Erythro-9-(2-hydroxy-3-nonyl)adenine Research Articles

Interleukin 2 responsive lymphocytes in patients with adenosine deaminase deficiency

We evaluated the effects of recombinant interleukin 2 (IL-2) on the proliferative responses to mitogens of peripheral blood mononuclear cells (PBMC) from three adenosine deaminase (ADA)-deficient patients. There was significant enhancement by IL-2 of the proliferative responses to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) of PBMC from all three patients. We found that normal PBMC respond with increased numbers of CD3-positive cells when exposed to PHA or PWM and that the response by normal CD8-positive cells was greater than that by CD4-positive cells. In contrast, we found that in ADA-deficient cells the response is almost entirely due to the CD3 CD4 -positive population of lymphocytes. These results could not be explained by either the culture conditions or the possibility of a mixed chimeric state. When we evaluated an in vitro cell model of ADA deficiency using an ADA inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), we found that the inhibitory effect of EHNA plus deoxy-adenosine on mitogen-stimulated PBMC could not be prevented by IL-2. These results suggest that the immunodeficiency in ADA deficiency includes the absence or failure of a subset of T cells to make IL-2 and the failure of the CD8-positive subset to respond to IL-2. Also, the in vitro cell model of ADA deficiency using EHNA as the ADA inhibitor is limited in its use in understanding the pathogenesis of this disease.

Adenosine deaminase and adenosine uptake inhibitions facilitate ventilation in rats.

The effects of intracarotid (i.c.) infusions of the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and of the adenosine uptake blocker, dipyridamole on spontaneous ventilation were studied in rats anaesthetized with sodium pentobarbitone. Both EHNA and dipyridamole mimicked the excitatory effect of adenosine on respiration increasing in a dose-dependent manner respiratory ventilation determined as increases in tidal volume (VT), respiratory frequency (f) and minute volume (VE). These excitatory effects were abolished after section of the carotid sinus nerves. The excitatory effect of EHNA on respiration was prevented by adenosine deaminase and antagonized by 1,3-dipropyl-8(p-sulfophenyl)xanthine (DPSPX). DPSPX also antagonized the excitatory effect of dipyridamole on respiration. Both EHNA and dipyridamole in doses virtually devoid of effect on respiration potentiated the excitatory effect of exogenous adenosine on respiration. Two different effects on respiration were observed during i.c. infusions of cumulative doses of DPSPX: one inhibitory, not present in glomectomized animals and another, excitatory, present in both glomectomized and non-glomectomized animals. It is concluded that endogenous adenosine could be involved in respiration mediated through carotid body chemoreceptors and that the nucleoside is inactivated at this level by deamination and uptake.

Inhibitors of adenosine catabolism improve recovery of dog myocardium after ischemia.

The effects of inhibitors of adenosine catabolism on contractile function and metabolites were assessed during 15 minutes of ischemia followed by 30 minutes of reperfusion in the open-chest dog heart. As compared to sham treatment, pretreatment with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and dipyridamole (DP) protected contractile function during ischemia, and improved recovery of high energy phosphate content and contractile function during reperfusion following ischemia. Testing EHNA and DP in a free-radical generating system indicated both compounds have some scavenging ability, suggesting the effect of EHNA + DP may not be on adenosine nucleotide metabolism alone. Comparison of end diastolic segment lengths to contractile function indicated the results were not affected by changes in preload resulting from peripheral vasodilation.

Characterization of a microtubule-stimulated adenosine triphosphatase activity associated with microtubule gelation-contraction

A microtubule-stimulated ATPase is associated with particles that are responsible for microtubule gelation-contraction in vitro. These particles have been proposed to be slow axonal transport, component a, particulates (SCAPs) [Weisenberg, R. C., Flynn, J. J., Gao, B., Awodi, S., Skee, F., Goodman, S., & Riederer, B. (1987) Science (Washington, D.C.) 238, 1119-1122]. The SCAP ATPase activity is stimulated approximately twofold by microtubules. The microtubule-stimulated ATPase activity correlates with the occurrence of microtubule gelation-contraction. Both microtubule-stimulated ATPase activity and microtubule gelation-contraction are inhibited by millimolar calcium, 0.3 M KCl plus 2 mM ethylenediaminetetraacetic acid (EDTA), 5 microM vanadate, and millimolar N-ethylmaleimide (NEM). Neither the ATPase activity nor microtubule gelation-contraction is affected by high magnesium concentrations (up to 8 mM) or by the anti-ATPase drugs ouabain, oligomycin, sodium azide, and erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). Magnesium is required for both ATPase activity and microtubule gelation-contraction. Microtubule-stimulated hydrolysis of GTP, CTP, ITP, and UTP is less than 50% of ATP hydrolysis, and microtubule gelation-contraction is reduced in these nucleotides. On the basis of these results we propose that the microtubule-stimulated ATPase activity associated with SCAPs is a previously undescribed enzyme that is responsible for microtubule gelation-contraction in vitro and that is the likely motor for component a of slow axonal transport.

