AU2004233597A1 - Angiotensin II receptor blockers for preventing the development or progression of microvascular disease due to diabetes - Google Patents

Description

WO 2004/096211 PCT/EP2004/004616 ANGIOTENSIN II RECEPTOR BLOCKERS FOR PREVENTING THE DEVELOPMENT OR PROGRESSION OF MICROVASCULAR DISEASE DUE TO DIABETES 5 The present invention relates to the field of inhibitors of angiotensin II receptor blockers and particularlyaddresses 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). 10 Diabetes is a disorder in which the body is unable to metabolize carbohydrates (e.g., food starches, sugars, cellulose) properly. The disease is characterized by excessive amounts of sugar in the blood (hyperglycemia) and urine, inadequate production and/or utilization of insulin, and by thirst, hunger, and loss of weight. Diabetes is affecting about 2% of the population. Of these 10-15% are insulin dependant (type 1) 15 diabetics and the remainder non insulin dependant (type 2) diabetics. Retinopathy is damage to the retina caused by microvascular changes. Diabetic retinopathy is a specific microvascular complication of both typel and type 2 diabetes. The prevalence of retinopathy is strongly linked to the duration of diabetes. 20 After 20 years of diabetes nearly all patients with type 1 diabetes and over 60% of patients with type 2 diabetes have some degree of retinopathy. A diabetic is 25 times more likely to go blind than a person in the general population. Upton a fifth of newly diagnosed diabetics have been found to have some retinopathy. Additionally, retinopathy develops earlier and is more severe in diabetics with elevated systolic 25 blood pressure levels. On average, a careful eye examination reveals mild retinal abnormalities about seven years after the onset of diabetes, but the damage that threatens vision usually does not occur until much later. Diabetic retinopathy is the most common cause of blindness in the working age population in many countries. 30 In the early phases of retinopathy, weakening of the small blood vessels in the retina produces bulges in the vessels (microaneurysms) and leakage of fluid (exudates) and blood (hemorrhages). Proliferative retinopathy, a later stage of the disease, involves the growth of fragile new blood vessels on the retina and into the vitreous, a jelly-like substance inside the eyeball. These vessels can rupture and release blood WO 2004/096211 PCT/EP2004/004616 -,2 into the vitreous, which causes blurred vision or temporary blindness. The scar tissue that may subsequently develop can pull on the retina and cause retinal detachment, which may lead to permanent vision loss. Macular edema-swelling due to fluid accumulating around the macular, the part of the retina most crucial for fine vision, 5 may also occur. If proliferative retinopathy is left untreated, about half of those who have it will become blind within five years, compared to just 5% of those who receive treatment. The condition can be treated with laser photocoagulation, if it is detected early. 10 Additionally, reduction in hyperglycemia at any time in the course of diabetes will result in a significant decrease in the long-term incidence and progression of retinopathy and in the development'of visual loss. In the EUCLID study, the angiotensin converting enzyme (ACE) inhibitor lisinopril reduced the risk of . progression of retinopathy by approximately 50%, and also significantly reduced the, 15 risk of progression to proliferative retinopathy. However, in thiei EUCLID Study retinopathy was not a primary endpoint and the study was not sufficiently powered for eye-related outcomes. Preventing the development or progression of the condition has the potential to save vision at a relatively low cost compared to the costs associated with a loss of vision. Thus, it is an object of the present invention to 20 provide further means which contribute to the prevention of the development or progression of diabetic retinopathy. Nephropathy is the deterioration of the kidneys. Diabetic nephropathy is a specific microvascular complication of both typel and type 2 diabetes. Type 1 diabetes is 25 more likely to lead to the final stage of nephropathy called end-stage renal disease (ESRD). There are five stages of diabetic nephropathy, the fifth stage is ESRD. Progress from one stage to the next can take many years, with 23 years being the average length of time to reach stage five. Diabetes is the most