Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4164—1,3-Diazoles
  • A61K31/4188—1,3-Diazoles condensed with other heterocyclic ring systems, e.g. biotin, sorbinil
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
  • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/4164—1,3-Diazoles
  • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
  • A61K31/425—Thiazoles
  • A61K31/426—1,3-Thiazoles
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
  • A61K31/438—The ring being spiro-condensed with carbocyclic or heterocyclic ring systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/50—Pyridazines; Hydrogenated pyridazines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/50—Pyridazines; Hydrogenated pyridazines
  • A61K31/502—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with carbocyclic ring systems, e.g. cinnoline, phthalazine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• Embodiments of this invention are related generally to physiology and medicine. More specifically, this invention is related to aldose reductase inhibitors (ARIs) and their use in treating chronic obstructive pulmonary disease (COPD).
• ARIs aldose reductase inhibitors
• COPD chronic obstructive pulmonary disease
• Aldose reductase catalyzes the reduction of a wide range of aldehydes (Bhatnager and Srivastava, Biochem Med Metab Biol. 48(2):91-121, 1992).
• the substrates of the enzyme range from aromatic and aliphatic aldehydes to aldoses such as glucose, galactose, and ribose.
• the reduction of glucose by AR is particularly significant during hyperglycemia and increased flux of glucose via AR has been etiologically linked to the development of secondary diabetic complications (Bhatnager and Srivastava, Biochem Med Metab Biol. 48(2):91-121, 1992; Yabe-Nishimura, Pharmacol Rev. 50(l):21-33, 1998).
• AR in contrast to its injurious role during diabetes, under normal glucose concentration, AR may be involved in protection against oxidative and electrophilic stress.
• the antioxidant role of AR is consistent with the observations that in a variety of cell types AR is upregulated by oxidants such as hydrogen peroxide (Spycher et al, FASEB J. 11(2): 181-8, 1997), lipid peroxidation-derived aldehydes (Ruef et al, Arterioscler Thromb Vase Biol. 20(7):1745-52, 2000; Rittner et al, J Clin Invest. 103(7): 1007-13, 1999), advanced glycosylation end products (Nakamura et al, Free Radio Biol Med.
• Inhibitors of aldose reductase have been indicated for some conditions and diseases, such as diabetes complications, ischemic damage to non-cardiac tissue, Huntington's disease. See U.S. Patents 6,696,407, 6,127,367, 6,380,200, which are all hereby incorporated by reference. In some cases, the role in which aldose reductase plays in mechanisms involved in the condition or disease is known. For example, in U.S. Patent 6,696,407 indicates that an aldose reductase inhibitors increase striatal ciliary neurotrophic factor (CNTF), which has ramifications for the treatment of Huntington's Disease. In other cases, however, the way in which aldose reductase or aldose reductase inhibitors work with respect to a particular disease or condition is not known.
• CNTF striatal ciliary neurotrophic factor
• Embodiments of the invention are directed to methods of treating chronic obstructive pulmonary disease (COPD) in a subject diagnosed with, exhibiting symptoms of, or at risk of developing COPD by administering a therapeutically effective amount of an aldose reductase inhibitor (ARI).
• COPD chronic obstructive pulmonary disease
• ARI aldose reductase inhibitor
• a "risk" of developing COPD is based on the subject's medical, personal, and/or family history.
• risk factors also include, but are not limited to exposure to smoke or other environmental hazards (e.g., mining or textile industry hazards, fumes, air pollution), genetic susceptibility, autoimmune disease, and bronchial hyperresponsiveness.
• a subject may also be one that exhibits one or more symptoms of COPD including, but not limited to: chronic cough, sputum production, dyspnea (shortness of breath), rhonchi (rattling breathing sounds), and airway limitation on pulmonary function testing.
• the aldose reductase inhibitor is administered to the patient as a prodrug.
• a prodrug is an inactive or less active form of a drug that is metabolized or converted in vivo to an active or more active form.
• the ARI can be administered by any route, including orally, endoscopically, intratracheally, intrabronchially, intravenously, intralesionally, intramuscularly, intraperitoneally, percutaneously, or subcutaneously.
• the ARI is administered orally or by inhalation or instillation, e.g. , by inhaler or other aerosol delivery devices.
• the aldose reductase inhibitor is a peptide, a peptide mimetic, a small molecule, or an inhibitory RNA.
• the aldose reductase inhibitor can be an siRNA or other inhibitory nucleic acid, a carboxylic acid, a hydantoin, a pyridazinone, or a pharmaceutically acceptable derivative thereof.
