CN118176005A - 用于护肤和伤口治疗的生物表面活性剂制剂 - Google Patents
用于护肤和伤口治疗的生物表面活性剂制剂 Download PDFInfo
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- CN118176005A CN118176005A CN202280073431.3A CN202280073431A CN118176005A CN 118176005 A CN118176005 A CN 118176005A CN 202280073431 A CN202280073431 A CN 202280073431A CN 118176005 A CN118176005 A CN 118176005A
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Abstract
本发明提供了用于增强伤口愈合的外用治疗性组合物和其使用方法,所述伤口包括烧伤和皮肤疤痕,其中,所述组合物和方法利用微生物生物表面活性剂。本发明缩短了皮肤伤口的愈合时间,减少了疤痕的出现,并改善了其它皮肤病况,例如痤疮、银屑病和湿疹。
Description
相关申请的交叉引用
本申请要求于2021年11月1日提交的美国临时专利申请第63/274,063号的优先权,其通过引用方式以其全文并入本文中。
背景技术
伤口是由切割或其它形式的撞击造成的组织损伤。伤口可以是闭合的(例如,挫伤、闭合性骨折)或开放的(例如,割伤、擦伤、溃疡、损伤、压伤、擦伤、刺伤、拉伤、烧伤、撕裂伤、手术切口、枪伤、咬伤、蜇伤或撕脱伤)。
特别是在烧伤伤口的情况下,伤口可能是由以下造成的损伤:热、冷、电、化学物质(例如酸或碱)、摩擦或辐射(例如晒伤)。一度烧伤通常限于损伤部位的发红(红斑)、白色斑块和轻微疼痛。此等烧伤通常只延伸到表皮。充满清液、皮肤表层起泡并且取决于神经受累程度包括或轻或重的疼痛的烧伤是二度烧伤。二度烧伤包括真皮浅层(乳头层),也可能包括真皮深层(网织层)。
皮肤烧焦并产生坚硬的皮革状焦痂的烧伤是三度烧伤。焦痂是从身体未受影响的部分分离出来的痂。通常,也存在紫色流体和/或脓。神经末梢、毛囊和汗腺可能会丧失。三度烧伤通常会留下疤痕。
四度、五度和六度烧伤可能包括烧焦和/或皮肤、肌肉和/或骨骼完全丧失,并且在某些情况下可能是致命的。
伤口和烧伤的愈合可能是漫长且连续的过程,但它通常被认为是分时期发生的。所述过程在受伤后立即开始,伴随发炎期。在这个通常持续两天到一周的时期期间,去除了伤口中的受损组织和异物。当血小板产生形成凝块和止血的物质时,血管收缩并封闭。接着,血管在实现止血后扩张,并允许营养物、白细胞、抗体和酶进入受影响的区域,以促进愈合和防止感染。
在发炎反应之后并与之重叠的增生期的特点是,成纤维细胞增殖,以及胶原蛋白和蛋白聚糖产生。在这一时期,细胞外基质发生合成以便开始闭合伤口并为组织提供结构完整性。身体将受损的间充质细胞转变为成纤维细胞以充当桥梁并帮助细胞在受影响的区域周围移动。此等成纤维细胞或肌成纤维细胞产生并沉积胶原蛋白以帮助巩固伤口。增生期通常持续约四天到几周。肥厚性疤痕通常就是在这一时期形成的。
与增生期重叠,愈合的最后时期包括重塑伤口。在重塑期期间,先前构建的和随机组织的颗粒状组织基质被重塑成高度交联和排列以提高机械强度的组织化结构。当胶原蛋白和弹性蛋白沉积时,伤口疤痕成熟,并当成纤维细胞成为肌成纤维细胞时,伤口疤痕连续产生。肌成纤维细胞采用可收缩表型,因此参与了伤口挛缩。从成纤维细胞到肌成纤维细胞的转化控制着收缩与再上皮化之间的微妙平衡,这部分地决定了已修复伤口的柔韧性。除了成纤维细胞转化之外,角质形成细胞和炎性细胞凋亡是伤口愈合终止和伤口整体最终外观的关键步骤。重塑可能要花费长达两年或更长的时间。
虽然烧伤在一些方面不同于其它伤口,但所有伤口的愈合都包括上述动态过程。烧伤伤口的愈合可能要花费大量时间,并且经常出现不适、疼痛和形成疤痕。像其它伤口一样,烧伤伤口愈合所需的时间量取决于烧伤的深度和严重程度。举例来说,对于一些更严重的烧伤,受伤的组织可能会被切除并用皮移植片再移植。这可能会延长愈合过程。(Rowan等人2015)。
在许多情况下,伤口,包括烧伤伤口,可能会愈合,但会形成疤痕。疤痕可以是良性的,但在一些情况下,它们可以是异常的或过度的,导致疼痛、不动性、功能丧失和/或不美观的外观。由过度疤痕形成或伤口愈合异常导致的病况包括例如纤维化、纤维瘤病、瘢痕瘤病、粘连(例如手术粘连)、肥厚性疤痕、纤维囊性病况和关节僵硬。挛缩也可能会发生,其中伤口周围的皮肤被肌成纤维细胞以可能会导致严重活动受限的方式拉在一起。(Tiwari2012)。
在异常疤痕形成中,在一些情况下,产生的胶原蛋白比降解的胶原蛋白多,这也可能伴随着细胞外基质组分、细胞、纤连蛋白、弹性蛋白和蛋白聚糖的过量产生而发生。因此,疤痕生长得比伤口愈合所需的要大。或者,胶原蛋白可能会被不充分地替代,结果,皮肤可能会形成有凹痕的外观。
对于肥厚性疤痕,形成的基础病理生理学尚不十分清楚。肥厚性疤痕是过度伤口愈合的副作用,并通常导致细胞、胶原蛋白和蛋白聚糖的过量产生。由于活性愈合细胞在增生期受到抑制,肉芽组织沉积未能减少,通常导致成纤维细胞、小血管和胶原蛋白纤维排列成结节型。通常,此等疤痕是凸起的,且其特点是组织束的随机分布。此等疤痕的外观(即大小、形状和颜色)因它们形成的身体部位和患者的种族而变化。肥厚性疤痕极为常见,并且可能在皮肤的任何全层损伤之后发生。
瘢痕瘤也可以在增生期期间形成,在此处肉芽组织的过度生长比肥厚性疤痕更严重。瘢痕瘤通常被表征为由出现在真皮和邻近皮下组织中的高度增生性肿块组成的肿瘤,最常见于创伤后。它们是位于皮肤上方的坚硬、有弹性的病灶或有光泽的纤维性结节。瘢痕瘤通常比肥厚性疤痕更严重,因为它们倾向于侵入正常的邻近组织,而肥厚性疤痕倾向于保持限制在原始疤痕边界内。
与大多数其它创伤相比,由烧伤导致的疤痕形成可能会更广泛,因为损伤表面积更大。如果深层真皮烧伤不进行切除和移植,那么几乎肯定会出现肥厚性疤痕形成。许多烧伤患者需要长期疤痕治疗,所述疤痕伴随并发症,包括瘙痒、干燥、溃疡、对阳光和化学物质敏感以及与外观相关的情绪问题。
通常,治疗疤痕和瘢痕瘤的方法是昂贵的,且成功的概率低。举例来说,治疗可以包括手术切除、接着为移植物应用,在切除后有出现新疤痕的风险,或者压力绷带,其通常需要维持至少25mm Hg的压力约六个月以实现视觉效果。其它治疗包括电离放射疗法;将硅胶垫应用于疤痕组织表面,有时在弹性绷带提供的压力下进行;硅胶片的局部应用,伴随或不伴随维生素E的添加;用皮质类固醇进行的局部或病灶内治疗;以及干扰已知会参与伤口愈合、皮肤生长和疤痕形成的蛋白质的各种药物乳膏、粉末和珠粒。因此,已经多次尝试改善疤痕愈合并减少疤痕形成的不利美学影响,但成效或便利性不高。
因此,由手术、创伤、病理状况、烧伤、运动损伤等导致的皮肤损伤通常以留疤的方式愈合。这种疤痕形成通常在美学上是非所期望的,而且其还可能会导致其它不利影响,包括功能丧失、活动受限、皮肤弹性降低以及可能地生活质量降低。尽管迄今为止已经尝试提供促进皮肤伤口愈合,例如以减少疤痕形成的方法,但在这方面仍需要不断改进。
因此,需要改善伤口、烧伤和疤痕愈合过程的用于治疗皮肤病况的制剂(formation)。此外,强烈需要解决皮肤管理的愈合和美容方面的皮肤治疗,其将伤口(包括烧伤)的治愈时间以及由此产生的疤痕的出现减到最少。
发明内容
本发明提供了基于微生物的产品以及其在外用护肤和治疗性组合物中的使用方法。更具体地说,本发明提供了使用包含微生物生长副产物的组合物促进伤口(包括烧伤)愈合,同时减少疤痕的可见性并改善皮肤的年轻外观的方法。有利的是,本发明的组合物和方法无毒且经济实惠。
在本发明的优选实施方案中,提供了一种外用治疗性基于微生物的护肤组合物,其中,所述组合物包含一种或多种微生物生长副产物。在某些实施方案中,所述组合物进一步包含转铁蛋白。
在某些实施方案中,微生物生长副产物是两亲分子(例如,生物表面活性剂)、酶和/或蛋白质。
在一个优选的实施方案中,微生物生长副产物是选自以下的生物表面活性剂:例如低分子量糖脂(例如槐糖脂、鼠李糖脂、纤维二糖脂、甘露糖赤藓糖醇脂和海藻糖脂)、脂肽(例如表面活性素、伊枯草菌素、丰原素、节活性素(arthrofactin)和地衣素)、黄脂(flavolipid)、磷脂(例如心磷脂)、脂肪酸酯化合物、脂肪酸醚化合物和高分子量聚合物(例如脂蛋白、脂多糖-蛋白质复合物和多糖-蛋白质-脂肪酸复合物)。
一种或多种生物表面活性剂可以进一步包括以下中的任一种或以下的组合:生物表面活性剂的改性形式、衍生物、级分、同工型、异构体或亚型,包括生物改性或合成改性的形式。在某些实施方案中,生物表面活性剂是盐形式的生物表面活性剂。
生物表面活性剂可以具有抗微生物和/或抗生物膜性质;皮肤回春性质(意味着它们有助于使皮肤看起来例如更年轻、更光滑和更明亮);抗炎性质;和/或在伤口愈合期间,例如在增生期和/或重塑期期间,可以增强成纤维细胞形成、增殖和/或功能。
在某些实施方案中,本发明的生物表面活性剂能够增强所述组合物中的活性成分和非活性成分的真皮渗透;因此,生物表面活性剂可以充当活性成分以及其它成分的皮肤渗透增强剂。
在某些实施方案中,本发明的组合物进一步包含转铁蛋白,包括其单体形式和聚合物形式。转铁蛋白是糖蛋白,因其与铁和其它金属离子结合并介导其转运通过血浆的能力而众所周知。转铁蛋白也能够通过受体介导的转运绕过血-脑屏障。示范性转铁蛋白类型包括血清转铁蛋白、乳转铁蛋白(乳铁蛋白)和黑素转铁蛋白。
在一个具体实施方案中,转铁蛋白是乳铁蛋白,一种在例如哺乳动物乳汁中发现的糖蛋白。除了其主要生物功能,即铁离子的结合和转运之外,乳铁蛋白还具有抗菌、抗病毒、抗寄生虫、催化、抗癌和抗过敏功能和性质,这些在护肤组合物中会有所助益。
在一个示范性实施方案中,本发明的组合物包含盐形式的生物表面活性剂,优选盐形式的槐糖脂(SLP)生物表面活性剂。在一个具体的优选的实施方案中,所述组合物包含金属盐形式的酸性(线性)槐糖脂,例如钠盐或钙盐形式。有利的是,在某些实施方案中,SLP盐相比于非盐形式的SLP具有增强的真皮渗透性质。
在一个示范性实施方案中,所述组合物包含内酯形式与线性形式的SLP的混合物,例如就总体SLP来说,50%内酯和50%线性。
