Pharmaceutical composition for the treatment of chronic kidney disease

Abstract

The purpose of the present invention is to establish a means for treating chronic kidney diseases including Alport's syndrome. The present invention provides a pharmaceutical composition characterized by containing: a) a renin / angiotensin-based inhibitor; and b) at least one omega-3 polyunsaturated fatty acid, an ester thereof, a metabolite thereof, a pharmaceutically acceptable salt thereof, or a triglyceride thereof.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for use in the treatment of chronic kidney diseases such as Alport syndrome. Background Art

[0002] Alport syndrome is an inherited disease in which progressive nephritis inevitably develops from early childhood due to mutations in the type IV collagen gene, and it is a rare disease sometimes accompanied by hearing impairment, ocular lesions, and diffuse leiomyomatosis. Since patients develop end-stage renal failure at a young age, it is designated as an intractable disease by the government.

[0003] In type IV collagen, α chains form a triple helix, and these multimers form a network structure. In Alport syndrome, the collagen mesh structure is not formed due to impaired synthesis of any or all of the type IV collagen α3, α4, and α5 chain proteins. The glomerular filtration membrane is formed by vascular endothelial cells, basement membrane, and podocyte foot processes of glomerular epithelial cells. In the glomeruli of Alport syndrome, the type IV collagen constituting the basement membrane is dysplastic or completely deficient, which causes abnormalities in glomerular filtration function and accompanying chronic decline in renal function.

[0004] Homozygous deficient mice for the type IV collagen α3 chain gene (Col4a3 - / - mice) are a pathological model of the autosomal recessive form of Alport syndrome, and their variation in pathological conditions is small, so their average lifespan is almost constant. Specifically, tubular dilation, inflammatory cell infiltration, crescent formation, and fibrosis appear from 6 weeks of age, the serum creatinine concentration rises rapidly, and individuals that die from end-stage renal failure appear after 8 weeks of age, with most individuals dying by 10 weeks of age. Due to such characteristics, these mice are widely used worldwide because the efficacy of pharmaceutical products can be evaluated in a short period of time.

[0005] In non-clinical studies using Col4a3- / - mice, it has been reported that survival time was significantly extended and urinary protein levels and serum urea concentrations were significantly reduced in groups treated with ramipril (an angiotensin-converting enzyme (ACE) inhibitor) or candesartan (an angiotensin II receptor antagonist), both of which are standard antihypertensive drugs (Non-Patent Literature 1). In clinical trials, Gross et al. retrospectively analyzed the relationship between oral administration of renin-angiotensin system inhibitors and renal dysfunction in 283 Alport syndrome patients and confirmed that the oral administration group significantly extended the median age at which renal replacement therapy was initiated (Non-Patent Literature 2). The Japanese Alport Syndrome Treatment Guidelines 2017 (Japanese Society of Pediatric Nephrology) also recommend the administration of renin-angiotensin system inhibitors to patients with Alport syndrome to suppress the progression of renal dysfunction. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Oliver Gross et al., Nephrol Dial Transplant (2004) 1716-23 [Non-Patent Document 2] Oliver Gross et al., Kidney Int (2012) 81:494-501 [Overview of the project] [Problems that the invention aims to solve]

[0007] As mentioned above, patients with Alport syndrome are administered renin-angiotensin system inhibitors, but there is a need for the development of drugs that can treat Alport syndrome more effectively. This invention was made against this backdrop and aims to provide a means of treating chronic kidney disease, including Alport syndrome. [Means for solving the problem]

[0008] As a result of diligent research to solve the above problems, the inventors of the present invention have found that administering an angiotensin II receptor antagonist and omega-3 polyunsaturated fatty acid ethyl ester to Alport syndrome model mice significantly improves the survival rate of these model mice, and have completed the present invention.

[0009] In other words, the present invention provides the following (1) to (15). (1) A pharmaceutical composition characterized by containing (a) a renin-angiotensin system inhibitor and (b) at least one omega-3 polyunsaturated fatty acid, its ester, its metabolite, its pharmaceutically acceptable salt, or its triglyceride derivative.

[0010] (2) A pharmaceutical composition for use in combination with a renin-angiotensin system inhibitor, characterized by containing at least one omega-3 polyunsaturated fatty acid, an ester thereof, a metabolite thereof, a pharmaceutically acceptable salt thereof, or a triglyceride thereof.

[0011] (3) A pharmaceutical composition for use in combination with an omega-3 polyunsaturated fatty acid, its ester, its metabolite, its pharmaceutically acceptable salt, or its triglyceride derivative, characterized in that it contains a renin-angiotensin system inhibitor.

[0012] (4) A pharmaceutical composition according to any one of (1) to (3), characterized in that it is used for the treatment of chronic kidney disease.

[0013] (5) The pharmaceutical composition according to (4), characterized in that the chronic kidney disease is a glomerulotoxic kidney disease.

[0014] (6) The pharmaceutical composition according to (4), characterized in that the chronic kidney disease is podocyte-mediated kidney disease or glomerular basement membrane-mediated kidney disease.

[0015] (7) The pharmaceutical composition according to (4), wherein the chronic kidney disease is tubular renal disease or interstitial renal disease.

[0016] (8) The pharmaceutical composition according to (4), wherein the chronic kidney disease is Alport syndrome.

[0017] (9) The pharmaceutical composition according to any one of (1) to (8), wherein the renin-angiotensin system inhibitor is an angiotensin II receptor antagonist.

[0018] (10) The pharmaceutical composition according to any one of (1) to (8), wherein the renin-angiotensin system inhibitor is candesartan cilexetil.

[0019] (11) The pharmaceutical composition according to any one of (1) to (8), wherein the renin-angiotensin system inhibitor is valsartan.

[0020] (12) The pharmaceutical composition according to any one of (1) to (11), wherein the at least one omega-3 polyunsaturated fatty acid is eicosapentaenoic acid, docosahexaenoic acid, or eicosapentaenoic acid and docosahexaenoic acid.

