Abstract:
A method of inhibiting TNFα or IL-1β expression with 5-hydroxymethyl furfural and its derivatives. Also disclosed is a method of treating TNFα or IL-1β related disorders using these compounds.

Description:
RELATED APPLICATION  
       [0001]     Pursuant to 35 U.S.C. § 119(e), this application claims the benefit of prior U.S. provisional application 60/499,258, filed Aug. 29, 2003. 
     
    
     BACKGROUND  
       [0002]     Tumor Necrosis Factor alpha (TNFα), a mononuclear cytokine, is predominantly produced by monocytes and macrophages. It possesses various biological activities: (1) killing cancer cells or inhibiting growth of cancer cells, (2) enhancing phagocytosis of neutrophilic granulocyte, (3) killing infectious pathogens, and (4) increasing expression of adhesion molecules on vascular endothelial cells during inflammatory responses.  
         [0003]     Disorders related to expression of TNFα include, but are not limited to, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, spondyloarthropathies, inflammatory bowel disease (including Crohn&#39;s disease and ulcerative colitis), chronic heart failure, systemic lupus erythematosus, scleroderma, sarcoidosis, polymyositis/dermatomyositis, psoriasis, multiple myeloma, myelodysplastic syndrome, acute myelogenous leukemia, Parkinson&#39;s disease, AIDS dementia complex, Alzheimer&#39;s disease, depression, sepsis, pyoderma gangrenosum, hematosepsis, septic shock, Behcet&#39;s syndrome, graft-versus-host disease, uveitis, Wegener&#39;s granulomatosis, Sjogren&#39;s syndrome, chronic obstructive pulmonary disease, asthma, acute pancreatitis, periodontal disease, cachexia, cancer, central nervous system injury, viral respiratory disease, and obesity. See, e.g., Ogata H. et al  Curr Pharm Des.  2003; 9(14): 1107-13; Moller D. R. et al  J Intern Med.  2003; 253(1): 31-40; Taylor P. C. et al  Curr Pharm Des.  2003; 9(14): 1095-106; Wilkinson N. et al  Arch Dis Child.  2003; 88(3): 186-91; Nishimura F. et al  J Periodontol.  2003; 74(1): 97-102; Weinberg J. M. et al  Cutis.  2003; 71(1): 41-5; Burnham E. et al  Crit Care Med.  2001; 29(3): 690-1; Sack M. et al  Pharmacol Ther.  2002; 94(1-2): 123-35; Barnes P. J. et al  Annu Rev Pharmacol Toxicol.  2002; 42:81-98; Mageed R. A. et al  Lupus  2002; 11 (12): 850-5; Tsimberidou A. M. et al  Expert Rev Anticancer Ther.  2002; 2(3): 277-86; Muller T. et al  Curr Opin Investig Drugs.  2002; 3(12): 1763-7; Calandra T. et al  Curr Clin Top Infect Dis.  2002; 22:1-23; Girolomoni G et al  Curr Opin Investig Drugs.  2002; 3(11): 1590-5; Tutuncu Z. et al  Clin Exp Rheumatol.  2002; 20(6 Suppl 28): S146-51; Braun J. et al  Best Pract Res Clin Rheumatol.  2002; 16(4): 631-51; Barnes P. J. et al  Novartis Found Symp.  2001; 234:255-67; discussion 267-72; Brady M. et al  Baillieres Best Pract Res Clin Gastroenterol.  1999; 13(2): 265-89; Goldring M. B. et al  Expert Opin Biol Ther.  2001; 1(5): 817-29; Mariette X.  Rev Prat.  2003; 53(5): 507-11; Sharma R. et al  Int J Cardiol.  2002; 85(1): 161-71; Wang C. X. et al  Prog Neurobiol.  2002; 67(2): 161-72; Van Reeth K. et al  Vet Immunol Immunopathol.  2002; 87(3-4): 161-8; Leonard B. E. et al  Int J Dev Neurosci.  2001; 19(3): 305-12; and Hays S. J. et al  Curr Pharm Des.  1998; 4(4): 335-48.  
         [0004]     Interleukin-1 beta (IL-1β), a cytokine secreted by cells such as monocytes, macrophages and dendritic cells, mediates a wide range of immune and inflammatory responses. One can modulate IL-1β production to treat a variety of disorders, such as rheumatoid arthritis, hematosepsis, periodontal disease, chronic heart failure, polymyositis/dermatomyositis, acute pancreatitis, chronic obstructive pulmonary disease, Alzheimer&#39;s disease, osteoarthritis, multiple myeloma, myelodysplastic syndrome, uveitis, central nervous system injury, viral respiratory disease, asthma, depression, and scleroderma. See, e.g., Taylor P. C. et al  Curr Pharm Des.  2003; 9(14): 1095-106; Dellinger R. P. et al  Clin Infect Dis.  2003; 36(10): 1259-65; Takashiba S. et al  J Periodontol.  2003; 74(1): 103-10; Diwan A. et al  Curr Mol Med.  2003; 3(2): 161-82; Lundberg I. E. et al  Rheum Dis Clin North Am.  2002; 28(4): 799-822; Makhija R. et al  J Hepatobiliary Pancreat Surg.  2002; 9(4): 401-10; Chung K. F. et al  Eur Respir J Suppl.  2001; 34:50s-59s; Hallegua D. S. et al  Ann Rheum Dis.  2002; 61(11): 960-7; Goldring M. B. et al  Expert Opin Biol Ther.  2001; 1(5): 817-29; Mrak R. E. et al  Neurobiol Aging.  2001; 22(6): 903-8; Brady M. et al  Baillieres Best Pract Res Clin Gastroenterol.  1999; 13(2): 265-89; Van der Meer J. W. et al  Ann N Y Acad Sci.  1998; 856:243-51; Rameshwar P. et al  Acta Haematol.  2003; 109(1): 1-10; de Kozak Y et al  Int Rev Immunol.  2002; 21(2-3): 231-53; Wang C. X. et al  Prog Neurobiol.  2002; 67(2): 161-72; Van Reeth K. et al  Vet Immunol Immunopathol.  2002; 87(3-4): 161-8; Stirling R. G et al  Br Med Bull.  2000; 56(4): 1037-53; Leonard B. E. et al  Int J Dev Neurosci.  2001; 19(3): 305-12; Allan S. M. et al  Ann N Y Acad Sci.  2000; 917:84-93; and Cafagna D. et al  Minerva Med.  1998; 89(5): 153-61.  
       SUMMARY  
       [0005]     This invention is based on the discovery that 5-(hydroxymethyl)furfural (5-HMF), unexpectedly, inhibited expression of both TNFα and IL-1β.  
         [0006]     Thus, an aspect of this invention relates to a method of inhibiting expression of TNF-α or IL-1β in a subject in need thereof. The method includes administering to the subject an effective amount of a furan compound of the following formula:  
                         
