CN102199206B - Insulin analogue having quick response and stability under acidic condition and preparation thereof - Google Patents

Insulin analogue having quick response and stability under acidic condition and preparation thereof Download PDF

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CN102199206B
CN102199206B CN 201110064530 CN201110064530A CN102199206B CN 102199206 B CN102199206 B CN 102199206B CN 201110064530 CN201110064530 CN 201110064530 CN 201110064530 A CN201110064530 A CN 201110064530A CN 102199206 B CN102199206 B CN 102199206B
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insulin
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acting
pharmaceutical
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CN102199206A (en
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王大梅
彭毅
金太河
王继胜
胡玉华
甘忠如
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甘李药业股份有限公司
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Abstract

本发明涉及一种快速起效且在酸性条件下稳定的胰岛素类似物及其药物组合物和药物制剂。 The present invention relates to insulin analogues and pharmaceutical compositions thereof, and one rapid-onset pharmaceutical formulation and stable under acidic conditions. 该胰岛素类似物的A链A21位的天冬酰胺(Asn)突变为甘氨酸(Gly),并可以与长效胰岛素类似物如甘精胰岛素混合,制成同时具有速效降糖和平稳长效降糖两种功能的预混制剂,解决现有天然胰岛素(猪、牛、人)和速效胰岛素类似物在酸性环境下不稳定,以及现有胰岛素预混制剂不澄清,需引入外源蛋白(例如鱼精蛋白)作缓释剂而易产生免疫反应,每日需早、晚注射两次的问题,使制剂安全性更高。 A chain at position A21 of the insulin analog of asparagine (Asn) is mutated to glycine (Gly), and may be mixed with insulin glargine as long-acting insulin analogues, antidiabetic made quick and smooth while having a long-acting hypoglycemic premix formulation two functions, where existing native insulin (porcine, bovine, human), and fast-acting insulin analog is unstable, and does not clarify the existing insulin premix formulation in an acidic environment, need to introduce an exogenous protein (e.g. fish protamine) as sustained release apparent immune response, the daily need early, late injection twice problem, the formulation higher security.

Description

快速起效且在酸性条件下稳定的胰岛素类似物及其制剂技术领域[0001] 本发明涉及人胰岛素类似物,具体地讲,涉及一种能快速起效且在酸性条件下稳定的胰岛素类似物,以及包含该胰岛素类似物的药物组合物和药物制剂。 Rapid onset and stable under acidic conditions and an insulin analog formulation Technical Field [0001] The present invention relates to a human insulin analogue, in particular, to a fast onset of action and stable in acidic conditions insulin analog , as well as pharmaceutical compositions and pharmaceutical preparations containing the insulin analogues. 背景技术[0002] 糖尿病是一种常见的内分泌代谢疾病。 [0002] Diabetes is a common endocrine and metabolic diseases. 近年来,全世界糖尿病的患病率都在迅猛增长。 In recent years, the prevalence of diabetes worldwide are increasing rapidly. 而在中国,随着人民生活方式的改变和老龄化进程的加速,糖尿病的患病率呈快速上升趋势,在2010年中国糖尿病患者总数已突破9000万人,成为继心脑血管疾病、肿瘤之后的另一个严重危害人民健康的重要慢性非传染性疾病。 In China, with the acceleration of change and the aging process of people's lifestyle, the prevalence of diabetes increased rapidly, in 2010 the total number of diabetics China has exceeded 90 million people, after becoming the heart and cerebrovascular diseases, cancer important chronic non-communicable diseases are another serious harm to people's health. 糖尿病的急、慢性并发症,尤其是慢性病并发症累及多个器官,致残、致死率高,严重影响患者的身心健康,并给个人、家庭和社会带来沉重的负担。 Diabetes acute and chronic complications, especially chronic complications involving multiple organs, disability, death rate, seriously affecting the patient's physical and mental health, and to individuals, families and society a heavy burden. [0003] 胰岛素治疗一直被当作是治疗糖尿病并使血糖得到良好控制的重要手段。 [0003] Insulin therapy has been used as an important means to treat diabetes and blood sugar well controlled. 正常人体的生理胰岛素分泌由餐时刺激相和基础平稳相组成,即人们进餐后外周血糖增高,刺激胰脏中贮存和新生的胰岛素释放,而平时为了维持机体的正常血糖水平,胰腺分泌少量基础胰岛素。 Normal human physiological insulin secretion stimulated by the meal-based phase and stationary phase, i.e. after meal blood glucose in people increased storage stimulate insulin release and newborn pancreas, but usually in order to maintain the body's normal blood sugar levels, the pancreas small amounts of base insulin. 因此,为了使糖尿病患者的餐时和基础血糖都得到良好控制,患者需要在每日餐前注射速效胰岛素(每日三次),并在每日早晨和睡前各注射一次中或长效胰岛素。 Therefore, in order to make the meals and diabetes patients are well controlled blood sugar basis, patients need rapid-acting insulin injections (three times daily), and once daily in the morning and before bed or long-acting insulin injections before meals daily. 这样, 糖尿病患者必须承受每天4-5次的注射次数。 In this way, diabetes patients must bear the number of injections 4-5 times a day. 为此,人们开发了在同一制剂中即有起速效作用的可溶胰岛素又有起延缓作用的结晶胰岛素(胰岛素-鱼精蛋白结晶)的预混胰岛素制剂(参见《百明顿制药科学》(Remington,s Pharmaceutical Sciences),第17版,第973-977页,1985年)。 To this end, it has developed a fast-acting role that is played in the same formulation of soluble insulin have played a role in delaying the crystallization of insulin (insulin - protamine crystallization) of premixed insulin preparations (see "one hundred Farmington Pharmaceutical Sciences" ( Remington, s Pharmaceutical Sciences), 17th edition, pages 973-977, 1985). 但是,这些预混胰岛素制剂仍然存在以下问题:1.制剂中含有外源蛋白一鱼精蛋白,易产生免疫反应;2.药效在注射后持续时间不足24小时,患者仍需每日注射两次;3.药效仍存在峰值,每天两次药效峰叠加,易造成午餐前和睡前低血糖;4.现有预混胰岛素制剂均为混悬液,如果患者注射前混合不均匀,易造成药液中可溶相和结晶相比例的持续差异,影响血糖控制。 However, these premixed insulin preparations still has the following problems: a formulation containing the exogenous protein a protamine, easy to produce an immune response; 2 less than the duration of effect 24 hours after injection, the patient still two daily injections. views; 3 peak efficacy still exist, twice daily peak efficacy superimposed, before lunch and before bedtime easily lead to hypoglycemia; 4 premixed prior insulin preparations are suspensions, if the patient prior to injection of uneven mixing, easily lead to continuous phase ratio of the difference in the soluble phase and liquid crystals affect blood glucose control. [0004]目前,市场上还有一种长效胰岛素类似物一甘精胰岛素,其由于能够模拟生理基础胰岛素分泌,平稳无峰,在血糖轻易控制达标的同时,几乎没有低血糖风险,且持续时间为24小时左右,符合人类生活作息周期,每天只需注射一次,而被患者和医生所青睐。 [0004] Currently on the market there is a long-acting insulin analog insulin glargine, which can simulate the physiological basis since insulin secretion, smooth no peak at the same time easily control the glucose standards, there is little risk of hypoglycemia, and the duration about 24 hours, in line with rest of human life cycle, a single injection once a day, and was favored by both patients and physicians. 但是,甘精胰岛素在临床应用时也存在一些问题:1.起效慢(起效时间约为I. 5小时),对餐时血糖过高的患者,或偶尔进餐较多的患者无法有效降低其餐后血糖;2.不能与其他胰岛素混合使用,这是由于各种胰岛素的等电点不同,甘精胰岛素在酸性条件下稳定,而天然胰岛素及现有速效胰岛素类似物在中性条件下稳定,如果将甘精胰岛素与其他胰岛素直接混合,会导致甘精胰岛素在注射前发生沉淀而不能施用,而其他胰岛素在混合后也由于受混合物酸碱度变化的影响,稳定性降低,安全性也随即降低。 