COMPOSITIONS AND METHODS FOR TREATING CARDIOVASCULAR RELATED DISORDERS
The present invention relates to nanoparticles complexed with therapeutic agents configured for treating cardiovascular related disorders, and methods of synthesizing the same. In particular, the present invention is directed to compositions comprising synthetic HDL (sHDL) nanoparticles carrying therapeutic agents configured for treating cardiovascular related disorders, methods for synthesizing such sHDL nanoparticles, as well as systems and methods utilizing such sHDL nanoparticles (e.g., in diagnostic and/or therapeutic settings (e.g., for the delivery of therapeutic agents, imaging agents, and/or targeting agents (e.g., in cardiovascular disease diagnosis and/or therapy, etc.))).
The present invention is a continuation of U.S. patent application Ser. No. 15/561,372, filed Sep. 25, 2017, allowed as U.S. Pat. No. 11,642,419, which is a national stage of International (PCT) Patent Application Serial No. PCT/US2016/024230, filed Mar. 25, 2016, which claims the priority benefit of U.S. Provisional Patent Application 62/138,193, filed Mar. 25, 2015, which are incorporated by reference in their entireties.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENTThis invention was made with government support under GM008353, HL068878 and HL117491 awarded by the National Institutes of Health. The government has certain rights in the invention.
SEQUENCE LISTINGThe text of the computer readable sequence listing filed herewith, titled “34360_303_SequenceListing”, created May 8, 2023, having a file size of 295,245 bytes, is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTIONThe present invention relates to nanoparticles complexed with therapeutic agents configured for treating cardiovascular related disorders, and methods of synthesizing the same. In particular, the present invention is directed to compositions comprising synthetic HDL (sHDL) nanoparticles carrying therapeutic agents configured for treating cardiovascular related disorders, methods for synthesizing such sHDL nanoparticles, as well as systems and methods utilizing such sHDL nanoparticles (e.g., in diagnostic and/or therapeutic settings (e.g., for the delivery of therapeutic agents, imaging agents, and/or targeting agents (e.g., in cardiovascular disease diagnosis and/or therapy, etc.))).
BACKGROUND OF THE INVENTIONThe target of action on many cardiovascular medicines or preventive substances is in the vascular wall. Increase of cholesterol efflux, reduction of inflammation, reduction of oxidation and thrombosis at the vascular wall prevent or alleviate many pathologies (e.g., atherosclerosis, thrombosis, vascular disease) and will reduce heart attacks, strokes and other acute disease manifestation. Yet, there are only select vehicles or particles that accumulate at the vascular wall, enter through endothelial cell layer and are capable of delivery drugs/nutrients to the local vascular areas where they are needed.
Improved compositions and techniques for delivering therapeutic agents targeting vascular regions for therapeutic purposes are needed.
SUMMARYExperiments conducted during the course of developing embodiments for the present invention demonstrated that sHDL nanoparticles selectively accumulate at specific vascular tissue regions (e.g., macrophages associated with atheromatous plaque regions), and improve the efficacy of therapeutic agents delivered to such vascular tissue regions with such sHDL nanoparticles. For example, increased sphingosine-1-phosphate (SIP) related nitric oxide release was demonstrated through its delivery within sHDL nanoparticices (see, Examples I and II). LXR, RXR, and PPARy agonist-encapsulated sHDL nanoparticles were shown to induce significant upregulation of ABC transporters, resulting in increased cholesterol efflux in macrophage (see, Examples XVI, XVII, XVIII and XIX). RXR and LXR agonist-encapsulated sHDL nanoparticles were shown to attenuate atherosclerosis development in vivo at a low dosage (Examples XX and XXI). In addition, LXR agonist-encapsulated sHDL nanoparticles were shown to induce in vivo atherosclerosis regression while avoiding liver toxicity (e.g., hepatic steatosis) (Examples III and IV).
Accordingly, the present invention relates to nanoparticles complexed with therapeutic agents configured for treating cardiovascular related disorders, and methods of synthesizing the same. In particular, the present invention is directed to compositions comprising synthetic HDL (sHDL) nanoparticles carrying therapeutic agents configured for treating cardiovascular related disorders, methods for synthesizing such sHDL nanoparticles, as well as systems and methods utilizing such sHDL nanoparticles (e.g., in diagnostic and/or therapeutic settings (e.g., for the delivery of therapeutic agents, imaging agents, and/or targeting agents (e.g., in cardiovascular disease diagnosis and/or therapy, etc.))).
In certain embodiments, the present invention provides methods for preparing a synthetic HDL-therapeutic agent nanoparticle (sHDL-TA). The present invention is not limited to particular methods for preparing a sHDL-TA. In some embodiments, such methods comprise combining at least one phospholipid having a transition temperature, at least one therapeutic agent, and at least one HDL apolipoprotein in a solvent to produce a mixture; lyophilizing the mixture to produce a dried mixture; hydrating the dried mixture in an aqueous buffer to produce an aqueous mixture; and heating (e.g., thermocycling or incubating) the aqueous mixture above and below the phospholipid transition temperature to produce a sHDL-TA. In some embodiments, the therapeutic agent is configured to treat a cardiovascular disorder. In some embodiments, the HDL apolipoprotein is an HDL apolipoprotein mimetic.
In some embodiments, the solvent is glacial acetic acid. In some embodiments, the aqueous buffer PBS. In some embodiments, the heating is thermocycling. In some embodiments, the thermocycling occurs between 25 and 50° C.
The sHDL-TA nanoparticles are not limited to a particular size. In some embodiments, the average particle size of the sHDL-TA nanoparticle is between 6-20 nm (e.g., 6-14) (e.g., 8-10 nm).
Such methods are not limited to a particular HDL apolipoprotein. For example, in some embodiments, the HDL apolipoprotein is selected from the group consisting of apolipoprotein A-I (apo apolipoprotein A-II (apo A-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), and apolipoprotein E (apo E). In some embodiments, the HDL apolipoprotein is selected from preproapoliprotein, preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, preproApoA-lV, proApoA-lV, ApoA-IV, ApoA-V, preproApoE, proApoE, ApoE, preproApoA-lMilano, proApoA-IMilano ApoA-lMilano preproApoA-IParis, proApoA-IParis, and ApoA-IParis and peptide mimetics of these proteins mixtures thereof.
In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic as described in Srinivasa, et al., 2014 Curr. Opinion Lipidology Vol. 25(4): 304-308, U.S. Pat. Nos. 6,743,778, 7,566,695, and/or U.S. Patent Application Publication Nos. 2003/0171277, 2006/0069030, 2009/0081293, 20110046056, 20130231459. In some embodiments, the the ApoA-I mimetic is described by any of SEQ ID NOs: 1-336. In some embodiments, the ApoA-I mimetic is an ApoA-I mimetic having the following amino acid sequence (PVLDLFRELLNELLEALKQKLK) (SEQ ID NO: 4) (the “22A” ApoA-I mimetic).
Such methods are not limited to a particular phospholipid. In some embodiments, the phospholipid is selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio) propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof.
Such methods are not limited to a particular therapeutic agent. In some embodiments, the therapeutic agent is selected from the group consisting of a liver X receptor agonist, retinoid X receptor agonist, sphingosine-1-phosphate (SIP), angiotensin-converting enzyme (ACE) inhibitors (e.g., benazepril, enalapril, Lisinopril, perindopril, Ramipril), adenosine, alpha blockers (alpha adrenergic antagonist medications) (e.g., clonidine, guanabenz, labetalol, phenoxybenzamine, terazosin, doxazosin, guanfacine, methyldopa, prazosin), angtiotensin II receptor blockers (ARBs) (e.g., candesartan, irbesartan, olmesartan medoxomil, telmisartan, eprosartan, losartan, tasosartan, valsartan), antiocoagulants (e.g., heparin fondaparinux, warfarin, ardeparin, enoxaparin, reviparin, dalteparin, nadroparin, tinzaparin), antiplatelet agents (e.g., abciximab, clopidogrel, eptifibatide, ticlopidine, cilostazol, dipyridamole, sulfinpyrazone, tirofiban), beta blockers (e.g., acebutolol, betaxolol, carteolol, metoprolol, penbutolol, propranolol, atenolol, bisoprolol, esmolol, nadolol, pindolol, timolol), calcium channel blockers (e.g., amlopidine, felodipine, isradipine, nifedipine, verapamil, diltiazem, nicardipine, nimodipine, nisoldipine), diuretics, aldosterone blockers, loop diuretics (e.g., bumetanide, furosemide, ethacrynic acid, torsemide), potassium-sparing diuretics, thiazide diuretics (e.g., chlorothiazide, chlorthalidone, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, metolazone, polythiazide, quinethazone, trichlormethiazide), inoptropics, bile acid sequestrants (e.g., cholestyramine, coletipol, colesevelam), fibrates (e.g., clofibrate, gemfibrozil, fenofibrate), statins (e.g., atorvastatinm, lovastatin, simvastatin, fluvastatin, pravastatin), selective cholesterol absorption inhibitors (e.g., ezetimibe), potassium channel blockers (e.g., amidarone, ibutilide, dofetilide), sodium channel blockers (e.g., disopyramide, mexiletine, procainamide, quinidine, flecainide, moricizine, propafenone), thrombolytic agents (e.g., alteplase, reteplase, tenecteplase, anistreplase, streptokinase, urokinase), vasoconstrictors, vasodilators (e.g., hydralazine, minoxidil, mecamylamine, isorbide dintrate, isorbide mononitrate, nitroglycerin).
In some embodiments, the liver X receptor agonist is selected from TO901317, ATI-111, LXR-623, XL-652, hypocholamide, GW3965, N,N-dimethyl-3beta-hydroxy-cholenamide (DMHCA), 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, (−)anthrabenzoxocinone and (−)bischloroanthrabenzoxocinone ((−)-BABX).
In some embodiments, the retinoid X receptor agonist is selected from Bexarotene, CD3254, Docosahexaenoic acid, fluorobexarotene, isotretinoin, retinoic acid, SR11237, fenretinide, HX630, liarozole dihydrochloride, LG100754 and LG101506.
In some embodiments, the combined LXR and RXR agonists are selected from TO901317, ATI-111, LXR-623, XL-652, hypocholamide, GW3965, N,N-dimethyl-3beta-hydroxy-cholenamide (DMHCA), 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, (−)anthrabenzoxocinone, (−)bischloroanthrabenzoxocinone ((−)-BABX), Bexarotene, CD3254, Docosahexaenoic acid, fluorobexarotene, isotretinoin, retinoic acid, SR11237, fenretinide, HX630, liarozole dihydrochloride, LG100754 and LG101506.
