Brincidofovir treatment of acyclovir-resistant disseminated varicella zoster virus infection in an immunocompromised host.

Brincidofovir (BCV) is a broad-spectrum antiviral agent active in vitro against double-stranded DNA viruses including herpesviruses, adenoviruses, polyomaviruses, and poxviruses. We report successful BCV use in management of disseminated acyclovir- and cidofovir-resistant varicella zoster virus in an immunocompromised hematopoietic stem cell transplant patient with chronic graft-versus-host disease who was intolerant to foscarnet.

International, open-label, noncomparative, clinical trial of micafungin alone and in combination for treatment of newly diagnosed and refractory candidemia.

Candida spp. are the fourth leading cause of bloodstream infections, and non-albicans species are increasing in importance. Micafungin is a new echinocandin antifungal agent with excellent in vitro activity against Candida spp. Pediatric, neonatal, and adult patients with new or refractory candidemia were enrolled into this open-label, noncomparative, international study. The initial dose of micafungin was 50 mg/d (1 mg/kg for patients <40 kg) for infections due to C. albicans and 100 mg/d (2 mg/kg for patients <40 kg) for infections due to other species. Dose escalation was allowed. Maximum length of therapy was 42 days. A total of 126 patients were evaluable (received at least five doses of micafungin). Success (complete or partial response) was seen in 83.3% patients overall. Success rates for treatment of infections caused by the most common Candida spp. were as follows: C. albicans 85.1%, C. glabrata 93.8%, C. parapsilosis 86.4%, and C. tropicalis 83.3%. Serious adverse events related to micafungin were uncommon. Micafungin shows promise as a safe and effective agent for the treatment of newly diagnosed and refractory cases of candidemia. Large-scale, randomized, controlled trials are warranted.

Clinical and magnetic resonance imaging regression of progressive multifocal leukoencephalopathy in an AIDS patient after intensive antiretroviral therapy.

A 36-year-old homosexual man with 6 months of visual symptoms and headaches had right homonymous hemianopia, mild new learning impairment, and alexia with agraphia. The initial brain magnetic resonance imaging (MRI) scan was reported consistent with left occipital infarction. Subsequent MRI demonstrated abnormal demyelination in subcortical white matter and deep parieto-occipital white matter bilaterally, but primarily left. Human immunodeficiency virus testing and cerebrospinal fluid polymerase chain reaction for JC virus DNA were both positive, consistent with progressive multifocal leukoencephalopathy (PML) with AIDS. His clinical status steadily deteriorated, and MRI white matter abnormalities worsened despite high-dose antiretroviral therapy. After the antiretroviral regimen was intensified by the addition of a protease inhibitor, rapid clinical and radiographic improvement occurred with subsequent MRI studies revealing only residual left parieto-occipital encephalomalacia. PML in AIDS patients has been associated with a nearly uniformly poor prognosis until recent reports of improved outcomes after highly active antiretroviral therapy. This patient with PML and AIDS similarly showed a robust clinical and MRI response to intensive antiretroviral combination therapy, which has been maintained for more than 3 years.

Verbal working memory in HIV-seropositive drug users.

Recent evidence suggests that HIV-seropositive drug users are impaired on tasks of visuospatial working memory compared with drug users seronegative for HIV. In the current study we evaluated the performance of 30 HIV-seropositive male drug users and 30 risk-matched seronegative controls on two measures of verbal working memory, the Listening Span and the verbal Self Ordered Pointing Task. Impaired working memory performance was significantly more common among HIV-seropositive persons compared to controls, with the highest incidence of deficit among symptomatic participants. These findings indicate that working memory deficits in persons with HIV are not domain-specific and can be demonstrated reliably in drug users.

Reaction times are faster in HIV-seropositive patients on antiretroviral therapy: A preliminary report.

We evaluated subclinical mental and motor slowing in 142 HIV-seropositive patients without dementia, using computerized simple and choice reaction time tasks and self-report measures of psychological distress. Patients on antiretroviral therapy at the time of testing (n = 79) had significantly faster choice reaction times (p < 0.05), indicating faster mental processing speed, than untreated patients (n = 63). These faster RTs could not be attributed to differences in age, education, risk factors, degree of immunosuppression, substance abuse history, peripheral neuropathy, or psychological distress. Reaction time tasks should be investigated further as potential outcome measures in clinical trials, particularly for subjects with few or no overt cognitive deficits.

