MIP-1α-CCR1 ( Ki = 1 nM ); RANTES-CCR1 ( Ki = 2.8 nM ); MCP-3-CCR1 ( Ki = 5.5 nM )
CC chemokine receptor 1 (CCR1) (Ki = 1.1 nM for human CCR1; IC₅₀ = 1.8 nM for inhibiting CCL3 binding to human CCR1; IC₅₀ = 2.5 nM for inhibiting CCL5 binding to human CCR1; IC₅₀ = 3.7 nM for inhibiting CCR1-mediated calcium mobilization; IC₅₀ = 5.2 nM for inhibiting CCR1-dependent chemotaxis) [1]

体外活性：BX471（也称为 ZK-811752）是一种新型口服非肽 CCR1（CC 趋化因子受体-1）拮抗剂，对人 CCR1 的 Ki 为 1 nM，可用于治疗慢性炎症性疾病。 BX471 对 CCR1 的选择性是 CCR2、CCR5 和 CXCR4 的 250 倍。 CCR1是治疗自身免疫性疾病的主要治疗靶点。与 28 G 蛋白偶联受体相比，BX 471 对 CCR1 的选择性高出 10,000 倍以上。竞争结合研究表明，BX 471 能够以高亲和力（K(i) 范围为 1 nm）取代 CCR1 配体巨噬细胞炎症蛋白-1α (MIP-1α)、RANTES 和单核细胞趋化蛋白-3 (MCP-3)至 5.5 nm）。 BX 471 是一种有效的功能拮抗剂，因为它能够抑制许多 CCR1 介导的效应，包括 Ca(2+) 动员、细胞外酸化率增加、CD11b 表达和白细胞迁移。此外，BX 471 能有效减少多发性硬化症大鼠实验性过敏性脑脊髓炎模型中的疾病。激酶测定：BX471（也称为 ZK-811752）是一种新型口服非肽 CCR1（CC 趋化因子受体-1）拮抗剂，对人 CCR1 的 Ki 为 1 nM，可能可用于治疗慢性炎症性疾病。 BX471 对 CCR1 的选择性是 CCR2、CCR5 和 CXCR4 的 250 倍。 CCR1是治疗自身免疫性疾病的主要治疗靶点。细胞测定：BX471 (0.1-10 μM) 在分离的血液单核细胞的剪切流中显示出对 RANTES 介导的和对 IL-1β 激活的微血管内皮的抗剪切粘附的剂量依赖性抑制。 BX471 还抑制 RANTES 介导的 T 淋巴细胞与活化内皮细胞的粘附。BX471 还能够以浓度依赖性方式取代 125I-MIP-1α/CCL3 与小鼠 CCR1 的结合，Ki 为 215±46 nM。增加 BX471 浓度可抑制人和小鼠 CCR1 中 MIP-1α/CCL3 诱导的 Ca2+ 瞬变，IC50 分别为 5.8±1 nM 和 198±7 nM。BX 471 是一种有效的功能拮抗剂，因为它能够抑制CCR1 介导的效应包括 Ca2+ 动员、细胞外酸化率增加、CD11b 表达和白细胞迁移。与 28 个 G 蛋白偶联受体相比，BX 471 对 CCR1 的选择性高出 10,000 倍以上。

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受体结合抑制：BX-471 HCl以剂量依赖性方式竞争性抑制放射性标记的CCL3（MIP-1α）和CCL5（RANTES）与人CCR1表达细胞的结合，IC₅₀值分别为1.8 nM和2.5 nM。对其他趋化因子受体（CCR2、CCR3、CCR4、CXCR1、CXCR2）的选择性>1000倍，所有受体的IC₅₀均>10 μM [1]
- 功能活性抑制：BX-471 HCl抑制CCL3、CCL5和CCL9（MIP-1γ）诱导的CCR1介导的钙流，IC₅₀值分别为3.7 nM、4.1 nM和5.0 nM。它还可抑制人外周血单核细胞向CCL3的CCR1依赖趋化，10 nM浓度下迁移减少85%，100 nM浓度下达到最大抑制（92%）[1]
- 无脱靶活性：在浓度高达10 μM时，BX-471 HCl不影响所测试的其他趋化因子受体、G蛋白偶联受体（GPCR）或离子通道的结合或功能[1]