However, insulin glargine there are some problems in clinical application: 1. slow onset (onset time of about I. 5 hours), in patients with high blood sugar meal, or a meal occasionally patients can not be more effectively reduced postprandial blood glucose; 2 can not be mixed with other insulin, due to the different isoelectric points of various insulin, insulin glargine stable under acidic conditions, conventional fast-acting insulin and native insulin analogues under neutral conditions stable, if the insulin glargine directly mixed with other insulin glargine cause precipitation prior to injection can not be administered, but also due to the influence of other insulin mixture pH changes, reducing stability after mixing, then security is also reduce. 因此,在甘精胰岛素的包装说明书上,生产商特别注明不要将甘精胰岛素与其他任何类型的胰岛素混合。 Thus, in a package insert insulin glargine, manufacturers do not otherwise stated glargine mixed with any other type of insulin. [0005] 在中国专利申请200780019450. 3中,提出通过添加特定的酸性稳定剂和螯合剂, 将常规人胰岛素制剂VIAjectTM在低pH的条件下与甘精胰岛素混合,以实现共同使用人胰岛素与甘精胰岛素。 [0005] In the Chinese patent application 200780019450.3, made acidic by adding a specific stabilizer and a chelating agent, a conventional VIAjectTM mixed with human insulin glargine at low pH, to achieve common use of human insulin and Gan fine insulin. 但是,该方法一方面需要在制剂中额外添加有机酸、螯合剂等辅料•'另一方面,在低PH的条件下,胰岛素A链21位的Asn非常容易脱氨降解(参见《胰岛素的稳定性》(Stability of insulin), Jens Brange, 1994年),使得产品极不稳定,而该专利申请并未对低PH条件下人胰岛素的稳定性、以及联合制剂的稳定性进行系统研究,产品的安全性数据不全。 However, this method requires an additional aspect of adding an organic acid, chelating agents and other materials in the formulation • 'On the other hand, under conditions of low PH, the A chain of insulin at position 21 is Asn readily degradable stable deaminase (see "insulin of "(stability of insulin), Jens Brange, 1994 years), so that the product is extremely unstable, and the stability of this patent application No Human insulin low PH conditions, and system stability studies combined preparation, product security incomplete data. 此外,该专利申请也没有从胰岛素的分子结构方面解决产品稳定性和保持产品临床特性的问题。 Furthermore, this patent application does not solve the molecular structure of insulin to maintain product stability and clinical characteristics of the product problems. 发明内容[0006] 本发明的目的是解决现有上述胰岛素预混制剂所存在的问题,提供一种在酸性条件下稳定且快速起效的胰岛素类似物或其可药用盐,使其能够与甘精胰岛素等长效胰岛素类似物混合,制成澄清制剂,该制剂在使用前无需混匀即可注射使用,同时具有速效降糖和平稳长效降糖两种功能,每天只需注射一次,即可模拟生理胰岛素的分泌模式,在血糖安全达标的同时可更大程度减少低血糖风险,并且不含外源蛋白和额外的辅料,安全性更高。 SUMMARY OF THE INVENTION [0006] The object of the present invention is to solve the above-described conventional insulin premix formulation problems, to provide a stable under acidic conditions and rapid-acting insulin analogue or a pharmaceutically acceptable salt thereof, and it is possible to long-acting insulin, glargine insulin analogue mixed to obtain a clear formulation, the formulation can be mixed prior to use without the use of injection, while having a quick and smooth long-lasting hypoglycemic hypoglycemic two functions, a single injection once per day, to simulate physiological insulin secretion patterns, to reduce the risk of hypoglycemia, while the degree of blood may be greater safety standards, and is free of exogenous protein and additional excipients, higher security. [0007] 在低pH的条件下,胰岛素A链A21位的Asn (天冬酰胺)非常容易发生脱氨降解反应,使得胰岛素分子在酸性条件下极不稳定。 [0007] under conditions of low pH, insulin A-chain at position A21 Asn (asparagine) is prone to degradation deamination reaction, such an insulin molecule is very unstable under acidic conditions. 本发明人通过大量研究发现,将胰岛素A链A21位的Asn替换为Gly (甘氨酸),所得到的全新序列的胰岛素类似物在酸性pH值时具有出人意料的稳定性,并且在酸性和中性PH值时都具有较高的溶解度,从而解决了上述问题。 The present inventors found that a large number of the A chain of insulin replace Asn at position A21 is Gly (glycine), the new insulin analogue having a sequence obtained unexpected stability at acidic pH and in acidic and neutral PH have a higher solubility value, so as to solve the above problems. [0008]因此,为了实现本发明的目的,本发明提供一种胰岛素类似物或其可药用盐,其在酸性条件下稳定且能快速起效,所述胰岛素类似物为至少将其A链A21位的天冬酰胺(Asn) 突变为甘氨酸(Gly)的人胰岛素,其中人胰岛素的结构如图I所示,人胰岛素A链序列为SEQ ID NO: I所示的氨基酸序列,人胰岛素B链序列为SEQ ID NO :2所示的氨基酸序列。 [0008] Accordingly, in order to achieve the object of the present invention, the present invention provides an insulin analogue or a pharmaceutically acceptable salt thereof, which is capable of rapid onset stable under acidic conditions, the insulin analog is at least the A chain which A21 bit asparagine (Asn) is mutated to glycine (Gly) human insulin, human insulin in which the structure shown in FIG. I, human insulin a-chain sequence is SEQ ID NO: I in the amino acid sequence of human insulin B chain sequence of SEQ ID NO: 2 amino acid sequence. [0009] 优选地,所述胰岛素类似物为仅在A21位上的Asn被Gly取代的人胰岛素,即GlyA21人胰岛素,其A链序列为SEQ ID NO :3所示的氨基酸序列,B链序列为SEQ ID NO :4 所示的氨基酸序列。 [0009] Preferably, the insulin analog is only Asn in position A21 is substituted with Gly human insulin, i.e., GlyA21 human insulin, the A chain sequence is SEQ ID NO: 3 the amino acid sequence shown in, B chain sequence of SEQ ID NO: 4 amino acid sequence. [0010] 优选地,所述胰岛素类似物为A21位上的Asn被Gly取代,B28位上的Pro被Lys 取代,并且B29位上的Lys被Pro取代的人胰岛素,即GlyA21-LysB28-Pr0B29人胰岛素,其A链序列为SEQ ID NO : 5所示的氨基酸序列,B链序列为SEQ ID NO :6所示的氨基酸序列。 [0010] Preferably, the insulin analog is Asn at position A21 is substituted with Gly, Pro at position B28 is substituted with the Lys and Lys at position B29 on the Pro is substituted human insulin, i.e., human GlyA21-LysB28-Pr0B29 insulin, the a chain sequence is SEQ ID NO: amino acid sequence of 5, B-chain sequence is SEQ ID NO: 6 amino acid sequence shown. [0011] 优选地,所述胰岛素类似物为A21位上的Asn被Gly取代,B28位上的Pro被Asp 取代的人胰岛素,即GlyA21-AspB28人胰岛素,其A链序列为SEQ ID NO :7所示的氨基酸序列, B链序列为SEQ ID NO :8所示的氨基酸序列。 [0011] Preferably, the insulin analog is Asn at position A21 is substituted with Gly, Pro on position B28 is substituted with Asp human insulin, i.e., GlyA21-AspB28 human insulin, the A chain sequence is SEQ ID NO: 7 amino acid sequence shown, B-chain sequence is SEQ ID NO: 8 amino acid sequence. [0012] 本发明还包括上述胰岛素类似物的可药用盐,所述可药用盐包括但不限定于锌盐、钠盐、钾盐、镁盐、钙盐。 [0012] Pharmaceutically acceptable salts of the present invention further comprises the above-described insulin analogs, the pharmaceutically acceptable salts include, but are not limited to zinc, sodium, potassium, magnesium, calcium. [0013] 本发明胰岛素类似物或其可药用盐可以采用任何一种公认的肽合成技术制备,例如溶液法、固相合成法(J. Stewart等,《固相肽合成》,Freeman and Co. , San Francisco, 1969)、半合成法、基因工程法、DNA重组法等。 [0013] The insulin analogs of the present invention or a pharmaceutically acceptable salt thereof can be prepared by any of the recognized peptide synthesis techniques using, for example, solutions, solid phase synthesis method (J. Stewart et al., "Solid Phase Peptide Synthesis", Freeman and Co ., San Francisco, 1969), semi-synthetic methods, genetic engineering, DNA rearrangement method and the like. [0014] 上述结构优化过的胰岛素类似物或其可药用盐从分子结构上消除了胰岛素在酸性条件下不稳定的因素,使得本发明胰岛素分子在酸性条件下不易发生脱氨基反应,具有非常高的稳定性。 [0014] The optimized structure of the insulin analogue or a pharmaceutically acceptable salt thereof to eliminate factors of instability of insulin under acidic conditions from the molecular structure, so that the insulin molecules of the present invention is less prone to deamination reaction under acidic conditions, with very high stability. 因此该胰岛素类似物或其可药用盐可以与长效胰岛素类似物例如甘精胰岛素一起组成药物组合物,或制成澄清的酸性预混胰岛素制剂。 Thus the insulin analogue or a pharmaceutically acceptable salt thereof may be composed of long-acting insulin analogs such as glargine pharmaceutical composition together, or acidic insulin preparations made clear premix. [0015]因此,本发明另一方面提供一种药物组合物,其包含本发明胰岛素类似物或其可药用盐,以及长效胰岛素类似物或其可药用盐,其中所述本发明胰岛素类似物为至少在其A 链A21位突变为甘氨酸(Gly)的人胰岛素。 [0015] Accordingly, another aspect of the present invention provides a pharmaceutical composition comprising an insulin analogue of the invention or a pharmaceutically acceptable salt thereof, and long-acting insulin analogue or a pharmaceutically acceptable salt thereof, wherein the insulin of the present invention analog is at least the a chain mutant in which position A21 is glycine (Gly) human insulin. [0016] 优选地,所述本发明胰岛素类似物选自GlyA21人胰岛素、GlyA21-LysB28-proB29人胰岛素或GlyA21-AspB28人胰岛素,所述长效胰岛素类似物选自甘精胰岛素。 [0016] Preferably, the insulin analogues of the present invention is selected from GlyA21 human insulin, GlyA21-LysB28-proB29 GlyA21-AspB28 human insulin or human insulin, a long acting insulin analogue selected from insulin glargine. [0017] 优选地,在上述药物组合物中,本发明胰岛素类似物和长效胰岛素类似物的含量百分比为30 : 70-70 : 30,更优选为50 : 50。 [0017] Preferably, in the pharmaceutical composition, the content percentage of long-acting insulin analogue and insulin analogues of the present invention is from 30: 70-70: 30, more preferably 50: 50. [0018] 本发明另一方面提供一种包含上述药物组合物的胰岛素药物制剂,优选地,该药物制剂的PH为3. 5-4. 5,更优选为3. 8-4. 2。 [0018] aspect of the invention provides a pharmaceutical formulation of insulin comprises the above pharmaceutical composition, preferably the pharmaceutical formulation PH 3. 5-4. 5, more preferably 3. 8-4. 2. [0019] 上述药物制剂还可进一步包含一种或多种可药用辅料,例如等渗剂、缓冲剂、增溶剂、防腐剂等。 [0019] The pharmaceutical formulations may further comprise one or more pharmaceutically acceptable adjuvants, such as isotonic agents, buffering agents, solubilizers, preservatives and the like. 其中等渗剂包括但不限于甘油、氯化钠等;缓冲剂包括但不限于磷酸盐缓冲液、乙酸盐缓冲液等;增溶剂包括但不限于生理可接受酸如盐酸、乙酸、柠檬酸等;防腐剂包括但不限于苯酚、间甲酚、对羟基苯甲酸甲酯等。 Wherein the isotonic agents include but are not limited to, glycerin, sodium chloride, and the like; buffering agents include, but are not limited to, phosphate buffer, acetate buffer, and the like; solubilizing agents include but are not limited to physiologically acceptable acid such as hydrochloric acid, acetic acid, citric acid and the like; preservatives include, but are not limited to, phenol, m-cresol, methyl p-hydroxybenzoate and the like. [0020] 上述药物制剂为澄清液体制剂,其可通过注射、口服、口腔、舌下、阴道、直肠或鼻腔施用的方式来施用。 [0020] The pharmaceutical formulation is a clear liquid formulation, which can be administered by way of injection, oral, buccal, sublingual, vaginal, rectal or nasal administration. 优选的,该药物制剂是通过注射来施用的。 Preferably, the pharmaceutical preparation is to be administered by injection. 在施用时,患者可以根据自身生活习惯选择在每日主餐前或主餐后注射一次。 When administered, the patient can choose the main injection once daily before or after meals according to their main habits. [0021] 在本发明酸性预混胰岛素制剂中,两种胰岛素类似物都有很好的溶解度,并且物理化学稳定性不变、临床作用特点也不变。 [0021] In the present invention, the acidic premix in insulin preparations, two insulin analogues have good solubility and physical and chemical stability unchanged, the same characteristics are also clinical effects. 在皮下注射后,长效胰岛素可在皮下注射后形成微小沉淀起长效作用,而本发明速效胰岛素类似物在体内中性PH下仍可保持较高的溶解度而发挥速效作用。 After subcutaneous injection, long-acting insulin may be formed from fine precipitate was long-lasting effect after subcutaneous injection, and fast-acting insulin analogs of the present invention can still maintain a relatively high solubility at neutral PH quick play role in vivo. [0022] 本发明预混胰岛素制剂具有以下优点:1.制剂澄清,因而在使用前无需混匀即可抽取准确剂量注射;2.临床作用时间上兼有现有速效胰岛素起效快、长效胰岛素平稳降糖的优点;3.患者可以根据自身生活习惯选择在每日主餐前或主餐后注射一次,即可模拟生理胰岛素的分泌模式;4.既能解决每日主餐后高血糖,又不易在其他时间特别是夜间出现低血糖,使血糖维持在一个相对平稳的状态,在使血糖安全达标的同时最大程度减少低血糖风险,保护糖尿病患者的微血管,防止或延缓糖尿病并发症的发生,进一步提高糖尿病患者的生活质量。 [0022] premixed insulin formulations of the invention has the following advantages: 1 formulation clarification, without mixing prior to use and thus can extract accurate dose injection; 2 on both the conventional fast-acting insulin clinical effects play time fast onset, long-acting. hypoglycemic advantage of stable insulin; 3 patients injected meal may be selected once, to mimic physiological insulin secretion pattern before or in the main daily according to their main habits; 4 daily solve both main postprandial hyperglycemia , but also difficult at other times especially at night hypoglycemia, blood sugar remained at a relatively stable state, blood safety standards at the same time minimizing the risk of hypoglycemia, diabetic microvascular protection, prevent or delay the complications of diabetes occur, to further improve the quality of life of diabetes patients. [0023] 本发明胰岛素类似物在动物体内未见明显毒性反应。 [0023] The insulin analogs of the present invention in animals no significant toxicity. 药效学评价结果显示,本发明预混胰岛素制剂在注射后10分钟开始起效,1-8小时内一直有平稳的降糖作用,12小时时作用仍明显,能满足临床糖尿病患者要求注射次数少、起效快、持续时间长、降糖平稳、低血糖发生率低的需求。 The evaluation results show pharmacodynamic, premixed insulin formulations of the invention was started 10 min after injection of onset, have been stable hypoglycemic effect within 1-8 hours, is still significant effect at 12 hours, the number of times to meet the requirements of clinical diabetes injection small, rapid onset, long duration, stable hypoglycemic, hypoglycemia low demand. 附图说明[0024] 图I :人胰岛素的结构。 BRIEF DESCRIPTION [0024] FIG. I: The structure of human insulin. [0025] 图2 :重组甘赖脯胰岛素各时点电泳表达检测图谱。 [0025] FIG. 2: Gan recombinant insulin lispro electrophoresis to detect the expression pattern of each time point. [0026] 图3 :重组甘赖脯胰岛素RP-HPLC检测图谱。 [0026] FIG. 3: Recombinant insulin lispro Gan detection RP-HPLC profiles. [0027] 图4:重组甘赖脯胰岛素与甘精胰岛素以50 : 50的百分比预混后的高效液相色谱的保留时间测定图谱。 [0027] FIG. 4: Gan recombinant insulin lispro and glargine 50: HPLC retention time profiles measured after 50 percentage premix. [0028] 图5 :不同胰岛素对四氧嘧啶高血糖大鼠作用的降糖百分率)。 [0028] FIG 5: different percentage of hypoglycemic action of insulin on alloxan induced hyperglycemic rats). 具体实施方式[0029] 实施例I本发明胰岛素类似物的制备和确认[0030]材料:[0031]菌株:[0032] 克隆菌种Escherichia coli(DH5a)是基因工程常用工具菌种, [0033] 表达菌种Escherichia coli(3110)购于美国模式培养物集存库(ATCC)。 [0031] Strain:: Example I Preparation and confirmation insulin analogue of the present invention [0030] Materials DETAILED DESCRIPTION [0029] Embodiment [0032] Cloning bacteria Escherichia coli (DH5a) is a genetically engineered strain common tool, [0033] The expression strain Escherichia coli (3110) were purchased from American Type culture collection library (ATCC). 