Metabolic intervention with glp-1 to improve the function of ischemic and reperfused tissue

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DESCRIPTION

Metabolic intervention with GLP -1 to improve the function of ischemic and reperfused tissue.

Field of the invention

The present invention relates to metabolic intervention with GLP-1 to therapeutically improve the function of ischemic and reperfused tissue.

Background of the invention

Cellular damage to aerobic organ tissues is well recognized as a consequence of ischemia, whether it is endogenous as in the case of a spontaneous coronary artery occlusion, or iatrogenic such as with open heart, coronary bypass surgery, or transplantation procedures with the heart or other organs such as the lung, liver, kidney, pancreas, and gastrointestinal tract. The degree and duration of the processes that produce ischemia are related to the amount of cell death and / or reversible alteration of cell function. It is also known that much of the tissue damage actually occurs after reperfusion (ie, resumption of blood flow) and reoxygenation of the previous anoxic tissue. Reperfusion injury has been the subject of considerable recent study driven by medical advances particularly in the treatment of reperfusion injury after myocardial infarction and other myocardial remedial procedures such as coronary bypass, other open heart surgeries, as well as transplants. of organs.

As a by-product of normal aerobic respiration, electrons are routinely lost from the mitochondrial electron transport chain. Such electrons can react with molecular oxygen to generate the reactive free radical superoxide, which through other reaction steps in the presence of hydrogen peroxide and iron produces the extremely reactive toxic hydroxyl radical. Metabolically active aerobic tissues possess defense mechanisms dedicated to degrading toxic free radicals before these reactive oxygen species can interact with cellular organelles, enzymes, or DNA, the consequences of which, without such protective mechanisms, could be fatal. Defense mechanisms include the enzymes superoxide dismutase (SOD), which provides superoxide, catalase which breaks down hydrogen peroxide, and glutathione peptide which is a non-specific free radical scavenger.

Although not yet fully understood, it is believed that with ischemia of metabolic tissues and subsequent reperfusion, a complex group of processes occurs. Initially during the ischemic period, the activity of intracellular antioxidant enzymes appears to decrease, including that of SOD, catalase, and glutathione. There is also an indication that the level of xanthine oxidase activity concomitantly increases in vascular endothelial tissue during the ischemic process. The combination of enhanced ability to produce oxygen free radicals (through enhanced xanthine oxidase activity) and reduced ability to scavenge the same oxygen radicals (through reduced activity of SOD, catalase, and glutathione) considerably sensitizes the ischemic cell to that an oxidative discharge and therefore damage occurs, if these cells are later reperfused with blood and, therefore, oxygen. This oxidative discharge that occurs within seconds to minutes from reperfusion could produce reversible and irreversible damage to endothelial cells and other cells that constitute the matrix of the ischemic-reperfused organ. If, for example, the heart is the organ under consideration, reversible oxidative damage may contribute to myocardial stunning, while irreversible damage presents itself as a myocardial infarction. Along with this oxidative discharge occurs oxidative damage to cell membranes. The oxidation of lipids in cell membranes appears to play a role in the chemotaxis of neutrophils towards postischemic areas. Such activated neutrophils adhere to the vascular endothelium, induce the conversion of xanthine dehydrogenase to xanthine oxidase within these endothelial cells, and subsequently aggravate the loss of endothelial integrity. Activated neutrophils also migrate out of the vasculature into myocardial interstitial spaces where inflammatory cells can directly destroy myocytes. Additionally, disturbances in the normal mobilization of calcium from the sarcoplasmic reticulum as a consequence of ischemia-reperfusion contribute to the reversible alteration of myocardial function called myocardial stunning.

The consequences of ischemia-reperfusion processes are reversible and irreversible cell damage, cell death and decreased functional efficiency of organs. More specifically, in the case of myocardial reperfusion injury, the consequences include myocardial stunning, arrhythmias, and infarction, and as a result, cariogenic shock and, potentially, congestive heart failure.

