EP1635857A1 - TREATMENT OF HAEMORRHAGIC SHOCK USING COMPLEMENT 5a RECEPTOR INHIBITORS - Google Patents

Abstract

This invention relates to methods of treatment of haemorrhagic shock, and especially to treatment of this condition with cyclic peptidic and peptidomimetic compounds which have the ability to act as antagonists of the C5a receptor. In one embodiment the compounds are active against C5a receptors on polymorphonuclear leukocytes and macrophages. Particularly preferred compounds for use in the invention are disclosed.

Description

TREATMENT OF HAEMORRHAGIC SHOCKUSING COMPLEMENT 5a RECEPTOR

INHIBITORS

This application claims priority from Australian provisional application No.2003902354 dated 15^th May 2003.

FIELD OF THE INVENTION

This invention relates to the treatment of haemorrhagic shock with novel cyclic peptidic and peptidomimetic compounds which have the ability to modulate the activity of G protein-coupled receptors. The compounds preferably act as antagonists of the C5a receptor, and are active against C5a receptors on polymorphonuclear leukocytes and macrophages .

BACKGROUND OF THE INVENTION

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents . It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

G protein-coupled receptors are prevalent throughout the human body, comprising approximately 60% of known cellular receptor types, and mediate signal transduction across the cell membrane for a very wide range of endogenous ligands . They participate in a diverse array of physiological and pathophysiological processes, including, but not limited to those associated with cardiovascular, central and peripheral nervous system, reproductive, metabolic, digestive, immunological, inflammatory, and growth disorders, as well as other cell- regulatory and proliferative disorders. Agents which selectively modulate functions of G protein-coupled receptors have important therapeutic applications . These receptors are becoming increasingly recognised as important drug targets, due to their crucial roles in signal transduction (G protein-coupled Receptors, IBC Biomedical Library Series, 1996) .

One of the most intensively studied G protein- coupled receptors is the receptor for C5a. C5a is one of the most potent chemotactic agents known, and recruits neutrophils and macrophages to sites of injury, alters their morphology; induces degranulation; increases calcium mobilisation, vascular permeability (oedema) and neutrophil adhesiveness; contracts smooth muscle; stimulates release of inflammatory mediators, including histamine, TNF-α, IL-1, IL-6, IL-8, prostaglandins, and leukotrienes, and of lysosomal enzymes; promotes formation of oxygen radicals; and enhances antibody production (Gerard and Gerard, 1994) . Agents which limit the pro-inflammatory actions of C5a have potential for inhibiting both acute and chronic inflammation, and its accompanying pain and tissue damage. Because such compounds act upstream from the various inflammatory mediators referred to above, and inhibit the formation of many of these compounds, they may have a more powerful effect in alleviating or preventing inflammatory symptoms .

In our previous applications No . PCT/AU98/ 00490 , we described the three-dimensional structure of some analogues of the C-terminus of human C5a, and used this information to design novel compounds which bind to the human C5a receptor (C5aR) , behaving as either agonists or antagonists of C5a. It had previously been thought that a putative antagonist might require both a C-terminal arginine and a C-terminal carboxylate for receptor binding and antagonist activity (Konteatis et al , 1994) . We showed that in fact a terminal carboxylate group is not generally required either for high affinity binding to C5aR or for antagonist activity. Instead we found that a hitherto unrecognised structural feature, a turn conformation, was the key recognition feature for high affinity binding to the human C5a receptor on neutrophils . As described in our International application PCT/AU0101427, we used these findings to design constrained structural templates which enable hydrophobic groups to be assembled into a hydrophobic array for interaction with a C5a receptor. The entire disclosures of these specifications are incorporated herein by this reference .

Shock is a condition of major haemodynamic and metabolic disturbance which may result from a number of causes, and is characterised by failure of the circulatory system to maintain adequate perfusion of vital organs with blood. It may result from inadequate blood volume, inadequate cardiac function or inadequate vasomotor tone. Haemorrhagic shock caused by inadequate blood volume, also known as hypovolaemic shock or volume deficiency shock, results from major haemorrhage, which can have a very wide range of underlying causes, such as trauma, uncontrollable bleeding in relation to childbirth or as a result of a nosebleed, blood-clotting disorders such as haemophilia, surgical interventions, congenital defects such as aneurysms, or gastrointestinal conditions such as perforated ulcers .

In many cases, major haemorrhage is very difficult to treat, and a variety of interventions has been employed in addition to transfusion, restoration of blood volume and other conventional supportive measures . These include arterial embolization, emergency surgery, and pharmacological agents such as sulprostone, somatostatin, and vasopressin. The primary interventions are directed to stopping the bleeding and to replacing the lost blood volume, for example using blood transfusion, infusion with isotonic or hypertonic saline, or blood substitutes, and the secondary treatment is related to alleviation or minimization of the sequelae of shock. Treatment of haemorrhagic shock involves maintaining blood pressure and tissue perfusion until bleeding is controlled. Different resuscitation strategies have been used to maintain the blood pressure in trauma patients until bleeding is controlled. However, while maintaining blood pressure may prevent shock, it may worsen bleeding. Consequently a fine balance between these considerations must be maintained. If shock is prolonged the cardiovascular system may suffer damage, so that cardiac output declines as the result of positive feedbacks and th4 shock may become irreversible.

The treatments may be of limited effectiveness, and may have serious side effects. For example, despite successful surgery and intensive care support, the repair of ruptured abdominal aortic aneurysm (RAAA) is associated with a mortality rate of 50-75% (Adam et al, 1999) . A variety of agents, including immune regulating hormones (Hollis-Eden Pharmaceuticals, Inc) and various blood substitutes, such as diaspirin cross-linked haemoglobin and other haemoglobin forms, are in various stages of clinical trial, with mixed success.

The combined injury of haemorrhagic shock and lower torso ischaemia-reperfusion injury initiates a systemic inflammatory response syndrome, which is characterised by increased microvascular permeability and neutrophil sequestration, leading to multiple organ dysfunction syndrome (MODS) . MODS is the primary cause of 70% of such deaths, and a major contributory cause of the remainder (Harris et al, 1991) . Using a rat model of ruptured abdominal aortic aneurysm, it has been shown that haemorrhagic shock paired with supramesenteric aortic clamping results in local intestinal and remote lung injury, and that this can be attenuated by reducing neutrophil adherence using a monoclonal antibody directed against CD18 integrin (Boyd et al, 1999) . Reperfusion of the ischaemic lower torso following prolonged ischaemia initiates a systemic inflammatory response syndrome, characterised by pro- inflammatory cytokines (Groeneveld et al, 1997) and increased circulating polymorphonuclear leucocyte (PMN) activation (Barry et al, 1997). Pulmonary sequestration of activated neutrophils is followed by acute pulmonary microvascular injury (Welbourne et al , 1991), acute respiratory distress syndrome (Paterson et al, 1989), and a high subsequent mortality. High circulating levels of pro-inflammatory cytokines responsible for leukocyte activation, such as tumour necrosis factor (TNF)-α, interleukin-6 and interleukin-8, and of endotoxin (Baigrie et al, 1993) have been demonstrated after repair of RAAA (Roumen et al, 1993). We and others have previously shown that lower limb ischaemia reperfusion injury is associated with increased intestinal permeability, endotoxaemia, and a systemic inflammatory response 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 haemorrhage and trauma, in conjunction with the syndrome of ischaemia-reperfusion injury, activate the complement cascade, and the degree of activation of the complement system correlates with the severity of injury, and the likelihood of development of multiple organ failure and ultimate death.

