CYTOKINE PRODUCTION INHIBITORS, FORMULATIONS THEREOF AND THEIR USE IN MEDICINE, AND METHODS FOR THEIR IDENTIFICATION

Abstract: Compounds or ligands which inhibit the binding of alpha -oxoaldehyde-modified arginine residues include those of formula (I) wherein R1 is an aryl or alkyl group; R2 is a hydrogen atom; and R3 and R4 are each independently selected from a hydrogen atom, and aryl and alkyl groups; including salts, esters or hydrated forms thereof. Such compounds may be formulated in association with a pharmaceutical carrier therefor and used to treat or prevent chronic clinical complications associated with diabetes mellitus, macrovascular disease, and amyloidosis associated with Alzheimer's disease and chronic renal dialysis. Certain alpha -oxoaldehyde-modified arginine residues are also disclosed together with their use in a method of screening test compounds for therapeutic activity.

Description: - - Cytokine Production Inhibitors, Formulations thereof and their Use in Medicine, and Methods for their Identification The present invention relates to cytokine production inhibitors, particularly antagonists to certain non-enzymatically-modif led proteins, and more particularly to their pharmacological activity and therapeutic effect especially in the form of a pharmaceutical formulation. The non-enzymatic modification of proteins by sacchaπdes, non-enzymatic glycosylation or glycation, is known to occur in physiological systems. For example, glucose reacts non-enzyma 11 cal 1 y under physiological conditions with lysine residue side chain- and N-termmal ammo groups in proteins to form a Schiff ' s base which undergoes an Amadori rearrangement to form N-( 1 -deoxy-D-f ructos-1 -yl )-amιno acid residues or f ructosamines . These f ructosamines undergo further reaction and slowly degrade to form advanced glycation end-products. The formation of advanced glycation end-products from glucose and other saccharide derivatives has been proposed as a process which provides a signal for the degradation of proteins or, more generally, for recognition and degradation of senescent macromolecules . Proteins modified by advanced glycation end-products are bound by cell surface receptors on phagocylic cells (such as macrophages , monocytes, Kuppfer cells or glial cells) , then internalised and degraded. This process also stimulates monocyte chemotaxis; transendothel lal movement and proliferation of macrophages; chemotaxis and angiogenesis of human endothelial cells; and the synthesis and secretion of pro-inflammatory cytokines and growth factors. The formation of advanced glycation end-products in vivo has been linked to the development of chronic clinical complications ( retinopathy , neuropathy and nephropathy) associated with diabetes mellitus, diabetic embryopathy, macrovascular disease, and amyloidosis associated with Alzheimer's disease and chronic renal dialysis. In each of these disease processes, an immune response mediated by the induction of the synthesis and secretion of inflammatory cytokines has been implicated in the biochemica l mechanism leading to these pathologies . Despite this, there has been no identification ol an advanced glycation end-product which acts both as a recognition factor for receptor- mediated endocytosis of glycated proteins and as a pharmacophore for the induction of the synthesis and secretion of cytokines by phagocytic cells. It has now been found that arginine residues are major sites of irreversible modification in glycated proteins and that the physiological modifying agents are reactive α-oxoaldehydes (RCOCHO) which are present in physiological systems. These α-oxoaldehydes are formed from the degradation of Amadori products and elsewhere (such as from the metabolism of ketone bodies, by catabolism of threonine or by lipid peroxidation) . Preferred α-oxoaldehydes are wherein R is H, C- | _6 alkyl, or a C- ] -£ > alcohol residue which itself may be substituted by further hydroxy groups, preferably α- or β- to the -COCHO terminus. Examples are: methylglyoxal MeCOCHO; pentosone HOCH 2 (CHOH) 2 COCHO; glyoxal (CH0) 2 ; 3-deoxγpentosone HOCH 2 -CHOH-CH 2 COCHO; 3-deoxyglucosone H0CH 2 (CHOH ) 2 CH 2 COCHO; and glucosone HOCH 2 (CHOH) 3COCHO. It has also been found that modified arginine residues provide the receptor recognition factor for the binding of