The paradox of cell damage associated with a limited period of ischemic anoxia, followed by reperfusion is that cell damage and death appears not only likely to be the result of the period of oxygen deprivation, but also as a consequence of reoxygenation. of tissues will become highly sensitive to oxidative damage during the ischemic period. Reperfusion damage begins with the initial oxidative discharge immediately after flow begins again and continues to worsen for a number of hours as inflammatory processes develop in the same postischemic tissues. Efforts have been shown

ES 2 233 366 T5 dedicated to reducing the sensitivity of post-toxic cells to oxidative damage and, additionally, efforts to reduce inflammatory responses in these same tissues reduce reversible and irreversible damage to post-toxic reperfused organs. A combination of procedures to reduce both the initial oxidative discharge and the subsequent inflammation associated with the damage can provide synergistic protection against reperfusion injury.

With respect to the treatment of ischemia coincident with MI patients, the treatments currently used are the administration of thrombolytics, such as streptokinase and t-PA, and angioplasty. US Patent No. 4,976,959 discloses the administration of t-PA and SOD to inhibit tissue damage during reperfusion and / or percutaneous transluminal coronary angioplasty coinciding with ischemia to restore regional blood flow. Thus, increasing numbers of patients are being exposed to the likelihood of reperfusion injury and its effects, particularly cardiac patients.

Reperfusion injury to organs other than the heart generally manifests as a considerable reduction in the effectiveness of function, a consequence of which may be premature degeneration of the organ, or it simply stops functioning. Additionally, transplanted organs experience increased rejection rates if there is significant underlying reperfusion injury.

As briefly described above, although the precise mechanism of reperfusion injury has not been clearly defined, a large body of data, most of which has been accumulated in various cardiac model studies, indicates that the generation of free radicals derivatives of oxygen, including superoxide anion (O₂)^-, the hydroxyl free radical (.OH) and H₂OR₂, occurs as a consequence of the reintroduction of molecular oxygen with reperfusion and plays an important role in tissue necrosis. Agents that decrease the production of these oxygen-derived free radicals (including allopurinol and deferroxamine) or increase the degradation of these materials, such as superoxide dismutase, catalase, glutathione, and copper complexes, appear to limit infarct size and may also enhance the recovery of left ventricular function from cardiac stunning.

The use of metabolic intervention as therapy specifically during acute myocardial infarction is well established, although not without controversy. There is abundant experimental and clinical evidence supporting the use of glucose-insulin-potassium (GIK) infusion, the main form of metabolic intervention, after acute MI, particularly after the success of the Swedish DIGAMI study (MaZmberg, K, and DIGAMI Study Group (1997) Prospective randomized study of intensive insulin treatment on long term survival alter acute myocardial infarction in patients with diabetes mellitus. Brit. Med. J. 314, 1512-1515). The DIGAMI study emphasized the efficacy of an insulin glucose infusion for acute MI in diabetic patients, but this type of therapy has never been suggested or used for reperfusion.

Therefore it can be seen that there is a need for a safe and effective composition that has broad applicability to prevent or ameliorate the deleterious effects of ischemia and reperfusion for tissues in general, especially organic tissues and, including but not limited to the myocardium. . A main object of the present invention is to satisfy this need.

Another object of the present invention is to provide a method for treating ischemia and reperfusion without the side effects that normally accompany currently available therapies.

Yet another object of the present invention is to provide a pharmaceutically acceptable carrier composition that can be used for intravenous administration of the compositions of the present invention without causing any undesirable side effects and without adversely affecting antigenic or immune stimulatory properties.

These and other objects and benefits of the present invention will be apparent to those skilled in the art from the following description and the appended claims.

In the publication "Circulation vol. 98, 1998, pp. 2223-2226 "the treatment of myocardial infarction with GIK (glucose-insulin-potassium) in the form of an acute reperfusion therapy is disclosed. International patent application No. WO 9808531 discloses the possibility of substituting GIK for GLP-1 in the treatment of acute myocardial infarctions (AMI), however such treatment still requires the administration of glucose and even potassium in some cases.