• the aldose reductase inhibitor is fidarestat, sorbinil, epalrestat, ponalrestat, methosorbinil, risarestat, imirestat, ALO-1567, quercetin, zopolrestat, AD-5467, NZ-314, M-16209, minalrestat, AS-3201, WP- 921, luteolin, tolrestat, EBPC, or a pharmaceutically acceptable derivative thereof.
• the aldose reductase inhibitor is fidarestat.
• treating includes treating a physiological cause of disease, treating a condition associated with the disease, and treating one or symptoms of the disease. Treating includes reducing the severity (including any measurable decrease to complete elimination), reducing the frequency, slowing or stopping the progression of, increasing the time until onset, or preventing the onset of one or more disease symptoms.
• FIG. 2 CSE-induced changes in cytokine levels in SAEC prevented by aldose reductase (AR) inhibition. *p ⁇ 0.01 Vs Control; **p ⁇ 0.05 Vs CSE 50%; #p ⁇ 0.001 Vs Control; ##p ⁇ 0.01 Vs CSE 50%
• FIG. 3 CSE-induced changes in chemokines and growth factors levels in SAEC prevented by AR inhibition. *p ⁇ 0.01 Vs Control; **p ⁇ 0.05 Vs CSE 50%; #p ⁇ 0.001 Vs Control; ##p ⁇ 0.01 Vs CSE 50%
• FIG. 4 Prevention of redox signaling by AR inhibition. Stimulation with Ragweed pollens, allergens, growth factors, or cytokines cause increased ROS production inside the cells.
• the ROS could oxidize membrane lipids to form lipid aldehydes such as 4- hydroxynonenal (FINE), which readily reacts with glutathione and form GS-conjugates such as GS-HNE.
• FINE 4- hydroxynonenal
• AR catalyzes the reduction of GS-HNE into GS-l,4-dihydroxynonene (GS- DHN).
• the latter is known to activate various downstream protein kinases such as PKC, PI3K and MAPK activating transcription factors NF- ⁇ and AP-1, which transcribes various inflammatory markers, including IL-13, which in paracrine fashion induces more ROS formation.
• the GS-DHN could also phosphorylate JAK-1 and MAPK such as ERK1/2, eventually phosphorylating and activating STAT-6, which translocate to the nucleus and transcribe genes including mucin genes - leading to goblet cell metaplasia.
• the continuously formed GS-DFTN may amplify the signaling loop by increased ROS production via PKC/NOX pathway.
• FIGs. 5A-5B Inhibition of AR prevents IL- 13 -induced reactive oxygen species (ROS) production in SAEC.
• A Approximately 5xl0 4 cells were seeded on 2-chambered slides and starved in serum- free basal medium without or with fidarestat overnight. The cells were washed with lx HBSS and incubated with 10 ⁇ H2DCF-DA at 37°C for 30min, washed again and treated with IL-13 (25 ng/ml) for lh. The cells were washed with cold lx HBSS twice and mounted using floursave mounting medium with DAPI. Photomicrographs were acquired using a fluorescence microscope (Nikon).
• FIGs. 6A-6B AR inhibition prevents IL-13-induced loss of cilia and ⁇ -tubulin in airway epithelial cell monolayer.
• Approximately 8.5xl0 4 SAEC were seeded per 12 mm diameter PET transparent insert and cultured for 7 days in differentiation media supplied from both lower and upper sides followed by culture on ALI for 11 days for differentiation into ciliated cells.
• the monolayer was treated with AR inhibitor for overnight and stimulated with IL-13 for 48 h.
• the monolayers on the membrane inserts were fixed for 24 h in z-fix (10% buffered formalin with zinc) at 4°C, paraffin embedded and 5 ⁇ sections were cut.
• FIGs. 7A-7C AR inhibition prevents IL-13-induced goblet cell metaplasia and expression of Muc5AC in airway epithelial monolayer.
• FIGs. 8A-8B AR inhibition prevents IL-13-induced expression of Mucin and transcription factor SPDEF in airway epithelial cell monolayer.
• FIG. 9 AR inhibition prevents IL-13 -induced phosphorylation of JAK-1, ER l/2 and STAT-6 in airway epithelial monolayer.
• the ciliated monolayer of airway cells at ALI was treated with AR inhibitor for 24 h and stimulated with IL-13 for different time periods as indicated.
• cells were lysed and cell lysate was subjected to western blotting using antibodies against phosphorylated and nonphosphorylated JAK-1, ERKl/2 and STAT-6 to analyze the activation of these signaling proteins.
• FIG. 10 FIG. 10.
• AR inhibition prevents RWE-induced expression of IL-13 in mouse lung.
• One microgram of total RNA from each sample was transcribed into first-strand cDNA and quantitative RT-PCR was conducted using IL-13 specific forward and reverse primers.
• the levels of RNA for the target sequences were determined by melting curve analysis. The values presented here are fold-change over the control. (**p ⁇ 0.001).
