CN104288756A - Glucagon-like peptide 1 (GLP-1) pharmaceutical formulations - Google Patents

Glucagon-like peptide 1 (GLP-1) pharmaceutical formulations Download PDF

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CN104288756A
CN104288756A CN201410514033.8A CN201410514033A CN104288756A CN 104288756 A CN104288756 A CN 104288756A CN 201410514033 A CN201410514033 A CN 201410514033A CN 104288756 A CN104288756 A CN 104288756A
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glp
fdkp
diketopiperazine
molecule
method
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CN201410514033.8A
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斯蒂芬尼·戈恩
大卫·布兰特
柯哈瓦·盖尔伯
马克·金
韦曼·温蒂尔·师特汉姆
凯斯·奥伯格
安德里亚·勒龙-贝
马克·J·豪肯森
玛丽·法瑞斯
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曼金德公司
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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/007Pulmonary tract; Aromatherapy
    • A61K9/0073Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy
    • A61K9/0075Sprays or powders for inhalation; Aerolised or nebulised preparations generated by other means than thermal energy for inhalation via a dry powder inhaler [DPI], e.g. comprising micronized drug mixed with lactose carrier particles
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22Hormones
    • A61K38/26Glucagons
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K45/00Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/0012Galenical forms characterised by the site of application
    • A61K9/0019Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/08Solutions
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/14Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/143Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with inorganic compounds
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K9/00Medicinal preparations characterised by special physical form
    • A61K9/14Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/145Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with organic compounds

Abstract

The invention relates to glucagon-like peptide 1 (GLP-1) pharmaceutical formulations. Specifically, the invention discloses a composition which is table both in vivo and in vitro. The composition comprises glucagon-like peptide 1 (GLP-I) particles in combination with diketopiperazine (DKP). The composition can be used as a pharmaceutical formulation for treating diseases such as diabetes, cancers, and obesity but is not limited to such diseases or conditions. In particularly, the composition can be used as a pharmaceutical formulation for pulmonary delivery.

Description

高血糖素样肽1 (GLP-1)药物制剂 Glucagon-like peptide 1 (GLP-1) a pharmaceutical formulation

[0001] 相关申请的夺叉引用 Wins fork [0001] Reference to Related Applications

[0002] 本申请是申请日为2007年4月16日、名称为"高血糖素样肽I(GLP-I)药物制剂" 的第200780013424. X号中国发明专利申请的分案申请。 [0002] This application is filed April 16, 2007, entitled "glucagon-like peptide I (GLP-I) a pharmaceutical formulation" No. 200780013424. X divisional application Chinese invention patent application.

[0003] 本申请是2003年7月22日递交的美国申请No. 10/632,878的部分继续申请(continuation-in-part),并根据35U.SC § 119(e)要求2006年4 月14 日递交的美国临时申请No. 60/744, 882的优先权。 [0003] This application is July 22, 2003 filed US Application 10 / 632,878 is No. continue to apply (continuation-in-part), and according to 35U.SC § 119 (e) requires 2006 April 14 filed US provisional application No. 60/744, 882. 每份上述优先权申请通过引用整体并入本文中。 Every such priority application is incorporated herein by reference in its entirety.

技术领域 FIELD

[0004] 本发明涉及药物制剂的领域。 [0004] The present invention relates to the field of pharmaceutical formulation. 本发明公开了包含与高血糖素样肽l(Glucagon-Like Peptide 1,GLP-1)组合的二酮哌嗪(DKP)颗粒的干粉制剂。 The present invention discloses a dry powder formulation comprising a diketopiperazine particles (the DKP) in combination with a glucagon-like peptide-l (Glucagon-Like Peptide 1, GLP-1). 本发明可用作治疗疾病的药物制剂,所述疾病如糖尿病、癌症和肥胖症但不仅限于这类疾病。 The present invention is useful as a pharmaceutical formulation for treating a disease, the disease such as diabetes, cancer and obesity but is not limited to such diseases. 本发明更尤其可用作肺部递送的药物制剂。 The present invention is more particularly useful as a pharmaceutical formulation for pulmonary delivery.

背景技术 Background technique

[0005] 文献中公开的高血糖素样肽I (GLP-I)是30或31个氨基酸的增泌素(incretin), 其响应来自进餐的脂肪、碳水化合物摄取和蛋白质而从肠内分泌L细胞中被释放。 [0005] Document disclosed glucagon-like peptide I (GLP-I) is 30 or 31 amino acid incretin (incretin), in response meal from fat, protein and carbohydrate intake and the enteroendocrine L cells It is released. 发现在患有2型糖尿病的个体中该肽激素的分泌受损,使其成为治疗该疾病和其他相关疾病的可能候选者。 Found in individuals with type 2 diabetes are impaired secretion of the peptide hormone, made possible candidate for the treatment of diseases and other related diseases.

[0006] 在无疾病的状态下,GLP-I响应经口摄取的养分(尤其是糖)从肠L细胞中分泌, 刺激来自胰腺的进餐诱导的胰岛素释放,抑制来自肝的高血糖素释放,以及对消化道和脑的其他效应。 [0006] In the non-disease state, GLP-I response to nutrient for oral intake (especially sugar) secreted from intestinal L cells, stimulating meal-induced insulin from the pancreas to release, inhibiting hyperglycemia from liver hormone releasing, and other effects on the digestive tract and brain. 胰腺中的GLP-I效应是依赖于葡萄糖的,使得外源肽施用期间的低血糖风险最小化。 GLP-I effect in the pancreas is glucose-dependent, so that the risk of hypoglycemia during exogenous peptide administration minimized. GLP-I还促进了胰岛素生物合成中的所有步骤,并直接刺激β-细胞生长和存活以及β_细胞分化。 GLP-I also promotes all steps in insulin biosynthesis and directly stimulate cell growth and survival as well as β- β_ cell differentiation. 这些效应的组合导致提高的β_细胞量。 The combination of these effects results in increased cell mass β_. 另外,GLP-I受体信号转导导致β-细胞凋亡的减少,这进一步有助于提高的β-细胞量。 Further, GLP-I receptor signaling results in a reduction of β- cell apoptosis, which further contributes to an increased β- cell mass.

[0007] 在胃肠道中,GLP-I抑制GI活动力,提高响应葡萄糖的胰岛素分泌,并降低高血糖素的分泌,从而有助于减少葡萄糖偏差(glucose excursion)。 [0007] In the gastrointestinal tract, GLP-I inhibition of GI motility, improve insulin secretion in response to glucose, and to reduce the secretion of glucagon, thereby contributing to reducing the variation of glucose (glucose excursion). 哨齿类中GLP-I的中枢施用已经显示抑制食物摄取,提示外周降低的GLP-I可直接影响脑。 Central post in rodents administered GLP-I has been shown to inhibit food intake, suggesting a reduced outer periphery of GLP-I may directly affect the brain. 这是可行的,因为已经显示循环的GLP-I能够达到某些脑区中的GLP-I受体;即穹隆下器官和最后区。 This is possible, because the circulation has been shown to achieve certain GLP-I brain regions GLP-I receptor; i.e. subfornical organ and area postrema. 已知脑的这些区域涉及食欲和能量稳态的调解。 These areas of the brain known appetite and energy homeostasis involves mediation. 有趣的是,胃膨胀激活孤束的动眼神经核中含GLP-I 的神经元,预示着中枢表达的GLP-I作为食欲抑制剂的作用。 Interestingly, gastric distension activates oculomotor nucleus of the solitary tract in the GLP-I containing neurons, indicating that the expression of central GLP-I as an appetite suppressant effect. 这些假设由使用GLP-I受体拮抗剂--毒蜥外泌肽(9-39)的研究支持,所述研究中观察到相反作用。 These assumptions are made using the GLP-I receptor antagonist - outer exendin (9-39) of the study support the opposite effect observed in this study. 在人中,施用的GLP-I具有饱足效应(Verdich et al.,2001),当在6周期间通过连续的皮下灌输提供时, 糖尿病患者显示食欲降低,这导致体重的显著减轻(Zander et al.,2002)。 In humans, administered GLP-I has a satiety effect (Verdich et al., 2001), when during the six weeks by continuous subcutaneous infusion to provide diabetic patients show decreased appetite, which results in a significant reduction in body weight (Zander et al., 2002).

[0008] GLP-I还已显示在患有2型糖尿病的患者中有效,当作为连续皮下灌输提供时,其提高胰岛素分泌并使得禁食和餐后血糖二者正常化(Nauck et al.,1993)。 [0008] GLP-I has also been shown to be effective in patients with type 2 diabetes, when provided as a continuous subcutaneous infusion, which increases insulin secretion and causes both fasting and postprandial blood glucose normalization (Nauck et al., 1993). 另外,GLP-I的灌输已经显示降低下述患者的葡萄糖水平:先前用非胰岛素口服药物治疗的患者和磺酰脲治疗失败后需要胰岛素治疗的患者(Nauck et al.,1993)。 Further, GLP-I infusion has been shown to decrease glucose levels in a patient following: previously insulin-treated patients after treatment failure in patients with non-insulin oral medication and sulfonylurea therapy required (Nauck et al, 1993.). 然而,如本领域中和下面本文中所记录到的,单一皮下注射GLP-I的作用提供了令人失望的结果。 However, in the art and as described herein below are recorded, the effect of a single subcutaneous injection of GLP-I provided disappointing results. 尽管达到了免疫反应性GLP-I的高血浆水平,但是胰岛素分泌快速地返回处理前的值,血糖浓度未被正常化(Nauck et al.,1996)。 While reaching high plasma levels of immunoreactive GLP-I, and insulin secretion but quickly return the value before the treatment, not normalize blood glucose concentration (Nauck et al., 1996). 只有进行重复的皮下施用时,对禁食血糖的作用才能够比得上静脉施用(Nauck et al.,1996)。 Repeated only for subcutaneous administration, fasted blood glucose comparable to intravenous administration to be able to effect (Nauck et al., 1996). 连续皮下施用6周显示降低禁食和餐后葡萄糖浓度并降低HbAlc 水平(Zander et al.,2002)。 Continuous subcutaneous administration of 6 weeks and shown to reduce fasting and postprandial glucose concentrations decrease HbAlc levels (Zander et al., 2002). 单一皮下注射GLP-I的短暂效果与其循环的不稳定性相关。 Short single subcutaneous injection of GLP-I effect its associated loop instability. 有证据表明GLP-I由血浆体外代谢,酶二肽基肽酶-IV (DPP-IV)负责该降解(Mentlein et al.,1993)。 There is evidence that plasma GLP-I In vitro metabolism by the enzyme dipeptidyl peptidase -IV (DPP-IV) is responsible for this degradation (Mentlein et al., 1993).

[0009] 考虑到GLP-I在糖尿病患者中的生理学显著性和外源GLP-I在健康受试者和2 型糖尿病受试者二者中被快速氨基酸末端降解的事实,许多研究已经致力于操作GLP-I的体内稳定性作为治疗糖尿病的抗糖尿病剂的新颖途径的可能性(Deacon et al.,2004)。 [0009] Considering the physiology GLP-I diabetes significantly and exogenous GLP-I is rapidly amino-terminal degradation of the fact that in both healthy subjects and type 2 diabetic subjects, many studies have been devoted in vivo stability of GLP-I as a novel way to the possibility of anti-diabetic agents for treatment of diabetes (Deacon et al., 2004) operation. 已经研究了两种独立的途径:1)开发对酶降解不敏感的GLP-I类似物,和2)使用选择性酶抑制剂防止体内降解并提高完整的、生物活性肽的水平。 Has investigated two separate ways: 1) the development of the enzymatic degradation of GLP-I is insensitive analogs, and 2) the use of selective enzyme inhibitors to prevent in vivo degradation and enhance full, the level of bioactive peptides. 已经在临床试验中研究了抗降解的、名为"增泌素模拟物"的长效GLP-I类似物(例如Liraglutide (Novo Nordisk, Copenhagen,丹麦);exenatide (毒晰外泌肤-4 ; Byetta® ) (Amylin Inc.,San Diego, CA)和exenatide-LAR, Eli Lilly, Indianapolis, IN)。 Resistant to degradation have been studied in clinical trials, named "incretin mimetic" long-acting GLP-I analogs (e.g., Liraglutide (Novo Nordisk, Copenhagen, Denmark); exenatide (Xi exosomes skin toxicity -4; Byetta®) (Amylin Inc., San Diego, CA) and exenatide-LAR, Eli Lilly, Indianapolis, IN). 抑制负责增泌素降解的酶的二肽基肽酶IV抑制剂(例如由Novartis, Basel,瑞士开发的Vildagliptin (Galvus))和由Merck, Whitehouse Station, New Jersey 开发的Januvia(sitagliptin))也在研究中(Deacon et al.,2004)。 Responsible for incretin degradation inhibiting the enzyme dipeptidyl peptidase IV inhibitors (for example Novartis, Basel, Switzerland developed Vildagliptin (Galvus)) and by the Merck, Whitehouse Station, New Jersey developed Januvia (sitagliptin)) are study (Deacon et al., 2004). 因此,GLP-I的多种作用模式(例如提高的胰岛素释放、延迟的胃排空和提高的饱满感)与其低的低血糖倾向似乎为它带来了超过目前可获得的疗法的优点。 Thus, GLP-I in a variety of modes of action (such as increased insulin release, delayed gastric emptying and increased satiety) and its low tendency to hypoglycemia appeared to bring the advantages over currently available therapies for it.

[0010] 然而,尽管GLP-I治疗中有这些途径/进步,目前糖尿病患者可获得的药物均不能够在所有的患者中达到目的(HbAlc、禁食血糖、葡萄糖偏差),且它们均不能避免副作用如毒性、低血糖、体重增加、恶心和来自呕吐的压力。 [0010] However, despite these approaches / advances GLP-I therapy, diabetics currently available drugs are not able to achieve in all patients (HbAlc, fasting blood sugar, glucose deviation), and they can not be avoided side effects such as toxicity, hypoglycemia, weight gain, nausea and stress from vomiting. 因此在本领域中,仍然需要作为药物施用时具有长期效用和最优吸收的稳定的GLP-I制剂。 Therefore, in the present art, there remains a need has long-term stability and utility of GLP-I formulation optimal absorption as a drug administration.

发明内容 SUMMARY

[0011] 用作药物的稳定的、可吸入的高血糖素样肽I (GLP-I)制剂是本领域缺乏的。 [0011] as stabilizing drugs, inhalable glucagon-like peptide I (GLP-I) formulations are lacking in the art. 为了克服本领域中的缺陷,本发明提供了与二酮哌嗪(DKP)颗粒组合的、作为药物的GLP-I制剂。 In order to overcome the deficiencies in the art, the present invention provides the diketopiperazine (the DKP) particles in combination, the GLP-I as a pharmaceutical formulation.

[0012] 因此,在本发明的具体的实施方案中,提供了包含GLP-I分子和二酮哌嗪或其可药用盐的干粉组合物。 [0012] Thus, in specific embodiments the present invention provides a dry powder composition comprising a pharmaceutically acceptable salt thereof GLP-I molecule and a diketopiperazine or a. 在其他实施方案中,本发明的干粉组合物包含选自下组的GLP-I 分子,所述组由天然GLP-1、GLP-I代谢产物、GLP-I类似物、GLP-I衍生物、二肽酰-肽酶-IV(DPP-IV)保护的GLP-I、GLP-I模拟物、毒蜥外泌肽(exendin)、GLP-I肽类似物或生物合成的GLP-I类似物组成。 In other embodiments, the dry powder compositions of the present invention comprises a GLP-I molecule is selected from the group, the group consisting of native GLP-1, GLP-I metabolites, GLP-I analogs, GLP-I derivative, dipeptidyl - peptidase -IV (DPP-IV) protected GLP-I, GLP-I mimetic, an exendin (exendin), GLP-I peptide analog, or a biosynthetic GLP-I analogs . 还在本发明的又一实施方案中,干粉组合物包含具有式2, 5-二酮-3, 6-二(4-X-氨丁基)哌嗪的二酮哌嗪,其中X选自由琥珀酰基、戊二酰基、马来酰基和富马酰基组成的组。 Still further embodiment of the present invention, comprising a dry powder composition having the formula wherein X is selected from the group consisting of 2, 5-dione 3,6-two (4-X- aminobutyl) piperazine diketopiperazine, succinyl group, glutaryl, maleyl and fumaryl thereof. 在另一实施方案中,干粉组合物包含二酮哌嗪盐。 In another embodiment, the dry powder composition comprising a diketopiperazine salt. 还在本发明的另一实施方案中提供了干粉组合物,其中二酮哌嗪为2, 5-二酮-3, 6-二(4-富马酰-氨丁基)哌嗪。 In yet another embodiment the present invention provides a dry powder composition, wherein the diketopiperazine is 2,5-dione 3,6-bis (4-fumaryl - aminobutyl) piperazine.

[0013] 本发明还包括干粉组合物,其中GLP-I分子为天然的GLP-I或酰胺化的GLP-I分子,其中酰胺化的GLP-I分子是GLP-I (7-36)酰胺。 [0013] The present invention further includes a dry powder composition, wherein the GLP-I molecule is native GLP-I or amidated GLP-I molecule wherein the amidated GLP-I molecule is GLP-I (7-36) amide.

[0014] 还在本发明的另一具体的实施方案中,提供了用于制备包含GLP-I分子和二酮哌嗪的颗粒的方法,所述方法包括步骤:提供包含GLP-I分子的GLP-I溶液;提供能形成颗粒的二酮哌嗪溶液或二酮哌嗪颗粒的悬浮液;和将GLP-I溶液与二酮哌嗪溶液或悬浮液组合。 [0014] In still another specific embodiment of the present invention, there is provided a method for preparing particles comprising a GLP-I molecule and a diketopiperazine, the method comprising the steps of: providing a GLP molecule comprising a GLP-I -I solution; providing a solution of a suspension of diketopiperazine particles or diketopiperazine capable of forming particles; and a GLP-I solution with the diketopiperazine solution or suspension fluid composition. 在本发明的其他具体的实施方案中,用于制备包含GLP-I分子和二酮哌嗪的颗粒的方法还包括通过冻干法、过滤或喷雾干燥从溶液或悬浮液中去除溶剂。 In other method embodiments of the present invention, for the preparation of particles comprising a GLP-I molecule and a diketopiperazine further comprises by lyophilization, filtration, or spray-drying removal of the solvent from a solution or suspension. 还在又一实施方案中, 本发明的颗粒通过去除溶剂形成,或在去除溶剂之前形成。 Still yet another embodiment, the particles of the present invention is formed by removing solvent or is formed prior to removing solvent.

[0015] 在本发明的一个实施方案中,在用于制备具有GLP-I分子和二酮哌嗪的颗粒的方法中,提供了选自下组的GLP-I分子,所述组由天然的GLP-UGLP-I类似物、GLP-I衍生物、 二肽基肽酶-IV(DPP-IV)保护的GLP-UGLP-I模拟物、毒蜥外泌肽、GLP-I肽类似物或生物合成的GLP-I类似物组成。 [0015] In one embodiment of the present invention, a method for preparing particles having a GLP-I molecule and a diketopiperazine, there is provided a GLP-I molecule is selected from the group, the group consisting of natural GLP-UGLP-I analogs, GLP-I derivative, a dipeptidyl peptidase -IV (DPP-IV) protected GLP-UGLP-I mimetic, an exendin, GLP-I peptide analogs, or biologically synthesis of GLP-I analogs. 在另一实施方案中,用于制备具有GLP-I分子和二酮哌嗪的颗粒的方法包括以颗粒悬浮液形式提供提供的二酮哌嗪。 In another embodiment, a method for preparing particles having a GLP-I molecule and a diketopiperazine comprises a diketopiperazine provided as a suspension provided in the form of particles. 在又一实施方案中,二酮哌嗪在溶液中提供,且该方法包括调节溶液的PH,从而沉淀二酮哌嗪并形成颗粒。 Diketopiperazine is provided in solution in a further embodiment, and the method includes adjusting the PH of the solution to precipitate the diketopiperazine and form particles.

[0016] 在本发明的其他具体实施方案中,GLP-I溶液处于约1 μ g/ml-50mg/ml、更优选地约0. lmg/ml-10mg/ml的浓度。 [0016] In other embodiments of the present invention, GLP-I solution is at from about 1 μ g / ml-50mg / ml, more preferably from about 0. lmg / ml-10mg concentration / ml. 还在本发明另一具体的实施方案中,GLP-I溶液处于约0· 25mg/ml 的浓度。 Still another specific embodiment of the present invention, GLP-I solution is at a concentration of about 0 · 25mg / ml of.

[0017] 在用于制备含GLP-I分子和二酮哌嗪颗粒的另一方法中,该方法还包括向溶液中添加一种试剂,其中所述试剂选自盐、表面活性剂、离子、渗透物(osmolyte)、离液剂(chaotrope)和感胶离子(Iyotrope)、酸、碱和有机溶剂。 [0017] In another method for preparing the GLP-I molecule and a diketopiperazine comprising the particles, the method further comprising adding to the solution an agent, wherein the agent is selected from salts, surfactants, ions, permeate (osmolyte), chaotropic agents (chaotrope) and lyotropic ions (Iyotrope), acids, bases and organic solvents. 该试剂促进GLP-I和二酮哌嗪颗粒之间的缔合,还改进GLP-I分子的稳定性和/或药物动力学。 The agent promotes association between the GLP-I and diketopiperazine particles, further improving the stability of GLP-I molecules and / or pharmacokinetics. 在本发明的一些实施方案中,该试剂为盐,例如但不限于氯化钠。 In some embodiments of the present invention, the agent is a salt, such as, but not limited to, sodium chloride. 还考虑了该试剂可以是表面活性剂,例如但不限于Tween、Triton、pluronic acid、CHAPS、cetrimide 和Bri j、H(CH2)7S04Na。 Also contemplated the agent may be a surfactant, such as, but not limited to, Tween, Triton, pluronic acid, CHAPS, cetrimide, and Bri j, H (CH2) 7S04Na. 该试剂可以是离子,例如阳离子或阴离子。 The agent may be ionic, for example cationic or anionic. 该试剂可以是渗透物(稳定剂),例如但不限于己二醇(Hexylene-Glycol, Hex-Gly)、海藻糖、甘氨酸、聚乙二醇(PEG)、三甲胺η-氧化物(TMAO)、 甘露醇和脯氨酸。 The agent may permeate (stabilizers) such as, but not limited to, hexane diol (Hexylene-Glycol, Hex-Gly), trehalose, glycine, polyethylene glycol (PEG), trimethylamine oxide η- (of TMAO) , mannitol and proline. 该试剂可以是离液剂或感胶离子,例如但不限于氯化铯、柠檬酸钠和硫酸钠。 The agent may be a chaotrope or lyotrope, such as, but not limited to, cesium chloride, sodium citrate and sodium sulfate. 该试剂可以是有机溶剂,例如选自甲醇(MeOH)、乙醇(EtOH)、三氟代乙醇(TFE)和六氟异丙醇(HFIP)的醇。 The agent may be an organic solvent, for example selected from methanol (MeOH), ethanol (EtOH), trifluoroethanol (TFE), and hexafluoroisopropanol (HFIP) alcohol.

[0018] 在本发明的另一具体实施方案中,考虑了用于制备含GLP-I分子和二酮哌嗪的颗粒的方法,其中该方法包括将颗粒悬浮液的PH调节至约4或更大。 [0018] In another embodiment of the present invention contemplates a method for preparing a GLP-I-containing molecule and a diketopiperazine particles, wherein the method comprises PH particle suspension is adjusted to about 4 or more Big. 在本发明的其他实施方案中,用于制备颗粒的方法包括GLP-I分子和二酮哌嗪,其中颗粒中的GLP-I分子具有更大的稳定性。 In other embodiments of the invention, a method for preparing particles comprising a GLP-I molecule and a diketopiperazine, wherein the GLP-I molecule in the particle has greater stability.

[0019] 本发明中还考虑了对需要的受试者施用有效量的GLP-I分子的方法,包括对受试者提供GLP-I/二酮哌嗪颗粒。 [0019] The present invention also contemplates a method of GLP-I molecule to a subject in need thereof an effective amount of a subject comprising providing GLP-I / diketopiperazine particle. 该施用方法可以是静脉内、皮下、经口、经鼻、经颊、经直肠或通过肺部递送,但不仅限于此。 The method of administration may be intravenous, subcutaneous, oral, nasal, buccal, rectally, or by pulmonary delivery, but is not limited thereto. 在一个实施方案中,施用方法是通过肺部递送。 In one embodiment, the method of administration is by pulmonary delivery. 还在本发明的又一实施方案中,施用的方法包括治疗选自下组的病症或疾病,所述组由糖尿病、局部缺血、再灌注组织损伤、血脂障碍、糖尿病性心脏病、心肌梗塞、急性冠状动脉综合征、肥胖症、 手术后的分解代谢改变、高血糖症、过敏性肠综合征、中风、神经变性病症、记忆和学习障碍、胰岛细胞移植和再生性治疗组成。 Still further embodiment of the present invention, the method comprises administering a disease treating a condition selected from the group or the group consisting of diabetes, ischemia, reperfusion injury, dyslipidemia, diabetes, heart disease, myocardial infarction , acute coronary syndrome, obesity, catabolic changes after surgery, hyperglycemia, irritable bowel syndrome, stroke, neurodegenerative disorders, memory and learning disorders, islet cell transplant and regenerative therapy composition.

[0020] 在本发明的另一实施方案中施用GLP-I/二酮哌嗪颗粒组合物的方法导致改进的GLP-I药物代谢动力学半衰期和生物利用度。 Method [0020] The administration of GLP-I / diketopiperazine particle composition In another embodiment of the present invention results in improved kinetic half-life and bioavailability of the drug metabolite GLP-I.

[0021] 还在本发明的又一具体实施方案中,提供了制备具有改进的药物代谢动力学模式的干粉组合物的方法,该方法包括步骤:提供GLP-I分子的溶液;提供能形成颗粒的二酮哌嗪;形成颗粒;和将GLP-I与二酮哌嗪组合;和之后通过干燥的方法去除溶剂,获得干粉,其中所述干粉具有改进的药物代谢动力学模式。 [0021] In still yet another embodiment of the present invention, there is provided a method of preparing a dry powder composition having an improved pharmacokinetic profile, the method comprising the steps of: providing a solution of GLP-I molecule; providing a particle-forming diketopiperazine; form particles; and a GLP-I in combination with diketopiperazine; and after removal of the solvent by drying, to obtain a dry powder, wherein the dry powder has improved pharmacokinetic profile. 经改进的药物代谢动力学模式包括提高的GLP-I半衰期和/或经改进的GLP-I生物利用度。 The improved pharmacokinetic profile of a drug including increased GLP-I half-life and / or improved bioavailability of GLP-I. 提高的GLP-I半衰期大于或等于7. 5分钟。 Increased GLP-I half-life greater than or equal to 7.5 minutes.

[0022] 在本发明的一个实施方案中,提供了包含GLP-I分子和二酮哌嗪组合物或其可药用盐的干粉组合物。 [0022] In one embodiment of the present invention, there is provided a dry powder composition may be a pharmaceutically acceptable salt thereof comprising GLP-I molecule and a diketopiperazine or a composition thereof. 在另一实施方案中,GLP-I分子选自由以下组成的组:天然GLP-1、 GLP-I代谢产物、GLP-I类似物、GLP-I衍生物、二肽酰-肽酶-IV(DPP-IV)保护的GLP-I、 GLP-I模拟物、GLP-I肽类似物或生物合成的GLP-I类似物。 In another embodiment, GLP-I molecule is selected from the group consisting of: native GLP-1, GLP-I metabolites, GLP-I analogs, GLP-I derivatives, dipeptidyl - peptidase -IV ( DPP-IV) protected GLP-I, GLP-I mimetics, GLP-I peptide analog, or a biosynthetic GLP-I analog.

[0023] 在本发明的一个实施方案中,二酮哌嗪是具有式2, 5-二酮-3,6-二(4-X-氨丁基)哌嗪的二酮哌嗪,其中X选自由琥珀酰基、戊二酰基、马来酰基和富马酰基组成的组。 [0023] In one embodiment of the present invention, the diketopiperazine having the formula 2,5-dione-3,6-di (4-X- aminobutyl) piperazine diketopiperazine, wherein X selected from the group consisting of succinyl, glutaryl, maleyl and fumaryl thereof. 在另一实施方案中,二酮哌嗪是二酮哌嗪盐。 In another embodiment, the diketopiperazine is a diketopiperazine salt. 在另一实施方案中,二酮哌嗪是2, 5-二酮-3, 6-二(4-富马酰-氨丁基)哌嗪。 In another embodiment, the diketopiperazine is 2,5-dione 3,6-bis (4-fumaryl - aminobutyl) piperazine.

[0024] 在本发明的一个实施方案中,GLP-I分子是天然的GLP-1。 [0024] In one embodiment of the present invention, GLP-I molecule is native GLP-1. 在另一实施方案中,GLP-I分子是酰胺化的GLP-I分子。 In another embodiment, GLP-I molecule is an amidated GLP-I molecule. 在另一实施方案中,酰胺化的GLP-I分子是GLP-I (7-36)酰胺。 In another embodiment, the amidated GLP-I molecule is GLP-I (7-36) amide.

[0025] 在本发明的一个实施方案中,提供了形成包含GLP-I分子和二酮哌嗪的颗粒的方法,该方法包括步骤:提供GLP-I分子;提供下述形式的二酮哌嗪,所述形式选自能形成颗粒的二酮哌嗪、二酮哌嗪颗粒及其组合;和将GLP-I分子与二酮哌嗪以共溶液的形式组合, 其中形成包含GLP-I分子和二酮哌嗪的颗粒。 [0025] In one embodiment of the present invention, there is provided a method of forming particles comprising a GLP-I molecule and a diketopiperazine, the method comprising the steps of: providing a GLP-I molecule; provides the following form diketopiperazine the form selected from particle-forming diketopiperazine, diketopiperazine particles, and combinations thereof; and a GLP-I molecule and the diketopiperazine in the form of a combination co-solution, wherein the form comprises a GLP-I molecule and diketopiperazine particles.

[0026] 在本发明的一个实施方案中,该方法还包括通过冻干法、过滤或喷雾干燥从所述共溶液中去除溶剂。 [0026] In one embodiment of the invention, the method further comprising by lyophilization, filtration, or spray drying said co-solvent is removed from the solution. 在另一实施方案中,通过去除溶剂形成包含所述GLP-I分子和二酮哌嗪的颗粒。 In another embodiment, the particles are formed comprising said GLP-I molecule and a diketopiperazine by removing the solvent. 在另一实施方案中,在去除溶剂之前形成包含所述GLP-I分子和二酮哌嗪的颗粒。 In another embodiment, the particles are formed comprising said GLP-I molecule and a diketopiperazine before the solvent was removed.

[0027] 在另一实施方案中,GLP-I分子选自由以下组成的组:天然的GLP-1、GLP-I类似物、GLP-I衍生物、二肽基肽酶-IV(DPP-IV)保护的GLP-UGLP-I模拟物、GLP-I肽类似物或生物合成的GLP-I类似物。 [0027] In another embodiment, GLP-I molecule is selected from the group consisting of: native GLP-1, GLP-I analogs, GLP-I derivative, a dipeptidyl peptidase -IV (DPP-IV ) protected GLP-UGLP-I mimetics, GLP-I peptide analog, or a biosynthetic GLP-I analog. 在另一实施方案中,GLP-I分子以溶液的形式提供,所述溶液包含约1 μ g/ml-50mg/ml的GLP-I浓度。 In another embodiment, GLP-I molecule is provided in the form of a solution, said solution comprising a GLP-I concentration of about 1 μ g / ml-50mg / ml of. 在另一实施方案中,GLP-I分子以溶液的形式提供, 所述溶液包含约〇. lmg/ml-10mg/ml的GLP-I浓度。 In another embodiment, GLP-I molecule is provided in the form of a solution, said solution comprising from about billion. GLP-I concentration of lmg / ml-10mg / ml of. 在另一实施方案中,GLP-I分子以溶液的形式提供,所述溶液包含约〇. 25mg/ml的GLP-I浓度。 In another embodiment, GLP-I molecule is provided in the form of a solution, said solution comprising from about billion. GLP-I concentration of 25mg / ml of.

[0028] 在本发明的另一实施方案中,二酮哌嗪以二酮哌嗪颗粒悬浮液的形式提供。 [0028] In another embodiment of the present invention, the diketopiperazine is a diketopiperazine provided as a suspension of particles. 在另一实施方案中,二酮哌嗪以包含能形成颗粒的二酮哌嗪的溶液的形式提供,该方法还包括调节溶液的PH以形成二酮哌嗪颗粒。 In another embodiment, the diketopiperazine comprises a diketopiperazine in the form of particle-forming solution provided, the method further comprising adjusting the PH of the solution to form diketopiperazine particles. 在另一实施方案中,该方法还包括向所述溶液或悬浮液中添加下述试剂,其中所述试剂选自由盐、表面活性剂、离子、渗透物、离液剂和感胶离子、酸、碱和有机溶剂组成的组。 In another embodiment, the method further comprising the following reagents were added to the solution or suspension, wherein the agent is selected from the group consisting of salts, surfactants, ions, the permeate, chaotropes and lyotropes, acid the group bases and organic solvents. 在另一实施方案中,该试剂促进GLP-I分子和二酮哌嗪颗粒或能形成颗粒的二酮哌嗪之间的缔合。 In another embodiment, the agent promotes association between the GLP-I molecule and the diketopiperazine particles or diketopiperazine particles can be formed. 在另一实施方案中,该试剂改善GLP-I分子的稳定性或药物动力学。 In another embodiment, the agent to improve the stability of the GLP-I molecule or pharmacokinetics. 在另一实施方案中,该试剂为氯化钠。 In another embodiment, the agent is sodium chloride.

[0029] 在本发明的另一实施方案中,该方法还包括调节悬浮液或溶液的pH。 [0029] In another embodiment of the invention, the method further comprising adjusting the pH of the suspension or solution. 在另一实施方案中,pH被调节至约4.0或更大。 In another embodiment, pH is adjusted to about 4.0 or greater. 还在另一实施方案中,颗粒中的GPL-I分子具有比天然GPL-I更大的稳定性。 In yet another embodiment, GPL-I molecule in the particle has a greater stability than natural GPL-I.

[0030] 在另一实施方案中,共溶液包含约1 μ g/ml-50mg/ml的GLP-I浓度。 [0030] In another embodiment, the co-solution comprises from about 1 μ g GLP-I concentration / ml-50mg / ml of. 在另一实施方案中,共溶液包含约〇. lmg/ml-10mg/ml的GLP-I浓度。 In another embodiment, the co-solution comprises approximately square. GLP-I concentration of lmg / ml-10mg / ml of. 在另一实施方案中,共溶液包含约0· 25mg/ml 的GLP-I 浓度。 In another embodiment, the co-solution comprises a GLP-I concentration of about 0 · 25mg / ml of.

[0031] 还在本发明的另一实施方案中,该方法还包括向共溶液中添加下述试剂,其中所述试剂选自由盐、表面活性剂、离子、渗透物、离液剂和感胶离子、酸、碱和有机溶剂组成的组。 [0031] Another embodiment of the present invention also, the method further comprises adding the following reagents to the common solution, wherein the agent is selected from the group consisting of salts, surfactants, ions, the permeate, chaotropes and lyotropes ions, an acid group, a base and an organic solvent. 在另一实施方案中,该试剂促进GLP-I分子和二酮哌嗪颗粒或能形成颗粒的二酮哌嗪之间的缔合。 In another embodiment, the agent promotes association between the GLP-I molecule and the diketopiperazine particles or diketopiperazine particles can be formed. 在另一实施方案中,该试剂改善GLP-I分子的稳定性或药物动力学。 In another embodiment, the agent to improve the stability of the GLP-I molecule or pharmacokinetics. 在另一实施方案中,该试剂为氯化钠。 In another embodiment, the agent is sodium chloride.

[0032] 在另一实施方案中,该方法还包括调节共溶液的pH。 [0032] In another embodiment, the method further comprising adjusting the pH of the solution co. 在另一实施方案中,pH被调节至约4. 0或更大。 In another embodiment, pH is adjusted to about 4.0 or greater.

[0033] 在本发明的一个实施方案中,提供了对需要的受试者施用有效量的GLP-I分子的方法,包括对受试者提供包含GLP-I和二酮哌嗪的颗粒。 [0033] In one embodiment of the present invention, there is provided a method of GLP-I molecule to a subject in need thereof an effective amount of a subject comprising providing a GLP-I and diketopiperazine particles. 在另一实施方案中,通过静脉内、 皮下、经口、经鼻、经颊、经直肠或通过肺部递送完成提供。 In another embodiment, intravenously, subcutaneously, orally, nasally, buccally, rectally, or by pulmonary delivery offers complete. 在其他实施方案中,通过肺部递送完成提供。 In other embodiments, the pulmonary delivery by providing complete.

