Patent Application
20120237617
This invention relates to methods and compositions useful for treating tissue damage, and more particularly to treating tissue necrosis.
Multicellular organisms are made up of tissues, i.e., organized aggregates of specialized groups of cells of similar form and function. Animal tissues include connective tissue, muscle tissue, nervous tissue and epithelial tissue. Regardless of the kind of tissue, tissue damage, whether by infection, trauma, or disease, contributes significantly to human morbidity and mortality. There is a continuing need for therapeutic strategies that lead to successful treatment of damaged tissue.
The present invention is based, in part, on our discovery of compositions and methods that can be used to treat tissue damage, including damage caused by cardiovascular disease, infection or trauma. Tissue damage generally occurs when some of the cells in a localized area given tissue become injured or die, so that the tissue itself looses part or all or its the ability to function. Severe tissue damage can result in tissue necrosis, i.e., the death of all or substantially all of the cells in a localized area. The tissue damage encompassed by the methods of the claims can stem from any a wide range of medical conditions that result in cellular injury or cell death, for example, disorders such as peripheral artery disease, type 1 or type 2 diabetes, atherosclerotic cardiovascular disease, intermittent claudication (which can manifest as cramping pain in the extremities due to inadequate blood supply), critical limb ischemic disease, stroke, myocardial infarction, inflammatory bowel disease, peripheral neuropathy, rheumatoid arthritis, lupus, bacterial infections, fungal infections, fasciitis, and cellulitis; traumatic injuries such as wounds, burns, lacerations, contusions, frostbite, envenomation, bone fractures, infections, or surgical procedures; congenital malformations such as hernias, cardiac defects and gastrointestinal defects. Thus, tissue damage can occur in a variety of tissue types including, for example, skeletal muscle, smooth muscle, cardiac muscle, neuronal tissue, skin, mesenchymal tissue, connective tissue, gastrointestinal tissue and bone.
Also contemplated are compositions and methods to stimulate tissue regeneration. The compositions and methods can be used to stimulate tissue regeneration following damage to a tissue or organ caused by such conditions as trauma, scarring, abnormal protein deposition, amyloidoses, ischemia or diabetes, infections, or surgical procedures; congenital malformations such as hernias, cardiac defects and gastrointestinal defects that result in damage to the tissue.
Also provided are compositions and methods to treat inflammatory diseases. The inflammatory diseases encompassed by the methods of this invention can stem from a wide range of medical conditions that cause inflammation. Inflammation refers to all phases of the process of inflammation, including the initial signaling events, antigen presentation, dendritic cell activation, antibody production, T-cell activation, B-cell activation and cytokine expression. One type of inflammatory disease which can be treated by the compositions and methods described herein are immunoinflammatory diseases. Examples of immunoinflammatory diseases include such conditions as rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, transplant rejection, sepsis, acute respiratory distress syndrome, asthma, and cancer. Another type of inflammatory disease which can be treated by the composition and methods described herein are autoimmune diseases. Examples of autoimmune diseases include such conditions as multiple sclerosis, psoriasis, inflammatory bowel disease, glomerulonephritis, lupus, uveitis, and chronic hepatitis. Other inflammatory diseases can also be treated by the compositions and methods described in this invention, including such conditions caused by trauma, oxidative stress, cell death, irradiation damage, ischemic, reperfusion, cancer, transplant rejection, and viral infection.
Regardless of whether the methods are described with respect to a particular medical condition or tissue type, the methods can be carried out by administering to a subject (e.g., a human patient) in need of treatment a pharmaceutically acceptable composition comprising inorganic nitrite or a pharmaceutically acceptable salt thereof. The inorganic nitrite or a pharmaceutically acceptable salt thereof can be formulated in various ways and can include pharmaceutically acceptable carriers. For ease of reading, we will not repeat the phrase “or a pharmaceutically acceptable salt thereof” on every occasion. It is to be understood that where inorganic nitrite can be used, a pharmaceutically acceptable salt of the compound may also be used.
Accordingly, the invention features physiologically acceptable compositions of inorganic nitrite and methods by which the compositions can be administered to a patient diagnosed as having, for example, tissue damage. These methods can include the steps of a) identifying a subject (e.g., a human patient) who is experiencing or is likely to experience tissue damage; and b) providing to the subject a composition including inorganic nitrite for a time and in an amount sufficient to reduce the tissue damage. The nitrite can result in the differential expression of genes involved in tissue repair responses, e.g., pathways or genes involved in cardiovascular system development and function, tissue morphology, carbohydrate metabolism; skeletal muscle development, tissue development; cellular assembly and organization, angiogenesis and molecular transport with the resulting remodeling of the damaged tissue. Such remodeling can include for example, the growth of new blood vessels in the damaged tissue and proliferation of muscle cells.
Patients amenable to being treated with inorganic nitrite can also be treated with nitrate. We may use the terms “subject,” “individual” and “patient” interchangeably. While the present methods are certainly intended for application to human patients, the invention is not so limited. Domesticated animals, including, for example cats, dogs, horses, cows and other domesticated animals can also be treated.
The pharmaceutically acceptable compositions of the invention include inorganic nitrite, e.g., a salt or ester of nitrous acid (HNO2) or a pharmaceutically acceptable salt thereof. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present invention include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred.
Suitable pharmaceutically acceptable salts can include, for example, sodium nitrite, potassium nitrite, or calcium nitrite. The invention is not so limited however and lists of exemplary salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety. It will also be understood that certain nitrite compounds of the present invention may exist in solvated, for example hydrated, as well as unsolvated forms. Nitrite has the chemical formula N02 and may exist as an ion in water. Sodium nitrite has the chemical formula NaN02 and typically dissolves in water to form the sodium ion Na+ and the nitrite ion N02. It will further be understood that the present invention encompasses all such solvated forms of the nitrite compounds.
The inorganic nitrite is administered for a time and in an amount sufficient to result the reduction of tissue damage. The effect on tissue damage may be monitored indirectly, i.e., by monitoring a clinical endpoint. For example, the nitrite may be administered until a symptom of tissue damage, e.g., pain, swelling, skin discoloration, ulceration, neuropathy, or defective tissue wound healing, improves. The assessment of clinical benefit may entail comparison of the damaged tissue with the corresponding non-damaged tissue. Choice of specific clinical endpoints may depend, in part, upon the nature of the underlying medical condition, e.g., cessation or amelioration of intermittent claudication may be useful for patients with peripheral artery disease or diabetes; healing of skin ulcers may be useful for patients with defective wound healing, and relief from gastrointestinal pain, diarrhea and constipation may be useful for patient suffering from bowel ischemia, enhancement of protein expression through profiling of a patient's blood might be used to monitor therapeutic benefit in patients with amyloidosis or ischemia; enhancement of cardiac function may be useful for patients with damage to heart tissue; and reduction in scarring in post-surgical patients. Revascularization can be monitored directly, via imaging techniques such as contrast angiography, contrast pulse sequence (CPS) ultrasound imaging for high-resolution perfusion or detection of biomarkers. The course of treatment can also be monitored by assessing the levels of expression of particular genes or gene families associated with tissue regeneration, tissue remodeling, tissue morphology, cardiovascular function, carbohydrate metabolism, antigen presentation, cell or humoral mediated immunity, skeletal muscle development, cardiovascular development, tissue development, cellular assembly and organization, carbohydrate metabolism, molecular transport, neurological disease, cancer, nervous system development, cellular development and/or inflammatory disease.
