ES2366538T3 - TREATMENT OF INTESTINAL INFLAMMATORY DISEASE. - Google Patents

Abstract

The use of an inhibitor of a G-protein coupled receptor in the production of a medicament for the treatment of inflammatory bowel disease, in which the inhibitor is a compound such that: (a) it is an antagonist of a protein-coupled receptor G, (b) has 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, NHSO3, NHSO2alkyl, NHSO2-aryl, OH, O-alkyl or O-aryl; B is an alkyl, aryl, phenyl, benzyl, naphthyl or indole group, or the side chain of an amino acid D or L, but it is not the side chain of glycine, D-phenylalanine, L-homophenylalanine, L-tryptophan, L- homotriptophan, L-tyrosine or Lhomothyrosine; C is the side chain of an amino acid D or L or a homoamino acid, but it is not the side chain of isoleucine, phenylalanine or cyclohexylalanine; D is the side chain of a neutral D amino acid, but it is not the glycine or D-alanine side chain, a bulky flat side chain or a bulky charged side chain; E is the side chain of an amino acid that is selected from the group consisting of L-phenylalanine, Ltriptophan and L-homotriptophan, or is L-1-naphthylalanine or L-3-benzothienylalanine; F is the side chain of L-arginine, L-homoarginine, L-citrulline or L-canavanine, or a bioister of these; and X 1 is - (CH2) nNH- or (CH2) n-S-, where n is an integer from 1 to 4; - (CH2) 2O-; - (CH2) 3O-; - (CH2) 3-; - (CH2) 4-; -CH2COCHRNH-; or -CH2-CHCOCHRNH-, where R is the side chain of any common or uncommon amino acid.

Description

FIELD OF THE INVENTION

This invention relates to the treatment of inflammatory bowel disease and especially to the treatment of this disease with novel cyclic peptide and peptidomimetic compounds that have the ability to modulate the activity of G-protein coupled receptors. The compounds preferably act as antagonists of the receptor of the C5a and are active against C5a receptors in macrophages and polymorphonuclear leukocytes.

BACKGROUND OF THE INVENTION

G protein-coupled receptors are spread throughout the human body, comprise approximately 60% of known cell receptor types and mediate signal transduction through the cell membrane to a wide variety of endogenous ligands. They participate in a wide selection of physiological and pathophysiological processes, which include, without limitation, those associated with cardiovascular, central and peripheral nervous system, reproductive, metabolic, digestive, immunological, inflammatory and growth disorders, as well as other regulatory disorders and cell proliferation. Agents that selectively modulate the functions of G protein-coupled receptors have important therapeutic applications. These receptors are increasingly being recognized as important targets for drugs, due to their crucial role in signal transduction (G protein-coupled Receptors, IBC Biomedical Library Series, 1996).

One of the G-protein coupled receptors studied in more detail is the C5a receptor. C5a is one of the most potent chemotactic agents known and is responsible for recruiting neutrophils and macrophages to the sites of injury, altering their morphology; induce degranulation; increase calcium mobilization, vascular permeability (edema) and neutrophilic adhesion; contract the smooth muscle; stimulate the release of inflammatory mediators, including histamine, TNF-α, IL-1, IL-6, IL-8, prostaglandins and leukotrienes, and lysosomal enzymes; promote the formation of oxygen radicals; and increase antibody production (Gerard and Gerard, 1994).

Agents that limit the proinflammatory action of C5a have the potential to inhibit chronic inflammation, as well as pain and damage to the accompanying tissue. Due to these reasons, molecules that prevent the binding of C5a to its receptors are useful for treating chronic inflammatory disorders caused by complement activation. Because such compounds act against the different inflammatory mediators mentioned above and inhibit the formation of many of these compounds, they can have a greater effect on the relief or prevention of inflammatory symptoms.

In the previous application No. PCT / AU98 / 00490, published as WO 99/00406, the inventors of the present described the three-dimensional structure of some analogs of the C-terminal end of the human C5a and used this information to design novel compounds that were join the human C5a receptor (C5aR) and act as agonists or antagonists of C5a. It was previously believed that a possible antagonist would require an arginine at the C-terminal end and a carboxylate at the C-terminal end for the receptor to bind and to have antagonistic activity (Konteatis et al. 1994). The inventors of the present have demonstrated that, in fact, a terminal carboxylate group is generally not required to obtain a high affinity binding to C5aR or for antagonistic activity. Instead, the inventors have discovered that a structural aspect not recognized to date, the shape of a turn, is the key recognition aspect to obtain a high affinity binding to the human C5a receptor in neutrophils. As described in International Patent Application No. PCT / AU02 / 01427 of the present inventors, filed on October 17, 2002 and published as WO 03/033528, said inventors used further refinements of these findings to design structural models. more restricted movement that allowed hydrophobic groups to be distributed in a hydrophobic arrangement to interact with the C5a receptor. The inventors have subsequently discovered that a preferred compound of this type is capable of inhibiting cardiac and pulmonary fibrosis, this is described in International Patent Application No. PCT / AU03 / 00415, filed on April 7, 2003 and published as WO 03/086448. Both documents WO 03/033528 and WO 03/086448 conform to the provisions of Article 54 (8) EPC.

Inflammatory bowel disease (IBD) consists of a group of serious, chronic and relapsing inflammatory diseases that affect the small intestine and the large intestine, which remain relatively resistant to current treatments. IBD is characterized by chronic and recurrent inflammation that appears spontaneously, of which the origin is unknown and for which current treatment options are inadequate (reviewed by van Deventer, 2002). Despite thorough research on the disease in humans and experimental animals, the exact mechanisms of the pathology have not yet been elucidated. It is believed that a host of immunological and inflammatory mediators should be involved, including biogenic amines, cynins, arachidonic acid metabolites, free radicals, nitric oxide, various proinflammatory cytokines and complement proteins (Nielsen et al. 1996).

Current therapies for IBD target one or more of these inflammatory mediators. Recent advances in the development of drugs for IBD involve the use of monoclonal antibodies to inhibit pro-inflammatory cytokines, such as interleukins, for example IL-8 (refer to US 5,677,426), interferons and the necrosis factor tumor α (TNF-α). In particular, anti-TNF-α CDP571 and infliximab antibodies have been used in medical centers to treat Crohn's disease with some success. However, these new protein therapies have major drawbacks, such as production costs, instability, low bioavailability, limited routes of administration and immunogenicity.

There are several main types of IBD, Crohn's disease (regional enteritis) and ulcerative colitis are the most common types of these diseases. Due to the nature of their pathology, there are several autoimmune diseases mediated by the immune system of the small intestine and the large intestine, which could benefit from treatment with these C5a antagonists. These include plasmacytic lymphocytic enteritis, celiac disease, collagen colitis, lymphocytic colitis and eosinophilic enterocolitis. Other less common types of IBD include undetermined colitis, infectious colitis (viral, bacterial or protozoan colitis, for example, amebic colitis), pseudomembranous colitis (necrotizing colitis) and ischemic inflammatory bowel disease. These diseases have been diagnosed in humans and in various animal species.

In 1999, approximately 1.7 million cases of this debilitating disease were diagnosed in the United States alone. Finding a satisfactory treatment for IBD is a medical necessity that has not yet been met, since existing therapeutic agents have not been effective in reducing the disease and avoiding the need for surgery. Up to 40% of all patients with ulcerative colitis are surgically operated, which usually includes the removal of part of the large intestine or a complete colostomy. Although surgery does not cure Crohn's disease, 75% of all patients will undergo at least one surgical operation throughout their life and up to 90% of patients will require additional operations. A therapeutic agent that could successfully treat inflammatory bowel disease could greatly improve the patient's quality of life, while potentially allowing the health system to save millions of dollars in costs associated with invasive surgery procedures.

In these diseases, chronic inflammation, caused by several inflammatory mediators, has been implicated in pathogenesis. The complement system has been recognized as one of these inflammatory mediators, since higher levels of complement products have been found in the colon of patients with IBD (Neilsen et al. 1996). Ulcerative colitis, also known as idiopathic colitis, is characterized by inflammation of the colon and rectum, which become inflamed and present with ulcers; its origin is not clear, although antibodies are usually found against the colon epithelium and against E. coli strain 0119 B14. Its severity varies and patients suffer frequent relapses. Crohn's disease, also called regional enteritis or regional ileitis, is characterized by inflammation, hardening and the appearance of ulcers in the intestinal wall, usually in the terminal part of the ileum, with edematous mucosa or hardened soft tissue, infiltration of the mesentery , hardened intestinal wall and frequently inflamed masses, abscesses or dilated loops filled with fluid. Complications include fistulas, intramural sinus tracts, abscesses, perforations, toxic megacolon, bowel obstruction or hydronephrosis and there is a higher risk of adenocarcinoma in the ileum or colon.

