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WO2025095862A1 - Composition, procédé de fabrication et utilisations de ladite composition - Google Patents

Composition, procédé de fabrication et utilisations de ladite composition Download PDF

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Publication number
WO2025095862A1
WO2025095862A1 PCT/SG2024/050699 SG2024050699W WO2025095862A1 WO 2025095862 A1 WO2025095862 A1 WO 2025095862A1 SG 2024050699 W SG2024050699 W SG 2024050699W WO 2025095862 A1 WO2025095862 A1 WO 2025095862A1
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WIPO (PCT)
Prior art keywords
active agent
composition
drug carrier
myriocin
lipid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/SG2024/050699
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English (en)
Inventor
Jiong-Wei WANG
Chenyuan HUANG
Giorgia Pastorin
Gerrit Storm
Suet Yen CHONG
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National University of Singapore
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National University of Singapore
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Application filed by National University of Singapore filed Critical National University of Singapore
Publication of WO2025095862A1 publication Critical patent/WO2025095862A1/fr
Pending legal-status Critical Current
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/201—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having one or two double bonds, e.g. oleic, linoleic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates generally to a composition and a method of preparing the composition as defined herein.
  • the present invention also relates to a method of delivering the composition, a method of treating using the composition and medical uses of the composition as defined herein.
  • Myocardial infarction occurs when the epicardial coronary artery is blocked and accounts for the highest morbidity and mortality globally.
  • myocardial protective drugs such as cardiotonic agents, 13 0-blockers, and anti-inflammatory drugs
  • cardiotonic agents such as cardiotonic agents, 13 0-blockers, and anti-inflammatory drugs
  • anti-inflammatory drugs are effective in reducing the infarct size
  • most of these pharmaceuticals lack the specificity to the heart and therefore, their bioavailability at the injured target site is limited.
  • high dosages of these drugs may also create nonspecific toxicity and other side effects.
  • Myriocin is a specific inhibitor of the ceramide de novo synthesis pathway. Since ceramides have been closely associated with cardiovascular and metabolic diseases, the therapeutic efficacy of myriocin has been tested in various animal diseases. As myriocin is insoluble in aqueous phase, most studies investigate the therapeutic efficacy via oral administration. Although oral administration is able to exert the therapeutic effect, the bioavailability of the drug is much lower compared to intravenous injection. Moreover, long term high dose usage through oral administration has been shown to cause gut and systemic toxicity.
  • myriocin a specific inhibitor to the toxic lipids, ceramides
  • myriocin a specific inhibitor to the toxic lipids, ceramides
  • another prevailing challenge would be the extremely low solubility of myriocin in water which hinders its application via intravenous injection.
  • delivery systems to deliver small molecules to cardiomyocytes are also a lack of delivery systems to deliver small molecules to cardiomyocytes.
  • a delivery system that can overcome, or at least ameliorate the disadvantages described above.
  • a delivery system to deliver an active agent to cardiomyocytes for the treatment of ischemic heart disease and atherosclerosis, as well as for sphingolipid-related metabolic diseases.
  • the present disclosure relates to a composition
  • a composition comprising an active agent and a drug carrier, wherein the drug carrier at least partially encapsulates the active agent, and wherein the molar ratio of the active agent to the drug carrier is between about 1:20 to about 1 :40.
  • the composition when the molar ratio of the active agent to the drug carrier is between about 1 :20 to about 1:40, the composition is able to reach a maximum encapsulation efficiency of about 98%. Furthermore, due to the smaller size of the composition in comparison with the solid lipid nanocarriers known in the art and at the molar ratio as defined above, the inventors have also found that the composition of the present invention is able to achieve a higher thermodynamic stability.
  • the present disclosure relates to a method of forming a composition as defined herein, comprising the step of dissolving a layer comprising an active agent and a drug carrier in an aqueous solvent to thereby at least partially encapsulate the active agent within the drug carrier to form the composition, wherein the molar ratio of the active agent to the drug carrier in the composition is between about 1 :20 to about 1 :40.
  • the above method is a one-step method that allows for a higher loading rate of the active agent (such as about 45% to about 55%, about 51% to about 52%, or about 51.7%) when compared to a two-step solvent assisted method to encapsulate the active agent within the drug carrier.
  • the method may further comprise, before the dissolving step, the steps of a) co-dissolving the active agent and the drug carrier in an organic solvent to form a solution; and b) removing the organic solvent from the solution formed to form the layer.
  • the composition may not be formed and this step provides a non-polar environment (such as the organic solvent) for the active agent and the drug carrier to freely dissolve in an unassembled state to form the solution.
  • the composition may be formed when dissolved in the aqueous solvent.
  • the active agent and the drug carrier when dissolved in the aqueous solvent, due to the amphiphilicity of the drug carrier, it may self-assemble into spherical particles with the hydrophilic end facing outwards (forming micelle outer shell) and the hydrophobic end facing inwards (forming micelle core). Therefore, this allows for the active agent to auto-assemble in the micelle core due to its hydrophobicity.
  • the present disclosure relates to a method of delivering an active agent to a target site, comprising the steps of:
  • composition comprising the active agent and a drug carrier as described herein or as formed according to the method as described herein, wherein in the composition, the drug carrier at least partially encapsulates the active agent and the molar ratio of the active agent to the drug carrier is about 1 :20 to about 1 :40, and
  • composition (b) administering the composition at a first site to allow the active agent to move to or be transported to the target site.
  • the present disclosure relates to a method of treating a disease in a subject, comprising administering to the subject an effective amount of the composition as described herein, wherein the disease is selected from the group consisting of an ischemic heart disease, atherosclerosis, a sphingolipid-related metabolic disease and a combination thereof.
  • the present disclosure relates to the composition as described herein for use in therapy.
  • the present disclosure relates to the composition as described herein for use in treating or preventing a disease selected from the group consisting of an ischemic heart disease, atherosclerosis, a sphingolipid-related metabolic disease and a combination thereof.
  • the present disclosure relates to the use of the composition as described herein in the manufacture of a medicament for the treatment of a disease selected from the group consisting of an ischemic heart disease, atherosclerosis, a sphingolipid-related metabolic disease and a combination thereof.
  • the term “encapsulate” refers to the drug carrier enclosing the active agent within its core or cavity, wherein the active agent is not covalently bonded to the drug carrier.