Summary of the invention

The present invention provides the use of a composition including GLP-1, or a biologically active analog thereof, and a pharmaceutically acceptable carrier, for the manufacture of a medicament for the treatment of individuals in need of amelioration of organic tissue damage caused by reperfusion of blood flow after a period of ischemia, such treatment does not include co-administration of glucose.

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Detailed description of the invention

GLP-1 is a glucose-dependent insulinotropic hormone that effectively enhances peripheral glucose uptake without inducing dangerous hypoglycemia. Furthermore, GLP-1 substantially suppresses glucagon secretion, independent of its insulinotropic action, and thus produces a potent reduction in plasma free fatty acid (FFA) levels substantially more than can be achieved with insulin. High levels of FFA have been suggested to be an important toxic mechanism during myocardial ischemia.

Currently, the inventors have developed the concept of GLP-1 as a metabolic therapy for ischemia reperfusion injury. This development was based on the existence of two clinical situations in which ischemia-reperfusion is a routine and potentially dangerous process: thrombolytic procedures for acute MI and cardiac reperfusion after ischemic cardioplegia during cardiac surgery. Furthermore, recent experimental and clinical data have established that the ischemia-reperfusion phenomenon responds particularly to metabolic therapy with GIK infusion, even more than isolated ischemia without reperfusion (Apstein, CS (1988) Glucose-insulin-potassium for acute myocardial infarction. Remarkable results from a new prospective, randomized trial. Circulation 98, 2223-2226).

The two most important therapeutic advances in the treatment of acute ischemia coinciding with MI in the past decade have been the introduction of thrombolysis and β-blockade. However, despite this global success, thrombolysis studies have revealed an excess of early mortality that has been attributed to reperfusion-induced injury and myocardial stunning. The mechanisms underlying stunning are complex, but an emerging consensus is that they are likely related to intracellular acidosis that impairs the function of Ca pumps.²⁺sarcolemmal and Ca overload²⁺cytosolic. The net result is impaired myocardial contractile function leading to decreased mechanical efficiency, as well as ventricular reperfusion arrhythmias. Furthermore, recent research has established that intracellular acidosis, in turn, is due to an imbalance between glycolysis and complete glucose oxidation, in the sense that the rate of glycolysis is not coupled to the oxidation of pyruvate (the final product glycolysis) in the TCA cycle. This lack of coupling results in the production of H⁺ net due to the conversion of pyruvate to lactate. The most likely cause of this imbalance is the presence of high levels of free fatty acids (FFA) in plasma, which preferentially enter the mitochondria and inhibit pyruvate oxidation, a mechanism that is carefully responsible for the well-established observation of that hearts perfused with LGA are less able to recover in the reperfusion phase than hearts perfused with glucose. It has been discovered, and is one of the bases of this therapeutic invention, that GLP-1 suppresses FFA beyond what is expected with insulin, which is at the level of 50% suppression, and the suppression of FFA produced by GLP-1 can up to 90%.

These considerations have strengthened the inventors' conviction to treat ischemia-reperfusion with glucagon-like peptides. It is well established that during normal perfusion and adequate oxygenation, the heart is dependent on aerobic metabolism and uses FFA as its preferred fuel. In contrast, during ischemia (reduced blood flow) or hypoxia (tension of O₂ reduced), the β-oxidation of fatty acids is altered because they are strict aerobes) and the continued supply of ATP is increasingly dependent on anaerobic glycolysis. During the ischemic period, glucose-insulin is beneficial because it enhances glucose uptake and stimulates glycolysis, thereby providing ATP for the maintenance of essential membrane functions, especially ion transport. Furthermore, glucose-insulin suppresses lipolysis in adipose tissue, in such a way that the levels of FFA and the uptake of FFA in the myocardium are reduced. High levels of FFA are toxic to the ischemic myocardium, both through direct detergent effects on the membrane and increases in APMc, and through the accumulation of acylcamitin, which inhibits Ca pumps.²⁺. The net effect is the alteration of ion exchange, Ca overload²⁺cytosolic, and resulting impaired contractile function and arrhythmias.

