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  Pharmaceutical Patents  

 

Title:  Therapeutic methods and agents for diseases associated with decreased expression of AOP-1 gene or AOP-1
United States Patent: 
7,598,228
Issued: 
October 6, 2009

Inventors: 
Hattori; Fumiyuki (Osaka, JP), Sugimura; Keijiro (Osaka, JP), Furuya; Mayumi (Osaka, JP)
Assignee: 
Asubio Pharma Co., Ltd. (Tokyo, JP)
Appl. No.: 
10/642,272
Filed: 
August 18, 2003

 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

A prophylactic or therapeutic method for a disease associated with decreased expression of AOP-1 gene or AOP-1, comprising (1) transfecting a nucleic acid encoding AOP-1 or a nucleic acid encoding a polypeptide having an addition, deletion or substitution of one or more amino acids as compared with the amino acid sequence of AOP-1 while retaining the function of AOP-1, or (2) administering a material enhancing the expression of AOP-1 gene, a material enhancing the production of AOP-1 or a material enhancing the function of AOP-1.

Description of the Invention

SUMMARY OF INVENTION

The present invention relates to:

(1) a prophylactic or therapeutic method for a disease associated with decreased expression of AOP-1 gene or AOP-1, comprising (1) transfecting a nucleic acid encoding AOP-1 or a nucleic acid encoding a polypeptide having an addition, deletion or substitution of one or more amino acids as compared with the amino acid sequence of AOP-1 while retaining the function of AOP-1, or (2) administering a material enhancing the expression of AOP-1 gene, a material enhancing the production of AOP-1 or a material enhancing the function of AOP-1, or (3) administering AOP-1 protein or a polypeptide having an addition, deletion or substitution of one or more amino acids as compared with the amino acid sequence of AOP-1 while retaining the function of AOP-1; as used herein, AOP-1 gene means AOP-1 mRNA unless otherwise specified though AOP-1 gene usually means to contain nucleic acid sequences encoding AOP-1 (exon sequences) and intervening nucleic acid sequences (intron sequences) and nucleic acid sequences regulating the transcription of AOP-1 gene;

(2) the prophylactic or therapeutic method as defined in (1) above comprising transfecting a nucleic acid for AOP-1 gene or a nucleic acid encoding a polypeptide having an addition, deletion or substitution of one or more amino acids as compared with the amino acid sequence of AOP-1 while retaining the function of AOP-1 into cells of an affected tissue;

(3) the prophylactic or therapeutic method as defined in (1) above comprising administering a material enhancing the expression of AOP-1 gene;

(4) the prophylactic or therapeutic method as defined in (1) above comprising administering a material enhancing the production of AOP-1;

(5) the prophylactic or therapeutic method as defined in (4) above wherein the material enhancing the production of AOP-1 is a nucleic acid encoding AOP-1 or a nucleic acid encoding a polypeptide having an addition, deletion or substitution of one or more amino acids as compared with the amino acid sequence of AOP-1 while retaining the function of AOP-1;

(6) the prophylactic or therapeutic method as defined in (1) above comprising administering a material enhancing the function of AOP-1;

(7) the prophylactic or therapeutic method as defined in any one of (1) to (6) above wherein the disease associated with decreased expression of AOP-1 gene or AOP-1 is chronic heart failure, ischemic heart failure, ischemic heart disease, rheumatoid arthritis, neurodegenerative disease, hepatic disease or renal failure;

(8) a prophylactic or therapeutic agent for a disease associated with decreased expression of AOP-1 gene or AOP-1, comprising as an active ingredient (1) a nucleic acid encoding AOP-1 or a nucleic acid encoding a polypeptide having an addition, deletion or substitution of one or more amino acids as compared with the amino acid sequence of AOP-1 while retaining the function of AOP-1, or (2) a material enhancing the expression of AOP-1 gene, a material enhancing the production of AOP-1 or a material enhancing the function of AOP-1, or (3) AOP-1 protein or a polypeptide having an addition, deletion or substitution of one or more amino acids as compared with the amino acid sequence of AOP-1 while retaining the function of AOP-1;

(9) the prophylactic or therapeutic agent as defined in (8) above comprising as an active ingredient a nucleic acid for AOP-1 gene or a nucleic acid encoding a polypeptide having an addition, deletion or substitution of one or more amino acids as compared with the amino acid sequence of AOP-1 while retaining the function of AOP-1;

(10) the prophylactic or therapeutic agent as defined in (8) above comprising as an active ingredient a material enhancing the expression of AOP-1 gene;

(11) the prophylactic or therapeutic agent as defined in (8) above comprising as an active ingredient a material enhancing the production of AOP-1;

(12) the prophylactic or therapeutic agent as defined in (11) above wherein the material enhancing the production of AOP-1 is a nucleic acid encoding AOP-1 or a nucleic acid encoding a polypeptide having an addition, deletion or substitution of one or more amino acids as compared with the amino acid sequence of AOP-1 while retaining the function of AOP-1;

(13) the prophylactic or therapeutic agent as defined in (8) above comprising as an active ingredient a material enhancing the function of AOP-1;

(14) the prophylactic or therapeutic agent as defined in any one of (8) to (13) above wherein the disease associated with decreased expression of AOP-1 gene or AOP-1 is chronic heart failure, ischemic heart failure, ischemic heart disease, rheumatoid arthritis, neurodegenerative disease, hepatic disease or renal failure;