common cause of ESRD accounting for more than 40 percent of cases in the US. 30 Treatment for diabetic nephropathy attempts to manage and slow the progression of the disease. Aggressive blood pressure control is by far the most important factor in WO 2004/096211 PCT/EP2004/004616 -.3 protecting kidney function. Angiotensin-converting enzyme inhibitors are considered to provide the best protection forthe kidneys. According to the RENAAL.Study (Brenner et al, The New England Journal of Medicine 345:861-869, 2001) the angiotensin II receptor blocker losartan might offer similar protection, but concerns 5 have been raised regarding to both the patient population as well as the outcome measures. Due to these methodological flaws and the incomplete data in the study, the question of the effectiveness and safety of this treatment in diabetic nephropathy remains unanswered (Fisman et al, Cardiovascular Diabetology 1:2, 2002). From the data of the similar IDNT study (Lewis et al, The New England Journal of Medicine 10 345:851-860, 2001) it has been concluded that the angiotensin-Il-receptor blocker irbesartan-is effective in protecting against the progression of nephropathy due to type II diabetes. Yet, preventing the development or progression of the condition has the potential -. to save kidney function Thus, it is another object of the present, invention to provide further means which contribute to the prevention of the, 15 development or,progression of diabetic: nephropathy. Angiotensin II plays a major role in pathophysiology, especially as the most potent blood pressure increasing agent in humans. Angiotensin 11 receptor blockers, particularly blockers of the type 1 receptor, are used for treating elevated blood 20 pressure and congestive heart failure in a mammal. 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 25 II receptor blockers are sartans such as candesartan, eprosartan, irbesartan, losartan, olmesartan, tasosartan, telmisartan or valsartan. The ongoing Diabetic Retinopathy Candesartan Trials (DIRECT) program has been established to determine whether AT1 -receptor blockade with candesartan can 30 prevent the incidence and progression of diabetic retinopathy. This program involves normotensive or treated hypertensive individuals and will assess the potential of an AT1-receptor blocker to protect against the pathological changes in the eye following WO 2004/096211 PCT/EP2004/004616 -4 diabetes. (Sjolie and Chaturvedi, Journal of Human Hypertension (August 2002) 16 Supply, pages 42-46). In the context of the present invention the effect of angiotensin II receptor blockers on 5 the development or progression of retinopathy is determined in a cell culture system avoiding extensive clinical trials. The system allows to determine, whether or not a selected or potential angiotensin II inhibitor is effective in the prevention of the development or progression of retinopathy. 10 Vessels of the microvasculature are composed of only two types of cells, endothelial cells and pericytes. Pericytes regulate the growth of co-cultured endothelial cells and serve a pivotal role in the maintenance of microvascular homeostasis. For instance they.preserve the ability of co-cultured endothelial pells-to produce prostacyclin)and to protect them against.lipid-peroxide-induced injuoryv.Pericyte loss and dysfunction, 15 are characteristic histopathological hallmarks ,observed in the early phase ofdiabetic retinopathy. The method according to the present invention allows to screen for angiotensin II receptor blockers, and in particular for angiotensin II receptor blockers which prevent 20 the development or progression of diabetic retinopathy or nephropathy. It comprises (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 intracellularly generated reactive oxygen species, and (c) identifying the compounds, which inhibit the intracellular generation of reactive 25 oxygen species induced by the presence of angiotensin II in the culture medium. The cell culture system used is based on pericytes isolated-from mammalian retina such as bovine retina. The cells are maintained in commercially available cell culture media such as Dulbecco's Eagle's medium usually supplemented with fetal bovine 30 serum. The term reactive oxygen species comprises molecules like hydrogen peroxide, ions like the hypochlorite ion, radicals like the hydroxyl radical which is the most reactive of them all, and the superoxide anion which