Enzyme-mediated drug-drug interactions: a review of in vivo and in vitro methodologies, regulatory guidance, and translation to the clinic.

Enzyme-mediated pharmacokinetic drug-drug interactions can be caused by altered activity of drug metabolizing enzymes in the presence of a perpetrator drug, mostly via inhibition or induction. We identified a gap in the literature for a state-of-the art detailed overview assessing this type of DDI risk in the context of drug development. This manuscript discusses in vitro and in vivo methodologies employed during the drug discovery and development process to predict clinical enzyme-mediated DDIs, including the determination of clearance pathways, metabolic enzyme contribution, and the mechanisms and kinetics of enzyme inhibition and induction. We discuss regulatory guidance and highlight the utility of in silico physiologically-based pharmacokinetic modeling, an approach that continues to gain application and traction in support of regulatory filings. Looking to the future, we consider DDI risk assessment for targeted protein degraders, an emerging small molecule modality, which does not have recommended guidelines for DDI evaluation. Our goal in writing this report was to provide early-career researchers with a comprehensive view of the enzyme-mediated pharmacokinetic DDI landscape to aid their drug development efforts.

CYP2J2-mediated metabolism of arachidonic acid in heart: A review of its kinetics, inhibition and role in heart rhythm control.

Cytochrome P450 2 J2 (CYP2J2) is primarily expressed extrahepatically and is the predominant epoxygenase in human cardiac tissues. This highlights its key role in the metabolism of endogenous substrates. Significant scientific interest lies in cardiac CYP2J2 metabolism of arachidonic acid (AA), an omega-6 polyunsaturated fatty acid, to regioisomeric bioactive epoxyeicosatrienoic acid (EET) metabolites that show cardioprotective effects including regulation of cardiac electrophysiology. From an in vitro perspective, the accurate characterization of the kinetics of CYP2J2 metabolism of AA including its inhibition and inactivation by drugs could be useful in facilitating in vitro-in vivo extrapolations to predict drug-AA interactions in drug discovery and development. In this review, background information on the structure, regulation and expression of CYP2J2 in human heart is presented alongside AA and EETs as its endogenous substrate and metabolites. The in vitro and in vivo implications of the kinetics of this endogenous metabolic pathway as well as its perturbation via inhibition and inactivation by drugs are elaborated. Additionally, the role of CYP2J2-mediated metabolism of AA to EETs in cardiac electrophysiology will be expounded.

Electrochemical Acetylcholinesterase Sensors for Anti-Alzheimer's Disease Drug Determination.

Neurodegenerative diseases and Alzheimer's disease (AD), as one of the most common causes of dementia, result in progressive losses of cholinergic neurons and a reduction in the presynaptic markers of the cholinergic system. These consequences can be compensated by the inhibition of acetylcholinesterase (AChE) followed by a decrease in the rate of acetylcholine hydrolysis. For this reason, anticholinesterase drugs with reversible inhibition effects are applied for the administration of neurodegenerative diseases. Their overdosage, variation in efficiency and recommendation of an individual daily dose require simple and reliable measurement devices capable of the assessment of the drug concentration in biological fluids and medications. In this review, the performance of electrochemical biosensors utilizing immobilized cholinesterases is considered to show their advantages and drawbacks in the determination of anticholinesterase drugs. In addition, common drugs applied in treating neurodegenerative diseases are briefly characterized. The immobilization of enzymes, nature of the signal recorded and its dependence on the transducer modification are considered and the analytical characteristics of appropriate biosensors are summarized for donepezil, huperzine A, rivastigmine, eserine and galantamine as common anti-dementia drugs. Finally, the prospects for the application of AChE-based biosensors in clinical practice are discussed.

Partial Reversible Inhibition of Enzymes and Its Metabolic and Pharmaco-Toxicological Implications.

Partial reversible inhibition of enzymes, also called hyperbolic inhibition, is an uncommon mechanism of reversible inhibition, resulting from a productive enzyme-inhibitor complex. This type of inhibition can involve competitive, mixed, non-competitive and uncompetitive inhibitors. While full reversible inhibitors show linear plots for reciprocal enzyme initial velocity versus inhibitor concentration, partial inhibitors produce hyperbolic plots. Similarly, dose-response curves show residual fractional activity of enzymes at high doses. This article reviews the theory and methods of analysis and discusses the significance of this type of reversible enzyme inhibition in metabolic processes, and its implications in pharmacology and toxicology.

