Calycosin Influences the Metabolism of Five Probe Drugs in Rats.

BACKGROUND: Calycosin (CAL), a type of O-methylated isoflavone extracted from the herb Astralagusmembranaceus (AM), is a bioactive chemical with antioxidative, antiphlogistic and antineoplastic activities commonly used in traditional alternative Chinese medicine. AM has been shown to confer health benefits as an adjuvant in the treatment of a variety of diseases. AIM: The main objective of this study was to determine whether CAL influences the cytochrome P450 (CYP450) system involved in drug metabolism. METHODS: Midazolam, tolbutamide, omeprazole, metoprolol and phenacetin were selected as probe drugs. Rats were randomly divided into three groups, specifically, 5% Carboxymethyl cellulose (CMC) for 8 days (Control), 5% CMC for 7 days + CAL for 1 day (single CAL) and CAL for 8 days (conc CAL), and metabolism of the five probe drugs evaluated using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). RESULTS: No significant differences were observed for omeprazole and midazolam, compared to the control group. T max and t1/2 values of only one probe drug, phenacetin, in the conc CAL group were significantly different from those of the control group (T max h: 0.50±0.00 vs 0.23±0.15; control vs conc CAL). C max of tolbutamide was decreased about two-fold in the conc CAL treatment group (conc vs control: 219.48 vs 429.56, P<0.001). CONCLUSION: Calycosin inhibits the catalytic activities of CYP1A2, CYP2D6 and CYP2C9. Accordingly, we recommend caution, particularly when combining CAL as a modality therapy with drugs metabolized by CYP1A2, CYP2D6 and CYP2C9, to reduce the potential risks of drug accumulation or ineffective treatment.

Development and validation of probe drug cocktails for the characterization of CYP450-mediated metabolism by human heart microsomes.

1. The objective of our study was to develop and validate a cocktail approach to allow the simultaneous characterization of various CYP450-mediated oxidations by human heart microsomes for nine probe drug substrates, namely, 7-ethoxyresorufin, bupropion, repaglinide, tolbutamide, bufuralol, chlorzoxazone, ebastine, midazolam and dodecanoic acid. 2. The first validation step was conducted using recombinant human CYP450 isoenzymes by comparing activity measured for each probe drug as a function of (1) buffer used, (2) selectivity towards specific isoenzymes and (3) drug interactions between probes. Activity was all measured by validated LC-MSMS methods. 3. Two cocktails were then constituted with seven of the nine drugs and subjected to kinetic validation. Finally, all probe drugs were incubated with human heart microsomes prepared from ventricular tissues obtained from 12 patients undergoing cardiac transplantation. 4. Validated cocktail #1 including bupropion, chlorzoxazone, ebastine and midazolam was used to characterize CYP2B6-, 2E1-, 2J2- and 3A5-mediated metabolism in human hearts. 5. Cocktail #2 which includes bufuralol, 7-ethoxyresorufin and repaglinide failed the validation step. Substrates in cocktail #2 as well as tolbutamide and dodecanoic acid had to be incubated separately because of their physico-chemical characteristics (solubility and ionization) or drug interactions. 6. Activity in HHM was the highest towards ebastine, chlorzoxazone and tolbutamide.

Impact of Herbal Medicines like Nigella sativa, Trigonella foenum-graecum, and Ferula asafoetida, on Cytochrome P450 2C11 Gene Expression in Rat Liver.

AIM: Combined use of herbs and drugs may result in clinically important herb-drug interactions. The majorities of these interactions are thought to be metabolism-based and involve induction or inhibition of cytochrome P450 (CYP). The current study was designed to investigate the effect of some commonly used herbs on rat CYP2C11 gene expression and metabolic activity. METHODS: Wistar rats were treated for 7 days with increasing doses of 3 herbs; Nigella sativa, Trigonella foenum-graecum, and Ferula asafoetida. Thereafter, CYP2C11 mRNA and protein levels were determined by real-time polymerase chain reaction (RT-PCR) and western blot analyses, respectively. In vitro metabolic activity of CYP2C11 was performed on rat hepatic microsomes using tolbutamide as specific substrate. RESULTS: Our results showed that all the 3 herbs significantly inhibited the mRNA and protein expression levels of CYP2C11 in a dose-dependent manner. Furthermore, the in vitro enzyme metabolic activity study showed a significant decrease in the formation of 4-hyroxy-tolbutamide, a tolbutamide metabolite, at the higher doses. The inhibitory effects of the investigated herbs on rat CYP2C11 was in the order: Nigella Sativa > Trigonella foenum-graecum > Ferula asafoetida. CONCLUSIONS: The 3 herbs are strong inhibitor of CYP2C11 expression, which can lead to an undesirable pharmacological effect of clinically used CYP2C11 substrate drugs with a low therapeutic index.

