WO2016095592A1 - 一种治疗实体肿瘤的卡莫司汀缓释植入剂及其制备方法 - Google Patents

一种治疗实体肿瘤的卡莫司汀缓释植入剂及其制备方法 Download PDF

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WO2016095592A1
WO2016095592A1 PCT/CN2015/091049 CN2015091049W WO2016095592A1 WO 2016095592 A1 WO2016095592 A1 WO 2016095592A1 CN 2015091049 W CN2015091049 W CN 2015091049W WO 2016095592 A1 WO2016095592 A1 WO 2016095592A1
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carmustine
lactide
implant
release
glycolide
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PCT/CN2015/091049
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English (en)
French (fr)
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孔庆忠
魏明星
苏红清
吴松芝
俞建江
姜俊敏
李兆文
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山东蓝金生物工程有限公司
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/17—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine
    • A61K31/175—Amides, e.g. hydroxamic acids having the group >N—C(O)—N< or >N—C(S)—N<, e.g. urea, thiourea, carmustine having the group, >N—C(O)—N=N— or, e.g. carbonohydrazides, carbazones, semicarbazides, semicarbazones; Thioanalogues thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers

Definitions

  • the invention relates to a carmustine sustained-release implant for treating solid tumors and preparation thereof, and belongs to the technical field of medicine.
  • the commonly used chemotherapy is mainly systemic chemotherapy, which has poor selectivity and large systemic side effects. Due to the histological features of the tumor, it is difficult for chemotherapeutic drugs to obtain an effective drug concentration at the tumor site and maintain sufficient time.
  • chemotherapeutic drugs such as the blood-brain barrier (BBB), blood tumor barrier (BTB) and blood-pancreatic barrier (BPB) and other natural barriers, increased intratumoral interstitial pressure (IFP), tumor vascular reduction and irregular changes, tumor cells Interstitial (ECM) barriers and the like greatly hinder the contact of chemotherapeutic drugs with tumor cells, and irregular small amount of drug contact is also likely to induce drug resistance.
  • BCNU carmustine
  • Gliadel implants have been approved for use in glioma treatment in many countries.
  • Animal experiments have shown that after topical placement, the BCNU concentration at the implant is 1300 times higher than that of intravenous administration (Fleming AB, Saltzman WM. Clin Pharmacokinet 2002, 41: 403-19).
  • the sustained release carrier used in Gliadel implants is polyphenylpropanol (p-CPP: azelaic acid (SA) copolymer, 80:20), and its release characteristics have many defects, such as It is not limited to: unstable release system, unstable release, obvious burst release, poor efficacy, and large side effects. The above defects limit the clinical application of its products.
  • BCNU In Gliadel implants, BCNU has a low drug content (3.85%), a short drug release period (5-7 days), and a very pronounced burst release. The body releases more than 50%-60% within 1-2 days. . Domb (1995) reported that the cumulative release of BCNU on days 3 and 6 after implantation of Gliadel implants in rabbit brain was 60% and >95%, respectively (Domb AJ et al Biomaterials 1995, Vol.16No.141069-1072); Dang (1996) reported that the cumulative release of BCNU on days 1, 2, and 5 after placement of Gliadel implants in the rat brain was 60%, 70%, and 100%, respectively (Dang W et Al Journal of Controlled Release 42 (1996) 83-92); Eric (1997) reported that in saline, 100% of BCNU was completely released within 7 days, and the burst on day 1 was very pronounced (Eric P.
  • Gliadel implants attempted to overcome their deficiencies by various means, such as increasing the drug loading and adjusting the ratio of p-CPP to SA in the carrier (Sipos EP et al Cancer Chemother Pharmacol (1997) 39: 383-389; Engelhard HH Surg Neurol 2000; 53: 458-64; Tamada, J. and Langer R. Proc Natl Acad Sci USA 1993; 90: 552-556), however, with 50:50 CPP: SA copolymer longest
  • the drug release cycle was only extended to 18 days, and the burst release was obvious within 24 hours. The toxicity was also obvious when the dose was large.
  • lactide glycolide copolymer (PLGA) has a long history and is highly regarded for its good biocompatibility and biodegradability (Benny O ,Menon LG,Ariel G,et al.Local delivery of poly lactic-co-glycolic acid microspheres containing imatinib mesylate inhibits intracranial xenograft glioma growth.Clin Cancer Res2009;15:1222-31;Zhang YH,Zhang H,Liu JM,et al.Temozolomide/PLGA microparticles: a new protocol for treatment of glioma in rats.
  • BCNU sustained-release implants with a molecular weight of 8,000 g/mol PLGA (50:50) have been studied (Zhu T, Shen Y, Tang Q, Chen L, Gao H, Zhu J. BCNU/PLGA microspheres). : a promising strategy for the treatment of gliomas in mice. Chin J Cancer Res. 2014 Feb; 26 (1): 81-8), however, there are many problems in many aspects such as drug loading and carrier viscosity selection and preparation methods. .
  • sustained release excipients are one of the key factors in determining the properties of the implant.
  • the sustained release excipients have been described in detail in the "Remote Release Excipients" (page 123, published by Sichuan Science and Technology Press in 1993, edited by Luo Mingsheng and Gao Tianhui).
  • a number of sustained release excipients are also listed in the Chinese patents (Application No. 96115937.5; 91109723.6; 9710703.3; 01803562.0) and the US invention patent (Patent No. 5,651,986).
  • sustained release excipient used in the sustained-release implant of carmustine is polyphenylpropanol (p-CPP: sebacic acid (SA) copolymer).
  • p-CPP polyphenylpropanol
  • SA sebacic acid copolymer
  • sustained-release implants made of polyphenylpropanol have many defects, such as but not limited to: unstable, need to be stored below -20 °C, short release period (about 5 days), and obvious burst release ( More than 60% in 2 days).
  • lactide-glycolide copolymer is not only biocompatible, but also completely degraded and absorbed after being placed in the body.
  • PLGA lactide-glycolide copolymer
  • sustained release implants not all of the sustained release excipients used are suitable for the release of a particular drug.
  • Different slow-release excipients, even different molecular weights of the same slow-release excipients and different combinations of drugs, or different ratios of the same drug and different preparation processes affect the drug release characteristics, the drug loading amount and the choice of production process It affects the metabolism of drugs in the body and has obvious relationship with its adverse reactions and therapeutic effects. It requires a lot of creative labor to achieve.
  • a sustained release implant composed of a lactide-glycolide copolymer and carmustine.
  • suitable excipients, drugs, ratios and production process selection is also a prerequisite for the successful development and application of drugs.
  • the present invention is directed to the deficiencies of the prior art, and provides a carmustine (BCNU) sustained release implant comprising carmustine and a sustained release adjuvant, wherein the sustained release adjuvant is a lactide-glycolide copolymer .
  • BCNU carmustine
  • a carmustine (BCNU) sustained release implant comprising carmustine and a sustained release adjuvant, said sustained release adjuvant being a lactide-glycolide copolymer; said carmustine in card
  • the weight percentage of the myestine sustained release implant is 7.5% to 30%; the weight average molecular weight of the lactide-glycolide copolymer is 25000 to 80,000, and the mass ratio of lactide to glycolide is (25-75): (75 ⁇ 25), viscosity of 0.2 ⁇ 1.3dl / g;
  • the carmustine is present in an amount of from 8% to 25% by weight. In a more preferred embodiment, the carmustine is present in an amount of from 10 to 20% by weight. In a most preferred embodiment, the carmustine is 10%, 15% or 20% by weight.
  • the lactide-glycolide copolymer may be formed by a ring-opening copolymer of lactide and glycolide, or may be obtained by polycondensation of lactic acid and glycolic acid, wherein lactide and glycolide are used.
  • a ring-opening copolymer is preferred.
  • the lactide-glycolide copolymer copolymer has a weight average molecular weight of preferably 30,000 to 60,000, most preferably 35,000 to 55,000, and a viscosity of 0.25 to 0.8 dl/g, preferably 0.3 to 0.6 dl/g is most preferable; the mass ratio of lactide (GA) to glycolide (LA) is (50 to 75): (25 to 50), preferably 50:50.
  • the invention finds through a lot of creative labor that the lactide-glycolide copolymer is a slow release carrier which is superior to polyphenylene benzoate, and the sustained release implant agent which is a carrier of lactide-glycolide copolymer is used as a carrier.
  • the composition ratio of carmustine and sustained-release excipients and the change of process are one of the main factors affecting drug release.
  • the sustained release property of a sustained release implant prepared by using a lactide-glycolide copolymer as a sustained release carrier is related to its molecular weight, and the weight average molecular weight may be 25,000 to 80,000, of which 30,000 to 60,000 is Preferably, it is most preferably from 35,000 to 55,000; the viscosity ranges from 0.2 to 1.3 dl/g, preferably from 0.25 to 0.8 dl/g, most preferably from 0.3 to 0.6 dl/g; and lactide (GA) and B.
  • the mass ratio of lactide (LA) is selected from (25 to 75): (75 to 25), preferably (50 to 75): (25 to 50), and most preferably 50:50.
  • the cumulative release in the first 2 days is not more than 20% of the total drug loading, preferably 5% to 12%.
  • the cumulative release in the first 12 days is greater than 30% of the total drug loading but less than 70% of the total drug loading. Preferably, it is between 40% and 60%.
  • the sustained-release implant prepared by the invention can release the drug slowly in the tumor site after being placed in the body, not only can maintain sufficient concentration and time locally, but also the effective drug concentration can reach 2 to 4 cm in the tissue, so Can better play the clinical therapeutic effect of carmustine, because most brain tumors recur within 2 cm of the primary site after surgery.
  • the invention provides the use of the above-described carmustine extended release implant in the treatment of cancer.
  • the cancer can be an intracranial tumor and an extracranial solid tumor.
  • Intracranial tumors are preferably gliomas and brain metastases.
  • Preferred for brain metastases are metastases derived from the lung, breast, kidney, skin, melanoma, digestive tract, and blood system.
  • the cancer is selected from the group consisting of a solid tumor that is primary to the extracranial and a variety of metastatic cancer.
  • the extracranial solid tumor is selected from the group consisting of liver cancer, lung cancer, esophageal cancer, gastric cancer, breast cancer, pancreatic cancer, thyroid cancer, nasopharyngeal cancer, oral tumor, ENT tumor, pharyngeal tumor, ocular tumor, ovarian cancer.
  • the solid tumor is selected from the group consisting of solid tumors such as pancreatic cancer, lung cancer, liver cancer, breast cancer, brain tumor, esophageal cancer, kidney cancer, and colorectal cancer.
  • solid tumors such as pancreatic cancer, lung cancer, liver cancer, breast cancer, brain tumor, esophageal cancer, kidney cancer, and colorectal cancer.
  • lung cancers non-small cell lung cancer (NSCLC) is preferred; in various extracranial metastatic cancer metastases, liver metastases are preferred, but are convenient, and the primary tumors of the lung, stomach, colon, and pancreas are transferred.
  • NSCLC non-small cell lung cancer
  • the amount of the sustained release implant of the present invention depends on many factors such as, but not limited to, tumor volume, tumor volume, patient weight, mode of administration, progression of the disease, drug content, and therapeutic response. However, the principle is that it can reduce the repair ability of tumor cells and increase the effect of chemotherapy without significantly increasing the toxicity of the drug.
  • the dose of BCNU in the sustained release implant of the present invention is from 1 to 1000 mg/time/person. Depending on the tumor volume, tumor volume, tumor type, and tumor site, each time it can be 1 to 19 mg, 20 to 200, 201 to 800, or 801 to 1000 mg.
  • Such as ocular tumors and brain stem tumors can choose low doses, such as 1 ⁇ 100 mg, intracranial tumors and brain metastases can choose 20 ⁇ 800 mg, breast and digestive tract tumors can choose 40 ⁇ 1000 mg.
  • the drug can be applied in a single application or repeated every 3 to 4 weeks.
  • sustained release implant of the invention in the manufacture of a medicament for the treatment of cancer.
  • treating includes inhibiting, curing, and alleviating cancer or its symptoms and preventing or delaying the metastasis of the primary cancer.
  • the invention can be made into various shapes or dosage forms such as, but not limited to, granules, tablets, spheres, lumps, needles, rods and patterns; can be administered by various routes, such as subcutaneous, intramuscular, intradermal , intracavitary, intratumoral, tumor, etc.
  • routes such as subcutaneous, intramuscular, intradermal , intracavitary, intratumoral, tumor, etc.
  • the route of administration depends on a variety of factors, such as the location of the tumor, whether surgery or metastasis, tumor size, tumor size, tumor type, patient age, physical condition, fertility status, and requirements.
  • the tumor For effective drug concentration at the site of the tumor, it can be selectively intraluminal, intraperitoneal (ip) or thoracic and intraspinal, or placed in the organ, such as the intestine, the bladder, the intrauterine, In the spinal canal, intravaginal, intragastric and esophageal.
  • local administration such as intratumoral and peritumoral injection, is preferred in the form of intratumoral, peritumoral or tumor cavity placement or injection, such as slow release agent, implant, and slow release. Release implants and sustained release injections.
  • the dosage can be selected according to the age of the patient, the characteristics of the disease, the tumor volume, the size of the tumor cavity, and the type of tumor. It can be administered in a single dose, and can be administered multiple times; it can be placed in the tumor cavity after surgery, and can be directly introduced into the tumor.
  • the single administration dose is preferably from 0.1 to 1000 mg, more preferably from 10 to 1000 mg, even more preferably from 20 to 800, and most preferably from 100 to 400 mg.
  • the sustained release implant is used in an amount of from 1 to 200 tablets, preferably from 3 to 50 tablets, and most preferably from 6 to 20 tablets.
