Abstract:
A risperidone sustained release microsphere formulation is provided. The microsphere formulation includes risperidone or 9-hydroxy risperidone or salts thereof, and a polymer blend having a first uncapped lactide-glycolide copolymer and a second uncapped lactide-glycolide copolymer, in which the first uncapped lactide-glycolide copolymer is a copolymer with a high intrinsic viscosity and the second uncapped lactide-glycolide copolymer is a copolymer with a low intrinsic viscosity. The sustained release micro sphere formulation according to an embodiment of the present disclosure is suitable for large-scale industrialized production with improved stability, the in vivo release behavior of which will not change after long-term storage.

Description:
FIELD 
     The present disclosure belongs to pharmaceutical preparation field, and more particularly relates to a risperidone long-acting sustained release microsphere composition, method for preparing the same and use of the same. 
     BACKGROUND 
     Schizophrenia is a serious disabling mental disorder. With the development of intense social competition, quick pace of life end changes in family structure, people face greater pressure than before, and consequently mental health problems become more and more prevalent. Schizophrenia is the most common disease in menial disorder. According to statistics, the prevalence of schizophrenia in China is 6.55%, there are more than 7.8 million schizophrenes, and the global disease rate is as high as 1.5%. 
     Antipsychotic drugs, also referred to as neuroleptic, may control mental symptoms of schizophrenia effectively. The commonly used antipsychotic drugs first appeared in 1950s, such as chlorpromazine or haloperidol, have a main pharmacological effect of blacking central dopamine D 2  receptor and are effective in the treatment of positive psychosis symptoms, but may cause extrapyramidal movement disorders, and are invalid for negative symptoms and cognitive function damage, accompanying with many adverse reactions, also have pester toxicity on cardiovascular and liver with larger administration dose, and significant side effects. In order so overcome these shortcomings, since 1980s, new antipsychotic drugs appeared, main pharmacological effect of which is to block 5-HT 2A  and D 2  receptors. The advantages of new antipsychotic drugs are that not only in the treatment of acute exacerbation of psychiatric patients, but also in the treatment of extrapyramidal symptoms and tardive dyskinesia, which have little side effects without the use of anticholinergic agents; tolerance and compliance of the treatment are good; therapeutic effects in improving positive and negative symptoms and cognitive function are strong, adverse reactions of extrapyramidal system (EPS) may be less or may not be caused, and endocrine adverse reactions may not be caused by the increase of prolactin levels. 
     Risperidone as a representative of new antipsychotic drugs was developed by Janssen Pharmaceutica in Belgium in 1984, with the chemical name of 3-[2-[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1-piperidyl]ethyl]-6,7,8,9-tetrahydro-2-methyl-4H-pyri do[1,2-a]pyrimidine-4-one, has a good therapeutic effect on positive symptoms and negative symptoms of schizophrenia, and the incidence rate of extrapyramidal adverse reactions is low. A metabolite of risperidone, i.e., 9-hydroxy risperidone (paliperidone) has pharmacological effects similar to those of risperidone. Risperidone and 9-hydroxy risperidone together constitute the active ingredients of antipsychotic drugs. 
     Commonly need clinical dosage forms of risperidone comprise tablets, oral solutions, capsules, and orally disintegrating tablets, etc. For common dosage forms of risperidone, drugs usually have to be taken every day, which is difficult for about 75% of psychiatric patients. This is also a very important factor contributing to deterioration during the treatment. 
     In order to solve such problems, researchers have actively developed risperidone long-acting sustained release preparations. For example, CN1137756, the entire content of which is incorporated herein by reference, disclosed a risperidone sustained release microsphere composition prepared by using a polymer matrix material with a molecular weight of 100,000 to 300,000. Long-acting antipsychotic drug Risperidal Consta (Chinese name: HENGDE), which developed based on the technology in CN1137756, came into market in August 2002. The product is prepared by encapsulating risperidone in a lactide-glycolide copolymer (PLGA) with a molecular weight of 150,000, suspended in a solution, and administrated by intramuscular injection once every 2 weeks, thus avoiding the peak-valley concentration of daily administration effectively. However, only a small amount of drug in the preparation is released on the first day, followed by a drug release lag phase after 3 weeks, and with the degradation of the microsphere skeleton, most of drugs are released in the 4th to 6th weeks [Chen Qinghua, Chan Gang, et al., pharmacokinetic characteristics and clinical application of risperidone long-acting injection, Chinese Pharmacy, 2006, 15 (15):1235-1238]. Therefore, while the drug is administrated to patients by injection in the first 3 weeks, patients also need to rely on oral risperidone tablets to achieve therapeutic effects, and subsequently the clinical use is not convenient and patient compliance is poor. 
     Chen Guoguang et al reported a risperidone microsphere composition prepared by using PLGA (50:50, molecular weight of 30,000) with a drug-loading rate of 18%, by which a stable drug blood concentration may be maintained in vivo for 5-20 days [Chen Guoguang. Tang Jun, et al, study on risperidone biodegradable microspheres. Journal of China Pharmaceutical University, 2006, 37 (6):512-515]. However, the drug-loading rate of this microsphere composition is low, and is also accompanied with a burst release when the drug-loading rate is low. 
     CN101653422, the entire content of which is incorporated herein by reference, disclosed a risperidone microsphere composition which may cause sustained release for more than 4 weeks, and the drug release lag phase was eliminated by improving the drug-loading rate (above 43%), substantially solve bust release problems. However, the patent application of CN101653422 only verifies that the laboratory level (5 L scale) may achieve the desired object. It has been found by the applicant of the present invention that drug crystals were precipitated out during the scaled-up production of risperidone microspheres provided in CN101653422, the preparation stability was poor, and in vivo release behavior of the microspheres will change substantially after long-term storage. 
     As is well known, large-scale industrialized production has always been the bottleneck of the industrialization of the microsphere preparation, and therefore there is an urgent need to provide a formulation of risperidone microspheres that is stable in quality and suitable for large-scale industrialized production. 
     SUMMARY 