modified proteins to phagocytic cells and provide the pharmacophoric group for the induction - - of synthesis and secretion of, for example, the following cytokines: ιnterleukιn-1 β (IL-1B), macrophage colony stimulating factor (MCSF) and tumour necrosis factor-α (TNFα) . Therefore, any compounds or ligands which inhibit the binding of such modified proteins to cell surface receptors such as those on human monocytes and/or which inhibit production of such cytokines will be useful in the treatment or prevention of the development of diseases or pathologies in which cytokines specifically or such modified arginine residues generally have a role. The present invention therefore provides a method of identifying a compound suitable for use in the treatment or prevention of a medical condition arising from the synthesis or secretion of a cytokine, which method comprises: (a) stimulating cells with a cytokme-synthesising or secreting amount of a protein modified with an α- oxoaldehyde (b) measuring the binding of the modified protein to the cells (c) repeating steps (a) and (b) m the presence of a test compound (d) comparing the binding of the modified protein resulting from steps (b) and (c) (e) identifying the test compound in cases where the binding resulting from step (b) is greater than that from step (c) . The present invention therefore further provides an antagonist to an α-oxoaldehyde-modified arginine residue. In particular, the present invention provides an antagonist to a glyoxal or methylglyoxal-modified arginine residue. Especially, the present invention provides an antagonist to a protein which comprises an arginine residue modified by an α-oxoaldehyde and which - - protein either (a) binds to receptors on phagocytes and/or (b) provides the pharmacophore which triggers the synthesis and/or secretion of cytokines. Chem. Abs . 115 8662f discloses certain lminohydantoms of formula (A) as being tested for anticonvulsant activity: wherein R ϋ and R are each independently H or CH3; R^° is H or, when R°=R 10 =H, COOCH 2 Ph; R 30 is H or Ph; and R 40 is H, Ph, CH 2 Ph or CH 2 CH(CH 3 ) 2 ; although when R°=R 10 =R 20 =H and R 30 =R 40 =Ph, the compound failed to show any significant activity. (Ph = phenyl). In particular, the present invention provides an antagonist to an α-oxoaldehyde-modi f ied arginine residue of formula (B): wherein R is as defined above. Preferably R is 2,3,4- trihydroxybutyl , H or methyl, more preferably H or methyl. Especially preferred is when R is methyl, namely, a 2-amιno-5- ( 2-amιno-5-hydro-5-methylimιdazol- 4-on-2-yl) pentanoic acid residue or an N § -( 5-hydro-5- methyl-4-imidazolon-2-yl ) ornithine residue. Examples of such antagonists are 2 , 5-substituted-2-amino-5-hydro-imidazol-4-ones . Therefore, the present invention further provides a 2, 5-substituted-2-amino-5-hydro-imidazol-4-one other than those of formula (A) above. In particular, the present invention provides a compound of formula (I): wherein R- | is an aryl or alkyl group; R 2 is a hydrogen atom; and R3 and R4 are each independently selected from a hydrogen atom, and aryl and alkyl groups; including salts, esters or hydrated forms thereof, provided that when R^ is methyl, then either R3 is other than H or Ph, or R^ is other than H, Ph, CH 2 Ph or CH 2 CH(CH 3 ) 2 . Herein, "aryl" groups may be substituted or unsubstituted and are preferably phenyl or phenylalkyl such as benzyl or phenylethyl. "Alkyl" groups may be substituted or unsubstituted, straight or branched chain or cyclic and are preferably C- j _g alkyl, more preferably Cη_4 alkyl such as methyl, ethyl, n- or iso- propyl, n- , iso- or sec-butyl, cyclopropyl or cyclobutyl. The groups are preferably unsubstituted or substituted by mildly hydrophobic radicals such as halo such as bromo. For example, aryl groups include p- bromophenyl or fluorophenyl. Alternatively, the groups may be substituted by amino, acylamino such as alkylcarbonylamino, carboxyl or alkyloxycarbonyl . For example, alkyl groups include 4-ace tarn 1 do- 4- alkoxycarbonyl butyl groups. Preferably, only one or two of Rι_4 is or are aryl. More preferably, R3 corresponds to R as defined with respect to the α-oxoaldehyde and R4 is H. The compounds of formula (I) can exist in three isomeric forms, each of which is encompassed by the present invention. (IA) (IB) (IC) The compounds of formula (I) may be prepared by reacting the corresponding guanidino derivative with the corresponding α-oxoaldehyde of formula RCOCHO (wherein R corresponds to R3 in formula (I)), by a method analgous to that described by Henle et. al . in Z. Lebensm. Unters . Forsch. 