is both ion and radical. An WO 2004/096211 PCT/EP2004/004616 -5 important aspect of the method is the finding that the intracellular generation of reactive oxygen species in pericytes increases in a dose-dependent manner after treating the cultured cells with angiotensin II. Simultaneously DNA synthesis as measured by the incorporation of [ 3 H]thymidine in pericytes is decreased, whereas 5 mRNAs for vascular permeability factor (VEGF), a specific mitogen to endothelial cells involved in the pathogenesis of proliferative diabetic retinopathy, and platelet derived growth factor-B (PDGF-B), a potent mitogen and chemoattractant for microvascular endothelial cells and glial cells in the retina, are increased. 10 Angiotensin 11 is a trigger of high blood pressure known as a major risk factor for diabetic retinopathy and nephropathy. Reactive oxygen species damage other molecules and, thus, the cell structures of which they are part. Generally cells use a variety of defenses against the.harmful effects of reactive oxygen species including small molecules:with antioxidative properties such asalpha-tocopherol (vitamin E), 15 uric acid, and:vitamin C or the two enzymes superoxide dismutase and catalase. Adding additional amounts of antioxidants like N-acetylcystein (NAC) during the treatment of pericytes with angiotensin II reverses the increase in the generation of reactive oxygen species induced by the presence of angiotensin 11. 20 Due to these findings, compounds which are devoid of antioxidative properties will prevent the development or progression of diabetic retinopathy or nephropathy, if they are capable of inhibiting in pericyte cell cultures the intracellular generation of reactive oxygen species induced by the presence of angiotensin II in the cell culture medium. Thus, compounds can be screened by treating pericytes 1-48 hours, 25 preferably 24 hours with or without angiotensin II in the presence or absence of such a compound. Following treatment the generation of reactive oxygen species is measured. Using this screening method the angiotensin 11 receptor blockers such as telmisartan were found to inhibit in pericyte cell cultures the increase in the generation of reactive oxygen species induced by angiotensin II, whereas treatment 30 with the receptor blocker alone did not affect the generation of reactive oxygen species. Thus, activation of angiotensin 11 receptor signaling in pericytes contributes to the pathogenesis of diabetic microvascular disease and antagonizing angiotensin WO 2004/096211 PCT/EP2004/004616 -6 II with compounds such as telmisartan prevent the development or progression of diseases such as diabetic retinopathy by attenuating pericyte loss and dysfunction. As a consequence of these results the present invention teaches a method of 5 preventing the development or progression of microvascular disease due to diabetes such diabetic retinopathy or nephropathy comprising administering to an individual in need thereof a pharmaceutically effective amount of an angiotensin II receptor blocker. The angiotensin II receptor blockers can be used for the production of a pharmaceutical composition to prevent the development or progression of 10 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 any receptor blocker can be used which is capable of inhibiting in. pericyte cell cultures the:increase in the 15 generation of reactive oxygen species induced by angiotensin tl.Individuals considered to be in need of such a treatment are affected by one or more risk factors of diabetic retinopathy. Examples of such risk factors are diabetes, elevated blood glucose level, proteinuria, elevated blood urea nitrogen, elevated blood creatinine, microalbuminuria or systemic hypertension. 20 The amount of receptor blocker used is dependent on the actual active ingredient and usually corresponds to the amount used to treat hypertension. The active compounds can be administered orally, bucally, parenterally, by inhalation spray, rectally or topically, the oral administration being preferred. Parenteral administration 25 may include subcutaneous, intravenous, intramuscular and intrasternal injections and infusion techniques. The pharmaceutical composition for preventing the development or progression of diabetic retinopathy comprising a pharmaceutically effective amount of an 30 angiotensin II receptor blocker is primarily dependent on the way 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.