  • the term “partially encapsulate” refers to the drug carrier enclosing parts of the active agent such that the un-enclosed parts of the active agent are exposed to an external environment.
  • the term “at least partially encapsulate” includes partial encapsulation of the active agent as well as total encapsulation of the active agent, the latter being the active agent being enclosed by the drug carrier in its entirety such that none of the active agent is exposed to the external environment.
  • the term “particle size” refers to the average diameter of the composition or the nanoparticle which is calculated based on an average of several diametral measurements of the composition or the nanoparticle.
  • the term “non-covalently bonded” refers to the active agent and the drug carrier having weak interactions such as hydrogen bonding, Van der Waals interaction or hydrophobic/hydrophilic interaction (particularly hydrophobic interaction) and are not bonded chemically through chemical reactions which may alter the structure of the active agent or the drug carrier.
  • the term “about”, in the context of concentrations of components of the formulations, typically means +/- 5% of the stated value, more typically +/- 4% of the stated value, more typically +/- 3% of the stated value, more typically, +/- 2% of the stated value, even more typically +/- 1% of the stated value, and even more typically +/- 0.5% of the stated value.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosed ranges. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range
  • composition comprises an active agent and a drug carrier, wherein the drug carrier at least partially encapsulates the active agent, and wherein the molar ratio of the active agent to the drug carrier is between about 1 :20 to about 1 :40.
  • the molar ratio of the active agent to the drug carrier may be between about 1 :20 to about 1 :40, about 1 :25 to about 1 :40, about 1 :28 to about 1 :40, about 1 :30 to about 1 :40, about 1:32 to about 1:40, about 1 :34 to about 1 :40, about 1 :25 to about 1 :28, about 1:25 to about 1 :30, about 1 :25 to about 1 :32, about 1 :20 to about 1 :25, or about 1 :25 to about 1 :34.
  • the weight ratio of the active agent to the drug carrier may be between about 1: 125 to about 1 :250 (w/w), about 1 :150 to about 1 :250 (w/w), about 1 :200 to about 1 :250 (w/w), about 1 :210 to about 1 :250 (w/w), about 1 :220 to about 1:250 (w/w), about 1:230 to about 1 :250 (w/w), about 1 :240 to about 1:250 (w/w), about 1 : 125 to about 1: 150 (w/w), about 1 :125 to about 1 :200 (w/w), about 1 : 125 to about 1 :210 (w/w), about 1 : 125 to about 1 :220 (w/w), about 1 : 125 to about 1 :230 (w/w) or about 1:125 to about 1 :240 (w/w).
  • the composition may be termed as a formulation.
  • the formulation may be a drug delivery system used to deliver the active agent (in the form of small molecules) to a target site for the treatment of a disease.
  • the composition may be in the form of a nanoparticle.
  • the nanoparticle may have a particle size in the range of about 15 nm to about 25 nm, about 17 nm to about 23 nm, about 19 nm to about 25 nm, about 19 nm to about 25 nm, about 21 nm to about 25 nm, about 23 nm to about 25 nm, about 15 nm to about 17 nm, about 15 nm to about 19 nm, about 15 nm to about 21 nm, or about 15 nm to about 23 nm.
  • the particle size may be measured by Dynamic Light Scattering (DLS) method or Transmission Electron Microscopy which are commonly used method known to a person skilled in the art.
  • the composition when the composition is in the nanoparticle size as defined above, the composition may be delivered to the target site in a targeted manner and with higher specificity.
  • the composition may have a polydispersity index (PDI) in the range of about 0.05 to about 0.3, about 0.1 to about 0.3, about 0.15 to about 0.3, about 0.2 to about 0.3, about 0.25 to about 0.3, about 0.05 to about 0.1, about 0.05 to about 0.15, about 0.05 to about 0.2, or about 0.05 to about 0.25.
  • the poly dispersity index (PDI) may be measured by Dynamic Light Scattering (DLS) method which is a commonly used method known to a person skilled in the art.
  • the composition may have a zeta potential in the range of about -9 mV to about -2 mV, about -7 mV to about -2 mV, about -5 mV to about -2 mV, about -3 mV to about -2 mV, about -9 mV to about -2 mV, about -9 mV to about -7 mV, about -9 mV to about -5 mV, or about -9 mV to about -3 mV.
  • the drug carrier may partially or fully encapsulate the active agent.
  • the drug carrier may have both hydrophobic and hydrophilic properties and may thus be considered as being amphiphilic.
  • the drug carrier may be a lipid.
  • the lipid may be selected from PEGylated lipid with amphiphilicity.
  • the PEGylated lipid with amphiphilicity may be PEGylated phospholipid and sphingolipid.
  • the lipid may be selected from 1, 2-Distearoyl-sn- glycero-3-phosphoethanolamine-Poly(ethylene glycol) (DSPE-PEG), dipalmitoyl phosphatidylcholine (DPPC), l,2-Distearoyl-.w?-glycero-3-phosphocholine (DSPC), cholesterol or a combination thereof.
  • the drug carrier may be DSPE-PEG.
  • the drug carrier when the drug carrier is made of the lipid as defined above, it may facilitate easier manufacturing of the composition with a total preparation time within a few hours (less than 3.5 hours) in comparison with a drug carrier made from other substances such as inorganic materials (silica).
  • the drug carrier may be labelled with a fluorescent dye.
  • the fluorescent dye may be any suitable dye that is not toxic to a living organism.
  • the fluorescent dye may be cyanine acid (Cy5).
  • the drug carrier may form micelles in an aqueous solution (such as phosphate buffered saline (PBS)) with a hydrophobic lipid core and a hydrophilic outer layer.
  • PBS phosphate buffered saline
  • the drug carrier is the lipid DSPE-PEG
  • the lipid forms micelles in the aqueous solution with DSPE as the hydrophobic lipid core and PEG as the hydrophilic outer layer.
  • the active agent is then at least partially encapsulated within the hydrophobic lipid core.
  • the drug carrier in the form of lipid micelles may facilitate the aqueous phase solubilization of the active agent, as well as targeted delivery and release of the active agent into the target site (such as cardiomyocytes) and infiltrate immune cells.
