CN101219217A - 佐剂组合物及其使用方法 - Google Patents
佐剂组合物及其使用方法 Download PDFInfo
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- CN101219217A CN101219217A CNA200810008718XA CN200810008718A CN101219217A CN 101219217 A CN101219217 A CN 101219217A CN A200810008718X A CNA200810008718X A CN A200810008718XA CN 200810008718 A CN200810008718 A CN 200810008718A CN 101219217 A CN101219217 A CN 101219217A
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- AOBORMOPSGHCAX-DGHZZKTQSA-N tocofersolan Chemical compound OCCOC(=O)CCC(=O)OC1=C(C)C(C)=C2O[C@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C AOBORMOPSGHCAX-DGHZZKTQSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
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- 125000003774 valeryl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
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Abstract
本文讨论了并要求保护一种稳定的水包油乳液的佐剂组合物，其中包括新陈代谢油、一种或多种表面活性剂、抗抗氧化剂以及使该乳液等渗的成分。该稳定的乳液的亲水－亲脂平衡值(HLB)为约7.5－10.5，粒径不到3微米。在优选的实施方案中，该稳定乳液包括10％体积/体积的角鲨烯、0.09％重量/体积的PLURONIC F68嵌段共聚物、1.9％重量/体积的蛋磷脂酰胆碱、1.75％体积/体积的甘油以及0.05％重量/体积的α－生育酚。优选的乳液的HLB为8.0，粒径约为0.2微米。在特别优选的实施方案中，该稳定的乳液同减弱的脂质A衍生物如单磷酰基脂质A或3－脱酰的单磷酰基脂质A结合，可以增强该组合物的辅佐性。
Description
本申请是申请号为99807591.4、申请日为1999年5月7日、发明名称为“佐剂组合物及其使用方法”的中国专利申请的分案申请。
疫苗包括抗原或混合抗原，当将其给予温血动物时可以防止、改善或治疗疾病。传染病的疫苗原来包括整个的减弱的或杀死的微生物。然而很快发现，微生物或细胞中只有少量的蛋白质或蛋白质片段刺激保护的免疫反应，事实上，整个细胞外来物质的包含会阻碍免疫反应。因此，疫苗的开发集中于找出会导出保护性免疫反应的具体的蛋白质、蛋白质片段和编码该抗原决定部位的DNA片段。但是，随着抗原鉴定越来越精确，疫苗的效率下降。鉴定出的抗原常是不能由抗原处理细胞识别的小分子。因此需要将这些抗原与能增强该抗原的抗原性以及给予卓越的免疫反应的物质结合起来。这些物质就是佐剂。
佐剂的作用有几种方法。某些帮助将抗原表给予抗原处理细胞(APC)。水包油乳液、油包水乳液、微脂粒和微玻璃细珠都帮助将抗原给予APC。小的抗原或半抗原常连接到更大的致免疫的蛋白质或多糖以促进由APC识别。某些佐剂具有将抗原就地固定直到身体有机会实现免疫反应的储存效果。其它的佐剂刺激免疫系统一般是增大实现抗原上的特异反应。
