Breast neoplasms in women treated with x-rays for acute postpartum mastitis.

Breast cancer has been studied by mail survey up to 34 years in 571 of 606 women treated with x-rays for acute postpartum mastitis. The incidence of neoplasms was compared with that of three nonirradiated control groups--nonirradiated sisters of the treated women, women with acute postpartum mastitis not treated with X-rays, and their nonirradiated sisters. For the irradiated group, with mean dose of 247 rads to both breasts, the overall relative risk of breast cancer was 2.2 for years 10-34 post irradiation and 3.6 for years 20-34. The dose response for malignant and benign breast neoplasms was compatible with a linear fit. For comparable total doses, fractionation of exposure did not reduce carcinogenic action. Women over age 30 years at radiation treatment had as great an excess risk of breast cancer as did younger women. The overall excess risk of developing breast cancer was about 8-10 cases per million women per rad per year, an increase of about 0.5% per rad.

Adenosine kinase inhibitors.

Adenosine (ADO) is an endogenous modulator of intercellular signaling that provides homeostatic reductions in cell excitability during tissue stress and trauma. The inhibitory actions of ADO are mediated by interactions with specific cell-surface G-protein coupled receptors regulating membrane cation flux, polarization, and the release of excitatory neurotransmitters. ADO kinase (AK; EC 2.7.1.20) is the key intracellular enzyme regulating intra- and extracellular ADO concentrations. Inhibition of AK produces marked increases in extracellular ADO levels that are localized to cells and tissues undergoing accelerated ADO release. Thus AK inhibition represents a mechanism to selectively enhance the protective actions of ADO during tissue trauma without producing the nonspecific effects associated with the systemic administration of ADO receptor agonists. During the last 10 years, specific inhibitors of AK based on the endogenous purine nucleoside substrate, ADO, have been developed. Potent AK inhibitors have recently been synthesized that demonstrate high specificity for this enzyme as compared to other ADO metabolic enzymes, transporters, and receptors. In both in vitro and in vivo models, AK inhibitors have been shown to potently increase ADO concentrations in a tissue and event specific fashion and to demonstrate potential clinical utility in animal models of epilepsy, ischemia, pain, and inflammation. AK inhibitors have demonstrated superior efficacy in these models as compared to other mechanisms of modulating ADO availability, and these agents exhibit reduced side-effect liabilities compared to direct acting ADO receptor agonists. The preclinical profile of AK inhibitors indicate that these agents may have therapeutic utility in a variety of central and peripheral diseases associated with cellular trauma and inflammation. Clinical trials are currently underway to evaluate the efficacy of AK inhibitors in seizure disorders and pain.

Nitroaromatic amino acids as inhibitors of neuronal nitric oxide synthase.

Nitric oxide (NO.) is an important biomodulator of many physiological processes. The inhibition of inappropriate production of NO. by the isoforms of nitric oxide synthase (NOS) has been proposed as a therapeutic approach for the treatment of stroke, inflammation, and other processes. In this study, certain 2-nitroaryl-substituted amino acid analogues were discovered to inhibit NOS. Analogues bearing a 5-methyl substituent on the aromatic ring demonstrated maximal inhibitory potency. For two selected inhibitors, investigation of the kinetics of the enzyme showed the inhibition to be competitive with l-arginine. Additionally, functional NOS inhibition in tissue preparations was demonstrated.

Discovery of 4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido[2,3-d]pyrimidine, an orally active, non-nucleoside adenosine kinase inhibitor..

Adenosine (ADO) is an endogenous homeostatic inhibitory neuromodulator that reduces cellular excitability at sites of tissue injury and inflammation. Inhibition of adenosine kinase (AK), the primary metabolic enzyme for ADO, selectively increases ADO concentrations at sites of tissue trauma and enhances the analgesic and antiinflammatory actions of ADO. Optimization of the high-throughput screening lead, 4-amino-7-aryl-substituted pteridine (5) (AK IC(50) = 440 nM), led to the identification of compound 21 (4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido [2,3-d]pyrimidine, ABT-702), a novel, potent (AK IC(50) = 1.7 nM) non-nucleoside AK inhibitor with oral activity in animal models of pain and inflammation.

Design of adenosine kinase inhibitors from the NMR-based screening of fragments.