在另一个示范性实施方案中,所述组合物包含内酯形式或线性形式的SLP、甘露糖赤藓糖醇脂(MEL)生物表面活性剂或此等中的任一种的组合。优选的是,SLP是盐形式的线性SLP。甚至更优选的是,盐形式的SLP与MEL结合使用。
在又另一个示范性实施方案中,所述组合物包含盐形式的SLP、MEL和转铁蛋白。优选的是,转铁蛋白是乳铁蛋白。有利的是,在某些实施方案中,这种组分组合协同运作以通过例如增强的伤口和手术愈合、增强的免疫功能和/或增强的成纤维细胞产生、增殖和/或功能来改善皮肤健康。
在一些实施方案中,所述组合物包含能够产生用于皮肤愈合和回春的生长副产物的活的或灭活的微生物。在某些实施方案中,所述微生物是酵母,例如熊蜂生假丝酵母(Starmerella bombicola)、异常威克汉姆酵母(Wickerhamomyces anomalus)、季也蒙迈耶氏酵母(Meyerozyma guilliermondii)和/或蚜虫拟酵母(Pseudozyma aphidis)。在某些实施方案中,所述微生物是细菌,例如乳杆菌属(Lactobacillus spp.)、双歧杆菌属(Bifidospp.)、乳球菌属(Lactococcus spp.)、链球菌属(Streptococcus spp.)和芽孢杆菌属(Bacillus spp.),例如凝结芽孢杆菌(Bacillus coagulans)、解淀粉芽孢杆菌(Bacillusamyloliquefaciens)NRRL B-67928或枯草芽孢杆菌(Bacillus subtilis)B4 NRRL B-68031。
在一些实施方案中,所述组合物包含由此等微生物中的一种的发酵产生的上清液,其中,上清液包含抗炎、抗微生物和/或增强免疫的组分,所述组分与所述组合物的生物表面活性剂协同运作以增强皮肤健康。
所述组合物可以具有其它组分,包括例如载体、pH调节剂、缓冲剂、局部麻醉剂、促进伤口愈合的药剂、帮助降解生物膜的药剂、抗微生物剂、止血和/或促进凝块形成的药剂,以及已知用于例如使皮肤愈合、补充皮肤、使皮肤回春、保湿皮肤、保护皮肤和/或改善皮肤的愈合、外观和/或运作的其它治疗性组分和非治疗性组分。举例来说,在某些实施方案中,所述组合物可以进一步包含维生素、矿物质、植物性药材、提取物、精油、维A酸、抗粉刺剂、保湿剂、抗氧化剂和/或遮光剂。
在一个实施方案中,所述组合物可以进一步包含皮肤病学上可接受的载体,例如油包水或水包油乳液,或含水血清。外用组合物可以进一步包含通常在外用护肤组合物中存在的佐剂和添加剂,例如有机溶剂、硅酮、稳定剂、增稠剂、软化剂、染料或香料。
在某些实施方案中,本发明提供了促进皮肤病况愈合和/或改善的方法,其中,将本发明的外用治疗性组合物直接施用于皮肤的存在这类病况的区域。在某些实施方案中,皮肤病况是损伤,例如伤口、烧伤、疤痕或切口,或例如老化、痤疮、感染、银屑病、老年斑、湿疹、皮炎或光化性角化病的病况。
根据本发明,“促进”愈合和/或改善意味着增强或加速愈合和/或改善速率和/或以其它方式在皮肤病况部位创造有利于其愈合和/或改善的条件。
在一些实施方案中,“施用”所述组合物包含将所述组合物留在伤口或疤痕上,和/或将其揉搓到里面以使得所述组合物被完全吸收到所述区域中。在一些实施方案中,将所述组合物施用到皮肤持续治疗有效的时间量,然后使用例如水或布将其从皮肤上冲洗或去除。在又其它实施方案中,使用可透气的聚合物基质来施用所述组合物,所述聚合物基质可以浸渍了所述组合物并用作伤口、疤痕或其它病况的敷料、贴片或覆盖物。
在某些实施方案中,例如每隔一天、每天一次、每天至多十次施用外用治疗性组合物。在一些实施方案中,每隔一天、每天一次、每天至多十次施用所述外用组合物,持续不确定的时间段,例如持续至少一周、两周、三周或更长时间,直到皮肤病况愈合和/或改善。
具体实施方式
本发明提供了基于微生物的产品以及其在外用美容和治疗性组合物中的使用方法。更具体地说,本发明提供了使用包含微生物生长副产物的组合物促进伤口(包括烧伤)愈合,同时减少疤痕的可见性并改善皮肤的年轻外观的方法。有利的是,本发明的组合物和方法无毒且经济实惠。
选定的定义
如本文所用,用语“愈合”和/或“改善”病况或病症是指根除病况或病症的征象或症状、将病况或病症的征象或症状减轻、改善或逆转至任何程度,并且包括但不要求完全治愈所述病况或病症。
如本文所用,“预防”病况或病症是指避免、延迟、预先阻止或最小化所述病况或病症的特定征象或症状的发作。预防可以是、但不要求是绝对的或完全的,这意味着征象或症状可能仍然会在一个未来时间出现。预防可以包括降低这类病况或病症发作的严重程度,和/或抑制所述病况或病症发展成更严重的病况或病症。
如本文所用,“促进”是指增强、提高或加速预期效果出现的速率。举例来说,促进伤口愈合可以意味着通过加快皮肤愈合的速度、防止疤痕形成和/或以其它方式在伤口部位创造有利于伤口愈合的条件来改善皮肤愈合过程。作为另一个实施例,促进疤痕愈合可以意味着减小疤痕的尺寸或可见性,减少与疤痕相关的阴性症状(例如,在瘢痕瘤或挛缩的情况下)和/或以其它方式在疤痕部位创造有利于疤痕愈合的条件。
如本文所用,用语“伤口”是指由例如切割、猛击或其它撞击造成的组织损伤。根据本发明,伤口包括被归类为“开放性伤口”的皮肤损伤,并且包括例如割伤、擦伤、溃疡、损伤、擦伤、压伤、刺伤、拉伤、烧伤、撕裂伤、切口、枪伤、咬伤、蜇伤和撕脱伤。
如本文所用,用语“烧伤”是指由热源(热或冷)、化学物质源(例如,来自酸或碱)、摩擦源、辐射源(例如,晒伤或UV)或电源造成的伤口。烧伤可以是“轻微”烧伤,包括伴随真皮外层浅层损伤的一度烧伤和伴随向下延伸到细胞表皮层的损伤的二度烧伤。烧伤的症状包括例如刺激、起泡、瘙痒、脱皮、皮疹、发红和肿胀。
如本文所用,用语“疤痕”是指皮肤上的纹路或增生,其中,损伤,例如伤口、烧伤、疮、手术切口或穿孔,没有真正地愈合,并且纤维结缔组织已经代替正常组织发展。疤痕可以包括肥厚性疤痕,其中,胶原蛋白的过量产生在周围皮肤上方产生凸起组织区域;瘢痕瘤,另一种形式的过度疤痕形成,其中,组织成形为大的突出的赘生物;萎缩性疤痕,其中,基层结构组织损失,造成有凹痕或凹陷的外观(例如痤疮疤痕);和妊娠纹,由例如怀孕、生长突增或皮肤再生期间皮肤的快速拉伸造成。
除了促进伤口(包括烧伤和/或疤痕)愈合之外,本发明的其它用途可以包括治疗和/或预防例如其它皮肤病况。如本文所用,用语“皮肤病况”涵盖影响外皮或皮肤的任何人类和动物病况、病症或疾病。这种皮肤病况包括但不限于涉及表皮、真皮(包括结缔组织、皮脂腺和毛囊)和皮下组织(subcutaneous tissue/hypodermis)的病况。在某些实施方案中可以使用本文所描述的组合物、产品和方法进行治疗和/或预防的皮肤病况包括但不限于伤口(包括例如烧伤)、疤痕、痤疮、瘢点、湿疹、银屑病、酒渣鼻、毛囊炎、癌瘤、黑色素瘤、口周皮炎、蜂窝组织炎、疖子、痈、光损伤、皮肤老化(例如皱纹、下垂、干燥)、老年斑、鱼鳞癣(ichtiosis)、特应性皮炎、皮疹(包括但不限于红斑、黄斑、丘疹和/或大疱病况)、晒伤、水疱、寻常性天疱疮、大疱性类天疱疮、获得性大疱性表皮松解症(EBA)、疱疹样皮炎、疣(人乳头瘤病毒)、营养不良性溃疡、慢性伤口、褥疮、毛发角化病、皮脂腺囊肿、白癜风、黄褐斑(melisma)、发炎性皮肤病、痛觉超敏、异位性皮炎、毛细血管扩张、炎症后色素沉着、角化病、干燥症、瘙痒症、扁平苔藓、硬化性苔藓、结节性痒疹、微生物感染、体臭、脱发、头皮病况和痱子。皮肤病况的症状可以包括例如皮肤刺激/敏感、瘢点和其它痤疮样症状、色素沉着或其损失、潮红、发炎、皱纹、干燥、松弛、增厚、脱皮、形成疤痕、片层剥落、皮疹、荨麻疹、水疱、溃疡、蜕皮、脱发以及皮肤健康、功能和外观的其它变化。
如本文所用,用语“受试者”是指动物,优选哺乳动物。在本发明的上下文中,优选的受试者是人。受试者可以是任何性别和任何年龄或发展阶段,包括婴儿、幼儿、青春期少年、青少年、年轻人、中年人或老年人。
如本文所用,“外用”意思指适合于皮肤外部的局部施用,或皮肤上施用。换句话说,外用组合物不打算用于通过口服、静脉内、肌肉内、鞘内、皮下、舌下、颊、直肠、阴道、吸入、眼部或耳部途径施用于受试者。
如本文所用,“皮肤病学上可接受的”、“美容上可接受的”和“外用可接受的”可互换使用,并旨在意味着特定组分对于以所用含量施用于外皮(例如皮肤)是安全且无毒的。在一个实施方案中,所述组合物的组分被认为是公认安全的(GRAS)。
如本文所用,用语“治疗有效量”、“有效量”和“有效剂量”用于指能够在受试者中实现期望量的愈合的某种事物(例如,化合物、组合物、时间)的量。实际量将取决于许多因素,包括但不限于需要愈合的特定病况或病症、病况的严重程度、受试者的尺寸、年龄和健康状况以及施用方式。
如本文所用,“基于微生物的组合物”是指包含因微生物或其它细胞培养物生长而产生的组分的组合物。因此,基于微生物的组合物可以包含微生物本身和/或微生物生长副产物(例如,生物表面活性剂、溶剂和/或酶)。细胞可能处于植物状态或呈孢子形式,或两者兼而有之。细胞可以是浮游的或呈生物膜形式,或两者兼而有之。细胞可以是活的或非活性的、完整的或裂解的。细胞可以从它们生长的培养基中移除,或以例如每毫升所述组合物至少1×103、1×104、1×105、1×106、1×107、1×108、1×109、1×1010或1×1011个或更多个细胞的浓度存在。在一个实施方案中,基于微生物的组合物可以仅包含细胞生长的培养基,或上清液,其中细胞已移除(但在一些情况下,一些残余细胞物质也可以保留在培养基中)。生长副产物可以存在于培养基中,并且可以包括例如代谢物、细胞膜组分、表达的蛋白质和/或其它细胞组分。在一个实施方案中,基于微生物的组合物仅包含微生物生长副产物。
本发明进一步提供了“基于微生物的产品”,其是在实践中应用以实现期望结果的产品。基于微生物的产品可以仅是从微生物培养过程中收获的基于微生物的组合物。或者,基于微生物的产品可以包含已经添加的另外的成分。此等附加成分可以包括例如稳定剂、缓冲剂、载体以及适用于特定应用的其它添加剂和/或佐剂。基于微生物的产品还可以包含基于微生物的组合物的混合物。基于微生物的产品还可以包含已经以某种方式,例如但不限于过滤、离心、裂解、干燥、纯化等加以处理的基于微生物的组合物的一种或多种组分。
“代谢物”是指通过代谢产生的任何物质(例如生长副产物)或参与特定代谢过程所必需的物质。代谢物的实施例包括但不限于酶、酸、溶剂、醇、蛋白质、碳水化合物、维生素、矿物质、微量元素、氨基酸、聚合物和生物表面活性剂。
