Tuning Surface Plasmon Resonance Responses through Size and Crosslinking Control of Multivalent Protein Binding-Capable Nanoscale Hydrogels.

Surface plasmon resonance (SPR) phenomena have been widely studied to detect biomolecules because of their high sensitivity and ability to determine biomolecular interactions with kinetic information. However, highly selective detection in specific concentration ranges relevant to target biomolecules is still a challenging task. Recently, we developed bioresponsive nanoscale hydrogels to selectively intensify SPR signals through multivalent protein binding (MPB) events with target biomolecules, including IL-2, where we were able to demonstrate exceptional selectivity for target biomolecules with minimal responses to nonspecific and monovalent binding events. In this work, we systematically explored the relationship between the physical properties of MPB-capable nanoscale hydrogels and their SPR response induced in the presence of the programmed cell death protein 1 antibody (PD-1Ab) as a model target biomolecule. First, we developed a synthetic protocol by controlling various reaction parameters to construct a library of nanoscale poly(N-isopropylacrylamide-co-acrylic acid) hydrogels (NHs) with different sizes (from 400 nm to 1 µm) and degrees of crosslinking (from 2 to 8%). Then, by incorporating MPB-capable PD-1 receptors onto the surface of NHs to form PD-1-responsive nanoscale hydrogels (PNHs), the hydrogel size and crosslinking dependency of their SPR responses were investigated. Our results reveal the appropriate hydrogel size regime and degree of crosslinking for effective PD-1Ab detection at specific concentrations range between a few nM and 1 µM. Overall, our study demonstrates that by tuning the physical properties of the nanoscale hydrogel matrix, the sensitivity and detection range of MPB-based SPR sensors can be modulated to potentially benefit clinical applications such as monitoring diverse therapeutic biomolecules.

Metabolite identification and profile of endosulfan sulfate in three human liver preparations using liquid chromatography-high resolution mass spectrometry.

In this study, we performed the metabolism of endosulfan sulfate in human liver preparations (human liver microsomes, S9 fractions and hepatocytes) to identify new metabolites using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Endosulfan sulfate is a major oxidized metabolite of the organochlorine insecticide endosulfan, and it exhibits a similar toxicity to endosulfan. Six metabolites, including 5 novel metabolites of endosulfan sulfate, were identified in the three different human liver reaction mixtures and metabolic pathways of endosulfan sulfate were proposed. The phase I metabolites M1 and M2 were observed in human liver microsomes, S9 fractions and hepatocytes. M1 was suggested to be an endosulfan diol monosulfate and M2 was identified as (1,4,5,6,7,7-hexachloro-3-formylbicyclo[2,2,1]hept-5-en-2-yl)methyl hydrogen sulfate through the interpretation of the HRMS spectrum. The phase II metabolite M3 was produced as an endosulfan sulfate-GSH conjugate in those three liver preparations and transformed to M5 (dipeptide) in S9 fractions and hepatocytes. M3 was the most predominant metabolite identified in the three liver preparations. M4 was only detected in microsomes as an M2-GSH conjugate and was metabolized to M6 (monopeptide) in hepatocytes. These results are different from the metabolic pathway of endosulfan and suggest the possible detoxification metabolic reaction of endosulfan sulfate in living organisms.

Simultaneous determination of 75 abuse drugs including amphetamines, benzodiazepines, cocaine, opioids, piperazines, zolpidem and metabolites in human hair samples using liquid chromatography-tandem mass spectrometry.

A liquid chromatography-tandem mass spectrometric method for the simultaneous determination of 75 abuse drugs and metabolites, including 19 benzodiazepines, 19 amphetamines, two opiates, eight opioids, cocaine, lysergic acid diethylamide, zolpidem, three piperazines and 21 metabolites in human hair samples, was developed and validated. Ten-milligram hair samples were decontaminated, pulverized using a ball mill, extracted with 1 mL of methanol spiked with 28 deuterated internal standards in an ultrasonic bath for 60 min at 50°C, and purified with Q-sep dispersive solid-phase extraction tubes. The purified extracts were evaporated to dryness and the residue was dissolved in 0.1 mL of 10% methanol. The 75 analytes were analyzed on an Acquity HSS T3 column using gradient elution of methanol and 0.1% formic acid and quantified in multiple reaction monitoring mode with positive electrospray ionization. Calibration curves were linear (r ≥ 0.9951) from the lower limit of quantitation (2-200 pg/mg depending on the drug) to 2000 pg/mg. The coefficients of variation and accuracy for intra- and inter-assay analysis at three QC levels were 4.3-12.9% and 89.2-109.1%, respectively. The overall mean recovery ranged from 87.1 to 105.3%. This method was successfully applied to the analysis of 11 forensic hair samples obtained from drug abusers.