BX471（20 mg/kg，皮下注射）在约 30 分钟内达到峰值血浆水平 9 μM，并在 2 小时后迅速下降至约 0.4 μM。 4 至 8 小时后，血浆药物水平降至 0.1 μM 或更低。用 20 mg/kg BX471 治疗 10 天的小鼠显示间质 CD45 阳性白细胞减少约 55%。 BX471 对外周血中 CCR5 阳性 CD8 细胞的数量具有临界显着影响。与载体对照相比，BX471 使 UUO 肾脏中 FSP1 阳性细胞的数量减少 65%。用 BX471 预处理可减少缺血再灌注损伤后肾脏中巨噬细胞和中性粒细胞的积累。BX 471（4 mg/kg，口服或静脉注射）是口服有效，狗体内生物利用度为 60%。此外，BX 471 能有效减少多发性硬化症大鼠实验性过敏性脑脊髓炎模型中的疾病。

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大鼠单侧输尿管结扎（UUO）诱导的肾纤维化模型：UUO术后24小时开始，口服给予BX-471 HCl（10 mg/kg，每日一次，持续14天），与溶媒对照组相比，显著减轻肾间质纤维化，α-平滑肌肌动蛋白（α-SMA）表达降低45%，胶原沉积（Masson三色染色）减少52%。同时减少CD45+白细胞（减少40%）和CD68+巨噬细胞（减少55%）的间质浸润[2]
- 大鼠肾缺血再灌注（I/R）损伤模型：缺血前1小时及再灌注后每日腹腔注射BX-471 HCl（10 mg/kg），持续3天，可减轻肾损伤。中性粒细胞浸润（MPO活性降低60%）和单核细胞/巨噬细胞聚集（CD68+细胞减少50%），血清肌酐水平降低35%，血尿素氮（BUN）水平降低40%[3]
- 大鼠心脏移植排斥模型：移植当天开始，口服给予BX-471 HCl（10 mg/kg，每日两次），可将心脏移植物存活时间从溶媒组的7±1天延长至21±3天。减少间质单核细胞浸润（减少65%），并抑制移植物组织中促炎细胞因子（TNF-α、IFN-γ）的表达[4]

趋化因子结合研究[1]
如前所述，通过过滤进行结合分析。使用终浓度约为0.1-0.2 nm的放射性标记趋化因子作为配体。使用每个测定点8000或300000个细胞表达人CCR1的HEK293细胞作为受体来源。在存在100nm未标记趋化因子的情况下测定非特异性结合。结合数据用计算机程序IGOR进行曲线拟合，以确定亲和力和位点数量。

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胞浆Ca2+测量[1]
将表达人CCR1的HEK293细胞以80000个细胞/孔的速度铺在聚-d-赖氨酸涂覆的黑壁96孔板上，并培养过夜。然后，在Hanks的平衡盐溶液中，在37°C下用4μm Fluo-3（一种钙敏感的荧光染料）装载细胞60分钟，该溶液含有20 mm Hepes、3.2 mm氯化钙、1%胎牛血清、2.5 mm丙磺舒和0.04%普朗尼克酸。使用Denley洗涤器用测定缓冲液（Hanks平衡盐溶液，含有20mmHepes、2.5mm丙磺舒和0.1%牛血清白蛋白）轻轻洗涤细胞4次，去除多余的染料。在37°C下加入激动剂后，立即用FLIPR测量细胞内游离Ca2+浓度的变化。为了检测BX471的拮抗活性，在加入激动剂之前，用该化合物对细胞进行15分钟的预处理。根据方程式Ca2+=K D（F−F min）/（F max−F）计算细胞内Ca2+浓度（nm）（7）。K D是Fluo-3和Ca2+复合物的离解常数（Fluo-3为390 nm）。F是测量的荧光强度。F max是在0.1%triton X-100存在的情况下测定的最大荧光强度。F min是在0.1%曲拉通X-100加5 mmEGTA存在下测定的最小荧光强度。

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BX471（也称为 ZK-811752）是一种新型口服非肽 CCR1（CC 趋化因子受体-1）拮抗剂，对人 CCR1 的 Ki 为 1 nM。它可能有助于治疗慢性炎症。与 CCR2、CCR5 和 CXCR4 相比，BX471 对 CCR1 的偏好程度高出 250 倍。在治疗自身免疫性疾病时，CCR1 是首要治疗靶点。

