WO2017007917A1 - Copolymères de polyoxyéthylène/polyoxypropylène et inhibiteurs fibrinolytiques, leurs utilisations et compositions - Google Patents

Copolymères de polyoxyéthylène/polyoxypropylène et inhibiteurs fibrinolytiques, leurs utilisations et compositions Download PDF

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WO2017007917A1
WO2017007917A1 PCT/US2016/041304 US2016041304W WO2017007917A1 WO 2017007917 A1 WO2017007917 A1 WO 2017007917A1 US 2016041304 W US2016041304 W US 2016041304W WO 2017007917 A1 WO2017007917 A1 WO 2017007917A1
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copolymer
poloxamer
molecular weight
composition
combination
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R. Martin Emanuele
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Mast Therapeutics, Inc.
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions

Definitions

  • This invention relates to a method of treating damaged tissue and/or damaged cell surfaces, unwanted bleeding, hemorrhagic shock or a precursor thereto with a polyoxyethylene/polyoxypropylene copolymer in conjunction with one or more fibrinolytic inhibitors.
  • the present invention further relates to pharmaceutical compositions comprising polyoxyethylene/polyoxypropylene copolymers and fibrinolytic inhibitors.
  • the present invention relates to kits comprising one or more pharmaceutical compositions of polyoxyethylene/polyoxypropylene copolymers and fibrinolytic inhibitors where the active agents are separate or combined pharmaceutical compositions. Also provided are said compositions for the treatment of damaged tissue and/or damaged cell surfaces, unwanted bleeding, hemorrhagic shock or a precursor thereto.
  • Fibrinolysis refers to the degradation of a fibrin blood clot.
  • plasmin is the primary fibrinolytic enzyme. Plasmin breaks down polymerized fibrin producing circulating fragments that are cleared by other proteases or by the kidney and liver. Plasmin is produced in an inactive form, plasminogen, in the liver. Plasminogen activators such as tissue plasminogen activator (t-PA) and urokinase convert plasminogen to the active enzyme plasmin, thus allowing fibrinolysis to occur. When there is a blockage of blood flow due to an occlusive thrombus (as in a heart attack), activators of plasmin such as tissue plasminogen activator (tPA) or streptokinase are used
  • Fibrinolytic inhibitors such as aminocaproic acid (AC A or EACA) and tranexamic acid (TXA) and lysine derivatives, are used as antagonists to fibrinolytic agents. They also are used to treat bleeding, such as occurs in hemorrhagic shock, bleeding during surgery, bleeding resulting from trauma and injury, bleeding disorders, excessive menstrual bleeding, and to counter the action of thrombolytic agents.
  • AC A or EACA aminocaproic acid
  • TXA tranexamic acid
  • lysine derivatives are used as antagonists to fibrinolytic agents. They also are used to treat bleeding, such as occurs in hemorrhagic shock, bleeding during surgery, bleeding resulting from trauma and injury, bleeding disorders, excessive menstrual bleeding, and to counter the action of thrombolytic agents.
  • Fibrinolytic inhibitors block the interaction of plasmin with its substrate allowing blood clots to remain intact. In damaged tissue, inhibitors of fibrinolysis can prevent or reduce the unwanted degradation of hemostatic blood clots. Inhibitors of fibrinolysis, however, have adverse effects. They can increase the risk for thromboembolic events and ischemic tissue injury. Thus, there is a need for reducing or preventing the adverse effects of fibrinolytic inhibitors.
  • Fibrinolytic inhibitors are employed for treatment of bleeding, such as hemorrhagic shock and precursors thereto and secondary effects, such as hypovolemia, as are poloxamers. Each, however, poses a risk of undesirable adverse effects, including thrombosis and bleeding.
  • a fibrinolytic inhibitor is administered to promote hemostasis but can have the undesirable consequence of thrombosis, especially in smaller blood vessels with sludged flow.
  • poloxamer 188 can promote bleeding.
  • poloxamer 188 and fibrinolytic inhibitors are administered together, such as sequentially, intermittently, simultaneously, and in the same composition. They can be administered together to treat or prevent the adverse effects from treatment by one or the other. They are administered for any treatment for which fibrinolytic inhibitors are administered (see, e.g., Tengborn in Treatment o/ Hemophilia, April 2007, no. 42 "Fibrinolytic Inhibitors In The Management Of Bleeding Disorders; Published by the World Federation of Hemophilia (WFH) ⁇ World Federation of Hemophilia 2007).
  • WFH World Federation of Hemophilia
  • the invention provides a poloxyethylene/polyoxypropylene copolymer alone or in combination with one or more fibrinolytic inhibitors for use in the treatment of damaged tissue and/or damaged cell surfaces, or the treatment and prevention of unwanted bleeding, hemorrhagic shock or a precursor thereto.
  • fibrinolytic inhibitors for use in the treatment of damaged tissue and/or damaged cell surfaces, or the treatment and prevention of unwanted bleeding, hemorrhagic shock or a precursor thereto.
  • the dosage of fibrinolytic inhibitors is the dosage for such treatment; and the dose of poloxamer is relatively low, for example, it is administered in an amount to achieve a circulating concentration of less than about 3.5 mg/ml in the circulation, or less than 2.5 mg/ml, such as about or at 0.25 to 2.5 mg/ml, and the dose of fibrinolytic inhibitor is the normal therapeutic dosage.
  • Fibrinolytic inhibitors also can be administered to mitigate bleeding that can occur with poloxamer 188 therapy for any disorder for which poloxamers are administered. They also can be administered together for any treatment in which poloxamer is administered, particularly at high doses, and more particularly at doses above 2.5 mg/ml and the dosage of poloxamer is the therapeutic dose for treatment of the condition for which the poloxamer is administered, and the dosage of the fibrinolytic inhibitor is sufficient to mitigate bleeding caused by or enhanced by the poloxamer.
  • 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 gel permeation chromatography (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).
  • GPC gel permeation chromatography
  • FIG. 7A-B shows enlarged HPLC-GPC chromatograms depicting the molecular weight distribution of components in plasma over time.
  • FIG. 8A-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 rV infusion of purified poloxamer 188 as described in Grindel et al. (2002) (Biopharmaceutics & Drug Disposition, 23:87-103).
  • FIG. 9 shows a Reverse Phase High Performance Liquid Chromatography (RP-HPLC) chromatogram comparing profiles of compositions of 15% LCMF 188 with 15% PI 88 (available under the trademark Flocor®), relative to other poloxamers and polymers (of different hydrophobicity / hydrophilicity) showing that the LCMF 188 is more hydrophilic than the PI 88.
  • RP-HPLC Reverse Phase High Performance Liquid Chromatography
  • FIG. 10 shows a RP-HPLC chromatogram comparing different lots of LCMF poloxamer 188 with purified poloxamer 188 confirming the difference in hydrophilicity.
  • FIG. 11 shows a control study of P188 addition to plasma. The results indicate a concentration dependent change in the rate of fibrin assembly.
  • FIG. 12 shows an effect of the addition of urokinase to plasma, which is a concentration dependent increase of clot lysis (decrease in OD).
  • FIG. 13 shows a concentration range of addition of urokinase to plasma with the addition of P188 as well. At all concentrations of urokinase, P188 shortened the time to onset of lysis and shortened the time to complete clot lysis (optical density of zero).
  • FIG. 14 shows a concentration range addition of a combination of urokinase with P188, along with the addition of tranexamic acid. There is no indication of fibrinolysis or fibrinogenolysis.
  • Poloxamer 188 3. Molecular Diversity of Poloxamer 188
  • fibrinolysis refers to the degradation of a fibrin blood clot.
  • plasmin is the primary fibrinolytic enzyme.
  • activators of plasmin such as tissue plasminogen activator (tPA) or streptokinase are used
  • fibrinolytic inhibitor refers to any compound that reduces the amount or activity of the plasmin protease in a subject.
  • exemplary of inhibitors of fibrinolysis are aminocaproic acid (ACA) or tranexamic acid (TXA) which block the interaction of plasmin with its substrate allowing blood clots to remain intact.
  • hemorhagic shock refers to conditions following the loss of blood. Precursors to hemorrhagic shock include trauma, injury and surgery.
  • polystyrene resin refers to synthetic block copolymers of ethylene oxide and propylene oxide.
  • a “polyoxyethylene/poloxypropylene copolymer,” “PPC” or “poloxamer” refers to a block copolymer containing a central block of polyoxypropylene (POP) flanked on both sides by blocks of polyoxyethylene (POE) having 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% 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 H60)3 ⁇ 4 (i.e., the polyoxypropylene portion of the copolymer) has a molecular weight of approximately 950 to 4,000 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.
  • Da molecular weight
  • 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, Flocor ® , Kolliphor ® and Lutrol ® ).
  • the nomenclature of the polyoxyethylene/polyoxypropylene copolymer relates to its monomeric composition.
  • poloxamer 188 describes a polymer containing a polyoxypropylene hydrophobe of about 1,800 Da with a hydrophilic polyoxyethylene block content of about 80% of the total molecular weight.
  • Poloxamers can be synthesized in two steps, first by building the
  • 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).
  • Polystyrene 188 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 (C3H6O) has a molecular weight of approximately 1,300 to 2,300 Da, such as 1,400 to 2,000 Da, for example approximately 1,750 Da.
  • a is about 79 and b is approximately or is 28.
  • the average total molecular weight of the compound is approximately, 7200-9700 Da, or approximately 7,680 to 9,510 Da, or 7350 to 8850 Da such as generally 8,400-8,800 Da, for example about or at 8,400 Da. or about 8500 Da.
  • the polyoxyethylene-polyoxypropylene- polyoxyethylene weight ratio of is approximately 4:2:4. According to specifications, P188 has a weight percent of polyoxyethylene of 81.8+1.9%, and an unsaturation level of about 0.010 to 0.034 mEq/g, or for example 0.026+0.008 mEq/g.
  • 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. Included among poloxamer 188 molecules are those that exhibit a species profile (e.g., determined by GPC) containing a main peak and "shoulder" peaks on both sides representing low molecular weight (LMW) polymer species and high molecular weight (HMW) polymer species. Poloxamer 188 also refers to materials that are purified to remove or reduce species other than the main component.
  • a species profile e.g., determined by GPC
  • LMW low molecular weight
  • HMW high molecular weight
  • main component or “main peak” with reference to a poloxamer 188 preparation refers to the species of copolymer molecules that have a molecular weight of less than about 13,000 Da and greater than about 4,500 Da, with an average molecular weight of between about 7200 to 9700 Da, or about 7,680 to 9,510 Da, or 7350 to 8850 Da, such as generally 8,400-8,800 Da, or about 8,200 -8,800 Da, for example about or at 8,400 Da or about 8500 Da.
  • Main peak species include those that elute by gel permeation chromatography (GPC) at between 14 and 15 minutes depending on the chromatography conditions (see U.S. Patent No. 5,696,298 and Grindel et al., Biopharm Drug Dispos. 2002; 23(3):87-103).
  • 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 4,500 Da. LMW species include those that elute by gel permeation chromatography (GPC) after 15 minutes depending on the
  • Such impurities can include low molecular weight 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 depending on the chromatography conditions (e.g., see U.S. Patent No. 5,696,298 and Grindel et al. (2002) Biopharmaceutics & Drug Disposition, 23:87-103).
  • 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. In such a case, the polydispersity (Mw/Mn) is 1.
  • 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. Hence, 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 chromato grams. 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 Powerline chromatographic system equipped with a column heater module, a Model 410 refractive index detector, Maxima 820 software package (all from Waters, Div.
  • purified poloxamer 188 or “P188-P” or “purified long circulating material (LCM)-containing poloxamer 188” refers to a poloxamer 188 that has polydispersity value of the poloxamer of less than or about 1.07, such as less than or 1.05 or less than or 1.03, and is a purified poloxamer 188 that has a reduced amount of low molecular weight components, but contains the long circulating material.
  • An embodiment of the disclosure herein are poloxamer 188 copolymers purified to remove or reduce low molecular weight components.
  • poloxamer 188 such as poloxamer 188NF (BASF) and purified poloxamer 188, have a long circulating material (LCM) that, when administered to a human, has a half-life that is more than 5.0 fold the circulating half-life of the main component in the distribution of the copolymer Grindel et al. (2002) Biopharmaceutics & Drug Disposition, 23:87-103 and WO1994/08596).
  • LCM long circulating material
  • An exemplary purified LCM-containing poloxamer 188 is poloxamer 188 available under the trademark FLOCOR ® (see, also U.S. patent No. 5,696,298, which describes LCM-containing poloxamer 188 and Grindel et al. (2002) Biopharmaceutics & Drug Disposition, 23:87-103).
  • the purified LCM-containing poloxamer 188 When the purified LCM-containing poloxamer 188 is administered as an intravenous injection to a mammal, particularly a human, GPC analysis of blood obtained from the treated subject exhibits two circulating peaks: a peak designated the main peak that comprises the main component of the polymeric distribution and a peak of higher molecular weight, compared to the main peak, that exhibits a substantially slower rate of clearance (more than 5-fold slower than the main peak, typically more than 30 hours and as much as 70 hours, as shown herein) from the circulation, i.e., a long circulating material (LCM) (Grindel et al. (2002)
  • long circulating material refers to material in prior poloxamer preparations that, upon administration to a subject, have a half-life in the subject, such as a human, that is substantially longer than the half-life of the main component of the poloxamer preparation.
