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J Appl Physiol 97: 1349-1357, 2004. First published May 28, 2004; doi:10.1152/japplphysiol.01161.2003
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Severity of sepsis alters the effects of superoxide anion inhibition in a rat sepsis model

Xizhong Cui,1 Chantal Parent,1 Heather Macarthur,2 Scott D. Ochs,2 Eric Gerstenberg,1 Steve Solomon,1 Yvonne Fitz,1 Robert L. Danner,1 Steven M. Banks,1 Charles Natanson,1 Daniela Salvemini,3 and Peter Q. Eichacker1

1Critical Care Medicine Department, Clinical Center, National Institutes of Health, Bethesda, Maryland 20892; 2Department of Pharmacological and Physiological Science, St. Louis University School of Medicine, St. Louis 63104; and 3Metaphore Pharmaceuticals, St. Louis, Missouri 63114

Submitted 28 October 2003 ; accepted in final form 21 May 2004


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
Previous analysis showed that selective inhibitors of five different host inflammatory mediators administered for sepsis, although beneficial with severe sepsis and high-control mortality rates, were ineffective or harmful with less severe sepsis. We hypothesized that severity of sepsis would also influence inhibition of superoxide anion, another inflammatory mediator. To test this, 6-h infusions of M40401, a selective SOD mimetic, or placebo were given to antibiotic-treated rats (n = 547) starting 3 h after challenge with differing doses of intravenous Escherichia coli designed to produce low- or high-control mortality rates. There was a positive and significant (P = 0.0008) relationship between the efficacy of M40401 on survival rate and control mortality rates. M40401 increased or decreased the log (odds ratio of survival) (means ± SE), dependent on whether control mortality rates were greater or less than the median (66%) (+0.19 ± 0.12 vs. –0.25 ± 0.10, P = 0.01). In a subset of animals examined (n = 152) at 9 h after E. coli challenge, M40401 increased (mean effect ± SE compared with control) mean arterial blood pressure (8 ± 5 mmHg) and decreased platelets (–37 ± 22 cells x 103/ml) with high-control mortality rates but had opposing effects on each parameter (–3 ± 3 mmHg and 28 ± 19 cells x 103/ml, respectively) with low rates (P ≤ 0.05 for the differing effects of M40401 on each parameter with high- vs. low-control mortality rates). A metaregression analysis of published preclinical sepsis studies testing SOD preparations and SOD mimetics showed that most (16 of 18) had control mortality rates >66%. However, across experiments from published studies, these agents were less beneficial as control mortality rate decreased (P = 0.03) in a relationship not altered (P = not significant) by other variables associated with septic challenge or regimen of treatment and which was similar, compared with experiments with M40401 (P = not significant). Thus, in these preclinical sepsis models, possibly related to divergent effects on vascular function, inhibition of superoxide anion improved survival with more severe sepsis and high-control mortality rates but was less effective or harmful with less severe sepsis. Extrapolated clinically, inhibition of superoxide anion may be most efficacious in septic patients with severe sepsis and a high risk of death.

septic shock; treatments; superoxide dismutase mimetic; M40401

SUPEROXIDE ANION IS A KEY regulator of normal leukocyte host defense and vascular endothelial functions (2, 29, 34, 49). However, excessive production of superoxide and its reactant products has been implicated in the pathogenesis of tissue injury and organ dysfunction occurring during sepsis and septic shock (4, 42, 45). Examination of tissue samples in animal models has also suggested that depletion of endogenous antioxidants, such as SOD, may potentiate tissue injury during sepsis (22, 28, 32, 46).

Based on the potential role of superoxide anion in the inflammatory injury associated with sepsis, both exogenous SOD preparations and SOD mimetics (SODm) have been proposed as adjunctive treatment for this potentially lethal condition (1, 5, 6, 14, 15, 17, 19, 20, 23, 30, 35, 36, 38, 39, 41, 47, 48). However, recent studies with other anti-inflammatory agents in sepsis suggest that the severity of sepsis, as reflected by control mortality rate, could have an important influence on agents directed against superoxide anion (10). An analysis of published preclinical and clinical trials as well as prospective experiments showed that selective inhibitors of five different host inflammatory mediators (i.e., TNF, IL-1, platelet activating factor, bradykinin, or prostaglandin metabolites), although very beneficial when mortality rate with sepsis was high, were less effective and potentially harmful as mortality rate decreased (10). Furthermore, human recombinant activated protein C, the first agent with recognized anti-inflammatory effects approved by the Food and Drug Administration for sepsis, has been restricted to patients with severe sepsis and a high risk of death based on phase III trial results (11). These findings are consistent with the divergent role that many inflammatory mediators are believed to have vis-à-vis tissue injury and host defense during infection and sepsis (18). During severe infection, excessive production of these mediators is likely to have net harmful effects. However, with less severe infection in which the host's response is better coordinated, the net effects of these mediators may be beneficial as they contribute to microbial clearance or other beneficial responses. Superoxide anion's beneficial contributions to host defense and vascular function and its opposing harmful effects on inflammatory tissue injury suggest that severity of infection may alter the outcome of its inhibition during sepsis as well.

