Central complement component C3 (KD = 0.5 nM)

Amyndas Pharmaceuticals临床开发了新一代高选择性和强效的C3抑制剂，称为compstatins Cp40/AMY-101，用于各种补体介导的适应症。这些小型肽C3抑制剂是灵长类/人类特异性的，与之前被评估为ARDS治疗选择的补体抑制剂TP-10等大型生物制剂相比，它们显示出更有利的药理学特征和更大的组织穿透能力[2]。

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鉴于基于compstatin-based抑制剂（如AMY-101）的C3阻断可以通过阻断参与SARS-CoV-2诱导的ARDS和血栓性微血管病的所有下游促炎介质的同时产生，提供比抗C5或抗C5a药物更广泛的治疗复盖面，AMY-101作为新冠肺炎感染严重病例中的抗炎剂，可以很好地进行临床评估[2]。

对于患有自然发生的慢性牙周炎的 NHP，AMY-101 可以帮助改善牙周健康 [1]。 AMY-101 可产生持久的抗炎作用 [1]。皮下注射 AMY-101（4 毫克/公斤体重，每 24 小时一次，持续 28 天）可显着且持续地降低 PPD（组织破坏的指标）[1]。在 UUO 诱导的肾纤维化中，AMY-101（Cp40，1 mg/kg，每 12 小时皮下注射一次，每天一次，持续 7 或 14 天）可减少纤维化和炎症细胞浸润 [3]。

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局部给药的AMY-101（0.1 mg/部位）不会对健康牙龈造成刺激[1]
为了确定在NHP中施用肽C3抑制剂AMY-101后可能出现的局部牙龈刺激，在五只动物的健康后牙牙龈中注射了治疗剂量的AMY-101（50μL 2mg/mL溶液，相当于0.1mg/部位）30。每只动物共接受四次注射，每象限一次；两次注射使用AMY-101，另外两次注射涉及含有5%葡萄糖的注射用水（WFI）（对照）。在每只动物中，在上颌和下颌象限（共2个部位）注射AMY-101，而在对侧的两个部位注射对照溶液。在第0、7和14天共注射AMY-101和对照溶液三次，然后进行2周的观察期，不再注射。在基线检查时（第0天）和每2-3天拍摄一次口腔照片，以记录注射部位周围的牙龈状况。仔细的日常临床检查显示，在整个观察期间注射AMY-101或对照溶液后没有刺激迹象（图1）。在第-1天和第15天采集血液样本，并进行血液学和生化分析。所有动物的所有测量值均在正常范围内（未显示）。

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AMY-101即使每3周服用一次，也能提供保护[1]
我们之前已经证明，每周龈内注射AMY-101可以改善患有自然慢性牙周病的NHP的牙周状况。30不那么频繁但仍然成功的给药将促进AMY-101在人类中的应用。为了探索这种可能性，我们测试了AMY-101在给药频率较低的情况下是否也有效。为此，5只动物每2周服用一次2-mg/mL的AMY-101溶液，另外5只动物则每3周服用一次AMY-101。具体而言，AMY-101被局部注射到上颌骨两侧的前牙和后牙的牙龈中（共17个部位；牙齿之间的腭乳头[15个部位]，第三磨牙的远端牙龈[2个部位]）。在整个研究过程中，在基线和1、2、4、6、7、8、10和12周进行了临床检查，以确定疾病的进展和AMY-101的潜在有益作用。AMY-101注射前的临床读数作为基线对照。下颌骨未接受治疗，但在整个研究过程中通过临床牙周检查进行监测，以进行比较。该研究包括6周的AMY-101治疗（治疗期），随后6周不进行AMY-101处理（随访期）。

无论给药频率如何，AMY-101都会显著降低测量牙周炎症（GI和探诊出血[BOP]）或组织破坏（PPD和CAL）的临床指标（图4和图5）。有趣的是，基线和后续读数之间的差异在6周时或之后达到了统计学意义（即，在AMY-101治疗停止的时间点）。即使在12周时，6周时观察到的许多差异仍然具有统计学意义（BOP、PPD和CAL）（图4B-4D和5B-5D）。在相同的12周间隔内，还监测了未经治疗的颌骨（下颌骨）的上述临床指标。与AMY-101治疗的上颌骨临床状况的改善相比，下颌骨的临床指标在研究过程中与基线值相比没有显示出显著差异（图4和图5）。综上所述，AMY-101可以诱导持久的临床抗炎作用。