199 55-58 (1994) or Kwon et . aJ in J. Med. Chem. 3_4, 1845-9 (1991 ). For example, 2- aminoalkyl-5-hydro-5-alkylιmιdazol-4-ones are prepared by the reaction of N-substituted guanidme derivatives R 1 NH-C( =NH )NH 2 with glyoxal derivatives R3COCHO in phosphate buffer, pH 7.4, at ambient or physiological temperature (37°C), and fractional crystallisation or chromatographic separation of the major desired product from the minor 1-N-alkyl isomer. - - Ma j or Minor 2-Amιnoalkyl-5, 5-dialkyliιnidazol -4-ones are prepared by the reaction of N-substituted guanidine derivatives R 1 NH-C(=NH)NH 2 with α, β-diketones R4COCOR3 in alcoholic potassium hydroxide solution under reflux, and fractional crystallisation or chromatographic separation of the major desired product from the minor 1-N-alkyl isomer. The formation of the α-oxoaldehydes glyoxal, methylglyoxal and 3-deoxyglucosone is increased in diabetes mellitus and has been linked to the development of chronic pathologies in diabetes (such as neuropathy, retinopathy, nephropathy) and embryonic malformations in diabetic pregnancy. The formation of glyoxal is also increased in alcohol intoxication and the formation of methylglyoxal is also increased in starvation. Methylglyoxal-modif ied proteins are also present in amyloid deposits associated with chronic renal dialysis and central neuropathies such as Alzheimer's disease. Methylglyoxal-modif ied proteins are normally degraded and modified arginine residues excreted in the urine; these may, however, accumulate in renal insufficiency. Diseases or pathologies m which the compounds or antagonists of the present invention are particularly useful are therefore: cataract, retinopathy, peripheral neuropathy, nephropathy and embryopathy in diabetes mellitus, coronary heart disease, chronic renal insufficiency, central neuropathies such as Alzheimer's disease, hepatitis and dementia associated with chronic alcohol intoxication, and inflammation and wasting associated with anorexia. More specifically, the increased formation and secretion of the cytokines IL- 1β, M-CSF and TNFα by monocytes/ macrophages and endothelial cells in response to modified arginine residues of α-oxoaldehyde-modif ied proteins m . vivo is expected to have a role in, for example: atherosclerosis associated with macrovascular disease; glomerulosclerosis associated with nephropathy; microang I opathy associated with retinopathy and peripheral neuropathy; and cytokine-mediated cytotoxicity associated with hepatitis and central neuropathies . The present invention further provides an antagonist as described hereinbefore, in particular, a 2, 5-substιtuted-2-amιno-5-hydroιmιdazol-4-one including those of formula (A) above for use in the treatment or prevention of any of the pathologies or diseases mentioned above. In particular, there is provided an antagonist as described hereinbefore, especially a compound of formula (I), including those also of formula (A), for treating patients with diabetes mellitus and/or Alzheimer's disease. Furthermore, the present invention provides the use of an antagonist as described hereinbefore, such as - a compound of formula (I), including those also of formula (A), for the preparation of a medicament for treating or preventing those pathologies or diseases. The present invention therefore also provides a pharmaceutical formulation comprising an antagonist as described hereinbefore, such as a compound of formula (I), in association with a pharmaceutically acceptable carrier therefor and the use of such formulations in the treatment or prevention of the aforementioned pathologies or diseases. Suitable formulations are well-known to those skilled m the art and include: tablets, capsules, mjectables (whether for intravenous, intramuscular, intraperitoneal or transdermal administration, or the like), suspensions, solutions, creams, powders, and the like. Preferably, the formulation is suitable for oral administration such as tablets and capsules, or for injection such as intravenous or intramuscular injection. The present invention