CN111228217B - Submicron emulsion containing clopidogrel or phospholipid complex of clopidogrel salt and preparation method thereof - Google Patents
Submicron emulsion containing clopidogrel or phospholipid complex of clopidogrel salt and preparation method thereof Download PDFInfo
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- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/107—Emulsions ; Emulsion preconcentrates; Micelles
- A61K9/1075—Microemulsions or submicron emulsions; Preconcentrates or solids thereof; Micelles, e.g. made of phospholipids or block copolymers
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- A61K31/00—Medicinal preparations containing organic active ingredients
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- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
- A61K31/4365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system having sulfur as a ring hetero atom, e.g. ticlopidine
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- 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
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- 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
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- 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
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
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- 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
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- 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
- A61P9/14—Vasoprotectives; Antihaemorrhoidals; Drugs for varicose therapy; Capillary stabilisers
Abstract
The invention belongs to the field of medicines, and discloses a submicron emulsion containing a phospholipid complex of clopidogrel or clopidogrel salt, which comprises the following components: phospholipid complex, vegetable oil, emulsifier, solubilizer, stabilizer, pH regulator and water; the phospholipid complex is prepared by the following method: dissolving clopidogrel and/or clopidogrel salt and phospholipid in an organic solvent, reacting, and removing the organic solvent to obtain the phospholipid complex. The invention utilizes clopidogrel and/or clopidogrel salt and phospholipid to form phospholipid complex, then prepares submicron emulsion, can be directly injected to patients, and after entering blood, the concentration of clopidogrel and/or clopidogrel salt rises rapidly, thus being capable of treating patients rapidly; the clopidogrel and/or clopidogrel salt and phospholipid are utilized to form a phospholipid complex, so that the stability of the clopidogrel and/or clopidogrel salt is improved, and the overall stability of the submicron emulsion is also improved.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a submicron emulsion containing a phospholipid complex of clopidogrel or clopidogrel salt and a preparation method thereof.
Background
Clopidogrel and its salts are platelet aggregation inhibitors that selectively and irreversibly inhibit the binding of Adenosine Diphosphate (ADP) to its platelet receptor and the subsequent activation of the ADP-mediated glycoprotein gpiib/iia complex, thereby inhibiting platelet aggregation. In addition, clopidogrel can also block platelet activation and amplification caused by ADP release, thereby inhibiting platelet aggregation induced by other agonists. Therefore, clopidogrel is often used for the treatment of cardiovascular diseases, such as myocardial infarction, ischemic cerebral thrombosis, thromboangiitis obliterans and complications caused by atherosclerosis and thromboembolism.
Currently, the clopidogrel product sold on the market is only a tablet, and because an oral preparation has long absorption time and slow response speed and cannot achieve the expected anti-platelet aggregation curative effect within the optimal treatment time range, the clopidogrel tablet has the limitation of clinical application in an emergency nursing environment, so that patients cannot be effectively treated at the optimal stage, and the risk of thrombosis is increased. Acute Coronary Syndrome (ACS) is a more serious coronary heart disease, is characterized by paroxysmal chest crush pain, and is acute in onset and serious in illness state, so that a patient is often died due to acute myocardial ischemia. When Acute Coronary Syndrome (ACS) occurs, the existing clopidogrel product can be taken before or after operation only when a patient is awake, and the clopidogrel has the following limitations: on one hand, if clopidogrel therapy is administered before cardiac catheterization, the risk of bleeding of a patient is increased during the coronary artery bypass surgery; on the other hand, postoperative administration of clopidogrel is limited to patients in a conscious and swallowable state, and oral administration of clopidogrel is reported to have symptoms of gastrointestinal irritation and bleeding, dizziness, nausea, conjunctival bleeding and the like. The situation shows that the existing clopidogrel oral preparation has obvious clinical deficiency aiming at the preventive treatment of the acute ACS postoperative antithrombotic, so that the development of a high-efficiency and low-toxicity clopidogrel preparation product for injection is imperative.
The solubility of clopidogrel and salts thereof has pH dependency, for example, the solubility of clopidogrel hydrogen sulfate is significantly reduced as the pH is increased to a physiological pH range, and clopidogrel hydrogen sulfate is easily degraded under alkaline conditions. At present, although the injection of clopidogrel and the salt thereof can be prepared by using a solubilizer or a cosolvent, the drug is extremely unstable in the sterilization process, and precipitates are easily separated out. Moreover, the solubilizer and cosolvent may be used in an excessive amount, which may cause hemolysis or potential allergic reactions in clinical use.
In addition, although clopidogrel and salts thereof and auxiliary agents such as an emulsifier are prepared into an emulsion injection in the prior art, the stability of the injection still needs to be improved, the change behavior and the pharmacodynamic rule of the blood drug concentration in vivo after the injection is administered cannot be known, and the rapidity of treating cardiovascular diseases still needs to be further clarified and improved.
Therefore, the invention hopes to provide a stable injection preparation of clopidogrel or the salt thereof, and the injection preparation can rapidly improve the effective concentration of the drug after entering blood, and has rapid and definite antithrombotic treatment effect on cardiovascular diseases.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a submicron emulsion of a phospholipid complex containing clopidogrel or clopidogrel salt, which is an injection, can be directly injected, has a quick treatment effect and good stability.
