Current Drug Metabolism - Volume 6, Issue 6, 2005

Dihydroergocristine (DHEC) is a semi-synthetic drug mainly used for age-related cognitive impairment. In this study, its major metabolite 8'-hydroxy-dihydroergocristine (8'-OH-DHEC) was produced in incubates of a bovine liver preparation using dihydroergocristine mesylate (DHECM) as substrate. Purification was achieved by flash silica gel column and reverse phase liquid chromatographies, and identification was based on accurate molecular mass measurements, mass fragmentation spectra and NMR ( 1H/13C) chemical shifts. By using the substance produced in vitro, a fast, sensitive, specific and robust LC/MS/MS method for the simultaneous determination of DHEC and its major metabolite in human plasma was developed and validated. Bromocriptine was used as internal standard and limits of quantification for DHEC and 8'-OH-DHEC were 10 pg/ml and 20 pg/ml, respectively. Pharmacokinetic parameters were investigated on 12 male healthy volunteers to whom a single dose of 18 mg DHECM was administered in tablets (Iskevert®). The peak of DHEC was 0.28 ± 0.22 μg/l, the tmax 0.46 ± 0.26 h, the AUClast 0.39 ± 0.41 μg/l.h and the terminal elimination half-life 3.50 ± 2.27 h. The peak of 8'-OH-DHEC was 5.63 ± 3.34 μg/l, the tmax 1.04 ± 0.66 h, the AUClast 13.36 ± 5.82 μg/l.h and the terminal elimination half-life 3.90 ± 1.07 h. Dosing of 18 mg DHECM was well tolerated, causing no adverse events.

Extracts of Hypericum perforatum are becoming increasingly popular for the treatment of mild to moderate depression, despite the lack of consensus on their efficacy. Although the mechanism(s) of this action are still debated, several components, including the naphthodianthrones hypericin and pseudohypericin, the acylphloroglucinol hyperforin and some flavonols, are believed to play major roles in the antidepressant-like effects. Some of these also increase the expression of the P-glycoprotein transporter and others the expression of cytochrome P450 enzymes, possibly contributing to the interactions involving the extracts and conventional drugs. However, few pharmacokinetic studies of naphthodianthrones and hyperforin have appeared and none has yet evaluated the exposure to unchanged quercetin and its glycosides after intake of extracts. There are no formal pharmacokinetic studies in special populations. Bioavailability appears low, giving variable steady-state plasma concentrations, whose prediction may be complicated by non-linearity for hypericin and hyperforin. Data on tissue distribution are scarce, and it appears that hypericin and hyperforin do not reach the central nervous system in appreciable concentrations in animals. Clearance is low-intermediate, with little or no unchanged compounds excreted with urine. Although some potentially active conjugated metabolites have been identified for quercetin and its glycosides after intake of authentic compounds or flavonol-rich foods, these too have been characterised little with regard to their pharmacokinetics and central activities. Thus, further pharmacokinetic and pharmacodynamic studies of the main components and their metabolites are urgently needed to clarify the role of each constituent and provide more rational and safe regimens for people preferring "natural" drugs.

Superfamily of cytochrome P450 enzymes (CYPs), a distinctive enzyme system by which human body defends itself against toxic compounds, is the subject of a complex regulation process involving various mechanisms, on the levels of expression and activity. Apart from physiological factors, several patho-physiological ones such as inflammation, infection, and stress affect CYP expression. The aim of this review is to summarize the current knowledge on the role of microtubules network in the regulation of drug metabolizing CYPs. Experiments on human and animal cell models revealed that microtubules disruption severely impaired basal and inducible expression of human CYP 1A1, 2B6, 2C8, 2C9, 2C19, and 3A4, and rat CYP 1A2, 2B1, 2B2, and 3A23. Inhibition of aryl hydrocarbon receptor (AhR) and glucocorticoid receptor (GR) transcriptional activity by microtubules disarray was found to be responsible for the suppressed CYP enzymes expression. However, the mechanism by which microtubules interfering agents (MIAs) inhibit GR and AhR transcriptional activities is not fully understood yet. Several lines of evidence indicate that: i) the cell cycle, G2/M phase in particular, has an influence on AhR and GR transcriptional activity, and ii) MIAs negatively modulate GR transcriptional activity via the activation of c-Jun-N-terminal kinase. In conclusion, down-regulation of major CYP enzymes by microtubules disarray is intriguing from the mechanistic point of view and in relation to the cell differentiation.

