8-fold increase in formation of the major metabolite of 25OHD3, 4β,25(OH)2D3. This inductive effect was blocked by the addition of 6′,7′-dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK-2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1 or CYP27B1 expression. 24R,25(OH)2D3 was the predominant monohydroxy metabolite produced from 25OHD3, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4β,25(OH)2D3 was increased 60% (p < 0.01) after short-term rifampin administration. This was accompanied by a statistically significant reduction in plasma 1α,25(OH)2D3 (−10%; p = 0.03), and a non-significant change in 24R,25(OH)2D3 (−8%; p = 0.09) levels. Further analysis revealed a negative correlation between the increase in 4β,25(OH)2D3 and decrease in 1α,25(OH)2D3 levels. Examination of the plasma monohydroxy metabolite/25OHD3 ratios indicated selective induction of the CYP3A4-dependent 4β-hydroxylation pathway of 25OHD3 elimination. These results suggest that induction of hepatic CYP3A4 may be important in the etiology of drug-induced osteomalacia.
PMCID: PMC3609874  PMID: 23212742
Cytochrome P450 3A4; Cytochrome P450 24A1; 25-Hydroxyvitamin D3; Pregnane X receptor; Osteomalacia
5.  Interpreting Tacrolimus Concentrations During Pregnancy and Postpartum 
Transplantation  2013;95(7):908-915.
Pregnancy following solid organ transplantation, although considered high risk for maternal, fetal and neonatal complications, has been quite successful. Tacrolimus pharmacokinetic changes during pregnancy make interpretation of whole blood trough concentrations particularly challenging. There are multiple factors that can increase the fraction of unbound tacrolimus, including but not limited to low albumin concentration and low RBC count. The clinical titration of dosage to maintain whole blood tacrolimus trough concentrations in the usual therapeutic range can lead to elevated unbound concentrations and possibly toxicity in pregnant women with anemia and hypoalbuminemia. Measurement of plasma or unbound tacrolimus concentrations for pregnant women might better reflect the active form of the drug, though these are technically-challenging and often unavailable in usual clinical practice. Tacrolimus crosses the placenta with in utero exposure being approximately 71% of maternal blood concentrations. The lower fetal blood concentrations are likely due to active efflux transport of tacrolimus from the fetus toward the mother by placental P-glycoprotein. To date, tacrolimus has not been linked to congenital malformations, but can cause reversible nephrotoxicity and hyperkalemia in the newborn. In contrast, very small amounts of tacrolimus are excreted in the breast milk and are unlikely to elicit adverse effects in the nursing infant.
PMCID: PMC3637974  PMID: 23274970
tacrolimus; pregnancy; nephrotoxicity; pharmacokinetics; protein binding
6.  CYP3A5 Gene Variation Influences Cyclosporine A Metabolite Formation and Renal Cyclosporine Disposition 
Transplantation  2013;95(6):821-827.
Higher concentrations of AM19 and AM1c9, secondary metabolites of cyclosporine A (CsA), have been associated with nephrotoxicity in organ transplant patients. The risk of renal toxicity may depend upon the accumulation of CsA and its metabolites in the renal tissue. We evaluated the hypothesis that CYP3A5 genotype, and inferred enzyme expression, affects systemic CsA metabolite exposure and intra-renal CsA accumulation.
An oral dose of CsA was administered to 24 healthy volunteers who were selected based on their CYP3A5 genotype. CsA and its six main metabolites in whole blood and urine were measured by LC-MS. In vitro incubations of CsA, AM1, AM9 and AM1c with recombinant CYP3A4 and CYP3A5 were performed to evaluate the formation pathways of AM19 and AM1c9.