therefore further provides a method for preparing a pharmaceutical formulation which method comprises bringing into intimate physical admixture an antagonist as described hereinbefore such as a compound of formula (I) together with a pharmaceutically acceptable carrier therefor. Suitable doses can be determined by those skilled in the art and depend upon the particular compound to be administered, the condition to be treated and the patient concerned. However, a suitable dose may fall in the range 0.1-1 mg/kg per dose, preferably administered in from one to four doses per week. The present invention will now be illustrated by reference to the following, by way of example only, in which: HSA = human serum albumin MG mιn -HSA = HSA minimally-modified with methylglyoxal - - M 9mm- pr human prothrombm minimally-modified with methylglyoxal MG Tr human transferrin minimally-modified with methylglyoxal RPMI Roswell Park Memorial Institute M-CSF macrophage colony stimulating factor TNFα tumour necrosis factor α IL-lβ ιnterleukm-1 β AGE mιn- HSA HSA minimally-modified with advanced glycation end products ELISA Enzyme-Linked Immunosorbent Assay - - Examp l e 1 : Prepa ra t i on and ch arac t er i sa t i on o f N g - acetyl-N δ - ( 5-hydro-5-methy l ιmιdazol-4-on-2-yl ) orm thine N α -Acetyl-Nδ-(5-hydro-5-methyl-4-ιmιdazolorr-2-yl)ornιthιne N α -acetyl-Ng-( 5-hydro-5-methylιmιdazol-4-on-2-yl ) ornithine was prepared by incubation of N Q - acetylarginme (16.7 mM) and methylglyoxal (20 mM) in sodium phosphate buffer (200 mM, pH 7.4) for 7 days at 37°C, then lyophilized to dryness . The residue was extracted with methanol (50ml ) ; the methanol was removed 1n vacuo and the residual solid product purified by reversed phase HPLC . The column was Nova- Pak™ octadecylsilica (ODS) cartridge (2.5 cm x 10 cm) fitted with a pre-column (2.5 cm x 1 cm) in a Waters 25 x 10 radial compression unit. The mobile phase was 500 mM potassium phosphate buffer, pH 7.4, the flow rate 9.9 ml/nun, the sample was 10 mg in 0.1 ml of mobile phase, and the eluate absorbance was monitored at 225 nm. A major peak was collected of retention time 5.7 mm, lyophilized, extracted with methanol ( 1 x 40 ml; 2 x 20 ml) and the methanol removed ι_n vacuo to yield the product. 1 H NMR(6 H ppm & J Hχ H Hz; D 2 0) : acetyl Me 2.00; - - ornithinyl 2-H 4.12, 3-H and 4-H(4H) 1.50-1.90(m) , 5- H(2H) 3.24 (t; J - 5.5; A,B) 3.32 ( t ; J = 5.8 ; C ) ; lmidazolone 5-methyl 1.32(d; J = 6.81 ), 5-H 4.14(m). 13 C NMR(6 C ppm; D 2 0) : acetyl Me 21.2, CO 160.3; ornithinyl C-1 172.9, C-2 54.1, C-3 28.1 , C-4 24.1 (A) 24.0(B) 24.9(C) , C-5 40.3(A) 40.1 (B) 41.2(C) ; lmidazolone C-2 154.5(A) 167.5(B,C), C-4 178.3(A) 178.0(B,C), C-5 56.6(A) 55.9(B) 51.6(C), 5-Me 15.8(A,C) 17.2(B). A,B and C relate 6 and J values to tautomeric forms shown above. FAB mass spectrometry gave an M-r1 peak of m/z = 271. The purity of the N α -acetyl-Ng-(5- hydro-5-methy1 lmidazol-4-on-2-y1 ) ornithine was assessed by analytical reversed phase HPLC (ODS cartridge 0.8 cm x 10 cm with pre-column in a Waters 8 x 10 radial compression unit, mobile phase 20 mM trifluoroacetic acid with 0.50% methanol from 5-25 min, flow rate 2 ml/mm, injection volume 10 μl, eluate absorbance 225 nm). The retention time was 3.9 mm. Example 2 (Reference): Preparation of 2-amιnomethyl- 5 , 5-dιphenylimidazol-4-one A mixture of methylguanidine hydrochloride (2.19g, 0.02 mol) with benzil (4.20g, 0.02 mol) in 40 ml of ethanol and potassium hydroxide (1.68g, 0.03 mol) in 3 ml of water was refluxed for 3.5 h. After cooling, the mixture was evaporated m . vacuo to near dryness . The resulting semi-solid residue was mixed with water (25 ml) and methylene chloride (25 ml) whereupon white precipitate was formed between the aqueous and organic layers. The precipitate was filtered and crystallized from hot methanol to give the product: mp. 324-326 °C, TLC (MeOH/CH 2 Cl 2 ) , 1 H NMR (DMSO-d 6 ) 6 7.30,S, 10H, ArH, δ 2.61 ,s,3H,NCH 3 ) . Example 3: Preparation of 2-Aminoethyl-5-hydro-5- methylιmidazol-4-one C Ethylguanidine sulphate (1 mmol, 272 mg) and methylglyoxal (1 mmol, 72 mg ) were dissolved in 1 OOmM sodium phosphate buffer, pH 7.4 (10 ml), and stirred at room temperature ( 18°C) for 4 days and a further 1 day at 37°C. The product mixture was lyophilised and the residual solid extracted with methanol (4 x 20 