     The present disclosure provides a pharmaceutical microsphere composition, containing an active ingredient and an uncapped poly(lactide-co-glycolide), in which the active ingredient is selected from risperidone or a salt thereof, and 9-hydroxy risperidone or a salt thereof, the uncapped poly(lactide-co-glycolide) consists of two copolymers; a weight content of the active ingredient in the pharmaceutical composition is within a range from 10% to 60%, preferably, from 35% to 55%, more preferably, from 40% to 50%; and a weight content of the uncapped poly(lactide-co-glycolide) in the pharmaceutical composition is within a range from 40% to 90%, preferably, from 43% to 65%, more preferably, from 50% to 60%. 
     The microspheres as disclosed herein is: Small spherical or spherical-like particles consist of drug dissolved and (or) dispersed homogeneously throughout a polymer material, with a particle sit ranging in 1-500 μm, and generally prepared as suspension for injection. 
     A lactide-glycolide copolymer is also referred to as poly(lactide-co-glycolide), abbreviated as PI.GA. As used herein, the term “uncapped poly(lactide-co-glycolide)” refers to poly(lactide-co-glycolide) having a carboxyl terminal group, below abbreviated as PLGA. 
     The two copolymers, i.e., the two PLGAs, are an uncapped PLGA with a high intrinsic viscosity of 0.4-0.9 dl/g, preferably, 0.45-0.8 dl/g, more preferably, 0.45-0.55 dl/g, and an uncapped PLGA with a low intrinsic viscosity of 0.1-035 dl/g, preferably, 0.1-0.3 dl/g, more preferably, 0.2-0.3 dl/g. A weight ratio of the uncapped PLGA with the high intrinsic viscosity to the uncapped PLGA with the low intrinsic viscosity is (50-95):(5-50), preferably, (70-90):(10-30), more preferably, 80:20. A molar ratio of lactide to glycolide in the uncapped PLGA with the high intrinsic viscosity is within a range from 65:35 to 90:10, preferably, 75:25; and a molar ratio of lactide to glycolide in the uncapped PLGA with the low intrinsic viscosity is within a range from 50530 to 75:25, preferably, 50:50. 
     The intrinsic viscosity of PLGA is determined by preparing an bout 0.5% (w/v) solution of PLGA in chloroform, and determining the intrinsic viscosity of PLGA at 30° C. using a Cannon-Fenske glass capillary viscometer. 
     The two PLGAs may also be a high molecular weight PLGA with a molecular weight of 50,000-145,000, preferably, 55,000-110,000, more preferably, 55,000-85,000 and a low molecular weight PLGA with a molecular weight of 4,000 to 45,000, preferably, 4,000-35,000, more preferably, 15,000-35,000. A weight ratio of the high molecular weight PLGA to the low molecular weight PLGA is (50.95):(5-50), preferably, (70-90):(10-30), more preferably, 80:20. A molar ratio of lactide to glycolide in the high molecular weight PLGA is within a range from 65:35 to 90:10, preferably, 75:25; and a molar ratio of lactide to glycolide in the low molecular weight PLGA is within a range from 50:50 to 75:25, preferably, 50:50. As used herein, the term “molecular weight” refers to “weight average molecular weight”, abbreviated as “molecular weight”. 
     For convenient description, hereinafter, the molar ratio of lactide to glycolide in PLGA and the intrinsic viscosity of PLGA are expressed in a bracket after PLGA. For example, “PLGA (75/25, 0.5 A)” refers to a lactide-glycolide copolymer having an Intrinsic viscosity of 0.5 dl/g and a carboxyl terminal group, in which a molar ratio of lactide to glycolide is 75:25. 
     Particularly, the preferred weight ratio of the uncapped PLGA (75/25, 0.5 A) with the high intrinsic viscosity to the uncapped PLGA (50/50, 0.25 A) with the low intrinsic viscosity in the present Invention is 80:20. 
     Specifically, in the microsphere composition of the present invention, the preferred weight content of risperidone is 45% and the weight content of uncapped PLGA is 55%, the weight ratio of the two PLGAs is 80-20, the molecular weight of the two PLGAs are 55,000-45,000 and 15,000-35,000, the intrinsic viscosity of the two PLGAs we 0.45-0.55 dL/g and 0.2-0.3 dL/g, and molar ratio of lactide to glycolide in the two PLGAs are 75:25 and 50:50, respectively. 
     As used herein, a drug-lading rate refers to a practical drug-loading rate, which is calculated by a formula: drug-loading rate=[amount of drug in microspheres/(amount of drug in microspheres+amount of polymer in microspheres)]×100%. 
     Risperidone or 9-hydroxy risperidone in the sustained release microspheres of the present invention may be present in the form of a salt. An acid which will form a salt with risperidone or 9-hydroxy risperidone comprises an inorganic acid, for example, halogen acid (e.g., hydrochloric acid or hydrobromic acid), nitric acid, sulfuric acid or phosphoric acid; or an organic acid, for example, acetic acid, propionic acid, hydroxy acetic acid, 2-hydroxy propionic acid pamoic acid, 2-oxo propionic acid, oxalic acid, malonic acid, succinic acid, 2-butenedioic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid or toluenesulfonic acid. 
     The risperidone sustained release microspheres of the present invention may be prepared by a conventional method, for example, an emulsion-solvent evaporation method, a spray drying method or a spray extraction method, etc. 
     Emulsion-Solvent Evaporation Method 
     Risperidone or a salt thereof or 9-hydroxy risperidone or a salt thereof and PLGA are dissolved in a suitable organic solvent, the organic solvent is injected into as aqueous solution prepared from a water soluble polymer to perform dispersion emulsifying, the organic solvent is evaporated, and the residue is washed and filtered to obtain microspheres. The organic solvent may be selected from halogenated hydrocarbons (e.g., dichloromethane, chloroform, ethyl chloride, dichloromethane, or trichloroethane), ethyl acetate, ethyl formate, diethyl ether, cyclohexane, benzyl alcohol or a combination thereof. The water soluble polymer may be selected from at least one of polyvinyl alcohol (PVA), sodium carboxymethyl cellulose (CMC-Na), polyvinyl pyrrolidone (PVP), sodium polymethacrylate and sodium polyacrylate, or a combination of two or more of them. The dispersion emulsifying may be performed by mechanical stirring or by a static mixer. 
     Spray Drying Method 
     Risperidone or a salt thereof or 9-hydroxy risperidone or a salt thereof and PLGA are dissolved in a suitable organic solvent and filtered, and a conventional spray drying method is used to prepare microspheres. The organic solvent may be selected from dichloromethane, chloroform, ethyl acetate, diethyl ether, acetone, benzyl alcohol, glacial acetic acid, or a combination thereof. 
     Spray Extraction Method 
     Risperidone or a salt thereof or 9-hydroxy risperidone or a salt thereof and PLGA are dissolved in a suitable organic solvent to form a solution, and then the solution is sprayed into an organic nonsolvent (i.e., an organic solvent in which risperidone or a salt thereof or 9-hydroxy risperidone or a salt thereof and PLGA we not dissolved) or water, and extracted the solvent to form the microspheres. The organic solvent may be selected from dichloromethane, chloroform, ethyl acetate, diethyl her acetone, benzyl alcohol, glacial acetic acid, or a combination thereof. The organic nonsolvent may be selected from methanol, ethanol, propanol, isopropanol, petroleum ether, alkane, paraffine, or a combination thereof. 