  • the LCM material in a poloxamer preparation has more than about or more than 5-fold the half-life of the main component (or main peak) of the poloxamer preparation.
  • the LCMF poloxamers as provided herein do not give rise to such long circulating material.
  • long circulating material free or "LCMF” with reference to poloxamer 188 refers to a purified poloxamer 188 preparation that has a reduced amount of low molecular weight components, as described above for purified poloxamer 188, and that, following intravenous administration to a subject, the components of the polymeric distribution clear from the circulation in a more homogeneous manner such that any long circulating material exhibits a half-life that is no more than 5- fold longer than the circulating half-life (ti/ 2 ) of the main peak.
  • an LCMF is a poloxamer 188 that does not contain components, such as a high molecular weight components or low molecular weight components as described herein, that are or gives rise to a circulating material with a ti/2 that, is more than 5.0-fold greater than the tm 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.
  • the LCMF poloxamer 188 has an unsaturation level of about 0.018 to about 0.034mEq/g.
  • an LCMF poloxamer is a poloxamer in which all of the components of the polymeric distribution clear from the circulation at a more homogeneous rate.
  • suitable LCMF poloxamer 188 are described in US patent application Serial No. 14/793,670, filed on July 7, 2015 which is incorporated in its entirety.
  • distribution of copolymer refers to the molecular weight distributions of the polymeric molecules 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 chromatography (GPC) methods can be employed that determine molecular weight distribution based on the polymer's hydrodynamic volume.
  • GPC gel permeation chromatography
  • the distribution of molecular weight or mass of a polymer can be summarized by polydispersity. For example, the greater the disparity of molecular weight distributions in a poloxamer, the higher the polydispersity.
  • half-life As used herein, “half-life,” “biological half-life,” “plasma half-life,” “terminal half-life,” “elimination half-life” or “tm” 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 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 measuring the concentration of the drug in the plasma at regular intervals. The concentration of the drug will reach a peak value in the plasma and will fall as the drug is cleared from the blood. In one embodiment, half-life is measured in a human subject.
  • Cmax refers to the peak or maximal plasma concentration of a drug after administration.
  • concentration of a drug at steady state refers to the concentration of drug at 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 13,000 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.
  • a poloxamer 188 contains 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 of polar solvents 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 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 form.
  • 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.
  • T c critical temperature
  • p c critical pressure
  • 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 is 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.
  • the 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. , Ci-Ce alkanols).
  • the alkane portion of alkanol can be branched or unbranched. Examples of alkanols include, but are not limited to, methanol, ethanol, isopropyl alcohol (2-propanol), and ie/ -butyl alcohol.
  • subcritical extraction refers to processes using fluid substances that would usually be gaseous at normal temperatures and pressures, that are 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.
  • isocratic refers to a system in which an 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
  • the extraction solvent does not remain constant.
  • plural 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.
  • an effective amount refers to the dose of poloxamer and/or fibrinolytic inhibitor that, when administered to patient, results in a desired biological effect.
  • pharmaceutical composition includes a composition comprising a polyoxyethylene/polyoxypropylene copolymer described herein, such as an LCMF poloxamer, formulated as a pharmaceutically acceptable formulation and/or with one or more pharmaceutically acceptable excipients. It can include a fibrinolytic inhibitor.
  • the pharmaceutical composition comprises an aqueous injectable solution of the poloxamer buffered at a desired pH, such as 4-8, 6-8 or 6-7 or 6 or about 6, with a suitable buffer.
  • exemplary of buffers 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 range from 0.005 to 0.05 M, particularly about 0.01 M 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 poloxamer (see, e.g. , Published International PCT Application No. WO 94/008596 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. Hence, treatment encompasses prophylaxis, therapy and/or cure. Treatment also encompasses any pharmaceutical use of the compositions herein.
  • treating means that a composition or other product provided or described herein is administered to the subject to thereby achieve 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 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 is readily determined by one of skill in the art following routine procedures, and depends upon the particular indication for which the composition is administered.
  • 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 sufficient to treat, such as prevent, cure ameliorate, arrest or otherwise treat a particular disease or disorder.
  • disease or “disorder” refers to a pathological condition in an organism resulting from cause or condition including, but not limited to, infections, acquired conditions, and genetic conditions, and characterized by identifiable symptoms.
  • Diseases and disorders of interest for which poloxamers have been indicated as potential therapeutics include, but are not limited to, any requiring membrane resealing and repair, tissue ischemia and reperfusion injury, decreasing inflammatory disorders, disorders related thrombolysis, and disorders related to hemostasis.
  • diseases and disorders include acute coronary syndromes, limb ischemia, shock, stroke, heart failure, including without limitation, systolic, diastolic, congestive, and cardiomyopathies, coronary artery disease, muscular dystrophy, circulatory diseases, pathologic hydrophobic interactions in blood, sickle cell disease, and associated syndromes such as venous occlusive crisis, and acute chest syndrome, inflammation, pain, neurodegenerative diseases, macular degeneration, thrombosis, kidney failure, burns, spinal cord injuries, ischemic/reperfusion injury, myocardial infarction, hemo-concentration, amyloid oligomer toxicity, diabetic retinopathy, diabetic peripheral vascular disease, sudden hearing loss, peripheral vascular disease, cerebral ischemia, transient ischemic attacks, critical limb ischemia, respiratory distress syndrome (RDS), and adult respiratory distress syndrome (ARDS).
  • systolic diastolic
  • congestive and cardiomyopathies
  • coronary artery disease muscular dystrophy
  • 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.
  • 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. The term is intended to encompass purified poloxamers contained in articles of packaging.
  • 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 purified poloxamers provided herein and another item for a purpose including, but not limited to,
  • 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.
  • 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.
  • an optionally substituted group means that the group is unsubstituted or is substituted.
  • retention time or means the time elapsed between the injection of a sample, such as an LCMF poloxamer 188 sample, onto a reverse phase column for reverse phase high performance liquid chromatography and the peak response by the evaporative light scattering detector.
  • the retention time is longer for more hydrophobic samples compared to less hydrophobic samples.
  • Capacity factor or V is determined by the following equation where to is equal to the void time or the time a non retained substance passes through a reverse phase HPLC column (see, Example 1 below):
  • LCM-containing purified poloxamer 188 such as the poloxamer sold under the trademark FLOCOR ® , has a mean retention time (tR) of 9.883 and a k' of 3.697; whereas the LCMF poloxamer 188 has a mean retention time (tR) of 8.897 and a mean k' of 3.202 (see, the Examples below).
  • Fibrinolytic inhibitors are employed for treatment of hemorrhagic shock and precursors thereto and secondary effects, such as hypovolemia, as are poloxamers. Each, however, poses a risk of undesirable adverse effects, including thrombosis and bleeding.
  • a fibrinolytic inhibitor is administered to promote hemostasis but can have the undesirable consequence of thrombosis especially in smaller blood vessels with sludged flow.
  • poloxamer 188 can promote bleeding, but at lower concentrations, such as less than about or less than 2.5 mg/ml, such as at or about 0.25 - 2.5 mg/ml circulating concentrations, the poloxamer has a potent rheologic effect that inhibits the pro-thrombotic effect of the fibrinolytic inhibitor.
  • concentrations such as less than about or less than 2.5 mg/ml, such as at or about 0.25 - 2.5 mg/ml circulating concentrations
  • the poloxamer has a potent rheologic effect that inhibits the pro-thrombotic effect of the fibrinolytic inhibitor.
  • atrheologic concentrations poloxamer can exacerbate bleeding.
  • a fibrinolytic inhibitor can antagonize this effect.
  • Administration of the poloxamer treats damaged tissue and also can prevent (reduce the risk of) ischemic tissue damage and thromboembolic events, including thrombosis and embolisms, that are associated with the administration of fibrinolytic inhibitors to a subject with hemorrhagic shock.
  • poloxamers and fibrinolytic inhibitors are used together.
  • a relatively low dose of poloxamer and therapeutic dosages such as those known to the skill of the art for the fibrinolytic inhibitor, of fibrinolytic inhibitor or other combination thereof, the adverse effects of each the poloxamer and fibrinolytic are mitigated and improved treatment results.
  • the fibrinolytic inhibitor and poloxamer when combined or administered together, mutually antagonize the unintended consequences of the other, resulting in improved outcomes.
  • a fibrinolytic inhibitor and a poloxamer generally a purified poloxamer PI 88.
  • the fibrinolytic inhibitors and the poloxamer can be administered separately, simultaneously, sequentially, in the same composition or in separate compositions.
  • the poloxamer can be administered to treat adverse events, such as from administration of fibrinolytic inhibitors, and fibrinolytic inhibitors can be administered to treat adverse effects of poloxamer treatment. Also provided are compositions containing a fibrinolytic inhibitor and a poloxamer.
  • Treatment is achieved by administering a polyoxyethylene/polyoxypropylene copolymer (poloxamer) and a fibrinolytic inhibitor.
  • the dosage of the poloxamer is relatively low, since higher dosages can promote bleeding or decrease clotting.
  • the methods, uses, combinations and compositions provided herein achieve therapeutic benefits of the fibrinolytic inhibitors and of the poloxamers and reduce the associated risks from each. These risks include, for example, ischemic tissue damage from the fibrinolytic inhibitor, and bleeding from or decreased clotting from the poloxamers.
  • the dosage of the poloxamer can be titrated so that it reduces the adverse effects of the fibrinolytic inhibitors without increasing the risk of bleeding.
  • the fibrinolytic inhibitors also permit administration of the poloxamer to treat damaged tissue, but avoid the side-effects of the poloxamer.
  • fibrinolytic activity is important.
  • inhibitors of fibrinolysis are administered to help prevent or reduce the unwanted degradation of hemostatic blood clots.
  • Fibrinolytic inhibitors also are administered to patients with bleeding disorders in which diminished and delayed thrombin generation leads to the formation of clots that have an abnormal fibrin network and are more soluble than normal clots.
  • Following treatment with a fibrinolytic inhibitor particularly after trauma or during surgery, there is an increased risk of ischemic tissue damage and thromboembolic events.
  • a poloxamer In accord with the methods and uses herein, a poloxamer, a
  • polyoxyethylene/polyoxypropylene copolymer such as a PI 88, is administeredto reducethe consequences associated with administration of fibrinolytic inhibitors.
  • the poloxamer for example, reduces the risk of ischemic tissue damage.
  • the poloxamer is not administered to dilute the blood, but rather has an effect on the blood to reduce the risks and consequences associated with administration of fibrinolytic inhibitors, such as during surgery or after trauma.
  • an effective amount of a poloxamer composition is administered.
  • the suitable dosage achieves a blood concentration that reduces the risks or consequences associated with administration of fibrinolytic inhibitors.
  • the particular dosage regimen depends upon the subject, and the severity and nature of the tissue damage or trauma or condition treated. The skilled physician can select an appropriate regimen.
  • the methods include administration of a fibrinolytic inhibitor and a poloxamer, a polyoxyethylene/polyoxypropylene copolymer, such that the administration of poloxamer sufficient to result in a concentration of the poloxamer in the circulation of the subject of from at or about 0.05 mg/mL to at or about 15 mg/mL, for example, from at or about 0.2 mg/mL to at or about 4.0 mg/mL, such as at or about at least 0.5 mg/mL.
  • the poloxamer when used to treat or prevent or mitigate adverse effects of fibrinolytic inhibitors, is present at a circulating concentration that is less than 2.5 mg/ml.
  • the concentration of the poloxamer in the circulation of the subject can be representative of a single time point or representative of a mean steady state concentration that is maintained for a period of time, for example, up to 72 hours or more after administration or by virtue of multiple doses.
  • an optimal steady- state plasma concentration range for treatment in conjunction with fibrinolytic inhibitors is a plasma concentration in the circulation of less than 3.0 ml/ml or 2.5 mg/ml, generally 0.25 to 2.5 mg/ml, such about 0.5 - 1.5 mg/ml or 0.5 - 1.5 mg/ml for a time sufficient to achieve treatment.
  • Treatment typically lasts for 12 hours to several days, such as 1, 2, 3 or 4 days or can be a onetime treatment, such as following an injury or during surgery.
  • the poloxamer can be administered by any suitable route and way of administration.
  • IV infusion intravenous
  • bolus injection a concentration of 0.5 mg/ml can be maintained by giving an IV infusion of between 30 mg/kg/hr - 50 mg/kg/hr depending on the renal function of the recipient; a plasma concentration of 1.0 mg/ml can be maintained by administering between 80 mg/kg/hr - 100 mg/kg/hr again depending on the renal function of the recipient.
  • the infusion can be continued for between 12-48 hours as needed.
  • repeat bolus injections can be administered.
  • 50 mg/kg as an IV bolus every 6 hours over 1 to 3 or 4 days can be administered to achieve a plasma concentration of about 0.5 mg/ml.
  • 100 mg/kg every 6 hours for 1 to 3 or 4 days would result in concentrations in the middle of the desired range.
  • the methods provided herein can be used in the treatment of hemorrhagic shock or any condition or consequence associated with hemorrhagic shock, in conjunction with administration of a fibrinolytic inhibitor, such as during surgery or after trauma.
  • ischemic tissue damage CADurosis
  • prothrombotic events for example, embolism or thrombosis
  • any other condition or unwanted consequence associated with hemorrhagic shock CADurosis
  • administration of the poloxamer is in combination with or subsequent to therapies for hemorrhagic shock.