减弱的脂质A衍生物(ALD)单磷酰基脂质A(MLA)和3-脱酰的单磷酰基脂质A(3D-MLA)是用于传染病的预防性疫苗和治疗恶性肿瘤和慢性感染的治疗疫苗的有效的免疫的佐剂。MLA和3D-MLA是细菌内毒素脂多糖(LPS)的改性形式，是众所周知的，并分别在美国专利4,436,727和4,912,094中讨论过。MLA和3D-MLA既能诱发服用具有抗原的该化合物的患者体液的抗体反应也诱发细胞-媒介的免疫反应。
有效的疫苗给予温血动物以抗原，使得动物能实现对这些抗原的保护的免疫反应。为实现这种作用，疫苗常需要包括佐剂，这些佐剂既刺激体液的和细胞的免疫反应并且是安全和无毒的，还会小、增进任何疫苗的效果。
本发明是新佐剂组合物。这种佐剂是包括有新陈代谢的油、表面活性剂、抗氧化剂和使该乳液等渗的成分的稳定的水包油乳液(SE)。这种稳定的乳液的粒径是不到130毫微米-3微米。70-200毫微米的乳液可以通过过滤杀菌。这种稳定乳液的亲水-亲脂平衡值约为7.5-10.5，优选约为8.0。
在优选的实施方案中，将佐剂组合物同减弱的脂质A衍生物(ALD)混合。ALD的加入增加了该组合物的辅佐性。可用于本发明的ALD包括单磷酰基脂质A和3-脱酰的单磷酰基脂质A。ALD可以浓度为约1微克-12,000微克/毫升包括在配方中。新的稳定的乳液的疫苗组合物也属于权利要求中。
本发明是稳定的水包油乳液的佐剂组合物，其中包括新陈代谢的油、表面活性剂、抗氧化剂以及使乳液等渗的成分。所得到的乳液经缓冲，具有粒径不到3微米，这种稳定乳液的亲水-亲脂平衡值约为7.5-10.5，优选为约8.0。
在优选的实施方案中，这种稳定的乳液包括约2％-15％，优选约为10％体积/体积的新陈代谢的油角鲨烯。在稳定乳液中约有2％表面活性剂。在本发明的稳定乳液中可以加入约50微克的抗氧化剂和约1.75％的使乳液等渗的试剂。
本发明可以使用的新陈代谢的油包括角鲨烯、豆油、芝麻油和MIGLYCOL 810油。角鲨烯是优选的。
本发明可以使用的表面活性剂是Tween 80、CAMPUL PEO-O低PV表面活性剂(ABITEC Corp.，Janesville，WI)、SOLITOL HS15表面活性剂(BASF Corp.，Chicago，IL)以及PLURONIC F68嵌段共聚物(BASFCorp.，Chicago，IL)、胆酸钠、甘油脱氧胆酸盐、磷脂酰胆碱，其中PLURONIC F68嵌段共聚物是优选的。已经发现，将Tween 80和CAMPULPEO-O低PV表面活性剂静脉内给狗时产生组胺型反应。其它适宜的表面活性剂包括神经鞘脂如神经磷脂、神经鞘氨醇，以及磷脂如蛋磷脂酰胆碱、1，2-二肉豆寇酰-顺-丙三氧基-3-.磷酸乙醇胺、L-α-磷脂酰乙醇胺以及1，2-二棕榈酰-顺-丙三氧基-3-磷酸胆碱(DPPC)或它们的混合物。DPPC可以用于人类。
本发明的稳定的乳液中所用的抗氧化剂包括α-生育酚和抗坏血酸，其中α-生育酚是优选的。
可以加到本发明的乳液中使佐剂等渗的试剂包括葡萄糖、甘油、甘露醇、山梨醇、PEG 300、PEG 400以及聚乙二醇。其中，甘油是优选的。
在特别优选的实施方案中，将减弱的脂质A衍生物(ALD)加到本发明的组合物中。ALD是象脂质A一样的分子，并已经改变或建造成使该分子显示较少的或不同的脂质A的不利的效应。这些不利的效应包括致热原性、局部Shwarzman反应性和毒性，如在鸡胚胎50％致死剂量试验(CELD50)中评价的。在本发明中所用的ALD包括单磷酰基脂质A(MLA)和3-脱酰的单磷酰基脂质A(3D-MLA)。MLA和3D-MLA是众所周知的，在此不需要详细讨论。例如可参看美国专利1984年3月13日发布并转让给Ribi immunoChem Research，Inc.的美国专利4,436,727，这篇专利公开了单磷酰基脂质A及其制造。同样转让给RibiimmunoChem Research，Inc.的Myers等人的美国专利4,912,094以及再审查证书B14,912,094中包括了3-脱酰的单磷酰基脂质A以及其制法。这些有关MLA和3D-MLA的专利内容在此引为参考。
