CN101163415B - 用于hiv患者的营养补充剂 - Google Patents
用于hiv患者的营养补充剂 Download PDFInfo
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- CN101163415B CN101163415B CN2006800132470A CN200680013247A CN101163415B CN 101163415 B CN101163415 B CN 101163415B CN 2006800132470 A CN2006800132470 A CN 2006800132470A CN 200680013247 A CN200680013247 A CN 200680013247A CN 101163415 B CN101163415 B CN 101163415B
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Abstract
本发明涉及一种用于HIV患者的营养产品。更具体地说,本发明涉及一种专门供给具有营养相关症状的HIV患者的营养组合物,该营养组合物提供仔细筛选的营养成分,包括寡糖和半胱氨酸和/或半胱氨酸源。本发明还涉及用于HIV患者的营养补充剂的制备。
Description
技术领域
本发明涉及一种用于HIV患者的营养产品。更具体地说,本发明涉及一种专门供给具有营养相关症状的HIV患者的营养组合物,该营养组合物提供仔细筛选的营养成分。本发明还涉及用于HIV患者的营养补充剂的制备。
背景技术
人体免疫缺陷病毒(HIV)的感染和获得性免疫缺陷综合征(AIDS)的发展已对国内和全球的健康、社会、政治以及经济成果产生了重大的影响。全世界感染HIV-1的人数已超过4000万,其中大部分居住在亚洲、撒哈拉以南的非洲地区和南美洲。尽管过去十年中取得了治疗上的进展,包括高效抗逆转录病毒疗法(HAART)的发展,但是一旦个体被感染,病毒的根除还是不可能的。
现在人们已经认识到HIV感染者营养供给的重要性。由于被感染患者受到的代谢压力,他们对基础能量、蛋白质和微量营养元素的需求可能增加。这种压力和与此疾病有关的厌食和吸收不良一起增进了营养不良。营养不良一般会影响例如免疫能力、(工作)表现和认知。提供额外的营养有助于这些患者改善其整体营养状况。
目前市面上有几种用于HIV患者营养供给的产品。不同的商品供应商销售多种临床营养产品,这些产品列出如下:
1.Advera,Ross Abbott
卡路里分配:
蛋白质:18.7%(大豆蛋白水解产物,酪蛋白酸钠)
碳水化合物:65.5%(麦芽糊精、蔗糖、大豆纤维)
脂肪:15.8%(芥花籽油(canola)、MCT、精炼脱味的沙丁鱼油1.5e%)
卡路里密度:1.28 kcal/mL
2.Resource,Novartis
卡路里分配:
蛋白质:14%(酪蛋白酸钠和酪蛋白酸钙,大豆蛋白分离物)
碳水化合物:64%(玉米糖浆、糖)
脂肪:22%(高油酸向日葵油、玉米油)
卡路里密度:1.06 kcal/mL
3.Benecalorie,Novartis
卡路里分配:
蛋白质:9%(酪蛋白酸钙)
碳水化合物:0%
脂肪:91%(高油酸向日葵油、甘油单酯和甘油二酯)
卡路里密度:7 kcal/mL
4.Bost,Mead Johnson现为Novartis出售的产品
卡路里分配:
蛋白质:24%(乳蛋白质浓缩物、酪蛋白酸钠和酪蛋白酸钙)
碳水化合物:55%(玉米糖浆固体、糖)
脂肪:21%(芥花籽油、高油酸向日葵油和玉米油)
卡路里密度:1.01 kcal/mL
但是,尽管向HIV感染患者供给一般营养需求的产品是可获得的,但是还没有既能改善营养状况又可另外显著减轻或预防特定的HIV感染相关的症状的营养产品,所述相关症状尤其是指受试者的免疫机能障碍、肠功能障碍和/或谷胱甘肽状态。
发明内容
现有的对HIV患者的营养治疗的缺点在于它们没有针对HIV患者营养相关的全部医学问题,尤其是与感染相关的免疫机能障碍、肠功能障碍和/或谷胱甘肽状态给出整体解决方案。在一个实施方案中,本发明涉及寡糖和半胱氨酸和/或半胱氨酸源在制备一种组合物中的用途,该组合物用于HIV或AIDS的治疗和/或预防方法,所述方法包括给予哺乳动物一种组合物,它包括治疗有效量的寡糖和半胱氨酸和/或半胱氨酸源。在另一个实施方案中,该组合物进一步包括一种或多种多不饱和脂肪酸(PUFA)和/或一种或多种生物活性化合物,尤其是乳源化合物。
本发明提供了适用于HIV患者营养治疗的全部营养补充剂。本发明的营养补充剂包括至少2种,优选至少3种组分,所述组分保证受试者的肠功能、谷胱甘肽(GSH)状态和/或免疫功能。我们惊奇地发现,通过仔细选择营养成分的组合,HIV感染的几种与营养相关的副作用(即感染相关的症状)可被预防和/或显著减少。我们发现,与目前实施的只给患者提供一种单独的成分时相比,当同时以几种疾病相关的症状为目标时,所述的效果要好得多。
健康的消化道和健康的肠道区系与健康的免疫功能复杂相关。特定纤维/寡糖的潜在免疫调节作用可能是对肠道区系组成(已记载了双歧杆菌和乳酸杆菌的免疫作用)和/或功能(纤维的发酵产生了如短链脂肪酸这样的化合物,所述化合物会影响消化道细胞的一般功能和免疫功能)影响的间接结果。令人惊奇地,本发明人发现树突细胞的DC-SIGN分子可被某些寡糖阻断。由于对该分子的阻断可以潜在地预防HIV的传染,在本发明的一个实施方案中提供了这些寡糖在阻断DN-SIGN受体和制备用于预防和/或治疗DC-SIGN介导的疾病(尤其是HIV和AIDS)的组合物方面的用途。
具体实施方式
一般定义
“寡糖”是指由单糖单元的碳水化合物链,链长介于1至5000之间,更优选介于2至250之间,更优选介于2至50之间,最优选介于2至10之间。
