Liposomal Copermeation Assay Reveals Unexpected Membrane Interactions of Commonly Prescribed Drugs.

The permeation of small molecules across biological membranes is a crucial process that lies at the heart of life. Permeation is involved not only in the maintenance of homeostasis at the cell level but also in the absorption and biodistribution of pharmacologically active substances throughout the human body. Membranes are formed by phospholipid bilayers that represent an energy barrier for permeating molecules. Crossing this energy barrier is assumed to be a singular event, and permeation has traditionally been described as a first-order kinetic process, proportional only to the concentration gradient of the permeating substance. For a given membrane composition, permeability was believed to be a unique property dependent only on the permeating molecule itself. We provide experimental evidence that this long-held view might not be entirely correct. Liposomes were used in copermeation experiments with a fluorescent probe, where simultaneous permeation of two substances occurred over a single phospholipid bilayer. Using an assay of six commonly prescribed drugs, we have found that the presence of a copermeant can either enhance or suppress the permeation rate of the probe molecule, often more than 2-fold in each direction. This can have significant consequences for the pharmacokinetics and bioavailability of commonly prescribed drugs when used in combination and provide new insight into so-far unexplained drug-drug interactions as well as changing the perspective on how new drug candidates are evaluated and tested.

A single-dose, randomized crossover study in healthy Chinese subjects to evaluate pharmacokinetics and bioequivalence of two capsules of calcium dobesilate 0.5 g under fasting and fed conditions.

OBJECTIVES: To compare the rate and extent of absorption of a launched generic calcium dobesilate capsule versus the branded reference formulation under fasting and fed conditions in healthy Chinese subjects, and to assess their bioequivalence and tolerability. METHODS: This single-dose, open-label, randomized-sequence, 2-period crossover bioequivalence study was conducted on healthy Chinese volunteers aged 18 to 45 years. Subjects received a single 0.5 g dose of calcium dobesilate capsule under fasting or fed conditions, with a 3-day washout period between doses of the test (T) and reference (R) formulations. Blood samples were collected before and up to 24 hours after administration. The plasma concentration of calcium dobesilate was determined by a validated Liquid chromatography-tandem mass spectrometry method. Non-compartmental analysis was applied to identify the pharmacokinetic (PK) properties. The primary PK parameters including the maximal plasma concentration (Cmax), the area under the plasma concentration-time curve (AUC0-t), and the AUC extrapolated to infinity (AUC0-inf) were used for bioequivalence evaluation. RESULTS: The mean of PK parameters for T and R capsules under fasting (fed) condition were: Cmax, 13.57 (6.71) and 12.59 (7.25) µg/mL; AUC0-t, 97.32 (79.74) and 96.97 (80.71) h*µg/mL; AUC0-inf, 101.68 (88.01) and 101.64 (87.81) h*µg/mL. The 90% confidence intervals (CIs) of GMRs under fasting (fed) condition were: Cmax, 97.91%-116.62% (88.63%-96.53%); AUC0-t, 97.15%-104.00% (96.58%-101.39%); and AUC0-inf, 97.19%-102.89% (98.67%-103.99%). These 90% CIs were all within the bioequivalence range of 80%-125%. All adverse events were mild. CONCLUSION: In this study, the T calcium dobesilate 0.5 g capsule was bioequivalent to the reference product under both fasting and fed conditions. Taking food would slow down its rate and reduce its amount of absorption. Both formulations were generally well tolerated.

Microphysiological systems in absorption, distribution, metabolism, and elimination sciences.

The use of microphysiological systems (MPS) to support absorption, distribution, metabolism, and elimination (ADME) sciences has grown substantially in the last decade, in part driven by regulatory demands to move away from traditional animal-based safety assessment studies and industry desires to develop methodologies to efficiently screen and characterize drugs in the development pipeline. The past decade of MPS development has yielded great user-driven technological advances with the collective fine-tuning of cell culture techniques, fluid delivery systems, materials engineering, and performance enhancing modifications. The rapid advances in MPS technology have now made it feasible to evaluate critical ADME parameters within a stand-alone organ system or through interconnected organ systems. This review surveys current MPS developed for liver, kidney, and intestinal systems as stand-alone or interconnected organ systems, and evaluates each system for specific performance criteria recommended by regulatory authorities and MPS leaders that would render each system suitable for evaluating drug ADME. Whereas some systems are more suitable for ADME type research than others, not all system designs were intended to meet the recently published desired performance criteria and are reported as a summary of initial proof-of-concept studies.

