CN1193915A - 用于治疗骨质疏松症化合物的鼻传递的药物组合物 - Google Patents
用于治疗骨质疏松症化合物的鼻传递的药物组合物 Download PDFInfo
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- CN1193915A CN1193915A CN96196522A CN96196522A CN1193915A CN 1193915 A CN1193915 A CN 1193915A CN 96196522 A CN96196522 A CN 96196522A CN 96196522 A CN96196522 A CN 96196522A CN 1193915 A CN1193915 A CN 1193915A
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Abstract
用于治疗骨质疏松症化合物的鼻传递的药物组合物,其中包含有效量的PTH或PTHrp的生理活性截短类似物或其盐,其中的氨基酸残基(22—31)形成一两亲α螺旋,所述的残基(22—31)选自(SEQ ID NO∶85、86、26、27、28、29和30);吸收增强剂,它选自二甲基-β-环糊精和胆酸表面活性剂;还包含水。
Description
发明背景
a)发明领域
本发明涉及药物组合物,用于经鼻给予药学上有效量的新的甲状旁腺素和甲状旁腺素相关肽的类似物来治疗骨质疏松症。
b)现有技术
骨质疏松症是代谢性骨病的最常见形式,并且可以被认为是骨损失(骨质减少症)的症状性、骨折性阶段。虽然骨质疏松症可能因许多潜在的疾病而继发,但是90%是自发性的。绝经后的妇女尤其可能患上自发性骨质疏松症(绝经性或I型骨质疏松症)。自发性骨质疏松症的另一高发人群是老年男性和女性(老年性或II型骨质疏松症)。骨质疏松症还被认为与以下情况相关:使用皮质类固醇、不活动或卧床过久、酗酒、糖尿病、性激素化疗、促乳激素过多症、神经性厌食、原发和继发性闭经和卵巢切除术。
在骨质疏松症的多种形式中,经常发生的是因骨损失达到机械衰竭点而导致的骨折。绝经性骨质疏松症多以腕骨和脊柱骨折为特征,而老年性骨质疏松症的突出特征则似乎是股颈骨折。
在骨质疏松症中引起骨损失的机制被认为与骨骼自我更新过程中的失衡有关。该过程被称为骨重塑。它发生在一系列分散的活性袋中。这些袋自发地出现在骨架的给定骨表面,作为骨吸收位点。破骨细胞(骨溶解或吸收细胞)负责部分骨的吸收,这些骨通常具有恒定的体积。在该吸收过程之后出现成骨细胞(骨形成细胞),这些细胞然后用新的骨重新填充由破骨细胞留下的空穴。
在健康成人体内,破骨细胞和成骨细胞的生成速度能够使得骨形成与骨吸收保持平衡。但是在骨质疏松症患者中,随着骨重塑过程中失衡的发展,造成骨损失的速度大于其生成速度。虽然多数个体随年龄增长都会发生一定程度的这种失衡,但是在绝经性骨质疏松症或卵巢切除术后,这种失衡严重得多,而且发生较早。
Adachi等在《关节炎和风湿病学会》(Seminars in Arthritis andRheumatism)22:6,375-84(1993,7)中报道,尽管有关皮质类固醇诱导的骨质疏松症的病理生理学很不一致,但获得普遍认同的是,发生了骨形成的相对减少和骨吸收的相对增加。皮质类固醇疗法的常见后果是骨损失和由此造成的骨折和骨坏死。有证据表明,在皮质类固醇疗法的最初6至12个月内,骨损失发生迅速;而且,骨损失速度似乎还与皮质类固醇的剂量密切相关。男性与女性同样易受到皮质类固醇的作用。骨折和骨坏死的估计发病率在30%至50%之间。
目前已经进行了多种尝试来治疗骨质疏松症,其目标为减慢骨的进一步损失,或者,获得骨质量的净增长则更好。有些药物,例如雌激素和磷酸二氢盐似乎能够减慢骨质疏松症中骨的进一步损失。因为骨吸收和骨形成的持续时间不同而减慢骨损失的药物可能表现出能够增加骨质量(3至7%)。但是,这种表观增加在时间上是有限的,并不是进展性的,而且是“重塑空间”减少的结果。此外,由于吸收和生成之间的密切关联,抑制骨吸收的治疗最终也会抑制骨形成。
已有人提出,甲状旁腺素(PTH)疗法能够同时提高骨转化和钙的正平衡。但是,人临床实验表明,小梁骨的增加全部被皮质骨的减少所抵消,总骨量因此没有净增长。
Hefrti等在《临床科学》(Clinical Science)62,389-396(1982)中报道,每日皮下给药bPTH(1-84)或hPTH(1-34)在正常和患骨质疏松症的成年雌性大鼠中都增加了总体钙量和各种骨的灰分重量。
Liu等在《骨的无机质研究杂志》(Bone Miner.Res.)6:10,1071-1080(1991)中提出,对雌性成年大鼠的卵巢切除术在邻近胫骨干骺端诱导47%的小梁骨损失,并伴随成骨细胞和小梁破骨细胞数量的显著增加。每日皮下注射hPTH(1-34)完全逆转了小梁骨的损失,并使得小梁骨量超过使用安慰剂的对照。成骨细胞数量增加而破骨细胞数量减少。
Hock等在《(骨的无机质研究杂志》(Bone Miner.Res.)7:1,65-71(1992)中报道,给健康的成年雄性大鼠皮下注射hPTH(1-34)12天,增加了小梁骨和皮质骨的钙含量和干重。总骨质量、小梁骨体积、小梁骨厚度和数量以及成骨性表面增加。
哺乳动物甲状旁腺素,例如人的(hPTH)、牛的(bPTH)和猪的(pPTH)是具有84个氨基酸残基的单多肽链,分子量约为9500。生物活性与N末端相关,至少必需有残基(1-34)。
人PTH的N末端片段与牛和猪PTH的N末端仅相差3至2个氨基酸残基,分别为: hPTH(1-34):Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu
1 5 10 15Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp
20 25 30Val His Asn Phe(SEQ ID NO:1);bPTH(1-34):Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His Leu
1 5 10 15Ser Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp
20 25 30Val His Asn Phe(SEQ ID NO:2);pPTH(1-34):Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu
1 5 10 15Ser Ser Leu Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp
20 25 30Val His Asn Phe(SEQ ID NO:3).
PTH的主要功能是激发适应性改变,从而维持胞外体液中恒定的Ca2+浓度。PTH作用于肾脏来增加肾小管从尿液中对Ca2+的再吸收,同时促进骨化二醇向钙三醇的转化,后者负责从肠中吸收Ca2+。突出的效果之一是促进了骨中的Ca2+的活化。PTH作用于骨来提高对Ca2+和磷酸盐的吸收速度。PTH提高破骨细胞的骨吸收速度,提高间质细胞分化为破骨细胞的速度,并延长后者的半衰期。随着PTH作用的延续,形成骨的成骨细胞数量也被增加;由此,骨转化和重塑的速度被提高。但是,个别成骨细胞的活性似乎低于正常水平。
Rosenblatt等在美国专利4,423,037、4,968,669和5,00l,223中公开了通过缺失N末端(1-6)氨基酸残基和选择性置换Phe7、Met8,18和Gly12得到的PTH拮抗剂。据报道,Tyr34-NH2提高以上化合物的活性和稳定性。
甲状旁腺素相关肽(PTHrp),一种140+氨基酸蛋白质,及其片段具有PTH的主要生物学作用。许多人和动物的肿瘤以及其它组织分泌PTHrp,它可能在恶性高钙血症中起一定作用。hPTHrp(1-34)rp的序列如下: Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu
20 25 30Ile His Thr Ala(SEQ ID NO:4).
hPTH和hPTHrp的序列同源性主要限于13个N末端氨基酸,其中8个是全同的;在hPTHrp中仅保留了hPTH(25-34)受体结合区域10个氨基酸中的1个。构象的类似可能是相同活性的基础。Cohen等在《生物化学杂志》(J.Biol.Chem.)266:3,1997-2004(1991)中提出,大多数PTH(1-34)和PTHrp(1-34)的序列,尤其在(5-18)和(21-34)区域具有一个α螺旋构型,同时提出,问题在于,在生理条件下,该构型是否是羧基端的主要构型。这一二级结构可能对与脂类的作用、与受体的作用和/或结构的稳定化是十分重要的。
为了开发出更好的用于在包括患骨质疏松症的那些哺乳动物体内保存骨量的药物,我们已经合成出了PTH和PTHrp的类似物。考虑到患者的使用方便和肽的高成本,急需尽可能最高效的这类药物的传递方式。看起来,似乎这类化合物即PTH类似物以搏动方式传递最为有效。虽然皮下注射就此目的而言效果相当,但是这种传递方式有明显的缺点,主要是日常注射带来的痛苦。经口传递特别地不适合肽类药物的传递,很大程度是由于被摄入的药物在肠胃道中的降解和由此造成的低生物利用率(<1%)。
或者,非常规传递方式,例如透皮、经肺和经鼻可能更适用于这些不稳定药物的传递。本发明发现并开发出了特别用于鼻传递的液体组合物。肽的鼻传递被成功地商品化的不多,例如Synarel滴鼻液(Syntex Corporation,Palo Alto,CA),适用于治疗子宫内膜异位的强效十肽纳发阮林(nafarelin),其生物利用率至多达到约3%。Anik在美国专利4,476,116和5,116,817中报道了生物利用率显著提高的LH-RH类似物,包括纳发阮林的鼻用制剂。Merkus等在PCT专利公开No.92/01440中还报道了用于经鼻给药肽,尤其是胰岛素,并包括二甲基-β-环糊精作为吸收增强剂的组合物。Aliverti等在美国专利No.5,183,802中公开了一种用于在骨质疏松症的治疗中经鼻给药的药物组合物,其中包含降钙素和甘草皂酯(glycyrrhizinate)吸收增强剂。但是,就传递肽化合物的同时具有可被认可的生物利用率而言,目前开发出的鼻用组合物都不能完全令人满意。
发明概述
本发明提供了一种用于治疗骨质疏松症的鼻传递化合物的药物组合物,其中包含有效量的PTH或PTHrp的生理活性截短类似物或其盐,其中的氨基酸残基(22-31)形成一两亲α螺旋,所述的残基选自(SEQ ID NO:85、86、26、27、28、29和30);吸收增强剂,它选自二甲基-β-环糊精和胆酸表面活性剂;还包含水。
详细说明缩写和定义
使用的各种普通核苷酸碱基和氨基酸的单字母和三字母缩写是根据《纯应用化学》(Pure Appl.Chem.)31,639-645(1972)和40,277-290(1974)和IUPAC-IUB生物化学命名委员会的建议,并符合37 CFR§1.822(55 FR 18245,1990年5月1日)。单字母和三字母缩写如下:
氨基酸的缩写
氨基酸 三字母符号 单字母符号
丙氨酸 Ala A
精氨酸 Arg R
天冬酰胺 Asn N
天冬氨酸 Asp D
Asn+Asp Asx B
半胱氨酸 Cys C
谷胺酰胺 Gln Q
谷氨酸 Glu E
Gln+Glu Glx Z
甘氨酸 Gly G
组氨酸 His H
异亮氨酸 Ile I
亮氨酸 Leu L
赖氨酸 Lys K
甲硫氨酸 Met M
苯丙氨酸 Phe F
脯氨酸 Pro P
丝氨酸 Ser S
苏氨酸 Thr T
色氨酸 Trp W
酪氨酸 Tyr Y
缬氨酸 Val V
其它氨基酸Xaa X
以上缩写代表L氨基酸,除非另外标示为D-或D、L-。有些天然和非天然的氨基酸是非手性的,例如甘氨酸。全部氨基酸序列的标示均以N末端在左而以C末端在右。
本文使用的其它氨基酸和化合物的缩写为:hSer 高丝氨酸hSerlac 高丝氨酸内酯Nle 正亮氨酸PEG2 二乙二醇甲基醚基,a.k.a.甲氧基二(亚乙氧基),
CH3O(CH2CH2O)2-,(分子量(MW)=119)PEG5000 聚(乙二醇甲基醚)基,a.k.a.甲氧基聚(亚乙氧基),
CH3O(CH2CH2O)110-,(分子量(MW)=5000)PEGX 聚(乙二醇甲基醚)基,a.k.a.CH3O(CH2CH2O)n-,n=2-225,
(平均分子量(avg.MW)=100至10,000)
“亲水性氨基酸(Haa)”指除了形成肽键所需的官能团之外至少具有一个亲水性官能团的氨基酸,例如精氨酸、天冬酰胺、天冬氨酸、谷氨酸、谷胺酰胺、组氨酸、赖氨酸、丝氨酸、苏氨酸,以及它们的同系物。
“亲脂性氨基酸(Laa)”指不带电的脂肪族或芳香族氨基酸,例如异亮氨酸、赖氨酸、甲硫氨酸、苯丙氨酸、色氨酸、酪氨酸、缬氨酸,以及它们的同系物。
出于本发明的目的,将丙氨酸归为“两亲氨基酸”,即,既能够表现出亲水性,又能够表现亲脂性。
“PTH或PTHrp的生理活性截短同系物或类似物”指其序列中包含的氨基酸比PTH或PTHrp中的全部氨基酸少,但是引发相似的生理应答的多肽。为了引发相似的生理应答,截短的PTH或PTHrp不必与PTH或PTHrp完全同源。这类化合物中代表性的、较好的的是PTH(1-34)和PTHrp(1-34),但也包括其它的。
“两亲α螺旋”指某些多肽表现出的二级结构,其中氨基酸呈α螺旋构型,沿着螺旋的长轴具有相的反极性和非极性表面。通过构建合适螺距的“Schiffer-Edmundson轮”(M.Schiffer和A.B.Edmundson,《生物物理学杂志》“(Biophys.J.),7,121(1967))并注意环绕螺旋的相对柱表面上亲水性和亲脂性残基的分离,可以在一定程度上探测目标多肽中α螺旋结构的几率。或者,可能有表明在给定多肽中存在α螺旋结构的经验性数据,例如环二色性或X光衍射数据。理想的α螺旋每转具有3.6个氨基酸残基,而且相邻侧链的分开弧度为100°。Eisenberg等在《自然》(Nature)299:371-374(1982)和《美国科学院院报》(Proc.Nat.Acad.Sci.USA)81:140-144(1984)将疏水性级数与螺旋轮组合来将两亲螺旋的概念定量化。平均疏水性力矩定义为构成螺旋的组分氨基酸的疏水性矢量和。以下氨基酸的疏水性是Eisenberg(1984)所报道的“交感”级数:
Ile 0.73;Phe 0.61;Val 0.54;Leu 0.53;Trp 0.37;
Met 0.26 Ala 0.25;Gly 0.16;Cys 0.04;Tyr 0.02;
Pro-0.07;Thr-0.18;Ser-0.26;His-0.40;Glu-0.62;
Asn-0.64;Gln-0.69;Asp-0.72;Lys-1.10;Arg-1.76.
