CN1925874B - Compositions and methods of delivering agents - Google Patents
Compositions and methods of delivering agentsDownload PDFInfo
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- CN1925874B CN1925874BCN200380109606.9ACN200380109606ACN1925874BCN 1925874 BCN1925874 BCN 1925874BCN 200380109606 ACN200380109606 ACN 200380109606ACN 1925874 BCN1925874 BCN 1925874B
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Abstract
Description
Translated fromChinese对相关专利申请的交叉引用Cross References to Related Patent Applications
本专利申请要求在2002年12月9日提出的美国临时专利申请60/432,317、在2003年12月3日提出的美国临时专利申请(律师备案号225519)、在2003年12月4日提出的美国临时专利申请(律师备案号225549)和在2003年12月5日提出的美国临时专利申请(律师备案号225585)的利益。This patent application claims U.S. Provisional Patent Application 60/432,317 filed December 9, 2002, U.S. Provisional Patent Application (Attorney Docket No. 225519) filed December 3, 2003, filed December 4, 2003 Benefit of U.S. Provisional Patent Application (Attorney Docket No. 225549) and U.S. Provisional Patent Application (Attorney Docket No. 225585) filed December 5, 2003.
发明领域field of invention
本发明涉及用于肠胃外或其他内部使用、包含药物活性剂的药物组合物，其与可获的类似药物制剂比较时具有降低在给药时某些不希望有的副作用的效果。The present invention relates to pharmaceutical compositions for parenteral or other internal use comprising pharmaceutically active agents which have the effect of reducing certain undesirable side effects upon administration when compared with similar pharmaceutical preparations available.
发明背景Background of the invention
公认许多用于肠胃外使用的药物，特别是静脉内给药的那些，导致不希望有的副作用如静脉刺激，静脉炎，注射时的烧灼感和疼痛，静脉血栓形成，外渗和其它给药相关的副作用。这些药物中的许多不溶于水，因此与增溶剂、表面活性剂、溶剂和/或乳化剂配制，当给药于患者时这些增溶剂、表面活性剂、溶剂和/或乳化剂是刺激性的、变应性的或有毒的(参见例如，Briggs等，Anesthesis 37，1099(1982)，和Waugh等，Am.J.Hosp.Pharmacists，48，1520(1991))。经常，存在于制剂中的游离药物在给药后诱导疼痛或刺激。例如，在作为晚期非小细胞肺癌的一线化学疗法接受外周静脉给药异环磷酰胺和长春瑞滨的患者中，患者中的50％观察到了静脉炎。(参见例如Vallejo等，Am.J.Clin.Oncol.，19(6)，584-8(1996))。此外，已经显示万古霉素诱导副作用如静脉炎(参见例如，Lopes Rocha等，Braz.J.Infect.Dis.，6(4)，196-200(2002))。在实体瘤患者中使用顺铂、吉西他滨和SU5416已经导致不利事件如深度静脉血栓形成和静脉炎(参见例如，Kuenen等，J.Clin.Oncol.，20(6)，1657-67(2002))。另外，丙泊酚(一种麻醉剂)在注射时可以诱导疼痛，烧灼感和静脉刺激，特别是当作为卵磷脂稳定的脂肪乳剂给药时(参见例如，Tan等，Anathesia，53，468-76，(1998))。其它显示给药相关副作用的药物包括例如泰素(紫杉醇)(参见例如，泰素的包装说明书I.V.)，codarone(盐酸胺碘酮)(参见例如，Codarone的包装说明书)，甲状腺激素T3或碘塞罗宁(可作为复方碘塞罗宁商购)，塞替派，博来霉素，和诊断放射造影剂。It is recognized that many drugs intended for parenteral use, especially those administered intravenously, cause undesirable side effects such as vein irritation, phlebitis, burning sensation and pain upon injection, venous thrombosis, extravasation and other administration associated side effects. Many of these drugs are insoluble in water and are therefore formulated with solubilizers, surfactants, solvents and/or emulsifiers which are irritating when administered to patients , allergic or toxic (see, eg, Briggs et al., Anesthesis 37, 1099 (1982), and Waugh et al., Am. J. Hosp. Pharmacists, 48, 1520 (1991)). Often, free drug present in the formulation induces pain or irritation after administration. For example, in patients who received peripheral intravenous ifosfamide and vinorelbine as first-line chemotherapy for advanced non-small cell lung cancer, phlebitis was observed in 50% of patients. (See eg Vallejo et al., Am. J. Clin. Oncol., 19(6), 584-8 (1996)). Furthermore, vancomycin has been shown to induce side effects such as phlebitis (see eg Lopes Rocha et al., Braz. J. Infect. Dis., 6(4), 196-200 (2002)). The use of cisplatin, gemcitabine and SU5416 in patients with solid tumors has resulted in adverse events such as deep vein thrombosis and phlebitis (see, e.g., Kuenen et al., J. Clin. Oncol., 20(6), 1657-67 (2002)) . In addition, propofol, an anesthetic, can induce pain, burning and venous irritation when injected, especially when administered as a lecithin-stabilized lipid emulsion (see, e.g., Tan et al., Anathesia, 53, 468-76 , (1998)). Other drugs showing administration-related side effects include, for example, taxol (paclitaxel) (see, for example, Taxol's package insert I.V.), codarone (amiodarone hydrochloride) (see, for example, Codarone's package insert), thyroid hormone T3 or iodine Ronine (commercially available as liothyronine compound), thiotepa, bleomycin, and diagnostic radiocontrast agents.
与制备注射用药物、特别是水不溶性药物相关的另一问题是确保无菌。通过在制备前将所有组分绝对消毒，接着通过在所有制备阶段中的绝对无菌技术可以完成药物乳剂/分散体的无菌制备。然而，这些方法耗时且昂贵。另外，在制备或贮存期间通过暴露于空气而氧化药物制剂可以导致例如降低的pH，药物降解，和变色，由此使药物制剂不稳定和/或减小了贮藏寿命。Another problem associated with the preparation of injectable drugs, especially water insoluble drugs, is ensuring sterility. The aseptic preparation of pharmaceutical emulsions/dispersions can be accomplished by absolute sterilization of all components prior to preparation, followed by absolute aseptic technique at all stages of preparation. However, these methods are time-consuming and expensive. Additionally, oxidation of the drug formulation by exposure to air during manufacture or storage can lead to, for example, lowered pH, drug degradation, and discoloration, thereby destabilizing the drug formulation and/or reducing shelf life.
为了避免药物制剂与给药相关的副作用有关的问题，已经尝试代替的制剂。例如关于丙泊酚，降低丙泊酚诱导的疼痛的方法包括增加溶剂的脂肪含量(例如长链甘油三酸酯(LCT))，术前用药法，用非甾族药物预处理，局部麻醉，阿片样物质，加入利多卡因，加入环糊精，和微量过滤法(参见例如，Mayer等，Anaesthesist，45(11)，1082-4(1996)，Davies，等Anaesthesia，57，557-61(2002)，Doenicke，等，Anaesth.Analg.，82，472-4(1996)，Larsen等，Anaesthesitis，50，842-5(2001)，Lilley等，Anaesthesia，51，815-8(1996)，Bielen等，Anaesth.Analg.，82(5)，920-4(1996)，和Knibbe等，Br.J.Clin.Pharmacol.，47(6)，653-60(1999))。然而，这些制剂诱导其它副作用(例如心血管并发症)或导致丙泊酚制剂的不稳定。In order to avoid the problems of pharmaceutical formulations with administration-related side effects, alternative formulations have been attempted. For example with respect to propofol, approaches to reduce propofol-induced pain include increasing the fat content of solvents (e.g. long-chain triglycerides (LCT)), premedication, pretreatment with non-steroidal drugs, local anesthesia, Opioids, addition of lidocaine, addition of cyclodextrin, and microfiltration (see, e.g., Mayer et al., Anaesthesist, 45(11), 1082-4 (1996), Davies, et al. Anaesthesia, 57, 557-61( 2002), Doenicke, et al., Anaesth. Analg., 82, 472-4 (1996), Larsen et al., Anaesthesitis, 50, 842-5 (2001), Lilley et al., Anaesthesia, 51, 815-8 (1996), Bielen et al., Anaesth. Analg., 82(5), 920-4 (1996), and Knibbe et al., Br. J. Clin. Pharmacol., 47(6), 653-60 (1999)). However, these formulations induce other side effects (such as cardiovascular complications) or lead to instability of propofol formulations.
为了克服细菌污染的问题，已经用抗菌剂如EDTA等价物(如乙二胺四乙酸盐)、喷替酸盐或含亚硫酸盐的试剂开发丙泊酚制剂，或者将它们用较低pH配制(参见例如，美国专利5,714,520、5,731,355、5,731,356、6,028,108、6,100,302、6,147,122、6,177,477、6,399,087、6,469,069和国际专利申请号WO 99/39696)。然而，由于乙二胺四乙酸盐和喷替酸盐是金属离子螯合剂，它们具有从身体细胞中清除必需金属离子的危险潜力。此外，将亚硫酸盐加入药物制剂对儿科人群和对硫敏感的普通人群中的那些带来了潜在的不利作用。To overcome the problem of bacterial contamination, formulations of propofol have been developed with antimicrobial agents such as EDTA equivalents (eg, ethylenediaminetetraacetate), pentetate, or sulfite-containing agents, or they have been formulated at a lower pH (See, e.g., U.S. Patents 5,714,520, 5,731,355, 5,731,356, 6,028,108, 6,100,302, 6,147,122, 6,177,477, 6,399,087, 6,469,069 and International Patent Application No. WO 99/39696). However, because EDTA and pentetate are metal ion chelators, they have the dangerous potential to remove essential metal ions from the body's cells. Furthermore, the addition of sulfites to pharmaceutical formulations poses potential adverse effects in the pediatric population and those in the sulfur-sensitive general population.
因此，仍需要降低或消除与肠胃外或体内给药药物有关的副作用的组合物和方法。还需要无菌的药物组合物和制备这种组合物的方法。另外，需要降低或消除药物制剂氧化以防止药物不稳定的药物组合物和方法。Accordingly, there remains a need for compositions and methods that reduce or eliminate side effects associated with parenterally or internally administered drugs. There is also a need for sterile pharmaceutical compositions and methods of preparing such compositions. Additionally, there is a need for pharmaceutical compositions and methods that reduce or eliminate oxidation of pharmaceutical formulations to prevent drug instability.
本发明提供这些组合物和方法。本发明的这些和其它优势以及另外的发明特征将从这里提供的本发明的描述中变得明显。The present invention provides these compositions and methods. These and other advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
发明概述Summary of the invention
本发明提供药物组合物的各种实施方案。各种实施方案中的一个，一些或所有性能可以在本发明不同的实施方案中发现并且仍然属于后附权利要求的范围内。The present invention provides various embodiments of pharmaceutical compositions. One of the various embodiments, some or all of the features may be found in different embodiments of the invention and still fall within the scope of the appended claims.
本发明提供一种包含药剂和药用载体的药物组合物，其中所述药用载体包含蛋白质如清蛋白，更优选人血清清蛋白，其量有效降低一种或多种将药物组合物给药于人的副作用，且其中所述药用载体包含去铁胺，其量有效抑制药物组合物中的微生物生长。本发明还提供包含药剂和药用载体的药物组合物，其中所述药用载体包含蛋白质如清蛋白，其量有效降低一种或多种将药物组合物给药于人的副作用，且其中所述药用载体包含去铁胺，其量有效抑制药物组合物中的氧化。The present invention provides a pharmaceutical composition comprising a medicament and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a protein such as albumin, more preferably human serum albumin, in an amount effective to reduce one or more of the effects of administering the pharmaceutical composition. side effects on humans, and wherein the pharmaceutical carrier comprises deferoxamine in an amount effective to inhibit the growth of microorganisms in the pharmaceutical composition. The present invention also provides a pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a protein, such as albumin, in an amount effective to reduce one or more side effects of administering the pharmaceutical composition to a human, and wherein the The pharmaceutical carrier comprises deferoxamine in an amount effective to inhibit oxidation in the pharmaceutical composition.
本发明提供降低一种或多种与将药物组合物给药于人相关的副作用的方法，该方法包含(a)向人给药包含药剂和药用载体的药物组合物，其中所述药用载体包含清蛋白和去铁胺。本发明还提供用于在药物组合物中抑制微生物生长，或抑制氧化，或抑制微生物生长和氧化的方法。这些方法包含制备包含药剂和药用载体的药物组合物，其中所述药用载体以有效抑制药物组合物中微生物生长的量或以有效抑制药物组合物氧化的量包含去铁胺。The present invention provides a method of reducing one or more side effects associated with administering a pharmaceutical composition to a human, the method comprising (a) administering to the human a pharmaceutical composition comprising a pharmaceutical agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable The carrier contains albumin and deferoxamine. The present invention also provides methods for inhibiting the growth of microorganisms, or inhibiting oxidation, or inhibiting the growth and oxidation of microorganisms in pharmaceutical compositions. These methods comprise preparing a pharmaceutical composition comprising a medicament and a pharmaceutically acceptable carrier, wherein the pharmaceutical carrier comprises deferoxamine in an amount effective to inhibit the growth of microorganisms in the pharmaceutical composition or in an amount effective to inhibit oxidation of the pharmaceutical composition.
本发明还提供用于增强药剂向虚弱部位转运的方法，该方法包含向人给药包含药剂和药用载体的药物组合物，其中所述药用载体包含清蛋白，且其中清蛋白与药物组合物中的药剂的比率为约18∶1或更小。本发明进一步提供用于增强药剂与体外或体内细胞结合的方法，该方法包含向所述体外或体内细胞给药包含药剂和药用载体的药物组合物，其中所述药用载体包含清蛋白，且其中清蛋白与药物组合物中的药剂的比率为约18∶1或更小。The present invention also provides a method for enhancing delivery of an agent to a site of infirmity, the method comprising administering to a human a pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises albumin, and wherein the albumin is combined with the agent The ratio of the agents in the drug is about 18:1 or less. The present invention further provides a method for enhancing the binding of an agent to a cell in vitro or in vivo, the method comprising administering to the cell in vitro or in vivo a pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises albumin, And wherein the ratio of albumin to agent in the pharmaceutical composition is about 18:1 or less.
本发明还提供包含药剂和药用载体的药物组合物，其中所述药用载体以有效增加药物向人虚弱部位转运的量包含清蛋白，且其中清蛋白与药剂的比率为约18∶1或更小。The present invention also provides a pharmaceutical composition comprising a medicament and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises albumin in an amount effective to increase the delivery of the medicament to a site of infirmity in a human, and wherein the ratio of albumin to the medicament is about 18:1 or smaller.
本发明进一步提供通过将所述药剂与蛋白质组合来增加药剂向体外或体内细胞转运的方法，其中所述蛋白质结合所述细胞上的特异细胞表面受体，其中所述蛋白质-药剂组合与所述受体的结合导致转运发生，且其中清蛋白与药剂的比率为约18∶1或更小。The invention further provides a method of increasing the transport of an agent to a cell in vitro or in vivo by combining said agent with a protein, wherein said protein binds to a specific cell surface receptor on said cell, wherein said protein-agent combination is associated with said Binding of the receptor results in translocation with a ratio of albumin to agent of about 18:1 or less.
发明详述Detailed description of the invention
本发明提供一种包含药剂和药用载体的药物组合物，其中所述药用载体包含蛋白质如清蛋白，优选人血清清蛋白，其量有效降低一种或多种将药物组合物给药于人的副作用，且其中所述药用载体包含去铁胺，其量有效抑制药物组合物中的微生物生长。本发明还提供包含药剂和药用载体的药物组合物，其中所述药用载体包含蛋白质如清蛋白，其量有效降低一种或多种将药物组合物给药于人的副作用，且其中所述药用载体包含去铁胺，其量有效抑制药物组合物中的氧化。The present invention provides a pharmaceutical composition comprising a medicament and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a protein such as albumin, preferably human serum albumin, in an amount effective to reduce one or more of the conditions in which the pharmaceutical composition is administered. side effects in humans, and wherein the pharmaceutically acceptable carrier comprises deferoxamine in an amount effective to inhibit the growth of microorganisms in the pharmaceutical composition. The present invention also provides a pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a protein, such as albumin, in an amount effective to reduce one or more side effects of administering the pharmaceutical composition to a human, and wherein the The pharmaceutical carrier comprises deferoxamine in an amount effective to inhibit oxidation in the pharmaceutical composition.
任何适当的药剂可以用于本发明的药物组合物中。适当的药剂包括但不限于抗癌剂或抗肿瘤药，抗微管剂，免疫抑制剂，麻醉剂，激素，用于心血管病症的药剂，抗心率失常药，抗生素，抗真菌剂，抗高血压药，抗喘药，镇痛药，抗炎药，抗关节炎剂，和血管活性剂。本发明还与许多其它药物类别有效。更具体地，适当的药剂包括但不限于紫杉烷类，(例如泰素
泰素
丙泊酚(2，6-二异丙基苯酚)是疏水性的、水不溶性油，广泛用作静脉麻醉药来诱导和保持人和动物的全身麻醉和镇静。丙泊酚典型地直接给药于血流中并通过血脑屏障。包含丙泊酚的药物组合物必须具有足够的脂溶性以通过该屏障和抑制大脑的相关机制。丙泊酚在水中在22.5℃具有的最大溶解度为1.0+/-0.02μM(参见例如，Tonner等，Anesthesiology，77，926-931(1992))。因此，丙泊酚通常作为含有增溶剂、表面活性剂、溶剂的乳剂配制，或作为水包油型乳剂配制(参见例如，U.S.专利6,150,423、6,326,406和6,362,234)。除了活性药剂以外，本发明的组合物还包括药物载体或赋形剂。载体的选择不一定是关键的，任何本领域已知的载体可以用于组合物中。载体的选择优选部分地通过药物组合物给药的具体部位和用于给药药物组合物的具体方法来确定。优选地，药用载体包含蛋白质。可以使用任何适当的蛋白质。适当蛋白质的实例包括但不限于清蛋白，包括IgA的免疫球蛋白，脂蛋白，载脂蛋白B，β-2-巨球蛋白，甲状腺球蛋白等。最优选地，药用载体包含清蛋白，最优选人血清清蛋白。适合于本发明的包括清蛋白的蛋白质可以是天然来源的或者合成制备的。Propofol (2,6-diisopropylphenol) is a hydrophobic, water-insoluble oil widely used as an intravenous anesthetic to induce and maintain general anesthesia and sedation in humans and animals. Propofol is typically administered directly into the bloodstream and across the blood-brain barrier. Pharmaceutical compositions containing propofol must be sufficiently lipid soluble to pass this barrier and inhibit the associated mechanisms of the brain. Propofol has a maximum solubility in water of 1.0 +/- 0.02 μΜ at 22.5°C (see eg Tonner et al., Anesthesiology, 77, 926-931 (1992)). Accordingly, propofol is typically formulated as an emulsion containing solubilizers, surfactants, solvents, or as an oil-in-water emulsion (see, eg, U.S. Patents 6,150,423, 6,326,406, and 6,362,234). In addition to the active agent, the compositions of the present invention include pharmaceutical carriers or excipients. The choice of carrier is not necessarily critical, and any carrier known in the art may be used in the composition. The choice of carrier is preferably determined in part by the particular site of administration of the pharmaceutical composition and the particular method used to administer the pharmaceutical composition. Preferably, the pharmaceutical carrier comprises a protein. Any suitable protein can be used. Examples of suitable proteins include, but are not limited to, albumin, immunoglobulins including IgA, lipoproteins, apolipoprotein B, beta-2-macroglobulin, thyroglobulin, and the like. Most preferably, the pharmaceutical carrier comprises albumin, most preferably human serum albumin. Proteins, including albumin, suitable for the present invention may be of natural origin or synthetically prepared.
人血清清蛋白(HSA)是Mr 65K的高度可溶的球蛋白，由585个氨基酸组成。HSA是血浆中最丰富的蛋白质并且占人血浆胶体渗透压的70-80％。HSA的氨基酸序列包含总共17个二硫桥，一个自由硫醇(Cys 34)，和单个色氨酸(Trp 214)。静脉使用HSA溶液已经显示用于预防和治疗低血容量性休克(参见例如，Tullis，JAMA，237，355-360，460-463，(1977))和Houser等，Surgery，Gynecology and Obstetrics，150，811-816(1980))和换血疗法一起用于治疗新生儿血胆红素过多(参见例如，Finlayson，Seminars in Thrombosisand Hemostasis，6，85-120，(1980))。Human serum albumin (HSA) is a highly soluble globulin of Mr 65K, consisting of 585 amino acids. HSA is the most abundant protein in plasma and accounts for 70-80% of the colloid osmotic pressure of human plasma. The amino acid sequence of HSA contains a total of 17 disulfide bridges, one free thiol (Cys 34), and a single tryptophan (Trp 214). Intravenous administration of HSA solutions has been shown to prevent and treat hypovolemic shock (see, e.g., Tullis, JAMA, 237, 355-360, 460-463, (1977)) and Houser et al., Surgery, Gynecology and Obstetrics, 150, 811-816 (1980)) together with exchange transfusion for the treatment of neonatal hyperbilirubinemia (see eg, Finlayson, Seminars in Thrombosis and Hemostasis, 6, 85-120, (1980)).
人血清清蛋白(HSA)具有多个疏水结合位点(总共8个针对脂肪酸，HSA的内源性配体)和结合各种各样的药物，特别是中性或带负电的疏水化合物(Goodman等，The Pharmacological Basis of Therapeutics，第9版，McGraw-Hill New York(1996))。在HSA的亚结构域IIA和IIIA已经提出了两个高亲和力结合位点，它们是高度延伸的疏水囊，在表面附近具有带电的赖氨酸和精氨酸残基，用作极性配体特征的连接点(参见例如，Fehske等，Biochem.Pharmcol.，30，687-92(1981)，Vorum，Dan.Med.Bull.，46，379-99(1999)，Kragh-Hansen，Dan.Med.Bull.，1441，131-40(1990)，Curry等，Nat.Struct.Biol.，5，827-35(1998)，Sugio等，Protein.Eng.，12，439-46(1999)，He等，Nature，358，209-15(1992)，和Carter等，Adv.Protein.Chem.，45，153-203(1994))。紫杉醇和丙泊酚已经显示结合HSA(参见例如，Paal等，Eur.J.Biochem.，268(7)，2187-91(2001)，Purcell等，Biochim.Biophys.Acta，1478(1)，61-8(2000)，Altmayer等，Arzneimittelforschung，45，1053-6(1995)，和Garrido等，Rev.Esp.Anestestiol.Reanim.，41，308-12(1994))。另外，多西他赛已经显示结合人血浆蛋白(参见例如，Urien等，Invest.NewDrugs，14(2)，147-51(1996))。因此，尽管不希望束缚于任何一种具体理论，但是认为将蛋白质如清蛋白包括在本发明的药物组合物中导致与给药药物组合物有关的副作用的降低，这至少部分是由于人血清清蛋白与存在于组合物中的任何游离药物的结合。Human serum albumin (HSA) has multiple hydrophobic binding sites (8 in total for fatty acids, endogenous ligands of HSA) and binds a wide variety of drugs, especially neutral or negatively charged hydrophobic compounds (Goodman et al., The Pharmacological Basis of Therapeutics, 9th ed., McGraw-Hill New York (1996)). Two high-affinity binding sites have been proposed in subdomains IIA and IIIA of HSA, which are highly extended hydrophobic pockets with charged lysine and arginine residues near the surface that serve as polar ligands Featured junctions (see for example, Fehske et al., Biochem.Pharmcol., 30, 687-92 (1981), Vorum, Dan. Med. Bull., 46, 379-99 (1999), Kragh-Hansen, Dan. Med .Bull., 1441, 131-40(1990), Curry et al., Nat.Struct.Biol., 5, 827-35(1998), Sugio et al., Protein.Eng., 12, 439-46(1999), He et al., Nature, 358, 209-15 (1992), and Carter et al., Adv. Protein. Chem., 45, 153-203 (1994)). Paclitaxel and propofol have been shown to bind HSA (see, e.g., Paal et al., Eur. J. Biochem., 268(7), 2187-91 (2001), Purcell et al., Biochim. Biophys. Acta, 1478(1), 61 -8 (2000), Altmayer et al., Arzneimittelforschung, 45, 1053-6 (1995), and Garrido et al., Rev. Esp. Anestestiol. Reanim., 41, 308-12 (1994)). In addition, docetaxel has been shown to bind human plasma proteins (see eg, Urien et al., Invest. New Drugs, 14(2), 147-51 (1996)). Thus, while not wishing to be bound by any one particular theory, it is believed that the inclusion of proteins such as albumin in the pharmaceutical compositions of the present invention results in a reduction in the side effects associated with administering the pharmaceutical compositions, at least in part due to human serum serum Binding of protein to any free drug present in the composition.
