Extension of the therapeutic window for recombinant tissue plasminogen activator with argatroban in a rat model of embolic stroke.

BACKGROUND AND PURPOSE: Argatroban, a specific thrombin inhibitor, has been shown to reduce ischemic lesion size after focal cerebral ischemia in rats. In addition, recombinant tissue plasminogen activator (rtPA) has been shown to reduce ischemic lesion size in both rats and humans if given within 3 hours of symptom onset. We tested the hypothesis that the administration of argatroban with rtPA could extend the treatment window of stroke to 4 hours without increasing gross cerebral hemorrhage rates or reducing efficacy. METHODS: Male Wistar rats were subjected to middle cerebral artery (MCA) occlusion by a single fibrin-rich clot. After embolization, rats were administered argatroban at the following dose levels: 2.08, 6.25, and 18.75 microgram . kg(-1). min(-1). In a second experiment, rats received argatroban (6.25 microgram . kg(-1). min(-1)) or argatroban in combination with rtPA 4 hours after MCA occlusion. Tissue sections were then analyzed for lesion volume, gross hemorrhage and fibrin deposition. RESULTS: The 6.25 microgram. kg(-1). min(-1) dose demonstrated a significant reduction (P<0.05) in lesion volume after 48 hours (27.2+/-6.3%) compared with controls (35.3+/-3.7%). A significant reduction (P<0.05) in lesion volume was observed in the argatroban-plus-rtPA group (17.1+/-10.4%) compared with controls (35.3+/-3.7%). No increase in hemorrhagic transformation was observed. Fibrin deposition in the ipsilateral cortical microvasculature was significantly decreased in the 4-hour combination argatroban-plus-rtPA group compared with the controls (P<0.05). CONCLUSIONS: This study demonstrates that the combination of argatroban and rtPA extends the window of opportunity for treatment of stroke to 4 hours without increasing hemorrhagic transformation.

Inhibition of adhesion of human neutrophils and eosinophils to P-selectin by the sialyl Lewis antagonist TBC1269: preferential activity against neutrophil adhesion in vitro.

BACKGROUND: Leukocyte rolling on vascular endothelium is mediated by selectins and their carbohydrate-containing counterligands. The tetrasaccharide sialyl Lewis(x) (sLe(x)) binds to all 3 selectins, so compounds that mimic sLe(x) are potential antagonists. OBJECTIVE: Our purpose was to examine the ability of the sLe(x) mimetic TBC1269 to inhibit binding of human neutrophils and eosinophils to P-selectin. METHODS: Expression of the primary P-selectin ligand, P-selectin glycoprotein ligand-1 (PSGL-1), was examined on neutrophils and eosinophils, and their adhesion to immobilized P-selectin was examined under both static and dynamic conditions in the presence and absence of TBC1269. RESULTS: Neutrophils and eosinophils expressed PSGL-1, with eosinophils expressing about twice as much as neutrophils. In the absence of TBC1269, both cell types adhered avidly to P-selectin under static and dynamic conditions. For neutrophils, preincubation of P-selectin-coated plates with TBC1269 (1 to 1000 microgram/mL) resulted in concentration-dependent decreases in neutrophil adhesion, with significant inhibition seen at concentrations >/=100 microgram/mL. Eosinophil adhesion to P-selectin was more refractory to inhibition by TBC1269 and was only partially inhibited at the highest concentration tested (1000 microgram/mL). Two structurally related control compounds, TBC1900 and TBC746, had no effect when tested at similar concentrations. CONCLUSION: These data indicate that an sLe(x) mimetic can exhibit cell type-specific differences in potencies with respect to antagonism of P-selectin adhesion. Although this may in part be the result of differences in PSGL-1 expression, the discrepancy in potencies may also be due to other differences, including carbohydrate composition and binding affinity of PSGL-1.

Pharmaceutical use by U.S. astronauts on space shuttle missions.

