WO2002026231A1 - Utilisation d'oxydes d'azote dans le traitement de troubles vasculaires chez un animal diabetique - Google Patents

Utilisation d'oxydes d'azote dans le traitement de troubles vasculaires chez un animal diabetique Download PDF

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WO2002026231A1
WO2002026231A1 PCT/US2001/029947 US0129947W WO0226231A1 WO 2002026231 A1 WO2002026231 A1 WO 2002026231A1 US 0129947 W US0129947 W US 0129947W WO 0226231 A1 WO0226231 A1 WO 0226231A1
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tempol
shr
wky
composition
diabetes mellitus
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PCT/US2001/029947
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Christopher S. Wilcox
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Georgetown University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine

Definitions

  • This invention is in the general field of methods for the treatment diabetes and complications arising therefrom including vascular disorders, endothelial dysfunction and renal dysfunction.
  • the present invention is directed to the treatment of oxidative stress associated with diabetes mellitus, the improvement of kidney function and preventing atherosclerotic disease which may be a consequence of endothelial dysfunction and nitric oxide deficiency in diabetes.
  • Oxidative stress is caused by other diseases such as hypertension. Indeed, hypertension frequently complicates diabetes mellitus. Both oxidative stress and hypertension contribute independently to the complications of diabetes mellitus such as vascular disease, retinopathy, neuropathy, coronary, cerebral and peripheral arterial disease and kidney failure.
  • the invention provides methods of treating these disorders by administering effective amounts of 4- hydroxy-2,2,6,6-tetramethyl-l-piperidine-l-oxyl (TEMPOL or TMPN), or derivatives and analogs thereof, to subjects with diabetes.
  • TMPN 4- hydroxy-2,2,6,6-tetramethyl-l-piperidine-l-oxyl
  • Systemic hypertension is the most prevalent cardiovascular disorder in the United States, affecting more than 50 million individuals. Accordingly, efforts to prevent, diagnose and treat hypertension remain an important concern of national health care. Although major advances have been made as to public awareness of the importance of hypertension, introducing antihypertensive therapies and in controlling hypertension, the adverse metabolic effects of some classes of antihypertensive drugs and the disappointing results in preventing associated coronary disease and kidney failure have challenged the traditional approaches of treating the antihypertensive patient.
  • Essential Hypertension Essential hypertension represents a collection of genetically based diseases and/or syndromes with a number of underlying inherited biochemical abnormalities which have yet to be elucidated. Hypertension leads to atherosclerosis and other forms of vascular pathology by damaging the endothelium.
  • BP blood pressure
  • ROS Reactive oxygen species
  • ROS superoxide anions
  • H 2 0 2 hydrogen peroxide
  • hypertensive patients may have higher plasma hydrogen peroxide, superoxide anion and lipid peroxides while having lower levels of the antioxidant, ascorbic acid (Lacy et al, 1998 J. Hypertens. 16: 291 303; Kumar et al., 1993 Free Rad. Res. Commun. 19: 59-66; Tse et al., 1994 J. Hum. HY12ertens. 89: 843 849; Bulpitt et al., J. Hypertens. 8: 1071-1075).
  • the molecular mechanism for oxygen toxicity in vascular diseases, such as essential hypertension remains to be elucidated.
  • Enzymes such as superoxide disumutase (SOD) catalyze the dismutation of superoxide radicals to remove the radicals from the subject's system where the radicals can destroy tissue.
  • SOD superoxide disumutase
  • SHR spontaneously hypersensitive rats
  • Nitric oxide which is one component of endothelium-derived relaxing factor (EDRF)
  • EDRF endothelium-derived relaxing factor
  • NOS nitric oxide synthase
  • Nitroxides Nitroxides have been used in ameliorating the deleterious effects of toxic oxygen- related species such as O 2 " (see Mitchell et al., 1995 U.S. Patent No. 5,462,946; Hsia 1997 and 1998 U.S.
  • Some nitroxides, such as Tempol (4-hydroxy-2,2,6,6-tetramethyl- 1 piperidinyloxy) have been indicated for use in treating renal hypertension disorders (Carney et al. , 1997 U.S. Patent No. 5,622,994; WO 92/22290).
  • renal hypertension occurs as a result of reduced blood flow to the kidney and is not essential hypertension. Endothelium-Dependent Vasodilation
  • Vascular diseases including hypertension, atherosclerosis, retinopathy, nephropathy and impotence are common complications in diabetes mellitus.
  • Reduced endothelium-dependent vasodilation (EDN) is a common characteristic of vascular dysfunction and has been demonstrated extensively in both human and animal models of diabetes (1-13).
  • EDN endothelium-dependent vasodilation
  • Previous studies using large, conduit vessels suggest that the endothelium- derived contracting factor superoxide (O 2 ' ⁇ ) contributes to impaired EDN in human and experimental diabetes (1, 3-5, 7, 11).
  • the endothelium modulates vascular function by releasing relaxant substances such as nitric oxide (NO) and constrictor substances such as O ' ⁇ NO is produced constitutively in conduit and resistance vessels and mediates the vasodilatory response to several agonists including acetylcholine (Ach). Ach-induced vasodilations are impaired in isolated renal arteries (3) and isolated, perfused kidneys (12, 13) of diabetic animals. However, studies in experimental and human diabetes show normal relaxation responses to endothelium-independent vasodilators such as NO donors (1, 7).
  • This invention proposes a new method of preventing the complications of diabetes mellitus in a subject, such as a human, using compositions containing nitroxides.
  • nitroxides contemplated for use in the treatment of essential hypertension include nitroxides selected from the group consisting of TEMPO, DOXYL or PROXYL nitroxides.
  • One preferred nitroxide is 4-hydroxy-2,2,6,6-tetramethyl- I piperidinyloxy (Tempol).
  • This invention also contemplates a method of treating a patient comprising the step of administering an effective amount of a nitroxide, preferably in admixture with a pharmaceutically acceptable carrier and/or an excipient.
  • Methods of administering pharmaceutical compositions containing nitroxides for treatment include oral, transdermal, parenteral and intravenous routes of administration.
  • This invention also provides for a method of treating patients with diabetes mellitus comprising the steps of administering to a subject, such as a human, any of the above pharmaceutical compositions in combination with an anti hypertensive agent (e.g., benzothiadiazine diuretics, loop diuretics, potassium-sparing diuretics, sympatholytic agents, angiotensin-converting enzyme inhibitors, calcium channel blocking agents, direct vasodilators, as well as other antioxidants).
  • an anti hypertensive agent e.g., benzothiadiazine diuretics, loop diuretics, potassium-sparing diuretics, sympatholytic agents, angiotensin-converting enzyme inhibitors, calcium channel blocking agents, direct vasodilators, as well as other antioxidants.
  • the invention likewise provides ranges of disclosed agents such as Tempol to be administered to a subject for the treatment of essential hypertension.
  • the pharmaceutical compositions of Tempol to be administered to a subject include an intravenous dose of from about 0.07 mg/kg/hr to about 750 mg/kg/hr; an intravenous bolus dose of from about 0.025 mg/kg/day to about 400 mg/kg/day; and an oral dose from about 0.05 mg/kg/day to about 1,000 mg/kg/day.
