WO2016007128A1 - Thérapie par poloxamères pour lutter contre l'insuffisance cardiaque - Google Patents

Thérapie par poloxamères pour lutter contre l'insuffisance cardiaque Download PDF

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Publication number
WO2016007128A1
WO2016007128A1 PCT/US2014/045627 US2014045627W WO2016007128A1 WO 2016007128 A1 WO2016007128 A1 WO 2016007128A1 US 2014045627 W US2014045627 W US 2014045627W WO 2016007128 A1 WO2016007128 A1 WO 2016007128A1
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Prior art keywords
poloxamer
molecular weight
heart failure
composition
copolymer
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PCT/US2014/045627
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English (en)
Inventor
R. Martin Emanuele
Santosh Vetticaden
Patrick Keran
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Mast Therapeutics, Inc.
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Priority to PCT/US2014/045627 priority Critical patent/WO2016007128A1/fr
Priority to US14/793,662 priority patent/US9757411B2/en
Publication of WO2016007128A1 publication Critical patent/WO2016007128A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • A61K31/765—Polymers containing oxygen
    • A61K31/77—Polymers containing oxygen of oxiranes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • a poloxamer 188 such as a long-circulating material free (LCMF) poloxamer.
  • LCMF long-circulating material free
  • Heart failure is a chronic, progressive condition in which heart muscle is unable to pump sufficient blood to meet the body's needs.
  • a healthy heart pumps blood continuously through the circulatory system to deliver oxygen- and nutrient- rich blood to the body's cells and permit normal functioning.
  • a variety of diseases and conditions can weaken the heart and reduce its ability to deliver an adequate blood supply, such as hypertension, coronary artery disease and others. It is estimated that more than 20 million individuals worldwide, including five to six million in the United States (U.S.), suffer from heart failure. It is the most common diagnosis for hospital admission in the U.S. for patients over the age of 65.
  • ACE inhibitors widen blood vessels (vasodilation) to lower blood pressure and reduce the resistance against which the heart must pump. These therapies, however, do not directly improve the heart's ability to contract normally.
  • Existing therapies also provide only short-term symptomatic relief, and therefore require multiple repeat infusions at a high frequency. Hence, there is a need for alternative treatments for heart failure.
  • the method for treating heart failure includes identifying a subject with heart failure; administering to the subject a composition comprising an amount of a polyoxyethylene/polyoxypropylene copolymer having the chemical formula
  • the copolymer preparation has been purified to remove low molecular weight impurities; a' and a are the same or different and each is an integer, whereby the hydrophile portion represented by (C 2 H 4 0) constitutes approximately 60% to 90% or 60%- 90%, by weight, of the compound; b is an integer, whereby the hydrophobe represented by (C 3 H 6 0) has a molecular weight of about 1200 Da to about 2,300 Da or 1,200 to 2,300 Da.
  • the copolymer is administered as a single infusion and after one week, repeating the infusion at least one more time.
  • the subject can have diastolic or systolic heart failure.
  • the subject having systolic heart failure is identified for treatment if the subject has a left ventricular ejection fraction (LVEF) of less than 40%.
  • LVEF left ventricular ejection fraction
  • FIG. 1 is a general process 100 for supercritical fluid extraction (SFE) of a poloxamer.
  • FIG. 2 is a specific exemplary process 100' for preparing a poloxamer, such as poloxamer 188, using the methods described herein.
  • FIG. 3 is a specific exemplary process 100" for preparing a poloxamer, such as poloxamer 188, using methods described herein.
  • FIG. 4 shows an extraction apparatus useful in the methods provided herein.
  • FIG. 5 shows one embodiment of the cross section of stainless spheres of different sizes in a solvent distribution bed.
  • FIG. 6A-B shows a GPC comparison of low molecular weight substance content in a commercially available poloxamer 188 (Panel A) versus a material purified according to an embodiment provided herein (Panel B).
  • FIG. 7 shows a GPC of long circulating material free (LCMF) poloxamer 188 purified according to an embodiment of the methods provided herein.
  • LCMF long circulating material free
  • FIG. 8A-B shows enlarged HPLC-GPC chromatograms depicting the molecular weight distribution of components in plasma over time.
  • FIG. 9A-B shows individual plasma concentrations of Poloxamer 188 (Panel A) and high molecular weight component (Panel B) in healthy humans during and following a 48 hour continuous IV infusion of purified poloxamer 188 as described in Grindel et al. ((2002) Biopharmaceutics & Drug Disposition, 23:87-103).
  • a "polyoxyethylene/polyoxypropylene copolymer,” “PPC” or “poloxamer” refers to a block copolymer containing a central block of
  • POP polyoxypropylene
  • POE polyoxyethylene
  • a' and a can be the same or different and each is an integer such that the hydrophile portion represented by (C 2 H 4 0) (i.e. the polyoxyethylene portion of the copolymer) constitutes approximately 60% to 90%, by weight, of the copolymer, such as 70% to 90%, by weight, of the copolymer; and b is an integer such that the hydrophobe represented by (C 3 H 6 0)6 (i.e.
  • the polyoxypropylene portion of the copolymer has a molecular weight of approximately 950 to 4000 daltons (Da), such as about 1 ,200 to 3,500 Da, for example, 1 ,200 to 2,300 Da, 1 ,500 to 2,100 Da, 1,400 to 2,000 Da or 1 ,700 to 1 ,900 Da.
  • the molecular weight of the hydrophile portion can be between 5,000 and 15,000 Da.
  • Exemplary poloxamers having the general formula described above include poloxamers wherein a or a' is an integer 5-150 and b is an integer 15-75, such as poloxamers wherein a is an integer 70-105 and b is an integer 15-75.
  • Poloxamers include poloxamer 188 (e.g.
  • the nomenclature of the polyoxyethylene/polyoxypropylene copolymer relates to its monomeric composition. The first two digits of a poloxamer number, multiplied by 100, gives the approximate molecular weight of the hydrophobic polyoxypropylene block. The last digit, multiplied by 10, gives the approximate weight percent of the hydrophilic polyoxyethylene content.
  • poloxamer 188 describes a polymer containing a polyoxypropylene hydrophobe of about 1 ,800 Da with the hydrophilic polyoxyethylene content being about 80% of the total molecular weight.
  • Poloxamers are synthesized in two steps, first by building the polyoxypropylene core, and then by addition of polyoxyethylene to the terminal ends of the polyoxypropylene core. Because of variation in the rates of polymerization during both steps, a poloxamer can contain heterogeneous polymer species of varying molecular weights. The distribution of polymer species can be characterized using standard techniques including, but not limited to, gel permeation chromatography (GPC).
  • GPC gel permeation chromatography
  • a "polyoxyethylene/polyoxypropylene copolymer,” “PPC” or “poloxamer” refers to a block copolymer containing a central block of
  • POP polyoxypropylene
  • POE polyoxyethylene
  • a' and a can be the same or different and each is an integer such that the hydrophile portion represented by (C 2 HLtO) (i.e.
  • the polyoxyethylene portion of the copolymer constitutes approximately 60% to 90% by weight of the copolymer, such as 70% to 90% by weight of the copolymer; and b is an integer such that the hydrophobe represented by (CiH fyb ( ⁇ the polyoxypropylene portion of the copolymer) has a molecular weight of approximately 950 to 4000 daltons (Da), such as about 1 ,200 to 3,500 Da, for example, 1 ,200 to 2,300 Da, 1 ,500 to 2, 100 Da, 1 ,400 to 2,000 Da or 1 ,700 to 1 ,900 Da.
  • the molecular weight of the hydrophile portion can be between 5,000 and 15,000 Da.
  • Exemplary poloxamers having the general formula described above include poloxamers wherein a or a' is an integer 5- 150 and b is an integer 15-75, such as poloxamers wherein a is an integer 70- 105 and b is an integer 15-75.
  • Poloxamers include poloxamer 188 (e.g. those sold under the trademarks Pluronic® F-68, FlocorTM, Kolliphor®, Lutrol®).
  • the nomenclature of the polyoxyethylene/polyoxypropylene copolymer relates to its monomeric composition. The first two digits of a poloxamer number, multiplied by 100, gives the approximate molecular weight of the hydrophobic polyoxypropylene block.
  • poloxamer 188 describes a polymer containing a polyoxypropylene hydrophobe of about 1 ,800 Da with the hydrophilic polyoxyethylene content being about 80% of the total molecular weight.
  • Poloxamers are synthesized in two steps, first by building the polyoxypropylene core, and then by addition of polyoxyethylene to the terminal ends of the polyoxypropylene core. Because of variation in the rates of polymerization during both steps, a poloxamer can contain heterogeneous polymer species of varying molecular weights. The distribution of polymer species can be characterized using standard techniques including, but not limited to, gel permeation chromatography (GPC).
  • Poloxamer 188 also called P-188 or PI 88 refers to a polyoxyethylene/polyoxypropylene copolymer that has the following chemical formula:
  • a' and a can be the same or different and each is an integer such that the hydrophile portion represented by (C 2 H 4 0) (i.e. the polyoxyethylene portion of the copolymer) constitutes approximately 60% to 90%, such as approximately 80% or 81 %; and b is an integer such that the hydrophobe represented by (C 3 H 6 0) has a molecular weight of approximately 1 ,300 to 2,300 Da, such as 1 ,400 to 2,000 for example
  • Poloxamer 188 is a preparation that can contain a heterogeneous distribution of polymer species that primarily vary in overall chain length of the polymer, but also include truncated polymer chains with unsaturation, and certain low molecular weight glycols.
  • poloxamer 188 molecules include those that exhibit a species profile
  • Poloxamer 188 also refers to materials that are purified to remove or reduce species other than the main component.
  • main component or “main peak” with reference to a poloxamer 188 preparation refers to a species of copolymer molecules that have a molecular weight of 7680 to 9510 Da, such as generally 8,400-8,800 Da, for example about or at 8,400 Da.
  • Main peak species include those that elute by gel permeation chromatography (GPC) between 14 and 15 minutes (see U.S. Patent No. 5,696,298).
  • low molecular weight or “LMW” with reference to species or components of a poloxamer 188 preparation refers to components that have a molecular weight generally less than 7,000 Da, less than 6,000 Da, less than 5,500 Da, less than 4500 Da or less.
  • LMW species include molecules having a molecular weight between 2,300 daltons and 5,000 daltons.
  • LMW species include those that elute by gel permeation chromatography (GPC) after 15 minutes (see U.S. Patent No. 5,696,298).
  • GPC gel permeation chromatography
  • poloxamers poloxamers, poloxamer degradation products (including alcohols, aldehydes, ketones, and hydroperoxides), diblock copolymers, unsaturated polymers, and oligomeric glycols including oligo(ethylene glycol) and oligo(propylene glycol).
  • high molecular weight or “HMW” with reference to species or components of a poloxamer 188 preparation refers to components that have a molecular weight generally greater than 13,000 Da, such as greater than 14,000 Da, greater than 15,000 Da, greater than 16,000 Da or greater.
  • HMW species include those that elute by gel permeation chromatography (GPC) at between 13 and 14 minutes (see U.S. Patent No. 5,696,298).
  • polydispersity refers to the breadth of the molecular weight distribution of a polymer composition.
  • a monodisperse sample is defined as one in which all molecules are identical.
  • the polydispersity (Mw/Mn) is 10.
  • Narrow molecular weight standards have a value of D near 1 and a typical polymer has a range of 2 to 5.
  • Some polymers have a polydispersity in excess of 20.
  • a high polydispersity value indicates a wide variation in size for the population of molecules in a given preparation, while a lower polydispersity value indicates less variation.
  • polydispersity can be determined from chromatograms. It is understood that polydispersity values can vary depending on the particular chromatogram conditions, the molecular weight standards and the size exclusion characteristics of gel permeation columns employed. For purposes herein, reference to polydispersity is as employed in U.S. Patent No. 5,696,298, as determined from chromatograms obtained using a Model 600E
  • purified poloxamer 188 or "P188-P” refers to a poloxamer 188 that has polydispersity value of the poloxamer of less than or about 1.07, such as less than or about 1.05 or less than or about 1.03.
  • the poloxamer 188 is purified to remove or reduce low molecular weight components.
  • An exemplary purified poloxamer 188 is described in U.S. patent No. 5,696,298.
  • a poloxamer 188 in which "low molecular weight material has been removed” or “low molecular weight material has been reduced,” or similar variations thereof refers to a poloxamer 188 in which there is a distribution of low molecular weight components, such as described above, of no more than or less than 3.0 %, and generally no more than or less than 2.0% or no more than or less than 1.5% of the total distribution of components.
  • it is a poloxamer 188 that contains a distribution of components less than 4,500 Da that is no more than 1.5% of the total distribution of components.
  • such a poloxamer 188 exhibits reduced toxicity compared to forms of poloxamer 188 that contain a higher or greater percentage of low molecular weight components.
  • long circulating material free or "LCMF" with reference to poloxamer 188 refers to a purified poloxamer 1 88 preparation that has a smaller percentage or amount of the LMW components as described above, and that, when administered to a subject, does not contain any component that is or gives rise in the plasma, of the subject, to a material or component that has a substantially or considerably longer residence time in the circulation (e.g. the circulating half-life) than the main peak components as described herein (see also Grindel et al. (2002) Journal of Pharmaceutical Sciences, 90: 1936- 1947 (Grindel et al. 2002a) or Grindel et al.
  • the poloxamer 188 (see, e.g., Grindel et al. 2002a and 2002b), which is purified to remove lower molecular weight components, contains components that, when administered to a subject, exhibit different pharmacokinetic profiles.
  • the main component exhibits a half-life (ti/ 2 ) in plasma of about 7 hours and a higher molecular weight component (i.e.
  • an LCMF is a poloxamer 188 that does not contain any component, such as a high molecular weight components or low molecular weight components as described herein, that is or gives rise to a circulating material with a t] /2 that is more than 5.0-fold greater than the t of the main component, and generally no more than 4.0, 3.0, 2.0 or 1 .5 fold greater than the half-life of the main component in the distribution of the copolymer.
  • an LCMF poloxamer is a poloxamer in which all of the components of the polymeric distribution clear from the circulation at approximately the same rate.
  • an LCMF poloxamer is a poloxamer 188 in which the percentage of high molecular weight components greater than 13,000 daltons is no more than or is less than 1%, such as less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5% or less of the total distribution of the components, and, when administered, does not result in the distinct component with the longer half-life.
  • distributed of copolymer refers to the molecular weight distributions of polymers in a poloxamer preparation.
  • the distribution of molecular masses can be determined by various techniques known to a skilled artisan, including but not limited to, colligative property measurements, light scattering techniques, viscometry and size exclusion chromatography. In particular, gel permeation
  • GPC chromatography
  • half-life or circulation half-life or terminal half-life As used herein, half-life or circulation half-life or terminal half-life,
  • elimination half-life or ti /2 refer to the time that a living body requires to eliminate one half of the quantity of an administered substance through its normal channels of elimination.
  • the normal channels of elimination generally include the body's cleansing through the function of kidneys and liver in addition to excretion functions to eliminate a substance from the body.
  • half-life can be described as the time it takes the blood plasma concentration of a substance to halve its steady state level, i.e. the plasma half-life.
  • a half-life can be determined by giving a single dose of drug, usually intravenously, and then the concentration of the drug in the plasma is
  • concentration of the drug will reach a peak value in the plasma and will then fall as the drug is broken down and cleared from the blood.
  • concentration of the drug can be determined as described herein by quantifying the plasma levels of copolymers, after intravenous
  • the experiments can be repeated over various time periods.
  • Cmax refers to the peak plasma concentration of a drug after administration.
  • the concentration of a drug at steady state refers to the concentration of drug in which the rate of drug elimination and drug administration are equal. It is achieved generally following the last of an infinite number of equal doses given at equal intervals.
  • the time required to achieve a steady state concentration depends on the half-life of the drug. The shorter the half-life, the more rapidly steady state is reached. Typically it takes 3-5 half-lives to accumulate to greater than 90% of the final steady state concentrations.
  • impurities refer to unwanted components in a poloxamer preparation. Typically impurities include LMW components less than 4,500 daltons and high molecular weight components greater than 1 3,000 daltons.
  • remove or reduce with reference to a poloxamer component in a preparation refers to decreasing the weight percentage of the component in the poloxamer preparation relative to the initial percentage of the component.
  • a poloxamer component is removed or reduced if the percentage, by weight, of the component to the total distribution of components is decreased by at least 1 %, and typically at least 2%, 3%, 4%, 5%, or more.
  • most commercial preparations of a poloxamer 188 contain a LMW component (less than 4,500 daltons) that is about 4%, by weight, of the total components in the distribution.
  • the LMW component is reduced in a purified product if there is less than 3%, by weight, of the component, such as less than 2% or 1 %.
  • solvent refers to any liquid in which a solute is dissolved to form a solution.
  • polar solvent refers to a solvent in whose molecules there is either a permanent separation of positive and negative charges, or the centers of positive and negative charges do not coincide. These solvents have high dielectric constants, are chemically active, and form coordinate covalent bonds. Examples are alcohols and ketones.
  • feed refers to a solute dissolved in a solvent.
  • extraction solvent refers to any liquid or supercritical fluid that can be used to solubilize undesirable materials that are contained in a poloxamer preparation. It is a solvent that can effect solvent extraction to separate a substance from one or more others based on variations in the solubilities.
  • an extraction solvent is immiscible or partially miscible with the solvent in which the substance of interest is dissolved.
  • an extraction solvent is one that does not mix or only partially mixes with a first solvent in which the substance of interest is dissolved, so that, when undisturbed, two separate layers forms.
  • Exemplary extraction solvents are supercritical liquids or high pressure liquids.
  • the terms "supercritical liquid” and “supercritical fluid” include any compound, such as a gas, in a state above its critical temperature (T c ; i.e. the temperature, characteristic of the compound, above which it is not possible to liquefy the compound) and critical pressure (p c ; i.e. , the minimum pressure which would suffice to liquefy the compound at its critical temperature). In this state, distinct liquid and gas phases typically do not exist.
  • a supercritical liquid typically exhibits changes in solvent density with small changes in pressure, temperature, or the presence of a co-modifier solvent.
  • critical carbon dioxide refers to a fluid state of carbon dioxide where it is held at or above its critical temperature (about 31° C) and critical pressure (about 74 bars). Below its critical temperature and critical pressure, carbon dioxide usually behaves as a gas in air or as a solid, dry ice, when frozen. At a temperature that is above 31° C and a pressure above 74 bars, carbon dioxide adopts properties midway between a gas and a liquid, so that it expands to fill its container like a gas but with a density like that of a liquid.
  • critical temperature or “critical point” refers to the temperature that denotes the vapor-liquid critical point, above which distinct liquid and gas phases do not exist. Thus, it is the temperature at and above which vapor of the substance cannot be liquified no matter how much pressure is applied.
  • critical temperature of carbon dioxide is about 31° C.
  • critical pressure refers to the pressure required to liquefy a gas at its critical temperature.
  • critical pressure of carbon dioxide is about 74 bars.
  • high pressure liquid includes a liquid formed by pressurizing a compressible gas into the liquid at room temperature or a higher temperature.
  • a "co-modifier solvent” refers to a polar organic solvent that increases the solvent strength of an extraction solvent (e.g. supercritical fluid carbon dioxide). It can interact strongly with the solute and thereby substantially increase the solubility of the solute in the extraction solvent.
  • co-modifier solvents include alkanols. Typically between 5% and 15%, by weight, of co-modified solvent can be used.
  • alkanol includes simple aliphatic organic alcohols.
  • the alcohols intended for use in the methods provided herein include six or fewer carbon atoms (i. e. , C i-C 6 alkanols).
  • the alkane portion of the alkanol can be branched or unbranched.
  • Examples of alkanols include, but are not limited to, methanol, ethanol, isopropyl alcohol (2-propanol), and 1 ⁇ 2r/-butyl alcohol.
  • subcritical extraction refers to processes using a fluid substance that would usually be gaseous at normal temperatures and pressures that is converted to liquids at higher pressures and lower temperatures. The pressures or temperatures are then normalized and the extracting material is vaporized leaving the extract. Extractant can be recycled.
  • extraction vessel or “extractor” refers to a high-pressure vessel that is capable of withstanding pressures of up to 10,000 psig and temperatures of up to 200° C.
  • the volume of the vessels can range from 2 mL to 200 L, and generally is 1 L to 200 L, such as 5 L to 150 L.
  • Extraction vessels generally are made out of stainless steel. Such devices are well known to a skilled artisan and available commercially.
  • isocrastic refers to a system in which a extraction solvent is used at a constant or near constant concentration.
  • gradient or “gradient steps” refers to a system in which two or more extraction solvents are used that differ in their composition of components, typically by changes in concentration of one or more components.
  • concentration of the alkanol solvent e.g. methanol
  • concentration of the alkanol solvent is successively increased during the course of the extraction.
  • the extraction solvent does not remain constant.
  • plurality refers to a number of iterations of a process or step. The number of repeats can be 2, 3, 4, 5, 6 or more.
  • extracted material refers to the product containing the removed materials.
  • raffinate refers to a product which has had a component or components removed.
  • the purified poloxamer in which extracted material has been removed is removed.
  • batch method or “batch extraction” refers to a process of extracting the solute from one immiscible layer by shaking the two layers until equilibrium is attained, after which the layers are allowed to settle before sampling.
  • a batch extraction can be performed by mixing the solute with a batch of extracting solvent.