In some embodiments, the methods further comprise combining an imaging agent (e.g., a lipophilic near infrared fluorescent dye or a nuclear imaging agent) with the combining of at least one phospholipid having a transition temperature, at least one therapeutic agent, and at least one HDL apolipoprotein in a solvent to produce a mixture. In some embodiments, the lipophilic near infrared fluorescent dye is DiD.
In certain embodiments, the present invention provides compositions comprising a synthetic HDL-therapeutic agent nanoparticle (sHDL-TA). In some embodiments, the sHDL-TA comprises a mixture of at least one phospholipid, at least one therapeutic agent, and at least one HDL apolipoprotein. In some embodiments, the therapeutic agent is configured to treat a cardiovascular disorder. In some embodiments, the HDL apolipoprotein is an HDL apolipoprotein mimetic.
Such compositions are not limited to a particular HDL apolipoprotein. In some embodiments, the HDL apolipoprotein is selected from the group consisting of apolipoprotein A-I (apo A-I), apolipoprotein A-II (apo A-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), and apolipoprotein E (apo E). In some embodiments, the HDL apolipoprotein is selected from preproapoliprotein, preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, preproApoA-lV, proApoA-lV, ApoA-IV, ApoA-V, preproApoE, proApoE, ApoE, preproApoA-lMilano, proApoA-IMilano ApoA-lMilano preproApoA-IParis, proApoA-IParis, and ApoA-IParis and peptide mimetics of these proteins mixtures thereof.
Such compositions are not limited to a particular phospholipid. In some embodiments, the phospholipid is selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio) propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof.
In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is 22A ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is described by any of SEQ ID NOs: 1-336.
Such compositions are not limited to a therapeutic agent. For example, in some embodiments, the therapeutic agent is as disclosed herein. In some embodiments, the therapeutic agent is a liver X receptor agonist (e.g., TO901317, ATI-111, LXR-623, XL-652, hypocholamide, GW3965, N,N-dimethyl-3beta-hydroxy-cholenamide (DMHCA), 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, (−)anthrabenzoxocinone and (−)bischloroanthrabenzoxocinone ((−)-BABX). In some embodiments, the therapeutic agent is a liver X receptor agonist (e.g., CD3254, Bexarotene, Docosahexaenoic acid, fluorobexarotene, isotretinoin, retinoic acid, SR11237, fenretinide, HX630, liarozole dihydrochloride, LG100754 and LG101506). In some embodiments, the therapeutic agent is a combination of LXR and RXR agonists.
The sHDL-TA nanoparticles are not limited to a particular size. In some embodiments, the average particle size of the sHDL-TA nanoparticle is between 6-20 nm (e.g., 6-14) (e.g., 8-10 nm).
In some embodiments, an imaging agent (e.g., a lipophilic near infrared fluorescent dye or a nuclear imaging agent) is contained within the sHDL-TA mixture of at least one phospholipid, at least one therapeutic agent, and at least one HDL apolipoprotein. In some embodiments, the lipophilic near infrared fluorescent dye is DiD.
In certain embodiments, the present invention provides methods of treating a subject having a cardiovascular related disorder, comprising administering to the subject a therapeutically effective amount of a composition comprising a synthetic HDL-therapeutic agent nanoparticle (sHDL-TA), wherein the sHDL-TA comprises a mixture of at least one phospholipid, at least one therapeutic agent, and at least one HDL apolipoprotein, wherein the therapeutic agent is configured to treat a cardiovascular disorder, wherein the HDL apolipoprotein is an HDL apolipoprotein mimetic.
Such methods are not limited to treating a particular cartdiovascular related disorder. In some embodiments, the cardiovascular related disorder is one or more disorders selected from the group consisting of atherosclerosis, coronary artery disease, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardiac dysrhythmias, inflammatory heart disease (e.g., endocarditis, inflammatory cardiomegaly, myocarditis), vulvular heart disease, cerebrovascular disease, peripheral arterial disease, congenital heart disease, and rheumatic heart disease.
In some embodiments, the HDL apolipoprotein is selected from the group consisting of apolipoprotein A-I (apo apolipoprotein A-II (apo A-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), and apolipoprotein E (apo E). In some embodiments, the HDL apolipoprotein is selected from preproapoliprotein, preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, preproApoA-lV, proApoA-lV, ApoA-IV, ApoA-V, preproApoE, proApoE, ApoE, preproApoA-lMilano, proApoA-IMilano ApoA-lMilano preproApoA-IParis , proApoA-IParis, and ApoA-IParis and peptide mimetics of these proteins mixtures thereof.
In some embodiments, the phospholipid is selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio) propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof.
In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is 22A ApoA-I mimetic.
In some embodiments, the average particle size of the sHDL-TA nanoparticle is between 6-20 nm (e.g., 8-10 nm).
Such methods are not limited to a therapeutic agent. For example, in some embodiments, the therapeutic agent is as disclosed herein. In some embodiments, the therapeutic agents are liver X receptor agonists (e.g., TO901317) and/or retinoid X receptor agonists (e.g., CD3254).
In some embodiments, the methods further comprise an imaging agent (e.g., a lipophilic near infrared fluorescent dye or a nuclear imaging agent) within the sHDL-TA mixture of at least one phospholipid, at least one therapeutic agent, and at least one HDL apolipoprotein. In some embodiments, the lipophilic near infrared fluorescent dye is DiD.
In certain embodiments, the present invention provides methods of targeting a therapeutic agent to an atheromatous plaque region within a biological sample comprising producing a therapeutic agent encapsulated within a synthetic HDL (sHDL) nanoparticle, wherein the sHDL nanoparticle accumulates at atheromatous plaque regions, and exposing the therapeutic agent encapsulated within the sHDL nanoparticle to the biological sample such that the therapeutic agent encapsulated within the sHDL nanoparticle accumulates at atheromatous plaque regions within the biological sample.
Such methods are not limited to a particular manner of producing the therapeutic agent encapsulated within a synthetic HDL (sHDL) nanoparticle. In some embodiments, such producing comprises combining at least one phospholipid having a transition temperature, at least one therapeutic agent, and at least one HDL apolipoprotein in a solvent to produce a mixture; lyophilizing the mixture to produce a dried mixture; hydrating the dried mixture in an aqueous buffer to produce an aqueous mixture; heating (e.g., thermocycling or incubating) the aqueous mixture above and below the phospholipid transition temperature to produce a therapeutic agent encapsulated within a sHDL nanoparticle. In some embodiments, the solvent is glacial acetic acid. In some embodiments, the aqueous buffer PBS. In some embodiments, the heating is thermocycling. In some embodiments, the thermocycling occurs between 25 and 50° C. In some embodiments, the average particle size of the therapeutic agent encapsulated within the sHDL nanoparticle is between 6-20 nm (e.g., 6-14) (e.g., 8-10 nm). In some embodiments, the producing further comprises combining an imaging agent (e.g., a lipophilic near infrared fluorescent dye or a nuclear imaging agent) with the combining of at least one phospholipid having a transition temperature, at least one therapeutic agent, and at least one HDL apolipoprotein in a solvent to produce a mixture. In some embodiments, the lipophilic near infrared fluorescent dye is DiD.
Such methods are not limited to a particular therapeutic agent. In some embodiments, the therapeutic agent is configured to treat a cardiovascular disorder. In some embodiments, the therapeutic agent is as disclosed herein. In some embodiments, the therapeutic agents are liver X receptor agonists (e.g., TO901317) and/or retinoid X receptor agonists (e.g., CD3254).
Such methods are not limited to a particular HDL apolipoprotein. In some embodiments, the HDL apolipoprotein is an HDL apolipoprotein mimetic.the HDL apolipoprotein is selected from the group consisting of apolipoprotein A-I (apo A-I), apolipoprotein A-II (apo A-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), and apolipoprotein E (apo E). In some embodiments, the HDL apolipoprotein is selected from preproapoliprotein, preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, preproApoA-lV, proApoA-lV, ApoA-IV, ApoA-V, preproApoE, proApoE, ApoE, preproApoA-lMilano, proApoA-IMilano ApoA-lMilano preproApoA-IParis , proApoA-IParis, and ApoA-IParis and peptide mimetics of these proteins mixtures thereof. In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is 22A ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is described by any of SEQ ID NOs: 1-336.
Such methods are not limited to a particular type of phospholipid. In some embodiments, the phospholipid is selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio) propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof.
Such methods are not limited a particular type of biological sample. In some embodiments, the biological sample is an in vivo, in vitro or an ex vivo sample. In some embodiments, the biological sample is a living mammal.
In certain embodiments, the present invention provides methods of upregulating ABCA1 and ABCG1 expression within a biological sample, comprising exposing a composition comprising a synthetic HDL-TA nanoparticle to the biological sample, wherein the exposing results in upregulation of ABCA1 and ABCG1 expression within the biological sample. In some embodiments, the sHDL-TA nanoparticle comprises a mixture of at least one phospholipid, at least one therapeutic agent, and at least one HDL apolipoprotein. In some embodiments, the therapeutic agent is a liver X receptor agonist and/or a retinoid X receptor agonist. In some embodiments, the HDL apolipoprotein is an HDL apolipoprotein mimetic. In some embodiments, the therapeutic agents are liver X receptor agonists (e.g., TO901317) and retinoid X receptor agonists (e.g., CD3254).
Such methods are not limited a particular type of biological sample. In some embodiments, the biological sample is an in vivo, in vitro or an ex vivo sample. In some embodiments, the biological sample is a living mammal.
Such methods are not limited to a particular HDL apolipoprotein. In some embodiments, the HDL apolipoprotein is selected from the group consisting of apolipoprotein A-I (apo A-I), apolipoprotein A-II (apo A-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), and apolipoprotein E (apo E). In some embodiments, the HDL apolipoprotein is selected from preproapoliprotein, preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, preproApoA-lV, proApoA-lV, ApoA-IV, ApoA-V, preproApoE, proApoE, ApoE, preproApoA-lMilano, proApoA-IMilano ApoA-lMilano preproApoA-IParis, proApoA-IParis, and ApoA-IParis and peptide mimetics of these proteins mixtures thereof. In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is 22A ApoA-I mimetic.
Such methods are not limited to a particular phospholipid. In some embodiments, the phospholipid is selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof.
In some embodiments, the average particle size of the sHDL-TA nanoparticle is between 6-20 nm (e.g., 6-14) (e.g., 8-10 nm).
In some embodiments, the methods further comprise an imaging agent (e.g., a lipophilic near infrared fluorescent dye or a nuclear imaging agent) within the sHDL-TA mixture of at least one phospholipid, at least one liver X receptor agonist, at least one retinoid X receptor agonist, and at least one HDL apolipoprotein. In some embodiments, the lipophilic near infrared fluorescent dye is DiD.