The mean CsA oral clearance was similar between CYP3A5 expressors and nonexpressors. However, compared to CYP3A5 nonexpressors, the average blood AUC for AM19 and AM1c9 was 47.4% and 51.3% higher in CYP3A5 expressors (P = 0.040 and 0.011, respectively), corresponding to 30% higher AUCmetabolite/AUCCsA ratios for AM19 and AM1c9 in CYP3A5 expressors. The mean apparent urinary CsA clearance, based on a 48-hour collection, was 20.4% lower in CYP3A5 expressors compared to CYP3A5 nonexpressors (4.2 ± 1.0 and 5.3 ± 1.3 mL/min, respectively, P = 0.037), which is suggestive of CYP3A5-dependent intra-renal CsA metabolism.
At steady-state, intra-renal accumulation of CsA and its secondary metabolites should depend on the CYP3A5 genotype of the liver and kidneys. This may contribute to inter-patient variability in the risk of CsA-induced nephrotoxicity.
PMCID: PMC3604156  PMID: 23354298
Cyclosporine A; CYP3A5 genotype; secondary metabolites; chronic calcineurin inhibitor nephrotoxicity; intra-renal metabolism
7.  Aspirin use and knowledge in the community: a population- and health facility based survey for measuring local health system performance 
Little is known about the relationship between cardiovascular risk, disease and actual use of aspirin in the community.
The Measuring Disparities in Chronic Conditions (MDCC) study is a community and health facility-based survey designed to track disparities in the delivery of health interventions for common chronic diseases. MDCC includes a survey instrument designed to collect detailed information about aspirin use. In King County, WA between 2011 and 2012, we surveyed 4633 white, African American, or Hispanic adults (45% home address-based sample, 55% health facility sample). We examined self-reported counseling on, frequency of use and risks of aspirin for all respondents. For a subgroup free of CAD or cerebral infarction that underwent physical examination, we measured 10-year coronary heart disease risk and blood salicylate concentration.
Two in five respondents reported using aspirin routinely while one in five with a history of CAD or cerebral infarction and without contraindication did not report routine use of aspirin. Women with these conditions used less aspirin than men (65.0% vs. 76.5%) and reported more health problems that would make aspirin unsafe (29.4% vs. 21.2%). In a subgroup undergoing phlebotomy a third of respondents with low cardiovascular risk used aspirin routinely and only 4.6% of all aspirin users had no detectable salicylate in their blood.
In this large urban county where health care delivery should be of high quality, there is insufficient aspirin use among those with high cardiovascular risk or disease and routine aspirin use by many at low risk. Further efforts are needed to promote shared-decision making between patients and clinicians as well as inform the public about appropriate use of routine aspirin to reduce the burden of atherosclerotic vascular disease.
PMCID: PMC3922250  PMID: 24507089
Aspirin; Prevention; Coronary disease
8.  Sulforaphane is not an effective antagonist of the human Pregnane X-Receptor in vivo 
Toxicology and applied pharmacology  2012;266(1):122-131.
Sulforaphane (SFN), is an effective in vitro antagonist of ligand activation of the human pregnane and xenobiotic receptor (PXR). PXR mediated CYP3A4 up-regulation is implicated in adverse drug-drug interactions making identification of small molecule antagonists a desirable therapeutic goal. SFN is not an antagonist to mouse or rat PXR in vitro; thus, normal rodent species are not suitable as in vivo models for human response. To evaluate whether SFN can effectively antagonize ligand activation of human PXR in vivo, a three-armed, randomized, crossover trial was conducted with 24 healthy adults. The potent PXR ligand – rifampicin (300 mg/d) was given alone for 7 days in arm 1, or in daily combination with 450 µmoles SFN (Broccoli Sprout extract) in arm 2; SFN was given alone in arm 3. Midazolam as an in vivo phenotype marker of CYP3A was administered before and after each treatment arm. Rifampicin alone decreased midazolam AUC by 70%, indicative of the expected increase in CYP3A4 activity. Co-treatment with SFN did not reduce CYP3A4 induction. Treatment with SFN alone also did not affect CYP3A4 activity in the cohort as a whole, although in the subset with the highest basal CYP3A4 activity there was a statistically significant increase in midazolam AUC (i.e., decrease in CYP3A4 activity). A parallel study in humanized PXR mice yielded similar results. The parallel effects of SFN between humanized PXR mice and human subjects demonstrate the predictive value of humanized mouse models in situations where species differences in ligand-receptor interactions preclude the use of a native mouse model for studying human ligand-receptor pharmacology.