  • the sustained release implant is used in an amount of from 1 to 1000, preferably from 5 to 200, and most preferably from 10 to 100.
  • the dose for one administration is preferably from 0.1 to 1000 mg, more preferably from 10 to 1000 mg, more preferably from 20 to 800, most preferably from 100 to 400 mg, and may be administered repeatedly, with a repetition period of 7 days or more. It is preferably 14 days or more.
  • a single dose can be administered.
  • the dose may be from 0.1 to 50 mg of BCNU sustained release stick, preferably Contains 2.5 to 25 mg of BCNU and is directly inserted into the tumor.
  • a larger dose of the implant can be administered again after the first dose. For example, a 550 mg dose is administered for the first time and repeated for 2 to 4 weeks.
  • the above medicament is administered by implantation, preferably, the medicament is administered topically.
  • This implant is applied topically to cancer treatment.
  • Topical administration includes the application of the drug to or within an organ and/or cancer cell or tumor, preferably within a tumor. Topical administration also includes surrounding the cancerous tumor with a drug or applying the drug to the surface of a cancerous tumor. In one embodiment, the drug is introduced directly into the cancerous area.
  • the implant is administered intratumorally, intracancer or post-cancer intratumoral, preferably via intracancer puncture.
  • the drug is administered by intratumoral implantation, peritumoral implantation, or intratumoral implantation after cancer surgery, preferably via intrathecal implantation.
  • the sustained-release implant of the invention can be directly applied to the cavity formed by the whole or partial resection of the primary or metastatic solid tumor, the tumor surrounding or the tumor body, the residual part of the suspected tumor cell after surgery, or directly placed or Injection into or near a primary or metastatic solid tumor that cannot be surgically removed can be used alone for the treatment of tumors or to prevent postoperative recurrence, or in combination with radiotherapy and/or chemotherapy.
  • the anti-cancer sustained-release implant of the invention can be combined with conventional chemotherapy, immunotherapy, hyperthermia therapy, photochemotherapy, electrotherapy, biological therapy, hormone therapy, magnetic therapy, ultrasound therapy, radiotherapy and gene therapy, etc., so that the effect is enhanced . Therefore, it can be combined with the above non-surgical treatment at the same time as the local slow release, thereby further enhancing the anticancer effect.
  • the anticancer sustained release implant of the present invention can be applied simultaneously with non-surgical therapy, or can be applied within a few days before the implementation of non-surgical therapy, with the aim of enhancing tumor sensitivity as much as possible. Therefore, it provides a more effective new method for eradicating all kinds of human and animal primary and metastatic solid tumors, with very high clinical application value and significant economic and social benefits.
  • the invention also provides a preparation method of carmustine (BCNU) sustained-release implant, comprising the following steps:
  • the mass ratio of the lactide (GA) to glycolide (LA) is selected from (25 to 75): (75 to 25);
  • the weight ratio of carmustine to lactide-glycolide copolymer in the mixture of lactide-glycolide copolymer and carmustine is (0.75 ⁇ 3): (7 ⁇ 9.25);
  • the weight percentage of carmustine in the sustained release implant is 7.5%-30%;
  • the sterile filtrate is aseptically dried to obtain a micropowder
  • a carmustine (BCNU) sustained release implant is prepared.
  • the organic solvent is selected from a combination of one or more of methyl chloride, chloroform, ethyl acetate or acetone; further preferably dichloromethane; the amount of the organic solvent is not strictly limited, It is suitable to dissolve completely.
  • the lactide-glycolide copolymer has a viscosity of 0.2 to 1.3 dl/g, preferably 0.25 to 0.8 dl/g, and is selected from the group consisting of 0.25 dl/g and 0.3. Dl/g, 0.35 dl/g, 0.4 dl/g, 0.45 dl/g, 0.50 dl / g, 0.55 dl / g, 0.6 dl / g or 0.65 dl / g, and more preferably 0.3 ⁇ 0.6 dl / g;
  • the mass ratio of lactide (GA) to glycolide (LA) is selected from (50 to 75): (25 to 50), and most preferably 50:50;
  • the weight average molecular weight of the lactide (GA) and glycolide (LA) copolymer is from 25,000 to 85,000, preferably from 30,000 to 65,000, and most preferably from 35,000 to 60,000.
  • the weight ratio of the carramustine to the lactide-glycolide copolymer in the lactide-glycolide copolymer and the carmustine mixture is (1 ⁇ ). 2): (8-9); that is, the weight percentage of carmustine in the sustained release implant is 10-20%.
  • the mixture of the lactide-glycolide copolymer and the carmustine mixture comprises a 0.45 ⁇ m microporous membrane filter for initial filtration and a 0.22 ⁇ m microporous membrane filter. Two fine filtrations. The main purpose of primary filtration is to remove impurities and residues, and fine filtration is used for sterilization purposes.
  • the sterile filtrate is prepared by aseptic drying to prepare a fine powder, and the method is applied under sterile conditions, and the method is selected from the group consisting of: an emulsification method, a spray drying method, a solvent evaporation method, Organic extraction or frozen powder method; preferably, frozen powder method or spray drying method; optimal, frozen powder method.
  • the mixture is dried prior to freeze comminution, the method used being selected from natural drying, heating and/or blast drying, freezing and/or vacuum drying, preferably by heated blast drying.
  • the BCNU-containing copolymer powder is dried to a powder, preferably having a particle diameter of 10 to 20 ⁇ m, more preferably 30 to 150 ⁇ m, and most preferably 40 to 100 ⁇ m, and a preferred liquid in the freeze-pulverization process.
  • Nitrogen is a refrigerant.
  • the powder formation after drying can be performed at room temperature, and the prepared implant can be in various shapes or dosage forms, such as, but not limited to, particle-like, sheet-like, Spherical, massive, needle-like, rod-like and shaped, with implants or implants being preferred.
  • the weight percentage of carmustine in the implanted tablet is 10% to 30%, preferably 10%, 15%, 20%, 25% or 30%; the implant piece may have a diameter of 0.5 to 2.0 cm, selected from 1.35 to 1.5.
  • the centimeter has a thickness of 0.8 to 3.0 mm, preferably 0.8 to 2.0 mm, and most preferably 0.8 to 1.2 mm.
  • the weight percentage of carmustine in the implanted rod is 10% to 30%, preferably 10%, 15%, 20%, 25% or 30%, and the diameter of the implant rod is selected from 0.8 to 3.8 mm and the length is 3 to 8.0 mm.
  • Preferred according to the invention also includes the step of aseptic packaging.
  • the packaging material is selected from medicinal packaging materials, preferably in an aluminum plastic or glass container.
  • the invention also provides a preparation method of another carmustine (BCNU) sustained-release implant, comprising the following steps:
  • the mass ratio of the lactide (GA) to glycolide (LA) is selected from (25 to 75): (75 to 25);
  • the weight ratio of the carmustine to the lactide-glycolide copolymer in the mixture of the lactide-glycolide copolymer and the carmustine is (0.75 to 4): (6 to 9.25);
  • the weight percentage of carmustine in the sustained release implant is 7.5% to 40%;
  • the organic solvent is selected from a combination of one or more of methyl chloride, chloroform, ethyl acetate or acetone; further preferably dichloromethane; the amount of the organic solvent is not strictly limited To dissolve fully.
  • the lactide-glycolide copolymer has a viscosity of 0.2 to 1.3 dl/g, preferably 0.25 to 0.8 dl/g, and is selected from 0.25 dl/g. , 0.3 dl / g, 0.35 dl / g, 0.4 dl / g, 0.45 dl / g, 0.50 dl / g, 0.55 dl / g, 0.6 dl / g or 0.65 dl / g, and again from 0.3 to 0.6 dl / g Most preferred;
  • the mass ratio of lactide (GA) to glycolide (LA) is (50 to 75): (25 to 50) is preferred, and 50:50 is Most preferably, the weight average molecular weight of the lactide (GA) and glycolide (LA) copolymer is from 25,000 to 85,000, preferably from 30,000 to 65,000, and most preferably from 35,000 to 60,000.
  • the weight ratio of the carramustine to the lactide-glycolide copolymer in the mixture of the lactide-glycolide copolymer and the carmustine is ( 1 to 3): (7 to 9); that is, the weight percentage of carmustine in the sustained release implant is 10 to 30%.
  • the mixture of the lactide-glycolide copolymer and the carmustine mixture is filtered by a 0.45 ⁇ m microporous membrane filter and 0.22 ⁇ m microporous membrane.
  • the filter is finely filtered twice.
  • the main purpose of primary filtration is to remove impurities and residues, and fine filtration is used for sterilization purposes.
  • the sterile filtrate is subjected to aseptic drying to prepare a micropowder, and the method is applied under sterile conditions, and the method is selected from the group consisting of: an emulsification method, a spray drying method, and a solvent evaporation.
  • Method organic extraction or frozen powder method; preferably, frozen powder method or spray drying method; optimal, frozen powder method.
  • the freeze pulverization method is cooled by means of liquid nitrogen, and the organic solvent should be removed before the freeze pulverization, and vacuum drying or low-temperature drying method or heating and drying can be selected.
  • the drying method is selected from the group consisting of: an emulsification method, a spray drying method, a solvent evaporation method, an organic extraction method or a frozen powder method; preferably, a frozen powder method or a spray drying method; , frozen powder method.
  • the freeze pulverization method is cooled by means of liquid nitrogen, and the organic solvent should be removed before the freeze pulverization, and vacuum drying or low-temperature drying method or heating and drying can be selected.
  • the prepared implant in the step (5) or 4, may be in various shapes or dosage forms, such as, but not limited to, particle-like, sheet-like, spherical, massive, needle-like, Rods and patterns, with implants or implants being preferred.
  • the weight percentage of carmustine in the implant is 10%, 15%, 20%, 25% or 30%; the implant may have a diameter of 0.5 to 2.0 cm, is selected from 1.35 to 1.5 cm, and has a thickness of 0.8 to 3.0.
  • the millimeter is preferably from 0.8 to 2.0 mm, most preferably from 0.8 to 1.2 mm.
  • the weight percentage of carmustine in the implanted rod is 10%, 15%, 20%, 25% or 30%, and the implant rod diameter is selected from 0.8 to 3.8 mm and the length is 3 to 8.0 mm.
  • the melting temperature is from 30 to 60 ° C, preferably from 40 to 60 ° C, for a period of from 1 to 8 minutes, preferably from 3 to 5 minutes.
  • the implants can be made in a variety of shapes or dosage forms such as, but not limited to, pellets, sheets, spheres, blocks, needles, rods, and patterns, with implants or implants being preferred.
  • the weight percentage of carmustine in the implant is 7.5% to 40%, preferably 10%, 15%, 20%, 25% or 30%; the implant may have a diameter of 0.5 to 2.0 cm, selected from 1.35 to 1.5.
  • the centimeter has a thickness of 0.8 to 3.0 mm, preferably 0.8 to 2.0 mm, and most preferably 0.8 to 1.2 mm.
  • the weight percentage of carmustine in the implanted rod is 7.5% to 40%, preferably 10%, 15%, 20%, 25% or 30%, and the implant rod diameter is selected from 0.8 to 3.8 mm and the length is 3 to 8.0 mm.
  • Preferred according to the invention also includes the step of aseptic packaging.
  • the inner packaging material is selected from aseptic packaging materials, preferably aluminum plastic or glass.
  • the above preparation method achieves the effect of on-line sterilization by filtering sterilization, aseptic drying, and aseptic molding, thereby achieving the sterility requirement for in vivo application.
  • the invention provides the use of a carmustine extended release implant for the preparation of a medicament for the treatment of cancer.
  • the carmustine (BCNU) sustained release implant which uses a lactide-glycolide copolymer as a sustained release carrier, and has a carmustine content, lactide-glycolide
  • the content of copolymer, the ratio of lactide to glycolide and other conditions were creatively selected, and the sustained release period was as long as 3-4 weeks, the drug release was stable, and there was no burst release (the release was not more than 20% on the next day).
  • Carmustine (BCNU) sustained-release implants with low side effects, stable formulation, non-fragile, biocompatible, and fully degradable absorption.
  • the sustained release implant of carmustine (BCNU) can achieve a cumulative release amount of 5%-12% of the total drug loading in the first 2 days, and the cumulative release amount in the first 12 days is greater than the total amount. 40% of the drug loading is less than 60% of the total drug loading.
  • the carmustine (BCNU) sustained-release implant prepared by the method of the invention not only has obvious clinical application effect, but also has mild systemic toxicity, can meet the requirements of drug production, and is more favorable for industrial development; Intraoperative treatment of solid tumors and metastases such as tumors can also be used for the treatment of postoperative or inoperable tumors and in combination with other non-surgical therapies.
  • BCNU carmustine
  • Example 1 is a drug release curve of the implant No. 1 A and the implant No. 1 B in Example 1, which can reflect the release profile of the product without burst release and compared with the burst release product;
  • Figure 2 is a drug release profile of implant A in Example 17;
  • Figure 3 is a drug release profile of the BCNU polyphenylene probiotic implant of Example 17;
  • the dried solid composition is freeze-pulverized, and after the organic residue is passed, the powder is placed in a tableting machine to form a sustained-release tablet at room temperature, and the cobalt 60-ray is sterilized after dispensing, and the obtained product is an "implant".
  • No. 1 a sustained-release implant with a diameter of 1.4 cm and a thickness of 1.0 mm, of which
  • Implant No. 1 A is: 10% carmustine, excipient is PLGA (the ratio of lactide to glycolide is 50:50, viscosity is 0.30 dl / g), implant No. 1 B is: 10 % Carmustine, excipient is polyphenylene (p-CPP: azelaic acid (SA) copolymer, 80:20).