The present study was designed to first investigate whether the severity of infectious challenge, as reflected by control mortality rate, would influence the efficacy of M40401, a highly specific inhibitor of superoxide anion, in a rat model of sepsis. M40401 belongs to a group of low-molecular-weight nonprotein membrane-permeable SODm (8, 9, 24, 37). These agents are metal-chelated macrocyclic ligand complexes that demonstrate free radical scavenging activities similar to SOD. M40401 shows high selectivity for superoxide anion due to its manganese (II) center, which resists oxidation by other potential free radicals such as peroxynitrite, hydrogen peroxide (H2O2), or hypochlorite (8, 9, 24, 37). Both M40401 and a similar compound, M40403, inhibit superoxide anion-mediated tissue injury related to ischemia-reperfusion, endotoxin, and other inflammatory stimuli (8, 9, 24, 37). Recent studies have also shown that M40401 increases endogenous serum catecholamine levels and IL-10 levels, decreases serum TNF and IL-1 levels, and improves arterial blood pressure and survival in rats challenged with highly lethal (i.e., 83% control mortality rate) intravenous (IV) Escherichia coli challenge (23). In the experiments reported here, animals were randomized to be challenged with doses of IV E. coli designed to produce high- or low-control mortality rates, following which they were treated with M40401 or placebo. Analysis then determined whether severity of infection as reflected by control mortality rates was related to variation in the efficacy of M40401. Such analysis in prior experiments demonstrated that, whereas control mortality rate significantly influenced the efficacy of anti-inflammatory agents, it did not influence the beneficial effects of fluid support or antibiotics, treatments without direct anti-inflammatory effects (10, 40). Experiments were also done to determine whether there was a relationship between the severity of septic challenge and the levels of mediators produced that M40401 is known to alter. Last, we performed a literature search and a metaregression analysis to assess the influence of control mortality rate on the efficacy of exogenous SOD enzyme preparations and on SODm in published preclinical sepsis trials.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
M40401 Experiments

Animal care.   The protocol used in this study was approved by the Animal Care and Use Committee of the Clinical Center of the National Institutes of Health. During the study, every effort was made to minimize the animals' suffering. The research protocol required the veterinary staff or principal investigators to euthanize any animal that experienced unexpected pain or distress.

Study design.   Sprague-Dawley rats (n = 547) with previously placed internal jugular venous catheters were anesthetized with ketamine (40 mg/kg) and xylazine (10 mg/kg) (10). Animals then received one of several increasing doses of IV E. coli, ranging from 0.5 to 2.5 x 109 colony-forming units (CFU)/kg, designed to produce a range of control mortality rates from low (<10%) to high (>90%) in 45 experiments. Some experiments (n = 15) investigating low or high doses of bacteria were done concurrently. Three hours after E. coli inoculation, animals were randomized to receive M40401 (25 µg·kg–1·h–1) or a corresponding volume of vehicle as placebo (26 mM sodium bicarbonate buffer, pH = 8.0–8.3) for 6 h as a continuous IV infusion (1 ml/h). A subset of animals (n = 152 with both jugular venous and carotid arterial catheters) had hemodynamic measurements, including mean blood pressure, heart rate (HR), and central venous pressure (CVP), arterial blood gases, and complete blood cell counts performed at 9 h (i.e., the end of drug infusion) and, in surviving animals, at 24 h after E. coli inoculation. Quantitative blood cultures were performed at 24 h only. Beginning 6 h after E. coli, all animals were treated with ceftriaxone (100 mg/kg IV, every day for 4 days; Roche Laboratories, Nutley, NJ). Animals were observed every 2 h for the first 24 h, every 4 h for the second 24 h, and then every 8 h. Animals alive after 7 days were considered survivors. To investigate the relationship between dose of infecting E. coli and blood and tissue glutathione and SOD levels and blood catecholamine and cytokine levels, in other experiments, animals (n = 46) were challenged with doses of E. coli [0 (diluent only), 0.5, or 2.5 x 109 CFU/kg]. At 9 h after E. coli challenge, animals were anesthetized for blood collection and then euthanized for liver sampling.