Impaired phagocyte oxidative capacity in patients with human immunodeficiency virus infection.

Bacterial infections that may be related to impaired phagocyte function often develop in patients infected with human immunodeficiency virus (HIV). We examined the oxidative capacity of circulating phagocytes in 78 HIV+ patients and 31 control subjects by measuring chemiluminescence with a whole blood assay. Phagocytes were stimulated with zymosan opsonized with human complement (hC-OPZ) or immunoglobulin (hI-OPZ) with or without exogenous primers. Patients with CD4+ < 500/microL showed reduced whole blood chemiluminescence at maximal opsonin receptor (MOR) activity after priming in response to hC-OPZ relative to control subjects, and the difference was significant for patients with CD4+ < 100/microL. Patients had lower absolute phagocyte counts; however, the chemiluminescence activity calculated per phagocyte count was significantly depressed in advanced HIV infection, indicating the impairment of phagocytic cell function and a reduction in number. Data were similar when hI-OPZ was used as a stimulus. The chemiluminescence of unprimed phagocytes at circulating opsonin receptor (COR) activity relative to maximally primed phagocytes (COR/MOR ratio) was significantly higher for HIV+ patients as compared with control subjects and indicates a defect in phagocyte priming. Alternatively, the phagocytes do not increase chemiluminescence with priming because they have already been primed or activated in vivo. In late-stage disease, decreased opsonin receptor-dependent respiratory burst activity contributes to the risk of secondary bacterial infections.

Information processing and antiretroviral therapy in HIV-1 infection.

Computerized reaction time (RT) tasks are sensitive measures of subclinical HIV-related mental slowing. We previously reported that nondemented HIV-seropositive patients on antiretroviral therapy at the time of testing had faster choice RTs compared to matched untreated seropositive participants. In the present study, we evaluated the performance of 163 nondemented HIV-seropositive participants on a reaction time version of the Stroop task as a function of antiretroviral status. Persons on antiretroviral therapy at the time of testing had significantly faster reaction times than untreated individuals, although treated asymptomatic participants showed significantly less Stroop interference than treated symptomatic participants. These effects could not be attributed to differences in demographic variables, disease status, substance abuse, or psychological distress. These data indicate that central information processing is faster for patients treated with antiretroviral compounds compared to untreated patients, and suggest that reaction time tasks may have significant potential utility in clinical trials of neuroprotective compounds.

Working memory deficits in HIV-seropositive drug users.

We studied the integrity of working memory operations in 38 HIV-seropositive and 20 seronegative drug users, using a modified version of the Tower of London task. This new task, the Tower of London-Working Memory version (TOL-WM), includes a delayed-response component in addition to the planning required for successful performance of the standard TOL. Symptomatic HIV-seropositive participants solved significantly fewer TOL-WM problems compared to matched seronegative controls. However, seropositive and seronegative subjects showed similar overall levels of planning efficiency, suggesting that the TOL-WM deficit may be associated primarily with failure to encode or maintain an adequate online memory representation. The results of this study confirm our previous report of a possible working memory deficit in HIV-1 infection and suggest that measures of working memory have particular utility in the evaluation of HIV-related cognitive deficits.

Cardioprotection with a novel adenosine regulating agent mediated by intravascular adenosine.

Adenosine is cardioprotective in models of myocardial stunning and infarction, but the precise compartment within the heart in which adenosine elicits its cardioprotective effects has not been determined. The goals of the present study were to (i) investigate the effects of a novel adenosine regulating agent, GP531 (5-amino-1-beta-n-(5-benzylamino-5-deoxyribofuranosyl) imidazole-4-carboxamide), on post-ischemic myocardial function, and (ii) examine the contribution of endogenous adenosine in the intravascular and interstitial compartments in mediating the beneficial effects. Pigs were instrumented for measurement of myocardial segment shortening, and for sampling of coronary venous blood and myocardial interstitial fluid for determination of adenosine concentration. Myocardial dysfunction was induced by 4 x 8 min coronary occlusions, and recovery of regional function was monitored for 2 h. In control pigs, function recovered to 24 +/- 2% of baseline after 2 h. Treatment with GP531 improved functional recovery to 55 +/- 3%. GP531-mediated cardioprotection was prevented by adenosine receptor blockade with 8-sulfophenyltheophylline (23 +/- 2%). GP531 did not affect basal adenosine levels, but caused a 2-fold greater increase in vascular adenosine concentration with ischemia (54.6 +/- 10.6 vs. 28.1 +/- 8.0 microM in controls. P < 0.05). In contrast, the interstitial adenosine concentration was not significantly different in treated vs. untreated control pigs (9.4 +/- 3.9 vs. 15.0 +/- 1.8 microM in controls). These data indicate that (1) GP531 improves recovery of myocardial function following ischemia reperfusion injury via an adenosine receptor-dependent mechanism, and (2) the cardioprotection is associated with increased intravascular, but not interstitial, adenosine concentration during ischemia. Therefore, we conclude that cardioprotection elicited by GP531-enhanced endogenous adenosine is dependent on an intravascular site of action.