  • exemplary of the treatments or conditions is treatment with fibrinolytic inhibitors. It is understood that the methods herein can be used to treat hemorrhagic shock or any conditions or consequences resulting from hemorrhagic shock, such as any condition or treatment or combination thereof that results in tissue damage, such as ischemic tissue damage.
  • Fibrinolysis is the process by which fibrin blood clots are prevented from growing and becoming problematic, and instead are degraded. Fibrinolysis also keeps blood vessels patent (i.e., open) and starts the process of remolding damaged tissue.
  • Fibrinolysis is either primary, a normal body process, or secondary, where clots are broken down as a result of medicine, a medical disorder, or some other cause.
  • the fibrinolytic system is activated when undesirable fibrin is formed or when a hemostatic thrombus becomes unnecessary, such as when tissue is damaged, vessels are ruptured, and the hemostatic mechanism is triggered.
  • the initial step in fibrinolysis is plasminogen activation to plasmin by plasminogen activators. In circulating blood, the fibrin blood clots are broken down by plasmin, a proteolytic enzyme whose primary role is to dissolve fibrin.
  • Plasmin degrades fibrin thrombi by cleaving fibrin at various places, leading to the production of circulating fragments (i.e., fibrin degradation products) that are then further degraded by other proteases and cleared by the kidneys and/or the liver.
  • fibrin degradation products i.e., fibrin degradation products
  • Plasmin is produced in the liver as its inactive form plasminogen, a proenzyme unable to cleave fibrin. Plasminogen has an affinity for fibrin and is incorporated into a fibrin clot when it is formed and later activated into plasmin by streptokinase (SK), urokinase (urokinase-type plasminogen activator, uPA), an enzyme found mainly in the urine, or tissue-type plasminogen activator (tPA).
  • SK streptokinase
  • uPA urokinase-type plasminogen activator
  • tPA tissue-type plasminogen activator
  • tPA is expressed in the endothelial cells of blood vessel walls and is slowly released into the blood by the damaged endothelium of blood vessels, activating plasminogen by binding to fibrin via its ly sine-binding sites (LBS). Plasmin production further stimulates additional plasmin generation by producing more active forms of both tPA and urokinase.
  • plasminogen activators such as tPA, SK, and uPA are often administered to facilitate clot degradation and restore blood flow.
  • tPA plasminogen activators
  • SK SK
  • uPA plasminogen activators
  • Fibrinolytic inhibitors are agents that result in either a decreased amount of plasmin production or a decrease in plasmin activity, and thus, degradation of the blood clot by fibrinolysis is prevented.
  • plasmin plays a central role in fibrin clot degradation and tissue remodeling
  • disruption of the tightly regulated fibrinolytic process e.g., disruption of the process of converting plasminogen to plasmin or the mechanism by which plasmin acts
  • administration of fibrinolytic inhibitors may have adverse consequences, for example, prothrombotic consequences (i.e., lead to the development of thrombosis).
  • inhibitors may also result in an increased risk for thromboembolic events, such as embolism and thrombosis, and ischemic tissue injury.
  • fibrinolytic activity is important.
  • inhibitors of fibrinolysis are administered to help prevent or reduce the unwanted degradation of hemostatic blood clots.
  • Fibrinolytic inhibitors also are administered to patients with bleeding disorders in which diminished and delayed thrombin generation leads to the formation of clots that have an abnormal fibrin network and are more soluble than normal clots.
  • Fibrinolytic inhibitors include, but are not limited to, endogenous and pharmaceutical (i.e., synthetic) inhibitors.
  • Endogenous fibrinolytic inhibitors include the plasminogen activator inhibitors (PAI) including plasminogen activator inhibitor- 1 (PAI- 1), which is the primary inhibitor of tPA and uPA and is synthesized in endothelial cells, adipocytes, and the liver; PAI-2, which is synthesized by the placenta, monocytes, and macrophages, and only occurs in significant amounts during pregnancy; and PAI-3 (also known as protein C inhibitor), which inhibits an array of proteases, including uPA, tPA, activated protein C, thrombin, and acrosin, and is synthesized in the liver and in numerous steroid-responsive organs.
  • PAI plasminogen activator inhibitors
  • PAI- 1 plasminogen activator inhibitor- 1
  • PAI-2 which is synthesized by the placenta,
  • endogenous inhibitors include the plasmin inhibitor alpha-2-antiplasmin (also known as a 2 -plasmin inhibitor), synthesized in the liver. It regulates fibrinolysis by forming a stoichiometric complex with plasmin, inhibiting plasmin adsorption on the fibrin clot, and preventing the binding of plasminogen to the fibrin clot.
  • alpha-2-antiplasmin also known as a 2 -plasmin inhibitor
  • Apha-2-macroglobulin primarily produced by the liver inhibits fibrinolysis by inhibiting plasmin and kallikrein; and thrombin-activatable fibrinolysis inhibitor (TAFI), an enzyme that circulates in plasma and suppresses fibrinolysis when activated to TAFIa by removing exposed lysine residues form the fibrin clot as it is degraded, thus restricting binding of plasminogen and further activation to plasmin.
  • TAFI thrombin-activatable fibrinolysis inhibitor
  • compositions include the polypeptide aprotinin and synthetic derivatives of lysine, such as ⁇ -aminocaproic acid (aminocaproic acid; EACA), and the more potent tranexamic acid (TA).
  • TA tranexamic acid
  • a commercial formulation of TA is available as Cyklokapron®.
  • An exemplary formulation is where each mL of Cyklokapron® contains 100 mg TA in water for injection.
  • Aminocaproic acid and TA are indirect plasmin inhibitors that bind to the LBS in a reversible and competitive manner, reducing plasminogen's affinity for binding to fibrin, thus reducing the activation of plasminogen to plasmin.
  • plasminogen competitively inhibits the activation of plasminogen to plasmin by binding to specific sites of both plasminogen and plasmin, allowing blood clots to remain intact.
  • Aprotinin derived from bovine lung tissue, is a direct inhibitor of plasmin as well as several other serine proteases, among them kallikrein. Accordingly, aprotinin and the synthetic lysine analogues and derivatives reduce fibrinolysis but via different mechanisms.
  • Fibrinolytic inhibitors for example, tranexamic acid, can be used to treat excessive blood loss during surgery and in various other medical conditions.
  • the fibrinolytic inhibitors can be administered before, after, or concomitant with administration of the
  • Poloxamers are a family of synthetic, linear, triblock copolymers composed of a core of repeating units of polyoxypropylene (PO or POP), flanked by chains of repeating units of polyoxyethylene (EO or POE). All poloxamers are defined by this EO- PO-EO structural motif. Specific poloxamers (e.g., poloxamer 188) 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 include POP/POE block copolymers having the following formula: HO(C2H 4 0)a'-(C 3 H 6 0)b-(C2H 4 0)aH, where "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) constitutes approximately 50% to 95% by weight of the compound, such as 60% to 90%, for example 70% to 90%, by weight of the compound; and the "b” is an integer such that the hydrophobe represented by (C 3 H 6 0) has a molecular weight of approximately 950 to 4,000 Da, such as 1,200 to 3,500 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 5,000 to 12,000 Da, for example 5,000 to 9,000 Da.
  • b is an integer of from about 15 to about 70, such as from about 15 to about 60, or from about 15 to about 30, or any of the numbers in between. In some instances, b is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In certain aspects, the integers for the flanking units with the subscript "a"' and "a" can differ or are the same values. In some instances, a or a' is an integer of about 45 to about 910, such as 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900.
  • a or a' is an integer from about 10 to about 215, such as 10, 20, 30, 40, 50, 60, 70, 80, 100, 125, 150, 175, 200 or 215.
  • a or a' is about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70.
  • the values for a', a and b represent an average; generally the polymeric molecules are a distribution or population of molecules. Therefore the actual values of a, a' and b within the population constitute a range of values.
  • poloxamer 407 describes a polymer containing a polyoxypropylene hydrophobe of about 4,000 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 1,800 Da and has a hydrophile that is about 80% of the total molecular weight of the copolymer.
  • Poloxamers are sold and referred to under trade names and trademarks including, but not limited to, ADEKA-NOL, SynperonicTM, Pluronic® and Lutrol®.
  • Exemplary poloxamers include, but are not limited to, poloxamer 188 (P188; sold under the trademarks Pluronic ® F-68, Kolliphor® P 188, 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 Pluronic ® F87, Kolliphor® P 237; 70% POE), poloxamer 338 (P338; sold under the trademark Kolliphor® P 338, Pluronic ® F- 108; 80% POE) and poloxamer 331 (Pluronic® L101; 10% POE).
  • non-purified PI 88 is commercially available or known under various names as described above. While the discussion below references using the methods herein to produce a more homogenous (LCMF) poloxamer 188, methods herein can be used to produce more homogenous preparations of any of the known poloxamers.
  • LCMF homogenous poloxamer
  • Poloxamers can be synthesized using standard polymer synthesis techniques. For example, 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 37,285, 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 Handbook of Biodegradable Polymers, Domb, A.J. et al. (eds.), Hardwood Academic Publishers, 1997).
  • 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, . Am. Oil Chem. Soc. 54 (1977) 110-116). 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.
  • a poloxamer 188 (P188) copolymer has the following chemical formula:
  • hydrophobe represented by (C 3 3 ⁇ 40) has a molecular weight of approximately 1,750 Daltons and the poloxamer 188 has an average molecular weight of 7,680 to 9,510 Da, or 7350 to 8850 Da such as generally approximately 8,400-8,800 Daltons.
  • the polyoxyethylene-polyoxypropylene-polyoxyethylene weight ratio is approximately 4:2:4. According to specifications, P188 has a weight percent of oxyethylene of 81.8+1.9% and an unsaturation level of about 0.010 to 0.034 mEq/g, or 0.026+0.008 mEq/g.
  • Unsaturation levels can be measured according to known techniques such as those described by Moghimi et al, Biochimica et Biophysica Acta 1689 (2004) 103- 113.
  • Various poloxamers are used for treatment of diseases and conditions in which resistance to blood flow is pathologically increased by injury due to the presence of adhesive hydrophobic proteins or damaged membranes. This adhesion is produced by pathological hydrophobic interactions and does not require the interaction of specific ligands with their receptors. Such proteins and/or damaged membranes increase resistance in the microvasculature by increasing friction and reducing the effective radius of the blood vessel. For example, it is believed that poloxamer 188 acts as a lubricant to increase blood flow through damaged tissues.
  • this blocks adhesion of hydrophobic surfaces to one another and thereby reduces friction and increases flow.
  • P188 binds to hydrophobic areas developed on injured cells and denatured proteins thereby restoring hydration lattices. Such binding facilitates sealing of damaged membranes and aborts the cascade of inflammatory mediators that could destroy the cell. 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 deformability of red blood cells, non-adhesiveness of platelets and granulocytes and the normal viscosity of blood, reducing apoptosis, and by multiple markers of inflammation including VEGF, various chemokines, and interleukins.
  • poloxamer 188 preparations are stated to have a molecular weight of approximately7680-9510 Daltons. Such poloxamer 188, however, is composed of molecules having a molecular weight from less than 3,000 Daltons to over 20,000 Daltons. The molecular diversity and distribution of molecules of commercial poloxamer 188 can be seen in the broad primary and secondary peaks detected using gel permeation chromatography (see, e.g., International PCT Published Application No. WO 94/08596).
  • the diversity in structure means that there is a diversity in biological activity.
  • the optimal rheologic, cytoprotective, anti-adhesive and antithrombotic effects are observed with molecules of P188 that are approximately 8,400 to 9,400 Daltons.
  • Such components can be identified as the main or predominant component in a poloxamer preparation using methods that separate components based on size, such as gel permeation chromatography (GPC).
  • GPC gel permeation chromatography
  • the distribution of components also typically show a smaller fraction of low molecular weight (LMW, i.e. generally below 4,500 Daltons) or high molecular weight (HMW, i.e. generally above 13,000 Daltons) components.
  • LMW low molecular weight
  • HMW high molecular weight
  • P188 components above 15,000 and below 4,500 Daltons are less effective rheologic or cytoprotective agents and exhibit unwanted side effects.
  • the other substances or components in a poloxamer preparation, such as a P188 preparation originate from two different sources, synthesis and degradation.
  • a primary mechanism contributing to the molecular diversity is the process by which poloxamers are synthesized.
  • the first step is the formation of the POP blocks. These are formed by reacting a propylene glycol initiator with propylene oxide monomer. Subsequently, ethylene oxide monomer is added to both ends forming the block copolymer.
  • the synthesis of poloxamers can result in a variation in the rates of polymerization during the steps of building the PO core and EO terminal ends.
  • HMW high molecular weight
  • high molecular weight substances can be formed due to inadequate cleaning of the polymerization reactor between batches of poloxamer 188 during a typical commercial manufacturing campaign. If the reactor is not completely cleaned to remove residual product after manufacturing a typical batch of poloxamer, such as poloxamer 188, the residual product will act as an initiator in the subsequent batch and form a "dimer like" poloxamer molecule. This substance is of higher molecular weight and would be part of the polymeric distribution observed on GPC as the HMW shoulder.
  • the degradation pathways for poloxamers include peroxidation leading to low molecular aldehydes and acids and thermal degradation leading to LMW
  • Oxidative degradation is the primary degradation pathway affecting stability of poloxamers. This process generates structural changes to the polymer chain and generates peroxides and carbonyls. Peroxides are transient in nature and quickly combine with butylated hydroxytoluene (BHT), which is typically added to commercial preparations as an antioxidant. Thermal degradation is another pathway that produces other substances. Glycols of various chain lengths are major degradation products of thermal degradation. Forced thermal degradation studies have shown that ethylene glycol, propylene glycol, diethylene glycol and triethylene glycol are formed.