(15) a diagnostic method for a disease associated with decreased expression of AOP-1 gene or AOP-1, comprising determining the expression level of AOP-1 gene or the production level of AOP-1 to make a diagnosis based on the expression level or production level;

(16) the diagnostic method as defined in (15) above wherein the disease associated with decreased expression of AOP-1 gene or AOP-1 is chronic heart failure, ischemic heart failure, ischemic heart disease, rheumatoid arthritis, neurodegenerative disease, hepatic disease or renal failure;

(17) a diagnostic agent or diagnostic kit for a disease associated with decreased expression of AOP-1 gene or AOP-1, comprising a means for determining the expression level of AOP-1 gene or the production level of AOP-1 as a measure;

(18) the diagnostic agent or diagnostic kit as defined in (17) above wherein the disease associated with decreased expression of AOP-1 gene or AOP-1 is chronic heart failure, ischemic heart failure, ischemic heart disease, rheumatoid arthritis, neurodegenerative disease, hepatic disease or renal failure;

(19) a non-human transgenic animal suitable for use as a pathologic model of a disease associated with decreased expression of AOP-1 gene or AOP-1 wherein the production of AOP-1 is suppressed or the expression of AOP-1 gene is suppressed or AOP-1 gene is deleted;

(20) the non-human transgenic animal as defined in claim 19 wherein the disease associated with decreased expression of AOP-1 gene or AOP-1 is chronic heart failure, ischemic heart failure, ischemic heart disease, rheumatoid arthritis, neurodegenerative disease, hepatic disease or renal failure;

(21) a transformed tissue or transformed cell suitable for use as a tissue model or a cell model of a disease associated with decreased expression of AOP-1 gene or AOP-1 wherein the production of AOP-1 is suppressed or the expression of AOP-1 gene is suppressed or AOP-1 gene is deleted;

(22) the transformed tissue or transformed cell as defined in (21) above wherein the disease associated with decreased expression of AOP-1 gene or AOP-1 is chronic heart failure, ischemic heart failure, ischemic heart disease, rheumatoid arthritis, neurodegenerative disease, hepatic disease or renal failure;

(23) a method for screening at least one selected from the group consisting of a material enhancing the expression of AOP-1 gene, a material enhancing the production of AOP-1 or a material enhancing the function of AOP-1, comprising administering or adding a synthesized or genetically engineered material or a natural material or a derivative thereof to the non-human transgenic animal or transformed tissue or transformed cell as defined in any one of (18) to (21) above to detect the expression level of AOP-1 gene or the production level of AOP-1;

(24) a method for screening at least one selected from the group consisting of a material enhancing the expression of AOP-1 gene, a material enhancing the production of AOP-1 or a material enhancing the function of AOP-1, comprising contacting a synthesized or genetically engineered material or a natural material or a derivative thereof with (1) a transformed cell or an in vitro expression system having a transcriptional regulatory region of AOP-1 gene and AOP-1 gene or a reporter gene to detect the expression level of AOP-1 gene or the reporter gene or with (2) AOP-1 or a target molecule of AOP-1 to detect the amount of AOP-1 or the target molecule of AOP-1;

(25) the screening method as defined in (24) above comprising constructing an expression vector having a transcriptional regulatory region of AOP-1 gene linked upstream or downstream of the translation region of a reporter gene, then culturing a suitable host cell transfected with said vector, adding a synthesized or genetically engineered material or a natural material or a derivative thereof to the cultured cell and detecting changes in the expression level of the reporter gene or the production level of the reporter protein after a given period;

(26) the screening method as defined in (24) above comprising contacting a synthesized or genetically engineered material or a natural material or a derivative thereof with AOP-1 or a target molecule of AOP-1 to detect the amount of AOP-1 or the target molecule of AOP-1 bound or unbound to said material;

(27) the screening method as defined in (24) above comprising immobilizing AOP-1 or a target molecule of AOP-1 on a substrate and adding a synthesized or genetically engineered material or a natural material or a derivative thereof and AOP-1 or target molecule of AOP-1 to the immobilized AOP-1 or target molecule of AOP-1 to detect the amount of AOP-1 or the target molecule of AOP-1 bound or unbound;

(28) the screening method as defined in (24) above comprising immobilizing a synthesized or genetically engineered material or a natural material or a derivative thereof on a substrate and adding AOP-1 or a target molecule of AOP-1 to the immobilized material to detect the amount of AOP-1 or the target molecule of AOP-1 bound or unbound;

(29) a method for screening a material enhancing the function of AOP-1, comprising contacting a synthesized or genetically engineered material or a natural material or a derivative thereof with AOP-1 or a target molecule of AOP-1 to determine the antioxidant or peroxynitrite scavenging activity of AOP-1;

(30) the screening method as defined in (29) above comprising adding a synthesized or genetically engineered material or a natural material or a derivative thereof and AOP-1 or a target molecule of AOP-1 to AOP-1 or the target molecule of AOP-1 to determine the antioxidant or peroxynitrite scavenging activity of AOP-1;

(31) the screening method as defined in (29) above comprising immobilizing AOP-1 or a target molecule of AOP-1 on a substrate and adding a synthesized or genetically engineered material or a natural material or a derivative thereof and AOP-1 or the target molecule of AOP-1 to the immobilized AOP-1 or target molecule of AOP-1 to determine the antioxidant or peroxynitrite scavenging activity of AOP-1;