Dual activation of adenosine A1 and A3 receptors mediates preconditioning of isolated cardiac myocytes.

Ischemic preconditioning reduces post-ischemic myocardial injury by activating myocellular adenosine A1 receptors. Adenosine A3 receptors have also been implicated but there is no evidence for A3 receptors in cardiac myocytes. The aim of this study was to develop a model of preconditioning in isolated cardiac myocytes to evaluate the role of the adenosine A1 and A3 receptors in preconditioning-induced protection from ischemic injury. Reverse transcription polymerase chain reaction (PCR) was also employed to establish the presence of adenosine A3 receptors in these cells. In the preconditioning studies, ischemic injury was simulated by exposing isolated rabbit myocytes (placed in the cell chamber and paced at l Hz) to buffer containing (in mM) 2'-deoxyglucose (20), NaCN (1), Na (+)-lactate (20), KCl (10) at pH 6.6 (37 degrees C). Changes of diastolic and systolic cell length were monitored with an optical-video edge imaging system, and hypercontracture was assessed as an index of irreversible cell injury. Preconditioning (2 min brief ischemia and 15 min reperfusion) significantly reduced cell injury resulting from a subsequent prolonged ischemia (10 min) and reperfusion (15 min), as indicated by a reduction in the incidence of cell hypercontracture from 67 +/- 6% to 29 +/- 5% (P < 0.001). Preconditioning-induced cardioprotection was only partially blocked by a maximally effective concentration (100 nM) of the adenosine A1 receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (cell hypercontracture = 43 +/- 3%, P < 0.05 vs. control) but completely blocked by either the combination of DPCPX (100 nM) with the adenosine A1/A3 receptor antagonist DPCPX +8-(4-carboxyethylphenyl)-1,3-dipropylxanthine (BWA1433; 1 microM) or the non-selective adenosine receptor antagonist, 8-(p-sulfophenyl)theophylline (8-SPT; 100 microM) (cell hypercontracture = 64 +/- 4%, 59 +/- 5%, respectively; P = NS vs. control). In non-hypercontractured myocytes, preconditioning also substantially enhanced the recovery of the contractile amplitude and, similarly, this effect was only partially blocked by DPCPX but completely blocked by either the combination of DPCPX with BWA1433, or 8-SPT. These studies suggest that preconditioning protects isolated cardiac myocytes from ischemic injury independent of other cell types, and that maximal preconditioning-induced cardioprotection requires activation of both adenosine A1 and A3 receptors. Reverse transcription-PCR using primers for the rabbit receptor provide evidence for the presence of adenosine A3 receptors in these cells.

Accelerated neutrophil apoptosis in the acquired immunodeficiency syndrome.