包括在本发明药物组合物中的清蛋白的量将根据药物活性剂、其它赋形剂和预期给药的途径和位点而变化。理想地，包括在组合物中的清蛋白的量是由于向人给药本发明的药物组合物有效降低一种或多种活性药剂的副作用的量。典型地，将药物组合物制备成液体形式，然后将清蛋白加入至溶液中。优选地，液体形式的药物组合物包含约0.1重量％至约25重量％(例如约0.5重量％，约5重量％，约10重量％，约15重量％，或约20重量％)的清蛋白。最优选地，液体形式的药物组合物包含约0.5重量％至约5重量％的清蛋白。药物组合物可以例如通过冻干、喷雾干燥、流化床干燥、湿式制粒法和其它本领域已知的适当方法脱水。当以固体形式，如通过湿式制粒法、流化床干燥和本领域技术人员已知的其它方法制备组合物时，优选将清蛋白作为溶液涂覆于活性药剂和其它赋形剂(如果存在)。HSA溶液优选有约0.1重量％至约25重量％(约0.5重量％，约5重量％，约10重量％，约15重量％，或约20重量％)的清蛋白。The amount of albumin included in the pharmaceutical compositions of the invention will vary depending on the pharmaceutically active agent, other excipients and the intended route and site of administration. Ideally, the amount of albumin included in the composition is an amount effective to reduce the side effects of one or more active agents upon administration of the pharmaceutical composition of the invention to a human. Typically, the pharmaceutical composition is prepared in liquid form and albumin is added to the solution. Preferably, the pharmaceutical composition in liquid form comprises from about 0.1% to about 25% by weight (e.g., about 0.5%, about 5%, about 10%, about 15%, or about 20% by weight) of albumin . Most preferably, liquid form pharmaceutical compositions comprise from about 0.5% to about 5% by weight albumin. The pharmaceutical composition can be dehydrated, for example, by lyophilization, spray drying, fluid bed drying, wet granulation and other suitable methods known in the art. When preparing the composition in solid form, such as by wet granulation, fluid bed drying and other methods known to those skilled in the art, the albumin is preferably applied as a solution to the active agent and other excipients (if present). ). The HSA solution preferably has from about 0.1% to about 25% by weight (about 0.5% by weight, about 5% by weight, about 10% by weight, about 15% by weight, or about 20% by weight) of albumin.
除了清蛋白以外，本发明的组合物优选还包含去铁胺。去铁胺是分离自Streptomyces pilous的天然产物，能够形成铁络合物。例如用于注射USP的甲磺酸去铁胺被食品与药物管理局批准为铁螯合剂且可供用于肌内、皮下和静脉内给药。甲磺酸去铁胺USP是白色至灰白色的粉末。它易溶于水并且它的分子量是656.79。甲磺酸去铁胺的化学名称是N-[5-[3-[(5-氨基戊基)-羟基氨基甲酰基]-丙-酰氨基]戊基]-3[[5-((N-羟基乙酰氨基)戊基]-氨基甲酰基]丙酰异羟肟酸一甲磺酸盐(盐)，它的结构式是C25H48N6O8.CH3SO3H。如实施例所述，去铁胺或其类似物、衍生物或盐(例如甲磺酸盐)抑制药物组合物中的微生物生长和氧化，认为它结合组合物中的游离药物。去铁胺还显示结合酚类化合物(参见例如，Juven等，J.Appl.Bacteriol.，76(6)，626-31(1994))。紫杉醇，多西他赛，丙泊酚等或者类似酚或者具有酚或苯基取代基。因此，认为去铁胺可以结合本发明药物组合物中的游离药物或减少本发明药物组合物中的游离药物的量，由此还降低或减轻了注射时的刺激或疼痛。In addition to albumin, the composition of the invention preferably also comprises deferoxamine. Desferoxamine is a natural product isolated from Streptomyces pilous capable of forming iron complexes. For example deferoxamine mesylate for injection USP is approved by the Food and Drug Administration as an iron chelator and is available for intramuscular, subcutaneous and intravenous administration. Desferoxamine mesylate USP is a white to off-white powder. It is easily soluble in water and its molecular weight is 656.79. The chemical name of deferoxamine mesylate is N-[5-[3-[(5-aminopentyl)-hydroxycarbamoyl]-propanylamino]pentyl]-3[[5-((N -Hydroxyacetamido) pentyl]-carbamoyl] propionyl hydroxamic acid monomethanesulfonate (salt), its structural formula is C25 H48 N6 O8 .CH3 SO3 H. As in the examples As stated, deferoxamine or its analogs, derivatives or salts (e.g. mesylate) inhibit microbial growth and oxidation in pharmaceutical compositions and it is believed to bind free drug in the composition. Deferoxamine has also been shown to bind phenolic (See, e.g., Juven et al., J.Appl.Bacteriol., 76(6), 626-31 (1994)). Paclitaxel, docetaxel, propofol, etc. or similar to phenols or with phenol or phenyl substitution Therefore, it is considered that deferoxamine can bind to or reduce the amount of free drug in the pharmaceutical composition of the present invention, thereby also reducing or alleviating irritation or pain during injection.
包括在组合物中的去铁胺或其优选盐(即去铁胺的甲磺酸盐)的量将取决于活性药剂和其它赋形剂。理想地，在组合物中去铁胺、其盐及其类似物的量是有效抑制微生物生长和/或抑制氧化的量。如上所述，典型地药物组合物以液体形式制备，然后将去铁胺及其类似物以溶液形式加入。优选地，液体形式的药物组合物包含约0.0001重量％至约0.5重量％(例如，约0.005重量％，约0.1重量％，或约0.25重量％)的去铁胺、其盐或其类似物。更优选地，液体形式的组合物包含类似量的优选的去铁胺盐，甲磺酸去铁胺。更优选地，液体形式的药物组合物包含约0.1重量％的甲磺酸去铁胺。当以固体形式，如上所述，如通过湿式制粒法、流化床干燥和本领域技术人员已知的其它方法制备组合物时，优选将甲磺酸去铁胺作为溶液加入活性药剂和其它赋形剂(如果存在)。甲磺酸去铁胺溶液优选有约0.0001重量％至约0.5重量％(例如，约0.005重量％，约0.1重量％，或约0.25重量％)的去铁胺。The amount of deferoxamine or its preferred salt (ie deferoxamine mesylate) included in the composition will depend on the active agent and other excipients. Desirably, the amount of deferoxamine, its salts, and analogs thereof in the composition is an amount effective to inhibit microbial growth and/or inhibit oxidation. As noted above, typically the pharmaceutical composition is prepared in liquid form and then deferoxamine and its analogs are added in solution. Preferably, liquid form pharmaceutical compositions comprise from about 0.0001% to about 0.5% by weight (eg, about 0.005%, about 0.1%, or about 0.25% by weight) of deferoxamine, a salt thereof, or an analog thereof. More preferably, the composition in liquid form comprises a similar amount of the preferred salt of deferoxamine, deferoxamine mesylate. More preferably, the pharmaceutical composition in liquid form comprises about 0.1% by weight of deferoxamine mesylate. When preparing the composition in solid form, as described above, such as by wet granulation, fluid bed drying and other methods known to those skilled in the art, deferoxamine mesylate is preferably added as a solution to the active agent and other Excipients (if present). The deferoxamine mesylate solution preferably has from about 0.0001% to about 0.5% by weight (eg, about 0.005%, about 0.1%, or about 0.25% by weight) of deferoxamine.
与本发明一致，药物组合物可以包括其它药剂，赋形剂或稳定剂以改善组合物的性能。例如，为了通过增加纳米粒或纳米滴的负ζ电位来增加稳定性，可以加入某些带负电组分。这些带负电组分包括但不限于胆汁酸的胆汁盐类，所述胆汁酸由下列各项组成：甘氨胆酸，胆酸，鹅脱氧胆酸，牛磺胆酸，甘鹅脱氧胆酸(glycochenodeoxycholic acid)，牛磺鹅脱氧胆酸(taurochenodeoxycholic acid)，石胆酸，乌索脱氧胆酸(ursodeoxycholic acid)，脱氢胆酸和其它；磷脂，包括卵磷脂(蛋黄)基磷脂，该卵磷脂(蛋黄)基磷脂包括下列磷脂酰胆碱：棕榈酰油酰磷脂酰胆碱，棕榈酰亚油酰磷脂酰胆碱，硬脂酰亚油酰磷脂酰胆碱，硬脂酰油酰磷脂酰胆碱，硬脂酰花生四烯酰磷脂酰胆碱，和二棕榈酰磷脂酰胆碱。其它磷脂包括L-α-二肉豆蔻酰磷脂酰胆碱(DMPC)，二油酰磷脂酰胆碱(DOPC)，二硬脂酰磷脂酰胆碱(DSPC)，氢化大豆磷脂酰胆碱(HSPC)，D-α-磷脂酰胆碱，β-乙酰基-γ-O-十六烷基，L-α-磷脂酰胆碱，β-乙酰基-γ-O-十六烷基，DL-α-磷脂酰胆碱，β-乙酰基-γ-O-十六烷基，L-α-磷脂酰胆碱，β-乙酰基-γ-O-十八烷基，L-α-磷脂酰胆碱，β-花生四烯酰-γ-O-十六烷基，L-α-磷脂酰胆碱，β-乙酰基-γ-O-(十八碳-9-顺-烯基)，D-α-磷脂酰胆碱，β-花生四烯酰-γ-O-棕榈酰，3-sn-磷脂酰胆碱，2-花生四烯酰-1-硬脂酰，L-α-磷脂酰胆碱，β-花生四烯酰-γ-硬脂酰，L-α-磷脂酰胆碱，二花生四烯酰，L-α-磷脂酰胆碱，二(山萮炔酰)，L-α-磷脂酰胆碱，β-(顺-8，11，14-二十碳三烯酰)-γ-O-十六烷基，L-α-磷脂酰胆碱，β-油酰-γ-肉豆蔻酰，L-α-磷脂酰胆碱，β-(芘-1-基)癸酰基-γ-棕榈酰，3-sn-磷脂酰-N，N-二甲基乙醇胺，1，2-二棕榈酰，L-α-磷脂酰乙醇胺，二(十七烷酰)，3-sn-磷脂酰乙醇胺，1，2-二月桂酰，3-sn-磷脂酰乙醇胺，1，2-二肉豆蔻酰，3-sn-磷脂酰乙醇胺，1，2-二油酰，3-sn-磷脂酰乙醇胺，1，2-二棕榈酰，L-α-磷脂酰乙醇胺，二棕榈酰，L-α-磷脂酰乙醇胺，二棕榈酰，N-丹酰，L-α-磷脂酰乙醇胺，二棕榈酰，N，N-二甲基，L-α-二肉豆蔻酰磷脂酰甘油(钠盐)(DMPG)，二棕榈酰磷脂酰甘油(钠盐)(DPPG)，二硬脂酰磷脂酰甘油(钠盐)(DSPG)，N-(羰基-甲氧基聚乙二醇2000)-1，2-二硬脂酰-sn-甘油-3-磷酸乙醇胺钠(MPEG-DSPE)，L-α-磷脂酸，二癸酰基钠盐，L-α-磷脂酸，二(十七碳酰)钠盐，3-sn-磷脂酸，1，2-二肉豆蔻酰钠盐，L-α-磷脂酸，二辛酰基钠盐，L-α-磷脂酸，二油酰钠盐，L-α-磷脂酸，二棕榈酰钠盐，L-α-磷脂酰-DL-甘油，二肉豆蔻酰钠盐，L-α-磷脂酰-DL-甘油，二油酰钠盐，L-α-磷脂酰-DL-甘油，二棕榈酰铵盐，L-α-磷脂酰-DL-甘油，二硬脂酰铵盐，L-α-磷脂酰-DL-甘油，β-油酰-γ-棕榈酰铵盐，L-α-磷脂酰肌醇铵盐，L-α-磷脂酰肌醇钠盐，L-α-磷脂酰-L-丝氨酸，二油酰钠盐，L-α-磷脂酰-L-丝氨酸，和二棕榈酰钠盐。带负电的乳化剂的表面活性剂也适合作为添加剂，例如胆甾醇基硫酸钠等。Consistent with the present invention, the pharmaceutical composition may include other agents, excipients or stabilizers to improve the properties of the composition. For example, to increase stability by increasing the negative zeta potential of nanoparticles or nanodroplets, certain negatively charged components can be added. These negatively charged components include, but are not limited to, bile salts of bile acids consisting of glycocholic acid, cholic acid, chenodeoxycholic acid, taurocholic acid, glycochenodeoxycholic acid ( glycochenodeoxycholic acid), taurochenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, dehydrocholic acid, and others; phospholipids, including lecithin (egg yolk)-based phospholipids, the lecithin (Egg yolk)-based phospholipids include the following phosphatidylcholines: palmitoyl oleoyl phosphatidyl choline, palmitoyl linoleoyl phosphatidyl choline, stearoyl linoleoyl phosphatidyl choline, stearoyl oleoyl phosphatidyl choline base, stearoylarachidonoylphosphatidylcholine, and dipalmitoylphosphatidylcholine. Other phospholipids include L-alpha-dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), distearoylphosphatidylcholine (DSPC), hydrogenated soybean phosphatidylcholine (HSPC ), D-α-phosphatidylcholine, β-acetyl-γ-O-hexadecyl, L-α-phosphatidylcholine, β-acetyl-γ-O-hexadecyl, DL- α-phosphatidylcholine, β-acetyl-γ-O-hexadecyl, L-α-phosphatidylcholine, β-acetyl-γ-O-octadecyl, L-α-phosphatidyl Choline, β-arachidonoyl-γ-O-hexadecyl, L-α-phosphatidylcholine, β-acetyl-γ-O-(octadec-9-cis-enyl), D-α-phosphatidylcholine, β-arachidonoyl-γ-O-palmitoyl, 3-sn-phosphatidylcholine, 2-arachidonoyl-1-stearoyl, L-α-phospholipid Acylcholine, β-Arachidonoyl-γ-Stearoyl, L-α-Phosphatidylcholine, Diarachidonoyl, L-α-Phosphatidylcholine, Di(behenynoyl), L -α-phosphatidylcholine, β-(cis-8,11,14-eicosatrienoyl)-γ-O-hexadecyl, L-α-phosphatidylcholine, β-oleoyl- γ-myristoyl, L-α-phosphatidylcholine, β-(pyrene-1-yl)decanoyl-γ-palmitoyl, 3-sn-phosphatidyl-N,N-dimethylethanolamine, 1, 2-dipalmitoyl, L-α-phosphatidylethanolamine, bis(heptadecanoyl), 3-sn-phosphatidylethanolamine, 1,2-dilauroyl, 3-sn-phosphatidylethanolamine, 1,2- Dimyristoyl, 3-sn-phosphatidylethanolamine, 1,2-dioleoyl, 3-sn-phosphatidylethanolamine, 1,2-dipalmitoyl, L-α-phosphatidylethanolamine, dipalmitoyl, L -α-phosphatidylethanolamine, dipalmitoyl, N-dansyl, L-α-phosphatidylethanolamine, dipalmitoyl, N,N-dimethyl, L-α-dimyristoylphosphatidylglycerol (sodium salt ) (DMPG), dipalmitoylphosphatidylglycerol (sodium salt) (DPPG), distearoylphosphatidylglycerol (sodium salt) (DSPG), N-(carbonyl-methoxypolyethylene glycol 2000)-1 , 2-Distearoyl-sn-glycero-3-phosphoethanolamine sodium (MPEG-DSPE), L-α-phosphatidic acid, didecanoyl sodium salt, L-α-phosphatidic acid, di(heptadecanoyl) Sodium salt, 3-sn-phosphatidic acid, 1,2-dimyristoyl sodium salt, L-alpha-phosphatidic acid, dicaprylyl sodium salt, L-alpha-phosphatidic acid, dioleoyl sodium salt, L-alpha - Phosphatidic acid, dipalmitoyl sodium salt, L-α-phosphatidyl-DL-glycerol, dimyristoyl sodium salt, L-α-phosphatidyl-DL-glycerol, dioleoyl sodium salt, L-α-phospholipid Acyl-DL-glycerol, dipalmitoyl ammonium salt, L-α-phosphatidyl-DL-glycerol, distearyl ammonium salt, L-α-phosphatidyl-DL-glycerol, β-oleoyl-γ-palmitoyl Ammonium salt, L-α-phosphatidylinositol ammonium salt, L-α-phosphatidylinositol sodium salt, L-α-phosphatidylinositol-L-serine, dioleoyl sodium salt , L-α-phosphatidyl-L-serine, and dipalmitoyl sodium salt. Negatively charged emulsifier surfactants are also suitable as additives, such as sodium cholesteryl sulfate and the like.
药剂(例如丙泊酚)可以单独使用或溶解在水不混溶的溶剂中。可以使用各种各样的水不混溶的溶剂如大豆油，红花油，棉籽油，玉米油，向日葵油，花生油，蓖麻油，橄榄油。优选的油是植物油，其中最优选大豆油。大豆油可以在组合物重量的1％-10％范围内使用。优选大豆油以约3重量％的量存在于组合物中。Agents such as propofol can be used alone or dissolved in a water-immiscible solvent. A variety of water-immiscible solvents can be used such as soybean oil, safflower oil, cottonseed oil, corn oil, sunflower oil, peanut oil, castor oil, olive oil. Preferred oils are vegetable oils, with soybean oil being most preferred. Soybean oil may be used in the range of 1% to 10% by weight of the composition. Preferably soybean oil is present in the composition in an amount of about 3% by weight.
本发明的药物组合物可以用药用表面活性剂稳定。这里使用的术语“表面活性剂”是指两性分子的表面活性基团。表面活性剂可以是阴离子型，阳离子型，非离子型和两性离子型。任何适当的表面活性剂可以包括在本发明的药物组合物中。适当的表面活性剂包括非离子型表面活性剂如磷脂，聚氧乙烯失水山梨醇酯，和生育酚聚氧乙烯琥珀酸酯。优选的表面活性剂是卵磷酯，吐温80和维生素E-t d-α-生育酚聚氧乙烯-1000琥珀酸酯(TPGS)。对于含有大豆油的制剂，优选卵磷脂且对于含有3％大豆油的制剂不超过组合物的1.2重量％，优选1.1重量％。对于不含大豆油的制剂，吐温80或维生素E-TPGS是优选的表面活性剂。典型，0.1-1.5重量％的吐温80或0.5-4重量％的维生素E-TPGS是适当的。优选地，使用1.5重量％的吐温80或1重量％的维生素E-TPGS。其它适当表面活性剂的实例描述于例如Becher，Emulsions：Theory and Practice，Robert E.KriegerPublishing，Malabar，Fla.(1965)中。The pharmaceutical compositions of the present invention can be stabilized with acceptable surfactants. The term "surfactant" as used herein refers to a surface-active group of an amphiphilic molecule. Surfactants can be anionic, cationic, nonionic and zwitterionic. Any suitable surfactant may be included in the pharmaceutical compositions of the present invention. Suitable surfactants include nonionic surfactants such as phospholipids, polyoxyethylene sorbitan esters, and tocopheryl polyoxyethylene succinates. Preferred surfactants are lecithin, Tween 80 and vitamin E-t d-alpha-tocopherol polyoxyethylene-1000 succinate (TPGS). For formulations containing soybean oil, lecithin is preferred and for formulations containing 3% soybean oil does not exceed 1.2% by weight of the composition, preferably 1.1% by weight. For formulations without soybean oil, Tween 80 or Vitamin E-TPGS are the preferred surfactants. Typically, 0.1-1.5% by weight Tween 80 or 0.5-4% by weight vitamin E-TPGS is suitable. Preferably, 1.5% by weight of Tween 80 or 1% by weight of Vitamin E-TPGS is used. Examples of other suitable surfactants are described, for example, in Becher, Emulsions: Theory and Practice, Robert E. Krieger Publishing, Malabar, Fla. (1965).
存在各种各样的本发明药物组合物的适当制剂(参见例如，U.S.专利5,916,596)。下列制剂和方法仅仅是例举性的而绝非限制性的。适于口服给药的制剂可以由以下各项组成：(a)液体溶液，如有效量的溶于稀释剂中的化合物，所述稀释剂如水，盐水或橙汁，(b)胶囊，小药囊或片剂，各自含有预定量的作为固体或颗粒的活性成分，(c)在适当液体中的混悬剂，和(d)适当的乳剂。片剂形式可以包括一种或多种乳糖，甘露糖醇，玉米淀粉，马铃薯淀粉，微晶纤维素，阿拉伯胶，明胶，胶体二氧化硅，交联羧甲基纤维素钠，滑石，硬脂酸镁，硬脂酸，和其它赋形剂，着色剂，稀释剂，缓冲剂，润湿剂，防腐剂，调味剂，和药理学上相容的赋形剂。锭剂形式可以在调味剂中包含活性成分，所述调味剂通常为蔗糖和阿拉伯胶或黄蓍胶，以及在惰性基质中包含活性成分的锭剂，所述惰性基质如明胶和甘油，或蔗糖和阿拉伯胶，除了活性成分以外还含有诸如本领域已知的赋形剂的乳剂、凝胶等。A wide variety of suitable formulations of the pharmaceutical compositions of the invention exist (see, eg, U.S. Patent 5,916,596). The following formulations and methods are illustrative only and not limiting in any way. Formulations suitable for oral administration may consist of (a) liquid solutions, such as an effective amount of the compound dissolved in a diluent, such as water, saline or orange juice, (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient as a solid or granules, (c) a suspension in a suitable liquid, and (d) a suitable emulsion. Tablet forms may include one or more of lactose, mannitol, corn starch, potato starch, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, stearin Magnesium acid, stearic acid, and other excipients, coloring agents, diluents, buffers, wetting agents, preservatives, flavoring agents, and pharmacologically compatible excipients. Lozenge forms may contain the active ingredient in a flavoring agent, usually sucrose and acacia or tragacanth, and lozenges contain the active ingredient in an inert base such as gelatin and glycerin, or sucrose. and gum arabic, emulsions, gels, etc. containing excipients such as those known in the art, in addition to the active ingredient.
适合于肠胃外给药的制剂包括水性和非水性的等渗无菌注射液，其可以含有抗氧化剂，缓冲剂，抑菌剂，和使得制剂与期望接受者的血液等渗的溶质，和可以包括悬浮剂、增溶剂、增稠剂、稳定剂和防腐剂的水性和非水性的无菌混悬剂。制剂可以以单位剂量或多剂量密封的容器提供，如安剖和小瓶，并且可以贮存在冷冻干燥(冻干)条件下，这在即刻使用前只要求加入无菌液体赋形剂，例如水以便注射。临时注射液和混悬剂可以从前述种类的无菌粉末、颗粒和片剂制备。优选注射制剂。Formulations suitable for parenteral administration include aqueous and nonaqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes to render the formulation isotonic with the blood of the intended recipient, and may contain Aqueous and non-aqueous sterile suspensions include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. The formulations may be presented in unit-dose or multi-dose sealed containers, such as ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition, which requires only the addition of a sterile liquid excipient, such as water, immediately prior to use for injection. Extemporaneous injections and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. Injectable formulations are preferred.
适于气溶胶给药的制剂包含本发明的药物组合物，其包括可以含有抗氧化剂、缓冲剂、抑菌剂和溶质的水性和非水性的等渗无菌溶液，以及可以包括悬浮剂、增溶剂、增稠剂、稳定剂和防腐剂的水性和非水性无菌混悬剂，这些组分单独或与其它适当组分组合，可以制备成气溶胶制剂以通过吸入给药。这些气溶胶制剂可以放入加压可接受的推进剂中，所述推进剂如二氯二氟甲烷，丙烷，氮气等。它们还可以配制成用于非加压制剂的药物，如在喷雾器或雾化器中。Formulations suitable for aerosol administration comprise pharmaceutical compositions of the invention comprising aqueous and non-aqueous isotonic sterile solutions which may contain antioxidants, buffers, bacteriostats and solutes, and which may include suspending agents, Aqueous and non-aqueous sterile suspensions of solvents, thickeners, stabilizers and preservatives, either alone or in combination with other suitable ingredients, can be prepared as aerosol formulations for administration by inhalation. These aerosol formulations can be placed into pressurized acceptable propellants such as dichlorodifluoromethane, propane, nitrogen and the like. They can also be formulated as medicaments for non-pressurized preparations, such as in a nebulizer or nebuliser.
