專利名稱:抗小細(xì)胞肺癌多肽混合物的制作方法
技術(shù)領(lǐng)域:
本發(fā)明涉及抗腫瘤的多肽,尤其是關(guān)于抗小細(xì)胞肺癌P物質(zhì)類似物的多肽混合物及其應(yīng)用。
目前治療小細(xì)胞肺癌方法主要是化療,藥物大多是小分子的DNA合成抑制劑,蛋白酶的抑制劑和細(xì)胞周期的抑制劑,這些藥物毒性高,特異性差,殺死腫瘤細(xì)胞的同時也殺死正常組織細(xì)胞。
實驗表明,小細(xì)胞肺癌細(xì)胞株H345和H69在細(xì)胞體外培養(yǎng)時過度稀釋,細(xì)胞密度過低,細(xì)胞會在24小時內(nèi)死亡。這一現(xiàn)象表明小細(xì)胞肺癌細(xì)胞株可能需要通過自分泌和旁分泌激素或活性神經(jīng)肽例如P物質(zhì)等來維持生存,(Sethi T.,et al.,Cancer Res,1992 May 1;52(9 Suppl)2737-2742;Heasley LE.,Oncogene 2001 Mar 26;20(13)1563-9)。在抗小細(xì)胞肺癌多肽藥物研究中,發(fā)現(xiàn)有很多P物質(zhì)的類似物和P物質(zhì)片段的類似物具有抗小細(xì)胞肺癌生長的功能及抑瘤效果顯著的特點如P物質(zhì)類似物(Substance P Analogues)SPDD[D-Arg1,D-Phe5,D-Trp7,9,Leu11]-P物質(zhì)(Nyeki O,et al.,J Pept Sci 1998 Dec;4(8)486-95);P物質(zhì)片段類似物(Substance P Fragment Analogues)SPDG[Arg5,D-Trp7,9,N-Me-Phe8]-P物質(zhì)片段6-11。
SPDG已在英國進(jìn)入臨床二期,(Jones DA.,et al.,BiochemPharmacol 1995 Aug 25;50(5)585-90)。
SPDD不但通過阻斷小細(xì)胞肺癌細(xì)胞通過自分泌和旁分泌神經(jīng)肽得到賴以生存的信號,抑制小細(xì)胞肺癌細(xì)胞株H315和H69生長,而且誘導(dǎo)細(xì)胞胞漿內(nèi)的過氧化物升高,引起H345細(xì)胞株的凋亡。聯(lián)合免疫缺陷小鼠靜脈注射SPDD,可以抑制移植的H315細(xì)胞在小鼠皮下生長。SPDD作為一種抗小細(xì)胞肺癌的多肽藥物,具有藥效顯著,特異性較高,毒副作用小的特點,不失為一種理想的抗腫瘤藥物。但其缺點是使用劑量較大,例如通過移植H69細(xì)胞株的小鼠(20克重),需每天腹膜注射240μgSPDD,(Jone DA,et al.,Peptides 1997;18(701073-7)。因此SPDD使用成本過高,也就限制了其臨床的應(yīng)用及商業(yè)上的開發(fā)。本發(fā)明以SPDD多肽結(jié)構(gòu)為基礎(chǔ)進(jìn)行發(fā)明創(chuàng)新,為了減少劑量,降低使用成本,以利于該多肽藥物的應(yīng)用與開發(fā)。
2.SPDD結(jié)構(gòu)趨同化組合的多肽文庫的多肽混合物(SPDDMIXO)以SPDD多肽結(jié)構(gòu)為基礎(chǔ),SPDD由11個D型或L型氨基酸組成,結(jié)構(gòu)式如下D-Arg1-Pro2-Lys3-Pro4-D-Phe5-Gln6-D-Trp7-Phe8-D-Trp9-Leu10-Leu11-NH2SPDDMIXO就是根據(jù)生物多樣性的原理,利用進(jìn)化上具有結(jié)構(gòu)類似的D型或L型氨基酸取代SPDD上對應(yīng)的氨基酸,具體改變?nèi)缦碌谝晃坏腄-Arg以D-Lys與之對應(yīng)取代;第三位的Lys以Arg與之對應(yīng)取代;第五位的D-Phe以D-Trp與之對應(yīng)取代;第六位的Glu以Asn與之對應(yīng)取代;第七位的D-Trp以D-Phe與之對應(yīng)取代;第八位的Phe以Trp與之對應(yīng)取代;第九位的D-Trp以D-Phe與之對應(yīng)取代;第十位的Leu以lle與之對應(yīng)取代;第十一位的Leu以11e與之對應(yīng)取代;第二位的Pro不變;第四位的Pro也不變。
SPDD取代前后的結(jié)構(gòu)式如下取代前 D-Arg1-Pro2-Lvs3-Pro4-D-Phe5-Gln6-D-Trp7Phe8-D-Trp9-Leu10-Leu11-NH2取代后D-Lys1-Pro2-Arg3-Pro4-D-Trp5-Asn6-D-Phe7-Trp8-D-Phe9-Ile10-Ile11-NH2這樣取代后,就列出了與SPDD結(jié)構(gòu)趨同化的另一條多肽鏈。
在SPDDMIXO人工合成時,將各取自于前述取代前后的二個SPDD多肽鏈的第一、第三、第五、第六、第七、第八、第九、第十、第十一位氨基酸的二個氨基酸同時接上,而第二及第四位的氨基酸僅是一個氨基酸接上,這樣按多肽中的氨基酸位置順序依次接上,通過排列組合就能得到29種的氨基酸排列順序,就是有512條11肽的多肽序列(見序列表)及它們不完整的多肽。512條多肽中,除第一條是SPDD外,其余511條都是與SPDD結(jié)構(gòu)趨同化的新的多肽鏈。同時對最終產(chǎn)物不進(jìn)行提純處理,直接冷凍干燥制得SPDDMIXO。512條多肽的各個肽鏈中11肽占75-96.99重量%,10肽及10肽以下的多肽占3.01-25重量%。SPDDMIXO為白色或微褐色粉末,512條11肽的平均分子量為1510.65,能溶于水。
3.SPDDMIXT和SPDDMIXO兩者混合的多肽混合物(SPDDMM)SPDDMIXT和SPDDMIXO可以以任意比例混合,例如兩者以1∶1重量比的多肽混合物的描述,詳見后面的實施例。
SPDDMIXT的合成工藝SPDD多肽混合物的合成工藝采用常用的固相合成法(參見黃惟德等,多肽合成,科學(xué)出版社,1985年),肽鏈的逐步延長合成是在不溶性的樹脂上進(jìn)行。樹脂用二異丙基乙胺活化,SPDD多肽C端第一氨基酸的氨基用叔丁氧羰基保護(hù)后,以二異丙基碳二亞胺為縮合劑,羧基即以共價鍵結(jié)合到樹脂上。用無水三氟醋酸解離氨基酸保護(hù)基團(tuán)后,多肽C端第二個氨基酸重復(fù)上述步驟循環(huán)合成,使肽鏈按照上述SPDD順序延長合成多肽。多肽合成到最后一步時,把樹脂懸浮在無水三氟乙酸中,通入干燥HBr,使多肽從樹脂上解離下來,同時一些保護(hù)基團(tuán)也被除去,得到的產(chǎn)物即為SPDD多肽混合物,不作提純,直接冷凍干燥,在-20℃下儲存?zhèn)溆谩?br>
SPDD多肽混合物合成工藝流程示意圖參見圖1。
SPDDMIXO的合成工藝SPDD結(jié)構(gòu)趨同化組合的多肽文庫的多肽混合物的合成工藝采用常用的固相合成法(參見黃惟德等,多肽合成,科學(xué)出版社,1985年),肽鏈的逐步延長合成是在不溶性的樹脂上進(jìn)行。將各取自于前述取代前后的二個SPDD多肽鏈的第一、第三、第五、第六、第七、第八、第九、第十、第十一位氨基酸的二個氨基酸同時接上,而第二及第四位的氨基酸僅是一個氨基酸接上。具體做法是樹脂用二異丙基乙胺活化,各取自于取代前后的二個SPDD多肽鏈C端的第一氨基酸的氨基用叔丁氧羰基保護(hù)后,以二異丙基碳二亞胺為縮合劑,這二個氨基酸的羧基即分別以共價鍵同時接合到樹脂上。用無水三氟醋酸解離氨基酸保護(hù)基團(tuán)后,再各自取C端的第二個氨基酸重復(fù)上述步驟循環(huán)合成,這樣按二個多肽鏈的氨基酸位置的逆向順序依次接上去,一直接到N端第四位氨基酸及進(jìn)而第二位氨基酸時,則僅是一個氨基酸接上去。由于在肽鏈順序延長合成過程中,有9次是二個氨基酸同時接上的,因此通過排列組合就得到29種的氨基酸排列順序,就是有512條多肽序列(見序列表)。除第一條是SPDD外,其余511條都是與SPDD結(jié)構(gòu)趨同化的新的多肽鏈。多肽合成到最后一步時,把樹脂懸浮在無水三氟醋酸中,通入干燥的HBr,使多肽從樹脂上解離下來,同時一些保護(hù)基也被除去,得到的產(chǎn)物即為SPDDMIXO,不作提純,直接冷凍干燥,在-20℃下儲存?zhèn)溆谩?br>
SPDDMIXO合成工藝流程示意圖參見圖1。
P物質(zhì)類似物抗腫瘤藥物的抗腫瘤機(jī)制包括兩個特點a)作用于線粒體,引起胞漿內(nèi)過氧化物升高,導(dǎo)致腫瘤細(xì)胞的凋亡;b)拮抗腫瘤細(xì)胞通過自分泌和旁分泌賴以生存的神經(jīng)肽激素,達(dá)到阻止腫瘤細(xì)胞生長,最終腫瘤細(xì)胞失去維持生存所必須的信號而導(dǎo)致死亡。
單一的SPDD能拮抗P物質(zhì)G蛋白受體,即SPDD對P物質(zhì)神經(jīng)肽激素有相對特異性。而本發(fā)明SPDDMIXO中,除第1條是SPDD外,其余511條11肽都是與SPDD結(jié)構(gòu)趨同化的新的多肽鏈,這些多肽鏈也具有SPDD類似的生物學(xué)功能,并且與SPDD有協(xié)同作用,不但可以拮抗P物質(zhì),而且可以拮抗小細(xì)胞肺癌細(xì)胞通過自分泌和旁分泌賴以生存的其他神經(jīng)肽激素,這些神經(jīng)肽激素的受體都是不同但類似的G蛋白偶聯(lián)受體,因而可以提高拮抗效率,是一種相對廣譜性神經(jīng)肽激素的拮抗劑。而且SPDD同時也作用于線粒體,引起胞漿內(nèi)過氧化物升高,直接誘導(dǎo)癌細(xì)胞凋亡。本發(fā)明SPDDMIXT,SPDDMIXO和SPDDMM都是多肽混合物,是在人工合成時不對最終產(chǎn)物進(jìn)行提純處理,直接冷凍干燥而得。該多肽混合物中具有不完整結(jié)構(gòu)的不完全多肽產(chǎn)物,例如10肽,9肽,8肽...等多肽產(chǎn)物,也具有拮抗作用和生物活性,因而也可以提高拮抗效率。本發(fā)明多肽混合物與單一的SPDD相比,拮抗效率可提高6-10倍。