     The present disclosure further provides a use of the risperidone microspheres in preparation of antipsychotic drugs, in which a psychosis comprises acute schizophrenia and chronic schizophrenia significant positive symptoms (e.g. hallucination, delusion, thought disorder, hostility, or suspicion) and significant negative symptoms (e.g., slow response, emotional indifference, social indifference, or hypologia) of other psychotic states, and affective symptoms (e.g., depression guilty feeling, or anxiety) related to schizophrenia, preferably, schizophrenia, anxiety, depression periodic headache, etc. 
     In another embodiment, the present disclosure provides a method of treating psychosis by administering a formulation of risperidone microspheres described herein. The psychosis comprises acute schizophrenia and chronic schizophrenia, significant positive symptoms (e.g., hallucination, delusion, thought disorder, hostility, or suspicion) and significant negative symptoms (e.g., slow response, emotional indifference, social indifference, or hypologia) of other psychotic states and affective symptoms (e.g., depression, guilty feeling, or anxiety) related to schizophrenia, preferably, schizophrenia, anxiety, depression, periodic headache, etc. 
     The microspheres according to an embodiment of the present disclosure may be present in the form of a sterile powder. The sterile powder may contain the risperidone microsphere composition and mannitol and may be prepared by washing the sustained release composition with water for injection, transferring the sustained release composition to a lyophilized plate, adding mannitol and an appropriate amount of injection water, placing the lyophilized plate in a lyophilizer for lyophilizing subjecting the lyophilized product to screening and mixing, sterile subpackaging, and capping to obtain the sterile powder. Before administering the drug to a patient, the sterile powder is suspended in a acceptable dispersion solvent. The dispersion solvent is selected from at least one of a suspending agent, a pH regulator, an isoosmotic adjusting agent, a surfactant, and water for injection. The suspending agent may be selected from at least one of sodium carboxyethyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, sodium alginate and glycerol. The isoosmotic adjusting agent may be selected from at least one of sodium chloride, glucose, mannitol, and glucitol. The surfactant is a nonionic surfactant, for example, polysorbate series (e.g., polysorbate 80 or polysorbate 60) or poloxamer series (e.g., poloxamer 188). 
     The risperidone sustained release microsphere composition according to an embodiment of the present disclosure is usually administrated parenterally, for example, intramuscular injection, subcutaneous injection, intradermal injection intraperitoneal injection and so on. For a patient with a body weight of 60 kg, an administration dose is 12.5-150 mg every time, based on risperidone. That is, a therapeutically effective amount of the risperidone sustained release microsphere composition is 0.2-2.5 risperidone/kg body weight, preferably, 0.4-1.7 m risperidone/kg body weight. 
     The sustained release microsphere composition has the following advantages: 1) it provides immediate release after entering into a body without drug release lag phase, in both high or low drug-loading; 2) its conducive to scaled-up production (scale above 75 L) and without drug crystals precipitating out during the production; 3) it is highly stable, and therefore in vivo release behavior will not change after long-term storage. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1-1  is a scanning electro microscope image of risperidone microspheres in CN101653422, in which drug crystals are precipitated out. 
         FIG. 1-2  is a scanning electron microscope mage of risperidone microspheres in Embodiment 6, in which no drug crystals are precipitated out, which indicates that the risperidone microspheres according to an embodiment of the present disclosure is suitable for large-scale industrialized production. 
         FIG. 2  shows in vivo release blood drug concentration-time curves of a risperidone microsphere composition (prepared according to CN101653422) before and after being stored for 6 months, which indicates that the in vivo drug release behavior of the risperidone microspheres in CN101653422 after being stored for 6 months changes substantially and the quality of the risperidone microspheres disclosed in CN101653422 is not stable. 
         FIG. 3  shows in vivo release blood drug concentration-time curves of risperidone microsphere in Embodiment 1 before and after being stored for 6 months, which indicates that in vivo drug release behavior of the risperidone microspheres in Embodiment 1 after being stored for 6 months does not change substantially and the quality of the risperidone microspheres according to an embodiment of the present disclosure is much more stable. 
         FIG. 4  shows in vivo release blood drug concentration-time curves of risperidone microspheres in Embodiment 3 before and after being stored for 6 months, which indicates that in vivo drug release behavior of the risperidone microspheres in Embodiment 3 after being stored for 6 months does not change substantially and the quality of the risperidone microspheres according to an embodiment of the present disclosure is much more stable. 
         FIG. 5  shows n vivo release blood drug concentration-time curves of risperidone microspheres in Embodiment 4 before and after being stored for 6 months, which indicates that in vivo drug release behavior of the risperidone microspheres in Embodiment 4 after being stored for 6 months does not change substantially and the quality of the risperidone microspheres according to an embodiment of the present disclosure is much more stable. 
         FIG. 6  shows in vivo release blood drug concentration-time curves of risperidone microspheres in Embodiment 6 before and after being stored for 6 months, which indicates that in vivo drug release behavior of the risperidone microspheres in Embodiment 6 after being stored for 6 months does not change submittals and the quality of the risperidone microspheres according to an embodiment of the present disclosure is much more stable. 
         FIG. 7  shows in vivo release blood drug concentration-time curves of risperidone microspheres in Embodiment 7 before and after being stored for 6 months, which indicates that in vie drug release behavior of the risperidone microspheres in Embodiment 7 after being stored for 6 months does not change substantially and the quality of the risperidone microspheres according to an embodiment of the present disclosure s much more stable. 
         FIG. 8  shows in vivo release blood drug concentration-time curves of risperidone microspheres in Embodiment 9 before and after being stored for 6 months, which indicates that in vivo drug release behavior of the risperidone microspheres in Embodiment 9 after being stored for 6 months does not change substantially and the quality of the risperidone microspheres according to an embodiment of the present disclosure is much more stable. 
         FIG. 9  shows in vivo release blood drug concentration-time curves of risperidone microspheres in Test embodiment 2, which indicates that a drug may be still released immediately after entering into a body even when the drug-loading rate of the risperidone microspheres according to an embodiment of the present disclosure as low as about 20%, without a release lag phase. 
     
