WO2017007917A1 - Polyoxyethylene/polyoxypropylene copolymers and fibrinolytic inhibitors, uses thereof and compositions - Google Patents

Abstract

Provided are uses of a poloxamer and methods of administering a poloxamer for treating hemorrhagic shock and other disorders with unwanted bleeding, such as in a subject that has been treated with a fibrinolytic inhibitor. Administration of a poloxamer prevents, treats or otherwise reduces adverse effects of administration of a fibrinolytic inhibitor to a subject with hemorrhagic shock or other disorders with unwanted bleeding.

Description

POLYOXYETHYLENE/POLYOXYPROPYLENE COPOLYMERS AND FIBRINOLYTIC INHIBITORS, USES THEREOF AND COMPOSITIONS

RELATED APPLICATIONS

[001] This application claims the benefit of priority to U.S. serial no. 62/189,705, filed July 7, 2015 and entitled POLYOXYETHYLENE/POLYOXYPROPYLENE COPOLYMERS AND FIBRINOLYTIC INHIBITORS, USES THEREOF AND

COMPOSITIONS, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[002] 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. Furthermore, 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.

BACKGROUND

[003] Fibrinolysis refers to the degradation of a fibrin blood clot. In circulating blood, 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

therapeutically to facilitate clot lysis and restore blood flow.

[004] 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.

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.

SUMMARY OF THE INVENTION

[005] 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.

[006] At high concentrations, poloxamer 188 can promote bleeding. The inventors surprisingly found that combining poloxamer 188 treatment with fibrinolytic inhibitor treatment exploits the advantages of each and mitigates the adverse effects of each.

Hence, provided herein are methods of combination therapy in which 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). In one aspect, 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. The following embodiments throughout the application apply to both the methods and the composition aspects. In such instances, 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.

[007] Hence provided is combination therapy with poloxamers and fibrinolytic inhibitors and uses of poloxamers and fibrinolytic inhibitors to treat or mitigate the adverse effects of each, and in particular, for example, to treat bleeding disorders, hemorrhagic shock and promote hemostasis.

BRIEF DESCRIPTION OF THE DRAWINGS

[008] The drawings described herein are for illustrative purposes of selected embodiments and not all possible implementations, and are not intended to limit the scope of the present disclosure.

[009] FIG. 1 is a general process 100 for supercritical fluid extraction (SFE) of a poloxamer.

[0010] FIG. 2 is a specific exemplary process 100' for preparing a poloxamer, such as poloxamer 188, using the methods described herein.

[0011] FIG. 3 is a specific exemplary process 100" for preparing a poloxamer, such as poloxamer 188, using methods described herein.

[0012] FIG. 4 shows an extraction apparatus useful in the methods provided herein.

[0013] FIG. 5 shows one embodiment of the cross section of stainless spheres of different sizes in a solvent distribution bed.

[0014] 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).

[0015] FIG. 7A-B shows enlarged HPLC-GPC chromatograms depicting the molecular weight distribution of components in plasma over time. [0016] 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).

[0017] 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.

[0018] FIG. 10 shows a RP-HPLC chromatogram comparing different lots of LCMF poloxamer 188 with purified poloxamer 188 confirming the difference in hydrophilicity.

[0019] FIG. 11 shows a control study of P188 addition to plasma. The results indicate a concentration dependent change in the rate of fibrin assembly.

[0020] FIG. 12 shows an effect of the addition of urokinase to plasma, which is a concentration dependent increase of clot lysis (decrease in OD).

[0021] 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).

[0022] 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.

DETAILED DESCRIPTION

[0023] Outline

A. DEFINITIONS

B. OVERVIEW OF THE METHODS, USES, COMBINATIONS AND COMPOSITIONS

C. FIBRINOLYSIS AND FIBRINOLYTIC INHIBITORS

1. Fibrinolysis

2. Fibrinolytic inhibitors

D. MOLECULAR DIVERSITY OF POLOXAMERS, POLOXAMER 188, LCM-CONTAINING POLOXAMER 188 AND LCMF POLOXAMERS

1. Poloxamers

2. Poloxamer 188 3. Molecular Diversity of Poloxamer 188

a. Low Molecular Weight Components

b. Components Resulting in Long Circulating Half -Life Material (LCM)

4. Long Circulating Material Free (LCMF) Poloxamer

5. Extraction Methods

a. Processes For Extraction

i) Supercritical Methods

ii) High Pressure Methods

b. Extraction Vessel and System

c. Extraction and Removal of Extractants d. Exemplary Methods for Preparation of Purified Poloxamers i) Removal of Low Molecular Weight (LMW)

Components

ii) Preparation of Long Circulating Material Free

(LCMF) Poloxamer

iii) Methods for Confirming the Identity of LCMF

Poloxamers

E. PHARMACEUTICAL COMPOSITIONS, FORMULATIONS AND COMBINATION THERAPY

1. Formulations

2. Dosage

3. Fibrinolytic inhibitors

4. Compositions, combinations and combination therapy

F. EXAMPLES

[0024] A. DEFINITIONS

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention(s) belong. All patents, patent applications, published applications and publications, Genbank sequences, databases, websites and other published materials referred to throughout the entire disclosure herein, unless noted otherwise, are

incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms herein, those in this section prevail. Where reference is made to a URL or other such identifier or address, it understood that such identifiers can change and particular information on the internet can come and go, but equivalent information can be found by searching the internet or in publications. Reference thereto evidences the availability and public dissemination of such information.

[0026] As used herein, "a", "an" and "the" mean one or more and include the plural unless the context is inappropriate.

[0027] As used herein, "fibrinolysis" refers to the degradation of a fibrin blood clot. In circulating blood, plasmin is the primary fibrinolytic enzyme. 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

therapeutically to facilitate clot lysis and restore blood flow.

[0028] As used herein, the term "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.

[0029] As used herein, "hemorrhagic shock" refers to conditions following the loss of blood. Precursors to hemorrhagic shock include trauma, injury and surgery.

[0030] As used herein, "poloxamers" 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:

HO(C₂H₄0)a^< -[C₃H₆0]b-(C₂H₄0)aH

[0031] where: a' and a can be the same or different and each is an integer such that the hydrophile portion represented by (C₂H₄0) (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 (C₃H60)¾ (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. For example, 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^®).

[0032] The nomenclature of the polyoxyethylene/polyoxypropylene copolymer relates to its monomeric composition. The first two digits of a poloxamer number, multiplied by 100, gives the approximate molecular weight of the hydrophobic polyoxypropylene block. The last digit, multiplied by 10, gives the approximate weight percent of the hydrophilic polyoxyethylene content. For example, 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.

[0033] Poloxamers can be synthesized in two steps, first by building the

polyoxypropylene core, and then by addition of polyoxyethylene to the terminal ends of the polyoxypropylene core. Because of variation in the rates of polymerization during both steps, a poloxamer can contain heterogeneous polymer species of varying molecular weights. The distribution of polymer species can be characterized using standard techniques including, but not limited to, gel permeation chromatography (GPC).

[0034] As used herein, "Poloxamer 188" (also called P-188 or P188) refers to a polyoxyethylene/polyoxypropylene copolymer that has the following chemical formula:

HO(CH₂CH20)a'-[CH(CH3)CH20]b-(CH2CH₂0)aH, where: a' and a can be the same or different and each is an integer such that the hydrophile portion represented by (C₂H₄0) (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. For example, 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. Unsaturation levels can be measured according to known techniques such as those described by Moghimi et al, Biochimica et Biophysica Acta (2004); 1689: 103- 113. [0035] 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.

[0036] As used herein, "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).

[0037] As used herein, "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

chromatography conditions (e.g., see U.S. Patent No. 5,696,298 and Grindel et al. (2002) Biopharmaceutics & Drug Disposition, 23:87-103). 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).

[0038] As used herein, "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). [0039] As used herein, "polydispersity" or "D" 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. Methods for assessing polydispersity are known in the art, and include methods as described in U.S. Patent No. 5,696,298. For example, 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. of Millipore, Milford, Mass.), two LiChrogel PS-40 columns and a LiChrogel PS-20 column in series (EM Science, Gibbstown, N.J.), and polyethylene glycol molecular weight standards (Polymer Laboratories, Inc.,

Amherst, Mass.). It is within the level of a skilled artisan to convert any polydispersity value that is obtained using a different separation method to the values described herein simply by running a single sample on both systems and then comparing the polydispersity values from each chromatogram.

[0040] As used herein, "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. A poloxamer 188 in which "low molecular weight material has been removed" or "low molecular weight material has been reduced," or similar variations thereof, refers to a purified poloxamer 188 in which there is a distribution of low molecular weight components of no more than or less than 3.0 %, and generally no more than or less than 2.0% or no more than or less than 1.5% or no more than or less than about 1% of the total distribution of components. Typically, such a poloxamer 188 exhibits reduced toxicity compared to forms of poloxamer 188 that contain a higher or greater percentage of low molecular weight components. An embodiment of the disclosure herein are poloxamer 188 copolymers purified to remove or reduce low molecular weight components.

[0041] Commercially available and prior preparations of 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).

[0042] 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). 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)

Biopharmaceutics & Drug Disposition, 23:87-103)..

[0043] As used herein, "long circulating material" or "LCM" 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. When administered to a human subject 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.

[0044] As used herein, "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. Thus, 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. In some embodiments the LCMF poloxamer 188 has an unsaturation level of about 0.018 to about 0.034mEq/g. Typically, 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. Examples of 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.

[0045] As used herein, "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.

[0046] 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.

[0047] 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.

[0048] As used herein "Cmax" refers to the peak or maximal plasma concentration of a drug after administration.

[0049] As used herein, the "concentration of a drug at steady state" or "Css" 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.

[0050] As used herein, "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.

[0051] As used herein, "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.

Generally, 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. For example, most commercial preparations of a poloxamer 188 contain a LMW component (less than 4,500 Daltons) that is about 4% by weight of the total components in the distribution. The LMW component is reduced in a purified product if there is less than 3% by weight of the component, such as less than 2% or 1%.

[0052] As used herein, "solvent" refers to any liquid in which a solute is dissolved to form a solution.

[0053] As used herein, a "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.

[0054] As used herein, "feed" refers to a solute dissolved in a solvent.

[0055] As used herein, "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. Generally an extraction solvent is immiscible or partially miscible with the solvent in which the substance of interest is dissolved. For example, 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. [0056] As used herein, the terms "supercritical liquid" and "supercritical fluid" include any compound, such as a gas, in a state above its critical temperature (T_c; i.e. the temperature, characteristic of the compound, above which it is not possible to liquefy the compound) and critical pressure (p_c; i.e., the minimum pressure which would suffice to liquefy the compound at its critical temperature). In this state, distinct liquid and gas phases typically do not exist. A supercritical liquid typically exhibits changes in solvent density with small changes in pressure, temperature, or the presence of a co-modifier solvent.

[0057] As used herein, "supercritical 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.

[0058] As used herein, "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. For example, the critical temperature of carbon dioxide is about 31° C.

[0059] As used herein, "critical pressure" refers to the pressure required to liquefy a gas at its critical temperature. For example, the critical pressure of carbon dioxide is about 74 bars.

[0060] As used herein, the term "high pressure liquid" includes a liquid formed by pressurizing a compressible gas into the liquid at room temperature or a higher temperature.

[0061] As used herein, 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. Examples of co-modifier solvents include alkanols. Typically, between 5% and 15% by weight of co-modified solvent can be used.

[0062] As used herein, the term "alkanol" includes simple aliphatic organic alcohols. In general, 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.

[0063] As used herein, "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.

[0064] As used herein, "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.

[0065] As used herein, "isocratic" refers to a system in which an extraction solvent is used at a constant or near constant concentration.

[0066] As used herein, "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. For example, the concentration of the alkanol solvent (e.g., methanol) is successively increased during the course of the extraction. Thus, the extraction solvent does not remain constant.

[0067] As used herein, "plurality" refers to a number of iterations of a process or step. The number of repeats can be 2, 3, 4, 5, 6 or more.

[0068] As used herein, "extracted material" refers to the product containing the removed materials.

[0069] As used herein, "raffinate" refers to a product which has had a component or components removed. For example, the purified poloxamer in which extracted material has been removed.

[0070] As used herein, "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. For example, 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. In this method, the spent solvent can be stripped and recycled by distillation or fresh solvent can be added continuously from a reservoir.

[0071] As used herein, 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. For example, 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.

[0072] As used herein, "effective amount" refers to the dose of poloxamer and/or fibrinolytic inhibitor that, when administered to patient, results in a desired biological effect.

[0073] As used herein, "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. In certain instances, 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. In certain instances, 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).

[0074] As used 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.

[0075] As used herein, "treating" a subject having a disease or condition means that a composition or other product provided or described herein is administered to the subject to thereby achieve treatment thereof. [0076] As used herein, "amelioration" of the symptoms of a particular disease or disorder by a treatment, such as by administration of a pharmaceutical composition or other therapeutic, 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.

[0077] As used herein, "prevention" or "prophylaxis" refers to methods in which the risk of developing a disease or condition is reduced. Prophylaxis includes reduction in the risk of developing a disease or condition and/or a prevention of worsening of symptoms or progression of a disease, or reduction in the risk of worsening of symptoms or progression of a disease.

[0078] As used herein, 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.

[0079] As used herein, "therapeutically effective amount" or "therapeutically effective dose" 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.

[0080] As used herein, "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. For example, 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)..

[0081] As used herein, "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. Thus, "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.

[0082] As used herein, 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.

[0083] As used herein, a "composition" refers to any mixture of two or more products or compounds (e.g., agents, modulators, regulators, etc.). It can be a solution, a suspension, liquid, powder, a paste, aqueous or non-aqueous formulations or any combination thereof.

[0084] As used herein, 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.

[0085] As used herein, "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.

