JP2006525269A - Angiotensin II receptor blocker for preventing the development or progression of microvascular disease due to diabetes - Google Patents

Abstract

The present invention relates to the field of angiotensin II receptor blockers and, in particular, the onset or progression of microvascular diseases (ie, diseases involving small blood vessels) that adversely affect the eyes (diabetic retinopathy) and kidneys (diabetic nephropathy) To deal with their use in diabetes to prevent.

Description

Detailed Description of the Invention

  The present invention relates to the field of inhibitors of angiotensin II receptor blockers, particularly for microvascular diseases (ie, small blood vessel related diseases) that adversely affect the eyes (diabetic retinopathy) and kidneys (diabetic nephropathy). Address their use in diabetes to prevent onset or progression.

  Diabetes is a disorder in which the body cannot properly metabolize carbohydrates (eg, carbohydrates, sugars, cellulose). This disease is characterized by insufficient sugar and insufficient production and / or utilization of excess sugar, insulin in the blood (hyperglycemia) and urine, and is also characterized by thirst, starvation and weight loss. About 2% of the population is affected by diabetes. 10-15% of these are insulin-dependent (type 1) diabetes and the rest are insulin-independent (type 2) diabetes.

  Retinopathy is retinal damage caused by microvascular changes. Diabetic retinopathy is a microvascular complication specific for both type 1 and type 2 diabetes. Retinopathy morbidity is strongly associated with the duration of diabetes. After the 20-year diabetic calendar, nearly 60% of all type 1 and type 2 diabetic patients have some degree of retinopathy. Diabetic patients are more than 25 times more likely to lose vision than ordinary people. One-fifth of newly diagnosed diabetics are known to have some retinopathy. In addition, retinopathy develops earlier and is more severe in patients with high systolic blood pressure levels. Careful eye examination, on average, reveals mild retinal abnormalities about 7 years after the onset of diabetes, but damage that threatens vision does not occur much later. Diabetic retinopathy is the most common cause of blindness in the working generation in many countries.

  In early retinopathy, weakening of small blood vessels in the retina results in bulges of the blood vessels (microaneurysms) and leakage of fluid (leachate) and blood (bleeding). Proliferative retinopathy, a later stage of the disease, involves the growth of fragile new blood vessels on the retina as well as the vitreous body, a jelly-like substance inside the eyeball. These blood vessels can rupture and release blood into the glass body, resulting in blurred vision and temporary blindness. Subsequent scar tissue can pull the retina and cause retinal detachment, which can lead to permanent vision loss. Macular edema swelling can be caused by fluid accumulated around the macula, the most important part for clear vision. If proliferative retinopathy is left untreated, about half of patients lose vision within 5 years, compared to only 5% of treated patients.

  If detected early, the condition can be treated with laser photocoagulation. In addition, a decrease in hyperglycemia at any point in the diabetic process will result in a significant reduction in the long-term pathogenesis and progression of retinopathy and vision loss. In the EUCLID study, the angiotensin converting enzyme (ACE) inhibitor lisinopril reduced the risk of progression to retinopathy by about 50% and also significantly reduced the risk of progression to proliferative retinopathy. However, in the EUCLID study, retinopathy was not the main end point and this study was not fully focused on eye-related outcomes. Preventing the onset or progression of symptoms has the ability to save vision at a relatively low cost compared to the cost associated with loss of vision. Accordingly, an object of the present invention is to provide additional means that contribute to the prevention of the onset or progression of diabetic retinopathy.

  A kidney disorder is a deterioration of the kidney. Diabetic nephropathy is a specific microvascular complication of type 1 and type 2 diabetes. Type 1 diabetes is likely to lead to the final stage of kidney injury called end stage renal disease (ESRD). There are five stages of diabetic nephropathy, and the fifth stage is ESRD. Progress from one stage to the next takes many years, and the average time to reach the fifth stage is 23 years. Diabetes is the most common cause of ESRD, accounting for over 40% of cases in the United States.