后述的體外細(xì)胞實驗證實本發(fā)明多肽混合物拮抗劑對小細(xì)胞肺癌細(xì)胞有誘導(dǎo)凋亡的作用,對小細(xì)胞肺癌細(xì)胞的生活力有影響,以及對P物質(zhì)受體介導(dǎo)的小細(xì)胞肺癌細(xì)胞的鈣離子信號有抑制作用。其中以SPDDMM 作用最強(qiáng),它們作用大小的順序是SPDDMM>SPDDMIXO>SPDDMIXT>SPDD。體外細(xì)胞試驗還證實了SPDDMIXO和SPDDMM不僅能抑制P物質(zhì)受體介導(dǎo)的小細(xì)胞肺癌細(xì)胞的鈣離子信號,而且還能抑制旁皮素(bombesin)受體介導(dǎo)的小細(xì)胞肺癌細(xì)胞的鈣離子信號,因此它們是一種廣譜性神經(jīng)肽激素的拮抗劑。體內(nèi)動物(小鼠)試驗顯示出本發(fā)明多肽混合物作為多肽拮抗劑時,使用劑量有望減少(尤其是SPDDMM),且因合成工藝簡單,容易生產(chǎn),使生產(chǎn)成本降低,也就降低了使用的成本,從而使臨床應(yīng)用成為可能。本發(fā)明多肽混合物SPDDMIXT,SPDDMIXO和SPDDMM可應(yīng)用于制造抗小細(xì)胞肺癌的多肽藥物。
通過下列附圖和實施例對本發(fā)明作進(jìn)一步闡述,但并不限制本發(fā)明范圍。
圖1是本發(fā)明抗小細(xì)胞肺癌多肽混合物合成工藝流程圖。
圖2是用四種多肽拮抗劑不同劑量處理后的小細(xì)胞肺癌細(xì)胞株H345凋亡的劑量動力曲線。
圖3是用四種多肽拮抗劑不同劑量處理后的小細(xì)胞肺癌細(xì)胞株H345生活力(以MTS光密度表示)的劑量動力曲線。
圖4是四種多肽拮抗劑不同劑量對P物質(zhì)受體介導(dǎo)的小細(xì)胞肺癌細(xì)胞株H345的鈣離子信號抑制的劑量動力曲線。
圖5是本發(fā)明多肽混合物拮抗劑對P物質(zhì)受體介導(dǎo)的小細(xì)胞肺癌細(xì)胞株H345的鈣離子信號的抑制作用。
圖6是本發(fā)明多肽混合物拮抗劑對旁皮素受體介導(dǎo)的小細(xì)胞肺癌細(xì)胞株H345的鈣離子信號的抑制作用。
圖7是四種多肽拮抗劑與對照組在體內(nèi)抑制小鼠H345細(xì)胞腫瘤生長的比較。
實施例2 SPDDMIXO多肽混合物的合成稱取對二甲基二苯甲胺樹脂(0.5-0.7meg)100mg,放入尼龍濾布袋中封口,一起放入50ml三角燒杯中,用5ml二氯甲烷洗滌樹脂,再用5%濃度的二異丙基乙胺每次5ml活化樹脂,處理二次。再用二氯甲烷每次5ml洗滌樹脂,洗滌二次。上述取代前后的二個SPDD多肽鏈的氨基酸順序C末端是Leu(亮氨酸)和Ile(異亮氨酸),用BOC-亮氨酸(叔丁氧羰基-亮氨酸)和BOC-異亮氨酸(叔丁氧羰基-異亮氨酸)的二氯甲烷溶液(0.26mol/l),2.2ml同時和偶聯(lián)縮合劑二異丙基碳二亞胺的二氯甲烷溶液(0.26mol/l)2.2ml在常溫下反應(yīng)4小時,接著用二氯甲烷洗滌樹脂及二甲基甲酰胺洗滌樹脂,用無水三氟醋酸5ml在常溫下反應(yīng)30分鐘,以解離氨基酸保護(hù)基團(tuán),再用二氯甲烷5ml洗滌樹脂及異丙醇5ml洗滌樹酯,以去除殘余的三氟醋酸。再按取代前后的二個SPDD多肽鏈C末端第二個氨基酸為亮氨酸和異亮氨酸,按上述步驟循環(huán)重復(fù)合成,這樣依次反復(fù)進(jìn)行,一直接到N端第四位氨基酸(脯氨酸)進(jìn)而第二位氨基酸(脯氨酸)時,則在反應(yīng)部分,用BOC-脯氨酸(叔丁氧羰基-脯氨酸)的二氯甲烷溶液之外,其余都按所述步驟進(jìn)行,肽鏈就可按SPDDMIXO多肽的順序延長,直到11個氨基酸全部接上去。多肽合成到最后一步時,把樹脂懸浮在無水三氟醋酸中,通入干燥HBr氣體,使多肽混合物與樹脂解離,同時一些保護(hù)基也被除去,該多肽混合物產(chǎn)物不作提純,直接于-20℃冷凍干燥,得到SPDDMIXO多肽混合物凍干粉,-20℃儲存?zhèn)溆谩F渲泄灿?12條多肽序列(見序列表)及它們的10肽及10肽以下的不完全多肽,在多肽混合物中11肽占85重量%,10肽及10肽以下的多肽占15重量%。
實施例3體外細(xì)胞培養(yǎng)體系中,四種多肽拮抗劑誘導(dǎo)小細(xì)胞肺癌細(xì)胞株H345(或H69或H510)凋亡試驗細(xì)胞株3種小細(xì)胞肺癌細(xì)胞株可以用于凋亡測定H345小細(xì)胞肺癌細(xì)胞株;H69小細(xì)胞肺癌細(xì)胞株;H510小細(xì)胞肺癌細(xì)胞株。
對照組用U937單核細(xì)胞株。
H345,H69,H510細(xì)胞株的培養(yǎng)和擴(kuò)增,使用RPMI1640培養(yǎng)液,加5%小牛血清,所有的細(xì)胞株培養(yǎng)在37℃,5%CO2。以下是每一細(xì)胞株理想的培養(yǎng)條件。
U937和細(xì)胞株可以稀釋到104-5×104/ml,生長3天-4天后即可使用。
H69和H345細(xì)胞取決于自分泌和旁分泌作用維持其生存和增殖。因此,細(xì)胞培養(yǎng)中保持一定的細(xì)胞密度非常重要。細(xì)胞必須保持106/ml,細(xì)胞培養(yǎng)稀釋時不要超過1∶4,避免過度稀釋細(xì)胞生長因子。
四種多肽拮抗劑的生物功效活性測定,在準(zhǔn)備測定以前,用顯微鏡觀察細(xì)胞,細(xì)胞必須健康。
1)準(zhǔn)備10x稀釋的96孔U形板,在稀釋時每一孔必須混勻,特別是在作系列稀釋。所有的肽化合物應(yīng)該稀釋在RPMI1640(5-10%小牛血清)培液中。
四種多肽拮抗劑的儲存液濃度是10mmol/l,稀釋在磷酸緩沖液中(PBS)。
11x稀釋四種多肽拮抗劑系列稀釋的第一孔終濃度為500μmol/l,體積為200μl,混合第一孔的溶液,轉(zhuǎn)移100μl到第二孔(100μlRPMI1640,10%小牛血清),在完成1∶1系列稀釋時,轉(zhuǎn)移100μl至第三孔,以此類推完成11x稀釋。稀釋濃度分別為500μmol/l,250μmol/l,125μmol/l,62μmol/l,31μmol/l,15μmol/l,8μmol/l,4μmol/l,2μmol/l,1μmol/l,0.5μmol/l。
2)用多通道移液器,從稀釋板的每一行,轉(zhuǎn)移20μl稀釋液至細(xì)胞培養(yǎng)板的相應(yīng)行中(含有180μl細(xì)胞懸液)以1∶10稀釋,得最終濃度為50μmol/l,25μmol/l,12.5μmol/l,6.2μmol/l,3.1μmol/l,1.5μmol/l,0.8μmol/l,0.4μmol/l,0.2μmol/l,0.1μmol/l,0.05μmol/l。
3)培養(yǎng)48小時37℃,5%CO2。
4)用多通道移液器將細(xì)胞轉(zhuǎn)移至96孔U形培養(yǎng)板,離心1000RMP,1分鐘,吸掉培養(yǎng)液,輕輕拍打培養(yǎng)板,加入冰凍70%酒精200μl,在冰上培養(yǎng)30分鐘,離心,吸掉酒精,加入200μl磷酸緩沖液(含有0.1%小牛血清),離心1000RMP,1分鐘,吸掉緩沖液,加入200μl磷酸緩沖液(含有propidium iodide 50μg/ml,10Unit/ml RNAse[無DNA酶]),然后轉(zhuǎn)移U形板孔內(nèi)的細(xì)胞懸液至流式細(xì)胞儀專用的試管內(nèi),管內(nèi)含有200μl緩沖液(propidium iodide50μg/ml,10Unit/ml RNase[無DNA酶]),保持室溫30分鐘,準(zhǔn)備用流式細(xì)胞儀測定細(xì)胞的DNA容量,對凋亡細(xì)胞進(jìn)行測定,測定結(jié)果見圖2。SPDDMM對小細(xì)胞肺癌細(xì)胞誘導(dǎo)凋亡的作用最強(qiáng),四種多肽拮抗劑作用大小的順序是SPDDMM>SPDDMIXO>SPDDMIXT>SPDD。
實施例4體外細(xì)胞培養(yǎng)體系中小細(xì)胞肺癌細(xì)胞株H345(或H69或H510)的生活力測定細(xì)胞培養(yǎng)和其他操作步驟1)-3)和實施例3相同測定方法如下在3)細(xì)胞培養(yǎng)完成以后對細(xì)胞使用MTS進(jìn)行細(xì)胞生活力測定。
加入40μl MTS (promega)溶液至每一孔內(nèi),把細(xì)胞培養(yǎng)板送培養(yǎng)箱37℃2小時-4小時。用酶標(biāo)儀讀(540nm-655nm)吸光度,測定結(jié)果見圖3。圖3中用光密度的測定值來表示小細(xì)胞肺癌細(xì)胞的生活力。光密度測定值越小,也就表示細(xì)胞生活力越小。四種多肽拮抗劑對小細(xì)胞肺癌細(xì)胞生活力降低作用的順序是SPDDMM>SPDDMIXO>SPDDMIXT>SPDD。
實施例5四種多肽拮抗劑選擇性抑制P物質(zhì)(或旁皮素)受體介導(dǎo)的小細(xì)胞肺癌細(xì)胞株H345(或H69或H510)的鈣離子信號試驗細(xì)胞培養(yǎng)的條件與實施例3相同。收集細(xì)胞,用磷酸緩沖液(無鈣離子,無血清)洗細(xì)胞一次。細(xì)胞密度為106/ml,細(xì)胞懸浮在磷酸緩沖液中(無鈣離子,無血清)。洗一次,加入含有熒光染料吲哚-1的磷酸緩沖液,吲哚-1終濃度為8μmol/l緩沖液中無鈣離子。將試管放入保溫?fù)u床37℃20-30分鐘,讓染料進(jìn)入細(xì)胞。
應(yīng)用備有紫外激光的流式細(xì)胞儀,測定390nm/480nm的發(fā)射光比值,此比值反應(yīng)細(xì)胞內(nèi)鈣離子信號的變化。用1μmol/lP物質(zhì)刺激細(xì)胞,可觀察到細(xì)胞鈣離子信號的變化。分別預(yù)先加入0.1μmol/l,0.3μmol/l,1μmol/l,3μmol/l,10μmol/l四種多肽拮抗劑至細(xì)胞懸液,然后讓細(xì)胞通過流式細(xì)胞儀兩分鐘,再加入1μmol/lP物質(zhì)刺激細(xì)胞,可觀察到細(xì)胞鈣離子信號被抑制。測定結(jié)果見圖4,圖5及圖6。圖4顯示SPDDMM對P物質(zhì)受體介導(dǎo)的小細(xì)胞肺癌細(xì)胞的鈣的抑制作用最強(qiáng),四種多肽拮抗劑作用大小的順序是SPDDMM>SPDDMIXO>SPDDMIXT>SPDD。
P物質(zhì)多肽和旁皮素多肽都是作用于不同的G蛋白偶聯(lián)的受體。圖5及圖6分別以P物質(zhì)和旁皮素作為對照,測定本發(fā)明多肽混合物對P物質(zhì)(或旁皮素)受體介導(dǎo)的小細(xì)胞肺癌細(xì)胞的鈣離子信號的抑制作用,測定結(jié)果顯示SPDD不能抑制旁皮素刺激引起的鈣離子信號,說明SPDD對P物質(zhì)刺激引起的小細(xì)胞肺癌細(xì)胞鈣離子信號的相對特異性抑制。而本發(fā)明SPDDMM和SPDDMIXO不僅能抑制P物質(zhì)刺激引起的細(xì)胞鈣離子信號,也能抑制旁皮素刺激引起的細(xì)胞鈣離子信號,因此它們是相對廣譜性神經(jīng)肽激素的拮抗劑。