  • the solute distributes between the two phases. Once equilibrium is achieved, the mixing is stopped and the extract and raffinate phases are allowed to separate.
  • the spent solvent can be stripped and recycled by distillation, or fresh solvent can be added continuously from a reservoir.
  • a “continuous method” or “continuous extraction” refers to a process in which there is a continuous flow of immiscible solvent through the solution or a continuous countercurrent flow of both phases.
  • a continuous extracting solvent is mixed with the solute.
  • the emulsion produced in the mixer is fed into a settler unit where phase separation takes place and continuous raffinate and extract streams are obtained.
  • heart failure refers to an abnormality of cardiac structure or function leading to failure of the heart to deliver oxygen at a rate commensurate with the requirements of the metabolizing tissues, despite normal filling pressure.
  • the underlying cause of heart failure can be due to systolic ventricular dysfunction or abnormalities of ventricular diastolic function.
  • the term "signs and symptoms of heart disease” or “signs and symptoms of heart failure” refers to signs and symptoms associated with heart failure as recognized by simple observation or by standard clinical tests. This, when combined with an individual's age and family history of heart disease, can lead to diagnosis of heart disease or heart failure.
  • signs of heart disease include, but are not limited to, dyspnea, chest pain (angina), palpitations, syncope, edema, cyanosis and fatigue. Among these are those that can be subject to quantitative analysis, such as palpitations, cyanosis and others.
  • Other symptoms include discomfort or pressure in the chest, radiating discomfort to the back, jaw, throat or arm, fullness or ingestion, sweating, nausea, vomiting, dizziness, weakness or shortness of breath and/or rapid or irregular heartbeats. It is within the level of a skilled artisan, such as a treating physician, to identify a sign or symptom of heart disease.
  • ADHF acute decompensated heart failure
  • left ventricular ejection fraction or LVEF or simply EF refers to the amount or percentage of blood pumped, out of the total amount of blood in the left ventricle, per beat. Thus, it is the percentage of blood pumped out of a filled left ventricle with each heartbeat. Generally, an LVEF > 55% is normal, and lower than 50% is reduced. A skilled artisan is familiar with methods to assess or measure LVEF. Exemplary methods to measure EF include, but are not limited to,
  • EF can be measured as the stroke volume divided by end-diastolic volume.
  • diastole refers to the cycle of heart pumping when the left ventricle fills with blood.
  • the filling phase occurs when the heart muscle relaxes, allowing blood to enter and fill the left ventricle.
  • systole refers to the cycle of heart pumping when the blood is forced out and the blood is emptied from the heart.
  • the emptying phase occurs when the heart muscle contracts or squeezes to pump out or eject blood.
  • stroke volume refers to the volume of blood pumped from one ventricle of the heart with each beat. Stroke volume is calculated as the end-diastolic volume minus the end-systolic volume.
  • end-diastolic volume refers to the volume of blood in the ventricle at end load or filling in (i.e. diastole). Hence, it is the volume of blood just prior to the beat.
  • end-systolic volume refers to the volume of blood in a ventricle at the end of contraction (i.e. systole) and the beginning of filling (i.e. diastole). Hence, it is the volume of the blood in the ventricle at the end of a beat.
  • ESV can be used to clinically measure systolic function. Methods of assessing or measuring ESV are well known to a skilled artisan and include, but are not limited to, an electrocardiogram (the end of the T wave), echocardiography, MRI or CT.
  • systolic ventricular dysfunction or “systolic heart failure” refers to reduced contraction and emptying of the left ventricle. It occurs when the left ventricle of the heart does not pump enough blood out into the body on each beat. Systolic heart failure is characterized by a reduced ejection fraction. Hence, systolic heart failure also is classified as heart failure with reduced ejection fraction.
  • reduced ejection fraction As used herein, “reduced ejection fraction,” “heart failure with reduced ejection fraction” or HFREF refers to an ejection fraction of less than or equal to 50%, and generally less than or equal to 40%.
  • diastolic ventricular dysfunction or “diastolic heart failure” refers to abnormal heart relaxation and filling of the left ventricle. It occurs when the heart does not relax properly so that the heart is not able to fill with blood. Diastolic heart failure is characterized by a preserved ejection fraction, and, hence, also can be classified as heart failure with preserved ejection fraction.
  • prefferved ejection fraction or “heart failure with preserved ejection fraction” or HFPEF refers to an ejection fraction of greater than or equal to 50%.
  • diseases and conditions associated with heart failure refer to any condition associated with signs or symptoms of heart failure and that is confirmed by a diagnostic test of heart failure. Signs, symptoms and diagnostic tests for heart failure are well known to a skilled artisan. Symptoms of heart failure include, but are not limited to, breathlessness, ankle swelling or fatigue. Signs of heart failure include, but are not limited to, elevated jugular venous pressure, pulmonary crackles and displaced apex beat.
  • Diagnostic tests for heart failure include, but are not limited to, abnormalities in the ability of the heart to pump blood as determined by an electrocardiogram (EKG), an enlarged heart as determined by a chest x-ray, elevated levels of BNP in the blood, abnormal characteristics of heart size or shape, or blood flow as determined by an echocardiography (echo), abnormalities in pressure and blood flow in heart chambers as determined by cardiac catheterization, or
  • IHD ischaemic heart disease
  • myocardial infarction myocardial infarction
  • cardiomyopathy high blood pressure
  • diseases of the heart valves diseases of the pericardium, or arrhythmias.
  • a single infusion refers to an infusion that provides a therapeutically effective dosage in only one infusion or administration.
  • pharmaceutical composition includes a composition comprising a polyoxyethylene/polyoxypropylene copolymer described herein, such as an LCMF poloxamer, formulated with one or more pharmaceutically acceptable excipients.
  • the pharmaceutical composition comprises an aqueous injectable solution of the poloxamer buffered at a desired pH, such as 6-7 or 6 or about 6, with a buffering agent.
  • buffering agents are any known to those of skill in the art to be biocompatible, such as citrate, including for example sodium citrate/citric acid. Suitable concentrations can be empirically determined, but typically ranged from 0.005 to 0.05 M, particularly about 0.01 , in an isotonic solution such as saline.
  • pharmaceutical compositions useful in the methods herein are known to those of skill in the art for formulating poloxamers (see, e.g., WO 94/08596 and other such references and publications described herein).
  • treatment refers to ameliorating or reducing symptoms associated with a disease or condition.
  • Treatment means any manner in which the symptoms of a condition, disorder or disease are ameliorated or otherwise beneficially altered.
  • treatment encompasses prophylaxis, therapy and/or a cure.
  • Treatment also encompasses any pharmaceutical use of the compositions described herein.
  • treating means that a composition or other product provided or described herein is administered to the subject to thereby effect treatment thereof.
  • amelioration of the symptoms of a particular disease or disorder by a treatment refers to any lessening, whether permanent or temporary, lasting or transient, of the symptoms that can be attributed to, or associated with, administration of the composition or therapeutic.
  • prevention or prophylaxis refers to methods in which the risk of developing a disease or condition is reduced.
  • Prophylaxis includes reduction in the risk of developing a disease or condition and/or a prevention of worsening of symptoms or progression of a disease, or reduction in the risk of worsening of symptoms or progression of a disease.
  • an "effective amount" of a compound or composition for treating a particular disease is an amount that is sufficient to ameliorate, or in some manner reduce symptoms to achieve the desired physiological effect. Such amount can be administered as a single dosage or can be administered according to a regimen, whereby it is effective.
  • the effective amount of a composition described herein is an amount which, when administered to a human or non-human subject, can effect hemostasis, and, thus, reduce bleeding or hemorrhage. The effective amount is readily determined by one of skill in the art following routine procedures.
  • therapeutically effective amount refers to an agent, compound, material, or composition containing a compound that is at least sufficient to produce a therapeutic effect.
  • An effective amount is the quantity of a therapeutic agent necessary to effect hemostasis, thereby preventing, curing, ameliorating, arresting or partially arresting hemostasis dysfunction.
  • subject refers to an animal, particularly human or a veterinary animal, including dogs, cats, pigs, cows, horses and other farm animals, zoo animals and pets.
  • patient or “subject” to be treated includes humans and/ or non-human animals, including mammals. Mammals include primates, such as humans, chimpanzees, gorillas and monkeys; domesticated animals, such as dogs, horses, cats, pigs, goats, cows; and rodents such as mice, rats, hamsters and gerbils.
  • a combination refers to any association between two or among more items.
  • the association can be spatial, such as in a kit, or refer to the use of the two or more items for a common purpose.
  • a composition refers to any mixture of two or more products or compounds (e.g., agents, modulators, regulators, etc.). It can be a solution, a suspension, liquid, powder, a paste, aqueous or non-aqueous formulations or any combination thereof.
  • an "article of manufacture” is a product that is made and sold.
  • fluid refers to any composition that can flow. Fluids thus encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.
  • kits refers to a packaged combination, optionally including reagents and other products and/or components for practicing methods using the elements of the combination.
  • kits containing a modified protease polypeptide or nucleic acid molecule provided herein and another item for a purpose including, but not limited to, administration, diagnosis, and assessment of a biological activity or property are provided. Kits optionally include instructions for use.
  • animal includes any animal, such as, but not limited to; primates including humans, gorillas and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, sheep; ovine, such as pigs, and other animals.
  • Non-human animals exclude humans as the contemplated animal.
  • the proteases provided herein are from any source, animal, plant, prokaryotic and fungal.
  • an optionally substituted group means that the group is unsubstituted or is substituted.
  • PI 88 poloxamer 188
  • the methods include repeat administration with a frequency of no more than once weekly.
  • administration of the poloxamer, such as PI 88 can be repeated one or more times beginning at least one week after the end of the prior administration.
  • the methods provided herein can be used in the treatment of any disease or condition associated with heart failure, such as coronary artery disease, hypertension, myocardial infarction and other conditions associated with heart failure, In particular, the methods provided herein can be used in the treatment of subjects in which the left ventricular ejection fraction is below normal, such as subjects with systolic heart failure.
  • Heart failure is caused by an impaired cardiac pump function that results in an inadequate systemic perfusion to meet the body's metabolic needs.
  • Common causes of heart failure are related to conditions that weaken the heart muscle, thereby limiting blood from reaching the heart muscle and/or causing the heart to pump harder to keep blood circulating.
  • coronary artery disease such as atherosclerosis
  • Myocardial infarction, or heart attack is associated with a block in arteries that supply blood to heart muscle, resulting in death of heart muscle tissue and weakening of the heart's ability to pump blood.
  • Hypertension, or high blood pressure causes the heart to pump harder to overcome increased resistance in order to achieve continued blood circulation, which causes the heart's chambers to enlarge and weaken.
  • Heart failure is generally divided into two types: systolic heart failure and diastolic heart failure.
  • systolic heart failure occurs when the heart fails to contract normally.
  • the heart can take in blood but cannot fully pump out adequate blood due to weakened cardiac muscles.
  • the volume of the blood pumping out to the whole body and lungs decreases and the heart, in particular the left ventricle, can become hypertrophic.
  • diastolic heart failure occurs when the heart wall becomes too stiff to fill up the heart with blood. As a result, blood dams up in the left atrium and lung blood vessels, which could cause congestion.
  • a mechanism underlying cardiac muscle dysfunction in heart failure is abnormalities in cycling of calcium into myocytes, which regulates the ability of the heart to pump blood.
  • calcium enters the cardiac myocyte from the outside through calcium channels, which triggers the release of calcium stored in the sarcoplasmic reticulum (SR) through calcium release channels called ryanodine receptors.
  • SR sarcoplasmic reticulum
  • the released calcium can bind to calcium-sensitive proteins that activate the interaction of actin and myosin in the myofilaments.
  • the release of calcium triggers the heart to contract.
  • Calcium is removed from the cytoplasm and shuttled back into the SR and also out through the plasma membrane, which shuts off contraction and initiates muscle relaxation. Impaired calcium release can cause decreased muscle contraction (systolic dysfunction) and defective calcium removal can hamper relaxation (diastolic dysfunction).
  • the body employs various compensatory mechanisms to assist a failing heart and overcome factors that otherwise can cause symptoms.
  • the heart can enlarge or develop more mass (pathologic hypertrophy) or pump faster (chronotropic response), which initially can increase the heart's ability to pump blood.
  • the body can respond by narrowing blood vessels (vasoconstriction), which maintains blood pressure and offsets the heart's loss of pumping power. This, however, also can put additional strain on the heart.
  • the body also can divert blood away from less important tissues and organs to maintain flow to the heart and brain.
  • these compensatory mechanisms will exacerbate the underlying problem and begin to fail, also called decompensation.
  • Symptoms of heart failure include shortness of breath, persistent coughing or wheezing, edema (buildup of excess fluid if body tissues), fatigue, lack of appetite or nausea, impaired thinking and increased heart rate. Everyday activities such as walking, climbing stairs or carrying groceries can become difficult.
  • fluid can accumulate and cause congestion in the body's tissues, resulting in congestive heart failure. Fluid accumulation in the lungs results in pulmonary edema, which can interfere with breathing, including shortness of breath. Left untreated, pulmonary edema can cause respiratory distress.
  • Patients with heart failure can die from end-organ failure resulting from inadequate systemic organ perfusion, progressive pump failure and congestion, or sudden cardiac death.
  • P188 and particularly non-purified P188, has been used to treat heart failure.
  • PI 88 is known to have cytoprotective, rheologic and antithrombotic effects.
  • PI 88 is a cytoprotective agent based on its ability to bind to damaged membranes, and restore the cell's natural, hydrated non-adhesive surface.
  • Cardiac stress in heart failure results in mechanical sheer stresses to the membrane of cardiac myocytes that can result in loss of membrane integrity. It is believed that PI 88 can seal damaged membranes of cardiac muscle cells, and thereby protect the heart from ongoing cardiomyocyte loss.
  • Pretreatment with PI 88 has been shown to reverse the cardiac injury caused by lysophosphatidylcholine (LPC), which is an amphiphilic metabolite of phosphatidylcholine that incorporates into lipid bilayers to affect the physiochemical properties of the membrane and the enzymes and ion channels embedded in the membrane (Watanabe and Okada (2003) Mol. Cell Biochem., 248:209-215).
  • LPC lysophosphatidylcholine
  • PI 88 is known to have a short plasma half-life of under 7.5 hours (Grindel et ah 2002a and Grindel et a 2002b).
  • purified 188 1510 mg/kg
  • drug was cleared within approximately 1 week after discontinuation of administration. It follows that the therapeutic benefit of PI 88 are expected during administration and shortly thereafter, but that benefits will decline after
  • a single acute infusion of PI 88 confers durable benefits in heart failure that provide immediate benefits and which last for at least one week.
  • the results provided herein demonstrate that a single, short infusion of a purified PI 88 provides a benefit for at least 168 hours (i.e. at least one week) in key hemodynamic parameters (i.e. ejection fraction, stroke volume and cardiac output).
  • left ventricular function was achieved with minimal effect on left ventricular end-diastolic pressure, end-diastolic volume, systemic vascular resistance or heart rate. This indicates that mechanisms other than vasodilation (i.e., alteration in cardiac loading conditions) could be involved. Also, the reduction in troponin indicates PI 88 is limiting ongoing cardiomyocyte loss, possibly through its membrane-sealing activity and limiting unregulated calcium entry into the cell and calcium overload. In particular, while the results show effects on both systolic and diastolic function, the data shows more of an effect on systolic function. Thus, the results indicate that PI 88 could particularly treat heart failure in subjects with abnormal systolic function by improving left ventricular ejection fraction and end- systolic volume.
  • PI 88 such as purified PI 88 (e.g. LCMF)
  • purified PI 88 can preserve heart tissue and directly improve heart function. This is an improvement over most existing treatments that target indirect methods that reduce workload on the heart and provide short-term symptomatic relief, but that do not directly improve heart function.
  • the effect of PI 88 could limit the accelerated cardiac damage that occurs during acute decompensation.
  • acute decompensation is a time of increased vulnerability, where disease progression accelerates and the risk of organ damage increases.
  • An acute intervention that alters the trajectory of decompensation, whether by decreasing cardiac workload or preserving heart tissue, has the potential to minimize organ damage and improve long-term outcomes.
  • PI 88 administration of PI 88, so that any repeat administrations are not given until at least one week or more after the prior administration.
  • the longer term benefits of PI 88, and ability to provide a less frequent dosage regime address problems in the art with existing chronic therapies related to fluid volume overload, renal insufficiency in subjects with heart failure, drug toxicity, and compliance of subjects when numerous repeat administrations are necessary, especially if subjects must return to hospitals on a frequent (e.g. daily) basis for treatments.
  • poloxamers such as poloxamer 188
  • compositions thereof for use in treating or ameliorating heart failure, including in the treatment of diseases or conditions associated with heart failure. Exemplary dosage regimes and methods are described.
  • a poloxamer and in particular a poloxamer 188 (PI 88), such as a purified P I 88 (e.g. LCMF), for treating or ameliorating heart failure.
  • Poloxamers are a family of synthetic, linear, triblock copolymers composed of a core of repeating units of poly(oxypropylene) (PO), flanked by chains of repeating units of (poly)oxyethylene (EO). All poloxamers are defined by this EO-PO-EO structural motif. Specific poloxamers (e.g. P I 88) are further defined by the number of repeating EO and PO units, which provide specific poloxamers with different chemical and physical characteristics, as well as unique pharmacodynamic properties.
  • Poloxamers for use in the methods provided herein include POP/POE block copolymers having the following formula: HO(C 2 H 4 0) a — (C 3 H 6 0) b -(C 2 H 4 0) a H ! wherein the numbers "a"' and “a” can be the same or different and each is an integer such that the hydrophile portion represented by (C 2 H 0) constitutes approximately 60% to 90%, such as 70% to 90%, by weight, of the compound; and the number "b” is an integer such that the hydrophobe represented by (C 3 H 6 0) has a molecular weight of approximately 950 to 4000 Da, such as 1200 to 3500 Da.
  • the hydrophobe has a molecular weight of 1 ,200 to 2,300 Da, such as generally 1 ,500 to 2,100 Da.
  • the average molecular weight of the copolymer is 5,000 to 15,000 Da, such as 5000 and 9000 Da.
  • Poloxamers including P I 88, for use in the methods herein are readily available from commercial sources, e.g. BASF.
  • poloxamers can be synthesized using standard polymer synthesis techniques.
  • poloxamers are formed by ethylene oxide-propylene oxide condensation using standard techniques know to those of ordinary skill in the art (see, e.g., U.S. Patent Nos. RE 36,665, RE 37285, RE 38,558, 6,747,064, 6,761 ,824 and 6,977,045; see also Reeve, L.E., The Poloxamers: Their Chemistry and Medical Applications, in the Handbook of Biodegradable Polymers, Domb, A.J. et al.
  • Poloxamers can be synthesized by sequential addition of POP and POE monomers in the presence of an alkaline catalyst, such as sodium or potassium hydroxide (See, e.g., Schmolka, J. Am. Oil Chem. Soc. 54 (1977) 1 10-1 16). The reaction is initiated by polymerization of the POP block followed by the growth of POE chains at both ends of the POP block. Methods of synthesizing polymers also are described in U.S. Patent No. 5,696,298.
  • poloxamer 407 describes a polymer containing a polyoxypropylene hydrophobe of about 4000 Da with the polyoxyethylene hydrophile comprising about 70% of the total molecular weight.
  • Poloxamer 188 (PI 88) has a hydrophobe with a molecular weight of about 1800 Da and has a hydrophile that is about 80% of the total molecular weight of the copolymer.
  • Poloxamers are sold and frequently referred to under trade names including, but not limited to, ADEKA NOL, SynperonicTM, Pluronic® and Lutrol®.
  • Exemplary poloxamers include, but are not limited to, poloxamer 188 (PI 88; sold under the trademarks Pluronic ® F-68, Kolliphor® P 188, RheothRx and FlocorTM; 80% POE), poloxamer 407 (P407; sold under the trademark Lutrol F-127, Kolliphor® P 188, Pluronic ® F-127; 70% POE), poloxamer 237 (P237; sold under the trademark Pluroni T F87, Kolliphor® P 237; 70% POE), and poloxamer 338 (P338; sold under the trademark Kolliphor® P 338, Pluronic® F-108; 80% POE).
  • Poloxamers including P I 88, for use in the methods herein also include purified preparations of a poloxamer.
  • poloxamers can be molecularly diverse. That is, specific poloxamers are composed of multiple chemical entities that have the EO-PO-EO structural motif, but vary in the number of repeating EO and PO units.
  • the molecular diversity is the product of the process by which poloxamers are synthesized. The result is material that is non-uniform (i.e. material that is polydisperse). Adding to this polydispersity is a variety of other substances that can form as a result of side reactions occurring during synthesis of the intended poloxamer compound. These other substances can be present and found within the overall poloxamer distribution.
  • the copolymer has been purified, for example to remove certain low molecular weight impurities and other components, so that the
  • polydispersity value is less than approximately 1.07.
  • Methods for purifying poloxamers are known, and include techniques such as supercritical fluid extraction methods (see e.g. U.S. Patent No. 5,567,859).
  • chemically modified forms of one or more poloxamers are utilized in the compositions and methods provided herein.
  • Chemical modifications of poloxamers include, but are not limited to, radiolabelling, acetylating,
  • poloxamer 188 PI 88
  • a P I 88 copolymer has the following chemical formula:
  • PI 88 has an average molecular weight of 7,680 to 9,510 Da, such as generally approximately 8,400-8,800 Daltons.