  Treatment for diabetic nephropathy is to manage and delay the progression of the disease. Aggressive blood pressure control is by far the most important factor in protecting kidney function. Angiotensin converting enzyme inhibitors are believed to provide the best protection for the kidney. According to the RENAAL study (Brenner et al, The New England Journal of Medicine 345: 861-869, 2001), the angiotensin II receptor blocker losartan may provide similar protection, but the patient population Concerns have also arisen regarding the outcome measures. Because of these methodological deficiencies and incomplete data in the study, the efficacy and safety issues of the treatment in diabetic nephropathy remain unanswered (Fisman et al, Cardiovascular Diabetology 1: 2 , 2002). From data from a similar IDNT study (Lewis etal, The New England Journal of Medicine 345: 851-860, 2001), the angiotensin II receptor blocker, irbesartan, is associated with the progression of kidney damage due to type II diabetes. It is concluded that it is effective in defense. Furthermore, preventing the onset or progression of the symptoms has the potential to save kidney function. Accordingly, another object of the present invention is to provide additional means that contribute to the prevention of the onset or progression of diabetic nephropathy.

  Angiotensin II plays a major role in pathophysiology, in particular as the most powerful blood pressure raising agent in humans. Angiotensin II receptor blockers, particularly type 1 receptor blockers, are used to treat elevated blood pressure and congestive heart failure in animals. Angiotensin II receptor blockers (also called angiotensin II antagonists) are EP-A-253310, EP-A-323841, EP-A-324377, EP-A-420237, EP-A-43983, EP-A-457136, EP-A-475206, EP-A-502314, EP-A-504888, EP-A-514198, WO 91/14679, WO 93/20816, US 4,355,040 and US 4,880,804. Particular angiotensin II receptor blockers are sultans such as candesartan, eprosartan, irbesartan, losartan, olmesartan, tasosartan, telmisartan, or valsartan.

  The ongoing diabetic retinopathy candesartan clinical trial (DIRECT) program was established to determine whether blocking of AT1 receptors with candesartan can prevent the onset and progression of diabetic retinopathy. is there. This plan will include individuals with normotensive or treated hypertension and will assess the ability of AT1 receptor blockers to protect against pathological changes in the eye that occur following diabetes (Sjφlie and Chaturvedi, Journal of Human Hypertension (August 2002) 16Suppl, pages 42-46).

  In the context of the present invention, the effect of angiotensin II receptor blockers on the development or progression of retinopathy is determined in cell culture systems to avoid extensive clinical trials. This system makes it possible to determine whether a selected or potential angiotensin II inhibitor is effective in preventing the onset or progression of retinopathy.

  Microvascular blood vessels are composed of only two types of cells: endothelial cells and pericytes. Pericytes regulate the proliferation of co-cultured endothelial cells and play a central role in maintaining microvascular homeostasis. For example, they preserve the ability of co-cultured endothelial cells to produce pericytes and protect them against lipid peroxide-induced damage. Pericyte loss and dysfunction are the prominent histopathological features observed in the early stages of diabetic retinopathy.

The method according to the invention allows the screening of angiotensin II receptor blockers, in particular angiotensin II receptor blockers that prevent the onset or progression of diabetic retinopathy or nephropathy. It comprises the following (a) to (c):
(A) treating tissue culture cells of pericytes with or without angiotensin II in the presence or absence of a potential angiotensin II receptor blocker compound;
(B) measuring the amount of reactive oxygen species generated in the cells; and (c) identifying compounds that inhibit the intracellular generation of reactive oxygen species induced by the presence of angiotensin II in the medium. .

The cell culture system used is based on pericytes isolated from mammalian retinas such as bovine retina. The cells are maintained in commercially available cell culture media, such as Dulbecco's Eagle's Modified Medium, usually supplemented with fetal calf serum. The term reactive oxygen species includes molecules such as hydrogen peroxide, ions such as hypochlorite ions, radicals such as the most reactive hydroxy radicals, and superoxide anions that are both radicals and radicals. included. The most important aspect of the method is that the intracellular generation of reactive oxygen species in the pericytes increases depending on the dose after the cultured cells are treated with angiotensin II. At the same time, DNA synthesis in pericytes, as measured by [ ³ H] thymidine incorporation, is reduced, while vascular permeability factor, a mitogen specific for endothelial cells involved in the pathogenesis of proliferative diabetic retinopathy ( VEGF) mRNA and platelet-derived growth factor B (PDGF-B), a potent mitogen and chemoattractant of microvascular endothelial and glial cells in the retina, are increased.

