Superficial basal cell carcinoma

摘要：大多数疫苗都是通过肌肉接种。皮肤含有大量的抗原提呈细胞，被认为是疫苗接种更具免疫原性的部位。皮内接种已被批准用于提高流感疫苗的效力和节省流感疫苗剂量。为了应对最近暴发的猴痘疫情，皮内接种也被批准通过延长有限的猴痘疫苗剂量，以免疫更多有风险的人。疫苗佐剂的加入有望进一步提高皮内接种效力，节省更多疫苗剂量。然而，与肌肉接种相比，皮内接种与更显著的局部反应有关。因此，适合于皮内接种的佐剂应具有良好的局部安全性，而不会引起明显的局部反应。本文介绍了目前已批准用于人类疫苗中的佐剂及其皮内给药的相对反应原性、具有良好局部安全性可用于皮内接种的新型化学和物理佐剂，同时对开发物理佐剂的合理性、物理佐剂的类型以及物理佐剂用于皮内接种的独特优势也进行了介绍。1.皮肤结构皮肤覆盖全身，具有重要的生理功能，如防止热量和水分流失，防止环境病原体感染。皮肤可分为3层：角质层(SC)、表皮层和真皮层。SC层位于皮肤的最外层，由角质细胞组成，是终末分化的角质细胞，并且具有高度的亲脂性。SC层承担皮肤的主要屏障作用。表皮位于SC层之下，主要由角质细胞（90%）组成，中间散布着朗格汉斯细胞（LC，3-5%），作为免疫哨兵）。表皮层还含有黑色素细胞，可根据其密度和黑色素含量赋予皮肤特殊的颜色。表皮层下面是真皮层，它比SC层和表皮层包含更多的解剖结构。真皮层中含有汗腺和毛囊，以及血液和淋巴管网和神经纤维。真皮层还包含多种类型的细胞，包括成纤维细胞和先天性免疫细胞，如树突状细胞（DC）、巨噬细胞、肥大细胞和嗜酸性粒细胞。有趣的是，皮肤还含有大量的T淋巴细胞，其中大部分是表皮和真皮层中皮肤驻留记忆T细胞。皮肤先天性免疫细胞是抵御病原体感染的第一道防线，也有助于诱导病原体或疫苗特异的适应性免疫应答。图1显示了带有常驻天然免疫细胞的简化皮肤模型。图1.皮肤结构和固有免疫细胞的图解：皮肤包含3个主要层：角质层、表皮和真皮。表皮含有朗格汉斯细胞，真皮含有树突状细胞、巨噬细胞、肥大细胞和嗜酸性粒细胞为主的天然免疫细胞。2.皮内疫苗接种及其好处由于肌肉注射的便利性，仅需要简单培训即能操作，因此大多数疫苗都是通过肌肉接种进行免疫。过去20-30年的大量研究发现，将疫苗接种途径从肌肉途径改为皮内途径可以诱导更有效的免疫反应。免疫原性更强的皮内接种在很大程度上可以归因于皮肤而不是肌肉中存在丰富的抗原提呈细胞，如表皮LC和真皮DC。抗原提呈细胞(主要是真皮DC)负责真皮内疫苗抗原在主要组织相容性复合体(MHC)I或II分子上的摄取和递呈，然后迁移到引流淋巴结，引发抗原特异性T和B细胞应答。皮内接种疫苗还可以激活局部先天性免疫细胞来分泌细胞因子和趋化因子，并招募循环中的先天免疫细胞，从而促进疫苗引起的局部反应。皮内接种有望能提高疫苗功效并节省疫苗剂量。最近的一项综述研究发现，在老年人中，皮内递送流感疫苗通常比肌内或皮下注射更具免疫原性。另外，在年轻人中，皮内接种低剂量流感疫苗（6或9µg）与肌肉注射全剂量疫苗（15µg）引起的免疫反应相当。除了流感疫苗外，皮内接种也能有效增强其他疫苗诱导的免疫反应，例如狂犬病疫苗和乙型肝炎疫苗。由于近期猴痘疫情暴发以及猴痘疫苗短缺，美国食品和药物管理局(FDA)授权皮内接种猴痘疫苗，以扩大疫苗剂量，让更多高危人群获得免疫接种。3.皮内接种的局限性皮内接种虽然提高了疫苗的免疫原性，但提高的程度相对较弱。皮内接种只能节省40-60%的流感疫苗剂量。在老年人中，皮内接种仅能将H1N1株的血清转换率从40%提高到52.3%，将H3N2株的血清转换率从49.1%提高到61.3%，将B型株的血清转换率从56.4%提高到61.3%。另一项研究也发现了类似的结果，即通过皮内接种流感疫苗，老年人的血清转换率仅略有增加。疫苗佐剂的加入有望进一步提高疫苗效力并节省更多的疫苗剂量。然而，添加佐剂的皮内疫苗面临着诱发严重局部反应的风险。研究一致发现，与肌肉接种疫苗相比，不含佐剂的皮内接种疫苗会诱发更频繁、更严重的局部不良反应。皮肤中存在的各种类型的先天性免疫细胞（如LCs、DCs、巨噬细胞）可以解释这种情况。这些细胞有助于增加抗原摄取和更有效的适应性免疫，同时通过合成和释放细胞因子和趋化因子以及招募外周中性粒细胞、单核细胞和嗜酸性粒细胞而引起局部不良反应。因此，用于皮内接种的佐剂需要比用于肌肉注射的佐剂具有更好的局部安全性。本综述侧重于介绍用于预防传染病疫苗的皮内接种佐剂，尽管这种途径对其他疾病（例如过敏和恶性肿瘤）的疫苗接种也很有吸引力。本综述还重点介绍佐剂如何增强皮内蛋白和亚单位疫苗诱导的免疫反应。此外，具有佐剂效应的疫苗/佐剂制剂（如聚合物纳米颗粒、脂质体）（至少有一部分是由二者联合引起的），不包括在本综述中。4.已获批准的佐剂及其皮内反应原性疫苗佐剂在开发新型疫苗和改良疫苗方面发挥着关键作用，因此在21世纪受到越来越多的关注。美国国家过敏与传染病研究所（NIAID）于2008年启动了"佐剂开发计划"，以支持新型佐剂的筛选、鉴定和临床前/临床开发。随着对这一领域的兴趣和投资不断增加，在过去二十年中，有五种佐剂被批准用于人类，而在过去八十年中，只有两种佐剂被批准用于人类（表1）。有趣的是，所有这些佐剂都被批准用于肌肉接种。如果没有皮内反应原性数据，则根据其肌肉注射后的局部不良反应来预测其皮内给药后诱发局部不良反应的可能。局部不良反应可能是皮肤红斑、肿胀、变色或组织学变化，如组织坏死或细胞死亡，或局部炎症反应，或细胞因子和趋化因子释放及免疫细胞浸润等。表1.目前批准的佐剂及其皮内反应原性l明矾佐剂明矾佐剂是一种铝盐类佐剂。临床上明矾佐剂主要以氢氧化铝为基础，由于纤维状纳米颗粒（图2A）或磷酸铝形成板状纳米颗粒的聚集而形成广泛的微米结构（0.5-10微米）。明矾佐剂是全球使用最广泛的佐剂，它已被用于多种儿童疫苗，如百白破（破伤风、白喉和百日咳）、乙型肝炎、乙型流感嗜血杆菌疫苗，明矾佐剂主要增强Th2细胞的抗体反应，但诱导细胞介导免疫反应的能力较弱。将明矾佐剂注射到肌肉组织中会引起明显的组织应激和细胞死亡。腹膜内注射明矾佐剂后也能观察到类似的现象。肌内注射明矾佐剂会诱导细胞因子/趋化因子的大量释放并快速募集中性粒细胞、单核细胞和嗜酸性粒细胞。研究发现，在小鼠皮肤中皮内注射Imject（一种实验性明矾佐剂）可诱导高密度炎症细胞浸润，并持续至少四天。由于明矾佐剂会诱发严重的组织应激和炎症，因此在皮内注射后很有可能诱发严重的局部反应。lAS04佐剂AS04佐剂是通过将MPL（3-O-去酰基-4'-单磷酰脂质A）吸附在氢氧化铝佐剂上制备的组合佐剂（图2B）。MPL是一种Toll样受体(TLR)4激动剂。MPL/TLR4结合激活骨髓分化初级反应88(MyD88)和下游信号通路，导致核因子-κB(NFκB)和激活蛋白(AP)-1激活以及促炎细胞因子基因表达。AS04被批准用于增强人乳头瘤病毒疫苗的效力。研究发现，与明矾佐剂疫苗相比，AS04佐剂疫苗可在局部注射部位诱导更高水平的细胞因子和趋化因子，并在引流淋巴结中募集更多数量的单核细胞和DC。研究发现，与不含佐剂的疫苗相比，含有AS04佐剂的疫苗会引起更频繁的局部反应，例如疼痛、发红和肿胀。局部不良反应可在几天内消失。研究发现AS04佐剂可诱导更平衡的辅助性T细胞1(Th1)/Th2免疫反应。我们之前的研究发现。皮内注射MPL/明矾佐剂会引起皮肤真皮组织红斑和肿胀，并伴有大量炎症细胞浸润。这些数据表明AS04佐剂在皮内递送后诱发严重局部反应的风险很高。lAS01佐剂AS01是一种含有MPL和QS21的脂质体制剂，QS21是从肥皂树（Quillajasaponaria）中纯化的皂苷成分（图2C）。AS01佐剂已被批准用于提高疟疾RTS,S疫苗的功效。研究发现AS01佐剂可诱导Th1为主的免疫反应，而对Th2反应的诱导较弱。研究发现肌肉注射AS01佐剂被快速清除，3h时AS01水平显著降低，24h后几乎检测不到AS01水平。AS01诱导细胞因子的短暂表达，该表达在24h内达到峰值，然后迅速消退。AS01还诱导中性粒细胞和单核细胞迅速聚集到注射佐剂的肌肉中。考虑到AS01含有MPL，预计局部细胞因子和趋化因子的刺激至少部分是由TLR4和下游MyD88信号通路的刺激引起的。研究发现MPL和QS21能协同激活驻留淋巴结的自然杀伤细胞(NK)和CD8+T细胞并促进早期干扰素(IFN)γ产生，该细胞通过白细胞介素(IL)-18分泌通过荚膜下的巨噬细胞控制白细胞介素(IL)-18分泌，并得到IL-12支持。MPL和QS21的协同作用以及对局部炎症的强烈诱导表明，AS01佐剂在皮内给药后诱发显著局部反应的风险很高。这种作用由巨噬细胞通过白细胞介素（IL）-18的分泌来控制，并得到IL-12的支持。MPL和QS21的协同作用以及对局部炎症的强烈诱导表明，AS01佐剂在皮内给药后诱发显著局部反应的风险很高。lMF59和AS03佐剂MF59和AS03是基于角鲨烯水包油型纳米乳液佐剂。角鲨烯是一种天然三萜油，作为动物和人类胆固醇合成过程中的一种生化中间体。角鲨烯的可生物降解性使其成为佐剂配方中的安全成分。MF59和AS03分别由Novartis和GlaxoSmithKline（GSK）开发，化学成分略有不同（图2D）。MF59的主要油相成分是角鲨烯，而AS03的油相中含有角鲨烯和α-生育酚（维生素E）。α-生育酚还具有可生物降解性和免疫调节功能。关于表面活性剂，MF59含有两种表面活性剂（Tween80和Span85），而AS03仅含有聚山梨醇酯80（Tween80）。MF59最初被批准用于提高欧洲老年人接种季节性流感疫苗功效。MF59最近在美国被批准用于增强老年人季节性流感疫苗（FluadQuadrivalent）的效力以及大流行前H5N1疫苗（Audenz）的效力。AS03最初被批准用于增强欧洲2009年甲型H1N1流感大流行期间的2009年甲型H1N1大流行流感疫苗(Pandemrix)效力，后来被批准用于增强美国大流行前H5N1疫苗的效力。研究发现MF59和AS03佐剂可诱导偏向Th2的免疫反应，而对Th1反应的诱导较弱。肌内注射MF59佐剂会诱导组织应激和细胞死亡。此外，MF59的清除不依赖于抗原，在肌肉中的清除半衰期为42h。研究发现，尽管时间招募模式相同，但发现肌内注射MF59比肌内注射明矾更能有力地募集中性粒细胞、单核细胞和嗜酸性粒细胞。与肌内注射后的明矾佐剂相比，肌肉注射MF59佐剂可诱导多种细胞因子和趋化因子释放模式。皮内注射MF59样AddaVax佐剂可强烈诱导中性粒细胞、单核细胞和嗜酸性粒细胞，注射后4d，中性粒细胞和单核细胞水平显著升高。皮内注射AddaVax佐剂还可诱导各种细胞因子和趋化因子的延长表达至少4d。这些数据表明MF59佐剂在皮内递送后诱发严重局部反应的风险很高。研究发现，肌内注射AS03佐剂可持续诱导至少7d的细胞因子表达（例如C-C基序趋化因子配体2(CCL2)、IL-1β）。AS03比明矾佐剂诱导更强的细胞因子表达。AS03佐剂中的α-生育酚可通过激活巨噬细胞和单核细胞显著增加细胞因子的表达,AS03佐剂可诱导脂质代谢快速且短暂的下调，导致细胞内脂质水平升高，诱导内质网(ER)应激并激活未折叠蛋白反应途径。此外，消耗髓系细胞中ER应激激酶传感器肌醇需要的跨膜激酶内切核糖核酸酶-1α(IRE1α)会减少AS03佐剂诱导的细胞因子表达和高亲和力抗体的诱导。局部炎性细胞因子和趋化因子以及ER应激的强烈诱导表明AS03佐剂也可能在皮内递送后诱导显著的局部反应。lMatrix-M佐剂Matrix-M佐剂由两种独特的40nm颗粒配方制成，来自皂苷提取物的两种组分（组分A和C）（图2E），并已被纳入最近批准的基于蛋白质的Novavax新冠疫苗（NVX-CoV2373），用于新型冠状病毒肺炎大流行期间的紧急使用。每种纳米颗粒制剂均由皂苷A或C部分以及胆固醇和磷脂制成。Matrix-M佐剂被发现可诱导偏向Th1的免疫反应。研究发现，含有Matrix-M佐剂的新冠疫苗与无效对照剂相比更容易引起局部反应（第一剂：57.6% vs 17.9%；第二剂：79.6% vs 16.4%）。大多数局部不良反应为轻度或中度，并在3天内消失。Matrix-M佐剂可刺激猪单核细胞和淋巴细胞表达IL-1β和C-X-C基序趋化因子配体8(CXCL8)，但不表达肿瘤坏死因子α(TNFα)或IL-6。猪体内肌肉注射Matrix-M佐剂可诱发以中性粒细胞、单核细胞、淋巴细胞浸润为特征的急性炎症。局部不良反应还包括大多数猪的出血和部分猪的坏死。Matrix-M佐剂组比生理盐水对照组出现发红的频率更高。