MXPA98008946A - Method of treatment or prevention of the injury caused by isquemia-reperfus - Google Patents

Abstract

A method for the manufacture of a pharmaceutical composition for use in the treatment of ischemia-reperfusion injury, which comprises mixing a pharmaceutically acceptable carrier and an effective amount of interleukin-

Description

METHOD OF TREATMENT OR PREVENTION OF INJURY CAUSED BY ISCHEMIA-REPERFUSION BACKGROUND OF THE INVENTION Ischemia-reperfusion injury often occurs when the blood flow in a region of the body is temporarily interrupted (ischemia) and then restored (reperfusion). The injury caused by ischemia-reperfusion can occur during certain surgical procedures, such as repair of aortic aneurysms and organ transplantation. Clinically, the injury caused by ischemia-reperfusion can be manifested by complications such as pulmonary dysfunction, including respiratory distress syndrome in adults, renal dysfunction, consumptive coagulopathies including thrombocytopenia, fibrin deposition in the microvasculature and disseminated intravascular coagulacopathy, transient and permanent spinal cord, cardiac arrhythmias and acute ischemic events, hepatic dysfunction, including acute hepatocellular damage and necrosis, multiple system organ dysfunction (DOMS) or acute systemic inflammatory response syndromes (SRIS). The injury can occur in parts of the body where the blood supply was interrupted, or it can occur in parts completely supplied with blood during the period of ischemia.

International Patent Publication No. WO 96/01318 refers to polypeptides other than interleukin-10 (IL-10) which are claimed to have one or more properties similar to those of IL-10. Among the very long list of diseases that are claimed to be treated with these proteins other than IL-10 are tissue damage as a result of "hypoxia / ischemia (infarction: reperfusion)", "ischemia", "reperfusion injury" ", and" reperfusion syndrome ". However, in this publication there is no evidence that proteins other than IL-10 really work for the treatment of all diseases in the long list.

SUMMARY OF THE INVENTION The present invention is a method for the treatment of the injury caused by ischemia-reperfusion comprising, administering to a patient in need of this treatment, an effective amount of IL-10. Another aspect of this invention comprises a method for preventing injury caused by ischemia-reperfusion in a patient who is going to be subjected to a procedure capable of causing ischemia-reperfusion injury or to a patient who has already undergone this procedure, wherein the ischemia-reperfusion injury has not yet occurred, and comprises administering to the patient an effective amount of IL-10. The preferred applications of this invention are the prevention of ischemia-reperfusion injury by administering IL-10 together with surgical repair of the thoracic or suprarenal aorta due to aneurysmal disease, but also together with those surgical procedures that induce or require occlusion or transient delivery of the visceral blood supply through the secondary hepatic, renal and / or enteric arteries to transplant a major organ, including the liver, kidney, small intestine and pancreas, as well as surgical procedures that give rise to the reduction or prevention Passage of the blood flow to the viscera, including surgical hepatic and biliary resections, total or partial pancreatectomy (Whipple procedure), partial or total gastrectomy, esophagectomy, colorectal surgery, vascular surgery for mesenteric vascular disease or abdominal insufflation during lapa surgical procedures roscópicos. Additional applications include blunt or penetrating injuries or wounds that result in the interruption of blood flow to the visceral organs including those arising from penetrating wounds to the abdomen resulting from bullet wounds, knife wounds or penetrating wounds or abdominal injuries. no secondary punctures until injuries due to deceleration and / or accidents in automotive vehicles. Other preferred applications include diseases or procedures that give rise to systemic hypotension and that interrupts or decreases the flow of blood to the visceral organs., including hemorrhagic shock due to blood loss, cardiogenic shock due to myocardial infarction or cardiac deficiency, neurogenic shock or anaphylaxis. Other applications of this invention include the prevention or treatment of injury caused by ischemia-reperfusion of the torso or lower extremities by administering IL-10 together with surgical procedures that induce or require transient occlusion or bypass of the blood supply to the upper or lower torso or extremities. This application is particularly important for the practice of vascular surgery that includes controlled periods of visceral ischemia, torso and extremities followed by reperfusion. The procedures included in this ischemia-reperfusion include, but are not limited to, repair of abdominal aortic aneurysms, femoral, popliteal or tibial aortic bypass or claudication, which puts the limb at risk, repair of popliteal or femoral aneurysms, referrals, thrombectomy or embolectomy due to acute ischemia of the limb or vascular trauma. The administration of IL-10 can improve the recovery and survival of the limb after significant ischemia of the torso or limb. The amount of IL-10 that can be administered preferably is between 0.1 to 500 μg / kg body weight, more preferably 1 to 50 μg / kg. IL-10 can be of human or viral origin produced biologically from mammalian cell sources or by recombinant DNA technology. The administration is preferably carried out by intravenous, intramuscular or subcutaneous injection. IL-10 is preferably administered from one to zero hours before blood flow is restored. In those surgical procedures in which temporary or sustained interruption of blood flow can be anticipated, such as before surgical repair of thoracoabdominal aneurysmal disease or supercelia, or surgical procedures for the abdomen that will necessarily include temporary reduction in blood flow visceral, or for organ transplantation, IL-10 is preferably given as a single injection in the bolus from one to zero hours before the ischemic event or as a? continuous intravenous injection that starts from one to zero hours before the ischemic event and that extends during the preoperative period and continues for at least 8 hours after the restoration of visceral blood flow.

