HK1081864B - Anti-inflammatory compositions and use of the same in the manufacture of medicaments - Google Patents

Description

Anti-inflammatory composition and pharmaceutical use thereof

Federally sponsored research or development

Defense Advanced Research Project Agency (DARPA) foundation No. n 65236-99-1-5420.

Background

The present invention relates to pharmaceutical compositions comprising active compounds and pharmaceutically acceptable salts thereof which have inhibitory effects on the binding of various chemokines, such as MIP-1 alpha and RANTES, to the CCR1 receptor. The invention also relates to methods of treating inflammatory and immunoregulatory disorders and diseases using the above pharmaceutical compositions.

Human health relies on the body's ability to discover, destroy, or otherwise eliminate foreign pathogens that may encroach on the available resources of an individual and/or induce disease. The immune system, which includes leukocytes (white blood cells (WBCs): T and B lymphocytes, monocytes, eosinophils, basophils, and neutrophils), lymphoid tissues and lymphatic vessels, is the body's defense system. To combat infection, B and T lymphocytes circulate in the body, interact with antigen presenting cells, and detect pathogens. Once the invader is detected, T lymphocytes accumulate at the site of infection, destroying the pathogen. Chemokines serve as molecular beacons for T lymphocytes, neutrophil and macrophage aggregation and activation, marking pathogen orientation.

The immune system also fails when protecting the body from pathogens. Inappropriate signaling by chemokines can lead to inflammatory disorders such as rheumatoid arthritis, multiple sclerosis and other diseases. In rheumatoid arthritis, unregulated chemokines in the bone joints attract and activate infiltrating macrophages and T-cells. The activation of these cells induces synovial cell proliferation, leading to inflammation and ultimately bone and cartilage loss (DeVries, Ran et al 1999). One hallmark of some demyelinating diseases, such as multiple sclerosis, is chemokine-mediated macrophage and T cell accumulation to the central nervous system (Kennedy and Karpus 1999). Chemokines of destructive WBCs that accumulate in grafts are involved in their subsequent rejection (DeVries, Ran et al 1999). Since chemokines play an extremely important role in the development of inflammation and lymphocytes, the ability to specifically manipulate their activity will have a tremendous impact on the amelioration and blockade of diseases for which no satisfactory therapeutic approach is currently available. In addition, graft rejection may be minimized without the systemic and concomitant effects of expensive immunosuppressant drugs.

Chemokines, a group of small peptides (7-10kD) larger than 40, are linked to receptors expressed by WBCs and signal their chemochemotaxis and chemostimulant functions through a G-protein-coupled signaling cascade. The receptor may be linked to more than one ligand; for example, the receptor CCR1 binds RANTES (a factor expressed by normal T cells that are regulated upon activation), MIP-1 α (macrophage inflammatory protein) and MIP-1 β chemokines. To date, 24 chemokine receptors are known. The absolute number of chemokines, multiple ligand-binding receptors, and different receptor properties of WBCs warrant tightly controllable and specific immune responses (Rossi and Zlotnik 2000). Chemokine activity can be controlled by modulating their respective receptors, treating related inflammatory and immune diseases, and facilitating organ and tissue transplantation.

The receptor CCR1 and its chemokine ligands, including, for example, MIP-1 α, MIP-1 β, and RANTES, are promising therapeutic targets because of their involvement in rheumatoid arthritis, transplant rejection (both reviewed in DeVries, Ran et al 1999), and multiple sclerosis (Fischer, Santambrogio et al 2000; IZikson, Klein et al 2000; Rottman, Slavin et al 2000). In fact, function-blocking antibodies, modified chemokine receptor ligands, and small organic compounds have been discovered and some of them have been successfully demonstrated to have utility in the prevention or treatment of certain chemokine-mediated diseases (reviewed in (Rossi and Zlotnik 2000)). In particular, in an experimental model of rheumatoid arthritis, disease progression is attenuated when a signal-blocking, modified-RANTES ligand is administered (Plater-Zyberk, Hoogerwf et al 1997). Although the use of function-blocking antibodies and small molecule peptide therapies is promising, it suffers from the risk of degradation, extremely short half-life upon administration, and the expense of most protein development and manufacture. Small molecule organic compounds are desirable because they have a long half-life in vivo, require a small dose to produce their effect, can be administered orally on a regular basis, and are therefore relatively inexpensive. Certain organic antagonists of CCR1 have been previously described (Hesselgesser, Ng et al 1998; Ng, May et al 1999; Liang, Mallaiet al 2000; Liang, Rosser et al 2000). Since these compounds have been shown to be effective in treating disease in certain animal models (Liang, Mallari et al 2000), there is a need in the art for more compounds useful in medicine. Applicants have discovered that potent organic antagonists of CCR1 are expected to be important tools in the art.

Piperazine derivatives of the type disclosed herein are known anti-inflammatory agents (see, e.g., WO 98/56771, W097/44329, WO 99/37651, WO 99/37619, WO 00/53600). The specific piperazine derivatives disclosed herein have not previously been identified as antagonists of CCR 1.