与对照相比，Matrix-M佐剂显着增加了384个基因表达并降低了162个基因表达。al,2012).在上调最多的基因中，23个中有10个与干扰素调节基因(IRGs)有关。尽管迄今为止还没有研究探索Matrix-M在促进皮内疫苗接种方面的有效性和安全性，但一项研究发现在猪模型中皮内给药脂质体中的QS-2（另一种基于皂素的佐剂）会剂量依赖性地诱发局部不良反应，如红斑、肿块和结痂。人类受试者肌肉注射免疫后出现较频繁的局部不良反应，至少一些猪出现强烈的局部炎症反应、出血和坏死，以及与之密切相关的QS-21佐剂皮内递送后诱发局部不良反应的剂量依赖性，都表明Matrix-M佐剂皮内递送后诱发严重局部反应的风险很高。lCpG 1018CpG 1018是一个未甲基化的含CpG基序的寡核苷酸。CpG 1018可激活TLR9和MyD88信号通路，从而激活各种转录因子，如NFκB、AP-1和干扰素调节因子（IRF），并控制细胞因子的表达，如TNFα、IL-1β、I型干扰素和IL-12。CpG1018刺激偏向Th1的抗体反应和细胞介导的免疫反应。CpG 1018已被纳入最近批准的乙肝疫苗。添加CpG 1018佐剂的乙肝疫苗被发现比添加明胶佐剂的乙肝疫苗诱导更有效的免疫反应。与物种特异性CpG寡核苷酸不同，CpG 1018在啮齿动物、非人类灵长类动物和人类中具有广泛的响应，从而在临床前和临床研究中不需要改变CpG序列。研究发现皮内注射小鼠特异性CpG只会诱发轻微的局部反应和低水平的免疫细胞招募。CpG 1018预计具有较低的皮内反应原性，有望安全地促进皮内疫苗接种。 图2.目前批准的颗粒状佐剂:A.氢氧化铝佐剂。B.AS04佐剂是由MPL吸附在氢氧化铝助剂上制备的。C.用脂质体包裹MPL和QS21制备AS01佐剂。D.角鲨烯纳米乳剂MF59和AS03佐剂。E.由两种皂素提取物(组分A和C)制成的两种纳米颗粒制剂组成的Matrix-M佐剂总之，大多数已批准的佐剂在皮内给药后诱发严重局部反应的风险很高。有趣的是，预测皮内反应原性较高的化学佐剂本质上都是自然界中微粒（表1），它们会强烈激活局部先天性免疫系统并诱发明显的局部反应。CpG1018可能是个例外，因为它能诱导轻微炎症，而不会引起明显的局部反应（图3A）。图3.皮内接种安全佐剂的化学结构A.CpG 1018。DNA骨架进行硫代修饰以提高佐剂的稳定性。B.具有六个14碳侧链的合成GLA。C.咪喹莫特。D.Poly(I：C)。E.cGAMP。F.PCPP聚合物。5.用于安全皮内接种的其他化学佐剂由于担心已获批准的佐剂具有较高的局部反应原性，人们还探索了其他化学佐剂的安全性和有效性，以促进皮内疫苗接种。其中几种佐剂已进入临床试验阶段。由于疫苗佐剂在皮内给药后有可能诱发明显的局部反应，因此本综述不包括未报告局部安全性数据的佐剂。lGLA-AF佐剂几种已获批准的佐剂，如AS01和AS04，都含有MPL作为提升疫苗接种效果的主要成分。虽然MPL还没有被批准作为一种独立的佐剂，但我们在小鼠模型中发现MPL皮内注射引起了轻微的炎症，没有明显的局部不良反应。Carter等人评价了一种合成的TLR4激动剂(GLA-AF)(图3B)以促进雪貂和人类皮内接种H5N1疫苗，并发现GLA-AF对于皮内接种H5N1疫苗在雪貂中诱导单针保护和在人类中诱导血清保护至关重要。此外，在人类临床研究中，加入GAL-AF的H5N1疫苗没有在豚鼠体内引起体温上升或体重下降，并没有引起一过性红斑和其他明显的不良反应。这项研究表明GLA-AF和其他可能的TLR4激动剂用于皮内接种的有效性和安全性。l外用咪喹莫特佐剂FDA批准外用咪喹莫特乳膏(5%阿尔达拉)用于治疗各种疾病，如光化性角化病、浅表性基底细胞癌和外生殖器疣。咪喹莫特是一种TLR7激动剂(图3C)并激活TLR7-MyD88信号通路，导致NFκB和IRF7的激活以及促炎细胞因子和I型干扰素基因的表达。有趣的是，外用咪喹莫特乳膏的载体也可能有助于观察到的疗效，因为在没有咪喹莫特的情况下，诱导激活炎症小体、角质细胞死亡和IL-1释放。在一项双盲、随机对照的临床试验中，在皮内接种流感疫苗前局部应用咪喹莫特乳膏可以获得更高的疫苗病毒株血清转换率和更好的非疫苗病毒株血清转换率。最常见的局部不良反应是1级红肿，尽管与其他组没有统计学差异，但局部应用咪喹莫特组红肿发生率更高。在炎症性肠病(IBD)患者中，外用咪喹莫特乳膏后皮下注射乙型肝炎疫苗的血清保护率明显高于外用水乳膏后肌肉注射疫苗的血清保护率。虽然局部咪喹莫特组引起更频繁的瘙痒和肿胀，但各组之间的局部不良反应无明显差异。这些结果表明，外用咪喹莫特乳膏可显著提高皮内疫苗的疗效，且总体上具有良好的局部安全性。lPoly(I:C)佐剂聚肌胞苷酸(Poly(I:C))是双链RNA(dsRNA，图3D)的合成类似物，可根据位置激活两种不同的信号通路。内质多聚体(I:C)激活TLR3并诱导IL-12和I型IFN产生，而胞质多聚体(I:C)可激活视黄酸诱导基因I(RIG-I)和黑色素瘤分化相关蛋白5(MDA-5）并增强I型干扰素的产生。Poly(I:C)还用于皮内或透皮疫苗接种，以诱导针对各种类型传染病的有效免疫反应。Weldon等人发现，当在小鼠模型中将Poly(I:C)佐剂亚单位流感疫苗涂覆在微针表面时，可引发与单独的亚单位疫苗类似的免疫反应和保护佐剂效果不明显，可能与该研究中使用的Poly(I:C)剂量相对较低有关（1µg）。在另一项研究中，Bardel等人发现25µg Poly(I:C)可以显著增强皮内HIV-1gp140或HSV-2gD糖蛋白诱导的全身和粘膜抗体反应以及在HSV-2攻击研究中对小鼠模型的保护作用，而不会明显增强小鼠模型中对皮肤反应原性的保护作用。这项研究表明，皮内接种添加Poly(I:C)佐剂的HIV-1gp140或HSV-2gD糖蛋白疫苗具有良好的安全性和效力，可诱导有效的粘膜免疫。Du等人发现，将白喉毒素（DT）和Poly(I:C)封装到脂质体中，然后以空心微针为基础进行皮内给药，与单独以空心微针为基础进行皮内单独接种DT相比，可显著增强DT特异性IgG2a抗体反应。有趣的是，该研究中使用了相对较低的poly(I:C)剂量（0.31µg），但并未观察到总IgG或IgG1抗体反应的显著增强。最近，在一项1期临床研究中测试了用聚-L-赖氨酸稳定的poly(I:C)与光化学内化（PCI）相结合增强人乳头瘤病毒多肽疫苗接种的效果，光化学内化是一种通过光诱导过程促进内吞抗原的胞体释放，从而促进诱导细胞介导的免疫反应的技术。尽管没有研究基于poly(I:C)佐剂在增强人乳头瘤病毒多肽疫苗接种方面的效力，但研究发现，当光敏剂剂量低于一定阈值时，整体对研究对象是安全的。上述研究表明，poly(I:C)佐剂用于加强皮内接种具有良好的安全性。lcGAMP佐剂2′3′-环GMP-AMP（cGAMP，图3E）是细胞内干扰素基因刺激物（STING）通路的刺激物，可导致NFκB和IRF3激活以及促炎细胞因子和I型IFN基因表达。cGAMP及其类似物，如cdAMP和cdGMP，已被探究为诱导Th1免疫应答对抗传染性疾病和癌症的有效佐剂。研究发现，在小鼠和猪模型中，皮内注射cGAMP与H5N1流感疫苗或2009年大流行H1N1疫苗可显著增强疫苗诱导的免疫反应，而不会引起明显的局部不良反应。此外，在小鼠模型中，cGAMP被发现是一种比CpG或MPL更有效的佐剂，可增强皮内对H5N1免疫。最近，Hernandez-Franco等人探索了cdAMP和一种以植物为基础的阳离子α-D-葡聚糖纳米粒(Nano-11)增强小鼠和猪皮内OVA免疫的安全性和有效性，发现cdAMP佐剂的皮内OVA免疫在注射后24小时诱发一过性变色，14天后消失，而Nano-11佐剂的皮内OVA免疫则不会诱发明显的不良反应。这些研究支持STING激动剂作为安全的佐剂来促进皮内疫苗接种。lPCPP佐剂微针是将疫苗递送至皮肤的一种极具吸引力的技术。疫苗可以涂布在金属或不可溶解聚合物微针的表面，或封装在可溶解聚合物微针结构中。考虑到微针通常会诱导与肌肉注射给药相当的免疫反应，人们探索了安全的佐剂来促进基于微针的皮内疫苗接种。Andrianov等人发现，PCPP（图3F）是一种合成的、水溶性、可生物降解的聚磷嗪聚合物，在药物输送和组织工程中有着广泛的应用，是一种安全的佐剂，可促进猪的基于微针的皮内乙型肝炎疫苗接种。此外，PCPP可作为一种关键的微针疫苗制造材料，以减少对表面活性剂的依赖。在这项研究中，只观察到轻微的皮肤红痕，没有发现严重的不良反应。除PCPP外，研究还发现一种新型聚磷腈聚合物PCEP可安全促进仔猪皮内接种H1N1流感疫苗，尽管对H3N2病毒缺乏异种亚型保护。总之，皮内反应原性低的化学佐剂通常是水溶性的或制成水性制剂，以便于从局部给药部位快速清除。这些佐剂大多基于病原体相关分子模式（PAMPs）或其合成类似物，可激活模式识别受体（PRRs），如TLR3、TLR4、TLR7、TLR9和STING，从而在不过度激活先天性免疫系统的情况下发挥佐剂作用。虽然PCPP不是PAMP，但研究发现PCPP可通过TLR3、TLR4和TLR9直接激活免疫细胞。这些研究表明，某些PRR激动剂作为皮内接种佐剂是安全的。6.安全用于皮内疫苗接种的物理佐剂除了注射化学佐剂，人们还探索了在皮内注射疫苗前在皮肤表面短暂施加物理能量，以促进皮内疫苗接种。l开发物理佐剂的合理性由于缺乏对佐剂作用原理的了解，过去的佐剂开发主要依赖于经验或试错方法，尤其是那些非PAMP型佐剂，如明矾、MF59和QS21。这些佐剂没有特定的细胞受体来介导其佐剂效应，因此不会直接激活先天性免疫细胞，如直流电和巨噬细胞。相反，这些佐剂更有可能刺激组织应激和细胞死亡，从而释放内源性危险信号或损伤相关分子模式（DAMPs）来介导其佐剂效应。20世纪90年代，Matzinger和她的同事提出了"危险理论"，证明免疫系统是由受压或受损细胞释放的危险信号激活的。"危险理论"解释了在没有外来病原体入侵的情况下诱导适应性免疫，如移植排斥、某些化疗诱导的全身性抗肿瘤免疫激活。在过去的二三十年中，人们发现了各种类型的DAMPs，其中包括尿酸和ATP等小分子化学物质以及dsDNA等大分子物质，它们能够根据"危险理论"调节免疫系统的功能。这些物质在生理条件下被封闭起来，无法被免疫系统识别，但在组织应激或细胞死亡时会释放出来，激活免疫系统。研究发现，明矾佐剂可刺激尿酸释放，从而激活NOD-、LRR-和含吡啶结构域蛋白3（NLRP3）炎性小体和Caspase-1激活，部分介导其促炎症反应。明矾佐剂还能刺激宿主DNA的释放，从而诱导IgG1抗体的产生和IgE类的转换，并延长DC和CD4+T细胞的相互作用。MF59佐剂可刺激ATP释放，从而增强疫苗接种的免疫反应。这些进展表明，刺激组织应激或细胞死亡的物理模式可能会诱导内源性危险信号的释放，从而促进疫苗接种。l物理佐剂类型研究最多的两种疫苗佐剂物理能量是激光和射频。图4总结了各种类型的激光佐剂和射频佐剂。图4.不同类型的物理佐剂。简要说明不同类型的激光佐剂(全表面、非消融部分、消融部分)和射频佐剂l激光佐剂激光发出窄波长的光，可被特定的组织发色团吸收。可见光主要被黑色素和血红蛋白吸收，而近红外光主要被组织水吸收。激光在美学和医学方面有着独特的应用。由于其超细的光束，高能激光已被用于无刀片激光辅助原位角膜磨镶术(LASIK)，术后并发症最少。激光还被用于低强度激光治疗，以刺激伤口愈合和缓解疼痛。激光还被用于皮肤表面处理、胎记和皱纹去除。已经探索了几种激光来促进皮内疫苗接种。Onikienko等人探索了一种发射两种波长(510nm和578nm)的铜蒸气激光，通过诱导持续的局部炎症来增强疫苗诱导的免疫反应，类似于化学佐剂。我们探索了非侵入性Q开关Nd：YAG532nm激光治疗来增强皮内疫苗诱导的免疫反应，并发现非侵入性激光治疗可以显著增强模型抗原OVA和流感疫苗诱导的小鼠模型的免疫反应，而不会导致可见或组织学皮肤损伤。激光治疗几乎没有诱导细胞因子的表达或免疫细胞的招募。进一步的研究发现，非炎症性激光治疗显著促进了MHCII+细胞(如巨噬细胞和DC)在皮肤内的迁移。MHCII+巨噬细胞和DC的局部运动增强可能有助于增强抗原摄取和增强获得性免疫。我们进一步发现，激光佐剂可以通过促进DC进入淋巴管和迁移到引流淋巴结来增强基于DC的抗肿瘤免疫治疗。激光佐剂被发现还能增强皮内尼古丁疫苗诱导的抗尼古丁抗体的产生。研究人员还开展了1064纳米近红外激光(NIR)增强疫苗皮内接种效果的研究。结果发现，无组织损伤的近红外治疗可以瞬时诱导一组明确的趋化因子，如CCL2和CCL20的表达，并增加表皮和真皮中DC的浓度。