We evaluated in-flight use of medications from astronaut debriefings after 79 U.S. Space Shuttle missions. From the 219 records obtained (each representing one person-flight), 94% included some medication being taken during flight; of that number, 47% were for space motion sickness, 45% for sleep disturbances, and smaller percentages for headache, backache, and sinus congestion. Drugs were taken most often orally, followed in decreasing order of frequency by intranasal, intramuscular, and rectal routes. Drugs for space motion sickness were taken mostly during the first 2 d of flight, drugs for pain during the first 4 d, and drugs for sleeplessness and sinus congestion were taken consistently for 9 flight days. About 85% of all doses had no reported side effects, and most of the side effects that were reported happened during the first mission day. About 80% of the drug-dose events were perceived effective by the recipients; most of the reports of ineffectiveness occurred during the first mission day. Promethazine, the only drug given by three different routes (orally, intramuscularly, and rectally), was most effective and had minimal side effects when taken intramuscularly. This information, although useful, should be expanded to include objective measures of effectiveness so that therapeutic efficacy can be assessed during flight.

Role of neutrophils and macrophages in experimental nephrosis of the rat.

The single dose administration of the aminonucleoside of puromycin (PAN) induces a nephrotic syndrome in rats characterized by massive proteinuria and progressive histologic changes. This model of acute parenchymal nephritis is thought to be mediated by the renal recruitment of monocytes and macrophages. To investigate the role of leukocytes in the experimental nephrotic syndrome model, the effects of two methylxanthines, pentoxifylline (45 mg/kg i.p. twice daily) and torbafylline (5 mg/kg i.p. twice daily) were compared with controls over a 2-week period. Pentoxifylline treatment was associated with 3- and 6-fold reductions in urinary protein excretion at 7 and 14 days, respectively, compared with PAN-treated control animals (p < .01). Similarly, 14-day dosing of torbafylline resulted in a 3-fold decrease in urinary protein excretion. Glomerular filtration and electrolyte excretion rates were similar between all treatment groups. There were significant reductions in glomerular neutrophil and macro-phage counts, but not T-cells (OX19+) or suppressor/cytotoxic T-cells (0X8+), in kidneys of rats given either treatment compared with PAN con-trol rats. In summary, both pentoxifylline and torbafylline attenuated the proteinuria and glomerular macrophage and neutrophil infiltration associated with PAN administration. These data support the role of macrophages and neutrophils in the pathogenesis of acute parenchymal injury and the potential role of pentoxifylline or related analogues in the attenuation of the ensuing renal deficit associated with minimal lesion disease.

Neutrophil sequestration and pulmonary dysfunction in a canine model of open heart surgery with cardiopulmonary bypass. Evidence for a CD18-dependent mechanism.

BACKGROUND: Previous studies documented an inflammatory reaction to cardiopulmonary bypass with neutrophil (PMN) sequestration in the lungs, contributing to microvascular injury and postoperative pulmonary dysfunction. This study explored the hypothesis that the beta 2 integrin CD18, a leukocyte adhesion molecule, mediates this response. METHODS AND RESULTS: Fifteen adult, mixed-breed dogs underwent 90 minutes of cardiopulmonary bypass with 3 hours of subsequent recovery. Seven additional dogs were treated before cardiopulmonary bypass with a 1-mg/kg IV bolus of R15.7 IgG, a monoclonal antibody to CD18. Both groups were compared with 5 sham bypass control dogs. Bypassed dogs demonstrated an increased number of PMNs sequestered in the lungs 3 hours after bypass compared with sham bypass control dogs (1466 +/- 75 versus 516 +/- 43 PMN/mm2 alveolar surface area, mean +/- SEM, P < .001). Also, when PMNs from bypass dogs were compared with those from sham dogs, they produced more H2O2 (305 +/- 45 versus 144 +/- 48 amol H2O2 per phagocyte per 20 minutes, P < .05). Bypass dogs had significantly decreased arterial oxygenation 3 hours after the procedure compared with shams (457 +/- 20 versus 246 +/- 49 mm Hg, P < .05), and they had a significantly increased lung wet-to-dry weight ratio (5.38 +/- 0.14 versus 4.54 +/- 0.15, P = .003), demonstrating a significant increase in lung water. R15.7 markedly attenuated pulmonary PMN accumulation in bypass dogs (412 +/- 73 PMN/mm2, P < .001) and significantly inhibited PMN production of H2O2 (146 +/- 18 amol H2O2 per phagocyte per 20 minutes, P < .05) Bypass dogs pretreated with R15.7 also had improved oxygenation (445 +/- 28 mm Hg, P < .05) and tended to have less lung water accumulation after bypass (4.99 +/- 0.20). CONCLUSIONS: Pulmonary dysfunction after cardiopulmonary bypass is caused, at least in part, by a neutrophil-mediated, CD18-dependent mechanism.