In some embodiments, the nitrite may be administered until a symptom of inflammation improves. The assessment of clinical benefit may entail comparison of the inflamed tissue or joint with the corresponding non-inflamed tissue or joint or comparing the inflamed tissue, joint or organ to a normal tissue joint or organ. Cytokine profiles can also be generated from blood samples obtained from patients and analyzed for reduction of cytokine levels to those found in normal conditions. Choice of specific clinical endpoints may depend, in part, upon the nature of the underlying medical condition, e.g., inhibition of cytokine expression through profiling of a patients blood might be used to monitor therapeutic benefit in patients with sepsis or asthma; reduction of swelling and pain in joints may be useful for patients with arthritis; and relief from gastrointestinal pain, diarrhea and constipation may be useful for patient suffering from irritable bowel disease.
The amount of inorganic nitrite per dose can vary. For example, a subject can receive from about 0.05 μg/kg up to about 5000 μg/kg., e.g., about 0.05, 1, 5, 10, 25, 50, 100, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1250, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 μg/kg. For example, a subject can receive up to or up to about 165 μg/kg, 16.5 μg/kg, or 8.25 μg/kg. Generally, we administer nitrite in an amount such that the circulating concentration does not exceed 1.0 μM (i.e., the nitrite is administered in a dose sufficient to produce a circulating concentration of nitrite in the subject that does not exceed 1.0 μM). For example, the nitrite can be administered in an amount such that the circulating concentration does not exceed 0.0005 μM, 0.001 μM, 0.002 μM, 0.003 μM, 0.004 μM, 0.005 μM, 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, 0.55 μM, 0.6 μM, 0.65 μM, 0.7 μM, 0.75 μM, 0.8 μM, 0.85 μM, 0.9 μM, 0.95 μM, or 1.0 μM. Thus, exemplary dosages can produce a circulating concentration of nitrite in the subject of up to or up to about 0.03 μM, 0.003 μM, or 0.0015 μM.
The frequency of treatment may also vary. The subject can be treated one or more times per day (e.g., once, twice, three, four, five, or six times per day) or every so-many hours (e.g., about every 2, 4, 6, 8, 12, or 24 hours). The time course of treatment may be of varying duration, e.g., for two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty five, thirty, thirty-five, forty, forty-five, fifty, fifty-five, sixty, sixty-five, seventy or more days. For example, the treatment can be twice a day for three days, twice a day for seven days, twice a day for ten days. Treatment cycles can be repeated at intervals, for example weekly, bimonthly or monthly, which are separated by periods in which no treatment is given. The treatment can be a single treatment or can last as long as the life span of the subject (e.g., many years).
The compositions can be administered to a subject in a variety of ways. For example, the compositions can be administered transdermally or injected (infused) intravenously, subcutaneously, sublingually, intracranially, intramuscularly, intraperitoneally, or intrapulmonarily. Oral formulations are also within the scope of the present invention. The treatment regime can vary depending upon various factors typically considered by one of ordinary skill in the art. These factors include the route of administration, the nature of the formulation, the nature of the patient's illness, the subject's size, weight, surface area, age, gender, other drugs being administered to the patient, and the judgment of the attending physician. The compositions can be administered along with or in addition to other treatments for tissue damage or, e.g., drug therapy, immunotherapy, or surgery (e.g., aspirin therapy, statin therapy, or antihypertensive therapy) or other treatments for the underlying cause of the tissue damage, e.g., antibiotics, antifungals.
Disorders amenable to the methods of the invention can include any disorder that presents with tissue damage. Conditions that present with tissue damage include, for example peripheral artery disease, diabetes, atherosclerotic cardiovascular disease, intermittent claudication, critical limb ischemic disease, defective wound healing, stroke, myocardial infarction, inflammatory bowel disease, a bone fracture, a bone infection, or peripheral neuropathy, avascular necrosis, rheumatoid arthritis, lupus, a bacterial infection, a fungal infection, fasciitis, or cellulitis, trauma, scarring, abnormal protein deposition, amyloidoses, or surgical procedures; congenital malformations such as hernias, cardiac defects and gastrointestinal defects.
Disorders amenable to the methods of the invention can include any disorder that presents with tissue necrosis or any disorder or condition that puts the patient at risk for tissue necrosis, for example peripheral artery disease, diabetes, atherosclerotic cardiovascular disease, intermittent claudication, critical limb ischemic disease, defective wound healing, stroke, myocardial infarction, inflammatory bowel disease, a bone fracture, a bone infection, or peripheral neuropathy, avascular necrosis, an autoimmune disease, a bacterial infection, a fungal infection, fasiculitis, or cellulitis.
Disorders amenable to the methods of the invention can include conditions that result in inflammation, for example, immunoinflammatory diseases such as rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, transplant rejection, sepsis, acute respiratory distress syndrome, asthma, and cancer; autoimmune diseases, such as multiple sclerosis, psoriasis, inflammatory bowel disease, glomerulonephritis, lupus, uveitis, and chronic hepatitis, or inflammation resulting from trauma, oxidative stress, cell death, irradiation damage, ischemia, reperfusion, cancer, transplant rejection, and viral infection.
Accordingly, disclosed herein is a method of treating tissue damage in a subject, the method comprising: identifying a subject who has tissue damage; and administering to the subject a pharmaceutical composition comprising inorganic nitrite or a pharmaceutically acceptable salt thereof, wherein the nitrite is administered for a time and in an amount sufficient to reduce said tissue damage. The method can further include monitoring whether the subject experiences reduced tissue damage. The subject can be a mammal; the mammal can be human.
The pharmaceutically acceptable salt of inorganic nitrite is sodium nitrite, potassium nitrite, or calcium nitrite. The nitrite can be administered one or more times a day for example, for at least about two to about twenty days; for at least about two days; at least about three days; at least about four days; at least about five days; at least about six days; at least about seven days; at least about 10 days; or at least about 15 days.
The nitrite can be administered until a symptom of tissue damage in the subject improves. The symptom of tissue damage can include pain, skin pallor, numbness, swelling, ulceration, neuropathy, erythema, decreased plantar sensation, or defective tissue wound healing.
The amount of nitrate can be a dose of about 1 μg/kg to about 5000 μg/kg; about 0.5 μg/kg to about 1000 μg/kg; about 0.5 μg/kg to about 500 μg/kg; about 0.5 μg/kg to about 250 μg/kg; about 0.5 μg/kg to about 100 μg/kg; or about 0.5 μg/kg to about 50 μg/kg; about 165 μg/kg; about 16.5 μg/kg; or about 8.25 μg/kg. The nitrite can be administered to a circulating concentration in the subject of about 0.0005 μM to about 1.0 μM; about 0.001 μM to about 0.03 μM, of about 0.01 μM to about 0.05 μM, or about 0.05 to about 0.5 μM; about 0.5 μM, 0.3 μM, 0.03 μM; of about 0.003 μM; or of about 0.0015 μM.