  Angiotensin II is a trigger for hypertension known as a major risk factor for diabetic retinopathy and nephropathy. Reactive oxygen species can damage other molecules and thus the cellular structure of which the molecule is a part. In general, cells use a variety of defenses against the harmful effects of reactive oxygen species, including small molecules with antioxidant properties such as alpha-tocopherol (vitamin E), uric acid and vitamin C, or super Two enzymes are included: oxiddismutase and catalase. During the treatment of pericytes with angiotensin II, the addition of an additional amount of antioxidants such as N-acetylcysteine (NAC) reverses the increased generation of reactive oxygen species induced by the presence of angiotensin II Is done.

  Due to these findings, compounds that are not antioxidant are able to inhibit the intracellular development of reactive oxygen species induced by the presence of angiotensin II in the cell culture medium in pericyte cell cultures. Will prevent the development or progression of diabetic retinopathy or nephropathy. Accordingly, compounds can be screened by treating pericytes for 1-48 hours, preferably 24 hours, with or without angiotensin II in the presence or absence of such compounds. . Following the treatment, the generation of reactive oxygen species is measured. Using this screening method, angiotensin II receptor blockers such as telmisartan inhibit the increased generation of reactive oxygen species induced by angiotensin II in pericytes, while treatment with the receptor blocker alone Was found not to affect the generation of reactive oxygen species. Thus, activation of angiotensin II receptor signaling in pericytes contributes to the pathogenesis of diabetic microvascular disease and antagonism of angiotensin with compounds such as telmisartan is due to pericyte loss and dysfunction Prevent the onset and progression of diseases such as diabetic retinopathy.

  As a result of these results, the present invention is a method for preventing the onset or progression of microvascular disease due to diabetes such as diabetic retinopathy or nephropathy, which is pharmaceutically effective against an individual in need thereof. A method comprising administering an amount of an angiotensin II receptor blocker is taught. This angiotensin II receptor blocker can be used to produce a pharmaceutical composition for preventing the onset or progression of microvascular disease due to diabetes in an individual in need thereof.

  Preferred examples of angiotensin II receptor blockers are candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, or valsartan, but inhibit the increased generation of reactive oxygen species induced by angiotensin II in pericyte cell cultures Any possible receptor blocker can be used. Individuals who appear to be in need of such treatment are adversely affected by one or more risk factors for diabetic retinopathy. Examples of such risk factors are diabetes, elevated blood sugar levels, proteinuria, elevated blood urea nitrogen, elevated blood creatinine, microalbuminuria, or systemic hypertension.

  The amount of receptor blocker used depends on the actual active ingredient and usually corresponds to the amount used to treat hypertension. The active compound can be administered orally, buccal, parenterally, rectally or topically by inhalation spray, oral administration being preferred. Parenteral administration may include subcutaneous injections, intravenous injections, intramuscular injections and substernal injections, and infusion techniques.

  A pharmaceutical composition for preventing the onset or progression of diabetic retinopathy containing a pharmaceutically effective amount of an angiotensin II receptor blocker mainly depends on the method of administration. The dosage range includes 0.5 to 500 mg / kg (oral), preferably 2 to 80 mg / kg (oral), and 3 mg / kg (intravenous).

  The active compound can be administered orally in a wide variety of different dosage forms. That is, they may be formulated with various pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozenges, troches, hard candy, powders, sprays, aqueous suspensions, elixirs, syrups and the like. Such carriers include solid diluents or fillers, sterile aqueous media, various non-toxic organic solvents, and the like. Furthermore, such oral formulations can be appropriately sweetened and / or flavored with various substances of the type normally used for such purposes. In general, the compound of the present invention will be in an amount sufficient to provide the desired unit dosage in such dosage forms at concentration levels ranging from about 0.5% to about 90% by weight of the total composition. Exists. Other suitable dosage forms of the compounds of the present invention include controlled release formulations and devices well known to those skilled in the art.