A submicron emulsion of a phospholipid complex containing clopidogrel and/or a clopidogrel salt comprises the following components: phospholipid complex, vegetable oil, emulsifier, solubilizer, stabilizer, pH regulator and water; the phospholipid complex is prepared by the following method: dissolving clopidogrel and/or clopidogrel salt and phospholipid in an organic solvent, reacting, and removing the organic solvent to obtain the phospholipid compound.
Preferably, the mass ratio of the clopidogrel and/or the clopidogrel salt to the phospholipid is (0.5-5): (2.5-10); further preferably, the mass ratio of the clopidogrel and/or the clopidogrel salt to the phospholipid is 2.1: 3.85 (the mass ratio means that at least one of clopidogrel and/or clopidogrel salt is present and then the mass ratio to the phospholipid is 2.1: 3.85). The mass ratio of the clopidogrel and/or the clopidogrel salt to the phospholipid is favorable for keeping the size of the prepared submicron emulsion at 160-200nm, is particularly favorable for preventing the phospholipid complex from being broken before and after the submicron emulsion is sterilized and improving the stability of the submicron emulsion.
Preferably, the clopidogrel salt is a hydrogen sulfate salt or a methanesulfonic acid salt of clopidogrel; further preferably, the clopidogrel salt is clopidogrel bisulfate.
Preferably, the phospholipid is egg yolk lecithin and/or soybean lecithin (soybean lecithin includes soybean lecithin).
Preferably, the organic solvent is an alcohol, such as methanol, ethanol, propanol (including n-propanol or isopropanol), butanol (including n-butanol or isobutanol); further preferably, the alcohol is a monohydric alcohol or a dihydric alcohol; more preferably, the alcohol is a monohydric alcohol.
Preferably, the amount ratio of the phospholipid to the organic solvent is (2.5-6.5g): 200-500 mL).
Preferably, the reaction temperature is 20-60 ℃, and the reaction time is 0.5-4 hours.
Preferably, the process for removing the organic solvent is as follows: removing the organic solvent by a rotary evaporation method, and then drying in vacuum; further preferably, the pressure of vacuum drying is less than-0.1 MPa, and the time of vacuum drying is 20-24 hours.
Preferably, the submicron emulsion also contains an isotonic agent; further preferably, the isotonic agent is glycerol and/or sucrose. The isotonic agent can reduce the stimulation of the submicron emulsion to human body and maintain the stability of the osmotic pressure inside and outside the cell.
A submicron emulsion containing phospholipid complex of clopidogrel and/or clopidogrel salt comprises the following components by 100 mL:
preferably, the vegetable oil is soybean oil or C6-C12Straight chain oil; further preferably, the vegetable oil is soybean oil.
Preferably, the emulsifier is selected from egg yolk lecithin and/or soybean lecithin.
Preferably, the solubilizer is tween and/or poloxamer.
Preferably, the stabilizer is oleic acid and/or oleate; further preferably, the oleate is sodium oleate.
Preferably, the submicron emulsion containing the phospholipid complex of clopidogrel and/or clopidogrel salt comprises the following components in 100 mL:
a method for preparing a submicron emulsion of a phospholipid complex containing clopidogrel and/or a clopidogrel salt, comprising the steps of:
(1) preparing an oil phase: weighing the components according to the formula ratio, stirring and mixing the phospholipid compound, the vegetable oil and the emulsifier, and heating to dissolve to prepare an oil phase mixture A for later use;
(2) preparing an aqueous phase: stirring and mixing a solubilizer, a stabilizer, an isotonic agent and water, and heating for dissolving to obtain a water-phase mixture B for later use;
(3) preparing a submicron emulsion: adding the oil phase mixture A prepared in the step (1) into the water phase mixture B prepared in the step (2) for dispersion, then adjusting the pH to 6.0-8.0 by using a pH regulator, adding water to a constant volume of 100mL, homogenizing, and sterilizing to prepare the submicron emulsion.
Preferably, the temperature for heating and dissolving in the step (1) and the step (2) is 65-75 ℃.
Preferably, in the step (2), the solubilizer, the stabilizer, the isotonic agent and the water are stirred and mixed, and are heated to be dissolved, so that the aqueous phase mixture B is prepared.
Preferably, the dispersion in step (3) is carried out by using a dispersing machine, wherein the dispersing machine carries out shearing on the oil phase mixture A and the water phase mixture B, the shearing speed is 10000-12000 r/min, and the shearing time is 10-15 min. High shear rates are beneficial for improving the stability of submicron emulsions.
Preferably, the pH regulator in step (3) is HCl or NaOH.
Preferably, the homogenization in the step (3) is carried out by a homogenizer, the pressure in the homogenization process is 80-100MPa, and the homogenization times are 2-6. Homogenization at 80-100MPa is advantageous for improving the stability of submicron emulsion, but if the pressure is further increased, the heat generated during homogenization will be increased, possibly leading to degradation of the drug. If the pressure is too low, the stability of the submicron emulsion is adversely affected.
Preferably, the sterilization process in step (3) is as follows: filling and sealing the homogenized product, and then autoclaving at 121 ℃ for 15 minutes.
A medicament comprising a sub-microemulsion according to the present invention.
