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Sex‐Dependent Effects of CYP2D6 on the Pharmacokinetics of Berberine in Humans
Abstract
An over‐the‐counter product berberine (a major alkaloid in goldenseal) is a substrate of the uptake transporter OCT1 and the metabolizing enzyme CYP2D6. The two genes exhibit common functional polymorphisms. Approximately 9% of Europeans and white Americans are either poor CYP2D6 metabolizers or poor OCT1 transporters. In this study, we investigated the effects of OCT1 and CYP2D6 polymorphisms on berberine pharmacokinetics in humans. We confirmed in vitro that berberine is an OCT1 substrate ( K M of 7.0 μM, CL int of 306 ± 29 μL/min/mg). Common OCT1 alleles *3 to *6 showed uptake reduced by at least 65% and Oct1/2 knockout mice showed 3.2‐fold higher AUCs in liver perfusion experiments. However, in humans, poor OCT1 transporters did not show any differences in berberine pharmacokinetics compared with reference participants. In contrast, CYP2D6 polymorphisms significantly affected berberine metabolism, but exclusively in females. Females who were poor CYP2D6 metabolizers had an 80% lower M1‐to‐berberine ratio. General linear model analyses suggest strong synergistic, rather than additive, effects between female sex and CYP2D6 genotype. Overall, berberine displayed low oral bioavailability, yet females had a 2.8‐fold higher AUC and a 3.6‐fold higher C max than males ( P < 0.001). These effects were only partially attributable to the sex‐ CYP2D6 genotype interaction. In conclusion, despite berberine being an OCT1 substrate, OCT1 deficiency did not affect berberine pharmacokinetics in humans. In contrast, CYP2D6 emerges as a critical enzyme for berberine metabolism in females, but not in males, highlighting sex‐specific differences. We suggest that factors beyond CYP2D6 metabolism are determining berberine's systemic exposure, especially in males (NCT05463003).
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Berberine is a plant-origin quaternary isoquinoline alkaloid with a vast array of biological activities, including antioxidant and blood-glucose-and blood-lipid-lowering effects. However, its therapeutic potential is largely limited by its poor oral bioavailability. The aim of this study was to investigate the in vitro solubility and Caco-2 cell permeability followed by pharmacokinetic profiling in healthy volunteers of a new food-grade berberine delivery system (i.e., Berberine LipoMicel ®). X-ray diffractometry (XRD), in vitro solubility, and Caco-2 cell permeability indicated higher bioavailability of LipoMicel Berberine (LMB) compared to the standard formulation. Increased aqueous solubility (up to 1.4-fold), as well as improved Caco-2 cell permeability of LMB (7.18 × 10 −5 ± 7.89 × 10 −6 cm/s), were observed when compared to standard/unformulated berber-ine (4.93 × 10 −6 ± 4.28 × 10 −7 cm/s). Demonstrating better uptake, LMB achieved significant increases in AUC 0-24 and C max compared to the standard formulation (AUC: 78.2 ± 14.4 ng h/mL vs. 13.4 ± 1.97 ng h/mL, respectively; p < 0.05; C max : 15.8 ± 2.6 ng/mL vs. 1.67 ± 0.41 ng/mL) in a pilot study of healthy volunteers (n = 10). No adverse reactions were reported during the study period. In conclusion, LMB presents a highly bioavailable formula with superior absorption (up to six-fold) compared to standard berberine formulation and may, therefore, have the potential to improve the therapeutic efficacy of berberine. The study has been registered on ClinicalTrials.gov with Identifier NCT05370261.
Goldenseal is a perennial plant native to eastern North America. A recent clinical study reported goldenseal decreased metformin Cmax and area under the blood concentration versus time curve (AUC) by 27% and 23%, respectively, but half-life and renal clearance were unchanged. These observations suggested goldenseal altered processes involved in metformin absorption. The underlying mechanism(s) remain(s) unknown. One mechanism for the decreased metformin systemic exposure is inhibition by goldenseal of intestinal uptake transporters involved in metformin absorption. Goldenseal extract and three goldenseal alkaloids (berberine, (–)-β-hydrastine, hydrastinine) were tested as inhibitors of organic cation transporter (OCT) 3, plasma membrane monoamine transporter (PMAT), and thiamine transporter (THTR) 2 using human embryonic kidney 293 cells overexpressing each transporter. The goldenseal extract, normalized to berberine content, was the strongest inhibitor of each transporter (IC50: 4.9, 13.1, and 5.8 μM for OCT3, PMAT, and THTR2, respectively). A pharmacokinetic study in mice compared the effects of berberine, (–)-β-hydrastine, goldenseal extract, and imatinib (OCT inhibitor) on orally administered metformin. Goldenseal extract and imatinib significantly decreased metformin Cmax by 31% and 25%, respectively, and had no effect on half-life. Berberine and (–)-β-hydrastine had no effect on metformin pharmacokinetics, indicating neither alkaloid alone precipitated the interaction in vivo. A follow-up murine study involving intravenous metformin and oral inhibitors examined the contributions of basolateral enteric/hepatic uptake transporters to the goldenseal–metformin interaction. Goldenseal extract and imatinib had no effect on metformin AUC and half-life, suggesting lack of inhibition of basolateral enteric/hepatic uptake transporters. Results may have implications for patients taking goldenseal with drugs that are substrates for OCT3 and THTR2.
SIGNIFICANCE STATEMENT
Goldenseal is used to self-treat respiratory infections and digestive disorders. We investigated potential mechanisms for the clinical pharmacokinetic interaction observed between goldenseal and metformin, specifically inhibition by goldenseal of intestinal uptake transporters (OCT3, PMAT, THTR2) involved in metformin absorption. Goldenseal extract inhibited all three transporters in vitro and decreased metformin systemic exposure in mice. These data may have broader implications for patients co-consuming goldenseal with other drugs that are substrates for these transporters.
Many organic cations (OCs) may be transported through membranes by a genetically still uncharacterized proton-organic cation (H + OC) antiporter. Here, we characterized an extended substrate spectrum of this antiporter. We studied the uptake of 72 drugs in hCMEC/D3 cells as a model of the human blood–brain barrier. All 72 drugs were tested with exchange transport assays and the transport of 26 of the drugs was studied in more detail concerning concentration-dependent uptake and susceptibility to specific inhibitors. According to exchange transport assays, 37 (51%) drugs were good substrates of the H + OC antiporter. From 26 drugs characterized in more detail, 23 were consistently identified as substrates of the H + OC antiporter in six different assays and transport kinetic constants could be identified with intrinsic clearances between 0.2 (ephedrine) and 201 (imipramine) mL × minute−1 × g protein−1. Excellent substrates of the H + OC antiporter were no substrates of organic cation transporter OCT1 and vice versa. Good substrates of the H + OC antiporter were more hydrophobic and had a lower topological polar surface area than non-substrates or OCT1 substrates. These data and further research on the H + OC antiporter may result in a better understanding of pharmacokinetics, drug–drug interactions and variations in pharmacokinetics.
OCT1 and OCT2 are polyspecific membrane transporters that are involved in hepatic and renal drug clearance in humans and mice. In this study, we cloned dog OCT1 and OCT2 and compared their function to the human and mouse orthologs. We used liver and kidney RNA to clone dog OCT1 and OCT2. The cloned and the publicly available RNA-Seq sequences differed from the annotated exon-intron structure of OCT1 in the dog genome CanFam3.1. An additional exon between exons 2 and 3 was identified and confirmed by sequencing in six additional dog breeds. Next, dog OCT1 and OCT2 were stably overexpressed in HEK293 cells and the transport kinetics of five drugs were analyzed. We observed strong differences in the transport kinetics between dog and human orthologs. Dog OCT1 transported fenoterol with 12.9-fold higher capacity but 14.3-fold lower affinity (higher KM) than human OCT1. Human OCT1 transported ipratropium with 5.2-fold higher capacity but 8.4-fold lower affinity than dog OCT1. Compared to human OCT2, dog OCT2 showed 10-fold lower transport of fenoterol and butylscopolamine. In conclusion, the functional characterization of dog OCT1 and OCT2 reported here may have implications when using dogs as pre-clinical models as well as for drug therapy in dogs.
Males and females present differences in complex traits and in the risk of a wide array of diseases. Genotype by sex (GxS) interactions are thought to account for some of these differences. However, the extent and basis of GxS are poorly understood. In the present study, we provide insights into both the scope and the mechanism of GxS across the genome of about 450,000 individuals of European ancestry and 530 complex traits in the UK Biobank. We found small yet widespread differences in genetic architecture across traits. We also found that, in some cases, sex-agnostic analyses may be missing trait-associated loci and looked into possible improvements in the prediction of high-level phenotypes. Finally, we studied the potential functional role of the differences observed through sex-biased gene expression and gene-level analyses. Our results suggest the need to consider sex-aware analyses for future studies to shed light onto possible sex-specific molecular mechanisms.
