Compositions and methods for treating glomerular disorders

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] The present invention relates to nephrotic disorders, and to compositions and methods of treating nephrotic disorders. In another aspect, the present invention relates to glomerular disorders, and to compositions and methods for treating glomerular disorders. In even another aspect, the present invention is related to compositions comprising an agent that inhibits a member of the cytochrome P-450 family, and to methods for using such compositions to treat glomerulonephritides. In yet another aspect, the present invention relates to nephrotic syndromes, to compositions comprising an agent that inhibits a cytochrome P450, and to methods of using such to treat nephrotic syndromes. In even yet another aspect, the present invention is related to compositions comprising an agent that inhibits a member of the cytochrome P-450 family, and to methods of using such compositions to reduce proteinuria associated with minimal change nephrotic syndrome.

[0004] 2. Description of the Related Art

[0005] Minimal change nephrotic syndrome (MCNS) is the most common nephrotic syndrome affecting children between 2 to 6 years of age, and accounts for more than 75% of the cases of nephrotic syndrome in children. In adults, MCNS accounts for about 30% of nephrotic syndrome. Unlike acute renal failure which affects the tubules, MCNS is a disease of the glomeruli, which function as filtering units of the kidneys. As is the case with many other glomerulonephritides, a hallmark of MCNS is proteinuria.

[0006] Current methods for treating individuals afflicted with glomerulonephritides, such as, MCNS, are empirical and consist mostly of corticosteroids and, at times, immunosuppressive agents. These traditional MCNS treatments are limited by their adverse side effects such as, growth failure, cushingoid features and bone deformities.

[0007] For acute renal failure, it has been suggested that the iron storage proteins ferritin, transferrin, iron-rich mitochondria, and extracellular heme proteins, such as hemoglobin and myoglobin, might serve as sources of catalytic iron. However, the actual sources of iron that catalyze free radical reactions in acute renal failure remains an actively researched area.

[0008] For example, Paller et al., 1994, PNAS 91:7000270006, disclose the hypothesis that hydroxyl radical formation during reoxygenation of the kidney is mediated by cytochrome P-450.

[0009] Ueda et al, 1993, Am. L. Physiol 265:F435-F439 disclose that isolated renal cortical mitochondria release iron when exposed to the nephrotoxic drug gentamicin.

[0010] Baliga et al, 1996, Kidney International 49:362-369, disclose evidence for cytochrome P-450 as a source of catalytic iron in a model of myoglobinuric acute renal failure induced by glycerol, wherein two cytochrome P450 inhibitors tested therein provided functional and histological protection against the acute renal failure.

[0011] Baliga et al., 1996, Kidney International 50:1118-1124, disclose a role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity in LLC-PK1 cells, wherein two cytochrome P450 inhibitors tested therein provided beneficial effects against hydrogen peroxide-induced cell injury.

[0012] Zager et al., 1997, Kidney International 51:728-738, suggest that the formation of iron/H2O2-based reactive intermediates in mitochondria may be responsible for the cell damage in an in vitro model of myoglobin cytotoxicity.

[0013] Baliga et al., 1998, Kidney International 53:394-401, disclose a hypothesis suggesting a role for iron in mediating tissue injury via hydroxy radicals in a cisplatin-induced model of nephrotoxicity.

[0014] Baliga et al., November 1998, Kidney International 54(5), 1562-1569, disclose a role for cytochrome P450 as a source of catalytic iron in cisplatin-induced nephrotoxicity.

[0015] Ueda et al, 1996, Kidney International 49:370-373, and Ueda et al, 1997, Kidney: A Current Survey of World Literature 6:143-146, disclose a pathogenic role for reactive oxygen metabolites (ROMS) in glomerular disease, a source of the glomerular catalytic iron is not disclosed. Thus, while ROMs are believed to be important in mediating renal injury, the molecular mechanisms underlying the activation of a catalytic iron source in glomerular disease, such as MCNS, are not known.

[0016] However, unlike models for acute renal failure, a role for a CYP in a glomerular disease model, such as, puromycin aminonucleoside (PAN)-induced MCNS, has not previously been disclosed.

[0017] Despite the advances made in the field of glomerular disease mechanisms, there remains a need for treatments for individuals having a glomerular disorder wherein the composition comprises an inhibitor of a cytochrome P450, glomerulonephritides. There is another need in the art for compositions and methods for treating an individual having a glomerular disorder, wherein the compositions and methods lack the adverse side effects associated with traditional therapies, such as, steroids and immunosuppressants. There is even another need in the art for compositions and methods for treating a patient having a proteinuric disorder, wherein the compositions comprise an agent that inhibits a cytochrome P450. There is even another need in the art for compositions and methods for treating a patient having a proteinuric disorder, such as minimal change nephrotic syndrome, wherein the compositions comprise an agent which inhibits a cytochrome P450 superfamily member.

