CN117530933B - Pirenpazide long-acting slow-release microsphere, preparation method and slow-release injection - Google Patents
Pirenpazide long-acting slow-release microsphere, preparation method and slow-release injection Download PDFInfo
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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
- A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
- A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
- A61K9/5005—Wall or coating material
- A61K9/5021—Organic macromolecular compounds
- A61K9/5031—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poly(lactide-co-glycolide)
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- 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
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
- A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
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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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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/08—Antiepileptics; Anticonvulsants
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Abstract
The invention belongs to the technical field of sustained release administration of pirenzepine, and particularly provides a sustained release microsphere with long-acting pirenzepine, a preparation method and a sustained release injection. The invention prepares the pirenzepine long-acting slow-release microsphere based on emulsion system research, has round microsphere appearance, good drug loading rate, high encapsulation efficiency, proper particle size and uniform distribution, and can continuously release the pirenzepine drug for 14-42 days. The prepared pirenzenepamine long-acting sustained-release microsphere can be further developed to prepare a pirenzenepamine long-acting sustained-release injection, and compared with the once-a-day administration of a pirenzenepamine commercial preparation, the pirenzenepamine long-acting sustained-release microsphere can obviously prolong the administration interval, reduce the times of taking medicine at a doctor, increase the convenience of medicine taking, improve the compliance of patients and reduce the risk of epileptic seizure induced by medicine leakage. Especially for some patients unsuitable for oral administration, such as cough, dysphagia, nasal feeding, fistulization patients, etc., the long-acting pirenzenenaphthalene microsphere and the preparation provide a new choice for convenient and safe administration.
Description
Technical Field
The invention relates to the technical field of sustained release administration of pirenzepine, in particular to a sustained release microsphere of pirenzepine, a preparation method and a sustained release injection.
Background
Epilepsy is a common chronic nervous system disease which is caused by multiple diseases and is next to cerebral apoplexy, and long-term administration (generally more than 3-5 years) is required. There are about 6000 ten thousand patients worldwide, of which about 340 ten thousand in the united states, about 100 ten thousand in japan, about 600 ten thousand in europe, and about 900 ten thousand in china. For newly diagnosed epileptic patients, treatment with an antiepileptic drug (ANTIEPILEPTIC DRUGS, AED) is still preferred, but about 30% of patients have unsatisfactorily controlled their condition, and many patients experience significant adverse effects. In the 21 st century, the development of antiepileptic drugs mainly aims at refractory epilepsy, and aims at improving the convenience of long-term administration and reducing the toxic and side effects of traditional drugs.
Pirenzenenape is a highly selective, non-competitive alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptor antagonist developed by Japanese guard. The pirenzeneb can reduce the hyperexcitation of neurons through non-competitive combination with an AMPA receptor, so as to achieve the aim of preventing and treating epilepsy. Pirenzeneb, the first highly selective AMPA receptor antagonist approved for marketing at present, has the characteristics of unique mechanism of action, good drug tolerance and broad spectrum antiepileptic, and has been applied to focal and comprehensive epileptic patients in more than 55 countries. The molecular formula of the pirenzenenaphthalene 3/4 hydrate is shown in figure 1.
The pirenzenenapestrap is prepared into tablets (with the specification of 2mg, 4mg, 6mg, 8mg, 10mg and 12 mg), granules (1 percent) and oral suspension (0.5 mg/ml) which are all administrated 1 time a day; the initial dose of the adult is 2 mg/day, the dosage can be added and titrated according to the increment of 2mg every 1 or 2 weeks, and the maintenance dose is 4-12 mg/day. Common adverse effects of pirenzenenaphthalene include dizziness, irritability, fatigue, aggression, nausea, weight gain, and the like. Studies suggest that adverse effects of pirenzenenaphthalene are dose dependent.
The existing pirenzeneb preparation has the following defects:
(1) Inconvenient oral administration: the dosage forms of pirenzenenaphthalene on the market all need to be orally administrated, and part of epileptic patients such as cough choking, dysphagia, nasal feeding, fistulization patients and the like are inconvenient to orally administrate every day.
(2) Once daily administration, the fluctuation of blood concentration is obvious, and the adverse reaction risk is high.
(3) The medicine leakage has great harm: patients with epilepsy generally need to take medicines for a long time, the frequency of once daily administration is more frequent, the compliance of the patients is poor, and the situation of medicine leakage is easy to occur. The risk of missed medication for epileptic patients is significantly higher than that of other patients, and if epileptic patients are missed several times in succession, the reduced curative effect of the medication is likely to induce epileptic seizures, which are generally more frequent than usual, more severe in symptoms, and even cause status epilepticus to endanger life.
In addition, according to the national drug administration bulletin, pirenzenenide (including its salts, isomers and single formulations) is administered as a class of psychotropic drugs from day 7, 2023. Later, the pirenzenenapestrap cannot be sold through a network, and the patient needs to go to a hospital to obtain the medicine offline after prescribing the corresponding narcotic medicine. Generally, the second class of psychotropic drugs is prescribed for no more than 7 days. This means that patients need to visit the clinic frequently to take medicine, which increases the medication cost.
The prior researchers develop new pirenzenenaphthalene preparation for reducing toxic and side effects and improving bioavailability and patient compliance, and the preparation comprises an oral tablet (CN 113440489A), a freeze-dried orally disintegrating tablet (CN 104706604A), a solid dispersion dry suspension (CN 107536805A), a dispersible tablet (CN 106619551A), an oral cavity film-dissolving agent (CN 109106696A), a nano suspension (CN 113069415B) and the like which are different from the original prescription process, and the preparation is an immediate release preparation. In addition, patent CN106667968A provides a sustained-release pirenzepine capsule which can prolong the maintenance time of the treatment concentration of pirenzepine in vivo by 12 hours, and the administration interval is still short, so that the problem of frequent administration is not solved.