[0021] (13) The pharmaceutical composition according to any one of (1) to (11), wherein the at least one omega-3 polyunsaturated fatty acid is eicosapentaenoic acid and docosahexaenoic acid.

[0022] (14) The pharmaceutical composition according to any one of (1) to (11), wherein the at least one omega-3 polyunsaturated fatty acid is docosahexaenoic acid.

[0023] (15) The pharmaceutical composition according to any one of (1) to (11), wherein the at least one omega-3 polyunsaturated fatty acid is eicosapentaenoic acid.

[0024] This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2020-219397, which is the priority basis of the present application. Effects of the Invention

[0025] The present invention provides a novel pharmaceutical composition. This pharmaceutical composition can be used for the treatment of chronic kidney diseases such as Alport syndrome. Brief Description of the Drawings

[0026] [Figure 1] A diagram showing the transition of the survival rate of each group of Alport syndrome model mice. In the diagram, PBS indicates the PBS group (untreated group), CAND indicates the candesartan alone group, PFD indicates the pirfenidone alone group, LOT indicates the loteplase alone group, CAND+PFD indicates the combined group of candesartan and pirfenidone, and CAND+LOT indicates the combined group of candesartan and loteplase. [Figure 2] A diagram showing the transition of the average body weight of each group of Alport syndrome model mice. In the diagram, WT indicates wild type, PBS indicates the PBS group (untreated group), CAND indicates the candesartan alone group, PFD indicates the pirfenidone alone group, LOT indicates the loteplase alone group, CAND+PFD indicates the combined group of candesartan and pirfenidone, and CAND+LOT indicates the combined group of candesartan and loteplase. [Figure 3] A diagram showing the transition of the total body weight of each group of Alport syndrome model mice. In the diagram, WT indicates wild type, PBS indicates the PBS group (untreated group), CAND indicates the candesartan alone group, PFD indicates the pirfenidone alone group, LOT indicates the loteplase alone group, CAND+PFD indicates the combined group of candesartan and pirfenidone, and CAND+LOT indicates the combined group of candesartan and loteplase. [Figure 4]This figure shows the results of blood biochemistry tests (kidney-related parameters) for each group of Alport syndrome model mice. From left to right, each bar graph represents: wild type (WT), PBS group (PBS), candesartan monotherapy group (CAND), pyrezoma monotherapy group (PFD), Lotriga monotherapy group (LOT), candesartan and pyrezoma combination group (CAND+PFD), and candesartan and Lotriga combination group (CAND+LOT). [Figure 5] This figure shows the results of bone mineral metabolism-related parameter tests for each group of Alport syndrome model mice. From left to right, each bar graph represents: wild type (WT), PBS group (PBS), candesartan monotherapy group (CAND), pyrezoma monotherapy group (PFD), Lotriga monotherapy group (LOT), candesartan and pyrezoma combination group (CAND+PFD), and candesartan and Lotriga combination group (CAND+LOT). [Figure 6] This figure shows the results of blood biochemistry tests (pancreatic and liver-related parameters) for each group of Alport syndrome model mice. From left to right, each bar graph represents: wild type (WT), PBS group (PBS), candesartan monotherapy group (CAND), pyrezoma monotherapy group (PFD), Lotriga monotherapy group (LOT), candesartan and pyrezoma combination group (CAND+PFD), and candesartan and Lotriga combination group (CAND+LOT). [Figure 7] HE staining of the kidneys of wild-type (WT) mice and Alport syndrome model mice from the PBS (untreated) group, the candesartan monotherapy group (CAND), and the candesartan and Lotriga combination group (CAND+LOT). [Figure 8] PAS staining of kidneys in wild-type (WT) mice and Alport syndrome model mice from the PBS (untreated) group, the candesartan monotherapy group (CAND), and the candesartan and Lotriga combination group (CAND+LOT). [Figure 9] MT staining images of the kidneys of wild-type (WT) mice and Alport syndrome model mice from the PBS (untreated) group, the candesartan monotherapy group (CAND), and the candesartan and Lotriga combination group (CAND+LOT). [Figure 10]PAM staining of the kidneys of wild-type (WT) mice and Alport syndrome model mice from the PBS (untreated) group, the candesartan monotherapy group (CAND), and the candesartan and Lotriga combination group (CAND+LOT). [Figure 11] This figure shows the degree of glomerular damage based on the renal pathology of each group of Alport syndrome model mice. In the figure, WT represents the wild type, PBS represents the PBS group (untreated group), CAND represents the candesartan monotherapy group, PFD represents the pirespa monotherapy group, LOT represents the Lotriga monotherapy group, CAND+PFD represents the candesartan and pirespa combination group, and CAND+LOT represents the candesartan and Lotriga combination group. [Figure 12] This figure shows the degree of tubular and interstitial damage based on renal pathology in each group of Alport syndrome model mice. In the figure, WT represents the wild type, PBS represents the PBS group (untreated group), CAND represents the candesartan monotherapy group, PFD represents the pirespa monotherapy group, LOT represents the Lotriga monotherapy group, CAND+PFD represents the candesartan and pirespa combination group, and CAND+LOT represents the candesartan and Lotriga combination group. [Figure 13] This figure shows the changes in survival rates between the candesartan monotherapy group and the omega-3 polyunsaturated fatty acid combination therapy group. [Figure 14] This figure shows the changes in survival rates in the group receiving candesartan in combination with either ethyl docosahexaenoic acid or ethyl eicosapentaenoic acid. [Figure 15] This figure shows the change in average body weight in the group receiving candesartan and either ethyl docosahexaenoic acid or ethyl eicosapentaenoic acid. ** P<0.01: Significant difference compared to the untreated group by t-test. When calculating average body weight, the body weight of deceased mice was not treated as zero; the body weight measured immediately before death was used in the calculation. [Figure 16] This graph shows the changes in the average weight of each treatment group. ** P<0.01: Significant difference compared to the untreated group as determined by the t-test. [Figure 17] This figure shows the average percentage change in body weight and survival rate for each group at the end of the study. [Figure 18] A graph showing the trend in survival rates for each test group. [Figure 19]This figure shows the average percentage change in body weight and survival rate for each group at the end of the study. [Modes for carrying out the invention]

[0027] The present invention will be described in detail below. The pharmaceutical composition of the present invention is a pharmaceutical composition for administering to a patient a) a renin-angiotensin system inhibitor and b) at least one omega-3 polyunsaturated fatty acid, its ester, its metabolite, its pharmaceutically acceptable salt, or its triglyceride form. The pharmaceutical composition of the present invention is sufficient to administer a) and b) to a patient, and may be, for example, a pharmaceutical composition characterized by containing a) and b), a pharmaceutical composition for use in combination with a) characterized by containing b), or a pharmaceutical composition for use in combination with b) characterized by containing a).