Summary of The Invention

In one embodiment, the invention provides compositions comprising a pharmaceutically acceptable carrier and an active compound that inhibits the binding of various chemokines, including, for example, MIP-1 α and RANTES, to the CCR1 receptor.

In another embodiment, the invention provides a method of blocking the CCR1 receptor comprising administering a compound that inhibits the activity of a variety of chemokines, including for example MIP-1 α and RANTES.

In another embodiment, the invention provides methods of treating inflammatory and immunoregulatory disorders and diseases comprising administering the compositions of the invention.

Detailed Description

The present invention provides compositions comprising a pharmaceutically acceptable carrier and an active compound that inhibits the binding of a variety of chemokines to the CCR1 receptor, including for example the major ligands MIP-1 α, MIP-1 β, MIP-1 γ, myeloid progenitor suppressor-I (XIF-I), hemofiltrate (hemofiltrate) C-I (HCC-1), leukopheromones and RANTES.

The compositions of the present invention are useful for treating inflammatory disorders.

Definition of

"alkyl" means a saturated aliphatic group including straight chain alkyl, branched chain alkyl, or cyclic alkyl. In preferred embodiments, the number of carbon atoms in the linear or branched alkyl backbone is 10 or less, more preferably 6 or less, and most preferably 4 or less. Likewise, preferred cycloalkyl groups have 3 to 10 carbon atoms in their ring structure, more preferably 3 to 6 carbon atoms in the ring structure. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, sec-butyl, cyclobutyl, pentyl, hexyl, cyclohexyl, and the like. Methyl and ethyl are preferred.

"alkoxy" means an alkyl group, as defined above, appended to the parent molecular moiety through an oxygen atom. Examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, tert-butoxy, neopentyloxy, and n-hexyloxy.

"aryl" refers to any monovalent aromatic carbocyclic group containing 5 to 10 carbon atoms. The aryl group may be bicyclic (i.e., phenyl (or Ph)) or polycyclic (i.e., naphthyl), and may be unsubstituted or substituted. Preferred aryl groups include phenyl, naphthyl, furyl, thienyl, pyridyl, indolyl, quinolinyl, or isoquinolinyl.

"haloalkyl" refers to an alkyl group as defined above substituted with one or more halogen atoms. Examples of haloalkyl groups include, but are not limited to, trifluoromethyl, difluoromethyl, trichloromethyl, chloroethyl, bromobutyl, 2-trifluoroethyl, 1-fluoromethyl-2-fluoroethyl, 3-bromo-2-fluoropropyl, 1-bromomethyl-2-bromoethyl, and the like. Trifluoromethyl is particularly preferred.

"halogen" refers to fluorine, chlorine, bromine and iodine.

"Heterocyclyl" means a stable, saturated, partially unsaturated or aromatic radical containing 5 to 10 ring atoms, preferably 5 or 6 ring atoms. The ring may be substituted one or more times with a substituent. The rings may be mono-, bi-or polycyclic. The heterocyclic group consists of carbon atoms and 1 to 3 heteroatoms, which may be independently selected from nitrogen, oxygen and sulfur. Examples of heterocyclyl groups include acridine, benzothiazoline, benzimidazole, benzofuran, benzopyran, benzoxazoline, benzothiophene, benzothiazole, benzothiophene, carbazole, cinnoline, furan, imidazole, 1H-indazole, indole, isoindole, isoquinoline, isothiazole, morpholine, oxazole (i.e., 1, 2, 3-oxadiazole), phenazine, phenothiazine, phenoxazine, phthalazine, piperidine, piperazine, pteridine, purine, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, thiazoline, quinoline, quinoxaline, tetrahydrofuran, tetrahydroquinolinyl, 1, 2, 3, 4-tetrahydroisoquinolinyl, tetrahydrothienyl and its sulfoxide and sulfone derivatives, thiomorpholine, thiazole, 1, 3, 4-thiadiazole, thiophene (thiene), thiophene, 1, 3, 5-triazine, triazole (i.e., 1, 2, 3, -triazoles) and the like.

"substituted" refers to a moiety comprising at least one, preferably 1-3 substituents. Suitable substituents include hydrogen (H) and hydroxyl (-OH), amino (-NH2), oxygen (-O-), carbonyl (-CO-), mercapto, alkyl, alkenyl, alkynyl, alkoxy, halogen, nitrile, nitro, aryl, and heterocyclyl. These substituents are optionally further substituted with 1 to 3 substituents. Examples of substituted substituents include amide, alkyl mercapto, alkyl sulfo, alkylamino, dialkylamino, carboxylate, alkoxycarbonyl, alkaryl, aralkyl, alkylheterocyclyl and the like.