SUMMARY OF THE INVENTION

[0018] It is an object of the present invention to provide compositions and methods for treating an individual having a glomerular disorder, wherein the compositions and methods lack the adverse side effects associated with traditional therapies, such as, asteroids and immunosuppressant.

[0019] It is even another object of the present invention to provide for compositions and methods comprising an agent which reduces proteinuria in a patient having glomerular disease.

[0020] It is still another object of the present invention to provide for compositions and methods comprising an agent which inhibit a cytochrome P450 superfamily member.

[0021] These and other objects of the present invention will become apparent to those of skill in the art upon review of this specification, including its drawings and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]
FIG. 1A shows the levels of proteinuria in PAN treated animals. A PAN-induced model of minimal change nephrotic syndrome was induced by a single intravenous injection of PAN (7.5 mg/100 g BW) to rats. The animals were sacrificed 7 days after PAN injection. Values are means ±SE. *p<0.01 comparing PAN-treated rats with control animals.

[0023]
FIG. 5 shows the effect of cytochrome P450 inhibitors on proteinuria in PAN-treated rats. Values are means ±SE. *p<0.05 compared with the rats treated with PAN alone.

[0024]
FIG. 6 shows the effect of cytochrome P450 inhibitors on serum albumin in rats treated with PAN. Values are means ±SE. *p<0.05 compared with the control rats.

[0025]
FIG. 7A shows renal function as measured by serum creatinine in rats injected with PAN with or without cytochrome P450 inhibitors. Values are means ±SE. *p<0.05 compared with the control rats.

[0026]
FIG. 7B shows renal function as measured by blood urea nitrogen in rats injected with PAN with or without cytochrome P450 inhibitors. Values are means ±SE. *p<0.05 compared with the control rats.

[0027]
FIG. 1B (6) shows the bleomycin detectable iron content levels in the glomeruli of PAN treated animals. A PAN-induced model of minimal change nephrotic syndrome was induced by a single intravenous injection of PAN (7.5 mg/100 g BW) to rats. The animals were sacrificed 7 days after PAN injection. Values are means ±SE. *p<0.01 comparing PAN-treated rats with control animals.

[0028]
FIG. 2A shows cytochrome P450 content in kidney in PAN-induced nephrotic syndrome in rats. Values are means ±SE.

[0029]
FIG. 2B shows cytochrome P450 content in the liver in PAN-induced nephrotic syndrome in rats. Values are means ±SE.

[0030]
FIG. 3 shows the effect of cytochrome P450 inhibitors on the bleomycin detectable iron content in the glomeruli of PAN-treated rats. Values are means ±SE. *p<0.05 compared with control rats. +p<0.05 compared with PAN treatment alone.

[0031]
FIG. 4 shows the effect of cytochrome P450 inhibitors on cytochrome P450 content in the glomeruli of PAN-treated rats. Values are means ±SE.

[0032]
FIG. 8 shows the effect of cytochrome P450 inhibitors on the release of catalytic iron in glomerular epithelial cells GEC exposed to PAN. Confluent GEC were incubated with a cytotoxic dose of PAN (1.5 mM) for a period of time less than the time at which substantial cell killing occurs (1 h). Cytochrome P450 inhibitors PB (25 μM), CM (2 mM) and RN (1 mM, as a control for CM) were preincubated with GEC for 30-60 min and then washed with chelexed HBSS followed by addition of PAN. Values are means ±SE. *p<0.05 compared with the control; +p<0.05 compared with GEC exposed to PAN alone.

[0033]
FIG. 9 shows the effect of cytochrome P450 inhibitors on hydroxyl radical generation in GEC exposed to PAN. Confluent GEC were incubated with cytotoxic dose of PAN (1.5 mM) for a period of time less than the time at which substantial cell killing occurs (1h). Cytochrome P450 inhibitors PB (25 μM), CM (2 mM) and RN (1 mM, as a control for CM) were preincubated with GEC for 30-60 min and then washed with HBSS followed by addition of PAN. Values are means ±SE. *p<0.05 compared with the control; +p<0.05 compared with PAN alone.

[0034]
FIG. 10A shows the concentration-dependent effect of PAN (0 to 2 mM for 48h) on cytotoxicity to GEC as measured by trypan blue exclusion assay. Values are means ±SE. N=2.

[0035]
FIG. 10B shows the time-dependent effect of PAN (0 to 4 days at PAN dose of 1.5 mM) on cytotoxicity in GEC as measured by trypan blue exclusion assay. Values are means ±SE. N=2.

