Thomas P. Zimmerman

Metabolism of 5-amino-1-β-d-ribofuranosyl- imidazole-4-carboxamide and related five-membered heterocycles to 5′-triphosphates in human blood and L5178Y cells

Abstract The facile metabolism of 5-amino-1-β- d -ribofuranosylimidazole-4-carboxamide (AICA ribonucleoside) and several of its structural analogs [5-fluoro-1-β- d -ribofuranosylimidazole-4-carboxamide (FICA ribonucleoside), pyrazofurin and ribavirin] to their corresponding 5′-triphosphates in human blood cells has been demonstrated in vitro . Evidence is presented that both the β- and α-anomeric forms of pyrazofurin nucleotides were present in the extracts of pyrazofurin-treated blood. Determination of the extent of incorporation of radioactivity from [U- 14 C] d -glucose into analog ribonucleoside 5′-triphosphates formed in human blood cells indicated that AICA ribonucleoside and ribavirin were metabolized to their 5′-monophosphates mainly (> 90 per cent) via a nucleoside kinase; however, FICA ribonucleoside appeared to be metabolized to its 5′-monophosphate both via a nucleoside kinase ( ca . 67 per cent) and via phosphorolytic cleavage followed by a phosphoribosyltransferase-mediated reaction ( ca. 33 per cent). The aglycones of AICA ribonucleoside and FICA ribonucleoside were also metabolized extensively to their corresponding ribonucleoside 5′-triphosphates. 5′-Aminoimidazole-4-thiocarboxamide and 3-aminopyrazole-4-thiocarboxamide were metabolized only slightly to their ribonucleoside 5′-triphosphates. [ 3 H]ribavirin was metabolized extensively to its 5′-triphosphate in L5178Y cells but was not detectably incorporated into RNA. Ribavirin caused a substantial decrease in the pool size of GTP in L5178Y cells and a concomitant increase in the pool size of both CTP and UTP.

Inhibition of lymphocyte-mediated cytolysis by adenosine analogs: Biochemical studies concerning mechanism of action

Abstract A number of adenosine (Ado) analogs have been found to inhibit lymphocyte-mediated cytolysis (LMC) in vitro at μM concentrations. Those analogs which are substrates for adenosine deaminase were more inhibitory to LMC in the presence of erythro-9-(2-hydroxy-3-nonyl)adenine, an inhibitor of the deaminase. The inhibitory activity of most of these analogs (the exceptions being 2′-deoxyadenosine, 9-β- d -arabinofuranosyladenine and 7-deazaadenosine) was markedly enchanced by an inhibitor (Ro 20-1724) of cyclic AMP phosphodiesterase. With the exceptions of 2-fluoroadenosine, 7-deazaadenosine and formycin A, the inhibition of LMC caused by the Ado analogs was fully reversible upon removal of the analogs from the medium. In general, the Ado analogs did not cause a reduction in the pool sizes of endogenous ribonucleoside 5′-triphosphates in the lymphocytes. Some inhibitory and non-inhibitory analogs were metabolized to their corresponding 5′-triphosphates. Lymphocytes pretreated with a reversible inhibitor of LMC, 2-aminoadenosine, retained most of the resultant 2-amino-ATP during subsequent incubation in analog-free medium. Most of the Ado analogs which were inhibitory to LMC caused a substantial elevation of lymphocyte cyclic AMP; the magnitude of this elevation was enhanced by Ro 20-1724. Collectively, these results suggest that Ado and many of its structural analogs inhibit LMC by reason of their ability to stimulate the formation and consequent build-up of cyclic AMP in the cytotoxic lymphocytes. This stimulation of adenylate cyclase appears to result from the binding of an appropriate nucleoside to an adenosine receptor located on the membrane of the lymphocytes.

Separation of 6-thiopurine derivatives on deae-sephadex columns and in the high-pressure liquid chromatograph

Conditions are described for the separation of 6-thiopurine derivatives of pharmacological interest, both on large columns of DEAE-Sephadex A-25 by a modified procedure of Caldwell and in the Varian Aerograph LCS-1000 high-pressure liquid chromatograph. It was found that the triethylammonium acetate buffer, pH 4.7, used by Caldwell caused extensive degradation of 6-thiopurines containing an unsubstituted thiol group, and that this decomposition could be prevented by the addition of 10 mM β-mercaptoethanol. Elution profiles are presented for a number of synthetic 6 thiopurine derivatives investigated by these two chromatographic procedures.