For individuals in whom interruption of visceral blood flow has already occurred, such as in those individuals with trauma or injury to the visceral organs or their blood supply, or in patients with systemic stress type due to shock, IL-10 would be preferably administered as a single bolus injection before or at the same time as the restoration of normal visceral blood flow or as a continuous intravenous injection before or at the same time as the restoration of normal visceral blood flow and extending for at least 8 hours after the restoration of visceral blood flow. For individuals in whom disruption of skeletal blood flow has already occurred, such as in those individuals with acute ischemia in the lower extremities due to embolic or thrombotic occlusion of peripheral blood vessels or acute ischemia due to vascular trauma, IL-10 would preferably be provided as a single injection before or at the same time with the restoration of normal blood flow or as a J-ravenous injection continues before or at the same time with the restoration of normal blood flow, and extending for at least 8 hours after the restoration of the Blood flow. Otherwise, IL-10 can be administered by therapy or gene transfer using liposomes and mammalian expression plasmids, mechanical delivery systems (gene gun) of viral transfection schemes, including, but not limited to, adenovirus , viruses associated with adeno, retroviruses or constructions of herpes simplex virus.

BRE ^ E DESCRIPTION OF THE DRAWINGS Figures 1 (a), 1 (b) and 1 (c) illustrate plasma concentrations of TNF-α, IL-1β and IL-8, respectively, after thoracoabdominal aortic aneurysm repair and infrarenal. Figures 2 (a), 2 (b) and 2 (c) illustrate the changes in IL-6 in plasma at p55 concentrations in plasma and changes in plasma p75 concentrations, respectively, after thoracoabdominal and infrarenal aortic aneurysm repair. Figure 3 illustrates the changes in myeloperoxidase levels in lung (neutrophil infiltration) in mice after superceliac transverse aortic subjection placement and treatment with inhibitors of TNF and IL-1. Figure 4 illustrates changes in lung permeability (passage of 125 I-albumin) in mice after placement of the supercelia aortic cross-section and treatment with TNF inhibitors and IL-1. Figure 5, which illustrates the appearance of IL-10 in the circulation of mice after using the superalflex aortic cross-clamp, shows plasma IL-10 concentrations in mice after superceliac aortic cross-clamping and treatment with Recombinant human IL-10. Figure 6 illustrates changes in myeloperoxidase levels in lung (neutrophil infiltration) in mice after superalflex aortic cross-clamping and treatment with recombinant human IL-10.

DETAILED DESCRIPTION OF THE INVENTION All references mentioned herein are incorporated by reference in their entireties. When used herein, "interleukin-10" or "IL-10" is defined as a protein that: (a) has an amino acid sequence of mature IL-10 (eg, absence of a leader secretory sequence) as described in U.S. Patent No. 5,231,012, and (b) has biological activity that is common to natural IL-10. Muteins and other analogs are also included, including the BCRF1 protein of Epstein-Barr virus (viral IL-10), which retains the biological activity of IL-10. IL-10 suitable for use in the invention can be obtained from culture medium conditioned by activated cells secreting the protein, and purified by the normal methods. In addition, IL-10, or active fragments of it, they can be chemically synthesized using standard techniques known in the art. See, Merrifield, Science 233: 341 (1986) and Atherton et al., Solid Phase Peptide Synthesis: A Practical Approach, 1989, I.R.L. Press, Oxford. See also U.S. Patent No. 5,231,012. Preferably, the protein or polypeptide is obtained by recombinant techniques using isolated nucleic acid encoding the IL-10 polypeptide. The general methods of molecular biology are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, New York, 2nd ed., 1989, and in Ausubel et al., (Eds) Current Protocols in Molecular Biology, Green / Woley, New York (1987 and periodic supplements). Suitable sequences can be obtained using standard techniques from genomic or cDNA libraries. It is possible to use polymerase chain reaction (PCR) techniques. See, for example, PCR Protocols: A Guide to Methods and Applications, 1990, Innis et al., (Ed.) Academic Press, New York, New York. Libraries are constructed from nucleic acid extracted from the appropriate cells. See, for example, U.S. Patent No. 5,231,012, which describes recombinant methods for preparing IL-10. It is possible to find useful sequences of genes, for example in various sequence databases, for example GenBank and BMPL or of nucleic acids and PIR and Swiss Prot for proteins, c / o Intelligenetics, Mountain View, California, or the Genetics Computer Group, University of Wisconsin Biotechnology Center, Madison, Wisconsin. Clones containing the sequences encoding human IL-10 have been deposited with the American Type Culture Collection (ATCC), Rockville, Maryland, with accession numbers 68191 and 68192. Identification of * other clones containing the sequences they encode IL-10 are performed by hybridization of the nucleic acid or by immunological detection of the encoded protein, if an expression vector is used. Oligonucleotide waves based on the deposited sequences described in U.S. Patent No. 5,231,012 are particularly useful. The sequences of the oligonucleotide waves can also be prepared from the conserved regions or related genes in other species. Otherwise, degenerate probes based on the amino acid sequences of IL-10 can be used. The normal methods can be used to produce transformed prokaryotic, mammalian, yeast or insect cell lines expressing large amounts of polypeptide. Exemplary E. coli strains suitable for expression and cloning include W3110 (ATCC Bi, 27325), X1776 (ATCC No. 31244. X2282 and RR1 (ATCC Mp / 31343). Mammalian cell lines, exemplary include COS-7 cell , mouse L cells and CHP cells See Sambrook (1989), supra and Ausubel et al., 1987 supplements, supra It is possible to use various expression vectors to express the DNA encoding IL-10. for the expression of recombinant proteins in prokaryotic or eukaryotic cells Preferred vectors include the pcD vectors described by Okaya a et al., Mol. Cell. Biol. 3: 180 (1983); and Takebe et al., Mol. Cell. Biol. 8: 466 (1988). Other mammalian expression vectors based on SV40 include those described in Kaufman et al., Mol. Cell. Biol. 2: 1304 (1982) and U.S. Patent No. 4,675,285. These SV40-based vectors are particularly useful in monkey cells COS-7 (ATCC No. CRL 1651), as well as in other mammalian cells such as mouse L cells, see also Powels et al. (1989 and supplements) Cloning Vectors: A Laboratory Manual, Elseiver, New York. IL-10 can be produced in soluble form, as a secreted product of transformed or transfected yeast, insect or mammalian cells. The peptides can then be purified by the normal procedures that are known in the art. For example, the purification steps could include precipitation with ammonium sulfate, ion exchange chromatography, gel filtration, electrophoresis, affinity chromatography and the like. See Methods in Enzymology Purification Principles and Practices (Springer-Verlag, New York). Otherwise, IL-10 can be produced insoluble, as aggregates or inclusion bodies. IL-10 in this form is purified by standard procedures that are well known in the art. Examples of the purification steps include separating the inclusion bodies from the disrupted host cells by centrifugation and then solubilizing the inclusion bodies with chaotropic agent and reducing agent so that the peptide takes a biologically active conformation. For the specificities of these procedures, see, for example Winkler et al., Biochemistry 25: 4041 (1986), Winkler et al., Bio / Technology 3: 9923 (1985); Koths et al., And U.S. Patent No. 4,569,790. The nucleotide sequences that are used to transfect the host cells can be modified using standard techniques to prepare IL-10 or fragments thereof with a variety of desired properties. This modified IL-10 may vary from the sequences that occur in the natural state at the level of the primary structure, for example by amino acids, insertions, substitutions, deletions and fusions. These modifications can be used in various combinations to produce the final modified protein chain. Variations in amino acid sequences can be prepared with various objectives in mind, including increasing the serum half-life, facilitating purification or preparation, improving therapeutic efficacy and reducing the severity or presence of side effects during therapeutic use. Variations in amino acid sequences are usually predetermined variants that are not found in nature, although others may be post-translational variants. These variants can be used in this invention as long as they retain the biological activity of IL-10. Modifications of the sequences encoding the polypeptides can be carried out easily by various techniques, such as site-directed mutagenesis (Gillman et al., Gene 8:81 (1987)). Most of the modifications are evaluated by routine exploration in a trial suitable for the desired characteristics. For example, U.S. Patent No. 5,231,012 describes various in vitro assays suitable for measuring the activity of IL-10.

