Classifications

• • 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
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/336—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having three-membered rings, e.g. oxirane, fumagillin
• • 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
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
  • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure

Definitions

• Heart failure is the leading cause of cardiovascular morbidity and mortality worldwide. About half of heart failure patients have heart failure with preserved ejection fraction (HFPEF). Distinct from traditional HF, i.e., heart failure with reduced ejection fraction in which the ventricle cannot contract, patients with HFPEF show declined performance of heart ventricle, not at the time of contraction, but during the phase of diastole. HFPEF patients show normal ejection fraction of blood pumped out of the ventricle, but the heart muscle does not quickly relax to allow efficient filling of blood returning from the body. Morbidity and mortality of HFPEF are similar to traditional HF; however, therapies that benefit traditional HF are not effective in treating or preventing HFPEF. See From et ah, Cardiovascular Therapeutics, 2011, 29:e6-21.
• cardiac fibrosis One of the causes of diastolic dysfunction is cardiac fibrosis. Cardiac fibrosis is characterized by pathological accumulation of fibrillar collagen throughout the myocardium, which results in reduced cardiac muscle compliance, impaired filling, and ultimately heart failure. In the heart, collagen is produced primarily by cardiac fibroblasts. Stress in the heart can lead to profibrotic cytokine-induced proliferation of cardiac fibroblast and transformation of cardiac fibroblasts into myofibroblasts. Myofibroblasts are responsible for excessive accumulation of extracellular matrix under pathological conditions. [005] Myofibroblasts secrete extracellular matrix to strengthen the tissue and are important in wound healing. Normally, myofibroblasts are eliminated by apoptosis after the healing is complete. Under pathological conditions, persistent proliferation of myofibroblasts and consequently expansion of the extracellular matrix can lead to tissue fibrosis, often seen in tissues such as liver, kidney, lung and heart.
• Omega-3 polyunsaturated fatty acids were shown to be beneficial in coronary artery disease and heart failure. See, e.g., Levitan et al, Eur Heart J. 2009, 30: 1495-1500,
• the invention thus provides methods and reagents directed toward treating or limiting development of heart failure with preserved ejection fraction (HFPEF) and fibrosis that are not hampered by the limitations found in conventional treatments.
• HFPEF preserved ejection fraction
• the methods and reagents provided in the instant application are capable of reversing or limiting development of HFPEF by inhibiting or preventing cardiac fibrosis.
• method for treating or limiting development of heart failure with preserved ejection fraction (HFPEF), comprising administering to a patient having or at risk of developing HFPEF an amount effective of a pharmaceutical composition comprising docosahexaenoic acid (DHA) or pharmaceutically acceptable salts, esters, amides, epoxides, and prodrugs thereof, to treat or limit development of HFPEF.
• DHA docosahexaenoic acid
• the method is for limiting development of HFPEF; while in other embodiments, the method is for treating HFPEF.
• the pharmaceutical composition does not include any omega- 3 fatty acid therapeutic other than DHA or a pharmaceutically acceptable salt, ester, amide, epoxide, or prodrug thereof. In certain other embodiments, the pharmaceutical composition does not include any fatty acid therapeutic other than DHA or a pharmaceutically acceptable salt, ester, amide, epoxide, or prodrug thereof. In yet other embodiments, the pharmaceutical composition comprises DHA or a pharmaceutically acceptable salt, ester, amide, epoxide, or prodrug thereof in an amount of at least 80%, at least 85%, at least 90%, at least 95% or at least 99.9% by weight of total fatty acids.
• the pharmaceutical composition does not include any fatty acid therapeutic or any ⁇ -3 fatty acid therapeutic, for the indication of the instant invention, other than DHA or a pharmaceutically acceptable salt, ester, amide, epoxide, or prodrug thereof.
• the pharmaceutical composition comprises purified or synthesized DHA or a pharmaceutically acceptable salt, ester, amide, epoxide, or prodrug thereof.
• DHA, or a pharmaceutically acceptable salt, ester, amide, epoxide, or prodrug thereof is administered to the patient at a concentration of from about 5 mg/kg of body weight/day to about 50 mg /kg of body weight/day.
• the dose is about 600 mg/day to about 1000 mg/day of DHA, or in certain other particular embodiments, about 800 mg/day.
• the pharmaceutical composition comprises DHA epoxides, including without limitation EpDPEs [epoxy docosapentaenoic acid]. In yet other preferred embodiments, the pharmaceutical composition comprises 19(20)EpDPE. In certain other embodiments, the patient to be administered the pharmaceutical composition is normotensive or hypotensive.
• the invention provides methods of treating or limiting development of fibrosis comprising administering to a patient having or at risk of developing fibrosis an amount effective of a pharmaceutical composition comprising docosahexaenoic acid (DHA) or pharmaceutically acceptable salts, esters, amides, epoxides, and prodrugs thereof, to treat or limit development of fibrosis.
• DHA docosahexaenoic acid
• the fibrosis is liver fibrosis, kidney fibrosis, cardiac fibrosis or lung fibrosis.
• the fibrosis is cardiac fibrosis.
• the cardiac fibrosis is fibrosis in the ventricle.
• the method is for limiting development of fibrosis; while in other embodiments, the method is for treating fibrosis. In certain particular embodiments, the method is for limiting development of cardiac fibrosis; while in other particular embodiments, the method is for treating cardiac fibrosis. In certain particular embodiments, the cardiac fibrosis is ventricular fibrosis.
