WO2019144868A1 - 用于保护缺血心肌的注射剂及其制备方法 - Google Patents

用于保护缺血心肌的注射剂及其制备方法 Download PDF

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WO2019144868A1
WO2019144868A1 PCT/CN2019/072710 CN2019072710W WO2019144868A1 WO 2019144868 A1 WO2019144868 A1 WO 2019144868A1 CN 2019072710 W CN2019072710 W CN 2019072710W WO 2019144868 A1 WO2019144868 A1 WO 2019144868A1
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injection
parts
emulsion
oil
hydroxamic acid
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PCT/CN2019/072710
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English (en)
French (fr)
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唐熠达
楚娜
秦艳
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唐熠达
楚娜
秦艳
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Priority to US16/964,995 priority Critical patent/US20210046025A1/en
Publication of WO2019144868A1 publication Critical patent/WO2019144868A1/zh

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    • 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
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • 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
    • 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
    • A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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 invention belongs to the field of pharmaceutical preparations, and relates to an injection for protecting an ischemic myocardium and a preparation method thereof, in particular to an injection emulsion for protecting an ischemic myocardium comprising N-suberanilide hydroxamic acid and Its preparation method.
  • Histone deacetylase inhibitor is a class of compounds that have the function of interfering with histone deacetylase, and has been widely used in the treatment of HIV, malignant tumors or chronic fibrotic diseases. Research reports on aspects.
  • SAHA suberoylanilide hydroxamic acid
  • HDAC histone deacetylase
  • N-suberanilide hydroxamic acid has been approved by the US Food and Drug Administration for sale under the trade name Vorinostat for the treatment of aggravation, persistence and relapse, or the use of two systemic drugs.
  • CTCL cutaneous T-cell lymphoma
  • N-suberanilide hydroxamic acid can be delivered in the context of clinically relevant reperfusion to reduce myocardial infarct size in large animal models.
  • N-suberanilide hydroxamic acid produces cardioprotection at least in part by inducing autophagic flux (Min Xie et al., Histone Deacetylase Inhibition Blunts Ischemia/Reperfusion Injury by Inducing Cardiomyocyte Autophagy, Circulation. 2014; 129: 1139-1151). Therefore, N-suberanilide hydroxamic acid has important clinical application value for myocardial protection under ischemia or reperfusion injury, for example, reducing the occurrence of myocardial infarction during cardiac interventional surgery.
  • N-suberanilide hydroxamic acid formulations are limited to anti-tumor oral dosage forms. Due to the poor water solubility of N-suberanilide hydroxamic acid, and the oral N-suberanilide hydroxamic acid also has obvious first-pass metabolism elimination, resulting in low oral bioavailability, unable to be in cardiovascular and other pro- An effective therapeutic concentration is formed in the fatty tissue.
  • N-suberanilide hydroxamic acid can only be used as a prototype to have a therapeutic effect, and its metabolites are inactive, so its oral bioavailability cannot be improved by chemical derivatization.
  • oral administration of N-suberanilide hydroxamic acid can cause systemic side effects before cardiac intervention.
  • an object of the present invention to provide an emulsion for injection for protecting an ischemic myocardium and a method for preparing the same.
  • the emulsion for injection can be effectively used for improving the local drug concentration of N-suberanilide hydroxamic acid in cardiovascular and other lipophilic organs and/or tissues before cardiac interventional surgery, and improving N-suberanilide hydroxamic acid
  • the bioavailability reduces systemic side effects, effectively protects the myocardium from ischemia or reperfusion injury, and reduces or avoids the occurrence of acute heart infarction.
  • the injectable emulsion also provides a new alternative dosage form for the application of N-suberanilide hydroxamic acid in the treatment of cancer and the like.
  • An injectable emulsion for protecting an ischemic myocardium, in parts by mass comprising:
  • N-suberanilide hydroxamic acid 1 to 5 parts
  • Emulsifier 0.2 ⁇ 12.5 parts
  • Oleic acid 0.03 to 0.4 parts
  • the injectable emulsion comprises, by mass part,:
  • Emulsifier 0.5 ⁇ 5 parts
  • Oleic acid 0.03 to 0.3 parts
  • the injectable emulsion comprises, by mass part,:
  • Emulsifier 1.0 ⁇ 2.5 parts
  • Oleic acid 0.05 ⁇ 0.15 parts
  • the injectable emulsion comprises, by mass part,:
  • Emulsifier 1.0 ⁇ 2.5 parts
  • Oleic acid 0.05 ⁇ 0.15 parts
  • the injectable emulsion comprises, by mass part,:
  • the emulsifier is preferably a phospholipid; more preferably, the phospholipid is selected from one or more of soybean phospholipid, lecithin, hydrogenated soybean phospholipid or hydrogenated lecithin; further preferably, The phospholipid is soybean phospholipid and/or lecithin.
  • the oil for injection may be selected from the group consisting of soybean oil for injection, safflower oil for injection, cottonseed oil for injection, sesame oil for injection, tea oil for injection, olive oil for injection or medium chain oil for injection.
  • the injectable oil is soybean oil for injection.
  • the solubilizing agent may be selected from one or more of Tween-80, propylene glycol, poloxamer 188 or polyethylene glycol 15 hydroxystearate (Solutol HS 15);
  • the solubilizing agent is Tween-80 and/or propylene glycol.
  • the injectable emulsion comprises, by mass fraction,:
  • the invention also provides a preparation method of the emulsion for injection, which comprises the following steps:
  • the colostrum is homogenized under pressurized conditions.
  • the heating is carried out to 50 to 90 ° C, more preferably to 70 to 80 ° C.
  • the mixing is performed by shear mixing; preferably, the shear mixing speed is 3000 to 10000 rpm; more preferably, the shearing The mixing speed is 5000 to 6000 rpm.
  • the heating is carried out to 70 ° C to 80 ° C and the shear rate of the high shear mixing is 3,000 to 10,000 rpm, preferably 5,000 to 6,000 rpm.
  • the high speed shear mixing is carried out for 10 to 40 minutes, more preferably for 25 to 40 minutes, and most preferably for 30 minutes.
  • the pressurization condition is 400 to 1200 bar, more preferably 700 to 900 bar; the homogenization may be carried out 1-6 times, preferably 5 to 6 times.
  • the preparation method further comprises the following steps:
  • the present invention provides the use of the injectable emulsion for the preparation of a medicament for protecting an ischemic myocardium, a medicament for treating myocardial ischemia-reperfusion injury or a medicament for treating myocardial infarction.
  • the invention provides the use of the N-suberanilide hydroxamic acid emulsion for the manufacture of a medicament for the treatment of ischemia-reperfusion injury.
  • the invention can also be used for therapeutic purposes in ischemia-reperfusion injury of the cerebral blood vessels, as well as ischemia-reperfusion injury of other blood circulation systems in the human body.
  • the present invention provides the use of the N-suberanilide hydroxamic acid emulsion in the preparation of a medicament for ischemic myocardial protection or a medicament for treating myocardial ischemia-reperfusion injury or Application in medicine for treating myocardial infarction.
  • the present invention provides the use of the N-suberanilide hydroxamic acid injection emulsion in the preparation of a medicament for ischemic myocardial protection or a medicament for treating myocardial ischemia-reperfusion injury Application in the application or treatment of drugs for myocardial infarction.
  • N-suberanilide hydroxamic acid (vorinostat) is a drug which is insoluble in both oil and water.
  • vorinostat is a drug which is insoluble in both oil and water.
  • the emulsion prepared by the conventional injection formulation has poor stability during the experiment. After the plastization is completed, obvious drug precipitation occurs. After the high-speed centrifugation, a clear layer of the drug powder can be seen.
  • the inventors have found that the addition of any solubilizing agent and/or stabilizer to such a particular poorly soluble drug can effectively improve the stability of the resulting injectable emulsion, while the solubilizing agent selected in the present invention is effective.
  • a stabilizer In the presence of a stabilizer, a stable and optimal emulsion for injection can be obtained.
  • the ratio of the various components in the emulsion for injection also has a significant influence on the stability of the emulsion for injection.
  • the present inventors conducted extensive research, screening, and optimization on the formulation of an injectable emulsion containing N-suberanilide hydroxamic acid, and finally obtained an injectable emulsion having excellent stability as described in the present invention. And its preparation method.
  • Sham oral represents a sham-operated oral gavage group
  • Sham iv represents a sham-operated intravenous group
  • I/R oral Pre represents an oral gavage group for ischemia-reperfusion.
  • I/R iv Pre indicates ischemia-reperfusion once intravenous group
  • I/R oral Pre+Reperfusion indicates ischemia-reperfusion divided oral gavage group
  • I/R iv Pre+Reperfusion indicates ischemia-reperfusion-division vein Injection group
  • MI oral indicates oral administration of myocardial infarction group
  • MI iv indicates myocardial infarction intravenous group.
  • Figure 1 is a photograph (24h) of the effect of oral gavage and intravenous administration of N-suberanilide hydroxamic acid on ischemia-reperfusion (I/R) mice by echocardiography.
  • Figure 2 shows the results of analysis of the effects of oral gavage and intravenous administration of N-suberanilide hydroxamic acid on ischemia-reperfusion (I/R) mice by echocardiography (24h); P ⁇ 0.05, compared to Sham oral; #P ⁇ 0.05, compared to I/R oral Pre; +P ⁇ 0.05, compared to I/R oral Pre+Reperfusion; ns, no significant difference.
  • Figure 3 is a photograph (7 days, 14 days, 28 days) of the effect of oral gavage and intravenous administration of N-suberanilide hydroxamic acid on mice in myocardial infarction (MI) group by echocardiography.
  • Figure 4 shows the results of analysis of the effects of oral gavage and intravenous administration of N-suberanilide hydroxamic acid on mice with myocardial infarction (MI) by echocardiography (7 days, 14 days, 28 days); Where *P ⁇ 0.05 compared to Sham oral; #P ⁇ 0.05 compared to MI oral.
  • Figure 7 is a photograph showing the effect of oral gavage and intravenous administration of N-suberanilide hydroxamic acid on mice in myocardial infarction group by Masson staining (28 days); wherein dark gray is normal myocardial tissue, shallow Gray is the fibrous structure (solid coil out area).
  • Figure 8 shows the results of analysis of the effect of oral administration of N-suberanilide hydroxamic acid on myocardial infarction mice by Masson staining (28 days); #P ⁇ 0.05, with MI oral compared to.
  • Figure 9 shows the results of Western blot analysis of the effects of oral administration of N-suberanilide hydroxamic acid on the levels of histone H3 and H4 acetylation in the heart of mice with ischemia-reperfusion injury.
  • *P ⁇ 0.05 compared to Sham oral #P ⁇ 0.05 compared to I/R oral Pre; +P ⁇ 0.05 compared to I/R oral Pre+Reperfusion; ns, no significant difference .