 
 wherein X is O or S; R 1  is —R, —C(O)H, —C(O)R, —C(O)OH, —C(O)OR, —RC(O)H, —RC(O)R′, —ROH, —ROR′, or —ROC(O)R′, and each of R 2 , R 3 , and R 4 , independently, is H, amino, halo, —R, —C(O)H, —C(O)R, —C(O)OH, —C(O)OR, —RC(O)H, —RC(O)R′, —ROH, —ROR′, or —ROC(O)R′, each of R and R′ independently being C 1-5  alkyl. 
 
         [0007]     The term “alkyl” refers to a straight or branched hydrocarbon, containing an indicated number of carbon atoms. Examples of alkyl groups include, but are not limited to, methyl, mehylene, ethyl, ethylene, n-propyl, i-propyl, n-butyl, i-butyl, and t-butyl.  
         [0008]     Shown below are examples of the above-described furan compounds:  
                         
 
         [0009]     Another aspect of this invention relates to a method for treating a TNFα-related disorder or an IL-1β-related disorder including administering to a subject in need thereof an effective amount of one of the above-described furan compounds.  
         [0010]     Examples of the TNFα-related disorders includes, but are not limited to, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, spondyloarthropathies, inflammatory bowel disease (including Crohn&#39;s disease and ulcerative colitis), chronic heart failure, diabetes mellitus, lupus (e.g., systemic lupus erythematosus), scleroderma, sarcoidosis, polymyositis/dermatomyositis, psoriasis, multiple myeloma, myelodysplastic syndrome, acute myelogenous leukemia, Parkinson&#39;s disease, AIDS dementia complex, Alzheimer&#39;s disease, depression, sepsis, pyoderma gangrenosum, hematosepsis, septic shock, Behcet&#39;s syndrome, graft-versus-host disease, uveitis, Wegener&#39;s granulomatosis, Sjogren&#39;s syndrome, chronic obstructive pulmonary disease, asthma, acute pancreatitis, periodontal disease, cachexia, cancer, central nervous system injury, viral respiratory disease, or obesity. In one embodiment, the TNFα-related disease is lupus, Crohn&#39;s disease, and psoriasis.  
         [0011]     Examples of the IL-1β related disorders include, but are limited to, rheumatoid arthritis, hematosepsis, periodontal disease, chronic heart failure, polymyositis/dermatomyositis, acute pancreatitis, chronic obstructive pulmonary disease, Alzheimer&#39;s disease, osteoarthritis, bacterial infections, multiple myeloma, myelodysplastic syndrome, uveitis, central nervous system injury, viral respiratory disease, asthma, depression, and scleroderma.  
         [0012]     A TNFα-related disorder and an IL-1β-related disorder may be induced by over-expression of TNFα and of IL-1β, respectively.  
         [0013]     Also within the scope of this invention is a pharmaceutical composition containing an effective amount of one of the above-described furan compounds and a pharmaceutically active carrier, as well as the use of such a composition for the manufacture of a medicament for treating a TNFα-related disorder or an IL-1β-related disorder.  
         [0014]     The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description, and from the claims.  
     
    
     DETAILED DESCRIPTION  
       [0015]     This invention includes methods of inhibiting expression of TNFα or IL-1β, treating a TNFα-related disorder, and treating an IL-1β-related disorder with an effective amount of one of the above-described furan compounds. The term “an effective amount” refers to the amount of the furan compound which is required to confer therapeutic effect in a subject. Effective amounts may vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and optional co-usage with another therapeutic agent. The term “treating” refers to administering a furan compound to a subject that has a TNFα-related disorder or an IL-1β-related disorder, or has a symptom of the disorder, or has a predisposition toward the disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptoms of the disorder, or the predisposition toward the disorder.  
         [0016]     Some of the furan compounds used in the above-described methods are naturally occurring. For example, 5-HMF can be isolated from  Cornus Officinalis , a medicinal plant (Xu L. et al  Chinese herb medicine  1995, 26(2) 62˜65). Others can be prepared by modification of the naturally occurring compounds or by total synthesis, using chemical transformations and protecting group methodologies (protection and deprotection) well known in the art. See, e.g., Larock R.  Comprehensive Organic Transformations , VCH Publishers, 1989; T. W. Greene and Wuts P. G. M.  Protective Groups in Organic Synthesis,  3 rd  Ed., John Wiley and Sons, 1999; Fieser L. and Fieser M.,  Fieser and Fieser&#39;s Reagents for Organic Synthesis , John Wiley and Sons, 1994; and Paquette L. ed.,  Encyclopedia of Reagents for Organic Synthesis , John Wiley and Sons, 1995 and subsequent editions thereof.  