Berberine, a well-known alkaloid, has been proved to possess various pharmacological activities. Previous studies demonstrated that berberine could be extensively metabolized and the metabolites also contributed to its therapeutic effects. However, as for berberine’s metabolites, especially phase II metabolites, pharmacokinetics and excretion studies were rarely reported. The objective of this study was to thoroughly investigate the pharmacokinetic and excretion profiles of berberine and its nine metabolites, namely, berberrubine (M1), demethyleneberberine (M2), jatrorrhizine (M3), jatrorrhizine-3-O-β-D-glucuronide (M4), jatrorrhizine-3-O-sulfate (M5), thalfendine-10-O-β-D-glucuronide (M6), berberrubine-9-O-β-D-glucuronide (M7), demethyleneberberine-2-O-sulfate (M8) and demethyleneberberine-2-O-β-D-glucuronide (M9) in rats. An accurate and reliable LC-MS/MS method was developed and validated for the determination of berberine and its nine metabolites in rat biosamples. Pharmacokinetic profiles of berberine and its nine metabolites were obtained after a single intravenous administration (4.0 mg/kg) and oral administration (48.2, 120 or 240 mg/kg) of berberine in rats. For excretion study, rats were intragastrically administered a single dose of 48.2 mg/kg berberine. Our results showed that berberine could be metabolized rapidly and all the nine metabolites could be detected in vivo. The absolute bioavailability of berberine was 0.37 ± 0.11%. As for the AUC0–48 h values, phase II metabolites were much higher than those of phase I metabolites, suggesting that phase II metabolites were the major metabolites exist in blood circulation. 18.6% of the berberine was excreted in feces as berberrubine (M1). The total recovery of berberine and its nine metabolites from urine, bile and feces was 41.2%. This is the first systematic study about the pharmacokinetics and excretion of berberine and its nine metabolites, which will be beneficial for both better understanding the clinical effects and further development of berberine.
Berberine is a plant metabolite belonging to the group of isoquinoline alkaloids with strong biological and pharmacological activity. Currently, berberine is receiving considerable interest due to its anticancer activity based on many biochemical pathways, especially its proapoptotic and anti-inflammatory activity. Therefore, the growing number of papers on berberine demands summarizing the knowledge and research trends. The efficacy of berberine in breast and colon cancers seems to be the most promising aspect. Many papers focus on novel therapeutic strategies based on new formulations or search for new active derivatives. The activity of berberine is very important as regards sensitization and support of anticancer therapy in combination with well-known but in some cases inefficient therapeutics. Currently, the compound is being assessed in many important clinical trials and is one of the most promising and intensively examined natural agents.
Background
Several small studies have previously investigated associations between the cytochrome P450 2D6 (CYP2D6) metabolism and response to opioids. We used a large sample of patients to study associations between CYP2D6 phenotypes and estimated CYP2D6 enzymatic activity scores with pain control and adverse reactions related to codeine and tramadol use. We conducted additional analyses to determine whether our results were consistent among men and women.
Methods
We used data from 2,877 participants in the RIGHT Protocol who were prescribed codeine and/or tramadol between 01/01/2005 and 12/31/2017 and who were not prescribed CYP2D6 inhibitors within 1 year prior to the opioid prescription. CYP2D6 phenotype categories were condensed into four groups: (1) Ultra-rapid and Rapid (n = 61), (2) Normal and Intermediate to Normal (n = 1,448), (3) Intermediate and Intermediate to Poor (n = 1,175), and (4) Poor metabolizer status (n = 193). Opioid-related outcomes included indications of poor pain control or adverse reactions related to medication use. We modeled the risk of each outcome using logistic regression, adjusting for age, sex, race, and ethnicity.
Results
The results revealed a trend from poor to ultra-rapid and rapid CYP2D6 phenotypes in which the risk of adverse reactions incrementally increased and the risk of poor pain control incrementally decreased. This trend reached statistical significance among female (but not male) participants. Among normal and intermediate to normal metabolizers, a larger proportion of women experienced adverse reactions relative to men.
Discussion
We replicated and extended the findings of previous research indicating associations between CYP2D6 phenotypes and response to opioids. In addition, the observed associations were stronger in women than in men. We recommend sex differences to be factored in future research investigating associations between pharmacogenomics and response to medications.
Objective
Yohimbine pharmacokinetics were determined after oral administration of a single oral dose of yohimbine 5 mg and a microdose of yohimbine 50 µg in relation to different cytochrome P450 (CYP) 2D6 genotypes. The CYP2D6 inhibitor paroxetine was used to investigate the influence on yohimbine pharmacokinetics. Microdosed midazolam was applied to evaluate a possible impact of yohimbine on CYP3A activity and the possibility of combining microdosed yohimbine and midazolam to simultaneously determine CYP2D6 and CYP3A activity.Methods
In a fixed-sequence clinical trial, 16 healthy volunteers with a known CYP2D6 genotype [extensive (10), intermediate (2) and poor (4) metaboliser] received an oral dose of yohimbine 50 µg, yohimbine 5 mg at baseline and during paroxetine as a CYP2D6 inhibitor. Midazolam (30 µg) was co-administered to determine CYP3A activity at each occasion. Plasma concentrations of yohimbine, its main metabolite 11-OH-yohimbine, midazolam and paroxetine were quantified using validated liquid chromatography-tandem mass spectrometry assays.ResultsPharmacokinetics of yohimbine were highly variable and a CYP2D6 genotype dependent clearance was observed. After yohimbine 5 mg, the clearance ranged from 25.3 to 15,864 mL/min and after yohimbine 50 µg, the clearance ranged from 39.6 to 38,822 mL/min. A more than fivefold reduction in clearance was caused by paroxetine in CYP2D6 extensive metabolisers, while the clearance in poor metabolisers was not affected. Yohimbine did not alter CYP3A activity as measured by microdosed midazolam.Conclusions
The pharmacokinetics of yohimbine were highly correlated with CYP2D6, which was further supported by the clearance inhibition caused by the CYP2D6 inhibitor paroxetine. With these data, yohimbine is proposed to be a suitable probe drug to predict CYP2D6 activity. In addition, the microdose can be used in combination with microdosed midazolam to simultaneously evaluate CYP2D6 and CYP3A activity without any interaction between the probe drugs and because the microdoses exert no pharmacological effects.Clinical Trial RegistrationEudraCT2017-001801-34.
The Pharmacogene Variation Consortium (PharmVar) provides nomenclature for the highly polymorphic human CYP2D6 gene locus. CYP2D6 genetic variation impacts the metabolism of numerous drugs and thus can impact drug efficacy and safety. This GeneReview provides a comprehensive overview and summary of CYP2D6 genetic variation and describes how the information provided by PharmVar is utilized by the Pharmacogenomics Knowledgebase (PharmGKB) and the Clinical Pharmacogenetics Implementation Consortium (CPIC).
Cardiovascular and metabolic diseases (CVMD) are the leading causes of death worldwide, underscoring the urgent necessity to develop new pharmacotherapies. Berberine (BBR) is an eminent component of traditional Chinese and Ayurvedic medicine for more than 2000 years. Recently, BBR has attracted much interest for its pharmacological actions in treating and/or managing CVMD. Recent discoveries of basic, translational and clinical studies have identified many novel molecular targets of BBR (such as AMPK, SIRT1, LDLR, PCSK9, and PTP1B) and provided novel evidences supporting the promising therapeutic potential of BBR to combat CVMD. Thus, this review provides a timely overview of the pharmacological properties and therapeutic application of BBR in CVMD, and underlines recent pharmacological advances which validate BBR as a promising lead drug against CVMD.
Background
Ranitidine (Zantac®) is a H2-receptor antagonist commonly used for the treatment of acid-related gastrointestinal diseases. Ranitidine was reported to be a substrate of the organic cation transporters OCT1 and OCT2. The hepatic transporter OCT1 is highly genetically variable. Twelve major alleles confer partial or complete loss of OCT1 activity. The effects of these polymorphisms are highly substrate-specific and therefore difficult to predict. The renal transporter OCT2 has a common polymorphism, Ala270Ser, which was reported to affect OCT2 activity.