In some embodiments, the upregulation of ABCA1 and ABCG1 expression within the biological sample occurs within macrophages associated with atheromatous plaque regions within the biological sample. In some embodiments, the upregulation of ABCA1 and ABCG1 expression further induces cholesterol efflux from the macrophages associated with atheromatous plaque regions within the biological sample.
In certain embodiments, the present invention provides methods of inducing cholesterol efflux within a biological sample, comprising exposing a composition comprising a synthetic HDL-TA nanoparticle to the biological sample, wherein the biological sample comprises cells comprising machrophages, wherein the exposing results in cholesterol efflux from the macrophages within the biological sample, wherein the sHDL-TA nanoparticle comprises a mixture of at least one phospholipid, at least one therapeutic agent, and at least one HDL apolipoprotein, wherein the therapeutic agent is a liver X receptor agonist;
wherein the HDL apolipoprotein is an HDL apolipoprotein mimetic.
In some embodiments, the HDL apolipoprotein is selected from the group consisting of apolipoprotein A-I (apo apolipoprotein A-II (apo A-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), and apolipoprotein E (apo E). In some embodiments, the HDL apolipoprotein is selected from preproapoliprotein, preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, preproApoA-lV, proApoA-lV, ApoA-IV, ApoA-V, preproApoE, proApoE, ApoE, preproApoA-lMilano, proApoA-IMilano ApoA-lMilano preproApoA-IParis, proApoA-IParis, and ApoA-IParis and peptide mimetics of these proteins mixtures thereof.
In some embodiments, the phospholipid is selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio) propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof.
In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is 22A ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is described by any of SEQ ID NOs: 1-336.
In some embodiments, the average particle size of the sHDL-TA nanoparticle is between 6-20 nm (e.g., 6-14) (e.g., 8-10 nm).
In some embodiments, the liver X receptor agonist is TO901317.
In some embodiments, the retinoid X receptor agonist is CD3254.
In some embodiments, the methods further comprise an imaging agent (e.g., a lipophilic near infrared fluorescent dye or a nuclear imaging agent) within the sHDL-TA mixture of at least one phospholipid, at least one liver X receptor agonist, at least one retinoid X receptor agonist, and at least one HDL apolipoprotein. In some embodiments, the lipophilic near infrared fluorescent dye is DiD.
In some embodiments, the cholesterol efflux from the macrophages within the biological sample occurs within macrophages associated with atheromatous plaque regions within the biological sample. In some embodiments, the cholesterol efflux coincides with upregulation of ABCA1 and ABCG1 expression within the macrophages associated with atheromatous plaque regions within the biological sample.
Such methods are not limited a particular type of biological sample. In some embodiments, the biological sample is an in vivo, in vitro or an ex vivo sample. In some embodiments, the biological sample is a living mammal.
In certain embodiments, the present invention provides methods for treating atherosclerotic lesions within a subject, comprising administering to the subject a composition comrprising a therapeutically effective amount of a composition comprising a synthetic HDL-therapeutic agent nanoparticle (sHDL-TA), wherein the sHDL-TA comprises a mixture of at least one phospholipid, at least one liver X receptor agonist, at least one retinoid X receptor, and at least one HDL apolipoprotein, wherein the HDL apolipoprotein is an HDL apolipoprotein mimetic, wherein the administering results in accumulation of the sHDL-TA at atherosclerotic lesions within the subject, wherein accumulation of the sHDL-TA at the atherosclerotic lesions within the subject results in cholesterol efflux from macrophages at the atherosclerotic lesions. In some embodiments, the Liver X Receptor agonist is TO901317. In some embodiments, the retinoid X receptor agonist is CD3254.
In some embodiments, administering the composition comprising a sHDL-TA nanoparticle results in reduced liver related lipogenesis in comparison to administration of a liver X receptor agonist not encapsulated within a sHDL nanoparticle. In some embodiments, the liver related lipogenesis is measured by SREBP1c expression.
In some embodiments, the cholesterol efflux from macrophages at the atherosclerotic lesions further involves upregulation of ABCA1 and ABCG1 expression within the macrophages.
Such methods are not limited to a particular HDL apolipoprotein. In some embodiments, the HDL apolipoprotein is selected from the group consisting of apolipoprotein A-I (apo A-I), apolipoprotein A-II (apo A-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), and apolipoprotein E (apo E). In some embodiments, the HDL apolipoprotein is selected from preproapoliprotein, preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, preproApoA-lV, proApoA-lV, ApoA-IV, ApoA-V, preproApoE, proApoE, ApoE, preproApoA-lMilano, proApoA-IMilano ApoA-lMilano preproApoA-IParis, proApoA-IParis, and ApoA-IParis and peptide mimetics of these proteins mixtures thereof. In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is 22A ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is described by any of SEQ ID NOs: 1-336.
Such methods are not limited to a particular phospholipid. In some embodiments, the phospholipid is selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio) propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof.
In some embodiments, the average particle size of the sHDL-TA nanoparticle is between 6-20 nm (e.g., 6-14) (e.g., 8-10 nm).
In some embodiments, the methods further comprise an imaging agent (e.g., a lipophilic near infrared fluorescent dye or a nuclear imaging agent) within the sHDL-TA mixture of at least one phospholipid, at least one liver X receptor agonist, at least one retinoid X receptor agonist, and at least one HDL apolipoprotein. In some embodiments, the lipophilic near infrared fluorescent dye is DiD.
In certain embodiments, the present invention provides methods of inducing nitric oxide efflux within a biological sample, comprising exposing a composition comprising a synthetic HDL-TA nanoparticle to the biological sample, wherein the biological sample comprises cells comprising endothelial cells, wherein the exposing results in nitric oxide efflux from the endothelial cells within the biological sample, wherein the sHDL-TA nanoparticle comprises a mixture of at least one phospholipid, at least one therapeutic agent, and at least one HDL apolipoprotein, wherein the therapeutic agent is sphingosine-1-phosphate; wherein the HDL apolipoprotein is an HDL apolipoprotein mimetic.
Such methods are not limited to a particular HDL apolipoprotein. In some embodiments, the HDL apolipoprotein is selected from the group consisting of apolipoprotein A-I (apo A-I), apolipoprotein A-II (apo A-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), and apolipoprotein E (apo E). In some embodiments, the HDL apolipoprotein is selected from preproapoliprotein, preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, preproApoA-lV, proApoA-lV, ApoA-IV, ApoA-V, preproApoE, proApoE, ApoE, preproApoA-lMilano, proApoA-IMilano ApoA-lMilano preproApoA-IParis, proApoA-IParis, and ApoA-IParis and peptide mimetics of these proteins mixtures thereof. In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is 22A ApoA-I mimetic.
Such methods are not limited to a particular phospholipid. In some embodiments, the phospholipid is selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio) propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof.
In some embodiments, the average particle size of the sHDL-TA nanoparticle is between 6-20 nm (e.g., 6-14) (e.g., 8-10 nm).
In some embodiments, the methods further comprise an imaging agent (e.g., a lipophilic near infrared fluorescent dye or a nuclear imaging agent) within the sHDL-TA mixture of at least one phospholipid, at least one liver X receptor agonist, at least one retinoid X receptor agonist, and at least one HDL apolipoprotein. In some embodiments, the lipophilic near infrared fluorescent dye is DiD.
Such methods are not limited a particular type of biological sample. In some embodiments, the biological sample is an in vivo, in vitro or an ex vivo sample. In some embodiments, the biological sample is a living mammal.
In certain embodiments, the present invention provides methods of detecting the presence of atherosclerotic lesions in a subject, comprising administering to the subject compositions comprising synthetic HDL-imaging agent (sHDL-IA) nanoparticles, wherein the synthetic HDL-IA nanoparticles are known to accumulate at atherosclerotic lesions, wherein the sHDL-IA nanoparticles comprise a mixture of at least one phospholipid, at least one imaging agent, and at least one HDL apolipoprotein, imaging the amount and location of the sHDL-IA within the subject, wherein determination of imaging within vascular regions indicates the presence of atherosclerotic lesions within the subject, wherein the HDL apolipoprotein is selected from the group consisting of apolipoprotein A-I (apo A-I), apolipoprotein A-II (apo A-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), and apolipoprotein E (apo E), wherein the phospholipid is selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio) propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof.
In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is 22A ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is described by any of SEQ ID NOs: 1-336. In some embodiments, the average particle size of the sHDL-IA nanoparticle is between 6-20 nm (e.g., 6-14) (e.g., 8-10 nm).
In some embodiments, the methods further comprise combining a therapeutic agent with the mixture of at least one phospholipid, at least one imaging agent, and at least one HDL apolipoprotein. In some embodiments, the therapeutic agent is as disclosed herein.
In some embodiments, the imaging agent is a lipophilic near infrared fluorescent dye. In some embodiments, the lipophilic near infrared fluorescent dye is DiD.
In certain embodiments, the present invention provides kits comprising at least one phospholipid, at least one therapeutic agent, and at least one HDL apolipoprotein. In som e embodiments, the kits further comprise at least one imaging agent.
In some embodiments, the therapeutic agent is as disclosed herein.
In some embodiments, the HDL apolipoprotein is selected from the group consisting of apolipoprotein A-I (apo apolipoprotein A-II (apo A-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), and apolipoprotein E (apo E). In some embodiments, the HDL apolipoprotein is selected from preproapoliprotein, preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, preproApoA-lV, proApoA-lV, ApoA-IV, ApoA-V, preproApoE, proApoE, ApoE, preproApoA-lMilano, proApoA-IMilano ApoA-lMilano preproApoA-IParis, proApoA-IParis, and ApoA-IParis and peptide mimetics of these proteins mixtures thereof. In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is 22A ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is described by any of SEQ ID NOs: 1-336.
In some embodiments, the phospholipid is selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio) propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof.
In certain embodiments, the present invention provides methods for preparing a synthetic HDL-therapeutic agent nanoparticle (sHDL-TA) comprising combining at least one phospholipid having a transition temperature, at least one therapeutic agent, and at least one HDL apolipoprotein in a solvent to produce a mixture; lyophilizing the mixture to produce a dried mixture; hydrating the dried mixture in an aqueous buffer to produce an aqueous mixture; incubating the aqueous mixture above and below the phospholipid transition temperature to produce a sHDL-TA; wherein the therapeutic agent is configured to treat a cardiovascular disorder; wherein the HDL apolipoprotein is an HDL apolipoprotein mimetic.
In some embodiments, the HDL apolipoprotein is selected from the group consisting of apolipoprotein A-I (apo apolipoprotein A-II (apo A-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), and apolipoprotein E (apo E). In some embodiments, the HDL apolipoprotein mimetic is an ApoA-I mimetic. In some embodiments, the ApoA-I mimetic is described by any of SEQ ID NOs: 1-336.