Compounds that inhibit chemokine, MIP-1 alpha and RANTES activity

In one embodiment, the active compounds of the invention are compounds of formula (1):

wherein n is 0, 1, or 2;

y is oxygen or sulfur;

R¹、R²and R³Each independently hydrogen, alkyl, alkoxy, halogen, haloalkyl or nitro.

Preferably, the active compound is of formula (2):

wherein n is 0, 1, or 2;

y is oxygen or sulfur; and is

R¹、R²And R³Each independently hydrogen, alkyl, alkoxy, halogen, haloalkyl or nitro.

Particularly preferably, R in the compound (1)¹And R²Is hydrogen, R³Is halogen (particularly preferably chlorine or fluorine) or hydrogen.

The compounds for use in the present invention are commercially available or can be prepared according to known methods (see, for example, FR 1,441, 071 and JP 63-41907).

Testing

It is to be demonstrated that the compounds of the present invention are antagonists of the CCR1 receptor, as can be determined if they inhibit the activity of the chemokines MIP-1 alpha and RANTES. Preferred such compounds have the following properties:

(1) can effectively inhibit the binding of chemokine MIP-1 alpha or RANTES and CCR1 receptor;

(2) significant inhibition of Ca for CCR1²⁺Responding; and

(3) limited nonspecific Ca²⁺And (6) responding.

An in vitro standard binding assay can be used to confirm the affinity of a compound to the CCR1 receptor (thereby inhibiting the activity of MIP-1 α and RANTES by competitive binding to the receptor). See the examples below. Preferably, the active compound IC₅₀Value of<10. mu.M, more preferably,<5 μ M, most preferably<1μM。

Compounds that inhibit the activity of MIP-1 alpha and RANTES affect MIP-1 alpha and RANTES stimulated intracellular Ca²⁺And (4) concentration. Binding of the ligand to the CCR1 receptor results in G-protein induced phospholipase C activation, which results in conversion of phosphatidylinositol phosphates to phosphoinositides and diacylglycerols. Phosphoinositides then bind to intracellular receptors, binding Ca²⁺Released into the cytoplasm. Remove Ca²⁺Release of Ca from intracellular stores²⁺In addition to the increased concentration, binding of phosphoinositides to their receptors results in an increased flux of extracellular calcium ions across the membrane into the cell. Thus, activation of the CCR1 receptor by MIP-1 α and RANTES, and subsequent inhibition of activation by the compounds of the invention, can be measured by measuring intracellular free Ca²⁺An increase in the level is determined. Representatively, this can also be accomplished by using a calcium-sensitive fluorescent probe such as quin-2, fura-2 and indo-1. See the examples below. Active compounds block Ca²⁺The effect of the response depends on the amount of active compound and chemokine present. In general, 10. mu.M of active compound is capable of producing 20-100% Ca when the chemokine is 10nM²⁺Response inhibition.

Determination of whether the active Compound produces non-specific Ca²⁺The response may be performed by: adding an active compound and measuring said Ca as described above²⁺Response followed by addition of a known additional chemotaxisFactor receptor antagonists (e.g., bradykinin or SDF-1, a CXCR4 ligand). The comparative response results indicate that the active compound is non-specific binding.

Pharmaceutical composition

Pharmaceutical compositions for the administration of active compounds may conveniently be presented in dosage unit form and may be prepared by any of the methods well known in the pharmaceutical arts. All methods include the step of bringing into association the active compound with the carrier which constitutes one or more accessory ingredients. In general, pharmaceutical compositions are prepared as follows: the active compound is mixed with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, the product is processed into the desired formulation. In the pharmaceutical composition, the active compound is present in an amount sufficient to produce the desired effect on the process or condition of the disease.

Pharmaceutical compositions containing the active compound may be in a form suitable for oral administration, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions for oral use may be prepared by any method known to the art for the manufacture of pharmaceutical compositions and such compositions may also include one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets comprise the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. Excipients are, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating or disintegrating agents, such as corn starch, or alginic acid; binding agents, such as starch, gelatin or acacia; and lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to achieve delayed disintegration, absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, the sustained release material may use glyceryl monostearate or glyceryl distearate. They may also be coated by techniques disclosed in U.S. Pat. Nos. 4,256,108, 4,166,452, and 4,265,874 to form osmotic therapeutic tablets with controlled release.

Oral formulations may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin; or soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; the dispersing or wetting agent may be a naturally-occurring phosphatide, for example lecithin, or a condensate of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or a condensate of ethylene oxide with long chain aliphatic alcohols, for example heptadecenoxycetyl alcohol, or a condensate of ethylene oxide with partial esters of fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or a condensate of ethylene oxide with partial esters of fatty acids and hexitol anhydrides, for example polyethylene sorbitan monooleate. Aqueous suspensions may also include one or more preservatives, for example ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may include a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those listed above, flavoring agents may be added to produce a good tasting preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Examples of suitable dispersing or wetting agents and suspending agents are as hereinbefore described. Other excipients, for example sweetening, flavoring and coloring agents, may also be used.