Mechanism of D-Cycloserine Inhibition of D-Amino Acid Transaminase from Haliscomenobacter hydrossis.

D-cycloserine inhibits pyridoxal-5'-phosphate (PLP)-dependent enzymes. Inhibition effect depend on organization of the active site and mechanism of the catalyzed reaction. D-cycloserine interacts with the PLP form of the enzyme similarly to the substrate (amino acid), and this interaction is predominantly reversible. Several products of the interaction of PLP with D-cycloserine are known. For some enzymes formation of a stable aromatic product - hydroxyisoxazole-pyridoxamine-5'-phosphate at certain pH - leads to irreversible inhibition. The aim of this work was to study the mechanism of D-cycloserine inhibition of the PLP-dependent D-amino acid transaminase from Haliscomenobacter hydrossis. Spectral methods revealed several products of interaction of D-cycloserine with PLP in the active site of transaminase: oxime between PLP and ß-aminooxy-D-alanine, ketimine between pyridoxamine-5'-phosphate and cyclic form of D-cycloserine, and pyridoxamine-5'-phosphate. Formation of hydroxyisoxazole-pyridoxamine-5'-phosphate was not observed. 3D structure of the complex with D-cycloserine was obtained using X-ray diffraction analysis. In the active site of transaminase, a ketimine adduct between pyridoxamine-5'-phosphate and D-cycloserine in the cyclic form was found. Ketimine occupied two positions interacting with different active site residues via hydrogen bonds. Using kinetic and spectral methods we have shown that D-cycloserine inhibition is reversible, and activity of the inhibited transaminase from H. hydrossis could be restored by adding excess of keto substrate or excess of cofactor. The obtained results confirm reversibility of the inhibition by D-cycloserine and interconversion of various adducts of D-cycloserine and PLP.

Investigating the relevance of CYP2J2 inhibition for drugs known to cause intermediate to high risk torsades de pointes.

Cardiac cytochrome P450 2J2 (CYP2J2) metabolizes endogenous polyunsaturated fatty acid, arachidonic acid (AA), to bioactive regioisomeric epoxyeicosatrienoic acid (EET) metabolites. This endogenous metabolic pathway has been postulated to play a homeostatic role in cardiac electrophysiology. However, it is unknown if drugs that cause intermediate to high risk torsades de pointes (TdP) exhibit inhibitory effects against CYP2J2 metabolism of AA to EETs. In this study, we demonstrated that 11 out of 16 drugs screened with intermediate to high risk of TdP as defined by the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative are concurrently reversible inhibitors of CYP2J2 metabolism of AA, with unbound inhibitory constant (Ki,AA,u) values ranging widely from 0.132 to 19.9 µM. To understand the physiological relevancy of Ki,AA,u, the in vivo unbound drug concentration within human heart tissue (Cu,heart) was calculated via experimental determination of in vitro unbound partition coefficient (Kpuu) for 10 CYP2J2 inhibitors using AC16 human ventricular cardiomyocytes as well as literature-derived values of fraction unbound in plasma (fu,p) and plasma drug concentrations in clinical scenarios leading to TdP. Notably, all CYP2J2 inhibitors screened belonging to the high TdP risk category, namely vandetanib and bepridil, exhibited highest Kpuu values of 18.2 ± 1.39 and 7.48 ± 1.16 respectively although no clear relationship between Cu,heart and risk of TdP could eventually be determined. R values based on basic models of reversible inhibition as per FDA guidelines were calculated using unbound plasma drug concentrations (Cu,plasma) and adapted using Cu,heart which suggested that 4 out of 10 CYP2J2 inhibitors with intermediate to high risk of TdP demonstrate greatest potential for clinically relevant in vivo cardiac drug-AA interactions. Our results shed novel insights on the relevance of CYP2J2 inhibition in drugs with risk of TdP. Further studies ascertaining the role of CYP2J2 metabolism of AA in cardiac electrophysiology, characterizing inherent cardiac ion channel activities of drugs with risk of TdP as well as in vivo evidence of drug-AA interactions will be required prior to determining if CYP2J2 inhibition could be an alternative mechanism contributing to drug-induced TdP.