The phospholipid complex (phospholipid complex) is a phospholipid complex formed by adding phospholipid and clopidogrel and/or clopidogrel salt and phospholipid in an aprotic transfer system solvent at a certain ratio through van der Waals force or hydrogen bonds. Clopidogrel hydrogen sulfate (CLP) has a strong pH-dependence of solubility, is unstable in an aqueous solution, and is easily degradable. Phospholipids are not only important constituents of cell membranes, but also very effective emulsifiers due to the amphiphilicity of the phospholipid molecules. After the clopidogrel and/or the clopidogrel salt and the phospholipid form the phospholipid complex, the physical and chemical properties and the biological characteristics are greatly changed compared with the original medicine, such as enhancing the lipid solubility of the medicine and increasing the permeability of a biological membrane, thereby obviously improving the bioavailability of the medicine. The improvement in solubility may be related to the amorphous nature of the complex and to the solubilization of the phospholipid to form micelles in water, which is masked by the interaction of the polar end of the drug with the phospholipid, on the one hand, due to its amorphous state and on the other hand.
Mixing clopidogrel or clopidogrel salt with phospholipid, stirring to prepare a phospholipid compound, increasing the lipid solubility of the phospholipid compound, dissolving the phospholipid compound in soybean oil, and preparing the submicron emulsion by a high-pressure homogenization mode. The preparation of clopidogrel hydrogen sulfate into submicron emulsion has many advantages. The clopidogrel or the clopidogrel salt medicine is prepared into the phospholipid compound, the medicine carrying amount is increased, the medicine carrying emulsion with various dosage specifications can be prepared, and a series of problems and clinical problems of the existing clopidogrel bisulfate preparation are expected to be overcome. Meanwhile, the phospholipid complex is dissolved in the soybean oil, so that the contact between the medicine and water can be avoided, the stability of the medicine can be effectively improved, the bioavailability can be improved, the toxic and side effects can be reduced, the treatment speed and the treatment effect can be improved, and the preparation can be used for treating acute thrombosis.
The submicron emulsion prepared by the invention has good platelet aggregation resisting effect, and is used for treating cardiovascular diseases, such as myocardial infarction, ischemic cerebral thrombosis, thromboangiitis obliterans, atherosclerosis and complications caused by thromboembolism.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention utilizes clopidogrel and/or clopidogrel salt and phospholipid to form phospholipid complex, then prepares submicron emulsion, can be directly injected to patients, and can quickly increase the concentration of clopidogrel and/or clopidogrel salt after entering blood, thereby being capable of quickly treating patients.
(2) The submicron emulsion prepared by the invention utilizes clopidogrel and/or clopidogrel salt and phospholipid to form phospholipid complex, thereby not only improving the stability of clopidogrel and/or clopidogrel salt, but also improving the overall stability of the submicron emulsion.
(3) When the vegetable oil used by the submicron emulsion is soybean oil, the prepared submicron emulsion has better stability; the submicron emulsion prepared by the invention is stable in the sterilization process, and does not have the phenomena of drug precipitation and precipitation.
Drawings
FIG. 1 is an XRD (X-ray diffraction) pattern of a phospholipid complex obtained in example 2 of the present invention;
FIG. 2 is a DSC (differential scanning calorimetry) curve of the phospholipid complex obtained in example 2 of the present invention;
FIG. 3 is a scanning electron micrograph of a submicron emulsion prepared according to example 7 of the present invention;
FIG. 4 is a graph of clopidogrel bisulfate concentration in blood of a submicron emulsion prepared in example 7 of the present invention as a function of time.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.
The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.
Example 1: preparation of phospholipid complexes
The phospholipid complex is prepared by the following method: dissolving clopidogrel and egg yolk lecithin in 400mL of ethanol according to the mass ratio of 1g to 6g, reacting for 3 hours at 30 ℃, removing the ethanol by using a rotary evaporation method, and then drying in vacuum at the pressure of less than-0.1 MPa for 24 hours to obtain the phospholipid compound.
Preferably, the process for removing the organic solvent is as follows: removing the organic solvent by a rotary evaporation method, and then drying in vacuum; further preferably, the pressure of vacuum drying is less than-0.1 MPa, and the time of vacuum drying is 20-24 hours.
Example 2: preparation of phospholipid complexes
The phospholipid complex is prepared by the following method: dissolving clopidogrel hydrogen sulfate and soybean phospholipid in 350mL of n-propanol according to the mass ratio of 2.1g to 3.85g, reacting for 3 hours at 40 ℃, removing the n-propanol by using a rotary evaporation method, and then performing vacuum drying under the vacuum drying pressure of less than-0.1 MPa for 22 hours to obtain the phospholipid compound.
Example 3: preparation of phospholipid complexes
The phospholipid complex is prepared by the following method: dissolving clopidogrel hydrogen sulfate and egg yolk lecithin in a mass ratio of 3g to 2.5g in 500mL of methanol, reacting for 3 hours at 55 ℃, removing the methanol by using a rotary evaporation method, and then performing vacuum drying under the vacuum drying pressure of less than-0.1 MPa for 24 hours to obtain the phospholipid compound.
Example 4: preparation of phospholipid complexes
Compared with the example 2, the clopidogrel hydrogen sulfate is replaced by clopidogrel mesylate in the example 4, and other components and a preparation process are the same as those in the example 2.