In recent years, the slow release microsphere has great industrial value and wide application prospect, so that the slow release microsphere becomes one of the research hot spots of pharmacy and is valued by the great importance of various international medicines. The microsphere is a tiny spherical entity formed by dissolving or dispersing the drug in carrier auxiliary materials, and the particle size is generally 1-250 mu m. After the microsphere is injected into the body, the slow medicine is released at a certain speed through medicine diffusion or carrier corrosion mechanism, and the stable blood concentration is maintained at the focus position.
However, despite extensive searching, no patent or paper literature has found an effective sustained release microsphere formulation of pirenzenepamil. Therefore, the development of the pirenzepine long-acting slow-release microsphere preparation reduces the administration frequency, reduces the times of patient visits, increases the convenience and compliance of patient medication, avoids peak valley phenomenon, reduces toxic and side effects and drug resistance, and has great clinical significance and economic significance.
Disclosure of Invention
The invention provides a sustained-release microsphere of pirenzepine, a preparation method and a sustained-release injection, which aim to reduce the administration frequency of the pirenzepine, improve the convenience of administration of patients and simultaneously reduce the toxic and side effects caused by the fluctuation of the concentration of the drug in the body.
The first aspect of the invention provides a sustained-release microsphere containing pirenzepine active ingredient and a high polymer carrier material, wherein the mass ratio of the pirenzepine active ingredient is 10% -35% in theory, and the drug load ratio is 1:9-7:13. Through actual measurement, the drug loading is 9-34%, the encapsulation efficiency is 45.8% -99.2% (preferably more than 90%), and the sustained release of the pirenzepine is generally realized for 14-42 days.
The active ingredient of the pirenzenepraline can be one or a mixture of a plurality of anhydrous substances, hydrates and salts thereof.
The high polymer carrier material is selected from polylactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA) or Polycaprolactone (PCL) with the weight average molecular weight of 12-145 KDa.
Among them, PLGA has flexible adjustability, PLGA can change end group type, lactic acid/glycolic acid monomer mole ratio (L/G) besides molecular weight, obtain microballoons of different characteristics, therefore carrier material is more preferably PLGA.
Further, PLGA preferably has a weight average molecular weight of 47-145 kDa, and the end groups may be ester groups, hydroxyl groups, carboxyl groups, and L/G may be 50/50-75/25.
Further, the particle size D90 of the pirenzenepraanese long-acting slow-release microsphere is less than or equal to 150 mu m, the SPAN SPAN is less than or equal to 2, the particle size is small, the distribution is uniform, and the needle penetrating property is good.
The second aspect of the invention provides a preparation method of a pirenzepine long-acting slow-release microsphere, which comprises the following steps:
s1, dissolving a high polymer carrier material in an oil phase solvent;
S2, dissolving the active ingredient of the pirenzepine into an oil phase solvent containing a high polymer carrier material to form an oil phase (O);
s3, dissolving a stabilizer in water to form a water phase (W);
S4, adding the oil phase (O) obtained in the step S2 into the water phase (W) obtained in the step S3 under the stirring condition, and uniformly mixing to form O/W emulsion;
S5, adding the O/W emulsion into a curing phase under the stirring condition, and curing emulsion drops in the curing phase to form microspheres to obtain a microsphere-containing suspension;
S6, extracting microspheres from the microsphere-containing suspension to obtain the pirenzenepamil long-acting slow-release microspheres.
Specifically, the oil phase solvent in S1 needs to include a main solvent component and a secondary solvent component, where the main solvent component is dichloromethane, and the secondary solvent component is one or more selected from methanol, ethanol, propylene glycol, and ethyl acetate.
In the implementation process of the invention, a large number of experiments prove that the type and the composition ratio of the oil phase solvent are critical to the preparation of the pirenzenenaphthalene microsphere. PLGA with the molar ratio (LA ratio) of more than or equal to 50 percent is known to be dissolved in methylene dichloride, and the methylene dichloride has low boiling point (about 39.8 ℃) and certain solubility in water, so that the methylene dichloride can diffuse from an oil phase to an aqueous phase and be volatilized and removed through the surface of liquid gas, and is an ideal solvent for preparing microsphere products. The pirenzenenaphthalene is almost insoluble in water, is easily dissolved in dichloromethane, is slightly soluble in acetonitrile and acetone, is slightly soluble in ethyl acetate, methanol, ethanol and propylene glycol, and is suitable for preparing microspheres by an O/W emulsion solvent volatilization method. However, experiments show that when only dichloromethane is used as an oil phase solvent, the medicine-carrier material mixture is separated out to obtain irregular particles, and the microspheres with round shapes and high encapsulation efficiency are difficult to prepare. Under the condition of adopting a single oil phase solvent of dichloromethane, the invention also tries several different polymer carrier materials, but the invention discovers that microsphere products with qualified comprehensive properties can not be prepared by adjusting schemes.
In the implementation process of the invention, the oil phase solvent is set to contain a main solvent component and a secondary solvent component, wherein the main solvent component is methylene dichloride, the secondary solvent component is selected from methanol, ethanol, propylene glycol and ethyl acetate, and a series of long-acting pirenzenepamine microspheres can be prepared by comprehensively optimizing and adjusting a carrier material, a preparation process and the like under the premise, the sustained and stable release period of the pirenzenepamine is generally more than 2 weeks, and the sustained and stable release period of the pirenzenepamine of a microsphere sample prepared by the preferred scheme can be up to 6 weeks. Compared with common preparations (such as tablets), the administration interval can be greatly prolonged, the administration convenience is improved, the patient compliance is improved, and the possibility of missed administration of the medicine is reduced.
Further, the secondary solvent component is preferably methanol, ethanol or propylene glycol, more preferably ethanol.
Further, in the oil phase solvent, the mass ratio of the main solvent component to the sub solvent component may be 4/1 to 6/1, preferably 5/1.
Further, the mass percentage of the polymer carrier material in the oil phase (O) is preferably 10-14%.
Further, the mass ratio (drug load ratio) of the active ingredients of the pirenzepine to the carrier material is 1:9-7:13, namely the theoretical drug load of the microsphere is 10-35%, and preferably 10-30%.