In another embodiment, the nitrite or pharmaceutically acceptable salt thereof can be administered intraperitoneally, intravenously, subcutaneously, intramuscularly, transdermally, sublingually, or orally. The subject can have peripheral artery disease, diabetes, atherosclerotic cardiovascular disease, intermittent claudication, critical limb ischemic disease, defective wound healing, stroke, myocardial infarction, inflammatory bowel disease, a bone fracture, a bone infection, or peripheral neuropathy, avascular necrosis, rheumatoid arthritis, lupus, a bacterial infection, a fungal infection, fasciitis, or cellulitis. The diabetes can be type 1 or type 2 diabetes.
In another aspect, the tissue damage includes skeletal muscle, smooth muscle, cardiac muscle, neuronal tissue, skin, mesenchymal tissue, connective tissue, gastrointestinal tissue or bone.
In another embodiment, the method further includes administering an anti-ischemic therapy, an antimicrobial agent, an analgesic agent, an anti-inflammatory agent, a chemotherapeutic agent or a growth factor.
In another aspect, the tissue damage can be a wound. The wound can include skin, muscle, or a connective tissue and be the result of a traumatic injury, a congenital malformation, or a surgical procedure. The nitrite can be administered until the wound substantially heals.
In another embodiment, disclosed is a method of treating tissue necrosis in a subject, the method comprising: a) identifying a subject who has or who is at risk for tissue necrosis; and b) administering to the subject a pharmaceutical composition comprising inorganic nitrite or a pharmaceutically acceptable salt thereof, wherein the nitrite is administered for a time and in an amount sufficient to reduce said necrosis. The method can further include monitoring whether the subject experiences reduced necrosis. The subject can be a mammal; the mammal can be human.
The pharmaceutically acceptable salt of inorganic nitrite is sodium nitrite, potassium nitrite, or calcium nitrite. The nitrite can be administered one or more times a day for example, for at least about two to about twenty days; for at least about two days; at least about three days; at least about four days; at least about five days; at least about six days; at least about seven days; at least about 10 days; or at least about 15 days.
The nitrite can be administered until a symptom of tissue damage in the subject improves. The symptom of tissue damage can include pain, skin pallor, numbness, swelling, ulceration, neuropathy, erythema, decreased plantar sensation, gangrene or defective tissue wound healing.
The amount of nitrate can be a dose of about 1 μg/kg to about 5000 μg/kg; about 0.5 μg/kg to about 1000 μg/kg; about 0.5 μg/kg to about 500 μg/kg; about 0.5 μg/kg to about 250 μg/kg; about 0.5 μg/kg to about 100 μg/kg; or about 0.5 μg/kg to about 50 μg/kg; about 165 μg/kg; about 16.5 μg/kg; or about 8.25 μg/kg. The nitrite can be administered to a circulating concentration in the subject of about 0.0005 μM to about 1.0 μM; about 0.001 μM to about 0.03 μM, of about 0.01 μM to about 0.05 μM, or about 0.05 to about 0.5 μM; about 0.5 μM, 0.3 μM, 0.03 μM; of about 0.003 μM; or of about 0.0015 μM.
In another embodiment, the nitrite or pharmaceutically acceptable salt thereof can be administered intraperitoneally, intravenously, subcutaneously, intramuscularly, transdermally, sublingually, or orally. The subject can have peripheral artery disease, diabetes, atherosclerotic cardiovascular disease, intermittent claudication, critical limb ischemic disease, defective wound healing, stroke, myocardial infarction, inflammatory bowel disease, a bone fracture, a bone infection, or peripheral neuropathy, avascular necrosis, an autoimmune disease, a bacterial infection, a fungal infection, fasiculitis, or cellulitis. The diabetes can be type 1 or type 2 diabetes.
In another aspect, the tissue necrosis includes skeletal muscle, smooth muscle, cardiac muscle, neuronal tissue, skin, mesenchymal tissue, connective tissue, gastrointestinal tissue or bone. In another embodiment, the method further includes administering an anti-ischemic therapy, an antimicrobial agent, an analgesic agent, an anti-inflammatory agent, a chemotherapeutic agent or a growth factor.
In another aspect, the tissue necrosis can be a wound. The wound can include skin, muscle, or a connective tissue and be the result of a traumatic injury, a congenital malformation, or a surgical procedure. The nitrite can be administered until the wound substantially heals. In another aspect, the method further includes administering a surgical treatment. The surgical treatment can be surgical debridement, surgical drainage or tissue grafting.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
Like reference symbols in the various drawings indicate like elements.
We further describe below the present methods for treatment of tissue damage. These methods can be applied to, and are expected to benefit subjects having any of a variety of medical conditions that can give rise to tissue damage. The methods are based, inter alia, on the inventor's discovery that administration of inorganic nitrite or a pharmaceutical composition comprising inorganic nitrite to a subject having tissue damage results in the selective reduction of symptoms of tissue damage and tissue necrosis and the inhibition or enhancement of genes associated with tissue remodeling and tissue regeneration.
Compositions
The pharmaceutically acceptable compositions of the invention include inorganic nitrite, e.g., a salt or ester of nitrous acid (HN02) or a pharmaceutically acceptable salt thereof. The nitrite ion is N02-. More generally, a nitrite compound is either a salt or an ester of nitrous acid. Nitrite salts can include, without limitation, salts of alkali metals, e.g., sodium, potassium; salts of alkaline earth metals, e.g., calcium, magnesium, and barium; and salts of organic bases, e.g., amine bases and inorganic bases. Compounds of the invention also include all isotopes of atoms occurring in the intermediate or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium. The term, “compound,” as used herein with respect to any inorganic nitrite or pharmaceutically acceptable salt thereof and is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted. All compounds, and pharmaceutically acceptable salts thereof, are also meant to include solvated or hydrated forms.
The compounds of the present invention can be prepared in a variety of ways known to one of ordinary skill in the art of chemical synthesis. The compounds of the present invention can be synthesized using the methods described below, together with synthetic methods known in the art of synthetic chemistry or variations thereon as appreciated by one of ordinary skill in the art. Methods for preparing nitrite salts are well known in the art and a wide range of precursors and nitrite salts are readily available commercially. Nitrites of the alkali and alkaline earth metals can be synthesized by reacting a mixture of nitrogen monoxide (NO) and nitrogen dioxide (N02) with a corresponding metal hydroxide solution, as well as through the thermal decomposition of the corresponding nitrate. Other nitrites are available through the reduction of the corresponding nitrates.
The present compounds can be prepared from readily available starting materials using the following general methods and procedures. It will be appreciated that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given, other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one of ordinary skill in the art by routine optimization procedures.
Regardless of their original source or the manner in which they are obtained, the compounds of the invention can be formulated in accordance with their use. For example, the compounds can be formulated within compositions for application to cells in tissue culture or for administration to a patient. When employed as pharmaceuticals, any of the present compounds can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known in the pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated.