  • PLGA the ratio of lactide to glycolide is 50:50, viscosity is 0.30 dl / g
  • implant No. 1 B is: 10 % Carmustine
  • excipient is polyphenylene (p-CPP: azelaic acid (SA) copolymer, 80:20).
  • the excipient is a carmustine implant made of polyphenylpropanoid
  • the drug release cycle is too short, less than 12 days, of which the cumulative release in the first 2 days is 68% of the total drug loading;
  • the carmustine implant made with the lactide-glycolide copolymer released smoothly, with a cumulative release of 14% in the first 12 days.
  • the cumulative release in the first 12 days was 34% of the total drug loading and the release time was approximately 4 weeks.
  • the sustained release implant tablets were prepared according to the preparation process of Example 1, except that the PLGA used was a copolymer of lactide and glycolide having a mass ratio of 50:50, and the weight average molecular weights were 15K, 45K and 75K, respectively.
  • Implant 2 (A, B, C), 1.35-1.45 cm in diameter and 0.8-1.2 mm in thickness, each containing 10% of carmustine.
  • the release results of implant 2 in pure water are shown in Table 2:
  • the release characteristics of the carmustine implant prepared by the lactide-glycolide copolymer are related to the molecular weight of the carrier, and the weight average molecular weight is 15K.
  • the agent (implant 2 A) was released too fast, 76.98% was released on the 12th day, and the release was completed in less than 20 days;
  • the implant with the weight average molecular weight of 75K (implant 2 C) was unstable, early Too fast, late is too slow, the cycle is too long (nearly 50 days);
  • the implant with a weight average molecular weight of 45K is ideal, and the drug release is stable with a cycle of about 4 weeks.
  • the sustained-release implant tablets were prepared according to the preparation process of Example 1, except that the PLGA was a lactide-glycolide copolymer having a mass ratio of 50:50, and the weight average molecular weights were 25K, 40K and 65K, respectively.
  • the diameter is 1.35 - 1.45 cm, and the thickness is 0.8 - 1.2 mm, both containing 10% of carmustine. See the release time in pure water
  • the release characteristics of the carmustine implant prepared by the PLGA being a lactide-glycolide copolymer having a mass ratio of 50:50 are related to the molecular weight of the carrier, along with the weight average molecular weight of the PLGA (or The increase in viscosity), the release rate of BCNU becomes slower.
  • the PLGA weight average molecular weight ranges from 25K to 65K.
  • Example 4 comparing the effects of different drug loadings on BCNU release
  • the sustained-release implant tablet was prepared according to the preparation process of Example 1.
  • the PLGA used was a lactide-glycolide copolymer having a mass ratio of 50:50, and the weight average molecular weight was 30K-35K, and the obtained product was an implant 4. No., diameter 1.4 cm, thickness 1.0 mm, containing 7.55% of BCNU (implant 4 A), 10% (implant 4 B), 15% (implant 4 C), 20% (implantant No. 4 D), 30% (implantant No. 4 E) and 40% (implantant No. 4 F). Its release time in pure water is shown in Table 4:
  • the release characteristics of the carmustine implant prepared by the lactide-glycolide copolymer are also related to the drug loading.
  • lactide and glycolide copolymers (50:50) with a weight average molecular weight of 30K-35K the drug release becomes faster as the drug loading increases, and the BCNU content is 7.5% (implant 4 A) ), 10% (implant 4 B), 15% (implant 4 C), 20% (implant 47 D) and 30% (implant 4 E) implants Ideally, the release on the second day is between 5% and 18%, and the release on the 12th day is between 40% and 60%. When the increase is 40%, such as the implant No. 4 F, the release begins. phenomenon.
  • Example 5 comparing the effects of different shapes on the release of implants
  • Example 2 Prepared according to the preparation process of Example 1, 9000mg PLGA (the ratio of lactide to glycolide is 50:50, weight average molecular weight is 35-45K) dissolved in dichloromethane, mixed and added, then 1000mg of carmustine was added. Shake it again, pour it into the tray and heat it to remove the organic solvent. The dried solid composition is freeze-pulverized, and after the organic residue is passed, the powder is separately placed in a tableting machine and an extruder, and a sustained-release tablet and a slow-release bar are prepared at room temperature, and the cobalt 60-ray sterilization is carried out after the separation. The obtained product was implant No.
  • Example 6 Comparison of the effect of different shapes of the implant on release.
  • the sustained release implant was prepared according to the preparation process of Example 1, except that the mass ratio of lactide to glycolide in the copolymer was 75:25, and the weight average molecular weight was 25K.
  • the obtained product was implant No. 6 A (sustained release implanted piece, diameter 14 mm, thickness 1.0 mm), implant No. 6 B (sustained release implant rod, diameter 1.0 mm, length 3.0 mm), planting Injectant No. 6 C (slow release implant rod, diameter 1.0 mm, length 5.5 mm), implant No. 6 D (sustained release implant rod, diameter 3.0 mm, length 5.5 mm).
  • Implant 6 C 9.0 47.8 90.4 Implant 6 D 8.2 50.6 92.6 Implant 6 E 9.4 54.3 92.2
  • the sustained-release implanted rods were prepared according to the same method steps of the present embodiment, and the drug loading amounts were 7.5%, 10%, 15%, 20%, 30%, and 40%, respectively.
  • the release test again showed that the drug release was related to the drug loading amount. The higher the release, the faster, but in the case of the same drug loading, the release characteristics are not significantly related to the shape of the implant.
  • the results of animal experiments showed that the 50:50 lactide and glycolide copolymer had a weight average molecular weight of 30K-35K, and the drug loading range was preferably 10%, 15%, 20%, 30%.
  • mice 2 ⁇ 10 5 brain tumor cells were subcutaneously injected into the ribs of mice (25 g), and after 7 days of tumor growth, they were divided into the following groups, 7 groups (10 per group, see Table 9). 10.0mm). The first group was the control group, the second group was the blank control group, and the third group was the treatment group.
  • Each animal was given an implant rod (prepared according to the preparation method of Example 7) (3.0 mm x 5.5 mm) and implanted by intratumoral puncture.
  • the BCNU content of the implanted rods was 5%, 10%, 20%, and 30%, respectively (equivalent to 0.05, 0.10, 0.20, 0.30, and 0.40 mg BCNU/kg, respectively) (see Table 9 for specific doses).
  • Tumor volume was measured on the 15th day after treatment, and the therapeutic effects of each group were compared (see Table 9).
  • the inhibitory effect of the implant on brain tumor growth was significantly dose-dependent with the drug loading of the implant.
  • 5% implants have no significant inhibitory effect on tumors (P>0.05), and can significantly inhibit tumor growth when the dose is increased to 10% or more.
  • the preferred drug loading is from 10% to 30%.
  • mice of the same sex and similar body weight were divided into the following three groups, and different numbers of brain tumor cells were injected subcutaneously into the ribs of the mice, and the first group was subcutaneously injected with 2 ⁇ 10 5 tumor cells, group 2 5 x 10 5 tumor cells were injected subcutaneously, and the third group was subcutaneously injected with 2 x 10 6 tumor cells.
  • Examples 9 and 10 are animal brain tumors
  • the invention is not limited to animal brain tumors such as, but not limited to, other solid tumors of humans and animals.
  • animal brain tumors such as, but not limited to, other solid tumors of humans and animals.
  • Lung cancer tumor cells (10 5 ) were inoculated subcutaneously into the right axilla of the mice, and the animals were randomly divided into 5 groups when the tumors grew to about 0.8-1.5 cm. Sterilize the surface of the tumor with 70% alcohol, select 1cm from the lower edge of the tumor, cut a 1mm long incision, implant the carmustine implant into the tumor tissue with a puncture needle, measure the tumor size every 3 days after treatment, plant The animals were sacrificed 21 days after the embedding, and the tumors were completely stripped and weighed. The tumor inhibition rate was calculated as %, and DAS.ver1.0 pharmacology software was used for statistical processing.
  • the anti-tumor effect of carmustine extended-release implant on lung cancer in mice was examined according to the above methods and procedures.
  • the implant adjuvant used was PLGA (molecular weight 30,000-35000, lactide and glycolide in a mass ratio of 50:50), and the implant was prepared according to the preparation method of Example 7. The results of this experiment are shown in Table 11.
  • Example 12 Antitumor effect of intramuscular implantation of carmustine sustained-release implant on mouse breast cancer
  • the anti-tumor effect of the sustained-release implant of carmustine on mouse breast cancer was examined according to the method and procedure described in Example 11.
  • the implants used were 5%, 10%, 20% and 30%, and the plants used were used.
  • the adjuvant was PLGA (molecular weight 25000-35000, mass ratio of lactide to glycolide 50:50).
  • the experimental results are shown in Table 12.
  • Example 13 Inhibition of tumor esophageal cancer by implantation of carmustine sustained-release implant in tumor
  • Example 14 Inhibition of tumor pancreatic cancer by implantation of carmustine sustained-release implant in tumor
  • the anti-tumor effect of the sustained-release implant of carmustine on pancreatic cancer in mice was examined according to the method and procedure described in Example 11.
  • the implant adjuvant used was PLGA (molecular weight 30000-35000, lactide and B-crossing).
  • the mass ratio of the ester is 50:50).
  • the content of carmustine in the sustained release implant is 5%, 10%, 20% and 30%.
  • the experimental results are shown in Table 14.
  • the anti-tumor effect of carmustine extended-release implants on rectal cancer in mice was examined according to the method and procedure described in Example 11.
  • the implant adjuvant used was PLGA (molecular weight 40000-45000, lactide and B-crossing).
  • the mass ratio of the ester is 50:50).
  • the content of carmustine in the sustained release implant is 5%, 10%, 15% and 25%.
  • the experimental results are shown in Table 15.
  • the anti-tumor effect of carmustine extended-release implant on liver cancer in mice was examined according to the method and procedure described in Example 11.
  • the implant adjuvant used was PLGA (molecular weight 40000-45000, lactide and glycolide).
  • the blend ratio is 50:50).
  • the content of carmustine in the sustained release implant is 5%, 10%, 20% and 30%.
  • the experimental results are shown in Table 16.
  • the slow-release implant containing 10% carmustine was prepared according to the preparation process of Example 7 using carmartestatin as raw material and PLGA (50:50) with different molecular weights as carriers.
  • the agent A (the carrier is a lactide-glycolide copolymer having a molecular weight of 65,000) and the sustained release implant B (the carrier is a lactide-glycolide copolymer having a molecular weight of 80,000).
  • implant B was too slow and has a sudden release of drug in the brain of rabbits, with a cumulative release of 31.2% ⁇ 4.0% for 3 days, showing significant burst release, and 21 days of cumulative release. At 80%, the cycle is significantly extended. This shows that PLGA The molecular weight is preferably less than or equal to 65,000.
  • the non-labeled carmustine was implanted into the brain of rabbits with BCNU polyphenylpropanoid implant.
  • the release amount of the sustained-release preparation was measured on days 3, 7, 14, 21 and 31 after administration.
  • the release amount was 72.4% ⁇ 5.0%, 98.9% ⁇ 1.3%, 100.0% ⁇ 0.0%, 100.0% ⁇ 0.0% and 100.0% ⁇ 0.0%, respectively.
  • Example 18 Effect of different carriers and different molecular weight PLGA on pharmacokinetics of sustained release implants
  • the PLGA implant takes the product of Example 7.
  • 14 C-calostine sustained release preparation 14 C-cartostin was prepared according to the method of Example 7;
  • Scintillation fluid Permafluor E+ liquid flash product manufactured by PerkinElmer.
  • Liquid scintillation counter Beckman LS6500 liquid scintillation
  • the rabbit brain was implanted with 14 C-cartosine sustained-release preparation, one capsule each, with a radiation dose of 185 kBq per capsule, and a chemical dose of 0.3 mg per carcinol.
  • the brain, plasma, heart, liver, spleen, lung, kidney and spinal cord tissues of the big white rabbits were longitudinally sectioned with a cryostat, each piece was 0.04 mm thick, and 10 pieces were collected as a sample, weighed, burned, and measured for radioactivity.
  • 2 specimens were collected from each animal for autoradiography, and 0.3 g of other tissues were burned to determine the radioactivity concentration.
  • Two animals killed on the 21st day collected feces and urine daily, measured radioactivity, and calculated cumulative excretion.
  • the release rate of sustained-release preparations of carnostatin was injected into the brain.
  • Each rabbit was implanted with one dose of sustained release of carmustine, and each group of animals was given 3 days, 7 days, and 14 days after administration. 21 days and 33 days of live killing, remove the residual drug particles in the brain, -20 ° C
  • the remaining amount of carmustine was determined by HPLC.
  • the in vitro release rate test of the carmustine implant was determined by HPLC method.
  • the sample containing 14 C-cartostin is placed in an oxidizing furnace, and under the action of oxygen and a combustion improver, it is completely converted into carbon dioxide and water. After absorption by the absorbent, it is mixed with the scintillation liquid to form a colorless and transparent solution.
  • the liquid scintillation counter measures radioactivity.
  • the oxidative combustion furnace has a radioactivity recovery rate of more than 97% for 3 H and 14 C, and a radioactive residue of less than 0.08%.
  • the counting efficiency of the 14 C was measured by a liquid scintillation meter to be 70%.
  • a certain number of cancer cells were subcutaneously injected into the ribs of the mice, and after 7 days of tumor growth, they were randomly divided into corresponding treatments.
  • the dose of the implant is calculated in kilograms of body weight (mg/kg) and administered once. Tumor volume was measured every two days after implantation and changes in body weight were recorded, and the therapeutic effects of each group were compared. Data processing and charting with Excel software.
  • the 14 C-labeled carmustine was used as a raw material, and PLGA (50:50) and polyphenylpropanol with different molecular weights were used as carriers to prepare a slow-release plant containing 10% carmustine according to the preparation process of Example 7.