  • Figure 10 shows the results of Western blot analysis of the effects of oral administration of N-suberanilide hydroxamic acid on the levels of histone H3 and H4 acetylation in the heart tissue of mice with myocardial infarction; , *P ⁇ 0.05, compared to Sham oral; #P ⁇ 0.05, compared to MI oral.
  • Figure 11 is a photograph (24h) of the effect of differently tested injectable emulsions on mice in the ischemia-reperfusion (I/R) group by ultrasound.
  • Figure 12 shows the results of an analysis of the effects of different formulations of injectable emulsions on mice in the ischemia-reperfusion (I/R) group by ultrasound (24h); wherein, *P ⁇ 0.05, prescriptions 1-6 and prescriptions 7-8 compared to.
  • Figure 13 is a photograph (7 days, 14 days, 28 days) of the effect of differently tested injectable emulsions on mice in the ischemia-reperfusion (I/R) group by ultrasound.
  • Figure 14 shows the results of analysis of the effects of different formulations of injectable emulsions on mice in the ischemia-reperfusion (I/R) group by ultrasound (7 days); wherein, *P ⁇ 0.05, prescriptions 1-6 and prescriptions 7- 8 compared.
  • Figure 15 shows the results of an analysis of the effects of different formulations of injectable emulsions on mice in the ischemia-reperfusion (I/R) group (14 days); wherein, *P ⁇ 0.05, prescriptions 1-6 and prescriptions 7- 8 compared.
  • Figure 16 shows the results of analysis of the effects of different formulations of injectable emulsions on mice in the ischemia-reperfusion (I/R) group by ultrasound (28 days); wherein, *P ⁇ 0.05, prescriptions 1-6 and prescriptions 7- 8 compared.
  • Figure 17 is a photograph (24h) of the effects of different formulations of injectable emulsions on mice in the ischemia-reperfusion (I/R) group using Evans blue and TTC double staining; wherein dark gray shows non-ischemic area; light gray The ischemic risk zone (solid coiled out area) is shown; white shows the post-ischemic infarct zone (dashed out region).
  • Figure 18 shows the results of double-stained area analysis of cardiac tissue in mice with ischemia-reperfusion (I/R) group by Evans blue and TTC double staining (24h); 0.05, prescription 1-6 compared to prescription 7-8.
  • Figure 19 is a photograph (28 days) of the effect of different formulations of injectable emulsions on mice in myocardial infarction (MI) group by Masson staining; where dark gray is the normal heart tissue region and light gray is the fibrous tissue formed after infarction Zone (solid coil out zone).
  • MI myocardial infarction
  • Figure 20 shows the results of analysis of the effect of different formulations of injectable emulsions on mice in myocardial infarction (MI) group by Masson staining (28 days); wherein, *P ⁇ 0.05, prescriptions 1-6 compared with prescriptions 7-8 .
  • Figure 21 shows the results of Western blot analysis of the effects of different formulations of injectable emulsion on the levels of histone H3 and H4 acetylation in the heart tissue of mice with ischemia-reperfusion (I/R); *P ⁇ 0.05 , prescription 1-6 compared to prescription 7-8.
  • Figure 22 shows the results of Western blot analysis of the effects of different formulations of injectable emulsion on the levels of histone H3 and H4 acetylation in heart tissue of mice with myocardial infarction (MI); *P ⁇ 0.05, prescription 1- 6 compared to prescription 7-8.
  • Prescription amount (g) N-suberanilide hydroxamic acid 15 Soy lecithin 10 Soybean oil for injection 100 Tween 80 3 Oleic acid 0.5 glycerin 12.5 Water for Injection Moderate amount
  • N-suberanilide hydroxamic acid add Tween-80 and glycerin, heat to 80 ° C, add about 100 ml of water, dissolve into the drug substance, add soy lecithin, and mix by shear (6000 rpm).
  • the aqueous phase is prepared; the oleic acid and the soybean oil for injection are weighed and uniformly mixed to prepare an oil phase; the oil and water phases are mixed at a high temperature of 70 ° C for 30 minutes at a high speed shear mixing (6000 rpm) to prepare colostrum.
  • the colostrum was homogenized under a pressure of 800 bar, cycled 6 times, filled into an ampoule, sealed, and sterilized at 115 ° C for 30 minutes.
  • N-suberanilide hydroxamic acid add poloxamer 188 and glycerin, heat to 80 ° C, add about 100 ml of water, dissolve the drug, add soy lecithin, and mix (6000 rpm).
  • the aqueous phase is prepared; the oleic acid and the soybean oil for injection are weighed and uniformly mixed to prepare an oil phase; the oil and water phases are mixed at a high temperature of 80 ° C for 40 minutes to form colostrum.
  • the colostrum was homogenized under a pressure of 700 bar, cycled 6 times, filled into an ampoule, sealed, and sterilized at 115 ° C for 30 minutes.
  • Prescription amount (g) N-suberanilide hydroxamic acid 15 Soy lecithin 12.5 Soybean oil for injection 50 Polyethylene glycol 15 hydroxystearate 5 Oleic acid 0.25 glycerin 11.5 Water for Injection Moderate amount
  • N-suberanilide hydroxamic acid add polyethylene glycol 15 hydroxystearate and glycerin, heat to 80 ° C, add water about 100ml, until the drug substance is dissolved, add soybean phospholipid, cut (5000 rpm) mixed evenly, made into an aqueous phase; weighed oleic acid and soybean oil for injection, mixed evenly to make an oil phase; oil and water two phases at 80 ° C, high pressure shear mixing (6000 rpm) for 40 minutes, made colostrum.
  • the colostrum was homogenized under a pressure of 800 bar, cycled 6 times, filled into an ampoule, sealed, and sterilized at 115 ° C for 30 minutes.
  • N-suberanilide hydroxamic acid add Tween-80 and glycerin, heat to 70 ° C, add about 100 ml of water, dissolve to the drug substance, add soy lecithin, and mix by shearing (6000 rpm).
  • the aqueous phase is prepared; the oleic acid and the olive oil for injection are weighed and uniformly mixed to prepare an oil phase; the oil and water phases are mixed at a high temperature of 70 ° C for 25 minutes at a high speed shear mixing (6000 rpm) to prepare colostrum.
  • the colostrum was homogenized under a pressure of 800 bar, cycled 6 times, filled into an ampoule, sealed, and sterilized at 115 ° C for 30 minutes.
  • Prescription amount (g) N-suberanilide hydroxamic acid 15 Lecithin 10 Medium chain oil for injection 100 Tween 80 0.5 Oleic acid 0.5 glycerin 12.5 Water for Injection Moderate amount
  • N-suberanilide hydroxamic acid add Tween-80 and glycerin, heat to 80 ° C, add about 100 ml of water, dissolve the drug, add lecithin, and mix by shear (5000 rpm).
  • the aqueous phase is prepared; the oleic acid and the medium-chain oil for injection are weighed and mixed uniformly to form an oil phase; the oil-water two-phase is mixed at a high temperature shear mixing (6000 rpm) for 30 minutes at 70 ° C to prepare colostrum.
  • the colostrum was homogenized under a pressure of 900 bar, cycled 5 times, filled into an ampoule, sealed, and sterilized at 115 ° C for 30 minutes.
  • Prescription amount (g) N-suberanilide hydroxamic acid 15 Soy lecithin 10 Soybean oil for injection 100 Propylene glycol 3 Oleic acid 0.5 glycerin 12.5 Water for Injection Moderate amount
  • N-suberanilide hydroxamic acid add glycerin and propylene glycol, heat to 70 ° C, add about 100 ml of water, dissolve to the drug substance, add soy lecithin, mix (6000 rpm) and mix to make water.
  • the oleic acid and the soybean oil for injection were weighed and mixed to form an oil phase; the oil and water phases were mixed at a high temperature of 70 ° C for 30 minutes at a high speed shear mixing (5000 rpm) to prepare colostrum.
  • the colostrum was homogenized under a pressure of 800 bar, cycled 6 times, filled into an ampoule, sealed, and sterilized at 115 ° C for 30 minutes.
  • Prescription amount (g) N-suberanilide hydroxamic acid 15 Soy lecithin 10 Soybean oil for injection 100 Tween 80 3 Vitamin E 0.5 glycerin 12.5 Water for Injection Moderate amount
  • N-suberanilide hydroxamic acid add glycerin and Tween-80, heat to 80 ° C, add about 100 ml of water, dissolve the drug, add soy lecithin, and mix by shear (6000 rpm).
  • the aqueous phase was prepared; the vitamin E and the soybean oil for injection were weighed and mixed uniformly to prepare an oil phase; the oil and water phases were mixed at a high temperature of 70 ° C for 30 minutes at a high speed shear mixing (6000 rpm) to prepare colostrum.
  • the colostrum was homogenized under a pressure of 800 bar, cycled 6 times, filled into an ampoule, sealed, and sterilized at 115 ° C for 30 minutes.
  • Prescription amount (g) N-suberanilide hydroxamic acid 15 Soy lecithin 10 Soybean oil for injection 100 Tween 80 3 Polyoxyethylene hydrogenated castor oil 0.5 glycerin 12.5 Water for Injection Moderate amount
  • N-suberanilide hydroxamic acid weigh the prescribed amount of N-suberanilide hydroxamic acid, add Tween-80 and glycerin, heat to 80 ° C, add about 100 ml of water, dissolve into the drug substance, add soy lecithin, and mix by shear (6000 rpm).
  • the aqueous phase is prepared; the polyoxyethylene hydrogenated castor oil and the soybean oil for injection are weighed and mixed uniformly to prepare an oil phase; the oil-water two-phase is mixed at a high temperature of 70 ° C for 30 minutes to form colostrum. .
  • the colostrum was homogenized under a pressure of 800 bar, cycled 6 times, filled into an ampoule, sealed, and sterilized at 115 ° C for 30 minutes.
  • mice received 2% isoflurane inhalation anesthesia
  • the surgeon cut a skin incision about 1.2 cm on the left chest wall and reserved a purse.
  • the fourth intercostal space of the left chest is exposed.
  • the pleural cavity and the pericardial cavity are gently and rapidly opened in the purse using the mosquito vascular forceps at the position of the fourth intercostal space to extrude the heart.
  • the suture was sutured at the anterior descending branch of the left coronary artery with a suture on the 6th.
  • the color of the myocardial tissue in the corresponding area was changed to gray, and the ligation was confirmed to be successful.
  • mice were placed in an air circulation environment to monitor their recovery. After 45 minutes of ischemia, the slip of the anterior descending branch was loosened by gently and gently dragging the indwelling head of the chest, and the blood flow was reperfused. The control sham-operated mice were operated with ischemia-reperfusion mice except that the nodule was not ligated at the anterior descending branch.