WO 2004/096211 PCT/EP2004/004616 -7 The active compounds can be orally administered in a wide variety of different dosage forms, i.e. they may be formulated with various pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozenges, troches, hard candies, 5 powders, sprays, aqueous suspensions, elixirs, syrups, and the like. Such carriers include solid diluents or fillers, sterile aqueous media and various non-toxic organic solvents, etc. Moreover, such oral pharmaceutical formulations can be suitably sweetened and/or flavored by means of various agents of the type commonly employed for such purposes. In general, the compounds of this invention are present 10 in such oral dosage forms at concentration levels ranging from about 0.5% to about 90% by weight of the total composition, in amounts which are sufficient to provide the desired unit dosages. Other suitable dosage forms for the compounds of this. invention include controlled release formulations and devices well known tothose who practice in the art. 15 For.purposes of oral administration, tablets containing various excipients such as sodium citrate, calcium carbonate and calcium phosphate may be employed along with various disintegrants such as starch and preferably potato or tapioca starch, alginic acid and certain complex silicate, together with binding agents such as 20 polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc or compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules; included lactose or milk sugar as well as high molecular weight polyethylene glycols. When aqueous suspensions and/or elixirs are desired for oral administration, the 25 essential active ingredient therein may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if so desired, emulsifying agents and/or water, ethanol, propylene glycol, glycerin and various like combinations thereof. For purposes of parenteral administration, solutions of the compounds in sesame or 30 peanut oil or in aqueous propylene glycol may be employed, as well as sterile aqueous solutions of the corresponding pharmaceutically acceptable salts. Such aqueous solutions should be suitably buffered if necessary, and the liquid diluent WO 2004/096211 PCT/EP2004/004616 -8 rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular and sub-cutaneous injection purposes. In this connection, the sterile aqueous media employed are readily obtained by standard techniques well known to those skilled in the art. For 5 instance, distilled water is ordinarily used as the liquid diluent and the final preparation is passed through a suitable bacterial filter such as a sintered glass filter or a diatomaceousearth or unglazed porcelain filter. Preferred filters of this type include the Berkefeld, the Chamberland and the Asbestos Disk-Metal Seitz filter, wherein the fluid is sucked into a sterile container with the aid of a suction pump. The 10 necessary steps should be taken throughout the preparation of these injectable solutions to insure that the final products are obtained in a sterile condition. For purposesof transdermal administration, the dosage form of the particular compound or compounds may include, by way of example, solutions, lotions, ointments, creams, gels, suppositories, rate-limiting sustained release formulations and devices therefor. 15 Such dosage forms comprise the particular compound or compounds and may include ethanol, water, penetration enhancer and inert carriers such as gel-producing materials, mineral oil, emulsifying agents, benzyl alcohol and the like. Angiotensin II receptor blockers may be administered in a daily dosage of 10 mg (or 20 0.143 mg/kg, based on a person of 70 kg) to 500 mg (7.143 mg/kg) orally and of about 20 mg (0.286 mg/kg) parenterally, preferably of 20 mg (0.286 mg/kg) to 100 mg (1.429 mg/kg) orally. Particularly preferred is an oral daily dosage of 40 mg (0.571 mg/kg) to 80 mg (1.143 mg/kg) or specifically of about 80 mg (1.143 mg/kg). 25 Several angiotensin II receptor blockers are already on the market and can be used for administration, e.g. Approvel®, Atacand®, Blopress@, Cozaar@, Diovan®, Karvea®, Lortaan®, Lorzaar®, Losaprex®, Micardis®, Neo-Lotan® or Oscaar®, and Teveten@. 30 WO 2004/096211 PCT/EP2004/004616 -9 Examples All values were presented as means + standard errors (SE). Statistical significance was evaluated using Student's ttest for paired comparison; P<0.05 was considered 5 significant. Example 1 - Measurement of reactive oxygen species in pericytes Pericytes were isolated from bovine retina and maintained in Dulbecco's Eagle's medium supplemented with 20% of fetal bovine serum (FBS) as described in 10 Yamagishi et al, Circulation 87:1969, 1993. Angiotensin II treatment was carried out in medium containing 2% FBS. Pericytes were treated with or without 10 -7 ' or 10-6 M angiotensin II in the presence or absence of 10- 7 M telmisacrtan for 24 hours. Then the intracellular formation of reactive oxygen species was detected as described in Yamagishi et al, A J Biol Chem 276:25096, 15 2001 by using the fluorescent probe CM-H 2 DCFDA (Molecular Probes Inc, Eugene, OR). Angiotensin II increased 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 angiotensin Il-induced increase 20 in the generation of reactive oxygen species in pericytes, while telmisartan alone did not affect the generation. Example 2 - Measurement of [ 3 H]thymidine incorporation in pericytes 25 Pericytes were treated with or without 1 0