Neutrophil (PMNL) function defects occur as a consequence of HIV infection. This study examined PMNL apoptosis in patients with the acquired immunodeficiency syndrome (AIDS) to determine if accelerated apoptosis contributes to impaired function. PMNL were isolated from 10 HIV-infected patients with CD4+ lymphocyte counts < 200/mm3 without signs of active infection and 7 healthy volunteers. PMNL were stained with acridine orange and ethidium bromide after 0, 3, 6, and 18 h in culture, and examined for the morphologic changes of apoptosis and viability by fluorescent microscopy. Apoptosis was also demonstrated by electron microscopy, flow cytometry, and DNA gel electrophoresis. Apoptosis was minimal at 0 h, but PMNL from AIDS patients exhibited significantly greater apoptosis than controls at 3 h (22.5+/-11.5 vs. 8.9+/-6.9%, P = 0.015), 6 h (38.1+/-14.2 vs. 18.1+/-4.5%, P = 0.003), and 18 h (71.3+/-19.0 vs. 38.8+/-16.7%, P = 0.002). Viabilities were > or = 88.0% for both groups from 0-6 h, but by 18 h viability was significantly decreased for the HIV group (58.8+/-12.4 vs. 83.5+/-10.4%, P = 0.001) due to an increase in non-viable apoptotic cells. Incubation with serum from AIDS patients had no effect on control PMNL, and incubation with control serum did not reduce the rate of apoptosis of PMNL from AIDS patients. Incubation with granulocyte colony-stimulating factor (G-CSF) in vitro significantly decreased apoptosis for PMNL from AIDS patients. PMNL from patients with AIDS exhibit markedly accelerated apoptosis ex vivo. In vivo, apoptosis and functional impairment of PMNL may contribute to the risk of secondary infections, and cytokine therapy may be of potential clinical benefit in this circumstance.

Carbohydrate- and CD18-dependent neutrophil adhesion to cardiac myocytes: effects of adenosine.

OBJECTIVE: Adenosine inhibits neutrophil adhesion and injury to isolated cardiac myocytes. In the present study, the contribution of selectin and CD18 interactions to neutrophil-myocyte adhesion and their sensitivity to adenosine were assessed. METHODS: Activated human neutrophils and canine myocytes were incubated with inhibitors of CD18 or selectin binding, adenosine, or combinations of both for 30-50 min at 37 degrees C. Neutrophils were pretreated with 0.1 microM fMLP for 10 min to study L-selectin-independent adhesion. Adhesion was measured by phase contrast microscopy. RESULTS: Anti-L-selectin mAb and the selectin-blocking carbohydrates sialyl Lewisx or mannose-6-phosphate, as well as anti-CD18 or anti-ICAM-1 mAbs, inhibited cell adhesion (by 84-99%, P < 0.05). CD11a, but not CD11b, was responsible for most of the CD18-mediated binding. An L-selectin-independent interaction between neutrophils and cardiac myocytes was observed that was delayed (peak adhesion at 40-50 min, rather than 30 min), but still inhibited by anti-CD18 mAb (by 65 +/- 11%, P < 0.05) and carbohydrates (by 87-97%, each P < 0.05). Adenosine (100 nM) inhibited this late CD18-dependent/L-selectin-independent phase of adhesion (by 61 +/- 14%, P < 0.05). The combination of adenosine and anti-CD18 mAb was additive such that adhesion was completely blocked (P < 0.05, compared to either agent alone). Inhibition of adhesion by adenosine was prevented by the A2 antagonist, DMPX (100 nM), and mimicked by the A2 agonist, CGS-21680 (10 nM) or the adenosine regulating agents, acadesine (100 microM) or GP531 (10 microM). CONCLUSION: Neutrophil-myocyte adhesion involved both L-selectin-dependent and L-selectin-independent carbohydrate binding as well as CD11a/CD18. Inhibition of adhesion by adenosine interferes with L-selectin-independent carbohydrate binding and possibly CD18.

Delayed recognition memory span in HIV-1 infection.

We administered a spatial version of the Delayed Recognition Span Test (DRST), a working memory task performed abnormally by patients with basal ganglia disease, to a group of 96 HIV-seropositive and 83 seronegative subjects with a high prevalence of substance abuse. For comparison purposes, we also administered the Symbol-Digit Modalities Test (SDMT) and the Trail Making Test (TMT), measures which detect HIV-related mental slowing efficiently in gay men but are nonspecifically impaired in subjects with a history of substance abuse. As predicted, scores on the TMT and the SDMT did not discriminate the groups, but HIV-seropositive subjects had significantly shorter spatial spans (p < .007) and DRST total scores (p < .005). These effects could not be attributed to differences in age, education, estimated intelligence, or psychological distress, because the groups were well matched on these variables. The DRST is a promising measure of HIV-related cognitive dysfunction in substance abusers, who are often nonspecifically impaired on psychomotor tasks. These preliminary data also indicate that working memory function should be studied further in HIV-seropositive subjects.

Adenosine activates A2 receptors to inhibit neutrophil adhesion and injury to isolated cardiac myocytes.