  • the polyoxyethylene- polyoxypropylene-polyoxyethylene weight ratio of P 188 is approximately 4:2:4.
  • PI 88 has a weight percent of oxyethylene of 81.8 ⁇ 1.9%, and an unsaturation level of 0.026 ⁇ 0.008 mEq/g.
  • PI 88 is a polyoxyethylene/polyoxypropylene linear copolymer that is a surface-active agent, or surfactant. As a surface active agent, PI 88 binds to
  • This polymer also inhibits hydrophobic adhesive interactions that cause deleterious aggregation of formed elements in the blood. P188's anti-adhesive and anti-inflammatory effects are exhibited by enhancing blood flow in damaged tissue by reducing friction, preventing adhesion and aggregation of formed elements in the blood, maintaining the
  • inflammation including VEGF, various chemokines, interleukins, and chemokines.
  • Non-purified PI 88 is commercially available or known under various names.
  • PI 88 As Pluronic® F68 (BASF, Florham Park, N.J. as Pluronic® F68) and RheothRx® (developed by Glaxo Wellcome Inc.). Due to the synthesis of PI 88, there can be variation in the rates of polymerization during the steps of building the PO core and EO terminal ends. Thus, most non-purified forms of PI 88 contain a bell-shaped distribution of polymer species, which vary primarily in overall chain length. In addition, various low molecular weight (LMW) components (e.g. glycols and truncated polymers) formed by incomplete polymerization, and high molecular weight (HMW) components (e.g. dimerized polymers) can be present.
  • LMW low molecular weight
  • HMW high molecular weight
  • characterization of PI 88 by gel permeation chromatography identifies a main peak of P 188 with "shoulder" peaks representing the unintended LMW and HMW components (Emanuele and Balasubramaniam (2014) Drugs R D, 14:73-83).
  • PI 88 Studies on the therapeutic potential of PI 88 led to the discontinuance of RheothRx® for therapeutic applications in part due to an acute renal dysfunction observed during clinical trial evaluation. It was found that these effects were due to the presence of various low molecular weight (LMW) substances that formed during synthesis process (Emanuele and Balasubramaniam (2014) Drugs R D, 14:73-83).
  • LMW low molecular weight
  • a component in PI 88 also has been identified that is or gives rise to a circulating material in the plasma or blood with a long circulating half-life and with longer retention time than the main or predominant poloxamer species. For example, in both non-clinical and clinical studies, analysis of plasma obtained following intravenous administration of purified PI 88 by high performance liquid
  • PI 88 are observed with the predominant or main copolymers of the distribution, which are approximately 8,400 to 9400 daltons and have a half-life of about 7 hours, the presence of other components that exhibit a long circulating half-life could result in unwanted side effects.
  • the desired activities of PI 88 is its rheologic effect to reduce blood viscosity and inhibit red blood cell (RBC)
  • the average molecular weight of P338 is 12,700 to 17,600. Therefore, since the HMW component observed in PI 88 is a block polymer with an estimated molecular weight of greater than 13,000 Da, such as about 16,000 Da, its presence as a long circulating material is believed to have a negative therapeutic effect by its opposing action compared to the main or predominant copolymer in the distribution.
  • the PI 88 is typically purified.
  • a purified PI 88 has a polydispersity value of the polyoxypropylene/polyoxyethylene block copolymer that is less than or equal to approximately 1.07, such as less than or equal to approximately 1.05, and generally less than or equal to approximately 1.03.
  • the purified PI 88 typically has reduced LMW components.
  • a purified PI 88 also can have reduced HMW components.
  • Exemplary of a purified PI 88 is a PI 88 purified to reduce or remove LMW components (e.g. as described in U.S. Patent No. 5,696,298 or known under the trademark FlocorTM) or a low material circulating free (LCMF) PI 88 as described herein or in in related application [Attorney Docket No.
  • a P188 can be purified using various extraction processes known in the art. Such methods include any methods provided herein. Such methods also can include, but are not limited to, high pressure extraction or supercritical fluid extraction (SFE) methods, such as any described in related application U.S.
  • SFE supercritical fluid extraction
  • Exemplary of a purified poloxamer 188 is a long circulating material free (LCMF) poloxamer.
  • LCMF is a poloxamer that is a purified PI 88 that has a polydispersity value less than 1.07; has no more than 1.5% of low molecular weight (LMW) components less than 4,500 daltons; no more than 1 .5% high molecular weight components greater than 13,000 daltons; a half-life of all components in the distribution of the co-polymer, when administered to a subject, that is no more than 5.0-fold longer half-life in the blood or plasma than the half-life of the main component in the distribution of the co-polymer; and has the following chemical formula:
  • the polyoxyethylene portion of the copolymer constitutes approximately 60% to 90%, such as approximately 80%> or 81%; and b is an integer such that the hydrophobe represented by (C 3 H 6 O) has a molecular weight of approximately 1,300 to 2,300 Da, such as approximately 1,750 Da; and the average total molecular weight of the compound is approximately 7680 to 9510 Da, such as generally 8,400-8,800 Da, for example about or at 8,400 Da, wherein the copolymer has been purified to remove impurities, including low molecular weight impurities or other impurities, so that the polydispersity value is less than 1.07.
  • the purified poloxamer designated LCMF 188
  • LCMF 188 is one in which all components of the polymeric distribution, when administered to a subject, clear from the circulation at approximately the same rate.
  • the LCMF poloxamer contains a substantially polydisperse composition of less than 1.07, and generally less than 1.05 or 1.03, wherein the half- life in the blood or plasma of any components in the distribution of the co-polymer, when administered to a subject, is no more than 5.0-fold longer than the half- life of the main component in the distribution of the co-polymer, and generally no more than 4.0-fold, 3.0-fold, 2.0-fold, 1.5-fold more longer.
  • the LCMF does not contain any component that exhibits a half-life in the blood or plasma, when administered to a subject, that is substantially more than or is more than the main component in the distribution of the co-polymer.
  • LCMF poloxamer when administered to a human subject, is such that no component has a half-life that is more than 30 hours, and generally is no more than 25 hours, 20 hours, 15 hours, 10 hours, 9 hours, 8 hours or 7 hours.
  • an LCMF preparation provided herein includes HMW components in the distribution that exhibit different properties that do not result in a longer circulating species.
  • HMW impurities greater than 13,000 Daltons in an LCMF preparation which generally is no more than 1.5%, by weight, of the total components, do not, when the LCMF preparation is administered to a subject, result in a circulating half-life that is substantially more than or is more than the main component in the distribution (see e.g. Figure 7 and Figures 8A and 8B).
  • the HMW impurities greater than 13,000 Daltons in an LCMF preparation which generally is no more than 1.5%, by weight, of the total components, do not, when the LCMF preparation is administered to a subject, result in a circulating half-life that is more than 5.0-fold longer than the half-life of the main component in the distribution, and generally no more than 4.0-fold, 3.0-fold, 2.0-fold, 1.5-fold more longer.
  • an LCMF poloxamer provided herein includes PI 88 poloxamers in which there are no more than 1.3% high molecular weight components greater than 13,000 daltons, such as no more than 1.2%, 1.1%, 1.0% or less.
  • an LCMF poloxamer provided herein includes PI 88 poloxamers in which there are less than 1.0 %, by weight, high molecular weight components greater than 13,000 daltons, and generally less than 0.9%, 0.8%, 0.7%, 0.6%, 0.5% or less.
  • the LCMF poloxamer provided herein can be prepared by methods as described herein below.
  • an LCMF poloxamer provided herein is made by a method that includes:
  • the temperature is above the critical temperature of carbon dioxide but is no more than 40° C;
  • the pressure is 220 bars to 280 bars
  • the alkanol is provided at an alkanol concentration that is 7% to 8%, by weight, of the total extraction solvent;
  • the alkanol concentration is increased 1 -2% compared to the previous concentration of the second alkanol.
  • any method known to a skilled artisan can be used to purify a poloxamer.
  • supercritical methods can be employed.
  • a supercritical extraction permits control of the solvent power by manipulation of temperature, pressure and the presence of a co-solvent modifier. It is found that carbon dioxide is not a particularly efficient extraction solvent of poloxamers, such as PI 88, but that the presence of a polar co-solvent, such as an alkanol, as a modifier can increase the solubilizing efficiency of the extraction solvent.
  • extraction methods described are performed in the presence of a polar co-solvent, such as an alkanol, whose
  • concentration is increased in a gradient fashion (e.g. a step-wise gradient or a continuously escalating gradient) as the extraction process progresses. It is found that by employing an alkanol co-solvent whose concentration is increased in this manner, the removal of impurities can be increased, and to a much greater extent than when carbon dioxide is used alone. For example, an extraction method that uses carbon dioxide alone is not capable of removing the unwanted components, such as the LMW components or HMW components described herein, to the same degree as that achieved by the provided method.
  • the LMW components or impurities of a poloxamer distribution can be selectively removed with a lower alkanol concentrations (e.g. methanol) and higher pressure than other HMW components in the distribution.
  • a lower alkanol concentrations e.g. methanol
  • polar solvent such as an alkanol (e.g. methanol)
  • a method is provided employing a gradient of higher concentrations of an alkanol (e.g. methanol), alone or in conjunction with a decrease in the pressure, that results in the removal of components (e.g. HMW components) in a poloxamer distribution that, when administered to a subject, is or gives rise to a longer circulating material in the plasma.
  • the methods achieve a yield such that the amount of the extracted or purified polymer obtained by the method is at least 55%, 60%, 70%, 75%, 80%, 85%, 90% or more of the starting amount of the poloxamer prior to performance of the method.
  • the resulting poloxamers exhibit a substantially greater purity with a higher percentage of main component in the distribution than the starting material, and without impurities that exhibit toxic side effects or that can result in a longer circulating material in the plasma when administered.
  • the methods can be performed on any poloxamer in which it is desired to increase the purity, for example by decreasing or reducing components that are undesired in the distribution of a polymer. It is within the level of a skilled artisan to choose a particular poloxamer for use in the methods.
  • An undesired component is any that is or gives rise to a material that is toxic or that has a biological activity that is counter or opposing to the desired activity.
  • the poloxamer can be one in which it is desired to reduce or remove LMW components in the poloxamer, for example, any LMW components that result in acute renal side effects, such as elevated creatine, when administered.
  • the poloxamer also can be one that contains any component, such as a HMW component, that, when administered, is or gives rise to a material that has a half-life in the blood that is different (e.g. longer) than the half-life of the main component in the distribution of the polymer.
  • Such components can increase blood viscosity and red blood cell aggregation, and hence are undesired.
  • the extraction methods provided herein can be employed to purify a PI 88 preparation, where the PI 88 preparation has the following chemical formula:
  • PI 88 has an average molecular weight of 7,680 to 9,510 Da, such as generally approximately 8,400-8,800 Daltons.
  • the polyoxyethylene- polyoxypropylene-polyoxyethylene weight ratio of PI 88 is approximately 4:2:4.
  • PI 88 has a weight percent of oxyethylene of 81.8 ⁇ 1.9%, and an unsaturation level of 0.026 ⁇ 0.008 mEq/g.
  • PI 88 preparations for use in the methods herein include
  • the supercritical fluid extraction process is essentially a solvent extraction process using a supercritical fluid as the solvent.
  • supercritical fluid multi- component mixtures can be separated by exploiting both the differences in component volatilities and the differences in the specific interactions between the component mixture and supercritical fluid solvent (solvent extraction).
  • solvent extraction solvent extraction
  • a compressible fluid such as carbon dioxide exhibits liquid-like density and much increased solvent capacity that is pressure dependent.
  • the tunable solvent power of a supercritical fluid changes rapidly around critical conditions within a certain range.
  • the solvent power of the supercritical fluid and thus the nature of the component that can be selectively removed during extraction, can be fine-tuned by varying the temperature and pressure of the supercritical fluid solvent.
  • Each supercritical fluid has a range of solvent power.
  • the tunable solvent power range can be selected by choosing an appropriate supercritical fluid.
  • supercritical fluids exhibit certain physico chemical properties making them more useful.
  • supercritical fluids exhibit liquid-like density, and possess gas-like transport properties such as diffusivity and viscosity. These characteristics also change rapidly around the critical region.
  • supercritical fluids also have zero surface tension. Since most of the useful supercritical fluids have boiling points around or below ambient temperature, the solvent removal step after purification is simple, energy efficient and does not leave any residual solvents.
  • solid matrices during extraction provides an additional dimension for a fractionation parameter.
  • Use of a suitable solid matrix provides solvent-matrix and solute-matrix interactions in addition to solute-solvent interaction to enhance the fractionation resolution.
  • the desirable transport properties of supercritical fluids make the process easily scalable for manufacturing. Heat transfer and mass transfer characteristics do not significantly change upon process scale up with supercritical fluid extraction processes. Since the extraction process conditions such as pressure, temperature, and flow rate can be precisely controlled, the purification process is reproducible in addition to highly tunable.
  • the extraction solvent can contain a supercritical liquid (e.g. supercritical carbon dioxide), as well as another co-modifier solvent, generally an alkanol, that is increased over time in the extraction.
  • a supercritical liquid e.g. supercritical carbon dioxide
  • another co-modifier solvent generally an alkanol
  • the method provided herein can include: a) providing or introducing a poloxamer (e.g. PI 88) solution into an extractor vessel, wherein the poloxamer solution is prepared by dissolving the poloxamer in a first alkanol to form the solution; b) admixing an extraction solvent containing a second alkanol and a supercritical liquid, under high pressure and high temperature sufficient to create supercritical liquid conditions, with the solution to form an extraction mixture, wherein the concentration of the second alkanol in the extraction solvent is increased over the time of extraction method; and c) removing the extraction solvent from the extractor vessel to thereby remove the impurities (e.g. LMW component or other components) from the poloxamer preparation.
  • a poloxamer e.g. PI 88
  • the first and second alkanol can be the same or different.
  • the step of dissolving the poloxamer solution in the first solvent can occur prior to charging the solution into an extraction vessel or at the time of charging the solution into an extraction vessel.
  • the poloxamer is dissolved in a separate vessel and then the solution is added to the extraction vessel.
  • FIG. 1 provided is a process 100 that is useful for removing impurities (e.g. LMW component or other components) from a poloxamer
  • the extraction system is pressurized, as shown in step 105, typically prior to dispensing a first alkanol into the feed mix tank, as shown in step 1 10.
  • the system is heated to a temperature suitable for the extraction process.
  • the temperature is typically a temperature that is above the critical temperature of the supercritical liquid (e.g. carbon dioxide). Generally, the temperature is no more than 40° C.
  • any suitable alkanol or combination of alkanols can be used in the methods of purifying a poloxamer.
  • suitable alkanols include, but are not limited to, methanol, ethanol, propanol, butanol, and the like.
  • the method provided herein includes an extraction method as described above, wherein the first and the second alkanol are each independently selected from methanol, ethanol, propanol, butanol, pentanol and a combination thereof.
  • the first alkanol is methanol.
  • methanol is selected as the purification solvent and is the second alkanol in practice of the method.
  • methanol has relatively low toxicity characteristics.
  • methanol has good solubility for poloxamer 188.
  • the first alkanol e.g. methanol
  • a poloxamer such as a PI 88 preparation
  • the amount of poloxamer that is added to the feed tank is a function of the scalability of the extraction method, the size of the extraction vessel, the degree of purity to achieve and other factors within the level of a skilled artisan.
  • non-limiting amounts of poloxamer (e.g. PI 88) per mL of an extraction vessel can be 0.1 kg to 0.5 kg or 0.2 kg to 0.4 kg.
  • non-limiting amounts of poloxamer in methods of extraction using a 3 L extraction vessel, can be 0.6 kg to 1.2 kg, such as 0.8 kg to 1.0 kg.
  • non-limiting amounts of poloxamer e.g. PI 88
  • non-limiting amounts of poloxamer can be 1.5 kg to 5 kg, such as 2 kg to 4 kg.
  • non-limiting amounts of poloxamer e.g. PI 88
  • non-limiting amounts of poloxamer e.g. PI 88
  • the ratio of poloxamer to alkanol, by weight can be, for example, from about 4:1 to about 1 :4, such as from about 3: 1 to about 1 :3, 2: 1 to about 1 :2. 1 : 1 to 4: 1 or 1 :2 to 1 :4.
  • the ratio of poloxamer to alkanol, by weight can be about 4 to 1 , or about 3 to 1 , or about 2 to 1 , or about 1 to 1 , or about 1 to 2, or about 1 to 3 or about 1 to 4.
  • a quantity of poloxamer, such as PI 88 can be mixed with an equal quantity, by weight, of alkanol (e.g.
  • alkanol e.g. methanol
  • One of skill in the art will appreciate that the appropriate poloxamer to alkanol ratio will depend on poloxamer properties such as solubility in a given alkanol.
  • step 120 After forming a poloxamer/alkanol mixture, all or part of the mixture is pumped into the extractor as shown in step 120.
  • the process of preparing the poloxamer solution is performed in a separate vessel from the extractor.
  • the poloxamer can also be introduced as a solid into the extractor prior to mixing with the first alkanol.
  • the process of preparing the poloxamer solution can be made directly in the extractor vessel.
  • the extractor is then pressurized and the extraction solvent is introduced into the extractor as shown in step 125 of process 100.
  • the extraction solvent contains the supercritical liquid.
  • supercritical fluids include, but are not limited to, carbon dioxide, methane, ethane, propane, ammonia, freon, water, ethylene, propylene, methanol, ethanol, acetone, and combinations thereof.
  • the supercritical liquid under pressure is a member selected from carbon dioxide, methane, ethane, propane, ammonia and freon.
  • the supercritical liquid under pressure is carbon dioxide (C0 2 ).
  • the extraction occurs under high pressure and high temperature to maintain a supercritical liquid condition (e.g. supercritical carbon dioxide). Typically, these are kept constant. At this pressure and temperature, the supercritical liquid (e.g.
  • the flow rate can be varied between 0.5 kg/h to 600 kg/h, such as 1 kg/h to 400 kg/h, 1 kg/h to 250 kg/h, 1 kg/h to 100 kg/h, 1 kg/h to 50 kg/h or 1 kg/h to 20 kg/h, or 1 kg/h to 10 kg/h, 10 kg/h to 400 kg/h, 10 kg/h to 250 kg/h, 10 kg/h to 100 kg/h, 10 kg/h to 50 kg/h, 10 kg/h to 20 kg/h, 20 kg/h to 400 kg/h, 20 kg/h to 250 kg/h, 20 kg/h to 100 kg/h, 20 kg/h to 50 kg/h, 50 kg/h to 400 kg/h, 50 kg/h to 250 kg/h, 50 kg/h to 100 kg/h, 100 kg/h to 400 kg/h, 50 kg/h to 100 kg/h, 100 kg/h to 400 kg/h, 100 kg/h to 200 kg/h or 200 kg/h to 400 kg/h, each inclusive.
  • the critical temperature of carbon dioxide is about 31° C.
  • the extractor vessel is kept at a temperature greater than 31° C.
  • the extractor vessel has a temperature of 32°C to 80°C, and generally 32° C to 60° C or 32° C to 60° C, each inclusive.
  • the temperature can be a temperature that is no more than 35° C, 36 ° C, 37° C, 38° C, 39° C, 40° C, 41 ° C, 42° C, 43° C, 44° C, 45° C, 50° C or 60° C.
  • the temperature is greater than 31 ° C but no more than 40 ° C.
  • the temperature can be varied, depending in part on the composition of the extraction solvent as well as the solubility of a given poloxamer in the solvents employed in the process.
  • any suitable pressure can be used in the methods of the present invention.
  • the system is pressurized at a level to ensure that the supercritical liquid remains at a pressure above the critical pressure.
  • the critical pressure of carbon dioxide is about 74 bars.
  • the extractor vessel is pressured to greater than 74 bars.
  • the particular degree of pressure can alter the solubility characteristics of the supercritical liquid. Therefore, the particular pressure chosen can affect the yield and degree of extraction of impurities.
  • the extractor vessel is pressurized in a range of 125 to 500 bars.
  • the extractor vessel is pressurized in a range of 200 bars to 400 bars, 200 bars to 340 bars, 200 bars to 300 bars, 200 bars to 280 bars, 200 bars to 260 bars, 200 bars to 240 bars, 200 bars to 220 bars, 220 bars to 400 bars, 220 bars to 340 bars, 220 bars to 300 bars, 220 bars to 280 bars, 220 bars to 260 bars, 220 bars to 240 bars, 240 bars to 400 bars, 240 bars to 340 bars, 240 bars to 300 bars, 240 bars to 280 bars, 240 bars to 260 bars, 260 bars to 400 bars, 260 bars to 340 bars, 260 bars to 300 bars, 260 bars to 280 bars, 280 bars to 400 bars, 280 bars to 340 bars, 280 bars to 300 bars or 300 bars to 340 bars.
  • the extraction vessel can be pressurized at about or at least 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, or 400 bars, but generally no more than 500 bars.
  • the extraction vessel can be pressurized, for example, at 310 ⁇ 15 bars.
  • the extraction solvent introduced into the extraction vessel also contains an alkanol.
  • the extraction solvent includes a second alkanol and a supercritical liquid under high pressure and high temperature.
  • the second alkanol acts as a co-solvent modifier of the supercritical liquid to change the solvent characteristics of the supercritical liquid and improve extractability of the solute in the method.