Further, the stabilizer in S3 is one or more selected from polyvinyl alcohol (PVA), tween, span, sodium dodecyl sulfate, sodium carboxymethyl cellulose, polyvinylpyrrolidone, and poloxamer, preferably polyvinyl alcohol (PVA). The PVA concentration is preferably 0.1 to 5%, more preferably 1%.
Further, the mass ratio of the oil phase to the water phase is selected from 1:1 to 1:100, preferably 1:1 to 1:30, and more preferably 1:10 to 1:30.
Further, the mode of "uniform mixing" in S4 is selected from stirring, homogenizing, ultrasound, static mixing. Preferably, the method is homogeneous, and the rotating speed is 1000-10000 rpm.
Specifically, the cured phase in S5 is a non-solvent for the carrier material, i.e. the carrier material is poorly soluble or insoluble therein.
Preferably, the solidification phase can be water or an aqueous solution containing additives, and the additives can be tween, span, sodium dodecyl sulfate, sodium dodecyl sulfonate, poloxamer, PVA and the like.
Further, the amount of the curing phase may be selected to be 0 to 1000 times, preferably 100 to 400 times, the theoretical weight of the microspheres.
Further, in the step S6, microspheres can be obtained by filtering, washing and drying from microsphere-containing suspension, and the dry and clean pirenzenepamil long-acting slow-release microspheres are obtained.
The third aspect of the invention provides a sustained release injection containing the sustained release microsphere of the pirenzepine.
Interpretation of the terms involved in the present invention
Theoretical drug loading rate: weight percent of drug contained in the theoretical microsphere. Theoretical drug loading= (theoretical drug dosage/(theoretical drug dosage+theoretical carrier weight)) ×100%.
Actual drug loading rate: the weight percentage of the drug contained in the microspheres was measured. The drug loading = (drug content in microsphere/weight of microsphere) ×100% was measured.
Encapsulation efficiency: weight percent of encapsulated drug in the microsphere. Encapsulation efficiency = (amount of encapsulated microspheres/total amount of encapsulated and unencapsulated microspheres) ×100% = (1-amount of unencapsulated in liquid/total amount of encapsulated and unencapsulated microspheres) ×100%.
D 10、D50、D90 is the particle size corresponding to 10%, 50% and 90% of the cumulative particle size distribution.
SPAN span= (D 90- D10）/ D50), the smaller the SPAN the narrower the distribution, i.e. the more uniform the particle size.
Advantageous effects
The study of the invention prepares the pirenzepine long-acting slow-release microsphere which has round microsphere shape, good drug loading rate, high encapsulation efficiency, proper particle size and uniform distribution, and can continuously release the pirenzepine drug for 14-42 days. The prepared pirenzenepamine long-acting sustained-release microsphere can be further developed to prepare a pirenzenepamine long-acting sustained-release injection, and compared with the once-a-day administration of a pirenzenepamine commercial preparation, the pirenzenepamine long-acting sustained-release microsphere can obviously prolong the administration interval, reduce the times of taking medicine at a doctor, increase the convenience of medicine taking, improve the compliance of patients and reduce the risk of epileptic seizure induced by medicine leakage. Especially for some patients unsuitable for oral administration, such as cough, dysphagia, nasal feeding, fistulization patients, etc., the long-acting pirenzenenaphthalene microsphere and the preparation provide a new choice for convenient and safe administration.
Drawings
Figure 1 shows the molecular structural formula of the pirenzenenaphthalene 3/4 hydrate.
Fig. 2 is a microscopic topography of examples 1 to 8 and comparative examples 1 to 4.
Fig. 3 shows the summary of the detection results of examples 1 to 8 and comparative examples 1 to 4.
FIG. 4 shows a summary of the test results of examples 2, 9, and 10.
Fig. 5 shows the summary of the detection results of examples 2, 11 to 14.
Fig. 6 shows the summary of the detection results of examples 13, 15 to 18.
Fig. 7 shows the summary of the detection results of examples 13, 19 to 21.
Fig. 8 shows the microscopic topography of examples 13, 22, 23.
Fig. 9 shows the summary of the detection results of examples 13, 22 and 23.
Figure 10 shows the in vitro release profile of examples 13, 22, 23.
FIG. 11 shows in vitro release profiles for comparative examples 5, 6.
Fig. 12 shows a summary of the detection results of examples 24 to 28.
FIG. 13 shows in vitro release profiles for examples 24-28.
Fig. 14 shows a summary of the detection results of examples 22, 29 to 31.
FIG. 15 shows in vitro release profiles of examples 22, 29-31.
Fig. 16 shows a summary of the detection results of examples 22, 32-38.
FIG. 17 shows in vitro release profiles of examples 22, 32-38.
Detailed Description
Unless defined otherwise, terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention relates.
The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The detection method comprises the following steps:
microsphere morphology: after a proper amount of microspheres were dispersed in 0.1% pva solution, the microspheres were added dropwise to a glass slide, and the morphology was observed under an optical microscope.
Drug loading rate: weighing 50-60 mg of microspheres, adding a certain amount of acetonitrile for dissolution, detecting the concentration of the pirenzepine by using an HPLC method, and calculating the drug loading.
Encapsulation efficiency: 50-60 mg of microspheres are weighed and dispersed in quantitative absolute ethyl alcohol for 5 minutes, then the concentration of pirenzenepamide in the absolute ethyl alcohol is detected to obtain free drug quantity, and the encapsulation efficiency is calculated according to an encapsulation efficiency= (1-free drug quantity/microsphere drug loading quantity) multiplied by 100% formula.
Particle size and particle size distribution: about 50mg of the microspheres are weighed, put into a 10mL penicillin bottle, added with 0.1% Tween 80 water solution, and subjected to ultrasonic treatment for 1min, and the particle size distribution of the microspheres are measured by a laser particle sizer.