Neutrophil activation and adhesion molecule expression in a canine model of open heart surgery with cardiopulmonary bypass.

OBJECTIVE: The aim was to determine whether, in a canine model, changes in surface expression of the neutrophil adhesion molecules CD11b/CD18 and L-selectin during and after open heart surgery with cardiopulmonary bypass can be used to identify subjects at risk for postoperative pulmonary dysfunction. METHODS: Adult mixed breed dogs underwent cardiopulmonary bypass and were compared to "sham bypass" controls. Flow cytometry was performed on blood from the two groups of dogs and changes in CD11b/CD18 adhesion molecules and L-selectin were investigated. RESULTS: Flow cytometry on blood from bypass dogs showed increased CD18 expression during and after cardiopulmonary bypass and a reciprocal decrease in L-selectin expression. Sham animals showed no significant change. In the bypass animals, changes in adhesion molecule expression were not evenly distributed across the population of circulating neutrophils; however, they were indicative of a percentage of activated cells. There was a significant negative linear relationship between the percentage of activated cells and arterial oxygenation 3 h after bypass (r = -0.80, P < 0.001). From this analysis, 11 animals were identified as "high" responders and seven as "low" responders, with different patterns of cellular activation and oxygenation during and after bypass. High responders had an average of 40(SEM 5)% activated cells during bypass with a persistently raised percentage of activated cells [38(3)%] 3 h later, whereas low responders had only 22(6)% activated cells during bypass and 11(2)% activated cells 3 h after bypass. High responder animals had a marked and continued deterioration in PO2 after bypass [to 25(6)% of baseline 3 h after bypass] whereas low responder animals showed recovery of oxygenation after the first hour postbypass and improved to 80(8)% of baseline at 3 h. CONCLUSIONS: Changes in adhesion molecule expression serve as a marker of neutrophil activation during cardiopulmonary bypass. The percentage of activated neutrophils in the circulation within 3 h after cardiopulmonary bypass may be predictive of an ongoing inflammatory process that is linked to pulmonary dysfunction.

Induction of intercellular adhesion molecule-1 by lipopolysaccharide in canine alveolar macrophages.

Previous studies have demonstrated that alveolar macrophages (AMphis) adherent to plastic release interleukin-8 in response to lipopolysaccharide (LPS). We sought to determine whether LPS could also alter surface adhesion molecule expression and thus modulate additional AMphi adhesive interactions. Canine AMphis obtained by bronchoalveolar lavage of excised lung were adhered onto tissue culture plastic and exposed to LPS (0.01 to 100 ng/ml). Expression of beta 2 integrins and intercellular adhesion molecule-1 (ICAM-1) was subsequently determined by flow cytometry, a cDNA probe to canine ICAM-1 was used to quantify ICAM-1 mRNA, and changes in adhesion molecule function were assessed by evaluating the extent of homotypic aggregation. ICAM-1 and CD11a/CD18 were present on freshly isolated AMphis. CD11b/CD18 and CD11c/CD18 were expressed at lower levels. Nonadherent AMphis expressed a pattern of beta 2 integrin and ICAM-1 comparable to adherent cells. During short-term LPS stimulation (3 h), adherent AMphis increased both the synthesis and expression of ICAM-1. CD18 expression was either decreased or remained unchanged with LPS stimulation. LPS stimulation in vitro (> 0.01 ng/ml) enhanced the homotypic aggregation of adherent AMphis. Aggregation was blocked by monoclonal antibodies to ICAM-1 (CL18/6) and CD11a (R7.1) and CD18 (R15.7). Similar kinetics were found for expression of ICAM-1 and homotypic aggregation, suggesting that up-regulation of ICAM-1 is a major determinant of the LPS-stimulated aggregation of AMphis.

Effects of suprofen on the isolated perfused rat kidney.