PMCID: PMC3538144  PMID: 23153560
Sulforaphane; cytochrome P450; CYP3A4; midazolam; pregnane X-receptor; NR1I2; phase I clinical trial; humanized PXR mice; isolated hepatocytes
9.  Innovations in preclinical biology: ex vivo engineering of a human kidney tissue microperfusion system 
Stem Cell Research & Therapy  2013;4(Suppl 1):S17.
Kidney disease is a public health problem that affects more than 20 million people in the US adult population, yet little is understood about the impact of kidney disease on drug disposition. Consequently there is a critical need to be able to model the human kidney and other organ systems, to improve our understanding of drug efficacy, safety, and toxicity, especially during drug development. The kidneys in general, and the proximal tubule specifically, play a central role in the elimination of xenobiotics. With recent advances in molecular investigation, considerable information has been gathered regarding the substrate profiles of the individual transporters expressed in the proximal tubule. However, we have little knowledge of how these transporters coupled with intracellular enzymes and influenced by metabolic pathways form an efficient secretory and reabsorptive mechanism in the renal tubule. Proximal tubular secretion and reabsorption of xenobiotics is critically dependent on interactions with peritubular capillaries and the interstitium. We plan to robustly model the human kidney tubule interstitium, utilizing an ex vivo three-dimensional modular microphysiological system with human kidney-derived cells. The microphysiological system should accurately reflect human physiology, be usable to predict renal handling of xenobiotics, and should assess mechanisms of kidney injury, and the biological response to injury, from endogenous and exogenous intoxicants.
PMCID: PMC4029535  PMID: 24564863
10.  Pharmacokinetics of Tacrolimus during Pregnancy 
Therapeutic drug monitoring  2012;34(6):660-670.
Information on the pharmacokinetics of tacrolimus during pregnancy is limited to case reports despite the increasing number of pregnant women being prescribed tacrolimus for immunosuppression.
Blood, plasma and urine samples were collected over one steady-state dosing interval from women treated with oral tacrolimus during early to late pregnancy (n = 10) and postpartum (n = 5). Total and unbound tacrolimus as well as metabolite concentrations in blood and plasma were assayed by a validated LC/MS/MS method. A mixed effect linear model was used for comparison across gestational age and using postpartum as the reference group.
The mean oral clearance (CL/F) based on whole blood tacrolimus concentration was 39% higher during mid- and late-pregnancy compared to postpartum (47.4 ± 12.6 vs. 34.2 ± 14.8 L/h, P < 0.03). Tacrolimus free fraction increased by 91% in plasma (fP) and by 100% in blood (fB) during pregnancy (P = 0.0007 and 0.002, respectively). Increased fP was inversely associated with serum albumin concentration (r = − 0.7, P = 0.003), which decreased by 27% during pregnancy. Pregnancy related changes in fP and fB contributed significantly to the observed gestational increase in tacrolimus whole blood CL/F (r2 = 0.36 and 0.47 respectively, P < 0.01). In addition, tacrolimus whole blood CL/F was inversely correlated with both hematocrit and red blood cell counts, suggesting that binding of tacrolimus to erythrocytes restricts its availability for metabolism. Treating physicians increased tacrolimus dosages in study participants during pregnancy by an average of 45% in order to maintain tacrolimus whole blood trough concentrations in the therapeutic range. This led to striking increases in unbound tacrolimus trough concentrations and unbound AUC, by 112% and 173%, respectively during pregnancy (P = 0.02 and 0.03, respectively).
Tacrolimus pharmacokinetics are altered during pregnancy. Dose adjustment to maintain whole blood tacrolimus concentration in the usual therapeutic range during pregnancy increases circulating free drug concentrations, which may impact clinical outcomes.