In both diseases, long periods of absolute rest are usually required and in severe cases the affected part of the intestine can be surgically removed, in which case the use of an ostomy bag is necessary. The only therapeutic agents available are corticosteroids such as prednisolone, monoclonal antibodies directed against tumor necrosis factor such as Remicade (infliximab), immunosuppressive agents such as methotrexate, azathioprine, cyclosporine, tacrolimus and mycophenolate mofetil or other anti-inflammatory agents such as sulfur. These agents, especially steroids, can have limited efficacy and can have serious side effects. There are a variety of other agents found in different phases of clinical trials, including corticosteroids such as budesonide, antagonists and cytokine agonists such as interleukin-10 and interleukin-11, and a nicotinic receptor agonist. To our knowledge, none of these agents approved or in experimentation and in particular no agent that is a small molecule, has as its objective the C5a receptor.

Therefore, effective non-toxic agents that do not require administration by injection and that can be produced at a reasonable cost are largely needed in the field.

SUMMARY OF THE INVENTION

Because the involvement of complement in IBD is unknown, the present inventors have studied the possible inhibitory effects of complement-specific inhibitors in an animal model of colitis. The inventors have just shown for the first time that a specific inhibitor of the C5a receptor is able to alleviate the harmful signs in trinitrobenzenesulfonic acid (TNBS) induced colitis in rats. This model has been used extensively to investigate the pathogenesis of IBD (Morris et al. 1989). The inventors have discovered great protective effects for C3a and C5a antagonists, which suggest that the complement has an important and previously undefined function as a mediator of IBD.

This is the first case described of a complement system inhibitor used to modulate pathology in an IBD model.

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Therefore, complement is a potential target for therapeutic intervention in inflammatory bowel disease.

According to a first aspect, the invention provides a treatment of inflammatory bowel disease (IBD) that uses an effective amount of an inhibitor of a G-protein coupled receptor in a subject in need of such treatment.

The inhibitor is a compound such that:

to)

is an antagonist of a G-protein coupled receptor,

b)

it has substantially no agonist activity and

C)

it is a cyclic peptide or peptidomimetic compound of formula I

image 1

where A is H, alkyl, aryl, NH2, NH-alkyl, N (alkyl) 2, NH-aryl, NH-acyl, NH-benzoyl, NHSO3, NHSO2alkyl, NHSO2-aryl, OH, O-alkyl or O-aryl ;

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

C is a small substituent, such as the side chain of an amino acid D or L or of a homoamino acid, such as glycine, alanine, leucine, valine, proline, hydroxyproline or thioproline, but 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, Dhomocyclohexylalanine, D-valine, D-norleucine, D-homonorleucine, D-phenylalanine, D-tetrahydroisoquinoline, D-glutamine , D-glutamate or D-tyrosine, but is preferably not a small substituent such as the glycine or Dalanine side chain, a bulky flat side chain such as D-tryptophan, or a bulky charged side chain such as D-arginine or D -lisin;

E is the side chain of an amino acid that is selected from the group consisting of L-phenylalanine, Ltriptophan and L-homotriptophan, or is L-1-naphthylalanine or L-3-benzothienylalanine;

F is the side chain of L-arginine, L-homoarginine, L-citrulline or L-canavanine, or a bioister of these, that is, a side chain in which the terminal guanidine or urea group is conserved, but the skeleton Carbonate is replaced by a group that has a different structure but is such that the side chain as a whole reacts with the target protein in the same way as the main group; Y

X is - (CH2) nNH- or (CH2) n-S-, where n is an integer from 1 to 4, preferably 2 or 3; - (CH2) 2O-;

- (CH2) 3O-; - (CH2) 3-; - (CH2) 4-; -CH2COCHRNH-; or

-CH2-CHCOCHRNH-, where R is the side chain of any common or uncommon amino acid.

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

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

Preferably, when A is a substituted sulfonamide, the substituent is an alkyl chain of 1 to 6, preferably 1 to 4 carbon atoms, or a phenyl or toluyl group.

In a particularly preferred embodiment, the compound has antagonistic activity against C5aR and has no agonist activity of C5a.

The compound is preferably an antagonist of C5a receptors in human and mammalian cells, which include, without limitation, polymorphonuclear human leukocytes and human macrophages. Preferably, the compound binds strongly and selectively to C5a receptors, and more preferably has a potent antagonistic activity at submicromolar concentrations. Even more preferably, the compound has an affinity for the IC50 receptor <25 µm and an antagonistic potency of IC50 <1 µM.

More preferably, the compound is compound 1, compound 33, compound 60 or compound 45 described in provisional application No. PCT / AU02 / 01427, published as WO 03/033528.

In a second aspect, the invention provides the use of a compound as defined above in the production of a medicament for the treatment of inflammatory bowel disease.

The inhibitor can be used in conjunction with one or more other agents for the treatment of IBD, including, but not limited to, corticosteroids such as prednisolone and budesonide, other immunosuppressive agents such as infliximab (Remicade, Johnson & Johnson), methotrexate or azathioprine , anti-inflammatory agents such as sulfasalacin, Colazal (balsalazide) and the like, and probiotic agents.

The inventors have demonstrated here that the C3a inhibitor, SB 290157, is also capable of modulating the pathology of IBD in an animal model system and, therefore, is considered to be a combination of a C5a inhibitor and a C3a inhibitor will be useful in the treatment of this disease.

The compositions of the invention may be formulated for oral, parenteral, inhalation, intranasal, rectal (intracolonic) or transdermal use, but oral or rectal formulations are preferred. Formulations suitable for any route of administration can be prepared by standard methods, for example, by reference to textbooks known as Remington: The Science and Practice of Pharmacy, Vol. II, 2000 (20th edition), AR Gennaro ( ed), Williams & Wilkins, Pennsylvania.

Preferred formulations include enteric coated capsules or rectal (intracolonic) pharmaceutical forms such as enemas, to facilitate absorption in the colon, the local area of inflammation.

The invention can be applied to the treatment of any type of inflammatory bowel disease, which includes, without limitation, Crohn's disease (regional enteritis); ulcerative colitis; autoimmune and mediated diseases of the immune system of the small and large intestine, such as plasmacytic lymphocytic enteritis, celiac disease, collagen colitis, lymphocytic colitis and eosinophilic enterocolitis; Indeterminate colitis, infectious colitis (viral, bacterial or protozoan colitis, for example, amebic colitis), pseudomembranous colitis (necrotizing colitis) and ischemic inflammatory bowel disease.

Although the invention is not restricted in any way to the treatment of any particular animal or species, it is particularly considered that the invention will be useful in the medical treatment of humans and will also be useful in the veterinary treatment, particularly of companion animals such as cats. and dogs, cattle such as cattle, horses and sheep, and zoo animals, which include nonhuman primates, large bovids, felids, ungulates and canids. For example, felines and particularly canines suffer from a variety of inflammatory enteropathies, which have some similarities with IBD in humans. These include enterocolitis, canine plasmacytic lymphocytic colitis, prototecal colitis and histiocytic ulcerative colitis.

The compound can be administered in any suitable dose and through any suitable route. Oral or rectal administration is preferred, for the greater convenience and acceptance of these pathways. It is expected that most, if not all of the compounds of the invention, are stable in the presence of metabolic enzymes, such as intestine, blood, lung or intracellular enzymes. Such stability can be easily evaluated by routine methods known to those skilled in the art.

The effective dose will depend on the nature of the disease to be treated, as well as the age, weight and underlying health status of the individual treatment. This will be left to the discretion of the doctor or veterinarian in charge. Suitable dose levels can be easily determined by trial and error experimentation, using methods known in the art.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the effect on the 8-day mortality rate. pre = pretreatment; post = post treatment.

Figure 2 shows the effect on food intake. The amount of food consumed was evaluated after 24 h [A] or at 8 days [B]. The data represent the mean + standard deviation of the mean (n = 4-12). Rats treated with drug * P <0.05 versus colitis control. pre = pretreatment; post = post treatment.

Figure 3 shows the effect on body weight. The change in body weight was evaluated after administration of TNBS after [A] 24 h or [B] 8 days. The data represent the mean + standard deviation of the mean (n = 4-12). Rats treated with drug * P <0.05 versus colitis control. pre = pretreatment; post = post treatment.

Figure 4 summarizes the macroscopic clinical scores. A score was awarded for each colon according to macroscopic damage on a scale of 0-13 after [A] 24 h or [B] 8 days. The data represent the mean + standard deviation of the mean (n = 4-12). Rats treated with drug * P <0.05 versus colitis control. pre = pretreatment; post = post treatment.