  • This invention also contemplates simultaneously treating hypertensive and oxidative stress that frequently coexist in patients with diabetes mellitus comprising the step of administering a pharmaceutical composition comprising an effective amount of a nitroxide, such as Tempol.
  • Such composition could ftirther comprise additional antihypertensives and/or NO providing agents.
  • the treatment of diabetes and complications arising therefrom including vascular disorders, endothelial dysfunction and renal dysfunction has long presented a serious problem to the medical profession. It is now found that administration of 4-hydroxy- 2,2,6,6-tetramethyl-l-piperidine-l-oxyl (TEMPOL) restores vasodilation in renal afferent arterioles of subjects with diabetes. As such, the present invention is directed to the treatment of the complications of diabetes mellitus that emanate from damage to the blood vessel.
  • TMPOL 4-hydroxy- 2,2,6,6-tetramethyl-l-piperidine-l-oxyl
  • the invention provides methods of treating these disorders by administering effective amounts of 4-hydroxy-2,2,6,6-tetramethyl-l-piperidine-l-oxyl (TEMPOL), or derivatives and analogs thereof, to subjects with diabetes.
  • Compositions containing effective amounts of TEMPOL, or derivatives and analogs thereof, in pharmaceutically acceptable carriers may be administered parenterally, orally or transdermally.
  • Figure 4. Mean arterial pressure (MAP) in WKY and SHR during control conditions (Control) and after two weeks of oral Tempol treatment (1 MM, Tempol, added to the drinking water). * p ⁇ 0.05 vs. WKY. # ⁇ 0.05 vs. Control.
  • FIG. 1 Whole Animal and Kidney Weights, mean Arterial Pressure (MAP), Heart Rate (HR), ad Tubuloglomerular Feedback Parameters in WKY and HR rats used for Functional Studies.
  • MAP Arterial Pressure
  • HR Heart Rate
  • FIG. 9 Values of Proximal stop flow pressure (PSF) as a function of rate of orthograde perfusion of artificial tubular fluid (ATF) in SHR and WKY: Effects of 7- nitroindazole (7-NI) or Renal perfusion pressure.
  • Figure 10 Values of proximal stop flow pressure (PSF) as a function of rate of orthograde perfusion of artificial tubular fluid (ATF) in SHR and WKY: Effects of L- arginine.
  • Figure 11 Values fo proximal stop flow pressure (PSF) as a function of rate of retrograde perfusion of artificial tubular fluid (ATF) in SHR and WKY: Effects of the nitroxide, superoxide dismutase mimetic, Tempol.
  • This invention provides a method of preventing oxidative stress, that not only controls hypertension, but also improves blood vessel function by administration of a nitroxide by itself in combination with another antihypertensive agent or antioxidants. Definitions
  • essential hypertension is meant essential primary or idiopathic hypertension which is a systemic hypertension of an unknown cause.
  • Essential hypertension is the cause of 95% of all cases of hypertension diagnosed.
  • the hypertension that frequently accompanies diabetes mellitus is often referred to as essential. It includes hypertension of all grades, including borderline, mild moderate and severe. It also includes hypertensive urgencies and emergencies or hypertensive crises, and indeed all cases of hypertension where there is not a known cause.
  • Secondary hypertension is systemic hypertension of a known and reversible cause. Secondary causes are largely those due to renal or renal artery diseases or disorders of the adrenal glands. These account for fewer than 2-10% of the diagnosed cases of hypertension.
  • blood pressure lowering amount is meant that amount of a compound that produces a therapeutically effective concentration significantly decreasing the blood pressure of a subject.
  • a clinically significant reduction of BP would be a fall in BP of greater than 5% relative to either the patient's normal base line BP or the patient's BPs under placebo therapy.
  • ⁇ concentration of TEMPOL or “effective amount of TEMPOL” or “effective concentration of a nitroxide” is meant that concentration of TEMPOL or nitroxide which significantly lowers the blood pressure of the hypertensive or diabetic subject.
  • BP normal diastolic blood pressure
  • a lower goal for BP of 120-125 (systolic) and 70-75 (diastolic) mm Hg is considered optimal currently.
  • nitroxide compounds which may be useful in the present invention, will be structurally diverse because the requisite property of the nitroxides is their ability to mimic superoxide dismutase (SOD) and catalase activity via the nitroxide free radical.
  • SOD superoxide dismutase
  • the main requirement of the nitroxide compound is the presence of a stable free radical. Therefore, the nitroxides described in this invention include stable nitroxide free radicals, their precursors, and their derivatives in a heterocyclic or linear structure, as represented by the general formula:
  • Ri and R 2 combine together with the nitrogen to form a heterocyclic group; and wherein the atoms in the heterocyclic group may be all carbon atoms, or may be carbon atoms as well as one or more N, O, and/or S atoms (such as, but not limited to a pyrrole, imidazole, oxazole, thiazole, pyrazole, 3-pyrroline, pyrrolidine, pyridine, pyrimidine, or purine, or derivatives thereof).
  • N, O, and/or S atoms such as, but not limited to a pyrrole, imidazole, oxazole, thiazole, pyrazole, 3-pyrroline, pyrrolidine, pyridine, pyrimidine, or purine, or derivatives thereof).
  • the heterocyclic group is preferably a 5-membered ring (such as PROXYL, or pyrroline) or a 6-membered ring (such as piperidinyl or TEMPO), with substitution at the carbon alpha to the nitrogen by electron donating groups, which may include straight or branched chain alkyl or aryl groups, preferably methyl or ethyl groups, although other longer carbon chain species could be used.
  • 5-membered ring such as PROXYL, or pyrroline
  • 6-membered ring such as piperidinyl or TEMPO
  • TEMPO DOXYL or PROXYL nitroxides or their derivatives may be used, as shown below:
  • the TEMPO, DOXYL or PROXYL nitroxides may or may not be substituted at any atom, other than the nitrogen bearing the oxygen free radical, with any combination of at least one of the following substituents: acetamido, aminomethyl, benzoyl, 2 bromoacetamido, 2-(2-(2-bromoacetamido)ethoxy)ethylcarbarnoyl, carbamoyl, carboxy, cyano, 5-(dimethylamino)-l-naphthalenesulfonamido, ethoxyfluorophosphinyloxy, ethyl, 5-fluoro-2, 4-dinitroanilino, hydroxy, 2- iodoacetarnido, isothiocyanato, isothiocyanatomethyl, methyl, maleimido, maleimidoethyl, 2-(2 maleimidoethoxy)ethylcarbamoyl, maleimi
  • the TEMPO, DOXYL or PROXYL nitroxides may also be substituents on, for example, 17b-hydroxy-5a-androstane, decane, nonadecane, 5 a- cholestane, stearic acid.
  • the TEMPO, DOXYL or PROXYL nitroxides may form the methyl, ethyl, or propyl ester with stearic acid. Additional nitroxides that are within the scope of the present invention are discussed in U.S. Patent Nos. 5,462,946 and 5,591,710, which are herein incorporated by reference.
  • the most preferred embodiment of the invention for the treatment of endothelial dysfunction in diabetes are the nitroxides, 4-hydroxy-2,2, 6, 6-tetramethyl- 1- piperidinyloxy (TEMPOL) or less preferred, 2,2,6, 6-tetramethyl- 1 -piperidinyloxy (TEMPO).