ml). Methanol was removed from the combined extracts and the solid dried to constant weight in a vacuum dessicator. H NMR spectrum of 2-ami noe t h y 1 - 5 -hy dro- 5 - methyl ιmidazol-4-one : δ H ppm (J Hz, integral) 2- aminoethyl: CH 3 - 1.18 (6.8, 3H), A,B and C; CH 2 - 3.20 (6.8, 2H), A,B and C; imidazolonyl 5-hydro: 3.71 (6.9, 0.7H), A and B; imidazolonyl 5-hydro: 3.99 (6.9, 0.3H), C; 5-methyl: 1.40 (6.9, 0.9H), A and B, 1.43 (6.9, 2.1H) . - Example 4: Preparation of 2-Amιnoethyl-5-hydro-5- phenylimidazol -4 -one C Ethylguanidine sulphate ( 1 mmol, 272mg) and pheny lgl y oxa 1 monohydrate (1 mmol, 152 mg ) were dissolved in 50mM sodium phosphate buffer, pH 7.4 , in 50% methanol (10 ml), and stirred at room temperature (18°C) for 4 days and a further 1 day at 37°C. The product was lyophilised and the residual solid extracted with methanol (4 x 20 ml) . Methanol was removed from the combined extracts and the solid dried to constant weight in a vacuum dessicator. H NMR spectrum of 2-aminoethyl-5-hydro-5-phenylimidazol-4- one : δ H ppm (J Hz) 2-amιnoethyl : CH3- 1.12 (7.3, 3H), A,B and C; CH 2 -3.14 (7.3, 2H), A,B and C; imidazolonyl 5-hydro: 3.30, A and B (0.6H); imidazolonyl 5-hydro: 3.48 (0.4H), C; 5-phenyl: 7.0 - 7.5 (5H), A,B and C. - Example 5: Preparation of 4- ( N , 2-Amino-5-hvdro-5- methylimidazol-4-on-2-yI )benzoic acid C 4-Guanidinobenzoic acid hydrochloride (1 mmol, 216 mg ) and methylglyoxal (1 mmol; 72 mg ) were dissolved in 50mM sodium phosphate buffer, pH 7.4 in 50% methanol (10 ml), and stirred at room temperature (18°C) for 4 days and a further 1 day at 37°C. The product mixture was lyophilised and the residual solid extracted with methanol (4 x 20 ml). Methanol was removed from the combined extracts and the solid dried to constant weight in a vacuum dessicator. H NMR spectrum of 4— ( N, 2-amino-5-hydro-5-methylimidazol-4-on-2-yl Jbenzoic acid : J U ppm (H Hz) phenyl 2,6-H: 7.90 (I 2H), A,B and C; phenyl 3, 5-H: 7.60 (8.8, 2H) , A,B and C; imidazolonyl 5-hydro: 3.46 (1H), Λ,B and C; 5-methyl : 1.25, A,B and C (3H) . Examp l e 6 : Prepa ra t i on of 4- ( N , 2-am i no- 5-hydro-5- phenyl imidazol-4-on-2-yl ) benzoic acid 4-Guanidinobenzoic acid hydrochloride (1 mmol, 216 mg) and phenylglyoxal monohydrate (1 mmol, 152 mg ) were dissolved in 50 mM sodium phosphate buffer, pH 7.4, in 50% methanol (10 ml), and stirred at room temperature (18°C) for 4 days and a further 1 day at 37°C. The product mixture was lyophilised and the residual solid extracted with methanol (4 x 20 ml). Methanol was removed from the combined extracts and the solid dried to constant weight in a vacuum dessicator. H NMR spectrum of 4-(N, 2-amino-5-hydro-5-phenylimidazol- 4-on-2-yl )benzoic acid: δ H ppm (J Hz) benzoic acid, phenyl 2,6-H: 7.98 (8.8, 2H), A,B and C; phenyl 3,5-H: 7.29 (8.8; 2H), A,B and C; imidazolonyl 5-hydro: 3.87 (1H), A,B and C; 5-phenyl: 7.3-7.9 (5H), A,B and C. Example 7: Preparation of and Cytokine Induction by MG mιn= HSA MG mιn -HSA was prepared by incubation of HSA (100 μM ) in sodium phosphate buffer (100 mM at pH 7.4 and 37°C) with 0.5 mM high purity methylglyoxal for 24 hours, dialysed against ammonium bicarbonate buffer (30 mM at pH 7.9 and 4°C) and lyophilized to dryness. The extent of modification was determined by preparation of MG mιn -HSA with 2-[ C Jmethylglyoxal , with assay of specific radioactivity content in the dialyzed protein by scintillation counting and assay for protein by the Lowry method using the unmodified protein as standard. This indicated the presence of 2.4 ± 0.3 methylglyoxal residues per molecule of MG mιn - HSA. Analysis by der l va t l za t I on with 9, 10- phenanthrenequmone gave 22.6 ± 0.9 residual arginine residues (n = 12), indicating that there were 1.4 modified arginine residues per MG mιn -HSA molecule; ammo acid analysis gave 22.1 ± 1.9 residual arginine residues, indicating that there were 1.9 arginine residues per MG mιn -HSA molecule; and preparation of wi th [ 14 C] methylglyoxal indicated the presence of 2.4 ± 0.25 methylglyoxal residues per MG mιn -HSA molecule. Demonstration of Induction of secretion of macrophage colony stimulating factor (M-CSF), tumour necrosis factor α (TNFα) and ιnterleukιn-1 β (IL-1B) from mature MG mιn -HSA Human blood mononuclear nuclear cells were isolated by centrifugation on a Ficoll-Hypaque density