Active metabolite screening

A good example of metabolite screening as part of a discovery PK study was reported by Tiller and Romanyshyn.123 They described a dog PK study in which a monohydroxylated metabolite was found at much higher levels than the dosed NCE it was active and fit the pharmacodynamic (PD) profile better than the dosed NCE. 

From this discussion, there is an obvious advantage to FAC in that the assay development approach is extremely flexible and adaptable to the requirements of the interaction to be studied. It is worth mentioning that the effort placed on creating a FAC cartridge is never wasted - it can be used as a simple capture/elute tool for alternative screening approaches, and even in preclinical studies that require methods for monitoring drug candidates and their active metabolites [30]. 

The information originating from preceding studies is needed for the proper design of a food interaction study safety and tolerance data has to be considered as well as the PK results including a food screen. The terminal half-life of the drug or its active metabolite(s) will provide the basis for the washout periods. Single dose linearity/proportionality will help to define the dose. Safety and tolerance data will justify the dose. The PK comparison in a food screen (FIM study, see below) will influence the sample size. 

Cromakalim (1) [2] and RP 49356 (4) [41] were both discovered during in vivo screening using SHR, when very potent vasodilator effects were observed on administration of their precursor pyrrolidine and tetrahy-drothiopyran compounds, respectively. Isolation of these active metabolites and their resolution led to levcromakalim (2) and aprikalim (5), respectively. SAR work for antihypertensive evaluation was generally performed in the SHR, but for evaluation as relaxants of airways tissue, in vitro models such as those outlined above were used initially, and further evaluation was only... 

As for any bioanalytical method, the extent of validation for an immunoassay should be related to the intended application of the assay. Thus, if an immunoassay is intended to support rapid screening in discovery R D, the characterization of specificity and the accuracy and precision specifications may be less stringent than if the assay is used to support pre-clinical and clinical development studies. Indeed, an assay for discovery support may be designed to detect active metabolites as well as parent molecule, so that... 

In an effort to uncover anticancer active metabolites in Brazilian plants, in early 1993 we initiated active collaboration with several Brazilian natural products chemists. Out of the large number of extracts brought to our laboratory by Dr. Vanderlan da S. Bolzani and screened in our mechanism-based yeast bioassay, extracts derived from two leguminous plants were selected for further studies. Based on preliminary investigation the bioactive methanolic chloroform extract of the leaves of Cassia leptophylla which was shown to contain bioactive alkaloidal constituents was further processed for alkaloids. The alkaloid fraction thus obtained was fractionated by column chromatography on alumina, followed by Silica gel preparative TLC and reversed-phase TLC to afford seven piperidine alkaloids [55] (-)-spectaline (68), (-)-spectalinine (69), canavaline (70), leptophyllin A (71), 3-acetylleptophyllin A (72), iso-... 

There are a number of advantages for screening drug candidates for active metabolites during drug discovery. The primary reason is that the process could lead to the discovery of a drug candidate with superior drug... 

However, this approach requires significant deconvolution efforts when activity is found in mixtures. To increase the success rate and decrease the number of compounds screened to a manageable size, the search for active metabolites could be limited to those compounds/chemotypes showing a high clearance rates in in vitro metabolic stability or in vivo exposure screens. 

Sewage water was extracted under acidic, neutral, and basic conditions with methylene chloride and these extracts, both free and methylated, were screened by gas chromatography-mass spectrometry. The presence of 2-(4-chlorophenoxy)-2-methylpropanoic acid (CPIB), the active metabolite of the widely used hypolipidemic drug, clofibrate, and 2-hydroxy benzoic acid (salicyclic acid), a metabolite of aspirin, was established in these screens. This is to our knowledge the first report of detection of drugs in water samples. A review describing the detection of other organic compounds has recently appeared (1). 

Hvastkovs EG, So M, Krishnan S, Bajrami B, Tarun M, Jansson 1, Schenkman JB, Rusling JF (2007) Electrochemiluminescent arrays for cytochrome P450-activated genotoxicity screening. DNA damage from Benzo[a]pyrene Metabolites. Anal Chem 79 1897—1906... 

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