實施例6 拮抗劑體內(nèi)抑制小鼠H345細(xì)胞瘤生長的試驗動物聯(lián)合免疫缺陷小鼠(SCID),4個星期小鼠10個,體重20g左右。
操作皮下注射106H345細(xì)胞,細(xì)胞懸浮在磷酸緩沖液(PBS)中,數(shù)天后皮下瘤開始形成,開始對每只小鼠分別腹腔注射四種多肽拮抗劑SPDD 240μg/200μl PBS,SPDDMIXT 160μg/200μlPBS,SPDDMIXO 120μg/200μlPBS,SPDDMM 40μg/200μlPBS為試驗組,另外以注射200μlPBS/小鼠作為對照組,每日注射一次,每日測量皮下腫瘤的大小,整個操作連續(xù)8天。每一種注射需做一組二個小鼠,測定結(jié)果見圖7。圖7中顯示以同樣體重的小鼠作試驗,試驗組對腫瘤生長的抑制作用顯著大于對照組。在圖7中顯示試驗組四種多肽拮抗劑對腫瘤生長的抑制作用差不多,但它們的注射劑量不相同,SPDD注射劑量是SPDDMIXT的1.5倍,是SPDDMIXO的2倍,是SPDDMM的6倍。本試驗表明本發(fā)明多肽混合物作為拮抗劑時,使用劑量有望大大減少,使臨床應(yīng)用成為可能。
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Leu1517.39108、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18109、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39110、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18111、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39112、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18113、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39114、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18115、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39116、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18117、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39118、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18119、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39120、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18121、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39122、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18123、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39124、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18(續(xù)后頁)125、D-Arg ProLysProD-Phe GlnD-TrpPheD-TrpLeu Leu1517.39126、D-Arg ProArgProD-Trp AsnD-PheTrpD-PheIle Ile1532.18127、D-Arg ProLysProD-Phe GlnD-TrpPheD-TrpLeu Leu1517.39128、D-Arg ProArgProD-Trp AsnD-PheTrpD-PheIle Ile1532.18129、D-Arg ProLysProD-Phe GlnD-TrpPheD-TrpLeu Leu1517.39130、D-Arg ProArgProD-Trp AsnD-PheTrpD-PheIle Ile1532.18131、D-Arg ProLysProD-Phe GlnD-TrpPheD-TrpLeu Leu1517.39132、D-Arg ProArgProD-Trp AsnD-PheTrpD-PheIle Ile1532.18133、D-Arg ProLysProD-Phe GlnD-TrpPheD-TrpLeu Leu1517.39134、D-Arg ProArgProD-Trp AsnD-PheTrpD-PheIle Ile1532.18135、D-Arg ProLysProD-Phe GlnD-TrpPheD-TrpLeu Leu1517.39136、D-Arg ProArgProD-Trp AsnD-PheTrpD-PheIle Ile1532.18137、D-Arg ProLysProD-Phe GlnD-TrpPheD-TrpLeu Leu1517.39138、D-Arg ProArgProD-Trp AsnD-PheTrpD-PheIle Ile1532.18139、D-Arg ProLysProD-Phe GlnD-TrpPheD-TrpLeu Leu1517.39140、D-Arg ProArgProD-Trp AsnD-PheTrpD-PheIle Ile1532.18141、D-Arg ProLysProD-Phe GlnD-TrpPheD-TrpLeu Leu1517.39142、D-Arg ProArgProD-Trp AsnD-PheTrpD-Ph Ile Ile1532.18143、D-Arg ProLysProD-Phe GlnD-TrpPheD-TrpLeu Leu1517.39144、D-Arg ProArgProD-Trp AsnD-PheTrpD-PheIle Ile1532.18145、D-Arg ProLysProD-Phe GlnD-TrpPheD-TrpLeu Leu1517.39146、D-Arg ProArgProD-Trp AsnD-PheTrpD-PheIle Ile1532.18147、D-Arg ProLysProD-Phe GlnD-TrpPheD-TrpLeu Leu1517.39148、D-Arg ProArgProD-Trp AsnD-PheTrpD-PheIle Ile1532.18149、D-Arg ProLysProD-Phe GlnD-TrpPheD-TrpLeu Leu1517.39150、D-Arg ProArgProD-Trp AsnD-PheTrpD-PheIle Ile1532.18151、D-Arg ProLysProD-Phe GlnD-TrpPheD-TrpLeu Leu1517.39152、D-Arg ProArgProD-Trp AsnD-PheTrpD-PheIle Ile1532.18153、D-Arg ProLysProD-Phe GlnD-TrpPheD-TrpLeu Leu1517.39154、D-Arg ProArgProD-Trp AsnD-PheTrpD-PheIle Ile1532.18155、D-Arg ProLysProD-Phe GlnD-TrpPheD-TrpLeu Leu1517.39156、D-Arg ProArgProD-Trp AsnD-PheTrpD-Ph Ile Ile1532.18(續(xù)后頁)157、D-Arg ProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39158、D-Arg ProArgProD-TrpAsnD-PheTrp8 D-PheIleIle1532.18159、D-Arg ProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39160、D-Arg ProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18161、D-Arg ProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39162、D-Arg ProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18163、D-Arg ProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39164、D-Arg ProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18165、D-Arg ProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39166、D-Arg ProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18167、D-Arg ProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39168、D-Arg ProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18169、D-Arg ProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39170、D-Arg ProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18171、D-Arg ProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39172、D-Arg ProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18173、D-Arg ProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39174、D-Arg ProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18175、D-Arg ProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39176、D-Arg ProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18177、D-Arg ProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39178、D-Arg ProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18179、D-Arg ProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39180、D-Arg ProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18181、D-Arg ProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39182、D-Arg ProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18183、D-Arg ProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39184、D-Arg ProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18185、D-Arg ProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39186、D-Arg ProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18187、D-Arg ProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39(續(xù)后頁)188、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIleIle1532.18189、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeuLeu1517.39190、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIleIle1532.18191、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeuLeu1517.39192、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIleIle1532.18193、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeuLeu1517.39194、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIleIle1532.18195、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeuLeu1517.39196、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIleIle1532.18197、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeuLeu1517.39198、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIleIle1532.18199、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeuLeu1517.39200、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIleIle1532.18201、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeuLeu1517.39202、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIleIle1532.18203、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeuLeu1517.39204、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIleIle1532.18205、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeuLeu1517.39206、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIleIle1532.18207、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeuLeu1517.39208、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIleIle1532.18209、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeuLeu1517.39210、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIleIle1532.18211、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeuLeu1517.39212、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIleIle1532.18213、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeuLeu1517.39214、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIleIle1532.18215、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeuLeu1517.39216、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIleIle1532.18217、D-Arg ProLysPro D-PheGlnD-TrpPheD-TrpLeuLeu1517.39218、D-Arg ProArgPro D-TrpAsnD-PheTrpD-PheIleIle1532.18(續(xù)后頁)219、D-ArgProLysPro D-PheGln6- D-TrpPheD-TrpLeu Leu1517.39220、D-ArgProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18221、D-ArgProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39222、D-ArgProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18223、D-ArgProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39224、D-ArgProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18225、D-ArgProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39226、D-ArgProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18227、D-ArgProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39228、D-ArgProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18229、D-ArgProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39230、D-ArgProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18231、D-ArgProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39232、D-ArgProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18233、D-ArgProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39234、D-ArgProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18235、D-ArgProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39236、D-ArgProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18237、D-ArgProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39238、D-ArgProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18239、D-ArgProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39240、D-ArgProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18241、D-ArgProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39242、D-ArgProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18243、D-ArgProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39244、D-ArgProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18245、D-ArgProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39246、D-ArgProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18247、D-ArgProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39248、D-ArgProArgPro