Patent Grant
10525070
Incorporated by reference in its entirety herein is a computer-readable nucleotide/amino acid sequence listing submitted concurrently herewith and identified as follows: One 21,165 Byte ASCII (Text) file named “730296ReplacementSequenceListing” created on Nov. 17, 2017.
The present invention relates to methods for treating heart failure.
Iron is required for oxygen transport, oxidative phosphorylation, DNA synthesis, and other cellular processes. Iron co-factors, for example, iron-sulfur (Fe—S) clusters and heme, are synthesized by mitochondria and needed for mitochondrial function. Mitochondria supply energy for cells, including the energy needed by cardiomyocytes for repeated heart muscle contraction. Accordingly, the maintenance of mitochondria, including the removal of dysfunctional mitochondria through mitophagy and synthesis of iron co-factors, is needed for cardiomyocyte function.
Improper cardiomyocyte function may lead to heart failure. Heart failure may be further complicated by the presence of iron deficiency. Up to 50% of patients with heart failure have iron deficiency, which is associated with poor outcomes despite current therapies.
Accordingly, a need exists for the identification and development of new therapies for heart failure.
The present invention relates to a method for treating heart failure in a subject in need thereof. The method may comprise administering a therapeutically effective amount of nicotinamide riboside to the subject.
The present invention also relates to a method for treating heart failure in a subject in need thereof. The method may comprise administering a therapeutically effective amount of nicotinamide riboside and a therapeutically effective amount of iron to the subject.
The present invention further relates to a method for treating heart failure associated with iron deficiency in a subject in need thereof. The method may comprise administering a therapeutically effective amount of nicotinamide riboside to the subject.
The present invention also relates to a method for treating heart failure associated with iron deficiency in a subject in need thereof. The method may comprise administering a therapeutically effective amount of nicotinamide riboside and a therapeutically effective amount of iron to the subject.
The present invention further relates to a method of identifying a subject suffering from heart failure as a candidate for treatment with nicotinamide riboside. The method may comprise (a) measuring in a sample obtained from the subject a level selected from the group consisting of: (i) a level of a ratio of NAD/NADH; (ii) a level of iron; (iii) a level of FGF21 protein; (iv) a level of Angiopoietin-like 4 (AngPtL4) protein; and (v) any combination thereof. The method may also comprise (b) comparing the measured level to a control level; and (c) identifying the subject as a candidate for treatment with nicotinamide riboside if (i) the measured level of the ratio of NAD/NADH is lower than the control level of the ratio of NAD/NADH; (ii) the measured level of iron is lower than the control level of iron; (iii) the measured level of FGF21 protein is higher than the control level of FGF21 protein; (iv) the measured level of AngPtL4 protein is higher than the control level of AngPtL4 protein; or (v) any combination thereof.
The present invention relates to a method for treating heart failure in a subject in need thereof. The heart failure may be associated with iron deficiency. The method includes administering nicotinamide riboside to the subject. Nicotinamide riboside is acted upon in the subject to induce the production of NAD, which in turn, increases the lifespan of the subject. The lifespan of the subject may be increased as compared to a lifespan of a subject suffering from heart failure and not administered nicotinamide riboside.
Nicotinamide riboside may also enhance or increase the mitochondrial unfolded-protein response, and thus, the method may enhance or increase the mitochondrial unfolded-protein response in the subject as compared to a mitochondrial unfolded-protein response in a subject suffering from heart failure and not administered nicotinamide riboside. Alternatively, nicotinamide riboside may inhibit or decrease the mitochondrial unfolded-protein response, and thus, the method may inhibit or decrease the mitochondrial unfolded-protein response in the subject as compared to a mitochondrial unfolded-protein response in a subject suffering from heart failure and not administered nicotinamide riboside.
The method may also include administering iron to the subject and thus, provides a combination therapy of nicotinamide riboside and iron for the subject suffering from heart failure.
The present invention also relates to a method of identifying a subject suffering from heart failure as a candidate for treatment with nicotinamide riboside.
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. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.
The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.
“Heart failure” as used herein refers to a chronic, progressive condition in which the heart muscle is unable to pump enough blood to meet the subject's needs for blood and oxygen. Heart failure is a clinical disorder characterized by congestion and decreased functional capacity. In some embodiments, the heart failure may be characterized by or associated with iron deficiency. Heart failure may include the left side, right side, or both sides of the heart. Accordingly, in some embodiments, heart failure may be left-sided heart failure (systolic failure or diastolic failure), right-sided heart failure, congestive heart failure, or any combination thereof. The heart failure may be classified by the New York Heart Association (NYHA) Functional Classification system. The NYHA Functional Classification system may include a functional capacity, which is a description of how the subject feels during physical activity, and an objective assessment. Functional capacity is ranked from class I to class IV, with increasing limitations of physical activity. Objective assessment is ranked from class A to class D, with increasing severity.
“Iron deficiency,” “Sideropenia,” and “Hypoferremia” as used herein may be used interchangeably to describe a condition in which a subject has a less than normal supply of iron. A normal Total Iron Binding Capacity (TIBC) test should range from 240 mcg/dL to 450 mcg/dL, resulting in a normal transferrin saturation range of 20 percent to 50 percent.
Iron deficiency may be accompanied with anemia, but need not be accompanied by anemia. Iron deficiency may exacerbate heart failure even when anemia is not present in the subject. In some embodiments, a subject suffering from heart failure may be considered iron deficient if ferritin is less than 100 μg/L, iron is less than 6 μmol/L, transferrin is less than 25 μmol/L, or transferrin saturation is less than 20%.
