Poloxamer composition free of long circulating material and methods for production and uses thereof

Abstract

To provide long circulating material free (LCMF) poloxamer compositions and uses thereof, in particular, LCMF poloxamer 188 compositions and uses thereof, and to provide supercritical fluid extraction (SFE) methods and high pressure (subcritical) methods for preparing poloxamer compositions, particularly the LCMF poloxamer compositions.SOLUTION: The LCMF poloxamer 188 is a polyoxyethylene / polyoxypropylene copolymer that has the formula HO(CH2CH2O)a'-[CH(CH3)CH2O]b-(CH2CH2O)aH, wherein: each of a and a' is an integer such that the percentage of (C2H4O) is between 60 to 90 wt.% of the total molecular weight of the copolymer; b is an integer such that the molecular weight of (C3H6O) is between 1,300 and 2,300 Da; 1.5% or less of the total components are low molecular weight components and 1.5% or less of the total components are high molecular weight components; the polydispersity value of the LCMF poloxamer is less than 1.07; and the circulating half-life of the LCMF poloxamer is less than 5.0-fold of the predetermined circulating half-life.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 021,697, filed July 7, 2014, to R. Martin Emanuele and Mannarsamy Balasubramanian, entitled "Long-Circulating Substance-Free Poloxamer Compositions, Methods for Producing Same, and Uses Thereof."

[0002] This application is related to U.S. Patent Application No. 38645.04003.US02 / 4003, filed on the same day by R. Martin Emanuele and Mannarsamy Balasubramanian, entitled "Long-Circulating Substance-Free Poloxamer Compositions, Methods for Producing Same, and Uses Thereof."

[0003] This application further relates to R. Martin Emanuele, Santosh Vetticaden, and Patrick Keran, 20 U.S. Provisional Patent Application No. 62 / 021,691, filed July 7, 2014, entitled "Poloxamer Therapy for Heart Failure"; U.S. Provisional Patent Application No. 62 / 021,691, filed February 27, 2015, entitled "Poloxamer Therapy for Heart Failure" by R. Martin Emanuele, Santosh Vetticaden and Patrick Keran; US Provisional Patent Application No. 62 / 126,400, filed July 7, 2014, by R. Martin Emanuele, Santosh Vetticaden and Patrick Keran, entitled “Poloxamer Therapy for Heart Failure.” No. PCT / US14 / 45627, filed on the same day by R. Martin Emanuele, Santosh Vetticaden and Patrick Keran, respectively. The present application also relates to International PCT Application No. (Attorney Docket No. 38645.04001.WO12 / 4001BPC) and U.S. Application No. (Attorney Docket No. 38645.04001.US12 / 4001) entitled "Poloxamer Therapy for Heart Failure."

[0004] Where permitted, the subject matter of each of the applications is incorporated by reference in its entirety.

[0005] Long-circulating substance-free (LCMF) poloxamer compositions and uses thereof are provided. Supercritical fluid extraction (SFE) and high pressure (subcritical) methods for preparing the LCMF poloxamer compositions are also provided. [Background technology]

[0006] Certain polyoxypropylene / polyoxyethylene (POP / POE) copolymers, called poloxamers, have beneficial biological effects when administered to humans or animals. These copolymers can be used alone or in combination with other agents, including, for example, anticoagulants, free radical scavengers, anti-inflammatory agents, antibiotics, membrane stabilizers, and perfusion media. It has been used to treat cardiovascular diseases, either in combination with compounds such as poloxamer 188 (P188) (see, for example, U.S. Pat. No. 5,696,298) is useful for treating pathological hydrophobic interactions in blood and other body fluids in humans and animals. Commercially available formulations of poloxamers contain a highly heterogeneous population of molecules that vary widely in size and structure of the constituent molecules. The molecular diversity present in commercially available poloxamers can make bioactivity difficult to predict and can lead to undesirable bioactivity. Therefore, alternative formulations of poloxamers are needed. Summary of the Invention [Problem to be solved by the invention]

[0007] Provided are alternative poloxamer formulations and methods of making and using them. Provided are poloxamers that are long-circulating substance free (LCMF), particularly LCMF poloxamer 188. When the LCMF poloxamer is administered to a subject, such as a human subject, the LCMF poloxamer is capable of inhibiting the production of human pulmonary circulation endothelial cells. When administered to a subject such as a subject, the LCMF poloxamer 188 does not produce a substance in the subject that has a half-life significantly longer than the main peak, such that the circulating half-life of all components in the copolymer distribution is 5.0 times or less longer than the circulating half-life of the main component in the copolymer distribution. Generally, the half-life of all components of LCMF poloxamer 188 in humans is less than 12 hours. Commercially available and conventional formulations of poloxamers, such as poloxamer 188, when administered to humans, contain long-circulating substances (LCMs) that have half-lives that are more than 5.0 times the circulating half-life of the main component in the copolymer distribution. Methods for preparing LCMF poloxamers and other poloxamers, including removal of low molecular weight impurities, are also provided.

[0008] LCMF poloxamers have the formula: HO(CH2CH2O) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) aH, where each of a and a' is an integer such that the percentage of hydrophilic material (C2H4O) is about 60%-90% by weight or 60%-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 hydrophobic material (C3H6O) is about 1,300-2,300 Daltons (Da); and no more than 1.5% of the total components in the distribution of the copolymer are 4,5 LCMF poloxamers are low molecular weight components having an average molecular weight of less than 1.00 Da; 1.5% or less of all components in the copolymer distribution are high molecular weight components having an average molecular weight of more than 13,000 Da; the polydispersity value of the copolymer is less than about 1.07 or less than 1.07, such as 1.06 or less, 1.05 or less, 1.04 or less, 1.03 or less, and 1.02 or less; and the circulating half-life of all components in the copolymer distribution is 5.0 times or less longer than the circulating half-life of the major component in the copolymer distribution when administered to a subject. LCMF poloxamers are more hydrophilic than formulations of poloxamer 188 known in the art, such as the poloxamers described in U.S. Patent No. 5,696,298, including long-circulating substances (LCMs), and their commercially available formulations. [Means for solving the problem]

[0009] poloxamer 188 having the formula HO(CH2CH2O) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) aH; each of a and a' are integers such that the percentage of hydrophilic material (C2H4O) is about 60% to 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 hydrophobic material (C3H6O) is about 1,300 to 2,300 Daltons; not more than 1.5% of all components in the distribution of the copolymer are low molecular weight components having an average molecular weight of less than 4,500 Daltons; and not more than 1.5% of all components in the distribution of the copolymer are low molecular weight components having an average molecular weight of less than 4,500 Daltons. 5% or less are high molecular weight components having an average molecular weight greater than 13,000 daltons; the polydispersity value of the copolymer is less than about 1.07 or less than 1.07, e.g., 1.06, 1.05, 1.04 or 1.03, or less than 1.06, 1.05, 1.04 or 1.03; and after intravenous administration to a human subject, the circulating plasma half-life of any component not constituting the main peak is no more than 5.0-fold longer than the circulating half-life of the main component in the copolymer distribution to provide a poloxamer 188 that is free of long circulating substances (LCMF).

[0010] Included are embodiments in which all components that make up the polymer distribution of the copolymer have a circulating half-life in the plasma of a subject, such as a human subject, that is 5.0 times or less, or 4.0 times or less, or 3.0 times or less longer than the circulating half-life of the major component of the copolymer following intravenous administration to the subject. For example, all components in the copolymer distribution, when administered to a human subject, have a circulating half-life in the plasma of the subject that is 4 times or less longer than the circulating half-life of the major component of the copolymer distribution. Embodiments in which all components in the copolymer distribution, when administered to a human subject, have a half-life in the plasma of a subject that is 30 hours or less, 25 hours or less, 20 hours or less, 15 hours or less, 12 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, or 7 hours or less, e.g., 10 or 12 hours or less. LCMF poloxamers therefore do not contain LCM.

[0011] Long-term circulating substance-free (LCMF) poloxamer 188: LCMF poloxamer 188 has the formula HO(CHCHO) a’ ─[CH(CH3)CH2O]b ─(CH2CH2O) a a and a' are each an integer such that the percentage of hydrophilic material (C2H4O) is about 60% to 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 hydrophobic material (C3H6O) is about 1,300 to 2,300 Daltons; no more than 1.5% of all components in the copolymer distribution are low molecular weight components having an average molecular weight of less than 4,500 Daltons; and Provided herein is a long circulating material free (LCMF) poloxamer 188 in which no more than 1.5% of all components in the copolymer are high molecular weight components having an average molecular weight greater than 13,000 daltons; the copolymer has a polydispersity value of less than about 1.07 or less than 1.07; and the LCMF poloxamer is more hydrophilic than purified poloxamer 188 with long circulating material (LCM), such that the circulating half-life of all components in the copolymer distribution is no more than 5.0-fold longer than the circulating half-life of the major component in the copolymer distribution.

[0012] Long-term circulating substance-free (LCMF) poloxamer 188: LCMF poloxamer 188 has the formula HO(CHCHO) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) awherein each of a and a' is an integer such that the percentage of hydrophilic material (C2H4O) is about 60%-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 hydrophobic material (C3H6O) is about 1,300-2,300 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 copolymer has a polydispersity value of less than about 1.07 or less than 1.07; and the LCMF has a mean retention time (t) as assessed by reversed-phase high performance liquid chromatography that is shorter than that of purified poloxamer 188 with LCM. R In some embodiments, for example, the RP-HPLC conditions have a mean t R is approximately 9.9-10 or 9.9-10; and the average t R is about 8.7 to 8.8 or 8.7 to 8.8.

[0013] Long-term circulating substance-free (LCMF) poloxamer 188: LCMF poloxamer 188 has the formula HO(CHCHO) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) aH; each of a and a' is an integer such that the percentage of hydrophilic material (C2H4O) is about 60%-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 hydrophobic material (C3H6O) is about 1,300-2,300 Daltons; not more than 1.5% of all components in the distribution of the copolymer are low molecular weight components having an average molecular weight of less than 4,500 Daltons; not more than 1.5% of all 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 about 1.07 or less than 1.07; and the capacity factor (k') as assessed by RP-HPLC is greater than or equal to 1.07 under the same conditions as in the case of The present invention provides a long-circulating substance-free (LCMF) poloxamer 188 having a lower k' than the manufactured LCM-containing poloxamer 188. For example, the RP-HPLC conditions are such that the LCMF poloxamer has a mean k' of about 3.2-3.3 or 3.2-3.3, and the LCM-containing poloxamer 188 has a mean k' of about 3.6-3.7 or 3.6-3.7.

[0014] In embodiments herein, the LCMF poloxamer can be a poloxamer having a hydrophobic portion having a molecular weight of about 1,400-2,000 Daltons (Da) or 1,400-2,000 Da, e.g., 1,750 Da, and a hydrophilic portion that constitutes about 70%-90% or 70%-90% by weight of the copolymer. The LCMF poloxamer can have an average molecular weight of 7,680-9,510 Daltons, e.g., 8,400-8,800 Daltons.

[0015] In some embodiments, the percentage of high molecular weight components in the formulation above 13,000 daltons constitutes less than 1% of the total distribution of components in the poloxamer formulation. In some embodiments, after intravenous administration to a human subject, the LCMF poloxamer does not result in components having a circulating half-life greater than four times the circulating plasma half-life of the main peak. In some embodiments, the percentage of high molecular weight components in the formulation above 13,000 daltons constitutes less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5% or less of the total distribution of components in the poloxamer formulation.

[0016] LCMF poloxamers differ from LCM-containing poloxamers in that when administered to a subject, all components are cleared in a shorter time than the components of the LCM-containing poloxamers, as described and demonstrated throughout this disclosure. When characterized by RP-HPLC, the LCMF poloxamers provided herein have mean k' and mean t R is less than the corresponding LCM-containing poloxamer. LCMF poloxamers are more hydrophilic than the corresponding LCM-containing poloxamers.

[0017] Provided are LCMF poloxamers produced by supercritical fluid extraction methods, including those described herein. For example, the present invention provides an LCMF poloxamer produced by a process comprising: a) introducing a poloxamer solution into an extraction vessel and dissolving the poloxamer in a first alkanol to form a solution; b) mixing the poloxamer solution with an extraction solvent comprising a second alkanol and supercritical carbon dioxide under a temperature and pressure that maintains supercritical carbon dioxide for a first period of time, wherein the temperature is above the critical temperature of carbon dioxide but not exceeding 40° C.; the pressure is between 220 bar and 280 bar; 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 the extraction solvent in step b) in gradient steps over the time of the extraction process multiple times, each multiple times during an additional period of time; wherein in each successive step, the alkanol concentration is increased by 1-2% compared to the previous concentration of the second alkanol; and d) removing the extraction solvent from the extraction vessel, thereby removing material extracted from the raffinate poloxamer formulation.

[0018] In some embodiments for producing the LCMF poloxamer, in a), the weight ratio of poloxamer to first alkanol is about 2:1-3:1 or 2:1-3:1 inclusive; and / or the multiple times in step c) are performed in 2, 3, 4 or 5 gradient steps; and / or step c) can be performed in two steps including: i) increasing the concentration of the second alkanol from about 7%-8% to about 8.2%-9.5% during a second period of time; and ii) increasing the concentration of the second alkanol from about 8.2%-9.5% to about 9.6%-11.5% during a third period of time. In certain embodiments, the LCMF poloxamer is: the alkanol concentration in step b) is about 7.4% by weight or 7.4% by weight; The alkanol concentration in step i) is about 9.1% by weight or 9.1% by weight; and The alkanol concentration in step ii) is about 10.7% by weight or 10.7% by weight.

[0019] Each of the first, second and third fixed periods can be performed for 2 hours to 12 hours; the fixed periods can be the same or different. For example, the first fixed period can be performed for 2 hours to 6 hours; the second fixed period can be performed for 2 hours to 6 hours; and the third fixed period can be performed for 4 hours to 10 hours. In the method of producing LCMF poloxamer, the first and second alkanols are each independently selected from methanol, ethanol, propanol, butanol, pentanol and combinations thereof. For example, the first and second alkanols can be the same or different, e.g., the first alkanol is methanol and the second alkanol is methanol or a different alkanol. Step d) of removing the extracted material can be performed throughout steps b) and c). The method can be performed, for example, as a batch or continuous process.

[0020] In embodiments herein, the LCMF poloxamer produced by the methods is LCMF poloxamer 188. Also provided are methods of producing LCMF poloxamers, including LCMF poloxamer 188, as described above and throughout this disclosure.

[0021] Compositions are provided that include the LCMF poloxamers provided herein. The compositions can be pharmaceutical compositions formulated in a pharma- ceutically acceptable vehicle. In particular, compositions and pharmaceutical compositions that contain or include the LCMF poloxamers provided herein are provided. Compositions are provided that include LCMF poloxamer 188. Compositions are provided that include long-term circulating substance-free (LCMF) poloxamer 188: the compositions are formulated for intravenous administration; and the compositions include 5-50 g of LCMF poloxamer. The LCMF poloxamer can be any LCMF poloxamer, including any provided herein. Use of the compositions and methods of treatment of any disease or disorder or condition in which a poloxamer is administered is provided. Exemplary diseases and conditions include, but are not limited to, heart failure, myocardial infarction, stroke, shock, and sickle cell disease, including, but not limited to, acute myocardial infarction, limb ischemia, shock, acute stroke, ischemic stroke, diabetic retinopathy, hemorrhagic shock, neurodegenerative diseases, macular degeneration, diabetic retinopathy, and congestive heart failure.

[0022] LCMF poloxamer compositions can be used, for example, but not limited to, for treating disorders treated by membrane resealing and repair; treating tissue ischemia and reperfusion injury; reducing inflammatory responses; reducing blood viscosity; promoting thrombolysis; and other disorders, conditions, diseases and uses including promoting or maintaining hemostasis; as vehicles for the delivery of drugs, nucleic acids or proteins; as emulsifiers to stabilize suspensions of hydrophobic drugs; for cleaning skin wounds; as surfactants in cosmetic formulations; for treating storage injury compromised blood or preventing storage injury in blood and blood products; to regulate the viscosity of personal care products and soaps; as a laxative, and other uses known to those of skill in the art.

[0023] The composition comprising LCMF poloxamer can be formulated into or comprise blood, red blood cells and / or blood products, such as packed red blood cells. Such compositions can be used for transfusion. Transfusion is used in the treatment of diseases including, but not limited to, sickle cell disease, acute chest syndrome, peripheral arterial disease, heart failure, stroke, peripheral vascular disease, macular degeneration, acute respiratory distress syndrome (ARDS), multiple organ failure, ischemia, shock, acidosis, hypothermia, anemia, surgery, trauma, blood loss and blood disorders; and in any treatment involving blood transfusion. These include hemorrhagic shock, septic shock and acute blood loss. Thus, the use of the composition for blood transfusion and administering the composition to a subject suffering from a disease or disorder selected from sickle cell disease, acute chest syndrome, peripheral arterial disease, heart failure, stroke, peripheral vascular disease, macular degeneration, acute respiratory distress syndrome (ARDS), multiple organ failure, ischemia, shock, including hemorrhagic shock and septic shock, acidosis, hypothermia, anemic breakdown, surgery, trauma, acute blood loss, and blood disorders. and wherein the treatment comprises a blood transfusion.

[0024] LCMF poloxamers can be prepared by any method that removes LCM, including, but not limited to, the described preparation methods for preparing LCMF poloxamer 188 or any suitable poloxamer, such as those described below, by a method that includes the steps of: a) introducing a poloxamer 188 solution into an extraction vessel and dissolving the poloxamer in a first alkanol to form a solution; b) mixing the poloxamer solution with an extraction solvent that includes a second alkanol and a supercritical fluid under a temperature and pressure that maintains the supercritical fluid, where the concentration of the second alkanol in the extraction solvent increases over the time of the extraction process; and c) removing the extraction solvent from the extraction vessel, thereby removing the extracted material from the poloxamer formulation, thereby producing the LCMF poloxamer.

[0025] The method includes: a) introducing a poloxamer 188 solution into an extraction vessel and dissolving the poloxamer in a first alkanol to form a solution; b) combining the poloxamer solution with an extraction solvent comprising a second alkanol and supercritical carbon dioxide at a temperature and pressure that maintains supercritical carbon dioxide for a first period of time, wherein the temperature is greater than the critical temperature of carbon dioxide but less than or equal to 40° C.; the pressure is between 220 bar and 280 bar; and the alkanol is provided at an alkanol concentration that is between 7% and 8% by weight of the total extraction solvent; c) and removing the extraction solvent from the extraction vessel, thereby removing extracted materials from the raffinate poloxamer formulation, thereby producing an LCMF poloxamer.

[0026] In some embodiments, step d) of removing extracted material can be performed throughout steps b) and c). As mentioned above, in step a), the weight ratio of poloxamer to first alkanol can be about 2:1-3:1 or 2:1-3:1 (inclusive); and / or the multiple times in step c) are performed in 2, 3, 4 or 5 gradient steps. Step c) can be performed in two steps including i) increasing the concentration of the second alkanol from about 7%-8% to about 8.1%-9.5% during a second period of time; and ii) increasing the concentration of the second alkanol from about 8.2%-9.5% to about 9.6%-11.5% during a third period of time. In certain embodiments, the alkanol concentration in step b) is about 7.4% or 7.4% by weight; the alkanol concentration in step i) is about 9.1% or 9.1% by weight; and / or the alkanol concentration in step ii) is about 10.7% or 10.7% by weight. Each of the first period of time, the second period of time, and the third period of time can be carried out for 2 hours to 12 hours; and the periods of time can be the same or different. For example, the first period of time can be carried out for 2 hours to 6 hours; the second period of time can be carried out for 2 hours to 6 hours; and the third period of time can be carried out for 4 hours to 10 hours. The first and second alkanols can each be independently selected from methanol, ethanol, propanol, butanol, pentanol, and combinations thereof. Each can be the same or different, for example, the first alkanol can be methanol, and / or the second alkanol can be methanol.

[0027] 1. An extraction process for preparing an LCMF poloxamer comprising: a) charging the poloxamer to an extraction vessel and dissolving the poloxamer in a first alkanol to form a solution; b) mixing the solution with an extraction solvent comprising a second alkanol and a supercritical fluid under pressure to form an extraction mixture, wherein the concentration of the second alkanol in the extraction solvent increases over the time of the extraction process; and c) removing the extraction solvent from the extraction vessel, thereby and removing low molecular weight material from the poloxamer by extraction with a solvent such as ethanol. In some embodiments, the method includes: a) charging the poloxamer to an extraction vessel and dissolving the poloxamer in a first solvent selected from the group consisting of alcohols, aliphatic ketones, aromatic ketones, amines, and mixtures thereof to form a solution; b) mixing the extraction solvent with the solution to form an extraction mixture, wherein the extraction solvent comprises high pressure carbon dioxide and the first solvent, and the concentration of the solvent in the extraction solvent increases over the time of the extraction method; and c) removing the extraction solvent from the extraction vessel, thereby removing low molecular weight impurities from the poloxamer. In some embodiments of the method, after step c, the method may further include repeating steps b and c.

[0028] The poloxamer used in the methods described herein can be any poloxamer, including but not limited to poloxamer 188, poloxamer 331, and poloxamer 407. As mentioned above, for all methods, the first and second alkanols are each independently selected from methanol, ethanol, propanol, butanol, pentanol, and combinations thereof. For example, one or both of the alkanols can be methanol. In all methods provided herein, the supercritical liquid under pressure can be any suitable supercritical liquid, including but not limited to carbon dioxide, methane, ethane, propane, ammonia, and freon. In certain embodiments, the supercritical liquid under pressure is carbon dioxide. In an embodiment of the method for preparing LCMF poloxamers, such as LCMF poloxamer 188, the extraction solvent comprises methanol and carbon dioxide. Exemplary methanol to carbon dioxide ratios are 1:100 to about 15:100 or 1:100 to 15:100, such as 2:100 to about 10:100 or 2:100 to 10:100. The methanol to carbon dioxide ratio can be increased over the course of practicing the method. The methods herein include batch and continuous methods. In exemplary embodiments of practicing the method, the extraction vessel can be pressurized in the range of 125 to 500 bar, such as 25 to 100 bar, or 200 bar to 400 bar or 280 bar to 340 bar. The temperature of the extraction vessel can be 10° C. to 80° C. In embodiments of the method, the second alkanol can be provided as a percentage (w / w) of the total extraction solvent of 3% to 20% or 3% to 15%.

[0029] In step b) of the above method, the concentration of the second alkanol of step b) in the extraction solvent can be increased multiple times in gradient steps over the time of the extraction method: each multiple time is performed for an additional predetermined period of time; and in each successive step, the alkanol concentration is increased, for example, by 1-2% compared to the previous second alkanol concentration; and the extraction solvent is removed from the extraction vessel, thereby removing the extracted material from the raffinate poloxamer formulation.

[0030] In other embodiments, a method for purifying LCMF poloxamers may include: a) introducing a poloxamer solution into an extraction vessel, dissolving the poloxamer in a solvent to form a solution, the solvent being selected from alcohols, aliphatic ketones, aromatic ketones, amines, and mixtures thereof; b) mixing the poloxamer solution with an extraction solvent comprising a solvent and high pressure carbon dioxide, the concentration of the solvent in the extraction solvent increasing over the time of the extraction process; and c) removing the extraction solvent from the extraction vessel, thereby removing the extracted material from the poloxamer to produce the LCMF poloxamer. The solvent in step a) may be methanol. The extraction solvent may comprise methanol and carbon dioxide. The extracted material comprises low molecular weight impurities less than 4,500 daltons. Methods including those described above and exemplified herein may be used to extract LCMF poloxamers, particularly poloxamers of the formula HO(CH2CHO) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) a H (wherein: each of a or a' is an integer such that the percentage of hydrophilic material (C2H4O) is about 60% to 90% by weight of the total molecular weight of the copolymer; a and a' may be the same or different. and b is an integer such that the molecular weight of the hydrophobe (CHO) is about 1,300 to 2,300 Daltons. The resulting poloxamers are LCMF poloxamers without the LCM material as described herein, such as poloxamer LCMF188: LCMF poloxamer 188 has the formula HO(CHCHO) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) aH; each of a and a' are integers such that the percentage of hydrophilic material (CHO) is about 60%-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 hydrophobic material (CHO) is between about 1,300 and 2,300 daltons; no more than 1.5% of all components in the distribution of the copolymer are low molecular weight components having an average molecular weight less than 4,500 daltons; no more than 1.5% of all components in the distribution of the copolymer are high molecular weight components having an average molecular weight greater than 13,000 daltons; the polydispersity value of the copolymer is less than about or equal to 1.07; and the poloxamer does not contain an LCM material such that, when administered to a subject, such as a human, the circulating half-life of all components in the distribution of the copolymer is no more than 5.0 times longer than the circulating half-life of the major component in the distribution of the copolymer. The resulting LCMF poloxamers, e.g., LCMF poloxamer 188, are more hydrophilic than the corresponding LCM-containing poloxamers, such as purified LCM-containing poloxamer 188. The resulting LCMF poloxamers have a lower average t than the corresponding LCM-containing poloxamers when evaluated under the same suitable conditions for RP-HPLC, such as those exemplified and described herein. R and has a low k'.

[0031] Methods are also provided to confirm or identify the resulting poloxamer as an LCMF poloxamer, including, for example, testing the LCMF poloxamer by RP-HPLC and comparing the material to standards known to contain the starting material and / or LCM, or assessing the hydrophilicity of the poloxamer and comparing it to standards known to contain the starting material and / or LCM material. [Brief description of the drawings]

[0032] The drawings described herein are intended to illustrate only selected embodiments rather than all possible implementations and are not intended to limit the scope of the present disclosure.