如本文所用,用语“分离的”或“纯化的”在与生物或天然材料例如核酸分子、多核苷酸、多肽、蛋白质、有机化合物例如小分子、微生物细胞/菌株或宿主细胞结合使用时意思指所述材料基本上不含在本质上与其相关的其它化合物例如细胞材料。也就是说,所述材料并非不含此等其它化合物而天然存在的,和/或与天然材料中发现的特征相比具有不同或独特的特征。
在某些实施方案中,纯化的化合物是至少60重量%感兴趣的化合物。优选的是,制剂(formulation)是期望化合物重量的至少75%(w/w)、更优选至少90%(w/w)并且最优选至少99%(w/w)或100%(w/w)。纯度是通过任何适当的标准方法,例如通过柱色谱、薄层色谱或高效液相色谱(HPLC)分析来进行测量。
如本文所用,“异构体”是指与另一个分子具有相同化学式、但具有独特结构的分子。异构体可以是结构异构体,其中,原子和官能团在不同的位置键结;和立体异构体(空间异构体),其中,键结构是相同的,但原子和官能团在空间中的几何定位是不同的。MEL异构体例如可以在碳水化合物、脂肪酸和/或乙酰基的键类型和键位置方面有所不同。
如本文所用,“表面活性剂”意思指表面活性物质或降低两相之间的表面张力(或界面张力)的化合物。表面活性剂用作例如洗涤剂、润湿剂、乳化剂、发泡剂和/或分散剂。“生物表面活性剂”意思指由活生物体产生和/或使用天然衍生的底物产生的表面活性剂。
过渡用语“包含”与“包括”或“含有”同义,是包容性的或开放式的,并且不排除附加的、未列举的元素或方法步骤。相比之下,过渡短语“由……组成”排除权利要求书中未规定的任何元素、步骤或成分。过渡短语“基本上由……组成”将权利要求书的范围限制为规定的材料或步骤“以及不会实质上影响所要求保护的发明的基本和新颖特征的材料或步骤”。用语“包含”的使用考虑了“由所叙述的组分组成”或“基本上由所叙述的组分组成”的其它实施方案。
除非具体说明或从上下文中显而易见,否则如本文所用的用语“或”被理解为是包容性的。除非具体说明或从上下文中显而易见,否则如本文所用的用语“一种(a/an)”和“所述”被理解为是单数或复数的。
除非具体说明或从上下文中显而易见,否则如本文所用的用语“约”被理解为在所属领域的正常公差范围内,例如在平均值的2个标准差内。“约”可以被理解为在规定值的10%、9%、8%、7%、6%、5%、4%、3%、2%、1%、0.5%、0.1%、0.05%或0.01%内。
本文中对变量的任何定义中的化学基团清单的叙述包括将所述变量定义为所列基团中的任何单个基团或组合。本文中对变量或方面的实施方案的叙述包括所述实施方案为任何单个实施方案或与任何其它实施方案或其部分的组合。
本文所提供的任何组合物或方法可以与本文所提供的任何其它组合物和方法中的一种或多种组合。本发明的其它特点和优点将从以下对本发明的优选实施方案的描述以及权利要求书中显而易知。本文引用的所有参考文献都通过引用并入在此。
外用护肤组合物
本发明提供了护肤组合物和其使用方法。特别地说,本发明提供了可以治疗和/或预防皮肤病况(包括伤口和疤痕)和/或本文所描述的其它病况的治疗性护肤产品。
在某些实施方案中,本发明利用微生物生长副产物。特别地说,本发明的实施方案提供了一种促进皮肤病况的愈合和/或改善的外用组合物,其中,所述组合物包含治疗有效量的通过微生物培养产生的一种或多种生物化学物质。
在某些实施方案中,微生物生长副产物是两亲分子、酶和/或蛋白质。在一个实施方案中,微生物生长副产物具有抗微生物、抗炎和/或抗生物膜性质。在一个实施方案中,微生物生长副产物具有皮肤回春性质,这意味着它们有助于使皮肤看起来更年轻、更光滑和更明亮。在一个实施方案中,微生物生长副产物可以改变成纤维细胞产生、增殖和/或功能以增强皮肤损伤的愈合。
在一个优选的实施方案中,所述组合物包含一种或多种两亲分子,其中,所述两亲分子是选自以下的生物表面活性剂:例如低分子量糖脂(例如槐糖脂、鼠李糖脂、纤维二糖脂、甘露糖赤藓糖醇脂和海藻糖脂)、脂肽(例如表面活性素、伊枯草菌素、丰原素、节活性素(arthrofactin)和地衣素)、黄脂(flavolipid)、磷脂(例如心磷脂)、脂肪酸酯化合物、脂肪酸醚化合物和高分子量聚合物(例如脂蛋白、脂多糖-蛋白质复合物和多糖-蛋白质-脂肪酸复合物)。
生物表面活性剂是两亲物,这意味着它们包含极性(亲水)部分和非极性(疏水)基团。由于其两亲性结构,生物表面活性剂降低了液体、固体和气体分子之间的表面张力和界面张力。
另外,生物表面活性剂在界面积聚,从而引起溶液中形成聚集胶束结构。生物表面活性剂形成孔并使生物膜不稳定的能力准许它们用作例如抗细菌剂和抗真菌剂以及使其它化合物穿过例如膜的递送剂。此外,生物表面活性剂是可生物降解的,具有低毒性,并且可以使用低成本的可再生资源产生。它们可以抑制微生物粘附到各种表面,防止生物膜形成,并可以具有强大的乳化和反乳化性质。
一种或多种生物表面活性剂可以进一步包括以下中的任一种或以下的组合:生物表面活性剂的改性形式、衍生物、级分、同工型、异构体或亚型,包括生物改性或合成改性的形式。在一个实施方案中,一种或多种生物表面活性剂是以临界胶束浓度(CMC)存在于所述组合物中。
有利的是,本发明的生物表面活性剂能够进行以下中的一种或多种:杀死皮肤内/皮肤上的病原体,调节皮肤的免疫系统,杀死黑素细胞以允许替代细胞生长,减少氧化应激,减少促炎细胞因子,增强角质形成细胞和成纤维细胞的增殖和功能,以及增强皮肤健康增强化合物的真皮渗透。因此,它们本身提供治疗益处,并且还可以增强外用组合物中可能存在的其它组分在治疗皮肤病况方面的有效性。
在优选的实施方案中,生物表面活性剂是槐糖脂(SLP)和/或甘露糖赤藓糖醇脂(MEL),两者都是由某些酵母产生的糖脂生物表面活性剂。糖脂通常包含连接到可以被乙酰化或烷基化的鞘脂或甘油基团的单糖或寡糖基团以及一种或多种脂肪酸。
SLP是由例如假丝酵母分支酵母,包括蜜生假丝酵母(Candida apicola)和熊蜂生假丝酵母产生。在一些实施方案中,异常威克汉姆酵母是SLP生产者(例如,异常威克汉姆酵母NRRL Y-68030)。SLP由连接到长链羟基脂肪酸的二糖槐糖组成。此等SLP是以β-糖苷连接到17-L-羟基十八烷酸或17-L-羟基-Δ9-十八烷酸的部分乙酰化的2-O-β-D-吡喃葡萄糖基-D-吡喃葡萄糖单元。羟基脂肪酸通常有16或18个碳原子,并且可以含有一个或多个不饱和键。脂肪酸羧基可以是自由的(酸性或开放形式)或在4″位内部酯化的(内酯形式)。
SLP具有在宽范围的温度、pH和盐度条件下的环境相容性、高生物降解性、低毒性、高选择性和比活性(specific activity)。此外,在一些实施方案中,SLP对于化妆品和皮肤病学中的应用可能有利,因为它们具有小胶束尺寸,这可以有助于促进胶束和包封在其中的化合物移动通过纳米级孔和空间(例如,在上皮细胞之间,并进入生物膜基质中)。在某些实施方案中,SLP的胶束尺寸是小于100nm、小于50nm、小于20nm、小于15nm、小于10nm或小于5nm。
在优选的实施方案中,本发明的SLP是由通式(1)和/或通式(2)表示,并且以多个结构同源物的集合形式获得:
其中,R1和R1’独立地表示饱和烃链或单个或多个,特别是单个,具有8到20个,特别是12到18个碳原子,更优选14到18个碳原子的不饱和烃链,所述不饱和烃链可以是线性的或分支的并且可以包含一个或多个羟基,R2和R2’独立地表示氢原子或饱和烷基官能团或单个或多个,特别是单个,具有1到9个碳原子、更优选1到4个碳原子的不饱和烷基官能团,所述不饱和烷基官能团可以是线性的或分支的并且可以包含一个或多个羟基,并且R3、R3’、R4和R4’独立地表示氢原子或-COCH3。R5可以是-OH或-H。
在某些实施方案中,本发明的SLP是由通式(2)表示的盐形式的SLP,其中,R5=-OX,并且X=金属离子,例如Na、K、Mg、Cu或Ca。
有利的是,SLP作为化妆品组合物中的活性或非活性成分可以具有几项益处。此等益处可以包括例如乳化水包油或油包水混合物;减轻发炎和氧化应激;从皮肤上层去除受损的角质形成细胞;通过抗微生物和抗炎作用增强伤口愈合;促进成纤维细胞代谢;改善胶原蛋白合成和调理/重建皮肤;刺激通过脂肪细胞进行的瘦素合成和帮助减少导致皮下脂肪团的皮下脂肪过载;抑制弹性蛋白酶活性并减少皱纹出现;使皮肤上的斑点脱落和褪色并抑制黑素生成;控制微生物头皮屑、痤疮和体臭;和/或减少发炎病况,例如皮炎、湿疹和银屑病。
在优选的实施方案中,外用化妆品组合物中的SLP浓度是总组合物重量的0.001%到90%、0.01%到50%、0.05%到10%、或0.1%到2.0%。
在某些实施方案中,就100%总体SLP来说,线性SLP与内酯SLP的比率是约10%:90%、20%:80%、30%:70%、40%:60%、50%:50%、60%:40%、70%:30%、80%:20%或90%:10%。
在一个实施方案中,外用组合物包含呈酸性形式的SLP。在一个优选的实施方案中,酸性SLP是盐形式的SLP。有利的是,在某些实施方案中,SLP盐相比于非盐形式的SLP具有增强的真皮渗透性质。
在一个实施方案中,糖脂是MEL。MEL包含作为亲水部分的4-O-B-D-吡喃甘露糖基-内消旋赤藓糖醇或1-O-B-D-吡喃甘露糖基-内消旋赤藓糖醇以及作为疏水部分的脂肪酸基团和/或乙酰基。通常在甘露糖残基的C4和/或C6处的一个或两个羟基可以被乙酰化。此外,可以存在一到三个链长为8到12个碳或更多个碳的酯化脂肪酸。
MEL和MEL样物质(例如基于甘露糖的物质)主要由蚜虫拟酵母属和黑粉菌属(Ustilago spp.)产生,并且每个物种产生的MEL结构中具有相当大的可变性。具有与MEL类似的性质的某些基于甘露糖的物质也可以由季也蒙迈耶氏酵母产生。
MEL无毒并且在宽范围的温度和pH下稳定。此外,MEL可以不伴随任何附加的防腐剂使用。
MEL可以超过93种不同的组合形式产生,所述组合分为5大类:MEL A、MEL B、MELD、三乙酰化MEL A和三乙酰化MEL B/C。此等分子可以被改性,无论合成地或天然地。举例来说,MEL可以包含不同碳长链或不同数量的乙酰基和/或脂肪酸基团。
本发明的MEL分子和/或它们的改性形式可以包括例如三酰化、二酰化、单酰化、三乙酰化、二乙酰化、单乙酰化和非乙酰化MEL以及它们的立体异构体和/或结构异构体。
根据本发明,也可以使用展现出类似结构和类似性质的其它基于甘露糖的物质/MEL样物质,例如甘露糖基-甘露醇脂(MML)、甘露糖基-阿拉伯糖醇脂(MAL)和/或甘露糖基-核糖醇脂(MRL)。