可由以下公式来计算每转3.6个残基(或者侧链分开100°弧度(360°/3.6))的理想α螺旋的疏水性力矩μH:
μH=[(∑HNsinδ(N-1))2+(∑HNcosδ(N-1))2]1/2,其中HN是Nth氨基酸的疏水性值,而总和则取自周期为δ=100°的N个氨基酸。可以通过将μH除以N得到<μH>将疏水性力矩标示为每个残基的平均疏水性力矩。100°±20°时的<μH>约为0.20或以上表示存在两亲螺旋构型。100°时hPTHrp(22-31)和hPTH(22-31)的<μH>分别为0.19和0.37。
Cornett等在《分子生物学杂志》(J.Mol.Biol.)195:659-685(1987)中通过引入“两亲指数”作为两亲性指示进一步扩展了对两亲α螺旋的研究。它们认为,全部已知α螺旋中约半数是两亲性的,主要频率是97.5°而非100°,每转的残基数更接近3.7而非3.6。虽然这种精确化具有科学上的意义,但是Eisenberg等的基本方法已足以将一给定序列归为两亲性,尤其是在从一开始就试图设计一段形成两亲α螺旋的序列时。
一段取代两亲α螺旋氨基酸序列与天然存在多肽中给定片段的序列可能不具有同源性,但能够在生理环境中形成相似的二级结构,即,具有相反极性或非极性表面的α螺旋。用替代序列置换天然存在的氨基酸序列可能改良被改变的亲代多肽的生理活性、稳定性或其它特性。有关设计和选择这样的序列可参见J.L.Krstenansky等(FEBS Letters)242:2,409-413(1989)和J.P.Segrest等《蛋白质:结构、功能和遗传》(Proteins:Structure,Function and Genetics)8:103-117(1990),以及其它。
本发明十个氨基酸的两亲α螺旋具有以下通式:
Haa(Laa Laa Haa Haa)2 Laa其中Haa选自前文定义的亲水性氨基酸,而Laa选自前文定义的亲脂性氨基酸。假设它是一个理想α螺旋,残基1、4、5、8和9分布在螺旋的一个平面(A)内,各自分开约140°弧度以下,而残基2、3、6、7和10则在螺旋的另一平面(B)上占据相反的140°弧度。较好的是,同一平面上的残基都具有相同的极性,而另一平面上的都具有相反的极性,即如果A平面上的残基都是亲水性的,B平面上的就都是亲脂性的,或者反过来。本领域技术人员将承认,虽然将本发明的螺旋描述成:
Haa(Laa Laa Haa Haa)2 Laa其反序列
Laa(Haa Haa Laa Laa)2 Haa也符合残基分布标准,是本发明螺旋的一种等同描述。
丙氨酸既可以取代亲水性氨基酸也可以取代亲脂性氨基酸,因为Ala能够方便地存在于两亲α螺旋的两种平面上,但是Ala10不形成两亲α螺旋。通常,不使用脯氨酸、半胱氨酸和酪氨酸;但是,它们在序列中的的存在和其它偶然误差是允许的,例如亲脂性平面上的一个亲水性残基,只要片段内的其它氨基酸符合亲水性平面-亲脂性平面区分。一种确定某序列是否具有足够的两亲性以成为本发明序列的简便方法是如前文所述计算疏水性力矩。如果在100°±20°时平均力矩/残基的峰值超过0.20,该序列将形成一个两亲螺旋,而且是本发明的序列。
例如,如下计算100°时(SEQ ID NO:26),Xaa=Glu的平均疏水性力矩/残基:
A.A. HN δ(N-1) H sinδ(N-1) H cosδ(N-1)
E -.62 0 0 -.62
L .53 100 .52 -.17
L .53 200 -.18 -.50
E -.62 300 .34 -.31
K -1.1 400 -.70 -.85
L .53 500 .34 -.41
L .53 600 -.46 -.27
E -.62 700 .21 -.58
K -1.1 800 -1.08 -.19
L .53 900 0 -.53
∑=0.81 ∑=-4.43
μH=[(0.81)2+(-4.43)2]1/2=4.50
<μH>=4.50/10=0.45
就此序列而言,平均峰值疏水性力矩出现在92°,为0.48。
将此概念用于甲状旁腺素和甲状旁腺素相关肽时,我们假设区域(7-16)和(22-31)其一或两者都表现出α螺旋二级结构,而且可以被具有相同结构趋向的非同源序列置换,但并不损失生物活性或诱导免疫反应。优选实施方式
本发明内容之一,提供了一种PTH、PTHrp的类似物,和PTH和PTHrp的生理活性截短类似物和同系物,或它们的盐,其中的氨基酸残基(22-31)来自一个两亲α螺旋,所述残基(22-31)的序列选自:0a)Xaa1Xaa2Leu Xaa4Xaa5Leu Xaa7Xaa8Xaa9Xaa10,其中Xaa1和Xaa4独立地选1 5 10自Glu、Glu(OCH3)、His或Phe;Xaa2是Leu或Phe;Xaa5是Lys或His;Xaa7和Xaa10独立地是Leu或Ile;Xaa8是Ala、Arg或Glu;Xaa9是Lys或Glu(SEQ ID NO:85);较好的是Glu Leu Leu Glu Lys Leu Leu Xaa Lys Leu,其中的Xaa是Glu或Arg1 5 10(SEQ ID NO:26);b)Xaa1Xaa2Leu Xaa4Arg Leu Leu Xaa8Arg Leu,其中Xaa1和Xaa4独立地选自1 5 10Glu、Glu(OCH3)、His或Phe;Xaa2是Leu或Phe;Xaa8是Glu、Lys或Lys(COCH2PEGX),而PEGX是分子量为100至10,000的聚(乙二醇甲基醚)基;(SEQ ID NO:86);较好的是Glu Leu Leu Glu Arg Leu Leu Xaa Arg Leu,其中的Xaa是Glu、Lys或1 5 10Lys(COCH2PEGX),而PEGX是分子量为100至10,000的聚(乙二醇甲基醚)基(SEQ ID NO:27);c)Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu(SEQ ID NO:28);1 5 10d)Ser Leu Leu Ser Ser Leu Leu Ser Ser Leu(SEQ ID NO:29);1 5 10e)Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu(SEQ ID NO:30)。1 5 10
本发明还提供了具有以下通式的PTH、PTHrp的类似物,和PTH和PTHrp的生理活性截短类似物和同系物,或它们的盐,Xaa1Xaa2Xaa3Xaa4Xaa5Xaa6Xaa7Leu His Xaa10Xaa11Gly Xaa13Ser Ile GlnXaa17Leu Xaa19Xaa20Xaa21Xaa22-31Xaa32Xaa33Xaa34Xaa35Xaa36Xaa37Xaa38末端,其中Xaa1缺失或者是Ala;Xaa2缺失或者是Val;Xaa3缺失或者是Ser;Xaa4缺失或者是Glu或Glu(OCH3);Xaa5缺失或者是His或Ala0;Xaa6缺失或者是Gln;Xaa7缺失或者是Leu;Xaa10和Xaa17独立地是Asp或Asp(OCH3);Xaa11是Lys、Arg或Leu;Xaa13是Lys、Arg、Tyr、Cys、Leu、Cys(CH2CONH(CH2)2NH(生物素),Lys(7-二甲基氨基-2-氧-2H-1-苯并吡喃基-4-乙酰基)或Lys(二氢肉桂酰基);Xaa20是Arg或Leu;Xaa19和Xaa21独立地是Lys、Ala、或Arg;Xaa22-31选自(SEQ ID NO:26、27、28、29或30);Xaa32是His、Pro或Lys;Xaa33缺失或者是Pro、Thr、Glu或Ala;Xaa34缺失或者是Pro、Arg、Met、Ala、hSer、hSer内酯、Tyr、Leu或1,4-二氨基丁酰基内酰胺;Xaa35缺失或者是Pro、Glu、Ser、Ala或Gly;Xaa36缺失或者是Ala、Arg、或Ile;Xaa37缺失或者是Arg、Trp或3-(-2-萘基)-L-丙氨酸;Xaa38缺失或者是Ala或hSer,或者,Xaa38-42是Thr Arg Ser Ala Trp;末端是OR或NR2,其中的R独立地为H、(C1-C4)烷基或苯基(C1-C4)烷基;以及它们的药学上认可的盐
本发明内容之一还提供了hPTHrp(1-34)的生理活性截短同系物的多肽类似物,如通式(I)所示:Ala Val Ser Glu Xaa5Gln Leu Leu His Asp Xaa11Gly Xaa13Ser Ile Gln Asp LeuXaa19Arg Xaa21Xaa22-31Xaa32Xaa33Xaa34末端,其中Xaa5是His或Ala;Xaa11和Xaa13独立地是Lys、Arg或Leu;Xaa19和Xaa21独立地是Ala或Arg;Xaa22-31选自:a)Glu Leu Leu Glu Lys Leu Leu Xaa Lys Leu,其中的Xaa是Glu或Arg1 5 10(SEQ ID NO:26);b)Glu Leu Leu Glu Arg Leu Leu Xaa Arg Leu,其中的Xaa是Glu、Lys或1 5 10Lys(COCH2PEGX),而PEGX是分子量为100至10,000的聚(乙二醇甲基醚)基(SEQ ID NO:27);c)Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu(SEQ ID NO:28);1 5 10d)Ser Leu Leu Ser Ser Leu Leu Ser Ser Leu(SEQ ID NO:29);1 5 10e)Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu(SEQ ID NO:30)。1 5 10Xaa32是His或Lys;Xaa33是Thr、Glu或Ala;Xaa34是Ala、hSer、Tyr或Leu;末端是GlyArgArg、内酯、OH或NR2,其中的R是H或(C1-C4)烷基;以及它们的药学上认可的盐。(通式I)
本发明更具体的内容还包括那些通式(I)所示的多肽,其中的Xaa22-31是(SEQID NO:26),100°时的<μH>超过0.45。本发明更具体的内容包括通式(I)所示、其中的Xaa22-31是(SEQ ID NO:26),Xaa11和Xaa13都是Lys;而且Xaa19和Xaa21都是Arg的多肽。
代表性的多肽包括但不限于: Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Arg Lys
20 25 30Leu His Thr Ala OH(SEQ ID NO:5);Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala OH(SEQ ID NO:6);Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala NH2(SEQ ID NO:7);Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr hSer NH2(SEQ ID NO:8);Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr hSerlac(SEQ ID NO:9);Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala Gly Arg Arg OH(SEQ ID NO:10);和
35
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu Lys Glu Leu NH2(SEQ ID NO:11).
本发明内容还包括通式(I)所示多肽,其中的Xaa22-31是(SEQ ID NO:26);Xaa11和Xaa13都是Lys;Xaa19和Xaa21其中之一是Arg,另一个是Ala。这一亚类的多肽包括但不限于:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Ala Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Ala NH2(SEQ ID NO:12)和
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Ala Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Ala NH2(SEQ ID NO:13).
本发明内容还包括通式(I)所示多肽,其中的Xaa22-31是(SEQ ID NO:26);Xaa11和Xaa13其中之一是Leu,另一个是Lys;Xaa19和Xaa21都是Arg。这一亚类的多肽包括但不限于:
Ala Val Ser Glu Ala Gln Leu Leu His Asp Leu Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Ala Leu OH(SEQ ID NO:14)
本发明内容还包括通式(I)所示多肽,其中的Xaa22-31是(SEQ ID NO:27);100°时的<μH>超过0.50。本发明更具体的内容包括通式(I)所示、其中的Xaa22-31是(SEQID NO:27),Xaa11和Xaa13都是Lys或者都是Arg;而且Xaa19和Xaa21都是Arg的多肽。这一亚类的多肽包括但不限于:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30
Leu His Thr Ala OH(SEQ ID NO:15);
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30
Leu His Thr Ala OH(SEQ ID NO:16);
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Lys Arg
20 25 30
Leu His Thr Ala OH(SEQ ID NO:17);
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu
20 25
Lys(COCH2PEG2)Arg Leu His Thr Ala OH(SEQ ID NO:18);和
30
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu
20 25
Lys(COCH2PEG5000)Arg Leu His Thr Ala OH(SEQ ID NO:19).
30
本发明内容还包括通式(I)所示、其中的Xaa22-31是(SEQ ID NO:28)的多肽,100°时的<μH>约为0.25。这一亚类的多肽包括但不限于:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Ala Leu Ala Glu Ala Leu Ala Glu Ala
20 25 30
Leu His Thr Ala NH2(SEQ ID NO:20).
本发明内容还包括通式(I)所示、其中的Xaa22-31是(SEQ ID NO:29)的多肽,100°时的<μH>约为0.28。这一亚类的多肽包括但不限于:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Ser Leu Leu Ser Ser Leu Leu Ser Ser
20 25 30
Leu His Thr Ala NH2(SEQ ID NO:21).
本发明内容还包括通式(I)所示、其中的Xaa22-31是(SEQ ID NO:30)的多肽,100°时的<μH>约为0.29。这一亚类的多肽包括但不限于:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Ala Phe Tyr Asp Lys Val Ala Glu Lys
20 25 30
Leu His Thr Ala NH2(SEQ ID NO:22).
本发明内容还包括bPTH(1-34)的生理活性截短同系物的多肽类似物,如通式(II)所示:Xaa1Val Ser Glu Ile Gln Xaa7Xaa8His Asn Leu Gly Lys His Leu Xaa16Ser Xaa18Xaa19Arg Xaa21Xaa22-31His Asn Xaa34末端,其中Xaa1是Ser或Ala;Xaa7是Leu或Phe;Xaa8是Met或Nle;Xaa16是Asn或Ser;Xaa18是Leu、Met或Nle;Xaa19是Glu或Arg;Xaa21是Val或Arg;Xaa22-31选自(SEQ ID NO:26、27、28、29和30);Xaa34是Phe或Tyr;末端是OH或NR2,其中的R是H或(C1-C4)烷基;以及它们的药学上认可的盐。(通式II)代表性的多肽包括但不限于:
Ala Val Ser Glu Ile Gln Phe Nle His Asn Leu Gly Lys His Leu
1 5 10 15
Ser Ser Nle Glu Arg Val Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Asn Tyr NH2(SEQ ID NO:23)和
Ala Val Ser Glu Ile Gln Phe Nle His Asn Leu Gly Lys His Leu
1 5 10 15
Ser Ser Nle Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Asn Tyr NH2(SEQ ID NO:24)
本发明还出人意料地发现,少于34个氨基酸的PTH和PTHrp的同系物和类似物也是强效的骨重塑药物。这类化合物的通式为:
Ala Val Ser Glu Xaa5Gln Leu Leu His Asp Xaa11Gly Xaa13Ser Ile Gln Asp LeuXaa19Arg Xaa21Xaa22-31Xaa32Xaa33Xaa34末端,
代表性的多肽包括但不限于:
化合物41:AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHP-NH2(SEQID NO:55)
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Pro NH2(SEQ ID NO:55).
物理数据
m.p.142.8-166.1℃ [α]D 25-53.80(c 0.38,H2O)
FAB(C173H295N55O49):[M+H]+ 3929
AAA:Asx 2.0(2) Glx 5.7(6) Ser 1.8(2)
His 3.0(3) Gly 1.1(1) Ala 0.9(1)
Arg 2.8(3) Val 1.2(1) Ile 0.9(1)
Leu 7.4(8) Lys 4.4(4) Pro 0.9(1)
化合物42:AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LP-NH2(SEQ IDNO:56)
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu Pro NH2(SEQ ID NO:56). 物理数据
m.p.161.0-177.0℃ [α]D 25-61.97(c 0.19,H2O)
FAB(C167H288N52O48):[M+H]+3792.0
AAA:Asx 2.2(2) Glx 5.9(6) Ser 1.9(2)
His 2.1(2) Gly 1.1(1) Ala 1.0(1)
Arg 3.0(3) Val 1.1(1) Ile 1.0(1)
Leu 7.9(8) Lys 4.3(4) Pro 0.9(1)
本领域技术人员将理解,可以人工进行许多多肽类似物的置换,它们都具有本文所述的要求的特性,条件是,在位置(22-31)插入的的氨基酸序列在100°±20°时的平均疏水性力矩/残基大于约0.20。多肽的经典合成法
本发明的多肽可以利用以下方法来合成:有关固相合成的J.M.Stewart和J.D.Yong,《固相肽合成法》(Solid Phase Peptide Synthesis),第二版,PierceChemical Co.,Rockford,Illinois(1984)和J.Meienhofer,《激素蛋白和肽》(HormonalProteins and Peptides),第2卷,Academic Press,New York(1973),以及有关液相合成的E.Schroder和K.Lubke,《肽》(The Peptides),第1卷,Academic Press,NewYork(1965)。
通常,以上方法是将被保护的氨基酸连续地加到不断延伸的肽链上。一般,被保护的是第一氨基酸的氨基或羧基以及任何反应性侧链基团。然后该被保护的氨基酸被固定在惰性固体载体上,或者以溶液形式使用,然后在形成酰胺键的相宜条件下,添加上同样被适当保护的、序列中的下一个氨基酸。在所有被要求的氨基酸以正确序列连接后,将保护基和各种固体载体去除,生成粗多肽。多肽经脱盐和纯化,最好是通过色谱法,生成最终产物。
一种较好的制备生理活性截短多肽类似物,即氨基酸少于40个的方法是固相肽合成法。在该方法中,α-氨基(Nα)官能团和任何反应性侧链用酸或碱敏感性基团保护。保护基必需能在肽键形成条件下稳定存在,通常易于被去除而不影响已生成的多肽链。合适的α-氨基保护基包括但不限于:叔丁氧基羰基Boc)、苄氧基羰基(Cbz)、邻氯苄氧基羰基、二苯基异丙氧基羰基、叔戊氧基羰基(Amoc)、异冰片基氧基羰基、α,α-二甲基-3,5-二甲氧基苄氧基-羰基、邻硝基苯基亚磺酰基、2-氰基-叔丁氧基羰基、9-芴基甲氧基羰基(Fmoc)等,较好的是叔丁氧基羰基(Boc)。合适的侧链保护基包括但不限于:乙酰基、苄基(Bzl)、苄氧基甲基(Bom)、邻溴苄氧基羰基、叔丁基、叔丁基二甲基甲硅烷基、2-氯苄基(Cl-z)、2,6-二氯苄基、环己基、环戊基、异丙基、新戊酰基、四氢吡喃-2-基、甲苯磺酰基(Tos)、三甲基甲硅烷基和三苯甲游基。