不用讨论这些参考专利文献的细节，这里所用的单磷酰基脂质A(MLA)衍生自脂质A，这是肠杆菌脂多糖(LPS)的成分，是有效的但高毒性的免疫系统调制器。Edgar Ribi和它们的同事实现了原称为精制的脱毒内毒素(RDE)的单磷酰基脂质A(MLA)的生产。MLA是通过在中度强度(0.1当量的HCl)的无机酸溶液中将由革兰阴性细菌的无庚糖突变体得到的内毒素浸出物(LPS或脂质A)回流约30分钟而得到。这种处理造成还原的末端葡(萄)糖胺1位的磷酸盐部分的失去。
同时，在处理中从非还原的葡(萄)糖胺的6位上除去芯碳水化合物。得到的产物(MLA)显示出通常与内毒素原料有关的内毒素活性(如致热源性、局部Shwarzman反应性以及如在鸡胚胎50％致死剂量试验(CELD50)评价的毒性)的大幅度的减弱。但是，未预料地保持了作为免疫调制剂的脂质A和LPSD功能。
用在本发明实际的另一种脱毒的内毒素称为3-脱酰的单磷酰基脂质A(3D-MLA)。3D-MLA在美国专利4,912,094、再审查证书B14,912,094中提到，它不同于MLA之处是，从MLA分子上选择地除去的B-羟基肉豆蔻酰残基是在对其它基团不产生不利影响的条件下连接到还原的末端葡(萄)糖胺3位的酯。3-脱酰的单磷酰脂质A可以从RibiImmunoChem Research，Inc.，Hamilton Montana 59840得到。
MLA和3D-MLA分子是许多脂肪酸取代基方式(即庚酰基、己酰基、戊酰基等具有不同的脂肪链长度)的复合和混合物。因此，本发明包括各种形式的MLA和3D-MLA，包括他们的混合物。在美国专利--094中举例的的脂质A主链相当于由S.minnesota R595的庚酰基脂质A经3-脱酰得到的产物。这篇专利包括其它脂肪酸取代方式，主要特征为材料是3-脱酰的。
用在本发明中的改性的3D-MLA的制备是将MLA在只有脂质A的主链的3位失去单一的脂肪酸的条件下进行碱水解。β-羟基肉豆蔻酸的3位在碱性介质中非常不稳定。只需很温和的碱处理就使脂质A完全3-脱酰，在发生水解前，需要稍强的条件使脂质A中的其它的酯键才能选择性地3位脱酰而不会明显响应该分子的其他部分。酯连接的β-羟基肉豆蔻酸3位在碱介质中的不寻常的敏感性的原因现在还不清楚。
尽管碱性水解的方法是熟知的，但是重要的是，选择除了在3位的酯键生成成β-羟基肉豆蔻酸外而不引起进一步水解的条件。
一般说来，水解可以在水或有机介质中进行。在后者情况下，溶剂包括甲醇(醇类)、二甲基亚砜(DMSO)、二甲基甲酰胺(DMF)、氯仿、二氯甲烷等以及他们的混合物。也可以使用水同一种或多种上述的有机溶剂的混合物。
碱可以选自各种氢氧化物、碳酸盐、磷酸盐以及胺。举例的碱包括无机碱如氢氧化钠、氢氧化钾、碳酸钠、碳酸钾、碳酸氢钠、碳酸氢钾等以及有机碱如烷基胺，包括但不限于二甲胺、三乙胺等。
在含水介质中，pH一般约介于10和14之间，而12-13.5是优选的。水解反应的温度一般约为20-80℃，优选约为50-60℃，反应时间约为10-30分钟。例如，水解可以在室温(22-25℃)下在3％的三乙胺水溶液中进行48小时。唯一的对水解温度和时间选择的要求是脱酰发生时只在3位除去β-羟基肉豆蔻酰。
在实际上已经发现，特别希望的水解方法包括将脂质A或单磷酰基脂质A溶解在用由0.5摩尔Na2CO3组成的pH 10.5的缓冲水溶液饱和的氯仿∶甲醇2∶1中，然后在吸气器(约100毫米汞柱)中真空下在40-50℃将溶剂闪蒸。将得到的材料在3位选择脱酰。这种方法也能用上述的任何无机碱进行。在某些情况下，在用缓冲水溶液饱和前，可以在有机溶液中加入相转移催化剂，如四丁基溴化铵。除了上述生产的MLA和3D-MLA外，也可以使用由合成或半合成制造的ALD。
本发明的组合物是佐剂。当给予有蛋白质抗原的患者以有效量的该组合物时，增强了患者对这种抗原的免疫反应。所谓的有效量的佐剂组合物是能刺激或增进免疫反应的量。熟悉该领域的人员知道需要刺激对这种抗原免疫反应的抗原的量。例如，2.5微克的乙肝表面抗原(HBsAg)服用本发明的优选的实施方案可诱导出鼠的体液反应。
