Methods for the preparation of phospholipid bilayer carriers

Abstract

The present disclosure concerns simplified methods for preparing crystalline therapeutic agents encapsulated in phospholipid particles. The methods include simpler and more effective steps that provide for more efficient and consistent particle formation. The methods include reduced steps and more straightforward homogenization that yield a more consistent lipid encapsulated crystalline therapeutic.

Description

Methods for the Preparationr CarriersBACKGROUND

[0001] The use of lipids to transport therapeutic materials and agents into cells is a desired vehicle as a carrier due to the ability to transport across the membrane with relative ease. Prior methods for preparing drug-loaded phospholipids are long and resource intensive. Further, they do not necessarily provide the most ideal result for in-tissue drug diffusion. Further, preparing phospholipid bilayers are susceptible to the chemicals utilized in their preparation. In particular, halogenated organic solvents such as dichloromethane can destroy or damage the phospholipid barrier as well as the cargo being enclosed therein. These effects in turn lead to ineffective and unreliable delivery to cells. Thus there is a need for a less corrosive approach to preparing phospholipid bilayer drug carriers.SUMMARY

[0002] A 1staspect of the present disclosure, either alone or in combination with any other aspect herein, concerns a method for preparing lipid microparticles comprising:(a) homogenizing a solution comprising a solvent with a therapeutic agent dissolved therein, a surfactant, and a phospholipid composition; and(b) isolating lipid particles from the solution.

[0003] A 2ndaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 1staspect, wherein the solution is prepared by adding a first solution to a second solution, wherein the first solution comprises a therapeutic agent dissolved in a solvent and the second solution comprises a surfactant, a phospholipid composition, and water.

[0004] A 3rdaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 1stor 2ndaspect, wherein the solvent is chosen from methanol, ethanol, propanol, acetone, ether, benzene, chloroform, ethyl acetate, and combinations thereof.

[0005] A 4thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 1stor 2ndaspect, wherein the therapeutic agent is chosen from paclitaxel, rapamycin, daunorubicin, 5-fluorouracil, doxorubicin, sunitinib, sorafenib, irinotecan, bevacizumab, cetuxamab, biolimus (biolimus A9), everolimus, zotarolimus , tacrolimus, dexamethasone, prednisolone, corticosterone, cisplatin, vinblastine, lidocaine, bupivacaine, bosutinib, ceritinib, crizotinib, gefitinib, ruxolitinib, imatinib, axitinib, nilotinib, trametinib, afatinib, ibrutinib, cabozantinib, imatinib, lenvatinib, sunitinib, re orafenib, sorafenib, vandetanib, dasatinib, pazopanib, triamciclone, tranilast, halofuginone, monte lukast, zafirlukast, pirfenidone, nintedanib, avapritinib, abemaciclib, erdafitinib, fedratinib, nilotinib, nintendanib, palbociclib, pemigatinib, xanthines, aminophylline, sildenafil, tadalafil, vardenafil, udenafil, avanafil, dipyridamole, quinazoline, paraxanthine, papaverine, mesembrenone, rolipram, ibudilast, piclamilast, luteolin, drotaverine, roflumilast, apremilast, crisaborole, inamrinone, milrinone, enoximone, anagrelide, cilostazol, pimobendan, erythro-9-(2-hydroxy-3- nonyl)adenine), (2-[(3,4-dimethoxyphenyl)methyl]-7-[(lR)-l-hydroxyethyl]-4-phenylbutyl]-5- methyl-imidazo[5, l-f][l,2,4]triazin-4(lH)-one), oxindole, (9-(6-phenyl-2-oxohex-3-yl)-2-(3 ,4- dimethoxybenzyl)- pur in-6- one), 3-isobutyl-l-methylxanthine, pentoxifylline, theobromine, theophylline, resveratrol, quercetin, curcumin, chrysin, myricetin, luteolin, apigenin, anthrocyanin, genistein, epigallocatechin gallate, fisetin, astaxanthin, tetrahydrocurcumin, imatinib, nintedanib, sorafenib, sunitinib, pazopanib, ROCK inhibitor (Y27632), YAP / TAZ inhibitor (CA3 and verteporfin), YAP / TAZ-TEAD interaction inhibitor (verteporfin, VGLL4 peptide), SRC inhibitor (dasatinib), and combinations thereof.