Enhanced Postischemic ATP Repletion by Pharmacological Inhibition of Nucleoside Washout and Catabolism

We tested the hypothesis that inhibition of adenosine transport by dipyridamole and inhibition of adenosine deamination by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) prevents nucleoside loss and stimulates postischemic ATP-repletion. In an open chest canine model, dipyridamole (0.5 mg/kg/h) and EHNA (5 mg/kg/h) were infused intra-atrially during a coronary occlusion period of 45 min and a reperfusion period of 180 min. Transmural needle biopsies, obtained during the ischemic period and within the reperfusion period, were analyzed using high performance liquid chromatography for adenine nucleotides and adenosine, inosine, xanthine, and hypoxanthine as well as creatine phosphate. During ischemia and under the influence of dipyridamole plus EHNA, 56% of the catabolized adenine nucleotides were recovered stoichiometrically as adenosine, whereas in the untreated group less than 10% of the nucleotides were recovered as adenosine because of rapid deamination to inosine. In the control group, ATP levels decreased during ischemia from control values of 5.25 +/- 0.28 microns/g to 2.01 +/- 0.18 microns/g. In the group treated with dipyridamole and EHNA, ATP levels fell to 2.2 +/- 0.22 microns/g but rose to 3.22 +/- 0.29 microns/g within 180 min of reperfusion, whereas in the untreated control group tissue levels of ATP did not increase. However, a significant proportion of the adenosine accumulated during ischemia under the influence of dipyridamole plus EHNA was not used for the restoration of the ATP level during reperfusion. A significant amount of adenosine was probably trapped in the interstitial space and could not be transported back into the myocytes in the presence of dipyridamole during reperfusion. In both groups, creatine phosphate levels were restored to normal levels during reperfusion.

On the adenosine receptor and adenosine inactivation at the rat diaphragm neuromuscular junction

1. The effects of adenosine and adenosine analogues 2-chloroadenosine (CADO), L-N6-phenylisopropyladenosine (L-PIA), D-N6-phenylisopropyladenosine (D-PIA), N6-cyclohexyladenosine (CHA) and 5'-N-ethylcarboxamide adenosine (NECA) on evoked endplate potentials (e.p.ps) and on twitch tension were investigated in innervated diaphragms of the rat. 2. Adenosine and its analogues decreased, in a concentration-dependent manner, the amplitude of both the e.p.ps and the twitch responses evoked by nerve stimulation. The order of potency in decreasing the twitch tension was CHA, L-PIA, NECA greater than D-PIA greater than CADO greater than adenosine. L-PIA was about 8 times more potent than D-PIA. Neither adenosine nor the adenosine analogues affected the twitch responses of directly stimulated tubocurarine-paralysed muscles. 3. 8-Phenyltheophylline (8-PT), theophylline and isobutylmethylxanthine (IBMX), in concentrations virtually devoid of effect on neuromuscular transmission, antagonized the inhibitory effect of 2-chloroadenosine. The order of potency of the alkylxanthines as antagonists of the adenosine receptor at the rat diaphragm neuromuscular junction was 8-PT greater than IBMX greater than theophylline. The antagonism by these xanthines was shown to be competitive, the pA2 value for 8-PT being 7.16. In concentrations slightly higher than those used to test its ability to antagonize the adenosine receptor, IBMX and 8-PT increased the amplitude of e.p.ps without modifying their decay phase or the resting membrane potential of the muscle fibre. 4. The adenosine uptake inhibitor, nitrobenzylthioinosine (NBI) and the adenosine deaminase inhibitor, erythro-9(2-hydroxy-3-nonyl)adenine (EHNA), in concentrations virtually devoid of effect on neuromuscular transmission, potentiated the inhibitory effect of adenosine at the rat diaphragm neuromuscular junction. The potentiation factors were about 2.6 for NBI (5 microM), 2.2 for EHNA (25 microM) and 4.6 for the combination of NBI (5 microM) and EHNA (25 microM). 5. It is concluded that both uptake and deamination contribute to the inactivation of adenosine at the rat diaphragm neuromuscular junction and that in this preparation the inhibitory effect of adenosine on transmission is mediated by a xanthine-sensitive adenosine receptor with an agonist profile which does not fit the criteria for its classification either as an A1 or A2-adenosine receptor.