  • mice received 2% isoflurane inhalation anesthesia
  • the surgeon cut a skin incision about 1.2 cm on the left chest wall and reserved a purse.
  • the fourth intercostal space of the left chest is exposed.
  • the pleural cavity and the pericardial cavity are gently and rapidly opened in the purse using the mosquito vascular forceps at the position of the fourth intercostal space to extrude the heart.
  • the left anterior descending coronary artery was directly ligated with a suture on the 6th. The color of the myocardial tissue in the corresponding area was visually observed to be grayish, and the ligation was confirmed to be successful.
  • the control sham-operated mice were operated with the same heart-throat mice except that the anterior descending branch was not ligated.
  • mice in the ischemia-reperfusion group 2% isoflurane anesthesia was inhaled 24 hours after surgery, and the short axis and long axis of the mouse heart were respectively applied by Vevo Rolling Imaging Cart (Canada, Vevo 770). Screening, followed by determination of various cardiac ultrasound indicators (including: ejection fraction (EF%), shortening fraction (FS%), left ventricular end-diastolic diameter (LVIDd), left ventricular end-systolic diameter (LVIDs), left ventricular diastolic Terminal volume (LV volume; d), left ventricular end-systolic volume (LV volume; s), etc.
  • EF% ejection fraction
  • FS% shortening fraction
  • LVIDd left ventricular end-diastolic diameter
  • LVIDs left ventricular diastolic Terminal volume
  • LV volume d
  • LV volume left ventricular end-systolic volume
  • mice were intraperitoneally injected with 1% sodium pentobarbital solution, the thoracic cavity was opened to expose the heart, and the left anterior descending artery of the left coronary artery was ligated again at the anterior descending branch of the left coronary artery that was ligated during modeling.
  • the 5% phthalocyanine blue staining solution was retrogradely injected along the ascending aorta root, and then the mouse heart was removed and placed in a cryotube for 20 minutes in a refrigerator at -80 °C. Thereafter, the frozen heart was taken out, and the heart was cut down from the ligated site, and the heart was quickly cut into 1-2 cm thick sections.
  • the sections were placed in a phosphate buffer solution of 2,3,5-triphenyltetrazolium chloride (TTC) and incubated in a 37 ° C water bath for 30 minutes in the dark. After that, the heart slices were soaked in formaldehyde for 4 hours, and then taken with a stereopsis.
  • TTC 2,3,5-triphenyltetrazolium chloride
  • the non-ischemic area of LV is dark gray
  • the ischemic danger area is light gray (solid coil out area)
  • the infarct area after ischemia is white (dashed circle area).
  • Images were quantified using Image J software, and the infarct area (IF), ischemic risk area (AAR), and left ventricular area (LV) were quantified and their ratios calculated.
  • mice were sacrificed by intraperitoneal injection of 1% sodium pentobarbital solution and the hearts of the mice were removed.
  • the heart was treated in paraformaldehyde (4 ° C) for 24 hours and then rinsed in running water for 1 hour.
  • the treated cardiac specimen tissue is embedded and sliced. Place the slices in the oven and bake for 50 minutes.
  • the sample was dewaxed and placed in a mixture of phosphomolybdic acid and potassium dichromate overnight. After the specimen was washed with distilled water, it was stained in hematoxylin dyeing solution for 4 minutes, and then fully washed with water, and then alcohol differentiated for 3 seconds. Specimens were stained with radiance red for 5 minutes.
  • the 1% aqueous solution of phosphomolybdic acid was differentiated for 3-5 minutes and then treated with 1% aniline blue for 15 seconds. After the specimens were thoroughly washed, they were soaked in 95% alcohol, absolute alcohol, and xylene for 3 seconds, and then sealed with a neutral gum. Observe and take pictures under the microscope. Image J software was used to quantify the images, and the myocardial infarct area (IF) and total left ventricular area (LV) were quantified and their ratios were calculated.
  • IF myocardial infarct area
  • LV total left ventricular area
  • mice were sacrificed by intraperitoneal injection of 1% sodium pentobarbital solution. The hearts of the mice were taken and the myocardium was cut with ophthalmology scissors and then fully ground. Thereafter, histones were extracted using a histone extraction kit, and the histone concentration of each group was measured, and the protein loading was adjusted to 50 ⁇ g. After 15% SDS-polyacrylamide gel electrophoresis, transfer to a 0.22 ⁇ m PVDF membrane, dilute the primary antibody (1:1000) with blocking solution, incubate for 15 h at 4 ° C, and dilute the secondary antibody (1:5000) after washing the membrane. Incubate in the greenhouse for 2 h. Chemiluminescence imaging after washing the membrane again. Image J software was used to analyze and quantify the gray value of acetylated histone 3, total histone 3, acetylated histone 4 and total histone 4 on protein bands, and the ratio was calculated and statistically analyzed.
  • Ischemia-reperfusion mice were administered with a single administration group (labeled I/R Pre in the drawing) and a separate administration group of ischemia-reperfusion mice (labeled I/R Pre+Reperfusion in the drawing).
  • the test agent was orally administered by oral gavage (labeled as I/R oral in the drawing) or tail vein injection at the dose of 50 mg/kg (labeled I/ in the drawing).
  • the I/R mice were administered in the manner of R iv), and all drugs were administered once an hour before the surgical operation.
  • test agent was administered by oral gavage (labeled as I/R oral in the drawing) or tail vein injection (labeled as I/R iv in the drawing).
  • I/R oral in the drawing oral gavage
  • I/R iv tail vein injection
  • mice were given a dose of 25 mg/kg of the drug one hour before the operation and the myocardial reperfusion procedure (total amount is the same as in the single administration group).
  • test agent was administered to Sham mice by oral gavage (labeled as oral in the drawing) or tail vein injection (labeled as i.v. in the drawing) at a dose of 50 mg/kg.
  • test agents were all prepared in accordance with the prescription 1 and the process of Example 1.
  • Figures 1 and 2 The results of the ultrasonic examination are shown in Figures 1 and 2.
  • Figure 1 shows the results of echocardiography of selected Sham and I/R mice.
  • Figure 2 is a quantitative analysis of cardiac ultrasound results.
  • Figures 2A and 2B show that the percentage of cardiac ejection fraction (EF%) and short axis shortening (FS%) of the mice after I/R operation was significantly reduced compared to the Sham group.
  • the EF% and FS% (I/R iv Pre and I/R iv Pre+Reperfusion group) of I/R mice administered by tail vein injection were significantly higher than those of oral I/R mice (I /R oral Pre and I/R oral Pre+Reperfusion group).
  • FIG. 2C-2G shows left ventricular end diastolic diameter (LVIDd), left ventricular end-systolic diameter (LVIDs), left ventricular end-diastolic volume (LV volume; d), left ventricular end-systolic volume (LV volume; s), and left ventricle LV mass These indicators did not show statistically significant differences between the tail vein administration group and the oral gavage administration group. In addition, different routes of administration did not have different effects on the EF% and FS% values of Sham mice.
  • Figure 5 shows the area of the light gray area (ischemic danger zone, real coiled out area) and white area (postischemic infarct zone, dotted circle area) in the heart tissue of the tail vein injection group compared with the oral gavage group. Smaller.
  • Figure 6 is a quantitative analysis result of a pathological staining picture.
  • Figure 6A-C shows the ratio of the risk zone to the left ventricular area (AAR/LV%), infarct zone, and risk zone area ratio in the tail vein injection group (I/R iv Pre and I/R iv Pre+Reperfusion groups) ( IF/AAR%), infarct area and left ventricular area ratio (IF/LV%) were significantly lower than the corresponding oral gavage group (I/R oral Pre and I/R oral Pre+Reperfusion group), indicating oral comparison
  • tail vein injection can further reduce the infarct area and ischemic injury area, significantly reducing ischemia and reperfusion injury. Consistent with the results of cardiac ultrasound, there was no difference in the area of infarct and ischemic areas between one dose and divided doses.
  • FIG. 9A is a western blot protein band.
  • Figures 9B and C are quantitative analysis results.
  • Figures 9B and C show that the ratio of acetylated histones 3 and 4 to total histones 3 and 4 decreased after I/R injury.
  • the levels of acetylation of histones 3 and 4 in the tail vein injection group (I/R iv Pre and I/R iv Pre+Reperfusion group) were higher than those of the corresponding oral gavage group (I/R oral Pre and I/R oral).
  • Pre+Reperfusion group compared with oral gavage, the tail vein medication has a stronger inhibitory effect on HDAC, thereby further increasing the level of histone acetylation.
  • test agent was administered to Sham and MI mice, respectively, by oral gavage (labeled as oral in the drawing) or tail vein injection (labeled as i.v. in the drawing) at a dose of 50 mg/kg.
  • test agents were prepared in accordance with the prescription 1 and the process of Example 1.
  • Figures 3 and 4 show the results of echocardiography at 7 days, 14 days, and 28 days in the Sham and MI groups of mice.
  • Figure 4 is a quantitative analysis of cardiac ultrasound results.
  • Figures 4A-F show that MI mice have a significantly reduced cardiac function compared to Sham mice and further decline over time. On day 7, 14 and 28, MI mice with tail vein injection had significantly lower cardiac ejection fraction (EF%) and short axis shortening (FS%) than oral administration. mouse.
  • EF% cardiac ejection fraction
  • FS short axis shortening
  • left ventricular end diastolic diameter (LVIDd), left ventricular end systolic diameter (LVIDs), left ventricular end diastolic volume (LV volume; d) and left ventricular end systolic volume (LV volume; s) are lower than oral gavage group. Similarly, different routes of administration did not have a differential effect on the cardiac function of Sham mice.
  • Figure 7 shows that the fibrous tissue (light gray, solid coiled out) formed after infarction in the heart tissue of the MI i.v group was significantly less than that of the MI oral group.
  • Figure 8 shows the results of quantitative analysis of Masson staining images. The results show that the ratio of infarct area to left ventricular area (IF/LV%) in MI iv group is significantly lower than that in MI oral group, indicating that compared with oral gavage, tail vein injection The drug has a stronger effect of reducing the infarct size after ischemia.
  • FIG. 10A is a western blot protein band.
  • Figures 10B and C quantify the results of the analysis.
  • Figures 10B and C show that the ratio of acetylated histones 3 and 4 to total histones 3 and 4 decreased after MI injury.
  • the acetylation level of histones 3 and 4 in the MI i.v group was higher than that in the corresponding MI oral group, indicating that the tail vein administration inhibited HDAC more strongly than oral administration, thereby further increasing the acetylation level of histones.
  • test agent was administered to MI mice by tail vein injection at a dose of 50 mg/kg.