  For oral administration, in addition to various disintegrants such as starch (preferably potato or tapioca starch), alginic acid and certain complex silicates, together with binders such as polyvinylpyrrolidone, sucrose, gelatin and acacia, citric acid Tablets containing various excipients such as sodium, calcium carbonate and calcium phosphate may be used. In addition, lubricants such as magnesium stearate, sodium lauryl sulfate and talc, or similar types of compositions may be used as fillers in soft and hard filled gelatin capsules; lactose or lactose and high molecular weight polyethylene Glycols may be included. When aqueous suspensions and / or elixirs are desired for oral administration, the essential active ingredients therein may be combined with various sweetening or flavoring agents, coloring substances or pigments, and when desired, emulsifiers and / or Or it may be combined with water, ethanol, propylene glycol, glycerin, and various similar formulations thereof.

  For parenteral administration, a solution of the compound in sesame oil or peanut oil or aqueous propylene glycol may be used, and a sterile aqueous solution of the corresponding pharmaceutically acceptable salt may be used. Such aqueous solutions should be appropriately buffered if necessary, and the liquid diluent should be made isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular and subcutaneous injection. In this regard, the sterile aqueous medium used is readily obtained by standard techniques well known to those skilled in the art. For example, distilled water is commonly used as the liquid diluent and the final formulation is passed through a suitable bacterial filter such as a sintered glass filter or a diatomaceous earth or unglazed porcelain filter. Preferred filters of this type include Berkefeld filters, Chamberland filters, and asbestos disc-metal Seitz filters, where the liquid is drawn into the sterile container by a suction pump. Throughout the preparation of these injectable solutions, the necessary steps should be taken to ensure that a sterile final product is obtained. Dosage forms of the particular compound (s) for transdermal administration may include, for example, solutions, lotions, ointments, creams, gels, suppositories, rate limiting sustained release formulations and devices therefor. Such dosage forms contain the specific compound (s) and may include ethanol, water, penetration enhancers, and inert carriers such as gel-forming substances, mineral oils, emulsifiers, benzyl alcohol, and the like.

  Angiotensin II receptor blockers are orally 10 mg (or 0.143 mg / kg based on a 70 kg person) to 500 mg (7.143 mg / kg), parenterally about 20 mg (0.286 mg / kg), Preferably, it may be administered orally at a daily dose of 20 mg (0.286 mg / kg) to 100 mg (1.429 mg / kg). Particularly preferred is an oral daily dose of 40 mg (0.571 mg / kg) to 80 mg (1.143 mg / kg), or especially about 80 mg (1.143 mg / kg).

  Several angiotensin II receptor blockers are already commercially available and can be used for administration. For example, Approve (R), Atacand (R), Blopress (R), Cozaar (R), Diovan (R), Karvea (R), Lortaan (R), Lorzaar (R), Losaprex (R), Micadis (R), Neo-Lotan (R), or Oscaar (R), and Teveten (R).

All example values are presented as mean ± standard error (SE). Statistical significance was assessed using Student's t test; P <0.05 is considered significant.
Example 1: Measurement of reactive oxygen species in pericytes Pericytes were isolated from bovine retina and 20% fetal bovine serum (FBS) as described in Yamagishi et al, Circulation 87: 1969, 1993. Maintained in Dulbecco's Eagle's modified medium supplemented with
Angiotensin II treatment was performed in medium containing 20% FBS. Pericytes were treated for 24 hours in the presence or absence of 10 ⁻⁷ M telmisartan with or without 10 ⁻⁷ or 10 ⁻⁶ M angiotensin II. The reactive oxygen species was then used by using the fluorescent probe CM-H ₂ DCFDA (Molecular Probes Inc, Eugene, OR) as described in Yamagishi et al, AJ Biol Chem 276: 25096, 2001. Intracellular formation was detected.
Angiotensin II increased the intracellular generation of reactive oxygen species in a dose-dependent manner. 10 ⁻⁶ M angiotensin II resulted in an increase of about 1.3 fold. Telmisartan was found to completely inhibit the increased generation of reactive oxygen species in pericytes induced by angiotensin II, but telmisartan alone had no effect on the development.

