KR20060009288A - Angiotensin ii receptor blockers for preventing the development or progression of microvascular disease due to diabetes - Google Patents

Abstract

The invention relates to the field of inhibitors of angiotensin II receptor blockers and particularly addresses their use in diabetes to prevent the development or progression of microvascular disease (i.e. disease involving small blood vessels) affecting eyes (diabetic retinopathy) and kidneys (diabetic nephropathy).

Description

Angiotensin II receptor blockers for preventing the development or progression of microvascular disease due to diabetes

FIELD OF THE INVENTION The present invention relates to the field of inhibitors of angiotensin II receptor blockers, in particular the development or progression of microvascular diseases (ie small vascular related diseases) that harm the eyes (diabetic retinopathy) and kidneys (diabetic nephropathy). It relates to its use in diabetes to prevent.

Diabetes is an abnormality in the body that cannot adequately metabolize carbohydrates (eg, food starch, sugars, cellulose). The disease is characterized by excessive amounts of sugar in blood (hyperglycemia) and urine, improper production and / or utilization of insulin, and loss of thirst, hunger and weight. About 2% of the population has diabetes. 10 to 15% of these are insulin dependent (type 1) diabetes mellitus and the rest are non-insulin dependent (type 2) diabetes mellitus.

Retinopathy is damage to the retina caused by microvascular changes. Diabetic retinopathy is a specific microvascular complication of type 1 and type 2 diabetes. The prevalence of retinopathy is deeply linked to the duration of diabetes. After 20 years of diabetes, almost all patients with type 1 diabetes and more than 60% of patients with type 2 diabetes have some degree of retinopathy. Diabetics are 25 times more likely to be blind than the general population. Some one-fifth of newly diagnosed diabetics have been found to have some retinopathy. In addition, retinopathy develops early and is more severe in diabetic patients with elevated systolic blood pressure levels. On average, a close eye examination revealed mild retinal abnormalities about seven years after the onset of diabetes, but vision-threatening damage usually appeared much later. Diabetic retinopathy is the most common cause of blindness among the workable population in many countries.

In the early stages of retinopathy, the weakening of small blood vessels in the retina causes swelling of blood vessels (microneurysms) and leakage of body fluids (exudation) and blood (bleeding). Proliferative retinopathy, a late state of the disease, involves the growth of vulnerable new blood vessels on the retina and into the vitreous, a substance like jelly in the eye. These blood vessels rupture and release blood into the vitreous, resulting in blurry or temporary blindness. Subsequently developing scar tissue may pull the retina and cause retinal detachment, which may cause permanent vision loss. Macular-edema swelling may also occur due to the accumulation of fluid around the macula, the part of the retina that is most important for fine vision. If left untreated, proliferative retinopathy will cause only 5% of the treated patients to be blinded, compared with about half of those who have blindness within five years.

The pathology, if found early, can be treated with laser photocoagulation. In addition, the reduction of hyperglycemia at any time during diabetes progression will significantly reduce the long-term development and progression of retinopathy and progression of vision loss. In the EUCLID study, angiotensin converting enzyme (ACE) inhibitor lisinopril reduced the risk of developing retinopathy by about 50% and also significantly reduced the risk of progressing to proliferative retinopathy. However, in the EUCLID study, retinopathy was not the primary endpoint and the study did not sufficiently inspire eye related outcomes. Preventing the development or progression of the pathology has the potential to obtain vision at a relatively low cost compared to the costs associated with vision loss. It is therefore an object of the present invention to provide another means that contributes to the prevention of the development or progression of diabetic retinopathy.

Nephropathy is a worsening condition of the kidney. Diabetic nephropathy is a specific microvascular complication of type 1 and type 2 diabetes. Type 1 diabetes is likely to cause the final stage of nephropathy called end stage kidney disease (ESRD). There are five stages of diabetic nephropathy, and the fifth stage is ESRD. The progression from one phase to the next can take years, and the average time to reach the fifth phase is 23 years. Diabetes is the most common cause of ESRD, accounting for over 40% of cases in the United States.