已经意外地发现，本发明的稳定的乳液同ALD混合时，可以明显降低ALD的致热原性。致热原性是由化合扬产生的发烧态。ALD、3D-MLA当同在40％聚乙二醇和10％乙醇中配制与由本发明的稳定的乳液配制相比，产生更高的发烧反应。组合物的致热原性可以以标准的三支兔USP热原试验评价。简言之，将化合物以不同的剂量给予三支兔。在4小时中，检测每支动物的体温。任何温度的下降记录作0。个别的温度升高不超过0.5被认为非热原的。如果该组合物引起温度的升高为0.5或更高，则用五支不同的兔再次对该组合物重新试验。如果总数八支兔中有不多于3支显示温度升高0.5或更高，以及如果八支兔中的每支的温度升高的总和不超过3.3，则该组合物被认为是非热原的。
下面的实施例说明实现本发明的方法。除非指出，则所有的百分数是指重量，所有的溶剂混合比例是指体积。
实施例1-3D-MLA/SE的制备
在特别优选的实施方案中，本发明的稳定的乳液包括下列物质：
材料 量
3D-MLA 1.200-0.005％重量/体积
角鲨烯 10.000％体积/体积
PLURONIC-F68嵌段共聚物 0.091％重量/体积
蛋磷脂酰胆碱 1.909％重量/体积
甘油 1.800％体积/体积
α-生育酚 0.050％重量/体积
注射用水 78.200％体积/体积
磷酸铵缓冲液 10.000％体积/体积
对熟悉该领域的人员显然知道如何制备上述溶液。但是我们发现，制备上述溶液最容易的是调制三种原料溶液：MLA/蛋磷脂酰胆碱原料、油原料溶液和原料水溶液。
为制备MLA/蛋磷脂酰胆碱原料，将3-脱酰的单磷酰基脂质A(3D-MLA)(Ribi ImmunoChem Research，Inc.Hamilton，MT)和蛋磷脂酰胆碱(egg PC)都溶解于4∶1氯仿∶甲醇(C∶M)中。然后将两种溶液混合，使C∶M蒸发。通过将此混合物放在冷冻干燥器中，在减压下放置约1-2小时，以除去剩余的溶剂C∶M。
通过将α-生育酚同角鲨烯混合并在热水浴中旋转直到溶解以制备油相原料溶液。
将PLURONIC F-68NF嵌段共聚物和甘油称重到螺帽瓶中，以制备水相原料溶液。在加温瓶中加入注射用水，并轻轻混合直到各成分溶解。将0.25摩尔的pH约5.1±0.005的磷酸胺缓冲液加到PLURONICF-68NF嵌段共聚物/甘油/水中。再加入水使达到所要求的体积。
为制备稳定的乳液，将原料油相和MLA/蛋磷脂酰胆碱混合物混合。将该混合物经声波处理直到MLA溶解。然后将油相加热到75℃，同时将水相加热到75℃。用Silverson乳化器将油相MLA/egg PC混合物乳化，同时缓慢加入水相。得到的SE乳液的HLB为8.0，在冰浴中冷至室温。在阀门压力22,000-25,000磅/平方英寸下用Avestin C-50均化器将乳液进一步均化到粒径达到≤0.2微米。最后的产物佐剂用0.2微米的亲水隔膜滤器过滤。进一步均化和最后的滤器消毒步骤不会影响组合物中的3D-MLA的量以及制备的辅佐性。
实施例2-用3D-MLA/SE产生抗体反应
在0天用实施例1中制备的佐剂配制的2.5微克乙肝表面抗原(HBsAg)使鼠初次免疫(初次免疫)，经静脉内注射(每次注射200微升)。在第21天使鼠得到第二次免疫(二次免疫)(每次注射200微升)。初次免疫后的第19天(第一次免疫后19天)和在二次免疫后第27天(第二次免疫后第27天)将鼠抽血。收集血清，用标准ELISA检测抗HBsAg抗体。表1表明，本发明的组合物在给予动物该抗原时在动物中诱导产生抗HBsAg抗体。
表1
用3D-MLA/SE产生的抗HBsAg的抗体滴定度
| 佐剂 | 3D-MLA微克 | 抗HBsAg滴定度-1 | ||||
| IgG1特异的 | IgG2a特异的 | |||||
| 初次免疫 后19天 | 二次免疫 后27天 | 初次免疫 后19天 | 二次免疫 后27天 | |||
| 预稀释1/10，第7天 | 3D-MLA/SE | 50 | 16K* | 256K | 64K | 1000K |
| 3D-MLA/SE | 25 | 32K | 512K | 64K | 1000K | |
| 3D-MLA/SE | 5 | 16K | 256K | 16K | 512K | |