• the pharmaceutical composition does not include any omega-3 fatty acid therapeutic other than DHA or a pharmaceutically acceptable salt, ester, amide, epoxide, or prodrug thereof. In certain other embodiments of this aspect, the pharmaceutical composition does not include any fatty acid therapeutic other than DHA or a pharmaceutically acceptable salt, ester, amide, epoxide, or prodrug thereof. In certain other embodiments, the pharmaceutical composition comprises DHA or a
• the pharmaceutical composition comprises purified or synthesized DHA or a pharmaceutically acceptable salt, ester, amide, epoxide, or prodrug thereof.
• DHA, or a pharmaceutically acceptable salt, ester, amide, epoxide, or prodrug thereof is administered to the patient at a concentration of from about 5 mg /kg of body weight/day to about 50 mg /kg of body weight/day.
• the dose is about 600 mg/day to about 1000 mg/day of DHA, and in certain other particular embodiments, about 800 mg/day.
• the pharmaceutical composition comprises DHA epoxide, including without limitation EpDPEs [epoxy docosapentaenoic acid].
• the pharmaceutical composition comprises 19(20)EpDPE.
• the patient to which the pharmaceutical composition is administered is normotensive or hypotensive.
• Figure 1 shows results demonstrating that fish oil prevented pressure overload- induced cardiac dysfunction.
• Fig. 1A shows a bar graph measuring the omega-3 content (EPA+DHA % of total fatty acids) of red blood cells or left ventricle (LV) determined by gas chromatography four weeks following transverse aortic constriction (TAC) surgery.
• Fig. IB shows the measurements of fractional shortening (FS) determined by echocardiography before and 1-, 2- and 4 weeks after TAC.
• Fig. 1C and ID show contractility ( ⁇ dP/dt) and end-diastolic pressure determined from hemodynamic measurements four weeks after TAC.
• FIG. 2 shows results demonstrating that fish oil prevented pressure overload- induced cardiac fibrosis in mice.
• Quantitative measurement of fibrosis in the left ventricle is shown in Fig. 2B using Image J and presented as the fibrosis area/total area.
• Fig. 2E shows the heart weight to body weight ratio (HW/BW) calculated four weeks after TAC.
• Figure 3 shows results demonstrating that fish oil blocked pressure overload- induced non-myocyte proliferation and myofibroblast transformation.
• Fig. 3A presents
• Fig. 3C shows the number of Ki67 positive cells from 15-20 fields per heart.
• FIG. 4 shows results demonstrating that fish oil did not block pressure overload- induced TGF- ⁇ production or phosphorylation of Smad2.
• Fig. 4B illustrates the levels of phospho- and total Smad2 measured by western blot analysis three days or four weeks after TAC.
• FIG. 5 presents results demonstrating that EPA and DHA inhibited the TGF- ⁇ - stimulated fibrotic response in isolated adult mouse cardiac fibroblasts.
• Fig. 5A shows the ⁇ - 3 PUFA content (EPA + DHA) in cultured cardiac fibroblasts treated for 48 hr with EPA (10 ⁇ ), DHA (10 ⁇ ), arachidonic acid (AA, 10 ⁇ ), or the oleic acid (
• Fig. 5D shows the levels of myofibroblast transformation in cardiac fibroblasts treated as in A, measured by a-SMA immunostaining (green, asterisks) with counterstains for fibroblast specific protein 1 (FSP1, red, arrows) and nuclei (DAPI, blue).
• Fig. 5E shows the quantification of a-SMA positive cells as a percentage of total FSP1 positive cells (4 slides for each group). Data were analyzed by one-way ANOVA with Dunnett's post-hoc test in A- C and Tukey's post-hoc test in E. Data are presented as mean ⁇ SEM.
• FIG. 6 presents the results demonstrating that EPA and DHA inhibited the TGF- ⁇ - stimulated fibrotic response through the cGMP/PKG pathway.
• EPA ⁇
• DHA 10 ⁇
• 8-bromo-cGMP 1 mM
• DT-3 guanylyl cyclase inhibitor
• FIG. 6C shows the levels of Smad2 and Smad3 phosphorylation (Smad2 Ser465/467; Smad3 Ser423/425) and total Smad2/3 detected by western blot analysis in cultured cardiac fibroblasts treated for 24 hr with EPA or DHA (10 ⁇ ) and for an additional 30 min or 24 hr with fatty acids and TGF- ⁇ (1 ng/ml).
• Fig. 6D and 6E show the
• FIG. 7A shows cellular localization of phospho-Smad detected by staining for phospho-Smad2 (green, first row), Smad4 (red, second row) or phospho-Smad3 (green, fourth row) with a nuclear counterstain (DAPI, merged images in the third and fifth rows) in cultured cardiac fibroblasts treated for 24 hr with EPA (10 ⁇ ) (images not shown), DHA (10 ⁇ ), 8-bromo- cGMP (1 mM), and/or the guanylyl cyclase inhibitor DT-3 (1 ⁇ ) and for an additional 30 min with fatty acids and TGF- ⁇ (1 ng/ml).
• EPA nuclear counterstain
• FIG. 8 shows results demonstrating that EPA and DHA increased NOx production, phosphor-eNOS expression and eNOS expression in cardiac fibroblasts.
• EPA ⁇
• DHA 10 ⁇
• 8-bromo-cGMP 0.1 mM
• 8-bromo-cGMP (1 mM
• FIG. 8B shows the levels of phospho-eNOS (endothelial nitric oxide synthase) and eNOS detected by western blot analysis in cultured cardiac fibroblasts treated for 24 hr with EPA or DHA (10 ⁇ ).