[0028] The renin-angiotensin system inhibitor used may be an angiotensin-converting enzyme inhibitor, an angiotensin II receptor antagonist, or a renin inhibitor, but preferably an angiotensin II receptor antagonist, more preferably candesartan cilexetil or valsartan, and even more preferably candesartan cilexetil. The aforementioned "renin-angiotensin system inhibitors," "angiotensin-converting enzyme inhibitors," "angiotensin II receptor antagonists," and "renin inhibitors" also include substances (so-called prodrugs) that undergo metabolism such as oxidation, reduction, or hydrolysis in the body to exhibit the effects of renin-angiotensin system inhibitors, angiotensin-converting enzyme inhibitors, angiotensin II receptor antagonists, or renin inhibitors.

[0029] Known angiotensin-converting enzyme inhibitors can be used, such as captopril, cilazapril, enalapril, hosinopril, lisinopril, quinapril, ramipril, zofenopril, imidapril, temocapril, perindopril, alacepril, delapril, benazepril, and trandolapril. Known angiotensin II receptor antagonists can also be used, such as candesartan, candesartan cilexetil, eprosartan, irbesartan, losartan, tasosartan, telmisartan, valsartan, azilsartan, and olmesartan.

[0030] Known omega-3 polyunsaturated fatty acids can also be used, such as eicosapentaenoic acid, docosahexaenoic acid, and alpha-linolenic acid. The pharmaceutical composition of the present invention may contain only one type of omega-3 polyunsaturated fatty acid, for example, only eicosapentaenoic acid or only docosahexaenoic acid, or it may contain two or more types of omega-3 polyunsaturated fatty acids, for example, eicosapentaenoic acid and docosahexaenoic acid.

[0031] Instead of omega-3 polyunsaturated fatty acids, their esters may be used. Specific examples of esters include methyl esters, ethyl esters, propyl esters, and esters to which phospholipids or lysophospholipids are bound. Suitable omega-3 polyunsaturated fatty acid esters include ethyl eicosapentaenoate and ethyl docosahexaenoate. The product may also contain metabolites of omega-3 polyunsaturated products, such as resolvin D1-D4, resolvin E1-E2, protectin D1, 17S-HDHA, and analogs of these metabolites. In addition, instead of omega-3 polyunsaturated fatty acids, pharmaceutically acceptable salts thereof, or derivatives such as their triglycerides, may also be included.

[0032] The omega-3 polyunsaturated fatty acids, their esters, their metabolites, their pharmaceutically acceptable salts, or their triglycerides are preferably 1) docosahexaenoic acid, its ester, its metabolite, its pharmaceutically acceptable salt, or its triglyceride, or 2) eicosapentaenoic acid, its ester, its metabolite, its pharmaceutically acceptable salt, or its triglyceride; more preferably, docosahexaenoic acid, its ester, its metabolite, its pharmaceutically acceptable salt, or its triglyceride; even more preferably, docosahexaenoic acid esters; and particularly preferably, docosahexaenoic acid ethyl ester.

[0033] The weight ratio of angiotensin-converting enzyme inhibitor to omega-3 polyunsaturated fatty acid in the pharmaceutical composition is not particularly limited. For example, the amount of omega-3 polyunsaturated fatty acid per 1 part angiotensin-converting enzyme inhibitor in the pharmaceutical composition can be 150 to 2000, preferably 150 to 1000, and more preferably 300 to 1000.

[0034] The pharmaceutical composition of the present invention can be used for the treatment of chronic kidney disease. Here, "treatment" includes not only inhibiting the progression of chronic kidney disease and curing chronic kidney disease, but also preventing chronic kidney disease.

[0035] In this invention, "chronic kidney disease" refers to a condition in which kidney function is reduced compared to that of a healthy person (for example, a glomerular filtration rate of 60 mL / min / 1.73 m³). 2This refers to a state in which kidney abnormalities persist, such as a decrease to less than a certain level, or the presence of proteinuria. The following kidney diseases are included in "chronic kidney disease" in this invention: Alport syndrome, focal segmental glomerulosclerosis, minimal change nephrotic syndrome, membranous nephropathy, HIV-1 related nephropathy, diffuse mesangial sclerosis, Finnish congenital nephrotic syndrome, lupus nephritis, collapsed glomerulosis, diabetic nephropathy, hypertensive nephrosclerosis, obesity-related nephropathy, IgA nephropathy, polycystic kidney disease (ADPKD, ARPKD), mesangial proliferative glomerulonephritis, Epstein syndrome, nail-patella syndrome, fibronectin nephropathy, lipoprotein glomerulopathy, chronic pyelonephritis, familial juvenile hyperuricemia nephritis, hereditary nephrotic syndrome, and tubulointerstitial nephritis. Furthermore, the following diseases are classified as rapidly progressive glomerulonephritis (RPGN), but since renal function decline often persists after treatment and progresses to chronic kidney disease, they are included in "chronic kidney disease" in this invention: crescentic glomerulonephritis, ANCA-associated glomerulonephritis, anti-GBM antibody-type glomerulonephritis, Henoch-Schönlein purpura glomerulonephritis, and cryoglobulinemia glomerulonephritis. "Suppression of the progression of chronic kidney disease" means, for example, suppression of glomerular damage and tubular damage. Glomerular damage includes, for example, glomerulosclerosis, crescent formation, and bilayered basement membrane, while tubular damage includes, for example, tubular dilation with hyaline casts, interstitial fibrosis, inflammatory cell infiltration into the interstitium, and tubular dilation with cellular casts.