[0036]
FIG. 11 shows the effect of cytochrome P450 inhibitors on GEC cell death in GEC exposed to PAN. Confluent GEC were incubated with a cytotoxic dose of PAN (1.5 mM) for a period of time necessary to induce consistent cytotoxicity (48 h). Cytochrome P450 inhibitors PB (25 μM), CM (2 mM) and RN (1 mM, as a control for CM) were preincubated with GEC for 30-60 min and then washed with HBSS followed by addition of PAN. Values are means ±SE. *p<0.05 compared with GEC exposed to PAN alone at concentration indicated in the figure. N=3.

DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention is directed to compositions and methods for treating a glomerular disorder. The compositions and methods of the present invention are advantageous over traditional treatments for glomerular disorders in that they are not associated with adverse side effects, yet they are effective in reducing proteinuria.

[0038] As used herein, the phrase “glomerular disorder” includes any glomerular disease and/or any glomerular injury such as, for example, minimal change disease, focal segmental sclerosis, membranous nephropathy, diabetic nepropathy, amyloidosis, and proliferative glomerular nephritis such as, anti-GBM antibody disease, and mesangioproliferative glomerular nephritis, and animals models such as passive Heymann nephritis, puromycin aminonucleoside-induced proteinuria.

[0039] One embodiment of the present invention is directed to compositions and methods comprising an agent that is useful in treating an individual having a glomerular disorder. Preferably, the compositions and methods of the invention reduce the level of proteinuria in an individual afflicted with a glomerular disease.

[0040] Even another embodiment of the present invention is directed to compositions and methods for inhibiting a cytochrome P450 superfamily member. This may comprise partially reducing or completely reducing the ability of a CYP to serve as a source of glomerular catalytic iron.

[0041] As used herein, the phrase “cytochrome P450” (CYP) is intended to include any member/isoenzyme of the CYP superfamily, which includes the mammalian CYP1, CYP2, CYP3 and CYP4 families (Omiecinski, et al., 1999, Toxicological Sciences 48:151-156). Thus, CYP includes the mammalian cytochrome P450 genes and their gene products, such as, P450 1A1, 1A2, 1B1, 2B1, 2B2, 2B4, 2B5, 2B6, 2B11, 2A6, 2C6, 2C8, 2C9, 2C11, 2C18, 2C19, 2D6, 2E1, 3A4, 3A5, 3A7, 4A1 and 4B1. Of particular interest is the CYP isozyme CYP2B1 which is in glomeruli. A complete mRNA sequence and translation sequence of a human CYP2B1, GenBank accession number M29874, is provided herein as SEQ.ID.NO.:1. The phrase “cytochrome P450” (CYP) also includes allelic variants, site-specific mutants, and chimeric constructs of members of the CYP superfamily (Chang et al., 1997, Pharmacogenetics 7:211-221; Szklarz et al., 1995, Biochemistry 34:14312-14322; He et al, 1997, Biochemistry 36:8831-8839).

[0042] There are numerous CYP inhibitors/agents useful in the present invention for inhibiting a CYP superfamily member, all of which are included in the present invention.

[0043] As used herein the phrase “CYP inhibitor” includes any agent that inhibits at least one function of a CYP superfamily member. According to the present invention, CYP inhibitors include agents that directly interact with a CYP, indirectly interact with a CYP, directly or indirectly reduce the expression of a CYP, function in a biochemical pathway upstream of a CYP, and agents that function in a biochemical pathway downstream of a CYP. The interaction between a CYP and the CYP inhibitor may be a direct or an indirect interaction. The inhibitors useful in the present invention may function by interacting with the heme iron of CYP, thereby blocking or reducing the availability of the iron to serve as a catalytic iron source. The inhibitor agents of the present invention may inhibit CYP at the level of gene expression, gene transcription, translation, or CYP function.

[0044] Non-limiting examples of agents suitable for use in the present invention as CYP inhibitors include, cimetidine (CM), piperonyl butoxide (PB), safrole, isosafrole, myristicinfurfylline, diethyldithiocarbamate, chlormethiazol, piperine, disulfiram, diallyl sulfid, malotilate, allylmercaptan, methylprazole, orphenadrine, arylacetylenes, clorgyline and diphenhydramine. Generally the CYP inhibitors useful in the present invention are selected from the group consisting of cimetidine, piperonyl butoxide, piperine, diethyldithiocarbamate, chlormethiazol, disulfiram and diallyl sulfid. Preferably, the CYP inhibitor used in the present invention is cimetidine, piperonyl butoxide, piperine, diethyldithiocarbamate, and chlormethiazol.

[0045] Without being limited by theory, it is possible that an increase in the generation of hydrogen peroxide may result in direct oxidative attack on the heme moiety of CYP promoting the heme destruction and the release of iron. One mechanism by which inhibitors of the present invention may function is to inhibit this release of iron.