  • the encapsulation efficiency of the drug carrier may be in the range of about 90% to about 98%, about 92% to about 98%, about 94% to about 98%, about 96% to about 98%, where encapsulation efficiency refers to the percentage of the amount of encapsulated active agent in the final product (composition) to the initial amount of active agent before encapsulation (that which is present in the aqueous solution)
  • encapsulation efficiency refers to the percentage of the amount of encapsulated active agent in the final product (composition) to the initial amount of active agent before encapsulation (that which is present in the aqueous solution)
  • the encapsulation of the active agent in the drug carrier reduces the systemic side effects of the active agent. More advantageously, a high encapsulation efficiency (up to about 98%) ensures that most of the active agent in the solution is encapsulated by the drug carrier, which enhances the cardiac-targeting property of the composition.
  • the loading rate of the drug carrier may be in the range of about 45% to about 55%, about 51% to about 52% or about 51.7%, where the loading rate refers to the weight percentage of the active agent present in the composition based on the total weight of the composition.
  • the method used in the present disclosure allows for a higher loading rate of the active agent in the drug carrier (of around 51.7%) when compared to a two-step solvent assisted method to encapsulate the active agent within a drug carrier.
  • the drug carrier may have a molecular weight in the range of about 2500 g/mol to about 6000 g/mol, about 3000 g/mol to about 6000 g/mol, about 4000 g/mol to about 6000 g/mol, about 5000 g/mol to about 6000 g/mol or about 2500 g/mol to about 5000 g/mol.
  • the drug carrier may be DSPE-PEG2000 or DSPE-PEG5000.
  • the composition may be a drug-lipid micelle.
  • the inventors have found that the cellular internalization of the composition (drug-lipid micelle) may be dependent upon the molecular weight of the drug carrier. Accordingly, a composition (drug-lipid micelle) where the drug carrier has a larger molecular weight (such as DSPE-PEG5000) was less internalized into the cells (cardiomycetes) compared to another composition having a drug carrier with a smaller molecular weight (such as DSPE-PEG2000) at the same molar concentration.
  • the inventors have also found that the composition (drug-lipid micelle) may have organ selectivity towards heart tissue during ischemia/repurfusion injury due to the enhanced permeability and retention effect.
  • the active agent may be a drug.
  • the active agent may be a hydrophobic compound
  • the hydrophobic compound may be selected from myriocin, fumonisin Bl, a derivative of myriocin, sphingofungin B, sphingofungin C, lipoxamycin or a combination thereof.
  • the active agent may be myriocin.
  • the hydrophilic outer layer (PEG) of the drug carrier (lipid micelle) facilitates long circulation in the blood and specific delivery of the active agent (myriocin) to the injured tissue (cardiomyocytes and infiltrated immune cells).
  • the method of forming the composition as defined herein comprises the step of dissolving a layer comprising an active agent and a drug carrier in an aqueous solvent to thereby at least partially encapsulate the active agent within the drug carrier to form the composition, wherein the molar ratio of the active agent to the drug carrier is between about 1:20 to about 1 :40.
  • the composition may only be formed when dissolved in the aqueous solvent.
  • the active agent and the drug carrier when dissolved in the aqueous solvent, due to the amphiphilicity of the drug carrier, it may self-assemble into spherical particles with the hydrophilic end facing outwards (forming micelle outer shell) and the hydrophobic end facing inwards (forming micelle core). Therefore, this allows for the active agent to auto-assemble in the micelle core due to its hydrophobicity.
  • the drug carrier may at least partially enclose the active agent within its core or cavity, wherein the active agent is not covalently bonded to the drug carrier. Accordingly, when the active agent is at least partially encapsulated or non-covalently bonded to the drug carrier forming a non-covalently bonded composition, the structure of the active agent remains unaltered chemically. Elowever, when the active agent is covalently bonded to the drug carrier, it may form a covalently bonded composition where the structure of the active agent is chemically altered, and the active/binding site of active agent might be hindered by the drug carrier. Furthermore, the active agent may not be released from the drug carrier due to the strong covalent bond and may not be catabolized.
  • the active agent and the drug carrier may be as defined above.
  • the aqueous solvent used may be selected from an aqueous buffer.
  • the aqueous solvent used may be water, phosphate buffered saline (PBS), HEPES buffered saline (HBS), sucrose buffer, saline or a combination thereof.
  • the solvent used may be PBS.
  • the dissolving step may include rotating the solution on a rotary evaporator at a speed in a range of about 150 rpm to about 200 rpm.
  • the dissolving step may be carried out at a temperature in the range of about 40°C to about 80°C.
  • the dissolving step may be carried out at a pressure in the range of about 40 mbar to about 100 mbar.
  • the dissolving step may be carried out for a period of time in the range of about 1 to 2 hours.
  • the rotary speed of the rotary evaporator, the temperature, pressure and time period provided above are guidelines and will depend on the drug carrier actually used.
  • the method may further comprise before the dissolving step, the steps of a) co-dissolving the active agent and the drug carrier in an organic solvent to form a solution; and b) removing the organic solvent from the solution formed to form the layer.
  • the composition may not be formed and this step advantageously provides a non-polar environment (such as the organic solvent) for the active agent and the drug carrier to freely dissolve in an unassembled state to form the solution.
  • the dissolving step may comprise the step of purifying to form the composition.
  • the organic solvent used for dissolving the active agent and the drug carrier may be an alcohol.
  • the organic solvent used may be methanol.
  • the solvent may be removed by means of rotary evaporation known in the art.
  • the temperature used to remove the solvent may be about 40°C to about 80°C.
  • the pressure used to remove the solvent may be in a range of about 40 mbar to about 100 mbar.
  • the speed of rotation used to remove the solvent may be in a range of about 150 rpm to about 200 rpm.
  • the solvent may be removed for a duration in a range of about 60 minutes to about 2 hours.
  • the rotary speed of the rotary evaporator, the temperature, pressure and time period provided above are guidelines and will depend on the drug carrier actually used.
  • the dissolving step may comprise the step of purifying to form the composition.
  • the purifying step may include removal processes such as fdtration and sterilization.
  • the removal processes may include removing unwanted materials from the solution to form the composition.
  • the unwanted materials may be in the form of aggregates, precipitates and bacteria.
  • the size of the filter used to remove aggregates and precipitates should be below about 0.45 pm; when an additional sterilization step is required or if the formulation is intended for injection, the size of the second filter should be below 0.22 pm.