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7 M angiotensin in the presence or absence of 1mM N-acetylcystein (NAC) for 24 hours, and then [ 3 H]thymidine incorporation in cells was determined as described in Yamagishi et al, FEBS Lett 384:103, 1996. Angiotensin II significantly inhibited DNA synthesis in pericytes. NAC significantly prevented the angiotensin II-induced decrease in DNA synthesis in pericytes. 30 WO 2004/096211 PCT/EP2004/004616 - 10 Example 3 - Quantitative reverse transcription PCR of VEGF m-RNA Sequences and primers for detecting VEGF and P-actin mRNAs are described in Yamagishi et al, J Biol Chem 272:8723, 1997. Poly(A)* RNAs were isolated from cells treated with or without 10 -7 M angiotensin 11 in 5 the presence or absence of 10-7 M telmisartan or 1 mM NAC for 4 hours, and analyzed by quantitative reverse transcription PCR (RT-PCR) as described in Yamagishi et al, Diabetologia 41:1435, 1998. The amounts of poly(A) + RNA templates (about 30ng) and cell cycle numbers for amplification (28 cycles for VEGF gene and 22 cycles for P-actin gene) were chosen in quantitative ranges were 10 reactions proceeded linearly, which had been determined by plotting signal intensities as functions of the template amounts and cell cycle numbers as described in Yamagishi et al, J Biol Chem 277:20309, 2002. It has been reported that there exist five alternatively spliced products from the single VEGF gene. They are designated as VEGF 121 , VEGF 145 , VEGF165s VEGF 189 , and 15 VEGF 2 0 6 . Since Northern blot analysis can not clearly discriminate these five mRNA products, we employed a more sensitive semi-quantitative RT-PCR technique as described in Okamoto et al, FASEB J 16:1928, 2002. In these experiments, cDNA products of 486 and 618 base pairs length are amplified from mRNAs for VEGF 121 and VEGF 16 5, respectively. Angiotensin II significantly up-regulated these secretory 20 forms of VEGF mRNA levels in pericytes. The VEGF mRNA level was about 1.5 fold higher than the basal level when exposed to 107 M angiotensin II. Telmisartan and NAC were found to completely inhibit the angiotensin II-induced up-regulation of VEGF mRNA levels in pericytes. 25 Example 4 - Molecular cloning of bovine PDGF-B partial cDNAs Partial cDNAs for bovine PDGF-B were cloned using primer sequences designed from the conserved amino acid sequences GELESL and NNRNVQ in human and sheep PDGF-B. The upstream and downstream primers were 30 5'-GGCGAGCTGGAGAGCTT-3' and 5'-CTGCACGTTGCGGTTGT-3', respectively. A 213-base pair RT-PCR product was amplified from 30 ng of bovine retinal pericyte WO 2004/096211 PCT/EP2004/004616 -11 poly(A) + RNA and cloned using the pGEM-T Easy Vector System according to the manufacturer's instructions (Promega, Madison, W1, USA). Cloned PCR products were sequenced by the chain termination method according to the manufacturer's instructions (DNA Sequencing Kit, Applied Biosystems, Foster, CA, USA). The 5 cloned bovine cDNA fragments showed strong sequence similarity with human and sheep PDGF-B. Nucleotide identities were 91% and 94%, amino acid identities 91% and 96% with human and sheep PDGF-B, respectively. 10 Example 5 - Quantitative reverse transcription PCR of PDGF-B m-RNA To investigate the effects of angiotensin II on PDGF-B gene expression in cultured retinal pericytes, poly(A) RNAs were isolated from cells treated with or without 10 7 M angiotensin II in the presence or absence of 10- 7 M telmisartan or 1mM NAC for 4 hours, and analyzed by RT-PCR as described in Yamagishi et al, Kidney Int 15 63:464, 2003. The amounts of poly(A)* RNA templates (about 30ng) and cell cycle numbers for amplification (28 cycles for PDGF-B gene and 22 cycles for P-actin gene) were chosen in quantitative ranges were reactions proceeded linearly, which had been determined by plotting signal intensities as functions of the template amounts and cell cycle numbers as described in Yamagishi et al, J Biol Chem 20 277:20309, 2002. Sequences of primers for detecting bovine P-actin mRNAs were the same as described in Okamoto et al, FASEB J 16:1928, 2002. PDGF-B has been implicated in vascular proliferative retinopathies, and hemizygous rhodopsin promoter/PDGF-B transgenic mice were shown to exhibit proliferation of vascular cells, glial cells and retinal pigment epithelial cells resulting in retinal 25 detachment. In the present experiment angiotensin II was found to significantly up regulate PDGF-B mRNA levels in pericytes. When exposed to 1 0 z M angiotensin II the PDGF-B mRNA level was about 5-fold higher than the basal level. Telmisartan or NAC were found to significantly inhibit the angiotensin 11 induced up-regulation of PDGF mRNA levels. From this it is concluded that angiotensin lI-type 1 receptor 30 interaction is involved in the pathogenesis of retinal detachment in proliferative diabetic retinopathy through overexpression of PDGF-B, and that antagonizing WO 2004/096211 PCT/EP2004/004616 -12 angiotensin II action by angiotensin II receptor blockers delays or even prevents the progression of diabetic retinopathy by.attenuating PDGF-B expression in vivo.