In some embodiments, the phospholipid is selected from the group consisting of dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio) propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof.
In some embodiments, the therapeutic agent is as disclosed herein.
In some embodiments, the solvent is organic solvent. In some embodiments, the aqueous buffer PBS. In some embodiments, the incubating occurs between 35 and 55° C.
In some embodiments, the average particle size of the sHDL-TA nanoparticle is between 6-20 nm (e.g., 6-14) (e.g., 8-10 nm).
In some embodiments, the methods further comprise combining an imaging agent with the combining of at least one phospholipid having a transition temperature, at least one therapeutic agent, and at least one HDL apolipoprotein in a solvent to produce a mixture. In some embodiments, the imaging agent is a lipophilic near infrared fluorescent dye or a nuclear imaging agent.
In certain embodiments, the present invention provides compositions comprising a synthetic HDL-therapeutic agent nanoparticle (sHDL-TA), wherein the sHDL comprises at least one HDL apolipoprotein and at least one phospholipid, wherein the sHDL-TA has a therapeutic agent to sHDL ratio of 0.1-10% wt/wt, 60-66% wt/wt, or 30/33% wt/wt.
In certain embodiments, the present invention provides compositions comprising a synthetic HDL-therapeutic agent/imaging agent nanoparticle (sHDL-TA/IA), wherein the sHDL comprises at least one HDL apolipoprotein and at least one phospholipid, wherein the sHDL-TA/IA has a (therapeutic agent/imaging agent) to (sHDL) ratio of 0.1-10% wt/wt, 60-66% wt/wt, or 30/33% wt/wt.
In certain embodiments, the present invention provides compositions comprising a synthetic HDL-therapeutic agent nanoparticle (sHDL-TA), wherein the sHDL comprises at least one HDL apolipoprotein and at least one phospholipid, wherein the therapeutic agent is between 0.01-20% by weight of the sHDL-TA, wherein the sHDL is between 80-99.99% by weight of the sHDL-TA. In some embodiments, the therapeutic agent is between 1-10% by weight of the sHDL-TA.
In certain embodiments, the present invention provides compositions comprising a synthetic HDL-therapeutic agent-imaging agent nanoparticle (sHDL-TA/IA), wherein the sHDL comprises at least one HDL apolipoprotein and at least one phospholipid, wherein the therapeutic agent-imaging agent is between 0.01-20% by weight of the sHDL-TA/IA, wherein the sHDL is between 80-99.99% by weight of the sHDL-TA/IA. In some embodiments, the therapeutic agent-imaging agent is between 1-10% by weight of the sHDL-TA/IA.
Additional embodiments will be apparent to persons skilled in the relevant art based on the teachings contained herein.
FIG.7 shows the effects of TO901317-encapsulated sHDL particles on cholesterol efflux in macrophage.
To facilitate an understanding of the present invention, a number of terms and phrases are defined below:
As used here, the term “lipids” refer to fatty substances that are insoluble in water and include fats, oils, waxes, and related compounds. They may be either made in the blood (endogenous) or ingested in the diet (exogenous). Lipids are essential for normal body function and whether produced from an exogenous or endogenous source, they must be transported and then released for use by the cells. The production, transportation and release of lipids for use by the cells is referred to as lipid metabolism. While there are several classes of lipids, two major classes are cholesterol and triglycerides. Cholesterol may be ingested in the diet and manufactured by the cells of most organs and tissues in the body, primarily in the liver. Cholesterol can be found in its free form or, more often, combined with fatty acids as what is called cholesterol esters.
As used herein the term, “lipoproteins” refer to spherical compounds that are structured so that water-insoluble lipids are contained in a partially water-soluble shell. Depending on the type of lipoprotein, the contents include varying amounts of free and esterified cholesterol, triglycerides and apoproteins or apolipoproteins. There are five major types of lipoproteins, which differ in function and in their lipid and apoprotein content and are classified according to increasing density: (i) chylomicrons and chylomicron remnants, (ii) very low density lipoproteins (“VLDL”), (iii) intermediate-density lipoproteins (“IDL”), (iv) low-density lipoproteins (“LDL”), and (v) high-density lipoproteins (“HDL”). Cholesterol circulates in the bloodstream as particles associated with lipoproteins.
As used herein, the term “HDL” or “high density lipoprotein” refers to high-density lipoprotein. HDL comprises a complex of lipids and proteins in approximately equal amounts that functions as a transporter of cholesterol in the blood. HDL is mainly synthesized in and secreted from the liver and epithelial cells of the small intestine. Immediately after secretion, HDL is in a form of a discoidal particle containing apolipoprotein A-I (also called apoA-I) and phospholipid as its major constituents, and also called nascent HDL. This nascent HDL receives, in blood, free cholesterol from cell membranes of peripheral cells or produced in the hydrolysis course of other lipoproteins, and forms mature spherical HDL while holding, at its hydrophobic center, cholesterol ester converted from said cholesterol by the action of LCAT (lecithin cholesterol acyltransferase). HDL plays an extremely important role in a lipid metabolism process called “reverse cholesterol transport”, which takes, in blood, cholesterol out of peripheral tissues and transports it to the liver. High levels of HDL are associated with a decreased risk of atherosclerosis and coronary heart disease (CHD) as the reverse cholesterol transport is considered one of the major mechanisms for HDL's prophylactic action on atherosclerosis.
As used herein, the terms “synthetic HDL,” “sHDL,” “reconstituted HDL”, or “rHDL” refer to a particle structurally analogous to native HDL, composed of a lipid or lipids in association with at least one of the proteins of HDL, preferably Apo A-I or a mimetic thereof, and which exhibits all of the known physiological functions of HDL. Typically, the components of sHDL may be derived from blood, or produced by recombinant technology.
As used herein, the term “atherosclerosis” refers to a cardiovascular related disorder. Generally, atherosclerosis begins with an injury to the inner wall of an artery (endothelium or endothelial cells). Once the inner wall is damaged, a combination of biological processes can lead to the accumulation of the plaque. In response to the injury, macrophages accumulate at the site and migrate beneath the inner layer. The macrophages then begin to absorb fatty substances from the blood and become foam cells. An accumulation of foam cells and other substances, such as proliferating smooth muscle cells, contribute to the formation of plaque and eventually forms bulges in the artery wall. Over time, as the bulges continue to absorb fatty substances, plaque accumulations (atheromatous plaque regions or atherosclerotic plaques) narrow the vessel lumen and occlude the blood flow. Further, plaque accumulation may cause blood vessel walls to harden and lose their elasticity, which can increase resistance to blood flow and raise blood pressure. As a result, vascular diseases are considered a progressive illness with symptoms often not evident until people are middle aged or older.
As used herein, the term “atheroma,” “atheromatous plaque region,” “atherosclerotic plaque,” or “atherosclerotic lesin” refers to an accumulation of degenerative material in the tunica intima (inner layer) of artery walls. The material consists of (mostly) macrophage cells, or debris, containing lipids (cholesterol and fatty acids), calcium and a variable amount of fibrous connective tissue. The accumulated material forms a swelling in the artery wall, which may intrude into the channel of the artery, narrowing it and restricting blood flow. Atheroma occurs in atherosclerosis.
As used herein, the term “liver X receptor” or “LXR” refers to a member of the nuclear receptor family of transcription factors and is closely related to nuclear receptors such as the PPARs, FXR and RXR. Liver X receptors (LXRs) are important regulators of cholesterol, fatty acid, and glucose homeostasis.
As used herein, the term “retinoid X receptors” or “RXR” refers to members of the nuclear receptor family of transcription factors and and are common binding partners to many other nuclear receptors, including PPARs, LXRs and FXR. RXR heterodimers act as ligand-dependent transcriptional regulators and increase the DNA-binding efficiency of its partner.
As used herein, the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
As used herein, the term “sample” is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Environmental samples include environmental material such as surface matter, soil, water, crystals and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present invention.
As used herein, the term “drug” or “therapeutic agent” is meant to include any molecule, molecular complex or substance administered to an organism for diagnostic or therapeutic purposes, including medical imaging, monitoring, contraceptive, cosmetic, nutraceutical, pharmaceutical and prophylactic applications. The term “drug” is further meant to include any such molecule, molecular complex or substance that is chemically modified and/or operatively attached to a biologic or biocompatible structure.
As used herein, the term “solvent” refers to a medium in which a reaction is conducted. Solvents may be liquid but are not limited to liquid form. Solvent categories include but are not limited to nonpolar, polar, protic, and aprotic.
DETAILED DESCRIPTION OF THE INVENTIONExperiments conducted during the course of developing embodiments for the present invention demonstrated that sHDL nanoparticles selectively accumulate at specific vascular tissue regions (e.g., macrophages associated with atheromatous plaque regions), and improve the efficacy of therapeutic agents delivered to such vascular tissue regions with such sHDL nanoparticles. For example, increased sphingosine-1-phosphate (SIP) related nitric oxide release was demonstrated through its delivery within sHDL nanoparticices (see, Examples I and II). In addition, LXR agonist-encapsulated sHDL nanoparticles were shown to induce in vivo atherosclerosis regression while avoiding liver toxicity (e.g., hepatic steatosis) (Examples III and IV). In addition, LXR agonist-encapsulated sHDL nanoparticles and RXR agonist-encapsulated sHDL nanoparticles were shown to attenuate in vivo atherosclerosis development (Example VI).
Accordingly, the present invention relates to nanoparticles complexed with therapeutic agents configured for treating cardiovascular related disorders, and methods of synthesizing the same. In particular, the present invention is directed to compositions comprising synthetic HDL (sHDL) nanoparticles carrying therapeutic agents configured for treating cardiovascular related disorders, methods for synthesizing such sHDL nanoparticles, as well as systems and methods utilizing such sHDL nanoparticles (e.g., in diagnostic and/or therapeutic settings (e.g., for the delivery of therapeutic agents, imaging agents, and/or targeting agents (e.g., in cardiovascular disease diagnosis and/or therapy, etc.))).
The present invention is not limited to specific types or kinds of sHDL nanoparticles carrying a therapeutic agent (e.g., sHDL-TA nanoparticles). Generally, sHDL-TA nanoparticles are composed of a mixture of HDL apolipoprotein, an amphipathic lipid, and a therapeutic agent.