[0036] The target of treatment is any chronic kidney disease, but preferably glomerulotoxic kidney disease, tubular kidney disease, or interstitial kidney disease; more preferably podocyte-mediated kidney disease or glomerular basement membrane-mediated kidney disease; and even more preferably Alport syndrome.

[0037] The pharmaceutical composition of the present invention is usually administered orally, but may be administered by other routes. For example, it may be administered sublingually, intradermally, subcutaneously, intramuscularly, via peripheral and central veins, arteries, lymphatic vessels, anus, nasal cavity, airway, or abdominal cavity.

[0038] The pharmaceutical composition of the present invention may contain other components such as pharmacoactive ingredients, nutrients, and excipients as needed. Examples of dosage forms of the pharmaceutical composition include tablets, capsules, granules, syrups, injections, intravenous infusions, patches, and suppositories. These can be formulated according to conventional methods using solvents, dispersion media, bulking agents, excipients, etc., as appropriate.

[0039] The pharmaceutical composition of the present invention may be in any form that allows the patient to receive both a) a renin-angiotensin system inhibitor and b) at least one omega-3 polyunsaturated fatty acid, its ester, its metabolite, its pharmaceutically acceptable salt, or its triglyceride. For example, both a) and b) may be formulated into a single dosage form (combination formulation), or a) and b) may be formulated into separate dosage forms (combination formulation). If formulated into separate dosage forms, they may be administered simultaneously or with a time difference. If administered with a time difference, a) may be administered first and b) later, or b) may be administered first and a) later. The administration methods may be the same or different.

[0040] The dosage of the pharmaceutical composition of the present invention is not particularly limited, but it is preferable that one of the active ingredients, a renin-angiotensin system inhibitor, be administered at a dose of 2 to 12 mg per adult per day, and more preferably at a dose of 4 to 8 mg. [Examples]

[0041] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0042] [Example 1] 1. Outsourcing of testing The following tests were outsourced to external facilities.

[0043] 2.Purpose Alport syndrome model mice (129-Col4a3tm1Dec / J) will be repeatedly administered each test substance orally for 10 weeks, after which serum biochemical tests will be performed. In addition, the kidneys will be histopathologically examined to confirm the effects of each test substance on the kidneys.

[0044] 3. Animal Welfare All animal experiments conducted in this example were planned in accordance with the laboratory animal welfare regulations of the contracted organization and carried out after approval by the Laboratory Animal Welfare Committee. Furthermore, the animal experiments conducted complied with the "Basic Guidelines on the Implementation of Animal Experiments, etc., in Research Institutions, etc. under the Jurisdiction of the Ministry of Agriculture, Forestry and Fisheries" (Notification from the Secretariat of the Agriculture, Forestry and Fisheries Research Council, 2006), etc.

[0045] 4. Test materials 4.1. Test substance (known compound) 4.1.1.Name [Table 1] Candesartan is a hypertension medication classified as an angiotensin II receptor antagonist, a type of renin-angiotensin system inhibitor. It is known to have renal protective effects. Pirespa is a drug used to treat idiopathic pulmonary fibrosis, possessing antifibrotic effects that suppress interstitial fibrosis in the lungs. Lotriga is an omega-3 polyunsaturated fatty acid ethyl preparation containing ethyl eicosapentaenoate and ethyl docosahexaenoate as its main components, and is used to treat hyperlipidemia.

[0046] 4.1.2. Preparation method of test substances, etc. The test substances were prepared for each minute of use on the day of the experiment. Candesartan was dissolved in phosphate-buffered saline (PBS) at a dose of 4 mg / ml to prepare the administration solutions for groups 6 and 7 (10 mg / 2.5 ml / kg). This administration solution was diluted with an equal volume of PBS to prepare the administration solution for group 3 (10 mg / 5 ml / kg). Pirespa was dissolved in PBS at a dose of 40 mg / mL to prepare the administration solution for group 6 (50 mg / 1.25 ml / kg). This administration solution was diluted with an equal volume of PBS to prepare the administration solution for group 4 (50 mg / 2.5 ml / kg). Lotriga was suspended in 0.5% methylcellulose solution (MC) at a dose of 600 mg / mL to prepare the administration solution for group 7 (1500 mg / 2.5 ml / kg). This administration solution was diluted with an equal volume of MC to obtain the administration solution for group number 5 (1500 mg / 5 ml / kg).

[0047] 4.2. Animal Experiments 4.2.1. Animals used For the experiment, male 129-Col4a3tm1Dec / J mice (hereinafter referred to as Homo mice) were introduced to the testing facility at 3 weeks of age, and healthy animals (4 weeks of age) that showed no abnormalities in their general condition during the approximately 1-week acclimatization period were used in the experiment.

[0048] 4.2.2. Rearing Environment The animals were kept in cages (189 mm x 298 mm x 128 mm) with a maximum of 5 animals per cage, in an animal enclosure controlled to a temperature of 20-25°C, humidity of 40-70%, ventilation of more than 10 times per hour, and lighting for 12 hours (7:00-19:00). They were given free access to solid feed CRF-1 (Oriental Yeast Co., Ltd.). They were also given free access to filtered water.

[0049] 5. Experimental Method 5.1. Group composition [Table 2]

[0050] 5.2. Grouping Animals identified as Homo (AS in Table 2) by genotyping were divided into groups 2 through 7 based on their 4-week-old body weight, with the mean values ​​between groups being similar. Group 1 consisted of four animals selected to have similar body weights to the Homo group. After grouping, individual identification was performed using ear piercings. Animals excluded from the experiment and those showing abnormalities during the acclimatization period were euthanized.