Aim
In this study we analyzed the effects of genetic polymorphisms in OCT1 and OCT2 on the uptake of ranitidine and on its potency to inhibit uptake of other drugs.
Methods and results
We characterized ranitidine uptake using HEK293 and CHO cells stably transfected to overexpress wild type OCT1, OCT2, or their naturally occurring allelic variants. Ranitidine was transported by wild-type OCT1 with a Km of 62.9 μM and a vmax of 1125 pmol/min/mg protein. Alleles OCT1*5, *6, *12, and *13 completely lacked ranitidine uptake. Alleles OCT1*2, *3, *4, and *10 had vmax values decreased by more than 50%. In contrast, OCT1*8 showed an increase of vmax by 25%. The effects of OCT1 alleles on ranitidine uptake strongly correlated with the effects on morphine uptake suggesting common interaction mechanisms of both drugs with OCT1. Ranitidine inhibited the OCT1-mediated uptake of metformin and morphine at clinically relevant concentrations. The inhibitory potency for morphine uptake was affected by the OCT1*2 allele. OCT2 showed only a limited uptake of ranitidine that was not significantly affected by the Ala270Ser polymorphism.
Conclusions
We confirmed ranitidine as an OCT1 substrate and demonstrated that common genetic polymorphisms in OCT1 strongly affect ranitidine uptake and modulate ranitidine’s potential to cause drug-drug interactions. The effects of the frequent OCT1 polymorphisms on ranitidine pharmacokinetics in humans remain to be analyzed.
We investigated the influence of gut microbiotal metabolism on the pharmacokinetics of berberine in healthy male Africans and Chinese. The Cmax and AUC in the Africans were 2.67-fold and 2.0-fold higher than the Chinese, respectively. Microbiotal compositions by 16S rRNA pyrosequencing showed higher abundance of the genera Prevotella, Bacteroides, and Megamonas (34.22, 13.88, and 10.68%, respectively) in the Chinese than the Africans (30.08, 9.43, and 0.48%, respectively). Scatter plot showed a strong negative correlation between the microbiotal abundance and the berberine AUC, especially for the genus Prevotella (r = −0.813) and its species. A more extensive metabolism was observed in Chinese with 1.83-fold higher metabolites, possibly contributing to the lower AUC than the Africans. In conclusion, significant PK differences of berberine were observed between Africans and Chinese, which is partly attributable to variations in gut microbiota and its corresponding metabolic capacity.
The organic cation transporter OCT1 (SLC22A1) mediates the uptake of vitamin B1, cationic drugs, and xenobiotics into hepatocytes. Nine percent of Caucasians lack or have very low OCT1 activity due to loss-of-function polymorphisms in OCT1 gene. Here we analyzed the global genetic variability in OCT1 to estimate the therapeutic relevance of OCT1 polymorphisms in populations beyond Caucasians and to identify evolutionary patterns of the common loss of OCT1 activity in humans.
We applied massively parallel sequencing to screen for coding polymorphisms in 1,079 unrelated individuals from 53 populations worldwide. The obtained data was combined with the existing 1000 Genomes data comprising an additional 1,092 individuals from 14 populations. The identified OCT1 variants were characterized in vitro regarding their cellular localization and their ability to transport 10 known OCT1 substrates. Both the population genetics data and transport data were used in tandem to generate a world map of loss of OCT1 activity.
We identified 16 amino acid substitutions potentially causing loss of OCT1 function and analyzed them together with five amino acid substitutions that were not expected to affect OCT1 function. The variants constituted 16 major alleles and 14 sub-alleles. Six major alleles showed improper subcellular localization leading to substrate-wide loss in activity. Five major alleles showed correct subcellular localization, but substrate-specific loss of activity. Striking differences were observed in the frequency of loss of OCT1 activity worldwide. While most East Asian and Oceanian individuals had completely functional OCT1, 80 % of native South American Indians lacked functional OCT1 alleles. In East Asia and Oceania the average nucleotide diversity of the loss-of-function variants was much lower than that of the variants that do not affect OCT1 function (ratio of 0.03) and was significantly lower than the theoretically expected heterozygosity (Tajima's D = -1.64, P < 0.01).
Comprehensive genetic analyses showed strong global variations in the frequency of loss of OCT1 activity with selection pressure for maintaining OCT1 activity in East Asia and Oceania. These results not only enable pharmacogenetically-based optimization of drug treatment worldwide, but may help elucidate the functional role of human OCT1.
Significance
This manuscript describes a previously unidentified mechanism for organic cation transporter 1 (OCT1), the major hepatic metformin transporter, in hepatic steatosis. Here we show that OCT1, long thought to function primarily as a transporter for drugs, functions as a major thiamine transporter in the liver, which has profound implications in cellular metabolism. Collectively, our results point to an important role of thiamine (through OCT1) in hepatic steatosis and suggest that the modulation of thiamine disposition by metformin may contribute to its pharmacologic effects.
We investigated whether morphine and its pro-drug codeine are substrates of the highly genetically polymorphic organic cation transporter OCT1 and whether OCT1 polymorphisms may affect morphine and codeine pharmacokinetics in humans. Morphine showed low transporter-independent membrane permeability (0.5×10(-6)cm/s). Morphine uptake was increased up to 4-fold in HEK293 cells overexpressing human OCT1. The increase was concentration-dependent and followed Michaelis-Menten kinetics (KM=3.4μM, VMAX=27pmol/min/mg protein). OCT1-mediated morphine uptake was abolished by common loss-of-function polymorphisms in the OCT1 gene and was strongly inhibited by drug-drug interactions with irinotecan, verapamil and ondansetron. Morphine uptake in primary human hepatocytes was strongly reduced by MPP(+), an inhibitor of organic cation transporters, and morphine was not a substrate of OCT3, the other organic cation transporter expressed in human hepatocytes. In concordance with the in vitro data, morphine plasma concentrations in healthy volunteers were significantly dependent on OCT1 polymorphisms. After codeine administration, the mean AUC of morphine was 56% higher in carriers of loss-of-function OCT1 polymorphisms compared to non-carriers (P=0.005). The difference remained significant after adjustment for CYP2D6 genotype (P=0.03). Codeine itself had high transporter-independent membrane permeability (8.2×10(-6)cm/s). Codeine uptake in HEK293 cells was not affected by OCT1 overexpression and OCT1 polymorphisms did not affect codeine AUCs. In conclusion, OCT1 plays an important role in the hepatocellular uptake of morphine. Carriers of loss-of-function OCT1 polymorphisms may be at higher risk of adverse effects after codeine administration, especially if they are also ultra-rapid CYP2D6 metabolizers.
Berberine is a widely used plant extract for gastrointestinal infections, and is reported to have potential benefits in treatment for diabetes and hypercholesterolemia. It has been suggested that interactions between berberine-containing products and cytochromes P450 (CYPs) exist, but little is known about which CYPs mediate the metabolism of berberine in vivo. In this study, berberine metabolites in urine and feces of mice were analyzed, and the role that CYPs play in producing these metabolites were characterized in liver microsomes from mice (MLM) and humans (HLM), as well as recombinant human CYPs. Eleven berberine metabolites were identified in mice, including 5 unconjugated metabolites, mainly in feces, and 6 glucuronide and sulfate conjugates, predominantly in urine. Three novel berberine metabolites were observed. Three unconjugated metabolites of berberine were produced by MLM, HLM, and recombinant human CYPs. CYP2D6 was the primary recombinant human CYP producing these metabolites, followed by CYP1A2, 3A4, 2E1 and CYP2C19. The metabolism of berberine in MLM and HLM was decreased the most by a CYP2D inhibitor, and moderately by inhibitors of CYP1A and 3A. CYP2D plays a major role in berberine biotransformation, therefore, CYP2D6 pharmacogenetics and potential drug-drug interactions should be considered when berberine is used.
The objective of the present research was to evaluate the physicochemical characteristics of berberine chloride and to assess the complexation of drug with 2-hydroxypropyl-β-cyclodextrin (HPβCD), a first step towards solution dosage form development. The parameters such as log P value were determined experimentally and compared with predicted values. The pH-dependent aqueous solubility and stability were investigated following standard protocols at 25°C and 37°C. Drug solubility enhancement was attempted utilizing both surfactants and cyclodextrins (CDs), and the drug/CD complexation was studied employing various techniques such as differential scanning calorimetry, Fourier transform infrared, nuclear magnetic resonance, and scanning electron microscopy. The experimental log P value suggested that the compound is fairly hydrophilic. Berberine chloride was found to be very stable up to 6 months at all pH and temperature conditions tested. Aqueous solubility of the drug was temperature dependent and exhibited highest solubility of 4.05 ± 0.09 mM in phosphate buffer (pH 7.0) at 25°C, demonstrating the effect of buffer salts on drug solubility. Decreased drug solubility was observed with increasing concentrations of ionic surfactants such as sodium lauryl sulfate and cetyl trimethyl ammonium bromide. Phase solubility studies demonstrated the formation of berberine chloride-HPβCD inclusion complex with 1:1 stoichiometry, and the aqueous solubility of the drug improved almost 4.5-fold in the presence of 20% HPβCD. The complexation efficiency values indicated that the drug has at least threefold greater affinity for hydroxypropyl-β-CD compared to randomly methylated-β-CD. The characterization techniques confirmed inclusion complex formation between berberine chloride and HPβCD and demonstrated the feasibility of developing an oral solution dosage form of the drug.