A Physiologically Based Pharmacokinetic Model of Ketoconazole and Its Metabolites as Drug-Drug Interaction Perpetrators.

The antifungal ketoconazole, which is mainly used for dermal infections and treatment of Cushing's syndrome, is prone to drug-food interactions (DFIs) and is well known for its strong drug-drug interaction (DDI) potential. Some of ketoconazole's potent inhibitory activity can be attributed to its metabolites that predominantly accumulate in the liver. This work aimed to develop a whole-body physiologically based pharmacokinetic (PBPK) model of ketoconazole and its metabolites for fasted and fed states and to investigate the impact of ketoconazole's metabolites on its DDI potential. The parent-metabolites model was developed with PK-Sim® and MoBi® using 53 plasma concentration-time profiles. With 7 out of 7 (7/7) DFI AUClast and DFI Cmax ratios within two-fold of observed ratios, the developed model demonstrated good predictive performance under fasted and fed conditions. DDI scenarios that included either the parent alone or with its metabolites were simulated and evaluated for the victim drugs alfentanil, alprazolam, midazolam, triazolam, and digoxin. DDI scenarios that included all metabolites as reversible inhibitors of CYP3A4 and P-gp performed best: 26/27 of DDI AUClast and 21/21 DDI Cmax ratios were within two-fold of observed ratios, while DDI models that simulated only ketoconazole as the perpetrator underperformed: 12/27 DDI AUClast and 18/21 DDI Cmax ratios were within the success limits.

Potential of Vitamin B6 Dioxime Analogues to Act as Cholinesterase Ligands.

Seven pyridoxal dioxime quaternary salts (1-7) were synthesized with the aim of studying their interactions with human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). The synthesis was achieved by the quaternization of pyridoxal monooxime with substituted 2-bromoacetophenone oximes (phenacyl bromide oximes). All compounds, prepared in good yields (43-76%) and characterized by 1D and 2D NMR spectroscopy, were evaluated as reversible inhibitors of cholinesterase and/or reactivators of enzymes inhibited by toxic organophosphorus compounds. Their potency was compared with that of their monooxime analogues and medically approved oxime HI-6. The obtained pyridoxal dioximes were relatively weak inhibitors for both enzymes (Ki = 100-400 µM). The second oxime group in the structure did not improve the binding compared to the monooxime analogues. The same was observed for reactivation of VX-, tabun-, and paraoxon-inhibited AChE and BChE, where no significant efficiency burst was noted. In silico analysis and molecular docking studies connected the kinetic data to the structural features of the tested compound, showing that the low binding affinity and reactivation efficacy may be a consequence of a bulk structure hindering important reactive groups. The tested dioximes were non-toxic to human neuroblastoma cells (SH-SY5Y) and human embryonal kidney cells (HEK293).

Synthesis and human monoamine oxidase inhibitory activity of novel C2-, C3- and C4-substituted phthalonitriles.

Development of new selective reversible monoamine oxidase (MAO) B inhibitors is still essential for the treatment of Alzheimer's and Parkinson's disease. Phthalonitrile compounds have been shown to display MAO inhibitory activity with MAO-B selectivity. In this study, we synthesized and evaluated the inhibitory activities of a new series of phthalonitrile compounds. Compound 3, 4 and 5 presented selective MAO-B inhibition, compound 5 being the most selective (75.16-fold). Additionally, molecular docking simulations were carried out. Investigation of binding modes of each compound with both isoforms were carried out to elaborate structure-activity relationships. Druglikeness was calculated for each compound, revealing that the lipophilicity of compound 5 (logP = 3.37) is optimal to cross membranes.

An Ultrasensitive and Selective Determination of Cadmium Ions at ppt Level Using an Enzymic Membrane with Colorimetric and Electrochemical Detection.