[0006] A 5thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of any one of the 1stto 4thaspects, wherein the surfactant is chosen from sodium stearate, sodium docusate, an alkyl ether phosphate, benzalkoaonium chloride, perfluorooctanesulfonate, PEG fatty esters, PEG omega-3 fatty esters and alcohols, glycerol fatty esters, sorbitan fatty esters, PEG glyceryl fatty esters, PEG sorbitan fatty esters, sugar fatty esters, PEG sugar esters, Tween 20, Tween 40, Tween 60, p- isononylphenoxy polyglycidol, PEG laurate, PEG oleate, PEG stearate, PEG glyceryl laurate, PEG glyceryl oleate, PEG glyceryl stearate, polyglyceryl laurate, polyglyceryl oleate, polyglyceryl myristate, polyglyceryl palmitate, polyglyceryl- 6 laurate, polyglyceryl-6 oleate, polyglyceryl-6 myristate, polyglyceryl-6 palmitate, polyglyceryl- 10 laurate, polyglyceryl-10 oleate, polyglyceryl-10 myristate, polyglyceryl- 10 palmitate , PEG sorbitan monolaurate, PEG sorbitan monolaurate, PEG sorbitan monooleate, PEG sorbitan stearate, PEG oleyl ether, PEG lauryl ether, Tween 20, Tween 40, Tween 60, Tween 80, octoxynol, monoxynol, tyloxapol, sucrose monopalmitate, sucrose monolaurate, decanoyl-N-methylglucamide, n-decyl -P -D-glucopyranoside, n-decyl - - D -maltopyranoside, n-dodecyl -P -D-glucopyranoside, n-dodecyl -P -D -maltoside, heptanoyl-N- methylglucamide, n-heptyl-P -D-glucopyranoside, n-heptyl -P -D-thioglucoside, n-hexyl -P -D- glucopyranoside, nonanoyl-N-methylglucamide, n-nonyl - -D-glucopyranoside, octanoyl-N- methylglucamide, n-octyl-P -D-glucopyranoside, octyl -P -D-thioglucopyranoside, sodium docusate, sorbitol, urea, BHT, BHA, PEG-sorbitan monolaureate, petrolatum, methyl stearate or a combination thereof.

[0007] A 6thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the any one of the 1stto 5thaspects, wherein the therapeutic is provided to the solvent at a weight to volume ratio of about 0.005g to about 0.5g in a volume of from about 2.5 mL to about 150 mL.

[0008] A 7thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 6thaspect, wherein the surfactant is provided to the second solution at a mass with respect to the first solution of about 1.5 mg to about 160 mg.

[0009] An 8thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 6thaspect, wherein water is to be at a ratio with the first solution of about 10:1 to about 100:1.

[0010] A 9thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 6thaspect, wherein the phospholipid composition is provided at a mass with respect to the first solution of about 0.05 g to about 5 g.

[0011] A 10thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 2ndor 9thaspect, wherein the phospholipid composition comprises a vegetable sourced phospholipid, a sunflower derived phospholipid, soy derived phospholipid, krill derived phospholipid, egg yolk derived phospholipid, milk derived phospholipid, fish derived phospholipid, or combination thereof.

[0012] An 11thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 2ndand 9thaspect, wherein the phospholipid composition comprises a phospholipid chosen from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin and combinations thereof.

[0013] A 12thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 10thor 11thaspect, wherein the phospholipid composition further comprises cholesterol, a sphingolipid, a glycolipids, or a combination thereof

[0014] A 13thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 1staspect, wherein the solution is prepared by dissolving the therapeutic agent in the solvent, and then adding in the surfactant, the phospholipid composition, and water.

[0015] A 14thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 1stor 2ndaspect, wherein the homogenization is at a temperature of from 10 to 30 °C.

[0016] A 15thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 1stor 2ndaspect, wherein the homogenization is for a period of from 1 to 180 min.

[0017] A 16thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 1stor 2ndaspect, wherein homogenization is performed with at least one rotating blade spinning at from 500 to 20,000 RPM.

[0018] A 17thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 1stor 2ndaspect, wherein homogenization is performed by application of ultrasound energy at from 20 to 50 kHz.

[0019] An 18thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 1stor 2ndaspect, wherein isolating the lipid particles comprises filtration of formed lipid nanoparticles.

[0020] A 19thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 18thaspect, wherein formed lipid particles are filtered with a pore or mesh size of from 50 to 300 pm.

[0021] A 20thaspect of the present disclosure, either alone or in combination with any other aspect herein, concerns the method of the 18thaspect, wherein isolating the lipid nanoparticles further comprises drying.BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 shows scanning electron microscopy (SEM) images of drug-encapsulated lipid particles of the present disclosure. Upper panel shows magnification at 500x, bottom panel is at 140x.DESCRIPTION

[0023] The present disclosure pertains to methods for the preparation of phospholipid carriers that encapsulate drugs, particularly crystalline drugs. A drug encapsulated by a nanocarrier is advantageous in drug delivery because it can provide a more controlled release profile than embedded polymer compositions with the drug more exposed during delivery. Furthermore, carriers that provide better in-tissue drug diffusion can potentially achieve optimal therapeutic results with lesser amount of drug required to be delivered to a target site. Prior methods for preparing drug-loaded phospholipids are long and resource intensive. Further, they do not necessarily provide the most ideal result for in-tissue drug diffusion and bioavailability. In aspects of the present disclosure, by using specific solvents in the manufacturing process, the final phase of the drug component can be optimized to yield a more crystalline form of the drug. A drug in a crystalline structure can offer greater stability and may sustain longer than a more amorphous drug dispersion. Thus, with the processes herein, a different portfolio of formulations and drug release profiles are possible.