Synergistic effect of 3'-deoxyadenosine N1-oxide and adenosine deaminase inhibitors on growth of Ehrlich ascites tumor cells in vivo.

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.

Study on the apparent resistant strains of herpes simplex virus type 1 against 9-beta-D-arabinofuranosyladenine.

The appearance of drug resistant strains of herpes simplex virus type 1 (HSV-1) against 9-beta-D-arabinofuranosyladenine (araA), in comparison with 5-iodo-2'-deoxyuridine (IUdR) and 9-(2-hydroxyethoxymethyl)guanine (acycloguanosine, ACG), has been investigated in green monkey kidney cell cultures (Vero) and human embryo lung cell cultures (HEL). Growth curves of HSV-1 in Vero cells and HEL cells in the presence or absence of 20 micrograms/ml of araA showed that not only the viruses produced by these cells in the presence of araA but also the viruses produced in the absence of araA were resistant to the same concentration of araA. However, both viruses were unable to grow in the presence of a combination of 20 micrograms/ml of araA and 3 micrograms/ml of erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) which is an inhibitor of adenosine deaminase. On the other hand, the viruses produced only in the presence of IUdR or ACG were resistant to each drug. All virus clones obtained from five serial clonings in the presence of araA alone or araA and EHNA in combination were not resistant to araA. These results show that the strain of HSV-1 resistant to araA is an apparently resistant strain, and a truly resistant strain to araA may not be established as easily as the truly resistant strains to IUdR and ACG.

Degradation of adenosine by extracellular adenosine deaminase in the rat duodenum

1. 1. Extracellular degradation of adenosine by adenosine deaminase was studied in the rat duodenum using high performance liquid chromatography (HPLC) and pharmacological techniques. 2. 2. Relaxant responses to adenosine (1–10 μM) were potentiated in a concentration-dependent manner by erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and deoxycoformycin, both of which are inhibitors of adenosine deaminase. 3. 3. Breakdown of adenosine, determined by HPLC, due to incubation with segments of rat duodenum was inhibited by both EHNA and deoxycoformycin. Cytosolic sources of adenosine deaminase were excluded. 4. 4. Relaxant responses to adenosine were also potentiated by the adenosine transport inhibitor dilazep, which did not inhibit adenosine deaminase activity. 5. 5. The extracellular adenosine deaminase activity (4 units/g tissue) was high compared with activity previously determined in other organs.

Stimulation-evoked release of purines from the rabbit retina

The evoked release of purines from rabbit retinae preloaded with [(3)H]adenosine was studied in vitro. Potassium (8.6-43.6 mM) and ouabain (1 or 10 microM) increased the release of radioactivity in a concentration-dependent manner. The K(+)-evoked release was significantly reduced when the superfusion was carried out at 2-4 degrees C. The effect of K(+) (8.6, 13.6 and 23.6 mM) and of ouabain (1 microM) were completely abolished when the retinae were superfused with a Ca(2+)-free medium containing 0.1 mM EGTA. Calcium removal only partially reduced the effect of higher K(+) and ouabain concentrations (43.6 mM and 10 microM, respectively). Further, the effect of K(+) was found to be independent of extracellular Ca(2+) when retinae were pretreated with ouabain for 30 min. Stimulation of the retina with light flashes induced a small, persistent increase in the release of radioactivity observable for several minutes after the end of stimulation. The superfusate contained mainly hypoxanthine and inosine. There were no significant changes in the relative proportions of the different purine compounds released before or in response to either K(+) (23.6 mM) or ouabain (10 microM) stimulation. Potassium stimulation significantly increased the release of adenosine, inosine and hypoxanthine. Addition of the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), significantly increased the relative proportions of released endogenous adenosine and inosine. The results indicate that K(+) stimulation induces the release of purines from the rabbit retina by a Ca(2+)- and energy-dependent process. Light flashes also induce a purine release. The results suggest an active role for adenosine in retinal neurotransmission.