Example 2: Measurement of [ ³ H] thymidine incorporation in pericytes with or without 10 ⁻⁷ M angiotensin for 24 hours in the presence or absence of 1 mM N-acetylcysteine (NAC) Skin cells were treated and then [ ³ H] thymidine incorporation in the cells was determined as described in Yamagishi etal, FEBS Lett 384: 103, 1996. Angiotensin II significantly inhibited DNA synthesis in pericytes. NAC significantly prevented angiotensin II-induced reduction of DNA synthesis in pericytes.

Example 3: Quantitative reverse transcription PCR of VEGF mRNA
Sequences and primers for detecting VEGF and β-actin mRNA are described in Yamagishi et al, J Biol Chem 272: 8723, 1997. Poly (A) ⁺ RNA was isolated from cells treated in the presence or absence of 10 ⁻⁷ M telmisartan or 1 mM NAC with or without 10 ⁻⁷ M angiotensin II, and Yamagishi et al. Analyzed by quantitative reverse transcription PCR (RT-PCR) as described in al, Diabetologia 41: 1435, 1998. The amount of poly (A) ⁺ RNA template (approximately 30 ng) and the number of cycles of amplification (28 cycles for the VEGF gene, 22 cycles for the β-actin gene) are selected within a quantitative range where the reaction proceeds linearly, The range was determined by plotting signal intensity as a function of template amount and cell cycle number as described in Yamagishi et al, J Biol Chem 277: 20309, 2002. It has been reported that there are five products spliced alternatively from one VEGF gene. They are named VEGF ₁₂₁ , VEGF ₁₄₅ , VEGF ₁₆₅ , VEGF ₁₈₉ , and VEGF ₂₀₆ . Since Northern blot analysis cannot clearly distinguish these five mRNA products, we used the more sensitive semi-quantitative RT-PCR technique described in Okamoto et al, FASEB J 16: 1928, 2002. . In these experiments, 486 and 618 base pair long cDNA products were amplified from VEGF ₁₂₁ and VEGF ₁₆₅ mRNA, respectively. Angiotensin II significantly upregulated these secreted forms of VEGF mRNA in pericytes. When exposed to 10 ⁻⁷ M angiotensin II, this VEGF mRNA level was 1.5 times higher than the basal level. Telmisartan and NAC were found to completely inhibit angiotensin II-induced upregulation of VEGF • MRNA levels in pericytes.

Example 4: Molecular cloning of partial cDNA of bovine PDGF-B Partial cDNA for bovine PDGF-B using a primer sequence designed from the conserved amino acid sequences GELSEL and NNRNVQ of human and sheep PDGF-B Has been cloned. The upstream and downstream primer were 5′-GGCGAGCTGGAGAGCTT-3 ′ and 5′-CTGCACGTTGCGGTTGT-3 ′, respectively. A 213 base pair RT-PCR product was amplified from 30 ng bovine retinal pericyte poly (A) ⁺ RNA and using the pGEM-T easy vector system according to the manufacturer's instructions (Promega, Madison, Wl, USA) Was cloned. Cloned PCR products were sequenced by the chain termination method according to the manufacturer's instructions (DNA Sequencing Kit, AppliedBiosystems, Foster, CA, USA). This cloned bovine cDNA fragment showed strong sequence similarity with human and sheep PDGF-B. Nucleotide identity with human and sheep PDGF-B was 91% and 94%, respectively, and amino acid identity was 91% and 96%.