Administration may be topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), ocular, oral or parenteral. Methods for ocular delivery can include topical administration (eye drops), subconjunctival, periocular or intravitreal injection or introduction by balloon catheter or ophthalmic inserts surgically placed in the conjunctival sac. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular administration. Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, and the like. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
This invention also includes pharmaceutical compositions which contain, as the active ingredient, one or more of the compounds described herein in combination with one or more pharmaceutically acceptable carriers. In making the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semisolid, or liquid material (e.g., normal saline), which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. As is known in the art, the type of diluent can vary depending upon the intended route of administration. The resulting compositions can include additional agents, such as preservatives. The compounds may also be applied to a surface of a device (e.g., a catheter) or contained within a pump, patch, or other drug delivery device. The therapeutic agents of the invention can be administered alone, or in a mixture, in the presence of a pharmaceutically acceptable excipient or carrier (e.g., physiological saline). The excipient or carrier is selected on the basis of the mode and route of administration. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.
Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The pharmaceutical compositions can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
The compositions can be formulated in a unit dosage form, each dosage containing, for example, from about 0.1 mg to about 50 mg, from about 0.1 mg to about 40 mg, from about 0.1 mg to about 20 mg, from about 0.1 mg to about 10 mg, from about 0.2 mg to about 20 mg, from about 0.3 mg to about 15 mg, from about 0.4 mg to about 10 mg, from about 0.5 mg to about 1 mg; from about 0.5 mg to about 100 mg, from about 0.5 mg to about 50 mg, from about 0.5 mg to about 30 mg” from about 0.5 mg to about 20 mg, from about 0.5 mg to about 10 mg, from about 0.5 mg to about 5 mg; from about 1 mg from to about 50 mg, from about 1 mg to about 30 mg” from about 1 mg to about 20 mg, from about 1 mg to about 10 mg, from about 1 mg to about 5 mg; from about 5 mg to about 50 mg, from about 5 mg to about 20 mg, from about 5 mg to about 10 mg; from about 10 mg to about 100 mg, from about 20 mg to about 200 mg, from about 30 mg to about 150 mg, from about 40 mg to about 100 mg, from about 50 mg to about 100 mg of the active ingredient. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.
For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid preformulation is then subdivided into unit dosage forms of the type described above 10 containing from, for example, 0.1 to about 500 mg of the active ingredient of the present invention.
The tablets or pills of the present invention can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. Exemplary methods of such formulations are described in U.S. application No. 61/251,483, which is herein incorporated by reference. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release.
A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
The liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described herein and/or known in the art. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in can be nebulized by use of inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device can be attached to a face masks tent, or intermittent positive pressure breathing machine.
Solution, suspension, or powder compositions can be administered orally or nasally from devices which deliver the formulation in an appropriate manner.
The compositions administered to a patient can be in the form of one or more of the pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques or may be sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the compound preparations typically will be between about 3 and 11, for example, between about 5 to 9, between 6 and 7, between 7 and 8. It will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of pharmaceutical salts.
The proportion or concentration of a compound of the invention in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, the compounds of the invention can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w/v of the compound for parenteral administration.
Methods of Treatment
Tissue damage is associated with a wide range of medical conditions that result in cellular injury and/or damage to the extracellular matrix which surrounds the cells in some tissue types and may be the result of disease, injury, or of an unknown cause, and may be influenced by one's genetic constitution. Regardless of the medical condition leading to tissue damage, a patient who has damaged tissue is a candidate for treatment with the pharmaceutically acceptable compositions comprising inorganic nitrite described herein. Treatment can completely or partially abolish some or all of the signs and symptoms of tissue damage, decrease the severity of the symptoms, delay their onset, or lessen the progression or severity of subsequently developed symptoms.
Tissue Damage and Regeneration
As described further below, the compositions of the invention are administered for a time and in an amount sufficient to reduce tissue damage or a symptom of tissue damage improves. Symptoms of tissue damage may vary depending upon the nature of the damage and the tissue type involved, but can include pain, skin pallor, numbness, swelling, ulceration, neuropathy, erythema, decreased plantar sensation, or defective tissue wound healing. We may use the terms “tissue regeneration,” “tissue repair” and “tissue remodeling” interchangeably. Tissue regeneration refers to all phases of the process of regeneration, including, for example the initial signaling events, induction of endothelial cell proliferation, enhancement of gene expression of genes involved in cell proliferation, tissue morphology and development, carbohydrate metabolism, cell assembly, molecular transport and induction of stem or progenitor cell growth and differentiation.
When tissues become damaged, an ordered series of physiological events must take place in a timely fashion for successful tissue regeneration to occur. The first events, termed the inflammatory phase, include blood clotting as well as the arrival at the site of damaged cells that remove bacteria, debris and damaged tissue. Later, circulating stem cells migrate to the site and differentiate into tissue-specific cell types. Finally, the differentiated cells begin to produce and deposit new extracellular matrix (ECM), a complex mixture of carbohydrates and proteins that provides support and anchorage for cells. The nature of tissue regeneration and repair depends in part on the extent of the damage, that is, the damaged cells themselves can be repaired in a process sometimes termed resolution, the dead cells can be replaced with the same cell type (regeneration), or the lost tissue can be replaced with scar tissue in a process termed fibrosis. These processes may occur simultaneously or sequentially.
The methods and compositions described herein can be used to treat any of a wide range of disorders in which amelioration or repair of tissue damage is needed. Tissue defects can arise from diverse medical conditions, including, for example, congenital malformations, traumatic injuries, infections, and oncologic resections. Thus, the compositions can be used to promote regeneration and repair in any soft tissue, e.g., tissues that connect, support, or surround other structures and organs of the body. The methods and compositions can also be used to promote bone repair, e.g., as a periosteal graft to support bone or an articular graft to drive cartilage repair. Soft tissue can be any non-osseous tissue. Soft tissue can also be epithelial tissue, which covers the outside of the body and lines the organs and cavities within the body. Examples of epithelial tissue include, but are not limited to, simple squamous epithelia, stratified squamous epithelia, cuboidal epithelia, or columnar epithelia.
Soft tissue can also be connective tissue, which functions to bind and support other tissues. One example of connective tissue is loose connective tissue (also known as areolar connective tissue). Loose connective tissue, which functions to bind epithelia to underlying tissues and to hold organs in place, is the most widely distributed connective tissue type in vertebrates. It can be found in the skin beneath the dermis layer; in places that connect epithelium to other tissues; underneath the epithelial tissue of all the body systems that have external openings; within the mucus membranes of the digestive, respiratory, reproductive, and urinary systems; and surrounding the blood vessels and nerves. Loose connective tissue is named for the loose “weave” of its constituent fibers which include collagenous fibers, elastic fibers (long, thread-like stretchable fibers composed of the protein elastin) and reticular fibers (branched fibers consisting of one or more types of very thin collagen fibers). Connective tissue can also be fibrous connective tissue, such as tendons, which attach muscles to bone, and ligaments, which joint bones together at the joints. Fibrous connective tissue is composed primarily of tightly packed collagenous fibers, an arrangement that maximizes tensile strength. Soft tissue can also be muscle tissue. Muscle tissue includes: skeletal muscle, which is responsible for voluntary movements; smooth muscle, which is found in the walls of the digestive tract, bladder arteries and other internal organs; and cardiac muscle, which forms the contractile wall of the heart.