| 3D-MLA/SE | 1 | 8K | 256K | 16K | 256K | |
| SE(媒介) | 0 | 4K | 128K | 2K | 64K | |
| PBS | 0 | 2K | 32K | 2K | 64K | |
| 稀释1/10，第0天 | 3D-MLA/SE3D-MLA/SE3D-MLA/SE3D-MLA/SESE(媒介)PBS正常血清 | 50255100- | 16K16K8K4K2K1K＜0.5K | 256K256K128K128K128K128K＜0.5K | 64K64K32K16K1K2K＜0.5K | 1000K1000K512K256K64K32K＜0.5K |
*K＝103
实施例3-细胞毒素的T-淋巴细胞反应的刺激
A)在服用本发明佐剂组合物和蛋白质抗原后用细胞毒素试验检测细胞毒素T-淋巴细胞(CTL)反应的诱导。C57/BL/6鼠各组用在本发明的稳定的乳液媒介(SE)和3D-MLA/SE中配制的1.0微克乙肝表面抗原给予静脉内初步免疫(腹股沟)。如在实施例1中制备MLA/SE佐剂。为试验本发明乳液的稳定性，在将其同抗原混合之前七天或在免疫之日将乳液稀释1/10。注射的体积为200微升。十四天后，杀死每试验组的三支鼠，并除去脾，作为单一细胞悬浮液集中并计数。在每组剩下的鼠中用在SE媒介和3D-MLA/SE佐剂中配制的1.0微克HbsAg给予静脉内二次免疫(腹股沟)。十四天后，将每试验组中所有的鼠杀死，除去脾，作为单一细胞的悬浮液集中并计数。
将试验各组的脾细胞(在3-4毫升介质中75X106细胞)放在25平方厘米的T烧瓶中。接着，将1.0毫升的经辐照过(20,000拉德)的E.G7(OVA)以5X106/毫升加到烧瓶中。使体积达到10毫升。通过将T-烧瓶直立放在37℃和5％CO2的恒温箱中培育4天。在第4天，从烧瓶中回收存活的细胞，洗1次，重新悬浮在5.0毫升中并计数。
将回收的效应细胞调节成5X106活细胞/毫升，用100微升/井的介质作为稀释剂在96孔的圆底盘(Corning 25850)将100微升体积依序稀释三倍。接着，在100微升体积的51Cr-标记的(见下面)靶[E.G7(OVA)-卵清蛋白基因感染的EL-4细胞系]以1X105细胞/毫升加入到井中。自发释放(SR)井含100微升的靶和100微升的介质。最大释放(MR)井含100微升的靶和100微升的洗涤剂(2％Tween20)。效应器/靶(E/T)比为50∶1、25∶1、12.5∶1、6.25∶1。将盘在400倍重力加速度下离心，并在37℃、5％CO2下培育4小时.培育后，用Skatron上清液收集系统收集井上清液。
靶细胞E.G7(OVA)用51Cr(铬酸钠)进行如下的标记。在总体积1.0毫升中，在15毫升的圆锥形管中将5X106靶细胞和250微居里的51Cr混合。将细胞悬浮液在37℃水浴中培育90分钟，每15分钟轻轻混合一次。培育后，标记的细胞通过离心和倾析用15毫升的介质体积洗3次。在第三次离心后，将细胞重新悬浮在10毫升新鲜介质中，并在室温放置30分钟，然后离心。最后将细胞重新悬浮在介质中达1X105细胞/毫升。细胞毒素试验的结果表示在表2和3中。
表2
初次免疫后14天细胞毒性反应
| 佐剂 | MLA微克 | ％细胞毒性E/T | ||||
| 50∶1 | 25∶1 | 12.5∶1 | 6.25∶1 | |||
| 预稀释第7天 | 3D-MLA/SE3D-MLA/SE3D-MLA/SE3D-MLA/SESE(介质)PBS | 50255100 | 44362927257 | 30171313135 | 18119772 | 764340 |
| 稀释第0天 | 3D-MLA/SE3D-MLA/SE3D-MLA/SE | 50255 | 214822 | 123614 | 5249 | 3134 |
| 3D-MLA/SE | 1 | 25 | 14 | 7 | 3 | |
| SE(媒介)PBS | 00 | 248 | 113 | 61 | 30 | |
| 正常 | -- | 6 | 3 | 2 | 0 | |