In some embodiments, the presence or absence of iron deficiency in the subject may be determined by examining red blood cells from the subject for characteristics of iron deficiency. In other embodiments, the presence or absence of iron deficiency in the subject may be determined by measuring the ratio of serum iron:total iron-binding capacity, this ratio is a dynamic measurement of how much iron is available to the tissues of the subject. In still other embodiments, the presence or absence of iron deficiency in the subject may be determined by measuring the level of serum ferritin in a sample obtained from the subject.
“Sample,” “test sample,” “specimen,” “sample from a subject,” and “patient sample” as used herein may be used interchangeably and may be a sample of blood, tissue, urine, serum, plasma, amniotic fluid, cerebrospinal fluid, placental cells or tissue, endothelial cells, leukocytes, or monocytes. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.
Any cell type, tissue, or bodily fluid may be utilized to obtain a sample. Such cell types, tissues, and fluid may include sections of tissues such as biopsy and autopsy samples, frozen sections taken for histologic purposes, blood (such as whole blood), plasma, serum, sputum, stool, tears, mucus, saliva, bronchoalveolar lavage (BAL) fluid, hair, skin, red blood cells, platelets, interstitial fluid, ocular lens fluid, cerebral spinal fluid, sweat, nasal fluid, synovial fluid, menses, amniotic fluid, semen, etc. Cell types and tissues may also include lymph fluid, ascetic fluid, gynecological fluid, urine, peritoneal fluid, cerebrospinal fluid, a fluid collected by vaginal rinsing, or a fluid collected by vaginal flushing. A tissue or cell type may be provided by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose). Archival tissues, such as those having treatment or outcome history, may also be used. Protein or nucleotide isolation and/or purification may not be necessary.
“Subject” and “patient” as used herein interchangeably refers to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc.) and a human). In some embodiments, the subject may be a human or a non-human. The subject or patient may be undergoing other forms of treatment.
“Treat”, “treating” or “treatment” are each used interchangeably herein to describe reversing, alleviating, or inhibiting the progress of a condition or disease, or one or more symptoms of such condition or disease, to which such term applies. Depending on the subject, the term also refers to preventing a condition or disease, and includes preventing the onset of a condition or disease, or preventing the symptoms associated with a condition or disease. A treatment may be either performed in an acute or chronic way. The term also refers to reducing the severity of a condition or disease, or symptoms associated with such condition or disease prior to affliction with the condition or disease. Such prevention or reduction of the severity of a condition or disease prior to affliction refers to administration of nicotinamide riboside with or without iron, or a pharmaceutical composition of the present invention to a subject that is not at the time of administration afflicted with the condition or disease. “Preventing” also refers to preventing the recurrence of a condition or disease or of one or more symptoms associated with such condition or disease. “Treatment” and “therapeutically,” refer to the act of treating, as “treating” is defined above.
For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.
Provided herein is a method of treating heart failure in a subject in need thereof. The heart failure may be associated with iron deficiency. The method may include administering nicotinamide riboside (NR) to the subject. The method may further comprise administering iron to the subject.
The subject may have iron deficiency. The subject may have iron deficiency in the heart. The subject may have iron deficiency with anemia. The subject may have iron deficiency without anemia.
The subject may have dysfunctional mitochondria. The subject may have dysfunctional mitochondria in the heart. The dysfunctional mitochondria may be caused by iron deficiency in the subject. In turn, the dysfunctional mitochondria may cause a reduced NAD/NADH ratio in the subject.
The subject may have reduced mitophagy. The subject may have dysfunctional mitophagy. The subject may have dysfunctional mitophagy in the heart. The dysfunctional mitophagy may be caused by iron deficiency in the subject, but may also exacerbate the iron deficiency in the subject because iron may not be recovered from dysfunctional mitochondria.
The subject may have a decreased NAD/NADH ratio as compared to a NAD/NADH ratio of a subject not suffering from heart failure. In some embodiments, the subject may have a decreased NAD/NADH ratio in the heart as compared to a NAD/NADH ratio in the heart of the subject not suffering from heart failure.
In some embodiments, the subject may have a decreased NAD/NADH ratio as compared to a NAD/NADH ratio of a subject not suffering from heart failure associated with iron deficiency. In other embodiments, the subject may have a decreased NAD/NADH ratio in the heart as compared to a NAD/NADH ratio in the heart of the subject not suffering from heart failure associated with iron deficiency.
The method may enhance or increase the mitochondrial unfolded-protein response in the subject. The method may enhance or increase the mitochondrial unfolded-protein response in the heart of the subject. The method may enhance or increase the mitochondrial unfolded-protein response in the subject as compared to a mitochondrial unfolded-protein response in a subject suffering from heart failure and not administered nicotinamide riboside. The enhanced or increased mitochondrial unfolded-protein response may correlate with an increase in expression of one or more UPRMT mRNAs. The method may inhibit or decrease the mitochondrial unfolded-protein response in the heart of the subject. The method may inhibit or decrease the mitochondrial unfolded-protein response in the subject as compared to a mitochondrial unfolded-protein response in a subject suffering from heart failure and not administered nicotinamide riboside. The inhibited or decreased mitochondrial unfolded-protein response may correlate with decrease or inhibition of expression of one or more UPRMT mRNAs.