  • TEMPOL 4-hydroxy-2,2, 6, 6-tetramethyl- 1- piperidinyloxy
  • TEMPO 2,2,6, 6-tetramethyl- 1 -piperidinyloxy
  • the compounds of the invention include nitroxide compounds that can be administered via either the oral, parenteral or topical routes and other routes of administration known to those skilled in the art.
  • these compounds are most desirably administered in the dosages discussed in Example 2, although variations will necessarily occur depending upon the weight, age, and condition of the subject being treated and the presence of co-morbid conditions that may affect the pharmokokinetics or pharmokodynamics of the agents. These will vary according to the particular route of administration chosen. Other variations may also occur depending upon the species of animal being treated and its individual response to said medicament, as well as on the type of pharmaceutical formulation chosen, and the time period and interval at which such administration is carried out.
  • dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effects, provided that such larger doses are first divided into several small doses for administration throughout the day or via sustained release formulations, or by continuous administration by intravenous infusion or dermal application.
  • tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract thereby providing a sustained action over a longer period.
  • Potential time delayed materials include glyceryl monostearate or glyceryl distearate. They may also be coated by the techniques described in U.S. Patent Nos.
  • the compounds of the invention may be administered alone or in combination with pharmaceutically acceptable carriers of diluents by any of the routes previously indicated, and such administration may be carried out in single or multiple doses.
  • novel therapeutic agents of this invention can be administered in a wide variety of different dosage forms, i.e., they may be combined with various pharmaceutically acceptable inert carriers in the form of tablets, capsules, lozenges, troches, hard candies, powders, sprays, creams, salves, suppositories, jellies, gels, pastes, lotions, ointments, aqueous suspensions, injectable solutions, elixirs, syrups, and the like.
  • Such carriers include solid diluents or fillers, sterile aqueous media and various non-toxic organic solvents, etc.
  • oral pharmaceutical compositions can be suitably sweetened and/or flavored.
  • tablets containing various excipients such as microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate and glycine may be employed along with various disintegrants such as starch (e.g., preferably corn, potato or tapioca starch), alginic acid and certain complex silicates, together with granulation binders such as polyvinylpyrrolidone, sucrose, gelatin, and acacia. Additionally, lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc are often very useful for tableting purposes.
  • Solid compositions of a similar type may also be employed as fillers in gelatin capsules; preferred materials in this connection also include lactose or milk sugar, as well as high molecular weight polyethylene glycols.
  • the active ingredient may be combined with various sweetening or flavoring agents, coloring matters or dyes, and, if so desired, emulsifying and/or suspending agents as well, together with such diluents as water, ethanol, propylene glycol, glycerin and various like combinations thereof.
  • Aqueous suspensions may also contain the active materials in admixture with excipients suitable for aqueous suspensions.
  • Useful suspending agents include, for example, sodium carboxymethyl cellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example, lecithin or condensation products of an alkylene oxide with fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide with long chain aliphatic alcohols (e.g., heptadecaethyleneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol (e.g., polyoxyethylene sorbitol monooleate), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene sorbitan monooleate
  • solutions of a therapeutic compound of the present invention could be formulated as a ready to use solution in an isotonic vehicle of normal saline containing suitable stabilizers.
  • the active agent may also be formulated as a dry, sterile powder or as a lyophilized powder which would require reconstitution with an acceptable isotonic, sterile liquid.
  • These aqueous solutions are suitable for intravenous, intramuscular, or subcutaneous injection purposes.
  • the preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art. These preparations can also be used in combination with other antihypertensive agents such as diuretics.
  • any of the above methods of administering the active ingredients are contemplated to treat subjects suffering from essential hypertension (also known as primary or idiopathic hypertension). It is contemplated to use these agents in patients with all grades of hypertension from borderline to severe, in patients with accelerated or malignant hypertension, and in those patients with hypertensive urgencies, emergencies or crises. In treating higher grades of hypertension or those categories outlined above, the combination of anti-hypertensive and free radical scavenging properties may be especially beneficial.
  • active ingredients e.g., nitroxides
  • Intracellular- and extracellular- oxidative stress is hypothesized to be a critical link between hypertension and the atherosclerotic complications of vascular disease that lead to myocardial infarction, stroke and peripheral vascular disease.
  • therapy directed simultaneously at hypertensive and intracellular and extracellular oxidative stress mediated by oxygen radicals (O " ) and other reactive oxygen species (ROS) is highly attractive.
  • the compounds and formulations containing these compounds are also considered for use to lower blood pressure and to reduce oxidative stress.
  • oxidative stress for example, vitamin E and C, selenium, chromium
  • general measures to improve oxygen homeostasis so as to limit ongoing oxygen radical production (e.g., vasodilator therapy, appropriate use of nitric oxide and NO donors) to further prevent complications arising from hypertension and oxidative stress.
  • the pharmaceutically acceptable composition comprising nitroxides can further be combined with other anti-hypertensive agents.
  • Antihypertensive agents include benzothiadiazine, diuretics (e.g., thiazides, phthalimidines and quinazolines), loop diuretics (e.g., furosemide, ethacrynic acid and bumetanide), potassium sparing diuretics (e.g., spironolactone, triamterene and amiloride), sympatholytic agents (e.g., centrally acting agents such as methyldopa; b-adrenergic blocking agents such as propranolol; a-adrenergic blocking agents such as prazosin; mixed a- and b-adrenergic blocking agents such as labetalol; ganglion blocking agents such as mecamylamine; and peripherally acting sympatholytic agents such as guanethidine), angiotensin converting enzyme inhibitors (e
  • compositions include combinations of a nitroxide and an NO providing reagent (e.g., NO generating agents and NO donors).
  • NO providing agents include: sodium nitroprusside (Nipride), S r-itrosoacetylpenacillamine (SNAP), 3-morpholino-synonimin-hydrochloride (SIN-1), 3-morpholino-N athoxycarbonyl-sydnonimin (molsidomin), amyl nitrite (isoamyl nitrite), nitroglycerin (glyceryl trinitrite), isosorbide dinitrate (Isodil), isosorbide-5-mononitrite (Imur), and erythrityl tetranitrate (car dilate).
  • Other agents which are NO generating or are NO donors could also be utilized in combination with nitroxides such as TEMPOL to treat essential hypertension and oxidative stress.
  • Tempol is a nonmetal, cell membrane-permeable superoxide dismutase (SOD) mimetic that can protect against cardiac reperfusion damage or cardiomyocyte oxidative damage (Iannone et aL, 1989 Biochem.
  • outer cortical kidney RNA For isolation of outer cortical kidney RNA, one kidney from 6 SHR and one from 6 WKY was cut longitudinally, and a segment of outer cortex removed and digested with collagenase (1%) for 30 min at 37'C. Glomeruli were dissected under a stereornicroscope in rinse solution at 4°C. This contained (200 pi volume): 170 [tl dissection solution, 20 PI of 5 pM DTT, 10 pi of 10 M vanadyl ribonucleoside complex. Dissected glomeruli were further cleaned in buffer under stereornicroscope at 4'C.
  • RNA PCR Kit Perkin Elmer, Inc., Branchburg, NJ.
  • the primers used for PCR of the bNOS gene product were those described previously.
