Interactions of midazolam and ketoconazole were studied in vivo and in vitro in rats. Ketoconazole (total dose of 15 mg/kg intraperitoneally) reduced clearance of intravenous midazolam (5 mg/kg) from 79 to 55 ml/min/kg (p Ͻ 0.05) and clearance of intragastric midazolam (15 mg/kg) from 1051 to 237 ml/min/kg (p Ͻ 0.05), increasing absolute bioavailability from 0.11 to 0.36 (p Ͻ 0.05). Presystemic extraction occurred mainly across the liver as opposed to the gastrointestinal tract mucosa. Midazolam increased electroencephalographic (EEG) amplitude in the ␤-frequency range. Ketoconazole shifted the concentration-EEG effect relationship rightward (increase in EC 50 ), probably because ketoconazole is a neutral benzodiazepine receptor ligand. Ketoconazole competitively inhibited midazolam hydroxylation by rat liver and intestinal microsomes in vitro, with nanomolar K i values. At a total serum ketoconazole of 2 g/ml (3.76 M) in vivo, the predicted reduction in clearance of intragastric midazolam by ketoconazole (to 6% of control) was slightly greater than the observed reduction in vivo (to 15% of control). However, unbound serum ketoconazole greatly underpredicted the observed clearance reduction. Although the in vitro and in vivo characteristics of midazolam in rats incompletely parallel those in humans, the experimental model can be used to assess aspects of drug interactions having potential clinical importance.

We evaluated the effect of age on CYP3A expression and function in the liver, intestine, and kidney from young (3-4 months), intermediate (13-14 months), and old (25-26 months) male Fischer-344 rats. The biotransformation of triazolam to its primary hydroxylated products, 4-OH-TRZ (triazolam) and ␣-OH-TRZ, was used as a marker of CYP3A activity in rat liver and intestine. Immunoactive CYP3A expression was evaluated by Western blot analysis in the rat intestine, liver, and kidney. Since testosterone and NADPH reductase levels may modulate CYP3A activity, we also examined free plasma testosterone concentrations and NADPH reductase expression in these rats. The effect of age on CYP3A expression was tissue-specific.Although both CYP3A activity and expression were reduced by approximately 50 to 70% in the old livers compared with the young animals, intestinal CYP3A activity and expression did not change significantly with age. The expression of one CYP3A isoform was increased by 1.5-fold in the old kidneys. NADPH reductase expression was reduced by 23 to 36% with age in all tissues; this reached statistical significance only in the liver. Plasma testosterone levels declined by 74% in the old animals. This study suggests that the effect of age on CYP3A expression and function is tissue-specific. In addition, changes in testosterone levels and NADPH reductase expression may contribute to age-related differences in hepatic CYP3A activity.

This article is available online at http://dmd.aspetjournals.org ABSTRACT:Sildenafil [SIL (Viagra); Pfizer, New York, NY] is a widely prescribed agent for erectile dysfunction in men older than 65 years. The present study evaluated experimental models to assess age-dependent changes in SIL biotransformation using hepatic microsomes from male rats and mice ranging from 6 weeks to 26 months of age. The role of specific isoforms in the conversion of SIL to its primary circulating metabolite, UK-103,320 (piperazine N-desmethyl sildenafil) in the mouse was also investigated using immunoinhibitory antibodies. Although CYP2C11 largely mediated UK-103,320 formation in the rat, UK-103,320 formation was principally inhibited by a CYP3A antibody in the mouse. An age-related decrement in metabolite formation rate was observed for both species, although this effect was more pronounced in the old rats (reduced to 7% of young) than in the old mice (reduced to 51% of young). CYP2C expression was assessed by Western blot analysis in rat and mouse livers. Age-related differences in hepatic CYP3A expression in the mouse were also compared with metabolite formation rates in the mouse model. Decrements with age in CYP2C and -3A expression in the aging rodents paralleled the decrements in SIL biotransformation, suggesting that age-related differences in SIL metabolic rate may, in part, reflect differences in expression. Although the role of specific CYP enzymes and the clearance values for this reaction may differ among species, age-related changes in these rodent models are consistent with the reduced clearance of SIL observed in human studies.

Objectives: Treatment with medications for opioid use disorder such as buprenorphine improves patient morbidity and mortality as well as treatment adherence, an important component of patient care. Buprenorphine is combined with naloxone to reduce misuse; and, when taken sublingually, naloxone is poorly absorbed. Urine testing for buprenorphine is a common way to monitor adherence. Some patients who want to appear adherent may directly tamper with their urine by adding buprenorphine to their urine to allow for the detection without ingestion. Practitioners may rely upon the concentration of buprenorphine and the metabolite, norbuprenorphine, and utilize the ratio of metabolite to parent compound (norbuprenorphine:buprenorphine – N:B ratio) to discern possible evidence of tampering; however, there remains debate as to what specific ratio may signify this practice. Testing for naloxone may also help determine if urine tampering occurred as only low naloxone concentrations are found in the urine when taken by a sublingual route. Methods: To determine a reliable N:B ratio that may be used to identify possible urine tampering by adding parent drug directly to urine, we examined 136,605 urine samples for quantitative concentrations of buprenorphine and norbuprenorphine by LC-MS/MS performed at a commercial laboratory. After identifying abnormal ratios (<0.02), we then compared them with naloxone concentrations and specimen validity testing, other markers that may coincide with specimen tampering of this type. Results: Correlating urinary buprenorphine and norbuprenorphine concentrations, we found 2 distinct patient populations, which could be distinguished by N:B ratios ranging from 0.01 to 0.2. In addition, while the distribution of urine naloxone concentrations itself did not demonstrate distinct populations, naloxone was able to further flag potential tampered specimens when combined with N:B ratios. Abnormal specimen validity testing was additionally found more commonly in cases with N:B ratios <0.02. Conclusions: This comprehensive study compared N:B ratios with naloxone concentrations and specimen validity testing. This study suggests that a N:B ratio of <0.02 in concert with high naloxone concentrations (>1000 ng/ml) can help to identify potential cases of tampered urine samples.