[0033] [Figure 1] FIG. 1 is a general process 100 for supercritical fluid extraction (SFE) of poloxamer. [Diagram 2] FIG. 2 is an example of a process 100' for preparing a poloxamer, such as poloxamer 188, using the methods described herein. [Diagram 3] FIG. 3 is an example of a process 100″ for preparing a poloxamer, such as poloxamer 188, using the methods described herein. [Figure 4] FIG. 4 shows an extraction apparatus useful in the methods provided herein. [Diagram 5] FIG. 5 shows one embodiment of a cross section of stainless steel spheres of different sizes in a solvent distribution bed. [Figure 6] 6A-6B show a gel permeation chromatography (GPC) comparison of low molecular weight material content in commercially available poloxamer 188 (panel A) and material purified according to embodiments provided herein (panel B). [Figure 7] 7A-B show zoomed-in HPLC-GPC chromatograms showing the molecular weight distribution of components in plasma over time. [Figure 8] 8A-B show the individual plasma concentrations of poloxamer 188 (panel A) and high molecular weight components (panel B) in healthy humans during and after a 48-hour continuous IV infusion of purified poloxamer 188 as described in Grindel et al. (2002) (Biopharmaceutics & Drug Disposition 23:87-103). [Figure 9] FIG. 9 is a reversed-phase high performance liquid chromatography (RP-HPLC) chromatogram comparing the composition profiles of 15% LCMF188 and 15% P188 (available under the trademark Flocor®) versus other poloxamers and polymers (of different hydrophobicity / hydrophilicity), showing that LCMF188 is more hydrophilic than P188. [Figure 10]FIG. 10 shows RP-HPLC chromatograms comparing different lots of LCMF poloxamer 188 with purified poloxamer 188 confirming the differences in hydrophilicity. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] overview A.Definition B. Molecular diversity of poloxamer, poloxamer 188, LCM-containing poloxamer 188, and LCMF poloxamer 1. Poloxamer 2. Poloxamer 188 3. Molecular Diversity of Poloxamer 188 a.Low molecular weight components b. Components that provide a long circulating half-life C. Poloxamer with Long Circulating Substance Free (LCMF) D. Extraction Methods for Purifying Poloxamer 1. Extraction process a.Supercritical method b. High pressure method 2. Extraction vessels and systems 3. Extraction and removal of extractant 4. Exemplary Methods for Preparation of Purified Poloxamers a. Removal of low molecular weight (LMW) components b. Preparation of long-circulating substance-free (LCMF) poloxamer 5. Methods for confirming the identity of LCMF poloxamers E. Pharmaceutical Compositions and Formulations 1. Formulation 2. Dosage 3. Dosage and Administration F. Methods and Therapeutic Uses of Poloxamer 188 and LCMFP188 G. Working Example

[0035] A.Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. All patents, patent applications, published applications and publications, Genbank sequences, databases, websites and other published materials mentioned throughout this disclosure are incorporated by reference in their entirety unless otherwise stated. In the event of multiple definitions for terms in this specification, those in this section shall prevail. When referring to a URL or other such identifier or address, it is understood that such identifiers may change and specific information on the Internet may come and go, but equivalent information may be found by searching the Internet. Reference thereto establishes the validity and public availability of such information.

[0036] As used herein, poloxamer is a copolymer of ethylene oxide and propylene oxide. A synthetic block copolymer. "Polyoxyethylene / polyoxypropylene copolymer," "PPC," or "poloxamer" has the following chemical formula: HO(C2H4O) a’ ─[C3H6O] b ─(C2H4O) a H wherein a' and a' may be the same or different and are each an integer such that the hydrophilic portion represented by (C2H4O) (i.e., the polyoxyethylene portion of the copolymer) constitutes about 60% to 90% by weight of the copolymer, e.g., 70% to 90% by weight of the copolymer; b is a (C3H6O) bis an integer such that the hydrophobic material (i.e., the polyoxypropylene portion of the copolymer) represented by has a molecular weight of approximately 950-4,000 Daltons (Da), e.g., about 1,200-3,500 Da, e.g., 1,200-2,300 Da, 1,500-2,100 Da, 1,400-2,000 Da, or 1,700-1,900 Da. For example, the molecular weight of the hydrophilic portion can be 5,000-15,000 Da. Exemplary poloxamers having the above general formula include poloxamers where a or a' is an integer between 5 and 150 and b is an integer between 15 and 75, e.g., poloxamers where a is an integer between 70 and 105 and b is an integer between 15 and 75. Poloxamers include poloxamer 188 (e.g., those sold under the trademarks Pluronic® F-68, Flocor®, Kolliphor®, and Lutrol®).

[0037] The nomenclature of polyoxyethylene / polyoxypropylene copolymers is related to the monomer composition. The first two digits of the poloxamer number are multiplied by 100 to give the approximate molecular weight of the hydrophobic polyoxypropylene block. The last digit is multiplied by 10 to give the approximate weight percent of the hydrophilic polyoxyethylene content. For example, poloxamer 188 represents a polymer containing approximately 1,800 Da polyoxypropylene hydrophobes with a hydrophilic polyoxyethylene block content of approximately 80% of the total molecular weight.

[0038] Poloxamers can be synthesized in two steps by first constructing a polyoxypropylene core and then adding polyoxyethylene to the ends of the polyoxypropylene core. Due to variations in the polymerization rate during both steps, poloxamers contain heterogeneous polymer species of various molecular weights. The distribution of polymer species can be characterized using standard techniques, including but not limited to gel permeation chromatography (GPC).

[0039] As used herein, poloxamer 188 (also referred to as P-188 or P188) has the following chemical formula: HO(CH2CH2O) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) a H, where a and a' can be the same or different, each being an integer such that the hydrophilic portion represented by (C2H4O) (i.e., the polyoxyethylene portion of the copolymer) constitutes about 60% to 90%, for example about 80% or 81%, and b is such that the hydrophobic portion represented by (C3H6O) has a molecular weight of about 1,300 to 2,300 Da, for example 1,400 to 2,000 Da, for example about 1,750 Da. For example, a is about 79 and b is about 28 or 28. The average total molecular weight of the compound is about 7,680 to 9,510 Da, for example generally 8,400 to 8,800 Da, for example about 8,400 Da or 8,400 Da. Poloxamer 188 is a preparation that may contain a heterogeneous distribution of polymer species that differ primarily in overall polymer chain length, but also includes truncated polymer chains with unsaturation, and some low molecular weight glycols. Poloxamer 188 molecules also include those that exhibit a species profile (e.g., as determined by GPC) that includes a main peak and two "shoulder" peaks representing low molecular weight (LMW) and high molecular weight (HMW) polymer species. Poloxamer 188 also refers to materials that have been purified to remove or reduce species other than the main component.

[0040] As used herein, "major component" or "main peak" in the context of a poloxamer 188 formulation refers to a species of copolymer molecule having a molecular weight less than about 13,000 Da and greater than about 4,500 Da, with an average molecular weight of about 7,680-9,510 Da, e.g., generally 8,400-8,800 Da, e.g., about 8,400 Da or 8,400 Da. Main peak species include those that elute by gel permeation chromatography (GPC) at 14-15 minutes depending on the chromatographic conditions (see U.S. Pat. No. 5,696,298).

[0041] As used herein, "low molecular weight" or "LMW" with respect to species or components of poloxamer 188 formulations generally refers to components having a molecular weight less than 4,500 Da. LMW species include those that elute by gel permeation chromatography (GPC) after 15 minutes depending on the chromatographic conditions. (See U.S. Patent No. 5,696,298). Such impurities may 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).

[0042] As used herein, "high molecular weight" or "HMW" with respect to species or components of a poloxamer 188 formulation generally refers to components having a molecular weight greater than 13,000 Da, e.g., greater than 14,000 Da, greater than 15,000 Da, greater than 16,000 Da or more. HMW species include those that elute by gel permeation chromatography (GPC) at 13-14 minutes depending on the chromatographic conditions (see U.S. Patent No. 5,696,298).

[0043] As used herein, "polydispersity" or "D" refers to the spread of a particular 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 D values ​​close to 1, while typical polymers have a range of 2-5. Some polymers have polydispersities greater than 20. Thus, a high polydispersity value means a wide variation in the size of the population of molecules in a given preparation, while a low polydispersity value means less variation. Methods for assessing polydispersity are known in the art and include methods such as those described in U.S. Pat. No. 5,696,298. For example, polydispersity can be determined from a chromatogram. It is understood that polydispersity values ​​can vary depending on the particular chromatographic conditions, molecular weight standards, and size exclusion characteristics of the gel permeation column employed. For purposes herein, references to polydispersity are as used in U.S. Patent No. 5,696,298 and are as determined from chromatograms obtained using a Model 600E Powerline chromatographic system equipped with a column heater module, a Model 410 refractive index detector, Maxima820 software (all from Waters Div., Millipore, Milford, Mass.), two LiChrogel PS-40 and LiChrogel PS-20 columns in series (EM Science, Gibbstown, NJ), and polyethylene glycol molecular weight standards (Polymer Laboratories, Inc., Amherst, Mass.). One skilled in the art can convert any polydispersity values ​​obtained using different separation methods to the values ​​described herein by simply testing one sample on both systems and then comparing the polydispersity values ​​obtained from each chromatogram.

[0044] As used herein, "purified poloxamer 188" or "P188-P" or "purified poloxamer 188 with long circulating material (LCM)" refers to poloxamer 188 having a poloxamer polydispersity value of about 1.07 or less, e.g., 1.05 or less or 1.03 or less, and is a purified poloxamer 188 that has a reduced amount of low molecular weight components but contains long circulating material. "Low molecular weight material-removed" or "Low molecular weight material-reduced" poloxamer Poloxamer 188 or similar variations thereof refers to purified poloxamer 188 having a distribution of low molecular weight components that is less than or equal to 3.0% and generally less than or equal to 2.0% or less than or equal to 1.5% of the total distribution of components. Typically, such poloxamer 188 exhibits reduced toxicity compared to forms of poloxamer 188 that contain a higher or greater percentage of low molecular weight components. Poloxamer 188 is purified to remove or reduce the low molecular weight components. Commercially available and conventional formulations of poloxamers, such as poloxamer 188, contain long circulating substances (LCMs) that, when administered to humans, have half-lives that are greater than 5.0 times the circulating half-life of the major component in the distribution of the copolymer.

[0045] An exemplary purified LCM-containing poloxamer 188 is poloxamer 188 available under the trademark FLOCOR® (see also, e.g., U.S. Patent No. 5,696,298, which describes LCM-containing poloxamer 188). When 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 shows two circulating peaks, a peak designated as the main peak containing the major component of the polymer distribution, and a peak of higher molecular weight than the main peak and indicative of a rate of clearance from the circulation (as shown herein, more than 5 times slower than the main peak, typically greater than 30 hours and as long as 70 hours), i.e., the long-circulating substance (LCM).

[0046] As used herein, long-circulating substances (LCM) refer to substances in conventional poloxamer formulations that, when administered to a subject, have a half-life in a subject, such as a human, that is substantially longer than the half-life of the main component of the poloxamer formulation. When administered to a human subject, the LCM substances in the poloxamer formulation have a half-life that is more than or about 5 times the half-life of the main component of the poloxamer formulation. The LCMF poloxamers provided herein do not produce such long-circulating substances. No component has a half-life that is 5 times longer than the main component. To compare poloxamers, the components of the corresponding poloxamers are compared, where the corresponding poloxamers have the same formula. For example, LCMF poloxamer 188 is compared to poloxamer 188.

[0047] As used herein, "free of long-circulating substances" or "LCMF" with respect to poloxamer 188 refers to a purified poloxamer 188 formulation having reduced amounts of low molecular weight components, as described above for purified poloxamer 188, that does not have any long-circulating substances after intravenous administration to a subject within the circulating half-life (t 1 / 2 LCMF refers to a purified poloxamer 188 formulation in which the polymer distribution components are more uniformly cleared from the circulation so as to exhibit a half-life (in human subjects) that is 5-fold longer than that of the primary component t 1 / 2 t greater than 5.0 times, and generally not greater than 4.0 times, not greater than 3.0 times, not greater than 2.0 times, or not greater than 1.5 times greater than the half-life of the major component in the copolymer distribution. 1 / 2 or is free of components, such as high or low molecular weight components, that arise when such circulating material is administered to a human subject. Typically, LCMF poloxamers are poloxamers in which all components of the polymer distribution are cleared from the circulation at a more uniform rate.

[0048] As used herein, "distribution of copolymer" refers to the molecular weight distribution of polymer molecules in a poloxamer formulation. The molecular weight distribution can be measured using standard techniques known to those skilled in the art, including but not limited to colligative measurement, light scattering techniques, viscometry, and size exclusion chromatography. In particular, gel permeation chromatography (GPC) can be used to determine the molecular weight distribution based on the hydrodynamic volume of the polymer. The molecular weight or mass distribution of a polymer can be summarized by polydispersity. For example, the greater the difference in molecular weight distribution in a poloxamer, the higher the polydispersity.

[0049] As used herein, half-life, biological half-life, plasma half-life, terminal half-life, excretion Half-life or t 1 / 2 Half-life refers to the time it takes for an organism to eliminate half of the amount of an administered substance through its normal elimination channels. Normal elimination channels generally include the excretory function of removing the substance from the body, as well as the body's cleansing by the kidneys and liver. Half-life can be described as the time it takes for the plasma concentration of a substance to be half of its steady-state level, i.e., plasma half-life. Half-life can be determined by administering a single dose of a drug, usually intravenously, and then measuring the concentration of the drug in the plasma at regular intervals. The concentration of the drug peaks in the plasma and then declines as the drug is degraded and removed from the blood.

[0050] As used herein, "Cmax" refers to the peak or maximum plasma concentration of a drug following administration.

[0051] As used herein, "concentration of drug at steady state" or "Css" refers to the concentration of drug at which the rate of drug elimination equals the rate of drug administration. It is achieved after an infinite number of doses, generally equally spaced apart. The time it takes to achieve a steady state concentration depends on the half-life of the drug. The shorter the half-life, the more quickly the steady state is reached. Typically, it takes 3-5 half-lives to accumulate to more than 90% of the final steady-state concentration.

[0052] As used herein, "impurities" refer to undesirable components in a poloxamer formulation. Typically, impurities include LMW components below 4,500 daltons and high molecular weight components above 13,000 daltons.

[0053] As used herein, "removing or reducing" in reference to a poloxamer component in a formulation refers to reducing the weight percentage of the component in a poloxamer formulation relative to the initial percentage of the component. In general, a poloxamer component is considered to be removed or reduced when the weight percentage of the component relative to the total distribution of the component is reduced by at least 1%, typically at least 2%, at least 3%, at least 4%, at least 5%, or more. For example, most commercial poloxamer 188 products contain LMW components (less than 4,500 Daltons) that are about 4% by weight of the total components in the distribution. LMW materials are considered to be reduced in a purified product when the component is less than 3% by weight, e.g., less than 2% by weight or less than 1% by weight.

[0054] As used herein, "solvent" refers to any liquid that dissolves a solute to form a solution.

[0055] As used herein, "polar solvent" refers to a solvent in whose molecule the positive and negative charges are permanently separated 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.

[0056] As used herein, "feed" refers to a solute dissolved in a solvent.

[0057] As used herein, "extraction solvent" refers to any liquid or supercritical fluid that can be used to solubilize undesirable substances contained in poloxamer formulations. An extraction solvent is a solvent that can perform solvent extraction to separate one or more other substances based on differences in solubility. In general, 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 the first solvent in which the substance of interest is dissolved, so that two separate layers are formed when left to stand. An exemplary extraction solvent is a supercritical liquid or a high-pressure liquid.

[0058] As used herein, the terms "supercritical liquid" and "supercritical fluid" refer to a liquid that is heated to a temperature above its critical temperature (T c (i.e., the temperature characteristic of a compound above which it is not possible to liquefy it) and the critical pressure (p c Supercritical fluids include any compound, such as a gas, in a state above the critical temperature (i.e., the minimum pressure sufficient to liquefy the compound at its critical temperature). In this state, distinct liquid and gas phases typically do not exist. Supercritical fluids typically exhibit a change in solvent density that is only slightly affected by changes in pressure, temperature, or the presence of a co-modifier solvent. It is.

[0059] As used herein, "supercritical carbon dioxide" refers to the fluid state of carbon dioxide held above its critical temperature (about 31°C) and critical pressure (about 74 bar). Below its critical temperature and pressure, carbon dioxide normally behaves as a gas in air, or as solid dry ice when frozen. At temperatures above 31°C and pressures above 74 bar, carbon dioxide exhibits properties intermediate between a gas and a liquid, so that it expands to fill its container like a gas, but has the density of a liquid.

[0060] As used herein, "critical temperature" or "critical point" refers to the temperature above which no distinct liquid or gas phases exist, meaning the vapor-liquid critical point. It is thus the temperature above which the vapor of a substance cannot be liquefied, no matter how much pressure is applied. For example, the critical temperature of carbon dioxide is about 31°C.

[0061] 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 bar.

[0062] As used herein, the term "high pressure liquid" includes liquids produced by compressing a compressible gas into a liquid at room temperature or at elevated temperatures.

[0063] 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). A co-modifier solvent can strongly interact with a solute, thereby substantially increasing the solubility of the solute in the extraction solvent. Examples of co-modifier solvents include alkanols. Typically, 5% to 15% by weight of a co-modifier solvent can be used.

[0064] As used herein, the term "alkanol" includes simple aliphatic organic alcohols. Generally, the alcohols contemplated for use in the methods provided herein contain six or fewer carbon atoms (i.e., C1-C6 alkanols). The alkane portion of the alkanol can be branched or unbranched. Examples of alkanols include, but are not limited to, methanol, ethanol, isopropyl alcohol (2-propanol), and tert-butyl alcohol.

[0065] As used herein, "subcritical extraction" refers to a process that uses a fluid substance that is normally gaseous at normal temperature and pressure, but turns into a liquid at higher pressure and lower temperature. The pressure or temperature is then normalized, and the extraction substance is evaporated, leaving the extract. The extractant can be recycled.

[0066] As used herein, "extraction vessel" or "extractor" refers to a high pressure vessel capable of withstanding pressures of up to 10,000 psig and temperatures of up to 200°C. The volume of the vessel can range from 2 mL to 200 L, typically from 1 L to 200 L, e.g., 5 L to 150 L. Extraction vessels are generally made of stainless steel. Such equipment is well known to those skilled in the art and is commercially available.

[0067] As used herein, isocratic refers to a system in which the extraction solvent is used at a constant or near constant concentration.

[0068] As used herein, "gradient" or "gradient stage" refers to a system using two or more extraction solvents that differ in composition, typically due to changes in the concentration of one or more components. For example, the concentration of an alkanol solvent (e.g., methanol) is continuously increased over the course of the extraction. Thus, the extraction solvent does not remain constant.

[0069] As used herein, "multiple" refers to several processes or steps. The number of repetitions can be 2, 3, 4, 5, 6 or more.

[0070] As used herein, "extracted material" refers to the product that contains the removed material.

[0071] As used herein, "raffinate" refers to a product from which a component or components have been removed, for example, purified poloxamer from which extracted materials have been removed.

[0072] As used herein, "batch method" or "batch extraction" refers to a process in which a solute is extracted from one immiscible layer by shaking the two layers until equilibrium is reached, followed by settling and sampling of the layers. For example, batch extraction can be performed by mixing the solute with a batch of extraction solvent. The solute distributes between the two phases. Once equilibrium is reached, mixing is stopped and the extract and raffinate phases are allowed to separate. In this method, the spent solvent can be removed by distillation and recycled, or fresh solvent can be added continuously from a reservoir.

[0073] As used herein, "continuous process" or "continuous extraction" refers to a process in which there is a continuous flow of an immiscible solvent through a solution or a continuous countercurrent flow of both phases. For example, a continuous extraction solvent is mixed with a solute. The emulsion that results in the mixer is fed into a settler unit where phase separation occurs, resulting in continuous raffinate and extract streams.

[0074] As used herein, a "pharmaceutical composition" encompasses compositions comprising a polyoxyethylene / polyoxypropylene copolymer as described herein, such as LCMF poloxamer, in a pharma- ceutically acceptable formulation and / or formulated with one or more pharma-ceutically acceptable excipients. In some cases, the pharmaceutical composition comprises an aqueous injection solution of the poloxamer, buffered with a suitable buffer at a desired pH, such as 6-7 or 6 or about 6. Examples of buffers are any known to those of skill in the art to be biocompatible, such as citrate, e.g., sodium citrate / citric acid. Suitable concentrations can be empirically determined and typically range from 0.005-0.05 M, particularly about 0.01 M, in an isotonic solution such as saline. In some cases, pharmaceutical compositions useful in the methods herein are known to those of skill in the art for formulation of poloxamers (see, e.g., International PCT Publication No. WO 94 / 008596 and other such references and publications described herein).

[0075] As used herein, the term "treatment" refers to improving or alleviating symptoms associated with a disease or condition. Treatment refers to any manner in which the symptoms of a condition, disorder, or disease are ameliorated or otherwise beneficially altered. Thus, treatment encompasses prophylaxis, therapy, and / or curative treatment. Treatment also encompasses any pharmaceutical use of the compositions herein.

[0076] As used herein, "treating" a subject with a disease or condition means providing the subject with the Treatment means administering a composition or other product as provided or described herein.

[0077] As used herein, amelioration of symptoms of a particular disease or disorder by treatment, such as by administration of a pharmaceutical composition or other therapeutic agent, refers to any alleviation, whether permanent or temporary, persistent or transient, of symptoms that can result from or be associated with the administration of the composition or therapeutic agent.

[0078] As used herein, "prevention" or "prophylaxis" refers to a method in which the risk of developing a disease or condition is reduced. Prevention includes reducing the risk of development of a disease or condition and / or preventing the worsening or progression of symptoms of a disease or reducing the risk of the worsening or progression of symptoms of a disease.

[0079] As used herein, an "effective amount" of a compound or pharmaceutical composition for treating a particular disease is an amount sufficient to improve or alleviate to some extent the symptoms to achieve the desired physiological effect. Such an amount can be administered as a single dose or according to a regimen, thereby being effective. The effective amount is readily determined by one of skill in the art following routine procedures and depends on the particular condition for which the composition is administered.

[0080] As used herein, a "therapeutically effective amount" or a "therapeutically effective dose" refers to a drug, compound, substance, or composition that contains at least a sufficient amount of compound to produce a therapeutic effect. An effective amount is an amount of a therapeutic agent sufficient to treat, e.g., prevent, ameliorate, inhibit, or otherwise treat a particular disease or disorder.

[0081] As used herein, "disease" or "disorder" refers to a pathological condition of an organism that results from a cause or condition, including, but not limited to, infection, acquired conditions, and genetic conditions, and is characterized by identifiable symptoms. Diseases and disorders of interest herein include, but are not limited to, any that require membrane resealing and repair, tissue ischemia and reperfusion injury, decreasing inflammatory disorders, disorders related to thrombolysis, and disorders related to hemostasis. For example, diseases and disorders include acute myocardial infarction, acute limb ischemia, shock, acute stroke, heart failure, sickle cell disease, neurodegenerative diseases, macular degeneration, diabetic retinopathy, and congestive heart failure.

[0082] As used herein, "subject" refers to animals, particularly humans or veterinary animals, such as dogs, cats, pigs, cows, horses and other farm animals, zoo animals and pets. Thus, the "patient" or "subject" to be treated includes humans and / or non-human animals, such as 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.

[0083] As used herein, "combination" refers to any relationship between two or more items. The relationship may be spatial, such as in a kit, or may refer to the use of two or more items for a common purpose.

[0084] As used herein, a "composition" refers to any mixture of two or more products or compounds (e.g., drugs, modulators, regulators, etc.). The composition may be a solution, suspension, liquid, powder, paste, aqueous or non-aqueous formulation, or any combination thereof.

[0085] As used herein, a "product" is a product that is made and sold. The term is intended to include purified poloxamer contained in a package.

[0086] As used herein, fluid refers to any composition that can flow. Fluids therefore encompass compositions that are in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams, and other such compositions.

[0087] As used herein, "kit" refers to a packaged combination that may optionally include reagents and other products and / or components for carrying out a method using the elements of the combination. For example, a kit is provided that includes the purified poloxamer provided herein and other items for purposes including, but not limited to, administration, diagnosis, and evaluation of biological activity or properties. The kit may optionally include instructions for use.

[0088] As used herein, animals include, but are not limited to, primates, including humans, gorillas, and monkeys; rodents, such as mice and rats; poultry, such as chickens; ruminants, such as goats, cows, deer, sheep; ovine, such as pigs and Non-human animals include any animal, such as mice, rabbits, and other animals. Non-human animals exclude humans as contemplated animals.

[0089] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0090] As used herein, ranges and amounts can be expressed as "about" or "approximately" a particular value or range. About also encompasses the exact amount. Thus, "about 0.05 mg / mL" also means "about 0.05 mg / mL" and "0.05 mg / mL."

[0091] As used herein, "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances when said event or circumstance occurs and instances when said event or circumstance does not occur. For example, an optionally substituted group means that the group is unsubstituted or substituted.

[0092] As used herein, "retention time" or "t R " refers to the time elapsed between injection of a sample, such as an LCMF poloxamer 188 sample, onto a reversed-phase column of a reversed-phase high performance liquid chromatography system and peak response by the evaporative light scattering detector. Retention times are longer for more hydrophobic samples than for less hydrophobic samples.

[0093] As used herein, the "capacity factor" or k' is determined by the formula: where t is equal to the void time, i.e., the time it takes for unretained material to pass through a reverse-phase HPLC column (see Example 7 below):

number

[0094] The LCM-containing purified poloxamer 188, such as the poloxamer sold under the trademark FLOCOR®, had a mean retention time (t R ) and has a k' of 3.697; On the other hand, LCMF poloxamer 188 had a mean retention time (t R ) and has an average k' of 3.202 (see Example 7).