  • the filter may be a hydrophobic filter. Where bacteria is meant to be removed, the size of the filter can be selected from 0.22pm; where aggregates and precipitates are to be removed, the size of the filter can be selected from 0.45 pm.
  • the purifying step is sterilization, the solution of the composition formed may be further sterilized to form the injectable composition.
  • the method of delivering the active agent to the target site comprises the steps of:
  • composition comprising the active agent and a drug carrier as described herein or as formed according to the method as described herein, wherein in the composition, the drug carrier at least partially encapsulates the active agent and the molar ratio of the active agent to the drug carrier is about 1 :20 to about 1 :40;
  • the administering step b) may be undertaken by injection at the first site.
  • the injection may be via intravenous, intraperitoneal, intraventricular, subcutaneous or combinations thereof.
  • the injection may be intravenous injection.
  • the aqueous phase solubilization of the active agent facilitated by the drug carrier allows for intravenous application and release of the active agent into the target site (such as cardiomyocytes) and infiltrate immune cells.
  • the composition may be absorbed into the body and the composition or the active agent may move to or be transported via the circulatory system to the target site (thus being delivered to the target site), the target site being the site where the disease happens or where a symptom of the disease is to be treated.
  • the target site can be a diseased organ or tissue.
  • composition may be administered at a dosage of about 1 mg/kg/day to about 2 mg/kg/day.
  • exemplary, non-limiting embodiments of a composition for use in therapy will now be disclosed The present disclosure relates to the composition as described herein for use in therapy.
  • the present disclosure relates to a method of treating or preventing a disease in a subject, comprising administering to the subject an effective amount of the composition as described herein, wherein the disease is selected from the group consisting of an ischemic heart disease, atherosclerosis, a sphingolipid-related metabolic disease and a combination thereof
  • the disease is selected from the group consisting of an ischemic heart disease, atherosclerosis, a sphingolipid-related metabolic disease and a combination thereof
  • the sphingolipid-related metabolic diseases may be obesity, diabetes, a fatty liver disease, nonalcoholic steatohepatitis or a combination thereof.
  • the administering step may be undertaken by injection.
  • the injection may be via intravenous, intraperitoneal, intraventricular, subcutaneous or combinations thereof.
  • the injection may be intravenous injection.
  • the present disclosure relates to the composition as described herein for use in the treatment or prevention of a disease selected from the group consisting of an ischemic heart disease, atherosclerosis, a sphingolipid-related metabolic disease and a combination thereof.
  • a disease selected from the group consisting of an ischemic heart disease, atherosclerosis, a sphingolipid-related metabolic disease and a combination thereof.
  • the sphingolipid-related metabolic diseases may be obesity, diabetes, a fatty liver disease, nonalcoholic steatohepatitis or a combination thereof.
  • compositions as described herein in the manufacture of a medicament for the treatment or prevention of a disease selected from the group consisting of an ischemic heart disease, atherosclerosis, a sphingolipid-related metabolic diseases and a combination thereof.
  • a disease selected from the group consisting of an ischemic heart disease, atherosclerosis, a sphingolipid-related metabolic diseases and a combination thereof.
  • the sphingolipid-related metabolic diseases may be obesity, diabetes, a fatty liver disease, non-alcoholic steatohepatitis or a combination thereof.
  • the present disclosure provides for a a composition
  • a composition comprising drug carrier (for example, lipid-based micelles which is a type of nanoparticle) encapsulating the active agent (for example, myriocin).
  • the composition achieved assists in the solubilization of the active agent (for example, myriocin) in water-based solution, the organ-selective (for example, infarcted heart tissue) delivery of the active agent (for example, myriocin) via injection (for example, intravenous); and is a form of cardiomyocyte-specific drug delivery.
  • the active agent (for example, myriocin) encapsulated may be replaced by other molecules with similar chemical properties (for example, hydrophobicity) such as sphingofungin B, sphingofungin C and lipoxamycin.
  • the composition as described herein may be regarded as a drug delivery system (for example, a myriocin delivery agent) for modulation of sphingolipid metabolism.
  • the composition may be used clinically as an adjunctive drug therapy for ischemic heart disease, atherosclerosis, and sphingolipid-related metabolic diseases (for example, obesity, diabetes, fatty liver disease, etc.)
  • the composition may be used as a myriocin delivery platform for the therapy of myocardial infarction and atherosclerosis, as well as achieving an organ or cell type-specific delivery of the active agent myriocin Accordingly, the delivery platform may also be applicable for other metabolic diseases such as diabetes and non-alcoholic steatohepatitis.
  • Fig. 1 shows a graph comparing the size (Z-ave) and polydispersity index (PDI) of the formulation with different drug:feed ratios.
  • Fig. 2a shows a schematic diagram of three synthetic/catabolic pathways of ceramide.
  • Fig. 2e shows a western blot image of the upregulation on expression levels (proteins) of SPTLC2 on ceramide de novo synthesis pathway in the infarct area of the I/R injured cardiac tissue; and a western blot image of the GAPDH (or “housekeeping”) gene used as a control for qPCR measurement of certain gene expression levels in the infarct area of the I/R injured cardiac tissue.
  • GAPDH or “housekeeping”
  • Fig. 2g shows images of immunohistochemistry staining of SPTLC2 in the sham or infarcted heart sections over periods of 3hours, 24 hours and 3 days. (Scale bar 50 pm).
  • Fig. 3a shows a schematic diagram of micelles loaded with myriocin.
  • Fig. 3b shows a graph plotting the size and polydispersity index (PDI) of micelle-myriocin.
  • Fig. 3c shows a graph of the encapsulation efficiency of myriocin-loaded micelle with different lipid/ drug feed ratio.
  • Fig. 3d shows an image of the Transmission Electron Microscopy (TEM) of micelles (scale bar 25 nm).
  • Fig. 3f is a FACS histogram showing Cy5 intensity from H9c2 cells co-incubated with Cy5- labeled micelles with different time periods.
  • Fig. 3g shows images of H9c2 cells co-incubated with unlabeled micelles (upper) and Cy5- labeled micelles (lower) for 24 hours (scale bar 50 pm).