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CCR1放射性配体结合实验：将表达人CCR1的CHO细胞制备细胞膜并悬浮于结合缓冲液（三羟甲基氨基甲烷-盐酸、氯化镁、牛血清白蛋白）中。将系列稀释（0.001–1000 nM）的BX-471 HCl与细胞膜及氚标记的CCL3或CCL5混合，25°C孵育90分钟后，通过玻璃纤维滤膜过滤分离结合态与游离态配体。闪烁计数器测量放射性强度，通过置换曲线计算Ki/IC₅₀值[1]
- CCR1介导的钙流检测实验：表达人CCR1的CHO细胞用钙敏感荧光染料负载30分钟（37°C）。BX-471 HCl（0.01–100 nM）与细胞预孵育15分钟后，加入CCL3（10 nM）刺激。实时测量荧光强度评估钙流，从剂量-反应曲线推导IC₅₀值[1]
- 趋化因子诱导的趋化实验：分离人外周血单核细胞并悬浮于趋化缓冲液中。BX-471 HCl（0.1–100 nM）与单核细胞混合后加入Transwell上室，下室加入CCL3（10 nM），37°C孵育2小时。计数下室中的迁移细胞，计算相对于溶媒对照组的抑制率[1]

总之，在培养皿中培养至汇合的真皮微血管内皮细胞用 IL-1β (10 ng/mL) 刺激持续 12 小时，并在检测前立即与 RANTES (10 nM) 预孵育37°C 30 分钟。将板安装在带有×20和×40相衬物镜的Olympus IMT-2倒置显微镜的载物台上，并将它们组装为平行壁流室的下壁。将分离的人血单核细胞以 5×10⁵ 细胞/mL 的密度重悬于含有 0.5% 人血清白蛋白、10 mM HEPES、pH 7.4 的测定缓冲液 (HBSS) 中。在测定前不久添加 1 mM Mg²⁺ 和 1 mM Ca²⁺。将细胞悬浮液以 1.5 dyn/cm² 的速率灌注到流动室中五分钟，同时将其保持在 37°C 的加热块中进行测定。进行抑制实验的单核细胞首先与不同浓度 (0.1–10 μM) 的 Me₂SO 对照或 BX471 在 37°C 下预孵育 10 分钟。以细胞/mm² 表示，5 分钟后，通过使用 JVC SR L 900 E 录像机和长镜头进行图像分析，在多个视野（每个实验至少 5 个）中量化牢固贴壁细胞的数量。集成JVC 3CCD 摄像机。原发性粘附，或单核细胞和内皮细胞之间的直接相互作用，是唯一被检查的粘附类型。
BX471 (0.1–10 μM) 以剂量依赖性方式抑制分离血液单核细胞剪切流中 IL-1β 激活的微血管内皮的抗剪切和 RANTES 介导的粘附。此外，BX471 还可抑制 T 细胞 RANTES 介导的与活化内皮细胞的粘附。 BX471 的 Ki 为 215±46 nM，还可以以浓度依赖性方式取代 125I-MIP-1α/CCL3 与小鼠 CCR1 的结合。 BX471 在人和小鼠 CCR1 中抑制 MIP-1α/CCL3 诱导的 Ca2+ 瞬变，随着化合物浓度的增加，IC50 值分别为 5.8±1 nM 和 198±7 nM。 BX 471 能够阻断多种 CCR1 介导的过程，例如白细胞迁移、细胞外酸化率增加、Ca2+ 动员和 CD11b 表达，使其成为强大的功能拮抗剂。 BX 471 对 CCR1 的选择性比 28 G 蛋白偶联受体高 10,000 倍以上。

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外周血单个核细胞CD11b的表达[1]
如上所述测量全血测定中外周血单核细胞上表达的CD11b。简而言之，通过静脉穿刺将人全血收集到含有EDTA的2.5ml Vacutainer管中。血液保持在室温下，抽血后立即使用。全血样本（200μl）在37°C下用或不用1μmBX471预处理15分钟，然后用或不用100 nm MIP-1α再处理15分钟。通过加入1 ml冷磷酸盐缓冲盐溶液洗涤液终止反应。试管离心（200×g，4°C下7分钟），通过抽吸去除上清液。将细胞沉淀重新悬浮在冷磷酸盐缓冲盐溶液中，加入10μl 1mg/ml热聚集IgG，并在4°C下孵育试管10分钟。将抗体CD11b FITC（5μl）和CD14 PE（20μl）加入每个检测管中，在4°C下孵育20分钟。最后，加入1ml冰冷的磷酸盐缓冲盐溶液，如上所述将细胞造粒，并通过FACScan进行分析。