  • Figures 11 and 12 The results of the ultrasound are shown in Figures 11 and 12, and Figure 11 is a simulation of the results of mouse echocardiography.
  • Figure 12 is a quantitative analysis of the results of each ultrasound.
  • Figures 12A and 12B show that the ejection fraction (EF%) and short axis shortening rate (FS%) of IR mice using intravenous prescriptions 1-6 were significantly higher than IR mice using intravenous prescriptions 7 and 8.
  • Figures 12C-12G show left ventricular end diastolic diameter (LVIDd), left ventricular end systolic diameter (LVIDs), left ventricular end diastolic volume (LV volume; d), left ventricular end systolic volume (LV volume; s), left ventricle LV mass These indicators did not show statistically significant differences between the intravenous formulation.
  • Figure 17 shows the light gray areas (ischemic risk area, solid coil out area) and white areas (post-ischemic infarct area, dotted circle area) in the cardiac tissue of I/R mice using intravenous prescriptions 1-6. The area is small.
  • Figure 18 is a quantitative result of a pathological staining picture.
  • Figure 18A and Figure 18B show that the ratio of infarct and area of risk (IF/AAR%), infarct area and left ventricular area ratio (IF/LV%) of I/R mouse hearts using intravenous prescriptions 1-6 are significant.
  • FIG. 21 Western blot results are shown in Figure 21.
  • Figure 21A is a western blot protein band.
  • 21B and 21C are quantitative analysis results.
  • Figure 21B and C show that the ratio of acetylated histones 3 and 4 to total histones 3 and 4 in I/R mouse myocardium with intravenous prescription 1-6 is significantly higher than I/R using intravenous prescriptions 7 and 8.
  • Mouse It is indicated that prescription 1-6 has a stronger inhibitory effect on HDAC, thereby further increasing the level of histone acetylation.
  • test agent was administered to MI mice by tail vein injection at a dose of 50 mg/kg.
  • Figures 13-16 The results of the ultrasound test are shown in Figures 13-16.
  • Figure 13 shows the results of cardiac ultrasound at 7 days, 14 days, and 28 days of the selected MI mice.
  • Figure 14, Figure 15, and Figure 16 are quantitative analyses of ultrasound results at 7 days, 14 days, and 28 days in MI mice, respectively.
  • a and B show that the cardiac ejection fraction (EF%) and short-axis shortening rate (FS%) of MI mice administered intravenous prescriptions 1-6 are evident at 7 days, 14 days, and 28 days. Higher than the corresponding MI mice with intravenous prescriptions 7 and 8.
  • CF showed left ventricular end-diastolic diameter (LVIDd), left ventricular end-systolic diameter (LVIDs), left ventricular end-diastolic volume (LV volume; d), and left ventricular end-systole in MI mice administered intravenous prescriptions 1-6.
  • Volume (LV volume; s) was significantly lower in 7 days, 14 days, and 28 days than MI mice using intravenous prescriptions 7 and 8.
  • Figure 22 The results of Western blot are shown in Figure 22.
  • Figure 22A is a western blot protein band.
  • 22B and 22C are quantitative analysis results.
  • 22B and 22C show that the ratio of acetylated histones 3 and 4 to total histones 3 and 4 in myocardial tissue of MI mice using intravenous prescriptions 1-6 was significantly higher than that of MI mice using intravenous prescriptions 7 and 8. It is indicated that prescription 1-6 has a stronger inhibitory effect on HDAC, thereby further increasing the level of histone acetylation.
  • the vorinostat content determination method uses reversed-phase high performance liquid chromatography, the chromatographic conditions are: C18 column (250mm * 4.6 ⁇ m * 5 ⁇ m), the mobile phase is acetonitrile - 0.1% phosphoric acid water (30: 70) (with three The pH of the ethylamine was adjusted to 3.0); the detection wavelength was 241 nm and the flow rate was 1.0 ml/min.
  • Table 2 The results of the stability study are shown in Table 2 below:
  • sample A has drug precipitation and fails to form milk; sample B has good indicators at the initial system, but the stability of the product subsequently shows a significant downward trend.
  • the encapsulation efficiency of the product is already low.
  • the drug demand could not be met; in comparison, the stability of sample 1 remained good.
  • Prescription 1 increases the proportion of stabilizers relative to prescription B, resulting in better stability of the product.
  • the stability of Sample C was somewhat lower than that of Sample 1.
  • N-suberanilide Hydroxamic acid is a kind of drug which is insoluble in oil and water.
  • the stability of sample A prepared by using traditional emulsion prescription A is poor. After homogenization, there is obvious drug precipitation. After high-speed centrifugation, A clear layer of drug powder can also be seen. It is therefore considered to add solubilizers and stabilizers.