Figure 5 shows the effect on myeloperoxidase (MPO) levels in the colon. MPO levels in the colon of the rats were evaluated after [A] 24 h or [B] 8 days. The data represent the mean + standard deviation of the mean (n = 4-12). Rats treated with drug * P <0.05 versus colitis control. pre = pretreatment; post = post treatment.

Figure 6 shows the effect on colon edema levels in 24-hour and 8-day studies. [A] 24 h; [B] 8 days. The data represent the mean + standard deviation of the mean (n = 4-12). Rats treated with drug * P <

0.05 versus colitis control. pre = pretreatment; post = post treatment.

Figure 7 shows the effect on TNF-α levels in serum and colon after 8 days. The data represent the mean + standard deviation of the mean (n = 4-12).

[A] serum; [B] colon tissue.

Rats treated with drug * P <0.05 versus colitis control. pre = pretreatment; post = post treatment.

Figure 8 shows the colon section of the rats on day 8 of the study, stained with hematoxylin and eosin (magnification of x 40). The images are common and representative of each study group.

[A] Rat with simulated operation injected with saline, with epithelial and mucosal layer (ML) intact;

[B] Drug-free TNBS colitis control rat, with extensive damage including inflammatory cell infiltration (IC), edema (E) and complete destruction of the ML architecture (indicated by arrows);

[C] Post-treated rat after TNBS with a C5a antagonist (10 mg / kg / day, oral), with infiltration of IC and E, but with ML intact; Y

[D] Post-treated rat after TNBS with prednisolone (1 mg / kg / day, SC), with infiltration of IC, E, extensive bleeding (H) and erosion of ML (indicated by arrows).

DETAILED DESCRIPTION OF THE INVENTION

In the claims that follow and in the previous description of the invention, except where the context requires otherwise by express language or by necessary implication, the term "understand" or variations such as "comprises / n" or "comprising / n "are used in an inclusive sense, that is, to specify the presence of the mentioned characteristics but not to rule out the presence or addition of other characteristics in various embodiments of the invention.

As used herein, the singular forms "a / o / a" and "el / la" include plural references, unless the context clearly indicates otherwise. Thus, for example, a reference to "an enzyme" includes a plurality of said enzymes, and a reference to "an amino acid" is a reference to one or more amino acids. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as an expert in the field to which this invention pertains usually means. 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 described below.

The abbreviations used herein are the following:

Cit

citrulline

dCha

D-cyclohexylamine

DPhe

D-phenylalanine

IBD

inflammatory bowel disease

ip

intraperitoneal

iv

intravenous

LPS lipopolysaccharide

MPO myeloperoxidase

PMN polymorphonuclear granulocyte

PMSF phenylmethylsulfonyl fluoride

vr rectal route

sc subcutaneous

TNBS trinitrobenzenesulfonic acid

TNF-α tumor necrosis factor α

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

For the purposes of this description, it should be understood that the term "alkyl" refers to a substituted or unsubstituted, linear, branched or cyclic alkyl chain of 1 to 6, preferably 1 to 4 carbons. More preferably, the alkyl group is a methyl group. It should be understood that the term "acyl" refers to a substituted or unsubstituted acyl of 1 to 6, preferably 1 to 4 carbon atoms. More preferably, the acyl group is acetyl. It should be understood that the term "aryl" refers to a substituted or unsubstituted homocyclic or heterocyclic aryl group, in which the ring preferably contains 5 or 6 members.

A "common" amino acid is an L amino acid that is 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, without limitation, D amino acids, homoamino acids, Nalkylamino 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, Lfluorenylalanine, L-3-benzothienylalanine and α, α-disubstituted amino acids.

Generally, the terms "treated", "treatment" and the like are used herein to refer to affecting a subject, tissue or cell to obtain a desired pharmacological and / or physiological effect. The effect can be prophylactic, in the sense that it completely or partially prevents a disease or signal or symptom of it, and / or it can be therapeutic, in the sense that it completely or partially cures a disease.

As used herein, the term "treated" encompasses any treatment or prevention of a disease in a vertebrate, a mammal, particularly a human, and includes: preventing the disease in a subject that may be predisposed to said disease, but that has not yet been diagnosed; inhibit the disease, that is, stop its development; or alleviate or improve the effects of the disease, that is, cause a regression of the effects of the disease.

The invention includes the use of various pharmaceutical compositions useful for alleviating the claimed disease. The pharmaceutical compositions according to an embodiment of the invention are prepared by introducing a compound of formula I or salts thereof and one or more pharmaceutically active agents or combinations of the compound of formula I and one or more pharmaceutically active agents, in a form suitable for be administered to a subject using carriers, excipients and additives or auxiliaries.

Some frequently used carriers or auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk proteins, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterilized water, alcohols, glycerol and polyhydric alcohols. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial agents, antioxidants, chelators and inert gases. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for example, in Remington's Pharmaceutical Sciences, 20th edition, Williams & Wilkins (2000) and The British National Formulary , 43rd edition (British Medical Association and Royal Pharmacy Society of the United Kingdom, 2002; http://bnf.rhn.net). The pH and the exact concentration of the different components of the pharmaceutical composition are adjusted according to the routine procedures in the field. Refer to The Pharmacological Basis for Therapeutics by Goodman and Gilman (7th edition, 1985).

The pharmaceutical compositions are prepared and preferably administered in unit doses. Solid unit doses include tablets, capsules and suppositories. To treat a subject, different daily doses can be used, depending on the activity of the compound, the route of administration, the nature and severity of the disorder, and the age and body weight of the subject. However, in certain circumstances, higher or lower daily doses may be appropriate. The administration of the daily dose can be carried out by a single

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administration in the form of an individual unit dose or by several smaller unit doses and also by multiple administration of doses subdivided into specific intervals.

The pharmaceutical compositions used in the invention can be administered locally or systemically in a therapeutically effective dose. The effective amounts for this use will obviously depend on the severity of the disease and the weight and general condition of the subject. Usually, the doses used in vitro can provide a useful guide to the amounts useful for the in situ administration of the pharmaceutical composition, and animal models can be used to determine the effective doses for the treatment of cytotoxic side effects. Several considerations are described, for example, in Science, 249: 1527 (1990) by Langer. Formulations for oral use may be presented 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 can also be presented in the form of soft gelatin capsules, in which the active substance is mixed with water or an oily medium, such as peanut oil, liquid paraffin or olive oil.

Normally, aqueous suspensions contain the active materials in a mixture with excipients suitable for the production of aqueous suspensions. Such excipients may be suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and acacia gum; dispersing or wetting agents, which can be

(a) a naturally occurring phospholipid 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, heptadecaethylene oxyethanol;

(d)

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

(and)

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 presented in the form of an aqueous or oleaginous injectable sterile suspension. This suspension can be formulated according to known methods using dispersing agents

or suitable humectants and suspending agents, such as those mentioned above. The injectable sterile preparation may also be a sterile solution or suspension for injection in a nontoxic parenterally acceptable diluent or solvent, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be used are water, Ringer's solution and an isotonic sodium chloride solution. In addition, sterilized fixed oils are conventionally employed as solvents or suspending media. Any single fixed oil can be used for this purpose, including synthetic mono- or diglycerides. In addition, fatty acids, such as oleic acid, can be used to prepare injectables.

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

The dose levels of the compound of formula I of the present invention will normally be of the order of about 0.5 mg to about 20 mg per kilogram of body weight, with a preferred dose ranging from about 0.5 mg to about 10 mg per kilogram of body weight per day (of approximately

0.5 g to approximately 3 g per patient per day). The amount of active ingredient that can be combined with the carrier materials to produce a single dose will vary depending on the host to be treated and the particular route of administration. For example, a formulation designed for oral administration in humans may contain from about 5 mg to 1 g of an active compound with a suitable and convenient amount of carrier material, which may vary from about 5 to 95 percent of the total composition. . Unit dosage forms will generally contain between about 5 mg and 500 mg of active substance.

However, it will be understood that the specific dose level for any particular patient will depend on several factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration, route of administration, Excretion rate, combination of drugs 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. Said solvates are encompassed within the scope of the invention.

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

The invention will be described below only with reference to the following general methods and experimental examples.

General methods

Peptide synthesis

The cyclic peptide compounds of formula I are prepared according to the methods described in detail in the previous applications of the inventors PCT / AU98 / 00490 (published as WO 99/00406) and PCT / AU02 / 01427 (published as WO 03 / 033528). In the provisional Australian application No. 2003902743 of the inventors, an alternative method of synthesis is described. With this reference all explanations of these descriptions are incorporated herein. Although the invention is specifically illustrated with reference to the AcF compound [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 described in international patent application No. PCT / AU98 / 00490 (published as WO 99/00406) and compounds 10-12, 14, 15 , 25, 33, 35, 40, 45, 48, 52, 58, 60, 66 and 68-70 described for the first time in the Australian provisional application No PCT / AU02 / 01427 (published as WO 03/033528) have a power appreciable antagonist (IC50 <1 M) against the C5a receptor in human neutrophils. PMX53 and compounds 33, 45 and 60 of application PCT / AU02 / 01427 (published as WO 03/033528) are most preferred.