“聚合度”或“DP”是指一个寡糖链中糖单元的总数。“平均DP”是指在不考虑可能的单糖或二糖(如果存在则优选将其排除)时,组合物中寡糖链的平均DP。可使用组合物的平均DP来区分组合物。寡糖混合物的平均聚合度优选介于2至100之间,更优选地介于3至250之间,例如,介于3至50之间。
两种或多种物质的“共同给药”是指将这些物质在同一时间(同时)或者在一小段时间内(例如,在几分钟内或几小时内分开使用或序贯使用)作为一种组合物或分开的组合物(各部分组成的药盒;作为结合的组合物)给予一个个体。
术语“包括”应被理解为特指存在已说明的部分、步骤或组分,但不排除存在一种或多种附加的部分、步骤或组分。
“百分数”或“均值”一般是指按重量计的均值百分数,除非另有指明或除非明确指出另一种计算基准。
“GOS”或“低聚半乳糖(galactooligosaccharide)”,或“反式-低聚半乳糖”或“TOS”是指由半乳糖单元构成的寡糖。
“HIV的治疗和/或预防”是指一种或多种选自免疫机能障碍、肠功能障碍和/或低谷胱甘肽状态的HIV感染相关的症状/功能障碍的显著减少或预防。在一个实施方案中,HIV的治疗或预防是指由于DC-SIGN受体的阻断引起的HIV扩散的明显减少(或完全阻止),这将会在上下文中阐明。
“显著的减少或阻止”是指,与未给予本发明的组合物的对照受试者相比,症状(或HIV扩散)减少了至少5%,10%,15%,30%,50%甚至是100%。症状可通过本领域公知的方法测量,例如,免疫机能障碍可通过测量CD4+细胞数测定。DC-SIGN受体的阻断可以按照实施例1中方法确定。
本发明的目的在于提供适用于治疗HIV患者的营养组合物,以改善他们的营养状况和至少两种,优选至少三种HIV相关的症状。本发明的组合物尤其适用于CD4+T细胞数低于约700个细胞/μL血液的临界水平的患者,此时通常还不需要HAART疗法,但患者确实是已经发展出或经受了免疫机能障碍、肠功能障碍和/或低谷胱甘肽状态中的一种或多种。
因此,本发明的组合物适用于预防和/或治疗一种或多种HIV感染相关的功能障碍,尤其是:
1.免疫机能障碍,即CD4+T细胞数的减少导致的免疫功能损伤;
2.肠功能障碍,即肠道问题,尤其是HIV诱发的吸收不良和腹泻;和/或
3.低谷胱甘肽状态,特别是在血液中和T细胞内的低谷胱甘肽水平。
在一个优选的实施方案中,该组合物至少适用于治疗或预防免疫机能障碍和低谷胱甘肽状态。这些组合物包括适量的寡糖和半胱氨酸和/或半胱氨酸源。在另一个实施方案中,组合物还包括适量的一种或多种PUFA和/或一种或多种生物活性化合物,且该组合物适用于治疗或预防所有上述三种功能障碍。
由于CD4+T-淋巴细胞被HIV感染和破坏,HIV的进展情况可通过测量血液循环中的CD4+T-淋巴细胞数定期且有规律地监测。HIV感染的初期(可持续三年到十年以上)的特点在于总CD4+T-细胞数缓慢却逐渐地降低,但没有出现感染抵抗力下降的明显症状。感染并发症的第一个迹象通常出现在CD4+T细胞数低于700个细胞/μL血液时。此时,HIV血清反应呈阳性的个体可能经受呼吸(咳嗽、感冒、流感)和/或胃肠(肠不适、腹泻)的症状。这些症状仍旧相对轻微并且可能被认为是临床症状不明显的;这些症状尽管对个体来说是令人烦恼的,但它们通常还没有严重到导致住院治疗或开始高效抗逆转录病毒疗法(HAART)的程度。
性传播后首先被I型人体免疫缺陷病毒(HIV-1)感染的细胞类型之一是树突细胞(DC)。DC通过C-型凝集素受体捕获HIV-1,所述受体中研究彻底的实例是DC-SIGN,它介导HIV-1的内在化。DC可将病毒的传染性保持几天,并能够向CD4(+)T细胞传播HIV-1。如实施例1中所描述的,本发明人惊奇地发现寡糖可与DC-SIGN结合。
本发明的组合物和用途
本发明的组合物适用于治疗和/或预防受试哺乳动物的HIV和/或AIDS。所述的受试者优选感染HIV的受试人,且他们含有的CD4+细胞数为约700个细胞/μL血液或更少、更优选地介于约200和700个细胞/μL血液之间,例如,介于约200至500个细胞,或约介于200至600个细胞,或介于500至700细胞/μL。在一个实施方案中,受试者的CD4+细胞数为700或更少,但未进行高效抗逆转录病毒疗法(HAART)。
在一个实施方案中,营养组合物优选为食品补充剂,并且包括寡糖和半胱氨酸和/或半胱氨酸源。
寡糖
本发明的组合物包括治疗有效量的寡糖,优选如下描述的酸性寡糖和/或中性寡糖。
酸性寡糖包括至少一种酸性基团,而中性寡糖没有此种酸性基团。由于膳食纤维有利于健康,人们已对其进行了广泛地研究。一些纤维是不溶的且不可发酵,通过肠道时无变化。其他的纤维类型可作为益生素,即它们被肠道细菌使用且刺激其生长。因此,已有记载表明诸如菊粉的纤维或诸如低聚半乳糖(GOS)和低聚果糖(FOS)的寡糖可刺激双歧杆菌和乳酸菌的生长,它们对于健康的肠道区系很重要。
酸性寡糖
术语“酸性寡糖”是指包括至少一种选自以下的酸性基团的寡糖:N-乙酰神经氨酸、N-乙醇酰神经氨酸、游离的或酯化的羧酸、硫酸基团和磷酸基团。在一个实施方案中,酸性寡糖优选是聚己糖。优选地,至少一个前述的酸性基团位于酸性寡糖的末端己糖单元上。优选地,该酸性寡糖具有如图1中所描绘的结构,其中末端己糖(左)优选含有一个双键。优选地,该酸性寡糖在其末端己糖单元上含有一个羧酸,其中所述的羧酸基团可以是游离的或酯化的。在WO 01/60378和/或WO 02/42484中提供了适用于本发明的方法和组合物的酯化果胶水解产物的制备方法,所述文献通过引用的方式纳入本说明书。除了末端己糖单元以外的己糖单元优选为糖醛酸单元,甚至更优选地为半乳糖醛酸单元。这些单元上的羧酸可以是游离的或(部分)酯化的,优选至少10%是甲基化的(见下)。
图1.聚合的酸性寡糖
其中:
R优选地选自氢、羟基或酸性基团,优选羟基:以及
R2、R3、R4和R5中至少一个代表N-乙酰神经氨酸、N-乙醇酰神经氨酸、游离的或酯化的羧酸、硫酸基团和磷酸基团,并且R2、R3、R4和R5中其余的代表羟基和/或氢。优选地,R2、R3、R4和R5中的一个代表N-乙酰神经氨酸、N-乙醇酰神经氨酸、游离的或酯化的羧酸、硫酸基团和磷酸基团,并且其余的代表羟基和/或氢。甚至更优选地,R2、R3、R4和R5中的一个代表游离的或酯化的羧酸,并且R2、R3、R4和R5中其余的代表羟基和/或氢;并且