(32) the screening method as defined in (29) above comprising immobilizing a synthesized or genetically engineered material or a natural material or a derivative thereof on a substrate and adding AOP-1 or a target molecule of AOP-1 to the immobilized material to determine the antioxidant or peroxynitrite scavenging activity of AOP-1;

(33) a use of (1) a nucleic acid for AOP-1 gene or a nucleic acid encoding a polypeptide having an addition, deletion or substitution of one or more amino acids as compared with the amino acid sequence of AOP-1 while retaining the function of AOP-1, or (2) a material enhancing the expression of AOP-1 gene, a material enhancing the production of AOP-1 or a material enhancing the function of AOP-1, or (3) AOP-1 protein or a polypeptide having an addition, deletion or substitution of one or more amino acids as compared with the amino acid sequence of AOP-1 while retaining the function of AOP-1, for the preparation of a prophylactic or therapeutic method for a disease associated with decreased expression of AOP-1 gene or AOP-1;

(34) the use as defined in (33) above comprising as an active ingredient a nucleic acid for AOP-1 gene or a nucleic acid encoding a polypeptide having an addition, deletion or substitution of one or more amino acids as compared with the amino acid sequence of AOP-1 while retaining the function of AOP-1;

(35) the use as defined in (33) above comprising as an active ingredient a material enhancing the expression of AOP-1 gene;

(36) the use as defined in (35) above wherein the material enhancing the production of AOP-1 is a nucleic acid encoding AOP-1 or a nucleic acid encoding a polypeptide having an addition, deletion or substitution of one or more amino acids as compared with the amino acid sequence of AOP-1 while retaining the function of AOP-1;

(37) the use as defined in (33) above comprising as an active ingredient a material enhancing the production of AOP-1;

(38) the use as defined in (33) above comprising as an active ingredient a material enhancing the function of AOP-1; and

(39) the use as defined in any one of (33) to (38) above wherein the disease associated with decreased expression of AOP-1 gene or AOP-1 is chronic heart failure, ischemic heart failure, ischemic heart disease, rheumatoid arthritis, neurodegenerative disease, hepatic disease or renal failure.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As used herein, "function of AOP-1" refers to the effect of activating/protecting organs and cells such as metabolic activation of heart/myocytes, protection of functions of heart/myocytes, suppression of heart/myocardial cellular death, activation of neurons, suppression of neuronal death and protection of renal function. Thus, the expression "having the function of AOP-1" as used herein means having the effect of activating/protecting organs and cells. Whether or not a material "has the function of AOP-1" is determined by various methods described in the section (6) Screening methods and in the examples below.

As used herein, "heart disease" means heart failure (including chronic heart failure, ischemic heart failure, diabetic heart failure, etc.), ischemic heart disease (including angina, myocardial infarction, etc.), etc. As used herein, "neurodegenerative disease" means cerebral infarction, Alzheimer's disease, Parkinson's disease, brain trauma, Huntington's chorea, cerebrovascular dementia, motor neuron degeneration, Binswanger's disease, etc. As used herein, "rheumatism" includes chronic rheumatoid arthritis, etc. As used herein, "renal disease" means renal failure such as diabetic nephropathy, hypertensive nephropathy, lupus nephropathy, etc. As used herein, "hepatic disease" means hepatitis (including viral hepatitis, alcoholic hepatitis and drug-induced hepatitis), cirrhosis, hepatic failure, etc.

We performed a series of experiments in order to find common causes of inducing cellular dysfunction or cellular death which is a common phenomenon in heart disease, neurodegenerative disease, rheumatism, renal disease and hepatic disease.

(1) Heart Diseases

In order to investigate the cause of breakdown at the compensatory stage in chronic heart failure, changes in the expression of various proteins in tissues with the progress from heart hypertrophy to chronic heart failure were first determined. A mixed protein solution in tissue extracts was separated into crude fractions consisting of a water-soluble fraction and a surfactant-soluble fraction, each of which was then analyzed by two-dimensional electrophoresis. Aortostenotic rats (heart pressure overload model), arteriovenous shunt rats (heart volume overload model), hereditary hypertensive rats (SHR) and Dahl salt-sensitive rats were used as pathologic models of chronic heart failure for comparative analysis. The proteins varying in the expression level with breakdown at the compensatory stage commonly in many models were searched to find common proteins related to chronic heart failure. Among them, AOP-1 protein was found to be decreased in expression with breakdown at the compensatory stage.

Gene expression analysis was performed to study whether or not changes in said protein with the progress of chronic heart failure result from any regulation at the level of gene expression. As a result, all the models tested showed suppressed expression of the gene, indicating that the decrease of AOP-1 with the progress of chronic heart failure results from the regulation of gene expression. Gene expression analysis using heart failure rats following myocardial infarction as an ischemic chronic heart failure model also showed suppressed expression of the gene. Thus, decreased expression of AOP-1 gene and therefore a decrease of AOP-1 was commonly found in chronic heart failure of a wide variety of causes. Analysis of changes in the gene expression of type 2 peroxiredoxin thiol-specific antioxidant (TSA) and the expression of CuZn-superoxide dismutase (CuZn-SOD), Mn-superoxide dismutase (Mn-SOD) and catalase genes having common antioxidant activity showed no change in the gene expression level of TSA, CuZn-SOD, Mn-SOD and catalase. Thus, AOP-1 was found to characteristically decrease expression with the transition to the heart failure stage among typical antioxidant proteins in the peroxiredoxin family. This shows that AOP-1 gene undergoes a different gene regulation from other antioxidant proteins.