• FIG. 9 shows the results demonstrating that EPA and DHA reduced the TGF- ⁇ - induced Smad-responsive promoter activity.
• Fig. 9B shows luciferase activity detected in cardiac fibroblasts transfected with VSV-g pseudotyped lentivirus particles expressing the firefly luciferase gene under the control of a CMV promoter and tandem repeats of the Smad transcriptional response element (TRE).
• TRE Smad transcriptional response element
• FIG. 10 shows results demonstrating that EPA and DHA did not block TGF- ⁇ - induced activation of ERK1/2.
• Fig. 10A shows the levels of phosphorylated ERK1/2 (Thr202/Tyr204) and total ERK1/2 detected by western blot analysis in cultured cardiac fibroblasts treated for 24 hr with EPA or DHA (10 ⁇ ) and for an additional 1 hr with fatty acids and TGF- ⁇ (1 ng/ml).
• FIG. 11 presents results demonstrating that only DHA inhibited the TGF- ⁇ - stimulated fibrotic response in isolated adult mouse cardiac fibroblasts.
• Cells were treated with no exogenous FAs (No FA), oleic acid (OA, 10 ⁇ ), arachidonic acid (AA, 10 ⁇ ), EPA (10 ⁇ ), or DHA (10 ⁇ ) and treated for a final 24 hr with TGF- ⁇ (1 ng/ml).
• Collagen production was measured by [ 3 H] -proline incorporation.
• CYV epoxygenase inhibitor a partial inhibitor of the epoxygenase
• Figure 12 shows the chemical structures of selected compounds.
• Fig. 12A shows the structure of docosahexaenoic acid
• Fig. 12B shows the structures of exemplary epoxide compounds
• Fig. 12C shows the structures of exemplary prodrugs of DHA or DHA epoxide.
• Heart failure as used herein means inability of the heart to supply sufficient blood flow to meet the needs of the body.
• Preserved ejection fraction means that the patient does not have a significant reduction in ventricular ejection fraction compared to that of a control (e.g., a healthy individual) or compared to an average value from a healthy population.
• ejection fraction in patients classified as HFPEF is > 45%.
• Treating" a patient having a disease or disorder means accomplishing one or more of the following: (a) reducing the severity of the disease; (b) arresting the development of the disease or disorder; (c) inhibiting worsening of the disease or disorder; (d) limiting or preventing recurrence of the disease or disorder in patients that have previously had the disease or disorder; (e) causing regression of the disease or disorder; (f) improving or eliminating the symptoms of the disease or disorder; and (f) improving survival.
• the disease or disorder is HFPEF.
• the inventive methods cause regression of HFPEF and improve patient survival by reducing cardiac fibrosis and reducing diastolic pressure.
• “Limiting development” means any reduction in development of a disease or disorder, including but not limited to (a) decreasing the rate of development of a disease or disorder; (b) delaying the onset of development of the disease or disorder; (c) reducing the risk of developing a disease or disorder; and (d) preventing development of the disease or disorder.
• the disease or disorder is HFPEF and the inventive methods described herein reduce the risk of developing HFPEF.
• the inventive methods reduce the risk of developing HFPEF by preventing developing cardiac fibrosis and maintaining the efficiency of blood filling into the heart ventricle during diastole.
• a patient at risk of developing HFPEF is any patient with one or more symptoms of HFPEF (including but not limited to shortness of breath, leg swelling, and exercise intolerance), or one or more other risk factors, including but not limited to, a genetic predisposition to HFPEF, a family member with HFPEF; and one or more disorders including, but not limited to, myocardial infarction, cardiomyopathy, ventricular diastolic dysfunction, ventricular systolic dysfunction, ventricular systolic stiffening, vascular stiffening and dysfunction, left atrial dysfunction, pulmonary hypertension, autonomic dysfunction (e.g., chronotropic incompetence; sympathetic hyperactivation), skeletal muscle dysfunction (e.g., impaired vasodilation; sympathetic hyperactivation and ergoreflex stimulation), anemia, and symptoms thereof.
• HFPEF including but not limited to shortness of breath, leg swelling, and exercise intolerance
• HFPEF a genetic predisposition to HFPEF
• a "subject" or “patient” refers to a mammal in need of the intervention of the inventive method.
• the mammal is a human.
• the patient is normotensive or hypotensive.
• the patient's heart muscle shows normal contractile capability that leads to normal ejection fraction of the heart.
• the term "amount effective,” “effective amount” or a “therapeutically effective amount” refers to an amount of a therapeutic compound sufficient to achieve the stated desired result, for example, treating or limiting development of HFPEF, tissue fibrosis or cardiac fibrosis.
• the amount of the compound which constitutes an "effective amount” or “therapeutically effective amount” may vary depending on the severity of the disease, the condition or age of the patient to be treated, or the route of administration, but can be determined routinely by one of ordinary skill in the art.
• DHA is administered to the patient at a concentration of from about 5 mg/kg of body weight/day to about 50 mg/kg of body weight/day.
• the pharmaceutical composition comprises DHA epoxide including without limitation epoxydocosapentaenoic acids (EpDPEs) at the 19(20) position or the 4(5), 7(8), 10(11), 13(14), or 16(17) position and all enantiomers thereof.