在固相合成法中,C末端氨基酸首先与合适的树脂载体结合。合适的树脂载体是那些对分步缩合反应和去保护反应中的反应剂和条件惰性,同时可溶于所用的介质的物质。市售树脂的实例包括:用反应性基团改性的苯乙烯/二乙烯基苯树脂等,例如氯甲基化的共聚(苯乙烯-二乙烯基苯)、羟甲基化的共聚(苯乙烯-二乙烯基苯)。其中以苄基化的和羟甲基化的苯乙酰氨甲基树脂(PAM)为佳。当化合物的C末端是酰胺时,树脂以对甲基二苯甲基氨基-共聚(苯乙烯-二乙烯基-苯)树脂为佳。
与PAM树脂的固定可如下进行:将Nα被保护氨基酸,以Boc-氨基酸为佳,以其铵、铯、三乙基铵、1,5-二氮杂环-[5.4.0]十一-5-烯、四甲基铵或类似的盐形式,在乙醇、乙腈、N,N-二甲基甲酰胺(DMF)等中,较好的是其铯盐在DMF中与树脂在较高温度,例如约40℃至60℃,以50℃为佳,反应约12至72小时,以约48小时为佳。
可以利用以下方法将Nα-Boc-氨基酸固定到二苯甲基胺树脂上:例如在二氯甲烷或二甲基甲酰胺之类,以二氯甲烷为佳的熔剂中,在约10℃至50℃,以25℃为佳的温度下,进行由N,N’-二异丙基碳化二亚胺(DIC)/1-羟基苯并三唑(HOBt)介导的偶合反应约2至24小时,以约2小时为佳。
可使用本领域熟知的方法,较典型的是利用自动化肽合成仪来成功地进行被保护氨基酸的偶合。在用三乙胺或类似碱中和后,加入最好过量约1.5至2.5倍摩尔的各被保护氨基酸,并在惰性、非水性、极性熔剂例如二氯甲烷、DMF或它们的混合物中,以二氯甲烷为佳,在环境温度下进行偶合。代表性的偶合剂是N,N’-二环己基碳化二亚胺(DCC)、N,N’-二异丙基-碳化二亚胺(DIC)或其它碳化二亚胺,它们或者单独使用,或者在1-羟基苯并三唑(HOBt)、O-酰基脲、六氟磷酸苯并-1-基-氧三(吡咯烷基)磷鎓(PyBop)、N-羟基琥珀酰胺、其它N-羟基酰胺或肟存在下使用。或者,可以使用被保护氨基酸活性酯(例如对硝基苯基、五氟苯基等)或对称酐。
在固相合成法的最后,将完全被保护氨基酸从树脂上分离。如果与树脂载体的结合键是苄酯型的,对具有烷基酰胺C末端的肽可通过使用烷基胺或氟烷基胺的氨解来切割,对具有非取代酰胺C末端的肽可通过使用氨/甲醇或氨/乙醇的氨解来切割,温度在约-10℃至50℃,以约25℃为佳,进行约12至24小时,以约18小时为佳。具有羟基C末端的肽可以利用HF或其它强酸性去保护法或通过皂化作用来切割。或者,可以通过先转酯,例如利用乙醇,然后进行氨解或皂化来将肽从树脂上分离。被保护的肽可利用硅胶色谱来纯化。
可以如下将侧链保护基从肽上去除:通过用例如无水氢氟酸在苯甲醚或其它碳鎓离子净化剂存在下处理氨解产物,用氟化氢/吡啶配合物处理,用三(三氟乙酰基)硼和三氟乙酸处理,通过用氢和钯碳或聚乙烯基吡咯烷酮进行还原,或者通过用钠在液氨中还原,较好的是用氢溴酸和苯甲醚在约-10℃至+10℃,以约0℃为佳,反应约15分钟至2小时,以约1.5小时为佳。对于二苯甲基胺树脂上的肽,如上所述利用氢溴酸和苯甲醚,从树脂上的切割和去保护步骤可以合并成一步。
可以通过使用了部分或全部以下步骤的一系列色谱步骤来将溶液脱盐(例如使用BioRad AG-3阴离子交换树脂)和纯化肽:乙酸盐型弱碱性树脂上的离子交换;非衍生共聚(苯乙烯-二乙烯基苯)上的疏水性吸附色谱,例如AmberliteXAD;硅胶吸附色谱;羧甲基纤维素上的离子交换色谱;在例如SephadexG-25上的分配色谱;逆流分布;或高性能液相色谱(HPLC),特别是辛基或十八烷基甲硅烷基二氧化硅(ODS)结合相充填柱上的反相HPLC。
所以,本发明的内容还涉及制备多肽及其药学上认可的盐的方法,这些方法包括在合适的树脂载体上依次缩合被保护氨基酸,去除保护基和树脂载体和纯化产物,由此提供PTH和PTHrp,以PTH(1-34)和PTHrp(1-34)为佳的生理活性截短同系物和类似物,其中(22-31)位的氨基酸形成一个前文所述的两亲α螺旋肽序列。多肽的重组合成
或者,本发明的多肽可以通过克隆和表达编码所需多肽的基因来制备。在这种方法中,包含所需DNA序列的质粒被制备并插入合适的宿主微生物中,较典型的是细菌,例如大肠杆菌或酵母,例如酿酒酵母,同时诱导宿主微生物产生质粒的多份拷贝,由此也产生编码本发明多肽类似物的cDNA的多份拷贝。
首先,设计一段带有常规限制性酶切位点以便于后继改变的选定PTH或PTHrp类似物的合成基因。可使用Mullis在美国专利4,683,195和4,683,202中所述的聚合酶链反应(PCR)来扩增此序列。
扩增后的合成基因可将其分离并与合适的质粒连接,例如Trp LE质粒,在该质粒中可串联插入四份基因拷贝。美国专利No.4,738,921和欧洲专利公开No.0212532中说明了Trp LE质粒的制备。Trp LE质粒通常比Trp E质粒产生多8至10倍的蛋白质。然后可在合适的宿主,例如大肠杆菌和酿酒酵母内表达多拷贝基因。
在此使用的具体的表达载体是包含以下元件的Trp LE 18 Prot(Ile3,Pro5):一段包含青霉素抗性基因和质粒复制起始点的pBR322片段(EcoRI-BamHI);包含trp启动子和trpE基因的一段EcoRI-SacII片段;一段HIV蛋白酶(Ile3,Pro5)基因片段(SacII-HindIII);一段bGRF基因片段(HindIII-BamHI);和大肠杆菌rpoc基因的转录终止子。HIV蛋白酶和bGRF基因片段并不是关键性的,可以根据需要以其它编码序列取代。
表达的多聚体融合蛋白在细胞内积聚成稳定的包涵体,它们可以通过离心与其它细胞蛋白分离。分离后的融合蛋白被转化成单体PTH或PTHrp类似物,它们可通过阳离子交换和/或反相HPLC来纯化。
其它克隆、扩增、表达和纯化方法对熟练技术人员来说显而易见的。代表性的方法被公开在Maniatis等的《分子克隆实验手册》(Molecular Cloning,aLaboratory Mannual)第2版,Cold Spring Harbor Laboratory(1989)。用途和给药方法
本发明的多肽可用于防止和治疗多种以骨质损失为特征的哺乳动物病症。具体地说,本发明的化合物适用于对人骨质疏松症和骨质减少症的预防和治疗。
通常,本发明的多肽或其盐按照每天约0.002至10微克/千克体重给药,以每天约0.04至约0.2微克/千克体重为佳。对一个50千克的女性来说,活性成份的日用剂量约为0.1至约500微克,以约2.0至约100微克为佳。在其它哺乳动物例如马、狗和牛中,可能需要更高的剂量。根据获取最佳药效的要求,该剂量可以以常规药物组合物的形式通过一次给药、多次给药或通过控释方法来传递,较好的是每天进行一次或多次注射。
对确切剂量、组成和最有效传递方法的选择同时受到所选多肽的药理特性、待治病情的特点和严重程度、以及患者的物理情况和精神状态的影响。
在治疗由皮质类固醇诱导的骨质减少症时,预计对于较高剂量的皮质类固醇将需要必需剂量更高的多肽。
代表性的传递方法包括口服,非肠胃道给药(包括皮下、肌内和静脉),经直肠、经颊(包括舌下),经肺,透皮和经鼻。
药学上认可的盐保留了本发明多肽所需的生物活性而没有毒性副作用。这类盐的实例是a)与无机酸例如盐酸、氢溴酸、硫酸、磷酸、硝酸等形成的酸加成盐;和与有机酸例如乙酸、草酸、酒石酸、琥珀酸、马来酸、富马酸、葡糖酸、柠檬酸、苹果酸、抗坏血酸、苯甲酸、鞣酸、双羟萘酸、藻酸、聚谷氨酸、萘磺酸、萘二磺酸、半乳糖醛酸等形成的盐;b)与多价金属阳离子例如锌、钙、钼、钡、镁、铝、铜、锆、镍、镉;或与N,N’-二苄基乙二胺或乙二胺形成的有机阳离子形成的碱加成盐;或c)、a)与b)的合并,例如鞣酸酯锌之类。
本发明的内容还涉及包含本发明多肽或其药学上认可的盐作为有效成份,并混合以药学上认可的非毒性载体的药物组合物。如前所述,这类组合物可以配制成用于非肠胃道给药的(皮下、肌内或静脉),具体为液体溶液或悬浮液的形式;用于口服或经颊给药,具体为片剂或胶囊;用于经肺或经鼻给药,具体为粉剂、滴鼻液或喷雾剂形式;以及用于经直肠或透皮给药。
较好的是,组合物以单位剂量形式给药,这样的组合物可以利用任意已知制药领域的常用方法来制备,例如在《雷明顿药物科学》(Remington’s PharmaceuticalSciences)第17版,Mack Publishing Company,Easton,PA(1985)中所述的方法。用于非肠胃道给药的制剂中可包含作为赋形剂的无菌水或生理盐水,丙二醇之类的烷二醇,聚乙二醇之类的聚烷二醇,植物油,氢化萘等。用于经口给药时,制剂可通过添加胆盐或酰基肉碱来强化。经鼻给药的制剂可以是固体的并包含以乳糖或葡聚糖为例的某些赋形剂,或者可以是适合以滴鼻液或定量喷雾剂形式使用的水溶液或油溶液。
在配制鼻用制剂时,可利用例如表面活性剂,例如甘氨胆酸、胆酸、牛黄胆酸、乙胆酸(ethocholic acid)、脱氢胆酸、鹅脱氧胆酸、脱氧胆酸、甘氨脱氧胆酸、环糊精等来加强通过鼻粘膜的吸收,表面活性剂的用量在约0.2至15重量百分比,以约0.5至4重量百分比为佳,约2重量百分比最好。
可以通过一次使用含有对要求的释放时间而言足量的活性成份的控释系统来达到在较长时间,例如一周至一年内向患者传递本发明的化合物。多种控释系统,例如骨架式或贮囊式微胶囊、缓释植入剂、渗透压泵、囊泡、胶团、脂质体、透皮贴片、离子透人装置和其它可注射剂型都可用于此目的。部分控释装置的附加特性之一是局限于需要传递活性成份的部位,这对于某些疾病的治疗来说可能是十分有利的。
一种形式的控释制剂中包含分散或包裹在缓慢降解、无毒、无抗原聚合物例如根据(乳酸/乙醇)酸中的多肽或其盐,正如Kent,Lewis,Sanders和Tice的前期工作即美国专利4,675,189中所述。这些化合物,更好的是它们相对难溶的盐还可以配制在甾醇或其它脂质基质药丸,或硅塑料基质植入剂中。对熟练技术人员来说,其它缓释剂、缓释植入剂或可注塑制剂是显而易见的。可参见例如,《缓释和控释药物传递系统》(Sustained and Controlled Release Drug Delivery Systems),J.R.Robinson编,Marcel Dekker,Inc.,New York,1978和R.W.Baker,《生物活性药物的控释》(Controlled Release of Biologically Active Agents),John Wiley &Sons,New York,1987。鼻传递系统
使用市售的喷雾泵和瓶来传递鼻用溶液,用于在cynomolgous猴中进行评价。传递体积为50至100微升的喷雾泵由Valois提供。在前期试验中使用成人尺寸的滴嘴。在后期试验中使用儿科尺寸的喷雾滴嘴。滴嘴的尺寸并不影响喷雾泵的性能。从各组喷雾泵中取样进行了测试,以便确定引发泵所需的激活次数、其传递体积的精确性及其喷雾方式的稳定性。
猴子在给药前被麻醉,并取背靠姿势给药。为了传递4毫克肽,在各鼻孔中滴入100微升100毫克/毫升的溶液。为了传递1毫克肽,在各鼻孔中滴入50微升20毫克/毫升的溶液。在以后的试验中,作为本身的对照,各猴被皮下给药以50微克的肽。与皮下注射比较来计算鼻生物利用率。在给药后的0、0.25、0.5、1、2、4、6和8小时采取血样并利用RIA测定肽含量。由血浆曲线计算药代动力学参数。利用梯形法则计算血浆浓度曲线的曲线下面积(AUC),通过与所有动物皮下给药平均AUC的比较来计算生物利用率。
本发明组合物的平均生物利用率接近50%。鼻用制剂的Tmax在0.3至0.7小时之间,与测得的皮下给药的Tmax(0.6小时)相近。
在本发明使用的胆酸表面活性剂中有甘氨胆酸、胆酸、牛黄胆酸、胆烷酸、乙胆酸、脱氧胆酸、鹅脱氧胆酸和它们药学上认可的盐,其中包括钠、钾、铵、钙和镁盐,以及其它无毒有机和无机阳离子。较好的是,胆酸表面活性剂是甘氨胆酸或牛黄胆酸的碱金属盐,最好的是钠盐。
在鼻用制剂中还可以加入其它物质,例如防腐剂、维持等渗的盐、缓冲液等。通常,肽的浓度约为1毫克/毫升至约100毫克/毫升,吸附增强剂的浓度约为0.5毫克/毫升至约50毫克/毫升。
以下具体实施例是为了说明本发明代表性化合物的合成和测试,而不应该被视为是对权利要求范围的限定。在实施例中“m.p.”表示熔点,“[α]D 25”是25℃时给定浓度下在所示溶剂中的光学活性,“FAB”是快速原子轰击质谱,“AAA”是氨基酸分析,同时在测定值后括弧内给出估计值。氨基酸分析用Hewlett-Packard Amino Quantw分析仪根据制造商的建议程序进行。用邻苯二甲酸衍生初级氨基酸;用Fmoc衍生次级氨基酸。利用对经衍生氨基酸的荧光检测来定量。被保护氨基酸由Applied Biosystems Inc.(Foster City,CA)提供。
实施例1
使用自动的Applied Biosystems 430A型肽合成仪在4-甲基二苯甲基胺树脂上制备0.5毫摩尔的化合物1(SEQ ID NO:7)。α-氨基用叔丁氧基羰基(Boc)保护。侧链保护基是:保护Asp,Glu和Ser的苄基(Bzl);Arg的苯磺酰基(Tos);His的苄氧基甲基(Bom);和Lys的2-氯苄基(Cl-z)。按照Stewart和Young(上文)所述用N,N’-二环己基碳化二亚胺/1-羟基苄氧基三唑(DCC/HOBt)来依次偶合氨基酸。每一个氨基酸偶合之后,用乙酸酐和二异丙基乙胺在N-甲基吡咯烷酮中的混合物酰化肽。在苯甲醚存在下(2.5毫升),用无水HF(25毫升)在-10℃反应30分钟,在0℃反应60分钟,将完成后的肽从树脂上切下,同时去保护侧链保护基。真空蒸发HF后,用无水醚洗涤残留物,然后用10%乙酸萃取粗肽。10%乙酸萃取液经冷冻干燥后得900毫克粗产物。通过中等压力ODS反相柱色谱纯化肽,使用的是CH3CN在0.1%TFA中的22-45%浓度梯度。将产物洗脱在三份流份中,经浓缩和冷冻干燥后得130毫克纯度高于98%的白色固体。
化合物1:
AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTA-NH2(SEQ ID NO:7)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala NH2(SEQ ID NO:7)物理数据:m.p.150-159 C[a]D25-34.88°(c 0.16,H2O)FAB(C175H300N56O51):[M+H]+4005.5AAA:Asp,1.9(2);Glu,5.6(6);Ser,1.6(2);His,2.7(3);Gly,1.0(1);Thr,0.9(1);
Ala,1.9(2);Arg,2.8(3);Val,1.0(1);Ile,0.9(1);Leu,7.3(8);Lys,4.0(4).
以同样方式制备和鉴定化合物2、5-18、21-27、29-36、38-48、50-54、58-64和66-70,在合成羟基末端多肽时代之以PAM树脂。
化合物2:AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTA-OH(SEQ ID NO:6)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala OH(SEQ ID NO:6)物理数据:m.p.154-170℃[α]D 25-49.35°(c 0.46,H2O)FAB(C175H301N57O50):[M+H]+4005.0AAA:Asp,2.1(2);Glu,5.9(6);Ser,1.7(2);His,2.9(3);Gly 1.1(1);Thr,1.0(1);
Ala,1.9(2);Arg,3.0(3);Val,1.2(1);Ile,1.0(1);Leu,7.8(8);Lys,4.2(4).
化合物5:AVSEHQLLHDKGKSIQDLRRRELLERLLERLHTA-OH(SEQ ID NO:15)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30Leu His Thr Ala OH(SEQ ID NO:15)物理数据:m.p.147-165℃[α]D 25-49.17(c 0.66,H2O)FAB(C175H299N59O52):[M+H]+4061AAA:Asp,2.1(2);Gly,6.1(6);Ser,1.8(2);His,3.1(3);Gly,1.1(1);Thr,1.0(1);
Ala,2.0(2);Arg,5.0(5);Val,1.0(1);Ile,0.9(1);Leu,7.7(8);Lys,1.9(2).
化合物6:AVSEHQLLHDRGRSIQDLRRRELLERLLERLHTA-OH(SEQ ID NO:16)
鞍Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30Leu His Thr Ala OH(SEQ ID NO:16)物理数据:m.p.150-170℃[α]D 25-48.65°(c 0.54,H2O)FAB(C175H299N63O52):[M+H]+4118.0AAA:Asp,2.1(2);Glu,6.1(6);Ser,1.8(2);His,3.2(3);Gly,1.2(1);Thr,1.0(1);
Ala,2.0(2);Arg,6.9(7);Val,1.0(1);Ile,1.0(1);Leu,7.8(8).
化合物7:AVSEHQLLHDRGRSIQDLRRRELLERLLKRLHTA-OH(SEQ ID NO:17)Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Lys Arg
20 25 30Leu His Thr Ala OH(SEQ ID NO:17)物理数据:m.p.177-182℃[α]D 25 -46.17°(c 0.14,H2O)FAB(C176H304N64O50):[M+H]+4117AAA:Asp,2.0(2);Glu,4.8(5);Ser,1.8(2);His,3.2(3);Gly,1.1(1);Thr,0.9(1);
Ala,1.9(2);Arg,6.7(7);Val,1.0(1);Ile,1.0(1);Lys,7.7(8);Lys,0.9(1).
化合物8:AVSEHQLLHDKGKSIQDLRRRELLEKLLRKLHTA-OH(SEQ ID NO:5)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Arg Lys
20 25 30Leu His Thr Ala OH(SEQ ID NO:5)物理数据:m.p.147-165℃[α]D 25-49.17°(c 0.66,H2O)FAB(C176H305N59O49):[M+H]+4033.0AAA:Asp,2.0(2);Glu,4.8(5);Ser,1.8(2);His,2.7(3);Gly,1.1(1);Thr,0.9(1);
Ala,2.0(2);Arg,3.9(4);Val,1.0(1);Ile,1.0(1);Leu,7.9(8);Lys,4.0(4).
化合物9:AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTAGRR-OH(SEQ ID NO:10)A1a Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala Gly Arg Arg OH(SEQ ID NO:10)
35物理数据:m.p.158-160℃ [α]D 25-44.76(c 0.1,H2O)FAB(C189H326N64O55):[M]+4375.0AAA:Asp,2.0(2);Glu,5.9(6);Ser,1.7(2);His,2.9(3);Gly,2.3(2);Thr,1.0(1);
Ala,1.9(2);Arg,5.0(5);Val,1.2(1);Ile,1.0(1);Leu,7.8(8);Lys,4.3(4).
化合物10:AVSEAQLLHDLGKSIQDLRRRELLEKLLEKLHAL-OH(SEQ ID NO:14)Ala Val Ser Glu Ala Gln Leu Leu His Asp Leu Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Ala Leu OH(SEQ ID NO:14)物理数据:m.p.170-175℃ [α]D 25-31.59°(c 0.54,H2O)FAB(C174H300N52O51):[M+H]+3936.0AAA:Asp,2.0(2);Glu,6.0(6);Ser,1.8(2);His,2.0(2);Gly,1.2(1);Ala,3.0(3);
Arg,2.8(3);Val,1.1(1);Ile,1.0(1);Leu,9.9(10);Lys,3.0(3).
化合物11:AVSEHQLLHDKGKSIQDLRRRELLEKLLELLKEL-NH2(SEQ ID NO:11)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu Lys Glu Leu NH2(SEQ ID NO:11) 物理数据:m.p.172-174℃ [α]D 25-43.29°(c 0.2,H2O)FAB(C179H311N55O52):[M+H]+4065.8AAA:Asp,2.2(2);Glu,7.7(7);Ser,1.7(2);His,2.0(2);Gly,1.0(1);Ala,1.0(1);
Arg,3.0(3);Val,1.1(1);Ile,1.0(1);Leu,9.3(9);Lys,5.1(5).
化合物12:
AVSEIQFXHNLGKHLSSXERVELLEKLLEKLHNY-NH2(X=Nle,SEQ IDNO:23)
Ala Val Ser Glu Ile Gln Phe Nle His Asn Leu Gly Lys His Leu
1 5 10 15
Ser Ser Nle Glu Arg Val Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Asn Tyr NH2(SEQ ID NO:23)
物理数据:m.p.178℃ [α]D 25-36.88°(c 0.4,H2O)
FAB(C182H295N50O51):[M+H]+4001.6
AAA:Asp,2.1(2);Glu,6.5(6);Ser,2.7(3);His,3.1(3);Gly,1.1(1);Ala,1.0(1);
Arg,1.0(1);Tyr,0.8(1);Val,2.0(2);Phe,1.0(1);Ile,0.9(1);Leu+Nle,
8.5(7+2);Lys,3.1(3).