n为整数,是指己糖单元的数目(也参见下面的聚合度),该己糖单元可以是任何己糖单元。适当地,n是介于1-5000之间的整数。优选地,该己糖单元是糖醛酸单元。
最优选地,R1,R2和R3代表羟基、R4代表氢,R5代表羧酸,n是介于1至250之间的任何数字,优选地介于1至10之间,且该己糖单元是半乳糖醛酸。
对本方法中所使用的酸性寡糖的检测、测量和分析已在申请人早期的涉及酸性寡糖的专利申请即WO 01/60378中给出,它通过引用的方式纳入本说明书。
优选地,酸性寡糖具有一个,优选两个末端糖醛酸单元,它可以是游离的或酯化的。优选地,该末端糖醛酸单元选自半乳糖醛酸、葡糖醛酸、古洛糖醛酸、艾杜糖醛酸、甘露糖醛酸、核糖醛酸(riburonic acid)和阿卓糖醛酸(alturonic acid)。这些单元可以是游离的或酯化的。在一个实施方案中,末端己糖单元具有一个双键,它优选地位于末端己糖单元的C4和C5位之间。优选地,末端己糖单元之一包括该双键。该末端己糖(例如糖醛酸)优选地具有图2的结构。
图2:优选的末端己糖酸性基团
其中;
R优选地选自氢、羟基或酸性基团,优选羟基(见上);并且
R2、R3、R4和R5中至少一个代表N-乙酰神经氨酸、N-乙醇酰神经氨酸、游离的或酯化的羧酸、硫酸基团和磷酸基团,并且R2、R3、R4和R5中其余的代表羟基和/或氢。优选地,R2、R3、R4和R5中的一个代表N-乙酰神经氨酸、N-乙醇酰神经氨酸、游离的或酯化的羧酸、硫酸基团和磷酸基团,并且R2、R3、R4和R5中其余的代表羟基和/或氢。甚至更优选地,R2、R3、R4和R5中的一个代表游离的或酯化的羧酸,并且R2、R3、R4和R5中其余的代表羟基和/或氢;并且n为整数,是指己糖单元的数目(也参见下面的聚合度),该己糖单元可以是任何己糖单元。适当地,n是1-5000之间的整数,代表己糖单元的数目。所述的己糖单元优选为糖醛酸,甚至更优选地,是半乳糖醛酸单元。这些单元上的羧酸基团可以是游离的或(部分)酯化的,且优选地至少是部分甲基化的。
最优选地,R2和R3代表羟基、R4代表氢且R5代表游离的或酯化的羧酸。
在一个实施方案中,该组合物包括单一类型的酸性寡糖(具有均一的聚合度),而在另一个实施方案中,该组合物包括具有不同的聚合度(DP)和/或包括饱和的和不饱和的末端己糖单元的酸性寡糖的混合物。优选地至少5%,更优选地至少10%,甚至更优选地至少25%的酸性寡糖的末端己糖单元为不饱和的己糖单元(例如,见图2)。由于每个单独的酸性寡糖优选地只包括一种不饱和的末端己糖单元,因此优选不多于50%的末端己糖单元是不饱和的末端己糖单元(即包括一个双键)。
一种酸性寡糖的混合物优选含有占己糖单元总量的2至50%的不饱和己糖单元,优选地介于10至40%之间。
用于本发明的方法中的酸性寡糖的聚合度(DP)介于1至5000之间,优选介于1至1000之间,更优选介于2至250之间,甚至更优选介于2至50之间,最优选介于2至10之间。如果使用一种具有不同聚合度的酸性寡糖的混合物,该酸性寡糖混合物的平均DP优选介于2至1000之间,更优选介于3至250之间,甚至更优选介于3至50之间。同样参见图1,其中“n”和末端单元的总和(即n+1)代表了聚合度。已发现,如果该酸性寡糖是以液体形式给予的,则稍低DP的寡糖改善了其口味并产生粘度降低的产品。该酸性寡糖可以是一种均一的或不均一的碳水化合物。
本发明中使用的酸性寡糖优选由果胶、果胶酸盐、藻酸盐、软骨素、透明质酸、肝素、类肝素、细菌碳水化合物、唾液酸聚糖(sialoglycan)、岩藻低聚糖(fucoidan)、岩藻寡糖(fucooligosaccharide)或角叉藻聚糖制备,优选由果胶和/或藻酸盐制备。酸性寡糖可通过WO 01/60378中描述的方法制备,例如,化学水解或酶解或部分水解,见第8页和第9页,其通过引用的方式纳入本文。
藻酸盐是线性非支链的聚合物,含有β-(1→4)-连接的D-甘露糖醛酸和α-(1→4)-连接的L-古洛糖醛酸残基,平均分子量范围较宽(100-100000个残基)。适当的藻酸盐源包括海藻和细菌藻酸盐。
果胶被分成两个主要的类别:甲氧基化程度高于50%的高度甲氧基化的果胶和甲氧基化程度低于50%的低度甲氧基化果胶。本文中使用的“甲氧基化程度”(也被称作DE或“酯化程度”)是指多聚半乳糖醛酸链上包含的游离羧酸基团被酯化(例如,通过甲基化)的程度。本发明中的酸性寡糖优选由高度甲氧基化的果胶制备。
优选地,酸性寡糖特征为甲氧基化程度高于20%,优选高于50%,甚至更优选高于70%。优选地,酸性寡糖具有高于20%的甲基化程度,优选地高于50%,甚至更优选地高于70%。
酸性寡糖的给药量优选地介于每天约10mg至100克之间,优选地介于每天约100mg至50克之间,甚至更优选地介于每天约0.5至20克之间。
中性寡糖
如上所述,该组合物还可以包括一种或多种中性寡糖,所述中性寡糖可以代替一种或多种酸性寡糖,也可以另外添加。一种或多种中性寡糖选自纤维二糖、纤维糊精、B-环糊精、不可消化的糊精、龙胆寡糖(gentiooligosaccharides)、葡糖寡糖、异麦芽糖寡糖、异麦芽糖、异麦芽三糖、潘糖、吡喃果糖-5-葡糖苷、帕拉金糖、theanderose、D-塔格糖、D-来苏-己酮糖、乳果糖、α-低聚半乳糖、β-低聚半乳糖、反式低聚半乳糖、乳果糖(lactulose)、4’-galatosyllactose、合成低聚半乳糖、果烷-果聚糖型、果烷-菊粉型、1f-β-果糖呋喃糖基真菌四糖(1f-β-fructofuranosylnystose)、乳糖-N-四糖、乳糖N-新四糖(neotetraose)、低聚木糖、lafinose、乳果糖和低聚阿拉伯糖(arabinooligosaccharide)。