The pharmaceutical compositions of the present invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures thereof. Suitable emulsifying agents may be natural gums such as gum acacia or gum tragacanth, natural phosphatides such as soya bean, lecithin, and esters or partial esters of fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensates of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsion also includes sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also include wetting agents, preservatives, flavoring and coloring agents.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. The suspension may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents as described hereinbefore. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. Among the acceptable carriers and solvents that can be employed are water, ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any non-irritating non-volatile oil may be used, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compounds of the present invention may also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. These materials are cocoa butter and polyethylene glycols.

For topical application, creams, ointments, jellies, solutions or suspensions, etc., containing the compounds of the present invention may be used. (for purposes of this application, topical applications include mouth washes and rinses.)

The pharmaceutical compositions and methods of the present invention may further comprise other therapeutically active compounds described herein, which are generally useful in the treatment of the pathological conditions described above.

In the treatment or prevention of conditions in which modulation of chemokine receptors is desired, appropriate dosage levels will generally be from about 0.01 to 500mg/kg patient body weight/day, and may be administered in single or multiple doses. Preferably, the dosage level is from about 0.1 to about 250 mg/kg/day; more preferably, from about 0.5 to about 100 mg/kg/day. Suitable dosage levels may be about 0.01 to 250 mg/kg/day, about 0.05 to 100 mg/kg/day, or about 0.1 to 50 mg/kg/day. Dosages within this range may be 0.05-0.5, 0.5-5 or 5-50 mg/kg/day. For oral administration, the preferred compositions are in the form of tablets containing 1.0 to 1000mg of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0mg of the active ingredient adjusted for the condition of the patient being treated. The compounds are administered 1-4 times per day, preferably once or twice per day.

It will be understood that the specific dose level and frequency of dosage for any particular patient will vary depending upon a variety of factors including the activity of the specific compound employed, the metabolic stability, the time of action of the compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

Methods of blocking CCR1 receptor

The invention also provides a method of inhibiting the binding of MIP-1 alpha or RANTES to the CCR1 receptor by contacting the compositions described above with cells expressing the CCR1 receptor under conditions suitable to inhibit the binding of chemokines to the CCR1 receptor.

Methods of treating inflammatory and immunoregulatory disorders and diseases

The present invention also provides a method of treating an inflammatory disease comprising administering to a patient in need thereof a therapeutically effective amount of a composition as described above for a time sufficient to treat the inflammatory disease. "treating" refers to preventing, inhibiting or ameliorating a disorder or symptom.

CCR1 provides a target for interfering with or promoting eosinophil and/or lymphocyte function in mammals, such as humans. Compounds that inhibit CCR1 are particularly effective for modulating eosinophil and/or lymphocyte function for therapeutic purposes. Accordingly, the present invention relates to compounds that are effective for the prevention and/or treatment of various inflammatory and immunoregulatory disorders and diseases.

For example, a compound that inhibits one or more of the functions of CCR1 can be used to inhibit (i.e., reduce or prevent) inflammation. As a result, one or more inflammatory processes, such as leukocyte migration, chemotaxis, exocytosis (e.g., of enzymes, histamine), or inflammatory mediator release, may be inhibited. For example, eosinophilic infiltration into inflammatory sites (e.g., asthma) can be inhibited according to the methods of the invention.

Similarly, a compound that promotes one or more of the functions of CCR1 may be used to stimulate (induce or enhance) an inflammatory response, such as leukocyte migration, chemotaxis, exocytosis (e.g., of enzymes, histamine), or inflammatory mediator release, resulting in beneficial stimulation of the inflammatory process. For example, eosinophils can be concentrated for use against parasitic infections.

In addition to primates, such as humans, a variety of other mammals can be treated according to the methods of the present invention. For example, mammals, including, but not limited to, cattle, sheep, goats, horses, dogs, cats, guinea pigs, mice or other bovine, ovine, equine, canine, feline, rodent, murine species may be treated. Moreover, the method can also be used to treat other species, such as avian species (e.g., chickens).

Diseases or conditions associated with inflammation or infection can be treated using the methods of the present invention. In a preferred embodiment, the disease or condition is one in which eosinophil and/or lymphocyte function is inhibited or enhanced for the purpose of modulating an inflammatory response,