  • Any suitable alkanol or combination of alkanols, as described above, can be used as the second alkanol in the methods provided herein.
  • the second alkanol is methanol.
  • the extraction solvent includes methanol and carbon dioxide.
  • the second alkanol is typically provided as a percentage (w/w) of the total extraction solvent that is 3% to 20%, and generally 3% to 15%, for example 5% to 12%, 5% to 10%, 5% to 9%, 5% to 8%, 5% to 7%, 7% to 1 5%, 7% to 12%, 7% to 10%, 7% to 9%, 7% to 8%, 8% to 1 5%, 8% to 12%, 8% to 10%, 8% to 9%, 9% to 15%, 9% to 12%, 9% to 10%, 10% to 15% or 10% to 12%, each inclusive.
  • the flow rate (kg h) of the alkanol is a function of the amount of alkanol introduced into the extractor.
  • a suitable ratio of the alkanol (e.g. methanol) to supercritical liquid (e.g. carbon dioxide) can be selected based on the identity and purity of the poloxamer starting material, or based on other extraction parameters such as temperature or pressure.
  • the ratio of alkanol (e.g. methanol) to supercritical liquid (e.g. carbon dioxide) can be about from about 1 : 100 to about 20: 100.
  • the ratio of alkanol (e.g. methanol) to supercritical liquid (e.g. carbon dioxide) is from about 1 : 100 to about 15 : 100.
  • the ratio of alkanol (e.g. methanol) to supercritical liquid (e.g. carbon dioxide) is from about 2: 100 to about 14: 100.
  • the ratio of alkanol (e.g.) methanol to supercritical liquid (e.g. carbon dioxide) can be about 3 : 100, or about 4: 100, or about 5 : 100, or about 6 : 100, or about 7: 100, or about 8: 100, or about 9: 100, or about 10: 100, or about 1 1 : 100, or about 12: 100, or about 13 : 100, or about 14: 100.
  • the extraction can be conducted in an isocratic fashion, wherein the composition of the extraction solvent remains constant throughout the extraction procedure.
  • the amount of supercritical liquid (e.g. carbon dioxide) and alkanol (e.g. methanol) are constant over the time of extraction, for example, by maintaining a constant flow rate of each.
  • the composition of the extraction solvent can be varied over time, typically, by altering (e.g. increasing or decreasing) the amount of the supercritical liquid and/or alkanol components that make up the extraction solvent.
  • the supercritical liquid e.g. carbon dioxide
  • the concentration of the alkanol (e.g. methanol) in the extraction solvent is altered (e.g. increased or decreased) over time of the extraction.
  • the concentrations of the components can be altered by adjusting the flow rate.
  • composition of the extraction solvent can be varied over time
  • a method in which the second alkanol is increased as the extraction process progresses, either as a step-wise gradient or continuously escalating gradient is beneficial to the method.
  • commercial grade poloxamers have both high molecular weight components and low molecular weight components along with the main product or component.
  • Low alkanol (e.g. methanol) concentrations in high pressure carbon dioxide extraction fluid can selectively remove low molecular weight components.
  • alkanol e.g. methanol
  • concentrations increase the solubility, and hence extraction, of higher molecular weight components.
  • a gradient with successively higher alkanol (e.g. methanol) concentrations of the extraction solvent can progressively extract low molecular weight components as well as eventually higher molecular weight components or components that were less soluble.
  • a lower alkanol e.g.
  • methanol concentration of about 6.6% w/w can remove low molecular weight components.
  • concentration of alkanol by 1 % to 3% will continue to effect extraction of low molecular weight components, but also result in removal of higher molecular weight components.
  • a further increase in the concentration of alkanol by 1 % to 3% will further remove these components as well as other components that have a higher molecular weight and/or were less soluble in the previous extraction solvents.
  • An extraction solvent with higher alkanol (e.g. methanol) concentrations is not as selective because it is provides more solubility for low molecular weight components, but also increases the solubility of other components including the main components. Therefore, the yield of purified product is reduced with high methanol concentrations.
  • concentration of the extraction solvent in a gradient fashion as provided in methods herein, the reduction of poloxamer yield is minimized and the purity of the final product is maximized.
  • a two-phase system forms inside the extractor.
  • a lower phase consists primarily of a mixture of poloxamer and methanol with some dissolved carbon dioxide.
  • the extraction solvent carbon dioxide with a lower methanol co- solvent fraction
  • An upper phase consists primarily of the extraction solvent and the components extracted from the poloxamer. The relative amount of the two phases depends upon methanol concentration in the solvent flow. In a typical extraction system there is adequate head space for proper phase separation of the upper phase.
  • concentration step-wise during the extraction process leads to higher feed charge into the extractor.
  • the composition of the extraction solvent can be varied as shown in steps 130- 140.
  • the percentage of alkanol (e.g. methanol), by weight, of the extraction solvent is increased over the course of the method.
  • the methanol content in a methanol/carbon dioxide mixture can be increased in a stepwise fashion or a continuous fashion as the extraction process progresses.
  • the extraction process for a poloxamer starts using about 3% to about 10%, by weight, (w/w) of an alkanol (e.g. methanol) in an extraction solvent with a supercritical liquid (e.g.
  • the alkanol (e.g. methanol) content of the extraction solvent is raised about 1 -3%, such as 1-2% ⁇ e.g. , to 7.6% or 9.1 %, respectively).
  • the alkanol (e.g. methanol) content is again subsequently raised about 1 -3%, such as 1 -2% ⁇ e.g. , to 8.6% or 10.7%, respectively) during a final period.
  • any suitable solvent gradient can be used in the methods of the invention.
  • the alkanol (e.g. methanol) concentration in supercritical liquid (e.g. carbon dioxide) can be increased from about 5% to about 20% over trie course ot extraction procedure.
  • the alkanol (e.g. methanol) concentration in supercritical liquid (e.g. carbon dioxide) can be increased from about 5% to about 20%, or from about 5% to about 15%, or from about 5% to about 10%.
  • concentration in supercritical liquid can be increased from about 6%) to about 18%), or from about 6%> to about 12%, or from about 6%> to about 10%>.
  • the alkanol (e.g. methanol) concentration in supercritical liquid (e.g. carbon dioxide) can be increased from about 7% to about 18%, or from about 7% to about 12%, or from about 7% to about 10%.
  • the alkanol (e.g. methanol) concentration can be increased in any suitable number of steps. For example, the alkanol (e.g. methanol) concentration can be increased over two steps, or three steps, or four steps, or five steps over the course of the extraction procedure. A skilled artisan will appreciate that other solvent ratios and solvent gradients can be used in the extraction processes.
  • Time of extraction of the process provided herein can be for any defined period that results in a suitable extraction of material in the preparation while minimizing poloxamer yield and maximizing purity.
  • the time is a function of the choice of pressure, temperature, second alkanol concentration, and process of providing the extraction solvent (e.g. isocratic or as a gradient of increasing alkanol concentration as described herein).
  • the extraction proceeds for 5 hours to 50 hours, and generally 10 hours to 30 hours, or 15 hours to 25 hours, each inclusive such as or about 15 hours or 24 hours.
  • the higher the alkanol (e.g. methanol) concentration employed in the method typically the shorter the time of the extraction.
  • the total time of extraction is divided as a function of the number of gradient steps in the procedure.
  • the extraction in each gradient step can be for the same amount of time or for different times. It is within the level of a skilled artisan to empirically determine the times of extraction to be employed.
  • Samples can be collected during the extraction process to monitor the removal of substances or to determine if adjustment of extraction parameters, such as temperature or the composition of the extraction solvent, is necessary.
  • the methods can be used to purify P188.
  • the process can be applied to other polymers as well.
  • the methods provided herein provide a method for preparing a purified
  • the method includes:
  • the second solvent contains a supercritical liquid under high pressure and high temperature and an alkanol that is methanol, ethanol, propanol, butanol, pentanol or a combination thereof, and the concentration of the second solvent in the extraction solvent is increased over the time of extraction method;
  • the mean or average molecular weight of the copolymer is from about 7680 to 9510 Da, such as generally 8,400-8,800 Da, for example about or at 8,400 Da.
  • the copolymer solution can be formed in the extractor vessel by the addition of the copolymer and by adding a first solvent to form a solution or a suspension of the copolymer, wherein the first solvent comprises an alkanol selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol and a combination thereof.
  • the addition of the first solvent to the copolymer to form a copolymer solution can be in a separate vessel and the copolymer solution, which is dissolved in the first solvent, is provided or introduced (i.e. charged) into the extractor vessel.
  • the method includes stirring the extraction mixture under high pressure and high temperature to extract impurities (e.g. low molecular weight extractable components and other components) from the copolymer composition,
  • system 200 represents one embodiment for practice of the provided methods.
  • System 200 is one system that can be used to extract impurities (e.g. LMW substances or other components) from the poloxamers using supercritical fluids or sub-supercritical methods.
  • impurities e.g. LMW substances or other components
  • Polymer feed pump 201 is charged with a poloxamer (e.g. PI 88) to be purified. Poloxamer is transported into polymer feed tank 207 through value 205.
  • the extractor vessel 215 is used to remove the extracted impurities from the sample, such as LMW substances or other components from the poloxamer.
  • Carbon dioxide (or other supercritical liquid or sub-supercritical liquid) pump 208 is charged with carbon dioxide from outside carbon dioxide supply 250 through value 243 and pre- cooler 203. Carbon dioxide is pumped from pump 208 into heat exchanger 210 and then into extractor 215. Methanol (or other suitable solvents) is pumped into extractor 215 through pump 209.
  • methanol and carbon dioxide extract impurities, such as LMW substances or other components, from the poloxamer in extractor 215.
  • the purified poloxamer mixture is discharged and collected via rapid depressurization processing.
  • the extracted components are isolated from the solvent stream using collector 225, pressure reduction vessel 227, and cyclone separator 231. Carbon dioxide vapor released during collection in collector 225 can be liquefied and recycled using condenser 232.
  • the extraction apparatus can include a solvent distribution system that contains particles of certain shapes forming a "fluidized" bed at the bottom of the extraction vessel.
  • the bed can be supported by a screen or strainer or sintered metal disk.
  • the particles used for the bed can be either perfectly shaped spheres or particles of irregular shape, such as pebbles. Having a smooth surface with less porosity or less surface roughness is preferred for easy cleaning.
  • the density of the particles forming the bed is selected to be higher than the solvent density so the bed remains undisturbed by the incoming solvent flow during the extraction process.
  • the size of the particles can be uniform or can have a distribution of different sizes to control the packing density and porosity of the bed.
  • the packing distribution arrangement is designed to provide for balanced, optimum extraction and subsequent coalescence of the solvent particles before exiting the extraction vessel. This facilitates maximum loading of the extractor with poloxamer charge. This can also maximize extraction efficiency, minimize the extraction time, and minimize undesirable carry-over of the purified product out of the extraction vessel.
  • the size of the spheres in the bed is selected based on one or more system properties including the dimensions of the extraction vessel, the residence time of the solvent droplets in the extraction vessel, and the ability of the solvent droplets to coalesce.
  • the diameter of the spheres can range from about 5 mm to about 25 mm.
  • the diameter can be an average diameter, wherein the bed contains spheres of different sizes. Alternatively, all of the spheres in the bed can have the same diameter.
  • An example of the cross section of stainless steel spheres of different sizes in a solvent distribution bed is shown in FIG. 5.
  • the apparatus includes:
  • the plurality of spheres includes metallic spheres, ceramic spheres, or mixtures thereof. In some embodiments, the plurality of spheres are the same size. In some embodiments, the plurality of spheres include spheres of different sizes. In some embodiments, the particle coalescence system includes one or more members selected from a demister pad, a static mister, and a temperature zone, c. Extraction and Removal of Extractants
  • Any of the methods provided herein can be performed as a batch method or as a continuous method.
  • the method is a batch method.
  • a batch method can be performed with extraction vessels of various dimensions and sizes as described above.
  • the equipment train can contain a 120-L high pressure extractor.
  • a Poloxamer (e.g. PI 88) solution which is a poloxamer dissolved in an appropriate solvent (e.g. an alkanol solvent, such as methanol), is provided or introduced into the extraction vessel.
  • the extraction solvents such as any described in the methods above (e.g. supercritical or high-pressure carbon dioxide and methanol) are independently and continuously pumped into the extraction vessel maintained at a controlled temperature, flow, and pressure. Substances are removed by varying the extraction solvent composition as described herein.
  • the extraction process conditions such as temperature and pressure can also be varied independently or in combination.
  • the purified product is discharged into a suitably designed cyclone separator to separate the purified product from carbon dioxide gas.
  • the product is dried to remove the residual alkanol solvent.
  • the extraction method is a continuous method.
  • a poloxamer (e.g. PI 88) solution which is a poloxamer dissolved in an appropriate solvent (e.g. an alkanol solvent, such as methanol), is loaded at the midpoint of a high pressure extraction column packed with a suitable packing material.
  • the extraction solvent is pumped through the extraction column from the bottom in counter current fashion.
  • the extracted material such as LMW substances or other components, are removed at the top of the column while purified product is removed from the bottom of the column.
  • the purified product is continuously collected at the bottom of the extractor column and periodically removed and discharged into a specially designed cyclone separator.
  • the purified polymer particles containing residual methanol are subsequently dried under vacuum.
  • the extraction step can be repeated for a given batch. That is, additional portions of the extraction solvent can be introduced into the extractor vessel and removed until a sufficient level of poloxamer purity is obtained.
  • the method further includes repeating steps b and c. Steps b and c can be repeated until the poloxamer is sufficiently pure. For example, steps b and c can be repeated one time, or two times, or three times, or four times, or five times, or in an iterative fashion. When the poloxamer material is sufficiently pure, the product is prepared for further processing.
  • the product is handled according to process 100 as summarized in Figure 1.
  • the product can be discharged from the extractor vessel and collected in an appropriate receiver, as shown in step 145.
  • the wet product can be sampled for testing with respect to purity, chemical stability, or other properties, as shown in step 150.
  • the product can be dried by removing residual solvents under vacuum. Vacuum level can be adjusted to control drying rates. Drying can be conducted at ambient temperature, or at elevated temperatures if necessary. In general, the drying temperature is held below the melting point of the poloxamer.
  • the wet product can be dried in a single lot or in smaller portions as sub- lots. As shown in steps 160-170, drying of the product can be initiated, for example on a sub-lot, under vacuum at ambient temperature.
  • any remaining portions of the wet product can be processed in a similar manner, as shown in step 175 of process 100.
  • the resulting product such as the various sub-lots that have been combined, are mixed in a suitable container, as shown in step 180, and the resulting product can be characterized, stored, transported, or formulated.
  • the methods disclosed herein effectively recycle carbon dioxide.
  • supercritical carbon dioxide or high-pressure carbon dioxide can be recovered by subjecting the extract phase to changes in temperature and pressure.
  • the methods employed herein have recycling efficiencies of greater than 80%, preferably greater than 90%, and most preferably greater than 95%.
  • the methods provided herein further include: d) passing the extract phase to a system consisting of several separation vessels; g) isolating the impurities (e.g. low molecular-weight impurities); h) processing the purified material or raffinate and i) recovering the compressed carbon dioxide for reuse.
  • impurities e.g. low molecular-weight impurities
  • parameters can be assessed in evaluating the methods and resulting products. For example, parameters such as methanol concentration, gradient profile, temperature, and pressure can be assessed for process optimization. Processes and suitable conditions for drying wet raffinate, such as vacuum level, mixing mode, time, and temperature, also can be assessed,
  • the methods provided herein above result in the generation of particular purified poloxamer preparations, and in particular PI 88 preparations.
  • the methods provided herein can be used to purify a PI 88 copolymer as described herein that has the formula: HO(CH2CH20)a-(CH 2 CH(CH 3 )0) b -(CH 2 CH 2 0)aH, and a mean or average molecular weight of the copolymer that is from 7680 to 9510 Da, such as generally 8,400-8,800 Da, for example about or at 8,400 Da, and that contains a plurality of low molecular weight substances having a molecular weight of less than 4000 Da, wherein the plurality of low molecular weight substances constitutes more that 4% of the total weight of the composition.
  • the present methods generate purified poloxamers with less than about 4% low molecular weight components such as less than about 3%, 2% or 1%.
  • the low molecular weight components include glycols, and volatile degradation impurities such as formaldehyde, acetaldehyde, propionaldehyde, acetone, methanol, and peroxides.
  • the processes herein produce poloxamer substantially free of low molecular weight components, ; ' . e. , less than 4%, 3%, 2% or 1% of the foregoing components.
  • the methods also can produce
  • poloxamer substantially free of long circulating material, such that when the purified poloxamer is administered to a subject, there are no components in the poloxamer that is or gives rise to a material that has a longer half-life in the blood or plasma more than 5.0-fold the half-life of the main component in the poloxamer distribution, such as generally no more than 4.0-fold, 3.0-fold, 2.0-fold, or 1.5-fold. Exemplary of such methods that produce these purified products are described below.
  • FIG. 2 certain embodiments of the methods herein provide a process 100' that is useful for removing LMW substances in a poloxamer.
  • the extraction system is pressurized, as shown in step 105', prior to dispensing a first alkanol (e.g. methanol) into the feed mix tank, as shown in step 1 10'.
  • a first alkanol e.g. methanol
  • the system is heated to a temperature suitable for the extraction process, which is a temperature above the critical temperature of carbon dioxide used in the process that is about 31° C. Typically, the temperature is no more than 40° C.
  • the temperature is generally kept constant through the process.
  • the first alkanol e.g. methanol
  • the first alkanol is used to form a poloxamer solution according to step 1 15' in process 100'.
  • poloxamer into the feed tank with the alkanol results in a P I 88 poloxamer solution that is dissolved in the alkanol (e.g. methanol).
  • the amount of poloxamer for use in the method can be any amount, such as any amount described herein above.
  • all or part of the mixture is pumped into the extractor as shown in step 120'.
  • the poloxamer solution can be formed in the extraction vessel by introducing the poloxamer as a solid into the extractor prior to mixing with the alkanol.
  • the extractor is then pressurized and the extraction solvent is introduced into the extractor as shown in step 125' of process 100'
  • the extraction solvent typically contains carbon dioxide and extraction is perform at a temperature greater than the critical temperature of 31 °C as described above and under high pressure greater than the critical pressure of 74 bars.
  • the extraction vessel is pressurized to about 310 ⁇ 15 bars, and the carbon dioxide is provided at a flow rate that is 20 kg/h to 50 kg/h, such as generally about or approximately 24 kg/h (i.e. 390 g/min).
  • the extraction is then conducted in the presence of a second alkanol acting as a co-solvent modifier of the carbon dioxide.
  • the second alkanol such as methanol
  • the second alkanol is added in a gradient step-wise fashion such that the concentration of the second alkanol in the extraction solvent is increased over the time of the extraction method.
  • the composition of the extraction solvent can be varied as shown in steps 130'-140'.
  • the extraction process for a poloxamer e.g. P188
  • starts using about 5% to 7%, by weight (w/w) of an alkanol e.g.
  • methanol in an extraction solvent with a supercritical liquid (e.g. carbon dioxide), (e.g. , about 6.6%).
  • a supercritical liquid e.g. carbon dioxide
  • the alkanol (e.g. methanol) content of the extraction solvent is raised about 1 -3%, such as 1 % (e.g. , to 7.6%).
  • the alkanol (e.g. methanol) content is again subsequently raised about 1 -3% such as 1 % (e.g. , to 8.6%) during a final period.
  • the total time of the extraction method can be 15 hours to 25 hours. Each gradient is run for a portion of the total time.
  • Residual low molecular weight components can be subsequently removed with high methanol concentrations in a short time. Therefore a stepwise methanol concentration profile where about 5-10% (e.g. , 6.6%) methanol is used for 12 hours, a higher methanol is used for 10 hours and finally an even higher methanol is used for 4 hours to produce purified product in high yields without significantly reducing the overall yield and not enriching the high molecular weight components.
  • the product is prepared for further processing as shown in process 100'.
  • the product can be discharged from the extractor vessel and collected in an appropriate receiver, as shown in step 145'.
  • the wet product can be sampled for testing with respect to purity, chemical stability, or other properties, as shown in step 150'.
  • the product can be dried by removing residual solvents under vacuum as described herein.
  • drying can be initiated with a sub-lot under vacuum at ambient temperature and drying can be then continued at higher temperatures and lower pressures as the process progresses.
  • Remaining sub-lots can be processed in a similar manner, as shown in step 175' of process 100.
  • Sub-lots can be combined and mixed in a suitable container, as shown in step 180, and the resulting product can be characterized, stored, transported, or formulated.
  • step 105 the poloxamer and first alkanol (e.g. methanol) are dispensed into the extractor vessel to form the poloxamer solution.
  • the alkanol e.g. methanol
  • step 105 dispensing of a P I 88 poloxamer into the extraction vessel with the alkanol (e.g. methanol) results in a P I 88 poloxamer solution that is dissolved in the alkanol (e.g. methanol).
  • the amount of poloxamer for use in the method can be any amount as described herein.
  • the poloxamer solution can be formed a separate vessel, and the poloxamer solution transferred to the extractor vessel.
  • the extraction system is pressurized, as shown in step 1 10", after dispensing a first alkanol (e.g. methanol) and poloxamer.