The treatment of diabetic nephropathy is to control and slow the progression of the disease. Active blood pressure control is the most important factor in protecting kidney function. Angiotensin-converting enzyme inhibitors are believed to best protect the kidneys. According to the RENAAL study [Brenner et al, The New England Journal of Medicine 345: 861-869, 2001], angiotensin II receptor blocker losartan can provide similar protection, but not only in the patient population. Concerns about the outcome have increased. Due to these methodological drawbacks and incomplete data in the study, questions about the efficacy and safety of the treatment in diabetic nephropathy have not been answered (Fisman et al, Cardiovascular Diabetology 1: 2, 2002). From data from a similar IDNT study (Lewi et al, The New England Journal of Medicine 345: 851-860, 2001), the angiotensin II receptor blocker, irbesartan, is a nephropathy caused by type II diabetes. It was concluded that it would be effective in protecting against Moreover, prevention of the development or progression of the pathology has the potential to relieve kidney function. Accordingly, another object of the present invention is to provide additional means that contribute to the prevention of the development or progression of diabetic nephropathy.

Angiotensin II plays a major role in pathophysiology, particularly as the most potent blood pressure booster in humans. Angiotensin II receptor blockers, particularly blockers of type 1 receptors, are used to treat elevated blood pressure and congestive heart failure in mammals. Examples of angiotensin II receptor blockers (also called angiotensin II antagonists) are described in EP-A-253310, EP-A-323841, EP-A-324377, EP-A-420237, EP-A-43983, EP -A-459136, 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. Specific angiotensin II receptor blockers candesartan, eprosartan, irbesartan, losartan, olmesartan, tasosartan, telmisartan Or sartan, such as valsartan.

Ongoing Diabetic Retinopathy Candesartan Trials (DIRECT) program was established to determine whether AT1-receptor blockade with candesartan can prevent the development and progression of diabetic retinopathy. This program includes individuals with normal or treated hypertension and will evaluate the potential for AT1-receptor blockers to protect against pathological changes in the eye after diabetes [Sjolie and Chaturvedi, Journal of Human Hypertension (August 2002). 16 Suppl, pages 42-46.

In the present invention, the effect of angiotensin II receptor blockers on the development or progression of retinopathy is measured in cell culture systems that avoid extensive clinical trials. Such a system can be used to determine whether a selected or potential angiotensin II inhibitor is effective in the development or progression of retinopathy.

Microvascular blood vessels consist of only two types of cells: endothelial cells and perivascular cells. Perivascular cells play a central role in regulating the growth of cocultured endothelial cells and maintaining microvascular homeostasis. For example, they preserve the ability of co-cultured endothelial cells to produce prostacyclin and protect them from lipid-peroxide-induced damage. Perivascular loss and dysfunction are characteristic histopathological phenomena observed in the early stages of diabetic retinopathy.

The method according to the invention enables the screening of angiotensin II receptor blockers, in particular angiotensin II receptor blockers which prevent the development or progression of diabetic retinopathy or nephropathy. this is,

(a) treating tissue culture cells of pericyte with or without angiotensin II receptor blocker compound, with or without angiotensin II;

(b) determining the amount of reactive oxygen species generated in the cell;

(c) identifying a compound that inhibits the intracellular production of reactive oxygen species induced in the presence of angiotensin II in the culture medium.

The cell culture system used utilizes perivascular cells isolated from mammalian retinas, for example bovine retinas. The cells are maintained in a commercial cell culture medium, such as Dulbecco's Eagle medium, generally supplemented with fetal bovine serum. The term 'reactive oxygen species' includes molecules such as hydrogen peroxide, ions such as hypochlorite, radicals such as hydroxyl radicals (the most reactive of radicals), and superoxides that are both ions and radicals. An important aspect of this method is the discovery that the generation of reactive oxygen species in perivascular cells increases in a dose-dependent manner after treatment of the cultured cells with angiotensin II. As measured by the incorporation of [ ³ H] thymidine in perivascular cells, simultaneous DNA synthesis is reduced, while specific mitotic material for endothelial cells involved in the pathogenesis of proliferative diabetic retinopathy ( mitogen), vascular permeability factor (VEGF), and mRNA for platelet-derived growth factor-B (PDGF-B), a potent mitotic and chemoattractant for endothelial and glial cells of microvessels in the retina, Is increased.

Angiotensin II is a high blood pressure trigger known as a major risk factor for diabetic retinopathy and nephropathy. Reactive oxygen species damage other molecules and the cellular structures that they are part of. In general, cells are composed of reactive oxygen species, including alpha-tocopherol (vitamin E), small molecules with anti-oxidative properties such as uric acid and vitamin C, and two enzymes: excess product dismutase and catalase. Use various defenses against harmful effects. Adding an additional amount of antioxidant such as N-acetylcysteine (NAC) during treatment of perivascular cells with angiotensin II reverses the increase in the production of reactive oxygen species induced in the presence of angiotensin II.