Different sampling techniques for collecting nasal mucus have been used. The eosinophil and its granule toxin proteins, especially the eosinophil cationic protein (ECP), is an important inflammatory cell.
In the healthy subjects (with no acute or chronic disease of the respiratory tract), no detailed quantitative data on mucus nasal proteins have been reported, so the aim of the study is to demonstrate a simple and reliable method for the collection of nasal mucus and for measuring nasal ECP.
Our method consists in collecting two specimens of mucus from each nasal fossa, using a square of sterile pre-humidified gauze, centrifuged at 2000g for 20 min and stored at -20 degrees C until tested. To evaluate the reproducibility of the technique, 30 healthy subjects were retested after 24 h.
The amounts of secretion gathered in the 89 collections did not show any statistical difference between the sexes. In the pooled subjects (n=59), the mean levels of ECP were 108 ng/mL and 325 ng/g and total protein (TP) levels were 701 mg/dL and 2.5 mg/g (expressed in absolute concentration and concentration related to the weight in grams). In the classes, according to age, no statistical differences were found on analysis of the groups by: sex, recovery rate, and ECP and TP concentrations. The Reproducibility Index was very good for the recovery rate (0.78), ECP (ng/mL=0.95, ng/g=0.83) and TP (mg/dL=0.89, mg/g=0.76).
With our method, the sample collections were taken with minimal stimulation of the mucosa and it was well tolerated by the subjects; the low known dilution factor is also adequate for analysing small quantities of mucus recovered, and the protein concentration can be measured as an absolute value.
The organic cation transporter, OCT1, is a major hepatic transporter that mediates the uptake of many organic cations from the blood into the liver where the compounds may be metabolized or secreted into the bile. Because OCT1 interacts with a variety of structurally diverse organic cations, including clinically used drugs as well as toxic substances (e.g., N-methylpyridinium, MPP(+)), it is an important determinant of systemic exposure to many xenobiotics. To understand the genetic basis of extensive interindividual differences in xenobiotic disposition, we functionally characterized 15 protein-altering variants of the human liver organic cation transporter, OCT1, in Xenopus oocytes. All variants that reduced or eliminated function (OCT1-R61C, OCT1-P341L, OCT1-G220V, OCT1-G401S, and OCT1-G465R) altered evolutionarily conserved amino acid residues. In general, variants with decreased function had amino acid substitutions that resulted in more radical chemical changes (higher Grantham values) and were less evolutionarily favorable (lower blosum62 values) than variants that maintained function. A variant with increased function (OCT1-S14F) changed an amino acid residue such that the human protein matched the consensus of the OCT1 mammalian orthologs. Our results indicate that changes at evolutionarily conserved positions of OCT1 are strong predictors of decreased function and suggest that a combination of evolutionary conservation and chemical change might be a stronger predictor of function.
The polyspecific organic cation transporters 1 and 2 (Oct1 and -2) transport a broad range of substrates, including drugs,
toxins, and endogenous compounds. Their strategic localization in the basolateral membrane of epithelial cells in the liver,
intestine (Oct1), and kidney (Oct1 and Oct2) suggests that they play an essential role in removing noxious compounds from
the body. We previously showed that in Oct1−/− mice, the hepatic uptake and intestinal excretion of organic cations are greatly reduced. Since Oct1 and Oct2 have extensively
overlapping substrate specificities, they might be functionally redundant. To investigate the pharmacologic and physiologic
roles of these proteins, we generated Oct2 single-knockout and Oct1/2 double-knockout mice. Oct2−/− and Oct1/2−/− mice are viable and fertile and display no obvious phenotypic abnormalities. Absence of Oct2 in itself had little effect
on the pharmacokinetics of tetraethylammonium (TEA), but in Oct1/2−/− mice, renal secretion of this compound was completely abolished, leaving only glomerular filtration as a TEA clearance mechanism.
As a consequence, levels of TEA were substantially increased in the plasma of Oct1/2−/− mice. This study shows that Oct1 and Oct2 together are essential for renal secretion of (small) organic cations. A deficiency
in these proteins may thus result in increased drug sensitivity and toxicity.
Metformin is among the most widely prescribed drugs for the treatment of type 2 diabetes. Organic cation transporter 1 (OCT1) plays a role in the hepatic uptake of metformin, but its role in the therapeutic effects of the drug, which involve activation of AMP-activated protein kinase (AMPK), is unknown. Recent studies have shown that human OCT1 is highly polymorphic. We investigated whether OCT1 plays a role in the action of metformin and whether individuals with OCT1 polymorphisms have reduced response to the drug. In mouse hepatocytes, deletion of Oct1 resulted in a reduction in the effects of metformin on AMPK phosphorylation and gluconeogenesis. In Oct1-deficient mice the glucose-lowering effects of metformin were completely abolished. Seven nonsynonymous polymorphisms of OCT1 that exhibited reduced uptake of metformin were identified. Notably, OCT1-420del (allele frequency of about 20% in white Americans), previously shown to have normal activity for model substrates, had reduced activity for metformin. In clinical studies, the effects of metformin in glucose tolerance tests were significantly lower in individuals carrying reduced function polymorphisms of OCT1. Collectively, the data indicate that OCT1 is important for metformin therapeutic action and that genetic variation in OCT1 may contribute to variation in response to the drug.
The natural product goldenseal is a clinical inhibitor of CYP3A activity, as evidenced by a 40-60% increase in midazolam AUC after coadministration with goldenseal. The predominant goldenseal alkaloids berberine and (-)-β-hydrastine were previously identified as time-dependent CYP3A inhibitors using human liver microsomes. Whether these alkaloids contribute to the clinical interaction, as well as the primary anatomical site (hepatic vs. intestinal) and mode of CYP3A inhibition (reversible vs. time-dependent), remain uncharacterized. The objective of this study was to mechanistically assess the pharmacokinetic goldenseal-midazolam interaction using an integrated in vitro-in vivo-in silico approach. Using human intestinal microsomes, (-)-β-hydrastine was a more potent time-dependent inhibitor of midazolam 1'-hydroxylation than berberine (KI and kinact: 8.48 µM and 0.041 min-1, respectively, vs. >250 µM and ~0.06 min-1, respectively). Both the AUC and Cmax of midazolam increased by 40-60% after acute (single 3 g dose) and chronic (1 g thrice daily x 6 days) goldenseal administration to healthy adults. These increases, coupled with a modest/no increase ({less than or equal to}23%) in half-life, suggested that goldenseal primarily inhibited intestinal CYP3A. A physiologically based pharmacokinetic interaction model incorporating berberine and (-)-β-hydrastine successfully predicted the observed goldenseal-midazolam interaction (within 20%) after both chronic and acute goldenseal administration. Simulations implicated (-)-β-hydrastine as the major alkaloid precipitating the interaction, primarily via time-dependent inhibition of intestinal CYP3A, after chronic and acute goldenseal exposure. Results highlight the potential interplay between time-dependent and reversible inhibition of intestinal CYP3A as the mechanism underlying natural product-drug interactions, even after acute exposure to the precipitant. Significance Statement Natural products can alter the pharmacokinetics of an object drug, potentially resulting in increased off-target effects or decreased efficacy of the drug. The objective of the current work was to evaluate fundamental mechanisms underlying the clinically observed goldenseal-midazolam interaction. Results support the use of an integrated approach involving established in vitro assays, clinical evaluation, and PBPK modeling to elucidate the complex interplay between multiple phytoconstituents and various pharmacokinetic processes driving a drug interaction.