The method may prolong the life span of the subject as compared to a lifespan of a subject suffering from heart failure and not treated with the method described herein. The method may prolong the life span of the subject by up to about 50% as compared to the lifespan of the subject suffering from heart failure and not treated with the method described herein. The method may prolong the life span of the subject by up to about 5% to about 50%, about 10% to about 50%, about 15% to about 50%, about 20% to about 50%, about 25% to about 50%, about 30% to about 50%, about 35% to about 50%, about 40% to about 500, about 5% to about 45%, about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 45%, about 15% to about 40%, or about 20% to about 35% as compared to the lifespan of the subject suffering from heart failure and not treated with the method described herein. The method may prolong the lifespan of the subject by up to about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 490/%, or 50% as compared to the lifespan of the subject suffering from heart failure and not treated with the method described herein.
The method may extend the lifespan of the subject as compared to the lifespan of the subject suffering from heart failure and not treated with the method described herein. The method may extend the lifespan of the subject by at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 months, 49 months, 50 months, 51 months, 52 months, 53 months, 54 months, 55 months, 56 months, 57 months, 58 months, 59 months, 60 months, 1 year, 2 years, 3 years, 4 years, or 5 years as compared to the lifespan of the subject suffering from heart failure and not treated with the method described herein.
a. Nicotinamide Riboside
As described above, the method of treating heart failure may include administering nicotinamide riboside to the subject. Nicotinamide riboside is a form of vitamin B3 and may be phosphorylated by Nmrk proteins to induce NAD production. Induction of NAD production may, in turn, increase the NAD/NADH ratio in the subject administered nicotinamide riboside. Administration of nicotinamide riboside may also increase the lifespan of the subject and the mitochondrial unfolded-protein response in the subject as described above. Alternatively, administration of nicotinamide riboside may decrease the mitochondrial unfolded-protein response in the subject as described above.
In some embodiments, the method may administer a therapeutically effective amount of nicotinamide riboside to the subject. The method may administer about 500 mg/day to about 2000 mg/day, about 550 mg/day to about 1900 mg/day, about 600 mg/day to about 1800 mg/day, about 650 mg/day to about 1700 mg/day, about 700 mg/day to about 1600 mg/day, about 750 mg/day to about 1500 mg/day, about 800 mg/day to about 1400 mg/day, about 850 mg/day to about 1300 mg/day, about 900 mg/day to about 1200 mg/day, about 950 mg/day to about 1100 mg/day, about 500 mg/day to about 1900 mg/day, about 500 mg/day to about 1800 mg/day, about 500 mg/day to about 1700 mg/day, about 500 mg/day to about 1600 mg/day, about 500 mg/day to about 1500 mg/day, about 500 mg/day to about 1400 mg/day, about 500 mg/day to about 1300 mg/day, about 500 mg/day to about 1200 mg/day, about 500 mg/day to about 1100 mg/day, about 500 mg/day to about 1000 mg/day, about 500 mg/day to about 950 mg/day, about 500 mg/day to about 900 mg/day, about 500 mg/day to about 850 mg/day, about 500 mg/day to about 800 mg/day, about 500 mg/day to about 750 mg/day, about 500 mg/day to about 700 mg/day, about 500 mg/day to about 650 mg/day, about 500 mg/day to about 600 mg/day, about 550 mg/day to about 2000 mg/day, about 600 mg/day to about 2000 mg/day, about 650 mg/day to about 2000 mg/day, about 700 mg/day to about 2000 mg/day, about 750 mg/day to about 2000 mg/day, about 800 mg/day to about 2000 mg/day, about 850 mg/day to about 2000 mg/day, about 900 mg/day to about 2000 mg/day, about 950 mg/day to about 2000 mg/day, about 1000 mg/day to about 2000 mg/day, about 1100 mg/day to about 2000 mg/day, about 1200 mg/day to about 2000 mg/day, about 1300 mg/day to about 2000 mg/day, about 1400 mg/day to about 2000 mg/day, about 1500 mg/day to about 2000 mg/day, about 1600 mg/day to about 2000 mg/day, about 1700 mg/day to about 2000 mg/day, about 1800 mg/day to about 2000, or about 1900 mg/day to about 2000 mg/day nicotinamide riboside to the subject.
In other embodiments, the method may administer about 500 mg/day, 550 mg/day, 600 mg/day, 650 mg/day, 700 mg/day, 750 mg/day, 800 mg/day, 850 mg/day, 900 mg/day, 950 mg/day, 1000 mg/day, 1100 mg/day, 1200 mg/day, 1300 mg/day, 1400 mg/day, 1500 mg/day, 1600 mg/day, 1700 mg/day, 1800 mg/day, 1900 mg/day, or 2000 mg/day nicotinamide riboside to the subject.
b. Iron
As described above, the treatment method may further administer iron to the subject. The iron may be administered to the subject in a therapeutically effective amount. The iron may be administered to the subject in an amount such that circulating transferrin (Tf) is further saturated with iron. Transferrin is a protein found in the blood and binds and transports iron throughout the bodies of mammals.
The iron administered to the subject may in the form of ferrous sulfate, ferrous fumarate, ferrous gluconate, ferric carboxymaltose, ferric saccharate, ferric gluconate, iron dextran, iron sucrose, or any combination thereof.