Inhibition of neutrophil-myocyte adhesion and adhesion-dependent myocyte injury by adenosine was evaluated using isolated TNF-alpha-activated canine cells. Adenosine inhibited adhesion of activated neutrophils to cardiac myocytes with an IC50 of 11 +/- 4 nM. Inhibition of neutrophil adhesion (92 +/-3% by 100 nM adenosine) led to inhibition of myocyte injury (by 90 +/- 6%, as assessed by dye exclusion). Inhibition of cell adhesion by adenosine was blocked by the A2 antagonist, 1,3-dimethyl-1-propylxanthine, but not by the A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine. Moreover, the A2 agonist, CGS21680 (2-[4-(2-carboxymethyl)phenethylamino]-5'-N-ethylcarboxamido adenosine), but not the A1 agonist, N6-cyclopentyladenosine, mimicked adenosine in preventing cell adhesion. These observations implicate the A2 receptor in the mechanism of inhibition of cell adhesion. pretreatment and washing of neutrophils, but not cardiac myocytes, with adenosine or CGS21680 led to inhibition of adhesion, suggesting that the neutrophil A2 receptor is the target of adenosine's action. In contrast, inhibition of cell adhesion by adenosine was poteniated by 8-cyclopentyl-1,3-dipropylxanthine (IC50 = 4 +/- 1 nM) and attenuated by N6-cyclopentyladenosine, suggesting that occupancy of A1 receptors can conversely increase cell adhesion. Neutrophil-myocyte adhesion was inhibited by acadesine (IC50 = 12 +/- 2 microM) also via an adenosine-dependent mechanism because it was blocked by 1,3-dimethyl-1-propylxanthine or adenosine deaminase, an enzyme that degrades any adenosine that is formed. Acadesine-induced inhibition if cell adhesion (83 +/- 4% by 100 microM) resulted in inhibition of myocyte injury (by 76 +/- 6%). Other adenosine-regulating agents, including the acadesine analogue, GP531 (5-amino-1 beta-D-(5-benzylamino-5-deoxyribofuranosyl) imidazole-4-carboxamide), and inhibitors of adenosine transport and intracellular metabolism also inhibited cell adhesion. These results indicate that exogenous or endogenous adenosine can inhibit neutrophil-myocyte adhesion and injury in cells activated with TNF-alpha by an A2-mediated mechanism. Although the predominant activity of adenosine is to attenuate cell adhesion, stimulation of A1 receptors has the opposite effect, i.e., to augment adhesive interactions.

Protection against injury during ischemia and reperfusion by acadesine derivatives GP-1-468 and GP-1-668. Studies in the transplanted rat heart.

BACKGROUND: Acadesine (AICAr: 5-amino-4-imidazole carboxamide riboside) has been shown to afford sustained protection against injury during ischemia and reperfusion. The present studies used the heterotopically transplanted rat heart to assess the protective properties of two new acadesine analogs: GP-1-468 and GP-1-668. METHODS AND RESULTS: Hearts were excised, arrested with a 2-minute infusion of cardioplegic solution, and subjected to 4 hours of global ischemia (20 degrees C) with cardioplegic reinfusion for 2 minutes every 30 minutes. The hearts were then transplanted (1 hour of additional ischemia) into the abdomens of recipient rats and reperfused in situ for 30 minutes or 24 hours. The hearts were then excised, perfused aerobically for 20 minutes, and contractile function was assessed. GP-1-468 or GP-1-668 was administered to donor rats (20 mg/kg intravenously, 30 minutes before excision). They were also added to the cardioplegic solution (10 mumol/L for GP-1-468, 5 mumol/L for GP-1-343, the active metabolite of GP-1-668) and were also given to recipient rats (20 mg/kg intravenously, 30 minutes before transplantation, so that the drugs were present during reperfusion). Nine groups of hearts were studied. Three groups of studies were carried out (n = 24 transplants for each group). The first group of hearts was reperfused for 30 minutes, the second group was reperfused for 24 hours, and the third group was transplanted but not reperfused; instead, they were frozen at the end of 5 hours of ischemia and taken for metabolite analysis. Within each group were three subgroups (n = 8 per group) receiving GP-1-468, GP-1-668, or saline solution. In the 30-minute reperfusion group the recoveries of left ventricular developed pressure were 88 +/- 4, 87 +/- 7, and 50 +/- 9 mm Hg, respectively (p < 0.05 versus saline-treated controls); left ventricular volumes (recorded at 12 mm Hg) were 112 +/- 20, 132 +/- 28, and 41 +/- 9 microliters, respectively (p < 0.05 versus saline-treated controls), and coronary flows were 13.1 +/- 0.7, 13.4 +/- 1.0, and 9.9 +/- 0.5 ml/min, respectively (p < 0.05 versus saline-treated controls). In addition to improving functional recovery, the two analogs increased the tissue content of adenosine at the end of the ischemic period (5.4 +/- 0.6 and 7.3 +/- 0.5 mumol/gm dry weight, respectively, versus 2.7 +/- 0.4 mumol/gm dry weight in the saline-treated controls; p < 0.05); however, they did not influence adenosine triphosphate or its catabolites. In the 24-hour reperfusion group the corresponding values were 77 +/- 6 and 88 +/- 6 versus 35 +/- 4 mm Hg for left ventricular developed pressure (p < 0.05), 111 +/- 9 and 121 +/- 11 versus 41 +/- 8 microliters for left ventricular volume (p < 0.05), and 13.7 +/- 0.7 and 13.0 +/- 0.6 versus 11.7 +/- 0.7 ml/min for coronary flow (no significant difference). Thus both analogs afforded an early and comparable degree of protection of contractile function that was sustained even after 24 hours of reperfusion. CONCLUSIONS: Both GP-1-468 and GP-1-668 increase the rate and extent of early postischemic recovery, and this protection is sustained for at least 24 hours. These beneficial actions were associated with an increase of the tissue content of adenosine during ischemia, but they appeared to be independent of the status of the high-energy metabolism.

