Jeffrey Grove

Anti-proliferative properties of DL-α-difluoromethyl ornithine in cultured cells. A consequence of the irreversible inhibition of ornithine decarboxylase

Abstract Both DL-α-Methyl orinithine (α-MeOrn), a competitive inhibitor of ornithine decarboxylase (ODC) (L-orinithine carboxy-lyase EC 4.1.1.17) and DL-α-difluoromethyl orinithine (α-DF MeOrn), a catalytic irreversible inhibitor of this enzyme, decrease the concentrations of putrescine and spermidine but not of spermine in rat hepatoma (HTC) cells and in mouse leukemia cells cultured in vitro . The depletion of the two amines is followed by a striking decrease in the rate of cell replication in both cell lines. Growth of human prostate adenoma cells is inhibited by α-DF MeOrn but not by α-MeOrn, illustrating the greater effectiveness of the irreversible inhibitor. These findings again support an essential function for putrescine and spermidine in cell division processes.

Biochemical and clinical effects of γ‐vinyl GABA in patients with epilepsy

Article abstract-In a pilot single-blind study, γ-vinyl GABA, an enzyme-activated irreversible inhibitor of GABA-transaminase (GABA-T), was administered orally to 10 epileptic patients who were refractory to conventional anticonvulsant therapy. Daily doses of 1 g and 2 g for 2 weeks each as add-on therapy were followed by 2 weeks of placebo treatment. CSF obtained from suboccipital and lumbar punctures demonstrated dose-related increases in concentrations of free and total GABA and homocarnosine with treatment, but no changes in 5-hydroxyindoleacetic acid or homovanillic acid levels, indicating effective and selective CNS GABA-T inhibition. These biochemical changes were associated with decreased seizure frequency in seven patients, decreased seizure severity in one, no change in one, and possible worsening in one. γ-Vinyl GABA may be useful in the therapy of epilepsy.

Increased gamma-aminobutyric acid (GABA), homocarnosine and β-alanine in cerebrospinal fluid of patients treated with γ-vinyl GABA (4-amino-hex-5-enoic acid)

Abstract γ-Vinyl GABA, an enzyme-activated irreversible inhibitor of GABA transaminase (GABA-T), was administered orally to 15 patients with various neurological conditions at daily doses of 0.5, 1, 2 or 6 g/day for 3 days. CSF samples were obtained by lumbar puncture before treatment and within 24 hours after the last dose and the CSF concentrations of free GABA, total GABA, homocarnosine, β-alanine and γ-vinyl GABA determined by ion-exchange chromatography with fluorometric detection. γ-Vinyl GABA treatment produced dose-dependent increases in free GABA, conjugated GABA (defined as total minus free GABA), homocarnosine and β-alanine. The concentrations of CSF γ-vinyl GABA also depended on the dose administered. These results indicate that γ-vinyl GABA enters the CNS after oral administration and alters GABA metabolism by inhibition of GABA-T and suggest that such treatment may achieve therapeutic benefit in conditions where such neurochemical alterations are desirable.

Effect of α-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase, on polyamine levels in rat tissues

Abstract α-Difluoromethylornithine (RMI 71.782), an enzyme-activated irreversible inhibitor of ornithine decarboxylase (E.C. 4.1.1.17) in vitro , causes a rapid, long-lasting, dose-dependent decrease of ornithine decarboxylase activity in prostate and, to a lesser extent, in thymus and testis of rats when injected intraperitoneally. Repeated doses (100 mg/kg or 1 g/kg) of α-difluoromethylornithine given to rats for two weeks markedly decreased polyamine concentrations in several rat tissues and selectively slowed down growth of ventral prostate and of thymus.

Kinetics of α‐difluoromethylornithine: An irreversible inhibitor of ornithine decarboxylase

We gave α‐difluoromethylornithine (DFMO), a selective, irreversible inhibitor of ornithine decarboxylase, to six healthy men in single intravenous doses of 5 and 10 mg/kg body weight and oral doses of 10 and 20 mg/kg. Plasma concentrations were monitored during the 24 hr after each dose. Urine was collected from 0 to 24 hr after drug and amount of unchanged drug excreted was determined. Peak plasma concentrations were reached within 6 hr after oral doses. The decay of the plasma concentrations followed first‐order kinetics with a mean half‐life (t½) for all four doses studied of 199 ± 6 min (±SD). Mean total body clearance (ClT) for the four doses was 1.20 ± 0.06 ml · min−1 · kg−1. Mean renal clearance was determined as 0.99 ± 0.03 ml · min−1 · kg−1, accounting for 83% of drug elimination. Mean apparent volume of distribution (aVD) was 0.337 ± 0.031 l/kg−1, corresponding to 24 l for 70 kg of body weight. The amount of unchanged drug in 24‐hr urine samples was 47 ± 7% and 40 ± 11% after 10 and 20 mglkg orally, and 78% and 81 ± 8% after 5 and 10 mglkg intravenously. Bioavailability of the 10 mg/kg dose was estimated as 58% from the urinary recoveries and as 54% from the areas under the plasma concentration curves (AUC0→∞). Since doubling of the dose resulted in a doubling of the mean AUC0→∞ and since other kinetic parameters, such as aVD, t½, ClT, and the urinary recovery of unchanged drug, were essentially the same at all doses, DFMO kinetics follow a dose‐linear model.