有利的是,MEL作为化妆品组合物中的活性或非活性成分可以具有几项益处。此等益处可以包括例如减轻皮肤发炎;预防因使用合成表面活性剂例如SDS所致的细胞损伤;刺激毛球细胞和毛发生长;修复和/或强化受损毛发(特别是例如MEL-A和MEL-B);提高成纤维细胞和乳头细胞的生存力(特别是例如MEL-A);减少排汗并帮助皮肤水分屏障发挥作用;和/或减少老年斑中的黑色素含量。
在优选的实施方案中,外用化妆品组合物中的MEL(或MEL样物质)浓度是总组合物重量的0.001%到90%、0.01%到50%、0.05%到10%、或0.1%到2.0%。在某些实施方案中,就所述组合物的总量来说,所述组合物包含小于10ppm MEL或小于5ppm MEL。
在一个具体的优选的实施方案中,本发明的外用组合物包含SLP和MEL,其中,MEL以所述组合物重量的约0.1%到2.0%、优选约1.0%存在;并且其中,SLP以所述组合物重量的约0.01%到约1.0%、优选约0.5%存在。
在一个实施方案中,生物表面活性剂可以包含一种或多种脂肽,例如表面活性素、伊枯草菌素、丰原素、节活性素(arthrofactin)、粘液菌素、两性霉素(amphisin)、丁香霉素和/或地衣素。
在一个具体的实施方案中,脂肽生物表面活性剂是表面活性素。脂肽由多种益生菌和非致病菌,例如纳豆芽孢杆菌(Bacillus natto)、凝结芽孢杆菌、枯草芽孢杆菌、解淀粉芽孢杆菌、乳酸菌等产生。在一些实施方案中,脂肽由特定芽孢杆菌菌株,例如解淀粉芽孢杆菌NRRL B-67928或枯草芽孢杆菌B4NRRL B-68031产生。
在一个实施方案中,表面活性剂可以包含一种或多种微生物产生的脂肪酸酯化合物和/或脂肪酸醚化合物,它们具有与生物表面活性剂的物理性质和/或行为类似的物理性质和/或行为,但通常不称为生物表面活性剂。
在某些实施方案中,脂肪酸酯化合物可以包括例如高度酯化的油酸脂肪酸,例如油酸脂肪酸乙酯和/或油酸脂肪酸甲酯(FAME)。
在一些实施方案中,外用组合物可以包含浓度为总组合物重量的约0.001%到90%、约0.01%到50%、约0.05%到10%、约0.1%到5.0%、或约0.01%到2.0%的此等其它两亲分子。
在一些实施方案中,两亲分子是以粗制物形式使用,其中,所述分子存在于生长培养基(例如培养液)中,在所述培养基中培养产生两亲物的微生物并不经纯化就从所述培养基中收集它。粗制物形式可以包含例如生长培养基中的至少0.001%、1%、5%、10%、20%、30%、40%、50%、60%、70%、80%、90%或99%的两亲分子。在替代实施方案中,两亲分子是从培养产物中纯化的。
在某些实施方案中,本发明的组合物进一步包含转铁蛋白,包括其单体形式和聚合物形式。转铁蛋白是糖蛋白,因其与铁和其它金属离子结合并介导其转运通过血浆的能力而众所周知。转铁蛋白也能够通过受体介导的转运绕过血-脑屏障。示范性转铁蛋白类型包括血清转铁蛋白、乳转铁蛋白(乳铁蛋白)和黑素转铁蛋白。
在一个具体实施方案中,转铁蛋白是乳铁蛋白,一种在例如哺乳动物乳汁中发现的糖蛋白。在优选的实施方案中,乳铁蛋白以总组合物重量的0.001%到90%、0.01%到50%、0.05%到10%、或0.1%到2.0%的浓度存在于外用组合物中。
在某些示范性实施方案中,外用组合物包含SLP、MEL和乳铁蛋白的组合。优选的是,SLP是线性SLP盐,例如钠盐SLP。有利的是,在某些实施方案中,这种组分组合协同运作以通过例如增强的伤口和手术愈合、减轻的发炎、增强的免疫功能和/或增强的成纤维细胞产生、增殖和/或功能来改善皮肤健康。在某些实施方案中,SLP有助于增加乳铁蛋白的透皮吸收以增强益处,例如细胞增殖、胶原蛋白合成和透明质酸合成。
在某些实施方案中,盐形式的SLP可以在乳铁蛋白周围形成胶束、脂质体或囊泡以促进其通过皮肤的吸收。在某些实施方案中,盐形式的SLP可以与乳铁蛋白结合以促进其通过皮肤的吸收。
在一些实施方案中,本发明的外用组合物包含能够产生用于皮肤愈合和回春的生长副产物的活的或灭活的微生物(例如生物表面活性剂)。在某些实施方案中,所述微生物是酵母,例如熊蜂生假丝酵母、异常威克汉姆酵母(例如NRRL Y-68030)、季也蒙迈耶氏(毕赤)酵母(Meyerozyma(Pichia)guilliermondii)和/或蚜虫拟酵母。在一个具体的实施方案中,所述组合物包含活的或灭活的异常威克汉姆酵母。
在某些实施方案中,所述微生物是细菌,例如乳杆菌属、双歧杆菌属、乳球菌属、链球菌属和芽孢杆菌属,例如酸快生芽孢杆菌、酸居芽孢杆菌、产酸芽孢杆菌、酸热芽孢杆菌、酸土芽孢杆菌、喜空芽孢杆菌、空气芽孢杆菌、嗜气芽孢杆菌、黏琼脂芽孢杆菌、土壤芽孢杆菌、艾丁氏芽孢杆菌(B.aidingensis)、秋白芽孢杆菌(B.akibai)、嗜碱芽孢杆菌、居藻芽孢杆菌、鲜藻芽孢杆菌、嗜碱固氮芽孢杆菌、碱性腈芽孢杆菌、碱性沉积物芽孢杆菌、碱土芽孢杆菌、高地芽孢杆菌、香鱼海槽芽孢杆菌、蜂房芽孢杆菌、解淀粉芽孢杆菌、解淀粉芽孢杆菌解淀粉亚种、解淀粉芽孢杆菌植生亚种、溶淀粉芽孢杆菌(B.mylolyticus)、安德里森芽孢杆菌(B.andreesenii)、解硫胺素芽孢杆菌、炭疽芽孢杆菌、海水芽孢杆菌、沙地芽孢杆菌、砷硒芽孢杆菌、砷芽孢杆菌、橙黄色芽孢杆菌、田地芽孢杆菌、阿氏芽孢杆菌(B.aryabhattai)、丰井氏芽孢杆菌(B.asahii)、萎缩芽孢杆菌、阿萨尔基亚芽孢杆菌(B.axarquiensis)、固氮芽孢杆菌、产氮芽孢杆菌、栗褐芽孢杆菌、罕见芽孢杆菌、巴达维亚芽孢杆菌(B.bataviensis)、北京芽孢杆菌(B.beijingensis)、食苯芽孢杆菌、柏林芽孢杆菌(B.beringensis)、伯克利芽孢杆菌(B.berkeleyi)、贝弗里奇芽孢杆菌(B.beveridgei)、博戈里亚芽孢杆菌(B.bogoriensis)、嗜硼芽孢杆菌、波茨坦芽孢杆菌(B.borstelensis)、短芽孢杆菌(B.brevis Migula)、食丁酸芽孢杆菌、卡纳维拉尔芽孢杆菌(B.canaveralius)、嗜碳芽孢杆菌、科研中心芽孢杆菌(B.cecembensis)、解纤维芽孢杆菌、中孢芽孢杆菌(B.centrosporus)、蜡样芽孢杆菌、恰甘诺湖芽孢杆菌(B.chagannorensis)、解几丁质芽孢杆菌、软骨酸芽孢杆菌、千叶芽孢杆菌、长安芽孢杆菌(B.chungangensis)、食物芽孢杆菌(B.cibi)、环状芽孢杆菌、克氏芽孢杆菌(B.clarkii)、克劳氏芽孢杆菌(B.clausii)、凝结芽孢杆菌、科阿韦拉芽孢杆菌(B.coahuilensis)、科氏芽孢杆菌(B.cohnii)、堆肥芽孢杆菌(B.composti)、解凝乳芽孢杆菌、环庚基脂环酸芽孢杆菌(B.cycloheptanicus)、细胞毒素芽孢杆菌、大理芽孢杆菌(B.daliensis)、腐叶芽孢杆菌、脱色芽孢杆菌、沙漠芽孢杆菌、蜥蜴芽孢杆菌、钻特省芽孢杆菌(B.drentensis)、环境芽孢杆菌(B.edaphicus)、爱媛芽孢杆菌(B.ehimensis)、赤子芽孢杆菌(B.eiseniae)、结束芽孢杆菌(B.enclensis)、植物内生芽孢杆菌、根尖内生芽孢杆菌、混料芽孢杆菌(B.farraginis)、苛求芽孢杆菌(B.fastidiosus)、封丘芽孢杆菌(B.fengqiuensis)、坚强芽孢杆菌(B.firmus)、弯曲芽孢杆菌(B.flexus)、小孔芽孢杆菌(B.foraminis)、福氏芽孢杆菌(B.fordii)、美丽芽孢杆菌(B.formosus)、强壮芽孢杆菌(B.fortis)、富马里奥利芽孢杆菌(B.fumarioli)、绳索状芽孢杆菌(B.funiculus)、纺锤芽孢杆菌(B.fusiformis)、嗜乳豆芽孢杆菌、解半乳糖芽孢杆菌、加里西亚芽孢杆菌(B.galliciensis)、明胶芽孢杆菌(B.gelatini)、吉氏芽孢杆菌(B.gibsonii)、人参芽孢杆菌(B.ginsengi)、人参土芽孢杆菌(B.ginsengihumi)、人参土壤芽孢杆菌(B.ginsengisoli)、圆芽孢杆菌(B.globisporus)、圆芽孢杆菌圆亚种、圆芽孢杆菌海洋亚种(B.g.subsp.marinus)、解葡糖芽孢杆菌、戈登氏芽孢杆菌(B.gordonae)、哥蒂氏芽孢杆菌(B.gottheilii)、草芽孢杆菌(B.graminis)、盐敏芽孢杆菌(B.halmapalus)、嗜盐碱芽孢杆菌(B.haloalkaliphilus)、盐虫芽孢杆菌(B.halochares)、盐反硝化芽孢杆菌(B.halodenitrificans)、耐盐芽孢杆菌(B.halodurans)、嗜盐芽孢杆菌(B.halophilus)、盐糖芽孢杆菌(B.halosaccharovorans)、解半纤维素芽孢杆菌(B.hemicellulosilyticus)、马粪海胆芽孢杆菌(B.hemicentroti)、黑布施泰因芽孢杆菌(B.herbersteinensis)、堀越氏芽孢杆菌(B.horikoshii)、堀崎芽孢杆菌(B.horneckiae)、花园芽孢杆菌(B.horti)、惠州芽孢杆菌(B.huizhouensis)、土地芽孢杆菌(B.humi)、花津滩芽孢杆菌(B.hwajinpoensis)、病研所芽孢杆菌(B.idriensis)、印度芽孢杆菌(B.indicus)、婴儿芽孢杆菌(B.infantis)、下层芽孢杆菌(B.infernus)、异常芽孢杆菌(B.insolitus)、无敌芽孢杆菌(B.invictae)、伊朗芽孢杆菌(B.iranensis)、伊氏芽孢杆菌(B.isabeliae)、伊斯隆恩西斯芽孢杆菌(B.isronensis)、咸海鲜芽孢杆菌(B.jeotgali)、好热地芽孢杆菌(B.kaustophilus)、哥本芽孢杆菌(B.kobensis)、郭霍氏芽孢杆菌(B.kochii)、木芥子形芽孢杆菌(B.kokeshiiformis)、韩国芽孢杆菌(B.koreensis)、库尔勒芽孢杆菌(B.korlensis)、胶冻样芽孢杆菌(B.kribbensis)、克鲁氏芽孢杆菌(B.krulwichiae)、左旋乳酸芽孢杆菌(B.laevolacticus)、幼虫芽孢杆菌(B.larvae)、侧孢芽孢杆菌(B.laterosporus)、灿烂芽孢杆菌(B.lautus)、列城芽孢杆菌(B.lehensis)、缓病芽孢杆菌(B.lentimorbus)、迟缓