[0046] Another embodiment of the present invention is directed to compositions and methods comprising an agent that reduces the ability of a CYP superfamily member to function as a glomerular catalytic iron source.

[0047] Additional inhibitors suitable for use in the compositions and methods of the invention include agents wherein the agent is a DNA, cDNA, RNA or polypeptide sequence. Suitable examples of such agents include, an antisense CYP sequence which inhibits transcription or translation of a CYP, transcription factors which decrease expression of a CYP gene, factors which affect translation of a CYP mRNA, factors which decrease the stability/half-life of a CYP mRNA molecule, factors which decrease the stability/half-life of a CYP polypeptide, and factors which interact with a CYP polypeptide, such as a polypeptide encoding an antibody which specifically interacts with an epitope of a CYP. The material and methods for producing these types of CYP inhibitors (DNA, cDNA, RNA and polypeptide) are known in the art and are included in the present invention.

[0048] For example, expression vectors comprising a sequence inhibitory to transcription of a CYP gene or comprising a sequence inhibitory to translation of a CYP mRNA are within the scope of the CYP inhibitors defined herein. Expression vectors suitable for the present invention may comprise an antisense CYP sequence, or a sequence encoding a negative regulator of transcription of a CYP gene.

[0049] Preferably the vectors of the invention comprise a CYP antisense sequence which will inhibit the mRNA of a CYP gene. Generally preferred targets for antisense technology are the CYP2 family members, more preferred are the CYP2B isozymes,and even more preferred is CYP2B1.

[0050] Vectors comprising a DNA sequence which encodes a polypeptide of a CYP dominant negative mutant, or a vetor comprising a DNA sequence encoding a CYP mutant may also be useful inhibitors in the present invention.

[0051] Suitable vectors are known in the art and include, for example, mammalian expression vectors and viral vectors. Examples of viral vectors suitable for use in the present invention include: retroviruses; adenoviruses; adenoviral/retroviral chimeras; adenoassociated viruses; herpes simples virus I or II; parvovirus; and reticuloendotheliosis virus. Other possible viral vectors may be derived from poliovirus, papillomavirus, vaccinia virus, lentivirus, as well as chineric vetors incorportation favorable aspects of any two or more of the the above viruses.

[0052] For guidance in the construction of gene therapy vectors and in the introduction thereof into affected individuals for therapeutic purposes, WO 99/05299 cites as well as U.S. Pat. Nos. 5,631,236; 5,688,773; 5,691,177; 5,670,488; 5,601,818; and WO 95/06486.

[0053] Generally, methods are known in the art for viral infection of the cells of interest. The virus can be injected into a patient bearing a neoplasm, either at, into, or near the site of neoplastic growth. Preferentially, the treatment will be by direct intraneoplastic inoculation.

[0054] The compositions of the present invention further comprise a pharmaceutically acceptable carrier/vehicle. Pharmaceutically acceptable carriers/vehicles are known in the art and include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, propylene glycol, polyethylene glycol, vegetable oil, injectable organic esters such as ethyloleate, water, saline solutions, parenteral vehicles such as sodium chloride and Ringer's dextrose, glycerol, lipids, alcohols.

[0055] Compositions of the present invention may be in any form known in the art, such as an orally digestible form, a sterile injectable form, forms suitable for delayed release, and forms that are enterically coated. Compositions of the invention may be in solid forms, including, for example, powders, tablets, pills, granules, capsules, sachets and suppositories, or may be in liquid forms including solutions, suspensions, gels and emulsions.

[0056] The compositions and methods of the present invention may be administered to a recipient/patient as a single dose unit, or may be administered in several dose units, for a period ranging from one day to several years. The dose schedule is dependent upon at least the severity of the glomerular disorder, as well as the mode of administration. The effective dose of the compositions of the present invention is further dependent upon the body weight (BW) of the recipient/patient and also upon the chosen inhibitor. Generally the compositions of the present invention are administered orally or intravenously.

[0057] Generally for a glomerular disease, such as minimal change disease, treatment takes place during a time period ranging from about one day to about four years, preferably about one week to about three years, and even more preferably from about two weeks to about two years.

[0058] Generally for chronic proteinuric diseases, treatment takes place during a time period ranging from about one day to about twenty years, preferably from about one week to about fifteen years, and even more preferably from about two weeks to about ten years.

[0059] Generally, cimetidine is administered via an oral or intravenous route at a concentration in the range of about 10 to about 50 mg per kilogram of body weight, preferably about 15 to about 45 mg per kilogram of body weight, and more preferably about 20 to about 40. Generally cimetidine is given every six hours in a twenty four hour period.

[0060] Generally, piperine is administered by an oral or intravenous route in an amount ranging from about 100 to about 300 mg, preferably from about 125 to about 250 mg, and more preferably from about 150 to about 200 mg. Generally piperine is administered orally once every twenty-four hours.