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

Activated products of the classical complement pathway, such as C5a and C3a, are potent inflammatory mediators with myriad effects, including alteration of blood vessel permeability and tone, leukocyte chemotaxis, and activation of multiple inflammatory cell types . The role of complement in some inflammatory tissue injury conditions is supported by the attenuation of such injury using anti-C5 antibody (Piccolo et al . , 1999) and a C5a receptor (C5aR) antagonist (Arumugam et al, 2003) . However, in contrast to this it has been reported that lung injury induced by limb ischaemia is mediated by leukotrienes, not by complement (Klausner et al, 1989) . Moreover, the role of complement in inflammatory tissue injury after ruptured abdominal aortic aneurysm is still largely unknown.

In studies using a rat model to examine haemodynamic and metabolic recovery from prolonged and profound haemorrhagic hypertension, it was found that haemorrhage and resuscitation resulted in complement consumption, and that prior depletion of circulating complement levels protected the animals from shock, as measured by mean arterial blood pressure and metabolic acidosis. In contrast, administration of an exogenous complement activator or inhibition of complement breakdown exacerbated the injury. Although the authors concluded that it was likely that C5a played a crucial role, they considered that until agents which specifically neutralise ,C5a without affecting the activities of the parent molecule C5 were available, this could not be confirmed. In particular, C3a was also potentially implicated (Younger et al, 2001) . Others have found that the intestinal and lung injury following haemorrhagic shock and reperfusion can be minimised by reducing neutrophil adherence with a monoclonal antibody directed against CD18 integrin (Boyd et al, 1999) . The plethora of inflammatory agents which have been identified in ischaemia-reperfusion syndrome means that it is very difficult to identify the most effective target for intervention. This difficulty is reflected in the wide variety of candidate targets and agents discussed at the 6^th World Congress on Trauma, Inflammation, Shock and Sepsis held in March 2004 (http: //www. trauma-shock-sepsis-congress-munich- 2004.org/lis.html.

Therefore there is a great need in the art for effective, non-toxic agents which preferably do not require administration by injection, and which can be produced at reasonable cost.

Glycoforms of the soluble complement receptor type 1 (CRl) have been proposed for use in the treatment of complement-mediated disorders and of shock. The soluble CRl fragments were functionally active, bound C3b and/or C4b, and demonstrated factor I cofactor activity, depending upon the regions they contained. Such constructs inhibited the consequences of complement activation, such as neutrophil oxidative burst, complement-mediated haemolysis, and C3a and C5a production (US patents No 5456909, No 5807844 and No 5858969) . However, to our knowledge none of these approved or experimental agents for treatment of shock, and in particular no small molecule agent, targets the C5a receptor.

SUMMARY OF THE INVENTION

Due to the current uncertainty as to the nature of the complement involvement in haemorrhagic shock, we tested the possible inhibitory effects of a specific complement inhibitor in an animal model of ruptured aortic aneurysm, a condition which causes haemorrhagic shock. We now show for the first time that a specific inhibitor of the C5a receptor is able to ameliorate signs of damage in an animal model of haemorrhagic shock. This is the first reported case of a small molecule inhibitor of the complement system being used to modulate pathology in a model of haemorrhagic shock.

According to a first aspect, the invention provides a method of treatment of haemorrhagic shock, comprising the step of administering an effective amount of an inhibitor of a C5a receptor to a subject in need of such treatment.

Preferably the inhibitor is a compound which

(a) is an antagonist of a C5a receptor,

(b) has substantially no agonist activity, and

(c) is a cyclic peptide or peptidomimetic compound of Formula I

where A is H, alkyl, aryl, NH₂, NH-alkyl, N(alkyl)₂, NH-aryl, NH-acyl, NH-benzoyl, NHS0₃, NHS0₂- alkyl, NHS0₂-aryl, OH, O-alkyl, or 0-aryl;

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

C is a small substituent, such as the side chain of a D-, L- or homo-amino acid such as glycine, alanine, leucine, valine, proline, hydroxyproline, or thioproline, but is preferably not a bulky substituent such as isoleucine, phenylalanine, or cyclohexylalanine;

D is the side chain of a neutral D-amino acid such as D-Leucine, D-homoleucine, D-cyclohexylalanine, D- homocyclohexylalanine, D-valine, D-norleucine, D-homo- norleucine, D-phenylalanine, D-tetrahydroisoquinoline, D- glutamine, D-glutamate, or D-tyrosine, but is preferably not a small substituent such as the side chain of glycine or D-alanine, a bulky planar side chain such as D- tryptophan, or a bulky charged side chain such as D- arginine or D-Lysine; E is a bulky substituent, such as the side chain of an amino acid selected from the group consisting of L- phenylalanine, L-tryptophan and L-homotryptophan, or is L- 1-napthyl or L-3-benzothienyl alanine, but is not the side chain of D-tryptophan, L-N-methyltryptophan, L-homophenylalanine, L-2-naphthyl L- tetrahydroisoquinoline, L-cyclohexylalanine, D-leucine, L- fluorenylalanine, or L-histidine;

F is the side chain of L-arginine, L- homoarginine, L-citrulline, or L-canavanine, or a bioisostere thereof, ie. a side chain in which the terminal guanidine or urea group is retained, but the carbon backbone is replaced by a group which has different structure but is such that the side chain as a whole reacts with the target protein in the same way as the parent group; and

X is -(CH₂)_nNH- or (CH₂)_n-S-, where n is an integer of from 1 to 4, preferably 2 or 3; -(CH₂)₂0-; -(CH₂)₃0-; -(CH₂)₃-; - (CH₂) ₄- ; . -CH₂COCHRNH- ; or -CH-CHCOCHRNH-, where R is the side chain of any common or uncommon amino acid.

In C, both the cis and trans forms of hydroxyproline and thioproline may be used.

Preferably A is an acetamide group, an aminomethyl group, or a substituted or unsubstituted sulphonamide group .

Preferably where A is a substituted sulphonamide, 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 has no C5a agonist activity.