In vitro release: (1) release medium: 1% sodium dodecyl sulfate and 25% ethanol, and the organic filter membrane is used for suction filtration. (2) 10mg of the microsphere is taken and placed in a 150ml glass bottle, 100ml of medium is added, in-vitro release test is carried out in water bath oscillation at 37 ℃ at the rotating speed of 100r/min, and sampling analysis and fluid infusion are carried out at each sampling point. The concentration of pirenzenenaphthalene in each sample was measured by HPLC, the cumulative release rate was calculated and the cumulative release rate (%) = cumulative release rate/theoretical microsphere drug loading x 100%, an in vitro cumulative release profile was made.
Example 1:
1.08gPLGA (L: g=75:25, mw=140: 140 KDa, ester end capped), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 6.30G of dichloromethane, 1.26G of methanol to obtain an oil phase solution. 27.00g of 1% PVA solution was weighed as an aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water was weighed as a solidification phase, the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. Then filtering the suspension with a sieve with the aperture of 90 μm and a sieve with the aperture of 20 μm, collecting particles between the sieve with the aperture of 90 μm and the sieve with the aperture of 20 μm, washing 3 times with water, and freeze-drying to obtain the powdery microspheres.
Example 2:
1.08gPLGA (L: G=75:25, mw=140: 140 KDa, ester end-capped), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 6.30G of dichloromethane, 1.26G of absolute ethanol to obtain an oil phase solution. 27.00g of 1% PVA solution was weighed as an aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water was weighed as a solidification phase, the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 3:
1.08gPLGA (L: g=75:25, mw=140: 140 KDa, ester end-capped), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 6.30G of dichloromethane, 1.26G of propylene glycol to obtain an oil phase solution. 27.00g of 1% PVA solution was weighed as an aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water was weighed as a solidification phase, the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 4:
1.08gPLGA (L: G=75:25, mw=140: 140 KDa, ester end-capped), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 6.30G of dichloromethane, 1.26G of ethyl acetate to obtain an oil phase. 27.00g of 1% PVA solution was weighed out as an aqueous phase, and the oil phase was added to the aqueous phase under a homogenization condition at a rotation speed of 5000rpm to form an O/W emulsion. 576.0g of water was weighed as a solidification phase, the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 5:
1.08gPLGA (L: G=75:25, mw=140: 140 KDa, ester end-capped), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 6.05G of dichloromethane, 1.51G of absolute ethanol to obtain an oil phase solution. 54.0g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water was weighed as a solidification phase, the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 6:
1.08gPLGA (L: G=75:25, mw=140: 140 KDa, ester end-capped), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 6.48G of dichloromethane, 1.08G of absolute ethanol to obtain an oil phase solution. 54.0g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water was weighed as a solidification phase, the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 7:
1.08gPLA g (Mw=180: 180 KDa) of pirenzenenaphthalene 3/4 hydrate (moisture 3.5%, equivalent to 0.36g of pirenzenenaphthalene) was weighed and dissolved in 6.30g of dichloromethane and 1.26g of absolute ethanol to obtain an oil phase solution. 27.00g of 1% PVA solution was weighed as an aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water was weighed as a solidification phase, the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 8:
1.08gPCL g (viscosity 1.75 dl/g) of pirenzenenaphthalene 3/4 hydrate (moisture 3.5%, equivalent to 0.36 g) was weighed and dissolved in 6.30g of dichloromethane and 1.26g of absolute ethanol to obtain an oil phase solution. 27.00g of 1% PVA solution was weighed as an aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water was weighed as a solidification phase, the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Comparative example 1:
1.08gPLGA (L: g=75:25, mw=140: 140 KDa, ester end-capped) was weighed and dissolved in 7.56G dichloromethane to give an oil phase solution. 45.00g of 1% PVA solution was weighed as an aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water was weighed as a solidification phase, the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. And filtering the suspension by using a 90 mu m screen, collecting solidified particles under the screen, washing for 3 times by using water, and drying to obtain the powdery microspheres.
Comparative example 2:
1.08gPLGA (L: g=75:25, mw=140: 140 KDa, ester end-capped), 0.373G of pirenzenepamine 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamine) was weighed and dissolved in 7.56G of dichloromethane to obtain an oil phase solution. 27.00g of 1% PVA solution was weighed as an aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water was weighed as a solidification phase, the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. And filtering the suspension by using a screen with the aperture of 90 mu m, collecting solidified particles below the screen, washing for 3 times by using water, and drying to obtain the powdery microspheres.
Comparative example 3:
1.08gPLA g (Mw=180: 180 KDa) of pirenzenenaphthalene 3/4 hydrate (moisture 3.5%, equivalent to 0.36g of pirenzenenaphthalene) was weighed and dissolved in 7.56g of methylene chloride to obtain an oil phase solution. 27.00g of 1% PVA solution was weighed as an aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water was weighed as a solidification phase, the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. And filtering the suspension by using a screen with the aperture of 90 mu m, collecting solidified particles below the screen, washing for 3 times by using water, and drying to obtain the powdery microspheres.
Comparative example 4:
1.08gPCL (viscosity 1.75 dl/g) and 0.373g of pirenzenenaphthalene 3/4 hydrate (moisture 3.5%, equivalent to 0.36 g) were weighed and dissolved in 7.56g of methylene chloride to obtain an oil phase solution. 27.00g of 1% PVA solution was weighed as an aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water was weighed as a solidification phase, the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. And filtering the suspension by using a screen with the aperture of 90 mu m, collecting solidified particles below the screen, washing for 3 times by using water, and drying to obtain the powdery microspheres.
Test example 1
Samples of examples 1-8 are taken to detect the morphology, drug loading and encapsulation efficiency of the microsphere, comparative example 1 is taken to detect the morphology, drug loading and encapsulation efficiency of the microsphere, and comparative example 2-4 is taken to detect the morphology, the drug loading and the encapsulation efficiency of the microsphere, and the results are shown in fig. 2 and 3. The morphologies shown by S1 to S8 in FIG. 2 are the morphologies corresponding to examples 1 to 8, respectively, and the morphologies shown by D1 to D4 in FIG. 2 are the morphologies corresponding to comparative examples 1 to 4, respectively.