Diseases or conditions of humans or other species that may be treated with CCR1 inhibitors include, but are not limited to: inflammatory or allergic diseases and conditions, including respiratory allergic diseases such as asthma, allergic rhinitis, allergic lung disease, hypersensitivity pneumonitis, eosinophilic pneumonia (e.g., lueffler's syndrome, chronic eosinophilic pneumonia), delayed-type hypersensitivity Interstitial Lung Disease (ILD) (e.g., idiopathic pulmonary fibrosis or ILD associated with rheumatoid arthritis, systemic lupus erythematosus, ankylosing spondylitis, systemic sclerosis, sjogren's syndrome, polymyositis, or dermatomyositis); systemic anaphylaxis or hypersensitivity, drug allergy (e.g., for penicillin, cephalosporins), insect sting allergy; autoimmune diseases, such as rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, leukoencephalopathy, encephalomyelitis, alzheimer's disease, systemic lupus erythematosus, myasthenia gravis, juvenile onset diabetes; glomerulonephritis, autoimmune thyroiditis, Behcet's disease; Guillian-Barre syndrome, acute cell-mediated transplant rejection (e.g., kidney transplant rejection), transplant rejection (as in transplantation), including allograft rejection or graft-versus-host disease; uricaria, cutaneous vasculitis, inflammatory bowel disease, such as crohn's disease and ulcerative colitis; spondyloarthropathy; scleroderma; psoriasis (including T-cell mediated psoriasis) and inflammatory dermatoses such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, urticaria; vasculitis (e.g., causing necrosis, epidermal, and allergic vasculitis); eosinophilic myositis, eosinophilic fasciitis; cancer in which leukocytes infiltrate the skin or organs. Other diseases or conditions in which an adverse inflammatory response can be inhibited can also be treated, including, but not limited to, reperfusion injury, restenosis, atherosclerosis, certain hematologic malignancies, cytokine-induced toxicity (e.g., septic shock, endotoxic shock), polymyositis, dermatomyositis. The compounds of the present invention are therefore useful in the prevention or treatment of a variety of inflammatory and immunoregulatory disorders and diseases.

In vitro standard tests can be used to confirm the effectiveness of the compositions of the invention in treating inflammatory disorders, including animal models of multiple sclerosis experimental autoimmune encephalomyelitis and adjuvant-induced arthritis models of rheumatoid arthritis.

The compositions of the present invention may be administered by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intracisternal injection or infusion, subcutaneous injection or implant), inhalation spray, nasal, vaginal, rectal, sublingual, or topical routes of administration, and may be formulated, alone or together, into suitable dosage unit formulations containing non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles appropriate for each route of administration. In addition to the treatment of warm-blooded animals such as rats, mice, horses, cattle, sheep, dogs, cats, monkeys, etc., the compositions of the invention are also effective in the treatment of humans.

Combination therapy

Combination therapies for the prevention and treatment of inflammatory and immunoregulatory disorders and diseases by modulating chemokine receptor activity as described above are exemplified by the combination of a compound of the invention with other compounds known to have the same effect.

For example, in the treatment or prevention of inflammation, the compounds of the present invention may be combined with an anti-inflammatory or analgesic agent, such as an opioid agonist; lipoxygenase inhibitors, such as 5-lipoxygenase inhibitors; cyclooxygenase inhibitors, such as cyclooxygenase-2 inhibitors; interleukin inhibitors, such as interleukin-1 inhibitors; an NMDA antagonist; a nitrogen oxide inhibitor or a nitrogen oxide synthesis inhibitor; a non-steroidal anti-inflammatory agent; or cytokine inhibitory anti-inflammatory agents, such as paracetamol, aspirin, codeine, fentanyl, ibuprofen, indomethacin, tollgate, morphine, naproxen, phenacetin, piroxicam; steroidal analgesics, sufentanil, sunlindac, tenidap, and the like. Similarly, the compounds of the present invention may be administered in combination with: a pain relieving agent; synergists such as caffeine, H2-antagonists, simethicone, aluminum hydroxide or magnesium hydroxide; decongestants such as phenylephrine, n-norephedrine, pseudoephedrine, oxymetazoline, epinephrine, naphazoline, xylometazoline, cyclopropylamine, or levo-deoxy-ephedrine; antitussives such as codeine, dihydrocodeinone, caramiphen, tobramon, or dextromethorphan; a diuretic; and sedative or non-sedative resistant histamine agents.

The following examples are given to illustrate the invention and are not intended to be limiting.

Examples

Example 1: materials and methods

A. Compound sample

The compound samples used herein include commercially available small molecules. The source plates included 1 or 5mg/ml of each compound in dimethyl sulfoxide (DMSO). According to these CLIP (Compound library), a plate was prepared in which 10 compounds were added per well and diluted with 20% DMSO to a concentration of 5-50. mu.g/ml. A20. mu.l aliquot of each mixture was added dropwise to the assay plate and stored at-20 ℃ until use.

B. Cells

CCR1 transfectants

CCR1-NSO cells

CCR1(CCR1-NSO) expressing a stable transfectant cell line was cultured in Iscove's Modified Dulbecco's Medium (IMDM) containing 4.5g/L glucose, 5% Fetal Bovine Serum (FBS), 10mN HCl, 250. mu.g/L xanthine (from 100X xanthine stock solution in 0.1N NaOH), 15. mu.g/L hypoxanthine (from 100X hypoxanthine stock solution in 0.1N NaOH), 10mg/L thymidine (from H NaOH)₂100X thymidine stock solution in O), 50. mu.M.beta. -mercaptoethanol (BME) (from H)₂1000X BME stock in O), and 1.5mg/L mycophenolic acid (from 2.5mg/ml mycophenolic acid stock in ethanol). Cells were in 5% CO₂95% air, 100% humidity, 37 deg.C growth, when the concentration is 0.5-1.0 × 10⁶Cells/ml were collected. Cells were subcultured twice weekly at 1: 4.