表3
二次免疫后第14天°细胞毒性反应
| 佐剂 | MLA微克 | ％细胞毒性E/T | ||||
| 50∶1 | 25∶1 | 12.5∶1 | 6.25∶1 | |||
| 预稀释第7天 | 3D-MLA/SE3D-MLA/SE3D-MLA/SE3D-MLA/SE | 502551 | 89796444 | 65644530 | 41402718 | 2523168 |
| SE(媒介)PBS | 00 | 6528 | 3918 | 3110 | 186 | |
| 稀释第0天 | 3D-MLA/SE3D-MLA/SE3D-MLA/SE3D-MLA/SE | 502551 | 80778663 | 56486836 | 39314323 | 26182811 |
| SE(媒介)PBS | 00 | 6317 | 4212 | 287 | 144 | |
| 正常 | -- | 7 | 2 | 0 | 0 | |
B)服用3D-MLA/SE和蛋白质抗原在处理的鼠中诱导出细胞毒素T-淋巴细胞反应和抗原产生。在0天(初次免疫)和21天(二次免疫)用2.0微克的HbsAg+2.5微克3D-MLA/SE使BALB/c鼠皮下免疫。如下面一样进行CTL试验。3D-MLA/SE佐剂如实施例1一样的制备。表4说明诱发细胞毒素T一淋巴细胞反应
表4
细胞毒素反应
| 佐剂 | 天 | ％细胞毒素的反应E/T | |||
| 50∶1 | 25∶1 | 12.5∶1 | 6.25∶1 | ||
| 3D-MLA/SE媒介SE | 初次免疫后17天 | 5532 | 2716 | 149 | 106 |
| 3D-MLA/SE媒介SE | 二次免疫后16天 | 8138 | 6223 | 4714 | 248 |
对HbsAg的抗体滴定度的结果示于表5。在二次免疫后28天取的血液的血清在涂以HbsAg或涂以28个氨基酸的多肽(p72)的ELISA盘进行滴定，该多肽在S区含HbsAg的B-细胞抗原决定部位的110-137的残基。
表5
在经处理的鼠中抗-肝炎抗体的滴定度
| 抗-HBsAg滴定度-1 | ||||
| HBsAg | p72-多肽 | |||
| 佐剂3D-MLA/SE媒介SE正常鼠的血清 | IgG11024K1024K＜0.5K | IgG2a2048K64K＜0.5K | IgG164K64K＜0.5K | IgG2a256K4K＜0.5K |
用3D-MLA/SE处理过的鼠对乙肝表面抗原既显示出体液的也显示出细胞毒素T-淋巴细胞的反应。
实施例4-3D-MLA/SE致热原性评价
3D-MLA/SE佐剂在标准的三兔USP热原试验(NAMSA，Northwood OH)中进行了评价。本发明的3D-MLA/SE佐剂同在40％丙二醇(PG)和10％乙醇(EtOH)中配制的3D-MLA(经过两次试验在剂量5、8、11、14、15、17、20、25、30、35微克/公斤下进行了评价)进行了对比。3D-MLA/SE配方经过两次试验用同样的兔热原试验中在剂量75、100、125、150、200、250、300、350微克/公斤下进行了评价。热原剂量和边界热原剂量由建立的USP说明中定义。边界热原剂量是三支兔中至少一支经三小时的施加剂量后具有峰值温度超过上述边界值≥0.5的剂量。
表6A
在40％PG/10％EtOH中的3D-MLA的致热原性最大的温度升高
| 剂量 | 兔 | 兔 | 兔 | 总计 |
| 微克/公斤 | 1 | 2 | 3 | ℃ |
| 35353025201715141185 | 1.11.10.90.70.50.40.40.40.10.00.1 | 0.60.90.61.10.60.30.30.30.20.10.0 | 0.92.40.70.90.60.30.40.30.10.10.0 | 2.64.32.22.71.71.01.11.00.40.20.1 |
表6B
3D-MLA的致热原性最大温度升高
| 剂量 | 兔 | 兔 | 兔 | 总计 |
| 微克/公斤 | 1 | 2 | 3 | ℃ |