[0061] The inhibitor DEDC is generally administered orally or intravenously at a concentration ranging from about 0.25 to about 2.5 g/m2, preferably from about 0.4 to about 2.0 g/m2, even more preferably from about 0.5 to about 1.8 g/m2, and still more preferably from about 0.6 to about 1.6 g/m2. Generally, DEDC is given once a day.

[0062] The inhibitor chlormethiazole is generally administered orally in an amount ranging from about 0.5 to about 3.5 g, preferably from about 0.75 to about 3.0 grams, even more preferably from about 1.0 to about 2.75 grams, and still more preferably from about 1.2 to about 2.4 grams. Generally, chlormethiazole is given once every twenty-four hours.

[0063] The compositions and methods of the present invention are suitable for any individual afflicted with a glomerular disorder. Suitable individuals include mammals such as, humans, dogs, cats, horses, cows, sheep, goats, pigs, rats and mice. The compositions and methods of the present invention are also suitable for use in any tissue or cell line that serves as a model for glomerular disorder, including glomeruli epithelial, endotheliea and mesanger cells. Thus the present invention is useful to medical and health care professionals including, medical doctors, and veterinarians, as well as research scientists.

[0064] Administration of the present invention to a recipient may be by any method known in the art. Thus, administration of the present invention to a recipient may be by a route selected from oral, parenteral (including, subcutaneous, intradermal, intramuscular, and intravenous) and rectal. For increased efficacy, the compositions of the present invention may be administered via localized delivery to the kidney. Generally the present invention is administered to a recipient intravenously.

[0065] One method the invention is directed to treating a patient having a glomerular disorder, such as a glomerular disease like minimal change disease. The method comprises the steps of administering a therapeutically effective amount of a composition to a patient, wherein the composition comprises an effective amount of an agent that inhibits a glomerular cytochrome P450, such as a cytochrome P450 2B family member. Preferably the agent is selected from the group consisting of cimetidine, pieronyl butoxide, piperine, diethyldithiocarbamate, chlormethiazole, and any combination thereof.

[0066] Another method in the invention is directed to treating a pateint having a glomerular disorder, such as a glomerular disease. The method comprises the steps of administering a therapeutically effective amount of a composition to the patient, wherein said composition comprises an effective amount of an expression vector comprising a sequence encoding an inhibitor of a cytochrome P450, such as an antisense cytochrome P450 sequence. Preferably the antisense cytochrome P450 sequence is a cytochrome P450 2B family member, such as SEQ.ID.NO.:1.

[0067] Another method of the invention is directed to treating a patient by administering a therapeutically effective amount of a composition to the patient, wherein the composition comprises an agent that inhibits a cytochrome P450 superfamily member and wherein said patient has a urinary protein excretion value of greater than about 300 mg in a twenty four hour time period. Preferably, the agent is selected from the group consisting of cimetidine, pieronyl butoxide, piperine, diethyldithiocarbamate, chlormethiazole, and any combination thereof. The cytochrome P450 superfamily member may be any cytochrome P450 of the 2B family. Treatment results in preservation of kidney function as measured by BUN, plasma creatinine, glomerular filtration rate, or any combination thereof.

[0068] Another method of the invention is directed to treating a patient by administering a therapeutically effective amount of a composition to the patient, wherein the composition comprises an expression vector comprising a sequence encoding a cytochrome P450 inhibitor, and wherein said patient has a urinary protein excretion value of greater than about 300 mg per twenty-four hour time period. Generally, the sequence is an antisense sequence of a cytochrome P450 2B family member, preferably the antisense sequence is a partial sequence of a cytochrome 2B family member.

[0069] Another method of the invention is directed to treating a patient by administering a therapeutically effective amount of a composition to the patient, wherein the composition comprises an agent that inhibits a cytochrome P450 superfamily member, and wherein said agent preserves kidney function as measured by BUN, plasma creatinine level, glomerular filtration rate, or any combination thereof.

[0070] Referring now to FIGS. 1 and 2, a glomerular disorder such as minimal change disease can be induced by a single injection of an effective dose of PAN to an animal, such as, a rat. The effective amount of PAN is dependent on the body weight (BW) of the animal and generally is administered at a concentration of 7.5 mg/100 g BW. A single intravenous injection of PAN to rats results in marked proteinuria and renal morphological changes similar to minimal change disease in humans.