Ontogeny of Drug-Metabolizing Enzymes.

Almost 50% of prescription drugs lack age-appropriate dosing guidelines and therefore are used "off-label." Only ~10% drugs prescribed to neonates and infants have been studied for safety or efficacy. Immaturity of drug metabolism in children is often associated with drug toxicity. This chapter summarizes data on the ontogeny of major human metabolizing enzymes involved in oxidation, reduction, hydrolysis, and conjugation of drugs. The ontogeny data of individual drug-metabolizing enzymes are important for accurate prediction of drug pharmacokinetics and toxicity in children. This information is critical for designing clinical studies to appropriately test pharmacological hypotheses and develop safer pediatric drugs, and to replace the long-standing practice of body weight- or surface area-normalized drug dosing. The application of ontogeny data in physiologically based pharmacokinetic model and regulatory submission are discussed.

Oleuropein-Induced Acceleration of Cytochrome P450-Catalyzed Drug Metabolism: Central Role for Nuclear Receptor Peroxisome Proliferator-Activated Receptor α.

Oleuropein (OLE), the main constituent of Olea europaea, displays pleiotropic beneficial effects in health and disease, which are mainly attributed to its anti-inflammatory and cardioprotective properties. Several food supplements and herbal medicines contain OLE and are available without a prescription. This study investigated the effects of OLE on the main cytochrome P450s (P450s) catalyzing the metabolism of many prescribed drugs. Emphasis was given to the role of peroxisome proliferator-activated receptor α (PPARα), a nuclear transcription factor regulating numerous genes including P450s. 129/Sv wild-type and Ppara-null mice were treated with OLE for 6 weeks. OLE induced Cyp1a1, Cyp1a2, Cyp1b1, Cyp3a14, Cyp3a25, Cyp2c29, Cyp2c44, Cyp2d22, and Cyp2e1 mRNAs in liver of wild-type mice, whereas no similar effects were observed in Ppara-null mice, indicating that the OLE-induced effect on these P450s is mediated by PPARα. Activation of the pathways related to phosphoinositide 3-kinase/protein kinase B (AKT)/forkhead box protein O1, c-Jun N-terminal kinase, AKT/p70, and extracellular signal-regulated kinase participates in P450 induction by OLE. These data indicate that consumption of herbal medicines and food supplements containing OLE could accelerate the metabolism of drug substrates of the above-mentioned P450s, thus reducing their efficacy and the outcome of pharmacotherapy. Therefore, OLE-induced activation of PPARα could modify the effects of drugs due to their increased metabolism and clearance, which should be taken into account when consuming OLE-containing products with certain drugs, in particular those of narrow therapeutic window. SIGNIFICANCE STATEMENT: This study indicated that oleuropein, which belongs to the main constituents of the leaves and olive drupes of Olea europaea, induces the synthesis of the major cytochrome P450s (P450s) metabolizing the majority of prescribed drugs via activation of peroxisome proliferator-activated receptor α. This effect could modify the pharmacokinetic profile of co-administered drug substrates of the P450s, thus altering their therapeutic efficacy and toxicity.

Methods Used for Pediatric Dose Selection in Drug Development Programs Submitted to the US FDA 2012-2020.

Dosing is a critical aspect of drug development in pediatrics that has led to trial failures and the inability to label the drug for pediatric use by the US Food and Drug Administration. Developing a structured approach for pediatric dose selection requires knowledge of the current approaches and their success or failure. This study describes the current experience with pediatric dosing methods from 2012 to 2020 and had 2 primary objectives: (1) to identify how the initial pediatric dose was selected and (2) to identify the pivotal dosing strategy used to identify the initially selected dose for safety and efficacy for pediatric clinical trials. Through September 2020, a total of 275 pediatric drug development programs were characterized for initial and pivotal dosing strategies. The success rate for labeling for pediatric use was 76.4%. The most common initial dosing strategy was previous experience with the product, followed by allometric scaling and exposure matching with adults. The most common pivotal dosing strategy was titration to target response in 33% of programs, with the second and third most common being pharmacokinetic/pharmacodynamic studies (30%) and exposure matching (20%), respectively. Additionally, about one-half of pediatric programs incorporated model-informed drug development. The emergence of titration to target response may signal a shift toward precision medicine in pediatric patients. Future work in pediatric drug dose selection should move toward the development of a structured pediatric dose selection approach.