The results of comparative examples 1 and 2 show that the pirenzepine drug substance itself adversely affects the formation of microspheres. Comparative example 1 was free of pirenzenenaphthalene drug to obtain PLGA microspheres with rounded morphology. The addition of pirenzenenaphthalene in comparative example 2 resulted in failure to form regular spherical microsphere particles, with an actual drug loading of 23.98%, but very low encapsulation efficiency of only 10.74%. This suggests that the presence of pirenzenenaphthalene as a drug substance not only inhibits the formation of regular microspheres, but also makes it difficult for the drug substance itself to be uniformly encapsulated within the microspheres during the formation of the microspheres, and to accumulate mainly in the surface layer of the microspheres in an adherent form. The extremely low encapsulation efficiency is not only unfavorable for prolonging the release period of the drug, but also easy to form burst release phenomenon at the initial stage of drug administration, so that the concentration of the pirenzepine in the body greatly deviates from the preset range.
Comparative examples 3, 4 are similar to the results of comparative example 2, in that the encapsulation efficiency is still very low, showing irregular particles under the microscope, failing to form a rounded sphere, although an attempt was made to replace different carrier materials, including PLA and PCL.
The results of examples 1-4 show that the oil phase solvent composition has a significant effect on the morphology and encapsulation efficiency of the pirenzenenaphthalene microsphere. The mixed solvent of dichloromethane, methanol, ethanol, propylene glycol and ethyl acetate is used as an oil phase solvent, and compared with comparative examples 2-4, the morphology and the encapsulation efficiency of the obtained microsphere are improved obviously. When the oil phase solvent is dichloromethane and ethanol, the obtained microsphere is round, has few surface protrusions, and has higher encapsulation efficiency.
The results of examples 5 and6 show that the mass ratio of dichloromethane to ethanol has an effect on the morphology and the encapsulation efficiency of the pirenzenenaphthalene microsphere, and the encapsulation efficiency is reduced when the ethanol ratio is higher or lower. However, compared with comparative examples 2-4, the microspheres obtained in examples 5 and6 still have significantly improved morphology and significantly improved encapsulation efficiency. Further, the results of examples 2, 7 and 8 show that the types of carrier materials have a certain influence on the morphology, the encapsulation efficiency and the drug loading of the pirenzepine microsphere.
Example 9:
1.08gPLGA (L: G=75:25, mw=140: 140 KDa, ester-terminated), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 7.79G of dichloromethane and 1.56G of absolute ethanol to obtain an oil phase solution. 27.00g of 1% PVA solution was weighed as an aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water was weighed as a solidification phase, the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 10:
1.08gPLGA (L: G=75:25, mw=140: 140 KDa, ester-terminated), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 5.22G of dichloromethane, 1.04G of absolute ethanol to obtain an oil phase solution. 27.00g of 1% PVA solution was weighed as an aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water was weighed as a solidification phase, the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Test example 2:
Samples of examples 9 and 10 were taken to test the drug loading and encapsulation efficiency of the microspheres, and the results are shown in fig. 4. The result shows that under the preparation process conditions of the scheme, when the PLGA concentration (PLGA concentration=PLGA mass/total oil phase mass multiplied by 100%) is between 10 and 14%, the influence on the encapsulation efficiency is small, and the drug loading rate is highest when the encapsulation efficiency is 14%.
Examples 11 to 14:
the preparation method of the pirrennet microspheres of examples 11-14 is basically the same as that of example 2, except that the weight of the aqueous phase 1% PVA solution is 9.00g, 54.00g, 90.00g and 270.0g of 1% PVA solution respectively.
Test example 3:
And (5) taking samples of examples 11-14 to detect the drug loading and encapsulation efficiency of the microspheres. The results of the oil-water ratio, drug loading rate and encapsulation efficiency of examples 2, 11-14 are shown in figure 5. The results show that the oil-water ratio has an effect on the drug loading rate and the encapsulation efficiency of the pirenzenenaphthalene microsphere. When the oil-water ratio is 1:1-1:30, the encapsulation rate can reach more than 70%. When the encapsulation efficiency is high, energy conservation and emission reduction are considered, and the oil-water ratio is more preferably 1:10.
Example 15:
The preparation method of the pirenzenenaphthalene microspheres of example 15 is basically the same as that of example 2, except that the weight of the aqueous phase is 90.00g and the aqueous phase is a 5% pva solution, specifically as follows.
1.08GPLGA (L: G=75:25, mw=140: 140 KDa, ester end-capped), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 6.30G of dichloromethane, 1.26G of absolute ethanol to obtain an oil phase solution. 90.00g of 5% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water was weighed as a solidification phase, the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Examples 16 to 18:
The preparation method of the pirenzenenaphthalene microspheres in examples 16-18 is basically the same as that in example 15, except that the aqueous phase composition is 0.1% PVA solution, 1% Tween 80 and 0.1% sodium dodecyl sulfate respectively.
Test example 4:
samples of examples 15-18 were taken to detect the drug loading and encapsulation efficiency of the microspheres, and samples of examples 13 and 15-18 were taken to detect the particle size and particle size distribution of the microspheres, and the results are shown in fig. 6. The results show that the concentration and the type of the aqueous phase stabilizer have little influence on the drug loading rate and the encapsulation efficiency of the pirenzenenape microsphere.
Example 19
1.08GPLGA (L: G=75:25, mw=140: 140 KDa, ester end-capped), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 6.30G of dichloromethane, 1.26G of absolute ethanol to obtain an oil phase solution. 90.00g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 144.0g of water was weighed as the solidification phase, and the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
The preparation method of the pirenzeneb microsphere in examples 20 and 21 is basically the same as that in example 19, except that the weight of the cured phase is 1440.0g and 72.0g respectively, specifically as follows.