  • the sense primer was: 5'
  • the primers used for P-actin mRNA were: sense primer 5' GATCAAGATCATTGCTCCTC-3'(SEQ ID NO:3) and antisense primer: 5'- TGTACAATCAAAGTCCTCAG-3'(SEQ ID NO:4).
  • the PCR product had a predicted length of 426 bp.
  • the amounts of NOS cDNAs were normalized by the amounts of actin cDNA.
  • the reaction mixture contained 50 pmol of each primer, 1.25 mM deoxynucleotide mixture, 2.5 [d Taq DNA polymerase, 10 mM Tris-HCl (pH 10), 50 mM KC1, 1.5mM MgCl 2 1 0.001% (w/v) gelatin in a final volume of 50 ⁇ l.
  • PCR was carried out as follows: after an initial melting temperature of 94 °C for 4 min, there was 30 sec of denaturation at 94°C; 45 sec of annealing at 60'C; and 45 sec of extension at 72°C for repeated cycles of amplification, followed by a final extension at 72°C for 7 min.
  • the PCR products were analyzed on a 1.5% agarose gel stained with ethidium bromide and visualized under UV light. The size of the products were compared to a rat kidney cDNA probe for bNOS.
  • the amplified bNOS cDNAs from the rat kidney cortex of an SHR and WKY rat were purified by ⁇ (Amicon Co., Beverly, -VIA) and sequenced with an AmliTaq cycle sequencing kit (Perkin Elmer, Inc., Branchburg, NJ).
  • the primers used for ecNOS were: sense primer 5'
  • Negative controls were undertaken by PCR without prior RT, and by RT-PCR of the buffer used.
  • TBST 1% Tween-20
  • a mouse monoclonal antibody for bNOS, iNOS, or ecNOS in a 1 :400 dilution.
  • membranes were incubated for I h with anti-mouse IgG antibody conjugated horseradish peroxidase at a 1: 1000 dilution.
  • Membranes were then rinsed with TBST, and bNOS, iNOS, or ecNOS protein was detected by diaminobenzidine (DAB) with 0.3% hydrogen peroxide.
  • DAB diaminobenzidine
  • PLP paraformaldehyde lysine periodate
  • the sections were incubated with the secondary antibody, biotinylated rabbit polyclonal antibody against mouse immunoglobin (Dako, Denmark), in a dilution of 1 :600 for 30 min, rinsed, and incubated for 20 min with horseradish peroxidase (HRP) labeled streptavidin.
  • HRP horseradish peroxidase
  • DAB diaminobenzidine
  • EM immunocytochernistry using the post embedding immunogold procedure
  • one MM3 blocks of kidney cortex was dehydrated and embedded in Lowicryl.
  • Ultrathin sections were cut on an ultramicrotome, mounted on colloidin coated nickel grids, and processed for immunogold labeling. The sections were incubated with 0. 1 M NH CI for 1 h, rinsed with buffer solution (0. 02 M Tris HCI, 0. 15 M NaCI, 0.05% Tween 20, adjusted to pH 7.2) for 15 min, and incubated with mouse monoclonal antibody against ecNOS (Transduction Laboratories Inc., Lexington, KY) at a concentration of 1 : 100 overnight at 4'C.
  • buffer solution 0. 02 M Tris HCI, 0. 15 M NaCI, 0.05% Tween 20, adjusted to pH 7.2
  • a catheter was placed in a jugular vein for fluid infusion and in a femoral artery for recording of mean arterial pressure (MAP) from the electrically damped output of a pressure transducer (Statham, Inc.).
  • MAP mean arterial pressure
  • a tracheotomy tube was inserted and the animals were allowed to breathe spontaneously.
  • the left kidney was exposed by a flank incision, cleaned of connective tissue, and stabilized in a Lucite cup. This kidney was bathed in 0. 154 M NaCI maintained at 37'C.
  • rats were infused with a solution of 0. 154 M NaCI and 1% albumin at 1. 5 ml - h-' to maintain a euvolemic state. Micropuncture studies were begun after 60 min for stabilization.
  • a micropipette (8 Ym OD) containing artificial tubular fluid (ATF) stained with FD&C dye was inserted into a late proximal tubule. Injections of the colored ATF identified the nephron and the direction of flow.
  • An immobile bone wax block was inserted into this micropuncture site via a micropipette (10- 15 kim) and connected to a hydraulic drive (Trent Wells, Inc., LaJolla, CA) to halt tubular fluid flow.
  • a perfusion micropipette (6-8 ym) containing ATF and test compounds or vehicle was inserted into the proximal tubule downstream from the wax block and connected to a nanoliter perfusion pump (VYTI, Sarasota, FL).
  • PSF proximal stop flow pressure
  • RT-PCR products corresponding to cDNAs for bNOS were obtained from outer cortex of 6 SHR and 6 WKY rat kidneys.
  • the density of the bands obtained ftom SHR was consistently greater than that for WKY, although similar densities were apparent for P-actin. This difference was confirmed by densitometric analysis.
  • Examination of bNOS immunoreactivity showed heavy staining of the macula densa cell plaque. There appeared to be less prominent stain in WKY compared to SHR. Kidneys from 5 SHR and 5 WKY rats were tested systematically for immunocytochemical staining. The results showed clearly stronger macula densa staining for bNOS in SHR compared to WKY in each pair examined by a blinded observer.
  • TGF tubuloglomerular feedback
  • TGF responses were contrasted in SHR and WXY during addition of the membrane-permeable nitroxide SOD mimetic, Tempol, to LH perfusates.
  • maximum TGF responses were again greater in SHR than in WKY during retrograde perfusion of ATF + vehicle.
  • the blunting of TGF was significantly (p ⁇ 0.01) greater in the SHR rats than in WKY rats.
  • the percentage reduction in TGF with Tempol was again greater in SHR (SHR: - 26 ⁇ ; 2 vs.
  • Tempol (10 "4 M artificial plasma) was microperfused into efferent arterioles of 7 SHR rats for 4-10 min periods. Whereas before Tempol admimstration there was no significant TGF response to microperftision of the NOS inhibitor 7-nitroindazole (7-NI) into the macula densa at the 10-' M concentration, during Tempol microperfusion there was a robust increase in TGF in the presence of 7 Nl of greater than 25%. This increase in TGF with 7-NI during Tempol administration is similar to the increase observed with 7-NI in WKY rats. Therefore, Tempol had normalized the response to NOS blockage in the hypertensive model.
  • the TGF responses of SHR are exaggerated and are unresponsive either to local blockade of nNOS by microperfusion of 7-NI into macula densa or to local provision of NOS substrate by microperfusion of L-arginine into the macula densa.
  • These enhanced responses persist after normalization of the renal perfusion pressure with a suprarenal aortic clamp and therefore are not a direct consequence of the elevated 13P.
  • Tempol is a low molecular weight, nontoxic compound that equilibrates rapidly between extra- and intracellular compartments, thereby conferring much greater protec tion against post-ischemic cellular damage than SOD. Unlike other SOD mimetics, it is not dependent on metals and therefore is stable in the intracellular environment that contains high Mg 2+ concentrations. Because endothelium-dependent vasodilatation is impaired in the SHR, in vitro studies were done in view of the SHR to further evaluate the effect of Tempol on renal vasoconstriction, vasodilatation and hypertension.