PMCID: PMC3498613  PMID: 23007747
Pregnancy; tacrolimus dose adjustment; serum albumin; hematocrit; tacrolimus unbound concentration
11.  Pulmonary Administration of a Water-Soluble Curcumin Complex Reduces Severity of Acute Lung Injury 
Local or systemic inflammation can result in acute lung injury (ALI), and is associated with capillary leakage, reduced lung compliance, and hypoxemia. Curcumin, a plant-derived polyphenolic compound, exhibits potent anti-inflammatory properties, but its poor solubility and limited oral bioavailability reduce its therapeutic potential. A novel curcumin formulation (CDC) was developed by complexing the compound with hydroxypropyl-γ-cyclodextrin (CD). This results in greatly enhanced water solubility and stability that facilitate direct pulmonary delivery. In vitro studies demonstrated that CDC increased curcumin’s association with and transport across Calu-3 human airway epithelial cell monolayers, compared with uncomplexed curcumin solubilized using DMSO or ethanol. Importantly, Calu-3 cell monolayer integrity was preserved after CDC exposure, whereas it was disrupted by equivalent uncomplexed curcumin solutions. We then tested whether direct delivery of CDC to the lung would reduce severity of ALI in a murine model. Fluorescence microscopic examination revealed an association of curcumin with cells throughout the lung. The administration of CDC after LPS attenuated multiple markers of inflammation and injury, including pulmonary edema and neutrophils in bronchoalveolar lavage fluid and lung tissue. CDC also reduced oxidant stress in the lungs and activation of the proinflammatory transcription factor NF-κB. These results demonstrate the efficacy of CDC in a murine model of lung inflammation and injury, and support the feasibility of developing a lung-targeted, curcumin-based therapy for the treatment of patients with ALI.
PMCID: PMC3488693  PMID: 22312018
cyclodextrin; LPS; turmeric; Calu-3; oxidative stress; TEER
12.  Use of Glucosamine and Chondroitin in Relation to Mortality 
European journal of epidemiology  2012;27(8):593-603.
Glucosamine and chondroitin are products commonly used by older adults in the US and Europe. There is limited evidence that they have anti-inflammatory properties, which could provide risk reduction of several diseases. However, data on their long-term health effects is lacking.
To evaluate whether use of glucosamine and chondroitin are associated with cause-specific and total mortality.
Participants (n = 77 510) were members of a cohort study of Washington State (US) residents aged 50–76 y who entered the cohort in 2000–2002 by completing a baseline questionnaire that included questions on glucosamine and chondroitin use. Participants were followed for mortality through 2008 (n = 5362 deaths). Hazard ratios for death adjusted for multiple covariates were estimated using Cox models.
Current (baseline) glucosamine and chondroitin use were associated with a decreased risk of total mortality compared to never use. The adjusted hazard ratio (HR) associated with current use of glucosamine (with or without chondroitin) was 0.82 (95% CI 0.75–0.90) and 0.86 (95% CI 0.78–0.96) for chondroitin (included in two-thirds of glucosamine supplements). Current use of glucosamine was associated with a significant decreased risk of death from cancer (HR 0.87 95% CI 0.76–0.98) and with a large risk reduction for death from respiratory diseases (HR 0.59 95% CI 0.41–0.83).
Use of glucosamine with or without chondroitin was associated with reduced total mortality and with reductions of several broad causes of death. Although bias cannot be ruled out, these results suggest that glucosamine may provide some mortality benefit.
PMCID: PMC3557824  PMID: 22828954
glucosamine; chondroitin; supplements; mortality; cohort; cancer
13.  Is ethnicity associated with morphine's side effects in children? morphine pharmacokinetics, analgesic response and side effects in children having tonsillectomy 
Paediatric Anaesthesia  2012;22(7):669-675.
To examine whether morphine pharmacokinetics (PK) and/or genetic polymorphisms in opioid-related genes, underlie differences in analgesic response and side effects to morphine in Latino (L) vs non-Latino Caucasian (NL) children.