Synthetic cannabinoids are substrates and inhibitors of multiple drug-metabolizing enzymes.

Synthetic cannabinoids, a new class of psychoactive substances, are potent agonists of cannabinoid receptors, which mimic the psychoactive effects of the principal psychoactive component of cannabis, ∆9-tetrahydrocannabinol. Despite governmental scheduling as illicit drugs, new synthetic cannabinoids are being produced. The abuse of synthetic cannabinoids with several drugs containing different chemical groups has resulted in large numbers of poisonings. This has increased the urgency for forensic and public health laboratories to identify the metabolites of synthetic cannabinoids and apply this knowledge to the development of analytical methods and for toxicity prediction. It is necessary to determine whether synthetic cannabinoids are involved in drug-metabolizing enzyme-mediated drug-drug interactions. This review describes the metabolic pathways of 13 prevalent synthetic cannabinoids and various drug-metabolizing enzymes responsible for their metabolism, including cytochrome P450 (CYP), UDP-glucuronosyltransferases (UGTs), and carboxylesterases. The inhibitory effects of synthetic cannabinoids on CYP and UGT activities are also reviewed to predict the potential of synthetic cannabinoids for drug-drug interactions. The drug-metabolizing enzymes responsible for metabolism of synthetic cannabinoids should be characterized and the effects of synthetic cannabinoids on CYP and UGT activities should be determined to predict the pharmacokinetics of synthetic cannabinoids and synthetic cannabinoid-induced drug-drug interactions in the clinic.

Simultaneous quantification of 18 saturated and unsaturated fatty acids and 7 sterols as their tert-butyldimethylsilyl derivatives in human saliva using gas chromatography-tandem mass spectrometry.

The profiling of fatty acids (FAs) or sterols has been applied in clinical studies, but still needs to be improved to enable their simultaneous quantification. Moreover, little progress has been made in determining the levels of FAs and sterols in human saliva in a single run. In this study, gas chromatography-tandem mass spectrometry (GC-MS/MS) using one-step tert-butyldimethylsilyl (TBDMS) derivatization was developed for comprehensive profiling of 18 FAs (eight saturated, five monounsaturated, and five polyunsaturated FAs) and 7 sterols (cholesterol and its precursors). The TBDMS derivatization process was also optimized in terms of reaction solvent, catalyst, temperature, and reaction time. The optimized conditions resulted in better derivatization efficiency with good chromatographic separation through a high-temperature column within 23 min. The present method provided good linearity (r > 0.993), precision (coefficient of variation, 2.7% to 10.4%), and accuracy (91.5% to 103.4%). The overall recovery ranged from 73.8% to 114.3% for the 18 FAs, and from 68.9% to 79.8% for the 7 sterols. The validated method was applied to characterize FAs and sterols in human saliva samples. This is the first report of a GC-MS/MS method for the simultaneous determination of various FAs and sterols from a small volume (100 µL) of saliva. This approach can be used as a primary screening tool to examine the levels of both FAs and sterols in saliva, providing detailed information about their homeostasis for diagnostic and prognostic purposes.

In Vitro Inhibitory Effects of Synthetic Cannabinoid EAM-2201 on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes.