The organic cation transporter 1 (OCT1) mediates the cell uptake and cytochrome P450 2D6 (CYP2D6) the metabolism of many cationic substrates. Activities of OCT1 and CYP2D6 are affected by enormous genetic variation and frequent drug-drug interactions. Single or combined deficiency of OCT1 and CYP2D6 might result in dramatic differences in systemic exposure, adverse drug reactions, and efficacy. Thus, one should know what drugs are affected to what extent by OCT1, CYP2D6 or both. Here, we compiled all data on CYP2D6 and OCT1 drug substrates. Among 246 CYP2D6 substrates and 132 OCT1 substrates, we identified 31 shared substrates. In OCT1 and CYP2D6 single and double-transfected cells, we studied which, OCT1 or CYP2D6, is more critical for a given drug and whether there are additive, antagonistic or synergistic effects. In general, OCT1 substrates were more hydrophilic than CYP2D6 substrates and smaller in size. Inhibition studies showed unexpectedly pronounced inhibition of substrate depletion by shared OCT1/CYP2D6 inhibitors. In conclusion, there is a distinct overlap in the OCT1/CYP2D6 substrate and inhibitor spectra, so in vivo pharmacokinetics and -dynamics of shared substrates may be significantly affected by frequent OCT1- and CYP2D6-polymorphisms and by comedication with shared inhibitors.
Berberine is widely used for the prevention of cancers and diabetes. However, the absorption rate of berberine is less than 1% in humans. The objective of this research was to determine whether emulsification improves the absorption and affects the metabolism of orally ingested berberine. Twelve healthy subjects, both men and women, received 800 mg berberine in a powder or emulsified form by vitamin E TPGS or Quillaja extract using a randomized crossover design. Blood samples were collected 12 hours after a dose. Berberine and its metabolites in plasma were analyzed with and without hydrolysis by glucuronidase and sulfatase on UHPLC-MS/MS. The area under the curve (AUC0-12 h) and peak plasma concentration (Cmax) of berberine was 6.7 nM h and 0.9 nM in participants who received berberine powder. They were increased to 12.6 nM h and 2.0 nM by TPGS emulsification and 28.0 nM h and 5.1 nM by Quillaja extract emulsification, respectively. Berberrubine and demethyleneberberine were detected as major phase-1 metabolites of berberine. The AUC0-12 of both free and total berberrubine was significantly increased by TPGS and Quillaja extract. Emulsification by Quillaja extract was more effective than TPGS to increase the plasma concentrations of free and total demethyleneberberine. However, the ratios of phase-1 metabolites and ratios of phase-2 conjugates were not affected by emulsification. Absorption increases of berberine by TPGS or Quillaja extract emulsification may lead to enhanced bioactivity in humans.
About 30% of all small molecular drugs are organic cations (OCs). If these are more or less hydrophilic, they require membrane transporters to pass through biological membranes. Here, the proton-organic cation (H+ OC) antiporter may play a physiologically most relevant role, particularly concerning passage through the blood-brain barrier. Membrane transport of about 70 OCs is significantly enhanced by this H+ OC antiporter. Surprisingly still today the gene coding for this antiporter was not yet identified. However, the H+ OC antiporter is characterized by concentration- and pH-dependent uptake, antiport with another OC, and susceptibility to inhibition by specific inhibitors. Moreover, in the studied tissues and cell types, transport is not mediated by already well-known organic cation transporters. The review explains the typically used assays to identify potential substrates of the H+ OC antiporter. Thus far, the gene encoding for this transporter has not yet been identified, but a better understanding of this protein may be most relevant because it may affect the pharmacokinetics of up to 10% of all low molecular substances. This review summarizes the known functional characteristics of the H+ OC antiporter, its cell and tissue expression, and its substrate spectrum. Summarizing the features of the substrates of the H+ OC antiporter may even suggest that for OCs, good penetration through the blood-brain barrier is almost synonymous with being a substrate of the H+ OC antiporter. In clinical studies, pharmacokinetics of typical substrates of the antiporter showed outstanding between-subject variability.
The botanical natural product goldenseal can precipitate clinical drug interactions by inhibiting cytochrome P450 (CYP) 3A and CYP2D6. Besides P‐glycoprotein, effects of goldenseal on other clinically relevant transporters remain unknown. Established transporter‐expressing cell systems were used to determine the inhibitory effects of a goldenseal extract, standardized to the major alkaloid berberine, on transporter activity. Using recommended basic models, the extract was predicted to inhibit the efflux transporter breast cancer resistance protein (BCRP) and uptake transporters organic anion transporting polypeptide (OATP) 1B1/3. Using a cocktail approach, effects of the goldenseal product on BCRP, OATP1B1/3, organic anion transporters (OATs), organic cation transporters (OCTs), multidrug and toxin extrusion (MATE) proteins, and CYP3A were next evaluated in 16 healthy volunteers. As expected, goldenseal increased the area under the plasma concentration‐time curve (AUC0‐inf) of midazolam (CYP3A; positive control), with a geometric mean ratio (GMR) [90% confidence interval] of 1.43 [1.35‐1.53]. However, goldenseal had no effects on the pharmacokinetics of rosuvastatin (BCRP, OATP1B1/3) and furosemide (OAT1/3); decreased metformin AUC0‐inf (OCT1/2, MATE1/2‐K; GMR, 0.77 [0.71‐0.83]); and had no effect on metformin half‐life and renal clearance. Results indicated that goldenseal altered intestinal permeability, transport, and/or other processes involved in metformin absorption, which may have unfavorable effects on glucose control. Inconsistencies between model predictions and pharmacokinetic outcomes prompt further refinement of current basic models to include differential transporter expression in relevant organs and intestinal degradation/metabolism of the precipitant(s). Such refinement should improve in vitro‐in vivo prediction accuracy, contributing to a standard approach for studying transporter‐mediated natural product‐drug interactions.
Bioavailability of orally administered drugs is partly determined by function of drug transporters in the liver and intestine. We therefore explored ABC and SLC family transporters expression (qPCR) and protein abundance (LC‐MS/MS) in human liver and duodenum, jejunum, ileum and colon in paired tissue specimens from 9 organ donors. The transporter proteins were detected in the liver (P‐gp, MRP2, MRP3, BCRP, OATP1B1, OATP1B3, OATP2B1, OCT1, OCT3, OAT2, NTCP, MCT1, MATE1) and the intestine (P‐gp, MRP2, MRP3, MRP4, BCRP, OATP2B1, OCT1, ASBT (only ileum), MCT1, PEPT1). Significantly higher hepatic gene expression and protein abundance of ABCC2/MRP2, SLC22A1/OCT1 and SLCO2B1/OATP2B1 were found, as compared to all intestinal segments. No correlations between hepatic and small intestinal protein levels were observed. These observations provide a description of drug transporters distribution without the impact of inter‐individual variability bias, and may help in construction of superior PBPK and humanized animal models. This article is protected by copyright. All rights reserved.
Sales of botanical dietary supplements and other purported medicinal natural products (NPs) have escalated over the past ~25 years, increasing the potential for NPs to precipitate clinically significant pharmacokinetic interactions with United States Food and Drug Administration (FDA)-approved medications (NP-drug interactions or NPDIs). Published NPDI studies to date often lack consistency in design, implementation, and documentation, which present difficulties in assessing the clinical significance of the results. Common hurdles include large variability in the admixture composition of phytoconstituents between and within batches of a given NP, limited knowledge on the pharmacokinetics of precipitant NP constituents, and use of animal and/or in vitro models which, in some cases, are not mechanistically appropriate for extrapolation to humans. The National Center for Complementary and Integrative Health has created a Center of Excellence for Natural Product-Drug Interaction (NaPDI Center) to address these unmet research needs. The NaPDI Center has two overarching goals: 1) develop Recommended Approaches to guide researchers in the proper conduct of NPDI studies, which will evolve over time concurrent with emerging technologies and new research data; and 2) apply the Recommended Approaches in evaluating four model NPs as precipitants of NPDIs with clinically relevant object drugs. The major objectives of this commentary are to 1) explain the rationale for creating the NaPDI Center; 2) describe the Decision Trees developed by the NaPDI Center to enhance the planning, rigor, and consistency of NPDI studies; and 3) provide a framework for communicating results to the multidisciplinary scientists interested in the NaPDI Center&rsquo's Interaction Projects.