优选地,中性寡糖选自低聚半乳糖、低聚果糖、反式低聚半乳糖、低聚木糖、乳果糖和低聚阿拉伯糖。甚至更优选地,中性寡糖选自低聚半乳糖、低聚果糖和反式低聚半乳糖。
优选地,该组合物包括两种化学上有区别的中性寡糖、一种选自基于半乳糖的中性寡糖,另外一种选自基于果糖和/或葡萄糖的寡糖。
更优选地,该组合物包括低聚果糖和至少一种选自反式低聚半乳糖和低聚半乳糖的寡糖。
优选的中性寡糖的每日用量介于每天约10mg至100克之间,优选地介于每天约100mg至50克之间,甚至更优选地介于每天约0.5至20克之间。
优选地使用一种包括中性和酸性寡糖的组合物,其中全部寡糖中至少15%是酸性寡糖,更优选介于10至90%之间,最优选地介于25至75%之间。优选地使用一种组合物,其中至少25%的寡糖是包括至少一个末端糖醛酸单元的酸性寡糖。
半胱氨酸或半胱氨酸源
所提供的组合物除了一种或多种上述的寡糖以外还包括适量的半胱氨酸和/或半胱氨酸源。短语“半胱氨酸源”在本文中是指以任何形式含有生物可用的半胱氨酸的所有化合物,它以存在于化合物中的半胱氨酸的量计算,或来自于消化后体内的化合物(基于摩尔量)。
本文下述的“半胱氨酸等价物”是指半胱氨酸本身的量或是指存在于半胱氨酸源中的半胱氨酸的量。例如,100mg NAC(N-乙酰半胱氨酸;MW=163.2)与74mg半胱氨酸(MW121.15)等价。因此,100mg NAC是74mg的半胱氨酸等价物。类似地,还可应用在蛋白质或肽中。当一种肽(MW=x Dalton)含有3个半胱氨酸氨基酸(3y Dalton),那么100mg这种肽与100×3Y/x mg半胱氨酸等价。因此,100mg这种肽是300y/x mg的半胱氨酸等价物。
适当的本发明半胱氨酸源是,例如,以变性的和/或未变性的形式存在的蛋白质,如乳蛋白(例如乳清或酪蛋白)。卵蛋白富含半胱氨酸,并因此也是适用的。诸如豌豆、马铃薯、大豆和稻的植物蛋白也可被用于提供半胱氨酸。也可使用这些蛋白质源的水解产物,或者富集富含半胱氨酸的蛋白或肽的组分(例如,如EP1201137中描述的)。此外,也可使用合成的半胱氨酸等价物(例如半胱氨酸衍生物),例如半胱氨酸、半胱氨酸盐、N-乙酰半胱氨酸和/或二乙酰半胱氨酸。
适合给予感染HIV的受试者的每日剂量至少约为100mg/天的半胱氨酸等价物,优选至少约每日200、400、或600mg/天的半胱氨酸等价物,更优选地为至少约每日1000mg/天半胱氨酸等价物。应理解,每日剂量可被分成在一天中分几次服用的2、3或更多个的剂量单位。
在另一个实施方案中,依据本发明的组合物包括一种或多种刺激谷胱甘肽水平的化合物。例如硫辛酸、丙酮酸盐、草酰乙酸盐、草酰天门冬氨酸盐能够刺激谷胱甘肽水平。这种刺激谷胱甘肽水平的化合物在半胱氨酸外额外加入使用,也可代替半胱氨酸使用。
在另一个实施方案中,包括一种或多种寡糖和半胱氨酸和/或半胱氨酸源的组合物进一步包括一种或多种PUFA和/或一种或多种生物学活性的化合物,例如乳或益生微生物中的化合物。
益生微生物
益生微生物是指一种微生物,它通过改善肠道微生物平衡有利地作用于HIV患者(Fuller,R.J.Applied Bacteriology,1989;66:365-378)。该益生微生物可选自一种或多种适于人类消耗的微生物,且能够改善肠道内的微生物平衡。优选地,对应于每克糖醛酸寡糖(DP介于2至100之间),本发明的组合物中含有104至1012,更优选为105至1011,最优选为107至5×1010菌落形成单位(cfu)的益生菌。对应于每克干重的本发明组合物,本发明的组合物优选地含有102至1013菌落形成单位(cfu)的益生菌,优选104至1012,更优选105至1010,最优选105至1×109cfu。依据本发明的益生菌的剂量优选为每天102至1013,更优选为每天105至1011,最优选为每天108至5×1010菌落形成单位(cfu)。优选地使用活的或可存活的细菌,但也可使用死亡的细菌或细菌片段。
优选地,本发明的组合物包括乳杆菌属(genus Lactobacillus)和/或双歧杆菌属(Bifidobacterium)的细菌。优选地,该组合物包括一种选自长双歧杆菌(B.longum)、短双歧杆菌(B.breve)、双歧双歧杆菌(B.bifidum)的双歧杆菌和/或一种选自干酪乳杆菌(L.casei)、类干酪乳杆菌(L paracasei)、鼠李糖乳杆菌(L.rhamnosus)、嗜酸乳杆菌(L.acidophilus)和植物乳杆菌(L.plantarum)的乳杆菌。最优选地,本发明的组合物包括短双歧杆菌和/或类干酪乳杆菌。
短双歧杆菌是一种厌氧棒状的革兰氏阳性细菌。如Stackebrandt&Goebel,194,Int.J.Syst.Bacteriol.44:846-849中所述,当与短双歧杆菌的ATCC 15700型菌株进行比较时,本发明的短双歧杆菌与16SrRNA序列优选地具有至少95%的核酸序列同一性,更优选至少97%、98%、99%或更多的序列同一性。核酸序列的同一性是在使用采用默认参数的GAP或BESTFIT程序进行最优比对时,对两个核苷酸序列计算得到的。使用GAP默认参数时,其中空位生成罚分(gap creation penalty)=50(核苷酸)/8(蛋白质),空位延伸罚分(gap extension penalty)=3(核苷酸)/2(蛋白质)。对于核苷酸,所使用的默认得分矩阵是nwsgapdna(Henikoff&Henikoff,1992,PNAS 89,915-919)。这比当RNA序列被称为是与DNA序列基本相似或具有一定程度的序列同一性时更清楚,DNA序列中的胸腺嘧啶(T)被认为等同于RNA序列中的尿嘧啶(U)。序列比对和序列同一性的百分数的计分可以使用计算机程序确定,例如可使用GCG Wisconsin Package,Version 10.3,可从Accelrys Inc.,9685Scranton Road,San Diego,CA 92121-3752,USA处获得或者使用EMBOSSwin v.2.1 .0确定。本发明中所使用的双歧杆菌优选与短双歧杆菌探针杂交,并通过5’核酸酶测定方法产生信号,该方法如本申请人共同未决的国际专利申请PCT/NL2004/000748和欧洲专利申请05075486.0中所述。根据一个优选的实施方案,本发明的组合物含有至少一种选自短双歧杆菌Bb-03(Rhodia)、短双歧杆菌M16-V(Morinaga)、短双歧杆菌R0070(Institute Rosell,Lallemand)、DSM 20091和LMG 11613的短双歧杆菌。最优选地,短双歧杆菌为短双歧杆菌M-16V(Morinaga)。