D-TrpAsnD-PheTrpD-PheIle Ile1532.18249、D-ArgProLysPro D-PheGlnD-TrpPheD-TrpLeu Leu1517.39(續(xù)后頁)250、D-ArgProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18251、D-ArgProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39252、D-ArgProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18253、D-ArgProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39254、D-ArgProArgProD-TrpAsnD-PheTrpD-PheIleIle1532.18255、D-ArgProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1517.39256、D-ArgProArgProD-TrpAsnD-PheTrp8 D-PheIleIle1532.18257、D-LysProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12258、D-LysProArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91259、D-LysProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12260、D-LysProArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91261、D-LysProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12262、D-LysProArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91263、D-LysProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12264、D-LysProArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91265、D-LysProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12266、D-LysProArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91267、D-LysProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12268、D-LysProArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91269、D-LysProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12270、D-LysProArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91271、D-LysProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12272、D-LysProArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91273、D-LysProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12274、D-LysProArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91275、D-LysProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12276、D-LysProArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91277、D-LysProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12278、D-LysProArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91279、D-LysProLysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12280、D-LysProArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91(續(xù)后頁)281、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeu Leu1489.12282、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIle Ile1503.91283、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeu Leu1489.12284、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIle Ile1503.91285、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeu Leu1489.12286、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIle Ile1503.91287、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeu Leu1489.12288、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIle Ile1503.91289、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeu Leu1489.12290、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIle Ile1503.91291、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeu Leu1489.12292、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIle Ile1503.91293、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeu Leu1489.12294、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIle Ile1503.91295、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeu Leu1489.12296、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIle Ile1503.91297、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeu Leu1489.12298、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIle Ile1503.91299、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeu Leu1489.12300、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIle Ile1503.91301、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeu Leu1489.12302、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIle Ile1503.91303、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeu Leu1489.12304、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIle Ile1503.91305、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeu Leu1489.12306、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIle Ile1503.91307、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeu Leu1489.12308、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIle Ile1503.91309、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeu Leu1489.12310、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIle Ile1503.91311、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeu Leu1489.12(續(xù)后頁)312、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91313、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12314、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91315、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12316、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91317、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12318、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91319、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12320、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91321、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12322、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91323、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12324、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91325、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12326、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91327、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12328、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91329、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12330、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91331、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12332、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91333、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12334、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91335、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12336、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91337、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12338、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91339、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12340、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91341、D-LysPro LysProD-PheGlnD-TrpPheD-TrpLeuLeu1489.12342、D-LysPro ArgProD-TrpAsnD-PheTrpD-PheIleIle1503.91(續(xù)后頁)343、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12344、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91345、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12346、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91347、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12348、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91349、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12350、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91351、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12352、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91353、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12354、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91355、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12356、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91357、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12358、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91359、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12360、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91361、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12362、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91363、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12364、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91365、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12366、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91367、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12368、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91369、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12370、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91371、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12372、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91373、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12(續(xù)后頁)374、D-LysProArg ProD-TrpAsnD-Phe TrpD-PheIle Ile1503.91375、D-LysProLys ProD-PheGlnD-Trp PheD-TrpLeu Leu1489.12376、D-LysProArg ProD-TrpAsnD-Phe TrpD-PheIle Ile1503.91377、D-LysProLys ProD-PheGlnD-Trp PheD-TrpLeu Leu1489.12378、D-LysProArg ProD-TrpAsnD-Phe TrpD-PheIle Ile1503.91379、D-LysProLys ProD-PheGlnD-Trp PheD-TrpLeu Leu1489.12380、D-LysProArg ProD-TrpAsnD-Phe TrpD-PheIle