JP2006528208A - Treatment of hemorrhagic shock using complement 5a receptor inhibitors - Google Patents

Abstract

The present invention relates to the treatment of hemorrhagic shock and in particular to the treatment of this condition with cyclic peptides and peptide-like compounds capable of acting as antagonists of C5a receptors. In one embodiment, these compounds are active against C5a receptors on polymorphonuclear leukocytes and macrophages. Particularly preferred compounds for use in the present invention are disclosed.

Description

Detailed Description of the Invention

  This application enjoys priority from Australian Provisional Application No. 2003902354 dated May 15, 2003.

The present invention relates to the treatment of hemorrhagic shock with novel cyclic peptides and peptide-like compounds that have the ability to modulate the activity of G protein coupled receptors. This compound preferably acts as an antagonist of the C5a receptor and is active on polymorphonuclear leukocytes and macrophage C5a receptors.

BACKGROUND OF THE INVENTION All documents, including all patents or patent applications cited herein, are hereby incorporated by reference. It is not an admission that these documents are prior art. The description of the documents states what their authors claim, and Applicant reserves the right to refute the correctness and correctness of the cited documents. Even though many previous publications are cited herein, it is clearly understood that these documents do not admit that they form part of the common general knowledge in technology in Australia or other countries. It will be.

  G protein-coupled receptors are prevalent in the human body, include about 60% of known cellular receptor types, and mediate signal conduction across the cell membrane for a very wide range of endogenous ligands. This is involved in various physiological and pathological processes. This process includes, but is not limited to, the neovasculature, central and peripheral nervous system, reproduction, metabolism, immunity, inflammation, and growth disorders, as well as other cell regulatory and proliferative disorders. Substances that selectively modulate the function of G protein-coupled receptors have important medical applications. These receptors are increasingly recognized as important drug targets due to their important role in signal conduction (G protein-coupled Receptors, IBC Biomedical Library Series, 1996).

  One of the best studied G protein-coupled receptors is the receptor for C5a. C5a is the most potent known chemotactic substance, carrying neutrophils and macrophages to the site of injury and changing their morphology; inducing granulocyte depletion; calcium mobilization, vascular permeability (edema) and neutrophil Increases ball adhesion; contracts smooth muscle; stimulates release of inflammatory mediators and lysosomal enzymes including histamine, TNF-α, IL-1, IL-6, IL-8, prostaglandins and leukotrienes; oxygen radicals The formation of antibodies; and increase antibody production (Gerard and Gerard, 1994).

  Substances that limit the pro-inflammatory effects of C5a have the potential to prevent both acute and chronic inflammation, as well as associated pain and tissue damage. Such compounds act upstream from the various inflammatory mediators described above and inhibit the formation of many of them so that they can have a powerful effect in relieving or preventing inflammatory signs.

  In applicant's previous application No. PCT / AU98 / 00490, the three-dimensional structure of several analogs of human C5a C-terminus is described and this information is used as either human C5a agonists or antagonists. Used to design a new compound that binds to the receptor (C5aR). Thus, it was believed that putative antagonists required both C-terminal arginine and C-terminal carboxylate for receptor binding and antagonist activity (Konteatis et al, 1994). In contrast, we have shown that terminal carboxylate groups are generally not required for high affinity binding or antagonist activity with C5aR. We have found that a structural feature that has not been recognized before, the turn conformation, is a key recognition feature for high affinity binding to the human C5a receptor on neutrophils. This information was used to design a stringent structural template that assembles hydrophobic groups into a hydrophobic array for interaction with the C5a receptor as described in International Application PCT / AU0101427. The entire disclosure of that specification is hereby incorporated by reference.

  Shock is a hemodynamic and metabolic change that results from many causes and is characterized by a failure of the circulatory system that maintains proper perfusion of living organs with blood. This can result from improper blood volume, improper cardiac function or improper vasomotor tone. Hemorrhagic shock caused by inadequate blood volume, also known as low-dose shock or underdose shock, results from massive bleeding with a wide range of causes: For example, trauma, bleeding related to childbirth or uncontrollable as a result of nasal bleeding, blood coagulation disorders such as hemophilia, surgery related, congenital defects such as aneurysms or gastrointestinal conditions such as perforated ulcers.

  In many cases, large amounts of bleeding are difficult to treat and various treatments have been adopted in addition to blood transfusion, blood volume recovery and other normal complementary means. It includes arterial embolization, emergency surgery, and the use of pharmacological agents such as sulprostone, somatostion and vasopressin. The first treatment is aimed at stopping bleeding and replenishing disappeared blood volume, for example, transfusion and injection of isotonic or hypertonic saline or blood substitute, and the second treatment is to reduce or reduce the shock sequence. About. Treatment of hemorrhagic shock involves maintaining blood pressure and tissue perfusion until bleeding is controlled. Various resuscitation techniques are used to maintain blood pressure in trauma patients until bleeding control. However, even if maintaining blood pressure can prevent shock, bleeding can be exacerbated. Therefore, a meticulous balance in consideration of these must be maintained. If the shock is prolonged, the cardiovascular system can be damaged, cardiac output can be reduced as a result of positive feedback, and the shock can be irreversible.

  This treatment has limited effects and may have serious side effects. For example, despite successful surgery and strong care retention, repair of a ruptured abdominal aortic aneurysm (RAAA) has a mortality rate of 50-75% (Adam et al, 1999). Immunomodulating hormones (Hollis-Eden Pharmaceuticals, Inc) and various blood substitutes, such as diaspirin cross-linked hemoglobin and other hemoglobin types, are in various stages of clinical trials and have been successful.

  Hemorrhagic shock combined injury and lower trunk ischemia-reperfusion injury initiate systemic inflammatory response syndrome, which is characterized by increased microvascular permeability and neutrophil segregation, resulting in multiple organ dysfunction syndrome (MODS) . MODS is the primary cause of 70% of such deaths and the remaining major contributor (Harris et al, 1991). Using a rat model of abdominal aortic aneurysm rupture, hemorrhagic shock coupled with superior mesenteric aortic clamping leads to local intestinal and distal lung damage, and using monoclonal antibodies to CD18 integrin It has been shown that remission can be reduced by reducing neutrophil adhesion (Boyd et al, 1999).

  Reperfusion of long ischemia following the ischemic lower trunk initiates a systemic inflammatory response syndrome characterized by pro-inflammatory cytokines (Groeneveld et al, 1997), which activates circulating polymorphonuclear leukocytes (PMN) Increase (Barry et al, 1997). After lung separation of activated neutrophils, acute lung microvascular injury (Welbourne et al, 1991), acute respiratory urgency syndrome (Paterson et al, 1989) and high rates of death. Tumor necrosis factor (TNF) -α, pro-inflammatory cytokines responsible for leukocyte activation such as interleukin-6 and interleukin-8, and high circulating levels of endotoxin (Baigrie et al, 1993) are found after repair of RAAA (Roumen et al, 1993). We and other researchers have previously shown that lower limb ischemia-reperfusion injury is associated with increased intestinal permeability, endotoxemia and systemic inflammatory responses associated with acute lung injury (Roumen et al, 1993 Harkin, D'Sa et al, 2001; Harkin, Barros et al, 2001; Yassin et al, 1997).

  Severe bleeding and trauma, along with the syndrome of ischemia-reperfusion injury, activates the complement cascade, and the degree of activation of the complement system depends on the severity of the injury, the progression of multiple organ failure and subsequent death Correlate.

