pentostatin and 9-(2-hydroxy-3-nonyl)adenine

Topics: Adenine; Adenosine Deaminase Inhibitors; Animals; Enzyme Inhibitors; Humans; In Vitro Techniques; Myocardial Ischemia; Pentostatin; Rabbits

Topics: Adenine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Animals; Cells, Cultured; Coformycin; Humans; Immunologic Deficiency Syndromes; Immunosuppressive Agents; Pentostatin; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Purine-Pyrimidine Metabolism, Inborn Errors

Topics: Adenine; Adenosine Deaminase Inhibitors; Adult; Animals; Carcinoma, Ehrlich Tumor; Clinical Trials as Topic; Coformycin; DNA; Drug Evaluation; Drug Synergism; Humans; Leukemia P388; Leukemia, Experimental; Leukemia, Lymphoid; Male; Mice; Nucleoside Deaminases; Pentostatin; Thymidine; Vidarabine

Adenosine deaminase is an enzyme involved in purine metabolism and its inhibitors are used as anticancer and antiviral drugs. In this study, we show that fast-scan cyclic voltammetry at carbon-fiber microelectrodes can be used to study the kinetics of adenosine deaminase by electrochemically monitoring decreases in adenosine concentration. Buffer and salt concentrations were shown to affect the enzyme kinetics and the inhibition by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and deoxycoformycin (DCF). In a Tris buffer containing salts that mimic cerebrospinal fluid, EHNA and DCF showed non-competitive inhibition with a K(i) of 1.7 +/- 0.6 nM and 1.2 +/- 0.2 nM, respectively. However, removing the divalent cations from the Tris buffer caused the inhibition to be competitive and reduced the K(i) for DCF by two orders of magnitude. In phosphate-buffered saline, the K(i) was 1.0 +/- 0.2 nM for EHNA and 3.6 +/- 0.3 pM for DCF, similar to literature values. Adenosine deaminase was also competitively inhibited by AgNO(3), showing it is susceptible to silver toxicity. Caffeine was found to increase adenosine deaminase activity. This is a fast, easy method for screening drug effects on enzyme kinetics and could be applied to other enzymatic reactions where there is a significant difference in the electroactivity of the reactant and product.

Topics: Adenine; Adenosine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Animals; Caffeine; Cattle; Electric Conductivity; Electrochemistry; Enzyme Assays; Kinetics; Oxidation-Reduction; Pentostatin; Purinergic P1 Receptor Antagonists; Receptors, Purinergic P1; Silver; Time Factors

Adenosine deaminase (ADA) is involved in purine metabolism and plays an important role in the mechanism of the immune system. ADA activity is composed of two kinetically distinct isozymes, which are referred to as ADA1 and ADA2. ADA1 is widely distributed in many animals and well characterized. On the contrary, relatively little is known about ADA2. In this study, we first purified ADA2 to homogeneity from chicken liver. The purified enzyme had a molecular mass of approximately 110 kDa on gel filtration. Also, the enzyme was shown to be a homodimer with an estimated molecular mass of 61 kDa on SDS-PAGE. Following treatment with N-glycosidase, the molecular mass of ADA2 changed to 55 kDa. Several properties of the highly purified ADA2 were also investigated in this study. Furthermore, the N-terminal amino acid sequence of ADA2 was determined.

Topics: Adenine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Amino Acid Sequence; Animals; Chickens; Enzyme Inhibitors; Enzyme Stability; Isoenzymes; Liver; Molecular Sequence Data; Pentostatin; Protein Structure, Quaternary; RNA-Binding Proteins; Sequence Analysis, Protein; Substrate Specificity

We searched for non-nucleoside inhibitors of adenosine deaminase by rational structure-based de novo design and succeeded in the discovery of 1-(1-hydroxy-4-phenyl-2-butyl)imidazole-4-carboxamide (FR221647: K(i)=5.9 microM to human ADA) as a novel inhibitor with moderate activity and good pharmacokinetics compared with the known inhibitors pentostatin and EHNA.

Topics: Adenine; Adenosine Deaminase Inhibitors; Administration, Oral; Animals; Area Under Curve; Drug Design; Enzyme Inhibitors; Humans; Hydrogen Bonding; Injections, Intraperitoneal; Mice; Models, Molecular; Pentostatin; Structure-Activity Relationship