Cadmium ions (Cd2+) are extremely toxic heavy metal pollutants found in the environment, and which endanger human health. Therefore, it is critical to develop a sensitive and simple method for rapidly detecting Cd2+ in water samples. Herein, an enzymic membrane was developed based on an easy and rapid immobilization method of horseradish peroxidase (HRP), for determination of Cd2+ in drinking water. Hence, for the first time, an enzymic membrane was applied for the detection of Cd2+ without being pretreated. In the first format, the inhibition of horseradish peroxidase was performed using a colorimetric microplate reader. Under optimal conditions, the achieved limit of detection was 20 ppt. In addition, an electrochemical biosensor was developed, by combining the enzymic membrane with screen printed electrodes, which showed a linear calibration range between 0.02-100 ppb (R2 = 0.990) and a detection limit of 50 ppt. The use of this enzymic membrane proved to be advantageous when reversible inhibitors such as the copper ion (Cu2+) were present in water samples, as Cu2+ can interfere with Cd2+ and cause erroneous results. In order to alleviate this problem, a medium exchange procedure was used to eliminate Cu2+, by washing and leaving only cadmium ions as an irreversible inhibitor for identification. The use of this membrane proved to be a simple and rapid method of immobilizing HRP with a covalent bond.

Studies on biochemical, oxidative and genotoxicity alterations following vas blockage with reversible inhibition of sperm under guidance and reversal in rats.

CONTEXT: Vas obstruction with reversible inhibition of sperm under guidance (RISUG) for contraception and its reversal, may cause oxidative stress or inimical effects on male reproductive functions. OBJECTIVE: To evaluate the biochemical and genotoxicity at the level of reactive oxygen species (ROS) following vas occlusion with RISUG and its reversal by Dimethyl sulphoxide (DMSO) and 5% NaHCO3 in Wistar albino rats. SETTINGS AND DESIGN: Animals were divided into seven groups (n = 10), namely sham-operated control, short-term vas occlusion with RISUG for 90 days, reversal with DMSO and 5% NaHCO3, long-term vas occlusion with RISUG for 360 days, reversal with DMSO and 5% NaHCO3. MATERIALS AND METHODS: Biochemical markers in reproductive tissues, hematology, serum biochemistry, serum electrolytes and ROS measuring indicators, e.g., lipid peroxidation, superoxide dismutase, catalase, glutathione peroxidase, and glutathione S-transferase were examined. STATISTICAL ANALYSIS USED: One-way analysis of variance test was performed for analyses of data obtained in this study using the SPSS 10.0 software (SPSS Inc., Chicago, IL, USA). RESULTS: The tissue and clinical chemistry did not show appreciable alterations in RISUG injected and reversal Groups (II-VII) as compared to sham control. The genotoxicity and various ROS markers fluctuated within control limits following short- and long-term vas occlusion and reversal. CONCLUSIONS: The study suggested that the reversal procedures, following RISUG contraception, were not associated with any kind of toxicological manifestations.

Molecular recognition of acetylcholinesterase and its subnanomolar reversible inhibitor: a molecular simulations study.

Recently, we designed and synthesized a subnanomolar, reversible, dual-binding site acetylcholinesterase (AChE) inhibitor which consists of the tacrine and aroylacrylic acid phenylamide moieties, mutually linked by eight methylene units. To further investigate the process of the molecular recognition between the AChE and its inhibitor, we performed six unconstrained molecular dynamics (MD) simulations, where the compound in three possible protonation states was placed inside binding sites of two available AChE crystal structures. In all six MD trajectories, the ligand generally occupied similar space inside the AChE active site, but the pattern of the interactions between the ligand functional groups and the amino acid residues was significantly different and highly dependent upon the crystal structure used to generate initial systems for simulation. The greatest differences were observed between the trajectories obtained with different AChE crystal structures used as starting target conformations. In some trajectories, several unusual positions and dynamic behavior of the tacrine moiety were observed. Therefore, this study provides important structure-based data useful in further optimization of the reversible, dual binding AChE inhibitors, and also emphasizes the importance of the starting crystal structure used for dynamics as well as the protonation state of the reversible inhibitors.Communicated by Ramaswamy H. Sarma.

Reversible Mechanisms of Enzyme Inhibition and Resulting Clinical Significance.

Inhibition of a drug-metabolizing enzyme by the reversible interaction of a drug with the enzyme, thus decreasing the metabolism of another drug, is a major cause of clinically significant drug-drug interactions. This chapter defines the four reversible mechanisms of inhibition exhibited by drugs: competitive, noncompetitive, uncompetitive, and mixed competitive/noncompetitive. An in vitro procedure to determine the potential of a drug to be a reversible inhibitor is also provided. Finally, a number of examples of clinically significant drug-drug interactions resulting from reversible inhibition are described.

Mutual inhibition in presence of a virus in continuous culture.