Lack of cross-resistance between cytosine arabinoside and a new halogenated nucleoside analogue, 2-bromo-2'-deoxyadenosine in human acute myeloid leukaemia cells.

2-Bromo-2'-deoxyadenosine (BdA) is one of a group of recently synthesised halogenated deoxyadenosine analogues that are relatively resistant to inactivation by adenosine deaminase (ADA). Its activity has been studied in human acute myeloid leukemia (AML) in vitro. In these studies BdA behaved as a cycle-active, phase-active agent that blocked cells at the G1-S transition. It did not exhibit significant cross-resistance with cytosine arabinoside (Ara-C) in either clinical AML samples (from patients who exhibited Ara-C resistance in vivo) or in HL60 in which Ara-C resistance had been induced in vitro. Deoxycytidine kinase levels were not reduced in resistant lines. Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), an adenosine deaminase (ADA) inhibitor, with BdA produced a simple additive response without the dramatic synergism reported when it is used with deoxyadenosine. This is consistent with the idea that BdA is a poor substrate for ADA. This group of compounds warrants further investigation to determine their suitability for clinical use, especially in situations where Ara-C resistance is likely to be a problem.

Isolation of a Ca2+-protease resistant high Mr microtubule binding protein from mammalian brain: Characterization of properties partially expected for a dynein-like molecule

A novel microtubule binding protein was isolated from cell extracts of pig brain after selective destruction of high Mr MAPs by Ca2+-activation of endogenous proteases. The protein had an apparent Mr of 300,000, but several criteria, including peptide maps, immunological crossreactivities, resistance to Ca2+-activated proteolysis, and inability to induce microtubule assembly, distinguished this protein from the major high Mr microtubule associated proteins MAP-1 and MAP-2. Certain molecular properties of the protein resembled those of dynein: its size under denaturing conditions, its crossreactivity with antibodies toTetrahymena dynein, the ATP-dependence of its microtubule binding, the erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA)—sensitivity of its ATPase activity and its resistance to Ca2+-activated proteolysis. However, in peptide maps and the insensitivity of its ATPase activity to vanadate the protein could be differentiated fromTetrahymena dynein heavy chain polypeptides. Based on the properties characterized so far, the protein seems qualified to function as a mechanochemical enzyme in the cytoplasm of mammalian brain cells and could represent a cytoplasmic dynein variant.

Effect of some drugs on thromboplastin activity in mouse trophoblast cells in vitro and in vivo

Mouse trophoblast cells are constitutive producers of the thromboplastin apoprotein in vitro. The effects on thromboplastin activity of the three transmethylation inhibitors 3-deazaadenosine (DZA), 3-deazaaristeromycin (DZAri) and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA), the four calcium antagonists TMB-8, verapamil, nifedipine and felodipine, the prostaglandin E 2 (PGE 2), the phosphodiesterase inhibitor 1-methyl 3-isobutylxanthine (MIX) and monensin have been studied. No cytotoxic effects were detected when trypan blue exclusion, release of lactic dehydrogenase, incorporation of 14C-leucine into protein and cell morphology were monitored. TMB-8, felodipine, nifedipine and verapamil all abolished the increase in thromboplastin when added after 68 hr or 90–96 hr in culture. EHNA and DZAri had the same effect (but were only added at 90–96 hr). DZA had a similar effect when added at 68 hr and an even more marked inhibitory effect when added at 90–96 hr. Monensin prevented the increase in thromboplastin activity at 68 hr as well as at 90–96 hr. The combination of DZA and 1-homocysteine thiolactone (Hcy) further increased the inhibition, indicating that in these cases synthesis as well as degradation of thromboplastin were altered. The combination of DZA/Hcy and one of the four calcium antagonists gave no additional inhibitory effect. PGE 2 had a biphasic dose-dependent effect. The increased thromboplastin activity at low concentrations of PGE 2 (10 ng/ml) was inhibited by addition of one of the compounds verapamil, felodipine, nifedipine or DZA/Hcy. PGE 2 at higher levels (10 μg/ml) significantly inhibited thromboplastin synthesis. Combination of PGE 2 (10 μg/ml) and one of the calcium antagonists, DZA/Hcy or MIX gave no significant additive inhibitory effect.