Because of these findings, if the intracellular production of reactive oxygen species induced in the presence of angiotensin II in the cell culture medium can be inhibited in the cell culture of perivascular cells, compounds without antioxidant properties may be used for diabetic retinopathy or nephropathy. Will prevent development or progression. Thus, perivascular cells can be screened by treatment with or without angiotensin II for 1 to 48 hours, preferably 24 hours, with or without such compounds. After treatment, the production of reactive oxygen species is measured. Using this screening method, angiotensin II receptor blockers, such as telmisartan, inhibit the increased production of reactive oxygen species induced in the presence of angiotensin II in cell culture of perivascular cells, whereas receptor blockers It was found that the treatments used alone did not affect the production of reactive oxygen species. Thus, activation of angiotensin II receptor signaling in perivascular cells is involved in the development of diabetic microvascular disease, and antagonizing angiotensin II with a compound such as telmisartan results in the loss of perivascular cells and attenuation of diabetes. Prevent the development or progression of diseases such as sexual retinopathy.

Because of these results, the present invention provides for the development or progression of microvascular disease due to diabetes, such as diabetic retinopathy or nephropathy, comprising administering to a subject in need thereof a pharmaceutically effective amount of angiotensin II receptor blocker. Teach you how to prevent this. The angiotensin II receptor blockers can be used in the manufacture of pharmaceutical compositions that prevent the development or progression of microvascular disease due to diabetes in a subject in need thereof.

Preferred examples of angiotensin II receptor blockers include candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan or valsartan, but induction in the presence of angiotensin II in cell culture of perivascular cells. Any receptor blocker can be used that can inhibit the increased production of reactive oxygen species. Subjects deemed to require such treatment will be at least one risk factor for diabetic retinopathy. Examples of such risk factors are diabetes, elevated blood sugar levels, proteinuria, elevated blood urea nitrogen, elevated creatinine in blood, microalbuminuria or systemic hypertension.

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

Pharmaceutical compositions for preventing the development or progression of diabetic retinopathy, comprising a pharmaceutically effective amount of angiotensin II receptor blockers, depend primarily on the mode of administration. Dosage ranges include 0.5 to 500 mg / kg p.o., preferably 2 to 80 mg / kg p.o., and 3 mg / kg i.v.

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

For oral administration, tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate may be used in various disintegrants, for example starch, preferably potato or tamioca starch, alginic acid and certain silicate complexes, and binders, For example, it can be used with polyvinylpyrrolidone, sucrose, gelatin and gum arabic. In addition, lubricants such as magnesium stearate, sodium lauryl sulfate and talc or similar types of compositions can also be used as fillers in soft and hard-filled gelatin capsules; Lactose or lactose as well as high molecular weight polyethylene glycols. Where aqueous suspensions and / or elixirs are contemplated for oral administration, the essential active ingredients of this genus are various sweetening or fragrances, coloring substances or dyes, and optionally emulsifiers and / or water, ethanol, propylene glycol, glycerin and It can be combined with various combinations thereof.

For parenteral administration purposes, solutions of the compounds in sesame or peanut oil or in aqueous propylene glycol, as well as sterile aqueous solutions of the corresponding pharmaceutically acceptable salts can be used. The aqueous solution should be suitably buffered as necessary and the liquid diluent imparts isotonicity with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular and subcutaneous injections. 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 a liquid diluent, and the final formulation is passed through a suitable bacterial filter, such as a sintered glass filter or diatomaceous earth or glazed magnetic filter. Preferred filters of this type include Berkefeld, Chamberland and Asbestos Disk-Metal Seitz filters, in which fluid is sucked into the sterile vessel with the aid of a suction pump. This essential step should be taken throughout the preparation of these injectable solutions to ensure that the final product is obtained sterile. For transdermal administration purposes, examples of dosage forms of specific compound (s) include solutions, lotions, research agents, creams, gels, suppositories, rate-limited sustained release formulations and devices therefor. Such dosage forms include specific compound (s) and may include ethanol, water, penetration enhancers, and inert carriers such as gel-producing materials, mineral oils, emulsifiers, benzyl alcohol, and the like.

Angiotensin II receptor blockers, at a daily dosage of 10 mg (or 0.143 mg / kg, based on 70 kg of human) to 500 mg (7.143 mg / kg) orally, about 20 mg (0.286 mg / kg) ) Can be administered orally, preferably 20 mg (0.286 mg / kg) to 100 mg (1.429 mg / kg). Particularly preferably, the daily oral dosage is 40 mg (0.571 mg / kg) to 80 mg (1.143 mg / kg), or specifically about 80 mg (1.143 mg / kg).