Then, we tested whether or not AOP-1 has some effect on the improvement or aggravation of heart function. We began by isolating the full-length gene of rat AOP-1 gene to construct a suitable expression vector, and then transfected the AOP-1 gene into cultured rat cardiac myocytes. AOP-1 gene-transfected group was tested under the following experimental conditions: cells were cultured without oxygen (hypoxia), or cultured under hypoxia followed by reperfusion for a period (reperfusion), or cultured under normal oxia (untreated). In view of the influence of forced gene expression and protein production, control group transfected with E. coli .beta.-galactosidase gene, which does neither harm nor good, was tested in parallel and comparative analyses were performed between both groups. Comparative method 1 involves counting the number of viable cells, comparative method 2 involves counting the number of autonomously pulsating cells among viable cells, and comparative method 3 involves confirming the number of viable cells and cellular metabolic activity according to the MTT assay (J. Immunol. Methods 1983 Vol. 65, 55-63). These three comparative analyses showed that the number of viable cells under hypoxia and reperfusion as well as the number of autonomously pulsating cells significantly increased in the AOP-1 gene transfected group as compared with the control group. In the MTT assay, the AOP-1 gene transfected group showed high levels under hypoxia, reperfusion and normal oxia as compared with the control group, indicating an increase in the number of viable cells during injury and an increase in metabolic activity during injury and normal states. Thus, it was found that AOP-1 shows high efficacy in both cell viability and cell function maintenance against hypoxic injury and reperfusion injury. We further constructed an adenovirus vector for expressing antisense AOP-1 (antisense AOP-1 vector) designed to express a complementary strand to AOP-1 gene. Generally, it has been reported that the mRNA expressed from this type of vector complementarily binds to mRNA expressed from endogenous AOP-1 gene to inhibit translation into a protein (Gene 1990 Vol. 91, 261-265). We cultured cardiac myocytes transfected with antisense AOP-1 vector, AOP-1 expression vector and .beta.-galactosidase expression vector for 3 days in parallel with untransfected cells. The MTT assay was performed on each group to reveal that chromogenesis was suppressed in cells transfected with antisense AOP-1 vector and the number of viable cells macroscopically decreased. Thus, the suppressed expression of the endogenous AOP-1 by antisense AOP-1 vector adversely affected cell viability. In order to further directly compare the efficacy of AOP-1, TSA and CuZn-SOD in cellular protection effect, we constructed adenovirus vectors capable of forced expression of these genes. These vectors were transfected into rat cardiac myocytes to increase the respective proteins and the resistance to the following stresses was analyzed. As a result, AOP-1 showed the most effective cellular protective effect against stresses of hydrogen peroxide, hypoxia and high glucose. This suggests that AOP-1 plays an especially important role in the cell maintenance mechanism. The finding of a cellular protective effect on high glucose injury suggests that AOP-1 may be effective for diabetic heart diseases (diabetes-induced heart failure and ischemic heart disease (angina, myocardial infarction, etc.)).

These results show that the decrease of AOP-1 in various heart diseases is a major cause of breakdown of compensatory mechanism (pathologic aggravation).

It should be especially noted that AOP-1 can protect against not only active oxygen-induced injury during reperfusion but also various hypoxic injuries such as energy depletion injury and intracellular oxygenation injury and that it even activated cellular metabolic function under normal oxia. Thus, it was strongly suggested that the cellular function protective effect and cellular death suppression effect of AOP-1 are based on not only antioxidant activity but also a new function including cellular metabolic activation (mitochondrial activation). This new function of AOP-1 (i.e., mitochondrial activation) has never been found in any other antioxidant proteins. Thus, this is a novel and specific function of AOP-1.

We further injected AOP-1 gene into the heart and performed the following analyses with the purpose of testing whether or not AOP-1 shows a protective effect in vivo. As soon as the protein was expressed after the gene was injected, the heart was rapidly isolated and connected to a perfusion device. The isolation-induced injury was controlled by perfusing the heart with a solution containing a gas in an amount equivalent to that of blood to try to maintain heart function under conditions as found in vivo. Perfusion was temporarily stopped to turn this heart into an ischemic state and then resumed to study the influence of reperfusion. In the heart with forced expression of AOP-1, ischemic rigidity during ischemia was significantly retarded and a significantly better functional recovery was shown during reperfusion as well as a significant cellular necrosis suppressing effect during reperfusion as compared with the negative control heart (sham). Ischemic rigidity is shown to highly correlate to ATP depletion in cardiac myocytes (J Mol Cell Cardiol 1996, Vol. 28, 1045-1057). The AOP-1 mediated prolongation of the time from ischemia to ischemic rigidity shows that the mitochodrial activation of AOP-1 found by us in cultured cardiac myocytes also works in vivo in the heart. This result showed that AOP-1 protects against both ischemic injury and reperfusion injury not only in cultured cells but also in vivo via novel mitochondrial activation in addition to antioxidation. Thus, it was shown that transfection of AOP-1 gene or administration of a material enhancing the expression of AOP-1 or the function of AOP-1 provides effective therapy for ischemic heart failure and ischemic heart disease. As hypertrophy-induced circulation failure in the myocardial tissue is reported in not only ischemic heart failure and ischemic heart disease but also chronic heart failure (Chin. Med. Sci. J. 1995 Vol. 10(3), 151-157), ischemia and ischemic reperfusion seem to be common causes of injury in pathologies of chronic heart failure. Therefore, administration of the protein to the pathologic heart showing chronic heart failure or a sign of chronic heart failure is expected to be an effective therapy for chronic heart failure by protecting against myocardial cellular death and keeping the heart pulsating function.