  • the emulsion formulation B was only used to solve the problem of drug dissolution, and a relatively stable emulsion was prepared, but the long-term stability of the prepared sample B was poor and needs to be improved.
  • Formulation 1 employs a suitable ratio of stabilizer which, together with the solubilizing agent, provides a significant improvement in the stability of the emulsion.
  • the use of the emulsion formulation C increased the proportion of stabilizer added, but did not further improve the stability of the emulsion as expected, but the stability of the emulsion was decreased, and the production cost was increased.

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Abstract

本发明涉及一种用于保护缺血心肌的注射剂及其制备方法，按质量份数计，该注射用乳剂包含：N-辛二酰苯胺异羟肟酸1～5份；乳化剂0.2～12.5份；注射用油2～100份；增溶剂0.02～5份；油酸0.03～0.4份；甘油0.4～12.5份，以及注射用水余量。该注射用乳剂可有效地用于提高心脏介入手术前N-辛二酰苯胺异羟肟酸在心血管等亲脂性器官和/或组织的局部药物浓度，提高N-辛二酰苯胺异羟肟酸的生物利用度，降低其全身性副作用，实现在缺血或再灌注损伤状态下对心肌的有效保护，减少或避免心急梗死的发生。同时，该注射用乳剂也为N-辛二酰苯胺异羟肟酸在治疗癌症的应用中提供一种新的可供选择的剂型。

Description

用于保护缺血心肌的注射剂及其制备方法 技术领域
本发明属于药物制剂领域，涉及一种用于保护缺血心肌的注射剂及其制备方法，具体涉及一种包含N-辛二酰苯胺异羟肟酸的用于保护缺血心肌的注射用乳剂及其制备方法。
背景技术
组蛋白去乙酰化酶抑制剂(histone deacetylase inhibitor，HDACI)是一类具有干扰组蛋白去乙酰化酶的功能的化合物，已有大量的关于其在治疗HIV、恶性肿瘤或慢性纤维化性疾病等方面的研究报道。
已知N-辛二酰苯胺异羟肟酸(suberoylanilide hydroxamic acid，SAHA，化学结构见下式I)作为一种组蛋白去乙酰酶(HDAC)抑制剂，能够通过诱导细胞分化、阻断细胞周期、诱导细胞调控而发挥治疗作用。体外研究表明，N-辛二酰苯胺异羟肟酸以纳摩尔级浓度(IC 50<86nmol/L)即可抑制HDAC 1、HDAC 2和HDAC 3(I型)以及HDAC 6(II型)酶的活性。在某些癌细胞内，抑制过量的HDAC酶可激活正常细胞。因此，N-辛二酰苯胺异羟肟酸通过降低HDAC活性有助于减缓或中止某些癌细胞生长基因的激活。
Figure PCTCN2019072710-appb-000001
目前，N-辛二酰苯胺异羟肟酸已经经美国食品药品监督管理局批准上市，商品名为伏立诺他(vorinostat)，用于治疗加重、持续和复发，或用两种全身性药物治疗后无效的皮肤T细胞淋巴瘤(CTCL，一种影响皮肤的白血细胞类型的T细胞癌)。
最新的研究还表明，N-辛二酰苯胺异羟肟酸可以在临床上相关的再灌注的情况下经递送来降低大型动物模型的心肌梗死面积。在缺血/再灌注期间，N-辛二酰苯胺异羟肟酸至少部分通过诱导自噬流(autophagic flux)产生心脏保护作用(Min Xie et al.,Histone Deacetylase Inhibition Blunts Ischemia/Reperfusion Injury by Inducing Cardiomyocyte Autophagy,Circulation.2014；129:1139-1151)。因此，N-辛二酰苯胺异羟肟酸对于缺血或再灌注损伤状态下的心肌保护，例如减少心脏介入手术期间心肌梗死的发生具有重要的临床应用价值。
然而，目前上市的N-辛二酰苯胺异羟肟酸制剂仅限于抗肿瘤的口服剂型。由于N-辛二酰苯胺异羟肟酸的水溶性差，而且口服N-辛二酰苯胺异羟肟酸还存在明显的首过代谢消除，导致其口服生物利用度很低，无法在心血管等亲脂性组织中形成有效的治疗浓度。另外，N-辛二酰苯胺异羟肟酸只有作为原型才能起到治疗作用，而其代谢产物均无活性，因此不能通过化学衍生化的方式来提高其口服生物利用度。此外，如果在心脏介入手术前，口服N-辛二酰苯胺异羟肟酸还会导致全身性副作用。
现有技术中存在的上述问题极大地限制了N-辛二酰苯胺异羟肟酸的临床应用，特别是其在缺血/再灌注 期间对心肌保护的应用，因为不能在心脏介入手术前在心脏组织中实现有效的局部药物浓度。因此，急需开发一种新的N-辛二酰苯胺异羟肟酸的药物剂型以解决N-辛二酰苯胺异羟肟酸在缺血心肌保护的过程中出现的心脏组织中局部药物浓度过低的问题。
发明内容
为了克服现有技术的上述缺陷，本发明的目的在于提供一种用于保护缺血心肌的注射用乳剂及其制备方法。该注射用乳剂可有效地用于心脏介入手术前提高N-辛二酰苯胺异羟肟酸在心血管等亲脂性器官和/或组织的局部药物浓度，提高N-辛二酰苯胺异羟肟酸的生物利用度，降低其全身性副作用，实现在缺血或再灌注损伤状态下对心肌的有效保护，减少或避免心急梗死的发生。同时，该注射用乳剂也为N-辛二酰苯胺异羟肟酸在治疗癌症等方面的应用提供了一种新的可供选择的剂型。
用于实现上述目的的技术方案如下：
一种用于保护缺血心肌的注射用乳剂，按质量份数计，其包含：
N-辛二酰苯胺异羟肟酸 1～5份；
乳化剂 0.2～12.5份；
注射用油 2～100份；
增溶剂 0.02～5份；
油酸 0.03～0.4份；
甘油 0.4～12.5份，以及
注射用水 余量。
优选地，按质量份数计，所述注射用乳剂包含：
N-辛二酰苯胺异羟肟酸 3份；
乳化剂 0.5～5份；
注射用油 5～40份；
增溶剂 0.05～2份；
油酸 0.03～0.3份；
甘油 1.0～5份，以及
注射用水 余量。
更优选地，按质量份数计，所述注射用乳剂包含：
N-辛二酰苯胺异羟肟酸 3份；
乳化剂 1.0～2.5份；
注射用油 10～20份；
增溶剂 0.1～1份；
油酸 0.05～0.15份；
甘油 2.0～2.5份，以及
注射用水 余量。
进一步优选地，按质量份数计，所述注射用乳剂包含：
N-辛二酰苯胺异羟肟酸 3份；
乳化剂 1.0～2.5份；
注射用油 10～20份；
增溶剂 0.6份；
油酸 0.05～0.15份；
甘油 2.0～2.5份， 以及
注射用水 余量。
再优选地，按质量份数计，所述注射用乳剂包含：
N-辛二酰苯胺异羟肟酸 3份；
乳化剂 2份；
注射用油 20份；
增溶剂 0.6份；
油酸 0.1份；
甘油 2.5份，以及
注射用水 余量。
在上述注射用乳剂中，所述乳化剂优选为磷脂；更优选地，所述磷脂选自大豆磷脂、卵磷脂、氢化大豆磷脂或氢化卵磷脂中的一种或多种；进一步优选地，所述磷脂为大豆磷脂和/或卵磷脂。
在上述注射用乳剂中，所述注射用油可以选自注射用大豆油、注射用红花油、注射用棉籽油、注射用芝麻油、注射用茶油、注射用橄榄油或注射用中链油中的一种或多种；优选地，所述注射用油为注射用大豆油。
在上述注射用乳剂中，所述增溶剂可以选自吐温-80、丙二醇、泊洛沙姆188或聚乙二醇15羟硬脂酸酯(Solutol HS 15)中的一种或多种；优选地，所述增溶剂为吐温-80和/或丙二醇。
根据本发明的一个具体实施方案，按质量份数计，所述注射用乳剂包含：
N-辛二酰苯胺异羟肟酸 3份；
大豆磷脂或卵磷脂 2份；
注射用大豆油 20份；
吐温-80或丙二醇 0.6份；
油酸 0.1份；
甘油 2.5份， 以及
注射用水 余量。
本发明还提供了所述注射用乳剂的制备方法，该制备方法包括以下步骤：
(1)向N-辛二酰苯胺异羟肟酸加入增溶剂和甘油，加热，再加注射用水至N-辛二酰苯胺异羟肟酸溶 解；
(2)向步骤(1)得到的溶液加入乳化剂，混匀，制得水相；
(3)将油酸与注射用油混匀，制得油相；
(4)将水相和油相在加热下高速剪切混合，制得初乳；
(5)将初乳在加压条件下进行均质化。
在所述制备方法的步骤(1)中，优选地，所述加热至50～90℃，更优选至70～80℃。
在所述制备方法的步骤(2)中，优选地，所述混匀采用剪切混合；优选地，所述剪切混合的速度为3000～10000转/分；更优选地，所述剪切混合的速度为5000～6000转/分。
在所述制备方法的步骤(4)中，优选地，所述加热至70℃～80℃且所述高速剪切混合的剪切速度为3000～10000转/分，优选为5000～6000转/分；优选地，所述高速剪切混合进行10～40分钟，更优选进行25～40分钟，最优选进行30分钟。
在所述制备方法的步骤(5)中，优选地，所述加压条件为400～1200巴，更优选为700～900巴；所述均质化可以进行1-6次，优选进行5～6次。
优选地，所述制备方法还包括以下步骤：
(6)封装；
(7)灭菌。
优选地，所述灭菌的条件为115℃下30分钟。
另一方面，本发明提供了所述的注射用乳剂在制备用于保护缺血心肌的药物、用于治疗心肌缺血再灌注损伤的药物或用于治疗心肌梗死的药物中的应用。
再一方面，本发明提供了所述N-辛二酰苯胺异羟肟酸乳剂在制备用于治疗缺血再灌注损伤的药物中的应用。在脑血管的缺血再灌注损伤，以及人体其他血液循环系统的缺血再灌注损伤，本发明也可以发挥治疗用途。
再一方面，本发明提供了所述N-辛二酰苯胺异羟肟酸乳剂在制备用于心血管疾病的药物中的应用。
再一方面，本发明提供了所述N-辛二酰苯胺异羟肟酸乳剂在制备用于心脏疾病的药物中的应用。
再一方面，本发明提供了所述N-辛二酰苯胺异羟肟酸乳剂在制备用于缺血心肌保护的药物中的应用或用于治疗心肌缺血再灌注损伤的药物中的应用或用于治疗心肌梗死的药物中的应用。
再一方面，本发明提供了所述N-辛二酰苯胺异羟肟酸注射用乳剂在制备用于缺血心肌保护的药物中的应用或用于治疗心肌缺血再灌注损伤的药物中的应用或用于治疗心肌梗死的药物中的应用。
本发明人经过大量研究发现，注射用乳剂可以作为一种有效提高N-辛二酰苯胺异羟肟酸在心血管等亲脂性器官和/或组织中的生物利用度的有效手段。然而，N-辛二酰苯胺异羟肟酸(伏立诺他)是一种油水均难溶的药物，本发明人在实验过程中发现使用传统注射用处方制备的乳剂稳定性很差，均质化完毕后出现明显的药物析出，高速离心后还能看到明显的药物粉末层，因此需要考虑加入增溶剂和稳定剂来改善所述注射用乳剂的稳定性，从而确保实现其提高N-辛二酰苯胺异羟肟酸在心脏组织中的有效浓度和生物利用度的效果。