• RWE ragweed pollen extract; ARI, aldose reductase inhibitor.
• FIGs. 11A-11B AR inhibition prevents phosphorylation of STAT-6 in mouse lung epithelium.
• the mice were sensitized and challenged with PBS or RWE, without or with AR inhibitor and 20 h later lungs were perfused and fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to 5 ⁇ .
• the sections were immunostained with p- STAT-6 specific antibodies using immunoflouroscence secondary antibodies (A) or DAB- based HRP conjugated antibodies counterstained with hematoxylin and eosin (B).
• Photomicrographs were acquired by fluorescence or light microscopy. A representative field for each group is shown (magnification: 200 x).
• RWE ragweed pollen extract
• ARI aldose reductase inhibitor.
• FIGs. 12A-12B Inhibition or deficiency of AR prevents RWE-induced goblet cell metaplasia in mice lungs.
• AR Aldose Reductase
• an aldose reductase inhibitor is used to treat COPD.
• COPD chronic obstructive pulmonary disease
• COPD chronic obstructive pulmonary disease
• Inflammation of the airway plays a major role in the pathogenesis of COPD, which involves infiltration of inflammatory cells in the airway and the accumulation of inflammatory mucous exudates.
• ROS reactive oxygen species
• aldose reductase prevents cigarette smoke extract (CSE)-induced cellular changes in human small airway epithelial cells (SAEC) that are characteristic of COPD including cellular apoptosis, and secretion of cytokines, chemokines, and other inflammatory markers.
• CSE cigarette smoke extract
• SAEC human small airway epithelial cells
• Aldose reductase (AR) a member of the aldo-keto reductase superfamily, is a cytosolic protein that catalyzes NADPH-dependent reduction of glucose to sorbitol in hyperglycemic conditions, which is implicated in diabetic complications.
• Aldose reductase a member of the aldo-keto reductase superfamily, is a cytosolic protein that catalyzes NADPH-dependent reduction of glucose to sorbitol in hyperglycemic conditions, which is implicated in diabetic complications.
• AR aldose reductase
• Evidence has recently been presented indicating that AR is crucial in the oxidative stress-induced molecular signal and that AR catalyzed metabolic product is an excellent mediator of redox signaling.
• the results described herein show that IL-13-induced goblet cell formation is prevented by AR inhibition in vitro as well as in vivo. These results suggest that AR inhibition is beneficial in preventing and treating the COPD as it prevented cell-death, cytokines release and mucus formation in human airway epithelial cells.
• aldose reductase inhibitor is any compound that inhibits the enzyme aldose reductase.
• Exemplary aldose reductase inhibitors are readily available or can be easily synthesized by those skilled in the art using conventional methods of organic synthesis. Many of these are well known to those of skill in the art, and a number of pharmaceutical grade AR inhibitors are commercially available, such as fidarestat (SNK-860), (2S,4S)-2-aminoformyl-6-fluoro- spiro[chroman-4,4'-imidazolidine]-2',5'-dione (CAS number 136087-85-9); Tolrestat, N-[[6- methoxy-5 -(trifluoromethyl)- 1 -naphthalenyljthioxomethyl] -N-methylglycine, [ Wyeth-
• Diabetes Complications Porte, ed., Ch. 5, pp. 325-353; Tomlinson et ah, 1992), such as spirohydantoins and related structures, spiro-imidazolidine-2',5'-diones; and heterocycloic alkanoic acids.
• aldose reductase inhibitors are ONO-2235; zopolrestat; SNK-860; 5-3- thienyltetrazol-l-yl (TAT); WAY-121,509; ZENECA ZD5522; M16209; (5-(3 * -indolal)-2- thiohydantoin; zenarestat; zenarestat 1 -O-acylglucuronide; SPR-210; (2S,4S)-6-fluoro-2',5'- dioxospiro-[chroman-4,4'-imidazolidine]-2-carboxami de (SNK-880); arylsulfonylamino acids; 2,7-difluorospirofluorene-9,5'-imidazolidine-2',4'-dione (imiriestat, All 1576, HOE 843); isoliquiritigenin; 3,4-dihydro-4-oxo-3-[[5
• the aldose reductase inhibitor is a compound that directly inhibits the bioconversion of glucose to sorbitol catalyzed by the enzyme aldose reductase.
• Such aldose reductase inhibitors are direct or specific inhibitors, which are contemplated as part of the invention. Direct inhibition is readily determined by those skilled in the art according to standard assays (Malone, 1980).
• the ARIs can also be classified by chemical structure.
• the ARI is a carboxylic acid, a hydantoin, a pyridazinone, or a pharmaceutically acceptable derivative thereof.
• the ARI is a synthetic chemical compound.