The inventors have discovered that all the compounds of formula I that have been studied so far have similar pharmacological activities in general, although the physicochemical properties, potency and bioavailability of the individual compounds vary somewhat, depending on the specific substituents. .

The general assays described below can be used as an initial screening of candidates to act as inhibitors 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 reverse phase HPLC and fully characterized by spectroscopy of proton NMR mass and spectroscopy. The C5a antagonist was prepared in olive oil (10 mg / mL) for oral doses and in a 30% solution in polyethylene glycol (0.6 mg / mL) for SC doses. The C3a antagonist SB 290157 (Ph2CHCH2OCH2CO-Arg-OH) has been described in detail above (Ames et al. 2001). It was synthesized and purified by reverse phase HPLC and characterized by mass spectroscopy and NMR spectroscopy. It was prepared in a 50% solution in propienglycol (30 mg / kg) for IP injections. Prednisolone glucocorticosteroid (Sigma, USA) was prepared in a 30% solution in polyethylene glycol for SC doses. The TNF-α infliximab inhibitor (Remicade) was prepared in a sterile saline solution (3 mg / mL) according to the indications for IV dose. No anti-inflammatory effects were observed for propylene glycol or polyethylene glycol in rats with TNBS-induced colitis.

Receptor Binding Assay

The assays are carried out with fresh human PMN, isolated as described above (Sanderson et al. 1995), using a 50 mM HEPES buffer solution, 1 mM CaCl2, 5 mM MgCl2, 0.5% bovine serum albumin, 0.1% bacitracin and phenylmethylsulfonyl fluoride (PMSF) 100 µM. In tests at 4 ° C, the buffer solution, the recombinant human unlabeled C5a (Sigma) or peptide, the C5a labeled with 125I is added sequentially using the Hunter / Bolton reagent (~ 20 pM) (New England Nuclear, MA) and PMN (0.2 x 106) in a Millipore Multiscreen test well (HV 0.45) with a final volume of 200 /L / well. After incubating for 60 min at 4 ° C, the samples are filtered and the well is washed once with the buffer solution. The filters are dried, punctured and evaluated in an LKB gamma counter. Non-specific binding is determined by inserting 1 mM peptide or 100 nM C5a, which usually causes a total 10-15% binding.

Data are analyzed using statistical and non-linear regression methods with Dunnett's post-test.

Myeloperoxidase release assay to determine antagonist activity

Cells are isolated as described above (Sanderson et al. 1995) and incubated with cytochalasin B (5 g / mL, 15 min, 37 C). Hank's balanced saline solution, containing 0.15% gelatin, and peptide is added to a 96-well plate (total volume of 100 /L / well) and then 25 L of cells (4x106 / mL) are added. To evaluate the ability of each peptide to act as a C5a antagonist, the cells are incubated for 5 min at 37 ° C with each peptide and then C5a (100 nM) is added and incubated for an additional 5 min. Then 50 L of sodium phosphate (0.1 M, pH 6.8) is added to each well, the plate is cooled to room temperature and

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Four. Five

Add 25 µL of a freshly prepared mixture of equal volumes of dimethoxybenzidine (5.7 mg / mL) and H2O2 (0.51%) to each well. The reaction is stopped at 10 min by the addition of 2% sodium azide. Absorbances at 450 nm are evaluated on a Bioscan 450 plate analyzer, corrected with control values (without peptide) and analyzed by nonlinear regression.

Example 1 Effect of PMX53 on inflammatory bowel disease

Because the implication of complement in IBD is currently unknown, the inventors here intend to evaluate the possible effects of inhibition of PMX53 (AcF- [OPdChaWR]) in rats with trinitrobenzenesulfonic acid-induced colitis (TNBS). For comparative purposes, the inventors also examine the efficacy of a C3a antagonist, SB 290157 (Ames et al. 2001), prednisolone corticosteroid and TNF-α infliximab antibody.

The ability of PMX53 (AcF- [OPdChaWR]) to relieve harmful signs in rats with trinitrobenzenesulfonic acid-induced colitis (TNBS) was evaluated. This model has been used extensively in the investigation of the pathogenesis of IBD (Morris et al. 1989).

Male Wistar rats (250-300 g) were deprived of food for 24 hours before being anesthetized with ketamine (80 mg / kg i.p.) and xylazine (8 mg / kg i.p.). A polyethylene catheter with an external diameter of 1.7 mm was inserted intracolonically, at a distance of 8 cm from the anus. Next, 120 mg / kg of TNBS (50 mg / mL solution) was infused together with 250 µL of ethanol (100%) in the rats, which remained upside down for 30 min to prevent losses. Rats with simulated operation (rats without colitis) received saline solution or 250 μL of ethanol and saline solution. Next, the rats were allowed to recover under observation.

At the end of each experiment, the rats were anesthetized with zolazapam (50 mg / kg, ip) and xylazine (12 mg / kg, ip), blood samples were taken and serum was stored at –20 C to subsequently determine the concentrations of TNF-α. The colon of each rat was dissected, the distal 8 cm was washed with saline solution and an independent observer blindly scored the clinical macroscopic damage, using a scale of 0-13, as discussed below (Bobin-Dubigeon et al. 2001) . A section of the affected colon was then taken, weighed, placed in an oven at 80 duranteC for 24 h, then weighed again and the proportion of wet weight versus dry weight was determined as a measure of edema of the colon (Rachmilewitz et al. 1989). A separate section of the affected colon was also taken and homogenized with 1 mL of a phosphate buffered saline solution, sonicated for 20 seconds and centrifuged (14,000 x g, 10 min). Then, the resulting supernatant was stored (-20 ° C) to subsequently determine TNF-α, or was immediately used in an assay to determine myeloperoxidase (MPO) levels. Simply put, the assay consisted of the addition of 20 L of substrate (o-dianisidine, 2.85 mg / mL, Sigma, USA; and 0.85% hydrogen peroxidase) at a 1:40 dilution of the supernatant in a phosphate buffered saline solution. The absorbances at 450 nm were then determined, 5 min after the substrate addition for the 24 h samples and 15 min after the addition for the 8 day samples. The results of the MPO content in the colon were converted into absorbance units / g of tissue. Additional sections of the colon were taken and stored in 10% formalin for histopathological analysis. Fixed colon samples were embedded in paraffin wax and the sections were stained using a hematoxylin and eosin dye, photographed and an independent observer examined them blindly. Tissue and serum levels of TNF-α in the stored samples were determined, using an enzyme-linked immunosorbent assay as described above (Arumugam et al. 2002), using undiluted samples. The results of the TNF-α content in the colon were subsequently converted into pg of TNF-α / g tissue.

Two time slots were selected for this study, a severe time slot (24 hours) and a chronic time slot (8 days). During the course of the study, body weight and food intake were evaluated daily. In the 24-hour study, all drug treatments were started 2 days before the infusion of TNBS (prevention). Rats treated with PMX53, with prednisolone and with a combination of these were treated once a day, and rats treated with the C3a antagonist received the dose twice daily. Rats treated with the anti-TNF-α monoclonal antibody, infliximab, received the dose intravenously once, 2 days before the TNBS infusion. In the 24-hour model, the following treatment groups were used:

(to)

PMX53 (10 mg / kg, in olive oil, oral),

(b)

PMX53 (0.3 mg / kg, in 30% polyethylene glycol, subcutaneous),

(C)

C3a antagonist (30 mg / kg, twice a day, IP),

(d)

prednisolone (1 mg / kg, in 80% polyethylene glycol, subcutaneous),

(and)

a combination of PMX53 (10 mg / kg, in olive oil, oral) and prednisolone (1 mg / kg, in 80% polyethylene glycol, subcutaneous), and

(F)

infliximab (1 mg / kg, in saline, intravenous).

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In the 8-day study, the pharmacologically treated rats were treated before inducing colitis (2 days before; prevention) or after induction of colitis (24 hours later; reversal). In the 8-day study, the following treatment groups were used:

(to)

PMX53 (10 mg / kg, in olive oil, oral, before and after induction),

(b)

PMX53 (0.3 mg / kg, in 30% polyethylene glycol, subcutaneous, only before induction),

(C)

PMX53 (1 mg / kg, in olive oil, oral, only after induction),

(d)

prednisolone (1 mg / kg, in 80% polyethylene glycol, subcutaneous, before and after induction),

(and)

a combination of PMX53 (10 mg / kg, in olive oil, oral) and prednisolone (1 mg / kg, in 80% polyethylene glycol, subcutaneous) (only before induction), and

(F)

infliximab (1 mg / kg, in saline, intravenous, only before induction).