[0051] 5.3. Observation of General Conditions Observations of the general condition were performed once a day from the start of the experiment (grouping day) to the end of the experiment (sample collection day).

[0052] 5.4. Weight Measurement Measurements were taken twice a week from the group assignment date and on the day of sample collection for each group, using an animal scale.

[0053] 5.5. Administration of the test substance The animals were restrained and administered orally using a 1 mL syringe and tube. The medication was administered according to the conditions in section 5.1, ensuring that each animal received approximately 0.1 ml of the drug. The dosage was calculated based on the animal's most recent body weight. For Pirespa, the daily dose was 100 mg / kg, administered in two divided doses. The interval between doses was at least 6 hours.

[0054] 5.6. Collection of biological materials On Day 45, the animals' weight was measured, and blood was drawn from the posterior vena cava under isoflurane anesthesia. Euthanasia was then performed by bleeding. Death was confirmed by visual inspection of cardiopulmonary arrest (euthanasia). Subsequently, the left and right kidneys, left and right femurs, left and right eyeballs, calf, heart, and pancreas were collected and trellised. The wet weights of the left and right kidneys, left and right femurs, and heart were measured. Of the collected biological tissues, the left and right femurs were immersed in a 70% ethanol solution. The rest were immersed in a 10% neutral buffered formalin solution. The kidneys were divided into two parts, with the renal pelvis of the right kidney being the central point, and then immersed.

[0055] 5.7. Blood Biochemistry Tests 5.7.1. Serum preparation The obtained blood was left to stand at room temperature for more than 4 hours, and then serum was obtained by centrifugation (4°C, 12000 rpm, 3 min). The serum was divided into two tubes and stored frozen.

[0056] 5.7.2. Measurement of each item Blood biochemistry parameters were measured using frozen serum samples sent to an external facility. The measured parameters were total protein, albumin, blood urea nitrogen (BUN), creatinine (CRE), uric acid, sodium (Na), potassium (K), chloride (Cl), calcium, phosphorus, amylase, lipase, AST (GOT), ALT (GPT), γ-GTP, LDH, triglycerides, total cholesterol, HDL cholesterol, total bilirubin, and glucose. Pooled samples from each group were used for measurement.

[0057] 5.8. Histopathological Examination Kidneys immersed in 10% neutral buffered formalin were sent to an external facility for pathological evaluation using Hematoxylin-Eosin (HE) staining, Periodic Acid-Schiff (PAS) staining, Masson's Trichrome (MT) staining, and Periodic Acid Methenamine Silver (PAM) staining.

[0058] 6. Data Analysis For each test group, the mean and standard error values ​​of body weight, kidney tissue weight, and femoral tissue weight were calculated.

[0059] 7. Humanitarian endpoints Animals excluded from the study based on group classification, or animals that deteriorated or became incurable, were euthanized. Euthanasia was performed using carbon dioxide inhalation. • Rapid weight loss: More than 25% loss in 7 days • Distressing symptoms: difficulty breathing, piloerection, hunching over, extreme cyanosis • Long-term external abnormalities with no signs of recovery: inability to change position, diarrhea, bleeding, genital soiling

[0060] 8. Experimental Results 8.1. Trends in Survival Rates Figure 1 shows the changes in survival rates for each group of Alport syndrome model mice.

[0061] The trial was terminated on day 45, the day after all-cause mortality was confirmed in the PBS group (untreated group). While all mice in the Lotriga monotherapy group and the Pirespa monotherapy group died early, all mice in the candesartan and Lotriga combination group survived with good overall condition. The survival rate in the candesartan and Pirespa combination group was 60%, which was higher than the survival rate in the candesartan monotherapy group (40%).

[0062] 8.2. Trends in average weight Figure 2 shows the changes in average body weight for each group of Alport syndrome model mice. Note that when calculating average body weight, the weight of deceased mice was not treated as zero; instead, the weight measured immediately before death was used in the calculation.

[0063] In the group receiving candesartan and Lotriga, the average body weight increased to wild-type levels until day 32 after administration. Although there was a slight decrease thereafter until the end of the experiment, it was significantly greater than in the other groups, and the overall condition was good. In the group receiving candesartan alone, weight loss began from day 25 after administration, and this weight loss was significant even in the surviving mice.

[0064] 8.3. Changes in total body weight Figure 3 shows the changes in the total body weight of each group of Alport syndrome model mice.

[0065] When the sum of the body weights of only the mice that survived at the time of weighing was plotted over time, the difference between the candesartan and Lotriga combination group and the candesartan monotherapy group became clearer. Ultimately, the candesartan and pirespa combination group outperformed the candesartan monotherapy group, but it did not surpass the candesartan and Lotriga combination group.

[0066] 8.4. Blood biochemistry tests (kidney-related items) Figure 4 shows the results of blood biochemistry tests (kidney-related parameters) for each group of Alport syndrome model mice.

[0067] In the candesartan and Lotriga combination group (100% survival rate) and the candesartan and Pirespa combination group (60% survival rate), BUN and creatinine levels were above the upper limit of normal, but were much lower than those in the untreated group and the candesartan monotherapy group, and Na, K, and Cl levels were all within the normal range. In the candesartan monotherapy group (40% survival rate), BUN and creatinine levels were very high, similar to the untreated group (0% survival rate), and the patient exhibited hyperkalemia. It is clear that the progression of renal failure was delayed in the candesartan and Lotriga combination group, but renal failure progressed at a similar rate to the untreated group with candesartan monotherapy.

[0068] 8.5. Bone Mineral Metabolism-Related Tests Figure 5 shows the results of bone mineral metabolism-related parameter tests for each group of Alport syndrome model mice.

[0069] In the group receiving candesartan and Lotriga (100% survival rate), serum calcium and inorganic phosphorus levels remained within the normal range, and femoral weight was maintained at a level comparable to wild-type individuals. The progression of renal failure was prolonged, renal osteodystrophy did not develop, and ectopic calcification of blood vessels and major organs did not occur, which is thought to have significantly contributed to the improved survival rate.