近红外激光治疗被发现显著提高皮内卵白蛋白和流感疫苗诱导的小鼠模型的免疫反应。进一步研究发现，近红外激光增加并激活皮肤中的Lang+和CD11b-DC亚群，并促进DC迁移到引流淋巴结，以增强疫苗诱导的免疫反应。与这项研究一致的是，皮肤中的CD103+DC亚群被发现在近红外佐剂效应中发挥关键作用。初步临床研究探索了近红外治疗在人类中的最大耐受量，并发现近红外激光促进CD1a+LC和CD11c+DC在真皮中的迁移。与非侵入性全表面激光治疗不同的是，非消融性部分激光(NAFL)也被用于增强皮内接种效果。NAFL发射高能微激光，并在皮肤表面形成微温区。在低温区域的死亡细胞招募浆细胞样树突状细胞(PDC)，它可以被局部咪喹莫特激活以增强皮内流感疫苗接种效果。NAFL治疗后皮内接种流感疫苗，然后局部注射咪喹莫特，在NAFL或局部咪喹莫特存在的情况下，比皮内接种流感疫苗产生更好的免疫反应，而在AddaVax佐剂存在的情况下，肌肉接种流感疫苗产生类似的免疫反应。有趣的是，NAFL/咪喹莫特佐剂的组合流感疫苗在小鼠和猪模型中显示出良好的局部和系统安全性。另一项研究发现，在小鼠模型中，与在NAFL或单独使用咪喹莫特的情况下皮内接种疱疹病毒多肽相比，局部应用咪喹莫特和NAFL治疗，然后皮内接种疱疹病毒多肽疫苗，可以诱导最有效的疫苗特异性CD4+和CD8+T细胞反应，并对生殖器疱疹病毒感染提供最显著的保护。上述研究表明，NAFL的使用顺序、局部应用咪喹莫特和皮内接种并不是该组高免疫原性的关键，只是在皮内接种疫苗之前使用NAFL以避免疫苗抗原的潜在热灭活。进一步研究发现，NAFL刺激死亡细胞释放dsDNA，并激活STING和IRF途径来介导其佐剂作用。NAFL还被发现在小鼠和猪模型中安全地加强基于微针的流感疫苗接种。关于激光疫苗佐剂更详细分类可参考其他综述。除了仅将激光用作佐剂外，最近开发了基于激光的粉末递送系统(LPD)，用于无需外部佐剂的无针经皮疫苗递送和辅助植入。LPD是基于消融部分激光(AFL)治疗产生皮肤微通道，然后局部应用粉末疫苗涂层阵列贴片，通过AFL产生的皮肤微通道将疫苗输送到皮肤。研究发现，能量为10mJ、覆盖率为10%的超脉冲CO2 AFL在促进OVA皮内免疫方面具有强大的佐剂作用。此外，基于AFL的粉末注射流感疫苗比皮内注射相同剂量的流感疫苗产生更强的抗体反应。在致命的病毒攻击后，流感疫苗的LPD也比皮内注射提供更显著的保护。AFL治疗诱导了显著的细胞因子和趋化因子的释放，并将中性粒细胞、单核细胞和嗜酸性粒细胞招募到微通道周围的组织中。由于消融了一小部分皮肤和靠近皮肤微通道的局部炎症反应，AFL治疗的皮肤没有明显的不良反应，并在几天内完全恢复。AFL的佐剂作用被认为是由于旁观者的光热效应。由于激光的脉冲宽度较长，在激光消融过程中产生的热量会扩散到周围组织，并导致周围一层薄薄的微通道组织的热损伤。dsDNA被发现从低温区域释放并诱导细胞因子表达。研究发现AFL还能激活NLRP3炎症体和Caspase1，导致IL-1β的活性释放。有趣的是，dsDNA、NLRP3炎症体和IL-1β对于激光佐剂效应是非必需的，而MyD88对激光佐剂有重要作用。与光热应激不同的是，在微针疫苗接种过程中施加的机械压力也被发现可以增强疫苗诱导的免疫反应。Ng等人发现，增加微投影阵列应用能量来诱导机械压力和细胞死亡增强疫苗(包被在微针表面)诱导的免疫反应，而不加入化学佐剂。这种方法被发现可以诱导与肿瘤坏死因子α和核因子-κB信号通路相关的基因表达。这些开创性的研究支持开发自佐剂疫苗递送技术，以便在不注射针头或化学佐剂的情况下进行更具免疫原性的疫苗接种。l射频佐剂射频(RFs)是交变电磁波。中高频(0.3-10MHz)的射频可产生组织加热，在美学和医学上有着广泛的应用。由于其热效应，RFs已被用于肿瘤治疗中的肿瘤组织消融或在皮肤表面修复中诱导新的胶原合成。为了寻求替代的物理能量来促进皮内疫苗接种，我们探索了在皮内疫苗注射之前对一小块皮肤进行双极射频治疗的潜在佐剂效果。射频治疗受到控制，以诱导显著的加热效应，但不会对皮肤造成损害。我们发现，非侵入性RF治疗诱导了一过性、低水平的局部炎症，而化学佐剂，如明矾、AddaVax和MPL，则诱导了持久且更强烈的局部炎症。RF治疗被发现显著增加真皮CD103+和CD11b-DC亚群的抗原摄取，并促进真皮DC向引流淋巴结的迁移。此外，射频治疗显著增加了引流淋巴结中传统DC和PDC的成熟。RF治疗显著增加了OVA诱导的抗体反应，佐剂效果与AddaVax相当，也增强了OVA特异性细胞免疫反应，佐剂效果与CpG佐剂相当。此外，研究发现，与0.06μg疫苗剂量的AddaVax佐剂疫苗相比，0.3μg疫苗剂量的RFA同样增强了2009年H1N1流感大流行疫苗诱导的抗体应答和保护作用。低剂量下的RFA效果较好，提示其具有优良的剂量节约效应。为了验证这一点，我们研究发现RFA可以帮助毫微克H3N2疫苗在小鼠模型中诱导有效的免疫反应和保护。考虑到先前的激光佐剂效果主要是与相对较弱的明矾佐剂进行比较，RFA是第一个与广泛使用的化学佐剂(类似MF59的AddaVax)相比的物理佐剂，可以增强流感疫苗接种。由于RFA能够帮助蛋白质或亚单位疫苗诱导细胞免疫反应，我们进一步探索了RFA在基于重组核蛋白(NP)和基质蛋白1(M1)的通用流感T细胞疫苗开发中的作用。通用流感T细胞疫苗主要针对保守的病毒细胞内抗原，如NP和M1。佐剂提供了一种方便的策略来帮助NP和M1诱导CD8+T细胞反应。我们发现RFA可以增强真皮内NP和M1诱导的CD8+T细胞反应。在小鼠模型中，在RFA存在的情况下，皮内NP/M1免疫对致死性病毒攻击具有显著的保护作用。在加强NP/M1免疫方面，RFA显示出与AddaVax相似的佐剂作用。最近发现，RFA还可以扩大流感疫苗诱导的针对异源病毒的免疫反应。这种交叉保护免疫可能是由于它诱导了交叉保护的细胞毒性T淋巴细胞。RFA还有效促进了雄性和雌性小鼠的H5N1流感疫苗皮内免疫。含有RFA的H5N1流感疫苗皮内接种(RFA/ID)诱导的血清血凝抑制(HI)效价和免疫球蛋白抗体效价显著高于单独皮内疫苗(ID)。在致死性病毒攻击后，RFA/ID组而不是ID组显示出显著的肺病毒滴度降低。有趣的是，与ID组相比，RFA/ID组攻击后肺组织中分泌肿瘤坏死因子α和干扰素γ的CD4+和CD8+T细胞显著增加。RFA/ID组对体重减轻也有显著的保护作用。RFA/ID组小鼠不分性别全部存活，ID组部分小鼠存活(雌鼠占25%，雄鼠占75%)。有趣的是，在这项研究中，与AddaVax佐剂相比，RFA对促进H5N1流感疫苗接种的作用较弱。比较组织蛋白质组学最近被用来鉴定RFA治疗后独特表达的蛋白质。我们发现，与化学佐剂处理(MPL、AddaVax、Alum、MPL/Alum)相比，RFA诱导了14种唯一表达的蛋白质。在唯一表达的蛋白质中，热休克蛋白70(HSP70)在RFA效应中发挥了关键作用。验证研究发现，与野生型小鼠相比，HSP70基因敲除小鼠显著降低了促进OVA和pdm09疫苗接种的RFA效应。有趣的是，这项研究发现RFA引起的局部组织蛋白质组变化最小，而AddaVax佐剂引起的局部组织蛋白质组变化最大。RFA对局部组织蛋白质组的最小变化与其诱导的一过性和低水平的局部炎症是一致的。l物理佐剂的优点物理佐剂将物理能量短暂地应用于皮肤表面，然后皮内注射疫苗，以增强疫苗诱导的免疫反应(图5)。物理佐剂主要由医疗器械输送，可在室温下保存，无需冷链储存。这与化学佐剂不同，化学佐剂通常与疫苗一起保存，并需要冷链储存。物理佐剂设备可以重复使用，每次应用的成本可能类似于化学佐剂。物理佐剂不需要与疫苗混合，因此不需要改变或修改疫苗制造或给药过程。物理佐剂不太可能引起显著的全身性或长期副作用，这可能是化学佐剂的一个问题。这是因为物理佐剂的作用主要局限于治疗部位，而化学佐剂可以迁移到引流淋巴结，甚至到达体循环，从而广泛激活免疫系统。物理佐剂可诱导良好控制的组织应激(例如，热)，并允许皮肤快速恢复，而不会产生持久的影响。物理佐剂经常引起短暂的、低水平的局部炎症，而化学佐剂往往引起持续的、更强烈的局部炎症。物理佐剂主要是改变局部环境以增加DC的运动性，诱导内源性危险信号的释放以增强抗原摄取和DC成熟，或促进DC迁移到引流淋巴结以增强疫苗诱导的免疫反应(图5)。经过适当的开发，物理佐剂的效果可以与化学佐剂一样强大。图5.化学佐剂和物理佐剂在皮内接种中的不同作用机制。皮内给药后，化学佐剂通过与PRRs结合直接激活DC，诱导DC成熟，诱导DC释放细胞因子/趋化因子，招募天然免疫细胞，或促进DC向引流淋巴结迁移，以增强疫苗诱导的免疫应答。物理佐剂传递激光或射频能量，诱导组织应激释放危险信号，刺激DC成熟，诱导DC释放细胞因子/趋化因子，并招募先天免疫细胞。物理佐剂还可以增加DC在真皮组织中的运动性，并促进DC迁移到引流淋巴结，以增强疫苗诱导的免疫反应。7.专家评论皮肤已被公认为疫苗接种的高免疫原性部位。与肌肉接种相比，皮内接种疫苗通常能诱导更强的免疫反应，在提高疫苗效力或节省疫苗剂量方面有很大作用。事实上，已有几种疫苗获得了皮内接种的许可，其中包括流感疫苗、狂犬病疫苗和最近的猴痘疫苗。然而，皮内接种面临着诱发严重局部反应的风险。目前获批的大多数佐剂都不适合皮内接种，因为它们具有诱发严重局部反应的高风险。我们需要新型佐剂来安全地增强皮内接种效果，同时避免皮肤出现明显的反应。最近的研究发现了几种有可能安全增强疫苗皮内接种效果的化学佐剂，包括CpG1018、GLA-AF、局部咪喹莫特、poly(I:C)、cGAMP和PCPP。CpG1018已被授权用于促进肌肉接种乙型肝炎疫苗，而GLA-AF和局部咪喹莫特已在临床试验中用于增强皮内接种流感疫苗。由于这些佐剂在人体中的安全性已得到证实，因此可随时用于皮内疫苗接种。poly(I:C)已在临床试验中被用于增强肿瘤内、肌内或皮下注射后的抗肿瘤免疫力。poly(I:C)其总体良好的耐受性支持对其进行评估，以增强皮内疫苗接种。皮内接种佐剂疫苗的好处是可以进一步提高疫苗效力，节省更多疫苗剂量，并有可能诱导交叉保护性免疫。流感疫苗可作为测试皮内佐剂接种的良好目标，以解决疫苗失配问题，因为疫苗失配会大大降低疫苗效力，或通过节省疫苗剂量来解决每年流感疫苗生产的负担。在批准皮内佐剂疫苗供大众使用时，需要仔细评估其效益/风险比。除化学佐剂外，人们还探索了不同类型的物理佐剂（激光、射频），以安全地促进皮内疫苗接种。物理佐剂是一种相对较新的佐剂类型，它利用物理能量诱导组织应激以促进皮内接种。虽然物理佐剂提高人类疫苗效力的潜力仍有待探索，但最近发现内源性危险信号在化学佐剂效应中发挥关键作用（如明矾、MF59），这支持了物理佐剂的发展，以诱导组织应激和释放内源性危险信号来促进疫苗接种。最近在临床前动物模型中研究了物理佐剂的概念及其增强皮内疫苗接种的安全性和有效性。这些研究支持物理佐剂在促进皮内疫苗接种方面具有良好的安全性和有效性。尽管取得了这些令人鼓舞的成果，但物理佐剂在人类使用方面仍面临着一些挑战。首先，物理佐剂是依靠设备的。目前，还没有指导物理佐剂开发和审批的指导方针。其次，物理佐剂的开发需要设备制造商和疫苗公司的共同努力，还需要引入佐剂开发和医疗设备审批方面的监管机构。多方的参与可能会给物理佐剂的开发带来新的挑战。第三，使用目前的物理佐剂进行皮内接种需要两个步骤：物理佐剂处理和皮内接种。与化学佐剂结合疫苗接种相比，两步接种过程需要花费更多时间，而且公众对这种新型疫苗接种的接受程度还有待进一步探讨。佐剂的开发在21世纪受到极大关注。在批准新佐剂供人类使用和阐明疫苗佐剂的内在机制方面取得了重大进展。所有这些努力都有望促进安全的化学和物理佐剂的开发，从而增强皮内接种疫苗的效果。参考来源：Chen X. Emerging adjuvants for intradermal vaccination. Int J Pharm. 2023 Feb 5;632:122559. doi: 10.1016/j.ijpharm.2022.122559. Epub 2022 Dec 28. PMID: 36586639; PMCID: PMC9794530.推荐学习：疫苗佐剂概述：发展现状和未来展望基于TLR激动剂佐剂的设计疫苗佐剂：选择标准、与免疫应答相关的作用机制和未来方向识别微信二维码，添加生物制品圈小编，符合条件者即可加入生物制品微信群！请注明：姓名+研究方向！版权声明本公众号所有转载文章系出于传递更多信息之目的，且明确注明来源和作者，不希望被转载的媒体或个人可与我们联系(cbplib@163.com)，我们将立即进行删除处理。所有文章仅代表作者观点，不代表本站立场。