Bacterial inoculation.   In brief, E. coli 0111:B4 was stored in 1-ml aliquots of bactopeptone broth (Difico, Detroit, MI) and glycerol at –70°C (10). For preparation of bacteria, an aliquot of frozen culture was thawed and inoculated into 500 ml of brain-heart infusion. The bacteria were incubated for 18 h, centrifuged, and then washed twice with sterile saline. The concentration of bacteria in the final suspension was estimated based on turbidity measurements, comparing the newly grown bacterial suspensions to known standards. Bacterial concentrations were verified by plating successive 10-fold dilutions of the bacterial suspensions onto MacConkey's agar and later scoring of visible colonies after 24 h of incubation at 37°C. Bacterial inoculation (0.5 ml per animal) was performed via central venous catheter.

Hemodynamic measures and blood sample collection.   Immediately after E. coli inoculation, catheters, protected with a coiled spring (Coiled Tether, Lomir, Malone, NY), were attached to the exteriorized arterial and central venous access ports on each animal. Central venous catheters were attached to a syringe pump (PHD 2000 syringe Pump, Harvard Apparatus, Holliston, MA) to provide M40401 or placebo infusion and to transducers (Pressure Transducer, Maxxim Medical, Athens, TX) to measure CVP. Arterial catheters were connected to transducers to determine arterial blood pressure and for blood collection. Animals were then allowed to awaken in a sternal position in a padded cage with absorbable tissue.

Nine hours after E. coli inoculation when M40401 or placebo infusions were completed, randomly selected animals had arterial and venous catheters connected via transducers to a hemodynamic monitoring system (BioSystem XA; BUXCO, Troy, NY) to measure CVP; diastolic, systolic, and mean arterial blood pressures (MBP); and HR. After equilibration, continuous measures of each parameter were collected for a 5-min period, and the mean of the measurements for that period was recorded. Arterial blood was collected for blood-gas analysis or complete blood counts, and, at 24 h, quantitative blood cultures.

Measurements of serum and liver glutathione and SOD levels, serum cytokines, and plasma catecholamines.   Serum and liver total glutathione and GSSG levels were measured by an enzymatic recycling method (Cayman Chemical glutathione assay kit, Cayman Chemical, Ann Arbor, MI). Levels of GSH were determined by subtracting GSSG from total glutathione levels. The GSSG-to-GSH ratio was determined as an index of oxidative stress. Serum and liver total SOD activity (Cu/Zn-, Mn-, and Fe-SOD) were measured by quantification of the dismutation of superoxide radicals generated by the combination of xanthine oxidase and hypoxanthine (Cayman Chemical superoxide dismutase assay kit, Cayman Chemical). Serum cytokine levels, including TNF-{alpha}, IL-1{alpha}, IL-1{beta}, IL-2, IL-4, IL-6, IL-10, INF-{gamma}, and granulocyte macrophage colony-stimulating factor, three migratory inhibitory proteins (MIP-1{alpha}, MIP-2, and MIP-3{alpha}), monocyte chemoattractant protein-1, and RANTES (regulated on activation, normal T-cell expressed and secreted) were measured by using the Searchlight Proteome Array Multiplex system (Pierce, Rockford, IL). Plasma nonoxidized and oxidized catecholamines (norepinephrine and epinephrine) were measured by HPLC with electrochemical detection, as previously described (24, 31).

SODm M40401.   M40401 (Metaphore Pharmaceuticals, St. Louis, MO) is a low-molecular-weight synthetic manganese containing SODm (8, 9, 24, 37). It is stable in vivo, has high activity, and is selective for superoxide with no activity toward H2O2, ONOO–, or hypochloride (OCl–). Its catalytic activity at pH = 7.4 exceeds 1 x 109 M–1/s, comparable with the native SOD enzyme. M40401 was mixed in 26 mM sodium bicarbonate buffer (pH = 8.0–8.3) to the final concentration of 6.25 µg/ml and was then administered at a rate of 1 ml/h IV for 6 h, starting 3 h after E. coli inoculation. Control animals received diluent only. The dose of M40401 chosen for the present study was based on published results in a rat model similar to ours, as well as on dose-response studies that we conducted with highly lethal E. coli challenge (23). Both in published studies (7) and in pilot experiments that we performed, M40401 did not result in lethality in sham-challenged controls.