[0095] As used herein, abbreviations for any protecting groups, amino acids and other compounds follow their common usage, accepted abbreviations, or those of the IUPAC-IUB Commission on Biochemical Nomenclature (see (1972) Biochem. 11:1726), unless otherwise noted.

[0096] B. Molecular diversity of poloxamer, poloxamer 188, LCM-containing poloxamer 188, and LCMF poloxamer 1. Poloxamer Poloxamers are a family of synthetic linear triblock copolymers composed of a core of repeating polyoxypropylene (PO or POP) units flanked on either side by chains of repeating polyoxyethylene (EO or POE) units. 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, providing specific poloxamers with distinct chemical and physical properties, as well as unique pharmacodynamic properties.

[0097] Certain polyoxyethylene / polyoxypropylene copolymers, including poloxamer 188, exert beneficial biological effects on several disorders when administered to humans or animals. These activities have been described, for example, in many publications and patents (e.g., U.S. Pat. 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, 5,030,102, and 5,040,101). Nos. 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, RE36,665 (reissue of No. 5,523,492), 5 Nos. 5,696,298, 6,359,014, 6,747,064, 8,372,387, and 8,580,245, U.S. Patent Publication Nos. 2011 / 0044935, 2011 / 0212047, and 2013 / 0177524, and International Application No. WO2006 / 037031 (PCT / US2012). (See, for example, WO2009 / 023177 (filed as PCT / US2005 / 037157) and WO2006 / 091941 (filed as PCT / US2006 / 006862), as well as PCT / US2014 / 45627, U.S. Provisional Patent Applications Nos. 62 / 021,691 and 62 / 021,676.) Activities that make poloxamers, such as poloxamer 188, useful as therapeutic agents include their ability to incorporate into cell membranes, thereby repairing damaged cell membranes. Poloxamers have the formula: HO(C2H4O) a’ ─(C3H6O) b ─(C2H4O) a H wherein "a'" and "a" may be the same or different, each being an integer such that the hydrophilic moiety represented by (C2H4O) constitutes approximately 50% to 95% by weight of the compound, such as 60% to 90% by weight, such as 70% to 90% by weight of the compound; and "b" is an integer such that the hydrophobic moiety represented by (C3H6O) has a molecular weight of about 950 to 4,000 Da, such as 1,200 to 3,500 Da. For example, the hydrophobic moiety has a molecular weight of 1,200 to 2300 Da, such as generally 1,500 to 2100 Da. The average molecular weight of the copolymer is 5,000 to 15,000 Da, such as 5,000 to 12,000 Da, 5,000 to 9000 Da.

[0098] In some examples, b is an integer from about 15 to about 70, such as from about 15 to about 60, or from about 15 to about 30, or any number therebetween. In some examples, b is about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30. In some embodiments, the integers of the flanking units with subscripts "a'" and "a" can be or are the same value. In some examples, a or a' is an integer from about 45 to about 910, such as 90, 100, 200, 300, 400, 500, 600, 700, 800, or 900. In some other examples, a or a' is an integer between about 10 and about 215, such as 10, 20, 30, 40, 50, 60, 70, 80, 100, 125, 150, 175, 200, or 215. In still other examples, 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. One of skill in the art will recognize that these values ​​are averages. The values ​​of a, a', and b are averages, and generally, the polymer molecules are a distribution or population of molecules. Thus, the actual values ​​of a, a', and b in the population constitute a range of values.

[0099] Poloxamer nomenclature is related to the composition of the various polymer members. The first two digits of the poloxamer number are multiplied by 100 to give the approximate molecular weight of the hydrophobe. The last digit is multiplied by 10 to give the approximate weight percent of the hydrophile (polyoxyethylene) content of the surfactant. For example, poloxamer 407 is a polymer containing a polyoxypropylene hydrophobe of about 4000 Da and the polyoxyethylene hydrophile makes up about 70% of the total molecular weight. Poloxamer 188 (P188) has a hydrophobe with a molecular weight of about 1800 Da and a hydrophile that is about 80% of the total molecular weight of the copolymer.

[0100] Poloxamers are sold and referred to under trade names and trademarks, including, but not limited to, ADEKA NOL, Synperonic™, Pluronic®, and Lutrol®. Examples of poloxamers include, but are not limited to, poloxamer 188 (P188; sold under the trademark Pluronic® F-68, Kolliphor® P188, 80% POE), poloxamer 407 (P407; sold under the trademark Lutrol F-127, Kolliphor® P188, Pluronic® F-127; 70% POE), poloxamer 237 (P237; sold under the trademark Pluronic® F87, Kolliphor® P237; 70% POE), poloxamer 338 (P338; sold under the trademark Kolliphor® P338, Pluronic® F-108; 80% POE), and poloxamer 331 (Pluronic® L101; 10% POE).

[0101] Thus, unpurified P188 is commercially available or sold or known under various names, such as those mentioned above. In the following discussion, reference is made to using the methods herein to produce more homogeneous (LCMF) poloxamer 188, but the methods herein can be used to produce more homogeneous preparations of any of the known poloxamers.

[0102] Poloxamers can be synthesized using standard polymer synthesis techniques. For example, poloxamers are formed by ethylene oxide-propylene oxide condensation using standard techniques known to those of skill in the art (see, e.g., U.S. Patent Nos. RE36,665; RE37,285; RE38,558; 6,747,064; 6,761,824; and 6,977,045; Reeve, LE, "The Poloxamers: Their Chemistry and Medical Applications," in Handbook of Biodegradable Polymers, Dombak, et al., J. Am. Soc., 1999, 144:1311-1315, 2001). , AJ et al. (eds.), Hardwood Academic Publishers, 1997). Poloxamers can be synthesized by the sequential addition of POP and POE monomers in the presence of an alkaline catalyst such as sodium hydroxide or potassium hydroxide (see, for example, Schmolka J. Am. Oil Chem. Soc. 54 (1977) 110-116). The reaction is initiated by polymerization of a POP block followed by growing POE chains at both ends of the POP block. Methods for synthesizing the polymers are also described in U.S. Pat. No. 5,696,298.

[0103] 2. Poloxamer 188 Poloxamer 188 (P188) copolymer has the following chemical formula: HO(CH2CH2O) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) a H where the hydrophobic material represented by (C3H6O) has a molecular weight of about 1,750 Daltons, and poloxamer 188 has an average molecular weight of 7,680-9,510 Da, for example, generally about 8,400-8,800 Daltons. The polyoxyethylene-polyoxypropylene-polyoxyethylene weight ratio is approximately 4:2:4. According to this specification, P188 has a weight percent of oxyethylene of 81.8±1.9%, and an unsaturation level of 0.026±0.008 mEq / g.

[0104] Various poloxamers, and especially P188, are used for the treatment of diseases and conditions in which there is a pathological increase in resistance to blood flow due to damage resulting from the presence of adhesive hydrophobic proteins or damaged membranes. This adhesion occurs through 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 decreasing the effective radius of blood vessels. For example, poloxamer 188 is believed to act as a lubricant to increase blood flow through damaged tissues. Advantageously, it prevents adhesion of hydrophobic surfaces to each other, thereby reducing friction and increasing flow rate.

[0105] P188 binds to hydrophobic moieties that occur on damaged cells and denatured proteins, thereby restoring the hydration lattice. It promotes sealing of damaged membranes and stops the cascade of inflammatory mediators that can destroy cells. The polymer also inhibits hydrophobic adhesive interactions that cause harmful aggregation of formed elements in the blood. The anti-adhesive and anti-inflammatory effects of P188 are shown by reducing friction, preventing adhesion and aggregation of formed elements in the blood, maintaining the deformability of red blood cells, the non-adhesiveness of platelets and granulocytes, and the normal viscosity of blood, reducing apoptosis, and enhancing blood flow in damaged tissues by multiple inflammatory markers, including VEGF, various chemokines, and interleukins.

[0106] 3. Molecular Diversity of Poloxamer 188 Commercially available poloxamer 188 preparations are said to have a molecular weight of about 8,400 daltons. However, such poloxamer 188 is composed of molecules having molecular weights ranging from less than 3,000 daltons to more than 20,000 daltons. The molecular diversity and distribution of molecules in commercially available poloxamer 188 can be seen in the broad primary and secondary peaks detected using gel permeation chromatography (see, e.g., International PCT Application Publication No. WO 94 / 08596).

[0107] Diversity of structure means diversity of biological activity. For example, optimal rheological, cytoprotective, anti-adhesive and anti-thrombotic effects are observed with the molecule P188, which is approximately 8400-9400 Da. Such components can be identified as the major components in poloxamer formulations using methods that separate components based on size, such as gel permeation chromatography (GPC). It can be identified as a component or predominant component. However, the distribution of components also typically exhibits minor low molecular weight (LMW, i.e. generally less than 4,500 Daltons) or high molecular weight (HMW, i.e. generally greater than 13,000 Daltons) components. P188 components greater than 15,000 and less than 4,500 Daltons are less effective rheological or cytoprotective agents and exhibit undesirable side effects. Other substances or components in poloxamer formulations, such as P188 formulations, originate from two different sources: synthesis and degradation.

[0108] The primary mechanism contributing to molecular diversity is the process by which poloxamers are synthesized. In a typical manufacturing process, the first step is the formation of POP blocks. These are formed by reacting propylene glycol initiator with propylene oxide monomers. Ethylene oxide monomers are then added to both ends to form the block copolymer. The synthesis of poloxamers can result in variability in polymerization rates during the steps that build the PO core and EO ends.

[0109] During the synthesis of POPs, two different reaction mechanisms limit POP chain growth, resulting in unintended diblock polymers. These materials typically have low molecular weights (relative to the P188 polymer distribution). In one mechanism, unsaturation is formed directly from propylene oxide by reacting with an alkaline catalyst. The base catalyzes the transfer of propylene oxide to allylic alcohol, which then initiates monofunctional chains with terminal unsaturation. These types of side reactions produce low molecular weight (LMW) materials over the course of the reaction. In gel permeation chromatography (GPC), the distribution of these impurities is in the main peak as well as in the LMW shoulder. In the second mechanism, a hydrogen atom located six carbon atoms away can be abstracted by a negative oxygen atom in the growing polymer chain, terminating and transferring the chain, resulting in an allyl end group. These back-biting reactions result in high molecular weight (H The distribution of these materials is mostly in the LMW shoulder.

[0110] In addition, high molecular weight material (relative to the polymer distribution of P188) can form due to improper cleaning of the polymerization reactor between batches of poloxamer 188 during a typical commercial manufacturing campaign. If the reactor is not thoroughly cleaned to remove residual product after the production of a typical batch of poloxamer such as P188, the residual product will act as an initiator in subsequent batches, forming "dimer-like" poloxamer molecules. This material has a high molecular weight and is part of the polymer distribution observed by GPC as an HMW shoulder.

[0111] Poloxamer degradation pathways include peroxidation leading to low molecular weight aldehydes and acids, and thermal degradation leading to LMW polyethylene glycols. Oxidative degradation is the main degradation pathway affecting poloxamer stability. This process results in structural changes to the polymer chains, producing peroxides and carbonyls. The peroxides are fugitive and rapidly combine with butylated hydroxytoluene (BHT), which is typically added to commercial products as an antioxidant. Thermal degradation is another pathway that produces other substances. Glycols of various chain lengths are the main degradation products of thermal degradation. Forced thermal degradation studies have demonstrated the formation of ethylene glycol, propylene glycol, diethylene glycol, and triethylene glycol.

[0112] Thus, a particular poloxamer is composed of multiple chemical entities that have the EO-PO-EO structural motif but differ in the number of repeating EO and PO units. A variety of truncated polymers and various other substances that have the EO-PO motif are of interest. These other substances may arise as a result of side reactions that occur during the synthesis of the poloxamer compounds shown. These other substances may be present and found within the overall poloxamer distribution. The result is a heterogeneous materials (i.e., materials that are polydisperse).

[0113] For example, for the synthesis of P188, there may be variations in polymerization rates during the steps that build the PO core and EO ends. Thus, most unpurified forms of P188 contain a distribution of bell-shaped polymer species that differ primarily in overall chain length. In addition, there may be various low molecular weight (LMW) components (e.g., glycols and truncated polymers) and high molecular weight (HMW) components (e.g., dimerized polymers) that arise from incomplete polymerization. Typically, characterization of P188 by gel permeation chromatography (GPC) identifies a main peak of P188 with "shoulder" peaks that represent unintended LMW and HMW components (see Emanuele and Balasubramanian (2014) Drugs RD 14:73-83). For example, an available formulation of P188 from BASF (Parsippany, NJ) has a published structure characterized by a hydrophobic block with a molecular weight of approximately 1,750 Da, a POE block that constitutes 80% by weight of the polymer, 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 with molecular weights ranging from less than 1,000 Da to more than 30,000 Da. The molecular diversity and distribution of molecules in commercially available poloxamer 188 is indicated by the broad primary and secondary peaks detected using gel permeation chromatography. The molecular diversity present in non-purified poloxamer preparations, including commercially available poloxamers, can result in diverse biological activities. Many of the observed biological activities are undesirable and / or can result in undesirable side effects that limit the therapeutic efficacy of poloxamers as drugs. Complement activation, phagocyte migration paralysis, and other side effects observed upon administration of artificial blood products have been reported. The toxicity and cytotoxicity are due in part to impurities in the poloxamer 188 component of these formulations. In addition, infusion of poloxamer 188 has resulted in elevated creatinine, indicative of renal damage, and increased organ weight (kidney) in toxicological animal studies. Histological evaluation of the kidneys showed dose-related cytoplasmic vacuolization of proximal tubular epithelial cells.

[0114] Poloxamer 188, which has been purified to remove low molecular weight components (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)), contains components that exhibit different pharmacokinetic profiles when administered to a subject. The major component has a plasma half-life (t 1 / 2 ), whereas the high molecular weight components (i.e., long retention time species) showed about a 10-fold or greater increase in half-life, with a t 1 / 2 and therefore exhibits a substantially longer plasma residence time and is cleared from the circulation more slowly than the major component, as shown herein (see, e.g., Figures 8A and 8B).

[0115] a.Low molecular weight components Substances in poloxamer 188 that are toxic to the kidney have been identified as having a lower molecular weight than the main component. Studies of the therapeutic potential of P188 have led to the discontinuation of therapeutic applications of poloxamer available under the trademark RheothRx®, in part due to acute renal insufficiency observed during clinical trial evaluation as evidenced by elevated serum creatinine. These effects were found to be due to the presence of various low molecular weight (LMW) substances generated during the synthesis process (Emanuele and Balasubramanian (2014) Drugs RD 14:73-83). The LMW substances were accumulated by proximal tubular epithelial cells in the kidney.

[0116] The molecular weights of LMW materials can range from hundreds to thousands of Da. The complex nature of these impurities with a wide range of solubility characteristics makes them difficult to selectively remove from the parent molecule. Traditional purification processes such as distillation, crystallization, and ultrafiltration do not effectively separate low molecular weight (LMW) materials from the main components. The use of chromatographic techniques for purification, such as preparative GPC, is costly and practically difficult to scale. Mixed solvent systems also require the use of large amounts of solvent that are difficult and expensive to recycle.

[0117] Supercritical fluid chromatography has been reported to reduce the amount of these low molecular weight materials present in P188 (see, e.g., U.S. Pat. No. 5,567,859). Supercritical fluid extraction has been performed using carbon dioxide to purify the copolymer and reduce the polydispersity to less than 1.17. However, that method does not sufficiently remove or reduce the LMW components as shown herein.

[0118] As described in more detail below, the methods provided herein produce poloxamer formulations that are substantially free of these LMW components. For example, purified P188 with reduced LMW components has less than about 5%, less than 4%, less than 3%, less than 2% or less than 1% LMW components. Thus, the poloxamer formulations provided herein, and in particular the P188 poloxamer formulations, generally exhibit reduced toxicity and do not elevate creatinine levels when administered. In addition, as described herein, the resulting LCMFP188 poloxamer formulations have other advantageous properties, including reduced long-term circulating substances when administered.

[0119] B. Components responsible for long circulating half-lives The components in P188 have been identified as being or giving rise to substances in plasma or blood that have a longer circulating half-life than the major or majority poloxamer species. This longer circulating half-life has been observed in non-clinical and clinical studies. Analysis by high performance liquid chromatography-gel permeation chromatography (HPLC-GPC) of plasma obtained after intravenous administration of purified P188 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). The peak has an average molecular weight of approximately 8,600 daltons. There is a major peak with an average molecular weight of approximately 16,000 daltons and a minor peak with an average molecular weight of approximately 16,000 daltons. The two peaks exhibit distinctly different pharmacokinetic profiles, with the higher molecular weight peak clearing from the circulation slower and exhibiting a distinctly longer plasma residence time (see Figures 8A and 8B). Similar observations have been reported in rats and dogs. A similar long-circulating component has been observed with native or unpurified poloxamer 188 (see International PCT Publication No. WO 94 / 008596).

[0120] For example, as shown in Figure 8A, after intravenous administration of purified P188 to healthy volunteers at a dose of 100 mg / kg / hr for 1 hour followed by a maintenance dose of 30 mg / kg / hr for 47 hours, the main or majority peak reached a mean maximum concentration (Cmax) of 0.9 mg / mL by the end of the 1-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. When the maintenance infusion was discontinued, plasma concentrations declined rapidly, with an elimination half-life (t1 / 2) of approximately 7 hours.

[0121] As shown in FIG. 8B, the HMW component was observed to exhibit a Cmax of 0.2 mg / mL, which was not achieved until the end of the maintenance infusion. As concentrations continued to accumulate during the infusion, steady state was not achieved. After cessation of the maintenance infusion, the plasma levels of the high molecular weight peak declined slowly, such that plasma levels declined by only about 33% during the 24-hour post-infusion monitoring period. This rate of elimination was approximately 1 / 10 that of the main peak, with a t 1 / 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). Long-circulating substances (or long-retention time substances) are identified in the HMW fraction of the P188 distribution (Grindel et al. (2002a)). This The HMW component was characterized by MALDI-TOF mass spectrometry to be approximately 16,000 Da, with a fragmentation pattern consistent with a block copolymer (see, e.g., Grindel et al. (2002a)).

[0122] The rheological, cytoprotective, anti-adhesive and anti-thrombotic effects of P188 are optimal within the majority or major copolymer of distribution, which is approximately 8,400-9400 Da and has a half-life of approximately 7 hours, so the presence of other components that exhibit long circulating half-lives is undesirable. For example, desirable activities of P188 include its rheological effect of reducing blood viscosity and inhibiting red blood cell (RBC) aggregation, which is responsible for its ability to improve blood flow in injured tissues. In contrast, high 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 P188, meaning that higher molecular weight poloxamer species may have undesirable biological effects.

[0123] As described in more detail below, poloxamer formulations are provided that are substantially reduced in components that are or give rise to long-circulating substances, i.e., they are long-circulating substance-free (LCMF). Exemplary methods for making LCMF poloxamers (see, e.g., Example 7) are also provided.

[0124] Thus, the LCMF poloxamer formulations provided herein, and in particular the LCMF poloxamer 188 formulation, exhibit a more uniform pharmacokinetic profile and therefore a more consistent therapeutic effect. The LCMF poloxamers are described in further detail in the following sections.

[0125] C. Poloxamer with Long Circulating Substance Free (LCMF) Provided herein is a long-circulating free (LCMF) P188, which is a purified P-188 having a polydispersity value of less than 1.07; 1.5% or less of a low molecular weight (LMW) component less than 4,500 Daltons; 1.5% or less of a high molecular weight component greater than 13,000 Daltons; and when administered to a subject, the half-life of all components in the distribution of the copolymer is less than 5.0 times longer in blood or plasma than the half-life of the major component in the distribution of the copolymer. Thus, when administered, LCMF poloxamer 188 does not produce a component that has a significantly longer half-life than the major component. LCMF P-188 has the following chemical formula: HO(CH2CH2O) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) a H where a' and a can be the same or different and each is an integer such that the hydrophilic portion represented by (C2H4O) (i.e., the polyoxyethylene portion of the copolymer) constitutes about 60% to 90%, for example about 80% or 81%; and b is an integer such that the hydrophobic portion represented by (C3H6O) has a molecular weight of about 1,300 to 2,300 Da, for example about 1,750 Da; and the average total molecular weight of the compound is about 7,680 to 9,510 Da, for example generally 8,400 to 8,800 Da, for example about 8,400 Da or 8,400 Da, and the copolymer has been purified to remove impurities, including low molecular weight or other impurities, such that the polydispersity value is less than 1.07.

[0126] Studies have shown that the major peak component of the purified (LCM-containing) P-188 formulation has a plasma half-life (t 1 / 2 ) (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)). Purified poloxamer was further purified to approximately 16,000 daltons. t, showing approximately a 10-fold or greater increase in half-life, 1 / 2 About It also yielded long-circulating substances (LCM) containing high molecular weight components up to 70 h.

[0127] For example, the purified P-188 ( In contrast to LCM-containing poloxamers, the purified poloxamers designated LCMF P-188 are those in which all components of the polymer distribution are cleared from the circulation at approximately the same rate when administered to a subject. Thus, LCMF P-188 differs from the prior art LCM-containing p188 poloxamers. Like the LCM-containing poloxamers, the LCMF poloxamers contain substantially less polydispersity compositions, less than 1.07, typically less than 1.05 or less than 1.03, but the half-life in blood or plasma of any component in the copolymer distribution is no more than 5.0 times longer, typically no more than 4.0 times, no more than 3.0 times, no more than 2.0 times, or no more than 1.5 times longer than the half-life of the major component in the copolymer distribution when administered to a human subject. Typically, the LCMF does not contain any component that, when administered to a subject, exhibits a half-life in blood or plasma that is substantially longer (more than 5 times) than the major peak in the copolymer distribution or longer than the major component in the copolymer distribution.

[0128] In certain instances, the blood or plasma half-life of all components in the LCMF poloxamer, when administered to a human subject, is greater than 30 hours, and generally, no component has a half-life that is 25 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, or 7 hours or less.

[0129] Without being bound by theory, it is believed that the higher molecular weight components of the poloxamer polymer distribution, such as those above 13,000 daltons, could be responsible for the longer circulating half-life material. The glomerular filtration rate of charged molecules is highly dependent on molecular size, as the glomerular filtration rate becomes increasingly limited above a size threshold (Chang et al., (1975) Biophysic. J. 15:887-906). Poloxamer 18, which has a molecular weight greater than 5,000 daltons, is a promising candidate for this purpose. 8 polymer distribution. Thus, it is likely that the higher molecular weight components of the poloxamer 188 polymer distribution (e.g., those greater than 13,000 daltons) are cleared from the circulation at a slower rate than the smaller sized components.

[0130] However, for LCMF formulations, the presence of HMW components in distribution does not result in long-term circulating species (i.e., species with half-life more than 5 times longer than that of the main peak).For example, the HMW impurities greater than 13,000 Daltons in LCMF formulations generally constitute 1.5% or less by weight of all components.When LCMF formulations are administered to a subject, these HMW impurities do not result in a circulating half-life that is more than 5.0 times longer than that of the main component in distribution, and generally is 4.0 times or less, 3.0 times or less, 2.0 times or less, or 1.5 times or less.When LCMF formulations are administered to a subject, they do not result in a component that is substantially longer (i.e., more than 5 times) than the main component in distribution or has a circulating half-life that is longer than that of the main component in distribution (see, for example, Figures 7A and 7B).

[0131] In LCMF formulations, the HMW content can be increased or decreased compared to other existing purified P-188 preparations. For example, the LCMF poloxamers provided herein include P-188 poloxamers with 1.3% or less of high molecular weight content greater than 13,000 daltons, such as 1.2% or less, 1.1% or less, 1.0% or less, or less. In certain examples provided herein, the LCMF poloxamers provided herein include P-188 poloxamers with less than 1.0% by weight, generally less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5% or less of high molecular weight content greater than 13,000 daltons.

[0132] The LCMF poloxamers provided herein are described in Section D herein, in particular Section The LCMF poloxamers can be prepared by the methods described below in Section D.1.b (see, e.g., FIG. 3). Given the description and illustration of the properties of the LCMF poloxamers, one of skill in the art can envision other methods of making the LCMF poloxamers. For example, the LCMF poloxamers provided herein can be prepared by: a) introducing a poloxamer solution into an extraction vessel and dissolving the poloxamer in a first alkanol to form a solution; b) contacting a second alkanol with an extraction solvent comprising supercritical carbon dioxide at a temperature and pressure to maintain the supercritical carbon dioxide for a first period of time, wherein: The temperature is above the critical temperature of carbon dioxide, but may typically be in the range of 35°C to 45°C; The pressure is 220bar~280bar; and providing the alkanol at a concentration of 7% to 8% by weight of the total extraction solvent; and c) increasing the concentration of the second alkanol from step b) in the extraction solvent multiple times in a gradient step over time of the extraction method, in this case: each of the plurality of times occurring within a further period of time; and In each successive step, the alkanol concentration is increased by 1-2% compared to the previous concentration of the second alkanol; and d) removing the extraction solvent from the extraction vessel, thereby removing the extracted materials from the raffinate poloxamer formulation.

[0133] D. Extraction Methods for Purifying Poloxamer Provided herein are supercritical fluid extraction (SFE) and high pressure procedures for purifying poloxamers such that the purified polymers are more uniform in terms of structure (diblock, triblock, etc.), percentage of molecules without unsaturation, distribution of molecular weights, and hydrophobic / hydrophilic distribution (HLB) ratio. The tunability of the process can be exploited to remove extraneous components and can be adjusted over time, thereby increasing the yield of purified product. The methods provided herein use solvent systems whose solvation properties can be varied to selectively remove various substances. The methods provide exemplary ways of producing LCMF poloxamer 188 products with the above properties.