Inhibition of murine embryonic development by α-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase

The activities of ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (SAMDC) and the concentrations of putrescine, spermidine and spermine were measured in mouse uterus placenta and foetus during gestation. The prominent post-implantation biochemical changes in the intact uterus were associated mainly with the deciduomata and significant ODC activity was located in the embryo. Administration of the irreversible inhibitor of ODC, alpha-difluoromethylornithine, DFMO, 2% inthe drinking water during days 5-8 of gestation, abolished the inareases in uterine ODC activity, putrescine and spermidine concentrations and enhanced the activity of SAMDC. Treated animals showed no signs of pregnancy when autopsied on day 18. The alterations in deciduomal weight and the changes in uterine DNA, RNA and protein content indicated that decidualization following DFMO took place normaly but that embryonic growth was arrested. Treatment on single days with DFMO, 200 mg/kg every six h, revealed optimal contragestational effects on day 8 which corresponded exactly to the time of the peak in deciduomal ODC activity. Treatment with DFMO at times other than during the vulnerable period of days 5-8 has less prominent effects on gestation. An increase in ODC activity appears to be an essential factor during a short, but critical, period after implantation for continued murine embryonal growth.

Assay of α-difluoromethylornithine in body fluids and tissues by automatic amino-acid analysis

A procedure is described for the measurement of dl-α-difluoromethylornithine (DFMO), a selective irreversible inhibitor of ornithine decarboxylase, in biological specimens. The drug is separated from other amino acids with a commercial amino-acid analyser and detected by formation of its alkylthio-isoindole derivative with o-phthalaldehyde. DFMO concentrations of 0.1 nmol can be determined in a sample volume of 100 μl. The assay has been used to determine the half-life of DFMO in serum of several species and the relationships between serum and tissue concentrations.

Endogenous GABA determines the characteristics of [3H]GABA-binding

Since the identification of sodium-independent GABA-binding sites in membrane preparations from the mammalian central nervous system numerous publications have dealt with the effects of putative endogenous inhibitors of GABA-binding. However, controversy has arisen over the existence and identity of such inhibitors and the possible artifactural role of residual endogenous GABA in the interpretation of GABA-binding data. Using direct determination of GABA concentrations we have demonstrated that it is extremely difficult to remove endogenous GABA from rat brain membrane preparations and have shown that, even in well-washed preparations, there is a source of GABA production which compounds this problem. When care has been taken to ensure that GABA concentrations are minimal, analysis of binding data reveals the presence of two classes of GABA-binding sites with dissociation constants of the order of 9 nM and 318 nM and maximum binding capacities of 0.63 and 1.65 pmol/mg protein respectively (i.e., 63 and 165 pmol/g original wet tissue). A theoretical analysis of the effects of endogenous GABA demonstrates how failure to take into account even very low concentrations of GABA leads to misinterpretation of the results.

Artifactual Increases in the Concentration of Free GABA in Samples of Human Cerebrospinal Fluid Are Due to Degradation of Homocarnosine

Abstract: Samples of untreated human cerebrospinal fluid (CSF) were kept at room temperature (20±1°C) up to 72 h, and changes in γ‐aminobutyric acid (GABA) and homocarnosine contents were measured. The concentration of free GABA increased with time, and concomitantly a similar decrease occurred in the concentration of homocarnosine. Total GABA after hydrolysis (present in human CSF at concentrations 40–100 times that of free GABA) did not change. After 2 h the increase in CSF GABA for seven subjects ranged from 42 to 244 pmol/ml. The rate of increase in CSF GABA was positively correlated with the initial homocarnosine concentration. Approximately 5% per h of the initial homocarnosine content was degraded during the first 7 h at room temperature; thereafter the rate gradually decreased. No free GABA was formed in CSF frozen at −70°C for 10 days. When this CSF was restored to room temperature, the formation of free GABA from homocarnosine occurred at essentially the same rate as that observed in fresh CSF. These results demonstrate that the well‐known artifactual increase in GABA concentration of untreated human CSF depends on the concentration of homocarnosine. The rapidity of this increase (up to 2 pmollmlimin) could account for disparities among CSF free GABA concentrations previously reported from normal subjects. It is suggested that measurement of concentrations of total GABA in the CSF would provide a better index of human brain GABA concentration than determination of CSF free GABA.

Secretin-induced accumulation of N1-acetylspermidine and putrescine in the rat pancreas

Abstract The effects of secretin on polyamine metabolism in rat pancreas were investigated. Single injections of secretin increased ornithine decarboxylase activity only very slightly. However a substantial time- and dose-dependent increase of acetyl CoA: polyamine N 1 -acetyltransferase activity was observed. The concentrations of N 1 -acetylspermidine, putrescine and β-alanine increased concomitantly, but spermidine and spermine remained unchanged. These results suggest that, in this model, the accumulated putrescine was formed from spermidine, via its acetylation, rather than from ornithine.