Tight junctions (TJs)

6 聚体合成肽 AT-1002 是新发现的一类药物的一部分，可以可逆地增强分子跨上皮屏障的细胞旁转运。 AT-1002[1] 可以发生 Cys-Cys 二聚化。通过测量细胞 ATP 浓度来评估细胞活力，并使用 AT-1002（0 至 5 mg/mL，3 或 24 小时）评估未分化的 Caco-2 细胞。通过 AT-1002 测量长达三个小时的处理，任何浓度的细胞活力都不会受到影响。具体来说，5 mg/mL 的 AT-1002 对 Caco-2 细胞的活力没有影响。 AT-1002 剂量为 2.5 mg/mL 及以上时，24 小时后会降低细胞活力。然而，在细胞暴露于 AT-1002 三小时后清洗细胞不会对细胞造成不可逆的损伤，因为细胞在二十四小时后仍然发挥作用[2]。

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AT-1002是一种六聚体肽，通过荧光显微镜观察到，它导致ZO-1从细胞连接处重新分布。AT-1002还激活了src和丝裂原活化蛋白（MAP）激酶途径，增加了ZO-1酪氨酸磷酸化和肌动蛋白丝的重排。从功能上讲，AT-1002导致Caco-2细胞单层跨上皮电阻（TEER）的可逆降低和荧光黄渗透性的增加。[2]

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使用CellTiter Glo®细胞活力测定法测量AT-1002对细胞活力的影响。处理3小时后，与未处理的对照细胞相比，AT-1002没有显著降低细胞存活率（图2）。这些数据表明，该化合物增强LY通透性是由于其通透性调节活性，而不是由于细胞存活率降低[1]。

体内活性[2]
确定上述AT-1002的体外作用是否可以转化为体内作用是非常有趣的。先前已经证明AT-1002能够增加体内给药到胃肠系统的有效载荷的递送（Motlekar等人，2006，Song等人，2008a，Song等，2008b）。在这里，我们想确定AT-1002是否可以增强应用于气道上皮的有效载荷的输送。因此，我们测试了AT-1002是否可以增加鲑鱼降钙素（sCT）的全身暴露，而鲑鱼降钙钙素本身的生物利用度非常低。向大鼠气管内滴注10μg sCT和越来越多的AT-1002，导致AT-1002含量最高的治疗组肺部吸收增加，sCT全身浓度升高（图8）。当在AT-1002给药后2小时给予sCT时，没有观察到吸收增强，表明AT-1002的作用是短暂的（数据未显示）。在300μg AT-1002的给药剂量以下，药代动力学参数之间没有观察到差异（数据未显示）。300和1000μg at-1002的sCT AUC0-240min明显高于对照组（0μg剂量）（表2）。sCT与300和1000μg AT-1002的联合给药分别使AUC比对照组增加1.6倍（161.8%）和5.2倍（522.5%）。与对照组相比，当1000μg AT-1002与sCT联合给药时，Cmax（衡量达到的最高浓度）也高出2.3倍。

TEER和荧光黄渗透性测定[2]
之前已经描述了这种方法和修改的细节（Artursson，1990，Ginski和Polli，1999）。对于经上皮电阻（TEER）和荧光黄（LY）渗透性测定，将Caco-2细胞以100000个细胞/cm2的密度接种到12孔Transwells™（孔径0.4μm）上，并生长21-28天，直至完全分化。Caco-2细胞单层的顶端和基底外侧隔室在37°C下在HBSS中预孵育30分钟。将含有0.4至5 mg/ml AT-1002或HBSS中5 mg/ml打乱肽的AT-1002浓度范围的处理溶液加入到每个单层的顶端隔室中，然后在37°℃、50 rpm下孵育180分钟。使用MilliCell ERS在0、30、60、120和180分钟下测量TEER。在180分钟时，顶端隔室用7.5 mM荧光黄替换AT-1002。在37°C下孵育1小时后，从基底外侧室中取出样品，在Tecan Spectrofluor荧光板读数器中以485 nm的激发波长和535 nm的发射波长分析LY。计算每种处理的TEER降低和LY渗透性增加，并相对于未处理的荧光黄对照表示。渗透率计算如下：Papp=[（dC/dt）×Vr]/（Co×A），其中dC/dt是渗透率，Vr是接收器的体积，A是膜过滤器的表面积，Co是供体室中的初始浓度，增强比定义为Papp AT-1002/Papp HBSS。