Because micromolar concentrations of adenosine (Ado) have been documented recently in the interstitial fluid of carcinomas growing in animals, we examined the effects of low concentrations of Ado on the growth of cultured human carcinoma cells. Ado alone had little effect upon cell growth. In the presence of one of a number of Ado deaminase (ADA) inhibitors, Ado led to significant growth inhibition of all cell lines tested. Similar effects were found when ATP, ADP, or AMP was substituted for Ado. Surprisingly, the ADA inhibitor coformycin (CF) had a much greater potentiating effect than did 2'-deoxycoformycin (DCF), although DCF is a more potent ADA inhibitor. The growth inhibition of the Ado/CF combination was not abrogated by pyrimidines or caffeine, a nonspecific Ado receptor blocker. Toxicity was prevented by the addition of the Ado transport inhibitor dipyridamole or the Ado kinase inhibitor 5'-amino 5'-deoxyadenosine. S-Adenosylhomocysteine hydrolase is not involved because neither homocysteine thiolactone nor an S-adenosylhomocysteine hydrolase inhibitor (adenosine dialdehyde) potentiated toxicity of the Ado/CF combination. Unexpectedly, substitution of 2'-deoxyadenosine (the toxic moiety in congenital ADA deficiency) for Ado, did not lead to equivalent toxicity. The Ado/CF combination inhibited DNA synthesis and brought about morphological changes consistent with apoptosis. Together, these findings indicate that the Ado-mediated killing proceeds via an intracellular route that requires the action of Ado kinase. The enhanced cofactor activity of CF may be attributable to its being a more potent inhibitor of AMP deaminase than is DCF.

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Apoptosis; Breast Neoplasms; Carcinoma, Squamous Cell; Cell Division; Cell Survival; Coformycin; Deoxyadenosines; Dipyridamole; Enzyme Inhibitors; Female; Humans; Kinetics; Ovarian Neoplasms; Pentostatin; Tumor Cells, Cultured

The adenine analog erythro-9-(2-hydroxy-3-nonyl)adenine, EHNA, a tight reversible inhibitor (KI = 1.6 x 10(-9) M) of adenosine deaminase (EC 3.5.4.4) (ADase), was modified into the fluorescent etheno derivative epsilon-EHNA. The latter is a competitive inhibitor of adenosine deaminase [KI = (2.80 +/- 0.01)10(-6) M], having the fluorescent properties of epsilon-adenines. Affinity to the active site, monitored by both steady-state and dynamic fluorescence polarization, was confirmed by competition experiments with 2'-deoxycoformycin, the substrate adenosine and EHNA. The epsilon-adenine moiety of epsilon-EHNA librates at the shallow active site of ADase. The low absorptivity of epsilon-EHNA required the measurement of fluorescence excitation spectra. Computer subtraction of fluorescence excitation spectrum of ADase from that of its equimolar complex with epsilon-EHNA revealed the corrected excitation spectrum of epsilon-EHNA at the active site of the enzyme. This spectrum mimics that of epsilon-EHNA at pH 5.5 in buffer solution, implying its protonation at the active site of the enzyme. These results are in agreement with the presence of acidic amino acids that are essential to the catalytic mechanism.

Topics: Adenine; Adenosine; Adenosine Deaminase; Animals; Binding Sites; Binding, Competitive; Catalysis; Cattle; Enzyme Inhibitors; Fluorescence Polarization; Fluorescent Dyes; Hydrogen-Ion Concentration; Intestines; Kinetics; Molecular Structure; Pentostatin; Protein Binding; Spectrometry, Fluorescence; Spectrophotometry

This study investigates the mechanisms of absorption and the role of intestinally localized purine salvage pathway enzymes on the ileal availabilities of 2',3'-dideoxyinosine (ddI), a substrate for purine nucleoside phosphorylase (PNP); 2'-fluoro-2',3'-dideoxyinosine (F-ddI), a non-PNP substrate; and 6-chloro-2',3'-dideoxypurine (6-Cl-ddP), an adenosine deaminase (ADA) activated prodrug of ddI. The potential for increasing the intestinal availability of 6-Cl-ddP through the use of ADA inhibitors, namely, 2'-deoxycoformycin (DCF) and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), is also explored. Drug permeability coefficients across the intestinal epithelium were determined in in situ perfusions in the mesenteric vein cannulated rat ileum based on both drug appearance in blood (Pblood) and disappearance from the lumen (Plumen) and their paracellular and transcellular components were estimated by comparison to the permeabilities of two paracellular markers, mannitol and urea. Values of Pblood for ddI were determined to be (1.1 +/- 0.3) x 10(-6) cm/s, in close agreement with the value of (1.0 +/- 0.3) x 10(-6) cm/s obtained for F-ddI, a PNP resistant analogue of ddI having virtually the same molecular size and lipophilicity as ddI. This indicates that PNP may not play an important role in the low intestinal absorption of ddI. The Pblood for 6-Cl-ddP, (19 +/- 2) x 10(-6) cm/s, was 4.5-fold lower than Plumen, (84 +/- 12) x 10(-6) cm/s, which means that 77 +/- 6% of 6-Cl-ddP was metabolized during its intestinal transport, thus qualitatively accounting for the low oral bioavailability (7%) of 6-Cl-ddP observed in vivo in rats. Extensive intracellular metabolism of 6-Cl-ddP by ADA was confirmed by the high concentrations of ddI found both in the intestinal lumen and blood during 6-Cl-ddP perfusions and by a rate of ddI appearance in blood which was approximately 10-fold higher than ddI controls. Co-perfusion of the potent, hydrophilic ADA inhibitor DCF (Ki = 0. 001-0.05 nM) with 6-Cl-ddP led to only partial inhibition of intestinal ADA, while complete inhibition was obtained using the less potent but more lipophilic inhibitor EHNA (Ki = 1-20 nM). Hence, EHNA may be used to improve intestinal absorption of 6-Cl-ddP in vivo.