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全身注射AMY-101可以改善NHPs的牙周状况[1]
鉴于AMY-101也被考虑用于全身性疾病，牙周炎是一种非常普遍的疾病，34我们测试了AMY-101在全身给药时是否有效。AMY-101以4mg/kg体重的浓度通过皮下注射在10只动物中给药，每24小时一次，共28天。为了确定疾病的进展和AMY-101的潜在有益作用，在基线（第0周）和整个研究期间（第1、2、3、4和11周的时间点）进行了临床检查。此外，在基线、4周和11周时对牙龈和骨骼进行了活组织检查。

系统给药AMY-101可显著长期降低PPD，PPD是衡量组织破坏的指标（图6A）。在第4周首次观察到保护作用。令人惊讶的是，即使在第4周后停药，保护作用仍持续至少7周（第11周）而没有下降（图6A）。还观察到评估牙周炎症的BOP有所改善；与基线相比的差异在第2周和第3周达到统计学意义（图6B）。4周时的组织学观察表明，与基线表达相比，AMY-101导致牙槽骨附近结缔组织中促炎和促破骨细胞因子（白细胞介素[IL]-17和核因子κB配体受体激活剂[RANKL]）的表达降低，骨保护素（OPG；RANKL的天然抑制剂）的表达升高（图7）。此外，AMY-101处理导致补体切割片段C3d和C5a减少，进一步证实了其抑制补体激活的能力。总之，全身性AMY-101改善了NHPs的牙周状况，在停药后至少7周内保持稳定。

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C3缺乏可减轻UUO诱导的肾纤维化中的纤维化和炎性细胞浸润[3]
为了研究C3在UUO发病机制中的作用，我们使用了一种肽C3抑制剂，Compstatin类似物AMY-101/Cp40来阻断C3的激活。Masson染色和α-SMA表达分析表明，注射1mg/kg Cp40的UUO小鼠的间质纤维化程度远低于注射对照肽的小鼠（补充图3A-D）。Western blot分析还表明，注射Cp40的UUO小鼠的α-SMA和PDGFR-β水平降低（补充图3E、F）。除了减轻的肾小管间质纤维化外，与注射肽的小鼠相比，注射Cp40的UUO小鼠的F4/80+巨噬细胞、CD3+T细胞、CD4+T细胞和CD8+T细胞的肾浸润显著减少（图5A-E）。同时，Cp40显著限制了UUO小鼠MCP-1、IL-6、IL-1β和TNF-αmRNA表达的升高（图5F）。这些数据表明，C3介导T细胞和巨噬细胞在梗阻性损伤时浸润肾脏。

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阻断C3-C3aR信号通过抑制UUO小鼠中IL-17A的产生来减轻肾纤维化[3]
如图7A所示，与假对照组小鼠相比，UUO小鼠肾脏IL-17A的mRNA水平显著升高。此外，与假对照小鼠血清中的IL-17A水平相比，UUO小鼠血清中IL-17A水平在早期和晚期显著升高（图7B）。与ELISA和mRNA数据一致，FACS结果显示，UUO后梗阻肾脏中8.48%和10.9%的CD4+肾细胞分别表达IL-17A，而假手术小鼠中≤5.3%的CD4+表达IL-17A+，这种作用被AMY-101/Cp40和SB290157强烈抑制（图7C-H）。此外，我们分析了C3阻断UUO小鼠和UUO小鼠肾脏中CD11b+F4/80+IL-17+细胞的比例。结果显示，两组单核细胞中CD11b+F4/80+IL-17+细胞约占1%，用CP40阻断C3后仅略有变化（补充图4A、B）。在14天的UUO小鼠中也证实了类似的结果（补充图4C，D）。因此，我们确定UUO小鼠中IL-17A的主要产生者是T细胞，单侧输尿管结扎后T细胞显著增加。

PBMC分离[3]
患者和正常受试者捐献了5毫升肝素化管中收集的血液。用PBS将血液1:1稀释，并覆盖在淋巴细胞分离培养基上。离心后，收集3ml含有PBMC的界面，用PBS稀释至6ml，然后用冷PBS洗涤两次并计数。收集PBMC进行流式细胞术分析。