HDL apolipoproteins include, for example apolipoprotein A-I (apo apolipoprotein A-II (apo A-II), apolipoprotein A4 (apo A4), apolipoprotein Cs (apo Cs), and apolipoprotein E (apo E). Preferably, the carrier particles are composed of Apo A-I or Apo A-II, however the use of other lipoproteins including apolipoprotein A4, apolipoprotein Cs or apolipoprotein E may be used alone or in combination to formulate carrier particle mixtures for delivery of therapeutic agents. In some embodiments, the HDL apolipoprotein is selected from preproapoliprotein, preproApoA-I, proApoA-I, ApoA-I, preproApoA-II, proApoA-II, ApoA-II, preproApoA-lV, proApoA-lV, ApoA-IV, ApoA-V, preproApoE, proApoE, ApoE, preproApoA-lMilano, proApoA-IMilano ApoA-lMilano preproApoA-IParis , proApoA-IParis, and ApoA-IParis and peptide mimetics of these proteins mixtures thereof. In some embodiments, mimetics of such HDL apolipoproteins are used.
ApoA-I is synthesized by the liver and small intestine as preproapolipoprotein which is secreted as a proprotein that is rapidly cleaved to generate a mature polypeptide having 243 amino acid residues. ApoA-I consists mainly of 6 to 8 different 22 amino acid repeats spaced by a linker moiety which is often proline, and in some cases consists of a stretch made up of several residues. ApoA-I forms three types of stable complexes with lipids: small, lipid-poor complexes referred to as pre-beta-1 HDL; flattened discoidal particles containing polar lipids (phospholipid and cholesterol) referred to as pre-beta-2 HDL; and spherical particles containing both polar and nonpolar lipids, referred to as spherical or mature HDL (HDL3 and HDL2). Most HDL particles in the circulating population contain both ApoA-I and ApoA-II (the second major HDL protein). However, the fraction of HDL containing only ApoA-I (referred to herein as the AI-HDL fraction) is more effective in reverse cholesterol transport.
In some embodiments, ApoA-I agonists or mimetics are provided. In some embodiments, such ApoA-I mimetics are capable of forming amphipathic α-helices that mimic the activity of ApoA-I, and have specific activities approaching or exceeding that of the native molecule. In some, the ApoA-I mimetics are peptides or peptide analogues that: form amphipathic helices (in the presence of lipids), bind lipids, form pre-β-like or HDL-like complexes, activate lecithin:cholesterol acyltransferase (LCAT), increase serum levels of HDL fractions, and promote cholesterol efflux.
The present invention is not limited to use of a particular ApoA-I mimetic. In some embodiments, any of the ApoA-I mimetics described in Srinivasa, et al., 2014 Curr. Opinion Lipidology Vol. 25(4): 304-308 are utilized. In some embodiments, any of the ApoA-I mimetics described in U.S. Patent Application Publication Nos. 20110046056 and 20130231459 are utilized.
In some embodiments, the “22A” ApoA-I mimetic is used (PVLDLFRELLNELLEALKQKLK) (SEQ ID NO: 4) (see, Examples I-IV) (see, e.g., U.S. Pat. No. 7,566,695). In some embodiments, any of the following ApoA-I mimetics shown in Table 1 as described in U.S. Pat. No. 7,566,695 are utilized:
In some embodiments, an ApoA-I mimetic having the following sequence as described in U.S. Pat. No. 6,743,778 is utilized: Asp Trp Leu Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu Lys Glu Ala Phe (SEQ ID NO: 256).
In some embodiments, any of the following ApoA-I mimetics shown in Table 2 as described in U.S. Patent Application Publication No. 2003/0171277 are utilized:
In some embodiments, an ApoA-I mimetic having the following sequence as described in U.S. Patent Application Publication No. 2006/0069030 is utilized: F-A-E-K-F-K-E-A-V-K-D-Y-F-A-K-F-W-D (SEQ ID NO: 333).
In some embodiments, an ApoA-I mimetic having the following sequence as described in U.S. Patent Application Publication No. 2009/0081293 is utilized: DWFKAFYDKVAEKFKEAF (SEQ ID NO: 334); DWLKAFYDKVAEKLKEAF (SEQ ID NO: 335); PALEDLRQGLLPVLESFKVFLSALEEYTKKLNTQ (SEQ ID NO: 336).
Amphipathic lipids include, for example, any lipid molecule which has both a hydrophobic and a hydrophilic moiety. Examples include phospholipids or glycolipids. Examples of phospholipids which may be used in the sHDL-TA nanoparticles include but are not limited to dipalmitoylphosphatidylcholine (DPPC), dioleoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio) propionate] (DOPE-PDP), 1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide], 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide], phosphatidylcholine, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, and combinations thereof.
In some embodiments, exemplary phospholipids include, but are not limited to, small alkyl chain phospholipids, egg phosphatidylcholine, soybean phosphatidylcholine, dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine 1-myristoyl-2-palmitoylphosphatidylcholine, 1-palmitoyl-2-myristoylphosphatidylcholine, 1-palmitoyl-2-stearoylphosphatidylcholine, 1-stearoyl-2-palmitoylphosphatidylcholine, dioleoylphosphatidylcholine dioleophosphatidylethanolamine, dilauroylphosphatidylglycerol phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylglycerols, diphosphatidylglycerols such as dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, dimyristoylphosphatidylserine, dipalmitoylphosphatidylserine, brain phosphatidylserine, brain sphingomyelin, egg sphingomyelin, milk sphingomyelin, palmitoyl sphingomyelin, phytosphingomyelin, dipalmitoylsphingomyelin, distearoylsphingomyelin, dipalmitoylphosphatidylglycerol salt, phosphatidic acid, galactocerebroside, gangliosides, cerebrosides, dilaurylphosphatidylcholine, (1,3)-D-mannosyl-(1,3)diglyceride, aminophenylglycoside, 3-cholesteryl-6′-(glycosylthio)hexyl ether glycolipids, and cholesterol and its derivatives. Phospholipid fractions including SM and palmitoylsphingomyelin can optionally include small quantities of any type of lipid, including but not limited to lysophospholipids, sphingomyelins other than palmitoylsphingomyelin, galactocerebroside, gangliosides, cerebrosides, glycerides, triglycerides, and cholesterol and its derivatives.
In some embodiments, the sHDL nanoparticles have a molar ratio of phospholipid/HDL apolipoprotein from 2 to 250 (e.g., 10 to 200, 20 to 100, 20 to 50, 30 to 40).
Therapeutic agents include drugs and/or medicaments known to be useful in treating and/or preventing cardiovascular related disorders (e.g., atherosclerosis, heart failure, arrhythmia, atrial fibrillation, hypertension, coronary artery disease, angina pectoris, etc.). Examples of therapeutic agents known to be useful in treating and/or preventing cardiovascular related disorders include, angiotensin-converting enzyme (ACE) inhibitors (e.g., benazepril, enalapril, Lisinopril, perindopril, Ramipril), adenosine, alpha blockers (alpha adrenergic antagonist medications) (e.g., clonidine, guanabenz, labetalol, phenoxybenzamine, terazosin, doxazosin, guanfacine, methyldopa, prazosin), angtiotensin II receptor blockers (ARBs) (e.g., candesartan, irbesartan, olmesartan medoxomil, telmisartan, eprosartan, losartan, tasosartan, valsartan), antiocoagulants (e.g., heparin fondaparinux, warfarin, ardeparin, enoxaparin, reviparin, dalteparin, nadroparin, tinzaparin), antiplatelet agents (e.g., abciximab, clopidogrel, eptifibatide, ticlopidine, cilostazol, dipyridamole, sulfinpyrazone, tirofiban), beta blockers (e.g., acebutolol, betaxolol, carteolol, metoprolol, penbutolol, propranolol, atenolol, bisoprolol, esmolol, nadolol, pindolol, timolol), calcium channel blockers (e.g., amlopidine, felodipine, isradipine, nifedipine, verapamil, diltiazem, nicardipine, nimodipine, nisoldipine), diuretics, aldosterone blockers, loop diuretics (e.g., bumetanide, furosemide, ethacrynic acid, torsemide), potassium-sparing diuretics, thiazide diuretics (e.g., chlorothiazide, chlorthalidone, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, metolazone, polythiazide, quinethazone, trichlormethiazide), inoptropics, bile acid sequestrants (e.g., cholestyramine, coletipol, colesevelam), fibrates (e.g., clofibrate, gemfibrozil, fenofibrate), statins (e.g., atorvastatinm, lovastatin, simvastatin, fluvastatin, pravastatin), selective cholesterol absorption inhibitors (e.g., ezetimibe), potassium channel blockers (e.g., amidarone, ibutilide, dofetilide), sodium channel blockers (e.g., disopyramide, mexiletine, procainamide, quinidine, flecainide, moricizine, propafenone), thrombolytic agents (e.g., alteplase, reteplase, tenecteplase, anistreplase, streptokinase, urokinase), vasoconstrictors, vasodilators (e.g., hydralazine, minoxidil, mecamylamine, isorbide dintrate, isorbide mononitrate, nitroglycerin), cholesteryl ester transfer protein inhibitors (e.g., anacetrapib, evacetrapib), PPAR agonists (e.g., K-877, CER-002, DSP-8658, INT131, GFT505), and apoA-I activators (e.g., RVX-208).
In some embodiments, the therapeutic agent is sphingosine-1-phosphate (SIP) (see, Examples I and II). In some embodiments, the therapeutic agent is a S1P receptor agonist. In some embodiments, the therapeutic agent is a S1P analogue.
In some embodiments, the retinoid X receptor agonist is selected from Bexarotene, CD3254, Docosahexaenoic acid, fluorobexarotene, isotretinoin, retinoic acid, SR11237, fenretinide, HX630, liarozole dihydrochloride, LG100754 and LG101506.
In some embodiments, the combined LXR and RXR agonists are selected from TO901317, ATI-111, LXR-623, XL-652, hypocholamide, GW3965, N,N-dimethyl-3beta-hydroxy-cholenamide (DMHCA), 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, (−)anthrabenzoxocinone, (−)bischloroanthrabenzoxocinone ((−)-BABX), Bexarotene, CD3254, Docosahexaenoic acid, fluorobexarotene, isotretinoin, retinoic acid, SR11237, fenretinide, HX630, liarozole dihydrochloride, LG100754 and LG101506.