| 35035030025020020015015012510075 | 0.70.30.80.41.00.20.30.10.10.10.0 | 0.60.60.70.51.00.10.20.30.10.20.1 | 0.80.60.60.31.10.10.20.20.10.10.1 | 2.11.52.11.23.10.40.70.60.10.40.2 |
进一步评价了3D-MLA的热原性并同在10％EtOH中配制的3D-MLA进行了对比。
表7
3D-MLA/SE与在10％EtOH中的3D-MLA的致热原性最大的温度升高
| 材料 | 剂量 | 兔 | 兔 | 兔 | 总计 |
| 微克/公斤 | 1 | 2 | 3 | ℃ | |
| 3D-MLA/Et0H3D-MLA/Et0H3D-MLA/EtOH3D-MLA/EtOH3D-MLA/EtOH3D-MLA/SE3D-MLA/SE3D-MLA/SE3D-MLA/SE3D-MLA/SE3D-MLA/SE3D-MLA/SE3D-MLA/SE3D-MLA/SE3D-MLA/SE | 1052.52.51.252001501005075503520100 | 1.50.70.30.10.00.60.50.20.00.10.00.30.00.00.4 | 1.50.90.40.20.00.60.20.30.00.10.20.10.00.00.0 | 1.50.40.10.10.01.00.40.10.00.30.00.10.00.10.0 | 4.52.00.80.40.02.21.10.60.00.50.20.50.00.10.4 |
是边界热原剂量的20微克/公斤剂量的在40％PG/10％EtOH中的3D-MLA和定义为阀热原剂量的剂量的200微克/公斤的3D-MLA/SE具有十倍的差别。3D-MLA/SE佐剂比其它配制的该化合物的热原性低的多(表6和7)。同样，本发明的佐剂组合物是安全的，在注射位置产生的与药物有关的伤害很低甚至没有，淋巴节将注射部位和脾脏引流。
应该理解，这里讨论的实施例和实施方案只是为了说明，对熟悉该领域的人可以提出各种改进和变化，这些改进和变化将包括在本申请的精神实质和范围以及所附的权利要求范围内。
Claims (24)
1.一种佐剂组合物，该组合物包括新陈代谢油、一种或多种表面活性剂、抗氧化剂以及使所说的组合物等渗的成分。
2.权利要求1的组合物，其中所说的新陈代谢油选自角鲨烯、豆油、芝麻油和MIGLYOL 810油。
3.权利要求1的组合物，其中所说的新陈代谢油是角鲨烯。
4.权利要求1的组合物，其中所说的一种或多种表面活性剂选自Tween 80、CAMPUL POE-O低PV表面活性剂、SOLITOL HS15表面活性剂、PLURONIC F68嵌段共聚物、胆酸钠、甘油脱氧胆酸盐、神经磷脂、神经鞘氨醇、1，2-二肉豆寇酰-顺-丙三氧基-3-.磷酸乙醇胺、L-α-磷脂酰乙醇、1，2-二棕榈酰-顺-丙三氧基-3-磷酸胆碱以及蛋磷脂酰胆碱或它们的混合物。
5.权利要求1的组合物，其中所说的一种或多种表面活性剂是蛋磷脂酰胆碱和PLURONIC F68嵌段共聚物的混合物。
6.权利要求1的组合物，其中所说的抗氧化剂选自α-生育酚和抗坏血酸。
7.权利要求1的组合物，其中所说的抗氧化剂是α-生育酚。
8.权利要求1的组合物，其中所说的使所说的乳液等渗的成分选自葡萄糖、甘油、甘露醇、山梨醇、PEG 300、PEG 400以及聚乙二醇。
9.权利要求1的组合物，其中所说的使所说的乳液等渗的成分是甘油。
10.权利要求1的组合物，其中所说的组合物是水包油乳液。
11.权利要求1的组合物，其中所说的乳液的亲水亲脂平衡值约为7.0-13.0。
12.权利要求1的组合物，其中所说的乳液的亲水亲脂平衡值约为8.0。
13.权利要求1的组合物，其中所说的稳定乳液包括约10％体积/体积的角鲨烯、0.09％重量/体积的PLURONIC F-68嵌段共聚物、1.9％重量/体积的蛋磷脂酰胆碱、1.75％体积/体积的甘油以及0.05％重量/体积的α-生育酚。