A strategy is described for designing high-affinity ligands using information derived from the NMR-based screening of fragments. The method involves the fragmentation of an existing lead molecule, identification of suitable replacements for the fragments, and incorporation of the newly identified fragments into the original scaffold. Using this technique, novel substituents were rapidly identified and incorporated into lead inhibitors of adenosine kinase that exhibited potent in vitro and in vivo activities. This approach is a valuable strategy for modifying existing leads to improve their potency, bioavailability, or toxicity profile and thus represents a useful technique for lead optimization.

Biochemical mechanism of organic nitrate action.

Increasing evidence suggests that organic nitrate action derives from their metabolic conversion to nitric oxide (NO) in the vascular smooth muscle cell. The primary catalytic activity of this process appears to reside at the cellular plasma membrane. There is no concrete evidence to indicate that NO formation is preceded by the production of inorganic nitrite ion or that the NO produced needs to form S-nitrosothiols before it can activate guanylate cyclase to produce cyclic guanosine 3',5'-monophosphate (cGMP). Although sulfhydryl donors can partially reverse nitroglycerin-induced tolerance in patients, this phenomenon (by itself) is not sufficient to implicate intracellular sulfhydryl depletion as an operating mechanism of clinical nitrate tolerance. This is because sulfhydryl donors can react with nitroglycerin extracellularly to form S-nitrosothiols, and nonsulfhydryl compounds, such as enalapril and hydralazine, can prevent the development of in vivo nitrate tolerance. In addition to the cellular biochemical reactions, organic nitrates also produce systemic biochemical effects through altering neurohormonal status. These systemic effects may contribute significantly to the development of nitrate tolerance in therapeutic situations.

The effect of ABT-702, a novel adenosine kinase inhibitor, on the responses of spinal neurones following carrageenan inflammation and peripheral nerve injury.

1. Adenosine (ADO) receptor activation modulates sensory transmission in the dorsal horn. Little is known about the circumstances underlying release of the purine. The present study was conducted to investigate the effect of a novel and potent non-nucleoside adenosine kinase (AK) inhibitor, ABT-702, on the responses of dorsal horn neurones to selected peripheral stimuli. ABT-702 is orally effective to reduce behavioural signs of nociception in models of acute, inflammatory, and neuropathic pain. 2. Electrophysiological recordings were made from wide dynamic range (WDR) neurones in halothane-anaesthetized rats. ABT-702 was given subcutaneously following either carrageenan inflammation or peripheral nerve injury (L5/L6 spinal nerve ligation). Comparisons were made between carrageenan and uninjected control animals, and similarly between spinal nerve ligated (SNL) and sham operated animals. 3. ABT-702 produced inhibition of the postdischarge, wind-up and C-fibre evoked responses in both carrageenan and nerve-injured animals. Furthermore, the mechanical and thermal evoked responses were similarly reduced in SNL rats. Overall, ABT-702 produced a significantly greater inhibition of these responses in SNL rats as compared to sham controls. Similarly ABT-702 tended to produce greater effects after carrageenan inflammation, however this did not reach significance. 4. Protection of endogenous adenosine by ABT-702 therefore produces a marked inhibition of the noxious evoked neuronal activity in inflamed and neuropathic rats. Our results demonstrate a plasticity in the endogenous adenosine-mediated inhibitory system following SNL and provide a possible basis for the use of this compound for the treatment of neuropathic and other persistent pain states.

Modulation of BzATP and formalin induced nociception: attenuation by the P2X receptor antagonist, TNP-ATP and enhancement by the P2X(3) allosteric modulator, cibacron blue.