Example 5: Quantitative reverse transcription PCR of PDGF-B mRNA
To study the effect of angiotensin II on PDGF-B gene expression in cultured retinal pericytes, 10 ⁻⁷ M telmisartan or 1 mM with or without 10 ⁻⁷ M angiotensin II Poly (A) ⁺ RNA was isolated from pericytes treated for 24 hours in the presence or absence of NAC and analyzed by RT-PCR as described in Yamagishi etal, Kidney Int 63: 464, 2003. It was. The amount of poly (A) ⁺ RNA template (approximately 30 ng) and the number of amplification cell cycles (28 cycles for the PDGF-B gene and 22 cycles for the β-actin gene) are selected within a quantitative range in which the reaction proceeds linearly. The range was determined by plotting signal intensity as a function of template amount and cell cycle number as described in Yamagishi et al, J Biol Chem 277: 20309, 2002. The primer sequences for detecting bovine β-actin mRNA were the same as described in Okamoto et al, FASEB J 16: 1928, 2002. PDGF-B has been implicated in vascular proliferative retinopathy, and the hemizygous rhodopsin promoter / PDGF-B transgenic mice show proliferation of vascular cells, glial cells and retinal pigment epithelial cells, and retinal detachment. It has been shown to occur. In this experiment, angiotensin II was found to significantly upregulate PDGF-B mRNA levels in pericytes. When exposed to 10 ⁻⁷ M angiotensin II, PDGF-B mRNA levels were 5 times higher than basal levels. Telmisartan or NAC was found to significantly inhibit angiotensin II-induced upregulation of PDGF mRNA levels. This suggests that type 1 receptor interaction of angiotensin II is involved in the pathogenesis of retinal detachment in proliferative diabetic retinopathy through overexpression of PDGF-B, and angiotensin II action by an angiotensin II receptor blocker It can be concluded that antagonizing can delay the progression of diabetic retinopathy and even prevent it by weakening PDGF-B expression in vivo.

Claims (11)

  A method for preventing the onset or progression of microvascular disease due to diabetes, comprising administering a pharmaceutically effective amount of an angiotensin II receptor blocker to an individual in need of preventing the onset or progression of the disease Comprising a method.   The method of claim 1, wherein the angiotensin II receptor blocker is a receptor blocker that inhibits intracellular generation of reactive oxygen species induced by the presence of angiotensin II in the medium in pericyte cell cultures. .   3. The method of claim 2, wherein the angiotensin II receptor blocker is selected from candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, or valsartan.   The method of claim 1, wherein the onset or progression of diabetic retinopathy is prevented.   The individual is affected by one or more risk factors of diabetes, elevated blood sugar levels, proteinuria, elevated blood urea nitrogen, elevated blood creatinine, microalbuminuria or systemic hypertension. Item 2. The method according to Item 1. A method of screening for an angiotensin II receptor blocker that prevents the onset or progression of diabetes-induced microvascular disease comprising:
(A) treating pericyte tissue culture cells with or without angiotensin II in the presence or absence of a potential angiotensin II receptor blocker compound;
(B) measuring the amount of reactive oxygen species generated in the cell;
(C) identifying a compound that inhibits the intracellular development of reactive oxygen species induced by the presence of angiotensin II in the medium;
Comprising a method.   A pharmaceutical composition for preventing the onset or progression of microvascular disease due to diabetes such as diabetic retinopathy, comprising a pharmaceutically effective amount of an angiotensin II receptor blocker.   Angiotensin II receptor for producing a pharmaceutical composition for preventing the onset or progression of a disease in an individual in need of preventing the onset or progression of diabetes due to diabetes such as diabetic retinopathy Use of body blockers.   9. The angiotensin II receptor blocker is a receptor blocker that inhibits intracellular generation of reactive oxygen species induced by the presence of angiotensin II in the medium in pericyte cell cultures. use.   10. Use according to claim 9, wherein the inhibitor of angiotensin II receptor blocker is selected from candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, or valsartan.   The individual is affected by one or more risk factors of diabetes, elevated blood sugar levels, proteinuria, elevated blood urea nitrogen, elevated blood creatinine, microalbuminuria or systemic hypertension. Item 9. Use according to Item 8.