重要的是，本发明人发现对于这种特殊的难溶性药物而言并非加入任何增溶剂和/或稳定剂均可以有效改 善所得到的注射用乳剂的稳定性，而在本发明选择的增溶剂和稳定剂的存在下，可以获得稳定最佳的注射用乳剂。此外，本发明人还注意到注射用乳剂中各种组成成分的配比也对注射用乳剂的稳定性存在显著的影响。为此，本发明人对包含N-辛二酰苯胺异羟肟酸的注射用乳剂的处方进行了大量研究、筛选和优化，最终获得了如本发明所述的具有优异稳定性的注射用乳剂及其制备方法。
实验表明，本发明所述注射用乳剂可有效提高N-辛二酰苯胺异羟肟酸在心血管等亲脂性器官和/或组织的局部有效浓度，改善了N-辛二酰苯胺异羟肟酸的生物利用度，有利于实现在缺血或再灌注损伤状态下对心肌的有效保护，减少或避免了心脏介入手术过程中心肌梗死的发生。同时，由于成功地提高了N-辛二酰苯胺异羟肟酸在心脏等组织的局部药物浓度，降低或避免了其全身性副作用，也改善了该药物对肿瘤等其他疾病的治疗效果。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：Sham oral表示假手术口服灌胃组；Sham i.v表示假手术静脉注射组；I/R oral Pre表示缺血再灌注一次口服灌胃组；I/R i.v Pre表示缺血再灌注一次静脉注射组；I/R oral Pre+Reperfusion表示缺血再灌注分次口服灌胃组；I/R i.v Pre+Reperfusion表示缺血再灌注分次静脉注射组；MI oral表示心肌梗死口服灌胃组；MI i.v表示心肌梗死静脉注射组。
图1为采用心脏超声检测经口服灌胃和静脉注射给予N-辛二酰苯胺异羟肟酸对缺血再灌注(I/R)组小鼠的影响的照片(24h)。
图2显示采用心脏超声检测经口服灌胃和静脉注射给予N-辛二酰苯胺异羟肟酸对缺血再灌注(I/R)组小鼠的影响的分析结果(24h)；其中，*P<0.05，与Sham oral相比；#P<0.05，与I/R oral Pre相比；+P<0.05，与I/R oral Pre+Reperfusion相比；n.s.，无显著性差异。
图3为采用心脏超声检测经口服灌胃和静脉注射给予N-辛二酰苯胺异羟肟酸对心肌梗死(MI)组小鼠的影响的照片(7天、14天、28天)。
图4显示采用心脏超声检测经口服灌胃和静脉注射给予N-辛二酰苯胺异羟肟酸对心肌梗死(MI)组小鼠的影响的分析结果(7天、14天、28天)；其中，*P<0.05，与Sham oral相比；#P<0.05，与MI oral相比。
图5为采用Evans blue和TTC双染色检测经口服灌胃和静脉注射给予N-辛二酰苯胺异羟肟酸对缺血再灌注组小鼠的影响的照片(24小时)；其中，深灰色显示非缺血区；浅灰色显示缺血危险区(实线圈出区域)；白色显示缺血后梗死区(虚线圈出区域)。
图6显示采用Evans blue和TTC双染色检测经口服灌胃和静脉注射给予N-辛二酰苯胺异羟肟酸对缺血再灌注组小鼠的影响的心脏组织双染色面积量化分析结果(24h)；其中，*P<0.05，与Sham oral相比；#P<0.05，与I/R oral Pre相比；+P<0.05，与I/R oral Pre+Reperfusion相比；n.s.，无显著性差异。
图7显示采用Masson染色检测经口服灌胃和静脉注射给予N-辛二酰苯胺异羟肟酸对心肌梗死组小鼠的影响的照片(28天)；其中，深灰色为正常心肌组织，浅灰色为纤维组织(实线圈出区域)。
图8显示采用Masson染色检测经口服灌胃和静脉注射给予N-辛二酰苯胺异羟肟酸对心肌梗死组小鼠的影响分析结果(28天)；其中，#P<0.05，与MI oral相比。
图9显示采用Western blot检测经口服灌胃和静脉注射给予N-辛二酰苯胺异羟肟酸对缺血再灌注组小鼠心脏组织组蛋白H3和H4乙酰化水平的影响的照片和分析结果；其中，*P<0.05，与Sham oral相比；#P<0.05，与I/R oral Pre相比；+P<0.05，与I/R oral Pre+Reperfusion相比；n.s.，无显著性差异。
图10显示采用Western blot检测经口服灌胃和静脉注射给予N-辛二酰苯胺异羟肟酸对心肌梗死组小鼠心脏组织组蛋白H3和H4乙酰化水平的影响的照片和分析结果；其中，*P<0.05，与Sham oral相比；#P<0.05，与MI oral相比。
图11为采用超声检测不同处方的注射用乳剂对缺血再灌注(I/R)组小鼠的影响的照片(24h)。
图12显示采用超声检测不同处方的注射用乳剂对缺血再灌注(I/R)组小鼠的影响的分析结果(24h)；其中，*P<0.05，处方1-6与处方7-8相比。
图13为采用超声检测不同处方的注射用乳剂对缺血再灌注(I/R)组小鼠的影响的照片(7天、14天、28天)。
图14显示采用超声检测不同处方的注射用乳剂对缺血再灌注(I/R)组小鼠的影响的分析结果(7天)；其中，*P<0.05，处方1-6与处方7-8相比。
图15显示采用超声检测不同处方的注射用乳剂对缺血再灌注(I/R)组小鼠的影响的分析结果(14天)；其中，*P<0.05，处方1-6与处方7-8相比。
图16显示采用超声检测不同处方的注射用乳剂对缺血再灌注(I/R)组小鼠的影响的分析结果(28天)；其中，*P<0.05，处方1-6与处方7-8相比。
图17为采用Evans blue和TTC双染色检测不同处方的注射用乳剂对缺血再灌注(I/R)组小鼠的影响的照片(24h)；其中，深灰色显示非缺血区；浅灰色显示缺血危险区(实线圈出区域)；白色显示缺血后梗死区(虚线圈出区域)。
图18显示采用Evans blue和TTC双染色检测不同处方的注射用乳剂对缺血再灌注(I/R)组小鼠的影响的心脏组织双染色面积量分析结果(24h)；其中，*P<0.05，处方1-6与处方7-8相比。
图19为采用Masson染色检测不同处方的注射用乳剂对心肌梗死(MI)组小鼠的影响的照片(28天)；其中，深灰色为正常心脏组织区域，浅灰色为梗死后形成的纤维组织区(实线圈出区域)。
图20显示采用Masson染色检测不同处方的注射用乳剂对心肌梗死(MI)组小鼠的影响的分析结果(28天)；其中，*P<0.05，处方1-6与处方7-8相比。
图21显示采用Western blot检测不同处方的注射用乳剂对缺血再灌注(I/R)组小鼠心脏组织组蛋白H3和H4乙酰化水平的影响的照片和分析结果；其中，*P<0.05，处方1-6与处方7-8相比。
图22显示采用Western blot检测不同处方的注射用乳剂对心肌梗死(MI)组小鼠心脏组织组蛋白H3和H4乙酰化水平的影响的照片和分析结果；其中，*P<0.05，处方1-6与处方7-8相比。
具体实施方式
以下参照具体的实施例来说明本发明。本领域技术人员能够理解，这些实施例仅用于说明本发明，其不以任何方式限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的原料、试剂材料等，如无特殊说明，均为市售购买产品。
实施例1
一、处方1
原辅料名称 处方量(g)
N-辛二酰苯胺异羟肟酸 15
大豆磷脂 10
注射用大豆油 100
吐温-80 3
油酸 0.5
甘油 12.5
注射用水 适量
制成500ml。
二、工艺：
称取处方量的N-辛二酰苯胺异羟肟酸，加入吐温-80和甘油，加热至80℃，加水约100ml，至原料药溶解，加入大豆磷脂，剪切(6000rpm)混合均匀，制成水相；称取油酸和注射用大豆油，混合均匀，制成油相；油水两相在70℃条件下，高速剪切混合(6000rpm)30分钟，制成初乳。将初乳在800bar压力下进行均质化，循环6遍，装入安瓿，封口，115℃下灭菌30分钟即得。
实施例2
一、处方2
原辅料名称 处方量(g)
N-辛二酰苯胺异羟肟酸 15
大豆磷脂 5
注射用大豆油 75
泊洛沙姆188 3
油酸 0.75
甘油 12.5
注射用水 适量
制成500ml。
二、工艺：
称取处方量的N-辛二酰苯胺异羟肟酸，加入泊洛沙姆188和甘油，加热至80℃，加水约100ml，至原料药溶解，加入大豆磷脂，剪切(6000rpm)混合均匀，制成水相；称取油酸和注射用大豆油，混合均匀，制成油相； 油水两相在80℃条件下，高速剪切混合(5000rpm)40分钟，制成初乳。将初乳在700bar压力下进行均质化，循环6遍，装入安瓿，封口，115℃下灭菌30分钟即得。
实施例3
一、处方3
原辅料名称 处方量(g)
N-辛二酰苯胺异羟肟酸 15
大豆磷脂 12.5
注射用大豆油 50
聚乙二醇15羟硬脂酸酯 5
油酸 0.25
甘油 11.5
注射用水 适量
制成500ml
二、工艺：
称取处方量的N-辛二酰苯胺异羟肟酸，加入聚乙二醇15羟硬脂酸酯和甘油，加热至80℃，加水约100ml，至原料药溶解，加入大豆磷脂，剪切(5000rpm)混合均匀，制成水相；称取油酸和注射用大豆油，混合均匀，制成油相；油水两相在80℃条件下，高压剪切混合(6000rpm)40分钟，制成初乳。将初乳在800bar压力下进行均质化，循环6遍，装入安瓿，封口，115℃下灭菌30分钟即得。
实施例4
一、处方4
原辅料名称 处方量(g)
N-辛二酰苯胺异羟肟酸 15
大豆磷脂 11.5
注射用橄榄油 85
吐温-80 3
油酸 0.6
甘油 10
注射用水 适量
制成500ml
二、工艺：
称取处方量的N-辛二酰苯胺异羟肟酸，加入吐温-80和甘油，加热至70℃，加水约100ml，至原料药溶解， 加入大豆磷脂，剪切(6000rpm)混合均匀，制成水相；称取油酸和注射用橄榄油，混合均匀，制成油相；油水两相在70℃条件下，高速剪切混合(6000rpm)25分钟，制成初乳。将初乳在800bar压力下进行均质化，循环6遍，装入安瓿，封口，115℃下灭菌30分钟即得。
实施例5
一、处方5
原辅料名称 处方量(g)
N-辛二酰苯胺异羟肟酸 15
卵磷脂 10
注射用中链油 100
吐温-80 0.5
油酸 0.5
甘油 12.5
注射用水 适量
制成500ml
二、工艺：
称取处方量的N-辛二酰苯胺异羟肟酸，加入吐温-80和甘油，加热至80℃，加水约100ml，至原料药溶解，加入卵磷脂，剪切(5000rpm)混合均匀，制成水相；称取油酸和注射用中链油，混合均匀，制成油相；油水两相在70℃条件下，高速剪切混合(6000rpm)30分钟，制成初乳。将初乳在900bar压力下进行均质化，循环5遍，装入安瓿，封口，115℃下灭菌30分钟即得。
实施例6
一、处方6
原辅料名称 处方量(g)
N-辛二酰苯胺异羟肟酸 15
大豆磷脂 10
注射用大豆油 100
丙二醇 3
油酸 0.5
甘油 12.5
注射用水 适量
制成500ml
二、工艺：
称取处方量的N-辛二酰苯胺异羟肟酸，加入甘油和丙二醇，加热至70℃，加水约100ml，至原料药溶解，加入大豆磷脂，剪切(6000rpm)混合均匀，制成水相；称取油酸和注射用大豆油，混合均匀，制成油相；油水两相在70℃条件下，高速剪切混合(5000rpm)30分钟，制成初乳。将初乳在800bar压力下进行均质化，循环6遍，装入安瓿，封口，115℃下灭菌30分钟即得。
对比例1
一、处方7
原辅料名称 处方量(g)
N-辛二酰苯胺异羟肟酸 15
大豆磷脂 10