EscharEx® Phase III study protocol: FDA/EMA-aligned; patient enrollment commencing early 2024; two key research collaborations with wound industry leaders NexoBrid® U.S. commercial launch timing not anticipated to impact revenues in 2023-2024Cash of $51.3 million; operating cash runway through profitability Conference call on Wednesday, August 16 at 8:30 a.m. Eastern Time YAVNE, Israel, Aug. 15, 2023 (GLOBE NEWSWIRE) -- MediWound Ltd. (Nasdaq: MDWD), the global leader in next-generation enzymatic therapeutics for tissue repair, today announced financial results for the second quarter ended June 30, 2023, and provided a corporate update. "We have achieved significant progress this quarter on many levels," stated Ofer Gonen, CEO of MediWound. "Our EscharEx Phase III study protocol is aligned with feedback from both FDA and EMA, and patient enrollment is planned for early 2024. Executing multiple collaborations on our EscharEx program with leading wound care companies, underscores the growing interest in this potential game-changing treatment and our commitment to maximize the likelihood of a successful study outcome.” Mr. Gonen added, “We dispatched initial batches of NexoBrid to both Japan and the U.S., with commercial availability already underway in Japan. Furthermore, our new manufacturing facility, set to be active by mid-2024, will increase our production capabilities to meet the rising global demand." Second Quarter 2023 Highlights and Recent Developments: Shipped NexoBrid finished product to Vericel for the U.S. commercial launch in June 2023. However, Vericel is unable to commercially release the product at this time due to a deviation associated with a third-party testing lab used during the manufacturing process. MediWound and Vericel are actively engaged with the U.S. Food and Drug Administration (FDA) to address this matter. Future production lots will not be impacted by this process deviation issue. Vericel expects to begin commercial sales of NexoBrid from a scheduled September 2023 production run, during the first quarter of 2024. MediWound believes that a possible delay in the U.S. launch, will not have an impact on NexoBrid revenues for the years 2023-2024. Received positive scientific advice from the Committee for Medicinal Products for Human Use (CHMP) within the European Medicine Agency (EMA) for the development of EscharEx. This advice aligns with feedback from the FDA, providing a clear path forward for the Company’s global Phase III study in patients with venous leg ulcers (VLUs). The Company is in the process of qualifying study sites, selecting vendors (including CRO, data management, and central labs), and producing final batches of EscharEx for the clinical study. These activities are to be completed by the fourth quarter of 2023, with patient enrollment in the Phase III study expected to begin in early 2024.Secured research and development collaborations with leading wound care companies, MIMEDX and Mölnlycke, to advance the EscharEx Phase III clinical study. MediWound is actively engaged with additional prominent companies to explore further collaboration opportunities.Awarded an additional $10 million in funding from BARDA to support NexoBrid’s $3 million replenishment, the pediatric indication sBLA submission, and the enrollment of an additional 50 patients in the expanded access treatment protocol (NEXT). Signed a turnkey scale-up agreement to bolster the Company’s manufacturing infrastructure supporting its long-term growth trajectory. The Company will establish, commission, and validate a cutting-edge, sterile, and GMP-compliant manufacturing facility to significantly increase the Company’s current production capacity. An estimated $12 million will be invested in this new state-of-the-art facility, which is projected to be completed by mid-2024, with full-scale manufacturing expected to commence in 2025.Announced commercial launch of NexoBrid in Japan for the treatment of deep partial thickness and full thickness burns in adults and pediatric patients with the Company’s strategic partner, Kaken Pharmaceutical Co. Ltd., a top ranked Japanese pharmaceutical company. Reported positive data from its Phase I/II study of MW005 in low-risk basal cell carcinoma (BCC). Results showed MW005 to be safe and well-tolerated, with eleven of the fifteen patients enrolled achieving complete clearance of their BCCs, with a majority of the patients also having histologically confirmed complete clearance. Appointed Mr. Shmuel (Milky) Rubinstein as an independent director to the Company’s Board of Directors. With a distinguished record of pharmaceutical and biotechnology leadership, Mr. Rubinstein previously held the position of CEO at Taro Pharmaceuticals (Nasdaq: TARO), which subsequently was acquired by Sun Pharmaceuticals. He currently holds the title of Chairperson of the Board at Trima Pharma and is a board member at Strata Skin Sciences (Nasdaq: SSKN), Medison Biotech, and Keystone Dental. Mr. Rubinstein will become a MediWound Board member, following the tenure of Mr. Assaf Segal, who stepped down from the MediWound Board of Directors.Cash and short-term deposits of $51.3 million as of June 30, 2023. Second Quarter 2023 Financial Highlights