Release of adenosine and its metabolites from activated human leucocytes

Human leucocytes whose adenine nucleotide pool was prelabelled with [3H]adenine were investigated for their capacity to release adenosine and its metabolites and histamine when activated with the calcium ionophore A23187, anti-immunoglobulin E and the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (f-MLP). Sixty-nine per cent of the 3H-label assimilated by the cells was incorporated into their adenine nucleotide pool in the ratio adenosine 5'-phosphate (AMP):adenosine 5'-pyrophosphate (ADP):adenosine 5'-triphosphate (ATP), 3:1:1. Spontaneous release of label from leucocytes plateaued at 5 min at 22.1 +/- 4.2% of the total radiolabel incorporated and mainly consisted of hypoxanthine, inosine and adenosine. Activation of cells with the calcium ionophore A23187 (1.0 mumol/l) caused a net increase in [3H]purine release above baseline of 27.9 +/- 4.6% accompanied by a net basophil histamine release of 46.6 +/- 9.4%. A23187 (1.0-3.0 mumol/l) caused a parallel concentration-dependent release of labelled purines and histamine. Purified mononuclear cells and granulocytes exhibited a similar ionophore-dependent capacity to release [3H]purines. The distribution of label did not significantly differ between supernatants of activated and non-activated cells. Preincubation of cells with dipyridamole (10 mumol/l) and erythro-9-(2-hydroxy-3)-nonyladenine (EHNA) (10 mumol/l) to inhibit uptake and catabolism of adenosine respectively, produced an increase in adenosine at the expense of hypoxanthine recovered from the supernatant of both ionophore-stimulated and resting cells. Activation of leucocytes with f-MLP (1.0 mumol/l) caused a net increase in release of basophil histamine, but was associated with only a transient net increase in release of [3H]purine in four of five experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Behavioral characteristics of centrally administered adenosine analogs

Mice were implanted with chronic indwelling cannulae in the lateral cerebral ventricle. A series of adenosine analogs and related compounds were injected into the lateral ventricle (ICVT) and their effects on spontaneous locomotor activity recorded. All analogs produced dose-related decreases in locomotor activity. 5′-N 6-ethyl-carboxamidoadenosine (NECA) was the most potent compound tested, with a number of N 6-substituted analogs also being effective depressants of activity. Caffeine, administratered either intracerebroventricularly or intraperitoneally, antagonized the depressant effects of the adenosine analogs. 3-Isobutyl-1-methylxanthine, administered ICVT, depressed locomotor activity. However, after caffeine, IBMX elicited behavioral stimulation. Agents which inhibit the transport of adenosine (dipyridamole, dilazep, papaverine) depressed locomotor activity, as did erythro-9-(2-hydroxy-3-nonyl)adenine(EHNA), an inhibitor of adenosine deaminase. The effects of dilazep, papaverine and EHNA, but not of dipyridamole, were antagonized by caffeine. These results further substantiate the notion that endogenous adenosine is involved in the regulation of central nervous system excitability.

ChemInform Abstract: IMPROVED SYNTHESIS OF 2′‐DEOXYFORMYCIN A AND STUDIES OF ITS IN VITRO ACTIVITY AGAINST MOUSE LYMPHOMA OF T‐CELL ORIGIN

AbstractDie ausgehend von Formycin A (I) erhaltene im Titel genannte Verbindung (V) besitzt cytotoxische Wirkung und inhibiert stark das Tumorwachstum des S49‐Lymphoms aus Mäusen in vitro.

Chemotherapy and trace element levels in blood and tissue of rats implanted with prostate tumor cells

Male Copenhagen rats with transplanted prostatic adeno-carcinoma were treated with different polyamine synthesis inhibitors, such as methylglyoxal-bis-guanylhydrazone (MGBG), erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) combined with 9-β-D-arabin-ofuranosyl-adenine (ARA-A), α-difluoromethyl-ornithine (DFMO), and some of their combinations. Levels of the essential trace elements-copper, zinc, magnesium, iron, selenium, and manganese -have been determined in blood, tumor, kidney, and liver of these animals and are discussed in terms of efficiency of the treatment. MGBG had the strongest effect on trace element levels in tissues of treated animals. MGBG combined with DFMO exhibited the highest antitumor activity of all treatment protocols. Selenium given as selenite with drinking water was used as an adjuvant with the most toxic combination, (ARA-A/EHNA, MGBG). Selenite reduced the toxicity of these therapeutic agents.