  The complement system is a major contributor to the inflammatory response in rupture of abdominal aortic aneurysms (Lindsay et al, 1999) and has been reported to mediate injury in experimental lower limb and intestinal ischemia-reperfusion injury ( Rubin et al, 1990; Williams et al, 1999).

  Activation products of normal complement pathways such as C5a and C3a are potent inflammatory mediators with numerous actions, including changes in vascular permeability and tension, leukocyte chemotaxis, and multi-inflammatory properties There is cell type activation. The role of complements in several inflammatory tissue injury states is supported by injury remission with anti-C5 antibodies (Piccolo et al., 1999) and C5a receptor (C5aR) antagonists (Arumugam et al, 2003). ing. However, it has been reported that lung injury due to ischemia is mediated by leukotrienes but not by complement (Klausner et al, 1989). Furthermore, the role of complements in inflammatory tissue damage after abdominal aortic aneurysm rupture is still largely unknown.

  In studies using a rat model to study hemodynamics and metabolic recovery of long and severe hemorrhagic hypertension, bleeding and resuscitation provide complement consumption, and prior depletion of circulating complement levels protects animals from shock It has been found in measurements of mean arterial blood pressure and metabolic acidosis. In contrast, administration of exogenous complement activator or inhibition of complement reduction exacerbates the damage. The authors conclude that C5a appears to play a major role, but until a substance that specifically neutralizes C5a is available without affecting the activity of the parent molecule C5, We thought that we could not confirm. In particular, C3a could be involved (Younger et al, 2001).

  According to other investigators, hemorrhagic shock and reperfusion following intestinal and lung injury can be reduced by reducing neutrophil adhesion with monoclonal antibodies to CD18 integrin (Boyd et al, 1999). The excess of inflammatory substances identified in ischemia-reperfusion syndrome means that it is very difficult to identify the most effective target for treatment. This difficulty was reflected in a wide range of candidate targets and substances discussed at the 6th International Conference on Injury, Inflammation, Shock, and Infectious Diseases in March 2004 (http: //www.trauma trauma). -Shock-sepsis-congress-munich-2004.org/lis.html).

  Therefore, there is a great demand for effective non-toxic substances that can be produced at a reasonable cost, preferably without the need for administration by injection.

  The soluble complement receptor type 1 (CR1) glycoform is proposed for use in the treatment of complement-mediated disorders and shock. The soluble CR1 fragment was a functionally active binding C3b and / or C4b and showed factor I cofactor activity depending on the region it contained. Such constructs inhibit the effects of complement activation, such as neutrophil oxidative burst, complement-mediated hemolysis and C3a and C5a production (US Pat. Nos. 5456909, No 5807844 and No 5858969). However, to our knowledge, these approved or experimental substances, particularly small molecule substances, for the treatment of shock do not target the C5a receptor.

SUMMARY OF THE INVENTION Because of the current uncertainty about the nature of the complement involved in hemorrhagic shock, an animal model of aortic aneurysm rupture, which is a condition that causes hemorrhagic shock, for possible inhibitory effects of specific complements Tested. For the first time, we show that a specific inhibitor of the C5a receptor can relieve signs of injury in an animal model of hemorrhagic shock. This is the first report on a small molecule inhibitor of the complement system used to adjust the pathogenesis in a model of hemorrhagic shock.

  In a first aspect, the present invention provides a method of treating hemorrhagic shock comprising administering an effective amount of an inhibitor of C5a receptor to a subject in need of treatment.

Preferably, the inhibitor is
(a) is an antagonist of the C5a receptor,
(b) substantially free of agonist activity, and
(c) a cyclic peptide or peptide-like compound of formula I:
Formula I

In the formula, A, H, alkyl, aryl, NH _2, NH- alkyl, N (alkyl) _2, NH- aryl, NH- acyl, NH- benzoyl, NHSO _3, NHSO ₂ - alkyl, NHSO ₂ - aryl, OH, O-alkyl or O-aryl;

B is an alkyl, aryl, phenyl, benzyl, naphthyl or indole group, or a side chain of a D- or L-amino acid such as L-phenylalanine or L-phenylglycine, but glycine, D-phenylalanine, L-homo Not the side chain of phenylalanine, L-tryptophan, L-homotryptophan, L-tyrosine or L-homotyrosine;

C is a small substituent such as a D-, L- or homo-amino acid side chain such as glycine, alanine, leucine, valine, proline, hydroxyproline or thioproline, but preferably isoleucine, phenylalanine or cyclohexylalanine Not large substituents such as side chains of

D is D-leucine, D-homoleucine, D-cyclohexylalanine, D-homocyclohexylalanine, D-valine, D-norleucine, D-homo-norleucine, D-phenylalanine, D-tetrahydroisoquinoline, D-glutamine, D -Neutral D-amino acid side chains such as glutamate or D-tyrosine, but preferably small substituents such as glycine or D-alanine side chains, large planar side chains such as D-tryptophan or Not large charge side chains such as D-arginine or D-lysine;

E is an amino acid selected from the group consisting of L-phenylalanine, L-tryptophan or L-homotryptophan, or a large substituent such as a side chain of L-1-naphthyl or L-3-benzothienylalanine, Not the side chain of D-tryptophan, LN-methyltryptophan, L homophenylalanine, L-2-naphthyl L-tetrahydroisoquinoline, L-cyclohexylalanine, D-leucine, L-fluorenylalanine or L-histidine;

F is a side chain of L-arginine, L-homoarginine, L-citrulline or L-canavanine, or a bioequivalent thereof, that is, a terminal guanidine or urea group is retained but the carbon skeleton is replaced with a different structure A side chain that reacts with the target protein in the same way as the parent group; and

X is-(CH ₂ ) _n NH- or (CH ₂ ) _n -S- (where n is an integer of 1 to 4, preferably 2 or 3);-(CH ₂ ) ₂ O-;-(CH _{2 ) 3 O-; - (CH 2} ) 3 - ;-( CH 2) 4 -; -CH 2 COCHRNH-; or CH ₂ CHCOCHRNH- (of which, R is normal or side chain of unconventional amino acids).

For C 1, both cis and trans forms of hydroxyproline and thioproline can be used.
Preferably, A is an acetamido group, an aminomethyl group or a substituted or unsubstituted sulfonamido group.
Preferably, when A is a substituted sulfonamide, the substituent is an alkyl chain of 1 to 6, preferably 1 to 4 carbon atoms or a phenyl or toluyl group.

In a particularly preferred embodiment, the compound has antagonist activity against C5aR and does not have C5a agonist activity.
The compound is preferably an antagonist of the C5a receptor on human and mammalian cells including human polymorphonuclear leukocytes and human macrophages. The compound preferably binds strongly and selectively to the C5a receptor, and more preferably has potent antagonist activity at sub-micromolar concentrations. Even more preferably, the compound has a receptor affinity IC50 <25 μM and an antagonist strength IC50 <1 μM.

  Most preferably, the compound is Compound 1 (PMX53), Compound 33 (AcF [OP-DPhe-WR]), Compound 60 (AcF [OP-DCha-FR]) described in International Patent Application No. PCT / AU02 / 01427 Or 45 (AcF [OP-DCha-WCit]), or HC- [OPdChaWR] (PMX205), AcF- [OPdPheWR] (PMX273), AcF- [OPdChaW citrulline] (PMX201) or HC- [OPdPheWR] (PMX218) It is.

  Inhibitors may be used with one or more other substances for the treatment of hemorrhagic shock. These include, but are not limited to, blood substitutes, vasopressin, somatostatin, telluripressin and nitric oxide agents.