Revenues: Revenues for the second quarter 2023 were $4.8 million, compared to $4.7 million in the second quarter of 2022.Gross Profit: Gross profit in the second quarter 2023 was $1.1 million, representing 24% of total revenue, unchanged from the second quarter 2022.Expenditures: Research and development expenses in the second quarter 2023 were $2.0 million compared to $2.2 million in the second quarter of 2022.Selling, general, and administrative expenses in the second quarter 2023 were $3.1 million, compared to $2.3 million in the second quarter of 2022. This increase is primarily attributed to the addition of several full-time employees to bolster future growth, along with greater share-based compensation expenses. Operating Results: Operating loss in the second quarter of 2023 was $4.0 million, compared to a $3.7 million loss in the second quarter of 2022.Net Profit/Loss: Net profit in the second quarter of 2023 was $0.9 million or $0.10 per share, compared to the net loss of $4.4 million, or $0.92 per share in the second quarter of 2022. This change is primarily attributed to a favorable adjustment from the revaluation of warrants.Adjusted EBITDA: Adjusted EBITDA in the second quarter of 2023 was a loss of $3.0 million, compared to a loss of $2.8 million in the second quarter of 2022. Year-to-Date 2023 Financial Highlights Revenues: Total revenues in the first half of 2023 were $8.6 million, compared to $9.1 million in the first half of 2022. The decline in revenues is primarily a result of the sales to BARDA's emergency stockpile procurement in 2022.Operating Results: Operating loss in the first half of 2023 was $8.4 million, up from the $7.0 million loss in the first half of 2022. This increase is primarily attributed to the addition of several full-time employees to bolster future growth, along with greater share-based compensation expenses.Net Loss: Net loss in the first half of 2023 was $2.8 million, or $0.32 per share, compared to a net loss of $7.9 million or $1.79 per share in the first half of 2022. This decrease is primarily attributed to a favorable adjustment from the revaluation of warrants.Adjusted EBITDA: Adjusted EBITDA in the first half of 2023, as further detailed below, was a loss of $6.4 million, compared to a $5.4 million loss in the first half of 2022. Balance Sheet Overview As of June 30, 2023, the Company's cash and short-term deposits were $51.3 million, compared to $34.1 million reported on December 31, 2022. In the first quarter of 2023 the Company raised a gross amount of $27.5 million through a registered direct offering. During the second quarter of 2023, the Company used $6.0 million to fund its operating activities. Existing cash and cash equivalents are expected to provide sufficient funds for the Company’s current operating plan through profitability. Conference Call MediWound management will host a conference call for investors on Wednesday, August 16, 2023, beginning at 8:30 a.m., Eastern Time to discuss these results and answer questions. Shareholders and other interested parties may participate in the conference call by dialing 1-833-630-1956 (in the U.S.), 1-80-921-2373 (Israel), or 1-412-317-1837 (outside the U.S. & Israel). The call will be available via webcast by clicking HERE or on the Events & Presentations page of Company’s website. A replay of the call will be available on the Company’s website at www.mediwound.com. Non-IFRS Financial Measures To supplement consolidated financial statements prepared and presented in accordance with IFRS, the Company has provided a supplementary non-IFRS measure to consider in evaluating the Company's performance. Management uses Adjusted EBITDA, which it defines as earnings before interest, taxes, depreciation and amortization, impairment, one-time expenses, restructuring and share-based compensation expenses. Although Adjusted EBITDA is not a measure of performance or liquidity calculated in accordance with IFRS, we believe the non-IFRS financial measures we present provide meaningful supplemental information regarding our operating results primarily because they exclude certain non-cash charges or items that we do not believe are reflective of our ongoing operating results when budgeting, planning and forecasting and determining compensation, and when assessing the performance of our business with our senior management. However, investors should not consider these measures in isolation or as substitutes for operating income, cash flows from operating activities or any other measure for determining the Company's operating performance or liquidity that is calculated in accordance with IFRS. In addition, because Adjusted EBITDA is not calculated in accordance with IFRS, it may not necessarily be comparable to similarly titled measures employed by other companies. The non-IFRS measures included in this press release have been reconciled to the IFRS results in the tables below. About MediWound MediWound Ltd. (Nasdaq: MDWD) is the global leader in next-generation enzymatic therapeutics focused on non-surgical tissue repair. Specializing in the development, production and commercialization of solutions that seek to replace existing standards of care. The Company is committed to providing rapid and effective biologics that improve patient