Gerhard Zürcher

Simultaneous radioenzymatic determination of plasma and tissue adrenaline, noradrenaline and dopamine within the femtomole range

A radiometric-enzymatic assay for measuring simultaneously femtomole quantities of adrenaline, noradrenaline and dopamine has been developed. The three catecholamines are first converted to their O-methylated analogues by catechol-O-methyltransferase in the presence of S-adenosyl-methionine-3H and thereafter extracted following addition of sodium tetraphenylborate. This extraction, together with an improved quick chromatographic separation and the oxidation of the adrenaline and noradrenaline derivatives to vanillin, yields an extremely high sensitivity and specificity of the method. The present assay allows the determination of adrenaline, noradrenaline and dopamine in tissue samples with a protein content of 100 μg or less and in plasma volumes of 20 – 100 μl. The amine content of 40 – 50 samples can be determined in two days by one person. Due to the high sensitivity achieved, this method promises to be a valid alternative to the gas chromatography-mass spectrometry technique.

Integrated pharmacokinetics and pharmacodynamics of the novel catechol‐O‐methyltransferase inhibitor tolcapone during first administration to humans

To assess the tolerability, pharmacokinetics and pharmacodynamics of single oral doses of the novel catechol‐O‐methyltransferase (COMT) inhibitor tolcapone in healthy volunteers.

Rapid and Sensitive Single‐Step Radiochemical Assay for Catechol‐O‐Methyltransferase

Abstract: A simple, rapid and reliable radiometric assay for the determination of catechol‐O‐methyltransferase activity is described. The method is based on the conversion of catechol to [3H]guaiacol by catechol‐O‐methyltransferase in the presence of Mg2+, adenosine deaminase and S‐adenosyl l‐[methyl‐3H]methionine. Incubation and direct extraction of [3H]guaiacol into organic scintillation fluid, as well as counting, are performed in the same standard scintillation vial. The assay is easy to perform and more sensitive than previous analogous procedures. The method has been applied to the assay of catechol‐O‐methyltransferase activity in discrete brain areas and also peripheral organs of rat and in human erythrocytes.

Multiple-dose clinical pharmacology of the catechol-O-methyl-transferase inhibitor tolcapone in elderly subjects.

Abstract.Objective: The purpose of this study was to assess the multiple-dose clinical pharmacology of tolcapone, a novel catechol-O-methyltransferase (COMT) inhibitor, in elderly subjects. Methods:The drug was administered orally t.i.d. for 7 days to four sequential groups of eight elderly subjects (gender ratio1:1) at doses of 100, 200, 400 and 800 mg in a double-blind, randomised, placebo-controlled, ascending-multiple-dose design. On days 2 and 7, a single dose of levodopa/benserazide 100/25 mg was given 1 h after the first intake of tolcapone. Plasma concentrations of tolcapone, its metabolite 3-O-methyltolcapone, levodopa and 3-O-methyldopa were determined during the course of the study in conjunction with COMT activity in erythrocytes. Results:Tolcapone was well tolerated at all dose levels, with a slight increase in gastrointestinal adverse events in females at higher doses. The drug was rapidly absorbed and eliminated and showed no changes in pharmacokinetics with time during multiple doses of 100 and 200 mg t.i.d. At doses of 400 and 800 mg t.i.d., tolcapone accumulated moderately as reflected in increased Cmax and AUC values. Despite the long half-life of 3-O-methyltolcapone (39 h), only minor accumulation occurred due to suppression of its formation by tolcapone. The pharmacodynamics of tolcapone did not change during the week of treatment as reflected in inhibition of COMT activity in erythrocytes, the derived parameters of the plasma concentration-effect relationship (inhibitory Emax model with constant EC50 values) and the effect on levodopa pharmacokinetics (1.6 to 2.5-fold increase in bioavailability). This suggests the absence of tolerance development and the insignificance of the altered pharmacokinetics at 400 and 800 mg t.i.d. with regard to the pharmacodynamics. Conclusion:The results of this study offer promising perspectives for the application of tolcapone as adjunct therapy to levodopa in the treatment of Parkinson’s disease.

Radioenzymatic assay of femtomole concentrations of DOPA in tissues and body fluids.

Abstract— A single isotope radioenzymatic procedure for the measurement of DOPA has been developed. The assay combines O‐methylation of DOPA by purified COMT using [3H]SAM as the methyl donor and subsequent purification as the DNFB derivative of 3‐O‐[methyl‐3H]DOPA. The present method is about 100 times more sensitive than currently available DOPA methods. This is due to decreased blank values and increased enzymatic conversion giving transmethylation values of 50% with tissue extracts and values of almost 100% with pure solutions. Although COMT methylates a wide variety of catechol compounds, specificity of the assay is achieved by selective extraction and purification of the final product by tlc.

Structure-based design, synthesis, and in vitro evaluation of bisubstrate inhibitors for catechol O-methyltransferase (COMT).

The enzyme catechol O-methyltransferase (COMT) catalyzes the Me group transfer from the cofactor S-adenosylmethionine (SAM) to the hydroxy group of catechol substrates. Potential bisubstrate inhibitors of COMT were developed by structure-based design and synthesized. The compounds were tested for in vitro inhibitory activity against COMT obtained from rat liver, and the inhibition kinetics were examined with regard to the binding sites of cofactor and substrate. One of the designed molecules was found to be a bisubstrate inhibitor of COMT with an IC50 = 2 microM. It exhibits competitive kinetics for the SAM and noncompetitive kinetics for the catechol binding site. Useful structure-activity relationships were established which provide important guidelines for the design of future generations of bisubstrate inhibitors of COMT.

