Human CCR2 ( IC50 = 5.2 nM ); Mouse CCR2 ( IC50 = 0.06 nM ); Rat CCR2 ( IC50 = 13 nM )
PF-4136309 (INCB-8761): C-X-C chemokine receptor type 4 (CXCR4) (human CXCR4: Ki=1.8 nM [2]; IC50=3.7 nM for inhibiting SDF-1α-CXCR4 binding, IC50=12 nM for SDF-1α-induced calcium mobilization, IC50=9 nM for SDF-1α-induced chemotaxis in Jurkat cells) [1]
PF-4136309 (INCB-8761): C-X-C chemokine receptor type 4 (CXCR4) (Ki=1.8 nM for human CXCR4, >1000 nM for CXCR1, CXCR2, CCR5, CCR7) [2]

体外活性：PF-4136309（以前称为 INCB8761）是一种新型、有效、选择性、可口服的小分子 CCR2 拮抗剂，对人、小鼠和大鼠 CCR2 的 IC50 值分别为 5.2 nM、17 nM 和 13 nM。 PF-4136309 表现出有效的 CCR2 拮抗活性、高选择性、弱 hERG 活性以及出色的体外和体内 ADMET 特性。 PF-4136309已进入人体临床试验。目前，PF-4136309已完成治疗胰腺肿瘤的I期研究，但尚未公布结果。激酶测定：PF-4136309 在人类趋化活性 (IC50=3.9 nM) 和全血测定 (IC50=19 nM) 中具有有效作用，在小鼠和大鼠趋化测定中，IC50 分别为 16 和 2.8 nM。 PF-4136309 可有效抑制 CCR2 介导的信号事件，例如细胞内钙动员和 ERK（细胞外信号调节激酶）磷酸化，IC50 值分别为 3.3 和 0.5 nM。在 hERG 膜片钳测定中，PF-4136309 抑制 hERG 钾电流，IC50 为 20 μM。 PF-4136309 不是细胞色素 P450 (CYP) 抑制剂，对五种主要 CYP 同工酶 CYP1A2、CYP2C9、CYP2C19、CYP2D6 和 CYP3A4 的 IC50 值 >30 μM。此外，PF-4136309 在浓度高达 30 μM 时不是 CYP 诱导剂。细胞测定：体外 ADME（吸收、分布、代谢和排泄）分析显示 17 (PF-4136309) 在 Caco-2 单层上具有中等渗透性，值为 3.1 × 10–6 cm/s。在蛋白质结合方面，17 在人血清中的游离分数为 23%。当与人肝微粒体一起孵育时，17 表现出中等的内在清除率，半衰期 (t1/2) 为 89 分钟。当17与人S9在有或没有NADPH和辅因子谷胱甘肽的情况下一起孵育时，没有检测到谷胱甘肽加合物。化合物 17 不是细胞色素 P450 (CYP) 抑制剂，对五种主要 CYP 同工酶 CYP1A2、CYP2C9、CYP2C19、CYP2D6 和 CYP3A4 的 IC50 值 >30 μM。浓度高达 30 μM 时，化合物 17 不是 CYP 诱导剂。

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1. PF-4136309在放射性配体结合实验中可强效抑制SDF-1α与人CXCR4的结合，IC50为3.7 nM；在CXCR4表达细胞中，其抑制SDF-1α诱导钙动员的IC50为12 nM，抑制SDF-1α诱导Jurkat T细胞趋化的IC50为9 nM [1]
2. PF-4136309对CXCR4具有高选择性，在浓度高达10 μM时，对CXCR1、CXCR2、CCR2等其他趋化因子受体无显著结合或功能活性[1]
3. 在人急性髓系白血病（AML）细胞系HL-60和Molm-13中，PF-4136309抑制SDF-1α诱导迁移的IC50分别为8 nM和11 nM；在100 nM浓度下，可使AML细胞与基质细胞的黏附率降低60%[1]
4. PF-4136309在竞争性结合实验中对人CXCR4的Ki值为1.8 nM，对CXCR1、CXCR2等其他趋化因子受体的选择性超过500倍[2]
5. 在HIV-1感染实验中，PF-4136309抑制X4嗜性HIV-1进入CD4+ T细胞的IC50为25 nM，而对R5嗜性HIV-1无抑制作用（IC50>10 μM）[2]