Fenoterol is widely used anti-asthmatic and tocolytic agent, but high plasma concentrations of fenoterol may lead to severe and even fatal adverse reactions. We studied whether heritable deficiency of the liver organic cation transporter OCT1, a trait observed in 3% of Europeans and White Americans, affects fenoterol plasma concentrations and toxicity. OCT1 transported fenoterol with high affinity. OCT1 inhibition in human hepatocytes reduced fenoterol uptake 3-fold. After administration of 180 µg fenoterol to 39 healthy individuals, the OCT1-deficicient individuals (zero active OCT1 alleles, n=5) showed 1.9-fold greater systemic fenoterol exposure (p=4.0x10(-5) ) and 1.7-fold lower volume of distribution (p=8.0x10(-5) ). Correspondingly, the OCT1-deficient individuals had a 1.5-fold stronger increase in heart rate in (p=0.002), a 3.4-fold greater increase in blood glucose (p=3.0x10(-5) ), and significantly lower serum potassium levels. In conclusion, heritable OCT1 deficiency significantly increases plasma concentrations of fenoterol and may be an important factor underlying the excess mortality associated with fenoterol. This article is protected by copyright. All rights reserved.
This study aims to characterize longitudinal blood pressure (BP) trajectories from childhood in black-white and sex groups and examine the association between childhood level-independent trajectories of BP and adult hypertension. The longitudinal cohort consisted of 2732 adults who had body mass index and BP measured 4 to 15 times from childhood (4-19 years) to adulthood (20-51 years). Model-estimated levels and linear slopes of BP and body mass index at childhood age points were calculated at 1-year intervals using the growth curve parameters and their first derivatives, respectively. Linear and nonlinear curve parameters differed significantly between race-sex groups; BP levels showed race and sex differences 15 years of age onward. Hypertensives had higher long-term BP levels than normotensives in race-sex groups. Although linear and nonlinear slope parameters of BP were race and sex specific, they differed consistently, significantly between hypertension and normotension groups. BP trajectories during young adulthood (20-35 years) were significantly greater in hypertensives than in normotensives; however, the trajectories during middle-aged adulthood (36-51 years) were significantly smaller in hypertensives than in normotensives. Level-independent linear slopes of systolic BP showed significantly negative associations (odds ratio=0.50≈0.76; P<0.001) during prepuberty period (4-11 years) but significantly positive associations (odd ratio=1.44≈2.80, P<0.001) during the puberty period (13-19 years) with adult hypertension, adjusting for covariates. These associations were consistent across race-sex groups. These observations indicate that adult hypertension originates in childhood, with different longitudinal BP trajectory profiles during young and middle-aged adulthood in black-white and sex groups. Puberty is a crucial period for the development of hypertension in later life.
HT3) receptor antagonists are used in the prevention of chemotherapy- induced, radiation-induced and postoperative nausea and vomiting. CYP2D6 polymorphisms can influence the metabolism of some of these drugs (i.e. ondansetron and tropisetron) thereby affecting drug efficacy. We summarize evidence from the published literature supporting these associations and provide therapeutic recommendations for ondansetron and tropisetron based on CYP2D6 genotype (updates at www.pharmgkb.org). This article is protected by copyright. All rights reserved.
Heritability of caffeine pharmacokinetics and CYP1A2 activity is controversial. Here we analyzed the pharmacokinetics of caffeine, an in vivo probe drug for CYP1A2 and arylamine N-acetyltransferase 2 (NAT2) activity, in monozygotic and dizygotic twins. In the entire group, common and unique environmental effects explained most variation in caffeine AUC. Apparently, smoking and hormonal contraceptives masked the genetic effects on CYP1A2 activity. However, when excluding smokers and users of hormonal contraceptives, 89% of caffeine AUC variation was due to genetic effects and even in the entire group, 8% of caffeine AUC variation could be explained by a CYP1A1/1A2 promotor polymorphism (rs2470893). In contrast, nearly all of the variation (99%) of NAT2 activity was explained by genetic effects. This study illustrates two very different situations in pharmacogenetics, from an almost exclusively genetic determination of NAT2 activity with no environmental modulation to only moderate genetic effects on CYP1A2 activity with strong environmental modulation. This article is protected by copyright. All rights reserved.
The low bioavailability of the anti-migraine drug sumatriptan is partially caused by first-pass hepatic metabolism. Here, we analyzed the impact of the hepatic organic cation transporter OCT1 on sumatriptan cellular uptake, and of OCT1 polymorphisms on sumatriptan pharmacokinetics. OCT1 transported sumatriptan with high capacity and sumatriptan uptake into human hepatocytes was strongly inhibited by the OCT1 inhibitor MPP(+) . Sumatriptan uptake was not affected by the Met420del polymorphism, but was strongly reduced by Arg61Cys and Gly410Ser, and completely abolished by Gly465Arg and Cys88Arg. Plasma concentrations in humans with two deficient OCT1 alleles were 215% of those with fully active OCT1 (P=0.0003). OCT1 also transported naratriptan, rizatriptan, and zolmitriptan, suggesting a possible impact of OCT1 polymorphisms on the pharmacokinetics of other triptans as well. In conclusion, OCT1 is a high-capacity transporter of sumatriptan and polymorphisms causing OCT1 deficiency have similar effects on sumatriptan pharmacokinetics as those observed in subjects with liver impairment. This article is protected by copyright. All rights reserved.
Berberine, the main active component of the herbal medicine Rhizoma Coptidis, has been reported to have hypoglycemic and insulin-sensitizing effects and, therefore, could be combined with metformin therapy. Thus, we assessed the potential drug-drug interactions between berberine and metformin. We investigated the in vitro inhibitory potency of berberine on metformin uptake in HEK293 cells overexpressing organic cation transporter (OCT) 1 and 2. To investigate whether this inhibitory effect of berberine on OCT1 and OCT2 could change the pharmacokinetics of metformin in vivo, we measured the effect of berberine co-administration on the pharmacokinetics of metformin at a single intravenous dose of 2 mg/kg metformin and 10 mg/kg berberine. In HEK293 cells, berberine inhibited OCT1- and OCT2-mediated metformin uptake in a concentration dependent manner and IC50 values for OCT1 and OCT2 were 7.28 and 11.3 μM, respectively. Co-administration of berberine increased the initial plasma concentration and AUC of metformin and decreased systemic clearance and volume of distribution of metformin in rats, suggesting that berberine inhibited disposition of metformin, which is governed by OCT1 and OCT2. Berberine inhibited the transport activity of OCT1 and OCT2 and showed significant potential drug-drug interactions with metformin in in vivo rats.
Debrisoquine is a probe drug for in vivo phenotyping of human CYP2D6 metabolic activity. However, debrisoquine is positively charged under physiological conditions and it is unclear how it enters the hepatocytes to undergo CYP2D6 metabolism. We analysed whether debrisoquine is a substrate of the hepatic organic cation transporter OCT1 and whether drug-drug interactions at OCT1, or polymorphisms in OCT1 gene, affect debrisoquine uptake. Debrisoquine showed low carrier-independent membrane permeability (P(e) of 0.01×10⁻⁶ cm/s in artificial PAMPA membranes) and strongly inhibited the uptake of the model OCT1 substrate MPP+ (IC₅₀ of 6.2 ± 0.8 μM). Debrisoquine uptake was significantly increased in HEK293 cells overexpressing OCT1 compared to control cells. The OCT1-mediated uptake of debrisoquine followed Michaelis-Menten kinetics (K(M) of 5.9 ± 1.5 μM and V(max) of 41.9 ± 4.5pmol/min/mg protein) and was inhibited by known OCT1 inhibitors and by commonly used drugs. OCT1-mediated debrisoquine uptake was reduced or missing in cells expressing loss-of-function OCT1 isoforms. Deletion of Met420 or substitution of Arg61Cys or Gly401Ser reduced V(max) by 48, 63 and 91%, respectively, but did not affect the K(M). The OCT1 isoforms carrying Cys88Arg or Gly465Arg substitutions completely lacked OCT1-mediated debrisoquine uptake. In conclusion, debrisoquine is a substrate of OCT1 and has the potential to be used as a phenotyping marker for OCT1 activity. Moreover, variations in debrisoquine metabolic phenotypes and their associations with diseases may be due not only to genetic variations CYP2D6, but also in OCT1.
The activity of various CYP isoforms is critical for maintaining the clinical effectiveness of many medications. Therefore, determining the sex-dependent activity of clinically relevant CYP families is highly important for optimal therapeutic effectiveness.
This review examined the sex-dependent activity of CYP3A, CYP1A2, CYP2D6, CYP2C9, CYP2C19 and CYP2E1.
This review searched for studies performed in humans and hormonal status was not a limiting factor.
The current evidence suggests that CYP2E1 and CYP1A2 activity is higher in males than females, while CYP3A, one of the most clinically relevant CYP isoforms, appears to have greater activity in females. Overall, more studies are needed to fully support these conclusions as there are many factors that influence drug metabolism and thus it is very difficult to isolate gender as a sole modulator of CYP activity.