The methods and compositions can be used to promote regeneration and repair of soft tissue in many different organ systems that fulfill a range of physiological functions in the body. These organ systems can include, but are not limited to, the muscular system, the genitourinary system, the gastroenterological system, the integumentary system, the circulatory system and the respiratory system. The compositions are particularly useful for support of regeneration of endothelial, muscle, cardiovascular tissue and epithelial tissue. The compositions and methods of the invention enhance the expression of genes associated with tissue remodeling and muscle growth, modulate the expression of genes associated with inflammation, and differentially stimulate new blood vessel growth as described in WO 2009/065142, which is herein incorporated by reference.
Tissue Necrosis
A particularly severe form of tissue damage is tissue necrosis. We tend to use the terms “tissue necrosis” and “necrosis” interchangeably. Symptoms of tissue necrosis may vary depending upon the nature of the damage and the tissue type involved, but can include pain, skin pallor, numbness, swelling, ulceration, neuropathy, erythema, decreased plantar sensation, defective tissue wound healing, skin blackening, gangrene and exudation. Tissue necrosis is associated with a wide range of medical conditions that result in cellular injury may be the result of disease, injury, infection, envenomation, toxins or of an unknown cause, and may be influenced by one's genetic constitution. Unlike apoptosis, which is a highly regulated, often beneficial process of cell death, necrosis typically begins with cell swelling, chromatin digestion, disruption of the plasma membrane and organelle membranes. Late necrosis is characterized by extensive DNA hydrolysis, vacuolation of the endoplasmic reticulum, organelle breakdown, and cell lysis. The release of intracellular content, particularly lysosomal enzymes, after plasma membrane rupture triggers death of surrounding cells. Tissue necrosis can require surgical removal, or debridement, and may in some cases necessitate amputation of the affected area; severe necrosis may in some cases, result in death.
Histologically, there are a number of different morphological patterns of necrosis: 1) coagulative necrosis, typically seen in hypoxic (low oxygen) environments, such as an infarction. Cell outlines remain after cell death and can be observed by light microscopy; 2) liquefactive necrosis (or colliquative necrosis) which is usually associated with cellular destruction and pus formation (e.g. pneumonia), is typical of bacterial or fungal infections because of their ability to stimulate an inflammatory reaction; 3) gummatous necrosis is restricted to necrosis involving spirochaetal infections (e.g. syphilis); 4) haemorrhagic necrosis, due to blockage of the venous drainage of an organ or tissue (e.g. in testicular torsion); 5) caseous necrosis, a specific form of coagulation necrosis typically caused by mycobacteria (e.g. tuberculosis), fungi, and some foreign substances; 6) fatty necrosis results from the action of lipases on fatty tissues (e.g. acute pancreatitis, breast tissue necrosis); and 7) fibrinoid necrosis, caused by immune-mediated vascular damage is marked by deposition of fibrin-like proteinaceous material in arterial walls.
Disorders or conditions associated with tissue necrosis or that put a patient at risk for tissue necrosis, for example, peripheral artery disease, diabetes, atherosclerotic cardiovascular disease, intermittent claudication, critical limb ischemic disease, defective wound healing, stroke, myocardial infarction, inflammatory bowel disease, a bone fracture, a bone infection, or peripheral neuropathy, avascular necrosis, an autoimmune disease, a bacterial infection, e.g., a Group A streptococcus infection, Bacillaceae infections such as Bacillus anthracis, a fungal infection, venomous spider bites, venomous snake bites, frostbite, osteonecrosis, fasiculitis, or cellulitis
Nitric Oxide
The methods and compositions provided herein can induce the preferential formation of nitric oxide in damaged tissue. Nitric oxide (NO) has been shown to positively regulate endothelial cell responses, can stimulate tissue regeneration, enhance stem cell growth and proliferation and alter inflammatory responses. NO increases the expression of various angiogenic factors, including YEOF, which, together with other mediators, increases NO levels via a positive feedback mechanism. In addition to stimulating the growth of endothelial cells, NO can also protect tissues against ischemic damage by slowing cellular respiration. NO has been shown to modulate several endothelial cell signaling pathways for example, Erk 112 and PKC. In the present invention, NO is found to accelerate tissue healing and recovery by augmenting angiogenic responses involving redox dependent pathways while diminishing inflammatory responses.
The primary enzyme responsible for NO production in the cardiovascular system is endothelial nitric oxide synthase (eNOS) which is regulated by numerous molecules and signaling pathways. Importantly, eNOS activity is also largely responsible for systemic NO production as the amount of enzyme expression is often directly proportional to NO metabolite levels. NO readily diffuses across lipid bilayers and its biological fate is dictated predominately by reactions with metalloproteins and other free radical species; the classic example being activation of the heme enzyme soluble guanylate cyclase (sGC) which initiates a signal cascade leading to vessel dilation and platelet inhibition. In addition, NO may also be oxidized through various mechanisms resulting in the formation of nitrite which can be further oxidized to nitrate (N03-).
Both nitrite and nitrate are involved in regulating production of NO from NOS independent pathways. Inorganic nitrite can undergo a one electron reduction back to NO through various mechanisms with oxygen-binding heme proteins (hemoglobin and myoglobin), deoxyhemoglobin, deoxymyoglobin, xanthine oxidoreductase, endothelial nitric oxide synthase, acidic disproportionation, and members of the mitochondrial electron transport chain, e.g., mitochondrial heme proteins all being potential electron donors. The ability of nitrite to be reduced back to NO classifies it as a unique NO donor under biological conditions, e.g., tissue ischemia, in which many of these potential reducing agents are active. NO interacts with several intracellular targets to form various NO-containing species including S-nitrosothiols, C— or N—S-nitroso compounds, and nitrosylheme adducts. Moreover, these nitro so-products may serve as a biological reservoir for NO, which can be liberated under certain conditions
Administration
The present methods for treating tissue damage are carried out by administering an inorganic nitrite for a time and in an amount sufficient to result a reduction in tissue damage.
The amount and frequency of administration of the compositions can vary depending on, for example, what is being administered, the state of the patient, and the manner of administration. In therapeutic applications, compositions can be administered to a patient suffering from tissue damage in an amount sufficient to relieve or least partially relieve the symptoms of tissue damage and its complications. In some embodiments, compositions can be administered to a patient suffering from a condition that carries a risk for tissue necrosis in an amount sufficient to reduce the likelihood of tissue necrosis. The dosage is likely to depend on such variables as the type and extent of progression of the tissue damage, any associated inflammatory conditions, the amount of damage in the tissue, the severity of the inflammation or tissue damage, the age, weight and general condition of the particular patient, the relative biological efficacy of the composition selected, formulation of the excipient, the route of administration, and the judgment of the attending clinician. Effective doses can be extrapolated from dose response curves derived from in vitro or animal model test system. An effective dose is a dose that produces a desirable clinical outcome by, for example, improving a sign or symptom of tissue damage or inflammation or reversing or slowing tissue damage progression.