EAM-2201, a synthetic cannabinoid, is a potent agonist of the cannabinoid receptors that is widely abused as an illicit recreational drug in combination with other drugs. To evaluate the potential of EAM-2201 as a perpetrator of drug−drug interactions, the inhibitory effects of EAM-2201 on major drug-metabolizing enzymes, cytochrome P450s (CYPs) and uridine 5'-diphospho-glucuronosyltransferases (UGTs) were evaluated in pooled human liver microsomes using liquid chromatography−tandem mass spectrometry (LC-MS/MS). EAM-2201 at doses up to 50 µM negligibly inhibited the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4) and five UGTs (1A1, 1A4, 1A6, 1A9 and 2B7) in human liver microsomes. EAM-2201 exhibited time-dependent inhibition of CYP2C8-catalyzed amodiaquine N-deethylation, CYP2C9-catalyzed diclofenac 4'-hydroxylation, CYP2C19-catalyzed [S]-mephenytoin 4'-hydroxylation and CYP3A4-catalyzed midazolam 1'-hydroxylation with Ki values of 0.54 µM (kinact: 0.0633 min−1), 3.0 µM (kinact: 0.0462 min−1), 3.8 µM (kinact: 0.0264 min−1) and 4.1 µM (kinact: 0.0250 min−1), respectively and competitively inhibited UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, with a Ki value of 2.4 µM. Based on these in vitro results, we conclude that EAM-2201 has the potential to trigger in vivo pharmacokinetic drug interactions when co-administered with substrates of CYP2C8, CYP2C9, CYP2C19, CYP3A4 and UGT1A3.

Targeted and non-targeted metabolite identification of MAM-2201 in human, mouse, and rat hepatocytes.

MAM-2201 is a fluorinated naphthoylindole synthetic cannabinoid with potent psychoactive properties that has been detected as an active ingredient in herbal incense blends. To gain a greater understanding of MAM-2201 metabolism and to compare its metabolic fate in humans with those in animals, the metabolism of MAM-2201 in human, mouse, and rat hepatocytes was investigated using liquid chromatography-high-resolution mass spectrometry combined with targeted and non-targeted metabolite profiling approaches. Nineteen phase I metabolites (M1-M19) reported previously in human liver microsomes and 13 novel metabolites were identified in human, mouse, and rat hepatocytes: 1 phase I metabolite (M20) and 12 phase II metabolites including 6 glucuronides (G1-G6), 1 sulfate (S1), and 5 glutathione (GSH) conjugates (GS1-GS5) of MAM-2201 metabolites. G3 was human-specific, but M20, G1, G2, and 5 GSH conjugates were rat-specific, indicating species-related differences in MAM-2201 metabolism. The findings in the present study can be useful for the experimental design and assessment of metabolism-mediated toxic risk of MAM-2201.

Inhibition of cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferases by MAM-2201 in human liver microsomes.

MAM-2201, a synthetic cannabinoid, is a potent agonist of the cannabinoid receptors and is increasingly used as an illicit recreational drug. The inhibitory effects of MAM-2201 on major drug-metabolizing enzymes such as cytochrome P450s (CYPs) and uridine 5'-diphospho-glucuronosyltransferases (UGTs) have not yet been investigated although it is widely abused, sometimes in combination with other drugs. We evaluated the inhibitory effects of MAM-2201 on eight major human CYPs (CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six UGTs (UGTs 1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) of pooled human liver microsomes; we thus explored potential MAM-2201-induced drug interactions. MAM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4'-hydroxylation, CYP3A4-catalyzed midazolam 1'-hydroxylation, and UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, with K i values of 5.6, 5.4 and 5.0 µM, respectively. MAM-2201 exhibited mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-de-ethylation with K i and k inact values of 1.0 µM and 0.0738 min-1, respectively. In human liver microsomes, MAM-2201 (50 µM) negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, UGT1A9, and UGT2B7. Based on these in vitro results, we conclude that MAM-2201 has the potential to trigger in vivo pharmacokinetic drug interactions when co-administered with substrates of CYP2C8, CYP2C9, CYP3A4, and UGT1A3.

AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5'-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes.

AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP) or uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) enzymes in pooled human liver microsomes using liquid chromatography-tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4'-hydroxylation, CYP3A4-catalyzed midazolam 1'-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 µM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 µM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 µM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.

Metabolic characterization of (1-(5-fluoropentyl)-1H-indol-3-yl)(4-methyl-1-naphthalenyl)-methanone (MAM-2201) using human liver microsomes and cDNA-overexpressed cytochrome P450 enzymes.