The compound is preferably an antagonist of C5a receptors on human and mammalian cells including, but not limited to, human polymorphonuclear leukocytes and human macrophages . The compound preferably binds potently and selectively to C5a receptors, and more preferably has potent antagonist activity at sub-micromolar concentrations . Even more preferably the compound has a receptor affinity IC50<25μM, and an antagonist potency IC50<lμM.

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

The inhibitor may be used in conjunction with one or more other agents for the treatment of haemorrhagic shock, including but not limited to blood substitutes, vasopressin, somatostatin, terlipresin and anti-nitric oxide agents.

The compositions of the invention may be formulated for oral, parenteral, inhalational, intranasal, rectal or transdermal use, but parenteral, and especially intravenous formulations are preferred. It is expected that most if not all compounds of the invention will be stable in the presence of metabolic enzymes, such as those of the gut, blood, lung or intracellular enzymes. Such stability can readily be tested by routine methods known to those skilled in the art. Suitable formulations for administration by any desired route may be prepared by standard methods, for example by reference to^p well-known textbooks such as Remington: The Science and Practice of Pharmacy, Vol. II, 2000 (20 edition) , A.R. Gennaro (ed) , Williams & Wilkins, Pennsylvania.

It is contemplated that the invention is applicable to the treatment of shock resulting from major haemorrhage of any origin, including but not limited to trauma, rupture of an aneurysm, uncontrollable epistaxis, viral haemorrhagic fevers such as dengue, Lassa, Marburg or Ebola virus, uterine haemorrhage during or after delivery, haemorrhage during or after surgery, haemorrhage resulting from gastrointestinal ulcers or oesophageal varices, or of the lower gastrointestinal tract, eg. diverticular haemorrhage, haemorrhage secondary to invasion of cancer, haemorrhage resulting from bleeding diatheses, eg. haemophilia, idiopathic thrombocytopaenic purpura and the like, and haemorrhage associated with thrombolytic therapy, eg. with agents such as warfarin, aspirin, plasminogen activator, streptokinase or urokinase. While the invention is not in any way restricted to the treatment of any particular animal or species, it is particularly contemplated that the method of the invention will be useful in medical treatment of humans, and will also be useful in veterinary treatment, particularly of companion animals such as cats and dogs, livestock such as cattle, horses and sheep, and zoo animals, including non-human primates, large bovids, felids, ungulates and canids .

The compound may be administered at any suitable dose and by any suitable route. The route of administration is preferably parenteral, for example i.v., so that effective blood concentrations of the drug are reached as quickly as possible, because of the gravity of the condition, and because shunting of the blood away from the non-vital organs such as the stomach would reduce absorption from enteral routes. In general i.v. administration is preferred.

The effective dose will depend on the nature of the condition to be treated, and the age, weight, and underlying state of health of the individual treatment.

This will be at the discretion of the attending physician or veterinarian. Suitable dosage levels may readily be determined by trial and error experimentation, using methods which are well known in the art .

BRIEF DESCRIPTION OF THE FIGURES Figure 1 summarises the mean arterial pressure results and fluid resuscitation requirements of the animals in each group. A. Mean arterial blood pressure. B. Fluid resuscitation requirements.

Figure 2 compares the lung permeability index (LPI) in rats from each group.

Figure 3 shows the change in intestinal permeability with time after removal of the clamp.

Figure 4 shows myeloperoxidase activity in samples of lung and intestin. A. Lung. B. Intestine. Figure 5 shows cytokine levels in samples of gut tissue from animals of each group. A. TNF-α. B. IL-6.

Figure 6 shows cytokine levels in lung tissue from animals of each group. A. TNF-α. B. IL-6.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described by way of reference only to the following general methods and experimental examples .

In the claims which follow and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features, but not to preclude the presence or addition of further features in various embodiments of the invention.

As used herein, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "an enzyme" includes a plurality of such enzymes, and a reference to "an amino acid" is a reference to 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 to practice or test the present invention, the preferred materials and methods are now described. 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 granulocyte

PMSF phenylmethylsulfonyl fluoride sc subcutaneous

TNF-α tumour necrosis factor-α

Throughout the specification conventional single- letter and three-letter codes are used to represent amino acids.

For the purposes of this specification, the term "alkyl" is to be taken to mean a straight, branched, or cyclic, substituted or unsubstituted alkyl chain of 1 to 6, preferably 1 to 4 carbons. Most preferably the alkyl group is a methyl group. The term "acyl" is to be taken to mean 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" is to be understood to mean a substituted or unsubstituted homocyclic or heterocyclic aryl group, in which the ring preferably has 5 or 6 members .

A "common" amino acid is a L-amino acid selected 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.

An "uncommon" amino acid includes, but is not restricted to, D-amino acids, homo-amino acids, N-alkyl amino acids, dehydroamino acids, aromatic amino acids other than phenylalanine, tyrosine and tryptophan, ortho-, meta- or para-aminobenzoic acid, ornithine, citrulline, canavanine, norleucine, γ-glutamic acid, aminobutyric acid, L-fluorenylalanine, L-3-benzothienylalanine, and α, α-disubstituted amino acids.

Generally, the terms "treating", "treatment" and the like are used herein to mean affecting a subject, tissue or cell to obtain a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure of a disease.

"Treating" as used herein covers any treatment of, or prevention of disease in a vertebrate, a mammal, particularly a human, and includes: preventing the disease from occurring in a subject who may be predisposed to the disease, but has not yet been diagnosed as having it; inhibiting the disease, ie., arresting its development; or relieving or ameliorating the effects of the disease, ie., cause regression of the effects of the disease.

The invention includes the use of various pharmaceutical compositions useful for ameliorating disease. The pharmaceutical compositions according to one embodiment of the invention are prepared by bringing a compound of formula I, analogue, derivatives or salts thereof and one or more pharmaceutically-active agents or combinations of compound of formula I and one or more pharmaceutically-active agents into a form suitable for administration to a subject using carriers, excipients and additives or auxiliaries.

Frequently used carriers or auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols. Intravenous vehicles include fluid and nutrient replenishers . Preservatives include antimicrobial, anti- oxidants, chelating agents and inert gases. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in 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), the contents of which are hereby incorporated by reference. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art.. See Goodman and Gilman's The Pharmacological Basis for Therapeutics (7th ed. , 1985). The pharmaceutical compositions are preferably prepared and administered in dosage units. Solid dosage units include tablets, capsules and suppositories. For treatment of a subject, depending on activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the subject, different daily doses can be used. Under certain circumstances, however, higher or lower daily doses may be appropriate. The dose can be administered either by single administration in the form of an individual dosage unit or in several smaller dosage units, or alternatively by multiple administration of subdivided doses at specific intervals . The pharmaceutical compositions according to the invention may be administered locally or systemically in a therapeutically effective dose. Amounts effective for this use will, of course, depend on the severity of the disease and the weight and general state of the subject. Typically, dosages used in vi tro may provide useful guidance in the amounts useful for in si tu administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of the cytotoxic side effects. Various considerations are described, eg. in Langer, Science, 249: 1527, (1990). Formulations for oral use may be in the form of hard gelatin capsules, in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin. They may also be in the form of soft gelatin capsules, in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions normally contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients may be suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents, which may be

(a) a naturally occurring phosphatide such as lecithin;

(b) a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate; (c) a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethylenoxycetanol;

(d) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol such as polyoxyethylene sorbitol monooleate, or

(e) a condensation product of ethylene oxide with a partial ester derived from fatty acids and hexitol anhydrides , for example polyoxyethylene sorbitan monooleate .