• EpDPEs epoxydocosapentaenoic acids
• a prodrug is an inactive or less active form of the active ingredient that is
• Prodrugs of DHA suitable for use in the instant invention include without limitation esters of DHA or esters of DHA epoxide. Exemplary prodrugs are shown in Figure 12 C, wherein R is without limitation ethyl, methyl, H, sugar or other carbohydrates, glycerol or as glycerol-lipid per phospholipids, triglycerides, or cholesterol.
• Omega-3 fatty acids are essential polyunsaturated fatty acids vital for normal metabolism and cannot be synthesized de novo by mammals.
• Omega-3 polyunsaturated fatty acids include without limitation a-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). These three polyunsaturated fatty acids have 3, 5, or 6 double bonds starting from the third carbon atom from the methyl group in a carbon chain of 18, 20, or 22 carbon atoms, respectively.
• Fish oils and plant oils are common sources of ⁇ -3 PUFAs. Although fish oils have been considered beneficial to cardiovascular health in general, to the best knowledge of the inventors, the effect of purified or synthetic DHA on heart failure and fibrosis has not been reported.
• the pharmaceutical composition comprises purified, isolated or synthetic DHA.
• the use of purified or synthetic DHA avoids the possibility of environmental or other sources of contamination.
• Purified or synthetic DHA is commercially available from, for example, Sigma (St. Louis, MO) or can be obtained based on the disclosure of, for example, Journal of the American Oil Chemists Society, 1997, 74: 1435-40.
• the DHA for use in the claimed invention is purified or synthesized DHA that is at least 90%, at least 95%, at least 98%, or at least 99% pure.
• the pharmaceutical composition comprising purified or synthesized DHA without any other ⁇ -3 PUFAs.
• the composition comprising purified or synthesized DHA without any other fatty acids are examples of the composition of purified or synthesized DHA without any other fatty acids.
• purified DHA and isolated DHA are used interchangeably throughout the application.
• the term refers to DHA obtained from a source, including but not limited to its natural source such as fish oils or plant oils, by one or more chemical and/or physical means.
• the purified or isolated DHA is separated from other components and impurities from its source or from the compounds or impurities introduced during the isolation process.
• a pharmaceutically acceptable salt, ester, amide or prodrug of DHA suitable for use in the instant invention can be naturally existing in the source and purified from the source or can be chemically synthesized or prepared.
• fatty acid therapeutic or “omega-3 fatty acid therapeutic” refers to a fatty acid (or omega-3 fatty acid) that is present in a pharmaceutical composition and exerts a therapeutic effect and is not merely present as an inert excipient or diluent or a minor contaminant without any therapeutic effect.
• the method comprising the step of administering to a patient in need thereof a pharmaceutical
• composition that does not include any fatty acid (or omega-3 fatty acid) therapeutic for the recited indication of the instant invention other than DHA or a pharmaceutically acceptable salt, ester, amide or prodrug thereof.
• minor contaminating fatty acids may be present in an amount not exceeding 0.01%, 0.05%, 0.1%, 0.15%, 0.5%, 1%, 2%, 3% or 5% of total fatty acids, wherein the contaminating fatty acids do not exert any therapeutic effect.
• the pharmaceutical composition comprises DHA or a pharmaceutically acceptable salt, ester, amide, epoxide, or prodrug thereof in an amount of at least 80%, at least 85%, at least 90%, at least 95% or at least 99.9% by weight of total fatty acids.
• the method comprising the step of administering to a patient in need thereof a pharmaceutical composition that does not include any fatty acid (or omega-3 fatty acid) therapeutic other than DHA or a pharmaceutically acceptable salt, ester, amide or prodrug thereof.
• the pharmaceutical composition does not include any fatty acid (or omega-3 fatty acid) other than DHA or a pharmaceutically acceptable salt, ester, amide or prodrug thereof.
• the inventors of the instant application unexpectedly discovered that, inter alia, the effect of DHA on heart failure is independent of blood pressure.
• the inventors unexpectedly discovered that ⁇ -3 PUFAs prevented pressure overload-induced cardiac dysfunction and cardiac fibrosis in animals that were normotensive.
• DHA reduced TGF- ⁇ -induced Smad responsive promoter activity and that DHA greatly inhibited TGF- ⁇ -induced collagen synthesis in cardiac fibroblasts.
• the invention provides methods of treating HFPEF comprising administering to a patient having or at risk of developing HFPEF an amount effective of a pharmaceutical composition comprising DHA wherein the
• the invention provides methods of limiting the development of HFPEF comprising administering to a patient at risk of developing HFPEF an amount effective of a pharmaceutical composition comprising DHA wherein the pharmaceutical composition differentially or selectively maintained normal diastolic pressure in the patient.
• methods for treating or limiting development of HFPEF, comprising the step of treating or limiting development of diastolic dysfunction by administering to a patient an amount effective of a pharmaceutical composition comprising DHA or pharmaceutically acceptable salts, esters, amides, epoxides, and prodrugs thereof, to treat or limit development of HFPEF.
• the invention provides methods of treating or limiting development of diastolic dysfunction comprising administering to a patient administering to a patient having or at risk of developing diastolic dysfunction an amount effective of a pharmaceutical composition comprising DHA or pharmaceutically acceptable salts, esters, amides, epoxides, and prodrugs thereof, to treat or limit development of diastolic dysfunction.
• normotensive refers to normal systolic and/or diastolic blood pressures.
• a normotensive patient has an end point systolic pressure of no more than 139 mmHg and no less than 90 mmHg.