Although suprofen has been associated with the development of acute renal failure in greater than 100 subjects, the mechanism of damage remains unclear. The direct nephrotoxic effects of a single dose of 15 mg of suprofen were compared in the recirculating isolated rat kidney perfused with cell-free buffer with or without the addition of 5 mg/dL of uric acid. There were no significant differences in renal sodium excretion, oxygen consumption, or urinary flow rates in kidneys perfused with suprofen compared with the drug-free control groups. In contrast, a significant decline in glomerular filtration rate was found after the introduction of suprofen to the kidney perfused with uric acid; no changes were found with suprofen in the absence of uric acid. A significant decrease in the baseline excretion rate of uric acid was found in rats given suprofen, compared with drug-free controls. However, the fractional excretion of uric acid was unchanged between the groups over the experimental period. In summary, suprofen causes acute declines in renal function, most likely by directly altering the intrarenal distribution of uric acid.

Influence of pentoxifylline and related analogues in endotoxemic renal failure.

Acute kidney dysfunction, manifested by reductions in renal blood flow and glomerular filtration rate with increased renal vascular resistance, is a common finding in septic shock. In an attempt to halt the progressive renal dysfunction, the hemorheologic methylxanthines, pentoxifylline (1, 5, or 10 mg/kg of PTX) and 2 structurally-related analogues, 5 mg/kg of HWA-138 and 5 mg/kg of HWA-448, or saline wee given 7.5 hr after endotoxin infusion in the rat. Renal function, assessed by single-dose inulin clearances (CIN), was measured at 6 hr after the infusion of endotoxin and also 1 hr following the drug treatment. The mean CIN at 6 and 9 hr after endotoxin infusion were 2- and 3-fold decreased compared with control rats given either saline or 5 mg/kg of PTX. Although renal function declined in all rats throughout the study period, the reduction in renal function was markedly slowed in endotoxemic rats given 10 mg/kg of PTX or 5 mg/kg of HWA-448 compared with untreated controls (74 +/- 9% and 77 +/- 9 vs. 47 +/- 12% of 6 hr CIN at 9 hr, respectively; P less than 0.01). Similar results were found with single doses of 5 mg/kg of PTX or HWA-138; PTX 1 mg/kg had a modest beneficial effect on renal function. There was no evidence of vascular congestion in endotoxemic kidneys upon histologic examination. These data suggest the potential benefit of PTX and related methylxanthines in stopping progressive renal damage associated with septic shock.

Attenuation of amphotericin-B nephrotoxicity in the candidiasis rat model.

Dose-limiting nephrotoxicity has hampered the clinical usefulness of amphotericin-B (AmpB). Recently, vascular decongestants, such as pentoxifylline, have shown benefit in reducing drug-associated renal damage of AmpB in rats. The present study investigated a dose-dependent protection of a structurally similar methylxanthine, HWA-448, in the candidiasis rat model given a single dose of AmpB. Forty-eight hours after inoculation with Candida albicans, each rat was treated with 0.8 mg/kg of AmpB or sterile water; animals were further randomized to receive 0.5, 1, 5, or 10 mg/kg i.v. of HWA-448 or saline given every 12 h for 3 doses. Kidney fungal counts, morphology, and renal function were compared between treatment groups upon completion of the study. Rats administered AmpB with HWA-448 had markedly improved renal function compared with those given AmpB alone; these effects were independent of the administered dose of HWA-448. The antifungal effect of AmpB was not impaired with concomitant HWA-448. Marked accumulation of granulomas and organisms was found in all rat groups. In summary, coadministration of low doses of HWA-448 attenuated the dose-limiting nephrotoxicity without impairing the antifungal effect of AmpB.

Benefit of vascular decongestion in glycerol-induced acute renal failure.

The post insult administration of vascular decongestants has resulted in attenuation of experimental acute renal failure (ARF) following the introduction of various nephrotoxins including drugs, heavy metals, and endotoxin. In the present study, the dose-dependent effects of a novel methylxanthine, HWA-138, were studied in the glycerol-induced murine model of ARF. Renal function, assessed by serial inulin clearances at 24 and 48 h after glycerol injection, urinary electrolyte excretion rates, and renal morphology, was compared between controls and those given glycerol and single i.v. doses of 0.1, 0.5, 1.0, 5.0, and 10.0 mg/kg of HWA-138, or physiologic saline. Whereas significant renal dysfunction was found in all animal groups given glycerol, the mean inulin clearance values of animals given HWA-138 1 mg/kg closely approximated values found in control rats. There were no changes in renal electrolyte excretion rates in animals given HWA-138 compared with relative natriuresis found in untreated glycerol ARF rat. Although a modest decrease in medullary congestion was associated with rats given 1 mg/kg of HWA-138, there was no obvious structural improvement found with HWA-138. The present data provide further evidence of the potential of methylxanthines in the glycerol-ARF murine model.