Example 5: preparation of phospholipid complexes
Compared with the example 2, the clopidogrel hydrogen sulfate and the soybean phospholipid in the example 5 have the mass ratio of 2g to 10g, and the rest components and the preparation process are the same as those in the example 2.
Example 6: preparation of submicron emulsions
A submicron emulsion comprises the following components in 100 mL:
a preparation method of a submicron emulsion comprises the following steps:
(1) preparing an oil phase: weighing the components according to the formula, stirring and mixing the phospholipid compound prepared in the example 1, the soybean oil and the egg yolk lecithin, and heating to 70 ℃ to dissolve the mixture to prepare an oil phase mixture A for later use;
(2) preparing an aqueous phase: mixing tween, sodium oleate, glycerol and water under stirring, heating to 70 deg.C for dissolving to obtain water phase mixture B;
(3) preparing a submicron emulsion: adding the oil phase mixture A prepared in the step (1) into the water phase mixture B prepared in the step (2), dispersing, and dispersing by using a dispersing machine, wherein the dispersing machine is used for shearing the oil phase mixture A and the water phase mixture B at the shearing speed of 12000 r/min for 15 min, then adjusting the pH to 7.4 by using NaOH, adding water to a constant volume of 100mL, homogenizing again by using a homogenizer, wherein the pressure in the homogenizing process is 100MPa, the homogenizing times are 3 times, and sterilizing at 121 ℃ for 15 min under high pressure to prepare the submicron emulsion.
Example 7: preparation of submicron emulsions
A submicron emulsion comprises the following components in 100 mL:
a preparation method of a submicron emulsion comprises the following steps:
(1) preparing an oil phase: weighing the components according to the formula, stirring and mixing the phospholipid compound prepared in the example 2, the soybean oil and the egg yolk lecithin, and heating to 65 ℃ for dissolving to prepare an oil phase mixture A for later use;
(2) preparing an aqueous phase: mixing tween, sodium oleate and water under stirring, heating to 65 deg.C for dissolving to obtain water phase mixture B;
(3) preparing a submicron emulsion: adding the oil phase mixture A prepared in the step (1) into the water phase mixture B prepared in the step (2) for dispersion, wherein the dispersion is realized by adopting a dispersion machine, the dispersion machine is used for shearing the oil phase mixture A and the water phase mixture B, the shearing speed is 11000 r/min, the shearing time is 15 min, then the pH value is adjusted to 7.4 by using NaOH, water is added for fixing the volume to 100mL, then homogenization is carried out, the homogenization is carried out by adopting a homogenizer, the pressure in the homogenization process is 90MPa, the homogenization frequency is 4 times, and the submicron emulsion is prepared by carrying out sterilization for 15 min under high pressure at 121 ℃.
FIG. 3 is a scanning electron micrograph of the submicron emulsion prepared in example 7 of the present invention, and it can be seen from FIG. 3 that the prepared submicron emulsion has a round shape and a uniform size, and the particle size is 160-170 nm.
Example 8: preparation of submicron emulsions
A submicron emulsion comprises the following components in 100 mL:
a preparation method of a submicron emulsion comprises the following steps:
(1) preparing an oil phase: weighing the components according to the formula, stirring and mixing the phospholipid complex prepared in the embodiment 2, the soybean oil and the soybean lecithin, and heating to 60 ℃ for dissolving to prepare an oil phase mixture A for later use;
(2) preparing an aqueous phase: mixing tween, sodium oleate, glycerol and water under stirring, heating to 60 deg.C for dissolving to obtain water phase mixture B;
(3) preparing a submicron emulsion: adding the oil phase mixture A prepared in the step (1) into the water phase mixture B prepared in the step (2), dispersing, shearing the oil phase mixture A and the water phase mixture B by a dispersion machine at the shearing speed of 10000 r/min for 12 min, adjusting the pH to 7.2 by NaOH, adding water to a constant volume of 100mL, homogenizing again by a homogenizer at the homogenization pressure of 100MPa for 5 times, and sterilizing at 121 ℃ for 15 min to prepare the submicron emulsion.
Example 9: preparation of submicron emulsions
In example 9, 2.5g of glycerin was added as compared with example 7, and other components and preparation process were the same as in example 7.
Example 10: preparation of submicron emulsions
A submicron emulsion comprises the following components in 100 mL:
a preparation method of a submicron emulsion comprises the following steps:
(1) preparing an oil phase: weighing the components according to the formula, stirring and mixing the phospholipid compound prepared in the embodiment 3, the soybean oil and the egg yolk lecithin, and heating to 70 ℃ for dissolving to prepare an oil phase mixture A for later use;
(2) preparing an aqueous phase: mixing tween, oleic acid, sucrose and water under stirring, heating to 70 deg.C for dissolving to obtain water phase mixture B;
(3) preparing a submicron emulsion: adding the oil phase mixture A prepared in the step (1) into the water phase mixture B prepared in the step (2), dispersing, shearing the oil phase mixture A and the water phase mixture B by a dispersion machine at the shearing speed of 12000 r/min for 10 min, adjusting the pH to 6.5 by HCl, adding water to a constant volume of 100mL, homogenizing again by a homogenizer at the homogenization pressure of 100MPa for 3 times, and sterilizing at 121 ℃ for 15 min to prepare the submicron emulsion.