Preferably, human IL-10 is used for the treatment of humans, although it may be possible to use viral IL-10. Most preferably, the IL-10 used is recombinant human IL-10. The preparation of human IL-10 has been described in U.S. Patent No. 5,231,012. The cloning and expression of viral IL-10 (BCRF1 protein) of Epstein-Barr virus has been described by Moore et al., Science 248: 1230 (1990). For examples of procedures and assays for determining the activity of IL-10, see U.S. Patent No. 5,231,012. This patent also provides proteins having IL-10 activity and the production of these proteins which includes recombinant and synthetic techniques. To prepare pharmaceutical compositions of IL-10 for the practice of this invention, IL-10 is mixed with a pharmaceutically acceptable carrier or excipient which is preferably inert. A pharmaceutical carrier can be any compatible non-toxic substance to deliver the polypeptide to a patient. The preparation of these pharmaceutical compositions is known in the art; see, for example, Remington's Pharmaceutical Sciences and the U.S. Pharmacopoeia: National Formulary, Mack Publishing Company, Easton, PA (1984). The compositions can be ingested orally or injected into the body. Formulations for oral use include compounds to protect the polypeptides of the proteases found in the gastrointestinal tract. Usually, the injections are intramuscular, subcutaneous, intradermal or intravenous. Otherwise, intra-articular injection or other routes can be used in appropriate circumstances. When administered parenterally, the compositions can be formulated in an injectable unit dosage form (solution, suspension, emulsion) in association with a pharmaceutical carrier. For example, IL-10 can be administered in aqueous vehicles such as water, saline or buffered vehicles with or without various additives and / or diluting agents. Examples of suitable carriers are normal saline, Ringer's solution, dextrose solution and Hank's solution. Non-aqueous carriers such as fixed oils and ethyl oleate can also be used. A preferred carrier is 5% dextrose / saline. The carrier may contain minor amounts of additives as substances that promote isotonicity and chemical stability, for example buffer and preservative solutions. However, the IL-10 in the composition is preferably formulated in purified form, substantially free of aggregates and other proteins, furthermore, it should be noted that a suspension, such as a zinc suspension, can be prepared to include the polypeptide. This suspension may be useful for subcutaneous (SC) or intramuscular (IM) injection. It is considered that the lesion caused by ischemia-reperfusion originates, at least in part, by the release of excessive amounts of proinflammatory cytokines, such as TNF-a, 11-1, IL-6 and IL-8. Examples 1 and 2 were performed to test this theory and the effect that IL-10 has on visceral injury due to ischemia-reperfusion. Example 3 establishes the application of the invention to a human patient suffering repair of aortic aneurysm. Example 4 was performed to demonstrate that IL-10 will attenuate lung and skeletal muscle injury after ischemia-reperfusion of the hind limbs in a rat model.

EXAMPLE 1 Initial studies prospectively investigated the associative relationship between proinflammatory cytokine response and morbidity and mortality following ischemia and re; -visceral fusion in humans by measuring proinflammatory cytokine levels in patients undergoing thoracoabdominal or infrarenal aortic aneurysm repair, and comparing these results for the incidence of postoperative organ dysfunction.