[0070] 8.6. Blood biochemistry tests (pancreatic and liver-related items) Figure 6 shows the results of blood biochemistry tests (pancreatic and liver-related parameters) for each group of Alport syndrome model mice.

[0071] In the group receiving candesartan and Lotriga (100% survival rate), amylase levels were significantly lower than in the untreated group and the candesartan-alone group, and lipase levels were normal. This suggests that the combination of candesartan and Lotriga suppressed the development of secondary pancreatitis by prolonging the progression of renal failure. Regarding the impact on liver function, both AST and ALT levels remained within the normal range in the candesartan and Lotriga combination group.

[0072] 8.7. Histopathological examination of the kidney (HE staining) Figure 7 shows HE staining images of kidneys from the wild-type, PBS group, candesartan monotherapy group, and candesartan and Lotriga combination group.

[0073] In the PBS group and the candesartan monotherapy group, glomerular sclerosis and crescent formation leading to adhesions with the glomeruli, tubular dilation, and lymphocyte infiltration into the interstitium were observed sporadically. However, in the candesartan and Lotriga combination therapy group, glomerular sclerosis and crescent formation were hardly observed, and there were no changes in the tubules or interstitium. The arrows indicate examples of normal glomeruli.

[0074] 8.8. Histopathological examination of the kidney (PAS staining) Figure 8 shows the PAS staining images of kidneys from the wild-type group, the PBS group, the candesartan monotherapy group, and the candesartan and Lotriga combination group.

[0075] In the PBS group and the candesartan monotherapy group, glomerular sclerosis with deposition of PAS-positive material in the mesangial matrix and crescent cell proliferation were observed, and PAS-positive hyaline casts were also confirmed in the tubular dilatation in the PBS group. In the candesartan and Lotriga combination therapy group, glomerular sclerosis and crescent formation were hardly observed, and there were no changes in the tubules or interstitium. The arrows indicate examples of normal glomeruli.

[0076] 8.9. Histopathological examination of the kidney (MT staining) Figure 9 shows the MT staining images of kidneys from the wild-type, PBS group, candesartan monotherapy group, and candesartan and Lotriga combination group.

[0077] Fibrosis was observed in the crescent-like hyperplasia and tubulointerstitium in the PBS group and the candesartan monotherapy group, but no fibrosis was observed in the candesartan and Lotriga combination therapy group.

[0078] 8.10. Histopathological examination of the kidney (PAM staining) Figure 10 shows the PAM staining images of kidneys from the wild-type group, the PBS group, the candesartan monotherapy group, and the candesartan and Lotriga combination therapy group.

[0079] Duplication of the capillary wall was observed in the PBS group, the candesartan monotherapy group, and the candesartan and Lotriga combination group. The arrows indicate examples of duplication of the capillary wall.

[0080] 8.11. Assessment of glomerular dysfunction The degree of glomerular damage was evaluated in each group of Alport syndrome model mice. Specifically, the degree of glomerular sclerosis, crescent formation, and bilaminate basement membrane was quantified on a 5-point scale (0; No change, 1; Slight, 2; Mild, 3; Moderate, 4; Severe; reference: "Guidelines for the Pathological Diagnosis of Diabetic Nephropathy and Hypertensive Nephrosclerosis," Tokyo Medical Publishing). The results are shown in Figure 11.

[0081] Excluding the wild-type group, the group receiving candesartan and Lotriga had the least degree of glomerular damage.

[0082] 8.12. Assessment of renal tubular dysfunction The degree of tubular damage was evaluated in each group of Alport syndrome model mice. Specifically, The degree of tubular dilation with hyaline casts, interstitial fibrosis, inflammatory cell infiltration into the interstitium, and tubular dilation with cellular casts was quantified on a 5-point scale (0; No change, 1; Slight, 2; Mild, 3; Moderate, 4; Severe; reference: "Guidelines for Pathological Diagnosis of Diabetic Nephropathy and Hypertensive Nephrosclerosis," Tokyo Medical Publishing). The results are shown in Figure 12.

[0083] Excluding the wild-type group, the group receiving candesartan and Lotriga had the least degree of tubular damage.

[0084] [Example 2] 1. Outsourcing of testing The following tests were outsourced to external facilities.

[0085] 2.Purpose We will investigate the effect of administering ethyl omega-3 fatty acid alone or in combination with a renin-angiotensin system inhibitor to Alport syndrome model mice (129-Col4a3tm1Dec / J) on inhibiting the progression of chronic renal failure.

[0086] 3. Animal Welfare All animal experiments conducted in this commissioned study were planned in accordance with the laboratory animal welfare regulations of the contracting organization and carried out after approval by the laboratory animal welfare committee. Furthermore, the animal experiments conducted complied with the "Basic Guidelines on the Implementation of Animal Experiments, etc., in Research Institutions, etc., under the Jurisdiction of the Ministry of Agriculture, Forestry and Fisheries" (Notification from the Secretariat of the Agriculture, Forestry and Fisheries Research Council, 2006), etc.

[0087] 4. Test materials 4.1. Test substance (known compound) 4.1.1.Name [Table 3]

[0088] 4.1.2. Preparation method of test substances, etc. The test substance was prepared for each minute of use on the day of testing. Candesartan cilexetil (hereinafter, candesartan: Tokyo Chemical Industry Co., Ltd.) The drug was dissolved in phosphate-buffered saline (PBS) at a dose of 1.12 mg / mL to prepare the administration solutions for group numbers (8), (9), and (10) (2.8 mg / 2.5 mL / kg). This administration solution was diluted with an equal volume of PBS to prepare the administration solution for group number (7) (2.8 mg / 5 mL / kg). In addition, the drug was dissolved in PBS at a dose of 4 mg / mL to prepare the administration solutions for group numbers (11) and (12) (10 mg / 2.5 mL / kg).