Example 11: preparation of submicron emulsions
A submicron emulsion comprises the following components in 100 mL:
a preparation method of a submicron emulsion comprises the following steps:
(1) preparing an oil phase: weighing the components according to the formula, stirring and mixing the phospholipid compound prepared in the embodiment 4, the soybean oil and the egg yolk lecithin, and heating to 70 ℃ for dissolving to prepare an oil phase mixture A for later use;
(2) preparing an aqueous phase: mixing tween, sodium oleate, glycerol and water under stirring, heating to 70 deg.C for dissolving to obtain water phase mixture B;
(3) preparing a submicron emulsion: adding the oil phase mixture A prepared in the step (1) into the water phase mixture B prepared in the step (2), dispersing, shearing the oil phase mixture A and the water phase mixture B by a dispersion machine at the shearing speed of 10000 r/min for 12 min, adjusting the pH to 7.0 by NaOH, adding water to a constant volume of 100mL, homogenizing again by a homogenizer at the homogenization pressure of 100MPa for 5 times, and sterilizing at 121 ℃ for 15 min to prepare the submicron emulsion.
Example 12: preparation of submicron emulsions
In comparison with example 7, the phospholipid complex prepared in example 12 was used in place of the phospholipid complex prepared in example 2 in example 7, and other components and preparation processes were the same as those in example 7.
Comparative example 1
Weighing 0.125g of clopidogrel hydrogen sulfate raw material medicine, placing the raw material medicine in a 25mL beaker, adding 0.3g of Tween 80, adding 20mL of water, adjusting the pH to 7.4 by NaOH, adding water to a constant volume of 25mL, filtering by a 0.22um microporous membrane, taking filtrate, and preparing the injection.
Comparative example 2
In comparison with example 7, in comparative example 2, the phospholipid complex prepared in example 2 was directly replaced with clopidogrel bisulfate (the amount of clopidogrel bisulfate in comparative example 2 was the same as that of clopidogrel bisulfate in the phospholipid complex in comparative example 7), and other components and preparation methods were the same as example 7 without performing a sterilization step (if sterilization is performed, a little precipitate occurs).
Product effectiveness testing
1. The XRD patterns of clopidogrel hydrogen sulfate (CLP), Phospholipids (PC), phospholipid complexes (CLPPC), physical mixtures of clopidogrel hydrogen sulfate and phospholipids (CLP + PC) were tested.
An XRD pattern was measured using clopidogrel bisulfate (CLP), soybean Phospholipid (PC), the phospholipid complex (CLPPC) prepared in example 2, and a physical mixture of clopidogrel bisulfate and soybean phospholipid (CLP + PC) under the same conditions, and the results are shown in fig. 1. FIG. 1 is an XRD (X-ray diffraction) pattern of a phospholipid complex obtained in example 2 of the present invention; as can be seen from FIG. 1, clopidogrel hydrogen sulfate (CLP) has many diffraction peaks characteristic of crystals, indicating that clopidogrel hydrogen sulfate is in a crystalline state. Soybean lecithin (PC) has a few diffraction peaks characteristic of crystals. In a physical mixture (CLP + PC) of clopidogrel bisulfate and soybean phospholipid, a characteristic diffraction peak of clopidogrel bisulfate is clear and visible, which shows that clopidogrel bisulfate still exists in a crystalline state. In the phospholipid complex (CLPPC) prepared in example 2, due to the interaction between soybean phospholipid and clopidogrel bisulfate, characteristic crystal diffraction peaks of soybean phospholipid and clopidogrel bisulfate disappeared to assume an amorphous state. As can be seen from fig. 1, clopidogrel hydrogen sulfate was complexed with soybean phospholipid in the phospholipid complex (CLPPC) prepared in example 2.
2. The clopidogrel hydrogen sulfate (CLP), Phospholipid (PC), phospholipid complex (CLPPC), physical mixture of clopidogrel hydrogen sulfate and phospholipid (CLP + PC) were tested for DSC (differential scanning calorimetry) curves.
DSC (differential scanning calorimetry) is a reliable method of screening for compatibility of drugs with excipients and provides information about the interaction between drugs and excipients.
DSC (differential scanning calorimetry) curves were measured under the same conditions using clopidogrel hydrogen sulfate (CLP), soybean Phospholipid (PC), the phospholipid complex (CLPPC) prepared in example 2, and a physical mixture of clopidogrel hydrogen sulfate and soybean phospholipid (CLP + PC), and the results are shown in fig. 2.
FIG. 2 is a DSC (differential scanning calorimetry) curve of the phospholipid complex obtained in example 2 of the present invention; as can be seen from FIG. 2, clopidogrel bisulfate (CLP) has an endothermic peak at 182.66 ℃, a physical mixture of clopidogrel bisulfate and phospholipids (CLP + PC) has endothermic peaks at 181.78 ℃ and 319.67 ℃, the endothermic peak of soybean Phospholipid (PC) is not very significant, and the phospholipid complex (CLPPC) has an endothermic peak only at 321.90 ℃. As can be seen from fig. 2, clopidogrel hydrogen sulfate was complexed with soybean phospholipid in the phospholipid complex (CLPPC) prepared in example 2.