• the ARI is a naturally-derived compound (e.g., plant extracts or endogenous antioxidants that inhibit aldose reductase).
• aldose reductase inhibitors include, but are not limited to: 3-(4-bromo-2-fluorobenzyl)-3,4-dihydro-4-oxo-l-phthalazineacetic acid (ponalrestat, U.S.
• Patent 4,251,528 N[[(5-trifluoromethyl)-6-methoxy-l- naphthalenyl]thioxomethyl ⁇ -N-methylglycine (tolrestat, U.S. Patent 4,600,724); 5-[( ⁇ , ⁇ )- ⁇ - methylcinnamylidene]-4-oxo-2-thioxo-3-thiazolideneacetic acid (epalrestat, U.S. Patent 4,464,382, U.S. Patent 4,791,126, U.S.
• Patent 4,831,045 3-(4-bromo-2-fluorobenzyl)-7- chloro-3,4-dihydro-2,4-dioxo-l(2H)-quinazolineacetic acid (zenarestat, U.S. Patent 4,734,419, and U.S. Patent 4,883,800); 2R,4R-6,7-dichloro-4-hydroxy-2-methylchroman-4- acetic acid (U.S. Patent 4,883,410); 2R,4R-6,7-dichloro-6-fluoro-4-hydroxy-2- methylchroman-4-acetic acid (U.S.
• Patent 4,883,410 3,4-dihydro-2,8-diisopropyl-3-oxo- 2H-l,4-benzoxazine-4-acetic acid (U.S. Patent 4,771,050); 3,4-dihydro-3-oxo-4-[(4,5,7- trifluoro-2-benzothiazolyl)methyl]-2H-l,4-benzothiazine-2-acetic acid (SPR-210, U.S. Patent 5,252,572); N-[3,5-dimethyl-4-[(nitromethyl)sulfonyl]phenyl]-2-methyl-benzeneacetamide (ZD5522, U.S. Patent 5,270,342 and U.S.
• Patent 5,430,060 (S)-6-fluorospiro[chroman-4,4 * - imidazolidine]-2,5'-dione (sorbinil, U.S. Patent 4,130,714); d-2-methyl-6-fluoro- spiro(chroman-4', 4'-imidazolidine)-2', 5'-dione (U.S. Patent 4,540,704); 2-fluoro-spiro(9H- fluorene-9,4'-imidazolidine)-2', 5'-dione (U.S.
• Patent 4,438,272 2,7-di-fhioro-spiro(9H- fluorene-9,4 * -imidazolidine)-2 * , 5 * -dione (U.S. Patent 4,436,745, U.S. Patent 4,438,272); 2,7- di-fluoro-5-methoxy-spiro(9H-fluorene-9,4'-imidazolidine)-2', 5'-dione (U.S. Patent 4,436,745, U.S. Patent 4,438,272); 7-fluoro-spiro(5H-indenol[l,2-b]pyridine-5,3 * - pyrrolidine)-2,5 * -dione (U.S.
• Patent 5,066,659 (2S,4S)-6-fluoro-2 * , 5 * -dioxospiro(chroman-4,4 * - imidazolidine)-2-carboxamide (fidarestat, U.S. Patent 5,447,946); and 2-[(4-bromo-2- fluorophenyl)methyl]-6-fluorospiro[isoquinoline-4(lH),3'-pyrrolidine]-l,2', 3,5'(2H)-tetrone (minalrestat, U.S. Patent 5,037,831).
• Other compounds include those described in U.S. Patents 6,720,348, 6,380,200, and 5,990,111, which are hereby incorporated by reference. Moreover, in other embodiments it is specifically contemplated that any of these may be excluded as part of the invention.
• compositions of the present invention may comprise an effective amount of one or more AR inhibitors dissolved or dispersed in a pharmaceutically acceptable carrier to a subject.
• pharmaceutically acceptable refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
• the preparation of a pharmaceutical composition that contains at least one AR inhibitor or additional active ingredient will be known to those of skill in the art in light of the present disclosure, and as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference.
• preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
• compositions of the aldose reductase inhibitors of this invention may be readily prepared by reacting the free acid form of the aldose reductase inhibitor with an appropriate base, usually one equivalent, in a co-solvent.
• bases are sodium hydroxide, sodium methoxide, sodium ethoxide, sodium hydride, potassium methoxide, magnesium hydroxide, calcium hydroxide, benzathine, choline, diethanolamine, piperazine and tromethamine.
• the salt is isolated by concentration to dryness or by addition of a non-solvent.
• salts are preferably prepared by mixing a solution of the acid with a solution of a different salt of the cation (sodium or potassium ethylhexanoate, magnesium oleate), and employing a solvent (e.g., ethyl acetate) from which the desired cationic salt precipitates, or can be otherwise isolated by concentration and/or addition of a non-solvent.