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents such as those mentioned above. The sterile injectable preparation may also a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol . Among the acceptable vehicles and solvents which may be employed are water. Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono-or diglycerides . In addition, fatty acids such as oleic acid may be used in the preparation of injectables . Compounds of formula I may 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 .

Dosage levels of the compound of formula I of the present invention will usually be of the order of about 0.5mg to about 20mg per kilogram body weight, with a preferred dosage range between about 0.5mg to about lOmg per kilogram body weight per day (from about 0.5g to about 3g per patient per day) . The amount of active ingredient which may be combined with the carrier materials to produce a single dosage will vary, depending upon the host to be treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain about 5mg to Ig of an active compound with an appropriate and convenient amount of carrier material, which may vary from about 5 to 95 percent of the total composition. Dosage unit forms will generally contain between from about 5mg to 500mg of active ingredient . It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

In addition, some of the compounds of the invention may form solvates with water or common organic solvents. Such solvates are encompassed within the scope of the invention.

The compounds of the invention may additionally be combined with other therapeutic compounds to provide an operative combination. It is intended to include any chemically compatible combination of pharmaceutically- active agents, as long as the combination does not eliminate the activity of the compound of formula I of this invention.

General Methods

Peptide synthesis

Cyclic peptide compounds of formula I are prepared according to methods described in detail in our earlier applications No. PCT/AU98/00490 and No. PCT/AU02/01427, the entire disclosures of which are incorporated herein by this reference. While the invention is specifically illustrated with reference to the compound AcF- [OPdChaWR] (PMX53), whose corresponding linear peptide is Ac-Phe-Orn-Pro-dCha-Trp-Arg, it will be clearly understood that the invention is not limited to this compound.

Compounds 1-6, 17, 20, 28, 30, 31, 36 and 44 - Un disclosed in International patent application NO.PCT/AU98/00490 and compounds 10-12, 14, 15, 25, 33, 35, 40, 45, 48, 52, 58, 60, 66, and 68-70 disclosed for the first time in International patent application PCT/AU02/01427 have appreciable antagonist potency (IC50 < 1 μM) against the C5a receptor on human neutrophils. PMX53 and compounds 33, 45 and 60 of PCT/AU02/01427 are most preferred.

We have found that all of the compounds of formula I which have so far been tested have broadly similar pharmacological activities, although the physicochemical properties, potency, and bioavailability of the individual compounds varies somewhat, depending on the specific substituents . The general tests described below may be used for initial screening of candidate inhibitor of G protein- coupled receptors, and especially of C5a receptors.

Drug preparation and formulation The human C5a receptor antagonist AcF- [OPdChaWR]

(AcPhe [Orn-Pro-D-Cyclohexylalanine-Trp-Arg] ) was synthesized as described above, purified by reversed phase HPLC, and fully characterized by mass spectrometry and proton NMR spectroscopy. The C5a antagonist was prepared in olive oil (10 mg/mL) for oral dosing and in a 30% polyethylene glycol solution (0.6 mg/mL) for SC dosing. It was prepared in a 50% propylene glycol solution (30 mg/kg) for IP injections.

Recepto -Binding Assay

Assays are performed with fresh human PMNs, isolated as previously described (Sanderson et al , 1995), using a buffer of 50 mM HEPES, 1 mM CaCl2 5 itiM MgCl2 , 0.5% bovine serum albumin, 0.1% bacitracin and 100 μM phenylmethylsulfonyl fluoride (PMSF) . In assays performed at 4°C, buffer, unlabelled human recombinant C5a (Sigma) or peptide, Hunter/Bolton labelled 125 _£5_a (_ 20 pM) (New England Nuclear, MA) and PMNs (0.2 x 10⁶) are added sequentially to a Millipore Multiscreen assay plate (HV 0.45) having a final volume of 200 μL/well. After incubation for 60 min at 4°C, the samples are filtered and the plate washed once with buffer. Filters are dried, punched and counted in an LKB gamma counter. Non-specific binding is assessed by the inclusion of ImM peptide or 100 nM C5a, which typically results in 10-15% total binding.

Data are analysed using non-linear regression and statistics with Dunnett post-test.

Myeloperoxidase Release Assay for Antagonist Activity

Cells are isolated as previously described (Sanderson efc al , 1995) and incubated with cytochalasin B (5μg/mL, 15 min, 37°C) . Hank's Balanced Salt solution containing 0.15% gelatin and peptide is added on to a 96 well plate (total volume 100 μL/well) , followed by 25 μL cells (4xl0^/mL) . To assess the capacity of each peptide to antagonise C5a, cells are incubated for 5 min at 37°C with each peptide, followed by addition of C5a (100 nM) and further incubation for 5 min. Then 50 μL of sodium phosphate (0.1M, pH 6.8) is added to each well, the plate was cooled to room temperature, and 25 μL of a fresh mixture of equal volumes of dimethoxybenzidine (5.7 mg/mL) and H2O2 (0.51%) is added to each well. The reaction is stopped at 10 min by addition of 2% sodium azide. Absorbances are measured at 450 nm in a Bioscan 450 plate reader, corrected for control values (no peptide) , and analysed by non-linear regression.

Statistical Analysis

Values are means ± standard error mean (SEM) , and differences between group means were considered significant at P<0.05. Data were analysed by a one-way ANOVA, and individual group comparisons by Student's t Test. EXAMPLE 1 : Animal Model of Ruptured Aortic Aneurysm Male Sprague-Dawley rats (350-500g) were used throughout the experiment. All animals were anesthetized with pentobarbital sodium (50 mg/kg ip) . For each rat, a tail vein and the right carotid artery were cannulated with 22-gauge angiocaths and sutured in place. The tail vein was used to administer supplemental doses of anesthetic, ¹²⁵I-labeled albumin, C5aR antagonist, and Ringer's lactate solution and for re-infusion of shed blood. The carotid artery cannula provided continuous monitoring of the mean arterial pressure (MAP) and was used to haemorrhage animals.

Animals were randomised into two groups: a) sham (n=6) ; and b) shock + clamp (n=19) .