[0134] The methods herein provide poloxamer formulations that differ from those produced by prior art methods, including LCMF poloxamer 188 formulations that, when administered, do not produce the long-circulating substances observed with purified poloxamer 188, such as those described in U.S. Patent No. 5,696,298. LCMF poloxamer 188 has a similar molecular size distribution as purified poloxamer 188, but produces formulations in which the component molecules are more hydrophilic than the purified poloxamer.

[0135] The absence of long-circulating substances (LCMs) results in improved properties of the poloxamer, including the rate of clearance and other pharmacological properties improved by the removal of long-circulating substances. The methods provided herein remove undesirable components in a poloxamer formulation, thereby preparing a more homogenous or uniform poloxamer formulation that exhibits desirable therapeutic activity while minimizing or reducing undesirable activity. As commercially available poloxamers have been reported to exhibit toxicity as well as variability in biological activity, a more homogenous and uniform poloxamer formulation would have reduced toxicity but retain the therapeutic efficacy of the main copolymer component.

[0136] Methods for preparing such poloxamers are provided herein, as are resultant poloxamers, including LCMF poloxamer 188. The methods provided herein, in addition to resulting in poloxamer formulations with reduced or eliminated low molecular weight (LMW) components, also provide for preparations of long-term circulating substances in plasma or blood that have reduced or eliminated any components that are or give rise to such circulating substances as described herein. Also provided herein are LCMF formulations of poloxamers, particularly LCMF poloxamer 188. The LCMF poloxamer 188 provided herein can be used for all of the known uses for poloxamer 188.

[0137] Provided herein are extraction methods for purifying poloxamers, such as P188, to remove or reduce components other than the major component, and thereby reduce the molecular diversity of the formulation. For example, the methods provided herein can remove or reduce LMW material in the poloxamer. In addition to removing or reducing LMW material, it has also been found herein that certain methods provided herein can also remove or reduce components in poloxamer formulations that are or give rise to long-circulating materials with half-lives substantially longer than the half-life of the major component in distribution. The degree of extraction, and the components extracted, are controlled by the specific temperature, pressure, and alkanol concentration used in the methods described herein.

[0138] The methods provided herein provide a method for reducing the solvent power in the presence of a co-solvent modifier. The extraction solvent used is a supercritical or subcritical solvent, controlled by the manipulation of temperature, pressure, etc. Carbon dioxide is not a particularly effective extraction solvent for poloxamers such as P188, but it has been found that the presence of a polar co-solvent, such as an alkanol, as a modifier increases the efficiency of solubilizing CO2 in the extraction solvent. In particular, the methods provided herein are carried out in the presence of a polar co-solvent, such as an alkanol, whose concentration increases in a gradient manner (e.g., a step gradient or a continuous ascending gradient) as the extraction process proceeds. It has been found that by employing an alkanol co-solvent, the concentration of which increases in this manner, the removal of impurities can be increased, and can be much more than when carbon dioxide is used alone. For example, extraction methods using carbon dioxide alone cannot remove undesirable components, such as LMW and HMW components, as described herein, to the same extent as achieved by the methods provided.

[0139] In the methods provided herein for purifying poloxamers using supercritical fluid extraction, the LMW components or impurities of the poloxamer distribution can be selectively removed at lower alkanol (e.g., methanol) concentrations and higher pressures than other HMW components in the distribution. As further described below, it is also possible to remove other impurities by increasing the solubilizing capacity of the extraction solvent, for example, by carefully controlling the pressure and concentration of a polar solvent such as an alkanol (e.g., methanol). In particular, the methods use a gradient of high concentrations of an alkanol (e.g., methanol), alone or in combination with reduced pressure, such that components in the poloxamer distribution (e.g., HMW components) are removed such that the resulting product, when administered to a subject, does not result in long-term circulating substances in plasma as observed with conventional P-188 products.

[0140] However, there is a trade-off 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 increases, solubilizing more compounds and increasing the extent of extraction. By increasing the concentration of the solvent in a gradient fashion, the decrease in poloxamer yield is minimized while maximizing the purity of the final product. Typically, the methods provided herein achieve a yield such that the amount of 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 poloxamer before the method is performed. However, the resulting poloxamer exhibits a substantially higher purity, has a higher percentage of the major component in the distribution than the starting material, and is free of impurities that may exhibit toxic side effects when administered or cause long-term circulation in plasma.

[0141] The method can be carried out for any poloxamer for which it is desirable to increase the purity, for example by reducing or reducing undesirable components in the distribution of the polymer. One skilled in the art can select a particular poloxamer for purification in this manner. Undesirable components include any that are or give rise to substances that are toxic or have an inverse or opposite biological activity to the desired activity. For example, the poloxamer may be one for which it is desirable to reduce or remove LMW components in the poloxamer, for example, any LMW components that, when administered, result in acute renal side effects such as elevated creatinine. The poloxamer may also include any components, such as HMW components, that are or give rise to substances that, when administered, have a half-life in the blood that is different (e.g., longer) than the half-life of the major components in the distribution of the polymer. Such components may increase blood viscosity and red blood cell aggregation, and are therefore undesirable.

[0142] Examples of poloxamers for use in the method 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 major component is within or about 4,700 Da to 12,800 Da, such as generally 7,680 Da to 9,510 Da, such as generally 8,400 to 8,800 Da. In particular, the poloxamer is P188. For example, the extraction methods provided herein can be used to purify P188 preparations, which have the following chemical formula: HO(CH2CH2O) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) a H: where the hydrophobic material represented by (C3H6O) has a molecular weight of about 1,750 Daltons and has an average molecular weight of 7,680-9,510 Da, for example, generally about 8,400-8,800 Daltons. The polyoxyethylene-polyoxypropylene-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 formulations for use in the methods herein include commercially available formulations. These include, but are not limited to, Pluronic® F68 (BASF, Florham Park, NJ) and RheothRx® (developed by Glaxo Wellcome Inc.).

[0143] In carrying out the extraction method provided herein, the method includes: a) providing a poloxamer (e.g., P188) solution in an extraction vessel, prepared by dissolving the poloxamer in a first solvent to form a solution; b) mixing an extraction solvent, including a supercritical fluid (e.g., supercritical carbon dioxide) or a subcritical fluid (e.g., high pressure carbon dioxide) and a co-modifier solvent, with the solution to form an extraction mixture, where the concentration of the co-modifier solvent in the extraction solvent increases over the time of the extraction method; and c) removing the extraction solvent from the extraction vessel, thereby removing impurities (e.g., LMW components or other components) from the poloxamer formulation. In the method, the step of dissolving the poloxamer solution in the first solvent can be performed before the solution is loaded into the extraction vessel or at the time the solution is loaded into the extraction vessel. For example, the poloxamer is dissolved in a separate vessel and then the solution is added to the extraction vessel.

[0144] The method can be a high pressure or supercritical fluid extraction method. Typically, the method is carried out using supercritical fluid extraction (SFE), which uses a supercritical fluid in an extraction solvent. A supercritical fluid is any liquid that is heated above its critical temperature and compressed above its critical pressure. For example, carbon dioxide has a critical temperature of 31.1° C. and a critical pressure of 73.8 bar. Thus, the extraction conditions for supercritical carbon dioxide are greater than the critical temperature of about 31° C. and the critical pressure of about 74 bar. In contrast, Typically, high pressure extraction can be achieved under subcritical conditions where the pressure exceeds the critical pressure but the temperature does not exceed the critical temperature.

[0145] 1. Extraction process a.Supercritical method In some instances, the supercritical fluid extraction process used in the methods provided herein is essentially a solvent extraction process using a supercritical fluid as a solvent.Using a supercritical fluid, a multi-component mixture can be separated (solvent extraction) by taking advantage of the difference in volatility of the components and the difference in the specific interactions between the component mixture and the supercritical fluid solvent.In the supercritical region of the phase diagram, compressible fluids such as carbon dioxide exhibit liquid-like density and a greatly increased solvent capacity depending on pressure.

[0146] Supercritical fluids exhibit many highly advantageous properties that make them excellent solvents. For example, the tunable solvent power of supercritical fluids changes rapidly around a certain range of critical states. The solvent power of supercritical fluids, and therefore the nature of the components that can be selectively removed during extraction, can be fine-tuned by varying the temperature and pressure of the supercritical fluid solvent.

[0147] Another beneficial property of different supercritical fluids is the difference in their critical temperatures and pressures. Each supercritical fluid has a different solvent power. A tunable solvent power range can be selected by choosing the appropriate supercritical fluid.

[0148] In addition to their unique dissolving properties, supercritical fluids exhibit certain physicochemical properties that make them even more useful. For example, supercritical fluids exhibit liquid-like densities and have gas-like transport properties, such as diffusivity and viscosity. These properties also change rapidly near the critical region. Supercritical fluids also have zero surface tension. Since most useful supercritical fluids have boiling points near or below ambient temperature, the solvent removal step after purification is simple, energy efficient, and does not leave any residual solvent.

[0149] The use of a solid matrix during extraction provides additional features to the fractionation parameters. The use of a suitable solid matrix provides solvent-matrix and solute-matrix interactions in addition to solute-solvent interactions to enhance fractionation resolution. The favorable transport properties of supercritical fluids make the process easily scalable for manufacturing. Heat and mass transfer properties do not change significantly with process scale-up in supercritical fluid extraction processes. Extraction process conditions such as pressure, temperature, and flow rate can be precisely controlled, making the purification process highly adjustable as well as reproducible.

[0150] In such methods, the extraction solvent may include a supercritical liquid (e.g., supercritical carbon dioxide) as well as another co-modifying solvent, typically an alkanol, which increases over time during the extraction. As noted above, the presence of the co-modifying solvent can improve the solubility of solutes, such as high molecular weight or even non-polar solutes, thereby increasing their extraction in the method.

[0151] For example, the methods provided herein may include: a) providing or introducing into an extraction vessel a poloxamer (e.g., poloxamer 188) solution prepared by dissolving the poloxamer in a first alkanol to form a solution; b) mixing an extraction solvent comprising a second alkanol and a supercritical fluid with the solution under high pressure and temperature sufficient to create a supercritical liquid state to form an extraction mixture, where the concentration of the second alkanol in the extraction solvent increases over time during the extraction method; and c) removing the extraction solvent from the extraction vessel, thereby removing impurities (e.g., LMW components or other components) from the poloxamer formulation. The first and second alkanols may be the same or different. In the method, the step of dissolving the poloxamer solution in the first solvent may be performed prior to loading the solution into the extraction vessel or after loading the solution into the extraction vessel. This can be done at the time of loading into the extraction vessel, for example by dissolving the poloxamer in a separate vessel and then adding the solution to the extraction vessel.

[0152] An exemplary process is detailed in Figure 1. Figure 1 shows a process 100 for removing impurities (e.g., LMW components or other components) from a poloxamer formulation. The extraction system is pressurized, as shown in step 105, typically prior to dispensing a first alkanol into a feed mix tank, as shown in step 110. The system is heated to a temperature suitable for the extraction process. The temperature is typically above the critical temperature of the supercritical liquid (e.g., carbon dioxide). Generally, the temperature is approximately 40°C.

[0153] Any suitable alkanol or combination of alkanols can be used in the method of purifying poloxamer. Examples of suitable alkanols include, but are not limited to, methanol, ethanol, propanol, butanol, and others. For example, the methods provided herein include the above-mentioned extraction methods, in which the first and second alkanols are each independently selected from methanol, ethanol, propanol, butanol, pentanol, and combinations thereof. In some embodiments, the first alkanol is methanol. In some examples, methanol is selected as the purification solvent and is the second alkanol in the implementation of the method. Those skilled in the art will understand that methanol has a relatively low toxicity. In addition, methanol has good solubility for poloxamer 188.

[0154] A 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 formulation, is dispensed into the feed tank and stirred until mixed with the first alkanol. The amount of poloxamer 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 be achieved, and other factors within the level of skill in the art. For example, an unlimited amount of poloxamer (e.g., P188) per mL of extraction vessel can be 0.1 kg to 0.5 kg or 0.2 kg to 0.4 kg. In some examples, in an extraction method using a 3 L extraction vessel, an unlimited amount 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 an extraction method using a 12 L extraction vessel, an unlimited amount 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 an extraction method using a 50 L extraction vessel, a non-limiting amount of poloxamer (e.g., P188) can be 8 kg to 20 kg, e.g., 10 kg to 16 kg or 12 kg to 15 kg. Variations in amounts are contemplated depending on the particular application, extraction vessel, purity of starting material, and other considerations within the level of one of ordinary skill in the art.

[0155] Any suitable ratio of poloxamer to alkanol is contemplated for use in the methods provided herein. The ratio of poloxamer to alkanol can be, for example, about 4:1 to about 1:4, for example, about 3:1 to about 1:3, 2:1 to about 1:2, 1:1 to 4:1, or 1:2 to 1:4, by weight. For example, the weight ratio of poloxamer to alkanol can be about 4:1, or about 3:1, or about 2:1, or about 1:1, or about 1:2, or about 1:3, or about 1:4. For example, a quantity of poloxamer, for example P188, can be mixed with an equal amount of alkanol (e.g., methanol) by weight. A quantity of poloxamer, for example P188, can be mixed with a lesser amount of alkanol by weight, for example, half the amount of alkanol (e.g., methanol) by weight. One of skill in the art will appreciate that the appropriate poloxamer to alkanol ratio will depend on the properties of the poloxamer, such as its solubility in a given alkanol.

[0156] After forming the poloxamer / alkanol mixture, all or a portion of the mixture is step The poloxamer is pumped into the extractor as shown at 120. In such an example, the process of preparing the poloxamer solution is carried out in a vessel separate from the extractor. One skilled in the art 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 carried out directly in the extraction vessel.

[0157] As shown in step 125 of process 100, the extractor is then pressurized and an extraction solvent is introduced into the extractor. The extraction solvent comprises a 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 refrigerants sold as Freon. In some embodiments, the supercritical liquid under pressure is carbon dioxide (CO2).

[0158] The extraction is carried out under high pressure and high temperature to maintain a supercritical liquid state (e.g., supercritical carbon dioxide). Typically, these are kept constant. At this pressure and temperature, a substantially constant flow rate of supercritical liquid (e.g., supercritical carbon dioxide) is provided. The flow rate may be from 0.5 kg / h to 600 kg / h, e.g., from 1 kg / h to 400 kg / h, from 1 kg / h to 250 kg / h, from 1 kg / h to 100 kg / h, from 1 kg / h to 50 kg / h, from 1 kg / h to 20 kg / h, from 1 kg / h to 10 kg / h, from 10 kg / h to 400 kg / h, from 10 kg / h to 250 kg / h, from 10 kg / h to 100 kg / h, from 10 kg / h to 50 kg / h, from 10 kg / h to 20 kg / h. g / h, 20kg / h to 400kg / h, 20kg / h to 250kg / h, 20kg / h to 100kg / h, 20kg / h to 50kg / h, 50kg / h to 400kg / h, 50kg / h to 250kg / h, 50kg / h to 100kg / h, 100kg / h to 400kg / h, 100kg / h to 200kg / h or 200kg / h to 400kg / h (each inclusive). For example, the flow rate is 20kg / h to 100kg / h (inclusive), for example generally about 100kg / h or 100kg / h.

[0159] Any suitable temperature that maintains the supercritical liquid in a supercritical state can be used to carry out the extraction process. For example, the critical temperature of carbon dioxide is about 31°C. Thus, the extraction vessel is maintained at a temperature above 31°C. In some embodiments, the extraction vessel has a temperature between 32°C and 80°C, generally between 32°C and 60°C, or between 32°C and 60°C, inclusive. For example, the temperature can be 35°C or less, 36°C or less, 37°C or less, 38°C or less, 39°C or less, 40°C or less, 41°C or less, 42°C or less, 43°C or less, 44°C or less, 45°C or less, 50°C or less, or 60°C or less. Generally, the temperature is greater than 31°C but not greater than 40°C. One of skill in the art will appreciate that the temperature can vary depending in part on the composition of the extraction solvent as well as the solubility of a given poloxamer in the solvent employed in the process.

[0160] Any suitable pressure can be used in the method. When using supercritical fluid extraction, the system is pressurized at a level to ensure that the supercritical liquid remains at a pressure higher than the critical pressure. For example, the critical pressure of carbon dioxide is about 74 bar. Thus, the extraction vessel is pressurized to above 74 bar. The particular degree of pressure can alter the solubility characteristics of the supercritical liquid. Thus, the particular pressure selected can affect the yield and the degree of extraction of impurities. Typically, the extraction vessel is pressurized within the range of 125-500 bar. In some embodiments, the extraction vessel is operated at 200 bar to 400 bar, 200 bar to 340 bar, 200 bar to 300 bar, 200 bar to 280 bar, 200 bar to 260 bar, 200 bar to 240 bar, 200 bar to 220 bar, 220 bar to 400 bar, 220 bar to 340 bar, 220 bar to 300 bar, 220 bar to 280 bar, 220 bar to 260 bar, 220 bar to 240 bar, 240 bar to Pressurize within the range of 400bar, 240bar~340bar, 240bar~300bar, 240bar~280bar, 240bar~260bar, 260bar~400bar, 260bar~340bar, 260bar~300bar, 260bar~280bar, 280bar~400bar, 280bar~340bar, 280bar~300bar or 300bar~340bar. For example, the extraction vessel can be pressurized to 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 bar, generally up to 500 bar. The extraction vessel can be pressurized, for example, to 310±15 bar.

[0161] Typically, in the method provided herein, the extraction solvent introduced into the extraction vessel also contains an alkanol. Thus, the extraction solvent contains 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, changing the solvent properties of the supercritical liquid and improving the extractability of the solute in the method. Any suitable alkanol or combination of alkanols can be used as the second alkanol in the method provided herein, as described above. As described above, in a particular example, the second alkanol is methanol.

[0162] Any suitable combination of second alkanol and supercritical liquid, such as any of those mentioned above, can be used in the extraction solvent in the methods provided herein. In some embodiments, the extraction solvent comprises methanol and carbon dioxide. The second alkanol is typically provided as a percentage (w / w) of the total extraction solvent that is between 3% and 20%, generally between 3% and 15%, for example between 5% and 12%, between 5% and 10%, between 5% and 9%, between 5% and 8%, between 5% and 7%, between 7% and 15%, between 7% and 12%, between 7% and 10%, between 7% and 9%, between 7% and 8%, between 8% and 15%, between 8% and 12%, between 8% and 10%, between 8% and 9%, between 9% and 15%, between 9% and 12%, between 9% and 10%, between 10% and 15%, or between 10% and 12%, inclusive. The flow rate (kg / h) of the alkanol is a function of the amount of alkanol introduced into the extractor.

[0163] For example, a suitable ratio of alkanol (e.g., methanol) to supercritical fluid (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 fluid (e.g., carbon dioxide) can be about 1:100 to about 20:100. In some embodiments, the ratio of alkanol (e.g., methanol) to supercritical fluid (e.g., carbon dioxide) is about 1:100 to about 15:100. In some embodiments, the ratio of alkanol (e.g., methanol) to supercritical fluid (e.g., carbon dioxide) is about 2:100 to about 14:100. The ratio of alkanol (e.g., methanol) to supercritical fluid (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.

[0164] In some embodiments, the extraction can be carried out in an isocratic manner, where the composition of the extraction solvent remains constant throughout the extraction procedure. For example, the amount of supercritical fluid (e.g., carbon dioxide) and alkanol (e.g., methanol) are constant over the time of the 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 varying (e.g., increasing or decreasing) the amount of supercritical liquid and / or alkanol components that make up the extraction solvent. In general, supercritical The liquid (e.g., carbon dioxide) is kept constant while the concentration of the alkanol (e.g., methanol) in the extraction solvent is varied (e.g., increased or decreased) over the time of the extraction. The concentration of the components can be varied by adjusting the flow rate.

[0165] In embodiments where the composition of the extraction solvent may change over time, it is beneficial for the method to increase the second alkanol as the extraction process proceeds, either as a stepwise gradient or as a continuous ascending gradient. In some cases, commercial grade poloxamer has both high and low molecular weight components along with the main product or component. The low alkanol (e.g., methanol) concentration in the high pressure carbon dioxide extraction fluid can selectively remove low molecular weight components. However, the solubility of the extractables containing a lot of impurities is low, and it takes time to significantly reduce low molecular weight components, making it ineffective. By increasing the alkanol concentration of the extraction solvent in a gradient manner (either as a stepwise gradient or as a continuous ascending gradient), the amount of low molecular weight impurities extracted increases.

[0166] Furthermore, higher alkanol (e.g., methanol) concentrations increase the solubility and therefore the extraction of higher molecular weight components. Thus, a gradient of successively higher alkanol (e.g., methanol) concentrations in the extraction solvent can progressively extract lower molecular weight components and ultimately higher molecular weight components, i.e., less soluble components. As a non-limiting example to illustrate this, a low alkanol (e.g., methanol) concentration of about 6.6% w / w is believed to be capable of removing low molecular weight components. Increasing the concentration of alkanol by 1% to 3% continues to perform the extraction of low molecular weight components, but also removes high molecular weight components. Further increasing the concentration of alkanol by 1% to 3% further removes these components, as well as other components that have a high molecular weight and / or are less soluble in the previous extraction solvent.

[0167] However, extraction solvents with higher alkanol (e.g., methanol) concentrations are not selective because they provide higher solubility for low molecular weight components, but also increase the solubility of other components, including the main component. Thus, the yield of purified product decreases at high methanol concentrations. By increasing the concentration of the extraction solvent in a gradient manner, as provided in the method herein, the decrease in poloxamer yield is minimized and the purity of the final product is maximized.

[0168] It has been found that a stepwise increase in methanol concentration increases the loading capacity of the extractor, thereby increasing the throughput in a given extraction system. A two-phase system constitutes the interior of the extractor. The lower phase consists mainly of a mixture of poloxamer and methanol with some dissolved carbon dioxide. The extraction solvent (carbon dioxide with a low methanol co-solvent fraction) permeates the lower phase. The upper phase consists mainly of the extraction solvent and components extracted from the poloxamer. The relative amounts of the two phases depend on the methanol concentration in the solvent stream. In a typical extraction system, there is adequate headspace for proper phase separation of the upper phase. The methanol co-solvent concentration is increased stepwise during the extraction process, leading to higher feed loadings into the extractor.

[0169] 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 weight percentage of the alkanol (e.g., methanol) of the extraction solvent is increased over the course of the method. The methanol content in the methanol / carbon dioxide mixture can be increased in a stepwise or continuous manner as the extraction process progresses. In some embodiments, for example, the extraction process of a poloxamer (e.g., P188) begins with about 3% to about 10% by weight (w / w), such as about 5% to about 10%, such as 6% to 8% (e.g., about 6.6% or 7.4%) of alkanol (e.g., methanol) in the extraction solvent with a supercritical fluid (e.g., carbon dioxide). After a predetermined period of time, the alkanol (e.g., methanol) content of the extraction solvent is increased to about 1-3% by weight (w / w). %, for example 1-2% (for example to 7.6% or 9.1%, respectively). The alkanol (e.g. methanol) content is then increased again during a final period by about 1-3%, for example 1-2% (for example to 8.6% or 10.7%, respectively).

[0170] Any suitable solvent gradient can be used in the method. For example, the alkanol (e.g., methanol) concentration in the supercritical fluid (e.g., carbon dioxide) can be increased from about 5% to about 20% during the extraction procedure. The alkanol (e.g., methanol) concentration in the supercritical fluid (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 fluid (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 the supercritical fluid (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, three, four, or five steps during the extraction procedure. One skilled in the art will appreciate that other solvent ratios and solvent gradients can be used during the extraction process.

[0171] The extraction time of the process provided herein can be any predetermined period that results in suitable extraction of the material in the formulation while minimizing the loss of poloxamer yield and maximizing purity. The time is a function of the selection of pressure, temperature, second alkanol concentration, and the 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, generally 10 hours to 30 hours, or 15 hours to 25 hours (each inclusive), for example, about 15 hours or 24 hours. The higher the alkanol (e.g., methanol) concentration used in the method, the shorter the extraction time will typically be. In examples where a gradient of alkanol is used in the method, it is also understood that the total extraction time is divided as a function of the number of gradient steps in the procedure. The extraction at each gradient step can be the same amount of time or a different amount of time. One skilled in the art can experimentally determine the extraction time to be used.

[0172] Samples can be collected during the extraction process to monitor the removal of materials or to determine whether adjustments to extraction parameters, such as temperature or composition of the extraction solvent, are necessary.

[0173] In particular, the method can be used to purify P188. The process can be applied to other polymers as well. For example, in some embodiments, the method provided herein provides a method for preparing a purified polyoxypropylene / polyoxyethylene composition. The method includes: a) providing or introducing into an extraction vessel a solution of a polyoxypropylene / polyoxyethylene block copolymer dissolved in a first solvent to form a copolymer solution, the first solvent being methanol, ethanol, propanol, butanol, pentanol or a combination thereof, and the composition being: i) Formula HO(CH2CH2O) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) a a polyoxypropylene / polyoxyethylene block copolymer having H, the copolymer having a mean or average molecular weight of about 4,000 to about 10,000 Da; ii) a plurality of low molecular weight substances having a molecular weight of less than 4,500 Da, said plurality of low molecular weight substances constituting more than 4% of the total weight of the composition; Contains; b) adding a second solvent to form an extraction mixture, the second solvent comprising a supercritical liquid under high pressure and temperature and an alkanol that is methanol, ethanol, propanol, butanol, pentanol or combinations thereof, and increasing the concentration of the second solvent in the extraction mixture over the time of the extraction method; and c) separating the extraction mixture to form a plurality of phases including a raffinate phase and an extract phase, wherein the raffinate phase and the extract phase are separately removed or isolated. Includes.