During the reperfusion period, glucose-insulin is beneficial because, as explained above, this therapy can alleviate the metabolic imbalance that produces lightheadedness. This is achieved by direct stimulation of PHD and hence pyruvate oxidation, and indirectly by reduced FFA uptake and therefore improved pyruvate-FFA oxidation ratio.

From the above description it is evident the dual action of glucose uptake and metabolism enhanced by glucose-insulin, and reduced levels of FFA have substantial therapeutic potential in reperfusion. Some authors have expressed concern that during deep, essentially zero-flow ischemia, end products of glycolysis, ie lactate, accumulate due to inadequate "flushing". Lactate accumulation, in turn, leads to high intracellular proton concentrations and the failure of NADH reoxidation; high levels of [H⁺] and NADH / NAD ratios⁺ inhibit productive glycolysis. Under these circumstances, glucose can be toxic to cells, because ATP is actually consumed in the production of fructose-1,6-bisphosphate, and [H⁺] high may aggravate myocyte necrosis (Neely, JR, and Morgan, HE (1974) Relationship betwenn carbohydrate and lipid metabolism and the energy balance of heart muscle. Ann. Rev. Physiol. 36, 413-459). However, these ideas have not been confirmed with experimental and clinical data, indicating that glucose-insulin produces beneficial results.

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Without wishing to be bound by theory, the likely explanation for this is that in humans, acute spontaneous ischemia is not a zero-flow ischemic condition, but rather represents a low-flow ischemic region in which residual perfusion It is suitable for substrate distribution and lactate washing. This idea has provided a powerful physiological rationale for the use of metabolic therapy in ischemia-reperfusion.

Modern cardiac surgery, whether it involves heart valve replacement or coronary artery bypass graft (CABG), typically requires hypodermic cardioplegic arrest, aortic clamping, and cardiopulmonary bypass during surgery. Thus, effectively, routine cardiac surgery induces a state of elective global ischemia after reperfusion, potentially exposing the heart to all the attendant risks and injuries peculiar to myocardial ischemia-reperfusion. Therefore, prevention of myocardial injury during and after cardiac operations is an important issue. Elective cardioplegic ischemia following reperfusion has obvious parallels with the ischemia-reperfusion found during acute MI after revascularization, and thus many of the pathophysiological principles considered in the previous sections also apply during cardiac surgery. However, there are notable differences between surgical cardioplegic ischemia-reperfusion and MI-associated ischemia-reperfusion. During surgery, the heart is stopped (cardioplegia) and infused with a cold (hypodermic) solution designed to optimize myocardial preservation. After the completion of the surgery, the heart is reactivated and reperfused with oxygenated blood at body temperature. This produces a sequence of hypodermic ischemia and normothermic reperfusion, which can prevent the accumulation of high tissue levels of H + and lactate. Furthermore, unlike acute MI, hypodermic cardioplegia represents a state of global ischemia, zero flow, followed by global reperfusion.

In the inventors' previous application (Serial No. 60 / 103,498), of which this is a continuation in part, the authors have reviewed the disadvantages of glucose-insulin infusions and the disadvantages of substituting these with GLP infusions. -1, which is safer than insulin. In summary, GIK infusions carry significant risks of both hypoglycemia and hyperglycemia, and are technically demanding and intensive care. The dangers of hypoglycemia are obvious.