Enhancement of transmission at the frog neuromuscular junction by adenosine deaminase: Evidence for an inhibitory role of endogenous adenosine on neuromuscular transmission

Adenosine deaminase reversibly increased the amplitude and the quantum content of the end-plate potentials (EPPs) recorded from superficial muscle fibers of frog sartorius preparations in which twitches have been prevented with high-magnesium solutions. Adenosine deaminase prevented the inhibitory effect of exogenously applied adenosine but not that of 2-chloroadenosine on the amplitude of EPPs. The effect of adenosine deaminase was abolished by erythro-9(2-hydroxy-3-nonyl)adenine (EHNA). The results suggest that endogenous adenosine exerts an inhibitory ‘tone’ over neuromuscular transmission.

The effects of an induced adenosine deaminase deficiency on T-cell differentiation in the rat

Inherited deficiency of the enzyme adenosine deaminase (ADA) has been found in a significant proportion of patients with severe combined immunodeficiency disease an inherited defect generally characterized by a deficiency of both B and T cells. Two questions are central to understanding the pathophysiology of this disease: (1) at what stage or stages in lymphocyte development are the effects of the enzyme deficiency manifested; (2) what are the biochemical mechanisms responsible for the selective pathogenicity of the lymphoid system. We have examined the stage or stages of rat T-cell development in vivo which are affected by an induced adenosine deaminase deficiency using the ADA inhibitors, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and 2′-deoxycoformycin (DCF). In normal rats given daily administration of an ADA inhibitor, cortical thymocytes were markedly depleted; peripheral lymphocytes and pluripotent hemopoietic stem cells (CFU-S) all were relatively unaffected. Since a deficiency of ADA affects lymphocyte development, the regeneration of cortical and medullary thymocytes and their precursors after sublethal irradiation was used as a model of lymphoid development. By Day 5 after irradiation the thymus was reduced to 0.10-0.5% of its normal size; whereas at Days 9 and 14 the thymus was 20–40% and 60–80% regenerated, respectively. When irradiated rats were given daily parenteral injections of the ADA inhibitor plus adenosine or deoxyadenosine, thymus regeneration at Days 9 and 14 was markedly inhibited, whereas the regeneration of thymocyte precursors was essentially unaffected. Thymus regeneration was at least 40-fold lower than in rats given adenosine or deoxyadenosine alone. Virtually identical results were obtained with both ADA inhibitors, EHNA and DCF. The majority of thymocytes present at Day 9 and at Day 14 in inhibitor-treated rats had the characteristics of subcapsular cortical thymocytes which are probably the most ancestral of the thymocytes. Thus, an induced ADA deficiency blocked the proliferation and differentiation of subcapsular cortical thymocytes which are the precursors of cortical and medullary thymocytes.

Improved synthesis of 2'-deoxyformycin A and studies of its in vitro activity against mouse lymphoma of T-cell origin.

7-Amino-3-(2'-deoxy-beta-D-ribofuranosyl)pyrazolo[4,3-d]pyrimidine (2'-deoxyformycin A) was synthesized from formycin A by a sequence consisting of (i) 3',5'-cyclosilylation with 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, (ii) 2'-acylation with phenoxythiocarbonyl chloride and 4-(N,N-dimethylamino)pyridine, (iii) N-trimethylsilylation with hexamethyldisilazane, (iv) reduction of the 2'-O-phenoxythiocarbonyl group with tri-n-butyltin hydride, and (v) desilylation with tetra-n-butylammonium fluoride. 2'-Deoxyformycin A was a potent inhibitor of the in vitro growth of S49 lymphoma, a murine tumor of T-cell origin. The IC50 of 2'-deoxyformycin A against S49 cells was 10-15 microM, whereas that of 2'-deoxyadenosine (dAdo) under the same conditions (72-h incubation in medium containing heat-inactivated horse serum) was 180 microM. In the presence of 10 microM erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) to block intracellular adenosine deaminase (ADA) activity, 2'-deoxyformycin A and dAdo both gave IC50's of 5-10 microM. When assayed against a mutant S49 subline lacking adenosine kinase (AK) or a subline with a combined deletion of AK and deoxycytidine kinase (dCK), 2'-deoxyformycin A in combination with 10 microM EHNA was inactive at concentrations of up to 50 microM. Similar lack of activity against kinase-deficient cells was shown by formycin A. Thus, phosphorylation of 2'-deoxyformycin A appears to be required for biological activity and is probably catalyzed by AK rather than dCK. 2'-Deoxyformycin A and related 2'-deoxyribo-C-nucleoside analogues of the purine type may be of interest as potential T-cell specific cytotoxic agents.