  The compositions of the invention can be formulated for oral, parenteral, inhalation, nasal, rectal or transdermal use. However, parenteral, particularly intravenous formulations are preferred. It can be assumed that most if not all of the compounds of the present invention are stable to metabolic enzymes such as enzymes in the digestive tract, blood, lung or cells. Such stability can be readily tested by conventional methods known to those skilled in the art.

  Formulations suitable for administration by any desired route can be prepared by standard methods. See, for example, the well-known textbook, Remington: The Science and Practice of Pharmacy, Vol. II, 2000 (20th edition), A.R. Gennaro (ed), Williams & Wilkins, Pennsylvania.

  The present invention is applicable to the treatment of shock resulting from heavy bleeding from any source. This includes, but is not limited to, trauma, aneurysm rupture, uncontrollable nasal bleeding, dengue fever, hemorrhagic fever such as Lassa, Marburg, Ebola virus, uterine bleeding after childbirth, bleeding during and after surgery, digestive ulcer or Bleeding from duodenal varices, bleeding from the lower gastrointestinal tract such as diverticular bleeding, secondary bleeding from cancer invasion, bleeding from haemorrhagic features such as hemophilia and idiopathic thrombocytopenic purpura, and warfarin There are bleedings associated with the treatment of thrombolysis with drugs such as aspirin, plasminogen activator, streptokinase or urokinase.

  The present invention is not limited in any way to the treatment of a particular animal or species, but is useful for human medical treatment, and veterinary treatments, particularly pets such as cats and dogs, cattle and horses. It is useful for the treatment of zoo animals, including livestock such as sheep, and non-human primates, large bovine, feline, ungulate and canine animals.

  The compound can be administered by any suitable dose and by any suitable route. The route of administration is preferably parenteral, for example intravenous, and an effective blood concentration of the drug is achieved as quickly as possible. This is because the severity of the condition and diverting blood from non-vital organs such as the stomach reduces absorption from the intestines. In general, intravenous administration is preferred.

  Effective doses will vary depending on the nature of the condition to be treated and the age, weight and health status of the individual subject to be treated. This is at the discretion of the attending physician or veterinarian. Appropriate dose levels can be determined by trial and error testing, using methods well known in the art.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 shows a summary of mean arterial pressure and resuscitation fluid requirements for each group of animals. A. Mean arterial pressure. B. Required amount of resuscitation fluid.
FIG. 2 compares the lung permeability index (LPI) in each group of rats.
FIG. 3 shows changes in intestinal permeability over time after unclamping.

FIG. 4 shows myeloperoxidase activity in the lung and intestine of the sample. A. lung. B. Intestines.
FIG. 5 shows the levels of cytokines in the gastrointestinal tissue of samples from each group of animals. A. TNF-α. B. IL-6.
FIG. 6 shows the levels of cytokines in the lung tissue of samples from each group of animals. A. TNF-α. B. IL-6.

DETAILED DESCRIPTION OF THE INVENTION The present invention will be described with reference to the following general methods and test examples only.
In the claims and specification of the present invention, the word “comprising” or variations thereof “including”, etc., is inclusive unless the content has another necessity for wording or necessary indication. It is used to embody the meaning, ie the presence of the described characteristics, and does not exclude the presence or addition of further characteristics in various embodiments of the present invention.

  As used herein, the singular forms “a”, “an”, and “the” include the plural unless the content clearly dictates otherwise. Thus, for example, the expression “an enzyme” includes a plurality thereof, and “an amino acid” includes one or more amino acids. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are now described.

The abbreviations used herein are as follows:
AAA abdominal aortic aneurysm
Cit Citrulline
dCha D-Cyclohexylamine
DPhe D-phenylalanine
IL-6 Interleukin-6
ip intraperitoneal
iv intravenous
LPS Lipopolysaccharide
MAP mean arterial pressure
MPO myeloperoxidase
PMN polymorphonuclear granulocytes
PMSF Phenylmethylsulfonyl fluoride
sc subcutaneous
TNF-α Tumor necrosis factor-α

  Throughout the specification, the usual one-letter code and three-letter code are used to represent amino acids.

  For the purposes of this specification, the term “alkyl” means a straight, branched or cyclic substituted or unsubstituted alkyl chain of 1 to 6, preferably 1 to 4 carbon atoms. Most preferably, the alkyl group is a methyl group. The term “acyl” means a substituted or unsubstituted acyl of 1 to 6, preferably 1 to 4 carbon atoms. Most preferably, the acyl group is acetyl. The term “aryl” means a substituted or unsubstituted homocyclic or heterocyclic aryl group, wherein the ring preferably has 5 or 6 members.

  “Normal” amino acids are from the group consisting of glycine, leucine, isoleucine, valine, alanine, phenylalanine, tyrosine, tryptophan, aspartate, asparagine, glutamate, glutamine, cysteine, methionine, arginine, lysine, proline, serine, threonine and histidine. L-amino acid chosen.

  “Unusual” amino acids include, but are not limited to, D-amino acids, homoamino acids, N-alkyl amino acids, dehydroamino acids, aromatic amino acids other than phenylalanine, tyrosine and tryptophan, ortho-, meta- or para-aminobenzoic acids, ornithine Citrulline, canavanine, norleucine, γ-glutamic acid, aminobutyric acid, L-fluorenylalanine, L-3-benzothienylalanine and α, α-disubstituted amino acids.

  In general, the terms “treat” or “treatment” and the like herein refer to affecting a subject, tissue or cell to obtain a desired pharmacological and / or physiological effect. Used in. The effect may be prophylactic in the sense of completely or partially preventing the disease or its signs or syndrome and / or therapeutic in the sense of completely or partially curing the disease.

  As used herein, “treating” encompasses the treatment or prevention of disease in vertebrates, mammals, particularly humans. Preventing the development of a disease in a subject who can become a disease but has not yet been diagnosed with disease, inhibiting the disease, ie, suppressing the progression of the disease, or reducing or ameliorating the effect of the disease, ie Including degrading the action.

  The present invention involves the use of various pharmaceutical compositions useful for ameliorating disease. A pharmaceutical composition according to an embodiment of the invention comprises a compound of formula I, an analog, derivative or salt thereof and one or more pharmacologically active substances, or a combination of formula I and one or more pharmacologically active substances The product is prepared by using carriers, excipients and additives or adjuvants into a form suitable for administration to the subject.

  Commonly used carriers or adjuvants include sugars such as magnesium carbonate, titanium dioxide, lactose, mannitol, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols, and sterile water. There are solvents such as glycerol and polyhydrated alcohol. Intravenous vehicles include fluid and nutritional supplements. Preservatives include antibacterial agents, antioxidants, chelating agents and inert gases. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients including salts, preservatives, buffers, etc., such as those described below: Remington's Pharmaceutical Sciences, 20th ed. Williams & Wilkins (2000) and The British National Formulary 43rd ed. (British Medical Association and Royal Pharmaceutical Society of Great Britain, 2002; http://bnf.rhn.net). Some). The pH and exact concentration for the various components of the pharmaceutical composition can be adjusted according to routine practice in the art. See, Goodman and Gilman's The Pharmacological Basis for Therapeutics (7th ed., 1985).