• the normotensive patient has an end point diastolic pressure of no more than 89 mmHg and no less than 60 mmHg.
• hypotensive refers to systolic and/or diastolic blood pressures that are lower than the normal values.
• a hypotensive patient has a systolic pressure lower than 90 mmHg.
• the hypotensive patient has a diastolic pressure lower than 60 mmHg.
• hypertensive refers to systolic and/or diastolic blood pressures that are higher than the normal values.
• a hypertensive patient has a systolic pressure of at least 140 mmHg. In certain other particular embodiments, the hypertensive patient has a diastolic pressure of at least 90 mmHg.
• the instant invention provides methods for treating or limiting development of fibrosis comprising the step of administering to a patient having or at risk of developing fibrosis an amount effective of a pharmaceutical composition comprising docosahexaenoic acid (DHA) or pharmaceutically acceptable salts, esters, amides, epoxides, and prodrugs thereof, to treat or limit development of fibrosis.
• Applicable tissue fibrosis includes without limitation fibrosis in the lung, kidney, liver, and heart.
• the invention provides methods for inhibiting cardiac fibrosis.
• the invention provides methods of treating or limiting development of fibrosis comprising the step of inhibiting cardiac fibroblast proliferation, myofibroblast
• the invention provides methods of treating or limiting
• the methods of treating or limiting development of diastolic dysfunction comprises the step of inhibiting cardiac fibrosis by administering to a patient having or at risk of developing fibrosis an amount effective of a pharmaceutical composition comprising docosahexaenoic acid (DHA) or pharmaceutically acceptable salts, esters, amides, epoxides, and prodrugs thereof, to treat or limit development of diastolic hypertension.
• DHA docosahexaenoic acid
• the invention provides uses of docosahexaenoic acid (DHA) or pharmaceutically acceptable salts, esters, amides, epoxides, and prodrugs thereof for the preparation for a medicament for treating or limiting development of HFPEF.
• DHA docosahexaenoic acid
• the invention provides uses of DHA or pharmaceutically acceptable salts, esters, amides, epoxides, and prodrugs thereof for the preparation for a medicament for treating or limiting development of fibrosis.
• the fibrosis is liver fibrosis, kidney fibrosis, cardiac fibrosis or lung fibrosis.
• the fibrosis is cardiac fibrosis.
• the fibrosis is fibrosis in the ventricle.
• compositions used in the inventive methods can be specially formulated for oral administration in solid or liquid form or for intravenous injection.
• compositions can be determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES, Id.
• the DHA or pharmaceutical acceptable salts, esters, amides, epoxides and prodrugs thereof can be incorporated in a conventional systemic dosage form, such as a tablet, capsule, soft gelatin capsule, elixir or injectable formulation.
• the dosage forms may also include the necessary physiologically acceptable carrier material, excipient, lubricant, buffer, surfactant, antibacterial, bulking agent (such as mannitol), antioxidants (ascorbic acid or sodium bisulfite) or the like.
• Oral dosage forms are preferred, although parenteral forms can be used as well.
• Suitable surfactants include without limitation Tween 20, Tween 80, a polyethylene glycol or a polyoxy ethylene polyoxypropylene glycol, such as Pluronic F-68.
• the salt or buffering agent may be any salt or buffering agent, such as for example sodium chloride, or sodium/potassium phosphate, respectively.
• the buffering agent maintains the pH of DHA or acceptable salts, esters, amides, epoxides and prodrugs thereof.
• the salt and/or buffering agent is also useful to maintain the osmolality at a level suitable for administration to a human.
• the drug is administered preferably orally, in particular in the form of soft gelatin capsules.
• Other types of formulation for oral administration are also suitable for the purposes of the invention; for example hard capsules or tablets, in which the ⁇ -3 fatty acid is adsorbed on solid supports or micro-particles.
• the dosage forms of capsules can be prepared with coatings and shells such as enteric coatings and other coatings well-known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
• coatings and shells such as enteric coatings and other coatings well-known in the pharmaceutical formulating art. They may optionally contain opacifying agents and may also be of a composition such that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
• embedding compositions which can be used include polymeric substances and waxes.
• Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs.
• the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan and mixtures thereof.
• inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as
• Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar, tragacanth and mixtures thereof.
• the pharmaceutical composition of the invention may be administered to a patient by sustained release, as is known in the art. Sustained release administration is a method of drug delivery to achieve a certain level of the drug over a particular period of time.
• compositions of this invention for parenteral injection comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
• suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol and the like), injectable organic esters (such as ethyl oleate) and suitable mixtures thereof.
• Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.
• compositions may also contain adjuvants such as preservative, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms can be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
• TAC transverse aortic constriction
• Echocardiography and hemodynamics were performed as described previously (Tang et al, Circulation, 2005, 1 12:3122-3130). Briefly, echocardiographic measurements were performed under anesthesia (3% isoflurane induction, 1% maintenance) using a Visual Sonics Vevo 660 High-Resolution Imaging System (Visual Sonics, Toronto, Canada) with a 30- MHz linear array transducer (model RMV-707). Left ventricular dimensions and heart rate were measured from 2-D short-axis M-mode tracings at the level of the papillary muscle. Left ventricular mass and functional parameters were calculated using the above primary measurements.
• Left ventricular hemodynamics were measured under anesthesia (3% isoflurane induction, 1% maintenance) using a Millar catheter inserted into the left ventricle via the carotid artery. After stabilization, heart rate (HR), left ventricular end-systolic pressure and end-diastolic pressure were measured from the left ventricular pressure waveform, and ⁇ dP/dt and tau were calculated.