Berberine chloride (BBR) is a natural isoquinoline alkaloid extracted from medicinal herbs. It has been reported that the intestinal absorption of BBR is very low. In this study, the absolute bioavailability of BBR was studied, and the enhancing effects of d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) on intestinal absorption were investigated in rats. BBR injection was administrated via the femoral vein at a dose of 1.0 mg kg−1 in intravenous group, and BBR oral formulations were administrated by oral gavage at a dose of 100 mg kg−1 in BBR control (control) group and BBR-TPGS (test) group, respectively. The result showed that BBR had a very low absolute bioavailability of 0.68%, and TPGS could enhance intestinal absorption of BBR significantly. TPGS at a concentration of 2.5% could improve peak concentration (C
max) and area under the curve (AUC0–36) of BBR by 2.9 and 1.9 times, respectively. The absorption enhancing ability of TPGS may be due to its ability to affect the biological activity of P-glycoprotein and thereby reduce the excretion of absorbed BBR into the intestinal lumen. This study indicated that absolute bioavailability of BBR was 0.68% in rats, and TPGS was a good absorption enhancer capable of enhancing intestinal absorption of BBR significantly.
Cytochrome P4501A2 (CYP1A2) is involved in the metabolism of several drugs and is induced by smoking. We aimed to determine the interindividual change in CYP1A2 activity after smoking cessation and to relate it to CYP1A2 genetic polymorphisms. CYP1A2 activity was determined from the paraxanthine:caffeine ratio in 194 smokers and in 118 of them who had abstained from smoking during a 4-week period. The participants were genotyped for CYP1A2*1F, *1D, and *1C polymorphisms. Smokers had 1.55-fold higher CYP1A2 activity than nonsmokers (P < 0.0001). The individual change in CYP1A2 activity after smoking cessation ranged from 1.0-fold (no change) to a 7.3-fold decrease in activity. In five participants with low initial CYP1A2 activity, an increase was observed after smoking cessation. Before smoking cessation, the following factors were found to influence CYP1A2 activity: CYP1A2*1F (P = 0.005), CYP1A2*1D (P = 0.014), the number of cigarettes/day (P = 0.012), the use of contraceptives (P < 0.001), and -163A/-2467T/-3860G haplotype (P = 0.002). After quitting smoking, only CYP1A2*1F (P = 0.017) and the use of contraceptives (P = 0.05) had an influence. No influence of CYP1A2 polymorphisms on the inducibility of CYP1A2 was observed.
Unlabelled:
An important function of hepatocytes is the biotransformation and elimination of various drugs, many of which are organic cations and are taken up by organic cation transporters (OCTs) of the solute carrier family 22 (SLC22). Because interindividual variability of OCT expression may affect response to cationic drugs such as metformin, we systematically investigated genetic and nongenetic factors of OCT1/SLC22A1 and OCT3/SLC22A3 expression in human liver. OCT1 and OCT3 expression (messenger RNA [mRNA], protein) was analyzed in liver tissue samples from 150 Caucasian subjects. Hepatic OCTs were localized by way of immunofluorescence microscopy. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and genome-wide single-nucleotide polymorphism microarray technology served to genotype 92 variants in the SLC22A1-A3/OCT1-3 gene cluster. Transport of metformin by recombinant human OCT1 and OCT3 was compared using transfected cells. OCT1 mRNA and protein expression varied 113- and 83-fold, respectively; OCT3 mRNA expression varied 27-fold. OCT1 transcript levels were on average 15-fold higher compared with OCT3. We localized the OCT3 protein to the basolateral hepatocyte membrane and identified metformin as an OCT3 substrate. OCT1 and OCT3 expression are independent of age and sex but were significantly reduced in liver donors diagnosed as cholestatic (P < or = 0.01). Several haplotypes for OCT1 and OCT3 were identified. Multivariate analysis adjusted for multiple testing showed that only the OCT1-Arg61Cys variant (rs12208357) strongly correlated with decreased OCT1 protein expression (P < 0.0001), and four variants in OCT3 (rs2292334, rs2048327, rs1810126, rs3088442) were associated with reduced OCT3 mRNA levels (P = 0.03).
Conclusion:
We identified cholestasis and genetic variants as critical determinants for considerable interindividual variability of hepatic OCT1 and OCT3 expression. This indicates consequences for hepatic elimination of and response to OCT substrates such as metformin.
Intraindividual variability and the effects of menstrual cycle phase on CYP2D6 activity were evaluated by dextromethorphan phenotyping in 20 Caucasian normal volunteers. Dextromethorphan 30 mg was administered to 10 men every 14 days for 3 months, and to 10 premenopausal women during the mid-follicular and mid-luteal phases of each menstrual cycle for three complete cycles. Urinary dextromethorphan/dextrorphan molar ratios were obtained after an overnight urine collection. Ten women and nine men were extensive metabolizer phenotypes, and one man was a poor metabolizer phenotype (confirmed by genotyping). There was no difference in dextromethorphan metabolic ratios between the mid-follicular (mean +/- SD: 0.00728+/-0.00717) and mid-luteal (0.00745+/-0.00815) phases of the menstrual cycle (P = 0.88). Also, no significant difference was found in the intraindividual variability of the metabolic ratios between the two phases (P = 0.80). No statistically significant sex difference in CYP2D6 activity was found between men (0.00537+/-0.00431) and women (0.00737+/-0.00983) extensive metabolizers (P = 0.84). For all individuals, intraindividual variability in dextromethorphan ratios ranged from 12.1-136.6% with a median of 36.7%. Because hormonal fluctuations within the mid-follicular and mid-luteal phases of the menstrual cycle do not appear to affect CYP2D6 activity, pharmacokinetic or clinical investigations of CYP2D6 substrate activity may not require menstrual cycle phase stratification. Because baseline metabolic ratios may fluctuate an average of 37%, repeat baseline and treatment phenotyping assessments should be obtained for accurate determination of a given drug's effect on CYP2D6 activity when measured by dextromethorphan.
Ezetimibe, a selective inhibitor of intestinal cholesterol absorption, is in clinical development for the treatment of hypercholesterolemia. It is rapidly absorbed and glucuronidated in the intestine. The parent compound and its conjugated metabolite undergo enterohepatic recirculation, resulting in multiple peaks in the plasma concentration-time profile.
The purpose of this study was to develop a population pharmacokinetic (PPK) model for ezetimibe that incorporates enterohepatic recirculation.
A population compartment model incorporating input from the gallbladder, consistent with food intake, was developed to account for enterohepatic recirculation. The amount recycled was allowed to vary within a subject and between subjects, accommodating variability in bile secretion. The data used consisted of 90 profiles from healthy subjects who received single or multiple doses of ezetimibe 10 or 20 mg. Modeling was carried out using a nonlinear mixed-effect function in the S-PLUS statistical program.
The amount of ezetimibe recycled into the central compartment was estimated to be approximately 17% to 20% of the total amount absorbed, independent of the volume of distribution. The intersubject coefficient of variation was 46% to 80% in the absorption rate constant, 27% in the distribution phase, and approximately 50% in the volume of distribution.
PPK models adapted for enterohepatic recirculation allowed a formal assessment of the magnitude and frequency of the enterohepatic recirculation process, and the associated intersubject and intrasubject variability in healthy subjects. The PPK approach also helped to assess the correlation between the observed maximum or minimum (24 hours postdose) concentration with the model-based area under the curve, confirming the appropriateness of the former measures as a surrogate of drug exposure for a possible correlation with pharmacodynamics.
By systematic mutation screening of the polyspecific organic cation transporter hOCT1 (SLC22A1) in 57 Caucasians, 25 genetic variations were identified and further analysed for population frequency. Five mutations resulting in the amino acid changes Arg61Cys, Cys88Arg, Phe160Leu, Gly401Ser, and Met420del, with respective allele frequencies of 9.1, 0.6, 22, 3.2, and 16%, were functionally characterized upon expression in Xenopus oocytes. Phe160Leu and Met420del exhibited substrate affinities and selectivites identical to hOCT1 wild-type. In contrast, uptake of 0.1 microm [3H]1-methyl-4-phenylpyridinium ([3H]MPP) by Arg61Cys, Cys88Arg and Gly401Ser were reduced to 30, 1.4 and 0.9% compared to wild-type, respectively. Since transport of 1 microm [3H]serotonin by Cys88Arg and Gly401Ser was reduced to only 13 and 12% of wild-type, these mutants exhibit a changed substrate selectivity. The data show that the mutants Arg61Cys, Cys88Arg and Gly401Ser could affect the disposition of OCT1 substrates and as a consequence may alter the duration and intensity of effects of drugs and neurotransmitters which are substrates for hOCT1.