  • the sustained release implant of 10% carmustine contained 0.84 mg of carmustine per dose and 80.7 kBq of radiation dose per capsule.
  • the effective concentration radius was 1.6 cm and maintained for more than 28 days.
  • the peak concentration was 2-3 weeks, and the drug concentration in the brain was maintained.
  • the maximum diffusion distance of more than 10 ⁇ g/g is 19mm, and the peak concentration is 7907.5 ⁇ 9112.9 ⁇ g ⁇ g -1 , which is more than 300 times of the concentration of brain tissue during systemic administration, which can fully meet the requirements of inhibiting tumor cells in vivo;
  • the drug concentration was the highest at 3 days after the administration of the agent C, and then gradually decreased.
  • the diffusion distance of the drug concentration in the brain was more than 5 ⁇ g ⁇ g -1 and the peak concentration was 557.5 ⁇ g ⁇ g -1 .
  • Sterile molding aseptic molding was carried out in a molten state at a melting temperature of 70 ° C for 1 minute.
  • the resulting sustained release implant has a diameter of 1.4 cm and a thickness of 1.0 mm.
  • the obtained carmustine (BCNU) sustained release implant was tested to meet the sterility requirements for postoperative application in the affected area.

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Abstract

一种治疗实体肿瘤的卡莫司汀缓释植入剂，该缓释植入剂包括缓释辅料及抗癌有效成分卡莫司汀，缓释辅料为丙交酯-乙交酯共聚物，该共聚物粘度范围为0.3-0.6dl/g，卡莫司汀的重量百分比为7.5%-30%，丙交酯与乙交酯的质量比例为（25-75）：(75-25)。该植入剂用于治疗胰腺癌、肺癌等实体肿瘤及肝脏、大脑转移瘤。

Description

一种治疗实体肿瘤的卡莫司汀缓释植入剂及其制备方法 技术领域
本发明涉及一种治疗实体肿瘤的卡莫司汀缓释植入剂及其制备，属于药物技术领域。
背景技术
2013年中国癌症新增病人高达有350万人，其中死亡人数高达250万人。预计2020年中国每年将有400万人死于癌症。美国等西方国家癌症的发病率也逐年上升，因此，探讨一种有效的治疗癌症的方法或药物已经成为目前研究的热点。
目前治疗癌症的主要方法为手术、放疗及化疗。由于多数病人就诊时已属晚期，因此失去手术机会，能手术切除者术后复发常是治疗失败的主要原因。因此有效的放化疗显得特别重要。
目前常用的化疗主要是全身化疗，该治疗方式选择性差，全身毒副作用大。由于肿瘤的组织学特点，化疗药物很难在肿瘤部位获得有效药物浓度并维持足够的时间。如血脑屏障(BBB)、血瘤屏障(BTB)及血胰屏障(BPB)等自然屏障的限制，瘤内间质压力(IFP)的升高，肿瘤血管的减少及不规则变化，肿瘤细胞外间质(ECM)的阻隔等极大地阻碍了化疗药物与肿瘤细胞的接触，而不规则的少量药物接触还容易诱发耐药性的产生。
药物局部缓释因其明显的优势已经成为近年的研究热点。其中卡莫司汀(BCNU)缓释植入剂已于1996年首先在美国批准上市(Lee JS等Eur J Pharm Biopharm 2005,59:169-75；Dang et al.,J Control Rel 1996；42:83-92))，已上市产品Gliadel植入片现已于许多国家批准用于脑胶质瘤的治疗。动物实验表明，局部放置后，植入剂处的BCNU浓度较静脉给药高出1300倍(Fleming AB,Saltzman WM.Clin Pharmacokinet 2002,41:403-19)。虽然与静脉给药相比具有一定优势，但总的疗效并不令人满意(Han L.等Oncol Rep 2012，27:121-8.)虽有一定的临床收益，病人生存期并无显著延长(Brem et al.,Lancet 1995；345:1008-12)，然而副作用较明显(McGovern PC,Lautenbach E,Brennan PJ,et al.Risk factors for postcraniotomy surgical site infection after 1,3-bis(2-chloroethyl)-1-nitrosourea(Gliadel)wafer placement.Clin Infect Dis2003；36:759-65)。
Gliadel植入片所用的缓释载体为聚苯丙生(对羧苯基丙烷(p-CPP)：癸二酸(SA)共聚物，80:20)，其释药特性存在很多缺陷，如但不限于：缓释体系不稳定，释放不稳定，突释明显，疗效差、毒副作用较大，上述缺陷限制了其产品的临床应用。
Gliadel植入片中BCNU的含药量低(为3.85％)，释药周期短(为5-7天)，且有非常明显的突释，体内放置1-2天内释放超过50％-60％。Domb(1995)报道，兔脑内放置Gliadel植入片后第3、6天BCNU的累积释放分别为60％和>95％(Domb AJ et al Biomaterials 1995， Vol.16No.141069-1072)；Dang(1996)报道，大鼠脑内放置Gliadel植入片后第1、2、5天BCNU的累积释放分别为60％、70％和100％(Dang W et al Journal of Controlled Release42(1996)83-92)；Eric(1997)报道，在生理盐水中，100％的BCNU于7天内完全释放,且第1天突释非常明显(Eric P.Sipos et al.,Cancer Chemother Pharmacol 39(1997)383-389；Lawrence(1998)报道，在PBS(pH 7.4)中，约有40％和70％BCNU在第1和4天释放，在猴脑内，约95％BCNU于5-7天释放(Lawrence K.Fung et al.,Cancer Research 58(1998)672-684).
为此，Gliadel植入片的研究者试图通过各种办法克服其缺陷，如加大其载药量、调整载体中p-CPP与SA的比例(Sipos EP et al Cancer Chemother Pharmacol(1997)39:383-389；Engelhard HH Surg Neurol 2000；53:458-64；Tamada,J.and Langer R.Proc Natl Acad Sci USA 1993；90:552-556)，然而，用50:50CPP:SA共聚物最长药物释放周期仅延长到18天，且24小时内突释明显，剂量大时毒性反应也很明显。为改善卡莫司汀(BCNU)缓释植入剂Gliadel植入片的治疗效果，其作者也在尝试同载其他药物有效成分如替莫唑胺(见美国专利：COMBINATION OF LOCAL TEMOZOLOMIDE WITH LOCAL BCNU，申请号：20110313010)，但效果仍因缓释载体的限制，药物释放周期没有明显改善。
为了克服上述诸多缺陷，不少研究尝试了选用其它辅料(Seong H et al Int J Pharm 2003,251(1-2):1-12；Qingzhong Kong,Bette.K.DeMasters and Kevin O.Lillehei:Intralesionally delivered cisplatin plus systemic BCNU in the treatment of brain tumor in rats.J Surg Oncol 1998；69:76-82；Qingzhong Kong,Bette K.DeMasters,and Kevin O.Lillehei:Intraletionally implanted cisplatin cures primary brain tumor in rats.J.Surg.Oncol.1997；64:268-273.)，其中丙交酯乙交酯共聚物(PLGA)历史悠久,因其良好的生物相容性和生物可降解性能而备受关注(Benny O,Menon LG,Ariel G,et al.Local delivery of poly lactic-co-glycolic acid microspheres containing imatinib mesylate inhibits intracranial xenograft glioma growth.Clin Cancer Res2009；15:1222-31；Zhang YH,Zhang H,Liu JM,et al.Temozolomide/PLGA microparticles:a new protocol for treatment of glioma in rats.Med Oncol 2011；28:901-6；Emerich EF et al.,Pharm Res 2000，17:767-775；Esther Gil-Alegre M,Gonzalez-Alvarez I,Gutierrez-Pauls L,et al.Three weeks release BCNU loaded hydrophilic-PLGA microspheres for interstitial chemotherapy:Development and activity against human glioblastoma cells.J Microencapsul 2008；25:561-8.11.Menei P,Daniel V,Montero-Menei C,et al.Biodegradation and brain tissue reaction to poly(D,Llactide-co-glycolide)microspheres.Biomaterials.1993；14:470-8；Allison SD.Effect of structural relaxation on the preparation and drug release behavior of poly(lactic-coglycolic)acid microparticle drug delivery systems.J Pharm Sci2008；97:2022-35；Onuki Y,Bhardwaj U,Papadimitrakopoulos F,et al.A review of the  biocompatibility of implantable devices:current challenges to overcome foreign body response.J Diabetes Sci Technol 2008；2:1003-15).
有动物实验结果表明，PLGA在脑内具有较好的生物相容性(Menei P.,等Biomaterials(Impact Factor:8.31).06/1993；14(6):470-8；Qingzhong Kong,Bette.K.DeMasters and Kevin O.Lillehei:Intralesionally delivered cisplatin plus systemic BCNU in the treatment of brain tumor in rats.J Surg Oncol 1998；69:76-82；Qingzhong Kong,Bette K.DeMasters,and Kevin O.Lillehei:Intraletionally implanted cisplatin cures primary brain tumor in rats.J.Surg.Oncol.1997；64:268-273.)，用其制成的顺铂缓释植入剂对动物脑肿瘤有明显的效果。