(2) Affected Organs other than Heart

Then, gene expression analysis was performed in other affected organs for the purpose of investigating whether or not the function of AOP-1 is responsible for pathogenesis in organs other than heart. The result showed that the expression of AOP-1 also decreases in the kidney of a nephritis model, the brain in a neurodegenerative disease model, and the liver in an infectious hepatitis (septic shock) model. This strongly suggested that decreased expression of AOP-1 commonly contributes to pathologenesis/aggravation in many diseases. AOP-1 gene showed decreased expression in not only the heart failure model but also the brain tissue of the neurodegenerative disease model in contrast to CuZn-SOD and catalase genes which showed no change in expression. This demonstrated that the expression of AOP-1 gene also undergoes a different regulation from other antioxidant proteins in diseases of organs other than heart.

a. Neurodegenerative Diseases

An increase of the extracellular glutamate level induces excessive activation of glutamate receptors (NMDA receptors) in cerebral infarction, cerebrovascular dementia, brain trauma, Alzheimer's disease, motor neuron degeneration, Parkinson's disease, etc. (Eur J Neurosci 2000 Vol. 12(8), 2735-45; Brain Res 1994 Vol. 11, No. 642(1-2), 117-122; Acta Neurochir Suppl. 2000 Vol. 76, 437-8; Neurosci Lett 2001 Vol. 299(1-2), 37-40;J Neurosci Res 2001 Vol. 63(5), 377-87; Drugs Aging 2001 Vol. 18(10), 717-24). Cells stimulated by the activation of NMDA receptors open their calcium channels to lead to influx of calcium into the cells. Excessive activation of NMDA receptors induces calcium overload in cells. This calcium overload results in apoptosis or necrosis of neurons, whereby nervous function disorder becomes irreversible. Administration of AOP-1 may also be effective for cerebral nerve degeneration because they are also associated with decreased expression of AOP-1 as already shown. When AOP-1 was actually tested for the protection of glutamate injury in cultured neurons, cellular death was significantly suppressed in cells transfected with AOP-1 gene. It was also shown that neurite outgrowth is promoted and neuronal network formation is activated under unloaded condition. This may be due to the metabolic activation (novel function of AOP-1) caused by the overproduction of AOP-1. Thus, AOP-1 was found to be very useful in cell protection against calcium overload and neuronal activation in neurons.

To verify efficacy in neurodegenerative disease animal models, analyses were performed on an ibotenic acid-induced brain damage model. Ibotenic acid is an NMDA receptor agonist, which induces excessive intracellular calcium influx to lead to neuronal death. After an AOP-1 expression gene was injected into the brain's hippocampus to induce the expression of AOP-1, ibotenic acid was injected into the same site. After 2 days, the brain was removed and neurons were observed. As compared with a control group untransfected with AOP-1 gene, neuronal death was clearly suppressed in the brain transfected with AOP-1 gene. Quantitative analysis of the proliferation/infiltration of glial cells showed higher suppression as compared with the control group. As the proliferation/infiltration of glial cells has been shown to correlate with the amount of neuronal death (Journal of Neuroimmunology 2000 Vol. 1009, 105-111; Brain Research 1991, Vol. 565, 312-320), this result shows that neurons were protected by the transfection of AOP-1 gene. Thus, AOP-1 was found to also have a protective effect on neurons in the tissue. Therefore, administration of AOP-1 may be effective for neurodegenerative diseases such as cerebral infarction, cerebrovascular dementia, brain trauma, Alzheimer's disease, motor neuron degeneration, Parkinson's disease, etc.

b. Renal Diseases

Administration of AOP-1 may be effective for renal diseases as well as heart diseases and neurodegenerative diseases because the renal failure model also showed decreased expression of AOP-1 as shown above. To verify the efficacy of AOP-1 gene against chronic renal failure, analysis was actually performed on a Thy-1 nephritis model. The Thy-1 nephritis model was prepared by injecting an antibody against the Thy-1 cell surface antigen (Thy-1) protein specifically expressed in mesangial cells constituting glomerular cells to induce autoimmunization against Thy-1, leading to cellular death of mesangial cells followed by injury in renal tubules. Generally, the Thy-1 nephritis model is considered to be a renal failure model primarily involving inflammation. AOP-1 gene was injected simultaneously with the Thy-1 antibody and renal function was determined over time on the basis of the blood urea nitrogen. Analysis in comparison with a group untransfected with AOP-1 showed that renal dysfunction was significantly suppressed in the group transfected with AOP-1 gene. Thus, AOP-1 seems to be effective for chronic renal failure.

c. Hepatic Diseases

Hepatic diseases progress to cirrhosis and hepatic failure via viral, alcoholic and drug-induced hepatitis. It can be said that hepatic failure such as cirrhosis is caused by chronic overstress on the liver, which is originally a regenerative organ but fails to regenerate hepatic function. Thus, protection of hepatic cells should be clearly effective for preventing hepatic failure. As shown above, a hepatitis model showed decreased expression of AOP-1, which suggests that administration of AOP-1 may be effective in the same manner as in heart diseases, neurodegenerative diseases and renal diseases.