14.权利要求1的组合物还包括减弱的脂质A衍生物。
15.权利要求14的组合物，其中所说的减弱的脂质A衍生物选自单磷酰基脂质A和3-脱酰的单磷酰基脂质A。
16.权利要求14的组合物，其中所说的减弱的脂质A衍生物是单磷酰基脂质A。
17.权利要求14的组合物，其中所说的减弱的脂质A衍生物是3D-单磷酰基脂质A。
18.权利要求14的组合物，其中所说的减弱的脂质A衍生物是约1.200％-0.005％重量/体积的所说的稳定的乳液。
19.一种疫苗组合物，其中包括抗原和佐剂组合物，该佐剂组合物包括新陈代谢油、一种或多种表面活性剂、抗氧化剂以及使所说的乳液等渗的成分。
20.权利要求19的疫苗组合物，其中所说的稳定乳液包括约10％体积/体积的角鲨烯、0.09％重量/体积的PLURONIC F68嵌段共聚物、1.9％重量/体积的蛋磷脂酰胆碱、1.75％体积/体积的甘油以及0.05％重量/体积的α-生育酚。
21.权利要求19的疫苗组合物，其中所说的稳定乳液还包括选自单磷酰基脂质A和3-脱酰的单磷酰基脂质A。
22.一种温血动物刺激免疫反应的方法，此法包括给该动物以抗原和有效量的稳定的水包油乳液，此乳液包括新陈代谢油、一种或多种表面活性剂、抗氧化剂以及使所说的乳液等渗的成分。
23.权利要求22的方法，其中所说的稳定乳液包括约10％体积/体积的角鲨烯、0.09％重量/体积的PLURONIC嵌段共聚物、1.9％重量/体积的蛋磷脂酰胆碱、1.75％体积/体积的甘油以及0.05％重量/体积的α-生育酚。
24.权利要求22的方法，其中所说的稳定乳液还包括选自单磷酰基脂质A和3-脱酰的单磷酰基脂质A的减弱的脂质A衍生物。
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| WO1999056776A2 (en) | 1999-11-11 |
| HU225844B1 (en) | 2007-10-29 |
| HUP0101804A3 (en) | 2003-11-28 |
| CY1107265T1 (el) | 2012-11-21 |
| ATE375803T1 (de) | 2007-11-15 |
| US6630161B1 (en) | 2003-10-07 |
| AU3973799A (en) | 1999-11-23 |
| JP2002513773A (ja) | 2002-05-14 |
| HUP0101804A2 (hu) | 2001-11-28 |
| DE69937343T2 (de) | 2008-07-24 |
| PT1075276E (pt) | 2008-01-22 |
| KR20010043404A (ko) | 2001-05-25 |
| BR9910269A (pt) | 2001-01-09 |
| ES2296390T3 (es) | 2008-04-16 |
| EP1075276B1 (en) | 2007-10-17 |
| CN1306438A (zh) | 2001-08-01 |
| NO20005596D0 (no) | 2000-11-06 |
| WO1999056776A3 (en) | 2000-01-06 |
| EP1075276A2 (en) | 2001-02-14 |
| KR100682154B1 (ko) | 2007-02-12 |
| DE69937343D1 (de) | 2007-11-29 |
| ZA200006379B (en) | 2002-01-07 |
| CA2330610A1 (en) | 1999-11-11 |
| NO20005596L (no) | 2001-01-05 |
| US20030215497A1 (en) | 2003-11-20 |
| HK1039072A1 (zh) | 2002-04-12 |
| AU755445B2 (en) | 2002-12-12 |
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