The colon of each rat was then evaluated macroscopically under blind conditions on a scale of 114, as shown below:

Ulcer formation

 Diarrhea  Adhesions

0-

No damage  0- Absent 3- Absent

one-

Focal hyperemia  one- Moderate 4- Moderate

2-3-4-5-

Hyperemia and hardening of the ulcer at one point Ulcer at two points Ulcer  1 cm stino 2- Serious 5- Serious

6-10 Ulcer  2 cm; the score increases by 1 for each additional cm

The results for several parameters are summarized in Figures 1-7. All experimental results are expressed as the mean + standard deviation of the mean (SEM). Data analysis was performed using GraphPad Prism 3.0 software (GraphPad Software, Inc. USA). Statistical comparisons were carried out between pharmacologically treated groups and colitis control animals using a one-way analysis of variance and then a Dunnett post-test comparison analysis. Statistical significance was determined at a value of P  0.05.

Mortality index: As shown in Figure 1, the induction of colitis in drug-free rats caused a high mortality rate, 39%, beginning 2 days after the administration of TNBS. All pharmacological treatments, except post-treatment with prednisolone, caused a decrease in mortality (n = 8-16) compared to drug-free colitis control rats (n = 13). No deaths were recorded in animals with simulated operation (n = 4). Rats treated with the C5a antagonist, both before induction (10 mg / kg / day oral and 0.3 mg / kg / day SC) and 24 h after induction (1 or 10 mg / kg / day oral), showed a greater survival, with mortality rates less than or equal to 20%. Rats pretreated with the prednisolone corticosteroid (1 mg / kg / day SC) showed a similar reduction in the death rate, up to 11%; however, post-treatment with prednisolone showed no significant effect in reducing mortality. Pretreatment with infliximab (3 mg / kg IV) reduced mortality to 20%, which proved to be less effective than the C5a antagonist (with 10 mg / kg oral or 0.3 mg / kg SC) or pretreatment with prednisolone. No deaths were recorded among rats pretreated with a combination of the C5a antagonist (10 mg / kg / day oral) and prednisolone (1 mg / kg / day SC) or in rats pretreated with the C5a antagonist with 0.3 mg / kg SC .

Food intake: No significant improvement in food intake was observed after 24 hours of drug treatment compared to drug-free colitis control rats. After 8 days, all treated rats, except those treated with prednisolone, ate significantly more food than the drug-free colitis control rats. As shown in Figure 2A, induction of colitis caused a decrease in food intake in all rats to which TNBS was administered compared to rats with simulated operation after 24 hours. Pharmacological treatment did not significantly improve food intake for 24 hours.

As illustrated in Figure 2B, after 8 days the drug-free rats still ate less than the rats with simulated operation. Rats treated with the C5a antagonist, both before induction (10 mg / kg / day oral and 0.3 mg / kg / day SC) and 24 h after induction (1 or 10 mg / kg / day oral), they ate significantly more food than colitis control rats and consumed similar levels of food to those of rats with simulated operation on day 8 (P  0.05). Rats pretreated with prednisolone (1 mg / kg / day SC) also ate significantly more food than colitis control rats after 8 days (P  0.05); however, rats post-treated with this drug showed no improvement in food intake (P  0.05). Rats pretreated with infliximab (3 mg / kg IV) also ate significantly more food than drug-free rats after 8 days, and so did rats pretreated with a combination of the C5a antagonist (10 mg / kg / day oral) ) and prednisolone (1 mg / kg / day SC) (P  0.05).

Body weight: No significant reduction in weight loss was observed after 24 hours of drug treatment compared to drug-free colitis control rats. After 8 days, only rats treated with the C5a antagonist gained weight significantly compared to drug-free colitis control rats. All rats that received TNBS showed considerable weight loss at 24 hours after induction compared to rats with simulated operation, as shown in Figure 3A. Pharmacological treatment did not significantly affect this weight loss for 24 hours.

As shown in Figure 3B, during the 8-day period of the study drug-free rats continued to lose weight, with an average weight loss on day 8 of –21 + 8 g (n = 8). Rats treated with the C5a antagonist, both before induction of colitis (10 mg / kg / day oral and 0.3 mg / kg / day SC) and 24 h after induction (1 or 10 mg / kg / day oral ), gained weight substantially during the 8 days, with significantly higher weights compared to colitis control rats (P  0.05). In contrast, rats both pretreated and post-treated with prednisolone (1 mg / kg / day SC) or rats pretreated with a combination of the C5a antagonist (10 mg / kg / day oral) and prednisolone (1 mg / kg / day SC) They lost weight during the 8 days and showed no significant difference compared to the colitis control rats on day 8 (P  0.05). Rats pretreated with infliximab (3 mg / kg IV) showed only a small weight gain on day 8; however, these levels were not significantly higher than in colitis control rats (P  0.05).

Gross scoring: An independent observer blindly examined each colon macroscopically for hemorrhages and ulcers, using a previously established scoring system (Bobin-Dubigeon et al. 2001). After 24 hours, all drug treatments, except infliximab, caused a significant improvement in macroscopic scores compared to drug-free colitis control rats. After 8 days, all pharmacological treatments, except for post-treatment with prednisolone, caused a significant improvement in macroscopic scores compared to drug-free colitis control rats. Animals with simulated operation showed no appreciable macroscopic damage.

As shown in Figure 4A, after 24 hours the colon of all rats treated with TNBS had large hemorrhages, edema and an onset of ulcer formation. No adhesions were observed and diarrhea was minimal. The drug-free rats had a mean macroscopic score of 7.2 + 0.5 (n = 12) at 24 h. Pretreatment of rats with any drug in the 24-hour study caused reductions in macroscopic scores and a significant improvement was observed in all treatment groups except with infliximab (P  0.05). As shown in Figure 4B, after 8 days the colon of rats treated with TNBS showed considerably greater damage than at 24 h, which indicates that the pathology was progressive. Although the colon presented less bleeding, ulcer formation was extensive and adhesions and diarrhea were common. Colitis control rats had a mean macroscopic score of 11.5 + 0.8 (n = 12) on day 8. Rats treated with the C5a antagonist, both before the induction of colitis (10 mg / kg / day oral and 0.3 mg / kg / day SC) as 24 h after induction (1 or 10 mg / kg / day oral), showed significantly improved macroscopic scores compared to colitis control rats, as did rats pretreated with prednisolone (1 mg / kg / day SC) (P  0.05). Rats pretreated with infliximab (3 mg / kg IV) also showed a significant reduction in scores on day 8 (P  0.05). Rats treated with prednisolone (1 mg / kg / day SC) did not show any improvement in macroscopic scores on day 8 (P  0.05).

MPO in the colon: After 24 h, only the rats treated with both the C5a antagonist and a combination of the C5a antagonist and prednisolone, showed a significant improvement in the MPO levels in the colon compared to the control rats of drug-free colitis. After 8 days, all pharmacological treatments, except for post-treatment with prednisolone, caused a significant improvement in the levels of MPO in the colon compared to drug-free colitis control rats. As shown in Figure 5A, the levels of MPO in the colon of drug-free rats were substantially higher at 24 hours than those of rats with simulated operation. The levels of this enzyme were reduced in all pharmacologically treated rats after 24 h and a significant improvement was observed in rats both pretreated with the C5a antagonist (10 mg / kg / day oral or 0.3 mg / kg / day SC) as with a combination of the C5a antagonist (10 mg / kg / day oral) and prednisolone (1 mg / kg / day SC) (P  0.05).

As shown in Figure 5B, the MPO levels in the colon after 8 days in the rats to which TNBS was administered were further reduced compared to those levels at 24 h. In rats treated with the C5a antagonist, both before induction (10 mg / kg / day oral and 0.3 mg / kg / day SC) and 24 h after induction (1 and 10 mg / kg / day oral) , or in rats pretreated with a combination of the C5a antagonist (10 mg / kg / day oral) and prednisolone (1 mg / kg / day SC), the levels of MPO in the colon were reduced to levels similar to those of animals with simulated operation after 8 days (P  0.05). Rats pretreated with both prednisolone (1 mg / kg / day SC) and infliximab (3 mg / kg IV) also showed a significant reduction in MPO levels after 8 days, although the degree of improvement was not as high as in rats treated with the C5a antagonist (P  0.05). Rats treated with prednisolone (1 mg / kg / day SC) did not show any improvement in MPO levels in the colon (P  0.05).