Status Toward the Implementation of Precision Dosing in Children.

Precision dosing is progressing beyond the conceptual and proof-of-concept stages toward implementation. As the availability of dosing algorithms, tools, and platforms increases, so do the investment in technology services and actual implementation of clinical services offering these solutions to patients. Nowhere is this needed more than in pediatric populations, which are still reliant on adult drug development and bridging strategies to support dosing, often in the absence of actual dose-finding studies in the target pediatric population. Still, there is more work to be done to ensure that proper governance of these services is maintained, and that sustainability of these early implementations is guided by new science as it evolves and meaningful outcome data to confirm that such services deliver on both clinical and economic return on investment. In addition, the field should ensure that all approaches beyond a therapeutic drug monitoring-driven, pharmacokinetic-centric approach should be considered as the tools and services evolve, especially when pediatric-specific pharmacokinetic/pharmacodyamic and pharmacogenetic data are available and shown to be useful to guide dosing. This review evaluates current pediatric precision dosing efforts, highlighting their utility, longevity, and sustainability and assesses the current process for implementing such approaches examining current barriers that stand in the way of broader implementation and the stakeholders that must engage to ensure its ultimate success.

Blood donor exposome and impact of common drugs on red blood cell metabolism.

Computational models based on recent maps of the RBC proteome suggest that mature erythrocytes may harbor targets for common drugs. This prediction is relevant to RBC storage in the blood bank, in which the impact of small molecule drugs or other xenometabolites deriving from dietary, iatrogenic, or environmental exposures ("exposome") may alter erythrocyte energy and redox metabolism and, in so doing, affect red cell storage quality and posttransfusion efficacy. To test this prediction, here we provide a comprehensive characterization of the blood donor exposome, including the detection of common prescription and over-the-counter drugs in blood units donated by 250 healthy volunteers in the Recipient Epidemiology and Donor Evaluation Study III Red Blood Cell-Omics (REDS-III RBC-Omics) Study. Based on high-throughput drug screenings of 1366 FDA-approved drugs, we report that approximately 65% of the tested drugs had an impact on erythrocyte metabolism. Machine learning models built using metabolites as predictors were able to accurately predict drugs for several drug classes/targets (bisphosphonates, anticholinergics, calcium channel blockers, adrenergics, proton pump inhibitors, antimetabolites, selective serotonin reuptake inhibitors, and mTOR), suggesting that these drugs have a direct, conserved, and substantial impact on erythrocyte metabolism. As a proof of principle, here we show that the antacid ranitidine - though rarely detected in the blood donor population - has a strong effect on RBC markers of storage quality in vitro. We thus show that supplementation of blood units stored in bags with ranitidine could - through mechanisms involving sphingosine 1-phosphate-dependent modulation of erythrocyte glycolysis and/or direct binding to hemoglobin - improve erythrocyte metabolism and storage quality.

"Little more than a gut feeling?"-considerations when prescribing psychotropic medications to patients undergoing bariatric surgery.

OBJECTIVE: Bariatric surgical procedures are being commonly performed increasingly, and many surgical candidates are concomitantly taking psychotropic medication. This paper aims to elucidate issues when prescribing psychiatric medication in this setting of substantial anatomical and physiological change. METHOD: A hand search of the literature to assess the current understanding of effects of various bariatric procedures on the bioavailability of psychotropic medication. RESULTS: Predominantly malabsorptive bariatric procedures may reduce bioavailability of some but not all commonly used psychiatric medications. There is minimal information about the effects of the most commonly performed surgery, vertical sleeve gastrectomy. Lithium prescription and monitoring requires caution. CONCLUSIONS: There is limited guidance for prescription for psychotropic medication in the bariatric surgery patient group, and vigilance for unexpected adverse effects or altered efficacy is warranted.

PK-DB: pharmacokinetics database for individualized and stratified computational modeling.