In some embodiments, the therapeutic agent is a liver X receptor (LXR) agonist (see, Examples III and IV). Generally, during the development of atherosclerosis, cholesterol deposited in macrophages at sites of atherosclerotic plaques (e.g., atheromatous plaque regions) converts the macrophages into foam cells, which account for the major fraction of lesion deposited cholesterol (see, e.g., Tangirala, R. K.; et al., Proceedings of the National Academy of Sciences of the United States of America 2002, 99 (18), 11896-11901; Rader, D. J.; et al., Cell Metabolism 2005, 1 (4), 223-230; Witztum, J. L.; et al., Journal of Clinical Investigation 1991, 88 (6), 1785-1792). In addition, macrophages produce proteolytic enzymes that can digest extracellular matrix, leading to plaque rupture, resulting in the recruitment of inflammatory cells (see, e.g., Libby, P.; et al., Nature 2011, 473 (7347), 317-325; Vanderwal, A. C.; et al., Circulation 1994, 89 (1), 36-44; Weber, C.; et al., Nature Medicine 2011, 17 (11), 1410-1422). Therefore, both promoting reverse cholesterol transport (RCT) and inhibiting inflammation at the plaque area have been widely pursued as therapeutic strategies for atherosclerosis. Recently, LXR agonists have been found to inhibit the atherosclerosis by reversing the above two pathological processes (see, e.g., Im, S. S.; et al., Circulation Research 2011, 108 (8), 996-1001). Specifically, LXR agonists can up-regulate ABCA1/ABCG1 transporters on macrophages and remove cholesterol from macrophages by reverse cholesterol transport (see, e.g., Briand, F.; et al., Journal of Lipid Research 2010, 51 (4), 763-770; Cuchel, M.; et al., Circulation 2006, 113 (21), 2548-2555), which reduces the lesion cholesterol content directly and prevents the conversion of macrophages into foam cells at sites of atherosclerotic lesions (see, e.g., Tangirala, R. K.; et al., Proceedings of the National Academy of Sciences of the United States of America 2002, 99 (18), 11896-11901). In addition, LXR agonists have been reported to attenuate the inflammatory response through a series of signaling cascades (see, e.g., Ghisletti, S.; et al., Genes & Development 2009, 23 (6), 681-693; Ghisletti, S.; Huang, W.; Ogawa, S.; Pascual, G.; Lin, M. E.; Willson, T. M.; Rosenfeld, M. G.; et al., Molecular Cell 2007, 25 (1), 57-70), which can also contribute to atherosclerosis inhibition (see, e.g., Joseph, S. B.; et al., Nature Medicine 2003, 9 (2), 213-219). However, treatment of experimental atherosclerotic mice with LXR agonists leads to liver toxicity, owing to unrestrained lipogenesis (see, e.g., Repa, J. J.; et al., Genes & Development 2000, 14 (22), 2819-2830; Schultz, J. R.; et al., Genes & Development 2000, 14 (22), 2831-2838). The present invention overcomes such limitations through encapsulation of the LXR agonist within a sHDL nanoparticle thereby ensuring efficient targeting of the drug to the desired atheromatous plaque regions while avoiding liver toxicity side effects.
Previously, high-density lipoproteins (HDLs) have been used for atherosclerotic plaque imaging and delivery of several different therapeutic molecules to the plaque (see, e.g., Skajaa, T.; et al., Biomaterials 2011, 32 (1), 206-213; Cormode, D. P.; et al., Nano Letters 2008, 8 (11), 3715-3723; Luthi, A. J.; et al., Acs Nano 2012, 6 (1), 276-285). HDL is able to target the plaque through two main mechanisms (see, e.g., Kingwell, B. A.; et al., Nat Rev Drug Discov 2014, 13 (6), 445-64). Firstly, the vasculature around atherosclerosis lesions becomes leaky due severe inflammation and endothelial injury, which would facilitate the infiltration of HDL into the vascular tissue (see, e.g., Moulton, K. S.; et al., Circulation 2004, 110 (10), 1330-1336; Zhang, W. L.; et al., International Journal of Pharmaceutics 2011, 419 (1-2), 314-321). Secondly, HDL can be retained in the plaque through ingestion by macrophages and macrophage-derived foam cells, mediated by the SR-BI receptor, ABCA1 and ABCG1 receptors expressed on macrophage surfaces (see, e.g., Rader, D. J., et al., Journal of Clinical Investigation 2006, 116 (12), 3090-3100; Tall, A. R.; et al., Cell Metabolism 2008, 7 (5), 365-375). In addition to the proven safety of HDL in previous clinical trials, the above properties of HDL indicate HDL as an efficient delivery vehicle for LXR agonists to the plaque while minimizing severe side effects (e.g., liver toxicity side effects (e.g., hepatic steatosis)).
As described in Example III, experiments conducted during the course of developing embodiments for the present invention utilized synthetic HDL composed of ApoAl mimetic peptide and lipids to deliver LXR agonists to atherosclerotic plaques.
The present invention is not limited to the use of particular LXR agonists. In some embodiments, the LXR agonist is TO901317. Additional examples of LXR agonist include, but are not limited to, TO901317, ATI-111, LXR-623, XL-652, hypocholamide, GW3965, N,N-dimethyl-3beta-hydroxy-cholenamide (DMHCA), 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, (−)anthrabenzoxocinone and (−)bischloroanthrabenzoxocinone ((−)-BABX)).
The present invention is not limited to the use of particular RXR agonists. In some embodiments, the RXR agonist is CD3254. Additional examples of RXR agonist include, but are not limited to, Bexarotene, CD3254, Docosahexaenoic acid, fluorobexarotene, isotretinoin, retinoic acid, SR11237, fenretinide, HX630, liarozole dihydrochloride, LG100754 and LG101506.
The present invention is not limited to the use of particular LXR or RXR agonists. In some embodiments, LXR and RXR agonists are combined. For example, in some embodiments, the combination of LXR and RXR agonists are from TO901317, ATI-111, LXR-623, XL-652, hypocholamide, GW3965, N,N-dimethyl-3beta-hydroxy-cholenamide (DMHCA), 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, (−)anthrabenzoxocinone and (−)bischloroanthrabenzoxocinone ((−)-BABX)), Bexarotene, CD3254, Docosahexaenoic acid, fluorobexarotene, isotretinoin, retinoic acid, SR11237, fenretinide, HX630, liarozole dihydrochloride, LG100754 and LG101506.
The present invention is not limited to a particular manner of generating sHDL-TA nanoparticles. In some embodiments, for example, such sHDL-TA nanoparticles are formed by mixing an amphipathic lipid and the therapeutic agent in a solvent. The solvent is then removed and the dried lipid mixture is hydrated with an aqueous buffer. HDL apolipoprotein is then added and the composition is mixed vigorously to effect the formation of the sHDL-TA nanoparticles. Example I describes the formation of sHDL-TA nanoparticles wherein the therapeutic agent is sphingosine-1-phosphate (SIP). Example III describes the formation of sHDL-TA nanoparticles wherein the therapeutic agent is an LXR agonist (TO901317). As described, phospholipids, apoA-I mimetic peptide (22A) and TO901317 were hydrated in an aqueous solution and the sHDL-TA nanoparticles were formed after three cydles of thermcal cycling between 25 and 50° C.
In some embodiments, the sHDL-TA nanoparticles are prepared by co-lyophilization methods. For example, in some embodiments, lipids, ApoA mimetic peptides and a therapuetuic agent will be dissolved (e.g., in glacial acetic acid) and lyophilized. The obtained powder will be hydrated in PBS (e.g., pH 7.4) and thermocycled above and below the phospholipid transition temperature to form drug-loaded sHDL (see,
Generally, the sHDL-TA nanoparticles so formed are spherical and have a diameter of from about 5 nm to about 20 nm (e.g., 4-22 nm, 6-18 nm, 8-15 nm, 8-10 nm, etc.). In some embodiments, the sHDL-TA nanoparticles are subjected to size exclusion chromatography to yield a more homogeneous preparation.
In some embodiments, the sHDL-TA nanoparticles are prepared by a thin-film dispersion method. For example, in some embodiments, lipid (e.g., approximately 15 mg lipid) is dissolved in chloroform (e.g., approximately 2 ml chloroform) and mixed with a drug stock DMSO solution (e.g., approximately 2.5 mg/mL drug stock DMSO solution). In some embodiments, organic solvent is evaporated and buffer (50 mM acetate buffer, pH 5.0) added into the lipid/drug mixture to hydrate the film by probe sonication in intervals (e.g., 30 second intervals) using an ultrasonic processor (e.g., a VibraCell ultrasonic processor (Sonics, Newtown, CT)). In some embodiments, apolipoprotein is dissolved in buffer and mixed with the lipid suspension. Next, in some embodiments, the mixture is incubated in water bath (e.g., 50° C. water bath for 5 min) and cooled (e.g., cooled at room temperature for 5 min). In some embodiments, the water bath/cooling is repeated (e.g., cycled three times) to form sHDL-TA nanoparticles.
In some embodiments, the sHDL-TA nanoparticles are prepared by mixing (e.g., vortexing) (e.g., ultraturrexing) buffer with powder formulations of peptide, lipid and therapeutic agent. In some embodiments, the mixture is further incubated at or about the lipid phase transition temperature until sHDL-TA assembly is complete.
Generally, the sHDL-TA nanoparticles so formed are spherical and have a diameter of from about 5 nm to about 20 nm (e.g., 4-22 nm, 6-18 nm, 8-15 nm, 8-10 nm, etc.). In some embodiments, the sHDL-TA nanoparticles are subjected to size exclusion chromatography to yield a more homogeneous preparation.
In some embodiments, the sHDL nanoparticles further encapsulate agents useful for determining the location of administered particles. Agents useful for this purpose include fluorescent tags, radionuclides and contrast agents.
Suitable imaging agents include, but are not limited to, fluorescent molecules such as those described by Molecular Probes (Handbook of fluorescent probes and research products), such as Rhodamine, fluorescein, Texas red, Acridine Orange, Alexa Fluor (various), Allophycocyanin, 7-aminoactinomycin D, BOBO-1, BODIPY (various), Calcien, Calcium Crimson, Calcium green, Calcium Orange, 6-carboxyrhodamine 6G, Cascade blue, Cascade yellow, DAPI, DiA, DID, Di1, DiO, DiR, ELF 97, Eosin, ER Tracker Blue-White, EthD-1, Ethidium bromide, Fluo-3, Fluo4, FM1-43, FM4-64, Fura-2, Fura Red, Hoechst 33258, Hoechst 33342, 7-hydroxy-4-methylcoumarin, Indo-1, JC-1, JC-9, JOE dye, Lissamine rhodamine B, Lucifer Yellow CH, LysoSensor Blue DND-167, LysoSensor Green, LysoSensor Yellow/Blu, Lysotracker Green FM, Magnesium Green, Marina Blue, Mitotracker Green FM, Mitotracker Orange CMTMRos, MitoTracker Red CMXRos, Monobromobimane, NBD amines, NeruoTrace 500/525 green, Nile red, Oregon Green, Pacific Blue. POP-1, Propidium iodide, Rhodamine 110, Rhodamine Red, R-Phycoerythrin, Resorfin, RH414, Rhod-2, Rhodamine Green, Rhodamine 123, ROX dye, Sodium Green, SYTO blue (various), SYTO green (Various), SYTO orange (various), SYTOX blue, SYTOX green, SYTOX orange, Tetramethylrhodamine B, TOT-1, TOT-3, X-rhod-1, YOYO-1, YOYO-3. In some embodiments, ceramides are provided as imaging agents. In some embodiments, S1P agonists are provided as imaging agents.