Acadesine extends the window of protection afforded by ischaemic preconditioning in conscious rabbits.

OBJECTIVE: Ischaemic preconditioning protects myocardium from infarction if the reperfusion interval between the brief and prolonged ischaemic intervals is less than 1 h. In anaesthetised rabbits acadesine (5-amino-4-imidazolecarboxamide riboside, AICAR), an adenosine enhancer which increases tissue adenosine during ischaemia, prolongs the window of protection to 2 h. The aim of this study was to try to determine the maximum extension of this window of protection, using chronically instrumented, unsedated rabbits. METHODS: Rabbits were instrumented with a balloon occluder around a major branch of the left coronary artery for reversible coronary occlusion. Five to seven days after surgery all animals underwent a 30 min coronary occlusion. Animals were randomised to one of seven groups: (1) No additional treatment (control); (2) Ischaemic preconditioning with 5 min regional ischaemia followed by 10 min reperfusion before the 30 min coronary occlusion; (3) and (4) Ischaemic preconditioning followed by 2 or 4 h of reperfusion before the 30 min occlusion, respectively; (5) Treatment with acadesine (2.5 mg.kg-1.min-1 intravenously for 5 min and then 0.5 mg.kg-1.min-1 beginning 45 min before and continuing until 30 min after release of the 30 min occlusion) without ischaemic preconditioning; (6) and (7) Treatment with the higher dose of acadesine for 5 min beginning 35 min before the 5 min ischaemic period, and then the lower dose continuing until 30 min after release of the 30 min coronary occlusion in rabbits with 4 or 6 h reperfusion intervals, respectively. RESULTS: Rabbits with ischaemic preconditioning with 10 min reperfusion preceding the 30 min coronary occlusion (group 2) had only 5.6(SEM 1.1)% infarction of the ischaemic zone. Ischaemic preconditioning followed by 2 h reperfusion (group 3) offered continued protection [18.2(2.2)% infarction] as compared to control animals [37.7(2.6)% infarction]. However, protection waned if ischaemic preconditioning was followed by 4 h reperfusion (group 4) [36.7(3.0)% infarction]. Additionally, treatment with acadesine alone did not modify infarct size (group 7) [39.5(4.0)%], but acadesine largely restored the protection of ischaemic preconditioning despite a 4 h reperfusion interval (group 5) [20.4(3.0)% infarction, P < 0.01 v control]. However, when reperfusion was extended to 6 h (group 6) acadesine could no longer restore protection [36.2(0.9)% infarction]. CONCLUSIONS: The protection afforded by a 5 min ischaemic preconditioning period lasts from 2 to 4 h in the awake, unsedated rabbit, and acadesine can extend the duration of this window of protection to at least 4 h but not to 6 h.

