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A Nonpeptidyl Mimic of Superoxide Dismutase with Therapeutic Activity in Rats

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Science  08 Oct 1999:
Vol. 286, Issue 5438, pp. 304-306
DOI: 10.1126/science.286.5438.304

Abstract

Many human diseases are associated with the overproduction of oxygen free radicals that inflict cell damage. A manganese(II) complex with a bis(cyclohexylpyridine)-substituted macrocyclic ligand (M40403) was designed to be a functional mimic of the superoxide dismutase (SOD) enzymes that normally remove these radicals. M40403 had high catalytic SOD activity and was chemically and biologically stable in vivo. Injection of M40403 into rat models of inflammation and ischemia-reperfusion injury protected the animals against tissue damage. Such mimics may result in better clinical therapies for diseases mediated by superoxide radicals.

Many diseases can be characterized as conditions in which the body fails to contain the overproduction of an undesired metabolic by-product. Although all mammalian life consumes O2 as the ultimate oxidant supporting cellular respiration, a considerable portion of this O2 is reduced, through one-electron paths, to the superoxide anion (O2 •−). Under normal circumstances, this radical burden is controlled by SOD enzymes in the mitochondria (Mn based), in the cytosol (Cu and Zn), or on extracellular surfaces (Cu and Zn). The SODs (1, 2) are oxidoreductases that contain Cu, Fe, or Mn at the active site and catalyze the dismutation of O2 •− to O2 and hydrogen peroxide (H2O2). In certain diseases, the production of O2 •− is enhanced, resulting in O2 •−-mediated cell injury. Examples of such oxidative stress-related diseases include reperfusion injury, such as that which occurs after acute myocardial infarction or stroke, and inflammatory processes, such as arthritis. Although administration of recombinant SOD has been beneficial in animal models of disease mediated, in part, by superoxide (for example, myocardial ischemia-reperfusion injury, inflammation, and cerebral ischemia-reperfusion injury), clinical trials of this enzyme had to be curtailed because of immunogenic responses (3). Further evidence implicating O2 •− as a mediator of diseases such as neuronal apoptosis, cancer, and acquired immunodeficiency syndrome (3) continues to accrue.

Because of the limitations associated with enzyme therapies (solution instability, limited cellular accessibility, immunogenicity, bell-shaped dose response curves, short half-lives, costs of production, and proteolytic digestion), we have synthesized SOD mimics with a low molecular weight (4). Through our previous work (5–7), we have discovered, using molecular modeling studies, a stable and active class of SOD mimic, exemplified by the prototypical complex, M40403. This mimic is derived from the macrocyclic ligand, 1,4,7,10,13-pentaazacyclopentadecane, containing the added bis(cyclohexylpyridine) functionalities. This complex catalyzes the dismutation of O2 •− with rates approaching that of the native Mn SOD enzyme (8). M40403 (Fig. 1) has a molecular weight of 484.4 and a catalytic SOD rate >2 × 107 M−1s−1, comparable to that of the Mn SODs at a pH of ∼6. It is thermodynamically stable (log K > 17;K, stability constant) and stable for up to 10 hours in whole rat blood at 37°C, although it was observed to partition into red blood cells. After intravenous (iv) injection into rats, M40403 distributes widely into the heart, lungs, brain, liver, and kidneys, while retaining its intact chemical identity. Moreover, the complex is excreted intact with no detectable dissociation and is recovered in urine and feces (8). M40403 does not react with nitric oxide (NO), H2O2, or peroxynitrite (OONO) (PN) (5, 7).

Figure 1

Structures of M40403 and M40404.

We evaluated the activity of M40403 in a rat model of inflammation. Intraplantar injection of carrageenan in rats (9) results in a time-dependent increase in paw volume that is maximal after 3 to 6 hours (10). Administration of M40403 (1 to 10 mg/kg, as iv bolus) 30 min before injection of carrageenan (n = 6) inhibited edema at subsequent time points (Fig. 2A), suggesting that O2 •− is a critical mediator in the development of the inflammatory response.

Figure 2

(A) Intraplantar injection of carrageenan causes a time-dependent increase in paw edema, and this is blocked in a dose-dependent manner by M40403 (1 to 10 mg/kg, given as an iv bolus). The catalytically inactive SOD mimic, M40404 (10 mg/kg, given as an iv bolus), had no effect. (B) Increases in neutrophil infiltration (indexed by MPO amounts in paw tissue), TNF-α, IL-1β, and LDH, but not in PGE2, at 6 hours after carrageenan injection are also inhibited by M40403 (1 to 10 mg/kg) (B). TNF-α, IL-1β, LDH, and PGE2 were measured in paw exudates. The error bars represent the mean ± SEM for six experiments.