[0024] In some aspects, the methods pertain to the preparation of phospholipid bilayers. In aspects, the methods pertain to the preparation of phospholipid bilayers encapsulating a payload or cargo for delivery to a subject. In aspects, the methods pertain to the preparation of phospholipid bilayer carriers that encapsulate crystalline particles of one or more therapeutic agents therein.

[0025] In some aspects, the present disclosure concerns preparing a first solution of a drug or therapeutic agent dissolved in a solvent. It will be appreciated that suitable solvents can beselected based on the solubility of a selected therapeutic therein. In some aspects, the present disclosure concerns preparing a first solution that includes one or more therapeutic agents and a non-halogenated organic solvent. In some aspects, the first solution is prepared by dissolving a therapeutic composition in an alcohol. Examples of suitable alcohols include methanol, ethanol, propanol, or other alkane-based alcohols. In some aspects, other compounds such as acetone, ether, benzene, chloroform, or ethyl acetate can be substituted for or added to the alcohol. In aspects, the therapeutic agent(s) is hydrophobic or rendered hydrophobic to increase solubility.

[0026] In some aspects, the therapeutic agent (s)need not be limited to a particular compound, particularly as the methods herein concern encapsulating crystalline therapeutic in a lipid particle (or microparticle or nanoparticle). In some aspects, cytostatic drugs and / or phosphodiesterase inhibitor drugs and / or anti-fibrosis drugs and / or kinase inhibitors and / or tyrosine kinase inhibitors and / or receptor tyrosine kinase inhibitors may be selected. Examples may include one or more of paclitaxel, rapamycin, daunorubicin, 5-fluorouracil, doxorubicin, sunitinib, sorafenib, irinotecan, bevacizumab, cetuxamab, biolimus (biolimus A9), everolimus, zotarolimus, tacrolimus, dexamethasone, prednisolone, corticosterone, cisplatin, vinblastine, lidocaine, bupivacaine, bosutinib, ceritinib, crizotinib, gefitinib, ruxolitinib, imatinib, axitinib, nilotinib, trametinib, afatinib, ibrutinib, cabozantinib, imatinib, lenvatinib, sunitinib, regorafenib, sorafenib, vandetanib, dasatinib, pazopanib, triamciclone, tranilast, halofuginone, monte Iuka st, zafirlukast, pirfenidone, nintedanib, avapritinib, abemaciclib, erdafitinib, fedratinib, nilotinib, nintendanib, palbociclib, pemigatinib, xanthines, aminophylline, sildenafil, tadalafil, vardenafil, udenafil, avanafil, dipyridamole, quinazoline, paraxanthine, papaverine, mesembrenone, rolipram, ibudilast, piclamilast, luteolin, drotaverine, roflumilast, apremilast, crisaborole, inamrinone, milrinone, enoximone, anagrelide, cilostazol, pimobendan, erythro-9-(2-hydroxy-3- nonyl)adenine), (2-[(3,4-dimethoxyphenyl)methyl]-7-[(lR)-l-hydroxyethyl]-4-phenylbutyl]-5- methyl-imidazo[5, l-f][l,2,4]triazin-4(lH)-one), oxindole, (9-(6-phenyl-2-oxohex-3-yl)-2-(3 ,4- dimethoxybenzyl)- pur in-6- one), 3-isobutyl-l-methylxanthine, pentoxifylline, theobromine, theophylline, resveratrol, quercetin, curcumin, chrysin, myricetin, luteolin, apigenin, anthrocyanin, genistein, epigallocatechin gallate, fisetin, astaxanthin, tetrahydrocurcumin, imatinib, nintedanib, sorafenib, sunitinib, pazopanib, ROCK inhibitor (Y27632), YAP / TAZ inhibitor (CA3 and verteporfm), YAP / TAZ-TEAD interaction inhibitor (verteporfin, VGLL4 peptide), SRC inhibitor (dasatinib), and combmations thereof.

[0027] In some aspects, the first solution can be prepared at an ambient or room temperature, such as about 25 °C. In aspects, the first solution can be prepared at a temperature of from about 4 °C to about 50 °C, including about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, and 45 °C. It will be appreciated that a higher temperature may increase the amount of therapeutic agent dissolved. However, it will also be appreciated that supersaturation of the solution may lead for some therapeutic to precipitate out prior to encapsulation in the phospholipid bilayer. It will also be appreciated that prior to any further use of the first solution, optional treatments such as filtration may be introduced to remove any undissolved compound or precipitate therein.