其它适当的制剂是可能的，例如可以通过使用各种基质(base)如乳化基质或水不溶性基质来制备栓剂。适于阴道给药的制剂可以提供为阴道栓剂，棉塞，乳膏剂，凝胶剂，糊剂，泡沫，或喷雾剂制剂，其除了活性成分以外还含有诸如本领域已知的适当载体。Other suitable formulations are possible, for example suppositories can be prepared by using various bases such as emulsifying bases or water-insoluble bases. Formulations suitable for vaginal administration can be presented as pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing in addition to the active ingredient such appropriate carriers as are known in the art.
在本发明的优选实施方案中，将药物组合物配制成具有4.5-9.0的pH，更优选5.0-8.0的pH。还可以通过加入适当渗透调节剂如甘油使得药物组合物与血液等渗。此外，药用载体优选还含有不含热原的水或注射用水，USP。优选地，本发明的药物组合物被制备成无菌水性制剂，纳米粒，水包油型乳剂，或油包水型乳剂。最优选地，药物组合物是水包油型乳剂。In a preferred embodiment of the invention, the pharmaceutical composition is formulated to have a pH of 4.5-9.0, more preferably 5.0-8.0. The pharmaceutical composition can also be made isotonic with the blood by the addition of suitable osmotic regulators, such as glycerol. In addition, the pharmaceutical carrier preferably also contains pyrogen-free water or water for injection, USP. Preferably, the pharmaceutical composition of the present invention is prepared as a sterile aqueous formulation, nanoparticles, oil-in-water emulsion, or water-in-oil emulsion. Most preferably, the pharmaceutical composition is an oil-in-water emulsion.
按照本发明，对于包含丙泊酚的药物组合物，可以通过将丙泊酚单独溶解在水不混溶的溶剂中和制备含有清蛋白、去铁胺、表面活性剂和其它水溶性成分的水相，将油与水相混合来制备水包油型乳剂。粗制乳剂在10,000至25,000psi的压力下高压均化并再循环5-20个周期以形成理想的乳剂。优选的压力是15,000至20,000psi，更优选10,000psi。粗制乳剂可以再循环7-15个周期且优选再循环15个周期。备选地，可以使用通过均化器的分开通道。According to the present invention, for pharmaceutical compositions containing propofol, it can be obtained by dissolving propofol alone in a water-immiscible solvent and preparing water containing albumin, deferoxamine, surfactant and other water-soluble ingredients. phase, the oil is mixed with the water phase to make an oil-in-water emulsion. The crude emulsion is high pressure homogenized at 10,000 to 25,000 psi and recirculated for 5-20 cycles to form the desired emulsion. A preferred pressure is 15,000 to 20,000 psi, more preferably 10,000 psi. The coarse emulsion can be recycled for 7-15 cycles and preferably for 15 cycles. Alternatively, a separate pass through the homogenizer can be used.
优选地，本发明的药物组合物可以含有尺寸小于约200纳米(nm)的颗粒或液滴。例如，在紫杉醇，多西他赛，雷帕霉素，环孢菌素，丙泊酚和其它的情形中，这些分散体的平均尺寸小于200nm。Preferably, the pharmaceutical compositions of the present invention may contain particles or droplets having a size of less than about 200 nanometers (nm). For example, in the case of paclitaxel, docetaxel, rapamycin, cyclosporine, propofol and others, the average size of these dispersions is less than 200 nm.
本发明另外提供降低一种或多种与向人给药药物组合物有关的副作用的方法。该方法包含向人给药包含药剂和药用载体的药物组合物，其中所述药用载体包含清蛋白和去铁胺。以上结合本发明药物组合物陈述的药物组合物、药剂和药用载体、及其成分的描述也适用于本发明方法的那些相同方面。The present invention additionally provides methods of reducing one or more side effects associated with administering a pharmaceutical composition to a human. The method comprises administering to a human a pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises albumin and deferoxamine. The descriptions of the pharmaceutical compositions, medicaments and pharmaceutically acceptable carriers, and ingredients thereof, set out above in connection with the pharmaceutical compositions of the present invention also apply to those same aspects of the methods of the present invention.
在本发明上下文中，本发明药物组合物给药于人的剂量将随着具体的药物组合物、给药方法和被治疗的具体部位而变化。该剂量应当足以在期望时间范围内实现期望反应，如对特定疾病的治疗或预防反应，或者当药剂是麻醉剂如丙泊酚时麻醉反应。In the context of the present invention, the dosage of the pharmaceutical composition of the present invention administered to humans will vary with the particular pharmaceutical composition, the method of administration and the particular site being treated. The dosage should be sufficient to achieve a desired response, such as a therapeutic or prophylactic response to a particular disease, or an anesthetic response when the agent is an anesthetic such as propofol, within a desired time frame.
尽管任何适当的向人给药药物组合物的方式可以用于本发明上下文中，优选将本发明的药物组合物通过静脉内给药、动脉内给药、肺内给药、口服给药、吸入、膀胱内给药、肌内给药、气管内给药、皮下给药、眼内给药、鞘内给药或透皮给药给药于人。例如，本发明的药物组合物可以通过吸入给药以治疗呼吸道病症。与吸入本发明药物组合物有关的副作用最小，因为清蛋白是呼吸道内层和分泌物的天然成分。本发明组合物可以用于治疗呼吸道病症如肺纤维化，闭塞性细支气管炎，肺癌，支气管肺泡癌等。Although any suitable means of administering a pharmaceutical composition to a human may be used in the context of the present invention, it is preferred that the pharmaceutical composition of the present invention be administered intravenously, intraarterially, intrapulmonarily, orally, by inhalation , intravesical, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, or transdermal. For example, the pharmaceutical compositions of the invention may be administered by inhalation to treat respiratory disorders. The side effects associated with inhalation of the pharmaceutical composition of the present invention are minimal since albumin is a natural component of the respiratory lining and secretions. The composition of the present invention can be used to treat respiratory diseases such as pulmonary fibrosis, bronchiolitis obliterans, lung cancer, bronchoalveolar carcinoma and the like.
本发明的方法导致与向人给药药物组合物有关的一种或多种副作用的减小。这些副作用包括例如，骨髓抑制，神经毒性，过敏，炎症，静脉刺激，静脉炎，疼痛，皮肤刺激，及其组合。然而这些副作用仅仅是例举性的，通过使用本发明的新组合物和方法可以减小或避免与各种药剂有关的其它副作用或副作用组合。The methods of the invention result in a reduction in one or more side effects associated with administering the pharmaceutical composition to a human. These side effects include, for example, myelosuppression, neurotoxicity, hypersensitivity, inflammation, vein irritation, phlebitis, pain, skin irritation, and combinations thereof. While these side effects are merely exemplary, other side effects or combinations of side effects associated with various agents can be reduced or avoided by using the novel compositions and methods of the invention.
本发明另外提供抑制药物组合物中微生物生长的方法。“抑制微生物生长”是指从药物组合物中完全消除微生物，或降低药物组合物中微生物生长的量或速率。该方法包含制备包含药剂和药用载体的药物组合物，其中所述药用载体包含去铁胺，其盐，其类似物，及其组合，它们的量有效抑制药物组合物中的微生物生长。另外，本发明提供抑制药物组合物氧化的方法。该方法包含制备包含药剂和药用载体的药物组合物，其中所述药用载体包含去铁胺，其盐，其类似物，及其组合，它们的量有效抑制药物组合物的氧化。以上结合本发明药物组合物阐明的药物组合物、药剂、和药用载体、及其组分的描述也适用于本发明方法的那些相同方面。The invention additionally provides methods of inhibiting the growth of microorganisms in pharmaceutical compositions. "Inhibiting the growth of microorganisms" means completely eliminating microorganisms from a pharmaceutical composition, or reducing the amount or rate of growth of microorganisms in a pharmaceutical composition. The method comprises preparing a pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier comprising deferoxamine, salts thereof, analogs thereof, and combinations thereof in an amount effective to inhibit the growth of microorganisms in the pharmaceutical composition. Additionally, the present invention provides methods of inhibiting oxidation of pharmaceutical compositions. The method comprises preparing a pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier comprising deferoxamine, salts thereof, analogs thereof, and combinations thereof in an amount effective to inhibit oxidation of the pharmaceutical composition. The descriptions of the pharmaceutical compositions, agents, and pharmaceutically acceptable carriers, and components thereof, set forth above in connection with the pharmaceutical compositions of the present invention also apply to those same aspects of the methods of the present invention.
包括在组合物中的去铁胺，或其优选盐(去铁胺的甲磺酸盐)的量将取决于活性药剂和其它赋形剂。理想地，组合物中去铁胺，其盐，其类似物的量是有效抑制微生物生长和/或抑制氧化的量。如上所述，典型地，药物组合物以液体形式制备，然后将去铁胺，其盐，和其类似物加入至溶液中。优选地，液体形式的药物组合物包含约0.0001重量％至约0.5重量％(例如约0.005重量％，约0.1重量％或约0.25重量％)的去铁胺，其盐，或其类似物。更优选地，液体形式的组合物包含类似量的优选去铁胺盐，甲磺酸去铁胺。最优选地，液体形式的药物组合物包含约0.5重量％的甲磺酸去铁胺。当组合物以固体形式制备时，如上所述，如通过湿式制粒法、流化床干燥和本领域技术人员已知的其它方法制备组合物时，优选将甲磺酸去铁胺作为溶液涂覆至活性药剂，和其它赋形剂(如果存在)。甲磺酸去铁胺溶液优选具有约0.0001重量％至约0.5重量％(例如约0.005重量％，约0.1重量％或约0.25重量％)的去铁胺。The amount of deferoxamine, or its preferred salt (deferoxamine mesylate), included in the composition will depend on the active agent and other excipients. Desirably, the amount of deferoxamine, its salts, and its analogs in the composition is an amount effective to inhibit microbial growth and/or inhibit oxidation. As noted above, typically, the pharmaceutical composition is prepared in liquid form, and deferoxamine, its salts, and its analogs are then added to the solution. Preferably, liquid form pharmaceutical compositions comprise from about 0.0001% to about 0.5% by weight (eg, about 0.005%, about 0.1% or about 0.25%) by weight of deferoxamine, a salt thereof, or an analog thereof. More preferably, the composition in liquid form comprises a similar amount of the preferred deferoxamine salt, deferoxamine mesylate. Most preferably, the pharmaceutical composition in liquid form comprises about 0.5% by weight deferoxamine mesylate. When the composition is prepared in solid form, as described above, such as by wet granulation, fluid bed drying and other methods known to those skilled in the art, deferoxamine mesylate is preferably applied as a solution. Top up with active agent, and other excipients if present. The deferoxamine mesylate solution preferably has from about 0.0001% to about 0.5% by weight (eg, about 0.005%, about 0.1%, or about 0.25% by weight) of deferoxamine.
本发明还提供用于增强药剂向虚弱部位转运的方法，该方法包含向人给药包含药剂和药用载体的药物组合物，其中所述药用载体包含清蛋白，且其中药物组合物中清蛋白与药剂的比率为约18∶1或更小。本发明进一步提供用于增强药剂与体外或体内细胞结合的方法，该方法包含向所述体外或体内细胞给药包含药剂和药用载体的药物组合物，其中所述药用载体包含清蛋白，且其中药物组合物中清蛋白与药剂的比率为约18∶1或更小。以上结合本发明药物组合物和本发明方法阐明的药物组合物、药剂、药用载体、给药途径、及其组分的描述也适用于转运和结合方法的那些相同方面。The present invention also provides a method for enhancing the delivery of an agent to a site of weakness, the method comprising administering to a human a pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises albumin, and wherein the albumin in the pharmaceutical composition is The ratio of protein to agent is about 18:1 or less. The present invention further provides a method for enhancing the binding of an agent to a cell in vitro or in vivo, the method comprising administering to the cell in vitro or in vivo a pharmaceutical composition comprising an agent and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises albumin, And wherein the ratio of albumin to agent in the pharmaceutical composition is about 18:1 or less. The descriptions of the pharmaceutical compositions, medicaments, pharmaceutical carriers, routes of administration, and components thereof set forth above in connection with the pharmaceutical compositions of the invention and the methods of the invention also apply to those same aspects of the methods of delivery and conjugation.
在用于将药剂转运给虚弱部位或用于增强药剂与细胞结合的方法中，药用载体优选包含清蛋白，最优选人血清清蛋白。不束缚于任何一种具体理论，认为蛋白质例如人血清清蛋白与药物组合物中的药剂的比率影响药剂与细胞结合的能力和药剂向细胞的转运。在这点上，蛋白质与药剂的较高比率通常与差的药剂的细胞结合和转运有关，这可能是受体在细胞表面上竞争的结果。蛋白质例如清蛋白与活性药剂的比率必须是使得足量药剂药剂与细胞结合或转运至细胞。蛋白质-药物制剂的例举性范围是蛋白质与药物比率(w/w)为0.01∶1至约100∶1。更优选地，比率为0.02∶1至约40∶1。尽管蛋白质与药剂的比率必须对不同的蛋白质和药剂组合优化，但是通常蛋白质例如清蛋白与药剂的比率为约18∶1或更小(例如约15∶1，约10∶1，约5∶1，或约3∶1)。更优选地，比率为约0.2∶1至约12∶1。最优选地，比率为约1∶1至约9∶1。优选地，制剂基本上不含聚氧乙烯蓖麻油，更优选地不含聚氧乙烯蓖麻油EL
药剂可以是本文描述的任何适当的药剂(例如，丙泊酚，紫杉醇，或多西他赛)。另外，药剂可以是核酸序列，最优选DNA序列。这方面，本发明的药物组合物可以通过受体介导/腔穴(caveolar)/囊转运将基因转运至细胞。为了将DNA序列如基因或其它遗传物质，包括但不限于质粒或c-DNA，转运到细胞(例如内皮细胞或肿瘤细胞)中，可以制备包含清蛋白结合遗传物质的药物组合物。因为在炎症部位的肿瘤细胞和其它细胞具有对蛋白质的高摄取，优选将遗传物质吸入至这些细胞类型中并且可以整合到细胞的遗传物质中以达到有效的治疗效果。蛋白质如人血清请蛋白的使用起作传递遗传物质的非病毒载体的作用而没有与病毒相关的疾病或副作用的风险。例如，可以制备包含编码β-半乳糖苷酶或绿色荧光蛋白(GFP)和清蛋白的核酸序列的药物组合物，并与来源于人脐静脉或人肺微脉管的内皮细胞接触，以促进核酸序列整合到内皮细胞中。核酸序列的整合可以使用本领域已知的方法检测，如例如荧光或染色。The agent can be any suitable agent described herein (eg, propofol, paclitaxel, or docetaxel). Additionally, the agent may be a nucleic acid sequence, most preferably a DNA sequence. In this regard, the pharmaceutical compositions of the present invention can deliver genes to cells via receptor-mediated/caveolar/vesicle transport. For the transfer of DNA sequences such as genes or other genetic material, including but not limited to plasmids or c-DNA, into cells (eg, endothelial cells or tumor cells), pharmaceutical compositions comprising albumin-binding genetic material can be prepared. Because tumor cells and other cells at sites of inflammation have a high uptake of proteins, the uptake of genetic material into these cell types is preferred and can be integrated into the genetic material of the cells to achieve an effective therapeutic effect. The use of proteins such as human serum protein acts as a non-viral vector for the delivery of genetic material without the risk of disease or side effects associated with the virus. For example, a pharmaceutical composition comprising nucleic acid sequences encoding β-galactosidase or green fluorescent protein (GFP) and albumin can be prepared and contacted with endothelial cells derived from human umbilical vein or human pulmonary microvessels to promote The nucleic acid sequence is integrated into endothelial cells. Integration of nucleic acid sequences can be detected using methods known in the art, such as, for example, fluorescence or staining.
在本发明用于增强药剂向虚弱部位转运的方法中，虚弱可以是任何适当的疾病或病症。优选地，虚弱是癌症，心血管疾病，或关节炎。In the methods of the invention for enhancing the delivery of agents to the site of infirmity, the infirmity may be any suitable disease or condition. Preferably, the infirmity is cancer, cardiovascular disease, or arthritis.
在本发明用于增强药剂与体外或体内细胞结合的方法中，将药物组合物给药于体外或体内细胞。理想地，细胞是动物细胞。更优选，细胞是哺乳动物细胞，最优选地，细胞是人细胞。优选将药物组合物给药于体内细胞。细胞可以是作为给药药物组合物的期望靶标的任何适当细胞。例如，细胞可以位于或来源于消化系统组织，包括例如食管，胃，小肠，结肠，直肠，肛门，肝，胆囊，和胰。细胞还可以位于或来源于呼吸系统组织，包括例如喉，肺，和支气管。细胞可以位于或来源于例如组成男性或女性生殖系统的子宫颈，子宫体，卵巢孔，阴道，前列腺，睾丸，和阴茎，和组成泌尿系统的膀胱，肾，肾盂，和输尿管。细胞可以位于或来源于心血管系统的组织，包括例如内皮细胞和心肌细胞。细胞还可以位于或来源于淋巴系统的组织(例如淋巴细胞)，神经系统(例如神经元或神经胶质细胞)，和内分泌系统(例如甲状腺细胞)。优选地，细胞位于或来源于心血管系统的组织。最优选地，细胞是内皮细胞。在本发明用于增强转运和药剂与细胞结合的方法的内容中，药物组合物理想地接触多于一种细胞。In the methods of the invention for enhancing the binding of an agent to a cell in vitro or in vivo, the pharmaceutical composition is administered to the cell in vitro or in vivo. Ideally, the cells are animal cells. More preferably, the cells are mammalian cells, most preferably, the cells are human cells. Preferably the pharmaceutical composition is administered to cells in vivo. The cell may be any suitable cell that is the desired target for administration of the pharmaceutical composition. For example, cells may be located in or derived from tissues of the digestive system, including, for example, the esophagus, stomach, small intestine, colon, rectum, anus, liver, gallbladder, and pancreas. Cells can also be located in or derived from tissues of the respiratory system, including, for example, the larynx, lungs, and bronchi. Cells may be located in or derived from, for example, the cervix, uterus, ovarian foramen, vagina, prostate, testes, and penis that make up the male or female reproductive system, and the bladder, kidneys, renal pelvis, and ureters that make up the urinary system. Cells may be located in or derived from tissues of the cardiovascular system, including, for example, endothelial cells and cardiomyocytes. Cells can also be located in or derived from tissues of the lymphatic system (eg, lymphocytes), nervous system (eg, neurons or glial cells), and endocrine system (eg, thyroid cells). Preferably, the cells are located in or derived from tissues of the cardiovascular system. Most preferably, the cells are endothelial cells. In the context of the methods of the invention for enhancing the transport and binding of agents to cells, the pharmaceutical combination desirably contacts more than one type of cell.
在本发明的另一方面，本发明用于增强转运和增强药剂与细胞结合的方法可以用于治疗肿瘤细胞。与正常细胞相比，肿瘤细胞显示增强的蛋白质摄取，该蛋白质包括清蛋白和转铁蛋白。由于肿瘤细胞快速分裂，所以与正常细胞相比，它们要求更多的营养来源。本发明含有紫杉醇和人血清清蛋白的药物组合物的肿瘤研究表明肿瘤对清蛋白-紫杉醇的高摄取。已经发现这是由于以前未认识到的通过糖蛋白60(“gp60”)受体的清蛋白-药物转运的现象，该糖蛋白60受体对于清蛋白是特异性的。In another aspect of the invention, the methods of the invention for enhancing transport and enhancing binding of agents to cells can be used to treat tumor cells. Tumor cells display enhanced uptake of proteins, including albumin and transferrin, compared to normal cells. Because tumor cells divide rapidly, they require more sources of nutrition than normal cells. Tumor studies of the pharmaceutical composition of the present invention containing paclitaxel and human serum albumin showed high tumor uptake of albumin-paclitaxel. This has been found to be due to a previously unrecognized phenomenon of albumin-drug transport through the glycoprotein 60 ("gp60") receptor, which is specific for albumin.
因此，按照本发明的另一方面，清蛋白特异性的gp60受体和肿瘤细胞上存在的其它蛋白转运受体可以用作抑制肿瘤生长的靶标。通过使用抗gp60受体的抗体或结合、阻断或失活gp60的其它大或小分子化合物阻断gp60受体，和阻断在肿瘤细胞或肿瘤内皮细胞上的其它蛋白转运受体，可以阻断蛋白质向这些细胞的转运，由此降低它们的生长速率和导致细胞死亡。因此，该机制的阻断导致患有癌症或另外的疾病的患者(例如人)的治疗。鉴定特异蛋白受体的阻断/结合是通过筛选任何数量的针对分离的gp60或其它受体如gp16或gp30的化合物或通过使用全细胞制备来完成的。另外，适当的动物模型也可以用于该目的，如例如含有编码gp60或小窝蛋白-1或对于转运特异的其它蛋白的基因“敲除”突变的小鼠。因此，鉴定阻断或结合gp60，gp16，gp30或其它蛋白受体的化合物的方法属于本发明的范围内。Thus, according to another aspect of the invention, albumin-specific gp60 receptors and other protein transport receptors present on tumor cells can be used as targets to inhibit tumor growth. By blocking the gp60 receptor with antibodies against the gp60 receptor or other large or small molecule compounds that bind, block or inactivate gp60, and by blocking other protein transport receptors on tumor cells or tumor endothelial cells, This disrupts the transport of proteins to these cells, thereby reducing their growth rate and causing cell death. Thus, blockade of this mechanism leads to the treatment of patients (eg, humans) suffering from cancer or another disease. Identification of specific protein receptors for blocking/binding is accomplished by screening any number of compounds against isolated gp60 or other receptors such as gp16 or gp30 or by using whole cell preparations. In addition, suitable animal models can also be used for this purpose, such as eg mice containing "knockout" mutations of genes encoding gp60 or caveolin-1 or other proteins specific for transport. Accordingly, methods of identifying compounds that block or bind gp60, gp16, gp30 or other protein receptors are within the scope of the present invention.
另外，阻断或结合gp60受体或其它蛋白受体的化合物可以用于治疗包括癌症的数种疾病。关于癌症的治疗，阻断或结合化合物可以用作单一药剂或与其它标准化疗或多种化疗结合。例如，它与常规化疗或与本发明的清蛋白-药物药物组合物(其在肿瘤中显示高累积)、接着与阻断蛋白质向肿瘤细胞转运的化合物用于治疗癌症。阻断化合物可以在其它化疗或抗癌剂之前或结合给药。因此，任何可以阻断或结合gp60受体或其它蛋白受体的化合物属于本发明的范围内。In addition, compounds that block or bind the gp60 receptor or other protein receptors can be used in the treatment of several diseases including cancer. With regard to the treatment of cancer, blocking or binding compounds may be used as single agents or in combination with other standard chemotherapy or chemotherapy. For example, it is used in the treatment of cancer with conventional chemotherapy or with the albumin-drug composition of the invention, which shows high accumulation in tumors, followed by compounds that block protein transport to tumor cells. Blocking compounds may be administered prior to or in conjunction with other chemotherapeutic or anticancer agents. Accordingly, any compound that blocks or binds to the gp60 receptor or other protein receptors is within the scope of the present invention.
本发明清蛋白-药物组合物，如例如清蛋白-紫杉醇，清蛋白-多西他赛，清蛋白-埃坡霉素，清蛋白-喜树碱，或清蛋白-雷帕霉素以及其它，可以用于治疗疾病。认为这些药物组合物有效是因为增加的蛋白质-药物组合物向所需部位(例如肿瘤)的受体介导的转运。不希望束缚于任何具体理论，通过导致治疗效果的受体介导的转运的蛋白质-药物组合物的转运被认为是例如清蛋白-紫杉醇组合物向肿瘤、以及清蛋白-紫杉醇和清蛋白-雷帕霉素转运透过肺的转运机制。转运受这些组织中gp60，gp16或gp30的存在的影响。因此，向疾病例如炎症(例如关节炎)或肿瘤位点的转运与gp60，gp16或gp30受体相关和导致治疗效果的药物和蛋白质-药物组合物被考虑作为本发明的组合物。Albumin-pharmaceutical compositions according to the invention, such as for example albumin-paclitaxel, albumin-docetaxel, albumin-epothilone, albumin-camptothecin, or albumin-rapamycin and others, Can be used to treat diseases. These pharmaceutical compositions are believed to be effective due to increased receptor-mediated transport of the protein-pharmaceutical composition to the desired site, such as a tumor. Without wishing to be bound by any particular theory, transport of the protein-drug composition by receptor-mediated transport leading to a therapeutic effect is thought to be, for example, albumin-paclitaxel compositions to tumors, and albumin-paclitaxel and albumin-paclitaxel. Transport Mechanism of Pamycin Transport Through the Lung. Translocation is affected by the presence of gp60, gp16 or gp30 in these tissues. Accordingly, drugs and protein-drug compositions whose transport to sites of disease such as inflammation (eg arthritis) or tumors are associated with gp60, gp16 or gp30 receptors and lead to a therapeutic effect are considered as compositions of the invention.