[0086] As used herein, a "kit" refers to a packaged combination, optionally including reagents and other products and/or components for practicing methods using the elements of the combination. For example, kits containing purified poloxamers provided herein and another item for a purpose including, but not limited to,

administration, diagnosis, and assessment of a biological activity or property are provided. Kits optionally include instructions for use.

[0087] As used herein, "animal" includes any animal, such as, but not limited to;

primates including humans, gorillas and monkeys; rodents, such as mice and rats; fowl, such as chickens; ruminants, such as goats, cows, deer, sheep; ovine, such as pigs and other animals. Non-human animals exclude humans as the contemplated animal.

[0088] As used herein, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0089] "About" or "approximately" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of +30% -+10%, more preferably +5%, even more preferably +1 %, and still more preferably +0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. About also includes the exact amount. Hence "about 0.05 mg/mL" means "about 0.05 mg/mL" and also "0.05 mg/mL."

[0090] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance does or does not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, an optionally substituted group means that the group is unsubstituted or is substituted.

[0091] As used herein "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.

[0092] As used herein "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):

tR - to

k' = .

to

[0093] 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).

[0094] As used herein, the abbreviations for any protective groups, amino acids and other compounds, are, unless indicated otherwise, in accord with their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see, (1972) Biochem. 11 : 1726). [0095] B. OVERVIEW OF THE METHODS, USES, COMBINATIONS AND COMPOSITIONS

[0096] 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. At high concentrations, 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. For example, data from animal models of hemorrhagic shock indicate that atrheologic concentrations poloxamer can exacerbate bleeding. As disclosed herein 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.

[0097] Provided herein are methods and uses, combinations and compositions in which poloxamers and fibrinolytic inhibitors are used together. When used together, particularly at the appropriate dosage, generally 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. As described herein, when combined or administered together, the fibrinolytic inhibitor and poloxamer mutually antagonize the unintended consequences of the other, resulting in improved outcomes.

[0098] Provided herein are methods of treatment of hemorrhagic shock, precursors thereto and consequences thereof, including bleeding during surgery, from trauma and other causes. The methods are practiced by administering 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.

[0099] 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.

[00100] 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. Thus, 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. To achieve these effects, 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.

[00101] When it is important to control bleeding and blood loss, such as during surgery and trauma or in patients with abnormally high fibrinolytic activity, reduction of fibrinolytic activity is important. For example, where there is damaged tissue, 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.

[00102] 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.

[00103] As described in the sections that follow, 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. There are many possible dosing regimens; the goal is to achieve an effective plasma concentration for a time sufficient to effect treatment of a subject. 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.

[00104] In particular, 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. Typically for the methods herein, 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.

[00105] Typically, 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.

[00106] 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.

Typically, it can be administered by intravenous (IV) infusion or bolus injection. For example, 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. In general, for treatment, the infusion can be continued for between 12-48 hours as needed. Alternatively, repeat bolus injections can be administered. For example, 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. To achieve a higher plasma concentration of about 1 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.

[00107] 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. These conditions include, but are not limited to, ischemic tissue damage, prothrombotic events, for example, embolism or thrombosis, and any other condition or unwanted consequence associated with hemorrhagic shock.

[00108] In some of the methods provided herein, 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.

[00109] C. FIBRINOLYSIS AND FIBRINOLYTIC INHIBITORS

[00110] 1. Fibrinolysis

[00111] When blood vessels are damaged, hemostasis, the process by which the body forms clots, is initiated, with the end goal of forming a stable fibrin clot at the site of vascular injury. 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.

[00112] 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). 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.

[00113] Where there is a blockage of blood flow due to a blood clot, such as in a heart attack, stroke, or pulmonary embolism, plasminogen activators such as tPA, SK, and uPA are often administered to facilitate clot degradation and restore blood flow. However, where there is damaged tissue, for example, after trauma or during surgery, vessels are more likely to rupture which can lead to problematic hemorrhaging and excessive bleeding. Thus, where active bleeding is a primary clinical concern, for example, in surgery- or trauma-related shock, excessive fibrinolysis or impairment of blood coagulation is undesirable.

[00114] 2. Fibrinolytic inhibitors

[00115] Reduction of fibrinolytic activity can be achieved through the administration of fibrinolytic inhibitors. 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. However, because 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) by administration of fibrinolytic inhibitors may have adverse consequences, for example, prothrombotic consequences (i.e., lead to the development of thrombosis). Thus, despite the benefits of using fibrinolytic inhibitors, particularly where bleeding must be controlled, such as during surgery or after trauma, such inhibitors may also result in an increased risk for thromboembolic events, such as embolism and thrombosis, and ischemic tissue injury.

[00116] When it is important to control bleeding and blood loss, such as during surgery and trauma or in patients with abnormally high fibrinolytic activity, reduction of fibrinolytic activity is important. For example, where there is damaged tissue, 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.

[00117] 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. Other endogenous inhibitors include the plasmin inhibitor alpha-2-antiplasmin (also known as a₂-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. 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.

[00118] Pharmaceutical fibrinolytic inhibitors include the polypeptide aprotinin and synthetic derivatives of lysine, such as ε-aminocaproic acid (aminocaproic acid; EACA), and the more potent tranexamic acid (TA). 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. These inhibitors competitively inhibit the activation of plasminogen to plasmin by binding to specific sites of both plasminogen and plasmin, allowing blood clots to remain intact. Aprotinin, on the other hand, 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. For purposes herein, the fibrinolytic inhibitors can be administered before, after, or concomitant with administration of the

polyoxyethylene/polyoxypropylene copolymer. [00119] D. MOLECULAR DIVERSITY OF POLOXAMERS,

POLOXAMER 188, LCM-CONTAINING POLOXAMER 188 AND LCMF

POLOXAMERS

[00120] 1. Poloxamers

[00121] 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.

[00122] Certain polyoxyethylene/polyoxypropylene copolymers, including poloxamer 188, have beneficial biological effects on several disorders when administered to a human or animal. These activities have been described, for example in numerous publications and patents (see, e.g., U.S. Patent Nos 4,801,452, 4,837,014, 4,873,083, 4,879,109, 4,897,263, 4,937,070, 4,997,644, 5,017,370, 5,028,599, 5,030,448, 5,032,394, 5,039,520, 5,041,288, 5,047,236, 5,064,643, 5,071,649, 5,078,995, 5,080,894, 5,089,260, RE 36,665 (Reissue of 5,523,492), 5,605,687, 5,696,298 6,359,014, 6,747,064,

8,372,387, 8,580,245, U.S. Patent Publication Nos. 2011/0044935, 2011/0212047, 2013/0177524, and International Applications WO2006/037031 (filed as

PCT/US2005/034790), WO2009/023177 (filed as PCT/US2005/037157) and

WO2006/091941 (filed as PCT/US2006/006862), and PCT/US2014/45627, U.S.

Provisional Application Serial Nos. 62/021,691 and 62/021,676). Among the activities of poloxamers, such as poloxamer 188, that make them useful as therapeutic agents is their ability to incorporate into cellular membranes, and thereby repair damaged cell membranes.

[00123] Poloxamers include POP/POE block copolymers having the following formula: HO(C2H₄0)a'-(C₃H₆0)b-(C2H₄0)aH, where "a"' and "a" can be the same or different and each is an integer such that the hydrophile portion represented by (C₂H₄0) 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 (C₃H₆0) has a molecular weight of approximately 950 to 4,000 Da, such as 1,200 to 3,500 Da. For example, 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.

[00124] In certain instances, 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. In some other instances, 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. In still other instances, 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. A skilled artisan will appreciate that these values are average values. 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.

[00125] The nomenclature of the poloxamer relates to the composition of the various polymer members. The first two digits of a poloxamer number, multiplied by 100, gives the approximate molecular weight of the hydrophobe. The last digit, times 10, gives the approximate weight percent of the hydrophile (polyoxyethylene) content of the surfactant. For example, 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.

[00126] Poloxamers are sold and referred to under trade names and trademarks including, but not limited to, ADEKA-NOL, Synperonic™, 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). [00127] Hence, 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.

[00128] 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.

[00129] 2. Poloxamer 188

[00130] A poloxamer 188 (P188) copolymer has the following chemical formula:

HO(CH₂CH₂0)a'-[CH(CH₃)CH₂0]b-(CH₂CH₂0)aH,

where the hydrophobe represented by (C₃¾0) 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.

[00131] Various poloxamers, and in particular P188, 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.

Advantageously, this blocks adhesion of hydrophobic surfaces to one another and thereby reduces friction and increases flow.

[00132] 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.

[00133] 3. Molecular Diversity of Poloxamer 188

[00134] Commercially available 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).

[00135] The diversity in structure means that there is a diversity in biological activity. For example, 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). The distribution of components, however, 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. 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. [00136] A primary mechanism contributing to the molecular diversity is the process by which poloxamers are synthesized. During the typical manufacturing process, 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.

[00137] During the synthesis of the POP, two different reaction mechanisms limit POP chain growth and result in unintended diblock polymers. These substances are typically of lower molecular weight (relative to the polymeric distribution of P188). In one mechanism, unsaturation is formed directly from propylene oxide by reacting with an alkali catalyst. The base catalyzes the rearrangement of the propylene oxide to an allyl alcohol, which then initiates a mono functional chain with terminal unsaturation. These types of side reactions will produce low molecular weight (LMW) substances throughout the time of the reaction. On gel permeation chromatography (GPC), the distribution of these impurities are located in the main peak as well as in the LMW shoulder. In a second mechanism, the abstraction of a hydrogen atom, located six carbon atoms away, by the negative oxygen atom in a growing polymer chain can terminate and transfer the chain, producing an allyl end group. These back-biting reactions are predominant with high molecular weight (HMW) POP blocks. The distribution of these substances is mostly in the LMW shoulder.

[00138] In addition, high molecular weight substances (relative to the polymeric distribution of P188) 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.

[00139] The degradation pathways for poloxamers include peroxidation leading to low molecular aldehydes and acids and thermal degradation leading to LMW

polyethylene glycols. 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.

[00140] Thus, 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).

[00141] For example, due to the synthesis of P188, there can be variation in the rates of polymerization during the steps of building the PO core and EO terminal ends. Thus, most non-purified forms of P188 contain a bell-shaped distribution of polymer species, which vary primarily in overall chain length. In addition, various low molecular weight (LMW) components (e.g., glycols and truncated polymers) formed by incomplete polymerization, and high molecular weight (HMW) components (e.g., dimerized polymers) can be present. Typically, characterization of P188 by gel permeation chromatography (GPC) 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). For example, the preparation of P188 that is available from BASF (Parsippany, N.J.) 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. Complement activation, phagocyte migration paralysis, and cytotoxicity observed upon administration of artificial blood preparations have been attributed in part to impurities in the poloxamer 188 component of those preparations. In addition, infusion of poloxamer 188 was shown to result in elevated creatinine, indicating kidney damage, and increased organ weights (kidney) in toxicological animal studies. Histologic evaluation of the kidney demonstrated a dose related cytoplasmic vacuolation of the proximal tubular epithelial cells.

[00142] 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. This is

demonstrated herein (see, e.g., Figure 8A and Figure8B).

[00143] a. Low Molecular Weight Components

[00144] Substances in poloxamer 188 that are toxic to kidneys have been identified as being of lower molecular weights than the main components. Studies on the therapeutic potential of PI 88 led to the discontinuance of the poloxamer available under the trademark RheothRx® for therapeutic applications in part due to an acute renal dysfunction observed during clinical trial evaluation as evidenced by elevated serum creatinine. It was found that these effects were due to the presence of various low molecular weight (LMW) substances that formed during the synthesis process (Emanuele and Balasubramanian (2014) Drugs R D, 14:73-83). The LMW substances were accumulated by the proximal tubule epithelial cells in the kidney.

[00145] 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.

[00146] Supercritical fluid chromatography that reduces the level of these low molecular weight substances present in P188 has been reported (see, e.g., U.S. Patent No. 5,567,859; and Emanuele and Balasubramanian (2014) Drugs R D, 14:73-83).

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) .

[00147] b. Components Resulting in Long Circulating Half-Life

[00148] 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). There is a main peak with an average peak molecular weight of about 8,600 Daltons and a smaller peak with an average molecular weight of about 16,000 Daltons. The two peaks exhibit distinctly different pharmacokinetic profiles with the higher molecular weight peak exhibiting a distinctly longer plasma residence time with slower clearance from the circulation (see Figure 8A and Figure 8B). Similar observations were reported in rats and dogs. A similar long circulating component is observed with native or unpurified poloxamer 188 (see PCT Published Application No. WO 94/008596).

[00149] For example, as shown in Figure 8A, following administration of a purified P188 intravenously to healthy volunteers as a loading dose of 100 mg/kg/hr for one hour followed by a maintenance dose of 30 mg/kg/hr for 47 hours, the main or predominant peak reached a mean maximum concentration (Cmax) of 0.9 mg/mL by the end of the one-hour loading infusion. A mean steady state concentration (Css) of 0.5 mg/mL was achieved essentially coincident with the start of the maintenance infusion. With the discontinuation of the maintenance infusion, plasma concentrations declined rapidly with an elimination half-life (tl/2) of about 7 hours. As shown in Figure 8B, a HMW component was identified that exhibited a Cmax of 0.2 mg/mL, which was not attained until the end of the maintenance infusion. Steady state was not attained as the

concentration continued to accumulate during infusion. Following discontinuation of the maintenance infusion, plasma levels of the high molecular weight peak declined slowly such that plasma levels had only declined by about 33% during the 24 hour post-infusion monitoring period. This elimination rate is approximately 1/10 that of the main peak and the ti/2 is approximately 70 hours. See, also Grindel et al. (2002) Journal of

Pharmaceutical Sciences, 90: 1936-1947 (Grindel et al. 2002a) and Grindel et al. (2002) Biopharmaceutics & Drug Disposition, 23:87-103 (Grindel et al. 2002b). The long circulating material (or long retention time material) is identified in the HMW fraction of the PI 88 distribution (Grindel et al. 2002a). This HMW component was determined to be approximately 16,000 Da as identified by MALDI-TOF mass spectrometry with a fragmentation pattern consistent with a block copolymer (see, e.g., Grindel et al. 2002a).