         [0017]     To practice one of the above-described methods, one administers to a subject in need thereof orally, rectally, parenterally, by inhalation spray, or via an implanted reservoir a composition that is either one of the above-described furan compounds alone or a mixture of the furan compound and a pharmaceutically acceptable carrier. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.  
         [0018]     An oral composition can be any orally acceptable dosage form including, but not limited to, tablets, capsules, emulsions and aqueous suspensions, dispersions and solutions. Commonly used carriers for tablets include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added to tablets. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added.  
         [0019]     A sterile injectable composition (e.g., aqueous or oleaginous suspension) can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation can 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 vehicles and solvents that can be employed are mannitol, water, Ringer&#39;s solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or di-glycerides). Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or similar dispersing agents.  
         [0020]     An inhalation composition can be prepared according to techniques well known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.  
         [0021]     A topical composition can be formulated in form of oil, cream, lotion, ointment and the like. Suitable carriers for the composition include vegetable or mineral oils, white petrolatum (white soft paraffin), branched chain fats or oils, animal fats and high molecular weight alcohols (e.g., containing more than 12 carbon atoms). The preferred carriers are those in which the active ingredient is soluble. Emulsifiers, stabilizers, humectants and antioxidants may also be included as well as agents imparting color or fragrance, if desired. Additionally, transdermal penetration enhancers may be employed in these topical formulations. Examples of such enhancers can be found in U.S. Pat. Nos. 3,989,816 and 4,444,762. Creams are preferably formulated from a mixture of mineral oil, self-emulsifying beeswax and water in which mixture the active ingredient, dissolved in a small amount of an oil, such as almond oil, is admixed. An example of such a cream is one which includes about 40 parts water, about 20 parts beeswax, about 40 parts mineral oil and about 1 part almond oil. Ointments may be formulated by mixing a solution of the active ingredient in a vegetable oil, such as almond oil, with warm soft paraffin and allowing the mixture to cool. An example of such an ointment is one which includes about 30% almond and about 70% white soft paraffin by weight.  
         [0022]     A carrier in a pharmaceutical composition must be “acceptable” in the sense of being compatible with the active ingredient of the formulation (and preferably, capable of stabilizing it) and not deleterious to the subject to be treated. For example, solubilizing agents, such as cyclodextrins can be utilized as pharmaceutical excipients for delivery of the active compounds. Examples of other carriers include colloidal silicon dioxide, magnesium stearate, cellulose, sodium lauryl sulfate, and D&amp;C Yellow # 10.  
         [0023]     A suitable in vitro assay can be used to preliminarily evaluate the efficacy of one of the above-described furan compounds in inhibiting expression of TNFα or IL-1β expression. The furan compound can further be examined for its efficacy in treating a TNFα related disorder or an IL-1β related disorder by in vivo assays. For example, it can be administered to an animal (e.g., a mouse model) having a TNFα or IL-1β related disorder and its therapeutic effect is then assessed. Based on the results, an appropriate dosage range and administration route can also be determined.  
         [0024]     Without further elaboration, it is believed that the above description has adequately enabled the present invention. The following specific examples are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All of the publications, including U.S. provisional application 60/499,258, cited herein are hereby incorporated by reference in their entirety.  
       EXAMPLE 1  
     Isolation and Identification of 5-HMF  
       [0025]    
       