[0174] In some of the above methods, the copolymer has a mean or average molecular weight of about 7,680-9,510 Da, such as generally 8,400-8,800 Da, such as about 8,400 Da or 8,400 Da. In the method, a copolymer solution can be formed in the extraction vessel by adding the copolymer and adding a first solvent to form a solution or suspension of the copolymer, the first solvent comprising an alkanol selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol, and combinations thereof. Alternatively, the first solvent can be added to the copolymer to form the copolymer solution in a separate vessel, and the copolymer solution dissolved in the first solvent is provided or introduced (i.e., charged) into the extraction vessel. Optionally, prior to step c), the method includes agitating 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.

[0175] b. High pressure method The method provided herein for purifying poloxamer (e.g., P188) can be a high-pressure fluid extraction method with a mixed solvent system. One of the solvents in the mixed system is a gaseous solvent that can be compressed to a liquid at moderate pressure, such as carbon dioxide. For example, the dissolving power of methanol or ethanol can be modified with high-pressure carbon dioxide (not necessarily supercritical carbon dioxide, i.e., subcritical) to obtain the exact solvating power required to selectively remove different fractions of poloxamer.

[0176] In such methods, the extraction solvent includes carbon dioxide provided under subcritical conditions, as well as another solvent that increases with time of extraction. Accordingly, certain embodiments of the methods provided herein provide an extraction method for removing impurities in a poloxamer formulation (e.g., low molecular weight components), the method comprising: a) providing or introducing into an extraction vessel a poloxamer dissolved in a first solvent to form a solution, the first solvent being selected from an alcohol, an aliphatic ketone, an aromatic ketone, an amine, and mixtures thereof; b) mixing an extraction solvent with the solution to form an extraction mixture, the extraction solvent comprising high pressure carbon dioxide and a solvent, and increasing the concentration of the solvent in the extraction solvent over the time course of the extraction; and c) removing the extraction solvent from the extraction vessel, thereby removing low molecular weight impurities from the poloxamer. Includes.

[0177] The first and second solvents can be the same or different. In the method, the step of dissolving the poloxamer solution in the first solvent can be performed before providing or introducing the solution into the extraction vessel or at the time the solution is provided or introduced into the extraction vessel. For example, the poloxamer is dissolved in a separate vessel and then the solution is added to the extraction vessel.

[0178] In an embodiment of the method, the extraction solvent is under subcritical conditions. In this process, one of the solvents is preferably a gas at (or near) room temperature, which can be compressed to a liquid at high pressure. Suitable gases that can be compressed to a gas include carbon dioxide, methane, ethylene glycol, ethylene glycol tetrahydrofuran ... Examples of suitable solvents include ethane, propane, ammonia, and the refrigerant sold as Freon®. Typical solvent pairs are selected such that one liquid is a solvent for the component to be removed by extraction and the other liquid is a non-solvent, or vice versa. The solvating power of the solvent pair is controlled primarily by the ratio of the solvents in the mixture. By passing the solvent pair through a product containing the substance, the relatively more soluble component can be extracted. The gaseous solvent can be pressurized at any suitable subcritical pressure. For example, carbon dioxide can be used at a pressure of about 25 bar 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 bar. In some embodiments, the pressure is about 60 to about 85 bar. In some embodiments, the pressure is about 75 bar.

[0179] Any suitable temperature may be used to carry out the extraction process. In some embodiments, the extraction vessel has a temperature of 10°C to 80°C. The temperature may 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 extraction vessel has a temperature of about 20°C to about 50°C. When purifying poloxamer 188, for example, the extraction vessel may have a temperature of about 20°C to about 60°C (e.g., about 40°C). Other temperatures may be suitable for purifying poloxamer 188 depending on the extraction equipment and extraction parameters selected. One of skill in the art will appreciate that the temperature may vary depending in part on the composition of the extraction solvent as well as the solubility of a given poloxamer in the solvent employed in the process.

[0180] Similar to the above-mentioned supercritical fluid extraction method, extraction can be carried out in an isocratic manner, where 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 is constant over the extraction time, for example by maintaining a constant flow rate of each. Alternatively, the composition of the extraction solvent can be changed over time, typically by changing (e.g., increasing or decreasing) the amount of carbon dioxide and / or other solvent (e.g., methanol) that constitutes the extraction solvent. Generally, carbon dioxide is kept constant, while the concentration of other solvent (e.g., methanol) in the extraction solvent is changed (e.g., increasing or decreasing) over the extraction time. The concentration of components can be changed by adjusting the flow rate. The specific concentration of the solvent, and the concentration gradient employed, can be similar to those described above for supercritical extraction. It is within the level of a person skilled in the art to appropriately adjust the concentration and extraction time to achieve the desired purity or yield.

[0181] Samples can be collected during the extraction process to monitor the removal of materials or to determine whether adjustments to extraction parameters, such as temperature or composition of the extraction solvent, are necessary.

[0182] In particular, the method can be used to purify P188. The process can be applied to other polymers as well. Benefits of mixed solvent systems include the effective removal of high molecular weight (HMW) and / or low molecular weight (LMW) materials using the mixed systems.

[0183] In one embodiment, provided is a method for preparing a purified polyoxypropylene / composition, the method comprising: a) providing or introducing a polyoxypropylene / polyoxyethylene block copolymer composition into an extraction vessel and dissolving it in a first solvent to form a copolymer solution, wherein the first solvent is an alcohol, an aliphatic ketone, an aromatic ketone, an amine, and mixtures thereof, and the composition comprises: i) a polyoxypropylene / polyoxyethylene block copolymer having a mean or average molecular weight of the copolymer of about 4,000 to about 10,000 Da; 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 than 4% of the total weight of the composition; b) adding a second solvent to form an extraction mixture, where the second solvent comprises high pressure carbon dioxide and the first solvent, and increasing the concentration of the first solvent in the extraction mixture over the extraction time period; and c) separating the extraction mixture to form multiple phases including a raffinate phase and an extract phase, and separately removing or isolating the raffinate phase and the extract phase.

[0184] If the poloxamer is purified poloxamer 188, the copolymer mean Or the average molecular weight is about 7,680 to 9,510 Da, for example, generally 8,40 0 to 8,800 Da, for example about 8,400 Da or 8,400 Da. In the method, a copolymer solution can be formed in the extraction vessel by adding the copolymer and by adding a first solvent to form a solution or suspension of the copolymer, the first solvent comprising an alkanol selected from the group consisting of methanol, ethanol, propanol, butanol, pentanol and combinations thereof. Alternatively, the addition of the first solvent to the copolymer to form the copolymer solution can be in a separate vessel, and the copolymer solution dissolved in the first solvent is provided or introduced (i.e., charged) into the extraction vessel. Optionally, prior to step c), the method includes agitating 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.

[0185] In some embodiments, this approach does not suffer from the density fluctuations and permeability characteristics of the supercritical fluid extraction process. Solvent recycling is easy and energy efficient. In a typical high pressure extraction, the exhaust stream containing the extracted components is subjected to low pressure, which causes phase separation and the more volatile solvent separates out as a gas, leaving the other solvent enriched with the extracted components. The extraction process continues until the mixture is substantially depleted of extractable components. The gaseous solvent can be compressed back to a liquid and available for continued extraction. This solvent recycling process is efficient because the compressible solvent is selected so that it is completely separated from the solvent mixture with minimal pressure changes.

[0186] 2. Extraction vessels and systems For any of the methods provided herein, system 200 in Figure 4 represents one embodiment for carrying out the provided methods. System 200 is one system that can be used to extract impurities (e.g., LMW materials and / or other components) from poloxamer using supercritical fluid or sub-supercritical methods. Polymer feed Pump 201 is loaded with the poloxamer (e.g., P188) to be purified. The poloxamer is transported through valve 205 to polymer feed tank 207. Extraction vessel 215 is used to remove extraction impurities from the sample, such as LMW material or other components from the poloxamer. Carbon dioxide (or other supercritical or near-supercritical fluid) pump 208 is charged with carbon dioxide from an external carbon dioxide supply 250 through valve 243 and precooler 203. Carbon dioxide is fed from pump 208 to heat exchanger 210 and then to extractor 215. Methanol (or other suitable solvent) is fed through pump 209 into extractor 215. In such an embodiment, the methanol and carbon dioxide extract impurities, such as LMW material or other components, from the poloxamer in extractor 215. After extraction, the purified poloxamer mixture is drained and collected by rapid depressurization. The extracted components are isolated from the solvent stream using a collector 225, a vacuum vessel 227, and a cyclone separator 231. Carbon dioxide vapor released during collection in collector 225 is condensed in condenser 23. 2 can be used, condensed, and recycled.

[0187] In some embodiments, the extraction equipment may include a solvent distribution system that includes particles of a certain shape that form a "fluidized" bed at the bottom of the extraction vessel. The bed may be supported by a screen or strainer or a sintered metal disk. The particles used in the bed may be either perfect spheres or irregularly shaped particles such as pebbles. The use of smooth surfaces with low porosity or low surface roughness is preferred for easy cleaning. These advantages may be demonstrated during pharmaceutical manufacturing processes.

[0188] The density of the particles forming the bed is selected to be higher than the solvent density, so that the bed remains unaffected by the inflowing solvent during the extraction process. The particle size can be uniform or have different size distributions to control the packing density and porosity of the bed. The packing distribution arrangement is designed to provide a balanced optimal extraction and subsequent coalescence of the solvent particles before leaving the extraction vessel. This facilitates maximum loading of the extractor loaded with poloxamer. This can also maximize the extraction efficiency, minimize the extraction time, and minimize the unwanted carryover of purified products from the extraction vessel.

[0189] The size of the spheres in the bed is selected based on one or more system characteristics 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 may range from about 5 mm to about 25 mm. The diameter may be the average diameter if the bed contains spheres of different sizes. Alternatively, the spheres in the bed may all have the same diameter. An example of a cross section of stainless steel spheres of different sizes in a solvent distribution bed is shown in FIG. 5.

[0190] Thus, an efficient solvent extraction apparatus is provided, the apparatus comprising: a) a distribution system at the bottom of the extractor, the distribution system including a plurality of spheres; and b) Particle coalescence system at the top of the extractor Equipped with.

[0191] In some embodiments, the plurality of spheres comprises metal spheres, ceramic spheres, or a mixture thereof. In some embodiments, the plurality of spheres are the same size. In some embodiments, the plurality of spheres comprises spheres of different sizes. In some embodiments, the particle coalescence system comprises one or more components selected from a demister pad, a stationary atomizer, and a temperature zone.

[0192] 3. Extraction and removal of extractant Any of the processes provided herein can be carried out as a batch or continuous process. In some embodiments, the process is a batch process. Batch processes can be carried out in extraction vessels of various dimensions and sizes, as described above. For example, an equipment train can be 120 L or 120 L. The extraction vessel may include a high-pressure extractor. A poloxamer (e.g., P188) solution, in which the poloxamer is dissolved in a suitable solvent (e.g., an alkanol solvent such as methanol), is provided or introduced into the extraction vessel. An extraction solvent, such as any of those described in the above method (e.g., supercritical or high-pressure carbon dioxide and methanol), is pumped independently and continuously into the extraction vessel, which is maintained at 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. After the substances are removed, the purified product is discharged into a cyclone separator suitably designed to separate the purified product from the carbon dioxide gas, as described below. The product is dried to remove residual alkanol solvent.

[0193] 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 a suitable 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 a countercurrent manner. Extraction materials such as LMW materials or other components are removed from the top of the column, while purified products are removed from the bottom of the column. The purified products are 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 then dried under vacuum.

[0194] 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 extraction solvent can be introduced into and removed from the extraction vessel until a sufficient level of poloxamer purity is obtained. Accordingly, certain embodiments of the methods provided herein provide an extraction method as described above, further comprising repeating steps b) and c) after step c). Steps b) and c) can be repeated until the poloxamer is sufficiently pure. For example, steps b) and c) can be repeated one, two, three, four, or five times, or in an iterative manner.

[0195] If the poloxamer material is sufficiently pure, the product is prepared for further processing. In some embodiments, the product is processed according to process 100 as outlined in FIG. 1. The product can be discharged from the extraction vessel and collected in a suitable receiver, as shown in step 145. The wet product can be sampled for testing for purity, chemical stability, or other properties, as shown in step 150. The product can be dried by removing residual solvent under vacuum. The vacuum level can be adjusted to control the drying rate. Drying can be carried out at ambient temperature, or at elevated temperature, if necessary. Generally, the drying temperature is kept below the melting point of the poloxamer. The wet product can be dried in a single lot, or in smaller quantities as sublots. Drying of the product can begin at ambient temperature under vacuum, for example, in sublots, as shown in steps 160-170. Drying can then be continued at higher temperatures and lower pressures as the process progresses. If necessary, for example, if collected in sublots, 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 various sublots combined, may be mixed in a suitable container, as shown in step 180, and the resulting product may be characterized, stored, shipped, or compounded.

[0196] Advantageously, the methods disclosed herein efficiently recycle carbon dioxide. In particular, supercritical or high pressure carbon dioxide can be recovered by subjecting the extract phase to temperature and pressure changes. In an embodiment, the methods employed herein have a recycle efficiency of greater than 80%, preferably greater than 90%, and most preferably greater than 95%.

[0197] In the methods provided herein (see, e.g., steps a)-c) above), the extract phase can be further processed. The methods can further include: passing the extract phase through a system of multiple separation vessels; isolating impurities (e.g., low molecular weight impurities); treating the purified material or raffinate; and recovering the compressed carbon dioxide for reuse.

[0198] In any of the methods provided herein, various parameters can be evaluated in screening the method and the resulting products. For example, parameters such as methanol concentration, gradient profile, temperature, and pressure can be evaluated for process optimization. The process and suitable conditions for drying the wet raffinate, such as vacuum level, mixing mode, time, and temperature, can also be evaluated.

[0199] 4. Exemplary Methods for Preparation of Purified Poloxamers The methods provided herein result in the production of certain purified poloxamer preparations, and in particular LCMFP188 preparations. In particular, the methods provided herein produce the P188 copolymer described herein, which has the formula: HO(CH2CH2O) a’ -(CH2CH(CH3)O) b -(CH2CH2O) a H, and a mean or average molecular weight of the copolymer of 7,680 to 9,510 Da, e.g., generally 8,400 to 8,800 Da, e.g., about 8,400 Da or 8,400 Da, and comprising 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 than 4% of the total weight of the composition.

[0200] In some embodiments, the methods of the invention produce purified poloxamers with less than about 4% low molecular weight components, e.g., less than about 3%, less than about 2%, or less than about 1% low molecular weight components. Typically, low molecular weight components include glycols and volatile degradation impurities, e.g., formaldehyde, acetaldehyde, propionaldehyde, acetone, methanol, and peroxides. In some examples, the processes herein produce poloxamers that are substantially free of low molecular weight components, i.e., less than 4%, less than 3%, less than 2%, or less than 1% of said components. The methods can also produce poloxamers that are substantially free of long-circulating substances, such that no component in the poloxamer is or gives rise to a substance that has a long half-life in blood or plasma that is greater than 5.0 times, e.g., generally 4.0 times or less, 3.0 times or less, 2.0 times or less, or 1.5 times or less than the half-life of the major component in the poloxamer distribution, when administered to a subject. The following discussion details examples of methods for producing such purified poloxamers.

[0201] a. Removal of low molecular weight (LMW) components 2 illustrates one embodiment of the methods herein that provide a process 100' useful for removing LMW material in poloxamers. The extraction system is pressurized as shown in step 105' prior to injecting 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 above the critical temperature of the carbon dioxide used in the process, i.e., about 31° C. Typically, the temperature is below 40° C. The temperature is generally kept constant throughout the process.

[0202] A first alkanol (e.g., methanol) is used to form a poloxamer solution according to step 115' in process 100'. In this process, P188 poloxamer is dispensed into a feed tank along with an alkanol (e.g., methanol), resulting in a P188 poloxamer solution dissolved in the alkanol (e.g., methanol). The amount of poloxamer for use in the process can be any amount, such as those amounts previously described herein. After the poloxamer / alkanol mixture is formed, all or a portion of the mixture is pumped into an 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.

[0203] As shown in step 125' of process 100', the extractor is then pressurized and an extraction solvent is introduced into the extractor. The extraction solvent typically comprises carbon dioxide, and the extraction is carried out at a temperature above the critical temperature of 31° C. and under high pressure above the critical pressure of 74 bar, as described above. For example, in an exemplary method, the extraction vessel is pressurized to about 310±15 bar, and carbon dioxide is provided at a flow rate of 20 kg / h to 50 kg / h, for example, generally about or approximately 24 kg / h (i.e., 390 g / min).

[0204] The extraction is then carried out in the presence of a second alkanol which acts as a co-solvent modifier for the carbon dioxide. The extraction process is carried out at 100° C. for 1 hour. A second alkanol, such as methanol, is added in a gradient step manner such that the concentration of the second alkanol in the extraction solvent increases over the time of the 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 of a poloxamer (e.g., P188) begins with about 5% to 7% by weight (w / w) of an alkanol (e.g., methanol) (e.g., about 6.6%) in an extraction solvent with a supercritical fluid (e.g., carbon dioxide). After a period of time, the alkanol (e.g., methanol) content of the extraction solvent is increased to about 1-3%, such as 1% (e.g., to 7.6%). The alkanol (e.g., methanol) content is then again increased to about 1-3%, such as 1% (e.g., to 8.6%) during a final period of time. The total time of the extraction method can be 15 hours to 25 hours. Each gradient is carried out for a portion of the total time.

[0205] For a commercially effective purification process, it is desirable to continuously increase the methanol concentration, with the profile suitably modified to selectively remove most of the low molecular weight components. The remaining low molecular weight components can then be removed with a higher methanol concentration for a shorter period of time. Thus, a stepwise methanol concentration profile using about 5%-10% (e.g., 6.6%) methanol for 12 hours, then higher methanol for 10 hours, and finally higher methanol for 4 hours produces a high yield of purified product without significantly reducing the overall yield and without concentrating the high molecular weight components.

[0206] If 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 extraction vessel and collected in a suitable receiver as shown in step 145'. The wet product can be sampled for testing for purity, chemical stability, or other properties as shown in step 150'. The product can be dried by removing residual solvent under vacuum as described herein. In an exemplary method, drying can begin on a sublot under vacuum at ambient temperature as shown in steps 160'-170', and drying can continue at higher temperatures and lower pressures as the process progresses. The remaining sublots can be similarly processed as shown in step 175' of process 100'. The sublots can be combined and mixed in a suitable container as shown in step 180', and the resulting product can be characterized, stored, shipped, or formulated.

[0207] b. Preparation of long-circulating substance-free (LCMF) poloxamer FIG. 3 illustrates an embodiment for preparing LCMF poloxamer. Certain embodiments of the methods herein provide a process 100" for producing a poloxamer that does not contain any components that result in long-term circulating substances in plasma or blood as described herein after administration to a subject. As shown in step 105", a poloxamer and a first alkanol (e.g., methanol) are dispensed into an extraction vessel and a poloxamer solution is formed. In this process, P188 poloxamer is dispensed into the extraction vessel with an alkanol (e.g., methanol), resulting in a P188 poloxamer solution 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, a poloxamer solution can be formed in a separate vessel and the poloxamer solution can be transferred to the extraction vessel.

[0208] After the first alkanol (e.g., methanol) and poloxamer are dispensed, as shown in step 110", the extraction system is pressurized. 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 the carbon dioxide used in the process, i.e., about 31°C. Typically, the temperature is between 35°C and 45°C. The temperature is generally kept constant throughout the process. A poloxamer solution forms under about 49 bar of pressurized carbon dioxide at a temperature between 35°C and about 45°C or between 45°C for a predetermined period of time, generally less than a few hours.

[0209] As shown in step 120" of process 100", the extractor is then pressurized and an extraction solvent is introduced into the extractor. The extraction solvent typically comprises carbon dioxide and a second alkanol, and the extraction is carried out at a temperature above the critical temperature of 31°C, as described above, and under high pressure, i.e., above the critical pressure of 74 bar. For example, in an exemplary method, the extraction vessel is pressurized to about 247±15 bar (range 240-260 bar) and carbon dioxide is provided at a flow rate of 50 kg / h to 120 kg / h (inclusive), e.g., generally about or approximately 100 kg / h.

[0210] The extraction is carried out in the presence of a second alkanol, which acts as a co-solvent modifier for the carbon dioxide. As shown in steps 125"-135", a second alkanol, such as methanol, is added in a gradient step manner such that the concentration of the second alkanol in the extraction solvent increases over the time of the extraction process. 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) begins with about 7% to 8% by weight (e.g., about 7.4% or 7.4% by weight) of an alkanol (e.g., methanol) in an extraction solvent with a supercritical fluid (e.g., carbon dioxide). After a period of time, the alkanol (e.g., methanol) content of the extraction solvent is increased to about 1-3%, such as 2% (e.g., to 9.1%). The alkanol (e.g., methanol) content is again subsequently increased to about 1-3%, such as 2% (e.g., to 10.7%) for a final period of time. The total duration of the extraction method can be 15 hours to 25 hours, inclusive. Each gradient is carried out for a portion of the total time.

[0211] For extraction processes that remove components other than low molecular weight components, including those that would result in long-circulating forms when administered, it is desirable to have a process that maximizes the purity and removal of these components while minimizing the loss of yield. Starting with a higher concentration of alkanol (e.g., methanol) than other methods, it has been found that successive increases in the alkanol (e.g., methanol) concentration, generally starting at 7%-8% by weight, suitably modify the profile to selectively remove these and low molecular weight components while minimizing the loss of yield. For example, such exemplary methods can result in yields of greater than 55%, generally greater than 60% or 65%. Residual low molecular weight components can be subsequently removed with higher methanol concentrations for a shorter period of time. Thus, a stepwise methanol concentration profile using about 7-8% (e.g., 7.4%) methanol for about 3 hours, higher methanol (e.g., 9.1%) for about 4 hours, and finally higher methanol (e.g., 10.7%) for about 8 hours, provides a high yield of purified product without a significant loss of overall yield.

[0212] If 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 extraction vessel and collected in a suitable receiver as shown in step 140". The product can be precipitated under reduced pressure by particles from gas saturated solutions (PGSS) techniques as shown in step 145". The product can be dried by removing residual solvent under vacuum as described herein. In an exemplary method as shown in steps 150"-165", drying can begin at an elevated temperature of 35°C-45°C under vacuum. The dried product can be collected as shown in step 160". The resulting product can be characterized, stored, shipped, or formulated as shown in step 165".

[0213] 5. How to confirm the identity of LCMF poloxamer Evaluate the properties of the poloxamer to confirm that poloxamer 188 formulations made by the methods herein or otherwise are LCMF poloxamer 188. The properties include, but are not limited to, the absence of long-circulating substances when administered to humans or animal models, the behavior of the poloxamer in reversed-phase (RP)-HPLC compared to formulations of poloxamers containing LCM substances such as poloxamers described in U.S. Patent No. 5,696,298 and commercially available poloxamer 188 (e.g., sold under the trademarks Pluronic® F-68, Flocor®, Kolliphor®, and Lutrol®), and the behavior in RP-HPLC under conditions exemplified herein (i.e., see Example 7). Any method can be used to confirm that the formulation is devoid of LCM substances.

[0214] E. Pharmaceutical Compositions and Formulations Compositions are provided that include poloxamer P188, such as any prepared by the methods provided herein. In particular, compositions are provided that include LCMF poloxamers, particularly LCMF poloxamer P188, that are used to treat or in methods of treating any disease or condition that P188 is known to treat or can treat, such as any of those described in Section F.

[0215] 1. Formulation Pharmaceutical compositions containing P188, such as LCMFP188, 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. The selection of the formulation, carrier and / or excipient is within the skill of the practitioner and can depend on many parameters. These include, for example, the mode of administration (i.e., systemic, oral, nasal, pulmonary, local, topical, or any other mode) and the condition, disorder, or disease to be treated.

[0216] An effective concentration of P188, e.g., LCMFP188, is administered systemically, topically or locally. For local administration, the copolymer is mixed with a suitable pharmaceutical carrier or vehicle. Suitable pharmaceutical carriers or vehicles for administration include any carrier known to those skilled in the art to be suitable for a particular mode of administration. A pharmaceutical composition containing a therapeutically effective amount of P188, e.g., LCMFP188, can also be provided as a lyophilized powder that is reconstituted with sterile water or the like immediately before administration.

[0217] The compound can be suspended in micronized or other suitable form, or derivatized to produce a more soluble active product. The form of the resulting mixture depends on many factors, including the intended mode of administration and the solubility of P188, such as LCMFP188, in the selected carrier or vehicle. The resulting mixture can be a solution, suspension, emulsion, and other such mixtures, and can be formulated as a non-aqueous or aqueous mixture, cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, irrigation, spray, suppository, bandage, or any other formulation suitable for systemic, topical, or local administration. For local internal administration, such as intramuscular, parenteral, or intraarticular administration, the poloxamer can be formulated as a solution suspension in an aqueous medium, such as isotonic buffered saline, or can be combined with a biocompatible support or bioadhesive intended for internal administration.