MAM-2201 is a synthetic cannabinoid that is increasingly found in recreational drug abusers and cases of severe intoxication. Thus, characterization of the metabolic pathways of MAM-2201 is necessary to predict individual pharmacokinetics and toxicity differences, and to avoid toxic drug-drug interactions. Collectively, 19 phase 1 metabolites of MAM-2201 were identified using liquid chromatography-Orbitrap mass spectrometry following human liver microsomal incubations in the presence of NADPH: 7 hydroxy-MAM-2201 (M1-M7), 4 dihydroxy-MAM-2201 (M8-M11), dihydrodiol-MAM-2201 (M12), N-(5-hydroxypentyl)-MAM-2201 (M13), hydroxy-M13 (M14), N-dealkyl-MAM-2201 (M15), 2 hydroxy-M15 (M16, M17), MAM-2201 N-pentanoic acid (M18), and hydroxy-M18 (M19). On the basis of intrinsic clearance values in human liver microsomes, hydroxy-MAM-2201 (M1), N-(5-hydroxypentyl)-MAM-2201 (M13), and hydroxy-M13 (M14) were the major metabolites. Based on an enzyme kinetics study using human cDNA-expressed cytochrome P450 (CYP) enzymes and an immunoinhibition study using selective CYP antibodies in human liver microsomes, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 enzymes were responsible for MAM-2201 metabolism. The CYP3A4 enzyme played a prominent role in MAM-2201 metabolism, and CYP1A2, CYP2B6, CYP2C8, and CYP2C9 enzymes played major roles in the formation of some metabolites. MAM-2201 is extensively metabolized by multiple CYP enzymes, indicating that MAM-2201 and its metabolites should be used as markers of MAM-2201 abuse and toxicity. Graphical abstract In vitro metabolic pathways of MAM-2201 were characterized in human liver microsomes and recombinant CYPs using LC-HRMS analysis. Total 19 phase I metabolites were identified with predominant contribution of CYP3A4.

Rapid analysis of drugs of abuse and their metabolites in human urine using dilute and shoot liquid chromatography-tandem mass spectrometry.

Liquid chromatography-tandem mass spectrometric method for analysis of 113 abuse drugs and their metabolites in human urine was developed and validated. A simple sample clean-up procedure using the "dilute and shoot" approach, followed by reversed phase separation, provided a fast and reliable method for routine analysis. Drugs were separated in a Capcell Pak MG-III C18 column using a gradient elution of 1 mM ammonium formate with 0.1% formic acid in water and acetonitrile. The total time for analysis was 32 min. The multiple reaction monitoring mode using two transitions (e.g., quantifier and qualifier) was optimized for both identification and determination. The calibration curves for each analyte were linear over the concentration ranges of 1-100, 5-100, or 10-100 ng/mL using 400 µL of human urine sample with the coefficient of determination above 0.9921. The coefficient of variation and accuracy for the intra- and inter-assays of the tested drugs at three QC levels were 1.1-14.6 and 86.7-106.8%, respectively. The present method was successfully applied to the analysis of forensic urine samples obtained from 17 drug abusers. This method is useful for the rapid and accurate determination of multiple drug abuse with a small amount of urine in forensic and clinical toxicology.

Comparative metabolism of honokiol in mouse, rat, dog, monkey, and human hepatocytes.

Honokiol has antitumor, antioxidative, anti-inflammatory, and antithrombotic effects. Here we aimed to identify the metabolic profile of honokiol in mouse, rat, dog, monkey, and human hepatocytes and to characterize the enzymes responsible for the glucuronidation and sulfation of honokiol. Honokiol had a high hepatic extraction ratio in all five species, indicating that it was extensively metabolized. A total of 32 metabolites, including 17 common and 15 different metabolites, produced via glucuronidation, sulfation, and oxidation of honokiol allyl groups were tentatively identified using liquid chromatography-high resolution quadrupole Orbitrap mass spectrometry. Glucuronidation of honokiol to M8 (honokiol-4-glucuronide) and M9 (honokiol-2'-glucuronide) was the predominant metabolic pathway in hepatocytes of all five species; however, interspecies differences between 4- and 2'-glucuronidation of honokiol were observed. UGT1A1, 1A8, 1A9, 2B15, and 2B17 played major roles in M8 formation, whereas UGT1A7 and 1A9 played major roles in M9 formation. Human cDNA-expressed SULT1C4 played a major role in M10 formation (honokiol-2'-sulfate), whereas SULT1A1*1, 1A1*2, and 1A2 played major roles in M11 formation (honokiol-4-sulfate). In conclusion, honokiol metabolism showed interspecies differences.