Animals in the shock + clamp group were further randomised into C5aR antagonist-treated (n=9) and control- treated groups (n=10) . In the treated group, the small molecule C5aR antagonist, AcF- (OPdChaWR) (Promics Pty Ltd, Queensland, Australia) was administered intravenously over two minutes at the end of a haemorrhagic shock at a dose of 1 mg/kg in endotoxin-free saline, whereas the control group received saline infusion. In all cases the operator was blinded to the treatment given. The abdominal aorta was exposed by midline laparotomy, and isolated at the superior mesenteric artery and just proximal to the iliac bifurcation. A 5-cm segment of jejunum, approximately 10 cm from the ligament of Trietz, was isolated and cannulated at its proximal end with an input cannula, and at its distal end with an output cannula. The cannulas were exteriorized via two incisions made in the right abdominal wall, and the abdomen was sutured closed. The cannulated intestinal segment was flushed with Ringer's lactate solution until the output was devoid of solid particles. The intestinal segment was perfused with Ringer's lactate solution at 37°C, at a rate of 0.3 ml/min with an infusion pump (model AVI 480, 3M, St. Paul, MN) throughout the duration of the experiment .

For the determination of intestinal and pulmonary permeability, animals then received ¹²⁵I-albumin (~lμCi) via the tail vein catheter, and were allowed to stabilize for 30 min to establish postoperative equilibrium. During the stabilization and experimental periods, intestinal perfusate was collected every 10 min. Throughout the experimental period, samples of blood (0.3 ml) were withdrawn at 1 h intervals. The blood samples were used for the measurement of total albumin concentration, and the specific activity of ¹²⁵I-albumin used for the calculation of intestinal albumin loss, as described below. In appropriate groups, shock was induced by withdrawal of blood into a plastic heparinized syringe (500 U) to reduce and maintain MAP at 50 mmHg for 1 h. The shed blood was maintained at room temperature on a tube rocker during the shock period. After 60 min of shock or the equivalent control period, clamps were applied to the abdominal aorta just proximal to the superior mesenteric artery and at the iliac bifurcation. At this point, one-half of the shed blood was reinfused into the tail vein. The clamps remained in place for 45 min. Just before clamp removal, the remainder of the shed blood was reinfused. Additional Ringer's lactate solution was also administered, as required, to resuscitate the animals and maintain MAP at 100 mmHg. Reperfusion was continued for 120 min, at which time the animals were killed with an overdose of pentobarbital sodium.

The perfused intestinal segment was harvested, weighed, and lyophilized to determine the intestinal dry weight. Portion of the lung and liver and of the intestine immediately distal to the perfused segment were excised, washed in ice-cold saline, and rapidly frozen in liquid nitrogen and stored at -70°C until analyzed for myeloperoxidase (MPO) and cytokine levels, respectively. The MAP and fluid resuscitation requirements for each group are summarised in Figure 1.

The mean arterial blood pressure (MAP) remained stable throughout the entire experimental period in sham animals, requiring minimal intravenous fluid resuscitation with Ringer's lactate solution, as summarised in Figure IB. In the shock and clamp animals the MAP was reduced during a haemorrhagic shock to < 50 mmHg for one hour as defined by the protocol. On application of the supramesenteric aortic clamp the MAP increased significantly compared to pre-shock levels (158±9.0 versus 117±3.0 mmHg, p<0.001) . In the shock and clamp animals after removal of the aortic clamp, the MAP dropped progressively during reperfusion to a nadir after 120 minutes of reperfusion (68+6.0 versus pre-shock 117+3.0, p<0.001), despite vigorous fluid resuscitation by intravenous infusion of Ringer's lactate solution (69.3+8.5 ml) .

Animals treated with C5a receptor antagonist maintained significantly better MAP during reperfusion compared to untreated shock and clamp animals (95±5.3 versus 68+6.0 mmHg, p<0.01), and required less intravenous fluid resuscitation (60.0+7.0 ml versus 69.3±8.5 ml, p<0.1, NS) . Throughout the experimental procedure the sham animals maintained a stable blood pressure, with minimal requirement for fluid resuscitation. Shock and clamp animals required significant fluid resuscitation from the start of the reperfusion period after aortic clamp release in order to maintain MAP. After the initial response to fluid resuscitation in the first hour of reperfusion, shock refractory to fluid resuscitation developed in the second hour of reperfusion, requiring large volumes of intravenous fluid to maintain a blood pressure. Treatment with the C5aR antagonist significantly prevented the severe hypotension seen in the untreated group, and the antagonist-treated animals required less fluid resuscitation.

EXAMPLE 2 : Determination of Pulmonary Permeability The heart and lungs were excised in toto, the left lung was lavaged three times with 3.5 ml Ringer's lactate solution, and the effluent bronchoalveolar lavage (BAL) fluid was collected. Blood and BAL fluid were weighed and counted for ¹²⁵I activity, and the lung permeability index (LPI) was calculated using the following formula:

LPI=BAL-^{1 5}I (cpm/g) /blood-¹²⁵I (cpm/g)

The results are summarised in Figure 2. The index of lung permeability (LPI) to ¹²⁵i- labelled albumin was significantly increased in- the shock and clamp group compared to the sham group (4.43+0.96 versus 1.30+0.17, p<0.01). This effect was blocked by treatment with C5a receptor antagonist (1.74±0.50, p<0.03) .

EXAMPLE 3 : Determination of Intestinal Permeability

Intestinal permeability was used as an index of intestinal injury, and was measured as previously described (Boyd et al, 1999) .

To calculate intraluminal intestinal albumin loss, all 10 min effluent collections from the intestinal perfusion were weighed, and a 1 ml sample of each was assayed for ¹²⁵I-albumin activity with a gamma counter. Each blood sample drawn during the experimental procedure was centrifuged at 100,000 rpm, and 100 μl of plasma were removed for determination of albumin content and ¹²⁵i- albumin activity. The level of ^{1 5}I in the blood samples was regressed against time, and the slope of the curve was used to determine the activity of this isotope in whole blood. This was used to determine the specific activity of ¹²⁵I per μ gram of total albumin to calculate intestinal albumin loss, expressed as milligrams per gram dry weight of the perfused intestinal segment. The results are shown in Figure 3.

The rate of intraluminal intestinal albumin loss, the intestinal permeability index (IPI) , remained stable throughout the entire experimental period in sham animals . In shock and clamp animals the IPI remained stable during the stabilization, haemorrhage and clamp periods; however, on reperfusion there was a statistically significant increase in IPI. After 30 minutes of reperfusion, the IPI was significantly increased in shock and clamp animals compared to pre-shock levels (8.05xl0^~2+3.59xl0^"2 versus 0.72xl0^_2+0.51xl0^~2, p<0.0001), and compared to control levels (8.05xl0^~2+3.59xl0^~2 versus 1.75xl0^~2±0.33xl0^~2, p<0.0001), and remained at similar levels throughout the 120-min reperfusion period.