Some angiotensin II receptor blockers are already commercially available and can be administered, for example, Approvel ^R , Atacand ^R , Blopress ^R , Cozaar ^R , Diovan ^R , Karvea ^R , Lortaan ^R , Lorzaar ^R , Losaprex ^R , Micardis ^R , Neo -Lotan ^R or Oscaar ^R , and Teveten ^R.

All values are expressed as mean ± standard error (SE). Statistical significance was assessed by Student's t-test for pair comparison; P <0.05 was considered significant.

Example 1 Measurement of Reactive Oxygen Species in Perivascular Cells

Perivascular cells were isolated from bovine retinas and maintained in Dulbecco's Eagle's medium supplemented with 20% fetal bovine serum (FBS) (Yamagishi et al, Circulation 87: 1969, 1993).

Angiotensin II treatment was performed in medium containing 2% FBS. Perivascular cells were treated with or without 10 ⁻⁷ or 10 ⁻⁶ M of angiotensin II for 24 hours in the presence or absence of 10 ⁻⁷ M of telmisartan. Intracellular formation of reactive oxygen species was then detected using fluorescent probe CM-H ₂ DCFDA (Molecular Probes Inc, Eugene, OR) as described in Yamagishi et al, AJ Biol Chem 276: 25096, 2001. It was.

Angiotensin II increased the intracellular production of reactive oxygen species in a dose-dependent manner. Angiotensin II of 10 ^-6 M resulted in an about 1.3-fold increase. It has been found that telmisartan completely inhibits the increase in production of angiotensin II-induced reactive oxygen species in perivascular cells, whereas telmisartan alone does not affect the production of reactive oxygen species.

Example 2 Measurement of [ ³ H] thymidine Incorporation in Perivascular Cells

Perivascular cells were treated with or without 10 ⁻⁷ M angiotensin for 24 hours in the presence or absence of 1 mM N-acetylcysteine (NAC), followed by [ ³ H] thymidine incorporation in cells. Yamagishi et al, FEBS Lett 384: 103, 1996]. Angiotensin II significantly inhibited DNA synthesis in perivascular cells. NAC significantly prevented a decrease in angiotensin II-induced DNA synthesis in perivascular cells.

Example 3- VEGF Quantitative Reverse Transcription PCR of 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 separating, from 10 ^-7 M of telmisartan or a use of angiotensin II of 1mM 4 sigan 10 ^-7 M for the presence or absence of NAC or without the use of treated cells, and quantitative reverse Transcription PCR (RT-PCR) was analyzed as described in Yamagishi et al, Diabetologia 41: 1435, 1998. The amount of poly (A) ⁺ RNA template (about 30 ng) and cell cycle recovery for amplification (28 cycles for the VEGF gene and 22 cycles for the β-actin gene) were selected from the quantitative range, see Yamagashi et al, J Biol Chem 277: 20309, 2002, as measured by plotting signal intensity as a function of template amount and cell cycle recovery, the reaction proceeded linearly.

It has been reported that there are five products that have been selectively spliced from a single VEGF gene. These are named VEGF ₁₂₁ , VEGF ₁₄₅ , VEGF ₁₆₅ , VEGF ₁₈₉ and VEGF ₂₀₆ . Since Northern blot analysis could not clearly identify these five mRNA products, we used the more sensitive semi-quantitative RT-PCR technique (Okamoto et al, FASEB J 16: 1928, 2002). In these experiments, 486 and 618 base pair length cDNA products were amplified from mRNA for VEGF ₁₂₁ and VEGF ₁₆₅ , respectively. Angiotensin II significantly up-regulated these levels of VEGF mRNA in perivascular cells. VEGF mRNA levels were about 1.5 times higher than basal levels when exposed to 10 ⁻⁷ M angiotensin II. It has been found that telmisartan and NAC completely inhibit up-regulation of angiotensin II-induced VEGF mRNA levels in perivascular cells.

