Assay, biological

Preliminary pharmacokinetic behavior can be tested through a number of whole cell assays. Most commercially successful drugs are administered orally, meaning the drug must be able to enter the bloodstream by crossing membranes in the intestines. The most common membrane permeability assay is performed by monitoring the absorption and secretion of a compound by colon carcinoma cells (Caco-2). Diffusion across Caco-2 cell membranes is considered to be a valid model for molecular transport in the small intestines.16 

Drugs are mostly metabolized by liver enzymes, especially the cytochrome P-450 enzyme family. The ability for cytochrome P-450 enzymes to metabolize a hit is tested with liver microsomes. Liver microsomes consist primarily of endoplasmic reticulum that contains metabolic enzymes. Hits are individually incubated in the presence of the liver microsomes. Monitoring changes in concentrations provides a sense of the rate of metabolism of each hit. Liver microsomes are also used to determine whether the hit inhibits metabolic processes. Hits that inhibit liver metabolism are shunned.17 

Acceptable hits do not need to show ideal behavior, but problem compounds will be removed from consideration. If all the hits fail initial pharmacokinetic screening, several options are possible. First, the search for hits could start over with screening of a new library. Second, the threshold for selection of hits could be lowered to enlarge the pool of hits, some of which may pass the permeability and metabolic screens. Third, the criteria for passing the Caco-2 and microsome screens may be softened to allow some hits to pass.18 

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Chemical compounds can also be represented by chemical data, or even by biological data. In fact, Briem et al. have used the results of a battery of biological assays to represent a compound for the modeling of other biological data [62-64]. 

The British Pharmacopoeia specifies a biological assay for the sodium salt of rifamycia SV [14897-39-3]. It also specifies a spectrophotometric assay for rifampicia (201). The United States Pharmacopeia requires an hplc assay for rifampin (202). 

Because of the time and expense involved, biological assays are used primarily for research purposes. The first chemical method for assaying L-ascorbic acid was the titration with 2,6-dichlorophenolindophenol solution (76). This method is not appHcable in the presence of a variety of interfering substances, eg, reduced metal ions, sulfites, tannins, or colored dyes. This 2,6-dichlorophenolindophenol method and other chemical and physiochemical methods are based on the reducing character of L-ascorbic acid (77). Colorimetric reactions with metal ions as weU as other redox systems, eg, potassium hexacyanoferrate(III), methylene blue, chloramine, etc, have been used for the assay, but they are unspecific because of interferences from a large number of reducing substances contained in foods and natural products (78). These methods have been used extensively in fish research (79). A specific photometric method for the assay of vitamin C in biological samples is based on the oxidation of ascorbic acid to dehydroascorbic acid with 2,4-dinitrophenylhydrazine (80). In the microfluorometric method, ascorbic acid is oxidized to dehydroascorbic acid in the presence of charcoal. The oxidized form is reacted with o-phenylenediamine to produce a fluorescent compound that is detected with an excitation maximum of ca 350 nm and an emission maximum of ca 430 nm (81). 

Both nicotinic acid and nicotinamide have been assayed by chemical and biological methods. Owing to the fact that niacin is found in many different forms in nature, it is important to indicate the specific analyte in question. For example, if biological assay procedures are used, it is necessary to indicate whether the analysis is to determine the quantity of nicotinic acid or if niacin activity is the desired result of the analysis. If nicotinic acid is desired, then a method specific for nicotinic acid should be used. If quantitation of niacin activity is the desired outcome, then all compounds (bound and unbound) which behave like niacin will assay biologically for this substance (1). 

As with many of the vitamins, biological assays have an important historical role and are widely used. For example, microbiological assays use l ctobacillusplantarum ATCC No. 8014 (57,59) or l ctobacillus arabinosus (60). These methods are appropriate for both nicotinamide and nicotinic acid. Selective detection of nictonic acid is possible if l euconostoc mesenteroides ATCC No. 9135 is used as the test organism (61). The use of microbiological assays have been reviewed (62). 

Many experimental approaches have been appHed to the deterrnination of stabihty constants. Techniques include pH titrations, ion exchange, spectrophotometry, measurement of redox potentials, polarimetry, conductometric titrations, solubiUty deterrninations, and biological assay. Details of these methods can be found in the Hterature (9,10). 

Table 1 Characteristics of biological assays for cyanobacterial toxins...

For this reason in this section we depart from our practice of discussing each synthesis in detail. Certain standard operations, for example, introduction of 9-fluoro substituents, are referred to as though they were a single reaction only the salient points of the syntheses are dwelt on. Examination of some of these syntheses strongly suggests that these were aimed at preparing compounds for biologic assay. It is more than likely that the commercial preparations have only the final product in common with the published route. 

It is critically important to capture biological assay data and allow the medicinal chemist to access the information for SAR analysis. Many software systems have been developed for this purpose, and we briefly describe two of them below. 

Di, L, Kerns, E. Biological assay challenges from compounds soluhility strategies for hioassay optimization. Drug Discov. Today 2006, 11, 446 51. 

Statistical analysis of results of biological assays and tests 5.3 (1997) European Pharmacopoeia 3rd Ed. Council of Europe, Strasbourg. 

There are indications that the variety of C substrates in the rhizosphere soil is basically too wide to be significantly affected by changes in quality of plant residues from the previous crop. For example, legumes as preceding crop were shown to increase significantly microbial diversity in the bulk soil, as estimated by Biolog assay, whereas in the rhizosphere soil this effect of legumes could not be detected (145). 

For example, use of 10 different isocyanides and amines, along with 40 different aldehydes and carboxylic acids has the potential to generate 160,000 different dipeptide analogs.65 This system was explored by synthesizing arbitrarily chosen sets of 20 compounds that were synthesized in parallel. The biological assay data from these 20 combinations were then used to select the next 20 combinations for synthesis. The synthesis-assay-selection process was repeated 20 times. At the end of this process the average inhibitory concentration of the set of 20 products had been decreased from 1 mM to less than 11xM. 

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