1. Exogenous ATP produces acute and localized pain in humans, and P2X receptor agonists elicit acute nociceptive behaviours in rodents following intradermal administration to the hindpaw. The predominant localization of P2X(3) mRNA in sensory neurones has led to the hypothesis that activation of P2X(3) and/or P2X(2/3) receptors contributes to nociception. 2. The local administration of the P2X receptor agonist, BzATP (100--1000 nmol paw(-1), s.c.) into the rat hindpaw produced an acute (<15 min) paw flinching response that was similar to that observed in the acute phase of the formalin (5%) test. 3. The co-administration of the potent P2X receptor antagonist, TNP-ATP (30--300 nmol paw(-1)), but not an inactive analogue, TNP-AMP, with BzATP into the rat hindpaw attenuated BzATP-induced nociception. Similarly, co-administration of TNP-ATP, but not TNP-AMP, with 5% formalin reduced both acute and persistent nociception in this test. 4. Co-administration of cibacron blue (30 and 100 nmol paw(-1)), a selective allosteric enhancer of P2X(3) and P2X(2/3) receptor activation, with BzATP (30 and 100 nmol paw(-1)) into the rat hindpaw produced significantly greater nociception as compared to the algogenic effects of BzATP alone. Intradermal co-administration of cibacron blue (30 and 100 nmol paw(-1)) with formalin (1 and 2.5%) into the rat hindpaw also produced significantly greater nociceptive behaviour as compared to formalin alone. 5. The ability of TNP-ATP and cibacron blue to respectively attenuate and enhance nociceptive responses elicited by exogenous BzATP and formalin provide further support for the hypothesis that activation of peripheral P2X(3) containing channels contributes specifically to both acute and persistent nociception in the rat.

Therapeutic potential of adenosine kinase inhibitors.

Adenosine kinase (AK; EC 2.7.1.20) is a key intracellular enzyme regulating intra and extracellular concentrations of adenosine (ADO), an endogenous modulator of intercellular signalling that reduces cell excitability during tissue stress and trauma. The inhibitory effects of ADO are mediated by interactions with specific cell-surface G-protein coupled receptors (GPCR), which regulate membrane cation flux, membrane polarisation and the release of excitatory neurotransmitters. Inhibition of AK potentiates local extracellular ADO levels at cell and tissue sites which are undergoing accelerated ADO release. Thus, AK inhibition represents a mechanism to selectively enhance the endogenous protective actions of ADO during cellular stress while potentially minimising the non-specific effects associated with the systemic administration of ADO receptor agonists. Novel, potent AK inhibitors have recently been synthesised that demonstrate high specificity for this particular enzyme as compared to other ADO metabolic enzymes, transporters and receptors. AK inhibitors have been shown to increase ADO concentrations in various systems in vitro, as well as in an in vivo model of neurotoxicity. In addition, AK inhibitors have demonstrated efficacy in animal models of epilepsy, cerebral ischaemia as well as pain and inflammation, thus suggesting their potential therapeutic utility for these conditions.

A-134974: a novel adenosine kinase inhibitor, relieves tactile allodynia via spinal sites of action in peripheral nerve injured rats.

Extracellular levels of adenosine (ADO) can be raised through inhibition of adenosine kinase (AK), a primary metabolic enzyme for ADO. AK inhibitors have shown antinociceptive activity in a variety of animal models of nociception. The present study investigated the antinociceptive actions of a novel and selective AK inhibitor, A-134974 (IC(50)=60 pM), in a rat model of neuropathic pain (ligations of the L5/L6 spinal nerves) and explored the relative contributions of supraspinal, spinal and peripheral sites to the actions of A-134974. Systemic A-134974 dose-dependently reduced tactile allodynia (ED(50)=5 micromol/kg, i.p.) for up to 2 h. Fall latencies in the rotorod test of motor coordination were unaffected by systemic administration of A-134974 (at doses up to 30 micromol/kg, i.p.). Administration of A-134974 intrathecally (i.t.) was more potent (ED(50)=10 nmol) in relieving tactile allodynia than delivering the compound by intracerebroventricular (ED(50)>100 nmol, i.c.v.) or intraplantar (ED(50)>500 nmol) routes suggesting that spinal sites of action are the primary contributors to the anti-allodynic action of A-134974. The anti-allodynic effects of systemic A-134974 (10 micromol/kg, i.p.) were antagonized by the non-selective ADO receptor antagonist, theophylline (30-500 nmol) administered i.t. These data demonstrate that the novel AK inhibitor A-134974 potently reduces tactile allodynia through interactions with spinal sites and adds to the growing evidence that AK inhibitors may be useful as analgesic agents in a broad spectrum of pain states.

Dissociation of nitrovasodilator-induced relaxation from cyclic GMP levels during in vitro nitrate tolerance.