In contrast, these risks do not exist with a GLP-1 infusion. Glucagon-like peptide amide (7-36) (GLP-1) is a naturally occurring insulinotropic peptide derived from the intestine that constitutes a major component of the so-called incretin effect. GLP-1 exerts its main effect on pancreatic endocrine cells, where (1) it regulates the expression and secretion of insulin by β cells in a glucose-dependent manner; (2) stimulates the secretion of somatostatin; and (3) suppress glucagon secretion by α cells. Although not formally resolved, the strong glucagonstatic effect is presumed to occur from one or all of the following: (1) direct suppression by stimulation of GLP-1 receptors on a cell, although this is unlikely; (2) paracrine suppression of glucagon secretion by release within the islets of somatostatin; or (3) paracrine suppression by release within the islets of insulin. Whatever the cellular mechanism, GLP-1 is unique in its ability to simultaneously stimulate insulin secretion and inhibit glucagon release. Although a therapeutic insulin infusion also inhibits glucagon release, this effect is not as potent as that of GLP-1, which exerts a direct, paracrine inhibition within glucagon secretion islets.

The dual ability of GLP-1 to energetically stimulate insulin release and inhibit glucagon secretion, along with strict dependence on glucose for its insulinotropic action, endows this molecule with a unique therapeutic potential in the direction of ischemia-reperfusion. First, GLP-1 strongly stimulates endogenous insulin secretion and therefore can be used to achieve all the beneficial actions attributed to insulin infusion in the metabolic treatment of ischemia reperfusion. Although high-dose GIK infusions typically contain 25 - 33% glucose and 50 - 100 U insulin / L, the requirement for the introduction of hyperglycemia per se to achieve therapeutic efficacy, versus only providing a metabolic means for safe administration from high doses of insulin, it is not clear. Adequate blood glucose levels are likely to be required to allow for substrate distribution, but this does not necessarily imply a need for hyperglycemia and should not be underestimated by the fact that insulin exerts important effects other than glucose uptake.

Glucose is not required as a safety measure, as blood levels <3.5 mM invalidate the insulin-stimulating GLP-1 activity, thus protecting against the dangers of hypoglycemia.

Second, GLP-1 exerts a powerful glucagonstatic effect, which together with its insulinotropic action will lead to a strong suppression of FFA. One of the main benefits of glucose-insulin infusions is the reduction of circulating FFA levels and the suppression of FFA uptake. FFA and their metabolites have direct toxic effects on the ischemic myocardium as well as during the reperfusion period, when they contribute to stunning, and therefore the reduction of FFA levels is a main therapeutic objective of metabolic intervention in ischemia-reperfusion, objective of metabolic intervention in ischemia - reperfusion. Since glucagon is a potent stimulus of adipose tissue lipolysis and FFA production, GLP-1-mediated glucagon suppression also

ES 2 233 366 T5 increases the insulin-induced reduction in circulating FFA. Thus, GLP-1 therapy is superior to glucose-insulin infusion in this regard. In fact, preliminary data with healthy volunteers indicate that an intravenous infusion of GLP-1 will reduce fasting plasma FFA levels to <10% of control values.

GLP-1 should be effective in most patients without requiring simultaneous glucose administration.

In addition, it may be necessary to administer potassium to correct deviations of excess potassium in the cell compartment.

In addition to GLP-1 or its biological analogs, therapy may include the use of free radical scavengers such as glutathione, melatonin, vitamin E, and superoxide dismutase (SOD). In such combinations the risk of reperfusion injury is further reduced.