在一个优选的实施方案中,本发明的组合物包括类干酪乳杆菌。优选地,如上所述,当与类干酪乳杆菌ATCC 25032型菌株相比时,本发明的类干酪乳杆菌株与16S rRNA序列具有至少95%、更优选至少97%、98%、99%或更多的核酸序列同一性。本发明中使用的乳杆菌优选与类干酪乳杆菌探针杂交,并通过5’核酸酶测定方法产生信号,该方法如本申请人的共同未决的欧洲专利申请05075486.0中所述。依据一个优选的实施方案,本发明的组合物含有至少一种选自类干酪乳杆菌F19(Arla,Sweden)、类干酪乳杆菌LAFTI L26(DSM Food specialties,the Netherlands)和类干酪乳杆菌CRL431(Chr.Hansen,Denmark)、LMG 12165和LMG 11407的类干酪乳杆菌。
多不饱和脂肪酸
本发明人发现二十碳五烯酸(EPA,n-3)和γ亚麻酸(GLA,n-6)可有效地降低炎症介导的肠道紧密连接的通透性。因此提供了一种适用于改善肠道屏障完整性的组合物,它包括(除寡糖和半胱氨酸和/或半胱氨酸源以外)EPA和/或GLA。基于生物化学途径可以假设,脂肪酸的其他组合也是有效的。因此,也提供了包括一种或多种其他PUFA或其混合物的组合物。例如,可以使用一种混合物,它是EPA、二十二碳六烯酸(DHA,n-3)、二高-γ亚麻酸(DGLA,C20:3n-6)、十八碳四烯酸(STA,C18:4n-4)、α-亚麻酸(ALA,C18:3n-3)、二十二碳四烯酸(DPA,C22:5n-3)、二十碳四烯酸(C20:4n-3)和/或花生四烯酸(AA,n-6)任一种的混合物。
适当地,应使用相对高的多不饱和脂肪酸的每日剂量。在一个实施方案中,使用至少约25en%,优选至少约30en%,更优选至少约35en%的包括n-3和/或n-6脂肪酸的脂肪混合物(en%是能量百分数的缩写,代表每种组分占制剂总卡路里值的相对量)。优选的每日剂量为至少1克PUFA,更优选1-50克PUFA,更优选5至25克PUFA,且最优选的量是7.5至15克PUFA。
一种优化的脂肪混合物可包括例如40%的琉璃苣油和60%的鱼油。那么n-3/n-6脂肪酸比例在1-2之间,且n-3的重量百分数占脂肪酸总含量的20-40,且n-6的重量百分数占脂肪酸总含量的15-35。琉璃苣油可部分地或完全地被月见草油替代。
因此,优选的每日剂量为至少0.1克EPA和0.05克GLA,更优选0.1至5克EPA和0.05至2.5克GLA,更优选0.5至2.5克EPA和0.25至1.25克GLA,且最优选的量是0.75至1.5克EPA和0.37至0.75克GLA。
生物学活性成分
本发明的组合物进一步可包括一种或多种生物学活性的分子,优选天然地存在于乳中的组分。这些组分包括生长因子、免疫球蛋白和其他乳组分或从乳中衍生的组分。
A.生长因子
已发现乳生长因子有益于消化道健康。转化生长因子β、胰岛素样生长因子和角质化细胞生长因子是乳生长因子最重要的实例。因此,在一个实施方案中,该组合物进一步包括一种或多种生长因子,例如每天约1-500μg生长因子。
B.免疫球蛋白
已显示免疫球蛋白可保护肠道免受感染,且本发明的组合物适当地包括每日剂量为0.1至10g的免疫球蛋白。
C.其他成分
还发现了其他从乳中获得的生物活性成分例如核苷酸、脂肪酸、寡糖对消化道屏障功能具有有益作用,因此它们可适用于该组合物的制备。
D.初乳
在一个实施方案中,组合物包括初乳。初乳是生产后的哺乳动物母体尤其是生完小牛后的母牛的乳腺分泌的前乳(pre-milk)流体。初乳含有多种生物活性的乳成分,因此是生物活性分子的极好来源。作为蛋白质源的初乳还具有可以提供半胱氨酸的附加优点。为对HIV患者起到有益的作用,至少提供每天约5克的初乳,优选至少约10克,更优选至少约每天20g或更多。
乳蛋白的提取物,例如EP0545946中所述的乳清生长因子提取物或WO02083164中描述的酪蛋白提取物,免疫球蛋白浓缩物,乳铁蛋白或其他浓缩的乳清部分也可被用于改善HIV患者的消化道屏障功能。
应理解生物活性分子或组分可使用一系列方法获得。许多是市售的,或可由重组DNA技术通过合成制备,或可从例如乳的天然来源中(部分)纯化或提取。也可使用生物活性分子或包括这些分子的组分的任一种的混合物。
适用于阻断DC-sign受体的组合物
在另一个实施方案中提供了适用于治疗和/或预防DC-sign介导的疾病例如HIV或AIDS的组合物。这类组合物包括适量的寡糖,尤其是如上文和实施例1中所述的酸性寡糖。优选的寡糖的IC50值为约1000、600、400,更优选的为200μg/ml或更少,例如150、100、50、25μg/ml或更少。IC50值可使用本领域公知的方法确定(参见实施例1)。
这些组合物可进一步包括半胱氨酸和/或半胱氨酸源、PUFA等,如本文其他部分所述。所述的寡糖可被制成药物组合物或食品或食品补充剂组合物(如本文中以下描述的包括寡糖和半胱氨酸和/或半胱氨酸源的组合物)。
适用于多种给药类型的药物制剂的指导可查阅Remington’sPharmaceutical Science,Mace Publishing Company,Philadelphia,PA,17th ed.(1985)。
营养组合物和食品补充剂