  • Tempol was administered intravenously at 4 mg/kg and the animals tested MAP:
  • Tempol is an effective treatment in the model of genetically transmitted essential hypertension
  • Tempol and its derivative forms, as well as other nitroxides can be used to treat essential hypertension in humans.
  • Tempol and other nitroxides have the special potential advantage of not only treating hypertension, but also correcting intra- and extra-cellular oxidative stress simultaneously .
  • TEMPOL is an effective vasodilator in the model of diabetes
  • TEMPOL and its derivative forms, as well as other nitroxides can be used to treat diabetes and complications arising therefrom including vascular disorders, endothelial dysfunction and renal dysfunction
  • the present invention is directed to the treatment of high blood pressure associated with diabetes mellitus, the improvement of kidney function and preventing atherosclerotic disease which may be a consequence of endothelial dysfunction and nitric oxide deficiency in diabetes
  • the invention provides methods of treating these disorders by administering effective amounts of 4-hydroxy-2,2,6,6-tetramethyl-l-piperidine-l-oxyl (TEMPOL), or derivatives and analogs thereof, as well as other nitroxides, to subjects with diabetes
  • TEMPOL 4-hydroxy-2,2,6,6-tetramethyl-l-piperidine-l-oxyl
  • Renal afferent arterioles from normal and insulin-treated, alloxan-diabetic rabbits were microdissected and microperfused in vitro for the study of luminal diameter responses to acetylcholine (Ach, 10 "11 - 10 "6 M)
  • the blood glucose concentration of insulin treated alloxan-diabetic rabbits was elevated four fold compared to normal rabbits (319 + 23 vs 79 + 6 mg/dl, p ⁇ 0 001)
  • compositions are suggestions only and are not meant to limit the scope of the invention.
  • Oral compositions may contain fillers and, additionally, preservatives along with other inert or active agents.
  • A. Compositions for oral administration may contain fillers and, additionally, preservatives along with other inert or active agents.
  • composition may be placed in capsules which may be enteric coated.
  • Other preparations can include concentrations of Tempol from about 0.01 mg/kg/day to about 500 mg/kg/day.
  • effective dosages administered orally include from about 0J to about 15,000 mg/kg/day orally, or more preferred from about 0J to about 1,500 mg/kg/day orally, or most preferred from about 7 to about 150 mg/kg/day.
  • the effective dosages of Tempol administered orally include from about 0.07 to about 7,500 mg/kg/day orally, or more preferred from about 0.07 to about 750 mg/kg/day orally, or most preferred from about 0.7 to about 75 mg/kg/day.
  • compositions for parenteral administration are described in B.
  • Tempol is added to from about 5.0 to about 100 mis of 5% dextrose or normal saline or other suitable isotonic solution for intravenous (i.v.) administration.
  • Additional compositions contemplated for parenteral use include from about 0.5 mM to about 100 mM Tempol. More preferred would be about 0.5 mM to about 10 mM Tempol administered in an isotonic vehicle intravenously (i. v.).
  • Tempol may be administered on a solid support.
  • patches patches.
  • Patches for the administration of Tempol can be formulated as adhesive patches containing a nitroxide.
  • the patch may be a discoid in which a pressure-sensitive silicone adhesive matrix containing the active agent may be covered with a non-permeable backing.
  • the discoid may either contain the active agent in the adhesive or may have attached thereto a support made of material such as polyurethane foam or gauze that will hold the active agent (e.g., Tempol). Before use, the material containing the active agent would be covered to protect the patch.
  • compositions for intravenous administration include: (1) from about 0.25 to about 800 mg/kg by i.v. bolus dosing, more preferred from about 0.25 to about 80 mg/kg by i.v. bolus dosing, and most preferred from about 2.5 mg/kg to about 8 mg/kg by i.v. bolus dosing; and (2) from about 0.5 to about 2,000 mg/kg/hr by Lv. infusion, more preferred from about 0. 5 to about 200 mg/kg/hour by i. v. infusion, and most preferred from about 5 to about 20 mg/kg/hour by i. v. infusion.
  • the effective dosages of Tempol administered intravenously include: (1) from about 0.025 to about 400 mg/kg by i.v. bolus dosing, more preferred from about 0.025 to about 40 mg/kg by i.v. bolus dosing, and most preferred from about 0.25 mg/kg to about 4 mg/kg by i.v. bolus dosing; and (2) from about 0.05 to about 1,000 mg/kg/hr by i.v. infusion, more preferred from about 0.05 to about 100 mg/kg/hour by i. v. infusion, and most preferred from about 0. 5 to about 10 mg/kg/hour by i. v. infusion.
  • compositions for Dermal Administration A patch or other solid support composed of trilarninate of an adhesive matrix sandwiched between a non-permeable backing and a protective covering layer is prepared in the following manner:
  • Tempol Two grams of Tempol is applied to from about 5 grams of a pressure sensitive silicone adhesive composition BIOPSA' Q7-2920 (Dow Coming Corp., Midland, Michigan, U.S.A.).
  • BIOPSA' Q7-2920 Dow Coming Corp., Midland, Michigan, U.S.A.
  • the adhesive is applied to a polyester film to provide in successive layers to provide about 200 mg of active agent per CM2 .
  • the film containing the adhesive is then made into a patch of 10 CM2 .
  • the patch is covered with a protective layer to be removed before application of the patch.
  • Patches may be prepared containing permeation enhancers such as cyclodextrin, butylated hydroxyanisole, or butylated hydroxytoluene.
  • permeation enhancers such as cyclodextrin, butylated hydroxyanisole, or butylated hydroxytoluene.
  • Example 3 groups of male SHR and WKY rats (200 to 300 g) were maintained on tap water and standard chow (Harlan Teklad Inc.).
  • Example 3 renal hemodynamics and MAP during bolus intravenous injection of Tempol were compared in anesthetized SHR and WKY.
  • a tracheostomy was performed with polyethylene PE-240 tubing, and the left jugular vein and carotid artery were cannulated with PE-50 tubing.
  • Intravenous infusion of 1% albumin dissolved in 0. 154 M NaCI solution was infused at 2 mL/h i. v. to maintain an euvolemic state. A midline incision was made, and the left renal artery was isolated. A blood-flow probe was placed around the renal artery and connected to a transit-time blood flowmeter (I RB, Transonic Systems Inc.). We have previously shown that this method of measuring real-time changes in RBF is valid in the rat (Welch et al., 1995 Am. J. Physiol. 3 7: F 175-F 178).
  • MAP was continuously recorded from the carotid artery using a Statham pressure transducer (model P23, Gould Instruments) and MACLab data acquisition program. After 60 minutes of equilibration, there was a basal period for measurement of MAP and RBF over 30 minutes. Then the MAP and RBF responses to Tempol at 24 and 72 ⁇ tmol/kg i.v. were determined.
  • Figure 1 shows the MAP during baseline conditions and after intravenous injections of Tempol at 24 and 72 ⁇ mol/kg in WKY and SHR.
  • Baseline MAP was significantly elevated in SHR compared with WKY (145 ⁇ ;4 versus 118+;3 mm Hg, respectively; P ⁇ 0.05).