Morphine has high interindividual variability in its analgesic response and side effects profile. Earlier studies suggest that morphine response may vary by race and ethnicity.
Prospective cohort study in L and NL children, 3–17 years of age comparing pain scores, occurrence of side effects, plasma morphine, morphine-6-and morphine-3-glucuronide concentrations measured after a single morphine IV bolus administration. Non-compartmental pharmacokinetic analysis and genotyping for 28 polymorphisms in 8 genes (UGT1A8, UGT2B7, ABCB1, COMT, STAT6, MC1R, OPRM1, and ARRB2) were done.
We enrolled 68 children (33 L, 35 NL). There were no differences in pain scores or need for rescue analgesia. Statistically significant differences in the occurrence of side effects were documented: While 58% of L children experienced at least one side effect only 20% of NL did (p=0.001). Pruritus was 4 times (p=0.006) and emesis 7 times (p=0.025) more frequent in L compared to NL. PK parameters were similar between groups. None of the assessed polymorphisms mediated the association between ethnicity and side effects.
We found statistically significant differences in occurrence of side effects after morphine administration between L and NL children. Neither differences in morphine or metabolite concentrations, nor the genetic polymorphisms examined, explain these findings. Studies are needed to further investigate reasons for the increase in morphine side effects by Latino ethnicity.
PMCID: PMC3366036  PMID: 22486937
14.  MDR1 (ABCB1) G1199A (Ser400Asn) polymorphism alters transepithelial permeability and sensitivity to anticancer agents 
P-glycoprotein (P-gp), encoded by MDR1 (or ABCB1), is important in anticancer drug delivery and resistance. We evaluated alterations in P-gp-mediated transport of anticancer agents due to the MDR1 G1199A polymorphism.
Using stable recombinant epithelial cells expressing wild-type (MDR1wt) or G1199A (MDR11199A), anticancer drug sensitivity and transepithelial permeability were evaluated.
The recombinant cells MDR1wt and MDR11199A displayed comparable doxorubicin resistance. However, MDR11199A cells displayed greater resistance to vinblastine, vincristine, paclitaxel, and VP-16 (11-, 2.9-, 1.9-, and 2.9-fold, respectively). Alterations in transepithelial permeability paralleled these changes. Efflux of doxorubicin was similar between MDR1wt- and MDR11199A-expressing cells, while P-gp-mediated transport was greater for vinblastine and vincristine in MDR11199A cells (2.9- and 2.0-fold, respectively).
The occurrence and magnitude of the MDR1 G1199A effect is drug specific. Overall, the MDR1 G1199A polymorphism may impact anticancer efficacy through modulation of drug distribution and delivery to target tumor cells.
PMCID: PMC3489915  PMID: 19123050
MDR1; ABCB1; P-Glycoprotein; Pharmacogenomics; Cancer chemotherapy; Transepithelial permeability
15.  A Prospective Open-Label Trial of Etanercept as Adjunctive Therapy for Kawasaki Disease 
The Journal of pediatrics  2010;157(6):960-966.e1.
To determine the safety and pharmacokinetics of etanercept (etanercept, Amgen inc. Thousands Oak, CA) a tumor necrosis factor-α (TNF-α) receptor blocker, in children with acute Kawasaki disease (KD). Standard therapy of acute KD includes IVIG and high dose aspirin, but a substantial number of patients are refractory and require additional treatment. TNF-α is elevated in children with KD suggesting a role for etanercept in treatment.
Study design
We performed a prospective open label trial of etanercept in patients with KD (6 months to 5 years, n = 17) meeting clinical criteria and with fever ≤ 10 days. All received IVIG and ASA. They received etanercept immediately after IVIG infusion and then weekly times two. For initial safety evaluation, the first five patients received 0.4 mg/kg/dose. Subsequent subjects received 0.8 mg/kg/dose.
Fifteen patients completed the study. Pharmacokinetics were similar to older children in published series. No serious adverse events related to etanercept occurred. No patient demonstrated prolonged or recrudescent fever requiring retreatment with IVIG. No patient showed an increase in coronary artery diameter, new coronary artery dilation /cardiac dysfunction.