已发现寡糖和半胱氨酸和/或半胱氨酸源可被方便地应用于食品中,例如婴儿食品和临床食品。这种食品优选包括脂质、蛋白质和碳水化合物,且可以液体或固体形式给予。本发明中使用的术语“液体食品”包括干燥的食品(例如粉末),附有关于将其与适当的液体(例如水)混合的说明书。固体食品包括水活度为0.2至0.4的补充棒剂形式的食品。水活度的定义是相同温度下一种产品的水蒸汽压与纯水的水蒸汽压的比率。固体产品必须满足目标水活度,否则该产品会不耐贮存。同样也可提供半固体食品和食品补充剂。
因此,本发明还涉及一种营养组合物,除本发明的寡糖和半胱氨酸和/或半胱氨酸源外,所述营养组合物优选地还包括5至50en%的脂质、10至60en%的蛋白质、15至85en%的碳水化合物。在本发明的上下文中,应理解本发明的组合物中的寡糖不会传递卡路里,因此不包含在本文提及的en%中。所有蛋白质、肽、氨基酸对卡路里有贡献,因此被包括在本文提及的en%中。在一个实施方案中,营养组合物包括15至50en%的脂质、25至60en%的蛋白质和15至45en%的碳水化合物。在另一个实施方案中,本发明的营养组合物包括15至50en%的脂质、35至65en%的蛋白质和15至45en%的碳水化合物。
优选使用的脂质具有高含量的EPA或GLA。鱼油和琉璃苣油或月见草油是这些多不饱和脂肪酸的优选来源。
一种可消化的碳水化合物的来源可被加入营养制剂中。它优选地提供约25%至约40%能量的营养组合物。可使用任何适当(来源)的碳水化合物,例如蔗糖、乳糖、葡萄糖、果糖、玉米糖浆固体和麦芽糖糊精,及其混合物。
优选地,维生素和矿物质存在的量是FSMP条例所要求的量。
腹泻是很多进食液体食物的HIV患者的主要问题。已发现通过给予本发明的寡糖可减少该排便问题,所述的寡糖存在于干燥营养组合物中或液体营养组合物中,所述液体营养组合物的摩尔渗透压浓度介于50至500mOsm/kg之间,更优选介于100至400mOsm/kg之间。
按照上述观点,该营养组合物优选地不递送过量的卡路里。因此,该营养组合物优选地不含有超过500kcal/每日剂量,更优选的是200至400kcal/每日剂量,且更优选地250至350kcal/每日剂量。
依据上文所述,本发明涉及一种营养组合物,包括:
a)寡糖,优选地,该寡糖至少包括酸性寡糖,所述的酸性寡糖的量是每日10mg至100克,优选地约每日100mg至50克,甚至更优选地所提供的每日剂量为约0.5至20克;和
b)半胱氨酸和/或半胱氨酸源,优选地,其中每日剂量中至少提供0.1g半胱氨酸等价物;以及任选地
c)一种或多种生物活性成分(例如初乳)和/或PUFA(例如EPA和/或GLA),优选地,其中每日剂量中至少提供1g PUFA,更优选地1-50克PUFA,更优选地5至2- 5克PUFA,甚至更优选地7.5至15克PUFA,同样优选地,每日剂量中至少提供0.1克EPA和0.05克GLA,更优选地0.1至5克EPA和0.05至2.5克GLA,更优选地0.5至2.5克EPA和0.25至1.25克GLA,甚至更优选地0.75至1.5克EPA和0.37至0.75克GLA。
在一个实施方案中,该营养组合物包括5至50en%的脂质、35至60en%的蛋白质、15至60en%的碳水化合物,酸性寡糖和半胱氨酸和/或半胱氨酸源,其中所述半胱氨酸源选自NAC、乳清、初乳、卵蛋白或其混合物。
在另一个实施方案中,该食品组合物包括15至50en%的脂质、35至60en%的蛋白质、15至45en%的碳水化合物、酸性寡糖和中性寡糖,以及半胱氨酸和/或半胱氨酸源,其中半胱氨酸源选自NAC、乳清、初乳、卵蛋白或其混合物。
该营养组合物优选为食品补充剂形式,或作为食品补充剂给予。该营养组合物或食品补充剂可以方便地用于治疗HIV患者的方法,所述的方法包括给予哺乳动物优选感染HIV的人所述组合物或补充剂。
同样提供的是一种用于治疗和/或预防HIV的组合物的制备方法,所述的方法包括:
-提供适量的一种或多种寡糖;
-提供适量的半胱氨酸和/或半胱氨酸源
-将上述两种组分制成适当的食品或食品补充剂或药物组合物。
下述实施例说明了本发明。除非另有说明,否则本发明的实施采用分子生物学、药理学、免疫学、病毒学、微生物学或生物化学的标准常规方法。这些技术描述于Sambrook and Russell(2001)Molecular Cloning:A Laboratory Manual,Third Edition,Cold Spring Harbor LaboratoryPress,NY,Volumes 1 and 2 of Ausubel et al.(1994)Current Protocolsin Molecular Biology,Current Protocols,USA和Remington’sPharmaceutical Science,Mack Publishing Company,Philadelphia,Pa.,17th ed.(1985),Microbiology:A Laboratory Manual(6th Edition)by James Cappuccino,Laboratory Methods in Food Microbiology(3rdedition)by W.Harrigan(作者),Academic Press,这些内容全部以引用的方式纳入本文。
实施例
实施例1.酸性寡糖和GOS对DC-SIGN-Fc结合的阻断
已知阻断DC-SIGN可以阻止病毒从树突细胞移位至CD4 T细胞上。本发明人惊奇地发现寡糖能够以不同的效能阻断DC-SIGN。如表1所示,酸性寡糖(AOS)如果胶水解产物是最有效的。这些结果显示AOS可阻止Fc片段与DC-SIGN以最低浓度结合。
表1.寡糖引起的DC-sign结合的功效
寡糖 | I.C.50(μg/ml) |
酸性寡糖(果胶水解产物) | 200 |
低聚半乳糖(反式低聚半乳糖) | 600 |
低聚果糖(菊粉HP) | >1000 |
材料与方法:
将系列稀释的寡糖制剂涂覆于ELISA板上。通过使用抗-DC-SIGN-Fc的ELISA测定DC-SIGN-Fc的结合,并通过加入标记的第二抗体对其进行观察。温育20分钟后用分光光度计(Becton Dickinson)测量OD值。结果表示为在50%抑制时的抑制浓度。
实施例2.一种营养棒剂组合物
原材料 | 代码 | g/天 | 蛋白质 | g/100g |
初乳琉璃苣油(Ropufa 25n-6)EPA-DHA油(Maruha)低聚乳糖酏剂或糖浆菊粉(Raftiline HP)酸性寡糖(果胶水解产物)N-乙酰基半胱氨酸Fructosestroop甘油 | SR2000342200129220011892001190SRSRJJJJ | 20.004.006.0015.380.798.541.8313.203.30 | 15.000.000.000.000.000.111.340.000.00 | 27.385.488.2121.061.0811.692.5018.074.52 |
每日kcal | En% | |||
蛋白质的能量碳水化合物的能量脂肪的能量 | 668297245 | 26.933.439.7 |
实施例3.一种营养棒剂组合物
原材料 | 代码 | g/天 | 蛋白质 | 碳水化合物 | 脂肪 | g/100g |
初乳琉璃苣油(Ropufa 25n-6)EPA-DHA油(Maruha)低聚半乳糖(酏剂或糖浆)菊粉(Raftiline HP)酸性寡糖(果胶水解产物)卵壳膜粉Fructosestroop甘油总量 | SR2000342200129220011892001190SRJJJJ | 20.004.006.0015.380.798.5421.0915.403.8595.05 | 15.000.000.000.000.000.1116.870.000.0031.98 | 2.100.000.004.780.000.090.0011.923.8322.72 | 0.804.006.000.000.000.000.000.000.0010.80 | 21.044.216.3116.180.838.9822.1916.204.05100.00 |
每日kcal | En% | 每100gkcal | ||||
蛋白质的能量碳水化合物的能量脂肪的能量总量 | 1289197316 | 40.528.830.8 | 13596102332 |
实施例4.粉剂组合物
原材料 | 代码 | g/天 | 蛋白质 | 碳水化合物 | 脂肪 | g/100g |
初乳琉璃苣油(Ropufa 25 n-6)EPA-DHA油(Maruha)GOS/MD DE2粉末菊粉(Raftiline HP)酸性寡糖(果胶水解产物)N-乙酰基-半胱氨酸MD DE47MD DE47SSL(乳化剂)总量 | SR2000342200129220011892001190SRSRMMMMSHS | 20.004.006.0014.780.798.541.837.005.000.1168.05 | 15.000.000.000.000.00O.111.340.010.010.0016.46 | 2.100.000.007.660.000.090.006.754.820.0021.41 | 0.804.006.000.000.000.000.000.020.01O.1110.94 | 29.395.888.8221.721.1612.552.6910.297.350.17100.0 |
每日Rcal | En% | 每100gkcal | ||||
蛋白质的能量碳水化合物的能量脂肪的能量总量 | 668698250 | 26.334.339.4 | 97126145367 |
实施例5.粉剂组合物
原材料 | 代码 | g/天 | 蛋白质 | 碳水化合物 | 脂肪 | g/100g |
初乳琉璃苣油(Ropufa 25 n-6)EPA-DHA油(Maruha)GOS/MaltoDex(DE2粉末)菊粉(Raftiline HP)酸性寡糖(果胶水解产物)α-乳清蛋白(Davisco)MaltoDex DE47MaltoDex DE47SSL(乳化剂)总量 | SR2000342200129220011892001190SRMMMMSHS | 20.004.006.0014.780.798.5434.037.005.00O.11100.25 | 15.000.000.000.000.000.1131.210.010.010.0046.33 | 2.100.000.007.660.000.090.176.754.820.0021.58 | 0.804.006.000.000.000.000.170.020.010.1111.11 | 19.953.995.9814.740.798.5233.946.984.990.11100.00 |
每日kcal | En% | 每100gkcal | ||||
蛋白质的能量碳水化合物的能量脂肪的能量总量 | 18586100372 | 49.923.226.9 | 18586100371 |
实施例6.液体营养组合物
原材料 | 代码 | g/天 | 蛋白质 | 碳水化合物 | 脂肪 | g/Itr |
琉璃苣油(Ropufa 25n-6)EPA-DHA油(Maruha)低聚半乳糖(酏剂或糖浆)菊粉(Raffiline HP)酸性寡糖(果胶水解产物)卵壳膜粉WPH(半胱氨酸肽)麦芽糊精(DE47)总量 | 2000342200129220011892001190SRSRSRMM | 4.006.0015.380.798.5421.0918.8074.60 | 0.000.000.000.000.1116.870.000.0217.00 | 0.000.004.780.000.090.000.0018.1222.99 | 4.006.000.000.000.000.000.000.0510.05 | 10.6716.0041.012.1122.7756.240.0050.13198.93 |