Sixteen human patients undergoing elective repair of a thoracoabdominal aortic aneurysm and nine patients undergoing elective infrarenal aortic aneurysm repair allowed arterial blood sampling for proinflammatory cytokine measurements. Each thoracoabdominal aortic aneurysm was repaired through an incision in the left flank using a retroperitoneal method. The diaphragm was divided in a circumferential way, allowing exposure of the descending thoracic aorta. Before transverse clamping, each patient received mannitol (0.5 g / kg) and solumedrol (15 mg / kg). Depending on the location of the aneurysm, the visceral arteries were cooked over the graft as a Carrel patch or as part of the proximal anastomosis with wide posterior tapering towards the graft. Once the repair was completed, the coagulation products (platelets and freshly frozen plasma) were infused as needed. After the operation, the catheter was placed in the lumbar spinal column and the cerebral spinal fluid was drained to maintain the intrathecal pressure at 5-10 cm dr. Water. The infrarenal abdominal aortic aneurysms were repaired through the peritoneum using normal surgical techniques, and the aorta was reconstructed using a straight tube graft for the aortic bifurcation or a bifurcated graft for the bifurcation of the internal / external iliac artery. In both groups of patients, arterial blood samples (7ml) were obtained after the induction of anesthesia, shortly before placing the aortic cross-clamp, before the clamp was released and at time intervals (1, 2). , 4, 6 to 8, 14 hours and daily for 7 days) after reperfusion. Clinical and laboratory data were collected as proposed from all patients to determine preoperative risk factors and postoperative organ dysfunction patterns. The data collected included operative parameters (total operative time, aortic cross-clamping time, calculated blood loss, intraoperative complications), the post-operative course (complications, organ dysfunction), and causes of death. Laboratory values were analyzed during the initial 7 days after surgery to focus on the lesion associated with tissue ischemia-reperfusion after repair of the thoracoabdominal and infrarenal aortic aneurysm. Postoperative pulmonary dysfunction was defined as the need for mechanical ventilation assistance with positive pressure for more than 7 days while postoperative hepatic dysfunction was defined as maximum levels of lactate dehydrogenase (LDH) greater than 500 U / L and transaminase levels in serum (AST / ALT) greater than 200 U / L or an increase in total bilirubin levels greater than 3mg / dl. Renal dysfunction was defined as an increase in serum creatinine of 2 mg / dL or more over the preoperative baseline, while a platelet content of less than 50000 / mm or a decrease in leukocyte count below 4500 / mm3 indicated the presence of hematopoietic dysfunction. Patients with two or more organ systems that met these criteria were designated with multiple system organ dysfunction (DOMS). Freshly frozen plasma samples were assayed for the TNF-α, IL-1, IL-6, IL-8 and TNF-α shed [sic] receptors (p55 and p75) by ELISA. The sensitivity of the TNF-α, IL-1, IL-6, IL-8, p55 and p75 assays are 14, 10, 27, 313, 14 and 17 pg / ml, respectively. Mortality and morbidity data of the sixteen patients who underwent thoracoabdominal aortic aneurysm repair and the nine patients undergoing infrarenal aortic aneurysm repair are reported in Table 1.

TABLE 1 Incidence of organ dysfunction after thoracoabdominal or infrarenal aortic aneurysm repair. The data presented show that the frequency of pulmonary dysfunction and DOMS after thoracoabdominal aortic aneurysm repair was significantly higher after abdominal aortic aneurysm repair.

Aortic aneurysm Aortic thoracoabdominal aneurysm (n = 16) infrarenal (n = 9) Mortality 19% 0% Pulmonary dysfunction 56% * 11% Tracheostomy 25% 0% Renal dysfunction 38% ** 0% Dialysis 13% 0% Hepatic dysfunction 31% 0% Hematopoietic dysfunction 38% ** 0% Leukopenia 13% 0% DOMS 44% 0% * p < 0.05 by Fisher's exact test ** 9 = 0.057 by Fischer's exact test Three patients died after thoracoabdominal aortic aneurysm repair, two from DOMS and one from cardiac arrest. Pulmonary dysfunction occurred in 9 patients and finally the placement of a temporary tracheostomy was necessary in 4 patients. Renal dysfunction developed in 6 patients and hemodialysis was necessary in two of them. Hepatic dysfunction, thrombocytopenia and leukopenia developed after repair of thoracoabdominal aortic aneurysm in 5, 6 and 2 patients, respectively, and lower limb dysfunction due to spinal cord injury occurred in 2 patients. On the other hand, there were no operative deaths after repair of infrarenal aortic aneurysm (Table 1). Pulmonary dysfunction occurred in only one patient and there was no evidence of renal, hepatic, hematopoietic or lower extremity dysfunction in any patient. The maximum plasma cytokine responses in groups of patients are reported in Table 2.