Treatment with the C5a receptor antagonist significantly reduced the increase in IPI in early reperfusion; after 30 minutes of reperfusion, the IPI was significantly reduced in C5aR antagonist-treated animals compared to untreated shock and clamp animals (2.82x10^" ±0.91xl0^~2 versus 8.05xl0^"2+3.59xl0^"2, p<0.01). However, as reperfusion progressed the IPI increased even in the C5a antagonist-treated group to mirror the' levels in untreated shock and clamp animals.

EXAMPLE 4 : Measurement of Neutrophil Sequestration

Lung and intestinal tissue samples were assayed for myeloperoxidase (MPO) activity, an index of neutrophil sequestration, as previously described (Boyd et al, 1999). In brief, MPO activity was assessed at 37°C by monitoring the change in absorbance at 655 nm over a 3-min period in a Cobas FARA II centrifugal analyzer (Roche Diagnostic Systems, Montclair, NJ) . The reaction mixture contained 16 mmol/1 3 , 3¹, 5 , 5'-tetramethylbenzidine dissolved in N,N- dimethylformamide in 0.22 mol/1 phosphate buffered saline which contained 0.11 mol/1 NaCl at pH 5.4. The reaction was initiated by the addition of 3 mmol/1 hydrogen peroxide. One unit of activity was defined as a one-unit change in absorbance per minute at 37°C. The protein content of pulmonary and intestinal samples was determined by the bicinchoninic acid protein assay system (Pierce, Rockford, IL) . MPO activity was expressed as units per milligram of protein. The results are shown in Figure 4.

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

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

EXAMPLE 5 : Measurement of Cytokines in Intestine and Lung

One hundred mg of each tissue was homogenised in 1 mL of PBS (0.4 mol/L NaCl and 10 mmol/L Na₂HP0 ) containing protease inhibitors (0.1 mmol/L phenylmethyl sulfonyl fluoride, 0.1 mmol/L benzethonium chloride, 10 mmol/L ethylenediaminetetraacetic acid, and 20 KI aprotinin A) and 0.05% Tween 20. The samples were then centrifuged for 10 minutes at 3000 g and the supernatant immediately used for enzyme-linked immunosorbent assay at a 1:2 dilution in assay dilution buffer. The concentrations of TNF-α and Interleukin-6 in samples were measured using commercially-available antibodies, according to the procedures supplied by the manufacturer (R&D Systems, Minneapolis, MN) . The protein content of intestine and lung samples was determined 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 Figure 6.

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

As shown in Figure 5b, intestinal IL-6 levels were elevated in the shock and clamp group compared to the sham group (280.91±35.95 versus 168.38+35.23 pg/mg protein, p<0.04). Interestingly, the increase in intestinal IL-6 levels was significantly less in the 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, lung TNF-α levels were significantly elevated in shock and clamp animals compared to sham animals (89.70+13.83 versus 47.57±11.22 pg/mg protein, p<0.03), but this increase was not prevented by treatment with C5a receptor antagonist (78.71+15.78 pg/mg protein, p=NS) .

Lung IL-6 levels, shown in Figure 6b, were elevated in the shock and clamp animals compared to the sham animals (227.98151.74 versus 144.81+26.31 U/mg protein, p=NS) . However, this increase did not reach statistical significance. Interestingly, lung IL-6 levels were significantly elevated in the shock and clamp group treated with C5a receptor antagonist compared to the sham groups (320.72±37.67 versus 144.81±26.31 U/mg protein, p<0.002) .

EXAMPLE 6 : Further pre-clinical studies

The effects of therapeutic agents such as those of the invention on haemorrhagic shock may be tested in a variety of experimental models in addition to the one described herein. The most common experimental species used are pigs and rats, and, to a lesser extent, sheep and mice. Because of their larger size and the strong similarities of their cardiovascular systems and parameters to those of humans, pigs are the most commonly used. Blood loss may be induced by a variety of methods, and the specific method does not appear to have any bearing on the outcome.

The C5a antagonist compounds of the invention may be used in any of these models, subject to the caveat that if the receptor affinity is lower in mice, sheep and pigs than the affinity observed in rats, this may reduce the potency or efficacy of the antagonist.

The test compound is administered following induction of haemorrhagic shock. The route of administration is preferably parenteral, for example i.v., so that effective blood concentrations of the drug are reached as quickly as possible, because of the gravity of the condition, and because shunting of the blood away from the non-vital organs such as the stomach would reduce absorption from enteral routes. In general i.v. administration is used in these experiments . The test compound is administered at various doses and at various times after induction of haemorrhagic shock, in order to ascertain the optimum regimen. Control animals are treated with a sham injection, are left untreated, or are treated with a comparative agent; this may be another ant-inflammatory agent such as infliximab, or may be another agent used for treatment of haemorrhagic shock.

The effect of each treatment is monitored using measurement of parameters such as cardiac output (stroke volume x heart rate) mean arterial pressure fluid resuscitation requirements neutrophil sequestration in tissues levels of circulating or tissue cytokines such as

TNFα and IL-1, IL-2, IL-6, and IL-8 reduction in intracellular ATP blood haemoglobin levels metabolic acidosis, and other changes such as intestinal permeability pulmonary permeability

Mean arterial pressure, fluid resuscitation requirements, neutrophil sequestration in tissues, intestinal permeability, pulmonary permeability, and levels of TNFα and IL-6 may be measured as described in the preceding examples, or using other methods known in the art. Physiological and biochemical parameters may be measured by standard methods; for example, blood haemoglobin is assessed by haematocrit, and metabolic acidosis is assessed by measurement of pH of arterial blood or by measurement of P_Co₂ • Levels of TNF-α, IL-6 and other cytokines may be measured using commercially- available assays, such as immunoassays .

As far as possible the parameters chosen for study are those which are accepted by regulatory authorities such as the US Food and Drug Administration, the European Agency for the Evaluation of Medicinal Products, and the Australian Therapeutic Goods Administration .