  It is preferred to prepare and administer the pharmaceutical composition in dosage units. Solid dose units include tablets, capsules and suppositories. Different daily doses may be used for subject treatment, depending on the activity of the compound, the method of administration, the nature and severity of the disorder, the age and weight of the subject. However, in certain situations, higher or lower daily doses are appropriate. The dose can be administered by single administration in individual dose units or in several dose units. Alternatively, subdivided doses can be administered multiple times at particular intervals.

  The pharmaceutical composition of the present invention can be administered locally or systemically in a medically effective amount. Effective amounts for this use will, of course, depend on the severity of the disease and the weight and general condition of the subject. In general, doses used in vitro provide useful guidance on the amount useful for in situ administration of pharmaceutical compositions, and animal models can be used to determine effective doses for the treatment of cytotoxic side effects . Various considerations have been described, for example, Langer, Science, 249: 1527, (1990). Oral use formulations may be in the form of hard gelatin capsules. There, the active ingredient is mixed with an inert solid diluent such as calcium carbonate, calcium phosphate or kaolin. The formulation can also be in the form of a soft gelatin capsule. The active ingredient is mixed with water or an oily medium such as peanut oil, liquid paraffin or olive oil.

  Aqueous suspensions usually contain the active materials in admixture with excipients suitable for the manufacture of aqueous buffers. These excipients can be suspensions, for example, carboxymethyl sodium, cellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tarragant gum and gum arabic; diffusion agents or wetting agents. The diffusing or wetting agent can be:

(a) Naturally occurring phosphatides such as lecithin;
(b) a condensation product of an alkylene oxide and a fatty acid, such as polyoxyethylene stearate;
(c) a condensation product of ethylene oxide and a long-chain aliphatic alcohol, such as heptadecaethyleneoxycetanol;
(d) a condensation product of a partial ester derived from ethylene oxide and a fatty acid and hexitol, such as polyoxyethylene sorbitol monooleate, or
(e) Condensation products of partial esters derived from ethylene oxide and fatty acids and hexitol anhydrides, such as polyoxyethylene sorbitan monooleate.

  The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension can be formulated according to known methods using diffusion or wetting agents and suspending agents as described above. The sterile injectable preparation may be a sterile injectable solution in a non-toxic parenterally applicable diluent or solvent, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be applied are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are usually used as a solvent or suspending medium. For this purpose, any brand of fixed oil can be applied, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can be applied to the injectable preparations.

  The compounds of formula I can also be administered in the form of liposome delivery systems such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.

  The dose level of the compound of formula I of the present invention is about 0.5 mg to 20 mg per kg body weight, and the preferred dose range is about 0.5 mg to 10 mg per kg body weight per day (patient daily, about 0.5 g to about 3 g). It is. The amount of active ingredient that may be combined with the carrier materials to produce a single dose will vary depending upon the host treated and the particular mode of administration. For example, formulations intended for oral administration to humans will contain from about 5 mg to 1 g of active compound with an appropriate and convenient amount of carrier material in the range of about 5 to 95% of the total composition. Dosage unit forms will generally contain about 5 mg to 500 mg of an active ingredient.

  However, it should be understood that the specific dose level for a particular patient will depend on various factors. This includes the activity of the specific compound used, age, weight, general health, sex, diet, time of administration, route of administration, excretion rate, drug combination, severity of the specific disease being treated, etc. There is.

In addition, some of the compounds of the present invention may form solvates with water or common organic solvents. Such solvates are within the scope of the present invention.
The compounds of the present invention provide operational combinations in combination with other therapeutic compounds. This is intended to include any chemically compatible combination of pharmaceutically active substances so long as the combination does not abolish the activity of Formula I of the present invention.

General Methods Peptide Synthesis Cyclic peptide compounds of formula I are described in detail in our previous applications No. PCT / AU98 / 00490 and No. PCT / AU02 / 01427 (incorporated herein by reference) Prepared according to published methods. The invention is specifically illustrated for the compound AcF- [OPdChaWR] (PMX53). The corresponding linear peptide is Ac-Phe-Orn-Pro-dCha-Trp-Arg. As will be clearly understood, the invention is not limited to this compound.

  Compounds 1-6, 17, 20, 28, 30, 31, 36 and 44 disclosed in International Application No. PCT / AU98 / 00490 and Compounds 10-12, 14 first disclosed in International Application PCT / AU02 / 01427 , 15, 25, 33, 35, 40, 45, 48, 52, 58, 60, 66 and 68-70 have an evaluable antagonist strength (IC50 <1 μM) for the C5a receptor on human neutrophils. Have. Most preferred are PMX53 and PCT / AU02 / 01427 compounds 33, 45 and 60.

It has been found that all compounds of formula I tested to date have broad pharmacological activity. Note that the physicochemical properties, titer, and bioavailability of individual compounds vary to some extent depending on the specific substituents.
The following general test may be used for initial screening for candidate inhibitors of G protein-coupled receptors, particularly C5a receptors.

Drug Preparation and Formulation Human C5a Receptor Antagonist AcF- [OPdChaWR] (AcPhe [Orn-Pro-D-Cyclohexylalanine-Trp-Arg]) was synthesized as described above, purified by reversed-phase HPLC, and mass spectrometry And fully characterized by proton NMR spectroscopy. C5a antagonists were prepared in olive oil solution (10 mg / mL) for oral administration and 30% polyethylene glycol solution (0.6 mg / mL) for SC administration. Prepared in 50% propylene glycol solution (30 mg / kg) for IP injection.

Receptor binding assay As previously described (Sanderson et al, 1995) An assay for isolated fresh human PMN was performed using 50 mM HEPES, 1 mM CaCl ₂ , 5 mM MgCl ₂ , 0.5% bovine serum. In assays performed at 4 ° C. using buffer of albumin, 0.1% bacitracin and 100 μM phenylmethylsulfonyl fluoride (PMSF), buffer, unlabeled human recombinant C5a (Sigma) or peptide, Hunter / Bolton Labeled ¹²⁵ I-C5a (˜20 pM) (New England Nuclear, MA) and PMN (0.2 × 10 ⁶ ) were sequentially added to the Millipore Multiscreen assay plate (HV 0.45) to a final volume of 200 μL / well. After incubation for 60 minutes at 4 ° C., the sample was filtered and the plate was washed once with buffer. Filters were dried, punched and measured with an LKB gamma meter. Non-specific binding was examined with 1 mM peptide or 100 nM C5a insertion and typically 10-15% total binding.
Data were analyzed using non-linear regression and Dunnett post-test statistical processing.

Myeloperoxidase release assay for antagonist activity Cells were isolated as previously described (Sanderson et al, 1995) and incubated with cytochalasin B (5 μg / mL, 15 min, 37 ° C.). Hank's Balanced Salt solution containing 0.15% gelatin and peptide was added to a 96-well plate (total volume 100 μL / well), followed by 25 μL cells (4 × 10 ⁶ / mL). To examine each peptide's ability to antagonize C5a, cells were incubated with each peptide for 5 minutes at 37 ° C, then C5a (100 nM) was added and incubated for an additional 5 minutes. Then 50 μL of sodium phosphate (0.1 M, pH 6.8) is added to each well, the plate is allowed to cool to room temperature, and an equal volume of dimethoxybenzidine (5.7 mg / mL) and H ₂ O ₂ (0.51%) fresh is added. 25 μL of the mixture was added to each well. The reaction was stopped in 10 minutes with the addition of 2% sodium azide. Absorbance was measured at 450 nm with a Bioscan 450 plate reader, corrected for control values (no peptide), and analyzed by non-linear regression.