    
    
     DETAILED DESCRIPTION 
     As described herein, various embodiments are directed to pharmaceutical compositions, which comprise: an active component selected from risperidone, a salt thereof 9-hydroxy risperidone and a salt thereof; and a polymer blend comprising a first uncapped poly(lactide-co-glycolide) and a second uncapped poly(lactide-co-glycolide), wherein a weight content of the active component in the pharmaceutical composition is within a range from 10% to 60%, preferably from 35% to 55%, more preferably from 40% to 50%; a weight content of the polymer blend in the pharmaceutical composition is within a range from 40% to 90%, preferably from 45% to 65%, more preferably from 50% to 60%; and the pharmaceutical composition is preset in the form of microspheres. 
     In the pharmaceutical composition of one embodiment of the present disclosure, the polymer blend consists of the first uncapped poly(lactide-co-glycolide) and the second uncapped poly(lactide-co-glycolide). 
     In the pharmaceutical composition of one embodiment of the present disclosure, the first uncapped poly(lactide-co-glycolide) ha high intrinsic viscosity of 0.4-0.9 dl/g, preferably 0.45-0.8 dug, more preferably 0.45-0.55 dl/g, and the second uncapped poly(lactide-co-glycolide) has a low intrinsic viscosity of 0.1-0.35 dl/g, preferably 0.1-0.3 dl/g, more preferably 0.2-0.3 dl/g; and a weight ratio of the first uncapped poly(lactide-co-glycolide) to the second uncapped poly(lactide-co-glycolide) is (50-95):(5-50), preferably (70-90):(10-30), more preferably 80-20; and a molar ratio of lactide to glycolide in the first uncapped poly(lacde-co-glycolide) is within a range from 65:35 to 90:10, preferably 75.25; and a molar ratio of lactide to glycolide in the second uncapped poly(lactide-co-glycolide) is within a mage from 50:50 to 75-25, preferably 50:50. 
     In the pharmaceutical composition of another embodiment of the present disclosure, the first uncapped poly(lactide-co-glycolide) has a weight average molecular weight of 50,000-145,000, preferably 55,000-110,000, more preferably 55,000-85,000 and the second uncapped poly(lactide-glycolide) has a weigh average molecular weight of 4,000 to 45,000, preferably 4,000-35,000, more preferably 15,000-35,000; and a weight ratio of the first uncapped poly(lactide-co-glycolide) to the second uncapped poly(lactide-co-glycolide) is (50-95):(5-50), preferably (70-90):(10-30), more preferably 80:20; and a molar ratio of lactide to glycolide in the first uncapped poly(lactide-co-glycolide) is within a range from 65:35 to 90:10, preferably 75:25; and a molar ratio of lactide to glycolide in the second uncapped poly(lactide-co-glycolide) is within a range from 50:50 to 75:25, preferably 50:50. 
     In the pharmaceutical composition of one preferred embodiment of the present disclosure, the weight content of risperidone is 45%, the weight content of the polymer blend is SS %, the weight ratio of the first uncapped PLGA to the second uncapped PLGA is 80:20, the molecular weigh of the first uncapped PLGA is 55,000-85,000 and the molecular weight of the second uncapped PLGA is 15,000˜35,000, the intrinsic viscosity of the first uncapped PLGA is 0.45˜0.55 dL/g and the intrinsic viscosity of the second uncapped PLGA is 0.2˜0.3 dL/g, and a molar ratio of lactide to glycolide in the first uncapped PLGA is 75:25 and a molar ratio of lactide to glycolide in the second uncapped PLGA is 50:50. 
     In the pharmaceutical composition of one embodiment of the present disclosure, a salt of risperidone or 9-hydroxy risperidone is selected from an inorganic acid salt and an organic acid salt; the inorganic acid salt being selected from hydrochlorate, hydrobromate, nitrate, sulfate and phosphate; and the organic acid salt being selected from acetate, propionate, hydroxy acetate, 2-hydroxy propionate, pamoate, 2-oxo propionate, oxalate; malonate, succinate, 2-butenedioate, methanesulfonate, ethanesulfonate, benzenesulfonate and toluenesulfonate. 
     The present disclosure further provides a use of any one of the above-mentioned pharmaceutical compositions in preparation of antipsychotics, wherein a psychosis comprises acute schizophrenia and chronic schizophrenia, significant positive symptoms and significant negative symptoms of other psychotic states, and affective symptoms related to schizophrenia. 
     Another embodiment of the present disclosure provides a sustained release microsphere formulation for injection, comprising any one of the above-mentioned pharmaceutical compositions; and the microspheres are suspended in a pharmaceutically acceptable dispersion solvent the dispersion solvent is selected from a suspending agent, a pH regulator, man isoosmotic adjusting agent, a surfactant, water, and physiological saline; and wherein the suspending agent is selected from sodium carboxymethyl cellulose, polyvinyl alcohol, polyvinyl pyrrolidone, sodium alginate, and glycerol; and wherein the isoosmotic adjusting agent is selected from sodium chloride, glucose, mannitol, and glucitol; and wherein the surfactant is a nonionic surfactant and is selected from polysorbate series and poloxamer series. 
     The present disclosure will be farther illustrated by the following embodiments and test embodiments, which will not limit the scope of the present invention in any way. 
     Embodiment 1 
     72 g of PLGA (75/25, 0.52 A) with a molecular weight of 74,000, 18 g of PLGA (50/50, 0.25 A) with a molecular weight of 25,000 and 110 g of risperidone were weighed and dissolved in 1000 ml of dichloromethane with stirring to prepare a clear solution. The clear solution was added into a microsphere preparation kettle containing a 100 L PVA solution (0.5%) cooled to 6° C. by a peristaltic pump. A stirrer and a homogenizer were started, and then the clear solution was homogeneously emulsified at 350 rpm for 1 min. Then, the rotation speed of the homogenizer was reduced, and an organic solvent was evaporated for 3-5 h. The residue was filtered with a screen, washed with deionized water, and lyophilized to obtain powdered microspheres. No crystals were precipitated out. The microspheres had a drug-loading rate of 45.9% and an encapsulation efficiency of 83.5%. 
     Embodiment 2 
     67.5 g of PLGA (75/25, 0.42 A) with a molecular weigh of 53,000, 7.5 g of PLGA (50/50, 0.25 A) with a molecular weight of 25,000 and 75 g of risperidone were weighed and dissolved in 750 ml of dichloromethane with stirring to prepare a clear solution. The clear solution was added into a microsphere preparation kettle containing a 75 L PVA solution (0.5%) cooled to 6° C. by a peristaltic pump. A stirrer and a homogenizer were started, and then the clear solution was homogeneously emulsified at 380 rpm for 1 min. Then, the rotation speed of the homogenizer was reduced, and an organic solvent was evaporated for 3-5 h. The residue was filtered with a screen, washed with deionized water, and lyophilized to obtain powdered microspheres. No crystals were precipitated out. The microspheres had a drug-loading rate of 40.2% and an encapsulation efficiency of 80.4%. 
     Embodiment 3 