Seong H等(2003)报道了用PLGA为载体制成的BCNU缓释植入片的体外抑瘤研究。所用辅料为丙交酯乙交酯共聚物，分子量分别为20,000及90,000，丙交酯与乙交酯的比例为75:25.植入片通过喷雾干燥法制粉后直接压片而成。所得植入片分别含3.85％、10％和20％BCNU。结果发现所有植入片的释放周期均太长，在60天以上，释放速率不稳，且有突释(Seong H.,et al.,Int J Pharmaceutics 2003；(251):1-12)。
最近有研究用分子量为8,000g/mol PLGA(50:50)为载体制成的BCNU缓释植入剂(Zhu T,Shen Y,Tang Q,Chen L,Gao H,Zhu J.BCNU/PLGA microspheres:a promising strategy for the treatment of gliomas in mice.Chin J Cancer Res.2014Feb；26(1):81-8)，然而其在载药量及载体粘度等选择及其制备方法等诸多方面存在很多问题。
缓释辅料的选择是决定植入剂特性的关键因素之一。在《缓释辅料大全》(第123页，四川科学技术出版社1993年出版，罗明生和高天惠主编)中对缓释辅料已有详细描述。另外，中国专利(申请号96115937.5；91109723.6；9710703.3；01803562.0)及美国发明专利(专利号5,651,986)也列举了许多缓释辅料。
美国专利(US4757128；US4789724)中，卡莫司汀缓释植入剂所用的缓释辅料为聚苯丙生(对羧苯基丙烷(p-CPP)：癸二酸(SA)共聚物)。然而，以聚苯丙生为辅料制成的缓释植入剂有很多缺陷，如但不限于：不稳定，需要在-20℃以下保存，释放周期短(5天左右)，突释明显(2天内超过60％)。
相对而言，丙交酯-乙交酯共聚物(PLGA)不仅生物相容性很好，而且体内放置后可完全降解吸收。作为药物载体，PLGA已经广泛应用于多种药物的释放，包括缓释微球的研究。
就缓释植入剂而言，并非所用的缓释辅料均适合用于特定药物的释放。不同的缓释辅料，乃至不同分子量的同一缓释辅料与不同药物的组合、或同一药物的不同配比及制备工艺的不同均影响着药物的释放特性，载药量大小和制作工艺的选择还影响着药物在体内的代谢并与其不良反应和治疗效果有着明显的关系，需经大量创造性的劳动才可实现。
丙交酯-乙交酯共聚物与卡莫司汀组成的缓释植入剂也是如此。另外，选择适合的辅料、药物、配比关系及制作工艺的选择也是保障药物成功开发应用的前提。
发明内容
本发明针对现有技术的不足，提供一种卡莫司汀(BCNU)缓释植入剂，其包含卡莫司汀及缓释辅料，其中缓释辅料为丙交酯-乙交酯共聚物。
本发明的技术方案如下：
一种卡莫司汀(BCNU)缓释植入剂，包括卡莫司汀及缓释辅料，所述的缓释辅料为丙交酯-乙交酯共聚物；所述卡莫司汀在卡莫司汀缓释植入剂中的重量百分比为7.5％～30％；所述丙交酯-乙交酯共聚物的重均分子量为25000～80000，丙交酯与乙交酯的质量比为(25～75):(75～25)，粘度为0.2～1.3dl/g；
在优选的实施方案中，卡莫司汀的重量百分比为8％～25％。在更优选的实施方案中，卡莫司汀的重量百分比10～20％。在最优选的实施方案中，卡莫司汀的重量百分比为10％、15％或20％。
在某些实施方案中，丙交酯-乙交酯共聚物可由丙交酯与乙交酯开环共聚物而成，也可由乳酸与羟基乙酸缩聚而成，其中以丙交酯与乙交酯开环共聚物为优选。
在某些实施方案中，丙交酯-乙交酯共聚物共聚物的重均分子量以30,000～60,000为优选，以35,000～55,000为最优选；粘度范围以0.25～0.8dl/g为优选，以0.3～0.6dl/g为最优选；丙交酯(GA)与乙交酯(LA)的质量比为(50～75):(25～50)，优选50:50。
本发明通过大量创造性劳动发现，用丙交酯-乙交酯共聚物为缓释载体较用聚苯丙生优越，表现为丙交酯-乙交酯共聚物为载体的缓释植入剂缓释周期长(3～4周)、释药平稳、无突释(第二天释放不超过20％)、毒副作用低、制剂稳定、不易碎、生物相容性好、可完全降解吸收。其中，卡莫司汀与缓释辅料的组成配比及工艺的变化是影响着释药的主要因素之一。
本发明还发现，用丙交酯-乙交酯共聚物为缓释载体制成的缓释植入剂的缓释特性与其分子量有关，重均分子量可以为25,000～80,000，其中以30,000～60,000为优选，以35,000～55,000为最优选；粘度范围为0.2～1.3dl/g，其中以0.25～0.8dl/g为优选，以0.3～0.6dl/g为最优选；丙交酯(GA)与乙交酯(LA)的质量比例选自(25～75):(75～25)，以(50～75):(25～50)为优选，以50:50为最优选。其中前2天的累计释放量为总载药量不超20％，优选为5％～12％，前12天的累计释放量大于总载药量的30％但小于总载药量的70％，优选为40％～60％间。
本发明所制成的缓释植入剂体内放置后能在肿瘤部位缓慢释放药物，不仅能在局部维持足够的浓度和时间，而且有效药物浓度在组织中的扩散范围可达到2～4cm，因此能够更好地发挥卡莫司汀的临床治疗效果，因为大多数脑肿瘤术后于原发部位2厘米范围内复发。
另一方面，本发明提供了上述卡莫司汀缓释植入剂在治疗癌症中的用途。
在某些实施方案中，所述癌症可以是颅内肿瘤和颅外实体肿瘤。颅内肿瘤优选胶质瘤及脑转移瘤。脑转移瘤中优选的是源于肺脏、乳腺、肾脏、皮肤、黑色素细胞瘤、消化道、血液系统的转移瘤。
在某些实施方案中，所述癌症选自原发于颅外的实体肿瘤及各种转移癌。优选地，所述颅外实体肿瘤选自肝癌、肺癌、食管癌、胃癌、乳腺癌、胰腺癌、甲状腺癌、鼻咽癌、口腔肿瘤、耳鼻喉肿瘤、咽部肿瘤、眼部肿瘤、卵巢癌、子宫内膜癌、子宫颈癌、肾癌、前列腺癌、膀胱癌、结肠癌、直肠癌、睾丸癌、皮肤癌、淋巴瘤、骨肿瘤、头颈部肿瘤。更优选地，所述实体肿瘤选自胰腺癌、肺癌、肝癌、乳腺癌、脑肿瘤、食道癌、肾癌、结直肠癌等实体肿瘤。在肺癌中，优选的是非小细胞肺癌(NSCLC)；在各种颅外转移癌转移瘤中，优选肝转移瘤，来自但便于，肺脏、胃、直结肠、胰腺的原发肿瘤转移而来。
本发明缓释植入剂的用量取决于很多因素，如但不限于：肿瘤体积、瘤腔体积、病人体重、给药方式、病情进展情况、药物含量及治疗反应。但其原则是在能够降低肿瘤细胞的修复能力，增加化疗作用效果的同时并不明显增加药物的毒性反应。本发明缓释植入剂中BCNU的剂量为1～1000毫克/次/人。根据肿瘤体积、瘤腔体积、肿瘤类型、肿瘤部位，每次每次可为1～19毫克、20～200、201～800或801～1000毫克。如眼部肿瘤及脑干部位肿瘤可选择低剂量，如1～100毫克，颅内肿瘤及脑转移瘤可选20～800毫克，乳腺及消化道肿瘤可选40～1000毫克。药物可单次应用，也可每3～4周重复应用。
另一方面，提供了本发明的缓释植入剂在制备用于治疗癌症的药物中的用途。
本文所用的“治疗”包括抑制、治愈和减轻癌症或其症状以及预防或延缓原发癌的转移。
本发明可制成各种形状或剂型，如，但不限于，颗粒样、片状、球形、块状、针状、棒状及模样；可经各种途径给药，如皮下、肌肉、皮内、腔内、瘤内、瘤周等。给药途径取决于多种因素，如肿瘤所在部位、是否手术或转移、肿瘤体积大小、瘤腔大小、肿瘤类别、病人年龄、身体状况、生育状况及要求等。为于肿瘤所在部位获得有效药物浓度，可选择性地腔内灌注,腹腔(i.p.)或胸腔及椎管内给药，也可脏器内放置，如肠腔内、膀胱内、宫腔内、椎管内、阴道内、胃内及食道内等。在各种途径中，以局部给药，如瘤内、瘤周注射为主，以瘤内、瘤周或瘤腔放置或注射的形式为优选，如可选用缓释剂、植入剂、缓释植入剂及缓释注射剂。
给药剂量可以按患者的年龄、病症的特性、肿瘤体积、瘤腔大小、肿瘤类型选择，可单次给药，可多次给药；可术后瘤腔内放置，可直接瘤内导入。
某些实施例中，一次给药剂量优选0.1～1000毫克，较优选为10～1000毫克，更优选20～800，最优选为100～400毫克。
某些实施例中，所述缓释植入剂的用量为每次1～200片，优选3～50片，最优选6～20片。
某些实施例中，所述缓释植入剂的用量为每次1～1000粒，优选5～200粒，最优选10～100。
某些实施例中，一次给药剂量优选0.1～1000毫克，较优选为10～1000毫克，更优选20～800，最优选为100～400毫克，可重复给药，重复周期为7天以上，优选14天以上。
例如，对小于1g的肿瘤，可以单次给药。剂量可以为0.1～50mg BCNU缓释棒，优选地 含有2.5～25mgBCNU，直接插入肿瘤。当肿瘤为5～10g时，在第一次剂量后还可以再次施用较大剂量的植入剂。例如，第一次施用550毫克剂量，2～4周后重复给药。
在某些实施方案中，上述药物以植入的方式给药，优选地，所述药物经局部给药。将此植入剂局部应用于癌症治疗。局部给药包括将药物应用于脏器和/或癌细胞或肿瘤内或其附近，优选为肿瘤内。局部给药还包括用药物包围癌性肿瘤或将药物应用于癌性肿瘤的表面。在一实施方案中，将药物直接导入癌症局部。
在优选的实施方案中，所述植入剂经癌灶内、癌周或癌症手术后瘤腔内给药，优选地经癌灶内穿刺给药。在其他优选的实施方案中，所述药物经癌灶内植入、癌周植入或癌症手术后瘤腔内植入给药，优选地经癌灶内植入给药。
本发明缓释植入剂可直接应用于原发或转移的实体肿瘤全部或部分切除后所形成的腔内、肿瘤周围或瘤体内、术后疑有瘤细胞残存的部位，也可直接放置或注射于不能手术切除的原发或转移的实体肿瘤内或附近，可单独用于肿瘤的治疗或用于防止术后复发，也可与放疗和(或)化疗量联合应用。
本发明抗癌缓释植入剂可与常规化疗、免疫治疗、高热治疗、光化学治疗、电疗、生物治疗、激素治疗、磁疗、超声治疗、放疗及基因治疗等方法合用，使其作用效果加强。因此在局部缓慢释放的同时可与上述非手术疗法合用，从而使其抗癌效果进一步加强。当与上述非手术疗法合用时，本发明抗癌缓释植入剂可与非手术疗法同时应用，也可在非手术疗法实施前几天内应用，其目的在于尽可能增强肿瘤的敏感性，从而为根治各种人体及动物原发和转移实体肿瘤提供一种更有效的新的方法，具有非常高的临床应用价值及显著的经济和社会效益。
本发明还提供了一种卡莫司汀(BCNU)缓释植入剂的制备方法，包括如下步骤：
a、称取处方量的丙交酯-乙交酯共聚物，溶解于有机溶剂中，制得丙交酯-乙交酯共聚物溶液；
所述丙交酯(GA)与乙交酯(LA)的质量比例选自(25～75):(75～25)；
b、称取处方量的卡莫司汀加入上述丙交酯-乙交酯共聚物溶液中充分溶解混合形成丙交酯-乙交酯共聚物与卡莫司汀混合液；
所述丙交酯-乙交酯共聚物与卡莫司汀混合液中卡莫司汀与丙交酯-乙交酯共聚物的重量比为(0.75～3):(7～9.25)；即卡莫司汀在缓释植入剂中的重量百分比为7.5％-30％；
c、将上述丙交酯-乙交酯共聚物与卡莫司汀混合液经过滤除菌，制得无菌滤液；
d、将无菌滤液经无菌干燥，制得微粉；
e、将上述微粉经无菌成形后，制得卡莫司汀(BCNU)缓释植入剂。
根据本发明优选的，所述步骤a中，有机溶剂选自氯甲烷、氯仿、乙酸乙酯或丙酮之一或两者以上的组合；进一步优选二氯甲烷；有机溶剂的量不严格限定，以充分溶解为宜。
根据本发明优选的，所述步骤b中，丙交酯-乙交酯共聚物的粘度为0.2～1.3dl/g，其中以0.25～0.8dl/g为优选，选自0.25dl/g、0.3dl/g、0.35dl/g、0.4dl/g、0.45dl/g、 0.50dl/g、0.55dl/g、0.6dl/g或0.65dl/g，又以0.3～0.6dl/g为最优选；
根据本发明优选的，所述步骤a中，丙交酯(GA)与乙交酯(LA)的质量比例选自(50～75):(25～50)，以50:50为最优选；丙交酯(GA)与乙交酯(LA)共聚物的重均分子量为25,000～85,000，其中以30,000～65,000为优选，以35,000～60,000为最优选。
根据本发明优选的，所述步骤b中，丙交酯-乙交酯共聚物与卡莫司汀混合液中卡莫司汀与丙交酯-乙交酯共聚物的重量比为(1～2):(8～9)；即卡莫司汀在缓释植入剂中的重量百分比为10～20％。
根据本发明优选的，所述步骤c中，丙交酯-乙交酯共聚物与卡莫司汀混合液过滤包括经0.45μm微孔滤膜过滤器初滤和0.22μm微孔滤膜过滤器两次精滤。初滤主要目的是除去杂质和残渣，精滤为除菌目的。
根据本发明优选的，所述步骤d中，无菌滤液经无菌干燥制成微粉所应用的方法均为在无菌条件下，方法选自：乳化法法、喷雾干燥法、溶剂挥发法、有机萃取法或冷冻粉粹法；优选，冷冻粉粹法或喷雾干燥法；最优，冷冻粉粹法。
根据本发明优选的，所述步骤d中，冷冻粉碎前将混合液干燥，所用的方法选自自然干燥、加热和/或鼓风干燥、冷冻和/或真空干燥，优选加热鼓风干燥。待有机溶剂残留合格后将干燥含BCNU共聚物粉粹成粉体，粒径优选10到20微米，更优选范围是30-150微米，最优选范围是40-100微米，冷冻粉碎过程中优选液氮为制冷剂。