芽孢杆菌(B.lentus)、地衣芽孢杆菌(B.licheniformis)、嗜木质素芽孢杆菌(B.ligniniphilus)、岸滨芽孢杆菌(B.litoralis)、盐地芽孢杆菌(B.locisalis)、路西法芽孢杆菌(B.luciferensis)、浅黄芽孢杆菌(B.luteolus)、藤黄芽孢杆菌(B.luteus)、澳门芽孢杆菌(B.macauensis)、软化芽孢杆菌(B.macerans)、马阔里芽孢杆菌(B.macquariensis)、马氏芽孢杆菌(B.macyae)、巨大芽孢杆菌(B.malacitensis)、解甘露醇糖芽孢杆菌(B.mannanilyticus)、黄海芽孢杆菌(B.marisflavi)、死海芽孢杆菌(B.marismortui)、马尔马里斯芽孢杆菌(B.marmarensis)、马赛芽孢杆菌(B.massiliensis)、巨大芽孢杆菌(B.megaterium)、仙草芽孢杆菌(B.mesonae)、甲醇芽孢杆菌(B.methanolicus)、甲基营养型芽孢杆菌(B.methylotrophicus)、米氏芽孢杆菌(B.migulanus)、莫海威芽孢杆菌(B.mojavensis)、胶胨样芽孢杆菌(B.mucilaginosus)、壁芽孢杆菌(B.muralis)、马丁教堂芽孢杆菌(B.murimartini)、蕈状芽孢杆菌(B.mycoides)、长野芽孢杆菌(B.naganoensis)、南海芽孢杆菌(B.nanhaiensis)、南海沉积物芽孢杆菌(B.nanhaiisediminis)、尼氏芽孢杆菌(B.nealsonii)、内德里芽孢杆菌(B.neidei)、雷州芽孢杆菌(B.neizhouensis)、农研所芽孢杆菌(B.niabensis)、烟酸芽孢杆菌(B.niacini)、休闲地芽孢杆菌(B.novalis)、海泥芽孢杆菌(B.oceanisediminis)、奥德赛芽孢杆菌(B.odysseyi)、奥哈芽孢杆菌(B.okhensis)、奥飞騨芽孢杆菌(B.okuhidensis)、蔬菜芽孢杆菌(B.oleronius)、稻壳芽孢杆菌(B.oryzaecorticis)、大岛芽孢杆菌(B.oshimensis)、饲料芽孢杆菌(B.pabuli)、巴基斯坦芽孢杆菌(B.pakistanensis)、苍白芽孢杆菌(B.pallidus)、苍白芽孢杆菌(B.pallidus)、人参地土壤芽孢杆菌(B.panacisoli)、人参地块芽孢杆菌(B.panaciterrae)、泛酸芽孢杆菌(B.pantothenticus)、副短芽孢杆菌(B.parabrevis)、近弯芽孢杆菌(B.paraflexus)、巴氏芽孢杆菌(B.pasteurii)、巴塔哥尼亚芽孢杆菌(B.patagoniensis)、蓼属植物芽孢杆菌(B.peoriae)、珀塞波尔芽孢杆菌(B.persepolensis)、桃色芽孢杆菌(B.persicus)、佩尔瓦格芽孢杆菌(B.pervagus)、海绵芽孢杆菌(B.plakortidis)、抱川芽孢杆菌(B.pocheonensis)、多边芽孢杆菌(B.polygoni)、多粘芽孢杆菌(B.polymyxa)、日本丽金龟芽孢杆菌(B.popilliae)、假嗜碱芽孢杆菌(B.pseudalcalophilus)、假坚强芽孢杆菌(B.pseudofirmus)、假真菌芽孢杆菌(B.pseudomycoides)、耐冷芽孢杆菌(B.psychrodurans)、噬冷芽孢杆菌(B.psychrophilus)、冷解糖芽孢杆菌(B.psychrosaccharolyticus)、耐寒芽孢杆菌(B.psychrotolerans)、尘埃芽孢杆菌(B.pulvifaciens)、短小芽孢杆菌(B.pumilus)、抗净化芽孢杆菌(B.purgationiresistens)、厚壁芽孢杆菌(B.pycnus)、青岛芽孢杆菌(B.qingdaonensis)、庆笙氏芽孢杆菌(B.qingshengii)、罗伊氏芽孢杆菌(B.reuszeri)、球状根芽孢杆菌(B.rhizosphaerae)、井水芽孢杆菌(B.rigui)、农庄芽孢杆菌(B.ruris)、沙福芽孢杆菌(B.safensis)、盐芽孢杆菌(B.salarius)、需盐芽孢杆菌(B.salexigens)、喜盐芽孢杆菌(B.saliphilus)、施氏芽孢杆菌(B.schlegelii)、沉积物芽孢杆菌(B.sediminis)、硒砷芽孢杆菌(B.selenatarsenatis)、还原硒酸盐芽孢杆菌(B.selenitireducens)、西岸芽孢杆菌(B.seohaeanensis)、莎车芽孢杆菌(B.shacheensis)、沙氏芽孢杆菌(B.shackletonii)、暹罗芽孢杆菌(B.siamensis)、森林狼芽孢杆菌(B.silvestris)、简单芽孢杆菌(B.simplex)、青贮窖芽孢杆菌(B.siralis)、史氏芽孢杆菌(B.smithii)、土壤芽孢杆菌(B.soli)、土壤红树芽孢杆菌(B.solimangrovi)、盐土芽孢杆菌(B.solisalsi)、宋克伦芽孢杆菌(B.songklensis)、索诺拉沙漠芽孢杆菌(B.sonorensis)、球形芽孢杆菌(B.sphaericus)、耐热芽孢杆菌(B.sporothermodurans)、嗜热嗜脂肪芽孢杆菌(B.stearothermophilus)、同温层芽孢杆菌(B.stratosphericus)、地下芽孢杆菌(B.subterraneus)、枯草芽孢杆菌、枯草芽孢杆菌沙漠亚种(B.s.subsp.inaquosorum)、枯草芽孢杆菌斯氏亚种(B.s.subsp.spizizenii)、枯草芽孢杆菌枯草亚种、台湾芽孢杆菌(B.taeanensis)、特基拉芽孢杆菌(B.tequilensis)、热南极芽孢杆菌(B.thermantarcticus)、嗜热嗜气芽孢杆菌(B.thermoaerophilus)、热嗜淀粉芽孢杆菌(B.thermoamylovorans)、热小链芽孢杆菌(B.thermocatenulatus)、热阴沟芽孢杆菌(B.thermocloacae)、嗜热粪生芽孢杆菌(B.thermocopriae)、嗜热脱氮芽孢杆菌(B.thermodenitrificans)、热葡糖苷芽孢杆菌(B.thermoglucosidasius)、热乳芽孢杆菌(B.thermolactis)、噬热芽孢杆菌(B.thermoleovorans)、嗜热芽孢杆菌(B.thermophilus)、热红芽孢杆菌(B.thermoruber)、热球状芽孢杆菌(B.thermosphaericus)、溶硫胺芽孢杆菌(B.thiaminolyticus)、产硫芽孢杆菌(B.thioparans)、苏云金芽孢杆菌(B.thuringiensis)、天神氏芽孢杆菌(B.tianshenii)、三脚芽孢杆菌(B.trypoxylicola)、托斯卡纳芽孢杆菌(B.tusciae)、强壮芽孢杆菌(B.validus)、死谷芽孢杆菌(B.vallismortis)、威氏芽孢杆菌(B.vedderi)、贝莱斯芽孢杆菌(B.velezensis)、越南芽孢杆菌(B.vietnamensis)、原野芽孢杆菌(B.vireti)、火山芽孢杆菌(B.vulcani)、和光芽孢杆菌(B.wakoensis)、威恩施蒂芽孢杆菌(B.weihenstephanensis)、厦门芽孢杆菌(B.xiamenensis)、小溪芽孢杆菌(B.xiaoxiensis)、湛江芽孢杆菌(B.zhanjiangensis)、解淀粉芽孢杆菌NRRL B-67928、枯草芽孢杆菌B4 NRRL B-68031和/或凝结芽孢杆菌(例如BC-30)。
在一些实施方案中,所述组合物包含由此等微生物中的一种的发酵产生的上清液,其中,上清液包含抗炎、抗微生物和/或增强免疫的组分,所述组分与所述组合物的生物表面活性剂协同运作以增强皮肤健康。
上清液可以占总组合物的0.001重量%到99.9重量%、约0.01重量%到约75重量%、或约0.1重量%到约25重量%。
根据本发明有用的附加的微生物生长副产物包括甘露糖蛋白、β-葡聚糖、酶和具有生物乳化和表面/界面张力降低性质的其它代谢物。
在一些实施方案中,外用化妆品组合物可以包含治疗有效量的由微生物产生的酶和/或蛋白质。举例来说,可以包括约0.001重量%到约20重量%、约0.01重量%到约15重量%或约0.05重量%到约10重量%的一种或多种酶和/或蛋白质。此等酶和/或蛋白质可以包括但不限于外切-β-1,3-葡聚糖酶;几丁质酶;酯酶;脂肪酶;糖苷酶;淀粉酶;和有益于改善皮肤健康的蛋白酶。
在一些实施方案中,外用治疗性组合物可以进一步包含皮肤病学上可接受的载体或媒剂。
所述载体或媒剂可以包括例如水;盐水;生理盐水;软膏;乳膏;油水乳液;油包水乳液;水包硅酮乳液;硅酮包水乳液;水包蜡乳液;水-油-水三重乳液;微乳液;凝胶;植物油;矿物油;酯油,例如棕榈酸辛酯、肉豆蔻酸异丙酯和棕榈酸异丙酯;醚,例如二辛基醚和二甲基异山梨醇;醇,例如乙醇和异丙醇;脂肪醇,例如鲸蜡醇、鲸蜡硬脂醇、硬脂醇和山嵛醇;异链烷烃,例如异辛烷、异十二烷(IDD)和异十六烷;硅油,例如环甲硅油、二甲硅油、二甲硅油交叉聚合物、聚硅氧烷和它们的衍生物,优选有机改性的衍生物,包括PDMS、二甲硅油共聚醇、二甲硅油醇和氨基二甲硅油醇;烃类油,例如矿物油、矿脂、异二十烷和聚烯烃(例如(氢化)聚异丁烯);多元醇,例如丙二醇、甘油、丁二醇、戊二醇、己二醇、辛二醇;蜡,例如蜂蜡、巴西棕榈蜡、地蜡、微晶蜡、聚乙烯蜡和植物蜡;或前述物质的任何组合或混合物。含水媒剂可以包括一种或多种与水混溶的溶剂,包括低级醇,例如乙醇、异丙醇等。所述媒剂可以占所述组合物重量的约1%到约99%、10%到约85%、25%到75%、或50%到约65%。
除非另有指示,否则如本文所用的用语“油”包括硅油。乳液可以包括乳化剂,例如非离子、阴离子或两性表面活性剂,或没食子酸盐,它们的量通常为约0.001重量%到约5重量%。
在一些实施方案中,外用组合物可以进一步包含外用或局部麻醉剂。外用麻醉剂可以包括例如丁卡因、苯佐卡因、丙美卡因、普鲁卡因、丙氧卡因、地布卡因、利多卡因、达克罗宁和丙嗪。麻醉剂可以以例如在约0.05重量%与25重量%之间、在约0.25重量%与10重量%之间、在约0.5重量%与10重量%之间以及在约1重量%与5重量%之间的不同的浓度存在。在给定制剂中采用的麻醉剂的确切剂量将取决于许多因素,例如待采用的特定麻醉剂。举例来说,在含有利多卡因的组合物中,示范性优选剂量范围是在约1重量%与20重量%之间,而在含有丁卡因的组合物中,示范性优选剂量是约0.5%。