The individualized medicine aims to identify the molecular basis of the individual's response to different therapeutic treatments. Individualized medicine is very relevant for human diseases such as cancer and it has become a major task to accomplish more efficient and specific therapeutics. An individualized response to treatment could underline therapeutic success or failure and, even more, could support the rationale for good or bad prognosis. The use of up to date genomic approaches is changing the way we understand modern medicine in terms of drug efficacy, toxicity and diagnosis. Results from genetic polymorphism studies, gene expression profiling and epigenetics illustrate how pharmacogenomic testing will contribute to the goal of individualized medicine. Antineoplastic drugs are designed to block the anomalous activity of specific molecules (therapeutic targets) that regulate cellular processes such as cell cycle. Understanding the relationship between molecular changes in therapeutic targets and enhanced antitumoral response or chemotherapeutic resistance is crucial to establish the clinical relevance of genomic approaches. The goal of this review is to discuss the basic and the clinical significance of genomic research on drug targets and its impact on the early diagnosis and treatment of cancer. We will also assess how these methodologies could contribute to individualized medicine in oncology. A special focus will be put on oncogenes and tumor suppressor genes. Aspects such as drug efficacy, side effects and the diagnostic value of antineoplastic pharmacogenomic research will be also considered.

In vitro models of the liver using isolated primary hepatocytes have been used as screens for measuring the metabolism, toxicity and efficacy of xenobiotics, for studying hepatocyte proliferation, and as bioartificial liver support systems. Yet, primary isolated hepatocytes rapidly lose liver specific functions when maintained under standard in vitro cell culture conditions. Many modifications to conventional culture methods have been developed to foster retention of hepatocyte function. Still, not all of the important functions -- especially the biotransformation functions of the liver -- can as yet be replicated at desired levels, prompting continued development of new culture systems. In the first part of this article, we review primary hepatocyte in vitro systems used in metabolism and enzyme induction studies. We then describe a scalable microreactor system that fosters development of 3D-perfused micro-tissue units and show that primary rat cells cultured in this system are substantially closer to native liver compared to cells cultured by other in vitro methods, as assessed by a broad spectrum of gene expression, protein expression and biochemical activity metrics. These results provide a foundation for extension of this culture model to other applications in drug discovery - as a model to study drug-drug interactions, as a model for the assessment of acute and chronic liver toxicity arising from exposure to drugs or environmental agents; and as a disease model for the study of viral hepatitis infection and cancer metastasis.

The metabolism of the bicyclic monoterpene Δ³-carene was investigated in vitro using human liver microsomes as well as human smoker/non-smoker lung microsomes and 12 different recombinant cytochrome P450 enzymes coexpressed with human CYP-reductase in Escherichia coli cells. We detected two metabolites using GC-MS analysis. The mass fragmentation indicated for one metabolite hydroxylation in the allyl position and for the other metabolite epoxidation at the double bond. For clear identification the suggested metabolites were synthesized in a four-step reaction. Comparison of GC retention times and mass spectra lead to the identification of the metabolites as Δ³-carene-10-ol ((1S, 6R)- 7,7-Dimethylbicyclo[4.1.0]hept-3-en-3-yl-methanol) and Δ³-carene-epoxide ((1S, 3S, 5R, 7R)-3,8,8-Trimethyl-4-oxatricyclo[ 5.1.0.03,5]octane). Δ³-carene-10-ol was formed by human liver microsomes and recombinant human CYP2B6, CYP2C19 and CYP2D6. Δ³-Carene-epoxide was obviously catalyzed only by CYP1A2. In both cases there was a clear correlation between the metabolite formation, incubation time and enzyme concentration, respectively. Further kinetic analysis revealed that CYP2B6 exhibited the highest activity for Δ³-carene 10-hydroxylation. Michaelis-Menten Km and Vmax for oxidation of Δ³-carene were 0.6 mM and 28.4 nmol/min/nmol P450 using human CYP2B6. For the formation of Δ³-carene-epoxide 98.2 mM and 3.9 nmol/min/nmol P450 were determined as Km and Vmax by using human CYP1A2. To our knowledge, this is the first time that Δ³-carene-10-ol and Δ³-carene-epoxide are described as human metabolites of Δ³- carene.