根据本发明优选的，所述步骤d中，干燥后的粉体成形可在室温下进行，制成的植入剂可为各种形状或剂型，如，但不限于，颗粒样、片状、球形、块状、针状、棒状及模样，其中以植入片或植入棒为优选。
植入片中卡莫司汀的重量百分比为10％～30％、优选10％、15％、20％、25％或30％；植入片直径可为0.5～2.0厘米，选自1.35～1.5厘米，厚度为0.8到3.0毫米，优选0.8到2.0毫米，最优选0.8-1.2毫米。
植入棒中卡莫司汀的重量百分比为为10％～30％、优选10％、15％、20％、25％或30％，植入棒直径选自0.8～3.8毫米，长度为3～8.0毫米。
根据本发明优选的，还包括无菌包装的步骤。
所述包装步骤中，包装材料选用药用包装材料，优选以铝塑或玻璃容器为内包材料。
本发明还提供另一种卡莫司汀(BCNU)缓释植入剂的制备方法，包括如下步骤：
(1)称取处方量的丙交酯-乙交酯共聚物，溶解于有机溶剂中，制得丙交酯-乙交酯共聚物溶液；
所述丙交酯(GA)与乙交酯(LA)的质量比例选自(25～75):(75～25)；
(2)称取处方量的卡莫司汀加入上述丙交酯-乙交酯共聚物溶液中充分溶解混合形成丙交酯-乙交酯共聚物与卡莫司汀混合液；
所述丙交酯-乙交酯共聚物与卡莫司汀混合液中卡莫司汀与丙交酯-乙交酯共聚物的重量比为(0.75～4):(6～9.25)；即卡莫司汀在缓释植入剂中的重量百分比为7.5％～40％；
(3)将步骤(2)中制得的丙交酯-乙交酯共聚物与卡莫司汀混合液经过滤除菌，制得无菌滤液；
(4)上述无菌滤液经无菌干燥后，制得微粉；
(5)将上述微粉进行无菌成型，无菌成型在熔融状态下进行，熔融温度为25～70℃，时间为1～10分钟，制得卡莫司汀(BCNU)缓释植入剂；
或者，
③将步骤(2)中制得的丙交酯-乙交酯共聚物与卡莫司汀混合液进行干燥，制得未灭菌微粉；
④将上述未灭菌微粉进行成型，成型在熔融状态下进行，熔融温度为25～70℃，时间为1～10分钟，经钴60照射灭菌，制得卡莫司汀(BCNU)缓释植入剂。钴60照射灭菌为本领域常规技术，可采用常规方法。
根据本发明优选的，所述步骤(1)中，有机溶剂选自氯甲烷、氯仿、乙酸乙酯或丙酮之一或两者以上的组合；进一步优选二氯甲烷；有机溶剂的量不严格限定，以充分溶解为宜。
根据本发明优选的，所述步骤(2)中，丙交酯-乙交酯共聚物的粘度为0.2～1.3dl/g，其中以0.25～0.8dl/g为优选，选自0.25dl/g、0.3dl/g、0.35dl/g、0.4dl/g、0.45dl/g、0.50dl/g、0.55dl/g、0.6dl/g或0.65dl/g，又以0.3～0.6dl/g为最优选；
根据本发明优选的，所述步骤(1)中，丙交酯(GA)与乙交酯(LA)的质量比例为(50～75):(25～50)为优选，以50:50为最优选；丙交酯(GA)与乙交酯(LA)共聚物的重均分子量为25,000～85,000，其中以30,000～65,000为优选，以35,000～60,000为最优选。
根据本发明优选的，所述步骤(2)中，丙交酯-乙交酯共聚物与卡莫司汀混合液中卡莫司汀与丙交酯-乙交酯共聚物的重量比为(1～3):(7～9)；即卡莫司汀在缓释植入剂中的重量百分比为10～30％。
根据本发明优选的，所述步骤(3)中，丙交酯-乙交酯共聚物与卡莫司汀混合液过滤包括经0.45μm微孔滤膜过滤器初滤和0.22μm微孔滤膜过滤器两次精滤。初滤主要目的是除去杂质和残渣，精滤为除菌目的。
根据本发明优选的，所述步骤(4)中，无菌滤液经无菌干燥制成微粉所应用的方法均为在无菌条件下，方法选自：乳化法法、喷雾干燥法、溶剂挥发法、有机萃取法或冷冻粉粹法；优选，冷冻粉粹法或喷雾干燥法；最优，冷冻粉粹法。冷冻粉碎法借助于液氮降温，冷冻粉碎前应去除有机溶剂，可选用真空干燥或低温干燥法或加热吹风干燥等方法。
根据本发明优选的，所述步骤③中，干燥方法选自：乳化法、喷雾干燥法、溶剂挥发法、有机萃取法或冷冻粉粹法；优选，冷冻粉粹法或喷雾干燥法；最优，冷冻粉粹法。冷冻粉碎法借助于液氮降温，冷冻粉碎前应去除有机溶剂，可选用真空干燥或低温干燥法或加热吹风干燥等方法。
根据本发明优选的，所述步骤(5)或④中，制成的植入剂可为各种形状或剂型，如，但不限于，颗粒样、片状、球形、块状、针状、棒状及模样，其中以植入片或植入棒为优选。
植入片中卡莫司汀的重量百分比为10％、15％、20％、25％或30％；植入片直径可为0.5～2.0厘米，选自1.35～1.5厘米，厚度为0.8～3.0毫米，优选0.8～2.0毫米，最优选0.8～1.2毫米。
植入棒中卡莫司汀的重量百分比为为10％、15％、20％、25％或30％，植入棒直径选自0.8～3.8毫米，长度为3～8.0毫米。
根据本发明优选的，所述步骤(5)或④中，熔融温度为30～60℃，优选40～60℃，时间为1～8分钟，优选3～5分钟。
制成的植入剂可为各种形状或剂型，如，但不限于，颗粒样、片状、球形、块状、针状、棒状及模样，其中以植入片或植入棒为优选。
植入片中卡莫司汀的重量百分比为7.5％～40％、优选10％、15％、20％、25％或30％；植入片直径可为0.5～2.0厘米，选自1.35～1.5厘米，厚度为0.8～3.0毫米，优选0.8～2.0毫米，最优选0.8～1.2毫米。植入棒中卡莫司汀的重量百分比为为7.5％～40％、优选10％、15％、20％、25％或30％，植入棒直径选自0.8～3.8毫米，长度为3～8.0毫米。
根据本发明优选的，还包括无菌包装的步骤。
所述的包装步骤，内包材料选用无菌包装材料，优选铝塑或玻璃材质。
上述制备方法，通过过滤除菌、无菌干燥、无菌成型的工艺，达到了在线灭菌的效果，从而使产品达到了体内施用的无菌要求。
另一方面，本发明提供了卡莫司汀缓释植入剂在制备用于治疗癌症药物中的应用。
有益效果
1、本发明所述的卡莫司汀(BCNU)缓释植入剂，用丙交酯-乙交酯共聚物为缓释载体，通过对卡莫司汀含量、丙交酯-乙交酯共聚物含量、丙交酯与乙交酯比例等条件进行创造性的选择，获得了缓释周期长达3-4周、释药平稳、无突释(第二天释放不超过20％)、毒副作用低、制剂稳定、不易碎、生物相容性好、可完全降解吸收的卡莫司汀(BCNU)缓释植入剂。
2、本发明所述的卡莫司汀(BCNU)缓释植入剂，可以达到前2天的累计释放量为总载药量的5％-12％，前12天的累计释放量大于总载药量的40％但小于总载药量的60％。
3、本发明所述方法制备的卡莫司汀(BCNU)缓释植入剂不仅临床应用效果更加明显，全身毒性反应轻，同时能满足药品生产要求、更有利于产业化开发；可用于脑肿瘤等治疗实体肿瘤及其转移瘤的术中治疗，也可用于术后或不能手术的肿瘤的治疗及与其它非手术疗法的联合应用。
附图说明
图1是实施例1中植入剂1号A、植入剂1号B的药物释放曲线，该曲线就能体现本产品无突释并与有突释现象产品对比的释放曲线图；
图2是实施例17中植入剂A的药物释放曲线；
图3是实施例17中BCNU聚苯丙生植入剂的药物释放曲线；
具体实施方式
上述公开内容总体上描述了本发明，通过下面的实施例进一步示例本发明。描述这些实施例仅为说明本发明，而不是限制本发明的范围。尽管本文中使用了特殊的术语和值，这些术语和值同样被理解为示例性的，并不限定本发明的范围。除非特别指明，本说明书中的实验方法和技术为本领域技术人员所公知的方法和技术。
实施例1、比较了本发明所选辅料与聚苯丙生的差异
将9.0gPLGA(丙交酯与乙交酯的质量比例为50:50，粘度范围为0.30dl/g)和9.0g聚苯丙生(对羧苯基丙烷(p-CPP)：癸二酸(SA)共聚物，80:20)分别放入容器“A”和”B”中，各加100ml二氯甲烷溶解混匀后加入1.0g卡莫司汀，重新摇匀后倒入托盘中加热干燥去除有机溶剂。将干燥后的固体组合物冷冻粉碎，待有机残留合格后将粉体放入压片机成形，室温条件下制成缓释片，分装后钴60射线灭菌，所得产品为“植入剂1号”，为缓释植入片，直径为1.4厘米，厚度1.0毫米，其中，
植入剂1号A为：10％卡莫司汀，辅料为PLGA(丙交酯与乙交酯的比例为50:50，粘度为0.30dl/g)，植入剂1号B为：10％卡莫司汀，辅料为聚苯丙生(对羧苯基丙烷(p-CPP)：癸二酸(SA)共聚物，80:20)。
将植入剂1号A和植入剂1号B分别放入纯水中测定其2天、12天及28天的累计释放量，结果见表1及图1：
表1
  2天释放百分含量 12天释放百分含量 28天释放百分含量
植入剂1号A 14％ 34％ 98％
植入剂1号B 68％ 100％  
以上结果表明，辅料为聚苯丙生制成的卡莫司汀植入剂，药物释放周期太短，不到12天，其中前2天的累计释放量为总载药量的68％；而在相同载药量(10％)情况下，以丙交酯-乙交酯共聚物制成的卡莫司汀植入剂释放平稳，前12天的累计释放量为14％。前12天的累计释放量为总载药量的34％，释药时间大约4周。
实施例2、不同分子量共聚物对BCNU释放的影响
按实施例1的制备工艺制备缓释植入剂片，只是所用PLGA为质量比50:50的丙交酯与乙交酯的共聚物，重均分子量分别为15K、45K和75K，所得产品为植入剂2号(A、B、C)，直径1.35-1.45厘米，厚度0.8-1.2毫米，均含10％的卡莫司汀。植入剂2号在纯水中的释药结果见表2：
表2
  2天释放百分含量(％) 12天释放百分含量(％) 28天释放百分含量(％)
植入剂2号A 9.03 76.98 100
植入剂2号B 9.66 48.1 92.31
植入剂2号C 13.91 38.92 78.52
以上结果表明，在相同载药量(10％)情况下，丙交酯-乙交酯共聚物制成的卡莫司汀植入剂的释放特性与载体分子量有关，重均分子量为15K的植入剂(植入剂2号A)释放太快，第12天释放76.98％，不到20天全部释放结束；重均分子量为75K的植入剂(植入剂2号C)不稳，早期太快，后期太慢，周期太长(近50天)；重均分子量为45K的植入剂(植入剂2号B)最为理想，药物释放平稳，周期为4周左右。
实施例3、比较不同分子量共聚物对BCNU释放的影响
按实施例1的制备工艺制备缓释植入剂片，所不同的是PLGA为质量比50:50的丙交酯与乙交酯共聚物，重均分子量分别为25K、40K和65K，所得产品为植入剂3号(A、B、C)，直径1.35-1.45厘米，厚度0.8-1.2毫米，均含10％的卡莫司汀。其在纯水中的释药时间见
表3:
表3
  2天释放百分含量(％) 12天释放百分含量(％) 28天释放百分含量(％)
植入剂3号A 9.20 58.98 100
植入剂3号B 9.48 46.1 94.31
植入剂3号C 12.61 40.92 84.52
以上结果表明，在相同载药量(10％)情况下，丙交酯-乙交酯共聚物制成的卡莫司汀植入剂的释放特性与载体分子量有关。重均分子量分别为25K的植入剂(植入剂3号A)和重均分子量为65K的植入剂(植入剂3号C)较为理想，第2天释放均在9％到13％之间，第12天释放,40％到60％之间。重均分子量为45K的植入剂(植入剂3号B)最为理想，平稳均匀，周期为4周左右。
以上实施例可以看出，PLGA为质量比50:50的丙交酯-乙交酯共聚物制成的卡莫司汀植入剂的释放特性与载体分子量有关，随着PLGA重均分子量(或粘度)的增加，BCNU的释放速度变慢。对于10％左右的载药量而言，优选PLGA重均分子量范围为25K-65K。
实施例4、比较不同载药量对BCNU释放的影响
按实施例1的制备工艺制备缓释植入剂片，所用PLGA为质量比50:50的丙交酯与乙交酯共聚物，重均分子量分为30K-35K，所得产品为植入剂4号，直径1.4厘米，厚度1.0毫米，所含BCNU分别为7.5％(植入剂4号A)、10％(植入剂4号B)、15％(植入剂4号C)、20％(植入剂4号D)、30％(植入剂4号E)和40％(植入剂4号F)。其在纯水中的释药时间见表4：
表4
  2天释放百分含量(％) 12天释放百分含量(％) 28天释放百分含量(％)
植入剂4号A 5.20 58.98 100
植入剂4号B 7.48 56.1 98.31
植入剂4号C 9.61 44.92 94.52
植入剂4号D 14.61 52.92 92.52
植入剂4号E 17.10 56.92 90.52
植入剂4号F 24.10 58.92 94.52
以上结果表明，丙交酯-乙交酯共聚物制成的卡莫司汀植入剂的释放特性还与载药量有关。对于重均分子量分为30K-35K的丙交酯与乙交酯共聚物(50:50)而言，药物释放随载药量增加而变快，BCNU含量为7.5％(植入剂4号A)、10％(植入剂4号B)、15％(植入剂4号C)、20％(植入剂47号D)和30％(植入剂4号E)的植入剂均较为理想，第2天释放均在5％到18％之间，第12天释放均在40％到60％之间，当增加到40％时，如植入剂4号F，开始有突释现象。
实施例5、比较不同形状对植入剂释放的影响
按实施例1的制备工艺制备，将9000mgPLGA(丙交酯与乙交酯的比例为50:50，重均分子量为35-45K)溶于二氯甲烷中溶解混匀后加入1000mg卡莫司汀,重新摇匀后倒入托盘中加热干燥去除有机溶剂。将干燥后的固体组合物冷冻粉碎，待有机残留合格后将粉体分别放入压片机和挤出机，室温条件下制成缓释片和缓释棒，分装后钴60射线灭菌，所得产品为植入剂5号A(缓释植入片，直径为为14毫米，厚度1.0毫米)、植入剂5号B(缓释植入棒，直径1.0毫米，长度3.0毫米)、植入剂5号C(缓释植入棒，直径1.0毫米，长度5.5毫米)、植入剂5号D(缓释植入棒，直径3.0毫米，长度5.5毫米)。