Example 20:
1.08gPLGA (L: G=75:25, mw=140: 140 KDa, ester end-capped), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 6.30G of dichloromethane, 1.26G of absolute ethanol to obtain an oil phase solution. 90.00g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 1440.0g of water was weighed as a solidification phase, the O/W emulsion was added to the solidification phase while stirring for 4 hours, and the microspheres were solidified to obtain a suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 21:
1.08gPLGA (L: G=75:25, mw=140: 140 KDa, ester end-capped), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 6.30G of dichloromethane, 1.26G of absolute ethanol to obtain an oil phase solution. 90.00g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 72.0g of water was weighed as a solidification phase, and the O/W emulsion was added to the solidification phase while stirring, and then stirring was continued for 4 hours to solidify the microspheres, to obtain a suspension. Filtering the suspension with a sieve with a pore diameter of 90 μm and a pore diameter of 20 μm, collecting particles between the sieve with a pore diameter of 90 μm and the sieve with a pore diameter of 20 μm, washing with water for 3 times, and drying to obtain the powdery microspheres.
Test example 5:
the drug loading, encapsulation efficiency, particle size and particle size distribution of examples 19-21 were tested and the results are shown in FIG. 7. The results of examples 13 and 19-21 show that the dosage of the curing phase has an effect on drug loading and encapsulation efficiency, and the encapsulation efficiency can reach more than 80% when the dosage of the curing phase is more than 100 times of the theoretical mass of the microsphere.
Example 22:
1.08gPLGA (L: G=75:25, mw=140: 140 KDa, ester base end), 0.373G of pirenzenenaphthalene 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenenaphthalene) was weighed and dissolved in 6.30G of dichloromethane and 1.26G of absolute ethanol to obtain an oil phase solution. 90.00g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water is weighed as a solidification phase, ice water bath is used for cooling to about 5 ℃ in advance, then O/W emulsion is added into the solidification phase while stirring, stirring is continued for 4 hours, and microspheres are solidified, so as to obtain suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 23:
1.08gPLGA (L: G=75:25, mw=140: 140 KDa, ester group end) and 0.36G of pirenzenenaphthalene were weighed and dissolved in 6.30G of dichloromethane and 1.26G of absolute ethanol to obtain an oil phase solution. 90.00g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water is weighed as a solidification phase, ice water bath is used for cooling to about 5 ℃ in advance, then O/W emulsion is added into the solidification phase while stirring, stirring is continued for 4 hours, and microspheres are solidified, so as to obtain suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Test example 6:
detecting the drug loading, encapsulation efficiency, particle size and particle size distribution of the samples of examples 22 and 23, and detecting the microsphere morphology and in-vitro release conditions of examples 13, 22 and 23; the results are shown in figures 8-10. The sample morphologies shown in fig. 8 as S13, S22, and S23 correspond to the sample morphologies shown in example 13, example 22, and example 23, respectively.
The pirenzenenaphthalene microspheres of examples 13 and 22 have round and uniform particle size, and the result shows that the curing temperature has influence on the drug loading and in vitro release of the pirenzenenaphthalene microspheres. Compared to example 13, example 22 uses a low temperature curing process, while maintaining high encapsulation efficiency, while maintaining higher drug loading. The curing temperatures are different so that the drug release rates are slightly different, but the drug release rates can be continuously released for 42 days.
The results of examples 22 and 23 show that microspheres with round morphology, good drug loading and high encapsulation efficiency can be prepared by using the pirenzenepamil 3/4 hydrate or the pirenzenepamil, and the in vitro release rates are slightly different, but can be continuously released for 42 days.
Comparative example 5:
Taking 2.5mg of the raw material drug pirenzenenaphthalene, placing the raw material drug pirenzenenaphthalene into a 150ml glass bottle, adding 100ml of release medium (1% sodium dodecyl sulfate and 25% ethanol phosphate buffer solution with pH 7.4), performing in-vitro release test in water bath oscillation at 37 ℃ at the rotating speed of 100r/min, sampling and analyzing after 5, 15, 30 and 60min, and supplementing liquid. The concentration of pirenzenenaphthalene in each sample is detected by an HPLC method, the accumulated release amount is calculated, the accumulated release rate (%) is calculated, and an in vitro accumulated release curve is made. The release profile results are shown in FIG. 11.
Comparative example 6:
taking 2.5mg of the drug substance pirenzeneb 3/4 hydrate, placing the drug substance into a 150ml glass bottle, adding 100ml of release medium (1% sodium dodecyl sulfate and 25% ethanol phosphate buffer solution with pH 7.4), performing in-vitro release test in water bath oscillation at 37 ℃ at the rotating speed of 100r/min, sampling and analyzing after 5,15, 30 and 60min, and supplementing liquid. The concentration of pirenzenenaphthalene in each sample is detected by an HPLC method, the accumulated release amount is calculated, the accumulated release rate (%) is calculated, and an in vitro accumulated release curve is made. The release profile results are shown in FIG. 11.
Comparative examples 5 and 6 under the same conditions as the release outside the microspheres, the drug was dissolved rapidly after the drug substance, pirenzepine 3/4 hydrate, was added to the release medium and released completely after 60 minutes. In contrast, the pirenzenenape microsphere prepared by the embodiment has a very remarkable slow release effect.
Example 24
1.08GPLGA (L: G=75:25, mw=140: 140 KDa, ester end-capped), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 6.30G of dichloromethane, 1.26G of absolute ethanol to obtain an oil phase solution. 90.00g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 1000rpm to form an O/W emulsion. 576.0g of water is weighed as a solidification phase, ice water bath is used for cooling to about 5 ℃ in advance, then O/W emulsion is added into the solidification phase while stirring, stirring is continued for 4 hours, and microspheres are solidified, so as to obtain suspension. Filtering the suspension with a screen with a pore diameter of 150 μm, collecting undersize suspension, subpackaging into a centrifuge tube, centrifuging at 5000rpm×5min, collecting precipitate, washing with water for 3 times, and drying to obtain powdery microsphere.
The preparation method of the pirenzenenaphthalene microspheres of examples 25-28 is basically the same as that of example 24, except that the homogenization conditions are 3000, 5000, 7000 and 10000rpm respectively.
Test example 7:
Samples of examples 24-28 were taken to detect the drug loading, encapsulation efficiency, particle size and particle size distribution of the microspheres, and release in vitro, and the results are shown in fig. 12 and 13. The results show that the homogenization rotational speed has a large influence on the particle size of the pirenzenenaphthalene microspheres, which in turn influences the in vitro release. The microsphere has good drug loading and encapsulation conditions within the rotation speed range of 1000-10000 rpm, and can be released for more than 28 days in vitro.
