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High concentrations of adenosine (Ado), when added to L1210 lymphocytic leukemia cells, resulted in apoptosis or programmed cell death. The apoptotic process was accompanied by distinct morphological changes including chromatin condensation and blebbing of plasma membranes. Extensive DNA fragmentation was correlated with Ado concentrations. Furthermore, apoptosis in these cells was preceded by an early but transient expression of c-myc proto-oncogene, and was not influenced by homocysteine thiolactone added to the cells. Since severe combined immunodeficiency (SCID) is associated with a deficiency of adenosine deaminase, leading to defects in both cellular and humoral immunity, Ado-induced apoptosis may thus be a contributing factor in the pathology of SCID. Copyright 1994 S. Karger AG, Basel

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Thymopentin prepared in 5, 15, and 20% 2-hydroxypropyl-beta-cyclodextrin (HPCD) was able to inhibit guinea-pig ileum contraction stimulated by anatoxin-a (3 x 10(-6) M) after fourteen months of storage at room temperature. Thus, in contrast to the instability of thymopentin prepared without HPCD, the pharmacological activity was retained and could be stored in a ready-to-use solution for extended periods without refrigeration.

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The preparation of 2-[N-(ethyl)-(N-beta-hydroxyethyl)]amino-ethyl 2,2-diphenylpropionate (1), a metabolite of aprophen [2-diethylaminoethyl 2,2-diphenylpropionate], is described. Hydrolysis of [2-(2-chloroethyl)ethylamino]ethyl acetate hydrochloride (2) in a basic solution, followed by acidic pH adjustment, gave the ethylcholineaziridinium ion (3) that upon treatment with 2,2-diphenylpropionic acid produced 1 in a 56% yield. Synthetic 1 was found to possess antimuscarinic activities, but was approximately 10-fold less potent than the parent compound aprophen.

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The metabolism of the anticholinergic drug aprophen was studied in rats after oral administration via stomach intubation. beta-Hydroxyethylaprophen, a major urinary metabolite of aprophen, was isolated and identified by normal-phase high-performance liquid chromatography and electron ionization mass spectrometry. More than 22% of the parent drug was recovered and quantified over a 72-h collection period. Results show that 2,2-diphenylpropionic acid, another major metabolite of aprophen which lacks anticholinergic properties, was also isolated and identified in this study. Experiments are currently underway to synthesize and test the anticholinergic properties of beta-hydroxyethylaprophen in mammals.

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(+)-Anatoxin-a (ANTX) stimulated guinea pig ileum contraction with a potency similar to that of acetylcholine (ACh); the stimulation was blocked by tubocurarine, hexamethonium, or atropine. Although the contraction stimulated by ANTX was blocked by atropine, no specific inhibition of the binding of [3H]N-methylscopolamine to ileum membranes was observed in the presence of ANTX. Furthermore, ANTX failed to stimulate the secretion of alpha-amylase from pancreatic acinar cells, a process that is activated by cholinergic agonists at the muscarinic receptors. When the ileum itself was stimulated by ACh, the contraction was not blocked by either hexamethonium or tubocurarine. Preincubation of the ileum with hemicholinium caused a 50% reduction in the ability of ANTX to stimulate contraction. Based upon these data, it was inferred that ANTX binds to postganglionic synaptic nicotinic receptors in the ileum, thus releasing endogenous ACh, which in turn causes ileum contraction by interacting with the postsynaptic muscarinic receptors. It was also observed that thymopentin (TP-5), a pentapeptide corresponding to positions 32-36 of thymopoietin, blocked the stimulation of ileum contraction by ANTX.

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The esters of cephalotaxine-harringtonine, homoharringtonine and deoxyharringtonine--have been reported by both Chinese and American oncologists as useful in the treatment of human nonlymphoblastic leukaemias and selected solid tumours of the head and neck. We report our results with homoharringtonine, currently a Phase II clinical trial drug with the National Cancer Institute, in the treatment of malaria. Homoharringtonine, 2.7-3.4 nM, was effective in causing 50% growth inhibition of two strains of chloroquine-resistant Plasmodium falciparum malaria in vitro. In vivo tests in mice infected with P. yoelii showed that this drug was effective in inhibiting parasite growth in this system as well. Histologically, the drug was associated with karyorrhexis. Drug-exposed cells showed decreased levels of putrescine and spermidine and increased spermine levels. Our findings not only demonstrate the potential usefulness of homoharringtonine in the treatment of chloroquine-resistant malaria, but also demonstrate the advantage of applying comparative biochemistry and an understanding of biological mechanisms in a rational approach to the development and treatment of diseases including malaria.

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Ethanol consumption retards the hepatic regenerative response to injury. This may contribute to the pathogenesis of liver injury in alcoholic individuals. The mechanisms responsible for ethanol-associated inhibition of liver regeneration are poorly understood. To determine if the antiregenerative effects of ethanol involve modulation of polyamine metabolism, parameters of polyamine synthesis were compared before and during surgically induced liver regeneration in ethanol-fed rats and isocalorically maintained controls. After partial hepatectomy, induction of the activity of ornithine decarboxylase (ODC), the rate limiting enzyme for polyamine synthesis, was delayed in rats that had been fed ethanol. This was correlated with reduced levels of putrescine, ODC's immediate product. Increases in hepatic spermidine and spermine were also inhibited. Differences in ODC activity between ethanol-fed and control rats could not be explained by differences in the expression of ODC mRNA or by differences in ODC apoenzyme concentrations, suggesting that chronic ethanol intake inactivates ODC posttranslationally. Supplemental putrescine, administered at partial hepatectomy and 4 and 8 h thereafter, increased hepatic putrescine concentrations and markedly improved DNA synthesis and liver regeneration in ethanol-fed rats. These data suggest that altered polyamine metabolism may contribute to the inhibition of liver regeneration that occurs after chronic exposure to ethanol.