Thrombolytic potency of acid‐stabilized plasmin: superiority over tissue‐type plasminogen activator in an in vitro model of catheter‐assisted thrombolysis

Summary.  Plasmin, the direct fibrinolytic enzyme, was compared with tissue plasminogen activator (t‐PA) in an in vitro thrombolysis model. Plasmin has been prepared in a highly pure form from human plasma and has been stabilized against auto‐degradation by low‐pH formulation. This acidified formulation of plasmin has been designed to have a low buffering capacity so that it can be directly infused into clots in a stable and latently active form. This low‐pH formulation has been shown to be equivalent to a neutral‐pH formulation of plasmin in its extent of clot lysis. An in vitro model of catheter‐assisted thrombolysis has been devised in which large (12 × 0.6 cm), retracted clots are treated with an intrathrombus thrombolytic agent via a multi‐sideport catheter. Plasmin dissolves these plasminogen‐deficient clots in a dose‐dependent manner and is clearly superior to t‐PA. In this model system, t‐PA exhibits efficacy only when retracted clots are replenished with plasminogen.

A more sensitive radioimmunoassay (RIA) for guanosine 3′, 5′-cyclic monophosphate (cGMP) involving prior 2′-O-succinylation of samples

Abstract The sensitivity of the radioimmunoassay (RIA) for cGMP has been increased approximately thirtyfold by prior 2′- O -succinylation of the assay samples according to the procedure of Cailla et al. ( Anal. Biochem. (1973) 56, 394). Assay samples (100 μl, in 50 m m sodium acetate, pH 6.2) were first reacted with succinic anhydride (50 μl, 100 mg/ml in tetrahydrofuran) in the presence of triethylamine (10 μl) for 10 min at 20–30°C. These reaction conditions were shown to result in quantitative derivatization of picomole amounts of [ 3 H]cGMP. The RIA was then carried out in the usual manner with these derivatized samples, employing heterologous anti-γ globulin to precipitate the [ 125 I] antigen-antibody complex. With this methodology, 1 fmol of cGMP could be detected using commercially available cGMP antiserum. This modified RIA exhibited excellent specificity for cGMP and routinely operated with a precision of ±10%. Tissue levels of cGMP determined with this modified RIA are generally in good agreement with those reported with other assay procedures.

Metabolic formation of nucleoside-modified analogues of S-adenosylmethionine.

Abstract Evidence is presented for the metabolism of six different adenosine analogues (formycin, 7-deazaadenosine, 8-azaadenosine, 2-fluoroadenosine, purine ribonucleoside and 3′-deoxyadenosine) to their corresponding S-nucleosidylmethionine derivatives in human erythrocytes and mouse lymphocytes. The identification of these novel metabolites was based upon chromatographic and enzymatic characterization of the unique radioactive substance found in cells incubated with each of these adenosine analogues plus L-[methyl-3H]methionine. The metabolic formation of analogues of S-adenosylmethionine may contribute to the inhibition of cellular methylation reactions caused by various purine analogues.

Immunosuppressive Effects of the S-Adenosylhomocysteine Hydrolase Inhibitor, 3-Deazaadenosine

Immunosuppressive effects of 3-deazaadenosine (3-DAA), an inhibitor of S-adenosylhomocysteine hydrolase, were tested in vivo in immune assays against sheep red blood cells (SRBC), involving serum titrations for hemagglutinins and hemolysins, cellular cytotoxicity tests and the direct plaque-forming cell assay. At daily doses up to 100 mg/kg, the compound was suppressive when injected before antigen and the effect appeared to be dose-dependent (ED50 = 52.6 +/- 4.9 mg/kg). When doses of 25 mg/kg of 3-DAA were given before antigen, co-injections of 250 mg/kg of L-homocysteine (L-HC) potentiated the suppressive effect, although L-HC alone was inactive. Daily administration of 100 mg/kg of 3-DAA or 250 mg/kg of L-HC alone was not suppressive when given after the antigen; however, in combination they were able to induce suppression. The possible biochemical mechanisms of the suppression, particularly those involving the inhibition of S-adenosylmethionine-dependent methylation reactions, are discussed.