  • 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.
  • Various truncated polymers with an EO-PO motif and a variety of other substances 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 result is material that is non-uniform (i.e. material that is polydisperse).
  • LMW low molecular weight
  • HMW high molecular weight
  • characterization of P188 by gel permeation chromatography identifies a main peak of P188 with "shoulder" peaks representing the unintended LMW and HMW components (Emanuele and Balasubramanian (2014) Drugs R D, 14:73-83).
  • the preparation of P188 that is available from BASF has a published structure that is characterized by a hydrophobic block with a molecular weight of approximately 1,750 Da, POE blocks making up 80% of the polymer by weight, and a total molecular weight of approximately 8,400 Da.
  • the actual compound is composed of the intended POE-POP-POE copolymer, but also contains other molecules which range from a molecular weight of less than 1,000 Da to over 30,000 Da.
  • the molecular diversity and distribution of molecules of commercial poloxamer 188 is illustrated by broad primary and secondary peaks detected using gel permeation chromatography.
  • the diversity of molecules present in the non-purified poloxamer preparations, including commercially available poloxamers, can result in diverse biological activities. Many of the observed biological activities are undesired or/and can result in unwanted side effects that limit the therapeutic efficacy of poloxamers as drugs.
  • Poloxamer 188 (see, e.g., Grindel et al. (2002) Journal of Pharmaceutical Sciences, 90:1936-1947 (Grindel et al. 2002a) or Grindel et al. (2002) Biopharmaceutics & Drug Disposition, 23:87-103 (Grindel et al. 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 (tm) in plasma of about 7 hours and a higher molecular weight component (i.e. the longer retention time species) exhibits about a 10-fold or more increase in half-life with a tm of approximately 70 hours or more and, thus, a substantially longer plasma residence time with slower clearance from the circulation than the main component.
  • the molecular weight of the LMW substances can range from a few hundred Da to a few thousand Da.
  • the complex nature of these impurities with wide solubility characteristics make it difficult to selectively remove them from the parent molecules.
  • Conventional purification processes such as distillation, crystallization, ultrafiltration, and the like, do not effectively separate the low molecular weight (LMW) substances from the main component.
  • Use of chromatographic techniques for purification, such as preparative GPC, are expensive and practically difficult to scale-up. Fine-tuning mixed solvent systems to differentially solubilize and remove various substances is also challenging and requires the use of large amounts of solvents that are costly to recycle.
  • Supercritical fluid extraction was performed using carbon dioxide to purify the copolymers to reduce the polydispersity to less than 1.17.
  • Purified PI 88 produced by these methods while having reduced renal toxicity still contain an accumulating long circulating material (Grindel et al. 2002b) .
  • a component in PI 88 has been identified that is or gives rise to a material in the plasma or blood with a longer circulating half-life compared to the main or predominant poloxamer species. This material with the longer circulating half-life is observed in non-clinical and clinical studies.
  • Analysis of plasma obtained following intravenous administration of purified P188 by high performance liquid chromatography - gel permeation chromatography (HPLC-GPC) shows two distinct peaks in the circulation (Grindel et al. (2002) Journal of Pharmaceutical Sciences, 90: 1936-1947 (Grindel et al. 2002a) or Grindel et al. (2002) Biopharmaceutics & Drug Disposition, 23:87-103 (Grindel et al. 2002b).
  • PI 88 Since the rheologic, cytoprotective, anti-adhesive and antithrombotic effects of PI 88 are optimal within 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 is not desirable.
  • PI 88 among the desired activities of PI 88 is its rheologic effect to reduce blood viscosity and inhibit red blood cell (RBC) aggregation, which account for its ability to improve blood flow in damaged tissues.
  • RBC red blood cell
  • poloxamer preparations that are substantially reduced in the component that is or gives rise to a long circulating material, i.e., they are long circulating material free (LCMF).
  • LCMF poloxamer preparations provided herein, and in particular LCMF poloxamer 188 preparations, exhibit a more uniform pharmacokinetic profile, and thus a more consistent therapeutic effect.
  • the LCMF poloxamer is described in more detail in the following section.
  • LCMF PI 88 Provided herein for use in thee compositions and methods is a long circulating material free (LCMF) PI 88 that is a purified PI 88 that has a polydispersity value less than 1.07; has no more than about 1.5% of low molecular weight (LMW) components less than 4,500 Daltons; no more than about 1.5% high molecular weight components greater than 13,000 Daltons; a half-life of all components in the distribution of the co-polymer that, when administered to a subject, 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.
  • LMW low molecular weight
  • LCMF Poloxamer 188 when administered, does not give rise to a component that has a significantly longer half-life than the main component.
  • the LCMF PI 88 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 (C3H6O) 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 7,680 to 9,510 Da, or 7350 to 8850 Da such as generally 8,400-8,800 Da, for example about or at 8,400 Da, where 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 also resulted in a long circulating material (LCM) containing higher molecular weight components that have an average molecular weight of about 16,000 Daltons, which exhibit about a 10-fold or more increase in half-life with a ti/ 2 of approximately 70 hours.
  • LCM long circulating material
  • the purified poloxamer designated LCMF PI 88
  • the purified poloxamer 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 PI 88 is different from prior LCM-containing pi 88 poloxamers.
  • LCMF poloxamer contains a substantially less polydisperse composition of less than 1.07, and generally less than 1.05 or 1.03, but where the half-life in the blood or plasma of any components in the distribution of the co-polymer, when administered to a human 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 (more than 5-fold) than or is more than the main component in the distribution of the co-polymer.
  • the half-life in the blood or plasma of all components in the 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.
  • HMW impurities greater than 13,000 Daltons in an LCMF preparation generally constitute no more than 1.5% by weight of the total component.
  • these HMW impurities do not 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 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 P188 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 in Section D, and in particular in Section D. l .b (see e.g., Figure 3).
  • an LCMF poloxamer provided herein is made by a method that includes:
  • the temperature is above the critical temperature of carbon dioxide but can typically range between 35°C - 45°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;
  • hydrophobic/hydrophilic (HLB) ratios The tunability of the processes can be leveraged to effectively remove extraneous components and can be adjusted over time, which can increase the yield of the purified product.
  • the method provided herein uses a solvent system that is variable in its solvation characteristics in order to selectively remove various substances.
  • the methods provide an exemplary way to produce the LCMF poloxamer 188 product, which has the above properties.
  • Methods herein provide poloxamer preparations that differ from those produced by prior methods. These include the LCMF poloxamer 188 preparation that, upon administration, does not give rise to long circulating material observed with purified poloxamer 188, such as that described in Grindel et al. 2002b.
  • the LCMF poloxamer 188 has the molecule size distribution similar to the purified poloxamer 188, but the component molecules produce a preparation that is more hydrophilic than purified poloxamer.
  • the absence of the long circulating material improves the properties of the poloxamer, including faster clearance and other such improved pharmacological properties by virtue of the elimination of the long circulating material.
  • the methods provided herein eliminate unwanted components in a poloxamer preparation, and thereby prepare a more homogenous or uniform poloxamer preparation that exhibits desired therapeutic activity while minimizing or reducing undesired activities. Because commercially available poloxamers have been reported to exhibit toxicity as well as variation in biological activity, a poloxamer preparation that is more uniform and homogenous has reduced toxicity but retains therapeutic efficacy of the main copolymer component.
  • LCMF poloxamers are provided herein.
  • the methods provided herein in addition to resulting in poloxamer preparations in which low molecular weight (LMW) components are reduced or removed, also result in long circulating material free (LCMF) preparations that are reduced or removed for any component that is or gives rise to a circulating material in the plasma or blood as described herein.
  • LMW low molecular weight
  • LCMF long circulating material free
  • LCMF poloxamer 188 are also provided herein.
  • the LCMF poloxamer 188 provided herein can be used for all of the uses known for poloxamer 188.
  • extraction methods for purifying poloxamers such as PI 88, in order to remove or reduce components other than the main component, and thereby decrease the molecular diversity of the preparation.
  • the methods provided herein can remove or reduce LMW substances in a poloxamer. It is also found herein, that, in addition to removing or reducing LMW substances, particular methods provided herein also can remove or reduce components in a poloxamer preparation that is or gives rise to a long circulating material that has a half-life that is substantially longer than the half-life of the main component in the distribution.
  • the degree of extraction, and components that are extracted, are controlled by the particular temperature, pressure and alkanol concentration employed in the methods as described herein.
  • the methods provided herein employ a supercritical or subcritical extraction solvent in which the solvent power is controlled by manipulation of temperature, pressure in 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 increases the solubilizing efficiency of C0 2 in the extraction solvent.
  • the methods provided herein 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.
  • 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 employing a gradient of higher concentrations of an alkanol (such as 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 such that, when the resulting product is administered to a subject, it does not result in a long circulating material in the plasma that is observed with the previous P188 products.
  • an alkanol such as methanol
  • the methods provided herein 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 long 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 purification in this manner.
  • Undesired components include any that are or give 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 creatinine, 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.
  • a HMW component such as a HMW component
  • Such components can increase blood viscosity and red blood cell aggregation, and hence are undesired.
  • poloxamers for use in the methods include, but are not limited to, poloxamer 188, poloxamer 331 and poloxamer 407.
  • the poloxamer is one in which the average molecular weight of the main component is within or about 4,700 Da to 12,800 Da, such as generally 7,680 Da to 9,510 Da, for example generally 8,400- 8,800 Da.
  • the poloxamer is PI 88.
  • the extraction methods provided herein can be employed to purify a PI 88 preparation, where the PI 88 preparation has the following chemical formula:
  • the hydrophobe represented by (C3H6O) has a molecular weight of approximately 1,750 Daltons and an average molecular weight of 7,680 to 9,510 Da, such as generally approximately 8,400-8,800 Daltons.
  • the polyoxyethylene:polyoxy- propylene:polyoxyethylene weight ratio of P188 is approximately 4:2:4.
  • P188 has a weight percent of oxyethylene of 81.8+1.9%, and an unsaturation level of 0.026+0.008 mEq/g.
  • P188 preparations for use in the extraction methods herein include commercially available preparations. These include, but are not limited to, Pluronic® F68 (BASF, Florham Park, N.J.) and RheothRx® (developed by Glaxo Wellcome Inc.).
  • the methods include: a) providing a poloxamer (e.g., P188) solution into an extractor vessel, where the poloxamer solution is prepared by dissolving the poloxamer in a first solvent to form the solution; b) admixing an extraction solvent containing a supercritical liquid (e.g., supercritical carbon dioxide) or sub-critical fluid (e.g., high pressure carbon dioxide) and a co-modifier solvent with the solution to form an extraction mixture, wherein the concentration of the co-modifier solvent 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 and/or other components), from the poloxamer.
  • a supercritical liquid e.g., supercritical carbon dioxide
  • sub-critical fluid e.g., high pressure carbon dioxide
  • 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.
  • the method can be a high pressure or supercritical fluid extraction method.
  • the method is performed using supercritical fluid extraction (SFE) using a supercritical liquid in the extraction solvent.
  • a supercritical liquid is any liquid that is heated above the critical temperature and compressed to above the critical pressure.
  • carbon dioxide has a critical temperature of 31.1° C. and a critical pressure of 73.8 bars.
  • extraction conditions for a supercritical carbon dioxide are above the critical temperature of about 31° C and critical pressure of about 74 bars.
  • high pressure extraction can be achieved under sub-critical conditions in which the pressure exceeds the critical pressure, but the temperature does not exceed the critical temperature.
  • the supercritical fluid extraction process employed in the methods provided herein is essentially a solvent extraction process using a supercritical fluid as the solvent.
  • supercritical fluid multi-component mixtures can be separated by exploiting 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 supercritical fluid exhibits a number of highly advantageous
  • 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 physicochemical 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.
  • a suitable solid matrix provides solvent-matrix and solute- matrix interactions in addition to solute-solvent interactions 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 presence of the co-modifier solvent can improve the solubility of solutes, such as higher molecular weight or more non-polar solutes, and thereby increase their extraction in the method.
  • the method provided herein can include: a) providing or introducing a poloxamer (e.g., a poloxamer 188) 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., a poloxamer 188
  • the poloxamer solution is prepared by dissolving the poloxamer in a first alkanol to form the solution
  • 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 depicts a process (100) that removes impurities (e.g., LMW component or other components) from a poloxamer preparation.
  • 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 110.
  • 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 approximately 40° C.
  • any suitable alkanol or combination of alkanols can be used in the methods provided herein.
  • suitable alkanols include, but are not limited to, methanol, ethanol, propanol and butanol.
  • 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 P188 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., P188) 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., P188
  • non-limiting amounts of poloxamer in methods of extraction using a 12 L extraction vessel, can be 1.5 kg to 5 kg, such as 2 kg to 4 kg.
  • non-limiting amounts of poloxamer in methods of extraction using a 50 L extraction vessel, non-limiting amounts of poloxamer (e.g., P188) can be 8 kg to 20 kg, such as 10 kg to 16 kg or 12 kg to 15 kg. Variations in the amounts are contemplated depending on the particular applications, extraction vessel, purity of the starting material and other considerations within the level of a skilled artisan.