The in-vitro effect of complementary and alternative medicines on cytochrome P450 2C9 activity.

OBJECTIVES: The aim of this study is to establish the inhibitory effects of 14 commonly used complementary and alternative medicines (CAM) on the metabolism of cytochrome P450 2C9 (CYP2C9) substrates 7-methoxy-4-trifluoromethyl coumarine (MFC) and tolbutamide. CYP2C9 is important for the metabolism of numerous drugs and inhibition of this enzyme by CAM could result in elevated plasma levels of drugs that are CYP2C9 substrates. Especially for anticancer drugs, which have a narrow therapeutic window, small changes in their plasma levels could easily result in clinically relevant toxicities. METHODS: The effects of CAM on CYP2C9-mediated metabolism of MFC were assessed in Supersomes, using the fluorometric CYP2C9 inhibition assay. In human liver microsomes (HLM) the inhibition of CYP2C9-mediated metabolism of tolbutamide was determined, using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). KEY FINDINGS: The results indicated milk thistle as the most potent CYP2C9 inhibitor. For milk thistle, silybin (main constituent of milk thistle) was mainly responsible for the inhibition of CY2C9. CONCLUSIONS: Milk thistle and green tea were confirmed as potent inhibitors of CYP2C9-mediated metabolism of multiple substrates in vitro. Clinical studies with milk thistle are recommended to establish the clinical relevance of the demonstrated CYP2C9 inhibition.

Enzyme kinetic and molecular docking studies on the metabolic interactions of 1-hydroxy-2,3,5-trimethoxy-xanthone, isolated from Halenia elliptica D. Don, with model probe substrates of human cytochrome P450 enzymes.

Halenia elliptica D. Don is a Tibetan herb and medicinal preparations containing Halenia elliptica have been commonly used for the treatment of hepatitis B virus infection in China. The metabolism of 1-hydroxy-2,3,5-trimethoxy-xanthone (HM-1) to its metabolites is mediated through cytochrome P450 enzymes. This study aimed to investigate the herb-drug interaction potential of HM-1 by studying its effects on the metabolism of model probe substrates of five major CYP450 isoforms in human liver microsomes. HM-1 showed moderate inhibitory effects on CYP1A2 (IC50 = 1.06 µM) and CYP2C9 (IC50 = 3.89 µM), minimal inhibition on CYP3A4 (IC20 = 11.94 µM), but no inhibition on model CYP2D6 (dextromethorphan) and CYP2E1 (chlorzoxazone) probe substrates. Inhibition kinetic studies showed that the K(i) values of HM-1 on CYP1A2, CYP2C9 and CYP3A4 were 5.12 µM, 2.00 µM and 95.03 µM, respectively. HM-1 competitively inhibited testosterone 6ß-hydroxylation (CYP3A4) but displayed mixed type inhibitions for phenacetin O-deethylation (CYP1A2) and tolbutamide 4-hydroxylation (CYP2C9). Molecular docking study confirmed the inhibition modes of HM-1 on these human CYP isoforms.

Effects of brucine combined with glycyrrhetinic acid or liquiritin on rat hepatic cytochrome P450 activities in vivo.