     56 g of PLGA (75/25, 0.90 A) with a molecular weight of 125,000, 24 g of PLGA (50/50, 0.25 A) with a molecular weight of 25,000 and 120 g of risperidone were weighed and dissolved in 1000 ml of dichloromethane with stirring to prepare a clear solution. The clear solution was added into a microsphere preparation kettle containing a 100 L PVA solution (0.5%) cooled to 6° C. by a peristaltic pump. A stirrer and a homogenizer were started, and then the clear solution was homogeneously emulsified at 380 rpm for 1 min. Then, the rotation speed of the homogenizer was reduced, and an organic solvent was evaporated for 3-5 h. The residue was filtered with a screen washed with deionized water, and lyophilized to obtain powdered microspheres. No crystals were precipitated out. The microspheres had a drug-loading rate of 51.5% and an encapsulation efficiency of 85.8%. 
     Embodiment 4 
     64.125 g of PLGA (75/25, 0.52 A) with a molecular weight of 74,000. 3.375 g of PLGA (50/50, 0.10 A) with a molecular weight of 4,200 and 82.5 g of risperidone were weighed and dissolved in 750 ml of dichloromethane with stirring to prepare a clear solution. The clear solution was added into a microsphere preparation kettle containing a 75 L PVA solution (0.5%) cooled to 6° C. by a peristaltic pump. A stirrer and a homogenizer were started, and then the clear solution was homogeneously emulsified at 380 rpm for 1 min. Then, the rotation speed of the homogenizer was reduced, and an organic solvent was evaporated for 3-5 h. The residue was filtered with a screen, washed with deionized water, and lyophilized to obtain powdered microspheres. No crystals were precipitated out. The microspheres had a drug-loading rate of 45.5% and an encapsulation efficiency of 82.7%. 
     Embodiment 5 
     63 g of PLGA (75/25, 0.52 A) with a molecular weight of 74,000, 27 g of PLGA (50/50, 0.35 A) with a molecular weight of 40,000 and 60 g of risperidone were weighed and dissolved in 750 ml of dichloromethane with stirring to prepare a clear solution. The clear solution was added into a microsphere preparation kettle containing a 75 L PVA solution (0.5%) cooled to 6° C. by a peristaltic pump. A stirrer and a homogenizer were started, and then the clear solution was homogeneously emulsified at 380 rpm for 1 min. Then, the rotation speed of the homogenizer was reduced, and an organic solvent was evaporated for 3-5 h. The residue was filtered with a seen, washed with deionized water, and lyophilized to obtain powdered microspheres. No crystals were precipitated out. The microspheres had a drug-loading rate of 33.1% and an encapsulation efficiency of 82.5%. 
     Embodiment 6 
     42 g of PLGA (65/35, 0.55 A) with a molecular weight of 85,000, 10.5 g of PLCA (50/50, 0.25 A) with a molecular weight of 25,000 and 97.5 g of risperidone we weighed and dissolved in 750 ml of dichloromethane with stirring to prepare a clear solution. The clear solution was added into a microsphere preparation kettle containing a 75 L PVA solution (0.5%) cooled to 6° C. by a peristaltic pump. A stirrer and a homogenizer were started, and then the clear solution was homogeneously emulsified at 380 rpm for 1 min. Then, the rotation speed of the homogenizer was educed, and an organic solvent was evaporated for 3-5 h. The residue was filtered with a screen, washed with deionized water, and lyophilized to obtain powdered microspheres. No crystals were precipitated our. The microspheres had a drug-loading rate of 55.0% and an encapsulation efficiency of 84.6%. 
     Embodiment 7 
     57.75 g of PLGA (90/10, 0.45 A) with a molecular weight of 67,000, 24.75 g of PLGA (50/50, 0.25 A) with a molecular weight of 25,000 and 67.5 g of risperidone were weighed and dissolved in 750 ml of dichloromethane with stirring to prepare a clear solution. The clear solution was added into a microsphere preparation kettle containing a 75 L PVA solution (0.5%) cooled to 6° C. by a peristaltic pump. A stirrer and a homogenizer were started, and then the clear solution was homogeneously emulsified at 380 rpm for 1 min. Then, the rotation speed of the homogenizer was reduced, and an organic solvent was evaporated for 3-5 h. The residue was filtered with a screen, washed with deionized water, and lyophilized to obtain powdered microspheres. No crystals were precipitated out. The microspheres had a drug-loading rate of 35.8% and an encapsulation efficiency of 79.6%. 
     Embodiment 8 
     68.25 g of PLGA (85/15, 0.71 A) with a molecular weight of 110,000, 36.75 g of PLGA (50/50, 0.25 A) with a molecular weight of 25,000 and 45 g of risperidone were weighed and dissolved in 750 ml of dichloromethane with stirring to prepare a clear solution. The clear solution was added into a microsphere preparation kettle containing a 75 L PVA solution (0.5%) cooled to 6° C. by a peristaltic pump. A stirrer and a homogenizer were started, and then the clear solution was homogeneously emulsified at 380 rpm for 1 min. Then, the rotation speed of the homogenizer was reduced, and an organic solvent was evaporated for 3-5 h. The residue was filtered with a screen, washed with deionized water, and lyophilized to obtain powdered microspheres. No crystals war precipitated out. The microspheres had a drug-loading rate of 23.9% and an encapsulation efficiency of 79.7%. 
     Embodiment 9 
     54 g of PLGA (75/25, 0.52 A) with a molecular weight of 74,000, 13.3 of PLGA (75/25, 0.20 A) with a molecular weight of 25,000 and 82.5 g of risperidone were weighed and dissolved in 750 ml of dichloromethane with stirring to prepare a clear solution. The clear solution was added into a microsphere preparation kettle containing a 75 L PVA solution (0.5%) cooled to 6° C. by a peristaltic pump. A stirrer and a homogenizer were started, and then the clear solution was homogeneously emulsified at 380 rpm for 1 min. Then, the rotation speed of the homogenizer was reduced, and an organic solvent was evaporated for 3-5 h. The residue was filtered with a screen, washed with deionized water, and lyophilized to obtain powdered microspheres. No crystals were precipitated out. The microspheres had a drug-loading rate of 45.3% and an encapsulation efficiency of 82.4%. 
     Embodiment 10 
     60 g of PLGA (85/15, 0.71 A) with a molecular weight of 110,000, 60 g of PLGA (50/50, 0.25 A) with a molecular weight of 25,000 and 30 g of risperidone were weighed and dissolved in 750 ml of dichloromethane with stirring to prepare a clear solution. The clear solution was added into a microsphere preparation kettle containing a 75 L PVA solution (0.5%) cooled to 6° C. by a peristaltic pump. A stirrer and a homogenizer were started, and then the clear solution was homogeneously emulsified at 380 rpm for 1 min. Then, the rotation speed of the homogenizer was reduced, and an organic solvent was evaporated for 3-5 h. The residue was filtered with a screen, washed with deionized water, and lyophilized to obtain powdered microspheres. No crystals were precipitated out. The microspheres had a drug-loading rate of 13.9% and an encapsulation efficiency of 69.5%. 
     Embodiment 11 
