Ketoconazole, a cytochrome P450 3A4 inhibitor, markedly increases concentrations of levo-acetyl-?-methadol in opioid-naive individuals*1

The opioid partial agonist medication, buprenorphine (BUP), and its primary metabolite, norbuprenorphine (NBUP), are extensively glucuronidated. Sensitive analytical methods that include determination of buprenorphine-3-glucuronide (BUPG) and norbuprenorphine-3-glucuronide (NBUPG) are needed to more fully understand the metabolism and pharmacokinetics of buprenorphine. A method has now been developed that uses solid-phase extraction followed by liquid chromatography-electrospray ionization-tandem mass spectrometry. BUP-d4, NBUP-d3, and morphine-3-glucuronide-d3 were used as internal standards. The lower limit of quantitation was 0.1 and 0.5 ng/mL for each of the analytes in 1-mL of human plasma and urine, respectively, except for NBUP in urine in which it was 2.5 ng/mL. The analytes were stable under the following conditions: plasma and urine at room temperature, up to 20 hours; plasma and urine at -20 degrees C for 119 and 85 days, respectively; plasma freeze-thaw, up to 3 cycles; processed sample, up to 96 hours at -20 degrees C and up to 48 hours on the autosampler; stock solutions at room temperature and at -20 degrees C, up to 6 hours and 128 days, respectively. In plasma collected from 5 subjects on maintenance daily sublingual doses of 16 mg BUP and 4 mg naloxone, respective 0- to 24-hour areas under the curve were 32, 88, 26, and 316 ng/mL x h for BUP, NBUP, BUPG, and NBUPG. In urine samples respective percent of daily dose excreted in the 24-hour urine were 0.014%, 1.89%, 1.01%, and 7.76%. This method allowed us to determine that NBUPG is a major metabolite present in plasma and urine of BUP. Because urinary elimination is limited ( approximately 11% of daily dose), the role of NBUPG in total clearance of buprenorphine is not yet known.

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“…Pharmacokinetic parameters were determined using noncompartmental analysis as previously described. 19 …”

Section: Clinical Samplesmentioning

confidence: 97%

The In Vivo Glucuronidation of Buprenorphine and Norbuprenorphine Determined by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

Huang

Moody

McCance‐Katz

2006

Therapeutic Drug Monitoring

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“…Inhibition of CYP3A4 by this class of drugs results in clinically relevant drug-drug interactions [20][21][22][23]. Antifungal binding to CYP3A4 is easily detected by optical spectroscopy as a shift from the substrate-free water-bound low spin heme spectrum toward the low spin, six-coordinate, nitrogen-iron complex [24, vide infra].…”

Section: Protein-ligand Interactions; Tight Binding Inhibitors; Drug mentioning

confidence: 99%

Surface Plasmon Resonance Analysis of Antifungal Azoles Binding to CYP3A4 with Kinetic Resolution of Multiple Binding Orientations

et al. 2006

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The heme-containing Cytochrome P450s (CYPs) are a major enzymatic determinant of drug clearance and drug-drug interactions. The CYP3A4 isoform is inhibited by antifungal imidazoles or triazoles, which form low spin heme iron complexes via formation of a nitrogen-ferric iron coordinate bond. However, CYP3A4 also slowly oxidizes the antifungal itraconazole (ITZ) at a site that is ∼ 25 Å from the triazole nitrogens, suggesting that large antifungal azoles can adopt multiple orientations within the CYP3A4 active site. Here, we report a surface plasmon resonance (SPR) analysis with kinetic resolution of two binding modes of ITZ, and the related drug ketoconazole (KTZ). SPR reveals a very slow off-rate for one binding orientation. Multiphasic binding kinetics are observed and one of the two binding components resolved by curve-fitting exhibits 'equilibrium overshoot'. Pre-loading of CYP3A4 with the heme ligand imidazole abolishes this component of the antifungal azole binding trajectories, and it eliminates the conspicuously slow off-rate. The fractional populations of CYP3A4 complexes corresponding to different drug orientations can be manipulated by altering the duration of the pulse of drug exposure. UV-vis difference absorbance titrations yield low spin spectra and K D values that are consistent with the high affinity complex resolved by SPR. These results demonstrate that ITZ and KTZ bind in multiple orientations, including a catalytically productive mode and a slowly-dissociating inhibitory mode. Most importantly, they provide the first example of an SPR-based method for the kinetic characterization of drug binding to any human CYP, including mechanistic insight not available from other methods. Keywords protein-ligand interactions; tight binding inhibitors; drug metabolism; drug interactionsThe hepatic and intestinal heme-containing Cytochrome P450s (CYPs) 1 oxidize most drugs, and thus play a critical role in their metabolism and disposition [1][2][3]. Among the various human CYP isoforms, CYP3A4 contributes most to drug metabolism. CYP3A4 exhibits complex allosteric kinetics, which may result from drug-drug interactions on a single CYP molecule through multiple drug binding, as suggested by steady state kinetic behavior [4][5][6], large active sites observed in crystal structures [7][8][9][10] and spectroscopic studies [11]. Complex kinetics † This work was supported by NIH grants GM32165 (WMA, NI, KLK) and GM07750 (JTP). * Corresponding author: Tel: (206) FAX: (206) [12][13][14][15], and equilibrium optical titrations that rely on the inhibitor-or substrate-dependent changes in ferric spin state equilibrium [16][17][18]. The complexity of CYP kinetics severely impedes prediction of drug clearance and drug-drug interactions [12][13][14][15]19]. Clearly, new methods are required to elucidate the molecular basis of CYP allosterism and complex inhibitor or substrate binding.The antifungal azoles, including ketoconazole (KTZ) and itraconazole (ITZ), historically have been considered to be potent inh...