A multitude of pharmacokinetics studies have been published. However, due to the lack of an open database, pharmacokinetics data, as well as the corresponding meta-information, have been difficult to access. We present PK-DB (https://pk-db.com), an open database for pharmacokinetics information from clinical trials. PK-DB provides curated information on (i) characteristics of studied patient cohorts and subjects (e.g. age, bodyweight, smoking status, genetic variants); (ii) applied interventions (e.g. dosing, substance, route of application); (iii) pharmacokinetic parameters (e.g. clearance, half-life, area under the curve) and (iv) measured pharmacokinetic time-courses. Key features are the representation of experimental errors, the normalization of measurement units, annotation of information to biological ontologies, calculation of pharmacokinetic parameters from concentration-time profiles, a workflow for collaborative data curation, strong validation rules on the data, computational access via a REST API as well as human access via a web interface. PK-DB enables meta-analysis based on data from multiple studies and data integration with computational models. A special focus lies on meta-data relevant for individualized and stratified computational modeling with methods like physiologically based pharmacokinetic (PBPK), pharmacokinetic/pharmacodynamic (PK/PD), or population pharmacokinetic (pop PK) modeling.

The Importance of Clinical Research in Pregnant Women to Inform Prescription Drug Labeling.

Pregnant women have historically been an understudied population and have been excluded from clinical trials. Recent efforts by stakeholders have raised awareness of the importance of clinical research in pregnant women to inform prescribing decisions. The Food and Drug Administration continues working to improve the format and content of prescription drug labeling for pregnant and lactating women, as demonstrated with the Pregnancy and Lactation Labeling Rule (PLLR), effective in 2015. The pregnancy labeling subsection now includes a subheading dedicated to the inclusion of pharmacokinetic (PK) data that inform the need for dose adjustments during pregnancy and the postpartum period. In addition, the PLLR also requires prescription drug labeling to be updated when important pregnancy information becomes available. Although PLLR improved the presentation of pregnancy-related information in labeling, there is a need to increase the quality and quantity of human data on the use of prescription drugs during pregnancy. PK studies in pregnant women should be incorporated into drug development programs and prioritized to obtain important information about safe and appropriate doses of a drug when used during pregnancy. In addition, opportunistic PK studies, postapproval pregnancy safety studies, ex vivo studies, and in silico modeling can be leveraged to better inform the risks and benefits of using a drug during pregnancy to inform study design and to further understand various mechanisms impacting pharmacokinetic/pharmacodynamic of drugs during pregnancy. It is important to address the significant existing data gaps and better inform the safety and dosing of prescription drugs for pregnant women.

Prescription of Colchicine with Other Dangerous Concomitant Medications: A Nation-Wide Survey Using the Japanese Claims Database.

The anti-inflammatory agent colchicine may cause toxic effects such as rhabdomyolysis, pancytopenia, and acute respiratory distress syndrome in cases of overdose and when patients have renal or liver impairment. As colchicine is a substrate for CYP3A4 and P-glycoprotein (P-gp), drug-drug interactions are important factors that cause fatal colchicine-related side effects. Thus, we conducted a nation-wide survey to determine the status of inappropriate colchicine prescriptions in Japan. Patients prescribed the regular use of colchicine from April 2014 to March 2017 were identified using the Japanese large health insurance claims database. As the primary endpoint, we evaluated the concomitant prescription proportions of strong CYP3A4 and/or P-gp inhibitors classified as "contraindications for co-administration" with colchicine in patients with renal or liver impairment. We defined these cases as "inappropriate colchicine prescriptions." Additionally, factors affecting inappropriate colchicine prescriptions were analyzed. Among the 3302 enrolled patients, 43 (1.30%) were inappropriately prescribed colchicine. Of these 43 patients, 11 had baseline renal and/or liver impairment. By multiple regression analysis, the primary diseases "gout" and "Behçet's disease" were extracted as independent factors for inappropriate colchicine prescriptions with odds ratios of 0.40 (95% confidence interval: 0.19-0.84) and 4.93 (95% confidence interval: 2.12-11.5), respectively. We found that approximately 1% of patients had important colchicine interactions. Particularly, Behçet's disease was a risk factor for inappropriate prescriptions, with approximately 25% of patients showing renal and/or liver impairment (classified as "contraindications for co-administration"). These findings may be useful for medical professionals who prescribe colchicine therapy.

Exploiting the placenta for nanoparticle-mediated drug delivery during pregnancy.

A major challenge to treating diseases during pregnancy is that small molecule therapeutics are transported through the placenta and incur toxicities to the developing fetus. The placenta is responsible for providing nutrients, removing waste, and protecting the fetus from toxic substances. Thus, the placenta acts as a biological barrier between the mother and fetus that can be exploited for drug delivery. Nanoparticle technologies provide the opportunity for safe drug delivery during pregnancy by controlling how therapeutics interact with the placenta. In this Review, we present nanoparticle drug delivery technologies specifically designed to exploit the placenta as a biological barrier to treat maternal, placental, or fetal diseases exclusively, while minimizing off-target toxicities. Further, we discuss opportunities, challenges, and future directions for implementing drug delivery technologies during pregnancy.

