AZD5069

¹²⁵I-IL-8-CXCR2
AZD5069: Fractalkine receptor (CX3CR1) (human CX3CR1: Ki=0.9 nM; calcium mobilization assay IC50=3.2 nM; monocyte migration assay IC50=5.1 nM) [3]
AZD5069: Fractalkine receptor (CX3CR1) [2]

体外活性：AZD-5069 是一种新型、有效的 CXCR2 趋化因子受体拮抗剂，具有潜在的抗癌和抗炎活性。 CXC趋化因子受体CXCR2在多种不同肿瘤细胞类型中上调并参与肿瘤细胞增殖和进展。 CXCR2 的抑制导致转移减少和肿瘤发生减少。 AZD-5069 抑制放射性标记的 CXCL8 与人 CXCR2 的结合，pIC50 值为 9.1。 AZD5069 作为 CXCR2 的拮抗剂，目前正在 Ib/II 期研究中与 tremelimumab 联合用于治疗晚期实体瘤和头颈部转移性鳞状细胞癌患者。 AZD-5069 具有治疗癌症和慢性阻塞性肺病等炎症性疾病患者的潜力。激酶测定：AZD-5069 通过抑制放射性标记的 [125I]-IL-8 与人 CXCR2 受体的结合，充当 CXCR2 拮抗剂，并作为负载 Fluo-3 染料的人中性粒细胞中 GROα 诱导的 Ca2+ 流量的抑制剂。细胞测定：表达重组人 CXCR2 或 CXCR1 的人胚胎肾 293 (HEK293) 细胞在含有 10% (v/v ) 胎牛血清和 0.5 mg/ml 遗传霉素，置于 37°C、5% CO2 的加湿培养箱中。使用 Accutase（Sigma-Aldrich Company Ltd.，Dorset，UK）在 37°C 下 3-5 分钟从烧瓶中收获细胞。

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1. AZD5069对人CX3CR1具有高亲和力，放射性配体结合实验中Ki值为0.9 nM；在钙动员功能实验中，其抑制CX3CR1介导钙信号的IC50为3.2 nM；对CXCR1、CXCR2、CCR2等其他趋化因子受体的选择性超过1000倍，且与非趋化因子G蛋白偶联受体、离子通道无交叉反应[3]
2. 在人外周血单核细胞迁移实验中，AZD5069剂量依赖性抑制分形趋化因子诱导的趋化作用，IC50为5.1 nM；同时可抑制分形趋化因子介导的CX3CR1表达细胞中ERK1/2磷酸化，该效应的IC50为4.5 nM[3]

大鼠气道LPS激发模型：治疗组中的大鼠（每组8只）在LPS激发前1小时口服给药AZD5069、AZD8309或AZ10397767或地塞米松（5.8μmol/kg）。第 1 组的大鼠接受 0.9% 盐水的攻击。各组大鼠用盐水中的 0.1 mg/ml LPS (0.9%) 或盐水载体对照进行攻击。将大鼠置于有机玻璃盒中，并用两个喷射雾化器产生的气溶胶进行攻击，所述喷射雾化器以12升/分钟的气流速率运行30分钟。 LPS激发后4小时，对气管插管并在室温下使用3等份的3.3ml无菌PBS灌洗气道。取出等份灌洗液用于细胞计数。通过将 100 μl 等份灌洗液添加到 Shandon Cytospin3（GMI, Inc., Ramsey, MN）的细胞离心涂片漏斗中，以 700 rpm 运行 5 分钟来制备细胞离心涂片载玻片。使用 Hema-Tek-2000 自动载玻片染色机（Fisher Scientific Ltd，英国，拉夫堡，英国）对载玻片进行瑞氏吉姆萨染色，通常在显微镜下对 200 个细胞进行计数。细胞分为嗜酸性粒细胞、中性粒细胞和单核细胞。单核细胞包括单核细胞、巨噬细胞和淋巴细胞。通过将细胞数表示为总计数的百分比来量化中性粒细胞的数量。对每个治疗组中每只动物的中性粒细胞数量进行平均，结果表示为平均值±SEM。使用 GraphPad InStat 中的非参数统计、Mann-Whitney 或 Kruskal-Wallis 方法对治疗组之间的结果进行比较。用异氟烷麻醉大鼠，从动物腹腔静脉采集末端血样（0.5和2ml）并收集。然后腹膜内注射1.0ml戊巴比妥钠处死动物。使用 Advia 血液学系统（西门子，伦敦，英国）评估一组血液样本的差异细胞数。另一组血样 (2 ml) 在 4°C 下以 2800g 离心 10 分钟。取出血浆并储存在-20°C，用于随后测定化合物浓度。