[0034] 在另一个实施方案中,该需要包括对选自下组的病症或疾病的治疗,所述组由糖尿病、局部缺血、再灌注组织损伤、血脂障碍、糖尿病性心脏病、心肌梗塞、急性冠状动脉综合征、肥胖症、手术后的分解代谢改变、高血糖症、过敏性肠综合征、中风、神经变性病症、记忆和学习障碍、胰岛细胞移植和再生性治疗组成。 [0034] In another embodiment, the need comprises the treatment of a disorder or disease is selected from the group consisting of the group consisting of diabetes, ischemia, reperfusion injury, dyslipidemia, diabetes, heart disease, myocardial infarction , acute coronary syndrome, obesity, catabolic changes after surgery, hyperglycemia, irritable bowel syndrome, stroke, neurodegenerative disorders, memory and learning disorders, islet cell transplant and regenerative therapy composition.

[0035] 在另一实施方案中,颗粒的提供导致与天然GLP-I分子相比改进的GLP-I药物代谢动力学半衰期和生物利用度。 [0035] In another embodiment, there is provided particles results in molecules with native GLP-I half-life compared to the improved kinetic drug metabolism and bioavailability using a GLP-I.

[0036] 在本发明的一个实施方案中,提供了形成具有改进的GLP-I药物代谢动力学模式的粉末组合物的方法,该方法包括步骤:提供GLP-I分子;提供溶液中的能形成颗粒的二酮哌嗪;形成二酮哌嗪颗粒;将GLP-I分子与溶液组合形成共溶液;和通过喷雾干燥从共溶液中去除溶剂,形成具有改进的GLP-I药物代谢动力学模式的粉末。 [0036] In one embodiment of the present invention, there is provided a method of forming a powder composition with an improved GLP-I pharmacokinetic model, the method comprising the steps of: providing a GLP-I molecule; providing a solution capable of forming the diketopiperazine particles; forming diketopiperazine particles; the GLP-I molecule was formed in combination with a co-solution; and removing the solvent from the co-solution by spray drying to form a GLP-I pharmacokinetic profile having the improved powder.

[0037] 在另一实施方案中,改进的GLP-I药物代谢动力学模式包括提高的GLP-I半衰期。 [0037] In another embodiment, the improved GLP-I pharmacokinetic profile of a drug include improved half-life of GLP-I. 在另一实施方案中,提高的GLP-I半衰期大于或等于7. 5分钟。 In another embodiment, the increased GLP-I half-life greater than or equal to 7.5 minutes. 在另一实施方案中,改进的GLP-I药物代谢动力学模式包括与天然GLP-I相比改进的GLP-I生物利用度。 In another embodiment, the improved pharmacokinetic profile comprises GLP-I as compared to native GLP-I improved bioavailability of GLP-I.

附图说明 BRIEF DESCRIPTION

[0038] 以下附图构成本说明书的一部分,用于进一步说明本发明的特定方面。 [0038] The following drawings form part of the present specification, it serves to further illustrate certain aspects of the invention. 通过以下附图,并结合具体实施方式的详细描述,可以更好地理解本发明。 The following drawings and detailed description of specific embodiments, the present invention may be better understood.

[0039] 图1A-1D. GLP-I在多种浓度下的结构分析(pH 4, 20°C )。 [0039] FIGS. 1A-1D. GLP-I structural analysis (pH 4, 20 ° C) at various concentrations. 图1A-GLP-1的远-UV圆二色性(CD)阐述了随着浓度的提高,肽的二级结构从主要为无组织的构象转化为螺旋构象。 FIGS. 1A-GLP-1 far -UV circular dichroism (CD) are set forth with increasing concentration, two from the primary structure of the peptide conformation unorganized into a helical conformation. 图IB-近-UV CD阐述了三级结构随着提高的肽浓度而增加,提示GLP-I的自身缔合。 FIG IB- near -UV CD tertiary structure as set forth increasing peptide concentration increased, suggesting that GLP-I self-association of. 图IC -由280nm处色氨酸激发引起的多种浓度下GLP-I (pH 4, 20°C )的荧光发射。 FIG IC - GLP-I (pH 4, 20 ° C) at various concentrations of tryptophan excitation at 280nm fluorescence emission caused. 图ID - 多种浓度下GLP-I (pH 4, 20°C )的透射FTIR。 FIG ID - GLP-I (pH 4, 20 ° C) at various concentrations transmission FTIR. 1656CHT1处的酰胺I条带表明在彡2mg/mL的浓度下GLP-I具有α -螺旋结构。 Article 1656CHT1 amide I band showed that at a concentration of at San 2mg / mL GLP-I has the α - helix structure.

[0040] 图2A-2D.在多种离子强度下(pH 4, 20°C )低浓度GLP-I的结构分析。 [0040] FIGS. 2A-2D. Analysis of Structure (pH 4, 20 ° C) of low concentration GLP-I at various ionic strength. 图2A-1. Omg/mL GLP-I的远-UV⑶阐明了提高盐浓度将无序的GLP-I结构转化为更有序的α -螺旋结构。 Figures 2A-1 Omg / mL GLP-I illustrates the increase of the salt concentration is far -UV⑶ disorderly structure of GLP-I into more ordered α - helical structure. 图2B - I. Omg/mL肽的近-UV⑶证实了提高NaCl浓度也增强GLP-I的三级结构。 FIG. 2B - I. Omg / mL peptide was confirmed near -UV⑶ increase NaCl concentration also enhances the tertiary structure of GLP-I. 图2C -在280nm处色氨酸激发后多种NaCl浓度下(pH 4, 20°C ) I. Omg/mL GLP-I 的内荧光发射。 FIG. 2C - under various NaCl concentration after tryptophan excitation at 280nm (pH 4, 20 ° C) the fluorescence I. Omg / mL GLP-I emission. 在高肽浓度下,最大值强度降低并迁移至更短的波长,这指示了明确定义的三级结构。 At high peptide concentrations, reducing the maximum strength and migrate to shorter wavelength, this indicates that a well-defined tertiary structure. 图2D -在多种离子强度下(pH 4,20°C)10mg/mL GLP-I的三级结构分析。 FIG. 2D - tertiary structure (pH 4,20 ° C) 10mg / mL GLP-I analysis in a variety of ionic strength. 近-UV CD谱证实提高的离子强度增强了自身缔合的GLP-I的三级结构。 Near -UV CD spectra confirmed that the increase of ionic strength enhances the tertiary structure of the self-association of GLP-I.

[0041] 图3A-3B.多种温度下(pH 4)10mg/mL GLP-I的结构分析。 [0041] Structural Analysis (pH 4) 10mg / mL GLP-I at 3A-3B. FIG various temperatures. 图3A-近-UV CD阐明了提高的温度下GLP-I寡聚物解离。 3A- FIG near -UV CD illustrates GLP-I oligomers dissociate at elevated temperatures. 图3B-多种温度下(pH 4) 10mg/mL GLP-I的结构分析。 (4 pH) Analysis of Structure 10mg / mL GLP-I at various temperatures 3B- FIG. 图3C-多种温度下(pH 4)0. 05mg/mL GLP-I的结构分析。 (4 pH) 0. Analysis of Structure 05mg / mL GLP-I at various temperatures 3C- FIG. 远-UV⑶阐明了肽对温度是不敏感的。 Far -UV⑶ clarify the peptide is insensitive to temperature.

[0042] 图4A-4B.多种pH 下(20°C )GLP-1 的结构分析。 Structural Analysis of GLP-1 [0042] Under 4A-4B. FIG various pH (20 ° C). 图4A-多种pH 下(20°C ) IOmg/ mL GLP-I的远-UV⑶。 Far -UV⑶ (20 ° C) IOmg / mL GLP-I at various pH 4A- FIG. 当pH被提高时,自身缔合的GLP-I在pH 6. 3和7. 6之间沉淀,但是在pH 1. 5和11. 7时保留螺旋结构。 When the pH is increased, self-associated GLP-I is between 7.6 and pH 6. 3 precipitation, but retains a helical structure at pH 1. 5 and 11.7 time. 图4B -放大pH 7. 6处的谱系揭示GLP-I的二级结构是无序的,由浓度降低引起。 FIG 4B - lineage enlarged at pH 7. 6 discloses secondary structure of GLP-I is unordered, caused by a reduced concentration.

[0043] 图5.通过HPLC证实的lmg/mL GLP-I对脱酰胺作用和氧化作用二者的抗性。 [0043] 5. confirmed by HPLC lmg / mL GLP-I in FIG resistance of both deamidation and oxidation pair. 通过将GLP-I在pH 10. 5时于40°C孵育5天达到脱酰胺条件。 By GLP-I at pH 10. 5 incubation at 40 ° C 5 days to deamidation conditions. 通过将GLP-I在0. 1 % H2O2中于室温下孵育2小时达到氧化条件。 GLP-I by incubation for 2 hours to reach the oxidizing conditions at room temperature in 0. 1% H2O2.

[0044] 图6A-6B.搅动对I. 5mg/mL GLP-I 和9. 4mg/mL GLP-I (pH 4)的三级结构的影响。 [0044] FIGS. 6A-6B. Effect of agitation on the tertiary structure I. 5mg / mL GLP-I and 9. 4mg / mL GLP-I (pH 4) of the. 近-UV CD (图6A)和GLP-I的荧光发射(图6B)均阐明了GLP-I肽的三级结构不由于搅动而显著改变。 Near -UV CD (FIG. 6A) and the fluorescence emission of GLP-I (FIG. 6B) both illustrate the tertiary structure of GLP-I peptide does not significantly change due to agitation. 样品在室温下搅动30和90分钟并在280nm处色氨酸激发后收集荧光发射谱。 Samples 30 and agitated for 90 minutes at room temperature and fluorescence emission spectra were collected after tryptophan excitation at 280nm.

[0045] 图7A-7C. 10次冻融循环对I. 6、5. 1和8. 4mg/mL GLP-I (pH 4)三级结构的影响。 Effect of 6,5. Tertiary structure I. 1 and 8. 4mg / mL GLP-I (pH 4) in [0045] FIGS. 7A-7C. 10 freeze-thaw cycles. 近-UV⑶(图7A)和GLP-I的荧光发射(图7B)均显示肽的三级结构不因为多重冻融循环而显著改变。 Near -UV⑶ (FIG. 7A) and fluorescence emission of GLP-I (FIG. 7B) showed no tertiary structure of the peptide as multiple freeze-thaw cycle significantly change. 样品冷冻于-20°C下并在室温下解冻。 Samples were frozen and thawed at room temperature and at -20 ° C. 在280nm处色氨酸激发后收集荧光发射谱。 Fluorescence emission spectra were collected after tryptophan excitation at 280nm. 通过远-UV⑶进行类似的实验,该实验显示11次冻融循环对10mg/mL GLP-UpH 4) 二级结构的影响(图7C)。 Similar experiments by far -UV⑶, this experiment shows the effect of 11 freeze-thaw cycles of 10mg / mL 4) of the secondary structure of GLP-UpH (FIG. 7C).

[0046] 图8A-8B.盐研究。 8A-8B. Salt [0046] FIG. 作为pH和NaCl浓度的函数的GLP-1/FDKP负载曲线(图8A)。 As a function of NaCl concentration and pH of the GLP-1 / FDKP load curve (Figure 8A). 在5mg/mL FDKP和0· 25mg/mL GLP-I下进行负载。 Load at 5mg / mL FDKP and 0 · 25mg / mL GLP-I. NaCl浓度以mM表示。 NaCl concentrations are expressed in mM. 图8B -作为pH 和NaCl浓度的函数,描述了在重建的无FDKP对照样品中检测的GLP-I量。 FIG. 8B - as a function of pH and NaCl concentration, describes GLP-I detected in the amount of FDKP-free control samples reconstructed.

[0047] 图9A-9B.表面活性剂研究。 . Surfactant [0047] FIGS. 9A-9B. 作为pH和表面活性剂的函数的GLP-1/FDKP负载曲线(图9A)。 As a function of pH and surfactant of GLP-1 / FDKP load curve (FIG. 9A). 在5mg/mL FDKP和0· 25mg/mL GLP-I下进行负载。 Load at 5mg / mL FDKP and 0 · 25mg / mL GLP-I. 图9B -作为pH和添加的表面活性剂的函数,描述了在重建的无FDKP对照样品中检测的GLP-I量。 FIG 9B - as a function of pH and surfactant added, describes GLP-I detected in the amount of FDKP-free control samples reconstructed.

[0048] 图10A-10D.离子研究。 10A-10D. Ion [0048] FIG. 作为pH和离子的函数的GLP-1/FDKP负载曲线。 As a function of pH and ions of GLP-1 / FDKP load curve. 在5mg/ mL FDKP和(λ 25mg/mL GLP-I下进行负载(图IOA和11C)。离子浓度在图例中示出(mM)。 右侧曲线描述IM NaCl的结果。图IOB和IOD -作为pH、离子和IM NaCl的函数,描述了在重建的无FDKP对照样品中检测的GLP-I量。 Carried out at a 5mg / mL FDKP and (λ 25mg / mL GLP-I load (IOA and FIG. 11C) ion concentration shown in the legend (mM) right graph of the results described in FIG IOB IM NaCl and IOD -... As function of pH, ionic and IM NaCl, describing the amount of GLP-I detected in the reconstituted FDKP-free control samples in.

[0049] 图11 A -11B.渗透物研究。 [0049] FIG 11 A -11B. Study permeate. 作为pH的函数和存在常见稳定剂(渗透物)时的GLP-1/FDKP 负载曲线(图11A)。 As a function of pH and GLP-1 in the presence of common stabilizers (permeate) / FDKP load curve (FIG. 11A). 在5mg/mL FDKP 和0. 25mg/mL GLP-I 下进行负载。 Load at 5mg / mL FDKP and 0. 25mg / mL GLP-I. 图IlB-作为pH和渗透物的函数,描述了在重建的无FDKP对照样品中检测的GLP-I量。 FIG IlB- as a function of pH and permeate, describes the amount of GLP-I detected in the reconstituted FDKP-free control samples in. "N/ A "表示样品中不存在渗透物。 "N / A" indicates the absence of a permeate sample.

[0050] 图12A-12B.离液剂/感胶离子研究。 [0050] FIGS. 12A-12B. Study lyotropic chaotrope / sense. 作为pH 3. 0(图12A)和pH 4. 0(图12C) 下离液剂或感胶离子浓度的函数的GLP-1/FDKP负载曲线。 As a function of pH sensitive or chaotrope gum ion concentration (FIG. 12A) and pH 4. 0 (FIG. 12C) 3. 0 of GLP-1 / FDKP load curve. 在5mg/mL FDKP和0. 25mg/mL GLP-I下进行负载。 Load at 5mg / mL FDKP and 0. 25mg / mL GLP-I. 图12B和12D -作为pH的函数和存在多种离液剂或感胶离子时,描述了在重建的无FDKP对照样品中检测的GLP-I量。 FIGS. 12B and 12D - as a function of pH and the presence of chaotropic agents or various lyotropic sensation when describing the amount of GLP-I detected in the reconstituted FDKP-free control samples in. "N/A"表示样品中不存在离液剂或感胶离子。 "N / A" indicates that the sample does not exist chaotropes or lyotropes.

[0051] 图13A-13B.醇研究。 [0051] FIGS. 13A-13B. Alcohol research. 作为pH和醇的函数的GLP-1/FDKP负载曲线。 As a function of pH and alcohols GLP-1 / FDKP load curve. 在5mg/mL FDKP和0. 25mg/mL GLP-I下进行负载。 Load at 5mg / mL FDKP and 0. 25mg / mL GLP-I. 对每种醇评价四种醇浓度,5%、10%、15%和20% v/v(图13A)。 Evaluation of Four for each alcohol concentration of the alcohol, 5%, 10%, 15% and 20% v / v (FIG. 13A). TFE =三氟代乙醇;HFIP =六氟异丙醇。 TFE = trifluoroethanol; HFIP = hexafluoroisopropanol. 图13B -作为pH和醇(20% )的函数,描述了在重建的无FDKP对照样品中检测的GLP-I量。 FIG 13B - as pH and alcohol (20%) of the functions described GLP-I detected in the amount of FDKP-free control samples reconstructed.

[0052] 图14A-14B.来自GLP-1/FDKP浓度研究的负载(图14A)。 [0052] FIGS. 14A-14B. Load from the FDKP concentration studies GLP-1 / (FIG. 14A). 负载在5mg/mL FDKP下进行,分析的GLP-I浓度列于X轴中。 Load at 5mg / mL FDKP, GLP-I concentration analyzed is listed in the X-axis. 图14B -多种GLP-1/FDKP配方(放大IOOOOx倍)的扫描电子显微术(SEM)图像显示球形和棒状GLP-1/FDKP颗粒组成。 FIG 14B - various GLP-1 / FDKP formulations (IOOOOx times enlarged) of a scanning electron microscopy (SEM) image of spherical and rod-shaped display GLP-1 / FDKP particles. (图A)0. 5mg/mL GLP-I 和2. 5mg/mL FDKP ;(图B) 0· 5mg/mL GLP-I 和10mg/mL FDKP ;(图C) 20mM 氯化钠、20mM 乙酸钾和20mM 磷酸钾,pH 4. O 中0· 5mg/mL GLP-I 和10mg/mL FDKP ;和(图D) 20mM 氯化钠、 20mM 乙酸钾和20mM 磷酸钾,pH 4. O 中10mg/mL GLP-I 和50mg/mL FDKP。 (Figure A) 0 5mg / mL GLP-I and 2. 5mg / mL FDKP; (FIG. B) 0 · 5mg / mL GLP-I and 10mg / mL FDKP; (FIG. C) 20mM NaCl, 20mM potassium acetate and 20mM potassium phosphate, pH 4. O in 0 · 5mg / mL GLP-I and 10mg / mL FDKP; and (D FIG) 20mM NaCl, 20mM potassium acetate, 20mM potassium phosphate, pH 4. O of 10mg / mL GLP-I and 50mg / mL FDKP.

[0053] 图15.展示胁迫对多种GLP-1/FDKP制剂的影响。 [0053] FIG. 15 shows stress on multiple GLP-1 / FDKP formulations. 图例指出冻干前GLP-I对FDKP 颗粒和溶液中存在的其他组分的质量-质量百分比。 Legend mass of other components noted GLP-I to FDKP particles and present in the solution before lyophilization - mass percent. 样品在40°C孵育10天。 Samples were incubated at 40 ° C for 10 days.

[0054] 图16A-16C. GLP-I的结构。 [0054] FIGS. 16A-16C. GLP-I structures. 图16A-描述GLP-I的甘氨酸-延伸的形式(SEQ ID NO. 1)和酰胺化的形式(SEQ ID NO. 2)。 And the amidated form (SEQ ID NO 2.) Extending in the form of - GLP-I described in FIG 16A- glycine (SEQ ID NO 1.). FIG. 16B-抑肽酶对DPPIV活性的抑制。 FIG. 16B- aprotinin inhibition of DPPIV activity. 图16C - DPPIV抑制剂对DPPIV活性的抑制。 FIG 16C - inhibitor of DPPIV DPPIV inhibition activity.

[0055] 图17.在肺灌洗液中孵育后检测GLP-I。 [0055] FIG. 17. After incubation the lung lavage detected GLP-I.

[0056] 图18A-18B.描述GLP-I在血浆中的定量。 [0056] FIGS. 18A-18B. Quantitative Description GLP-I in plasma. 图18A显示1:2血浆稀释液中的定量。 Figure 18A shows 1: 2 quantified in plasma dilutions. 图18B显示1:10血楽稀释液中的定量。 Figure 18B shows quantification of blood yue 1:10 dilutions.

[0057] 图19A-19B. GLP-I和GLP-I类似物对细胞存活的影响。 [0057] FIGS. 19A-19B. Effect of GLP-I and GLP-I analogs on cell survival. GLP-I对大鼠胰腺上皮(ARIP)细胞死亡的影响(图19A)。 Effects of GLP-I on rat pancreatic epithelial (ARIP) cell death (FIG. 19A). 在单独存在和组合存在GLP-I和十字孢碱(Stau)时,锚定蛋白V染色显示抑制细胞凋亡(图19B)。 In the presence and the presence of GLP-I and staurosporine (Stau) composition alone, annexin V staining inhibition of apoptosis (FIG. 19B). GLP-I的浓度为15nM,十字孢碱的浓度为1 μ M。 GLP-I concentration was 15nM, staurosporine concentrations of 1 μ M.

[0058] 图20. GLP-I类似物毒蜥外泌肽-4对细胞存活力的影响。 Effect [0058] FIG 20. GLP-I analog exendin outer -4 on cell viability. 用0、10、20和40ηΜ毒蜥外泌肽将ARIP细胞处理16、24和48小时。 The ARIP cells were treated with 0, 10 lizard and exendin 40ηΜ drug outer 16, 24 and 48 hours.

[0059] 图21.多种GLP-1/FDKP制剂对十字孢碱诱导的细胞死亡的影响。 Effect [0059] FIG 21. more GLP-1 / FDKP formulations on staurosporine-induced cell death. 用GLP-I样品预处理的ARIP细胞被暴露于5 μ M十字孢碱中4小时,并用Cell Titer-Glo™分析测定细胞存活力。 With GLP-I samples pretreated ARIP cells were exposed to 5 μ M staurosporine for 4 hours, and treated with Cell Titer-Glo ™ Determination of cell viability. 在4°C和40°C将样品胁迫处理4周。 At 4 ° C and 40 ° C for 4 weeks Samples stress. 右侧显示的对照样品(培养基、GLP-USTAU、 GLP+STAU)示出了培养基(不含GLP-I或十字孢碱)中、含GLP-I、含十字孢碱和含GLP-I 与十字孢碱时细胞的存活力(注意:图例不适用于对照样品)。 Right control sample (medium, GLP-USTAU, GLP + STAU), illustrate the display media (without GLP-I or staurosporine), and containing GLP-I, including staurosporine and containing GLP-I and the viability of the cells when staurosporine (note: the legend does not apply to the control sample). 显示的所有结果是一式三份运行的平均值。 All results shown are the average of triplicate runs.

[0060] 图22A-22B.药物代谢动力学研究描述使用多种浓度的GLP-1/FDKP制剂在大鼠中单一静脉注射(IV ;图22A)和肺吹入法(IS ;图22B)。 [0060] FIGS. 22A-22B pharmacokinetic studies describe the use of various concentrations of GLP-1 / FDKP formulations in rat single intravenous injection (IV; FIG. 22A) and pulmonary insufflation (the IS; FIG. 22B). 图例指出分析的制剂中GLP-I对FDKP颗粒的质量-质量百分比。 Legend noted that the quality of GLP-I FDKP particle formulations analyzed - mass percent.

[0061] 图23A-23B.给药后2小时(图23A)和6小时(图23B)用GLP-1/FDKP制剂给药的大鼠中累积食物消耗的减少。 [0061] FIGS. 23A-23B. 2 hours (FIG. 23A) and 6 hours post dose (FIG. 23B) to reduce cumulative food consumption in rats administered GLP-1 formulation / FDKP in.

[0062] 图24.雄性肥胖Zucker大鼠中通过肺吹入法施用的GLP-1/FDKP的药物动力学研究。 Pharmacokinetic studies in male obese Zucker rats administered by pulmonary insufflation GLP-1 / FDKP in [0062] 24. FIG. 数据描述了对照组(空气;第1组)和GLP-1/FDKP处理组(第2组)在第0、15、30、 45、60和90分钟的葡萄糖测量结果。 Data describing control (air; group 1) of 15, 30, 45, 60 and 90 minutes and the glucose measurements GLP-1 / FDKP treated (group 2) in.

[0063] 图25.雄性肥胖Zucker大鼠中通过肺吹入法施用的GLP-1/FDKP的药物动力学研究。 Pharmacokinetic studies in male obese Zucker rats administered by pulmonary insufflation GLP-1 / FDKP in [0063] 25. FIG. 数据描述了对照组(空气;第1组)和GLP-1/FDKP处理组(第2组)在第0、15、30、 45、60和90分钟的GLP-I测量结果。 Data describing control (air; group 1) and GLP-1 / FDKP treated (group 2) At 15, 30, 45, 60 and 90 minutes of GLP-I measurements.

[0064] 图26.雄性肥胖Zucker大鼠中通过肺吹入法施用的GLP-1/FDKP的药物动力学研究。 Pharmacokinetic studies in male obese Zucker rats administered by pulmonary insufflation GLP-1 / FDKP in [0064] 26. FIG. 数据描述了对照组(空气;第1组)和GLP-1/FDKP处理组(第2组)在第0、15、30、 45、60和90分钟的胰岛素测量结果。 Data describing control (air; group 1) and GLP-1 / FDKP treated (group 2) At 15, 30, 45, 60 and 90 minutes of insulin measurements.

[0065] 图27.雌性大鼠中用通过肺吹入法施用的多种GLP-I浓度进行的GLP-1/FDKP药物动力学研究。 Kinetic studies using GLP-1 female rats by various GLP-I concentrations administered pulmonary insufflation / FDKP Drug [0065] 27. FIG. 数据描述了对照组(空气;第1组)和分别施用了5%、10%和15% GLP-I 的GLP-1/FDKP处理第2、3和4组在第0、2、5、10、20、30、40和60分钟的GLP-I测量结果。 Data describing control (air; group 1) and were administered 5%, 10% and 15% GLP-I is GLP-1 / FDKP treated groups 2, 3 and 4 at its 0,2,5,10 , 20, 30 and 60 minutes of GLP-I measurements.

[0066] 图28.雌性大鼠中用通过肺吹入法施用的多种GLP-I浓度进行的GLP-1/FDKP药物动力学研究。 Kinetic studies using GLP-1 female rats by various GLP-I concentrations administered pulmonary insufflation / FDKP Drug [0066] 28. FIG. 数据描述了对照组(空气;第1组)和分别施用了5%、10%和15% GLP-I 的GLP-1/FDKP处理第2、3和4组在第0、2、5、10、20、30、40和60分钟的FDKP测量结果。 Data describing control (air; group 1) and were administered 5%, 10% and 15% GLP-I is GLP-1 / FDKP treated groups 2, 3 and 4 at its 0,2,5,10 , 20, 30 and 60 minutes FDKP measurements.

[0067] 图29.雌性大鼠(η = 10)中的GLP-1/FDKP药物动力学研究,所述大鼠连续4天通过单一每日肺吹入法施用了含15%61^-1(0.31^61^-1)的61^-1作0即。 [0067] FIG 29. GLP-1 female rats (η = 10) is / FDKP pharmacokinetic studies, the rats continuously administered four days containing 15% -1 61 single daily pulmonary insufflation (0.31 ^ 61 ^ -1), i.e., 61 -1 for 0. 数据显示连续4天在给药前、给药后1、2、4和6小时测量的平均食物消耗。 Data show that 4 consecutive days prior to dosing, after dosing, 2, 4 and 6 hours average food consumption measured.

[0068] 图30.雌性大鼠(η = 10)中的GLP-1/FDKP药物动力学研究,所述大鼠连续4天通过单一每日肺吹入法施用了含15%61^-1(0.31^61^-1)的61^-1作0即。 [0068] FIG 30. The female rats (η = 10) of GLP-1 / FDKP pharmacokinetic studies, the rats administered 4 days -1 61 containing 15% by a single daily pulmonary insufflation (0.31 ^ 61 ^ -1), i.e., 61 -1 for 0. 数据显示连续4天在给药前、给药后1、2、4和6小时测量的平均体重。 Data show that 4 consecutive days prior to dosing, after dosing, 2, 4 and 6 hours mean body weight measurements.

[0069] 图31.在猴子中的GLP-1/FDKP毒物代谢动力学研究,所述猴子连续5天通过每日一次(每天30分钟)口鼻施用施用了GLP-1/FDKP。 In metabolic kinetics of FDKP [0069] FIG. 31. In monkeys GLP-1 /, the monkeys for 5 consecutive days by one (30 minutes per day) administered daily oronasal administration of GLP-1 / FDKP. 数据显示雄性和雌性中GLP-I的血浆浓度峰值(Cmax)。 Data show that the peak plasma concentration males and females of GLP-I (Cmax). 动物接受对照(空气;第1组)、2mg/kg FDK(第2组)或0· 3、I. 0或2. Omg/kg GLP-1/FDKP (分别为第3、4 和5 组)。 Animals received control (air; group 1)., 2mg / kg FDK (Group 2), or 0 · 3, I 0 or 2. Omg / kg GLP-1 / FDKP (respectively 3, 4 and 5 groups) .

[0070] 优诜的实施方案详沭 [0070] The preferred embodiment details Shen Shu

[0071] 用作药物的稳定的、可吸入的高血糖素样肽I(GLP-I)制剂是本领域缺乏的。 [0071] as stabilizing drugs, inhalable glucagon-like peptide I (GLP-I) formulations are lacking in the art. 这归因于GLP-I肽体内的不稳定性。 This is due to the instability of GLP-I peptide in vivo. GLP-I化合物在大量条件下趋向于保留在溶液中,并且当作为溶液制剂被施用时具有相对短的体内半衰期。 GLP-I compounds in a large number of conditions tends to remain in solution and has a relatively short half-life in vivo when administered as a solution formulation. 另外,发现存在于多种生物学流体如肺和血中的二肽基-肽酶IV(DPP-IV)极大地降低GLP-I分子的生物学半衰期。 Also, it found in various biological fluids such as lung and blood dipeptide - biological half-peptidase IV (DPP-IV) greatly reduced GLP-I molecule. 例如, GLP-I (7-37)的生物学半衰期已经显示为3到5分钟;见美国专利No. 5, 118, 666。 For example, GLP-I (7-37) has a biological half life of the display 3 to 5 minutes; see U.S. Patent No. 5, 118, 666. GLP-I也已显示在胃肠外施用后经历快速的体内吸收。 GLP-I has also been shown in parenteral undergo rapid absorption in vivo after administration. 类似地,酰胺GLP-l(7-36)被皮下施用时具有约50分钟的半衰期;也参见美国专利No. 5, 118, 666。 Similarly, when an amide having GLP-l (7-36) was administered subcutaneously half-life of about 50 minutes; see also U.S. Pat. No. 5, 118, 666.

[0072] 本领域现有技术中GLP-I组合物的快速清除和短半衰期代表了本发明克服的缺陷。 [0072] The present state of the art in rapid clearance of the GLP-I and the short half-life of the compositions of the present invention overcomes represent defects. 本发明通过提供特别适用于肺部递送的最优化的天然GLP-1/FDKP (富马酰二酮哌嗪) 制剂克服了本领域现有技术的缺陷。 Natural optimized by providing the present invention is particularly suitable for pulmonary delivery of GLP-1 / FDKP (fumaryl diketopiperazine) formulation overcomes the drawbacks of the prior art in this field. 在其他具体的方面,本发明提供了天然GLP-I分子的制剂,所述制剂可在体内引起GLP-I应答。 In other specific aspects, the present invention provides formulations of native GLP-I molecule, the formulation may cause GLP-I response in vivo. 还考虑了在这类制剂中使用天然GLP-I的变体。 Also contemplated is the use of native GLP-I in such formulations variants. [0073] 为了克服本领域现有技术的缺陷,本发明提供了与二酮哌嗪(DKP)颗粒组合的GLP-I的制剂。 [0073] In order to overcome the drawbacks of the prior art in this field, the present invention provides formulations of GLP-I with diketopiperazine (the DKP) particles in combination. 在本发明具体的实施方案中,提供了GLP-1/DKP制剂用于施用给受试者。 In a specific embodiment of the invention, there is provided a GLP-1 / DKP formulation for administration to a subject. 在其他具体的实施方案中,GLP-1/DKP制剂包含富马酰二酮哌嗪(FDKP)但不仅限于此,并可包括其他KDP (不对称的DKP、xDKP)如2, 5-二酮-3, 6-二(4-琥珀酰基-氨丁基)哌嗪(SDKP),不对称的二酮哌嗪,包括仅在DKP环上一个位置取代的那些(例如FDKP的"单臂" 类似物),和DKP盐。 In other specific embodiments, GLP-1 / DKP formulations comprise fumaryl diketopiperazine (the FDKP), but is not limited thereto, and may include other of KDP (asymmetric DKP, xDKP) such as 2, 5-dione 3,6-di (4-succinyl - aminobutyl) piperazine (SDKP), asymmetrical diketopiperazines including only one position on the DKP ring substituents as those (e.g. FDKP the "single arm" similar thereof), and DKP salts. 在本发明的其他具体的实施方案中,通过肺部递送施用GLP-1/FDKP制剂。 In other specific embodiments of the present invention, GLP-1 / FDKP formulations administered by pulmonary delivery.

[0074] 开发GLP-I分子的治疗制剂时,通过使用多种的生物物理学和分析技术评价溶液中GLP-I的结构特征,所述技术包括远紫外线圆二色性(远-UV⑶)、近紫外线圆二色性(近-UV CD)、内荧光、傅立叶变换红外光谱学(FTIR)、高压液相色谱(HPLC)和质谱(MS)。 [0074] When developing therapeutic formulations of GLP-I molecule, the structural characteristics of GLP-I was analyzed by using biophysical techniques and evaluation of a variety of techniques including far-ultraviolet circular dichroism (far -UV⑶), near-ultraviolet circular dichroism (near -UV CD), intrinsic fluorescence, fourier transform infrared spectroscopy (FTIR), high pressure liquid chromatography (HPLC) and mass spectrometry (MS). 圆二色性(CD)技术是用于分析多种实验条件下蛋白质结构改变的有力工具,并是本领域公知的。 Circular dichroism (CD) technology is a powerful tool for analyzing the structure of the protein changes under various experimental conditions, and are known in the art. 进行这些分析的实验条件包括:浓度、离子强度、温度、pH、氧化胁迫、搅动和多重冻融循环对GLP-I肽的影响。 The experimental conditions for these analyzes include: concentration, ionic strength, temperature, pH, oxidative stress, agitation, and multiple freeze-thaw cycles Effects of GLP-I peptide. 这些分析被设计为表征降解的主要途径以及确立操作GLP-I 肽结构的条件从而达成优选的GLP-1/DKP制剂,所述制剂具有想要的药物代谢动力学(PK) 和药物动力学(PD)特征。 These analyzes were designed to characterize the major routes of degradation and to establish GLP-I peptide structures operating conditions so as to achieve preferred GLP-1 / DKP formulation pharmacokinetic (PK) having the desired pharmaceutical and pharmacokinetic ( PD) characteristics.

[0075] 观察到随着GLP-I浓度提高,肽的二级结构从主要是无组织的构象转化为更螺旋的构象。 [0075] observed with increasing concentrations of GLP-I, the secondary structure of the peptide from the primary conversion unorganized conformation to a more helical conformation. 提高溶液中的离子强度引起GLP-I的结构增加,直到其可逆地沉淀为止。 Increasing the ionic strength in solution causes an increase in the structure of GLP-I until it reversibly precipitated. NaCl的存在提高了GLP-I的三级结构,这通过图2D中所示的近⑶条带的强度提高显示。 The presence of NaCl increased the tertiary structure of GLP-I, which increases the strength displayed by the FIG. 2D strip near ⑶ shown. 这甚至在不显示自身缔合的低肽浓度下发生。 This is not displayed even at low peptide concentrations occur association itself. 提高的离子强度容易地将无组织的GLP-I转化为α -螺旋形式(如图2A中朝向208nm和222nm的远⑶最小值迁移所示)和自身缔合的构象(如使用提高的盐和近⑶模式的图2B和2D中向更短波长的色氨酸激发迁移所示)。 Increased ionic strength readily unstructured GLP-I into α - helix (Figure 2A toward 208nm and 222nm ⑶ minimum migrate far shown) and self-associated conformations (e.g., increased use of salts and FIG near ⑶ 2B and 2D mode excitation migrate shown) to the shorter wavelength tryptophan. 温度和pH有差异地影响GLP-I构象,因为GLP-I的无序结构不被这些参数中任一个所改变。 Temperature and pH differentially affect the conformation of GLP-I, GLP-I because of the disordered structure is not changed by one of any of these parameters. 另一方面,发现GLP-I的自身缔合构象对热变性是敏感的并其其溶解度对pH是敏感的,如图4A和4B中所示,该图显示在10mg/ml的肽浓度下,在pH 6. 3-7. 6之间GLP-I肽可逆地沉淀。 On the other hand, we found that GLP-I self-associated conformation is sensitive to pH and its solubility sensitive to heat denaturation, as shown in FIG. 4A and 4B, which shows at a peptide concentration of 10mg / ml of GLP-I peptide reversibly precipitates between pH 6. 3-7. 6. 发现GLP-I的多种构象一般对搅动和多重冻融循环是稳定的。 GLP-I is found in a variety of conformations general agitation and multiple freeze-thaw cycles are stable. 对GLP-I未观察到脱酰胺,也未观察到氧化。 Of GLP-I was not observed deamidation nor oxidation was observed.

[0076] 还在多种条件下观察了GLP-I对FDKP颗粒的吸附,所述条件包括pH、GLP-I浓度的改变,和多种表面活性剂、盐、离子、离液剂和感胶离子、稳定剂和醇浓度的改变。 The [0076] still more conditions of GLP-I adsorption on FDKP particles was observed, the conditions including pH, change in GLP-I concentration, and a variety of surfactants, salts, ions, chaotropes and lyotropes changing the ion, stabilizer and concentration of alcohol. 发现GLP-I对FDKP颗粒的吸附受pH的强影响,特别是在约pH 4. O或更大时发生结合。 I found that GLP-I adsorption to FDKP particles was strongly affected by the influence of pH, especially at about pH 4. O or greater when the binding occurs. 发现其他赋形剂对GLP-I对FDKP颗粒的吸附具有有限的影响。 Other excipients found to have limited effect on the adsorption on FDKP particles of GLP-I.