d. Rheumatism

In rheumatism, synovial cells differentiated into immune cells and proliferated and inflammatory cells in peripheral blood infiltrating into joints induce destruction of cartilage/bone to irreversibly destruct joints. Recently, it has become known that the regeneration of cartilages may be hampered by the apoptosis of chondroblasts under the stress of peroxynitrite (Arthritis & Rheumatism 1999 Vol. 42, No. 7, 1528-1537;J Agric Food Chem. 2001 Vol. 49(8), 3614-21). Analytic results on heart diseases, neurodegenerative disease, renal failure and hepatic failure suggest that the expression of AOP-1 may be decreased under stress threatening the life of cells. Thus, it is anticipated that the expression of AOP-1 may be decreased in chondroblasts and osteoblasts in rheumatic pathologies. Mitochondria are deeply involved in the intracellular cascade of apoptosis. Decreased mitochondrial function may directly trigger apoptosis (General Review: Advanced Medicine, Vol. 54, No. 4, 1999). Our analyses showed that AOP-1 has a protective effect on various cells and tissues. Administration of AOP-1 to chondroblasts and osteoblasts may protect and activate mitochondrial function and suppress the death of chondroblasts and osteoblasts. This makes it possible to maintain the regenerating ability of cartilage/bone.

Consequently, we found novel AOP-1 functions to related cellular protection/activation such as myocardial cellular metabolic activation, protection of myocardial cell function and suppression of myocardial cellular death, neuronal activation and suppression of neuronal death. We also found that administration of AOP-1 is effective for treating diseases associated with decreased expression of AOP-1 gene or AOP-1 such as chronic heart failure, ischemic heart failure, ischemic heart disease, chronic renal failure, neurodegenerative disease and rheumatism, and finally achieved the present invention.

As used herein, "diseases associated with decreased expression of AOP-1 gene or AOP-1" refers to those which contain cells showing decreased expression of AOP-1 gene or AOP-1 in affected tissue (e.g., heart in case of heart failure, neurons in case of brain damage, joints in case of rheumatism). A decrease of AOP-1 is synonymous with decreased intracellular AOP-1 function, so that diseases associated with AOP-1 function decreased by genetic variation can also be included in the scope of the present invention.

The following materials (1) to (4) can be used as active ingredients of prophylactic or therapeutic agents for diseases associated with decreased expression of AOP-1 gene or AOP-1 according to the present invention.

(1) Materials Having the Effect of Enhancing the Expression of AOP-1 Gene

Materials having the effect of enhancing the expression of AOP-1 gene may be any of synthesized or genetically engineered compounds or natural compounds or derivatives thereof, including materials acting on a promoter or enhancer region of AOP-1 gene to enhance the transcription of AOP-1 gene into mRNA or materials having a similar effect via transcriptional factors or the like in cells (e.g. materials binding to a transcriptional factor or a co-activator to promote binding to DNA or another transcriptional factor or co-activator, or materials binding to a transcriptional factor or a co-activator to inhibit binding to DNA or another transcriptional factor or co-activator).

(2) Materials Having the Effect of Enhancing the Production of AOP-1

Materials having the effect of enhancing the production of AOP-1 may be any of synthesized or genetically engineered compounds or natural compounds or derivatives thereof, including a nucleic acid encoding AOP-1 (RNA or DNA; SEQ ID NOS: 1 to 3) or a nucleic acid (RNA or DNA) encoding a polypeptide having an addition, deletion or substitution of one or more amino acids as compared with the amino acid sequence of AOP-1 while retaining the function of AOP-1. A nucleic acid (RNA or DNA) encoding a polypeptide hybridizing to a complementary strand of a nucleic acid encoding AOP-1 (RNA or DNA; SEQ ID NOS: 1 to 3) under stringent conditions while retaining the function of AOP-1 is also included. For example, a nucleic acid hybridizing to it in a solution containing 6.times.SSC, 0.5% SDS, 10 mM EDTA, 5.times. Denhardt's solution, 10 mg/ml denatured salmon sperm DNA at 68.degree. C. as described in the literature as standard methods (Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory Press, 1989) is included. Such a nucleic acid preferably has a sequence identity of 90% or more, more preferably 95% or more to a nucleic acid encoding AOP-1 (RNA or DNA; SEQ ID NOS: 1 to 3). Other materials such as modified virus vectors containing these nucleic acids are also suitable. Suitable examples are virus vectors, preferably lentivirus vectors, adeno-associated virus vectors, more preferably adenovirus vectors, or chemically synthesized liposomes, virus envelops or complexes of a virus envelop and a synthetic liposome containing a nucleic acid sequence integrated with AOP-1 gene downstream of a promoter sequence functional in a host cell such as cytomegalovirus promoter (CMV promoter).

Materials binding to AOP-1 mRNA or proteins or nucleic acids inhibiting the degradation of AOP-1 mRNA or materials having the activity of increasing the translation efficiency may also be included similarly to the production of TNF-.alpha. own to be regulated by the post-transcriptional stability of mRNA and stabilized by binding of HuR protein.