  • Low-dose Tempol (24 ⁇ mol/kg IV) had no effect in either the WKY (1 14+;5 mm Hg) or SHR (147 ⁇ ;4 mm Hg).
  • higher-dose Tempol normalized the MAP of the SHR to the level of WKY.
  • Tempol (72 ⁇ mol/kg IV) significantly (P ⁇ 0.05) decreased MAP by 11% in WKY (96+6 mm Hg) and by 28% in SHR (104 ⁇ ;9 mm Hg).
  • Renal hemodynamics were studied during basal conditions and infusion of Tempol at 24 and 72 ⁇ mol/kg in WKY and SHR.
  • Baseline RBF was similar between groups (WKY, 7.1+; 0.7; SHR, 6.8+; 1.0 mL/min) and was not affected during Tempol (WKY, 6.6+; 0.7; SHR, 6J ⁇ ;0.8 mL/min).
  • baseline RVR was significantly increased in SH compared with WKY (24+;3 versus 17+; 1 mm Hg/mL/min respectively; P ⁇ 0.05).
  • Low-dose Tempol had no effect on RVR in either group (WKY, 17+; 1; SHR, 24 ⁇ ;3 mm Hg/mL/min).
  • Figure 2 illustrates the dose-response relationship between Tempol at 1, 8, 18, 180, 1,800 ⁇ mol-kg "1 - ⁇ 1 and MAP in WKY and SHR.
  • Baseline MAP was again significantly (P ⁇ 0.05) elevated in the SHR (166+;7 mm Hg) compared with WKY (121+4 mm Hg).
  • Tempol dose-dependently decreased MAP in WKY and SHR, with SHR having a greater sensitivity and responsiveness to Tempol infusion.
  • the highest dose of Tempol (1,800 ⁇ mol-kg-'-h-') normalized the MAP of SHR (72+;10 mm Hg) to the level of WKY (71 ⁇ ;3 mm Hg).
  • Example 5 the role of NO in the MAP response to constant Tempol infusion in SHR was investigated.
  • Tempol decreases MAP through interaction with the NO pathway
  • L-NAME NO synthase inhibitor
  • MAP was measured during basal conditions; during 20 minutes of pretreatment with either saline vehicle, L-
  • this example shows that the antihypertensive response must depend on NOS, because it was blocked by NO synthesis inhibition.
  • the intravenous infusion of Tempol decreases MAP by 32% in SHR, and this response is blocked in SHR rats pretreated with the NO synthase inhibitor L-NAME.
  • the negative response to Tempol during L-NAME was not merely due to an increase in systemic vascular resistance and blood pressure, because of the MAP response to TEMPOL persisted in SHR infused with norepinephrine.
  • Tempol reduced MAP by 14%.
  • Tempol may inhibit the production of vasoconstrictor endoperoxides that are stimulated by peroxynitrite in macrophages (Landino et al., 1996). Nevertheless, this is the first study to show that scavenging of Of " both extracellularly and intracellularly with a membrane permeable SOD mimetic, such as Tempol, normalizes the RVR and MAP of SHR. Other SOD nitroxide mimetics are also considered (see Schnackenberg et al., 1998).
  • Urine was collected in containers with 10 ⁇ L of 2 mm ethylenediaminetetraacetic acid (EDTA) to prevent ex vivo production of 8-iso prostaglandin F2. (8-ISO). Urine was centrifliged at 1,000 rpm for 10 min at 4°C and stored in aliquots at -80°C until assayed.
  • EDTA ethylenediaminetetraacetic acid
  • WKY and SHR were anesthetized with thiobutabarbital (1 00 mg/kg, i.p., Inactin, Research Biochemicals International) and maintained at 3°C on a servo controlled heated rodent operating table.
  • a tracheostomy was performed with polyethylene PE-240 tubing and the left jugular vein and carotid artery were cannulated with PE-50 tubing.
  • a 1% albumin solution in 0.154 M NaCI was infused at 2 ml h, i.v. to maintain a euvolemic state.
  • a midline incision was made and the left renal artery was isolated.
  • a blood flow probe was placed around the renal artery and connected to a transit-time blood flowmeter (IRB, Transonic Systems, Inc.). We have previously shown that this method of measuring real-time changes in renal blood flow (RBF) is valid in the rat.
  • Mean arterial pressure (MAP) and heart rate (HR) were continuously recorded from the carotid artery using a Statham pressure transducer (model P23, Gould Instruments) and MACLab data acquisition software.
  • Glomerular filtration rate (GFR) was determined from the clearance of [ 3 H]-inulin. Following surgery and a 60 min equilibration period, MAP, HR, GFR, and RBF were measured over 30 minutes and the data was averaged.
  • Mean arterial pressure in WKY and SHR is represented in Figure 4.
  • MAP in SHR was increased by 41 % compared to WKY (SHR: 162+;8 vs. WKY: 115+;5 min Hg, p ⁇ 0.001).
  • MAP was reduced in SHR to a value that was not significantly different from WKY (SHR: 134 ⁇ ;6 vs. WKY: 118+;7 min Hg).
  • MAP in SHR given Tempol was significantly lower by 18% compared to normal SHR. Analysis of variance showed that Tempol specifically and significantly (p ⁇ 0.05) decreased MAP in SHR.
  • Heart rate was significantly (p ⁇ 0.001) elevated in SHR (420+;6 bts/min) compared to WKY (374+9 bts/min) during control conditions and was not changed by Tempol (SHR: 414 ⁇ ;9 vs. WKY: 373+;8 bts/min).
  • Figure 12 depicts renal hemodynamic and excretory function during normal conditions and after 2 weeks of Tempol administration in the drinking water.
  • the RBF of SHR was decreased by 34% (SHR: 5.8+;0.6 vs. WKY: 8.8+;0J ml/min, p ⁇ O. 0 1)
  • the GFR was decreased by 47% (SHR: 1. 6 ⁇ ;0.2 vs. WKY: 3.0+;0.4 ml/min, p ⁇ 0.05)
  • the RVR was increased by 117% (SHR: 29.4+; 7 vs. WKY:13.5 ⁇ ; 1. 0 mm Hg/ml/min, p ⁇ 0.00 1).
  • the drug of choice currently is usually the NO donor compound sodium nitroprusside (Nipride).
  • NO donor compound sodium nitroprusside Napride
  • Tempol acts synergistically with S-nitrosopenacillamine (SNAP) in our rat studies to reduce glomerular capillary pressure by relaxing the efferent arteriole of the kidney.
  • SNAP S-nitrosopenacillamine
  • SNAP is a NO donor compound.
  • This provides a potential basis for combining Tempol with NO providing agents, such as Nipride, in certain patients.
  • Tempol has some potential advantages over drugs such as Nipride that act as direct NO donors.
  • Tempol can restore responses to endogenously generated and released NO, thereby restoring the normal physiology and minimizing the adverse effects from a uniform increase in NO on the body produced by drugs such Nipride or SNAP.
  • the present invention is directed to the treatment of high blood pressure associated with diabetes mellitus, the improvement of kidney function and preventing vascular and atherosclerotic disease which may be a consequence of endothelial dysfunction and nitric oxide deficiency and which contributes to heart attacks, strokes, peripheral vascular disease, neuropathy, eye disease and retinopathy.