The iron may be administered orally to the subject, for example, in tablet form. In some embodiments, the method may administer to the subject an oral daily dose from about 150 mg/day to about 200 mg/day of elemental iron. In other embodiments, the method may administer an oral daily dose from about 100 mg/day to about 300 mg/day or about 125 mg/day to about 250 mg/day of elemental iron. In some embodiments, the method may administer to the subject an oral daily dose of about 100 mg/day, 105 mg/day, 110 mg/day, 115 mg/day, 120 mg/day, 125 mg/day, 130 mg/day, 135 mg/day, 140 mg/day, 145 mg/day, 150 mg/day, 155 mg/day, 160 mg/day, 165 mg/day, 170 mg/day, 175 mg/day, 180 mg/day, 185 mg/day, 190 mg/day, 195 mg/day, 200 mg/day, 205 mg/day, 210 mg/day, 215 mg/day, 220 mg/day, 225 mg/day, 230 mg/day, 235 mg/day, 240 mg/day, 245 mg/day, 250 mg/day, 255 mg/day, 260 mg/day, 265 mg/day, 270 mg/day, 275, mg/day, 280 mg/day, 285 mg/day, 290 mg/day, 295 mg/day, or 300 mg/day of elemental iron.
The iron may be administered intravenously to the subject. In some embodiments, the method may administer to the subject up to about 1000 mg of elemental iron per week. In other embodiments, the method may administer to the subject up to about 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, or 950 mg of elemental iron per week.
c. Combinatorial Therapy
As described above, the treatment method administers nicotinamide riboside to the subject and may further include administering iron to the subject. The nicotinamide riboside and iron may be administered simultaneously. In other embodiments, the nicotinamide riboside and iron may be administered sequentially, in which nicotinamide riboside administration may precede iron administration or iron administration may precede nicotinamide riboside administration.
d. Pharmaceutical Composition
The nicotinamide riboside and/or iron may be incorporated into a pharmaceutical composition suitable for administration to a subject (such as a patient, which may be a human or non-human).
The pharmaceutical composition may include a “therapeutically effective amount” or a “prophylactically effective amount” of nicotinamide riboside and/or iron. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of nicotinamide riboside and/or iron may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of nicotinamide riboside and/or iron are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.
The pharmaceutical composition may include pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.
Also provided herein is a method for identifying a subject suffering from heart failure as a candidate for treatment with nicotinamide riboside. In some embodiments, the method may also identify a subject suffering from heart failure associated with iron deficiency as a candidate for treatment with nicotinamide riboside. In still other embodiments, the method may further identify a subject suffering from heart failure associated with iron deficiency as a candidate for treatment with nicotinamide riboside, in which anemia is present with the iron deficiency.
The method may include obtaining a sample from the subject and measuring in the sample a level of a ratio of NAD/NADH, a level of iron, a level of a secreted or serum protein marker, or any combination thereof.
The secreted or serum protein marker may be secreted from a heart cell such as a cardiomyocyte. The secreted or serum protein marker may be secreted from a cell having the mitochondrial unfolded-protein response. The secreted or serum protein marker may be FGF21 protein, or Angiopoietin-like 4 (AngPtL4) protein, or the combination thereof. Accordingly, the method may measure a level of FGF21, AngPtL4, or the combination thereof.
Measuring the level of iron may include measuring serum ferritin, transferrin, transferrin saturation, serum iron, total iron-binding capacity, or any combination thereof.
The method may also include comparing the measured level to a control level. In some embodiments, the control level may be a predetermined or reference value, which is employed as a benchmark against which to assess the measured result. The predetermined or reference value may be a level in a sample from a subject not suffering from heart failure.
The method may further include identifying the subject as a candidate for treatment with nicotinamide riboside if measured level of the ratio of NAD/NADH is lower than the control level of the ratio of NAD/NADH, the measured level of iron is lower than the control level of iron, the measured level of FGF21 protein is higher than the control level of FGF21 protein, the measured level of AngPtL4 protein is higher than the control level of AngPtL4 protein, or any combination thereof. The nicotinamide riboside may be administered, alone or in combination with iron as described above, to the subject identified as a candidate for treatment with nicotinamide riboside.
Also provided herein a kit which may be used with the diagnostic method described above. The kit may include a positive control and/or a negative control. The kit may include material(s), which may be desirable from a user standpoint, such as a buffer(s), a diluent(s), a standard(s), and/or other material useful in sample processing, washing, or conducting any other step of the diagnostic method described herein.
The kit according to the present disclosure may also include instructions for carrying out the diagnostic method. Instructions included in the kit of the present disclosure may be affixed to packaging material or may be included as a package insert. While instructions are typically written or printed materials, they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” can include the address of an internet site which provides instructions.
The present invention has multiple aspects, illustrated by the following non-limiting examples.
Animal Experiments.
129/SvEv mice bearing a floxed Tfrc allele were crossed with transgenic C57BL/6 mice expressing Cre recombinase under the control of the a-MyHC promoter. Backcrossing was with 129/SvEv mice for more than 10 generations and after the initial phenotypic characterization, all studies used mice with a homogeneous 129/SvEv background. Animals were genotyped by polymerase chain reaction (PCR) using genomic DNA from toe clips. Primers for Tfrc alleles, Cre, Hjv alleles, and Rosa26 are described in Table 1.
For iron rescue experiments, mice were injected intraperitoneal (IP) with 5 mg of iron dextran (Uniferon® 100 (25 μl)) at P3, or at both P3 and P7. For nicotinamide riboside (NR) rescue experiments, mice were injected IP with NR dissolved in PBS at 750 mg/kg daily from P5. Volume was based on the body weight, with less than 100 μl at postnatal day 10. Control animals were injected with PBS only as described in Yang et al. (2007) J. Med. Chem. 50:6458-6461, the entire contents of which are incorporated herein by reference.
Histopathological Analysis.
4 μm paraffin sections were stained with hematoxylin and eosin and examined by light microscopy. Cardiomyocyte dimensions were measured by digital morphometry of paraffin-embedded myocardial cross-sections stained with Alexa Fluor® 594 WGA (Life Technologies, Invitrogen) using ImageJ image processing. Apoptotic cells were identified and quantified using the In Situ Cell Death Detection Kit (Roche) on paraffin sections according to the manufacturer's instructions. Quantitation was done by ImageJ using three consecutive sections for each mouse.