  • a first alkanol e.g. methanol
  • poloxamer e.g. methanol
  • the system is heated to a temperature suitable for the extraction process, which is a temperature above the critical temperature of carbon dioxide used in the process that is about 31 ° C. Typically, the temperature is no more than 40° C.
  • the temperature is generally kept constant through the process.
  • the poloxamer solution is formed under pressurized carbon dioxide of about 49 bars and a temperature of no more than 40° C or about 40° C for a defined period, generally less than several hours.
  • the extractor is then pressurized and the extraction solvent is introduced into the extractor as shown in step 120" of process 100"'.
  • the extraction solvent typically contains carbon dioxide and a second alkanol and extraction is perform at a
  • the extraction vessel is pressurized to about 247 ⁇ 15 atm bars, and the carbon dioxide is provided at a flow rate that is 50 kg/h to 100 kg/h, inclusive, such as generally about or approximately 100 kg/h.
  • the extraction is conducted in the presence of the second alkanol, which acts as a co-solvent modifier of the carbon dioxide.
  • the second alkanol such as methanol
  • the composition of the extraction solvent can be varied as shown in steps 125"- 135".
  • the extraction process for a poloxamer e.g. PI 88
  • an alkanol e.g. methanol
  • a supercritical liquid e.g.
  • the alkanol (e.g. methanol) content of the extraction solvent is raised about 1 -3%, such as up to 2% (e.g. , to 9.1%).
  • the alkanol (e.g. methanol) content is again subsequently raised about 1 -3%, such as up to 2% (e.g. , to 10.7%), during a final period.
  • the total time of the extraction method can be 15 hours to 25 hours, inclusive. Each gradient is run for a portion of the total time.
  • a stepwise methanol concentration profile where about a 7-8% (e.g. , 7.4%) methanol is used for about 3 hours, a higher methanol (e.g. 9.1%) is used for about 4 hours and finally an even higher methanol (e.g. 10.7%) is used for about 8 hours to produce purified product in high yields without significantly reducing the overall yield.
  • the product is prepared for further processing as shown in process 100".
  • the product can be discharged from the extractor vessel and collected in an appropriate receiver, as shown in step 140".
  • the product can be precipitated under reduced pressure via particles from gas saturated solutions (PGSS) techniques as shown in step 145".
  • PGSS gas saturated solutions
  • the product can be dried by removing residual solvents under vacuum as described herein. In an exemplary method as shown in steps 150"- 165", drying can be initiated under vacuum at high temperatures no more than 40° C.
  • the dried product can be collected as shown in step 160".
  • the resulting product can be characterized, stored, transported, or formulated as shown in step 165".
  • compositions containing a poloxamer PI 88 provided herein such as any prepared by methods provided herein, are provided.
  • compositions containing an LC F poloxamer are provided.
  • the compositions are used in methods for treating heart failure as described in Section F. 1.
  • compositions containing PI 88 can be formulated in any conventional manner by mixing a selected amount of the poloxamer with one or more physiologically acceptable carriers or excipients. Selection of the carrier or excipient is within the skill of the administering professional and can depend upon a number of parameters. These include, for example, the mode of administration (i.e., systemic, oral, nasal, pulmonary, local, topical, or any other mode) and the symptom, disorder, or disease to be treated.
  • Effective concentrations of PI 88, such as an LCMF PI 88, are mixed with a suitable pharmaceutical carrier or vehicle for systemic, topical or local administration.
  • Pharmaceutical carriers or vehicles suitable for administration of the copolymers include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • the compound can be suspended in micronized or other suitable form or can be derivatized to produce a more soluble active product.
  • the form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of PI 88, such as LCMF PI 88, in the selected carrier or vehicle.
  • the resulting mixtures are solutions, suspensions, emulsions and other such mixtures, and can be formulated as an non-aqueous or aqueous mixtures, creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, or any other formulation suitable for systemic, topical or local administration.
  • aqueous or aqueous mixtures creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, or any other formulation suitable for systemic, topical or local administration.
  • the polypeptides can be formulated as a solution suspension in an aqueous-based medium, such as isotonically buffered saline or are combined with a biocompatible support or bioadhesive intended for internal administration.
  • an aqueous-based medium such as isotonically buffered saline or are combined with a biocompatible support or bioadhesive intended for internal administration.
  • compositions are prepared in view of approvals for a regulatory agency or are prepared in accordance with generally recognized pharmacopeia for use in animals and in humans.
  • Pharmaceutical compositions can include carriers such as a diluent, adjuvant, excipient, or vehicle with which an isoform is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water is a typical carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions also can be employed as liquid carriers, particularly for injectable solutions.
  • Compositions can contain along with an active ingredient: a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; a lubricant, such as magnesium stearate, calcium stearate and talc; and a binder such as starch, natural gums, such as gum acacia gelatin, glucose, molasses, polyvinylpyrrolidone, celluloses and derivatives thereof, povidone, crospovidones and other such binders known to those of skill in the art.
  • a diluent such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose
  • a lubricant such as magnesium stearate, calcium stearate and talc
  • a binder such as starch, natural gums, such as gum acacia gelatin, glucose, molasses, polyvinylpyrrolidone, celluloses and derivatives thereof, povidone, crospovid
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, and ethanol.
  • a composition if desired, also can contain minor amounts of wetting or emulsifying agents, or pH buffering agents, for example, acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents.
  • These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, and sustained release formulations.
  • Capsules and cartridges of e.g. , gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of a therapeutic compound and a suitable powder base such as lactose or starch.
  • a composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
  • Such compositions will contain a therapeutically effective amount of P I 88, in a form described herein, including the LCMF form, together with a suitable amount of carrier so as to provide the form for proper administration to a subject or patient.
  • compositions containing P I 88 can be formulated for parenteral administration by injection (e.g., by bolus injection or continuous infusion).
  • the injectable compositions can take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles.
  • the sterile injectable preparation also can be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1 ,4- butanediol.
  • Sterile, fixed oils are
  • any bland fixed oil can be employed, including, but not limited to, synthetic mono- or diglycerides, fatty acids (including oleic acid), naturally occurring vegetable oils like sesame oil, coconut oil, peanut oil, cottonseed oil, and other oils, or synthetic fatty acid vehicles like ethyl oleate. Buffers, preservatives, antioxidants, and the suitable ingredients, can be incorporated as required, or, alternatively, can comprise the formulation.
  • Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions, which can contain anti-oxidants, buffers, bacteriostats and solutes that render the formulation compatible with the intended route of administration.
  • the formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, prefilled syringes or other delivery devices and can be stored in an aqueous solution, dried or freeze-dried (lyophilized) condition, requiring only the addition of the sterile liquid carrier, for example, water for injection, immediately prior to use.
  • Liposomal suspensions including tissue-targeted liposomes, also can be suitable as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposome formulations can be prepared as described in U.S. Patent No. 4,522,81 1. Liposomal delivery also can include slow release formulations, including pharmaceutical matrices such as collagen gels and liposomes modified with fibronectin (see, for example, Weiner et al. (1 85) J Pharm Sci. 74(9): 922-925).
  • compositions provided herein further can contain one or more adjuvants that facilitate delivery, such as, but not limited to, inert carriers, or colloidal dispersion systems.
  • inert carriers can be selected from water, isopropyl alcohol, gaseous
  • fluorocarbons ethyl alcohol, polyvinyl pyrrolidone, propylene glycol, a gel-producing material, stearyl alcohol, stearic acid, spermaceti, sorbitan monooleate,
  • methylcellulose as well as suitable combinations of two or more thereof.
  • the PI 88 such as LCMF PI 88, is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the subject treated.
  • the therapeutically effective concentration can be determined empirically by testing the compounds in known in vitro and in vivo systems, such as the assays provided herein.
  • compositions containing PI 88 can be formulated for single dosage (direct) administration, multiple dosage administration or for dilution or other modification.
  • PI 88 such as LCMF PI 88
  • concentrations of the compounds in the formulations are effective for delivery of an amount, upon administration, that is effective for the intended treatment, and in particular as a single infusion treatment according to the dosage regime provided herein.
  • Those of skill in the art readily can formulate a composition for administration in accord with the methods herein. For example, to formulate a composition, the weight fraction of a compound or mixture thereof is dissolved, suspended, dispersed, or otherwise mixed in a selected vehicle at an effective concentration such that hemostasis is improved.
  • the precise amount or dose of the therapeutic agent administered depends on the route of administration, and other considerations, such as the severity of the bleeding to be stopped or slowed and the weight and general state of the subject and the subject.
  • Local administration of the therapeutic agent will typically require a smaller dosage than any mode of systemic administration, although the local concentration of the therapeutic agent can, in some cases, be higher following local administration than can be achieved with safety upon systemic administration.
  • a particular dosage and duration and treatment protocol can be empirically determined or extrapolated.
  • exemplary doses of PI 88, such as LCMF PI 88 provided herein, if necessary, can be used as a starting point to determine appropriate dosages for a particular subject and condition.
  • the duration of treatment and the interval between injections will vary with the severity of the disease or condition and the response of the subject to the treatment, and can be adjusted
  • LCMF PI 88 can be taken into account when making dosage determinations.
  • the poloxamer can be formulated at a concentration ranging from about 10.0 mg/mL to about 300.0 mg/mL or 10.0 mg/mlto 200.0 mg/ml, such as at or at least 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0,
  • the concentration is not more than 22.5%, i.e.
  • the poloxamer is administered at a
  • the poloxamer can be administered in an amount between about 0.5% and about 20%, by weight/volume, such as 0.5%, 1%,
  • the poloxamer is administered in an amount between about 0.5% to about 10% by weight/volume, such as 0.5%, 1%, 1.5%, 2%,
  • the poloxamer is administered in an amount between about 5% and about 15%, by weight/volume, such as 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10.0%, 10.5%, 1 1%, 1 1.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, or 15%, by weight/volume.
  • the concentration is 10% to 22.5%, such as 10% to 20% or 15% to 20%.
  • the poloxamer can be formulated as a sterile, non-pyrogenic solution intended for administration with or without dilution.
  • the final dosage form can be a prepared in a 100 mL vial where the 100 mL contains 15 g (150 mg/mL) of purified poloxamer 188, such as LCMF PI 88, 308 mg sodium chloride USP, 238 mg sodium citrate USP, 36.6 mg citric acid USP and water for injection USP Qs to 100 mL.
  • the pH of the solution is approximately 6.0 and has an osmolarity of about 312 mOsm/L.
  • at least 500 mis is prepared with a concentration of 10% to 20%, such as about or at 15% weight of poloxamer preparation/volume of the composition.
  • poloxamer 188 such as a purified poloxamer 188 described herein
  • poloxamer 188 such as a purified poloxamer 188 described herein
  • a poloxamer 188 provided herein is used to treat heart disorders and muscular disorders such as acute, chronic, ischemic heart failure, congestive heart failure, stroke, myocardial infarction, high blood pressure, disorders of the heart which cellular damage occurs, arrhythmias and fibrillations, angina, peripheral artery disease, and stroke.
  • Arrhythmias include tachycardias, bradycardias and true arrhythmias of disturbed rhythm.
  • Arrhythmias are classified as lethal if they cause a severe decrease in the pumping function of the heart
  • Treatment of diseases and conditions, such as any described in Section F, with poloxamer 188, such as a purified poloxamer 188 described herein, can be effected by any suitable route of administration using suitable formulations as described herein including, but not limited to, injection, pulmonary, oral and transdermal
  • Treatment typically is effected by intravenous administration.
  • Active agents for example a poloxamer 188, an LCMF P I 88, are included in an amount sufficient that they exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.
  • the amount of a P I 88, such as a LCMF PI 88, to be administered for the treatment heart failure, for example for treating a patient with acute decompensation, can be determined by standard clinical techniques.
  • in vitro assays and animal models can be employed to help identify optimal dosage ranges.
  • the precise dosage which can be determined empirically, can depend on the particular composition, the route of administration, the type of disease to be treated and the seriousness of the disease.
  • Poloxamer 188 such as a purified poloxamer 188 described herein, is designed to retain therapeutic activity without overt toxicity, but exhibit modified properties, particularly long-lasting improvement of diastolic or systolic cardiac function. Such modified properties, for example, can improve the therapeutic effectiveness of the poloxamer 188 and can exhibit improvement of in vivo activities and therapeutic effects compared to previously administered Poloxamer 188, including fewer and/or less frequent administrations and longer lasting improvement in therapeutic effects .
  • poloxamer 188 that was manufactured according to National Formulary specification (P188-NF) (Emanuele and Balsubramaniam. Drugs R D 14(2):73-83 (2014)) due to renal dysfunction in a subset of patients enrolled in early clinical trials.
  • P188-NF National Formulary specification
  • animal studies reveal that P188-NF increases the levels of serum creatine and creatine is not efficiently cleared from the kidneys at the end of the drug infusion.
  • the purified poloxamer 188 described herein has been modified to address the limitations of PI 88- NF. To prevent elevation of creatine levels and renal toxicity, poloxamer 188 was purified to remove low and high molecular weight species contaminants.
  • the dosing regimen of poloxamer 188 has been modified to address the limitations of clinical use of previous poloxamer 188. Any poloxamer 188 described herein is administered in a single dose, or in a low number of doses in a limited time frame, to produce a long lasting effect on cardiac function without the toxicity associated with a previously analyzed poloxamer 188.
  • methods of treatment with poloxamer 188 requires a longer duration of action in order to effect a sustained therapeutic effect. This is particularly true in treatment of chronic heart failure. As discussed elsewhere herein, the half-life of poloxamer 188 is 18 hours. However, despite a relatively short half-life the effects of poloxamer 188, such as a purified poloxamer 188, are long lasting. Thus, the poloxamer 188 described herein can be used to deliver longer lasting therapies for cardiac disorders.
  • a particular dosage and duration and treatment protocol can be empirically determined or extrapolated.
  • Dosages for poloxamer 188 previously administered to human subjects and used in clinical trials can be used as guidance for determining dosages for poloxamer 188, such as a purified poloxamer 188 described herein.
  • Dosages for poloxamer 188 can also be determined or extrapolated from relevant animal studies. Factors such as the level of activity and half- life of poloxamer 188 can be used in making such determinations.
  • Particular dosages and regimens can be empirically determined based on a variety of factors.
  • the active ingredient, poloxamer 188 typically is combined with a pharmaceutically effective carrier.
  • the amount of active ingredient that can be combined with the carrier materials to produce a single dosage form or multi-dosage form can vary depending upon the host treated and the particular mode of administration.
  • the poloxamer such as PI 88 (e.g. LCMFP188) is formulated for administration to a patient at a dosage of about 100 to 675 mg/kg patient body weight, such as 100 to 500 mg/kg patient body weight, for example 100 mg/kg to 450 mg/kg, 100 to 400 mg/kg, 100 mg/kg to 300 mg/kg, 100 mg/kg to 200 mg/kg, 200 mg/kg to 500 mg/kg, 200 mg/kg to 450 mg/kg, 200 mg/kg to 400 mg/kg, 200 mg/kg to 300 mg/kg, 300 mg/kg to 500 mg/kg, 300 mg/kg to 450 mg/kg 300 mg/kg to 400 mg/kg, 400 mg/kg to 500 mg/kg, 400 mg/kg to 450 mg/kg or 450 mg/kg to 500 mg/kg patient body weight, such as about 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, or 600 mg/kg patient body weight.
  • the poloxamer is formulated
  • the goal is to administer the dose in the smallest volume possible.
  • the volume to be administered is not greater than 3.0 mL/kg of a subject.
  • the volume in which the dose is administered to a subject can be 0.4 mL/kg to 3.0 mg/kg, 0.4 mL/kg to 2.5 mL/kg, 0.4 mL/kg to 2.0 mL/kg, 0.4 mL/kg to 1.8 mL/kg.
  • a composition with a concentration of 22.5% (i.e. 225 mg/mL) that is administered at 100 mg/mL would be administered in a volume of about 0.4 mg/mL to achieve that dose.
  • the particular volume chosen is one that provides a dosage over an appropriate time period to meet the patient's needs.
  • the administered dose is typically administered as an infusion.
  • the infusion is an intravenous infusion.
  • the infusion to provide the appropriate dosage, can be provided to the subject over a time period that is 1 hour to 24 hours, 1 hour to 12 hours, 1 hour to 6 hours, 1 hour to 3 hours, 1 hour to 2 hours, 2 hours to 24 hours, 2 hours to 12 hours, 2 hours to 6 hours, 2 hours to 3 hours, 3 hours to 24 hours, 3 hours to 12 hours, 3 hours to 6 hours, 6 hours to 24 hours, 6 hours to 12 hours, or 12 hours to 24 hours, such as generally over a time period that is up to or is about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 12 hours, 15 hours, 18 hours, 20 hours, 22 hours or more. It is within the level of a treating physician to determine the appropriate time and rate of infusion that can be tolerated by a subject.
  • poloxamer such as P188 (e.g. LCMF P188)
  • P188 e.g. LCMF P188
  • PI 88 the effects of PI 88 on parameters of heart function lasts for more than a week and up to 2 weeks.
  • the dosage can be repeated once every week, once every 2 weeks, once every three weeks or once every 4 weeks.
  • the dose can be repeated between 1 week to 4 weeks after the previous dose, such that the dose is repeated within 7 days, 8 days, 9 days, 10 days, 1 1 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 or 30 days following completion of the prior dose.
  • the dose that is administered in the repeated dosing can be the same or different than the prior dose. For example, it can be increased or decreased from the prior dose. It is within the level of the treating physician to determine the appropriate frequency of administration and level or amount of dosages in repeated dosings.
  • the length of time of the cycle of administration can be empirically determined, and is dependent on the disease to be treated, the severity of the disease, the particular patient, and other considerations within the level of skill of the treating physician.
  • the length of time of treatment with P188, such as a LCMF P188 can be one week, two weeks, one months, several months, one year, several years or more.
  • a PI 88, such as an LCMF PI 88 is administered no more than once weekly, such as every 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days or more.
  • a PI 88 such as an LCMF P188 can be administered no more than once weekly, such every 7 days, 8 days, 9 days, 10 days, 1 1 days, 12 days, 13 days, 14 days or more, over a period of a year or more. If disease symptoms persist in the absence of discontinued treatment, treatment can be continued for an additional length of time. Over the course of treatment, evidence of disease and/or treatment-related toxicity or side effects can be monitored.
  • the cycle of administration can be tailored to add periods of discontinued treatment in order to provide a rest period from exposure to the treatment.
  • the length of time for the discontinuation of treatment can be for a predetermined time or can be empirically determined depending on how the patient is responding or depending on observed side effects.
  • the treatment can be discontinued for one week, two weeks, one month or several months.
  • dosings will typically start when the patient is admitted to the hospital, but it can be started any time during
  • hospitilization to meet the subjects needs. More generally, the dosing will start during the first 72 hours of hospitilization. For chronic heart failure, dosing generally is provided based on the needs of the subject, since such subjects generally do not undergo hospitilization.
  • the formulations used in the methods provided herein can be administered by any appropriate route, for example, orally, nasally, pulmonary, parenterally, intravenously, intradermally, subcutaneously, intraarticularly, intracisternally, intraocularly, intraventricularly, intrathecally, intramuscularly, intraperitoneally, intratracheally or topically, as well as by any combination of any two or more thereof, in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration.
  • Multiple administrations, such as repeat administrations described herein can be effected via any route or combination of routes. The most suitable route for administration will vary depending upon the disease state to be treated.
  • the compositions are fonnulated for intravenous infusions.
  • a poloxamer such as PI 88 and in particular an LCMF PI 88 as provided herein, can be delivered alone or in combination with other agents for treating a disease or condition. It is within the level of a skilled artisan to choose a further additional treatment to administer in conjunction with a therapeutic regime employing LCMF PI 88. Such a decision will depend on the particular disease or condition being treated, the particular subject being treated, the age of the subject, the severity of the disease or condition and other factors.
  • Assays for heart failure using clinical assessment including assays for breathlessness, orthopnoea (shortness of breath experienced when lying flat), paroxysmal nocturnal dyspnoea (severe shortness of breath that occurs most often at night), exercise tolerance including time to fatigue, edema, including ankle and abdomen swelling, and cyanosis.
  • Assays to evaluate heart failure can also be used to diagnose cough, wheezing, weight, to assess for weight loss or gain and/or change in appetite, personality changes, palpitations and fainting.
  • a number of such assays known to those of skill in the art are subject to quantitative analysis (e.g. palpitations, cyanosis, etc.).
  • Systolic heart failure or diastolic heart failure share the same clinical phenotype, but differ with respect to the morphological and functional changes that occur in the heart. Assays to differentiate between systolic and diastolic dysfunction can be used to assess ejection volume by one of skill in the art, such as a skilled physician. Systolic heart failure presents with a decrease in ejection volume, while diastolic heart failure retains an intact ejection volume; moreover, systolic and diastolic heart failure are most effectively distinguished from each other using internal measurements of heart volume, such as, for example, M-Mode, two- dimensional or three-dimensional echocardiography, cardiac magnetic resonance imaging (CMRI), doppler, or pulsed reflected ultrasound.
  • M-Mode two- dimensional or three-dimensional echocardiography
  • CMRI cardiac magnetic resonance imaging
  • doppler or pulsed reflected ultrasound.