  • Fig. 3h shows a graph plot of relative MFI measured by FACS from H9c2 cells co-incubated with Cy5-labeled micelles and cell uptake inhibitors, indicating that the micelles are internalized by cardiomyocytes in vitro via clathrin-mediated endocytosis or micropinocytosis.
  • Fig. 3i is a FACS histogram showing Cy5 intensity from H9c2 cells co-incubated with DSPE- PEG2000 and DSPE-PEG5000 micelles.
  • Fig. 4a shows a graph of relative MFI to untreated measured by FACS for genistein. H9C2 heart cells were co-incubated with Cy5-labeled micelles and inhibitors of cellular uptake.
  • Fig. 4b shows a graph of relative MFI to untreated measured by FACS for genistein. H9C2 heart cells were co-incubated with Cy5-labeled micelles and inhibitors of cellular uptake.
  • Fig. 4b shows a graph of relative MFI to untreated measured by FACS for genistein. H9C2 heart cells were co-incubated with Cy5-labeled micelles and inhibitors of cellular uptake.
  • Fig. 4b shows a graph of relative MFI to untreated measured by FACS for genistein. H9C2 heart cells were co-incubated with Cy5-labeled micelles and inhibitors of cellular uptake.
  • Fig. 4b shows a graph of relative MFI to untreated measured by FACS for
  • Fig. 4b shows a graph of relative MFI to untreated measured by FACS for chloropromazine.
  • H9C2 heart cells were co-incubated with Cy5-labeled micelles and inhibitors of cellular uptake.
  • Fig. 4c shows a graph of relative MFI to untreated measured by FACS for MbCD.
  • H9C2 heart cells were co-incubated with Cy5-labeled micelles and inhibitors of cellular uptake.
  • Fig. 5 shows photographs of micelles protect encapsulated molecules (dyes as model drug) from leakage.
  • Micelles containing dyes were incubated in BSA-PBS solution at 37 °C for up to 24 hours.
  • Rho refers to rhodamine free dye.
  • Fig. 6a shows photographs of IVIS imaging showing the biodistribution of intravenously injected Cy5-labeled micelles in healthy and I/R injured mice.
  • Fig. 6b shows a bar chart plot of biodistribution of intravenously injected Cy5-labeled micelles in healthy and I/R injured mice.
  • Fig. 6c shows a graph plot of FACS analysis showing the uptake percentage of Cy5-labeled micelles in cardiac immune cells in sham and I/R injured mice.
  • Fig. 6d shows a bar chart plot of the measurement of myriocin extracted from hearts by LC- MS.
  • Fig. 7a shows a schematic diagram of LC-MS/MS based lipidomics performed on heart tissue harvested from different regions.
  • Fig. 7b shows a heatmap of the overall infarcted heart tissue sphingolipid profile from different timepoints and treatment groups, all data were presented as fold changes to baseline levels.
  • Fig. 7d shows a graph of the dynamics of infarct total ceramides in the first 7 days.
  • Fig. 7e shows a graph of the dynamics of infarct total sphingomyelins in the first 7 days.
  • Fig. 7f shows a graph of the dynamics of remote total ceramides in the first 7 days.
  • Fig. 8a shows a graph of the dynamic profiles of total ceramides in the liver within first 7 days after heart I/R injury.
  • Fig. 8b shows a graph of the dynamic profiles of total sphingomyelins in the liver within first 7 days after heart 1/R injury.
  • Fig. 9a shows a schematic timeline diagram of I/R heart surgery.
  • Fig. 9b shows a bar chart of the heart function as determined by left ventricular ej ection fraction (LVEF). Animals were treated either with empty micelles (control) or micelle-myriocin (LM- Myriocin) after I/R heart surgery.
  • LVEF left ventricular ej ection fraction
  • Fig. 9c shows photographs of the heart scars (white color, which is indicated in white circles) at 8 weeks post-surgery in mice treated with empty micelles (control) or micelle-myriocin.
  • Fig. 9d shows a schematic timeline diagram of MI surgery (permanent ligation of left coronary artery).
  • Fig. 10a and 10b are IVIS images showing the biodistribution of intravenously injected Cy5- labeled micelles in ApoE" ' mice fed with high-fat diet.
  • Fig. 10c is an image showing the aortic root cross-section of atherosclerotic ApoE"'" mice treated with Cy5-labeled micelle (White/lightest grey areas (1) : macrophage; Medium dark grey areas (2) : nucleus; Medium dark grey areas surrounding the lightest grey areas (3) : Cy5- labeled micelle; scale bar 500 pm).
  • Fig. lOd is an enlarged image of the white box area in Fig. 10c (scale bar 50 pm).
  • Fig. 1 la shows a schematic timeline diagram of high-fat diet and treatment.
  • Fig. 11b is representative images of aortic arches from different treatment groups imaged during harvesting.
  • Fig. 12a is a bar chart showing the percentage of at risk and infarcted areas of ER mice hearts treated or untreated with SLN-myriocin of a previous work.
  • Fig. 12b is a bar chart showing percentages of area at risk (AAR) to total area (TA) and infarct area (IA) to AAR of I/R mice treated with control micelle and myriocin micelle, respectively.
  • Fig. 13a is a bar chart showing percentages of area at risk (AAR) to total area (TA) and infarct area (IA) to AAR of I/R mice treated with control micelle and myriocin micelle, respectively.
  • Fig. 13a is a bar chart showing percentages of area at risk (AAR) to total area (TA) and infarct area (IA) to AAR of I/R mice treated with control micelle and myriocin micelle, respectively.
  • thermostability of formulation with different drug-feed ratios at room temperature as shown in Fig. 1 was tested.
  • the experiment was performed based on fixed amounts of DSPE- PEG2000 (50mg) (obtained from Avanti, United Kingdom) in 1ml aqueous buffer with different drug-feed amounts.
  • DSPE- PEG2000 50mg
  • Fig. 3b from the size (Z-ave) and poly dispersity index (PDI)
  • the formulation of 0.4 mg myriocin obtained from Cayman Chemical Company, Michigan, United States of America
  • 50 mg DSPE-PEG2000 achieved the best thermostability.
