Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock

Definitions

• 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 .
• 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) .
• 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- ⁇ , 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 .
• 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 .
• 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 .
• interventions 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.
• the treatments may be of limited effectiveness, and may have serious side effects.
• RAAA abdominal aortic aneurysm
• 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) .
• 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)- ⁇ , interleukin-6 and interleukin-8, and of endotoxin (Baigrie et al, 1993) have been demonstrated after repair of RAAA (Roumen et al, 1993).
• 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) .
• C5a and C3a 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) .
• C5aR C5a receptor
• lung injury induced by limb ischaemia is mediated by leukotrienes, not by complement (Klausner et al, 1989) .
• the role of complement in inflammatory tissue injury after ruptured abdominal aortic aneurysm is still largely unknown.
• soluble complement receptor type 1 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) .
• complement activation such as neutrophil oxidative burst, complement-mediated haemolysis, and C3a and C5a production
• C3a and C5a production US patents No 5456909, No 5807844 and No 5858969
• 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.
• the inhibitor is a compound which
• (a) is an antagonist of a C5a receptor
• (c) is a cyclic peptide or peptidomimetic compound of Formula I
• A is H, alkyl, aryl, NH 2 , NH-alkyl, N(alkyl) 2 , NH-aryl, NH-acyl, NH-benzoyl, NHS0 3 , NHS0 2 - alkyl, NHS0 2 -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
• 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 -(CH 2 ) n NH- or (CH 2 ) n -S-, where n is an integer of from 1 to 4, preferably 2 or 3; -(CH 2 ) 2 0-; -(CH 2 ) 3 0-; -(CH 2 ) 3 -; - (CH 2 ) 4 - ; . -CH 2 COCHRNH- ; or -CH-CHCOCHRNH-, where R is the side chain of any common or uncommon amino acid.
• A is an acetamide group, an aminomethyl group, or a substituted or unsubstituted sulphonamide group .
• 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.
• 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 ⁇ 25 ⁇ M, and an antagonist potency IC50 ⁇ l ⁇ M.
• 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.
• 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.
• 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.
• agents such as warfarin, aspirin, plasminogen activator, streptokinase or urokinase.
• 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.
• 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.
• Suitable dosage levels may readily be determined by trial and error experimentation, using methods which are well known in the art .
• Figure 1 summarises the mean arterial pressure results and fluid resuscitation requirements of the animals in each group.
• 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.
• Figure 5 shows cytokine levels in samples of gut tissue from animals of each group.
• Figure 6 shows cytokine levels in lung tissue from animals of each group.
• 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.
• 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 .
• 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, ⁇ -glutamic acid, aminobutyric acid, L-fluorenylalanine, L-3-benzothienylalanine, and ⁇ , ⁇ -disubstituted amino acids.
• 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 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.
• 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.
• 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.
• an inert solid diluent for example, calcium carbonate, calcium phosphate or kaolin.
• 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.
• 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
• 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 .
• the acceptable vehicles and solvents which may be employed are water. Ringer's solution, and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil may be employed, including synthetic mono-or diglycerides .
• 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.
• 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 .
• 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.
• solvates 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.
• 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.
• 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.
• 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 ⁇ M phenylmethylsulfonyl fluoride (PMSF) .
• PMSF phenylmethylsulfonyl fluoride
• Cells are isolated as previously described (Sanderson efc al , 1995) and incubated with cytochalasin B (5 ⁇ g/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 ⁇ L/well) , followed by 25 ⁇ L 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.
• 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, 125 I-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.
• 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 .
• mice For the determination of intestinal and pulmonary permeability, animals then received 125 I-albumin ( ⁇ l ⁇ Ci) 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 125 I-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.
• 125 I-albumin ⁇ l ⁇ Ci
• 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.
• MPO myeloperoxidase
• cytokine levels respectively.
• the MAP and fluid resuscitation requirements for each group are summarised in Figure 1.
• MAP mean arterial blood pressure
• 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) .
• 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 125 I activity, and the lung permeability index (LPI) was calculated using the following formula:
• Intestinal permeability was used as an index of intestinal injury, and was measured as previously described (Boyd et al, 1999) .
• IPI intestinal permeability index
• 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.
• 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 , 3 1 , 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.
• 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) .
• 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).
• 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
• Tween 20 0.05% Tween 20
• TNF- ⁇ 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.
• 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).
• 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).
• 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.
• 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.
• cardiac output stroke volume x heart rate
• neutrophil sequestration in tissues levels of circulating or tissue cytokines neutrophil sequestration in tissues levels of circulating or tissue cytokines
• Mean arterial pressure, fluid resuscitation requirements, neutrophil sequestration in tissues, intestinal permeability, pulmonary permeability, and levels of TNF ⁇ 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 C o 2 •
• Levels of TNF- ⁇ , IL-6 and other cytokines may be measured using commercially- available assays, such as immunoassays .
• Intestinal injury in this model was associated with a significant increase in intestinal capillary permeability to 125 I-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.
• 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) .
• 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).
• 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- ⁇ and IL-6.
• 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 .
• 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) .
• activated complement is known to cause the release of TNF- ⁇ 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- ⁇ 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) .
• ARDS acute respiratory distress syndrome
• 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 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).
• 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.
• 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.
• 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.
• a small molecule C5a receptor antagonist protects kidneys from ischemia/reperfusion injury in rats. Kidney Int 2003; 63 (1) : 134-142.
• Soluble complement receptor type 1 ameliorates the local and remote organ injury after intestinal ischemia- reperfusion in the rat. J Immunol 1992; 149 (5) : 1723-1728.
• Paterson IS Klausner JM, Pugatch R, Allen P, Mannick JA, Shepro D et al . Noncardiogenic pulmonary edema after abdominal aortic aneurysm surgery. Ann Surg 1989; 209(2) :231-236. Piccolo, MT, Wang, Y, Sannomiya, P et al . , Chemotactic mediator requirements in lung injury following skin burns. Exp. Mol. Pathol. 199; 66: 220-226

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Public Health (AREA)
• Engineering & Computer Science (AREA)
• Veterinary Medicine (AREA)
• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• Medicinal Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Pharmacology & Pharmacy (AREA)
• Organic Chemistry (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Diabetes (AREA)
• Hematology (AREA)
• Gastroenterology & Hepatology (AREA)
• Immunology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Epidemiology (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=31501223

Family Applications (1)

Country Status (5)

Families Citing this family (11)

Family Cites Families (1)

• 2003
  • 2003-05-15 AU AU2003902354A patent/AU2003902354A0/en not_active Abandoned
• 2004
  • 2004-05-14 CA CA002557615A patent/CA2557615A1/en not_active Abandoned
  • 2004-05-14 WO PCT/AU2004/000642 patent/WO2004100975A1/en not_active Application Discontinuation
  • 2004-05-14 EP EP04732886A patent/EP1635857A1/en not_active Withdrawn
  • 2004-05-14 JP JP2006529441A patent/JP2006528208A/ja not_active Withdrawn

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events