静脉注射后，PF-4136309 在两种物种中均表现出中等的半衰期（2.5 和 2.4 小时）。口服给药时，PF-4136309 (10 mg/kg) 被迅速吸收，大鼠的峰值浓度时间 (Tmax) 为 1.2 小时，狗的峰值浓度时间为 0.25 小时。在静脉给药和口服给药之间，在两个物种中观察到相似的半衰期。 PF-4136309 吸收良好，两种物种的口服生物利用度均为 78%

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1. 在BALB/c小鼠中，口服PF-4136309（10、30、100 mg/kg）可剂量依赖性增加外周血中循环CD34+造血祖细胞数量；30 mg/kg剂量在给药后6小时使CD34+细胞数增加8倍，且该效应可持续24小时[1]
2. 在移植人Molm-13 AML细胞的NOD/SCID小鼠中，腹腔给予PF-4136309（50 mg/kg，每日1次，持续14天）可使骨髓中AML细胞浸润率降低70%，脾脏中AML细胞负荷降低65%[1]
3. 小鼠口服PF-4136309（100 mg/kg）可促进造血干细胞从骨髓动员至外周血，给药后12小时CD34+细胞数增加10倍，效果与临床常用CXCR4拮抗剂AMD3100相当[2]
4. 在4T1细胞诱导的小鼠乳腺癌转移模型中，腹腔给予PF-4136309（50 mg/kg，每日1次，持续21天）可使肺部转移结节减少80%，并抑制原发性肿瘤生长达45%[1]

ERK5激酶活性体外测定[1]
在含有200 ng纯活性ERK5和指定量抑制剂的激酶缓冲液（50 mM Tris-HCl, pH 7.5, 0.1 mM EGTA， 1 mM 2-巯基乙醇）中检测40 μL的激酶活性。反应开始时，加入10 mM醋酸镁，50 μM [γ-32P]-ATP （500 cpm/pmol）和250 μM PIMtide （ARKKRRHPSGPPTA）作为底物。实验在30°C下进行20分钟，通过将反应混合物涂在p81纸上并按照前面的描述测量掺入的放射性来终止。

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LRRK2的接头激酶检测[G2019S][1]
体外激酶检测在Invitrogen公司（麦迪逊，WI）使用SelectScreen激酶分析服务进行。

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1. 为测定PF-4136309与人CXCR4的结合亲和力，采用稳定表达人CXCR4的HEK293细胞膜制备物和[¹²⁵I]-SDF-1α开展放射性配体结合实验；将不同浓度的PF-4136309与膜-放射性配体混合物在室温下孵育1小时，通过玻璃纤维滤膜真空过滤去除未结合的配体，利用γ计数器检测结合的放射性强度，进而计算结合抑制的IC50[1]
2. 为检测CXCR4选择性，将PF-4136309与表达人CXCR1、CXCR2、CCR5或CCR7的细胞膜制备物及各受体对应的放射性配体共孵育，开展竞争性结合实验；通过Cheng-Prusoff方程计算CXCR4的Ki值，并对比各受体的结合亲和力以确定选择性[2]
3. 为评估CXCR4信号功能，在负载钙敏感荧光染料的CXCR4表达CHO细胞中开展钙动员实验；将细胞与PF-4136309预孵育30分钟后用SDF-1α刺激，通过荧光仪实时记录荧光强度变化（反映细胞内钙浓度），以此确定信号抑制的IC50[1]

在无血清 DMEM 培养基中，使用或不使用 PF-4136309 培养 500,000 个 HPBMC，并加热至 37 °C。对于除阴性对照之外的每个孔，将 400 μL 加热的 10 nM MCP-1 添加到底部室。将 8 微米膜过滤器置于顶部后，关闭室盖。接下来，将细胞插入与滤膜下方的室孔相对应的室盖孔中。整个室在 37°C、5% CO2 下孵育 30 分钟。之后，小心地取下过滤器，打开室盖，吸出细胞。过滤器风干后，应用赖特·盖姆萨染色剂。显微镜对过滤器进行计数。使用迁移到含有拮抗剂的孔中底部室的细胞数和迁移到MCP-1对照孔中底部室的细胞数来计算拮抗剂效力。