3. And (5) testing the long-term stability.
The submicron emulsions prepared in examples 7, 8, 12 and 2 were filled with nitrogen and sealed in ampoules, left to stand at 4 ℃ and 20 ℃ for 9 months in the dark, and sampled at 1 month, 3 months, 6 months and 9 months, respectively, to examine the changes in the drug content (content: mass of clopidogrel bisulfate/input amount of clopidogrel bisulfate in the submicron emulsion), the average particle size, the pH value and the encapsulation efficiency (encapsulation efficiency: mass of clopidogrel bisulfate after ultrafiltration of the submicron emulsion/mass of clopidogrel bisulfate before ultrafiltration of the submicron emulsion × 100%, and the effect of ultrafiltration was to filter out small molecular substances not coated in the prepared submicron emulsion). The results of the examination were compared with those of the submicron emulsion just prepared, and the storage stability of the submicron emulsion was examined, and the results are shown in tables 1 and 2.
Table 1: stability test at 4 ℃ in the dark
As can be seen from Table 1, the stability of the submicroemulsion prepared in example 7, example 8 and example 12 is significantly better than that of the submicroemulsion prepared in comparative example 2 under the condition of being protected from light at 4 ℃. As can be seen from the stability data of example 7 and example 12, the stability of the submicron emulsion prepared in example 7 is better, indicating that the dosage of clopidogrel bisulfate and soybean phospholipid during the preparation of the phospholipid complex has a certain influence on the stability of the finally prepared submicron emulsion.
Table 2: stability test at 25 ℃ in the dark
As can be seen from Table 2, the stability of the submicroemulsion prepared in example 7, example 8 and example 12 is significantly better than that of the submicroemulsion prepared in comparative example 2 under the condition of being protected from light at 25 ℃. The submicron emulsion prepared in comparative example 2 has coalescence between emulsion droplets with time (coalescence refers to a process in which emulsion film of emulsion droplets in the emulsion is broken to enlarge the emulsion droplets), so that the particle size becomes large. As can be seen from the data in tables 1 and 2, the 25 ℃ conditions have a greater effect on the stability of the submicron emulsion than the 4 ℃ conditions. In other words, the stability of submicron emulsions is more critical at 25 ℃.
3. Testing of the concentration of the agent into the blood.
18 male rats were randomly divided into three groups (tablet group, comparative example 1 injection group and sub-microemulsion group), and all rats were fasted for 12 hours before administration and allowed free access to water. Clopidogrel bisulfate was administered to each male rat at a dose of 10 mg/kg. For the tablet group, clopidogrel bisulfate tablets were ground prior to oral administration, suspended in a 5% solution of carboxymethyl cellulose, and administered by gavage. The control group of comparative example 1 (injection of the injection solution prepared in comparative example 1) and the submicroemulsion group (injection of the submicroemulsion prepared in example 7) were injected directly into the tail vein of rats. At different set time points (5, 10, 15, 20, 30, 45, 60, 90, 120, 180, 240min), 0.3mL of blood was taken out, centrifuged at 3000r/min at 4 ℃ for 5 minutes, supernatant plasma was aspirated, and clopidogrel hydrogen sulfate concentration was measured, with the results shown in fig. 4. FIG. 4 is a graph showing the change of clopidogrel hydrogen sulfate concentration in blood of the submicron emulsion prepared in example 7 of the present invention with time (in FIG. 4, the abscissa indicates time in unit of minute, and the ordinate indicates clopidogrel hydrogen sulfate concentration in unit of ng/mL), in which curve (i) indicates the relationship between the concentration of the submicron emulsion prepared in example 7 in blood and time, curve (ii) indicates the relationship between the concentration of the injection prepared in comparative example 1 in blood and time, and curve (iii) indicates the relationship between the concentration of the tablet in blood and time, as can be seen from FIG. 4, the concentration of the drug in blood of the submicron emulsion prepared in example 7 of the present invention in 0 to 40 minutes is significantly higher than that in 0 to 40 minutes, which is advantageous for the rapid treatment of cardiovascular diseases such as myocardial infarction and cerebral thrombosis, obliterative vasculitis and complications caused by atherosclerosis and thromboembolism.
4. The submicron emulsion prepared by the invention is used for researching the treatment effect.
When the carotid artery loop of the model rat is established, platelets in blood flow contact the rough surface of the silk thread in the bypass circulation, adhesion and aggregation can occur, and platelet aggregates surround the surface of the silk thread to form thrombus. The thrombus formed by the method is similar to white thrombus, further fibrin is formed, a large number of red blood cells are netted, and red thrombus is formed. Comparing the weights of the thrombi allows to examine the inhibitory effect of the drugs on the adhesion and aggregation functions of the platelets.