TABLE 2 Maximal concentrations of proinflammatory cytokine after thoracoabdominal or infrarenal aortic aneurysm repair. Plasma samples were obtained at 0, 1, 2, 4, 6-8, 24, 48, 72 hours and daily for up to 7 days after thoracoabdominal or infrarenal aortic aneurysm repair. The maximum concentrations are reported below. The levels of all proinflammatory cytokines were significantly higher in patients after thoracoabdominal and infrarenal aortic aneurysm repair. (p <0.05) Aortic aneurysm Aortic thoracoabdominal aneurysm (n = 16) infrarenal (n = 9) TNF-α pgs / ml 161 ± 58 IO-IO IL-lb pgs / ml 133 ± 59 24 ± 10 IL-6, pgs / ml 1,280 ± 664 181 ± 108 11-8, pgs / ml 410 ± 139 137 ± 77 p55, line change 751 ± 668 204 ± 218 base in pgs / ml p75, line change 5,201 ± 1,983 3831171 base in pgs / ml C3a, μg / ml 111 ± 21 30 ± 7 All values are significantly different between the two groups, by ANOVA in two senses, p < 0.05 The concentrations of TNF-a, IL-1. IL-6, IL-8 in plasma were not detectable before surgery. After surgical repair of thoracoabdominal aortic aneurysms, a monophasic TNF-α response was detected in 11 of 16 patients (69%), figures l (a), l (b) and l (c)). The levels of TNF-a reached a maximum 4 hours after reperfusion and then gradually decreased towards the baseline during the following 24 hours. The levels of IL-6 and IL-8 were also increased in a monophasic pattern with maximum levels again present 4 hours after reperfusion in 16 (100%) and 11 (70%) of the patients, respectively; however, unlike the pattern seen with TNF-α, levels of circulating IL-6 and IL-8 decreased to baseline within 8 hours. IL-1 was also detected in a monophasic pattern in 50% of patients with thoracoabdominal aortic aneurysm, but their maximum levels were presented one hour after reperfusion and IL-1 levels returned to baseline levels of 4 to 6 hours after reperfusion. Plasma concentrations of soluble TNF-α receptors, p55 and p75, were increased after repair of the thoracoabdominal aortic aneurysm in 12 (75%) and 16 (100%) of the patients tested., respectively (figure 2). The p55 receptor concentrations reached a zenith at 24 hours and remained elevated for several days while the p75 receptor concentrations continued to increase during the initial 48 hours after reperfusion. Contrary to patients undergoing thoracoabdominal aortic aneurysm repair, the maximum serum levels of TNF-α, IL-1, IL-6, IL-8, p55 and p75 were 3 to 15 times less in patients undergoing repair. of infrarenal aortic aneurysm (Table 2 and figures 1 (a), 1 (b), l (c) and 2 (a), 2 (b) and 2 (c)).

In an effort to establish an associative relationship between the patient's clinical response and the concentrations of various proinflammatory cytokines, a retrospective analysis was made. Patients undergoing thoracoabdominal aortic aneurysm repair in whom maximal TNF-a levels were less than 150 pg / ml did not experience single or multiple organ dysfunction, while individual organ dysfunction and DOMS were common in patients whose maximum levels of TNF-a were greater than 150 pg / ml (Table 3).

TABLE 3 Relationship between postoperative organ dysfunction and maximum levels of circulating TNF-a. TNF-a < 150 pgs / ml TNF-a > 150 pgs / ml Mortality 1 cardiac death 2 deaths-DOMS Pulmonary dysfunction 0% 57% ** Renal dysfunction 0% 71% * Dialysis 0% 29% Hepatic dysfunction 0% 71% * Hematopoietic dysfunction 0% 71% * Leukopenia 0% 28% DOMS 0% 86% * * p < 0.05 by Fisher's exact test ** p = 0.07 by Fisher's exact test In addition, patients who developed DOMS after thoracoabdominal aortic aneurysm repair had higher circulating levels of all tested cytokines and soluble TNF-a receptors ( p55 and p75) compared to patients who did not have DOMS (Table 4); however, only TNF-a and p55 receptor levels were statistically different (p <0.05) while there was a trend towards higher levels of IL-1, IL-6, IL-8 and p75 receptors in patients who developed DOMS compared to patients who did not have DOMS (Table 4).

TABLE 4 Plasma concentrations of proinflammatory cytokine in patients with and without evidence of organ dysfunction in multiple systems (DOMS). The maximum plasma concentrations of TNF, IL-6, p55 and p75 were significantly higher in patients after thoracoabdominal aortic aneurysm repair with DOMS in patients after thoracoabdominal aortic aneurysm repair without DOMS or in patients after repair of the aortic aneurysm. infrarenal aortic aneurysm The values for p55 and p75 are changes from the baseline.

All values are in pgs / ml. * p < 0.05 versus no DOMS by ANOVA in two senses. nr = not reported The results presented in this table demonstrate that surgical repair of thoracoabdominal aortic aneurysms that cause visceral injury due to ischemia-reperfusion gives rise to the systemic response of the proinflammatory cytokine characterized by the appearance of TNF-a, IL-1 , IL-6 and IL-8 in blood from 1 to 4 hours after the release of the transverse fixation. In addition, the presence and magnitude of this proinflammatory cytokine response is associated with the incidence of postoperative organ dysfunction after thoracoabdominal aortic aneurysm repair. Ischemic injury and subsequent reperfusion of the viscera appears to be crucial for the induction of this systemic response of the proinflammatory cytokine, because the magnitude of the proinflammatory cytokine response is 3 to 15 times lower in patients undergoing repair. of the infrarenal aorta where visceral ischemia / reperfusion does not occur, compared with the response after thoracoabdominal aortic repair. In addition, patients with infrarenal aortic aneurysm repair, in whom visceral ischemia is prevented, have a significantly lower incidence of postoperative organ dysfunction. To further explore the direct role of acute visceral ischemia in mediating this proinflammatory cytokine response and associated organ dysfunction, 8 additional patients were studied after elective thoracoabdominal aortic aneurysm repair. However, in this case, the duration of visceral ischemia was reduced by the derivation of the left atrial-femoral artery (DAFI) and the retrograde perfusion of the visceral arteries. The DAFI provides distal blood flow during the repair of thoracoabdominal aneurysms and reduces the time of visceral ischemia. The effect of DAFI on patients undergoing thoracoabdominal aortic repair (n = 8) was prospectively examined and compared with the cytokine response, morbidity and mortality in patients undergoing repair of normal thoracoabdominal aortic aneurysm (n = 16) without the benefit of DAFI. Timely measurements of cytokine levels were made during the 48 hours of the perioperative period and cytokine levels were measured by the ELISA test. Clinical data related to postoperative pulmonary, hepatic, renal and hematopoietic dysfunction were also collected prospectively. ' Patients undergoing repair of thoracoabdominal aortic aneurysms with DAFI had shorter visceral ischemia times (1815 min compared to 45112 min) and statistically significant reductions in circulating levels of TNF-a, IL-10 and p75 (p <0.05 per ANOVA in two ways) when compared with the control group (Table 5).