     48 g of PLGA (75/25, 0.61 A) with a molecular weight of 92,000, 12 g of PLGA (65/35, 0.12 A) with a molecular weight of 5,000 and 140 g of risperidone were weighed and dissolved in 1000 ml of dichloromethane with stirring to prepare a clear solution. The clear solution was added into a microsphere preparation kettle containing a 100 L PVA solution (0.5%) cooled to 6° C. by a peristaltic pump. A stirrer and a homogenizer were started, and then the clear solution was homogeneously emulsified at 380 rpm for 1 min. Then, the rotation speed of the homogenizer was reduced, and a organic solvent was evaporated for 3-5 h. The residue was filtered with a screen, washed with deionized water, and lyophilized to obtain powdered microspheres. No crystals were precipitated out. The microspheres had a drug-loading rte of 60.6% and an encapsulation efficiency of 84.3%. 
     Embodiment 12 
     54 g of PLGA (75/25, 0.52 A) with a molecular weight of 74,000, 13.5 g of PLGA (50/50, 0.25 A) with a molecular weight of 25,000 and 85.65 g of 9-hydroxy risperidone were weighed and dissolved is 750 ml of dichloromethane with stirring to prepare a clear solution. The clear solution was added into a microsphere preparation kettle containing a 75 L PVA solution (0.5%) cooled to 6° C. by a peristaltic pump. A stirrer and a homogenizer were started, and then the clear solution was homogeneously emulsified at 380 rpm for 1 min. Then, the rotation speed of the homogenizer was reduced, and a organic solvent was evaporated for 3-5 h. The residue was filtered with a screen, washed with deionized water, and lyophilized to obtain powdered microspheres. No crystals were precipitated out. The microspheres had a drug-loading rate of 45.9% and an encapsulation efficiency of 83.5%. 
     Embodiment 13 
     64.8 g of PLGA (75/25, 0.52 A) with a molecular weight of 74,000, 16.2 g of PLGA (50/50, 0.25 A) with a molecular weight of 25,000 and 192.6 g of pamoic acid risperidone were weighed and dissolved in 750 ml of dichloromethane with stirring to prepare a clear solution. The clear solution was added into a microsphere preparation kettle containing a 75 L PVA solution (0.5%) cooled to 6° C. by a peristaltic pump. A stirrer and a homogenizer were started, and then the clear solution was homogeneously emulsified at 380 rpm for 1 min. Then, the rotation speed of the homogenizer was reduced, and an organic solvent was evaporated for 3-5 h. The residue was filtered with a screen, washed with deionized water, and lyophilized so obtain powdered microspheres. No crystals were precipitated out. The microspheres had a drug-loading rate of 45.9% and an encapsulation efficiency of 83.5%. 
     Embodiment 14 
     The microspheres obtained in Embodiment 1 were washed with water for injection and transferred to a lyophilized plate. 4 g of mannitol and an appropriate amount of water were added, and the lyophilized plate was placed in a lyophilizer for lyophilizing. The lyophilized product was subjected to screening and mixing, sterile subpackaging, and capping to obtain risperidone sustained release microspheres for injection. 
     Comparative Test 1 Scaled-up Production Of Risperidone Microspheres Disclosed In CN101653422 (75 L) 
     1) Test Materials 
     Risperidone, PLGA (75/25, 0.52 A) with a molecular weight of 74,000 
     2) Method and Results: 
     60 g of PLGA (75/25, 0.52 A) with a molecular weight of 74,000 and 90 g of risperidone were weighed and dissolved in 750 ml of dichloromethane with stirring to prepare a clear solution. The clear solution was added into a microsphere preparation kettle containing a 75 L PVA solution (0.5%) cooled to 6° C. by a peristaltic pump. A stirrer and a homogenizer were started, and than the clear solution was homogeneously emulsified at 380 rpm for 1 min. Then, the rotation speed of the homogenizer was reduced, and an organic solvent was evaporated for 3-5 h. The residue was filtered with a screen, washed with deionized water, and lyophilized to obtain powdered microspheres. After observed by a microscope, drug crystals we discovered, as shown in  FIG. 1-1 . 
     In contrast, the microspheres obtained in Embodiments 1-10 according to the present disclosure, when observed by a microscope, showed no drug crystals that precipitated out.  FIG. 1-2  is a scanning electron microscope image of risperidone microspheres in Embodiment 6. 
     The results indicate that the risperidone microspheres according to an embodiment of the present disclosure are more suitable for large-scale industrialized production. 
     Comparative Test 2 Stability Test Of An Embodiment of The Present Disclosure As Compared With CN101653422 
     1) Test Materials 
     Test Drugs: 
     The present disclosure: the risperidone microspheres obtained in Embodiments 1, 3, 4, 6, 7, 9 were stored for 0 month and 6 months respectively. 
     CN101653422: the risperidone microspheres obtained in Embodiment 3 in CN101653422 was stored for 0 month and 6 months. 4.0 g of PLGA (75/25, 0.52 A) with a molecular weight of 74,000 and 6.0 g of risperidone were weighed and dissolved in 50 ml of dichloromethane with stirring to prepare a clear solution. The clear solution was added into a microsphere preparation kettle containing a 5000 ml PVA solution (0.5%) cooled to 6° C. with high-speed stirring by a peristaltic pump, and dispersion emulsified at 1000 rpm for 1 min. Then, the rotation speed was adjusted to 300 rpm, the rotation speed of a stirring paddle was 150 rpm, and any organic solvent was removed by evaporation for 6 h. The residue was filtered with a screen, washed with deionized water 5 times, and lyophilized to obtain powdered microspheres. The microspheres had a drug-loading rat of 50.7% and an encapsulation efficiency of 84.5%. 
     Test Animals: 56 healthy beagles dogs, 4 dogs in each group, 28-female-28-male, with a body weight of 9.5-10.5 kg. 
     Test Instruments: an API 4000 triple quadrupole tandem mass spectrometer equipped with an ion spray ionization source and an Analyst 1.4 data processing software, U.S., Applied Biosystem company; Agilent 1100 high performance liquid chromatograph. 
     2) Method and Results 
     The test animals were randomly divided into 2 groups (0-month group and 6-month group) with 4 dogs in each group, a dose of 1.5 mg/kg (based on risperidone) was administrated by intramuscular injection each beagle, and after administrating for 0 h, 1 h, 3 h, 6 h, 1 d, 2 d, 3 d, 5 d, 7 d, 9 d, 11 d, 14 d, 16 d, 18 d, 21 d, 23 d, 25 d and 28 d, 3 ml of blood was sampled via the forelimb vein of each beagle, placed in the heparinization centrifuge tube immediately, and centrifugated for 10 min (3600 rpm). A plasma was separated, and stored in a refrigerator at −37° C. to be measured. Drug blood concentration of risperidone and a metabolite thereof i.e., 9-hydroxy risperidone, in the plasma were measured respectively, and the results were shown in Table 1 and  FIGS. 2-8 . 
     It may be seen from the results that there was substantial changes in the in who drug release behavior of the risperidone microspheres disclosed in CN101653422 after being stored for 6 months; but the in vivo drug release behavior of the risperidone microspheres according to an embodiment of the present disclosue after being stored for 6 months does not change substantially due to improved stability. 
     