The term "GLP-1", or glucagon-like peptide, includes mimetics, and, as used in the context of the present invention, may comprise glucagon-like peptides and related peptides and glucagon-like peptide-1 analogs that are bind to a glucagon-like peptide receptor protein (GLP-1) such as GLP-1 amide receptor protein (7 36) and has a corresponding biological effect on insulin secretion as GLP-1 amide (7- 36), which is a native form, biologically active GLP-1, see Goke, B and Byrne, M, Diabetic Medicine, 1996, 13: 854-860. GLP-1 receptors are cell surface proteins found, for example, on pancreatic β-producing cells insulin. Glucagon-like peptides and analogs will include species that have insulinotropic activity and that are agonists, i.e., activate, of the GLP-1 receptor molecule and its second messenger activity on, among others, insulin-producing pancreatic β cells. Glucagon-like peptide agonists showing activity through this receptor have been described: EP 0708179A2; Hjorth, SA et al., J. Biol. Chem. 269 (4B): 30121-30124 (1994); Siegel, EG et al., Amer. Diabetes Assoc. issue 57^to Scientific Sessions, Boston (1997); Hareter, A. et al., Amer. Diabetes Assoc. 57th Edition Scientific Sessions, Boston (1997); Adelhorst, K. et al., J. Biol. Chem. 269 (9): 6275-6278 (1994); Deacon CF et al., 16th International Diabetes Federation Congress Abstracts, Diabetologia Supplement (1997); Irwin, DM et al., Proc. Natl. Acad. Sci. USA 94: 7915-7920 (1997); Mosjov, S., Int. J. Peptide Protein Res. 40: 333-343 (1992). Glucagon-like molecules include polynucleotides that express GLP-1 agonists, that is, activators of the GLP-1 receptor molecule and its second messenger activity found, among others, in insulin-producing pancreatic β cells. GLP-1 mimetics that are also agonists include, for example, chemical compounds specifically designed to activate the GLP-1 receptor. Glucagon-like peptide 1 antagonists are also known, see, for example, Watanabe, Y et al., J. Endocrinol. 140 (1): 45-52 (1994), and include exendin (9-39) amine, an analog of exendin, which is a potent antagonist of GLP-1 receptors (see, for example, WO 97 / 46584). Recent publications disclose the GLP-1 Black Widow and the GLP-1 Ser², see GG Holz, JF Hakner / Comparative Biochemistry and Physiology, part B 121 (1998) 177-184 and Ritzel, et al., A synthetic glucagon - like peptide-1 analog with improved plasma stability, J. Endocrinol October 1998 159 (1): 93-102.

Additional embodiments include chemically synthesized glucagon-like polypeptides, as well as any polypeptides or fragments thereof that are substantially homologous. "Substantially homologous", which can refer to both nucleic acid and amino acid sequences, means that a particular target sequence, eg, a mutant sequence, varies from a reference sequence by one or more substitutions, deletions, or additions. , the net effect of which does not result in an adverse functional difference between the reference and object sequences. For the purposes of the present invention, in sequences having more than 50% homology, and preferably more than 90% homology, the equivalent biological activity with respect to the enhancement of β-cell responses to the Plasma glucose levels and equivalent expression characteristics are considered substantially homologous. For the purposes of determining homology, truncation of the mature sequence must be ruled out. Sequences that have lower degrees of homology, comparable bioactivity, and equivalent expression characteristics are considered equivalent.

Mammalian GLP and glucagon peptides are encoded by the same gene. In ileus the phenotype is processed into two main classes of GLP peptide hormones, designated GLP-1 and GLP-2. Four GLP-1 related peptides are known to be processed from phenotypic peptides. GLP-1 (1-37) has the sequence His Asp Glu Phe Glu Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly ( SEQ ID NO: 1). GLP-1 (1-37) is amidated by post-translational processing to produce GLP-1 (1-36) NH2 having the sequence His Asp Glu Phe Glu Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg (NH2) (SEQ ID NO: 2); or it is enzymatically processed to produce GLP-1 (7-37) having the sequence His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly ( SEQ ID NO: 3). GLP-1 (7-37) can also be amidated to produce GLP-1 (7-36) amide which is the natural form of the GLP-1 molecule, and which has the sequence His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg (NH2) (SEQ ID N °: 4) and in the same natural form of the GLP-1 molecule.

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Intestinal L cells secrete GLP-1 (7-37) (SEQ ID NO: 3) and GLP-1 (7-36) NH₂ (SEQ ID NO: 4) and a ratio of 1 to 5, respectively. These truncated forms of GLP-1 have short half-lives in situ, that is, less than 10 minutes, and are inactivated by an aminodipeptidase IV to produce Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly (SEQ ID NO: 5); and Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg (NH2) (SECID N °: 6), respectively. The peptides Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg Gly (SEQ ID No: 5) and Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly Arg (NH₂) (SEQ ID NO: 6), it has been speculated that they affect hepatic glucose production, but do not stimulate the production or release of insulin by the pancreas.