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AT-1002对Caco-2细胞影响的可逆性[2]
如上所述，将Caco-2细胞接种在transwell膜上，并在37°C、5%CO2和95%湿度的DMEM中生长21天，每隔一天更换一次培养基。在生长期结束时，从上部（顶端）和下部（基底外侧）隔室中取出培养基。细胞在预热（37°C）的HBSS（含Ca和Mg）中与10 mM HEPES pH 7.4一起孵育。在HBSS中，用或不用浓度为5mg/ml的AT-1002对转运井进行不同时间的顶部处理。15、30、45和60分钟后，AT-1002要么被HBSS替换，要么根本不移除。在不同时间点使用欧姆表监测TEER读数。

细胞活力测定[2]
细胞类型： Caco-2 细胞
测试浓度： 0 至 5 mg/mL
孵育时间：3或24小时
实验结果：处理长达3小时不会影响任何浓度的细胞活力。浓度为 2.5 mg/mL 或更高时，24 小时后细胞活力降低。

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体外细胞毒性试验[2]
通过使用发光ATP测定法测量细胞中ATP的量来确定细胞存活率。ATP的浓度由甲虫荧光素在Mg2+和ATP依赖的反应中被萤光素酶单氧化时发出的光量决定。在去除生长培养基后，将100μl Hank's平衡盐溶液（HBSS）中0至5mg/ml的浓度范围内的AT-1002添加到96孔组织培养板上生长的30000个Caco-2细胞中。3小时后，将等体积的细胞滴度Glo试剂加入孔中，在Tecan Spectrafluor plus平板阅读器中孵育15分钟后测量化学发光。生成ATP的标准曲线，并用于计算用AT-1002处理后的ATP浓度。

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免疫荧光[2]
IEC6电池以每室60000个电池的速度镀在8室载玻片上。在接种后24小时，在无血清培养基中洗涤细胞，并在37°C下用稀释于无血清培养基中的AT-1002（5mg/ml）孵育60分钟。处理后，在PBS中洗涤细胞，并在室温下在含有4%多聚甲醛的PBS中固定15分钟。在PBS中洗涤细胞，在室温下在含有0.5%Triton X-100的PBS中透化5分钟，并在室温下用含有2%山羊血清的PBS封闭30分钟。然后将细胞与稀释在阻断缓冲液（pMLC（1:50））中的一抗在37°C下孵育1-2小时。在PBS中洗涤细胞，并在室温下与FITC标记的抗兔抗体一起孵育45分钟。分别使用Alexa Fluor555鬼笔环肽和FITC标记的抗ZO-1抗体检测肌动蛋白和ZO-1。将载玻片洗涤并安装在含有DAPI的Vectashield中，并在Nikon-TE2000荧光显微镜上成像。 Caco-2 BBE细胞在37°C下用AT-1002（5 mg/ml）在顶部处理3小时。将以下处理细胞固定在甲醇：丙酮（1:1）中，并用含有2%山羊血清的PBS封闭。将过滤器与FITC标记的抗ZO-1抗体在室温下孵育1小时，洗涤并如上所述安装在载玻片上。