[0071] Intravenous administration of PAN results in nephrotic range proteinuria by the seventh day following administration, as shown in FIG. 1A. The catalytic iron content, as measured by the bleomycin detectable iron assay, is significantly elevated from a control value of 39 to 160 nmol/mg protein (N=3, P<0.01) in the glomeruli obtained from rats injected with PAN, as shown in FIG. 1B. In the liver, there is no difference in the level of CYP between the untreated and the PAN treated animals (FIG. 2B). There is a difference in the kidney CYP content of untreated and PAN treated animals however, wherein the CYP content of PAN treated rats was not dectable (FIG. 2A). Thus, administration of PAN results in a significant increase in the catalytic iron accompanied, at least, by a loss of CYP content in the glomeruli. This loss of CYP content is specific to the glomeruli as there is no difference detected in the levels of CYP content in the liver between PAN and control group of animals.

[0072] Referring now to FIGS. 3 and 4, the effects of two CYP inhibitors of the invention, cimetidine (CM) and piperonyl butoxide (PB), on the bleomycin detectable iron content in the glomeruli in PAN treated rats are shown. Without being limited by theory, it is believed that CM and PB function as inhibitors of CYP by interacting with the heme moiety of CYP. As shown in FIG. 3, both CM and PB significantly prevent the increase of bleomycin detectable iron in the glomeruli of PAN-treated rats. CM and PB also significantly preserve the loss of CYP content in the glomeruli of PAN-treated rats, as shown in FIG. 4. As discussed previously, CYP inhibitors in addition to CM and PB, are useful in the invention. These additional inhibitors include piperine, DEDC, and chlormethiazole.

[0073] Referring now to FIG. 5, the protective effect of two CYP inhibitors of the invention, CM and PB, on PAN-induced proteinuria is shown. Administration of PAN results in significant proteinuria on the fourth day with marked increases thereafter. The CYP inhibitor cimetidine is adminstered intraperitoneally one hour prior to PAN injection, and twice a day thereafter. The CYP inhibitor, PB, is administered intraperitoneally four hours prior to PAN injection, and every other day thereafter. As shown in FIG. 5, preincubation of the cells with the CYP inhibitor CM or the CYP inhibitor PB provides substantial protection against PAN-induced proteinuria.

[0074] Referring now to Table 1 and FIGS. 6-8, the effect of two CYP inhibitors, CM and PB, on serum albumin, and renal function is shown. PAN treated rats exhibit significant weight gain at the time of sacrifice compared to control animals and those treated with the CYP inhibitors CM and PB (Table 1).

[0075] INSERT TABLE 1 FROM Liu et al, manuscript, HERE.

[0076] The serum albumin of PAN treated animals is markedly decreased, and this decrease is prevented by CYP inhibitors, such as CM and PB (FIG. 6). Renal function as measured by serum creatinine is similar in all the groups, while the BUN value is significantly elevated in the rats treated with both PAN and CYP inhibitors, compared to that of the control animals (FIG. 7).

[0077] Referring now to FIG. 8, a role of CYP in an in vitro model of PAN induced cytotoxicity is shown. GEC cells are at least one of the specific cell-types injured in MCNS-induced nephrotoxicty. Exposure of GEC cells to a dose of 1.5 mM PAN results in a significant increase in the bleomycin detectable iron content. Treatment with either 2 mM CM or 25 μM PB drastically prevents the increase in the bleomycin detectable iron content. As a control, treatment with 1 mM RN, which has a similar structure as CM but is a weak inhibitor of CYP, is used to indicate this particular weak inhibitor of CYP does not prevent the marked increase in the bleomycin detectable iron content, and is not suitable for use alone. As weak CYP inhibitors may exxert a combinatory effect with respect to CYP inhibition, it is within the scope of the invention to combine the use of more than one weak CYP inhibitor in the compositions and methods described herein to result in a less weak CYP inhibitor.

[0078] Referring now to FIG. 9, a potential role of hydroxyl radicals in PAN induced cytotoxicity in GEC is shown. Iron has been shown to participate in the generation of powerful oxidant species, such as the hydroxyl radical via the metal catalyzed Haber-Weiss reaction. As shown in FIG. 9, exposure of GEC to PAN leads to a significant increase in hydroxyl radical formation. The CYP inhibitors CM and PB markedly reduce the PAN induced hydroxyl radical formation in GEC. The weak CYP inhibitor RN does not reduce the PAN induced hydroxy radical formation.

[0079] Referring now to FIGS. 10-11, PAN is cytotoxic to GEC in a manner that is both time and a dose dependent, as measured by the trypan blue exclusion assay (FIG. 10). However, two CYP inhibitors of the present invention, CM and PB, significantly reduce PAN induced ctotoxicity to the GEC (FIG. 11). RN, which has three times the H2 receptor blocking activity as CM but is a weak inhibitor of CYP, does not exhibit protection against PAN-induced proteinuria.