[0218] Generally, pharma- ceutically acceptable compositions are prepared in consideration of approval from a regulatory agency or in accordance with generally accepted pharmacopoeial standards for animal and human use. Pharmaceutical compositions can include a carrier, such as a diluent, adjuvant, excipient, or vehicle, with which the isoform is administered. Such pharmaceutical carriers can be sterile liquids, such as water and those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, and the like. and oils, including sesame oil. Water is a typical carrier when pharmaceutical compositions are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. The composition can contain, together with the active ingredient: diluents, such as lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose; lubricants, such as magnesium stearate, calcium stearate, and talc; and binders, such as starch, natural gums, such as acacia gum, gelatin, glucose, molasses, polyvinylpyrrolidone, cellulose and its derivatives, povidone, crospovidone, and other such binders known to those skilled 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, skimmed milk powder, glycerol, propylene, glycol, water, and ethanol. The compositions may also optionally contain minor amounts of wetting or emulsifying agents, or pH buffering agents, such as acetates, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, and other such agents. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, and sustained release formulations. Capsules and cartridges (e.g., gelatin) for use in an inhaler or insufflator can be formulated containing a powder mix of the therapeutic compound and a suitable powder base, such as lactose or starch. Such compositions contain a therapeutically effective amount of P188, in the form described herein, including LCMF form, together with a suitable amount of carrier to provide the form for proper administration to the subject or patient.

[0219] The formulation is selected to suit the mode of administration. For example, a composition containing P188, such as LCMFP188, can be formulated for parenteral administration by injection (e.g., bolus injection or continuous infusion). Injectable compositions can take the form of suspension, solution or emulsion in oily or aqueous vehicles. Buffers, preservatives, antioxidants, and suitable ingredients can be incorporated as necessary or can constitute the formulation.

[0220] Formulations suitable for parenteral administration include, but are not limited to, aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation compatible with the intended route of administration. The formulations can be presented in unit-dose or multi-dose containers, e.g., sealed ampoules and vials, pre-filled syringes or other delivery devices, and can be stored in aqueous solution, in a dry or freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, e.g., water for injections, immediately prior to use.

[0221] P188, such as LCMFP188, can be formulated as the sole pharmacoactive ingredient in the composition or can be combined with other active ingredients. Liposomal suspensions, including tissue-targeted liposomes, can further be suitable as pharmacologic acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, liposomal formulations can be prepared as described in U.S. Pat. No. 4,522,811. Liposomal delivery can also include sustained release formulations that include pharmaceutical matrices such as collagen gels and fibronectin-modified liposomes (see, e.g., Weiner et al. (1985) J. Pharm. Sci. 74(9):922-925). The compositions provided herein can further include one or more adjuvants that facilitate delivery, e.g., The carrier may further include, for example, but not limited to, an inert carrier or a colloidal dispersion system. Representative and non-limiting examples of such inert carriers may be selected from water, isopropyl alcohol, gaseous fluorocarbons, ethyl alcohol, polyvinylpyrrolidone, propylene glycol, gel-producing substances, stearyl alcohol, stearic acid, spermaceti, sorbitan monooleate, and methylcellulose, and suitable combinations of two or more thereof.

[0222] P188, such as LCMF P188, is included in a pharma- ceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect without undesirable side effects on the subject being treated. Therapeutically effective concentrations can be empirically determined by testing the compounds in known in vitro and in vivo systems, such as the assays provided herein.

[0223] 2. Dosage Pharmaceutical compositions containing P188, such as LCMF P188 provided herein, can be formulated for single dose (direct) administration, multiple dose administration, or for dilution or other modification. The concentration of the compound in the formulation is effective to deliver an effective amount for the intended treatment when administered. Those skilled in the art can formulate the composition for administration according to the methods herein. For example, to formulate the composition, the weight fraction of the compound or mixture is dissolved, suspended, dispersed, or otherwise mixed in the selected vehicle at an effective concentration such that the intended effect is observed.

[0224] The exact amount or dose of therapeutic agent to be administered will depend on the condition being treated, the route of administration, and other considerations such as the weight and physiological condition of the subject and the subject.

[0225] If necessary, specific dosage and duration and treatment protocol can be empirically determined or estimated.For example, the exemplary dose of P188, such as LCMFP188, provided herein can be used as a starting point to determine the dosage suitable for a specific subject and condition, if necessary.The duration of treatment and the interval between injections vary with the severity of disease or condition and the subject's response to treatment, and can be adjusted accordingly.Factors such as the activity level and half-life of P188, such as LCMFP188, can be taken into consideration when determining dosage.Specific dosage and regimen can be empirically determined by those skilled in the art.

[0226] In particular, poloxamers can be used at concentrations ranging from about 10.0 mg / mL to about 300.0 mg / mL, such as (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. It can be formulated for administration at 0, 155.0, 160.0, 165.0, 170.0, 175.0, 180.0, 185.0, 190.0, 195.0, 200.0, 205.0, 210.0, 215.0, 220.0, 225.0, 230.0, 235.0, 240.0, 245.0, 250.0, 255.0, 260.0, 265.0, 270.0, 275.0, 280.0, 285.0, 290.0, 295.0 or 300.0 mg / mL. Typically, the concentration is 22.5% or less, i.e., 225 mg / mL.

[0227] For example, when administered separately or as a component of a pharmaceutical composition described herein, the poloxamer is generally administered at a concentration of about 0.5% to 25.0%, e.g., 0.5% to 20% or 25%, although lesser or higher concentrations can be used. For example, the poloxamer is administered at a concentration of about 0.5% to about 25% weight / volume, e.g., at least 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%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 8 Poloxamer can be administered at a concentration of about 0.5% to about 10% by weight / volume. In yet another embodiment, the poloxamer is administered at a concentration of 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. In other embodiments, the poloxamer is administered at a concentration of 16-25%, such as 22.5% weight / volume, for example, the concentration is 10%-22.5%, such as 10%-20% or 15%-20%.

[0228] 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 100 mL contains 15 g (150 mg / mL) of purified poloxamer 188, e.g., LCMFP188, 308 mg of sodium chloride USP, 238 mg of sodium citrate USP, 36.6 mg of citric acid USP, and water for injection USP, Qs to 100 mL. The pH of the solution is about 6.0 and has an osmolality of about 312 mOsm / L. For other applications, at least 500 ml is prepared at a concentration of 10%-20%, e.g., about 15% by weight or 15% by volume of the poloxamer formulation / composition. For example, for intravenous administration, the target C is 100 mg / kg per hour, followed by a maintenance infusion of 30 mg / kg / hr for 48 hours, when the composition is infused. ss The composition is formulated to achieve the following: A skilled physician or pharmacist or other person skilled in the art can select the appropriate concentration for the particular subject, the condition being treated, and the target blood concentration.

[0229] 3. Dosage and Administration In the methods herein, poloxamer 188, such as LCMFP188 described herein, can be administered to a subject to treat a disease or condition, including any of the diseases or conditions described in Section F. In particular, poloxamer 188, such as the purified poloxamer 188 described herein, is intended for use in therapeutic methods for which other P188 compositions can or have been used for treatment.

[0230] Treatment of diseases and conditions, such as any of those described in Section F with respect to poloxamer 188, such as the purified poloxamer 188 described herein, can include, but is not limited to, intravenous and intra-arterial (catheter directed administration or other routes). Administration may be by any suitable route using suitable formulations as described herein, including parenteral administration, including intravenous, intravenous, pulmonary, oral or transdermal administration. Treatment is typically administered intravenously.

[0231] The active agent, for example, poloxamer 188, such as LCMF P188, is included in an amount sufficient to exert a therapeutically useful effect on the treated patient in the absence of undesirable side effects. The amount of P188, such as LCMFP188, to be administered for any disease or condition can be determined by standard clinical techniques. In addition, in vitro assays and animal models can be used to help identify optimal dosage ranges. The exact dosage, which can be determined empirically, can depend on the specific composition, route of administration, desired administration period, type of disease being treated, and severity of disease.

[0232] A practical limitation is that renal insufficiency in a subset of patients enrolled in early-stage clinical trials has led to the inclusion of 1,000 mg of cyclosporine in the National Drug Specification (P188-NF) (Emanuele and Balasubramanian, Drugs RD 14 (2):73-83 (2014)) In addition, animal studies reveal that P188-NF increases serum creatinine levels, and creatinine is not efficiently cleared by the kidney at the end of the drug infusion. The purified poloxamer 188 described herein has been modified to address the limitations of P188-NF. To prevent elevated creatinine levels and renal toxicity, poloxamer 188 was purified to remove low and high molecular weight species contaminants. In clinical trials, such as the C97-1248 study, researchers found that intravenous administration of P188-P did not result in a significant increase in serum creatinine above placebo levels. The reduction in low and high molecular weight species, based on assessment by high performance liquid chromatography, reduces or eliminates the renal risks associated with non-purified (P188-NF) treatment. Thus, purified poloxamer 188, such as the LCMF poloxamer 188 described herein, does not exhibit the practical limitations present in the non-purified forms previously evaluated.

[0233] If necessary, the specific dosage and duration and treatment protocol can be empirically determined or estimated. The dosage of poloxamer 188 previously administered to human subjects and used in clinical trials can be used as a guide to determine the dosage of poloxamer 188, such as the purified poloxamer 188 described herein. The dosage of poloxamer 188 can also be determined or estimated from relevant animal studies. Factors such as the activity level and half-life of poloxamer 188 can be used in such determination. The specific dosage and regimen can be empirically determined based on various factors. Such factors include the individual's weight, general health, age, activity of the specific compound employed, sex, diet, time of administration, rate of excretion, drug combination, severity and course of the disease, and the patient's disposition to the disease, and the judgment of the treating physician. The active ingredient poloxamer 188 is typically combined with a pharmacologic effective carrier. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form or multiple dosage forms can vary depending on the host treated and the specific mode of administration.

[0234] In certain examples, poloxamers such as P188 (e.g., LCMFP188) are formulated for administration at a dosage of about 100 mg / kg or between 100 mg / kg and 2000 mg / kg, depending on the condition being treated. Doses may range from, for example, 100-500 mg / kg of patient weight, e.g., 100 mg / kg-450 mg / kg, 100-400 mg / kg, 100 mg / kg-300 mg / kg, 100 mg / kg-200 mg / kg, 200 mg / kg-500 mg / kg, 200 mg / kg-450 mg / kg, 200 mg / kg-400 mg / kg, 200 mg / kg-300 mg / kg, 300 mg / kg-500 mg / kg, 300 mg / kg-450 mg / kg, 300 mg / kg-400 mg / kg, 300 mg / kg-500 mg / kg, 300 mg / kg-45 ... 400 mg / kg, 400 mg / kg to 500 mg / kg, 400 mg / kg to 450 mg / kg or 450 mg / kg to 500 mg / kg body weight of the patient, such as at least or at least about 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 and 2000 mg / kg body weight of the patient.

[0235] Doses of poloxamer are administered in concentrations and fluid volumes consistent with the mode of administration and the physiological needs of the patient. In general, for chronic infusions (e.g., 12, 24, or 48 hour continuous infusions), the volume administered is typically about 5.0 mL / kg / hr or less, e.g., 4.5 ml / kg / hr, 4.0 ml / kg / hr, 3.5 ml / kg / hr, 3.0 ml / kg / hr, 2.5 ml / kg / hr, 2.0 ml / kg / hr, 1.5 ml / kg / hr, 1.0 ml / kg / hr, 0.5 ml / kg / hr, 0.25 ml / kg / hr, or 0.125 ml / kg / hr. For short-term administration (e.g., bolus administration or short-term infusion), the dose of poloxamer can be administered in volumes greater than 5.0 ml / kg / hr, e.g., 7.5 ml / kg / hr or 10.0 ml / kg / hr or 12.5 ml / kg / hr or 15 ml / kg / hr or even higher, depending on the needs of the patient. The poloxamer can be administered as a single dose or hourly, daily, weekly, monthly, or intravenously. The administration can be in multiple doses repeated at various intervals, such as 1 hour to 72 hours or longer. With regard to infusions, the infusion can provide a suitable dosage to a subject over a period that is typically between 1 hour and 72 hours, for example 12 hours, 24 hours or 48 hours.

[0236] The formulations used in the methods provided herein can be administered by any suitable route, for example, orally, nasally, pulmonary, parenterally, intravenously, intradermally, subcutaneously, intraarticularly, intracisternally, intraocularly, intracerebroventricularly, intrathecally, intramuscularly, intraperitoneally, intratracheally or topically, and any combination of any two or more thereof, in liquid, semi-liquid or solid form, and are formulated in a manner suitable for each administration route. Multiple administrations, such as repeated administrations as described herein, can be carried out by any route or combination of routes. The most suitable administration route depends on the condition to be treated. Typically, the composition is formulated for intravenous infusion.

[0237] An effective amount of a poloxamer, such as P188, particularly LCMF P188 provided herein, can be delivered alone or in combination with other agents for treating a disease or condition. Those skilled in the art can select additional treatments to administer in combination with a treatment regimen employing LCMFP188. Such a decision will depend on the particular disease or condition being treated, the particular subject being treated, the age of the subject, the severity of the disease or condition, and other factors.

[0238] F. Methods and Therapeutic Uses of Poloxamer 188 and LCMFP188 Poloxamer 188 (P188) and compositions thereof, such as LCMFP188 and compositions thereof, can be used in a variety of applications, including cytoprotective, hemorheological, anti-inflammatory, and antithrombotic / profibrinolytic applications, with clinical utility in a variety of diseases, including, but not limited to, acute myocardial infarction, acute limb ischemia, shock, acute stroke, heart failure, sickle cell disease, and neurodegenerative diseases. Any P188 provided herein, such as LCMFP188 or any P188 produced by the methods provided herein, can be used to treat any disease or condition for which P188 has previously been used or is known to be effective. Some applications of P188 are reviewed, for example, in Moloughney et al., (2012) Recent Pat Biotechnol. 6(3):200-211 and Karmarker, "Poloxamers and their applications" Pharmainfo.net Published Oct. 27, 2008. and the URL is: pharmainfo.net / pharma-student-magazine / poloxamers-and-their-applications-0. Examples of uses of P188 include, but are not limited to, use in applications requiring membrane resealing and repair, treating tissue ischemia and reperfusion injury, reducing inflammatory responses, reducing blood viscosity, promoting thrombolysis, promoting hemostasis, use as a vehicle for drugs, delivery of nucleic acids or proteins, use as an emulsifier, use as a suspension stabilizer for hydrophobic drugs, cleansing skin wounds, use as a surfactant in cosmetic formulations, use to adjust the viscosity of personal care products and soaps, and pharmaceutical use as a laxative (see, e.g., European Publication No. EP0682946).

[0239] P188, such as LCMFP188, can be used in applications requiring membrane resealing, stability, and / or repair. Such use prevents cell loss in tissues, such as damaged tissues, by protecting cells from apoptosis and necrosis. For example, P188 can be used to repair cell membranes after electrical or free radical damage (see, e.g., U.S. Pat. No. 5,605,687, U.S. Pat. Publication No. 2006 / 0121016, and Lee et al., (1992) Proc. Natl. Acad. Sci. USA 89:4524-4528). P188 can also be used to reduce cell death after cartilage injury, such as after external joint injury (see, e.g., Isaac et al., (2010) J Orthop Res. (4):553-558), for example, to help prevent osteoarthritis. P188 has also been used for neuroprotection in response to excitotoxicity, e.g., glutamate toxicity (Frim et al., (2004) Neuro Reports. 15: 171-174). P188 has also been shown to inhibit misfolded proteins. By repairing membranes damaged by plaque, it is possible to treat degenerative diseases such as Alzheimer's disease. It has been used as a therapeutic agent (see, for example, US Patent Publication No. 20100316590).

[0240] P188 has also been used to treat and prevent cardiomyopathy and heart disease associated with decreased expression of dystrophin (see, e.g., U.S. Patent Nos. 7,846,426 and 8,580,245), and to treat chronic heart failure due to mechanisms other than dystrophin loss (see, e.g., U.S. Patent Publication No. 2009 / 0246162). Compositions containing P188 can further be used to inhibit thrombosis, reduce myocardial infarct size, reduce blood viscosity, and improve perfusion of damaged tissues after myocardial infarction (Justicz et al., (1991) Am Heart J. 122(3 Pt 1):671-680; O'Keefe et al., (1996) Am. J. Cardiol. 78:747-750).

[0241] P188 can also be used in cell transplantation methods to minimize damage to cell membrane of transplanted cells during testing.For example, compositions comprising P188 can be used to improve the survival of adipocytes during transplantation of adipose tissue, adipocytes, stem cells and other cells derived from adipose tissue (see, for example, U.S. Patent No. 8,512,695 and U.S. Patent Publication No. 2010 / 0104542), such as for soft tissue reconstruction or augmentation applications.P188 has also been used to improve the survival and neural regeneration of transplanted dopaminergic cells for Parkinson's disease therapy (Quinn et al., (2008) Eur J Neurosci. 27(1):43-52).

[0242] P188 can also be used as a therapeutic agent to treat chronic microvascular diseases, including but not limited to macular degeneration, diabetic retinopathy and congestive heart failure (see, e.g., U.S. Patent Publication No. US2011 / 0212047). The use of P188 for the treatment of tissue ischemia and reperfusion injury, such as in a model of superior mesenteric artery occlusion (SMAO), has also been described (see, e.g., Hunter et al., (2010) Ann Clin Lab Sci. 40(2): 115-125). Compositions containing P188 have also been used to treat skeletal muscle disorders, such as Duchenne muscular dystrophy (DMD) and related disorders (see, e.g., U.S. Patent Publication No. 2011 / 0033412).

[0243] P188 has also been used to enhance blood flow, for example by reducing blood viscosity, for example by preventing adhesive interactions in blood (see, for example, US Publication No. 2010 / 0183519). Such use can reduce problems associated with reduced blood flow, such as the occurrence of fat emboli (see, for example, Adams et al., (1960) Surg. Forum 10:585 and Danielson et al., (1970) J Thorac Cardiovasc Surg. 59(2):178-184), and erythrocyte sedimentation (Hoppensteadt et al., (2014) FASEB J.28(1):suppl. 1139.6). P188 has also been used to treat hemorrhagic shock (Mayer et al., (1994) Ann Clin Lab Sci. 24(4):302-311).

[0244] P188 has also been used to treat sickle cell disease (SD), which is homozygous sickle cell anemia (SS) as well as mixed heterozygous states such as SC, SD, and S-β thalassemia (see, e.g., Adams-Graves et al., (1997) Blood 90:2041-2046; Ballas et al., Hemoglobin 2004, 28(2):85-102; Gibbs and Hagemann, (2004) Ann. Pharmacother. 38:320-324; Orringer JAMA. 2001;286(17):2099-2106).

[0245] Inflammatory responses can further be reduced by use of P188, e.g., by inhibiting phagocyte migration and, e.g., by reducing neutrophil influx and adhesion (see, e.g., Lane et al., (1984) Blood. 64:400-405; Schaer et al., (1994) Circulation. 90(6):2964-2975).

[0246] P188 may also be used in wound healing and sealing therapies (e.g. , U.S. Provisional Patent Application No. 62 / 021,676, U.S. Patent No. 8,758,738, and U.S. Patent Publication No. 20140056839).

[0247] Further uses of P188 include as a tissue culture medium additive, for example as a supplement for cryostorage media (see, e.g., Kerleta et al., (2010) ALTEX. 27(3):191-197), and as an additive to blood and blood products, such as packed red blood cells, to prevent or reduce stored damaged, compromised blood.

[0248] P188 has also been used in various cosmetic and pharmaceutical formulations. P188 has been used in pharmaceutical compositions (see, e.g., U.S. Patent Publication Nos. 20040258718, 20090214685, 20100087501, 20100249240, 20110008266, 20120277199, and U.S. Patent Nos. 8,133,918, 8,460,644, and 8,709,385). Nos. 20030206910 and 20060013883, antimicrobial agents (see, e.g., U.S. Patent Publication Nos. 20060078616), and proteins (see, e.g., U.S. Patent Publication Nos. 20100310669 and 2012 / 0141619, U.S. Patent No. 8,137,677, and Jeong B., et al., (2002) Adv Drug Del Rev, 54(1); 37-51) Can be used to increase P188 is useful for drug delivery, such as ophthalmic delivery (Qi et al. (2007) Int. J. Pharm. 337:178-187), mucoadhesive delivery (Chang et al., (2002) J. Controlled Rel. 82:39-50), and rectal delivery (Choi et al. (1998)). Int. J. Pharm. 165:23-32; Yong et al, (2006) Int. J. Pharm. 321:56-61; ElHady et al., (2003) Saudi Pharmaceutical Journal. 11:159-171; Yong et al., (2004) Eur. J. Pharm. Sci. 23:347-353; Yun et al, (1999) Int. J. Pharm. 189:137-145; and Paek et al., (2006) Biological & Pharmaceutical Bulletin. 29:1060-1063), and as a pharmaceutical carrier for transdermal delivery (Cappel et al., (1991) Int. J. Pharm. 69:155-167). P188 can further be used as an emulsifier, suspension stabilizer, and solubilizer for hydrophobic drugs in liquid oral, parenteral, and topical dosage forms. In solid dosage forms, P188 can be used as a wetting agent, plasticizer, or tablet lubricant, and has broad application in the formulation of gels due to its thermoreversible gelation behavior (e.g., Desai et al, (2007) Drug Deliv. 14(7):413-426 and Muzikova et al., (2013) Acta Pol Pharm. 70(6):1087-1096). EXAMPLES

[0249] G. Working Example

[0250] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention herein.

[0251] Example 1 Continuous purification of poloxamer 188 by extraction with methanol / supercritical CO2 co-solvent Continuous purification of poloxamer 188 by extraction with methanol / supercritical CO2 co-solvent was evaluated. Continuous processing allows for high throughput. A feed solution of poloxamer 188 (Asahi Denka Kogyo, Japan) in methanol was pumped to the midpoint of a high-pressure extraction column packed with a suitable packing material. Supercritical CO2 (Carboxyque, France) mixed with methanol was pumped through the extraction column from the bottom in a countercurrent manner (flow rate = 30 kg / h-40 kg / h). The average concentration of methanol was 13% and was fed as a gradient from 9 wt% to 13.2 wt%. The gradient was adjusted by adjusting the methanol, CO2 and poloxamer flow rates at the inlet in the middle of the column and the CO2 / methanol flow rate introduced at the bottom of the column. The column pressure was 200 ± 15 bar. The feed solution and The temperature of the column jacket and the extraction temperature were gradients from 36° C. to 54° C.

[0252] The purified product, containing methanol, was removed from the bottom of the extraction column while low molecular weight (LMW) polymers were removed at the top of the column. The purified product was collected hourly and precipitated under reduced pressure by Particles from Gas Saturated Solution (PGSS) technique. The purified product was dried under vacuum below 40°C to remove residual methanol.

[0253] The approximate yield of purified poloxamer per feed was approximately 60%. The peak average molecular weight was approximately 9,000 Daltons. The low molecular weight component (less than 4,500 Daltons) was approximately 1.0%. The polydispersity was approximately 1.0.

[0254] Example 2 Estimation of equilibrium concentration of methanol and its effect on product emissions. To minimize handling of purified poloxamer 188 in an open environment after purification, a suitable concentration of methanol in the purified poloxamer 188 product was determined to allow the product to be pushed smoothly out of the extractor without opening the lid. Two batches of poloxamer 188 (approximately 8,000 and 9,000 Da) were evaluated to identify the poloxamer 188 / methanol / CO2 concentration in the extraction vessel. The extraction vessel was charged with 0.25 grams of poloxamer 188 per mL of extraction cell volume at 3,000 psig (208 bar) and 4,500 psig (311 bar). The equilibrium concentration of methanol in the charged polymer 188 product inside the extraction vessel was measured during a 26-hour run in a dedicated experiment. The results showed that the equilibrium concentration of methanol in the charged poloxamer 188 product in the extraction vessel at 6.6%-8.6% methanol / CO2 was approximately 25-35% methanol.

[0255] To evaluate what refinery product concentration was suitable for discharging the product from the extractor without opening the lid, the concentration of the raffinate remaining in the 12 L extraction vessel was adjusted to various levels between 25 and 35%. The product was discharged through a rapid decompression system. The correlation between dryness and discharge conditions was evaluated. Results showed that at higher methanol concentrations, at the high end of the 25% to 35% concentration range, approximately 2600 grams of wet product was released in less than 10 minutes to give a free-flowing fine powder. The appearance of the product did not change with a change in head pressure from 4500 to 1000 psi (311 to 70 bar). Slower discharge rates and lower methanol concentrations gave coarser particles.

[0256] Example 3 The effect of solvent distribution systems on supercritical fluid extraction(SFE). The effect of a solvent distribution system on supercritical fluid extraction (SFE) using a methanol / supercritical CO2 co-solvent was determined. The distribution system may contain metal or ceramic spheres of various sizes packed at the bottom. The porosity of this bed can be precisely adjusted by selecting different sized spheres or by using a mixture of different sized spheres. The effective rate of the system controls the bubble size and extraction efficiency. The spheres can be easily removed and cleaned. Various distribution systems were compared for their effect on the SFE process and their efficiency in extracting low molecular weight (LMW) materials relative to the total material extracted. Efficiency is typically determined by the yield and throughput of a particular target low molecular weight species (% of LMW components less than 4,500 Daltons).

[0257] A. Method The SFE process was carried out using a 3.08 L cell under the following conditions: T = 40 °C, pressure = 300 bar, methanol flow rate = 6.6% of the total flow rate (CO of 95–100 g / min). Methanol flow rate of 6.5-7g / min for CO2 flow rate; methanol flow rate of 10-10.5g / min for CO2 flow rate of 140-148g / min), cell length = 5', and ID=2". A series of experiments was performed by running the process using different solvent distribution systems: no system; stainless steel (SS) balls at the bottom of the cell; aluminum suction strainer with SS balls; suction wire screen (40 mesh) with SS balls; bend with 6 holes (1 / 16" diameter); or bend with 12 holes (1 / 16" diameter).

[0258] Extract samples collected at the first, second, third and fourth extraction times were analyzed by GPC and gravimetric analysis. Weight average molecular weight (Mw), molecular weight of the highest peak (Mp) and polydispersity index (PD) (defined as Mw / number average molecular weight) were calculated.