注射用大豆油 100
吐温-80 3
维生素E 0.5
甘油 12.5
注射用水 适量
制成500ml
二、工艺：
称取处方量的N-辛二酰苯胺异羟肟酸，加入甘油和吐温-80，加热至80℃，加水约100ml，至原料药溶解，加入大豆磷脂，剪切(6000rpm)混合均匀，制成水相；称取维生素E和注射用大豆油，混合均匀，制成油相；油水两相在70℃条件下，高速剪切混合(6000rpm)30分钟，制成初乳。将初乳在800bar压力下进行均质化，循环6遍，装入安瓿，封口，115℃下灭菌30分钟即得。
对比例2
一、处方8
原辅料名称 处方量(g)
N-辛二酰苯胺异羟肟酸 15
大豆磷脂 10
注射用大豆油 100
吐温-80 3
聚氧乙烯氢化蓖麻油 0.5
甘油 12.5
注射用水 适量
制成500ml
二、工艺：
称取处方量的N-辛二酰苯胺异羟肟酸，加入吐温-80和甘油，加热至80℃，加水约100ml，至原料药溶解，加入大豆磷脂，剪切(6000rpm)混合均匀，制成水相；称取聚氧乙烯氢化蓖麻油和注射用大豆油，混合均匀，制成油相；油水两相在70℃条件下，高速剪切混合(6000rpm)30分钟，制成初乳。将初乳在800bar压力下进行均质化，循环6遍，装入安瓿，封口，115℃下灭菌30分钟即得。
药理实验例
1.动物模型的建立
1.1实验材料：
眼科直剪和弯剪各1把，带齿弯镊和直镊各1把，无齿弯镊和直镊各1把，蚊式血管钳1把，6号带针缝线若干，弯盘，纱布和无菌棉签若干，生理盐水，吸入式气体麻醉机，10ml和50ml注射器，小鼠灌胃针，小鼠手术台，保温垫，1.5ml离心管，15ml冻存管，细胞培养皿，手术刀片，手术刀柄，2％异氟烷。
1.2假手术(Sham)和缺血再灌注小鼠模型(I/R)的建立
小鼠接受2％异氟烷吸入麻醉后，术者在其左侧前胸壁上切开一个1.2cm左右的皮肤切口，并预留置一荷包缝合。在荷包内钝性分离胸部肌肉组织后，暴露左胸第四肋间隙。接下来，在该荷包内使用蚊式血管钳在第四肋间隙的位置轻柔而迅速地撑开胸膜腔和心包腔，挤出心脏。使用6号带针缝线于心脏左冠状动脉前降支处结扎一活结，肉眼观察相应区域心肌组织颜色转为灰白，确认结扎成功。其后，迅速将心脏放置回胸腔内，排净腔内空气，收紧荷包关闭胸腔，肌肉和皮肤，并留置结扎左前降支的活结的线头于胸外。将小鼠置于空气流通的环境中，监控其复苏情况。缺血45分钟后，通过轻柔平缓地拖拽胸外留置的线头松解结扎前降支的滑结，心肌组织获得血流再灌注。对照的假手术小鼠除了不于前降支处结扎活结外，其余操作同缺血再灌注小鼠。
1.3假手术(Sham)和心梗小鼠模型(MI)的建立
小鼠接受2％异氟烷吸入麻醉后，术者在其左侧前胸壁上切开一个1.2cm左右的皮肤切口，并预留置一荷包缝合。在荷包内钝性分离胸部肌肉组织后，暴露左胸第四肋间隙。接下来，在该荷包内使用蚊式血管钳在第四肋间隙的位置轻柔而迅速地撑开胸膜腔和心包腔，挤出心脏。使用6号带针缝线直接结扎心脏左冠状动脉前降支，肉眼观察相应区域的心肌组织颜色转为灰白，确认结扎成功。其后，迅速将心脏放置回胸腔内，排净腔内空气，通过收紧荷包关闭胸腔，肌肉和皮肤。对照的假手术小鼠除了不结扎前降支外，其余操作同心梗小鼠。
2.检测方法
2.1心脏功能的超声检测
缺血再灌注组小鼠：术后24小时后再次吸入2％异氟烷麻醉，应用小动物超声波影像诊断仪(Vevo Rolling Imaging Cart，Canada，Vevo 770)分别沿小鼠心脏短轴和长轴采图，其后测定各项心脏超声指标(包括：射血分数(EF％)、缩短分数(FS％)、左室舒张末期内径(LVIDd)、左心室收缩末期内径(LVIDs)、左室舒张末期容积(LV volume；d)、左室收缩末期容积(LV volume；s)等)
心梗组小鼠：分别于术后7天、14天、28天进行心脏超声检查，麻醉和超声测定步骤同上所述。
2.2Evans blue和TTC双染色检测
1％戊巴比妥钠溶液腹腔注射麻醉小鼠后，打开胸腔暴露心脏，将造模时结扎的左冠状动脉前降支部位再次 结扎左冠状动脉前降支。以5％酞菁蓝染色液沿升主动脉根部逆行注射，之后摘取小鼠心脏，置于冻存管中于-80℃冰箱冻存20分钟。其后，取出冰冻的心脏，从结扎部位起向下，迅速将心脏切为1-2cm厚的切片。将切片置于2,3,5-氯化三苯基四氮唑(TTC)的磷酸盐缓冲溶液中，在37℃的水浴箱中避光孵育30分钟。之后将心脏切片用甲醛浸泡4小时固定后用体视仪采图。经过上述步骤，在展示的病理图中，LV的非缺血区域为深灰色，缺血危险区域为浅灰色(实线圈出区域)，缺血后梗死区域为白色(虚线圈出区域)。图像使用Image J软件进行量化分析，定量梗死区域(IF)、缺血危险区(AAR)和左室面积(LV)并计算其比值。
2.3Masson染色检测
1％戊巴比妥钠溶液腹腔注射麻醉处死小鼠后摘取小鼠心脏。心脏在多聚甲醛(4℃)中处理24小时，然后在流动水中冲洗1小时。将处理后的心脏标本组织包埋并切片。将切片放置于烤箱中，烘烤50分钟。标本脱蜡后置于磷钼酸及重铬酸钾混合液中过夜。标本经蒸馏水冲洗后，在苏木素染液中染4分钟，其后充分水洗，再酒精分化3秒。标本经立春红染色5分钟。1％磷钼酸水溶液分化3-5分钟，后用1％苯胺蓝处理15秒。标本经充分冲洗后，用95％酒精、无水酒精、二甲苯顺序浸泡3秒，后用中性树胶封固。显微镜下观察并拍照。应用Image J软件量化分析图像，定量小鼠心肌梗死区面积(IF)和左心室总面积(LV)并计算其比值。
2.4Western blot
1％戊巴比妥钠溶液腹腔注射麻醉处死小鼠，取材小鼠心脏，使用眼科剪剪碎心肌组织后再进行充分研磨。其后，应用组蛋白提取试剂盒提取组蛋白，检测各组的组蛋白浓度，并调整蛋白上样量为50μg。通过15％SDS-聚丙烯酰胺凝胶电泳后转至0.22μm的PVDF膜上，封闭液稀释一抗(1:1000)，4℃孵育15h，洗膜后封闭液稀释二抗(1:5000)，温室孵育2h。再次洗膜后化学发光成像。应用Image J软件分析并定量蛋白条带上乙酰化组蛋白3、总组蛋白3、乙酰化组蛋白4和总组蛋白4的灰度值，计算比值并进行统计学分析。
3.检测结果
3.1注射给药与口服给药的比较
3.1.1待测药剂在缺血再灌注模型中的作用
分别设置缺血再灌注小鼠一次给药组(附图中标注为I/R Pre)和缺血再灌注小鼠分次给药组(附图中标注为I/R Pre+Reperfusion)。其中，对于缺血再灌注小鼠一次给药组，待测药剂按照50mg/kg的剂量通过口服灌胃(附图中标注为I/R oral)或尾静脉注射(附图中标注为I/R i.v)的方式给予I/R小鼠，于手术操作一小时前一次给予所有药物。对于缺血再灌注小鼠分次给药组，待测药剂通过口服灌胃(附图中标注为I/R oral)或尾静脉注射(附图中标注为I/R i.v)的方式给予I/R小鼠，在手术操作一小时前和心肌再灌注操作时分别给予25mg/kg剂量的药物(总量与一次给药组相同)。
另外，待测药剂按照50mg/kg的剂量通过口服灌胃(附图中标注为oral)或尾静脉注射(附图中标注为i.v)的方式给予Sham小鼠。
上述待测药剂均按照实施例1的处方1和工艺制备。
超声检测结果见图1和2。图1为选取展示的Sham和I/R组小鼠的心脏超声结果。图2为对心脏超声结果的量化分析。图2A和2B显示，相比Sham组，I/R操作后小鼠心脏射血分数百分比(EF％)和短轴缩短率(FS％)明显下降。尾静脉注射给药的I/R小鼠的EF％和FS％(I/R i.v Pre和I/R i.v Pre+Reperfusion组)要明显高于口服 灌胃给药的I/R小鼠(I/R oral Pre和I/R oral Pre+Reperfusion组)。但EF％和FS％值在I/R i.v Pre组和I/R i.v Pre+Reperfusion组之间并未表现出统计学差异，说明一次给药和分次给药对小鼠心脏射血功能的影响没有差异。图2C-2G显示，左室舒张末期内径(LVIDd)、左室收缩末期内径(LVIDs)、左室舒张末期容积(LV volume；d)、左室收缩末期容积(LV volume；s)和左室质量(LV mass)这些指标在尾静脉给药组和口服灌胃给药组之间并未出现有统计学意义的差异。此外，不同给药途径对Sham小鼠的EF％和FS％值并未产生不同的影响。
Evans blue和TTC双染色的病理结果见图5。图5显示，相比口服灌胃组，尾静脉注射组的心脏组织中浅灰色区域(缺血危险区，实线圈出区域)和白色区域(缺血后梗死区，虚线圈出区域)的面积较小。图6为病理染色图片的量化分析结果。图6A-C显示，尾静脉注射组(I/R i.v Pre和I/R i.v Pre+Reperfusion组)的危险区和左室面积的比值(AAR/LV％)、梗死区和危险区面积比值(IF/AAR％)、梗死区和左室面积比值(IF/LV％)要明显低于对应的口服灌胃组(I/R oral Pre和I/R oral Pre+Reperfusion组)，说明相比口服灌胃给药，尾静脉注射给药能进一步缩小梗死区域和缺血损伤区域，显著减轻缺血和再灌注损伤。同心脏超声指标结果一致，一次给药和分次给药对梗死和缺血区域的面积未有差异的影响。
Western blot结果见图9。图9A为western blot蛋白条带。图9B和C为定量分析结果。图9B和C显示，乙酰化的组蛋白3和4与总组蛋白3和4的比值在I/R损伤后降低。尾静脉注射组(I/R i.v Pre和I/R i.v Pre+Reperfusion组)的组蛋白3和4的乙酰化水平要高于对应的口服灌胃组(I/R oral Pre和I/R oral Pre+Reperfusion组)，说明相比口服灌胃，尾静脉用药对HDAC的抑制作用更强，从而进一步提高组蛋白的乙酰化水平。
3.1.2待测药剂在心梗动物模型中的作用
待测药剂按照50mg/kg的剂量通过口服灌胃(附图中标注为oral)或尾静脉注射(附图中标注为i.v)的方式分别给予Sham和MI小鼠。
待测药剂均按照实施例1的处方1和工艺制备。
超声检测结果见图3和4。图3为选取展示的Sham和MI组小鼠7天、14天和28天的心脏超声结果。图4为心脏超声结果的量化分析。图4A-F显示，相比Sham小鼠，MI小鼠的心功能明显下降，并且随着时间的推移进一步下降。在第7天、14天和28天，尾静脉注射用药的MI小鼠的心脏射血分数百分比(EF％)和短轴缩短率(FS％)都要显著高于口服灌胃用药的MI小鼠。而左室舒张末期内径(LVIDd)、左室收缩末期内径(LVIDs)、左室舒张末期容积(LV volume；d)和左室收缩末期容积(LV volume；s)等值要低于口服灌胃组。同样地，不同给药途径对Sham小鼠的心功能并未出现有差异的影响。
Masson病理染色结果参见图7。图7显示MI i.v组小鼠心脏组织中梗死后形成的纤维组织(浅灰色，实线圈出区域)要明显少于MI oral组小鼠。图8为Masson染色图片的量化分析结果，结果显示MI i.v组的梗死区和左室面积的比值(IF/LV％)要明显低于MI oral组，说明相比口服灌胃，尾静脉注射给药具有更强的缩小缺血后梗死面积的作用。
Western blot结果见图10。图10A为western blot蛋白条带。图10B和C定量分析结果。图10B和C显示，乙酰化的组蛋白3和4与总组蛋白3和4的比值在MI损伤后降低。MI i.v组的组蛋白3和4的乙酰化水平要高于对应的MI oral组，说明相比口服灌胃，尾静脉用药对HDAC的抑制作用更强，从而进一步提高组蛋白的乙酰化水平。
