Effects of a standardized meal on the pharmacokinetics of the new cardiotonic agent piroximone.

The influence of food on the pharmacokinetics of piroximone (MDL 19.205, CAS 84490-12-0) was evaluated in two groups of 6 healthy male volunteers receiving either 25 or 50 mg of the drug. Single doses were administered intravenously and orally under fasting conditions or orally with a standard breakfast on 3 different days with a washout period of at least 3 days in-between doses, according to an open, 3-way crossover, randomized design. Pharmacokinetic parameters (Cmax, tmax, AUC, t1/2, Cl, aVd, UEx) were not affected by food administration, but significant differences were found in t1/2 calculated from the decay of plasma concentrations in response to oral administration of 25 mg and 50 mg treatment doses. The urinary excretion of piroximone was significantly reduced after oral administration, when compared with the values obtained after intravenous application. In addition, extra-renal clearance was significantly reduced in the 50 mg treatment group, when compared with the values obtained in response to 25 mg. Bioavailability of piroximone calculated from AUC data compared favorably with data obtained from urinary recovery results.

Piroximone, dobutamine and nitroprusside: comparative effects on haemodynamics in patients with congestive heart failure.

Four intravenous doses of piroximone, an imidazolone derivative, were administered to 12 patients with congestive heart failure to produce a four-point dose-response curve. The haemodynamic effects were compared with those of dobutamine and nitroprusside, the substances being given sequentially and in randomized order. Piroximone and dobutamine significantly and similarly increased cardiac index (CI) and stroke volume index (SVI). Nitroprusside produced no such effect. By contrast, piroximone and nitroprusside significantly and similarly decreased mean pulmonary artery pressure (MPAP), pulmonary capillary wedge pressure (PCWP), right atrial pressure (RAP) and pulmonary vascular resistance (PVR), while such changes were not seen following dobutamine. Direct comparisons between the agents were made at doses that lowered systemic vascular resistance (SVR) to the same extent. The major difference between dobutamine and piroximone was an apparent additional vasodilator activity displayed by piroximone as demonstrated by a significantly greater decrease in MPAP, PCWP and RAP for a matched reduction in SVR and a similar increase in CI. The major difference between nitroprusside and piroximone was the significantly higher increase in CI and SVI elicited by piroximone for a matched reduction in SVR and a similar decrease in PCWP and RAP. The changes in loading conditions being equivalent, the higher increase in CI is likely to be accounted for by a direct inotropic activity.

Phase I study of methylacetylenic putrescine, an inhibitor of polyamine biosynthesis.

In a phase I clinical trial, nine patients with advanced malignancies not amenable to alternative therapy received alpha-methyl-delta-acetylenic putrescine (MAP), an enzyme-activated, irreversible inhibitor of ornithine decarboxylase (ODC). MAP was given orally in increasing doses to successive groups of three patients as follows: 375 mg, 750 mg and 1500 mg/day, given as three equally divided doses for 4 weeks. Doses of 375 and 750 mg/day were well tolerated, with no detectable toxicity. Of three patients receiving 1500 mg/day, two experienced moderate to severe myelosuppression; one of these also became anuric, requiring the discontinuation of therapy after 9 days. Both effects were reversible after treatment was stopped. No objective responses were observed, with five patients having stable disease and four, progressive disease during the study period. In the seven patients in whom it could be calculated, the plasma elimination half-life t1/2 of MAP measured on the last day of treatment was between 3.9 and 9.2 h in six patients (mean, 5.6 h) and 26.1 h in the seventh. Mean steady-state trough concentrations of MAP were 2.3 mumol after the 375 mg/day dose, 7.1 mumol after 750 mg/day and 16.6 mumol after dosing with 1500 mg/day for 4 weeks, the levels after each treatment schedule being sufficient to inhibit ODC as demonstrated by increases in the urinary excretion of decarboxylated S-adenosylmethionine (dc-SAM). MAP treatment was associated with mean maximal increases in the urinary excretion of dc-SAM of 2.6-, 9.3- and 17.9-fold after 375, 750 and 1500 mg/day for 4 weeks, respectively, but no consistent changes in the urinary excretion of the polyamines, putrescine, spermidine or spermine were observed. Thus, the 24-h urinary excretion of dc-SAM may be used as a conveniently accessible marker of ODC inhibition in cancer patients.

Double-blind study of oral gamma-vinyl GABA in the treatment of dystonia.