Claims (7)

1. A method of preventing the development or progression of microvascular disease due to diabetes comprising administering to an individual in need 5 thereof a pharmaceutically effective amount of an angiotensin II receptor blocker.

2. The method of claim 1, wherein the angiotensin II receptor blocker is a receptor blocker which in pericyte cell culture inhibits the intracellular generation of 10 reactive oxygen species induced by the presence of angiotensin II in the culture medium.

3. The method of claim 2, wherein the angiotensin II receptor blocker is selected from candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan or 15 valsartan.

4. The method of claim 1 preventing the development or progression of diabetic retinopathy. 20 5. The method of claim 1, wherein the individual is affected by one or more of the risk factors diabetes, elevated blood glucose level, proteinuria, elevated blood urea nitrogen, elevated blood creatinine, microalbuminuria or systemic hypertension. 25 6. A method to screen for angiotensin II receptor blockers which prevent the development or progression of microvascular disease due to diabetes, comprising (a) treating pericyte tissue culture cells with or without angiotensin II in the presence or absence of a potential angiotensin II receptor blocker 30 compound, (b) measuring the amount of intracellularly generated reactive oxygen species, and WO 2004/096211 PCT/EP2004/004616 -14 (c) identifying the compounds, which inhibit the intracellular generation of reactive oxygen species induced by the presence of angiotensin 11 in the culture medium. 5 7. 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 an angiotensin 11 receptor blocker.

8. Use of an angiotensin II receptor blocker for the production of a pharmaceutical 10 composition to prevent the development or progression of microvascular disease due to diabetes such as diabetic retinopathy in an individual in need thereof. 9: The use according to claim 8, wherein the angiotensin 11 receptor blocker is a 15 receptor blocker which in pericyte cell culture inhibits the intracellular generation of reactive oxygen species induced by the presence of angiotensin 11 in the culture medium.

10. The use according to claim 9, wherein the inhibitor of the angiotensin II receptor 20 blocker is selected from candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan or valsartan.

11. The use of claim 8, wherein the individual is affected by one or more of the risk factors diabetes, elevated blood glucose level, proteinuria, elevated blood urea 25 nitrogen, elevated blood creatinine, microalbuminuria or systemic hypertension.