CCR1-293 cell

Human embryonic kidney adenovirus-transformed cell line 293 (American type culture Collection (ATCC); Manassas, Va.) was stably transfected with human CCR 1. pIRESpuro vectors (Clontech; Palo Alto, Calif.) were engineered to contain a prolactin signal sequence, the FLAG-epitope cloned into the EcoRV-Not I restriction site. The human CCR1cDNA clone was subcloned into the Not I site so that the insert was operably linked to the strong pCMV promoter. Cells were selected under 2. mu.g/ml puromycin in Dubecco's modified Eagle's medium at 5% CO₂Growth was carried out in 95% air, 100% humidity, 37 ℃ and the medium was supplemented with 2mM L-glutamine, 1.5g/L sodium bicarbonate, 0.1mM nonessential amino acids, 1.0mM sodium pyruvate, 10% fetal bovine serum. The cells were cultured in a 1: subcultured twice a week at 1X 10⁶Cells/ml were collected.

THP-1 cells

THP-1 cells were obtained from ATCC and cultured as a suspension in RPMI-1640 medium supplemented with 2mM L-glutamine, 1.5g/L sodium bicarbonate, 4.5g/L glucose, 10mM HEPES, 1mM sodium pyruvate, 0.05% 2-mercaptoethanol and 10% FBS. Cells were in 5% CO₂Growth at 37 ℃ in 95% air, 100% humidity, in a ratio of 1: subcultured twice a week at 1X 10⁶Cells/ml were collected.

C. Measurement of

CCR1 ligand binding inhibition

Cells expressing CCR1 were centrifuged and assayed in assay buffer (20mM HEPES pH7.1, 140mM NaCl, 1mM CaCl₂，5mM MgCl₂And 0.2% bovine serum albumin) to a concentration of 5.6X 10⁶Cells/ml (CCR1-NSO) or 2.2X 10⁶Cells/ml (CCR 10293). Screening experiments were performed as follows. Firstly, the method0.09ml of cells (5X 10)⁵CCR1-NSO cells/well or 2X 10⁵CCR1-293 cells/well) were added to test plates containing the compounds to give a final concentration of about 2-10 μ M for each compound. Then 0.09ml of test buffer was added to dilute to a final concentration of about 50pM, with a yield of about 30,000cpm per well¹²⁵I-labeled MIP-1 α (purchased from Amersham; Piscataway, N.J.), sealed plates, and incubated at 4 ℃ for about 3 hours in a shaking platform. The reaction was aspirated to a GF/B glass filter pre-soaked in a 0.3% Polyethyleneimine (PEI) solution on a vacuum cell harvester (Packard Instruments; Meiden, CT). Scintillation fluid (50. mu.l, Microscint 20, Packard Instruments) was added to each well, the plates were sealed and the radioactivity measured in a high-count scintillation counter (Packard Instruments). Control wells containing only diluent (for total counts) or excess MIP-1. alpha. or MIP-1. beta. (1. mu.g/ml for non-specific binding) were used to calculate the percentage of total inhibition for each group of compounds. After completing the CLIP plate test, wells with an inhibition rate of 40% or more were confirmed. Once confirmed, each compound of CLIP was subjected to a reactivity (deconvolution step) test. IC (integrated circuit)₅₀The value is the concentration required to reduce the binding of labeled MIP-1 alpha by 50% to the receptor.

Calcium mobilization

To detect the release of calcium stored in the cells, the cultured cells were incubated with 3. mu. MINDO-1AM dye (molecular probe; Eugene, OR) in cell culture medium for 45 minutes at room temperature and washed with Phosphate Buffered Saline (PBS). After addition of INDO-1AM, cells were resuspended in flow-through buffer (Hank's Balanced salt solution (HBSS) and 1% FBS). Calcium mobilization was measured using an international photon technology spectrophotometer (international photon technology; new jersey), with excitation at 350nm and dual simultaneous recordings of fluorescence emission at 400nm and 490 nm. The relative intracellular calcium level was expressed as 400nm/490nm emission ratio. The test was carried out at 37 ℃ with continuous mixing in sample cups each containing 10⁶Cells/2 ml flow-through buffer. The chemokine ligands used ranged from 1-100 nM. The emission ratio (typically 2-3 minutes) is plotted against time. At 10 secondsCandidate ligand blocking compounds (10-20. mu.M) were added followed by chemokine (MIP-1. alpha.; R) at 60 seconds&A system D; minneapolis, MN), at 150 seconds with addition of a control chemokine (bradykinin; ICN pharmaceuticals, Costa Mesa, CA). In some experiments, candidate blocking compound and cells were added simultaneously, and MIP-1 α was added after 40 seconds.