In this paper, we consider two species competing for a limiting substrate such that each species impedes the growth of the other one (Mutual inhibition) in presence of a virus inhibiting one bacterial species. A system of ordinary differential equations is proposed as a mathematical model for this competition. A detailed local qualitative analysis of the system is carried out. We proved that for a general nonlinear growth rates, the Competitive Exclusion Principle still valid, that at least one species goes extinct. For some cases where we have two locally stable equilibrium points, initial species concentrations are important in determining which is the winning species. Obtained results were confirmed by some numerical simulations using Matlab software.

Atypical kinetics of cytochrome P450 2J2: Epoxidation of arachidonic acid and reversible inhibition by xenobiotic inhibitors.

Extrahepatic CYP2J2 metabolism of arachidonic acid (AA) to bioactive regioisomeric epoxyeicosatrienoic acids (EETs) is implicated in both physiological and pathological conditions. Here, we aimed to characterize atypical substrate inhibition kinetics of this endogenous metabolic pathway and its reversible inhibition by xenobiotic inhibitors when AA is used as the physiologically-relevant substrate vis-à-vis conventional probe substrate astemizole (AST). As compared to typical Michaelis-Menten kinetics observed for AST, complete substrate inhibition was observed for CYP2J2 metabolism of AA to 14,15-EET whereby velocity of the reaction declined significantly at concentrations of AA above 20-30 µM with an estimated substrate inhibition constant (Ks) of 31 µM. In silico sequential docking of two AA substrates to orthosteric (OBS) and adjacent secondary binding sites (SBS) within a 3-dimensional homology model of CYP2J2 revealed favorable and comparable binding poses of glide-scores -3.1 and -3.8 respectively. Molecular dynamics (MD) simulations ascertained CYP2J2 conformational stability with dual AA substrate binding as time-dependent root mean squared deviation (RMSD) of protein Cα atoms and ligand heavy atoms stabilized to a plateau in all but one trajectory (n=6). The distance between heme-iron and ω6 (C14, C15) double bond of AA in OBS also increased from 7.5 ± 1.4 Å to 8.5 ± 1.8 Å when CYP2J2 was simulated with only AA in OBS versus the presence of AA in both OBS and SBS (p<0.001), supporting the observed in vitro substrate inhibition phenomenon. Poor correlation was observed between inhibitory constants (Ki) determined for a panel of nine competitive and mixed mode xenobiotic inhibitors against CYP2J2 metabolism of AA as compared to AST, whereby 4 out of 9 drugs had a greater than 5-fold difference between Ki values. Nonlinear Eadie-Hofstee plots illustrated that complete substrate inhibition of CYP2J2 by AA was not attenuated even at high concentrations of xenobiotic inhibitors which further corroborates that CYP2J2 may accommodate three or more ligands simultaneously. In light of the atypical kinetics, our results highlight the importance of using physiologically-relevant substrates in in vitro enzymatic inhibition assays for the characterization of xenobiotic-endobiotic interactions which is applicable to other complex endogenous metabolic pathways beyond CYP2J2 metabolism of AA to EETs. The accurate determination of Ki would further facilitate the association of xenobiotic-endobiotic interactions to observed therapeutic or toxic outcomes.

Taxifolin and gastro-adhesive microparticles containing taxifolin promotes gastric healing in vivo, inhibits Helicobacter pylori in vitro and proton pump reversibly in silico.

Taxifolin (3,5,7,3,4-pentahydroxy flavanone or dihydroquercetin, Tax) was identified as a gastroprotective compound and a gastroadhesive formulation was recently developed to prolong its residence time and release in the stomach. So, the gastric healing effectiveness of Tax and gastro-mucoadhesive microparticles containing Tax (MPTax) against the acetic acid induced-gastric ulcer in rats was investigated in this study. Moreover, the interactions between Tax and H+/K+-ATPase were investigated in silico, and its anti- H. pylori activity was determined in vitro. The oral treatment with MPTax (81.37 mg/kg, containing 12.29% of Tax) twice a day for seven days reduced the ulcer area by 63%, compared to vehicle-treated group (Veh: 91.9 ± 10.3 mm2). Tax (10 mg/kg, p.o) reduced the ulcer by 40% but with a p = 0.07 versus Veh group. Histological analysis confirmed these effects. Tax and MPTax increased the gastric mucin amount, reduced the myeloperoxidase activity, and increased the glutathione reduced content at ulcer site. However, only MPTax decreased the lipoperoxide accumulation at ulcer site. Besides, Tax and MPTax normalize the catalase and glutathione S-transferase activity. Tax showed reversible interaction with H+/K+-ATPase in silico and its anti-H. pylori effects was confirmed (MIC = 625 µg/mL). These results suggest that the antiulcer property of Tax involves the strengthening of the gastric protective factors in parallel to its inhibitory interaction with H+/K+-ATPase and H. pylori. Considering that ulcer healing action displayed by Tax was favored by gastroadhesive microparticles, this approach seems to be promising for its oral delivery to treat acid-peptic diseases.