Example 29:
1.08gPLGA (L: g=75:25, mw=140: 140 KDa, ester end-capped), 0.124G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.12G of pirenzenepamil) was weighed and dissolved in 6.30G of dichloromethane, 1.26G of absolute ethanol to obtain an oil phase solution. 90.00g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water is weighed as a solidification phase, ice water bath is used for cooling to about 5 ℃ in advance, then O/W emulsion is added into the solidification phase while stirring, stirring is continued for 4 hours, and microspheres are solidified, so as to obtain suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 30:
1.08gPLGA (L: g=75:25, mw=140: 140 KDa, ester end-capped), 0.477G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.46G of pirenzenepamil) was weighed and dissolved in 6.30G of dichloromethane, 1.26G of absolute ethanol to obtain an oil phase solution. 90.00g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water is weighed as a solidification phase, ice water bath is used for cooling to about 5 ℃ in advance, then O/W emulsion is added into the solidification phase while stirring, stirring is continued for 4 hours, and microspheres are solidified, so as to obtain suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 31:
1.08gPLGA (L: g=75:25, mw=140: 140 KDa, ester end-capped), 0.60G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.58G of pirenzenepamil) was weighed and dissolved in 6.30G of dichloromethane, 1.26G of absolute ethanol to obtain an oil phase solution. 90.00g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water is weighed as a solidification phase, ice water bath is used for cooling to about 5 ℃ in advance, then O/W emulsion is added into the solidification phase while stirring, stirring is continued for 4 hours, and microspheres are solidified, so as to obtain suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Test example 8:
The samples of examples 29-31 were tested for drug loading, encapsulation efficiency, particle size, and in vitro release, and the results are shown in FIGS. 14 and 15. The results show that the theoretical drug loading has an effect on encapsulation efficiency and in vitro release. When the theoretical drug loading rate is between 10% and 35%, the encapsulation rate can reach more than 77%, and the sustained release in vitro can be realized for more than 35 days, but the release rates are different, and when the theoretical drug loading rate is 35%, the initial burst release is highest and the release is fastest.
Example 32:
1.08gPLGA (L: G=75:25, mw=140: 140 KDa, hydroxyl end), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 6.30G of dichloromethane and 1.26G of absolute ethanol to obtain an oil phase solution. 90.00g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water is weighed as a solidification phase, ice water bath is used for cooling to about 5 ℃ in advance, then O/W emulsion is added into the solidification phase while stirring, stirring is continued for 4 hours, and microspheres are solidified, so as to obtain suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 33:
1.08gPLGA (L: G=75:25, mw=145: 145 KDa, carboxyl terminal), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 6.30G of dichloromethane and 1.26G of absolute ethanol to obtain an oil phase solution. 90.00g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water is weighed as a solidification phase, ice water bath is used for cooling to about 5 ℃ in advance, then O/W emulsion is added into the solidification phase while stirring, stirring is continued for 4 hours, and microspheres are solidified, so as to obtain suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 34:
1.08gPLGA (L: G=75:25, mw=120: 120 KDa, ester base end), 0.373G of pirenzenenaphthalene 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenenaphthalene) was weighed and dissolved in 6.30G of dichloromethane and 1.26G of absolute ethanol to obtain an oil phase solution. 90.00g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water is weighed as a solidification phase, ice water bath is used for cooling to about 5 ℃ in advance, then O/W emulsion is added into the solidification phase while stirring, stirring is continued for 4 hours, and microspheres are solidified, so as to obtain suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 35:
1.08gPLGA (L: G=75:25, mw=47: 47 KDa, ester base end) and 0.373G of pirenzenenaphthalene 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenenaphthalene) were weighed and dissolved in 6.30G of dichloromethane and 1.26G of absolute ethanol to obtain an oil phase solution. 90.00g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water is weighed as a solidification phase, ice water bath is used for cooling to about 5 ℃ in advance, then O/W emulsion is added into the solidification phase while stirring, stirring is continued for 4 hours, and microspheres are solidified, so as to obtain suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 36:
2.16gPLGA (L: G=75:25, mw=12: 12 KDa, ester base end), 0.746G of pirenzenenaphthalene 3/4 hydrate (moisture 3.5%, equivalent to 0.72G of pirenzenenaphthalene) was weighed and dissolved in 12.60G of dichloromethane and 2.52G of absolute ethanol to obtain an oil phase solution. 90.00g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 1152.0g of water is weighed as a solidification phase, ice water bath is used for cooling to about 5 ℃ in advance, then O/W emulsion is added into the solidification phase while stirring, stirring is continued for 4 hours, and microspheres are solidified, so as to obtain suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 37:
1.08gPLGA (L: G=65:35, mw=60: 60 KDa, carboxyl terminal), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 6.30G of dichloromethane and 1.26G of absolute ethanol to obtain an oil phase solution. 90.00g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water is weighed as a solidification phase, ice water bath is used for cooling to about 5 ℃ in advance, then O/W emulsion is added into the solidification phase while stirring, stirring is continued for 4 hours, and microspheres are solidified, so as to obtain suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Example 38:
1.08gPLGA (L: G=50:50, mw=55: 55 KDa, carboxyl terminal), 0.373G of pirenzenepamil 3/4 hydrate (moisture 3.5%, equivalent to 0.36G of pirenzenepamil) was weighed and dissolved in 6.30G of dichloromethane and 1.26G of absolute ethanol to obtain an oil phase solution. 90.00g of 1% PVA solution was weighed as the aqueous phase, and the oil phase solution was slowly added to the aqueous phase while homogenizing at 5000rpm to form an O/W emulsion. 576.0g of water is weighed as a solidification phase, ice water bath is used for cooling to about 5 ℃ in advance, then O/W emulsion is added into the solidification phase while stirring, stirring is continued for 4 hours, and microspheres are solidified, so as to obtain suspension. Then filtering the suspension by using a sieve with the aperture of 90 mu m and 20 mu m, collecting particles between the sieve with the aperture of 90 mu m and the sieve with the aperture of 20 mu m, washing the particles with water for 3 times, and drying the particles to obtain the powdery microspheres.