酶和试剂:[0034] 分子克隆工具酶和试剂购于北京经科宏达公司。 Enzymes and reagents: [0034] Molecular cloning tool enzymes and reagents were purchased from Beijing Branch Hongda Company. [0035] 质粒抽提试剂盒、PCR纯化试剂盒购于北京天根公司(TianGen)。 [0035] The plasmid extraction kit, PCR purification kit was purchased from Beijing days Allergan (TianGen). [0036] 定点突变试剂盒(Fast Mutagenesis System)购于全式金公司(TransGene)。 [0036] Directed Mutagenesis Kit (Fast Mutagenesis System) available from the full CICC formula (TransGene). [0037] 培养基:[0038] LB培养基、氨苄抗性LB培养基、M9培养基和富集培养基为基因工程领域常用培养基,各培养基的配方可参见分子克隆第三版下册附录2。 [0037] Medium: [0038] LB medium, ampicillin-resistant LB media, and M9 media enriched media commonly used for genetic engineering media, each medium formulation may be found in Molecular Cloning, Third Edition Volume Appendix 2. [0039]方法:[0040] 质粒提取扩增、聚合酶链反应、PCR产物纯化、质粒回收、连接和转化大肠杆菌, 这些在基因工程研究领域都是常规操作方法,可参见Sambrook J, Fristsh EF, Maniatis T. MolecularCloning ;A Laboiatory Manual 2nd ed. NY :Cold Spring Harbor Laboratory Piess,1989,pp.16-340。 [0039] Method: [0040] plasmid extraction amplification, polymerase chain reaction, the PCR product was purified, recovered plasmid, ligation and transformation of E. coli, these are conventional genetic engineering operations research methods, see Sambrook J, Fristsh EF , Maniatis T. MolecularCloning; A Laboiatory Manual 2nd ed NY:. Cold Spring Harbor Laboratory Piess, 1989, pp.16-340. [0041] I. GlyA21-LysB28-ProB29人胰岛素(甘赖脯胰岛素)的制备:[0042] (I)重组甘赖脯胰岛素质粒的构建[0043] 赖脯胰岛素质粒的扩增:将根据现有LySB28-PiOB29人胰岛素(赖脯胰岛素)序列制得的赖脯胰岛素质粒转入到Escherichia coli (DH5a)中,然后加入到氨节抗性LB培养基中,在摇床中225rpm,37°C培养16小时,对赖脯胰岛素质粒进行扩增,然后用质粒抽提试剂盒提取质粒。 Preparation I. GlyA21-LysB28-ProB29 human insulin (insulin lispro Gan) is [0041]: [0042] Construction [0043] insulin lispro plasmid amplification (I) insulin lispro Gan recombinant plasmids: according to the prior LySB28-PiOB29 human insulin (insulin lispro) sequence of insulin lispro prepared plasmid was transferred to the Escherichia coli (DH5a), then added to the section of the ammonia-resistant LB media, 225rpm, 37 ° C shaker in culture 16 hours of insulin lispro plasmid was amplified, and plasmid was extracted using a plasmid extraction kit. [0044] PCR定点突变:使用定点突变试剂盒(Fast Mutagenesis System),将扩增的赖脯胰岛素质粒稀释60倍作为定点突变的模板,以设计的两条引物为上下游引物,进行PCR扩 士SS >曰O [0045] 其中引物序列如下:[0046] N-Gf : CTCGAGAACTACTGCGGCTAATGAAGCTTGATC[0047] N-Gr : CCGCAGTAGTTCTCGAGCTGGTACAGGCTG[0048] PCR体系为:[0049] 质粒模板(5ng) lul,[0050] 上游引物N-Gf(IOuM)O. 5uL,[0051] 下游引物N-Gr (IOuM) O. 5uL,[0052] 5倍Trans Start快速Pfu缓冲液5uL,[0053] dNTPs(IOmM)IuL,[0054] TransStart 快速Pfu DNA 聚合酶O. 7uL,[0055] 无菌水16. 3uL,[0056] 总体积:25uLo[0057] 扩增条件为-MV 4min ;[0058] 94°C 30sec,55°C 30sec,72°C 2. 5min 25 个循环;[0059] 72°C IOmin。 [0044] PCR site-directed mutagenesis: using site-directed mutagenesis kit (Fast Mutagenesis System), the amplification of insulin lispro plasmid was diluted 60 times as a template for site-directed mutagenesis in order to design two primers upstream and downstream primers for PCR amplification disabilities SS> said O [0045] wherein the primer sequences are as follows: [0046] N-Gf: CTCGAGAACTACTGCGGCTAATGAAGCTTGATC [0047] N-Gr: CCGCAGTAGTTCTCGAGCTGGTACAGGCTG [0048] PCR system is: [0049] plasmid template (5ng) lul, [0050] Forward primer N-Gf (IOuM) O. 5uL, [0051] reverse primer N-Gr (IOuM) O. 5uL, [0052] 5-fold Trans Start fast Pfu buffer 5uL, [0053] dNTPs (IOmM) IuL, [0054] TransStart fast Pfu DNA polymerase O. 7uL, [0055] sterile water 16. 3uL, [0056] total volume: 25uLo [0057] The amplification conditions -MV 4min; [0058] 94 ° C 30sec, 55 ° C 30sec , 72 ° C 2. 5min 25 cycles; [0059] 72 ° C IOmin. [0060] PCR产物的纯化和消化:将上述PCR产物用TianGen公司的PCR纯化试剂盒进行纯化,然后将lul DMT酶加于40ul PCR纯化产物中,混匀,37°C孵育I小时进行消化。 Digestion and Purification [0060] PCR products: PCR products were performed with the above-described companies TianGen PCR purification kit, and then added to 40ul enzyme lul DMT PCR product was purified, mixed, 37 ° C incubation for I hr digestion. [0061] 甘赖脯胰岛素质粒的构建和确认:[0062] 将2ul的DMT酶消化产物加到50ul的DMT感受态细胞中(来自定点突变试剂盒(Fast Mutagenesis System),在感受态细胞刚刚解冻时加入消化产物),轻弹混勻,冰浴30 分钟;于42°C准确热激45秒,立即置于冰上2分钟;加到250ul室温的LB培养基中,在摇床中225rpm,37°C培养I小时。 [0061] Gan insulin lispro plasmid constructs and confirmed: [0062] The enzymatic digestion products 2ul of DMT DMT was added in 50ul competent cells (from site-directed mutagenesis kit (Fast Mutagenesis System), in competent cells were thawed just digestion product is added), flick mixing, on ice for 30 min; at 42 ° C accurate heat shock for 45 seconds immediately placed on ice for 2 min; room temperature was added 250ul LB medium, 225rpm in a shaker, 37 ° C I h culture. 然后取200ul菌液铺于氨苄抗性LB培养基上,培养过夜。 Then take 200ul ampicillin-resistant bacteria were plated on LB medium and incubated overnight. [0063] 挑选10个长出的转化子,分别接入氨苄抗性LB培养基中,培养过夜。 [0063] The selection of 10 transformants grown were ampicillin-resistant access LB medium and incubated overnight. 第二天再从这10个过夜培养物中用质粒抽提试剂盒提取质粒;[0064] 把提取的10个质粒送到北京诺赛公司进行序列测定,以鉴定突变基因的正确性。 10 from the next day the overnight culture was used to extract plasmid Plasmid extraction kit; [0064] The extracted plasmid 10 to match company Beijing Sino sequenced to identify the mutant gene correctness. 选出载有正确突变基因的重组甘赖脯胰岛素质粒。 Gan elected contains a recombinant plasmid insulin lispro correct gene mutations. [0065] (2)重组甘赖脯胰岛素表达工程菌的构建[0066] 将2ul筛选出来的载有正确突变基因的重组甘赖脯胰岛素质粒加入到IOOul Escherichia coli (3110)的感受态细胞中(在感受态细胞刚刚解冻时加入产物),轻弹混勻,冰浴30分钟;于42°C准确热激90秒,立即置于冰上2分钟;加到300ul室温的LB培养基中,在摇床中225rpm,37°C培养I小时,然后取50ul菌液铺于氨苄LB培养基上,培养过夜。 [0065] (2) a recombinant Gan insulin lispro Expression Bacteria constructs [0066] The 2ul screened contained the competent cells correct mutant recombinant gene Gan insulin lispro plasmid was added to IOOul Escherichia coli (3110) of ( when added just competent cells were thawed product), flick mixing, on ice for 30 min; at 42 ° C accurate heat shock for 90 seconds immediately placed on ice for 2 min; 300ul was added to an LB medium at room temperature, the shaker 225rpm, 37 ° C culture I h, then take 50ul bacteria plated on ampicillin-LB medium and cultured overnight. [0067] 从表达菌种Escherichia coli (3110)的培养平板上挑取单菌落,接种于5ml氨苄LB培养基中,在摇床中225rpm,37°C培养8小时,再从中吸取IOOul菌液接种于IL含有20%富集培养基的M9培养基中,在摇床中225rpm,37°C培养16小时,制成大量甘赖脯胰岛素的表达工程菌。 [0067] The culture plates were picked from the strain expressing Escherichia coli (3110) A single colony was inoculated in 5ml LB medium with ampicillin, 225rpm, 37 ° C for 8 hours in a shaking culture, then draw IOOul inoculated broth in M9 medium containing 20% ​​IL-enriched culture media, 225rpm, 37 ° C shaker in culture for 16 hours to prepare a large number of insulin lispro Gan expression bacteria. [0068] (3)重组甘赖脯胰岛素工程菌的发酵表达[0069] 将上述合格的工程菌接种至2L氨苄LB液体培养基中,在摇床中32°C,250rpm培养约6小时,转种到5L发酵罐中,培养至菌密度为0D600值为1.5-2. O时,再转接入250L 发酵罐中,并在以下条件下进行发酵培养:pH控制在约7. O ;溶氧控制在7-65%之间,温度前期控制在32°C,待0D600到20以后升温至35°C,保持2小时后升温至37°C,直至发酵结束。 [0068] (3) insulin lispro Gan recombinant engineering bacteria Expression of the fermentation [0069] The above qualified engineers to 2L ampicillin-inoculated LB liquid medium in a shaker 32 ° C, 250rpm for about 6 hours culture, transfected seeded into 5L fermenter, bacteria grown to a density of 1.5 when the value of O 0D600, the access sub-250L fermenter, and fermentation under the following conditions:. pH controlled at about 7. O; oxygen controlled between 7-65%, pre-temperature control 32 ° C, after heating to be 0D600 20 to 35 ° C, 2 hours later warmed to 37 ° C, until the end of the fermentation. 发酵过程中PH值靠补加氨水量控制;溶氧靠补料、通入压缩空气和调整搅拌速度控制。 PH value of the fermentation process by controlling the amount of additional ammonia; fed by dissolved oxygen, and pass into the air adjusting stirring speed control. 图2为所得重组甘赖脯胰岛素在250L发酵罐中的各时点电泳表达检测图谱。 Figure 2 is the resulting recombinant insulin lispro Gan electrophoresis to detect the expression pattern of each time point in 250L fermentor. 由电泳图谱可以看出甘赖脯胰岛素在发酵15小时后,13kDa处可见明显的目的蛋白表达条带,26小时电泳扫描表达量为27. 37%。 As can be seen by the electrophoretic patterns of insulin lispro Gan after fermentation for 15 hours at 13kDa protein expression showed obvious bands 26 hours scanning expression electrophoresis 27.37%. [0070] (4)甘赖脯胰岛素原的复性与转化[0071] 将诱导表达后的工程菌经离心回收菌体,将菌体悬浮在菌体洗涤液(pH 8. 0,50mM Tris-Cl)中,4-15°C搅拌洗涤I小时,再次离心回收菌体,然后将菌体悬浮在冰水中,在高压匀浆机中进行细胞破碎(750-800个大气压),通过离心收集甘赖脯胰岛素包涵体,包涵7体再通过洗涤(pH8. 0,50mM Tris-Cl,O. 2% Triton X_100),离心回收沉淀。 [0070] (4) compound and the conversion of proinsulin Gan lispro [0071] The engineered bacteria after induction of expression cells were collected by centrifugation, cells were suspended in a cell washing solution (pH 8. 0,50mM Tris- cl) in, 4-15 ° C was stirred for I h and washed again centrifuged to collect the cells, then the cells were suspended in ice water in a high pressure homogenizer for cell crushing machine (750-800 atm), collected by centrifugation Gan insulin lispro inclusion bodies, washed inclusion body 7 through the (pH8. 0,50mM Tris-Cl, O. 2% Triton X_100), the precipitate was recovered by centrifugation. 而后将所得包涵体在高浓度尿素(8M)下进行蛋白质变性,低浓度尿素(1.6M)下进行蛋白质复性,得到有正确构象的甘赖脯胰岛素原。 After the inclusion body protein denaturation resulting in high concentrations of urea (8M), Protein renaturation at a low concentration of urea (1.6M), to give the correct conformation Gan lispro proinsulin. [0072] 将甘赖脯胰岛素原溶液除盐后用50mM Tris调pH至10. 0,按质量比I : 500-1 : 1000(E/S,即酶/前体胰岛素原)分别加入胰蛋白酶和羧肽酶B,37°C反应30min,得到有正确构象的甘赖脯胰岛素。 [0072] The insulin lispro Gan original solution after desalting with 50mM Tris pH was adjusted to 10.0 mass ratio I: 500-1: 1000 (E / S, i.e., the enzyme / precursor proinsulin) were added to trypsin and carboxypeptidase B, 37 ° C the reaction 30min, to obtain the correct conformation Gan insulin lispro. [0073] (5)甘赖脯胰岛素的纯化及确认[0074] 转化后的甘赖脯胰岛素经阳离子交换层析(CM)纯化,层析用缓冲液包含pH5. O, IOmM柠檬酸,梯度洗脱用0-0. 8M NaCl梯度进行,甘赖脯胰岛素层析后纯度在90%以上。 [0073] (5) insulin lispro Gan Purification and confirmation [0074] The transformed Gan insulin lispro by cation exchange chromatography (CM) was purified by chromatography comprising pH5 buffer. O, IOmM citric acid, gradient elution removal performed with 0-0. 8M NaCl gradient, Gan insulin lispro chromatography to a purity above 90%. 再经反相高压液相层析后,经RP-HPLC检测纯度达到99. 77% (参见图3),符合国内外药典重组人胰岛素项下的标准要求。 And then by reverse phase high pressure liquid chromatography, detected by RP-HPLC purity 99.77% (see FIG. 3), the domestic and international standard requirements of Pharmacopoeia term recombinant human insulin. 将所得重组甘赖脯胰岛素经氨基酸序列分析,所得结构与预期一致。 The resulting recombinant insulin lispro Gan by amino acid sequence analysis, the resulting structure is consistent with expectations. [0075] 2. Gl/21人胰岛素(甘胰岛素)和GlyA21-AspB28人胰岛素(甘门冬胰岛素)的制备:[0076] 甘胰岛素和甘门冬胰岛素的制备过程与甘赖脯胰岛素的制备过程一致,只是在质粒构建的步骤中,分别用现有的人胰岛素质粒和门冬胰岛素质粒代替赖脯胰岛素质粒进行。 Preparation [0075] 2. Gl / 21 human insulin (insulin Gan), and GlyA21-AspB28 human insulin (insulin aspart Gan) is: [0076] Preparation process Gan Gan insulin aspart and insulin and insulin lispro GAN preparation of same, except that in step in plasmid construction were performed with conventional insulin aspart and human insulin plasmid plasmid instead of insulin lispro plasmid. [0077] 实施例2本发明胰岛素类似物在酸性条件下的稳定性[0078] 将通过实施例I方法制备得到的本发明胰岛素类似物分别置于pH值为4. 0±0. 2 的溶液中,所得溶液为澄清状态。 Example 2 Stability of insulin analogues under acidic conditions of the invention [0077] Embodiment [0078] A were placed in a pH of 4. 0 ± 0. 2 was prepared by the insulin analogs of the present invention obtained in Example I Method , the resulting solution was a clear state. 将上述溶液在2_8°C贮存9个月后,各种胰岛素类似物溶液中的相关蛋白的总增加量不超过I. 26% ;高分子蛋白的增加量不超过O. 55% ;效价的降低量不超过1%,都在95% -105%的范围内。 The above solution was 2_8 ° C 9 months after storage, the total increased amount of the insulin analog solution of various related proteins does not exceed I. 26%; molecular proteins increase in the amount of not more than O. 55%; titer reducing the amount of not more than 1%, are within the range of 95% to 105% by weight. [0079] 上述结果说明,由于本发明胰岛素类似物将酸性条件下易脱氨的A21位天冬酰氨替换为甘氨酸,因此在酸性条件下表现出了较好的稳定性。 [0079] The above results indicated that, due to the insulin analogs of the present invention will be easily under acidic conditions deamination replaced asparagine at position A21 is glycine, thus showing a good stability under acidic conditions. [0080] 实施例3本发明药物制剂的制备方法及成分分析[0081] I.本发明药物制剂的制备方法[0082] < 原料> :[0083] 甘赖脯胰岛素:通过实施例I的方法制备得到。 [0080] Preparation and composition of three embodiments of the invention the pharmaceutical formulation Analysis [0081] I. Preparation of pharmaceutical formulations of the present invention [0082] <Materials>: [0083] Gan Lispro: prepared by the method of Example I get. [0084] 甘精胰岛素:甘李药业有限公司提供,批号GLGB09001。 [0084] Insulin glargine: Gan Li Pharmaceutical Co., Ltd. to provide, lot GLGB09001. [0085] 盐酸(批号20070802)、氢氧化钠(批号20090804)购自湖南尔康制药有限公司; 氯化锌(批号20090522)、间甲酚(07496PK)购自上海京华有限公司;甘油购自浙江遂昌惠康药业有限公司,批号20090921。 [0085] hydrochloric acid (batch 20070802), sodium hydroxide (lot 20090804) was purchased from Hunan Kang Pharmaceutical Co., Ltd.; chloride (batch 20090522), m-cresol (07496PK) were purchased from Shanghai Co., Beijing; glycerol available from Zhejiang Suichang Wellcome Pharmaceutical Co., Ltd., batch number 20090921. [0086] <制备步骤> :[0087] 胰岛素母液的制备:分别精密称取甘赖脯胰岛素和甘精胰岛素各50万单位,加入到3升蒸馏水中,搅拌混悬均匀,滴加3M盐酸使其完全溶解后,补入适量氯化锌溶液,使最终胰岛素混合液中每毫升含锌量约为30ug,然后补水至5L。 [0086] <Preparation Step>: Preparation of [0087] Insulin stock solution: Gan were accurately weighed insulin lispro and glargine, 50 million units, added to 3 liters of distilled water, uniformly stirred suspension, so 3M hydrochloric acid was added dropwise after complete dissolution, into the amount of zinc chloride solution up to a final zinc insulin mixture is about 30 ug per ml, and then pay to 5L. [0088] 其他辅料母液的制备:精密称取甘油160克,间甲酚27. O克,放于适量注射用水中,搅拌至全部溶解后补水至4升。 [0088] Preparation of other excipients mother liquor: Precision Weigh 160 g of glycerol, metacresol 27. O g, placed in an appropriate amount of water for injection, and stirred until complete dissolution replenishment to 4 liters. [0089] 混合方法:将其他辅料母液加入到胰岛素母液中,再用10%氢氧化钠或10%盐酸调溶液的PH值至4. 0±0. 2,补水至终体积10升,贮存于2-8°C的条件下。 [0089] The mixing process: the mother liquor was added to the other excipients insulin mother liquor, and then the PH value of 10% sodium hydroxide or 10% hydrochloric acid solution was adjusted to 4. 0 ± 0 2, water supplement to a final volume of 10 l, stored at. conditions of 2-8 ° C. [0090] 最终所得澄清溶液中甘赖脯胰岛素和甘精胰岛素的浓度各为50IU/ml,总活性胰岛素成分的浓度为100IU/ml。 [0090] The final concentration of the resulting clear Gan insulin lispro and glargine solution each 50IU / ml, a concentration of total active ingredient is insulin 100IU / ml. [0091] 2.本发明药物制剂的成分分析[0092] 分析方法:高效液相色谱法[0093] 分析条件:使用烷基硅烷键合硅胶为填充物的C4柱(3·5μπι,15(3πιχ4·6πιπι ID),用高氯酸钠盐缓冲液(精密称取无水磷酸二氢钠5. 9998g,加水900ml溶解,再加高氯酸钠14. 046g溶解,混均,用高氯酸调其pH值为2. 5,加水至1L,过滤)为流动相A ;再取高氯酸钠盐缓冲液-乙腈(50 : 50)为流动相B,流速为l.Oml/min,柱温为40°C,检测波长为214nm。洗脱梯度为60% B平衡,初始状态为60% B,然后在49分钟内将B %增至74%, 50分钟时为100% B,持续10分钟,61分钟时为60% B,持续5分钟。[0094] 分析结果:图4为本发明甘赖脯胰岛素与甘精胰岛素以50 : 50的百分比预混后的高效液相色谱的保留时间测定图谱。从图4可以看出,在pH4. O时,速效和长效胰岛素类似物的含量比例基本为50 : 50,且两样品峰之间的 [0091] 2. The composition analysis of the present invention is a pharmaceutical formulation [0092] Analysis method: High performance liquid chromatography [0093] Analysis conditions: silane bonded C4 column (3 · 5μπι silica gel as filler, 15 (3πιχ4 · 6πιπι ID), sodium perchlorate buffer (accurately weighed anhydrous monosodium phosphate 5. 9998g, 900ml water was added to dissolve, plus sodium perchlorate 14. 046g was dissolved, mixed, adjusted with perchloric acid a pH of 2.5, add water to 1L, filtration) as mobile phase A; then take perchlorate salt buffer - acetonitrile (50: 50) as mobile phase B, flow rate was l.Oml / min, column temperature to 40 ° C, detection wavelength of 214nm. 60% B elution gradient equilibrium, initial state is 60% B, then 49 min 74% B% increase, 50 minutes 100% B, 10 minutes when 61 minutes to 60% B, 5 min [0094] analysis: HPLC retention time measured after the percentage of premixed 50: 4 Gan insulin lispro and glargine present invention in 50 . maps can be seen from Figure 4, when pH4 O, content ratio of fast-acting and long-acting insulin analog is substantially 50: 50, and between the two peaks of the sample 离度大于2.0,说明两种类似物在该条件下可以得到充分的分离,即可以进行有效含量分析,保证生产过程中各组份的含量准确控制。[0095] 本发明实施例I中所制备的Gl/21人胰岛素、GlyA21-AspB28人胰岛素也可按上述方法与甘精胰岛素混合,并可按照需要以不同比例投料,制成含量百分比为30 : 70-70 : 30 的混合药物制剂。所制得制剂通过高效液相色谱法分析也可以在PH4. O时得到充分的分离,即可以进行有效含量分析,保证生产过程中各组份的含量准确控制。[0096] 实施例4本发明药物制剂的安全性评价试验[0097] <试验药品> :实施例3制备的本发明药物制剂。[0098] <试验动物> :[0099] Wistar大鼠,体重200〜220g,由吉林大学实验动物中心提供,许可证号: SCXK-(吉)2003-0007。[0100] 昆明种小白鼠,体重18〜22g,由长春生物制品研究所提供,许可证号: SCXK-(吉)2006-0001。 From greater than 2.0, illustrate two analogs can be fully separated at this condition, the content of which can be analyzed effectively, the production process to ensure that the content of each component accurately. Prepared as in Example I [0095] The present invention the Gl / 21 human insulin, GlyA21-AspB28 human insulin insulin glargine above-described method can also be mixed with sweet, and may need to be different according to the feeding ratio, the percentage content is made 30: 70-70: 30 by mixing the drug formulation formulations may also be prepared. adequately separated when the O PH4, i.e., the content analysis may be performed efficiently, the production process to ensure that the content of each component is accurately controlled by HPLC analysis. [0096] Example 4 medicament invention safety evaluation test formulation [0097] <test drugs>: pharmaceutical formulation of the present invention prepared in Example embodiment 3 [0098] <test animals>:. [0099] Wistar rats weighing 200~220g, by the experimental animal Center of Jilin University provided, license number:. SCXK- (guitar) 2003-0007 [0100] Kunming mice, weighing 18~22g, provided by the Changchun Institute of biological products, license number: SCXK- (Kyrgyzstan) 2006-0001. [0101] 豚鼠,体重300〜350g,吉林大学实验动物中心,许可证号:SCXK-(吉)2008-0004。[0102] 家兔,体重2. O〜3. Okg,吉林大学实验动物中心,许可证号: SCXK-(吉)2008-0004。[0103] <试验方法和结果> :[0104] I.急性毒性研究:[0105] 按照现有药物急性毒性研究方法(参见徐叔云,《药理实验方法学》第二版,人民卫生出版社,第201页),观察小鼠禁食后皮下注射本发明药物制剂引起的急性毒性反应,记录14日内试验动物的活动情况及死亡数,计算LD50。 [0101] guinea pigs, weighing 300~350g, Jilin University Experimental Animal Center, License number:.. SCXK- (guitar) 2008-0004 [0102] rabbits, weighing 2. O~3 Okg, Jilin University Experimental Animal Center, license number: SCXK- (Ji) 2008-0004 [0103] <test methods and results>:. [0104] I. acute toxicity: [0105] acute toxicity study in accordance with conventional pharmaceutical methods (see Xu Shuyun, "pharmacological experimental methodology "second edition, people's Medical Publishing House, page 201), acute toxicity in mice after subcutaneous injection of a pharmaceutical formulation fasting induced the present invention, and the number of deaths recorded activity of the test animals within 14 days, calculated LD50. [0106] 所得计算结果为:[0107]雄性小鼠 LD50 = 3. 346U/Kg,其95%的可信限为2. 372 〜4. 719U/kg ;[0108]雌性小鼠 LD50 = 4. 767U/kg,其95%的可信限为3. 556 〜6. 391U/kg ;[0109]雄性大鼠 LD50 = 7. 447U/kg,其95% 的可信限为6. 159 〜9. 004U/kg。 [0106] The results obtained are: [0107] Male mice LD50 = 3. 346U / Kg, 95% confidence limit of 2. 372 ~4 719U / kg; [0108] female mouse LD50 = 4.. 767U / kg, 95% confidence limit of 3. 556 ~6 391U / kg;. [0109] male rats LD50 = 7. 447U / kg, 95% confidence limit of 6.159 ~ 9. 004U / kg. [0110] 对死亡动物解剖肉眼观察,主要脏器无异常。 [0110] naked eye observation of dead animals anatomy, major organs without exception. [0111] 2.长期毒性研究:[0112] 试验方法:分别给大鼠皮下注射3.72U/kg(LD50的1/2)、2. OU/kg(大鼠药效剂量)、1. OU/kg(LD50的1/8)剂量的本发明药物制剂,以及生理盐水(对照组),每日I次,连续5周,再停药观察2周。 [0111] 2. Long-term toxicity studies: [0112] Test Method: Rats were injected subcutaneously 3.72U / kg (LD50 of 1/2), 2 OU / kg (dose pharmacodynamic rat), 1 OU / kg (LD50 1/8) dose pharmaceutical formulation of the present invention, and physiological saline (control group), once a day I, 5 consecutive weeks, then discontinued observed for 2 weeks. [0113] 试验结果:在给药期间、停药恢复期间,本发明药物制剂各剂量组大鼠的体重、饮食量、饮水量及一般活动等与生理盐水组(对照组)比较均无明显差异。 [0113] Test Results: During the period of administration, drug discontinuation, the pharmaceutical formulations of the present invention the weight in each dose group of rats, food intake, water intake and general activities with the saline group (control group) showed no significant difference . 给药5周、停药2 周时,本发明药物制剂各剂量组大鼠血常规指标、凝血项指标、血生化指标、血清抗体、脏器系数及脏器组织病理学检查等指标与生理盐水组(对照组)比较均无明显差异。 5 wks after administration, when the withdrawal two weeks, blood indicators of the present invention the pharmaceutical formulation in each dose group, coagulation indices, biochemical indices of blood serum antibodies, organs and organ coefficient index and histopathology examination saline There were no significant difference between group (control group). [0114] 3.免疫毒性试验:[0115] 按照现有动物免疫毒性试验方法(参见《中药、天然药物免疫毒性(过敏性、光敏反应)研究的技术指导原则》,国家药监局,2005年)进行豚鼠的主动全身过敏试验(ASA) 和大鼠的被动皮肤过敏试验(PCA)。 [0114] 3. immunotoxicity test: [0115] in accordance with the existing animal immune toxicity test methods (see "traditional Chinese medicine, technical guidelines for the study of natural medicine immunotoxicity (hypersensitivity, photosensitivity reactions)," the State Food and Drug Administration, 2005 ) initiative allergy test guinea pig whole body (ASA) and rat passive cutaneous anaphylaxis test (PCA). [0116] ①主动全身过敏试验(ASA)-豚鼠试验:[0117] 将本发明药物制剂O. 87U/kg (豚鼠药效剂量的1/2)、3. 48U/kg (豚鼠药效剂量的2倍)经皮下注射于豚鼠,隔日I次,共5次,末次致敏后的第10天由静脉注射上述药物制剂的2倍量进行激发,观察并记录激发注射后30分钟内豚鼠有无过敏症状:两个剂量组的结果皆呈阴性。 [0116] ① Test active systemic anaphylaxis (ASA) - guinea pig test: [0117] The pharmaceutical formulations of the present invention is O. 87U / kg (guinea pig dose efficacy 1/2), 3 48U / kg (guinea pig efficacy dose 2-fold) in guinea pig every other day I, a total of 5 times, 10 days after the last sensitizing excited by the intravenous injection 2 times the amount of the above pharmaceutical preparations by subcutaneous injection, to observe and record the presence or absence of excitation in guinea pigs within 30 minutes of injection allergy symptoms: results of the two dose groups were highly negative. [0118] ②被动皮肤过敏试验(PCA)-大鼠试验:[0119] 将本发明药物制剂I. OU/kg(大鼠药效剂量的1/2)、4. OU/kg(大鼠药效剂量的2 倍)经皮下注射于大鼠,隔日I次,共5次,于末次致敏后第11天,腹主动脉采血,制备抗体血清。 [0118] ② passive cutaneous anaphylaxis test (PCA) - Test in Rats: [0119] The pharmaceutical formulations of the invention I. OU / kg (dose pharmacodynamic rat 1/2), 4 OU / kg (rat poison large. 2 times the effective dose) were injected subcutaneously in rats, I every other day, a total of five times, after the last sensitization to 11 days, the abdominal aorta blood, serum antibody preparation. 将上述抗体血清用生理盐水稀释后,在大鼠皮内注射O. Iml,48小时后,静脉注射与致敏剂量相同的抗原(内含O. 75%的伊文思蓝染料)激发,30分钟后测量皮肤内侧的蓝色反应斑直径并判定阴阳性:蓝色反应斑直径大于5mm者判定为阳性,本发明药物制剂两个剂量组的结果皆呈阴性。 After the above-described antibodies in the serum diluted with saline, intradermal injection in rats O. Iml, after 48 hours, the same doses of antigen sensitized with intravenous (containing O. 75% of Evans Blue dye) excitation 30 minutes after measuring the inside diameter of the skin blue spot and determines the reaction of yin and yang: reaction blue spot diameter greater than 5mm were judged to be positive, the results of the present invention is a pharmaceutical formulation two dose groups were highly negative. [0120] 4.刺激性、溶血性试验:[0121] 按照现有动物刺激性、溶血性试验方法(参见《中药、天然药物刺激性、溶血性研究的技术指导原则》,国家药监局,2005年)对家兔进行刺激性试验和溶血试验。 [0120] 4. irritating, hemolytic test: [0121] in accordance with the existing animal irritating, hemolytic test methods (see "traditional Chinese medicine, natural medicine irritating, technical guidelines for the research hemolytic", the State Food and Drug Administration, 2005) conducted on rabbit irritation test and hemolysis test. [0122] ①刺激性试验[0123] 将本发明药物制剂O. 5U/kg(家兔药效剂量的1/2)、2. OU/kg(家兔药效剂量的2 倍)单次经皮下注射于家兔,从注射时至72h,注射局部没有红斑、水肿、充血等反应症状, 其组织病理学检查结果也表明本发明药物制剂的两个剂量组对家兔皮下组织无刺激性。 [0122] ① irritation test [0123] The pharmaceutical formulations of the present invention is O. 5U / kg (rabbits efficacy dose 1/2), 2. OU / kg (2 times rabbit efficacy dose) by a single subcutaneous injection in rabbits to 72h from the time of injection, injection site reactions no symptoms of erythema, edema, hyperemia, which results also show histopathological pharmaceutical formulation of the present invention, two non-irritating dose subcutaneous tissue of rabbits. [0124] ②溶血试验[0125] 本发明药物制剂在3小时内都没有发生溶血和凝聚现象。 [0124] ② hemolysis test pharmaceutical formulation [0125] The present invention is not hemolysis and agglomeration within 3 hours. [0126] 由以上安全性评价试验结果可以看出:本发明药物制剂对动物均未见明显毒副作用影响,没有过敏现象,注射部位无刺激反应,药物制剂本身无溶血现象。 [0126] As can be seen from the above results of safety evaluation test: pharmaceutical formulation of the present invention to an animal showed no obvious toxic side effects, no allergies, no irritation at the injection site, the pharmaceutical formulation itself without hemolysis. 说明本发明药物制剂具有很高的安全性。 DESCRIPTION The pharmaceutical formulation of the present invention has high safety. [0127] 实施例5本发明药物制剂的药效学试验[0128] I、试验药品:[0129] 实施例3制备的本发明药物制剂;[0130] 重组赖脯胰岛素注射液(100IU/ml,批号:22080301,由甘李药业有限公司提供);[0131] 重组甘精胰岛素注射液(100IU/ml,批号:12080303,由甘李药业有限公司提供)。 [0127] Pharmacodynamic Test 5 of the present invention is a pharmaceutical formulation [0128] I, Test Example Drug: [0129] Pharmaceutical formulations of the present invention prepared in Example 3 embodiment; [0130] The recombinant insulin lispro injection (100IU / ml, lot: 22080301, by Gan Li Pharmaceutical Co., Ltd.); [0131] the recombinant insulin glargine injection (100IU / ml, batch number: 12080303, by Gan Li Pharmaceutical Co., Ltd.). [0132] 2、试验动物:Wistar大鼠,雄性,50只,体重250〜280g,由吉林大学实验动物中心提供,许可证号:SCXK-(吉)2003-0007。 [0132] 2, the test animals: Wistar rats, male, 50, weight 250~280g, by the Experimental Animal Center of Jilin University, license number: SCXK- (Kyrgyzstan) 2003-0007. [0133] 3、试验试剂:葡萄糖试剂盒,中生北控生物科技股份有限公司,批号: 081251. 200802。 [0133] 3, test reagents: Glucose kit, Biosino Biotechnology Co., Ltd., batch number: 081,251.200802. [0134] 4、试验仪器:GF_D800型半自动生化分析仪,山东高密彩虹分析仪器有限公司。 [0134] 4, test equipment: GF_D800 semi-automatic biochemical analyzer, analytical instruments Ltd. Shandong Gaomi rainbow. TGL-16B台式离心机。 TGL-16B desktop centrifuge. [0135] 5、试验设计,具体方法参见徐叔云,《药理实验方法学》第三版,人民卫生出版社, 1517 页:[0136] ①动物分组和建模:[0137] 将50只Wistar大鼠随机分为5组,每组10只,分别为:[0138] 正常组(不建立高血糖模型,给药生理盐水);[0139] 模型组(建立高血糖模型,给药生理盐水);[0140] 赖脯胰岛素组(建立高血糖模型,给药赖脯胰岛素);[0141] 甘精胰岛素组(建立高血糖模型,给药甘精胰岛素组);[0142] 本发明药物制剂组(建立高血糖模型,给药本发明药物制剂)。 [0135] 5, test design, specific methods see Xu Shuyun, "pharmacological experiments methodology" third edition, People's Health Press, 1517: [0136] ① Animal grouping and modeling: [0137] 50 Wistar rats They were randomly divided into 5 groups of 10, respectively: [0138] normal (not established diabetic model, physiological saline); [0139] group (established model hyperglycemia, physiological saline); [ 0140] insulin lispro group (established diabetic model, administration of insulin lispro); [0141] glargine (established diabetic model, administration of insulin glargine); [0142] pharmaceutical formulations group (the present invention establishes diabetic model, administration of a pharmaceutical formulation of the present invention). [0143] 其中高血糖模型通过以下方法建立:先令Wistar大鼠禁食不禁水24小时,尾部静脉注射四氧卩密唳60mg/kg,48小时后形成永久性高血糖模型。 [0143] wherein hyperglycemia model by the following method: Shilling water Wistar rats fasted for 24 hours, four tail vein injection of oxygen-tight Jie Li 60mg / kg, after 48 hours in a permanent model of hyperglycemia. [0144] ②给药和采血:[0145] 高血糖模型建立后,令所有Wistar大鼠禁食不禁水16h,并在给药前先采血测药前血糖,然后对赖脯胰岛素组、甘精胰岛素组、本发明药物制剂组的动物皮下注射相应胰岛素,剂量均选为2. 0IU/kg ;对模型组、正常对照组的动物皮下注射等体积生理盐水。 [0144] ② administration and blood collection: [0145] After the hyperglycemia model, so that all water Wistar rats fasted for 16 h, prior to dosing and prior to blood glucose test drug, then insulin lispro group, glargine insulin group, the group of animals injected subcutaneously pharmaceutical formulation of the present invention, the respective insulin doses are preferably 2. 0IU / kg; model group, normal control group of the animals injected subcutaneously volume of normal saline. 然后于给药后10min、30min、lh、2h、3h、4h、8h、12h、16h 眼眶米血,3000rpm/min 离心5min,取血清, 终点法测血糖。 Then after administration 10min, 30min, lh, 2h, 3h, 4h, 8h, 12h, 16h meters orbital blood, 3000rpm / min centrifuge 5min, the serum, blood sugar endpoint method. [0146] ③数据处理:[0147] 降糖百分率通过以下公式计算:[0148] 降糖百分率(<%) = ((药前血糖值-药后血糖值)/药前血糖值)X 100 %[0149] 试验结果采用[士s表示,相同时间点血糖及降糖百分率进行组间t检验。 [0146] ③ Data Processing: [0147] hypoglycemic percentage calculated by the formula: [0148] hypoglycemic percentage (<%) = ((blood glucose level before drug - the blood glucose level after administration) / blood glucose level before drug) X 100% [0149] test results using [Shi s represents the same time the percentage of glucose and hypoglycemic t test between groups. 计算结果见表I、表2和图5。 The results in Table I, Table 2 and FIG. [0150] 表I不同胰岛素对四氧卩密唳高血糖大鼠血糖值(mmol/L)的影响(Xt1S1,n = 10)[0151] [0150] TABLE I Effect of insulin on four different oxygen blood glucose level (mmol / L) density Jie Li hyperglycemic rats (Xt1S1, n = 10) [0151]

Figure CN102199206BD00111
Figure CN102199206BD00121

[0152]注与模型组比较:*P < O. 05,**P < O. 01,***P < O. 001[0153] 表2不同胰岛素对四氧喃唳高血糖大鼠降糖百分率(% )的影响(x±s,n = 10)[0154] [0152] Note compared with model group: * P <O. 05, ** P <O. 01, *** P <O. 001 [0153] Table 2 for four different oxygen insulin hyperglycemic rats hypoglycemic thiopyran Li Effect of percentage (%) of the (x ± s, n = 10) [0154]

Figure CN102199206BD00131

[0155]注与模型组比较:*P < O. 05,**P < O. 01,***P < O. 001[0156] 从上述结果可以看出,赖脯胰岛素组注射后IOmin开始起效,30min〜Ih降糖作用达峰值,4h作用仍明显,8h以后无降糖作用;甘精胰岛素组注射后Ih开始起效,Ih〜8h降糖作用平稳,12h作用仍明显;本发明药物制剂组注射后IOmin开始起效,Ih〜8h —直有平稳的降糖作用,12h作用仍明显,同时具有速效降糖和平稳长效降糖两种功能。 [0155] Note compared with model group: * P <O. 05, ** P <O. 01, *** P <O. 001 [0156] As can be seen from the above results, insulin lispro injection group starts after IOmin onset, 30min~Ih of peak hypoglycemic effect, 4h still significantly effect, hypoglycemic effect after 8h; after injection of insulin glargine Ih start onset, steady Ih~8h hypoglycemic effect, 12h still significantly effect; present invention pharmaceutical formulations were injected after the onset begins IOmin, Ih~8h - have a straight smooth hypoglycemic effect, 12h still significantly effect, while having a quick and smooth long-lasting hypoglycemic hypoglycemic two functions.

Figure CN102199206BD00151
Figure CN102199206BD00161

<223> GlyA21-LysB28-ProB29人胰岛素A链:将人胰岛素A链中第21位替换为Gly <400> 5Gly He Val Glu Gln Cys Cys Thr Ser He Cys Ser Leu Tyr Gln Leu 15 10 15Glu Asn Tyr Cys Gly 20<210> 6<211> 30<212> PRT <213>人I:序列<220><223> GlyA21-LysB28-ProB29人胰岛素B链:将人胰岛素B链中第28位替换为Lys,第20位替换为Pro<400> 6Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr I 5 10 15Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Lys Pro Thr 20 25 30<210> 7<211> 21<212> PRT<213> 人I:序列<220><223> GiyA21-AspB28人胰岛素A链:将人胰岛素A链中第21位替换为Gly <400> 7Gly He Val Glu Gln Cys Cys Thr Ser He Cys Ser Leu Tyr Gln Leu 15 10 15[0004]17 <223> GlyA21-LysB28-ProB29 human insulin A-chain: The human insulin A-chain position 21 is replaced with Gly <400> 5Gly He Val Glu Gln Cys Cys Thr Ser He Cys Ser Leu Tyr Gln Leu 15 10 15Glu Asn Tyr Cys gly 20 <210> 6 <211> 30 <212> PRT <213> human I: sequence <220> <223> GlyA21-LysB28-ProB29 human insulin B chain: the human insulin B-chain at position 28 replaced by Lys, position 20 is replaced with Pro <400> 6Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr I 5 10 15Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Lys Pro Thr 20 25 30 <210> 7 < 211> 21 <212> PRT <213> human I: sequence <220> <223> GiyA21-AspB28 human insulin A-chain: the human insulin A-chain position 21 is replaced with Gly <400> 7Gly He Val Glu Gln Cys Cys thr Ser He Cys Ser Leu Tyr Gln Leu 15 10 15 [0004] 17

Figure CN102199206BD00171
Figure CN102199206BD00181

Claims (7)

1. 一种胰岛素类似物或其可药用盐,其特征在于,所述胰岛素类似物为A21位上的Asn被Gly取代,B28位上的Pro被Lys取代,并且B29位上的Lys被Pro取代的人胰岛素。 1. An insulin analog or a pharmaceutically acceptable salt thereof, wherein said insulin analog is Asn at position A21 is substituted with Gly, Pro is substituted on position B28 Lys, and position B29 is Lys on Pro substituted human insulin.
2. 一种胰岛素药物组合物,包含:权利要求I所述的胰岛素类似物或其可药用盐,以及长效胰岛素类似物或其可药用盐,其中所述长效胰岛素类似物选自甘精胰岛素。 Insulin A pharmaceutical composition, comprising: an insulin analogue according to claim I or a pharmaceutically acceptable salt thereof, and long-acting insulin analogue or a pharmaceutically acceptable salt thereof, wherein the long-acting insulin analog is selected from glargine.
3.根据权利要求2所述的药物组合物,其中所述胰岛素类似物和长效胰岛素类似物的含量百分比为30:70-70:30。 3. A pharmaceutical composition according to claim 2, wherein the percentage content of the long-acting insulin analogues and insulin analogues is 30: 70-70: 30.
4.根据权利要求3所述的药物组合物,其中所述胰岛素类似物和长效胰岛素类似物的含量百分比为50:50。 4. The pharmaceutical composition according to claim 3, wherein the percentage content of the long-acting insulin analogues and insulin analogue is 50:50.
5. 一种胰岛素药物制剂,包含权利要求2-4中任一项所述的药物组合物和可药用辅料。 A pharmaceutical formulation of insulin, the pharmaceutical compositions comprising pharmaceutically acceptable excipients and may be in any one of claims 2-4.
6.根据权利要求5所述的药物制剂,该药物制剂的pH为3. 5-4. 5。 6. A pharmaceutical formulation as claimed in claim 5, pH of the pharmaceutical formulation is 3. 5-4. 5.
7.根据权利要求6所述的药物制剂,该药物制剂的pH为3. 8-4. 2。 7. A pharmaceutical formulation according to claim 6, pH of the pharmaceutical formulation is 3. 8-4. 2.
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