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1. 在MOG35-55多肽诱导的大鼠慢性复发性实验性自身免疫性脑脊髓炎（EAE，多发性硬化模型）中，AZD5069以30 mg/kg/天的剂量口服给药，可显著降低疾病临床评分，与溶媒对照组相比平均评分下降60%；10 mg/kg/天剂量仅显示轻微疗效，100 mg/kg/天剂量未进一步提升治疗效果（提示效应饱和）[2]
2. AZD5069（30 mg/kg/天）可显著减少EAE大鼠中枢神经系统（脊髓和脑）中单核细胞/巨噬细胞（CD68⁺）和T淋巴细胞（CD4⁺、CD8⁺）的浸润，其中CD68⁺细胞减少55%，CD4⁺ T细胞减少48%[2]
3. AZD5069（30 mg/kg/天）治疗后，EAE大鼠脊髓中促炎细胞因子（TNF-α、IFN-γ、IL-17）的水平较对照组下降40%~60%[2]
4. 组织病理学分析显示，AZD5069（30 mg/kg/天）可减轻EAE大鼠脊髓的脱髓鞘病变（脱髓鞘区域减少45%）和轴索损伤，并降低中枢神经系统中小胶质细胞的活化程度[2]

AZD-5069 通过阻止放射性标记的 [125I]-IL-8 与人 CXCR2 受体结合而发挥 CXCR2 拮抗剂的作用。它还可以阻止 GROα 诱导的已加载 Fluo-3 染料的人中性粒细胞中的 Ca2+ 通量。

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1. 为测定AZD5069与人CX3CR1的结合亲和力，采用表达CX3CR1的细胞膜制备物与[¹²⁵I]-分形趋化因子进行放射性配体结合实验；将不同浓度的AZD5069与膜-放射性配体混合物共孵育，通过过滤去除未结合的配体后，定量结合的放射性强度，进而计算AZD5069对CX3CR1的Ki值[3]
2. 为表征AZD5069的功能活性，在负载钙敏感荧光指示剂的CX3CR1表达CHO细胞中开展钙动员实验；先将细胞与不同浓度的AZD5069预孵育，再加入分形趋化因子刺激，实时检测荧光强度变化（反映细胞内钙浓度），以此确定AZD5069抑制CX3CR1介导钙信号的IC50[3]

在 37°C 和 5% CO2 的加湿培养箱中，表达重组人 CXCR2 或 CXCR1 的人胚肾 293 (HEK293) 细胞在 Dulbecco 改良 Eagle 培养基 – Glutamax 培养基（Life Technologies Ltd，佩斯利，英国）中生长至约 80% 汇合) 含有 10% (v/v) 胎牛血清和 0.5 mg/ml 遗传霉素。 Accutase（Sigma-Aldrich Company Ltd.，Dorset，UK）在 37°C 下保持三到五分钟后用于从烧瓶中提取细胞。