[0259] B. Results The run conditions and parameters of each experiment for each of the distribution systems tested are summarized as well as the GPC results and gravimetric analyses.

[0260] 1. Distribution System – None

[0261] [Table 1]

[0262] [Table 2]

[0263] 2. Distribution system – 1000 stainless steel balls at the bottom of the cell

[0264] [Table 3]

[0265] [Table 4]

[0266] The results show that adding 1000 SS balls at the bottom of the cell for better CO2 and methanol distribution reduces the main product carryover and increases the process efficiency by 59%. Therefore, the results show that the system provides effective CO2 / MeOH solvent distribution using stainless steel SS balls at the bottom of the extractor.

[0267] 3. Distribution system – 1000 stainless steel balls at the bottom of the cell for even higher CO2 flow and linear velocities

[0268] [Table 5]

[0269] [Table 6]

[0270] The results show that this process was not as efficient as that described in previous experiments using lower CO2 flow and linear velocities.

[0271] 4. Distribution system-Aluminum suction strainer with SS balls

[0272] [Table 7]

[0273] [Table 8]

[0274] The results showed that using an aluminum suction strainer with SS balls in it as a distribution system instead of just the 1000-SS balls at the bottom resulted in a higher linear velocity and It can be seen that the process was four times more efficient. For example, the method removed less extracted material but still removed a similar level of LMW fraction, i.e., 31.09 grams of extracted material in this example compared to 120.88 grams in the previous experiment, and 17.1 grams of extracted LMW fraction in this example compared to 23.09 grams in the previous experiment. Thus, the results show that the system provided an effective CO2 / MeOH solvent distribution using an aluminum suction strainer at the bottom of the extractor.

[0275] 5. Distribution system - suction wire mesh 40 mesh with SS balls

[0276] [Table 9]

[0277] [Table 10]

[0278] The results show that the extraction efficiency in the distribution system using the 40 mesh suction wire screen was similar to the aluminum suction strainer with SS balls described in the previous example.

[0279] 6. Distribution system - 6 holes 1 / 16" bend

[0280] [Table 11]

[0281] [Table 12]

[0282] The results show that the 6-hole bend of 1 / 16" diameter was less efficient than the previous distribution system.

[0283] 7. Distribution system – Bend pipe with 12 holes, 1 / 16” diameter

[0284] [Table 13]

[0285] [Table 14]

[0286] The results show that the bend with 12 holes of 1 / 16" diameter is slightly more efficient than previous experiments using a distribution system with 6 holes, but is less efficient than the suction screen and stainless steel balls.

[0287] C. Summary The highest purification efficiency was observed with the method where the distribution system was an aluminum suction strainer with stainless steel balls or a suction wire mesh with stainless steel balls (see, e.g., Experiments 4 and 5). Increasing the number of holes in the bent pipe distribution system also increased the purification efficiency to a certain extent (see, e.g., Experiment 7). Thus, the results show that the distribution system plays a role in SFE equipment and its design can make the process more or less efficient. The preferred design is a suction wire mesh with stainless steel balls or a suction strainer. A bent pipe can also be used as a distribution system, but with as many holes as possible. For most of the other experiments described, the experiments were performed with a suction wire mesh distribution system.

[0288] Example 4 Two-step extraction batch purification of poloxamer 188 at 12L scale The process is designed to enrich for high molecular weight components (>13,000 Daltons). Typical extraction profiles show that the increase in high molecular weight components after purification is not significant. Poloxamer 18 A two-step batch purification of 8 is carried out at a 12 L scale by adjusting the methanol concentration. Poloxamer 188 before extraction contains approximately 1% high molecular weight components and 5% low molecular weight components as determined by gel permeation chromatography (GPC).

[0289] A 12 L extraction system including a stirred extraction vessel, cyclone separator, CO2 solvent circulation, and methanol co-solvent system is tested for leaks. The extraction system is pressurized with CO2 to 310±15 bar at the start of the campaign. Methanol (2 kg) is dispensed into the lined feed mix tank and warmed to 40°C. Approximately 3700 grams of poloxamer 188 is added to the feed tank and stirred until thoroughly mixed. 5100 grams of the mixed solution is pumped into the extractor. The CO2 flow rate is maintained at 390 gm / min. Two consecutive extractions are performed by adjusting the methanol concentration. The extraction is performed for 12 hours ± 30 minutes at 7.6% MeOH / CO2 with a methanol flow rate of 27.6 ± 1.0 gm / min. The extraction continues for 12 hours ± 15 minutes at 8.6% MeOH / CO2 with a methanol flow rate of 36.6 ± 1.0 gm / min.

[0290] After 24 hours of purification, the extractor is evacuated through a rapid vacuum system (Particles from Gas Saturated Solution (PGSS)) and the wet product is collected in a liner. A sample of the wet product (~600 gm) is transferred to a flask and dried using a rotary evaporator at room temperature under moderate vacuum for about 3 hours, followed by high vacuum at room temperature for 30 minutes and at 35°C for another 30 minutes. The dried product is collected and tested by gel permeation chromatography (GPC) for molecular weight distribution. No low molecular weight (LMW) components are detected in the purified product. The purified product contained approximately 4.5% high molecular weight (HMW) components.

[0291] Example 5 Batch purification of poloxamer 188 by extraction with methanol / supercritical CO2 co-solvent Batch purification of poloxamer 188 by extraction with methanol / supercritical CO2 co-solvent was evaluated. Poloxamer 188 (Asahi Denka Kogyo, Japan) was purified by adjusting the solvent properties by controlling the extraction solvent temperature, pressure, and methanol co-solvent content. Processes varied in pressure and co-solvent content.

[0292] Poloxamer 188 (13–14 kg) was mixed with methanol solvent in a high-pressure extraction vessel. Methanol and supercritical CO2 co-solvents (BOC Gas, USA) were mixed and pumped through the extraction vessel. The extraction was started with a low methanol concentration, which was increased continuously while monitoring the composition of the fractions removed during the extraction. The average methanol concentration was 7.3% (by weight). The concentration was increased stepwise from 6.6% to 7.6% to 8.6%. The extraction vessel pressure was 300 ± 15 bar. The methanol / supercritical CO2 solvent temperature and the extractor jacket temperature were 40 ± 5 °C. The extraction temperature was adjusted to 35–45 °C. The eluted fractions were analyzed by gel permeation chromatography (GPC). The molecular weight distribution of the purified poloxamer 188 recovered from the extraction vessel was narrower than that of the starting material.

[0293] The resulting yield was about 75%. The peak average molecular weight was approximately 9,000 Daltons. The low molecular weight component (less than 4,500 Daltons) was about 1.0%. The polydispersity was about 1.0.

[0294] Example 6 12 L-scale multi-extraction batch purification of poloxamer 188 and analysis by gel permeation chromatography (GPC)

[0295] A. Supercritical fluid extraction (SFE) method Four batches of poloxamer 188 were purified by SFE batch processing in a 12 liter extraction vessel. Each batch was purified as described below. The system was pressurized with CO2 and the pressure was maintained above 900 psig (63 bar) between batches. Methanol (2000 ± 20 gm) was dispensed into a lined feed mix tank and warmed to 40°C. Poloxamer 188 (3696 ± 20 gm) was dispensed into the feed tank and stirred until mixed. Ninety percent (90%) poloxamer 188 solution was pumped into the extractor and the system was pressurized to 310 ± 15 bar. The CO2 flow rate was maintained at 390 gm / min. Three consecutive extractions were performed by adjusting the methanol concentration in controlled stepwise increments of 6.6%, 7.6% or 8.6% by weight. At each methanol concentration, the extraction was performed for a given time as described in Table 15. In-process samples were collected from the bottom of the extractor at designated times between each extraction.

[0296] [Table 15]

[0297] At the end of the 26-hour purification process, the extractor was discharged through a rapid vacuum system and the wet product was collected in a liner. A sublot of the wet product (~600 g) was transferred to a flask and dried using a rotary evaporator at room temperature and moderate vacuum for approximately 3 hours, followed by room temperature and high vacuum for 30 minutes, and then at 35°C and high vacuum for an additional 30 minutes. The dried product was collected as a sublot. This drying process was repeated with the remaining wet product to create three sublots of dried product. The three sublots were combined in a 10 L drum and mixed for 30 minutes to produce purified poloxamer 188. The yield per feed was approximately 55%.

[0298] B. Characterization of Purified Products The starting and purified poloxamer 188 products were evaluated by gel permeation chromatography (GPC), and the results are depicted in Figures 6A-6B.

[0299] FIG. 6A shows the GPC profile of the starting poloxamer 188. In the GPC diagram, retention time is plotted against the relative amounts of different molecular weight species. Increasing retention time on the x-axis corresponds to decreasing molecular weight. The GPC diagram of the starting poloxamer 188 shows a narrow molecular weight distribution with a small additional peak on the low molecular weight side. The area under the curve for the low molecular weight component is approximately 4-7%, with an average molecular weight below 4,500 daltons.

[0300] Figure 6B shows the GPC profile of purified poloxamer 188. Purified poloxamer 188 has a narrower molecular weight distribution than commercial poloxamer 188. In comparison, the GPC diagram of purified poloxamer 188 shows a narrower molecular weight distribution with significantly fewer low molecular weight peaks (less than 1.5% of the area of ​​the main peak).

[0301] Example 7 Preparation and administration of long-circulating substance-free (LCMF) poloxamer 188 A. Supercritical fluid extraction (SFE) process A multistage extraction batch process of poloxamer 188 was performed with extractions performed at a pressure of 247 ± 15 atm (approximately 200-260 bar) and increasing methanol in a controlled stepwise manner of 7.4, 9.1, and 10.7 wt% methanol. Prior to purification, the poloxamer 188 feedstock (BASF Corporation, Washington, NJ) was characterized by gel permeation chromatography (GPC). Molecular weight analysis demonstrated that the feedstock had a main peak average molecular weight of approximately 8,500 ± 750 Da, 6.0% or less of low molecular weight (LMW) species below 4,500 Da, and 1% or less of high molecular weight species (HMW) above 13,000 Da. In addition, the polydispersity was 1.2 or less.

[0302] A 50 L high pressure stainless steel extraction vessel was charged with 14 kg of commercial grade poloxamer 188 (BASF Corporation, Washington, NJ) and 7 kg of methanol (Messer France, SAS, Lavera, France) pressurized with CO2 (49 ± 10 atm, i.e. 720 ± 147 psi) and heated to 35 °C-50 °C for 40-80 min until a homogenous solution was obtained. CO2 (supplied from the main supply tank or via recycle through the 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 the 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 feed tank into the CO2 solvent stream to generate the extraction methanol / CO2 co-solvent, which was delivered as a fine mist through an inlet system into the extraction vessel at a pressure of 247 ± 15 atmospheres (3600 ± psi) or 240-260 bar and a temperature of 40 °C.

[0303] The 7.4% methanol / CO2 extraction co-solvent was filtered through the poloxamer solution for 3 hours, typically at a methanol flow rate of 8 kg / hr (range 6.8 kg / hr to 9.2 kg / hr; total flow rate of 108 kg / hr). Extraction continued with 9.1% methanol / CO2 co-solvent for another 4 hours, typically at a methanol flow rate of 10 kg / hr (range 8.5 kg / hr to 11.5 kg / hr; total flow rate of 110 kg / hr). Extraction continued with 10.7% methanol / CO2 co-solvent for another 8 hours, typically at a methanol flow rate of 12 kg / hr (range 10.2 kg / hr to 13.8 kg / hr; total flow rate of 112 kg / hr). Throughout the extraction process, the extract of soluble species was continuously extracted from the top of the extractor. The extraction solvent is removed from the top of the extractor and passed through two high pressure stainless steel cyclone separators in series to reduce the system pressure from 247 atmospheres (3600 psi) to 59 atmospheres (870 psi), then from 59 atmospheres to 49 atmospheres (720 psi), and The CO2 was separated from the methanol vapor. The separated CO2 was condensed, passed through a heat exchanger, and stored in a solvent reservoir. The pressure of the methanol exhaust stream was further reduced by passing it through another cyclone separator. The purified poloxamer 188 remained in the extractor.

[0304] 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 by Particle from Gas Saturated Solution (PGSS) technology. The precipitate contained approximately 20% to 35% methanol. The purified poloxamer 188 was dried under vacuum below 40 or 45°C to remove residual methanol. The product feed yield gave an average yield of 65%.

[0305] Molecular weight analysis of the purified product as measured by GPC showed that the purified product met the accepted specifications. There was a main peak average molecular weight of about 8,500±750 Da and an average molecular weight of 8,500±750 Da, 1.5% or less of low molecular weight (LMW) species below 4,500 Da, and 1.5% or less of high molecular weight (HMW) species above 13,000 Da. In addition, the polydispersity was 1.05 or less. Thus, the results show that the treatment resulted in a measurable reduction in LMW species and improved polydispersity of the purified product.

[0306] The resulting purified poloxamer 188 was formulated into a clear, colorless, sterile, non-pyrogenic aqueous solution containing 150 mg / ml purified poloxamer, 3.08 mg / ml sodium chloride, 2.38 mg / ml sodium citrate (dihydrate), and 0.366 mg / ml anhydrous citric acid in water for injection. The solution was sterile filtered and filled into 100 ml glass vials, blanketed with nitrogen, and closed with butyl rubber stoppers and aluminum overseals. The osmolality of the resulting solution was approximately 312 mOsm / L. The LCMF poloxamer 188 composition did not contain any bacteriostatic or preservative agents.

[0307] B. Characterization of the plasma concentration time course of purified (LCMF) poloxamer 188 following intravenous administration using HPLC-GPC (Method 1) Purified LCMF poloxamer 188 produced as described above was administered intravenously to 62 healthy volunteers as part of an evaluation to determine its effect on the QT / QTc interval. Eight of the 62 subjects were randomly selected for quantitative analysis of plasma poloxamer levels using HPLC-GPC methods. After dosing, blood samples were obtained by venipuncture into heparin-anticoagulated tubes at baseline, between drug doses (1, 2, 3, 4, 5, and 6 hours), and 1, 1.5, 2, 2.5, 5, 6, and 18 hours after dosing. Plasma was separated by centrifugation and stored frozen until analysis. Purified poloxamer 188 was administered at either a high dose of 300 mg / kg / hr for 1 hour followed by a maintenance dose of 200 mg / kg / hr for 5 hours, or a low dose of 100 mg / kg for 1 hour followed by 30 mg / kg / hr for 5 hours. A mean maximum concentration (Cmax) of 0.9 mg / mL of administered purified poloxamer 188 was achieved by the end of the 1-hour loading infusion. A mean concentration at steady state (Css) of approximately 0.4 mg / mL was achieved during the maintenance infusion. Plasma concentrations declined rapidly after cessation of the maintenance infusion. The LCMF product purified as described above did not exhibit long-circulating high molecular weight material in plasma as observed with prior art poloxamer 188 and as defined herein.

[0308] To confirm the absence of such long-term circulating substances in the plasma, plasma from subjects receiving the high dose was similarly examined using HPLC-GPC. Figures 7A and 7B show a series of HPLC-GPCs of plasma obtained at various time points following administration of purified LCMF poloxamer 188 from 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 chromatograms are enlarged and The high molecular weight portion of the polymer distribution (19.8 kilodaltons to 12.4 kilodaltons) is shown. The main peak portion (12.8 to 4.7 KDa) and the low molecular weight portion (4.7 to 2.5 KDa) are also shown. The HPLC-GPC method quantifies plasma levels based on the height of the elution peak relative to standards of known concentration (i.e., the higher the elution peak, the higher the plasma level). The GPC method also identifies molecular weight ranges by comparing sample elution times to the elution times of standards of known molecular weight.

[0309] The chromatograms show that over time, the high molecular weight portion of the poloxamer 188 polymer distribution decreases in proportion to the main peak and the low molecular weight components. Thus, the polymer distribution shows that the high molecular weight portion clears from the circulation in substantially the same manner. The results also show that the higher molecular weight species do not exhibit a longer circulating half-life (relative to the other polymer components) and do not accumulate in the circulation after intravenous administration.

[0310] C. Comparison of plasma concentration time courses following intravenous administration of purified LCMF poloxamer 188 and purified LCM-containing poloxamer 188 by HPLC-GPC. 1. Administration of Poloxamer 188 containing long-term circulating substances (LCM) As part of a safety and pharmacokinetic study, purified poloxamer 188 (containing LCM) was administered to six healthy volunteers as an intravenous dose of 100 mg / kg / hr for 1 hour, followed by 30 mg / kg / hr for 48 hours (Grindel et al.). Blood samples were obtained by venipuncture into EDTA anticoagulant tubes at the following times: baseline, mid-dose (1, 6, 12, 18, 24, 36, and 48 hours), and 30 minutes, 1, 1.5, 2, 4, 6, 8, 12, 14, 20, and 24 hours after drug administration. Plasma was separated and lyophilized until analysis using HPLC-GPC methods. Analysis of plasma samples revealed the clearance kinetics of the main and HMW peaks of purified poloxamer 188 (containing LCM).

[0311] HMW peak (long-term circulating substances) After administration at these doses, the HMW components (detected as a peak at approximately 16,000 daltons in the HPLC-GPC assay) accumulated during the drug administration period and did not reach a mean Cmax concentration of 225 μg / ml (n=6) until 2 hours after drug administration. By 6 hours after cessation of infusion, the mean plasma level remained at 202 ug / ml, a concentration that was only about 10% reduced from the Cmax value. Over the 24-hour post-infusion blood collection period, the mean plasma level only decreased 22.5% to a plasma concentration of 165 μg / ml. Based on these changes in plasma concentration time course, an elimination half-life of more than 48 hours is estimated.

[0312] Main Peak After administration at the above doses, the main peak achieved an apparent mean steady-state concentration of 522 μg / ml (n=6), which was maintained for the duration of the drug infusion. One hour after cessation of infusion, plasma levels declined 52% from the steady-state concentration to 255 μg / ml. By 6 hours after cessation, plasma levels had declined 85% to 81 μg / ml. By 24 hours after infusion, plasma levels had declined 96% to a plasma concentration of approximately 19 μg / ml (n=6). Based on these changes in plasma concentration time course, the half-life is estimated to be approximately 5 hours.

[0313] 2. LCMF Poloxamer 188 (prepared as described above) As part of the evaluation to determine its effect on the QT / QTc interval previously described, LCMF poloxamer was administered to 62 healthy volunteers at a dose of 300 mg / kg for 1 hour followed by 200 mg / kg / hr for 5 hours. Eight of the 62 subjects were randomly selected for quantitative analysis of plasma poloxamer levels using an HPLC-GPC method similar to that described in part (B) but with improved linearity at low plasma levels.

[0314] HMW peak After administration at the above doses, the HMW component, detected as a peak at approximately 16,000 daltons in the HPLC-GPC assay, accumulated slightly during drug administration and achieved its Cmax (mean value of 117 μg / ml, n=8) by the last infusion. By 1 hour after cessation of drug administration, the plasma level had decreased by 27% from the Cmax value to 86 μg / ml. By 6 hours after the end of drug administration, the mean plasma level had decreased by 71% from the Cmax value to 34 μg / ml. By 18 hours after the end of infusion, the mean plasma level had decreased by 82% to a concentration of 19 μg / ml (n=8). Based on these changes in plasma concentration over time, the elimination half-life of the HMW component was estimated to be 6-9 hours.

[0315] Main Peak After administration at these doses, the main peak achieved an apparent mean steady-state concentration of 2,637 μg / ml, which was maintained for the 6-hour infusion period (n=8). One hour after cessation of infusion, the mean plasma level decreased 67% from steady state to 872 μg / ml, and by 6 hours after cessation, the mean plasma level decreased 93% (from steady state) to 184 μg / ml. By 18 hours after cessation of infusion, the mean plasma level decreased more than 98% (from steady state) to a plasma concentration of approximately 34 μg / ml (n=6). Based on these changes in plasma concentration time course, the elimination half-life is estimated to be approximately 3 hours.

[0316] c. Summary comparison table A comparison of the relative clearance rates from plasma at similar time points after administration is shown in Table 1 below. The data shows the difference in the rate of decline of plasma concentrations of purified (LCM-containing) poloxamer 188 and LCMF poloxamer 188, indicating that LCMF poloxamer 188 is eliminated more quickly. The difference is evident for the HMW peak and the main peak. The difference is most evident for the HMW peak. This indicates that LCMF poloxamers are different from the LCM-containing poloxamers of the prior art.

[0317] [Table 16]

[0318] D. Analytical Data Supporting That Purified LCMF Poloxamer 188 is Different from Purified Poloxamer 188 Containing LCM

[0319] 1. Analytical studies to compare various poloxamers (RP-HPLC assay) In reversed-phase chromatography, there is a hydrophobic stationary phase (column) and a more polar mobile phase. Because of this "reversed" phase, RP-HPLC is typically used to separate compounds based on their relative hydrophobicity. Hydrophobic compounds exhibit longer column retention times than hydrophilic compounds.

[0320] The column retention times of various poloxamers with known differences in hydrophilic / lipophilic balance (HLB) were compared along with purified poloxamer 188-containing LCM and LCMF poloxamer 188 using the following HPLC conditions:

[0321] [Table 17]

[0322] result The results show that LCMF poloxamer 188 is different from the prior art purified poloxamer 188. It has different pharmacokinetic properties, which reflects it being more hydrophilic than the prior art material, including the long-circulating material.

[0323] Figure 9 shows RP-HPLC chromatograms of a highly hydrophilic polymer (PEG 8000), LCMF poloxamer 188, LCM-containing purified poloxamer 188, and two poloxamers of decreasing HLB value (increasing hydrophobicity), namely, poloxamer 338 and poloxamer 407, respectively. PEG 8000, the most hydrophilic polymer, shows low retention on the column consistent with its high hydrophilicity. Poloxamer 338 (HLB>24) and poloxamer 407 (HLB 18-23) show much longer retention times (t Rand k' values). LCMF-purified poloxamer 188 elutes more rapidly than LCM-containing purified poloxamer 188 (mean t R and k' are 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 the LCM-containing purified poloxamer), which means that the LCMF poloxamer 188 is relatively more hydrophilic than the LCM-containing purified poloxamer 188.

[0324] Figure 10 shows chromatograms of three different lots of purified LCMF poloxamer 188 and two different lots of purified (LCM-containing) poloxamer 188. These results demonstrate robust reproducibility for the different lots of material and show that the differences between the two materials cannot be explained by assay variability. These results demonstrate that the polymer distribution of LCMF poloxamer 188 is more hydrophilic than purified poloxamer 188. 2. The different pharmacokinetic behavior of LCMF-purified and LCM-containing poloxamers correlates with their differences in hydrophilicity

[0325] As described herein (e.g., see Example 7B above, and Figures 9-10) and Table 1), LCMF poloxamer 188 exhibits significantly different pharmacokinetic behavior following administration to human subjects when compared to purified poloxamer 188 that contains long-circulating material (LCM) following in vivo administration. The data provided in this example imply that LCMF poloxamer 188 is more hydrophilic than purified poloxamer 188 that gives rise to long-circulating material.

[0326] The polymer size distributions of the purified form of poloxamer 188, purified LCM-containing poloxamer 188, and LCMF poloxamer 188 are similar in terms of size as shown by HPLC-GPC. Both meet the criteria:

[0327] [Table 18]

[0328] While the polymer size distributions of both purified poloxamers are similar as shown by HPLC-GPC, the molecules comprising the polymer distribution of LCMF poloxamer 188 are more hydrophilic as evidenced by RP-HPLC herein.

[0329] When injected into animals, the more hydrophilic polymer distribution is cleared from the circulation at a faster rate. This accounts for the reduced presence of long-term circulating material in LCMF poloxamer 188 formulations. The results further imply that the main peak of the polymer distribution is cleared faster, as observed and described above. For example, plasma concentration time course data from clinical trials shows a shorter elimination half-life of the main and high molecular weight peaks of LCMF poloxamer 188 compared to purified poloxamer 188 containing LCM.

[0330] The rheological, cytoprotective, anti-adhesive and anti-thrombotic effects of P188 are optimal within the majority or major copolymer range of distribution, which is about 8,400-9,400 daltons (with a circulatory half-life of about 4-7 hours), so the presence of the larger, more hydrophobic, long-circulating half-life components of poloxamer 188 is undesirable. For example, desirable activities of P188 include its rheological effect of reducing blood viscosity and inhibiting red blood cell (RBC) aggregation, which is responsible for its ability to improve blood flow in injured tissues. In contrast, more hydrophobic high 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 P188, implying that high molecular weight hydrophobic poloxamer species may have undesirable biological effects.

[0331] Therefore, the results imply that the hydrophobic components contained in the high molecular weight peak of purified (LCM-containing) poloxamer 188 are undesired impurities. A poloxamer such as an LCMF poloxamer with reduced amounts of 188 is preferred.

[0332] Example 8 The effect of controlling methanol concentration in multistage extraction batch processes employing controlled stepwise increases in methanol. The process was carried out substantially as described in the examples above, except that the extraction was carried out by varying the methanol concentration in controlled stepwise increments of methanol to perform a multi-stage extraction batch process of poloxamer 188 as follows: The maximum pressure feasible is typically 250 atmospheres. Thus, the process of adjusting the methanol concentration and extraction time can be used to adjust the pressure limit.

[0333] A. Controlled stepwise increase: 6.3, 7.1 and 8.1 wt% methanol Poloxamer 188 (14 kg) was purified as described in Example 8, except that the 6.3% methanol / CO2 extraction co-solvent was filtered through the poloxamer solution for 3 hours at a methanol flow rate of 107 kg / hr. Extraction continued with 7.1% methanol / CO2 co-solvent for an additional 8 hours at a methanol flow rate of 108 kg / hr. Extraction continued with 8.1% methanol / CO2 co-solvent for an additional 7 hours at a methanol flow rate of 109 kg / hr. At the end of the process, the extractor contents were drained and dried to remove residual methanol as described in Part A. The yield of purified poloxamer 188 was 66%. The results further demonstrated that the process resulted in a measurable reduction of LMW species.