Etanercept appears to be safe and well tolerated in children with KD. The data support performance of a placebo-controlled trial.
PMCID: PMC2970727  PMID: 20667551
Kawasaki’s disease; IVIG; TNF-α antagonist; Etanercept; Coronary artery aneurysm
16.  Pharmacogenomic Trial Design: Use of a PK/PD Model to Explore Warfarin Dosing Interventions through Clinical Trial Simulation 
Pharmacogenetics and Genomics  2009;19(12):965-971.
Variants of two genes, CYP2C9 and VKORC1, explain approximately 30% of variability in warfarin maintenance dose requirements. However, the clinical utility of using this information in addition to clinical and demographic data (‘pharmacogenomic-guidance’) is unclear, as few comparative clinical trials have been conducted to date. The objective of this study was to explore the incremental effect of pharmacogenomic-guided warfarin dosing under various conditions using clinical trial simulation.
We utilized an existing PK/PD model to perform clinical trial simulations of pharmacogenomic-guided versus standard of care warfarin therapy. The primary outcome was the percentage of patient time spent in therapeutic range over the first month of therapy. We assessed the influence of the frequency of INR monitoring, and the use of a loading dose and dose increase delay in patients with CYP2C9 variants.
Pharmacogenomic guidance resulted in a 3-4% absolute increase in time spent in therapeutic range over the first month of therapy compared to standard of care. The improvement in time in range was greater when the frequency of INR monitoring in both arms was assumed to be lower. The absolute difference increased to 6-8% with use of a loading dose and dose increase delay in patients with a CYP2C9 variant.
Our initial results imply that pharmacogenomic-guided warfarin dosing may be more useful in settings with less intensive patient follow-up, and when adjustments are made for slower therapeutic response in patients with a CYP2C9 variant. Further PK/PD model development may be useful for warfarin pharmacogenomic trial design.
PMCID: PMC3164437  PMID: 19881396
warfarin; trial simulation; pharmacogenomic; pharmacokinetic; pharmacodynamic
17.  Effects of Garlic on Cytochromes P450 2C9- and 3A4-Mediated Drug Metabolism in Human Hepatocytes 
Scientia pharmaceutica  2010;78(3):473-481.
Several reports suggest garlic supplements may inhibit the metabolism of cytochrome P450 (CYP) 2C9 and CYP3A4 substrates, such as warfarin and saquinavir. To characterize the effects of garlic extract on CYP2C9 and CYP3A4 enzyme activity immortalized human hepatocytes (Fa2N-4 cells) were exposed to garlic extract (0–200 μg/mL). CYP2C9 and CYP3A4 enzyme activities were evaluated in parallel with enzymatic activities, expression of respective RNA transcripts was also assessed.
Exposure to increasing concentrations of garlic extract led to progressive reduction in Fa2N-4 CYP2C9 activity as detected by diclofenac hydroxylation. CYP2C9 mRNA expression also revealed a concentration-dependent reduction. Greater than 90% reduction in CYP2C9 activity was observed following four days of exposure to 50 μg/mL garlic extract. In contrast, exposure to garlic extract had no effect on the CYP3A4 enzymatic activity or RNA transcript concentration in Fa2N-4. Therefore, suppression of CYP2C9 expression and activity is a heretofore unrecognized mechanism by which garlic extract may modulate CYP activity. Exposure of hepatocytes to garlic extract may reduce the expression and activity of CYP2C9 with no detectible effects on CYP3A4.
PMCID: PMC2951329  PMID: 20936048
Garlic; Cytochrome P450 2C9; Drug Interactions
18.  Effects of Garlic on Cytochromes P450 2C9- and 3A4-Mediated Drug Metabolism in Human Hepatocytes 
Scientia Pharmaceutica  2010;78(3):473-481.