Statistical analysis Values were mean ± standard error (SEM). Differences between groups were significant at P <0.05. Data were analyzed by one-way ANOVA, and individual groups were compared by Student's t test.

Example 1: Animal model of aortic aneurysm rupture Male Sprague-Dawley rats (350-500 g) were used in the experiments. All rats were anesthetized with sodium pentobarbital (50 mg / kg ip). For each rat, a 22-gauge vascular catheter was inserted into the tail vein and right carotid artery and sutured in situ. Using the tail vein, supplemental doses of anesthetic, 125I-labeled albumin, C5aR antagonist and Ringer's lactate solution were administered and the spilled blood was reinfused. A carotid catheter was used for continuous monitoring of mean arterial pressure (MAP) used in bleeding animals.

The animals were randomly divided into the following two groups:
a) pseudo (sham) (n = 6); and
b) Shock + clamp (n = 19).

  The animals in the shock + clamp group were further randomly divided into a C5aR antagonist treatment group (n = 9) and a control treatment group (n = 10). In the treatment group, a small molecule C5aR antagonist, AcF- (OPdChaWR) (Promics Pty Ltd, Queensland, Australia) was administered intravenously in saline without endotoxin at a dose of 1 mg / kg for 2 minutes after the end of the hemorrhagic shock, A saline solution was injected into the control group. In all cases, the operator is not informed about the action taken.

  The abdominal aorta was exposed by centerline laparotomy and isolated at the proximal mesenteric artery and iliac bifurcation. A 5-cm segment of the jejunum, approximately 10 cm from the Trietz ligament, was isolated and an input cannula was inserted at the proximal end and the distal end of the output cannula. The cannula was removed through two incisions made in the right abdominal wall and the abdomen was sewn together. The cannula intestinal segment was washed with Ringer lactate until there were no solid particles in the output. The intestinal segment was perfused with Ringer lactate at 37 ° C at a rate of 0.3 ml / min with an infusion pump (model AVI 480, 3M, St. Paul, MN) for the duration of the study.

To measure intestinal and pulmonary permeability, animals were given ¹²⁵ I-albumin (˜1 μCi) via a tail vein catheter and allowed to stand for 30 minutes to maintain post-treatment balance. Intestinal perfusates were collected every 10 minutes during the stability and testing period. During the test period, blood samples (0.3 ml) were taken at 1 hour intervals. Blood samples were used to determine the specific activity of ¹²⁵ I-albumin used to calculate total albumin concentration and intestinal albumin loss as described below.

  In appropriate groups, shocks were induced by drawing blood into plastic heparinized syringes (500 U) and MAP was reduced to 50 mmHg and held for 1 hour. Outflow blood was kept at room temperature on a tube shaker for the duration of the shock. After a 60 minute shock or equivalent control period, a clamp was used at the iliac bifurcation in the abdominal aorta immediately adjacent to the upper mesenteric aorta. At this point, half of the shed blood was reinfused into the tail vein. The clamp was held in place for 45 minutes. The remaining spilled blood was reinfused just before unclamping. Additional Ringer's lactate was administered as needed to re-wake the animals and keep the MAP at 100 mmHg. Reperfusion was continued for 120 minutes before the animals were killed with excess sodium pentobarbital.

  Perfused intestinal segments were collected, weighed, lyophilized, and dry gut weight was measured. The immediate intestinal segment of the liver / lung and perfusion segment was removed, washed with ice-cold saline, immediately frozen in liquid nitrogen at 70 ° C., and stored until myeloperoxidase (MPO) and cytokine levels were measured.

The required amount of MAP and resuscitation fluid is summarized in Figure 1 for each group.
Mean arterial blood pressure (MAP) was stable in sham animals throughout the entire study and required minimal venous resuscitation fluid of Ringer's lactate solution, as shown in FIG. 1B. In shock and clamp animals, MAP decreased to < 50 mmHg for 1 hour during hemorrhagic shock as defined in the protocol. In the application of the upper mesenteric aortic clamp, MAP was significantly increased compared to pre-shock levels (158 ± 9.0 vs. 117 ± 3.0 mmHg, p <0.001). In shock and clamped animals after aortic clamp release, MAP gradually decreased during reperfusion to a minimum after 120 minutes of reperfusion, despite strong resuscitation fluid by Ringer lactate infusion (68 ± 6.0 vs. pre-shock) 117 ± 3.0, p <0.001).

  Animals treated with C5a receptor antagonists retained significantly better MAP during reperfusion than untreated shock and clamp animals (95 ± 5.3 vs. 68 ± 6.0 mmHg, p <0.01) and venous resuscitation Less liquid was needed (60.0 ± 7.0 ml vs. 69.3 ± 8.5 ml, p <0.1, NS).

  Throughout the study period, sham animals maintained stable blood pressure with the minimum resuscitation fluid required. Shock and clamp animals required significant resuscitation fluid from the beginning of the reperfusion period until after the aortic clamp was released to retain the MAP. After an initial response to the resuscitation fluid in the first hour of reperfusion, a shock that was difficult to respond to the resuscitation fluid occurred in the next hour of reperfusion, requiring a large amount of venous fluid to maintain blood pressure. Treatment with the C5aR antagonist significantly prevented the severe hypotension seen in the untreated group, and antagonist treated animals had less need for resuscitation fluid.

Example 2 Measurement of Lung Permeability The entire heart and lungs were excised, the left lung was washed 3 times with 3.5 ml Ringer's lactate solution, and bronchoalveolar lavage fluid (BAL) was collected. Blood and BAL fluid were weighed, ¹²⁵ I activity was examined, and lung permeability index (LPI) was calculated using the following formula:

^{LPI = BAL- 125 I (cpm /} g) / Blood - ¹²⁵ I (cpm / g)

The results are summarized in FIG.

The lung permeability index for ¹²⁵ I-labeled albumin was significantly increased in the shock and clamp groups compared to the sham group (4.43 ± 0.96 vs 1.30 ± 0.17, p <0.01). This effect was significantly blocked by treatment with C5a receptor antagonist (1.74 ± 0.50, p <0.03).

Example 3: Measurement of intestinal permeability Intestinal permeability was measured using the intestinal damage index as previously reported (Boyd et al, 1999).
To calculate intraluminal intestinal albumin loss, the effluent collection from the intestinal perfusion was weighed for a total of 10 minutes and each 1 ml sample was measured for ¹²⁵ I-albumin activity with a gamma meter. Each blood sample during the test process was centrifuged at 100,000 rpm and 100 μl of plasma was used to measure albumin content and ¹²⁵ I-albumin activation. The ¹²⁵ I level in the blood sample was regressed against time and the slope of the curve was used to determine the activity of this isotope in whole blood. Using this, the specific activity of ¹²⁵ I per μg of total albumin was determined to calculate mg intestinal albumin loss per gram dry weight of perfused intestinal segment. The result is shown in FIG.

  The rate of intraluminal intestinal albumin loss, the intestinal permeability index (IPI), was stable in sham animals during the entire study period. In shock and clamp animals, IPI was constant during stabilization, bleeding and clamp. However, with reperfusion, IPI increased statistically significantly. After 30 minutes of reperfusion, IPI was compared to pre-shock levels (8.05x10-2 ± 3.59x10-2 vs. 0.72x10-2 ± 0.51x10-2, p <0.0001) and control in shock and clamp animals Compared to the level (8.05x10-2 ± 3.59x10-2 vs. 1.75x10-2 ± 0.33x10-2, p <0.0001), it remained significantly similar during the 120 minute reperfusion period.