The effects of nitroglycerin tolerance on relaxation and cyclic GMP accumulation by nitroglycerin, nitric oxide and S-nitroso-N-acetylpenicillamine were examined in isolated rate aortic rings. Cyclic GMP accumulation by nitroglycerin, S-nitroso-N-acetylpenicillamine and nitric oxide was diminished in nitroglycerin-tolerant aorta. In contrast, only relaxation by nitroglycerin, but not S-nitroso-N-acetylpenicillamine and nitric oxide, was attenuated. These data suggest that cyclic GMP levels might represent an inadequate index for mechanistic studies of nitroglycerin relaxation tolerance.

Tolerance to relaxation in rat aorta: comparison of an S-nitrosothiol with nitroglycerin.

Tolerance and cross-tolerance to the relaxant effects of S-nitroso-N-acetylpenicillamine (SNAP) and nitroglycerin were examined in rat aortic rings. Nitroglycerin-tolerant rings remained fully responsive to SNAP and sodium nitroprusside. Thus, reduced metabolic activation of nitroglycerin, rather than impaired guanylate cyclase activity, appeared to be the operating mechanism for nitrate tolerance in this preparation. Under similar conditions, SNAP incubation induced less tolerance than nitroglycerin. S-nitrosothiols, therefore, appear to be less tolerance-producing than nitroglycerin.

An adenosine kinase inhibitor attenuates tactile allodynia in a rat model of diabetic neuropathic pain.

The present study was conducted to characterize the development of tactile allodynia in the streptozotocin-induced rat model of diabetes, and to evaluate the antinociceptive effects of systemically administered morphine and the adenosine kinase inhibitor, 5'-deoxy-5-iodotubercidin (5'd-5IT) in this model. Rats were injected with 75 mg/kg streptozotocin (i.p.), and blood glucose levels were determined 3-4 weeks later. Diabetic (blood glucose levels > or = 250 mg/dl) and vehicle-injected rats were examined weekly for the development of tactile allodynia by measuring the threshold for hind paw withdrawal using von Frey hairs. Withdrawal thresholds were reduced to 6.8+/-0.6 g (mean+/-S.E.M.) in approximately one-third of streptozotocin-treated rats 7 weeks after streptozotocin treatment as compared to control thresholds (13.2+/-0.1 g), and this allodynia persisted for at least an additional 7 weeks. In additional experiments, morphine sulfate (5-21 micromol/kg, i.p.) produced dose-dependent antinociceptive effects on tactile allodynia for up to 2 h post-dosing. The adenosine kinase inhibitor, 5'd-5IT (2.5 and 5 micromol/kg, i.p.) also dose-dependently attenuated tactile allodynia. Pretreatment with the opioid receptor antagonist, naloxone (27 micromol/kg, i.p.) or the non-selective adenosine receptor antagonist, theophylline (111 micromol/kg, i.p.) significantly diminished the anti-allodynic effects of morphine and 5'd-5IT, respectively. The present study demonstrates that the potent and selective adenosine kinase inhibitor, 5'd-5IT, is equally effective as morphine in blocking tactile allodynia in this model.

Adenosine kinase inhibition protects brain against transient focal ischemia in rats.

Endogenous adenosine released locally during cerebral ischemia is neuroprotective, and agents which decrease adenosine inactivation may potentiate its protective effects. The effects of 5'-deoxy-5-iodotubercidin (5'd-5IT), an inhibitor of the adenosine-catabolizing enzyme, adenosine kinase, were studied in male Wistar rats subjected to 2 h of transient middle cerebral artery occlusion. 5'd-5IT or the vehicle (10% DMSO in saline) was administered i.p. 30 min before, and 2 h and 6 h after the induction of middle cerebral artery occlusion. The infarct volume was determine using 2,3,5-triphenyltetrazolium chloride staining 48 h after middle cerebral artery occlusion. The infarct volume was significantly reduced in rats treated with 1.85 mg/kg x 3 (57% reduction, P < 0.001) or 1.0 mg/kg x 3 (34% reduction, P < 0.05), but not 0.3 mg/kg x 3 5'd-5IT compared to vehicle-treated rats. The reduction of infarct volume was accompanied by a significant improvement in behavioral measures of neurological deficit. These data further support a role of adenosine in neuroprotection and suggest that adenosine kinase inhibition may be a useful approach to the treatment of focal cerebral ischemia.