每日kcal | En% | 每Itrkcal | ||||
蛋白质的能量碳水化合物的能量脂肪的能量总量 | 689290250 | 27.236.736.1 | 181245241668 |
实施例7.液体营养组合物
原材料 | 代码 | g/天 | 蛋白质 | 碳水化合物 | 脂肪 | g/Itr |
琉璃苣油Ropufa25 n-6Maruha EPA-DHA油低聚半乳糖(酏剂或糖浆)Raftiline HP(菊粉)AOS(果胶水解产物)WPH(半胱氨酸肽)MD DE47总量 | 2000342200129220011892001190SRSRMM | 4.006.0015.380.798.5424.1913.5072.40 | 0.000.000.000.000.1120.830.0220.95 | 0.000.004.780.000.090.9213.0118.80 | 4.006.000.000.000.000.020.0310.06 | 10.6716.0041.012.1122.7764.5136.00193.07 |
每日kcal | En% | 每Itrkcal | ||||
蛋白质的能量碳水化合物的能量脂肪的能量总量 | 847591250 | 33.630.136.3 | 223201241665 |
Claims (11)
1.一种或多种酸性和中性寡糖和半胱氨酸和/或半胱氨酸源在制备一种组合物中的用途,所述组合物用于哺乳动物中HIV或AIDS的治疗和/或预防,所述的组合物包括酸性和中性寡糖,其中所述酸性寡糖是果胶水解产物,而所述中性寡糖选自低聚半乳糖、低聚果糖、反式低聚半乳糖、低聚木糖、乳果糖和低聚阿拉伯糖,且其中所述的半胱氨酸源是N-乙酰半胱氨酸和/或二乙酰半胱氨酸、乳清、初乳、卵蛋白或其组合物,其中所述酸性寡糖提供每日量10 mg至100 g之间的酸性寡糖,所述中性寡糖提供每日量10 mg至 100 g之间的中性寡糖,并且所述半胱氨酸和/或半胱氨酸源提供每日剂量至少100 mg的半胱氨酸等价物。
2..根据权利要求1的用途,其中所述乳清、初乳或卵蛋白是至少部分水解的。
3.根据权利要求1或2的用途,其中所述半胱酸和/或半胱氨酸源提供每日剂量至少600 mg的半胱氨酸等价物。
4.根据权利要求3的用途,其中所述半胱酸和/或半胱氨酸源提供每日剂量至少1000 mg的半胱氨酸等价物。
5.根据权利要求1的用途,其中所述中性寡糖是低聚果糖和低聚半乳糖的混合物。
6.根据权利要求1的用途,其中所述全部寡糖的至少15%含有酸性寡糖。
7.根据权利要求1的用途,其中所述组合物还包括多不饱和脂肪酸。
8.根据权利要求7的用途,其中所述多不饱和脂肪酸包括占所述总脂肪酸含量至少20%的γ亚麻酸和二十碳五烯酸。
9.一种食品组合物,包括15至50能量%的脂质、25至60能量%的蛋白质、15至45能量%的碳水化合物、果胶水解产物、中性寡糖和半胱氨酸和/或半胱氨酸源,其中所述半胱氨酸源选自N-乙酰半胱氨酸和/或二乙酰半胱氨酸、乳清、初乳、卵蛋白或其组合物,所述的中性寡糖选自低聚半乳糖、低聚果糖、反式低聚半乳糖、低聚木糖、乳果糖和低聚阿拉伯糖。
10.一种食品组合物,包括15至50能量%的脂质、35至60能量%的蛋白质、15至45能量%的碳水化合物、果胶水解产物、中性寡糖和半胱氨酸和/或半胱氨酸源,其中所述半胱氨酸源选自N-乙酰半胱氨酸和/或二乙酰半胱氨酸、乳清、初乳、卵蛋白或其组合物,所述的中性寡糖选自低聚半乳糖、低聚果糖、反式低聚半乳糖、低聚木糖、乳果糖和低聚阿拉伯糖。
11.根据权利要求9或10的食品组合物,其中所述的脂质包括二十碳五烯酸和/或γ亚麻酸。
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PCT/NL2006/050091 WO2006112716A2 (en) | 2005-04-21 | 2006-04-19 | Nutritional supplement for hiv patients |
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李毅敏等,1.N-乙酰半胱氨酸的研究进展.《天津药学》.2003,第15卷(第02期), |
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PL1871181T5 (pl) | 2017-10-31 |
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AU2006237738A2 (en) | 2006-10-26 |
EP1871181B2 (en) | 2016-12-21 |
RU2007143061A (ru) | 2009-05-27 |
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RU2421077C2 (ru) | 2011-06-20 |
EP1871181A2 (en) | 2008-01-02 |
NO20075009L (no) | 2008-01-18 |
ATE471665T1 (de) | 2010-07-15 |
AU2006237738A1 (en) | 2006-10-26 |
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CN101163415A (zh) | 2008-04-16 |
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AU2006237738B2 (en) | 2011-09-29 |
CA2605960A1 (en) | 2006-10-26 |
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