  • assays for hemodynamic and ventriculographic measurements including those used to assess aortic and left- ventricular (LV) pressures, peak rate of change of LV pressure during isovolumic contraction and relaxation, LV end-diastolic pressure, cardiac output (CO), LV stroke volume (SV), systemic vascular resistance (SVR), LV end- systolic (ESV) and end-diastolic (EDV) volumes, and LV ejection fraction (EF) (Sabbah et al. (1991) Am. J. Physiol. 260 (Heart Circ. Physiol. 20): H1379-H1384; Zaca et al. (2007) J. Am. Coll. Cardiol, 50:551 -557; Dodge, H.T., et al.
  • LV left- ventricular
  • assays for heart failure include those that assess hemodynamic performance, left ventricular-end diastolic volume, left ventricular-end systolic volume, and ejection fraction.
  • assays such as pulsed-wave Doppler echocardiography can be used to measure mitral inflow velocity.
  • in vivo measurement of cardiac function can be measured using cardiac ventriculography.
  • ventriculograms are performed using power injection of liquid contrast material, and movement of the contrast liquid into the heart is recorded on digital media.
  • Image correction can be performed using radiopaque markers placed on the distal end of the LV ventriculographic catheter and LV end-systolic and LV end-diastolic volumes can be calculated.
  • a poloxamer 188 such as a purified poloxamer 188
  • the ability of a poloxamer 188, such as a purified poloxamer 188, to exhibit therapeutic activity for reducing orpreventing systolic heart dysfunction can be assessed using any one or more of the assays described above.
  • the ability of a poloxamer 188, such as a purified poloxamer 188, to reduce end systolic volume can be assessed using any one or more of the assays above in vivo.
  • a purified poloxamer 188 can be administered to a subject with heart failure, or an appropriate animal model, and the effect on end-systolic volume can be assessed using cardiac ultrasound and compared to subjects or animal models not administered a purified poloxamer 188.
  • the ability of a poloxamer 188, such as a purified poloxamer 188, to exhibit therapeutic activity for reducing orpreventing diastolic heart dysfunction can be assessed using any one or more of the assays described above.
  • the ability of a poloxamer 188, such as a purified poloxamer 188, to increase Ei/Ai and DCT can be assessed using any one or more of the assays above in vivo.
  • a purified poloxamer 188 can be administered to a subject with diastolic heart failure, or an appropriate animal model, and the effect Ei/Ai and DCT can be assessed and compared to subjects or animal models not administered a purified poloxamer 188.
  • the ability of a poloxamer 188, such as a purified poloxamer 188, to exhibit therapeutic activity for reducing or blocking heart dysfunction can be assessed using any one or more of the assays described above.
  • the ability of a poloxamer 188, such as a purified poloxamer 188, to modify LV fractional area of shortening (FAS), a measure of LV systolic fuction can be assessed using any one or more of the assays above in vivo.
  • a purified poloxamer 188 can be administered to a subject with heart failure, or an appropriate animal model, and the effect on FAS can be assessed using
  • a poloxamer 188 such as a purified poloxamer 188
  • the ability of a poloxamer 188, such as a purified poloxamer 188, to affect any one or more of the structural or physiological properties associated with heart failure described above, or any other associated phenotypes, can be assessed using any one or more of the assays described above.
  • the methods can be used to assess heart function in any subject with heart failure, including heart failure induced by diastolic or systolic dysfunction.
  • Biomarkers in blood serum and the effects of administration of a poloxamer 188, such as a purified poloxamer 188, can be measured using various ex vivo techniques that are well known in the art.
  • biomarkers can be utilized for diagnostic purposes to assess cardiac risk and acute cardiac damage or failure, and vary depending on the disease with which they are associated. By determining the presence, absence, increase or decrease of such a biomarker in, for example, the blood or serum of an individual or animal model, it is also possible to detennine whether cardiac failure is progressing or is being suppressed.
  • Exemplary biomarkers for heart failure include, but are not limited to: N-terminal pro-brain natriuretic peptide (nt-pro BNP), troponin-I (Tn-I), matrix metalloproteinase-2 (MMP-2), Interleukin 6 (IL6), C-reactive protein (CRP) and Tumor necrosis factor-alpha (TNFa).
  • one of skill in the art may identify and determine the level of expression of one or more of these biomarkers, or functional variants thereof, in the patient, wherein an alteration, including an increase or a decrease, in expression indicates that the patient may have a more severe form of heart failure.
  • an alteration, including an increase or a decrease, in expression indicates that the patient may have a more severe form of heart failure.
  • a patient with increased Tn-I expression indicates that the patient has experienced cardiomyocyte injury and death, and thus, may have a more severe form of heart failure.
  • Additional biomarkers that may be used to assay for heart failure include: measures of oxidative stress, including isoprostane and derivatives of reactive oxygen metabolites (D-ROMs), urinary excretion of 8-iso-prostaglandin F2 alpha (IPGF2), which is a chemically stable and quantitative measure of oxidative stress, and is known to correlate with the severity of systolic heart failure, Bilirubin, a scavenger of ROS, 8-OHdG, a marker of systemic DNA damage whose level correlates with the severity of ischemic systolic heart failure, as assayed by the number of diseased vessels visualized using coronary angiography.
  • measures of oxidative stress including isoprostane and derivatives of reactive oxygen metabolites (D-ROMs), urinary excretion of 8-iso-prostaglandin F2 alpha (IPGF2), which is a chemically stable and quantitative measure of oxidative stress, and is known to correlate with the severity
  • a poloxamer 188 such as a purified poloxamer 188, to affect any one or more of the markers associated with heart failure described above, or any other associated markers or phenotypes, can be assessed using any one or more of the assays described above.
  • the methods can be used to assess heart function in any subject with heart failure, including heart failure induced by diastolic or systolic dysfunction.
  • MMP-2 Matrix Metalloproteinase-2
  • inflammatory signals such as infl ammation of the heart, regul ate matrix metailoproteinase activity.
  • Activated metalloproteinases facilitate changes in the extracellular matrix and, ultimately, tissue remodeling in conditions of inflammation and injury and are highly expressed in unstable plaques.
  • Exemplary matrix metalloproteinases are MMP-9, MMP-1, MMP-3, and MMP-2.
  • MMP-2 (Shirakabe, A. et al. Int. Heart J. 51(6): 404-10 (2010)).
  • Exemplary assays for heart failure include determining the expression level of the MMP-2 gene, wherein increased MMP-2 expression indicates that the patient may have heart disease or actively progressing heart failure.
  • the ability of a poloxamer 188, such as a purified poloxamer 188, to exhibit therapeutic activity for reducing or blocking heart dysfunction can be assessed using any one or more of the biomarkers described above.
  • the ability of a poloxamer 188, such as a purified poloxamer 188, to ameliorate heart failure can be assessed using any one or more of the assays above in vivo.
  • a purified poloxamer 188 can be administered to a subject with heart failure, or an appropriate animal model, and the effect on MMP-2 expression can be assessed using a biochemical assay, such as an assay known to those of skill in the art, and MMP-2 levels in, for example, the blood or serum can be compared to subjects or animal models not administered a purified poloxamer 188.
  • a biochemical assay such as an assay known to those of skill in the art
  • MMP-2 levels in, for example, the blood or serum can be compared to subjects or animal models not administered a purified poloxamer 188.
  • administration of a poloxamer 188 such as a purified poloxamer 188, may result in a decrease in MMP-2 levels in blood of a model animal subjected to heart failure.
  • IL-6 Interleukin 6
  • Interleukin-6 is a pro-inflammatory member of the family of cytokine proteins which increase their expression in response to tissue injury or inflammation, for example cardiac injury or inflammation.
  • Blood serum measurement of IL-6 expression using, for example, the enzyme-linked immunosorbent assay (ELISA) can be used to measure IL-6 levels.
  • IL-6 levels increase in heart failure patients and IL-6 expression promotes production of C-reactive protein.
  • the ability of a poloxamer 188, such as a purified poloxamer 188, to exhibit therapeutic activity for reducing or blocking heart dysfunction can be assessed using any one or more of the biomarkers described above.
  • the ability of a poloxamer 188, such as a purified poloxamer 188, to ameliorate heart failure can be assessed using any one or more of the assays above in vivo.
  • a purified poloxamer 188 can be administered to a subject with heart failure, or an appropriate animal model, and the effect on IL-6 expression can be assessed using a biochemical assay, such as an assay known to those of skill in the art, and IL-6 levels in, for example, the blood or serum can be compared to subjects or animal models not administered a purified poloxamer 188.
  • administration of a poloxamer 188, such as a purified poloxamer 188 may result in a decrease in IL-6 levels in blood of a model animal subjected to heart failure.
  • CRP C-reactive protein
  • CRP C-reactive protein
  • Increase in serum CRP is a risk factor for heart failure and myocardial infarction (Kiechl, et al. Circulation, 103, 1064-1070. (2001 )) and a positive prognosis in heart failure patients inversely correlates with CRP levels (Lourneco P. et al. Clin Cardiol. 33(1 1 ):708-14 (2010)).
  • Blood serum measurement of CRP levels can be assayed, for example, using biochemical methods such as ELISA or western blotting.
  • a poloxamer 188 such as a purified poloxamer 188
  • a purified poloxamer 188 can be administered to a subject with heart failure, or an appropriate animal model, and the effect on CRP expression can be assessed using a biochemical assay, such as an assay known to those of skill in the art, and CRP levels in, for example, the blood or serum can be compared to subjects or animal models not administered a purified poloxamer 188.
  • administration of a poloxamer 188, such as a purified poloxamer 188 may result in a decrease in CRP levels in blood of a model animal subjected to heart failure.
  • TNFa Tumor necrosis factor-alpha
  • TNFa is a proinflammatory cytokine involved in the regulation of immune function.
  • TNFa expression is used as a marker for inflammation, including cardiac inflammation; moreover, TNFa is not expressed at detectable levels in normal heart tissue, but increases dramatically in heart failure patients and its expression correlates with the severity of the disease (Feldman et al. J Am Coll Cardiol. 35(3):537-544 (2000)). Therefore, TNFa serves as an effective biomarker for heart failure and a decrease in TNFa expression may indicate an improvement in heart failure progression and symptoms.
  • the ability of a poloxamer 188, such as a purified poloxamer 188, to exhibit therapeutic activity for reducing or blocking heart dysfunction can be assessed using TNFa expression as a biomarker for heart failure.
  • a purified poloxamer 188 can be administered to a subject with heart failure, or an appropriate animal model, and the effect on TNFa expression can be assessed using a biochemical assay, such as an assay known to those of skill in the art, and TNFa levels in, for example, the blood or serum can be compared to subjects or animal models not administered a purified poloxamer 188.
  • a decrease in TNFa levels after administration of poloxamer 188, such as a purified poloxamer 188 may indicate an improvement in heart failure symptoms and a decrease in heart failure progression.
  • nt-pro BNP N-terminal pro-brain natriuretic
  • nt-pro BNP N-terminal pro-brain natriuretic
  • nt-pro BNP N-terminal pro-brain natriuretic
  • a poloxamer 188 such as a purified poloxamer
  • nt-pro BNP expression as a biomarker for heart failure or heart failure severity.
  • a purified poloxamer 188 can be administered to a subject with heart failure, or an appropriate animal model, and the effect on nt-pro BNP expression can be assessed using a biochemical assay, such as an assay known to those of skill in the art, and nt-pro BNP levels in, for example, the blood or serum can be compared to subjects or animal models not administered a purified poloxamer 188.
  • a decrease in nt-pro BNP levels after administration of poloxamer 188, such as a purified poloxamer 188 may indicate an improvement in heart failure symptoms and a decrease in heart failure symptoms or disease progression.
  • poloxamer 188 The activities and properties of poloxamer 188 can be assessed in vivo.
  • Non-human animal models can be used to assess activity, efficacy and safety of poloxamer 188.
  • non-human animals can be used as models for a disease or condition.
  • Animal models can include, but are not limited to, mice, rats, rabbits, dogs, guinea pigs and non-human primate models, such as cynomolgus monkeys or rhesus macaques.
  • Animal models include genetic models as well as induced heart failure models, including acute, chronic, ischemic, stress-induced and other types of heart disease or heart failure that can be induced in non-human animals, such as by multiple sequential intracoronary embolizations with microspheres, prior to administration of a poloxamer 188, such as a purified poloxamer 188, to monitor the effects on acute or chronic heart failure.
  • poloxamer 188 Animal models also can be used to monitor stability, half-life, and clearance of poloxamer 188. Such assays are useful for comparing different forms of poloxamer 188 and for calculating doses and dose regimens for further non-human animal and human trials.
  • poloxamer 188 such as any poloxamer 188 described herein, can be infused into an animal's bloodstream through intravenous
  • mice can be generated which mimic a disease or condition by the overexpression, underexpression or knock- out of one or more genes, such as, for example, dystrophin deficient MDX mice (also known as Dmd mdx mice) that display muscular dystrophy phenotypes, and dobutamine challenged MDX mice. Such animals can be generated by transgenic animal production techniques well-known in the art or using naturally-occurring or induced mutant strains. MDX mice exhibit reduced left ventricular end diastolic volume (LVEDV). LVEDV can be measured upon intravenous infusion of poloxamer 188.
  • LVEDV left ventricular end diastolic volume
  • Poloxamer 188 activity can also be assessed in MDX mice subject to the severe insult of dobutamine treatment, which serves as a model for acute stress, and which typically causes acute heart failure in the mice. Moreover, pre-treatment with poloxamer 188 can be provided to MDX mice challenged with dobutamine to induce heart failure, and heart function can be assessed using a variety of readouts.
  • Additional small animal models of cardiac dysfunction can be used to assess the effect of poloxamer 1 88, such as any poloxamer 188 described herein, on acute, chronic, ischemic or other types of heart failure.
  • an animal model such as a rat can be used. Both permanent and temporary ligation of the left coronary artery can be performed in rats to produce an infarction of greater than 40%. The rats become stable after 1 to 3 weeks, in 3 weeks exhibit significant left ventricular dysfunction, and after 8 weeks exhibit significant loss of dystrophin, which results in heart myopathy.
  • Poloxamer 188 such as any Poloxamer 188 described herein can be administered prior to or following artery ligation and the animals can be assessed for heart function using a variety of assays described above.
  • Poloxamer activity can be assessed in rats with myocardial damage induced by chemical means, ie. administering the beta-one adrenergic receptor agonist isoproterenol, by electrical means, ie. generating overlapping burns to the ventricle epicardium, or surgically, ie. ligation of the left coronary artery.
  • Non-rodent models of heart failure also exist. Poloxamer 188, such as a purified poloxamer 188, activity can be assessed in dogs with progressive
  • GRMD muscular dystrophy
  • DMD Duchenne muscular dystrophy
  • afflicted golden retrievers may be infused with a treatment composition through a vascular access port connected to an indwelling jugular catheter (Townsend et al. J. Clin Invest 120(4): 1 140-50 (2010)).
  • animals may be infused with a treatment composition or a placebo such as saline.
  • Drug or placebo administration may be continual (ie. 24 hours/day), or consist of a single or multiple infusion. The effect of drug administration can be assessed using known assays, such as assays described here previously, for heart function.
  • heart dysfunction include pressure overloading to induce ventricular hypertrophy and failure, produced by a variety of techniques including corticosteroid administration, renal artery occlusion, unilateral nephrectomy with contralateral occlusion of the renal artery, and most extensively banding of major outflow tracts such as the aorta, which have been used in a variety of species including rat, cat and dog (Smith and Nutall,
  • a poloxamer 188 such as a purified poloxamer 188 described herein, can be administered to the dogs prior to, during, or following cardiac injury or dysfunction to assess effects on cardiac function. For example, prior to, and following
  • a poloxamer 188 assays for cardiac function, such as, for example, systolic or diastolic heart function, can be assessed or measured using any of the assays described above.
  • the effects of administering a poloxamer 188 on biomarkers for heart failure can be assessed.
  • a single dose of a poloxamer 188 can be administered to dogs exhibiting advanced heart failure (HF), as defined as a stable (for at least 2 weeks) left ventricular ejection fraction (EF) of ⁇ 30% , produced by multiple sequential intracoronary microembolizations.
  • HF advanced heart failure
  • EF left ventricular ejection fraction
  • dogs can be assessed for long- lasting, significant or moderate improvements to several assays of systolic and diastolic heart functions, including: systolic and diastolic AoP, mean AoP, Peak LV, LV ESV, LV EF, CO, SV, LV FAS Ei/Ai and DCT, when assessed by ultrasound, echocardiographic and/or Doppler studies. Degree of heart failure and improvements to heart function may also be assessed using assays for biomarkers of inflammation, including cardiac inflammation.
  • An improvement in cardiac function may be indicated by an decrease in the serum levels of biomarkers for heart failure, including: N-terminal pro-brain natriuretic peptide (nt-pro BNP), troponin-I (Tn-I), matrix metalloproteinase-2 (MMP-2), Interleukin 6 (IL6), C-reactive protein (CRP) and Tumor necrosis factor-alpha (TNFa).
  • nt-pro BNP N-terminal pro-brain natriuretic peptide
  • Tn-I troponin-I
  • MMP-2 matrix metalloproteinase-2
  • IL6 Interleukin 6
  • CRP C-reactive protein
  • TNFa Tumor necrosis factor-alpha
  • Pharmacokinetic or pharmacodynamic studies can be performed using animal models or can be performed during studies with patients to assess the pharmacokinetic properties of a poloxamer 188, such as a purified poloxamer 188.
  • Animal models include, but are not limited to, mice, rats, rabbits, dogs, guinea pigs and non-human primate models, such as cynomolgus monkeys or rhesus macaques.
  • pharmacokinetic or pharmacodynamic studies are performed using healthy animals.
  • the studies are performed using animal models of a disease or disorder for which therapy with a poloxamer 188 is considered, such as animal models of heart failure, for example animal models of diastolic or systolic heart failure.
  • the pharmacokinetic properties of a poloxamer 188 can be assessed by measuring such parameters as the maximum (peak) concentration (C niax ), the peak time (i.e. when maximum concentration occurs; Tmax), the minimum concentration (i.e. the minimum concentration between doses; C mm ), the elimination half-life (Ti /2 ), and area under the curve (i.e. the area under the curve generated by plotting time versus concentration; AUC), following
  • a range of doses and different dosing frequency of dosing can be administered in the pharmacokinetic studies to assess the effect of increasing or decreasing concentrations poloxamer 188, such as a purified poloxamer 188,
  • Adverse reactions can include, but are not limited to, injection site reactions, such as edema or swelling, headache, fever, fatigue, chills, flushing, dizziness, urticaria, wheezing or chest tightness, nausea, vomiting, rigors, back pain, chest pain, muscle cramps, seizures or convulsions, changes in blood pressure and anaphylactic or severe hypersensitivity responses.
  • injection site reactions such as edema or swelling, headache, fever, fatigue, chills, flushing, dizziness, urticaria, wheezing or chest tightness, nausea, vomiting, rigors, back pain, chest pain, muscle cramps, seizures or convulsions, changes in blood pressure and anaphylactic or severe hypersensitivity responses.
  • a range of doses and different dosing frequencies can be administered in the safety and tolerability studies to assess the effect of increasing or decreasing concentrations of poloxamer 188 in the dose.
  • Heart failure is a chronic, progressive condition in which heart muscle is unable to pump sufficient blood to meet the body's needs.
  • a healthy heart pumps blood continuously through the circulatory system to deliver oxygen- and nutrient-rich blood to the body's cells and enable normal functioning.
  • a variety of diseases and conditions can weaken the heart and reduce its ability to deliver an adequate blood supply.
  • the methods and uses provided herein are for treating subjects that typically exhibit symptom(s) associated with heart failure. Generally, prior to treatment, patients are selected that exhibit one or more signs or symptoms associated with heart failure. It is within the level of a skilled physician to diagnose heart failure.
  • Heart failure most typically exhibit breathlessness, orthopnoea (shortness of breath experienced when lying flat), paroxysmal nocturnal dyspnoea (severe shortness of breath that occurs most often at night), reduced exercise tolerance, fatigue and tiredness, chest pain, palpitations, edema, including ankle and abdomen swelling, and cyanosis.
  • Heart failure can also be associated with one or more other symptoms such as nocturnal cough, wheezing, weight gain, weight loss, bloated feelings, loss of appetite, confusion, depression, palpitations and fainting.
  • a number of such symptoms are subject to quantitative analysis (e.g. palpitations, cyanosis, etc.).
  • Other symptoms include diastolic dysfunction, decreased hemodynamic performance and decreased left ventricular-end diastolic volume.
  • Heart failure is typically classified as either systolic heart failure or diastolic heart failure. Both presentations are common in hypertensive patients, and both are associated with high mortality and morbidity rates. Although diastolic and systolic heart failure share the same clinical phenotype, they differ with respect to the morphological and functional changes that occur in the heart, and represent distinct diseases (Borlaug et al . Circulation 123:2006-2014 (201 1 )). Systolic heart failure presents with a decrease in ejection volume, while diastolic heart failure retains an intact ejection volume; moreover, systolic and diastolic heart failure are most effectively distinguished from each other using internal measurements of heart volume.
  • poloxamer 188 such as a purified poloxamer 188, can be used to increase ejection fraction, LV end-systolic volume, and/or stroke volume in subjects with decreased ejection fraction, LV end-systolic volume, and/or stroke volume associated with systolic heart failure.
  • a poloxamer 188 such as a purified poloxamer 188
  • a poloxamer 188 can be used to treat systolic heart failure of diastolic heart failure.
  • poloxamer 188 is found to have a particular effect on treatment of systolic heart failure.