  • 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
  • alkanol e.g., methanol
  • a quantity of poloxamer, such as P188 can be mixed with a lesser amount, by weight, of alkanol, such as half the amount, by weight, of alkanol (e.g., methanol).
  • alkanol e.g., methanol
  • P188 a quantity of poloxamer
  • alkanol e.g., methanol
  • alkanol e.g., methanol
  • the appropriate poloxamer to alkanol ratio will depend on poloxamer properties, such as solubility, in a given alkanol.
  • 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 liquids 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 the refrigerants sold as freons.
  • 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., supercritical carbon dioxide) is provided at a substantially constant flow rate.
  • a supercritical liquid condition e.g., supercritical carbon dioxide
  • the supercritical liquid e.g., supercritical carbon dioxide
  • 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, 1 kg/h to 20 kg/h, 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, 100 kg/h to 200 kg/h or 200 kg/h to 400 kg/h, each inclusive.
  • the flow rate is 20 kg/h to 100 kg/h, inclusive, such as generally
  • any suitable temperature that maintains the supercritical liquid in the supercritical state can be used to conduct the extraction processes.
  • 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 about 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 0 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 0 C but no more than 40 0 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.
  • 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 pressurized 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 typically is 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 15%, 7% to 12%, 7% to 10%, 7% to 9%, 7% to 8%, 8% to 15%, 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 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 11 : 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
  • the concentrations of the components can be altered by adjusting the flow rate.
  • a method in which the second alkanol is increased as the extraction process progresses 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.
  • the solubility of impurity enriched extractables is low and it takes time to significantly reduce the low molecular weight components, making it less efficient.
  • higher 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 in the extraction solvent can progressively extract low molecular weight components, as well as eventually higher molecular weight components, or components that are 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 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 the methanol concentration in the solvent flow. In a typical extraction system there is adequate head space for proper phase separation of the upper phase.
  • Increasing the methanol co-solvent 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., carbon dioxide), such as about 5% to about 10%, such as 6% to 8% (e.g. , about 6.6% or 7.4%).
  • a supercritical liquid e.g., carbon dioxide
  • the alkanol (e.g., methanol) content of the extraction solvent is raised about 1-3%, such as 1-2% (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.
  • the alkanol (e.g., methanol) concentration in the supercritical liquid (e.g., carbon dioxide) can be increased from about 5% to about 20% over the course of extraction procedure.
  • the alkanol (e.g., methanol) concentration in the 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%.
  • methanol concentration in the 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
  • the alkanol (e.g., methanol) concentration 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.
  • 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.
  • 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 reductions in 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:
  • a polyoxypropylene/polyoxyethylene block copolymer having the formula HO(CH 2 CH20)a'-[CH(CH3)CH20]b-(CH2CH 2 0)aH, the mean or average molecular weight of the copolymer is from about 4,000 to about 10,000 Da;
  • 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 7,680 to 9,510 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.
  • the method provided herein to purify a poloxamer can be a high pressure fluid extraction method with mixed solvent systems.
  • One of the solvents in the mixed system is a gaseous solvent that can be compressed to liquid at moderate pressures, such as carbon dioxide.
  • the solvent power of methanol or ethanol can be modified with high pressure carbon dioxide (although not necessarily supercritical carbon dioxide i.e. , sub-critical) to give the precise solvating power required to selectively remove different fractions of poloxamers.
  • the extraction solvent contains carbon dioxide that is provided under sub-critical conditions, as well as another solvent that is increased over time in the extraction. Accordingly, some embodiments of methods provided herein provide an extraction method for removing impurities in a poloxamer preparation (e.g., low molecular weight components), wherein the method includes:
  • the first and second solvent can be the same or different.
  • the step of dissolving the poloxamer solution in the first solvent can occur prior to providing or introducing the solution into an extraction vessel or at the time of providing or introducing the solution into an extraction vessel.
  • the poloxamer is dissolved in a separate vessel and then the solution is added to the extraction vessel.
  • the extraction solvent is under sub-critical conditions.
  • one of the solvents is preferably a gas at room temperature (or close to room temperature) that can be compressed to a liquid at high pressures.
  • gases that can be compressed to liquids are carbon dioxide, methane, ethane, propane, ammonia, and refrigerants sold as Freon®.
  • a typical solvent pair is chosen in such a way that one is a solvent for the component to be removed by extraction, while the other liquid is a non-solvent, or vice-versa.
  • the solvating capacity of the solvent pair is primarily controlled by the ratio of the solvents in the mixture.
  • Gaseous solvents can be pressurized at any suitable sub-critical pressure.
  • carbon dioxide can be employed at a pressure of from about 25 bars to about 100 bars.
  • the pressure can be about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 bars.
  • the pressure is from about 60 to about 85 bars. In some embodiments, the pressure is about 75 bars.
  • the extractor vessel has a temperature of 10°C to 80°C.
  • the temperature can be, for example, about 10°C, or about 15°C, or about 20°C, or about 25°C, or about 30°C, or about 35°C, or about 40°C, or about 45°C, or about 50°C, or about 55°C, or about 60°C, or about 65°C, or about 70°C, or about 75°C, or about 80°C.
  • the extractor vessel has a temperature of from about 20°C to about 50°C.
  • the extractor vessel can have a temperature of from about 20°C to about 60°C (e.g., about 40°C). Other temperatures can be suitable for purification of poloxamer 188 depending on the extraction apparatus and the chosen extraction parameters. One of skill in the art will appreciate that 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. [00212] Similar to supercritical fluid extraction methods discussed above, 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 carbon dioxide and solvent (e.g., methanol) in the extraction solvent 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 carbon dioxide and/or other solvent (e.g., methanol) that make up the extraction solvent.
  • the carbon dioxide is kept constant while the concentration of the other solvent (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.
  • the particular concentration of solvent, and the gradient of concentrations employed, can be similar to those discussed above with respect to the supercritical extraction methods. It is within the level of a skilled artisan to adjust concentrations and extraction time appropriately to achieve a desired purity or yield.
  • 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 benefits of the mixed solvent system include effective removal of high molecular weight (HMW) substances and/or low molecular weight (LMW) substances using the mixed system.
  • HMW high molecular weight
  • LMW low molecular weight
  • the provided methods provide a method for preparing a purified polyoxypropylene/composition.
  • the method includes:
  • a plurality of low molecular weight substances having a molecular weight of less than 4,000 Da, wherein the plurality of low molecular weight substances constitutes more that 4% of the total weight of the composition; b) adding a second solvent to form an extraction mixture, wherein the second solvent comprises high-pressure carbon dioxide and the first solvent, and the
  • concentration of the first 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 7,680 to 9,510 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.
  • this approach does not have the density variation and permeability characteristics of the supercritical fluid extraction process.
  • the solvent recycling is easy and energy efficient.
  • the exit stream containing the extracted component is subjected to lower pressure that causes phase separation and separation of the more volatile solvent as a gas. This leaves the other solvent enriched with the extracted component.
  • the extraction process continues until the extractable component is substantially depleted from the mixture.
  • the gaseous solvent is compressed back into liquid and is available for continued extraction.
  • This solvent recycling process is efficient because the compressible solvent is selected to have complete separation from the solvent mixture with minimum change in the pressure.
  • system 200 in FIG. 4 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 and/or other components) from the poloxamers using supercritical fluids or sub-supercritical methods.
  • Polymer feed pump 201 is charged with a poloxamer (e.g., P188) to be purified. Poloxamer is transported into polymer feed tank 207 through valve 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 valve 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. In such embodiments, methanol and carbon dioxide extract impurities, such as LMW substances or other components, from the poloxamer in extractor 215. After extraction, 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.
  • 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.
  • 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., P188) 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 examples of the solvents.
  • 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., P188) 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.
  • 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.
  • drying of the product can be initiated, for example on a sub-lot, under vacuum at ambient temperature. Drying can be then continued at higher temperatures and lower pressures as the process progresses. If necessary, for example if collection was made in sub-lots, 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 extract phase can be further processed.
  • the methods further can include: passing the extract phase to a system consisting of several separation vessels; isolating the impurities (e.g., low molecular-weight impurities); processing the purified material or raffinate; and 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.
  • poloxamer 188 including purified LCM-containing poloxamer 188, as well as other poloxamers are known, including commercial sources therefor.
  • the LCMF poloxamer and exemplary methods of preparation are described herein (see also International PCT Application No. (PCT/US2015/039418) and U.S. Application Serial No. (14/793,670)). Both incorporated herein by reference in their entireties.
  • the methods provided herein above result in the generation of particular purified poloxamer preparations, and in particular LCMF PI 88 preparations.
  • the methods provided herein can be used to purify a P188 copolymer as described herein that has the formula: HO(CH 2 CH 2 0) a -(CH 2 CH(CH3)0)b-(CH2CH 2 0)aH, and a mean or average molecular weight of the copolymer that is from 7,680 to 9,510 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 4,000 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, i.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 are or give 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.
  • the following discussion details an exemplary of method that produces such purified poloxamer.
  • FIG. 2 depicts certain embodiments of the methods herein that 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 110'.
  • 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
  • a poloxamer solution is used to form a poloxamer solution according to step 115' in process 100'.
  • dispensing of a P188 poloxamer into the feed tank with the alkanol (e.g., methanol,) results in a P188 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 performed 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 then is 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 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
  • 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.
  • 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.
  • FIG. 3 depicts embodiments for preparation of LCMF poloxamer.
  • Certain embodiments of the methods herein provide a process 100" that generates a poloxamer that does not contain any components that, after administration to a subject, results in a long circulating material in the plasma or blood as described herein.
  • the poloxamer and first alkanol e.g., methanol
  • the poloxamer and first alkanol are dispensed into the extractor vessel and to form the poloxamer solution.
  • a P188 poloxamer into the extraction vessel with the alkanol (e.g., methanol,) results in a P188 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 110", after dispensing a first alkanol (e.g., methanol) and poloxamer. As shown in step 115", 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.
  • a first alkanol e.g., methanol
  • poloxamer e.g., methanol
  • the temperature is between 35° C and 45° 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 between 35° C to about or at 45°C for a defined period, generally less than several hours.
  • the extractor then is 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 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 247 + 15 atm bars (range between 240 to 260 bar), and the carbon dioxide is provided at a flow rate that is 50 kg/h to 120 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., P188
  • an alkanol e.g., methanol
  • an extraction solvent with a supercritical liquid e.g., carbon dioxide
  • 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 produces a 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 of between 35°C to 45°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".
  • LCMF poloxamer 188 preparations have different properties from poloxamer
  • the LCMF poloxamer 188 which lacks the LCM material, is more hydrophilic, and can be distinguished based on this property.
  • the properties of the poloxamer can be assessed. The properties include, but are not limited to, the absence of a long circulating material upon administration to a human or an animal model, the behavior of the poloxamer in reverse phase (RP)-HPLC compared to a preparation of poloxamer that contains the LCM material such as the poloxamer described in U.S. Patent No.5, 696,298 and commercially available poloxamer
  • Example 1 Any method that confirms that the preparation lacks LCM material can be used.
  • compositions containing poloxamers particularly a poloxamer 188, including any prepared by methods described herein and/or known to those of skill in the art, are provided.
  • Compositions containing an LCMF poloxamer 188 are provided.
  • the concentration of poloxamer is such that it achieves a target plasma concentration for a time sufficient to effect treatment. The particular time and concentration depends upon the target plasma concentration, the mode of administration, the duration of
  • low doses of poloxamer generally are used, so that the target circulating concentrations typically are at or about 0.25 - 2.5 mg/ml.
  • the poloxamer composition is administered in conjunction with fibrinolytic inhibitor treatment, such as for treating hemorrhagic shock.
  • the poloxamer and fibrinolytic inhibitors can be administered in separate compositions, simultaneously, sequentially or intermittently or can be administered in the same composition.
  • the fibrinolytic inhibitors are any known to those of skill in the art, and the dosage is the therapeutic dosage for the particular fibrinolytic inhibitor.
  • compositions containing the poloxamer and a fibrinolytic inhibitor are provided herein.
  • the poloxamer is in an amount that is therapeutically effective to mitigate adverse effects of the fibrinolytic inhibitor, which is present in the composition in an amount that is therapeutically effective for treatment.
  • the fibrinolytic inhibitor When administered separately, the fibrinolytic inhibitor is administered in a therapeutically effective dosage, and the poloxamer is administered to achieve a circulating concentration that mitigates any adverse effects or potential adverse effects of the fibrinolytic inhibitor.
  • the poloxamer can be administered first, and can be used to prevent the adverse effects.
  • the poloxamer can be administered with the fibrinolytic inhibitor, or after the fibrinolytic inhibitor. It can be administered after adverse effects are observed. Generally it is administered shortly before, with or shortly after to prevent the potential adverse effects of the fibrinolytic inhibitor.
  • the fibrinolytic inhibitor is administered to mitigate adverse effects of poloxamer, particularly poloxamer 188 therapy.
  • the dosage of poloxamer is appropriate for treatment of any particular disorder.
  • the fibrinolytic inhibitor is administered to mitigate any adverse effects, particularly bleeding.
  • compositions containing P188 can be formulated in any conventional manner by mixing a selected amount of the poloxamer with one or more physiologically acceptable carriers or excipients to produce a formulation. Selection of the formulation carrier and/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.
  • mode of administration i.e., systemic, oral, nasal, pulmonary, local, topical, or any other mode
  • Effective concentrations of PI 88 are mixed with a suitable pharmaceutical carrier or vehicle for systemic, topical, or local administration.