ABT-627, an endothelin ET(A) receptor-selective antagonist, attenuates tactile allodynia in a diabetic rat model of neuropathic pain.

Tactile allodynia, the enhanced perception of pain in response to normally non-painful stimulation, represents a common complication of diabetic neuropathy. The activation of endothelin ET(A) receptors has been implicated in diabetes-induced reductions in peripheral neurovascularization and concomitant endoneurial hypoxia. Endothelin receptor activation has also been shown to alter the peripheral and central processing of nociceptive information. The present study was conducted to evaluate the antinociceptive effects of the novel endothelin ET(A) receptor-selective antagonist, 2R-(4-methoxyphenyl)-4S-(1,3-benzodioxol-5-yl)-1-(N, N-di(n-butyl)aminocarbonyl-methyl)-pyrrolidine-3R-carboxylic acid (ABT-627), in the streptozotocin-induced diabetic rat model of neuropathic pain. Rats were injected with 75 mg/kg streptozotocin (i. p.), and drug effects were assessed 8-12 weeks following streptozotocin treatment to allow for stabilization of blood glucose levels (>/=240 mg/dl) and tactile allodynia thresholds (

Pharmacological characterization of recombinant human and rat P2X receptor subtypes.

ATP functions as a fast neurotransmitter through the specific activation of a family of ligand-gated ion channels termed P2X receptors. In this report, six distinct recombinant P2X receptor subtypes were pharmacologically characterized in a heterologous expression system devoid of endogenous P2 receptor activity. cDNAs encoding four human P2X receptor subtypes (hP2X1, hP2X3, hP2X4, and hP2X7), and two rat P2X receptor subtypes (rP2X2 and rP2X3), were stably expressed in 1321N1 human astrocytoma cells. Furthermore, the rP2X2 and rP2X3 receptor subtypes were co-expressed in these same cells to form heteromultimeric receptors. Pharmacological profiles were determined for each receptor subtype, based on the activity of putative P2 ligands to stimulate Ca2+ influx. The observed potency and kinetics of each response was receptor subtype-specific and correlated with their respective electrophysiological properties. Each receptor subtype exhibited a distinct pharmacological profile, based on its respective sensitivity to nucleotide analogs, diadenosine polyphosphates and putative P2 receptor antagonists. Alphabeta-methylene ATP (alphabeta-meATP), a putative P2X receptor-selective agonist, was found to exhibit potent agonist activity only at the hP2X1, hP2X3 and rP2X3 receptor subtypes. Benzoylbenzoic ATP (BzATP, 2' and 3' mixed isomers), which has been reported to act as a P2X7 receptor-selective agonist, was least active at the rat and human P2X7 receptors, but was a potent (nM) agonist at hP2X1, rP2X3 and hP2X3 receptors. These data comprise a systematic examination of the functional pharmacology of P2X receptor activation.

Inhibition of adenosine kinase during oxygen-glucose deprivation in rat cortical neuronal cultures.

Adenosine kinase (AK) inhibitors potentiate the actions of endogenous adenosine (ADO) and ameliorate cerebral ischemic damage in animal models. The present study examined the effects of the AK inhibitor, 5-iodotubercidin (5-IT) in an in vitro model of neuronal ischemia, specifically, combined oxygen-glucose deprivation of rat cortical mixed neuronal-glial cultures. Oxygen-glucose deprivation caused extensive neuronal loss which was accompanied by a marked increase in ADO release into the extracellular medium, was ameliorated by exogenous ADO (10 microM(-1) mM), and was exacerbated by a high concentration of the selective A1 receptor antagonist 8-cyclopentyl-1,3-dimethylxanthine (CPT; 10 microM). 5-IT (1 microM) had no effect on extracellular ADO levels nor on neuronal loss. However, AK activity in these cultures was markedly suppressed during oxygen-glucose deprivation. Taken together, these data demonstrate a marked down-regulation of AK activity during oxygen-glucose deprivation in this in vitro model, providing an endogenous mechanism contributing to the accumulation of extracellular ADO, which exerts neuroprotective effects by activating the ADO A1 receptor.

Site and event specific increase of striatal adenosine release by adenosine kinase inhibition in rats.