experiences and outcomes, while reducing costs and unnecessary surgeries. MediWound’s first drug, NexoBrid®, is an FDA-approved orphan biologic for eschar removal in severe burns that can replace surgical interventions and minimize associated costs and complications. Utilizing the same core biotherapeutic enzymatic platform technology, MediWound has developed a strong R&D pipeline including the Company’s lead drug under development, EscharEx®. EscharEx is a Phase III biologic for debridement of chronic wounds with significant advantages over the $300 million monopoly legacy drug and an opportunity to expand the market. MediWound’s pipeline also includes MW005, a topical therapeutic for the treatment of basal cell carcinoma that has demonstrated positive results in a recently completed Phase I/II study. For more information, please visit www.mediwound.com and follow the Company on LinkedIn. Cautionary Note Regarding Forward-Looking Statements MediWound cautions you that all statements other than statements of historical fact included in this press release that address activities, events, or developments that we expect, believe, or anticipate will or may occur in the future are forward-looking statements. Although we believe that we have a reasonable basis for the forward-looking statements contained herein, they are based on current expectations about future events affecting us and are subject to risks, assumptions, uncertainties, and factors, all of which are difficult to predict and many of which are beyond our control. Actual results may differ materially from those expressed or implied by the forward-looking statements in this press release. These statements are often, but are not always, made through the use of words or phrases such as “anticipates,” “intends,” “estimates,” “plans,” “expects,” “continues,” “believe,” “guidance,” “outlook,” “target,” “future,” “potential,” “goals” and similar words or phrases, or future or conditional verbs such as “will,” “would,” “should,” “could,” “may,” or similar expressions. Specifically, this press release contains forward-looking statements concerning the anticipated progress, development, study design, expected data timing, objectives anticipated timelines, expectations and commercial potential of our products and product candidates, including EscharEx® and NexoBrid®. Among the factors that may cause results to be materially different from those stated herein are the inherent uncertainties associated with the uncertain, lengthy and expensive nature of the product development process; the timing and conduct of our studies of our products and product candidates, including the timing, progress and results of current and future clinical studies, and our research and development programs; the approval of regulatory submission by the FDA, the European Medicines Agency or by any other regulatory authority, our ability to obtain marketing approval of our products and product candidates in the U.S. or other markets; the clinical utility, potential advantages and timing or likelihood of regulatory filings and approvals of our products and products; our expectations regarding future growth, including our ability to develop new products; risks related to our contracts with BARDA; market acceptance of our products and product candidates; our ability to maintain adequate protection of our intellectual property; competition risks; the need for additional financing; the impact of government laws and regulations and the impact of the current global macroeconomic climate on our ability to source supplies for our operations or our ability or capacity to manufacture, sell and support the use of our products and product candidates in the future. These and other significant factors are discussed in greater detail in MediWound’s annual report on Form 20-F for the year ended December 31, 2022, filed with the Securities and Exchange Commission (“SEC”) on March 16, 2023 and Quarterly Reports on Form 6-K and other filings with the SEC from time-to-time. These forward-looking statements reflect MediWound’s current views as of the date hereof and MediWound undertakes, and specifically disclaims, any obligation to update any of these forward-looking statements to reflect a change in their respective views or events or circumstances that occur after the date of this release except as required by law. Contacts: Hani LuxenburgChief Financial OfficerMediWound Ltd.ir@mediwound.com Monique KosseManaging Director, LifeSci Advisors212-915-3820monique@lifesciadvisors.com MediWound, Ltd. CONDENSED CONSOLIDATED STATEMENT OF FINANCIAL POSITIONS (UNAUDITED) U.S. dollars in thousands June 30, Dec 31, 2023 2022 2022 Unaudited AuditedCash and cash equivalents and short-term bank deposits 51,122 10,406 33,895Trade and other receivable 3,818 4,412 9,982Inventories 3,113 1,991 1,963Total current assets 58,053 16,809 45,840 Other receivables 277 230 364Property, plant and equipment, net 4,705 2,439 2,366Right of use assets 1,133 1,364 1,215Intangible assets, net 198 264 231Total non-current assets 6,313 4,297 4,176 Total assets 64,366 21,106 50,016 Current maturities of long-term liabilities 1,961 2,479 2,242Trade payables and accrued expenses 3,531 4,877 5,656Other payables 2,817 3,060 4,159Total current liabilities 8,309 