纯水中测定其2天、12天及28天的累计释放量，结果见表5
表5
  2天释放百分含量(％) 12天释放百分含量(％) 28天释放百分含量(％)
植入剂5号A 9.68 45.1 98.3
植入剂5号B 8.98 48.9 97.8
植入剂5号C 9.2 48.8 96.4
植入剂5号D 8.8 54.6 98.6
植入剂5号E 10 50.3 96.2
以上结果表明，在相同载药量(10％)情况下，丙交酯-乙交酯共聚物制成的卡莫司汀植入剂的释放特性与植入剂的形状关系不明显。
实施例6、比较植入剂不同形状对释放的影响。
按实施例1的制备工艺制备缓释植入剂，所不同是共聚物中丙交酯与乙交酯的质量比例为75:25，重均分子量为25K。所得产品为植入剂6号A(缓释植入片，直径为为14毫米，厚度1.0毫米)、植入剂6号B(缓释植入棒，直径1.0毫米，长度3.0毫米)、植入剂6号C(缓释植入棒，直径1.0毫米，长度5.5毫米)、植入剂6号D(缓释植入棒，直径3.0毫米，长度5.5毫米)。
纯水中测定其2天、12天及28天的累计释放量，结果表6:
表6
  2天释放百分含量(％) 12天释放百分含量(％) 28天释放百分含量(％)
植入剂6号A 8.68 50.1 88.3
植入剂6号B 7.98 46.9 92.8
植入剂6号C 9.0 47.8 90.4
植入剂6号D 8.2 50.6 92.6
植入剂6号E 9.4 54.3 92.2
以上结果表明，在相同载药量(10％)情况下，丙交酯-乙交酯共聚物制成的卡莫司汀植入剂的释放特性与植入剂的形状关系不明显。
按照本实施例同样的方法步骤制作缓释植入棒，载药量分别为7.5％、10％、15％、20％、30％和40％，释放试验再次表明药物释放与载药量有关，越高释放越快，但在相同载药量情况下，释放特性与植入剂的形状关系不明显。动物实验结果表明，50:50的丙交酯与乙交酯共聚物，重均分子量分为30K-35K时，载药量优选的范围是10％、15％、20％、30％。
实施例7、熔融温度下成形对药物释放的影响
将9000、8000、7000、6000mgPLGA(丙交酯与乙交酯的质量比例为50:50，粘度为0.30-0.35dl/g)分别放入A、B、C、D容器中，各加一定量的二氯甲烷溶解混匀后分别加入1000、2000、3000和4000mg卡莫司汀，重新摇匀后加热干燥去除有机溶剂。将干燥后的固体组合物冷冻粉碎，待二氯甲烷残留合格后将200毫克粉体放入金属模具中，压片温度控制在50-60℃，待粉体软化后压片，分装后钴60射线灭菌，所得产品为直径为1.4厘米，厚度1.0毫米缓释植入片，其中，植入剂7号A到D的BCNU含量分别为10％、20％、30％和40％。其在纯水中的释药时间见表7：
表7
  2天释放百分含量(％) 12天释放百分含量(％) 28天释放百分含量(％)
植入剂7号A 8.20 40.98 100
植入剂7号B 9.48 46.10 96.30
植入剂7号C 10.61 48.02 98.52
植入剂7号D 14.61 59.92 98.56
以上结果表明，丙交酯-乙交酯共聚物粉体在熔融状态下成形使得所制成的卡莫司汀植入剂的药物释放更加平稳，但也和载药量有关。药物释放随载药量增加而变快，BCNU含量为10％(植入剂7号A)、20％(植入剂7号B)、30％(植入剂7号C)和40％(植入剂7号E)的植入剂均非常理想，第2天释放均在8％到15％之间，第12天释放均在40％到50％之间。当载药量增加到40％时(植入剂7号D)，第12天释放接近60％。
实施例8、熔融状态对药物释放的影响
将6000mgPLGA(丙交酯与乙交酯的质量比例为50:50，粘度为0.55dl/g)分别放入容器中，加一定量的二氯甲烷溶解混匀后分别加入4000mg卡莫司汀,重新摇匀后加热干燥去除有机溶剂。将干燥后的固体组合物冷冻粉碎，待二氯甲烷残留合格后将200毫克粉体放入金属模具中，分别在室温和50-60℃环境下压片，所得产品为直径为1.4厘米，厚度1.0毫米缓释植入片，其中，植入剂8号A为室温片，植入剂8号B为熔融片，BCNU含量均40％。其 在纯水中的释药时间见表8：
表8
  2天释放百分含量(％) 12天释放百分含量(％) 28天释放百分含量(％)
植入剂8号A 21.20 70.98 98.2
植入剂8号B 14.48 56.1 86.30
以上结果表明，丙交酯-乙交酯共聚物粉体在熔融状态下成形使得所制成的卡莫司汀植入剂的药物释放更加平稳缓慢。相同载药量(40％)下，熔融状态成形有利于药物平稳释放，第2天释放均在14.48％，第12天释放均在56.1％，而室温下成形片第2天释放均在21.2％，第12天释放均在70.98％。
实施例9、不同剂量的植入剂对脑肿瘤生长的影响
将2×105个脑肿瘤细胞皮下注射于小白鼠(25克)的季肋部，待肿瘤生长7天后将其分为以下，7组(每组10只，见表9)(肿瘤直径约10.0mm)。第1组为对照组，第2组为空白对照组，第3-7组为治疗组。每只动物给1只植入棒(按实施例7的制备方法制备)(3.0mmx5.5mm)，经瘤内穿刺植入。植入棒的BCNU含量分别为5％、10％、20％和30％(分别相当于0.05、0.10、0.20、0.30和0.40毫克BCNU/公斤)(具体剂量见表9)。治疗后第15天测量肿瘤体积大小，比较各组的治疗效果(参见表9)。
表9
Figure PCTCN2015091049-appb-000001
如表9的结果所示，植入剂对脑肿瘤生长的抑制作用与植入剂的载药量呈明显量效关系。例如，5％植入剂对肿瘤的抑制作用不明显(P>0.05)，当剂量增到10％及以上时就能够显著抑制肿瘤生长。值得注意的是40％虽然抑瘤作用明显，但局部皮肤溃烂及动物早期体重明显减轻则说明该剂量可能存在突释。因此优选的载药量为10％-30％。
实施例10 卡莫司汀缓释植入剂对不同体积脑肿瘤的抑制作用比较
将相同性别、相近体重的小白鼠分为以下3组，分别将不同数量的脑肿瘤细胞经皮下注射于小白鼠季肋部，其中第1组皮下注射2×105个瘤细胞，第2组皮下注射5×105个瘤细胞，第3组皮下注射2×106个瘤细胞。待肿瘤生长14天后测量瘤体大小，然后每组再随机分成 两小组(n＝6)，其中一小组接受10％BCNU植入剂(按实施例7的制备方法制备)。治疗后第10天再次测量肿瘤体积大小，比较肿瘤生长抑制率(见表10)。
表10
Figure PCTCN2015091049-appb-000002
表10的数据表明，应用等量的BCNU植入剂对不同体积的肿瘤生长抑制程度不同。肿瘤体积越大，实现相同的抑制效果需要的药物剂量越大。
可以理解，尽管本发明实施例以某种形式被说明，如实施例9和10是动物脑肿瘤，但本发明并不局限于动物脑肿瘤，如，但不限于，人及动物的其他实体肿瘤。对本领域的技术人员显而易见的是，在不偏离本发明的范围的前提下还可做出各种变化。这些变化都在本发明要求保护的范围内。
实施例11、肿瘤内植入卡莫司汀对颅外实体肿瘤的抑制作用
将肺癌肿瘤细胞(105)接种于小鼠的右侧腋窝皮下，当肿瘤生长至0.8-1.5cm左右时将动物随机分为5组。用70％酒精消毒肿瘤表面皮肤，选择距肿瘤下缘1cm处，剪开1mm长切口，用穿刺针将卡莫司汀植入剂植入肿瘤组织中，治疗后每3天测量肿瘤大小，植入剂包埋后21天处死动物，称体重后完整剥离肿瘤并称瘤重。计算肿瘤抑制率％，DAS.ver1.0药理学软件做统计学处理。
Figure PCTCN2015091049-appb-000003
按照上述方法和步骤检验卡莫司汀缓释植入剂对小鼠肺癌的抑瘤作用。所用的植入剂辅料为PLGA(分子量为30000-35000,丙交酯和乙交酯的质量比为50：50)，植入剂按实施例7的制备方法制备。本次实验结果见表11。
表11
组别 治疗 肿瘤抑制率(％) P值
1 对照组    
2 i.p.组 22 〉0.05
3 5％组 39 〉0.05
4 10％组 58 <0.05
5 20％组 82 <0.01
结果表明，卡莫司汀植入剂较腹腔注射效果明显增强，不同剂量的卡莫司汀植入剂能明显抑制小鼠肺癌肿瘤生长，肿瘤抑制率与药物剂量呈明显量效关系。优选的载药量为10％到20％。由于20％的载药量为表现出明显的毒副反应，更高的载药量也适用于肺癌的治疗。
实施例12、肿瘤内植入卡莫司汀缓释植入剂对小鼠乳腺癌的抑瘤作用
按照实施例11所述方法和步骤检验卡莫司汀缓释植入剂对小鼠乳腺癌的抑瘤作用，所用的植入剂为5％、10％、20％和30％，所用的植入剂辅料为PLGA(分子量为25000-35000，丙交酯与乙交酯的质量比50：50)。实验结果见表12。
表12
组别 治疗 肿瘤抑制率(％) P值
1 对照组    
2 5％组 26 〉0.05
3 10％组 56 <0.05
4 20％组 68 <0.01
5 30％组 74 <0.01
结果表明，不同剂量的卡莫司汀植入剂植入小鼠乳腺癌中能明显抑制肿瘤生长，肿瘤抑制率与药物剂量呈明显量效关系。10％、20％和30％卡莫司汀植入剂的肿瘤抑制率分别为56％、68％和70％，显著高于5％的实验组。
实施例13、肿瘤内植入卡莫司汀缓释植入剂对小鼠食道癌的抑瘤作用
按照实施例11所述方法和步骤检验卡莫司汀缓释植入剂对小鼠食道癌的抑瘤作用。实验结果见表13。
表13
组别 治疗 肿瘤抑制率(％) P值
1 对照组    
2 5％组 20 〉0.05
3 10％组 60 <0.05
4 20％组 68 <0.01
5 30％组 76 <0.01
结果表明，不同剂量的卡莫司汀植入剂植入裸鼠模型人食管癌(9706)实体肿瘤中，均能明显抑制肿瘤生长，肿瘤抑制率与药物剂量呈明显量效关系。载药量为10％、20％和30％卡莫司汀植入剂肿瘤的抑制率分别为60％、68％、76％，显著高于5％的实验组，P值均小于0.05。
实施例14、肿瘤内植入卡莫司汀缓释植入剂对小鼠胰腺癌的抑瘤作用
按照实施例11所述方法和步骤检验卡莫司汀缓释植入剂对小鼠胰腺癌的抑瘤作用，所用的植入剂辅料为PLGA(分子量为30000-35000，丙交酯和乙交酯的质量比为50：50)。卡莫司汀在缓释植入剂中的含量为5％、10％、20％和30％。实验结果见表14。
表14
组别 治疗 肿瘤抑制率(％) P值
1 对照组    
2 5％组 29 〉0.05
3 10％组 56 <0.05
4 20％组 72 <0.01
5 30％组 78 <0.01
结果证明不同剂量的卡莫司汀植入剂植入裸鼠模型人胰腺癌(JF305)实体肿瘤中，能明显抑制肿瘤生长，肿瘤抑制率与药物剂量呈明显量效关系。其中10％、20％和30％载药量效果显著高于5％的实验组，P值均小于0.05。
实施例15、肿瘤内植入卡莫司汀缓释植入剂对小鼠直肠癌的抑瘤作用
按照实施例11所述方法和步骤检验卡莫司汀缓释植入剂对小鼠直肠癌的抑瘤作用，所用的植入剂辅料为PLGA(分子量为40000-45000,丙交酯和乙交酯的质量比为50：50)。卡莫司汀在缓释植入剂中的含量为5％、10％、15％和25％。实验结果见表15。
表15
组别 治疗 肿瘤抑制率(％) P值
1 对照组    
2 5％组 18 〉0.05
3 10％组 40 <0.05
4 15％组 66 <0.01
5 25％组 87 <0.01
结果表明，不同剂量的卡莫司汀植入剂，能明显抑制肿瘤生长，肿瘤抑制率与药物剂量呈明显量效关系。10％、20％和30％卡莫司汀植入剂的肿瘤抑制率分别为40％、66％和87％，显著高于5％的实验组，与对照组比较P值均小于0.05。
实施例16、肿瘤内植入卡莫司汀缓释植入剂对小鼠肝癌的抑瘤作用
按照实施例11所述方法和步骤检验卡莫司汀缓释植入剂对小鼠肝癌的抑瘤作用，所用的植入剂辅料为PLGA(分子量为40000-45000，丙交酯和乙交酯的共混比为50：50)。卡莫司汀在缓释植入剂中的含量为5％、10％、20％和30％。实验结果见表16。
表16
组别 治疗 肿瘤抑制率(％) P值
1 对照组    
2 5％组 22 〉0.05
3 10％组 58 <0.05
4 20％组 66 <0.01
5 30％组 84 <0.001
结果表明，不同剂量的卡莫司汀植入剂植入裸鼠模型人肝癌肿瘤中，能明显抑制肿瘤生长，肿瘤抑制率与药物剂量呈明显量效关系。10％、20％和30％卡莫司汀植入剂肿瘤抑制率分别为58％、66％和84％，显著高于5％的实验组，与对照组比较P值均小于0.05。
进一步研究发现，肿瘤内植入卡莫司汀缓释植入剂对胃癌、结肠癌、淋巴瘤、子宫颈癌、肾癌等其它实体肿瘤也具有很好的治疗作用，其作用明显超过卡莫司汀腹腔注射组和局部注射组。另外，毒性反应明显低于卡莫司汀腹腔注射组和局部注射组。这一意外发现构成本发明的又一主要技术特征，为实体肿瘤的治疗提供了又一新的选择。
实施例17、不同分子量PLGA对缓释植入剂体内释放的的影响