化合物13:
AVSEIQFXHNLGKHLSSXRRRELLEKLLEKLHNY-NH2(X=Nle,SEQ IDNO:24)
Ala Val Ser Glu Ile Gln Phe Nle His Asn Leu Gly Lys His Leu
1 5 10 15
Ser Ser Nle Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Asn Tyr NH2(SEQ ID NO:24)
物理数据:m.p.260℃[α]D 25-37.02°(c 0.2,H2O)
FAB(C184H304N56O49):[M+H]+4084
AAA:Asp,2.1(2);Glu,5.5(5);Ser,2.6(3);His,3.1(3);Ala,1.0(1);Gly,1.1(1);
Arg,3.2(3);Tyr,1.0(1);Val,1.0(1);Phe,1.0(1);Ile,1.0(1);Leu,9.0(9);
Lys,3.0(3).
化合物14:AVSEHQLLHDKGKSIQDLRRRALAEALAEALHTA-NH2(SEQ ID NO:20)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Ala Leu Ala Glu Ala Leu Ala Glu Ala
20 25 30Leu His Thr Ala NH2(SEQ ID NO:20)物理数据:m.p.190-225℃[α]D 25-56.58(c 0.36,H2O)FAB(C161H272N54O49):[M+H]+3747.0AAA:Asp,2.1(2);Glu,4.9(5);Ser,1.7(2);His,2.6(3);Gly,1.1(1);Thr,1.0(1);
Ala,7.6(7);Arg,2.8(3);VaI,1.2(1);Ile,1.0(1);Leu,6.6(6);Lys,1.9(2).
化合物15:AVSEHQLLHDKGKSIQDLARRELLEKLLEKLHTA-NH2(SEQ ID NO:12)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Ala Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala NH2(SEQ ID NO:12)物理数据:m.p.170-180℃[α]D 25-48.19°(c 0.2,H2O)FAB(C172H293N53O51):[M+H]+3919.0AAA:Asp,2.1(2);Glu,6.1(6);Ser,1.7(2);His,3.1(3);Gly,1.1(1);Thr,1.0(1);
Ala,3.0(3);Arg,2.1(2);Val,1.1(1);Ile,1.0(1);Leu,8.0(8);Lys,4.4(4).
化合物16:AVSEHQLLHDKGKSIQDLRRAELLEKLLEKLHTA-NH2(SEQ ID NO:13)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Ala Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala NH2(SEQ ID NO:13)物理数据:m.p.190-195℃[α]D 25-50.50°(c 0.4,H2O)FAB(C172H293N53O51):[M+H]+3919.0AAA:Asp,2.1(2);Glu,6.0(6);Ser,1.8(2);His,3.1(3);Gly,1.1(1);Thr,1.0(1);
Ala,3.0(3);Arg,2.1(2);Val,1.1(1);Ile,1.0(1);Leu,7.5(8);Lys,4.2(4).
化合物17:AVSEHQLLHDKGKSIQDLRRRSLLSSLLSSLHTA-NH2(SEQ ID NO:21)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Ser Leu Leu Ser Ser Leu Leu Ser Ser
20 25 30Leu His Thr Ala NH2(SEQ ID NO:21)物理数据:m.p.195-204℃[α]D 25-67.11°(c 0.3,H2O)FAB(C163H280N54O50):[M+H]+3796.0AAA:Asp,2.1(2);Glu,2.9(3);Ser,6.8(7);His,3.1(3);Gly,1.2(1);Thr,1.0(1);
Ala,2.0(2);Arg,3.0(3);Val,1.0(1);Ile,1.0(1);Leu,8.2(8);Lys,2.0(2).
化合物18:AVSEHQLLHDKGKSIQDLRRRAFYDKVAEKLHTA-NH2(SEQ ID NO:22)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Ala Phe Tyr Asp Lys Val Ala Glu Lys
20 25 30Leu His Thr Ala NH2(SEQ ID NO:22)物理数据:m.p.200-207℃[α]D 25-60.26°(c 0.6,H2O)FAB(C174H284N56O50):[M+H]+3960.0AAA:Asp,2.9(3),Glu,3.5(4);Ser,1.4(2);His,2.6(3);Gly,0.9(1);Thr,1.0(1);
Ala,4.0(4);Arg,3.0(3);Tyr,0.9(1);Val,1.9(2);Phe,1.1(1);Ile,0.9(1);Leu,
3.6(4);Lys,4.1(4).
化合物21:AVSEIQFLHN LGKHLSSLRR RELLEKLLEK LHNY-NH2(SEQ ID NO:35)Ala Val Ser Glu Ile Gln Phe Leu His Asn Leu Gly Lys His Leu1 5 10 15Ser Ser Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Asn Tyr NH2(SEQ ID NO:35)物理数据:m.p.148-155℃[α]D 25-45.97(c 0.26,H2O)FAB(C184H304N56O49):[M+H]+ 4084AAA:Asx,2.1(2);Glx,5.0(5);Ser,2.7(3);His,3.0(3);Gly,1.0(1);Ala,0.9(1);
Arg,3.1(3);Tyr,0.9(1);Val,1.0(1);Phe,0.9(1);Ile,0.9(1);Leu 9.3(9);Lys,
3.2(3).
化合物24:AVSEHQLLHD KGKSIQDLKL KELLEKLLEK LHTA-NH2(SEQ ID NO:38)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Lys Leu Lys Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala NH2(SEQ ID NO:38)物理数据:m.p.175-182℃[α]D 25-49.99(c 0.47,H2O)FAB(C175H299N49O51):[M+H]+ 3906.5AAA:Asx,2.1(2);Glx,6.5(6);Ser,1.8(2);His,3.1(3);Gly,1.1(1);Thr,1.0(1);
Ala,2.1(2);Val,1.1(1);Ile,1.0(1);Leu,9.1(9);Lys,6.5(6).
化合物25:AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTA-NH2(SEQ ID NO:39)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30Leu His Thr Ala-NH2(SEQ ID NO:39)物理数据:m.p.136.5-153.5℃[α]D 25-32.57(c 0.13,H2O)FAB(C175H300N60O51):[M+H]+ 4060.8AAA:Asx,2.2(2);Glx,6.2(6);Ser,1.8(2);His,3.2(3);Gly,1.1(1);Thr,1.0(1);
Ala,2.1(2);Arg,5.2(5);Val,1.1(1);Ile,1.1(1);Leu,8.4(8);Lys,2.2(2).
化合物26:AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTAP-OH(SEQ ID NO:40)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30Leu His Thr Ala Pro OH(SEQ ID NO:40)
35物理数据:m.p.125.8-127.2℃[α]D 25-54.62(c 0.23,H2O)FAB(C180H306N60O53):[M+H]+ 4158.0AAA:Asx,2.1(2);Glx,6.2(6);Ser,1.8(2);His,2.9(3);Gly,1.1(1);Thr,1.0(1);
Ala,2.0(2);Arg,5.1(5);Val,1.0(1);Ile,1.0(1);Leu,8.0(8);Lys,2.1(2);Pro,
1.1(1).
化合物27:AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTAGRR-OH(SEQ ID NO:41)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30Leu His Thr Ala Gly Arg Arg OH(SEQ ID NO:41)
35物理数据:m.p.106-137.3℃[α]D 25-39.55(c 0.67,H2O)FAB(C189H326N68O55):[M+H]+ 4430.5AAA:Asx,2.1(2);Glx,5.9(6);Ser,1.6(2);His,2.7(3);Gly,2.2(2);Thr,1.0(1);
Ala,1.8(2);Arg,7.3(7);Val,0.8(1);Ile,1.0(1);Leu,8.1(8);Lys,2.1(2).
化合物29:AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTY-NH2(SEQ ID NO:43)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Tyr NH2(SEQ ID NO:43)物理数据:m.p.160-172℃[α]D 25-49.85(c 0.34,H2O)FAB(C181H304N56O52):[M+H]+ 4096.9AAA:Asx,2.0(2);Glx,5.6(6);Ser,1.7(2);His,3.1(3);Gly,1.1(1);Thr,0.9(1);
Ala,0.9(1);Arg,3.0(3);Tyr,0.9(1);Val,1.0(1);Ile,1.0(1);Leu,7.7(8);Lys,
4.4(4).
化合物30:AVSEHQLLHD KGYSIQDLRR RELLEKLLEK LHTA-NH2(SEQ ID NO:44)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Tyr Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala NH2(SEQ ID NO:44)物理数据:m.p.130-171℃[α]D 25 -40.65(c 0.34,H2O)FAB(C178H297N55O52):[M+H]+ 4039.4AAA:Asx,2.0(2);Glx,5.5(6);Ser,1.8(2);His,3.4(3);Gly,1.1(1);Thr,1.0(1);
Ala,2.0(2);Arg,2.9(3);Tyr,0.8(1);Val,1.0(1);Ile,0.9(1);Leu,7.9(8);Lys,
3.4(3).
化合物31:AVSEHQLLHD KGCSIQDLRR RELLEKLLEK LHTA-NH2(SEQ ID NO:45)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Cys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala NH2(SEQ ID NO:45) 物理数据:m.p.140-160℃[α]D 25-44.48(c 0.25,H2O)FAB(C172H293N55O51S1):[M+H]+3979AAA:Asx+Cys, 3.0(2+1);Glx,5.6(6);Ser,1.7(2);His,3.0(3);Gly,1.0(1);Thr,
0.9(1);Ala,1.9(2);Arg,2.5(3);Val,1.0(1);Ile,0.9(1);Leu,7.5(8);Lys,
3.3(3).
化合物32:AVSEHQLLHD KGXSIQDLRR RELLEKLLEK LHTA-NH2(SEQ ID NO:46)
(X=Cys(CH2CONH(CH2)2NH(生物素)))Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Xaa Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala NH2,(Xaa=Cys(CH2CONH(CH2)2NH(生物素))),(SEQ IDNO:46)物理数据:m.p.(未测定)[α]D 25(未测定)FAB(C186H316N59O54S2):[M+H]+ 4306.6AAA:Asx,2.2(2);Glx,6.1(6);Ser,1.8(2);His,3.8(3);Gly,1.0(1);Thr,1.0(1);
Ala,2.0(2);Arg,3.1(3);Val,1.1(1);Ile,0.9(1);Leu,8.3(3);Lys,3.3(3).
化合物33:AVSEHQLLHD KGXSIQDLRR RELLEKLLEK LHTA-NH2(SEQ ID NO:47)
(X=Lys(7-二甲基氨基-2-氧-2H-l-苯并吡喃-4-乙酰基))Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Xaa Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala NH2,(Xaa=Lys(7-二甲基氨基-2-氧-2H-1-苯并吡哺-4-乙酰基),(SEQ ID NO:47)物理数据:m.p.135-205℃[α]D 25-26.92(c 0.104,50%aq.HOAc)FAB(C188H311N57O54):[M+H]+ 4233AAA:Asx,1.9(2);Glx,6.3(6);Ser,1.7(2);His,3.2(3);Gly,1.0(1);Thr,1.1(1);
Ala,2.0(2);Arg,3.2(3);Val,1.1(1);Ile,0.9(1);Leu,8.2(8);Lys,4.5(4).
化合物34:
AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAG-OH(SEQ ID NO:48)
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Ala Gly OH(SEQ ID NO:48)
35
物理数据:m.p.92.1-146.6℃[α]D 25-40.76(c 0.34,H2O)
FAB(C177H302N56O53):[M+H]+ 4062.0
AAA:Asx,2.0(2);Glx,5.7(6);Ser,1.8(2);His,3.0(3);Gly,2.2(2);Thr,0.9(1);
Ala,1.9(2);Arg,2.8(3);Val,1.2(1);Ile,0.9(1);Leu,7.5(8);Lys,4.2(4).
化合物35:
AVSX1HQLLHX2 KGKSIQX2LRR RX1LLX1KLLX1K LHA-OH(SEQ ID NO:49)
(X1=Glu(OCH3);X2=Asp(OCH3))
Ala Val Ser Xaa1His Gln Leu Leu His Xaa2Lys Gly Lys Ser Ile
1 5 10 15
Gln Xaa2Leu Arg Arg Arg Xaa1Leu Leu Xaa1Lys Leu Leu Xaa1Lys
20 25 30
Leu His Ala OH(Xaa1=Glu(OCH3);Xaa2=Asp(OCH3))(SEQ ID NO:49)
物理数据:m.p.(未测定)[α]D 25-21.96(c 0.132,H2O)
FAB(C181H311N55O52):[M+H]+ 4089.0
AAA:Asx,2.1(2);Glx,6.3(6);Ser,1.8(2);His,3.3(3);Gly,1.1(1);Thr,1.0(1);
Ala,2.0(2);Arg,3.1(3);Val,1.1(1);Ile,0.9(1);Leu,8.0(8);Lys,4.2(4).
化合物36:
AVSX1HQLLHX2 KGKSIQX2LRR RX1LLX1KLLX1K LHA-OCH3(SEQ IDNO:50)
(X1=Glu(OCH3);X2=Asp(OCH3))
Ala Val Ser Xaa1His Gln Leu Leu His Xaa2Lys Gly Lys Ser Ile
1 5 10 15 Gln Xaa2Leu Arg Arg Arg Xaa1Leu Leu Xaa1Lys Leu Leu Xaa1Lys
20 25 30Leu His Ala OCH3(Xaa1=Glu(OCH3);Xaa2=Asp(OCH3))(SEQ ID NO:50)物理数据:m.p.(未测定)[α]D 25-46.80(c 0.07,H2O)FAB(C182H313N55O52):[M+H]+ 4103AAA:Asx,2.1(2);Glx,6.2(6);Ser,1.4(2);His,3.0(3);Gly,1.1(1);Thr,1.1(1);
Ala,1.7(2);Arg,3.2(3);Val,0.6(1);Ile,0.9(1);Leu,8.0(8);Lys,4.1(4).
化合物38:AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAP-OH(SEQ ID NO:52)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala Pro OH(SEQ ID NO:52)
35物理数据:m.p.152.1-186.5℃[α]D 25-55.91(c 0.33,H2O)FAB(C180H306N56O53):[M+H]+ 4102.6AAA:Asx,2.0(2);Glx,5.6(6);Ser,1.7(2);His,2.9(3);Gly,1.1(1);Thr,0.9(1);
Ala,1.9(2);Arg,2.9(3);Val,1.2(1);Ile,1.0(1);Leu,7.7(8);Lys,4.3(4).
化合物39:AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTP-OH(SEQ ID NO:53)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Pro OH(SEQ ID NO:53)物理数据:m.p.120-148.2℃[α]D 25-52.78(c 0.47,H2O)FAB(C177H301N55O52):[M+H]+ 4031.0AAA:Asx,2.0(2);Glx,5.5(6);Ser,1.8(2);His,2.9(3);Gly,1.0(1);Thr,1.0(1);
Ala,0.9(1);Arg,2.9(3);Val,1.2(1);Ile,0.9(1);Leu,7.5(8);Lys,3.6(3);Pro,
0.9(1).
化合物40:AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTP-NH2(SEQ ID NO:54)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Pro NH2(SEQ ID NO:54)物理数据:m.p.133.9-155.1℃[α]D 25-54.22(c 0.37,H2O)FAB(C177H302N56O51):[M+H]+ 4030.7AAA:Asx,2.0(2);Glx,5.6(6);Ser,1.9(2);His,2.9(3);Gly,1.1(1);Thr,0.9(1);
Ala,0.9(1);Arg,2.8(3);Val,1.2(1);Ile,1.1(1);Leu,7.8(8);Lys,4.2(4);
Pro,0.9(1).
化合物41:AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHP-NH2(sEQ ID NO:55)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Pro NH2(SEQ ID NO:55)物理数据:m.p.142.8-166.1℃[α]D 25-53.80(c 0.38,H2O)FAB(C173H295N55O49):[M+H]+ 3929AAA:Asx,2.0(2);Glx,5.7(6);Ser,1.8(2);His,3.0(3);Gly,1.1(1);Ala,0.9(1);
Arg,2.8(3);Val,1.2(1);Ile,0.9(1);Leu,7.4(8);Lys,4.4(4);Pro,0.9(1).
化合物42:AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LP-NH2(SEQ ID NO:56)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu Pro NH2(SEQ ID NO:56)
物理数据:m.p.161.0-177.0℃[α]D 25-61.97(c 0.19,H2O)
FAB(C167H288N52O48):[M+H]+ 3792.0
AAA:Asx,2.2(2);Glx,5.9(6);Ser,1.9(2);His,2.1(2);Gly,1.1(1);Ala,1.0(1);
Arg,3.0(3);Val,1.1(1);Ile,1.0(1);Leu,7.9(8);Lys,4.3(4);Pro,0.9(1).
化合物43:
AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTRSAW-OH(SEQ ID NO:57)
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Arg Ser Ala Trp OH(SEQ ID NO:57)
35
物理数据:m.p.181-202℃[α]D 25-45.14(c 0.19,H2O)
FAB(C195H326N62O56):[M+H]+ 4435.2
AAA:Asx,2.0(2);Glx,5.8(6);Ser,2.8(3);His,2.8(3);Gly,1.1(1);Thr,0.9(1);
Ala,1.9(2);Arg,3.7(4);Ile,0.9(1);Leu,7.5(8);Lys,4.3(4);Trp,0.9(1).
化合物44:
AVSEHQLLHD RGRSIQDLRR RELLERLLER LHTAGRRTRSAW-OH(SEQ IDNO:58)
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30
Leu His Thr Arg Gly Arg Arg Thr Arg Ser Ala Trp OH(SEQ ID NO:58)
35 40物理数据:m.p.130-132.2℃[α]D 25-46.66(c 0.195,H2O)FAB(C216H365N81O62):[M+H]+ 5088.8AAA:Asx,2.2(2);Glx,6.0(6);Ser,2.7(3);His,3.0(3);Gly,2.2(2);Thr,2.1(2);
Ala,3.0(3);Arg,10.5(10);Val,0.9(1);Ile,1.0(1);Leu,8.2(8);Trp,1.0(1).