• HR heart rate
• left ventricular end-systolic pressure and end-diastolic pressure were measured from the left ventricular pressure waveform, and ⁇ dP/dt and tau were calculated.
• Fibroblasts were resuspended in Dulbecco's Modification of Eagle's Medium (DMEM, Fisher Scientific, Pittsburgh, PA) with 15% fetal bovine serum (FBS, Fisher Scientific) and plated on laminin-coated 60 mm dishes (Becton Dickinson, Franklin Lakes, NJ). After one hour, the culture medium was changed, which removed weakly adherent cells, including any myocytes and endothelial cells. After 24 hours, the culture medium was changed again, and the fibroblast cultures were grown to 85% confluence, at which point the medium was replaced with DMEM containing 0.1% FBS. After 24 hours in reduced serum medium, fibroblast cultures were treated with fatty-acids or other agonist/antagonists as indicated.
• DMEM Dulbecco's Modification of Eagle's Medium
• FBS fetal bovine serum
• Free fatty acids (DHA, EPA, arachidonic acid and oleic acid, Sigma, St. Louis, MO) for in vitro studies were dissolved in 100% ethanol to make a stock solution (100 mg/ml). Aliquots (5 ⁇ /tube) of the stock solution were stored in PCR tubes under a blanket of nitrogen and sealed with parafilm and stored at -80 °C. Before use, the aliquots were diluted with 0.2% BSA/PBS and then further diluted in culture medium.
• DHA Free fatty acids
• EPA arachidonic acid and oleic acid
• RNA from the left ventricle was extracted using the RNeasy Fibrous Tissue Mini Kit (QIAGEN, Valencia, CA).
• Oligo (dT) primed cDNA synthesis was performed using Superscript III (Invitrogen, Carlsbad, CA).
• the heart tissues were excised, cannulated through the aorta and perfused with buffer (1% adenosine, 2% BDM, and 0.04% heparin in PBS). Hearts were fixed in formalin overnight, embedded in paraffin and sectioned (5 ⁇ ) by the Histology Core at The Burnham Institute for Medical Research (La Jolla, CA). To measure fibrosis, tissue sections were stained with picrosirius red. High resolution images were obtained through Aperio
• tissue sections were incubated with antibodies to a-SMA (1 : 100, Dako, Carpinteria, CA) and an HRP-labeled secondary antibody. Dako EnVision+ System-HRP (DAB) was used to visualize positive a-SMA staining. The sections were counterstained with hematoxylin to visualize nuclei. Images were captured using an inverted microscope (Olympus 1X71). The positive areas of a-SMA staining were quantified by Image-Pro plus (Media Cybernetics, Bethesda, MD).
• Ki67 staining for proliferation was performed on formalin-fixed, paraffin-embedded left ventricular sections (5 ⁇ ). After deparaffinization, re-hydration and heat-induced epitope retrieval, sections were incubated with antibodies to Ki67 (1 :200, Abeam, Cambridge, MA) and a goat anti-rabbit AlexaFluor 488 secondary antibody (1 :400, Invitrogen). Images were captured using confocal microscopy (FV1000, Olympus). Quantitative data were obtained by measuring co-localization of 4',6-diamidino-2-phenylindole (DAPI, nuclear staining) with Ki67 in the interstitial area of the left ventricle.
• DAPI 4',6-diamidino-2-phenylindole
• FSP-1 fibroblast-specific protein-1
• a-SMA or Smad was performed on cultured cardiac fibroblasts fixed with 4% paraformaldehyde and permeabilized with ice-cold methanol.
• fibroblasts were incubated with primary antibody [phospho-Smad3 (Ser423/425) (1 :400, Cell Signaling Technology, Danvers, MA); phospho-Smad2 (ser465/467) (1 :500, Millipore, Billerica, MA); Smad4 (1 : 100, Santa Cruz Biotechnology, Inc, Santa Cruz, CA); FSP1 (1 : 100, Abeam); and a-SMA (1 :200, Dako)] and fluorochrome-conjugated secondary antibodies. Fibroblasts were counterstained with DAPI to visualize nuclei. Images were captured using confocal microscopy (FV1000, Olympus).
• Proteins were transferred to PVDF membranes. Membranes were incubated overnight at 4°C with primary antibodies specific for phospho-Smad2, phospho-Smad3, Smad2/3, phospho- eNOS, total eNOS, phospho-ERKl/2, total ERK1/2 ( all from Cell Signaling Technology, Inc., Danvers, MA) in 5% BSA. A horseradish peroxidase conjugated secondary antibody was incubated for 1 h at room temperature in 5% milk and processed for chemiluminescent detection using an ECL Advanced Western Blotting Kit (GE Healthcare, Piscataway, NJ). Protein abundance on western blots was quantified by densitometry with the Quantity One program from Bio-Rad (Hercules, CA).
• mice were fed a diet supplemented with ⁇ -3 PUFAs or control diet for eight weeks.
• Omega-3 content in the red blood cells and left ventricle was measured as previously described (Duda et ah, Cardiovasc Res. 2009, 81 :319- 327).
• ⁇ -3 PUFAs-supplemented diet increased the ⁇ -3 content (EPA+DHA % of total fatty acids) in both red blood cells (3.4 fold) and left ventricle (2.4 fold) relative to mice on the control diet.