[0077] 在开发本发明的GLP-1/DKP制剂时,评价了可能影响或冲击其体内可递送性和吸收的大量参数。 [0077] In developing the present invention, GLP-1 / DKP formulation was evaluated a number of parameters that may affect or impact its deliverability and absorption in vivo of. 这类参数包括例如GLP-I肽的结构、在某配制条件下分子上的表面电荷、作为制剂的溶解度和稳定性,以及对丝氨酸蛋白酶降解的易感性和体内稳定性;这些均在产生可被容易地吸收且显示延长的生物学半衰期的制剂时起关键的作用。 Such parameters include, for example, the structure of GLP-I peptide, the surface charges on the molecule under certain formulation conditions, solubility and stability as a preparation, as well as in vivo stability, and susceptibility to serine protease degradation; may be generated which are It plays a key role when the formulation is easily absorbed and prolonged biological half life of the display.

[0078] 在多种条件下体外和体内测试了获得的GLP-1/FDKP制剂的稳定性。 [0078] under various conditions was tested in vivo and in vitro stability of GLP-1 / FDKP formulations obtained. 通过HPLC分析和基于细胞的测定法分析了GLP-I的稳定性。 And analyzed by HPLC analysis of the stability of GLP-I cell-based assays. 另外,在肺灌洗液(其含有DPP-IV)中检查了GLP-I的稳定性。 Further, in lung lavage fluid (which contains DPP-IV) examined the stability of GLP-I. 还发现天然GLP-I的稳定性在溶液中是浓度依赖性的。 Also I found that the stability of native GLP-I was concentration dependent in solution.

[0079] 还使用体外GLP-I生物活性研究用于GLP-1/FDKP负载的研究,并测定了体内效力。 [0079] GLP-I biological activity in vitro studies using further GLP-1 / FDKP a load, and measured in vivo efficacy. 该策略有助于进一步鉴定领先的GLP-1/FDKP配制方法。 This strategy helps to further identify the leading GLP-1 / FDKP formulation methods. 另外,基于GLP-I已经显示通过抑制细胞凋亡、刺激β-细胞增殖和胰岛再生在提高β-细胞量中起作用的事实,通过基于细胞的测定法检查本发明的GLP-1/FDKP制剂的增殖和抗细胞凋亡潜能。 Further, based on GLP-I it has been shown by inhibition of apoptosis and stimulation of cell proliferation β- islet cell regeneration in fact increase in the amount of β- act by GLP-1 / FDKP formulations cell-based assay of the present invention to check proliferation and anti-apoptotic potential.

[0080] 因此,本发明提供了包含与富马酰基二酮哌嗪(FDKP)组合的天然人GLP-I的最优化的制剂,该制剂是稳定的并且抗降解。 [0080] Accordingly, the present invention provides a formulation comprising a combination of optimized with fumaryl diketopiperazine (the FDKP) of native human GLP-I, the formulation is stable and resistant to degradation.

[0081] II. GLP-I 分子 [0081] II. GLP-I molecules

[0082] 在本发明具体的实施方案中,提供了最优化的制剂,其包含与二酮哌嗪如富马酰基二酮哌嗪(FDKP)组合的天然人高血糖素样肽I (GLP-I)。 [0082] In a particular embodiment of the invention, there is provided optimized formulations comprising native human in combination with a diketopiperazine such as fumaryl diketopiperazine (the FDKP) glucagon-like peptide-I (GLP- I). 本发明的这类GLP-1/FDKP制剂是稳定的并且抗降解。 Such GLP-1 / FDKP formulations of the invention are stable and resistant to degradation.

[0083] 人GLP-I是本领域公知的,并且来源于远端回肠(distal ileum)中、胰腺中和脑中L-细胞合成的前高血糖素原(preproglucagon)多肽。 [0083] human GLP-I are known in the art, and from the distal ileum (distal ileum), the pancreas and brain cells synthesize L- front proglucagon (Preproglucagon) polypeptide. GLP-I是30-31个氨基酸的肽,其以两种分子形式存在:7-36和7-37,其中7-36形式是主要的。 GLP-I is a 30-31 amino acid peptide that exists in two molecular forms: 7-36 and 7-37, 7-36 wherein the main form. 前高血糖素原成为GLP-I (7-36)酰胺和GLP-I (7-37)延长形式的加工主要发生在L-细胞中。 Before the proglucagon be GLP-I (7-36) amide and GLP-I (7-37) extended form occurs mainly in processing L- cells. 本领域已经显示在禁食状态下,GLP-I的血浆水平为约40pg/ml。 This art has been shown in the fasted state, plasma levels of GLP-I is about 40pg / ml. 进餐后,GLP-I血浆水平迅速提高至约50_165pg/ml。 After a meal, GLP-I plasma levels rapidly increase to about 50_165pg / ml.

[0084] 本文使用的术语"GLP-1分子"是指GLP-I蛋白质、肽、多肽、类似物、模拟物、衍生物、同种型、片段等等。 [0084] As used herein, the term "GLP-1 molecule" means GLP-I proteins, peptides, polypeptides, analogs, mimetics, derivatives, isoforms, fragments and the like. 这类GLP-I分子可包括天然存在的GLP-I多肽(GLP-1 (7-37)0H、 GLP-I (7-36)NH2)和GLP-I代谢产物如GLP-I (9-37)。 Such GLP-I molecules may include naturally occurring GLP-I polypeptides (GLP-1 (7-37) 0H, GLP-I (7-36) NH2) and GLP-I metabolites such as GLP-I (9-37 ). 因此,在本发明具体的实施方案中, GLP-I分子包括:天然的GLP-1、GLP-I类似物、GLP-I衍生物、二肽基肽酶-IV(DPP-IV)保护的GLP-I、GLP-I模拟物、GLP-I肽类似物或生物合成的GLP-I类似物。 Thus, in particular embodiments of the present invention, GLP-I molecules include: a native GLP-1, GLP-I analogs, GLP-I derivative, a dipeptidyl peptidase -IV (DPP-IV) protected GLP -I, GLP-I mimetics, GLP-I peptide analog, or a biosynthetic GLP-I analog.

[0085] 本文使用的术语"类似物"包括与另一化合物的结构相似的化合物。 [0085] As used herein, the term "analog" includes a compound of the structure of the other similar compounds. 例如,抗病毒化合物阿昔洛韦(acyclovir)是一种核苷类似物,其与衍生自碱基鸟嘌呤的核苷鸟苷结构上类似。 For example, an anti-viral compound acyclovir (acyclovir) is a nucleoside analogue, which is derived from the nucleoside guanosine similar structure of guanine. 因此,阿昔洛韦模拟鸟苷(生物学上与鸟苷类似),并且通过置换病毒核酸中的鸟苷残基(或与之竞争)来干扰DNA合成并阻止翻译/转录。 Thus, acyclovir analog of guanosine (is biologically analogous to a guanosine), and interferes with DNA synthesis by replacement of guanosine residues (or competing) viral nucleic acid and prevents translation / transcription. 因此,与另一(亲本化合物) 具有结构类似性的化合物是类似物,其模拟亲本化合物的生物学或化学活性。 Thus, the compounds of structural similarity to another (a parent compound) is like having that mimic the parent biologically or chemically active compound present. 将化合物确定为类似物不需要最大或最小的基本或功能基团取代数量,只要类似物能够以一些相关的模式(同一地、补充地或竞争地)模拟亲本化合物的生物学特性或化学特性即可。 Compound analogs determined substantially not require minimum or maximum number of substituents or functional group, as long as a number of related analogs can be mode (identically, complementarily or competitively) simulated biological properties of the parent compound or chemical properties i.e. can. 类似物可以是并且通常是亲本化合物的衍生物(见下文"衍生物")。 And the like may be derivatives of the parent compound is usually (see below "derivatives"). 本文公开的化合物的类似物可以具有等于、小于或大于它们亲本化合物的活性。 Analogs of the compounds disclosed herein may have equal, less than or greater than the activity of the parent compound thereof.

[0086] 本文使用的"衍生物"是指天然地或合成地从亲本化合物制成(或衍生)的化合物。 [0086] "Derivatives" as used herein refers to a natural or synthetic compound made of (or derived) from the parent compound. 衍生物可以是类似物(见上文"类似物")并从而可具有类似的化学或生物学活性。 Derivative may be an analog (see above "analog") and so may have a similar chemical or biological activity. 然而,与类似物不同,衍生物不必须模拟亲本化合物的生物学或化学活性。 However, with the different analogs, derivatives of the parent does not have to simulate biological or chemical activity of the present compounds. 将化合物确定为衍生物不需要最大或最小的基本或功能基团取代数量。 Determining a derivative of the compound need not substantially minimum or maximum number of functional groups or substituents. 例如,尽管抗病毒化合物甘克洛韦(ganclovir)是阿昔洛韦的衍生物,但是甘克洛韦具有与阿昔洛韦不同的抗病毒活性谱系和不同的毒物学特性。 For example, while the antiviral compound Gan Ke Luowei (ganclovir) is a derivative of acyclovir, acyclovir but has a different Gan Ke Luowei antiviral activity of different lineages and toxicological properties. 本文公开的化合物的衍生物与其亲本化合物比较时可以具有相等、 更小、更大或甚至不相似的活性。 Derivatives of the compounds disclosed herein, when compared to their parent compound may have equal, less, greater or even no similar activity.

[0087] 本文使用的术语"代谢产物"是指代谢的任何中间产物或产物,并包括大分子和小分子。 [0087] As used herein, the term "metabolite" refers to any metabolic intermediate or product, and comprising large and small molecules. 本文使用时和适当时,该定义适用于初级和次级代谢产物。 As used herein and where appropriate, the definition applies to both primary and secondary metabolites. 初级代谢产物直接涉及活体生物正常的生长、发育和繁殖。 Primary metabolites directly involved in the normal growth of living organisms, development and reproduction. 次级代谢产物不直接涉及这些过程,但是典型地具有重要的生态学功能(例如抗生素)。 Secondary metabolites not directly involved in those processes, but typically has important ecological function (e.g., antibiotics).

[0088] 本文使用的术语"生物合成的"是指活体生物对化合物的任何生产。 [0088] As used herein, the term "biosynthesis" refers to any living organism to produce the compound.

[0089] 本文使用的术语"能形成颗粒的"是指化学的、生物合成的或生物学实体或化合物,其能够形成固体颗粒,通常在液体培养基中形成。 Refers to chemical, biosynthetic, or biological entities or compounds [0089] As used herein, the term "capable of forming particles", capable of forming solid particles, usually formed in a liquid medium. 颗粒的形成典型地发生在能形成颗粒的实体暴露于某条件下时,所述条件例如为pH、温度、湿度和/或摩尔渗透压浓度/重量摩尔渗透压浓度。 Grain formation typically occurs at a particle-forming entity is exposed to certain conditions, the conditions such as pH, temperature, humidity and / osmolality / osmolality or from. 对所述条件的暴露可导致例如结合、聚结、凝固和/或脱水,从而形成颗粒。 The exposure can result in conditions such as binding, coalescence, solidification and / or dehydration, to form granules. 沉淀反应是能形成颗粒事件的一个例子。 Precipitation reaction is one example of a particle-forming event.

[0090] 本文使用的"共溶液"是由至少两种化学、生物学和/或生物合成的实体组成的任何介质。 [0090] As used herein, "co-solution" is any medium by the at least two chemical, biological and / or biosynthetic entity thereof. 例如,可以通过将包含至少一种化学、生物学和/或生物合成的实体的液体与包含化学、生物学和/或生物合成的实体的固体组合来形成共溶液。 For example, by including at least one chemical, biological and / or biosynthetic entity liquid with a co-solution comprises a chemical, biological solids and / or biosynthetic entity combined to form. 在另一个例子中,可以通过将包含至少一种化学、生物学和/或生物合成的实体的液体与包含化学、生物学和/或生物合成的实体的另一种液体组合来形成共溶液。 In another example, by comprising at least one chemical, biological and / or biosynthetic entity liquid with a co-solution comprises a chemical, biological, and / or another liquid compositions biosynthesis entity formed. 在又一个例子中,可以通过向单个溶液中添加至少两种固体形成共溶液,所述每种固体包含至少一种化学、生物学和/或生物合成的实体。 In yet another example, by addition of at least two solid solutions were formed into a single solution, each of said solid comprising at least one chemical, biological and / or biosynthetic entity.

[0091] 本发明中考虑的天然GLP-I是具有SEQ ID NO. 1或SEQ ID NO. 2的氨基酸序列的多肽。 [0091] native GLP-I contemplated in the present invention having SEQ ID NO. 1 polypeptide or amino acid sequence of SEQ ID NO. 2 in. 天然GLP-I肽在体内于数分钟内被快速的切割和灭活。 Native GLP-I peptide within minutes and is rapidly inactivated in vivo cleavage.

[0092] 本发明的GLP-I类似物可包括毒蜥外泌肽,其为被发现是GLP-I受体激动剂的肽; 这类类似物可还包括毒蜥外泌肽1到4。 [0092] GLP-I analogs of the present invention may include exendin peptide, which is found to be GLP-I receptor agonist peptide; Such analogs may further include an outer exendin 1-4. 毒蜥外泌肽在毒蜥(Gila-monster)的毒液中被发现,并与哺乳动物GLP-I分享约53%的氨基酸同源性。 Exendin peptides are found in the Gila (Gila-monster) venom, and share about 53% amino acid homology with mammalian GLP-I. 毒蜥外泌肽还具有对GLP-I受体的类似亲合力。 Exendin peptides also have similar affinity for the GLP-I receptor. 据报道毒蜥外泌肽-3和毒蜥外泌肽_4刺激胰腺腺泡细胞中的cAMP生产和淀粉酶从胰腺腺泡细胞中的释放(Malhotra et al.,1992 ;Raufman et al.,1992 ;Singh et al.,1994)。 It has been reported outside the outer exendin-3 and exendin _4 pancreatic acinar cells stimulated cAMP production and amylase release from pancreatic acinar cells (Malhotra et al, 1992;. Raufman et al,. 1992; Singh et al, 1994).. 已经提出了毒蜥外泌肽-3作为促胰岛素剂用于治疗糖尿病和预防高血糖症的用途(美国专利No. 5, 424, 286)。 It has been proposed -3 exendin peptides for treating diabetes and the prevention of hyperglycemia (U.S. Patent No. 5, 424, 286) as an insulinotropic agent.

[0093] 毒蜥外泌肽的羧基末端片段如毒蜥外泌肽[9-39](羧基酰胺化的分子)和穿过9-39的片段3-39已经被报道为是GLP-I的有力的和选择性的拮抗剂(Goke et al.,1993 ; Raufman et al.,1991 ;Schepp et al.,1994 ;Montrose_Rafizadeh et al.,1996)。 [0093] exendin peptide carboxy-terminal fragment peptide as exendin [9-39] (carboxyl amidated molecule) and fragments 9-39 through 3-39 have been reported to be a GLP-I potent and selective antagonist (Goke et al, 1993;. Raufman et al, 1991;. Schepp et al, 1994;. Montrose_Rafizadeh et al, 1996.). 文献还证实了毒蜥外泌肽[9-39]在体内封闭内源GLP-1,导致减少的胰岛素分泌(Wang et al.,1995 ;D' Alessio et al.,1996)。 Document also confirms the outer exendin [9-39] enclosed in vivo endogenous GLP-1, resulting in reduced insulin secretion (Wang et al, 1995;. D 'Alessio et al, 1996.). 毒蜥外泌肽-4有效结合分泌胰岛素的β -TCl细胞上的GLP-I、来自胰腺的分散的腺泡细胞和来自胃的壁细胞。 Outer exendin -4 effectively bind the GLP-I β -TCl insulin secreting cells, dispersed acinar cells from pancreas, and parietal cells from stomach. 毒蜥外泌肽-4肽也在经分离的胃中刺激促生长素抑制素释放和抑制胃泌素释放中起作用(Goke et al.,1993; Sch印ρ et al.,1994;Eissele et al. ,1994)。 Toxicity outer lizard exendin-4 peptide also somatostatin release and inhibiting gastrin releasing function (Goke et al, 1993 isolated stomach irritation;. Sch printed ρ et al, 1994;. Eissele et al., 1994). 在用克隆的GLP-I受体转染的细胞中,毒蜥外泌肽-4 被报道为激动剂(即其促进cAMP),而毒蜥外泌肽[9-39]被定义为拮抗剂(即其阻断毒蜥外泌肽-4和GLP-I的刺激作用)。 In cells transfected with the cloned receptor of the GLP-I, exendin agonist peptide is reported to -4 (i.e., promotion of cAMP thereof), and the outer exendin [9-39] is defined as an antagonist (i.e., it blocks external stimulation -4 exendin and GLP-I) is. 还发现毒蜥外泌肽是抗降解的。 Also found that exendin peptide is resistant to degradation.

[0094] 本发明的另一实施方案考虑了肽模拟物的用途。 [0094] Another embodiment of the present invention contemplates the use of peptide mimetics. 如本领域技术人员已知的,肽模拟物是生物学上模拟激素、细胞因子、酶底物、病毒或其他生物分子上活性决定簇的肽, 并可拮抗、刺激或以其他方式调节天然配体的生理活性。 As known to the skilled person, peptidomimetic analog is biologically active peptide determinants on hormones, cytokines, enzyme substrates, viruses or other biological molecules, and may antagonize, stimulate, or otherwise adjust the natural ligand physiological activity thereof. 参阅例如BIOTECHNOLOGY AND PHARMACY, Pezzuto et al·,Eds. ,Chapman and Hall, New York(1993)中的Johnson et al·,"Peptide Turn Mimetics"。 See, for example BIOTECHNOLOGY AND PHARMACY, Pezzuto et al ·, Eds., Chapman and Hall, New York Johnson et al · (1993) in, "Peptide Turn Mimetics". 使用肽模拟物的潜在原理是蛋白质的肽主链主要以确定氨基酸侧链方向有助于分子相互作用的方式存在。 Underlying principles use of peptide mimetics is that the peptide backbone of proteins exists chiefly to determine the direction of the amino acid side chain interactions contribute to the way the presence of the molecule. 预计肽模拟物允许与天然分子相似的分子相互作用。 Peptide mimetic is expected to permit molecular interactions similar to the natural molecule.

[0095] 在其他实施方案中,本发明的GLP-I分子会具有天然GLP-I的至少一种生物活性, 例如与GLP-I受体结合并引发导致促胰岛素活性的信号转导途径的能力。 Capacity of at least one biological activity [0095] In other embodiments, GLP-I molecules of the invention may have the native GLP-I such as GLP-I receptor binding and signal transduction pathways resulting in insulinotropic activity induced . 在本发明的其他实施方案中,GLP-I分子可以是肽、多肽、蛋白质、类似物、模拟物、衍生物、同种型、片段等等,其维持天然存在的GLP-I的至少一种生物活性。 In other embodiments of the present invention, GLP-I molecules can be peptides, polypeptides, proteins, analogs, mimetics, derivatives, isoforms, fragments, etc., which maintains at least one naturally occurring GLP-I of biological activity. GLP-I分子还可包括可药用盐和前药,和前药的盐,天然存在的人GLP-I的多形体(polymorph)、水合物、溶剂合物、生物活性片段、生物活性变体和立体异构体,以及天然存在的人GLP-I的激动剂、模拟物和拮抗剂变体。 GLP-I molecules may further comprise pharmaceutically acceptable salts may be salts and prodrugs, and prodrugs, polymorphs naturally occurring human GLP-I of (PolyMorph), hydrates, solvates, biologically-active fragments, biologically active variants agonist and stereoisomers thereof, as well as naturally occurring human GLP-I, the mimetic, and antagonist variants. 包括毒蜥外泌肽1到4的毒蜥外泌肽家族,及其多肽融合物。 They include exendin peptide exendin peptide family 1 to 4, and polypeptide fusions thereof. 本发明的GLP-I分子也可以包括二肽基肽酶-IV(DPP-IV)保护的GLP-1,其阻止或抑制GLP-I的降解。 GLP-I molecules of the invention may also include dipeptidyl peptidase -IV (DPP-IV) protected GLP-1, which prevents or inhibits the degradation of GLP-I.

[0096] 本发明的GLP-I分子包括肽、多肽、蛋白质及其衍生物,其含有氨基酸取代、改进溶解度、赋予对氧化的抗性、提高生物效力,或提高循环中的半衰期。 GLP-I molecule [0096] the present invention include peptides, polypeptides, proteins and derivatives thereof which contain amino acid substitutions, improve solubility, confer resistance to oxidation, increase biological potency, or increase half-life in circulation. 因此,本发明中考虑的GLP-I分子包含氨基酸取代、删除或添加,其中所述氨基酸选自本领域公知的那些。 Thus, GLP-I molecules of the invention are contemplated comprising amino acid substitutions, deletions or additions, wherein said amino acid is selected from those known in the art. 分子的N端或C端也可以被修饰,例如但不限于被酰化、乙酰化、酰胺化。 N-terminus or C-terminus of the molecule may also be modified, for example, but not limited to be acylated, acetylated, amidated. 因此,在本发明中,术语"氨基酸"是指天然存在的和非天然存在的氨基酸,以及氨基酸类似物和氨基酸模拟物, 其以与天然存在的氨基酸相似的方式发挥功能。 Accordingly, in the present invention, the term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics, which is a similar manner as naturally occurring amino functions. 天然编码的氨基酸是20种常见的氨基酸(丙氨酸、精氨酸、天冬酰氨、天冬氨酸、半胱氨酸、谷氨酰氨、谷氨酸、甘氨酸、组氨酸、异亮氨酸、亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸和缬氨酸)和焦赖氨酸(pyrolysine)和硒代半胱氨酸(selenocysteine)。 Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, iso leucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrophosphoric lysine ( pyrolysine) and selenocysteine ​​(selenocysteine). 氨基酸类似物是指具有与天然存在的氨基酸相同的基本化学结构的化合物,所述基本化学结构即与氢、羧基、氨基和R基团结合的α碳,例如高丝氨酸、正亮氨酸、正缬氨酸、甲硫氨酸亚砜、甲硫氨酸甲基锍、瓜氨酸、羟基谷氨酸、羟基脯氨酸和praline。 Amino acid analogs refers to compounds that have the same basic chemical structure as the amino acid naturally occurring in the basic chemical structure is bound to a hydrogen, a carboxyl group, an amino group, and R α carbons, e.g., homoserine, norleucine, n valine, methionine sulfoxide, methionine methyl sulfonium, citrulline, hydroxyl glutamic acid, hydroxyproline, and praline. 这类类似物具有经修饰的R基团(例如正亮氨酸),但是保留与天然存在的氨基酸相同的基本化学结构。 Such analogs have modified R groups (e.g., norleucine), but retain the same amino acid naturally occurring basic chemical structure. 本发明中考虑的氨基酸还包括与α-氨基酸相似的β-氨基酸,因为它们含有氨基端和羧基端。 Amino acids contemplated in the present invention further comprises the α- β- amino acids similar because they contain amino and carboxy termini. 然而,在β-氨基酸中,两个碳原子分开这些功能性末端。 However, β- amino acids, two carbon atoms separate these functional termini. 带有特定侧链的β-氨基酸能够作为a (C2)碳或β (C3)碳上的R或S异构体存在。 a particular amino acid side chains with β- can be present as a (C2) carbon or β (C3) R on carbon or S isomer. 这导致对任何给定的侧链而言总共四种可能的非对映异构体。 This results in a total of four possible diastereoisomers for any given body in terms of side-chain.

[0097] 本发明的GLP-I分子也可包括杂种GLP-I蛋白、其融合蛋白、寡聚体和多聚体、同系物、糖基化模式变体和突变蛋白质,其中GLP-I分子保持天然分子的至少一种生物活性, 并且与其合成或制造方法无关,所述方法包括但不限于重组(无论是从cDNA、基因组DNA、 合成的DNA或其它形式的核酸产生)、合成和基因激活方法。 [0097] GLP-I molecules of the present invention may also include hybrid GLP-I proteins, fusion proteins, oligomers and multimers, homologues, glycosylation pattern variants, and muteins, wherein the GLP-I molecule remains at least one biological activity of the native molecule, or synthetic and regardless of their method of manufacturing, including, but not limited to, recombinant (whether from cDNA, genomic DNA, synthetic DNA or other form of nucleic acid is generated), the synthesis method of gene activation and . 重组DNA技术是本领域普通技术人员公知的(见Russell, DW,et al. ,Molecular Cloning:A Laboratory Manual, Cold Spring Harbor, NY,2001)。 Recombinant DNA techniques are well known to those of ordinary skill in the art (see Russell, DW, et al, Molecular Cloning:. A Laboratory Manual, Cold Spring Harbor, NY, 2001).

[0098] III.二酮哌嗪 [0098] III. Diketopiperazine

[0099] 二酮哌嗪因为其形成微粒的能力而是本领域公知的,所述能力适用于药物递送和稳定。 [0099] Because of its ability to diketopiperazine microparticles known in the art but is formed of a suitable drug delivery capability and stability. 在本发明中,使用二酮哌嗪有助于GLP-I分子的吸收,从而提供抗降解的稳定制剂。 In the present invention, the diketopiperazine help absorb GLP-I molecules thereby providing a stable formulation resistant to degradation. [0100] 可使用多种方法,其中二酮哌嗪可以形成整和GLP-I分子的颗粒,或GLP-I分子能够吸附在上面的颗粒。 [0100] Various methods may be used, wherein the diketopiperazine particles may be formed integral, and GLP-I molecule or GLP-I molecules can be adsorbed on the above particles. 这可涉及将二酮哌嗪溶液与GLP-I分子的溶液或悬浮液混合然后沉淀,随后形成包含二酮哌嗪和GLP-I的颗粒。 This may involve diketopiperazine and GLP-I solution of molecules in solution or suspension is then mixed precipitate subsequently formed comprising a diketopiperazine and GLP-I particles. 或者,可以沉淀二酮哌嗪形成颗粒,并随后与GLP-I分子的溶液混合。 Alternatively, the diketopiperazine can be precipitated to form particles, and then mixed with a solution of the GLP-I molecule. 二酮哌嗪颗粒与GLP-I分子之间的缔合可以通过溶剂去除来进行, 或在干燥之前可以包括特定的步骤(如pH调节),从而促进缔合。 Association between the diketopiperazine particle and the GLP-I molecule can be removed by a solvent, or may include a particular prior to drying step (such as pH adjustment) to facilitate the association.

[0101] 在一个优选的实施方案中,本发明的二酮哌嗪包括但不限于3, 6-二(4-富马酰-氨丁基)-2,5_二酮哌嗪,也已知为(E)-3,6-二[4-(N-羧基-2-丙烯基)氨丁基]-2, 5-二酮哌嗪(其也被称作富马酰二酮哌嗪或FDKP)。 [0101] In a preferred embodiment, diketopiperazines of the present invention include, but not limited to 3, 6- di (4-fumaryl - aminobutyl) in -2,5_ diketopiperazine, also known as (E) -3,6- two [4- (N--carboxy-2-propenyl) aminobutyl] -2,5-diketopiperazine (which may also be referred to as fumaryl diketopiperazine or FDKP).

[0102] 本发明中考虑的其他二酮哌嗪包括,但不限于3, 6-二(4-氨丁基)-2,5-二酮哌嗪的衍生物例如:3, 6-二(琥珀酰-4-氨丁基)-2, 5-二酮哌嗪(在本文中也称作3, 6-二(4-羧丙基)氨丁基-2, 5-二酮哌嗪;琥珀酰二酮哌嗪或SDKP) ;3, 6-二(马来酰-4-氨丁基)-2, 5-二酮哌嗪;3, 6-二(柠康酰-4-氨丁基)-2-5-二酮哌嗪;3, 6-二(戊二酰-4-氨丁基)-2,5-二酮哌嗪;3,6_二(丙二酰-4-氨丁基)-2,5-二酮哌嗪;3, 6-二(草酰-4-氨丁基)-2, 5-二酮哌嗪及其衍生物。 [0102] Other diketopiperazines contemplated in the present invention include, but are not limited to, 3, 6-bis (4-aminobutyl) -2,5-diketopiperazine derivative, for example: 3, 6-bis ( -4- succinyl-aminobutyl) -2, 5-diketopiperazine (also referred to herein as 3, 6-bis (4-carboxypropyl) amino-butyl-2, 5-diketopiperazine; succinyl diketopiperazine or SDKP); 3, 6-bis (maleimido -4- aminobutyl) -2, 5-diketopiperazine; 3, 6-bis (citraconimidomethyl -4- ammonia butoxy yl) -2-5- diketopiperazine; 3, 6-bis (glutaryl -4- aminobutyl) -2,5-diketopiperazine; 3,6_ di (malonyl -4- aminobutyl) -2,5-diketopiperazine; 3, 6-bis (oxalyl -4- aminobutyl) -2, 5-diketopiperazine and derivatives thereof. 在其它实施方案中,本发明考虑了二酮哌嗪盐的用途。 In other embodiments, the present invention contemplates the use of diketopiperazine salts. 这类盐可包括例如任何可药用的盐,如二酮哌嗪的Na、K、Li、Mg、Ca、铵或单、二或三烷基铵(如衍生自三乙胺、丁胺、二乙醇胺、三乙醇胺或吡啶等等)盐。 Such salts may comprise any pharmaceutically acceptable salts, for example, such as diketopiperazine Na, K, Li, Mg, Ca, ammonium or mono-, di- or trialkylammonium (such as those derived from triethylamine, tributylamine, diethanolamine, triethanolamine, pyridine or the like) salt. 盐可以是单盐、二盐或混合的盐。 Salt may be a mono-salts, di-salts or mixed salts. 也考虑了二酮哌嗪的更高级的盐,其中R基团含有多于一个酸基。 Also contemplated salts of the higher diketopiperazine, wherein the R groups contain more than one acid group. 在本发明的其它方面,试剂的基本形式可以与二酮哌嗪混合形成二酮哌嗪的药物盐,从而该药物是二酮哌嗪的平衡阳离子(counter cation)。 In other aspects of the present invention, the basic form of a pharmaceutical agent may be mixed to form diketopiperazine salts and diketopiperazine, such that the drug is the counter cation of the diketopiperazine (counter cation). 本文考虑的盐的一个例子以非限制性的方式包括FDKP 二钠。 Examples of the salt of a contemplated herein comprises a non-limiting manner FDKP disodium. 美国专利申请N〇:ll/210, 710教导了使用DKP的药物递送,其涉及DKP盐的所有内容通过参考并入本文。 U.S. Patent Application N〇: ll / 210, 710 teaches the use of drug delivery DKP, DKP covering all salts are incorporated herein by reference.

[0103] 如本文中别处所公开的,本发明还使用了新颖的FDKP不对称类似物xDKP,例如(E) -3- (4- (3, 6-二氧代哌嗪-2-基)丁基氨基甲酰基)-丙烯酸;(E) -3- (3- (3, 6-二氧代哌嗪-2-基)丙基-氨基甲酰基)丙烯酸;和伍)-3-(4-(5-异丙基-3,6-二氧代哌嗪-2_基)-丁基氨基甲酰基)丙烯酸,并在题为〃Asymmetrical FDKP Analogs for Use as Drug Delivery Agents"的美国临时专利申请中公开,该申请在与此一致的日期提交并以其整体并入本文(代理人案号No. 51300-00041)。 [0103] As disclosed elsewhere herein, the present invention also uses a novel asymmetrical analogs of FDKP xDKP, e.g. (E) -3- (4- (3, 6- dioxo-piperazin-2-yl) butylcarbamoyl) - acrylic acid; (E) -3- (3- (3, 6- dioxo-piperazin-2-yl) propyl - carbamoyl) acrylic acid; and Wu) -3- (4 - (5-isopropyl-3,6-dioxo-piperazin--2_ yl) - butylcarbamoyl) acrylic acid, and entitled 〃Asymmetrical FDKP Analogs for Use as Drug Delivery Agents "U.S. provisional Patent open application, filed and in its entirety (Attorney docket No. 51300-00041) this paper is consistent with this date.

[0104] 二丽脈嚷可以如下形成:如1^1:(3]13181^,6七31.,(]\六11161'.〇16111.3〇(3· 68:879-80 ; 1946)所述通过氨基酸酯衍生物的环二聚体作用j[JKopple,etal.,(J.0rg· Chem. 33:862-64 ; 1968)所述通过二肽酯衍生物的环化或通过氨基酸衍生物在高沸点溶剂中的热脱水,所述文件的教导被引入本文。 [0104] Li two shouted pulse can be formed: such as 1 ^ 1: (3] ^ 13181, seven 31 6, (] \ 11161'.〇16111.3〇 six (3 * 68: 879-80; 1946) the cyclic dimer by the action of amino acid ester derivative of j [JKopple, etal, (J.0rg · Chem 33:. 862-64; 1968). by the cyclization of dipeptide ester derivatives, or by an amino acid derivative high-boiling solvent to thermal dehydration, the teachings of the document is incorporated herein by reference.

[0105] 用于合成和制备二酮哌嗪的方法是本领域普通技术人员公知的,并公开于美国专利5, 352, 461 ;5, 503, 852 ;6, 071,497 ;6, 331,318 ;6, 428, 771 和美国专利申请No. 20060040953中。 [0105] The method of synthesis and preparation of diketopiperazines for those of ordinary skill in the art is well known and is disclosed in U.S. Patent No. 5, 352, 461; 5, 503, 852; 6, 071,497; 6, 331, 318; 6, 428, 771 and in U.S. Patent application No. 20060040953. 美国专利No. 6, 444, 226和6, 652, 885描述了在水性悬浮液中制备和提供二酮哌嗪微粒,所述水性悬浮液中被添加了活性试剂的溶液,从而使活性试剂与颗粒结合。 U.S. Patent No. 6, 444, 226 and 6, 652, 885 describes the preparation of an aqueous suspension and the diketopiperazine microparticles provide the aqueous suspension was added a solution of the active agent, whereby the active agent with granule-bound. 这些专利还描述了通过冻干法去除液体介质产生包含活性剂的微粒的方法,改变这类悬浮液的溶剂条件以促进活性试剂与颗粒的结合则在美国专利申请系列N 〇:60/717, 524 和11/532,063(二者均题为"Method of Drug Formulation Based on Increasing the Affinity of Active Agents for Crystalline Microparticle Surfaces")和11/532, 065(题为"Method of Drug Formulation Based on Increasing the Affinity of Active Agents for Crystalline Microparticle Surfaces·")中教导。 These patents also describe methods to produce a liquid medium of microparticles containing an active agent is removed by lyophilization, changing the solvent conditions of such suspension to promote binding of the active agent with the particles is in U.S. Patent Application Serial N ○: 60/717, 524 and 11 / 532,063 (both of which are entitled "Method of Drug Formulation Based on Increasing the Affinity of Active Agents for Crystalline Microparticle Surfaces") and 11/532, 065 (entitled "Method of Drug Formulation Based on Increasing the Affinity of teach Surfaces · ") in Active Agents for Crystalline Microparticle. 还参阅美国专利No. 6, 440,463和2005年8月23日提交的美国专利申请系列No: 11/210, 709和美国专利申请No. 11/208,087。 See also US Patent No. 6, 440,463 and 2005 August 23 filed US Patent Application Serial No: 11/210, 709 and US Patent Application No. 11 / 208,087. 在一些情况下,考虑到通过例如2006年2月22日提交的题为〃A Method For Improving the Pharmaceutic Properties of Microparticles Comprising Diketopiperazine and an Active Agent. "的美国专利申请系列No. 11/678, 046 中公开的喷雾干燥方法干燥本发明的被负载的二酮哌嗪颗粒。每份专利和专利申请针对它们涉及二酮哌嗪的内容通过参考并入本文。 In some cases, for example, by taking into account entitled Method For 〃A February 22, 2006, filed Improving the Pharmaceutic Properties of Microparticles Comprising Diketopiperazine and an Active Agent. "US Patent Application Serial No. 11/678, 046 in was loaded diketopiperazine particles of the spray-drying process disclosed in the present invention is dried. patents and patent applications for each content thereof relates to a diketopiperazine incorporated herein by reference.

[0106] IV. GLP-1/DKP颗粒的治疗制剂 Therapeutic formulations [0106] IV. GLP-1 / DKP particles

[0107] 本发明还提供了用于施用给需要治疗的受试者的GLP-1/FDKP制剂。 [0107] The present invention further provides a GLP-1 / FDKP formulation for administration to a subject in need of treatment. 本发明中考虑的受试者可以是家居宠物或人。 The subject of the present invention may be considered household pet or human. 在某些实施方案中,治疗是针对II型糖尿病、肥胖症、癌症或其任何相关疾病和/或病症。 In certain embodiments, the treatment is for Type II diabetes, obesity, cancer or any related diseases and / or disorders. 人是尤其优选的受:试者。 Especially preferred is a human subject: subjects were.