Statistics.   The relationship between dose of E. coli inoculation and control mortality rate was determined with linear regression weighted based on the number of animals studied. One-way ANOVA was used to compare the difference in the mean E. coli doses between the animals with control mortality rate less than or equal to or greater than the median.

The relationship between control mortality rate and the efficacy of M40401 was analyzed, as described previously (10). For each experiment, treatment mortality rate was plotted on the y-axis and the corresponding control mortality rate on the x-axis, yielding a graph on the unit square in which each axis represents fully independent measures. To avoid constraining data at the extremes of the mortality ranges, we reparameterized the unit square to occupy the whole plane. The y-axis was transformed to become the log of the odds of treatment mortality and the x-axis to become the log of the odds of control mortality. A weighted linear regression based on the number of animals in each experiment was performed. Although the analysis was always done with the y-axis expressed as the odds of treatment mortality, for ease of presentation, the results are presented with the y-axis expressed as the log (odds ratio of survival). The odds ratio of survival is the odds of survival in the treatment group divided by the odds of survival in the control group. The odds ratio of survival is mathematically equivalent and is a commonly used measure of efficacy. One-way ANOVA was used to compare the difference in the odds ratio of survival between the animals with control mortality rate less than or equal to or greater than the median.

Three-way ANOVA, accounting for treatment (M40401 vs. vehicle), time (9 vs. 24 h), and median control mortality rate (less than or equal to or greater than the median) was used to analyze hemodynamics, arterial blood gases, and complete blood cell counts. Analysis of glutathione, SOD, cytokine, and catecholamine levels across E. coli doses was performed by using one-way ANOVA. A level of 5% was chosen for statistical significance (i.e., P ≤ 0.05). For ease of presentation, the treatment effect and its standard error are shown and were calculated by subtracting the mean of the treatment group from the mean of the control group with the appropriate error.

Metaregression Analysis of Published Preclinical Trials Testing Exogenous SOD Preparations and SODm

With the methods described previously (10), a literature search using Medline and Embase was conducted from 1980 to the present by using the search terms sepsis, septic shock, superoxide anion, antioxidant, superoxide dismutase, superoxide dismutase mimetic, and treatment. The published studies retrieved were also reviewed for additional references. All reported preclinical experiments were analyzed in which the survival rates with SOD enzyme preparations or SODm were directly compared with the survival rate of placebo. Survival data were analyzed by using a Cochran-Mantel-Haenszel and Breslow-Day test to estimate the pooled effect of SOD preparations or SODm (10). In experiments in which several different doses of treatment were investigated, these doses were analyzed separately. The relationship between the odds ratio of survival and control mortality rate in the individual experiments was then examined, as had been done with M40401. For all experiments, the type and route of septic challenge, the regimen of treatment employed, including the type, timing, and dose, the species studied, and the duration of observation were recorded, and their influences on the correlation between odds ratio of survival and control odds of dying were assessed by using a two-way ANOVA.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 ACKNOWLEDGMENTS
 REFERENCES
 
M40401 Experiments

Clinical manifestations and survival comparing M40401 and placebo.   By 4–6 h after intravascular E. coli inoculation, animals appeared weak and lethargic. A weighted linear regression showed that increasing doses of E. coli inoculation (0.5, 1.0, 1.5, 2.0, or 2.5 x 109 CFU/kg) produced increased control mortality rates [13% (4/30), 43% (13/30), 61% (52/85), 75% (58/77), and 79% (38/48), respectively] (r = 0.62, P = 0.0001). As control mortality rates (i.e., control odds) increased, the odds ratio of survival with M40401 increased, and the slope for this relationship was highly significant (P = 0.0008; Fig. 1). Based on the significance of this relationship, the differing effects of treatment with M40401 were further analyzed by grouping experiments based on the median control mortality rate noted over the course of study. The mean dose (1.96 ± 0.01 x 109 CFU/kg) of bacteria resulting in control mortality rates greater than the median was significantly higher than the one producing control mortality rates less than the median (1.06 ± 0.11 x 109 CFU/kg) (P < 0.001, one-way ANOVA for the effect of bacteria dose on control mortality rate). In contrast to experiments with control mortality rates equal to or greater than the median (66%) in which M40401 increased survival rates (Fig 2B) and the odds ratio of survival (Fig. 3), in experiments with control mortality rates less than the median, M40401 decreased these (Fig. 2A and Fig. 3) in patterns that were significantly different (P = 0.01, one-way ANOVA for the differing effects of M40401 with high- vs. low-control mortality rates). Results in experiments employing additional batches of M40401 in fewer animals (n = 198) were similar [P = not significant (NS)] with these findings (data not shown).