[0089] Enalapril maleate (hereinafter, Enalapril: Tokyo Chemical Industry Co., Ltd.) The drug was dissolved in PBS at a dose of 3.28 mg / mL to prepare the administration solution for group number (4) (8.2 mg / 2.5 mL / kg). This administration solution was then diluted with an equal volume of PBS to prepare the administration solution for group number (3) (8.2 mg / 5 mL / kg).

[0090] Valsartan (Tokyo Chemical Industries) The drug was dissolved in PBS at a dose of 53.44 mg / mL to prepare the administration solution for group number (6) (133.6 mg / 2.5 mL / kg). This administration solution was then diluted with an equal volume of PBS to prepare the administration solution for group number (5) (133.6 mg / 5 mL / kg).

[0091] Omega-3 fatty acid ethyl (1) (hereinafter referred to as Lotriga: Takeda Pharmaceutical Company) The drugs were suspended in a 0.5% methylcellulose solution (hereinafter referred to as MC), and the administration solutions for group numbers (4) and (6) were prepared at 1671.4 mg / 2.5 mL / kg, while the administration solution for group number (8) was prepared at 501.0 mg / 2.5 mL / kg.

[0092] Omega-3 fatty acid ethyl (2) (hereinafter referred to as DHA97E: Bizen Kasei) The solution was suspended in 0.5% MC, and the administration solution for group number (9) was prepared at 421.0 mg / 2.5 mL / kg, and the administration solution for group number (11) was prepared at 1503.6 mg / 2.5 mL / kg. This administration solution was diluted with an equal volume of PBS to prepare the administration solution for group number (13) (1503.6 mg / 5 mL / kg).

[0093] Omega-3 fatty acid ethyl (3) (hereinafter referred to as EPA97E: Bizen Kasei) The solution was suspended in 0.5% MC, and the administration solution for group number (10) was prepared at 421.0 mg / 2.5 mL / kg, while the administration solution for group number (12) was prepared at 1503.6 mg / 2.5 mL / kg. [Table 4] [Table 5]

[0094] 4.2. Animal Experiments 4.2.1. Animals used For the experiment, male 129-Col4a3tm1Dec / J mice (hereinafter referred to as Alport syndrome; AS mice) and wild-type mice of the same strain (WT mice) were introduced to the experimental facility at 3 weeks of age. Healthy animals (4 weeks of age) that showed no abnormalities in their general condition during the acclimatization period were used in the experiment.

[0095] 4.2.2. Rearing Environment The animals were kept in cages (189 mm x 298 mm x 128 mm) with a maximum of 5 animals per cage, in an animal enclosure controlled to a temperature of 20-25°C, humidity of 40-70%, ventilation of more than 10 times per hour, and lighting for 12 hours (7:00-19:00). They were given free access to solid feed CRF-1 (Oriental Yeast Co., Ltd.). They were also given free access to filtered water.

[0096] 5. Experimental Method 5.1. Group composition [Table 6]

[0097] 5.2. Grouping AS mice identified as Homo by genotyping were divided into groups (2) to (13) based on their body weight at 4 weeks of age, with the mean values ​​between groups being similar. Group (1) consisted of 5 mice selected to have similar body weights to the AS mouse group. After group division, individual identification was performed using ear piercings. Animals excluded from the study and animals showing abnormalities during the acclimatization period were euthanized.

[0098] 5.3. Observation of General Conditions General conditions were assessed once a day from the start of the experiment (group assignment day) to the end of the experiment (sample collection day).

[0099] 5.4. Weight Measurement Measurements were taken twice a week from the grouping date and daily from the date of the first individual's death until the date of sample collection, using an animal scale.

[0100] 5.5. Administration of the test substance The animals were restrained, and the drug was administered directly into the stomach using a 1 mL syringe and a tube (Fuchigami Kiki Co., Ltd., CAT No. 5200). The drug was administered according to the conditions in Section 5.1, ensuring that each animal received a dose of 5 mL / kg. The dose was calculated based on the most recent (prior to the administration day) body weight.

[0101] 5.6. Collection of biological materials 5.6.1. Implementation Date The date for collecting biological samples was determined in consultation with the research sponsor when all animals in group number (2) were euthanized or died.

[0102] 5.6.2. Collection method Death was confirmed by visual inspection of cardiopulmonary arrest (euthanasia). Subsequently, the left and right kidneys, left and right femurs, left and right eyeballs, cochlea, heart, liver, and pancreas were collected and trimmed, and the wet weight of the left and right kidneys, left and right femurs, and heart was measured.

[0103] Of the collected biological tissues, the left and right femurs were immersed in a 70% ethanol solution (Fujifilm Wako Pure Chemical Industries, Ltd.). The rest were immersed in a 10% neutral buffered formalin solution (Fujifilm Wako Pure Chemical Industries, Ltd.). The kidney was divided into two parts, centering on the renal pelvis of the right kidney, and immersed in each part.

[0104] 6. Data Analysis For each test group, the mean and standard error values ​​of body weight and wet weight of each organ were calculated. Furthermore, for each measurement, group (2) comparisons were performed between the group numbers and each individual group. Statistical analysis was performed using the statistical analysis add-in software "Excel Statistics 2015" (manufactured by Social Information Service Co., Ltd.), and a significance level (P-value) of less than 0.05 was considered statistically significant.

[0105] 7. Humanitarian endpoints Animals excluded from the study based on group classification, or animals whose condition deteriorated or became irrecoverable, were euthanized. Euthanasia was performed using carbon dioxide inhalation. • Rapid weight loss: More than 25% loss in 7 days • Distressing symptoms: difficulty breathing, piloerection, hunching over, extreme cyanosis • Long-term external abnormalities with no signs of recovery: inability to change position, diarrhea, bleeding, genital soiling

[0106] 8. Experimental Results 8.1. Trends in Survival Rates Figures 13, 14, and 18 show the changes in survival rates for each treatment group. As shown in Figure 13, the combination of candesartan and DHA ethyl ester showed a higher survival rate than the combination with Lotriga, suggesting that DHA ethyl ester may be a potential treatment for Alport syndrome. On the other hand, no difference was observed when EPA ethyl ester was added compared to candesartan alone. As shown in Figure 14, DHA ethyl ester, even at normal doses, was more effective than high doses of EPA ethyl ester. As shown in Figure 18, Lotriga was also effective when added to valsartan, an active ARB, and the survival rate in the valsartan (high dose)-Lotriga (high dose) combination group was significantly higher than that of the valsartan (high dose) alone group. On the other hand, no effect was observed when Lotriga was added to enalapril, an ACE inhibitor.