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The metabolic fate of aprophen hydrochloride (2-diethylaminoethyl 2,2-diphenylpropionate) was studied in rats after intravenous administration. Both 14C-labeled and unlabeled aprophen were used in these studies. Blood samples were collected and analyzed to determine the identities of the metabolites formed. Utilizing high-performance liquid chromatography, desethylaprophen was identified as a major metabolite in ether-extracted samples from rats, and could be detected in blood samples 1 min after intravenous administration. It was most likely formed by N-de-ethylation of aprophen by a cytochrome P-450-dependent monooxygenase. Synthetic desethylaprophen was found to possess cholinolytic activity (i.e., it functioned as a muscarinic antagonist by blocking the contraction of acetylcholine-stimulated guinea pig ileum, the release of alpha-amylase from pancreatic acinar cells stimulated by carbachol, and also by inhibiting the binding of [3H]N-methyl scopolamine to the muscarinic receptors of guinea pig ileum). It was interesting that although the biological effects of desethylaprophen were 100-fold lower than those of aprophen, it was equally able to compete for the binding sites of muscarinic receptors of the guinea pig ileum.

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The pharmacokinetics of [14C]aprophen and its distribution were determined after intravenous administration to rats. The drug was distributed rapidly with a t1/2 (alpha) of 4 min to highly perfused organs like the brain, kidney and adrenals. An elimination phase was apparent 10 min after injection with a t1/2 (beta) of 23.5 min. The high plasma clearance of the drug was due both to a large volume of distribution and to a high metabolic rate. Aprophen could be hydrolysed to diphenylpropionic acid and diethylaminoethanol in-vivo and in-vitro. Diethylaminoethanol competed with [3H]QNB binding to muscarinic receptors of N4TG1 cells, whereas diphenylpropionic acid did not. The lower plasma concentrations and lower binding activity of diethylaminoethanol compared with aprophen indicate that unchanged aprophen is largely responsible for the in-vivo actions.

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Human retroplacental serum (RPS) containing polyamine oxidase inhibited the growth of the Camp strain of Plasmodium falciparum in vitro as assayed by the parasite's decreased incorporation of 3H-hypoxanthine. Inhibition was dose-dependent on the concentrations of serum polyamine oxidase and added polyamines. Almost complete inhibition was seen in 96-hr asynchronous cultures containing 10% RPS and in those containing 1.2% RPS plus 50 microM polyamine. Subtle morphologic changes in mature stages and decreased numbers of new rings were associated with inhibition seen in 19-hr synchronous cultures initiated at the trophozoite stage. These incubation times were longer than in previous reports showing inhibition of malaria parasites by bovine polyamine oxidase but not by human polyamine oxidase. Macrophages contain polyamine oxidase, the reaction products of which are known to be similar to those of RPS polyamine oxidase but different from those of bovine polyamine oxidase. It remains to be determined whether human polyamine oxidase, acting upon ubiquitous polyamines, contributes to host defenses against malaria.

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Neplanocin A, a novel carbocyclic analog of adenosine, was found to be a mixed type inhibitor of S-adenosylhomocysteine hydrolase of human red blood cells with a Ki of 2 nM and an inactivating constant, Ki, of 3 nM. When tested against Plasmodium falciparum cultured in human red blood cells, neoplanocin A inhibited malarial growth with an ED50 of 3 microM. Above 2.5 microM, some red blood cells showed morphological aberrations and became echinocytes (spiculated red blood cells). In infected red blood cells, neplanocin A caused an elevation of the concentrations of S-adenosylmethionine and S-adenosylhomocysteine in a dose-dependent manner. Concurrently, a new analog of S-adenosylmethionine, S-neplanocinylmethionine, was formed. Analysis of polyamines showed that only putrescine was decreased significantly; the others were not changed. Purine analyses showed two putative neplanocinyl nucleotides, possibly the di-and the triphosphates. Neplanocin A most likely exerted its in vitro antimalarial effect via the inhibition of S-adenosylhomocysteine hydrolase and the attendant perturbation of methylation reactions.

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The addition of DL-alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase, to human Plasmodium falciparum-infected red cells in continuous culture decreased both parasite growth and intracellular polyamine concentrations. Growth of P. falciparum in infected red cells was assessed by parasite counts and by assays for macromolecular syntheses of protein, DNA and RNA. Polyamine concentrations were measured by an automated high-performance liquid chromatography method. Concentrations of DL-alpha-difluoromethylornithine greater than 0.3 mM decreased intracellular levels of putrescine and spermidine, reduced ornithine decarboxylase activity and inhibited growth and maturation of the intracellular parasite at the trophozoite stage. There was a concomitant decrease in the synthesis of protein and nucleic acids in the infected cells. The use of this parasitized red cell model system together with polyamine inhibitors provide means of studying polyamine synthesis and cell growth in the design of new antimalarial therapy.

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