Abstract: The activities of four CYP450 enzymes (CYP3A, 1A2, 2El and 2C) and the mRNA expression levels of CYP1A2, 2El, 2Cll and 3A1 in rat liver were determined after Wistar rats were orally administered with brucine (BR) at three dosage levels (3, 15 and 60 mg.kg-1 per day) and the high dose of BR combined with glycyrrhetinic acid (GA, 25 mg.kg-1 per day) or liquiritin (LQ, 20 mg.kg-1 per day) for 7 consecutive days. Compared with the control, brucine caused 24.5% and 34.6% decrease of CYP3A-associated testosterone 6beta-hydroxylation (6betaTesto-OH) and CYP2C-associated tolbutamide hydroxylation (Tol-OH), respectively, and 146.1% increase of CYP2El-associated para-nitrophenol hydroxylation (PNP-OH) at the high dose level. On the other hand, (BR+GA) caused 51.4% and 33.5% decrease, respectively, of CYP2El-associated PNP-OH and CYP1A2-associated ethoxyresorufin-O-de-ethylation (EROD) as compared with the high dose of BR group. Meanwhile, (BR+LQ) caused 41.1% decrease of CYP2El-associated PNP-OH and 37.7% increase of CYP2C-associated Tol-OH. The results indicated that the co-administration of BR with GA or LQ had effect on mRNA expression and activities of the CYP450 enzymes mentioned above to some extent, and the in vivo antagonism of LQ on BR-induced CYPs adverse effects and the in vivo inhibitory action of GA on CYP2E1 and 1A2 might play an important role in the detoxification of Radix Glycyrrhizae against Strychnos nux-vomica L.

Polysaccharide peptides from Coriolus versicolor competitively inhibit tolbutamide 4-hydroxylation in specific human CYP2C9 isoform and pooled human liver microsomes.

Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy in China. Previous studies have shown that PSP decreased antipyrine clearance and inhibited CYP2C11-mediated tolbutamide 4-hydroxylation in the rat both in vitro and in vivo. In this study, the effects of water extractable fraction of PSP on tolbutamide 4-hydroxylation was investigated in pooled human liver microsomes and in specific human CYP2C9 isoform. PSP (2.5-20µM) dose-dependently decreased the biotransformation of tolbutamide to 4-hydroxy-tolbutamide. Enzyme kinetics studies showed inhibition of tolbutamide 4-hydroxylase activity was competitive and concentration-dependent. In pooled human liver microsomes, PSP had a K(i) value of 14.2µM compared to sulfaphenazole, a human CYP2C9 inhibitor, showed a K(i) value of 0.32µM. In human CYP2C9 isoform, the K(i) value of PSP was 29.5µM and the K(i) value of sulfaphenazole was 0.04µM. This study demonstrated that PSP can competitively inhibit tolbutamide 4-hydroxylation in both pooled human liver microsomes and specific human CYP2C9 in vitro. This study compliments previous findings in the rat that PSP can inhibit human tolbutamide 4-hydroxylase, but the relatively high K(i) values in human CYP2C9 would suggest a low potential for PSP to cause herb-drug interaction.

In vitro modulatory effects on three major human cytochrome P450 enzymes by multiple active constituents and extracts of Centella asiatica.

ETHNOPHARMACOLOGICAL RELEVANCE: Centella asiatica (CA) has been widely cultivated as a vegetable or spice in China, Southeast Asia, India, Sri Lanka, Africa, and Oceanic countries and traditionally used for wound healing and maintaining normal blood pressure. AIM OF THE STUDY: The present study was carried out to examine the potential modulatory effects of three commercially available active components (asiaticoside, asiatic acid and madecassic acid) and four extracts (aqueous, ethanol, dichloromethane and hexane) of CA on three major cDNA-expressed human cytochrome P450 (CYP) isoforms. MATERIALS AND METHODS: High-performance liquid chromatography (HPLC)-based enzyme assays, namely tolbutamide 4-methyhydroxylase, dextromethorphan O-demethylase and testosterone 6beta-hydroxylase assays were developed to probe activities of CYP2C9, CYP2D6 and CYP3A4, respectively. Probe substrates were incubated with or without each active component and extract for each isoform, followed by examination of the kinetics parameters, IC(50) and K(i), to characterize modulatory effects. RESULTS: CYP2C9 was more susceptible to inhibitory effects by CA extracts compared to CYP2D6 and CYP3A4. Moderate degree of inhibition was observed in ethanol (K(i)=39.1 microg/ml) and dichloromethane (K(i)=26.6 microg/ml) extracts implying potential risk of interaction when CYP2C9 substrates are consumed with CA products. The two extracts however showed negligible inhibition towards CYP2D6 and CYP3A4 (IC(50)'s of 123.3 microg/ml and above). Similarly CA aqueous and hexane extracts did not significantly inhibit all three isoforms investigated (IC(50)'s of 117.9 microg/ml and above). Among the active constituents investigated, asiatic acid and madecassic acid appeared to selectively inhibit CYP2C9 and CYP2D6 more than CYP3A4. Of particular interest is the potent inhibitory effect of asiatic acid on CYP2C9 (K(i)=9.1 microg/ml). This signifies potential risk of interaction when substrates for this isoform are taken together with CA products with high asiatic acid content. Inhibitions of asiatic acid with the other isoforms and that of madecassic acid with all isoforms were only moderate (K(i)'s ranged from 17.2 to 84.4 microg/ml). On the other hand, the IC(50) values for asiaticoside were high (1070.2 microg/ml or above) for all three isoforms, indicating negligible or low potential of this compound to modulate CYP enzymatic activity. CONCLUSION: Centella asiatica extracts and active constituents inhibited CYP2C9, CYP2D6 and CYP3A4 activities with varying potency with CYP2C9 being the most susceptible isoform to inhibition. Significant inhibition was observed for asiatic acid and CA ethanol and dichloromethane extracts, implying involvement of semipolar constituents from CA in the effect. This study suggested that CA could cause drug-herb interactions through CYP2C9 inhibition.

