Novel benzamide-based histamine h3 receptor antagonists: the identification of two candidates for clinical development.

The preclinical characterization of novel phenyl(piperazin-1-yl)methanones that are histamine H3 receptor antagonists is described. The compounds described are high affinity histamine H3 antagonists. Optimization of the physical properties of these histamine H3 antagonists led to the discovery of several promising lead compounds, and extensive preclinical profiling aided in the identification of compounds with optimal duration of action for wake promoting activity. This led to the discovery of two development candidates for Phase I and Phase II clinical trials.

Human liver cytochrome P450 2D6 genotype, full-length messenger ribonucleic acid, and activity assessed with a novel cytochrome P450 2D6 substrate.

OBJECTIVE: The goal of this study was to develop and validate a cytochrome P450 (CYP) 2D6 probe substrate with improved sensitivity to elucidate the relationship of CYP2D6 ribonucleic acid transcript levels, genotype, and enzyme activity in human liver biopsy samples. METHODS: CYP2D6 activity in tissue homogenates of liver biopsy specimens collected from control subjects (with no apparent liver disease), liver biopsy subjects, liver transplant subjects, and liver bank specimens was assessed with a calcimimetic, R-568, a high-clearance and specific substrate of CYP2D6. The livers were genotyped for the 6 most common CYP2D6 genetic variants (ie, *3, *4, *5, *6, *7, and *8). The 1.5-kilobase CYP2D6 messenger ribonucleic acid (referred to as full-length) transcripts were estimated with a semiquantitative reverse transcription-polymerase chain reaction assay. RESULTS: As a CYP2D6-specific catalytic probe, R-568 offers a 20-fold higher sensitivity compared with that of dextromethorphan. The improved assay sensitivity allowed evaluation of CYP2D6 enzyme activity in a few milligrams of tissue collected from biopsy specimens. The ratio of CYP2D6 enzyme activity to transcript remained relatively constant within each group of subjects, especially within the control group. However, mean activity to transcript varied greatly across the 4 groups of subjects. The liver samples in the control group showed significantly higher enzyme activity but a lower transcript level. CONCLUSIONS: A combination of genotyping and messenger ribonucleic acid level determination could allow a quantitative estimation of functional CYP2D6 activity in healthy human livers with a reasonable degree of confidence. Kinetic study with R-568 indicates that this compound is probably the most sensitive CYP2D6 probe substrate available.

In vitro studies in drug discovery and development: an analysis of study objectives and application of good laboratory practices (GLP).

In vitro drug metabolism studies play a dual role along the path from drug discovery to preclinical development. By analyzing the objectives of each type of study the question of whether to apply good laboratory practices (GLP) requirements is clarified. This review outlines the various in vitro techniques available and categorizes the goals for which they are applied as either supporting drug discovery or influencing decisions of clinical safety. Based on this categorization it is proposed that studies performed to explore the utility of a potential drug candidate be conducted non-GLP, while studies used to support IND and post-IND submissions be considered for GLP.

Only truncated, not complete cytochrome p450 2D6 RNA transcript and no detectable enzyme activity are expressed in human lymphocytes.

Genotyping of the highly polymorphic cytochrome p450 2D6 (CYP2D6) permits a gross classification of individual phenotype (viz. ultra-rapid, extensive, and poor metabolizers). It does not, however, provide a precise prediction of CYP2D6 activity, particularly in an individual possessing at least one functional CYP2D6 allele. It has been suggested that the level of mRNA expression or enzyme activity in lymphocytes, isolated from blood, could potentially provide a better quantitative estimate of in vivo hepatic enzymatic activity in human subjects. Although short sequences of CYP2D6 mRNA have been detected in human lymphocytes by reverse transcriptase-polymerase chain reaction (RT-PCR) that suggests the potential use of lymphocyte RNA as a readily accessible biomarker, it is not known whether a functional enzyme is expressed in human lymphocytes. In this study, human lymphocyte activity was assessed with a CYP2D6-specific, high-turnover probe substrate that is severalfold more sensitive than traditional markers of CYP2D6 (e.g., dextromethorphan). CYP2D6 catalytic activity could not be detected in homogenates of human lymphocytes, even at high protein concentrations and with supplementation of enzyme cofactors. Further RT-PCR analysis of lymphocytes collected from eight human donors revealed the presence of only a fragment, but not the complete transcript, of CYP2D6 mRNA. Northern blot RNA transcript analysis also failed to indicate the presence of the full-length transcript in lymphocytes. Collectively, these data indicate that human lymphocytes express neither the full-length CYP2D6 mRNA transcript nor functional enzyme activity. Therefore, the utility of lymphocytes as a functional biomarker for CYP2D6 enzyme activity is not clear at present.