Topics: Adenine; Adenosine Deaminase Inhibitors; Animals; Anti-HIV Agents; Biological Availability; Biological Transport; Catheterization; Didanosine; Enzyme Inhibitors; Ileum; Intestinal Absorption; Male; Mesenteric Veins; Pentostatin; Permeability; Prodrugs; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Rats; Rats, Sprague-Dawley

Diarrhea induced by Escherichia coli heat-stable enterotoxin (STa) is mediated by a receptor guanylyl cyclase cascade. The present study establishes that an intracellular nucleotide-dependent pathway disrupts toxin-induced cyclic GMP (cGMP) production and the associated chloride (Cl-) flux that underlie intestinal secretion. Incubation of Caco 2 human intestinal epithelial cells with the nucleoside analog 2-chloroadenosine (2ClAdo) resulted in a concentration- and time-dependent inhibition of toxin-induced cGMP production. Inhibition of cGMP production correlated with the metabolic conversion of 2ClAdo to 2-chloroadenosine triphosphate. The effect of 2ClAdo did not reflect activation of adenosine receptors, inhibition of adenosine deaminase, or modification of the binding or distribution of STa receptors. Guanylyl cyclase activity in membranes prepared from 2ClAdo-treated cells was inhibited, in contrast to membranes from cells not exposed to 2ClAdo, demonstrating that inhibition of guanylyl cyclase C (GCC) was mediated by a noncompetitive mechanism. Treatment of Caco 2 cells with 2ClAdo also prevented STa-induced Cl- current. Application of 8-bromo-cGMP, the cell-permeant analog of cGMP, to 2ClAdo-treated cells reconstituted the Cl- current, demonstrating that inhibition of Cl- flux reflected selective disruption of ligand stimulation of GCC rather than the chloride channel itself. Thus, the components required for adenine nucleotide inhibition of GCC signaling are present in intact mammalian cells, establishing the utility of this pathway to elucidate the mechanisms regulating ST-dependent guanylyl cyclase signaling and intestinal fluid homeostasis. In addition, these data suggest that the adenine nucleotide inhibitory pathway may be a novel target to develop antisecretory therapy for enterotoxigenic diarrhea.

Topics: 2-Chloroadenosine; Adenine; Adenosine; Adenosine-5'-(N-ethylcarboxamide); Bacterial Toxins; Caco-2 Cells; Chloride Channels; Enterotoxins; Enzyme Inhibitors; Escherichia coli Proteins; Guanylate Cyclase; Humans; Models, Biological; Pentostatin; Purinergic P1 Receptor Agonists; Signal Transduction

Postmitotic sympathetic neurons are known to undergo a programmed cell death (apoptosis) when they are deprived of nerve growth factor (NGF) or treated with arabinofuranosyl nucleoside antimetabolites. Here we report the existence of a biochemical mechanism for the induction of neuronal death by an endogenous nucleoside in the presence of NGF. In support of such a mechanism we show that 2-deoxyadenosine (dAdo) induces apoptosis in chick embryonic sympathetic neurons supported in culture by NGF, excess K+, phorbol 12,13-dibutyrate, or forskolin. Neuronal death was related to a dramatic increase in the dATP content of sympathetic neurons exposed to dAdo (34.96 +/- 5.98 versus 0.75 +/- 0.16 pmol/micrograms protein in untreated controls, n = 9), implicating dATP in the toxicity. Supportive evidence for a central role of dATP was gained by inhibition of kinases necessary for phosphorylation of dAdo. 5'-Iodotubercidin in nanomolar concentrations completely and dose-dependently inhibited formation of dATP and also protected against toxicity of submillimolar concentrations of dAdo in sympathetic neurons. Although some of these actions of dAdo were remarkably similar to those reported for human lymphoid cells, several were uniquely different. For example, [3H]dAdo was not transported into neurons by the nucleoside transporter, and therefore inhibition of the transporter (dilazep, nitrobenzylthioinosine) did not prevent neurotoxicity by dAdo. Precursors of pyrimidine synthesis (2'-deoxycytidine, uridine) or NAD+ synthesis (nicotinamide) were ineffective in protecting sympathetic neurons against dAdo toxicity. Finally, inhibition of adenosine deaminase by deoxycoformycin or erythro-9-(2-hydroxy-3-nonyl) adenine did not potentiate the toxic effects of dAdo. Our results provide evidence for the first time that neuronal cells are as susceptible to nucleoside lethality as human lymphocytes are, and provide a new model to study the salvage pathway of deoxyribonucleosides in controlling neuronal populations through programmed cell death.