  • DSPE-PEG and 0.4 mg of myriocin were dissolved in 2 mL of methanol in a round bottom flask. The organic phase was then completely removed by rotary evaporation at 60°C, 50mbar, 175 rpm for 1 hour in order to form a thin layer of lipid on the bottom of the flask. Subsequently, 1 mL of PBS was introduced to dissolve the lipid layer by rotating at 175 rpm for 1 hour. 0.45 pm filter (nylon-based or polyethylene based) (obtained from Sartorius AG, Gottingen, Germany) was then used to remove big aggregates and precipitations. Empty lipid micelles were prepared without myriocin. Cy5 labelled micelle was prepared by adding 0.1% of Cy5 DSPE-PEG. Prior to further experiments, micelles were filtered through 0.2 pm filter.
  • MI myocardial infarction
  • I/R ischemia/reperfusion
  • mice were anesthetized by intraperitoneal injection of 0.5 mg/kg medetomidine, 5.0 mg/kg dormicum and 0 05 mg/kg fentanyl Subsequently, MI was induced by ligating the left descending coronary artery (LAD) with 8-0 suture (obtained from Ethilon, Cornelia, Georgia). I/R injury was further induced by releasing the LAD ligation after 30 minutes of ischemia. After the surgery, 0.5 mg/kg atipamezole and 5 mg/kg flumazenil were applied intraperitoneally to recover the mice from anesthesia, and 0.1 mg/kg buprenorphine were applied subcutaneously twice daily for three days.
  • LAD left descending coronary artery
  • mRNA was extracted from frozen tissue with the RNeasy Mini Kit (obtained from Qiagen, Netherlands) and cDNA was converted from 125 ng mRNA with the QuantiTect- Reverse-Transcription Kit (obtained from Qiagen, Netherlands) according to the manufacturer’s instructions.
  • cDNA triplicates with iTaq Universal SYBR Green Supermix obtained from Bio-Rad, California, United States
  • QuantStudio 5 obtained from ThermoFisher, Massachusetts, United States of America
  • GAPDH was used as the housekeeping gene.
  • Acid sphingomyelinase protein was detected with acid sphingomyelinase rabbit polyclonal antibody (1 :500; bs-6318R obtained from ThermoFisher, Massachusetts, United States of America) and anti-rabbit IgG, HRP-linked (1:10000, ab6721 obtained from Abeam, Cambridge, United Kingdom).
  • HIF-1 alpha was detected with HIF-1 alpha rabbit polyclonal antibody (1 : 1000; ab216842 obtained from Abeam, Cambridge, United Kingdom) and antirabbit TgG, HRP-linked (1 :10000, ab6721 obtained from Abeam, Cambridge, United Kingdom).
  • SPTLC2 was detected with SPTLC2 rabbit polyclonal antibody (1 :2000; PAS- 31130 obtained from ThermoFisher, Massachusetts, United States of America) and anti-rabbit IgG, HRP-linked (1: 10000, ab6721 obtained from Abeam, Cambridge, United Kingdom).
  • ceramide synthesis pathways (Fig. 2a) in the I/R injured mouse hearts at the timepoints of 3 hours, 24 hours, 3 days and 7 days post myocardial ischemia reperfusion (I/R) heart injury was characterized.
  • a significant upregulation of ceramide de novo synthesis pathway related mRNA such as SPTLC2 and DEGS 1 were observed in the heart infarct region (Fig. 2b to 2c), peaking at 3-day post I/R injury.
  • the total concentration of ceramides (Cers), sphingomyelins (SMs) and hexosylceramides (HexCers) were all found elevated (Fig. 2d).
  • ceramides have been reported to be upregulated upon heart I/R injury, the exact cell type producing ceramides remains unknown. To assess this, the I/R injured heart was stained with SPTLC2 antibody and observed a clear and gradual increase of SPTLC2 protein in specifically cardiomyocytes across time after I/R (Fig. 2g).
  • LC-MS/MS was used to determine the concentration of myriocin in micelle and tissue.
  • micelle internal standard with external calibration curve was used.
  • Drug-loaded lipid micelles were firstly diluted in methanol to a theoretical concentration of 0.4 gg/mL and samples for calibration curve were prepared. Internal standard arachidonic acid was then added at a final concentration of 1 LIM in each sample.
  • tissue samples only external calibration curve was used for measurement.
  • Heart samples were firstly lyophilized in the lyophilizer overnight to completely dry the tissue. The samples were then smashed and mixed with 100 uL of methanol and sonicated under boost mode for 30 minutes and centrifuge at 21,000 g for 10 minutes. The supernatant was collected for LC-MS/MS.
  • LC-MS/MS was performed using an Acquity UPLC BEH C18 column (1.7 gm, 2.1x100 mm) in ESI negative mode on an Agilent 1290 Infinity ultra-high pressure liquid chromatography (UHPLC) system, coupled to an AB SCIEX QTRAP 5500 tandem mass spectrometry.
  • Eluent A consisted of 0.1% formic acid in MilliQ water and eluent B consisted of 0. 1% formic acid in acetonitrile. Column and sample temperatures were set at 45°C and 4°C, respectively. Injection volume was 2 gl and flow was set at 0.65 ml/min.
  • Micelles were characterized for size and size distribution (polydisperse index, PDI) by DLS at the backscatter mode on a Zetasizer Ultra (Malvern Panalytical, Malvern, United Kingdom). Zeta potential was measured by ELS on a Zetasizer Ultra. Each sample was measured three times (DLS) and five times (ELS), and data were acquired and analyzed with XS EXPLORER (Malvern Panalytical).
  • PDI polydisperse index
  • Myriocin has been reported in some studies and showed good effectiveness in reducing ceramide production and preserving cardiac function. Due to the highly hydrophobic nature of myriocin, a lipid-based micellar system was developed to solubilize this drug in aqueous buffer (Fig. 3a). This formulation of myriocin can be used for intravenous injection, which will increase the drug bioavailability and potentially yield a higher therapeutic efficacy.
  • Various myriocin-lipid feed ratios were tested to develop the lipid micelles. Size and polydispersity index (PDI) were measured by Dynamic Light Scattering (DLS) (Fig. 3b) and encapsulation efficiency was determined by LC-MS/MS (Fig. 3c).
  • the optimized formulation with myriocin-lipid feed ratio at 0.4/50 displayed 19.9 nm in size, 0.24 in PDI, -2.6 mV in zeta potential, and yielded a 51.7% loading rate (Table 1)
  • the morphology was viewed under a TEM showing an averaged 20 nm sized dense-core particles (Fig. 3d). Next, the in vitro releasing profile was investigated.