Natural Products: Experimental Approaches to Elucidate Disposition Mechanisms and Predict Pharmacokinetic Drug Interactions.

Natural products have been used by humans since antiquity for both egregious and beneficial purposes. Regarding the latter, these products have long been valued as a rich source of phytochemicals and developed into numerous life-saving pharmaceutical agents. Today, the sales and use of natural products with purported medicinal qualities continue to increase worldwide. However, natural products are not subject to the same premarket testing requirements as pharmaceutical agents, creating critical gaps in scientific knowledge about their optimal use. In addition, due to the common misperception that "natural" means "safe," patients may supplement or replace their prescription medications with natural products, placing themselves at undue risk for subefficacious pharmacotherapy or potentially toxic exposure. Collectively, with few exceptions, researchers, health care providers, and educators lack definitive information about how to inform consumers, patients, and students in the health professions on the safe and optimal use of these products. Recognition of this deficiency by key stakeholders, including the three pillars of biomedical research-industry, academia, and government-has facilitated multiple collaborations that are actively addressing this fundamental knowledge gap. This special issue contains a collection of articles highlighting the challenges faced by researchers in the field and the use of various experimental systems and methods to improve the mechanistic understanding of the disposition and drug interaction potential of natural products. Continued refinement of existing, and development of new, approaches will progress toward the common overarching goal of improving public health. SIGNIFICANCE STATEMENT: Natural products with purported medicinal value constitute an increasing share of the contemporary health care market. Natural products are not subject to the same premarket testing requirements as drug products, creating fundamental scientific knowledge gaps about the safe and effective use of these products. Collaborations among industrial, academic, and governmental researchers in multiple disciplines are anticipated to provide the definitive information needed to fill these gaps and improve public health.

Prediction of maternal pharmacokinetics using physiologically based pharmacokinetic models: assessing the impact of the longitudinal changes in the activity of CYP1A2, CYP2D6 and CYP3A4 enzymes during pregnancy.

Concerns over gestational effects on the disposition of drugs has highlighted the need for a better understanding of drug distribution and elimination during pregnancy. This study aimed at predicting maternal drug kinetics using a physiologically based pharmacokinetic (PBPK) modelling approach focusing on the observed gestational changes in three important Cytochrome P450 metabolizing enzymes, namely, CYP1A2, CYP2D6 and CYP3A4 at different gestational weeks (GWs). The Pregnancy PBPK model within the Simcyp Simulator V19 was used to predict the pharmacokinetics of sensitive probes to these enzymes; namely caffeine, theophylline, metoprolol, propranolol, paroxetine, midazolam, nifedipine and rilpivirine. PBPK model predictions were compared against clinical data collated from multiple studies for each compound to cover a wide spectrum of gestational ages. Pregnancy PBPK model predictions were within 2-fold error and indicated that CYP1A2 activity is approximately 0.70, 0.44 and 0.30 fold of the non-pregnant level at the end of the first, second and third trimesters, respectively. On the other hand, CYP2D6 activity increases by 1.36, 2.16 and 3.10 fold of the non-pregnant level at the end of the first, second and third trimesters, respectively. Likewise, CYP3A4 activity increases by 1.25, 1.75 and 2.32 fold of the non-pregnant level at the end of the first, second and third trimesters, respectively. The enzymes activity have been qualified throughout pregnancy. Quantified changes in drug dosing are most relevant during the third trimester, especially for drugs that are mainly eliminated by CYP1A2, CYP2D6 and CYP3A4 enzymes. The provided functions describing the continuous changes to the activity of these enzymes during pregnancy are important when modelling long term pharmacokinetic studies where longitudinal modelling or time-varying covariates are used.

Potential drug-drug interactions associated with drugs currently proposed for COVID-19 treatment in patients receiving other treatments.

Patients with COVID-19 are sometimes already being treated for one or more other chronic conditions, especially if they are elderly. Introducing a treatment against COVID-19, either on an outpatient basis or during hospitalization for more severe cases, raises the question of potential drug-drug interactions. Here, we analyzed the potential or proven risk of the co-administration of drugs used for the most common chronic diseases and those currently offered as treatment or undergoing therapeutic trials for COVID-19. Practical recommendations are offered, where possible.