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CFSE标签[3]
通过将冻干的CFSE溶解在DMSO中新鲜制备CFSE储备溶液（5mM）。从幼年小鼠的脾脏中获得脾细胞，并在37°C下用PBS中5μM的CFSE标记15分钟。通过加入三倍体积的冰冷FBS并在冰上孵育细胞5分钟来淬灭过量的CFSE。然后用PBS洗涤CFSE标记的细胞三次，并在有或没有刺激的情况下培养。

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T细胞活化[3]
将预涂有抗CD3和抗CD28抗体的96孔检测板在37°C下孵育4小时。从幼年小鼠的脾脏中获得脾细胞（1×106个细胞/孔），并在含有IL-2（10 ng/mL）、IL-12（10μg/mL）或IL-4（4 ng/mL的培养基中在96孔板中培养3天。

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流式细胞术分析[3]
制备单个肾细胞悬浮液，用PMA/离子霉素/高尔基体塞刺激4小时。将细胞与不同的一抗或适当的同种型对照抗体在4°C下孵育30分钟。使用PerCP/Cy5.5结合的抗人CD14抗体；PerCP/Cy5.5联合抗小鼠CD4；APC偶联抗小鼠F4/80；以及PerCP/Cy5.5结合的抗小鼠CD11b。细胞表面染色后，用Cytofix/Cytoperm Soln试剂盒固定和渗透细胞，用Alexa Fluor 488偶联的抗人C3和PE偶联的抗小鼠IL-17A进行细胞内染色。所有流式细胞术分析均使用LSR II流式细胞仪和Flowjo软件进行。

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ELISA[3]
为了量化肾脏中的IL-17A水平，根据制造商的说明使用小鼠IL-17A ELISA分析样本。所有测量均进行了两次。

Animal/Disease Models: 15 adult male cynomolgus monkeys (Macaca fascicularis) (7-15 years old; weight 5.0-7.6 kg) [1].
Doses: 0.1 mg/site; 50 μL of 2 mg/mL solution.
Route of Administration: local injection. (3 times a week or 1 time a week for 6 weeks, then 6 weeks of follow-up, no treatment required.)
Experimental Results: No irritation to healthy gums.

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Animal/Disease Models: UUO and sham-operated mice [3].
Doses: 1 mg/kg.
Route of Administration: Inject subcutaneously (sc) (sc) every 12 hrs (hrs (hours)) one time/day for 7 or 14 days.
Experimental Results: 1 mg/kg Cp40 produced Dramatically less severe interstitial fibrosis than control mice injected with the peptide.

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C3 Inhibitor AMY-101 [1]
The 14-residue compstatin analog AMY-101 [(D)Tyr-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Sar-Ala-His-Arg-Cys]-mIle-NH2, where Sar is sarcosine/N-methyl glycine and mIle is N-methyl isoleucine] was produced as a disulfide-bridged, cyclic peptide by solid-phase peptide synthesis methodology as previously described. AMY-101 was injected locally into the gingiva (50 μL volume) at different concentrations (2–200 mg/mL) using a 30G short needle. Alternatively, for systemic administration, AMY-101 was given by subcutaneous injection (4 mg/kg bodyweight) using a 1-mL insulin safety syringe with a 28G × 1/2-inch needle.

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Clinical Examination and Observation [1]
Clinical periodontal examinations were performed and the diagnosis was established according to the criteria of the American Academy of Periodontology for human periodontal disease. Examinations using a periodontal probe were performed at baseline and throughout the study to monitor the progression of the disease and the effect of AMY-101 treatment. The examinations included determination of PPD (by measuring the distance [in millimeters] from the gingival margin to the base of the pocket), CAL (distance from the cementoenamel junction to the base of the pocket), GI (using a scale of 0–3, according to Löe), BOP (percentage of positive sites), and plaque index (PI; scale of 0–3 according to Löe). PPD, CAL, and BOP were measured at six sites: mesio-buccal, mid-buccal, disto-buccal, mesio-lingual, mid-lingual, and disto-lingual aspects of each tooth. GI and PI were assessed at four sites (buccal, lingual, mesial, and distal). GI and BOP are measures of periodontal inflammation, while CAL and PPD assess tissue destruction. The PI is a clinical measure of biofilm accumulation on tooth surfaces. In the irritation study, injection sites were clinically observed daily for signs of inflammation or the formation of an abscess, redness, itching, hematoma, bruising, bleb, or nodules. The degree of gingival inflammation was assessed as healthy, mild (slight change in color, no BOP), moderate (redness, BOP), or severe (marked redness, tendency to spontaneous bleeding).