Chemotaxis assay

Chemotaxis assays were performed in a 96-well chemotaxis chamber (Neuroprobe; Gaithersburg, Md.) using 5 μm-well polycarbonate, polyvinylpyrrolidone-coated filters. Matrix-derived factor (SDF-1) was used as a specific control. The lower chamber contained 29. mu.l, 0.1nM MIP-I.alpha.and various amounts of inhibitor, and the upper chamber contained 100,000THP-1 cells in 20. mu.l. The chemotaxis chamber at 37 degrees C temperature 1-2 hours, using CyQuant test (Molecular Probes), measuring nucleic acid content of fluorescent dye method and microscope observation to determine the cell number in the lower chamber.

Example 2: assay for inhibitors of CCR1 binding to MIP-I alpha

A. Testing

To test for small organic molecules that prevent binding of the receptor CCR1 to the ligand, an assay was performed to detect radioligand (MIp-I α) bound on the cell surface to cells expressing exogenous CCR 1. If a compound is capable of inhibiting binding, whether or not it is competitively inhibited, a lower radioactive count will be observed when compared to an uninhibited control.

Construction of NSO murine myeloma cell line constitutively expressing human CCR1 on the cell surface (CCR1-NSO) and Human Embryonic Kidney (HEK) cancer cell line (CCR1-293) these cells lack other chemokine receptors that bind MIP-I α. The same number of cells was added to each well in CLIP plates, where each well contained 10 candidate organic compounds. Cells were incubated with radiolabeled MIP-I α. Cells were washed to remove unbound ligand and bound ligand was determined by quantitative radioactive counting. Determining a total count by cells not incubated with any organic compound; non-specific binding was determined by incubating the cells with unlabeled ligand and labeled ligand. Percent inhibition was calculated as follows:

% inhibition of 1- [ (sample cpm) - (non-specific cpm) ]/[ (total cpm) - (non-specific cpm) ] × 100

B. Inhibitors identified from compound libraries using CCR1-NSO cells

In group 1 of test compounds, the normalized standard deviation is 19%; thus, significant inhibition was confirmed to be greater than 38%; 40% inhibition is the threshold of choice. Of the number of wells tested, the content of 58 wells inhibited MIP-la binding by 40% or more. The contents of these 58 wells were retested in CLIP, with 6 inhibiting binding by 40% or more. This result indicates that at least one of the 10 compounds in each of the 6 wells was able to inhibit MIP-la binding to CCR 1. To confirm which of the 10 compounds in each well inhibited the CCR1 ligation of MIP-la, the inhibitory activity of each individual compound in the assay was tested to deconvolute the complex. Since some compounds may act together to inhibit binding, whereas the deconvolution test is only suitable for compounds tested alone, no compounds that are effective in combination but not in isolation are identified in this assay. Three candidate compounds were identified:

and

in the screening of the second group of compounds, the normalized standard deviation was 17%, indicating that 34% or more of the inhibitory activity was significant: further, the threshold value is 40%. These CLIPs produced 39 wells that showed greater than 40% inhibition. When screened again as CLIPs, 14 wells were depleted in ligand by more than 40%. Testing compounds individually to identify 13 inhibitory candidates;

C. inhibitors identified from compound libraries using CCR1-293 cells

CCR1-293 cells were used, which expressed CCR1 at higher levels, resulting in an increase in the standard deviation of noise to signal ratio normalization of approximately 10%. Screening group 1 compounds, 46 wells showed greater than 20% inhibition of ligand binding in the initial screen, and 10 wells in CLIP inhibited ligand binding in the second screen. Compounds were tested individually and 6 candidates were identified:

for the tests performed using CCR1-293 cells for group 1 compounds, the standard deviation for normalization for group 2 compound screening was smaller, most likely due to the use of these cells. Individual testing of 8 of the 35 CLIP wells that inhibited binding in assay 1 to obtain validated compounds identified 3 candidates;

and

example 3: dose response curve

To determine the affinity of a candidate compound for CCR1, and to determine its ability to inhibit ligand binding, the affinity was determined at 1X 10^-8-1×10^-4Compound concentrations in the M range titrate the inhibitory activity. This test is essentially identical to the CLIP screen except that the amount of compound used was varied; the number of cells and the ligand concentration remained unchanged. Only commercially available compounds were titrated at this time. Of the 22 candidates identified, 16 of the following were subjected to dose-response tests:

CCX-3343, CCX-1057, CCX-1307, CCX-1513, CCX-238, CCX-3345, CCX-3493, CCX-4425, CCX-4462, CCX-4682, CCX-469, CCX-5062, CCX-5119, CCX-541, CCX-6019 and CCX-6530.

Test compounds that do not inhibit MIP-I α binding in a dose-dependent manner are CCX-238, CCX-4425, and CCX-4462. Compounds whose inhibitory activity varies with their affinity for CCR1 are shown in table 1.