Treatment with a C5a receptor antagonist significantly reduced the increase in IPI at initial reperfusion, and after 30 minutes of reperfusion, IPI was significantly greater in C5aR antagonist treated animals compared to untreated shock and clamp animals. Decreased (2.82x10 ^-2 ± 0.91x10 ^-2 vs. 8.05x10 ^-2 ± 3.59x10 ^-2 , p <0.01). However, as reperfusion progressed, IPI also increased in the C5a antagonist-treated group, reflecting levels in untreated shock and clamp animals.

Example 4: Measurement of Neutrophil Separation Lung and intestinal tissue samples were assayed for myeloperoxidase (MPO) activity, neutrophil separation index as previously reported (Boyd et al, 1999). Briefly, MPO activity was assayed on a Cobas FARA II centrifuge (Roche Diagnostic Systems, Montclair, NJ) at 37 ° C and absorbance changes at 655 nm for 3 minutes. The reaction mixture consisted of a solution of 16 mmol / l 3,3 ', 5,5'-tetramethylbenzidine in N, N-dimethylformamide, 0.22 mol / l phosphate buffer containing 0.11 mol / l NaCl at pH 5.4. Contained in the liquid. The reaction was started with the addition of 3 mmol / l hydrogen peroxide. One unit of activity was defined as an absorbance change of 1 unit per minute at 37 ° C. The protein content of lung and intestine samples was measured with a bicinchoninic acid protein assay system (Pierce, Rockford, IL). MPO activity was expressed as units for milligrams of protein. The results are shown in FIG.

  As shown in Figure 4a, lung tissue MPO activity was significantly increased in the shock and clamp groups compared to the sham group (2.41 ± 0.34 vs. 1.03 ± 0.29 U / mg, p <0.009), and this increase was C5a Blocked by treatment with receptor antagonist (1.11 ± 0.09 U / mg, p <0.006).

  As shown in Figure 4b, intestinal tissue MPO activity is not significantly increased in the shock and clamp groups compared to the sham group (3.93 ± 0.66 vs. 3.34 ± 0.53 U / mg, p = NS). Interestingly, intestinal MPO activity was (1.86 ± 0.26 vs. 3.93 ± 0.66 U / mg, p <0.01) as well as sham levels (3.34 ±±) in C5a receptor antagonist treated animals compared to untreated shock and clamp animals. Compared to 0.53 U / mg, p <0.017), it was significantly reduced.

Example 5: Measurement of cytokines in the intestine and lung 100 mg of each tissue was treated with protease inhibitors (0.1 mmol / L phenylmethylsulfonyl fluoride, 0.1 mmol / L benzethonium chloride, 10 mmol / L ethylenediaminetetraacetic acid and 20 KI aprotinin. Homogenized in 1 mL PBS (0.4 mol / L NaCl and 10 mmol / L Na ₂ HP0 ₄ ) containing A) and 0.05% Tween 20. Samples were then centrifuged at 3000 g for 10 minutes and the supernatant was immediately used in an enzyme-linked immunosorbent assay at a 1: 2 dilution in assay dilution buffer. The concentrations of TNF-α and interleukin-6 in the samples were measured using commercially available antibodies (R & D Systems, Minneapolis, Minn.) According to the method provided by the manufacturer. The protein content of intestinal and lung samples was measured by the bicinchoninic acid protein assay system (Pierce, Rockford, IL). Cytokine concentrations were expressed as picograms per milligram of protein. The results are shown in Table 6.

  As shown in Figure 5a, intestinal TNF-α levels were significantly increased in shock and clamp animals compared to sham animals (73.02 ± 10.12 vs. 45.42 ± 6.23 pg / mg protein, p <0.038), but C5a receptor Not affected by treatment with body antagonists (72.00 ± 13.95 pg / mg protein, p = NS).

  As shown in FIG. 5b, intestinal IL-6 levels were significantly increased in shock and clamp animals compared to sham animals (280.91 ± 35.95 vs. 168.38 ± 35.23 pg / mg protein, p <0.04). Interestingly, the increase in intestinal IL-6 levels was significantly less in C5a receptor antagonist treated animals than in untreated shock and clamp animals (196.30 ± 23.68 pg / protein, p <0.05).

  As shown in Figure 6a, intestinal TNF-α levels were significantly increased in shock and clamp animals compared to sham animals (89.70 ± 13.83 vs 47.57 ± 11.22 pg / mg protein, p <0.03). Was not prevented by treatment with a C5a receptor antagonist (78.71 ± 15.78 pg / mg protein, p = NS).

  As shown in FIG. 6b, pulmonary IL-6 levels were significantly increased in shocked and clamped animals compared to sham animals (227.98 ± 51.74 vs. 144.81 ± 26.31 U / mg protein, p = NS). However, this increase did not reach statistical significance. Interestingly, pulmonary IL-6 levels were significantly increased in the C5a receptor antagonist-treated group compared to the sham group (320.72 ± 37.67 vs. 144.81 ± 26.31 U / mg protein, p <0.002).

Example 6: Further preclinical studies In addition to the above, the effects of medical agents such as the present invention on hemorrhagic shock can be tested in various experimental models. The most common experimental species used are pigs and rats, followed by sheep and mice. Pigs are most often used because of their size and the great similarity of their cardiovascular system and parameters to humans. Blood loss can be induced in various ways, and special methods do not appear to affect the results.

  The C5a antagonist compounds of the present invention can be used in any of these models. However, if receptor affinity is lower in mice, sheep and pigs than that observed in rats, this may reduce the strength and potency of the antagonist.

  Test compound is administered after hemorrhagic shock. The route of administration is preferably parenteral, eg, intravenous, and an effective blood concentration of the drug can be achieved as soon as possible. Because of the severity of the symptoms, avoidance of blood from non-vital organs such as the stomach may reduce absorption from the intestinal route. In general, intravenous administration is used in these experiments.

  Test compound administration was performed at various doses and at various times after the hemorrhagic shock to identify the best therapy. Control animals are treated with sham injections, left untreated, or treated with comparative substances. This material includes other anti-inflammatory agents such as infliximab or other materials commonly used in the treatment of hemorrhagic shock.

Monitor the effect of each treatment by measuring parameters such as:
Cardiac drainage (beat volume x heart rate)
Mean arterial pressure Resuscitation requirement Neutrophil separation in tissues
Circulating or tissue cytokine levels such as TNFα and IL-1, IL-2, IL-6 and IL-8 Decrease in intracellular ATP Blood hemoglobin levels Metabolic acidosis Others such as intestinal and lung permeability change.

Mean arterial pressure, resuscitation fluid requirement, neutrophil segregation in tissue, intestinal permeability, lung permeability, and levels of TNFα and IL-6, as described in the examples above or known in the art It can be measured by another method. Physiological and biochemical parameters can be measured by standard methods. Examples are metabolic acidosis by measuring blood hemoglobin and arterial blood using the hematocrit method, or measuring _{CO 2} . TNF-α, IL-6 and other cytokines can be measured by commercially available assays, such as immunoassays.
The parameters chosen for testing are those that are accepted by supervisory authorities such as the US Food and Drug Administration, the European Medicine Evaluation Agency and the Australian Medical Products Agency as much as possible.