10,416 12,057 Deferred revenues - 61 -Warrants 9,683 - 15,606Liabilities in respect of IIA grants 7,806 8,131 7,445Liability in respect of TEVA 2,529 3,361 2,788Lease liabilities 677 1,053 846Severance pay liability, net 433 319 360Total non-current liabilities 21,128 12,925 27,045 Total equity (deficit) 34,929 (2,235) 10,914 Total liabilities and equity 64,366 21,106 50,016 MediWound, Ltd. CONDENSED CONSOLIDATED STATEMENT OF PROFIT OR LOSS AND OTHET COMPREHENSIVE INCOME OR LOSS (UNAUDITED)U.S. dollars in thousands Six months ended Three months ended June 30, June 30, 2023 2022 2023 2022 Total revenues 8,572 9,075 4,773 4,668 Total cost of revenues 6,609 6,502 3,636 3,555 Gross profit 1,963 2,573 1,137 1,113 Research and development 4,126 4,599 2,024 2,191 Selling, general & administrative6,208 4,623 3,120 2,287 Other expenses - 309 - 309 Total operating expenses 10,334 9,531 5,144 4,787 Operating loss (8,371) (6,958) (4,007) (3,674) Financial income (expenses), net5,611 (977) 4,935 (676) Profit (loss) before taxes on income (2,760) (7,935) 928 (4,350) Taxes on income (17) (8) (12) (4) Net profit (loss) (2,777) (7,943) 916 (4,354) Foreign currency translation adjustments (9) 22 - 17 Total comprehensive profit (loss) (2,786) (7,921) 916 (4,337) Basic and diluted net profit (loss) per share: (0.32) (1.79) 0.10 (0.92) Weighted average number of ordinary 8,803 4,440 9,209 4,734 shares used in calculation basic and diluted net profit (loss) per share MediWound, Ltd. ADJUSTED EBITDA U.S. dollars in thousands Six months ended Three months ended June 30, June 30, 2023 2022 2023 2022 Net profit (loss) for the period (2,777) (7,943) 916 (4,354) Adjustments: Financial income (expenses), net5,611 (977) 4,935 (676) Other expenses - (309) - (309) Tax expenses (17) (8) (12) (4) Depreciation and amortization(618) (650) (315) (329) Share-based compensation expenses(1,331) (597) (712) (252) Total adjustments 3,645 (2,541) 3,896 (1,570) Adjusted EBITDA (6,422) (5,402) (2,980) (2,784) MediWound, Ltd. CONDENSED CONSOLIDATED STATEMENT OF CASH FLOW (UNAUDITED)U.S. dollars in thousands Six months ended Three months ended June 30, June 30, 2023 2022 2023 2022 Cash Flows from Operating Activities: Net income (loss)(2,777) (7,943) 916 (4,354) Adjustments to reconcile net profit (loss) to net cash used in operating activities: Adjustments to profit and loss items: Depreciation and amortization618 650 315 329 Share-based compensation1,331 597 712 252 Revaluation of warrants accounted at fair value(5,923) - (4,990) - Revaluation of liabilities in respect of IIA grants492 482 233 248 Revaluation of liabilities in respect of TEVA241 272 119 135 Revaluation of lease liabilities(22) (152) (9) (138) Increase (decrease) in severance liability, net67 55 (10) 35 Net financing income(1,005) (11) (759) (11) Un-realized foreign currency loss466 528 120 283 (3,735) 2,421 (4,269) 1,133 Changes in asset and liability items: Decrease (increase) in trade receivables6,115 (2,024) (707) (1,445) Increase in inventories(1,162) (747) (579) (37) Decrease in other receivables122 330 435 205 Increase (decrease) in trade payables and accrued expenses(1,636) 11 312 (272) Increase (decrease) in other payables and deferred revenues(1,526) (1,367) (1,359) (484) 1,913 (3,797) (1,898) (2,033) Net cash (used in) operating activities(4,599) (9,319) (5,251) (5,254) Cash Flows from Investment Activities: Purchase of property and equipment(2,570) (298) (1,065) (138) Interest received879 - 577 - Investment in short term bank deposits, net(31,830) (2,499) (25,590) (2,499) Net cash used in investing activities(33,521) (2,797) (26,078) (2,637) Cash Flows from Financing Activities: Repayment of lease liabilities(334) (350) (157) (172) Proceeds from (repayment of) issuance of shares and warrants, net24,909 9,861 (248) (556) Repayments to IIA, net(310) (162) - - Repayment of liabilities in respect of TEVA(417) - - - Net cash provided by (used in) financing activities23,848 9,349 (405) (728) Exchange rate differences on cash and cash equivalent balances(457) (550) (120) (303) Increase (decrease) in cash and cash equivalents from continuing activities(14,729) (3,317) (31,854) (8,922) Balance of cash and cash equivalents at the beginning of the period33,895 11,046 51,020 16,651 Balance of cash and cash equivalents at the end of the period19,166 7,729 19,166 7,729

Trial Results Expected in Mid-2024 WOBURN, MA / ACCESSWIRE / August 10, 2023 / Biofrontera AG Inc. (NASDAQ:BFRI) (the "Company"), a biopharmaceutical company specializing in the commercialization of dermatological products, announces that patient enrollment is now complete in the Phase 3 clinical study evaluating Ameluz®-PDT in combination with the BF-RhodoLED® lamp for the treatment of basal cell carcinoma (BCC). This study is being conducted by Biofrontera Bioscience GmbH. "Completing patient enrollment in this Phase 3 clinical study is an important milestone in our strategy to expand the Ameluz® label beyond actinic keratosis (AK). As awareness about skin cancer increases, so does demand for non-surgical BCC treatments without ionizing radiation. According to the Skin Cancer Foundation, of the 5.4 million annual cases of non-melanoma skin cancer in the U.S., approximately 3.6 million, or 67%, are BCC. There is a significant unmet