Pentoxifylline in the isolated perfused rat kidney.

The effects of pentoxifylline, a new methylxanthine with marked hemorrheologic properties, were studied following brief renal artery occlusion in the isolated rat kidney model perfused with cell-free Krebs-Henseleit buffer. Anuria was observed in 3 of 6 control kidneys within 5 min after reperfusion; urine flow was maintained in all rat kidneys perfused with pentoxifylline (2500 ng/ml). Glomerular filtration rate was significantly greater in kidneys administered pentoxifylline compared with controls following 40 min of postocclusion reperfusion (460 +/- 100 vs. 100 +/- 110 microliters/min/gKW; P less than 0.01). Pretreatment of kidneys with indomethacin, a nonspecific cyclooxygenase inhibitor, blocked the protective effects of pentoxifylline in this setting. These data suggest that the addition of pentoxifylline may prevent hypoxia-related changes in renal function of transplanted kidneys. Stimulation of renal prostaglandin synthesis, as well as an interaction at the level of the adenosine receptors, were most likely responsible for the observed beneficial effects of pentoxifylline.

Influence of lovastatin on the pharmacokinetics, toxicity and immunologic response of cyclosporine in the obese Zucker rat.

The combined use of lovastatin, a hypolipidemic agent effective in the reduction of cholesterol levels, and the lipophilic immunosuppressant, cyclosporine, was studied in the obese rat model. Pharmacokinetics, immunosuppressive activity, lipid levels, and creatinine clearances were compared between groups administered drug-free vehicle, lovastatin or cyclosporine alone, or concomitant cyclosporine and lovastatin. All groups were pre-treated with either oral lovastatin 2.5 mg kg-1 day-1 or propylene glycol vehicle for 1 week. Although no differences in renal function were observed in rat groups administered cyclosporine or lovastatin alone, there was a significant reduction in baseline creatinine clearance following combination therapy compared to placebo controls (70 +/- 18 vs 121 +/- 16 per cent of baseline; p less than 0.05). No differences in trough cyclosporine concentrations were observed between groups. Similarly, mean areas under the whole blood concentration-time profiles were not significantly different with or without concomitant lovastatin (61823 +/- 27295 vs 41470 +/- 10312 ng h ml-1; p = 0.13). No differences in systemic clearance or volume of distribution of parent cyclosporine were observed with combination therapy. Furthermore, lipid levels and T-lymphocyte activity were unchanged with the addition of lovastatin. Per cent increases in creatine kinase were significantly correlated with percentage drop in baseline renal function, suggesting the development of rhabdomyolysis. The present data support the interaction between cyclosporine and lovastatin observed clinically, resulting in acute renal dysfunction. Caution should be exercised in their combined use.

Impact of time and feeding habits on lipid levels in Zucker obese rats.

The influences of time and hyperphagia on cholesterol, triglyceride, glucose and insulin levels were compared in the obese Zucker rat and compared to its lean litter-mates. Following a 28 day acclimation period in a 12 hr light/dark cycle (08-20-08) animal facility, blood samples were obtained every 2 hr in both obese and lean rats over a 24 hr period (N = 48; Dec 1988); serum was measured enzymatically for cholesterol, triglyceride and glucose and by radioimmunoassay for insulin and cortisol levels. Synchronization with other animal studies was established by endogenous serum cortisol measurements (acrophase 18-20 HALO in both groups). Cholesterol, triglyceride, insulin and glucose concentrations were significantly greater per time interval in obese vs. lean rats. No circadian pattern was observed in glucose concentrations in either rat group. Insulin levels peaked in both rat groups during the dark cycle; however, glucose and insulin levels were not correlated. Cholesterol concentrations were unchanged over time in obese as well as lean rats. Although triglyceride levels showed an acrophase at 13 HALO in lean rats, no circadian pattern was found in obese rats. Triglyceride levels remained elevated throughout the 24 hour period in obese rats whereas significant increases were observed in lean rats during the dark cycle. The present results suggest that triglyceride levels, and not insulin and cholesterol levels, are most likely dependent on feeding and activity patterns.