Additionally radionuclides can be used as imaging agents. Suitable radionuclides include, but are not limited to radioactive species of Fe(III), Fe(II), Cu(II), Mg(II), Ca(II), and Zn(I1) Indium, Gallium and Technetium. Other suitable contrast agents include metal ions generally used for chelation in paramagnetic T1-type MIR contrast agents, and include di- and tri-valent cations such as copper, chromium, iron, gadolinium, manganese, erbium, europium, dysprosium and holmium. Metal ions that can be chelated and used for radionuclide imaging, include, but are not limited to metals such as gallium, germanium, cobalt, calcium, indium, iridium, rubidium, yttrium, ruthenium, yttrium, technetium, rhenium, platinum, thallium and samarium. Additionally metal ions known to be useful in neutron-capture radiation therapy include boron and other metals with large nuclear cross-sections. Also suitable are metal ions useful in ultrasound contrast, and X-ray contrast compositions.
Examples of other suitable contrast agents include gases or gas emitting compounds, which are radioopaque.
In some embodiments, the sHDL-TA nanoparticles further encapsulate a targeting agent. In some embodiments, targeting agents are used to assist in delivery of the sHDL-TA nanoparticles to desired body regions (e.g., bodily regions affected by a cardiovascular related disorder). Examples of targeting agents include, but are not limited to, an antibody, receptor ligand, hormone, vitamin, and antigen, however, the present invention is not limited by the nature of the targeting agent. In some embodiments, the antibody is specific for a disease-specific antigen. In some embodiments, the receptor ligand includes, but is not limited to, a ligand for CFTR, EGFR, estrogen receptor, FGR2, folate receptor, IL-2 receptor, glycoprotein, and VEGFR. In some embodiments, the receptor ligand is folic acid.
In some embodiments, the sHDL-TA nanoparticles further encapsulate transgenes for delivery and expression to a target cell or tissue, in vitro, ex vivo, or in vivo. In such embodiments, rather than containing the actual protein, the sHDL-TA nanoparticles encapsulate an expression vector construct containing, for example, a heterologous DNA encoding a gene of interest and the various regulatory elements that facilitate the production of the particular protein of interest in the target cells.
In some embodiments, the gene is a therapeutic gene that is used, for example, to treat cardiovascular related disorders, to replace a defective gene, or a marker or reporter gene that is used for selection or monitoring purposes. In the context of a gene therapy vector, the gene may be a heterologous piece of DNA. The heterologous DNA may be derived from more than one source (i.e., a multigene construct or a fusion protein). Further, the heterologous DNA may include a regulatory sequence derived from one source and the gene derived from a different source. Tissue-specific promoters may be used to effect transcription in specific tissues or cells so as to reduce potential toxicity or undesirable effects to non-targeted tissues. The nucleic acid may be either cDNA or genomic DNA. The nucleic acid can encode any suitable therapeutic protein.
The nucleic acid may be an antisense nucleic acid. In such embodiments, the antisense nucleic acid may be incorporated into the nanoparticle of the present invention outside of the context of an expression vector.
In some embodiments, the sHDL-TA nanoparticles of the present invention may be delivered to local sites in a patient by a medical device. Medical devices that are suitable for use in the present invention include known devices for the localized delivery of therapeutic agents. Such devices include, but are not limited to, catheters such as injection catheters, balloon catheters, double balloon catheters, microporous balloon catheters, channel balloon catheters, infusion catheters, perfusion catheters, etc., which are, for example, coated with the therapeutic agents or through which the agents are administered; needle injection devices such as hypodermic needles and needle injection catheters; needleless injection devices such as jet injectors; coated stents, bifurcated stents, vascular grafts, stent grafts, etc.; and coated vaso-occlusive devices such as wire coils.
Exemplary devices are described in U.S. Pat. Nos. 5,935,114; 5,908,413; 5,792,105; 5,693,014; 5,674,192; 5,876,445; 5,913,894; 5,868,719; 5,851,228; 5,843,089; 5,800,519; 5,800,508; 5,800,391; 5,354,308; 5,755,722; 5,733,303; 5,866,561; 5,857,998; 5,843,003; and 5,933,145; the entire contents of which are incorporated herein by reference. Exemplary stents that are commercially available and may be used in the present application include the RADIUS (SCIMED LIFE SYSTEMS, Inc.), the SYMPHONY (Boston Scientific Corporation), the Wallstent (Schneider Inc.), the PRECEDENT II (Boston Scientific Corporation) and the NIR (Medinol Inc.). Such devices are delivered to and/or implanted at target locations within the body by known techniques.
As noted, the sHDL-TA nanoparticles of the present invention are useful in treating cardiovascular related disorders. Examples of cardiovascular related disorders include, but are not limited to, atherosclerosis, coronary artery disease, cardiomyopathy, hypertensive heart disease, heart failure, pulmonary heart disease, cardiac dysrhythmias, inflammatory heart disease (e.g., endocarditis, inflammatory cardiomegaly, myocarditis), vulvular heart disease, cerebrovascular disease, peripheral arterial disease, congenital heart disease, and rheumatic heart disease.
The present invention is not limited to a particular method or technique for treating a cardiovascular related disorder. In some embodiments, the methods involve administering to a subject (e.g., a human subject suffering from or at risk for developing a cardiovascular related disorder) a therapeutically effective amount of a composition comprising a sHDL-TA nanoparticle as described herein. The utilized therapeutic agent will depend on the type of condition being treated. For example, if the cardiovascular related disorder is atherosclerosis, the therapeutic agent (in some embodiments) is an LXR agonist (e.g., TO901317) or a RXR agonist (e.g., CD3254).
In some embodiments, the present invention also provides kits comprising sHDL-TA nanoparticles as described herein. In some embodiments, the kits comprise one or more of the reagents and tools necessary to generate sHDL-TA nanoparticles, and methods of using such sHDL-TA nanoparticles.
The sHDL-TA nanoparticles of the present invention may be characterized for size and uniformity by any suitable analytical techniques. These include, but are not limited to, atomic force microscopy (AFM), electrospray-ionization mass spectroscopy, MALDI-TOF mass spectroscopy, 13C nuclear magentic resonance spectroscopy, high performance liquid chromatography (HPLC) size exclusion chromatography (SEC) (equipped with multi-angle laser light scattering, dual UV and refractive index detectors), capillary electrophoresis and get electrophoresis. These analytical methods assure the uniformity of the sHDL-TA nanoparticle population and are important in the production quality control for eventual use in in vivo applications.
In some embodiments, gel permeation chromatography (GPC), which can separate sHDL nanoparticles from liposomes and free ApoA-I mimetic peptide, is used to analyze the sHDL-TA nanoparticles. In some embodiments, the size distribution and zeta-potential is determined by dynamic light scattering (DLS) using, for example, a Malven Nanosizer instrument.
In some embodiments, the encapsulation efficiency of the therapeutic agent will be determined by a desalting column method. Briefly, a sHDL-TA nanoparticle will be passed through a desalting column (cut off=7000 Da) to remove any unencapsulated therapeutic agent, and an equal volume of a sHDL-TA nanoparticle that is not subject to desalting will be used as a comparison. All samples will be incubated with ethanol to break sHDL and subsequently analyzed by HPLC equipped with a C18 column39. In some embodiments, an equation is used to calculate encapsulation efficiency. In some embodiments, the following equation is used to calculate the encapsulation efficiency: Encapsulation efficiency (%)=(the content of drug in sHDL passed through the desalting column)/(the content of therapeutic agent in sHDL not passed through the desalting column)×100%.
In some embodiments, to learn the release profile of therapeutic agent from sHDL, sHDL-TA nanoparticles and free therapeutic agent are placed into a dialysis bag (6-8kda), which will be put in 200 ml PBS (pH 7.4) containing 0.1% Tween 8040. The release media will be put in a 37° C. air bath shaker at 100 rpm. In some embodiments, at predetermined time points, 2 ml of the medium will be sampled and replaced with an equal volume of fresh release media. The amount of therapeutic agent in the media will be quantified by reverse-phase HPLC.
In some embodiments, the sHDL-TA nanoparticles of the present invention are configured such that they are readily cleared from a subject (e.g., so that there is little to no detectable toxicity at efficacious doses).
Where clinical applications are contemplated, in some embodiments of the present invention, the sHDL-TA nanoparticles are prepared as part of a pharmaceutical composition in a form appropriate for the intended application. Generally, this entails preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals. However, in some embodiments of the present invention, a straight sHDL-TA nanoparticle formulation may be administered using one or more of the routes described herein.
In preferred embodiments, the sHDL-TA nanoparticles are used in conjunction with appropriate salts and buffers to render delivery of the compositions in a stable manner to allow for uptake by target cells. Buffers also are employed when the sHDL-TA nanoparticles are introduced into a patient. Aqueous compositions comprise an effective amount of the sHDL-TA nanoparticles to cells dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. The phrase “pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the compositions.
In some embodiments of the present invention, the active compositions include classic pharmaceutical preparations. Administration of these compositions according to the present invention is via any common route so long as the target tissue is available via that route. This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
The active sHDL-TA nanoparticles may also be administered parenterally or intraperitoneally or intratumorally. Solutions of the active compounds as free base or pharmacologically acceptable salts are prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
In some embodiments, a therapeutic agent is released from the sHDL-TA nanoparticles within a target cell (e.g., within a vascular region) (e.g., within an atheroscleroma).
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it may be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active sHDL-TA nanoparticles in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
Upon formulation, sHDL-TA nanoparticles are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the solution is suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, “Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). In some embodiments of the present invention, the active particles or agents are formulated within a therapeutic mixture to comprise about 0.0001 to 1.0 milligrams, or about 0.001 to 0.1 milligrams, or about 0.1 to 1.0 or even about 10 milligrams per dose or so. Multiple doses may be administered.
Additional formulations that are suitable for other modes of administration include vaginal suppositories and pessaries. A rectal pessary or suppository may also be used. Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum, vagina or the urethra. After insertion, suppositories soften, melt or dissolve in the cavity fluids. In general, for suppositories, traditional binders and carriers may include, for example, polyalkylene glycols or triglycerides; such suppositories may be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably 1%-2%. Vaginal suppositories or pessaries are usually globular or oviform and weighing about 5 g each. Vaginal medications are available in a variety of physical forms, e.g., creams, gels or liquids, which depart from the classical concept of suppositories. The sHDL-TA nanoparticles also may be formulated as inhalants.