3.2不同注射用乳剂处方的比较
3.2.1待测药剂在缺血再灌注动物模型中的作用
待测药剂按照50mg/kg的剂量通过尾静脉注射的方式给予MI小鼠。
超声结果见图11和12，图11为选取展示的小鼠心脏超声结果。图12为各超声结果的量化分析。图12A和图12B显示应用静脉处方1-6的IR小鼠的射血分数(EF％)和短轴缩短率(FS％)要明显高于应用静脉处方7和8的IR小鼠。图12C-12G显示，左室舒张末期内径(LVIDd)、左室收缩末期内径(LVIDs)、左室舒张末期容积(LV volume；d)、左室收缩期末容积(LV volume；s)、左室质量(LV mass)这些指标在各静脉剂型处方间未出现有统计学意义的差异。
Evans blue和TTC双染色的病理结果见图17。图17显示应用静脉处方1-6组的I/R小鼠的心脏组织中浅灰色区域(缺血危险区，实线圈出区域)和白色区域(缺血后梗死区，虚线圈出区域)的面积较小。图18为病理染色图片的量化结果。图18A和图18B图显示应用静脉处方1-6的I/R小鼠心脏的梗死区和危险区面积比值(IF/AAR％)、梗死区和左室面积比值(IF/LV％)要明显低于应用静脉处方7和8的I/R小鼠，说明对比处方7和8，处方1-6能进一步缩小梗死和缺血损伤区域，显著减少缺血再灌注损伤。
Western blot结果见图21。图21A为western blot蛋白条带。图21B和图21C为定量分析结果。图21B和C显示应用静脉处方1-6的I/R小鼠心肌组织的乙酰化组蛋白3和4与总组蛋白3和4的比值要明显高于应用静脉处方7和8的I/R小鼠。说明处方1-6对HDAC的抑制作用更强，从而进一步提高组蛋白的乙酰化水平。
3.2.2待测药剂在心梗动物模型中的作用
待测药剂按照50mg/kg的剂量通过尾静脉注射的方式给予MI小鼠。
超声检测结果见图13-16，图13为选取展示的MI小鼠7天、14天、28天心脏超声结果。图14、图15、图16分别为MI小鼠7天、14天、28天超声结果的量化分析。图14-16中A和B显示，应用静脉处方1-6的MI小鼠的心脏射血分数(EF％)和短轴缩短率(FS％)在7天、14天、28天均要明显高于应用静脉处方7和8对应的MI小鼠。各图中C-F显示应用静脉处方1-6的MI小鼠的左室舒张末期内径(LVIDd)、左室收缩末期内径(LVIDs)、左室舒张末期容积(LV volume；d)、左室收缩期末容积(LV volume；s)在7天、14天、28天均要明显低于应用静脉处方7和8的MI小鼠。
Masson的病理结果见图19。图19显示，应用静脉处方1-6的MI小鼠心脏组织中的梗死后纤维组织(浅灰色，实线圈出区域)要明显少于应用静脉处方7和8的MI小鼠。图20为Masson染色的量化分析结果，结果显示应用静脉处方1-6的MI小鼠心脏组织的梗死区和左心室面积比值(IF/LV％)要明显低于应用静脉处方7和8的MI小鼠，说明相比静脉处方7和8，静脉处方1-6具有更强的缩小缺血后梗死面积的作用。
Western blot结果见图22。图22A为western blot蛋白条带。图22B和图22C为定量分析结果。图22B和图22C显示应用静脉处方1-6的MI小鼠心肌组织的乙酰化组蛋白3和4与总组蛋白3和4的比值要明显高于应用静脉处方7和8的MI小鼠。说明处方1-6对HDAC的抑制作用更强，从而进一步提高组蛋白的乙酰化水平。
稳定性实验例
采用以下表1所示的各个处方制备注射用乳剂样品并进行稳定性比较：
表1进行稳定性实验的处方
  处方A 处方B 处方C 处方1
N-辛二酰苯胺异羟肟酸(g) 15 15 15 15
大豆磷脂(g) 10 10 10 10
注射用大豆油(g) 100 100 100 100
吐温-80(增溶剂)(g) 0 3 3 3
油酸(稳定剂)(g) 0 0.1 2.5 0.5
甘油(g) 12.5 12.5 12.5 12.5
注射用水 适量 适量 适量 适量
对上述四种处方按照实施例1的制备工艺制成的样品(处方A、处方B、处方C和处方1的样品在下表2中分别显示为样品A、样品B、样品C和样品1)进行了6个月的稳定性考察，即室温条件下，分别在0个月、1个月、3个月和6个月对样品的外观、平均粒径、Zata电位、包封率(含量)进行持续考察，其中平均粒径和zata电位采用激光粒径仪测定，包封率采用超速离心法将油相、乳化层和水相分开，通过测定水相中伏立诺他的含量计算包封率，伏立诺他含量测定方法采用反相高效液相色谱法，色谱条件为：C18色谱柱(250mm*4.6μm*5μm)，流动相为乙腈-0.1％磷酸水(30:70)(用三乙胺调pH至3.0)；检测波长为241nm，流速为1.0ml/min。稳定性考察的结果如下表2：
表2稳定性考察的结果
Figure PCTCN2019072710-appb-000002
Figure PCTCN2019072710-appb-000003
由上述数据可以看到，样品A有药物析出，未能成乳；样品B初制时各项指标良好，但产品的稳定性随后呈明显下降趋势，6个月时，产品的包封率已经低于80％，不能满足用药需求；相比较下，样品1稳定性一直保持良好。处方1相对处方B而言，增加了稳定剂的比例，使产品达到更好的稳定性。但是，尽管处方C中的稳定剂比例更高，样品C的稳定性相对样品1还是有所下降。
N-辛二酰苯胺异羟肟酸是一种油水均难溶的药物，使用传统的乳剂处方A制备的样品A稳定性较差，均质化完毕后有明显的药物析出，高速离心后，还能看到明显的药物粉末层。因此考虑加入增溶剂和稳定剂。
使用乳剂处方B仅解决药物溶解问题，制成相对稳定的乳剂，但是制备的样品B的长期稳定性不佳，有待于改善。处方1采用适合比例的稳定剂，其与增溶剂一起对乳剂的稳定性有显著改善。相比之下，使用乳剂处方C加大了稳定剂的加入比例，但是并未使得乳剂的稳定性如所预期那样得到进一步改善，反而使乳剂的稳定性有所下降，且生产成本增加。

Claims (15)

  1. 一种用于保护缺血心肌的注射用乳剂，按质量份数计，其包含：
    N-辛二酰苯胺异羟肟酸 1-5份；
    乳化剂 0.2-12.5份；
    注射用油 2-100份；
    增溶剂 0.02-5份；
    油酸 0.03-0.4份；
    甘油 0.4-12.5份，以及
    注射用水余量。
  2. 根据权利要求1所述的注射用乳剂，按质量份数计，其包含：
    N-辛二酰苯胺异羟肟酸3份；
    乳化剂 0.5～5份；
    注射用油 5～40份；
    增溶剂 0.05～2份；
    油酸 0.03～0.3份；
    甘油 1.0～5份，以及
    注射用水余量。
  3. 根据权利要求1或2所述的注射用乳剂，按质量份数计，其包含：
    N-辛二酰苯胺异羟肟酸3份；
    乳化剂 1.0～2.5份；
    注射用油 10～20份；
    增溶剂 0.1～1份；
    油酸 0.05～0.15份；
    甘油 2.0～2.5份，以及
    注射用水余量。
  4. 根据权利要求1至3中任一项所述的注射用乳剂，其中，按质量份数计，所述注射用乳剂包含：
    N-辛二酰苯胺异羟肟酸3份；
    乳化剂 1.0～2.5份；
    注射用油 10～20份；
    增溶剂 0.6份；
    油酸 0.05～0.15份；
    甘油 2.0～2.5份，以及
    注射用水余量；
    优选地，其中，按质量份数计，所述注射用乳剂包含：
    N-辛二酰苯胺异羟肟酸 3份；
    乳化剂 2份；
    注射用油 20份；
    增溶剂 0.6份；
    油酸 0.1份；
    甘油 2.5份，以及
    注射用水余量。
  5. 根据权利要求1至4中任一项的注射用乳剂，其中所述乳化剂为磷脂；优选地，所述磷脂选自大豆磷脂、卵磷脂、氢化大豆磷脂或氢化卵磷脂中的一种或多种；进一步优选地，所述磷脂为大豆磷脂和/或卵磷脂；
    优选地，所述注射用油选自注射用大豆油、注射用红花油、注射用棉籽油、注射用芝麻油、注射用茶油、注射用橄榄油或注射用中链油中的一种或多种；更优选地，所述注射用油为注射用大豆油；
    优选地，所述增溶剂选自吐温-80、丙二醇、泊洛沙姆188或聚乙二醇15羟硬脂酸酯(Solutol HS 15)中的一种或多种；更优选地，所述增溶剂为吐温-80和/或丙二醇。
  6. 根据权利要求1至5中任一项所述的注射用乳剂，其中，按质量份数计，所述注射用乳剂包含：
    N-辛二酰苯胺异羟肟酸 3份；
    大豆磷脂或卵磷脂 2份；
    注射用大豆油 20份；
    吐温-80或丙二醇 0.6份；
    油酸 0.1份；
    甘油 2.5份，以及
    注射用水余量。
  7. 根据权利要求1至6中任一项所述的注射用乳剂的制备方法，该制备方法包括以下步骤：
    (1)向N-辛二酰苯胺异羟肟酸加入增溶剂和甘油，加热，再加注射用水至N-辛二酰苯胺异羟肟酸溶解；
    (2)向步骤(1)得到的溶液加入乳化剂，混匀，制得水相；
    (3)将油酸与注射用油混匀，制得油相；
    (4)将水相和油相在加热下高速剪切混合，制得初乳；
    (5)将初乳在加压条件下进行均质化。
  8. 根据权利要求7所述的制备方法，其中，在步骤(1)中，所述加热至50～90℃，优选至70～80℃；
    在步骤(2)中，所述混匀采用剪切混合；优选地，所述剪切混合的速度为3000～10000转/分；更优选地，所述剪切混合的速度为5000～6000转/分；
    在步骤(4)中，所述加热至70℃～80℃且所述高速剪切混合的速度为3000～10000转/分，优选为5000～6000转/分；优选地，所述高速剪切混合进行10～40分钟，更优选进行25～40分钟，最优选进行30分钟；
    在步骤(5)中，所述加压条件为400～1200巴，优选为700～900巴；所述均质化可以进行1～6次，优选进行5～6次。
  9. 根据权利要求7或8所述的制备方法，其中，所述制备方法还包括以下步骤：
    (6)封装；
    (7)灭菌；
    优选地，所述灭菌的条件为115℃下30分钟。
  10. 根据权利要求1至6中任一项所述的注射用乳剂或根据权利要求7至9中任一项所述的制备方法制备的注射用乳剂在制备用于缺血心肌保护的药物、用于治疗心肌缺血再灌注损伤的药物或用于治疗心肌梗死的药物中的应用。
  11. N-辛二酰苯胺异羟肟酸乳剂在制备用于治疗缺血再灌注损伤的药物中的应用。
  12. N-辛二酰苯胺异羟肟酸乳剂在制备用于心血管疾病的药物中的应用。
  13. N-辛二酰苯胺异羟肟酸乳剂在制备用于心脏疾病的药物中的应用。
  14. N-辛二酰苯胺异羟肟酸乳剂在制备用于缺血心肌保护的药物中的应用或用于治疗心肌缺血再灌注损伤的药物中的应用或用于治疗心肌梗死的药物中的应用。
  15. N-辛二酰苯胺异羟肟酸注射用乳剂在制备用于缺血心肌保护的药物中的应用或用于治疗心肌缺血再灌注损伤的药物中的应用或用于治疗心肌梗死的药物中的应用。
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