TAL • '' 5 Concentrations of proinflammatory cytokine in plasma in patients with thoracoabdominal aortic aneurysm with left atrial femoral bypass (DAFI) or without DAFI. The maximum plasma concentrations of TNF-α, IL-10 and p75 were significantly higher in patients after repair of thoracoabdominal aortic aneurysm without LAFB than in patients after thoracoabdominal aortic aneurysm repair with LAFB. * p < 0.05 In addition, the incidence of pulmonary dysfunction, renal dysfunction, thrombocytopenia, organ dysfunction in multiple systems and mortality were reduced in patients undergoing DAFI, although the numbers were too small to show any statistical difference. These findings suggest that acute visceral ischemia-reperfusion injury secondary to repair of the aortic-thoracoabdominal aneurysm is associated with a high rate of morbidity and organ dysfunction in multiple systems that is not seen with similar surgical procedures that do not cause visceral ischemia. In addition, techniques aimed at reducing the duration of ischemia during aortic cross-clamping (left atrial-femoral shunt) appear to reduce the magnitude of the TNF-α and IL-1 responses.

EXAMPLE 2 Experiments were performed in mice demonstrating that pretreatment with recombinant human IL-10 can reduce lesion of distant organs in a clinically relevant model of acute visceral ischemia-reperfusion injury. The initial objective of these studies was to develop a clinically relevant model of acute ischemia-reperfusion injury that demonstrates evidence of organ injury that was dependent on an endogenous proinflammatory cytokine response that would be inhibited by a TNF-a receptor construct or a monoclonal antibody against the IL-1 receptor type I (p80) (35F5, Hoffmann-LaRoche, Nutley, NJ; .30 mice (C57BL / 6, approximately 20 grams) were anesthetized with pentobarbital.In 16 of these animals, the supercelia aorta was clamped for 30 minutes, 6 animals had their infrarenal aorta held for 30 minutes, while 8 other animals received anesthesia, incision, and bowel mobilization without aortic fixation. 16 animals were pretreated with the intreperitoneal injection of 10 mg / kg BW of TNF-bp (a TNF-a binding protein that is composed of domains extracellular of two p55 TNF-a receptors covalently linked to polyethylene glycol). Two hours after removing the aortic fixation and closing the abdominal wound, the animals were sacrificed and the infiltration of neutrophils in the lung was evaluated by the MPO content. The results are shown in figure 3. The supercelia aortic cross-clamping gave rise to a significant increase in the infiltration of neutrophils in the lung at two hours, which was not observed in animals that had the infrarenal pinched aorta. The pretreatment of the animals subjected to supercelia aortic cross-clamping with TNF-bp significantly attenuated this increase. To determine the effect of visceral ischemia-reperfusion on lung capillary function, 50 mice were anesthetized with pentobarbital, and in 34 animals the supercelia aorta was pinched for 30 minutes. 11 of these animals were pretreated with TNF-bp (10 mg / kg) while 9 were pretreated with 150 μg of a monoclonal antibody directed against the murine IL-1 receptor type I (35F5). Previously it had been reported that this antibody blocks the binding of IL-1 to the functional receptor of IL-1 type I and attenuates inflammation mediated by IL-1. The control groups consisted of a group with simulated operation (n = 10) and a group. After removing the transverse aortic clamp and the initiation of reperfusion, the animals were injected with 1 μCi of albumin labeled with I12 intravenously through the inferior vena cava. At the end of 4 hours of reperfusion the animals were sacrificed and the lungs were treated with bronchoalveolar lavage (BAL) with 1.75 ml of normal saline. The average lung permeability index was calculated as the ratio of CPM / gm LBA to CPM / gm in blood. The results are shown in Figure 4. Pretreatment with TNF-bp and with 35F5 decreased pulmonary capillary injury (p <; 0.05) presenting with 35F5 a more pronounced effect. In this way, these findings demonstrate that the lung injury secondary to transverse superalflex clamping in the mouse is a result of the endogenous production of TNF-a or IL-1. Inhibition of any of these cytokines with novel inhibitors of the TNF-a receptor or type I IL-1 can minimize lung injury secondary to visceral ischemia-reperfusion injury.

In order to demonstrate that similar effects can be obtained by immediate pretreatment with human recombinant IL-10, an additional study was performed in mice subjected to superceliac transverse aortic subjection. Visceral scans were induced in 90 C57BL / 6 female mice (20-22g) by subalveal cross-clamping supercelia 25-30 minutes. A group of 38 additional mice received sham procedures. Plasma IL-10 levels were measured by ELISA at 1, 2, 4 and 8 hours after reperfusion, and infiltration of neutrophil in lung was determined by the MPO test at two hours, as previous studies had revealed that the maximum infiltration of neutrophils occurs in the lung at two hours. 36 of the mice undergoing visceral ischemia-reperfusion were pretreated with 0.2 μg (n = 7), 2 μg (n = 13), 5 μg (n = 6), or 20 μg (n = 10) of human IL-10 recombinant. The average concentrations of IL-10 in plasma had a maximum at 9, 120 pg / ml 2 hours after 25-30 minutes of superceliac aortic cross-clamping (Figure 5). Visceral ischemia-reperfusion injury also resulted in a 6-fold increase in infiltration of neutrophils in the lung (p <0.05) (Figure 6). When mice were pretreated with exogenous IL-10, infiltration of neutrophils was significantly reduced (p <0.05 for all doses). The maximum improvements in pulmonary infiltration of neutrophils were achieved with 5 μg / mouse (250 μg / kg PC) of IL-10. Visceral ischemia-reperfusion injury associated with superalflex aortic cross-clamping favors the release of IL-10, whereas administration of exogenous IL-10 before aortic cross-clamping limits lung injury in this model of acute visceral injury due to ischemia -reperfusion. In this way, exogenous IL-10 may offer a novel therapeutic approach for the reduction of complications associated with thoracoabdominal aortic aneurysm repair and other ischemia-reperfusion injuries. The hypothetical example 3 illustrates a preferred application of the invention contemplated for the treatment of humans.