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流式细胞术[2]
Caco-2 BBE细胞在37°C下用AT-1002（5 mg/ml）在顶部处理3小时。处理后，使用胰蛋白酶将细胞从过滤器中分离出来。分离的细胞在PBS中洗涤，在室温下在含有4%多聚甲醛的PBS中固定15分钟，在含有0.5%Triton X-100的PBS中在室温下渗透5分钟，并在含有2%山羊血清的PBS中封闭30分钟。细胞在室温下与Alexa Fluor555鬼笔环肽一起孵育1小时，在PBS中洗涤，并使用FACSCAN通过流式细胞术进行分析。

Intratracheal delivery of salmon calcitonin [1]
Male Sprague–Dawley rats were used for this study and were approximately 12 weeks of age at the initiation of the study. All rats were instilled intratracheally with 10 μg of sCT in 200 μl of saline containing 0, 300 or 1000 μg of AT-1002 (n = 6 per dose group). Blood samples (200 μl) were collected and placed into EDTA coated tubes prior to dosing and at 2.5, 5, 10, 15, 30, 60, 120 and 240 min following dosing. Plasma was harvested and stored at ≤−70 °C until assayed for sCT. The DSL 10–3600 ACTIVE® Salmon Calcitonin Enzyme-Linked Immunosorbent (ELISA) kit was used with slight modifications to determine concentrations of sCT in rat plasma. This assay is an enzymatically amplified “two-step” sandwich-type immunoassay involving the biotin-streptavidin bridging detection system. For these studies standards were prepared in a rat serum matrix and the curve ranged from 15.6 to 1000 pg/ml. The LLOQ was 31.3 pg/ml. The sample volume used was 50 μl. When necessary, samples (concentrations expected or measured above 1000 pg/ml) were diluted with the rat serum matrix. Standard curves were calculated using a four parameter fit model using the KC4 software available on a BioTek Plate reader. Assay performance did not appear to be influenced by the difference between the sample (rat plasma) and standard (rat serum) matrix. Microsoft Excel® was used for calculation of AUC using the linear trapezoidal rule and data were plotted using GraphPad Prism version 4.01. Cmax and Tmax for each condition were also determined.

[1]. Structure-activity relationship studies of permeability modulating peptide AT-1002. Bioorg Med Chem Lett. 2008 Aug 15;18(16):4584-6.

[2]. Mechanism of action of ZOT-derived peptide AT-1002, a tight junction regulator and absorption enhancer. Int J Pharm. 2009 Jan 5;365(1-2):121-30.

AT-1002 a 6-mer synthetic peptide belongs to an emerging novel class of compounds that reversibly increase paracellular transport of molecules across the epithelial barrier. The aim of this project was to elaborate on the structure-activity relationship of this peptide with the specific goal to replace the P2 cysteine amino acid. Herein, we report the discovery of peptides that exhibit reversible permeability enhancement properties with an increased stability profile.[1]

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Tight junctions (TJs) are intercellular structures that control paracellular permeability and epithelial polarity. It is now accepted that TJs are highly dynamic structures that are regulated in response to exogenous and endogenous stimuli. Here, we provide details on the mechanism of action of AT-1002, the active domain of Vibrio cholerae's second toxin, zonula occludens toxin (ZOT). AT-1002, a hexamer peptide, caused the redistribution of ZO-1 away from cell junctions as seen by fluorescence microscopy. AT-1002 also activated src and mitogen activated protein (MAP) kinase pathways, increased ZO-1 tyrosine phosphorylation, and rearrangement of actin filaments. Functionally, AT-1002 caused a reversible reduction in transepithelial electrical resistance (TEER) and an increase in lucifer yellow permeability in Caco-2 cell monolayers. In vivo, co-administration of salmon calcitonin with 1 mg of AT-1002 resulted in a 5.2-fold increase in AUC over the control group. Our findings provide a mechanistic explanation for AT-1002-induced tight junction disassembly, and demonstrate that AT-1002 can be used for delivery of other agents in vivo.[2]