The amount of inorganic nitrite per dose can vary. For example, a subject can receive from about 0.05 μg/kg to about 5000 μg/kg., e.g., about 0.05, 1, 5, 10, 25, 50, 100, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 1000, 1250, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 5000 μg/kg. Generally, we administer nitrite in an amount such that the circulating concentration does not exceed 1.0 μM, e.g., 0.0005 μM, 0.001 μM, 0.002 μM, 0.003 μM, 0.004 μM, 0.005 μM, 0.01 μM, 0.02 μM, 0.03 μM, 0.04 μM, 0.05 μM, 0.1 μM, 0.15 μM, 0.2 μM, 0.25 μM, 0.3 μM, 0.35 μM, 0.4 μM, 0.45 μM, 0.5 μM, 0.55 μM, 0.6 μM, 0.65 μM, 0.7 μM, 0.75 μM, 0.8 μM, 0.85 μM, 0.9 μM, 0.95 μM, or 1.0 μM. Thus, exemplary dosages can include 8.25 μg/kg, 16.5 μg/kg or 165 μg/kg and exemplary circulating plasma concentrations can include 0.0015 μM, 0.003 μM or 0.030 μM, 0.30 μM or 0.50 μM.
The frequency of treatment may also vary. The subject can be treated one or more times per day (e.g., once, twice, three, four or more times) or every so-many hours (e.g., about every 2, 4, 6, 8, 12, or 24 hours). The time course of treatment may be of varying duration, e.g., for two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty five, thirty, thirty-five, forty, forty-five, fifty, fifty-five, sixty, sixty-five, seventy or more days. For example, the treatment can be twice a day for three days, twice a day for seven days, twice a day for ten days. Treatment cycles can be repeated at intervals, for example weekly, bimonthly or monthly, which are separated by periods in which no treatment is given. The treatment can be a single treatment or can last as long as the life span of the subject (e.g., many years).
Method of the invention are applicable to any of a wide range of medical conditions which have as their underlying feature a persistent or progressive tissue damage due in part to reduction of oxygen levels in a tissue or organ. Thus, the methods are applicable to treatment of tissue damage associated with a disorder, with a trauma or an environmental stress. The reduction in oxygen levels can be, for example, the result of a progressive blockage of an artery due to hardening and/or loss of elasticity due to an atheromatous plaque or the presence of a clot. Reduction of oxygen levels in a tissue can also be the result of an environmental insult, for example, a traumatic injury or surgical procedure that interrupts the blood flow to a tissue or organ. Typically, the oxygen tension of a wound quickly and progressively decreases with the development of varying degrees of hypoxia throughout the wound region. Environmental conditions that induce hypoxia are also within the scope of the invention.
Disorders encompassed by the invention include, for example, cardiovascular disease, peripheral artery disease, arteriosclerosis, atherosclerotic cardiovascular disease, myocardial infarction, critical limb ischemic disease, stroke, acute coronary syndrome, intermittent claudication, diabetes, including type 1 and type 2 diabetes, skin ulcers, peripheral neuropathy, inflammatory bowel disease, ulcerative colitis, Crohn's disease, intestinal ischemia, and chronic mesenteric ischemia.
The methods of the invention are also applicable to tissue damage associated with a trauma, for example, a traumatic injury such as a wound, laceration, burn, contusion, bone fracture or chronic infection. Also encompassed by the invention are tissue injuries sustained as part of any surgical procedure, for example, endarterectomy. Procedures involving tissue or organ transplantation are within the scope of the invention. Examples include vascular bypass grafts, heart, liver, lung, pancreatic islet cell transplantation as well as transplantation of tissues generated ex vivo for implantation in a host. The methods of the invention are also useful for treating tissue damage brought about by exposure to an environmental insult, for example, chronic exposure to hypoxic conditions e.g., high altitude, or sustained aerobic exertion.
The methods provided herein are applicable to any of a wide range of tissue types including, for example, muscle, smooth muscle, skeletal muscle, cardiac muscle, neuronal tissue, skin, mesenchymal tissue, connective tissue, gastrointestinal tissue or bone. Soft tissue, such as epithelial tissue, e.g., simple squamous epithelia, stratified squamous epithelia, cuboidal epithelia, or columnar epithelia, loose connective tissue (also known as areolar connective tissue), fibrous connective tissue, such as tendons, which attach muscles to bone, and ligaments, which join bones together at the joints.
The methods of the invention can include the steps of identifying a subject (e.g., a human patient) who is experiencing tissue damage or who is experiencing or is likely to experience tissue necrosis. Since tissue damage can result from a wide range of medical conditions all of which have as their underlying feature a persistent reduction of oxygen levels in a tissue, the specific signs and symptoms will vary depending upon factor or factors responsible for the reduction of oxygen levels.
Tissue damage can result from chronic tissue ischemia in peripheral artery disease (PAD), a form of peripheral vascular disease in which there is partial or total blockage of an artery, usually due to atherosclerosis in a vessel or vessels leading to a leg or arm, can include intermittent claudication, that is, fatigue, cramping, and pain in the hip, buttock, thigh, knee, shin, or upper foot during exertion that goes away with rest, claudication during rest, numbness, tingling, or coldness in the lower legs or feet, neuropathy, or defective tissue wound healing. PAD in the lower limb is often associated with diabetes, particularly type 2 diabetes. Arm artery disease is usually not due to atherosclerosis but to other conditions such as an autoimmune disease, a blood clot, radiation therapy, Raynaud's disease, repetitive motion, and trauma. Common symptoms when the arm is in motion include discomfort, heaviness, tiredness, cramping and finger pain. PAD can be diagnosed by performing one or more diagnostic tests including, for example, an ankle brachial index (ABI) test, angiography, ultrasound, or MRI analysis.
Myocardial ischemia can have few or no symptoms, although typically, it is associated with a symptoms such as angina, pain, fatigue elevated blood pressure. Diagnostic tests for myocardial ischemia include: angiography, resting, exercise, or ambulatory electrocardiograms, scintigraphic studies (radioactive heart scans); echocardiography; coronary angiography; and, rarely, positron emission tomography.
The method of the invention can also be used in conjunction with other remedies known in the art that are used to treat tissue damage including, drug therapy, surgery, anti-inflammatory agents, antibodies, exercise, or lifestyle changes. The choice of specific treatment may vary and will depend upon the severity of the tissue damage, the subject's general health and the judgment of the attending clinician. The present compositions can also be formulated in combination with one or more additional active ingredients, which can include any pharmaceutical agent such antihypertensives, anti-diabetic agents, statins, anti-platelet agents (clopidogrel and cilostazol), antibodies, immune suppressants, anti-inflammatory agents, antibiotics, chemotherapeutics, and the like. The compositions and methods described herein can be administered in conjunction with standard therapies for tissue necrosis including removal of the necrotic tissue by surgical means, chemical removal, via an enzymatic debriding agent, or removal by maggot therapy.
Animals and Reagents.
Unless otherwise stated, male wild type (C57BL16J) mice weighing 20-25 gm and age 2-3 months were used. The mice were bred and housed at the Association for Assessment and Accreditation of Laboratory Animal Care, International accredited LSUHSC-Shreveport animal resource facility and maintained according to the National Research Council's Guide for Care and Use of Laboratory Animals. All experimental protocols were approved by the LSU Institutional Animal Care and Use Committee. Sodium nitrite, sodium nitrate, phosphate buffered saline (PBS), and all other chemicals were purchased from Sigma Chemical (St Louis, Mo.).
Hind-Limb Ischemia Model.