  • a suitable pharmaceutical carrier or vehicle for systemic, topical, or local administration.
  • the poloxamer is administered by IV, such as by continuous infusion or a series of bolus injections.
  • compositions 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.
  • Pharmaceutical compositions that include a therapeutically effective amount of a P188, such as an LCMF P188, also can be provided as a lyophilized powder that is reconstituted, such as with sterile water, immediately prior to administration.
  • the compositions can be prepared for dilution prior to administration or for direct administration. In general, for the methods herein, the compositions are
  • the target circulating concentration is at least 0.5 mg/mL, and can be as high as 15 mg/mL, but generally is up to and includes 1.5 mg/mL or 2 mg/mL. This level is maintained for a sufficient number of hours to effect treatment, typically at least 12 hours to 1 to 3 days or 4 days to reduce or eliminate undesirable risks and complications associated with administration of a fibrinolytic inhibitor and/or to prevent the risk of developing such risks/complications .
  • the compound can be suspended in micronized form 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 P188, such as LCMF P188, 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.
  • the poloxamers can be formulated as a solution suspension in an aqueous-based medium, such as isotonically buffered saline or can be combined with a biocompatible support or bioadhesive intended for internal administration.
  • an aqueous-based medium such as isotonically buffered saline
  • the compositions typically are aqueous solutions, suspensions, or emulsions for IV administration.
  • compositions are prepared in view of approvals for a regulatory agency or are prepared in accordance with generally recognized standards for use in animals and in humans.
  • the methods provided herein have applications for both human and animal use.
  • 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 and saline solutions are typical carriers 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 a poloxamer, such as P188, such as LCMF P188: a diluent, such as lactose, sucrose, dicalcium phosphate, or
  • carboxymethylcellulose such as a 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,
  • 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
  • compositions can take the form of solutions, suspensions, or emulsions for IV administration.
  • 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 " E. W. Martin (ed.), Mack Publishing Co., Easton, PA, 19 th Edition (1995).
  • Such compositions will contain a therapeutically effective amount of P188, 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.
  • the 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.
  • compositions containing 188 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 saline, such as citrate buffered saline.
  • Sterile, fixed oils can be employed as a solvent or suspending medium.
  • 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 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, but are not limited to, aqueous and non-aqueous sterile injection solutions, which can contain antioxidants, buffers, bacteriostats and solutes that render the formulation compatible with the intended route of administration.
  • the formulations can be prepared in unit-dose or multi-dose form by conventional pharmaceutical techniques, for example, including bringing the active ingredient, e.g., P188, such as LCMF P188, into association with the pharmaceutical carrier(s) or excipient(s).
  • 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 or in a dried or freeze- dried (lyophilized) conditions, requiring only the addition of the sterile liquid carrier, for example, water or saline for injection, immediately prior to use.
  • sterile liquid carrier for example, water or saline for injection
  • PI 88 such as LCMF PI 88
  • LCMF PI 88 can be formulated as the sole pharmaceutically active ingredient in the composition or can be combined with other active ingredients.
  • 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. 4,522,811. 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. (1985) J. Pharm. Sci.
  • the P 188 such as LCMF P 188 , 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 P188 can be formulated for single dosage (direct) administration, multiple dosage administration, or for dilution or other modification.
  • the compositions containing poloxamer PI 88, such as LCMF PI 88 provided herein are formulated to achieve a targeted circulating concentration of poloxamer, e.g., LCMF P188, in the circulation of the subject at a desired time point after administration.
  • This target for the uses and methods herein is at least 0.05 mg/mL, typically 0.25 mg/mL to 2.5 mg/mL, or higher, if needed to mitigate adverse effects of a fibrinolytic inhibitor.
  • compositions for administration can readily formulate a composition for administration in accord with the methods herein.
  • 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 risks and consequences associated with administration of a fibrinolytic inhibitor are improved and/or the intended effect is observed.
  • the precise amount or dose of the therapeutic agent administered depends on the condition being treated, the route of administration, and other considerations, such as the weight and physiological state of the subject, and the subject. Routine procedures that adjust for physiological variables (including, but not limited to, kidney and liver function, age, and body weight and or body surface area) can be used to determine appropriate dosing regimens.
  • Local administration of the therapeutic agent typically requires 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 safely upon systemic administration.
  • a particular dosage and duration and treatment protocol can be empirically determined or extrapolated.
  • exemplary doses P188 such as LCMF P188 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 disorder or condition and the response of the subject to the treatment, and can be adjusted
  • LCMF P188 Factors such as the level of activity and half-life of the P188, such as LCMF P188, can be taken into account when making dosage determinations. Particular dosages and regimens can be empirically determined by one of skill in the art.
  • the poloxamer can be formulated at a concentration ranging from about 10.0 mg/mL to about 300.0 mg/mL or 10.0 to 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, 85.0, 90.0, 95.0, 100.0, 105.0, 110.0, 115.0, 120.0,125.0, 130.0, 135.0, 140.0, 145.0, 150.0, 155.0, 160.0, 165.0, 170.0, 175.0, 180.0, 185.0, 190.0, 195.0 or 200.0 mg/mL, for direct administration.
  • the concentration is not more than 22.5%, i.e., 225 mg/mL.
  • the selected amount to administer can be determined for a particular target plasma concentration and duration.
  • the poloxamer is administered at a concentration of between about 0.5% to 20%, although more dilute or higher
  • the poloxamer can be administered in an amount between about 0.5% to about 20% by weight/volume, such as 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, 10.0%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5% or 20% by weight/volume.
  • 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%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10.0% by weight/volume.
  • the poloxamer is administered in an amount between about 5% to 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%, 11%, 11.5%, 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 is formulated so that administration of the poloxamer to a subject results in an effective amount of poloxamer, such as a P188, such as LCMF P188, in the circulation of the subject.
  • a P188 such as LCMF P188
  • the poloxamer such as a PI 88, such as LCMF PI 88, is formulated so that administration of a single dose of the poloxamer to a subject results in an effective amount of poloxamer in the circulation of the subject to prevent, treat or mitigate adverse effects of administration of a fibrinolytic inhibitor.
  • the poloxamer is formulated so that repetitive
  • administration of the poloxamer to a subject results in an effective amount of poloxamer in the circulation of the subject.
  • the repetitive treatment is sufficient to result in a concentration of the poloxamer in the circulation of the patient of from about 0.05 mg/mL to about 15.0 mg/mL, or about 0.05 mg/mL to about 10.0 mg/mL, or about 0.5 mg/mL to about 2 mg/mL, for example, from about 0.2 mg/mL to about 4.0 mg/mL.
  • the concentration of the poloxamer, such as LCMF P188, in the circulation of the subject is from about 0.2 mg/mL to about 4.0 mg/mL, such as 0.5 mg/mL to about 2.0 mg/mL, e.g. , about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5 or 4.0 mg/mL.
  • repetitive administration of poloxamer, e.g., LCMF P188 results in a concentration of the poloxamer in the circulation of the subject of about 0.5 mg/niL.
  • the poloxamer can be formulated as a sterile, non-pyrogenic solution intended for administration with or without dilution.
  • the final dosage form can be prepared in a 100 mL vial, where the 100 mL contains 15g (150 mg/ml) of a purified poloxamer 188 such as LCMF P188, 308 mg sodium chloride USP, 238 mg sodium citrate USP, 36.6 mg citric acid USP, and water for injection USP, Qs (quantity sufficient) to 100 mL.
  • the pH of the solution is approximately 6.0 and has an osmolality of about 312 mOsm/L.
  • the solution is sterilized prior to administration to a subject.
  • 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.
  • This dosage ranges provided herein are not intended to be limiting, and vary based on the needs and response of the individual subject, the particular subject, as well as the properties of the particular poloxamer chosen for administration.
  • Fibrinolytic inhibitors are used to treat or prevent excessive bleeding, in bleeding disorders, during surgery, from trauma and injury, and hemorrhagic shock. Fibrinolytic inhibitors are agents that result in either a decreased amount or a decrease in the activity of the proteolytic enzyme plasmin.
  • the fibrinolytic inhibitors can include endogenous and pharmaceutical fibrinolytic inhibitors.
  • Endogenous fibrinolytic inhibitors include: plasminogen activator inhibitors 1, 2, and 3 (PAI-1, PAI-2, PAI-3), alpha 2 antiplasmin, alpha 2 macroglobulin and thrombin-activatable fibrinolysis inhibitor.
  • Pharmaceutical fibrinolytic inhibitors include, but are not limited to, the polypeptide aprotinin and synthetic derivatives of lysine such as ⁇ -aminocaproic acid (EACA or ACA) and the more potent tranexamic acid (TXA).
  • EACA or ACA ⁇ -aminocaproic acid
  • TXA more potent tranexamic acid
  • a commercial formulation of TXA is available as Cyklokapron® TXA. Each ml of Cyklokapron contains 100 mg TXA in water for injection.
  • the fibrinolytic inhibitor is tranexamic acid.
  • Fibrinolytic inhibitors such as tranexamic acid
  • Tranexamic acid is an antifibrinolytic that competitively inhibits the activation of plasminogen to plasmin by binding to specific sites of both plasminogen and plasmin, a molecule responsible for the degradation of fibrin.
  • Typical doses of EACA for an adult is an infusion of 4-5 g in 250 ml of diluent during the first hour, followed by a continuous infusion of 1 g per hour in 50 ml of diluent until the bleeding situation has been controlled.
  • EACA is available for oral administration, such as 5 g tablets and 20 ml syrup (25%), each administered during the first hour, followed by 1 g per hour until the bleeding situation has been controlled.
  • TA is administered intravenously, orally, or topically.
  • the intravenous dosage is generally 10 mg/kg, 3 to 4 times daily. Orally the dosage is 15 to 20 mg/kg, 3 to 4 times daily.
  • the first intravenous dose is given immediately before starting; if the first dose is administered orally, it is administered two hours before the procedure.
  • TA for topical administration is available as a mouthwash, 10 ml of a 5% aqueous solution is equal to 0.5 g if swallowed.
  • TA also is used as a constituent in some types of fibrin glue.
  • any suitable dosage and regimen of the fibrinolytic inhibitor is contemplated.
  • Suitable compositions for injection, by IV or bolus, containing the poloxamer and the fibrinolytic inhibitor can be administered. Such compositions are provided. The skilled medical practitioner can determine appropriate concentrations and amounts of each.
  • compositions that contain a poloxamer, particularly poloxamer 188, and a fibrinolytic inhibitor. Also provided are combinations, and kits, containing two compositions: a first composition containing the fibrinolytic inhibitor as described herein and known to those of skill in the art; and second composition containing a poloxamer, particularly a poloxamer 188, including an LCMF poloxamer 188. Uses of the compositions and combinations for treatment and methods of treatment of
  • hemorrhagic shock and precursors thereto, and any bleeding disorder and hemostatic dysfunction are provided.
  • the uses and therapy include combination therapy with a fibrinolytic inhibitor and the poloxamer, each which mitigate adverse effects of the other.
  • any suitable ratio of poloxamer to fibrinolytic inhibitor is used in the methods, uses and compositions herein.
  • the amount of poloxamer 188 administered in conjunction with fibrinolytic inhibitor therapy is an amount that achieves a circulating concentration of less than about or less than 2.5 mg/ml, such as about or at 0.25 to 2.5 mg/ml or 0.5 mg/ml to 1.5 mg/ml, but lower or higher concentrations can be used if appropriate as ascertained by the skilled person.
  • the amount of fibrinolytic inhibitor typically is a therapeutic dosage thereof. The precise ratios and dosages readily can be determined.
  • the ratio of poloxamer 188 to fibrinolytic inhibitor is from about 0.001: 1 to about 1000: 1 by weight.
  • the ratio of poloxamer 188 to fibrinolytic inhibitor can be, for example, about 0.001: 1, or about 0.01: 1, or about 0.1: 1, or about 1: 1 or about 10: 1, or about 100: 1 or about 1000: 1.
  • the ratio of poloxamer 188 to fibrinolytic inhibitor is from about 1:500 to about 500: 1 by weight.
  • the ratio of poloxamer 188 to fibrinolytic inhibitor can be, for example, about 1:500, or about 1:50, or about 50: 1, or about 500: 1 by weight.
  • the ratio of poloxamer to fibrinolytic inhibitor can be about 1:5, or about 1 :4, or about 1:3, or about 3: 1, or about 4: 1, or about 5: 1 by weight.
  • the target concentration of the poloxamer and fibrinolytic inhibitor in the circulation is generally maintained for 4 - 72 hours, although this time is not meant to be limiting.
  • the amount of poloxamer and fibrinolytic inhibitor dosed to achieve the target concentration can be readily determined by one of ordinary skill in the art. Routine procedures that adjust for physiological variables (including, but not limited to, kidney and liver function, age, and body weight) can be used to determine appropriate dosing regimens.
  • the effective amounts of a poloxamer and a fibrinolytic inhibitor may be delivered by administration of either agent alone or in combination immediately prior to, concomitant with or immediately following the other agent.
  • the effective amount may result from administration either once or multiple times by various routes of
  • the effective amount of poloxamer generally leads to a plasma concentration of between about 0.1 mg/ml and about 10.0 mg/ml in the subject depending upon its application.