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Drug absorption is thought to occur predominantly via passive transcellular and paracellular transport mechanisms (Behrens et al., 2001). Lipophilic drugs are transported primarily by the transcellular route and by means of transporters such as channels, pumps and carriers on the plasma membrane. However, the paracellular route is usually the main route of absorption for hydrophilic drugs (proteins, peptides, etc.). Here we confirm that AT-1002, a small peptide derived from ZOT, causes opening of the TJs of a Caco-2 cell monolayer (Motlekar et al., 2006, Song et al., 2008a) and show that this effect is reversible. Additional experiments showed that co-administration of AT-1002 with salmon calcitonin intratracheally increased the systemic exposure of salmon calcitonin by up to 5.2-fold suggesting that we can use AT-1002 to deliver antigens and other payloads systemically. In fact, previous studies have shown that AT-1002 enhances the delivery of small molecules (Motlekar et al., 2006, Song et al., 2008a, Song et al., 2008b). Another study showed that peptides from the extracellular loops of the TJ protein occludin could be used for TJ modulation (Tavelin et al., 2003) by increasing the permeability of the TJs without causing short-term toxicity. However, these peptides had an effect only when added to the basolateral side of the monolayer. Agents that enhance drug or antigen delivery have to be applied to the apical side of epithelial surfaces to be useful. Here we show that AT-1002 is such an agent and thus represents a prototype of a new class of TJ modulators.
Two main applications of TJ-opening molecules such as AT-1002 can be envisioned: classical drug delivery and antigen delivery for vaccination. Recently, a peptide from the capsid of rotaviruses was shown to facilitate insulin uptake in rats (Nava et al., 2004). Other TJ-modulating (TJM) peptides and peptide YY (PYY) improved drug transfer across epithelial tissues (Chen et al., 2006, Gonzalez-Mariscal and Nava, 2005). Therefore, compounds that enable efficient, non-toxic and non-invasive drug delivery would revolutionize the treatment of multiple diseases. Thus, a TJ-modulating peptide such as AT-1002 represents a promising advancement in mucosal drug delivery.[2]

C34H54F3N9O9S

821.91

821.37172

AT-1002;835872-35-0

138319696

L-phenylalanyl-L-cysteinyl-L-isoleucyl-glycyl-L-arginyl-L-leucine trifluoroacetic acid; H-Phe-Cys-Ile-Gly-Arg-Leu-OH.TFA

FCIGRL

White to off-white solid powder

312Ų

11

15

22

56

1210

6

CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC1=CC=CC=C1)N.C(=O)(C(F)(F)F)O

JDVTZXDTPSAPFV-CACDTQBQSA-N

InChI=1S/C32H53N9O7S.C2HF3O2/c1-5-19(4)26(41-29(45)24(17-49)40-27(43)21(33)15-20-10-7-6-8-11-20)30(46)37-16-25(42)38-22(12-9-13-36-32(34)35)28(44)39-23(31(47)48)14-18(2)3;3-2(4,5)1(6)7/h6-8,10-11,18-19,21-24,26,49H,5,9,12-17,33H2,1-4H3,(H,37,46)(H,38,42)(H,39,44)(H,40,43)(H,41,45)(H,47,48)(H4,34,35,36);(H,6,7)/t19-,21-,22-,23-,24-,26-;/m0./s1

(2S)-2-[[(2S)-2-[[2-[[(2S,3S)-2-[[(2R)-2-[[(2S)-2-amino-3-phenylpropanoyl]amino]-3-sulfanylpropanoyl]amino]-3-methylpentanoyl]amino]acetyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-4-methylpentanoic acid;2,2,2-trifluoroacetic acid

AT-1002 (TFA); AT1002 TFA;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~33.33 mg/mL (~40.55 mM)
H2O :~1 mg/mL (~1.22 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (3.04 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (3.04 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (3.04 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 8.33 mg/mL (10.13 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶 (<60°C).

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
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7、 以上所有助溶剂都可在 Invivochem.cn网站购买。