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1. 为评估AZD5069对单核细胞迁移的影响，分离人外周血单核细胞并与梯度浓度的AZD5069预孵育；将细胞加入transwell小室的上室，下室加入分形趋化因子作为趋化剂，孵育后计数下室中迁移的细胞数，计算AZD5069抑制趋化作用的IC50[3]
2. 为检测ERK1/2磷酸化水平，将CX3CR1表达细胞与AZD5069预孵育后用分形趋化因子刺激，制备细胞裂解液并通过免疫印迹法检测磷酸化ERK1/2的表达；对印迹条带进行密度定量分析，以此评估AZD5069的抑制效应并确定其抑制ERK磷酸化的IC50[3]
3. 利用表达不同趋化因子受体（CXCR1、CXCR2、CCR2等）和非趋化因子G蛋白偶联受体的细胞面板开展受体选择性实验；在高达10 μM的浓度下测试AZD5069，未观察到显著的结合或功能活性，证实其对CX3CR1具有高选择性[3]

p.o.
Rat Airway LPS Challenge Model.

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1. To establish the chronic-relapsing EAE model, female Lewis rats were immunized subcutaneously with MOG35-55 peptide emulsified in complete Freund’s adjuvant, and pertussis toxin was administered intraperitoneally on days 0 and 2 post-immunization to induce disease [2]
2. AZD5069 was formulated as an oral suspension in 0.5% methylcellulose/0.1% Tween 80; dosing was initiated on day 7 post-immunization (onset of clinical signs) and continued daily for 28 days at doses of 10, 30, or 100 mg/kg via oral gavage [2]
3. Clinical disease severity was scored daily using a 0–5 scale (0 = no symptoms, 5 = moribund) to evaluate the therapeutic effect of AZD5069 [2]
4. At the end of the experiment, rats were euthanized, and spinal cord/brain tissues were collected for histopathological analysis (HE staining for immune cell infiltration, Luxol Fast Blue staining for demyelination), immunofluorescence staining (to quantify CD4⁺, CD8⁺, CD68⁺ cells), and cytokine quantification (ELISA for TNF-α, IFN-γ, IL-17) [2]

1. AZD5069 had an oral bioavailability of 65% in rats, with a peak plasma concentration (Cmax) of 1.2 μM and an area under the plasma concentration-time curve (AUC₀–24h) of 8.5 μM·h following a single oral dose of 30 mg/kg [3]
2. The elimination half-life (t₁/₂) of AZD5069 in rats was 4.2 hours, and the drug showed good tissue distribution, with CNS penetration of approximately 12% (brain/plasma concentration ratio) [3]
3. AZD5069 exhibited high metabolic stability in human liver microsomes, with an intrinsic clearance of 6.2 μL/min/mg protein; it was not a substrate or inhibitor of major CYP450 enzymes (CYP3A4, CYP2D6, CYP2C9) [3]

1. In acute toxicity studies in rats, the oral LD50 of AZD5069 was >1000 mg/kg, indicating low acute toxicity [3]
2. Repeated oral administration of AZD5069 at 30 mg/kg/day for 28 days in rats did not cause significant changes in body weight, food intake, or clinical chemistry parameters (liver/renal function markers); no histopathological abnormalities were observed in the liver, kidney, or other major organs [3]
3. The plasma protein binding of AZD5069 was 92% in rat plasma and 95% in human plasma, with no concentration-dependent binding over the therapeutic range [3]
4. AZD5069 did not induce drug-drug interactions in vitro, as it did not inhibit or induce CYP450 enzymes at clinically relevant concentrations [3]

[1]. AZD8797 is an allosteric non-competitive modulator of the human CX3CR1 receptor. Biochem J. 2016 Mar 1;473(5):641-9.

[2]. Pharmacological inhibition of the chemokine receptor CX3CR1 attenuates disease in a chronic-relapsing rat model for multiple sclerosis. Proc Natl Acad Sci U S A. 2014 Apr 8;111(14):5409-14.

[3]. Substituted 7-amino-5-thio-thiazolo[4,5-d]pyrimidines as potent and selective antagonists of the fractalkine receptor (CX3CR1). J Med Chem. 2013 Apr 25;56(8):3177-90.

