Identification of Novel High-Affinity Substrates of OCT1 Using Machine Learning-Guided Virtual Screening and Experimental Validation.

OCT1 is the most highly expressed cation transporter in the liver and affects pharmacokinetics and pharmacodynamics. Newly marketed drugs have previously been screened as potential OCT1 substrates and verified by virtual docking. Here, we used machine learning with transport experiment data to predict OCT1 substrates based on classic molecular descriptors, pharmacophore features, and extended-connectivity fingerprints and confirmed them by in vitro uptake experiments. We virtually screened a database of more than 1000 substances. Nineteen predicted substances were chosen for in vitro testing. Sixteen of the 19 newly tested substances (85%) were confirmed as, mostly strong, substrates, including edrophonium, fenpiverinium, ritodrine, and ractopamine. Even without a crystal structure of OCT1, machine learning algorithms predict substrates accurately and may contribute not only to a more focused screening in drug development but also to a better molecular understanding of OCT1 in general.

Differences in Metformin and Thiamine Uptake between Human and Mouse Organic Cation Transporter 1: Structural Determinants and Potential Consequences for Intrahepatic Concentrations.

The most commonly used oral antidiabetic drug, metformin, is a substrate of the hepatic uptake transporter OCT1 (gene name SLC22A1). However, OCT1 deficiency leads to more pronounced reductions of metformin concentrations in mouse than in human liver. Similarly, the effects of OCT1 deficiency on the pharmacokinetics of thiamine were reported to differ between human and mouse. Here, we compared the uptake characteristics of metformin and thiamine between human and mouse OCT1 using stably transfected human embryonic kidney 293 cells. The affinity for metformin was 4.9-fold lower in human than in mouse OCT1, resulting in a 6.5-fold lower intrinsic clearance. Therefore, the estimated liver-to-blood partition coefficient is only 3.34 in human compared with 14.4 in mouse and may contribute to higher intrahepatic concentrations in mice. Similarly, the affinity for thiamine was 9.5-fold lower in human than in mouse OCT1. Using human-mouse chimeric OCT1, we showed that simultaneous substitution of transmembrane helices TMH2 and TMH3 resulted in the reversal of affinity for metformin. Using homology modeling, we suggest several explanations, of which a different interaction of Leu155 (human TMH2) compared with Val156 (mouse TMH2) with residues in TMH3 had the strongest experimental support. In conclusion, the contribution of human OCT1 to the cellular uptake of thiamine and especially of metformin may be much lower than that of mouse OCT1. This may lead to an overestimation of the effects of OCT1 on hepatic concentrations in humans when using mouse as a model. In addition, comparative analyses of human and mouse orthologs may help reveal mechanisms of OCT1 transport. SIGNIFICANCE STATEMENT: OCT1 is a major hepatic uptake transporter of metformin and thiamine, but this study reports strong differences in the affinity for both compounds between human and mouse OCT1. Consequently, intrahepatic metformin concentrations could be much higher in mice than in humans, impacting metformin actions and representing a strong limitation of using rodent animal models for predictions of OCT1-related pharmacokinetics and efficacy in humans. Furthermore, OCT1 transmembrane helices TMH2 and TMH3 were identified to confer the observed species-specific differences in metformin affinity.

Variability and Heritability of Thiamine Pharmacokinetics With Focus on OCT1 Effects on Membrane Transport and Pharmacokinetics in Humans.

Thiamine is substrate of the hepatic uptake transporter organic cation transporter 1 (OCT1), and pathological lipid metabolism was associated with OCT1-dependent thiamine transport. However, it is unknown whether clinical pharmacokinetics of thiamine is modulated by OCT1 genotype. We analyzed thiamine transport in vitro, thiamine blood concentrations after high-dose and low-dose (nutritional) intake, and heritability of thiamine and thiamine-phosphate blood concentrations. The variant OCT1*2 had reduced and OCT1*3 to OCT1*6 had deficient thiamine uptake activity. However, pharmacokinetics of thiamine did not differ depending on OCT1 genotype. Further studies in primary human hepatocytes indicated that several cation transporters, including OCT1, OCT3, and THTR-2, contribute to hepatic uptake of thiamine. As much as 54% of the variation in thiamine and 75% in variation of thiamine monophosphate plasma concentrations was determined by heritable factors. Apparently, thiamine is not useful as a probe drug for OCT1 activity, but the high heritability, particularly of thiamine monophosphate, may stimulate further genomic research.

An in vitro study on interaction of anisodine and monocrotaline with organic cation transporters of the SLC22 and SLC47 families.