  • Studies relevant to the instant disclosure have been published as International
  • the inventors show for the first time that Ach-induced EDV is impaired in afferent arterioles in diabetes. These results are similar to other reports of impaired Ach-induced EDN in large conduit vessels of the renal (3) and systemic circulations (1, 2, 5-7, 9-11) of diabetic humans and animals. These studies showed that stimulated Ach-induced EDV is impaired in large vessels after several weeks of diabetes. Some investigators suggest, however, that impaired Ach-induced EDV in diabetic vessels is time-dependent. Peiper et al (8) found that Ach-induced EDV in rat aorta is increased, unaltered, and impaired after 24 hours, 1 or 2 weeks, and 8 weeks of diabetes, respectively.
  • the impaired stimulated EDV of mesenteric arteries (9) and aorta (5) of diabetic rat and forearm vessels of diabetic patients (11) is improved by scavenging oxygen radicals.
  • the results of the present studies extend those observations revealing that the stimulation of NO-dependent EDV is improved in diabetic afferent arterioles treated with the SOD mimetic TEMPOL. Since TEMPOL has no time-dependent effect (19) and no effect on basal vascular tone but significantly improved Ach-induced EDV of afferent arterioles, the data support the hypothesis that Ach stimulates the release of Of " in diabetic vessels.
  • TEMPOL could improve endothelium-dependent vasodilation by reducing the tissue levels of both O " and H 2 O 2 .
  • H 2 O 2 levels after TEMPOL were sufficient to impair endothelium-dependent vasodilation responses in the diabetic rabbit afferent arterioles.
  • H 2 O 2 directly to the rabbit isolated perfused afferent arterioles and shown that it causes only a weak contraction.
  • Other studies have shown that blockade of TxA 2 /PGH 2 (TP) receptors improves the relaxant response to Ach in diabetic renal arteries (3) and aorta (10).
  • renal afferent arterioles are the primary site of resistance in the renal circulation, their response to stimulated EDV in diabetes is of central importance to kidney function.
  • afferent arteriolar tone is a result of a number of factors. Afferent arterioles respond rapidly to changes in perfusion pressure (myogenic response) and to changes in NaCI delivery to the macula densa (tubuloglomerular feedback response). In the present study, perfusion pressure was held constant and the macula densa was not present in order to eliminate the myogenic and tubuloglomerular feedback responses.
  • Hyperfiltration is a common characteristic of renal hemodynamics during the early stage of diabetes (31).
  • mice Male New Zealand White rabbits (1.8-2.1 kg) were maintained on tap water and standard chow. All protocols were approved by the Institutional Animal Care and Use Committee of Georgetown University Medical Center and were performed according to the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health as well as the guidelines of the Animal Welfare Act. Rabbits were divided into two groups: normal and diabetic. Rabbits were made diabetic using a single bolus injection of alloxan (100 mg/kg, iv) as previously described (2). Insulin (HumulinN, Eli Lilly, 1.0 - 2.0 U/day sq) was administered and blood glucose concentrations were monitored (Accu-Chek, Boehringer Mannheim Corp.) daily in diabetic rabbits. Ten days after induction of diabetes, rabbits were sacrificed for the study of afferent arteriolar function.
  • a single superficial afferent arteriole with glomerulus attached was microdissected under a stereornicroscope (model SZ40, Olympus Corp.) on a stage maintained at 4 °C.
  • the arteriole was transferred to a temperature-regulated chamber mounted on the stage of an inverted microscope (model 1X70, Olympus Corp.) modified with micromanipulators.
  • the afferent arteriole was cannulated with a series of concentric glass pipettes including holding, perfusion, and exchange pipettes and perfused at 60 mmHg with alpha modification of minimum essential media (MEMa).
  • the arteriole was superfused at ⁇ 1 ml/min with MEMa bubbled with 95% O 2 /5% C0 2 .
  • microperfused arteriole was displayed at 400x magnification (Nomarski 7 OPtics, Olympus Corp.) on a video monitor via a black and white camera (model NC 70, Dage-MTI, Inc.) attached to the inverted microscope and recorded on VHS tape. Rabbit microperfused afferent arterioles were gradually warmed to 37 °C and allowed to equilibrate for 30 min. All drugs were added to the superfusate. We have shown previously that extraluminal application of increasing concentrations of vasoactive agents obviates the confounding, effect of myogenic adjustments that are caused by interrupting the luminal perfusion to change the luminal concentration of a drug (19). L-NAME was added to both the superfusate and perfusate.
  • All agents including alloxan, norepinephrine, TEMPOL (4- hydroxy TEMPOL) and L-NAME were purchased from Sigma Chemical Co. and prepared fresh daily. Heparin was dissolved in 0.9% NaCI at 1000 USP/ml. The preservation solution consisted of 150 mM sucrose, 52 mM NaHP0 (anhydrous), 16 mM NaH 2 PO 4 , and 5% bovine serum albumin (BSA). It was filtered (0.8 mm), saturated with 95% 0 2 /5% C0 2 (pH 7.40-7.45), and prepared fresh daily.
  • BSA bovine serum albumin
  • MEMa solution containing standard concentrations of glucose (100 mg/dl) and L-arginine (126.40 mg/L) and an additional 26 M NaHC0 3 and 5% BSA for perfusion and 26 mM NaHCO 3 and 0.15% BSA for superfusion were filtered (0.2 mm), saturated with 95% O 2 /5% CO 2 , and buffered to pH 7.40 - 7.45 before use daily.
  • FIG. 5 illustrates the afferent arteriolar luminal diameter response to Ach in normal rabbits.
  • Afferent arterioles were pretreated with either NE or L-NAME to examine the role of NO in the vasodilator response to Ach.
  • NE significantly (p ⁇ 0.001) decreased resting luminal diameter (16.63 + 1.42 ⁇ m) by 45 ⁇ 6 % in afferent arterioles of normal rabbits.
  • Ach dose-dependently vasodilated (p ⁇ 0.001) NE-preconstricted afferent arterioles.
  • Ach maximally increased luminal diameter by 165 ⁇ 44 % from baseline (9.17 + 1.43 mm, p ⁇ 0.001).
  • Ach dose-dependently vasoconstricted afferent arterioles pretreated with L- NAME decreased resting luminal diameter (14.76 + 0J2 ⁇ m) by 28 + 9 %, which was not significantly different from NE pretreatment.
  • Ach administration after L-NAME maximally reduced luminal diameter by 56 + 12 % from baseline (10.42 + 0.85 ⁇ m).
  • the overall luminal diameter response to Ach of L- NAME-pretreated afferent arterioles was significantly (p ⁇ 0.05) less thanNE- pretreated afferent arterioles.
  • Series 9 The blood glucose concentration of insulin-treated alloxan-diabetic rabbits was elevated four-fold compared to normal rabbits (319 ⁇ 23 vs.
  • Figure 6 shows the luminal diameter response to Ach of NE-preconstricted afferent arterioles from diabetic rabbits and is compared to the response from normal rabbits (previously shown in Figure 5).
  • Ach significantly (p ⁇ 0.001) vasodilated NE-preconstricted afferent arterioles of normal rabbits Ach significantly (p ⁇ 0.01) vasoconstricted NE-preconstricted afferent arterioles of diabetic rabbits.
  • NE significantly (p ⁇ 0.001) decreased resting luminal diameter (14.74 ⁇ 0.33 ⁇ m) by 26 + 3 %.