• a solvent e.g., ethyl acetate
• the acid addition salts of the aldose reductase inhibitors of this invention may be readily prepared by reacting the free base form of said aldose reductase inhibitor with the appropriate acid.
• the salt is of a monobasic acid (e.g., the hydrochloride, the hydrobromide, the p-toluenesulfonate, the acetate)
• the hydrogen form of a dibasic acid e.g., the hydrogen sulfate, the succinate
• the dihydrogen form of a tribasic acid e.g., the dihydrogen phosphate, the citrate
• salts as the sulfate, the hemisuccinate, the hydrogen phosphate, or the phosphate
• the appropriate and exact chemical equivalents of acid will generally be used.
• the free base and the acid are usually combined in a co-solvent from which the desired salt precipitates, or can be otherwise isolated by concentration and/or addition of a non-solvent.
• aldose reductase inhibitors that may be used in accordance with this invention, prodrugs thereof, and pharmaceutically acceptable salts thereof or of said prodrugs, may occur as hydrates or solvates. These hydrates and solvates are also within the scope of the invention.
• a pharmaceutical composition of the present invention may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it needs to be sterile for such routes of administration as injection.
• a pharmaceutical composition of the present invention can be administered intravenously, intradermally, intraarterially, intraperitoneally, intraarticularly, intrapleurally, intrabronchially, intrapleurally, intranasally, topically, intramuscularly, intraperitoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, orally, topically, locally, inhalation (e.g., aerosol inhalation), instillation, injection, infusion, continuous infusion, via a catheter, via a lavage, in lipid compositions (e.g., liposomes), or by other method or any combination of the forgoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference).
• Carriers include any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the therapeutic or pharmaceutical compositions is contemplated.
• preservatives e.g., antibacterial agents, antifungal agents
• isotonic agents e.g., absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders,
• the actual dosage amount of a composition of the present invention administered to a subject can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration.
• the number of doses and the period of time over which the dose may be given may vary.
• the practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s), as well as the length of time for administration for the individual subject.
• An amount of an aldose reductase inhibitor that is effective for inhibiting aldose reductase activity is used.
• an effective dosage for the inhibitors is in the range of about 0.01 mg/kg/day to 100 mg/kg/day in single or divided doses, preferably 0.1 mg/kg/day to 20 mg/kg/day in single or divided doses. Doses of about, at least about, or at most about 0.01, 0.05, 0.1, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90.
• compositions may comprise, for example, at least about 0.1% of an active compound.
• the an active compound may comprise between about 2%> to about 75 %> of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
• a dose may also comprise from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight, to about 1000 mg/kg/body weight or more per administration, and any range derivable therein.
• a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
• the composition may comprise various antioxidants to retard oxidation of one or more component. Additionally, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
• the AR inhibitors are prepared for administration by such routes as oral ingestion.
• the solid composition may comprise, for example, solutions, suspensions, emulsions, tablets, pills, capsules (e.g., hard or soft shelled gelatin capsules), sustained release formulations, buccal compositions, troches, elixirs, suspensions, syrups, wafers, or combinations thereof.
• Oral compositions may be incorporated directly with the food of the diet.
• Preferred carriers for oral administration comprise inert diluents, assimilable edible carriers or combinations thereof.
• the oral composition may be prepared as a syrup or elixir.
• a syrup or elixir and may comprise, for example, at least one active agent, a sweetening agent, a preservative, a flavoring agent, a dye, a preservative, or combinations thereof.
• an oral composition may comprise one or more binders, excipients, disintegration agents, lubricants, flavoring agents, and combinations thereof.
• a composition may comprise one or more of the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, corn starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.; or
• the dosage unit form When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, carriers such as a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both.
• Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and/or the other ingredients.
• the preferred methods of preparation are vacuum-drying or freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered liquid medium thereof.
• the liquid medium should be suitably buffered if necessary and the liquid diluent first rendered isotonic prior to injection with sufficient saline or glucose.
• the preparation of highly concentrated compositions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents to a small area.
• prolonged absorption of an injectable composition can be brought about by the use in the compositions of agents delaying absorption, such as, for example, aluminum monostearate, gelatin or combinations thereof.
• compositions of the present invention such as an AR inhibitor
• This process may involve contacting the cell(s) with an AR inhibitor and a therapeutic agent at the same time or within a period of time wherein separate administration of the modulator and an agent to a cell, tissue or organism produces a desired therapeutic benefit.
• the terms "contacted” and "exposed,” when applied to a cell, tissue or organism, are used herein to describe the process by which a AR inhibitor and/or therapeutic agent are delivered to a target cell, tissue or organism or are placed in direct juxtaposition with the target cell, tissue or organism.