  • poloxamer 188 such as purified Poloxamer 188
  • Heart failure can be caused by any conditions that reduces the efficiency of the heart to pump blood.
  • exemplary diseases and conditions associated with heart failure include, but are not limited to, ischemic heart disease (IHD; also called coronary heart disease), myocardial infarction, cardiomyopathy, high blood pressure, hypertensive heart disease, diseases of the heart valves, diseases of the pericardium, arrhythmias (irregular heartbeats), endocarditis, myocarditis, cerebrovascular disease, peripheral arterial disease, congenital heart disease, and rheumatic heart disease.
  • IHD ischemic heart disease
  • myocardial infarction also called coronary heart disease
  • cardiomyopathy high blood pressure
  • hypertensive heart disease diseases of the heart valves
  • diseases of the pericardium diseases of the pericardium
  • arrhythmias irregular heartbeats
  • endocarditis myocarditis
  • cerebrovascular disease cerebrovascular disease
  • peripheral arterial disease congenital heart disease
  • Ischemic heart disease coronary artery disease
  • a poloxamer 188 such as a purified poloxamer 188
  • Ischemic heart disease also termed coronary heart disease, is the most common form of heart disease and accounts for 80% of cases, or hypertension, often precluding systolic dysfunction and other clinical symptoms.
  • ischemic heart disease is attributable to the buildup of plaque, a heterogeneous material made up of macrophages, lipids, such as cholesterol and fatty acids, calcium and other products, which accumulate on the inner walls of arteries, which results in reduced blood flow and increased blood pressure.
  • plaque a heterogeneous material made up of macrophages, lipids, such as cholesterol and fatty acids, calcium and other products, which accumulate on the inner walls of arteries, which results in reduced blood flow and increased blood pressure.
  • poloxamer 188 such as a purified poloxamer 188
  • Subjects or patients with ischemic heart disease can be administered a poloxamer 188, such as a purified poloxamer 188.
  • the methods and uses provided herein are for treating subjects that typically exhibit symptom(s) associated with ischemic heart disease.
  • the methods herein can be used to treat patients with chronic or acute ischemic heart disease.
  • ischemic heart disease Prior to treatment, patients are selected that exhibit one or more signs or symptoms associated with ischemic heart disease. It is within the level of a skilled physician to diagnose ischemic heart disease. Subjects that have ischemic heart disease, including chronic or acute ischemic heart disease, generally exhibit decreased blood supply which slows ventricular relaxation and can impair
  • Ischemic heart disease also can be associated with one or more other symptoms such as chest pain (angina), shortness of breath, increased pro-inflammatory cytokines, and in cases of an acute 'heart attack' severe chest pressure and pain in the shoulder or arm.
  • Selection of a subject having ischemic heart failure for treatment with a poloxamer 188, such as a purified poloxamer 188, in the methods provided herein can be based on clinical symptoms, hemodynamic and ventriculographic measurements, or levels of proinflammatory biomarkers for ischemic heart failure, for example in the blood.
  • a poloxamer 188 such as a purified poloxamer 188
  • methods of using a poloxamer 188 for treating, ameliorating or reducing ischemic heart failure induced by the reduced blood supply to the heart.
  • Subjects or patients with ischemic heart disease can be administered a poloxamer 188, such as a purified poloxamer 188.
  • a method of treating or ameliorating heart failure caused by myocardial infarction by administering a poloxamer 188, such as a purified poloxamer 188, to a subject.
  • Myocardial infarction also termed heart attack, is the acute, secondary effect of prolonged ischemia, or lack of blood flow, to the heart, and presents as an irreversible necrosis, or cell death, of heart tissue.
  • a poloxamer 188 such as a purified poloxamer 188
  • Subjected or patients with myocardial infarction can be administered a poloxamer 1 88, such as a purified poloxamer 1 88.
  • the methods and uses provided herein are for treating subjects that typically exhibit symptom(s) associated with myocardial infarction.
  • patients are selected that exhibit one or more signs or symptoms associated with myocardial infarction. It is within the level of a skilled physician to myocardial infarction.
  • Subjects that have myocardial infarction generally exhibit an episode of angina, or chest pain, and may experience jaw pain, toothache, shortness of breath, nausea, vomiting, sweating, heartburn and/or indigestion, arm pain, upper back pain, and general malaise.
  • Myocardial infarction commonly precipitates acute decompensated heart failure and may also be present in patients experiencing chronic heart failure.
  • Selection of a subject having myocardial infarction for treatment with a poloxamer 188, such as a purified poloxamer 188, in the methods provided herein may be based on the clinical symptoms listed above, the results of an
  • a poloxamer 188 such as a purified poloxamer 188
  • subjects or patients with myocardial infarction can be administered a poloxamer 188, such as a purified poloxamer 188.
  • a method of treating or ameliorating heart failure caused by hypertension, or high blood pressure by administering a poloxamer 188, such as a purified poloxamer 188, to a subject.
  • a poloxamer 188 such as a purified poloxamer 188
  • Hypertension or high blood pressure
  • hypertension is characterized by an increase of blood pressure on the artery walls. Due to this increased pressure, hypertension results in remodeling of the cardiac and vascular tissue in an attempt to normalize the stress on the heart and arterial walls, which can impact proper heart functioning.
  • Hypertension is a risk factor for hypertensive heart disease and coronary artery disease due to the strain on the heart muscle from the increased blood pressure, and is a probable contributing factor to the majority of cases of systolic heart failure and in at least 25% of the incidence of diastolic heart failure.
  • Provided herein are methods of using a poloxamer 188, such as a purified poloxamer 188, for treating, ameliorating or reducing hypertension
  • Poloxamer 188 such as a purified Poloxamer 188.
  • the methods and uses provided herein are for treating subjects that typically exhibit symptom(s) associated with hypertension.
  • the methods herein can be used to treat patients with chronic or acute hypertension.
  • patients are selected that exhibit one or more signs or symptoms associated with hypertension. It is within the level of a skilled physician to diagnose
  • hypertension The most significant indicator of hypertension is the blood pressure measurement when assessed using a sphygmomanometer, typically used in conjunction with a stethoscope.
  • Selection of a subject having hypertension failure for treatment with a poloxamer 188, such as a purified poloxamer 188, in the methods provided herein can be based on arterial pressure as assessed using a sphygmomanometer, clinical symptoms, hemodynamic and ventriculographic measurements, or levels of proinflammatory biomarkers for hypertension, for example in the blood.
  • a poloxamer 188 such as a purified poloxamer 188
  • methods of using a poloxamer 188 for treating, ameliorating or reducing hypertension induced by increased blood volume or narrowing of the blood vessels, which increase pressure on blood vessel walls.
  • Subjects or patients with hypertension can be administered a poloxamer 188, such as a purified poloxamer 188.
  • Selection of a subject having heart failure for treatment with poloxamer 188, such as a purified poloxamer 188, in the methods provided herein can be based on clinical symptoms, such as breathlessness, orthopnoea (shortness of breath
  • natriuretic peptides including B-type Natriuretic peptide (BNP) and N- terminal pro B-type natriuretic peptide (nt-proBNP) (Krishnaswamy et al.
  • Selection of a subject having heart failure for treatment with poloxamer 188, such as a purified poloxamer 188, in the methods provided herein can be based on hemodynamic and ventriculographic measurements, including aortic and LV pressures , peak rate of change of LV pressure during isovolumic contraction and relaxation, LV end-diastolic pressure, cardiac output (CO), LV stroke volume (SV), systemic vascular resistance (SVR), LV end-systolic (ESV) and end-diastolic (EDV) volumes, and LV ejection fraction (EF).
  • Hemodynamic and ventriculographic measurements can be assessed or calculated using standard methods (Sabbah et al. ( 1991 ) Am, J. Physiol. 260 (Heart Circ. Physiol. 20): H 1379-H 1384; Zaca et al.
  • Selection of a subject having heart failure for treatment with poloxamer 188, such as a purified poloxamer 188, in the methods provided herein can be assessments using on echocardiograms and Doppler echocardiography, and include LV fractional area of shortening (FAS), which is a measure of LV systolic function, LV thickness, which can be used to calculate cardiac wall stress, mitral inflow velocity, which can be used to calculate peak mitral flow velocity in early diastole (PE), peak mitral inflow velocity during left atrial (LA) contraction (PA), the ratio of PE to PA, the time-velocity integral of the mitral inflow velocity waveform representing early filling (Ai), the time- velocity integral representing LA contraction (Ai), the ratio of Ei/Ai, and deceleration time (DCT) of early mitral inflow velocity.
  • Echocardiographic and doppler measurements can be assessed or calculated using standard methods (Sabbah et al. (2007) Am. J. Cardiol., 99:41 A
  • Selection of a subject having heart failure for treatment with poloxamer 188, such as a purified poloxamer 1 88, in the methods provided herein can be based on assessments of subjects at risk for heart disease, including subjects with comorbidities that increase the incidence of heart disease such as metabolic syndrome (ie. hypertension, dyslipidemia, obesity and diabetes), increased age, those with a genetic predisposition for the disease, such as those with heritable disorders, and those susceptible to environmental factors.
  • metabolic syndrome ie. hypertension, dyslipidemia, obesity and diabetes
  • a genetic predisposition for the disease such as those with heritable disorders, and those susceptible to environmental factors.
  • left ventricular diastolic dysfunction may represent the first stage of diabetic cardiomyopathy (Raev, Diabetes Care 17:633-639 ( 1994)).
  • harvesting of peripheral venous blood samples are generally performed prior to, during, or following treatment of the subject with a poloxamer 188, such as a purified poloxamer 188.
  • harvesting of the peripheral venous blood samples from the subject can be performed before, during or after the subject has received one or more treatments with a poloxamer 188, such as a purified poloxamer 188.
  • Selection of a subject having heart failure for treatment with poloxamer 1 88, such as a purified poloxamer 188, in the methods provided herein can be based on the presence of biomarkers in venous blood samples, such as, for example, blood serum.
  • biomarkers for heart failure include, but are not limited to, N-terminal pro-brain natriuretic (nt-pro BNP), troponin-I (Tn-I), matrix metalloproteinase-2 (MMP-2), MMP-9, Interleukin 6 (IL6), C-reactive protein (CRP), TNF-alpha (TNFa), measures of oxidative stress, including isoprostane, derivatives of reactive oxygen metabolites, IPGF2, bilirubin, and 8-OHdG. Selection of subjects may be based on presentation with a change in biomarker protein expression from normal levels, including an increase or a decrease. For example, a patient with increased Tn-I expression indicates that the patient has experienced cardiomyocyte injury and death, and thus, may have a more severe form of heart failure.
  • nt-pro BNP N-terminal pro-brain natriuretic
  • Tn-I troponin-I
  • MMP-2 matrix metalloproteinase-2
  • Assays for use in the methods provided herein are those in which a biomarker of heart dysfunction, or heart failure, present in the sample is detected using an antibody sandwich enzyme-linked immunosorbent assay (ELISA).
  • ELISA is a biochemical experimental method based on enzymatic reactions using a binding partner, such as an antibody (e.g. monoclonal or polyclonal antibodies) or other binding partner, to detect the plasma levels of specific proteins such as inflammatory proteins, or biomarkers, for example, troponin-I (Tn-1).
  • ELISA based methods can be used for quantitative or semi-quantitative detection of the amount of troponin-I that binds to a troponin-I antibody in a sample, such as a fluid sample from a subject having heart failure or suspected of having heart failure.
  • a sample such as a fluid sample from a subject having heart failure or suspected of having heart failure.
  • the use of solid phase binding assays can be used when troponin-I is detected in a bodily fluid.
  • ELISA assays for use in the methods herein include those where an anti-troponin-I antibody is used as a binding partner to detect troponin-I in blood plasma.
  • ELISA protocols include detection systems that make the presence of the markers visible, to either the human eye or an automated scanning system, for qualitative or quantitative analyses.
  • Biomarkers for heart failure include, but are not limited to, 8-iso-prostaglandin F2 alpha (IPGF2), which is a chemically stable and quantitative measure of oxidative stress, N-terminal pro-BNP (N-BNP), collagen alphal (I and III) (Zimmerli et al. 7:290-298 (2008)) and are well known to those of skill in the art. Selection of subjects may be based on presentation with a change in biomarker protein expression from normal levels, including an increase or a decrease.
  • IPGF2 8-iso-prostaglandin F2 alpha
  • Systolic dysfunction also known as heart failure with reduced (left ventricular) ejection fraction (HFREF) develops from the interactions between genetic factors and accumulated cardiac insults.
  • the selection of a subject having systolic heart failure for treatment with poloxamer 188, such as a purified poloxamer 188, in the methods provided herein can be based on echocaridographic evidence of depressed left ventricular systolic function.
  • systolic dysfunction may be assessed in subjects who present with eccentric hypertrophy, which is a disproportionate increase in ventricle volume coupled with little increase in wall thickness that can result in both volume and pressure overload. Such subjects can be selected based on alterations to LV end systolic volume and/or LV end-systolic pressure.
  • the selection of a subject having subtypes of non-ischemic systolic heart failure for treatment with poloxamer 188, such as a purified poloxamer 188, in the methods provided herein can be based on a collection of clinical features that reflect their underlying pathophysiology, including, but not limited to, their clinical course and rate of response to treatments.
  • the selection of a subject having systolic heart failure for treatment with poloxamer 188, such as a purified poloxamer 188, in the methods provided herein can be based on presence of biomarkers in venous blood samples, such as, for example, blood serum or urinary excretions.
  • biomarkers for systolic heart failure include, but are not limited to, 8-iso-prostaglandin F2 alpha (IPGF2), which is known to correlate with the severity of systolic heart failure and is inversely correlated with ejection fraction.
  • an exemplary biomarker to ischemic systolic heart failure is 8-hydroxy-2'-deoxyguanosine (8-OHdG), a marker of systemic oxidatively generated DNA damage, whose level in urine correlates with the severity of ischemic systolic heart failure as assayed by the number of diseased vessels visualized on coronary angiography (Nagayoshi et al.. Free Radic Res
  • Selection of subjects may be based on presentation with a change in biomarker protein expression from normal levels, including an increase or a decrease.
  • the selection of a subject having systolic heart failure for treatment with poloxamer 188, such as a purified poloxamer 188, in the methods provided herein can be based on changes to hemodynamic, ventriculographic and doppler- echocardiographic readings including, but not limited to systolic aortic pressure, mean aortic pressure, peak rate of change of LV pressure during isovolumic contraction, left ventricular ejection fraction, left ventricular end- systolic volume, cardiac output, stroke volume, fractional area of shortening, and the ratio the ratio of integral of mitral inflow velocity in early diastole (Ei) to integral of mitral inflow velocity during left atrial contraction (Ai) (Ei/Ai).
  • Diastolic dysfunction refers generally to a condition in which abnormalities in mechanical function are present during diastole, and which can occur in the presence or absence of heart failure. Diastolic dysfunction presents as concentric hypertrophy where, although there is no change or a slight decrease in the radius of the ventricular chamber, the walls of the heart thicken and are, thus, capable of generating increased pressure with greater force. The heart muscle subsequently becomes 'stiff, which can impair ventricle filling. Diastolic dysfunction is most commonly a chronic condition and may be well tolerated by the affected individual.
  • Diastolic dysfunction is most often diagnosed in asymptomatic patients using Doppler echocardiography.
  • the selection of a subject having diastolic heart failure for treatment with a poloxamer 188, such as a purified poloxamer 188, in the methods provided herein can be based on a normal or preserved left ventricular (LV) end diastolic volume, normal ejection fraction, delayed active relaxation, and increased passive stiffness of the left ventricle.
  • LV left ventricular
  • the selection of a subject having diastolic heart failure can be characterized by dilated cardiomyopathy in which the ventricles are dilated, resulting in thinner muscle, reduced contractility and failure of the ventricle to fill properly. Furthermore, this diastolic dysfunction can co-exist both with or without
  • the selection of a subject having diastolic heart failure for treatment with poloxamer 188, such as a purified poloxamer 188, in the methods provided herein can be based on the expression of biomarkers for heart failure or heart dysfunction.
  • biomarkers for heart failure or heart dysfunction For example, interleukin- 16 (IL- 16) levels can be used to determine the type of heart failure. IL- 16 levels were specifically elevated in diastolic heart dysfunction, compared to systolic dysfunction and controls, in a rat model of heart failure and in human patients (Tamaki et al. PloS ONE 8(7):e68893 (2013)) and IL-16 levels positively correlated with LVEDP.
  • IL-16 interleukin- 16
  • the selection of a subject having diastolic heart failure can be based on the elevated IL-16 in the presence of elevated LVEDP.
  • the selection of a subject having diastolic heart failure can be based on the elevated BNP in the blood plasma in the presence of normal ejection fraction.
  • systolic dysfunction Like all heart failure patients, patients with systolic dysfunction are heterogenous with respect to etiology, prognosis, and response to therapy, and the ability to identify patients likely to respond to medical therapy remains limited. However, several characteristics distinguish systolic dysfunction and diastolic dysfunction patients from each other.
  • the selection of a subject having diastolic heart failure for treatment with Poloxamer 188, such as a purified Poloxamer 188, in the methods provided herein can be based on demographic characteristics, for example, patients with diastolic dysfunction are more likely to be women, older, less likely to have ischemia and more likely to have comorbid systolic hypertension (Little and Zile. Circulation 5:669-671 (2012)).
  • selection of patients with diastolic dysfunction can be based on hemodynamic, ventriculographic and doppler- echocardiographic readings.
  • the selection of a subject having diastolic heart failure for treatment with poloxamer 188, such as a purified poloxamer 188 can be based on changes to hemodynamic, ventriculographic and doppler-echocardiographic readings including, but not limited to mean aortic pressure, peak rate of change of LV pressure during isovolumic relaxation, left ventricular ejection fraction, left ventricular end- diastolic volume, cardiac output, stroke volume, fractional area of shortening, and the ratio the ratio of integral of mitral inflow velocity in early diastole (Ei) to integral of mitral inflow velocity during left atrial contraction (Ai) (Ei/Ai).
  • the poloxamer 188 may reduce, lessen or ameliorate heart dysfunction, and thereby also can prevent or ameliorate diseases and conditions associated with heart disease, including, but not limited to, diastolic and/or systolic dysfunction, ischemic heart failure, myocardial infarction, and hypertension.
  • Heart function of the subject can be monitored over time to assess whether a decrease in cardiac failure has been achieved over the course of therapy with a poloxamer 188, such as a purified poloxamer 188, provided herein.
  • Poloxamer 188 such as any Poloxamer 188 described herein, may be administered in combination with therapeutics previously utilized to treat heart failure, in order to improve the efficacy of the Poloxamer 188 compound on its own.
  • treatments include, but are not limited to, methods of treatment of physiological and medical conditions described and listed herewith.
  • compositions provided herein can be further co-formulated or co-administered together with, prior to, intermittently with, or subsequent to, other therapeutic or pharmacologic agents or treatments, such as treatments where normalized or improved left ventricular end-diastolic or end-systolic pressure and volume are desired.
  • Poloxamer 1 88 such as any Poloxamer 188 described herein, can be used in the treatments of heart failure, for example chronic, acute or ischemic heart failure. Poloxamer 188 is particularly well suited for treating patients with heart failure, including but not limited to systolic heart failure because of its ability to impart long-lasting, significant improvements to a variety of measures of systolic heart failure including.
  • Poloxamer 188 such as any Poloxamer 188 described herein, also can be used to treat heart failure related to a primary disease state such as, for example duchenne muscular dystrophy.
  • a preparation of a second agent or agents or treatment or treatments can be administered at once, or can be divided into a number of smaller doses to be administered at intervals of time.
  • Selected agent / treatment preparations can be administered in one or more doses over the course of a treatment time for example over several hours, days, weeks, or months. In some cases, continuous administration is useful. It is understood that the precise dosage and course of administration depends on the indication and patient's tolerability. Generally, dosing regimes for second agents/treatments herein are known to one of skill in the art.
  • Poloxamer 188 such as a purified poloxamer 188 described herein, can also be used in conjunction with currently available therapeutics, including, but not limited to: a diuretic, loop diuretic, a potassium sparing agent, a vasodilator, an ACE inhibitor, ARBs (angiotensin receptor blockers), an angiotensin II antagonist, Aldosterone antagonist, a positive inotrophic agent, a phosphodiesterase inhibitor, a beta-adrenergic receptor antagonist, a calcium channel blocker, a nitrate, an alpha blocker, a central alpha antagonist, a statin, Digoxin, Nitrates, chlorthalidone, amiodipine, lisinopril, doxazosin, or a combination of these agents.
  • poloxamer 188 such as a purified poloxamer 188 described herein, also can be used in conjunction with mechanical devices, including: implantable pacemakers, defibrillators, and left ventricular assist devices (LVAD). With the possible exception of the LVAD, when used individually, these therapies prolong life, but do not stop, or reverse, disease progression and deterioration of heart function.
  • mechanical devices including: implantable pacemakers, defibrillators, and left ventricular assist devices (LVAD).
  • LVAD left ventricular assist devices
  • a multi-step extraction batch process of poloxamer 188 was performed with extraction conducted at a pressure of 247 ⁇ 15 atm (approximately 250 bar) and a controlled step-wise increase of methanol of 7.4, 9.1 and 10.7 weight % methanol.
  • the poloxamer 188 raw material BASF Corporation,
  • polydispersity was no more than 1.2.