DISCUSSION

The development of specific monoclonal anti- complement antibodies has renewed interest in complement as a therapeutic target in the critically ill (Matis et al, 1995) . In the RAAA model used herein, haemorrhagic shock for 1 hour at a MAP of 50 mmHg followed by 45 minutes of supramesenteric aortic clamping and 120 minutes of reperfusion resulted in significant intestinal and pulmonary injury, and refractory shock despite vigorous fluid resuscitation. In this study we have demonstrated for the first time that the local and systemic injury associated with RAAA can be attenuated using a specific small molecule C5a receptor, the cyclic peptide AcF- (OPdChaWR) , in a rat model. Intestinal injury in this model was associated with a significant increase in intestinal capillary permeability to ¹²⁵I-albumin immediately after release of the supra-mesenteric aortic clamp, an increase which persisted throughout the 120 minute reperfusion period. Treatment with the C5aR antagonist significantly prevented the increase in intestinal permeability in early reperfusion. However, in late reperfusion intestinal permeability increased to levels similar to those in non- treated shock and clamp animals. Increased intestinal permeability has been reported after RAAA and elective 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) . Injury to the intestine is two-fold, in that the initial global hypoxia during haemorrhagic shock is compounded by the direct ischaemia-reperfusion injury during and after release of the supramesenteric aortic clamp. Intestinal ischaemia-reperfusion injury is associated with neutrophil sequestration and increased microvascular permeability, and can be modulated by neutrophil depletion or by antibodies directed against neutrophil adhesion molecules (Hernandez et al, 1987). In a recent study in the rat, the same C5a receptor antagonist as that used in the present study was reported to be effective in reducing intestinal ischaemia- reperfusion injury and reduced the neutrophilic response to ischaemia-reperfusion injury (Arumugam et al, 2002).

Complement activation occurs in the early stages of inflammation, releasing the anaphylatoxins C3a, C4a, C5a and the C5b-C9 membrane attack complex. These activated complement components alter vascular tone and permeability, and have been shown to be integral to intestinal reperfusion injury (Williams et al, 1999) , while the membrane attack complex is directly lytic to cells. The anaphylatoxins, particularly C5a, chemotactically recruit and activate inflammatory cells and lead to the release of the cytokines TNF-α and IL-6. By inhibiting the early interaction between the activated complement component, C5a, and its target cells on the intestinal vascular endothelium, and circulating immune cells, we have reduced the severity of the initial gut injury in this model.

Direct or indirect intestinal ischaemia- reperfusion injury induces functional and morphological changes in the gut associated with translocation of bacterial fragments across a damaged intestinal capillary barrier, with the resultant endotoxaemia producing an exaggerated inflammatory response . This suggests that the gut drives the inflammatory response to a variety of critical illnesses (Baue et al, 1997) . Complement has 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 blunts the oxidative burst in PMNs following exposure to E. coli (Mollnes et al. , 2002) . We hypothesized that C5a receptor blockade could decrease the pro-inflammatory effects of endotoxin in early reperfusion. However, as reperfusion continues intestinal injury increases, most probably due to the cellular effects of ischaemia-reperfusion injury and parallel activation of cytokine and pro-inflammatory mediator cascades . Our findings are in agreement with previous reports that direct intestinal ischaemia- reperfusion injury can be attenuated by blocking the complement cascade at a variety of points (Hill et al, 1992; Arumuga et al, 2002) .

The reduction in intestinal myeloperoxidase concentration by the C5a antagonist compared to untreated shock and clamp animals suggests that local complement- induced neutrophil chemotaxis and activation, or target cell opsonisation, may be crucial to neutrophil sequestration and intestinal injury in this model. Complement activation has been shown in vi tro to stimulate the rapid adhesion of neutrophils to endothelial target cells (Marks et al, 1989) , and this effect is mediated via the actions of C5a. In the present study, shock and clamp animals have significantly increased intestinal levels of the pro-inflammatory cytokine TNF-α compared to sham animals, and this is not altered by treatment with C5aR antagonist. Although activated complement is known to cause the release of TNF-α from a variety of cell types, including immune cells, by a receptor-mediated effect (Barton et al, 1993), and intravenously-administered C5a increases circulating TNF-α levels in rats (Strachan et al, 2000), a variety of other mediators released after ischaemia-reperfusion injury such as arachidonic acid metabolites, also stimulate cytokine release. IL-6 is an important pleiotrophic cytokine, with a variety of pro- and anti-inflammatory effects. High serum levels of IL-6 have been associated with increased morbidity and mortality after abdominal aortic aneurysm repair (Groeneveld et al, 1997) . In the present study we have found that in this model, intestinal injury is associated with increased intestinal tissue levels of IL- 6, and that treatment with C5aR antagonist prevents this increase. This is important, as IL-6 has been shown to upregulate the pro-inflammatory effects of TNF-α in response to complement activation (Platel et al, 1996) . IL-6 has been shown to inhibit apoptosis of neutrophils, prolonging their functional longevity and potential for tissue injury (Biffl et al, 1996) ; therefore inhibition of its release may reduce further neutrophil-mediated intestinal injury. These data suggest that C5a and immune cell receptor interaction may be integral to the release of IL-6 in this model, or alternatively that this may also reflect the decreased tissue sequestration of neutrophils due to C5aR antagonism.

Ruptured abdominal aortic aneurysm is associated with a non-cardiac acute interstitial pulmonary oedema and associated hypoxemia, termed acute respiratory distress syndrome (ARDS) , which carries a grave prognosis. In our model of RAAA the shock and clamp group had significantly increases in pulmonary permeability to ¹²⁵I-albumin and in lung myeloperoxidase levels . The suggestion that lung injury in this syndrome is primarily due to neutrophil adherence, sequestration and subsequent respiratory burst- induced oxidative injury is supported by the observation that lung injury can be attenuated with anti-CDl8 monoclonal antibodies (Boyd et al, 1999). In the model used in the present study, antagonism of the C5a receptor on target cells reduces neutrophil sequestration and subsequent microvascular hyperpermeability, and the combination of the systemic haemorrhagic shock injury compounded by lower torso ischaemia-reperfusion produces a severe acute lung injury. Abrogation of C5a-induced neutrophil chemotaxis and activation in the pulmonary circulation (Solokin et al, 1985) may in part explain the attenuation of injury in this study. The C5a receptor antagonist does not inhibit the formation of the membrane attack complex (Arumugam et al, 2003). It is unlikely that C5a receptor blockade affects the formation of the C5b-9 membrane attack complex, but, regardless of this, it is unlikely that this large complex could travel unmolested to the pulmonary circulation to exert its lytic effects. In this acute model, modulation of the early complement-dependent injury may be sufficient to convert a lethal acute lung failure to a recoverable acute lung dysfunction. As with the intestine, TNF-α levels in the lung significantly increase after shock and clamp, and this is not affected by treatment with C5aR antagonist. TNF-α is produced after a variety of stresses, and has been shown to induce direct lung injury (Welbourn et al, 1991) . We found that IL-6 levels in the lung are also increased after shock and clamp compared to sham animals, although this increase did not reach significant levels.

Interestingly, shock and clamp animals treated with C5aR antagonist have a highly significant increase in lung tissue IL-6 compared to sham animals, and this suggests that protection from complement-induced remote injury may be independent of IL-6. Alternatively this may suggest that IL-6 release has a beneficial anti-inflammatory effect in the lung in this model, perhaps through paracrine inhibition of inflammatory mediator release.