Current study systematically investigated the interaction of two alkaloids, anisodine and monocrotaline, with organic cation transporter OCT1, 2, 3, MATE1 and MATE2-K by using in vitro stably transfected HEK293 cells. Both anisodine and monocrotaline inhibited the OCTs and MATE transporters. The lowest IC50 was 12.9 µmol·L-1 of anisodine on OCT1 and the highest was 1.8 mmol·L-1 of monocrotaline on OCT2. Anisodine was a substrate of OCT2 (Km = 13.3 ± 2.6 µmol·L-1 and Vmax = 286.8 ± 53.6 pmol/mg protein/min). Monocrotaline was determined to be a substrate of both OCT1 (Km = 109.1 ± 17.8 µmol·L-1, Vmax = 576.5 ± 87.5 pmol/mg protein/min) and OCT2 (Km = 64.7 ± 14.8 µmol·L-1, Vmax = 180.7 ± 22.0 pmol/mg protein/min), other than OCT3 and MATE transporters. The results indicated that OCT2 may be important for renal elimination of anisodine and OCT1 was responsible for monocrotaline uptake into liver. However neither MATE1 nor MATE2-K could facilitate transcellular transport of anisodine and monocrotaline. Accumulation of these drugs in the organs with high OCT1 expression (liver) and OCT2 expression (kidney) may be expected.

Population nutrikinetics of green tea extract.

Green tea polyphenols may contribute to the prevention of cancer and other diseases. To learn more about the pharmacokinetics and interindividual variation of green tea polyphenols after oral intake in humans we performed a population nutrikinetic study of standardized green tea extract. 84 healthy participants took green tea extract capsules standardized to 150 mg epigallocatechin-gallate (EGCG) twice a day for 5 days. On day 5 catechin plasma concentrations were analyzed using non-compartmental and population pharmacokinetic methods. A strong between-subject variability in catechin pharmacokinetics was found with maximum plasma concentrations varying more than 6-fold. The AUCs of EGCG, EGC and ECG were 877.9 (360.8-1576.5), 35.1 (8.0-87.4), and 183.6 (55.5-364.6) h*µg/L respectively, and the elimination half lives were 2.6 (1.8-3.8), 3.9 (0.9-10.7) and 1.8 (0.8-2.9) h, respectively. Genetic polymorphisms in genes of the drug transporters MRP2 and OATP1B1 could at least partly explain the high variability in pharmacokinetic parameters. The observed variability in catechin plasma levels might contribute to interindividual variation in benefical and adverse effects of green tea polyphenols. Our data could help to gain a better understanding of the causes of variability of green tea effects and to improve the design of studies on the effects of green tea polyphenols in different health conditions. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01360320.

Rationale, objectives, and design of the EUTrigTreat clinical study: a prospective observational study for arrhythmia risk stratification and assessment of interrelationships among repolarization markers and genotype.

AIMS: The EUTrigTreat clinical study has been designed as a prospective multicentre observational study and aims to (i) risk stratify patients with an implantable cardioverter defibrillator (ICD) for mortality and shock risk using multiple novel and established risk markers, (ii) explore a link between repolarization biomarkers and genetics of ion (Ca(2+), Na(+), K(+)) metabolism, (iii) compare the results of invasive and non-invasive electrophysiological (EP) testing, (iv) assess changes of non-invasive risk stratification tests over time, and (v) associate arrythmogenomic risk through 19 candidate genes. METHODS AND RESULTS: Patients with clinical ICD indication are eligible for the trial. Upon inclusion, patients will undergo non-invasive risk stratification, including beat-to-beat variability of repolarization (BVR), T-wave alternans, T-wave morphology variables, ambient arrhythmias from Holter, heart rate variability, and heart rate turbulence. Non-invasive or invasive programmed electrical stimulation will assess inducibility of ventricular arrhythmias, with the latter including recordings of monophasic action potentials and assessment of restitution properties. Established candidate genes are screened for variants. The primary endpoint is all-cause mortality, while one of the secondary endpoints is ICD shock risk. A mean follow-up of 3.3 years is anticipated. Non-invasive testing will be repeated annually during follow-up. It has been calculated that 700 patients are required to identify risk predictors of the primary endpoint, with a possible increase to 1000 patients based on interim risk analysis. CONCLUSION: The EUTrigTreat clinical study aims to overcome current shortcomings in sudden cardiac death risk stratification and to answer several related research questions. The initial patient recruitment is expected to be completed in July 2012, and follow-up is expected to end in September 2014. Clinicaltrials.gov identifier: NCT01209494.