Example 4 Molecular Cloning of Bovine PDGF- B Partial cDNA

Partial cDNA for bovine PDGF-B was cloned using primer sequences designed from the conserved amino acid sequences GELESL and NNRNVQ in bovine and sheep PDGF-B. The upstream and downstream primers were 5'-GGCGAGCTGGAGAGCTT-3 'and 5'-CTGCACGTTGCGGTTGT-3', respectively. 213-base pairs of RT-PCR products were amplified from 30 ng of bovine retinal perivascular poly (A) ⁺ RNA and pGEM-T Easy Vector System was prepared according to the manufacturer's instructions (Promega, Madison, WI, USA). Cloned using. The cloned PCR product was sequenced by chain termination according to the manufacturer's instructions (DNA Sequencing Kit, Applied Biosystems, Foster, Calif., USA). The cloned bovine cDNA fragment showed marked 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 with 91% and 96%, respectively.

Example 5 Quantitative Reverse Transcription of PDGF- B mRNA PCR

To investigate the effects of angiotensin II on PDGF-B gene expression in perivascular cells of cultured retinas, poly (A) ⁺ RNA was added to the presence or absence of 10 ^-7 M of telmisartan or 1 mM NAC. Isolates from cells treated with or without 10 ⁻⁷ M of angiotensin II for an hour were analyzed by RT-PCR as described in Yamagishi et al, Kidney Int 63: 464, 2003. The amount of poly (A) ⁺ RNA template (about 30 ng) and cell cycle recovery (28 cycles for VEGF and 22 cycles for β-actin gene) for amplification were chosen in the quantitative range, see Yamagashi et al, J. Biol Chem 277: 20309, 2002, as measured by plotting signal intensity as a function of template amount and cell cycle recovery, the reaction proceeded linearly. Primer sequences for detecting bovine β-actin mRNA are as described in Okamoto et al, FASEB J 16: 1928, 2002.

PDGF-B is associated with vascular proliferative retinopathy and is associated with vascular cells, glial cells and retinal pigment epithelial cells in which hemizygous rhodopsin promoter / PDGF-B transgenic mice cause retinal detachment. It was found to indicate proliferation. In this experiment, it was found that angiotensin II significantly up-regulated PDGF-B mRNA levels in perivascular cells. When exposed to 10 ⁻⁷ M angiotensin II, PDGF-B mRNA levels were about 5 times higher than baseline levels. It has been shown that telmisartan or NAC significantly inhibits up-regulation of angiotensin II-induced PDG-F mRNA levels. From this, angiotensin II-1 receptor interactions are associated with the pathogenesis of retinal detachment in proliferative diabetic retinopathy due to overexpression of PDGF-B, and when antagonizing angiotensin II action by angiotensin II receptor blockers, In conclusion, we conclude that attenuated PDGF-B expression may delay or even prevent the progression of diabetic retinopathy.

Claims (11)

A method for preventing the development or progression of microvascular disease due to diabetes, comprising administering a pharmaceutically effective amount of angiotensin II receptor blocker to an individual in need thereof. The method of claim 1, wherein the angiotensin II receptor blocker is a receptor blocker that inhibits intracellular production of reactive oxygen species induced in the presence of angiotensin II in the culture medium during cell culture of perivascular cells. 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 development or progression of diabetic retinopathy is prevented. The method of claim 1, wherein the subject is at least one risk factor of diabetes, elevated blood sugar levels, proteinuria, elevated urea nitrogen, elevated creatinine in blood, microalbuminuria, or systemic hypertension. (a) treating tissue culture cells of perivascular cells with or without angiotensin II receptor blocker compounds, with or without angiotensin II, (b) determining the amount of reactive oxygen species generated in the cell; (c) screening for angiotensin II receptor blockers that prevent the development or progression of microvascular disease due to diabetes, including identifying compounds that inhibit the intracellular production of reactive oxygen species induced in the presence of angiotensin II in the culture medium. How to. A pharmaceutical composition for preventing the development or progression of microvascular disease due to diabetes, such as diabetic retinopathy, comprising a pharmaceutically effective amount of angiotensin II receptor blocker. Use of an angiotensin II receptor blocker in the manufacture of a pharmaceutical composition for preventing the development or progression of microvascular disease due to diabetes, such as diabetic retinopathy, in an individual. The use according to claim 8, wherein the angiotensin II receptor blocker is a receptor blocker that inhibits the intracellular production of reactive oxygen species induced in the presence of angiotensin II in the culture medium during cell culture of perivascular cells. The use of claim 9, wherein the angiotensin II receptor blocker is selected from candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan or valsartan. The use of claim 8, wherein the subject is at least one risk factor of diabetes, elevated blood sugar levels, proteinuria, elevated blood urea nitrogen, elevated creatinine, microalbuminuria, or systemic hypertension.