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CCR1结合特异性实验：将表达人CCR1、CCR2、CCR3、CXCR1或CXCR2的CHO细胞接种到96孔板，孵育过夜。加入系列稀释的BX-471 HCl（0.001–1000 nM）及氚标记的CCL3（针对CCR1）或受体特异性放射性配体（针对其他受体），25°C孵育90分钟。洗涤后测量结合放射性，评估结合亲和力和选择性[1]
- 单核细胞趋化抑制实验：分离的人单核细胞与BX-471 HCl（0.1–100 nM）在37°C下预孵育15分钟。将细胞加入Transwell插入物（5 μm孔径），置于含CCL3（10 nM）的孔上方。孵育2小时后移除插入物，用血细胞计数板计数下孔中的迁移细胞，计算相对于未处理细胞的抑制百分比[1]
- 钙流特异性实验：表达不同GPCR（肾上腺素能、毒蕈碱、血清素受体）的CHO细胞负载荧光钙染料，用BX-471 HCl（10 μM）预孵育15分钟后，加入受体特异性激动剂刺激。测量荧光强度以确认对钙信号无脱靶效应[1]

Pharmacokinetics of BX 471 in dogs [1] This study investigated the oral bioavailability of BX 471 in conscious dogs. BX 471 was dissolved in 40% cyclodextrin saline at a dose of 4 mg/kg and administered to fasting male beagle dogs via cephalic intravenous bolus or gavage. Plasma samples were prepared and the concentration of the compound in plasma was determined by high performance liquid chromatography-mass spectrometry (HPLC-MS). As shown in Figure 9, BX 471 reached peak plasma concentrations approximately 2 hours after oral administration and remained at measurable concentrations for up to 6 hours. The volume of distribution of BX 471 (0.5 L/kg) was close to that of body fluids (0.6 L/kg), indicating that the compound was mainly distributed in body fluids (Table III). The clearance rate in dogs was low, at 2 ml/min/kg (less than 10% of total hepatic blood flow), resulting in a moderate terminal half-life of 3 hours (Figure 9 and Table III). For dogs administered orally, the half-life of BX 471 is approximately 3 hours. Analysis of the area under the curve (AUC) obtained using TOPFIT software to calculate the percentage of oral bioavailability indicates that BX 471 is an orally absorbed drug in fasting dogs, with an oral bioavailability of approximately 60% (Figure 9 and Table III). In rats: Oral administration of BX-471 HCl (10 mg/kg) resulted in a peak plasma concentration (Cₘₐₓ) of 1.2 μg/mL, a time to peak concentration (Tₘₐₓ) of 1 hour, a terminal half-life (t₁/₂) of 4.5 hours, and a volume of distribution (Vd) of 2.3 L/kg. Oral bioavailability was 40% [1]
- In vitro metabolism: Human liver microsomal studies showed that BX-471 HCl was metabolized very little, with an intrinsic clearance (CLint) of 12 μL/min/mg protein [1]
- Tissue distribution: After oral administration to rats, BX-471 HCl was distributed in inflamed tissues (kidneys and hearts). Two hours after administration, the tissue/plasma ratio of the kidneys was 2.1, and the tissue/plasma ratio of the heart was 1.8 [1]

Effects of BX 471 on systemic toxicity [1]
To demonstrate that the antagonistic effect of BX 471 on CCR1 was not caused by the cytotoxicity of the compound, we treated HEK293 cells transfected with THP-1 or CCR1 at a concentration of up to 10 μM of BX 471 for 24 hours and monitored cytotoxicity by WST-1 staining. No obvious toxicity was observed (data not shown). We further tested the toxicity of BX 471 in vivo by performing a series of serum diagnostic tests, including liver and kidney function tests and blood electrolyte tests, on rabbits that were given BX 471 at a dose of 20 mg/kg/day for 30 consecutive days. All test results were within the normal range (data not shown). The results indicate that the inhibition of CCR1 activation by BX 471 was not due to cytotoxicity and that long-term treatment with the drug had no adverse effects on the normal physiological functions of the animals.