Topics: Adenine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Animals; Apoptosis; Cell Division; Cell Survival; Cells, Cultured; Chick Embryo; Deoxyadenosines; DNA; Nerve Growth Factors; Neurons; Pentostatin; Sympathetic Nervous System

Field and intracellular potentials were recorded from CA1 pyramidal stratum in submerged slices (at 33 degrees). During "normal" oxygenation (95% O2 + 5% CO2), tonic depression of population spikes and field excitatory postsynaptic potentials by endogenous adenosine was demonstrated by (i) the marked enhancement by the adenosine antagonists 8-(p-sulfophenyl)theophylline (10 microM) and caffeine (0.2 mM), (ii) depression by the transport blocker dipyridamole (5 microM), and (iii) enhancement by exogenous adenosine deaminase (all tested by bath application). Thus, adenosine deaminase (0.5 units/ml) reduced by 10.7 +/- 3.0% (S.E.) the half-maximal stimulus intensity (for population spikes). The effects of adenosine deaminase were prevented by the specific inhibitor, deoxycoformycin (30 microM). In intracellular recordings, excitatory postsynaptic potentials were enhanced in a comparable manner by adenosine deaminase. By contrast, neither deoxycoformycin (5 and 30 microM) nor erythro-9-(2-hydroxy-3-nonyl)adenine (another adenosine deaminase inhibitor; 10 and 50 microM) had significant effects on population spikes. Superfusion with anoxic medium (saturated with 95% N2 + 5% CO2) for 2-3 min suppressed population spikes reversibly, by a mechanism involving adenosine, because 8-(p-sulfophenyl)theophylline (10 microM) and caffeine (0.2 mM) delayed the onset of anoxic block and accelerated the subsequent recovery, and the recovery was much slower or incomplete in the presence of dipyramidole (0.5 microM). However, the anoxic suppression of population spikes was not affected by deoxycoformycin (30 microM) or erythro-9-(2-hydroxy-3-nonyl)adenine (10 microM); the corresponding 50% postanoxic recovery times were also unchanged (e.g. 4.0 +/- 0.2 min for controls and 4.1 +/- 0.3 min in deoxycoformycin).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Animals; Electrophysiology; Hippocampus; Hypoxia; Pentostatin; Rats; Rats, Sprague-Dawley; Reference Values; Synaptic Transmission; Theophylline

The synthesis of various chiral derivatives of (+)-erythro-9-(2-hydroxy-3-nonyl)adenine, (+)-EHNA, from (2S,3R)-3-amino-1,2-O-isopropylidene-1,2-nonanediol by condensation with 5-amino-4,6-dichloropyrimidine is described. The compounds synthesized were C1'- and nor-C1'-(+)-EHNA derivatives. When tested with calf spleen ADA, C1'-OH- and nor-C1'-(+)-EHNA had comparable inhibitory activity that was 1 order of magnitude lower than that of (+)-EHNA. Potency was reduced further in nor-C1' derivatives.

Topics: Adenine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Animals; Binding Sites; Cattle; In Vitro Techniques; Stereoisomerism; Structure-Activity Relationship

Near total inhibition of brain adenosine deaminase (ADA) activity in rats injected with the potent ADA inhibitor 2'-deoxycoformycin (DCF) was previously shown to reduce enzyme activity for up to 50 days during which time the enzyme exhibited reduced sensitivity to in vivo inhibition by DCF. Here, we investigated the biochemical properties of ADA and the basis for its reduced activity after DCF treatment. It was found that much higher doses of DCF were required to inhibit ADA in DCF-treated compared with drug-naive animals. Fourteen days after DCF administration, reduced ADA activity in brain homogenates was due to a decrease in Vmax, rather than to an altered Km of ADA for adenosine. DCF treatment had no effect on Ki values for erythro-9-(2-hydroxy-3-nonyl)adenine inhibition of ADA. The IC50 value for DCF inhibition of ADA in hypothalamus was unchanged. However, the Ki for DCF inhibition of ADA in whole brain increased by fivefold. Sucrose gradient analysis of brain ADA revealed only one corresponding peak of activity and [3H]DCF-labeled ADA in DCF-treated and control rats. A radioligand filtration assay with [3H]DCF was developed to assess the effects of DCF on ADA protein levels. Over a roughly 200-fold range of ADA activities the binding of [3H]DCF was highly correlated with deaminase activity (r = 0.99). In brain tissues taken 8 and 33 days after treatment of rats with DCF, [3H]DCF binding was reduced to 27% and 48% of control levels, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adenine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Animals; Brain; Cattle; Centrifugation, Density Gradient; Intestinal Mucosa; Ligands; Male; Pentostatin; Rats; Rats, Inbred Strains