Test example 9:
The samples of examples 32-38 were tested for drug loading, encapsulation efficiency, particle size, and in vitro release, and the results are shown in figures 16 and 17. The results show that the end groups, molecular weight, L/G of PLGA have a greater effect on in vitro release. When L/G=75/25 and the ester group end is adopted, the smaller the molecular weight is, the faster the release is, the encapsulation efficiency is obviously reduced when the molecular weight is 12kDa, the release is rapid, the release is complete after about 1 week, the encapsulation efficiency can reach more than 90% when the molecular weight is 47kDa and above, and the release can last for 14 days and more. When the molecular weight is 140-145 kDa and L/G=75/25, the release is the fastest when the end group is carboxyl, the hydroxyl is the next slowest, and the ester group is the slowest. When the molecular weight is 55-60 kDa and carboxyl ends are present, the larger the G ratio is, the faster the release is.
The PLGA molecular weight, L/G and end group can regulate the drug release speed of the pirenzenenaphthalene microsphere, has potential to develop into sustained release preparations with different drug administration periods, has wide application prospect, for example, example 36 can be developed into preparations with 1-week/time drug administration period, example 38 can be developed into preparations with 2-week/time drug administration period, example 37 can be developed into preparations with 3-week/time drug administration period, example 35 can be developed into preparations with 4-week/time drug administration period, examples 32, 33 and 34 can be developed into preparations with 5-week/time drug administration period, and example 22 can be developed into preparations with 6-week/time drug administration period.
Claims (10)
1. A preparation method of a pirenzenenaphthalene long-acting slow-release microsphere is characterized by comprising the following steps of: the method comprises the following steps:
s1, dissolving a high polymer carrier material in an oil phase solvent;
S2, dissolving the active ingredients of the pirenzepine into an oil phase solvent containing a high polymer carrier material to form an oil phase O;
s3, dissolving a stabilizer in water to form a water phase W;
S4, adding the oil phase O obtained in the S2 into the water phase W obtained in the S3, and uniformly mixing to form O/W emulsion;
S5, adding the O/W emulsion into a curing phase, and curing the emulsion drops in the curing phase to form microspheres to obtain a microsphere-containing suspension;
s6, screening out microspheres from the microsphere-containing suspension, washing with water, and drying to obtain the pirenzenepamil long-acting slow-release microspheres;
The high polymer carrier material is selected from polylactic acid-glycolic acid copolymer, polylactic acid or polycaprolactone;
the stabilizer is polyvinyl alcohol, tween 80 or sodium dodecyl sulfate;
The solidifying phase is water or an aqueous solution containing an additive;
In the step S4, the oil phase O and the water phase W are mixed according to the mass ratio of 1:1-1:30;
the mass percentage of the polymer carrier material in the oil phase O is 10-14%;
the oil phase solvent comprises a main solvent component and a secondary solvent component, wherein the main solvent component is dichloromethane, the secondary solvent component is selected from any one or more of methanol, ethanol, propylene glycol and ethyl acetate, and the mass ratio of the main solvent component to the secondary solvent component is 4/1-6/1;
the mass ratio of the active ingredients of the pirenzepine to the polymer carrier material is 1:9-7:13.
2. The method for preparing the pirenzepine long-acting slow-release microspheres according to claim 1, which is characterized in that: the secondary solvent component is ethanol.
3. The method for preparing the pirenzepine long-acting slow-release microspheres according to claim 1, which is characterized in that: the oil phase solvent is a mixture formed by mixing dichloromethane and ethanol according to the mass ratio of 4/1-6/1.
4. The method for preparing the pirenzepine long-acting slow-release microspheres according to claim 1, which is characterized in that: the polymer carrier material is a polylactic acid-glycolic acid copolymer with weight average molecular weight of 47-145 kDa.
5. The method for preparing the pirenzenene long-acting slow release microsphere according to claim 4, which is characterized in that: the terminal group of the polylactic acid-glycolic acid copolymer is selected from one or more of ester group, hydroxyl group and carboxyl group, and the molar ratio of lactic acid to glycolic acid monomer in the polylactic acid-glycolic acid copolymer is 50/50-75/25.
6. The method for preparing the pirenzepine long-acting slow-release microspheres according to claim 1, which is characterized in that: the stabilizer is polyvinyl alcohol, and the mass content of the stabilizer in the water phase W is 0.1-5%.
7. The method for preparing the pirenzepine long-acting slow-release microspheres according to claim 1, which is characterized in that:
the solidifying phase is water, and the dosage of the solidifying phase is 100-400 times of the theoretical weight of the microsphere.
8. A pirenzenenape long-acting slow-release microsphere, which is characterized in that: comprises a high polymer carrier material and a pirenzenenaphthalene active ingredient embedded in the high polymer carrier material, and is prepared by the preparation method of the pirenzenenaphthalene long-acting slow-release microsphere according to any one of claims 1 to 7.
9. The pirenzeneb long-acting slow release microsphere according to claim 8, wherein: the active ingredient of the pirenzenenaphthalene is one or a mixture of more of a pirenzenenaphthalene anhydride, a hydrate and a salt thereof; and/or
The drug loading rate of the active ingredients of the pirenzepine in the pirenzepine long-acting slow-release microspheres is 9-34%, and the encapsulation rate is 90% -99.2%; and/or
The particle diameter D90 of the pirenzenepraline long-acting slow release microsphere is less than or equal to 150 mu m, and the SPAN SPAN is less than or equal to 2; and/or
The time for continuously releasing the active ingredients of the pirenzepine is more than 14 days.
10. A pirenzenenape long-acting sustained-release injection is characterized in that: the pirenzepine long-acting slow-release injection contains the pirenzepine long-acting slow-release microsphere according to claim 8 or 9.
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| CN117530933A (en) | 2024-02-09 |
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