化合物45:
AVSEHQLLHD RGRSIQDLRR RELLERLLER LHTAGRRTRSAW-NH2(SEQID NO:59)
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30
Leu His Thr Arg Gly Arg Arg Thr Arg Ser Ala Trp NH2(SEQ ID NO:59)
35 40
物理数据:m.p.158-174℃[α]D 25-43.57(c 0.53,H2O)
FAB(C216H366N82O61):[M+H]+ 5087.4
AAA:Asx,1.9(2);Glx,5.6(6);Ser,2.6(3);His,3.3(3);Gly,2.1(2);Thr,2.0(2);
Ala,2.9(3);Arg,10.1(10);Val,0.9(1);Ile,1.0(1);Leu,8.3(8);Trp 1.1(1).
化合物46:
AVSEHQLLHD RGXSIQDLRR RELLERLLER LHTAGRRTRSAW-OH(SEQID NO:60)
(X=Lys(二氢肉桂酰基))
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Xaa Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30
Leu His Thr Arg Gly Arg Arg Thr Arg Ser Ala Trp OH(Xaa=
35 40
Lys(二氢肉桂酰基),(SEQ ID NO:60)
物理数据:m.p.165.4-175.2℃[α]D 25-40.43(c 0.20,H2O)
FAB(C225H374N80O62):[M+H]+ 5191
AAA:Asx,2.1(2),Glx,6.3(6);Ser,2.8(3);His,3.2(3);Gly,2.1(2),Thr,2.0(2);
Ala,3.2(3);Arg,9.9(9);Val,1.0(1),Ile,0.9(1);Leu,8.6(8);Lys,1.1(1);Trp,
1.1(1).
化合物47:AVSEIQFXHN LGKHLSSXTR SAWLRKKLQD VHNY-NH2(SEQ ID NO:61)
(X=正亮氨酸)Ala Val Ser Glu Ile Gln Phe Nle His Asn Leu Gly Lys His Leu1 5 10 15Ser Ser Nle Thr Arg Ser Ala Trp Leu Arg Lys Lys Leu Gln Asp
20 25 30Val His Asn Tyr NH2(SEQ ID NO:61)物理数据:m.p.140-160℃[α]D 25-56.88(c 0.16,H2O)FAB(C180H287N55O58):[M+H]+ 3989.8AAA:Asx,3.0(3);Glx,2.9(3);Ser,3.7(4);His,2.8(3);Gly,1.1(1);Thr,0.9(1);
Ala,1.9(2);Arg,2.0(2);Tyr,1.0(1);Val,1.7(2);Phe,0.9(1);Ile,0.9(1);
Leu+Nle 5.8(2+4);Lys,3.4(3);Trp,1.1(1).
化合物48:AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTMA-NH2(SEQ ID NO:62)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Met Ala NH2(SEQ ID NO:62)
35物理数据:m.p.140-210℃[α]D 25-47.75(c 0.178,H2O)FAB(C180H309N57O52S1):[M+H]+ 4135.0AAA:Asx,2.3(2);Glx,6.6(6);Ser,1.4(2);His,3.2(3);Gly,1.1(1);Thr,1.0(1);
Ala,2.0(2);Arg,3.1(3);Val,0.9(1);Met,1.1(1);Ile,1.0(1);Leu,8.8(8);
Lys,4.4(4).
化合物50:AVSEHQLLHD KGKSIQDLRR RFFLEKLLEK LHTA-NH2(SEQ ID NO:64)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Phe Phe Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala NH2(SEQ ID NO:64)
35物理数据:m.p.136.5-156.8℃[α]D 25-49.89(c 0.24,H2O)FAB(C182H300N56O49):[M+H]+ 4056.0AAA:Asx,2.2(2);Glx,5.0(5);Ser,1.9(2);His,3.3(3);Gly,1.0(1);Thr,1.0(1);
Ala,2.1(2);Arg,3.1(3);Val,1.0(1);Phe,2.0(2);Ile,0.9(1);Leu,7.2(7);Lys,
3.5(4).
化合物51:AVSEHQLLHD KGKSIQDLRR RELLHKLLEK LHTA-NH2(SEQ ID NO:65)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu His Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala NH2(SEQ ID NO:65)物理数据:m.p.80.7-141.0℃[α]D 25-55.38(c 0.23,H2O)FAB(C176H300N58O49):[M+H]+ 4012.8AAA:Asx,2.2(2);Glx,4.9(5);Ser,1.8(2);His,4.3(4);Gly,1.1(1);Thr,1.0(1);
Ala,2.0(2);Arg,3.1(3);Val,1.1(1);Ile,1.0(1);Leu,8.1(8);Lys,3.9(4).
化合物52:AVSEHQLLHD KGKSIQDLRR RELLEHLLEK LHTA-NH2(SEQ ID NO:66)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu His Leu Leu Glu Lys
20 25 30Leu His Thr Ala NH2(SEQ ID NO:66)物理数据:m.p.134.3-157.9℃[α]D 25-50.72(c 0.45,H2O)FAB(C175H295N57O51):[M+H]+ 4012.8AAA:Asx,2.1(2);Glx,5.9(6);Ser,1.8(2);His,4.2(4);Gly,1.1(1);Thr,1.0(1);
Ala,2.0(2);Arg,3.0(3);Val,1.1(1);Ile,0.9(1);Leu,8.1(8);Lys,3.1(3).
化合物53:
AVSEHQLLHD KGKSIQDLRR RELLEKLIAK LHTA-NH2(SEQ ID NO:67)
Ala Val Ser Glu His Gln Leu Leu His Asp Lys G1y Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Ile Ala Lys
20 25 30
Leu His Thr Ala NH2(SEQ ID NO:67)
物理数据:m.p.142.7-159.8℃[α]D 25-54.01(c 0.21,H2O)
FAB(C173H298N56O49):[M+H]+ 3946.0
AAA:Asx,2.2(2);Glx,4.9(5);Ser,1.8(2);His,3.1(3);Gly,1.1(1);Thr,1.0(1);
Ala,3.1(3);Arg,3.1(3);Val,1.0(1);Ile,1.9(2);Leu,7.0(7);Lys,4.3(4).
化合物54:
AVSEHQLLHD KGKSIQDLRR RELLEKLLEE IHTA-NH2(SEQ ID NO:68)
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Glu
20 25 30
Ile His Thr Ala NH2(SEQ ID NO:68)
物理数据:m.p.138-185℃[α]D 25-50.17(c 0.14,H2O)
FAB(C174H295N55O53):[M+H]+ 4005
AAA:Asx,2.2(2);Glx,7.1(7);Ser,1.7(2);His,2.8(3);Gly,1.0(1);Thr,1.0(1);
Ala,2.1(2);Arg,3.1(3);Val,1.1(1);Ile,1.7(2);Leu,7.1(7);Lys,2.7(3).
化合物58:
AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTRSAW-NH2(SEQ IDNO:72)
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Arg Ser Ala Trp NH2(SEQ ID NO:72)
35物理数据:m.p.158-163℃ [α]D 25-46.06(c 0.17,H2O)FAB(C195H327N63O55):[M+H]+ 4434.8AAA:Asx,2.0(2);Glx,5.5(6);Ser,2.7(3);His,3.1(3);Gly,1.0(1);Ala,1.8(2);
Arg,4.0(4);Thr,0.9(1);Val,0.9(1);Ile,0.9(1);Leu,7.5(8);Lys,3.9(4);
Trp,1.0(1).
化合物59:AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTRSAX-OH(SEQ ID NO:73)
(X=Nal(2)=3-(2-萘基)-L-丙氨酸)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Arg Ser Ala 3-(2-萘基)-L-丙氨酸-OH(SEQ ID NO:73)
35物理数据:m.p.156-162℃ [α]D 25-44.44(c 0.189,H2O)FAB(C197H328N62O55):[M+H]+ 4445.6AAA:Asx,2.1(2);Glx,5.5(6);Ser,2.8(3);His,2.9(3);Gly,1.0(1);Ala,2.0(2);
Arg,4.0(4);Thr,0.9(1);Val,1.0(1);Ile,0.9(1);Leu,7.5(8);Lys,4.2(4);
Nal,1.1(1).
化合物60:AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTASAW-OH(SEQ ID NO:74)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala Ser Ala Trp OH(SEQ ID NO:74)
35物理数据:m.p.159-164℃ [α]D 25-50.94(c 0.29,H2O)FAB(C192H320N60O55):[M+H]+ 4349.0AAA:Asx,2.0(2);Glx,5.6(6);Ser,2.7(3);His,3.2(3);Gly,1.0(1);Ala,3.1(3);
Arg,2.8(3);Thr,1.0(1);Val,1.l(1);Ile,0.9(1);Leu,7.6(8);Lys,4.0(4);Trp,
1.0(1).
化合物6l:AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAEIRA-OH(SEQ ID NO:75)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala Glu Ile Arg Ala OH(SEQ ID NO:75)
35物理数据:m.p.155-210℃ [α]D 25-46.15(c 0.12,H2O)FAB(C195H334N62O58):[M+H]+ 4475.8AAA:Asx,2.2(2);Glx,6.9(7);Ser,1.7(2);His,3.2(3);Gly,1.1(1);Ala,3.1(3);
Arg,4.0(4);Thr,0.9(1);Val,1.1(1);Ile,1.9(2);Leu,8.1(8);Lys,4.1(4).
化合物62:AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAEIR-OH(SEQ ID NO:76)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala Glu Ile Arg OH(SEQ ID NO:76)
35物理数据:m.p.186-218℃[α]D 25-52.73(c 0.265,H2O)FAB(C192H329N61O57):[M+H]+ 4404.4AAA:Asx,2.0(2);Glx,6.6(7);Ser,1.9(2);His,3.4(3);Gly,1.1(1);Ala,2.0(2);
Arg,3.8(4);Thr,1.0(1);Val,1.1(1);Ile,1.7(2);Leu,7.9(8);Lys,4.0(4).
化合物63:AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAEI-OH(SEQ ID NO:77)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala Glu Ile OH(SEQ ID NO:77)
35物理数据:m.p.169-205℃ [α]D 25-50.78(c 0.51,H2O)FAB(C186H317N57O56):[M+H]+ 4248.0AAA:Asx,2.2(2);Glx,6.8(7);Ser,1.8(2);His,3.3(3);Gly,1.0(1);Ala,2.0(2);
Arg,3.0(3);Thr,1.0(1);Val,1.0(1);Ile,1.8(2);Leu,7.8(8);Lys,3.6(4).
化合物64:AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTAE-OH(SEQ ID NO:78)Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala Glu OH(SEQ ID NO:78)
35物理数据:m.p.199-205℃ [α]D 25-52.47(c 0.41,H2O)FAB(C180H306N56O55):[M+H]+ 4135.0AAA:Asx,2.0(2);Glx,6.6(7);Ser,1.9(2);His,3.3(3);Gly,1.1(1);Ala,2.0(2);
Arg,2.9(3);Thr,1.0(1);Val,1.1(1);Ile,1.0(1);Leu,8.2(8);Lys,3.8(4).
化合物66:SEHQLLHD KGKSIQDLRR RELLEKLLEK LHTA-NH2(SEQ ID NO:80)Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln Asp1 5 10 15Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu His
20 25 30Thr Ala NH2(SEQ ID NO:80)物理数据:m.p.134.2℃[α]D 25-48.12(c 0.36,H2O)FAB(C167H286N54O49):[M+H]+ 3834.4AAA:Asx,2.0(2);Glx,5.7(6);Ser,1.7(2);His,2.9(3);Gly,1.0(1);Thr,0.9(1);
Ala,1.0(1);Arg,2.8(3);Ile,0.9(1);Leu,7.4(8);Lys,4.3(4).
化合物67:
LLHD KGKSIQDLRR RELLEKLLEK LHTA-NH2(SEQ ID NO:81)
Leu Leu His Asp Lys Gly Lys Ser Ile Gln Asp Leu Arg Arg Arg
1 5 10 15
Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu His Thr Ala NH2(SEQ ID NO:81)
20 25
物理数据:m.p.128.5-184.5℃[α]D 25-6.53(c 0.69,MeOH)
FAB(C148H259N47O41):[M+H]+ 3353
AAA:Asx,2.0(2);Glx,4.1(4);Ser,0.9(1);His,2.1(2);Gly,1.0(1);Thr,0.9(1);
Ala,1.0(1);Arg,3.0(3);Ile,1.0(1);Leu,8.1(8);Lys,4.2(4).
化合物68:
LHD KGKSIQDLRR RELLEKLLEK LHTA-NH2(SEQ ID NO:82)
Leu His Asp Lys Gly Lys Ser Ile Gln Asp Leu Arg Arg Arg Glu
1 5 10 15
Leu Leu Glu Lys Leu Leu Glu Lys Leu His Thr Ala NH2(SEQ ID NO:82)
20 25
物理数据:m.p.165-210℃[α]D 25-36.05(c 0.12,H2O)
FAB(C142H248N46O40):[M+H]+ 3239.0
AAA:Asx,2.0(2);Glx,3.9(4);Ser,0.9(1);His,1.9(2);Gly,1.0(1);Thr,1.0(1);
Ala,1.0(1);Arg,2.9(3);Ile,0.9(1);Leu,6.8(7);Lys,4.2(4).
化合物69:
SEHQLLHD RGRSIQDLRR RELLERLLER LHAGRRTRSAW-OH(SEQ IDNO:83)
Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile Gln Asp
1 5 10 15
Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg Leu His
20 25 30
Leu His Arg Gly Arg Arg Thr Arg Ser Ala Trp OH(SEQ ID NO:83)
35 40
物理数据:m.p.150-210℃[α]D 25-18.0(c 0.64,H2O)
FAB(C208H351N79O60):[M+H]+ 4918.6
AAA:Asx,2.2(2);Glx,6.2(6);Ser,2.8(3);His,3.1(3);Glv,2.2(2);Thr,2.2(2);
Ala,2.2(2);Arg,10.4(10);Ile,1.0(1);Leu,8.0(8);Trp,1.1(1).
化合物70:
LLHD RGRSIQDLRR RELLERLLER LHAGRRTRSAW-OH(SEQ ID NO:84)
Leu Leu His Asp Arg Gly Arg Ser Ile Gln Asp Leu Arg Arg Arg
1 5 10 15
Glu Leu Leu Glu Arg Leu Leu Glu Arg Leu His Leu His Arg Gly
20 25 30
Arg Arg Thr Arg Ser Ala Trp OH(SEQ ID NO:84)
35
物理数据:m.p.150-210℃ [α]D 25-41.70(c 0.36,H2O)
FAB(C189H324N72O52):[M+H]+ 4437.14
AAA:Asx,2.1(2);Glx,4.1(4);Ser,1.9(2);His,2.0(2);Gly,2.1(2);Thr,2.0(2);
Ala,2.1(2);Arg,9.7(10);Ile,0.9(1);Leu,7.4(8).
如上所述合成侧链环化的类似物(化合物57),所不同的是在第13位和第17位分别是Nα-Boc-Nε-Fmoc-Lys和Nα-Boc-Nγ-Fmoc-Asp。在Boc氨基酸合成完成后,在室温下在20%的哌啶DMF溶液中处理树脂30分钟。过滤树脂并用DMF、MeOH和CH2Cl2洗涤。将树脂(1.1克)悬浮在10毫升含有250毫克PyBOP的DMF中。用DIEA将pH调节至8到9,并搅拌树脂1小时。过滤树脂,用DMF和CH2Cl2洗涤,然后重悬在DMF中。用125毫克PyBOP重复偶合。过滤树脂,用DMF、MeOH和CH2Cl2洗涤,并干燥。然后如前文所述用HF处理树脂和纯化肽。
物理数据:m.p.142.5-163.5℃[α]D 25-34.31(c 0.17,H2O)
FAB(C175H298N56O50):[M+H]+ 3986.4
AAA:Asx,1.9(2);Glx,5.9(6);Ser,1.8(2);His,3.2(3);Gly,1.1(1);Ala,2.0(2);
Arg,3.0(3);Thr,1.0(1);Val,1.1(1);Ile,0.9(1);Leu,8.0(8);Lys,4.0(4).
实施例II
按照实施例I的方法制备和纯化[Met34,Ala35]化合物1,AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTMA-NH2,(SEQ ID NO:25)。如下所述将该多肽转化为高丝氨酸内酯。将纯化肽(160毫克)溶解在44%甲酸(4毫升)中。在0℃,将该溶液与溴化氰(700毫克)和苯酚(1.6毫克)在44%甲酸(4毫升)中预先混制而成的溶液合并。该溶液在0℃搅拌2小时,在室温下搅拌2小时。通过HPLC(VydacC-18,300埃、4.6×250毫米,流速为1.2毫升/分钟,浓度梯度为乙腈在0.1%TFA中形成的25-45%)检测产物的形成。4小时后反应完全。取一半样品浓缩并利用制备RP-HPLC(VydacC-18,浓度梯度为乙腈在0.1%TFA中形成的25-45%)纯化。汇集高丝氨酸内酯肽流份,经冷冻干燥得28毫克纯度高于95%的白色固体,化合物4。
化合物4
AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTX(X=hSerlac,SEQ ID NO:9)
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr hSerlac(SEQ ID NO:9)
物理数据:
m.p.138-142℃ [α]D 25-50.66°(c 0.1,H2O)
FAB(C176H299N55O52):[M+H]+4017.61
AAA:Asp,2.1(2);Glu,6.1(6);Ser,1.8(2);His,3.0(3);Thi,1.1(1);Ala,1.1(1);
Arg,2.7(3);Val,1.0(1);Ile,1.0(1);Leu,8.2(8);Lys,3.8(4);Gly 1.09(1);
hSer,1.09(1).
同样,用此方法制备化合物65。
化合物65:
AVSEIQFX1HN KGKHLSSX1ER VEWLRKKLQD VHNX2(SEQ ID NO:79)
(X1=L-正亮氨酸;X2=高丝氨酸内酯)
Ala Val Ser Glu Ile Gln Phe Nle His Asn Lys Gly Lys His Leu
1 5 10 15
Ser Ser Nle Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp
20 25 30
Val His Asn hSerlac(SEQ ID NO:79)
物理数据:m.p.166-176℃ [α]D 25-52.22(c 0.25,H2O)
FAB(C180H288N54O50):[M+H]+ 4008.6
AAA:Asx,3.1(3);Glx,4.8(5);Ser,2.9(3);His,2.9(3);Gly,1.1(1);Ala,1.1(1);
Arg,2.0(2);Val,2.7(3);Phe,1.0(1);Ile,1.0(1);Leu+Nle 5.9(4+2);Lys,
2.8(3).