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  		Fig. 1. Relationship between the log (control odds of dying) and the effect of M40401 on the log (odds ratio of survival) for all experiments individually. Circles, individual experiments with differing control odds; solid line, weighted regression. As shown by the regression line, M40401, although most beneficial in experiments with the highest control odds, was less beneficial or harmful as control odds decreased. The slope of this relationship was highly significant (P = 0.0008).

 


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  		Fig. 2. Proportion of animals (n) treated with M40401 or placebo surviving over time in experiments with control mortality rates less than the median (66%) (A) compared with experiments with control mortality rates equal to or greater than the median (B). Although M40401 increased survival rates in experiments with higher control mortality rates, it decreased survival rates in experiments with lower control mortality rates.

 


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  		Fig. 3. Mean effect (±SE) of M40401 on the odds ratio of survival (log) in experiments with control mortality rates equal to or greater than the median (66%) compared with experiments with control mortality rates less than the median. In patterns that were significantly different, M40401 increased survival rates overall in experiments with high-control mortality rates but decreased survival rates in experiments with low ones.

 
Hemodynamic measurements comparing M40401 and placebo.   To investigate mechanisms underlying the opposing effects of M40401 on survival in experiments with control mortality rates (i.e., control odds) equal to or greater than the median vs. less than the median, its effects on hemodynamic and other laboratory parameters were compared in animals having these measures. At 9 h (Table 1) but not 24 h (data not shown) after bacterial inoculation, MBP and CVPs in control animals were lower (both P ≤ 0.05, three-way ANOVA accounting for treatment, treatment time, and median control mortality rate) in experiments with E. coli challenges, producing high- vs. low-control mortality rates. At both 9 h (Table 1) and 24 h (data not shown), HRs were lower (P = 0.05, three-way ANOVA accounting for treatment, treatment time, and median control mortality rate) in experiments with high- vs. low-control mortality rates. At 9 h (Fig. 4) but not 24 h (data not shown) in patterns that were significantly different (P = 0.05, three-way ANOVA accounting for treatment, treatment time, and median control mortality rate), M40401 increased MBP in experiments with high-control mortality rates but decreased MBP with low ones. Although M40401 showed similarly different effects at 9 h on CVP comparing experiments with high- vs. low-control mortality rates, these differences did not reach significance (P = 0.16).


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  		Table 1. Mean blood pressure, heart rate, and central venous pressure in animals treated with placebo (control) or M40401 9 h after challenge in experiments with doses of E. coli producing control mortality rates less than or greater than or equal to the median

 


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  		Fig. 4. Mean effect (±SE) of M40401 on mean arterial blood pressure (MBP) 9 h after E. coli challenge in experiments with control mortality rates equal to or greater than the median (66%) compared with experiments with control mortality rates less than the median. In patterns that were significantly different (P = 0.05), M40401 increased MBP with E. coli challenges producing high-control mortality rates but decreased MBP with low ones.

 
Blood measurements comparing M40401 and placebo.   At 9 h (Table 2) but not 24 h (data not shown) after bacterial inoculation, arterial base excess levels were lower, and lactate and hemoglobin levels were higher (all P ≤ 0.05) in experiments with E. coli challenges producing high- vs. low-control mortality rates (Table 2). At both 9 h (Table 2) and 24 h (data not shown), total leukocyte counts and platelets were lower (both P ≤ 0.05, three-way ANOVA accounting for treatment, treatment time, and median control mortality rate) in experiments with high- vs. low-control mortality rates. At 24 h, blood bacteria counts [mean log (CFU/ml) ± SE] were higher (P < 0.05, three-way ANOVA accounting for treatment, treatment time, and median control mortality rate) in experiments with high- vs. low-control mortality rates (8.5 ± 1.3 vs. 5.6 ± 0.5 in controls and 9.5 ± 0.3 vs. 5.3 ± 0.5 CFU/ml with M40401). At 9 h (Fig. 5) and 24 h (data not shown) after bacterial inoculation, in patterns that were significantly different (P < 0.01, three-way ANOVA accounting for treatment, treatment time, and median control mortality rate), M40401 decreased platelets in experiments with high-control mortality rates but increased them with low ones. M40401 did not have significant effects on any other parameter, including bacteria counts during the study (P = NS for all).