Several reports suggest garlic supplements may inhibit the metabolism of cytochrome P450 (CYP) 2C9 and CYP3A4 substrates, such as warfarin and saquinavir. To characterize the effects of garlic extract on CYP2C9 and CYP3A4 enzyme activity immortalized human hepatocytes (Fa2N-4 cells) were exposed to garlic extract (0–200 μg/mL). CYP2C9 and CYP3A4 enzyme activities were evaluated in parallel with enzymatic activities, expression of respective RNA transcripts was also assessed.
Exposure to increasing concentrations of garlic extract led to progressive reduction in Fa2N-4 CYP2C9 activity as detected by diclofenac hydroxylation. CYP2C9 mRNA expression also revealed a concentration-dependent reduction. Greater than 90% reduction in CYP2C9 activity was observed following four days of exposure to 50 μg/mL garlic extract. In contrast, exposure to garlic extract had no effect on the CYP3A4 enzymatic activity or RNA transcript concentration in Fa2N-4. Therefore, suppression of CYP2C9 expression and activity is a heretofore unrecognized mechanism by which garlic extract may modulate CYP activity. Exposure of hepatocytes to garlic extract may reduce the expression and activity of CYP2C9 with no detectible effects on CYP3A4.
PMCID: PMC2951329  PMID: 20936048
Garlic; Cytochrome P450 2C9; Drug Interactions
19.  Effects of Cranberry Juice on Pharmacokinetics of β-Lactam Antibiotics following Oral Administration▿  
Cranberry juice consumption is often recommended along with low-dose oral antibiotics for prophylaxis for recurrent urinary tract infection (UTI). Because multiple membrane transporters are involved in the intestinal absorption and renal excretion of β-lactam antibiotics, we evaluated the potential risk of pharmacokinetic interactions between cranberry juice and the β-lactams amoxicillin (amoxicilline) and cefaclor. The amoxicillin-cranberry juice interaction was investigated in 18 healthy women who received on four separate occasions a single oral test dose of amoxicillin at 500 mg and 2 g with or without cranberry juice cocktail (8 oz) according to a crossover design. A parallel cefaclor-cranberry juice interaction study was also conducted in which 500 mg cefaclor was administered with or without cranberry juice cocktail (12 oz). Data were analyzed by noncompartmental methods and nonlinear mixed-effects compartmental modeling. We conclude that the concurrent use of cranberry juice has no significant effect on the extent of oral absorption or the renal clearance of amoxicillin and cefaclor. However, delays in the absorption of amoxicillin and cefaclor were observed. These results suggest that the use of cranberry juice at usual quantities as prophylaxis for UTI is not likely to alter the pharmacokinetics of these two oral antibiotics.
PMCID: PMC2704661  PMID: 19398645
20.  Comparison of Blood and Brain Mercury Levels in Infant Monkeys Exposed to Methylmercury or Vaccines Containing Thimerosal 
Environmental Health Perspectives  2005;113(8):1015-1021.
Thimerosal is a preservative that has been used in manufacturing vaccines since the 1930s. Reports have indicated that infants can receive ethylmercury (in the form of thimerosal) at or above the U.S. Environmental Protection Agency guidelines for methylmercury exposure, depending on the exact vaccinations, schedule, and size of the infant. In this study we compared the systemic disposition and brain distribution of total and inorganic mercury in infant monkeys after thimerosal exposure with those exposed to MeHg. Monkeys were exposed to MeHg (via oral gavage) or vaccines containing thimerosal (via intramuscular injection) at birth and 1, 2, and 3 weeks of age. Total blood Hg levels were determined 2, 4, and 7 days after each exposure. Total and inorganic brain Hg levels were assessed 2, 4, 7, or 28 days after the last exposure. The initial and terminal half-life of Hg in blood after thimerosal exposure was 2.1 and 8.6 days, respectively, which are significantly shorter than the elimination half-life of Hg after MeHg exposure at 21.5 days. Brain concentrations of total Hg were significantly lower by approximately 3-fold for the thimerosal-exposed monkeys when compared with the MeHg infants, whereas the average brain-to-blood concentration ratio was slightly higher for the thimerosal-exposed monkeys (3.5 ± 0.5 vs. 2.5 ± 0.3). A higher percentage of the total Hg in the brain was in the form of inorganic Hg for the thimerosal-exposed monkeys (34% vs. 7%). The results indicate that MeHg is not a suitable reference for risk assessment from exposure to thimerosal-derived Hg. Knowledge of the toxicokinetics and developmental toxicity of thimerosal is needed to afford a meaningful assessment of the developmental effects of thimerosal-containing vaccines.