[0028] In some aspects, the therapeutic is provided to the solvent at a weight to volume ratio of about 0.005g to about 0.5g in a volume of from about 2.5 mL to about 250 mL. In some aspects, the mass of the therapeutic (s) can be of about 0.005 to about 0.4 g, about 0.005 to about 0.3 g, about 0.005 to about 0.2 g, about 0.005 to about 0.1 g, about 0.005 to about 0.05 g, about 0.01 to about 0.5 g, about 0.01 to about 0.4 g, about 0.01 to about 0.3 g, about 0.01 to about 0.2 g, about 0.01 to about 0.1 g, about 0.01 to about 0.05 g, about 0.05 to about 0.5 g, about 0.05 to about 0.4 g, about 0.05 to about 0.3 g, about 0.05 to about 0.2 g, about 0.05 to about 0.1 g, about 0.1 to about 0.5 g, about 0. 1 to about 0.4 g, about 0. 1 to about 0.3 g, and about 0. 1 to about 0.2 g. In some aspects, the solvent volume can be of about 2.5 to about 250 mL, about 2.5 to about 225 mL, about 2.5 to about 200 mL, about 2.5 to about 175 mL, about 2.5 to about 150 mL, about 2.5 to about 125 mL, 2.5 to about 110 mL, about 2.5 to about 100 mL, about 2.5 to about 90 mL, about 2.5 to about 75 mL, about 2.5 to about 60 mL, about 2.5 to about 50 mL, about 2.5 to about 25 mL, about 2.5 to about 10 mL, about 2.5 to about 5 mL, about 5 to about 250 mL, about 5 to about 225 mL, about 5 to about 200 mL, about 5 to about 175 mL, about 5 to about 150 mL, about 5 to about 125 mL, about 5 to about 110 mL, about 5 to about 100 mL, about 5 to about 90 mL, about 5 to about 75 mL, about 5 to about 60 mL, about 5 to about 50 mL, about 5 to about 25 mL, about 5 to about 10 mL, about 10 to about 250 mL, about 10 to about 225 mL, about 10 to about 200 mL, about 10 to about 175 mL, about 10 to about 150 mL, about 10 to about 125 mL, about 10 to about 110 mL, about 10 to about 100 mL, about 10 to about 90 mL, about 10 to about 75 mL, about 10 to about 60 mL, about 10 to about 50 mL, about 10 to about 25 mL, about 50 to about 250 mL, about 50 to about 225 mL, about 50 to about 200 mL, about 50 to about 175 mL, about 50 to about 150 mL, about 50 to about 125 mL, about 50 to about 110 mL, about 50 to about 100 mL, about 50 to about 90 mL, about 50 to about 75 mL, and about 50 to about 60 mL. It will be appreciated that suchratios of mass to volume can be scalable as well. For example, a ratio of 0.01 g to 10 mL can be scaled to 1 g to 1000 mL.

[0029] In aspects, the present disclosure concerns preparation of a second solution. In aspects, the second solution includes a surfactant, water, and one or more phospholipids. The water is to be at a ratio with the first solution of about 10 to about 100 mL of water to every 1 mL of solvent, including about 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, and 95 mL to every I mL of solvent. In some aspects, the water is purified, such as by filtration, reverse osmosis, distillation, heat sterilization, combinations thereof, and similar.

[0030] In aspects, the surfactant is provided at a mass with respect to the first solution of about 1.5 mg to about 160 mg, including about 2, 5, 10, 15, 20, 25, 50, 75, 100, 125, and 150 mg. In some aspects the surfactant is a polysorbate, such as polysorbate 20, polysorbate 80, or similar polysorbate with varying aliphatic tail lengths. In some aspects, the surfactant is sodium stearate, sodium docusate, an alkyl ether phosphate, benzalkonium chloride, perfluorooctanesulfonate, combinations thereof, or similar. The surfactant can be an anionic surfactant, a cationic surfactant, a non-ionic surfactant, or an amphoteric surfactant. Exemplary surfactants may be chosen from PEG fatty esters, PEG omega-3 fatty esters and alcohols, glycerol fatty esters, sorbitan fatty esters, PEG glyceryl fatty esters, PEG sorbitan fatty esters, sugar fatty esters, PEG sugar esters, Tween 20, Tween 40, Tween 60, p-isononylphenoxypolyglycidol, PEG laurate, PEG oleate, PEG stearate, PEG glyceryl laurate, PEG glyceryl oleate, PEG glyceryl stearate, polyglyceryl laurate, polyglyceryl oleate, polyglyceryl myristate, polyglyceryl palmitate, polyglyceryl-6 laurate, polyglyceryl-6 oleate, polyglyceryl-6 myristate, polyglyceryl-6 palmitate, polyglyceryl- 10 laurate, polyglyceryl- 10 oleate, polyglyceryl- 10 myristate, polyglyceryl- 10 palmitate , PEG sorbitan monolaurate, PEG sorbitan monolaurate, PEG sorbitan monooleate, PEG sorbitan stearate, PEG oleyl ether, PEG lauryl ether, Tween 20, Tween 40, Tween 60, Tween 80, octoxynol, monoxynol, tyloxapol, sucrose monopalmitate, sucrose monolaurate, decanoyl-N-methylglucamide, n-decyl - P -D-glucopyranoside, n-decyl -P -D-maltopyranoside, n-dodecyl -P -D-glucopyranoside, n- dodecyl -P -D-maltoside, heptanoyl-N-methylglucamide, n-heptyl-P -D-glucopyranoside, n- heptyl -P -D-thioglucoside, n-hexyl -P -D-glucopyranoside, nonanoyl-N-methylglucamide, n- nonyl -P -D-glucopyranoside, octanoyl-N-methylglucamide, n-octyl-P -D-glucopyranoside, octyl -P -D-thioglucopyranoside and their derivatives. In some aspects, the excipients may include oneof sodium docusate sorbitol, urea, BHT, BHA, PEG-sorbitan monolaureate, petrolatum, methyl stearate or a combination thereof.