gradient. The cells (2 x 10 6 cells, 1 ml) in RMPI 1640 with 5μg/ml polymyxm B were added to wells of 12-well plates and incubated for one hour at 37°C under aseptic conditions. Non-adherent cells were removed by washing in RPMI 1640 with 5 μg/ml polymyxm B. The cells were then stimulated with MG mιn -HSA glycated protein (20 μM) - - in RPMI 1640 with 5 μg/ml polymyxm B and 5% autologous plasma and incubated for 24 hours. M-CSF, TNFα and IL- 1β were then assayed in the extracellular medium by ELISA. Data in the bar charts in Figures 1 to 3 are mean ± SD of three determinations. These data show that the modified arginine residue MG m ^ n -HSA induces synthesis of the cytokines M-CSF, TNFα and TL-1 β. Example 8: Preparation of human prothrombin minimally- modified by methyl glyoxal (MG rn ^ n ~Pr) Human prothrombin minimally-modified by methylglyoxal (MG mιn -Pr) was prepared by incubation of human prothrombin (100 μM ) in sodium phosphate buffer (100 mM, pH 7.4 and 37°C) with 500 μM methylglyoxal for 24 h. The modified protein was then dialyzed against ammonium bicarbonate buffer (30 mM, pH 7.9 and 4°C) and lyophilized to dryness. Preparation of MG m ^ n -Pr with 2- [ C 1 me th y lgl yoxal indicated the presence of 4.1 ± 0.1 methylglyoxal moieties per molecule of MG min- pr ( n - 3 ) . Example 9: Preparation of human transferrin minimally- modified by methylglyoxal (Mq min -Tr) Human transferrin minimally-modified by methylglyoxal (MG m ^ n -Tr) was prepared by incubation of human transferrin (100 μM ) in sodium phosphate buffer (100 μM, pH 7.4 and 37°C) with 500 μM methylglyoxal for 24 h. The modified protein was then dialyzed against ammonium bicarbonate buffer (30 mM, pH 7.9 and 4°C) and lyophilized to dryness. Preparation of Mg min -Tr with 2- [ 14 C ]methy lglyoxal indicated the presence of 3.7 ± 0.5 methylglyoxal moieties per molecule of Mg min -Tr (n = 3). Example 10- Binding of human serum albumin modified by methylglyoxal (MG mιn -HSA) to human THP-1 monocvtic cells and displacement by Nα-Acety1-Nδ- ( 5-hydro-5- methyl-4-ιmιdazolon-2-y3 )ornιthine THP-1 cells (2 x IO 6 ) were incubated in a culture medium of RPMI 1640 with 3% (w/v) human serum albumin (HSA) with [ 125 I]MG rnιn -HSA at 4°C, total volume 100 μl. The cells were then sedimented by centrifugation (at 2,300g for 0.5 min); washed three times in the culture medium (1 ml); re-suspended in 100 μl of culture medium; transferred to plastic vials, and counted for radioactivity on an LKB 1260 Multigamma counter. MG rnιn -HSA was prepared according to the method of 1 or. Example 7 and was labelled with l iJ ι as described by Westwood et al in J. Biol. Chem 169, 32293-8 (1994). The graphs in Figures 4 to 6 show, respectively: 4. Time course of the binding of 250 nM MG mιn -HSA to THP-1 cells. Data are mean i SEM of 3 determinations. 5. Concentration dependence of the binding of MG rnιn -HSA to THP-1 cells after incubation for 60 mm at 4°C in the absence (P-) and presence (■) of 30 μM MG mιn -HSA (to correct for non-specific binding) 6. Competitive binding of MG mιn -HSA and Nα-acetyl-Nδ- ( 5-methyl-5-hydro-4-ιmιdazolon-2 yl)ornιthιne ("model ligand") to THP-1 cells. Example 11 : Inhibition of advanced glycated protein- induced ιnterleukm-1 β secretion of human monocvtic THP-1 cells in vitro by 2-amι noe t hy 1- 5-hydro- 5- methyl ιmιdazol-4-one and 4- ( N , 2-amιno-5-hydro-5- methylιmιdazol-4-on-2-yl )benzoιc acid Human serum albumin minimally-modified by methylglyoxal (MG mιn -HSA) was prepared by incubation of human serum albumin (6.6 mg/ml) in sodium phosphate buffer ( 100 mM, pH 7.4 and 37°C) with 0.5 mM methylglyoxal for 24 h. The solution of MG mιn -HSA was - - then dialyzed against ammonium bicarbonate buffer (30 mM, pH 7.9 and 4°C) and the modified protein solution lyophilized to dryness. THP-1 cells, 5 x 10 cells/ml, were incubated for 24 h in RPMI 1640 medium containing 20% foetal calf serum under an atmosphere of 5% C0 2 in air with 100% humidity with 20 μM MG mιn -HSA and 0-10 μM 2-amιnoethyl- 5-hydro-5-methylιmιdazol-4-one and 4- (N, 2-amino-5- hydro-5-methylιmιdazol-4-on-2-yl ) benzoic acid. The cells were then sedimented by centrifugation and the supernatant removed and assayed for IL-1 β by ELISA. The median inhibitory concentration EC Q values were: 2-amιnoethyl-5-hydro-5-methylιmidazol-4-one 0.37 μM and 4-(N, 