• the cell, tissue or organism may be contacted (e.g., by administration) with a single composition or pharmacological formulation that includes both a AR inhibitor and one or more agents, or by contacting the cell with two or more distinct compositions or formulations, wherein one composition includes an AR inhibitor and the other includes one or more agents.
• the AR inhibitor may precede, be concurrent with and/or follow the other agent(s) by intervals ranging from minutes to weeks.
• the AR inhibitor and other agent(s) are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the inhibitor and agent(s) would still be able to exert an advantageously combined effect on the cell, tissue or organism.
• one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i.e., within less than about a minute) as the modulator.
• one or more agents may be administered within of from substantially simultaneously, about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, or more hours, or about 1 day or more days, or about 4 weeks or more weeks, or about 3 months or more months, or about one or more years, and any range derivable therein, prior to and/or after administering the AR inhibitor.
• AR inhibitors and other active agents may be administered together or separately.
• the administration of one agent may be prior to, concurrent to, or subsequent to the administration of other agent(s).
• CSE caused a dose-dependent cell-death in SAEC that was 23, 26, 37 and 40% in 24 h at 12.5, 25, 50 and 75% concentration of CSE, respectively.
• the treatment of SAEC with fidarestat prevented CSE-induced cell death such that at 50%> CSE >85%> cells were alive and at 12.5% more than 95% cells were alive (FIG. 1).
• TNF-a-induced cell death in SAEC was used as a positive control.
• the airway epithelial cells which are the point of first contact between the allergens and the respiratory system, plays an important function as a barrier to foreign particles including environmental gases and particles, cigarette smokes and other xenobiotics which disturb cellular redox homeostasis leading to changes in cell viability, morphology and physiology of airway epithelial cells resulting in COPD. Therefore we chose primary human small airway epithelial cells.
• SAEC Primary human Small Airway Epithelial Cells obtained from Lonza (WalkersviUe, MD) were normal human SAEC harvested from distal airspace of 18 yrs old male donor. The cells were cultured according to the supplier's instructions at 37°C in humidified atmosphere containing 95% air and 5% C0 2 in small airway epithelial basal medium (SABM) with supplements containing 52 ⁇ g/ml bovine pituitary extract, 0.5 ng/ml human recombinant epidermal growth factor (EGF), 0.5 ⁇ g/ml epinephrine, 1 ⁇ g/ml hydrocortisone, 10 ⁇ g/ml transferrin, 5 ⁇ g/ml insulin, 0.1 ng/ml retinoic acid (RA), 6.5 ng/ml triiodothyronine, 50 ⁇ g/ml Gentamicin/Amphotericin-B (GA-1000), and 50 ⁇ g/ml
• CSE cigarette smoke extract
• Cell viability assays The SAEC were plated at the density of 5000 cells/well in a 96-well plate and growth-arrested for 24 h by replacing complete medium with fresh basal medium containing fidarestat (20 ⁇ ) or carrier. The cells were incubated with CSE (75, 50, 25, 12.5, 0%) for an additional 24 h with or without fidarestat, after which 10 ⁇ of MTT (5 mg/ml) was added to each well and incubated at 37°C for an additional 2 h. The medium was removed and the formazan granules were dissolved in 100% DMSO. Absorbance was read at 570 nm using a 96-well ELISA plate reader.
• AR inhibition prevents IL-13 -induced ROS levels in SAEC: Since cytokines- induced oxidative stress is known to mediate molecular signaling that leads to differentiation of airway epithelial cells into mucus cells, so we determined the effects of AR inhibition on IL-13-induced ROS levels in SAEC by two different methods. As shown in FIG. 3A and 3B, stimulation with IL-13 caused approximately twofold increase in the ROS levels over the control and treatment of the cells with fidarestat significantly (80%) prevented the increase. These results suggest that cytokine -induced oxidative stress could be prevented by inhibition of AR.
• AR inhibition prevents IL-13-induced goblet cell metaplasia in SAEC:
• the inventors used an air-liquid interface culture system that mimics in-vivo airway environment.
• the inventors cultured primary human airway epithelial cells on polyethylene terephthalate (PET) membrane insert with collagen type-1 coating.
• PET polyethylene terephthalate
• the cells on the collagen-coated membrane insert formed a consistent 2-3 cells thick layer of airway cells.
• the monolayer cells fully differentiated into ciliated cells when cultured on the ALI in EGF-containing medium for 11 days (FIG.