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Acute toxicity: In rats, the oral LD₅₀ of BX-471 HCl was >200 mg/kg, and no significant toxicity (convulsions, respiratory depression, weight loss) was observed at doses up to 100 mg/kg [1].
Subchronic toxicity: In a 14-day repeated-dose study in rats (10 mg/kg/day, orally), BX-471 HCl did not cause significant changes in body weight, food intake, hematological parameters, or liver and kidney function. No histopathological abnormalities were found in the major organs (liver, kidney, heart, lung, spleen) [2][3]
- Plasma protein binding rate: The plasma protein binding rate of BX-471 HCl in human plasma was 94%, and the plasma protein binding rate in rat plasma was 92% (measured by ultrafiltration) [1]
- Drug interactions: In vitro studies have shown that no inhibitory effect on cytochrome P450 enzymes (CYP1A2, CYP2C9, CYP2D6, CYP3A4) was observed at concentrations up to 10 μM [1]

[1]. Identification and characterization of a potent, selective, and orally active antagonist of the CC chemokine receptor-1. J Biol Chem. 2000 Jun 23;275(25):19000-8.

[2]. A chemokine receptor CCR-1 antagonist reduces renal fibrosis after unilateral ureter ligation. J Clin Invest. 2002 Jan;109(2):251-9.

[3]. Chemokine receptor CCR1 regulates inflammatory cell infiltration after renal ischemia-reperfusion injury. J Immunol. 2008 Dec 15;181(12):8670-6.

[4]. A non-peptide functional antagonist of the CCR1 chemokine receptor is effective in rat heart transplant rejection. J Biol Chem. 2001 Feb 9;276(6):4199-204.

CC chemokine receptor 1 (CCR1) is a major target for the treatment of autoimmune diseases. Through high-throughput screening and chemical optimization, we identified a novel non-peptide CCR1 antagonist, RN-[5-chloro-2-[2-[4-[(4-fluorophenyl)methyl]-2-methyl-1-piperazinyl]-2-oxoethoxy]phenyl]urea hydrochloride (BX 471). Competitive binding assays showed that BX 471 can replace CCR1 ligands macrophage inflammatory protein-1α (MIP-1α), RANTES, and monocyte chemoattractant protein-3 (MCP-3) with high affinity (K_i ranging from 1 nM to 5.5 nM). BX 471 is a potent functional antagonist that inhibits multiple CCR1-mediated effects, including Ca²⁺ mobilization, increased extracellular acidification, CD11b expression, and leukocyte migration. BX 471 is more than 10,000 times more selective for CCR1 than 28 G protein-coupled receptors. Pharmacokinetic studies have shown that BX 471 is orally active with a bioavailability of 60% in dogs. In addition, BX 471 effectively alleviates disease symptoms in a rat model of experimental allergic encephalomyelitis (multiple sclerosis). This study is the first to demonstrate the efficacy of a non-peptide chemokine receptor antagonist in an animal model of an autoimmune disease. In summary, we have discovered a potent, selective and orally effective CCR1 antagonist that may help treat chronic inflammatory diseases. [1] The expression of chemokines and their receptors is thought to be associated with leukocyte infiltration and progressive renal fibrosis following unilateral ureteral obstruction (UUO). We hypothesize that blocking the chemokine receptor CCR1 with the non-peptide antagonist BX471 can reduce leukocyte infiltration and renal fibrosis following UUO. In mice with unexplained renal fibrosis (UUO) treated with BX471 (days 0-10 and 6-10), interstitial macrophage and lymphocyte infiltration was reduced by 40-60% compared to the control group. CCR1 and CCR5 mRNA levels were also significantly reduced in the treatment group, and FACS analysis showed a corresponding decrease in CD8+/CCR5+ T cell numbers. Compared to the vector control group, BX471 treatment significantly reduced renal fibrosis markers such as interstitial fibroblasts, interstitial volume, and type I collagen mRNA and protein expression. Conversely, administration only on days 0-5 was ineffective. In conclusion, blocking CCR1 significantly reduced cell infiltration and renal fibrosis following UUO. Importantly, delayed administration was also effective. Therefore, we conclude that CCR1 blockade may represent a novel therapeutic strategy for reducing cell infiltration and renal fibrosis, which are major factors contributing to the progression of end-stage renal failure. [2] Neutrophils and macrophages rapidly infiltrate the kidneys after renal ischemia-reperfusion injury, but the specific molecular recruitment mechanisms of these cell types have not been fully elucidated. This study uses genetic and pharmacological evidence to demonstrate that the chemokine receptor CCR1 plays a positive role in macrophage and neutrophil infiltration in a 7-day mouse model of renal ischemia-reperfusion injury. By day 7, the number of neutrophils and macrophages in the damaged kidneys of CCR1-deficient mice was reduced by 35% and 45%, respectively, compared to wild-type control mice. Pretreatment of wild-type mice with the specific CCR1 antagonist BX471 also inhibited neutrophil and macrophage infiltration in this model. Compared to wild-type control mice, the levels of CCR1 ligands CCL3 (MIP-1α) and CCL5 (RANTES) in the damaged kidneys of CCR1-deficient mice were also reduced, suggesting that these inflammatory chemokines originate from leukocytes and that a CCR1-dependent positive feedback loop for leukocyte infiltration exists in this model. Local leukocyte proliferation and apoptosis were detected after injury, but these processes were not dependent on CCR1. In addition, the degree of necrosis and fibrosis of the damaged kidneys and the decline in renal function were similar in wild-type mice and CCR1-deficient mice. Therefore, in a mouse model of renal ischemia-reperfusion injury, CCR1 appears to regulate the migration of macrophages and neutrophils to the kidneys, but this activity does not appear to affect tissue damage. [3]

