Ronald R. Bowsher

Method Validation and Measurement of Biomarkers in Nonclinical and Clinical Samples in Drug Development: A Conference Report

No HeadingBiomarkers are increasingly used in drug development to aid scientific and clinical decisions regarding the progress of candidate and marketed therapeutics. Biomarkers can improve the understanding of diseases as well as therapeutic and off-target effects of drugs. Early implementation of biomarker strategies thus promises to reduce costs and time-to-market as drugs proceed through increasingly costly and complex clinical development programs. The 2003 American Association of Pharmaceutical Sciences/Clinical Ligand Assay Society Biomarkers Workshop (Salt Lake City, UT, USA, October 24–25, 2003) addressed key issues in biomarker research, with an emphasis on the validation and implementation of biochemical biomarker assays, covering from preclinical discovery of efficacy and toxicity biomarkers through clinical and postmarketing implementation. This summary report of the workshop focuses on the major issues discussed during presentations and open forums and noted consensus achieved among the participants on topics from nomenclature to best practices. For example, it was agreed that because reliable and accurate data provide the basis for sound decision making, biomarker assays must be validated in a manner that enables the creation of such data. The nature of biomarker measurements often precludes direct application of regulatory guidelines established for clinical diagnostics or drug bioanalysis, and future guidance on biomarker assay validation should therefore be adaptable enough that validation criteria do not stifle creative biomarker solutions.

A sensitive radioenzymatic assay for the simultaneous determination of salsolinol and dopamine

Abstract A sensitive radioenzymatic assay for the simultaneous quantitation of salsolinol and dopamine in tissues and fluids has been developed. Salsolinol and dopamine were radiolabeled by 0-methylation using the enzyme catechol-0-methyltransferase and its cosubstrate, [3H]-S-adenosylmethionine, as the methyl donor. Specificity was achieved by alumina adsorption, selective solvent extraction, thin layer chromatography, primary amine precipitation and ion pair solvent extraction. The assay was linear over a 1000 fold concentration range. Sensitivities of 2 and 3 picograms were obtained for dopamine and salsolinol, respectively. Separate assay of standard samples had a coefficient of variation of 5%. Salsolinol was formed in vitro in dopamine enriched plasma and whole brain homogenates following incubation with physiologic concentrations of acetaldehyde.

Dopamine in rat adrenal glomerulosa.

There is increasing evidence that dopamine (DA) inhibits aldosterone production, but the source of DA for this dopaminergic influence is not known. In the present study we examined the adrenals zona glomerulosa for the presence of DA. Rats maintained on an intake of regular food were killed by decapitation and the adrenal capsule (containing zona glomerulosa) and the remainder of the gland (containing both cortex and medulla) were examined for their content of DA and also for norepinephrine (NE) and epinephrine (E). DA was found in adrenal glomerulosa in substantial quantity, 1.92 +/- 0.17 (SEM) ng/mg wet weight, representing an approximate concentration of DA of 1-100 microM. DA in adrenal capsule represented 12.2% of the total adrenal content of DA. NE and E were also present in glomerulosa, 3.46 +/- 0.32 and 18.7 +/- 2.1 ng/mg respectively, but, unlike DA, about 98% of the total adrenal content of NE and E was contained in adrenal medulla. The NE/E ratio in capsule and medulla were similar, although slightly higher in adrenal medulla, suggesting that the medulla is the source of the NE and E found in glomerulosa. On the other hand, the DA/E ratio was several-fold higher in glomerulosa than medulla--suggesting that glomerulosa DA was derived at least partially from a source other than adrenal medulla. We also found that short-term culturing of the adrenal reduced DA levels to 1/3 that observed in fresh tissue. This could explain in part why cultured glomerulosa has been shown to be more responsive to administered stimuli. In summary, the findings indicate a significant concentration of DA in adrenal glomerulosa, and suggest that the effects of DA on aldosterone production are mediated locally within the adrenal.