24 adult rats were divided into three groups and administered with each of 0.3% CMC (sodium carboxymethylcellulose) in the control group, 10mg/kg clopidogrel bisulfate in the tablet group by intragastric administration, and 10mg/kg clopidogrel bisulfate submicron emulsion prepared in example 7 of the present invention in the submicron emulsion group by intravenous injection. The administration is carried out continuously for three days, and after the administration for 2 hours on the third day, rats are anesthetized with phenytoin sodium, fixed in a supine position, and the neck skin is unhaired and disinfected. An incision is made in the middle of the neck of a rat, muscles are separated in a blunt mode, the left common carotid artery and the right external jugular vein are exposed, three sections of polyethylene tubes are inserted into a semicircle, and a fourth suture cotton thread is sleeved in the middle of the semicircle. Filling a polyethylene tube with 1% heparin normal saline solution by mass fraction, inserting the polyethylene tube into the right external jugular vein, and then inserting the other end of the polyethylene tube into the left common carotid artery to form an arteriovenous loop. Loosening the artery clamp, accurately opening the blood flow for 30 minutes, then interrupting the blood flow, quickly taking out the suture cotton thread, removing superficial blood on the surface, and weighing. The weights before and after the suture experiment were recorded separately, and the wet weight of the thrombus was calculated. The thrombus was placed in a plate, dried in an oven at 60 ℃ for 4 hours, cooled and weighed to obtain the dry weight of the thrombus, and the results are shown in table 3 (inhibition rate ═ control thrombus weight-administration group thrombus weight)/control thrombus weight ═ 100%).
Table 3:
remarking: indicates that p is less than 0.05, with significant differences.
As can be seen from Table 3, compared with the control group, the wet weight and the dry weight of the thrombus were significantly reduced in the submicron emulsion group and the tablet group, indicating that the thrombus formation of the arteriovenous bypass of the rat was significantly inhibited. The submicron emulsion group had a stronger inhibitory effect on thrombosis than the tablet group.
5. The submicron emulsion prepared by the invention is used for measuring the platelet aggregation rate, the cAMP (cyclic adenosine monophosphate) content and the content of p-selectin (glycoprotein with the relative molecular mass of 140000).
Platelets play a major role in the formation of arterial thrombi. FeCl3The infiltrated filter paper strip covers the abdominal aorta of the ratThrough the generation of oxygen free radicals, the lipid peroxidation injury of the intima of blood vessels is caused, and the intrinsic coagulation system is activated, so that the formation of thrombus in the blood vessels is promoted. The induction of thrombus formation in vivo by intimal injury is pathologically similar to clinical. The method can be used for inducing rats with arterial thrombosis, so that the following model groups and low, medium and high dose submicron emulsion groups can be conveniently tested.
48 adult rats, sham operation group (no thrombus induction and no drug injection), model group (thrombus induction and no drug injection), low, medium and high dose submicron emulsion group (thrombus induction and submicron emulsion injection prepared by the invention in the example 8), and tablet group. The dosage of the submicron emulsion for intravenous injection of the low, medium and high dosage submicron emulsion group is 5mg/kg, 10mg/kg and 20mg/kg in sequence, and the clopidogrel hydrogen sulfate of 10mg/kg is administrated by intragastric administration of the tablet group for 3 days continuously.
5.1 platelet aggregation Rate determination
After the administration of the rats in each group, the rats were anesthetized, 4.5mL of blood was collected from the aorta in the abdominal cavity into a vacuum anticoagulation tube containing 3.8% by mass of trisodium citrate, the anticoagulation was centrifuged at 800 rpm (200g) for 8 minutes, and then the plasma was separated to obtain platelet-rich plasma (PRP), the remaining portion was centrifuged at 3000 rpm (2200g) for 10 minutes, and the supernatant was collected to obtain platelet-poor plasma (PPP). Starting platelet aggregation instrument, measuring platelet number with platelet counter, and adjusting platelet number to 3.0 × 108One cell/mL, stored at room temperature for later use, and zeroed with PPP. The platelet aggregation function was measured by a platelet aggregation meter, and after adjusting the baseline to be stable, 5 μmol/L ADP (adenosine diphosphate) agonist was added to calculate the platelet aggregation rate and platelet inhibition rate (%) of each group (for example, platelet inhibition rate of tablet group ═ platelet aggregation rate of model group-platelet aggregation rate of tablet group)/platelet aggregation rate of model group: "100%), and the results are shown in table 4.
Table 4:
| group of | Dosage to be administered | Platelet aggregation Rate (%) | Platelet inhibition (%) |
| Artificial operation group | - | 56.5±10.8 | 17.75 |
| Model set | - | 68.7±9.70 | - |
| Tablet set | 10mg/kg | 36.4±9.36*# | 47.01 |
| Low dose sub-microemulsion group | 5mg/kg | 42.2±5.43*# | 38.57 |
| Medium dose submicroemulsion group | 10mg/kg | 35.2±5.42**# | 48.76 |
| High dose sub-microemulsion group | 20mg/kg | 30.3±7.86**# | 55.89 |
Remarking:#P<0.05，##P<0.01，###P<0.001 to sham group,. P<0.05，**P<0.01，***P<0.001 to model group ratio.
As can be seen from Table 4, both the tablet group and the microemulsion group inhibited platelet aggregation as compared with the model group, and the low, medium and high dose of the microemulsion group gradually increased the platelet aggregation inhibition ability with the increase of the administration dose.
5.2 measurement of cAMP (cyclic adenosine monophosphate) content
The rats after the administration of the above groups are anesthetized, the abdominal aorta is used for blood sampling, the blood is added into a centrifuge tube, a whole blood specimen is placed for 4 hours at 20 ℃, then the centrifugation is carried out for 20 minutes at 2500 rpm, the supernatant is taken, and the supernatant is placed in a refrigerator at-20 ℃ for storage, so that repeated freeze thawing is avoided. The specific procedures were performed according to the cAMP ELISA kit (commercially available) using enzyme-linked immunosorbent assay (ELISA), and the cAMP content was measured, the results are shown in Table 5.