                 
         
             
             
         
       
     
         [0026]     500 g of  Cornus Officinalis  fruit was suspended in a 0.36% HCl aqueous solution (5000 mL). The suspension was slightly boiled for 2 hrs and then filtered. The residue was extracted twice in the same manner described above. The filtrates were combined, concentrated, and charged onto an HP-20 column (6.5×100 mm), eluted with water, 10% ethanol, and 15% ethanol, respectively. The collected 15% ethanol eluant was concentrated and purified by a RP-18 column, eluted with water. The water eluant was concentrated and further purified by a Gilson semi-preparation HPLC instrument (Zorbax C18 column, 9.4×250 mm) with 8:92 CH 3 OH:H 2 O at a flow rate of 8 ml/min (λ=280 nm). Fractions were collected at the retention time of 5 mins and concentrated to afford 100 mg of 5-HMF (yielded 0.02%) as a light yellow to yellow-tan power.  
         [0027]     Boiling point: 110° C.; melting point 31.5° C.;  
         [0028]     UV (λ max , nm): 284, 230, 195;  
         [0029]     IR (cm −1 , KBr): 3350, 1673, 1522, 1399, 1193, 1024, 777;  
         [0030]      1 H NMR (600 MHz, D 2 O) δ: 4.60 (2H, s, H-9), 6.58 (1H, d, J=3.5 Hz, H-6), 7.43 (1H, d, J=3.8 Hz, H-8), 9.35 (1H, s, H-4);  
         [0031]      13 C NMR (100 MHz, D 2 O) δ: 58.5, 113.3, 129.2, 154.0, 163.5, 182.5; EIMS (m/z): 126 (M + ).  
       EXAMPLE 2  
     Inhibition of the Expression of TNFα and IL-1β 
       [0032]     Peripheral blood monocytes (PBMCs) were isolated from fresh blood using a Ficoll-Paque Plus reagent (Amersham Bioscience) according to the protocol recommended by the manufacturer. The cells were suspended in a RPMI 1640 medium containing 10% FBS at a concentration of 1×10 5  cells/ml and seeded in a 96-well plate (1×10 4  cells total in each well). Each reaction was carried out in three wells.  
         [0033]     10 μl of 5-HMF in DMSO was added to wells to obtain the final concentrations at 0.1, 0.3, 1, 3, 10, and 30 μg/ml. In a positive control, dexamethason (DEX), an anti-inflammatory agent, instead of 5-HMF, was added (final concentration 10 μM). In a positive control, 10 μl of the medium, instead of 5-HMF, was added. The plate was incubated at 37° C. under 5% CO 2  for 15 minutes. After 10 μl aliquots of 100 μg/ml of lipopolysaccharide (LPS) were added to all wells except for the negative control, the plate was incubated at 37° C. under 5% CO 2  overnight.  
         [0034]     The plate was spun at 1000 rpm for 15 minutes and the supernatants were collected. The concentrations of TNFα and IL-1β were measured by the TNFα ELISA (Enzyme Linked Immunosorbent Assay) Kit and IL1-β ELISA Kit (Jingmei Bioengineer Technology). The inhibition ratios of TNFα and IL1-β were calculated as follows and the results are shown in Tables 1 and 2: 
 