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“…To confirm the role of P450 in both the intestinal and the hepatic metabolism, slices were incubated with ketoconazole, a broad rat P450 inhibitor (Kobayashi et al, 2003). In the clinic, ketoconazole affects largely the pharmacokinetics of drugs that are primarily metabolized by CYP3A4, resulting in a substantial decrease of first-pass metabolism (Moody et al, 2004). In intestinal slices from both mouse and rat, ketoconazole strongly inhibited the metabolism of all tested compounds, whereas in liver, the inhibitory effect was more variable.…”

Section: Metabolite Formation By Rat Intestinal Slices and Rat Intestmentioning

confidence: 99%

Comparison of Mouse and Rat Cytochrome P450-Mediated Metabolism in Liver and Intestine

Martignoni¹,

Groothuis²,

Kanter³

2006

Drug Metab Dispos

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ABSTRACT:The liver is considered to be the major site of first-pass metabolism, but the small intestine is also able to contribute significantly. The improvement of existing in vitro techniques and the development of new ones, such as intestinal slices, allow a better understanding of the intestine as a metabolic organ. In this paper, the formation of metabolites of several human CYP3A substrates by liver and intestinal slices from rat and mouse was compared. The results show that liver slices exhibited a higher metabolic rate for the majority of the studied substrates, but some metabolites were produced at a higher rate by intestinal slices, compared with liver slices. Coincubation with ketoconazole inhibited the metabolic conversion in intestinal slices almost completely, but inhibition was variable in liver slices. To better understand the role of CYP3A in mice, we studied the relative mRNA expression of different CYP3A isoforms in intestine and liver from mice because, in this species, CYP3A expression has not been well described in these organs. It was found that in mice, CYP3A13 is more expressed in the intestine, whereas CYP3A11, CYP3A25, and CYP3A41 are more expressed in the liver, comparable to similar findings in the rat.Altogether, these data demonstrate that, in addition to liver, the intestine from mouse and rat may have an important role in the process of first-pass metabolism, depending on the substrate. Moreover, we show that intestinal slices are a useful in vitro technique to study gut metabolism.Although it is widely believed that the liver is the major site of first-pass metabolism, recent studies have indicated that the small intestine is able to contribute significantly to the overall first-pass metabolism of many drugs. Apart from the metabolic capacity, there are several other factors that play a role in the contribution of the intestine to the first-pass effect of drugs. First, the intestine is exposed to high concentrations of orally administered xenobiotics (Doherty and Charman, 2002). Second, anatomically, the small intestine has a serial relationship, with the liver in the process of absorption being the anterior organ. Third, in addition to drug-metabolizing enzymes such as cytochrome P450 (P450) isoenzymes, efflux proteins, such as P-glycoprotein and multidrug resistance-associated proteins, present in the apical membrane of the enterocytes, influence the metabolism process by recycling drugs between enterocytes and lumen, with the consequent higher drug exposure to intestinal metabolic enzymes (Doherty and Charman, 2002). Thus, the amount of an orally administered drug that reaches the systemic circulation can be reduced by both intestinal and hepatic metabolism. For some substrates, it has even been suggested that the role of intestinal metabolism is quantitatively greater than that of hepatic metabolism in the overall firstpass effect (Wu et al., 1995;Paine et al., 1996). Therefore, the important question is raised as to which factors determine the role of intestinal metabolism ...

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Ketoconazole, a cytochrome P450 3A4 inhibitor, markedly increases concentrations of levo-acetyl-?-methadol in opioid-naive individuals*1

Cited by 25 publications

References 30 publications

The In Vivo Glucuronidation of Buprenorphine and Norbuprenorphine Determined by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

The In Vivo Glucuronidation of Buprenorphine and Norbuprenorphine Determined by Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry

Surface Plasmon Resonance Analysis of Antifungal Azoles Binding to CYP3A4 with Kinetic Resolution of Multiple Binding Orientations

Comparison of Mouse and Rat Cytochrome P450-Mediated Metabolism in Liver and Intestine

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