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  		Table 2. Actual base excess, lactate, white blood cell count platelets, and hemoglobin 9 h after E. coli challenge in experiments with control mortality rates less than or greater than or equal to the median

 


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  		Fig. 5. Mean effect (±SE) of M40401 on platelet count 9 h after E. coli challenge in experiments with control mortality rates equal to or greater than the median (66%) compared with experiments with control mortality rates less than the median. In patterns that were significantly different (P = 0.01), M40401 decreased platelets with E. coli challenges producing high-control mortality rates but decreased them with low ones.

 
Relationship between increasing dose of E. coli challenge and glutathione, SOD, cytokine, and catecholamine levels.   Although animals were killed at 9 h for these measures, the doses of E. coli with which they were challenged (0, 0.5, or 2.5 x 109 CFU/kg) were associated with mortality rates of 0, 13, and 79%, respectively, in other experiments where observation was continued for 168 h. In blood samples, compared with noninfected controls (E. coli dose of 0), at 9 h after bacterial inoculation, GSSG levels and the GSSG-to-GSH ratio, the cytokines TNF-{alpha}, IL-1{alpha} and -{beta}, IL-2, IL-6, IL-10, IFN-{gamma}, granulocyte macrophage colony-stimulating factor, MIP-1{alpha}, MIP-2, MIP-3{alpha}, and nonoxidized catecholamines (norepinephrine and epinephrine combined) were all increased, but these increases were greater with the E. coli dose producing the highest mortality rate (all P ≤ 0.05 for the effect of dose of E. coli on each of these parameters, one-way ANOVA; Table 3). In blood and liver samples at this same time point, compared with noninfected controls, SOD levels were decreased, but these decreases were greater with the more lethal dose of E. coli (P < 0.05 for the effect of dose of E. coli, one-way ANOVA; Table 3). In contrast to blood, liver GSSG and GSH levels were decreased similarly with increasing E. coli dose, and there was no difference in the GSSG-to-GSH ratio between noninfected and infected animals (data not shown). In blood, oxidized catecholamine levels and other cytokines measured were not altered significantly (P = NS for all) by the dose of E. coli inoculated.


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  		Table 3. Serum GSSG and GSH, GSSG-to-GSH ratio, SOD and cytokines, and plasma catecholamine in animals challenged with E. coli doses of 0 (diluent only), 0.5, or 2.5 x 109 CFU/kg

 
Metaregression Analysis of Published Preclinical Trials Testing SOD and SODm

A literature search identified 409 studies in which 16 compared the effects of exogenous SOD enzyme preparations or SODm alone to placebo on survival in preclinical sepsis models (Refs. 1, 5, 6, 14, 17, 19, 20, 23, 30, 35, 36, 38, 39, 41, 47, 48; also see supplemental material at http://jap.physiology.org/cgi/content/full/01161.2003/DC1). These 16 studies included 18 experiments, 6 of which tested more than a single dose of a SOD preparation or SODm (Tables 4 and 5). Individual doses were compared separately, resulting in a total of 32 comparisons. In 16 of these 18 experiments, control mortality rates were higher than the median [66% (25–75% range; 25–89%)] in our prospective experiments, and the median control mortality rates in published experiments [85% (25 to 75% range; 82 to 97%)] were significantly greater than in ours (P < 0.0001). Even over this higher and more restricted range, however, independent of the site or type of septic challenge, regimen of treatment, duration of observation, or species studied, the odds ratio of survival with SOD preparations or SODm significantly correlated to control mortality rate (i.e., control odds; P = 0.03) and was similar (P = NS) to the one with M40401 (Fig. 6). A weighted linear regression (based on the number of animals in each experiment) showed that the slope for this relationship was significant (P = 0.03). Furthermore, this slope was similar (P = NS) to the one noted in the experiments with M40401. Neither the type nor route of septic challenge, the timing and dose of treatment, the species studied, nor the duration of observation influenced this relationship significantly (P = NS based on two-way ANOVA, accounting for each variable and control odds of dying).