Edema of the colon: Edema of the colon was determined at 24 hours and 8 days, based on the proportions of wet weight versus dry weight. The results are shown in Figure 6. Both after 24 h and after 8 days, only rats treated with both the C5a antagonist and a combination of the C5a antagonist and prednisolone, showed a significant improvement in weight ratios. wet versus dry weight in the colon, compared to drug-free colitis control rats. As shown in Figure 6A, the proportions of wet weight versus dry weight in the colon of the rats to which TBNS was administered increased significantly compared to those of rats with simulated operation after 24 h, which indicates the presence of edema. At this point, only rats pretreated with the C5a antagonist (10 mg / kg / day oral or 0.3 mg / kg / day SC) or with a combination of the C5a antagonist (10 mg / kg / day oral) and prednisolone ( 1 mg / kg / day SC) showed a significant reduction in the proportions of wet weight versus dry weight compared to colitis control rats (P  0.05). The C3a antagonist showed no significant effect.

As shown in Figure 6B, the level of edema of the colon after 8 days in the rats to which TNBS was administered was still considerably higher than that in rats with simulated operation, although it was lower than that observed at 24 h. Significant reductions in the proportions of wet weight versus dry weight were observed in rats treated with the C5a antagonist, both before induction (10 mg / kg / day oral and 0.3 mg / kg / day SC) and 24 h after of induction (1 and 10 mg / kg / day oral), as well as in rats pretreated with a combination of the C5a antagonist (10 mg / kg / day oral) and prednisolone (1 mg / kg / day SC) in this 8 day time slot (P  0.05). Post-treatment with prednisolone (1 mg / kg / day SC) showed no effect in reducing edema, while rats pretreated with prednisolone (1 mg / kg / day SC) and infliximab (3 mg / kg IV) did not show No significant reduction compared to colitis control rats (P  0.05).

TNF-α levels in serum and tissue: TNF-α levels were then evaluated after 8 days in both serum and colon tissue. The results are illustrated in Figure 7. Serum TNF-α levels increased in drug-free colitis control animals and all drug treatments significantly reduced these levels. All pharmacological treatments, except prednisolone after treatment, significantly improved levels of TNF-α in the colon compared to drug-free colitis control rats. The levels of TNF-α in serum and colon tissue did not increase in rats treated with TNBS compared to rats with simulated operation after 24 h. As shown in Figure 7A, serum TNF-α levels after 8 days in colitis control rats increased compared to rats with simulated operation. All pharmacologically treated rats presented serum TNF-α levels similar to those of simulated operation rats and these levels were significantly reduced compared to the control levels of colitis (P  0.05). The levels of TNF-α in the colon tissue homogenate, shown in Figure 7B, also increased in drug-free rats compared to simulated operation animals. All pharmacologically treated rats had significantly lower levels of this cytokine in the tissue, compared to colitis control rats (P  0.05), except for rats treated with prednisolone (1 mg / kg / day SC), which did not showed no significant reduction (P  0.05).

Histopathology: An independent observer blindly examined the stained sections of the colon for signs of pathology. 24 hours after administration of TNBS, sections of the colon showed signs of severe inflammation. In moderate cases, edema of the submucosa layers of the intestine and infiltration, predominantly by neutrophils, were observed in the mucosa and submucosa. In the most severe cases, necrosis of the colon mucosa cells and more severe inflammatory edema and cell infiltration were observed. No improvements were observed in the colon sections of pharmacologically treated rats compared to drug-free colitis control rats in this severe time slot.

8 days after the TNBS challenge, evidence of healing was observed, since it was observed that the affected mucosa was replaced by fibrous tissue. PMN infiltration was observed in the affected tissues. When discrete ulcers were identified, the limiting mucosa appeared normal, although generalized submucosal inflammation was observed, which consisted of inflammatory cells and moderate edema. The colon sections of the drug-free colitis control rats showed the most severe pathology after 8 days (Figure 8B). In comparison, rats colon sections with simulated operation showed minimal signs of damage (Figure 8A). After 8 days, the colon sections of all rats pretreated with both the C5a antagonist and prednisolone, infliximab or a combination of the C5a antagonist / prednisolone showed an improvement in the pathology compared to drug-free rats. The colon sections of the rats post-treated with the C5a antagonist (10 mg / kg / day oral) also showed an improvement in the disease pathology after 8 days, indicated by reduced ulcer formation, edema, inflammatory cell infiltration. , without perforations and with the mucosal layers intact compared to the drug-free rat sections (Figure 8C). In contrast, the colon sections of rats treated with prednisolone (1 mg / kg / day SC) did not show any improvement in disease pathology compared to drug-free rats (Figure 8D).

In the 24-hour study, rats treated with PMX53 had significantly lower macroscopic colon scores and higher body weights and food consumption. The level of edema of the colon, neutrophil accumulation and levels of TNF-α in the colon were also significantly reduced. Compared with prednisolone and infliximab, which are currently used in IBD therapy, rats treated with PMX53 showed a greater inhibition of the parameters evaluated. These results show that the blocking of the inflammatory C5a protein by PMX53 significantly improves the disease pathology in the severe 24-hour model of IBD, to a degree at least as high as that achieved by prednisolone and infliximab.

In the 8-day study, both pre-treated and post-treated rats with PMX53 lost significantly less body weight and ate significantly more food compared to colitis control animals. These rats also had macroscopic colon scores, colon edema, neutrophil accumulation and significantly lower levels of TNF-α in the colon. Importantly, rats treated with PMX53 also showed reduced mortality compared to colitis control rats. In comparison, only rats pretreated with prednisolone showed some improvement in the disease parameters evaluated. Rats treated with infliximab also showed a significant reduction in disease parameters, however, to a lesser extent than that observed in rats treated with PMX53.

Histologically, the colon sections of rats treated with PMX53 showed a reduction in inflammatory cell accumulation and hemorrhage formation, and this is shown in Figure 8. Surprisingly, these results show that the blocking of the C5a inflammatory protein by Part of PMX53 both prevents and reverses the disease pathology in a model with chronic colitis rats. This pathology reversal effect was not observed in rats treated with prednisolone.

In summary, these studies demonstrate for the first time that a complement system inhibitor has beneficial effects in an established model of IBD. The improvement observed with PMX53 was greater than that observed with prednisolone or infliximab. This indicates that PMX53 will be useful in the clinical treatment of IBD.

Example 2 Other studies in animal models

An 8-day time slot is used in other animal studies, as this provides the most significant and clinically relevant pathology. All drugs are administered 24 hours after the induction of colitis (reversal therapy) and then daily throughout the study period. PMX53 is administered with doses of 0.1, 0.3, 1 and 10 mg / kg in mineral oil rectally (vr). PMX53 is also administered orally with doses of 0.1, 0.3, 1 and 10 mg / kg in an enteric coated capsule.

PMX53 analogues are also evaluated with various doses, ranging from 0.1-10 mg / kg, in olive oil as a solvent, to determine their effectiveness compared to PMX53. In the first example, the following analogues are evaluated:

PMX205: Hydrocinamate- [OPdChaWR]

PMX73: AcF- [OPdPheWR]

PMX201: AcF- [OPdChaWCitrulina]

These compounds are administered after treatment with a dose of 0.3, 1, 3 and 10 mg / kg / day in the 8-day study.

In addition to the TNBS model system, felines and particularly canines suffer from a variety of inflammatory enteropathies, which have some similarities with IBD in humans (Tams, 1993; German et al. 2003). These include enterocolitis, canine plasmacytic lymphocytic colitis, prototecal colitis and histiocytic ulcerative colitis. PMX53 and its analogues were also evaluated in these conditions.

Example 3 Evaluation of clinical efficacy

For example, a randomized phase II placebo-controlled trial in the treatment of patients with moderate to severe Crohn's disease usually employs at least two dose levels of the test compound. A decrease greater than 70 points in the Crohn's disease activity index (CDAI), a standardized tool designed to specifically assess disease activity, is a primary evaluation criterion to indicate efficacy. Disease remission is a secondary evaluation criterion. An advantage is established in the treatment effect if significantly more recipients of the test compound compared to placebo recipients have CDAI scores consistent with the resolution of symptoms during a severe exacerbation of inflammatory bowel disease.

In ulcerative colitis, the effects of treatment with the test compound are evaluated in patients with symptoms of active ulcerative colitis which have either not been previously treated or are also receiving standard medical treatment. The evaluation criteria are the induction of complete remission or the significant improvement of the signs and symptoms of ulcerative colitis, as reflected in the changes in the colitis activity index (CAI) score. The IAC score assesses the frequency of deposition, rectal bleeding, the endoscopic appearance of the colon and includes a doctor's overall evaluation.

The profiles of the adverse effect and tolerance of the test compound for each condition are also recorded.