[0031] In aspects, a phospholipid composition is provided at a mass with respect to the first solution of about 0.05 g to about 5 g, including about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, and 4.5 g. In aspects, the phospholipid composition may include, but is not limited to, a vegetable sourced phospholipid, such as from sunflower, soy, or krill, or from egg yolk, milk, or fish. The phospholipid includes a hydrophilic head and a lipophilic tail. In aspects, the phospholipid is biocompatible and / or biodegradable. The phospholipid composition may include one or more of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin and combinations thereof. The phospholipid composition may further include membrane co-factors such as cholesterol, sphingolipids, and glycolipids. In aspects where the phospholipids are sourced from a plant or animal, natural incidental cofactors from membrane therein may also be included. Tn some aspects, the percentage of certain phospholipids within the phospholipid composition may vary, such as between about 25 to about 90 percent phosphatidylcholine, including about 50, 65, and 75. Other phospholipids may similarly vary over similar or the same ranges within the phospholipid composition. In some aspects, the phospholipid may be derived from a natural source, such as from Gallus gallus domesticus or an Anatidae species eggs. Such may in some aspects provide an advantage over polymer-based nano- and micro- particles as polymers can elicit side effects that a biologic does not incur, such as inflammation etc.

[0032] In aspects, the methods of the present disclosure may forgo the preparation of a second solution. In such instance, the methods may include adding directly to the first solution the surfactant and the phospholipid composition. In other aspects, the method may include adding to the first solution the phospholipid composition and then the surfactant.

[0033] In some aspects, the first solution is not homogenized prior to combining with the second solution. In some aspects, the second solution is not homogenized prior to combining with the first solution. It is understood that prior approaches to encapsulation would homogenize the second and / or the first solution prior to any combining thereof. It is an aspect of the present disclosure that the therapeutic dissolved in solvent, the surfactant, and the phospholipid composition are combined prior to homogenization. In some aspects, the therapeutic dissolved insolvent, the surfactant, and the phospholipid composition are combined without a particular rate of application, such as dropwise. It is an aspect of the present disclosure that all are present in the same solution prior to homogenization.

[0034] In aspects, the methods of the present disclosure include preparing a solution of a solvent with a therapeutic agent dissolved therein with a surfactant and a phospholipid composition added therein. In aspects, the method includes homogenizing the solution to prepare the lipid particle. Homogenization refers to the application of energy to disrupt the solution. In aspects, homogenization is achieved through application of a shear force, such as through a rotating blade or clades. In other aspects, homogenization is achieved through application of ultrasonic energy. In aspects, the homogenization is applied for a period of time such as from about 1 minute (min) to about 180 min, including from about 1 min to about 150 min, from about 1 min to about 120 min, from about 1 min to about 90 min, from about 1 min to about 60 min, from about 1 min to about 45 min, from about 1 min to about 30 min, from about 1 min to about 15 min, from about 1 min to about 10 min, from about 1 min to about 5 min, from about 5 min to about 180 min, including from about 5 min to about 150 min, from about 5 min to about 120 min, from about 5 min to about 90 min, from about 5 min to about 60 min, from about 5 min to about 45 min, from about 5 min to about 30 min, from about 5 min to about 15 min, from about 5 min to about 10 min, from about 10 min to about 180 min, including from about 10 min to about 150 min, from about 10 min to about 120 min, from about 10 min to about 90 min, from about 10 min to about 60 min, from about 10 min to about 45 min, from about 10 min to about 30 min, from about 10 min to about 15 min, from about 15 min to about 180 min, including from about 15 min to about 150 min, from about 15 min to about 120 min, from about 15 min to about 90 min, from about 15 min to about 60 min, from about 15 min to about 45 min, from about 15 min to about 30 min, from about 30 min to about 180 min, including from about 30 min to about 150 min, from about 30 min to about 120 min, from about 30 min to about 90 min, from about 30 min to about 60 min, from about 30 min to about 45 min, from about 45 min to about 180 min, including from about 45 min to about 150 min, from about 45 min to about 120 min, from about 45 min to about 90 min, from about 45 min to about 60 min, from about 60 min to about 180 min, including from about 60 min to about 150 min, from about 60 min to about 120 min, from about 60 min to about 90 min, from about 90 min to about 180 min, including from about 90 min to about 150 min, from about 90 min to about 120 min, from about 120 min to about 180 min, including from about 120 min to about150 min, and from about 150 min to about 180 min. It will be appreciated that homogenization can be of about 1, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, and 180 min.