[0108] 本发明中考虑的其它疾病或病症包括,但不限于肠易激综合征、心肌梗死、缺血、 再灌注组织损伤、血脂障碍、糖尿病心肌病、急性冠状动脉综合征、代谢综合征、手术后分解代谢改变、神经变性病症、记忆和学习障碍、胰岛细胞移植和再生治疗或中风。 [0108] Other diseases or conditions contemplated in the present invention include, but are not limited to, irritable bowel syndrome, myocardial infarction, ischemia, reperfusion injury, dyslipidemia, diabetic cardiomyopathy, acute coronary syndrome, metabolic syndrome , catabolic changes after surgery, neurodegenerative disorders, memory and learning disorders, islet cell transplant and regenerative therapy or stroke. 本发明考虑的其它疾病和/或病症包括与上文所列的相关的任何疾病和/或病症,其可以通过对需要的受试者施用GLP-1/FDKP干粉制剂来治疗。 Other diseases contemplated by the invention and / or associated disorders include any disease and / or disorders listed above and which can be treated by a subject in need of GLP-1 / FDKP dry powder formulation. 本发明的GLP-1/FDKP干粉制剂也可以用于治疗II型糖尿病和高血糖症的人细胞中β细胞分化的诱导。 Human cell GLP-1 / FDKP dry powder formulation of the present invention may also be used to treat type II diabetes and hyperglycemia in β cell differentiation induction.

[0109] 还在本发明的又一个实施方案中,考虑到受试者可以是家居宠物或动物,包括大鼠、兔子、仓鼠、荷兰猪、沙土鼠(gerbil)、旱獭、猫、犬、绵羊、山羊、猪、牛、马、猴子和猿(包括猩猩、长臂猿和狒狒)。 [0109] Yet another embodiment of the present invention, also in consideration of the subject may be a household pet or animal, including rats, rabbits, hamsters, guinea pigs, gerbils (Gerbil), woodchucks, cats, dogs, sheep , goats, pigs, cattle, horses, monkeys and apes (including chimpanzees, gibbons and baboons).

[0110] 还考虑到本发明的GLP-1/FDKP颗粒制剂可以通过本领域普通技术人员公知的和用于临床或非临床目的的多种施用途径施用。 [0110] also contemplated GLP-1 / FDKP particle formulations of the invention can be prepared by those of ordinary skill in the art and known for a variety of clinical or non-clinical purposes routes of administration. 本发明的GLP-1/FDKP组合物可以被施用至任何靶向的生物膜,优选受试者的粘膜。 GLP-1 / FDKP compositions of the invention may be administered to any targeted biological membrane, preferably the mucosa of the subject. 施用可以通过任何途径进行,所述途径包括但不限于经口、经鼻、经颊、全身性静脉注射、皮下、通过血或淋巴提供的分区施用、直接施用至受侵袭的位点或甚至通过局部手段进行。 Administration can be carried out by any of routes including, but not limited to oral, nasal, buccal, systemic intravenous injection, subcutaneously, by blood or partition administration lymphatic provided, applied directly to the affected site or even by local means of. 在本发明优选的实施方案中,GLP-1/FDKP组合物的施用通过肺部递送。 In a preferred embodiment of the invention, administration of GLP-1 / FDKP composition is by pulmonary delivery.

[0111] 本发明中可以使用的其它备选的施用途径可包括:真皮内、动脉内、腹膜内、病灶内、颅内、关节内、前列腺内、胸膜内、气管内、玻璃体内、阴道内、直肠的、瘤内、肌内、血管内(intravesicular)、粘膜的、心包内、支气管局部施用,使用气雾剂,注射、灌输、连续灌输、集中灌注直接沐浴靶细胞,通过导管、通过灌洗,在霜剂中、在液体组合物(例如脂质体)中,或通过本领域常规计数人员会制造的其它方法或前述的任何组合(参阅例如Remington's Pharmaceutical Sciences, 1990,其涉及给药方法的全部内容通过参考并入本文)。 [0111] Other alternative routes of administration may be used in the present invention may include: intradermal, intraarterial, intraperitoneal, intralesional, intracranial, intraarticular prostatic intrapleural intratracheal, intravitreal, the,,,, vaginal , rectal, intratumoral, intramuscular, intravascular (intravesicular), mucosa, intrapericardial, bronchial administration local, using aerosol, injection, infusion, continuous infusion, bathing target cells directly concentrated infusion, via a catheter, by oral wash, in creams, in liquid compositions (such as liposomes), or in the present art will be produced by conventional counting art or any other combination of the foregoing methods (see e.g. Remington's Pharmaceutical Sciences, 1990, a method which involves the administration of the entire contents of which are incorporated herein by reference).

[0112] 作为干粉制剂,本发明的GLP-1/DKP颗粒可以通过吸入被递送至呼吸系统的特定区域,这取决于颗粒的大小。 [0112] As a dry powder formulation, GLP-1 / DKP particles of the present invention may be delivered to a particular region of the respiratory system by inhalation, depending on the size of the particles. 另外,GLP-1/DKP颗粒可以被制造成足够小而能掺入静脉注射悬浮液剂型中。 Further, GLP-1 / DKP particles can be made sufficiently small and can be incorporated into intravenous suspension dosage forms. 对于经口递送而言,可以可以被掺入悬浮液、片剂或胶囊中。 For purposes of oral delivery, it may be incorporated into a suspension, tablets or capsules. GLP-1/DKP组合物可以从吸入装置被递送,所述吸入装置例如雾化器、压力定量气雾剂、干粉吸入器和喷雾器。 GLP-1 / DKP composition may be delivered from the suction means, said suction means such as a nebulizer, a metered-dose aerosols, dry powder inhaler and a nebulizer.

[0113] 在其它实施方案中,考虑向需要的患者施用"有效量"的GLP-1/DKP制剂。 [0113] In other embodiments, consider administering an "effective amount" of a GLP-1 / DKP formulation to a patient in need thereof. 本发明中考虑的"有效量"的GLP-1/DKP干粉制剂是指GLP-I化合物、类似物或肽模拟物等等的量, 该用量会在一些程度上缓解被治疗的疾病、病症或障碍的一个或多个症状。 An "effective amount" of the present invention is contemplated GLP-1 / DKP dry powder formulation refers to an amount of a compound of GLP-I, analog or peptide mimetic or the like, an amount which will alleviate the disease to be treated to some degree, disorder or or more symptoms of a disorder. 在一个实施方案中,"有效量"的GLP-1/DKP干粉制剂应当是通过将血浆胰岛素水平提高、禁食血糖水平减少或降低和胰腺β-细胞量提高至少约1%、2%、3%、4%、5%、6%、7%、8%、9%、10%、 15%、20%、25%、30%、35%、40%、45%、50%或更多(但不限于此)来治疗糖尿病的61^-1 分子的量。 In one embodiment, an "effective amount" of a GLP-1 / DKP dry powder formulation should by increasing plasma insulin levels, reducing or lowering fasting blood glucose levels and pancreatic β- cells increase an amount of at least about 1%, 2%, 3 %, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or more ( but the amount is not limited thereto) for the treatment of diabetes 61 -1 molecules. 在另一优选的实施方案中,本发明考虑通过对需要这类治疗的受试者施用药物有效量的GLP-I分子来治疗肥胖症。 In another preferred embodiment, the present invention contemplates treating obesity by administering the drug GLP-I molecule to a subject in need of such treatment a therapeutically effective amount. 在这类情况下,"有效量"的GLP-1/DKP干粉制剂应当是通过将体重减少或降低至少约5%、10%、15%、20%、25%、30%、35%、40%、45%、50% 或更多(但不限于此)的GLP-I分子的量。 In such cases, an "effective amount" of a GLP-1 / DKP dry powder formulation should be reduced by reducing the weight or at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40 %, 45%, 50% or more of an amount of (but not limited to) the GLP-I molecule. 本发明还考虑了施用"有效量"的GLP-1/DKP 干粉制剂用于通过对需要这类治疗的受试者施用药物有效量的GLP-I分子来控制饱满感。 The present invention also contemplates GLP-1 / DKP dry powder formulation administered in an "effective amount" of a GLP-I molecule for by administering the drug to a subject in need of such treatment an effective amount to control satiety. 就非限制性的方式而言,GLP-I分子可以是毒蜥外泌肽分子如毒蜥外泌肽-1或-4。 For a non-limiting manner to, GLP-I molecule may be a peptide exendin molecule such as exendin peptide-1 or -4. 在这类情况下,"有效量"的GLP-1/DKP干粉制剂应当是将饥饿感受和食物摄入(例如通过质量或卡路里含量测量)减少至少约5% ,6%、7%、8%、9%、10%、11 %、12%、13%、14%、15%、 16 %U7 %U8 %U9 %,20 %,21 %,22 %,23 %,24 %,25 %,26 %,27 %,28 %,29 %,30 %, 35%、40%、45%、50%或更多(但不限于此)的61^-1分子的量。 In such cases, an "effective amount" of a GLP-1 / DKP dry powder formulation should feel hunger and food intake (e.g., by measuring the mass or caloric content) by at least about 5%, 6%, 7%, 8% , 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16% U7% U8% U9%, 20%, 21%, 22%, 23%, 24%, 25%, 26 %, 27%, 28%, 29%, 30%, 35%, 40%, 45%, 50% or more (but not limited to) an amount of 61 -1 molecules. "有效量"的61^-1/1)即干粉制剂还可以被定义为足够可检测地和可重复地改善、减少、最小化或限制疾病或病症或其症状的程度的量。 An "effective amount" of 61 -1 / 1), i.e. a dry powder formulation can also be defined as sufficiently detectably and repeatedly to improve, reduce, minimize or limit the amount of a disease or disorder or a symptom thereof degree. 也可能使用"有效量"的本发明制剂消除、清除或治愈疾病或病症。 Also possible to use an "effective amount" of the formulation of the present invention to eliminate, remove or cure the disease or disorder.

[0114] 在对需要的受试者施用本发明的GLP-1/FDKP组合物时,组合物的实际剂量可以以物理和生理因素为基础确定,所述因素如体重、病症的严重性、被治疗的疾病类型、先前或共存的治疗干涉、患者的特发病(idiopathy)和施用途径。 [0114] When a subject in need thereof, the present invention GLP-1 / FDKP composition, the actual dosage of the composition may be physical and physiological factors is determined based on factors such as the severity of the body weight, condition, and is the type of disease being treated, previous or concurrent therapeutic intervention, idiopathy of the patient (idiopathy) and route of administration. 技术人员应当能够基于一个或多个这些因素确定实际的剂量。 Skilled person should be able to determine actual dosages based on one or more of these factors.

[0115] 本发明的GLP-1/DKP制剂可以被施用一次或多于一次,取决于待治疗的疾病或病症。 GLP-1 / DKP formulation [0115] The present invention may be administered once or more than once, depending the disease or condition to be treated. GLP-1/DKP制剂的施用可以以下述间隔被提供给受试者,所述间隔在数分钟、数小时、数天、数周或数月的范围内变化。 GLP-1 / DKP formulation can be administered at to a subject is provided by the following intervals, said intervals of minutes, hours, days, weeks, or within the range of variation months. 在一些情况下,治疗方案的计时可涉及施用后GLP-I分子的半衰期。 In some cases, the timing treatment regimen may involve administration of GLP-I half-life of the molecule. 在其它实施方案中,例如在治疗具体的或复杂的疾病或病症如癌症时,例如可期望施用含药物赋形剂或试剂的本发明的GLP-1/DKP制剂。 In other embodiments, for example, in treating particular or complex diseases or conditions such as cancer, for example, it may be desirable administered GLP-1 / DKP formulation of the present invention containing a pharmaceutical excipient or agent. 在这类情况下,可以由药物赋形剂或试剂指导施用方案。 In such cases, a pharmaceutical excipient or agent may be administered program guide.

[0116] V.实施例 [0116] V. Example

[0117] 以下的实施例包括在本文中用于阐述本发明的某些实施方案。 Example [0117] The following includes a certain embodiments of the invention set forth herein. 本领域技术人员应当明白,实施例中公开的技术阐明了在本发明的实践中适当地发挥功能的代表性技术。 Those skilled in the art will appreciate, the techniques disclosed in the examples elucidate representative techniques that function properly in the practice of the present invention. 然而,本领域的技术人员在本发明的启发下应当明白,可以在公开的特定实施方案中进行许多改变,并且仍然获得相似或类似的结果,而不偏离本发明的宗旨和范围。 However, those skilled in the art in light of the present disclosure should be understood that many changes may be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0118] 实施例1 [0118] Example 1

[0119] GLP-I结构的牛物物理学分析和分析法分析 [0119] and physical analysis were bovine GLP-I analysis Structure Analysis

[0120] 为了分析GLP-I的结构和行为,使用了大量的生物物理学技术和分析技术。 [0120] In order to analyze the structure and behavior of GLP-I, the use of a large number of biophysical and analytical techniques. 这些技术包括远紫外线圆二色性(远-UV⑶)、近紫外线圆二色性(近-UV⑶)、内荧光、傅立叶变换红外光谱学(FTIR)、高压液相色谱(HPLC)和质谱(MS);其均是本领域普通技术人员公知的。 These techniques include far-ultraviolet circular dichroism (far -UV⑶), near-ultraviolet circular dichroism (near -UV⑶), intrinsic fluorescence, fourier transform infrared spectroscopy (FTIR), high pressure liquid chromatography (HPLC) and mass spectrometry (MS ); which it is known to those of ordinary skill in the art. 使用大范围的条件研究浓度、离子强度、温度、pH、氧化胁迫、搅动和多重冻融循环对GLP-I肽的影响;其均在下文更详细地描述。 Conditions of use of a wide range of concentrations, ionic strength, temperature, pH, oxidative stress, agitation, and multiple freeze-thaw cycles Effects of GLP-I peptide; both of which are described in more detail below. 也使用这些分析表征降解的主要途径,并确立操作GLP-I的肽结构从而达成某GLP-1/DKP制剂的条件。 These analyzes also be used to characterize the major routes of degradation and to establish the operating structure of GLP-I peptide so as to achieve conditions of a GLP-1 / DKP formulation.

[0121] 实骀步骤 [0121] Step solid forworn

[0122] GLP-1 购自American Peptide (Sunnyvale, CA)或AnaSpec (San Jose, CA),或内部制造(MannKind Corporation, Valencia, CA)。 [0122] GLP-1 was purchased from American Peptide (Sunnyvale, CA) or AnaSpec (San Jose, CA), or internal manufacturing (MannKind Corporation, Valencia, CA). 在pH 4.0 和2〇°C (除非另有说明)下分析多种浓度的水性GLP-I样品。 Analysis of Aqueous GLP-I samples at various concentrations (unless otherwise noted) at pH 4.0 and 2〇 ° C. 样品一般是新鲜制备的并在每次实验前与适当的添加剂(例如盐、pH缓冲液、H2O 2等,如果有的话)混合。 Fresh samples are typically prepared with suitable additives (e.g. salts, pH buffers, H2O 2, etc., if any) before each experiment mixing. 用远-UV⑶和透射傅立叶转换红外光谱(FTIR)收集多种条件下GLP-I的二级结构测量结果。 -UV⑶ and transmission using far infrared Fourier transform spectroscopy (FTIR) measurements collected by the secondary structure of GLP-I results under various conditions. 另外,使用近-UV⑶和内荧光,通过监测其芳香族残基(即色氨酸)周围的环境来分析GLP-I的三级结构。 In addition, near -UV⑶ and the fluorescence, which is monitored by an aromatic residue (i.e., Trp) to analyze the environment around the tertiary structure of GLP-I.

[0123] GLP-I的浓度依赖件结构 [0123] GLP-I concentration dependence piece structure

[0124] 使用圆二色性(CD)谱分析分子(如蛋白质或肽)可能显示的α -螺旋、随机卷曲、β-折叠片层、β-转角和随机卷曲。 α dichroic [0124] A circle (CD) spectroscopy molecules (e.g., proteins or peptides) that may be displayed - helix, random coil, [beta] pleated sheet, [beta] turn and random coil. 具体地,使用远-UV⑶测定蛋白质和肽中二级结构的类型,例如纯α-螺旋、β-片层等。 Specifically, the determination of protein and peptide far -UV⑶ the type of secondary structure, such as pure α- helix, [beta] sheet and the like. 另一方面,使用近-UV⑶分析分子的三级结构。 On the other hand, the use of tertiary structure near -UV⑶ analyze molecules. 因此,为了检查浓度对GLP-I的影响,使用远-UV⑶和近-UV⑶技术二者。 Accordingly, in order to examine the effect of concentration on GLP-I, the use of both near and far -UV⑶ -UV⑶ technology.

[0125] 图IA中的远UV-CD证实了在大范围的浓度(例如L 8、4· 2、5· 1、6· 1、7· 2和8. 6mg/ ml)下,GLP-I形成两种不同的结构,所述结构包括α -螺旋和随机卷曲。 [0125] FIG IA far UV-CD is demonstrated at a wide range of concentrations (e.g. L 8,4 · 2,5 · 1,6 · 1,7 · 2 and 8. 6mg / ml), GLP-I forming two different structures, said structure comprising α - helix and random coil. 通过205nm处的巨大单个最小值确定,在低浓度(< 2mg/mL)下GLP-I主要是无组织的。 Determined by the minimum value at 205nm great individual, at low concentrations (<2mg / mL) GLP-I is primarily unstructured. 通过208nm和224nm 处的两个最小值确定,当浓度提高时肽采取α -螺旋结构(图1A)。 By two minima at 208nm and 224nm to determine, when increasing the concentration of peptide taken α - helical structure (FIG. 1A).

[0126] 三级结构分析提示高浓度的GLP-I结构是自身缔合的构象(即寡聚物)。 [0126] tertiary structure analysis suggests that high concentrations of GLP-I are self-associated structure conformations (i.e., oligomers). 近-UV ⑶和荧光发射数据均支持该假设。 Near -UV ⑶ and fluorescence emission data support this hypothesis. 近-UV⑶(图1Β)中250-300nm之间的阳性条带揭示了GLP-I具有确定的三级结构,该结构在更高的浓度下增加。 Near -UV⑶ (FIG. L [beta]) between the positive band 250-300nm band tertiary structure disclosed GLP-I has a defined, this structure increases at higher concentrations. 更特别地,这些条带表明肽的芳香族残基被大量地固定,并存在于明确定义的环境中。 More specifically, these bands indicate that the aromatic residues of the peptide are largely fixed, well-defined and exist in the environment.

[0127] 类似地,多种浓度下(pH 4. 0, 20°C )GLP_1的荧光发射显示芳香族残基色氨酸(其也在近-UV⑶谱中显示强烈的条带)存在于明确定义的三级结构中;显示的数据得自280nm处的色氨酸激发(图1C)。 [0127] Similarly, at various concentrations (pH 4. 0, 20 ° C) Fluorescence emission display GLP_1 aromatic residue tryptophan (which also displayed intense near -UV⑶ spectrum bands) present in the clear defined tertiary structure; the data obtained excitation of tryptophan (FIG. 1C) from at 280nm. 对低浓度GLP-I而言355nm处的荧光最大值表明色氨酸被暴露于溶剂中,且不存在显著的三级结构。 For low concentrations of GLP-I fluorescence maximum at 355nm indicated that the tryptophan is solvent exposed, a significant tertiary structure and there. 在高肽浓度下,最大值强度降低并迁移至更短的波长,表明更多确定的三级结构。 At high peptide concentrations, reducing the maximum strength and migrate to shorter wavelengths, show more tertiary structure determined.

[0128] 为了进一步测定GLP-I自身缔合构象的潜在的二级结构,在多种浓度下(pH 4. 0, 20°C )进行FTIR分析。 [0128] To further determine the underlying secondary structure of GLP-I self-associated conformation, FTIR analysis was performed at various concentrations (pH 4. 0, 20 ° C). 1656cm-1处的酰胺I条带清楚地表明在彡2mg/mL的浓度下GLP-I具有α -螺旋结构(图1D)。 Article amide I band at 1656cm-1 clearly show that at a concentration of San 2mg / mL GLP-I has the α - helix structure (FIG. 1D). 因此,GLP-I不形成β -片层结构;取而代之,在高浓度下肽更可能产生螺旋束。 Thus, GLP-I does not form β - sheet structure; instead, the peptide at a high concentration is more likely helix bundle.

[0129] 另外,实验显示这些不同的GLP-I结构不是通过样品处理产生的。 [0129] Further, these experiments showed different GLP-I structures are not generated by the sample processing. 与将肽直接溶于缓冲液中制备的GLP-I相比,来自浓缩的储存溶液的稀释液产生类似的远-UV CD、近-UV CD和荧光发射谱。 The peptide was dissolved directly compared to GLP-I prepared in buffer, dilution from a concentrated stock solution of a similar far -UV CD, near -UV CD and fluorescence emission spectra.

[0130] 离子强度对GLP-I的影响 Effects [0130] Ionic Strength on GLP-I of

[0131] 还进行了一些研究来测定离子强度对GLP-I肽的影响。 [0131] also conducted to determine the effect of ionic strength on GLP-I peptide in some studies. 图2A (远-UV-⑶)阐述了提高盐的浓度(从IOOmM到IOOOmM)将无序的GLP-I结构转化为α -螺旋构象,如208nm 和224nm处的最小值所揭示的那样。 FIG 2A (far -UV-⑶) describes increasing the concentration of salt (from IOOmM to IOOOmM) the disordered structure into a GLP-I α - helical conformation, such as minimum at 208nm and 224nm as disclosed. 将NaCl浓度提高至IM后,很多肽(在I. Omg/mL时) 从溶液中沉淀出来(图2A)。 After the NaCl concentration was increased to IM, is a polypeptide (when I. Omg / mL) precipitates out of solution (FIG. 2A). 然而,这种类型的沉淀显示用水稀释后溶解,从而确定了在高离子强度下GLP-I可以被可逆地沉淀。 However, this type of display after dilution with water to dissolve the precipitate, in order to determine the GLP-I can be reversibly precipitated at high ionic strength.

[0132] 盐还显示产生并促进GLP-I的三级结构。 [0132] and shown to produce salts also facilitate the tertiary structure of GLP-I. 这在图2B(近-UV-⑶)中例证,其中I. Omg/mL GLP-I在无盐时不显示信号,但是显示清楚的三级结构,该三级结构随着提高的离子强度而强化。 This (near -UV-⑶) illustrated in Figure 2B, there I. Omg / mL GLP-I displays no signal in the absence of salt, but showed a clear tertiary structure, the tertiary structure with increasing ionic strength strengthen. 280nm处色氨酸激发后在多种NaCl浓度下(pH 4. 0, 20°C ) I. 0mg/mL GLP-I的荧光发射(图2C)证实了这些结果。 After tryptophan excitation at 280nm at various concentrations of NaCl (pH 4. 0, 20 ° C) I. 0mg / mL GLP-I fluorescence emission (FIG. 2C) confirmed these results. 提高的离子强度引起荧光最大值迁移至更短的波长,表明1.0mg/mL GLP-I的三级结构既被产生又被增强。 Increased ionic strength caused the fluorescence maximum to migrate to shorter wavelengths, show the tertiary structure of 1.0mg / mL GLP-I is both generated and enhanced.

[0133] 另外,使用近-UV⑶谱在多种离子强度下(pH 4. 0, 20°C )对10mg/mL GLP-I的三级结构分析证实提高的离子强度也增强了GLP-I的自身缔合构象(图2D)。 [0133] Further, the use of a variety of spectra near -UV⑶ ionic strength (pH 4. 0, 20 ° C) the tertiary structure of 10mg / mL GLP-I analysis confirms improving ionic strength GLP-I also enhances the self-associated conformations (FIG. 2D).

[0134] 因此,数据提示离子强度对GLP-I的结构具有巨大的影响,引起蛋白质既采取α-螺旋构象又缔合成为寡聚体。 [0134] Thus, the data suggests that ionic strength has a great influence on the structure of GLP-I, causing the protein taken both α- helical conformation and associate into an oligomer. 另外,提高溶液中的离子强度引起GLP-I的寡聚化提高, 直到其可逆地沉淀为止。 Further, increasing the ionic strength in solution causes the oligomerization of GLP-I increase until it reversibly precipitated. 该事件在低肽浓度(其中最初不存在三级结构)以及高肽浓度(其已经显示大量二级和三级结构)下是明显的。 In the event of low peptide concentration and a high concentration of the peptide (which initially no tertiary structure present) (which has been shown that a large number of secondary and tertiary structure) are under significant. 因此,提高的离子强度容易地将无组织的GLP-I转化为α -螺旋和自身缔合的构象。 Thus, increased ionic strength readily unstructured GLP-I into α - helix and self-associated conformations. 另外,观察到的分光镜结果与先前显示的提高的肽浓度的影响是可比较的。 Further, the dichroic mirror was observed with improved impact previously displayed peptide concentrations are comparable.

[0135] 淵度和DH对GLP-I的影响 [0135] Effect of Yuan and DH of the GLP-I

[0136] 还进行了研究来测定GLP-I的自身缔合的构象是否对温度或pH的改变敏感。 [0136] Studies were also conducted to determine the GLP-I self-associated conformation of whether to change the temperature or pH sensitive. 图3A(近-UV-⑶)证实随着温度提高,lOmg/mL GLP-I的三级结构显著地离解。 Figure 3A (near -UV-⑶) confirmed that with increasing temperature, the tertiary structure of lOmg / mL GLP-I significantly dissociates. 另一方面, 在多种温度和pH 4. 0下,温度对低浓度(0. 05mg/mL)的GLP-I不具有影响;见图3B和3C(远-UV-⑶)。 On the other hand, at various temperatures and pH 4. 0, the temperature of the low concentration (0. 05mg / mL) of GLP-I does not have an effect; see FIG. 3B and 3C (far -UV-⑶). 远-UV⑶阐明了肽对温度是不敏感的。 Far -UV⑶ clarify the peptide is insensitive to temperature. 因此,提高的分子运动显著地妨碍了GLP-I的自身缔合。 Therefore, increased molecular motion significantly hinders self-association of GLP-I.

[0137] 相反,图4A (远-UV-⑶)证实α -螺旋GLP-I构象的溶解度是pH敏感的。 [0137] In contrast, FIG. 4A (far -UV-⑶) demonstrated α - helix GLP-I conformation is pH sensitive solubility. 尽管在pH 4. 4和以下时,lOmg/mL GLP-I的结构是相对均一的(即GLP-I保持螺旋),但是当pH提高至接近或处于中性(pH 6. 3和7. 6之间)时,一些沉淀发生并产生无序的谱系。 Although pH 4. 4 and less, the structure lOmg / mL GLP-I is relatively uniform (i.e., GLP-I remains helical), but when the pH is raised to near or at neutral (pH 6. 3 and 7.6 It is between), and generates a number of lineage precipitated disorder. 其中发生了沉淀的样品具有更低的强度,因为溶液中存在更少的可溶性GLP-1。 Where precipitation occurred have less intensity of the sample, as present in the solution less soluble GLP-1. 该无序的结构通过图4A(远-UV-⑶)中208nm处观察到的单个最小值确定,这在图4B(近-UV-⑶)中进一步描述并且有可能得自沉淀后溶液中GLP-I的减少。 The disordered structure (far -UV-⑶) 208nm was observed at the minimum value determined by a single FIGS. 4A, which is further described and may precipitate from FIG. 4B (near -UV-⑶) After a solution of GLP -I reduced. 当pH提高至高于GLP-I的5. 5的pi时, 可发生该沉淀。 When the pH is increased to above the pi GLP-I is 5.5, the precipitation may occur. 然而,随着pH从接近中性提高至11. 7,大部分沉淀再溶解,表明该沉淀是可逆的。 However, as the pH increased from near neutral to 11.7, most of the precipitate re-dissolved, indicating the precipitation is reversible. 在PHIL 7剩余的未溶解的GLP-I沉淀会引起溶液中肽量降低,从而降低远-UVCD谱系的强度,如图4A中所观察到的。 PHIL 7 remaining in the undissolved precipitate GLP-I peptide solution can cause a decrease in the amount, thereby reducing the strength of the far -UVCD lineage, as shown in FIG. 4A observed. 还观察到当冻干的GLP-I粉末与pH 9缓冲液混合为高浓度GLP-I时,GLP-I是极端不溶的。 Also observed when lyophilized GLP-I buffer 9 and the pH of the mixed powder of a high concentration while GLP-I, GLP-I is extremely insoluble.

[0138] GLP-I的稳定件 [0138] GLP-I is stable member

[0139] 通过测定GLP-I肽对脱酰胺作用和氧化作用的抗性以及对搅动和冻融循环影响的抗性来测定其稳定性。 [0139] Stability was determined by measuring its resistance GLP-I peptide deamidation and oxidation resistance as well as agitation and freeze-thaw cycles of the impact.

[0140] 将处于pH 10.5的GLP-l(lmg/mL)在40°C孵育5天,之后进行反相HPLC和电喷射质谱(MS)用于脱酰胺作用和氧化作用分析。 [0140] The pH 10.5 in the GLP-l (lmg / mL) were incubated at 40 ° C 5 days, followed by reverse phase HPLC and electrospray mass spectrometry (MS) deamidation and oxidation for analysis. 也使用HPLC和MS对GLP-I样品(lmg/mL)进行氧化研究,所述样品在存在〇. 1% H2O2时孵育2小时。 HPLC and MS use to GLP-I samples (lmg / mL) to study the oxidation, in the presence of the sample square. 1% H2O2 for 2 hours.

[0141] 图5描述了GLP-I在脱酰胺和氧化条件下的稳定性。 [0141] FIG. 5 depicts the stability of GLP-I under conditions of deamidation and oxidation. HPLC色谱图阐明了GLP-I在相同的滞留时间下洗脱,并且所分析的失稳条件没有产生降解峰。 HPLC chromatogram illustrates GLP-I was eluted at the same retention time, and the analyzed condition of instability no degradation peaks. 另外,MS分析对所有样品产生了类似的质量3297g/mol,表明该质量未被改变。 Further, the MS analysis yielded a similar mass of 3297g / mol for all samples, indicating that the mass is not changed. 数据还阐明了在多种条件下孵育时肽保持纯净和完整。 The data also illustrates the peptides incubated under conditions when kept clean and complete. 因此,未观察到GLP-I的脱酰胺。 Thus, not observed deamidation of GLP-I. GLP-I也显示对氧化胁迫是稳定的,如在存在0. 1% H2O2时所观察的那样,其中GLP-I的纯度和质量保持完整,如通过HPLC 和MS分别测定的那样。 GLP-I is also shown to be stable to oxidative stress as observed in the presence as time 0. 1% H2O2, wherein the GLP-I remained intact purity and quality, respectively, as determined by HPLC and the MS. 总之,不存在滞留时间或质量值的改变,并且未产生降解峰,从而证实了GLP-I肽对脱酰胺和氧化均是抗性的。 In short, changing the residence time or the mass value does not exist, and does not produce degradation peaks, confirming the GLP-I peptide are deamidation and oxidation resistant.

[0142] 搅动和连续的冻融循环对多种浓度的GLP-I的影响用近-UV⑶和内荧光分析。 [0142] continuous agitation and freeze-thaw cycles on various concentrations affect GLP-I was analyzed by the fluorescence and near -UV⑶. 9. 4和I. 5mg/mL GLP-I的搅动不产生肽的显著改变,如通过近-UV⑶(图6A)和荧光发射(图6B)所观察到的那样。 9.4 and I. 5mg / mL GLP-I produced no significant agitation of the altered peptides, such as by near -UV⑶ (FIG. 6A) and as fluorescence emission (FIG. 6B) observed. 将样品在室温下搅动30和90分钟,并在280nm处色氨酸激发后收集荧光发射谱。 Samples were agitated for 30 and 90 minutes at room temperature, and fluorescence emission spectra were collected after tryptophan excitation at 280nm. 在独立的冻融研究中,将含I. 6、5. 1和8. 4mg/mL GLP-I (pH 4. 0)的溶液在-20°C冷冻并在室温下熔化。 Solution was freeze-thaw separate study, containing I. 6,5. 1 and 8. 4mg / mL GLP-I (pH 4. 0) at room temperature and melts at -20 ° C freezer. 通过近-UV⑶(图7A)和荧光发射(图7B)指导和分析10 次冻融循环对GLP-I的影响。 By near -UV⑶ (FIG. 7A) and fluorescence emission (FIG. 7B) to guide and Analysis of 10 freeze-thaw cycles on the GLP-I. 在280nm处色氨酸激发后收集荧光发射谱。 Fluorescence emission spectra were collected after tryptophan excitation at 280nm. 两种分析均显示肽的三级结构不由于多重冻融循环而可观地改变。 Both analyzes showed the tertiary structure of the peptide due to multiple freeze-thaw cycles and does not appreciably change. 在类似的实验中,分析11次冻融循环对10mg/mL GLP-UpH 4. 0)二级结构的影响(图7C)。 In similar experiments, 11 freeze-thaw cycles Influence of 10mg / mL GLP-UpH 4. 0) secondary structure (FIG. 7C). 远-UV⑶阐明了肽的二级结构不因为多重冻融循环而显著改变。 It illustrates the far -UV⑶ peptide secondary structure because multiple freeze-thaw cycles and does not change significantly.

[0143] 总之,得自以上实验的生物物理学分析显示GLP-I肽的结构受其在溶液中浓度的强烈影响。 [0143] In summary, from the above experimental biophysical analysis shows the structure of GLP-I peptide is strongly influenced by its concentration in the solution. 当GLP-I的浓度被提高时,α-螺旋更加突出。 When the concentration of GLP-I is increased, α- helical more prominent. 另外,提高离子强度增强(在一些情况下产生)三级GLP-I结构。 Further, increasing the ionic strength enhanced (generated in some cases) three GLP-I structures.

[0144] 实施例2 [0144] Example 2

[0145] GLP-I/FDKP 吸附研究 [0145] GLP-I / FDKP Adsorption Studies

[0146] 通过进行吸附研究评价悬浮液中GLP-I与二酮哌嗪(DKP)颗粒的相互作用。 [0146] interaction of the particles by adsorption Evaluation suspension of GLP-I with diketopiperazine (DKP). 吸附研究中的变量探究了静电、氢键、水结构、蛋白质柔性和特异的盐-配对相互作用对GLP-I/ DKP相互作用的影响。 Adsorption of variables explored electrostatic, hydrogen bonding, water structure, protein flexibility, and specific salts - Effect of GLP-I / DKP interaction pairing interactions. 另外,测试了若干种常见的蛋白质稳定剂对GLP-I与DKP表面吸附的影响。 Further, we tested the effect of several common protein stabilizers on GLP-I adsorption to DKP surfaces.

[0147] 使用预先形成的DKP悬浮液颗粒(即FDKP),研究了GLP-I吸附至预先形成的DKP 颗粒表面的条件。 DKP suspension of particles [0147] using the previously formed (i.e., FDKP), studied the adsorption of GLP-I to DKP particle surfaces preformed condition. FDKP颗粒悬浮液(其中FDKP颗粒是预先形成的)与3Χ pH缓冲液和3Χ 添加剂或赋形剂的溶液组合。 And the combination with the FDKP particle suspension (wherein FDKP preformed particles) 3Χ pH buffer solution 3Χ additives or excipients. 最终溶液具有5, mg/ml的FDKP浓度和0. 25, mg/ml (5% w/w) 的GLP-I浓度。 The final solution has a 5, FDKP concentration mg / ml and 0. 25, mg / ml (5% w / w) of the GLP-I concentration. 上清液中未结合的GLP-I从悬浮液中滤除。 Supernatant Unbound GLP-I from the suspension was filtered. 用100, mM碳酸氢铵溶解FDKP 颗粒与结合的GLP-I蛋白质,并过滤以分离任何聚集的GLP-I蛋白质。 With 100, mM ammonium bicarbonate dissolved GLP-I with FDKP particles was protein bound, and filtered to separate any aggregated GLP-I protein. 通过HPLC定量上清液和重建的级分中的GLP-I量。 GLP-I and the amount of supernatant is quantified by HPLC fractions reconstruction. 进行一系列实验,其中使用的条件包括使用添加剂(例如盐、表面活性剂、离子、渗透物、离液齐U、感胶离子)和多种浓度的GLP-1。 A series of experiments in which conditions include the use of additives (such as salts, surfactants, ions, the permeate, from homogeneous solution U, lyotropic) and various concentrations of GLP-1. 得自这些研究的结果在下文描述。 The results from these studies are described below.

[0148] 盐研究。 [0148] Salt. -通过HPLC分析观察盐对GLP-I与FDKP颗粒结合的影响。 - To observe the effect of GLP-I with a salt FDKP particles bound analyzed by HPLC. 在存在0、 25、50、100、250、500、1000 和1500mM NaCl 时于5mg/mL FDKP 和0· 25mg/mL GLP-I 下进行GLP-1/FDKP颗粒的负载(图8A)。 At 5mg / mL FDKP and 0 · 25mg / mL GLP-I load GLP-1 / FDKP particles (FIG. 8A) in the presence of 0, 25,50,100,250,500,1000 and 1500mM NaCl. 还作为pH和NaCl浓度的函数,评价了在重建的无FDKP 对照样品中检测的GLP-I量(图8B)。 Also as a function of pH and NaCl concentration, we evaluated the amount of GLP-I (FIG. 8B) detected in the FDKP-free control samples reconstructed. 两组数据组中的pH均用20mM磷酸盐/20mM乙酸盐混合物控制。 pH group are two sets of data with 20mM phosphate / 20mM acetate mixture control.