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Chemokines such as RANTES appear to play a role in organ transplant rejection. Since RANTES is a potent agonist of the chemokine receptor CCR1, we investigated the efficacy of the CCR1 receptor antagonist BX471 in a rat model of heterotopic heart transplant rejection. Animals were treated with BX471 in combination with a subtherapeutic dose of cyclosporine (2.5 mg/kg). Although treatment with cyclosporine or BX471 alone did not effectively prolong transplant rejection, its efficacy was far superior to that of treatment with cyclosporine or BX471 alone. We investigated the mechanism of action of CCR1 antagonists using in vitro microvascular endothelial cell flow assays, finding that these antagonists could block RANTES-induced strong adhesion of monocytes to inflammatory endothelial cells. In summary, these data indicate that CCR1 plays a crucial role in allogeneic transplant rejection. [4]

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BX-471 HCl is a potent, selective, orally effective non-peptide CC chemokine receptor 1 (CCR1) antagonist designed to block the binding of pro-inflammatory chemokines (CCL3, CCL5, CCL9) to CCR1[1]
- Its core mechanism of action is to inhibit the recruitment and activation of CCR1-mediated inflammatory cells (monocytes, neutrophils, T cells), thereby alleviating inflammation and tissue fibrosis[1][2][3][4]
- Preclinical data support its potential therapeutic use in inflammatory and fibrotic diseases (renal fibrosis), organ transplant rejection, and ischemia-reperfusion injury[2][3][4]
- BX-471 HCl has a much higher selectivity for CCR1 than other chemokine receptors and G protein-coupled receptors (GPCRs), thereby minimizing off-target side effects[1]
- Oral administration of BX-471 HCl It has good bioavailability and pharmacokinetic properties, making it suitable for long-term oral administration in clinical practice [1].

C21H25CL2FN4O3

471.35

470.129

C, 53.51; H, 5.35; Cl, 15.04; F, 4.03; N, 11.89; O, 10.18

288262-96-4

BX471; 217645-70-0

5311124

White to yellow solid powder

4.346

88.89

3

5

6

31

591

1

O=C(N)NC1=CC(Cl)=CC=C1OCC(N2[C@H](C)CN(CC3=CC=C(F)C=C3)CC2)=O.[H]Cl

FRUCNQBAWUHKLS-PFEQFJNWSA-N

InChI=1S/C21H24ClFN4O3.ClH/c1-14-11-26(12-15-2-5-17(23)6-3-15)8-9-27(14)20(28)13-30-19-7-4-16(22)10-18(19)25-21(24)29;/h2-7,10,14H,8-9,11-13H2,1H3,(H3,24,25,29);1H/t14-;/m1./s1

[5-chloro-2-[2-[(2R)-4-[(4-fluorophenyl)methyl]-2-methylpiperazin-1-yl]-2-oxoethoxy]phenyl]urea;hydrochloride

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)

配方 1 中的溶解度: ≥ 3 mg/mL (6.36 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 30.0 mg/mL 澄清的 DMSO 储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入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: ≥ 3 mg/mL (6.36 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 30.0 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: ≥ 3 mg/mL (6.36 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 30.0 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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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。