2-amιno-5-hydro-5-methylιmidazol-4-on-2-yl)benzoic 2-Amιnoetyl-5-hydro-5-methylιmιdazol-4-one and 4- ( N, 2-amino-5-hydro-5-methylιmιdazol-4-on-2-yl ) benzoic acid had no significant toxicity to human monocytic THP-1 cells under these conditions in the concentration range tested (0-10 μM ) by the trypan blue exclusion test . Example 12: Inhibition of advanced glycated protein- induced macrophage-colony stimulating factor secretion of human monocytes in vitro by 2-aminoethyl-5-hydro-5- methy 1 imidazol-4-one and 4- ( N , 2-amιno-5~hydro-5- methylimidazol-4-on-2-yl )benzoιc acid Human monocytes were isolated from venous human blood (50 ml) collected with sterile acid-ci trate- dextrose anticoagulant. The red blood cells were allowed to sediment and the leukocytes collected from the leukocyte-rich plasma by centrif ugation (200g, 15 min). Leukocytes were re-suspended in RMPI 1640 medium (4 ml), layered on Ficoll-Hypaque 1077 (4 ml) and centrifuged (400g, 30 min). The mononuclear layer at the interface was removed, washed with RPMI 1640 and re-suspended in RPMI 1640 at a density of 2 x 10 6 cells/ml. Cell suspension (0.25 ml) was added to each well of a 96-well plate and incubated for 60 min when the monocytes had adhered to the well surface. Non- adherent cells were removed by washing with RPMI 1640, and the cells were finally immersed in RPMI 1640 containing 5% (v/v) autologous plasma, 20 μM MG m;Ln -HSA and 0-10 μM 2-amιnoethyl-5-hydro-5-methylιmιdazol-4-one and 4-(N, 2-amιno-5-hydro-5-methylιmιdazol-4-on-2-yl) benzoic acid, total volume 0.25 ml. The monocytes were incubated with MG mιn -HSA and compounds for 24 h. The concentration of M-CSF in the extracellular medium was assayed by removal of the culture medium from the wells, centrifugation of the medium (200g, 15 min) to sediment suspended cells and assy of the supernatant for M-CSF by ELISA. The median inhibitory cencentration EC^ Q values were 2-amιnoethyl- 5-hydro-methylιmιdazol-4-one 7.6 μM and 4-(N, 2-amιno-5- hydro-5-methylιmιdazol-4-on-2-tl)benzoιc acid 64 nM. 2-Amιnoethyl-5-hydro~5-methylιmιdazol-4-one and 4- (N, 2-amιno-5-hydro-5-methylιmιdazol-4-on-2-yl)benzoic acid had no significant toxicity to human monocytes under these conditions in the concentration range tested (0-10 μM) by the trypan blue exclusion test.

Claims: - CLAIMS 1. The use of a compound which is an antogomst to an α-oxoaldehyde-mod I f I ed arginine residue in the manufacture of a medicament for the treatment or prevention of a disease or pathology involving a cytokine. 2. The use according to claim 1, wherein the cytokine is selected from : nterleukιn-1 β ; macrophage colony stimulating [actor; tumour necrosis factor; and mixtures of these. 3. The use according to claim 1 or claim 2, characterised m that the compound is an antagonist to a glyoxal- or methylglyoxal-modif ied arginine residue. 4. The use according to any preceding claim, characterised in that the compound is an antagonist to an α-oxoaldehyde-modif ied arginine residue of formula (B): wherein R is an α-oxoaldehyde residue. 5. The use according to any preceding claim characterised in that the compound is selected from the class consisting of 2 , 5-subs t l tu ted-2-amino- 5- hydroιmιdazol-4-ones . 6. The use according to claim 5 characterised in that the compound is selected from: 2-aminomethyl-5, 5-diρhenylimιdazol-4-one; N a -acetyl-N 6 - ( 5-hydro-5-methy lιmιdazol-4-on-2-y 1 ) orni thine ; 2-amιnoethy 1- 5-hydro- 5-me thy lιmιdazol-4-one; 2-amιnoethyl-5-hydro-5-phenylιmιdazol-4-one; 4-(N, 2-amιno-5-hydro-5-methylιmιdazol-4-on-2-yl )benzoιc acid; and 4-(N, 2-amιno-5-hydro-5-phenylιmιdazol-4-on-2-yl )benzoιc 7. A compound for use m the treatment or prevention of a disease or pathology involving cytokine production, characterised in that the compound is an antagonist to an α-oxoaldehyde-mod I fi ed arginine residue provided that the compound is other than an lminohydantom of formula (A) : 8. A compound according to claim 7, characterised in that the compound is an antagonist to a glyoxal- or methylglyoxal-modif ied arginine residue. 9. A compound according to any of claims 7 to 9 characterised m that the compound is an antagonist to an α-oxoaldehyde-modif ied arginine residue of formula (B): wherein R is an α-oxoaldehyde residue. 