Echocardiography.
Echocardiography was performed without anesthesia on 7 or 8 age-, sex- and body weight-matched mice per genotype by an investigator blinded to genotypes. Left ventricular dimension and fractional shortening were calculated based on data generated by echocardiography as described in Esposito et al. (2000) Am. J. Physiol. Heart Circ. Physiol. 279:H3101-3112, the entire contents of which are incorporated herein by reference.
Electron Microscopy.
Hearts were removed and rinsed quickly in cold Krebs-Henseleit buffer, and then immersed in 5% glutaraldehyde buffer. Samples were prepared for transmission electron microscopy and imaged by Duke University Medical Center Research Electron Microscopy Services.
Tissue Iron Analysis.
Heart non-heme iron was measured as described in Levy et al. (1999) Nat. Genetics 21:396-399 and Torrance and Bothwell (1980) Methods in Hematology,
J. D. Cook, ed. (New York, Churchill Livingstone Press) pp. 104-109, the entire contents of both of which are incorporated herein by reference.
Enzymatic Analysis of Respiratory Chain Complexes I-IV.
Tissues were homogenized in 250 mM sucrose, 40 mM potassium chloride, 1 mM EGTA, 1 mg/ml fatty acid free BSA, 20 mM Tris-HCl (pH 7.2) and homogenates were centrifuged at 600×g for 10 min at 4° C. Steady state activities of enzyme complexes in the supernatant were determined as described in Janssen et al. (2007) Clin. Chem. 53:729-734 and Spinazzi et al. (2011) Mitochondrion 11:893-904, the entire contents of both of which are incorporated herein by reference.
Preparation of Mitochondrial Lysates and Acetyl-Lysine Analysis.
Mouse hearts were homogenized (40-50 strokes) in 15 volumes of ice-cold homogenization buffer [320 mM sucrose, 50 mM KH2PO4 (pH 7.4), 10 mM Tris-HCl (pH 7.4), 1 mM EDTA] in the presence of protease inhibitors, phosphatase inhibitors (Roche), and deacetylase inhibitors (2.5 μM Trichostatin A, 5 mM nicotinamide, 5 mM sodium butyrate) using glass homogenizers. Crude mitochondria were isolated by differential centrifugation. Homogenates were centrifuged at 1600 rpm for 10 min at 4° C. The supernatant was centrifuged again at 1600 rpm for 10 min at 4° C. The supernatant was subsequently centrifuged at 10,000×g for 10 min at 4° C. The pellet was collected, rinsed with 1 ml homogenization buffer and centrifuged at 10,000×g again for 10 min at 4° C. The pellet was resuspended in 100 μl 20 mM HEPES (pH 7.4), 150 mM NaC, and 1% Triton-X-100 with protease, phosphatase and deacetylase inhibitors. Western blot analysis was performed with 70 μg protein per lane. Antibodies recognizing acetyl-lysine were purchased from Cell Signaling Technology.
Statistical Analysis.
One-way ANOVA was performed for comparisons between two means, including when the comparison was between the means of WT and Tfr1hrt/hrt. Survival analysis was performed using Logrank in Graphpad software. P<0.05 was considered statistically significant.
An independent statistical analysis for survival analysis was also performed as described below.
Survival time was modeled as follows:
Tij=μ+Bj+sij
Where Tij was the survival time (in days) for the i-th mouse in the j-th group. The model explained this in terms of a baseline mean m, an effect bj for the j-th group (relative to baseline) and inter-mouse variation eij. The model was fit using ordinary least squares (1-way ANOVA).
For the Tfr1R654A rescue experiment, the baseline was Tfr1hrt/hrt There was a significant improvement in mean survival in the Tfr1hrt/hrt; Tfr1R654A group (2.7 days, p-value<0.001, Table 2). In WT, the improvement was over 49 days (all observations were censored in this group).
For the NR rescue experiment, the baseline was Tfr1hrt/hrt with PBS. There was no significant improvement in mean survival when using just Tfr1hrt/hrt (0.625 days, p-value=0.26, Table 3). There was, however, a significant improvement in the Tfr1hrt/hrt with NR group (2.29 days, p-value<0.001). For statistical calculations, lifespan was over 19.6 days for wild-type mice.
Western Blot Analysis.
Mouse hearts were homogenized in 15 volumes of ice-cold RIPA buffer in the presence of protease inhibitors and phosphatase inhibitors (Roche) using glass homogenizers. Protein lysates were collected as the supernatant after centrifugation at 14,000 rpm for 15 min at 4° C. Protein concentrations were determined using the DC protein assay (Bio-Rad). Antibodies were used recognizing Dpyd, Cathepsin D, Tom20 (Santa Cruz Biotechnology), LC3, Gabarap, p62, Beclin-1, Ulk1, Atg3, Atg4B, Atg7, Atg5, Atg12, Atg16L1, Bnip3, Nix, H-Ferritin (Cell Signaling Technology), Atg10 (ThermoFisher Scientific), Fundc1 (Aviva Systems Biology), Rcan1 (Abgent), Rpl19 (Sigma-Aldrich), Tfr1 (Invitrogen), Ppat and Atg9A (Novus Biologicals). Total OXPHOS Rodent WB Antibody Cocktail was purchased from Abcam.
RNA Extraction, Microarray Gene Expression Analysis, and PCR.