medical need for more effective, less invasive and cost-efficient therapies that treat BCC as well as underlying premalignancies. We look forward to sharing results from this Phase 3 study in mid-2024," said Hermann Luebbert, Chief Executive Officer and Chairman of Biofrontera Inc. "In addition to treating individual lesions, Ameluz® is indicated by its prescribing information* for the lesion-directed and field-directed treatment of skin areas with multiple AKs. Superficial BCC is another, more severe form of neoplastic damage that such a field may carry, and inclusion into the label broadens the use of Ameluz® in field-directed treatment, starting a new chapter of PDT for non-melanoma skin cancer", he continued. This randomized, double-blind, placebo-controlled study aimed at enrolling 186 patients at 19 U.S. sites. With 183 patients now treated, and four more patients screened and awaiting histological results and treatment, the clinical sites have been informed that the enrollment is complete. Each patient had one or more clinically and histologically confirmed superficial BCC. Patients receive one cycle of two PDTs (either Ameluz®-PDT or placebo-PDT) 1-2 weeks apart, which may be repeated after three months if required. The last assessment of the patient will take place three months after the final PDT cycle. The primary endpoint is the composite complete clinical and histological clearance of a main BCC lesion, which was selected at the beginning of the study. In addition, data on drug safety as well as secondary efficacy parameters of all BCCs will be evaluated. After completion of the trial, patients will be followed for an additional five years. *For full prescribing information for Ameluz®, please see About Biofrontera Inc. Biofrontera Inc. is a U.S.-based biopharmaceutical company commercializing a portfolio of products for the treatment of dermatologic conditions with a focus on photodynamic therapy (PDT) and topical antibiotics. The Company's licensed products are used for the treatment of actinic keratoses, which are pre-cancerous skin lesions, as well as impetigo, a bacterial skin infection. For more information, visit and follow Biofrontera on LinkedIn and Twitter. Forward-Looking Statements Certain statements in this press release may constitute "forward-looking statements" within the meaning of the United States Private Securities Litigation Reform Act of 1995, as amended to date. These statements include, but are not limited to, statements relating to Biofrontera Inc.'s (the "Company") preliminary revenues for the three and six months ended June 30, 2023, expectations for revenue growth in 2023, the adoption of PDT and sBCC for the treatment of actinic keratoses by dermatologists, the achievements of our salesforce and the performance of the Company's new hires. We have based these forward-looking statements on our current expectations and projections about future events, nevertheless, actual results or events could differ materially from the plans, intentions and expectations disclosed in, or implied by, the forward-looking statements we make. These risks and uncertainties, many of which are beyond our control, including, but not limited to, the impact of any extraordinary external events; any changes in the Company's relationship with its licensors; the ability of the Company's licensors to fulfill their obligations to the Company in a timely manner; the Company's ability to achieve and sustain profitability; whether global disruptions in supply chains will impact the Company's ability to obtain and distribute its licensed products; changes in the practices of healthcare providers, including any changes to the coverage, reimbursement and pricing for procedures using the Company's licensed products; the uncertainties inherent in the initiation and conduct of clinical trials; availability and timing of data from clinical trials; whether results of earlier clinical trials or trials of Ameluz® in combination with BF-RhodoLED® in different disease indications or product applications will be indicative of the results of ongoing or future trials; uncertainties associated with regulatory review of clinical trials and applications for marketing approvals; whether the market opportunity for Ameluz® in combination with BF-RhodoLED® is consistent with the Company's expectations; the Company's ability to comply with public company requirements; the Company's ability to regain compliance with Nasdaq continued listing standards, the Company's ability to retain and hire key personnel; the sufficiency of cash resources and need for additional financing and other factors that may be disclosed in the Company's filings with the SEC, which can be obtained on the SEC website at . Readers are cautioned not to place undue reliance on the forward-looking statements, which speak only as of the date on which they are made and reflect management's current estimates, projections, expectations and beliefs. The Company does not plan to update any such forward-looking statements and expressly disclaims any duty to update the information contained in this press release except as required by law. Contact: LHA Investor Relations Tirth T. Patel 212-201-6614 tpatel@lhai.com SOURCE: Biofrontera Inc. View source version on accesswire.com: