Nikolaus Seiler

Pharmacological properties of the natural polyamines and their depletion by biosynthesis inhibitors as a therapeutic approach

The structures of the natural aliphatic di-, tri- and tetramines, which are commonly designated “polyamines”, are shown in Figure 1. From a chemical point of view this designation is incorrect, because the natural polyamines are in fact small molecules.

Is ammonia a pathogenetic factor in Alzheimer's disease?

An attempt was made to review experimental evidence in favor of the idea that ammonia plays a role in dementia of the Alzheimer type (DAT). Hyperammonemia causes biochemical and cellular dysfunctions in the brain, which can be found in brains of DAT patients. The most conspicuous among these findings are astrocytosis, impairment of glucose utilization, and a decreased rate of energy metabolism, and the impairment of neurotransmission, with a net increase in excitability and glutamate release. The derangement of lysosomal processing of proteins is another potential site of ammonia action. This aspect is especially important in view of the growing evidence for the role of the endosomal-lysosomal system in the formation of amyloidogenic fragments from β-amyloid precursor protein. Ammonia is not considered a primary factor of the disease. However, since hyperammonemia and release of ammonia from the brains of DAT patients is well supported by published observations, ammonia should be taken into account as a factor that contributes to manifestations and the progression of DAT. If elevated ammonia concentrations turn out to be indeed as important in DAT, as is suggested in this review, rational therapeutic avenues can be envisaged that lead to the amelioration of symptoms and progression of the disease.

The Growth of MAT-LyLu Rat Prostatic Adenocarcinoma can be Prevented in Vivo by Polyamine Deprivation

The combination of inhibitors of ornithine decarboxylase and polyamine oxidase, and of antibiotics suitable for the (partial) decontamination of the gastrointestinal tract, with a polyamine deficient diet, is responsible for the almost complete inhibition of the growth of MAT-LyLu prostatic adenocarcinoma. In the tumor-bearing animals, erythrocyte spermidine levels were reduced, but spermine concentrations were increased. As has been previously observed, the increase in erythrocyte spermine level was associated with an enhancement of malignant cell death. Adriamycin administration did neither diminish tumor growth, nor potentiate the antitumor effect of polyamine deprivation treatment. Interruption of the polyamine deprivation treatment was accompanied by a significant enhancement of tumor growth. Since polyamine deprivation causes only reversible growth inhibition, it seems not appropriate as a monotherapy.

Effects of inhibition of ornithine aminotransferase on thioacetamide-induced hepatogenic encephalopathy

Repeated administration of thioacetamide (TAA) to CD1 mice produced hepatic failure and biochemical and behavioral effects characteristic of hepatogenic encephalopathy (HE). The symptoms in mice resembled those previously observed in rats after similar treatments. It is, howeve, obvious that both in rats and mice the severity of symptoms depends not only on dose and dosing schedule of TAA, but also on strain and body weight (age). Administration of 5-fluoromethylornithine (5FMOrn), a selective inactivator of ornithine aminotransferase (OAT), significantly reduced mortality, and it ameliorated most of the TAA-induced pathologic symptoms, such as hypothermia, decreased locomotor and exploratory behavior, pathologic liver function and amino acid patterns. The most prominent biochemical consequence of 5FMOrn administration is the elevation of ornithine concentrations in tissues, including the brain, and in body fluids. Elevated ornithine concentrations are, therefore, the most likely basis for the therapeutic effects of 5FMOrn. In agreement with this notion is the enhancement of citrulline and urea formation. These findings and the observation that administration of ornithine in combination with a branched-chain 2-oxoacid ameliorated the pathologic symptoms of portal-systemic encephalopathy suggest inhibition of OAT in the treatment of this disease. The liver protective effect of 5FMOrn is not yet understood; the enhancement of regenerative processes is a likely explanation.

Endogenous ornithine in search for CNS functions and therapeutic applications.

The vertebrate brain has the machinery to transport arginine and ornithine, and to form within nerve endings from these amino acids glutamate and GABA, the major excitatory and inhibitory neurotransmitters. Ornithine aminotransferase is a key enzyme of the Arg -> Om-> Glu -> GABA pathway; the physiological significance of this pathway is still unclear. With 5-fluoromethylornithine, a selective inactivator of ornithine aminotransferase, a tool is in our hands that allows us to study biochemical and behavioral consequences of elevated tissue ornithine concentrations. Increase of the rate of hepatic urea formation, and of ornithine decarboxylation are the most important changes in vertebrates following inactivation of ornithine aminotransferase. Administration of 5-fluoromethylornithine prevented the accumulation of lethal concentrations of ammonia in brain, and ameliorated pathological consequences of thioacetamide intoxication. Inhibition of ornithine catabolism has, therefore, potentials in the therapy of those hyperammonemic states which are characterized by a conditional deficiency of ornithine. The enhancement of polyamine formation due to elevated ornithine concentrations may allow us to favorably affect tissue regeneration following injury.

Protection against lethal ammonia intoxication: Synergism between endogenous ornithine and L-carnitine

The protective effects of combinations of 5-fluoromethylornithine (5FMOrn), a selective inhibitor of ornithine aminotransferase, and of compounds known to antagonize ammonia toxicity, were studied in acute, lethal ammonia intoxication in mice. Two test conditions were used : (a) Mice were pretreated with 5FMOrn at a dose (5 µmol.kg−1) which partially protects against 13 mmol.kg−1 ammonium acetate. (b) Mice were pretreated with a maximally protective dose of 5FMOrn (0.1 mmol.kg−1), however, 15 mmol.kg−1 ammonium acetate was used for intoxication. Under these conditions treatment with 5FMOrn alone protected only marginally. Under condition (a), administration of L-citrulline, L-carnitine, and L-acetylcarnitine improved the protective effect of 5FMOrn significantly, in an additive manner. N-acetyl-L-glutamate administration was ineffective. Under condition (b), ornithine, arginine and citrulline did not improve the protective effect of 5FMOrn, even when these amino acids were given at doses, which were effective in preventing ammonia toxicity induced with 13 mmol.kg−1 ammonium acetate. The inability to improve the effect of 5FMOrn by these compounds is most probably due to the fact that 5FMOrn and these amino acids enhance urea formation by the same mechanism, namely by increasing the concentration of substrates of the urea cycle. In contrast, L-carnitine and L-acetylcarnitine, which are assumed to stimulate urea production by different mechanisms, or compounds which antagonize ammonia toxicity by a urea cycle-independent mechanism, such as antagonists of the NMDA-type glutamate receptor (MK-801; MDL 100,453), potentiated the effects of 5FMOrn. The principle reason for the observed protective effects of the treatments described in this work seems to be the prevention of accumulation of lethal concentrations of ammonia in the brain. But other effects may also contribute.

Antileukemic effects of non-metabolizable derivatives of spermidine and spermine

To determine whether non-metabolizable derivatives of spermidine and spermine exert anticancer effects, L1210 leukemic mice were treated with 5,8-dimethylspermidine and 5,8-dimethylspermine. Both derivatives cured 5% of the leukemic mice. The increase in median survival time, however, was slight. In combination with alpha-difluoromethylornithine (DFMO), an ornithine decarboxylase inhibitor, only 5,8-dimethylspermine had a favorable effect. Treatment with DFMO is known to increase the uptake of extracellular polyamines and presumably their derivatives, by depleting the intracellular putrescine and spermidine content. However, treatment of L1210 leukemia cells in vitro with DFMO did not affect the uptake of the methyl-substituted polyamines added to the growth medium. 5,8-Dimethylspermidine and 5,8-dimethylspermine repressed the ornithine decarboxylase activity when added to cultures of L1210 leukemia cells. S-Adenosylmethionine decarboxylase activity was only repressed by 5,8-dimethylspermine. This finding may explain the potentiation by this derivative and not by 5,8-dimethylspermidine, of the antileukemic effect of DFMO.

Effects of ornithine aminotransferase inactivation by 5-fluoromethylornithine in rats following portacaval anastomosis

Abstract5-Fluoromethylornithine (5FMOrn) is a selective inactivator of ornithine aminotransferase. Administration of this compound to rodents causes a prominent increase of tissue ornithine concentrations, and prevents the neurological consequences of acute ammonia intoxication. However, long-term treatment with 5FMOrn of rats with portacaval shunts did not result in decreased circulating ammonia concentrations, nor did it prevent other pathologic manifestations of shunting. The sensitivity to ammonia intoxication of rats with portacaval shunts was also unaffected by pretreatment with 5FMOrn, although liver ornithine concentrations were significantly elevated; specific activities of urea cycle enzymes were slightly higher in portacaval shunted compared to sham-operated controls following 5-FMOrn treatment. Administration of 5FMOrn dramatically elevated urinary excretion of several amino acids in rats with portacaval shunts, but not in sham-operated animals, suggesting that the reabsorption of amino acids from the glomerular filtrate may be impaired in shunted rats. These results suggest that, in contrast to acute hyperammonemic syndromes, 5-FMOrn may be of limited therapeutic value in chronic hyperammonemia syndromes in which there is significant portal-systemic shunting.

Characterization of a COS cell line deficient in polyamine transport

In the present study, we describe the isolation and characterization of a COS cell line deficient in polyamine uptake that may provide an important tool for the molecular cloning of polyamine transporter(s). The cells were selected by isolation for resistance against the cytotoxic agent, methylglyoxal bis(guanylhydrazone) (MGBG), which is entering the cells using the same transport system as the polyamines. The isolated cell line was capable of growing in the presence of 100 microM MGBG, which totally inhibited the growth of the wild-type cells. The transport of putrescine and spermidine was markedly decreased in the COS-MGBGr cells. The decrease in putrescine transport was mainly a result of a 14-fold decrease in Vmax, whereas the reduced spermidine uptake was due to a 3-4-fold decrease in Vmax as well as 12-fold increase in Km, indicating the existence of at least two separate transport systems. No major difference in polyamine content was seen between the parental and the COS-MGBGr cells when grown without MGBG. In the presence of MGBG, both cell lines exhibited an increase in putrescine content. Treatment with MGBG also resulted in a decrease in spermidine and spermine contents in the wild-type cells. In the COS-MGBGr cells, on the other hand, there were no statistically significant effects on the spermidine and spermine contents by MGBG treatment. In the wild-type cells, depletion of polyamines, e.g., by treatment with the ornithine decarboxylase inhibitor 2-difluoromethylornithine (DFMO), stimulated the uptake of polyamines (3-7-fold), whereas in the COS-MGBGr cells the effect of DFMO treatment on polyamine transport was only minor. In contrast to the growth-medium of the wild-type cells, large amounts of polyamines accumulated in the medium of the COS-MGBGr cells, presumably indicating that COS cells normally excrete polyamines and then salvage them using the polyamine transport system.

Decreased hyperammonaemia and orotic aciduria due to inactivation of ornithine aminotransferase in mice with a hereditary abnormal ornithine carbamoyltransferase.

SummaryMice with the X-chromosomal sparse-fur (spf) mutation are an animal model of some hereditary deficiencies of ornithine carbamoyltransferase (OCT) in man. Orotic aciduria and hyperammonaemia are the most conspicuous metabolic changes in these diseases. Selective inactivation of ornithine aminotransferase (OAT) by 5-fluoromethylornithine raises endogenous ornithine concentrations so that citrulline formation is effectively catalysed by the aberrant OCT, in spite of its low affinity for ornithine. As a consequence, blood and tissue ammonia concentrations and orotic acid excretion are reduced near to normal values, and the abnormal amino acid patterns in blood, brain and liver are normalized. Selective inactivation of OAT seems a promising therapeutic approach in some hereditary OCT deficiencies, and a tool that may allow us to clarify the role of ammonia and orotic acid in the development of nanism and abnormal behaviour in spf mutant mice.