Total heart RNA was isolated from flash-frozen tissue using the RNeasy Fibrous Tissue Mini Kit (Qiagen) and reverse transcribed as described in Schmidt et al. (2008) Cell Metab. 7:205-214, the entire contents of which are incorporated herein by reference. For the microarray analysis, the Affymetrix GeneChip® Mouse Genome 430 2.0 Array was used. A minimum of 200 ng RNA was used for each sample. RNA samples were processed by the Duke Microarray Shared Resource Services. Transcript abundance was quantified by 2-step real-time PCR, using amplification conditions as described in Schmidt et al. (2008) Cell Metab. 7:205-214, the entire contents of which are incorporated herein by reference. Primers are summarized in Table 2 and Rpl19 was used as an internal control.
Spectrophotometric Analysis of Single Respiratory Chain Complexes I-IV.
All reactions were performed using a computer-tunable spectrophotometer (Spectramax Plus Microplate Reader, Molecular Devices, Sunnyvale, Calif.) at 20-s intervals for 5 minutes at room temperature. The reaction conditions for assaying activity of each complex were described in Janssen et al. (2007) Clin. Chem. 53:729-734 and Spinazzi et al. (2011) Mitochondrion 11:893-904, the entire contents of both of which are incorporated herein by reference. The total volume of each reaction was 200 μl. Each sample had three replicates for total activity and inhibitor-insensitive activity. For complexes I and II, 2,6-dichloroindophenol (DCIP) reduction by electrons accepted from decylubiquinol was measured. The reduction of DCIP was followed by spectrophotometry at 600 nm. 20 μg heart homogenates were used in each reaction for both complex I and complex II activity assays. Complex III activity was measured by following the reduction of cytochrome c at 550 nm. Complex IV activity was measured by following the oxidation of reduced cytochrome c at 550 nm. 3.6 μg and 1 μg of heart homogenate were used in each reaction for complex III and complex IV activity assays, respectively. The addition of standard respiratory chain inhibitors for each complex was used to ascertain the specificity of the enzymatic assays. The same amount of protein was used for each sample.
Murine Tfr1 was inactivated in cardiomyocytes by expressing a Myh6-Cre transgene to recombine loxP sites flanking Tfrc exons 3 to 6 (
Echocardiography of Tfr1hrt/hrt mice showed left ventricular dilatation in both systole and diastole, and decreased fractional shortening (
The canonical function of Tfr1 is to supply iron to meet cellular needs. Non-heme cardiac iron was measured to determine whether loss of Tfr1 resulted in iron deficiency. At P5, cardiac iron concentration was decreased in Tfr1hrt/hrt mice. Between P5 and P10, non-heme iron nearly doubled in WT hearts, but increased only slightly in Tfr1hrt/hrt hearts (
Mitochondria from Tfr1hrt/hrt hearts were slightly abnormal at P5, and severely disrupted and enlarged at P10 (
At P5, complex II was decreased in Tfr1hrt/hrt hearts and complex IV was increased, but the other ETC complexes did not differ from controls (
Iron-containing complexes I through IV were diminished in hearts from Tfr1hrt/hrt mice at P10, but complex V, which does not contain iron, was unchanged (
Expression of mitochondrial RNA polymerase (Polrmt) and mitochondria-encoded mRNA for Nd4, Cytb and Cox3 was similar to WT at P5 (not shown), but by P10 all were decreased in Tfr1hrt/hrt hearts (
mRNA profiling was used to examine gene expression changes in Tfr1hrt/hrt hearts and patterns were looked for using Gene Set Enrichment Analysis. Genes downregulated in the mutants were significantly associated with PPAR (particularly PPARa) and PGC1-a signaling, myogenesis, insulin signaling and cardiomyopathy. Upregulated genes were significant for hypoxia-inducible targets, Myc targets and glycolytic enzymes.
Decreased expression of mRNA encoding both PGC1-a (Ppargc1a) and PGC1-B (Ppargc1b) in Tfr1hrt/hrt hearts (
Cardiomyopathy is associated with a switch to fetal-like metabolism, with glucose, rather than fatty acids, as the preferred energy source. The switch has been attributed to decreased activity of PPARa (Ppara), which forms a heterodimer with Rxr. Expression of Ppara and Rxrg was decreased in Tfr1-null hearts, as was fatty acid transport protein (Fatp1/Slc27a1,
Increased mRNA expression of hypoxia-inducible genes (
Accumulation of damaged mitochondria should activate mitophagy to clear dysfunctional organelles and recover iron for re-use. Glycolytic enzyme Hk2 promotes autophagy during energy deprivation and was upregulated in Tfr1hrt/hrt hearts (
Cardiomyocytes from young mice cannot be cultured, requiring mitophagy to be characterized using tissue samples. Expression of putative mitophagy receptors, Nix (Bnip31) and Fundc1, was decreased, rather than increased, in Tfrhrt/hrt hearts. Ulk1, a kinase that phosphorylates Fundc1 for clearance of damaged mitochondria, was also decreased (
Cisd2 (Naf-1), an Fe—S cluster protein associated with the mitochondrial membrane, was depleted in Tfr1hrt/hrt mice (
Map1lc3 (LC3) and Gabarap are also involved in the cargo recognition step of autophagy (
Other proteins involved in early steps of autophagy were evaluated to explore why mitophagy was ineffective (
Sqstm1 (p62) is a molecule that links the phagophore to cargo. During normal autophagic flux, p62 is degraded by lysosomal enzymes. However, it was increased in Tfr1hrt/hrt hearts (
Lipin1 (Lpin1) enhances transcription regulated by Ppar-a and PGC-1a and controls autophagic clearance in skeletal muscle. Lipin1 deficiency in skeletal muscle leads to accumulation of p62, similar to what was observed and described above. Lpin1 mRNA was decreased in hearts from Tfr1hrt/hrt mice (
Ndrg1 is induced by iron depletion and was upregulated in Tfr1hrt/hrt hearts (
Atg9 traffics with Tfr1 and may deliver membrane components to developing autophagosomes. Atg9 was increased in Tfr1hrt/hrt hearts (
While it was observed that Tfr1 deletion resulted in cardiac iron deficiency and mitochondrial dysfunction, it was also possible that the mutant phenotype resulted from abrogation of an alternative role of Tfr1, or from both loss of such a role and iron deficiency. As such, two approaches were used to address this question—it was attempted to rescue the Tfr1hrt/hrt phenotype by co-expressing a Tfr1 mutant protein that cannot bind Tf and, in parallel, by inducing iron overload in Tfr1hrt/hrt mice.