[0149] 如在图8A中所观察到的,GLP-I对FDKP的最佳结合(吸附)受到悬浮液pH的强烈影响。 [0149] As observed in FIG. 8A, GLP-I is strongly affected by the pH of the suspension of FDKP optimal binding (adsorption). 在4和以上的pH下,溶液中GLP-1/FDKP比例为5% w/w时,观察到约3. 2%到约4%的GLP-I对FDKP的结合。 When at least 4 and pH, a solution of GLP-1 / FDKP ratio of 5% w / w, binding was observed from about 3.2% to about 4% of GLP-I to FDKP. 在存在0和25mM NaCl时,在pH 2.0下基本上没有明显的GLP-I对FDKP颗粒的吸附,但是用提高的离子强度观察到一些明显的负载。 0 and in the presence of 25mM NaCl, at pH 2.0 substantially no significant GLP-I adsorption on FDKP particles was observed but some apparent loading with increasing ionic strength. 在含> IM NaCl 的无FDKP对照中观察到GLP-I沉淀,图8B。 GLP-I precipitation was observed, in FIG. 8B containing> IM NaCl FDKP-free controls. 在彡IM NaCl下该明显的负载是GLP-I肽在高离子强度下可逆沉淀(盐析)的结果。 San IM NaCl in the load is a significant GLP-I peptide at high ionic strength reversible precipitation (salting out) of the result. 不含FDKP颗粒的高盐GLP-I对照也在重建的样品中显示高GLP-I水平,表明收集上清液时GLP-I已经被滤器捕获。 FDKP particles was high salt-free GLP-I control showed high levels of GLP-I samples are reconstructed, when the supernatant was collected indicates that GLP-I has been captured filter. 低于IM NaCl时,不存在FDKP颗粒的情况下没有GLP-I沉淀的迹象。 No sign of precipitated GLP-I lower than the case where the IM NaCl, the absence of FDKP particles.

[0150] 表面活件剂研究.-通过HPLC分析研究表面活性剂对GLP-I与FDKP颗粒结合的影响。 [0150] Research member surfactant agent by HPLC analysis .- Effects of Surfactants on GLP-I to FDKP particles was bound. 负载在存在表面活性剂时5mg/ml FDKP和0. 25mg/mL GLP-I下进行(图9A)。 Load (FIG. 9A) in the presence of a surfactant at 5mg / ml FDKP and 0. 25mg / mL GLP-I. 也作为pH和表面活性剂的函数评价了在重建的无FDKP对照样品中检测的GLP-I量(图9B)。 Also evaluated the amount of GLP-I (FIG. 9B) detected in the reconstituted FDKP-free control samples as a function of pH and surfactant. pH和对照样品条件与上面描述的用于离子强度研究的条件相同。 The same conditions as for the above ionic strength study described pH conditions and a control sample. 该研究中使用的表面活性剂包括:〇· 〇9mM 的fcij 78、0· OlmM 的Tween 80、0· 2mM 的Triton X、0. 12mM 的Pluronic F68、0. 9mM 的H(CH2)7S04Na、0. 9mM 的CHAPS、0. 9 mM 的Cetrimide。 Surfactants used in this study include: a square-〇9mM fcij 78,0 · OlmM of Tween 80,0 · 2mM of Triton X, Pluronic 0 12mM F68,0 9mM of the H (CH2) 7S04Na, 0 . 9mM of CHAPS, 0. 9 mM of Cetrimide. 存在每种表面活性剂时GLP-I的负载曲线作为pH的函数针对GLP-1/FDKP显示。 GLP-I load curve as a function of pH for the display of GLP-1 / FDKP the presence of each surfactant.

[0151] 数据显示针对GLP-1/FDKP颗粒的pH吸附曲线不受接近其临界胶团浓度(CMC)的表面活性剂的存在影响,所述临界胶团浓度即分开下述界限的小范围的浓度:在其以下事实上检测不到聚集物/胶团的界限,且在其以上事实上所有额外的表面活性剂分子均形成聚集物的界限。 [0151] Data show the effect of the presence of the surfactant adsorption curve for pH GLP-1 / FDKP particles is not close to its critical micelle concentration (CMC) of the critical micelle concentration i.e. below the limits of separate small-scale concentration: less than detection limit in fact aggregates / micelles in its less, and in fact all additional surfactant molecules are formed at the boundaries above its aggregates. 因此,还提示了任何这些表面活性剂可被用于最优化如下所述的稳定性和/或药物代谢动力学(PK)。 Thus, further suggesting the stability and / or pharmacokinetics of any of these surfactants can be used to optimize the following of the drug (PK). 如上文盐研究中可证实的,GLP-I与FDKP颗粒的相互作用受悬浮液的pH影响。 As described above salt demonstrable study, GLP-I interaction with FDKP particles was influenced by the pH of the suspension.

[0152] 离子研究。 [0152] Ion Research. 对该实验而言,进行两种不同的离子研究来测定离子对GLP-I与FDKP 颗粒结合的影响。 Two different studies to determine the effect of ions on the ion FDKP and GLP-I bound to the particles in terms of experiments performed. 在两种研究中,Cr为阳离子的平衡离子,Na+为阴离子的平衡离子。 In both studies, Cr cation counter ion, Na + is an anionic counter ion. 如先前实验所述进行GLP-1/FDKP颗粒的负载。 As previous experiments the load GLP-1 / FDKP particles. 如上文所述控制pH。 As described above, control pH. 在存在和不存在NaCl (其被用于更好地评价高离子强度的情况)时用pH3. 0、3. 5、4. 0或5. 0的pH缓冲液制备样品。 Samples were prepared with buffer pH pH3. 0,3. 5,4. 0, or 5.0 in the presence and NaCl (which is the case for better evaluation of high ionic strength) does not exist. 个体样品中如下包含其他离子:20或250mM的LiCl、20或250mM的NH4C1、20或250mM的NaF 和20 或250mM 的NaCH3COO15 Other individual sample comprises the following ions: NaF 20 or of 250mM LiCl, 20 or 250mM and 250mM of NH4C1,20 or 20 or 250mM of NaCH3COO15

[0153] 如图IOA所示,来自第一个离子研究的数据显示作为pH和离子的函数的GLP-I/ FDKP负载曲线。 [0153] As shown in FIG IOA, data from the first ion study are shown as a function of pH and ions of GLP-I / FDKP load curve. 在无NaCl时,20或250mM浓度的氟化物强烈地影响(增强)低pH下的吸附,其中250mM的NaF浓度显示与pH无关的最大的结合。 In the absence of NaCl, 20 or 250mM concentration of fluoride strongly influenced (enhanced) adsorption at low pH, NaF concentration of 250mM maximum binding to display pH-independent. 观察到该模式是由于溶液中的氟而不是钠,因为碳酸氢钠在20和250nM不具有相同的作用。 This pattern was observed due to the fluoride in the solution instead of sodium bicarbonate as not have the same effect at 20 and 250nM. 另外,这些作用不是样品中钠的结果,因为如图8中所示,类似浓度的盐不显示该作用。 Further, these effects are not sodium sample results because as shown in FIG. 8, a salt concentration similar to that role is not displayed. 存在IM NaCl时,所有的离子给出了高的"表观"负载。 The presence of IM NaCl, all ions are given the high "apparent" load. 对IM NaCl,而言该"表观"负载由存在高离子强度时GLP-I肽从溶液中盐析引起。 To IM NaCl, in terms of the "apparent" load caused by the GLP-I peptide salting out of solution in the presence of high ionic strength. 这在图IOB中进一步阐明,所述图IOB显示GLP-I存在于含IM NaCl的重建的无FDKP对照样品中。 This is further elucidated in FIG IOB, the IOB FIG display GLP-I present in the FDKP-free control samples containing IM NaCl in the reconstruction. 针对这些对照样品检测的GLP-I的量在更大的离子浓度下提高, 因为它们增加了样品中的总离子强度。 Increase in ion concentration for a larger amount of GLP-I detected in the control samples, because they increase the total ionic strength in the sample.

[0154] 在第二个离子实验(图10C)中,在存在20或250mM KC1、20或250mM咪唑、20或250mM Nal、或20或250mM NaPO4时制备GLP- 1/FDKP样品。 [0154] In the second ion experiment (FIG. 10C) in the presence of 20 or 250mM or 250mM KC1,20 imidazole, 250mM Nal or 20, prepared GLP- 1 / FDKP sample or 20 or 250mM NaP04 time. 数据显示250mM咪唑在存在IM NaCl时降低负载,250mM磷酸盐和250mM均给出高的"表观"负载(图10C)。 Reduce the load data 250mM imidazole in the presence of IM NaCl, 250mM and 250mM phosphate are given a high "apparent" load (FIG. 10C). 根据在OM 和IM NaCl浓度下重建的无FDKP对照样品中检测的GLP-I量(图10D),这些影响是由离子对GLP-I肽自身的影响而不是对肽与FDKP颗粒相互作用的影响引起。 (FIG. 10D), these effects according to the amount of GLP-I reconstituted FDKP-free control samples at OM and IM NaCl concentrations detected in the peptides themselves are affected by the plasma GLP-I peptide rather than affecting the interaction with FDKP particles cause. 磷酸钠和碘化钠引起不存在NaCl时一些GLP-I的盐析。 Sodium phosphate and sodium iodide caused some of salting GLP-I is the absence of NaCl. 另外,咪唑帮助将GLP-I溶解于IM NaCl样品中,从而给出更低的"表观"负载。 Additionally, imidazole helped to GLP-I was dissolved in IM NaCl samples, giving lower "apparent" load. 在250mM磷酸盐和碘化物的OM NaCl对照中也观察到了沉淀。 OM NaCl in 250mM phosphate and control of iodide precipitate was also observed.

[0155] 渗诱物研究。 [0155] Nitriding ATTRACTION. 也通过HPLC分析观察GLP-I对FDKP颗粒的结合。 Also observed that GLP-I binding to FDKP particles was analyzed by HPLC. 图IlA显示存在常见的稳定剂(渗透物)时作为pH函数的GLP-1/FDKP负载曲线。 FIG IlA display GLP-1 / FDKP load curve as a function of pH in the presence of common stabilizers (permeate). 如先前实验中所述进行GLP-1/FDKP颗粒的负载。 As in previous experiments the load GLP-1 / FDKP particles. 类似地,如上文所述控制pH。 Similarly, as described above control pH. 在pH 3. 0和存在20、50、100、150、 200或300mM的渗透物(稳定剂)时制备样品。 Samples were prepared in the presence of 3.0 when the permeate 20,50,100,150, or 200 of 300mM (stabilizer) pH. 渗透物为己二醇(Hex-Gly)、海藻糖、甘氨酸、PEG、TMAO、甘露醇或脯氨酸;N/A表示无渗透物。 The permeate is hexanediol (Hex-Gly), trehalose, glycine, PEG, TMAO, mannitol or proline; N / A indicates no permeate. 在类似的实验中,样品中渗透物(稳定齐U)的浓度被保持恒定在lOOmM,PH从2. O到4. O变化。 In a similar experiment, the concentration of the sample permeate (stable homogeneous U) is kept constant at lOOmM, PH from 2. O 4. O to change.

[0156] 研究的渗透物(稳定剂)均对GLP-I到FDKP表面的吸附没有可观的影响,无论pH 被维持在pH 3. 0而渗透物的浓度变化(图IlA ;左侧曲线)还是渗透物浓度被维持恒定在IOOmM而改变pH(图IlA ;右侧曲线)。 [0156] Study of the permeate (stabilizer) were adsorbed on the surface of the FDKP GLP-I to no appreciable effect, regardless of the pH is maintained at pH 3. 0 and concentration permeate (Fig IlA; left graph) or permeate concentration is maintained at a constant change IOOmM pH (FIG IlA; right graph). 在重建的无FDKP对照样品中未检测到GLP-I沉淀(图11B)。 In the reconstituted FDKP-free control samples was not detected in precipitated GLP-I (FIG. 11B). 这些渗透物可被用于最优化稳定性和/或药物代谢动力学。 The permeate can be used to optimize stability and / or pharmacokinetics.

[0157] 离液剂和感胶离子研究。 [0157] Research gum and ionic chaotrope sense. 研究影响水和蛋白质结构的离子种类(离液剂和感胶离子),确定这些因素在GLP-I对FDKP吸附中的作用。 Effect of ion species of water and protein structure (chaotropes and lyotropes), FDKP adsorption determine the role of these factors in the GLP-I pair. 如先前的实验中所述进行GLP-I/ FDKP颗粒的负载。 As in the previous experiments, the load GLP-I / FDKP particles. 类似地,如上文所述控制pH。 Similarly, as described above control pH. 在pH 3. 0和存在0、20、50、100、150、200或300mM下述离液剂或感胶离子时制备样品,所述离液剂或感胶离子为:NaSCN、CsCl、Na 2S04、 (CH3)3N-HCU Na2NO3、柠檬酸钠和NaClO4。 PH 3. 0 and in the presence of the following samples were prepared 300mM 0,20,50,100,150,200 or when chaotropes or lyotropes, a chaotrope or lyotrope as: NaSCN, CsCl, Na 2S04 , (CH3) 3N-HCU Na2NO3, sodium citrate, and NaClO4. 在类似的实验中,样品中离液剂或感胶离子的浓度被保持恒定在lOOmM,PH从2. 0到4. 0变化。 In a similar experiment, the concentration of the sample from the lyotropic liquid or feeling is kept constant at lOOmM, PH from 2.0 to 4.0 change.

[0158] 图12A显示作为pH和离液剂和/或感胶离子的函数的GLP-1/FDKP负载曲线。 [0158] FIG. 12A shows GLP-1 / FDKP loading curve as a function of pH and chaotrope and / or lyotropic ions. 在低pH( < 3)时,对于分析的不同离液剂发生了负载中的显著变化,特别是在较高的离液剂浓度下。 At low pH (<3) while, for the different chaotropes analyzed undergone significant changes in the load, especially at higher chaotrope concentrations. 然而在PH 4时,未观察到变化(图12C)。 However, when PH 4, no change was observed (FIG. 12C). 因此,这些试剂显示在不利的较低pH下促进GLP-I对FDKP颗粒的结合,但是在对结合有利的较高pH条件下具有非常少的影响。 Thus, these agents shown to promote binding of GLP-I to FDKP particles at unfavorable lower pH, but have very little impact at the higher pH conditions of binding advantageous. 来自重建的无FDKP对照样品的数据提示在pH 3.0下观察到的负载改变部分归因于特定的离液剂在多种程度上影响GLP-I肽的盐析(沉淀)(图12B和12D)。 Data FDKP-free control samples from the reconstructed tips observed at pH 3.0 in part due to the load change from the specific liquid salting affect GLP-I peptide in a variety of extent (precipitate) (FIGS. 12B and 12D) . 这对强离液剂如NaSCN 和NaClO4而言是显著的。 This strong chaotropes such as NaSCN and NaClO4 terms are significant.

[0159] 有机物研究。 [0159] Studies of organic matter. 评价下述醇来确定螺旋构象在GLP-I对FDKP的吸附中所起的作用, 所述醇已知通过提高氢键的键合强度在无组织的肽中诱导螺旋构象。 Evaluation is determined by the following alcohol helical conformation in GLP-I adsorption to FDKP role in the alcohol are known to induce helical conformation in unstructured peptides by increasing hydrogen-bonding strength. 如先前实验中所述进行GLP-1/FDKP颗粒的负载。 As in previous experiments the load GLP-1 / FDKP particles. 类似地,如上文所述控制pH。 Similarly, as described above control pH. 在pH 2· 0、3· 0、4· 0和5. 0下观察每种醇的作用。 Effect of pH 2 · 0,3 · 0,4 · 0 and the observation of each 5.0 ol. 使用的醇为:甲醇(MeOH)、乙醇(EtOH)、三氟代乙醇(TFE)或六氟异丙醇(HFIP)。 Alcohols used were: methanol (MeOH), ethanol (EtOH), trifluoroethanol (TFE) or hexafluoroisopropanol (HFIP). 在5%、10%、15%和20% v/v的浓度下评价每种醇。 5%, 10%, evaluated for each alcohol at a concentration of 15% and 20% v / v of.

[0160] 图13A显示对每种浓度的每种醇而言作为pH函数的GLP-1/FDKP负载曲线。 [0160] FIG. 13A shows each concentration of each alcohol in terms of GLP-1 / FDKP load curve as a function of pH. 在pH 3. 0下,低浓度的HFIP (5% )导致高吸附,如GLP-I对FDKP颗粒的质量比所证实的那样。 At pH 3. 0, the low concentration of HFIP (5%) results in a high adsorption, such as GLP-I to FDKP particle mass ratio demonstrated. 只有最强的H-键增强(成螺旋)醇HFIP对缓冲的悬浮液中的吸附具有影响。 Only the strongest bond reinforcing H- (in a spiral) HFIP alcohol has an influence on the buffered suspension adsorption. 在更高浓度的HFIP(20%)下,GLP-1/FDKP吸附被抑制。 At higher concentrations of HFIP (20%), GLP-1 / FDKP adsorption was inhibited. 图13B显示在20%的醇浓度下,在重建的无FDKP 对照样品中未记录到显著的GLP-I沉淀。 Figure 13B shows at an alcohol concentration of 20%, the reconstituted FDKP-free control samples unrecorded significant precipitation of GLP-I.

[0161] 这提示药物的构象柔性(即能够形成的FDKP-接触物的熵和数量)可在吸附中起作用。 [0161] This suggests that conformational flexibility of a drug (FDKP- entropy and the number thereof, i.e. the contact can be formed) may play a role in adsorption. 数据提示H-键合在上述条件下GLP-I与FDKP表面的相互作用中可起作用。 Data suggests that H- bonding interactions GLP-I with FDKP surfaces can function under the conditions described above. 基于该数据,还推测如果H-键合作为FDKP-GLP-I相互作用中主要的和一般的力作用,则应期待更多和更强的作用。 Based on this data, also speculated that if the H- key cooperation and interaction in the major general of force FDKP-GLP-I, you should expect more and stronger role.

[0162] 浓度研究。 [0162] Studies concentration. -在多种GLP-I浓度下研究了GLP-I对FDKP颗粒表面的吸附。 - Study of the GLP-I adsorption to FDKP particle surfaces at various GLP-I concentrations. 图14A 显示多种PH下作为GLP-I浓度函数的来自GLP-1/FDKP的负载曲线。 14A shows the load curve from GLP-1 / FDKP as a function of the concentration of GLP-I at various PH. GLP-I浓度为0. 15、 0· 25、0· 4、0· 5、0· 75、1· 0、1· 5、2· 0、5· 0 或10mg/mL。 GLP-I concentration of 0.15, 0.5 · 25,0 75,1 · 4,0 · 5,0 · 0,1 · 5,2 · 0,5 · 0 or 10mg / mL. 样品的pH 为2· 5、3· 0、3· 5、4· 0、4· 5 或5. 0〇 and a pH of 2 · 5,3 · 0,3 · 5,4 · 0,4 · 5 or 5. 0〇

[0163] 当FDKP浓度被保持恒定在5mg/mL而GLP-1浓度提高时,观察到FDKP颗粒上GLP-I负载的提高。 [0163] When the FDKP concentration was held constant at 5mg / mL and the GLP-1 concentrations increase, FDKP particles was observed to increase the GLP-I load. 在pH 4下当GLP-I的浓度为10mg/mL时,观察到FDKP颗粒上接近20% 的GLP-I吸附。 At pH 4 when the concentration of GLP-I to 10mg / mL, was observed nearly 20% of the GLP-I adsorption on FDKP particles. 惊人的是,在高浓度GLP-I下未观察到FDKP颗粒上负载的GLP-I吸附饱和。 Surprisingly, the load on FDKP particles was observed at high concentrations of GLP-I GLP-I adsorption saturation. 该观察结果可能归因于GLP-I自身缔合成为多层体。 This observation may be attributed to self-associated GLP-I is a synthetic multilayer body.

[0164] 通过扫描电子显微镜(SEM)对GLP-1/FDKP制剂形态学的分析显示GLP-1/FDKP颗粒作为晶体或平板样结构存在,其能够形成包含多于一个GLP-1/FDKP颗粒的聚集物(图14B)。 [0164] by a scanning electron microscope (SEM) analysis of the GLP-1 / FDKP formulations show morphological GLP-1 / FDKP particles as a crystal or a tablet-like structures are present, which can be formed comprising more than one GLP-1 / FDKP particles aggregates (FIG. 14B). 通过冻干溶液制备这些制剂,所述溶液含有:(图A)0. 5mg/mL GLP-I和2. 5mg/mL FDKP ;(图B) 0· 5mg/mL GLP-I 和10mg/mL FDKP ;(图C) 20mM 氯化钠、20mM 乙酸钾和20mM 磷酸钾,pH 4. 0 中0· 5mg/mL GLP-I 和10mg/mL FDKP ;和(图D)20mM 氯化钠、20mM 乙酸钾和20mM 磷酸钾,pH 4. 0 中10mg/mL GLP-I 和50mg/mL FDKP These formulations are prepared by lyophilization from solution, the solution contains Fig :( A) 0 5mg / mL GLP-I and 2. 5mg / mL FDKP;. (FIG. B) 0 · 5mg / mL GLP-I and 10mg / mL FDKP ; (FIG. C) 20mM NaCl, 20mM and 20mM potassium phosphate, potassium acetate, pH 4. 0 of 0 · 5mg / mL GLP-I and 10mg / mL FDKP; and (D FIG) 20mM NaCl, 20mM potassium acetate and 20mM potassium phosphate, pH 4. 0 of 10mg / mL GLP-I and 50mg / mL FDKP

[0165] 结果概沭 [0165] The results are summarized Shu

[0166] 总之,对GLP-I与FDKP颗粒相互作用的吸附研究显示GLP-I以pH-依赖性的方式与DKP颗粒表面结合,在pH 4或之上具有高吸附。 [0166] In summary, Adsorption of GLP-I to FDKP particle interaction display GLP-I in combination with pH- dependent manner the DKP particle surfaces, with high adsorption at 4 or above pH. 发现DKP颗粒表面对GLP-I的吸附受到pH最强影响,在pH 2. 0时基本没有吸附,在pH > 4. 0时有重大的相互作用。 DKP particle surfaces was found by the strongest effect of pH on the adsorption of GLP-I, substantially no pH 2. 0 adsorption at pH> 4. 0 when there is a significant interaction. 根据观察, 在低PH下钠和氟离子增强吸附。 The observed at low PH sodium and fluoride ions enhanced adsorption. 其他添加剂如表面活性剂和常用的稳定剂仅对GLP-I与FDKP颗粒表面的吸附具有轻微影响。 Other additives such as surfactants, and common stabilizers adsorbed only to GLP-I with FDKP particle surfaces having a slight effect.

[0167] 另外,GLP-I自身的特性影响这些实验的结果。 [0167] In addition, GLP-I own characteristics affect the results of these experiments. 发现GLP-I的行为是非典型并且惊人的,因为未观察到饱和吸附,这归因于高浓度下GLP-I的自身缔合。 GLP-I was found behavior is atypical and surprising, because the saturation adsorption was not observed, due to self-associated GLP-I at high concentration. 高浓度下GLP-I的自身缔合允许可能的多层GLP-I肽对DKP颗粒的包裹,从而促进更高百分比的GLP-I肽负载。 At high concentrations of GLP-I self-association may allow a multilayer wrapping GLP-I peptide DKP particles, thereby promoting a higher percentage of GLP-I peptide loading. 该惊人的自身缔合性质证实在制备稳定的GLP-I施用形式中是有益的。 The striking association properties justify their beneficial in preparing stable GLP-I administration in the form. 另外,GLP-I的自身缔合构象可以能够减少或延迟其在血中的降解。 Further, GLP-I self-associated conformation may be able to reduce or delay its degradation in blood. 然而,注意到当操作缔合的GLP-I时必须小心,因为其对温度和高pH是敏感的。 However, it is noted when the operation of the association must be careful when GLP-I since it is sensitive to temperature and high pH.

[0168] 实施例3 [0168] Example 3

[0169] GLP-1/FDKP制剂的完整件分析 Full member Analysis [0169] GLP-1 / FDKP formulations

[0170] 以来自实施例1和2中实验的结果为基础,选择了具有表1中所述特征的一系列GLP-I制剂用于本文讨论的细胞存活力测定。 [0170] The results from Examples 1 and 2 is based on experimental embodiment, a selected series of GLP-I formulations having the characteristics in Table 1 for the cell viability assay as discussed herein. 大部分制剂含有GRAS ("一般被认为安全的") 赋形剂,但是一些被选择以允许研究稳定性和吸附之间的关系。 Most formulations contain GRAS ( "generally regarded as safe") excipients, but some were selected to allow the study of the relationship between stability and adsorption.

[0171] 表L 针对完整性相分析(Integrity Phase Analysis)选择的GLP-1/FDKP。 [0171] Table L for Integrity Phase Analysis (Integrity Phase Analysis) selected GLP-1 / FDKP.

[0172] [0172]

Figure CN104288756AD00261

[0173] 另外,以实施例1和2中获得的结果为基础,还选择了一系列制剂用于GLP-l/FDKP 的相II完整性研究。 [0173] Further, the results of Examples 1 and 2 obtained on the basis, a series of further formulation selected GLP-l / FDKP for phase II integrity of the study. 下表2显示选择用于相II完整性的六种GLP-I制剂。 Table 2 below shows selected for the six kinds of GLP-I formulation phase II integrity. 制备粉末后, 将它们与空白的FDKP掺合,在每种制剂中产生类似的大量GLP-I肽和FDKP。 After preparing a powder, they were blended with blank FDKP, produce a similar large number of GLP-I peptide and FDKP in each formulation.

[0174] 表2.针对II期完整性选择的GLP-1/FDKP制剂。 [0174] Table 2. Phase II for the integrity of the selected GLP-1 / FDKP formulations. 从20mM NaCl和pH 4. 0缓冲液中10mg/ml GLP-I制成的制剂被描述为盐-缔合制剂。 From 20mM NaCl and buffer pH 4. 0 10mg / ml GLP-I formulation was prepared as described salt - association formulation.

[0175] [0175]

Figure CN104288756AD00262

[0176] 通过HPLC分析胁迫对表2中GLP-1/FDKP制剂的影响(图15)。 Stress on Table 2. GLP-1 / FDKP formulations (FIG. 15) [0176] analyzed by HPLC. 含负载在H2O中5%、10%或20% GLP-1/FDKP 的样品;或负载在NaCl+pH4.0 缓冲液中5%或10% GLP-I/ FDKP的样品在40°C孵育10天。 Sample load containing 5% H2O, 10%, or 20% GLP-1 / FDKP; a load in a sample or NaCl + pH4.0 buffer, 5% or 10% GLP-I / FDKP was incubated at 40 ° C 10 day. HPLC色谱图证实GLP-I肽以相同的滞留时间洗脱并且不存在降解峰。 HPLC chromatograms confirm GLP-I peptide was eluted at the same retention time and no degradation peaks exist. 另外,MS分析产生对所有样品而言类似的质量3297g/mol,表明该质量对所有分析的样品而言是均一的。 Further, the MS analysis yielded a similar mass of 3297g / mol for all samples, indicating that the quality of the samples analyzed in terms of all is uniform. 数据显示冻干前GLP-I对Π )ΚΡ颗粒和溶液中存在其他成分的质量-质量比例。 Data show that GLP-I pre-lyophilization of the presence of other ingredients ΚΡ Π particles in solution and mass) - mass ratio. 总之,GLP-1/FDKP制剂显示对胁迫是稳定的。 Overall, GLP-1 / FDKP formulations showed to be stable to stress.

[0177] 实施例4 [0177] Example 4

[0178] 在肺灌洗液中孵育的GLP-I的稳定件 [0178] incubated in lung lavage fluid of the GLP-I stabilizer

[0179] 考虑到在生物流体中发现二肽基-肽酶IV(DPP-IV)切割并灭活GLP-I,分析生物流体(如肺流体和血)中GLP-I的稳定性。 [0179] Considering the biological fluids found in the dipeptidyl - peptidase IV (DPP-IV) cleavage and inactivation of GLP-I, the stability of a biological fluid analysis (such as lung fluid and blood) of the GLP-I.

[0180] 二肽基-肽酶IV(DPP-IV)是一种细胞外膜结合的丝氨酸蛋白酶,其在若干种细胞类型(尤其是⑶4+T细胞)的表面上表达。 [0180] dipeptidyl - peptidase IV (DPP-IV) is a cell membrane bound serine protease expressed in the cell type (especially ⑶4 + T cells) on the surface of several species. 还在血和肺流体中发现了DPP-IV。 Blood and lung fluid also found DPP-IV. DPP-IV已经涉及葡萄糖代谢的控制,因为其底物包括促胰岛素激素GLP-1,所述GLP-I通过其N-端氨基酸的去除而被灭活;见图16A。 DPP-IV has been implicated in the control of glucose metabolism because its substrates include the insulinotropic hormone GLP-1, a GLP-I amino acid by removal of its N- terminal to be inactivated; Figure 16A. DPP-IV切割人GLP-UGLP-I (7-36))的主要循环形式的Ala-Glu键,释放N-末端的两个残基。 Major circulating form of human DPP-IV cleavage GLP-UGLP-I (7-36)) of the Ala-Glu bond, releasing the N- terminal two residues. DPP-IV显示通过降解GLP-I从而降低肠降血糖素对胰腺β-细胞的作用来负调节葡萄糖处理。 DPP-IV degradation of GLP-I through the display to reduce the incretin effect on pancreatic β- cells to negative regulation of glucose disposal.

[0181] 进行一些研究测定存在抑肽酶或DPP-IV抑制剂时大鼠血和肺流体中的GLP-I降解。 [0181] There are some studies for determination of GLP-I in rat blood and lung fluid degradation during aprotinin or DPP-IV inhibitors. 向收集后的样品中以1、2、3、4和5TIU/ml添加天然存在的丝氨酸蛋白酶抑制剂抑肽酶, 其在本领域已知抑制蛋白质降解。 1,2, 3,4 and 5TIU / ml serine protease inhibitor aprotinin naturally occurring after the sample collection, which is known in the art to inhibit protein degradation. 然后通过检测发光底物的切割测量DPP-IV活性,所述底物含有DPP-IV识别的Gly-Pro序列。 DPP-IV activity was then measured by detecting the cleavage of a luminescent substrate, said substrate comprising DPP-IV recognized Gly-Pro sequence. 将支气管肺洗出液与前-荧光底物孵育30分钟, 并通过发光检测切割产物。 The former eluate bronchopulmonary and - a fluorogenic substrate for 30 min, and the cleavage product was detected by luminescence.

[0182] 如在具有递增的抑肽酶浓度的多种生物流体(如本文所述)中通过肽降解的抑制所检测的,数据显示DPP-IV活性抑制的提高(图16B)。 [0182] The inhibition by peptide degradation in various biological fluids (as described herein) with increasing aprotinin concentration in the detected data showed an increase (FIG. 16B) DPP-IV inhibition activity. 向收集后样品中以1.25、2. 5、5、10 和20 μ Ι/ml添加DPP-IV抑制剂观察到类似的结果(图16C)。 After the sample was collected to 1.25,2. 5,5,10 and 20 μ Ι / ml was added to the DPP-IV inhibitor Similar results were observed (FIG. 16C). 样品收集后抑制剂的添加允许更精确的样品评价。 More accurate evaluation of the sample after allowing the sample collection inhibitor added.

[0183] 还使用捕获ELISA mAb (其识别GLP-I氨基酸7-9)在肺洗出液中检查了GLP-I的稳定性。 [0183] Also using a capture ELISA mAb (which recognizes GLP-I amino acids 7-9) eluted in lung fluid check the stability of GLP-I. GLP-I在肺洗出液(LLF)中孵育2、5、20和30分钟。 GLP-I in the lung eluate (the LLF) 2,5,20 and incubated for 30 min. 如图17所示,孵育条件为:1 或10 μ g (w/w) LLF和1或10 μ g (w/w) GLP-I。 17, the incubation conditions were: 1 or 10 μ g (w / w) LLF and 1 or 10 μ g (w / w) GLP-I. 单独在LLF中未检测到GLP-I。 LLF alone is not detected in GLP-I. 使用多种浓度的LLF和GLP-I的组合,存在可与单独的GLP-I相比的高GLP-I检测,表明在肺洗出液中GLP-I是随时间稳定的(图17)。 Using a combination of various concentrations of LLF and GLP-I, GLP-I high presence detector indicates eluate GLP-I is stable over time (FIG. 17) alone in the lung compared to GLP-I. 在类似的研究中证实了未稀释的肺洗出液中GLP-I的稳定性;标注了20分钟时70-72%的GLP-I完整性(数据未示出)。 Similar studies demonstrated the undiluted lung lavage fluid stability of GLP-I; marked 70-72% of GLP-I integrity (data not shown) at 20 minutes.

[0184] 另外,检查了大鼠血浆中GLP-I的稳定性。 [0184] Further, examined in rat plasma stability of GLP-I. 血浆得自不同的大鼠(如图例中血浆1和血浆2所示)并被1:2或l:10(v/v)稀释。 Plasma was obtained from various rats (as an illustration of plasma and plasma 2) and 1: 2 or l: 10 (v / v) dilution. 向ΐομΐ血浆或PBS中添加1毫克GLP-1。 Was added to 1 mg of GLP-1 ΐομΐ plasma or PBS. 将样品在37°C孵育5、10、30或40分钟。 Samples were incubated at 37 ° C 5,10,30 or 40 minutes. 在冰上终止反应并添加0. IU的抑肽酶。 The reaction was stopped on ice and adding 0. IU of aprotinin. 数据显示在测试的所有时间点1:2和1:10的血浆稀释液中高浓度的GLP-I (图18A和18B)。 Data show that at all time points tested 1: 2 and 1:10 dilutions of plasma in high concentrations of GLP-I (FIG. 18A and 18B). 总之, 数据表明GLP-I在发现了丝氨酸蛋白酶DPP-IV的肺洗出液和血浆中均是惊人的稳定的。 Overall, the data indicate that GLP-I was found in the lungs of the serine protease DPP-IV eluate and plasma are amazing stability.

[0185] 实施例5 [0185] Example 5

[0186] GLP-I分子对细朐凋亡和细朐增葙的影响 [0186] Effects of GLP-I molecules and fine fine Qu Qu increased apoptosis of the suitcase

[0187] 为了检查GLP-I是否抑制细胞凋亡,进行筛选测定来确定GLP-I对β -细胞死亡抑制的影响。 [0187] To examine whether GLP-I inhibits apoptosis, screening assays for determining GLP-I β - cell death suppressing effect. 用〇、2、5、10、15或20ηΜ浓度的GLP-I将大鼠胰腺上皮(ARIP)细胞(用作胰腺β -细胞模型;购自ATCC,Manassas, VA)预处理10分钟。 A square, or 2,5,10,15 20ηΜ the concentrations of GLP-I rat pancreatic epithelial (ARIP) cells (used as a pancreatic β - cell model; purchased from ATCC, Manassas, VA) pretreated for 10 min. 然后使细胞未处理或用5 μ M 十字孢碱(细胞凋亡诱导物)处理4. 5小时。 The cells were then untreated or treated with 5 μ M staurosporine (an inducer of apoptosis) 4.5 hours. 使用Cell Titer-Glo™(Promega,Madison, WI) 评价细胞存活力。 Using the Cell Titer-Glo ™ (Promega, Madison, WI) Evaluation of cell viability. 在十字孢碱处理的细胞中,用高达IOnM的GLP-I浓度提高记录到细胞死亡百分比的降低(图19A) In staurosporine treated cells with high concentrations of GLP-I IOnM the recording to reduce the percentage increase cell death (FIG. 19A)

[0188] 使用锚定蛋白V染色通过FACS分析确定了GLP-I对细胞凋亡的影响的进一步检查。 By FACS analysis to determine the influence of further examination GLP-I on apoptosis Annexin V staining [0188] Using anchored. 锚定蛋白V染色是检测凋亡细胞的一个有用工具,并是本领域技术人员公知的。 Annexin V staining is a useful tool in detecting apoptotic cells and is well known to those skilled in the art. 锚定蛋白与细胞膜的结合允许在与细胞凋亡相关的形态学改变发生之前和膜完整性丧失之前分析磷脂(PS)不对称的改变。 Anchoring protein binding to the cell membrane permits analysis of the phospholipids (PS) asymmetry before the change and before membrane integrity loss associated with apoptosis morphological changes. 因此,在用15nm GLP-1、1 μ M十字孢碱4小时、1 μ M十字孢碱+15nmGLP-l或既无十字孢碱又无GLP-I (实验对照)处理的细胞中测定GLP-I对细胞凋亡的影响。 Thus, cell 1 μ M staurosporine + 15nmGLP-l or neither staurosporine nor GLP-I (experimental control) treated with GLP- measured 15nm GLP-1,1 μ M staurosporine 4 hours I on apoptosis. 数据显示GLP-I将十字孢碱诱导的细胞凋亡抑制约40% (图19B)。 The data shows that GLP-I inhibited by about 40% (FIG. 19B) staurosporine induced apoptosis.