  • Myriocin-loaded micelle was dialyzed against PBS at 37 °C. The residual myriocin in the micelle was collected at different timepoints and measured. A burst release in the first 6 hours and a sustained release up to 3 days which coincide with the timing of the upregulation of SPTLC2 after I/R injury was observed (Fig. 3e). The releasing profile indicates the myriocin-loaded micelle could be applied right after the I/R injury.
  • H9c2 cell line a rat myoblast cell line simulating the cardiomyocytes in vitro was used for investigation of the cellular internalization of the lipid micelle.
  • Cells were incubated with Cy5- labeled lipid micelles for different time periods up to 24 hours.
  • a clear time-dependent manner of cellular uptake was observed by flow cytometry (Fig. 3f). This is further verified by immunofluorescence staining where clear Cy5 signals were seen from H9c2 co-incubated with Cy5 lipid micelles for 24 hours (Fig. 3g). Further, it was observed that the median fluorescence intensity (MFI) decreased when the cells were pre-incubated with dynamin and amiloride in a dose-dependent manner (Fig.
  • MFI median fluorescence intensity
  • DSPE-PEG5000 micelle with same molar concentration were less internalized compared to DSPE-PEG2000 lipid micelle (Fig. 3i).
  • the DSPE-PEG5000 shared similar structure of DSPE-PEG2000 with slightly different surface modification which led to larger size ( ⁇ 30 nm), suggesting the internalization might also be size-dependent.
  • composition/formulation lipid micelle
  • Cy5 was used to label the lipid carrier of the micelles, and Rhodamine B (obtained from Sigma-Aldrich, Missouri of the United States of America, Cat: 81-88-9) was encapsulated in the Cy5-micelles to serve as the model drug mimicking myriocin.
  • Cy5 rhodamine dual-labeled micelles were dialyzed against 30 mg/ml bovine serum albumin (BSA) solution at 37 °C simulating physiological environment in plasma was used to incubate the Cy5 rhodamine dual-labeled micelle (Fig. 5). 24 hours after incubation, the mixture of micelle and BSA-PBS solution were centrifuged in an Amicron filter to collect the free rhodamine in the solution. Although free rhodamine was observed in the dual-labeled micelle, it was far lower compared to the free-rhodamine control (Fig. 5). This indicated good stability of the drug-loaded micelle in the plasma-like environment.
  • BSA bovine serum albumin
  • Fig. 6d shows myriocin concentration in the homogenized tissue separated from the whole heart, which was not merely cardiomyocytes. Therefore, Fig. 6a and Fig. 6d show the drug-lipid micelle having organ selectivity towards heart tissue during the I/R injury, which is possibly caused by EPR effect. Thus, it is observed that the organ selectivity is due to the enhanced permeability and retention (EPR) effect, since the leaky vasculature in the ER injured site may trap particles, especially small particles in circulation.
  • EPR enhanced permeability and retention
  • the gradient started with a flow rate of 0.4 mL/min at 20% B and increased to 60% B at 2 minutes, 100% B at 7 minutes, held at 100% B until 9 minutes, followed by equilibration with 20% B from 9.01 minutes until 10.8 minutes.
  • the column effluent was introduced to the mass spectrometer via AJS-ESI ion source operating under the following conditions: Gas temperature, 200 °C, gas flow, 14 L/min; nebulizer, 20 psi; sheath gas temperature, 250 °C; sheath gas flow, 1 1 L/min; capillary, 3500 V.
  • Mass spectrometry analysis was performed in positive ion mode with dynamic scheduled multiple reaction monitoring (dMRM).
  • lipid metabolism was severely hindered after 1/R injury, especially for the sphingolipid family.
  • LC-MS/MS based lipidomics were performed as illustrated in Fig. 7a.
  • Fig. 7b the infarct tissue from untreated I/R mice illustrated a significantly upregulated sphingolipid profile in the first week after the injury.
  • myriocin lipid micelle treatment distinctively reversed the profile (Fig. 7c).
  • Ceramides are the most crucial bioactive sphingolipids in the cardiac I/R injury. A significant inhibition of all ceramides in the infarct area was observed, especially for dl8: 1/16:0, dl8:l/24:0 and dl8: l/24:l (Fig. 7c), which were widely reported and considered as ‘toxic species’.
  • the myriocin lipid micelle treatment achieved a suppression of the total ceramide and sphingomyelin concentration at the baseline level in both infarct and remote areas of the I/R hearts up to 7-day post injury (Fig. 7d to Fig.
  • LV trace mode left ventricular ejection fraction
  • LVEF left ventricular ejection fraction
  • MI myocardial infarction
  • Example 7 Characterization of the composition/formulation (myriocin lipid micelle) in treating atherosclerosis
  • mice Female apolipoprotein E deficient mice (ApoE") were purchased from Jackson Laboratory (Maine, USA). All mice were housed under specific pathogen-free conditions with a 12/12- hour light-dark cycle at the Comparative Medicine Animal Vivarium at the National University of Singapore. ApoE-/-mice were fed with high-fat diet (HFD, 42% from fat, TD.88137, Envigo) starting from 8 weeks old to accelerate the development of atherosclerosis.
  • HFD high-fat diet
  • the whole aorta was fixed in 10% formalin for 72 hours and followed by incubating in 78% isopropanol for 5 minutes prior to staining with ORO working solution for 1 hour on a rotating platform at room temperature.
  • the aorta was washed twice with 78% isopropanol for 5 minutes and re-immersed in PBS.
  • ORO-stained aorta was cut open longitudinally and pinned on a dissection plate with a dark background, then imaged with a stereo microscope (Nikon Instrument Inc., Tokyo, Japan) connected to a digital camera.
  • Plaques at aortic roots were sectioned serially at 5 pm thick and fixed with ice-cold 10 % formalin for 15 minutes and washed once with PBS before blocking with blocking buffer (goat or donkey serum with 0.5 % BSA in PBS) for one hour at room temperature.
  • Primary antibodies against macrophages (CD68), collagen (collagen type I), smooth muscle cells (a-sma), ceramides, serine palmitoyltransferase (SPTLC2), and proto-oncogene tyrosine-protein kinase MER (MerTK) were applied for overnight in staining buffer (0.2 % Triton X-100 with 0.3 % of BSA in PBS).