The present invention also includes methods involving co-administration of the sHDL-TA nanoparticles as described herein with one or more additional active agents. Indeed, it is a further aspect of this invention to provide methods for enhancing prior art therapies and/or pharmaceutical compositions by co-administering the sHDL-TA nanoparticles of this invention. In co-administration procedures, the agents may be administered concurrently or sequentially. In some embodiments, the sHDL-TA nanoparticles described herein are administered prior to the other active agent(s). The agent or agents to be co-administered depends on the type of condition being treated. For example, when the condition being treated is a cardiovascular related disorder, the additional agent includes angiotensin-converting enzyme (ACE) inhibitors (e.g., benazepril, enalapril, Lisinopril, perindopril, Ramipril), adenosine, alpha blockers (alpha adrenergic antagonist medications) (e.g., clonidine, guanabenz, labetalol, phenoxybenzamine, terazosin, doxazosin, guanfacine, methyldopa, prazosin), angtiotensin II receptor blockers (ARBs) (e.g., candesartan, irbesartan, olmesartan medoxomil, telmisartan, eprosartan, losartan, tasosartan, valsartan), antiocoagulants (e.g., heparin fondaparinux, warfarin, ardeparin, enoxaparin, reviparin, dalteparin, nadroparin, tinzaparin), antiplatelet agents (e.g., abciximab, clopidogrel, eptifibatide, ticlopidine, cilostazol, dipyridamole, sulfinpyrazone, tirofiban), beta blockers (e.g., acebutolol, betaxolol, carteolol, metoprolol, penbutolol, propranolol, atenolol, bisoprolol, esmolol, nadolol, pindolol, timolol), calcium channel blockers (e.g., amlopidine, felodipine, isradipine, nifedipine, verapamil, diltiazem, nicardipine, nimodipine, nisoldipine), diuretics, aldosterone blockers, loop diuretics (e.g., bumetanide, furosemide, ethacrynic acid, torsemide), potassium-sparing diuretics, thiazide diuretics (e.g., chlorothiazide, chlorthalidone, hydrochlorothiazide, hydroflumethiazide, methyclothiazide, metolazone, polythiazide, quinethazone, trichlormethiazide), inoptropics, bile acid sequestrants (e.g., cholestyramine, coletipol, colesevelam), fibrates (e.g., clofibrate, gemfibrozil, fenofibrate), statins (e.g., atorvastatinm, lovastatin, simvastatin, fluvastatin, pravastatin), selective cholesterol absorption inhibitors (e.g., ezetimibe), potassium channel blockers (e.g., amidarone, ibutilide, dofetilide), sodium channel blockers (e.g., disopyramide, mexiletine, procainamide, quinidine, flecainide, moricizine, propafenone), thrombolytic agents (e.g., alteplase, reteplase, tenecteplase, anistreplase, streptokinase, urokinase), vasoconstrictors, vasodilators (e.g., hydralazine, minoxidil, mecamylamine, isorbide dintrate, isorbide mononitrate, nitroglycerin). The additional agents to be co-administered can be any of the well-known agents in the art, including, but not limited to, those that are currently in clinical use.
EXPERIMENTALThe following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
Example IThis example describes S1P-sHDL compositions.
Sphingosine-1-phosphate (SIP, CAS 26993-30-6) was obtained from Cayman Chemical Company (Ann Arbor, MI). 22A mimetic peptide (SEQ?) and dipalmitoylphosphatidylcholine (DPPC) was obtained from NOF (Japan). Cell culture reagents were obtained from Lonza (Walkersville, MD).
S1P stock solution was prepared in methanol, heating to 55° C. and using bath sonication to help dissolve. 22A mimetic peptide and DPPC were weighed and added to separate vials, followed by the addition of acidified methanol (containing 10-20% glacial acetic acid) to fully dissolve both peptide and lipid. Various amounts (1-500 nmol) of S1P was then added to DPPC solution, followed by the addition of peptide solution to create a 1:2 peptide:lipid ratio by weight. Samples were flash frozen in liquid nitrogen and lyophilized for 24 hours to remove solvents. Once the solvents evaporated, samples were removed from lyophilizer and hydrated to desired concentrations using 1X PBS (pH 7.4). The solutions were vortexed and thermocycled above and below the lipid glass transition temperature (between 25° C. and 50° C. for DPPC). Thermocycling was repeated 3 times, at which point the solution transitioned from cloudy to clear, indicating formation of HDL. Samples were adjusted to pH 7.4 with NaOH and sterile filtered with either a 0.22 or 0.45 μm syringe filter. sHDL particles containing S1P weare characterized by gel permeation chromatography (GPC), as seen in
This example describes a S1P-HDL in vitro assay.
Human umbilical vein endothelial cells (HUVEC) C2519A Clonetics were recovered from cryopreservation and cultured per manufacturer instruction. At passage 5 the cells were trypsinized, counted, re-plated into 12-well plates at 106 cells per well and incubated for 48 hours at 37° C. and 5% CO2. Media was aspirated and replaced with fresh media containing sHDL, S1P-sHDL, free 22A peptide, or PBS. Cells were placed back into incubator for 10 minutes, after which the media was collected and analyzed for nitric oxide (NO) content via ozone chemiluminescence (
This example demonstrates that LXR agonist-sHDL successfully upregulated ABCA1 expression in macrophages.
Incorporation of LXR agonist into sHDL is the first and most important step for all subsequent studies. Phospholipids, apoA-I mimetic peptide (22A) and LXR agonist TO901317 (TO) were hydrated in an aqueous solution and the sHDL nanoparticles were formed after 3 cycles of thermal cycling between 25 and 50° C. (see, e.g., Di Bartolo, B. A.; et al., Atherosclerosis 2011, 217 (2), 395-400; Dasseux, J.-L. Peptide/lipid complex formation by co-lyophilization. 2001). The homogeneity of the size distribution was confirmed by gel permeation chromatography (GPC) and scanning electron microscopy (SEM) (
This example shows synthetic HDL (sHDL) nanomedicines can accumulate in atherosclerotic lesions, with less side effects than free LXR agonists.
To visualize whether sHDL can deliver its cargo to the plaque efficiently, a lipophilic near-infrared fluorescent dye, DiD, was incorporated into sHDL. DiD was chosen because it exhibits little auto-fluorescence and possesses a low phototoxicity. The DiD-labeled sHDL nanoparticles were administrated by tail vein injection at a dose of 100 μg DiD and 10 mg/Kg sHDL to a murine atherosclerosis model. 24 hours post injection, animals were sacrificed and aortas were removed. DiD fluorescence associated with aortas were analyzed using the Xenogen IVIS optical imaging system. This study clearly showed that the sHDL nanoparticles accumulated in atherosclerotic lesions (
This example describes the materials and methods used in conducting the experiments described in Example 6.
Preparation of Drug-Loaded sHDL Nanoparticles
Drug-loaded sHDL nanoparticles were prepared by a co-lyophilization method. Briefly, lipids, ApoA mimetic peptides and a therapeutic agent (TO901317, Rosiglitazone, or CD3254) were dissolved in glacial acetic acid and lyophilized. The obtained powder was hydrated in PBS (pH 7.4) and thermocycled above and below the phospholipid transition temperature to form drug-loaded sHDL nanoparticles.
Characterization of Drug-Loaded sHDL Nanoparticles
Size and Morphology
The drug-loaded sHDL nanoparticles were characterized for purity by gel permeation chromatography (GPC), which can separate sHDL nanoparticles from liposomes and free
ApoA-I mimetic peptide. The size distribution and zeta-potential were determined by dynamic light scattering (DLS) using a Malven Nanosizer instrument after proper dilution of samples.
To observe the morphology of drug-loaded sHDL nanoparticles, they were further characterized by transmission electron microscopy (TEM). Briefly, 3 μL of the sample solution was deposited on carbon film-coated 400 mesh copper grids (Electron Microscopy Sciences) and dried for 1 minute. The samples were then negatively-stained with 5 droplets of 1% uranyl acetate solution, excessive solutions on the grid were blotted and the grid was dried before TEM observation.
Encapsulation Efficiency
The encapsulation efficiency of the therapeutic agent was determined by a desalting column method. Briefly, drug-loaded sHDL was passed through a desalting column (MWCO=7000 Da) to remove any unencapsulated drug, and an equal vole of a sHDL-TA nanoparticle not passed through the desalting column was used as a control to calculate the total amount of unencapsulated and encapsulated drug. All samples were incubated with ethanol to break sHDL and subsequently analyzed by HPLC equipped with a C18 column. The following equation was used to calculate the encapsulation efficiency: Encapsulation efficiency (%)=(the content of drug in sHDL passed through the desalting column)/(the content of therapeutic agent in sHDL not passed through the desalting column)×100%.
Drug Release from sHDL Nanoparticles
To learn the release profile of therapeutic agent from sHDL, drug-loaded sHDL nanoparticles or free therapeutic agent was placed into a dialysis bag (6-8 kda), which was put in 200 ml PBS (pH 7.4) containing 0.1% Tween 80. The release medium was put in a 37° C. air bath shaker at 100 rpm. At predetermined time points, 2 ml of the medium was sampled and an equal volume of fresh release medium was added back. The amount of therapeutic agent in the release medium was quantified by reverse-phase HPLC.
Example VIThis example demonstrates the successful encapsulation of six compounds and fatty acids in sHDL nanoparticles. For example, the average particle sizes of sHDL and sHDL-TO (TO901317, Rosiglitazone, or CD3254) nanoparticles 8-12 nm measured by transmission electron microscopy. The sHDL-TO encapsulation efficiency was more than 85%. Four of those compounds were shown to have the ability to upregulate the expression of ABC transporters, which predominately control cholesterol efflux activity in macrophages. sHDL-compound nanoparticle treatment can significantly increase the upregulative effects of those compounds.
All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the relevant fields are intended to be within the scope of the present invention.
Claims
1. A method for preparing a synthetic HDL-therapeutic agent nanoparticle (sHDL-TA) comprising
- combining at least one phospholipid having a transition temperature, at least one therapeutic agent, and at least one HDL apolipoprotein in a solvent to produce a mixture;
- lyophilizing the mixture to produce a dried mixture;
- hydrating the dried mixture in an aqueous buffer to produce an aqueous mixture;
- thermocycling the aqueous mixture above and below the phospholipid transition temperature to produce a sHDL-TA;
- wherein the therapeutic agent is configured to treat a cardiovascular disorder;
- wherein the HDL apolipoprotein is an HDL apolipoprotein mimetic.
Type: Application
Filed: May 8, 2023
Publication Date: Oct 19, 2023
Inventors: Anna A. Schwendeman (Ann Arbor, MI), Yuqing Eugene Chen (Superior Township, MI), Yanhong Guo (Ann Arbor, MI), Jifeng Zhang (Ann Arbor, MI), Wenmin Yuan (Ann Arbor, MI), Emily Morin (Ann Arbor, MI)
Application Number: 18/313,856