A New Data Repository for Pharmacokinetic Natural Product-Drug Interactions: From Chemical Characterization to Clinical Studies.

There are many gaps in scientific knowledge about the clinical significance of pharmacokinetic natural product-drug interactions (NPDIs) in which the natural product (NP) is the precipitant and a conventional drug is the object. The National Center for Complimentary and Integrative Health created the Center of Excellence for NPDI Research (NaPDI Center) (www.napdi.org) to provide leadership and guidance on the study of pharmacokinetic NPDIs. A key contribution of the Center is the first user-friendly online repository that stores and links pharmacokinetic NPDI data across chemical characterization, metabolomics analyses, and pharmacokinetic in vitro and clinical experiments (repo.napdi.org). The design is expected to help researchers more easily arrive at a complete understanding of pharmacokinetic NPDI research on a particular NP. The repository will also facilitate multidisciplinary collaborations, as the repository links all of the experimental data for a given NP across the study types. The current work describes the design of the repository, standard operating procedures used to enter data, and pharmacokinetic NPDI data that have been entered to date. To illustrate the usefulness of the NaPDI Center repository, more details on two high-priority NPs, cannabis and kratom, are provided as case studies. SIGNIFICANCE STATEMENT: The data and knowledge resulting from natural product-drug interaction (NPDI) studies is distributed across a variety of information sources, rendering difficulties to find, access, and reuse. The Center of Excellence for NPDI Research addressed these difficulties by developing the first user-friendly online repository that stores data from in vitro and clinical pharmacokinetic NPDI experiments and links them with study data from chemical characterization and metabolomics analyses of natural products that are also stored in the repository.

Modulation of Major Human Liver Microsomal Cytochromes P450 by Component Alkaloids of Goldenseal: Time-Dependent Inhibition and Allosteric Effects.

Botanical and other natural products (NPs) are often coconsumed with prescription medications, presenting a risk for cytochrome P450 (P450)-mediated NP-drug interactions. The NP goldenseal (Hydrastis canadensis) has exhibited antimicrobial activities in vitro attributed to isoquinoline alkaloids contained in the plant, primarily berberine, (-)-ß-hydrastine, and to a lesser extent, hydrastinine. These alkaloids contain methylenedioxyphenyl rings, structural alerts with potential to inactivate P450s through formation of metabolic intermediate complexes. Time-dependent inhibition experiments were conducted to evaluate their ability to inhibit major P450 activities in human liver microsomes by using a cocktail of isozyme-specific substrate probes. Berberine inhibited CYP2D6 (dextromethorphan O-demethylation; K I = 2.7 µM, kinact = 0.065 minute-1) and CYP3A4/5 (midazolam 1'-hydroxylation; K I = 14.8 µM, kinact = 0.019 minute-1); (-)-ß-hydrastine inhibited CYP2C9 (diclofenac 4'-hydroxylation; K I = 49 µM, kinact = 0.036 minute-1), CYP2D6 (K I > 250 µM, kinact > 0.06 minute-1), and CYP3A4/5 (K I = 28 µM, kinact = 0.056 minute-1); and hydrastinine inhibited CYP2D6 (K I = 37 µM, kinact = 0.049 minute-1) activity. Berberine additionally exhibited allosteric effects on midazolam hydroxylation, showing both positive and negative heterotropic cooperativity. Experiments with recombinant isozymes showed that berberine activated midazolam 1'-hydroxylation by CYP3A5, lowering K m(app), but showed mixed inhibition and negative cooperativity toward this reaction when catalyzed by CYP3A4. Berberine inactivated CYP3A4 at a much faster rate than CYP3A5 and was a noncompetitive inhibitor of midazolam 4-hydroxylation by CYP3A4 but a strong mixed inhibitor of the CYP3A5 catalyzed reaction. These complex kinetics should be considered when extrapolating the risk for NP-drug interactions involving goldenseal. SIGNIFICANCE STATEMENT: Robust kinetic parameters were determined for the reversible and time-dependent inhibition of CYP2C9, CYP2D6, and CYP3A4/5 activities in human liver microsomes by major component isoquinoline alkaloids contained in the botanical natural product goldenseal. The alkaloid berberine also exhibited opposing, isozyme-specific allosteric effects on midazolam hydroxylation mediated by recombinant CYP3A4 (inhibition) and CYP3A5 (activation). These data will inform the development of a physiologically based pharmacokinetic model that can be used to predict potential clinically relevant goldenseal-drug interactions.