• AR inhibition prevents IL-13-induced phosphorylation and activation of signaling intermediates: Since AR inhibition in airway epithelial cells monolayer on ALI successfully prevented the expression of transcription regulator SPDEF and subsequent expression of mucin, the effect of AR inhibition on the molecular mechanism(s) that regulates the expression of these mediators of asthma was studied. Cells grown on ALI were stimulated with IL-13 in presence or absence AR inhibitor and determined the phosphorylation of JAK1, ER l/2 and STAT-6 proteins. As shown in FIG. 7, IL-13 caused a time-dependent increase in the phosphorylation of STAT-6 and upstream mediators such as JAK1 and ERKl/2.
• AR inhibition/deficiency prevents RWE-induced goblet cell metaplasia in mice lung: The inventors next examined the effect of AR inhibition on goblet cell metaplasia in RWE-sensitized and-challenged mice. After 72 h of challenge the lung sections were obtained and stained with PAS and changes in the airway epithelia were examined. As shown in FIG. 10A, there was a significant increase in the PAS positive cells in the airway of RWE-challenged mice and absent in the lungs of mice treated with fidarestat prior to RWE challenge. Similar to the inhibitor-treated mice, AR-null mice challenged with RWE showed absence of PAS positive cell in the airway (FIG. 10B). These results further confirm that AR plays a significant role in the goblet cell metaplasia and AR inhibition could prevent metaplasia in allergic asthma.
• Reagents Small airway epithelial basal medium (SABM), and small airway epithelial growth media (SAGMTM) bulletkit; and ReagentpackTM containing Trypsin 0.025%/EDTA 0.01%, Trypsin neutralizing solution and HEPES buffered-saline solution were purchased from Lonza Walkersvillle Inc. (Walkersville, MD). Dulbecco's modified Eagle's medium (DMEM) and phosphate buffered saline (PBS) were purchased from Gibco, Invitrogen (Grand Island, NY). AR inhibitor, fidarestat, was a gift from Sanwa-Kayagu (South Korea).
• SABM Small airway epithelial basal medium
• SAGMTM small airway epithelial growth media
• ReagentpackTM containing Trypsin 0.025%/EDTA 0.01%, Trypsin neutralizing solution and HEPES buffered-saline solution were purchased from Lonza Walkersvillle Inc.
• Human recombinant IL-13 was from R & D systems (Minneapolis, MN). Dimethyl sulfoxide (DMSO) was obtained from Fischer scientific (Pittsburg, PA). Human mucin5AC ELISA kit was from Cosmo Bio USA (Carlsbad, CA). Antibodies against STAT- 6, phospho-STAT- 6, phospho-JAKl, JAK1, ERK1/2, phospho-ERKl/2 were from Cell Signaling Tech (Danvers, MA) and mucin 5 subtypes A and C (Muc5AC), Muc5B, GAPDH and ⁇ -actin antibodies were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA).
• SPDEF SAM pointed domain-containing ETS transcription factor
• SAEC Primary human Small Airway Epithelial Cells obtained from Lonza Walkersville, Inc. (Walkersville, MD) were normal human SAEC harvested from distal airspace. The cells were cultured and maintained according to the supplier's instructions at 37°C in humidified atmosphere containing 95% air and 5% C0 2 in small airway epithelial basal medium (SABM) supplemented with 52 ⁇ g/ml bovine pituitary extract, 0.5 ng/ml human recombinant epidermal growth factor (EGF), 0.5 ⁇ g/ml epinephrine, 1 ⁇ g/ml hydrocortisone, 10 ⁇ g/ml transferrin, 5 ⁇ g/ml insulin, 0.1 ng/ml retinoic acid (RA), 6.5 ng/ml triiodothyronine, 50 ⁇ g/ml Gentamicin/Amphotericin-B (GA-1000), and 50 ⁇ g/ml
• ROS levels determination Approximately 5xl0 4 SAEC were seeded on 2- chambered culture slides in triplicate or 10,000 SAEC per well were plated in a 96-well plate. After they attached, cells were starved in basal medium containing 0.1% serum without or with fidarestat (10 ⁇ ) for overnight. Next day cells were washed with lx HBSS buffer and incubated with 10 ⁇ H2DCF-DA at 37°C for 30 min, washed again to remove excess H2DCF-DA and treated with IL-13 (25 ng/ml) for lh. At the end of incubation, cells were washed twice with cold lx HBSS buffer.
• the cells on the culture slide were mounted using floursave mounting medium with DAPI after which photomicrographs were acquired using a fluorescence microscope (Nikon). Fluorescence was determined using 485 nm excitation and 538 nm emission wavelengths for cells in 96-well plate and relative ROS production is expressed as mean fluorescence intensity (MFI) (arbitrary units).