Inhibition of Myeloperoxidase.

Myeloperoxidase participates in innate immune defense mechanism through formation of microbicidal reactive oxidants and diffusible radical species. A unique activity is its ability to use chloride as a cosubstrate with hydrogen peroxide to generate chlorinating oxidants such as hypochlorous acid, a potent antimicrobial agent. However, chronic MPO activation can lead to indiscriminate protein modification causing tissue damage, and has been associated with chronic inflammatory diseases, atherosclerosis, and acute cardiovascular events. This has attracted considerable interest in the development of therapeutically useful MPO inhibitors. Today, based on the profound knowledge of structure and function of MPO and its biochemical and biophysical differences with the other homologous human peroxidases, various rational and high-throughput screening attempts were performed in developing specific irreversible and reversible inhibitors. The most prominent candidates as well as MPO inhibitors already studied in clinical trials are introduced and discussed.

Discovery of novel potential plant growth regulators from Corydalis mucronifera.

Three pairs of enantiomers mucroniferals A-C (1-3), with a novel skeleton of 1,4-epoxynaphthalene-2,3-dicarboxylic acid first reported from nature source, were isolated from Corydalis mucronifera. Their structures were elucidated based on extensive spectroscopic data analysis of MS, 1D and 2D NMR, and their absolute configurations were confirmed by single-crystal X-ray diffraction analysis and comparison of the experimental and calculated ECD data. Mucroniferals A-C showed broad-spectrum inhibitory activities on seedling growth of all plants tested (Lepidium apetalum, Raphanus sativus, Lactuca sativa, and Arabidopsis thaliana) with a dose-dependent relationship. Additionally, mucroniferals A and B exhibited significant inhibitory effects on germination of most seeds at concentration of 80 µg/mL, and the inhibition was reversible.

N-Sulfonyl dipeptide nitriles as inhibitors of human cathepsin S: In silico design, synthesis and biochemical characterization.

A library of cathepsin S inhibitors of the dipeptide nitrile chemotype, bearing a bioisosteric sulfonamide moiety, was synthesized. Kinetic investigations were performed at four human cysteine proteases, i.e. cathepsins S, B, K and L. Compound 12 with a terminal 3-biphenyl sulfonamide substituent was the most potent (Ki = 4.02 nM; selectivity ratio cathepsin S/K = 5.8; S/L = 67) and 24 with a 4'-fluoro-4-biphenyl sulfonamide substituent the most selective cathepsin S inhibitor (Ki = 35.5 nM; selectivity ratio cathepsin S/K = 57; S/L = 31). In silico design and biochemical evaluation emphasized the impact of the sulfonamide linkage on selectivity and a possible switch of P2 and P3 substituents with respect to the occupation of the corresponding binding sites of cathepsin S.

Deoxyshikonin reversibly inhibits cytochrome P450 2B6.

Deoxyshikonin, a natural shikonin derivative, is the major component of Lithospermum erythrorhizon and exhibits various pharmacological effects such as lymphangiogenetic, antibacterial, wound healing, and anticancer effects. To investigate the herb-drug interaction potential associated with deoxyshikonin, the inhibitory effects of deoxyshikonin on eight major cytochrome P450 (CYP) enzymes were examined using cocktail substrate-incubated human liver microsomes. Deoxyshikonin strongly inhibited CYP2B6-catalyzed bupropion hydroxylation, with a Ki value of 3.5 µM, and the inhibition was confirmed using purified human CYP2B6. The inhibition was reversible because the inhibitory effect of deoxyshikonin was not dependent on the preincubation time. The results indicated that deoxyshikonin-induced drug-drug interaction should be considered when any herb containing deoxyshikonin is used for conventional medications.