[00150] 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. For example, 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. In contrast, higher molecular weight poloxamers such as P338 (also called Pluronic® F108) and P308(Pluronic® F98), increase blood viscosity and RBC aggregation (Armstrong et al. (2001) Biorheology, 38:239-247). Thus higher molecular weight poloxamer species may have undesirable biological effects.

[00151] As described in more detail below, provided are 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). Also provided are exemplary methods (see, e.g., Example 1) for production of LCMF poloxamer. Thus, the 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.

[00152] 4. Long Circulating Material Free (LCMF) Poloxamer

[00153] 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. Hence the 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:

HO(CH₂CH20)a'-[CH(CH3)CH20]b-(CH2CH₂0)aH,

where a' and a can be the same or different and each is an integer such that the hydrophile portion represented by (C₂H₄0) (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.

[00154] Studies have demonstrated that the main peak component of a purified (LCM-containing) P188 preparation, when administered to a human subject, has a half- life (ti/₂) in (human) plasma of about 7 hours (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)). 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.

[00155] In contrast to the purified PI 88 (LCM-containing) characterized, for example, in the studies of Grindel et al., (2002a and 2002b), the purified poloxamer, designated LCMF PI 88, is one in which all components of the polymeric distribution, when administered to a subject, clear from the circulation at approximately the same rate. Thus, the LCMF PI 88 is different from prior LCM-containing pi 88 poloxamers. Like LCM containing 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. Typically, 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.

[00156] In some examples, 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.

[00157] Without being bound by theory, higher molecular weight components of the poloxamer polymeric distribution, such as those greater than 13,000 Daltons could account for the longer circulating half-life material. The rate of glomerular filtration of uncharged molecules like poloxamer 188 and purified poloxamer 188 is highly dependent upon molecular size. This is observed for components of the poloxamer 188 polymeric distribution with molecular weights greater than 5,000 Daltons since, the rate of glomerular filtration becomes increasingly restricted above that size threshold (Chang et ah, (1975) Biophysic. J. 15:887 - 906). Accordingly, the higher molecular weight components of the poloxamer 188 polymeric distribution (such as those greater than 13,000 Daltons) would be more likely to be cleared from the circulation at a slower rate than those of smaller size.

[00158] For the LCMF preparations, however, the presence of HMW components in the distribution does not result in a longer circulating species {i.e., a species with a half- life more than 5-fold longer than the main peak). For example, HMW impurities greater than 13,000 Daltons in an LCMF preparation generally constitute no more than 1.5% by weight of the total component. When the LCMF preparation is administered to a subject, 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. When the LCMF preparation is administered to a subject, they do not result in any component with a circulating half-life that is substantially more {i.e., more than 5-fold) than or is more than the main component in the distribution (see, e.g., Figures 7A and 7B). [00159] In the LCMF preparation, the HMW components can be either increased or decreased compared to other existing purified PI 88 preparations. For example, 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. In particular examples provided herein, 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.

[00160] 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). In view of the description and exemplification of the properties of the LCMF poloxamer, those of skill in the art can envision other methods for producing an LCMF poloxamer. For example, an LCMF poloxamer provided herein is made by a method that includes:

a) introducing a poloxamer solution into an extractor vessel, where the poloxamer is dissolved in a first alkanol to form a solution;

b) contacting the poloxamer solution with an extraction solvent comprising a second alkanol and supercritical carbon dioxide under a temperature and pressure to maintain the supercritical carbon dioxide for a first defined period, wherein:

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; and

the alkanol is provided at an alkanol concentration that is 7% to 8% by weight of the total extraction solvent; and

c) increasing the concentration of the second alkanol in step b) in the extraction solvent a plurality of times in gradient steps over time of the extraction method, wherein:

each plurality of times occurs for a further defined period; and

in each successive step, the alkanol concentration is increased 1-2% compared to the previous concentration of the second alkanol; and

d) removing the extraction solvent from the extractor vessel to thereby remove the extracted material from the raffinate poloxamer preparation.

[00161] 5. Extraction Methods [00162] Provided herein are supercritical fluid extraction (SFE) and high-pressure procedures for purifying poloxamers such that the purified polymer is more homogenous with regard to structure (diblock, triblock, etc.), the percentage of molecules without unsaturation, the distribution of molecular weights, and distribution of

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.

[00163] 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.

[00164] The absence of the long circulating material (LCM) 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.

[00165] Provided herein are methods for preparing such poloxamers, and provided are the resulting poloxamers, including the LCMF poloxamer 188. 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. Hence, also provided herein are LCMF preparations of poloxamers, and in particular LCMF poloxamer 188. The LCMF poloxamer 188 provided herein can be used for all of the uses known for poloxamer 188.

[00166] Provided herein are 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. For example, 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.

[00167] 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 C0₂ in the extraction solvent. In particular, 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. It is found that by employing an alkanol co-solvent whose concentration is increased in this manner, the removal of impurities can be increased, and to a much greater extent than when carbon dioxide is used alone. For example, an extraction method that uses carbon dioxide alone is not capable of removing the unwanted components, such as the LMW components and HMW components as described herein, to the same degree as that achieved by the provided method.

[00168] In the methods provides herein for purifying a poloxamer using supercritical fluid extraction, 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. As described further below, by increasing the solubilizing power of the extraction solvent, for example by carefully controlling the pressure and concentration of polar solvent, such as an alkanol (e.g., methanol), it also is possible to remove other impurities. In particular, a method is provided 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.

[00169] There, however, can be a tradeoff with respect to the yield of poloxamer. Generally, as the concentration of the alkanol (e.g., methanol) co-solvent increases, the solvating power of the extraction solvent is increased so that more compounds are solubilized and the degree of extraction increases. By increasing the concentration of extraction solvent in a gradient fashion, the reduction of poloxamer yield is minimized, while the purity of the final product is maximized. Typically, 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, however, 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.

[00170] 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. For example, 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. Such components can increase blood viscosity and red blood cell aggregation, and hence are undesired.

[00171] Exemplary of poloxamers for use in the methods include, but are not limited to, poloxamer 188, poloxamer 331 and poloxamer 407. Typically, 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. In particular, the poloxamer is PI 88.

[00172] For example, the extraction methods provided herein can be employed to purify a PI 88 preparation, where the PI 88 preparation has the following chemical formula:

HO(CH₂CH20)a'-[CH(CH3)CH20]b-(CH2CH₂0)aH, where:

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.).

[00173] In practicing the extraction methods provided herein, 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. In the method, 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. For example, the poloxamer is dissolved in a separate vessel and then the solution is added to the extraction vessel.

[00174] The method can be a high pressure or supercritical fluid extraction method. Typically, 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. For example, carbon dioxide has a critical temperature of 31.1° C. and a critical pressure of 73.8 bars. Thus, extraction conditions for a supercritical carbon dioxide are above the critical temperature of about 31° C and critical pressure of about 74 bars. In contrast, 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.

[00175] a. Processes For Extraction

[00176] i) Supercritical Methods

[00177] In certain instances, the supercritical fluid extraction process employed in the methods provided herein is essentially a solvent extraction process using a supercritical fluid as the solvent. With 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). In the supercritical region of the phase diagram, a compressible fluid such as carbon dioxide exhibits liquid-like density and much increased solvent capacity that is pressure dependent.

[00178] The supercritical fluid exhibits a number of highly advantageous

characteristics making it a superior solvent. For example, 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.

[00179] Another beneficial property of various supercritical fluids is the difference in their critical temperatures and pressures. Each supercritical fluid has a range of solvent power. The tunable solvent power range can be selected by choosing an appropriate supercritical fluid.

[00180] In addition to its unique solubility characteristics, supercritical fluids exhibit certain physicochemical properties making them more useful. For example, 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.

[00181] The use of 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.

[00182] In such a method, 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. As described above, 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.

[00183] For example, 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. The first and second alkanol can be the same or different. In the method, 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. For example, the poloxamer is dissolved in a separate vessel and then the solution is added to the extraction vessel.

[00184] An exemplary process is detailed in FIG. 1. 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.

[00185] Any suitable alkanol or combination of alkanols can be used in the methods provided herein. Examples of suitable alkanols include, but are not limited to, methanol, ethanol, propanol and butanol. For example, 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. In some embodiments, the first alkanol is methanol. In certain instances, methanol is selected as the purification solvent and is the second alkanol in practice of the method. A skilled artisan will appreciate that methanol has relatively low toxicity characteristics. Moreover, methanol has good solubility for poloxamer 188.

[00186] The first alkanol (e.g., methanol) is used to form a poloxamer solution according to step 115 in process 100. A poloxamer, such as a P188 preparation, is dispensed into the feed tank and is stirred until mixed with the first alkanol. 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. For example, 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. In some examples, in methods of extraction using a 3 L extraction vessel, non-limiting amounts of poloxamer (e.g., P188) can be 0.6 kg to 1.2 kg, such as 0.8 kg to 1.0 kg. In another example, in methods of extraction using a 12 L extraction vessel, non-limiting amounts of poloxamer (e.g., P188) can be 1.5 kg to 5 kg, such as 2 kg to 4 kg. In a further example, 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.

[00187] Any suitable ratio of poloxamer and alkanol is contemplated for use in the methods provided herein. 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. For example, 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. For example, a quantity of poloxamer, such as PI 88, can be mixed with an equal quantity, by weight, of 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). One of skill in the art will appreciate that the appropriate poloxamer to alkanol ratio will depend on poloxamer properties, such as solubility, in a given alkanol. [00188] After forming a poloxamer/alkanol mixture, all or part of the mixture is pumped into the extractor as shown in step 120. In such examples, the process of preparing the poloxamer solution is performed in a separate vessel from the extractor. A skilled artisan will appreciate that the poloxamer can also be introduced as a solid into the extractor prior to mixing with the first alkanol. Thus, the process of preparing the poloxamer solution can be made directly in the extractor vessel.

[00189] 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. Examples of supercritical liquids include, but are not limited to, carbon dioxide, methane, ethane, propane, ammonia, Freon®, water, ethylene, propylene, methanol, ethanol, acetone, and combinations thereof. In some embodiments, the supercritical liquid under pressure is a member selected from carbon dioxide, methane, ethane, propane, ammonia and the refrigerants sold as freons. In some embodiments, the supercritical liquid under pressure is carbon dioxide (C0₂).

[00190] 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. 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. For example, the flow rate is 20 kg/h to 100 kg/h, inclusive, such as generally about or 100 kg/h.

[00191] Any suitable temperature that maintains the supercritical liquid in the supercritical state can be used to conduct the extraction processes. For example, the critical temperature of carbon dioxide is about 31° C. Thus, the extractor vessel is kept at a temperature greater than 31° C. In some embodiments, 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. For example, 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. Generally the temperature is greater than 31 ⁰ C but no more than 40 ⁰ C. 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.

[00192] Any suitable pressure can be used in the methods. When supercritical fluid extraction is employed, the system is pressurized at a level to ensure that the supercritical liquid remains at a pressure above the critical pressure. For example, the critical pressure of carbon dioxide is about 74 bars. Thus, 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. Typically, the extractor vessel is pressurized in a range of 125 to 500 bars. In some embodiments, 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. For example, 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.

[00193] Typically, in the methods provided herein, the extraction solvent introduced into the extraction vessel also contains an alkanol. Thus, 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. As described above, in particular examples, the second alkanol is methanol.

[00194] Any suitable combination of the second alkanol and the supercritical liquid, such as any described above, can be used in the extraction solvent in the methods provided herein. In some embodiments, 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.

[00195] For example, 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. For example, the ratio of alkanol (e.g., methanol) to supercritical liquid (e.g., carbon dioxide) can be from about 1 : 100 to about 20: 100. In some embodiments, the ratio of alkanol (e.g., methanol) to supercritical liquid (e.g., carbon dioxide) is from about 1 : 100 to about 15: 100. In some embodiments, 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.

[00196] In certain aspects, the extraction can be conducted in an isocratic fashion, wherein the composition of the extraction solvent remains constant throughout the extraction procedure. For example, 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. Alternatively, 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. Generally, the supercritical liquid (e.g., carbon dioxide) is kept constant while the concentration of the alkanol (e.g., methanol) in the extraction solvent is altered (e.g., increased or decreased) over time of the extraction. The concentrations of the components can be altered by adjusting the flow rate.

[00197] In aspects in which the composition of the extraction solvent can be varied over time, a method in which the second alkanol is increased as the extraction process progresses, either as a step-wise gradient or continuously escalating gradient, is beneficial to the method. In certain instances, 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, however, is low and it takes time to significantly reduce the low molecular weight components, making it less efficient. By increasing the alkanol concentration of the extraction solvent in a gradient fashion (either as a step-wise gradient or as a continuously escalating gradient), the amount of low molecular weight impurities that are extracted increases.

[00198] Also, higher alkanol (e.g., methanol) concentrations increase the solubility, and hence extraction, of higher molecular weight components. Thus, 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. As a non-limiting example to illustrate this, it is believed that a lower alkanol (e.g., methanol) concentration of about 6.6% w/w can remove low molecular weight components. Increasing the 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.

[00199] An extraction solvent with higher alkanol (e.g., methanol) concentrations, however, 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. By increasing the 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.

[00200] It was found that increasing the methanol concentration step-wise increases the loading capacity of the extractor, thereby increasing the throughput in a given extraction system. 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) permeates through the lower phase. 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.