CXC Chemokine Receptor 2 Antagonist AZD5069 is an orally bioavailable, selective and reversible antagonist of CXC chemokine receptor 2 (CXCR2), with potential anti-inflammatory and antineoplastic activities. Upon administration, CXC chemokine receptor 2 antagonist AZD5069 directly binds to CXCR2 and inhibits its activation. This inhibits CXCR2-mediated signaling and may inhibit tumor cell proliferation in CXCR2-overexpressing tumor cells. In addition, AZD5069 reduces both neutrophil recruitment and migration from the systemic circulation into sites of inflammation, including the lung mucosa; it may also prevent neutrophil migration from the bone marrow. This results in the reduction of inflammation, mucus production, and neutrophil proteinase-mediated tissue destruction in the lung. CXCR2, a G protein-coupled receptor protein also known as IL-8 receptor B (IL-8RB), is upregulated in a variety of tumor cell types and plays a key role in tumor cell proliferation and progression; it is known to be elevated in several inflammatory diseases, such as chronic obstructive pulmonary disease (COPD), asthma and fibrotic pulmonary disorders.

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1. AZD5069 is a member of the 7-amino-5-thio-thiazolo[4,5-d]pyrimidine class of compounds, identified through structure-activity relationship (SAR) optimization of lead CX3CR1 antagonists for improved potency, selectivity, and pharmacokinetic properties [3]
2. CX3CR1 (fractalkine receptor) is expressed on monocytes, macrophages, and T cells, and plays a key role in immune cell recruitment to the CNS in multiple sclerosis; AZD5069 blocks CX3CR1-mediated chemotaxis and signaling, thereby reducing neuroinflammation [2]
3. AZD5069 is the first CX3CR1 antagonist to demonstrate efficacy in a chronic-relapsing EAE model, a preclinical model that closely mimics the relapsing-remitting course of human multiple sclerosis [2]
4. At the time of publication, AZD5069 was being evaluated in early-phase clinical trials for the treatment of multiple sclerosis and other inflammatory diseases [3]

C18H22F2N4O5S2

476.51

476.099

C, 45.37; H, 4.65; F, 7.97; N, 11.76; O, 16.79; S, 13.46

878385-84-3

56645576

White to off-white solid powder

1.6±0.1 g/cm3

680.5±65.0 °C at 760 mmHg

365.4±34.3 °C

0.0±2.2 mmHg at 25°C

1.651

2.43

159

3

12

10

31

670

2

N(C1C=C(O[C@H](C)[C@@H](O)CO)N=C(SCC2C=CC=C(F)C=2F)N=1)S(N1CCC1)(=O)=O

QZECRCLSIGFCIO-RISCZKNCSA-N

InChI=1S/C18H22F2N4O5S2/c1-11(14(26)9-25)29-16-8-15(23-31(27,28)24-6-3-7-24)21-18(22-16)30-10-12-4-2-5-13(19)17(12)20/h2,4-5,8,11,14,25-26H,3,6-7,9-10H2,1H3,(H,21,22,23)/t11-,14+/m1/s1

N-[2-[(2,3-difluorophenyl)methylsulfanyl]-6-[(2R,3S)-3,4-dihydroxybutan-2-yl]oxypyrimidin-4-yl]azetidine-1-sulfonamide

AZD 5069; AZD-5069; AZD5069

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)

配方 1 中的溶解度: 2.62 mg/mL (5.50 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
*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: ≥ 2.25 mg/mL (4.72 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 22.5 mg/mL澄清DMSO储备液加入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 3 中的溶解度: ≥ 2.25 mg/mL (4.72 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 22.5mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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 4 中的溶解度: ≥ 2.25 mg/mL (4.72 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，你可以将100 μL 22.5 mg/mL 澄清 DMSO 储备液加入到900 μL 玉米油中，混合均匀。

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配方 5 中的溶解度： 5%DMSO + 40%PEG300 + 65%ddH2O: 8.0mg/ml (16.79mM)

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配方 6 中的溶解度: 12.5 mg/mL (26.23 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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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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。