The effects of the systemically administered adenosine kinase (AK) inhibitor, 5'-deoxy-5-iodotubercidin (5'd-5IT) on the striatal adenosine (ADO) release evoked by the excitotoxin, kainic acid (KA) were examined using rat bilateral striatal microdialysis. Local KA perfusion of one rat striatum caused a significant ipsilateral elevation of striatal ADO levels compared to basal and contralateral (artificial CSF-perfused) striatal ADO levels. KA-evoked striatal ADO release was augmented in animals receiving systemic 5'd-5IT treatment (cumulative dose of 7.5 micromol/kg, i.p.) compared with i.p. vehicle controls. In contrast, 5'd-5IT administration had no significant effect on basal or contralateral (artificial CSF-perfused) striatal ADO levels. Thus, consistent with the hypothesis of 'site and event specific' potentiation of ADO by AK inhibitors, 5'd-5IT unilaterally enhanced ADO levels in the striatum where KA-induced excitotoxic injury evoked endogenous ADO release, but not at the contralateral uninjured striatum.

Effect of intermittent exposure and drug-free intervals on the in vitro vascular tolerance to nitroglycerin.

Recent reports suggest that intermittent nitroglycerin (NTG) treatment, incorporating a daily nitrate-free interval, can avoid the tolerance associated with continuous NTG therapy. This study has investigated whether an in vitro model of NTG tolerance could be used to examine the mechanisms of tolerance avoidance (by intermittent NTG exposure) and tolerance reversal. Isolated rat abdominal aortic rings were exposed to 55 microM NTG at varying intervals over a 60 min period, and the concentration-relaxation curves to NTG were subsequently determined. Intermittent NTG exposure (either 12 x 0.5 min or 3 x 2 min) significantly reduced NTG tolerance compared to continuous exposure over the same period of time (60 min). The diminished tolerance was apparently due to the reduced total exposure time, since the NTG responsiveness of aortic rings exposed to NTG intermittently or continuously for 6 min of the incubation period was not significantly different. Under the present conditions, in vitro NTG tolerance could be reversed if sufficient washout time was allowed. Thus, aortic rings exposed to NTG for the initial, but not the final, 6 min of incubation were not tolerant to NTG. In addition, rings exposed to NTG for 3 min exhibited near-maximal tolerance after 5 min washout, but no tolerance after 60 or 120 min washout. It appears, therefore, that the isolated vessel retains the "repair" mechanism responsible for tolerance reversal under the present conditions. This study suggests that the in vitro model of NTG tolerance may be useful for investigating the characteristics and mechanisms of tolerance avoidance and reversal, as well as tolerance induction.

Dynamics of clomethiazole edisylate interaction with plastic infusion systems.

The dynamics of the interaction of clomethiazole edisylate (1) with polyvinyl chloride and cellulose propionate, the main plastics used in the manufacture of infusion bags and sets, was examined. An experimental system in which the plastic was either open or closed to the environment was used to determine the relative contribution of the sorption and permeation processes to loss from solutions of clomethiazole edisylate (I) in contact with the plastic infusion systems. Sorption by the plastic infusion materials accounted for most of the drug loss, while permeation into the external environment accounted for the remainder. The sorption and permeation into and through polyvinyl chloride was temperature dependent. The diffusion coefficient and permeation rate constant both increased with temperature, while the polyvinyl chloride-water partition coefficients were independent of temperature. The activation energy for the diffusion in polyvinyl chloride was 13.5 kcal/mol. The permeability of the infusion bag plastic and the evaporation across an unstirred air boundary layer adjacent to the external surface of the plastic both appeared to contribute to the overall diffusional resistance encountered in the permeation process. The plastic-water partition coefficients are independent of initial concentration, suggesting that the concentration-dependent loss of the drug from solutions stored in plastic infusion bags and burets is a result of the greater diffusivity of the drug in the plastic at the higher initial concentrations. Plasticization of the polymers by the drug is indicated by the increase in the diffusivity of the drug in polyvinyl chloride and cellulose propionate, the increase in the rate and extent of sorption of a radiolabeled marker (diazepam) by the plastic, and the decreased stiffness of polyvinyl chloride exposed to higher concentrations of the drug.