[0035] In aspects, the homogenization occurs at a temperature of from about -10 to about 30 °C, including about -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29 °C. In some aspects, the temperature can be maintained within ± 10 °C or within ± 5 °C or within ± 2 °C or within ± 1 °C during homogenization.

[0036] In aspects, the homogenization occurs at a speed of from about 500 to about 20 000 RPM, including about 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10 000, 11 000, 12 000, 13 000, 14 000, 15 000, 16 000, 17 000, 18 000, and 19 000 RPM. It will be appreciated that homogenization can occur with the speed of a rotating blade or series of blades. In aspects, the higher the speed of the homogenizer, the smaller the particle size. Accordingly, the methods of the present disclosure further allow for the production of desired particle sizes. In other aspects, the homogenization can be achieved through the application of ultrasonic energy. In aspects, ultrasonic energy is applied at a rate of about 20 kHz or higher, such as about 25, 30, 35, 40, 45, and 50 kHz.

[0037] In aspects, the present disclosure includes the step of isolating the particles formed during homogenization from the remainder of the solution. In some aspects, the particles can be isolated by filtration or evaporation of the remaining solution. In some aspects, the particles can be filtered using a mesh or pore filtration, wherein the pores or mesh offer varying size or cross- sectional width parameters, such that particles over a threshold size will be retained on the filtration medium. For example, a pore or mesh may allow for retention of particles greater than 50 pm, greater than 100 pm, greater than 125 pm, greater than 150 pm, greater than 175 pm, greater than 200 pm, greater than 225 pm, greater than 250 pm, and so on. It is understood that particle smaller that the threshold will either pass through or become entangled in the pores, likely depending on how close in size they are to the pore / mesh selected.

[0038] In aspects, the methods of the present disclosure concerns drying recovered lipid particles. Such may be accelerated through application of an air flow, the presence of elevatedheat, such as an oven, placement within a desiccator, and so on. In further aspects, the recovered lipid particles canthen be further characterized and / or sorted.EXAMPLES

[0039] Experiment 1

[0040] A first solution was prepared by dissolving 0.2 g of sirolimus in 20 rnL of methanol (MeOH). 80 rnL of reverse osmosis (RO purified water was added and the mixture was homogenized in an ice bath for 60 min. Then, 6.3 g of Tween80 and 1.2 g of Sunlipon65 were added into the mixture and the total mixture was homogenized for a further 60 min. still within the ice bath / ice cold water.

[0041] The results were then examined by scanning electron microscopy (SEM) No clear particles seemed have been formed.

[0042] Experiment 2

[0043] A second solution was then prepared. In the revised approach, the surfactant and the phospholipids were to be added prior to homogenization of the therapeutic in solvent. Accordingly, 0.02 g of sirolimus was dissolved in 8 g of MeOH. 6.3 mg of Tween 80 and 0.2 g of Sunlipon65 were prepared in 80 mL of water and the resulting solution was combmed with the sirolimus / MeOH solution and homogenized for 60 min in an ice bath (ice being changed ~ every 30 min).

[0044] The results were assessed and pronounced particles of about 250 pm were recovered (see, FIG. 1). These data suggest that the sirolimus particles form within the oil phase.

[0045] Experiment 3

[0046] Further experiments were then conducted to assess the role of the therapeutic agent and the phospholipid in the formation of the particles. A solution of 2.5 mL of MeOH was mixed with a solution of 80 rnL water with 1.58 mg of Tween80 therein. After combining the solutions, the mixture was homogenized in an ice bath for 60 min (ice changed ~ every 15-30 mins).

[0047] SEM analysis showed there to be an absence of the microstructures seen in the second experiment, though some smaller structures were observed that are likely of the surfactant with water therein.

[0048] Experiment 4

[0049] A final series of experiments were arranged to assess more common techniques, particularly where the two solutions are homogenized prior to combining. 200 mg of egg phospholipid were prepared in 5 mL of methanol and then 5 mg of Tween80 and 100 mL of water were added. This solution was homogenized in an ice bath for 20-25 mins. A second solution of 10 mg of sirolimus in 5 mL of methanol was prepared, followed by addition of a further 100 mL of water to obtain an aqueous solution of sirolimus by recrystallization. The second solution was then homogenized for 100-200 mins in an ice bath. 5 mL of first solution was added to the second dropwise during homogenization. Homogenization was then continued an additional 15 mins and then maintained in an ultrasonic cleaner for 20 minutes. The resulting product was centrifuged and assessed by dynamic light scattering. The results identify that particles of about 250 pm were generated. SEM analysis however showed significant variety in material size with much smaller particles observed than in the second experimental setup herein.