Table 5:
remarking:#P<0.05，##P<0.01，###P<0.001 to sham group,. P<0.05，**P<0.01，***P<0.001 to model group ratio.
Platelet function includes activation, aggregation, adhesion and release, and this process is not isolated, and they interact with cAMP as a second messenger for platelets, with the effect of modulating platelet function. In normal state, the cAMP content is low, and the activation of platelets is not caused. The increase in cAMP content can further inhibit platelet activation. As can be seen from Table 5, the tablet group and the microemulsion group both enhanced the cAMP expression compared to the model group, and the low, medium and high dose of the microemulsion group increased the cAMP content with increasing dose (the higher the cAMP content, the more the inhibition of thrombus formation).
5.3p selectin assay
The rats after the administration of the above groups are anesthetized, the abdominal aorta is used for taking blood, the blood is added into a centrifuge tube, a whole blood specimen is placed for 4 hours at room temperature, then centrifugation is carried out for 20 minutes at 2500 rpm, supernatant is taken, and the supernatant is placed in a refrigerator at the temperature of-20 ℃ for storage, so that repeated freeze thawing is avoided. The enzyme-linked immunosorbent assay (ELISA) was used, the specific procedures were performed according to the instructions of the P-selectin kit (commercially available), and the results of the assay for P-selectin are shown in Table 6.
Table 6:
| group of | Dosage to be administered | p selectin (pg/mL) |
| Artificial operation group | - | 4.36±1.42 |
| Model set | - | 6.75±1.53# |
| Tablet set | 10mg/kg | 5.75±0.73* |
| Low dose sub-microemulsion group | 5mg/kg | 5.65±1.52* |
| Medium dose submicroemulsion group | 10mg/kg | 5.39±1.71* |
| High dose sub-microemulsion group | 20mg/kg | 4.60±0.73* |
Remarking:#P<0.05，##P<0.01，###P<0.001 to sham group,. P<0.05，**P<0.01，***P<0.001 to model group ratio.
The p-selectin only distributes on the alpha-particle membrane in a resting state, the expression is less on the platelet membrane, after the platelet is activated, the p-selectin is rapidly combined with a plasma membrane along with the release of the particle content, and the p-selectin exists on the surface of the platelet and in the plasma, so that the p-selectin becomes a specific marker for identifying whether the platelet is activated. An increase in the content of p-selectin indicates increased activation and release of platelets, and is also seen in hypercoagulable states of blood and thrombotic diseases. As can be seen from Table 6, the expression of p-selectin was reduced in both the tablet group and the microemulsion group as compared with the model group, and the p-selectin content was gradually decreased in the low, medium and high dose microemulsion groups with increasing administration dose, indicating that the tablet and the microemulsion had therapeutic effects, and that the microemulsion dosage form was superior to the tablet effect (the lower the p-selectin content, the more the inhibition of thrombus formation).
Claims (9)
1. A submicron emulsion of a phospholipid complex comprising the following components: phospholipid complex, vegetable oil, emulsifier, solubilizer, stabilizer, pH regulator and water; the phospholipid complex is prepared by the following method: dissolving clopidogrel and/or clopidogrel salt and phospholipid in an organic solvent, reacting, and removing the organic solvent to obtain the phospholipid compound; the mass ratio of the clopidogrel and/or the clopidogrel salt to the phospholipid is (0.5-5): (2.5-10); the stabilizer is oleic acid and/or oleate; the organic solvent is methanol, ethanol, propanol or butanol.
2. The submicron emulsion of claim 1, wherein said clopidogrel salt is the bisulfate or mesylate salt of clopidogrel.
3. The submicron emulsion of claim 1, wherein the vegetable oil is soybean oil or C6-C12Straight chain oil.
4. The submicron emulsion according to claim 1, characterized in that it contains an isotonicity agent.
5. The submicron emulsion according to any one of claims 1-4, characterized by comprising the following components in 100 mL:
6. a method for preparing a submicron emulsion, comprising the steps of:
(1) preparing an oil phase: weighing the components according to the formula ratio, stirring and mixing the phospholipid compound, the vegetable oil and the emulsifier, and heating to dissolve to prepare an oil phase mixture A for later use;
(2) preparing an aqueous phase: stirring and mixing a solubilizer, a stabilizer, an isotonic agent and water, and heating for dissolving to obtain a water-phase mixture B for later use;
(3) preparing a submicron emulsion: adding the oil phase mixture A prepared in the step (1) into the water phase mixture B prepared in the step (2) for dispersion, then adjusting the pH to 6.0-8.0 by using a pH regulator, adding water to reach 100mL, homogenizing, and sterilizing to obtain the submicron emulsion of any one of claims 1-5.
7. The method of claim 6, wherein the step (3) comprises dispersing the mixture with a dispersing machine, wherein the oil phase mixture A and the water phase mixture B are sheared at 10000-12000 rpm for 10-15 min.
8. The method of claim 6, wherein the homogenizing step (3) is performed with a homogenizer at a pressure of about 80 MPa to about 100 MPa.
9. A medicament comprising a microemulsion according to any one of claims 1 to 5.
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