10. A compound according to any preceding claim - - selected from the class consisting of 2, 5-substituted-2-amino-5-hydroimidazol-4-ones . 11. A compound of formula (I) : wherein R- ] is an aryl or alkyl group; R 2 is a hydrogen atom; and R3 and R4 are each independently selected from a hydrogen atom, and aryl and alkyl groups; including salts, esters or hydrated forms thereof, provided that when R- | is methyl, then either R3 is other than H or Ph, or R 4 is other than H, Ph, CH 2 Ph or CH 2 CH(CH 3 ) 2 . 12. A compound according to claim 11, wherein one or two of R- | , R 2 , R3 or R4 is or are aryl. 13. A compound according to claim 11 or claim 12 wherein R corresponds to an α-oxoaldehyde residue (R) 14. A compound according to any of claims 11 to 13 wherein R- j is C- ] _^alkyl, phenyl or phenyl C- ] _g alkyl, each of which may be optionally substituted by one or more of carboxyl, alkoxy carbony 1 , amino or alkylcarbonylamino . 15. A compound according to claim 11 selected from: N α -acetyl-N Q -( 5-hydro-5-methylimidazol-4-on-2-yl ) ornithine 2-aminoethyl-5-hydro-5-methylimidazol-4-one 2-aminoethyl-5-hydro-5-phenylimidazol-4-one 4-(N, 2-amino-5-hydro-5-methylimidazol-4-on-2-yl)benzoic acid 4-(N, 2-amino-5-hydro-5-phenylimidazol-4-on-2-yl)benzoic 16. The use of a compound according to any preceding claim in the treatment or prevention of: cataract, retinopathy, peripheral neuropathy, nephropathy or embryopathy, coronary heart disease, chronic renal insufficiency, Alzheimer' s disease, hepatitis, dementia, inflammation or wasting associated with anorexia, atherosclerosis, glomerulosclerosi s , microangiopathy or cytokine-mediated cytotoxicity . 17. The use of a compound according to any of claims 7 to 15 for the preparation of a medicament suitable for use according to any of claims 1 to 6 or 16. 18. A pharmaceutical formulation comprising a compound according to any of claims 7 to 15 in association with a pharmaceutically acceptable carrier therefor. 19. A method of identifying a compound suitable for use in the treatment or prevention of a medical condition arising from the synthesis or secretion of cytokine, which method comprises: (a) stimulating cells with a cytokine-synthesismg or secreting amount of a protein modified with an α- oxoaldehyde (b) measuring the binding of the modified protein to the cells (c) repeating steps (a) and (b) in the presence of a test compound (d) comparing the binding of the modified protein resulting from steps (b) and (c) (e) identifying the test compound in cases where the binding resulting from step (b) is greater than that from step (c) . 20. A compound or ligand for use in a method according to claim 19, which compound or ligand is a protein comprising an α-oxoaldehyde-modif ied arginine residue of formula (B): wherein R is an α-oxoaldehyde residue. 21. A compound according to claim 20 which is selected from MG min -HSA; MG mιn -Pr; and MG min -Tr, as defined hereinbefore. 22. A method of preparing a compound of formula (I ) as defined in any of claims 11 to 15, which method comprises : reacting the corresponding N-substituted guanidine derivative of formula R-,NH-C(=NH)NH 2 (a) with the corresponding glyoxal derivative R3COCHO; or (b) with the corresponding α,β-diketone of formula R 4 COCOR 3 wherein R- | , R3 and R4 are as defined in formula (I). 23. A method for the treatment or prevention of a disease or pathology involving a cytokine, which method comprises administration to a patient in need thereof of a non-toxic, effective amount of an antagonist to an α-oxoaldehyde-arginine residue. 24. A method according to claim 23 wherein the antagonist antagonises a residue of formula (B): wherein R is an α-oxoaldehyde residue. 25. A method for the treatment or prevention of a disease or pathology involving a cytokine, which method comprises administration to a patient in need thereof of a non-toxic, effective amount of a compound of formula ( I ) : wherein R- | is an aryl or alkyl group; R 2 is a hydrogen atom; and R3 and R4 are each independently selected from a hydrogen atom, and aryl and alkyl groups; including salts, esters or hydrated forms thereof.

Inventors: THORNALLEY PAUL, THORNALLEY, PAUL
Applicants: THORNALLEY PAUL, WIVENHOE TECHN LTD, THORNALLEY, PAUL, WIVENHOE TECHNOLOGY LIMITED
Application year: 1997
Main classification: C07D233
IPC codes: A61K31, A61P1, A61P3, A61P9, A61P13, A61P25, A61P27, C07D233