[0050] While particular aspects have been illustrated and described herein, it should be understood that various other changes and modifications may be made without departing from the spirit and scope of the claimed subject matter. Moreover, although various aspects of the claimed subject matter have been described herein, such aspects need not be utilized in combination. It is therefore intended that the appended claims cover all such changes and modifications that are within the scope of the claimed subject matter.

[0051] It is appreciated that all reagents are obtainable by sources known in the art unless otherwise specified.

[0052] It is also to be understood that this disclosure is not limited to the specific aspects and methods described herein, as specific components and / or conditions may, of course, vary. Furthermore, the terminology used herein is used only for the purpose of describing particular aspects of the present disclosure and is not intended to be limiting in any way. It will be also understood that, although the terms “first,” “second,” “third” etc. may be used herein to describe various elements, components, regions, layers, and / or sections, these elements, components,regions, layers, and / or sections should not be limited by these terms. These terms are only used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. Thus, “a first element,” “component,” “region,” “layer,” or “section” discussed below could be termed a second (or other) element, component, region, layer, or section without departing from the teachings herein. Similarly, as used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms, including “at least one,” unless the content clearly indicates otherwise. “Or” means “and / or.” As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items. It will be further understood that the terms “comprises” and / or “comprising,” or “includes” and / or “including” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and / or groups thereof. The term “or a combination thereof’ means a combination including at least one of the foregoing elements.

[0053] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. It will be further understood that terms such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure, and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

[0054] Reference is made in detail to exemplary compositions, aspects and methods of the present disclosure, which constitute the best modes of practicing the disclosure presently known to the inventors. The Figures are not necessarily to scale. However, it is to be understood that the disclosed aspects are merely exemplary of the disclosure that may be embodied in various and alternative forms. Therefore, specific details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for any aspect of the disclosure and / or as a representative basis for teaching one skilled in the art to variously employ the present disclosure.

[0055] Patents, publications, and applications mentioned in the specification are indicative of the levels of those skilled in the art to which the disclosure pertains. These patents, publications, and applications are incorporated herein by reference to the same extent as if each individualpatent, publication, or application was specifically and individually incorporated herein by reference.

[0056] The foregoing description is illustrative of particular embodiments of the disclosure, but is not meant to be a limitation upon the practice thereof. The following claims, including all equivalents thereof, are intended to define the scope of the disclosure.

Claims

CLAIMSWe claim:

1. A method for preparing lipid microparticles comprising:(a) homogenizing a solution comprising a solvent with a therapeutic agent dissolved therein, a surfactant, and a phospholipid composition; and(b) isolating lipid particles from the solution.

2. The method of claim 1, wherein the solution is prepared by adding a first solution to a second solution, wherein the first solution comprises a therapeutic agent dissolved in a solvent and the second solution comprises a surfactant, a phospholipid composition, and water.

3. The method of claim 1 or 2, wherein the solvent is chosen from methanol, ethanol, propanol, acetone, ether, benzene, chloroform, or ethyl acetate.