[00201] For example, returning to process 100, the composition of the extraction solvent can be varied as shown in steps 130-140. In some embodiments, 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. In some embodiments, for example, the extraction process for a poloxamer (e.g., P188) 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%). After a defined period, 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. For example, 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%. The alkanol (e.g,. methanol) concentration in the supercritical liquid (e.g., carbon dioxide) can be increased from about 6% to about 18%, or from about 6% to about 12%, or from about 6% to about 10%. The alkanol (e.g., methanol) concentration in supercritical liquid (e.g., carbon dioxide) can be increased from about 7% to about 18%, or from about 7% to about 12%, or from about 7% to about 10%. The alkanol (e.g., methanol) concentration can be increased in any suitable number of steps. For example, the alkanol (e.g., methanol) concentration can be increased over two steps, or three steps, or four steps, or five steps over the course of the extraction procedure. A skilled artisan will appreciate that other solvent ratios and solvent gradients can be used in the extraction processes.

[00202] 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). Generally, 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. It also is understood that in examples in which a gradient of alkanol is employed in the method, 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.

[00203] 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.

[00204] In particular, the methods can be used to purify P188. The process can be applied to other polymers as well. For example, in some embodiments, the methods provided herein provide a method for preparing a purified

polyoxypropylene/polyoxyethylene composition. The method includes:

a) providing or introducing a polyoxypropylene/polyoxyethylene block copolymer solution into an extractor vessel that is dissolved in a first solvent to form the copolymer solution, wherein the first solvent is methanol, ethanol, propanol, butanol, pentanol or a combination thereof, and the composition comprises:

i) a polyoxypropylene/polyoxyethylene block copolymer having the formula HO(CH₂CH20)a'-[CH(CH3)CH20]b-(CH2CH₂0)aH, the mean or average molecular weight of the copolymer is from about 4,000 to about 10,000 Da; and

ii) a plurality of low molecular weight substances having a molecular weight of less than 4,500 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 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; and

c) allowing the extraction mixture to separate to form a plurality of phases comprising a raffinate phase and an extract phase, wherein the raffinate phase and extract phase are separately removed or isolated. [00205] In some cases of the above 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. In the method, 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. Alternatively, 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. In some cases, prior to step c) 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.

[00206] ii) High Pressure Methods

[00207] The method provided herein to purify a poloxamer (e.g., P188) 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. For example, 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.

[00208] In such a method, 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:

a) providing or introducing a poloxamer into an extractor vessel that is dissolved in a first solvent to form a solution, wherein the first solvent is selected from among alcohols, aliphatic ketones, aromatic ketones, amines, and mixtures thereof;

b) admixing an extraction solvent with the solution to form an extraction mixture, wherein the extraction solvent comprises high-pressure carbon dioxide and the solvent, and the concentration of the 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 low molecular weight impurities from the poloxamer.

[00209] The first and second solvent can be the same or different. In the method, 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. For example, the poloxamer is dissolved in a separate vessel and then the solution is added to the extraction vessel.

[00210] In aspects of the method, the extraction solvent is under sub-critical conditions. In this process, 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. Suitable 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. By passing the solvent pair through the product containing the substances, the relatively more soluble component can be extracted. Gaseous solvents can be pressurized at any suitable sub-critical pressure. For example, 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. In some embodiments, the pressure is from about 60 to about 85 bars. In some embodiments, the pressure is about 75 bars.

[00211] Any suitable temperature can be used to conduct the extraction processes. In some embodiments, 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. In some embodiments, the extractor vessel has a temperature of from about 20°C to about 50°C. When purifying poloxamer 188, for example, 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. For example, 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. Alternatively, 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. Generally, 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.

[00213] 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.

[00214] In particular, 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.

[00215] In certain embodiments, the provided methods provide a method for preparing a purified polyoxypropylene/composition. The method includes:

a) providing or introducing a polyoxypropylene/polyoxyethylene block copolymer composition into an extractor vessel that is dissolved in a first solvent to form the copolymer solution, wherein the first solvent is an alcohol, aliphatic ketone, aromatic ketone, amines and mixtures thereof, and the composition contains:

i) a polyoxypropylene/polyoxyethylene block copolymer wherein the mean or average molecular weight of the copolymer is from about 4,000 to about 10,000 Da; and

ii) 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; and

c) allowing the extraction mixture to separate to form a plurality of phases including a raffinate phase and an extract phase, and the raffinate phase and extract phase are separately removed or isolated.

[00216] When the poloxamer is a poloxamer 188 that is purified, 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. In the method, 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. Alternatively, 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. In some cases, prior to step c) 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.

[00217] In certain aspects, 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. In a typical high pressure extraction, 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.

[00218] b. Extraction Vessel and System

[00219] For any of the methods provided herein, 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.

[00220] In some embodiments, 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. These advantages can be validated in a pharmaceutical manufacturing process.

[00221] 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.

[00222] The size of the spheres in the bed is selected based on one or more system properties including the dimensions of the extraction vessel, the residence time of the solvent droplets in the extraction vessel, and the ability of the solvent droplets to coalesce. The diameter of the spheres can range from about 5 mm to about 25 mm. The diameter can be an average diameter, wherein the bed contains spheres of different sizes.

Alternatively, all of the spheres in the bed can have the same diameter. An example of the cross section of stainless steel spheres of different sizes in a solvent distribution bed is shown in FIG. 5.

[00223] Accordingly, an efficient solvent extraction apparatus is provided. The apparatus includes:

a) a distribution system at the bottom of the extractor, wherein the distribution system comprises a plurality of spheres; and

b) a particle coalescence system at the top of the extractor.

In some embodiments, 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.

[00224] c. Extraction and Removal of Extractants

[00225] Any of the methods provided herein can be performed as a batch method or as a continuous method. In some embodiments, the method is a batch method. A batch method can be performed with extraction vessels of various dimensions and sizes as described above. For example, 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

independently and continuously pumped into the extraction vessel maintained at a controlled temperature, flow, and pressure. Substances are removed by varying the extraction solvent composition as described herein. Alternatively, the extraction process conditions such as temperature and pressure can also be varied independently or in combination. As described below, after substances are removed, 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.

[00226] In some embodiments, the extraction method is a continuous method. In a typical continuous extraction, 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.

[00227] Depending on the level of purity desired in the purified poloxamer product, 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. Accordingly, some embodiments of methods provided herein provide extraction methods as described above, wherein after step c), 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.

[00228] When the poloxamer material is sufficiently pure, the product is prepared for further processing. In some embodiments, the product is handled according to process 100 as summarized in Figure 1. The product can be discharged from the extractor vessel and collected in an appropriate receiver, as shown in step 145. The wet product can be sampled for testing with respect to purity, chemical stability, or other properties, as shown in step 150. The product can be dried by removing residual solvents under vacuum. Vacuum level can be adjusted to control drying rates. Drying can be conducted at ambient temperature, or at elevated temperatures if necessary. In general, the drying temperature is held below the melting point of the poloxamer. The wet product can be dried in a single lot or in smaller portions as sub-lots. As shown in steps 160-170, drying of the product can be initiated, for example on a sub-lot, under vacuum at ambient temperature. 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. [00229] Advantageously, the methods disclosed herein effectively recycle carbon dioxide. In particular, supercritical carbon dioxide or high-pressure carbon dioxide can be recovered by subjecting the extract phase to changes in temperature and pressure. In certain embodiments, the methods employed herein have recycling efficiencies of greater than 80%, preferably greater than 90%, and most preferably greater than 95%.

[00230] In the methods provided herein (see, e.g., steps a)-c) above), 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.

[00231] In any of the methods provided herein, various 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.

[00232] d. Exemplary Methods for Preparation of Purified Poloxamers

[00233] As described herein, 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.

[00234] The methods provided herein above result in the generation of particular purified poloxamer preparations, and in particular LCMF PI 88 preparations. In particular, the methods provided herein can be used to purify a P188 copolymer as described herein that has the formula: HO(CH₂CH₂0)_a -(CH₂CH(CH3)0)b-(CH2CH₂0)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.

[00235] In some embodiments, the present methods generate purified poloxamers with less than about 4% low molecular weight components such as less than about 3%, 2% or 1%. Typically, the low molecular weight components include glycols, and volatile degradation impurities such as formaldehyde, acetaldehyde, propionaldehyde, acetone, methanol, and peroxides. In certain instances, 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.

[00236] i) Removal of Low Molecular Weight (LMW) Components

[00237] 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.

[00238] The first alkanol (e.g., methanol) is used to form a poloxamer solution according to step 115' in process 100'. In this process, 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. After forming a poloxamer/alkanol mixture, all or part of the mixture is pumped into the extractor as shown in step 120'. In some cases, 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.

[00239] 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. For example, in an exemplary method, 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).

[00240] 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, 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. For example, the composition of the extraction solvent can be varied as shown in steps 130'- 140'. For example, as shown in step 130', the extraction process for a poloxamer (e.g., P188) starts using about 5% to 7%, by weight (w/w) of an alkanol (e.g. , methanol) in an extraction solvent with a supercritical liquid (e.g. , carbon dioxide), (e.g. , about 6.6%). After a defined period, 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.

[00241] For a commercially efficient purification process, it desirable to have successively increasing methanol concentrations where the profile is suitably modified to selectively remove most of the low molecular weight components. Residual low molecular weight components can be subsequently removed with high methanol concentrations in a short time. Therefore a stepwise methanol concentration profile where about a 5- 10% (e.g. , 6.6%) methanol is used for 12 hours, a higher methanol is used for 10 hours and finally an even higher methanol is used for 4 hours is used to produce purified product in high yields without significantly reducing the overall yield and not enriching the high molecular weight components.

[00242] When the poloxamer material is sufficiently pure, 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. In an exemplary method, as shown in steps 160'-170', 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.

[00243] ii) Preparation of Long Circulating Material Free (LCMF)

Poloxamer

[00244] 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. As shown in step 105", the poloxamer and first alkanol (e.g., methanol) are dispensed into the extractor vessel and to form the poloxamer solution. In this process, dispensing of 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. In some cases, the poloxamer solution can be formed a separate vessel, and the poloxamer solution transferred to the extractor vessel.

[00245] 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.

Typically, 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.

[00246] 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. For example, in an exemplary method, 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.

[00247] The extraction is conducted in the presence of the second alkanol, which acts as a co-solvent modifier of the carbon dioxide. As shown in steps 125"- 135", the second alkanol, such as methanol, 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. For example, the composition of the extraction solvent can be varied as shown in steps 125"-135". For example, as shown in step 125", the extraction process for a poloxamer (e.g., P188) starts using about 7% to 8% (e.g. , about or 7.4%), by weight (w/w) of an alkanol (e.g., methanol) in an extraction solvent with a supercritical liquid (e.g., carbon dioxide). After a defined period, 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.

[00248] For an extraction process that removes components other than low molecular weight components, including components that, when administered, give rise to long circulating forms, it desirable to have a process that maximizes the purity and removal of these components while minimizing reductions in yield. It is found that successively increasing alkanol (e.g., methanol) concentrations when starting from a higher concentration of alkanol (e.g., methanol) than in other methods, generally starting at 7% to 8% by weight, the profile is suitably modified to selectively remove these components and low molecular weight components, while minimizing reductions in yield. For example, such an exemplary method can produce yields greater than 55%, and generally greater than 60% or 65%. Residual low molecular weight components can be

subsequently removed with high methanol concentrations in a short time. Therefore a stepwise methanol concentration profile where about 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.

[00249] When the poloxamer material is sufficiently pure, 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". 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".

[00250] iii) Methods for Confirming the Identity of LCMF Poloxamers

[00251] LCMF poloxamer 188 preparations have different properties from poloxamer

188 preparations of the prior art and commercially available preparations that contain the

LCM material. In particular, the LCMF poloxamer 188, which lacks the LCM material, is more hydrophilic, and can be distinguished based on this property. To confirm that a poloxamer 188 preparation made by the methods herein or other methods is an LCMF poloxamer 188, 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

188 (e.g., those sold under the trademarks Pluronic^® F-68, Flocor®, Kolliphor® and

Lutrol^®), and the behavior in RP-HPLC under the conditions exemplified herein (see e.g. ,

Example 1). Any method that confirms that the preparation lacks LCM material can be used.

[00252] E. PHARMACEUTICAL COMPOSITIONS, FORMULATIONS AND COMBINATION THERAPY

[00253] 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

administration, and the regimen. For purposes herein, low doses of poloxamer generally are used, so that the target circulating concentrations typically are at or about 0.25 - 2.5 mg/ml.

[00254] 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. Provided herein are compositions containing the poloxamer and a fibrinolytic inhibitor. 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. 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.

[00255] In other embodiments, the fibrinolytic inhibitor is administered to mitigate adverse effects of poloxamer, particularly poloxamer 188 therapy. In such instances, the dosage of poloxamer is appropriate for treatment of any particular disorder. The fibrinolytic inhibitor is administered to mitigate any adverse effects, particularly bleeding.

[00256] 1. Formulations

[00257] Pharmaceutical compositions containing P188, such as LCMF 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.

[00258] Effective concentrations of PI 88, such as an LCMF PI 88, are mixed with a suitable pharmaceutical carrier or vehicle for systemic, topical, or local administration. In particular, for the methods herein, the poloxamer is administered by IV, such as by continuous infusion or a series of bolus injections.

[00259] Pharmaceutical carriers or vehicles suitable for administration of the copolymers include any such carriers known to those skilled in the art to be suitable for the particular mode of administration. 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. [00260] The compositions can be prepared for dilution prior to administration or for direct administration. In general, for the methods herein, the compositions are

administered by IV, either continuous infusion or a series of bolus injections. 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 .

[00261] 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. For local internal administration, such as, intramuscular, parenteral or intra-articular 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. For purposes herein, the compositions typically are aqueous solutions, suspensions, or emulsions for IV administration.

[00262] Generally, pharmaceutically acceptable 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. For example, the methods provided herein have applications for both human and animal use.

[00263] Pharmaceutical compositions can include carriers such as a diluent, adjuvant, excipient, or vehicle with which an isoform is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, and sesame oil. Water 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. [00264] Compositions can contain, along with a poloxamer, such as P188, such as LCMF P188: a diluent, such as lactose, sucrose, dicalcium phosphate, or

carboxymethylcellulose; a lubricant, such as a magnesium stearate, calcium stearate, and talc; and a binder, such as starch, natural gums, such as gum acacia, gelatin, glucose, molasses, polyvinylpyrrolidone, celluloses and derivatives thereof, povidone,

crospovidones, and other such binders known to those of skill in the art. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol. A composition, if desired, also can contain minor amounts of wetting or emulsifying agents or pH buffering agents, for example, acetate, sodium citrate, cyclodextrin derivatives, sorbitan

monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. These compositions can take the form of solutions, suspensions, 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.