[0189] 使用GLP-I类似物毒蜥外泌肽-4观察到类似的细胞凋亡抑制结果,所述毒蜥外泌肽-4以类似GLP-I的方式与GLP-I受体结合。 [0189] A GLP-I analog exendin -4 drug outer lizard similar to that observed apoptosis suppression result, the exendin peptide -4 GLP-I in a similar manner to GLP-I receptor binding. 在存在0、10、20或40nM毒蜥外泌肽时用5 μ M十字孢碱将ARIP细胞分别处理16、24或48小时。 The ARIP cells were treated for 16, 24 or 48 hours with 5 μ M staurosporine in the presence of 0, 10 or 40nM exendin peptide. 数据(图20)显示在IOnM时,毒蜥外泌肽对抑制细胞凋亡是完全无效的,因为100%细胞死亡。 Data (FIG. 20) displayed when IOnM, exendin peptide inhibition of apoptosis completely ineffective, as 100% cell death. 在20和40ηΜ下,毒蜥外泌肽相同程度抑制细胞凋亡,存在40ηΜ毒蜥外泌肽-4时48小时约50%抑制。 And 40ηΜ at 20, to the same extent exendin peptide inhibits apoptosis is present from about -4 at 48 hours 50% inhibition of toxic 40ηΜ exendin outer lizard.

[0190] 实施例6 [0190] Example 6

[0191] 候诜物GLP-1/FDKP制剂对细朐死亡的影响 Effects [0191] Shen was waiting GLP-1 / FDKP formulations on fine Qu death

[0192] 进行基于细胞的测定实验来评价GLP-1/FDKP制剂(如实施例3、上表1中公开的)抑制细胞死亡的能力。 [0192] Cell-based assay to evaluate experimental GLP-1 / FDKP formulation (as in Example 3, disclosed in the above table 1) inhibit cell death. 这些GLP-1/FDKP颗粒制剂处于悬浮液中或被冻干。 These GLP-1 / FDKP particle formulations in suspension or lyophilized. 分析制剂在ARIP细胞中抑制十字孢碱诱导的细胞死亡的能力。 Ability to staurosporine-induced cell death inhibition analysis of formulations in ARIP cells. 用GLP-I样品预处理的ARIP细胞被暴露于5 μ M十字孢碱中4小时,并用Cell Titer-Glo™(Promega,Madison, WI)分析以确定细胞存活力。 ARIP cells with GLP-I samples were exposed to pretreated 5 μ M staurosporine for 4 hours, and treated with Cell Titer-Glo ™ (Promega, Madison, WI) to determine cell viability analysis.

[0193] 将多种GLP-1/FDKP制剂的样品不胁迫或在4°C或40°C下胁迫4周。 [0193] The sample was more GLP-1 / FDKP formulations or not stress or at 4 ° C for 40 ° C 4 circumferential stress. 在基于细胞的测定法中以45nM使用每种GLP-1/FDKP样品,来确定它们抑制十字孢碱诱导的细胞死亡的能力。 In a cell-based assay using each to 45nM GLP-1 / FDKP samples to determine their ability to staurosporine-induced cell death inhibition. 右侧所示对照样品阐明单独的培养基中,单独含GLP-I的、单独含十字孢碱的、或存在GLP-I和十字孢碱二者的培养基中细胞的存活力(注意:图例不适用于对照样品。每条代表独立的一式三份)。 As shown in the right side of the control sample set forth in medium alone, containing the GLP-I alone, containing staurosporine alone, or in the presence of both the viability of the culture medium GLP-I cells and staurosporine (note: Legend does not apply to the control samples. each bar represents a separate triplicate). 所有所示的结果是一式三份进行的平均值。 All results shown are the average of triplicate performed.

[0194] 数据显示所有胁迫的GLP-1/FDKP冻干制剂抑制十字孢碱诱导的细胞死亡(图21)。 [0194] Data show all Stress GLP-1 / FDKP lyophilized formulations inhibiting staurosporine-induced cell death (FIG. 21). 然而,许多GLP-1/FDKP悬浮液制剂不抑制细胞死亡。 However, many GLP-1 / FDKP suspension formulations does not inhibit cell death.

[0195] 实施例7 [0195] Example 7

[0196] GLP-1/DKP颗粒的肺吹入法 [0196] GLP-1 / DKP particles pulmonary insufflation

[0197] 为了检查GLP-1/FDKP的药物代谢动力学,在雌性Sprague Dawley大鼠中评价GLP-I的血浆浓度,所述雌性大鼠通过静脉(IV)注射或肺吹入法施用了多种GLP-1/FDKP制齐[J。 [0197] To examine the pharmacokinetics of GLP-1 / FDKP drug plasma concentrations of GLP-I evaluated in female Sprague Dawley rats, female rats the pulmonary insufflation or injection administered by multiple intravenous (IV) species GLP-1 / FDKP homogeneous system [J. 在初步的研究中,使用占GLP-1/FDKP颗粒制剂约4%和16% (w/w)的GLP-1。 In preliminary studies, the use of accounting GLP-1 / FDKP particle formulations of about 4% and 16% (w / w) of the GLP-1. 大鼠被随机化为12组,其中第1、4、7和10组接受通过肺液体滴注或IV注射施用的GLP-I溶液。 Rats were randomized into 12 groups, wherein the groups 1,4,7 and 10 receiving by pulmonary liquid instillation or IV injection administered GLP-I solution. 第2、5、8和11组接受通过肺吹入法或IV注射施用的GLP-1/FDKP盐结合的制剂(图表2 中公开的)。 5, 8 and 11 receiving group by pulmonary insufflation or IV injection administered GLP-1 / FDKP salt binding agents (disclosed in Figure 2). 第3、6、9、12组接受通过肺吹入法或IV注射施用的GLP-1/FDKP盐结合的混合制剂。 The first group received 3,6,9,12 by pulmonary insufflation or IV injection administered GLP-1 formulation mixed / FDKP salt bound. GLP-1/FDKP制剂是约16%负载的盐结合的制剂。 GLP-1 / FDKP formulations are salts of binding of about 16% load. 为了达到约4%的负载,将16%的制剂与DKP颗粒在3:1的混合物中掺合。 In order to achieve about 4% load, 16% of the particles DKP formulation 3: 1 mixture blended. 对0. 08mg的总GLP-I剂量而言,肺吸入法或静脉注射为0· 5或2. Omg的颗粒(分别为16 %或4% GLP-I负载)。 Total GLP-I dose of 0. 08mg terms, pulmonary inhalation or intravenous injection is 2. Omg 0 · 5 or particles (16% or 4% GLP-I load, respectively).

[0198] 在独立的动物组(第7-12组)中,在第2天重复施用。 [0198] In a separate group of animals (Groups 7-12) was repeated on day 2 of administration. 对第1、4、7和10组施用80 μ g GLP-I溶液。 10 and 80 μ administered to group 1,4,7 g GLP-I solution. 对第2、5、8和11组施用GLP-1/DKP盐结合的制剂(〜16% GLP-I负载)。 5, 8 and 11 formulations group administered GLP-1 / DKP salt binding (~16% GLP-I load). 第3、6、9、12组接受GLP-1/DKP盐结合的掺合制剂(〜4% GLP-I负载)。 The first group received 3,6,9,12 blend formulation GLP-1 / DKP salt binding (~4% GLP-I load).

[0199] 使用相同的制剂将实验进行两次,在连续的两天中给药和收集血。 [0199] The same experiment was performed twice formulations, administration and blood was collected in two consecutive days. 对每组而言在给药当天,在给药前(时间0)、给药后2、5、10、20、30、60和120分钟取血样。 For each group, the administration day, prior to dosing (time 0), and 120 minutes after administration 2,5,10,20,30,60 blood samples are taken. 在每个时间点,将来自尾缘静脉的约150 μ L全血收集在含约3U/mL抑肽酶和0. 3 % EDTA的cyro-vial 管中,倒置并储存于冰上。 At each time point, approximately 150 μ L whole blood was collected from the tail vein containing from about 3U / mL aprotinin and 0. 3% cyro-vial tube in EDTA, inverted and stored on ice. 将血样在4000rpm离心并将40 μ 1血楽用移液管移至96-孔平板中,所述平板被储存于-80°C,之后按照制造商的推荐(Linco Research, St Charles, MO)通过ELISA分析GLP-I水平。 Blood samples were centrifuged at 4000rpm and 40 μ 1 with blood yue pipetted into 96-well plates, the plates are stored at -80 ° C, then in accordance with the manufacturer's recommendations (Linco Research, St Charles, MO) by ELISA analysis of GLP-I level. 测定了最佳条件是只存在血清(5% FBS)而无基质的情况下当测定缓冲液为GLP-I时。 When measuring the optimum conditions are present only serum (5% FBS) as measured when no substrate buffer was GLP-I.

[0200] 静脒施用:第5、6、10、11和12组静脉(IV)接受了多种GLP-1/FDKP制剂和GLP-I 溶液(图22A)。 [0200] Static amidine administration: 5,6,10,11 and 12 groups of intravenous (IV) received various GLP-1 / FDKP formulations and GLP-I solution (FIG. 22A). 第5组和第6组被施用给15. 8% GLP-1/FDKP,第11和12组在相连的一天被施用给15. 8% GLP-1/FDKP的另一种剂量;第10组被施用给作为对照的GLP-I溶液。 Group 5 and Group 6 was administered to a 15. 8% GLP-1 / FDKP, day was administered 12 and 11 in the group is connected to a further dose 15. 8% GLP-1 / FDKP; a second group 10 It is administered to a GLP-I as a control solution. 在第0、2、5、10、20、40、60、80、100和120分钟的时间点检测GLP-1/FDKP的浓度。 Detecting the concentration of GLP-1 / FDKP at its 0,2,5,10,20,40,60,80,100 and 120 min time point. 所有的组在静脉施用后显示GLP-I血浆水平的可检测提高,在处理后2分钟观察到最大的浓度。 All GLP-I group display a detectable increase plasma levels after intravenous administration, it was observed after 2 minutes to a maximum concentration. 对所有组而言处理后20分钟活性GLP-I的血浆水平返回背景水平。 It returns the background level of all the groups in terms of the plasma levels after 20 minutes of active GLP-I. 通过静脉注射施用时,这些多种GLP-1/FDKP制剂和GLP-I溶液的动力学未观察到显著差异。 When administered by intravenous injection, these various GLP-1 / FDKP formulations and kinetics GLP-I was not observed significant differences. 注意到在通过静脉注射处理的大鼠中,给药后10-20分钟GLP-I的血浆水平回复至基线水平,提示生理动力学(即约95%的GLP-I在10分钟内被消除)。 Note that in rats treated by intravenous injection, 10 to 20 minutes after administration the plasma levels of GLP-I returned to baseline levels, suggesting physiological kinetics (i.e., about 95% of GLP-I was eliminated within 10 minutes) .

[0201] 单一吹入法施用:第1、2、3、7、8和9组通过肺吹入法接受了多种61^-1/^即配方或GLP-I溶液(图22B)。 [0201] Single insufflation administration: 1,2,3,7,8 and 9 receiving a plurality of group 61 by pulmonary insufflation -1 / ^ i.e. formulation or solution of GLP-I (FIG. 22B). 第1组通过肺液体吹入法(LIS)施用了80 μ g的GLP-I对照;第2 组通过肺吹入法(IS)施用了15. 8% GLP-1/FDKP ;第3组通过肺吹入法(IS)施用了3. 8% GLP-1/FDKP ;第7组通过肺液体吹入法(LIS)施用了80 μ g的GLP-I对照;第8组通过肺吹入法(IS)施用了15. 8% GLP-1/FDKP ;和第9组通过肺吹入法(IS)施用了3. 8% GLP-I/ FDKP。 Group 1 was administered 80 μ liquid by pulmonary insufflation (LIS) g of the GLP-I control; the second group was administered a 15. 8% GLP-1 / FDKP by pulmonary insufflation (the IS); Group 3 by pulmonary insufflation (iS) administered 3. 8% GLP-1 / FDKP; group 7 was administered 80 μ g of GLP-I control by pulmonary insufflation fluid (the LIS); group 8 by pulmonary insufflation (IS) administration of 15. 8% GLP-1 / FDKP; 9 administered group and a 3. 8% GLP-I / FDKP by pulmonary insufflation (IS). 在第0、2、5、10、20、40、60、80、100 和120 分钟的时间点测量GLP-1/FDKP 的浓度。 Measuring the concentration of GLP-1 / FDKP at its 0,2,5,10,20,40,60,80,100 and 120 min time point.

[0202] 所有的组在肺施用后显示血浆GLP-I浓度的可检测提高。 [0202] All groups of plasma GLP-I concentrations display a detectable increase in the lung after administration. GLP-I最大血浆浓度随使用的制剂/组合物而变化。 Maximum plasma concentration of GLP-I varied with the formulation used / composition. 如AUC所指示的,第2组和第8组显示在处理后10-20分钟的最大GLP-I血浆水平,而第3组和第9组显示在第5-10分钟的显著活性GLP-I水平,第1 组和第7组显示活性GLP-I血浆水平的更快速和瞬时的提高。 As indicated by AUC, the second group and the 8 groups shown in the maximum plasma levels of GLP-I 10-20 minutes after treatment, and Group 3 and Group 9 show significant activity of GLP-I at 5-10 minutes level, group 1 and group 7 show activity GLP-I plasma levels more rapid and transient increase. 在第2、3、7和8组中,处理后60分钟活性GLP-I的血浆水平回归至背景水平,而第1和7组在处理后20分钟达到背景水平。 In group 3, 7 and 8, after treatment of plasma levels of active GLP-I 60 minutes to return to background levels, while groups 1 and 7 after 20 minutes to reach the background level.

[0203] 在糖尿病大鼠模型中8纳摩尔的GLP-I似乎是有效的;GLP-I剂量为80 μ g(大于报道的有效剂量3000倍);在给药后30分钟使用肺递送的血浆GLP-I水平与3小时灌输(Chelikani et al.,2005)相比高10倍;通过肺吹入法递送的GLP-1/FDKP的生物利用度为71%。 [0203] In a diabetic rat model 8 nanomolar GLP-I appears to be effective; GLP-I dose of 80 μ g (greater than 3000 times the effective dose reported); at 30 minutes after administration pulmonary delivery using plasma GLP-I levels and three hours infusion (Chelikani et al, 2005.) compared to 10 times higher; bioavailability delivered by pulmonary insufflation GLP-1 / FDKP was 71%. 这些结果在下表4中进一步报道。 These results are reported in Table 4 below further. 在通过肺递送处理的大部分大鼠中,给药后30-60 分钟GLP-I的血浆水平回复至基线水平。 In most rats treated via pulmonary delivery, 30-60 minutes after administration plasma levels of GLP-I returned to baseline levels. 除了第2组中的1号大鼠外,所有的大鼠在静脉注射或肺吹入多种GLP-1/FDKP制剂后显示GLP-I血浆浓度的提高。 In addition to No. 1 in the second group of rats, all rats showed an increase in plasma concentrations of GLP-I after intravenous administration or pulmonary insufflation of various GLP-1 / FDKP formulations.

[0204] 益论:在GLP-1/FDKP制剂的药物代谢动力学模式中观察到了与GLP-I溶液相比的差异。 [0204] On the benefits: observed difference compared to GLP-I pharmacokinetic profile of a drug solution in the GLP-1 / FDKP formulations. 相对于用GLP-I溶液处理的大鼠而言,通过GLP-1/FDKP制剂肺吹入法处理的大鼠中GLP-I的血浆浓度更加持久。 With respect to rat GLP-I solution in terms of process, the plasma concentration of GLP-I in rats treated by the GLP-1 / FDKP formulation is more persistent pulmonary insufflation. 在给药后20到60分钟之间,所有的动物显示GLP-I血浆浓度的降低。 Between 20 to 60 minutes after administration, all the animals showed decreased plasma concentration of GLP-I. 这些结果显示连续2天进行的2个实验的相对一致性。 These results show that the experiment 2 relative consistency in 2 consecutive days.

[0205] 表4. GLP-1/FDKP制剂的生物利用度 [0205] Table 4. GLP-1 bioavailability / FDKP formulation

[0206] [0206]

Figure CN104288756AD00301

[0207] *与FDKP颗粒3:1掺合 [0207] * with FDKP particles was 3: 1 blend

[0208] 实施例8 [0208] Example 8

[0209] GLP-1/FDKP减少大鼠的食物摄入 [0209] GLP-1 / FDKP reduced food intake in rats

[0210] 本领域还已知GLP-I在大脑中作用,以触发饱满的感觉并减少食物摄入。 [0210] It is also known in the art GLP-I action in the brain to trigger the feeling of fullness and reduce food intake. 根据GLP-I在饱满感和食物摄入中的该作用,进行实验确定本发明的GLP-1/FDKP制剂是否作为减少饲养的试剂是有效的并从而具有控制肥胖症的潜能。 The effect of the GLP-I in satiety and food intake, and experiments to determine whether the present invention, GLP-1 / FDKP formulation is effective as a reducing agent and kept so as to have the potential to control obesity.

[0211] 通过肺吹入法用对照(空气)或2mg/天剂量的15. 8% GLP-1/FDKP制剂(0· 32mg GLP-I/剂)对两组雌性Sprague Dawley大鼠给药。 [0211] administered by pulmonary insufflation with control (air) or 2mg / 15. 8% day dose of GLP-1 / FDKP formulations (0 · 32mg GLP-I / agent) female Sprague Dawley rats of both groups. 对照组由五只大鼠组成,测试组由十只大鼠组成。 Control group of five rats and the test group consists of ten rats composition. 每个大鼠连续5天被提供给单一剂量,并在每次给药后2和6小时测量食物摄入。 5 consecutive days each rat is supplied to a single-dose, food intake was measured at 2 and 6 hours after each dose. 每天收集每只大鼠的体重。 Body weight of each rat was collected daily.

[0212] 初步的数据显示在给药后2和6小时,在用GLP-1/FDKP制剂给药的大鼠中存在累积食物消耗的整体减少(图23A和23B)。 [0212] Preliminary data show that the 2 and 6 hours after administration, there is an overall reduction (FIGS. 23A and 23B) cumulative food consumption in rats dosed with GLP-1 / FDKP formulations. 在第4天给药后2小时该减少更显著(p = 0. 01)。 Two hours after administration of the 4 days more significantly reduced (p = 0. 01). 第1天和第2天6小时时该减少更显著(p〈0. 02)。 The first day of the more significant decrease (p <0. 02) When the day 26 hours. 给药后24小时没有对食物消耗的影响。 24 hours after dosing had no effect on food consumption.

[0213] 实施例9 [0213] Example 9

[0214] 毒件研究 [0214] toxicity study pieces

[0215] 进行重复的剂量毒性研究,以评价多次施用后GLP-1/DKP的可能的毒性作用和毒物代谢动力学模式。 [0215] Repeat dose toxicity studies to evaluate the potential toxic effects and toxicokinetic mode GLP-1 / DKP after multiple administrations. 进行了大鼠中14天的研究和猴子中28天的研究。 Studied 28 days of a 14-day study in rats and monkeys. 通过吸入途径每天进行GLP-1/DKP给药。 By inhalation pathway / DKP GLP-1 is administered per day. 在对动物给药28天的研究中,一部分动物要在给药方案后立即处死, 而其它动物允许在处死前有至多一个月的恢复周期。 In the study animals were dosed for 28 days, a part of the animal to be sacrificed immediately after the dosing regimen while other animals are allowed up to a one month recovery period prior to sacrifice. 评价所有动物的临床体征,多种生理参数,包括GLP-1、葡萄糖、胰岛素、器官重量和临床病理学和多种器官的组织病理学。 Evaluation of all clinical signs of animals, a variety of physiological parameters, including GLP-1, glucose, insulin, organ weights, and clinical pathology and histopathology of various organs.

[0216] 进行一系列GLP诱变性研究,以评价二酮哌嗪颗粒的诱变潜能。 [0216] A series of mutagenicity studies GLP, to evaluate the mutagenic potential of diketopiperazine particles. 这些研究包括体外Ames和染色体畸变测定法,其均是本领域技术人员公知的。 These studies include in vitro Ames chromosomal aberration assays, which are well known to those skilled in the art. 另外,也进行了技术人员已知的体内小鼠微核测定法。 Further, also it is known to the skilled in vivo mouse micronucleus assay. 遗传毒性数据显示没有证据表示二酮哌嗪颗粒有诱变性或遗传毒性的潜能。 Genotoxicity data indicate that there is no evidence showing diketopiperazine particles or mutagenic potential genotoxicity.

[0217] 还进行一些研究评价二酮哌嗪颗粒对繁殖毒性的影响。 [0217] Evaluation is also some influence diketopiperazine particles on reproductive toxicity. 这些研究包括在大鼠和兔子中的生育力、胚胎-胎儿发育和出生后发育研究。 These studies included fertility in rats and rabbits, the embryo - Developmental fetal development and after birth. 通过皮下注射施用的二酮哌嗪颗粒在大鼠中不损伤生育力或植入,并且没有在大鼠或兔子中致畸性的证据。 Administered by subcutaneous injection diketopiperazine particles without damaging fertility or implantation in rats and there is no evidence of teratogenicity in rats or rabbits. 二酮哌嗪颗粒并未不利地影响生育力和早期胚胎发育、胚胎发育或出生前或出生后发育。 Diketopiperazine particles not adversely affect fertility and early embryonic development, embryo development, or prenatal or postnatal development.

[0218] 考虑到大量药物已经由于它们引起LQT综合征(获得性LQTS或长期QT综合征是一种在用药人群中发生的罕见的心脏电节律的遗传性异常)的倾向而从临床市场中被去除,使用hERG测定法检查二酮哌嗪颗粒的药理学。 [0218] Taking into account a large number of drugs have been due to their cause LQT syndrome (LQTS or acquired long QT syndrome is a rare hereditary heart's electrical rhythm that occurs in the drug crowd abnormal) tend to be from the clinical market removing, using pharmacological assays hERG check diketopiperazine particles. 考虑到引起获得性LQT的大部分药物通过封闭人醚-ά-go-go相关基因(hERG)钾通道而引起获得性LQT的,因此使用hERG测定法,所述钾通道负责心室贲门动作电位(ventricular cardiac action potential)的复极化。 Considering that most drugs cause acquired LQT caused by closed acquired LQT human ether -ά-go-go related gene (hERG) potassium channel, hERG assay so using the potassium channel responsible for cardiac action potential of ventricular ( ventricular cardiac action potential) repolarization. 来自hERG测定法的结果表明二酮哌嗪颗粒的IC5ciMOOyMtj另外,来自二酮哌嗪非临床研究的结果显示对QTc间隔(心率校正的QT间隔)没有影响,因为在犬中未观察到延长(9个月或安全药理学心血管研究)。 Results from the hERG assay indicated that IC5ciMOOyMtj diketopiperazine particles Further, the results of non-clinical studies show diketopiperazine from no effect on QTc interval (the QT interval corrected for heart rate), because not observed in dogs extension (9 months or cardiovascular safety pharmacology studies). 静脉内施用时,二酮哌嗪颗粒对在安全药理学核心电池组中评价的CND或心血管系统没有影响。 When administered intravenously, diketopiperazine particles CND has no effect on the cardiovascular system or in the evaluation of the safety pharmacology core battery pack.

[0219] 实施例10 [0219] Example 10

[0220] GLP-I对β -细朐质量的影响 [0220] GLP-I on β - Effect of fine quality Qu

[0221] 已知GLP-I促进胰岛素生物合成中的所有步骤并直接刺激β-细胞生长和存活以及β-细胞分化。 [0221] GLP-I is known to promote all steps in insulin biosynthesis and directly stimulate cell growth and survival β- and β- cell differentiation. 这些作用的组合导致增加的β-细胞质量。 The combination of these effects results in increased cell mass β-. 另外,GLP-I受体信号转导导致β-细胞凋亡的减少,这进一步有助于增加β-细胞质量。 Further, GLP-I receptor signaling results in a reduction of β- cell apoptosis, which further contributes to increased β- cell mass. 已知GLP-I通过三种可能的途径调节β -细胞质量:增强β -细胞增殖;抑制β -细胞凋亡;和分化导管上皮中推定的干细胞。 GLP-I is regulated by known three possible approaches β - cell mass: Enhanced β - cell proliferation; inhibition of β - cell apoptosis; and differentiation of the ductal epithelium of putative stem cells.

[0222] 为了证实GLP-I对β -细胞的影响,在第1、3和5天用GLP-1/FDKP处理细胞并与未处理的细胞比较。 [0222] In order to confirm GLP-I β - Effect of cell 1, 3 and 5 days the cells were treated with GLP-1 / FDKP and compared to untreated cells. 如文献中所述,活性GLP-I的施用将β-细胞质量至多提高2倍(Sturis et al.,2003)。 As described in the literature, administration of the active GLP-I will β- cell mass of at most 2-fold increase (Sturis et al., 2003). 另外,多种GLP-I受体(GLP-IR)激动剂对糖尿病的影响的检查证实GLP-IR激动剂预防或延迟了糖尿病的发生或发展。 Further, examine the influence of various GLP-I receptor (GLP-IR) agonists on diabetes demonstrated GLP-IR agonists prevent or delay the development or progression of diabetes.

[0223] 在雄性Zucker糖尿病肥胖/肥胖(ZDF)大鼠(η = 8/组)中评价GLP-1/FDKP对β -细胞增殖、胰岛素和葡萄糖的影响。 [0223] In obese male Zucker Diabetic / Obese (ZDF) rats (η = 8 / group) Evaluation of GLP-1 / FDKP on β - cell proliferation, insulin and glucose. 动物连续3天接受对照(空气)或含15% (0. 3mg) GLP-I的2mg GLP-1/FDKP。 Control animals receiving 3 consecutive days (air), or containing 15% (0. 3mg) GLP-I of 2mg GLP-1 / FDKP. 进行腹膜内(IP)葡萄糖耐受测试,并在给药前、给药后15、30、 45、60和90分钟收集血样用于血浆GLP-I和葡萄糖分析。 Intraperitoneal (IP) glucose tolerance test, and before administration after administration 15, 30, 45, 60 and 90 minutes Blood samples were collected for plasma GLP-I and glucose analysis. 收集胰腺组织用于通过免疫组织化学分析胰岛素分泌、β-细胞质量和细胞凋亡。 Pancreatic tissue was collected for insulin secretion, apoptosis and cell mass β- chemical analysis by immunohistochemistry.

[0224] 在给药的第4天进行IP葡萄糖耐受测试(IPGTT,图24)。 [0224] the IP glucose tolerance test (IPGTT, FIG. 24) on the 4th day of administration. 过夜禁食后在第3天, 动物通过腹膜内注射接受葡萄糖推注,随后立即通过肺吹入法接受对照(空气)或GLP-I/ FDKP施用。 After an overnight fast on day 3, the animals receiving glucose by intraperitoneal injection bolus followed immediately received the control (air) or GLP-I / FDKP administered by pulmonary insufflation. 在葡萄糖激发前和至给药后90分钟的多个时间点收集血。 Blood was collected prior to the glucose challenge, and 90 minutes after the plurality of time points to administration. 在给药后30分钟, 第1组显示与给药前相比47 %的葡萄糖水平提高,而第2组(GLP-1/FDKP)显示与给药前值相比17%的葡萄糖水平提高。 30 minutes after the administration, Group 1 showed an increase of 47% compared to pre-dosing glucose levels, while the second group (GLP-1 / FDKP) showed that compared glucose levels of 17% and increase the value before administration. 在葡萄糖耐受测试后所有的时间点中,处理与对照动物相t匕,葡萄糖水平显著更低(Ρ〈〇. 05)。 At all time points after the glucose tolerance test and control animals treated with t dagger, glucose levels were significantly lower (Ρ <square. 05).

[0225] 还在给药的第3天测量GLP-I水平(图25)。 [0225] Day 3 of dosing also measured the level of GLP-I (FIG. 25). 第2组中血浆GLP-I水平的最大浓度是给药后15分钟的10, 643ρΜ。 The second group the maximum plasma concentration of GLP-I levels after administration of 10, 643ρΜ 15 minutes.

[0226] 另外,与IP葡萄糖耐受测试后的葡萄糖测量一起,在第3天的多个时间点测量胰岛素水平。 [0226] Further, after the glucose measurement with the IP glucose tolerance test, insulin levels are measured on day 3 of a plurality of time points. 对照(空气)第1组和第2组(GLP-/DKP)均证实给药后15分钟相对于给药前水平而言分别46%和30%的胰岛素浓度初始降低(图26)。 Control (air) Group 1 and Group 2 (GLP- / DKP) were confirmed 15 minutes relative to the level before the administration were 46% and 30% of the initial insulin concentration decreased (FIG. 26) after dosing. 然而,在给药后30分钟,第2 组中的胰岛素水平回归至基线,而第1组中的胰岛素水平持续降低至给药前值的64%。 However, 30 minutes after administration, insulin levels in Group 2 return to baseline, whereas insulin levels in Group 1 continued to decrease to 64% of the value before administration. 在被处理的动物中,45分钟、60分钟和90分钟的胰岛素水平接近给药前值,偏差少于1. 5%。 In the animals treated, 45 minutes, 60 minutes and 90 minutes before the administration of insulin levels close value, the deviation is less than 1.5%.

[0227] 制备了用于胰岛素免疫染色和显微镜评价胰岛素表达的载玻片。 [0227] a slide for insulin immunostaining and microscopic evaluation of insulin expression was prepared. 根据通过光学显微镜对胰岛素表达的定量评价,雄性ZDF大鼠的胰中存在处理相关的胰岛素表达提高(其为剂量相关的),尽管未达到统计学显著性(P = 0.067);这通过表达胰岛素的β胰岛细胞的百分比所确定。 The quantitative assessment of insulin expression by light microscopy, pancreas in male ZDF rats present in treatment-related increase in insulin expression (which is dose-related), although not reach statistical significance (P = 0.067); This insulin expression by the percentage of the determined β islet cells.

[0228] 还对ZDF大鼠的胰腺组织进行了细胞凋亡分析。 [0228] pancreatic tissue of ZDF rats also were analyzed for cell apoptosis. 通过TUNEL测定法评价外分泌和内分泌的胰腺细胞(Tornusciolo DR et al.,1995)。 Evaluated by TUNEL assay outer exocrine and endocrine cells of the pancreas (Tornusciolo DR et al., 1995). 对胰中约10, 000个细胞(外分泌的和内分泌的)评分。 Pancreatic about 10, 000 cells (endocrine and exocrine) score. 大部分TUNEL-阳性细胞是外分泌的。 Most TUNEL- positive cells were exocrine. 在处理和对照组中没有细胞凋亡标记指数的差异。 No differences in apoptosis labeling index in treated and control groups.

[0229] 另外,在Zucker糖尿病肥胖大鼠的胰中评价了β细胞增殖,所述大鼠通过肺吹入法用对照(空气)或GLP-1/FDKP每天一次给药3天。 [0229] Furthermore, cell proliferation was evaluated pancreatic β in Zucker Diabetic obese rats, the rats by pulmonary insufflation with control (air) or GLP-1 / FDKP administered for three days once per day. 制备了使用免疫组织化学对胰岛素和Ki67(增殖标记物)共定位的载玻片。 The use of immunohistochemical slides of insulin and of Ki67 (proliferation marker) colocalization prepared. 在总计17只ZDF大鼠的胰岛素阳性的胰岛和外分泌胰腺中进行细胞增殖的显微镜评价。 Microscopic evaluation of cell proliferation of insulin-positive islets and in the exocrine pancreas in a total of 17 ZDF rats. 根据细胞增殖的定量评价,雄性ZDF大鼠的胰岛β-细胞或胰腺外分泌细胞中没有对细胞增殖的处理相关的影响。 The quantitative evaluation of cell proliferation, treatment-related effects on cell proliferation Cells no outer β- islet cells or pancreatic secretion in male ZDF rats.

[0230] 总之,该研究显示通过肺吹入法以2mg或0· 3mg GLP-I施用的GLP-1/FDKP在葡萄糖耐受测试后降低糖尿病肥胖小鼠(II型糖尿病的模型)中的血糖水平,并提高每个胰岛的胰岛素分泌细胞数量。 [0230] In summary, this study shows that by pulmonary insufflation or 2mg to 0 · 3mg GLP-I administered GLP-1 / FDKP reduced blood glucose (type II diabetes model) in obese diabetic mice after a glucose tolerance test levels, and increase the amount of insulin secreting cells per islet.

[0231] 实施例11 [0231] Example 11

[0232] GLP-I/FDKP颗粒制剂的制各 [0232] GLP-I / FDKP particle formulations prepared for each

[0233] 还使用了制备GLP-1/FDKP颗粒制剂的备选方法。 [0233] An alternative method also uses GLP-1 / FDKP particle formulations prepared. 制剂如下制备:通过将1份GLP-I (按重量计)添加进9份去离子水中并添加小量的冰醋酸形成澄清溶液来制备10wt% GLP-I储存溶液。 Formulation prepared as follows: 1 part by GLP-I (by weight) added to 9 parts of deionized water was prepared and added 10wt% GLP-I stock solution was a small amount of glacial acetic acid to form a clear solution. 将FDKP颗粒的储存悬浮液(约10wt%的颗粒)分为三部分。 The FDKP particle suspension storage (about 10wt% of the particles) is divided into three parts. 向每部分悬浮液中添加合适量的GLP-I储存溶液,提供按干燥粉末计5和15wt % GLP-I的靶组合物。 Appropriate amount to each part of the suspension in GLP-I stock solution to provide a dry powder and from 5 to 15wt% GLP-I target composition. 添加蛋白质溶液后,悬浮液的pH约为3. 5。 After the addition of the protein solution, pH of the suspension was about 3.5. 然后将悬浮液调节至约pH 4.4-4. 5, 之后将悬浮液在液氮中制成小球并冻干除去冰。 The suspension was then adjusted to about pH 4.4-4. 5, then the suspension was pelletized in liquid nitrogen and lyophilized to remove the ice.

[0234] 粉末的气体动力学用填充量可吸入级分比(respirable fraction on fill) (RF Based on Fill)来表征,即以药筒中粉末的量计可吸入范围内的粉末百分比(% ),其如下测定:用5mg的粉末手工填充五个药筒并通过MannKind' s MedTone®吸入器(描述于美国专利申请No. 10/655, 153中)排放。 [0234] Powder aerodynamic filled with an amount of respirable fraction ratio (respirable fraction on fill) (RF Based on Fill) characterized, i.e., in an amount of cartridge powder meter powder percentage (%) within a respirable range, which is determined as follows: 5mg filled with powder by hand and five cartridges MannKind 's MedTone® inhaler (described in U.S. Patent application No. 10/655, 153) is discharged.

[0235] 该方法产生具有良好的填充量RF的制剂。 [0235] The preparation method produces a good filling amount of RF. 具有5wt % GLP-I的粉末被测量为48. 8% RF/填充量,而含约15wt% GLP-I的粉末为32. 2% RF/填充量。 Powder having 5wt% GLP-I was measured to be 48. 8% RF / fill amount, and powder contain about 15wt% GLP-I was 32. 2% RF / fill amount.

[0236] 实施例12 [0236] Example 12

[0237] 含多种GLP-I浓度的GLP-1/FDKP的药物代谢动力学 [0237] Pharmacokinetic containing various GLP-I concentrations of GLP-1 / FDKP pharmaceutical

[0238] 为了评价具有多种GLP-I浓度的GLP-1/FDKP的药物代谢动力学,将称重在192. 3 克到211. 5克之间的十八只雄性Sprague Dawley大鼠分为四个处理组:对照GLP-I (第1 组,η = 3) ;GLP-1/FDKP制剂(第2-4组,η = 5/组)。 Pharmacokinetic GLP-1 / FDKP in [0238] In order to evaluate a variety of GLP-I concentration, weighing between 192.3 grams to 211.5 grams of eighteen male Sprague Dawley rats were divided into four treatment groups: control GLP-I (group 1, η = 3); GLP-1 / FDKP formulations (groups 2-4, η = 5 / group). 动物接受以下的测试物品之一:通过肺吹入法的对照(空气);含5% GLP-I的2. 42mg GLP-1/FDKP(0. 12mg GLP-1);通过肺吹入法的含10% GLP-I 的I. 85mg GLP-1/FDKP(0. 19mg GLP-1),或含15% GLP-I 的2. 46mg GLP-1/FDKP(0. 37mg GLP-1)。 Test animals received one of the following items: control by pulmonary insufflation (air) method; containing 5% GLP-I is 2. 42mg GLP-1 / FDKP (0 12mg GLP-1.); By pulmonary insufflation containing 10% GLP-I in I. 85mg GLP-1 / FDKP (0. 19mg GLP-1), or containing 15% GLP-I is 2. 46mg GLP-1 / FDKP (0. 37mg GLP-1). 收集血样并测定给药前和给药后多个时间点(2、5、10、20、30、 40和60分钟)血清FDKP和血浆GLP-I水平。 Blood samples were collected and assayed for serum FDKP and plasma GLP-I levels of a plurality of time points (2,5,10,20,30, 40 and 60 minutes) before and after administration.