Ile1503.91381、D-LysProLys ProD-PheGlnD-Trp PheD-TrpLeu Leu1489.12382、D-LysProArg ProD-TrpAsnD-Phe TrpD-PheIle Ile1503.91383、D-LysProLys ProD-PheGlnD-Trp PheD-TrpLeu Leu1489.12384、D-LysProArg ProD-TrpAsnD-Phe TrpD-PheIle Ile1503.91385、D-LysProLys ProD-PheGlnD-Trp PheD-TrpLeu Leu1489.12386、D-LysProArg ProD-TrpAsnD-Phe TrpD-PheIle Ile1503.91387、D-LysProLys ProD-PheGlnD-Trp PheD-TrpLeu Leu1489.12388、D-LysProArg ProD-TrpAsnD-Phe TrpD-PheIle Ile1503.91389、D-LysProLys ProD-PheGlnD-Trp PheD-TrpLeu Leu1489.12390、D-LysProArg ProD-TrpAsnD-Phe TrpD-PheIle Ile1503.91391、D-LysProLys ProD-PheGlnD-Trp PheD-TrpLeu Leu1489.12392、D-LysProArg ProD-TrpAsnD-Phe TrpD-PheIle Ile1503.91393、D-LysProLys ProD-PheGlnD-Trp PheD-TrpLeu Leu1489.12394、D-LysProArg ProD-TrpAsnD-Phe TrpD-PheIle Ile1503.91395、D-LysProLys ProD-PheGlnD-Trp PheD-TrpLeu Leu1489.12396、D-LysProArg ProD-TrpAsnD-Phe TrpD-PheIle Ile1503.91397、D-LysProLys ProD-PheGlnD-Trp PheD-TrpLeu Leu1489.12398、D-LysProArg ProD-TrpAsnD-Phe TrpD-PheIle Ile1503.91399、D-LysProLys ProD-PheGlnD-Trp PheD-TrpLeu Leu1489.12400、D-LysProArg ProD-TrpAsnD-Phe TrpD-PheIle Ile1503.91401、D-LysProLys ProD-PheGlnD-Trp PheD-TrpLeu Leu1489.12402、D-LysProArg ProD-TrpAsnD-Phe TrpD-PheIle Ile1503.91403、D-LysProLys ProD-PheGlnD-Trp PheD-TrpLeu Leu1489.12404、D-LysProArg ProD-TrpAsnD-Phe TrpD-PheIle Ile1503.91(續(xù)后頁)405、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12406、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91407、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12408、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91409、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12410、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91411、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12412、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91413、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12414、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91415、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12416、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.914l7、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12418、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91419、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12420、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91421、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12422、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91423、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12424、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91425、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12426、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91427、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12428、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91429、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12430、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91431、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12432、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91433、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12434、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91435、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12(續(xù)后頁)436、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91437、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12438、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91439、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12440、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91441、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12442、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91443、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12444、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91445、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12446、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91447、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12448、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91449、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12450、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91451、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12452、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91453、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12454、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91455、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12456、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91457、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12458、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