按照本发明的另一方面，内皮细胞可以与具有特定功能的细胞共培养。内皮细胞与其它细胞类型如胰岛细胞、肝细胞、神经内分泌细胞和其它细胞的温育允许所需的组分如蛋白质和其它有益组分向这些细胞的转移。内皮细胞提供这些组分向培养的细胞类型的转移以便模拟体内条件，即其中这些细胞类型通常与内皮细胞紧密接近和将依赖于内皮细胞转运营养，生长因子，激素信号等，而这些对于它们的正常功能是需要的。当内皮细胞不存在时，先前不可能充分地培养这些不同细胞类型和获得生理性能。在具有期望细胞类型的培养物中内皮细胞的存在允许体外或来自体内的胰岛、肝细胞、或神经内分泌组织的分化和适当功能。因此发现与在缺少内皮细胞下培养的那些相比，内皮细胞和胰岛的共培养导致具有改善的生理性能(例如分泌胰岛素的能力)的胰岛。该组织然后可以在体外使用或移植到体内以治疗由于缺少适当细胞功能而导致的疾病(例如在胰岛细胞的情形中糖尿病，在肝细胞情形中的肝功能失常，和在神经内分泌细胞的情形中的神经内分泌紊乱或疼痛减轻)。来源于其它组织和器官的细胞(如上所述)也可以与内皮细胞共培养以提供相同的益处。另外，共培养可以用于将遗传物质整合到靶细胞类型中。发现在这些培养物中清蛋白的存在非常有益。According to another aspect of the present invention, endothelial cells can be co-cultured with cells having specific functions. Incubation of endothelial cells with other cell types such as islet cells, hepatocytes, neuroendocrine cells and others allows the transfer of desired components such as proteins and other beneficial components to these cells. Endothelial cells provide for the transfer of these components to cultured cell types to mimic in vivo conditions, i.e., where these cell types are normally in close proximity to endothelial cells and will depend on endothelial cells for translocation of nutrients, growth factors, hormonal signals, etc. for their normal function is required. In the absence of endothelial cells, it has previously not been possible to adequately culture these different cell types and obtain physiological properties. The presence of endothelial cells in culture with the desired cell type allows differentiation and proper function of islets, hepatocytes, or neuroendocrine tissue in vitro or ex vivo. It was thus found that co-culture of endothelial cells and islets resulted in islets with improved physiological properties, such as the ability to secrete insulin, compared to those cultured in the absence of endothelial cells. This tissue can then be used in vitro or transplanted into the body to treat diseases due to lack of proper cellular function (such as diabetes in the case of islet cells, liver dysfunction in the case of hepatocytes, and hepatic dysfunction in the case of neuroendocrine cells) neuroendocrine disturbances or pain relief). Cells derived from other tissues and organs (as described above) can also be co-cultured with endothelial cells to provide the same benefits. Additionally, co-culture can be used to incorporate genetic material into target cell types. The presence of albumin in these cultures was found to be very beneficial.
下列实施例进一步举例说明本发明，但是当然不应当被认为是以任何方式限制其范围。The following examples further illustrate the invention but, of course, should not be construed in any way as limiting its scope.
实施例1Example 1
本实施例举例说明了含有紫杉醇和清蛋白的药物组合物的制备。紫杉醇-清蛋白组合物的制备描述于美国专利5,439,686和5,916,596中，它们通过引用完整地结合于此。具体地，将30mg紫杉醇溶解在3.0ml二氯甲烷中。将该溶液加入至27.0ml人血清清蛋白溶液(2％w/v)中。根据需要加入去铁胺。将该混合物在低RPM下均化5分钟(Vitris均化器，型号TempestI.Q.)以便形成粗乳剂，然后转移到高压均化器(Avestin)中。在9000-40,000psi下进行乳化，同时将乳剂再循环至少5个周期。将获得的系统转移至旋转式蒸发器，在40℃、减压(30mm Hg)下快速去除二氯甲烷20-30分钟。获得的分散体是半透明的，获得的紫杉醇颗粒的典型平均直径为50-220nm(Z-平均值，Malvem Zetasizer)。将该分散体进一步冻干48小时。通过加入无菌水或盐水，获得的饼块可以容易地重构为原有分散体。在重构后的粒径与冻干前相同。This example illustrates the preparation of a pharmaceutical composition containing paclitaxel and albumin. The preparation of paclitaxel-albumin compositions is described in US Patent Nos. 5,439,686 and 5,916,596, which are hereby incorporated by reference in their entirety. Specifically, 30 mg of paclitaxel was dissolved in 3.0 ml of dichloromethane. This solution was added to 27.0 ml of human serum albumin solution (2% w/v). Add deferoxamine as needed. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000 psi while recirculating the emulsion for at least 5 cycles. The resulting system was transferred to a rotary evaporator and the dichloromethane was rapidly removed at 40 °C under reduced pressure (30 mm Hg) for 20-30 minutes. The obtained dispersion is translucent and the paclitaxel particles obtained have a typical mean diameter of 50-220 nm (Z-mean, Malvem Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted into the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization.
应当认识到用于本实施例的药物、溶剂、蛋白质的量、类型和比率不受任何方式的限制。当与溶于聚氧乙烯蓖麻油制剂中的紫杉醇的毒性相比时，本发明含有清蛋白的药物组合物显示显著更低的毒性。It should be appreciated that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The albumin-containing pharmaceutical composition of the invention showed significantly lower toxicity when compared to the toxicity of paclitaxel dissolved in a polyoxyethylene castor oil formulation.
实施例2Example 2
本实施例举例说明了含有胺碘酮和清蛋白的药物组合物的制备。将30mg胺碘酮溶于3.0ml二氯甲烷中。将溶液加入至27.0ml人血清清蛋白溶液(1％w/v)中。根据需要加入去铁胺。将混合物在低RPM下均化5分钟(Vitris均化器，型号Tempest I.Q.)以便形成粗乳剂，然后转移到高压均化器(Avestin)中。在9000-40,000psi下进行乳化，同时将乳剂再循环至少5个周期。将获得的系统转移至旋转式蒸发器，在40℃、减压(30mm Hg)下快速去除二氯甲烷20-30分钟。获得的分散体是半透明的，获得的胺碘酮颗粒的典型平均直径为50-220nm(Z-平均值，Malvern Zetasizer)。将该分散体进一步冻干48小时。通过加入无菌水或盐水，获得的饼块可以容易地重构为原有分散体。在重构后的粒径与冻干前相同。This example illustrates the preparation of a pharmaceutical composition containing amiodarone and albumin. 30 mg of amiodarone was dissolved in 3.0 ml of dichloromethane. The solution was added to 27.0 ml of human serum albumin solution (1% w/v). Add deferoxamine as needed. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000 psi while recirculating the emulsion for at least 5 cycles. The resulting system was transferred to a rotary evaporator and the dichloromethane was rapidly removed at 40 °C under reduced pressure (30 mm Hg) for 20-30 minutes. The dispersion obtained is translucent and the amiodarone particles obtained have a typical mean diameter of 50-220 nm (Z-mean, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted into the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization.
应当认识到用于本实施例的药物、溶剂、蛋白质的量、类型和比率不受任何方式的限制。当与溶于吐温制剂中的胺碘酮的毒性相比时，本发明含有清蛋白的药物组合物显示显著更低的毒性。It should be appreciated that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The albumin-containing pharmaceutical composition of the present invention showed significantly lower toxicity when compared to the toxicity of amiodarone dissolved in a Tween formulation.
实施例3Example 3
本实施例举例说明了含有碘塞罗宁和清蛋白组合物的药物组合物的制备。将碘塞罗宁(或适当盐)以0.5-50mg/ml的浓度溶于水性醇溶液或碱性溶液中。将醇(或碱性)溶液加入至清蛋白溶液(0.1-25％w/v)中并搅拌。搅拌是使用搅拌器的低剪切或使用超声波仪或均化器的高剪切。在低浓度的碘塞罗宁下，获得(5-1000μg/ml)澄清溶液。当浓度增加时，获得稳定的乳状混悬剂。这些溶液或混悬剂通过无菌滤器过滤。通过蒸发或其它适当方法去除有机溶剂。This example illustrates the preparation of a pharmaceutical composition containing liothyronine and albumin composition. Dissolve liothyronine (or an appropriate salt) in an aqueous alcohol solution or an alkaline solution at a concentration of 0.5-50 mg/ml. The alcoholic (or alkaline) solution is added to the albumin solution (0.1-25% w/v) and stirred. Agitation is either low shear using a stirrer or high shear using a sonicator or homogenizer. At low concentrations of liothyronine (5-1000 μg/ml) clear solutions were obtained. As the concentration increases, a stable milky suspension is obtained. These solutions or suspensions are filtered through sterile filters. The organic solvent is removed by evaporation or other suitable methods.
实施例4Example 4
本实施例举例说明了含有雷帕霉素和清蛋白的药物组合物的制备。将30mg雷帕霉素溶于2ml氯仿/乙醇中。将溶液加入至27.0ml人血清清蛋白溶液(3％w/v)中。将混合物在低RPM下均化5分钟(Vitris均化器，型号Tempest I.Q.)以便形成粗乳剂，然后转移到高压均化器(Avestin)中。在9000-40,000psi下进行乳化，同时将乳剂再循环至少5个周期。将获得的系统转移至Rotavap，在40℃、减压(30mm Hg)下快速去除溶剂20-30分钟。获得的分散体是半透明的，获得的颗粒的典型平均直径为50-220nm(Z-平均值，Malvern Zetasizer)。将该分散体进一步冻干48小时。通过加入无菌水或盐水，获得的饼块可以容易地重构为原有分散体。在重构后的粒径与冻干前相同。应当认识到用于本实施例的药物、溶剂、蛋白质的量、类型和比率不受任何方式的限制。This example illustrates the preparation of a pharmaceutical composition comprising rapamycin and albumin. Dissolve 30 mg rapamycin in 2 ml chloroform/ethanol. The solution was added to 27.0 ml of human serum albumin solution (3% w/v). The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000 psi while recirculating the emulsion for at least 5 cycles. The resulting system was transferred to a Rotavap for rapid solvent removal at 40 °C under reduced pressure (30 mm Hg) for 20-30 min. The dispersion obtained is translucent and the particles obtained have a typical average diameter of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted into the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be appreciated that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way.
实施例5Example 5
本实施例举例说明了含有埃坡霉素B和清蛋白的药物组合物的制备。将30mg埃坡霉素B溶于2ml氯仿/乙醇中。将溶液加入至27.0ml人血清清蛋白溶液(3％w/v)中。根据需要加入去铁胺。将混合物在低RPM下均化5分钟(Vitris均化器，型号Tempest I.Q.)以便形成粗乳剂，然后转移到高压均化器(Avestin)中。在9000-40,000psi下进行乳化，同时将乳剂再循环至少5个周期。将获得的系统转移至Rotavap，在40℃、减压(30mm Hg)下快速去除溶剂20-30分钟。获得的分散体是半透明的，获得的颗粒的典型平均直径为50-220nm(Z-平均值，Malvern Zetasizer)。将该分散体进一步冻干48小时。通过加入无菌水或盐水，获得的饼块可以容易地重构为原有分散体。在重构后的粒径与冻干前相同。应当认识到用于本实施例的药物、溶剂、蛋白质的量、类型和比率不受任何方式的限制。当与溶于聚氧乙烯蓖麻油制剂中的埃坡霉素B的毒性相比时，本发明含有清蛋白的药物组合物显示显著更低的毒性。This example illustrates the preparation of a pharmaceutical composition comprising epothilone B and albumin. Dissolve 30 mg of epothilone B in 2 ml of chloroform/ethanol. The solution was added to 27.0 ml of human serum albumin solution (3% w/v). Add deferoxamine as needed. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000 psi while recirculating the emulsion for at least 5 cycles. The resulting system was transferred to a Rotavap for rapid solvent removal at 40 °C under reduced pressure (30 mm Hg) for 20-30 min. The dispersion obtained is translucent and the particles obtained have a typical average diameter of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted into the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be appreciated that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The albumin-containing pharmaceutical composition of the present invention showed significantly lower toxicity when compared to the toxicity of epothilone B dissolved in a polyoxyethylene castor oil formulation.
实施例6Example 6
本实施例举例说明了含有秋水仙碱二聚体和清蛋白的药物组合物的制备。将30mg秋水仙碱-二聚体溶于2ml氯仿/乙醇中。将溶液加入至27.0ml人血清清蛋白溶液(3％w/v)中。根据需要加入去铁胺。将混合物在低RPM下均化5分钟(Vitris均化器，型号Tempest I.Q.)以便形成粗乳剂，然后转移到高压均化器(Avestin)中。在9000-40,000psi下进行乳化，同时将乳剂再循环至少5个周期。将获得的系统转移至Rotavap，在40℃、减压(30mm Hg)下快速去除溶剂20-30分钟。获得的分散体是半透明的，获得的颗粒的典型平均直径为50-220nm(Z-平均值，Malvern Zetasizer)。将该分散体进一步冻干48小时。通过加入无菌水或盐水，获得的饼块可以容易地重构为原有分散体。在重构后的粒径与冻干前相同。应当认识到用于本实施例的药物、溶剂、蛋白质的量、类型和比率不受任何方式的限制。当与溶于吐温中的秋水仙碱二聚体的毒性相比时，本发明含有清蛋白的药物组合物显示显著更低的毒性。This example illustrates the preparation of a pharmaceutical composition containing colchicine dimer and albumin. 30 mg of colchicine-dimer was dissolved in 2 ml of chloroform/ethanol. The solution was added to 27.0 ml of human serum albumin solution (3% w/v). Add deferoxamine as needed. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000 psi while recirculating the emulsion for at least 5 cycles. The resulting system was transferred to a Rotavap for rapid solvent removal at 40 °C under reduced pressure (30 mm Hg) for 20-30 min. The dispersion obtained is translucent and the particles obtained have a typical average diameter of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted into the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be appreciated that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. When compared to the toxicity of colchicine dimer dissolved in Tween, the albumin-containing pharmaceutical composition of the present invention showed significantly lower toxicity.
实施例7Example 7
本实施例举例说明了含有多西他赛和清蛋白的药物组合物的制备。将30mg多西他赛溶于2ml氯仿/乙醇中。将溶液加入至27.0ml人血清清蛋白溶液(3％w/v)中。根据需要加入去铁胺。将混合物在低RPM下均化5分钟(Vitris均化器，型号Tempest I.Q.)以便形成粗乳剂，然后转移到高压均化器(Avestin)中。在9000-40,000psi下进行乳化，同时将乳剂再循环至少5个周期。将获得的系统转移至Rotavap，在40℃、减压(30mm Hg)下快速去除溶剂20-30分钟。获得的分散体是半透明的，获得的颗粒的典型平均直径为50-220nm(Z-平均值，Malvern Zetasizer)。将该分散体进一步冻干48小时。通过加入无菌水或盐水，获得的饼块可以容易地重构为原有分散体。在重构后的粒径与冻干前相同。应当认识到用于本实施例的药物、溶剂、蛋白质的量、类型和比率不受任何方式的限制。当与溶于该药物的标准溶剂--吐温/乙醇中的多西他赛的毒性相比时，本发明含有清蛋白的药物组合物显示显著更低的毒性。This example illustrates the preparation of a pharmaceutical composition containing docetaxel and albumin. Dissolve 30 mg of docetaxel in 2 ml of chloroform/ethanol. The solution was added to 27.0 ml of human serum albumin solution (3% w/v). Add deferoxamine as needed. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000 psi while recirculating the emulsion for at least 5 cycles. The resulting system was transferred to a Rotavap for rapid solvent removal at 40 °C under reduced pressure (30 mm Hg) for 20-30 min. The dispersion obtained is translucent and the particles obtained have a typical average diameter of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted into the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be appreciated that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The albumin-containing pharmaceutical composition of the present invention showed significantly lower toxicity when compared to the toxicity of docetaxel dissolved in Tween/Ethanol, the standard solvent of the drug.
实施例8Example 8
本实施例举例说明了含有多西他赛和清蛋白的药物组合物的制备。将150mg多西他赛溶于1ml乙酸乙酯/乙酸丁酯和0.5ml油例如大豆油或维生素E油中。使用其它比率的溶剂和油，这些组合物也被考虑为本发明的一部分。还任选地加入将少量带负电组分，例如苯甲酸(0.001％-0.5％)。然后将溶液加入至27.0ml人血清清蛋白溶液(5％w/v)中。根据需要加入去铁胺。将混合物在低RPM下均化5分钟(Vitris均化器，型号Tempest I.Q.)以便形成粗乳剂，然后转移到高压均化器(Avestin)中。在9000-40,000psi下进行乳化，同时将乳剂再循环至少5个周期。将获得的系统转移至Rotavap，在40℃、减压(30mm Hg)下快速去除溶剂20-30分钟。获得的分散体是半透明的，获得的颗粒的典型平均直径为50-220nm(Z-平均值，MalvernZetasizer)。将该分散体进一步冻干48小时。通过加入无菌水或盐水，获得的饼块可以容易地重构为原有分散体。在重构后的粒径与冻干前相同。应当认识到用于本实施例的药物、溶剂、蛋白质的量、类型和比率不受任何方式的限制。当与溶于该药物的标准溶剂--吐温/乙醇中的多西他赛的毒性相比时，本发明含有清蛋白的药物组合物显示显著更低的毒性。This example illustrates the preparation of a pharmaceutical composition containing docetaxel and albumin. Dissolve 150 mg docetaxel in 1 ml ethyl/butyl acetate and 0.5 ml oil such as soybean oil or vitamin E oil. Using other ratios of solvent and oil, these compositions are also contemplated as part of the invention. Small amounts of negatively charged components such as benzoic acid (0.001%-0.5%) are also optionally added. The solution was then added to 27.0 ml of human serum albumin solution (5% w/v). Add deferoxamine as needed. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000 psi while recirculating the emulsion for at least 5 cycles. The resulting system was transferred to a Rotavap for rapid solvent removal at 40 °C under reduced pressure (30 mm Hg) for 20-30 min. The dispersions obtained are translucent and the particles obtained have a typical average diameter of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted into the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be appreciated that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The albumin-containing pharmaceutical composition of the present invention showed significantly lower toxicity when compared to the toxicity of docetaxel dissolved in Tween/Ethanol, the standard solvent of the drug.
实施例9Example 9
本实施例举例说明了含有紫杉烷IDN5390和清蛋白的药物组合物的制备。将150mg IDN5390溶于1ml乙酸乙酯/乙酸丁酯和0.5ml油例如大豆油或维生素E油中。使用其它比率的溶剂和油，这些组合物也被考虑为本发明的一部分。还任选地加入将少量带负电组分，例如苯甲酸(0.001％-0.5％)。然后将溶液加入至27.0ml人血清清蛋白溶液(5％w/v)中。根据需要加入去铁胺。将混合物在低RPM下均化5分钟(Vitris均化器，型号Tempest I.Q.)以便形成粗乳剂，然后转移到高压均化器(Avestin)中。在9000-40,000psi下进行乳化，同时将乳剂再循环至少5个周期。将获得的系统转移至Rotavap，在40℃、减压(30mm Hg)下快速去除溶剂20-30分钟。获得的分散体是半透明的，获得的颗粒的典型平均直径为50-220nm(Z-平均值，Malvern Zetasizer)。将该分散体进一步冻干48小时。通过加入无菌水或盐水，获得的饼块可以容易地重构为原有分散体。在重构后的粒径与冻干前相同。应当认识到用于本实施例的药物、溶剂、蛋白质的量、类型和比率不受任何方式的限制。当与溶于吐温中的IDN5390的毒性相比时，本发明含有清蛋白的药物组合物显示显著更低的毒性。This example illustrates the preparation of a pharmaceutical composition containing taxane IDN5390 and albumin. Dissolve 150 mg IDN5390 in 1 ml ethyl acetate/butyl acetate and 0.5 ml oil such as soybean oil or vitamin E oil. Using other ratios of solvent and oil, these compositions are also contemplated as part of the invention. Small amounts of negatively charged components such as benzoic acid (0.001%-0.5%) are also optionally added. The solution was then added to 27.0 ml of human serum albumin solution (5% w/v). Add deferoxamine as needed. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000 psi while recirculating the emulsion for at least 5 cycles. The resulting system was transferred to a Rotavap for rapid solvent removal at 40 °C under reduced pressure (30 mm Hg) for 20-30 min. The dispersion obtained is translucent and the particles obtained have a typical average diameter of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted into the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be appreciated that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The albumin-containing pharmaceutical composition of the invention showed significantly lower toxicity when compared to the toxicity of IDN5390 dissolved in Tween.
实施例10Example 10
本实施例举例说明了含有紫杉烷IDN5109和清蛋白的药物组合物的制备。将150mg IDN5109溶于2ml氯仿/乙醇。使用其它比率的溶剂和油，这些组合物也被考虑为本发明的一部分。还任选地加入将少量带负电组分，例如苯甲酸(0.001％-0.5％)。然后将溶液加入至27.0ml人血清清蛋白溶液(5％w/v)中。根据需要加入去铁胺。将混合物在低RPM下均化5分钟(Vitris均化器，型号Tempest I.Q.)以便形成粗乳剂，然后转移到高压均化器(Avestin)中。在9000-40,000psi下进行乳化，同时将乳剂再循环至少5个周期。将获得的系统转移至Rotavap，在40℃、减压(30mm Hg)下快速去除溶剂20-30分钟。获得的分散体是半透明的，获得的颗粒的典型平均直径为50-220nm(Z-平均值，Malvern Zetasizer)。将该分散体进一步冻干48小时。通过加入无菌水或盐水，获得的饼块可以容易地重构为原有分散体。在重构后的粒径与冻干前相同。应当认识到用于本实施例的药物、溶剂、蛋白质的量、类型和比率不受任何方式的限制。当与溶于吐温中的IDN5109的毒性相比时，本发明含有清蛋白的药物组合物显示显著更低的毒性。This example illustrates the preparation of a pharmaceutical composition containing taxane IDN5109 and albumin. Dissolve 150mg IDN5109 in 2ml chloroform/ethanol. Using other ratios of solvent and oil, these compositions are also contemplated as part of the invention. Small amounts of negatively charged components such as benzoic acid (0.001%-0.5%) are also optionally added. The solution was then added to 27.0 ml of human serum albumin solution (5% w/v). Add deferoxamine as needed. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000 psi while recirculating the emulsion for at least 5 cycles. The resulting system was transferred to a Rotavap for rapid solvent removal at 40 °C under reduced pressure (30 mm Hg) for 20-30 min. The dispersion obtained is translucent and the particles obtained have a typical average diameter of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted into the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be appreciated that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The albumin-containing pharmaceutical composition of the invention showed significantly lower toxicity when compared to the toxicity of IDN5109 dissolved in Tween.
实施例11Example 11
本实施例举例说明了含有10-羟基喜树碱(10HC)和清蛋白的药物组合物的制备。将30mg 10HC溶于2.0ml DMF/二氯甲烷/大豆油。然后将溶液加入至27.0ml人血清清蛋白溶液(3％w/v)中。将混合物在低RPM下均化5分钟(Vitris均化器，型号Tempest I.Q.)以便形成粗乳剂，然后转移到高压均化器(Avestin)中。在9000-40,000psi下进行乳化，同时将乳剂再循环至少5个周期。将获得的系统转移至Rotavap，在40℃、减压(30mm Hg)下快速去除溶剂20-30分钟。获得的分散体是半透明的，获得的颗粒的典型平均直径为50-220nm(Z-平均值，Malvern Zetasizer)。将该分散体进一步冻干48小时。通过加入无菌水或盐水，获得的饼块可以容易地重构为原有分散体。在重构后的粒径与冻干前相同。应当认识到用于本实施例的药物、溶剂、蛋白质的量、类型和比率不受任何方式的限制。This example illustrates the preparation of a pharmaceutical composition containing 10-hydroxycamptothecin (10HC) and albumin. Dissolve 30 mg 10HC in 2.0 ml DMF/dichloromethane/soybean oil. The solution was then added to 27.0 ml of human serum albumin solution (3% w/v). The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000 psi while recirculating the emulsion for at least 5 cycles. The resulting system was transferred to a Rotavap for rapid solvent removal at 40 °C under reduced pressure (30 mm Hg) for 20-30 min. The dispersion obtained is translucent and the particles obtained have a typical average diameter of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted into the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be appreciated that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way.