Discussion The development of specific monoclonal anti-complement antibodies has a new interest in complement as a medical target in clinical pathology (Matis et al, 1995). In the RAAA model used in the present invention, for 1 hour, MAP 50 mmHg hemorrhagic shock, 45 minutes upper mesenteric aortic clamp and 120 minutes of severe intestinal and lung injury reperfusion and strong resuscitation fluid The respiratory shock continues regardless. For the first time, this study demonstrated that local and systemic damage associated with RAAA can be ameliorated in a rat model by a specific small molecule C5a receptor, the cyclic peptide AcF- (OPdChaWR).

Intestinal damage in this model was associated with a significant increase in intestinal capillary permeability for ¹²⁵ I-albumin immediately after release of the upper mesenteric aortic clamp, a sustained increase through a 120 minute reperfusion period. Treatment with C5aR antagonist significantly prevented the increase in intestinal permeability during early reperfusion. However, in late reperfusion, intestinal permeability increased to a level similar to that in untreated shock and clamp animals. Increased intestinal permeability has been reported after RAAA and after selective abdominal and thoracoabdominal aneurysm repair in humans and is associated with increased morbidity and mortality (Van Damme et al, 2000; Lau et al, 2000; Harward et al, 1996).

  The damage to the intestine is double and the first general hypoxia during hemorrhagic shock is exacerbated by direct ischemia-reperfusion injury during and after the upper mesenteric aortic clamp. Intestinal ischemia-reperfusion injury is associated with increased neutrophil segregation and microvascular permeability and can be modulated by neutrophil depletion or by antibodies to neutrophil adhesion molecules (Hernandez et al, 1987) . In a recent study in rats, the same C5a receptor antagonist used in this study was effective in reducing intestinal ischemia-reperfusion injury and neutrophil response to ischemia-reperfusion injury. It has been reported (Arumugam et al, 2002).

  Complement activation occurs in the early stages of inflammation, releasing the anaphylatoxins C3a, C4a, C5a and C5b-C9 membrane attack complexes. These active complement components alter vascular tone and permeability and are considered essential for intestinal reperfusion injury (Williams et al, 1999) and membrane attack complexes are directly degradable to cells. Work. Anaphylatoxins, particularly C5a, chemotactically recruit and activate inflammatory cells, leading to the release of cytokines TNF-α and IL-6. By inhibiting the initial interaction of the activated complement component with its target cells on intestinal vascular endothelium and circulating immune cells, the severity of the first gastrointestinal injury was reduced in this model.

  Direct or indirect intestinal ischemia-reperfusion injury results in functional and morphological changes in the gastrointestinal tract associated with the movement of bacterial fragments across the blocked intestinal capillary barrier, resulting in an excessive inflammatory response Trigger with blood. This suggests that the gastrointestinal tract drives an inflammatory response to various important diseases (Baue et al, 1997). Complements have been shown to be important in neutrophil activation in response to endotoxin (van Deventer et al, 1991), and the C5a antagonist used in this study was found in PMN after exposure to E. coli. Blunt the oxidative burst (Mollnes et al., 2002).

  We hypothesized that blocking C5a receptors could reduce the pro-inflammatory effects of endotoxin in early reperfusion. However, as reperfusion continues to increase intestinal damage, it is likely due to the cellular action of ischemia-reperfusion injury and parallel activation of the cascade of cytokines and pro-inflammatory mediators. Our findings are consistent with previous reports that direct intestinal ischemia-reperfusion injury can be relieved by blocking at various points in the complement cascade (Hill et al, 1992; Arumugam et al , 2002).

  The decrease in intestinal myeloperoxidase levels by C5a antagonists compared to untreated shock and clamp animals is due to local complement-induced neutrophil chemotaxis and activation, ie, target cell opsonization opsonisation. It is essential for neutrophil segregation and intestinal damage. Complements have been shown to promote rapid adhesion of neutrophils to endothelial target cells in vitro (Marks et al, 1989) and this effect is mediated by the action of C5a. In this study, shock and clamp animals significantly increased intestinal levels of the pro-inflammatory cytokine TNF-α compared to sham animals. This is not changed by treatment with the C5aR antagonist. Activation supplements cause the release of TNF-α from various types of cells, including immune cells, by receptor-mediated action (Barton et al, 1993) and intravenously administered C5a is circulating TNF-α Although increasing levels in rats are known (Strachan et al, 2000), various other mediators released after ischemia-reperfusion injury, such as arachidonic acid metabolites, also promote cytokine release.

  IL-6 is an important pleiotropic cytokine and has various pro-inflammatory and anti-inflammatory effects. High serum levels of IL-6 are associated with morbidity and mortality after repair of abdominal aortic aneurysms (Groeneveld et al, 1997). In this study, it was discovered in this model that intestinal damage was associated with increased intestinal tissue levels of IL-6 and that treatment with C5aR antagonists prevented this increase. This is important because IL-6 has been shown to up-regulate the pro-inflammatory effect of TNF-α in response to complement activation (Platel et al, 1996). IL-6 has been shown to inhibit neutrophil apoptosis and prolong its functional life and intensity of tissue damage (Biffl et al, 1996). Thus, inhibition of this release can reduce further neutrophil-mediated intestinal damage. These data indicate that the interaction between C5a and immune cell receptors is essential for IL-6 release in this model, or that this may also reflect a decrease in neutrophil tissue segregation by C5aR antagonism. Suggest.

Abdominal aortic aneurysm rupture is associated with noncardiac acute interstitial pulmonary edema and associated hypoxemia, called acute respiratory distress syndrome (ARDS), which has a significant prognosis. In our model for RAAA, the lung permeability and the level of lung myeloperoxidase for ¹²⁵ I-albumin were significantly increased in the shock and clamp groups. The suggestion that lung injury in this syndrome is mainly due to neutrophil adhesion, sequestration, and subsequent respiratory burst-induced oxidative damage has been observed that lung injury can be ameliorated by anti-CD18 monoclonal antibodies (Boyd et al, 1999).

  In the model used in this study, antagonism of the C5a receptor to target cells reduced neutrophil segregation and subsequent hyperpermeability of microvessels, and systemic hemorrhagic shock injury combined with lower chest ischemia-reperfusion The combination causes severe acute lung injury. Eliminating C5a-induced neutrophil chemotaxis and activation in the pulmonary circulation (Solokin et al, 1985) may partially explain the remission of injury in this study.

  C5a receptor antagonists do not inhibit the formation of membrane attack complexes (Arumugam et al, 2003). C5a receptor blocking does not appear to affect the formation of the C5b-9 membrane attack complex, but nevertheless this large complex does not appear to be able to move undisturbed into the pulmonary circulation and cause its degradative effects . In this acute model, the regulation of early complement-dependent damage may be sufficient to convert fatal acute lung failure into a recovering acute lung abnormality.

In relation to the gut, TNF-α levels in the lung are significantly increased after shock and clamp, which is not affected by treatment with C5aR antagonists. TNF-α has been shown to occur after various stresses and induce direct lung injury (Welbourn et al, 1991). We have now discovered that IL-6 levels in the lung also increase compared to sham animals after shock and clamp. However, this increase did not reach a significant level. Interestingly, shock and clamp animals treated with C5aR antagonists have a highly significant increase in lung tissue IL-6 compared to sham animals. This suggests that protection from complement-induced distal damage is independent of IL-6. Alternatively, in this model, it is suggested that IL-6 release would have a favorable anti-inflammatory effect in the lung, possibly through paracrine inhibition of inflammatory mediator release.