Intraplantar injection of carrageenan also provokes a time-dependent infiltration of neutrophils at the inflamed site (10); a profound release of proinflammatory mediators, such as prostaglandin E2 (PGE2), tumor necrosis factor–α (TNF-α), and interleukin-1β (IL-1β) (10–12); and tissue damage, as evidenced by the release of lactate dehydrogenase (LDH) (13). Because these events are maximal at 6 hours after carrageenan injection (8), we evaluated the effects of M40403 at this time point. M40403 (1 to 10 mg/kg; n = 6), given 30 min before carrageenan injection, inhibited neutrophil infiltration and the release of TNF-α, IL-1β, and LDH in a dose-dependent manner (Fig. 2B). Thus, the biological properties of M40403 mimic those of the native enzymes that have also been shown to attenuate edema (10, 14) and neutrophil infiltration. This is consistent with a role for O2 •− in eliciting neutrophil adhesion and infiltration (10, 15). Similar to results obtained with polyethylene glycol (PEG) SOD (10), M40403 had no effect on the release of PGE2 (Fig. 2B), indicating that inhibition of proinflammatory prostaglandins (PGs) (11) does not account for its beneficial effects. This contrasts with our observations of inhibitors of NO synthase, in which inhibition of inflammation was associated with a reduction in NO and PGs (10,16). In addition, a monoclonal antibody to PGE2 inhibits edema after intraplantar injection of carrageenan (17), indicating that PGE2plays a role in edema. We cannot explain at this stage why M40403 is anti-inflammatory, despite high levels of PGE2. Nevertheless, these findings highlight the complex interactions between various mediators in inflammation. The SOD-inactive M40404, a structural analog of M40403 (Fig. 1) (8), at 10 mg/kg (n = 6) had no effect on edema (Fig. 2A), nor did M40404 have any effect on neutrophil recruitment or TNF-α, IL-1β, and LDH release (n = 6) (8).

M40403 was also evaluated in a rat model of ischemia-reperfusion injury and shock, namely the splanchnic artery occlusion (SAO) model. In this model, circulatory shock occurs when reperfusion follows prolonged ischemia of the splanchnic circulation (18). The end result is a high mortality, with most animals dying within the first 2 hours after reperfusion (18). Occlusion of the splanchnic arteries produced an increase in mean arterial pressure (from 115 ± 4 to 127 ± 4 mm of Hg), which, upon reperfusion, decreased until death (mean survival time was 90 ± 5 min; n = 27) (8).

To evaluate the effects of M40403 on local and systemic changes associated with reperfusion injury, we collected blood and tissue samples after the period of ischemia or 70 min after reperfusion. The local alterations in this model include neutrophil infiltration into the intestine (Fig. 3B) and a profound peroxidation of membranes resulting in high plasma levels of lipid peroxidation products, such as malondialdehyde (MDA) (Fig. 4A). The systemic alterations include an increase in plasma levels of TNF-α and IL-1β (Fig. 4, B and C), infiltration of neutrophils into the lung and intestine (Fig. 3), and severe hypotension. These events are most likely triggered by O2 •− generated during the reperfusion phase, because no changes were observed when blood or tissues were removed after the period of ischemia before reperfusion (n = 6) (8). When infused for 15 min before reperfusion, M40403 (0.1 to 1 mg/kg; n = 6), but not M40404 (1 mg/kg; n = 6), inhibited, in a dose-dependent manner, the increase in plasma levels of MDA (45, 61, and 67% at 0.1, 0.3, and 1 mg/kg, respectively), TNF-α (88, 95, and 100% at 0.1, 0.3, and 1 mg/kg, respectively), and IL-1β (68, 96, and 98% at 0.1, 0.3, and 1 mg/kg, respectively) (Fig. 4, A through C), as well as the infiltration of neutrophils, as measured by myeloperoxidase (MPO) levels, into the ileum (62, 75, and 76% at 0.1, 0.3, and 1 mg/kg, respectively) and lung (42, 56, and 61% at 0.1, 0.3, and 1 mg/kg, respectively) (Fig. 3, A and B).

Figure 3

Reperfusion of the ischemic splanchnic circulation (IR) results in the infiltration of neutrophils in the (A) lung and (B) ileum, as evaluated by MPO levels, and this is inhibited in a dose-dependent manner by M40403 (0.1 to 1 mg/kg), but not by M40404 (1 mg/kg). The error bars represent the mean ± SEM for six experiments. *P < 0.05 when compared to basal values. Sham, animals that underwent the same surgical procedure but did not undergo ischemia and reperfusion (controls).

Figure 4

Reperfusion of the ischemic splanchnic circulation results in increases in (A) plasma MDA, (B) TNF-α, and (C) IL-1β, and this is inhibited in a dose-dependent manner by M40403 (0.1 to 1 mg/kg), but not by M40404 (1 mg/kg). The error bars represent the mean ± SEM for six experiments. *P < 0.05 when compared to basal values.

These results with M40403 are consistent with observations from transgenic mice overexpressing human SOD (Cu and Zn) (19), indicating that neutrophil infiltration in the lung and intestine in the SAO model is prevented. Likewise, administration of PEG SOD also exhibited a protective effect in this model (20). Furthermore, M40403 (n = 8), but not M40404 (n = 4), both given at 1 mg/kg, prevented the fall in blood pressure seen after reperfusion (8) and increased the survival time (90 ± 5% survival at 4 hours for rats treated with M40403 versus 0% survival at 4 hours in untreated rats and those treated with M40404).

In summary, our results demonstrate that M40403 is a stable SOD mimic with therapeutic activity in models of inflammation and ischemia. In addition to the direct effects of O2 •− in these models, there are likely to be indirect effects mediated by the formation of PN. It is possible that some of the beneficial anti-inflammatory and cytoprotective effects of M40403 are due to the prevention of PN formation by the removal of O2 •− before it reacts with NO (21). The mechanism or mechanisms by which O2 •− modulates events such as neutrophil influx at inflamed sites or cytokine production and release have yet to be defined.

Understanding the signal transduction mechanisms used by free radicals to modify the course of disease will undoubtedly elucidate important molecular targets for future pharmacological intervention. SOD mimics such as M40403 can serve as tools to dissect these mechanisms. In addition, these molecules may have potential for the treatment of diseases ranging from acute and chronic inflammation to cardiovascular disease and cancer.

  • * To whom correspondence should be addressed. E-mail: dsalvemini{at}metaphore.com

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