实施例12Example 12
本实施例举例说明了含有环孢菌素和清蛋白的药物组合物的制备。将30mg环孢菌素溶于3.0ml二氯甲烷。然后将溶液加至入27.0ml人血清清蛋白溶液(1％w/v)中。将混合物在低RPM下均化5分钟(Vitris均化器，型号Tempest I.Q.)以便形成粗乳剂，然后转移到高压均化器(Avestin)中。在9000-40,000psi下进行乳化，同时将乳剂再循环至少5个周期。将获得的系统转移至Rotavap，在40℃在减压(30mm Hg)下快速去除溶剂20-30分钟。获得的分散体是半透明的，获得的颗粒的典型平均直径为50-220nm(Z-平均值，Malvern Zetasizer)。将该分散体进一步冻干48小时。通过加入无菌水或盐水，获得的饼块可以容易地重构为原有分散体。在重构后的粒径与冻干前相同。This example illustrates the preparation of a pharmaceutical composition containing cyclosporine and albumin. Dissolve 30 mg of cyclosporine in 3.0 ml of dichloromethane. The solution was then added to 27.0 ml of human serum albumin solution (1% w/v). The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000 psi while recirculating the emulsion for at least 5 cycles. The resulting system was transferred to a Rotavap and the solvent was rapidly removed under reduced pressure (30mm Hg) at 40°C for 20-30 minutes. The dispersion obtained is translucent and the particles obtained have a typical average diameter of 50-220 nm (Z-average, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted into the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization.
实施例13Example 13
本实施例举例说明了含有油和包含环孢菌素和清蛋白的药物组合物的制备。将30mg环孢菌素溶于3.0ml适当油(含有10％橙油的芝麻油)中。然后将溶液加入至27.0ml人血清清蛋白溶液(1％w/v)中。将混合物在低RPM下均化5分钟(Vitris均化器，型号Tempest I.Q.)以便形成粗乳剂，然后转移到高压均化器(Avestin)中。在9000-40,000psi下进行乳化，同时将乳剂再循环至少5个周期。获得的颗粒的典型平均直径为50-220nm(Z-平均值，Malvern Zetasizer)。直接使用分散体或通过任选地加入适当防冻剂冻干48小时。通过加入无菌水或盐水，获得的饼块可以容易地重构为原有分散体。应当认识到用于本实施例的药物、溶剂、蛋白质的量、类型和比率不受任何方式的限制。This example illustrates the preparation of a pharmaceutical composition comprising an oil and comprising cyclosporin and albumin. Dissolve 30 mg of cyclosporine in 3.0 ml of appropriate oil (sesame oil with 10% orange oil). The solution was then added to 27.0 ml of human serum albumin solution (1% w/v). The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000 psi while recirculating the emulsion for at least 5 cycles. The typical mean diameter of the particles obtained is 50-220 nm (Z-mean, Malvern Zetasizer). The dispersion was used directly or lyophilized for 48 hours by optionally adding a suitable antifreeze. The cake obtained can be easily reconstituted into the original dispersion by adding sterile water or saline. It should be appreciated that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way.
实施例14Example 14
本实施例举例说明了含有两性霉素和清蛋白的药物组合物的制备。将30mg两性霉素溶于3.0ml甲基吡咯烷酮/二氯甲烷。将溶液加入于27.0ml人血清清蛋白溶液(1％w/v)中。将混合物在低RPM下均化5分钟(Vitris均化器，型号Tempest I.Q.)以便形成粗乳剂，然后转移到高压均化器(Avestin)中。在9000-40,000psi下进行乳化，同时将乳剂再循环至少5个周期。将获得的系统转移至旋转式蒸发器，在40℃、减压(30mm Hg)下快速去除溶剂20-30分钟。获得的分散体是半透明的，获得的两性霉素颗粒的典型平均直径为50-220nm(Z-平均值，Malvern Zetasizer)。将该分散体进一步冻干48小时。通过加入无菌水或盐水，获得的饼块可以容易地重构为原有分散体。在重构后的粒径与冻干前相同。应当认识到用于本实施例的药物、溶剂、蛋白质的量、类型和比率不受任何方式的限制。加入其它组分如脂质、胆盐等也产生适当的制剂。This example illustrates the preparation of a pharmaceutical composition comprising amphotericin and albumin. 30 mg amphotericin was dissolved in 3.0 ml methylpyrrolidone/dichloromethane. The solution was added to 27.0 ml of human serum albumin solution (1% w/v). The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000 psi while recirculating the emulsion for at least 5 cycles. The resulting system was transferred to a rotary evaporator and the solvent was rapidly removed at 40 °C under reduced pressure (30 mm Hg) for 20-30 min. The dispersion obtained is translucent and the amphotericin particles obtained have a typical mean diameter of 50-220 nm (Z-mean, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted into the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization. It should be appreciated that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. Addition of other components such as lipids, bile salts etc. also results in suitable formulations.
实施例15Example 15
本实施例举例说明含有清蛋白和紫杉醇的药物组合物的临床前的药代动力学和药效学。This example illustrates the preclinical pharmacokinetics and pharmacodynamics of a pharmaceutical composition containing albumin and paclitaxel.
进行小鼠和大鼠中的几个临床前药代动力学研究以评估清蛋白-紫杉醇药物组合物相对于聚氧乙烯蓖麻油-紫杉醇(泰素)药物组合物的可能优势。这些研究证明：(1)在大鼠中清蛋白-紫杉醇的药代动力学是线性的，而泰素的药代动力学相对于剂量是非线性的，(2)包含清蛋白和紫杉醇的药物组合物显示更低的血浆AUC和Cmax，提示与泰素相比清蛋白-紫杉醇组合物对组织的更快的分布(排泄是类似的)，(3)包含清蛋白和紫杉醇的药物组合物显示更低的Cmax，这可能解释了与泰素相比与血液峰值水平相关的降低的毒性，(4)与泰素相比，包含清蛋白和紫杉醇的药物组合物显示的半衰期在大鼠中高约2-倍和在携带肿瘤的小鼠中高4-倍，和(5)包含清蛋白和紫杉醇的药物组合物中紫杉醇的代谢低于泰素药物组合物中的紫杉醇的代谢。在大鼠中注射后24小时，对于包含清蛋白和紫杉醇的药物组合物，44％的总放射性仍与紫杉醇相关，相比较下对于泰素仅22％。对于含有清蛋白和紫杉醇的药物组合物显示的上述药代动力学的最终效果，即增强的细胞内摄取，延长的半衰期和更低的代谢导致与泰素相比在携带肿瘤的小鼠中肿瘤AUC高1.7倍，肿瘤Cmax高1.2倍，和肿瘤半衰期长1.7倍。Several preclinical pharmacokinetic studies in mice and rats were performed to assess the possible advantages of the albumin-paclitaxel pharmaceutical composition over the polyoxyethylene castor oil-paclitaxel (Taxol) pharmaceutical composition. These studies demonstrate that: (1) the pharmacokinetics of albumin-paclitaxel in rats is linear, whereas the pharmacokinetics of taxol is nonlinear with respect to dose, and (2) drug combinations containing albumin and paclitaxel (3) the pharmaceutical composition comprising albumin andpaclitaxel showed Lower Cmax , which may explain the reduced toxicity associated with peak blood levels compared to Taxol, (4) Pharmaceutical compositions containing albumin and paclitaxel exhibited higher half-lives in rats compared to Taxol About 2-fold and 4-fold higher in tumor-bearing mice, and (5) the metabolism of paclitaxel in the pharmaceutical composition comprising albumin and paclitaxel was lower than the metabolism of paclitaxel in the taxol pharmaceutical composition. 24 hours after injection in rats, for the pharmaceutical composition comprising albumin and paclitaxel, 44% of the total radioactivity was still associated with paclitaxel, compared to only 22% for Taxol. The net effects of the above pharmacokinetics shown for pharmaceutical compositions containing albumin and paclitaxel, namely enhanced intracellular uptake, prolonged half-life and lower metabolism resulted in tumors in tumor-bearing mice compared to taxol AUC was 1.7-fold higher, tumorCmax was 1.2-fold higher, and tumor half-life was 1.7-fold longer.
实施例16Example 16
本实施例举例说明了与包含紫杉醇和清蛋白的药物组合物相关的降低的副作用和降低的毒性。This example illustrates the reduced side effects and reduced toxicity associated with a pharmaceutical composition comprising paclitaxel and albumin.
由于在没有聚氧乙烯蓖麻油下包含紫杉醇和清蛋白的药物组合物的独特性质，包含紫杉醇和清蛋白的药物组合物的毒性显著低于泰素。在小鼠和大鼠中的临床前研究中，在小鼠中的单一剂量急性毒性研究显示对于含有紫杉醇和清蛋白的药物组合物，其LD50剂量比泰素大约59倍。在小鼠中的多剂量毒性研究中，对于含有紫杉醇和清蛋白的药物组合物，其LD50剂量比泰素大约10倍。进一步的研究评估在用含有紫杉醇和清蛋白的药物组合物和泰素处理的大鼠中骨髓抑制的程度。结果显示在等剂量下，包含紫杉醇和清蛋白的药物组合物在大鼠中比泰素产生显著更小的骨髓抑制。在大鼠中的急性毒性研究中，在接受9mg/kg泰素的动物中观察到脑皮层坏死或严重的神经毒性，但在接受高达120mg/kg剂量的包含紫杉醇和清蛋白的药物组合物的动物中未观察到。因此，与包含紫杉醇的常规药物组合物相比，包含紫杉醇的药物组合物中清蛋白的存在导致副作用和毒性的显著减小。Due to the unique properties of pharmaceutical compositions comprising paclitaxel and albumin in the absence of polyoxyethylene castor oil, the toxicity of the pharmaceutical composition comprising paclitaxel and albumin was significantly lower than taxol. In preclinical studies in mice and rats, a single-dose acute toxicity study in mice showed anLD50 dose approximately 59-fold higher than Taxol for a pharmaceutical composition containing paclitaxel and albumin. In a multiple-dose toxicity study in mice, theLD50 dose was approximately 10-fold higher than Taxol for a pharmaceutical composition containing paclitaxel and albumin. A further study evaluated the degree of myelosuppression in rats treated with a pharmaceutical composition containing paclitaxel and albumin and taxol. The results showed that the pharmaceutical composition comprising paclitaxel and albumin produced significantly less myelosuppression than taxol in rats at equivalent doses. In an acute toxicity study in rats, cortical necrosis or severe neurotoxicity was observed in animals receiving 9 mg/kg Taxol, but not in animals receiving doses up to 120 mg/kg of a pharmaceutical composition comprising paclitaxel and albumin. Not observed in animals. Thus, the presence of albumin in pharmaceutical compositions comprising paclitaxel results in a significant reduction in side effects and toxicity compared to conventional pharmaceutical compositions comprising paclitaxel.
实施例17Example 17
本实施例举例说明了包含紫杉醇和清蛋白的药物组合物在人中的临床效果。This example illustrates the clinical effect in humans of a pharmaceutical composition comprising paclitaxel and albumin.
迄今在超过500位人患者中的临床研究为含紫杉醇和清蛋白(“清蛋白-紫杉醇”)的药物组合物提供了支持与聚氧乙烯蓖麻油-紫杉醇组合物(泰素)相比降低的毒性和副作用的证据。在19位患者的I期研究中，每3周提供清蛋白-紫杉醇的最大耐受剂量为300mg/m2。这显著高于通常给药的聚氧乙烯蓖麻油-紫杉醇的剂量，即每3周提供一次175mg/m2。在这些患者中血液毒性是温和的，没有过敏，轻度神经病，和没有给药相关的副作用如静脉刺激等。Clinical studies to date in more than 500 human patients provide support for a pharmaceutical composition containing paclitaxel and albumin ("albumin-paclitaxel") to reduce the Evidence of toxicity and side effects. In a phase I study of 19 patients, albumin-paclitaxel was provided every 3 weeks at a maximum tolerated dose of 300 mg/m2 . This is significantly higher than the commonly administered dose of ricor-paclitaxel, which is 175 mg/m2 delivered every 3 weeks. Hematological toxicity was mild in these patients, with no hypersensitivity, mild neuropathy, and no drug-related side effects such as venous irritation.
在另一对27位患者的I期研究中，按照每周一次进度表提供的清蛋白-紫杉醇的最大耐受剂量为125-150mg/m2。这显著高于通常给药的聚氧乙烯蓖麻油-紫杉醇的剂量，即按照每周一次进度表提供的80mg/m2。在这些患者中血液毒性是温和的，没有过敏，轻度神经病，和没有给药相关的副作用如静脉刺激等。In another phase I study of 27 patients, the maximum tolerated dose of albumin-paclitaxel was 125-150 mg/m2 given on a weekly schedule. This is significantly higher than the usual dose of ricor-paclitaxel administered, ie 80 mg/m2 given on a weekly schedule. Hematological toxicity was mild in these patients, with no hypersensitivity, mild neuropathy, and no drug-related side effects such as venous irritation.
分别在43和63位患者中的每3周提供175或300mg/m2的清蛋白-紫杉醇的两个II期研究中，血液毒性低，在175mg/m2和300mg/m2下分别仅有7％和24％患者ANC＜500/mm3。在175mg/m2和300mg/m2下分别有0％和14％的患者发生严重的神经病。没有发生严重的过敏，没有发生给药相关的副作用如静脉刺激，注射疼痛等，这些副作用显著低于使用泰素所经历的。In two Phase II studies of albumin-paclitaxel at 175 or 300 mg/m2 every 3 weeksin 43 and 63 patients, hematological toxicity was low, with only 7% and 24% of patients had ANC < 500/mm3 . Severe neuropathy occurred in 0% and 14% of patients at 175 mg/m2 and 300 mg/m2 , respectively. No severe allergies occurred, and no administration-related side effects such as vein irritation, injection pain, etc. occurred, which were significantly lower than those experienced with Taxol.
在将清蛋白-紫杉醇组合物ABI-007与泰素(含有聚氧乙烯蓖麻油-紫杉醇)比较的III期试验中，ABI-007的剂量显著更高(260mg/m2，比较泰素的175mg/m2)，表明它更好地被耐受。当与聚氧乙烯蓖麻油-紫杉醇相比时，清蛋白-紫杉醇组合物还显示显著减少的中性粒细胞减少症。In a Phase III trial comparing the albumin-paclitaxel combination ABI-007 to Taxol (containing polyoxyethylene castor oil-paclitaxel), the dose of ABI-007 was significantly higher (260 mg/m2 compared to Taxol's 175 mg /m2 ), indicating that it is better tolerated. The albumin-paclitaxel composition also showed significantly reduced neutropenia when compared to polyoxyethylene castor oil-paclitaxel.
实施例18Example 18
本实施例举例说明了使用含有清蛋白和紫杉醇的药物组合物的增强的临床前功效。This example illustrates the enhanced preclinical efficacy of using a pharmaceutical composition comprising albumin and paclitaxel.
比较清蛋白-紫杉醇和泰素对子宫颈鳞状细胞癌A431的作用的体外细胞毒性研究显示：清蛋白-紫杉醇细胞毒活性增加约4倍，清蛋白-紫杉醇和泰素的IC50分别为0.0038和0.012μg/ml。The in vitro cytotoxicity study comparing the effects of albumin-paclitaxel and taxol on cervical squamous cell carcinoma A431 showed that the cytotoxic activity of albumin-paclitaxel increased by about 4 times, and the IC50 of albumin-paclitaxel and taxol were 0.0038, respectively and 0.012 μg/ml.
在无胸腺小鼠中的五个不同人异种移植肿瘤模型(MX-1乳房，NCI-H522肺，SK-OV-3卵巢，PC-3前列腺，和HT-29结肠)中，ABI-007的MTD或等毒剂量比泰素高1.5-3.4倍，在除了肺肿瘤(p＝0.15)以外的所有肿瘤中导致肿瘤生长延迟方面统计学显著的改善(p＜0.05)。In five different human xenograft tumor models (MX-1 breast, NCI-H522 lung, SK-OV-3 ovary, PC-3 prostate, and HT-29 colon) in athymic mice, the The MTD, or equivalent dose, was 1.5-3.4 times higher than Taxol, resulting in a statistically significant improvement in tumor growth delay (p<0.05) in all tumors except lung tumors (p=0.15).
在MX 1乳房模型中，百分之百(100％)的清蛋白-紫杉醇治疗的动物存活103天，相比较下在用等价剂量的泰素治疗的组中20-40％存活。In the MX 1 breast model, one hundred percent (100%) albumin-paclitaxel-treated animals survived 103 days, compared to 20-40% survival in groups treated with equivalent doses of Taxol.
实施例19Example 19
本实施例举例说明了使用动脉内给药的包含清蛋白和紫杉醇的药物组合物增强的临床功效。This example illustrates the enhanced clinical efficacy of a pharmaceutical composition comprising albumin and paclitaxel administered intra-arterially.
在动脉内给药包含清蛋白和紫杉醇的药物组合物的I/II期研究中，如本文所述，招募头颈癌患者(N＝31)和肛管癌患者(N＝12)。通过经皮超选择性动脉内输注，剂量在30分钟内给药的剂量从120mg/m2逐步增加到300mg/m2，q 3-4周。头颈癌患者显示76％(N＝29)的应答率，而肛管癌患者显示64％(N＝11)的应答率。In a Phase I/II study of intra-arterial administration of a pharmaceutical composition comprising albumin and paclitaxel, as described herein, patients with head and neck cancer (N=31 ) and anal canal cancer (N=12) were recruited. Through percutaneous superselective intra-arterial infusion, the dose is gradually increased from 120 mg/m2 to 300 mg/m2 within 30 minutes, q 3-4 weeks. Head and neck cancer patients showed a 76% (N=29) response rate, while anal canal cancer patients showed a 64% (N=11) response rate.
实施例20Example 20
本实施例举例说明了含有3％油和包含丙泊酚和清蛋白的药物组合物的制备。This example illustrates the preparation of a pharmaceutical composition containing 3% oil and comprising propofol and albumin.
如下制备含有1重量％的丙泊酚的水包油型乳剂。通过将甘油(2.25重量％)和人血清清蛋白(0.5重量％)加入至注射用水中并搅拌直至溶解来制备水相。将水相通过滤器(0.2um滤器)。通过在约50℃-60℃下，将卵磷脂(0.4重量％)和丙泊酚(1重量％)溶解在大豆油(3重量％)中并搅拌直至溶解来制备油相。将油相加入至水相中并在10,000RPM均化5分钟。将粗乳剂在20,000psi下高压均化并在5℃再循环15个周期。备选地，使用通过均化器的分离通道。将最终乳剂过滤(0.2μm滤器)并在氮下贮存。获得的药物组合物包含下列通常范围的组分(按重量％计)：丙泊酚0.5-5％；人血清清蛋白0.5-3％；大豆油0.5-3.0％；卵磷脂0.12-1.2％；甘油2.25％；注射用水适量至100；pH 5-8。任选地加入适当的螯合剂，例如去铁胺(0.001-0.1％)。An oil-in-water emulsion containing 1% by weight of propofol was prepared as follows. The aqueous phase was prepared by adding glycerol (2.25% by weight) and human serum albumin (0.5% by weight) to water for injection and stirring until dissolved. Pass the aqueous phase through a filter (0.2um filter). The oil phase was prepared by dissolving lecithin (0.4% by weight) and propofol (1% by weight) in soybean oil (3% by weight) at about 50°C-60°C and stirring until dissolved. Add the oil phase to the water phase and homogenize at 10,000 RPM for 5 minutes. The coarse emulsion was autoclaved at 20,000 psi and recirculated at 5°C for 15 cycles. Alternatively, a separation channel through a homogenizer is used. The final emulsion was filtered (0.2 μm filter) and stored under nitrogen. The obtained pharmaceutical composition comprises the following components (by weight %): 0.5-5% of propofol; 0.5-3% of human serum albumin; 0.5-3.0% of soybean oil; 0.12-1.2% of lecithin; Glycerin 2.25%; water for injection in appropriate amount to 100; pH 5-8. A suitable chelating agent such as deferoxamine (0.001-0.1%) is optionally added.
实施例21Example 21
本实施例举例说明了含有5％油和包含丙泊酚和清蛋白的药物组合物的制备。This example illustrates the preparation of a pharmaceutical composition containing 5% oil and comprising propofol and albumin.
如下制备含有1重量％的丙泊酚的水包油型乳剂。通过将甘油(2.25重量％)和人血清清蛋白(0.5重量％)加入至注射用水中并搅拌直至溶解来制备水相。将水相通过滤器(0.2um滤器)。通过在约50℃-60℃下，将卵磷脂(0.8重量％)和丙泊酚(1重量％)溶解在大豆油(5重量％)中并搅拌直至溶解来制备油相。将油相加入至水相中并在10,000RPM均化5分钟。将粗乳剂在20,000psi下高压均化并在5℃再循环15个周期。备选地，使用通过均化器的分离通道。将最终乳剂过滤(0.2μm滤器)并在氮下贮存。获得的药物组合物包含下列通常范围的组分(按重量％计)：丙泊酚0.5-5％；人血清清蛋白0.5-3％；大豆油0.5-10.0％；卵磷脂0.12-1.2％；甘油2.25％；注射用水适量至100；pH 5-8。任选地加入适当的螯合剂，例如去铁胺(0.001-0.1％)。An oil-in-water emulsion containing 1% by weight of propofol was prepared as follows. The aqueous phase was prepared by adding glycerol (2.25% by weight) and human serum albumin (0.5% by weight) to water for injection and stirring until dissolved. Pass the aqueous phase through a filter (0.2um filter). The oil phase was prepared by dissolving lecithin (0.8% by weight) and propofol (1% by weight) in soybean oil (5% by weight) at about 50°C-60°C and stirring until dissolved. Add the oil phase to the water phase and homogenize at 10,000 RPM for 5 minutes. The coarse emulsion was autoclaved at 20,000 psi and recirculated at 5°C for 15 cycles. Alternatively, a separation channel through a homogenizer is used. The final emulsion was filtered (0.2 μm filter) and stored under nitrogen. The obtained pharmaceutical composition comprises the following components (by weight %): 0.5-5% of propofol; 0.5-3% of human serum albumin; 0.5-10.0% of soybean oil; 0.12-1.2% of lecithin; Glycerin 2.25%; water for injection in appropriate amount to 100; pH 5-8. A suitable chelating agent such as deferoxamine (0.001-0.1%) is optionally added.
实施例22Example 22
本实施例举例说明了含有丙泊酚和清蛋白、不含油的药物组合物的制备。This example illustrates the preparation of an oil-free pharmaceutical composition containing propofol and albumin.
使用类似于实施例18所述方法，制备含有清蛋白和吐温80的丙泊酚组合物。通过将甘油(2.25重量％)、人血清清蛋白(0.5重量％)，吐温80(1.5重量％)和甲磺酸去铁胺(0.1重量％)加入至注射用水中并搅拌直至溶解来制备水相。将水相通过滤器(0.2μm滤器)。将丙泊酚(1重量％)加入至水相中并在10,000RPM均化5分钟。将粗乳剂在20,000psi下高压均化并在5℃再循环15个周期。备选地，使用通过均化器的分离通道。将最终乳剂过滤(0.2μm滤器)并在氮下贮存。获得的药物组合物包含下列通常范围的组分(按重量％计)：丙泊酚0.5-5；人血清清蛋白0.5-3％；吐温80 0.1-1.5％；甲磺酸去铁胺0.0001-0.1％；甘油2.25％；注射用水适量至100；pH 5-8。Using a method similar to that described in Example 18, a propofol composition containing albumin and Tween 80 was prepared. Prepared by adding glycerol (2.25% by weight), human serum albumin (0.5% by weight), Tween 80 (1.5% by weight) and deferoxamine mesylate (0.1% by weight) to water for injection and stirring until dissolved water box. The aqueous phase was passed through a filter (0.2 μm filter). Propofol (1% by weight) was added to the aqueous phase and homogenized at 10,000 RPM for 5 minutes. The coarse emulsion was autoclaved at 20,000 psi and recirculated at 5°C for 15 cycles. Alternatively, a separation channel through a homogenizer is used. The final emulsion was filtered (0.2 μm filter) and stored under nitrogen. The pharmaceutical composition obtained comprises the following components (in % by weight): propofol 0.5-5; human serum albumin 0.5-3%; Tween 80 0.1-1.5%; deferoxamine mesylate 0.0001 -0.1%; Glycerin 2.25%; Water for injection in appropriate amount to 100; pH 5-8.
实施例23Example 23
本实施例举例说明了含有丙泊酚、清蛋白和维生素E-TPGS、不含油的药物组合物的制备。This example illustrates the preparation of an oil-free pharmaceutical composition containing propofol, albumin and vitamin E-TPGS.