在一些实施方案中,外用组合物可以进一步包含通常包括在护肤组合物中的附加佐剂和添加剂,例如有机溶剂、稳定剂、硅酮、增稠剂、软化剂、遮光剂、保湿剂、香料或本文所描述的其它佐剂和添加剂。每种成分(无论是活性的还是非活性的)的量都是在化妆品领域中常规使用以实现其预期目的的量,并且通常在所述组合物的约0.0001%到约25%或约0.001%到约20%的范围内,但所述量可以落在此等范围之外。此等成分的性质和其量必须与本公开的组合物的产生和功能相容。
在一个实施方案中,所述组合物可以包括附加的皮肤活性剂,仅举几例,所述皮肤活性剂包括但不限于角质溶解剂、剥离溶解剂、角质细胞增殖增强剂、胶原酶抑制剂、弹性蛋白酶抑制剂、褪色剂、抗炎剂、类固醇、抗痤疮剂、抗氧化剂和晚期糖基化终末产物(AGE)抑制剂。
在一个实施方案中,所述组合物可以包括抗老化组分,所述抗老化组分包括但不限于植物性药材(例如,甄叔迦树(Butea frondosa)提取物);植醇;植烷酸;磷脂;硅酮;矿脂;甘油三酯;ω脂肪酸;维A酸;羟基酸(包括α-羟基酸和β-羟基酸)、水杨酸和烷基水杨酸盐;去角质剂(例如乙醇酸、3,6,9-三噁十一烷二酸等)、雌激素合成酶刺激化合物(例如咖啡因和衍生物);能够抑制5α-还原酶活性的化合物(例如亚麻酸、亚油酸、非那雄胺和它们的混合物);和屏障功能增强剂(例如神经酰胺、甘油酯、胆固醇和其酯、α-羟基脂肪酸和ω-羟基脂肪酸以及它们的酯)。
示范性维A酸包括但不限于视黄酸(例如,全反式或9-顺式或13-顺式)和其衍生物、视黄醛、视黄醇(维生素A)和其酯,例如棕榈酸视黄酯、乙酸视黄酯和丙酸视黄酯,以及它们的盐。当存在时,维A酸通常以约0.0001重量%到约5重量%、更通常约0.01重量%到约2.5重量%、或约0.1重量%到约1.0重量%的量包括在内。
在一个实施方案中,所述组合物可以包括去角质剂。合适的去角质剂包括例如α-羟基酸、β-羟基酸、氧杂酸、氧杂二酸和它们的衍生物例如酯、酸酐和它们的盐。合适的羟基酸包括例如乙醇酸、乳酸、苹果酸、酒石酸、柠檬酸、2-羟基链烷酸、扁桃酸、水杨酸和它们的衍生物。一种示范性去角质剂是乙醇酸。当存在时,去角质剂可以占所述组合物的约0.001重量%到约20重量%。
在一个实施方案中,所述组合物可以包含一种或多种抗氧化剂。合适的抗氧化剂包括例如具有酚羟基官能团的化合物,例如抗坏血酸和其衍生物/酯;ω-3脂肪酸(例如DHA、EPA);β-胡萝卜素;儿茶素;姜黄素;阿魏酸衍生物(例如阿魏酸乙酯、阿魏酸钠);没食子酸衍生物(例如没食子酸丙酯);番茄红素;还原酸;迷迭香酸;单宁酸;四氢姜黄素;生育酚和其衍生物,包括生育酚乙酸酯;尿酸;或它们的任何混合物。其它合适的抗氧化剂是呈还原或非还原形式的具有一个或多个硫醇官能团(-SH)的抗氧化剂,例如谷胱甘肽、硫辛酸、巯基乙酸和其它巯基化合物。抗氧化剂可以是无机的,例如亚硫酸氢盐、偏亚硫酸氢盐、亚硫酸盐或其它含硫的无机盐和酸。抗氧化剂可以单独或共同占所述组合物总重量的约0.001%(w/w)到约10%(w/w)或约0.01%(w/w)到约5%(w/w)。
非生物表面活性剂也可以被添加到制剂中。表面活性剂的实施例包括但不限于烷基硫酸盐、烷基醚硫酸盐(例如,月桂基硫酸钠/铵和月桂醇聚醚硫酸钠/铵)、两性物(例如,两性乙酸酯和两性丙酸酯)、磺基琥珀酸酯、烷基聚葡糖苷、甜菜碱(例如,椰油酰胺丙基甜菜碱(CAPB))、磺基甜菜碱、沙可劳塞酯(sacrosinate)、羟乙基磺酸盐、牛磺酸盐、乙氧基化脱水山梨醇酯、链烷醇酰胺和基于氨基酸的表面活性剂。
粘度调节剂也可以被添加到所述组合物中,包括例如椰油酰胺DEA、油酰胺DEA、氯化钠、纤维素聚合物、聚丙烯酸酯、乙氧基化酯、醇、乙二醇、二甲苯磺酸盐、聚山梨醇酯20、链烷醇酰胺和纤维素衍生物(例如羟丙基甲基纤维素和羟乙基纤维素)。
聚合物也可以被添加,包括例如黄原胶、瓜尔胶、聚季铵盐-10、PEG-120甲基葡萄糖二油酸酯、PEG-150二硬脂酸酯、PEG-150聚甘油-2三硬脂酸酯和PEG-150季戊四醇四硬脂酸酯。
遮光剂或遮光剂的组合可以被包括在内以保护皮肤不受UVA和UVB射线之害。可用于本发明组合物中的遮光剂是阿伏苯宗、肉桂酸衍生物(例如肉桂酸辛基甲氧酯)、水杨酸辛酯、羟苯甲酮、奥克立林、二氧化钛、氧化锌或它们的任何混合物。遮光剂可以以所述组合物总重量的约1wt%到约30wt%存在。
所述组合物可以任选地包含所属领域的技术人员已知的其它组分、添加剂或佐剂,所述其它组分、添加剂或佐剂包括但不限于:皮肤渗透增强剂;润肤剂(例如肉豆蔻酸异丙酯、矿脂、挥发性或非挥发性硅油(例如甲硅油和二甲硅油)、酯油、矿物油和脂肪酸酯);湿润剂(例如甘油、己二醇、辛二醇);皮肤饱满剂(例如棕榈酰寡肽、胶原蛋白、胶原蛋白和/或糖胺聚糖(GAG)增强剂);抗炎剂(例如芦荟、生物类黄酮、双氯芬酸、水杨酸);螯合剂(例如EDTA或其盐,例如EDTA二钠);维生素(例如生育酚和抗坏血酸);维生素衍生物(例如抗坏血酸单棕榈酸酯、生育酚乙酸酯、维生素E棕榈酸酯);增稠剂(例如羟烷基纤维素、羧甲基纤维素、卡波姆和植物胶(例如黄原胶));胶凝剂(例如酯封端的聚酯酰胺);结构化剂;蛋白质;免疫调节剂(例如皮质类固醇和非类固醇免疫调节剂)。
可以包括在内的其它组分是成膜剂、保湿剂、矿物质、粘度调节剂和/或流变改性剂、驱虫剂、皮肤冷却化合物、皮肤保护剂、润滑剂、防腐剂、珍珠、chromalite、云母、调理剂、抗过敏剂、抗微生物剂(例如,抗真菌剂、抗病毒剂、抗菌剂)、杀菌剂(antiseptic)、药剂、光稳定剂、表面平滑剂、光学扩散剂和去角质促进剂。关于此等和其它合适的化妆品成分的细节可以在以下中找到:《国际化妆品原料字典和手册》第10版(2004),由化妆品、盥洗品和芳香品协会(CTFA)出版,第2177-2299页,其全文通过引用并入本文中。此等不同物质的量是化妆品或药物领域中常规地使用的量,例如,它们可以占所述组合物总重量的约0.01%到约20%。
所述组合物可以包括待在宽范围的pH水平内配制的pH调节剂(例如,柠檬酸、乙醇胺、氢氧化钠等)。在一个实施方案中,外用组合物的pH范围是1.0到13.0。在一些实施方案中,外用组合物的pH范围是2.0到12.0。适用于本发明组合物的其它pH范围包括3.5到7.0或7.0到10.5。可以添加合适的pH调节剂例如氢氧化钠、柠檬酸和三乙醇胺以使pH在期望范围内。
所述组合物可以配制为悬浮液、乳液、水凝胶、多相溶液、囊泡分散液或呈任何其它已知形式的外用皮肤组合物。
在某些实施方案中,外用组合物可以被配制成使得其可以例如通过笔、管、瓶、刷、棒、海绵、棉签、湿巾(擦拭巾)、喷雾器、滴管、手或手指来施用。
所述组合物可以配制成多种产品形式,例如乳液、乳膏、浆液、喷剂、气雾剂、液体蛋糕、软膏、香精、凝胶、糊状物、贴片、铅笔、粉末、湿巾、肥皂、条皂、洗发剂、护发剂、沐浴露、棒状物、泡沫、摩丝、酏剂或浓缩物。在优选的实施方案中,所述组合物被配制成使得其特别适合局部施用于皮肤。
在一个实施方案中,所述组合物可以被掺入伤口敷料、贴片或绷带中,所述伤口敷料、贴片或绷带可以被施用、附着或结合到受试者的一层或多层皮肤或组织。举例来说,所述组合物可以被施用于伤口敷料,然后可以将所述伤口敷料放置在所治疗的皮肤区域上方。
伤口敷料可以例如由皮肤病学上可接受并且适合放置在伤口上的任何材料制成。在示范性实施方案中,伤口敷料可以由合成纤维或非合成纤维的织物或无纺布、或它们的任何组合制成。敷料还可以包括支撑物,例如可以吸收组合物或已经在其上布置组合物的聚合物泡沫、天然或人造海绵、凝胶或膜。再次举例来说,支撑物可以是膜、天然或合成聚合物、或刚性或延展性材料(例如,纱布)。伤口敷料可以是吸收性的,并且可以例如在将纱布施用到伤口、疤痕或其它部位之前用本发明的组合物加以润湿。
在一个实施方案中,伤口敷料由聚合物聚羟基丁酸酯(PHB)做成。PHB是由细菌产生的可生物降解的聚酯。所述化合物可以被做成柔性的、可呼吸的基质,当浸渍了本发明的组合物时,所述基质向底层皮肤提供愈合和再生化合物(healing and rejuvenatingcompounds)的逐渐释放。
在一个实施方案中,伤口敷料可以浸渍本发明的组合物,并且任选地进行干燥。这允许将浸渍的敷料储存起来以备后续使用,或避免过度弄湿受伤区域。可以通过用所述组合物的溶液润湿敷料的表面并干燥敷料以使所述组合物沉积在其上来将所述组合物施用于敷料的表面。
促进皮肤病况愈合和/或改善的方法
在某些实施方案中,本发明提供了促进皮肤病况愈合和/或改善的方法,其中,将本发明的外用化妆品组合物直接施用于皮肤的存在这类病况的区域。在优选的实施方案中,皮肤病况是伤口,例如烧伤或手术切口,或疤痕。在一些实施方案中,将所述组合物施用于患有另一类皮肤病况例如痤疮、银屑病、湿疹、皱纹、老化、下垂和干燥的皮肤。所述组合物可以被施用于皮肤的任何外部区域,所述外部区域包括例如面部、耳朵、头皮、颈部、背部、肩部、手臂、手、手指、胸部、躯干、腹部、腋下、脚、脚趾、臀部和腿部的皮肤。
在一些实施方案中,“施用”所述组合物可以包含将所述组合物留在皮肤的所述区域上,和/或将其揉搓到里面以使得所述组合物被完全吸收到所述区域中。在一些实施方案中,可以将所述组合物施用到皮肤持续治疗有效的时间量,然后使用例如水或布将其从皮肤上冲洗或去除。
在又另一个实施方案中,所述组合物可以被浸渍到贴片或伤口敷料中,并根据标准敷料程序通过用浸渍的敷料覆盖皮肤来施用于皮肤。在一个具体实施方案中,伤口敷料由PHB制成。
在某些实施方案中,将外用组合物每天施用零到十次,优选每天至少一次或每隔一天至少一次。在一些实施方案中,每天或每隔一天施用外用组合物,持续不确定的时间段,例如持续至少一周、两周、三周或更长时间,以便实现和/或维持对皮肤病况的治疗。
优选的是,当施用于伤口时,伤口已经进入愈合的增生期或此后的愈合期。在一些实施方案中,伤口已经进入愈合的哪个时期的判定可以视觉上或使用用于作出这类判定的标准分析或测试来判定。
在一个实施方案中,所述组合物可以被大量施用于皮肤,优选以覆盖期望治疗的整个区域;然而,应该只需要一个薄涂层就能实现期望效果。在一个实施方案中,所述组合物是以约0.001到约100mg/cm2皮肤、更通常约0.01到约20mg/cm2皮肤或约0.1到约10mg/cm2皮肤的量施用。然而,取决于待治疗的皮肤的区域的大小,可以或多或少地使用。