[00265] The formulation is selected to suit the mode of administration. For example, compositions containing 188 such as LCMF P188, 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. For this purpose 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.

[00266] 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.

[00267] PI 88, such as 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.

74(9):922-925).

[00268] 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.

[00269] 2. Dosage

[00270] The pharmaceutical compositions containing P188, such as LCMF P188 provided herein, can be formulated for single dosage (direct) administration, multiple dosage administration, or for dilution or other modification. Typically, 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. Those of skill in the art can readily formulate a composition for administration in accord with the methods herein. For example, to formulate a composition, the weight fraction of a compound or mixture thereof is dissolved, suspended, dispersed, or otherwise mixed in a selected vehicle at an effective concentration such that risks and consequences associated with administration of a fibrinolytic inhibitor are improved and/or the intended effect is observed.

[00271] 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.

[00272] If necessary, a particular dosage and duration and treatment protocol can be empirically determined or extrapolated. For example, 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

accordingly. 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.

[00273] In particular, 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. Typically, 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. [00274] For example, when administered separately or as a component of the pharmaceutical composition described herein, the poloxamer is administered at a concentration of between about 0.5% to 20%, although more dilute or higher

concentrations can be used. For example, 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. In other embodiments, 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. In yet other embodiments, 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. For example, the concentration is 10% to 22.5%, such as 10% to 20% or 15% to 20%.

[00275] Typically, 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.

[00276] In some examples, 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.

[00277] In other examples, the poloxamer is formulated so that repetitive

administration of the poloxamer to a subject, for example, administration a second, third, or multiple times, results in an effective amount of poloxamer in the circulation of the subject. For example, 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. For example, 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. In one example, 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.

[00278] In one example, 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. For other applications, 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.

[00279] 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.

[00280] 3. Fibrinolytic inhibitors

[00281] 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.

[00282] 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 ε-aminocaproic acid (EACA or ACA) and the more potent tranexamic acid (TXA). For example, a commercial formulation of TXA is available as Cyklokapron® TXA. Each ml of Cyklokapron contains 100 mg TXA in water for injection.

[00283] In some embodiments, the fibrinolytic inhibitor is tranexamic acid.

Fibrinolytic inhibitors, such as tranexamic acid, are used to treat excessive blood loss during surgery and in various other medical conditions. 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. [00284] 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. For surgery, 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.

[00285] 4. Compositions, combinations and combination therapy

[00286] Provided are 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.

[00287] For the methods and uses herein any suitable ratio of poloxamer to fibrinolytic inhibitor is used in the methods, uses and compositions herein. In general, as discussed above, 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. [00288] For example, the ratio of poloxamer 188 to fibrinolytic inhibitor is from about 0.001: 1 to about 1000: 1 by weight. In some embodiments, 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. In some embodiments, 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.

[00289] 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.

[00290] 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

administration. 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. For use in conjunction with a fibrinolytic inhibitor administered for bleeding, such as hemorrhagic shock, 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. Generally, the poloxamer is administered by the intravenous route either by bolus or by continuous infusion although other routes may be used.

[00291] One of skill in the art will appreciate that the effective amount of the fibrinolytic inhibitor depends on the potency of the fibrinolytic inhibitor. When using the pharmaceutical fibrinolytic inhibitor TXA, for example, 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.

[00292] 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.

Generally the fibrinolytic inhibitor is administered either by the intravenous or oral route although other routes of administration can be employed. When administered as the pharmaceutical composition described herein, the route generally preferred is intravenous administration although other routes may be used. In this case, the fibrinolytic inhibitor is typically at a concentration of between 0.1% and 10% although more dilute or higher concentrations can be used. When the fibrinolytic inhibitor is administered individually, a commercially available preparation (either as an oral tablet or intravenous injection) can be used.

[00293] In the methods herein, 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. In particular, 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.

[00294] Prevention of the risks and consequences associated with administration of a fibrinolytic inhibitor with poloxamer 188, such as a purified poloxamer 188 and LCMF P188 described herein, can be effected by any suitable route of administration using suitable formulations as described herein including, but not limited to, injection, pulmonary, oral, and transdermal administration. Treatment typically is effected by intravenous administration of the poloxamer.

[00295] 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.

[00296] 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

administration of a fibrinolytic inhibitor, such as, for example, administration of a fibrinolytic inhibitor during surgery or following trauma. The need for such treatment, if not readily apparent, can be determined by standard clinical techniques. In addition, if needed, 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.

[00297] In some examples, 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. Despite the relatively short half-life, the effects of a poloxamer, such as a purified poloxamer 188, can be long lasting. Thus, 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. In general, 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.

[00298] If necessary, 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.

[00299] In general, a goal is to administer the dose in the smallest volume possible. Typically, the volume to be administered is not greater than 3.0 mL/kg of a subject. For example, 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 to 1.0 mL/kg, 1 mL/kg to 3 mL/kg, 1 mL/kg to 2.5 mL/kg, 1 mL/kg to 2.0 mL/kg, 1 mL/kg to 1.8 mL/kg, 1 mL/kg to 1.4 mL/kg, 1.4 mL/kg to 3.0 mL/kg, 1.4 mL/kg to 2.5 mL/kg, 1.4 mL/kg to 2.0 mL/kg, 1.4 mL/kg to 1.8 mL/kg, 1.8 mL/kg to 3.0 mL/kg, 1.8 mL/kg to 2.5 mL/kg, 1.8 mL/kg to 2.0 mL/kg, 2.0 mL/kg to 3.0 mL/kg, 2.0 mL/kg to 2.5 mL/kg, or 2.5 mL/kg to 3.0 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. Again, 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.

[00300] The 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.

[00301] Typically, the administered dose is administered as an infusion. Generally, 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. In some examples, 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.

[00302] In some examples, 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.

[00303] 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. For example, 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. For example, 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

risk/consequences. Exemplary fibrinolytic inhibitors for use in the methods provided herein include any known to those of skill in the art, and those described above.

Exemplary of these are, but are not limited to, 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 ε-aminocaproic acid (ACA) and tranexamic acid (TA; Cyklokapron®), and combinations thereof.

[00304] 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(C₂H₄0)_a'— (C₃H60)b— (C₂H₄0)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. In the methods provided herein, 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.

[00305] 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. For example, dosages for fibrinolytic inhibitors are typically the recommended doses for such inhibitors, for example, dosages described in standard manuals, including the Physician's Desk

Reference and Remington 's Pharmaceutical Sciences (Mack Publishing Co., Easton, PA). As described, the provided methods include administration of the poloxamer where a subject suffering from hemorrhagic shock has been administered a fibrinolytic inhibitor. In some methods, the poloxamer mitigates a consequence of administering a fibrinolytic inhibitor, including ischemic tissue damage and prothrombotic events, such as embolism and thrombosis.

[00306] In methods provided herein, 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

administered in combination with therapy, such as administration of fibrinolytic inhibitors, for an underlying condition. In other embodiments of the methods, the polyoxyethylene/polyoxypropylene copolymer is administered after the fibrinolytic inhibitor is administered or symptoms occur.

[00307] In the provided methods, administration of the poloxamer can be repeated, for example, a second, third, fourth time, or more. For example, 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. In methods where the poloxamer is administered in combination with a fibrinolytic inhibitor, administration of the inhibitor can be repeated, for example, a second, third, fourth time, or more.

[00308] F.Examples

[00309] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[00310] Example 1

[00311] Preparation and administration of long circulating material free

(LCMF) poloxamer 188

[00312] A. Supercritical Fluid Extraction (SFE) Process

[00313] 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.

Before purification, the poloxamer 188 raw material (BASF Corporation, Washington, New Jersey) 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.

[00314] 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 C0₂ (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), was cooled in a heat exchanger and fed into a temperature-controlled, high pressure, stainless steel, solvent reservoir. 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) 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.

[00315] 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). Throughout the extraction process, 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.

[00316] After extraction, 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%.

[00317] Molecular weight analysis of the purified product as determined by GPC demonstrated that the purified product met the acceptance specifications. There was an average molecular weight of the main peak of about 8,500 + 750 Da and an average molecular weight average of 8,500 + 750 Da, no more than 1.5 % low molecular weight (LMW) species of less than 4,500 Da and no more than 1.5 % high molecular weight species (HMW) greater than 13,000 Da. In addition, the polydispersity was no more than 1.05. Thus, the results showed that the procedures resulted in a measurable reduction in the LMW species, and an improvement in the polydispersity of the purified product.

[00318] 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.

[00319] B. Characterization of the plasma concentration time course following intravenous administration of purified (LCMF) poloxamer 188 using HPLC-GPC (method 1)

[00320] Purified LCMF poloxamer 188 generated as described above was

administered intravenously to 62 healthy volunteers as part of assessment to determine its effect on the QT/QTc interval. Eight of the 62 subjects were randomly selected for quantitative analysis of the plasma poloxamer levels using an HPLC-GPC method.

Following LCMF poloxamer 188 administration, 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. A mean maximum concentration (Cmax) of the administered purified poloxamer 188 of 0.9 mg/mL was attained by the end of the one hour loading infusion. The mean concentration at steady state (Css) was about 0.4 mg/ml and was attained during maintenance infusion. The plasma concentration declined rapidly following

discontinuation of the maintenance infusion. The LCMF product purified as described above did not demonstrate the long circulating higher molecular weight material, observed with prior poloxamer 188 and as defined herein, in the plasma. [00321] To confirm the absence of such long circulating material in plasma, plasma from subjects receiving the higher dose were similarly studied using HPLC-GPC.

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. In both figures, 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.

[00322] 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.

[00323] C. Comparison of the plasma concentration time course following intravenous administration of purified LCMF poloxamer 188 and purified LCM- containing poloxamer 188 by HPLC-GPC

[00324] 1. Administration of the long circulating material (LCM)-containing poloxamer 188

[00325] 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.

Plasma was separated and stored frozen until analysis using an HPLC-GPC method. Analysis of the plasma samples revealed the clearance kinetics of the main peak and the HMW peak for the (LCM-containing) purified poloxamer 188

[00326] HMW peak (the long circulating material)

[00327] Following administration at the above dose, the HMW component (detected in the HPLC-GPC assay as a peak of approximately 16,000 Daltons) was accumulating during the drug administration period and did not reach its mean Cmax concentration of 225 ug/ml ( n = 6) until 2 hours after the end of drug administration. By 6 hours after discontinuation of infusion, mean plasma levels remained at 202 ug/ml, a concentration that had declined by only about 10% from the Cmax value. 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.

[00328] Main peak

[00329] Following administration at the dose above, the main peak achieved an apparent mean steady state concentration of 522 ug/ml (n=6) that was maintained during drug infusion. One hour after discontinuation of infusion, plasma levels dropped from the steady state concentration by 52 % to 255 ug/ml. By 6 hours after discontinuation, plasma levels had dropped by 85% to 81 ug/ml. By 24 hours post infusion, plasma levels declined by 96% to a plasma concentration of about 19 ug/ml (n=6). Based on these changes in the plasma concentration time course the half-life is estimated to be about 5 hours

[00330] 2. LCMF poloxamer 188 (prepared as described above)

[00331] 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.

[00332] HMW peak

[00333] Following administration at the above dose, the HMW component, which was detected in the HPLC-GPC assay as a peak of approximately 16,000 Daltons, accumulated to a small extent during drug administration, and achieved its Cmax (mean value of 117 ug/ml, n = 8) by end infusion. By 1 hour after discontinuation of drug administration, plasma levels had declined by 27 % from the Cmax value to 86 ug/ml. By 6 hours after the end of drug administration, mean plasma levels had declined by 71 % from the Cmax value to 34 ug/ml. By 18 hours after the end of infusion, the mean plasma level had declined by 82 % to a concentration of 19 ug/ml (n = 8). Based on these changes in the plasma concentration over time, the elimination half-life for the HMW component was estimated to be between 6 - 9 hours.

[00334] Main Peak

[00335] Following administration at the dose above, the main peak achieved an apparent mean steady state concentration of 2,637 ug/ml that was maintained during the 6 hour infusion period (n=8). One hour after discontinuation of infusion, 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. By 18 hours after discontinuation of infusion, mean plasma levels declined by over 98 % (from steady state) to a plasma concentration of about 34 ug/ml (n=6). Based on these changes in the plasma concentration time course, the elimination half-life is estimated to be about 3 hours.

a. Summary comparison table

[00336] A comparison of the relative rates of clearance from the plasma at similar time points following administration is shown in TABLE 1 below. The data demonstrate a marked difference in the rate of decline in plasma concentration between (LCM- containing) purified poloxamer 188 and the LCMF poloxamer 188, demonstrating that LCMF poloxamer 188 clears faster. The difference is apparent for the HMW peak and for the main peak. The difference is most apparent for the HMW peak. This shows that the LCMF poloxamer is different from the LCM-containing poloxamer of the prior art.

TABLE 1

[00337] D. Analytical Data Confirming That Purified LCMF Poloxamer 188 Is Different From Purified Poloxamer 188 containing LCM [00338] 1. Analytical test (RP-HPLC assay) to compare various poloxamers

[00339] In reversed phase chromatography there is a hydrophobic stationary phase (the column) and a more polar mobile phase. Because of this "reversed" phase condition, RP-HPLC is commonly used to separate compounds based on relative hydrophobicity. More hydrophobic compounds exhibit a longer column retention time compared to more hydrophilic compounds.

[00340] The following 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:

*ELS = evaporative light scattering

Results

[00341] The results show that the LCMF poloxamer 188 is different from the prior art purified poloxamer 188. It has different pharmacokinetic properties, which reflect that it is more hydrophilic than the prior art material that contains the long circulating material.