  • the plasma concentration is less than about or less than or at 3.5, 3.0, 2.5 mg/ml, such as 0.25-2.5 mg/ml, as noted above. This range is not intended to be limiting, however, and varies based on the needs and response of the individual patient, the condition treated, as well as the properties of a particular poloxamer and fibrinolytic inhibitor chosen for administration.
  • the poloxamer is administered by the intravenous route either by bolus or by continuous infusion although other routes may be used.
  • the effective amount of the fibrinolytic inhibitor depends on the potency of the fibrinolytic inhibitor.
  • the target plasma concentrations can be between 0.05 mg/mL and 3.0 mg/mL.
  • Target plasma concentrations for ACA a less potent fibrinolytic inhibitor, can be between 0.5 mg/niL and 30 mg/niL. These concentrations are not intended to be limiting; the concentration of fibrinolytic inhibitor vary based on the needs and response of the individual patient.
  • the poloxamer When administered separately or as a component of the pharmaceutical composition described herein, the poloxamer is administered at a concentration of between 0.5% to 15% although more dilute or higher concentrations can be used.
  • the fibrinolytic inhibitor is administered either by the intravenous or oral route although other routes of administration can be employed.
  • the route generally preferred is intravenous administration although other routes may be used.
  • the fibrinolytic inhibitor is typically at a concentration of between 0.1% and 10% although more dilute or higher concentrations can be used.
  • a commercially available preparation can be used.
  • the poloxamer such as a purified poloxamer 188 or LCMF P188 described herein, is administered to a subject for reducing or preventing the risks or complications associated with administration of a fibrinolytic inhibitor.
  • these risks can be associated with administration of a fibrinolytic inhibitor, and in particular any risk or consequence associated with administration of a fibrinolytic inhibitor during surgery or after trauma.
  • poloxamer 188 such as a purified poloxamer 188 and LCMF P188 described herein, is intended for use in methods in which administration of a fibrinolytic inhibitor, such as known fibrinolytic inhibitors, for controlling blood loss, particularly during surgery or after trauma, results in ischemic tissue damage and subsequently causes unwanted consequences.
  • a fibrinolytic inhibitor such as known fibrinolytic inhibitors
  • a fibrinolytic inhibitor with poloxamer 188 such as a purified poloxamer 188 and LCMF P188 described herein
  • a fibrinolytic inhibitor with poloxamer 188 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 administration. Treatment typically is effected by intravenous administration of the poloxamer.
  • Active agents for example a poloxamer 188, such as an LCMF PI 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 poloxamer results in a concentration of the poloxamer in the circulation of the subject, i.e., a targeted plasma concentration, of between about 0.05 mg/mL and about 15.0 mg/mL in the subject, particularly 0.05-3 mg/ml, such as less than 2.5 mg/ml, including 0.25 mg/ml to 2.5 mg/ml or 0.5 mg/ml to 1.5 mg/ml, and the concentrations described elsewhere herein.
  • the poloxamer such as poloxamer 188, such as an LCMF PI 88, can be administered for the prevention or reduction of the risks associated with the
  • a fibrinolytic inhibitor such as, for example, administration of a fibrinolytic inhibitor during surgery or following trauma.
  • the need for such treatment 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, and the seriousness of the risk of ischemic tissue damage and/or thromboembolic events.
  • methods of treatment with poloxamer 188 require a longer duration of action in order to effect a sustained therapeutic effect.
  • the half-life of the purified poloxamer 188 and of the LCMF poloxamer 188 is described in detail elsewhere herein.
  • the effects of a poloxamer, such as a purified poloxamer 188 can be long lasting.
  • the poloxamer 188 described herein can be used to deliver longer lasting therapies for the prevention of the risks associated with administration of a fibrinolytic inhibitor, for example, including ischemic tissue damage.
  • the poloxamer is administered by IV to achieve and maintain a target concentration of at least 0.25 mg/mL up to about 3.5 mg/mL, 3.0 mg/mL or 2.5 mg/mL for sufficiently long to effect treatment and mitigate, treat or prevent adverse effects of administration of the fibrinolytic inhibitor. This includes at least 12 hours, 1 day, 2 days, 3 days, and up to 4 days.
  • a particular dosage and duration and treatment protocol can be empirically determined or extrapolated.
  • the amount depends on various parameters including the dosage of the fibrinolytic inhibitor.
  • Particular dosages and regimens can be empirically determined based on a variety of factors. Such factors include body weight of the individual, general health, age, the activity of the specific compound employed, sex, diet, time of administration, rate of excretion, drug combination, the severity and course of the side effects, and the patient's disposition to the side effects and the judgment of the treating physician.
  • 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.
  • a 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, 0.4 mL/kg to 1.4 mL/kg, 0.4 mL/kg to 1.0 mL/kg, 0.4 mL/kg to 0.6 mL/kg, 0.6 mL/kg to 3.0 mL/kg, 0.6 mL/kg to 2.5 mL/kg, 0.6 mL/kg to 2.0 mL/kg, 0.6 mL/kg to 1.8 mL/kg, 0.6 mL/kg to 1.4 mL/kg, 0.6 mL/kg,
  • the particular volume chosen is one that results in the desired target concentration of poloxamer in the circulation of the subject after administration.
  • the particular volume and dosage is a function of the target circulating concentration, which for preventing or reducing the risks or consequences associated with administration of a fibrinolytic inhibitor, is described herein.
  • formulations used in the methods provided herein can be administered by any appropriate route, for example, orally, nasally, pulmonary, intrapulmonary, 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 routes 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 as described herein, can be effected via any route or combination of routes. The most suitable route for administration depends upon the condition treated and the needs of the individual and other parameters.
  • the administered dose is administered as an infusion.
  • the infusion is an intravenous (IV) infusion.
  • the poloxamer such as P188, such as an LCMF P188, can be administered as a single continuous IV infusion, a plurality of continuous IV infusions, a single IV bolus administration, or a plurality of IV bolus administrations.
  • the poloxamer is administered by other routes of administration, for example, subcutaneous or intraperitoneal injection, to achieve the desired concentration of poloxamer in the circulation in the subject after administration.
  • the poloxamer is administered as an IV 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.
  • the poloxamer is administered to the subject in combination with a fibrinolytic inhibitor, which is administered for treatment of the underlying condition.
  • the poloxamer can be administered to the subject prior to, concomitant with, or after administration of the other agent, for example, a fibrinolytic inhibitor.
  • a poloxamer such as PI 88, such as LCMF PI 88, can be administered in combination with one or more fibrinolytic inhibitors, such as therapeutically effect amounts of a fibrinolytic inhibitor.
  • the methods in which the poloxamer PI 88, such as LCMF PI 88, is administered in combination with a fibrinolytic inhibitor are where treatment with a fibrinolytic inhibitor results in or may increase the risk of consequences, such as undesirable consequences, e.g., ischemic tissue damage or thromboembolic events.
  • the poloxamer can be administered before or with the other agent to prevent the
  • fibrinolytic inhibitors for use in the methods provided herein include any known to those of skill in the art, and those described above.
  • endogenous fibrinolytic inhibitors for example, plasminogen activator inhibitors 1, 2, and 3 (PAI- 1, PAI-2, PAI-3), alpha-2- antiplasmin, alpha-2-macroglobulin, and thrombin-activatable fibrinolysis inhibitor (TAFI), and pharmaceutical fibrinolytic inhibitors, for example, polypeptide aprotinin (Ap) and synthetic derivatives of lysine, such as ⁇ -aminocaproic acid (ACA) and tranexamic acid (TA; Cyklokapron®), and combinations thereof.
  • PAI- 1, PAI-2, PAI-3 plasminogen activator inhibitors 1, 2, and 3
  • TAFI thrombin-activatable fibrinolysis inhibitor
  • pharmaceutical fibrinolytic inhibitors for example, polypeptide aprotinin (Ap) and synthetic derivatives of lysine, such as ⁇ -aminocaproic acid (ACA) and tranexamic acid (TA; Cyklokapro
  • the provided methods include administering to the subject a therapeutically effective amount of a composition that contains the polyoxyethylene/polyoxypropylene copolymer (poloxamer) having the chemical formula HO(C 2 H 4 0) a '— (C 3 H60)b— (C 2 H 4 0)aH, as described herein and/or known to those of skill in the art, to treat damaged or injured tissue; and administering a therapeutically effective amount of a fibrinolytic inhibitor.
  • the poloxamer can be administered to the subject prior to, concomitant with, or after administration of a fibrinolytic inhibitor or other treatment, or any combination thereof. The amount and duration of poloxamer administration is sufficient to maintain a target blood concentration that effects treatment.
  • Target blood concentrations can depend upon the particular poloxamer, the subject to whom it is administered, the condition treated, underlying conditions and the severity of the tissue damage or injury. Dosages are described herein and also can be determined empirically by the skilled artisan. Generally, the target dosage is one that achieves a circulating concentration of at least 0.05 mg/mL, typically at least 0.5 mg/mL, and generally a range of 0.5 mg/mL to 1.5 mg/mL.
  • the therapeutically effective amount of poloxamer is an amount that results in a concentration of poloxamer in the circulation of the subject of from about or at 0.2 mg/mL to about or at 4.0 mg/mL, for example, about 0.5 mg/mL to 1.5 mg/mL or at least 0.5 mg/mL, at a desired time point, typically steady- state, after administration of the poloxamer.
  • Other ranges are contemplated as well, such as 0.05 mg/mL to 3.0 mg/mL, 0.05 mg/mL to 10 mg/mL, 0.5 mg/mL to 10 mg/mL, and others described herein.
  • Dosages for other treatments and therapeutics that are concomitantly administered or administered prior to administration of the poloxamer depend upon the particular therapeutic and condition treated and the regimen.
  • dosages for fibrinolytic inhibitors are typically the recommended doses for such inhibitors, for example, dosages described in standard manuals, including the Physician's Desk
  • the provided methods include administration of the poloxamer where a subject suffering from hemorrhagic shock has been administered a fibrinolytic inhibitor.
  • the poloxamer mitigates a consequence of administering a fibrinolytic inhibitor, including ischemic tissue damage and prothrombotic events, such as embolism and thrombosis.
  • the polyoxyethylene/polyoxypropylene copolymer poloxamer can be administered to treat, prevent or reduce the risk of the complications of treatment with fibrinolytic inhibitors.
  • the poloxamer can be
  • the polyoxyethylene/polyoxypropylene copolymer is administered after the fibrinolytic inhibitor is administered or symptoms occur.
  • administration of the poloxamer can be repeated, for example, a second, third, fourth time, or more.
  • the method can be repeated until administration of the poloxamer is sufficient to result in a concentration of the poloxamer in the circulation of the subject of from about 0.05 mg/mL to about 10 mg/mL, about 0.05 mg/mL to about 4.0 mg/mL, or about 0.2 mg/mL to about 2.0 mg/mL.
  • administration of the inhibitor can be repeated, for example, a second, third, fourth time, or more.
  • a multi-step extraction batch process of poloxamer 188 was performed with extraction conducted at a pressure of 247 + 15 atm (approximately 200 - 260 bars) and a controlled step-wise increase of methanol of 7.4, 9.1 and 10.7 weight % methanol.
  • the poloxamer 188 raw material was characterized by Gel Permeation Chromatography (GPC). Molecular weight analysis demonstrated that raw material had an average molecular weight of the main peak of about 8,500 + 750 Da, no more than 6.0 % low molecular weight (LMW) species of less than 4,500 Da and no more than 1 % high molecular weight species (HMW) greater than 13,000 Da. In addition, the 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 35°C to 50°C for 40-80 minutes until a homogenous solution was obtained.
  • CO2 supplied either from a main supply tank or via recycling through an extraction system
  • a high-pressure pump increased the pressure of liquid CO2 to the desired extraction pressure.
  • the high pressure CO2 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 CO2 solvent stream to produce the extraction methanol/CC cosolvent, which was fed through inlet systems into the extractor vessel as a fine mist at a pressure of 247 + 15 atm (3600 + psi) or 240 to 260 bars and a temperature of 40 °C.
  • a 7.4% methanol/C extraction cosolvent was percolated through the poloxamer solution for 3 hours at a methanol flow rate typically at 8 kg/hr (range 6.8 kg/hr to 9.2 kg/hr; 108 kg/hr total flow rate). The extraction continued with a 9.1% methanol/C cosolvent for 4 more hours at a methanol flow rate typically at 10 kg/ hour (range of 8.5 kg/hr to 11.5 kg/hr; 110 kg/hr total flow rate).
  • the extraction further continued with a 10.7% methanol/CC cosolvent for 8 more hours at a methanol flow rate typically at 12 kg per hour (range of 10.2 kg/hr to 13.8 kg/hr; 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 CO2 from the methanolic stream.
  • the separated CO2 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 20% to 35% methanol.
  • the purified poloxamer 188 was dried under vacuum at not more than 40 or 45°C to remove residual methanol.
  • the feed yield of the product gave an average yield of 65%.
  • the resulting purified poloxamerl88 was formulated into a clear, colorless, sterile, non-pyrogenic, aqueous solution containing the purified poloxamer at 150 mg/ml, sodium chloride at 3.08 mg/ml, sodium citrate (dihydrate) at 2.38 mg/ml, citric acid anhydrous at 0.366 mg/ml in water for injection.
  • the solution was sterile filtered and filled into 100 ml glass vials, covered with a nitrogen blanket, and closed with a butyl rubber stopper and aluminum overseal.
  • the resulting osmolality of the solution was approximately 312 mOsm/L.
  • the LCMF poloxamer-188 composition did not contain any bacteriostatic agents or preservatives.