Haemorrhagic shock itself initiates a cascade of pro-inflammatory mediator induction (Abraham, 1991) , and oxidative injury is associated with the degree of complement activation in cardiac patients (Cavarocchi et al, 1986) . Aortic clamp release is associated with a variety of vasoactive effects, including hypovolaemia due to peripheral vasodilation and increased vascular permeability, reperfusion of ischaemic tissues with circulation of vasoactive mediators and metabolites, and myocardial depressant factors (Barry et al, 1997) . The effect of the C5a receptor antagonist on reducing microvascular permeability and immune cell activation via complement receptor-specific pathways reduces the degree and duration of hypotension in the present model. This is probably a reflection of the reduced third space fluid loss due to the prevention of complement activation and complement-dependent increases in microvascular permeability. Complement activation is also known to have effects on vascular tone and histamine release (Ellis et al, 1991) , prevention of which may also help maintain vascular resistance. Reduced organ injury, and perhaps reduced myocardial depression, may also allow the animal to better handle the fluid load required to maintain target blood pressure. In conclusion, we have shown for the first time that a small molecule C5a receptor antagonist can reduce local intestinal and remote lung injury in a model of ruptured abdominal aortic aneurysm. This treatment appears to mediate its effects by reducing activated complement-immune cell interaction, thus in turn reducing the inflammatory stimulus to tissue neutrophil sequestration. Antagonism of the human C5a receptor therefore represents a realistic therapeutic target in patients with haemorrhagic shock, and potentially addresses a major clinical need.

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

References cited herein are listed on the following pages, and are incorporated herein by this reference.

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Claims

1. A method of treatment of haemorrhagic shock, comprising the step of administering an effective amount of an inhibitor of a C5a receptor to a subject in need of such treatment .

2. A method according to claim 1, in which the inhibitor is a compound which

(d) is an antagonist of a C5a receptor,

(e) has substantially no agonist activity, and (f) is a cyclic peptide or peptidomimetic compound of formula I

where A is H, alkyl, aryl, NH₂, NH-alkyl, N(alkyl)₂, NH-aryl, NH-acyl, NH-benzoyl, NHS0₃, NHS0₂- alkyl, NHS0₂-aryl, OH, 0-alkyl, or 0-aryl;

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

C is the side chain of a D-, L- or homo-amino acid, but is not the side chain of isoleucine, phenylalanine, or cyclohexylalanine;

D is the side chain of a neutral D-amino acid, but is not the side chain of glycine or D-alanine, a bulky planar side chain, or a bulky charged side chain;

E is a bulky substituent, but is not the side chain of D-tryptophan, L-N-methyltryptophan, L-homophenylalanine, L-2-naphthyl L-etrahydroisoquinoline, L-cyclohexylalanine, D-leucine, L-fluorenylalanine, or L-histidine;

F is the side chain of L-arginine, L- homoarginine, L-citrulline, or L-canavanine, or a bioisostere thereof; and X is -(CH₂)_nNH- or (CH₂)_n-S-, where n is an integer of from 1 to 4; -(CH₂)₂0-; -(CH₂)₃0-; -(CH₂)₃-_; -(CH₂)₄-; -CH₂COCHRNH- ; or -CH₂_CHCOCHRNH- , where R is the side chain of any common or uncommon amino acid.

3. A method according to claim 2, in which n is 2 or 3.

4. A method according to claim 2 or claim 3, in which A is an acetamide group, an aminomethyl group, or a substituted or unsubstituted sulphonamide group.

5. A method according to claim 3, in which A is a substituted sulphonamide, and the substituent is an alkyl chain of 1 to 6 carbon atoms, or a phenyl or toluyl group.

6. A method according to claim 5 , in which the substituent is an alkyl chain of 1 to 4 carbon atoms.

7. A method according to any one of claims 2 to 6, in which B is the side chain of L-phenylalanine or L- phenylglycine .

8. A method according to any one of claims 2 to 7, in which C is the side chain of glycine, alanine, leucine, valine, proline, hydroxyproline, or thioproline.

9. A method according to any one of claims 2 to 8 , in which D is the side chain of D-Leucine, D-homoleucine, D-cyclohexylalanine, D-homocyclohexylalanine, D-valine, D- norleucine, D-homo-norleucine, D-phenylalanine, D- tetrahydroisoquinoline, D-glutamine, D-glutamate, or D- tyrosine.

10. A method according to any one of claims 2 to 9 , in which E is the side chain of an amino acid selected from the group consisting of L-phenylalanine, L-tryptophan and L-homotryptophan, or is L-1-napthyl or L-3- benzothienyl alanine.

11. A method according to any one of claims 1 to 10, in which the inhibitor is a compound which has antagonist activity against C5aR, and has no C5a agonist activity.

12. A method according to any one of claims 1 to 11, in which the inhibitor has potent antagonist activity at sub-micromolar concentrations .

13. A method according to any one of claims 1 to 12, in which the compound has a receptor affinity IC50< 25μM, and an antagonist potency IC50< lμM.

14. A method according to any one of claims 1 to 13 , in which the compound is selected from the group consisting of compounds 1 to 6, 10 to 15, 17, 19, 20, 22, 25, 26, 28, 30, 31, 33 to 37, 39 to 45, 47 to 50, 52 to 58 and 60 to 70 described in PCT/AU02/01427.

15. A method according to claim 14, in which the compound is AcF [OP-DCha-WR] , AcF [OP-DPhe-WR] , AcF [OP-DCha- FR] , AcF[OP-DCha-WCit] ) , HC- [OPdChaWR,

AcF-[OPdPheWR, AcF- [OpdChaWCitrulline] or HC- [OPdPheWR.

16. A method according to any one of claims 1 to 15, in which the inhibitor is used in conjunction with one or more other agents for the treatment of haemorrhagic shock.

17. A method according to any one of claims 1 to 16, in which the shock results from major haemorrhage caused by a condition selected from the group consisting of trauma, rupture of an aneurysm, uncontrollable epistaxis, haemorrhagic fever, uterine haemorrhage during or after delivery, haemorrhage during or after surgery, haemorrhage resulting from gastrointestinal ulcers or oesophageal varices, haemorrhage of the lower gastrointestinal tract, haemorrhage secondary to invasion of cancer, haemorrhage resulting from bleeding diatheses, and haemorrhage associated with thrombolytic therapy.

18. A method according to any one of claims 1 to 17, in which the sub ect is a human.

19. A method according to any one of claims 1 to 18, in which the inhibitor is administered parenterally, orally, transdermally or intranasally .

20. A method according to claim 19, in which the inhibitor is administered intravenously.

21. Use of an inhibitor of a C5a receptor in the manufacture of a medicament for the treatment of haemorrhagic shock.

22. Use according to claim 21, in which the inhibitor is a compound as defined in any one of claims 1 to 15.

23. Use according to claim 21 or claim 22, in which the medicament is suitable for intravenous administration.