[00342] 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 ¾ 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 ¾· 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.

[00343] Figure 10 shows the chromatograms for 3 different lots of purified LCMF poloxamer 188 and 2 different lots of purified (LCM-containing) poloxamer 188. These results demonstrate a robust reproducibility for the different lots of materials, and show that the difference between the two materials cannot be accounted for by assay variability. These results demonstrate that the polymeric distribution of LCMF poloxamer 188 is more hydrophilic than purified poloxamer 188.

[00344] 2. The different pharmacokinetic behavior of the LCMF purified poloxamer and the LCM-containing poloxamer correlate with the differences in their hydrophilicity

[00345] As described herein (see, e.g., Example IB, above, and FIGs. 7-10) and TABLE 1), 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.

[00346] The polymeric size distribution of purified variants of poloxamer 188 (purified LCM-containing poloxamer 188, and the LCMF poloxamer 188) is similar with regard to size as shown by HPLC-GPC. Both meet the criteria:

* NMT= Not More Than [00347] While the polymeric size distribution, as shown by HPLC-GPC, of both purified poloxamers is similar, as demonstrated by the RP-HPLC herein, the molecules that comprise the polymeric distribution of LCMF poloxamer 188 are more hydrophilic.

[00348] When injected into an animal a more hydrophilic polymeric distribution clears from the circulation at a faster rate. This accounts for the decreased presence of a long circulating material in the LCMF poloxamer 188 preparation. The results also indicate that, as observed and described above, the main peak of the polymeric distribution clears faster. For example, the plasma concentration time course data from a clinical trial show a shorter elimination half-life for the main peak and the high molecular weight peak of the LCMF poloxamer 188 compared to the purified poloxamer 188 containing LCM.

[00349] 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. For example, 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. In contrast, more hydrophobic, higher molecular weight poloxamers such as P338 (also called Pluronic® F108) and P308(Pluronic® F98), increase blood viscosity and RBC aggregation (Armstrong et al. (2001) Biorheology, 38:239-247). This is the opposite effect of PI 88 and indicates that higher molecular weight, hydrophobic poloxamer species can have undesirable biological effects.

[00350] The results, thus, indicate that the hydrophobic components contained in the high molecular weight peak of purified (LCM-containing) poloxamer 188 are an unwanted impurity. Thus a poloxamer 188 such as LCMF poloxamer with a reduced amount of these components is desirable.

[00351] Example 2

[00352] In vitro fibrinolytic assay assessing the effect of poloxamer 188 and a fibrinolytic inhibitor on fibrinolysis

[00353] Following traumatic injury, coagulation enzymes promote clot formation (hemostasis) while the fibrinolytic system promotes the competing mechanism of clot dissolution (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. During the assembly of a fibrin clot, optical density increases, while during clot dissolution (fibrinolysis), optical density decreases.

[00354] 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). In each assay, clotting was initiated by sequential addition of 0.25 μΜ calcium chloride followed by 0.5 μg/mL thrombin. Following addition of thrombin, the change in optical density (405 nm) was measured using a

spectrophotometer in 1 minute intervals for 15 minutes. Details and results of each assay are discussed below.

[00355] Poloxamer 188 in plasma

[00356] The poloxamer 188 at concentrations of 0.3125, 0.625, 1.25, 2.5, 5 and 10 mg/mL was added to citrated human plasma, followed by the addition of 0.25 μΜ calcium chloride and 0.5 μg/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 11, there was a modest concentration dependent increase in the rate of fibrin assembly (indicated by the rate of increase in OD) and maximum turbidity (indicated by the peak OD) for samples containing at least 2.5 mg/mL poloxamer 188 compared to plasma containing none or low concentrations, less than 2.5 mg/mL, of the poloxamer. There was no evidence of fibrin dissolution at any

concentration.

[00357] Urokinase in plasma

[00358] 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 μΜ calcium chloride and 0.5 μg/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. As shown in Figure 12, at the highest concentration of urokinase (2,500 U/mL), complete clot lysis (OD = 0) was achieved in about 6 minutes. At the lowest concentration of urokinase (312 U /mL), complete clot lysis was not achieved even after 15 minutes.

[00359] Poloxamer 188 and urokinase in plasma

[00360] Varying concentrations of urokinase (312.5, 625, 1250, and 2500 U/mL) and 10 mg/mL poloxamer 188 were added to citrated human plasma, followed by the addition of 0.25 μΜ calcium chloride and 0.5 μg/mL thrombin. The rate of fibrin assembly and dissolution was assessed at 1 minute intervals for 15 minutes after thrombin addition. As shown in Figure 13, which depicts the change in turbidity over time for each sample as measured by optical density at 405 nm, at all concentrations of Poloxamer 188, the time until the onset of lysis, indicated by the point at which turbidity decreased, and the time to complete clot lysis, OD = 0, were shortened. For each sample, the final optical density following complete lysis decreased below baseline values, indicating that poloxamer 188 increases the lytic activity of urokinase on fibrinogen (fibrinogenolysis).

[00361] Poloxamer 188, urokinase, and tranexamic acid in plasma

[00362] Urokinase (1500 U/mL), the fibrinolytic inhibitor tranexamic acid

(10 mg/mL), and either 0.3125, 0.625, 1.25, 2.5, 5, or 10 mg/mL of poloxamer 188 were added to citrated human plasma, followed by the addition of 0.25 μΜ calcium chloride and 0.5 μg/mL thrombin. The rate of fibrin assembly and dissolution was assessed at 1 minute intervals for 15 minutes after thrombin addition. The results show that any fibrinolytic and/or fibrinogenolytic enhancement due to poloxamer 188 (see Figure 13) was offset by the addition of the fibrinolytic inhibitor tranexamic acid, as shown in Figure 14. Figure 14 depicts the change in turbidity over time for each sample as measured by optical density at 405 nm. There was no evidence of fibrinolysis and/or fibrinogenolysis in any sample.

[00363] These results indicate that the combination of the fibrinolytic inhibitor, such as tranexamic acid, and poloxamer 188 mitigate or overcome the enhanced fibrinolysis from the poloxamer 188, resulting in coagulation. This effect would decrease

hemorrhagic risk in a subject to whom the poloxamer and fibrinolytic inhibitor are administered compared to administration of fibrinolytic inhibitor alone.

[00364] Example 3

[00365] Preparation and administration of a composition containing poloxamer 188 and a fibrinolytic inhibitor to human subjects [00366] A. Preparation of a composition containing poloxamer 188 and a fibrinolytic inhibitor

[00367] 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. To prepare the composition, 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.

[00368] B. Administration of a composition containing LCMF poloxamer 188 and a fibrinolytic inhibitor to a patient with trauma and hemorrhagic shock

[00369] A 22-year-old male weighing 70 kg (154 pounds), admitted to the emergency room with multiple abdominal stab wounds and severe hypotension, receives hypotensive resuscitation and is quickly transported to the operating room for exploratory surgery. Following surgery, the patient is relatively stable with a blood pressure of 108/68 mm Hg and tissue oxygenation (St0₂) of 85%. Two hours later he is hypotensive and is oliguric (low urine output), and his St0₂ decreases to 49%. Sublingual intravital microscopy reveals a severely impaired microcirculation. Blood chemistry reveals acidosis, significant base excess, and high lactate. 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 St0₂ increases to 85%. Three hours after administration of the composition, his St0₂ 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.

[00370] C. Administration of a composition containing poloxamer 188 and a fibrinolytic inhibitor to a patient who had undergone surgery

[00371] A 78-year-old woman, weighing 80 kg and a history of diabetes, has severe three-vessel coronary artery disease with left main coronary trunk stenosis upon cardiac catheterization. She has emergency coronary artery bypass surgery. Prior to

cardiopulmonary bypass, she is treated with the composition containing poloxamer 188, described above, and TA, administered as a loading infusion of 53 mL over 15 minutes, followed by a maintenance infusion of 27 mL/hr, which continued during the 4-hour surgery. The surgery is successful and uneventful. Blood samples taken 1, 6, 12 and 24 hours post-surgery show that her serum creatine kinase-MB (CK-MB), lactate and lactate dehydrogenase levels are moderately elevated at hour 1, but almost within normal range by 24 hours post-surgery. The patient's interleukin 6 (IL-6), tumor necrosis factor (TNF) alpha, and high sensitivity C-reactive protein (hs-CRP) levels remain within normal limits at all time points. Despite her age and other pre-existing risk factors, the patient's clinical course is uncomplicated. 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.

[00372] The various features and embodiments of the present invention, referred to in individual sections above apply, as appropriate, to other sections, mutatis mutandis.

Consequently features specified in one section may be combined with features specified in other sections, as appropriate. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for treating damaged tissue and/or damaged cell surfaces, or unwanted bleeding or hemorrhagic shock or a precursor thereto, comprising

administering to a subject in need thereof a therapeutically effective amount of a polyoxyethylene/polyoxypropylene copolymer and a therapeutically effective amount of a fibrinolytic inhibitor.

2. The method of claim 1, wherein the bleeding is consequent to treatment with a fibrinolytic inhibitor or the copolymer.

3. The method of claim 1, wherein the bleeding occurs during surgery or from trauma or injury.

4. The method of claim 1 wherein the bleeding is associated with

hemorrhagic shock or a precursor thereto.

5. The method of claim 1, wherein the damaged tissue or bleeding results from a condition selected from among blunt trauma, anemia, aneurysm, hypovolemia, burns, shock, hemorrhage, surgery and tissue infarction.

6. The method of any of claims 1-5, wherein the fibrinolytic inhibitor and copolymer are co-administered, or are administered sequentially or intermittently, or in the same composition.

7. The method of any of claims 1-6, wherein the fibrinolytic inhibitor is administered before the copolymer.

8. The method of any of claims 1-7, wherein the copolymer is administered before the fibrinolytic inhibitor.

9. The method of any of claims 1-8, wherein the fibrinolytic inhibitor and copolymer are administered via intravenous infusion or bolus injection.

10. The method of any of claims 1-8, wherein the fibrinolytic inhibitor is administered orally.

11. The method of claim 8, wherein the copolymer is administered by intravenous infusion or bolus injection.

12. The method of claim 10, wherein the fibrinolytic inhibitor is administered as a tablet or as a mouthwash.

13. The method of any of claims 1-12, wherein the fibrinolytic inhibitor is an endogenous inhibitor selected from the group consisting of a plasminogen activator inhibitor (PAI), a plasmin inhibitor, alpha-2-macroglobulin, and thrombin-activatable fibrinolysis inhibitor (TAFI) and modified forms thereof, or a pharmaceutical or synthetic inhibitor that is a synthetic derivative of lysine.

14. The method of claim 13, wherein the fibrinolytic inhibitor is an

endogenous inhibitor selected from the group consisting of PAI-1, PAI-2, PAI-3, alpha-2- antiplasmin, alpha-2-macroglobulin and TAFI.

15. The method of claim 14, wherein the fibrinolytic inhibitor is a

pharmaceutical or synthetic inhibitor selected from among aprotinin, ε-aminocaproic acid and tranexamic acid (TA).

16. The method of any of claims 1-15, wherein the fibrinolytic inhibitor is administered in an amount between 1 mg/kg and 30 mg/kg, 5 mg/kg and 25 mg/kg, 7.5 mg/kg and 20 mg/kg, 10 mg/kg and 15 mg/kg, or at least 1, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, up to 30 mg/kg subject body weight.

17. The method of any of claims 1-16, wherein the copolymer is administered in an amount to result in an amount of copolymer in the circulation of from 0.25 mg/mL to 2.5 mg/mL, or 0.4 mg/mL to 2.0 mg/mL, or 0.5 mg/mL to 1.5 mg/mL in less than 24 hours, 20 hours, 15 hours, 12 hours, 10 hours, 6 hours or between 2 and 6 hours, inclusive.

18. The method of claim 17, wherein the concentration of the copolymer in the circulation is the peak concentration.

19. The method of any of claims 17 - 18, wherein the concentration of the copolymer is the concentration in the circulation at steady state.

20. The method of any of claims 17-18, wherein the concentration of the copolymer in the circulation is administered so that it remains at the target concentration for up to 72 hours following administration.

21. The method of any of claims 1-20, wherein the copolymer is administered to prevent the adverse effects of administration of fibrinolytic inhibitor, wherein the adverse effects are thromboembolic or ischemic events.

22. The method of any of claims 1- 21, wherein the fibrinolytic inhibitor and the copolymer are administered prior to surgery and/or to treat or prevent hemorrhagic shock.

23. The method of any of claims 1-22, wherein:

the polyoxyethylene/polyoxypropylene copolymer has the formula:

HO(CH₂CH20)a'-[CH(CH3)CH20]b-(CH2CH₂0)aH;

each a' and a are the same or different and each is an integer, whereby the hydrophile portion represented by (C2H₄0) constitutes approximately 60% to 90% or 60%- 90% by weight of the compound; and

b is an integer, whereby the hydrophobe represented by (¾Η₆0) has a molecular weight of about 1,200 Da to about 2,300 Da or 1,200 to 2,300 Da.

24. The method of any of claims 1-23, wherein:

the polyoxyethylene/polyoxypropylene copolymer has the formula:

HO(CH₂CH20)a'-[CH(CH3)CH20]b-(CH2CH₂0)aH;

a' and a can be the same or different and each is an integer from 5 to 150, inclusive; and

b is an integer from 15 to 75, inclusive.

25. The method of any of claims 23 - 24, wherein:

the copolymer has the formula:

HO(CH₂CH20)a'-[CH(CH3)CH20]b-(CH2CH₂0)aH;

a' and a are the same or different and each is an integer from 70 to 105, inclusive; and

b is an integer from 15 to 75, inclusive.

26. The method of any of claims 23-25, wherein the hydrophobe represented by (C₃H₆0) has a molecular weight of about 1,400 to 2,000 Da or 1,400 to 2,000 Da, and the hydrophile portion constitutes approximately 70% to 90% or 70% to 90% by weight of the copolymer.