实施例III
为了制备高丝氨酸酰胺,将粗hSer内酯类似物即化合物4浓缩并用25毫升NH3饱和甲醇溶液处理。溶液在0℃搅拌2小时,在室温下搅拌16小时。用HPLC(VydacC-18,300埃、4.6×250毫米,流速为1.2毫升/分钟,浓度梯度为乙腈在0.1%TFA中形成的20-45%)监测反应,18小时后反应完全。将溶液浓缩,并利用制备RP-HPLC(VydacC-18,浓度梯度为乙腈在0.1%TFA中形成的25-45%)纯化。汇集高丝氨酸酰胺肽流份,经冷冻干燥得30毫克纯度高于98%的白色固体,化合物3。
化合物3
AVSEHQLLHDKGKSIQDLRRRELLEKLLEKLHTX-NH2(X=hSer,SEQ IDNO:8)
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr hSer NH2(SEQ ID NO:8).
物理数据:
m.p.138-142℃ [α]D 25-45.97°(c 0.25,H2O)
FAB(C176H302N56O52):[M+H]+ 4033.9AAA:Asp,2.1(2);Glu,6.1(6);Ser,1.6(2);His,2.8(3);Gly,0.97(1);hSer,0.97(1);
Thi,1.0(1);Ala,1.0(1);Arg,2.9(3);Val,1.0(1);Ile,1.0(1);Leu,7.6(8);Lys,
3.9(4).同样,以此方法制备化合物22、23和28。
化合物22:AVSEIQFLHN LGKHLSSLRR RELLEKLLEK LHNX-NH2(SEQ ID NO:36)
(X=高丝氨酸)Ala Val Ser Glu Ile Gln Phe Leu His Asn Leu Gly Lys His Leu1 5 10 15Ser Ser Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Asn hSer NH2(SEQ ID NO:36)物理数据:m.p.69.4-128℃ [α]D 25-43.93(c 0.15,H2O)FAB(C179H302N56O49):[M+H]+ 4022.9AAA:Asx,2.0(2);Glx,4.9(5);Ser,2.6(3);His,2.8(3);Gly,1.0(1);Ala,1.0(1);
Arg,3.0(3);Val,1.0(1);Phe,1.0(1);Ile,0.9(1);Leu,8.8(9);Lys,3.4(3);
化合物23:AVSEIQFLHN KGKHLSSLRR RELLEKLLEK LHNX-NH2(SEQ ID NO:37)
(X=高丝氨酸)Ala Val Ser Glu Ile Gln Phe Leu His Asn Lys Gly Lys His Leu1 5 10 15Ser Ser Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Asn hSer NH2(SEQ ID NO:37)物理数据:m.p.87.1-142.1℃ [α]D 25-52.14(c 0.41,H2O)FAB(C179H303N57O49):[M+H]+ 4038AAA:Asx,2.1(2);Glx,4.9(5);Ser,2.7(3);His,2.8(3);Gly,1.0(1);hSer,1.0(1);
Ala,1.0(1);Arg,3.0(3);Val,1.1(1);Phe,0.9(1);Ile,0.9(1);Leu,7.9(8);Lys,
3.7(4).
化合物28:AVSEHQLLHD KGKSIQDLRR RELLERLLER LHTAGRRX-NH2(SEQ IDNO:42)
(X=高丝氨酸)
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30
Leu His Thr Ala Gly Arg Arg hSer NH2(SEQ ID NO:42)
35
物理数据:m.p.80℃ [α]D 25-48.64(c 0.09,H2O)
FAB(C193H334N70O56):[M+H]+ 4530.0
AAA:Asx,2.2(2);Glx,6.1(6);Ser,1.7(2);His,3.0(3);Gly,1.9(2);hSer,1.0(1);
Thr,1.0(1);Ala,2.1(2);Arg,7.2(7);Val,0.8(1);Ile,1.0(1);Leu,8.4(8);Lys,
2.1(2).
将高丝氨酸内酯溶解在含有过量对应烷基的DMF中,以同样的方法由高丝氨酸内酯制备高丝氨酸烷酰胺。在室温下搅拌数日后(利用分析HPLC监测反应),将混合物蒸发至干燥,然后利用制备HPLC纯化肽。代表性高丝氨酸烷酰胺是化合物55和56。
化合物55:
AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTX-NHCH2CH3(SEQ IDNO:69)
(X=高丝氨酸)
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr hSer NHCH2CH3(SEQ ID NO:69)
物理数据:m.p.(未测定)[α]D 25(未测定)
FAB(C178H306N56O52):[M+H]+ 4063.0
AAA:Asx,2.1(2);Glx,5.8(6);Ser,1.7(2);His,3.1(3);Gly,0.9(1);Thr,
1.0(1);Ala,0.9(1);Arg,3.0(3);Val,1.1(1);Ile,1.0(1);Leu,8.4(8);Lys,
3.7(4);hSer,0.9(1).
化合物56:
AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTX-NHCH2CH2C6H5(SEQID NO:70)
(X=高丝氨酸)
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr hSer NHCH2CH2C6H5(SEQ ID NO:70)
物理数据:m.p.(未测定)[α]D 25(未测定)
FAB(C184H310N56O52):[M+H]+ 4138.8
AAA:Asx,2.0(2);Glx,5.9(6);Ser,1.7(2);His,2.9(3);Gly,0.9(1);Thr,1.0(1);
Ala,0.9(1);Arg,3.0(3);Val,1.0(1);Ile,0.9(1);Leu,8.0(8);Lys,4.1(4);
hSer,0.9(1).
实施例IV
将Nα-Boc-Nγ-Fmoc-L-2,4-二氨基丁酸的铯盐固定到Merrifield树脂上(DMF,50℃,48小时),并代替实施例I中的Boc-Ala用于固相合成。合成完成后,肽在室温下用20%哌啶的DMF溶液处理30分钟以去除侧链的Fmoc保护基。被保护的肽自发地环化成内酰胺,由此将自身从树脂上分割。从树脂中滤出溶液,真空蒸发至油状残留。如实施例I所述用液体HF处理残留物得到粗非保护肽。如实施例I所述处理和纯化该肽。
化合物49:
AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTX(SEQ ID NO:63)
(X=L-2,4-二氨基丁酰基内酰胺)
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr L-2,4-二氨基丁酰基内酰胺(SEQ ID NO:63)物理数据:m.p.161-181℃[α]D 25-48.38(c 0.25,H2O)FAB(C176H300N56O51):[M+H]+ 4016.8
AAA:Asx,2.1(2);Glx,6.3(6);Ser,1.7(2);His,3.3(3);Gly,1.1(1);Thr,1.0(1);
Ala,2.1(2);Arg,2.9(3);Val,0.9(1);Ile,0.9(1);Leu,8.0(8);Lys,3.8(4).
实施例V
用猪肝酯酶(Sigma Chemical Company,St.Louis,MO)处理实施例II制备的高丝氨酸内酯类似物水溶液。用分析HPLC来对内酯水解成C末端高丝氨酸进行监测。判断水解完全后,如实施例I所述利用制备HPLC进行纯化。
化合物37:
AVSEHQLLHD KGKSIQDLRR RELLEKLLEK LHTX-OH(SEQ ID NO:51)
(X=高丝氨酸)
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr hSer OH(SEQ ID NO:51)
物理数据:m.p.(未测定)[α]D 25(未测定)
FAB(C176H301N55O53):[M+H]+ 4035.1
AAA:Asx,2.1(2);Glx,5.9(6);Ser,2.0(2);His,3.1(3);Gly,0.8(1);hSer,0.8(1);
Thr,1.0(1);Ala,1.0(1);Arg,3.0(3);Val,1.3(1);Ile,1.0(1);Leu,8.1(8);Lys,
3.8(4).
实施例VI
按照实施例I所述,被保护肽-树脂BocAVS(Bzl)E(OBz)H(Bom)QLLHD(OBzl)R(Ts)GR(Ts)S(Bzl)IQD(OBz)LR(Ts)R(Ts)E(OBz)LLE(OBzl)R(Ts)LLK(Fmoc)R(Ts)LH(Bom)T(Bzl)AO-PAM的合成规模为0.35毫摩尔。全部Nα基团都用叔丁氧基羰基(Boc)保护;侧链保护基如文中所述。合成完成后,肽树脂在室温下用50毫升20%哌啶的二甲基甲酰胺(DMF)溶液处理30分钟,以去除赖氨酸上的芴基甲氧基羰基(Fmoc)保护基。依次用DMF,MeOH,CH2Cl2洗涤树脂,经干燥得1.6克部分被保护的肽。在室温下,在20毫升DMF中,在0.16克(0.3毫摩尔)六氟磷酸苯并三唑基氧基-三(吡咯烷基)磷鎓(PyBop)和0.067克(0.525毫摩尔)二异丙基乙胺(DIEA)的存在下,0.8克(0.175毫摩尔)部分被保护的肽在赖氨酸位置用0.44克(0.3毫摩尔)甲氧基(亚乙氧基)乙酸[PEG(2)CH2COOH]进行5小时的酰化。5小时后,过滤树脂,并依次用DMF、MeOH和CH2Cl2洗涤。酰化步骤重复两次,直至树脂上的茚三酮试验结果为阴性。如实施例I所述从树脂上分离最终的肽,同时去除侧链保护基,并纯化;由此得到100毫克化合物19。
化合物19
AVSEHQLLHDRGRSIQDLRRRELLERLLKRLHTA-OH(SEQ ID NO:18)
CH3O(CH2CH2O)2CH2C=O
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu
20 25
Lys(COCH2PEG2)Arg Leu His Thr Ala OH(SEQ ID NO:18)
30
物理数据:
m.p.145-195℃[α]D 25-44.60°(c 0.2,H2O)
FAB(C183H316N64O54):[M+H]+ 4276.2
AAA:Asp,2.1(2);Glu,5.0(5);Ser,1.6(2);His,2.9(3);Gly,0.9(1);Thr,1.9(2);
Arg,7.1(7);Val,1.1(1);Ile,1.0(1);Leu,8.0(8);Lys,0.9(1).
实施例VII
如实施例IV所述合成、分离和纯化肽,所不同的是使用2-甲氧基聚(亚乙基氧基)乙酸[PEG(5000)CH2CO2H]作为酰化剂。由0.8克(0.175毫摩尔)部分被保护的肽得到300毫克化合物20。
化合物20
AVSEHQLLHDRGRSIQDLRRRELLERLLKRLHTA-OH(SEQ ID NO:19)
CH3O(CH2CH2O)110CH2C=O
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu
20 25
Lys(COCH2PEG5000)Arg Leu His Thr Ala OH(SEQ ID NO:19).
30
物理数据:
m.p.105℃[α]D 25-22.95°(c 0.1l,50%aq.HOAc)
AAA:Asp,2.0(2);Glu,4.8(5);Ser,1.6(2);His,2.6(3);Gly,1.1(1);Thr,1.1(1);
Arg,7.3(7);Val,0.8(1);Ile,0.9(1);Leu,8.3(8);Lys,1.1(1);Ala,1.8(2).
实施例VIII
hPTHrp(1-34)类似物基因的合成
设计了一段编码hPTHrp(1-34)类似物化合物4(SEQ ID NO:9)的合成基因。使用Sinha等在《核酸研究》(Nucleic Acid Research)12,4539-4557(1984)中所述的亚磷酰胺法,用DNA合成仪(Milligen/Biosearch)制备必需寡脱氧核苷酸。去保护后,通过在15%聚丙烯酰胺制备凝胶上进行的凝胶电泳来纯化粗寡核苷酸。利用UV定位寡核苷酸,将其从凝胶中切取出来,在Waters c18 Sep-pak柱上脱盐,然后冷冻干燥。
在Perkin-Elmer Cetus热循环仪上通过聚合酶链反应(PCR)进行扩增,共25轮:94℃1分钟,50℃2分钟和72℃3分钟,使用“GeneAmp”DNA扩增试剂盒(Perkin-Elmer Cetus)中的反应剂,包括Taq聚合酶。
制备两段重迭寡核苷酸,一段为88聚体(2微克),PTH3,(SEQ ID NO:31):
CCTCTAGATC TCCGCGGCGC TAGC ATG GCT GTT TCT GAA CAT CAG 45
Met Ala Val Ser Glu His Gln
1 5
CTG CTT CAT GAC AAA GGT AAA TCG ATT CAA GAT CTG AGA CGT C 88
Leu Leu His Asp Lys Gly Lys Ser Ile Gln Asp Leu Arg Arg
10 15 20和一段反义99聚体(2微克),PTH4,(SEQ ID NO:32):
CCTCGAAGCT TATGCATCAT TATCTAGA CAT AGT ATG CAG CTT TTC 46
Met Thr His Leu Lys Glu
30
AAG CAG TTT CTC CAG CAG CTC GCG ACG TCT CAG ATC TTG AAT 88
Leu Leu Lys Glu Leu Leu Glu Arg Arg Arg Leu Asp Gln Ile
25 20 15
CG 90.
作为hPTHrp(1-34)类似物基因的模板DNA序列。利用以下侧翼引物:PTHPCR1:CCTCTAGATC TCCGCGGCGC TAG(SEQ ID NO:33)和PTHPCR2:CCTCGAAGCT TATGCATCAT TATC(SEQ ID NO:34)通过PCR扩增完整的基因。通过4%NuSieve琼脂糖凝胶上的凝胶电泳来纯化扩增得到的DNA产物。从凝胶中切取包含长约150碱基对的hPTHrp(1-34)类似物合成基因的条带,经Elu-Quik玻璃凝胶DNA萃取(Schleicher & Schuell,Kaene,NH),分离得到约200纳克DNA。
实施例IX
一段hPTHrp(1-34)1类似物基因的分子克隆
为了亚克隆实施例VI中的hPTHrp(1-34)类似物基因,分离出200纳克扩增DNA并用限制性内切酶HinD III和Sac II切割。将DNA与2微克事先用HinD III和Sac II切割的TrpLE 18 Prot(Ile3,Pro5)质粒连接。
生成的含单拷贝hPTHrp(1-34)类似物基因的质粒TrpLE 18 hPTHrp(1-34)1然后被转化到感受态大肠杆菌HB101细胞(CLONTECH,Palo Alto,CA)中。对转化子进行PCR分析来确定是否插入。挑选出转化的细胞集落在200微升水中煮沸5分钟;取2微升用插入片段两侧的引物进行PCR。然后在1%琼脂糖凝胶上分析PCR产物以证实有单拷贝hPTHrp(1-34)基因的插入。然后在自动DNA测序仪(Applied Biosystems Model 373A,Foster City,CA)上,用vendor染料脱氧终止剂测序试剂盒对TrpLE 18 hPTHrp(1-34)1构建物进行测序鉴定。
实施例X
含有hPTHrp(1-34)类似物基因多拷贝的TrpLE 18载体的构建
单一的Nhe I和Xba I限制性位点位于hPTHrp(1-34)类似物基因序列的开头和结尾处。这两个识别不同序列但产生相同单链粘端的位点使得在TrpLE 18载体内构建hPTHrp(1-34)基因的多拷贝成为可能。
分别在反应中用Bam HI+Nhe I和Xba I+Bam HI切割5微克含有基因单拷贝的质粒TrpLE 18 hPTHrp(1-34)1。从各消化产物中分离300纳克含hPTHrp(1-34)类似物基因的片段。将两份片段混合而连接成TrpLE 18 hPTHrp(1-34)2质粒。将该质粒用于转化感受态大肠杆菌HB101细胞。利用在1%琼脂糖凝胶上测定PCR产物的大小来确定是否存在hPTHrp(1-34)二拷贝插入片段。然后用DNA测序来鉴定含有基因两份拷贝的质粒TrpLE 18 hPTHrp(1-34)2。两份hPTHrp(1-34)基因的正确融合消除了连接处的Nhe I和Xba I位点。这样,使得留下的Nhe I和Xba I位点仅存在于串联基因的两侧。
通过重复该过程,构建出了包含hPTHrp(1-34)基因四拷贝的最终质粒TrpLE18 hPTHrp(1-34)4。利用DNA序列分析来证实TrpLE 18 hPTHrp(1-34)4的序列是否正确。
实施例XI
TrpLE 18 hPTHrp(1-34)4的表达和纯化
TrpLE 18 hPTHrp(1-34)4的诱导
在50毫升起始培养液,即含有50微克/毫升青霉素和100微克/毫升色氨酸的LB培养基(J.H.Miller,“分子遗传试验”“Experiments in Molecular Genetics”,P.431(1972),在此引用参考)中接种含有TrpLE 18 hPTHrp(1-34)4质粒的大肠杆菌细胞,在37℃剧烈振荡培养过夜,直至A550约为6。将2升用于生产的LB培养基预热至37℃,接种以20毫升起始培养物,至A550约为0.06。然后该培养物剧烈振荡培养直至A550约为0.6至0.8,然后加入2毫升10毫克/毫升的吲哚丙烯酸溶液(IAA)。在良好的通气条件下继续培养约16小时,直至最终的A550约为6(通常,在4至10之间)。细胞经离心浓缩后重悬在500毫升50毫摩尔浓度的Tris-HCl,pH7.5,0.1毫摩尔浓度EDTA缓冲液(Tris缓冲液)中。
用Heat Systems-Ultrasonicx,Inc的220F型超声仪(配备有3/4”的喇叭)对悬浮液进行超声处理,为了防止过热,使用50%的全功率。
为了确定诱导的程度,利用SDS-PAGE进行全细胞分析。可以看到,由TrpLE 18 hPTHrp(1-34)4生成的基因产物为预计分子量约17,000的一条主条带。这占总细胞蛋白的10%。
融合蛋白的分离
将细胞裂解物以3600×g离心15分钟,沉淀出TrpLE 18 hPTHrp(1-34)4融合蛋白;弃去上清液。将沉淀重悬在200毫升Tris缓冲液中。(通常为40至80A550/毫升)。
实施例XII
融合蛋白的加工和高丝氨酸内酯hPTHrp(1-34)肽的纯化
用CNBr切去hPTHrp(1-34)多元融合蛋白侧区的甲硫氨酸残基得到所需的高丝氨酸内酯hPTHrp(1-34)多肽,如下所述将其纯化。用CNBr处理融合蛋白
通过轻微搅拌将TrpLE 18 hPTHrp(1-34)4融合蛋白沉淀重悬在60毫升70%的甲酸中(约20毫克/毫升总蛋白;通常,将得自1000A550单位细胞的物质溶解在3毫升体积中)。加入数滴辛醇,在溶液中用N2鼓泡20分钟,然后加入5.5克CNBr。该反应在25℃进行6小时,然后将等体积的50∶50的MeOH∶H2与样品混合,再经旋转蒸发去除。该过程重复2至4次后,基本去除甲酸和CNBr。然后将样品蒸发至干燥,重溶在200毫升水中,冷冻干燥保存。高丝氨酸内酯hPTHrp(1-34)的纯化
用50毫摩尔浓度pH6.5的KH2PO4透析经CNBr切割的上清液24小时,中途换液几次。透析过程中,将pH维持在6.5。透析后,利用高速离心去除沉淀。上清液利用Gelman 0.45微米过滤仪(Acrodisc 4184)来澄清。阳离子交换色谱
利用Bio-Gel TSK-SP-5PW HPLC色谱柱(21.5×150毫米)上的阳离子交换色谱来进行初纯化。流速为8毫升/分钟,得率约12毫克高度纯化高丝氨酸内酯hPTH(1-34)肽的条件为:
1.在50毫摩尔浓度的KH2PO4,pH6.5中平衡色谱柱
2.上样10毫升澄清过的上清液(约1.5升培养液或2.4克包涵体)
3.用含有50毫摩尔浓度NaCl的50毫摩尔浓度KH2PO4,pH6.5洗柱,直至基线稳定
4.用含有90毫摩尔浓度NaCl的50毫摩尔浓度KH2PO4,pH6.5洗脱。收集流份约45分钟。
5.用C18 HPLC分析90毫摩尔浓度NaCl流份中的高丝氨酸hPTH(1-34)浓度,然后汇集保存反相HPLC色谱
将反相Poros R/H 4.6×100毫米色谱柱(Perseptive Biosystems,Cambridge,MA)用于最终纯化步骤。色谱条件如下:移动相A:0.1%三氟乙酸(TFA)/水移动相B:0.1%三氟乙酸(TFA)/CH3CN
时间 流速 %B
0分钟 4毫升/分钟 15
5.0分钟 4毫升/分钟 40
5.2分钟 4毫升/分钟 100
6.8分钟 4毫升/分钟 100
7.0分钟 4毫升/分钟 15
高丝氨酸hPTH(1-34)即化合物4的滞留时间约为2.943分钟。经质谱测定,纯化肽的纯度约为98%。
实施例XIII
大致按照Gunness-Hey和Hock,《代谢骨疾病》(Metab.Bone Dis.)5:177181(1984)中所述的方法,在切除卵巢的大鼠中评价本发明化合物对骨质的作用。
使成年Sprague-Dawley雌鼠适应环境,按体重分组(n=9、10或12),并切除双侧卵巢(OVX),或者进行假手术。在手术后第17天开始给药,持续20天。将测试化合物溶解与2%鼠血清/生理盐水媒质中,每天皮下给药一次。
给药20天后,将鼠杀死,取出右侧股骨。将股骨从中间切开,通过钻取小梁将远半端股骨(DHF)再分离成小梁骨(TB)和皮质骨(CB)。利用Calcette钙分析仪测定钙质,并以毫克/DHF/100克体重为单位表示为平均骨钙。
利用双样品t测试来比较OVX组和假处理组。用单向ANOVA来比较OVX组,然后用Fisher LSD多元比较法来将各处理组与媒质比较。
卵巢切除诱导了严重的骨质损失,主要是小梁骨中的骨质损失。总骨钙比假手术对照低47至54%。
80微克/千克/天的bPTH(1-34)和hPTHrp(1-34)显著提高了被治疗OVX大鼠的总骨钙质,在数据上分别由53%提高至95%,由18%提高至40%;但是,皮质骨钙质并没有明显的增加。
按80微克/千克/天给药的本发明化合物将总骨钙质由66%提高至138%,将小梁骨钙质由87%提高至128%。与未治疗的OVX大鼠相比,皮质骨钙质、小梁骨厚度和骨体积也显著增加。
在该测试中,测试了以下化合物:
n 小梁骨钙质 总骨钙质
化合物编号 (测试次数) (相对OVX鼠的增%) (相对OVX鼠的增%)化合物1(SEQ ID NO:7) 6 101-128% 88-138%化合物2(SEQ ID NO:6) 3 87-102% 66-114%化合物4(SEQ ID NO:9) 3 - 88-114%
用相同的研究方法,卵巢切除后的大鼠按40微克/千克/天的剂量给药5,10和20天,结果如下:
n 总骨钙质
化合物编号 (测试次数) 给药天数(d) (相对OVX鼠的增%)化合物 1(SEQ ID NO:7) 3 20d 73-109%化合物 4(SEQ ID NO:9) 5 20d 79-105%化合物 4(SEQ ID NO:9) 1 10d 79%化合物49(SEQ ID NO:63) 1 10d 93%化合物 4(SEQ ID NO:9) 1 5d 55%化合物42(SEQ ID NO:56) 1 5d 60%
实施例XIV
给成年新西兰白色雌兔(Hazelton Research Products,Inc.,3.6千克,n=9-11/处理组)每日注射媒质或0.15毫克/千克强的松,持续13个月。利用双能X光吸光测定法(Hologic QDR-1500W型,Hologic,Inc.,Waltham,MA)测定骨无机质密度(BMD),然后进行骨代谢的生物标记。9个月后,分亚组另外接受0.05、0.15或0.5微克/千克/天的化合物1(SEQ ID NO:7)皮下注射。2个月后,将剂量提高至0.5、3和10微克/千克/天。强的松处理引起快速的早期损失(2-4个月),然后在以后的试验期内BMD保持稳定。在腰脊A/P视图、侧视图和远端股骨中可观察到偏离基线12.4±1.3%,18.1±1.9%和11.8±2.5%的最终BMD损失。在股骨骨干中的损失较少(7.8±2.2%)。低剂量的早期给药并没有改变以上情况。在30至60天后,给继续用强的松处理的动物10微克/千克的化合物1引起全部评价位点的BMD恢复,但与仅用媒质处理了12个月的对照兔相比没有明显差别。在用强的松处理的兔中,血清骨钙蛋白从50±8纳克/毫升的基线下跌6.4倍,但通过同时使用10微克/千克化合物1恢复至正常。
实施例XV
制备了本发明化合物1(SEQ ID NO:7)的鼻用制剂,并评价了其高生物利用率地传递化合物的效率。
在无菌水中制备含20毫克/毫升化合物1,0.2毫克/毫升柠檬酸,2.6毫克/毫升柠檬酸钠,0.2毫克/毫升氯苄烷铵和不同量的山梨醇和不同增强剂的溶液,并用NaOH将pH调节至6.0。
给cynomolgous猴经鼻使用该溶液,并如下评价药物动力学。
表1列出测试制剂组成以及体外测试的结果,从中可以看出,本发明制剂的生物利用率接近皮下注射的50%,因而适用于骨质疏松症的临床治疗。
表1
PTHrp类似物(SEQ ID NO:7)的鼻用制剂
配方 A B C D E F G化合物SEQ ID NO:7 20 20 20 20 20 20 20柠檬酸 0.2 0.2 0.2 0.2 0.2 0.2 0.2柠檬酸钠 2.6 2.6 2.6 2.6 2.6 2.6 2.6氯苄烷铵 0.2 0.2 0.2 0.2 0.2 0.2 0.2山梨醇 30 30 12 - 35 40 35甲基纤维素1500 - 2.5 - - - - -丙二醇 - - 10 50 - - -二甲基-β-环糊精 - - - - 50 - -牛黄胆酸钠 - - - - - 10 -甘氨胆酸钠 - - - - - - 10剂量 N Tmax Cmax 平均AUC 平均生物利用率(毫克) (小时) (纳克/毫升) (纳克/毫升小时) 百分比B 4 3 0.3 2.7 1.7±0.6 0.6±0.2C 4 6 0.3 2.8 2.7±2.8 1.0±1.0D 4 3 0.3 4.0 3.3±2.6 1.2±1.0D 1 6 0.4 0.5 1.1±2.1 1.7±3.0E 4 8 0.3 40 36±40 13±15F 4 6 0.6 119 128±48 47±18G 4 6 0.3 157 133±77 49±29皮下注射:0.05 23 0.6 2.0 3.4±2.8 100±82