• Aortic constriction decreased the ⁇ -3 content in red blood cells and heart in mice on the control diet (p ⁇ 0.01), whereas aortic constriction had no effect on the fish oil groups ( Figure 1A and Tables 1-2).
• fractional shortening a repeated measures model was used.
• aortic constriction induced contractile dysfunction in mice fed with the control diet as evidenced by a 38% decrease in fractional shortening (FS), a 29% decrease in dP/dt max, a 27% decrease in dP/dt min, and a 6.5-fold increase in end-diastolic pressure (all at least p ⁇ 0.05 relative to sham).
• TAC- induced contractile dysfunction was mitigated or prevented in mice fed the fish oil diet ( Figures 1B-D).
• Linoleic acid (n-6) C18:2n6 11.1 ⁇ 0.53 10.5 ⁇ 0.34 10.2 ⁇ 0.12 10.3 ⁇ 0.16 trans Linoleic acid (n-6) C18:2n6t 0.24 ⁇ 0.02 0.28 ⁇ 0.02 0.32 ⁇ 0.04 0.17 ⁇ 0.02 B,c a-Linolenic acid (n-3) C18:3n3 0.05 ⁇ 0.01 0.05 ⁇ 0.00 0.04 ⁇ 0.01 * 0.03 ⁇ 0.00 0
• Eicosapentaenoic acid (EPA, n-3) C20:5n3 0.05 ⁇ 0.01 0.08 ⁇ 0.04 3.59 ⁇ 0.14* 3.64 ⁇ 0.11 c
• Omega-9, omega-6 and omega-3 FAs are indicated as n9, n6, and n3, respectively
• Linoleic acid (n-6) C18:2n6 17.4 ⁇ 0. 51 16.6 ⁇ 0.33 11.7 ⁇ 0.14
• a 12.4 ⁇ 0.45 c trans Linoleic acid (n-6) C18:2n6t 0.07 ⁇ 0.00 0.07 ⁇ 0.00 0.06 ⁇ 0.00 0.07 ⁇ 0.00
• a-Linolenic acid (n-3) C18:3n3 0.04 ⁇ 0.00 0.04 ⁇ 0.00 0.03 ⁇ 0.00 0.04 ⁇ 0.00
• Eicosapentaenoic acid (EPA, n-3) C20:5n3 0.01 ⁇ 0.00 0.01 ⁇ 0.00 0.49 ⁇ 0.02 A 0.47 ⁇ 0.03 c
• Nervonic acid C24:1n9 0.06 ⁇ 0.00 0.07 ⁇ 0.00 0.07 ⁇ 0.01 0.11 ⁇ 0.01 BC
• Omega-9, omega-6 and omega-3 FAs are indicated as n9, n6, and n3, respectively
• BW body weight
• HW heart weight
• IVSth intraventricular septal thickness
• LVPWth left ventricular posterior wall thickness
• LVEDD left ventricular end-diastolic dimension
• LVESD left ventricular end-systolic dimension
• FS fractional shortening
• HR heart rate
• ESP end-systolic pressure
• EDP end-diastolic pressure
• PG pressure gradient.
• Aortic constriction induced a hypertrophic response as evidenced by a 42% increase in heart weight-to-body weight (HW/BW) ratio in mice fed the control diet and a 31% increase in HW/BW ratio in mice fed the fish oil diet (Figure 2E). See also Duda et ah, Cardiovasc Res. 2007, 76:303-310. However, after adjusting for the effect of fish oil, aortic constriction increased the HW/BW ratio by 1.9 ⁇ 0.3 in both control and fish oil groups (Figure 2E).
• mice fed the control diet aortic constriction significantly induced expression of the hypertrophic marker genes atrial and brain natriuretic peptide (ANP and BNP, respectively), which was not observed in mice fed the fish oil diet ( Figures 2F-G, the slight increase in ANP and BNP expression after constriction as compared to sham treatment is not statistically significant).
• TGF- ⁇ levels were measured by ELISA (R&D Systems, Minneapolis, MN) as directed by the product insert. Absorbance was measured at 450 nm on a ThermoMax microplate reader (Molecular Devices, Sunnyvale, CA) and data collected using SoftMax (Molecular Devices) software for data analysis. Cyclic GMP levels were quantified using the acetylation protocol for a competitive Cyclic GMP EIA kit (Cayman Chemical, Ann Arbor, MI).
• aortic constriction increased TGF- ⁇ levels and Smad2 phosphorylation to a similar degree in both control and fish oil groups ( Figures 4A-C).
• the Smad3 phosphorylation levels were not detected in the heart tissue at three days or four weeks post-TAC (data not shown). Therefore, dietary supplementation with fish oil did not interrupt TGF- ⁇ production and Smad2 phosphorylation induced by aortic constriction.
• Example 5 EPA and DHA Inhibited TGF-pi-stimulated Fibrotic Response in Isolated Adult Mouse Cardiac Fibroblasts [0082] To explain the anti-fibrotic effects of dietary fish oil observed in vivo, EPA- and DHA-mediated inhibition of pro-fibrotic TGF- ⁇ signaling was investigated in isolated adult mouse cardiac fibroblasts. EPA (10 ⁇ ) and DHA (10 ⁇ ) significantly increased the ⁇ -3 content (EPA + DHA) in cardiac fibroblasts by 2.3-fold and 1.65-fold, respectively. Neither arachidonic acid ( ⁇ -6 PUFA) nor oleic acid ( ⁇ -9 PUFA) altered the ⁇ -3 content ( Figure 5A).