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 Drug blood concentrations (ng/mL) at different time after the microspheres according 
               
               
                 to an embodiment of the present disclosure and in CN101653422 after stored for 
               
               
                 6 months were administrated by intramuscular injection on each beagle 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Time 
                 Embodiment 1 
                 Embodiment 3 
                 Embodiment 4 
                 Embodiment 6 
               
             
          
           
               
                 (day) 
                 0 month 
                 6 month 
                 0 month 
                 6 month 
                 0 month 
                 6 month 
                 0 month 
                 6 month 
               
               
                   
               
               
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 0.042 
                 2.619 
                 2.593 
                 2.126 
                 2.739 
                 2.635 
                 2.817 
                 4.792 
                 4.469 
               
               
                 0.125 
                 1.665 
                 2.326 
                 1.156 
                 1.512 
                 1.049 
                 1.55 
                 1.186 
                 2.202 
               
               
                 0.25 
                 1.79 
                 3.449 
                 1.209 
                 2.665 
                 1.174 
                 2.673 
                 3.311 
                 3.325 
               
               
                 1 
                 1.268 
                 1.406 
                 0.786 
                 0.607 
                 0.652 
                 0.630 
                 5.662 
                 1.282 
               
               
                 2 
                 1.589 
                 1.860 
                 1.096 
                 1.058 
                 0.973 
                 1.084 
                 3.110 
                 4.739 
               
               
                 3 
                 1.73 
                 3.928 
                 1.205 
                 3.122 
                 1.114 
                 6.569 
                 4.265 
                 7.221 
               
               
                 5 
                 10.561 
                 8.32 
                 10.262 
                 7.536 
                 9.945 
                 7.544 
                 10.082 
                 10.298 
               
               
                 7 
                 16.548 
                 19.789 
                 13.099 
                 15.621 
                 15.932 
                 16.336 
                 16.069 
                 15.223 
               
               
                 9 
                 12.837 
                 18.237 
                 12.337 
                 17.433 
                 13.229 
                 15.623 
                 13.366 
                 16.275 
               
               
                 11 
                 10.125 
                 14.214 
                 9.625 
                 13.414 
                 9.509 
                 13.438 
                 9.646 
                 14.09 
               
               
                 14 
                 13.641 
                 17.38 
                 13.114 
                 16.33 
                 13.025 
                 16.604 
                 13.162 
                 17.256 
               
               
                 16 
                 12.326 
                 12.427 
                 11.865 
                 11.627 
                 11.71 
                 11.651 
                 11.847 
                 11.301 
               
               
                 18 
                 13.582 
                 14.195 
                 13.006 
                 13.656 
                 12.966 
                 13.419 
                 13.103 
                 14.071 
               
               
                 21 
                 8.48 
                 6.581 
                 7.980 
                 5.781 
                 7.864 
                 5.805 
                 5.007 
                 6.457 
               
               
                 23 
                 6.155 
                 5.027 
                 5.565 
                 5.227 
                 5.539 
                 4.251 
                 1.676 
                 2.903 
               
               
                 25 
                 3.004 
                 1.866 
                 2.069 
                 1.066 
                 2.388 
                 1.090 
                 1.230 
                 1.742 
               
               
                 28 
                 0.713 
                 1.386 
                 0.231 
                 0.583 
                 0.097 
                 0.610 
                 0.234 
                 0.213 
               
               
                   
               
             
          
           
               
                 Time 
                 Embodiment 7 
                 Embodiment 9 
                 CN101653422 
               
             
          
           
               
                 (day) 
                 0 month 
                 6 month 
                 0 month 
                 6 month 
                 0 month 
                 6 month 
               
               
                   
               
               
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 0.042 
                 2.098 
                 2.661 
                 4.496 
                 4.112 
                 2.919 
                 2.369 
               
               
                 0.125 
                 1.097 
                 1.434 
                 0.89 
                 1.845 
                 2.014 
                 1.628 
               