 TNF α Inhibition Ratio (%)=[ TNFα ] control   −[TNFα]   5-HMF   /[TNFα]   control ×100% 
 
 IL 1-β Inhibition Ratio (%)=[ IL 1-β] control   −[IL 1-β] 5-HMF   /[IL 1-β] control ×100% 
 
         [0035]     where [TNFα] 5-HMF  is the concentration of TNFα in PBMCs treated with 5-HMF and LPS, [TNFα] control  is the concentration of TNFα in PBMCs treated with LPS and the medium, [IL1-β] 5-HMF  is the concentration of IL1-β in PBMCs treated with 5-HMF and LPS, and [IL1-β] control  is the concentration of IL1-β in PBMCs treated with LPS and the medium.  
                                           TABLE 1                           Inhibition of TNFα expression by 5-HMF                Concentration           Final concentration of   of TNFα in   Inhibition ratio of production       5-HMF (μg/ml)   PBMCs (pg/ml)   of TNFα in PBMCs (%)                    3   124.4 ± 15.2    15.7 ± 10.3       10   107.9 ± 20.0    26.8 ± 13.6       30   82.9 ± 27.9   43.8 ± 18.9       100   39.3 ± 19.7   73.4 ± 13.4       300   0.3 ± 6.4   99.3 ± 4.3        LPS   147.5 ± 10.0    —       10 μM DEX   39.5 ± 9.0    73.2 ± 6.1                   
 
         [0036]    
       
         
               
             
               
               
               
             
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                   
               
               
                 Inhibition of IL-1β expression by 5-HMF 
               
             
          
           
               
                   
                 Concentration 
                   
               
               
                 Final concentration of 
                 of IL-1β in 
                 Inhibition ratio of production 
               
               
                 5-HMF (μg/ml) 
                 PBMCs (pg/ml) 
                 of IL-1β in PBMCs (%) 
               
               
                   
               
             
          
           
               
                 3 
                 69.9 ± 12.6 
                 40.2 ± 10.8 
               
               
                 10 
                 62.7 ± 10.5 
                 46.4 ± 9.0  
               
               
                 30 
                 40.1 ± 2.4  
                 65.7 ± 2.0  
               
               
                 100 
                 13.0 ± 3.7  
                 88.8 ± 3.1  
               
               
                 300 
                   0 ± 5.0 
                  100 ± 4.3  
               
               
                 LPS 
                 116.9 ± 28.5  
                 — 
               
               
                 10 μM DEX 
                 20.2 ± 7.0  
                 82.7 ± 6.0  
               
               
                   
               
             
          
         
       
     
         [0037]     The results show that 5-HMF inhibited expression of both TNFα and IL-1β production in a concentration-dependent manner. It was unexpected that 5-HMF completely inhibited expression of TNFα and IL-1β at the concentration of 300 μg/ml.  
       OTHER EMBODIMENTS  
       [0038]     A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are also within the scope of the following claims.  
         [0039]     The details one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.