[0080] The present invention discloses possible existence of a close relationship between catalytic iron formation and the content of microsomal CYP, which is mainly involved in drug metabolism (Glassock et al, 1991, In the Kidney, 4th Edition, B. M Brenner and F. C. Rector Editors, 1182-1279.). The CYP inhibitors of the present invention are markedly effective in reducing PAN-induced proteinuria in rats, and also in PAN-induced cytotoxicity to GEC. It is suggested herein, that microsomal CYP is a major source of catalytic iron.

[0081] It is not outside the scope of the present invention that other intracellular sources of iron participating in ROM mediated glomerular injury may exist. For instance, mitochondrial cytochromes, iron-sulfur protein and other iron containing proteins may also serve as alternative sources of iron.

[0082] All references cited herein, including research articles, all U.S. and foreign patents and patent applications, are specifically and entirely incorporated by reference.

EXAMPLES

[0083] The following Examples are provided merely to illustrate the present invention, and are not meant to limit the scope of the claims in any way. Examples 1-5 were carried out in vivo, and Examples 610 were carried out in vitro.

Example 1

PAN-Induced Nephrotic Syndrome

[0084] Male Sprague-Dawley rats weighing 200-250 g were injected either saline or a single intravenous injection of PAN (Sigma, St. Louis) in a dose of 7.5 mg/100 g BW (day 0) as described in Ueda et al, 19996, Kidney Intl. 49:370-373. Animals were housed in separate metabolic cages and allowed free access to rat chow (Purina). Daily urine protein excretion was determined and animals were sacrificed on day 7. Blood was obtained for the measurement of serum albumin and the evaluation of renal function as measured by blood urea nitrogen (BUN) and plasma creatinine. Cell fraction of the glomeruli was prepared for bleomycin detectable iron assay. The microsome fraction was utilized for the measurement of CYP content.

Example 2

Inhibition of Cytochrome P450

[0085] Two different inhibitors of CYP were used. cimetidine (CM) has imidazole and cyano groups that inhibit CYP by interacting with the heme moiety. This effect of CM is specific for CYP, as it does not interact with other heme enzymes. To determine the effect of the CYP inhibitors in PAN-induced nephrotic syndrome, CM (120 mg/kg BW) was administered intraperitoneal 1h prior to PAN injection and then twice a day. Another CYP inhibitor, piperonyl butoxide (PB) which yields a metabolite that binds to the heme moiety of CYP, was given intraperitoneal (400 mg/kg BW) 4h before PAN injection and then every other day.

Example 3

Isolation of Glomeruli

[0086] Glomeruli were isolated by a combination of sieving and differential centrifugation as described in Ueda et al, 19996, Kidney Intl. 49:370-373, and Baliga et al, 1992, Am.J.Physiol. 263:214-221.

Example 4

Bleomycin Detectable Iron Assay

[0087] Iron capable of catalyzing free radical reactions was measured by bleomycin detectable iron assay as described by Gutteridge et al, 1981, Biochem.J.199:263-265, Gutteridge et al, 1982, Biochem.J. 206:605-609, Baliga et al, 1993, Bioche.J. 291:901-905, Baliga et al, 1996, Kidney Int. 49:362-369, Baliga et al, Kidney Int. 53:394-401, Ueda et al, 1996, Kidney Int.49:370373, Blaiga et al, 1998, Kidney Int. 54:1562-1569.

Example 5

Cytochrome P450 Content in Microsomes

[0088] Glomeruli isolated from rat kidneys were suspended in an extraction buffer containing 20 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 1 mM EDTA and protease inhibitor cocktail (5 μl/100 mg glomeruli wet weight, Sigma, St. Louis, Mo.) and frozen at −80° C. Subsequently, the glomeruli were thawed and sonicated. The homogenate was centrifuged at 15,000 g for 20 min at 4° C. and the precipitate discarded. The microsomes were sedimented by centrifugation of the supernatant at 105,000 g for 60 min at 4° C. The firmly packed pellet of microsomes was resuspended in above extracting buffer at a concentration of approximately 10 mg protein per ml.

[0089] CYP content was measured by the method of Omura and Sato, 1964, JBC 239:2370-2378. In brief, suspension of microsome from the glomeruli was diluted to about 1 mg of protein per ml with the assay buffer (0.1 M potassium phosphate buffer, pH 7.25, 20% glycerol, 0.2% tergitol). After recording the baseline, the sample was reduced with a few crystals of dithionite, and followed by CO bubbling for about 1 min. The CO difference spectrum of reduced microsomes was recorded on a Shimadzu UV-2101PC spectrophotometer. The peak absorbance at A450nm was measured, and the amount of CYP was determined using the extinction coefficient of 91 mM−1cm−1.