5-Ethynyl-2(1H)-pyrimidinone: Aldehyde oxidase-activation to 5-ethynyluracil, a mechanism-based inactivator of dihydropyrimidine dehydrogenase

5-Ethynyluracil is a potent mechanism-based inactivator of dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) in vitro (Porter et al., J Biol Chem 267: 5236-5242, 1992) and in vivo (Spector et al., Biochem Pharmacol, 46: 2243-2248, 1993. 5-Ethynyl-2(1H)-pyrimidinone was rapidly oxidized to 5-ethynyluracil by aldehyde oxidase. The substrate efficiency (kcat/Km) was 60-fold greater than that for N-methylnicotinamide. In contrast, xanthine oxidase oxidized 5-ethynyl-2(1H)-pyrimidinone to 5-ethynyluracil with a substrate efficiency that was only 0.02% that of xanthine. Because 5-ethynyl-2(1H)-pyrimidinone did not itself inactivate purified DPD in vitro and aldehyde oxidase is predominately found in liver, we hypothesized that 5-ethynyl-2(1H)-pyrimidinone could be a liver-specific inactivator of DPD. We found that 5-ethynyl-2(1H)-pyrimidinone administered orally to rats at 2 micrograms/kg inactivated DPD in all tissues studied. Although 5-ethynyl-2(1H)-pyrimidinone produced slightly less inactivation than 5-ethynyluracil, the two compounds showed fairly similar patterns of inactivation of DPD in these tissues. At doses of 20 micrograms/kg, however, 5-ethynyl-2-pyrimidinone and 5-ethynyluracil produced equivalent inactivation of DPD. Thus, 5-ethynyl-2(1H)-pyrimidinone appeared to be an efficient, but not highly liver-selective prodrug of 5-ethynyluracil.

ATP depletion in human platelets caused by permeabilization with saponin does not prevent serotonin secretion induced by collagen

Saponin (5 to 25 micrograms/ml) produced a concentration-dependent decrease in the cellular content of total ATP and [32P]ATP in 32P-labeled human platelets. In platelets whose ATP had been profoundly decreased by saponin, Ca2+ produced phosphomonoesteratic cleavage of the polyphosphoinositides with a concomitant accumulation of phosphatidylinositol. Collagen still induced secretion of serotonin in platelets that had been treated with saponin in the presence or absence of Ca2+. This effect of collagen occurred in the absence of the formation of cyclooxygenase metabolites. In platelet permeabilized with saponin, agonist-induced secretion and aggregation seems to be unrelated to protein phosphorylation, breakdown of the inositol phospholipids by phospholipase C and formation of cyclooxygenase metabolites.

Potentiation by homocysteine of adenosine-stimulated elevation of cellular adenosine 3′,5′-monophosphate

Abstract The novel ability of l -homocysteine (Hcy) to potentiate the cellular elevation of adenosine 3′,5′-monophosphate (cAMP) caused by adenosine (Ado) is described. This effect of Hcy is highly selective in that it is not mimicked by l -cysteine, and Hcy does not potentiate the elevation of cellular cAMP caused by either 2-chloroadenosine or prostaglandin E1. Hcy also augments the Ado-stimulated increase in 2-fluoroadenosine 3′,5′-monophosphate in cells preloaded with nucleotides of 2-fluoroadenosine. Addition of Hcy to cells during their incubation with radioactive Ado results in a decrease in the cellular content of radioactive Ado and a concomitant buildup of S-adenosylhomocysteine. The enhancive effect of Hcy on the Ado-stimulated elevation of cAMP may be due to this associated reduction in the intracellular pool of Ado (due to condensation of Ado with Hcy via S-adenosylhomocysteinase) and to a resultant reduction in inhibition of adenylate cyclase by intracellular Ado, thereby allowing greater net stimulation of the cyclase by extracellular Ado.