27. The method of any of claims 23-26, wherein the hydrophobe represented by (C₃H₆0) has a molecular weight of 1,500 Da to 2,100 Da or 1,700 Da to 1,900 Da.

28. The method of any of claims 25-27, wherein:

the hydrophile portion is about 80% of the total molecular weight of the copolymer; and

the hydrophobe portion has a molecular weight of at or about 1,800 Da.

29. The method any of claims 23-28, wherein the molecular weight of the hydrophobe portion (¾Η₆0) is approximately or is 1,750 Da and the total molecular weight of the copolymer is approximately or is 7350 Da to 8,850 Da.

30. The method of any of claims 23-29, wherein the copolymer comprises a poloxamer 188.

31. The method of any of claims 23-30, wherein the copolymer has reduced impurities, whereby the polydispersity value is less than or equal to 1.07.

32. The method of any of claims 23-31, wherein the polydispersity value is less than or equal to 1.06, 1.05, 1.04, 1.03, 1.02 or 1.01.

33. The method of any of claims 23-32, wherein no more than 1.5% or 1% of the total components in the distribution of the copolymer are low molecular weight components having an average molecular weight of less than 4,500 Daltons.

34. The method of any of claims 23-33, wherein the

polyoxyethylene/polyoxypropylene copolymer has the formula:

HO(CH₂CH20)a'-[CH(CH3)CH20]b-(CH2CH₂0)aH, wherein a' and a are the same and are 78, 79 or 80, and b is 27, 28, 29 or 30.

35. The method of claim 34, wherein: a' and a are 80; and b is 27.

36. The method of any of claims 23-35, wherein the

polyoxyethylene/polyoxypropylene copolymer is purified to reduce low molecular weight substances.

37. The method of any of claims 23-36, wherein the copolymer comprises poloxamer 188 (P188).

38. The method of any of claims 23-37, wherein the copolymer is a long- circulating material-free (LCMF) poloxamer.

39. The method claim 38, wherein the copolymer is a long-circulating material-free (LCMF) poloxamer that, when administered to a human subject, does not contain a component that is or gives rise to in the plasma of the subject to a material or component that has a circulating half-life (ti/₂) that is more than about 1.5-fold or 1.5-fold greater than the half-life of the main component in the distribution of the copolymer preparation, or is such that all components have a circulating half-life that is within 5-fold of the half-life of the main component.

40. The method of any of claims 38 - 39, wherein:

the long circulating material free (LCMF) poloxamer 188 is a polyoxyethylene/polyoxypropylene copolymer that has the formula

HO(CH₂CH20)a'-[CH(CH3)CH20]b-(CH2CH₂0)aH;

each of a and a' is an integer such that the percentage of the hydrophile (C₂H₄0) is between approximately 60% and 90% by weight of the total molecular weight of the copolymer;

a and a' are the same or different;

b is an integer such that the molecular weight of the hydrophobe (C3H6O) is between approximately 1,300 and 2,300 Daltons;

no more than 1.5% of the total components in the distribution of the copolymer are low molecular weight components having an average molecular weight of less than 4,500 Daltons;

no more than 1.5% of the total components in the distribution of the copolymer are high molecular weight components having an average molecular weight of greater than 13,000 Daltons;

the polydispersity value of the copolymer is less than approximately 1.07 or less than 1.07; and

following intravenous administration to a human subject, the circulating plasma half-life of any components not comprising the main peak in the polymeric distribution of copolymer is no more than 5.0-fold the circulating half-life of the main component in the distribution of the copolymer.

41. The method of any of claims 38-40, wherein all components comprising the polymeric distribution of the poloxamer copolymer have a circulating half-life in the plasma of the subject that is no more than 4.0-fold or 3.0 -fold longer than the circulating half-life of the main component of the copolymer following intravenous administration to a subject.

42. The method of any of claims 38-41, wherein all components in the distribution of the copolymer, when administered to a human subject, have a circulating half-life in the plasma of the subject that is no more than 3-fold longer than the circulating half-life of the main component in the distribution of the copolymer.

43. The method of any of claims 38-42, wherein all components in the distribution of the copolymer, when administered to a human subject, have a half-life in the plasma of the subject that is no more than 10 or 12 hours.

44. The method of any of claims 38-43, wherein the

polyoxyethylene/polyoxypropylene copolymer is a poloxamer with a hydrophobe having a molecular weight of about 1,400 Da to 2,000 Da or 1,400 to 2,000 Da, and a hydrophile portion constituting approximately 70% to 90% or 70% to 90% by weight of the copolymer.

45. The method of claim 44, wherein:

the molecular weight of the hydrophobe (C3H6O) is about or is 1,750 Da; and the average molecular weight of the polyoxyethylene/polyoxypropylene copolymer is 7,350 to 9,510 Daltons.

46. The method of any of claims 38-45, wherein the average molecular weight of the polyoxyethylene/polyoxypropylene copolymer is 7350-8,850 Daltons.

47. The method of any of claims 38-40, wherein:

the LCMF poloxamer is a polyoxyethylene/polyoxypropylene copolymer that has the formula HO(CH₂CH20)a'-[CH(CH3)CH20]b-(CH2CH₂0)aH;

each of a and a' is an integer such that the percentage of the hydrophile (C₂H₄0) is between approximately 60% and 90% by weight of the total molecular weight of the copolymer;

a and a' are the same or different;

b is an integer such that the molecular weight of the hydrophobe (C3H6O) is between approximately 1,300 and 2,300 Daltons;

no more than 1.5% of the total components in the distribution of the co-polymer are low molecular weight components having an average molecular weight of less than 4,500 Daltons;

no more than 1.5% of the total components in the distribution of the co-polymer are high molecular weight components having an average molecular weight of greater than 13,000 Daltons;

the polydispersity value of the copolymer is less than approximately 1.07 or less than 1.07; and

the LCMF poloxamer is more hydrophilic than a purified poloxamer 188 that contains the long circulating material (LCM).

48. The method of any of claims 36-40, wherein:

the LCMF poloxamer is a polyoxyethylene/polyoxypropylene copolymer that has the formula HO(CH₂CH₂OV-[CH(CH₃)CH₂0]b-(CH₂CH₂0)aH;

each of a and a' is an integer such that the percentage of the hydrophile (C₂H₄0) is between approximately 60% and 90% by weight of the total molecular weight of the copolymer;

a and a' are the same or different;

b is an integer such that the molecular weight of the hydrophobe (C3H6O) is between approximately 1,300 and 2,300 Daltons;

no more than 1.5% of the total components in the distribution of the co-polymer are low molecular weight components having an average molecular weight of less than 4,500 Daltons;

no more than 1.5% of the total components in the distribution of the co-polymer are high molecular weight components having an average molecular weight of greater than 13,000 Daltons;

the polydispersity value of the copolymer is less than approximately 1.07 or less than 1.07;

the LCMF poloxamer 188 has a mean retention time (½) as assessed by reverse phase-high performance liquid chromatography (RP-HPLC) that is shorter than purified LCM-containing poloxamer 188 assessed under the same RP-HPLC conditions; and

the capacity factor (k') of the LCMF poloxamer 188 as assessed by RP-HPLC is less than the k' for purified LCM-containing poloxamer 188 under the same RP-HPLC conditions.

49. The method of any of claims 38-48, wherein the copolymer is produced by a method comprising supercritical fluid extraction pressurized with C0₂.

50. The method of any of claims 23-49, wherein the copolymer is produced by a method comprising:

a) introducing a poloxamer 188 solution into an extractor vessel, wherein the poloxamer is dissolved in a first alkanol to form a solution;

b) admixing the poloxamer solution with an extraction solvent comprising a second alkanol and supercritical carbon dioxide under a temperature and pressure to maintain the supercritical carbon dioxide for a first defined period, wherein:

the temperature is above the critical temperature of carbon dioxide but is no more than 40° C; the pressure is 220 bars to 280 bars; and

the alkanol is provided at an alkanol concentration that is 7% to 8% by weight of the total extraction solvent;

c) increasing the concentration of the second alkanol in step b) in the extraction solvent a plurality of times in gradient steps over time of the extraction method, wherein: each plurality of times occurs for a further defined period; and

in each successive step, the alkanol concentration is increased 1-2% compared to the previous concentration of the second alkanol; and

d) removing the extraction solvent from the extractor vessel to thereby remove the extracted material from the raffinate poloxamer preparation.

51. The method of claim 50, wherein:

in step a), the ratio of poloxamer to first alkanol, by weight, is about or is from 2: 1 to 3: 1, inclusive; and

the plurality of times in step c) occurs in two, three, four or five gradient steps.

52. A composition, comprising:

a polyoxyethylene/polyoxypropylene copolymer; and

a fibrinolytic inhibitor.

53. A combination, comprising:

a polyoxyethylene/polyoxypropylene copolymer that is formulated as a pharmaceutical composition; and

a second separate composition, comprising a fibrinolytic inhibitor.

54. The composition of claim 52 that is formulated as a pharmaceutical composition.

55. The composition or combination of any of claims 52-54, wherein:

the polyoxyethylene/polyoxypropylene copolymer has the formula:

HO(CH₂CH20)a'-[CH(CH3)CH20]b-(CH2CH₂0)aH;

each of a' and a are the same or different and each is an integer, whereby the hydrophile portion represented by (C2H₄0) constitutes approximately 60% to 90% or 60%- 90% by weight of the compound; and

b is an integer, whereby the hydrophobe represented by (¾Η₆0) has a molecular weight of about 1,200 Da to about 2,300 Da or 1,200 to 2,300 Da.

56. The composition or combination of claim 55, wherein:

the polyoxyethylene/polyoxypropylene copolymer has the formula:

HO(CH₂CH20)a'-[CH(CH3)CH20]b-(CH2CH₂0)aH;

a' and a can be the same or different and each is an integer from 5 to 150, inclusive; and

b is an integer from 15 to 75, inclusive.

57. The composition or combination of claim 55 or claim 56, wherein:

the copolymer has the formula:

HO(CH₂CH₂0)a'-[CH(CH₃)CH₂0]b-(CH₂CH₂0)aH;

a' and a are the same or different and each is an integer from 70 to 105, inclusive; and

b is an integer from 15 to 75, inclusive.

58. The composition or combination of any of claims 55-57, wherein the hydrophobe represented by (C₃H₆0) has a molecular weight of about 1,400 to 2,000 Da or 1,400 to 2,000 Da, and the hydrophile portion constitutes approximately 70% to 90% or 70% to 90% by weight of the copolymer.

59. The composition or combination of any of claims 55-57, wherein the hydrophobe represented by (C₃H₆0) has a molecular weight of 1,500 Da to 2,100 Da or 1,700 Da to 1,900 Da.

60. The composition or combination of any of claims 55-59, wherein:

the hydrophile portion is about 80% of the total molecular weight of the copolymer; and

the hydrophobe portion has a molecular weight of at or about 1,800 Da.

61. The composition or combination of any of claims 55-59, wherein the molecular weight of the hydrophobe portion (C₃H₆0) is approximately or is 1,750 Da and the total molecular weight of the copolymer is approximately or is 7350 to 8,850 Da.

62. The composition or combination of any of claims 52-61, wherein the copolymer comprises a poloxamer 188.

63. The composition or combination of any of claims 52-62, wherein the copolymer has reduced impurities, whereby the polydispersity value is less than or equal to 1.07.

64. The composition or combination of any of claims 52-62, wherein the polydispersity value is less than or equal to 1.06, 1.05, 1.04, 1.03, 1.02 or 1.01.

65. The composition or combination of any of claims 52-64, wherein no more than 1.5% or 1% of the total components in the distribution of the copolymer are low molecular weight components having an average molecular weight of less than 4,500 Daltons.

66. The composition or combination of any of claims 52-65, wherein the polyoxyethylene/polyoxypropylene copolymer has the formula:

HO(CH₂CH20)a'-[CH(CH3)CH20]b-(CH2CH₂0)aH, wherein:

a' and a are the same and are 78, 79 or 80; and

b is 27, 28, 29 or 30.

67. The composition or combination of claim 66, wherein:

a' and a are 80; and

b is 27.

68. The composition or combination of any of claims 52-65, wherein the polyoxyethylene/polyoxypropylene copolymer is purified to reduce the amount of low molecular weight substances.

69. The composition or combination of any of claims 52-68, wherein the copolymer comprises poloxamer 188 (P188).

70. The composition or combination of any of claims 52-69, wherein the copolymer is a long-circulating material-free (LCMF) poloxamer.

71. The composition or combination claim 70, wherein the copolymer is a long-circulating material-free (LCMF) poloxamer that, when administered to a human subject, does not contain a component that is or gives rise to in the plasma of the subject to a material or component that has a circulating half-life (ti/₂) that is more than about 1.5- fold or 1.5-fold greater than the half-life of the main peak in the distribution of the copolymer preparation, or is such that all components have a circulating half-life that is within 5-fold of the half-life of the main peak.

72. The composition or combination of claim 70 or 71, wherein:

the LCMF poloxamer is a polyoxyethylene/polyoxypropylene copolymer that has the formula HO(CH₂CH₂0)a'-[CH(CH₃)CH₂0]b-(CH₂CH₂0)aH;

each of a and a' is an integer such that the percentage of the hydrophile (C₂H₄0) is between approximately 60% and 90% by weight of the total molecular weight of the copolymer;

a and a' are the same or different; b is an integer such that the molecular weight of the hydrophobe (C3H6O) is between approximately 1,300 and 2,300 Daltons;

no more than 1.5% of the total components in the polymeric distribution of the co-polymer are low molecular weight components having an average molecular weight of less than 4,500 Daltons;

no more than 1.5% of the total components in the polymeric distribution of the copolymer are high molecular weight components having an average molecular weight of greater than 13,000 Daltons;

the polydispersity value of the copolymer is less than approximately 1.07 or less than 1.07; and

following intravenous administration to a human subject, the circulating plasma half-life of any components not comprising the main peak in the distribution of copolymer is no more than 5.0-fold the circulating half-life of the main component in the distribution of the copolymer.