4. The method of claim 1 or 2, wherein the therapeutic agent is chosen from paclitaxel, rapamycin, daunorubicin, 5 -fluorouracil, doxorubicin, sunitinib, sorafenib, irinotecan, bevcizumab, cetuxamab, biolimus (biolimus A9), everolimus, zotarolimus, tacrolimus, dexamethasone, prednisolone, corticosterone, cisplatin, vinblastine, lidocaine, bupivacaine, bosutinib, ceritinib, crizotinib, gefitinib, ruxolitinib, imatinib, axitinib, nilotinib, trametinib, afatinib, ibrutinib, cabozantinib, imatinib, lenvatinib, sunitinib, regorafenib, sorafenib, vandetanib, dasatinib, pazopanib, triamciclone, tranilast, halofuginone , monte lukast, zafirlukast, pirfenidone, nintedanib, avapritinib, abemaciclib, erdafitinib, fedratinib, nilotinib, nintendanib, palbociclib, pemigatinib, xanthines, aminophylline, sildenafil, tadalafil, vardenafil, udenafil, avanafil, dipyridamole, quinazoline, paraxanthine, papaverine, mesembrenone, rolipram, ibudilast, piclamilast, luteolin, drotaverine, roflumilast, apremilast, crisaborole, inamrinone, milrinone, enoximone, anagrelide, cilostazol, pimobendan, erythro-9-(2-hydroxy-3-nonyl)adenine), (2-[(3,4- dimethoxyphenyl)methyl]-7-[(lR)-l-hydroxyethyl]-4-phenylbutyl]-5-methyl-imidazo[5,l-f][l,2,4]triazin-4(lH)-one), oxindole, (9-(6-phenyl-2-oxohex-3-yl)-2-(3,4-dimethoxybenzyl)- purin-6-one), 3-isobutyl-l-methylxanthine, pentoxifylline, theobromine, theophylline, resveratrol, quercetin, curcumin, chrysin, myricetin, luteolin, apigenin, anthrocyanin, genistein, epigallocatechin gallate, fisetin, astxanthin, tetrahydrocurcumin, imatinib, nintedanib, sorafenib, sunitinib, pazopanib, ROCK inhibitor (Y27632), YAP / TAZ inhibitor (CA3 and verteporfin), YAP / TAZ-TEAD interaction inhibitor (verteporfin, VGLL4 peptide), SRC inhibitor (dasatinib), and combinations thereof5. The method of claim 1 or 2, wherein the surfactant is chosen from sodium stearate, sodium docusate, an alkyl ether phosphate, benzalkonium chloride, perfluorooctane sulfonate, PEG fatty esters, PEG omega-3 fatty esters and alcohols, glycerol fatty esters, sorbitan fatty esters, PEG glyceryl fatty esters, PEG sorbitan fatty esters, sugar fatty esters, PEG sugar esters, Tween 20, Tween 40, Tween 60, p-isononylphenoxypolyglycidol, PEG laurate, PEG oleate, PEG stearate, PEG glyceryl laurate, PEG glyceryl oleate, PEG glyceryl stearate, polyglyceryl laurate, polyglyceryl oleate, polyglyceryl myristate, polyglyceryl palmitate, polyglyceryl-6 laurate, polyglyceryl-6 oleate, polyglyceryl-6 myristate, polyglyceryl-6 palmitate, polyglyceryl- 10 laurate, polyglyceryl- 10 oleate, polyglyceryl- 10 myristate, polyglyceryl- 10 palmitate , PEG sorbitan monolaurate, PEG sorbitan monolaurate, PEG sorbitan monooleate, PEG sorbitan stearate, PEG oleyl ether, PEG lauryl ether, Tween 20, Tween 40, Tween 60, Tween 80, octoxynol, monoxynol, tyloxapol, sucrose monopalmitate, sucrose monolaurate, decanoyl-N-methylglucamide, n-decyl - P -D-glucopyranoside, n-decyl -P -D-maltopyranoside, n-dodecyl -P -D-glucopyranoside, n- dodecyl -P -D-maltoside, heptanoyl-N-methylglucamide, n-heptyl- -D-glucopyranoside, n- heptyl -P -D-thioglucoside, n-hexyl -P -D-glucopyranoside, nonanoyl-N-methylglucamide, n- nonyl -P -D-glucopyranoside, octanoyl-N-methylglucamide, n-octyl-P -D-glucopyranoside, octyl -P -D-thioglucopyranoside, sodium docusate, sorbitol, urea, BHT, BHA, PEG-sorbitan monolaureate, petrolatum, methyl stearate or a combination thereof.

6. The method of claim 1 or 2, wherein the therapeutic is provided to the solvent at a weight to volume ratio of about 0.005g to about 0.5g in a volume of from about 2.5 mL to about 150 mL.

7. The method of claim 6, wherein the surfactant is provided to the second solution at a mass with respect to the first solution of about 1.5 mg to about 160 mg.

8. The method of claim 6, wherein water is to be at a ratio with the first solution of about 10: 1 to about 100:1.

9. The method of claim 6, wherein the phospholipid composition is provided at a mass with respect to the first solution of about 0.05 g to about 5 g.

10. The method of claim 2 or 9, wherein the phospholipid composition comprises a vegetable sourced phospholipid, a sunflower derived phospholipid, soy derived phospholipid, krill derived phospholipid, egg yolk derived phospholipid, milk derived phospholipid, fish derived phospholipid, or combinations thereof.

11. The method of claim 2 or 9, wherein the phospholipid composition comprises a phospholipid chosen from phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin and combinations thereof.

12. The method of claim 10 or 11, wherein the phospholipid composition further comprises cholesterol, a sphingolipid, a glycolipid, or a combination thereof.

13. The method of claim 1, wherein the solution is prepared by dissolving the therapeutic agent in the solvent, and then adding in the surfactant, the phospholipid composition, and water.

14. The method of claim 1 or 2, wherein the homogenization is at a temperature of from 10 to15. The method of claim 1 or 2, wherein the homogenization is for a period of from 1 to 180 min.

16. The method of claim 1 or 2, wherein homogenization is performed with atleast one rotating blade spinning at from 500 to 20,000 RPM.

17. The method of claim 1 or 2, wherein homogenization is performed by application of ultrasound energy at from 20 to 50 kHz.

18. The method of claim 1 or 2, wherein isolating the lipid particles comprises filtration of formed lipid nanoparticles.

19. The method of claim 18, wherein formed lipid particles are filtered with a pore or mesh size of from 50 to 300 pm.

20. The method of claim 18, wherein isolating the lipid nanoparticles further comprises drying.

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