Mice expressing a mutant Tfr1R654A protein from the ubiquitously transcribed Rosa26 locus was developed and it was shown that Tfr1R654A was unable to bind Tf for iron uptake. Breeding was used to obtain Tfr1hrt/hrt; Tfr1R61A mice, which had cardiomyocytes lacking WT Tfr1 but expressing mutant Tfr1R654A, and compared them to Tfr1fl/fl(+); Tfr1R654A control mice. Because R654A Tfr1 cannot assimilate Tf-bound iron, hearts from Tfr1hrt/hrt; Tfr1654A mice should be iron deficient, but should express an otherwise intact form of Tfr1 that differed from WT Tfr1 by only one amino acid in the extracellular domain. If the phenotype of Tfr1hrt/hrt mice was due wholly or in part to loss of a function of Tfr1 unrelated to iron uptake, expression of Tfr1R654A might fully or partially rescue the mutant phenotype.
Tfr1hrt/hrt; Tfr1R64A mice died at P13 to P14, later than Tf1hrt/hrt mice (
To test whether cardiac iron repletion could rescue Tfr1hrt/hrt mice, a large dose of iron dextran was administered at P3, to supersaturate circulating Tf and induce non-Tf-bound iron uptake. This prolonged survival, but Tfr1hrt/hrt mice still died at 4 to 5 weeks with severe cardiomegaly. It was confirmed that the hearts had assimilated iron by immunoblotting for Fe—S cluster proteins. In contrast to untreated Tfr1hrt/hrt mice, Dpyd and Ppat levels were similar at P10 in Tfr1hrt/hrt and WT mice treated with iron dextran (
It was hypothesized that the heart might require continuous iron uptake, and that iron administered early might no longer be available. To sustain elevated plasma iron concentrations, the hemojuvelin knockout (Hjv−/−) mice were used, which persistently have increased non-Tf-bound iron. Tfr1hrt/hrt; Hjv−/− mice were generated, in which Tfr1 was deleted in the heart and Hjv was deleted globally. These mice also died at P11, similar to Tfr1hrt/hrt mice. However, the Hjv−/− background did not allow for substantial iron accumulation during the neonatal period. Therefore, Tfr1hrt/hrt and control mice were treated with iron dextran at P3 to support the animals until the Hjv mutation caused elevated iron levels. With this strategy, the Tfr1hrt/hrt; Hjv−/− mice had lifespans (not shown) and heart to body weight ratios (
It was confirmed that this experimental protocol had restored cardiomyocyte iron by immunoblotting for Dypd and ferritin at 10-11 weeks of age (
Mitochondrial dysfunction can cause a decreased NAD/NADH ratio and inactivation of sirtuin deacetylases. A decreased NAD/NADH ratio may block signals for mitochondrial biogenesis while also causing defective mitophagy. Deacetylase activity is important for deacetylation of autophagy-related proteins; in the absence of Sirt1, LC3-II is decreased and p62 accumulates. This was similar to what was observed in Tfr1hrt/hrt mice as described above. It was hypothesized that augmentation of NAD levels might modify the mutant phenotype.
Induction of mRNA encoding nicotinamide riboside kinase 2 (Nmrk2/Itgb1bp3,
NR can be phosphorylated by Nmrk proteins to induce NAD production, activating sirtuins and mitochondrial biogenesis. How NR feeds into the synthetic pathway for NAD production is shown in
It was hypothesized that NR might improve the NAD/NADH ratio by increasing NAD. NAD/NADH ratios were measured with and without NR treatment but measurements from tissue were inconclusive. Alternatively, it was hypothesized that NR may enhance the mitochondrial unfolded protein response (UPRMT). Although not apparent at P5 (not shown), increased mRNA for multiple genes associated with the UPRMT in P10 hearts was observed, including Atf4, Lonp1, Ddit3 and Fgf21 (
In summary, Examples 1-7 demonstrated that mice lacking Tfr1 in the heart died in the second week of life, with cardiomegaly, poor cardiac function, failure of mitochondrial respiration, and interrupted mitophagy. In this interrupted mitophagy, mitophagy was induced, but failed due to a block at the cargo recognition step. This failure of mitophagy led to iron not being recovered from dysfunctional mitochondria for re-use by cardiomyocytes. Accordingly, the interrupted mitophagy was caused by iron deficiency, but also contributed to ongoing iron deficiency (
The phenotype of the mice lacking Tfr1 in the heart was only rescued by aggressive iron therapy, which overwhelmed the capacity of serum Tf to bind iron. The phenotype was ameliorated by expression of an iron uptake-incompetent Tfr1 (i.e., Tfr1R564A) or administration of nicotinamide riboside, an NAD precursor. Nicotinamide riboside increased the lifespan of treated mice in the absence of iron therapy, and thus, may be a therapeutic approach for patients with heart failure complicated by iron deficiency.