(3) Materials Having the Effect of Enhancing the Function of AOP-1

AOP-1 scavenges oxidation and peroxynitrite activity by enzymatically reversing redox of active site cysteine residue. Materials having the effect of enhancing the function of AOP-1 may be any of synthesized or genetically engineered compounds or natural compounds or derivatives thereof, including materials binding to this activated cysteine residue to promote redox cycle or materials binding to other than active sites of AOP-1 to promote redox cycle of active sites by allosteric effect. Materials promoting binding of AOP-1 to a target molecule (e.g. receptor) of AOP-1 protein on which AOP-1 protein acts or intracellular signal transduction to enhance the activity of AOP-1 protein are also suitable. For example, thioredoxin protein is known to bind to AOP-1 to increase biochemical antioxidant activity. Materials having efficacy by indirectly enhancing the function of AOP-1 via introduction of proteins enhancing the function of AOP-1 or genes thereof or synthetic induction are also suitable. Materials inhibiting the activity of proteases specifically degrading AOP-1 are also included.

(4) AOP-1 Proteins and Polypeptides Having the Function of AOP-1.

In the present invention, AOP-1 proteins per se or polypeptides having an addition, deletion or substitution of one or more amino acids as compared with the amino acid sequence of AOP-1 while retaining the function of AOP-1 can also be used as active ingredients of prophylactic or therapeutic agents for diseases associated with decreased expression of AOP-1 gene or AOP-1.

(5) Pharmaceutical Preparations

Pharmaceutical preparations containing the materials as described above as active ingredients are prepared using carriers or excipients or other additives used for standard formulation.

Active ingredients of pharmaceutical compositions of the present invention may be free or pharmaceutically acceptable salts thereof. Suitable salts include salts with inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid or salts with organic acids such as formic acid, acetic acid, butyric acid, succinic acid and citric acid. Salts with metals such as sodium, potassium, lithium and calcium or salts with organic bases are also suitable.

Preferably, active ingredients are mixed with known pharmacologically acceptable carriers, excipients, diluents or the like and administered in a pharmaceutically conventional manner such as oral administration or parenteral administration including intravenous, intramuscular or subcutaneous administration. Pharmaceutical compositions of the present invention can be prepared by, for example, appropriately mixing an active ingredient with physiologically acceptable carriers, flavoring agents, excipients, stabilizers, diluents, emulsifiers, solubilizing agents, suspending agents, syrups or the like and can be used as tablets, powders, granules, solutions, etc. Tablets can contain additives including, for example, binders such as gelatin and lubricants such as cornstarch, or can be coated with sugar or a gastric or enteric film. Capsules can be prepared by further including liquid carriers in said compositions. Sterile compositions for injection can also be prepared by applying conventional formulations. Aqueous solutions for injection include isotonic solutions containing glucose or the like and can be combined with suitable solubilizers such as polyethylene glycol. They may also contain buffers, stabilizers, preservatives, antioxidants, soothing agents and the like. When active ingredients are susceptible to degradation in the gastrointestinal tract via oral administration, they can be orally administered as formulations less susceptible to degradation in the gastrointestinal tract such as microcapsules enclosing active ingredients in liposomes, for example. They can also be adsorbed via non-gastrointestinal mucosa such as rectal, nasal or sublingual administration. In this case, they can be administered in the form of suppositories, nasal sprays or sublingual tablets.

For use in gene therapy, known vehicles suitable for gene therapy such as virus vectors, preferably lentivirus vectors, adeno-associated virus vectors, more preferably adenovirus vectors, or chemically synthesized liposomes, virus envelops or complexes of a virus envelop and a synthetic liposome can be used in which AOP-1 gene or a nucleic acid for a material enhancing the expression of AOP-1 gene or a material enhancing the production of AOP-1 or a material enhancing the function of AOP-1 has been integrated downstream of a promoter sequence functional in a host cell such as cytomegalovirus promoter (CMV promoter).

When pharmaceutical compositions of the present invention are therapeutically used, therapeutically effective doses are determined for each case depending on the age and the weight of the subject, the severity of the disease and the route of administration and other factors. Typically, the oral dose is about 0.1-1000 mg/adult/day, which may be administered once or divided into several subdoses.

(6) Screening Methods

Materials that can be used as active ingredients of prophylactic or therapeutic agents of the present invention can be screened as follows, for example.

AOP-1 gene or AOP-1 or derivatives thereof used for screening materials enhancing the expression of AOP-1 gene or enhancing the production of AOP-1 may be derived from any sources including mammals such as human (AOP-1 gene: SEQ ID NO: 1, AOP-1: SEQ ID NO: 4), rat (AOP-1 gene: SEQ ID NO: 2, AOP-1: SEQ ID NO: 5) and mouse (AOP-1 gene: SEQ ID NO: 3, AOP-1: SEQ ID NO: 6). Among them, those derived from human are preferably used for studies/developments of prophylactic or therapeutic agents for human. Those derived from animals such as mouse and rat are preferably used for studies/developments using animal models, i.e. non-human transgenic animals having chronic heart failuredue to suppressed or deleted expression of AOP-1 gene. However, those derived from human are desirably used for drug screening using animal models.