TABLE 1 affinity values for CCR 1-MIP-Ia binding, ranging from highest to lowest

Compounds CCX-541 and CCX-469 show CCR1 affinity values below 1. mu.M. Compounds CCX-5062, CCX-3345, and CCX-3343 have blocking activity, but are not able to completely inhibit MIP-la binding at the highest concentrations tested; their affinity value was estimated to be about 2. mu.M. Although compounds CCX-6019, CCX-4682, CCX-1307 and CCX-1057 showed blocking activity, their affinity values for CCR1 were lower, ranging from 18 to 45. mu.M. Although many compounds have been identified that inhibit the binding of CCR1 to MIP-la ligands, only two compounds have high CCR1 affinity values: CCX-541 and CCX-469. Other MIP-ia binding inhibitory compounds with lower affinity values were identified, but since the inhibitory properties competitive or non-competitive are not known, it is not clear whether these compounds are more effective at inhibiting the binding of other CCR1 ligands such as RANTES.

Example 4: CCR1 functional assay

CCR1 is a seven transmembrane G-protein linked receptor. The hallmark of the signaling cascade induced by these receptor connections is a pulse-like release of calcium ions from intracellular depots. Calcium mobilization tests were performed to determine whether candidate MIP-la inhibitory compounds also blocked CCR1 signaling. For use, it is desirable that the candidate compound be capable of inhibiting specific ligand binding and delivering the delivery.

Calcium ion release in response to MIP-I α binding is measured by cell permeable INDO-1/AM visualizers, which fluoresce in the presence of free, rather than chelated, calcium ions. CCR1-293 or THP-1 cells were loaded with INDO-1 and tested for calcium ion release in response to the addition of MIP-I α. For the specificity control, another non-CCR 1-binding chemokine, bradykinin, was added, which also signals through a seven transmembrane receptor. A pulse of fluorescent signal was seen with the addition of MIP-I α without the compound. If a compound specifically inhibits CCR1-MIP-I α signaling, no pulse of signal will be seen upon addition of MIP-I α, but one pulse will be seen upon addition of bradykinin. However, if a compound non-specifically inhibits signaling, no pulse will be seen upon addition of MIP-I α and bradykinin.

As shown in Table.2, of the selected compounds tested, CCX-469, CCX-541, CCX-1513 and CCX-3493, only CCX-469 was able to significantly and specifically inhibit signaling of CCR 1. CCX-541 and CCX-1513 have non-specific effects on calcium ion levels, and CCX-3493 is unable to affect signaling, whether MIP-I α or bradykinin induced.

TABLE 2 calcium signalling inhibition

Example 5 (inference)

One of the major functions of chemokines is their ability to attract WBCs to the site of pathogen invasion. To confirm that the compounds are not only capable of inhibiting MIP-la binding and CCR1 signaling as determined by calcium mobilization assays; and can inhibit CCR1 function, and a chemotaxis test is carried out. THP-1 myeloid monocytic leukemia cells, which, like monocytes, can be used as targets for MIP-I α chemoattraction. THP-1 cells are located in the upper chamber of the microwell migration chamber, while MIP-I α and the compound at increasing concentration are loaded in the lower chamber. In the absence of inhibitor, THP-1 cells migrate into the lower compartment in response to MIP-ia chemokines; if a compound inhibits CCR1 function, then the majority of TIP-1 cells are maintained in the upper compartment.

From the above detailed description, numerous variations of the present invention will be suggested to those skilled in the art and such obvious variations are intended to be included within the scope of the following claims.

Reference to the literature

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Hesselgesser，J.，H.P.Ng，et al.(1998).“Identifioation and characterization of smallmoleoule functional antagonists of the CCR1 chemokine receptor.＂J Riol Chem273(25)；15687-92.

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Claims (9)

1. A composition comprising a pharmaceutically acceptable carrier and a compound of formula (1):

wherein:

n is 2;

y is oxygen or sulfur;

R¹、R²and R³Each independently hydrogen or halogen.

2. The composition of claim 1, wherein the compound is a compound of formula (2):

wherein:

n is 2;

y is oxygen or sulfur; and is

R¹、R²And R³Each independently hydrogen or halogen.

3. The composition according to claim 1, wherein R¹And R²Is hydrogen.

4. The composition according to claim 1, wherein R³Is a halogen.

5. A composition according to claim 4, wherein R³Is chlorine or fluorine.

6. The composition according to claim 1, wherein R³Is hydrogen.

7. A composition comprising a pharmaceutically acceptable carrier and the compound CCX-469:

8. use of a composition according to claim 1 or 2 for the manufacture of a medicament for inhibiting the binding of a chemokine, which is MIP-1 α or RANTES, to a CCR1 receptor.

9. Use of a composition according to claim 1 or 2 in the manufacture of a medicament for the treatment of an inflammatory disease.