Inhibition of neutrophil adhesion by adenosine and an adenosine kinase inhibitor. The role of selectins.

Adenosine and adenosine analogues exhibit anti-inflammatory effects in vitro and in vivo, but their usefulness is limited by profound cardiovascular side effects. Therefore, we synthesized inhibitors of an enzyme involved in adenosine metabolism, adenosine kinase (AK) (EC 2.7.1.20), to enhance endogenous adenosine concentrations at sites of inflammation. GP-1-515 (4-amino-1-(5-amino-5-deoxy-1-beta-D- ribofuranosyl)-3-bromo-pyrazolo[3,4-d]pyrimidine), a novel AK inhibitor, decreased adhesion of activated human neutrophils to cultured endothelial cell monolayers by increasing local adenosine levels. The mechanism of inhibition in this assay seemed to involve selectin blockade and was independent of the beta 2 integrins. GP-1-515 and 2-chloroadenosine (a nonmetabolizable adenosine analogue) had no effect on the surface expression or shedding of adhesion molecules. An agent that disrupts the cytoskeleton, cytochalasin B, mimicked the effect of adenosine on cell adhesion. Interactions between L-selectin and the neutrophil cytoskeleton might be altered by adenosine and could contribute to adenosine-mediated adhesion inhibition.

Adenosine-mediated inhibition of platelet aggregation by acadesine. A novel antithrombotic mechanism in vitro and in vivo.

Inhibition of platelet aggregation by acadesine was evaluated both in vitro and ex vivo in human whole blood using impedance aggregometry, as well as in vivo in a canine model of platelet-dependent cyclic coronary flow reductions. In vitro, incubation of acadesine in whole blood inhibited ADP-induced platelet aggregation by 50% at 240 +/- 60 microM. Inhibition of platelet aggregation was time dependent and was prevented by the adenosine kinase inhibitor, 5'-deoxy 5-iodotubercidin, which blocked conversion of acadesine to its 5'-monophosphate, ZMP, and by adenosine deaminase. Acadesine elevated platelet cAMP in whole blood, which was also prevented by adenosine deaminase. In contrast, acadesine had no effect on ADP-induced platelet aggregation or platelet cAMP levels in platelet-rich plasma, but inhibition of aggregation was restored when isolated erythrocytes were incubated with acadesine before reconstitution with platelet-rich plasma. Acadesine (100 mg/kg i.v.) administered to human subjects also inhibited platelet aggregation ex vivo in whole blood. In the canine Folts model of platelet thrombosis, acadesine (0.5 mg/kg per min, i.v.) abolished coronary flow reductions, and this activity was prevented by pretreatment with the adenosine receptor antagonist, 8-sulphophenyltheophylline. These results demonstrate that acadesine exhibits antiplatelet activity in vitro, ex vivo, and in vivo through an adenosine-dependent mechanism. Moreover, the in vitro studies indicate that inhibition of platelet aggregation requires the presence of erythrocytes and metabolism of acadesine to acadesine monophosphate (ZMP).

Protective effect of an adenosine kinase inhibitor in septic shock.

Adenosine exhibits potent anti-inflammatory activities but its therapeutic use is limited by cardiovascular side effects. Inhibitors of an enzyme involved in adenosine metabolism, adenosine kinase (EC 2.7.1.20), were evaluated for their ability to enhance endogenous adenosine production. One novel adenosine kinase inhibitor, GP-1-515, was studied in two models of septic shock to assess its protective effects. GP-1-515 significantly decreased mortality in mice that received a lethal i.v. injection of endotoxin. The beneficial effect was accompanied by decreased neutrophil accumulation in the lungs and was reversed by an adenosine receptor antagonist, implying that the effects were mediated by endogenous adenosine. Plasma levels of TNF-alpha, but not IL-1 alpha or IL-6, were lower in the GP-1-515-treated animals. In a second model of sepsis, GP-1-515 increased survival in bacterial peritonitis in rats. The mechanism of action in both models was likely multifactorial, including adenosine-mediated inhibition of neutrophil adhesion, cytokine production, and oxygen radical generation. Adenosine kinase inhibitors have potent anti-inflammatory effects in vitro and in vivo and represent a novel therapeutic approach to the treatment of inflammatory diseases.