The adenosine deaminase inhibitors deoxycoformycin and erythro-9-(2-hydroxy-3 nonyl) adenine (EHNA) induce single-strand DNA breaks in cultured human lymphocytes. Deoxycoformycin produced a significant number of strand breaks (4-fold increase compared to controls) and EHNA induced strand breaks in a dose-dependent manner. Strand breaks stimulate repair by poly(ADP-ribosylation) which requires NAD+ as a cofactor. Niacin is a precursor of NAD+ and when preincubated with human lymphocytes prior to exposure to adenosine deaminase inhibitors, strand breakage was reduced significantly. The administration of niacin may represent an approach to decreasing the toxicity associated with these agents.

Topics: Adenine; Adenosine Deaminase Inhibitors; Cell Line; Cells, Cultured; DNA; DNA Damage; Flow Cytometry; Humans; Kinetics; Lymphocytes; Niacin; Nucleoside Deaminases; Pentostatin

The simultaneous administration of 3'-deoxyadenosine N1-oxide (3'-dANO) and the adenosine deaminase inhibitors erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) or 2'-deoxycoformycin (2'-dCF) to mice bearing Ehrlich ascites tumor cells resistant to 3'-dANO resulted in 80%-90% inhibition of tumor growth in vivo. 3'-dANO and 2'-dCF increased the survival time of tumor-bearing mice by a factor of 2. In vitro studies showed that the 3'-dANO resistant Ehrlich cells initiate the metabolism of 3'-dANO by a reduction to 3'-deoxyadenosine, which is converted primarily to 3'-deoxyinosine by adenosine deaminase and, to a small extent, phosphorylated to the cell toxic agent 3'-dATP. By the addition of EHNA or 2'-dCF it was possible to block the formation of 3'-deoxyinosine, resulting in a profound stimulation in the accumulation of 3'-dATP. The development of resistance to 3'-dANO was studied in cell cultures and found to be accompanied by changes in the enzyme activities of the reductase, the adenosine kinase, and the adenosine deaminase.

Topics: Adenine; Adenosine Deaminase Inhibitors; Animals; Antineoplastic Agents; Carcinoma, Ehrlich Tumor; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Drug Resistance; Drug Synergism; Female; Mice; Nucleoside Deaminases; Pentostatin; Ribonucleosides

DL-ethionine increases the activity of liver biotinidase, an enzyme which hydrolyzes biotinylesters and biotinylpeptides. Chronic DL-ethionine feeding increases transiently the activity of biotinidase in mouse and rat liver, after which it remains elevated in the serum. In the present work we show that both isomers of DL-ethionine are equally good enhancers of the liver biotinidase, while, 3-ethylthiopropionate, the toxic metabolite of DL-ethionine, has no effect on the biotinidase activity of either liver or serum. We have also employed two different combinations of inhibitors of the hydrolytic pathway of SAH, a transmethylation product and potent inhibitor of methylation. It was found that these inhibitors (EHNA and Ara-A, 2-deoxycoformycin and adenosine) increase the activity of serum biotinidase as was the case with ethionine. Because SAH does not ethylate biomolecules, these changes in biotinidase activity, which can not be prevented by adenine, biotin or lecithin are most probably related to the inhibition of methylation.

Topics: Adenine; Adenosine; Amidohydrolases; Animals; Biotinidase; Coformycin; Ethionine; Female; Liver; Methylation; Mice; Pentostatin; Vidarabine

Adenosine deaminase is a purine salvage enzyme that catalyzes the deamination of adenosine and deoxyadenosine. Deficiency of the enzyme activity is associated with T-cell and B-cell dysfunction. Mutant adenosine deaminase has been isolated from heterozygous and homozygous deficient lymphoblast cell lines with the aid of an affinity matrix consisting of coformycin (a potent inhibitor of the enzyme) as the affinity ligand, bound to 3,3'-iminobispropylamine-derivatized Sepharose. Routinely, 80-90% of adenosine deaminase in crude cell homogenates could be bound to the material. Adenosine deaminase was specifically eluted by enzyme inhibitors or less efficiently by high substrate concentrations. Protein preparations isolated from several different deficient cell lines were highly purified and exhibited molecular weights identical to wild-type adenosine deaminase. This method produces a protein that is suitable for structural studies.