Materials enhancing the expression of AOP-1 gene or materials enhancing the production of AOP-1 or materials enhancing the function of AOP-1 are generally screened by methods using a reporter gene. Suitable reporter genes include, for example, chloramphenicol acetyl transferase (CAT), .beta.-galactosidase (.beta.-Gal) and luciferase. Materials enhancing the expression of AOP-1 gene can be screened by, for example, constructing an expression vector having an expression regulatory region (promoter, enhancer or the like region) of AOP-1 gene linked upstream or downstream of the translation region of a reporter gene, transfecting said vector into a suitable culture cell, adding a test material to the culture cell (said material may be any of synthesized or genetically engineered compounds or natural compounds or derivatives thereof) and determining the expression level of the reporter gene or the amount of the reporter protein after a given period. The expression regulatory region of AOP-1 gene (promoter, enhancer and the like region) can be obtained from commercially available genomic libraries by plaque hybridization using a fragment of AOP-1 cDNA as a probe. The amount of the reporter protein can be determined as enzymatic activity or the expression level of the protein using antibodies or the like.

Materials enhancing the production of AOP-1 include DNA containing an AOP-1 sequence or RNA, which can be integrated into a liposome or modified virus vector with similar effects.

Materials enhancing the function of AOP-1 can also be screened by mixing 4 components, i.e. purified AOP-1 or a cell lysate, desirably a mitochondrial fraction and hydrogen peroxide and a reducing agent having a thiol group (e.g. dithiothreitol) and a monitor enzyme having measurable enzymatic activity, and after a given period, determining the activity of the monitor enzyme (Biochemical and Biophysical Research Communications 1994 Vol. 199, No. 1, 199-206; Journal of Biological Chemistry 1996 Vol. 271, No. 26, 15315-15321). Peroxynitrite (obtained by e.g. mixing an acidified nitrite and hydrogen peroxide) is screened by mixing 3 components, i.e. purified AOP-1 or a cell lysate, desirably a mitochondrial fraction and peroxynitrite and a monitor enzyme having measurable enzymatic activity or a substance (e.g. DNA) modified with peroxynitrite, and after a given period, determining the activity of the monitor enzyme or the amount of the substance modified (Nature 2000 Vol. 407, No. 14, 211-215). Thus, materials enhancing the function of AOP-1 can be screened by determining the protective activity of AOP-1 against the deactivation of a monitor enzyme by free radical injury.

(7) Diagnostic Methods, Diagnostic Agents and Diagnostic Kits

AOP-1 gene was shown by us to have an expression level decreased with aggravation of various diseases. The extent of aggravation of chronic heart failure can be known by determining the expression level of AOP-1 gene using a biopsy sample of a patient. For example, the expression level of AOP-1 gene can be determined by isolating total RNA from 100 mg of a biopsy sample of a patient using ISOGEN (Nippon Gene) and treating it with DNase, then synthesizing cDNA, amplifying AOP-1 gene by PCR using suitable primers and determining the strength of the band corresponding to AOP-1 by gel electrophoresis. The expression of AOP-1 gene can also be assayed by any other techniques for assaying RNA or DNA such as the method described in Example 3, Northern hybridization or cDNA array techniques.

As AOP-1 gene was shown by the present invention to be an improving factor of various diseases, it can be readily predicted that individuals having some variation in AOP-1 gene or a regulatory region thereof tend to be susceptible to such disease as chronic heart failure or aggravation in such disease if the function of AOP-1 is lowered or the gene expression level is decreased by the presence of such variation. Thus, risk factors can be diagnosed by testing these variations on the gene. Variations on the gene can be known by isolating DNA from a blood sample of a patient according to standard methods, determining the nucleotide sequence according to the method described in Examples 1-5 and comparing it with the normal sequence. Once the relation between a variation and chronic heart failure has been clarified, the DNA chip or SSCP technique can be applied to detect only such a variation.

Moreover, a biopsy sample of a patient affected with a disease accompanied by chronic heart failure can be used to know the extent of aggravation of chronic heart failure or said disease by determining the AOP-1 level in cardiac myocytes. Suitable assay methods include ELISA or RIA using antibodies against AOP-1, or HPLC or mass spectrometric assays. AOP-1 here may not be in a whole form but may be fragmented so far as it can be assayed.

The present invention also include diagnostic agents or diagnostic kits for chronic heart failure comprising a means for determining the expression level of AOP-1 gene or the production level of AOP-1 using the assay means as described above.

(8) Non-Human Animals and Cells or Tissues Transformed with AOP-1 Gene

Materials of the present invention can be identified by not only the methods as described above in the section (6) Screening methods, but also by using non-human transgenic animals having chronic heart failure or other condition due to suppressed expression of AOP-1. Non-human host animals include small animals such as mouse, rat and rabbit as well as large animals such as dog, pig, sheep and cattle and any other animals expressing the gene and showing the function or physiological action. Gene expression can be suppressed by introducing variation or deletion or the like into a transcriptional regulatory region of AOP-1 or any other means capable of suppressing gene expression. The production of AOP-1 can be suppressed by introducing DNA or RNA complementary to AOP-1 mRNA or a gene encoding the complementary sequence or any other means capable of suppressing the production of AOP-1. Methods for deleting the gene include techniques using embryonic stem cells from knockout mice or any other systems for suppressing the expression of the gene.
 

Claim 1 of 5 Claims

1. A method for treating a heart disease, comprising administering by direct injection into a heart an expression vector comprising a nucleic acid sequence encoding AOP-1 operably linked to a promoter, wherein expression of said nucleic acid sequence within cells of the heart enhances the production of AOP-1, and wherein said expression of AOP-1 protects against myocardial cellular death or maintains the heart pulsating function in said heart.

 

 

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