Protocol for minimizing the risk of metachronous adenomas of the colorectum with green tea extract (MIRACLE): a randomised controlled trial of green tea extract versus placebo for nutriprevention of metachronous colon adenomas in the elderly population.

BACKGROUND: Prevention of colorectal cancer is a major health care issue. People who have undergone colonoscopy screening and had colorectal polyps removed have a higher risk of being diagnosed with polyps again compared to the normal population. Therefore, it would be ideal to find appropriate means that effectively help to prevent the reoccurrence of polyps after polypectomy. So far, pharmaceutical chemoprevention with NSAIDs including aspirin has been shown to be effective but not gained general acceptance due to side effects. Nutraceuticals such as polyphenols from tea plants have demonstrated remarkable therapeutic and preventive effects in molecular, epidemiological and clinical trials. However, placebo-controlled trials demonstrating the efficacy of nutraceuticals for the (secondary) prevention of colorectal polyps as precursors for colorectal cancer are missing. METHODS/DESIGN: We present the design of a randomized, placebo controlled, multicentre trial to investigate the effect of diet supplementation with green tea extract containing 300 mg epigallocatechin gallate (EGCG), the major polyphenol in green tea, on the recurrence of colon adenomas. Patients who have undergone polypectomy for colonic polyps will be randomized to receive either green tea extract containing 150 mg EGCG two times daily or a placebo over the course of three years. After a one month run-in period in which all patients will receive the active intervention, 2534 patients will be randomized, and 2028 patients are expected to complete the whole study course. Incidence, number and histology of adenoma at endpoint colonoscopy at three years will be compared in both groups. DISCUSSION: The beneficial safety profile of decaffeinated green tea extract, the quantifiable and known active content EGCG, and the accumulating evidence of its cancer preventive potential require, in our view, a validation of this compound for the nutriprevention of colorectal adenoma. Good accessibility and low costs might render this neutraceutical a top candidate for wider use as food supplement in colon cancer prevention. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01360320.

Impact of cytochrome P-450 inhibition by cimetidine and induction by carbamazepine on the kinetics of hypericin and pseudohypericin in healthy volunteers.

This study evaluated the influence of cimetidine and carbamazepine on the pharmacokinetics of the St. John's wort (SJW) ingredients hypericin and pseudohypericin. In a placebo-controlled, double blind study, 33 healthy volunteers were randomized into three treatment groups that received SJW extract (LI160) with different comedications (placebo, cimetidine, and carbamazepine) for 7 days after a run-in period of 11 days with SJW alone. Hypericin and pseudohypericin pharmacokinetics were measured on days 10 and 17. Between-group comparisons showed no statistically significant differences in AUC(0-24), C(max), and t(max) values for hypericin and pseudohypericin. Within-group comparisons, however, revealed a statistically significant increase in hypericin AUC(0-24) from a median of 119 (range 82-163 microg h/l) to 149 microg h/l (61-202 microg h/l) with cimetidine comedication and a decrease in pseudohypericin AUC(0-24) from a median of 51.0 (16.4-102.9 microg h/l) to 36.4 microg h/l (14.0-102.0 microg h/l) with carbamazepine comedication compared to the baseline pharmacokinetics in each group. Hypericin and pseudohypericin pharmacokinetics were only marginally influenced by comedication with the enzyme inhibitors and inducers cimetidine and carbamazepine.

Differential effects of Saint John's Wort (hypericum perforatum) on the urinary excretion of D-glucaric acid and 6beta-hydroxycortisol in healthy volunteers.

OBJECTIVE: We investigated the effects of treatment with Saint John's wort (hypericum perforatum) extract on the urinary excretion of D-glucaric acid, 6beta-hydroxycortisol, and free cortisol in order to assess the effect of this extract on the activity of hepatic xenobiotic metabolizing enzymes. METHODS: Forty-eight healthy volunteers (25 male and 23 female) received a daily dose of 1800 mg hypericum extract for 14 days. Urinary excretion of D-glucaric acid, 6beta-hydroxycortisol, and free cortisol was measured in 24-h urine samples on the day preceding the initiation of hypericum treatment and after 14 days of treatment. D-Glucaric acid was measured enzymatically. Cortisol and 6beta-hydroxycortisol were quantified using high-performance liquid chromatography with ultraviolet detection. RESULTS: Urinary excretion of D-glucaric acid was unaffected after a 14-day treatment with Saint John's wort extract (26.7 micromol/day vs 27.7 micromol/day; 95% confidence interval of the difference: -1.9 to 3.8). The urinary excretion of 6beta-hydroxycortisol increased from a mean baseline value of 254 microg/day to 369 microg/day (P<0.0001) indicating induction of CYP3A. While the excretion of free cortisol was unaltered, the ratio of 6beta-hydroxycortisol to free cortisol changed significantly from 9.9 at baseline to 14.3 (95% confidence interval of the difference: 2.3-6.5) after Saint John's wort treatment. CONCLUSIONS: High-dose treatment with Saint John's wort extract induced CYP3A activity in healthy volunteers as evidenced by increased 6beta-hydroxycortisol excretion. This enzyme induction most likely contributes to the decreased bioavailability observed upon co-administration of various drugs with Saint John's wort extract. The D-glucuronic acid pathway appeared unaffected by Saint John's wort.