以卡莫司汀作原料，分别以不同分子量的PLGA(50:50)为载体，按实施例7的制备工艺制成含10％卡莫司汀的缓释植入剂，分别为缓释植入剂A(载体为丙交酯-乙交酯共聚物，分子量为65,000)和缓释植入剂B(载体为丙交酯-乙交酯共聚物，分子量为80,000)。
将30只新西兰大白兔分为两大组，每大组含15只动物，进一步分为5个小组，每小组3只。脑内放置植入剂A和植入剂B，动物分别于给药后3天、7天、14天、21天和33活杀，取出脑内残余药粒，-20℃保存，用HPLC的方法测定卡莫司汀的剩余量，结果表明，植入剂A在家兔脑内药物释放平稳缓慢，3、7、14、21和33天的累积释药量分别为19.2％±2.0％、37.2％±0.5％、75.3％±4.2％、97.6％±1.6％和100.0％±0.0％。见表17及图2。
表17、卡莫司汀缓释植入剂在新西兰大白兔脑内的释药速度
Figure PCTCN2015091049-appb-000004
相比而言，植入剂B在家兔脑内药物释放太慢且有突释，其中3天的累积释药量为31.2％±4.0％，表现出明显突释，21天的累计释放约为80％，周期明显延长。由此可见，PLGA 的分子量以低于或等于65,000为优选。
把非标记卡莫斯汀制成BCNU聚苯丙生植入剂植入家兔脑内，给药后3、7、14、21和31天测缓释制剂的释药量，上述时间的累积释药量分别为72.4％±5.0％，98.9％±1.3％，100.0％±0.0％，100.0％±0.0％和100.0％±0.0％，药物在7天内释放绝大部分，14天内基本释放完毕，见图3。
实施例18、不同载体及不同分子量PLGA对缓释植入剂药代动力学的影响
试验材料和方法
(一)实验材料
1、药品和试剂
14C-卡莫斯汀：美国Moravek生物化学试剂公司生产，批号327-195-053，2004年12月22日生产，放化纯度98.4％，-20℃保存。
卡莫斯汀原料按现有技术制备
PLGA按现有技术制备
PLGA植入剂取实施例7的产品；
14C-卡莫斯汀缓释制剂、将14C-卡莫斯汀按实施例7的方法制备产品；
14C-卡莫斯汀与聚苯丙生(CPP和SA的混合物)按现有技术混合制成缓释制剂。
闪烁液：PerkinElmer公司生产的Permafluor E+液闪产品。
2、设备
冷冻切片机：Bright 8250型
氧化燃烧炉：PerkinElmer公司生产的307Packard样品氧化炉
液体闪烁记数器：      Beckman LS6500液闪仪
高效液相：            Agilent 1100系统
3、动物
昆明种大耳白家兔，雌雄各半，体重1.5kg～2.0kg，购自军事医学科学院实验动物中心，实验动物合格证号：SCXK(军)2002-001，动物实验设施使用许可证号：SYXK(军)2002-001。
(二)实验方法
1、药代实验设计及体外释放试验
家兔脑内植入14C-卡莫斯汀缓释制剂，每只1粒，放射剂量每粒185kBq，化学剂量每粒含卡莫斯汀0.3mg。大耳白家兔脑、血浆、心脏、肝脏、脾脏、肺脏、肾脏和脊髓组织用冷冻切片机纵向切片，每片厚度0.04mm，集合10片作为一个样品，称重，燃烧，测放射性，在药粒植入位置每只动物采集2个标本用于放射自显影，其他组织取0.3g燃烧，测定放射性浓度。21天活杀的2只动物每天收集粪便和尿液，测放射性，计算累积排泄量。
卡莫斯汀脑内植入缓释制剂的释放速度试验，每只兔脑内植入卡莫斯汀缓释制剂1粒，各组动物分别于给药后3天、7天、14天，21天和33天活杀，取出脑内残余药粒，-20℃保 存，用HPLC的方法测定卡莫斯汀的剩余量。
卡莫斯汀植入剂体外释放速度试验采用HPLC方法测定。
2、放射性测定
含有14C-卡莫斯汀的样品放入氧化燃烧炉中，在氧气和助燃剂的作用下，全部转化成二氧化碳和水，吸收剂吸收后，与闪烁液混合成无色透明的溶液，用液体闪烁计数仪测定放射性。氧化燃烧炉对3H和14C的放射性回收率大于97％，放射性的残留小于0.08％。液闪仪测定14C的计数效率计为70％。
3、药效试验
将一定数量癌瘤细胞经皮下注射于小白鼠季肋部，待肿瘤生长7天后将其随机分组接受相应治疗。植入剂剂量按公斤体重(mg/kg)计算，给药一次。植入后每两天测量肿瘤体积大小并记录动物体重变化，比较各组的治疗效果。用Excel软件进行数据处理和制图。
将14C标记的卡莫司汀作原料，分别以不同分子量的PLGA(50:50)和聚苯丙生为载体，按实施例7的制备工艺制备含10％卡莫司汀的缓释植入剂，分别为缓释植入剂A(载体为丙交酯-乙交酯共聚物，分子量为25,000)、缓释植入剂B(载体为丙交酯-乙交酯共聚物，分子量为20,000)和缓释植入剂C(聚苯丙生80:20)。10％卡莫司汀的缓释植入剂每粒含卡莫司汀0.84mg，放射剂量每粒80.7kBq。
将45只新西兰大白兔分为三大组，每大组含15只动物，进一步分为5个小组，每小组3只。脑内放置植入剂A、植入剂B和植入剂C，动物分别于给药后3天、7天、14天、21天和28活杀，取脑组织，横向切片测定脑组织切片的放射性。以药粒的植入部位为零距离，药物以浓度梯度的方式向周围扩散。用放射自显影和液体闪烁计数方法测定不同时间，不同扩散距离的药物浓度。
结果发现，植入剂A在兔脑组织内缓慢释放，扩散半径可达2cm，有效浓度的半径可达1.6cm，并维持28天以上；其中峰浓度在2-3周间，脑内药物浓度大于10μg/g的最大扩散距离为19mm，峰浓度为7907.5±9112.9μg·g-1，该浓度为全身给药时脑组织浓度的300倍以上，完全可以满足体内抑制肿瘤细胞的要求；植入剂B在兔脑组织内释放较快，给药后3天释放近50％，仅维持8天左右，其中峰浓度在4到6天之间，有效浓度的半径为0.6cm；缓释植入剂C给药后3天时药物浓度最高，以后逐渐下降，脑内药物浓度大于5μg·g-1的扩散距离为4.6mm，峰浓度为557.5μg·g-1。
实施例19
将9.0gPLGA(丙交酯与乙交酯的质量比例为50:50，粘度范围为0.30dl/g)放入容器中，加100ml二氯甲烷溶解混匀后加入1.0g卡莫司汀，混合均匀，制得的丙交酯-乙交酯共聚物与卡莫司汀混合液经经0.45μm微孔滤膜过滤器初滤和0.22μm微孔滤膜过滤器两次精 滤，制得无菌滤液，然后经无菌干燥，无菌成型，制得卡莫司汀(BCNU)缓释植入剂；
无菌成型无菌成型在熔融状态下进行，熔融温度为70℃，时间为1分钟。
所得缓释植入片，直径为1.4厘米，厚度1.0毫米。
经检测，制得的卡莫司汀(BCNU)缓释植入剂符合术后患处体内施用的无菌要求。

Claims (20)

  1. 一种卡莫司汀缓释植入剂，包括卡莫司汀及缓释辅料，所述的缓释辅料为丙交酯-乙交酯共聚物；所述卡莫司汀在卡莫司汀缓释植入剂中的重量百分比为7.5％～30％；所述丙交酯-乙交酯共聚物的重均分子量为25000～80000，丙交酯与乙交酯的质量比为(25～75):(75～25)，粘度为0.2～1.3 dl/g。
  2. 如权利要求1所述的卡莫司汀缓释植入剂，其特征在于，卡莫司汀的重量百分比为8％～25％；优选，卡莫司汀的重量百分比10～20％；最优，卡莫司汀的重量百分比为10％、15％或20％。
  3. 如权利要求1所述的卡莫司汀缓释植入剂，其特征在于，丙交酯-乙交酯共聚物共聚物的重均分子量为30000～60000；优选，35000～55000。
  4. 如权利要求1所述的卡莫司汀缓释植入剂，其特征在于，粘度为0.25～0.8 dl/g，优选0.3-0.6 dl/g；丙交酯与乙交酯的质量比为(50～75):(25～50)；优选50:50。
  5. 一种卡莫司汀缓释植入剂的制备方法，包括如下步骤：
    a、称取处方量的丙交酯-乙交酯共聚物，溶解于有机溶剂中，制得丙交酯-乙交酯共聚物溶液；
    所述丙交酯与乙交酯的质量比例选自(25～75):(75～25)；
    b、称取处方量的卡莫司汀加入上述丙交酯-乙交酯共聚物溶液中充分溶解混合形成丙交酯-乙交酯共聚物与卡莫司汀混合液；
    所述丙交酯-乙交酯共聚物与卡莫司汀混合液中卡莫司汀与丙交酯-乙交酯共聚物的重量比为(0.75～3):(7～9.25)；
    所述丙交酯-乙交酯共聚物的粘度为0.2～1.3 dl/g，重均分子量为25000～85000；
    c、将上述丙交酯-乙交酯共聚物与卡莫司汀混合液经过滤除菌，制得无菌滤液
    d、将无菌滤液经无菌干燥，制得微粉；
    e、将上述微粉经无菌成形后，制得卡莫司汀缓释植入剂。
  6. 如权利要求5所述的制备方法，其特征在于，所述步骤a中，有机溶剂选自氯甲烷、氯仿、乙酸乙酯或丙酮之一或两者以上的组合；进一步优选二氯甲烷。
  7. 如权利要求5所述的制备方法，其特征在于，所述步骤b中，丙交酯-乙交酯共聚物的粘度为0.25～0.8，优选0.3～0.6，最优0.3、0.35、0.4、0.45、0.50、0.55或0.6；
    根据本发明优选的，所述步骤a中，丙交酯与乙交酯的质量比例选自(50～75):(25～50)，优选50:50；
    优选的，丙交酯与乙交酯共聚物的重均分子量为30000～65000，优选35000～60000。
  8. 如权利要求5所述的制备方法，其特征在于，所述步骤b中，丙交酯-乙交酯共聚物与卡莫司汀混合液中卡莫司汀与丙交酯-乙交酯共聚物的重量比为优选(1～3):(7～9)，优选(1～2):(8～9)。
  9. 如权利要求5所述的制备方法，其特征在于，所述步骤c中，丙交酯-乙交酯共聚物与卡莫司汀混合液过滤包括经0.45μm微孔滤膜过滤器初滤和0.22μm微孔滤膜过滤器两次精滤。
  10. 如权利要求5所述的制备方法，其特征在于，所述步骤d中，无菌滤液经无菌干燥制成微粉所应用的方法均为在无菌条件下，方法选自：乳化法、喷雾干燥法、溶剂挥发法、有机萃取法或冷冻粉粹法；优选，冷冻粉粹法或喷雾干燥法；最优，冷冻粉粹法。
  11. 一种卡莫司汀缓释植入剂的制备方法，其特征在于，包括如下步骤：
    (1)称取处方量的丙交酯-乙交酯共聚物，溶解于有机溶剂中，制得丙交酯-乙交酯共聚物溶液；
    所述丙交酯(GA)与乙交酯(LA)的质量比例选自(25～75):(75～25)；
    (2)称取处方量的卡莫司汀加入上述丙交酯-乙交酯共聚物溶液中充分溶解混合形成丙交酯-乙交酯共聚物与卡莫司汀混合液；
    所述丙交酯-乙交酯共聚物与卡莫司汀混合液中卡莫司汀与丙交酯-乙交酯共聚物的重量比为(0.75～4):(6～9.25)；即卡莫司汀在缓释植入剂中的重量百分比为7.5％～40％；
    (3)将步骤(2)中制得的丙交酯-乙交酯共聚物与卡莫司汀混合液经过滤除菌，制得无菌滤液；
    (4)上述无菌滤液经无菌干燥后，制得微粉；
    (5)将上述微粉进行无菌成型，无菌成型在熔融状态下进行，熔融温度为25～70℃，时间为1～10分钟，制得卡莫司汀缓释植入剂；
    或者，
    ③将步骤(2)中制得的丙交酯-乙交酯共聚物与卡莫司汀混合液进行干燥，制得未灭菌微粉；
    ④将上述未灭菌微粉进行成型，成型在熔融状态下进行，熔融温度为25～70℃，时间为1～10分钟，经钴60照射灭菌，制得卡莫司汀缓释植入剂。
  12. 如权利要求11所述的制备方法，其特征在于，所述步骤(1)中，有机溶剂选自氯甲烷、氯仿、乙酸乙酯或丙酮之一或两者以上的组合；进一步优选二氯甲烷。
  13. 如权利要求11所述的制备方法，其特征在于，所述步骤(2)中，丙交酯-乙交酯共聚物的粘度为0.25～0.8 dl/g，优选0.3～0.6 dl/g，最优0.3 dl/g、0.35 dl/g、0.4 dl/g、0.45 dl/g、0.50 dl/g、0.55 dl/g或0.6 dl/g。
  14. 如权利要求11所述的制备方法，其特征在于，所述步骤(1)中，丙交酯与乙交酯的质量比例为(50～75):(25～50)，优选50:50；
    丙交酯与乙交酯共聚物的重均分子量为25000～85000，优选30000～65000，最优35000～60000。
  15. 如权利要求11所述的制备方法，其特征在于，所述步骤(2)中，丙交酯-乙交酯 共聚物与卡莫司汀混合液中卡莫司汀与丙交酯-乙交酯共聚物的重量比为(1～3):(7～9)；优选(1～2):(8～9)。
  16. 如权利要求11所述的制备方法，其特征在于，所述步骤(3)中，丙交酯-乙交酯共聚物与卡莫司汀混合液过滤包括经0.45μm微孔滤膜过滤器初滤和0.22μm微孔滤膜过滤器两次精滤。
  17. 如权利要求11所述的制备方法，其特征在于，所述步骤(4)或③中，干燥方法选自：乳化法、喷雾干燥法、溶剂挥发法、有机萃取法或冷冻粉粹法；优选，冷冻粉粹法或喷雾干燥法；最优，冷冻粉粹法。
  18. 如权利要求11所述的制备方法，其特征在于，所述步骤(5)或④中，熔融温度为30～60℃，优选40～60℃，时间为1～8分钟，优选3～5分钟。
  19. 如权利要求5或11所述的制备方法，其特征在于，还包括无菌包装的步骤。
  20. 权利要求1～4所述的卡莫司汀缓释植入剂在制备用于治疗癌症药物中的应用。
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