使用类似于实施例19所述方法，制备含有清蛋白和维生素E-TPGS的丙泊酚组合物。通过将甘油(2.25重量％)、人血清清蛋白(0.5重量％)，维生素E-TPGS(1重量％)和甲磺酸去铁胺(0.1重量％)加入至注射用水中并搅拌直至溶解来制备水相。将水相通过滤器(0.2μm滤器)。将丙泊酚(1重量％)加入至水相中并在10,000RPM均化5分钟。将粗乳剂在20,000psi下高压均化并在5℃再循环15个周期。备选地，使用通过均化器的分离通道。将最终乳剂过滤(0.2μm滤器)并在氮下贮存。获得的药物组合物包含下列通常范围的组分(按重量％计)：丙泊酚0.5-5；人血清清蛋白0.5-3％；维生素E-TPGS 0.5-4.0％；任选地甲磺酸去铁胺0.0001-0.1％；甘油2.25％；注射用水适量至100；pH 5-8。Using a method similar to that described in Example 19, a propofol composition containing albumin and vitamin E-TPGS was prepared. By adding glycerol (2.25% by weight), human serum albumin (0.5% by weight), vitamin E-TPGS (1% by weight) and deferoxamine mesylate (0.1% by weight) to water for injection and stirring until dissolved Prepare the aqueous phase. The aqueous phase was passed through a filter (0.2 μm filter). Propofol (1% by weight) was added to the aqueous phase and homogenized at 10,000 RPM for 5 minutes. The coarse emulsion was autoclaved at 20,000 psi and recirculated at 5°C for 15 cycles. Alternatively, a separation channel through a homogenizer is used. The final emulsion was filtered (0.2 μm filter) and stored under nitrogen. The pharmaceutical composition obtained comprises the following components (by weight %) in the usual ranges: propofol 0.5-5; human serum albumin 0.5-3%; vitamin E-TPGS 0.5-4.0%; optionally methanesulfonic acid Desferrioxamine 0.0001-0.1%; Glycerin 2.25%; Water for injection up to 100; pH 5-8.
实施例24Example 24
本实施例举例说明了含有丙泊酚、清蛋白、维生素E-TPGS和1％油的药物组合物的制备。This example illustrates the preparation of a pharmaceutical composition containing propofol, albumin, vitamin E-TPGS and 1% oil.
通过下列方法制备含有1重量％的丙泊酚的乳剂。通过将甘油(2.25重量％)和人血清清蛋白(0.5重量％)加入至注射用水中并搅拌直至溶解来制备水相。将水相通过滤器(0.2μm滤器)。将表面活性剂例如维生素E-TPGS(0.5％)加入至水相中。油相由丙泊酚(1重量％)和1％大豆油组成。将油相加入至水相中并在10,000RPM均化5分钟。将粗乳剂在20,000psi下高压均化并在5℃再循环最多15个周期。备选地，使用通过均化器的分离通道。将最终乳剂过滤(0.2μm滤器)并在氮下贮存。An emulsion containing 1% by weight of propofol was prepared by the following method. The aqueous phase was prepared by adding glycerol (2.25% by weight) and human serum albumin (0.5% by weight) to water for injection and stirring until dissolved. The aqueous phase was passed through a filter (0.2 μm filter). A surfactant such as vitamin E-TPGS (0.5%) is added to the aqueous phase. The oily phase consisted of propofol (1% by weight) and 1% soybean oil. Add the oil phase to the water phase and homogenize at 10,000 RPM for 5 minutes. The coarse emulsion was high pressure homogenized at 20,000 psi and recirculated at 5°C for a maximum of 15 cycles. Alternatively, a separation channel through a homogenizer is used. The final emulsion was filtered (0.2 μm filter) and stored under nitrogen.
获得的药物组合物包含下列通常范围的组分(按重量％计)：丙泊酚0.5-5％；人血清清蛋白0.01-3％；维生素E-TPGS 0.1-2％；大豆油或其它油(0.1％-5％)；甘油2.25％；注射用水适量至100；pH 5-8。任选地加入去铁胺(0.001重量％-0.1重量％)。The obtained pharmaceutical composition comprises the following components (in % by weight): propofol 0.5-5%; human serum albumin 0.01-3%; vitamin E-TPGS 0.1-2%; soybean oil or other oils (0.1%-5%); glycerol 2.25%; water for injection in appropriate amount to 100; pH 5-8. Optionally deferoxamine (0.001% to 0.1% by weight) is added.
实施例25Example 25
本实施例举例说明了含有丙泊酚、清蛋白、维生素E-TPGS、1％油和带负电组分的药物组合物的制备。This example illustrates the preparation of a pharmaceutical composition containing propofol, albumin, vitamin E-TPGS, 1% oil and negatively charged components.
通过下列方法制备含有1重量％的丙泊酚的乳剂。通过将甘油(2.25重量％)和人血清清蛋白(0.5重量％)加入至注射用水中并搅拌直至溶解来制备水相。将水相通过滤器(0.2μm滤器)。将表面活性剂例如维生素E-TPGS(0.5％)加入至水相中。油相由丙泊酚(1重量％)和1％大豆油组成。加入少量带负电组分(0.001％-1％)，例如磷脂或胆盐。将油相加入至水相中并在10,000RPM均化5分钟。将粗乳剂在20,000psi下高压均化并在5℃再循环最多15个周期。备选地，使用通过均化器的分离通道。将最终乳剂过滤(0.2μm滤器)并在氮下贮存。An emulsion containing 1% by weight of propofol was prepared by the following method. The aqueous phase was prepared by adding glycerol (2.25% by weight) and human serum albumin (0.5% by weight) to water for injection and stirring until dissolved. The aqueous phase was passed through a filter (0.2 μm filter). A surfactant such as vitamin E-TPGS (0.5%) is added to the aqueous phase. The oily phase consisted of propofol (1% by weight) and 1% soybean oil. Small amounts of negatively charged components (0.001%-1%), such as phospholipids or bile salts, are added. Add the oil phase to the water phase and homogenize at 10,000 RPM for 5 minutes. The coarse emulsion was high pressure homogenized at 20,000 psi and recirculated at 5°C for a maximum of 15 cycles. Alternatively, a separation channel through a homogenizer is used. The final emulsion was filtered (0.2 μm filter) and stored under nitrogen.
获得的药物组合物包含下列通常范围的组分(按重量％计)：丙泊酚0.5-5％；人血清清蛋白0.01-3％；维生素E-TPGS 0.1-2％；大豆油或其它油(0.1％-5％)；甘油2.25％；注射用水适量至100；pH 5-8。任选地加入去铁胺(0.001重量％-0.1重量％)。The obtained pharmaceutical composition comprises the following components (in % by weight): propofol 0.5-5%; human serum albumin 0.01-3%; vitamin E-TPGS 0.1-2%; soybean oil or other oils (0.1%-5%); glycerol 2.25%; water for injection in appropriate amount to 100; pH 5-8. Optionally deferoxamine (0.001% to 0.1% by weight) is added.
实施例26Example 26
本实施例举例说明了含有丙泊酚、清蛋白、维生素E-TPGS、1％油和带负电组分(脱氧胆酸钠)的药物组合物的制备。This example illustrates the preparation of a pharmaceutical composition containing propofol, albumin, vitamin E-TPGS, 1% oil and a negatively charged component (sodium deoxycholate).
通过下列方法制备含有1重量％的丙泊酚的乳剂。通过将甘油(2.25重量％)和人血清清蛋白(0.5重量％)加入至注射用水中并搅拌直至溶解来制备水相。将水相通过滤器(0.2μm滤器)。将表面活性剂例如维生素E-TPGS(0.5％)加入至水相中。油相由丙泊酚(1重量％)和1％大豆油组成。加入少量带负电组分(0.001％-1％)，例如脱氧胆酸钠。将油相加入至水相中并在10,000RPM均化5分钟。将粗乳剂在20,000psi下高压均化并在5℃再循环最多15个周期。备选地，使用通过均化器的分离通道。将最终乳剂过滤(0.2μm滤器)并在氮下贮存。An emulsion containing 1% by weight of propofol was prepared by the following method. The aqueous phase was prepared by adding glycerol (2.25% by weight) and human serum albumin (0.5% by weight) to water for injection and stirring until dissolved. The aqueous phase was passed through a filter (0.2 μm filter). A surfactant such as vitamin E-TPGS (0.5%) is added to the aqueous phase. The oily phase consisted of propofol (1% by weight) and 1% soybean oil. Small amounts of negatively charged components (0.001%-1%), such as sodium deoxycholate, are added. Add the oil phase to the water phase and homogenize at 10,000 RPM for 5 minutes. The coarse emulsion was high pressure homogenized at 20,000 psi and recirculated at 5°C for a maximum of 15 cycles. Alternatively, a separation channel through a homogenizer is used. The final emulsion was filtered (0.2 μm filter) and stored under nitrogen.
获得的药物组合物包含下列通常范围的组分(按重量％计)：丙泊酚0.5-5％；人血清清蛋白0.01-3％；维生素E-TPGS 0.1-2％；大豆油或其它油(0.1％-5％)；甘油2.25％；注射用水适量至100；pH 5-8。任选地加入去铁胺(0.001重量％-0.1重量％)。The obtained pharmaceutical composition comprises the following components (in % by weight): propofol 0.5-5%; human serum albumin 0.01-3%; vitamin E-TPGS 0.1-2%; soybean oil or other oils (0.1%-5%); glycerol 2.25%; water for injection in appropriate amount to 100; pH 5-8. Optionally deferoxamine (0.001% to 0.1% by weight) is added.
实施例27Example 27
本实施例举例说明了含有丙泊酚、清蛋白、维生素E-TPGS、1％油和带负电组分(磷脂，胆盐，聚氨基酸等)的药物组合物的制备。This example illustrates the preparation of a pharmaceutical composition containing propofol, albumin, vitamin E-TPGS, 1% oil and negatively charged components (phospholipids, bile salts, polyamino acids, etc.).
通过下列方法制备含有1重量％的丙泊酚的乳剂。通过将甘油(2.25重量％)和人血清清蛋白(0.5重量％)加入至注射用水中并搅拌直至溶解来制备水相。将水相通过滤器(0.2μm滤器)。将表面活性剂例如维生素E-TPGS(0.5％)加入至水相中。油相由丙泊酚(1重量％)和1％大豆油组成。加入少量带负电组分(0.001％-1％)，例如磷脂酰胆碱。将油相加入至水相中并在10,000RPM均化5分钟。将粗乳剂在20,000psi下高压均化并在5℃再循环最多15个周期。备选地，使用通过均化器的分离通道。将最终乳剂过滤(0.2μm滤器)并在氮下贮存。An emulsion containing 1% by weight of propofol was prepared by the following method. The aqueous phase was prepared by adding glycerol (2.25% by weight) and human serum albumin (0.5% by weight) to water for injection and stirring until dissolved. The aqueous phase was passed through a filter (0.2 μm filter). A surfactant such as vitamin E-TPGS (0.5%) is added to the aqueous phase. The oily phase consisted of propofol (1% by weight) and 1% soybean oil. Small amounts of negatively charged components (0.001%-1%), such as phosphatidylcholine, are added. Add the oil phase to the water phase and homogenize at 10,000 RPM for 5 minutes. The coarse emulsion was high pressure homogenized at 20,000 psi and recirculated at 5°C for a maximum of 15 cycles. Alternatively, a separation channel through a homogenizer is used. The final emulsion was filtered (0.2 μm filter) and stored under nitrogen.
获得的药物组合物包含下列通常范围的组分(按重量％计)：丙泊酚0.5-5％；人血清清蛋白0.01-3％；维生素E-TPGS 0.1-2％；大豆油或其它油(0.1％-5％)；甘油2.25％；注射用水适量至100；pH 5-8。任选地加入去铁胺(0.001重量％-0.1重量％)。The pharmaceutical composition obtained comprises the following components (by weight %): propofol 0.5-5%; human serum albumin 0.01-3%; vitamin E-TPGS 0.1-2%; soybean oil or other oils (0.1%-5%); glycerol 2.25%; water for injection in appropriate amount to 100; pH 5-8. Optionally deferoxamine (0.001% to 0.1% by weight) is added.
实施例28Example 28
本实施例举例说明了丙泊酚与清蛋白的结合。This example illustrates the binding of propofol to albumin.
如下测定丙泊酚与清蛋白的结合。在水中和在含有清蛋白的溶液中测试丙泊酚的溶解度。将250μL丙泊酚加入至10mL水或清蛋白溶液中并在闪烁管中搅拌2小时。然后将溶液转移至15mL聚乙烯离心管中并保持在40℃约16小时。测定水和清蛋白溶液样品中的丙泊酚。丙泊酚在水中的溶解度被测定为0.12mg/ml。丙泊酚在清蛋白溶液中的溶解度取决于清蛋白的浓度，当清蛋白的浓度为2％(20mg/ml)时增加至0.44mg/ml。将溶液超滤通过30kD MWCO滤器并测定滤液中的丙泊酚。发现对于丙泊酚/水溶液，61％的丙泊酚可以在滤液中回收，而对于丙泊酚/清蛋白溶液，仅14％被回收在滤液中，显示丙泊酚与清蛋白的相当大的结合。基于这些结果，由于清蛋白与丙泊酚结合，将清蛋白加入包含丙泊酚的药物组合物导致游离丙泊酚的量的减少。Binding of propofol to albumin was determined as follows. The solubility of propofol was tested in water and in solutions containing albumin. 250 μL of propofol was added to 10 mL of water or albumin solution and stirred in scintillation vials for 2 hours. The solution was then transferred to a 15 mL polyethylene centrifuge tube and maintained at 40°C for approximately 16 hours. Determination of propofol in water and albumin solution samples. The solubility of propofol in water was determined to be 0.12 mg/ml. The solubility of propofol in albumin solution depends on the concentration of albumin, increasing to 0.44 mg/ml when the concentration of albumin is 2% (20 mg/ml). The solution was ultrafiltered through a 30 kD MWCO filter and the filtrate was assayed for propofol. It was found that for the propofol/water solution, 61% of propofol could be recovered in the filtrate, while for the propofol/albumin solution, only 14% was recovered in the filtrate, showing a considerable interaction between propofol and albumin. combined. Based on these results, the addition of albumin to a pharmaceutical composition comprising propofol results in a decrease in the amount of free propofol due to the binding of albumin to propofol.
实施例29Example 29
本实施例举例说明了通过过滤/膜接触在药物组合物中游离丙泊酚的减少。This example illustrates the reduction of free propofol in pharmaceutical compositions by filtration/membrane contact.
如在实施例28中所述实验中观察到的，包含丙泊酚的药物组合物的过滤或超滤导致游离丙泊酚的量的减少。双异丙酚和按照本发明制备的含有清蛋白的药物组合物，各自含有1％丙泊酚(10mg/ml)，使用30kD膜超滤。使用HPLC测量在滤液中游离丙泊酚的量。对于双异丙酚在滤液中游离丙泊酚的浓度为约17μg/ml，而对于本发明的药物组合物在滤液中的游离丙泊酚的浓度为约7μg/ml。结果对应于对于包含丙泊酚和清蛋白的药物组合物的游离丙泊酚的有效减少大于2倍。As observed in the experiments described in Example 28, filtration or ultrafiltration of a pharmaceutical composition comprising propofol resulted in a reduction in the amount of free propofol. Dipropofol and albumin-containing pharmaceutical compositions prepared according to the present invention, each containing 1% propofol (10 mg/ml), were ultrafiltered using a 30 kD membrane. The amount of free propofol in the filtrate was measured using HPLC. The concentration of free propofol in the filtrate was about 17 μg/ml for dipropofol and about 7 μg/ml for the pharmaceutical composition of the invention. The results correspond to an effective reduction of free propofol of greater than 2-fold for pharmaceutical compositions comprising propofol and albumin.
实施例30Example 30
本实施例举例说明了向人给药包含丙泊酚和清蛋白的药物组合物。This example illustrates the administration to humans of a pharmaceutical composition comprising propofol and albumin.
进行随机的双盲临床试验，以比较包含丙泊酚和清蛋白的药物组合物与商购的丙泊酚制剂--双异丙酚的不利的皮肤感觉。按照良好临床实践(Good Clinical Practices)进行试验并且从受试者获得知情同意书。任何性别的成人受试者都可以参加，前提是他们具有未破损的、表面正常的手背面皮肤。A randomized double-blind clinical trial was performed to compare the adverse skin sensation of a pharmaceutical composition comprising propofol and albumin with a commercial propofol formulation, dipropofol. The trial was conducted in accordance with Good Clinical Practices and informed consent was obtained from the subjects. Adult subjects of any gender may participate provided they have unbroken, apparently normal skin on the dorsal side of the hand.
原来贮存在冰箱中的制剂被带到室温环境中，然后将10μL制剂同时缓慢放置在受试者两只手的手背。记录他们的手对于制剂的总体反应和感觉。该研究的结果在表1中列出。The preparation originally stored in the refrigerator was brought to room temperature, and then 10 μL of the preparation was slowly placed on the backs of both hands of the subject at the same time. The general reaction and feel of their hands to the formulation was recorded. The results of this study are listed in Table 1.
表1Table 1
实施例31Example 31
本实施例举例说明了在包含丙泊酚的药物组合物中作为抗氧化剂的去铁胺的应用。This example illustrates the use of deferoxamine as an antioxidant in a pharmaceutical composition comprising propofol.
包含丙泊酚和甲磺酸去铁胺，和含有吐温或TPGS的药物组合物被贮存在4℃，25℃或40℃，以检测甲磺酸去铁胺在防止丙泊酚氧化中的作用。对于这些制剂随时间测量丙泊酚的浓度以，测定去铁胺的抗氧化活性。该数据在下表2和3中作为相对于时间零点的效力％报道。Pharmaceutical compositions comprising propofol and deferoxamine mesylate, and containing Tween or TPGS were stored at 4°C, 25°C, or 40°C to examine the role of deferoxamine mesylate in preventing propofol oxidation. effect. The concentration of propofol was measured over time for these formulations to determine the antioxidant activity of deferoxamine. This data is reported in Tables 2 and 3 below as % potency relative to time zero.
表2.清蛋白/吐温制剂Table 2. Albumin/Tween Formulations
表3.清蛋白/TPGS制剂Table 3. Albumin/TPGS Formulations
在这些条件下，去铁胺足以降低丙泊酚的氧化水平。该效果在越高的温下越显著。在4℃未发生显著氧化。使用没有被惰性材料或聚四氟乙烯涂覆的塞子进行该研究。Under these conditions, deferoxamine was sufficient to reduce oxidation levels of propofol. This effect becomes more remarkable at higher temperatures. No significant oxidation occurred at 4°C. The study was performed using stoppers not coated with inert material or Teflon.
实施例32Example 32
本实施例举例说明了含有紫杉醇和清蛋白的药物组合物(ABI-007)的肺内传递。This example illustrates the intrapulmonary delivery of a pharmaceutical composition (ABI-007) containing paclitaxel and albumin.
本研究的目的是测定[3H]ABI-007在血液中的时程和在向SpragueDawley大鼠气管内滴注后选择组织。The purpose of this study was to determine the time course of [3 H]ABI-007 in blood and select tissues after intratracheal instillation into Sprague Dawley rats.
基于每kg体重1.5mL的剂量体积，计算向动物给药的气管内剂量制剂的目标体积。剂量给药装置由与1-mL气密的luer-锁型注射器连接的Penn-Century微型喷雾器(型号1A-1B；Penn-Century，Inc.，Philadelphia，PA；购自DeLong Distributors，Long Branch，NJ)。将适当体积的剂量制剂吸入剂量给药装置，称量填充的装置并记录重量。将导管放入被麻醉的动物的气管中，将剂量给药装置的微型喷雾器部分通过导管放置在气管内，给药剂量。在剂量给药后，重新称量空的剂量给药装置，作为剂量给药前后剂量给药装置的重量差计算给药的剂量。对于所有动物平均剂量为4.7738±0.0060(CV 1.5059)mg紫杉醇/kg体重。Target volumes of intratracheal dose formulations administered to animals were calculated based on a dose volume of 1.5 mL per kg body weight. Dosing devices consisted of Penn-Century micronebulizers (Models 1A-1B; Penn-Century, Inc., Philadelphia, PA; available from DeLong Distributors, Long Branch, NJ) connected to 1-mL airtight luer-lock syringes. ). An appropriate volume of dosage formulation is drawn into the dosing device, the filled device is weighed and the weight is recorded. A catheter is placed in the trachea of an anesthetized animal, the micronebulizer portion of the dosing device is placed through the catheter into the trachea, and the dose is administered. After dosing, the empty dosing set is reweighed and the dose administered is calculated as the difference in weight of the dosing set before and after dosing. The mean dose for all animals was 4.7738±0.0060 (CV 1.5059) mg paclitaxel/kg body weight.
在下列预定剂量给药后的时刻从JVC大鼠的留置的颈静脉套管中收集约250μL的血样：1，5，10，15，30，和45分钟(min)，和1，4，8，和24小时(h)。24小时血样，以及在10分钟、45分钟和2小时从处死的动物中收集的血样是在处死时通过心穿刺从麻醉的大鼠中收集的。将用于总放射性分析的所有血样分散到预称重的样品管中，将样品管重新称重，通过减法计算每个样品的重量。测定从颈静脉收集的血样以及在处死时从每个动物收集的250-μL的血液等分试样的总氚含量。Blood samples of approximately 250 μL were collected from the indwelling jugular vein cannula of JVC rats at the following predetermined dosing times: 1, 5, 10, 15, 30, and 45 minutes (min), and 1, 4, 8 , and 24 hours (h). 24 hour blood samples, and blood samples collected from sacrificed animals at 10 minutes, 45 minutes and 2 hours were collected from anesthetized rats by cardiac puncture at the time of sacrifice. Disperse all blood samples for total radioactivity analysis into pre-weighed sample tubes, reweigh the sample tubes, and calculate the weight of each sample by subtraction. The total tritium content was determined in blood samples collected from the jugular vein as well as in 250-μL blood aliquots collected from each animal at sacrifice.
对于所有大鼠，血液中氚的最大浓度在剂量给药后5分钟(0.0833小时)时观察到。在从4h至24h的时间间隔内测定的氚的消除半衰期为19.73h至43.02h。应当注意该间隔仅包括三个数据点，其可以解释该参数的变异性。氚从血液中的表观清除率为约0.04L/h。这些实验的结果在下表4中列出。For all rats, the maximum concentration of tritium in the blood was observed at 5 minutes (0.0833 hours) after dosing. The elimination half-life of tritium was determined from 19.73h to 43.02h in the time interval from 4h to 24h. It should be noted that this interval only includes three data points, which may account for the variability of this parameter. The apparent clearance rate of tritium from the blood is about 0.04L/h. The results of these experiments are listed in Table 4 below.
表4.在气管内滴注[3H]ABI-007后在大鼠中血液氚浓度(mg-eq/L)对于时间曲线的非区室分析Table 4. Non-compartmental analysis of blood tritium concentration (mg-eq/L) versus time curves in rats following intratracheal instillation of [3 H]ABI-007
作为时间的函数分析在向大鼠静脉内给药后来源于[3H]ABI-007的放射性的平均血浓度，以便评估来源于气管内给药[3H]ABI-007的氚的生物利用度。该分析导致6.1354mg-eq□hr/L的24-小时AUC(AUC终)。基于这些数据，来源于气管内给药[3H]ABI-007的放射性是高生物利用度的。这些分析是基于总放射性。Analysis of mean blood concentrations of radioactivity derived from [3 H]ABI-007 after intravenous administration to rats as a function of time in order to assess bioavailability of tritium derived from intratracheal administration of [3 H]ABI-007 Spend. This analysis resulted in a 24-hour AUC (AUC final) of 6.1354 mg-eq hr/L. Based on these data, radioactivity from intratracheal administration of [3 H]ABI-007 is highly bioavailable. These analyzes are based on total radioactivity.
来源于[3H]ABI-007的氚在气管内滴注以后被快速吸收。在气管内给药[3H]ABI-007后在血液中氚的平均吸收和消除半衰期(分别为k01半衰期和k10半衰期)分别为0.0155+/-0.0058小时和4.738+/-0.366小时。氚从血液中的平均表观清除率为0.1235+/-0.0180L/小时(参见以上表4)。Tritium derived from [3 H]ABI-007 was rapidly absorbed after intratracheal instillation. After intratracheal administration of [3 H]ABI-007, the mean absorption and elimination half-lives of tritium in blood (k01 half-life and k10 half-life, respectively) were 0.0155+/-0.0058 hours and 4.738+/-0.366 hours, respectively. The mean apparent clearance of tritium from the blood was 0.1235 +/- 0.0180 L/hour (see Table 4 above).