[0334] B. Controlled stepwise increase: 3.8, 9.1 and 10.7 wt% methanol Poloxamer 188 (14 kg) was purified as described in Example 8, except that the 3.8% methanol / CO2 extraction co-solvent was filtered with the poloxamer solution for 4 hours at a methanol flow rate of 104 kg / hr. Extraction was continued with 9.1% methanol / CO2 co-solvent for an additional 3 hours at a methanol flow rate of 108 kg / hr. Extraction was further continued with 10.7% methanol / CO2 co-solvent for an additional 8 hours at a methanol flow rate of 112 kg / hr. At the end of the process, the extractor contents were drained and dried to remove residual methanol as described in Part A. The yield of purified poloxamer 188 was 57%. The results further demonstrated that the process resulted in a measurable reduction of LMW species.

[0335] Example 9 Batch purification of poloxamer 188 by extraction with methanol / pressure CO2 co-solvent Batch purification of poloxamer 188 by extraction with methanol / high pressure CO2 co-solvent is evaluated. Poloxamer 188 (13-14 kg) is purified by extraction with methanol / high pressure CO2 solvent. Poloxamer 188 is stirred with methanol in a high pressure extraction vessel until mixed. Methanol and high pressure CO2 co-solvent are pumped through the extraction vessel. The solvent properties of the extraction solvent are adjusted by adjusting the extraction solvent temperature, pressure and the amount of methanol co-solvent. Specifically, a combination of three parameters is selected to remove low molecular weight (LMW) and high molecular weight (HMW) components from commercial grade poloxamer 188. The starting concentration of methanol is approximately 2.5 wt% and is gradually increased continuously to 25 wt%. The extraction vessel pressure is 75±10 bar, and the extraction temperature, methanol / CO2 co-solvent temperature and extractor jacket temperature are 20-25°C. The extraction process is carried out in a sequential manner, with the various components being continuously removed from the extractor.

[0336] The extraction solvent is removed and the eluted fractions are analyzed by gel permeation chromatography (GPC). After purification, the purified poloxamer 188 is recovered from the extraction vessel and analyzed by GPC. Low molecular weight (LMW) components are initially removed during extraction and the main fraction is removed with high concentrations of methanol. High molecular weight components are removed later in the extraction process. The molecular weight distribution of the purified poloxamer 188 is narrower than that of the starting material.

[0337] The polymer yield is estimated to be 60-80%, with less than 1.5% low molecular weight components (less than 4,500 Daltons). The peak average molecular weight is approximately 8,500 ± 750 Daltons. Since modifications will be apparent to those of skill in this art, it is intended that this invention be limited only by the scope of the appended claims. Examples of embodiments of the present invention are as follows. [Section 1] Poloxamer 188 that is free of long circulating substances (LCMF): The LCMF poloxamer 188 has the formula HO(CH2CH2O) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) a H, wherein: each of a and a' is an integer such that the percentage of said hydrophilic material (CHO) is about 60% to 90% by weight of the total molecular weight of said copolymer; a and a' are the same or different; b is an integer such that the molecular weight of the hydrophobic material (CHO) is about 1,300 to 2,300 daltons; no more than 1.5% of all components in the polymer 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 all components in the polymer distribution of the copolymer are high molecular weight components having an average molecular weight greater than 13,000 Daltons; the polydispersity value of the copolymer is less than about 1.07 or less than 1.07; The long-circulating substance-free (LCMF) poloxamer 188, following intravenous administration to a human subject, has a circulating plasma half-life of any component not including the major peak in the copolymer distribution that is less than or equal to 5.0 times the circulating half-life of the major component in the copolymer distribution. [Section 2] Item 2. The LCMF poloxamer of item 1, wherein all components constituting the polymer distribution of the copolymer have a circulating half-life in the plasma of the subject that is 4.0 times or less, or 3.0 times or less longer than the circulating half-life of the main component of the copolymer after intravenous administration to the subject. [Section 3] 3. The LCMF poloxamer of claim 1 or 2, wherein all components in the distribution of the copolymer have a circulating half-life in the plasma of a human subject that is no more than 3 times longer than the circulating half-life of the major component in the distribution of the copolymer when administered to the subject. [Section 4] 4. The LCMF poloxamer according to any one of items 1 to 3, 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 of 30 hours or less, 25 hours or less, 20 hours or less, 15 hours or less, 10 hours or less, 9 hours or less, 8 hours or less, or 7 hours or less. [Section 5] 5. The LCMF poloxamer of item 4, wherein all components in the distribution of the copolymer have a half-life of 10 hours or less or 12 hours or less in the plasma of a human subject when administered to the subject. [Section 6] Item 6. The LCMF poloxamer according to any one of Items 1 to 5, wherein the polyoxyethylene / polyoxypropylene copolymer is a poloxamer having a hydrophobic moiety having a molecular weight of about 1,400 to 2,000 Da or 1,400 to 2,000 Da and a hydrophilic portion that constitutes about 70% to 90% or 70% to 90% by weight of the copolymer. [Section 7] Item 7. The LCMF poloxamer according to any one of items 1 to 6, wherein the molecular weight of the hydrophobic substance (CHO) is about 1,750 Da or 1,750 Da. [Section 8] Item 8. The LCMF poloxamer according to any one of items 1 to 7, wherein the average molecular weight of the polyoxyethylene / polyoxypropylene copolymer is 7,680 daltons to 9,510 daltons or 8,400 to 8,800 daltons. [Section 9] the percentage of high molecular weight components in the formulation greater than 13,000 daltons constitutes less than 1% of the total distribution of components in the poloxamer formulation; 9. The LCMF poloxamer according to any one of items 1 to 8, wherein intravenous administration to a human subject does not result in a component having a circulating plasma half-life greater than four times the circulating plasma half-life of the major component in the distribution of the copolymer. [Section 10] Item 11. The LCMF poloxamer of item 9, wherein the percentage of high molecular weight components in the formulation greater than 13,000 daltons constitutes less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, or less of the total distribution of components in the poloxamer formulation. 11. The LCMF poloxamer according to any one of items 1 to 10, wherein the polydispersity value is less than 1.06, less than 1.05, less than 1.04 or less than 1.03. [Section 12] Poloxamer 188 that is free of long circulating substances (LCMF): The LCMF poloxamer 188 has the formula HO(CH2CH2O) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) a H; each of a and a' is an integer such that the percentage of said hydrophilic material (CHO) is about 60% to 90% by weight of the total molecular weight of said copolymer; a and a' are the same or different; b is an integer such that the molecular weight of the hydrophobic substance (CHO) is approximately 1,300 to 2,300 daltons; no more than 1.5% of all components in the distribution of said copolymer are low molecular weight components having an average molecular weight of less than 4,500 daltons; no more than 1.5% of all components in the distribution of said copolymer are high molecular weight components having an average molecular weight greater than 13,000 daltons; the polydispersity value of the copolymer is less than about 1.07 or less than 1.07; the LCMF poloxamer is more hydrophilic than purified poloxamer 188 or a standard that contains the long circulating substance (LCM), and the LCM substance in the poloxamer has a half-life that is about 5-fold or greater than the half-life of the major component of the poloxamer formulation when administered to a human subject; The long-circulating substance-free (LCMF) poloxamer 188. [Section 13] Poloxamer 188 that is free of long circulating substances (LCMF): The LCMF poloxamer 188 has the formula HO(CH2CH2O) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) a H; each of a and a' is an integer such that the percentage of said hydrophilic material (CHO) is about 60% to 90% by weight of the total molecular weight of said copolymer; a and a' are the same or different; b is an integer such that the molecular weight of the hydrophobic substance (CHO) is approximately 1,300 to 2,300 daltons; no more than 1.5% of all components in the distribution of said copolymer are low molecular weight components having an average molecular weight of less than 4,500 Daltons; No more than 1.5% of all components in said copolymer distribution have an average molecular weight greater than 13,000 Daltons. a high molecular weight component having a molecular weight of; the polydispersity value of the copolymer is less than about 1.07 or less than 1.07; The LCMF poloxamer had a shorter mean retention time (t ) as assessed by reversed-phase high performance liquid chromatography (RP-HPLC) than purified LCM-containing poloxamer 188 under the same RP-HPLC conditions. R) the long circulating substance-free (LCMF) poloxamer 188. [Section 14] The RP-HPLC conditions are R is about 9.9 to 10 or 9.9 to 10; and R Item 14. The LCMF poloxamer according to item 13, wherein the LCMF poloxamer is such that the LCMF poloxamer is about 8.7 to 8.8 or 8.7 to 8.8. [Section 15] Poloxamer 188 that is free of long circulating substances (LCMF): The LCMF poloxamer 188 has the formula HO(CH2CH2O) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) a H, wherein: each of a and a' is an integer such that the percentage of said hydrophilic material (CHO) is about 60% to 90% by weight of the total molecular weight of said copolymer; a and a' are the same or different; b is an integer such that the molecular weight of the hydrophobic substance (CHO) is about 1,300 to 2,300 daltons; no more than 1.5% of all components in the distribution of said copolymer are low molecular weight components having an average molecular weight of less than 4,500 Daltons; no more than 1.5% of all components in the distribution of said copolymer are high molecular weight components having an average molecular weight greater than 13,000 Daltons; the polydispersity value of the copolymer is less than about 1.07 or less than 1.07; The long-circulating substance-free (LCMF) poloxamer 188, wherein the capacity factor (k') of the LCMF poloxamer 188, as assessed by RP-HPLC, is less than the k' of purified LCM-containing poloxamer 188 under the same RP-HPLC conditions. [Section 16] 16. The LCMF poloxamer according to any one of items 13 to 15, wherein the RP-HPLC conditions are such that the average k' of the LCMF poloxamer 188 is about 3.2 to 3.3 or 3.2 to 3.3, and the average k' of the purified LCM-containing poloxamer 188 is about 3.6 to 3.7 or 3.6 to 3.7. [Section 17] a) introducing a poloxamer 188 solution into an extraction vessel and dissolving said poloxamer in a first alkanol to form a solution; b) combining the poloxamer solution with an extraction solvent comprising a second alkanol and supercritical carbon dioxide at a temperature and pressure that maintains supercritical carbon dioxide for a first period of time: said temperature being greater than the critical temperature of carbon dioxide but less than or equal to 40°C; The pressure is between 220 bar and 280 bar; the alkanol is provided at a concentration of 7% to 8% alkanol by weight of the total extraction solvent; and c) increasing the concentration of the second alkanol of step b) in the extraction solvent multiple times in gradient steps over the time of the extraction method: each of the plurality of times occurring over a further period of time; In each successive step, the concentration of said alkanol is increased by 1 to 2% compared to the previous concentration of said second alkanol; and d) removing the extraction solvent from the extraction vessel, thereby removing the raffinate poloxamer. Removing extractable materials from drug products [Item 18] The LCMF poloxamer according to any one of items 1 to 16, which is produced by a method comprising the steps of: Item 18. The LCMF poloxamer of item 17, wherein in step a) the weight ratio of poloxamer to first alkanol is about 2:1 to 3:1 or 2:1 to 3:1, inclusive. [Section 19] 19. The LCMF poloxamer of clause 17 or clause 18, wherein the multiple times in step c) are performed in 2, 3, 4 or 5 gradient steps. [Section 20] Step c): i) increasing the concentration of the second alkanol from about 7% to 8% to about 8.2% to 9.5% over a second period of time; and ii) increasing the concentration of said second alkanol from about 8.2% to 9.5% to about 9.6% to 11.5% over a third period of time. Item 18. The LCMF poloxamer according to item 19, which is carried out in two steps including: [Section 21] the alkanol concentration in step b) is about 7.4% by weight or 7.4% by weight; the alkanol concentration in step i) is about 9.1% by weight or 9.1% by weight; and 21. The LCMF poloxamer of item 20, wherein the alkanol concentration in step ii) is about 10.7% by weight or 10.7% by weight. [Section 22] 20. The LCMF poloxamer according to any one of items 17 to 19, wherein the first fixed period, the second fixed period, and the third fixed period are each carried out for 2 hours to 12 hours. [Section 23] 23. The LCMF poloxamer of item 22, wherein the fixed periods are the same or different. [Section 24] The first period of time is carried out for 2 hours to 6 hours; The second period of time is carried out for 2 hours to 6 hours; Item 24. The LCMF poloxamer according to item 22 or 23, wherein the third fixed period is carried out for 4 hours to 10 hours. [Section 25] 25. The LCMF poloxamer according to any one of items 17 to 24, wherein the first and second alkanols are each independently selected from methanol, ethanol, propanol, butanol, pentanol, and combinations thereof. [Section 26] 26. The LCMF poloxamer according to any one of items 17 to 25, wherein the first and second alkanols are the same or different. [Section 27] 26. The LCMF poloxamer of claim 25, wherein the first alkanol is methanol. [Section 28] 26. The LCMF poloxamer of claim 25, wherein the second alkanol is methanol. [Section 29] 29. The LCMF poloxamer according to any one of items 25 to 28, wherein the first alkanol is methanol and the second alkanol is methanol. [Section 30] 30. The LCMF poloxamer according to any one of items 25 to 29, wherein the step d) of removing the extracted material is carried out throughout steps b) to c). [Section 31] Item 32. The LCMF poloxamer according to any one of items 17 to 30, wherein the method is a batch method. 31. The LCMF poloxamer according to any one of items 17 to 30, wherein the method is a continuous method. [Section 33] Item 33. A pharmaceutical composition comprising the LCMF poloxamer according to any one of items 1 to 32. [Section 34] Item 34. The pharmaceutical composition according to item 33, comprising a pharma- ceutical acceptable formulation. [Section 35] 35. A method for treating a disease, disorder or condition in a subject, comprising administering the pharmaceutical composition according to claim 33 or 34, wherein the disease, disorder or condition is selected from heart failure, myocardial infarction, limb ischemia, shock, stroke, ischemic stroke, sickle cell disease, neurodegenerative diseases, macular degeneration, diabetic retinopathy and congestive heart failure. [Section 36] Item 35. The pharmaceutical composition according to item 33 or 34, for use in the treatment of acute myocardial infarction, acute limb ischemia, septic shock, hemorrhagic shock, acute stroke, ischemic stroke, heart failure, sickle cell disease, neurodegenerative disease, macular degeneration, diabetic retinopathy and congestive heart failure. [Section 37] Treating disorders that are treated by membrane resealing and repair; Treating tissue ischemia and reperfusion injury; Reducing inflammatory responses; Reducing blood viscosity; Promoting thrombolysis; Promoting hemostasis; As a vehicle for delivery of drugs, nucleic acids or proteins; As an emulsifier to stabilize suspensions of hydrophobic drugs; Cleansing skin wounds; As a surfactant in cosmetic formulations; For processing stored damaged and compromised blood To control the viscosity of personal care products and soaps; and As a laxative Item 35. The pharmaceutical composition according to item 33 or 34, for use in treating a disease or condition selected from the group consisting of: [Section 38] 1. A method for preparing a long circulating substance free (LCMF) poloxamer comprising: a) introducing a poloxamer 188 solution into an extraction vessel and dissolving said poloxamer in a first alkanol to form a solution; b) mixing the poloxamer solution with an extraction solvent comprising a second alkanol and supercritical carbon dioxide for a first period of time under a temperature and pressure to maintain supercritical carbon dioxide: said temperature being greater than the critical temperature of carbon dioxide but less than or equal to 40°C; The pressure is between 220 bar and 280 bar; providing said alkanol at a concentration of alkanol that is between 7% and 8% by weight of the total extraction solvent; c) increasing the concentration of the second alkanol in step b) in the extraction solvent multiple times in gradient steps over the time of the extraction method: conducting a plurality of times each over a further period of time; In each successive step, the concentration of said alkanol is increased by 1 to 2% compared to the previous concentration of said second alkanol; and d) removing the extraction solvent from the extraction vessel, thereby removing the extracted material from the raffinate poloxamer preparation, thereby producing the LCMF poloxamer. The method of producing the same. [Section 39] Item 39. The method of item 38, wherein in step a), the weight ratio of poloxamer to first alkanol is about 2:1 to 3:1 or 2:1 to 3:1, inclusive. [Section 40] 40. The method of claim 38 or 39, wherein said multiple times in step c) are carried out in 2, 3, 4 or 5 gradient steps. [Section 41] Step c): i) increasing the concentration of the second alkanol from about 7% to 8% to about 8.1% to 9.5% for a second period of time; and ii) increasing said concentration of said second alkanol from about 8.2% to 9.5% to about 9.6% to 11.5% for a third period of time. Item 38. The method according to Item 39, which is carried out in two steps comprising: [Section 42] the alkanol concentration in step b) is about 7.4% by weight or 7.4% by weight; the alkanol concentration in step i) is about 9.1% by weight or 9.1% by weight; and 42. The method of claim 41, wherein the alkanol concentration in step ii) is about 10.7% by weight or 10.7% by weight. [Section 43] Item 43. The method according to any one of Items 38 to 42, wherein each of the first certain period, the second certain period and the third certain period is carried out for 2 hours to 12 hours. [Section 44] Item 44. The method of item 43, wherein the fixed periods are the same or different. [Section 45] The first period of time is carried out for 2 hours to 6 hours; The second period of time is carried out for 2 hours to 6 hours; Item 46: The method according to item 43 or 44, wherein the third certain period is carried out for 4 hours to 10 hours. 46. ​​The method according to any one of items 38 to 45, wherein the first and second alkanols are each independently selected from methanol, ethanol, propanol, butanol, pentanol, and combinations thereof. [Section 47] 47. The method according to any one of items 38 to 46, wherein the first and second alkanols are the same or different. [Section 48] 47. The method of claim 46, wherein the first alkanol is methanol. [Section 49] 47. The method of claim 46, wherein the second alkanol is methanol. [Section 50] 50. The method according to any one of items 46 to 49, wherein the first alkanol is methanol and the second alkanol is methanol. [Section 51] 51. The method according to any one of items 46 to 50, wherein the step d) of removing the extracted material is carried out through steps b) and c). [Section 52] 1. A method for preparing a purified LCMF poloxamer, comprising: a) introducing a poloxamer 188 solution into an extraction vessel and eluting said poloxamer with a first alkanoate; 2. Dissolving in water to form a solution; b) combining the poloxamer solution with an extraction solvent comprising a second alkanol and a supercritical fluid at a temperature and pressure that maintains the supercritical fluid, and increasing the concentration of the second alkanol in the extraction solvent over the time of the extraction process; and c) removing the extraction solvent from the extraction vessel, thereby removing the extracted material from the poloxamer formulation, thereby producing an LCMF poloxamer. The method comprising: [Section 53] 53. The method of claim 52, wherein the first and second alkanols are each independently selected from methanol, ethanol, propanol, butanol, pentanol, and combinations thereof. [Section 54] 54. The method of claim 52 or 53, wherein the first alkanol is methanol. [Section 55] 54. The method of claim 52 or 53, wherein the second alkanol is methanol. [Section 56] 56. The method according to any one of items 52 to 55, wherein the supercritical liquid under high temperature and pressure is selected from carbon dioxide, methane, ethane, propane, ammonia and freon. [Section 57] 57. The method according to any one of items 52 to 56, wherein the supercritical liquid under high temperature and pressure is carbon dioxide. [Section 58] Item 58. The method according to any one of items 52 to 57, wherein the extraction solvent comprises methanol and carbon dioxide. [Section 59] 59. The method of any of claims 52 to 58, wherein the second alkanol is provided as a percentage (w / w) of the total extraction solvent of 3% to 20% or 3% to 15%. [Section 60] increasing the concentration of the second alkanol of step b) in the extraction solvent multiple times in gradient steps over the time of the extraction method, each of the multiple times over a further period of time; In each successive step, increasing the concentration of said alkanol by 1-2% compared to the previous concentration of said second alkanol; 60. The method of any of claims 52 to 59, comprising removing the extraction solvent from the extraction vessel, thereby removing the extracted material from the raffinate poloxamer formulation. [Section 61] 61. The method according to any one of items 52 to 60, wherein the pressure is between 125 bar and 500 bar or about 125 bar to 500 bar. [Section 62] Item 62. The method according to any one of items 52 to 61, wherein the pressure is about 200 bar to 400 bar, or about 280 bar to 340 bar, or about 280 bar to 340 bar. [Section 63] 1. An extraction method for purifying LCMF poloxamer, comprising: a) introducing a poloxamer solution into an extraction vessel and dissolving the poloxamer in a solvent to form a solution, the solvent being selected from an alcohol, an aliphatic ketone, an aromatic ketone, an amine, and mixtures thereof; b) mixing the poloxamer solution with an extraction solvent comprising a solvent and high pressure carbon dioxide, and increasing the concentration of the solvent in the extraction solvent over the time of the extraction process; and c) removing the extraction solvent from the extraction vessel, thereby removing the extracted material from the poloxamer to produce the LCMF poloxamer. The method comprising: [Section 64] 64. The method of claim 63, wherein the solvent in step a) is methanol. [Section 65] 65. The method of claim 63 or 64, wherein the extraction solvent comprises methanol and carbon dioxide. [Section 66] Item 66. The method according to any one of Items 38 to 65, wherein the extracted material contains low molecular weight impurities of less than 4,500 daltons. [Section 67] The purified poloxamer has the formula HO(CH2CH2O) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) a H, a or a' is an integer such that the percentage of said hydrophile (CHO) is about 60% to 90% by weight of the total molecular weight of said copolymer; a and a' are the same or different; b is an integer such that the molecular weight of the hydrophobic substance (CHO) is approximately 1,300 to 2,300 daltons; no more than 1.5% of all components in the distribution of said copolymer are high molecular weight components having an average molecular weight greater than 13,000 daltons; and Item 67. The method according to any one of items 38 to 66, wherein the polydispersity value of the copolymer is less than 1.07. [Section 68] Item 38. The method according to any one of items 38 to 67, wherein the poloxamer is poloxamer 188 (P188). [Section 69] The LCMF poloxamer has the formula: HO(CH2CH2O) a’ ─[CH(CH3)CH2O] b ─(CH2CH2O) a H, wherein: each of a and a' is an integer such that the percentage of said hydrophilic material (CHO) is approximately 60% to 90% by weight of the total molecular weight of said copolymer; a and a' are the same or different; b is an integer such that the molecular weight of the hydrophobic substance (CHO) is approximately 1,300 to 2,300 daltons; no more than 1.5% of all components in the distribution of said copolymer are low molecular weight components having an average molecular weight of less than 4,500 Daltons; no more than 1.5% of all components in the distribution of said copolymer are high molecular weight components having an average molecular weight greater than 13,000 Daltons; 69. The method of any of claims 38 to 68, wherein the polydispersity value of the copolymer is less than about 1.07 or less than 1.07. [Section 70] Item 70. The method according to any one of items 38 to 69, wherein the method is a batch method. [Section 71] 71. The method according to any one of items 38 to 70, wherein the method is a continuous method. [Section 72] Item 38 to 71. A poloxamer free of long-circulating substances (LCMF), produced by the method according to any one of items 38 to 71. [Section 73] 1. A composition comprising long circulating substance free (LCMF) poloxamer 188: the composition is formulated for intravenous administration; and The composition comprises 5-50 g of the LCMF poloxamer. [Section 74] A composition comprising the LCMF poloxamer according to any one of items 1 to 32 and 72, comprising: Formulated for intravenous administration; The composition comprising 5 to 50 g of the LCMF poloxamer. [Section 75] A composition comprising blood or a blood product and the LCMF poloxamer according to any one of items 1 to 32 and 72. [Section 76] 76. The composition of claim 75, wherein the blood or blood product is packed red blood cells or platelets. [Section 77] 77. The composition of claim 75 or 76 for transfusion to treat a disease or disorder or condition, wherein the treatment comprises transfusion. [Section 78] 78. The composition of claim 77, wherein the disease or disorder or condition is selected from sickle cell disease, acute chest syndrome, peripheral arterial disease, heart failure, stroke, peripheral vascular disease, macular degeneration, respiratory distress syndrome (ARDS), multiple organ failure, ischemia, shock, acidosis, hypothermia, anemic breakdown, surgery, trauma, blood loss and blood disorders. [Section 79] 79. The composition of claim 78, wherein the disease or disorder or condition is selected from hemorrhagic shock, septic shock, acute ARDS, anemic cataract and acute blood loss. [Section 80] Item 33. The LCMF poloxamer according to any one of items 1 to 32, which is produced by a method including a supercritical fluid extraction method using CO2 pressure.

Claims

[Claim 1] 2. Poloxamer 188 that is long circulating substance free (LCMF): The LCMF poloxamer 188 has the formula HO(CH ２ CH ２ O) ａ’ ─[CH (CH ３ ) CH ２ O] ｂ ─(CH ２ CH ２ O) ａ H, wherein: Each of a and a′ is the hydrophilic substance (C ２ H ４ O) is an integer such that the percentage is about 60% to 90% by weight of the total molecular weight of said copolymer; a and a' are the same or different; b is the hydrophobic substance (C ３ H ６ O) is an integer such that the molecular weight of about 1,300 to 2,300 daltons; 1.5% or less of all components in the polymer 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 all components in the polymer distribution of the copolymer are high molecular weight components having an average molecular weight greater than 13,000 Daltons; the polydispersity value of said copolymer is less than about 1.07 or less than 1.07; The long-circulating substance-free (LCMF) poloxamer 188, following intravenous administration to a human subject, has a circulating plasma half-life of any component not including the major peak in the copolymer distribution that is less than or equal to 5.0 times the circulating half-life of the major component in the copolymer distribution.

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