Six patients with different forms of dystonia were treated with gamma-vinyl GABA, a specific enzyme-activated inhibitor of GABA-transaminase, in a double-blind, placebo-controlled crossover study. gamma-Vinyl GABA therapy, 2 g daily for 2 weeks, was compared with placebo by weekly assessments. There were no consistent changes in three evaluation scores. Agents that augment CNS GABA are unlikely to benefit patients with generalized dystonia.

Effects of (2R, 5R)-6-heptyne-2,5-diamine, a potent inhibitor of L-ornithine decarboxylase, on rat hepatoma cells cultured in vitro.

DL-alpha-Difluoromethylornithine (F2MeOrn), the most widely-used inhibitor of L-ornithine decarboxylase, has been a useful tool to demonstrate that polyamine biosynthesis is required to maintain maximum rates of cell proliferation. However, in most eukaryotic cell systems, F2MeOrn exerts cytostatic rather than cytotoxic effects. This may be due to the fact that this inhibitor creates only incomplete polyamine deficiency. In particular, F2MeOrn scarcely depletes intracellular spermine levels. We now demonstrate in rat hepatoma tissue culture (HTC) cells that (2R, 5R)-6-heptyne-2,5-diamine, a more potent irreversible inhibitor of L-ornithine decarboxylase than F2MeOrn, decreases the concentrations of all polyamines including spermine. In parallel with the depletion of these amines, there is a progressive decrease in the rate of cell proliferation and in cell viability. Restoration of the intracellular polyamine content, by addition to the medium of polyamines or a high concentration of L-ornithine, the substrate of L-ornithine decarboxylase, further demonstrates that the antiproliferative effects of (2R, 5R)-6-heptyne-2,5-diamine do result from polyamine deficiency. These findings support the concept that polyamines play an essential function in the cell division processes and emphasize the vital function of spermine in mammalian cells.

Metabolism of N8-monoacetylspermidine in rat hepatoma cells. Investigation of its effect on the activity of L-ornithine decarboxylase.

Recent evidence has indicated a role for the acetyl derivatives of polyamines, particularly N8-monoacetylspermidine, as activators of L-ornithine decarboxylase in rat hepatoma tissue culture (HTC) cells. This is in contrast with the well-described negative regulatory control of ornithine decarboxylase exerted by their non-acetylated counterparts. Because of the possibility of a rapid extracellular and intracellular catabolism of the acetyl derivatives of polyamines, the metabolism of N8-monoacetylspermidine and its effect on HTC cell ornithine decarboxylase have been investigated, under conditions which eliminate its extracellular catabolism. Differing from previous reports, we demonstrate that N8-monoacetylspermidine does not elevate ornithine decarboxylase activity when added at low concentrations to the culture medium of HTC cells. Higher concentrations decrease ornithine decarboxylase activity in a dose-dependent manner. This effect cannot be unambiguously attributed to the effect of the acetyl derivative itself, because of the presence in situ of a very active N8-monoacetylspermidine deacetylase, which generates spermidine intracellularly.

Accumulation of decarboxylated S-adenosyl-L-methionine in mammalian cells as a consequence of the inhibition of putrescine biosynthesis.

Biological transmethylation reactions and polyamine biosynthesis share the substrate S-adenosyl-L-methionine. Under normal conditions, decarboxylated S-adenosyl-L-methionine, the aminopropyl donor for polyamine biosynthesis, does not accumulate because of its rapid utilization in spermidine and spermine synthesis. Alteration of polyamine synthesis by DL-alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of L-ornithine decarboxylase, leads to a striking accumulation of decarboxylated S-adenosyl-L-methionine in rat hepatoma cells cultured in vitro and in rat ventral prostate. This increase is due both to lack of putrescine and spermidine for the aminopropyltransferase reactions and to the elevation of S-adenosyl-L-methionine decarboxylase activity. The biological implications of accumulation of decarboxylated S-adenosyl-L-methionine are discussed with regard to the regulation of S-adenosyl-L-methionine decarboxylase activity and to the antiproliferative effects of DL-alpha-difluoromethylornithine.

Metabolism of acetyl derivatives of polyamines in cultured polyamine-deficient rat hepatoma cells.