PMCID: PMC1280342  PMID: 16079072
brain and blood distribution; elimination half-life; ethylmercury; infant nonhuman primates; methylmercury; thimerosal
21.  Development and characterization of a recombinant madin-darby canine kidney cell line that expresses rat multidrug resistance-associated protein 1 (rMRP1) 
AAPS PharmSci  2004;6(1):77-85.
Multidrug resistance-associated protein 1 (MRP1) is one of the major proteins shown to mediate efflux transport of a broad range of antitumor drugs, glucuronide conjugates, and glutathione, in addition to endogenous substrates. Significant differences in substrate selectivity were reported for murine and human MRP1. As preclinical drug disposition and pharmacokinetics studies are often conducted in rats, we have recently cloned the rat MRP1 (rMRP1) and demonstrated that rMRP1 expressed in transfected cells effluxes calcein, a commonly used fluorescence substrate for human MRP1. To further characterize the rat ortholog of MRP1, we isolated a cell line stably expressing recombinant rMRP1. These cells were tested for their ability to transport calcein and a range of chemotherapeutic drugs. Our results showed that cells expressing rMRP1 consistently efflux calcein at a rate 5-fold greater than control cells. The rMRP1 transfected cells, like their human ortholog, can confer drug resistance to vinca alkaloid (vinblastine and vincristine) and anthracycline drugs (daunorubcin and doxorubicin), and the resistance conferred by the MRP1 can be partially abolished by the MRP-specific inhibitors. The transepithelial permeability due to rMRP1 expression in differentiated Madin-Darby canine kidney cells (MDCK) cells was also investigated. The MRP1 transport activity is directional, as demonstrated by directional vinblastine transport. Collectively, our results demonstrate that the cellular expression of rMRP1, like its human ortholog, could confer resistance to anticancer drugs.
PMCID: PMC2750943  PMID: 18465260
rat; MRP1; drug resistance; chemotherapeutic agents; cytotoxicity; transport; ATP-binding cassette; transwell
22.  Cloning and characterization of the rat multidrug resistance-associated protein 1 
AAPS PharmSci  2002;4(3):31-37.
Multidrug resistance-associated protein 1 (MRP1) was originally shown to confer resistance of human tumor cells to a broad range of natural product anticancer drugs. MRP1 has also been shown to mediate efflux transport of glutathione and glucuronide conjugates of drugs and endogenous substrates. An ortholog of MRP1 in the mouse has been cloned and characterized. Significant functional differences between murine and human MRP1 have been noted. Since drug disposition and pharmacology studies often are conducted in rats, there is a need to clone and characterize the rat ortholog of MRP1. We isolated a rat MRP1 (rMRP1) cDNA from rat brain astrocytes, characterized its coding sequences, and verified the transport activity of the protein expressed in MRP1 cDNA-transfected Madin-Darby canine kidney (MDCK) cells. Our results showed that rMRP1 has a coding sequence of 4599 bp, which predicts a polypeptide of 1533 amino acids with an apparent molecular weight of 190 kd by Western immunoblot analysis. rMRP1-transfected MDCK cells are capable of efflux transport of a fluorescent MRP1 marker-calcein-that is inhibitable by known MRP1 inhibitors, indomethacin, and MK571. Sequence analysis indicates that rMRP1 is more closely related to mouse MRP1 than human MRP1.
PMCID: PMC2751354  PMID: 12423064
multidrug resistance gene; ABC transporter; MRP1; cloning; functional characterization

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