本发明的外用组合物和方法可以用于促进被归类为开放性伤口的伤口的愈合,所述伤口包括烧伤和烧伤相关刺激、水疱和皮疹。优选的是,伤口已经过了愈合的发炎期,并且已经进入愈合的增生期或愈合的后一时期。
此外,本发明的外用组合物和方法可以用于促进疤痕(例如,肥厚性疤痕、痤疮疤痕、挛缩和/或瘢痕瘤)的愈合。举例来说,本发明可以用于缓解或减少疤痕的出现和/或存在,或缓解或减少疤痕的负面影响,例如不动、疼痛或瘙痒。
此外,本发明的外用组合物和方法可以用于改善皮肤病况的症状,例如痤疮、银屑病和湿疹。所述组合物可以作为例如清洁剂、去角质剂、免洗型斑点治疗或保湿治疗来施用。
微生物生长和微生物生长副产物生产
本发明提供了培养微生物和生产微生物代谢物和/或其它微生物生长副产物的方法。如本文所用,“发酵”是指细胞在受控条件下的生长。生长可以是好氧的或厌氧的。
在一个实施方案中,本发明提供了生产生物质(例如,活细胞材料)、细胞外代谢物(例如,小分子和排泄的蛋白质)、残余营养物和/或细胞内组分(例如,酶和其它蛋白质)的材料和方法。
根据本发明使用的微生物生长容器可以是任何工业用发酵罐或培养反应器。在一个实施方案中,容器可以具有功能性控制件/传感器,或可以连接到功能性控制件/传感器,以测量培养过程中的重要因素,例如pH、氧气、压力、温度、搅拌器轴功率、湿度、粘度和/或微生物密度和/或代谢物浓度。
在另一个实施方案中,容器还能够监测容器内的微生物生长(例如,细胞数和生长期测量)。或者,可以从容器中取出每日样品,并通过所属领域中已知的技术例如稀释平板技术进行计数。
在一个实施方案中,所述方法包括在培养中补充氮源。氮源可以是例如硝酸钾、硝酸铵、硫酸铵、磷酸铵、氨、尿素和/或氯化铵。此等氮源可以独立地或以两种或更多种的组合形式使用。
所述方法可以为生长培养物提供氧合。一个实施方案利用空气的慢速运动来去除含低氧空气并引入含氧空气。含氧空气可以是每天通过机械装置补充的环境空气,所述机械装置包括用于机械搅拌液体的叶轮和用于向液体供应气体气泡以将氧气溶解到液体中的空气喷射器。
所述方法可以进一步包含在培养中补充碳源。碳源通常是碳水化合物,例如葡萄糖、蔗糖、乳糖、果糖、海藻糖、甘露糖、甘露醇和/或麦芽糖;有机酸,例如乙酸、富马酸、柠檬酸、丙酸、苹果酸、丙二酸和/或丙酮酸;醇,例如乙醇、丙醇、丁醇、戊醇、己醇、异丁醇和/或甘油等;脂肪和油,例如大豆油、米糠油、橄榄油、玉米油、芝麻油和/或亚麻籽油;等等。此等碳源可以独立地或以两种或更多种的组合形式使用。
在一个实施方案中,培养基中包括用于微生物的生长因子和微量营养物。当所生长的微生物不能产生它们所需的所有维生素时,这是特别优选的。培养基中也可以包括无机营养物,所述无机营养物包括微量元素,例如铁、锌、铜、锰、钼和/或钴。此外,维生素、必需氨基酸和微量元素的源可以例如以面粉或膳食例如玉米粉的形式、或以提取物例如酵母提取物、马铃薯提取物、牛肉提取物、大豆提取物、香蕉皮提取物等的形式、或以纯化的形式包括在内。也可以包括氨基酸,例如对蛋白质的生物合成有用的氨基酸。
在一个实施方案中,也可以包括无机盐。可用的无机盐可以是磷酸二氢钾、磷酸氢二钾、磷酸氢二钠、硫酸镁、氯化镁、硫酸铁、氯化铁、硫酸锰、氯化锰、硫酸锌、氯化铅、硫酸铜、氯化钙、碳酸钙和/或碳酸钠。此等无机盐可以独立地或以两种或更多种的组合形式使用。
在一些实施方案中,培养方法可以进一步包含在培养过程之前和/或期间在液体培养基中添加附加的酸和/或抗微生物剂。抗微生物剂或抗生素可用于保护培养物免受污染。此外,还可以添加消泡剂来防止培养过程期间泡沫的形成和/或积聚。
混合物的pH应适合感兴趣的微生物。缓冲剂和pH调节剂例如碳酸酯和磷酸酯可以用于将pH稳定在优选值附近。当金属离子以高浓度存在时,可能需要在液体培养基中使用螯合剂。
用于培养微生物和生产微生物副产物的方法和设备可以在分批、准连续或连续过程中执行。
在一个实施方案中,培养微生物的方法是在约5℃到约100℃、优选15℃到60℃、更优选25℃到50℃下进行。在另一个实施方案中,培养可以在恒温下连续进行。在另一个实施方案中,培养可能会经历温度变化。
在一个实施方案中,所述方法和培养过程中使用的设备是无菌的。培养设备例如反应器/容器可以与灭菌单元例如高压釜分隔开、但连接。培养设备还可以具有在开始接种之前原位灭菌的灭菌单元。空气可以通过所属领域中已知的方法进行灭菌。举例来说,环境空气可以在被引入容器之前通过至少一个过滤器。在其它实施方案中,可以对培养基进行巴氏法灭菌,或任选地,根本不加热培养基,其中可以利用低水活性和低pH来控制细菌生长。
在一个实施方案中,本发明提供了通过在适合代谢物生长和生产的条件下培养本发明的微生物菌株来生产微生物代谢物的方法,所述微生物代谢物包括例如生物表面活性剂、酶和/或其它蛋白质。所产生的培养物的代谢物含量可以是例如至少20%、30%、40%、50%、60%、70%、80%或90%。
发酵培养液的生物质含量可以是例如5g/l到180g/l或更高、或10g/l到150g/l。
由感兴趣的微生物产生的微生物生长副产物可以保留在微生物中或分泌到液体培养基中。在另一个实施方案中,产生微生物生长副产物的方法可以进一步包含浓缩并纯化感兴趣的微生物生长副产物的步骤。在另一个实施方案中,液体培养基可以含有使微生物生长副产物的活性稳定的化合物。
在一个实施方案中,在培养完成时(例如,在例如实现期望细胞密度或培养基中特定代谢物的密度时),移除所有微生物培养物。在这一分批程序中,在收获第一批后,开始全新的批次。
在另一个实施方案中,在任何一个时间仅移除一部分发酵产物。在这一实施方案中,具有活细胞的生物质保留在容器中作为新培养批次的接种剂。所移除的组合物可以是无细胞培养液或含有细胞。以这种方式创建了准连续系统。
有利的是,所述方法不需要复杂设备或高能耗。可以在现场小规模或大规模培养感兴趣的微生物并利用,甚至与它们的培养基仍然混合。类似地,也可以在需要的位置大量产生微生物代谢物。
微生物可以是例如细菌、酵母和/或真菌。此等微生物可以是天然或转基因微生物。举例来说,可以用特定基因使微生物转型以展现出特定特征。微生物也可以是期望菌株的突变体。如本文所用,“突变体”意思指参考微生物的菌株、遗传变体或亚型,其中,与参考微生物相比,突变体具有一种或多种遗传变异(例如,点突变、错义突变、无义突变、缺失、复制、移码突变或重复扩增)。制造突变体的程序在微生物学领域中众所周知。举例来说,UV诱变和亚硝基胍被广泛用于这一目的。
在某些实施方案中,微生物能够产生两亲分子、酶、蛋白质和/或生物聚合物。特别地说,微生物生物表面活性剂由多种微生物例如细菌、真菌和酵母产生,所述细菌、真菌和酵母包括例如:土壤杆菌属(Agrobacterium spp.)(例如放射形土壤杆菌(A.radiobacter));节杆菌属(Arthrobacter spp.);曲霉菌属(Aspergillus spp.);短梗霉属(Aureobasidium spp.)(例如出芽短梗霉(A.pullulans));固氮菌(Azotobacter)(例如维氏固氮菌(A.vinelandii)、褐球固氮菌(A.chroococcum));固氮螺菌属(Azospirillumspp.)(例如巴西固氮螺菌(A.brasiliensis));芽孢杆菌属(例如枯草芽孢杆菌、解淀粉芽孢杆菌、凝结芽孢杆菌、短小芽孢杆菌、蜡样芽孢杆菌、地衣芽孢杆菌、坚强芽孢杆菌、侧孢芽孢杆菌、巨大芽孢杆菌(B.megaterium));布拉氏霉(Blakeslea);假丝酵母属(Candidaspp.)(例如白假丝酵母(C.albicans)、皱褶假丝酵母(C.rugosa)、热带假丝酵母(C.tropicalis)、解脂假丝酵母(C.lipolytica)、白球拟酵母(C.torulopsis));梭菌(Clostridium)(例如丁酸梭菌(C.butyricum)、酪丁酸梭菌(C.tyrobutyricum)、醋酪酸梭菌(C.acetobutyricum)和拜氏梭菌(C.beijerinckii));弯曲杆菌属(Campylobacterspp.);棒状杆菌属(Cornybacterium spp.);隐球菌属(Cryptococcus spp.);德巴利酵母属(Debaryomyces spp.)(例如汉逊德巴利酵母(D.hansenii));虫霉属(Entomophthoraspp.);黄杆菌属(Flavobacterium spp.);戈登氏菌属(Gordonia spp.);汉逊酵母属(Hansenula spp.);有孢汉逊酵母属(Hanseniaspora spp.)(例如葡萄有孢汉逊酵母(H.uvarum));伊萨酵母属(Issatchenkia spp);克鲁维酵母属(Kluyveromyces spp.);迈耶氏酵母属(Meyerozyma spp.)(例如季也蒙迈耶氏酵母);被孢霉属(Mortierella spp.);菌根属(Mycorrhiza spp.);分支杆菌属(Mycobacterium spp.);诺卡菌属(Nocardiaspp.);毕赤酵母属(Pichia spp.)(例如异常毕赤酵母(P.anomala)、季也蒙毕赤酵母(P.guilliermondii)、西方毕赤酵母(P.occidentalis)、库德里阿兹氏毕赤酵母(P.kudriavzevii));须霉属(Phycomyces spp.);腐霉菌属(Phythium spp.);假单胞菌属(Pseudomonas spp.)(例如铜绿假单胞菌(P.aeruginosa)、绿叶假单胞菌(P.chlororaphis)、恶臭假单胞菌(P.putida)、荧光假单胞菌(P.florescens)、莓实假单胞菌(P.fragi)、丁香假单胞菌(P.syringae));拟酵母属(Pseudozyma spp.)(例如蚜虫拟酵母(P.aphidis));罗尔斯通菌属(Ralslonia spp.)(例如富养罗尔斯通菌(R.eulropha));红球菌属(Rhodococcus spp.)(例如红平红球菌(R.erythropolis));红螺菌属(Rhodospirillum spp.)(例如深红红螺菌(R.rubrum));根瘤菌属(Rhizobium spp.);根霉属(Rhizopus spp.);酵母属(Saccharomyces spp.)(例如酿酒酵母(S.cerevisiae)、布拉迪酵母(S.boulardii sequela)、圆酵母(S.torula));鞘氨醇单胞菌属(Sphingomonasspp.)(例如少动鞘氨醇单胞菌(S.paucimobilis));假丝酵母属(Starmerella spp.)(例如熊蜂生假丝酵母);破囊壶菌属(Thraustochytrium spp.);球拟酵母属(Torulopsisspp.);黑粉菌属(Ustilago spp.)(例如玉米黑粉菌(U.maydis));威克汉姆酵母属(例如异常威克汉姆酵母);魏氏酵母属(Williopsis spp.);和/或接合酵母属(Zygosaccharomycesspp.)(例如拜耳接合酵母(Z.bailii))。