[0239] 施用GLP-1/FDKP (5 %制剂)后的最大血浆GLP-I浓度(Cmax)为:给药后5分钟Tmax处的2321pM ;给药后10分钟Tmax处的4, 887?1(10%制剂);和给药后10分钟Tmax处的10, 207pM(15%制剂)。 The maximum plasma GLP-I concentration (Cmax) after the [0239] administration of GLP-1 / FDKP (5% formulation) as follows: 5 minutes at Tmax after administration 2321pM; 10 minutes after administration at 4 Tmax, 8871? (10% formulation); Tmax at 10 minutes after administration of 10, 207pM (15% formulation). 如图27所示,直到给药后30分钟仍观察到显著的GLP-I水平。 27, still observed 30 minutes until a significant GLP-I levels after administration. 第1-4组的GLP-I水平的曲线下面积(AUC)分别为10622、57101、92606、227873口]\#分钟。 Area under the curve GLP-I levels 1-4 group (AUC), respectively 10622,57101,92606,227873 port] \ # minutes. 对10%或15% GLP-I负载的GLP-1/FDKP估计的GLP-I半衰期为10分钟。 To 10% or 15% GLP-I load GLP-1 / FDKP estimated half-life of GLP-I was 10 minutes.

[0240] 如图28所示,5%、10%和15% GLP-I的GLP-1/FDKP制剂的最大FDKP浓度分别被测定为8· 5 μ g/mL (第2组)、4· 8 μ g/mL (第3组)和7· 1 μ g/mL (第4组)。 [0240] As shown in FIG, 5%, maximum FDKP concentrations of GLP-1 / FDKP formulations of 10% and 15% GLP-I respectively to be measured 28 8 · 5 μ g / mL (Group 2) 4 · 8 μ g / mL (group 3) and 7 · 1 μ g / mL (group 4). 到最大浓度的时间(Tmax)为1〇分钟。 Time to maximum concentration (Tmax) of 1〇 minutes. 该数据显示FDKP和GLP-I显示类似的吸附动力学,类似量的FDKP 被吸附而与颗粒上的GLP-I负载无关。 This data shows FDKP and GLP-I exhibited similar adsorption kinetics, similar amounts of FDKP adsorption is independent of the GLP-I load on the particles.

[0241] 总之,研究显示在Sprague Dawley大鼠中通过肺吹入法单一剂量施用GLP-I/ FDKP后,血浆GLP-I水平被检测为显著水平。 [0241] In summary, studies have shown that in Sprague Dawley rats after a single dose pulmonary insufflation administration of GLP-I / FDKP, plasma GLP-I levels were detected as a significant pass. 观察到血浆GLP-I水平的剂量相关提高约在给药后10分钟达到最大浓度,在给药后40分钟具有可观察的GLP-I水平。 Dose was observed in plasma GLP-I levels after administration related to an increase of about 10 minutes to reach maximum concentration for 40 minutes with observable GLP-I levels after administration. 所有的动物存活,直到研究结束时为止。 All animals survived until the end of the study so far.

[0242] 实施例13 [0242] Example 13

[0243] 通讨肺吹入法施用的GLP-1/FDKP的药物动力学特件 [0243] administered via pulmonary insufflation method discussed GLP-1 / FDKP pharmacokinetic member Laid

[0244] 为了评价GLP-1/FDKP的药物动力学特性,将雌性Sprague Dawley大鼠分为2个处理组。 [0244] To assess the pharmacokinetic properties of GLP-1 / FDKP will female Sprague Dawley rats were divided into two treatment groups. 动物(η = 10)连续4天通过单一的每日肺吹入法接受对照(空气;η = 5)或含15% GLP-1(0. 3mg GLP-1)的2mg GLP-1/FDKP。 Animals (η = 10) 4 consecutive days by a single control daily pulmonary insufflation (air; η = 5) or containing 2mg 15% GLP-1 (0 3mg GLP-1.) The GLP-1 / FDKP.

[0245] 连续4天在暗循环中于给药前、给药后1、2、4、和6小时测量食物消耗(图29)。 [0245] 4 consecutive days before the administration, 2, 4, and 6 hours post dose measuring food consumption in the dark cycle (FIG. 29). 与对照(空气)组相比,受处理的动物中在第1、2和3天通过肺吹入法每日单一剂量施用GLP-1/FDKP后食物消耗减少(p〈0. 05)。 Compared to the control (air) group, the animals treated by administration of GLP-1 / FDKP by pulmonary insufflation daily single dose at days 1, 2 and 3, decreased food consumption (p <0. 05). 第1天的1小时和6小时时间点上和第2天的4 小时、6小时和第3天的给药前,受处理组中的动物相对于对照(空气)存在统计学显著的食物消耗减少。 Day 1 1 hour and 6 hour time points day 2 and 4 hours, 6 hours before dosing and on day 3, the animals receiving treatment groups with respect to statistically significant food consumption control (air) cut back.

[0246] 连续4天在给药前每天测量体重(图30)。 [0246] Body weight was measured for 4 consecutive days (FIG. 30) prior to dosing each day. 给药起始时的体重在从约180到209 克的范围内变化。 Change in body weight when administered starting from the range of about 180-209 grams. 尽管处理和对照(空气)动物之间未达到统计学显著性,但是处理的动物中体重更低。 Although the treated and control (air) between the animals it did not reach statistical significance, but lower body weight of treated animals. 所有的动物存活直到预定的处死为止。 All animals survived until scheduled sacrifice.

[0247] 实施例14-16 [0247] Examples 14-16

[0248] 毒物代谢动力学(TK)研究 [0248] Pharmacokinetics and Toxicology (TK) study

[0249] 下文实施例14到16公开了为了评价GLP-1/FDKP吸入粉末可能的毒性作用和毒物代谢动力学模式而在大鼠和猴子中进行的重复给药毒性研究。 Discloses a repeated-dose toxicity study of GLP-1 / FDKP inhalation may be toxic and toxicokinetic powder pattern in rats and monkeys in order to evaluate the Example 14 to 16 [0249] below. 数据表明在高于计划的临床用途剂量若干倍的剂量下,GLP-1/FDKP吸入粉末没有明显的毒性。 Data show that at doses several times higher than the dose for clinical use plan, GLP-1 / FDKP inhalation powder without significant toxicity. 另外,在每个物种中显示雄性和雌性动物之间没有差异。 Further, in each species show no difference between male and female animals.

[0250] 实施例14 [0250] Example 14

[0251] 通讨猴子中肺吹入法施用5天的GLP-I/FDKP的毒物代谢动力学 Pharmacokinetics [0251] via pulmonary insufflation discussion monkeys administered 5 days GLP-I / FDKP poison

[0252] 进行研究测定GLP-1/FDKP的毒性和毒物代谢动力学模式,所述GLP-1/FDKP连续5天每天一次(每天30分钟)通过口鼻施用(预期的人治疗施用途径)给短尾猴(Macaca fascicularis)。 [0252] Determination for GLP-1 / toxicity and toxicokinetic mode of FDKP, the GLP-1 / FDKP. 5 consecutive days once a day (30 minutes per day) via oronasal administration (the intended human therapeutic route of administration) to cynomolgus monkeys (Macaca fascicularis). 口鼻施用涉及在猴子的口和鼻上佩戴面具并呼吸测试制剂30分钟。 Oronasal administration involving a mask worn over the mouth and nose and breathing the test formulation monkey 30 minutes.

[0253] 在开始处理前十四天使动物适应限制(restraint)和给药步骤。 [0253] Before starting treatment accommodate the limitations of fourteen animals angel (restraint) and administration step. 在处理开始时(第1天),雄性动物在30和56个月龄之间,体重范围从2. 3到4. Okg ;雌性在31和64个月龄之间,体重范围从1. 6到3. 4kg。 When processing is started (Day 1), male animals between 30 and 56 months of age, body weight ranging from 2.3 to 4. Okg; females between 31 and 64 months of age, body weight ranging from 1.6 to 3. 4kg. 如下文表5和表6中所示,将十只(5只雄性和5只雌性)非首次实验的短尾猴指定为5组(每组2只动物)。 And the table shown below in Table 56, the ten (5 male and 5 female) non-designated first experiments cynomolgus 5 groups (2 animals per group). 非首次实验的猴子是先前已接受待测试制剂的群体动物。 Not the first time the experimental monkeys that have been previously accepted test group of animals to be preparation. 然而,这些制剂具有短半衰期,并且预期在本文公开的给药实验期间不会显示或具有对猴子的任何影响。 However, these formulations have short half-life, or the display and is not expected to have any effect on the monkeys during the dosing experiments disclosed herein. 动物接受对照(空气)、2mg/kg FDKP或0. 3 (0. 04mg GLP-1)、1· 0(0· 13mg GLP-1)或2. 0(0· 26mg GLP-l)mg/kg GLP-1/FDKP。 Animals received control (air), 2mg / kg FDKP or 0. 3 (0. 04mg GLP-1), 1 · 0 (0 · 13mg GLP-1), or 2. 0 (0 · 26mg GLP-l) mg / kg GLP-1 / FDKP.

[0254] 表5:目标的和评估的实际剂量水平(通过重量分析Ί则定): [0254] Table 5: Actual dosage levels and assessment target (Ί is scheduled by gravimetry):

[0255] [0255]

Figure CN104288756AD00341

[0256] #通过在给药前、给药期间和给药后称重吸入腔中的滤纸进行重量分析,来计算腔中气溶胶的浓度和测定给药的持续时间。 [0256] # by prior administration, for administration during the suction chamber and weighed filter paper weight analysis after administration to calculate the aerosol concentration in the chamber and measuring the duration of administration.

[0257] 1基于假定的2. 5kg体重。 [0257] Based on an assumed body weight 2. 5kg.

[0258] 2基于测量的体重(对雄性和雌性平均)。 [0258] 2 based on the measured weight (average of males and females).

[0259] 3列出的目标和实际的剂量水平假定产生的气体中GLP-I的比例为13%。 GLP-I ratio in the gas target and actual dosage level [0259] 3 lists generated assumed 13%. 总吸入剂量的估值假定在呼吸道中100%沉积。 The total inhaled dose assumed valuation deposition in the respiratory tract of 100%.

[0260] 表6:目标的和实际的平均气溶胶浓度(通过重量分析Ί则定): [0260] Table 6: Target and actual mean aerosol concentrations (Ί is given by gravimetry):

[0261] [0261]

Figure CN104288756AD00342

[0262] #通过在给药前、给药期间和给药后称重吸入腔中的滤纸进行重量分析,来计算腔中气溶胶的浓度和测定给药的持续时间。 [0262] # gravimetrically prior to administration by weighing the filter paper and the suction chamber after the administration during the administration period to calculate the aerosol concentration in the chamber and measuring the duration of administration.

[0263] 1列出的目标和实际的气雾剂浓度假定产生的气体中GLP-I的比例为13%。 GLP-I ratio of the gas and the actual target aerosol concentrations [0263] 1 lists generated is assumed 13%. 总吸入剂量的估值假定在呼吸道中100%沉积。 The total inhaled dose assumed valuation deposition in the respiratory tract of 100%.

[0264] 在第5天的以下时间点获得全血样品(I. 4mL/血样):给药前、给药后10、30、45、 60、90、120分钟。 [0264] Whole blood samples (I. 4mL / blood) on day 5 of the following time points: pre-dose, after administration 10,30,45, 60,90,120 minutes. 通过静脉穿刺从股静脉中收集血。 Blood was collected from the femoral vein by venipuncture. 将血样分为2等分;一份用于血浆GLP-I分析(0. 8mL),另一份(0. 6mL)用于血清FDKP分析。 Blood samples were divided into 2 aliquots; one for plasma GLP-I analysis (0. 8mL), another (0. 6mL) for serum FDKP analysis. 对于血浆GLP-I分析而言,在每个时间点将全血(〇. 8mL)收集进I. 3mL EDTA管(0. 1% EDTA)中。 For plasma GLP-I analysis, whole blood at each time point (square. 8 mL) were collected into tubes I. 3mL EDTA (0. 1% EDTA) in. 在血收集后约5-10 秒向管中添加(1(^171^血)0?卩-1¥抑制剂(]\^111口〇代^1161^〇3,嫩),得到100 4]\1的DPP-IV浓度。将管反转数次并立即置于湿润的冰上。全血样品保持在湿润的冰上,直到在4000rpm离心(2° -8°C )约10分钟产生血浆为止。将血浆样品转移至适当的管中,并在储存于-70 (± 10) °C冰箱前保持在干冰上。测定GLP-I的须将浓度(Cmax)、Tmax、AUC和T 1/2。 Add approximately 5-10 seconds after blood collection to the tube (1 (blood ^ 171 ^) 0? -1 ¥ Jie inhibitors (] \ ^ 111 ^ 1161 ^ 〇3 substituting square, soft), to give 1004] \ DPP-IV concentration of 1. the tube was inverted several times and immediately placed on ice wetted whole blood samples were kept on wet ice until the plasma is generated in the centrifugation 4000rpm (2 ° -8 ° C) for about 10 minutes so far. the plasma samples were transferred to the appropriate tube, and stored at -70 (± 10) ° C freezer on dry ice before holding. shall assay concentration (Cmax) GLP-I's, Tmax, AUC, and T 1 / 2.

[0265] 连续4天吸入施用GLP-1/FDKP后,第5天在所有给药前样品中发现可检测的GLP-I水平。 After [0265] 4 consecutive days inhalation GLP-1 / FDKP, 5 day GLP-I was found in detectable levels in all samples prior to administration. 在第5天,给药施用后约10分钟内达到了GLP-I的血浆浓度峰值(Cmax)(图31)。 On day 5, the administration after administration of about 10 minutes to reach the peak plasma concentration of GLP-I (a Cmax) (FIG. 31).

[0266] 第5天在雄性和雌性猴子中均观察到作为剂量函数的GLP-ICmax和AUC last (从零时间到最后可计量浓度时间的浓度-时间曲线下的面积)的剂量相关增加。 [0266] Day 5 male and female monkeys was observed as a function of dose of GLP-ICmax and AUC last (from time zero to the last quantifiable concentration time concentration - area under time curve) increased dose-related. 在研究的剂量范围中,在雄性和雌性猴子中用递增的剂量观察到少于与剂量成比例的GLP-IAUClast增加, lmg/kg/天剂量水平的雄性除外。 In the dose range studied in male and female monkeys was observed with increasing doses of less than dose proportional increases GLP-IAUClast except lmg / kg / day dose level in male. 从0. 3到2. Omg/kg/天的6. 7倍剂量增加仅导致雄性中AUClast的2. 9倍增加和雌性中AUClast的I. 1倍增加。 Doses from 6.7 to 0.3 2. Omg / kg / day results in only a 2.9 fold increase in AUClast in males and 1-fold increase in AUClast in females I. increases.

[0267] 当分别以0· 3、L 0和2. Omg/kg/天的剂量水平施用GLP-1/FDKP时,平均的GLP-I 浓度峰值在雄性中为17. 2、93. 1和214pg/mL,在雌性中为19. 3、67· 9和82. 8pg/mL。 [0267] When 0 · 3, respectively, dosage levels L 0 and 2. Omg / kg / day administered GLP-1 / FDKP, the mean peak concentration of GLP-I in males was 17. 2,93. 1 and 214pg / mL, in females of 3,67 · 19 9 and 82. 8pg / mL. GLP-I 的血浆水平快速下降,其表观清除半衰期范围在4分钟到24分钟。 GLP-I plasma levels rapidly decreased, the apparent elimination half-life in the range of 4 minutes to 24 minutes.

[0268] 当分别以0· 3、I. 0和2. Omg/kg/天的剂量水平施用GLP-1/FDKP时,GLP-I的AUC 值在雄性中为21. 6、105 和62. 3pg*h/mL,在雌性中为33. 4、23. 7 和35. 4pg*h/mL。 [0268] When respectively 0 · 3, I. Dose level 0 and 2. Omg / kg / day administered GLP-1 / FDKP, AUC values ​​of GLP-I was 21 and 62 in 6,105 males. 3pg * h / mL, 33. 4, 23 is in females. 7 and 35. 4pg * h / mL.

[0269] 在最低剂量水平观察的GLP-ITK参数中不存在明显的性别差异。 [0269] there is no significant gender differences in GLP-ITK parameters observed at the lowest dose level of. 然而,在研究的中和高剂量水平下,雄性猴子始终展示比雌性猴子更高的AUC last值。 However, in the medium and high dose levels studied, male monkeys consistently demonstrated higher than female monkeys AUC last values. 来自运载体对照和对照(空气)猴子的一些样品显示可测量的GLP-I水平。 Some samples from the vehicle control and control (air) monkeys show measurable levels of GLP-I. 这可由动物吸入的空气污染引起, 或可以是这些具体猴子中内源GLP-I的度量。 This may be caused by air pollution animal inhaled, or may be a measure of these particular monkeys of endogenous GLP-I. 应当注意对照动物被暴露在与GLP-1/FDKP 处理的动物不同的房间。 It should be noted that control animals were exposed to different animal GLP-1 / FDKP treated rooms.

[0270] 因为GLP-I的生物半衰期少于15分钟,所以来自GLP-1/FDKP施用的GLP-I在24 小时内应当被完全清除。 [0270] For GLP-I biological half-life less than 15 minutes, so that from the GLP-1 / FDKP administered GLP-I should be completely removed within 24 hours. 因此,GLP-I的内源水平可能解释第5天在所有GLP-1/FDKP处理的动物中收集的零时间样品中始终可计量的GLP-I水平。 Therefore, endogenous levels of GLP-I could explain the GLP-I level on day 5 all animals were collected in the GLP-1 / FDKP treated samples at time zero always quantifiable. 从观察到的给药后GLP-I浓度中减去零时间值会反映由于GLP-1/FDKP施用引起的GLP-I改变。 Observed after administration of GLP-I concentrations by subtracting the zero time value will reflect the changes since GLP-I GLP-1 / FDKP administered caused.

[0271] 对于血清FDKP分析而言,在每个时间点将全血(0. 6mL)收集进不含抗凝血剂的管中,允许在室温下凝结最少30分钟,并通过离心分离获得血清。 [0271] For serum FDKP analysis, at each time point of whole blood (0. 6mL) was collected into tubes containing no anticoagulant, allowed to clot at room temperature for minimum of 30 minutes, and centrifuged to obtain serum . FDKP分析并测定血清浓度(C max)、Tmax、AUC和T1/2。 FDKP analysis and serum concentrations (C max), Tmax, AUC, and T1 / 2. 连续四天吸附施用GLP-1/FDKP后,第5天在所有给药后样品中发现可检测的FDKP水平。 Adsorption of four consecutive days after administration of GLP-1 / FDKP, FDKP day 5 found detectable levels after administration of all the samples. 第5天,在给药施用后约10到30分钟达到FDKP的血浆浓度峰值(Cmax) 〇 On day 5, after administration is administered for about 10-30 minutes to reach peak plasma concentration of FDKP (Cmax) square

[0272] 第5天在雄性和雌性猴子中均观察到作为剂量函数的FDKP AUC °〇(从零时间推至无限时间的浓度-时间曲线下的面积)的剂量相关增加。 [0272] Day 5 male and female monkeys was observed as a function of dose of FDKP AUC ° billion (concentration from time zero to infinite time is pushed - the area under the time curve) increased dose-related. 然而在雄性中,〇. 3和I. Omg/ kg/天之间没有FDKP AUC °°的差异,但是在1和2mg/kg/天之间记录到剂量相关的提高。 However, in males, square. There was no difference in FDKP AUC °° between 3 and I. Omg / kg / day, in between 1 and 2mg / kg / day dose-related increase recorded. 在观察到增加的所有情况下,其小于与剂量成比例。 In all cases the increase was observed, which is less than dose proportional. 从0. 3到2. Omg/kg/天的6. 7倍剂量增加仅导致雄性中AUClast的2. 7倍增加和雌性中AUC c«的3. 0倍增加。 Doses from 6.7 to 0.3 2. Omg / kg / day results in only a 2.7 fold increase in AUClast in males and 3.0 fold increase in females AUC c «increases. 当分别以0. 3、I. 0 和2. Omg/kg/天的剂量水平施用GLP-1/DKP时,平均的FDKP浓度峰值(Cmax)在雄性中为200、451 和339ng/mL,在雌性中为134、161 和485ng/mL。 When respectively 0. 3, I. Dose level 0 and 2. Omg / kg / day administered GLP-1 / DKP, FDKP mean peak concentration (Cmax) of 200,451 in males and 339ng / mL, in 134,161 for females and 485ng / mL. 当分别以0· 3、L 0 和2. Omg/kg/ 天的剂量水平施用GLP-1/FDKP时,平均的FDKP AUC c«值在雄性中为307、578和817ng. h/ mL,在雌性中为268、235和810ng. h/mL。 When 3 · 0, respectively, dosage levels L 0 and 2. Omg / kg / day administered GLP-1 / FDKP, the average FDKP AUC c «value of 307,578 males and 817ng. H / mL, at 268,235 for females and 810ng. h / mL. 仅以2. lmg/kg/天施用FDKP的动物(第2组)中AUC c«和Cmax水平与接受2. 13mg/kg/天GLP-1/FDKP的动物处于相同的数量级,例外是给药施用后30到45分钟的Tmax轻微更长。 Only 2. lmg / kg / day animals administered FDKP (group 2) AUC c «animals receiving and Cmax levels 2. 13mg / kg / day GLP-1 / FDKP at the same order of magnitude, with the exception of administration Tmax of 30 to 45 minutes after administration slightly longer.

[0273] 总之,GLP-1/FDKP被良好耐受,没有临床体征或影响体重、食物消耗、临床病理学参数、肉眼或显微镜的评价。 [0273] Overall, GLP-1 / FDKP was well tolerated with no clinical signs or affect body weight, food consumption, clinical pathology parameters evaluated, the naked eye or microscope. 还记录到对短尾猴每天30分钟施用5天的GLP-1/FDKP吸入施用不具有任何剂量限制的毒性,所述施用的评估的实际剂量高达2. 13mg/kg/天(对应于0· 26mg/kg/ 天的GLP-I 剂量)。 Is also recorded on cynomolgus administered 30 minutes to 5 days per day GLP-1 / FDKP inhalation without any dose-limiting toxicity, the actual dose administered up evaluation 2. 13mg / kg / day (corresponding to 0.5 26mg / kg / day GLP-I dose).

[0274] 实施例15 [0274] Example 15

[0275] 通讨肺吹入法在大鼠中施用14天的GLP-1/FDKP的毒物代谢动力学 [0275] Pharmacokinetic administered via pulmonary insufflation discussion of 14 days in the rat GLP-1 / FDKP poison

[0276] 该研究评价了连续14天通过肺吹入法每日施用后GLP-1/FDKP的可能毒性。 [0276] This study evaluated the potential toxicity of GLP-1 / FDKP after daily administration for 14 consecutive days via pulmonary insufflation. 大鼠连续14天(η = 24/性别/组)接受对照(空气)、10mg/kg的FDKP颗粒,或1 (0· 15mg GLP-l)、3(0.45mg GLP-1)或10(1.5mg GLP-l)mg/kg GLP-1/FDKP 作为每日肺吹入。 Rats for 14 consecutive days (η = 24 / sex / group) received control (air), 10mg / kg of FDKP particles, or 1 (0 · 15mg GLP-l), 3 (0.45mg GLP-1) or 10 (1.5 mg GLP-l) mg / kg GLP-1 / FDKP as a daily pulmonary insufflation. 每天观察动物的毒性临床体征;也记录体重和食物消耗。 Clinical signs of toxicity observed in animals every day; also recorded body weight and food consumption.

[0277] 在第1天和第14天,在所有剂量组中于给药施用后约10到15分钟内达到了GLP-ICmax。 [0277] In the first and 14 days after administration to administration in all dose groups in the range of about 10 to 15 minutes to reach the GLP-ICmax. 在雄性和雌性中,10mg/kg/天GLP-1/FDKP下平均的GLP-I的浓度峰值分别为第1天6714和6270pg/mL和第14天2979和5834pg/mL。 In males and females, the mean peak concentration of GLP-I at 10mg / kg / day GLP-1 / FDKP day respectively 16,714 and 6270pg / mL and 2979 day 14 and 5834pg / mL. GLP-I的血浆水平下降,表观清除半衰期范围从0. 7小时到4. 4小时。 Plasma levels of GLP-I decreases, the apparent elimination half-lives ranging from 0.7 hours to 4.4 hours. 在10mg/kg/天GLP-1/FDKP的最高剂量下,GLP-I的平均AUC水平在雄性中为2187pM*h,在雌性中为2703pM*h。 At the highest dose of 10mg / kg / day GLP-1 / FDKP, the mean AUC levels of GLP-I in males is 2187pM * h, in females is 2703pM * h. 观察到最小或没有GLP-I蓄积, 并且C max、半衰期和Tmax没有性别差异。 We observed minimal or no accumulation of GLP-I, and C max, half-life and no gender difference Tmax. 在所有剂量下,雌性大鼠中GLP-I的AUC值轻微高于雄性大鼠中。 At all doses, AUC values ​​of GLP-I female rats than in male rats slight. 通过肺吹入法连续14天施用GLP-1/FDKP的大鼠中不可见的不良作用水平(NOAEL)为10mg/kg/ 天GLP-l/FDKP(I. 5mg/kg/ 天GLP-1)。 Rats administered by pulmonary insufflation GLP-1 / FDKP is not visible adverse effect level (NOAEL) 14 consecutive days of 10mg / kg / day GLP-l / FDKP (I. 5mg / kg / day GLP-1) .

[0278] 最终给药后约24小时处死动物(12/性别/组);进行临床病理学、肉眼的和显微镜评价。 [0278] Approximately 24 hours after the final administration the animals were killed (12 / sex / group); clinical pathology, macroscopic and microscopic evaluation. 给药的第14天在最终血收集后处死动物代谢动力学(TK)随体动物(12/性别/ 组)。 Dosing on day 14 animals were sacrificed pharmacokinetic (TK) after the final blood collection satellite animals (12 / sex / group). 不存在与GLP-1/DKP相关的死亡或临床观察结果。 Death or clinical observations related to GLP-1 / DKP does not exist. 对照和受处理的动物之间没有体重或食物消耗的差异。 No difference in body weight or food consumption between control and treatment of affected animals. 仅在l〇mg/kg GLP-1/FDKP的雌性中,肝重量和肝对体重的比例与对照(空气)组相比明显更低。 Only female l〇mg / kg GLP-1 / FDKP, the liver weights and liver to body weight ratio control (air) group was significantly lower compared.

[0279] 来自血液学、凝固、化学、尿液分析或尿化学的结果中,未记录到施用了运载体的大鼠和空气对照之间有明显的差异。 [0279] From hematology, coagulation, chemistry, or urinalysis results of urine chemistry, unrecorded administered to a significant difference between the vehicle and the air in the control rats. 组织中不存在下述肉眼发现或组织病理学发现,所述发现被确定为由于施用了GLP-1/FDKP而具有可能的毒性。 Find the naked eye or not there is a histopathological findings, the tissue is determined to be found since the administration of GLP-1 / FDKP have possible toxicity.

[0280] 实施例16 [0280] Example 16

[0281] 猴子中通讨肺吹入法施用28天的GLP-1/FDKP的毒物代谢动力学 [0281] toxicokinetics monkeys administered via pulmonary insufflation discussed Day 28 GLP-1 / FDKP kinetics

[0282] 该研究评价了至少4周通过吸入每天施用GLP-1/FDKP的毒性和毒物代谢动力学。 [0282] This study evaluated toxicity and is administered at least 4 weeks toxicokinetic GLP-1 / FDKP per day by inhalation. 为了评价任何作用的可逆性、持久性或晚发,存在4周的恢复周期。 In order to evaluate any effect reversibility, persistence or late onset, presence of 4-week recovery period.

[0283] 动物接受以下的处理之一:第1组:对照(空气);第2组:3. 67mg/kg/天FDKP颗粒;第3 组:0· 3mg/kg/ 天GLP-1/FDKP (0· 045mg/kg/ 天GLP-1);第4 组:lmg/kg/ 天GLP-I/ FDKP (0· 15mg/kg/ 天GLP-1)或第5 组:2. 6mg/kg/ 天GLP-1/FDKP (0· 39mg/kg/ 天GLP-1)。 [0283] Animals received one of the following process: Group 1: control (air); Group 2:. 3 67mg / kg / day FDKP particles; Group 3: 0 · 3mg / kg / day GLP-1 / FDKP (0 · 045mg / kg / day GLP-1); group 4:. lmg / kg / day GLP-I / FDKP (0 · 15mg / kg / day GLP-1) or group 5: 2 6mg / kg / day GLP-1 / FDKP (0 · 39mg / kg / day GLP-1).

[0284] 将四十二只短尾猴分为2组:第1、2和5组中的恢复(η = 2/性别/组)和主要研究(η = 3/性别/组)。 [0284] The forty-two cynomolgus monkeys were divided into 2 groups: group 5 and recovery of 1,2 (η = 2 / sex / group) and main (η = 3 / sex / group). 第1组:空气对照,第2组:FDKP (〜4mg/kg/天);第3组:0· 3mg/ kg/ 天GLP-l/FDKP (低剂量);第4 组:· Omg/kg/ 天GLP-l/FDKP (中剂量);第5 组:2. 6mg/ kg/天GLP-l/FDKP (高剂量)。 Group 1: air control Group 2: FDKP (~4mg / kg / day); Group 3: 0 · 3mg / kg / day GLP-l / FDKP (low dose); Group 4: · Omg / kg / day GLP-l / FDKP (dose); group 5:. 2 6mg / kg / day GLP-l / FDKP (high dose). 典型地,在猴子研究中只有高剂量和对照在恢复时评价。 Typically, only the high-dose evaluation and control in the recovery in the monkey study.

[0285] 每天两次观察动物的死亡率或发病率,至少每天一次在给药后30分钟观察异常和毒性症状。 [0285] observed twice daily mortality or morbidity animal, at least once a day for 30 minutes and abnormal signs of toxicity were observed after dosing. 每周收集体重数据,每天评价定性的食物消耗。 Weekly weight data collection, evaluation of qualitative food consumption every day. 在第1、28和56天收集血用于毒物代谢动力学。 Blood was collected for pharmacokinetics poisons 1,28 and 56 days. 在第29天将三只动物/性别/组麻醉、称重、放血和尸体剖检。 In the first 29 days to three animals / sex / group anesthetized, weighed, bled and necropsy. 在第57 天将第1、2和5组中的剩余动物(η = 2/性别/组)麻醉、称重、放血和尸体剖检。 In the first 57 days and in the 5 remaining Group 2 animals (η = 2 / sex / group) were anesthetized, weighed, exsanguinated, and necropsied. 尸体剖检时,将选定的器官称重,收集选定的组织并防腐。 At necropsy, selected organs were weighed, and tissue collection selected corrosion. 显微镜检查来自每个动物的所有组织。 Microscopic examination of all tissues from each animal.

[0286] 所有组的体重存在偶然的波动;但是不存在与处理相关的对体重的影响。 [0286] body weight of all groups present accidental fluctuations; however, the absence of treatment-related effects on body weight. 一般地, 所有的动物在研究过程中维持体重或增加小量体重。 In general, all the animals maintain body weight during the study or a small increase in body weight. 在高剂量下观察到更高发病率和频率的松弛或液体粪便。 We observed a higher incidence and frequency of loose or liquid feces at high doses. 未记录到被认为是与处理相关的任何临床化学参数的显著改变,例外是第29天(处理结束时)高剂量雌性中乳酸脱氢酶(LDH)和天冬氨酸氨基转移酶(AST) 的中度增加;见表7。 Is considered to be unrecorded and any significant change processing related to clinical chemistry parameters, with the exception on day 29 (end of treatment) in high dose females lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) moderate increase; Table 7. 雄性中LDH的水平也轻微提高。 LDH levels in males is also slightly improved. 这些改变在恢复周期结束时已经消除,并且与肝中任何显微镜发现不相关。 These changes at the end of the recovery period has been eliminated, and found that the liver is not related to any microscope. 高剂量雄性组中AST水平的改变主要归因于五个动物之一。 Change the high-dose male group AST levels primarily due to one of the five animals.

[0287] 表7:ALT、AST和LDH的均值改变% [0287] Table 7: ALT, AST and LDH mean% change

Figure CN104288756AD00371

[0288] [0288]

[0289] [0289]

[0290] 在高达2. 6mg/kg/天GLP-1/FDKP的剂量水平下,不存在任何与处理相关的肉眼可见的改变或组织学改变的迹象。 [0290] In the up 2. 6mg / kg / day GLP-1 / FDKP dose level of, or change in sign of histological changes related to the processing of any tissue visible absent. 在高达2. 6mg/kg/天GLP-1/FDKP(0. 39mg/kg/天GLP-1) 的剂量下,GLP-1/FDKP被良好耐受,没有显著的临床体征,不影响体重、食物消耗、血液学、 尿液分析、胰岛素分析、检眼镜检查、ECG,没有观察到的肉眼可见的或显微镜下的改变。 Up to 2. 6mg / kg / day GLP-1 / FDKP at (0. 39mg / kg / day GLP-1) dose, GLP-1 / FDKP was well tolerated with no significant clinical signs, does not affect the weight, food consumption, hematology, urinalysis, insulin analysis, ophthalmoscopy, ECG, no change was observed at the macroscopic or microscopic. 28 天内以高达3. 67mg/kg/天的评估的实际剂量用高达每天30分钟吸入施用FDKP也不与任何毒性相关。 28 days to assess the actual dose of up to 3. 67mg / kg / day up to 30 minutes with FDKP inhalation administration per day is not associated with any toxicity.

[0291] 第1天在雄性和雌性猴子中均观察到作为剂量函数的GLP-I和FDKPCmax和AUC last 的剂量相关增加。 [0291] Day 1 in both male and female monkeys was observed as a function of dose and dose FDKPCmax GLP-I and the associated increase in AUC last. 在研究的剂量范围中,第28天在雄性和雌性猴子中用递增的剂量均观察到少于与剂量成比例的GLP-ICmax增加,而未发现AUC last增加。 In the dose range studied, 28 male and female monkeys were observed with increasing doses of less than proportional to dose increase GLP-ICmax, without found to increase AUC last. 2. 6mg/kg/天GLP-1/FDKP 下平均的GLP-I浓度峰值在雄性中为259pg/mL,在雌性中为164pg/mL。 The average concentration of GLP-I 2. 6mg / kg / day GLP-1 / FDKP as a peak in males 259pg / mL, in females was 164pg / mL. GLP-I的血浆水平降低,清除半衰期范围从0. 6到2. 5小时变化。 Plasma levels of GLP-I is reduced, elimination half-life range varies from 0.6 to 2.5 hours. 高剂量下GLP-I的平均AUC值在雄性中为103pg*hr/mL,在雌性中为104pg*hr/mL。 At high doses the mean AUC values ​​of GLP-I in males as 103pg * hr / mL, in females as 104pg * hr / mL. 2. 6mg/kg/ 天GLP-1/FDKP 下平均的FDKP 浓度峰值在雄性中为1800ng/mL,在雌性中为1900pg/mL。 Under 2. 6mg / kg / day GLP-1 / FDKP averaged FDKP concentration peak in males of 1800ng / mL, in females was 1900pg / mL.

[0292] 总而言之,对短尾猴的GLP-1/FDKP吸入施用被临床良好地耐受,所述施用以高达2. 6mg/kg/天GLP-1/FDKP或0· 39mg/kg/天GLP-I的评估的实际剂量、以高达每天30分钟吸入施用GLP-l/FDKP。 [0292] In summary, for the GLP-1 / FDKP to cynomolgus inhalation is well tolerated clinically, the administration of up to 2. 6mg / kg / day GLP-1 / FDKP or 0 · 39mg / kg / day GLP -I actual dose assessment of up to 30 minutes a day inhalation GLP-l / FDKP. NOAEL 为2. 6 mg/kg/ 天GLP-1/FDKP (0· 39mg/kg/ 天GLP-1)。 NOAEL was 2. 6 mg / kg / day GLP-1 / FDKP (0 · 39mg / kg / day GLP-1). 如下文实施例19中所述,I期研究中的最大人剂量应为每天I. 5mg GLP-1/FDKP或〜0. 021mg/ kg GLP-1(假定为70kg的人)。 As described in Example 19, the maximum human dose in the Phase I study day should I. 5mg GLP-1 / FDKP or ~0. 021mg / kg GLP-1 (assumed to be 70kg person). 其它的研究剂量应为每天3. Omg GLP-1/FDKP或〜0. 042mg/ kg GLP-I。 Other studies dose should day 3. Omg GLP-1 / FDKP or ~0. 042mg / kg GLP-I.

[0293] 实施例17 [0293] Example 17

[0294] 制各毒蜥外泌狀/FDKP制剂 [0294] each manufactured by exendin-like / FDKP formulation

[0295] 通过将酸性毒蜥外泌肽-4肽(SEQ ID No. 3)溶液与FDKP颗粒悬浮液组合来制备毒蜥外泌肽-4/FDKP。 [0295] By the outer acidic exendin-4 peptide (SEQ ID No. 3) solution with a FDKP particle suspension composition is prepared exendin peptide -4 / FDKP. 酸性肽溶液为溶解在2%乙酸中的10% (w/w)肽。 Acidic peptide solution dissolved in 2% acetic acid in 10% (w / w) peptide. FDKP悬浮液含有约10 % (w/w) FDKP颗粒。 FDKP suspension contained about 10% (w / w) FDKP particles. 将酸性毒蜥外泌肽-4肽溶液添加进FDKP颗粒悬浮液中,同时柔和地混合。 The outer acidic exendin-4 peptide solution was added to the FDKP particle suspension, while mixing gently. 用25%氨溶液将毒蜥外泌肽-4/FDKP混合物逐步滴定至pH 4.50。 With 25% ammonia solution -4 / FDKP mixture exendin peptide titrated to pH 4.50. 然后将混合物在液氮中制成小球并冻干。 The mixture is then pelletized in liquid nitrogen and lyophilized.