Hind limb ischemia was induced by ligating the left common femoral artery proximal to origin of profunda femoris artery according to Senthilkumar, A., Smith, R. D., Khitha, J., Arora, N., Veerareddy, S., Langston, W., Chidlow, I H., Jr., Barlow, S. c., Teng, x., Patel, R. P., et al. 2007. Arterioscler Thromb Vasc BioI 27: 1947-1954. Mice were anesthetized with intraperitoneal injection of ketamine (100 mg/kg) and xylazine (8 mg/kg); surgery was performed under aseptic conditions. The common femoral vein and femoral nerve were dissected away from the artery. Two ligatures were placed in the common femoral artery proximal to the profunda femoris artery and then transected between the two ligations. The incision was then closed and the ligation was immediately verified by laser Doppler measurement of tissue blood flow. Following Hind-limb ligation was performed on mice which received either PBS or sodium nitrite (165 μg/kg) twice daily injection.
Gene Analysis.
RNA Isolation and Microarray Hybridization:
Total RNA was isolated from either PBS or sodium nitrite (165 μg/kg) treated gastrocnemius muscle tissues specimens at day 3 or day 7 post-ischemia using the RNeasy Midi Kit (Qiagen, Chatswort, Calif.). RNA integrity was checked using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Calif.). The RNA integrity number (RIN) for all specimens used for gene array analysis was between 8-9. Double stranded cDNA was synthesized from 5 ug total RNA using a Superscript cDNA synthesis Kit (Invitrogen, Carlsbad, Calif.) and purified using the Gene Chip Sample Cleanup Module (Affymetrix, Santa Vlara, Calif.). After biotin-labeling, the cDNA were fragmented at 94° C. and hybridized to the GeneChip® Mouse Genome 430 2.0 Array (Affymetrix), which contains 39,000 fully annotated transcripts of the mouse genome. After washing and staining, the arrays were scanned using GeneChip Scanner 3000 (Affymetrix) and hybridization efficiency determined by housekeeping control and spike control probe sets. Analysis of all control probe sets received a pass call demonstrating consistent hybridization efficiency across all specimens.
Microarray Data Normalization and Analysis.
Array data were globally scaled to a target intensity value of 500 to compare individual array experiments. To determine differentially expressed genes between PBS and nitrite treatment, data were log transformed and uploaded into Genesifter program (www.genesifter.net). Identification of significant changes in gene expression were determined using a pair-wise comparison of PBS versus nitrite therapy gene array data at each individual time point with the following criteria: a minimal 2-fold difference in expression with no maximal threshold limit, a hybridization quality cutoff of 1, and an unpaired student t-test between treatment groups followed by Benjamini and Hochberg post test to limit false discovery rates.
Genes which were significantly altered as identified by Genesifter analysis were uploaded into Ingenuity software (www.ingenuity.com) to perform network analysis using the Ingenuity Pathway Knowledge Base, which is a data base created from data mining for expression and functional relationships between molecules extracted from previously published peer reviewed papers found in NCBI Pubmed, Medline, and several other databases. The resulting output of network analysis was broken down according to the genetic composition of networks, the biological function of the networks, the number of interrelated network relationships, as well as statistical analysis of individual networks.
Cell System
A contained hypoxia tissue culture system was used allowing the long term culturing of cells in hypoxia (24 hrs). A fluorescent plate reader in hypoxic chamber measured NO formation using the fluorescent dye DAF-FM. The primary human vascular cell cultures used to monitor sodium nitrite activity were iliac vein smooth muscle cell, aortic smooth muscle cell, coronary artery endothelial cell and umbilical vein endothelial cell.
We examined the effect of chronic sodium nitrite treatment on ischemic tissue gene expression in the mouse hind-limb ischemia model described in Example 1. Induction of chronic hind limb ischemia was performed according to the method in Example 1. Sodium nitrite treatment (165 μg/kg) or PBS control treatments was begun the afternoon after hind limb ligation via intraperitoneal injection. Nitrite doses were given once in the morning (8 am) and a second in the afternoon (5 pm) with muscle tissue harvested on the morning of the 4th and 8th days (to give an entire 3 and 7 day period for study). Four muscle specimens from individual mice were harvested per treatment cohort per time point for a total of 16 individual specimens for gene array analysis. Harvested tissue was immediately frozen in liquid nitrogen for subsequent total RNA isolation. Laser doppler blood flow measurements were taken from non-ischemic and ischemic hind limbs prior to tissue collection to determine the amount of ischemic tissue perfusion using a Vasamedics Laserflo BPM2 deep tissue penetrating doppler device. Percent restoration of blood flow was determined by dividing the ischemic hind limb blood flow by the non-ischemic hind limb blood flow and multiplying by 100. Statistical analysis of changes in tissue blood flow between treatment groups at specific time points were performed with an unpaired students t-test using Prism software (Graphpad).
Heat map results of PBS control and sodium nitrite gene expression profiles at day 3 are shown in
Results of the Ingenuity network analysis are shown in
Human iliac vein smooth muscle cells, aortic smooth muscle cells, coronary artery endothelial cells and umbilical vein endothelial cells were cultured in normal 21% or low oxygen (1%) conditions in the presence or absence of increasing doses of sodium nitrite and intracellular nitric oxide levels were determined as described in Example 1. The effect of specific inhibitors of NO production, carboxy PTIO (2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide), allopurinol and L-NAME, was also analyzed. Both smooth muscle and endothelial cells reduced nitrite to NO under hypoxic conditions. Results for iliac vein smooth muscle cells are shown in
The effect of chronic sodium nitrite treatment on ischemic hind limb blood flow was assayed in an aged diabetic mouse model according to the experimental design shown in
B6.BKS-Leprdb/db diabetic mice were purchased from Jackson's laboratory and housed in the animal center of LSU Health Sciences Center for 6 months. Surgical hind limb ischemia was created in 9 month-old male (Db/Db) type-2 diabetic mice. The mice were anesthetized by isoflurane inhalation and the left groin area was shaved and naired. The left external iliac artery and vein, left femoral artery and vein, left saphenous artery and vein and deep femoral and circumflex arteries and veins were ligated, cut, and excised to obtain a mouse model of critical hind limb ischemia. Five days after surgery, animals received either sodium nitrite (165 ug/kg) or PBS control treatment as described in example 1. All treatments were performed in a double-blinded manner.
Laser Doppler blood flows were measured using a Vasamedics Laserflo BPM2 device in the gastrocnemius pre-ligation, post-ligation and on days 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 post-ligation. Blood vessels visible through the skin were avoided to ensure readings indicative of muscle blood flow. Measurements were recorded as milliliter of blood flow per 100 grams of tissue per minute. Percent blood flows were calculated as:
where a=ischemic limb average flow and b=non-ischemic limb average flow.
As shown in
We analyzed the effect of chronic sodium nitrite treatment on weight, blood glucose and blood lipids in an aged diabetic mouse model. Blood glucose measurements were taken using Ascensia Elite blood glucose test strips and a Bayer Glucometer Elite. Delayed chronic sodium nitrite treatment did not alter body weight or blood glucose levels in aged diabetic mice, as shown in
Plasma total cholesterol and LDL levels were determined using a Siemens Vista Clinical Chemistry analyzer. Delayed chronic sodium nitrite treatment significantly decreased serum cholesterol in aged diabetic mice as shown in
The effect of delayed chronic sodium nitrite treatment on tissue vascular density and cell proliferation was assayed in the aged diabetic mouse model described in Example 4.