Influence of GSTT1 and GSTM1 genotypes on sunburn sensitivity.

BACKGROUND: Exposure to sunlight may cause sunburn, skin cancer or phototoxic reactions to certain drugs such as Hypericum extract. All these are ultraviolet B (UVB)-mediated reactions which may be modulated by individual genetic susceptibility. UVB exposure results in oxidative stress. Many products of oxidative stress are detoxified by glutathione-S-transferases mu 1 (GSTM1) and theta 1 (GSTT1). Deletion polymorphisms (genotype *0/*0) of GSTM1 and GSTT1 occur in 50% and 20% of Caucasians, respectively. By affecting the individual ability to detoxify oxidative stress-related products, they may influence the severity of the cutaneous photoreaction. METHODS: Minimal erythema doses (MED) of UVB irradiation on the skin were determined in 110 subjects who were selected according to their GSTT1 genotype (28 GSTT1*0/*0, 54 GSTT1*A/*0, and 28 GSTT1*A/*A). Genotypes were detected with novel polymerase chain reaction (PCR) assays that allow the differentiation between homozygous and heterozygous GSTT1 and GSTM1 deletions. RESULTS: In the absence of GSTT1 enzyme, the susceptibility of individuals to UVB-induced inflammatory skin reactions increased significantly (p = 0.02, ANCOVA). 'Gene-equivalents' were calculated from the number of functional GSTM1 and GSTT1 alleles as a measure of the gene-dose. UVB sensitivity correlated with gene dose up to a threshold above which additional GSTT1 or GSTM1 alleles did not provide additional protection. Volunteers who were homozygously deficient in GSTT1 and GSTM1 were most sensitive to UVB. Interestingly, individuals with high GSTM1 gene-doses showed increased photosensitization after administration of Hypericum extract (St. John's wort). CONCLUSION: Individuals harboring the *0/*0 genotype of GSTT1 and/or GSTM1 showed enhanced UVB-induced cutaneous damage. Moreover, GST genotypes modulated Hypericum-induced photosensitization.

Decreased plasma levels of amitriptyline and its metabolites on comedication with an extract from St. John's wort ( Hypericum perforatum ).

Extracts of St. John's wort ( Hypericum perforatum ) became increasingly popular as easily available remedies for mild to moderate depression. Comedication with hypericum extract was recently shown to drastically reduce plasma concentration of ciclosporin, digoxin, and indinavir. We investigated the possible interaction of hypericum extract LI160 with amitriptyline. Both antidepressants have a high probability of concomitant use. Twelve patients requiring amitriptyline treatment received a single dose of hypericum extract (900 mg) at day 1, continued by a 12-to 14-day treatment with retarded amitriptyline (75 mg twice daily). Then hypericum (900 mg/day) was added for another 14 to 16 days. Steady-state pharmacokinetics of amitriptyline were compared before and after multiple-dose treatment with hypericum extract. Furthermore, comparisons were made for single-dose kinetics of hypericum-extract ingredients hypericin, pseudohypericin, and hyperforin between the first day of concomitant treatment and LI160 alone. Multiple-dose comedication with LI160 led to a statistically significant decrease in the area under the plasma concentration-time curve within one dosing interval of amitriptyline by 22% ( p = 0.03) and nortriptyline by 41% ( p = 0.002), as well as of all hydroxylated metabolites, except for 10-E-hydroxynortriptyline. Plasma levels of amitriptyline and hydroxylated metabolites gradually decreased, whereas nortriptyline concentrations were already markedly decreased after 3 days of cotreatment with hypericum. Cumulative urinary amounts of amitriptyline and metabolites decreased to the same extent as plasma concentrations upon hypericum comedication. Induction of cytochrome P-450 enzymes or drug transporters (P-glycoprotein) by St. John's wort extract may explain this pharmacokinetic interaction. Physicians should be aware of this interaction when treating patients with amitriptyline.