• MFI mean fluorescence intensity
• Air-liquid interface culture For air-liquid interface (ALI) culture, SAEC were seeded at 8.5xl0 4 cells/ 12-mm-diameter PET transparent insert with 1.0 ⁇ pores (12-well millicell culture insert; Millipore) and pre-coated with rat tail collagen type-1 (Sigma- Aldrich). The cells were grown submerged in the differentiation medium as described earlier by Zhen et al. ⁇ Am J Respir Cell Mol Biol 36: 244-253, 2007).
• the differentiation medium contained a 1 : 1 mixture of DMEM and small airway epithelial growth medium supplemented as described above, except that gentamycin sulfate, amphotericin B, and triiodothyronine were replaced with 1% penicillin / streptomycin, and 50 nM all-trans retinoic acid.
• the SAEC were maintained submerged for the first 7 days, after which the apical medium was removed and an air-liquid interface culture was established. The cells were maintained at ALI for the remainder of the culture period. Medium was refreshed every third day and once in a week the apical surface of the cells was rinsed with PBS to remove accumulated mucus and debris.
• Cells were maintained at 37°C in 95% air and 5% C0 2 in a humidified incubator.
• the cells on the ALI were pre -treated with AR inhibitor, fidarestat (10 ⁇ ), for over-night from the basal side in differentiation medium without EGF beginning day 11 after establishment of ALI and stimulated with IL-13 by addition of recombinant human IL-13 (25 ng/ml) to the medium for varying time periods as indicated.
• Antibodies against ⁇ -tubulin and Muc5AC (Santa Cruz Biotechnology, Santa Cruz, CA) and matched control IgG were used for immunocytochemistry. Antibodies were detected using the Vector LSAB kit (Vector Laboratories, Burlingame, CA) as suggested by the manufacturer.
• oligonucleotide primer sequences were as follows: Muc5AC: 5'- TCCGGCCTCATCTTCTCC-3' (SEQ ID NO: l) (sense) and 5'- ACTTGGGC ACTGGTGCTG-3 ' (SEQ ID NO:2) (Antisense); SPDEF: 5'- CGAAGTGCTCAAGGACATCGAG-3 ' (SEQ ID NO:3) (sense) and 5'- CGGTATTGGTGCTCTGTCCACA-3 ' (SEQ ID NO:4) (anti-sense) and GAPDH: 5'- GACCCCTTCATTGACCTCAAC-3' (SEQ ID NO:5) (sense) and 5'- C AT AC C AGG A AATG AGCTTG- 3' (SEQ ID NO:6) (antisense).
• RT-PCR reaction was carried out in a PCR Sprint thermal cycler (Thermo electron corporation, Milford, MA) under the following conditions: initial denaturation at 95°C for 15 min followed by 35 cycles at 94°C for 1 min, 60°C for 1 min, 72°C for 1 min, followed by 72°C for 10 min for final extension.
• the RT-PCR products were subjected to electrophoresis on a 1.5% agarose-lX TAE gels containing 0 ⁇ g/ml ethidium bromide. The densitometry analysis of the gel was performed using NIH image analysis software.
• Muc5AC ELISA Muc5AC levels in the culture medium were assessed by ELISA using commercially available human anti-Muc5AC ELISA essentially as described by the manufacturer (Cosmo Bio USA; Carlsbad, CA).
• mice Wild type C57BL/6 and Balb/cJ mice were purchased from Harlan Sprague-Dawley (San Diego, CA, USA) and AR null mice on C57BL/6 background were bread by us at Animal resource center, UTMB, Galveston, TX. Six-eight weeks old female mice were sensitized with RWE as previously described (Hwang et al, FASEB J. 19: 795-797, 2005). Briefly, mice were sensitized with two intraperitoneal administrations of 100 ⁇ of endotoxin-free RWE (150 ⁇ g) combined with Alum adjuvant (1 mg) in a 3 : 1 ratio (v/v), on days 0 and 4.
• mice (n 6) were challenged intranasally with RWE (100 ⁇ g).
• Control groups of mice were challenged with equivalent volumes of PBS.
• the animals were euthanized at different time points as mentioned, with ketamine (135 mg/kg body wt) and xylazine (15 mg/kg body wt), the lungs were perfused and fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned to 5 ⁇ . Lung sections were stained with PAS and the representative fields were observed and photographed with a Photometrix CoolSNAP Fx camera mounted on a NIKON Eclipse TE 200 UV microscope.
• mice lungs were harvested 16 h after RWE-challenge and total RNA was isolated. From each sample, one microgram of total RNA was transcribed into first-strand cDNA and quantitative RT-PCR was conducted using IL-13 specific forward and reverse primers 5 'AGACCAGACTCCCCTGTGCA (SEQ ID NO:7), 3 'TGGGTCCTGTAGATGGCATTG (SEQ ID NO:8); GAPDH specific primers (5 'TGTGTCCGTCGTGGATCTGA (SEQ ID NO:9),

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