The acetyl derivatives of polyamines, N1-acetylspermine (N1-AcSPM) and N1-acetylspermidine (N1-AcSPD), are in vitro better substrates of tissue polyamine oxidase than the corresponding non-acetylated polyamines. Rat hepatoma tissue culture (HTC) cells, depleted of their putrescine (PUT) and spermidine (SPD) content by the use of DL-alpha-difluoromethylornithine (DFMeOrn), an irreversible inhibitor of L-ornithine decarboxylase, were used to study in situ the catabolism of these acetyl derivatives of polyamines. Normal intracellular spermidine content was restored by the addition of N1-acetylspermidine to polyamine-deficient cells. Addition of spermine (SPM) did not restore the spermidine content, although this polyamine elevated the spermine content of the cells. N1-Acetylspermidine reestablished normal spermidine levels of the cells and elevated the cellular putrescine content more efficiently and more rapidly than spermidine. Monoacetylputrescine and N1, N12-diacetylspermine (di-AcSPM) were ineffective in restoring putrescine and spermidine contents. These findings support the concept that N1-acetylspermine and N1-acetylspermidine are natural substrates of tissue polyamine oxidase and suggest poor membrane permeability of monoacetylputrescine (AcPUT) and N1, N12-diacetylspermine. Furthermore, they indicate that acetylation of polyamines by the cytosolic acetyl CoA: polyamine N1-acetyltransferase is the rate-limiting step of polyamine catabolism in rat hepatoma cells. Growth inhibition by DL-alpha-difluoromethylornithine was reversed by N1-acetylspermine and N1-acetylspermidine but not by monoacetylputrescine and N1, N12-diacetylspermine. These results suggest again that the antiproliferative effect of DL-alpha-dilfuoromethylornithine is related to inhibition of polyamine biosynthesis.

Indirect evidence for a strict negative control of S-adenosyl-L-methionine decarboxylase by spermidine in rat hepatoma cells.

1. Direct or indirect inhibitors of l-ornithine decarboxylase (EC 4.1.1.17), structurally related or unrelated to l-ornithine, including dl-alpha-difluoromethylornithine, alpha-methylornithine and 1,3-diaminopropane, used alone or in combination, decreased polyamine concentrations in rat hepatoma tissue culture (HTC) cells and increased S-adenosyl-l-methionine decarboxylase activity (EC 4.1.1.50). 2. Comparison of the catalytic properties of S-adenosyl-l-methionine from cells with elevated and normal activities revealed no apparent modification of the catalytic site as judged by affinity for the substrate, stimulation by di- and tri-amines and inhibition by methylglyoxal bis-(guanylhydrazone). 3. Actinomycin D and cycloheximide, and RNA and a proteinsynthesis inhibitor respectively, blocked the increase of S-adenosyl-l-methionine decarboxylase activity elicited by alpha-difluoromethylornithine. In polyamine-depleted cells the apparent half-life of elevated S-adenosyl-l-methionine decarboxylase activity, determined by inhibition of protein synthesis, was 2.5-fold longer than in control cells. The present results suggest that elevation of S-adenosyl-l-methionine decarboxylase activity by alpha-difluoromethylornithine is due to stabilization of the enzyme. 4. Restoration of the normal intracellular putrescine content, by addition of putrescine to the medium of polyamine-deficient cells, transiently increased S-adenosyl-l-methionine decarboxylase activity. Thereafter, intracellular conversion of putrescine into spermidine was accompanied by inactivation of the enzyme at a rate that was similar to that found on addition of spermidine itself. No relationship between total intracellular spermine content and S-adenosyl-l-methionine decarboxylase activity could be established. 5. Addition of 1mm-1,3-diaminopropane to polyamine-deficient cells did not cause a decrease in the activity of S-adenosyl-l-methionine decarboxylase, whereas addition of 1,5-diaminopentane (cadaverine) did. 1,3-Diamino-N-(3-aminopropyl)propane did not accumulate in cells treated with alpha-difluoromethylornithine and 1,3-diaminopropane, whereas addition of 1,5-diaminopentane led to the accumulation of 1,5-diamino-N-(3-aminopropyl)pentane. 1,3-Diamino-N-(3-aminopropyl)propane (10mum) was as effective as spermidine in decreasing S-adenosyl-l-methionine decarboxylase activity. Thus effectiveness of a diamine in decreasing enzyme activity is related to its capability of being converted into a closely structurally related homologue of spermidine by spermidine synthase. 6. The spermidine site of action appears to be post-translational since (a) the spermidine-induced decrease of S-adenosyl-l-methionine activity was not prevented by actinomycin D and (b) spermidine in the presence of cycloheximide led to a synergistic inactivation of the enzyme with a decay rate that progressively approached control values. Altogether these results are indirect evidence for a strict negative control of S-adenosyl-l-methionine decarboxylase by spermidine and substantiate previous findings [Mamont, Duchesne, Grove & Tardif (1978) Exp. Cell Res.115, 387-393]. Spermidine appears to act on some processes involved in denaturation and/or degradation of the enzyme protein. Putrescine appears to decrease the rate of these processes. The physiological significance of the regulatory control of S-adenosyl-l-methionine decarboxylase is discussed.