• TGF- ⁇ The effect of TGF- ⁇ on fibroblast proliferation and collagen synthesis was tested.
• Cardiac fibroblasts (3 x 10 5 cells/dish) were cultured and treated with TGF- ⁇ as above. After 48 hours, fibroblasts were harvested by trypsinization and the cell number was determined by trypan blue exclusion. Collagen synthesis was measured by incorporation of [ 3 H]-proline.
• Cardiac fibroblasts were cultured in 24-well plates (2.5 x 10 4 cells/well), and after 24 hours of culture in reduced serum medium, fibroblasts were treated with TGF- ⁇ (lng/ml) to induce collagen synthesis. After 8 hours, [ 3 H]-proline (1 ⁇ / ⁇ , Perkin Elmer, Waltham, MA) was added.
• fibroblasts were rinsed three times with PBS and fixed with ice cold 10% TCA for 30 min. Cell precipitates were washed with phosphate- buffered saline and solubilized in 0.2 N NaOH at room temperature for one hour.
• Example 6 EPA and DHA Inhibited TGF-pi-stimulated Fibrotic Response Through the cGMP/PKG Pathway [0085]
• the cGMP/PKG signaling pathway plays a counter-regulatory role against TGF- ⁇ - induced cardiac fibrosis.
• cultured cardiac fibroblasts were treated with either EPA or DHA (10-50 ⁇ ).
• Smad2 and Smad3 phosphorylation and subsequent translocation to the nucleus are required for TGF- ⁇ signaling. Since EPA and DHA demonstrated an inhibitory effect on TGF- ⁇ -induced fibrosis ( Figure 5B-E), the effects of EPA and DHA on the TGF- ⁇ -induced phosphorylation of Smad2 and Smad3 were tested. In cultured cardiac fibroblasts, TGF- ⁇ treatment for 30 minutes induced robust phosphorylation of Smad2 (2.7 fold, O.01) and Smad3 (2.2 fold, O.01) ( Figures 6C-E). Longer exposure (24 hours) to TGF- ⁇ induced phosphorylation of Smad2 but not Smad3 ( Figure 6C-E).
• TGF- ⁇ -induced phosphorylation of Smads was not blocked by DHA and EPA, the ability of EPA and DHA to block TGF- ⁇ -induced nuclear translocation of phospho- Smad2 and -Smad3 was analyzed.
• TGF- ⁇ increased the nuclear localization of phospho-Smad2 (66%, PO.01) and phospho-Smad3 (83%, P ⁇ 0.01) ( Figure 7).
• TGF- ⁇ -induced nuclear translocation of both Smads was significantly blocked by EPA and DHA (phospho-Smad2: reduced to 15% with DHA; to 13% with EPA, and phospho-Smad3 : reduced to 14% with DHA; to 13% with EPA) (Figure 7).
• phosphodiesterases hydrolyze cGMP.
• DHA increases NO synthesis and phospho-eNOS and eNOS expression levels (see also Stebbins et al, J Cardiovasc Pharmacol Ther. 2008, 13 :261-268).
• the results presented above also show that EPA and DHA did not increase particulate guanylyl cyclase activity or ANP/BNP production. Additionally, EPA and DHA did not inhibit cGMP-specific phosphodiesterase activity.
• EPA (10 ⁇ ) and DHA (10 ⁇ ) did not increase particulate guanylyl cyclase activity and did not inhibit cGMP- specific phosphodiesterase activity (data not shown).
• EPA (10 ⁇ ) and DHA (10 ⁇ ) significantly decreased ANP mRNA and did not change the BNP mRNA in cardiac fibroblasts (data not shown).
• TGF- ⁇ -induced Smad-responsive promoter activity was also determined by measuring Smad-induced luciferase activity. Passage one cardiac fibroblasts were seeded into a 96-well tissue culture plate (5 x 10 3 cells/well) and were transduced with commercially available lentivirus particles (SABiosciences, Frederick, MD) expressing the firefly luciferase gene under the control of a minimal (m) CMV promoter and tandem repeats of the SMAD transcriptional response element (AGCCAGACA). After 24 hours of culture in reduced serum medium, fibroblasts were pretreated with vehicle or fatty acids for 24 hours, followed by TGF- ⁇ (1 ng/ml) treatment for 48 hours.
• TGF- ⁇ -induced phosphorylation of ERKl/2 is also involved in the TGF- ⁇ -induced fibrotic response in fibroblasts (Liu et al, Mol Pharmacol. 2006, 70: 1992-2003).
• omega-3 PUFAs on the TGF- ⁇ -induced phosphorylation of ERKl/2 was tested in cardiac fibroblasts after one hour treatment of TGF- ⁇ with or without pretreatment of EPA and DHA.
• TGF- ⁇ (1 ng/ml) induced significant phosphorylation of ERKl/2 in cardiac fibroblasts whereas EPA and DHA did not block this effect (Figure 10A-B).
• TGF- ⁇ -induced collagen synthesis was measured in the absence of exogenous FAs (no FA), in the presence of oleic acid (OA, 10 ⁇ ), arachidonic acid (AA, 10 ⁇ ), EPA (10 ⁇ ), or DHA (10 ⁇ ) and treated for a final 24 hr with TGF- ⁇ (1 ng/ml) and incubation with [ 3 H] -proline.
• OA oleic acid
• AA arachidonic acid
• EPA ⁇
• DHA DHA

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