  Hemorrhagic shock itself initiates a pro-inflammatory mediator-induced cascade (Abraham, 1991), and oxidative damage is associated with the degree of complement activation in patients with heart disease (Cavarocchi et al, 1986). Aortic clamp release is associated with various vasoactive effects. This includes low blood volume due to peripheral vasodilation and increased vascular permeability, reperfusion of ischemic tissue with vasoactive mediators and metabolites, and myocardial function inhibitors (Barry et al, 1997). The effect of C5a receptor antagonists on reduced microvascular permeability and immune cell activation via complement receptor-specific pathways reduces the extent and time of hypotension in this model. This probably reflects a decrease in the third space fluid loss by preventing complement activation and complement-dependent increase in microvascular permeability. It is also known that complement activation has an effect on vascular tone and histamine release (Ellis et al, 1991) and its prevention can also help maintain vascular resistance. Reduced organ damage, and possibly reduced myocardial depression, may also allow animals to successfully handle the amount of fluid needed to maintain the target blood pressure.

  In conclusion, this is the first time that small molecule C5a receptor antagonists can reduce local and distal lung injury in a model of abdominal aortic aneurysm rupture. This treatment seems to mediate its effects by reducing activated complement-immune cell interactions, in other words, reducing inflammatory stimuli on tissue neutrophil segregation. Thus, the human C5a receptor antagonism represents a real medical target in patients with hemorrhagic shock and provides a major clinical need.

While the present invention has been described in some detail for purposes of clarity and ease of understanding, it will be apparent to those skilled in the art that various modifications and changes may be made herein to the embodiments and methods described herein. This may be done without departing from the scope of the disclosed inventive concept.
References are displayed on the following pages. These are incorporated herein by reference.

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FIG. 1 shows a summary of mean arterial pressure and resuscitation fluid requirements for each group of animals. A. Mean arterial pressure. B. Required amount of resuscitation fluid. FIG. 2 compares the lung permeability index (LPI) in each group of rats. FIG. 3 shows changes in intestinal permeability over time after unclamping. FIG. 4 shows myeloperoxidase activity in the lung and intestine of the sample. A. lung. B. Intestines. FIG. 5 shows the levels of cytokines in the gastrointestinal tissue of samples from each group of animals. A. TNF-α. B. IL-6. FIG. 6 shows the levels of cytokines in the lung tissue of samples from each group of animals. A. TNF-α. B. IL-6.

Claims (23)

  A method of treating hemorrhagic shock comprising administering an effective amount of an inhibitor of a C5a receptor to a subject in need of treatment. An inhibitor
(d) is an antagonist of the C5a receptor,
(e) substantially free of agonist activity, and
(f) Formula I, which is a cyclic peptide or peptide-like compound of Formula I

[Wherein A is H, alkyl, aryl, NH ₂ , NH-alkyl, N (alkyl) ₂ , NH-aryl, NH-acyl, NH-benzoyl, NHSO ₃ , NHSO ₂ -alkyl, NHSO ₂ -aryl, OH, O-alkyl or O-aryl;
B is an alkyl, aryl, phenyl, benzyl, naphthyl or indole group, or a D- or L-amino acid side chain, but glycine, D-phenylalanine, L-homophenylalanine, L-tryptophan, L-homotryptophan, Not the side chain of L-tyrosine or L-homotyrosine;
C is the side chain of D-, L- or homo-amino acid, but not the side chain of isoleucine, phenylalanine or cyclohexylalanine;
D is a neutral D-amino acid side chain, but not a glycine or D-alanine side chain, a large planar side chain or a large charged side chain;
E is a large substituent, but D-tryptophan, LN-methyltryptophan, L homophenylalanine, L-2-naphthyl L-tetrahydroisoquinoline, L-cyclohexylalanine, D-leucine, L-fluorenylalanine or L Not the side chain of histidine;
F is the side chain of L-arginine, L-homoarginine, L-citrulline or L-canavanine, or a bioequivalent thereof; and
X is-(CH ₂ ) _n NH- or (CH ₂ ) _n -S- (where n is an integer from 1 to 4);-(CH ₂ ) ₂ O-;-(CH ₂ ) ₃ O-;- _{(CH 2) 3 - ;-( CH} 2) 4 -; -CH 2 COCHRNH-; or CH ₂ CHCOCHRNH- (of which, R is normal or side chain of a non-conventional amino acids)
The method of claim 1, wherein   The method of claim 2, wherein n is 2 or 3.   The method of claim 2 or 3, wherein A is an acetamido group, an aminomethyl group, or a substituted or unsubstituted sulfonamide.   4. The method of claim 3, wherein A is a substituted sulfonamide and the substituent is an alkyl chain of 1 to 6 carbon atoms, or a phenyl or toluyl group.   6. The method of claim 5, wherein the substituent is an alkyl chain of 1 to 4 carbon atoms.   The method according to claim 2, wherein B is a side chain of L-phenylalanine or L-phenylglycine.   The method according to any of claims 2 to 6, wherein C is a side chain of glycine, alanine, leucine, valine, proline, hydroxyproline or thioproline.   D is D-leucine, D-homoleucine, D-cyclohexylalanine, D-homocyclohexylalanine, D-valine, D-norleucine, D-homonorleucine, D-phenylalanine, D-tetrahydroisoquinoline, D-glutamine, D- The method according to claim 2, which is a side chain of glutamate or D-tyrosine.   E is an amino acid selected from the group consisting of L-phenylalanine, L-tryptophan and L-homotryptophan, or a side chain of L-1-naphthyl or L-3-benzothienylalanine. the method of.   The method according to any one of claims 1 to 10, wherein the inhibitor is a compound having antagonist activity against C5aR and not having C5a agonist activity.   12. The method of any of claims 1-11, wherein the inhibitor has potent antagonist activity at submicromolar concentrations.   13. The method of any of claims 1-12, wherein the compound has a receptor affinity IC50 <25 μM and an antagonist strength IC50 <1 μM.   The compounds are compounds 1-6, 10-15, 17, 19, 20, 22, 25, 26, 28, 30, 31, 33-37, 39-45, 47-50, described in PCT / AU02 / 01427 The method according to any one of claims 1 to 13, which is a compound selected from the group consisting of 52-58 and 60-70.   The compounds are AcF [OP-DCha-WR], AcF [OP-DPhe-WR], AcF [OP-DCha-FR], AcF [OP-DCha-WCit]), HC- [OPdChaWR], AcF- [OPdPheWR ], AcF- [OpdChaW citrulline] or HC- [OPdPheWR].   16. The method of any of claims 1-15, wherein the inhibitor is used in combination with one or more other substances for the treatment of hemorrhagic shock.   Shock, trauma, ruptured aneurysm, uncontrollable nasal bleeding, hemorrhagic fever, uterine bleeding after childbirth, bleeding during and after surgery, bleeding from digestive ulcers or duodenal varices, bleeding in the lower gastrointestinal tract, 17. Any one of claims 1-16, which is due to a major bleeding resulting from a condition selected from the group consisting of secondary bleeding from cancer invasion, bleeding from hemorrhagic predisposition, bleeding associated with the treatment of thrombolysis. That way.   18. The method of any of claims 1-17, wherein the subject is a human.   19. The method of any of claims 1-18, wherein the inhibitor is administered parenterally, orally, transdermally or nasally.   20. The method of claim 19, wherein the inhibitor is administered intravenously.   Use of an inhibitor of the C5a receptor in the manufacture of a medicament for the treatment of hemorrhagic shock.   Use according to claim 21, wherein the inhibitor is a compound as defined in any of claims 1-15.   23. Use according to claim 21 or 22, wherein the medicament is suitable for intravenous administration.

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