There are six peptides in Gila monster venoms that are homologous to GLP-1. Their sequences are compared to the GLP-1 sequences in Table 1.

TABLE 1

to. HAEGTFTSDVSSYLEGQAAKE FIAWLVKGR NH2

b. HSDGTFTSDLSKQMEEEAVRL FIEWLKNGGPSSGAPPPSN H2

c. DLSKQMEEEAVRLFIEWLKNG GPSSGAPPPS NH2

d. HGEGTFTSDLSKQMEEEAVRL FIEWLKNGGPSSGAPPPS NH2

and. HSDATFTAEYSKLLAKLALQK YLESILGSSTSPRPPSS

F. HSDATFTAEYSKLLAKLALQK YLESILGSSTSPRPPS

g. HSDA 1FT EEYSKLLAKLALQKY LAS 1 LGSRTSPPP NH2

h. HSDA 1FT QQYSKLLAKLALQKY LAS ILGSRTSPPP NH2 a = GLP-1 (SEQ ID No. 4).

b = Exendin 3 (SEQ ID No. 7).

c = Exendina 4 (9-39 (NH₂(SEQ ID No. 8).

d = Exendin 4 (SEQ ID No. 9).

e = Helospectin I (SEQ ID No. 10).

f = Helospectin II (SEQ ID No. 11).

g = Helodermine (SEQ ID NO: 12).

h = Q⁸, Q⁹ Helodermine (SEQ ID No. 13)

The main homologies as indicated in the areas summarized in Table 1 are: peptides c and h are derived from b and g, respectively. The 6 naturally occurring peptides (a, b, d, e, f and g) are homologous at positions 1, 7, 11 and 18. GLP1 and exendins 3 and 4 (a, b and d) are also homologous at positions 4 , 5, 6, 8, 9, 15, 22, 23, 25, 26 and 29. At position 2, A, S and G are structurally similar. At position 3, residues D and E (Asp and Glu) are structurally similar. At positions 22 and 23 F (Phe) and I (Ile) are structurally similar to Y (Tyr) and L (Leu), respectively. Similarly, at position 26 L and I are structurally equivalent.

Thus, of the 30 GLP-1 residues, exendins 3 and 4 are identical in 15 positions and equivalent in 5 additional positions. The only positions where radical structural changes are evident are at residues 16, 17, 19, 21, 24, 27, 28, and 30. Exendins also have 9 extra residues at the carboxyl terminus.

GLP-1-like peptides can be prepared by solid phase chemical peptide synthesis. The GLP-1 also

ES 2 233 366 T5 can be prepared by conventional recombinant techniques using standard procedures described in, for example, Sambrook and Maniatis. "Recombinant", as used herein, means that a protein is derived from recombinant expression systems (eg, microbial or mammalian) that can be genetically modified to contain an expression gene for GLP-1 or its biologically active analogs.

GLP-1-like peptides can be recovered and purified from recombinant cell cultures by procedures including, but not limited to, ethanol or ammonium sulfate precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, interaction chromatography. hydrophobic, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (HPLC) can be used for final purification steps.

The polypeptides of the present invention can be a naturally purified product, or a product of chemical synthesis procedures, or they can be produced by recombinant techniques from prokaryotic or eukaryotic hosts (for example, by bacteria, yeast, plant cells superiors, insects and mammals in culture or in vivo). Depending on the host employed in a recombinant production process, the polypeptides of the present invention are generally non-glycosylated, but may be glycosylated.