               
                 0.25 
                 1.15 
                 1.562 
                 3.015 
                 2.968 
                 2.795 
                 1.76 
               
               
                 1 
                 0.727 
                 0.529 
                 5.366 
                 0.925 
                 1.213 
                 1.138 
               
               
                 2 
                 1.037 
                 0.98 
                 2.814 
                 4.382 
                 1.497 
                 0.804 
               
               
                 3 
                 1.146 
                 3.044 
                 3.969 
                 5.621 
                 1.8595 
                 0.62 
               
               
                 5 
                 9.203 
                 7.458 
                 9.786 
                 9.941 
                 7.2195 
                 5.335 
               
               
                 7 
                 8.268 
                 12.361 
                 15.773 
                 14.866 
                 15.9145 
                 5.314 
               
               
                 9 
                 12.278 
                 10.695 
                 13.07 
                 11.902 
                 14.361 
                 8.079 
               
               
                 11 
                 9.566 
                 13.336 
                 9.35 
                 10.356 
                 12.0665 
                 7.719 
               
               
                 14 
                 13.055 
                 11.252 
                 12.866 
                 13.231 
                 16.868 
                 13.095 
               
               
                 16 
                 11.806 
                 11.549 
                 11.551 
                 10.944 
                 11.955 
                 17.679 
               
               
                 18 
                 12.947 
                 13.578 
                 10.32 
                 9.967 
                 12.998 
                 20.781 
               
               
                 21 
                 7.921 
                 5.703 
                 4.711 
                 6.1 
                 6.044 
                 18.068 
               
               
                 23 
                 5.506 
                 5.149 
                 1.38 
                 2.546 
                 3.026 
                 9.215 
               
               
                 25 
                 3.056 
                 2.988 
                 0.934 
                 1.385 
                 1.727 
                 4.123 
               
               
                 28 
                 1.265 
                 1.658 
                 0.063 
                 0.123 
                 1.3495 
                 2.136 
               
               
                   
               
             
          
         
       
     
     Comparative Test 3 Release Results Of Risperidone Microspheres Of The Present Disclosure With Different Drug-Loading Rates In Dog Bodies Compared With Release Results Of Risperidone Microspheres In CN101653422 With Different Drug-Loading Rates In Dog Bodies 
     1) Test Materials 
     Test Drugs: 
     The present disclosure: the risperidone microspheres with drug-loading rates of 13.9%, 23.9%, 33.1%, 40.2% obtained in Embodiments 2, 5, 510 respectively. 
     CN101653422: the risperidone microspheres with drug-loading rates of 45.5%, 40.3%, 35.6% obtained according to Embodiments 7-9 of CN101653422, respectively. 
     Test Animals: 24 healthy beagles dogs, 4 dogs in each group, 12-female-12-male, with a body weight of 9.5-10.5 kg. 
     Test Instruments: the same as those in Test Embodiment 2. 
     2) Method and Results 
     The test method is the same as that in Test Embodiment 2. 
     The test results were shown in Table 2 and  FIG. 9 . 
     The results show that, for the risperidone microspheres in CN101653422, the drug may not be released immediately after mitering into a body when the drug-loading ruse is below 45% i.e., there is a release lag phase. In contrast, for the risperidone microspheres according to an embodiment of the present disclosure, the drug may still be released immediately after entering into a body even when the drug-loading rate of is as low as about 10%, i.e., there is no a release lag phase. 
     
       
         
               
             
               
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 Drug blood concentrations (ng/mL) at different times after the microspheres 
               
               
                 according to an embodiment of the present disclosure and in CN101653422 
               
               
                 were administrated by intramuscular injection on each beagle 
               
             
          
           
               
                   
                 Drug-loading rate 
               
             
          
           
               
                 Time 
                 The present disclosure 
                 CN101653422 
               
             
          
           
               
                 (day) 
                 13.9% 
                 23.9% 
                 33.1% 
                 40.2% 
                 35.6% 
                 40.3% 
                 45.5% 
               
               
                   
               
             
          
           
               
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 0.042 
                 1.011 
                 1.028 
                 1.059 
                 1.428 
                 0.635 
                 0.233 
                 3.023 
               
               
                 0.125 
                 2.365 
                 2.846 
                 2.991 
                 1.367 
                 0.621 
                 0.412 
                 2.566 
               
               
                 0.25 
                 1.652 
                 1.899 
                 1.051 
                 1.423 
                 0.619 
                 0.411 
                 2.651 
               
               
                 1 
                 2.368 
                 2.486 
                 2.628 
                 3.577 
                 0.617 
                 0.405 
                 3.553 
               
               
                 2 
                 2.356 
                 2.786 
                 2.938 
                 2.509 
                 0.539 
                 0.455 
                 4.065 
               
               
                 3 
                 2.669 
                 2.895 
                 3.047 
                 3.416 
                 0.432 
                 0.636 
                 4.322 
               
               
                 5 
                 6.659 
                 7.296 
                 8.104 
                 8.473 
                 0.612 
                 1.323 
                 7.587 
               
               
                 7 
                 11.026 
                 12.018 
                 12.169 
                 12.538 
                 1.321 
                 6.036 
                 14.852 
               
               
                 9 
                 14.011 
                 14.058 
                 15.179 
                 15.548 
                 2.365 
                 7.229 
                 19.286 
               
               
                 11 
                 13.102 
                 13.022 
                 15.469 
                 13.838 
                 5.691 
                 11.292 
                 16.963 
               
               
                 14 
                 13.561 
                 12.804 
                 17.697 
                 15.838 
                 13.665 
                 20.552 
                 16.665 
               
               
                 16 
                 14.667 
                 15.556 
                 17.707 
                 18.076 
                 29.053 
                 30.026 
                 18.337 
               
               
                 18 
                 19.223 
                 18.696 
                 17.808 
                 17.102 
                 30.658 
                 29.199 
                 20.544 
               
               
                 21 
                 14.003 
                 13.085 
                 10.822 
                 10.191 
                 20.511 
                 15.236 
                 12.802 
               
               
                 23 
                 12.325 
                 9.236 
                 8.407 
                 6.776 
                 10.664 
                 11.813 
                 7.801 
               
               
                 25 
                 9.166 
                 8.805 
                 6.957 
                 5.364 
                 6.366 
                 5.221 
                 4.503 
               
               
                 28 
                 6.076 
                 5.016 
                 4.196 
                 4.535 
                 4.112 
                 2.323 
                 2.209