Topics: Adenine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Cell Line; Chromatography, Affinity; Coformycin; Humans; Lymphocytes; Mutation; Nucleoside Deaminases; Pentostatin

Topics: 5'-Nucleotidase; Adenine; Adenosine Monophosphate; AMP Deaminase; Animals; Bone Marrow; Cells, Cultured; Coformycin; Cytoplasm; Deoxyadenine Nucleotides; Deoxyadenosines; Humans; Lymph Nodes; Lymphocytes; Male; Mice; Nucleotidases; Nucleotide Deaminases; Pentostatin; Phosphotransferases; Spleen; Thymus Gland

Cerebral blood flow in the rat was monitored by a venous outflow technique with an extracorporeal circulation, which allows for the continuous recording of flow over periods of several hours. The adenosine deaminase inhibitors erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) (1.0-100 micrograms/kg) and deoxycoformycin (0.1-1 micrograms/kg) potentiated the reactive hyperemia elicited by a brief (24-s) anoxic challenge. Basal flow rate was unaltered by EHNA administration and slightly enhanced by deoxycoformycin. The results are consistent with the hypothesis that adenosine plays a significant role in cerebral vascular regulation and suggest that low doses of these deaminase inhibitors may be useful in the treatment of cerebral vascular insufficiency.

Topics: Adenine; Adenosine Deaminase Inhibitors; Animals; Brain; Cerebrovascular Circulation; Coformycin; Hyperemia; Hypoxia, Brain; Male; Pentostatin; Rats; Rats, Inbred Strains; Ribonucleosides

Topics: Adenine; Adenosine Deaminase Inhibitors; Animals; Cerebrovascular Circulation; Coformycin; Dogs; Hypoxia; Nucleoside Deaminases; Pentostatin; Rats; Ribonucleosides; Vasodilation

Four compounds that inhibit adenosine deaminase, erythro-9-(2-hydroxy-3-nonyl)adenine, 2'-deoxycoformycin, coformycin, and 9-(1-hydroxy-2-octyl)adenine have been studied in an in vitro lymphocyte-mediated cytolysis assay. At low concentration (congruent to 10 microM) these agents enhance the activities of a number of inhibitory purine nucleosides, including adenosine and 2'-deoxyadenosine. The LMC-inhibitory activity of Ado but not dAdo is further enhanced by 5-iodotubercidin, uridine, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone, or L-homocysteine and is antagonized by theophylline. The inhibition of LMC by Ado and dAdo is increased by nitrobenzyl-thioinosine. Lymphocyte-mediated cytolysis was inhibited by EHNA or HOA alone (IC50 congruent to 150 microM), but not by dCF and CF (even at 400 microM). Inhibition of LMC by EHNA, HOA, Ado, or dAdo could not be attributed to changes in nucleoside 5'-triphosphate or S-adenosylhomocysteine levels. Inhibition of LMC by Ado appears to be related to increases in lymphocyte cAMP levels, while the mechanism of action of dAdo remains obscure. Lymphocyte-mediated cytolysis may be inhibited by EHNA and HOA through modulation of cAMP metabolism.

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Animals; Cell Line; Coformycin; Cytotoxicity, Immunologic; Immunity, Cellular; Immunosuppressive Agents; Leukemia; Mice; Nucleoside Deaminases; Pentostatin; T-Lymphocytes, Cytotoxic

Analysis of the response of baby hamster kidney cells to adenosine in the presence of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine has revealed two distinct mechanisms of toxicity. The first is apparent at low concentrations of adenosine (less than 5 microM) and is dependent upon the presence of a functional adenosine kinase. The initial toxicity is abolished by uridine, is unrelated to the inhibition of ribonucleotide reductase, and is accompanied by a decrease in the size of the pyrimidine nucleotide pool. Toxicity at higher concentrations of adenosine is adenosine kinase independent and is potentiated by homocysteine thiolactone. An elevation in the intracellular level of S-adenosylhomocysteine, which was observed following treatment with higher concentrations of adenosine (greater than 10 microM), is believed to mediate toxicity at these levels. Interestingly, BHK cells were resistant to intermediate levels of adenosine. The mechanism of resistance is currently unknown, but appears unrelated to a lack of inhibition of adenosine deaminase. It is proposed that substrate inhibition of adenosine kinase may be a determinant of this property.