Phytochemical-mediated modulation of cytochrome P450 (CYP) activity may underlie many herb-drug interactions. Single-time point phenotypic metabolic ratios were used to determine whether long-term supplementation of goldenseal ( Hydrastis canadensis ), black cohosh ( Cimicifuga racemosa ), kava kava ( Piper methysticum ), or valerian ( Valeriana officinalis ) extracts affected CYP1A2, CYP2D6, CYP2E1, or CYP3A4/5 activity.
Twelve healthy volunteers (6 women) were randomly assigned to receive goldenseal, black cohosh, kava kava, or valerian for 28 days. For each subject, a 30-day washout period was interposed between each supplementation phase. Probe drug cocktails of midazolam and caffeine, followed 24 hours later by chlorzoxazone and debrisoquin (INN, debrisoquine), were administered before (baseline) and at the end of supplementation. Presupplementation and postsupplementation phenotypic trait measurements were determined for CYP3A4/5, CYP1A2, CYP2E1, and CYP2D6 by use of 1-hydroxymidazolam/midazolam serum ratios (1-hour sample), paraxanthine/caffeine serum ratios (6-hour sample), 6-hydroxychlorzoxazone/chlorzoxazone serum ratios (2-hour sample), and debrisoquin urinary recovery ratios (8-hour collection), respectively. The content of purported "active" phytochemicals was determined for each supplement.
Comparisons of presupplementation and postsupplementation phenotypic ratio means revealed significant inhibition (approximately 40%) of CYP2D6 (difference, -0.228; 95% confidence interval [CI], -0.268 to -0.188) and CYP3A4/5 (difference, -1.501; 95% CI, -1.840 to -1.163) activity for goldenseal. Kava produced significant reductions (approximately 40%) in CYP2E1 only (difference, -0.192; 95% CI, -0.325 to -0.060). Black cohosh also exhibited statistically significant inhibition of CYP2D6 (difference, -0.046; 95% CI, -0.085 to -0.007), but the magnitude of the effect (approximately 7%) did not appear to be clinically relevant. No significant changes in phenotypic ratios were observed for valerian.
Botanical supplements containing goldenseal strongly inhibited CYP2D6 and CYP3A4/5 activity in vivo, whereas kava inhibited CYP2E1 and black cohosh weakly inhibited CYP2D6. Accordingly, serious adverse interactions may result from the concomitant ingestion of goldenseal supplements and drugs that are CYP2D6 and CYP3A4/5 substrates. Kava kava and black cohosh may interact with CYP2E1 and CYP2D6 substrates, respectively. Valerian appears to be less likely to produce CYP-mediated herb-drug interactions.
Cytochrome P450 2D6 (CYP2D6) is one of the most important enzymes catalyzing biotransformation of xenobiotics in the human liver. This enzyme's activity shows a high degree of interindividual variability caused in part by its genetic polymorphism, the so-called debrisoquine/sparteine polymorphism. The genetic component influencing CYP2D6 activity can be determined by genotyping. However, genotyping alone is not sufficient to accurately predict an individual's actual CYP2D6 activity, as this is also influenced by other factors. For the determination of the exact actual enzymatic activity ("phenotyping"), adequate probe drugs have to be administered prior to measurements of these compounds and/or their metabolites in body fluids. PROBE DRUGS: Debrisoquine, sparteine, metoprolol or dextromethorphan represent well-established probe drugs while tramadol has been recently investigated for this purpose. The enzymatic activity is reflected by various pharmacokinetic metrics such as the partial clearance of a parent compound to the respective CYP2D6-mediated metabolite or metabolic ratios. Appropriate metrics need to fulfill pre-defined validation criteria.
In this review, we have compiled a list of such criteria useful to select the best metrics to reflect CYP2D6 activity. A comprehensive Medline search for reports on CYP2D6 phenotyping trials with the above mentioned probe drugs was carried out.
Application of the validation criteria suggests that dextromethorphan and debrisoquine are the best CYP2D6 phenotyping drugs, with debrisoquine having the problem of very limited availability as a therapeutic drug. However, the assessment of the best dextromethorphan CYP2D6 phenotyping metric/procedure is still ongoing.
A liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) method for the determination of berberine in human plasma using chlorobenzylidine as the internal standard (IS) has been developed and validated. The plasma samples were prepared by LLE and the analytes were chromatographically separated on a Hanbon Lichrospher 5-C18 HPLC column under gradient elution with a mobile phase consisted of acetonitrile and 10mm ammonium acetate buffer containing 0.1% formic acid. Berberine was determined with electrospray ionisation-mass spectrometry (ESI-MS). LC-ESI-MS was performed in the selected-ion monitoring (SIM) mode using target ions at M(+)m/z 336.1 for berberine and M(+)m/z 464.1 for the IS. Calibration curve was linear over the range of 0.020-3.0 ng/ml. The lower limit of quantification (LLOQ) was 0.020 ng/ml. The intra- and inter-run variability values were less than 6.7 and 7.7%, respectively. The method has been successfully applied to determine the plasma concentration of berberine in healthy Chinese volunteers.
An important function of hepatocytes is the biliary elimination of endogenous and xenobiotic small molecules, many of which are organic cations. To study this vectorial transport of organic cations, we constructed a double-transfected Madin-Darby canine kidney strain II (MDCKII) cell line permanently expressing the human organic cation transporter 1 (OCT1, SLC22A1) in the basolateral membrane and MDR1 P-glycoprotein (MDR1 P-gp, ABCB1), an adenosine triphosphate (ATP)-dependent efflux pump for organic cations, in the apical membrane. Additionally, MDCKII single transfectants stably expressing OCT1, MDR1 P-gp, or human organic cation transporter 2 (OCT2, SLC22A2) were generated. Antisera directed against OCT1 or OCT2 specifically detected OCT1 in the basolateral membrane of human hepatocytes, OCT2 in tubular epithelial cells of human kidney, and the respective recombinant transporter in the basolateral membrane of MDCKII transfectants. We identified the lipophilic organic cation berberine, a fluorescent plant alkaloid exhibiting a broad range of biological activities, as substrate of OCT1 and OCT2 with Michaelis-Menten constants of 14.8 microM and 4.4 microM, respectively. Berberine also inhibited the uptake of the prototypic cations tetraethylammonium and 1-methyl-4-phenylpyridinium by MDCK-OCT1 and MDCK-OCT2 transfectants. When transfected cells were grown polarized on permeable filter supports, berberine was transferred from the basolateral to the apical compartments many times faster by MDCK-OCT1/MDR1 P-gp double transfectants than by MDCK-OCT1 or MDCK-MDR1 P-gp single transfectants. The specific MDR1 P-gp inhibitor, zosuquidar trihydrochloride (LY335979), strongly inhibited berberine efflux into the apical compartment. The MDCK-OCT1/MDR1 P-gp double transfectants may be useful to identify additional cationic substrates and inhibitors of OCT1 and MDR1 P-gp, including drug candidates.
Cytochrome P450 2D6 (CYP2D6), an important CYP isoform with regard to drug-drug interactions, accounts for the metabolism of approximately 30% of all medications. To date, few studies have assessed the effects of botanical supplementation on human CYP2D6 activity in vivo. Six botanical extracts were evaluated in three separate studies (two extracts per study), each incorporating 16 healthy volunteers (eight females). Subjects were randomized to receive a standardized botanical extract for 14 days on separate occasions. A 30-day washout period was interposed between each supplementation phase. In study 1, subjects received milk thistle (Silybum marianum) and black cohosh (Cimicifuga racemosa). In study 2, kava kava (Piper methysticum) and goldenseal (Hydrastis canadensis) extracts were administered, and in study 3 subjects received St. John's wort (Hypericum perforatum) and Echinacea (Echinacea purpurea). The CYP2D6 substrate, debrisoquine (5 mg), was administered before and at the end of supplementation. Pre- and post-supplementation phenotypic trait measurements were determined for CYP2D6 using 8-h debrisoquine urinary recovery ratios (DURR). Comparisons of pre- and post-supplementation DURR revealed significant inhibition (approximately 50%) of CYP2D6 activity for goldenseal, but not for the other extracts. Accordingly, adverse herb-drug interactions may result with concomitant ingestion of goldenseal supplements and drugs that are CYP2D6 substrates.
Effects of alcohol consumption and tobacco smoking on the composition of the ensemble of drug metabolizing enzymes and transporters in human liver
- K A Gaither