Pharmacokinetic interaction studies of tanshinones with tolbutamide, a model CYP2C11 probe substrate, using liver microsomes, primary hepatocytes and in vivo in the rat.

The effects of Danshen and its active components (tanshinone I, tanshinone IIA, dihydrotanshinone and cryptotanshinone) on tolbutamide 4-hydroxylation was investigated in the rat. Danshen (0.125-2mg/ml) decreased 4-hydroxy-tolbutamide formation in vitro and in vivo. Enzyme kinetics studies showed that inhibition of tolbutamide 4-hydroxylase activity was competitive and concentration-dependent. The K(i) values of the tanshinones were: dihydrotanshinone (8.92microM), cryptotanshinone (24.5microM), tanshinone I (80.3microM) and tanshinone IIA (242.9microM). In freshly prepared primary rat hepatocytes, tanshinones inhibited tolbutamide 4-hydroxylation in a concentration-dependent manner, with EC(40) values in the order: cryptotanshinone (15.8microM), tanshinone IIA (16.2microM), dihydrotanshinone (20.1microM) and tanshinone I (48.2microM). In whole animal studies, single dose Danshen treatment (50 or 200mg/kg, i.p.) increased tolbutamide clearance (17-26.9%), decreased AUC (14.4-20.9%) and increased the Vd (7.26%). Three-day Danshen treatment (200mg/kg/day, i.p.) decreased the C(initial), increased T(1/2) and Vd but did not affect tolbutamide clearance and AUC. Tolbutamide-4-hydroxylation in vivo was decreased by Danshen after acute and after 3-day treatment, with decreases in the AUC of 4-hydroxy-tolbutamide (15-28%) over the time period studied. Despite competitive inhibition of rat CYP2C11 in vitro and in vivo, as shown by the decrease in tolbutamide 4-hydroxylation, only minor changes in tolbutamide pharmacokinetics was observed. This study illustrated that the herb-drug interaction potential should be monitored by both in vitro and in vivo biotransformation/ pharmacokinetic parameters.

Ocimum sanctum leaf extracts stimulate insulin secretion from perfused pancreas, isolated islets and clonal pancreatic beta-cells.

Ocimum sanctum leaves have previously been reported to reduce blood glucose when administered to rats and humans with diabetes. In the present study, the effects of ethanol extract and five partition fractions of O. sanctum leaves were studied on insulin secretion together with an evaluation of their mechanisms of action. The ethanol extract and each of the aqueous, butanol and ethylacetate fractions stimulated insulin secretion from perfused rat pancreas, isolated rat islets and a clonal rat beta-cell line in a concentration-dependent manner. The stimulatory effects of ethanol extract and each of these partition fractions were potentiated by glucose, isobutylmethylxanthine, tolbutamide and a depolarizing concentration of KCl. Inhibition of the secretory effect was observed with diazoxide, verapamil and Ca2+ removal. In contrast, the stimulatory effects of the chloroform and hexane partition fractions were associated with decreased cell viability and were unaltered by diazoxide and verapamil. The ethanol extract and the five fractions increased intracellular Ca2+ in clonal BRIN-BD11 cells, being partly attenuated by the addition of verapamil. These findings indicated that constituents of O. sanctum leaf extracts have stimulatory effects on physiological pathways of insulin secretion which may underlie its reported antidiabetic action.