EXAMPLE 3 A 58-year-old white man presents to the emergency room of a local university hospital with 7 months of periumbilical abdominal pain and intermittent acute epigastric pain, without other significant symptoms. The patient has no history of any significant medical problem in addition to a history of atherosclerotic disease. In the physical examination it is observed that the patient has a non-soft mass, throbbing in the middle abdomen, with an audible noise or murmur. Laboratory tests include hematology, biochemistry, liver function tests, urinalysis and amylase all are within normal limits. The horizontal and vertical x-rays of the abdomen, as well as the chest X-rays are unbeatable. An abdominal CT scan with cuts through the lower thorax reveals an aortic aneurysm extending from the level of the diaphragmatic hiatus to the aortic bifurcation, 6.5 cm in the longest diameter. After obtaining informed consent, the patient prepares for surgery. One hour before the skin incision, the patient is given a single bolus administration of recombinant human IL-10 in a dose of 10 μg / kg of body weight through a permanent catheter in the middle ulnar vein. In addition, a lumbar catheter is placed to drain brain spinal fluid to maintain intrathecal pressure in 5-10 cm of water pressure. With general anesthetic inhalation, an incision is made in the left flank to obtain access to the aorta through a retroperitoneal route. The diaphragm is circumferentially divided to allow exposure of the thoracic aorta. After the patient receives intravenous doses of mannitol (0.5 g / kg) and edrol solution (15 mg / kg), the aorta is fastened in the transverse direction near the head of the aneurysm and distal to the aortic bifuration at the level of. Proximal external iliac arteries. The aorta is then reconstructed using a bifurcated graft from the level of the caudal thoracic aorta and laterally to the external iliac arteries. The celiac and superior mesenteric arteries are then sewn to the graft as a Carrel patch. The transverse clamping time and the visceral ischemia period is 42 minutes. Transverse aortic attachments after this are removed, restoring the perfusion of the viscera, pelvis and upper extremities. 3 units of packed erythrocytes and 2 units of freshly frozen plasma are infused. After the incisions are closed, the patient is transported to the intensive care unit, is intubated and receives ventilatory assistance, but is hemodynamically stable. After an unbeatable night, the patient is separated from the tubes on 'postoperative day 1. On the second day, the patient is transferred from the intensive care unit to the surgery room. The patient has returned to bowel function on postoperative day 5, and is sent home, travels without difficulty, tolerates a regular diet with his incision, healing acceptably without evidence of infection in the eL !. 7 postoperative day. Another preferred application of this invention is the administration of IL-10 to a patient from 1 to 0 hours before the patient receives a major organ transplant.

This invention is especially applicable to the treatment of ischemia-reperfusion that occurs in the visceral section of the body. Regardless of the procedure that causes or is expected to cause ischemia-reperfusion injury, the method of treatment of the invention will be considered successful if one or more of the signs or symptoms of ischemia-reperfusion injury align or do not occur.

EXAMPLE 4 The following experiments in rats demonstrate that pretreatment with exogenous human IL-10 can decrease lung and soleus muscle injury in a clinically relevant model of ischemia-reperfusion injury in hind limbs. 28 male Sprague-Dawley rats (Charles River Laboratories Wilmington, MA, approximately 350 gm) were anesthetized with pentobarbital intraperitoneally (40 mg / kg, Abbott Laboratories, Chicago, IL.) In 20 of the rats bilateral limb ischaemia occurred posterior by placing a tourniquet with rubber band across the upper thigh of both lower extremities. The interruption of arterial blood flow was confirmed by the absence of a Doppler signal in the superficial femoral artery. The remaining 8 rats received only anesthesia.

Half of the animals in each group (10 in the case group and 4 of the non-ischemic controls) were pretreated with 10 μg of recombinant IL-10. After induction of anesthesia, a catheter was placed in the right atrium through the external jugular vein to sample the blood and infusion of normal saline (1 cc / h). Recombinant IL-10 (rhIL-10, 10 μg approximately 30 μg / kg PC IV) or a comparable volume of normal saline was administered 20 minutes before the onset of ischemia or at comparable times for non-ischemic controls. After 4 hours of the ischemia, the tourniquets were removed and the extremity was reperfused. The restoration of arterial blood flow was confirmed by the presence of a Doppler signal in the superficial femoral artery. Blood (0.5 ce) was sampled at the time of central venous line placement, on reperfusion, 30 minutes after reperfusion, 60 minutes after reperfusion and every hour after reperfusion. Blood was sampled at comparable time periin the non-ischemic controls. Animals were sacrificed (pentobarbital 100 mg / kg PC IV) after 4 hours of reperfusion or at comparable times for non-ischemic controls. The soleus muscle of a posterior limb and lung were analyzed for the evaluation of neutrophil infiltration. The soleus muscle and the sequestration of pulmonary neutrophils were quantified by tissue myeloperoxidase (MPO) levels (Warren et al., 1989, J. Clin, Invest 84: 1873). The soleus muscle and the remaining lung tissue were analyzed to quantify capillary and / or cellular cellular injury. The cell lesions of the skeletal muscle and the capillary endothelium of the lung were quantified by the uptake of albumin labeled with 125 I (Welbourn et al., 1991 J. Appl. Physiol. 70: 2645). Skeletal muscle cell injury was quantified by the absorption of Tc-labeled pyrophosphate (Blebea et al., 1988 J. Vasc. Surg. 8 ': 117). The average capillary permeability index (CPI) and the skeletal muscle injury index (ILME) were calculated using the following formulas: CPI = (I125 muscle / muscle mass) / (I125 blood / blood mass) ILME = (Tc99 muscle / muscle mass) / (Tc99 blood / blood mass) Circulating bioactive TNF was measured using TNF-sensitive murine fibrosis sarcoma cells line (Van Zeed et al., 1992, PNAS 89: 4845).