GLP-1 activity can be determined by standard procedures, generally by screening procedures for receptor-bound activity that involve providing appropriate cells that express the GLP1 receptor on their surface, for example, insulinoma cell lines such as RINmSF cells. oo INS 1 cells. See also Mosjov, S. (1992) and EP708170A2. In addition to measuring specific binding of the tracer to the membrane using radioimmunoassay procedures, cAMP activity or glucose-dependent insulin production can also be used. In one method, a polynucleotide encoding the receptor of the present invention is used to transfect cells, which therefore express the GLP-1 receptor protein. Thus, for example, these procedures can be employed to select a receptor agonist by contacting such cells with the compounds to be screened and determining whether such compounds generate a signal, ie activate the receptor.

Polyclonal and monoclonal antibodies can be used to detect, purify, and identify GLP-1-like peptides for use in the procedures described in this specification. Antibodies such as ABGA1178 detect intact unspliced ​​GLP-1 (1-37) or N-terminally truncated GLP-1 (7-37) or (7-38) amide. Other antibodies are detected at the same end of the C-terminal of the precursor molecule, a procedure that allows by subtraction to calculate the amount of biologically active truncated peptide, that is, GLP-1 (7-37) or (7-36) amide ( Orskov et al., Diabetes, 1993, 42: 658-661; Orskov et al., J. Clin. Invest. 1991, 87: 415-423).

Other selection techniques include the use of cells that express the GLP-1 receptor, eg, transfected CHO cells, in a system that measures extracellular pH or ionic changes produced by receptor activation. For example, potential agonists can be contacted with a cell that expresses the GLP-1 protein receptor and a second messenger response, for example signal translation or ionic or pH changes, can be measured to determine whether it is or the potential agonist is not effective.

The glucagon-like peptide-1 receptor binding proteins of the present invention can be used in combination with a pharmaceutically acceptable carrier. Such compositions comprise a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes, but is not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, lactose, phosphate, mannitol, arginine, trehalose, and combinations thereof. Formulations should follow the mode of administration and are readily determined by those skilled in the art. The GLP-1-like peptide can also be used in combination with agents known in the art that enhance the in vivo half-life of the peptide in order to enhance or prolong the biological activity of the peptide. For example, a molecule or chemical moiety can be covalently attached to the composition of the present invention prior to administration thereof. Alternatively, the enhancing agent can be administered simultaneously with the composition. Still further, the agent may comprise a molecule known to inhibit the enzymatic degradation of GLP-1-like peptides can be administered simultaneously with or after administration of the GLP-1 peptide composition. Such a molecule can be administered, for example, orally or by injection.

Patients who are administered GLP-1 or its analogues in combination with the vehicle systems listed here, especially those treated before a planned process or within the first 4 hours after an ischemic process, are observed to have fewer arrhythmias. , less tissue damage and less discomfort without side effects.

From these considerations it is clear that a GLP-1 infusion can be expected to exert a major therapeutic effect in myocardial reperfusion. It is expected that GLP-1 can be administered by IV or subcutaneous administration for continuous infusion by intravenous (IV) injection 0.1 pmol / kg / min to 10 pmol / kg / min and subcutaneous

ES 2 233 366 T5 (SC) 0.1 pmol / kg / min at 75 pmol / kg / min, and single injection (bolus) by IV 0.005 nmol / kg at 20 nmol / kg and SC 0.1 nmol / kg at 100 nmol / kg are suitable levels of administration. The GLP-1 infusion can be co-administered with glucose (5%) if it is required to maintain blood glucose levels> 5 mM (to maintain effective insulin secretion). Similarly, the co-administration of potassium (K⁺), depending on the degree to which the activation of the N⁺/ K⁺ Membrane ATPase leads to K shift⁺ in the intracellular space. GLP-1 treatment will begin as early in the post-ischemic period as possible after, for example, acute spontaneous ischemia in the home or ambulance setting and before reperfusion therapies, and are continued thereafter. In the case of cardiac surgery, the GLP-1 infusion should begin 12 - 24 hours before surgery, during surgery from the start of anesthesia until aortic clamping, and immediately after clamping for a period of at least 10 less 72 hours after the operation. As explained above, co-administration of a free radical scavenger will further aid reperfusion recovery.

From the foregoing it can be seen that the invention achieves all of its stated objectives.