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Adenosine Kinase; Animals; Cell Adhesion; Cell Line; Coformycin; Drug Resistance; Drug Synergism; Guinea Pigs; Homocysteine; Kidney; Nucleotides; Pentostatin; Ribonucleotide Reductases; S-Adenosylhomocysteine; Uridine

We have presented a discussion on the relevance of ADA to organ transplantation with regard to whether or not purine analogue inhibitors of ADA can prevent allograft rejection, and whether or not cells with high ADA activity may be involved in the rejection of allografts. It is clear that ADA inhibitors do have a modest amount of immunosuppressive activity. The potentiation of this modest effect by the addition of a deoxyadenosine analogue supports studies by others suggesting that it is the metabolism of deoxyadenosine and the presence of this compound as a substrate that is biochemically responsible for the immunosuppressive effects observed. In other studies that we are currently conducting, we have found that the ADA inhibitor EHNA causes a rapid and severe depletion of ATP in resting murine lymphocytes and that EHNA potentiates a similar effect of the new immunosuppressive agent cyclosporine in the same model. Investigations are currently underway to see if ADA inhibitors may potentiate the immunosuppressive effect of cyclosporine in vivo. It appears that cells with high ADA activity that are detectable in the peripheral blood mononuclear cells of renal allograft patients may indeed be involved in the rejection of allografts. However, from murine studies allogeneic cells alone do not seem to generate the appearance of these cells nearly as strongly as infection with murine cytomegalovirus. It must be determined if the ADA-rich cells that appear at the time of CMV infection are involved in viral functions or are the ontologic appearance of cytotoxic T cells representing the host's response to the antigens of the virus. The attack of these host cells against allograft cells infected with the virus may then explain the long-standing observation that viral infections seem to trigger allograft rejection responses.

Topics: Adenine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Animals; Coformycin; Female; Graft Rejection; Humans; Immunosuppressive Agents; Kidney Transplantation; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nucleoside Deaminases; Pentostatin; Skin Transplantation; Splenectomy; T-Lymphocytes; Vidarabine

Structural analogues of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), in which the adenine moiety of the molecule was modified, were prepared in order to investigate the structural requirement of EHNA as an inhibitor of adenosine deaminase (ADA). Thus, 1- and 3-deaza-EHNA and their 6-deamino analogues were synthesized and evaluated as inhibitors of ADA from calf intestine. Inhibition studies indicated that isosteric substitution of pyrimidine nitrogens by carbons could be tolerated at the enzymatic binding site. In fact, 3-deaza-EHNA was found to have an inhibitory activity comparable to EHNA itself, and 1-deaza-EHNA, though less potent, is a good inhibitor. The 6-amino group gives an important contribution to the enzymatic binding if the N1 nitrogen is also present, conferring on the compound the characteristic of a semitight inhibitor.

Topics: Adenine; Adenosine Deaminase Inhibitors; Animals; Cattle; Intestines; Nucleoside Deaminases

Enzyme inhibitors used to simulate the inherited immunodeficiency diseases, adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP) deficiency, have been assessed in cultured human lymphocytes. Only 2'-deoxycoformycin (dCF) completely inhibited ADA in T and B cells at concentrations in excess of 5 microM. Erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and 8-amino guanosine (8-NH2GR) did not inhibit ADA or PNP completely at any concentration. Detailed metabolic experiments comparing viability and deoxynucleotide accumulation showed that B cell lines of malignant origin also accumulated high levels of dATP from 2'-deoxyadenosine (dAR), and dGTP from 2'-deoxyguanosine (dGR) as effectively as T cells--even without inhibitors, however, dAR reduced cell viability only when ADA was inhibited by dCF, whilst dGR was equally toxic with or without inhibitor, even to a line which accumulated no dGTP. These experiments indicate that cultured lymphocytes, using either EHNA or 8-NH2GR as enzyme inhibitor, are not valid models of the toxicity to the immune system in inherited ADA or PNP deficiency. They demonstrate that the ability to accumulate high levels of dATP or dGTP is not exclusive to T cells and that the in vitro toxicity of dAR or dGR could relate to the use of excess substrate and/or accumulation in different nucleotide, not deoxynucleotide pools.

Topics: Adenine; Adenosine Deaminase Inhibitors; B-Lymphocytes; Cell Line; Cell Survival; Coformycin; Deoxyadenine Nucleotides; Deoxyadenosines; Deoxyguanine Nucleotides; Deoxyguanosine; Dose-Response Relationship, Drug; Guanosine; Humans; Pentostatin; Purine-Nucleoside Phosphorylase; T-Lymphocytes