  • a 50-L, high pressure, stainless steel, extractor vessel was charged with 14 kg of commercial grade poloxamer 188 (BASF Corporation, Washington, New Jersey) and 7 kg of methanol, pressurized with C0 2 (49 ⁇ 10 atm, i.e. 720 ⁇ 147 psi) (Messer France, S.A.S., Lavera, France) and heated to 40 °C for 40-80 minutes until a homogenous solution was obtained.
  • C0 2 supplied either from a main supply tank or via recycling through an extraction system
  • a high- pressure pump increased the pressure of liquid C0 2 to the desired extraction pressure.
  • the high pressure C0 2 stream was heated to the process temperature by a second heat exchanger.
  • Methanol Merck KGaA, Darmstadt, Germany
  • Methanol was fed from a main supply tank into the C0 2 solvent stream to produce the extraction methanol/C0 2 cosolvent, which was fed through inlet systems into the extractor vessel as a fine mist at a pressure of 247 ⁇ 15 atm (3600 ⁇ psi) and a temperature of 40 °C.
  • a 7.4% methanol/C0 2 extraction cosolvent was percolated through the poloxamer solution for 3 hours at a methanol flow rate of 8 kg/hr (108 kg/hr total flow rate).
  • the extraction continued with a 9.1% methanol/C0 2 cosolvent for 4 more hours at a methanol flow rate of 10 kg/ hour (110 kg/hr total flow rate).
  • the extraction further continued with a 10.7% methanol/C0 2 cosolvent for 8 more hours at a methanol flow rate of 12 kg per hour (112 kg/hr total flow rate).
  • extraction of soluble species were continuously extracted from the top of the extractor.
  • the extraction solvent was removed from the top of the extractor and passed through two high pressure, stainless steel, cyclone separators arranged in series to reduce system pressure from 247 atm (3600 psi) to 59 atm (870 psi) and then from 59 atm to 49 atm (720 psi) and to separate C0 2 from the methanolic stream.
  • the separated C0 2 was condensed, passed through the heat exchanger and stored in the solvent reservoir. Pressure of the methanol waste stream was further reduced by passing through another cyclone separator.
  • the purified poloxamer 188 remained in the extractor.
  • the purified poloxamer 188 solution was discharged from the bottom of the extractor into a mixer/dryer unit equipped with a stirrer.
  • the poloxamer 188 product was precipitated under reduced pressure via a Particle from Gas Saturated Solutions (PGSS) technique.
  • the precipitate contained approximately 25% to 35% methanol.
  • the purified poloxamer 188 was dried under vacuum at not more than 40°C to remove residual methanol.
  • the feed yield of the product gave an average yield of 65%.
  • the resulting Poloxamer 188 was a clear, colorless, sterile, non-pyrogenic, aqueous solution in 100 mL glass vials containing 15 g of purified poloxamer drug substance (150 mg/mL).
  • the composition contained 0.01 M citrate buffer and sodium chloride to adjust the total sodium content to be equivalent to that in 0.45% sodium chloride solution for injection.
  • the resulting osmolarity of the solution was approximately 312 mOsm/L.
  • the LCMF poloxamer-188 composition did not contain any bacteriostatic agents or preservatives.
  • Purified poloxamer 188 is reported to result in two distinct peaks in the circulation that exhibit different pharmacokinetic profiles, a main peak with an average peak molecular weight of 8,600 daltons and a smaller high molecular weight (HMW) peak with an average molecular weight of about 16,000 daltons (Grindel et al. (2002) Biopharmaceutics and Drug Disposition, 23:87-103). As reported by
  • the higher molecular weight peak exhibits a longer plasma residence time with slower clearance from the circulation such that it is cleared at
  • Purified poloxamer 188 generated as described above was administered intravenously to healthy human volunteers.
  • the purified poloxamer 188 was administered as a loading dose of 100 mg/kg/hr for one hour followed by a maintenance dose of 30 mg/kg/hr for 5 hours.
  • Plasma was collected at various time points and the plasma concentration of poloxamer 188 was determined using HPLC- GPC.
  • the results are set forth in Figure 7. Consistent with reported studies regarding the half-life of the main peak, the results demonstrate a mean maximum concentration (Cmax) of the administered purified poloxamer 188 of 0.9 mg/mL was attained by the end of the one hour loading infusion.
  • the chromatogram is enlarged to show the high molecular weight portion (19.8 K daltons - 12.4 daltons) of the polymeric distribution. Also shown are the main peak portion (12.8 - 4.7 K Daltons) and the lower molecular weight portion (4.7 - 2.5 K Da).
  • the HPLC-GPC method quantifies plasma levels based on the height of the eluting peak relative to standards of known concentration (i.e. the higher the eluting peak, the higher the plasma level).
  • the GPC method also identifies the molecular weight range by comparison of the sample elution time to that of standards of known molecular weight.
  • the chromatograms show that over time the high molecular weight portion of the poloxamer 188 polymeric distribution declines in relative proportion to the main peak and lower molecular weight components. Thus, the polymeric distribution shows a substantially uniform pharmacokinetic profile. Thus, the results show that the higher molecular weight species do not exhibit a longer circulating half-life (relative to the other polymeric components) and do not accumulate in the circulation following intravenous administration. Thus, the poloxamer 188 is designated longer circulating material free (LCMF) poloxamer 188.
  • LCMF circulating material free
  • HF left ventricular ejection fraction
  • EF left ventricular ejection fraction
  • Dogs treated as described in Example 2 were evaluated for left ventricular (LV) systolic and diastolic cardiac function and other heart functions to assess the effect of a single infusion of low dose and high dose of the poloxamer product for treating advanced heart failure.
  • LV left ventricular
  • hemodynamic, ventriculographic, echocardiographic, and electrocardiographic measurements were made at baseline prior to drug administration, and at the end of 2 hours of drug infusion. Measurements also were repeated 24 hours, 48 hours, 1 week and 2 weeks after completion of the 2 hours of drug infusion. All measurements were performed under general anesthesia and sterile conditions and all dogs fasted for 12 hours prior to cardiac catheterization. Induction of anesthesia was initiated with IV hydromorphone (0.22 mg/kg) and diazepam (0.17 mg/kg) and plane of anesthesia was maintained with 1 - 1 .5% isofluorane.
  • LV end-systolic (ESV) and EDV) volumes were calculated from angiographic silhouettes using the area length method (Dodge et al. (1966) The American Journal of Cardiology, 1 8: 10-24).
  • LV ejection fraction was calculated as the ratio of the difference of end-diastolic and end-systolic volumes to end-diastolic volume times 100.
  • Echocardiographic and Doppler studies were performed in all dogs at all specified study time points using a VIVID 7 ultrasound system (General Electric) with a 3.5 MHz transducer. All echocardiographic measurements were made with the dog placed on its right side and recorded on digital media for subsequent off-line analysis.
  • LV fractional area of shortening (FAS) a measure of LV systolic function, was measured from a short axis view at the level of the papillary muscles.
  • LV thickness of the posterior wall and interventricular septum were measured, summed, and divided by 2 to obtain average LV wall thickness (h), used for calculation of wall stress.
  • LV major and minor semi axes were measured and used for calculation of LV end-diastolic circumferential wall stress (EDWS; Sabbah et al. (2007) The American Journal of Cardiology, 99:41A-46A)). Wall stress was calculated as follows:
  • P is LV end-diastolic pressure
  • a is LV major semiaxis
  • b is LV minor semiaxis
  • h is LV wall thickness
  • Mitral inflow velocity was measured by pulsed-wave Doppler
  • the velocity waveforms were used to calculate 1) peak mitral flow velocity in early diastole (PE), 2) peak mitral inflow velocity during left atrial (LA) contraction (PA), 3) ratio of PE to PA, 4) time- velocity integral of the mitral inflow velocity waveform representing early filling
  • Electrocardiogram Lead-II of the electrocardiogram was monitored throughout the study and recorded at all specified study time points. If de-no vo ventricular arrhythmias were to develop at any time during the study, the electrocardiogram would be recorded continuously. If at any time arrhythmias developed and were associated with hemodynamic compromise, drug infusion would be stopped and the study terminated for that day.
  • the change ( ⁇ ) in each measurement among study groups (intragroup) was assessed using one way ANOVA with alpha set at p ⁇ 0.05. If significance was attained, comparisons between the Group III (control group) and each of the two active treatment groups, Group II (low dose LCMF Poloxamer-188) and Group I (high dose LCMF Poloxamer-188) animals, were made using the Student-Newman-Keuls Test with significance set at p ⁇ 0.05. All data are reported as the mean ⁇ standard error of the mean (SEM).
  • Tables 1-3 The results of the hemodynamic, ventriculographic, and Doppler- echocardiographic results in each of the treatment groups are shown in Tables 1-3, and summarized below.
  • the Tables set forth measured values for the following assessed variables: systolic and mean aortic pressure (AoP); left ventricular end- diastolic pressure (LV EDP); peak rate of change of LV pressure during isovolumic contraction (peak +dP/dt) and relaxation (peak -dP/dt); LV end-diastolic volume (LV EDV); LV endsystolic volume (LV ESV); LV ejection fraction (LV EF); cardiac output (CO); stroke volume (SV); systemic vascular resistance (SVR); fractional area of shortening (FAS); ratio of peak mitral inflow velocity in early diastole (PE) to peak mitral inflow velocity during left atrial contraction (PA) (PE/PA); ratio of integral of mitral inflow velocity in early diastole (E
  • Hemodynamic, ventriculographic, and Doppler-echocardiographic results in control dogs are shown in Table 1.
  • systolic aortic pressure mean aortic pressure or LV end-diastolic pressure. Both systolic and mean aortic pressures tended to increase at 1 week, but the increase did not reach statistical significance. Peak LV + dP/dt and LV -dP/dt increased at 1 week. This increase was most likely driven by an increase in aortic blood pressure also seen at 1 week.
  • EDV tended to increase but the change did not reach statistical significance.
  • LV ESV also tended to increase during the course of 2 weeks. The increase reached statistical significant at the 24-hours, 1 -week and 2-week time points compared to pre-treatment (Table 1 ).
  • EDV decreased at all study time points but the change did not reach statistical significance.
  • ESV decreased during the follow-up period.
  • the decrease compared to pre-treatment was significant at 2 hours, 24 hours and 1 week.
  • LV EF increased during the follow-up period reaching statistical significance at 2 hours, 24 hours and 1 week post treatment (Table 2).
  • High Dose LCMF poloxamer- 188 increased CO and SV at all study time points compared to pre-treatment and the increase reached statistical significance at 2 hours and 1 week for SV, and at 1 week for CO.
  • FAS increased significantly at all study time points compared to pre- treatment with the exception of 2 weeks.
  • Indexes of LV diastolic function improved modestly for up to 1 week post treatment.
  • the ratio Ei/Ai increased significantly at 2 hours post treatment and DCT increased significantly at 2 hours, 24 hours and 1 week post treatment.
  • EDV tended to decrease at all study time points and reached significance at 24 hours post treatment.
  • ESV tended to decrease during the follow-up period.
  • the decrease compared to pre-treatment was significant at 2 hours, 24 hours and 1 week.
  • LV EF increased during the follow-up period reaching significance at 2 hours, 24 hours and 1 week post treatment (Table 3).
  • Systolic AoP 90 ⁇ 1.1 99 ⁇ 4.5 95 ⁇ 3.6 105 ⁇ 4.3* 100 ⁇ 3.9
  • LV EDWS (g/cm2) 59 ⁇ 3 61 ⁇ 5 61 ⁇ 5 60 ⁇ 5 62 ⁇ 4
  • the treatment effect ( ⁇ ) for each measured variable at various time points after infusion compared to the pretreatment value for each of the study groups are set forth in Table 4.
  • the Table sets forth the difference in the assessed parameter between baseline and 2 hours ( ⁇ 2 hrs Post), baseline and 24 hours ( ⁇ 24 hrs Post), baseline and 1 week ( ⁇ 1 Wk Post) and baseline and 2 weeks ( ⁇ 2 Wks Post).
  • the results show that treatment with both low and high dose of the LCMF poloxamer-188 improved LV systolic and diastolic functions compared to the control group treated with normal saline.
  • low dose LCMF poloxamer-188 increased Ei/Ai and DCT at 2 hours with improvements lasting for at least 1 week post drug administration.
  • Low dose LCMF poloxamer-188 tended to decrease EDV but the changes were not significant.
  • ES V decreased significantly at 2 hours and the significant decrease persisted for 2 weeks.
  • EF, FAS, SV and CO all increased significantly at 2 hours and remained elevated for at least 1 week post drug administration.
  • High dose LCMF poloxamer-188 increased Ei/Ai and DCT significantly at 2 hours and 24 hours, with improvements lasting for at least 1 week post drug
  • High dose LCMF poloxamer-188 tended to decrease EDV but the changes were not significant compared to control. In contrast, ESV decreased significantly at 2 hours and the significant decrease persisted for 2 weeks. EF, FAS, SV and CO all increased significantly at 2 hours and remained elevated for at least 1 week post drug administration. Like low dose LCMF poloxamer-188, high dose LCMF poloxamer-188 also had no effect on heart rate, aortic pressure, LV end- diastolic pressure, LV + dP/dt and LV -dP/dt, and end-diastolic wall stress at all time points compared to the control group.
  • Table 4 Treatment Effect ( ⁇ ) of Control, Low Dose LCMF Poloxamer-188 (225 mg/kg), and High Dose LCMF Poloxamer-188 (450 mg/kg) in Dogs
  • treatment with low dose or high dose poloxamer-188 resulted in reduced ESV, and increased EF, CO, SV and FAS.
  • the results also show a more moderate effect of treatment on LV diastolic function, for example, as evidenced by increased
  • Heart rate was essentially unchanged during each of the study time points and, therefore, the improvements in LV function could not be attributed to changes in the chronotropic state.
  • Administration of the single infusion of a poloxamer- 188 had minimal or no effects on LV end-diastolic pressure and end-diastolic volume.
  • Tnl is an intracellular protein that is released from cardiomyocytes (heart muscle cells) following injury to and/or death of these cells, and thus is a biomarker of cardiomyocyte injury and death.
  • cardiomyocytes heart muscle cells
  • nt- pro BNP N-terminal pro-brain natriuretic peptide
  • NT-pro BNP is released from the heart during periods of increased
  • nt-pro BNP cardiac wall stress, typically as a result of the increased fluid volumes that are common in heart failure.
  • higher levels of nt-pro BNP correlates to poor prognosis and increased mortality.
  • Peripheral venous blood samples were obtained at baseline, at the end of 2 hours of drug infusion and at 24 hours, 1 week and 2 weeks after drug infusion to assess Tnl and nt-pro BNP.
  • the blood samples were centrifuged at 3000 rpm for 10 minutes and plasma withdrawn and placed in cryo-storage tubes and stored upright at -70°C until needed.
  • Plasma samples from 6 normal dogs (same breed, age and weight as study dogs) were also obtained and stored for comparison.
  • Plasma levels of Tnl and nt-pro BNP were determined by an antibody sandwich enzyme-linked immunosorbent assay (ELISA) using commercially available kits for Tnl (ALPCO Diagnostics, Salem, NH) or for nt-pro BNP (Kamiya Biomedical Company; Cat# KT -23770). Concentrations were determined from standard curves and expressed as ng/mL (Tnl) and pg/mL (nt-pro BNP). Data analysis of Tnl and nt-pro BNP levels, and determination of statistical significance, was performed as described in Example 3.
  • ELISA antibody sandwich enzyme-linked immunosorbent assay
  • the pre-treatment levels of plasma Tnl were significantly elevated in the dogs with induced advanced heart failure compared to levels found in normal dogs.
  • concentration of Tnl in normal dogs was on average about 0.1 - 0.2 ng/mL, but was elevated to about 0.4 ng/mL in dogs with induced heart failure.
  • the treatment effect ( ⁇ ), i.e. difference between pre-treatment values and subsequent values obtained at 2 hours, 24 hours, 1 week and 2 weeks, for the treated Groups also is depicted in Table 6.
  • plasma Tnl levels were unchanged at 2 hours but tended to decrease at 24 hours and the reduction reached statistical significance at 1 week and 2 weeks for low dose treatment, and at 24 hours, 1 week and 2 weeks after treatment with high dose.
  • results show that a single infusion of a poloxamer-188 resulted in statistically significant and progressive reductions in Tnl, at one week and two weeks after administration. Specifically, at two weeks post-administration, compared to baseline values, mean reduction (improvement) in Tnl was 46.7% for the low dose group and 48.8% for the high dose group. In contrast, in the control group, Tnl increased 7.7%.
  • nt-pro BNP plasma nt-pro BNP
  • concentration of nt-pro BNP in normal dogs was on average about 300 pg/mL, but was elevated to about 1 100- 1300 pg/mL in dogs with induced advanced heart failure.
  • the treatment effect ( ⁇ ), i.e. difference between pre-treatment values and subsequent values obtained at 2 hours, 24 hours, 1 week and 2 weeks, for the treated Groups also is depicted in Table 8.
  • plasma nt-pro BNP levels tended to decrease at 2 hours and 24 hours and the reduction reached statistical significance at 2 weeks after treatment with low dose and at 1 week and 2 weeks after treatment with high dose.
  • results show that a single infusion of LCMF poloxamer- 188 administered over two hours resulted in a statistically significant and progressive reduction in hemodynamic stress as evidenced by a reduction in plasma nt-pro BNP, with such effect persisting for at least two weeks after administration. Specifically, at two weeks post-administration, compared to baseline values, mean reduction
  • nt-pro BNP (improvement) in nt-pro BNP was 54.5% for the low dose group and 61.4% for the high dose group. In contrast, in the control group, nt-pro BNP increased 3.5%.
  • the dogs treated as described in Example 2 were assessed for plasma levels of various inflammatory biomarkers of heart failure, including matrix metalloproteinase- 2 (MMP-2), Interleukin 6 (IL-6), C-reactive protein (CRP) and TNF-alpha (TNFct). Elevated levels of these biomarkers are known to be associated with cardiac risk.
  • MMP-2 matrix metalloproteinase- 2
  • IL-6 Interleukin 6
  • CRP C-reactive protein
  • TNF-alpha TNF-alpha
  • cytokine TNFa increases levels of the proinflammatory cytokine TNFa
  • IL-6 is an acute phase cytokine that promotes production of other inflammatory mediators and is associated with decreased cardiac functional status
  • elevated levels of CRP are known to be associated with an increased risk for coronary artery disease and acute coronary syndromes (ACS)
  • MMP-2 can degrade collagen fibrils and its upregulation results in unstable plaques and is associated with cardiovascular disease progression.
  • biomarker Procollagen type I N-terminal propeptide ( ⁇ ) also was assessed, which is an indicator of collagen synthesis and remodeling of the myocardium.
  • Peripheral venous blood samples were obtained at baseline, at the end of 2 hours of drug infusion and at 24 hours, 1 week and 2 weeks after drug infusion.
  • the blood samples were centrifuged at 3000 rpm for 10 minutes and plasma withdrawn and placed in cryo-storage tubes and stored upright at -70°C until assay.
  • Plasma samples from 6 normal dogs (same breed, age and weight as study dogs) were also obtained and stored for comparison.
  • ELISA antibody sandwich enzyme-linked immunosorbent assay
  • results show that the pre-treatment plasma levels of all tested biomarkers were elevated in dogs with induced heart failure compared to levels found in normal dogs. Infusion of normal saline into heart failure dogs had no effect on the levels of any of the biomarkers.
  • Treatment with low and high poloxamer-188 reduced plasma levels of TNFa, IL-6, CRP and MMP- 1 at 1 week and 2 weeks post-treatment.
  • the results were dose-dependent, with increased reduction in plasma levels occurring in the group of dogs treated with a high dose of poloxamer-188. No effect on plasma levels of ⁇ was observed after treatment at either tested dose compared to controls.
  • the results show that a single, 2 hour infusion of a poloxamer-188 in dogs with heart failure elicits improvements in biomarkers of inflammation, and to a lesser extent collagen deposition, that persisted for at least 2 weeks post-treatment .

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Abstract

La présente invention concerne des procédés et des utilisations d'un traitement de l'insuffisance cardiaque par perfusion unique d'un poloxamère 188, tel qu'un poloxamère exempt de substance à circulation longue (LCMF).
PCT/US2014/045627 2014-07-07 2014-07-07 Thérapie par poloxamères pour lutter contre l'insuffisance cardiaque WO2016007128A1 (fr)

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Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Press Releases - Mast Therapeutics - Mast Therapeutics Announces Positive Data In Model Of Heart Failure", 6 January 2014 (2014-01-06), pages 1 - 3, XP055133893, Retrieved from the Internet <URL:http://www.masttherapeutics.com/investors/news/?releaseid=1887966> [retrieved on 20140808] *
ANONYMOUS: "Press Releases - Mast Therapeutics - Mast Therapeutics Press Release 2014, February 18", 18 February 2014 (2014-02-18), pages 1 - 3, XP055133891, Retrieved from the Internet <URL:http://www.masttherapeutics.com/investors/news/?releaseid=1900672> [retrieved on 20140808] *
MARTIN EMANUELE ET AL: "Differential Effects of Commercial-Grade and Purified Poloxamer 188 on Renal Function", DRUGS IN R&D, vol. 14, no. 2, 11 April 2014 (2014-04-11), pages 73 - 83, XP055133853, ISSN: 1174-5886, DOI: 10.1007/s40268-014-0041-0 *

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