The gastrocnemius muscles from ischemic and non-ischemic hind limbs were removed, dissected, and embedded in OCT freezing medium. Then tissues were fixed with 95% ethanol and 5% glacial acetic acid at −20 C for 1 hour. Endogenous Peroxidase quenching is performed in 20% hydrogen peroxide in methanol for 20 minutes. Slides are then washed with PBS three times. Sections were blocked with 10 cc of 0.1% PBS-BSA (Bovine serum albumin) (0.5 gram BSA in 10 cc of PBS=5% PBS-BSA, then 9.8 cc of PBS+0.2 cc of 5% PBS-BSA=0.1% PBS-BSA) and blocking serum (VECTASTAIN ABC—Mouse IgG kits with horseradish peroxidase enzymes) for one hour. Primary antibody (CD 31 antibody ab28364 Abcam) was diluted in 00.1% PBS-BSA (1:50 dilution) and tissue was incubated overnight at 4 C in a humidifier box.
Slides were then washed in PBS thrice, incubated in 0.5% biotinylated secondary antibody and 0.1% PBS-BSA for one hour at room temperature in humidifier box. In the mean time, Avidin-Biotin complex (ABC) was prepared (2 drops of A, 2 drops of B in 5 cc of 0.1% PBS-BSA) and incubated for one hour. Slides were washed in PBS after one hour incubation with secondary antibody, and incubated in ABC for one hour at room temperature in a humidifier box. Tissue was then again washed in PBS. NovaRED stain (Vector NovaRED substrate kit for peroxidase; catalog number SK 4800) was then prepared by adding 3 drops of reagent 1, 2 drops of each reagent 2, reagent 3 and hydrogen peroxide in 5 cc of distilled water. Tissues were then incubated in NovaRed stain for 10 min at room temp in a humidifier box, and then washed in distilled water for 5 minutes. Counterstain was then performed with hematoxylin (Vector Hematoxylin QS, H-3404) for 45 seconds and then washed with distilled water.
Tissue was then dehydrated with ethanol (95%, 100% for 10 and 15 min respectively) and xylene for 30 minutes. Slides were mounted with permount and cover slips. From each mouse, 5 random fields on 2 different sections (approximately 3 mm apart) were photographed with a digital camera (Olympus, Tokyo, Japan). The number of CD31-positive vessels and muscle fibers were counted manually in a blind fashion in per mm2. Finally ratio between CD31 and muscle fibers was calculated.
The tissue vascular density/angiogenic index was measured as follows. The gastrocnemius muscles from ischemic and non-ischemic hind limbs were removed, dissected, and embedded in OCT freezing medium. Frozen tissue blocks were cut into 5 μm sections and slides prepared. A primary antibody against PECAM-1 was added at a 1:200 dilution and incubated at 37° C. for 1 hour. Slides were then washed and a Cy3 conjugated secondary antibody was added at a 1:250 dilution and incubated at room temperature for 1 hour. Slides were once again washed and mounted with cover slips using Vectashield DAPI. A minimum of four slides per hind limb with three sections per slide were prepared for vascular density analysis. A minimum of two fields were acquired per section of muscle. Images were captured using a Hamamatsu digital camera in conjunction with a Nikon TE-2000 epifluorescence microscope (Nikon Corporation, Japan) at 200× magnification for CD31 and DAPI staining. Simple PCI software version 6.0 (Compix Inc., Sewickly, Pa., USA) was used to determine the area CD31 and DAPI positive staining. Tissue vascular density was determined as the ratio between CD31 positive areas and DAPI positive regions.
As shown in
Endothelial Cell Proliferation Assay.
The gastrocnemius muscles from ischemic and non-ischemic hind limbs were removed, dissected, and embedded in OCT freezing medium. Frozen tissue blocks were cut into 5 um sections and slides prepared. A primary antibody against PECAM-1 was added at a 1:200 dilution along with anti-Ki67 (1:350 dilutions) and incubated at 37° C. for 1 hour. Slides were then washed and a Cy3 conjugated secondary antibody was added at a 1:250 dilution along with anti-rabbit Ig antibody at a 1:150 dilution and incubated at room temperature for 1 hour. Slides were once again washed and mounted with cover slips using Vectashield DAPI. A minimum of four slides per hind limb with three sections per slide were prepared for endothelial cell proliferation analysis. A minimum of two fields were acquired per section of muscle. Images were captured using a Hamamatsu digital camera in conjunction with a Nikon TE-2000 epifluorescence microscope (Nikon Corporation, Japan) at 200× magnification for CD31 and DAPI staining. Simple PCI software version 6.0 (Compix Inc., Sewickly, Pa., USA) was used to determine the area CD31 and DAPI positive staining as well as area Ki67 and DAPI staining. Cellular proliferation was determined as the ratio between regions positive for Ki67 and DAPI positive area.
Tunnel Assay:
Hind limb ischemic tissue and non-ischemic tissues (gastrocnemius muscles) were collected at day 21. Apoptosis was determined (groups PBS and Na2S) using the Tunel staining kit (Promega, Inc) according to manufacturer's recommendations. 5 μm formalin fixed sections was prepared and mounted with Vectashield plus 4,6-diamidino-2 phenylindole (DAPI) (Vector Laboratories, Burlingame, Calif.). Immunofluorescence (green Tunnel staining at 488 nm) was measured from five random fields per slide for quantification of the extent of apoptosis. Images were captured using a Hamamatsu digital camera in conjunction with a Nikon TE-2000 epifluorescence microscope (Nikon Corporation, Japan). Simple PCI software version 6.0 (Compix Inc., Sewickly, Pa., USA) was used to determine the area tunnel positive and DAPI positive staining. Apoptosis of cells was determined as the ratio between regions positive for tunel and DAPI positive area.
As shown in
The effect of delayed chronic sodium nitrite treatment on gross footpad morphology in the aged diabetic mouse model described in Example 4 is shown in
C57B1/6J mice were treated with delayed chronic sodium nitrite treatment according to the method in example 1. Representative fluorescence angiograms using the SPY imaging system are shown in
C2C12 myoblasts were incubated in sodium nitrite under conditions of normoxia and hypoxia according to the methods in Example 1. As shown in
Gene array analysis (performed according to the methods of Examples 1 and 3) of ischemic mouse hindlimb grastrocnemius muscle showed that myogenin mRNA levels were significantly increased tissue from nitrite-treated animals, as shown in
A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
This application claims the benefit of the filing date of U.S. Provisional Application No. 61/268,926, which was filed on Jun. 18, 2009. For the purpose of any U.S. application that may claim the benefit of U.S. Provisional Application No. 61/268,926, the contents of that earlier filed application are hereby incorporated by reference in their entirety.
The work described below was support by Grant No. HL80482, which was awarded by the National Institutes of Health. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US10/36269 | 5/26/2010 | WO | 00 | 6/5/2012 |
| Number | Date | Country | |
|---|---|---|---|
| 61268926 | Jun 2009 | US |