In vitro metabolism of a novel synthetic cannabinoid, EAM-2201, in human liver microsomes and human recombinant cytochrome P450s.

In vitro metabolism of a new synthetic cannabinoid, EAM-2201, has been investigated with human liver microsomes and major cDNA-expressed cytochrome P450 (CYP) isozymes using liquid chromatography-high resolution mass spectrometry (LC-HRMS). Incubation of EAM-2201 with human liver microsomes in the presence of NADPH resulted in the formation of 37 metabolites, including nine hydroxy-EAM-2201 (M1-M9), five dihydroxy-EAM-2201 (M10-M14), dihydrodiol-EAM-2201 (M15), oxidative defluorinated EAM-2201 (M16), two hydroxy-M16 (M17 and M18), three dihydroxy-M16 (M19-M21), N-dealkyl-EAM-2201 (M22), two hydroxy-M22 (M23 and M24), dihydroxy-M22 (M25), EAM-2201 N-pentanoic acid (M26), hydroxy-M26 (M27), dehydro-EAM-2201 (M28), hydroxy-M28 (M29), seven dihydroxy-M28 (M30-M36), and oxidative defluorinated hydroxy-M28 (M37). Multiple CYPs, including CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2J2, 3A4, and 3A5, were involved in the metabolism of EAM-2201. In conclusion, EAM-2201 is extensively metabolized by CYPs and its metabolites can be used as an indicator of EAM-2201 abuse.

Inhibitory effects of cedrol, ß-cedrene, and thujopsene on cytochrome P450 enzyme activities in human liver microsomes.

Cedrol, ß-cedrene, and thujopsene are bioactive sesquiterpenes found in cedar essential oil and exert antiseptic, anti-inflammatory, antispasmodic, tonic, astringent, diuretic, sedative, insecticidal, and antifungal activities. These compounds are used globally in traditional medicine and cosmetics. The aim of this study was to investigate the inhibitory effects of cedrol, ß-cedrene, and thujopsene on the activities of eight major human cytochrome P-450 (CYP) enzymes using human liver microsomes to assess potential ß-cedrene-, cedrol-, and thujopsene-drug interactions. Cedrol, ß-cedrene, and thujopsene were found to be potent competitive inhibitors of CYP2B6-mediated bupropion hydroxylase with inhibition constant (Ki) values of 0.9, 1.6, and 0.8 µM, respectively, comparable with that of a selective CYP2B6 inhibitor, thioTEPA (Ki, 2.9 µM). Cedrol also markedly inhibited CYP3A4-mediated midazolam hydroxylation with a Ki value of 3.4 µM, whereas ß-cedrene and thujopsene moderately blocked CYP3A4. Cedrol, ß-cedrene, and thujopsene at 100 µM negligibly inhibited CYP1A2, CYP2A6, and CYP2D6 activities. Only thujopsene was found to be a mechanism-based inhibitor of CYP2C8, CYP2C9, and CYP2C19. Cedrol and thujopsene weakly inhibited CYP2C8, CYP2C9, and CYP2C19 activities, but ß-cedrene did not. These in vitro results indicate that cedrol, ß-cedrene, and thujopsene need to be examined for potential pharmacokinetic drug interactions in vivo due to their potent inhibition of CYP2B6 and CYP3A4.

Evaluation of the transporter-mediated herb-drug interaction potential of DA-9801, a standardized dioscorea extract for diabetic neuropathy, in human in vitro and rat in vivo.