Skeletal muscle injury: The results are shown in Table 6. The I / R of the hind limbs resulted in significant skeletal muscle injury. Both the average rate of capillary permeability of the soleus muscle (IPCM) and the average soleus skeletal muscle injury index (ILME) after the I / R of the hind limbs were significantly greater than the non-ischemic controls. Pretreatment of the animals with recombinant human IL-10 before ischemia of the hind limbs resulted in significantly less capillary injury of the skeletal muscle than that not significantly different from the non-ischemic control. Pretreatment with human IL-10 before ischemia also gave rise to a decrease in skeletal muscle cell injury, although the difference did not reach significant. However, again the skeletal muscle cell injury in the ischemic animals pretreated with recombinant human IL-10 was not different from that of the non-ischemic controls. Infiltration of neutrophils in skeletal muscle was not detected by the MPO assay for any of the 4 treatment groups.

Table 6 Skeletal muscle injury * significantly different from I / R (ANOVA, Duncan's multiple range test, p < .05).

Lung injury The results are shown in Table 7. Ischemia-reperfusion in the hind limbs also resulted in significant pulmonary vascular injury determined by the passage of albumin I 125 to the lungs. Both the average pulmonary capillary permeability index and the average infiltration of pulmonary neutrophils in the animals subjected to ischemia-reperfusion of the hind limbs were significantly greater than the non-ischemic controls. Pretreatment with human recombinant IL-10 significantly reduced capillary injury in lung after ischemia-reperfusion of hind limbs and PCPI values in pretreated animals were not different from non-ischemic controls. In contrast, pretreatment with recombinant human IL-10 resulted in a significant increase in myeloperoxidase content in lung after ischemia-reperfusion in hind limbs. Although it is not possible to obtain a quick explanation for these latter findings and in no sense is essential for this invention, it may well have been that IL-10 prevented the activation and degranulation of neutrophils in the lung. In this model, IL-10 may not have prevented the recruitment of neutrophils in the lung, but avoided degranulation of its toxic content, thus explaining both higher MPO levels and reducing endothelial injury. The treatment of non-ischemic controls with recombinant human IL-10 also increased the infiltration of pulmonary neutrophils, although this difference was not significant.

Table 7 Lung injury, * significantly different from I / R (ANOVA, Duncan; p < .5) # Significantly different from I / R + IL-10 (Duncan multiple range test ANOVA, p <0.05).

TNF assay: serum was assayed for circulating TNF in 6/10 rats undergoing ischemia-reperfusion and TNF levels > 50 pg / ml were detected in 77% (4/6). In contrast, significant circulating TNF levels were found in only 30% (3/10) of the ischemic animals pretreated with recombinant human IL-10. Serum TNF levels of 50 pg / ml were not detected in any of the non-ischemic control animals. These findings demonstrate that anti-inflammatory cytokine IL-10 attenuates local and distal organ injury resulting from ischemia-reperfusion of hind limbs.

The findings therefore provide direct evidence that the associated lesions are mediated in part by proinflammatory cytokines and are suitable for IL-10-based treatments.

Claims (6)

1. The use of IL-10 for the manufacture of a medically for treatment or prevention of injury caused by ischemia-reperfusion.

2. A method for the manufacture of a pharmaceutical composition for the treatment or prevention of injury caused by ischemia-reperfusion, comprising mixing a pharmaceutically acceptable carrier and an effective amount of IL-10.

The use or method of claim 1 or 2, wherein the ischemia-reperfusion injury is caused by a major organ transplant or repair of an aneurysm.

4. The use or method of claim 1 or 2, wherein the ischemia-reperfusion injury is caused by surgical repair of a thoracic aortic aneurysm, a suprarenal aortic aneurysm, liver, kidney, small intestine or pancreas transplant, hepatic surgical resections and biliary, total or partial pancreatectomy, total and partial gastrectomy, esophagectomy, colorectal surgery, vascular surgery for mesenteric vascular disease, abdominal insufflation during surgical laparoscopic procedures, blunt or punctured trauma to the abdomen including gunshot wounds, knife wounds or penetrating wounds or trauma abdominal contusion secondary to deceleration injury and / or motor vehicle accidents, hemorrhagic shock due to blood loss, cardiogenic shock to myocardial infarction or cardiac deficiency, neurogenic shock or anaphylaxis.

The use or method of claim 1 or 2, wherein the ischemia-reperfusion injury is caused by surgical repair of an abdominal aortic aneurysm, femoral, popliteal or tibial aortic bypass or by claudication or ischemia that threatens a limb, repair of popliteal or femoral aneurysm, shunts, thrombectomy or embolectomy for acute limb ischaemia or vascular trauma.

6. The method or • use of any of claims 1-5, wherein the IL-10 is human or viral IL-10.