Parent compounds assay

Degradation product(s) co-eluting with the parent compound in the parent compound assay. Check for peak homogeneity via PDA-UV, LC MS, and/or use an orthogonal separation technique, as discussed earlier. Modify conditions to separate if necessary. 

Parent compound assayed using the bromine oxidation protocol... 

Neu5Ac2en 4, a micromolar inhibitor of influenza virus sialidase 4 x 10 M (A/N2)] (Holzer et al. 1993), was first identified as a very good inhibitor in the late 1960s (Meindl and Tuppy 1969). A series of C-5 modified Neu5Ac2en derivatives provided the first improved in vitro inhibitors compared with the parent compound 4. The replacement of the C-5 A-acetyl moiety with a A-trifluoroacetyl group resulted in the most potent inhibitor of this series, 2-deoxy-2,3-didehydro-A-trifluoroacetylneuraminic acid 10 [A] 8 x 10 M (A/Nl)] (Meindl et al. 1974). While these C-5 modified compounds were also very effective in cell culture assays (Palese et al. 1974a Palese and Compans 1976), none, including the parent... 

Agarwal et al. 1978), the quantification of these specific enzymes may indicate that exposure to endosulfan has occurred. Blood tests, such as decay curves for aminopyrine in plasma, which are semiquantitative indices of liver enzyme induction, have been used successfully in the past to demonstrate enzyme induction in pesticide-exposed workers. Because numerous chemicals found at hazardous waste sites also induce these hepatic enzymes, these measurements are not specific for endosulfan exposure. However, measurements of enzyme activity, together with the detection of the parent compound or its metabolites in tissue or excreta, can be useful indicators of exposure. All of these potential biomarkers require further verification in epidemiological studies. Further studies with focus on the development of methods to separate and measure the estrogenicity of endosulfan in in vitro assays would be valuable since these assays are more sensitive and discriminative than other conventional biomarkers. Preliminary results have been presented by Sonnenschein et al. (1995). 

With this focus on CYP and fiver metabolism, most companies have established high throughput assays to measure compound stability in the presence of human (or preclinical species) fiver microsomes [49]. Disappearance of starting compound from an incubation with microsomes is monitored. Measurement at a single time point enables a rank-ordering of compounds for stability based on percent of parent compound remaining acquisition of data at multiple time points allows determination of half-life, intrinsic clearance, and extrapolation to a predicted in vivo clearance [50]. 

It is well known that 1,10-phenanthrolines are highly active ironchelating agents. The parent compound itself has recently been shown to increase HIF-la levels in ocular tissue and to suppress 02-mediated epithelial cell proliferation when administered to mice [29]. A quantitative assay was developed to measure transcriptional potency of certain HIF stabilizers via an HRE-mediated (3-lactamase production in which the EC50 of 1,10-phenanthroline was measured to be approximately 8 pM. In addition, VEGF was dose-dependently produced in mouse embryonic fibroblasts by 1,10-phenanthroline with an EC50 of... 

Table 7.5 provides a complete list of common metabolites and their mass shifts relative to parent compounds.117 While the concept of metabolite profiling is not new,20 multiple advances in MS hardware and software allow researchers to look more easily for metabolites and include them in PK assays.118... 

Phenothiazines The phenothiazines (PTZs) undergo extensive metabolism. Metabolic routes include S-oxidation, aromatic hydroxylation, N-dealkylation, N-oxidation, and a combination of these processes. Chlorpromazine, for example, possesses 168 possible metabolites, a large proportion of which are pharmacologically active compounds. The development of an HPLC assay capable of resolving a large number of these metabolites is virtually impossible and assays that permit the simultaneous determination of the parent compound and a selected number of active metabolites must suffice. The PTZ group of compounds includes chlorpromazine, thioridazine, fluphenazine, and perphenazine. 

In addition to ARSAC approval, the protocol must also be approved by ethics committees in the normal manner for studies in man. The study should be conducted in between four and eight consenting subjects, in facilities where any spills of radiolabelled materials can be contained and monitored. Normally, subjects will be required to provide blood samples and to collect all excreta for a period determined by the known or estimated half-lives of the parent compound and metabolite. With cooperative subjects, recoveries of radioactivity should be close to 100%. Samples will be assayed for radioactivity and by cold chromatographic methods, and every attempt should be made to identify major metabolites... 

In man, dorzolamide is metabolized by A-deethylation. Sensitive HPLC assays for both entities in whole blood, plasma, and urine have been developed to support clinical pharmacokinetic studies [13]. Both the parent compound and /V-desethyl-dorzolamide are excreted in the urine. 

Reporting false-positive results may send an innocent person to prison, so everything possible must be done to avoid such an outcome. Assays with enhanced sensitivity to detect both parent compound and metabolites will allow for detection of drugs over longer periods of time and for more accurate determination of when an individual was first exposed to a chemical. 

Methods for Determining Biomarkers of Exposure and Effect. Section 2.6.1 reported on biomarkers used to identify or quantify exposure to diazinon. Some methods for the detection of the parent compound in biological samples were described above. The parent chemical is quickly metabolized so the determination of metabolites can also serve as biomarkers of exposure. The most specific biomarkers will be those metabolites related to 2-isopropyl-6-methyl-4-hydroxypyrimidine. A method for this compound and 2-(r-hydroxy-l -methyl)-ethyl-6-methyl-4-hydroxypyrimidine in dog urine has been described by Lawrence and Iverson (1975) with reported sensitivities in the sub-ppm range. Other metabolites most commonly detected are 0,0-diethylphosphate and 0,0-diethylphosphorothioate, although these compounds are not specific for diazinon as they also arise from other diethylphosphates and phosphorothioates (Drevenkar et al. 1993 Kudzin et al. 1991 Mount 1984 Reid and Watts 1981 Vasilic et al. 1993). Another less specific marker of exposure is erythrocyte acetyl cholinesterase, an enzyme inhibited by insecticidal organophosphorus compounds (see Chapter 2). Methods for the diazinon-specific hydroxypyrimidines should be updated and validated for human samples. Rapid, simple, and specific methods should be sought to make assays readily available to the clinician. Studies that relate the exposure concentration of diazinon to the concentrations of these specific biomarkers in blood or urine would provide a basis for the interpretation of such biomarker data. 

In 1989 Cutler et al.1 reported the isolation of 3,7-dimethyl-8-hydroxy-6-methoxyisochro-man (1) from Penicillium corylophilum and demonstrated that it inhibited etiolated wheat coleoptiles at 10-3 and 1(T4M, as did the acetoxy (2) and methoxy (3) derivatives (Figure 5.1).2 The Parent compound had originally been isolated from moldy millet hay implicated in the death of cattle,3 but the metabolite had not been tested in plants. Because of the encouraging results obtained in the wheat coleoptile bioassay, isochromans 1,2, and 3 were assayed on greenhouse-grown bean, com, and tobacco plants. The methyl ether exhibited the greatest herbicidal activity in all the plants treated, while the parent and its acetoxy derivative were active only on corn. 

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