73. The composition or combination of any of claims 70-72, wherein all components comprising the polymeric distribution of the poloxamer copolymer have a circulating half-life in the plasma of the subject that is no more than 4.0-fold, or 3.0-fold longer than the circulating half-life of the main component of the co-polymer following intravenous administration to a subject.

74. The composition or combination of any of claims 70-72, wherein all components in the distribution of the co-polymer, when administered to a human subject, have a circulating half-life in the plasma of the subject that is no more than 3-fold longer than the circulating half-life of the main component in the distribution of the co-polymer.

74. The composition or combination of any of claims 70-73, wherein all components in the distribution of the co-polymer, when administered to a human subject, have a half-life in the plasma of the subject that is no more than 10 or 12 hours.

76. The composition or combination of any of claims 70-75, wherein the polyoxyethylene/polyoxypropylene copolymer is a poloxamer with a hydrophobe having a molecular weight of about 1,400 to 2,000 Da or 1,400 to 2,000 Da, and a hydrophile portion constituting approximately 70% to 90% or 70% to 90% by weight of the copolymer.

77. The composition or combination of claim 72, wherein:

the molecular weight of the hydrophobe (C3H6O) is about or is 1,750 Da; and the average molecular weight of the polyoxyethylene/polyoxypropylene copolymer is 7,680 to 9,510 Daltons.

78. The composition or combination of any of claims 52-77, wherein the average molecular weight of the polyoxyethylene/polyoxypropylene copolymer is 7350- 8,850 Daltons.

79. The composition or combination of any of claims 70-72, wherein:

the LCMF poloxamer is a polyoxyethylene/polyoxypropylene copolymer that has the formula HO(CH₂CH20)a'-[CH(CH3)CH20]b-(CH2CH₂0)aH;

each of a and a' is an integer such that the percentage of the hydrophile (C₂H₄0) is between approximately 60% and 90% by weight of the total molecular weight of the copolymer;

a and a' are the same or different;

b is an integer such that the molecular weight of the hydrophobe (C3H6O) is between approximately 1,300 and 2,300 Daltons;

no more than 1.5% of the total components in the distribution of the co-polymer are low molecular weight components having an average molecular weight of less than 4,500 Daltons;

no more than 1.5% of the total components in the distribution of the co-polymer are high molecular weight components having an average molecular weight of greater than 13,000 Daltons;

the polydispersity value of the copolymer is less than approximately 1.07 or less than 1.07; and

the LCMF poloxamer 188 is more hydrophilic than a purified poloxamer 188 that contains the long circulating material (LCM).

80. The composition or combination of any of claims 70-72, wherein:

the LCMF poloxamer is a polyoxyethylene/polyoxypropylene copolymer that has the formula HO(CH₂CH₂0)_a'-[CH(CH₃)CH₂0]b-(CH₂CH₂0)aH;

each of a and a' is an integer such that the percentage of the hydrophile (C₂H₄0) is between approximately 60% and 90% by weight of the total molecular weight of the copolymer;

a and a' are the same or different; b is an integer such that the molecular weight of the hydrophobe (C3H6O) is between approximately 1,300 and 2,300 Daltons;

no more than 1.5% of the total components in the distribution of the co-polymer are low molecular weight components having an average molecular weight of less than 4,500 Daltons;

no more than 1.5% of the total components in the distribution of the co-polymer are high molecular weight components having an average molecular weight of greater than 13,000 Daltons;

the polydispersity value of the copolymer is less than approximately 1.07 or less than 1.07;

the LCMF poloxamer has a mean retention time (½) as assessed by reverse phase- high performance liquid chromatography (RP-HPLC) that is shorter than purified LCM- containing poloxamer 188 under the same RP-HPLC conditions; and

the capacity factor (k') of the LCMF poloxamer as assessed by RP-HPLC is less than the k' for purified LCM-containing poloxamer 188 under the same RP-HPLC conditions.

81. The composition or combination of any of claims 52-80, wherein the copolymer is produced by a method comprising supercritical fluid extraction pressurized with C0₂.

82. The composition or combination of any of claims 52-81, wherein the copolymer is produced by a method comprising:

a) introducing a poloxamer 188 solution into an extractor vessel, wherein the poloxamer is dissolved in a first alkanol to form a solution;

b) admixing the poloxamer solution with an extraction solvent comprising a second alkanol and supercritical carbon dioxide under a temperature and pressure to maintain the supercritical carbon dioxide for a first defined period, wherein:

the temperature is above the critical temperature of carbon dioxide but is no more than 40° C;

the pressure is 220 bars to 280 bars; and

the alkanol is provided at an alkanol concentration that is 7% to 8% by weight of the total extraction solvent; and

c) increasing the concentration of the second alkanol in step b) in the extraction solvent a plurality of times in gradient steps over time of the extraction method, wherein: each plurality of times occurs for a further defined period; and in each successive step, the alkanol concentration is increased 1-2% compared to the previous concentration of the second alkanol; and

d) removing the extraction solvent from the extractor vessel to thereby remove the extracted material from the raffinate poloxamer preparation.

83. The composition or combination of claim 82, wherein:

in step a), the ratio of poloxamer to first alkanol, by weight is about or is from 2: 1 to 3: 1, inclusive; and

the plurality of times in step c) occurs in two, three, four or five gradient steps.

84. The composition or combination of any of claims 52-83, wherein the copolymer is formulated such that the amount of copolymer is sufficient to result in an amount of copolymer in the circulation from 0.25 mg/mL to 2.5 mg/mL, or 0.4 mg/mL to 2.0 mg/mL, or 0.5 mg/mL to 1.5 mg/mL in less 24 hours, 20 hours, 15 hours, 12 hours, 10 hours, 6 hours or between 2 and 6 hours, inclusive.

85. The composition or combination of claim 84, wherein the concentration of the copolymer in the circulation is the peak concentration.

86. The composition or combination of claim 84 or claim or 85, wherein the concentration of the copolymer is the concentration in the circulation at steady state.

87. The composition or combination of any one of claims 84-86, wherein the concentration of the copolymer in the circulation is administered so that it remains at the target concentration for up to 72 hours following administration.

88. The composition or combination of any of claims 52-87, wherein the fibrinolytic inhibitor is an endogenous inhibitor selected from among a plasminogen activator inhibitor (PAI), a plasmin inhibitor, alpha-2-macroglobulin, and thrombin- activatable fibrinolysis inhibitor (TAFI) and modified forms thereof, or a pharmaceutical or synthetic inhibitor that is a synthetic derivative of lysine.

89. The composition or combination of claim 88, wherein the fibrinolytic inhibitor is an endogenous inhibitor selected from among PAI-1, PAI-2, PAI-3, alpha-2- antiplasmin, alpha-2-macroglobulin and TAFI.

90. The composition or combination of claim 89, wherein the fibrinolytic inhibitor is a pharmaceutical or synthetic inhibitor selected from among aprotinin, ε- aminocaproic acid and tranexamic acid (TA).

91. The composition or combination of any of claims 52-90 that is formulated for multidose or single dose administration, wherein a single dose of the fibrinolytic inhibitor is therapeutically effective to prevent or treat hemorrhagic shock or a precursor thereto or any bleeding or bleeding disorder, and the single dose of the copolymer is effective to prevent or mitigate any adverse side effects of the fibrinolytic inhibitor.

92. The composition or combination of any of claims 52-90 that is formulated for single dose or multidose administration, wherein the a single dose of the fibrinolytic inhibitor is sufficient to prevent or treat any adverse effect of poloxamer administration that can or does result in bleeding.

93. The composition or combination of any of claims 52-92 that is a composition, wherein the copolymer and fibrinolytic inhibitor are present in the composition at a ratio of copolymer to fibrinolytic inhibitor that is between 5: 1 and 7.5: 1, 5: 1 and 10: 1, 5: 1 and 15: 1, 5: 1 and 20: 1, 7.5: 1 and 10: 1, 7.5: 1 and 15: 1, 7.5: 1 and 20: 1, 10: 1 and 15: 1, 10: 1 and 20: 1, and 15: 1 and 20: 1 copolymer to fibrinolytic inhibitor.

94. The composition or combination of any of claims 52-93, wherein the fibrinolytic inhibitor is ε-aminocaproic acid or tranexamic acid (TA).

95. The composition or combination of claim 94, wherein the LCMF poloxamer is an LCMF P188 that, when administered to a subject, does not contain a component that is or gives rise to in the plasma of the subject a material or component that has a circulating half-life (tm) that is more than about 1.5-fold or 1.5-fold greater than the half-life of the main component in the distribution of the copolymer preparation or such that all components have a circulating half-life that is within 5-fold of the half-life of the main peak.

96. The composition or combination of any of claims 52-96, wherein the composition or copolymer in the combination is formulated for administration by intravenous infusion.

97. The composition or combination of any of claims 52-96, wherein the composition or copolymer in the combination is formulated for administration by bolus injection.

98. The composition or combination of any of claims 52-96, wherein the composition or copolymer in the combination is formulated for topical or local administration.

99. The composition or combination of any of claims 52-98, wherein the fibrinolytic inhibitor in the combination is formulated for systemic or local

administration.

100. The composition or combination of any of claims 52-98, wherein the fibrinolytic inhibitor in the combination is formulated for intravenous infusion or bolus injection.

101. The composition or combination of any of claims 52-98, wherein the fibrinolytic inhibitor in the combination is formulated for oral administration.

102. The composition or combination of any of claims 52-98, wherein the fibrinolytic inhibitor in the combination is formulated for oral administration as a tablet or mouthwash.

103. The composition or combination of any of claims 52-95, wherein the composition or copolymer in the combination is formulated for administration as a single continuous IV infusion, a plurality of continuous IV infusions, a single IV bolus administration, a plurality of IV bolus administrations, or a combination thereof.

104. The composition or combination of any of claims 52-95, wherein the composition or fibrinolytic inhibitor and copolymer in the combination is/are formulated for administration as a single continuous IV infusion, a plurality of continuous IV infusions, a single IV bolus administration, a plurality of IV bolus administrations, or a combination thereof.

105. The composition or combination of any of claims 52-95, wherein the copolymer is administered in an amount to treat or prevent unwanted side effects or consequences from administration of the fibrinolytic inhibitor.

106. The composition or combination of claim 105, wherein the unwanted side effects or consequences are selected from ischemic tissue damage, prothrombotic events, embolism, thrombosis, and combinations thereof.

107. The composition or combination of any of claims 52-106, wherein the fibrinolytic inhibitor is administered in an amount to treat or prevent unwanted side effects or consequences from administration of the copolymer for treatment of a disease or condition.

108. The composition or combination of any of claims 52-107 that is a combination, wherein the combination is packaged as a kit.

109. The composition or combination of any of claims 52-108 for use for treating damaged tissue, damaged cell surfaces and/or unwanted bleeding in a subject.

110. The composition or combination of claim 109, wherein the bleeding is consequent to treatment with a fibrinolytic inhibitor or the copolymer.

111. The composition or combination of claim 109, wherein the bleeding occurs during surgery or from trauma or injury.

112. The composition or combination of claim 109 for use for treating hemorrhagic shock or a precursor thereto.

113. The composition or combination of claim 109, wherein the damaged tissue results from a condition selected from among blunt trauma, anemia, aneurysm, hypovolemia, burns, shock, hemorrhage, surgery and tissue infarction.

114. A method for treating damaged tissue and/or damaged cell surfaces, or unwanted bleeding or hemorrhagic shock or a precursor thereto, comprising

administering a composition or combination of any of claims 52-113 to a subject.

115. The method of claim 1 wherein said subject has unwanted bleeding or hemorrhagic shock or a precursor thereto,

said copolymer is the long circulating material free (LCMF) poloxamer 188 that has the formula HO(CH₂CH20)a'-[CH(CH3)CH20]b-(CH2CH₂0)aH;

each of a and a' is an integer such that the percentage of the hydrophile (C₂H₄0) is between approximately 60% and 90% by weight of the total molecular weight of the copolymer;

a and a' are the same or different;

b is an integer such that the molecular weight of the hydrophobe (C3H6O) is between approximately 1,300 and 2,300 Daltons;

no more than 1.5% of the total components in the distribution of the copolymer are low molecular weight components having an average molecular weight of less than 4,500 Daltons;

no more than 1.5% of the total components in the distribution of the copolymer are high molecular weight components having an average molecular weight of greater than 13,000 Daltons;

the polydispersity value of the copolymer is less than approximately 1.07 or less than 1.07; and

following intravenous administration to a human subject, the circulating plasma half-life of any components not comprising the main peak in the polymeric distribution of copolymer is no more than 5.0-fold the circulating half-life of the main component in the distribution of the copolymer.

116. The method of claim 1 wherein said subject has unwanted bleeding or hemorrhagic shock or a precursor thereto,

said copolymer is purified poloxamer 188 that has the formula

HO(CH₂CH20)a'-[CH(CH3)CH20]b-(CH2CH₂0)aH;

each of a and a' is an integer such that the percentage of the hydrophile (C2H₄0) is between approximately 60% and 90% by weight of the total molecular weight of the copolymer;

a and a' are the same or different;

b is an integer such that the molecular weight of the hydrophobe (C3H6O) is between approximately 1,300 and 2,300 Daltons;

no more than 1.5% of the total components in the distribution of the copolymer are low molecular weight components having an average molecular weight of less than 4,500 Daltons;

no more than 1.5% of the total components in the distribution of the copolymer are high molecular weight components having an average molecular weight of greater than 13,000 Daltons;

the polydispersity value of the copolymer is less than approximately 1.07 or less than 1.07.

117. The method of claim 116 wherein said purified poloxamer 188 is a LCMF poloxamer and is more hydrophilic than a purified poloxamer 188 that contains the long circulating material (LCM).