在一个实施方案中,所述方法利用酵母,例如异常威克汉姆酵母、蚜虫拟酵母、熊蜂生假丝酵母、库德里阿兹威毕赤酵母(Pichia kudriavzevii)或季也蒙毕赤酵母(季也蒙迈耶氏酵母)。此等酵母是各种两亲分子的有效生产者,所述两亲分子包括糖脂、酶和其它有用的代谢物。
根据本发明可以使用其它微生物菌株,包括例如能够积聚大量例如两亲分子的其它菌株。根据本发明有用的附加的代谢物包括甘露糖蛋白、β-葡聚糖和具有生物乳化和表面/界面张力降低性质的其它分子。
基于微生物的产品的制备
本发明的一种基于微生物的产品仅仅是含有微生物和/或由微生物产生的微生物代谢物和/或任何残余营养物的发酵培养液。发酵产物可以不经提取或纯化即直接使用。
然而,使用文献中描述的标准提取和/或纯化方法或技术可以容易地实现提取和纯化。举例来说,在某些实施方案中,基于微生物的产品仅仅包含呈粗制物或纯化形式的微生物生长副产物。在特定的实施方案中,副产物是由本发明的所生长微生物产生的生物表面活性剂。
由微生物生长产生的微生物和/或培养液可以从生长容器中移除,并通过例如管子转移以立即使用。
在其它实施方案中,考虑到例如预期用途、预期施用方法、发酵罐大小以及从微生物生长设施到使用位置的任何转运模式,所述组合物(微生物、培养液或微生物和培养液)可以被放置在适当大小的容器中。因此,放置基于微生物的组合物的容器可以是例如1加仑到1,000加仑或更大。在其它实施方案中,容器是2加仑、5加仑、25加仑或更大。
在某些实施方案中,本发明的组合物相比于例如单独的生物表面活性剂具有优势,所述优势包括以下中的一种或多种:作为酵母细胞壁外表面一部分的高浓度甘露糖蛋白(甘露糖蛋白是能够达到高达80%乳化指数的高效生物乳化剂);酵母细胞壁中存在生物聚合物β-葡聚糖(一种乳化剂);培养物中存在能够降低表面张力和界面张力的生物表面活性剂;以及存在代谢物(例如,乳酸、乙醇等)。
当从生长容器中收获基于微生物的组合物后,在将所收获的产物放置到容器和/或管子中(或以其它方式转运以供使用)时,可以添加另外的组分。添加剂可以是例如缓冲剂、载体、在相同或不同设施中生产的其它基于微生物的组合物、粘度调节剂、防腐剂、用于微生物生长的营养物、追踪剂、溶剂、杀生物剂、其它微生物和特定于预期用途的其它成分。
可以含于本发明的制剂中的其它合适的添加剂包括通常用于这类制剂的物质。这类添加剂的实施例包括表面活性剂、乳化剂、润滑剂、缓冲剂、溶解度控制剂、pH调节剂、防腐剂、稳定剂和抗紫外线剂。
在一个实施方案中,所述组合物可以进一步包含缓冲剂,所述缓冲剂包括有机酸和氨基酸或它们的盐。合适的缓冲剂包括柠檬酸盐、葡萄糖酸盐、酒石酸盐、苹果酸盐、乙酸盐、乳酸盐、草酸盐、天冬氨酸盐、丙二酸盐、葡庚糖酸盐、丙酮酸盐、半乳糖酸盐、葡萄糖二酸盐、酒石酸盐、谷氨酸盐、甘氨酸、赖氨酸、谷氨酰胺、蛋氨酸、半胱氨酸、精氨酸和它们的混合物。还可以使用磷酸和亚磷酸或它们的盐。合成缓冲剂适合使用,但优选的是使用天然缓冲剂,例如上文列出的有机酸和氨基酸或它们的盐。
在另一个实施方案中,pH调节剂包括氢氧化钾、氢氧化铵、碳酸钾或碳酸氢钾、盐酸、硝酸、硫酸或它们的混合物。
在一个实施方案中,制剂中可以包括附加的组分,例如盐的含水制剂,例如碳酸氢钠或碳酸钠、硫酸钠、磷酸钠、磷酸二氢钠。
有利的是,根据本发明,基于微生物的产品可以包含微生物所生长的培养基。所述产品可以是例如至少1重量%、5重量%、10重量%、25重量%、50重量%、75重量%或100重量%的生长培养基。所述产品中的生物质的量(按重量计)可以是例如0%到100%的任意值,包括两者之间的所有百分比。
任选地,所述产品可以在使用前储存起来。储存时间优选是短的。因此,储存时间可以是小于60天、45天、30天、20天、15天、10天、7天、5天、3天、2天、1天或12小时。在一个优选的实施方案中,如果所述产品中存在活细胞,则将所述产品在低温例如低于20℃、15℃、10℃或5℃下储存。另一方面,生物表面活性剂组合物通常可以在环境温度下储存。
实施例
从以下通过举例给出的实施例中可以更好地理解本发明和其许多优点。以下实施例说明了本发明的一些方法、应用、实施方案和变型。它们不被认为限制了本发明。可以对本发明作出许多改变和修改。
实施例1-护肤制剂
在一个实施方案中,将所述组合物配制成包含以下示范性组分的任何组合的护肤制剂:
参考文献
Rowan,M.P.,et al.(2015).Burn wound healing and treatment:review andadvancements.Critical Care.12June 2015.doi:10.1186/s13054-015-0961-2.(“Rowanet al.2015”).
Tiwari,V.K.(2012).Burn wound:How it differs from other wounds?IndianJ Plast Surg.2012May-Aug;45(2):364-373.doi:10.4103/0970-0358.101319.(“Tiwari2012”).
Claims (27)
1.一种外用护肤组合物,包含治疗有效量的槐糖脂、甘露糖赤藓糖醇脂和皮肤病学上可接受的载体。
2.根据权利要求1所述的组合物,其中,所述槐糖脂是盐形式的线性槐糖脂。
3.根据权利要求2所述的组合物,其中,所述盐形式的线性槐糖脂是钠盐形式的槐糖脂。
4.根据权利要求1所述的组合物,进一步包含乳铁蛋白。
5.根据权利要求1所述的组合物,进一步包含选自有机溶剂、硅酮、pH调节剂、螯合剂、胶凝剂、蛋白质、维生素、润肤剂、油、羟基酸、去角质剂、粘度调节剂、聚合物、矿物质、驱虫剂、润滑剂、防腐剂、植物性药材、精油、澄清剂、非生物表面活性剂、抗氧化剂、增稠剂、软化剂、遮光剂、保湿剂、着色剂和香料的一种或多种皮肤病学佐剂和/或添加剂。
6.根据权利要求1所述的组合物,进一步包含选自麻醉剂、角质溶解剂、剥离溶解剂、角质形成细胞增殖增强剂、胶原酶抑制剂、弹性蛋白酶抑制剂、褪色剂、抗炎剂、类固醇、抗痤疮剂和晚期糖基化终末产物AGE抑制剂的一种或多种皮肤活性物质。
7.根据权利要求1所述的组合物,进一步包含衍生自乳杆菌属、双歧杆菌属、乳球菌属、链球菌属和/或芽孢杆菌属的发酵的上清液,其中,所述芽孢杆菌属选自凝结芽孢杆菌、解淀粉芽孢杆菌NRRL B-67928和枯草芽孢杆菌B4 NRRL B-68031。
8.根据权利要求1所述的组合物,其中,所述组合物被配制为洗剂、乳膏、凝胶、软膏、液体、擦布、肥皂、洗发剂、护发剂或喷剂,并且其中,制剂适用于直接施用于人外皮。
9.一种伤口敷料,包含皮肤病学上可接受的敷料材料,所述敷料材料已经浸渍了根据权利要求1至8中任一项所述的组合物,并任选地进行干燥以储存。
10.根据权利要求9所述的伤口敷料,其中,所述皮肤病学上可接受的敷料材料选自合成纤维或非合成纤维的织物或无纺布、或它们的任何组合。
11.根据权利要求9所述的伤口敷料,其中,所述皮肤病学上可接受的敷料材料是聚合物聚羟基丁酸酯PHB。
12.一种促进皮肤病况愈合和/或改善的方法,其中,将包含槐糖脂、甘露糖赤藓糖醇脂和皮肤病学上可接受的载体的组合物施用于受试者皮肤的存在这类病况的区域。
13.根据权利要求12所述的方法,其中,所述槐糖脂是盐形式的线性槐糖脂。
14.根据权利要求13所述的方法,其中,所述盐形式的线性槐糖脂是钠盐形式的线性槐糖脂。
15.根据权利要求12所述的方法,进一步包含将乳铁蛋白施用于所述皮肤。
16.根据权利要求12所述的方法,其中,施用于所述皮肤的所述组合物进一步包含选自麻醉剂、角质溶解剂、剥离溶解剂、角质形成细胞增殖增强剂、胶原酶抑制剂、弹性蛋白酶抑制剂、褪色剂、抗炎剂、类固醇、抗痤疮剂和晚期糖基化终末产物AGE抑制剂的一种或多种皮肤活性物质。
17.根据权利要求12所述的方法,其中,施用于所述皮肤的所述组合物进一步包含衍生自乳杆菌属、双歧杆菌属、乳球菌属、链球菌属和/或芽孢杆菌属的发酵的上清液,其中,所述芽孢杆菌属选自凝结芽孢杆菌、解淀粉芽孢杆菌NRRL B-67928和枯草芽孢杆菌B4 NRRLB-68031。
18.根据权利要求12所述的方法,其中,施用于所述皮肤的所述组合物被配制为洗剂、乳膏、凝胶、软膏、液体、擦布、肥皂、洗发剂、护发剂或喷剂,并且其中,制剂适用于直接施用于人外皮。
19.根据权利要求12所述的方法,其中,所述皮肤病况是伤口、烧伤、手术切口或疤痕。
20.根据权利要求19所述的方法,其中,所述皮肤病况是处于愈合的增生期或更晚时期的伤口。
21.根据权利要求12所述的方法,其中,所述皮肤病况是痤疮、银屑病、湿疹、皱纹、下垂、干燥或感染。
22.根据权利要求12所述的方法,其中,所述皮肤病况在比未用所述组合物治疗的相同皮肤病况更短的时间内愈合和/或改善。
23.根据权利要求12所述的方法,其中,将所述组合物每天至少一次或每隔一天至少一次施用于所述受试者皮肤。
24.根据权利要求12所述的方法,其中,将所述组合物直接施用于所述受试者皮肤的存在所述皮肤病况的所述区域上,并任选地揉搓到所述皮肤中。
25.根据权利要求12所述的方法,其中,通过用已经浸渍了所述组合物的贴片或伤口敷料覆盖皮肤的存在所述皮肤病况的所述区域来施用所述组合物。
26.根据权利要求25所述的方法,其中,所述伤口敷料是PHB基质。
27.一种缩短受试者皮肤的伤口愈合所需的时间的方法,其中,所述方法包括将包含钠盐形式的线性槐糖脂、甘露糖赤藓糖醇脂和乳铁蛋白的组合物施用于所述伤口,每天至少一次,持续数天,直到所述伤口愈合。
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