Clause 1. A method for treating heart failure in a subject in need thereof, the method comprising administering a therapeutically effective amount of nicotinamide riboside to the subject.
Clause 2. The method of clause 1, wherein the subject has iron deficiency.
Clause 3. The method of clause 1, wherein the subject has iron deficiency in the heart.
Clause 4. The method of clause 1, wherein the subject has iron deficiency with anemia.
Clause 5. The method of clause 1, wherein the subject has a decreased NAD/NADH ratio as compared to a NAD/NADH ratio of a subject not suffering from heart failure.
Clause 6. The method of clause 1, wherein the subject has mitochondrial dysfunction.
Clause 7. The method of clause 1, wherein the subject has reduced or dysfunctional mitophagy.
Clause 8. The method of clause 1, further comprising administering iron to the subject.
Clause 9. The method of clause 1, wherein a lifespan of the subject is extended as compared to a lifespan of a subject suffering from heart failure and not administered nicotinamide riboside.
Clause 10. A method for treating heart failure in a subject in need thereof, the method comprising administering a therapeutically effective amount of nicotinamide riboside and a therapeutically effective amount of iron to the subject.
Clause 11. The method of clause 10, wherein the heart failure is associated with iron deficiency.
Clause 12. A method for treating heart failure associated with iron deficiency in a subject in need thereof, the method comprising administering a therapeutically effective amount of nicotinamide riboside to the subject.
Clause 13. The method of clause 12, wherein the iron deficiency is in the heart of the subject.
Clause 14. The method of clause 12, wherein anemia is present with the iron deficiency.
Clause 15. The method of clause 12, wherein the subject has a decreased NAD/NADH ratio as compared to a NAD/NADH ratio of a subject not suffering from heart failure associated with iron deficiency.
Clause 16. The method of clause 12, wherein the subject has mitochondrial dysfunction.
Clause 17. The method of clause 12, wherein the subject has reduced or dysfunctional mitophagy.
Clause 18. The method of clause 12, further comprising administering iron to the subject.
Clause 19. The method of clause 12, wherein a lifespan of the subject is extended as compared to a lifespan of a subject suffering from heart failure associated with iron deficiency and not administered nicotinamide riboside.
Clause 20. A method for treating heart failure associated with iron deficiency in a subject in need thereof, the method comprising administering a therapeutically effective amount of nicotinamide riboside and a therapeutically effective amount of iron to the subject.
Clause 21. A method of identifying a subject suffering from heart failure as a candidate for treatment with nicotinamide riboside, the method comprising: (a) measuring in a sample obtained from the subject a level selected from the group consisting of: (i) a level of a ratio of NAD/NADH; (ii) a level of iron; (iii) a level of FGF21 protein; (iv) a level of Angiopoietin-like 4 (AngPtL4) protein; and (v) any combination thereof; (b) comparing the measured level to a control level; and (c) identifying the subject as a candidate for treatment with nicotinamide riboside if (i) the measured level of the ratio of NAD/NADH is lower than the control level of the ratio of NAD/NADH; (ii) the measured level of iron is lower than the control level of iron; (iii) the measured level of FGF21 protein is higher than the control level of FGF21 protein; (iv) the measured level of AngPtL4 protein is higher than the control level of AngPtL4 protein; or (v) any combination thereof.
Clause 22. The method of clause 21, wherein the heart failure is associated with iron deficiency.
Clause 23. The method of clause 22, wherein the iron deficiency is in the heart of the subject.
Clause 24. The method of clause 22, wherein anemia is present with the iron deficiency.
Clause 25. The method of clause 21, wherein the subject has mitochondrial dysfunction.
Clause 26. The method of clause 21, wherein the subject has reduced or dysfunctional mitophagy.
Clause 27. The method of clause 21, further comprising administering a therapeutically effective amount of nicotinamide riboside to the subject identified as a candidate for treatment with nicotinamide riboside.
Clause 28. The method of clause 27, wherein administration of nicotinamide riboside includes administration of iron to the subject identified as a candidate for treatment with nicotinamide riboside.
It is understood that the foregoing detailed description and accompanying examples are merely illustrative and are not to be taken as limitations upon the scope of the invention, which is defined solely by the appended claims and their equivalents.
Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications, including without limitation those relating to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations, or methods of use of the invention, may be made without departing from the spirit and scope thereof.
This patent application is a U.S. National Phase of International Patent Application No. PCT/US2016/017371, filed Feb. 10, 2016, which claims priority to U.S. Provisional Application No. 62/216,698, filed Sep. 10, 2015, U.S. Provisional Application No. 62/215,502, filed Sep. 8, 2015, and U.S. Provisional Application No. 62/114,501, filed Feb. 10, 2015, all of which are incorporated herein by reference in their entirety.
This invention was made with government support under contract number R01 DK089705 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2016/017371 | 2/10/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2016/130691 | 8/18/2016 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20020128205 | Stamler | Sep 2002 | A1 |
| 20120135091 | Roth | May 2012 | A1 |
| 20120172584 | Sauve et al. | Jul 2012 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 2006105440 | Oct 2006 | WO |
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| Number | Date | Country | |
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| 20190038647 A1 | Feb 2019 | US |
| Number | Date | Country | |
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| 62216698 | Sep 2015 | US | |
| 62215502 | Sep 2015 | US | |
| 62114501 | Feb 2015 | US |