[0296] 15%毒蜥外泌肽-4/FDKP粉末的填充量可吸入级分% RF on Fill)含量为36%,药筒排空百分比(Percent Cartridge Emptying)为99%。 Filling amount [0296] 15% exendin peptide -4 / FDKP powder inhalable fraction% RF on Fill) content of 36% cartridge emptying percentage (Percent Cartridge Emptying) was 99%. 以类似规模生产的15% GLP-1/FDKP粉末显示34%的% RF on Fill,药筒排空百分比为100%。 Display a similar scale production 15% GLP-1 / FDKP powder was 34% of the% RF on Fill, cartridge emptying percentage is 100%.

[0297] 实施例18 [0297] Example 18

[0298] 通讨肺吹入法施用的毒蜥外泌狀/FDKP的药物代谢动力学 [0298] Exendin-like / FDKP administered drug metabolism via pulmonary insufflation Discussion Kinetics

[0299] 进行重复的剂量初始毒性研究,以检查多种浓度下和通过肺部途径多次施用后毒蜥外泌肽-4(GLP-1类似物)的药物动力学和药物代谢动力学模式。 [0299] The initial repeated dose toxicity studies to examine various concentrations and pharmacokinetic and pharmacokinetic profile after administration of exendin peptides via the pulmonary route multiple -4 (analogue GLP-1) is .

[0300] 在大鼠和猴子中进行了28天的研究。 [0300] 28 days studies conducted in rats and monkeys. 通过吸入途径每天进行毒蜥外泌肽/FDKP 给药。 By inhalation pathway / FDKP administered daily exendin peptide. 在对动物给药28天的研究中,一部分动物要在给药方案后立即处死,而其它动物允许在处死前有多达一个月的恢复周期。 In the study animals were dosed for 28 days, a part of the animal to be sacrificed immediately after the dosing regimen while other animals are allowed up to one month recovery period prior to sacrifice. 评价所有动物的临床体征,多种生理参数,包括毒蜥外泌肽-4、葡萄糖和胰岛素的血水平;器官重量和多种器官的临床病理学和组织病理学。 Evaluation of all the animals clinical signs, various physiological parameters including -4, blood glucose and insulin levels of exendin peptide; clinical pathology and histopathology of various organs and organ weights.

[0301] 初始的研究组由每组五只动物组成并具有两个对照组:空气和静脉施用毒蜥外泌肽。 [0301] Initial Study Groups of five animals per group and having two control groups: air and Exendin administered intravenously peptide. 存在六个肺吹入组,其接受约2. Omg剂量的,5%、10%、15%、20%和25%和30%毒蜥外泌肽负载(w/w)的毒蜥外泌肽/FDKP。 There are six pulmonary insufflation groups which receives about 2. Omg dose, 5%, 10%, 15%, 20% and 25% and 30% exendin exendin peptide loading (w / w) of peptide / FDKP. 收集全血用于血糖和毒蜥外泌肽浓度,直到8小时的时间点。 Whole blood was collected for blood glucose and exendin peptide concentrations for the 8 hour time point.

[0302] 收集数据(Cmax、T1/2和Tmax),其证实毒蜥外泌肽/FDKP制剂具有相对于GLP-1/FDKP 可比较的或更好的药物代谢动力学。 [0302] collecting data (Cmax, T1 / 2 and Tmax), which was confirmed exendin peptide / FDKP formulation with respect to the GLP-1 / FDKP or may be better pharmacokinetic comparison.

[0303] 实施例19 [0303] Example 19

[0304] 大鼠中通讨肺吹入法施用的GLP-1/xDKP的药物代谢动力学 [0304] Pulmonary administration via insufflation discuss GLP-1 / xDKP pharmacokinetics in rats

[0305] 为了确定不同的DKP是否可以影响GLP-1/FDKP制剂的药物代谢动力学模式,如题为"Asymmetrical FDKP Analogs for Use as Drug Delivery Agents"的美国临时专利申请所公开,制造多种GLP-l/xDKP制剂,所述申请在与本文一致的日期提交并以其整体并入本文(代理人案号No. 51300-00041)。 [0305] To determine whether different modes DKP can affect the pharmacokinetics of the drug GLP-1 / FDKP formulations, such as, entitled "Asymmetrical FDKP Analogs for Use as Drug Delivery Agents" U.S. Provisional Patent Application Publication, manufactured more GLP- l / xDKP formulation, and the filed is incorporated in its entirety (Attorney docket No. 51300-00041) herein consistent with the date herein.

[0306] 在被分为6个处理组的大鼠中进行研究,所述组由每组5只动物组成。 [0306] In the study rats were divided into six treatment groups, the group consisting of 5 animals per group. 对照组(η =3)通过液体滴注接受GLP-1。 Controls (η = 3) by the liquid instillation receiving GLP-1. 还使用通过肺吹入施用的GLP-1/FDKP(0. 3mg GLP-1)作为第二对照。 Also be used by pulmonary insufflation administered GLP-1 / FDKP (0. 3mg GLP-1) as a second control. 每个GLP-1/xDKP处理的组通过肺吹入以负载10%和15% GLP-I的〜2. Omg的xDKP剂量接受GLP-1/xDKP制剂。 Each group of GLP-1 / xDKP treated by pulmonary insufflation at 10% load and 15% GLP-I of ~2. Omg dose of xDKP receiving GLP-1 / xDKP formulation. 使用的xDKP为(E) -3- (4- (3, 6-二氧代哌嗪-2-基)丁基氨基甲酰基)-丙烯酸)、(3, 6-二(4-羧丙基)氨基丁基-2, 5-二酮哌嗪)和((E) -3, 6-二(4-(羧基-2-丙烯基)氨基丁基)-2,5-二酮哌嗪二钠盐)负载。 XDKP used is (E) -3- (4- (3, 6- dioxo-piperazin-2-yl) butylcarbamoyl) - acrylic acid), (3, 6-bis (4- carboxypropyl ) aminobutyl-2,5-diketopiperazine), and ((E) -3, 6- bis (4- (carboxy-2-propenyl) aminobutyl) -2,5-diketopiperazine two sodium salt) load. 在给药后5、10、20、30、 45、60直到90分钟收集全血用于评价GLP-I浓度。 After administration 5,10,20,30, 45, 60, up to 90 minutes to collect the whole blood GLP-I concentration for evaluation.

[0307] 实施例20 [0307] Example 20

[0308] 健康成年雄件夸试者中GLP-1/FDKP吸入粉末的Ia期、单一剂量、开放标签、递增剂量、夸控的安全件和耐夸件试骀 [0308] boast subjects were healthy adult male member GLP-1 / FDKP inhalation powder Ia period, single dose, open-label, ascending dose, the safety member and the resistance member boast boast tired controlled trial

[0309] 通过静脉(iv)或皮下(sc)灌输或通过多重皮下注射给予时,GLP-I已经显示在人中控制提高的血糖。 When [0309] administered by multiple subcutaneous injections or infusion by intravenous (iv) or subcutaneously (sc), GLP-I has been shown to control elevated blood glucose in humans. 因为激素的半衰期非常短,可能需要连续皮下灌输或多次每日皮下注射。 Because the half-life of the hormone is very short, you may need a continuous subcutaneous infusion or multiple daily subcutaneous injection. 这些途径均不适用于长期临床用途。 These pathways are not applicable to long-term clinical use. 动物实验显示通过吸入施用GLP-I时可达到治疗水平。 Animal experiments show that therapeutic levels can be achieved by inhalation administration of GLP-I.

[0310] GLP-I的若干种作用(包括减少胃排空、提高饱满感和抑制不适当的高血糖素分泌)似乎与进餐开始时的GLP-I释放爆发有关。 [0310] Several of the role of GLP-I (including reducing gastric emptying and increase satiety and inhibition of inappropriate glucagon secretion) seems meal GLP-I released at the start of outbreaks. 通过用GLP-1/FDKP吸入粉末补充GLP-I 的该早期波动,可以在糖尿病动物中引起药物动力学应答。 By treatment with GLP-1 / FDKP inhalation powder was added to the GLP-I early fluctuation may cause drug response kinetics in diabetic animals. 另外,可以通过餐后施用GLP-I/ FDKP吸入粉末模拟与提高的胰岛素分泌相关的天然GLP-I的晚期波动。 Further, by postprandial administration of GLP-I / FDKP inhalation powder analog late secretory phase fluctuation related to the native GLP-I with increased insulin.

[0311] GLP-1/FDKP吸入粉末的Ia期临床试验被设计为在人受试者中第一次测试选定剂量的新型吸入式血糖控制治疗产品的安全性和可耐受性。 Safety and tolerability [0311] GLP-1 / FDKP inhalation powder Ia clinical trial is designed to be the first time in human subjects selected dose tested new inhaled glycemic control therapeutic product. 给药利用了先前测试过的MedTone®吸入器设备。 Administration using the previously tested MedTone® inhaler device. 该临床试验的主要目的在于鉴定通过肺吸入的GLP-1/FDKP吸入粉末的剂量范围,所述剂量范围是安全的、可耐受的并可在临床试验中进一步用于建立有效性和安全性的证据。 The main purpose of the clinical trial that was identified by pulmonary inhalation a dose range of GLP-1 / FDKP inhalation powder, the dosage range is safe, tolerable and can be further used to establish the effectiveness and safety in clinical trials evidence of. 针对Ia期临床试验选择的剂量是以下述动物安全性结果为基础的, 所述动物安全性结果来自上述实施例中描述的GLP-1/FDKP吸入粉末在大鼠和灵长类中的非临床试验。 Ia clinical trial doses for selected animal safety results based on the following basis, the animal safety results from GLP-1 / FDKP described in the above embodiments nonclinical inhalation powder in rats and primates test.

[0312] 二十六(26)个受试者被登记在5队中,达到第1和2队中每队多达4个可评估的受试者,第3到5队中每队多达6个可评估的受试者,所述可评估的受试者满足合格标准并且完成临床试验。 [0312] bis-six (26) subjects are registered in the 5 team, 2 teams to reach each of the first team and up to 4 evaluable subjects, the team of 3 to 5 up to each team 6 may be evaluated in a subject, the subject may be evaluated to meet the eligibility criteria and complete the clinical trial. 每个受试者用作为GLP-1/FDKP吸入粉末的高血糖素样肽-I(GLP-I)给药一次,剂量水平如下:第1队:0. 05mg ;第2队:0. 45mg ;第3队:0. 75mg ;第4队:1. 05mg 和第5队:1. 5mg的GLP-1。 Each subject used as the GLP-1 / FDKP inhalation powder of glucagon-like peptide -I (GLP-I) is administered at a dose level as follows: 1 Team: 0 05mg; 2 Team:.. 0 45mg ; 3 team:. 0 75mg; 4 teams:.. 1 05mg teams and 5: 1 5mg of GLP-1. 不替换中途退出者。 Not replacing those who dropped out. 这些剂量假定70kg的体重。 These doses assume 70kg weight. 本领域技术人员能够根据上文公开的研究确定额外的剂量水平。 Those skilled in the art can determine additional dosage levels according to the studies disclosed above.

[0313] 该试验的目的是确定健康成年的男性受试者中递增剂量的GLP-1/FDKP吸入粉末的安全性和耐受性。 [0313] The purpose of this test is to determine healthy adult male subjects incremental doses of GLP-1 / FDKP inhalation safety and tolerability of the powder. 将评价递增剂量的GLP-1/FDKP吸入粉末的耐受性,其通过监测药理学或对变量的不利作用确定,包括报道的不利事件(AE)、生命体征、物理检查、临床实验室测试和心电图(ECG)。 The evaluation of increasing doses of GLP-1 / FDKP inhalation powder tolerated, as determined by monitoring pharmacological or adverse effect on the variables, including adverse events reported (AE), vital signs, physical examination, clinical laboratory tests and electrocardiogram (ECG).

[0314] 第二个目的是评价另外的安全性和药物动力学参数。 [0314] The second objective was to evaluate additional safety and pharmacokinetic parameters. 这包括另外的安全性参数, 表述为肺和其它AE的发病率和调查1(筛选)与调查3 (随访)之间肺功能的改变;用GLP-1/FDKP吸入粉末给药后血浆GLP-I和血清富马酰二酮哌嗪(FDKP)的药物代谢动力学(PK)参数,通过AUC ch12ck分,+ )血浆GLP-I和AUCch48ci分钟血清FDKP测量;和另外的血浆GLP-IPK参数,包括:tmax血浆GLP-I ;Cmax血浆GLP-I ;和T1/2血浆GLP-I。 These include additional safety parameters, as expressed in AE incidence of lung and other surveys and 1 (screening) changes in lung function between 3 (follow-up) to the inquiry; with GLP-1 / FDKP inhalation powder administration plasma GLP- I and serum fumaryl diketopiperazine (FDKP) pharmacokinetic (PK) parameters, AUC ch12ck by points, +) plasma GLP-I and serum FDKP measurements AUCch48ci minutes; and other parameters of plasma GLP-IPK, comprising: tmax of plasma GLP-I; Cmax plasma GLP-I; and T1 / 2 of plasma GLP-I. 另外的血清FDKP PK参数包括:Tmax血清FDKP ;Cmax血清FDKP ;和Τ1/2血清FDKP。 Additional serum FDKP PK parameters include: Tmax serum FDKP; Cmax serum the FDKP; and Τ1 / 2 serum FDKP.

[0315] 试验终末点(endpoint)基于在试验受试者群体中测定的以下药理学和安全性参数的比较。 [0315] The following comparison is based on pharmacological and safety parameters determined in the trial subject population test endpoint (endpoint). 初级终末点应包括:安全性终末点将根据报道的AE(包括咳嗽和呼吸困难、恶心和/或呕吐)发病率和严重性、来自生命体征筛选的改变、临床实验室测试和物理检查来评价。 Primary endpoints should include: end-point security based on AE reports (including cough and difficulty breathing, nausea and / or vomiting) incidence and severity change from screening in vital signs, clinical laboratory tests and physical examination evaluated. 次级终末点应包括:血浆GLP-I和血清FDKP的PK分布(AUC ch12c^肖血浆GLP-I和AUC〇_分钟血清FDKP);另外的血浆GLP-IPK参数(Tmax血浆GLP-I、Cmax血浆GLP-I、T 1/2血浆GLP-1);另外的血清FDKP PK参数(Tmax血清FDKP,Cmax血清FDKP);和另外的安全性参数(肺功能测试(PFTs))和ECG。 Secondary endpoints include: plasma GLP-I and serum FDKP PK profile (AUC ch12c ^ Shore plasma GLP-I and serum FDKP AUC〇_ min); additional plasma GLP-IPK parameters (Tmax plasma GLP-I, Cmax plasma GLP-I, T 1/2 plasma GLP-1); additional serum FDKP PK parameters (Tmax serum FDKP, Cmax serum the FDKP); and additional safety parameters (pulmonary function tests (of PFTs)) and ECG.

[0316] Ia期、单一剂量试验整合了开放标签、递增剂量结构和符合21CFR312, Good Clinical Practice :Consolidated Guidance(ICH-E6)和Guidance on General Considerations for Clinical Trials (ICH-E8)的设计策略,用于测定研究的药物产品(MP)安全性和耐受性。 [0316] Ia period, single dose trial incorporates open-label, ascending dose structure and meet the 21CFR312, Good Clinical Practice: Consolidated Guidance (ICH-E6) and Guidance on General Considerations for Clinical Trials (ICH-E8) design strategy, with in the determination of the drug product (MP) safety and tolerability.

[0317] 临床试验应由3个临床调查组成:1) 一个筛选调查(调查1) ;2) -个治疗调查(调查2);和3)调查2后8-14天的一个随访调查(调查3)。 [0317] Clinical trials Clinical Investigation 3 shall be composed of: 1) a screening visit (Investigation 1); 2) - two treatment investigation (investigation 2); and 3) a follow-up survey investigation 2 (8-14 days of the investigation 3). 单一剂量的GLP-1/FDKP吸入粉末的施用将在调查2期间发生。 Administration of a single dose of GLP-1 / FDKP inhalation powder will occur during the investigation 2.

[0318] 该临床试验会评价每队中的安全性参数。 [0318] The clinical trial will evaluate the safety parameters of each team. 在主要调查人(PI)回顾第一次给药或先前给药的所有安全性和耐受性数据之前,不对计划接受下一剂量浓度的小队进行给药。 Before the principal investigator (PI) review all safety and tolerability data for the first time the administration or previous administration, are not scheduled to receive the next dose concentration squad administered. 应在每队的受试者之间执行半小时的给药滞后时间,以确保受试者安全。 It should perform half-hour lag time between the administration of each subject team, to ensure subject safety. 如果小队中3个或更多的受试者经受严重的恶心和/或呕吐,或当达到最大剂量时,或在PI的判断下,可中止给药。 If three or more teams in the subject experience severe nausea and / or vomiting or when the maximum dose, or in the determination of PI, can suspend delivery.

[0319] 要评价5 种剂量的GLP-1/FDKP 吸入粉末(0· 05、0· 45、0· 75、L 05 和I. 5mg 的GLP-1)。 [0319] To rating of doses of GLP-1 / FDKP inhalation powder (0 · 05,0 · 45,0 · 75, L GLP-1 05 and the I. 5mg). 为了适应所有的剂量,配制好的GLP-1/FDKP将与FDKP吸入粉末混合。 To accommodate all doses, formulated GLP-1 / FDKP will be mixed with FDKP inhalation powder. 将原样使用或与适当量的FDKP吸入粉末混合使用含IOmg干粉的单一剂量药筒,所述干粉由GLP-I/ FDKP吸入粉末组成(15%重量比重量的GLP-l/FDKP),以获得想要的GLP-l(0.05mg、0.45 mg、0. 75mg、l. 05mg和I. 5mg)剂量:1.最低的2个剂量水平将在每队4个受试者的2队中评价,3个较高的剂量食品将在每队6个受试者的3队中评价。 Will be used as a powder mixture or a single dry powder dose cartridge containing IOmg with an appropriate amount of FDKP inhalation, the dry powder of GLP-I / FDKP inhalation powder composition (ratio by weight 15% by weight of a GLP-l / FDKP), to obtain desired GLP-l (0.05mg, 0.45 mg, 0 75mg, l 05mg and I. 5mg..) dose: 1 2 lowest dose levels will be evaluated every 2 team 4 team subject, 3 higher doses of the food will be evaluated for each team three teams six subjects. 每个受试者仅接受待评价的5个剂量水平中的1个剂量。 Each subject received only 5 dose levels to be evaluated in a dose. 除了取血进行GLP-I (活性的和总体的)和FDKP测量外, 取样用于高血糖素、葡萄糖、胰岛素和C-肽测定。 In addition to blood for GLP-I (active and total) and FDKP measurements, the sample was taken for glucagon, glucose, insulin, and C- peptide determination.

[0320] 在该说明书全文中已经引用了大量专利和印刷的出版物。 [0320] In the already cited throughout this specification to a number of patents and printed publications. 每个上述参考文献和印刷的出版物通过引用以其整体个别地并入本文。 Each of the above cited references and printed publications are individually incorporated herein by reference in its entirety.

[0321] 除非另有指明,本说明书和权利要求书中使用的表示成分数量,以及分子量、反应条件等性质的所有数字都应当被理解为:在所有情况下,用术语"约"加以了修饰。 All figures represent the number of component properties [0321] Unless otherwise specified, the specification and the claims used, and the molecular weight, reaction conditions, etc. are to be understood as follows: In all cases, by the term "about" is to be modified . 因此,除非有相反含义的说明,本说明书和所附权利要求书中示出的数量参数都是约数,它们可以根据本发明想要获得的性质而变动。 Accordingly, unless indicated to the contrary explanation, this specification and the appended claims the number of parameters are illustrated in the book about the number of requests, which may vary according to the nature of the invention sought to be obtained. 至少,并且并非对权利要求书范围等同原则的应用加以限制,每个数量参数至少应按照报道的有效数字的数,以及应用普通的凑整技术来解释。 At least, and not to limit the scope of application of the doctrine of equivalents of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. 虽然示出本发明宽广范围的数字范围和参数是约数,但是特别实施例中所示的数值却被尽可能地精确报道。 Although a broad scope of the present invention, the numerical ranges and parameters are approximations, the numerical values ​​shown in the particular embodiment of the reports was precisely as possible. 但是,任何数值,必然含有一定误差,这是它们各自的检验测量方法中发现的标准偏差必然导致的。 However, any value, contains certain errors necessarily, a standard deviation found in their respective testing measurements methods will inevitably lead to the discovery.

[0322] 本领域技术人员容易理解,在不脱离本发明的范围和精神的前提下,可对本发明进行各种改进。 [0322] Those skilled in the art will readily appreciate, without departing from the scope and spirit of the present invention is the premise of the present invention may be variously modified.

[0323] 在本文中,在权利要求书和/或说明书中与"包括"一起使用的"一个"一般表示"一个",但其表示的意思与"一个或多个"、"至少一个"和"一个或多于一个"也是一致的。 [0323] Herein, in the claims and / or specification and "comprising" "a" for use with a general mean "one," but it represents the meaning "one or more", "at least one" and "one or more" is also the same.

[0324] 本文所述的任何方法或组合物可通过本文所述的任何其它方法或组合物来实现。 [0324] Any of the methods described herein or compositions may be achieved by any other method or composition described herein.

[0325] 除非明确指出某个可选方式或可选方式之间互相排斥,权利要求中所用术语"或" 是指"和/或"。 [0325] repel each other unless explicitly stated or alternatively an alternative embodiment, the claims, the term "or" means "and / or."

[0326] 在本文中,术语"约"所表示的值包括确定该值所用设备或方法的标准偏差。 [0326] Herein, the value of the term "about" is represented by the value includes determining a standard deviation with a device or method.

[0327] 根据实施例以及权利要求书提供的先前描述,可以清楚地看出本发明的其它目的、特征和优点。 [0327] According to the embodiment described and claimed in the previous claims is provided, it is clear that other objects, features and advantages of the present invention. 然而应当理解,详细描述和具体实施例仅为示例性,而本领域技术人员根据这些详细描述在本发明的精神和范围内可以作出各种改变和改进。 However, it should be understood that the detailed description and specific embodiments are merely exemplary, and those skilled in the art according to the detailed description can make various changes and modifications within the spirit and scope of the invention.

[0328] 参考文献 [0328] Reference

[0329] 在提供补充本文公开内容的示范性步骤或其它细节的程度上,以下的参考文献通过引用被明确地并入本文。 [0329] the extent that they provide exemplary disclosures herein complementary steps or on other details, the following references are expressly incorporated herein by reference.

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Claims (38)

1. 一种干粉组合物,其包含含有吸附了GLP-1分子的多肽的二酮哌嗪的微粒,其中所述GLP-1分子选自由以下物质组成的组:天然的GLP-1、GLP-1代谢产物、GLP-1类似物、 GLP-1衍生物、二肽基肽酶-IV(DPP-IV)保护的GLP-UGLP-1模拟物、GLP-1肽类似物或生物合成的GLP-1类似物。 1. A dry powder composition comprising particles comprising diketopiperazine adsorbed polypeptide GLP-1 molecule, consisting of the following substances wherein the GLP-1 molecule selected from the group consisting of: native GLP-1, GLP 1 metabolites, GLP-1 analogs, GLP-1 derivative, a dipeptidyl peptidase -IV (DPP-IV) protected GLP-UGLP-1 mimetics, GLP-1 peptide analog, or a biosynthetic GLP- 1 analogs.
2. 权利要求1的干粉组合物,其中所述二酮哌嗪是具有式2, 5-二酮-3, 6-二(4-X-氨丁基)哌嗪的二酮哌嗪,其中X选自由琥珀酰基、戊二酰基、马来酰基和富马酰基组成的组。 2. The dry powder composition of claim 1, wherein said diketopiperazine having the formula 2,5-dione 3,6-two (4-X- aminobutyl) piperazine diketopiperazine, wherein X selected from the group consisting of succinyl, glutaryl, maleyl and fumaryl thereof.
3. 权利要求2的干粉组合物,其中所述二酮哌嗪是2, 5-二酮-3, 6-二(4-富马酰-氨丁基)哌嗪。 Dry powder composition of claim 2, wherein said diketopiperazine is 2,5-dione 3,6-bis (4-fumaryl - aminobutyl) piperazine.
4. 权利要求1的干粉组合物,其中所述GLP-1分子是天然的GLP-1。 4. The dry powder composition of claim 1, wherein said GLP-1 molecule is native GLP-1.
5. 权利要求1的干粉组合物,其中所述GLP-1分子是酰胺化的GLP-1分子。 5. The dry powder composition of claim 1, wherein said GLP-1 molecule is an amidated GLP-1 molecule.
6. 权利要求5的干粉组合物,其中所述酰胺化的GLP-1分子是GLP-1 (7-36)酰胺。 6. The dry powder composition of claim 5, wherein the amidated GLP-1 molecule is GLP-1 (7-36) amide.
7. 用于形成包含吸附了GLP-1分子的二酮哌嗪的颗粒的方法,所述方法包括步骤: 提供GLP-1分子; 提供下述形式的二酮哌嗪,所述形式选自能形成颗粒的二酮哌嗪、二酮哌嗪颗粒及其组合;和将所述GLP-1分子与所述二酮哌嗪以共溶液的形式组合,其中形成包含所述吸附了GLP-1分子的二酮哌嗪的所述颗粒,并且其中所述GLP-1分子选自由以下物质组成的组:天然的GLP-1、GLP-1代谢产物、GLP-1类似物、GLP-1衍生物、二肽基肽酶-IV(DPP-IV)保护的GLP-1、GLP-1模拟物、GLP-1肽类似物或生物合成的GLP-1类似物。 7. A method for forming diketopiperazine comprising adsorbed particles GLP-1 molecule, said method comprising the steps of: providing a GLP-1 molecule; providing a diketopiperazine of the form, the form can be selected forming diketopiperazine particles, diketopiperazine particles, and combinations thereof; and the GLP-1 molecule and the diketopiperazine in the form of a combination of co-solution, wherein said adsorbent comprises forming a GLP-1 molecule the diketopiperazine particles, and wherein said GLP-1 molecule is selected from the group consisting of: native GLP-1, GLP-1 metabolites, GLP-1 analogs, GLP-1 derivatives, dipeptidyl peptidase -IV (DPP-IV) protected GLP-1, GLP-1 mimetics, GLP-1 peptide analog, or a biosynthetic GLP-1 analog.
8. 权利要求7的方法,其还包括通过冻干法、过滤或喷雾干燥从所述共溶液中去除溶剂。 8. A method as claimed in claim 7, further comprising a by lyophilization, filtration, or spray drying said co-solvent is removed from the solution.
9. 权利要求8的方法,其中通过去除所述溶剂形成包含所述吸附了GLP-1分子的二酮哌嗪的所述颗粒。 9. The method of claim 8, wherein the solvent is removed by forming the particles of the adsorbent comprising diketopiperazine GLP-1 molecule.
10. 权利要求8的方法,其中在去除所述溶剂之前形成包含所述吸附了GLP-1分子的二酮哌嗪的所述颗粒。 10. The method as claimed in claim 8, comprising the adsorbent particles of the diketopiperazine, wherein the GLP-1 molecule is formed prior to removing the solvent.
11. 权利要求7的方法,其中所述GLP-1分子以溶液的形式提供,所述溶液包含约1 μ g/ml_50mg/ml 的GLP-1 浓度。 11. The method of claim 7, wherein said GLP-1 molecule provided in the form of a solution, said solution comprising a GLP-1 concentrations from about 1 μ g / ml_50mg / ml of.
12. 权利要求7的方法,其中所述GLP-1分子以溶液的形式提供,所述溶液包含约0· lmg/ml-10mg/ml 的GLP-1 浓度。 12. The method of claim 7, wherein said GLP-1 molecule provided in the form of a solution, said solution comprising a GLP-1 concentrations from about 0 · lmg / ml-10mg / ml of.
13. 权利要求7的方法,其中所述GLP-1分子以溶液的形式提供,所述溶液包含约0· 25mg/ml 的GLP-1 浓度。 13. The method of claim 7, wherein said GLP-1 molecule provided in the form of a solution, said solution comprising a GLP-1 concentrations from about 0 · 25mg / ml of.
14. 权利要求7的方法,其中所述二酮哌嗪以二酮哌嗪颗粒悬浮液的形式提供。 14. The method of claim 7, wherein said diketopiperazine is a diketopiperazine provided as a suspension of particles.
15. 权利要求7的方法,其中所述二酮哌嗪以包含能形成颗粒的二酮哌嗪的溶液的形式提供,所述方法还包括调节所述溶液的pH以形成二酮哌嗪颗粒。 15. The method of claim 7, wherein said diketopiperazine comprises a diketopiperazine in the form of particle-forming solution provided, said method further comprising adjusting the pH of said solution to form diketopiperazine particles.
16. 权利要求14或15的方法,其还包括向所述溶液或悬浮液中添加试剂,其中所述试剂选自由盐、表面活性剂、离子、渗透物、离液剂和感胶离子、酸、碱和有机溶剂组成的组。 16. A method as claimed in claim 14 or 15, further comprising adding a reagent to the solution or suspension, wherein the agent is selected from the group consisting of salts, surfactants, ions, the permeate, chaotropes and lyotropes, acid the group bases and organic solvents.
17. 权利要求16的方法,其中所述试剂促进所述GLP-1分子和所述二酮哌嗪颗粒或所述能形成颗粒的二酮哌嗪之间的缔合。 17. The method of claim 16, wherein said agent promoting the GLP-1 molecule and the diketopiperazine particles or the association between can form diketopiperazine particles.
18. 权利要求16的方法,其中所述试剂改善所述GLP-1分子的稳定性或药物动力学。 18. The method of claim 16, wherein the agent to improve the stability of the GLP-1 molecule or pharmacokinetics.
19. 权利要求16的方法,其中所述试剂为氯化钠。 19. The method of claim 16, wherein said agent is sodium chloride.
20. 权利要求14或15的方法,其还包括调节所述悬浮液或溶液的pH。 20. The method of claim 14 or claim 15, further comprising adjusting the pH of the suspension or solution.
21. 权利要求20的方法,其中所述pH被调节至约4或更大。 21. The method of claim 20, wherein the pH is adjusted to about 4 or greater.
22. 权利要求7的方法,其中所述颗粒中的所述GLP-1分子具有更高的稳定性。 22. A method as claimed in claim 7, wherein said particles of said GLP-1 molecule has a higher stability.
23. 权利要求7的方法,其中所述共溶液包含约1 μ g/ml-50mg/ml的GLP-1浓度。 23. The method of claim 7, wherein said co-solution comprises a GLP-1 concentrations from about 1 μ g / ml-50mg / ml of.
24. 权利要求7的方法,其中所述共溶液包含约0. lmg/ml-10mg/ml的GLP-1浓度。 24. The method of claim 7, wherein said co-solution comprises a GLP-1 concentrations from about 0. lmg / ml-10mg / ml of.
25. 权利要求7的方法,其中所述共溶液包含约0. 25mg/ml的GLP-1浓度。 25. The method of claim 7, wherein said co-solution comprises / GLP-1 ml a concentration of about 0. 25mg.
26. 权利要求7的方法,其还包括向所述共溶液中添加试剂,其中所述试剂选自由盐、 表面活性剂、离子、渗透物、离液剂和感胶离子、酸、碱和有机溶剂组成的组。 26. A method as claimed in claim 7, further comprising adding a reagent to said co-solution, wherein the agent is selected from the group consisting of salts, surfactants, ions, the permeate, chaotropes and lyotropes, acids, bases and organic group solvents.
27. 权利要求26的方法,其中所述试剂促进所述GLP-1分子和所述二酮哌嗪颗粒或所述能形成颗粒的二酮哌嗪之间的缔合。 27. The method of claim 26, wherein said agent promoting the GLP-1 molecule and the diketopiperazine particles or the association between can form diketopiperazine particles.
28. 权利要求26的方法,其中所述试剂改善所述GLP-1分子的稳定性或药物动力学。 28. The method of claim 26, wherein the agent to improve the stability of the GLP-1 molecule or pharmacokinetics.
29. 权利要求26的方法,其中所述试剂为氯化钠。 29. The method of claim 26, wherein said agent is sodium chloride.
30. 权利要求7的方法,其还包括调节所述共溶液的pH。 30. A method as claimed in claim 7, further comprising adjusting the pH of said co-solution.
31. 权利要求30的方法,其中所述pH被调节至约4或更大。 31. The method of claim 30, wherein the pH is adjusted to about 4 or greater.
32. 吸附了GLP-1分子的二酮哌嗪微粒在制造用于治疗病症或疾病的药物中的用途, 其中所述病症或疾病选自由糖尿病、局部缺血、再灌注组织损伤、血脂障碍、糖尿病性心脏病、心肌梗塞、急性冠状动脉综合征、肥胖症、手术后的分解代谢改变、高血糖症、过敏性肠综合征、中风、神经变性病症、记忆和学习障碍、胰岛细胞移植和再生性治疗组成的组,其中所述GLP-1分子选自由以下物质组成的组:天然的GLP-1、GLP-1代谢产物、GLP-1类似物、 GLP-1衍生物、二肽基肽酶-IV(DPP-IV)保护的GLP-1、GLP-1模拟物、GLP-1肽类似物或生物合成的GLP-1类似物。 32. diketopiperazine microparticles adsorbed GLP-1 molecule in the manufacture of medicament for treating a disorder or disease, wherein said disorder or disease is selected from the group consisting of diabetes, ischemia, reperfusion injury, dyslipidemia, diabetes, heart disease, myocardial infarction, acute coronary syndrome, obesity, catabolic changes after surgery, hyperglycemia, irritable bowel syndrome, stroke, neurodegenerative disorders, memory and learning disorders, islet cell transplant and regenerative the group consisting of treatment, wherein the substance consisting of the following GLP-1 molecule is selected from the group consisting of: native GLP-1, GLP-1 metabolites, GLP-1 analogs, GLP-1 derivative, a dipeptidyl peptidase -IV (DPP-IV) protected GLP-1, GLP-1 mimetics, GLP-1 peptide analog, or a biosynthetic GLP-1 analog.
33. 权利要求32的用途,其中所述药物通过静脉内、皮下、经口、经鼻、经颊、经直肠或通过肺部递送进行给药。 33. The use as claimed in claim 32, wherein said medicament intravenously, subcutaneously, orally, nasally, buccally, or rectally administered by pulmonary delivery.
34. 权利要求32的用途,其中所述药物通过肺部递送进行给药。 34. The use as claimed in claim 32, wherein said medicament is administered by pulmonary delivery.
35. 形成具有改善的GLP-1药物代谢动力学模式的粉末组合物的方法,所述方法包括步骤: 提供GLP-1分子,其中所述GLP-1分子选自由以下物质组成的组:天然的GLP-UGLP-1 代谢产物、GLP-1类似物、GLP-1衍生物、二肽基肽酶-IV(DPP-IV)保护的GLP-1、GLP-1模拟物、GLP-1肽类似物或生物合成的GLP-1类似物; 提供溶液中的能形成颗粒的二酮哌嗪; 形成二酮哌嗪颗粒; 将所述GLP-1分子与所述溶液组合形成共溶液;和通过喷雾干燥从所述共溶液中去除溶剂,形成具有改善的GLP-1药物代谢动力学模式并且包含下述微粒的粉末,所述微粒含有吸附了GLP-1分子的二酮哌嗪。 35. A method of forming powder composition GLP-1 having improved pharmacokinetic profile, said method comprising the steps of: providing a GLP-1 molecule, consisting of the following substances wherein the GLP-1 molecule is selected from the group consisting of: natural GLP-UGLP-1 metabolites, GLP-1 analogs, GLP-1 derivative, a dipeptidyl peptidase -IV (DPP-IV) protected GLP-1, GLP-1 mimetics, GLP-1 peptide analogs or a biosynthetic GLP-1 analogs; provide particle-forming diketopiperazine in a solution; forming diketopiperazine particles; the GLP-1 molecule co-form the solution composition and solution; and by spray drying removed from the co-solvent in the solution to form a GLP-1 has an improved pharmacokinetic profile and comprises the following powder particles, said particles containing adsorbed diketopiperazine GLP-1 molecule.
36. 权利要求35的方法,其中所述改善的GLP-1药物代谢动力学模式包含提高的GLP-1半衰期。 GLP-1 pharmacokinetic model 36. The method of claim 35, wherein the improvement comprises an increased GLP-1 half-life.
37. 权利要求36的方法,其中所述提高的GLP-1半衰期大于或等于7. 5分钟。 37. The method of claim 36, wherein said increased GLP-1 half-life greater than or equal to 7.5 minutes.
38. 权利要求35的方法,其中所述改善的GLP-1药物代谢动力学模式包括与天然GLP-1相比改善的GLP-1生物利用度。 38. The method of claim 35, wherein said GLP-1 improved pharmacokinetic profile compared to native comprising a GLP-1 improved bioavailability of GLP-1.
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