  • LCMF poloxamer 188 blood samples were obtained by venipuncture into heparin anti-coagulated tubes at baseline, during drug administration (hours 1, 2, 3, 4, 5, and 6) and post administration at hours 1, 1.5, 2, 2.5, 5, 6, and 18. Plasma was separated by centrifugation and stored frozen until analysis.
  • the purified poloxamer 188 was administered as either a high dose of a loading dose of 300 mg/kg/hr for one hour followed by a maintenance dose of 200 mg/kg/hr for 5 hours or a lower dose of 100 mg/kg for 1 hour followed by 30 mg/kg/hr for 5 hours.
  • the plasma concentration time course observed following administration of the low dose are set forth in Figure 7.
  • Cmax mean maximum concentration of the administered purified poloxamer 188 of 0.9 mg/mL was attained by the end of the one hour loading infusion.
  • the mean concentration at steady state (Css) was about 0.4 mg/ml and was attained during maintenance infusion.
  • the plasma concentration declined rapidly following
  • Figures 7A and 7B show serial HPLC-GPC of plasma obtained at various time points following administration of the purified LCMF poloxamer 188 for a single subject.
  • Figure 7A shows the chromatograms at all time points, while Figure 7B shows selected time points for comparison.
  • the chromatogram is enlarged to show the high molecular weight portion (19.8 K Daltons - 12.4 K 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 clearance from the circulation in a substantially uniform manner. The results also 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.
  • the (LCM-containing) purified poloxamer 188 was administered to 6 healthy volunteers as an intravenous loading dose of 100 mg/kg/hr for one hour followed by 30 mg/kg/hr for 48 hours as part of a safety and pharmacokinetics study (Grindel et al). Blood samples were obtained by venipuncture into EDTA anticoagulated tubes prior to drug administration (baseline), during administration (at 1 hour, 6 hours, 12 hours 18 hour 24 hours 36 and 48 hours) and at 30 minutes, 1 hour, 1.5 hours, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 14 hours, 20 hours and 24 hours post drug administration.
  • mean plasma levels remained at 202 ug/ml, a concentration that had declined by only about 10% from the Cmax value.
  • mean plasma levels Over the 24 hour post infusion blood collection period, mean plasma levels only declined by 22.5 % to a plasma concentration of 165 ug/ml. Based on these changes in the plasma concentration time course the elimination half-life of > 48 hours is estimated.
  • plasma levels dropped from the steady state concentration by 52 % to 255 ug/ml.
  • plasma levels had dropped by 85% to 81 ug/ml.
  • LCMF poloxamer was administered to 62 healthy volunteers at a dose of 300 mg/kg for one hour followed by 200 mg/kg/hr for 5 hours as part of the assessment to determine its effect on the QT/QTc interval as previously described. Eight of the 62 subjects were randomly selected for quantitative analysis of the plasma poloxamer levels using a similar HPLC-GPC method as described in part (B) above but with improved linearity at lower plasma levels.
  • plasma levels had declined by 27 % from the Cmax value to 86 ug/ml.
  • mean plasma levels had declined by 71 % from the Cmax value to 34 ug/ml.
  • mean plasma levels had decreased from steady state by 67% to 872 ug/ml and by 6 hours after discontinuation, mean plasma levels had declined by 93% (from steady state) to 184 ug/ml.
  • HPLC conditions were used to compare column retention times for various poloxamers with known differences in their hydrophilic/lipophilic balance (HLB), along with purified poloxamer 188 containing LCM and the LCMF poloxamer 188:
  • Figure 9 shows the RP-HPLC chromatograms for a highly hydrophilic polymer (PEG 8000), the LCMF poloxamer 188, the LCM-containing purified poloxamer 188 , and two poloxamers with decreasing HLB values (increasing hydrophobicity), Poloxamer 338 and Poloxamer 407, respectively.
  • the most hydrophilic polymer, PEG 8000 exhibits little retention on the column consistent with its highly hydrophilic nature.
  • Poloxamer 338 (HLB > 24) and Poloxamer 407 (HLB 18-23) exhibit far longer retention times (add the 3 ⁇ 4 and V values) in accord with their known HLB values.
  • the LCMF purified poloxamer 188 elutes more quickly than the LCM-containing purified poloxamer 188, (the average 3 ⁇ 4 ⁇ and ⁇ for LCMF purified poloxamer is about 8.8 (8.807) and about 3.2 (3.202), respectively, compared to about 10.0 (9.883) and 3.7 (3.697) for LCM containing purified poloxamer) indicating that the LCMF poloxamer 188 is relatively more hydrophilic than the LCM containing purified poloxamer 188.
  • Figure 10 shows the chromatograms for 3 different lots of purified LCMF poloxamer 188 and 2 different lots of purified (LCM-containing) poloxamer 188.
  • the LCMF poloxamer 188 exhibits a markedly different pharmacokinetic behavior following administration to human subjects when compared to purified poloxamer 188, which contains the long circulating material (LCM) following in vivo administration.
  • the data provided in this example indicate that LCMF poloxamer 188 is more hydrophilic compared to purified poloxamer 188 that gives rise to the long circulating material.
  • PI 88 Since the rheologic, cytoprotective, anti-adhesive and antithrombotic effects of PI 88 are optimal within the predominant or main copolymers of the distribution, which are approximately 8,400 to 9,400 Daltons (which have a circulating half life of about 4 - 7 hours), the presence of larger, more hydrophobic, longer circulating half-life components of poloxamer 188 is not desirable.
  • P188 among the desired activities of P188 is its rheologic effect to reduce blood viscosity and inhibit red blood cell (RBC) aggregation, which account for its ability to improve blood flow in damaged tissues.
  • RBC red blood cell
  • coagulation enzymes promote clot formation (hemostasis) while the fibrinolytic system promotes the competing mechanism of clot dissolution (fibrinolysis).
  • fibrinolysis The effect on fibrinolysis in plasma of a poloxamer 188 in combination with a fibrinolytic inhibitor was assessed in vitro.
  • the assays assessed the kinetics of fibrin assembly (i.e., clot formation) and fibrin clot dissolution (i.e., fibrinolysis) by measuring the change in turbidity, measured as change in optical density at 405 nm, resulting from fibrin monomer assembly or, alternatively, dissolution.
  • optical density increases, while during clot dissolution (fibrinolysis), optical density decreases.
  • Assays were performed using citrated human plasma containing various concentrations of either poloxamer 188 (prepared as described in Example 1 above); urokinase a serine protease that converts plasminogen to plasmin; poloxamer 188 and urokinase; and poloxamer 188, urokinase, and the fibrinolytic inhibitor tranexamic acid (TA; Cyklokapron®, Pfizer).
  • TA fibrinolytic inhibitor tranexamic acid
  • clotting was initiated by sequential addition of 0.25 ⁇ calcium chloride followed by 0.5 ⁇ g/mL thrombin. Following addition of thrombin, the change in optical density (405 nm) was measured using a
  • Urokinase was added to citrated human plasma at concentrations of 312.5, 625, 1250, and 2500 U/mL, followed by the addition of 0.25 ⁇ calcium chloride and 0.5 ⁇ g/mL thrombin. The rate of fibrin assembly and dissolution was assessed at 1 minute intervals for 15 minutes after thrombin addition.
  • Figure 11 depicts the change in turbidity over time for each sample as measured by optical density at 405 nm. As shown in Figure 12, the urokinase had little or no effect on clot formation (i.e., fibrin assembly); and there was a concentration dependent increase in clot dissolution (fibrinolysis), as indicated by decreasing OD values.
  • Poloxamer 188 urokinase, and tranexamic acid in plasma
  • Urokinase (1500 U/mL), the fibrinolytic inhibitor tranexamic acid
  • a composition containing the purified poloxamer 188 and a fibrinolytic inhibitor is formulated as a sterile, non-pyrogenic solution for intravenous administration, with or without dilution.
  • a 100 mL glass vial is filled with: 15 g (150 mg/mL) of purified LCMF poloxamer 188, prepared as described above in Example 1; 0.75 g (7.5 mg/mL) of the fibrinolytic inhibitor tranexamic acid (TA;
  • Cyklokapron®, Pfizer 308 mg sodium chloride USP; 238 mg sodium citrate USP; 36.6 mg citric acid USP; and water for injection USP q.s. to 100 mL.
  • the pH of the solution is adjusted to approximately 6.0 before administration.
  • Example 1 At that point he is treated with 1 liter of crystalloid and 1 unit of packed red cells, and 90 mis of the composition of Example 1 is administered as an intravenous bolus over about 15 minutes. One hour later his blood pressure is 130 /70 and St0 2 increases to 85%. Three hours after administration of the composition, his St0 2 values rises to 91%, he is producing urine, and sublingual intravital microscopy shows a nearly normal microcirculation. By six hours after treatment, base excess and lactate are clearing. He continues to recover and is discharged from the hospital approximately 2 weeks post injury.
  • Post-operative cognitive assessment on day 4 is normal for her age, and a chest X-ray on day 5 (post-op) shows no signs of pulmonary congestion.
  • the patient is discharged from the hospital on the fifth post-operative day.

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Abstract

L'invention concerne des utilisations d'un poloxamère et des méthodes d'administration d'un poloxamère pour traiter un choc hémorragique et d'autres troubles présentant des saignements non voulus, par exemple chez un sujet qui a été traité avec un inhibiteur fibrinolytique. L'administration d'un poloxamère prévient, traite ou autrement réduit les effets négatifs de l'administration d'un inhibiteur fibrinolytique à un sujet atteint de choc hémorragique ou d'autres troubles présentant des saignements non voulus.
PCT/US2016/041304 2015-07-07 2016-07-07 Copolymères de polyoxyéthylène/polyoxypropylène et inhibiteurs fibrinolytiques, leurs utilisations et compositions WO2017007917A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2020122745A1 (fr) * 2018-12-10 2020-06-18 Arshintseva Elena Valentinovna Nouvelle utilisation du poloxamère en tant que substance pharmacologiquement active
BE1026546B1 (fr) * 2018-09-06 2020-09-28 RTU Pharma SA Solution intraveineuse d'acide tranexamique prete a l'emploi
RU2773153C1 (ru) * 2019-05-06 2022-05-31 Елена Валентиновна АРШИНЦЕВА Способ повышения уровня гемоглобина в крови у пациента, больного раком
EP3965734A4 (fr) * 2019-05-06 2022-12-14 Arshintseva, Elena Valentinovna Méthode destinée à augmenter le taux d'hémoglobine d'un patient atteint de cancer
US11622893B2 (en) 2020-04-09 2023-04-11 Bio 54, Llc Devices for bleeding reduction and methods of making and using the same
US11642324B1 (en) 2022-03-01 2023-05-09 Bio 54, Llc Topical tranexamic acid compositions and methods of use thereof

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US20020099075A1 (en) * 2001-01-25 2002-07-25 Tracey Wayne R. Combination therapy
WO2015058013A1 (fr) * 2013-10-16 2015-04-23 Mast Therapeutics, Inc. Modifications de volume plasmatique induites par un diurétique
WO2015067549A1 (fr) * 2013-11-05 2015-05-14 Bayer Pharma Aktiengesellschaft (aza)pyridopyrazolopyrimidinones et indazolopyrimidinones utilisées comme inhibiteurs de la fibrinolyse

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Publication number Priority date Publication date Assignee Title
US20020099075A1 (en) * 2001-01-25 2002-07-25 Tracey Wayne R. Combination therapy
WO2015058013A1 (fr) * 2013-10-16 2015-04-23 Mast Therapeutics, Inc. Modifications de volume plasmatique induites par un diurétique
WO2015067549A1 (fr) * 2013-11-05 2015-05-14 Bayer Pharma Aktiengesellschaft (aza)pyridopyrazolopyrimidinones et indazolopyrimidinones utilisées comme inhibiteurs de la fibrinolyse

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BE1026546B1 (fr) * 2018-09-06 2020-09-28 RTU Pharma SA Solution intraveineuse d'acide tranexamique prete a l'emploi
US10980757B2 (en) 2018-09-06 2021-04-20 RTU Pharma SA Ready-to-use tranexamic acid intravenous solution
US11696905B2 (en) 2018-09-06 2023-07-11 RTU Pharma SA Ready-to-use tranexamic acid intravenous solution
WO2020122745A1 (fr) * 2018-12-10 2020-06-18 Arshintseva Elena Valentinovna Nouvelle utilisation du poloxamère en tant que substance pharmacologiquement active
RU2760324C1 (ru) * 2018-12-10 2021-11-24 Елена Валентиновна Аршинцева Новое применение полоксамера в качестве фармакологически активного вещества
RU2773153C1 (ru) * 2019-05-06 2022-05-31 Елена Валентиновна АРШИНЦЕВА Способ повышения уровня гемоглобина в крови у пациента, больного раком
EP3965734A4 (fr) * 2019-05-06 2022-12-14 Arshintseva, Elena Valentinovna Méthode destinée à augmenter le taux d'hémoglobine d'un patient atteint de cancer
US11622893B2 (en) 2020-04-09 2023-04-11 Bio 54, Llc Devices for bleeding reduction and methods of making and using the same
US11654057B2 (en) 2020-04-09 2023-05-23 Bio 54, Llc Devices for bleeding reduction and methods of making and using the same
US11642324B1 (en) 2022-03-01 2023-05-09 Bio 54, Llc Topical tranexamic acid compositions and methods of use thereof

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