来源于[3H]ABI-007的氚在气管内给药后被吸收和分配。氚在血液中的时程很好地通过二区室模型描述，平均吸收和消除半衰期分别为0.0155和4.738小时。在气管内给药后10分钟在肺中回收约28％的给药剂量。除了胃肠道以外，在所有检查的时刻，在其它组织中回收到最大小于1％的剂量。Tritium derived from [3 H]ABI-007 was absorbed and distributed after intratracheal administration. The time course of tritium in blood is well described by a two-compartment model, with mean absorption and elimination half-lives of 0.0155 and 4.738 hours, respectively. Approximately 28% of the administered dose was recovered in the lungs 10 minutes after intratracheal administration. With the exception of the gastrointestinal tract, a maximum of less than 1% of the dose was recovered in other tissues at all times examined.
基于来自先前使用[3H]CapxolTM进行的静脉内剂量给药研究结果，对于该剂量组的三只动物，来源于气管内给药的氚的生物利用度为1.229±0.268(平均值±SD)。然而，应当注意，这个对于生物利用度的估计是基于总放射性。令人惊奇的是，通过使用本发明含有清蛋白的组合物的肺途径传递的紫杉醇快速可生物利用，显示极好的跨过肺内皮的转运。在动物中未注意到毒性，这是令人惊奇的，因为已知肺传递细胞毒素类导致肺毒性。Based on results from a previous intravenous dosing study using [3 H]Capxol™ , the bioavailability of tritium derived from intratracheal administration was 1.229±0.268 (mean±SD ). It should be noted, however, that this estimate of bioavailability is based on total activity. Surprisingly, paclitaxel delivered by the pulmonary route using the albumin-containing composition of the invention is rapidly bioavailable, showing excellent transport across the lung endothelium. No toxicity was noted in animals, which is surprising since pulmonary delivery of cytotoxins is known to lead to pulmonary toxicity.
在剂量给药(27％的气管内给药)后24小时，相当量的放射性存在于胃肠道(包括内含物)中。在胃肠道中氚的存在可能是由于胆汁排泄或氚从呼吸道通过粘膜纤毛清除和随后吞咽的清除。A substantial amount of radioactivity was present in the gastrointestinal tract (including contents) 24 hours after dosing (27% of intratracheal administration). The presence of tritium in the gastrointestinal tract may be due to biliary excretion or clearance of tritium from the respiratory tract via mucociliary and subsequent ingestion.
实施例33Example 33
本实施例举例说明了用于肺部传递包含紫杉醇和清蛋白的药物组合物的Aerotech II和Pari喷雾器。This example illustrates the Aerotech II and Pari nebulizers for pulmonary delivery of a pharmaceutical composition comprising paclitaxel and albumin.
本研究是使用紫杉醇-清蛋白药物组合物ABI-007在下列条件下进行的：室温(20-23℃)，相对湿度(48-54％)，环境压力(629mmHg)，喷雾器流率(对于Aerotech II为10L/min；对于Pari为7L/min)，总流率(28.3L/min)，喷雾器压力下降(对于Aerotech II为23lb/in2；对于Pari为32lb/in2)，运行时间(15-60秒)，样品体积(1.5mL)，ABI-007紫杉醇浓度(5，10，15和20mg/mL)。This study was carried out using the paclitaxel-albumin pharmaceutical composition ABI-007 under the following conditions: room temperature (20-23°C), relative humidity (48-54%), ambient pressure (629mmHg), nebulizer flow rate (for Aerotech 10 L/min for Aerotech II; 7 L/min for Pari), total flow rate (28.3 L/min), nebulizer pressure drop (23 lb/in2 for Aerotech II; 32 lb/in2 for Pari), run time (15 -60 seconds), sample volume (1.5 mL), ABI-007 paclitaxel concentrations (5, 10, 15 and 20 mg/mL).
当将ABI-007以5-15mg/mL的浓度重构时，Aerotech II和Pari喷雾器都提供了可接受的总效率(30％-60％)。Pari喷雾器效率具有比Aerotech II喷雾器更高的效率。Pari喷雾器效率随着ABI-007浓度增加而有稍微下降。观察到极好的细粒部分(74％-96％)。Aerotech II喷雾器具有比Pari喷雾器更高的细粒部分。细粒部分与浓度无关。Both Aerotech II and Pari nebulizers provided acceptable overall efficiencies (30%-60%) when ABI-007 was reconstituted at concentrations of 5-15 mg/mL. Pari sprayer efficiency has a higher efficiency than Aerotech II sprayer. Pari nebulizer efficiency decreased slightly with increasing concentration of ABI-007. An excellent fines fraction (74%-96%) was observed. The Aerotech II sprayer has a higher fines fraction than the Pari sprayer. The fine particle fraction is independent of concentration.
使用15mg/mL的ABI-007溶液，在小于30分钟内Pari喷雾器传递100mg紫杉醇。使用10mg/mL或15mg/mL的ABI-007溶液，在约65分钟内Aerotech II喷雾器传递100mg紫杉醇。对于Aerotech II和Pari喷雾器检测性能稳定性。直至药物排尽，两种喷雾器的气溶胶浓度和效率是稳定的。在15mg/mL，Pari喷雾器消耗药物的速率是Aerotech II喷雾器的两倍，产生比Aerotech II喷雾器更高的气溶胶浓度。Using a 15 mg/mL solution of ABI-007, the Pari nebulizer delivered 100 mg of paclitaxel in less than 30 minutes. Using a 10 mg/mL or 15 mg/mL solution of ABI-007, the Aerotech II nebulizer delivered 100 mg of paclitaxel over approximately 65 minutes. Performance stability was tested for Aerotech II and Pari nebulizers. Aerosol concentrations and efficiencies were stable for both nebulizers until the drug was exhausted. At 15 mg/mL, the Pari nebulizer consumed drug at twice the rate of the Aerotech II nebulizer and produced higher aerosol concentrations than the Aerotech II nebulizer.
总之，当通过肺途径给药时，紫杉醇的纳米粒/清蛋白制剂(ABI-007)在大鼠中显示极好的生物利用度。在给药剂量下，没有早期毒性的明显标记。纳米粒紫杉醇(ABI-007)的肺传递可以使用常规喷雾器实现。In conclusion, the nanoparticle/albumin formulation of paclitaxel (ABI-007) showed excellent bioavailability in rats when administered by the pulmonary route. At administered doses, there were no overt markers of early toxicity. Pulmonary delivery of nanoparticulate paclitaxel (ABI-007) can be achieved using conventional nebulizers.
实施例34Example 34
本实施例描述了包含清蛋白和雷帕霉素的药物组合物的肺内传递。本研究的目的是测定在向Sprague Dawley大鼠气管内滴注以后在血液中雷帕霉素的肺吸收，并与静脉内滴注比较。This example describes the intrapulmonary delivery of a pharmaceutical composition comprising albumin and rapamycin. The purpose of this study was to determine the pulmonary absorption of rapamycin in the blood following intratracheal instillation in Sprague Dawley rats and compare it with intravenous infusion.
基于每kg体重1mL的剂量体积计算向动物给药的气管内剂量制剂的目标体积。剂量给药装置由与1-mL气密的luer-锁型注射器连接的Penn-Century微型喷雾器(型号1A-1B；Penn-Century，Inc.，Philadelphia，PA；购自DeLong Distributors，Long Branch，NJ)。将适当体积的剂量制剂吸入剂量给药装置，称量填充的装置并记录重量。将导管放入被麻醉的动物的气管中，将剂量给药装置的微型喷雾器部分通过导管放置在气管内，给药剂量。在剂量给药后，重新称量空的剂量给药装置，作为剂量给药前后剂量给药装置的重量差计算给药的剂量。Target volumes of intratracheal dose formulations administered to animals were calculated based on a dose volume of 1 mL per kg body weight. Dosing devices consisted of Penn-Century micronebulizers (Models 1A-1B; Penn-Century, Inc., Philadelphia, PA; available from DeLong Distributors, Long Branch, NJ) connected to 1-mL airtight luer-lock syringes. ). An appropriate volume of dosage formulation is drawn into the dosing device, the filled device is weighed and the weight is recorded. A catheter is placed in the trachea of an anesthetized animal, the micronebulizer portion of the dosing device is placed through the catheter into the trachea, and the dose is administered. After dosing, the empty dosing set is reweighed and the dose administered is calculated as the difference in weight of the dosing set before and after dosing.
在下列预定剂量给药时刻从大鼠的留置的颈静脉套管中收集约250μL的血样：1，5，10，15，30，和45分钟(min)，和1，4，8，和24小时(h)。将所有分析的血样分配到预先称重的样品管中，将样品管重新称重，通过减法计算每个样品的重量。使用LC/MS/MS测定收集的血样的总雷帕霉素浓度。Blood samples of approximately 250 μL were collected from rats' indwelling jugular vein cannula at the following scheduled dosing times: 1, 5, 10, 15, 30, and 45 minutes (min), and 1, 4, 8, and 24 hours (h). All analyzed blood samples were dispensed into pre-weighed sample tubes, the tubes were reweighed, and the weight of each sample was calculated by subtraction. Total rapamycin concentrations in collected blood samples were determined using LC/MS/MS.
令人惊奇的是，结果显示在通过肺途径和通过静脉内传递的雷帕霉素的血浓度之间没有显著差异。使用包含清蛋白的药物组合物，通过肺途径传递的雷帕霉素的生物利用度被计算为109％，显示极好的跨过肺内皮的转运。Surprisingly, the results showed no significant difference between the blood levels of rapamycin delivered by the pulmonary route and by the intravenous route. Using a pharmaceutical composition comprising albumin, the bioavailability of rapamycin delivered by the pulmonary route was calculated to be 109%, showing excellent transport across the lung endothelium.
实施例35Example 35
本实施例举例说明在肺内给药按照本发明制备的包含雷帕霉素和清蛋白的药物组合物以后，清蛋白-雷帕霉素的组织分布。本研究的目的是测定在向Sprague Dawley大鼠气管内滴注以后雷帕霉素在组织中的肺吸收，并与静脉内滴注相比较。This example illustrates the tissue distribution of albumin-rapamycin following intrapulmonary administration of a pharmaceutical composition comprising rapamycin and albumin prepared according to the present invention. The purpose of this study was to determine the lung absorption of rapamycin in tissues following intratracheal instillation in Sprague Dawley rats and compare it with intravenous infusion.
基于每kg体重1mL的剂量体积计算向动物给药的气管内剂量制剂的目标体积。剂量给药装置由与1-mL气密的luer-锁型注射器连接的Penn-Century微型喷雾器(型号1A-1B；Penn-Century，Inc.，Philadelphia，PA；购自DeLong Distributors，Long Branch，NJ)。将适当体积的剂量制剂吸入剂量给药装置，称量填充的装置并记录重量。将导管放入被麻醉的动物的气管中，将剂量给药装置的微型喷雾器部分通过导管放置在气管内，给药剂量。在剂量给药后，重新称量空的剂量给药装置，作为剂量给药前后剂量给药装置的重量差计算给药的剂量。Target volumes of intratracheal dose formulations administered to animals were calculated based on a dose volume of 1 mL per kg body weight. Dosing devices consisted of Penn-Century micronebulizers (Models 1A-1B; Penn-Century, Inc., Philadelphia, PA; available from DeLong Distributors, Long Branch, NJ) connected to 1-mL airtight luer-lock syringes. ). An appropriate volume of dosage formulation is drawn into the dosing device, the filled device is weighed and the weight is recorded. A catheter is placed in the trachea of an anesthetized animal, the micronebulizer portion of the dosing device is placed through the catheter into the trachea, and the dose is administered. After dosing, the empty dosing set is reweighed and the dose administered is calculated as the difference in weight of the dosing set before and after dosing.
在10分钟、45分钟、2小时和24小时的每个时刻从每组3只大鼠的脑，肺和肝中收集样品。收集样品并使用LC/MS/MS分析总雷帕霉素浓度。结果显示当通过肺传递时与静脉内传递相比雷帕霉素浓度在肺组织中更大。然而，与静脉内(IV)传递相比，当通过气管内(IT)传递时雷帕霉素在脑中的总浓度较低。在肝中，无论IT或IV传递，雷帕霉素的浓度似乎没有差别。基于这些结果，肺内传递雷帕霉素可以适用于治疗病症(即肺移植)，其中高局部浓度的雷帕霉素将是有益的。Samples were collected from the brain, lung and liver of 3 rats per group at each time of 10 minutes, 45 minutes, 2 hours and 24 hours. Samples were collected and analyzed for total rapamycin concentration using LC/MS/MS. The results showed that rapamycin concentrations were greater in lung tissue when delivered through the lungs compared to intravenous delivery. However, the total concentration of rapamycin in the brain is lower when delivered intratracheally (IT) compared to intravenous (IV) delivery. In the liver, there appeared to be no difference in the concentration of rapamycin regardless of IT or IV delivery. Based on these results, intrapulmonary delivery of rapamycin may be suitable for the treatment of conditions (ie, lung transplantation) where high local concentrations of rapamycin would be beneficial.
实施例36Example 36
本实施例举例说明了口服传递含有紫杉醇和清蛋白的药物组合物(ABI-007)。This example illustrates the oral delivery of a pharmaceutical composition (ABI-007) containing paclitaxel and albumin.
将氚化的ABI-007用于在大鼠中在口服管饲法后测定紫杉醇的口服生物利用度。在过夜禁食后，将5只大鼠给予5.5mg/kg在ABI-007中的紫杉醇(组A)且另5只大鼠(组B)用环孢菌素(5.0mg/kg)预处理，接着用5.6mg/kg在ABI-007中的紫杉醇处理。在通过燃烧测定血样中的放射性以后，进行在0.5，1，2，3，4，5，6，8，12，和24小时抽取的血样的药代动力学分析。通过与先前获得的静脉内数据相比较来测定口服生物利用度。结果在下表5中列出。Tritiated ABI-007 was used to determine the oral bioavailability of paclitaxel following oral gavage in rats. After an overnight fast, 5 rats were given 5.5 mg/kg paclitaxel in ABI-007 (group A) and another 5 rats (group B) were pretreated with cyclosporine (5.0 mg/kg) , followed by treatment with 5.6 mg/kg paclitaxel in ABI-007. Pharmacokinetic analysis of blood samples drawn at 0.5, 1, 2, 3, 4, 5, 6, 8, 12, and 24 hours was performed after determination of radioactivity in the blood samples by combustion. Oral bioavailability was determined by comparison with previously obtained intravenous data. The results are listed in Table 5 below.
表5.在口服给药后3H-紫杉醇来源的放射性的平均AUC 0-24，Cmax，Tmax和％吸收Table 5. Mean AUC0-24,Cmax ,Tmax and % Absorption of 3H-paclitaxel-Derived Radioactivity Following Oral Administration
组 处理 剂量/途径 AUC0-24 吸收 Cmax(mg/kg) TmaxGroup Treatment Dose/Route AUC0-24 Absorption Cmax(mg/kg) Tmax
mg/kg (μg eq×hr/mL) (％) (μg×eq/mL) (hr)mg/kg (μg eq×hr/mL) (%) (μg×eq/mL) (hr)
A 在生理盐水中的 5.5/PO(P) 2.92 44.3 0.245 1A 5.5/PO(P) 2.92 44.3 0.245 1 in normal saline
ABI-007ABI-007
B 在含有CsA的生 5/PO(C)， 8.02 121.1 0.565 0.5B in raw 5/PO(C) containing CsA, 8.02 121.1 0.565 0.5
理盐水中的 5.6/PO(P)5.6/PO(P) in saline
ABI-007ABI-007
将AUC 0-24IV(6.06μg×hr./mL)和IV剂量(5.1mg/kg)用于计算吸收百分率(基于ABI-007的IV剂量的数据)。AUC 0-24 IV (6.06 μg x hr./mL) and IV dose (5.1 mg/kg) were used to calculate percent absorption (based on data for the IV dose of ABI-007).
对于单独ABI-007观察到44％的口服生物利用度。这显著高于对于其它紫杉醇制剂所观察到的。当用环孢菌素(CsA)处理动物时，生物利用度增加到121％。这是预料到的，因为CsA是p-糖蛋白泵的已知抑制剂，其通常将抑制化合物如紫杉醇从GI道吸收。大于100％的生物利用度可以通过在将紫杉醇胆汁排泄到GI道以后的再吸收来解释。其它已知的吸收抑制剂或增强剂也可以用于该目的。An oral bioavailability of 44% was observed for ABI-007 alone. This is significantly higher than that observed for other paclitaxel formulations. When animals were treated with cyclosporine (CsA), the bioavailability increased to 121%. This is expected since CsA is a known inhibitor of the p-glycoprotein pump, which would normally inhibit the absorption of compounds such as paclitaxel from the GI tract. The greater than 100% bioavailability can be explained by the reabsorption of paclitaxel following bile excretion into the GI tract. Other known absorption inhibitors or enhancers may also be used for this purpose.
实施例37Example 37
本实施例举例说明了在给药包含紫杉醇和清蛋白的药物组合物后改善的紫杉醇向红细胞和肿瘤细胞的穿透。This example illustrates the improved penetration of paclitaxel into erythrocytes and tumor cells following administration of a pharmaceutical composition comprising paclitaxel and albumin.
将人MX-1乳腺瘤碎片皮下移植到无胸腺小鼠中。用3H紫杉醇紫杉醇制备如上所述的包含紫杉醇和清蛋白的药物组合物(“紫杉醇-清蛋白”)和泰素，比放射性为25μCi/mg紫杉醇。当肿瘤体积达到约500mm3时，将20mg/kg放射标记的紫杉醇-清蛋白或泰素在盐水中静脉内给药。将血浆、血液和肿瘤组织采样并在给药后5，15，和30分钟和在1，3，8，和24小时显示分析放射性。使用WinNonlin，Pharsight，USA分析肿瘤药代动力学(AUC和吸收常数)。Fragments of human MX-1 mammary tumors were subcutaneously transplanted into athymic mice. A pharmaceutical composition comprising paclitaxel and albumin ("paclitaxel-albumin") as described above was preparedusing3H paclitaxel paclitaxel and taxol with a specific activity of 25 μCi/mg paclitaxel. When the tumor volume reached approximately 500mm3 , 20 mg/kg radiolabeled paclitaxel-albumin or taxol was administered intravenously in saline. Plasma, blood, and tumor tissue were sampled and assayed for radioactivity at 5, 15, and 30 minutes and at 1, 3, 8, and 24 hours post-dose. Tumor pharmacokinetics (AUC and absorption constant) were analyzed using WinNonlin, Pharsight, USA.
紫杉醇-清蛋白显示向红细胞(RBC)的快速分配，如在静脉内给药药物以后血浆/血液放射性比率快速降低至一致所示。向RBC中的完全分配早在给药紫杉醇-清蛋白后1小时就发生了。相比之下，配制为泰素的紫杉醇向RBC的分配要缓慢地多，并且直至超过8小时还未完成。Paclitaxel-albumin showed rapid partitioning to red blood cells (RBCs), as indicated by a rapid decrease in the plasma/blood radioactivity ratio to coincide after intravenous administration of the drug. Complete distribution into RBCs occurred as early as 1 hour after paclitaxel-albumin administration. In contrast, the distribution of Paclitaxel formulated as Taxol to RBC was much slower and was not complete until more than 8 hours.
紫杉醇-清蛋白显示向肿瘤组织的快速分配，吸收常数(Ka)比泰素大3.3X。Ka对于紫杉醇-清蛋白和泰素分别为0.43hr-1和0.13hr-1。紫杉醇的快速摄取导致紫杉醇-清蛋白的肿瘤AUC比泰素高33％。AUC对于紫杉醇-清蛋白和泰素分别为3632nCi*hr/g和2739nCi*hr/g。Paclitaxel-albumin showed rapid partition to tumor tissue with an absorption constant (Ka ) 3.3X greater than Taxol.Ka for paclitaxel-albumin and taxol were 0.43hr-1 and 0.13hr-1 , respectively. Rapid uptake of paclitaxel resulted in a 33% higher tumor AUC for paclitaxel-albumin than taxol. AUC was 3632 nCi*hr/g and 2739 nCi*hr/g for paclitaxel-albumin and taxol, respectively.
实施例38Example 38
本实施例举例说明了向小鼠给药的含有紫杉醇和清蛋白的药物组合物的安全性。This example illustrates the safety of a pharmaceutical composition containing paclitaxel and albumin administered to mice.
每天用逐渐增加剂量的紫杉醇-清蛋白或泰素处理无胸腺小鼠，连续5天。将存活率对剂量作图以测定LD50。与泰素相比，紫杉醇-清蛋白极大增加了存活率(p＝0.017，ANOVA)。对于q1d×5进度表，紫杉醇-清蛋白和泰素的LD50分别计算为47mg/kg/天和30mg/kg/天。在13.4mg/kg/天的剂量水平下，紫杉醇-清蛋白和泰素都被很好地耐受，死亡率分别为1％(72只小鼠中1只死亡)和4％(47只小鼠中2只死亡)。在20mg/kg/天的剂量水平下，紫杉醇-清蛋白的死亡率为1％(72只小鼠中1只死亡)，泰素死亡率为17％(47只小鼠中8只死亡)(p＝0.0025)。在30mg/kg/天的剂量水平下，紫杉醇-清蛋白的死亡率为4％(72只小鼠中3只死亡)，泰素死亡率为49％(47只小鼠中23只死亡)(p＜0.0001)。Athymic mice were treated daily for 5 consecutive days with increasing doses of paclitaxel-albumin or taxol. Survival was plotted against dose to determineLD50 . Paclitaxel-albumin greatly increased survival compared to Taxol (p=0.017, ANOVA). For the q1d x 5 schedule, the LD50s for paclitaxel-albumin and taxol were calculated to be 47 mg/kg/day and 30 mg/kg/day, respectively. At a dose level of 13.4 mg/kg/day, both paclitaxel-albumin and taxol were well tolerated, with mortality rates of 1% (1 death in 72 mice) and 4% (47 mice), respectively. 2 of the mice died). At the dose level of 20mg/kg/day, the mortality rate of paclitaxel-albumin was 1% (1 death in 72 mice), and the death rate of taxol was 17% (8 deaths in 47 mice) ( p=0.0025). At the dose level of 30 mg/kg/day, the mortality rate of paclitaxel-albumin was 4% (3 out of 72 mice) and Taxol was 49% (23 out of 47) ( p<0.0001).
实施例39Example 39
本实施例举例说明了对于紫杉醇-清蛋白组合物，跨过微脉管内皮细胞(EC)的新紫杉醇转运机制。This example illustrates a novel paclitaxel transport mechanism across microvascular endothelial cells (EC) for paclitaxel-albumin compositions.
由于肿瘤中“渗漏”脉管导致的EPR作用，纳米粒和清蛋白-紫杉醇组合物可以在肿瘤组织中累积。清蛋白特异的gp60受体(albondin)通过细胞表面处的细胞膜穴样内陷内的受体的胞转，将清蛋白转运通过EC。该胞转机制允许清蛋白-紫杉醇转运至底层的胞间隙中。相反，泰素中的聚氧乙烯蓖麻油抑制紫杉醇与清蛋白的结合，极大地降低了紫杉醇向肿瘤的转运。另外，gp16和gp30受体还涉及含有结合的紫杉醇的修饰的清蛋白的胞内转运，导致增加的紫杉醇与内皮细胞的结合，与泰素相比抗血管生成作用更大。Nanoparticles and albumin-paclitaxel compositions can accumulate in tumor tissue due to the EPR action caused by "leaky" vessels in the tumor. The albumin-specific gp60 receptor (albondin) transports albumin through ECs through transcytosis of the receptor in caveolae at the cell surface. This transcytosis mechanism allows the transport of albumin-paclitaxel into the underlying interstitial space. In contrast, polyoxyethylene castor oil in Taxol inhibits the binding of paclitaxel to albumin, greatly reducing the transport of paclitaxel to tumors. In addition, gp16 and gp30 receptors are also involved in the intracellular transport of modified albumin containing conjugated paclitaxel, leading to increased binding of paclitaxel to endothelial cells and a greater anti-angiogenic effect than taxol.
实施例40Example 40
本实施例举例说明了与泰素相比，含有紫杉醇和清蛋白的药物组合物的内皮胞转的增加。This example illustrates the increase in endothelial translocation of a pharmaceutical composition containing paclitaxel and albumin compared to taxol.
将人肺微脉管内皮细胞(HLMVEC)在transwell上培养至汇合。将浓度为20μg/mL本发明含有紫杉醇和清蛋白的药物组合物或泰素加入至上transwell室中。Human lung microvascular endothelial cells (HLMVEC) were cultured on transwell until confluent. The pharmaceutical composition containing paclitaxel and albumin or taxol of the present invention at a concentration of 20 μg/mL was added into the upper transwell chamber.