The effects of adenosine were studied on human neutrophils with respect to their generation of superoxide anion, degranulation, and aggregation in response to soluble stimuli. Adenosine markedly inhibited superoxide anion generation by neutrophils stimulated with N-formyl methionyl leucyl phenylalanine (FMLP), concanavalin A (Con A), calcium ionophore A23187, and zymosan-treated serum; it inhibited this response to PMA to a far lesser extent. The effects of adenosine were evident at concentrations ranging from 1 to 1,000 microM with maximal inhibition at 100 microM. Cellular uptake of adenosine was not required for adenosine-induced inhibition since inhibition was maintained despite the addition of dipyridamole, which blocks nucleoside uptake. Nor was metabolism of adenosine required, since both deoxycoformycin (DCF) and erythro-9-(2-hydroxy-3-nonyl) adenine did not interfere with adenosine inhibition of superoxide anion generation. The finding that 2-chloroadenosine, which is not metabolized, resembled adenosine in its ability to inhibit superoxide anion generation added further evidence that adenosine metabolism was not required for inhibition of superoxide anion generation by neutrophils. Unexpectedly, endogenously generated adenosine was present in supernatants of neutrophil suspensions at 0.14-0.28 microM. Removal of endogenous adenosine by incubation of neutrophils with exogenous adenosine deaminase (ADA) led to marked enhancement of superoxide anion generation in response to FMLP. Inactivation of ADA with DCF abrogated the enhancement of superoxide anion generation. Thus, the enhancement was not due to a nonspecific effect of added protein. Nor was the enhancement due to the generation of hypoxanthine or inosine by deamination of adenosine, since addition of these compounds did not affect neutrophil function. Adenosine did not significantly affect either aggregation or lysozyme release and only modestly affected beta-glucuronidase release by neutrophils stimulated with FMLP. These data indicate that adenosine (at concentrations that are present in plasma) acting via cell surface receptors is a specific modulator of superoxide anion generation by neutrophils.

Topics: 2-Chloroadenosine; Adenine; Adenosine; Adenosine Deaminase; Calcimycin; Cell Aggregation; Coformycin; Cytochalasin B; Cytoplasmic Granules; Depression, Chemical; Dipyridamole; Humans; Methylation; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Pentostatin; Superoxides

Topics: Adenine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Cell Survival; Cells, Cultured; Coformycin; Deoxyadenosines; Humans; Interphase; Leukemia; Lymphopenia; Nucleoside Deaminases; Pentostatin; Purines; Ribonucleosides; T-Lymphocytes

Topics: Adenine; Adenosine; Adenosine Deaminase Inhibitors; Blood Proteins; Coformycin; DNA; Humans; In Vitro Techniques; Lymphocyte Activation; Lymphocytes; Pentostatin; Phytohemagglutinins

Topics: Adenine; Adenosine Deaminase Inhibitors; Chemical Phenomena; Chemistry; Nucleoside Deaminases

Topics: Adenine; Adenosine Deaminase; Adenosine Deaminase Inhibitors; Adenylate Kinase; AMP Deaminase; Cells, Cultured; Coformycin; Humans; Lymphocytes; Nucleoside Deaminases; Nucleotide Deaminases; Pentostatin; Phosphotransferases

Topics: Adenine; Adenosine Deaminase Inhibitors; Cell Line; Coformycin; Deoxyadenosines; Enzyme Inhibitors; Humans; Leukemia, Lymphoid; Nucleoside Deaminases; Pentostatin; Phosphorylation; Vidarabine

The finding of elevated intracellular levels of adenosine deaminase (ADA) in some patients with acute lymphoblastic leukemia has led to attempts to control this disease with the adenosine deaminase inhibitor 2'-deoxycoformycin (dCF). Because of clinical reports indicating its relative freedom from myelotoxicity, we have tested the effects of this drug on erythroid, granulocytic, and T-lymphocyte colony formation by normal marrow and peripheral blood cells. While clinically the drug has been found to be active at serum concentrations of approximately 10 microM, we have tested it at concentrations up to and including 1 mM. It was found that both erythroid and granulocytic colony growth was completely unaffected by 1 mM dCF, a concentration at least 2 magnitudes higher than that necessary to totally ablate intracellular ADA levels. T-lymphocyte colony growth was unaffected by 100 microM dCF, but at 1 mM some inhibition was observed. These findings therefore indicate that dCF, while able to cause leukemic cell lysis in vivo, has no inhibitory effect on the proliferative capacity of normal hematopoietic cells.

Topics: Adenine; Adenosine Deaminase Inhibitors; Coformycin; Colony-Forming Units Assay; Dose-Response Relationship, Drug; Erythrocytes; Granulocytes; Growth Inhibitors; Humans; Pentostatin; Phytohemagglutinins; Ribonucleosides; T-Lymphocytes

Topics: Adenine; Adenosine Deaminase Inhibitors; Animals; Cell Survival; Coformycin; Concanavalin A; Female; Horses; Lipopolysaccharides; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mitogens; Nucleoside Deaminases; Pentostatin; Phytohemagglutinins; Spleen; T-Lymphocytes