Isolation-induced renal functional changes in rats from four breeders.

The investigation of drug-induced nephrotoxicity depends on the adequate estimation of renal function at baseline and upon completion of the study. Typically, this procedure requires housing of the animal in an individual wire-bottom metabolic cage to facilitate complete urine collection. The present study compared the effects of 4 consecutive days of isolation on Sprague-Dawley rats from four breeders: Harlan Sprague-Dawley, Charles River Laboratories, BioLab and TIMCO Breeders. Following 4 days of isolation, weight loss was not significantly different between groups. However, urine flow rate declined significantly (p less than 0.0005) in TIMCO and Charles River breeder rat groups during the study period compared to baseline values and other groups. Serum creatinine levels were 63% greater (p less than 0.01) with a 40% decline in creatinine clearance (p less than 0.0001) after 4 days of isolation in TIMCO rats. Although a 59% decrease in baseline creatinine clearance was found in Charles River rats after 96 hours of isolation (p less than 0.0005), the mean baseline value was 38% greater than other rat groups (p = 0.04). Fractional reabsorption of sodium was 4.4% less (p less than 0.001) in TIMCO rats compared to baseline. Fractional excretion of potassium was highly variable in all rat groups. We conclude that animal isolation was associated with a significant change in renal function in TIMCO rats which was not observed in others. Caution is required to consider the source of the rat, and also duration of isolation, in studies requiring the passive assessment of renal function.

Role of vascular decongestion in ischemic acute renal failure defined by postinsult administration of pentoxifylline.

The patholophysiologic significance of vascular congestion in the mechanism of ischemic acute renal failure following postocclusive reflow was studied with a novel hemorheologic probe, pentoxifylline. Using the autoperfused rat kidney model, inulin clearances (CIN), urine flow rates (UFR), renal electrolyte excretions, and renal hemodynamic parameters (RVR, RBP, RBF) were compared in saline- and pentoxifylline-treated anesthetized rats prior to and following a 45-min occlusive period. Renal functional and hemodynamic parameters were significantly altered in saline controls. In contrast, postischemia treatment with pentoxifylline was associated with significant recovery in CIN and UFR, and stable RVR, RBF, and RBP. Kidneys treated with saline infusion had pronounced vascular congestion, in contrast to those administered pentoxifylline. Coupled with the absence in medullary hyperemia, the present experiments support the role of vascular congestion in ischemic acute renal failure. Pentoxifylline, administered in pharmacologic doses after the insult, provided benefit during the initiation phase of postischemic acute renal failure. These data strengthen the opinion that ischemic insult results in vascular congestion, and that restoration of blood flow will prevent further deterioration in renal function.

Circadian assessment of lipids in the hyperphagic obese rat compared with lean litter-mates.

Time and feeding influences on cholesterol, triglyceride, glucose and insulin levels, and serum cholinesterase activity were assessed in a genetically-hyperlipidemic hyperphagic obese rat model, and compared with its lean litter-mate. Following a 28-day acclimation to a 12-hr light/dark cycle, blood samples were obtained every 2 hr from rats via tail bleed for a 24-hr period. Synchronization with other animal studies was established by endogenous serum cortisol levels [acrophase 18-20 hr after light onset (HALO) in both groups]. Triglycerides cholesterol, insulin and glucose levels were significantly elevated in obese versus lean rats. Obese rats were observed to feed throughout the 24-hr cycle, whereas lean litter-mates ate only during the dark cycle. No circadian rhythmicity was found in glucose levels with either rat group. Insulin levels were not correlated. Although triglyceride levels peaks at 13 HALO in lean rats, no pattern was observed in obese rats. Cholesterol levels were unchanged with time in either group. Cholinesterase activity followed a circadian rhythm in the lean, but not obese, rats with an acrophase estimated at 8 HALO. In contrast to previous reports, enzyme activity was not correlated with triglyceride levels in either rat group. Circadian similarities in insulin levels between rat groups suggest changes in insulin metabolism and/or secretion which are likely to be independent of feeding or activity. Conversely, triglyceride levels remained elevated throughout the 24-hr period in obese rats, whereas significant increases were observed in lean rats during the dark active cycle. These data suggest that triglyceride levels, and not insulin and cholesterol levels, are most likely dependent on feeding patterns.