DISCUSSION

Cyclic peptides have several important advantages over acyclic peptides as drug candidates (Fairlie et al. 1995, Fairlie et al. 1998, Tyndall and Fairlie, 2001). The cyclic compounds described in this description are stable against proteolytic degradation for at least several hours at 37 ° C in human blood or plasma, in human or rat gastric juices or in the presence of digestive enzymes such as pepsin, trypsin and chymotrypsin. In contrast, short linear peptides composed of L-amino acids rapidly degrade into their component amino acids in a few minutes when they undergo these conditions. A second advantage is based on the unique conformations of restricted mobility adopted by cyclic and non-peptide molecules, contrary to linear or acyclic peptides, which are flexible enough to adopt multiple dissolving structures other than those required for binding with the receiver. Third, cyclic compounds such as those described in this invention are usually more fat-soluble and more pharmacologically bioavailable as drugs than acyclic peptides, which can rarely be administered orally. Fourth, the plasma half-lives of cyclic molecules are normally longer than those of peptides.

IBD models usually involve administration within the colon of an anti-inflammatory or haptenization agent. The TNBS-induced colitis model used herein is a simple and reliable model that has been widely used to examine the efficacy of several drug candidates for the treatment of IBD.

The inventors have discovered that 24 h after administration of TNBS, the rat colon showed large areas of hemorrhage, edema and inflammatory cell infiltration. However, in this serious model it was difficult for the inventors to detect any significant therapeutic effect of the drugs compared to rats with drug-free colitis. On the contrary, 8 days after the induction of colitis, the colon of the rats showed considerably greater damage than that observed at 24 h and it was easier to buy the effects of the different drugs. The greatest damage observed at 8 days confirms that the pathology continues to develop, even after the initiating agent has been metabolized. In this chronic model, a substantial proportion of the rats died and elevated levels of TNF-α were observed in the serum and in the colon tissue of the rats that survived. The 8-day experiment also allowed pharmacological treatments to be included after induction, to determine if the drugs could be used to treat and perhaps reverse the developing disease.

The inventors used both time bands, the 24-hour and the 8-day, to compare the efficacy of two newly developed antagonists of the C3a and C5a receptor with that of the prednisolone steroid and that of the TNF-α infliximab inhibitor. All the drugs evaluated were effective in reducing some or all of the parameters evaluated. However, it was determined that the C5a antagonist was superior to the other drugs.

Corticosteroids, such as prednisolone, are therapies that are usually prescribed in the treatment of IBD. They are potent drugs that act at the genetic level and cause the regulation by decrease of several proinflammatory mediators, such as cytokines, as well as immunological pathways. Due to their non-specific actions, corticosteroids have numerous side effects and should be used with caution in patients, particularly in chronic treatment. In the present study, pretreatment with prednisolone was effective in reducing most of the parameters evaluated, as expected. However, rats treated with prednisolone lost considerable body weight. This was attributed to catabolism, one of the main side effects of steroid therapy. Although steroids are widely used in colitis therapy, it should be noted that rats treated with steroids 24 h after induction of colitis showed no appreciable therapeutic effects. Due to the time necessary for the steroids to be effective, it could be too late to observe any appreciable effect of prednisolone on the disease pathology.

Alternatively, the absence of effect could be caused by the tendency of steroid treatments to inhibit the natural healing processes associated with the disease pathology.

It is believed that TNF-α proinflammatory cytokine contributes significantly to the pathology of IBD. Recent advances in drug development have produced several antibodies that block the effects of this cytokine (Muller, 2002). It has been described that one of these antibodies, infliximab, is effective in reducing the pathology of IBD and is currently marketed in several countries for the treatment of IBD. Such antibody-based therapies present supply problems in hospitals and are significantly more expensive than therapies with other drugs (Valle et al. 2001).

According to the inventors, the efficacy of infliximab in an animal model of IBD has not been described above. Accordingly, the inventors have used this compound as a comparative drug for C5a and C3a receptor antagonists. Although pretreatment of rats with infliximab reduced the severity of the lesions, it was not as effective as the C5a antagonist or prednisolone. Infliximab was more effective in rats treated for 8 days than in rats in the 24-hour study. Since only detectable increases in TNF-α levels were observed in serum and in colon tissue in the 8-day study, one might expect that the effect of this compound on TNF-α inhibition would be minimal during the stages serious of this disease.

It is suspected that abnormal complement activity is important in the pathology of IBD, although there is limited evidence today. The efficacy of the C3a SB 290157 antagonist was examined in this study. It has recently been described that this compound is a human C3a receptor antagonist and shows activity in several in vitro and in vivo tests (Mollnes et al. 2002, Ames et al. 2001). The inventors have discovered that this antagonist is effective in reducing some of the parameters evaluated in the 24-hour study. It should be noted that it reduced the intensity of macroscopic damage compared to colitis control rats. The dose regimen selected in the study of the inventors (30 mg / kg ip twice daily) was the same as that used in previous studies in vivo and it seems that this high dose is necessary to obtain therapeutic effects with this compound ( Ames et al. 2001). Due to the need to inject animals intraperitoneally twice daily, the drug was not evaluated in the 8-day model. Despite the inhibitory effects observed with SB 290157 after 24 h, additional studies should be carried out over a longer period of time to identify the exact role of C3a in TNBS-induced colitis.

The cyclic peptide of the C5a PMX53 antagonist (AcF- [OPdChaWR] proved to be the most effective agent used in this study. The inventors have found that the pretreatment of rats with the C5a antagonist both orally and SC significantly reduced all indicators of the disease evaluated Rats treated orally 24 hours after establishing colitis also showed a significant reduction of all disease indicators.This C5a antagonist has been shown to be specific for the C5a receptor in humans, rats and dogs (Woodruff et al. 2001) and which is specific for the C5a receptor compared to other related human receptors (Finch et al. 1999) In particular, PMX53 does not bind to the C3a receptor (Finch et al. 1999) and has no effect on the formation of the membrane attack complex (Arumugam et al. 2003). The highest efficacy observed with the C5a antagonist against the C3a antagonist, at least In the 24-hour study, it is consistent with the highest potency and the highest proinflammatory activity of C5a compared to C3a in vivo.

The greater efficacy of the C5a antagonist compared to infliximab can be explained by the central role of C5a in the inflammatory cascade. There is evidence that C5a induces the release not only of TNF-α, but also of a host of other inflammatory cytokines and several mediators, which are also supposedly involved in IBD (Haynes et al. 2002, Mollnes et al. 2002 ). There is also evidence that this C5a antagonist prevents the formation of several of these mediators (Finch et al. 1999, Haynes et al. 2002), which probably explains its greater efficacy compared to infliximab, which only inhibits TNF- α. The inventors have just shown that a human C5a receptor antagonist is also an effective and potent treatment for IBD in a rat model.

When rats were pretreated with a combination of the C5a antagonist and prednisolone, no more significant reduction in disease indicators was observed compared to the C5a antagonist alone. This suggests a regulation by habitual decrease of inflammatory mediators.

In summary, this study demonstrates that complement components C3a and C5a, and especially C5a, have a significant role in the pathogenesis of TNBS-induced colitis in rats. Therefore, complement can play a fundamental role in the development of IBD in humans. This should encourage the development of anti-complement-based therapies. The potent therapeutic effects of the orally active human C5a antagonist AcF- [OPdChaWR] and its greater efficacy compared to prednisolone and infliximab, support the fact of considering the use of C5a antagonists in the treatment of IBD in humans.

References cited herein are listed on the following pages.

REFERENCES

Ames RS, Lee D, Foley JJ, Jurewicz AJ, Tornetta MA, Bautsch W, Settmacher B, Klos A, Erhard KF, Cousins RD, Sulpizio AC, Hieble JP, McCafferty G, Ward KW, Adams JL, Bondinell WE, Underwood DC , Osborn RR, Badger AM, Sarau HM.

J. Immunol. 2001, 166 (10): 6341-8. Fairlie, D.P., Wong, A.K .; West, M.W. Curr. Med. Chem. 1998, 5, 29-62. Fairlie, D.P., Abbenante, G. and March, D. Curr. Med. Chem. 1995, 2, 672-705. Gerard, C. and Gerard, N.P. Ann. Rev. Immunol. 1994, 12, 775-808. German, A.J., Hall, E.J. and Day, M.J.

J. Vet. Intern. Med. 2003 January-February; 17 (1): 8-20.

Konteatis, Z.D., Siciliano, S.J., Van Riper, G., Molineaux, C.J., Pandya, S., Fischer, P., Rosen, H., Mumford, R.A. and Springer, M.S.