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Animal model [3]
Unilateral ureteral obstruction (UUO) is a popular experimental model of renal injury. Mice aged 6–8 weeks were anesthetized followed by a lateral incision on the back of the mouse. Subsequently, the left ureter was exposed and tied off with two 4.0 silk suture. Sham-operated mice underwent an identical procedure but without ureteric ligation. The therapeutic experiment was performed with the Compstatin analog AMY-101/Cp40 (dTyr-Ile-[Cys-Val-Trp(Me)-Gln-Asp-Trp-Sar-His-Arg-Cys]-mIle-NH2;1.7kDa) which was produced by solid-phase peptide synthesis, and SB290157, a C3a receptor antagonist, which was purchased from Sigma-Aldrich. UUO and sham-operated mice were treated with Cp40 (1 mg/kg) via subcutaneous injection every 12 h and SB290157 (30 mg/kg) via intraperitoneal injection daily. After 7 or 14 days, the mice were sacrificed by cervical vertebra dislocation, and then, peripheral blood, spleen, and renal tissues were collected. The mouse kidneys were fixed in 4% formalin for 24 h, processed through dehydration in a graded series of alcohol and embedded in paraffin The remaining sample was frozen in liquid nitrogen for later use.

Dose-Escalation Study for Local Injection of AMY-101 [1]
This study was designed to determine possible local gingival irritation after local injection of increasing concentrations of AMY-101 in NHPs with naturally occurring periodontitis. Escalating doses of AMY-101 tested were 2, 10, 50, 100, and 200 mg/mL in a total volume of 50 μL, thus corresponding to 0.1, 0.5, 2.5, 5, and 10 mg/site, respectively. The injected sites involved posterior teeth on both sides of the maxilla (palatal interdental papillae) and mandible (buccal interdental papillae). Five animals were used and all injections were given in a single session followed by a 2-week observation period. The animals were examined daily for the possible presence of local gingival irritation in response to AMY-101 injections. Doses equal to or higher than 10 mg/mL caused mild to moderate inflammation, which was observed more often with the highest doses (100 and 200 mg/mL) (Figure 2). No irritation was observed with the 2-mg/mL dose at any treated site, consistent with the data discussed above. Clinical examinations to determine periodontal disease activity and intraoral photography were performed at baseline and after 1 and 2 weeks. In terms of efficacy, doses up to 50 mg/mL caused a reduction in periodontal clinical parameters (probing pocket depth [PPD], clinical attachment level [CAL], and gingival index [GI]) (Figure 3). In contrast, the highest doses (100 and 200 mg/mL) caused deterioration in the same clinical parameters (Figure 3). Therefore, among the different AMY-101 concentrations tested, the 2-mg/mL dose appears to be an optimal dose fulfilling both safety and protection requirements.

[1]. Safety and Efficacy of the Complement Inhibitor AMY-101 in a Natural Model of Periodontitis in Non-human Primates. Mol Ther Methods Clin Dev. 2017 Aug 18;6:207-215.

[2]. The first case of COVID-19 treated with the complement C3 inhibitor AMY-101. Clin Immunol. 2020 Apr 29:108450.

[3]. Complement C3 Produced by Macrophages Promotes Renal Fibrosis via IL-17A Secretion. Front Immunol. 2018 Oct 22;9:2385.

AMY-101, also known as compstatin 40, is a peptidic inhibitor of the central complement component C3. AMY-101 is under investigation in clinical trial NCT03694444 (A Study of the C3 Complement Inhibitor AMY-101 in Adults With Gingivitis).
C3 Complement Inhibitor AMY-101 is a compstatin-based inhibitor of human complement component C3, with potential use as a treatment for various diseases in which excessive complement activation plays a key role, including paroxysmal nocturnal hemoglobinuria (PNH) and complement 3 glomerulopathy (C3G). Upon administration, C3 complement inhibitor AMY-101 selectively binds to C3 and inhibits C3 activity. This prevents complement pathway activation, and inhibits complement-mediated inflammation and cell lysis. Excessive complement activation plays a key role in various inflammatory and autoimmune diseases, and leads to tissue destruction. C3 is a crucial and central component of the complement system, and the complement system is an integral component of the innate immune response.