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  		Table 4. Summary of the experiments from published studies testing exogenous superoxide dismutase preparations in preclinical models of sepsis

 

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  		Table 5. Summary of the experiments from published studies testing superoxide dismutase mimetics in preclinical models of sepsis

 


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  		Fig. 6. Relationship between the log [control odds of dying (i.e., control mortality rate)] and the effect of exogenous SOD preparations and SOD mimetics on the log (odds ratio of survival) in individual published preclinical experiments (using the same format as Fig. 1). Sixteen published studies included 18 experiments, 6 of which examined more than a single dose of SOD preparation or SOD mimetics. This resulted in a total of 32 comparisons between the effects of SOD preparations or SOD mimetics on the odds ratio of survival and control mortality rate (i.e., control odds).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
ACKNOWLEDGMENTS
 REFERENCES
 
Administration of M40401, a superoxide anion scavenger, in this rat model of E. coli sepsis altered survival rates differently, depending on control mortality rate. In experiments with E. coli challenges producing high-control mortality rates, M40401 was beneficial and increased the odds ratio of survival. However, with E. coli challenges producing lower control mortality rates, M40401 was ineffective or harmful. Severity of infection altered the effects of M40401 on MBP in a pattern similar to its effects on survival rates. Also, in opposing patterns that were significant, platelet counts were decreased or increased with M40401, depending on whether control mortality rates were high or low, respectively. Finally, metaregression analysis of published preclinical sepsis models showed that, although most were conducted in models with high-control mortality rates, independent of other variables, the efficacy of exogenous SOD preparations and SODm appeared potentially dependent on control mortality rate in a relationship that was significant and similar to M40401.

Increased survival rates with M40401 during IV E. coli challenge producing high-control mortality rates in the present study are consistent with the protective effects this agent and a similar one (i.e., M40403) had, either with highly lethal IV endotoxin or E. coli challenge or with ischemia reperfusion injury (8, 9, 24, 37). In combination, these findings with two selective inhibitors of superoxide anion support the central role of this mediator in the pathogenesis of tissue injury related to several different stimuli, including bacterial sepsis. Although superoxide anion contributes to important components in host defense, this and a previous study show that, even with infection, inhibition of superoxide may have beneficial effects if the infectious challenge is sufficiently severe (23). This benefit may, in part, relate to the inhibitory effect that superoxide anion may have on catecholamines and vascular tone (13, 26, 50). Very consistent with this, in a prior study also in Sprague-Dawley rats challenged with a highly lethal dose of IV E. coli, an identical regimen of M40401 to the one that we employed was shown to increase circulating levels of nonoxidized catecholamines and, as in our study, to improve hemodynamics and survival rates (23). In other published studies, M40403 has been shown to similarly increase nonoxidized catecholamines with highly lethal intravascular endotoxin challenge and to improve hemodynamics and survival rates (24). Decreased levels of circulating platelets in treated animals with more severe infection in the present study are also consistent with greater availability of active endogenous catecholamines with M40401. In prior studies, increases in plasma epinephrine levels have been shown to augment platelet deposition along injured vascular endothelium, as may have been present with systemic inflammation after IV E. coli challenge in this rat model (3, 12, 44). An alternative explanation for the beneficial effects of M40401 on survival in experiments with high-control mortality rates in the present study may relate to the inhibition of TNF and IL-1. In the prior study employing a rat model of highly lethal intravascular E. coli sepsis similar to ours, the protective effects of M40401 treatment were also associated with reductions in TNF and IL-1 levels (9). Our laboratory showed previously that selective inhibitors of TNF also improved survival with E. coli challenges producing high-control mortality rates in our model (10).

In contrast to its beneficial effects with highly lethal E. coli doses, M40401 was ineffective or harmful in experiments with doses of E. coli challenge producing low-control mortality rates. Under these conditions, superoxide anion may contribute more to a coordinated host defense response than to deleterious inflammatory tissue injury. However, in the present study, blood bacteria counts were not altered by M40401, suggesting that this agent's effect on survival was not directly related to microbial clearance. Reductions in systemic arterial blood pressures with M40401 suggest that superoxide anion was important for the maintenance of vascular tone in experiments with less severe infection. Such an effect would be consistent with the role that superoxide anion also has on the inactivation of nitric oxide (16, 21, 25). Because nitric oxide is known to interfere with endothelial platelet deposition, increases in platelet counts with M40401 also suggest that superoxide anion was functioning to inactivate nitric oxide in experiments with less severe infection (33, 43). Experiments have shown directly that potentiation of endogenous nitric oxide with SOD can inhibit platelet adhesion and aggregation (27). Worsened outcome with M40401 in experiments with a low-control mortality rate is also consistent with this agent's ability to suppress TNF levels (23). Our laboratory demonstrated previously that inhibition of TNF with a TNF-soluble receptor was harmful in animals challenged with doses of E. coli producing low-control mortality rates (10).

Just as varying the dose and lethality of E. coli challenge altered the effects of M40401, this variation also altered levels of many of the mediators that M40401 has the potential to influence. Compared with noninfected controls (E. coli dose of 0), challenge with E. coli increased oxidant activity in the vascular space, as reflected by increases in both GSSG levels and the ratios of oxidized glutathione to GSH, as well as by decreases in SOD levels. In the liver, although the ratio of oxidant glutathione to GSH was not changed with E. coli challenge, SOD levels were decreased as in serum. All of these changes with E. coli, however, were greater with doses of E. coli producing high- compared with low-control mortality rates. Also, whereas E. coli challenge increased 10 different cytokines associated with the inflammatory response compared with noninfected controls, these increases were again all greater with E. coli doses producing high- compared with low-control mortality rates. Thus M40401 may have been most beneficial with the highest doses of E. coli, where increased oxidant activity and cytokine release and decreased SOD levels were most pronounced. M40401 may have been less beneficial or potentially harmful in less severe infection, where each of the changes was less pronounced and better controlled.

Compared with a noninfectious control challenge, E. coli inoculation also increased nonoxidized catecholamines, and these increases were greater with E. coli doses producing high- compared with low-control mortality rates. Oxidized catecholamines were not altered by a dose of infecting bacteria. Overall, however, compared with oxidized catecholamines, nonoxidized catecholamines were decreased in noninfected controls, equal in infected animals with low-control mortality rates, and increased in animals with high ones. Our findings, in combination with those previously published, show that, with highly lethal E. coli in this model, although nonoxidized catecholamines are already greater than oxidized ones, increasing the former to even higher levels is protective (23). With minimally lethal infection, however, where nonoxidized and oxidized catecholamine levels appear equal in this model, these findings suggest that disturbing this balance and increasing nonoxidized catecholamines is harmful. It is noteworthy that, based on the published studies noted, animals with minimally lethal E. coli challenge may have had nonoxidized catecholamine levels increased almost fivefold with the present dose of M40401 (23). In sick animals still managing to compensate for this minimally but still potentially lethal infectious challenge, such an increase may not have been tolerable. Titrating the dose of M40401 or other antioxidants based on the relationship between nonoxidized to oxidized catecholamines or other parameters deserves further study, especially if sufficiently rapid assay techniques can be developed.

A literature search and metaregression analysis of published preclinical studies testing exogenous SOD preparations and SODm demonstrate several points (1, 5, 6, 14, 17, 19, 20, 23, 30, 35, 36, 38, 39, 41, 47, 48). In these studies, inhibition of superoxide anion appeared beneficial in most cases. However, the control mortality rates in these published studies were high and significantly greater than those in our studies. Even over this higher and more restricted range, however, these agents appeared most beneficial in the experiments with the highest control mortality rates and less beneficial when control mortality rates decreased. This relationship, in addition to being very similar to the one we observed with M40401, is also consistent with our laboratory's meta-analysis of other anti-inflammatory agents in sepsis (10).

In conclusion, the present prospective studies with M40401, as well as a review of published preclinical studies testing exogenous SOD preparations and SODm, suggest that superoxide anion is harmful during very severe sepsis with high-control mortality rates. Although few published preclinical studies have assessed the role of this mediator during less severe sepsis with lower control mortality rates, studies with M40401 in this rat model suggest that superoxide anion is not harmful and may be protective. Extrapolated clinically, inhibition of superoxide anion with M40401 or other agents directed at superoxide anion may be safest and most efficacious in patients with severe sepsis and the highest risk of death. However, determining the potential risk of agents like M40401, if administered over a wide range of control mortality rates before their clinical application, is likely to be important for their safe and effective usage.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
REFERENCES
 
The writers thank Jennifer Candotti for preparation of the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: X. Cui, Critical Care Medicine Dept., National Institutes of Health, Bldg. 10, Rm. 7D43, Bethesda, MD 20892 (E-mail: cxizhong{at}mail.cc.nih.gov).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGMENTS
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