Optimizing levodopa pharmacokinetics with multiple tolcapone doses in the elderly

The multiple‐dose tolerability, pharmacokinetics, and pharmacodynamics of tolcapone, a novel catechol‐O‐methyltransferase (COMT) inhibitor, were assessed in healthy elderly volunteers receiving concomitant carbidopa and levodopa.

Expression of functional membrane-bound and soluble catechol-O-methyltransferase in Escherichia coli and a mammalian cell line.

Abstract: Human catechol‐O‐methyltransferase (hCOMT) cDNA was used to express the recombinant hCOMT enzyme in sufficient quantities in prokaryotic as well as in eukaryotic cells to allow kinetic studies. When human membrane‐bound catechol‐O‐methyltransferase (MB‐COMT; amino acids 1‐271) and the soluble catechol‐O‐methyltransferase COMT (S‐COMT; Δ membrane anchor hCOMT; amino acids 27‐271), with the latter lacking the first 26 hydrophobic amino acids, were expressed in Escherichia coli, a relatively high‐level synthesis of catalytically active enzymes was obtained. Insertion of the human MB‐COMT‐coding sequence into an eukaryotic expression vector under transcriptional control of the cytomegalovirus (CMV) promoter and enhancer yielded large quantities of hCOMT in human kidney 293 cells. Subcellular fractionation of 293 cells transfected with pBC12/CMV‐hCOMT showed hCOMT to be located predominantly in the membrane fraction. The catechol‐O‐methyltransferase (COMT) activity was measured in cytosolic and membrane fractions at 37°C, giving values of 33 and 114 units/mg of protein, respectively (1 unit produces 1 nmol of guaiacol/h). Km values were 10 μM for MB‐COMT and 108 μM for S‐COMT, indicating that recombinant MB‐COMT exhibits a higher affinity for catechol as the substrate than the soluble form. RNA blot analysis of human hepatome cells (Hep G2), kidney, liver, and fetal brain revealed only one species of hCOMT mRNA of ∼ 1.4 kb. Its level in these various tissues was similar to those of COMT protein in each tissue. Using the polymerase chain reaction (PCR) with primers surrounding the putative membrane anchor region, we have clearly identified a single‐size PCR product generated from hCOMT mRNA of various human tissues. Hence, the two forms of the enzyme cannot be the products of an alternative splicing of transcripts. We suggest that S‐COMT is generated by proteolytic cleavage between the NH2‐terminal membrane anchor and the catalytic domain of the membrane‐bound form. Lack of the N‐terminal fragments reduces the catalytic activity of the enzyme.

Bisubstrate inhibitors for the enzyme catechol-O-methyltransferase (COMT): influence of inhibitor preorganisation and linker length between the two substrate moieties on binding affinity.

Inhibition of the enzyme catechol-O-methyltransferase (COMT) is an important approach in the treatment of Parkinsons disease. A series of new potent bisubstrate inhibitors for COMT, resulting from X-ray structure-based design and featuring adenosine and catechol moieties have been synthesised. Biological results show a large dependence of binding affinity on inhibitor preorganisation and the length of the linker between nucleoside and catechol moieties. The most potent bisubstrate inhibitor for COMT has an IC50 value of 9 nM. It exhibits competitive kinetics for the SAM and mixed inhibition kinetics for the catechol binding site. Its bisubstrate binding mode was confirmed by X-ray structure analysis of the ternary complex formed by the inhibitor, COMT and a Mg2+ ion.

Effects of tolcapone, a novel catechol-O-methyltransferase inhibitor, on striatal metabolism of L-DOPA and dopamine in rats

In vivo brain microdialysis was used to assess the effects of tolcapone, a novel central and peripheral inhibitor of catechol-O-methyltransferase on striatal 3,4-dihydroxyphenyl-L-alanine (L-dopa) and dopamine metabolism. The oral administration of 30 mg/kg of tolcapone failed to change dopamine output but elicited a marked and long-lasting decrease of the extracellular levels of homovanillic acid (HVA) and 3-methoxytyramine with a concomitant increase of 3,4-dihydroxyphenylacetic acid (DOPAC). The administration of L-dopa (20 and 60 mg/kg p.o.) + benserazide (15 mg/kg p.o.) resulted in dose-dependent increase of dialysate levels of L-dopa and 3-O-methyl-DOPA. Tolcapone (30 mg/kg p.o.), given as adjunct to both doses of L-dopa, markedly enhanced the elevation or extracellular L-dopa, while it completely prevented the formation of 3-O-methyl-DOPA. In another experiment, the administration of L-dopa + benserazide (30 + 15 mg/kg p.o.) resulted in increased extracellular levels of dopamine, DOPAC, HVA and 3-methoxytyramine. The co-administration of tolcapone (30 mg/kg p.o.) further increased dopamine and DOPAC levels, whereas HVA and 3-methoxytyramine effluxes were reduced. These findings support the notion that tolcapone has the ability to enhance striatal dopamine neurotransmission by increasing L-dopa bioavailability through peripheral and central inhibition of L-dopa O-methylation, as well as by blocking the central conversion of dopamine into 3-methoxytyramine.