Ginkgo biloba extract modifies hypoglycemic action of tolbutamide via hepatic cytochrome P450 mediated mechanism in aged rats.

We examined hepatic cytochrome P450 (CYP)-mediated interactions between Ginkgo biloba extract (GBE) and tolbutamide, an oral anti-diabetic agent, in aged and young rats. Tolbutamide was orally given to rats with or without GBE treatment, and time-dependent changes in blood glucose were monitored. The basal activity of six CYP subtypes in liver was lower in the aged rats than in the young rats, while the inductions of these enzymes by 5 day pretreatment of 0.1% GBE diet were more in the aged rats. Further, the pretreatment of GBE significantly attenuated the hypoglycemic action of tolbutamide in the aged rats, corresponding well to the enhanced activity of (S)-warfarin 7-hydroxylase, which is responsible for CYP2C9 subtype, a major isoform metabolizing tolbutamide. In contrast, the simultaneous administration of GBE with tolbutamide potentiated the hypoglycemic action of this drug. The in vitro experiments revealed that GBE competitively inhibited the metabolism of tolbutamide by (S)-warfarin 7-hydroxylase in the rat liver microsomes. In the young rats, the 5 day pretreatment with GBE significantly attenuated the hypoglycemic action of tolbutamide, but a simultaneous treatment had little influence on the tolbutamide effect. In conclusion, the present study has shown that the simultaneous and continuous intake of GBE significantly affects the hypoglycemic action of tolbutamide, possibly via a hepatic CYP enzyme-mediated mechanism, particularly in the aged rats. Therefore, it is anticipated that the intake of GBE as a dietary supplement with therapeutic drugs should be cautious, particularly in elderly people.

The inhibitory effects of herbal components on CYP2C9 and CYP3A4 catalytic activities in human liver microsomes.

Herbal medicines are widely consumed by patients in different clinical settings in the United States and all over the world. In this study, 7 herbal components ginsenosides Rb1, Rb2, Rc, and Rd (from ginseng quercetin) ginkgolides A and B (from ginkgo biloba) were investigated for their inhibitory effects on hepatic CYP2C9 and CYP3A4 catalytic activities in human liver microsomes. Tolbutamide 4-methylhydroxylation and testosterone 6beta-hydroxylation were used as index reactions of CYP2C9 or CYP3A4 catalytic activities, respectively. The metabolites of both reactions were measured by high-performance liquid chromatography and used as indicators of whether enzymes were inhibited or unaffected by these agents. Herbal components were studied at various concentrations (0.1, 1, 10, 100, 200 micromol/L). The herbal compounds investigated were capable of inhibiting CYP2C9 and CYP3A4 catalytic activities, but the potencies differed. Quercetin showed marked inhibitory effects on both tolbutamide 4-methylhydroxylation and testosterone 6beta-hydroxylation with IC(50) values of 35 and 38 micromol/L, respectively. Ginsenoside Rd also had significant inhibitory potency on both CYP2C9- and CYP3A4-mediated index reactions with IC(50) values of 105 and 62 micromol/L, respectively. Ginsenosides Rb1, Rb2, and Rc had limited inhibitory activities on both enzyme reaction systems, whereas the effects of ginkgolides A and B appeared negligible. It is concluded that the components of ginseng and ginkgo biloba screened are capable of inhibiting CYP2C9- and CYP3A4-mediated metabolic reactions. Our findings suggest that quercetin and ginsenoside Rd have the potential to interact with conventional medicines that are metabolized by CYP2C9 and CYP3A4 in vivo.

CYP2C44, a new murine CYP2C that metabolizes arachidonic acid to unique stereospecific products.