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Periodontitis is a chronic inflammatory disease associated with overactivation of the complement system. Recent preclinical studies suggest that host-modulation therapies may contribute to effective treatment of human periodontitis, which may lead to loss of teeth and function if untreated. We previously showed that locally administered AMY-101 (Cp40), a peptidic inhibitor of the central complement component C3, can inhibit naturally occurring periodontitis in non-human primates (NHPs) when given once a week. This study was undertaken to determine the local safety of increasing doses of the drug as well as its efficacy when given at a reduced frequency or after systemic administration. Our findings have determined a local dose of AMY-101 (0.1 mg/site) that is free of local irritation and effective when given once every 3 weeks. Moreover, a daily subcutaneous dose of AMY-101 (4 mg/kg bodyweight) was protective against NHP periodontitis, suggesting that patients treated for systemic disorders (e.g., paroxysmal nocturnal hemoglobinuria) can additionally benefit in terms of improved periodontal condition. In summary, AMY-101 appears to be a promising candidate drug for the adjunctive treatment of human periodontitis, a notion that merits investigation in human clinical trials. [1]

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Acute respiratory distress syndrome (ARDS) is a devastating clinical manifestation of COVID-19 pneumonia and is mainly based on an immune-driven pathology. Mounting evidence suggests that COVID-19 is fueled by a maladaptive host inflammatory response that involves excessive activation of innate immune pathways. While a "cytokine storm" involving IL-6 and other cytokines has been documented, complement C3 activation has been implicated as an initial effector mechanism that exacerbates lung injury in preclinical models of SARS-CoV infection. C3-targeted intervention may provide broader therapeutic control of complement-mediated inflammatory damage in COVID-19 patients. Herein, we report the clinical course of a patient with severe ARDS due to COVID-19 pneumonia who was safely and successfully treated with the compstatin-based complement C3 inhibitor AMY-101. [2]

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Complement synthesis in cells of origin is strongly linked to the pathogenesis and progression of renal disease. Multiple studies have examined local C3 synthesis in renal disease and elucidated the contribution of local cellular sources, but the contribution of infiltrating inflammatory cells remains unclear. We investigate the relationships among C3, macrophages and Th17 cells, which are involved in interstitial fibrosis. Here, we report that increased local C3 expression, mainly by monocyte/macrophages, was detected in renal biopsy specimens and was correlated with the severity of renal fibrosis (RF) and indexes of renal function. In mouse models of UUO (unilateral ureteral obstruction), we found that local C3 was constitutively expressed throughout the kidney in the interstitium, from which it was released by F4/80+macrophages. After the depletion of macrophages using clodronate, mice lacking macrophages exhibited reductions in C3 expression and renal tubulointerstitial fibrosis. Blocking C3 expression with a C3 and C3aR inhibitor provided similar protection against renal tubulointerstitial fibrosis. These protective effects were associated with reduced pro-inflammatory cytokines, renal recruitment of inflammatory cells, and the Th17 response. in vitro, recombinant C3a significantly enhanced T cell proliferation and IL-17A expression, which was mediated through phosphorylation of ERK, STAT3, and STAT5 and activation of NF-kB in T cells. More importantly, blockade of C3a by a C3aR inhibitor drastically suppressed IL-17A expression in C3a-stimulated T cells. We propose that local C3 secretion by macrophages leads to IL-17A-mediated inflammatory cell infiltration into the kidney, which further drives fibrogenic responses. Our findings suggest that inhibition of the C3a/C3aR pathway is a novel therapeutic approach for obstructive nephropathy. [3]

C83H117N23O18S2

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C, 55.72; H, 6.59; N, 18.01; O, 16.10; S, 3.58