序列表(1)一般信息:(i)申请人:Syntex(U.S.A)Inc.,(ii)发明名称:用于治疗骨质疏松症化合物的鼻传递的药物组合物(iii)序列数量:86(iv)通讯地址:
(A)地址:Heller Ehrman White & McAuliffe
(B)街道:525 University Ave.
(C)城市:Palo Alto
(D)州:CA
(E)国家:USA
(F)邮编:94301(v)计算机可读形式:
(A)介质类型:软盘
(B)计算机:IBM PC兼容机
(C)操作系统:PC-DOS/MS-DOS
(D)软件:PatentIn Release#1.0,1.25版(vi)本申请信息:
(A)申请号:
(B)申请日:
(C)分类:(viii)代理人信息:
(A)姓名:Chow,Y.Ping
(B)登记编号:30,740
(C)参考文献/卷宗编号:27610-04(ix)电讯信息:
(A)电话:415-324-7078
(B)电传:415-324-0638(2)SEQ ID NO:1的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:1:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu
1 5 10 15
Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp
20 25 30
Val His Asn Phe(2)SEQ ID NO:2的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:2:
Ala Val Ser Glu Ile Gln Phe Met His Asn Leu Gly Lys His Leu
1 5 10 15
Ser Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp
20 25 30
Val His Asn Phe(2)SEQ ID NO:3的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:3:
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu
1 5 10 15
Ser Ser Leu Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp
20 25 30
Val His Asn Phe(2)SEQ ID NO:4的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:4:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Phe Phe Leu His His Leu Ile Ala Glu
20 25 30
Ile His Thr Ala(2)SEQ ID NO:5的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:5:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Arg Lys
20 25 30
Leu His Thr Ala(2)SEQ ID NO:6的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:6:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Ala(2)SEQ ID NO:7的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:7:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Ala(2)SEQ ID NO:8的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:被修饰位点
(B)位置:34
(D)其它信息:/注:“Xaa34=高丝氨酸”(xi)序列描述:SEQ ID NO:8:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Xaa(2)SEQ ID NO:9的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:被修饰位点
(B)位置:34
(D)其它信息:/注:“Xaa34=高丝氨酸内酯”(xi)序列描述:SEQ ID NO:9:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Xaa(2)SEQ ID NO:10的信息:(i)序列特征:
(A)长度:37氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:10:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Ala Gly Arg Arg
35(2)SEQ ID NO:11的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:11:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu Lys Glu Leu(2)SEQ ID NO:12的信息: (i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:12:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Ala Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Ala(2)SEQ ID NO:13的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:13:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Ala Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Ala(2)SEQ ID NO:14的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:14:
Ala Val Ser Glu Ala Gln Leu Leu His Asp Leu Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Ala Leu(2)SEQ ID NO:15的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:15:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30
Leu His Thr Ala(2)SEQ ID NO:16的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:16:
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30
Leu His Thr Ala(2)SEQ ID NO:17的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:17:
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Lys Arg
20 25 30
Leu His Thr Ala(2)SEQ ID NO:18的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:被修饰位点
(B)位置:29
(D)其它信息:/注:“Xaa29=赖氨酸-(OCCH2(OCH2CH2)2OCH3)”(xi)序列描述:SEQ ID NO:18:
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu
20 25
Lys(COCH2PEG2)Arg Leu His Thr Ala
30(2)SEQ ID NO:19的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:被修饰位点
(B)位置:29
(D)其它信息:/注:“Xaa29=赖氨酸-(OCCH2(OCH2CH2)110OCH3)”(xi)序列描述:SEQ ID NO:19:
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu
20 25
Lys(COCH2PEG5000)Arg Leu His Thr Ala
30(2)SEQ ID NO:20的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:20:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Ala Leu Ala Glu Ala Leu Ala Glu Ala
20 25 30
Leu His Thr Ala(2)SEQ ID NO:21的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:21:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Ser Leu Leu Ser Ser Leu Leu Ser Ser
20 25 30
Leu His Thr Ala(2)SEQ ID NO:22的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:22:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Ala Phe Tyr Asp Lys Val Ala Glu Lys
20 25 30
Leu His Thr Ala(2)SEQ ID NO:23的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:被修饰位点
(B)位置:8
(D)其它信息:/注:“Xaa8=正亮氨酸”(ix)特征:
(A)名称/代码:被修饰位点
(B)位置:18
(D)其它信息:/注:“Xaa18=正亮氨酸”(xi)序列描述:SEQ ID NO:23:
Ala Val Ser Glu Ile Gln Phe Nle His Asn Leu Gly Lys His Leu
1 5 10 15
Ser Ser Nle Glu Arg Val Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Asn Tyr(2)SEQ ID NO:24的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽 (iii)段设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:被修饰位点
(B)位置:8
(D)其它信息:/注:“Xaa8=正亮氨酸”(ix)特征:
(A)名称/代码:被修饰位点
(B)位置:18
(D)其它信息:/注:“Xaa18=正亮氨酸”(xi)序列描述:SEQ ID NO:24:
Ala Val Ser Glu Ile Gln Phe Nle His Asn Leu Gly Lys His Leu
1 5 10 15
Ser Ser Nle Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Asn Tyr(2)SEQ ID NO:25的信息:(i)序列特征:
(A)长度:35氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:肽(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:25:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Asp Leu Arg Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Met Ala
35(2)SEQ ID NO:26的信息:(i)序列特征:
(A)长度:10氨基酸
(B)类型:氨基酸
(D)拓扑:螺旋(ii)分子类型:肽(iii)假设:非(v)片段类型:内部(ix)特征:
(A)名称/代码:被修饰位点
(B)位置:8
(D)其它信息:/注:“Xaa8=谷氨酸或精氨酸”(ix)特征:
(A)名称/代码:区域
(B)位置:1..10
(D)其它信息:/注:“序列26被插入在序列5、6、7、8、9、10、11、
12、13和14的22至31位。”(xi)序列描述:SEQ ID NO:26:
Glu LeuLeu Glu Lys Leu Leu Xaa Lys Leu
1 5 10(2)SEQ ID NO:27的信息:(i)序列特征:
(A)长度:10氨基酸
(B)类型:氨基酸
(D)拓扑:螺旋(ii)分子类型:肽(iii)假设:非(v)片段类型:内部(ix)特征:
(A)名称/代码:被修饰位点
(B)位置:8
(D)其它信息:/注:“Xaa8=谷氨酸赖氨酸或赖氨酸-(OCCH2PEGX)”(ix)特征:
(A)名称/代码:区域
(B)位置:1..10
(D)其它信息:/注:“序列27被插入在序列15、16、17、18和19的22
至31位。”(xi)序列描述:SEQ ID NO:27:
Glu Leu Leu Glu Arg Leu Leu Xaa Arg Leu
1 5 10(2)SEQ ID NO:28的信息:(i)序列特征:
(A)长度:10氨基酸
(B)类型:氨基酸
(D)拓扑:螺旋(ii)分子类型:肽(iii)假设:非(v)片段类型:内部(ix)特征:
(A)名称/代码:肽
(B)位置:1..10
(D)其它信息:/注:“序列28被插入在序列20的22至31位。”(xi)序列描述:SEQ ID NO:28:
Ala Leu Ala Glu Ala Leu Ala Glu Ala Leu
1 5 10(2)SEQ ID NO:29的信息:(i)序列特征:
(A)长度:10氨基酸
(B)类型:氨基酸
(D)拓扑:螺旋(ii)分子类型:肽(iii)假设:非(v)片段类型:内部(ix)特征:
(A)名称/代码:肽
(B)位置:1..10
(D)其它信息:/注:“序列29被插入在序列21的22至31位。”(xi)序列描述:SEQ ID NO:29:
Ser Leu Leu Ser Ser Leu Leu Ser Ser Leu
1 5 10(2)SEQ ID NO:30的信息:(i)序列特征:
(A)长度:10氨基酸
(B)类型:氨基酸
(D)拓扑:螺旋(ii)分子类型:肽(iii)假设:非(v)片段类型:内部(ix)特征:
(A)名称/代码:肽
(B)位置:1..10
(D)其它信息:/注:“序列30被插入在序列22的22至31位。”(xi)序列描述:SEQ ID NO:30:
Ala Phe Tyr Asp Lys Val Ala Glu Lys Leu
1 5 10(2)SEQ ID NO:31的信息:(i)序列特征:
(A)长度:88碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑:线性(ii)分子类型:cDNA(iii)假设:非(iv)反义链:非(xi)序列描述:SEQ ID NO:31:CCTCTAGATC TCCGCGGCGC TAGCATGGCT GTTTCTGAAC ATCAGCTGCT TCATGACAAA 60GGTAAATCGA TTCAAGATCT GAGACGTC 88(2)SEQ ID NO:32的信息:(i)序列特征:
(A)长度:90碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑:线性(ii)分子类型:cDNA(iii)假设:非 (iv)反义链:是(xi)序列描述:SEQ ID NO:32:CCTCGAAGCT TATGCATCAT TATCTAGACA TAGTATGCAG CTTTTCAAGC AGTTTCTCCA 60GCAGCTCGCG ACGTCTCAGA TCTTGAATCG 90(2)SEQ ID NO:33的信息:(i)序列特征:
(A)长度:23碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑:线性(ii)分子类型:cDNA(iii)假设:非(iv)反义链:非(xi)序列描述:SEQ ID NO:33:
CCTCTAGATC TCCGCGCGCT AGC 23(2)SEQ ID NO:34的信息:(i)序列特征:
(A)长度:24碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑:线性(ii)分子类型:cDNA(iii)假设:非(iv)反义链:是(xi)序列描述:SEQ ID NO:34:
CCTCGAAGCT TATGCATCAT TATC 24(2)SEQ ID NO:35的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:35:Ala Val Ser Glu Ile Gln Phe Leu His Asn Leu Gly Lys His Leu1 5 10 15Ser Ser Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Asn Tyr(2)SEQ ID NO:36的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:修饰位点
(B)位置:34
(D)其它信息:/注:“Xaa=高丝氨酸”(xi)序列描述:SEQ ID NO:36:
Ala Val Ser Glu Ile Gln Phe Leu His Asn Leu Gly Lys His Leu
1 5 10 15
Ser Ser Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Asn Xaa(2)SEQ ID NO:37的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:修饰位点
(B)位置:34
(D)其它信息:/注:“Xaa=高丝氨酸”(xi)序列描述:SEQ ID NO:37:
Ala Val Ser Glu Ile Gln Phe Leu His Asn Lys Gly Lys His Leu
1 5 10 15
Ser Ser Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Asn Xaa(2)SEQ ID NO:38的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:38:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Lys Leu Lys Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Ala(2)SEQ ID NO:39的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:39:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30
Leu His Thr Ala(2)SEQ ID NO:40的信息:(i)序列特征:
(A)长度:35氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:40:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30
Leu His Thr Ala Pro
35(2)SEQ ID NO:41的信息:(i)序列特征:
(A)长度:37氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:41:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30
Leu His Thr Ala Gly Arg Arg
35(2)SEQ ID NO:42的信息:(i)序列特征:
(A)长度:38氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:修饰位点
(B)位置:38
(D)其它信息:/注:“Xaa=高丝氨酸”(xi)序列描述:SEQ ID NO:42:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30
Leu His Thr Ala Gly Arg Arg Xaa
35(2)SEQ ID NO:43的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:43:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Tyr(2)SEQ ID NO:44的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:44:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Tyr Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Ala(2)SEQ ID NO:45的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:45:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Cys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Ala(2)SEQ ID NO:46的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:修饰位点
(B)位置:13
(D)其它信息:/注:“Xaa是Cys(CH-2CONH(CH-2)-2NH(生物素))”(xi)序列描述:SEQ ID NO:46:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Xaa Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Ala(2)SEQ ID NO:47的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非 (v)片段类型:N末端(ix)特征:
(A)名称/代码:修饰位点
(B)位置:13
(D)其它信息:/注:“Xaa是Lys(7-二甲基氨基-2-氧-2H-1-苯并吡喃-4-乙酰基”(xi)序列描述:SEQ ID NO:47:Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Xaa Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala(2)SEQ ID NO:48的信息:(i)序列特征:
(A)长度:35氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:48:Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala Gly
35(2)SEQ ID NO:49的信息:(i)序列特征:
(A)长度:33氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(ix)特征:(A)名称/代码:修饰位点(B)位置:4(D)其它信息:/注:“Xaa4是Glu(OCH-3)”(ix)特征:(A)名称/代码:修饰位点(B)位置:10(D)其它信息:/注:“Xaa10是Asp(OCH-3)”(ix)特征:(A)名称/代码:修饰位点(B)位置:17(D)其它信息:/注:“Xaa17是Asp(OCH-3)”(ix)特征:(A)名称/代码:修饰位点(B)位置:22(D)其它信息:/注:“Xaa22是Glu(OCH3)”(ix)特征:(A)名称/代码:修饰位点(B)位置:25(D)其它信息:/注:“Xaa25是Glu(OCH-3)”(ix)特征:(A)名称/代码:修饰位点
(B)位置:29(D)其它信息:/注:“Xaa29是Glu(OCH-3)”(xi)序列描述:SEQ ID NO:49:Ala Val Ser Xaa1 His Gln Leu Leu His Xaa Lys Gly Lys Ser Ile1 5 10 15Gln Xaa Leu Arg Arg Arg Xaa1 Leu Leu Xaa Lys Leu Leu Xaa Lys
20 25 30Leu His Ala(2)SEQ ID NO:50的信息:(i)序列特征:
(A)长度:33氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:修饰位点
(B)位置:4
(D)其它信息:/注:“Xaa4是Glu(OCH-3)”(ix)特征:
(A)名称/代码:修饰位点
(B)位置:10
(D)其它信息:/注:“Xaa10是Asp(OCH-3)”(ix)特征:
(A)名称/代码:修饰位点
(B)位置:17(D)其它信息:/注:“Xaa17是Asp(OCH-3)”(ix)特征:
(A)名称/代码:修饰位点
(B)位置:22(D)其它信息:/注:“Xaa22是Glu(OCH3)”(ix)特征:
(A)名称/代码:修饰位点
(B)位置:25(D)其它信息:/注:“Xaa25是Glu(OCH-3)”(ix)特征:
(A)名称/代码:修饰位点
(B)位置:29(D)其它信息:/注:“Xaa29是Glu(OCH-3)”(xi)序列描述:SEQ ID NO:50:Ala Val Ser Xaa His Gln Leu Leu His Xaa Lys Gly Lys Ser Ile1 5 10 15Gln Xaa Leu Arg Arg Arg Xaa Leu Leu Xaa Lys Leu Leu Xaa Lys
20 25 30Leu His Ala(2)SEQ ID NO:51的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:修饰位点
(B)位置:34
(D)其它信息:/注:“Xaa=高丝氨酸”(xi)序列描述:SEQ ID NO:51:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Xaa(2)SEQ ID NO:52的信息:(i)序列特征:
(A)长度:35氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:52:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Ala Pro
35(2)SEQ ID NO:53的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:53:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Pro(2)SEQ ID NO:54的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:54:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Pro(2)SEQ ID NO:55的信息:(i)序列特征:
(A)长度:33氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质 (iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:55:Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser I1e1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Pro(2)SEQ ID NO:56的信息:(i)序列特征:
(A)长度:32氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:56:Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu Pro(2)SEQ ID NO:57的信息:(i)序列特征:
(A)长度:37氨基酸
(B)类型:氨基酸
(D)拓扑:线性 (ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:57:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Arg Ser Ala Trp
35(2)SEQ ID NO:58的信息:(i)序列特征:
(A)长度:42氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:58:
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30
Leu His Thr Arg Gly Arg Arg Thr Arg Ser Ala Trp
35 40(2)SEQ ID NO:59的信息:(i)序列特征:
(A)长度:42氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:59:
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30
Leu His Thr Arg Gly Arg Arg Thr Arg Ser Ala Trp
35 40(2)SEQ ID NO:60的信息:(i)序列特征:
(A)长度:42氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:修饰位点
(B)位置:13
(D)其它信息:/注:“Xaa13是Lys(二氢肉桂酰基)”(xi)序列描述:SEQ ID NO:60:
Ala Val Ser Glu His Gln Leu Leu His Asp Arg Gly Xaa Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg
20 25 30
Leu His Thr Arg Gly Arg Arg Thr Arg Ser Ala Trp
35 40(2)SEQ ID NO:61的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:修饰位点
(B)位置:8
(D)其它信息:/注:“Leu8是正亮氨酸”(ix)特征:
(A)名称/代码:修饰位点
(B)位置:18
(D)其它信息:/注:“Leu18是正亮氨酸”(xi)序列描述:SEQ ID NO:61:
Ala Val Ser Glu Ile Gln Phe Nle His Asn Leu Gly Lys His Leu
1 5 10 15
Ser Ser Nle Thr Arg Ser Ala Trp Leu Arg Lys Lys Leu Gln Asp
20 25 30
Val His Asn Tyr(2)SEQ ID NO:62的信息:(i)序列特征:
(A)长度:35氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:62:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Met Ala
35(2)SEQ ID NO:63的信息:(i)序列特征:
(A)长度:33氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:修饰位点
(B)位置:33
(D)其它信息:/注:“Xaa是Thr1,4-二氨基丁酰基内酰胺”(xi)序列描述:SEQ ID NO:63:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Xaa(2)SEQ ID NO:64的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:64:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Phe Phe Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Ala(2)SEQ ID NO:65的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:65:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile 1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu His Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala(2)SEQ ID NO:66的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:66:Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu His Leu Leu Glu Lys
20 25 30Leu His Thr Ala(2)SEQ ID NO:67的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:67:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Ile Ala Lys
20 25 30
Leu His Thr Ala(2)SEQ ID NO:68的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:68:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Glu
20 25 30
Ile His Thr Ala(2)SEQ ID NO:69的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端 (ix)特征:
(A)名称/代码:修饰位点
(B)位置:34
(D)其它信息:/注:“Xaa是高丝氨酸”(xi)序列描述:SEQ ID NO:69:Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Xaa(2)SEQ ID NO:70的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:修饰位点
(B)位置:34
(D)其它信息:/注:“Xaa是高丝氨酸”(xi)序列描述:SEQ ID NO:70:Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Xaa(2)SEQ ID NO:71的信息: (i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:71:Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala(2)SEQ ID NO:72的信息:(i)序列特征:
(A)长度:37氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:72:Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Arg Ser Ala Trp
35(2)SEQ ID NO:73的信息:(i)序列特征:
(A)长度:36氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:修饰位点
(B)位置:36
(D)其它信息:/注:“Xaa是Ala3-(2-萘基)-L-丙氨酸”(xi)序列描述:SEQ ID NO:73:Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Arg Ser Xaa
35(2)SEQ ID NO:74的信息:(i)序列特征:
(A)长度:37氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端 (xi)序列描述:SEQ ID NO:74:Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala Ser Ala Trp
35(2)SEQ ID NO:75的信息:(i)序列特征:
(A)长度:38氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:75:Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala Glu Ile Arg Ala
35(2)SEQ ID NO:76的信息:(i)序列特征:
(A)长度:37氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质 (iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:76:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Ala Glu Ile Arg
35(2)SEQ ID NO:77的信息:(i)序列特征:
(A)长度:36氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:77:
Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile
1 5 10 15
Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30
Leu His Thr Ala Glu Ile
35(2)SEQ ID NO:78的信息:(i)序列特征:
(A)长度:35氨基酸
(B)类型:氨基酸
(D)拓扑:线性 (ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(xi)序列描述:SEQ ID NO:78:Ala Val Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile1 5 10 15Gln Asp Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys
20 25 30Leu His Thr Ala Glu
35(2)SEQ ID NO:79的信息:(i)序列特征:
(A)长度:34氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:N末端(ix)特征:
(A)名称/代码:修饰位点
(B)位置:8
(D)其它信息:/注:“Leu8是正亮氨酸”(ix)特征:
(A)名称/代码:修饰位点
(B)位置:18(D)其它信息:/注:“Leu18是正亮氨酸”(ix)特征:
(A)名称/代码:修饰位点
(B)位置:34(D)其它信息:/注:“Xaa是高丝氨酸内酯”(xi)序列描述:SEQ ID NO:79:
Ala Val Ser Glu Ile Gln Phe Nle His Asn Lys Gly Lys His Leu
1 5 10 15
Ser Ser Nle Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp
20 25 30
Val His Asn Xaa(2)SEQ ID NO:80的信息:(i)序列特征:
(A)长度:32氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:内部(xi)序列描述:SEQ ID NO:80:
Ser Glu His Gln Leu Leu His Asp Lys Gly Lys Ser Ile Gln Asp
1 5 10 15
Leu Arg Arg Arg Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu His
20 25 30
Thr Ala(2)SEQ ID NO:81的信息:(i)序列特征:
(A)长度:28氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:内部(xi)序列描述:SEQ ID NO:81:Leu Leu His Asp Lys Gly Lys Ser Ile Gln Asp Leu Arg Arg Arg1 5 10 15Glu Leu Leu Glu Lys Leu Leu Glu Lys Leu His Thr Ala
20 25(2)SEQ ID NO:82的信息:(i)序列特征:
(A)长度:27氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:内部(xi)序列描述:SEQ ID NO:82:
Leu His Asp Lys Gly Lys Ser Ile Gln Asp Leu Arg Arg Arg Glu
1 5 10 15
Leu Leu Glu Lys Leu Leu Glu Lys Leu His Thr Ala
20 25(2)SEQ ID NO:83的信息:(i)序列特征:
(A)长度:41氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:内部(xi)序列描述:SEQ ID NO:83:
Ser Glu His Gln Leu Leu His Asp Arg Gly Arg Ser Ile Gln Asp
1 5 10 15
Leu Arg Arg Arg Glu Leu Leu Glu Arg Leu Leu Glu Arg Leu His
20 25 30
Leu His Arg Gly Arg Arg Thr Arg Ser Ala Trp
35 40(2)SEQ ID NO:84的信息:(i)序列特征:
(A)长度:35氨基酸
(B)类型:氨基酸
(D)拓扑:线性(ii)分子类型:蛋白质(iii)假设:非(v)片段类型:内部(xi)序列描述:SEQ ID NO:84:
Leu Leu His Asp Arg Gly Arg Ser Ile Gln Asp Leu Arg Arg Arg
1 5 10 15
Glu Leu Leu Glu Arg Leu Leu Glu Arg Leu His Leu His Arg Gly
20 25 30
Arg Arg Thr Arg Ser Ala Trp
35(2)SEQ ID NO:85的信息:(i)序列特征:
(A)长度:10氨基酸
(B)类型:氨基酸 (D)拓扑:螺旋(ii)分子类型:肽(iii)假设:非(v)片段类型:内部(ix)特征:(A)名称/代码:修饰位点(B)位置:1和4(D)其它信息:/注:“Xaa1和Xaa4=Glu、Glu(OCH3)、His或Pro”(ix)特征:(A)名称/代码:修饰位点(B)位置:2(D)其它信息:/注:“Xaa2=Leu或Phe”(ix)特征:(A)名称/代码:修饰位点(B)位置:5(D)其它信息:/注:“Xaa5=Lys或His”(ix)特征:(A)名称/代码:修饰位点(B)位置:7和10(D)其它信息:/注:“Xaa7和Xaa10=Leu或Ile”(ix)特征:(A)名称/代码:修饰位点(B)位置:8(D)其它信息:/注:“Xaa8=Ala、Arg或Glu”(ix)特征:(A)名称/代码:修饰位点(B)位置:9(D)其它信息:/注:“Xaa9=Lys或Glu”(xi)序列描述:SEQ ID NO:85:
Xaa Xaa Leu Xaa Xaa Leu Xaa Xaa Xaa Xaa
1 5 10(2)SEQ ID NO:86的信息:(i)序列特征:
(A)长度:10氨基酸
(B)类型:氨基酸
(D)拓扑:螺旋(ii)分子类型:肽(iii)假设:非(v)片段类型:内部(ix)特征:
(A)名称/代码:修饰位点
(B)位置:1和4
(D)其它信息:/注:“Xaa1和Xaa4=Glu、Glu(OCH3)、His或Pro”(ix)特征:
(A)名称/代码:修饰位点
(B)位置:2
(D)其它信息:/注:“Xaa2=Leu或Phe”(ix)特征:
(A)名称/代码:修饰位点
(B)位置:8
(D)其它信息:/注:“Xaa8=Glu、Lys或Lys(COCH2PEGX)”(xi)序列描述:SEQ ID NO:86:
Xaa Xaa Leu Xaa Arg Leu Xaa Xaa Arg Leu
1 5 10
Claims (11)
1.一种用于治疗骨质疏松症化合物的鼻传递的药物组合物,其中包含有效量的PTH或PTHrp的生理活性截短类似物或它们的盐,其中氨基酸残基(22-31)形成一个两亲α螺旋,所述的残基(22-31)选自(SEQ ID NO:85、86、26、27、28、29和30);吸收增强剂,它选自二甲基-β-环糊精和胆酸表面活性剂;还包含水。
2.根据权利要求1所述的组合物,其中的胆酸表面活性剂选自甘氨胆酸和牛黄胆酸。
3.根据权利要求2所述的组合物,其中的胆酸表面活性剂是牛黄胆酸钠。
4.根据权利要求2所述的组合物,其中的胆酸表面活性剂是甘氨胆酸钠。
5.根据权利要求1所述的组合物,其中PTH或PTHrp类似物的浓度为1毫克/毫升至约100毫克/毫升。
6.根据权利要求1所述的组合物,其中吸收增强剂的浓度为约0.5至约50毫克/毫升。
7.根据权利要求1所述的组合物,其中的PTHrp类似物是化合物(SEQ ID NO:7)。
8.根据权利要求7所述的组合物,其中的吸收增强剂是二甲基-β-环糊精。
9.根据权利要求7所述的组合物,其中的吸收增强剂是牛黄胆酸钠。
10.根据权利要求7所述的组合物,其中的吸收增强剂是甘氨胆酸钠。
11.一种药物组合物,其中包含约1至约100毫克/毫升化合物(SEQ ID NO:7),约0.5至约50毫克/毫升选自二甲基-β-环糊精、甘氨胆酸钠和牛黄胆酸钠的吸收增强剂,以及水。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US08/521,097 US5977070A (en) | 1992-07-14 | 1995-08-29 | Pharmaceutical compositions for the nasal delivery of compounds useful for the treatment of osteoporosis |
US08/521,097 | 1995-08-29 |
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Publication Number | Publication Date |
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CN1193915A true CN1193915A (zh) | 1998-09-23 |
Family
ID=24075345
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Application Number | Title | Priority Date | Filing Date |
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CN96196522A Pending CN1193915A (zh) | 1995-08-29 | 1996-08-22 | 用于治疗骨质疏松症化合物的鼻传递的药物组合物 |
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US (1) | US5977070A (zh) |
EP (1) | EP0858344A2 (zh) |
JP (1) | JPH11511462A (zh) |
CN (1) | CN1193915A (zh) |
AU (1) | AU6958996A (zh) |
BR (1) | BR9610331A (zh) |
CA (1) | CA2230929A1 (zh) |
TR (1) | TR199800335T1 (zh) |
WO (1) | WO1997007815A2 (zh) |
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1995
- 1995-08-29 US US08/521,097 patent/US5977070A/en not_active Expired - Fee Related
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- 1996-08-22 TR TR1998/00335T patent/TR199800335T1/xx unknown
- 1996-08-22 BR BR9610331-0A patent/BR9610331A/pt unknown
- 1996-08-22 CN CN96196522A patent/CN1193915A/zh active Pending
- 1996-08-22 EP EP96930607A patent/EP0858344A2/en not_active Ceased
- 1996-08-22 AU AU69589/96A patent/AU6958996A/en not_active Abandoned
- 1996-08-22 JP JP9510521A patent/JPH11511462A/ja active Pending
- 1996-08-22 WO PCT/US1996/013769 patent/WO1997007815A2/en not_active Application Discontinuation
- 1996-08-22 CA CA002230929A patent/CA2230929A1/en not_active Abandoned
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101489408B (zh) * | 2006-05-19 | 2013-10-30 | 索莱有限责任公司 | 蛋白组合物及其在重组肉制品和食品中的用途 |
CN114057863A (zh) * | 2020-08-06 | 2022-02-18 | 珠海联邦制药股份有限公司 | 一种甲状旁腺激素相关肽类似物及其应用 |
CN114057863B (zh) * | 2020-08-06 | 2023-10-20 | 珠海联邦制药股份有限公司 | 一种甲状旁腺激素相关肽类似物及其应用 |
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US5977070A (en) | 1999-11-02 |
JPH11511462A (ja) | 1999-10-05 |
BR9610331A (pt) | 2005-05-10 |
TR199800335T1 (xx) | 1998-05-21 |
EP0858344A2 (en) | 1998-08-19 |
CA2230929A1 (en) | 1997-03-06 |
AU6958996A (en) | 1997-03-19 |
WO1997007815A3 (en) | 1997-09-18 |
MX9801630A (es) | 1998-08-30 |
WO1997007815A2 (en) | 1997-03-06 |
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