  • the slides were rinsed thrice in PBS, incubated with fluorescently labeled secondary antibodies for 2 hours, and mounted with DAPI-mounting media.
  • the fluorescent images were captured with an inverted epifluorescence microscope (Nikon Eclipse Ti-E inverted microscope, Nikon Instrument Inc. Tokyo, Japan). Regions of plaque without any DAPI or fluorescent staining were identified as necrotic cores
  • the images were analyzed and quantified with Nikon AR element analysis software version 4.5.0 (Nikon Instrument Inc. Tokyo, Japan).
  • the information on the antibodies used is listed in Table SI in the supplemental information.
  • myriocin lipid micelle Another application of myriocin lipid micelle is in treating atherosclerosis.
  • the plaque formed in the coronary artery resulting from atherosclerosis is the major contributor to myocardial infarction. Therefore, reducing plaque is a promising therapy in the prevention of MI among high-risk patients.
  • Cy5-labeled lipid micelles were firstly applied to ApoE" ' mice on high-fat diet, which have significant plaques formed in the arteries, in order to profile the biodistribution of the micelles in the atherosclerosis context. As shown in Fig. 10a and Fig. 10b, a significant deposition of Cy5 signal in the aortic arch and descending aorta was observed, co-localizing with the position of plaques. Furthermore, on the cellular level, Cy 5-labeled micelles were found internalized by macrophages inside the plaques, indicated by the highly overlapped CD68 and Cy5 signals in the plaque area of the aortic root (Fig. 10c and Fig. lOd). These suggested that micelles could achieve a targeted drug delivery towards plaques, specifically macrophages.
  • the myriocin lipid micelles were injected intravenously at the dose of 1 .3 mg/kg to ApoF.' ' mice on the high-fat diet on a weekly basis (timeline of the study is shown in Fig. Ila).
  • plaque sizes were measured by ORO staining as an indicator of the treatment efficacy.
  • myriocin lipid micelles significantly reduced -48% of the plaque size and -6.2% body weight when compared to saline-treated control, suggesting a potent anti- atherogenesis effect of the myriocin lipid micelle.
  • FIG. 13a to Fig. 13d Further analysis of plaque composition with ORO and immunofluorescent staining (Fig. 13a to Fig. 13d) revealed that the mice receiving myriocin micelle treatment have reduced lipids (Fig. 13a), inflammation (as indicated by reduced macrophage infiltration in Fig. 13b), and enhanced collagen (Fig. 13c) and alpha-smooth muscle actin expression (Fig. 13d) in the plaques. This points to a more stable plaque phenotype, hence reduced chances of plaque rupture, and ultimately reduced risk of ischemic heart and brain diseases.
  • Example 8 Characterization of the comnosition/formulation (myriocin linid micelle) in determining infarct areas
  • mice similar to that used in the I/R injury model were sacrificed 24 hours after reperfusion and the hearts were isolated for analysis.
  • the hearts were stained using 2,3,5-triphenyltetraxolium (TTC) to determine the infarct areas (IA) and Evans blue to determine the areas at risk (AAR).
  • TTC 2,3,5-triphenyltetraxolium
  • AAR areas at risk
  • the IA and AAR are quantified using ImageJ (version 1.53e) and expressed as a percentage of IA to total heart area (1A/TA) and IA to AAR (IA/AAR).
  • Fig. 12b shows that the treatment by micellar myriocin can effectively reduce around 25% infarct area 24 hours after myocardial I/R injury.
  • Fig. 12b also shows that the result is comparable with the formulation in Fig. 12a (based on solid lipid nanocarriers (SLN-myriocin) of a previous work) which was injected intra- ventricularly.
  • composition comprising an active agent and a drug carrier may be used for drug delivery which is applicable to industries such as medical, phannaceutical and cosmetic
  • the disclosure has great clinical potential as an adjunctive drug therapy especially, but not limited to, for metabolic diseases.

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Abstract

La présente divulgation concerne une composition comprenant un agent actif et un vecteur de médicament, le vecteur de médicament encapsulant au moins partiellement l'agent actif, et le rapport molaire entre l'agent actif et le vecteur de médicament étant compris entre environ 1:20 et environ 1:40. La présente divulgation concerne également un procédé de formation de la composition telle que définie dans la description. L'invention concerne également une méthode d'administration de la composition, une méthode de traitement au moyen de la composition, ainsi que les utilisations médicales de la composition telle que définie dans la description.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020034538A1 (en) * 2000-06-09 2002-03-21 Gilead Sciences, Inc. Liposomal benzoquinazolne thymidylate synthase inhibitor formulations
US20060051406A1 (en) * 2004-07-23 2006-03-09 Manjeet Parmar Formulation of insoluble small molecule therapeutics in lipid-based carriers
US20120309780A1 (en) * 2005-04-12 2012-12-06 Wisconsin Alumni Research Foundation Micelle composition of polymer and passenger drug
US9925160B1 (en) * 2017-01-16 2018-03-27 Universita' Degli Studi Di Milano Methods for treating cardiac reperfusion injury

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020034538A1 (en) * 2000-06-09 2002-03-21 Gilead Sciences, Inc. Liposomal benzoquinazolne thymidylate synthase inhibitor formulations
US20060051406A1 (en) * 2004-07-23 2006-03-09 Manjeet Parmar Formulation of insoluble small molecule therapeutics in lipid-based carriers
US20120309780A1 (en) * 2005-04-12 2012-12-06 Wisconsin Alumni Research Foundation Micelle composition of polymer and passenger drug
US9925160B1 (en) * 2017-01-16 2018-03-27 Universita' Degli Studi Di Milano Methods for treating cardiac reperfusion injury

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ESCUDé MARTINEZ DE CASTILLA POL; TONG LINGJUN; HUANG CHENYUAN; SOFIAS ALEXANDROS MARIOS; PASTORIN GIORGIA; CHEN XIAOYUAN; STO: "Extracellular vesicles as a drug delivery system: A systematic review of preclinical studies", ADVANCED DRUG DELIVERY REVIEWS, vol. 175, 18 May 2021 (2021-05-18), NL, XP086704680, ISSN: 0169-409X, DOI: 10.1016/j.addr.2021.05.011 *

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