BACKGROUND: Drug transporters play important roles in the absorption, distribution, and elimination of drugs and thereby, modulate drug efficacy and toxicity. With a growing use of poly pharmacy, concurrent administration of herbal extracts that modulate transporter activities with drugs can cause serious adverse reactions. Therefore, prediction and evaluation of drug-drug interaction potential is important in the clinic and in the drug development process. DA-9801, comprising a mixed extract of Dioscoreae rhizoma and Dioscorea nipponica Makino, is a new standardized extract currently being evaluated for diabetic peripheral neuropathy in a phase II clinical study. METHOD: The inhibitory effects of DA-9801 on the transport functions of organic cation transporter (OCT)1, OCT2, organic anion transporter (OAT)1, OAT3, organic anion transporting polypeptide (OATP)1B1, OATP1B3, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP) were investigated in HEK293 or LLC-PK1 cells. The effects of DA-9801 on the pharmacokinetics of relevant substrate drugs of these transporters were also examined in vivo in rats. RESULTS: DA-9801 inhibited the in vitro transport activities of OCT1, OCT2, OAT3, and OATP1B1, with IC50 values of 106, 174, 48.1, and 273 µg/mL, respectively, while the other transporters were not inhibited by 300 µg/mL DA-9801. To investigate whether this inhibitory effect of DA-9801 on OCT1, OCT2, and OAT3 could change the pharmacokinetics of their substrates in vivo, we measured the pharmacokinetics of cimetidine, a substrate for OCT1, OCT2, and OAT3, and of furosemide, a substrate for OAT1 and OAT3, by co-administration of DA-9801 at a single oral dose of 1,000 mg/kg. Pre-dose of DA-9801 5 min or 2 h prior to cimetidine administration decreased the Cmax of cimetidine in rats. However, DA-9801 did not affect the elimination parameters such as half-life, clearance, or amount excreted in the urine, suggesting that it did not inhibit elimination process of cimetidine, which is governed by OCT1, OCT2, and OAT3. Moreover, DA-9801 did not affect the pharmacokinetic characteristics of furosemide, as evidenced by its unchanged pharmacokinetic parameters. CONCLUSION: Inhibitory effects of DA-9801 on OCT1, OCT2, and OAT3 observed in vitro may not necessarily translate into in vivo herb-drug interactions in rats even at its maximum effective dose.

Effect of honokiol on cytochrome P450 and UDP-glucuronosyltransferase enzyme activities in human liver microsomes.

Honokiol is a bioactive component isolated from the medicinal herbs Magnolia officinalis and Magnolia grandiflora that has antioxidative, anti-inflammatory, antithrombotic, and antitumor activities. The inhibitory potentials of honokiol on eight major human cytochrome P450 (CYP) enzymes 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4, and four UDP-glucuronosyltransferases (UGTs) 1A1, 1A4, 1A9, and 2B7 in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. Honokiol strongly inhibited CYP1A2-mediated phenacetin O-deethylation, CYP2C8-mediated amodiaquine N-deethylation, CYP2C9-mediated diclofenac 4-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4-hydroxylation, and UGT1A9-mediated propofol glucuronidation with K(i) values of 1.2, 4.9, 0.54, 0.57, and 0.3 µM, respectively. Honokiol also moderately inhibited CYP2B6-mediated bupropion hydroxylation and CYP2D6-mediated bufuralol 1'-hydroxylation with K(i) values of 17.5 and 12.0 µM, respectively. These in vitro results indicate that honokiol has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP1A2, CYP2C8, CYP2C9, CYP2C19, and UGT1A9.

Evaluation of DA-9801, a new herbal drug for diabetic neuropathy, on metabolism-mediated interaction.

DA-9801, the mixture extract of Dioscoreae rhizoma and Dioscorea nipponica Makino, is a new herbal drug currently being evaluated in a phase II clinical study for the treatment of diabetic peripheral neuropathy in Korea. The inhibitory potentials of DA-9801, D. rhizoma extract, D. nipponica Makino extract, and dioscin, an active component of DA-9801, on eight human cytochrome P450 (CYP) enzymes and four UDP-glucuronosyltransferase (UGT) enzymes were investigated in human liver microsomes using liquid chromatography-tandem mass spectrometry. DA-9801 showed slight inhibition of CYP1A2, CYP2C8, UGT1A1, and UGT1A9 enzyme activities with IC(50) values of 396.4, 449.9, 226.0, and 408.8 µg/mL, respectively. D. rhizoma extract showed negligible inhibition of CYP and UGT activities, but D. nipponica extract slightly inhibited CYP1A2, CYP2C8, CYP2C9, UGT1A1, and UGT1A9 activities with IC(50) values of 264.2, 237.1, 206.8, 302.4, and 383.1 µg/mL, respectively. DA-9801 showed volume per dose index values of 0.44-0.88 L for a 200-mg dose, suggesting that they may not cause the inhibition of the metabolism of CYP1A2, CYP2C8, UGT1A1, and UGT1A9-catalyzed drugs in humans. These results suggest that the administration of DA-9801 in human may not cause clinically relevant inhibition of these enzymes.