  • FIG. 7 displays the role of Of " in the vasoconstrictor response to Ach of NE-preconstricted afferent arterioles of diabetic rabbits. NE-preconstricted afferent arterioles were pretreated with TEMPOL and compared to the data shown in Figure 6 for diabetic rabbits.
  • Ach significantly (p ⁇ 0.01) vasoconstricted NE-preconstricted diabetic afferent arterioles pretreated with vehicle Ach significantly (p ⁇ 0.001) vasodilated NE-preconstricted diabetic afferent arterioles pretreated with TEMPOL.
  • TEMPOL pretreatment had no affect on luminal diameter of NE-preconstricted diabetic afferent arterioles (NE: 12.53 ⁇ 1.61 ⁇ m; NE + TEMPOL: 12.98 + 1.59 ⁇ m, ns).
  • Ach maximally (p ⁇ 0.001) increased luminal diameter by 25 ⁇ 6 % in diabetic afferent arterioles pretreated with TEMPOL.
  • the overall luminal diameter response to Ach of diabetic afferent arterioles pretreated with TEMPOL was significantly (p ⁇ 0.001) greater than diabetic afferent arterioles pretreated with vehicle.
  • Timimi FK Ting HH, Haley EA, Roddy M, Ganz P, Creager MA: Vitamin C improvesendothehum-dependent vasodilation in patients with insulin-dependent diabetes mellitus. JAm Coll Cardiol 31 :442-447, 1998 12. Costa e Forti A, Fonteles MC: Decreased endothelium dependent relaxation (nitric oxide) in diabetic kidneys. HormMetab Res 30:55-57, 1998
  • Rubanyi GM Vascular effects of oxygen-derived free radicals. Free Rad Biol Med 4: 107-120,1988 22.
  • Krishna MC Samuni A, Taira J, Goldstein S, Mitchell JB, Russo A: Stimulation bynitroxides of catalase-like activity of hemeproteins. JBiol Chem 271:26018-26025, 1996
  • Juncos LA, Garvin J, Carretero OA, Ito-S Flow modulates myogenic responses in isolatedmicroperfused rabbit afferent arterioles via endothelium-derived nitric oxide. J Clin Invest95.2741-2748, 1995

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Abstract

L'invention concerne des méthodes destinées au traitement du diabète sucré et des complications entraînées par le diabète sucré, y compris les troubles vasculaires, le dysfonctionnement endothélial et le dysfonctionnement rénal. A ce titre, la présente invention concerne des méthodes destinées au traitement de l'hypertension associée au diabète sucré, à l'amélioration de la fonction rénale et à la prévention de la maladie artérioscléreuse qui peut être une conséquence du dysfonctionnement endothélial et de la carence en oxyde nitrique dans le diabète sucré. L'invention concerne des méthodes destinées au traitement de ces troubles, consistant à administrer des doses efficaces de 4-hydroxy-2,2,6,6-tetraméthyl-1-pipéridine-1-oxyl (TEMPOL ou TMPN), ou de dérivés et d'analogues de ce composé, à des sujets souffrant du diabète sucré.
PCT/US2001/029947 2000-09-26 2001-09-26 Utilisation d'oxydes d'azote dans le traitement de troubles vasculaires chez un animal diabetique WO2002026231A1 (fr)

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RU2751414C1 (ru) * 2020-06-17 2021-07-13 федеральное государственное бюджетное образовательное учреждение высшего образования "Саратовский государственный медицинский университет имени В.И. Разумовского" Министерства здравоохранения Российской Федерации (ФГБОУ ВО Саратовский ГМУ им. В.И. Разумовского Минздрава России) Способ коррекции дисфункции эндотелия при нарушениях углеводного обмена, сопровождающихся абсолютной недостаточностью инсулина
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WO2003053442A1 (fr) * 2001-12-06 2003-07-03 Yissum Research Development Company Of The Hebrew University Of Jerusalem Compositions de tempamine et leur utilisation
EP2052720A3 (fr) * 2003-11-20 2009-05-06 Othera Holding, Inc. Utilisation d'au moins un composé d'hydroxylamine pour le traitement de maladies oculaires
AU2004293105B2 (en) * 2003-11-20 2010-09-09 Othera Holding, Inc. Amelioration of macular degeneration and other ophthalmic diseases
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US9522144B2 (en) 2005-02-02 2016-12-20 Mitos Pharmaceuticals, Inc. Nitroxides for use in treating or preventing diabetes and obesity
JP2008528702A (ja) * 2005-02-02 2008-07-31 ミトス・ファーマシューティカルズ・インコーポレーテッド 免疫疾患の治療又は予防において使用するためのニトロキシド類
WO2006084200A3 (fr) * 2005-02-02 2006-12-14 Mitos Inc Oxydes d'azote destines a etre utilises pour le traitement ou la prevention de maladies cardiovasculaires
WO2006084200A2 (fr) * 2005-02-02 2006-08-10 Mitos Pharmaceuticals, Inc. Oxydes d'azote destines a etre utilises pour le traitement ou la prevention de maladies cardiovasculaires
US9101619B2 (en) 2005-02-02 2015-08-11 Mitos Pharmaceuticals, Inc. Nitroxides for use in treating or preventing diabetes and obesity
US10245256B2 (en) 2005-02-02 2019-04-02 Mitos Pharmaceuticals, Inc. Nitroxides for use in treating or preventing diabetes and obesity
JP2008528694A (ja) * 2005-02-02 2008-07-31 ミトス・ファーマシューティカルズ・インコーポレーテッド 糖尿病の治療または予防において使用するためのニトロキシド類
US9522143B2 (en) 2005-02-02 2016-12-20 Mitos Pharmaceuticals, Inc. Nitroxides for use in treating or preventing diabetes and obesity
US9937162B2 (en) 2014-06-19 2018-04-10 The University Of Utah Research Foundation Methods of treating and preventing vascular instability diseases
US9314457B2 (en) 2014-06-19 2016-04-19 The University Of Utah Research Foundation Methods of treating and preventing vascular instability diseases
RU2751414C1 (ru) * 2020-06-17 2021-07-13 федеральное государственное бюджетное образовательное учреждение высшего образования "Саратовский государственный медицинский университет имени В.И. Разумовского" Министерства здравоохранения Российской Федерации (ФГБОУ ВО Саратовский ГМУ им. В.И. Разумовского Минздрава России) Способ коррекции дисфункции эндотелия при нарушениях углеводного обмена, сопровождающихся абсолютной недостаточностью инсулина
US11510913B1 (en) 2021-05-25 2022-11-29 Louis Habash Modulating expression level of a gene encoding an apurinic/apyrimidinic endodeoxyribonuclease protein by treating a human subject with a nitroxide
WO2022251390A1 (fr) * 2021-05-25 2022-12-01 Louis Habash Modulation du niveau d'expression d'un gène codant pour une protéine endodésoxyribonucléase apurinique/apyrimidinique par traitement d'un sujet humain avec un nitroxyde
WO2022251381A1 (fr) * 2021-05-25 2022-12-01 Louis Habash Modulation du niveau d'expression d'un gène codant pour une protéine de découplage par traitement d'un sujet humain avec un nitroxyde

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