使用荧光计连续监视紫杉醇通过胞转从上室转运到下室中的转运。还使用仅含有Flutax而无清蛋白的对照。含有Flutax的对照未显示转运，证明汇合HLMVEC单层的完整性。在5％HSA(生理浓度)的存在下，来自清蛋白-紫杉醇组合物的紫杉醇的转运要比来自泰素的紫杉醇快得多。转运速率常数(Kt)对于清蛋白-紫杉醇组合物和泰素分别为1.396hr-1和0.03hr-1。清蛋白-紫杉醇组合物的转运跨过单层的紫杉醇的总量比泰素的转运跨过单层的紫杉醇的总量高3倍。The transport of paclitaxel by transcytosis from the upper chamber into the lower chamber was continuously monitored using a fluorometer. A control containing only Flutax without albumin was also used. Controls containing Flutax showed no translocation, demonstrating the integrity of the confluent HLMVEC monolayer. In the presence of 5% HSA (physiological concentration), paclitaxel from the albumin-paclitaxel composition was transported much faster than paclitaxel from Taxol. The transport rate constant (Kt ) was 1.396 hr−1 and 0.03 hr−1 for the albumin-paclitaxel composition and Taxol, respectively. The total amount of paclitaxel transported across the monolayer was 3-fold higher for the albumin-paclitaxel composition than for Taxol.
实施例41Example 41
本实施例举例说明了与泰素相比，含有紫杉醇和清蛋白的药物组合物改善的内皮细胞(EC)结合。This example illustrates the improved endothelial cell (EC) binding of a pharmaceutical composition containing paclitaxel and albumin compared to Taxol.
将人脐静脉内皮细胞(HUVEC)培养在96-孔微量滴定板上。在一个实验中，在增加浓度的聚氧乙烯蓖麻油EL/EtOH(泰素的载体)的存在下，将HUVEC与紫杉醇(Flutax-Oregon Green标记的紫杉醇)反应。在另一实验中，含有清蛋白和Flutax的药物组合物和泰素-Flutax组合物在各种终浓度下与HUVEC反应。紫杉醇与细胞的结合受聚氧乙烯蓖麻油的抑制。通过聚氧乙烯蓖麻油EL/EtOH的0.02％的IC50显示抑制。该浓度的聚氧乙烯蓖麻油已经显示在泰素化疗期间持续至少24小时。因此，它是体内相关过程。在所有检测浓度下，显著量的来自清蛋白-紫杉醇组合物的紫杉醇与细胞结合。相比之下，对于泰素观察到很少或未观察到结合。Human umbilical vein endothelial cells (HUVEC) were cultured in 96-well microtiter plates. In one experiment, HUVECs were reacted with paclitaxel (Flutax-Oregon Green labeled paclitaxel) in the presence of increasing concentrations of polyoxyethylene castor oil EL/EtOH (vehicle of Taxol). In another experiment, pharmaceutical compositions containing albumin and Flutax and Taxol-Flutax compositions were reacted with HUVECs at various final concentrations. Binding of paclitaxel to cells is inhibited by polyoxyethylene castor oil. Inhibition was shown by anIC50 of 0.02% of polyoxyethylene castor oil EL/EtOH. This concentration of polyoxyethylene castor oil has been shown to persist for at least 24 hours during taxol chemotherapy. Therefore, it is a relevant process in vivo. Significant amounts of paclitaxel from the albumin-paclitaxel composition were bound to cells at all concentrations tested. In contrast, little or no binding was observed for Taxol.
实施例42Example 42
本实施例举例说明了与泰素相比，含有紫杉醇和清蛋白的药物组合物的改善的清蛋白结合。This example illustrates the improved albumin binding of a pharmaceutical composition containing paclitaxel and albumin compared to taxol.
将人血清清蛋白(HSA)固定在塑料ELISA板上。将紫杉醇(Flutax-Oregon Green标记的紫杉醇)与固定的HSA在增加浓度的聚氧乙烯蓖麻油EL/EtOH的存在下反应。在另一实验中，清蛋白-紫杉醇-Flutax组合物和泰素-Flutax组合物与固定的HSA在20μg紫杉醇/mL的终浓度下反应。紫杉醇与清蛋白的结合受聚氧乙烯蓖麻油的抑制。通过聚氧乙烯蓖麻油EL/EtOH的0.003％的IC50显示抑制。该浓度的聚氧乙烯蓖麻油已经显示在泰素化疗期间持续至少24小时。因此，它是体内相关过程。在相关药物紫杉醇浓度(20μg/mL)下，显著量的来自清蛋白-紫杉醇组合物的紫杉醇与固定的HSA结合。相比之下，对于泰素未观察到结合。Human serum albumin (HSA) was immobilized on plastic ELISA plates. Paclitaxel (Flutax-Oregon Green labeled paclitaxel) was reacted with immobilized HSA in the presence of increasing concentrations of polyoxyethylene castor oil EL/EtOH. In another experiment, albumin-paclitaxel-Flutax compositions and Taxol-Flutax compositions were reacted with immobilized HSA at a final concentration of 20 μg paclitaxel/mL. Binding of paclitaxel to albumin is inhibited by polyoxyethylene castor oil. Inhibition was shown by anIC50 of 0.003% of polyoxyethylene castor oil EL/EtOH. This concentration of polyoxyethylene castor oil has been shown to persist for at least 24 hours during taxol chemotherapy. Therefore, it is a relevant process in vivo. At the relevant drug paclitaxel concentration (20 μg/mL), a significant amount of paclitaxel from the albumin-paclitaxel composition was bound to immobilized HSA. In contrast, no binding was observed for taxol.
实施例43Example 43
本实施例举例说明了与泰素相比，对于含有紫杉醇和清蛋白的药物组合物增加的紫杉醇向清蛋白的转运。This example illustrates the increased transport of paclitaxel to albumin for a pharmaceutical composition containing paclitaxel and albumin compared to taxol.
将泰素-Flutax和清蛋白-紫杉醇-Flutax组合物以20μg/mL，40μg/ml，和80μg/ml与在Hanks缓冲液中的5％HSA或与血清混合。立即在天然3-14％聚丙烯酰胺凝胶上分离混合物，通过扫描荧光计测定与清蛋白结合的紫杉醇的量。与泰素相比，对于清蛋白-紫杉醇组合物，紫杉醇向HSA的转运更快。当将血清或5％HSA与清蛋白-紫杉醇-Flutax组合物或泰素-Flutax组合物温育时，更多的紫杉醇与HSA共电泳。与泰素-Flutax组合物相比，20μg/ml，40μg/ml，和80μg/ml的清蛋白-紫杉醇-Flutax组合物在接触5％HSA后，分别有45％，60％，和33％更多的紫杉醇转运至HSA。对于260mg/m2的ABI-007，Cmax为约20μg/mL，因此这是重要的体内过程。Taxol-Flutax and Albumin-Paclitaxel-Flutax compositions were mixed at 20 μg/mL, 40 μg/ml, and 80 μg/ml with 5% HSA in Hanks buffer or with serum. The mixture was immediately separated on a native 3-14% polyacrylamide gel, and the amount of paclitaxel bound to albumin was determined by scanning fluorometry. The transport of paclitaxel to HSA was faster for the albumin-paclitaxel composition compared to taxol. When serum or 5% HSA was incubated with albumin-paclitaxel-Flutax composition or taxol-Flutax composition, more paclitaxel co-electrophoresed with HSA. Compared with the Taxol-Flutax composition, the 20 μg/ml, 40 μg/ml, and 80 μg/ml albumin-paclitaxel-Flutax compositions were 45%, 60%, and 33% more effective after exposure to 5% HSA, respectively. More paclitaxel is transported to HSA. For 260 mg/m2 of ABI-007, theCmax is about 20 μg/mL, so this is an important in vivo process.
实施例44Example 44
本实施例举例说明了糖蛋白受体gp60负责清蛋白-紫杉醇的结合和胞转。This example illustrates that the glycoprotein receptor gp60 is responsible for albumin-paclitaxel binding and transcytosis.
荧光标记的紫杉醇(Flutax)清蛋白组合物与培养物中的微脉管内皮细胞接触。在显微镜下观察到荧光染色，证据是斑点区域，这推测是与清蛋白-紫杉醇结合的gp60受体。这通过使用罗丹明标记的清蛋白证实，罗丹明标记的清蛋白与紫杉醇的斑点荧光位置相同。A fluorescently labeled paclitaxel (Flutax) albumin composition is contacted with microvascular endothelial cells in culture. Fluorescent staining was observed under the microscope, evidenced by areas of dots, which were presumed to be gp60 receptors bound to albumin-paclitaxel. This was confirmed by using rhodamine-tagged albumin, which has the same spot fluorescence as paclitaxel.
实施例45Example 45
本实施例举例说明了增加量的清蛋白可以与紫杉醇的结合竞争。This example demonstrates that increasing amounts of albumin can compete with paclitaxel binding.
将清蛋白固定在微量滴定板上。将荧光紫杉醇加入孔中，使用扫描荧光计测量紫杉醇的结合。将增加量的清蛋白加入孔中并测量与固定的清蛋白结合的紫杉醇的抑制水平。数据显示随着加入的清蛋白的量增加，观察到结合的相应降低。对于与内皮细胞的结合观察到类似效果。这显示更高的清蛋白浓度抑制紫杉醇的结合。优选本发明具有较低量的清蛋白的组合物。Albumin was immobilized on microtiter plates. Fluorescent paclitaxel was added to the wells and paclitaxel incorporation was measured using a scanning fluorometer. Increasing amounts of albumin were added to the wells and the level of inhibition of paclitaxel binding to immobilized albumin was measured. The data show that as the amount of albumin added increases, a corresponding decrease in binding is observed. Similar effects were observed for binding to endothelial cells. This shows that higher albumin concentrations inhibit paclitaxel binding. Compositions of the invention having lower amounts of albumin are preferred.
实施例46Example 46
本实施例举例说明了在本发明药物组合物中较低量的清蛋白导致稳定的组合物。This example illustrates that lower amounts of albumin in pharmaceutical compositions of the invention lead to stable compositions.
为了研究组合物中较低量的清蛋白是否影响本发明药物组合物的稳定性，制备含有低量清蛋白的清蛋白-紫杉醇组合物。当在不同温度(2-8℃，25℃和40℃)下检查紫杉醇功效、杂质形成、粒径、pH和其它稳定性的典型参数时，发现这些组合物与具有更高量的清蛋白的组合物一样稳定。因此优选具有较低量的清蛋白的组合物，因为这可以极大地降低成本和允许增加的结合和向细胞的转运。In order to investigate whether the lower amount of albumin in the composition affects the stability of the pharmaceutical composition of the present invention, an albumin-paclitaxel composition containing a low amount of albumin was prepared. When typical parameters of paclitaxel efficacy, impurity formation, particle size, pH and other stability were examined at different temperatures (2-8°C, 25°C and 40°C), it was found that these compositions were comparable to those with higher amounts of albumin. The composition is equally stable. Compositions with lower amounts of albumin are therefore preferred as this can greatly reduce costs and allow increased binding and transport to cells.
实施例47Example 47
本实施例举例说明了含有清蛋白和紫杉醇的药物组合物，其具有高的清蛋白与紫杉醇比率。This example illustrates a pharmaceutical composition containing albumin and paclitaxel, which has a high ratio of albumin to paclitaxel.
将30mg紫杉醇溶解在3.0ml二氯甲烷中。将溶液加入至27.0ml人血清清蛋白溶液(3％w/v)中(相当于清蛋白与紫杉醇的比率为27)。根据需要加入去铁胺。将该混合物在低RPM下均化5分钟(Vitris均化器，型号Tempest I.Q.)以便形成粗乳剂，然后转移到高压均化器(Avestin)中。在9000-40,000psi下进行乳化，同时将乳剂再循环至少5个周期。将获得的系统转移至旋转式蒸发器，在40℃、减压(30mm Hg)下快速去除二氯甲烷20-30分钟。获得的分散体是半透明的，获得的紫杉醇颗粒的典型平均直径为50-220nm(Z-平均值，Malvern Zetasizer)。将该分散体进一步冻干48小时。通过加入无菌水或盐水，获得的饼块可以容易地重构为原有分散体。在重构后的粒径与冻干前相同。30 mg paclitaxel was dissolved in 3.0 ml dichloromethane. The solution was added to 27.0 ml of human serum albumin solution (3% w/v) (equivalent to a ratio of albumin to paclitaxel of 27). Add deferoxamine as needed. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000 psi while recirculating the emulsion for at least 5 cycles. The resulting system was transferred to a rotary evaporator and the dichloromethane was rapidly removed at 40 °C under reduced pressure (30 mm Hg) for 20-30 minutes. The dispersion obtained was translucent and the paclitaxel particles obtained had a typical mean diameter of 50-220 nm (Z-mean, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted into the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization.
应当认识到用于本实施例的药物、溶剂、蛋白质的量、类型和比率不受任何方式的限制。当与溶于聚氧乙烯蓖麻油制剂中的紫杉醇的毒性相比时，本发明含有清蛋白的药物组合物显示显著更低的毒性。It should be appreciated that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The albumin-containing pharmaceutical composition of the invention showed significantly lower toxicity when compared to the toxicity of paclitaxel dissolved in a polyoxyethylene castor oil formulation.
实施例48Example 48
本实施例举例说明了含有清蛋白和紫杉醇的药物组合物，其具有低的清蛋白与紫杉醇比率。This example illustrates a pharmaceutical composition containing albumin and paclitaxel, which has a low ratio of albumin to paclitaxel.
具体地，将300mg紫杉醇溶解在3.0ml二氯甲烷中。将溶液加入至27ml人血清清蛋白溶液(5％w/v)中(相当于清蛋白与紫杉醇的比率为4.5)。根据需要加入去铁胺。将该混合物在低RPM下均化5分钟(Vitris均化器，型号Tempest I.Q.)以便形成粗乳剂，然后转移到高压均化器(Avestin)中。在9000-40,000psi下进行乳化，同时将乳剂再循环至少5个周期。将获得的系统转移至旋转式蒸发器，在40℃、减压(30mm Hg)下快速去除二氯甲烷20-30分钟。获得的分散体是半透明的，获得的紫杉醇颗粒的典型平均直径为50-220nm(Z-平均值，Malvern Zetasizer)。将该分散体进一步冻干48小时。通过加入无菌水或盐水，获得的饼块可以容易地重构为原有分散体。在重构后的粒径与冻干前相同。Specifically, 300 mg of paclitaxel was dissolved in 3.0 ml of dichloromethane. The solution was added to 27 ml of human serum albumin solution (5% w/v) (equivalent to a ratio of albumin to paclitaxel of 4.5). Add deferoxamine as needed. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000 psi while recirculating the emulsion for at least 5 cycles. The resulting system was transferred to a rotary evaporator and the dichloromethane was rapidly removed at 40 °C under reduced pressure (30 mm Hg) for 20-30 minutes. The dispersion obtained was translucent and the paclitaxel particles obtained had a typical mean diameter of 50-220 nm (Z-mean, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted into the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization.
应当认识到用于本实施例的药物、溶剂、蛋白质的量、类型和比率不受任何方式的限制。当与溶于聚氧乙烯蓖麻油制剂中的紫杉醇的毒性相比时，本发明含有清蛋白的药物组合物显示显著更低的毒性。It should be appreciated that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The albumin-containing pharmaceutical composition of the invention showed significantly lower toxicity when compared to the toxicity of paclitaxel dissolved in a polyoxyethylene castor oil formulation.
实施例49Example 49
本实施例举例说明了含有清蛋白和紫杉醇的药物组合物，其具有中等的清蛋白与紫杉醇比率。This example illustrates a pharmaceutical composition containing albumin and paclitaxel, which has an intermediate ratio of albumin to paclitaxel.
具体地，将135mg紫杉醇溶解在3.0ml二氯甲烷中。将溶液加入至27ml人血清清蛋白溶液(5％w/v)中。根据需要加入去铁胺。将该混合物在低RPM下均化5分钟(Vitris均化器，型号Tempest I.Q.)以便形成粗乳剂，然后转移到高压均化器(Avestin)中。在9000-40,000psi下进行乳化，同时将乳剂再循环至少5个周期。将获得的系统转移至旋转式蒸发器，在40℃在减压(30mm Hg)下快速去除二氯甲烷20-30分钟。获得的分散体是半透明的，获得的紫杉醇颗粒的典型平均直径为50-220nm(Z-平均值，MalvernZetasizer)。将该分散体进一步冻干48小时。通过加入无菌水或盐水，获得的饼块可以容易地重构为原有分散体。在重构后的粒径与冻干前相同。在本发明组合物中，清蛋白与紫杉醇的计算比率(w/w)为约10。Specifically, 135 mg of paclitaxel was dissolved in 3.0 ml of dichloromethane. The solution was added to 27 ml of human serum albumin solution (5% w/v). Add deferoxamine as needed. The mixture was homogenized at low RPM for 5 minutes (Vitris homogenizer, model Tempest I.Q.) to form a coarse emulsion and then transferred to a high pressure homogenizer (Avestin). Emulsification was performed at 9000-40,000 psi while recirculating the emulsion for at least 5 cycles. The resulting system was transferred to a rotary evaporator and dichloromethane was rapidly removed under reduced pressure (30 mm Hg) at 40 °C for 20-30 min. The dispersion obtained was translucent and the paclitaxel particles obtained had a typical mean diameter of 50-220 nm (Z-mean, Malvern Zetasizer). The dispersion was further lyophilized for 48 hours. The cake obtained can be easily reconstituted into the original dispersion by adding sterile water or saline. The particle size after reconstitution was the same as before lyophilization. In the composition of the present invention, the calculated ratio (w/w) of albumin to paclitaxel is about 10.
应当认识到用于本实施例的药物、溶剂、蛋白质的量、类型和比率不受任何方式的限制。当与溶于聚氧乙烯蓖麻油制剂中的紫杉醇的毒性相比时，本发明含有清蛋白的药物组合物显示显著更低的毒性。It should be appreciated that the amounts, types and ratios of drugs, solvents, proteins used in this example are not limited in any way. The albumin-containing pharmaceutical composition of the invention showed significantly lower toxicity when compared to the toxicity of paclitaxel dissolved in a polyoxyethylene castor oil formulation.
实施例50Example 50
本实施例举例说明了用清蛋白-紫杉醇组合物治疗动物模型中的类风湿性关节炎。This example illustrates the treatment of rheumatoid arthritis in an animal model with an albumin-paclitaxel composition.
将在Louvain大鼠中胶原蛋白诱导的关节炎模型用于检测清蛋白-紫杉醇组合物对关节炎的治疗效果。监视实验动物的爪尺寸以评估关节炎的严重性。The collagen-induced arthritis model in Louvain rats was used to examine the therapeutic effect of the albumin-paclitaxel composition on arthritis. The paw size of experimental animals was monitored to assess the severity of arthritis.
在关节炎完全发展以后(通常在胶原蛋白注射后约9-10天)，将实验动物分成不同组以腹膜内接受清蛋白-紫杉醇1mg/kg q.o.d，或清蛋白-紫杉醇0.5mg/kg+泼尼松0.2mg/kg q.o.d.(联合治疗)达6个剂量，然后每周一个剂量达三周。在治疗开始(第0天)和每次注射药物时测量爪尺寸。仅接受生理盐水的一组作为对照。在实验结束时，接受清蛋白-紫杉醇的组爪尺寸实现42％的减小，联合治疗组显示爪尺寸减小33％，而对照组爪尺寸与治疗开始时相比增加约20％。After the arthritis has fully developed (usually about 9-10 days after collagen injection), the experimental animals are divided into different groups to receive albumin-paclitaxel 1mg/kg q.o.d intraperitoneally, or albumin-paclitaxel 0.5mg/kg+prednisolone Loose 0.2 mg/kg q.o.d. (combination therapy) for up to 6 doses, then one dose per week for three weeks. Paw dimensions were measured at the beginning of treatment (day 0) and at each drug injection. A group that received only saline served as a control. At the end of the experiment, the group receiving albumin-paclitaxel achieved a 42% reduction in paw size, the combination treatment group showed a 33% reduction in paw size, while the control group increased paw size by about 20% compared to the beginning of treatment.
总之，清蛋白-紫杉醇组合物对关节炎显示治疗效果。清蛋白-紫杉醇的组合可能通过受体介导的机制如gp60的转运定位于关节炎损伤的部位。In conclusion, the albumin-paclitaxel composition showed a therapeutic effect on arthritis. The albumin-paclitaxel combination may localize to sites of arthritic lesions through receptor-mediated mechanisms such as gp60 translocation.
实施例51Example 51
本实施例举例说明了清蛋白-紫杉醇组合物在治疗心血管再狭窄中的应用。This example illustrates the application of the albumin-paclitaxel composition in the treatment of cardiovascular restenosis.
动物中紫杉醇流出支架(Paclitaxel eluting stents)导致不完全的愈合，在有些情形中，导致在动脉中缺少对新内膜生长的持续抑制。本研究检验了新的系统传递清蛋白-紫杉醇本发明组合物对于降低支架内再狭窄的功效。Paclitaxel eluting stents in animals resulted in incomplete healing and, in some cases, a lack of sustained inhibition of neointimal growth in arteries. This study examined the efficacy of the novel systemic delivery of the albumin-paclitaxel composition of the invention for reducing in-stent restenosis.
在接受双侧髂动脉支架的38只新西兰白兔中检测盐水重构的清蛋白-紫杉醇。将清蛋白-紫杉醇的剂量(1.0-5.0mg/kg紫杉醇剂量)作为10-分钟的动脉内输注给药；对照动物接受载体(0.9％生理盐水)。Saline-reconstituted albumin-paclitaxel was tested in 38 New Zealand white rabbits receiving bilateral iliac artery stents. Doses of albumin-paclitaxel (1.0-5.0 mg/kg paclitaxel dose) were administered as a 10-minute intra-arterial infusion; control animals received vehicle (0.9% saline).
在接下来的慢性实验中，将5.0mg/kg清蛋白-紫杉醇提供给支架，在第28天静脉内重复剂量给药3.5-mg/kg清蛋白-紫杉醇；这些研究在3个月终止。在第28天，通过剂量给药清蛋白-紫杉醇＞＝2.5mg/kg降低了平均新内膜厚度(p＜＝0.02)，证据为延长的愈合。然而，单剂量的5.0mg/kg清蛋白-紫杉醇的功效在90天前丧失。相反，在上支架后28天第二次重复给药3.5mg/kg清蛋白-紫杉醇导致在90天时新内膜厚度的持续抑制(与单剂量5.0mg/kg清蛋白-紫杉醇和对照相比，p＜＝0.009)，新内膜几乎完全愈合。In subsequent chronic experiments, 5.0 mg/kg albumin-paclitaxel was given to the stent, followed by repeated intravenous doses of 3.5-mg/kg albumin-paclitaxel on day 28; these studies were terminated at 3 months. On day 28, mean neointimal thickness was reduced (p<=0.02) by dosing albumin-paclitaxel >= 2.5 mg/kg, evidenced by prolonged healing. However, the efficacy of a single dose of 5.0 mg/kg albumin-paclitaxel was lost before 90 days. In contrast, a second repeat dose of 3.5 mg/kg albumin-paclitaxel at 28 days after stenting resulted in sustained suppression of neointimal thickness at 90 days (compared to a single dose of 5.0 mg/kg albumin-paclitaxel and control, p<=0.009), the neointima was almost completely healed.
尽管系统的清蛋白-紫杉醇在28天降低了新内膜生长，但是对于持续新内膜抑制要求单次重复剂量。因此，本发明组合物适用于治疗心血管疾病如再狭窄。本发明包含除了紫杉醇以外的药剂(例如雷帕霉素，其它紫杉烷类，埃坡霉素等)的组合物全都适用于治疗血管或人造血管移植物如在需要血液透析的患者中用于动脉-静脉入口的那些中的再狭窄。Although systemic albumin-paclitaxel reduced neointimal growth at 28 days, a single repeat dose was required for sustained neointimal suppression. Therefore, the compositions of the present invention are useful in the treatment of cardiovascular diseases such as restenosis. Compositions of the invention comprising agents other than paclitaxel (e.g. rapamycin, other taxanes, epothilone, etc.) are all suitable for use in the treatment of blood vessels or artificial vascular grafts such as in patients requiring hemodialysis Restenosis in those of arterio-venous entry.
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| US52717703P | 2003-12-05 | 2003-12-05 | |
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| US4425319A (en)* | 1980-03-08 | 1984-01-10 | Nihon Medi-Physics Co., Ltd. | Radioactive diagnostic agent and non-radioactive carrier therefor |
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