The human CYP2Cs have been studied extensively with respect to the metabolism of clinically important drugs and endogenous chemicals such as arachidonic acid (AA). Five members of the mouse CYP2C family have previously been described that metabolize arachidonic acid into regio- and stereospecific epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids, which have many important physiological roles. Herein, we describe the cloning and characterization of a new mouse cytochrome P450 (P450), CYP2C44, which has the lowest homology with other known mouse CYP2Cs. Western blotting and real-time polymerase chain reaction detected CYP2C44 mRNA and protein in liver >> kidney > adrenals. Kidney contained approximately 10% of the CYP2C44 mRNA content of liver. CYP2C44 metabolized AA to unique stereospecific products, 11R,12S-EET and 8R, 9S-EET, which are similar to those produced by rat CYP2C23. CY2C23 is highly expressed in rat kidney and has been suggested to be important in producing compensatory renal artery vasodilation in response to salt-loading in this species. Immunohistochemistry showed the presence of CYP2C44 in hepatocytes, biliary cells of the liver, and the proximal tubules of the kidney. Unlike mouse CYP2C29, CYP2C38, and CYP2C39, CYP2C44 did not metabolize the common CYP2C substrate tolbutamide. CYP2C44 was not induced by phenobarbital or pregnenolone-16alpha-carbonitrile, two prototypical inducers of hepatic P450s. The presence of CYP2C44 in mouse liver, kidney, and adrenals and the unique stereospecificity of its arachidonic acid metabolites are consistent with the possibility that it may have unique physiological roles within these tissues, such as modulation of electrolyte transport or vascular tone.

Interaction of drugs and Chinese herbs: pharmacokinetic changes of tolbutamide and diazepam caused by extract of Angelica dahurica.

The inhibitory effects of Angelica dahurica root extract on rat liver microsomal cytochrome P450 and drug-drug interactions were studied. The 2alpha- and 16alpha-hydroxylase activity of testosterone were most strongly inhibited, with 17.2% and 28-5% of their activity remaining, respectively, after oral administration of A. dahurica extract at a 1 g kg(-1) dose. 6beta-Hydroxylase activity was also inhibited, with 70% of its activity remaining, under the same conditions. In addition, treatment with the extract inhibited the metabolism of tolbutamide, nifedipine and bufuralol. These results showed that the extract inhibited the various isoforms of cytochrome P450 such as CYP2C, CYP3A and CYP2D1. The A. dahurica extract delayed elimination of tolbutamide after intravenous administration at a 10 mg kg(-1) dose to rats. Thus, the extract altered the liver intrinsic clearance. It had little effect, however, on the pharmacokinetic parameters of diazepam after intravenous administration at 10 mg kg(-1). Since diazepam showed high clearance, it underwent hepatic blood flow rate-limited metabolism. Therefore, the change of intrinsic clearance had little effect on hepatic clearance. However, the Cmax value after oral administration of diazepam with extract treatment was four times that with non-treatment. It was suggested that the first-pass effect was changed markedly by the extract. High-dose (1 g kg(-1)), but not low dose (0.3 g kg(-1)), administration of A. dahurica extract increased significantly the duration of rotarod disruption following intravenous administration of diazepam at 5 mg kg(-1). It was concluded that administration of A. dahurica extract has the potential to interfere with the metabolism, by liver cytochrome P450, of other drugs.

Characterisation of tolbutamide hydroxylase activity in the common brushtail possum, (Trichosurus vulpecula) and koala (Phascolarctos cinereus): inhibition by the eucalyptus terpene 1,8-cineole.

Plant constituents such as terpenes are major constituents of the essential oil in Eucalyptus sp. 1,8-Cineole and p-cymene (Terpenes present in high amounts in Eucalyptus leaves) are potential substrates for the CYP family of enzymes. We have investigated tolbutamide hydroxylase as a probe substrate reaction in both koala and terpene pretreated and control brushtail possum liver microsomes and examined inhibition of this reaction by Eucalyptus terpenes. The specific activity determined for tolbutamide hydroxylase in the terpene treated brushtails was significantly higher than that for the control animals (1865+/-334 nmol/mg microsomal protein per min versus 895+/-27 nmol/mg microsomal protein per min). The activity determined in koala microsomes was 8159+/-370 nmol/mg microsomal protein per min. Vmax values and Km values for the terpene treated possum, control, possum and koala were 1932-2225 nmol/mg microsomal protein per min and 0.80 0.81 mM; 1406-1484 nmol/mg microsomal protein per min and 0.87-0.92 mM and 5895-6403 nmol/mg microsomal protein per min and 0.067-0.071 mM, respectively. Terpenes were examined as potential inhibitors of tolbutamide hydroxylase activity. 1,8-Cineole was found to be a competitive inhibitor for the enzyme responsible for tolbutamide hydroxylation (Ki 15 microM) in the possum. In koala liver microsomes stimulation of tolbutamide hydroxylase activity was observed when concentrations of cineole were increased. Therefore, although inhibition was observed, the type of inhibition could not be determined.