J. Immunol. 1994, 153, 4200-4204. Mollnes TE, Brekke OL, Fung M, Fure H, Christiansen D, Bergseth G, Videm V, Lappegard KT, Kohl J, Lambris JD. Blood. 2002 September 1; 100 (5): 1869-77. Morris GP, Beck PL, Herridge MS, Depew WT, Szewczuk MR, Wallace JL. Gastroenterol 1989; 96: 795-803. Nielsen OH, Rask-Madsen J. Scand. J. Gastroenterol. Suppl. 1996; 31: 149-159. Sanderson, S.D., Kirnarsky, L., Sherman, S.A. Vogen, S.M., Prakesh, O., Ember, J.A., Finch, A.M. and Taylor, S.M.

J. Med. Chem. 1995, 38, 3669-3675. Tams, T.R. Vet. Clin. North Am. Small Anim. Pract. 1993, 23: 569-86. Tyndall, J.D.A .; Fairlie, D.P. Curr. Med. Chem. 2001, 8, 893-907.

Claims (23)

1. The use of an inhibitor of a G-protein coupled receptor in the production of a medicament for the treatment of inflammatory bowel disease, in which the inhibitor is a compound such that:

(to)

is an antagonist of a G-protein coupled receptor,

(b)

has no agonist activity and

(C)

it is a cyclic peptide or peptidomimetic compound of formula I

image 1

where A is H, alkyl, aryl, NH2, NH-alkyl, N (alkyl) 2, NH-aryl, NH-acyl, NH-benzoyl, NHSO3, NHSO2

alkyl, NHSO2-aryl, OH, O-alkyl or O-aryl;

10

B is an alkyl, aryl, phenyl, benzyl, naphthyl or indole group, or the side chain of an amino acid D or L, but not

it is the side chain of glycine, D-phenylalanine, L-homophenylalanine, L-tryptophan, L-homotriptophan, L-tyrosine or L

homothyrosine;

C is the side chain of an amino acid D or L or a homoamino acid, but it is not the side chain of

isoleucine, phenylalanine or cyclohexylalanine;

fifteen

D is the side chain of a neutral D amino acid, but it is not the side chain of glycine or D-alanine, a

bulky flat side chain or a bulky loaded side chain;

E is the side chain of an amino acid that is selected from the group consisting of L-phenylalanine, L

tryptophan and L-homotriptophan, or is L-1-naphthylalanine or L-3-benzothienylalanine;

F is the side chain of L-arginine, L-homoarginine, L-citrulline or L-canavanine, or a bioister of these; Y

twenty

X1 is - (CH2) nNH- or (CH2) n-S-, where n is an integer from 1 to 4; - (CH2) 2O-; - (CH2) 3O-; - (CH2) 3-;

- (CH2) 4-; -CH2COCHRNH-; or -CH2-CHCOCHRNH-, where R is the side chain of any amino acid

common or not common.

2.

An inhibitor of a G-protein coupled receptor for use in the treatment of inflammatory disease

intestinal, in which the inhibitor is a compound such that:

25

(to) is an antagonist of a G-protein coupled receptor,

(b)

has no agonist activity and

(C)

it is a cyclic peptide or peptidomimetic compound of formula I

image 1 where A is H, alkyl, aryl, NH2, NH-alkyl, N (alkyl) 2, NH-aryl, NH-acyl, NH-benzoyl, NHSO3, NHSO2alkyl, NHSO2-aryl, OH, O-alkyl or O-aryl ; B is an alkyl, aryl, phenyl, benzyl, naphthyl or indole group, or the side chain of an amino acid D or L, but it is not the side chain of glycine, D-phenylalanine, L-homophenylalanine, L-tryptophan, L- homotriptophan, L-tyrosine or Lhomothyrosine; C is the side chain of an amino acid D or L or a homoamino acid, but it is not the side chain of isoleucine, phenylalanine or cyclohexylalanine; D is the side chain of a neutral D amino acid, but it is not the glycine or D-alanine side chain, a bulky flat side chain or a bulky charged side chain; E is the side chain of an amino acid that is selected from the group consisting of L-phenylalanine, Ltriptophan and L-homotriptophan, or is L-1-naphthylalanine or L-3-benzothienylalanine; F is the side chain of L-arginine, L-homoarginine, L-citrulline or L-canavanine, or a bioister of these; Y X1 is - (CH2) nNH- or (CH2) n-S-, where n is an integer from 1 to 4; - (CH2) 2O-; - (CH2) 3O-; - (CH2) 3-; - (CH2) 4-; -CH2COCHRNH-; or -CH2-CHCOCHRNH-, where R is the side chain of any common or uncommon amino acid.

3.

The use according to claim 1 or the inhibitor according to claim 2, wherein n is 2 or 3.

Four.

The use according to claim 1 or 3 or the inhibitor according to claim 2 or 3, wherein A is an acetamide group, an aminomethyl group or a substituted or unsubstituted sulfonamide group.

5.

The use according to claim 3 or the inhibitor according to claim 3, wherein A is a substituted sulfonamide and the substituent is an alkyl chain of 1 to 6 carbon atoms or a phenyl or toluyl group.

6.

The use according to claim 5 or the inhibitor according to claim 5, wherein the substituent is an alkyl chain of 1 to 4 carbon atoms.

7.

The use according to any of claims 1 and 3 to 6 or the inhibitor according to any of claims 2 to 6, wherein B is the side chain of L-phenylalanine or L-phenylglycine.

8.

The use according to any of claims 1 and 3 to 7 or the inhibitor according to any of claims 2 to 7, wherein C is the side chain of glycine, alanine, leucine, valine, proline, hydroxyproline or thioproline .

9.

The use according to any of claims 1 and 3 to 8 or the inhibitor according to any of claims 2 to 8, wherein D is the side chain of D-leucine, D-homoleucine, D-cyclohexylalanine, D -homocyclohexylalanine, D-valine, D-norleucine, D-homonorleucine, D-phenylalanine, D-tetrahydroisoquinoline, Dglutamine, D-glutamate or D-tyrosine.

10.

The use according to any of claims 1 and 3 to 9 or the inhibitor according to any of claims 2 to 9, wherein the inhibitor is a compound that has antagonistic activity against C5aR and has no agonist activity of C5a .

eleven.

The use according to any of claims 1 and 3 to 10 or the inhibitor according to any of claims 2 to 10, wherein the inhibitor has a potent antagonistic activity at submicromolar concentrations.

12.

The use according to any of claims 1 and 3 to 11 or the inhibitor according to any of claims 2 to 11, wherein the compound has an affinity for the IC50 receptor <25 µM and an antagonistic potency of IC50 <1 µM.

13.

The use according to any of claims 1 and 3 to 12 or the inhibitor according to any of claims 2 to 12, wherein 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 WO 03/033528.

14.

The use according to claim 13 or the inhibitor according to claim 13, wherein the compound is PMX53 (compound 1), compound 33, compound 60 or compound 45 described in WO 03/033528.

fifteen.

The use according to any of claims 1 and 3 to 14 or the inhibitor according to any of claims 2 to 14, wherein the inhibitor is used in conjunction with one or more other agents for the treatment of inflammatory bowel disease .

16.

The use according to claim 15 or the inhibitor according to claim 15, wherein the other agent is infliximab or is a C3a inhibitor.

17.

The use according to any of claims 1 and 3 to 16 or the inhibitor according to any of claims 2 to 16, wherein the treatment is to prevent or alleviate the serious recurrences of inflammatory bowel disease.

18.

The use according to any of claims 1 and 3 to 16 or the inhibitor according to any of claims 2 to 16, wherein the treatment is to prevent or alleviate a primary onset of inflammatory bowel disease.

19.

The use according to any of claims 1 and 3 to 18 or the inhibitor according to any of claims 2 to 18, wherein the inflammatory bowel disease is selected from the group consisting of ulcerative colitis, Crohn's disease, enteritis Plasmocytic lymphocytic, celiac disease, collagen colitis, lymphocytic colitis and eosinophilic enterocolitis, indeterminate colitis, infectious colitis, pseudomembranous colitis (necrotizing colitis) and ischemic inflammatory bowel disease.

twenty.

The use according to any of claims 1 and 3 to 18 or the inhibitor according to any of claims 2 to 18, wherein the inflammatory bowel disease is ulcerative colitis.

twenty-one.

The use according to any of claims 1 and 3 to 18 or the inhibitor according to any of claims 2 to 18, wherein the inflammatory bowel disease is Crohn's disease.

22

The use according to any of claims 1 and 3 to 18 or the inhibitor according to any of claims 2 to 18, wherein the inflammatory bowel disease is selected from the group consisting of enterocolitis, canine plasmacytic lymphocytic colitis, colitis Prototecal and histiocytic ulcerative colitis.

2. 3.

The use according to any of claims 1 and 3 to 20 or the inhibitor according to any of claims 2 to 20, wherein the inhibitor is administered in an enteric coated capsule or rectally.