[0107] 8.2. Trends in average weight Figures 15 and 16 show the changes in average body weight for each test group. As shown in Figure 15, DHA ethyl ester and EPA ethyl ester, when used in combination with candesartan, each showed an effect of suppressing weight loss. The effect of DHA ethyl ester significantly outweighed that of EPA ethyl ester, and in particular, the high-dose group showed a weight change comparable to that of the wild type. As shown in Figure 16, only the average body weight of the group administered DHA ethyl ester increased significantly compared to the untreated group.

[0108] 8.3. Relationship between survival rate and average body weight Figures 17 and 19 show the average weight change rate and survival rate for each group at the end of the study. Figure 17 shows the test group and control group for candesartan dose of 10 mg / kg / d in Example 1 (*) and Example 2. As shown in Figure 17, the candesartan-DHA ethyl ester combination group had a 100% survival rate and an average weight change rate comparable to the wild type, confirming the extremely excellent effect of DHA ethyl ester in suppressing the progression of chronic renal failure. In addition, EPA ethyl ester, when administered in combination with candesartan, significantly improved the survival rate compared to candesartan monotherapy, confirming an excellent effect in suppressing the progression of chronic renal failure. As shown in Figure 19, not only the prodrug candesartan but also the active form valsartan showed improved survival rates and high weight change rates when combined with Lotriga.

[0109] All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety. [Industrial applicability]

[0110] Since this invention relates to pharmaceuticals, it can be used in industries such as pharmaceutical manufacturing.

Claims

1. a) an angiotensin-converting enzyme inhibitor or an angiotensin II receptor antagonist and b) docosahexaenoic acid, an ester of docosahexaenoic acid, a triglyceride of docosahexaenoic acid, resolvin D1-D4, protectin D1, 17S-HDHA, or a pharmaceutically acceptable salt thereof, wherein the pharmaceutical composition is used for the treatment of glomerulotoxic kidney disease, and the angiotensin-converting enzyme inhibitor is captopril, cilazapril, enalapril, hosinopril, lisinopril, quinapril, ramipril, zofenopril, imidapril, temocapril, perindopril, alacepril, delapril, benazepril, or trandolapril, and the angiotensin II receptor antagonist is candesartan, candesartan A pharmaceutical composition comprising cilexetil, eprosartan, irbesartan, losartan, tasosartan, telmisartan, valsartan, azilsartan, or olmesartan.

2. A pharmaceutical composition for administration to a person who has been administered an angiotensin-converting enzyme inhibitor or an angiotensin II receptor antagonist, characterized in that it contains docosahexaenoic acid, an ester of docosahexaenoic acid, a triglyceride of docosahexaenoic acid, resolvin D1 to D4, protectin D1, 17S-HDHA, or a pharmaceutically acceptable salt thereof, the pharmaceutical composition used for the treatment of glomerulotoxic kidney disease, wherein the angiotensin-converting enzyme inhibitor is captopril, cilazapril, enalapril, hosinopril, lisinopril, quinapril, ramipril, zofenopril, imidapril, temocapril, perindopril, alacepril, delapril, benazepril, or trandolapril, and the angiotensin II receptor antagonist is candesartan, candesartan A pharmaceutical composition comprising cilexetil, eprosartan, irbesartan, losartan, tasosartan, telmisartan, valsartan, azilsartan, or olmesartan.

3. A pharmaceutical composition for administration to a person who has been administered docosahexaenoic acid, an ester of docosahexaenoic acid, a triglyceride of docosahexaenoic acid, resolvin D1-D4, protectin D1, 17S-HDHA, or a pharmaceutically acceptable salt thereof, characterized in that it contains an angiotensin-converting enzyme inhibitor or an angiotensin II receptor antagonist, a pharmaceutical composition used for the treatment of glomerulotoxic kidney disease, wherein the angiotensin-converting enzyme inhibitor is captopril, cilazapril, enalapril, hosinopril, lisinopril, quinapril, ramipril, zofenopril, imidapril, temocapril, perindopril, alacepril, delapril, benazepril, or trandolapril, and the angiotensin II receptor antagonist is candesartan, candesartan A pharmaceutical composition comprising cilexetil, eprosartan, irbesartan, losartan, tasosartan, telmisartan, valsartan, azilsartan, or olmesartan.

4. The pharmaceutical composition according to any one of claims 1 to 3, characterized in that the glomerulotoxic kidney disease is Alport syndrome.

5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that the angiotensin-converting enzyme inhibitor or angiotensin II receptor antagonist is an angiotensin II receptor antagonist.

6. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that the angiotensin-converting enzyme inhibitor or angiotensin II receptor antagonist is candesartan cilexetil.

7. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that the angiotensin-converting enzyme inhibitor or angiotensin II receptor antagonist is valsartan.

8. A pharmaceutical composition according to any one of claims 1 to 7, characterized in that docosahexaenoic acid, an ester of docosahexaenoic acid, a triglyceride of docosahexaenoic acid, resolvin D1 to D4, protectin D1, 17S-HDHA, or a pharmaceutically acceptable salt thereof is docosahexaenoic acid.

9. The pharmaceutical composition according to any one of claims 1 to 8, further characterized by containing eicosapentaenoic acid, an ester of eicosapentaenoic acid, a triglyceride of eicosapentaenoic acid, resolvin E1 to E2, or a pharmaceutically acceptable salt thereof.

Images