Example 6

Cell Culture

[0090] Rat GEC (kindly provided by Dr. Saulo Klahr, Washington University School of Medicine, St. Louis, Mo.) were maintained in RPMI-1640 medium supplemented with 10% FBS, 15 mM HEPES, insulin, penicillin, streptomycin and L-glutamine in a humidified atmosphere of 95% air-5% CO2 at 37° C. and fed at intervals of 3 days (Kasinath et al, 1995, Biochem.Biophys. 318(2):286-294). The cells were maintained in 75 cm2 tissue culture flasks and the monolayers were subcultured using 0.05% trypsin-0.53 mM EDTA in Hank's balanced salt solution (HBSS). For the experimental study, the cells were grown in 12-well tissue culture plate until confluence.

Example 7

PAN-Induced Cytotoxicity

[0091] On the day of experiment, the medium was discarded and the confluent GEC monolayer was washed twice with HBSS. The cells were then incubated with various concentrations of PAN (0, 0.1, 0.5, 1.0, 1.5, and 2.0 mM in HBSS) for different periods of time (0, 6h, 1, 2, 3 and 4 day) at 37° C. At the end of the incubation, the incubation medium was discarded and the GEC monolayer was harvested by trypsinization with 0.05% trypsin-0.53 mM EDTA for 5 min at 37° C. Isolated cells were suspended in HBSS to give about 106 cells/ml. Cell viability was determined by use of trypan blue exclusion assay (Baliga et al, 1996, Kidney Int. 50:1118-1124).

Example 8

Effect of CYP Inhibitors on the PAN Induced Cytotoxicity

[0092] Confluent GEC monolayers were washed twice with HBSS and then incubated with various concentrations of CM, ranitidine (RN, as a control of CM) for 30 min and PB for 60 min at 37° C. After the incubation, the cell monolayers were washed twice with HBSS and then incubated with cytotoxic dose of PAN in HBSS for a period of time necessary to induce consistent cytotoxicity (1.5 mM/ml, 48 h, based on the concentration and time course studies) at 37° C. PAN induced cytotoxicity on GEC was measured by trypan blue exclusion assay.

Example 9

Effect of CYP Inhibitors on PAN Induced Catalytic Iron Release

[0093] Confluent cell monolayer was washed three times with Chelex-treated HBSS to remove as much contaminating iron as possible. The GEC monolayer was then incubated in Chelex-treated HBSS with cytotoxic dose of PAN for a period time at 37° C. before substantial cell killing and after significant iron release occurs (1.5 mM, 60 min, based on a time course study on iron release induced by PAN, data not shown) The incubation medium was then collected for the measurement of catalytic iron using bleomycin detectable iron assay as mentioned above. To determine the effect of CYP inhibitors on the iron release, GEC monolayer was preincubated with CM (2 mM), RN (1 mM, as a control of CM) for 30 min or PB (25 μM) for 60 min in Chelex-treated HBSS at 37° C. After the incubation, the medium with CYP inhibitor was discarded and then the cell monolayers were washed twice with chelex-treated HBSS prior to the incubation of PAN.

Example 10

Effect of CYP Inhibitors on PAN Induced Hydroxyl Radical Formation

[0094] Confluent GEC monolayer was washed twice with HBSS and then incubated with 1.5 mM PAN in HBSS for a period of time before substantial cell killing occurs (1 h) at 37° C. 2-deoxy-D-ribose in a final concentration of 3 mM was added to the medium just prior to the incubation. At the end of the incubation, the incubation medium was collected for the measurement of hydroxyl radical formation by deoxyribose degradation method described in Baliga et al, 1996, Kidney Int. 50:1118-1124. To determine the effect of CYP inhibitors on the hydroxyl radical formation, GEC monolayers were preincubated with CM, RN or PB as mentioned above.

Example 11

Statistical Analysis

[0095] The values shown in FIG. 10 are expressed as mean ±standard error (SE). Statistical analysis was performed using unpaired t-test (for only two groups) and analysis of variance (for more than two groups) Statistical significance was considered at P<0.05.

[0096] While the illustrative embodiments of the invention have been described with particularity, it will be understood that various other modifications will be apparent to and can be readily made by those skilled in the art without departing from the spirit and scope of the invention. Accordingly, it is not intended that the scope of the claims appended hereto be limited to the examples and descriptions set forth herein but rather that the claims be construed as encompassing all the features of patentable novelty which reside in the present invention, including all features which would be treated as equivalents thereof by those skilled in the art to which this invention pertains. Thus, the specification and examples should be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.

	Number	Date	Country
Parent	09672296	Mar 2000	US
Child	10059763	Jan 2002	US

Compositions and methods for treating glomerular disorders

Information

Publication Number

Date Filed

Date Published

Inventors

CPC

US Classifications

International Classifications

Abstract

Description

Claims

REFERENCE TO GOVERNMENT FUNDING

Divisions (1)