91459、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12460、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91461、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12462、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91463、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12464、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91465、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12466、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91(續(xù)后頁)467、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12468、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91469、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12470、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91471、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12472、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91473、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12474、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91475、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12476、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91477、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12478、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91479、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12480、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91481、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12482、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91483、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12484、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91485、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12486、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91487、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12488、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91489、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12490、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91491、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12492、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91493、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12494、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91495、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12496、D-LysProArg Pro D-TrpAsnD-PheTrpD-PheIleIle1503.91497、D-LysProLys Pro D-PheGlnD-TrpPheD-TrpLeuLeu1489.12(續(xù)后頁)498、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91499、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12500、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91501、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12502、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91503、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12504、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91505、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12506、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91507、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12508、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91509、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12510、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91511、D-Lys Pro Lys Pro D-Phe Gln D-Trp Phe D-Trp Leu Leu 1489.12512、D-Lys Pro Arg Pro D-Trp Asn D-Phe Trp D-Phe Ile Ile 1503.91512條多 1510.65肽平均分子量
權(quán)利要求
1.一種抗小細(xì)胞肺癌P物質(zhì)類似物的多肽混合物,其特征在于該多肽混合物具有三種形式(1)SPDD([D-Arg1,D-Phe5,D-Trp7,9,Leu11]-P物質(zhì))的多肽混合物,其中11肽占75~96.99重量%,10肽及10肽以下的多肽占3.01~25重量%;(2)SPDD結(jié)構(gòu)趨同化組合的多肽文庫的多肽混合物SPDD多肽結(jié)構(gòu)取代前為D-Arg1-Pro2-Lys3-Pro4-D-Phe5-Gln6-D-Trp7-Phe8-D-Trp9-Leu10-Leu11-NH2取代后為D-Lys1-Pro2-Arg3-Pro4-D-Trp5-Asn6-D-Phe7-Trp8-D-Phe9-Ile10-Ile11-NH2在人工合成多肽時,將各取自于取代前后的二個SPDD多肽鏈的第一、第三、第五、第六、第七、第八、第九、第十、第十一位氨基酸的二個氨基酸同時接在樹脂上,而第二及第四位的氨基酸僅是一個氨基酸接上,這樣按多肽中氨基酸位置順序依次接上,通過排列組合就得到29即512條11肽的多肽序列(SPDD多肽除外),以及它們的10肽及10肽以下多肽的多肽混合物,其中11肽占75~96.99重量%,10肽及10肽以下的多肽占3.01~25重量%;(3)SPDD的多肽混合物和SPDD結(jié)構(gòu)趨同化組合的多肽文庫的多肽混合物兩者混合的多肽混合物。
2.按權(quán)利要求1所述的多肽混合物,其特征在于所述SPDD結(jié)構(gòu)趨同化組合的多肽文庫的多肽混合物中512條11肽的多肽序列(SPDD除外)見序列表。
3.權(quán)利要求1所述多肽混合物的應(yīng)用,其特征在于所述多肽混合物在制備抗小細(xì)胞肺癌的多肽藥物中的應(yīng)用。
全文摘要
一種抗小細(xì)胞肺癌P物質(zhì)類似物的多肽混合物有三種形式(1)SPDD多肽混合物,其中11肽占75~96.99重量%,10肽及10肽以下的多肽占3.01~25重量%;(2)SPDD結(jié)構(gòu)趨同化組合的多肽文庫的多肽混合物,是包含有512條11肽的多肽序列以及他們的10肽及10肽以下多肽的多肽混合物,其中11肽占75~96.99重量%,10肽及10肽以下的多肽占3.01~25重量%;(3)前兩者混合的多肽混合物。在合成多肽時,不對最終產(chǎn)物進(jìn)行提純處理,直接冷凍干燥制得。作為拮抗劑,其拮抗效率是單一的SPDD的6~10倍,使用劑量有望大大減少,且合成工藝簡單,生產(chǎn)和使用成本都降低,這樣使本發(fā)明多肽混合物的臨床應(yīng)用成為可能,可用于制備抗小細(xì)胞肺癌的多肽藥物。
文檔編號A61K38/16GK1405184SQ01126429
公開日2003年3月26日 申請日期2001年8月9日 優(yōu)先權(quán)日2001年8月9日
發(fā)明者王志勤, 賀堅慧 申請人:上海億康生物化學(xué)工程有限公司