1427001-89-5

AMY-101 TFA;1789738-04-0;AMY-101 acetate

131634231

{D-Tyr}-Ile-Cys-Val-{Trp(Me)}-Gln-Asp-Trp-{Sar}-Ala-His-Arg-Cys-{N(Me)Ile}-NH2 (Disulfide bridge:Cys3-Cys13); H-D-Tyr-Ile-Cys(1)-Val-Trp(Me)-Gln-Asp-Trp-Sar-Ala-His-Arg-Cys(1)-N(Me)Ile-NH2; D-tyrosyl-L-isoleucyl-L-cysteinyl-L-valyl-N1-methyl-L-tryptophyl-L-glutaminyl-L-alpha-aspartyl-L-tryptophyl-sarcosyl-L-alanyl-L-histidyl-L-arginyl-L-cysteinyl-N2-methyl-L-isoleucinamide (3->13)-disulfide

YICVXQDWGAHRCI; {D-Tyr}-ICV-{Trp(Me)}-QDW-{Sar}-AHRC-{N(Me)Ile}-NH2 (Disulfide bridge:Cys3-Cys13)

White to off-white solid powder

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CC[C@H](C)[C@@H](C(=O)N[C@H]1CSSC[C@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)CN(C(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@@H](NC1=O)C(C)C)CC2=CN(C3=CC=CC=C32)C)CCC(=O)N)CC(=O)O)CC4=CNC5=CC=CC=C54)C)C)CC6=CN=CN6)CCCNC(=N)N)C(=O)N(C)[C@@H]([C@@H](C)CC)C(=O)N)NC(=O)[C@@H](CC7=CC=C(C=C7)O)N

MUSGYEMSJUFFHT-UWABRSFTSA-N

InChI=1S/C83H117N23O18S2/c1-11-43(5)68(103-72(114)53(84)30-46-23-25-50(107)26-24-46)80(122)100-61-39-125-126-40-62(82(124)106(10)69(70(86)112)44(6)12-2)101-73(115)55(21-17-29-90-83(87)88)94-76(118)58(33-49-36-89-41-92-49)96-71(113)45(7)93-65(109)38-105(9)81(123)60(31-47-35-91-54-20-15-13-18-51(47)54)99-77(119)59(34-66(110)111)97-74(116)56(27-28-64(85)108)95-75(117)57(98-79(121)67(42(3)4)102-78(61)120)32-48-37-104(8)63-22-16-14-19-52(48)63/h13-16,18-20,22-26,35-37,41-45,53,55-62,67-69,91,107H,11-12,17,21,27-34,38-40,84H2,1-10H3,(H2,85,108)(H2,86,112)(H,89,92)(H,93,109)(H,94,118)(H,95,117)(H,96,113)(H,97,116)(H,98,121)(H,99,119)(H,100,122)(H,101,115)(H,102,120)(H,103,114)(H,110,111)(H4,87,88,90)/t43-,44-,45-,53+,55-,56-,57-,58-,59-,60-,61-,62-,67-,68-,69-/m0/s1

2-((4R,7S,10S,13S,19S,22S,25S,28S,31S,34R)-10-((1H-imidazol-5-yl)methyl)-19-((1H-indol-3-yl)methyl)-34-((2S,3S)-2-((R)-2-amino-3-(4-hydroxyphenyl)propanamido)-3-methylpentanamido)-4-(((2S,3S)-1-amino-3-methyl-1-oxopentan-2-yl)(methyl)carbamoyl)-25-(3-amino-3-oxopropyl)-7-(3-guanidinopropyl)-31-isopropyl-13,17-dimethyl-28-((1-methyl-1H-indol-3-yl)methyl)-6,9,12,15,18,21,24,27,30,33-decaoxo-1,2-dithia-5,8,11,14,17,20,23,26,29,32-decaazacyclopentatriacontan-22-yl)acetic acid

AMY-101; AMY 101; AMY101; Compstatin 40; 1427001-89-5; UNII-4Z4DFR9BX7; 4Z4DFR9BX7; peptide 14 [PMID: 22795972]; S3,S13-Cyclo(D-tyrolsyl-L-isoleucyl-L-cysteinyl-L-valyl-1-methyl-L-tryptophyl-L-glutaminyl-L-aspartyl-L-tryptophyl-N-methyl-L-glycyl-L-alanyl-L-histidyl-L-arginyl-L-cysteinyl-N-methyl-L-isoleucinamide); Compstatin 40; CP40; CP 40; CP-40;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。