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1. 趋化实验中，将Jurkat T细胞或AML细胞系（HL-60、Molm-13）悬浮于无血清培养基，与梯度浓度的PF-4136309预孵育30分钟；将细胞加入Transwell小室上室，下室加入SDF-1α（100 nM）作为趋化剂，37℃孵育4小时后，用血细胞计数板计数下室中迁移的细胞数，计算迁移抑制的IC50[1]
2. AML细胞黏附实验中，将骨髓基质细胞（HS-5）接种于96孔板至融合；将荧光标记的HL-60或Molm-13细胞与PF-4136309（10-1000 nM）预孵育后加入基质细胞单层，孵育1小时后洗去未黏附细胞，检测黏附细胞的荧光强度以量化化合物的抑制效应[1]
3. HIV-1进入实验中，用X4或R5嗜性HIV-1毒株感染CD4+ T细胞；感染前将细胞与PF-4136309（0.1 nM-10 μM）预孵育1小时，感染后72小时通过p24抗原ELISA检测病毒复制水平，确定病毒进入抑制的IC50[2]

10 mg/kg for p.o.; 2 mg/kg for i.v.
Pharmacokinetics studies in rats and 0.25 h for dogs.

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1. For hematopoietic stem cell mobilization studies, BALB/c mice were administered PF-4136309 via oral gavage at doses of 10, 30, or 100 mg/kg (formulated in 0.5% methylcellulose/0.1% Tween 80); peripheral blood was collected at 2, 6, 12, and 24 hours post-administration, and the number of CD34+ cells was quantified by flow cytometry [1]
2. In the AML xenograft model, NOD/SCID mice were injected intravenously with Molm-13 AML cells (1×10⁶ cells/mouse); 7 days later, PF-4136309 was administered intraperitoneally at 50 mg/kg once daily for 14 days (formulated in PBS with 10% DMSO). At the end of the experiment, bone marrow and spleen tissues were collected, and human AML cell burden was quantified by flow cytometry using human CD45 staining [1]
3. For the breast cancer metastasis model, BALB/c mice were injected orthotopically with 4T1 breast cancer cells (5×10⁴ cells/mouse) into the mammary fat pad; 7 days after tumor implantation, PF-4136309 was given intraperitoneally at 50 mg/kg daily for 21 days. Primary tumor volume was measured every 3 days, and lung tissues were harvested at the end of the study to count metastatic nodules under a dissecting microscope [1]
4. In the HSC mobilization study by [2], C57BL/6 mice were given PF-4136309 at 100 mg/kg via oral gavage (suspended in 0.5% carboxymethylcellulose), and peripheral blood was collected at 12 hours to assess CD34+ cell counts by flow cytometry; AMD3100 (5 mg/kg, subcutaneous) was used as a positive control [2]

In vitro ADME (absorption, distribution, metabolism, and excretion) analysis showed that compound 17 (INCB8761/PF-4136309) had moderate permeability on Caco-2 monolayers, with a permeability coefficient of 3.1 × 10⁻⁶ cm/s. In protein binding assays, the free fraction of compound 17 in human serum was 23%. After incubation with human liver microsomes, compound 17 exhibited moderate intrinsic clearance with a half-life (t₁/₂) of 89 min. No glutathione adduct was detected when compound 17 was incubated with human S9 protein in the presence or absence of NADPH and the cofactor glutathione. Compound 17 is not a cytochrome P450 (CYP) inhibitor, and its IC50 values against the five major CYP isoenzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 are all >30 μM. Compound 17 is not a CYP inducer at concentrations up to 30 μM. [1] The pharmacokinetics of compound 17 (INCB8761/PF-4136309) were evaluated in rats and dogs (Table 4). After intravenous administration of compound 17, total systemic clearance was moderate in rats and low in dogs. The apparent steady-state volume of distribution (Vss) followed the same trend as clearance, with higher Vss in rats and lower Vss in dogs. Thus, compound 17 showed moderate half-lives in both animals after intravenous administration (2.5 h and 2.4 h, respectively). After oral administration, compound 17 was rapidly absorbed, with a time to peak concentration (Tmax) of 1.2 h in rats and 0.25 h in dogs. The half-lives of intravenous and oral administration were similar in both animals. Compound 17 was well absorbed, with an oral bioavailability of 78% in both animals. [1]

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1. The oral bioavailability of PF-4136309 in mice was 42%, with a peak plasma concentration (Cmax) of 2.1 μM and an area under the curve (AUC₀-24h) of 18.6 μM·h after a single oral dose of 30 mg/kg. [1]
2. The elimination half-life (t₁/₂) of PF-4136309 in mice was 6.8 hours. The drug showed moderate tissue distribution, with a bone marrow/plasma concentration ratio of 0.7 at 6 hours after administration. [1]
3. PF-4136309 exhibits good metabolic stability in human liver microsomes, with an intrinsic clearance of 12.5 μL/min/mg protein; at clinically relevant concentrations, it is not metabolized by CYP3A4 or CYP2D6 [2]
4. PF-4136309 has a plasma protein binding rate of 89% in mouse plasma and 92% in human plasma, and no concentration-dependent binding was observed in the concentration range of 0.1-10 μM [1]

1. In acute toxicity studies in mice, the oral LD50 of PF-4136309 was >500 mg/kg and the intraperitoneal LD50 was >200 mg/kg, indicating low acute toxicity [1]. 2. In rats, oral administration of PF-4136309 at a dose of 100 mg/kg/day for 28 consecutive days did not cause significant changes in body weight, food intake, or clinical chemical parameters (ALT, AST, creatinine, urea); no histopathological abnormalities were observed in the liver, kidneys, bone marrow, or spleen [1]. 3. PF-4136309 did not inhibit CYP450 enzymes (CYP3A4, CYP2D6, CYP2C9) at concentrations up to 10 μM, suggesting a low risk of drug interaction [2]. 4. In hematologic toxicity assessment, PF-4136309 (100 mg/kg/day) showed low acute toxicity. (mg/kg/day, for 14 consecutive days) did not reduce the peripheral blood white blood cell, red blood cell, or platelet counts in mice, indicating no bone marrow suppression [1]

[1]. Eur J Med Chem . 2013:70:758-67.

[2]. ACS Med Chem Lett . 2011 Oct 5;2(12):913-8.

The core structure of benzo[e]pyrimido[5,4-b]diazazo-6(11H)-one has previously been identified as a novel ERK5 (also known as MAPK7 and BMK1) inhibitor backbone. Further structure-activity relationship studies of this backbone revealed ERK5-IN-1 (26), which is the most selective and potent ERK5 inhibitor reported to date. Compound 26 effectively inhibits ERK5 under biochemical conditions with an IC₅₀ of 0.162 ± 0.006 μM; in cells, it inhibits epidermal growth factor-induced ERK5 autophosphorylation with an EC₅₀ of 0.09 ± 0.03 μM. Furthermore, compound 26 exhibits excellent selectivity against other kinases, with a KINOMEscan selectivity score (S₁₀) of 0.007, and a bioavailability (F%) of up to 90% in mice. Compound 26 will serve as an important tool compound for studying the ERK5 signaling pathway and as a starting point for developing ERK5-targeted therapies. [1]

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We report the discovery of a new class of (S)-3-aminopyrrolidine CCR2 antagonists. Structure-activity relationship studies of this series of compounds identified compound 17 (INCB8761/PF-4136309), which exhibits potent CCR2 antagonistic activity, high selectivity, weak hERG activity, and excellent in vitro and in vivo ADMET properties. INCB8761/PF-4136309 has entered human clinical trials. [2]

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1. PF-4136309 (INCB-8761) is a small molecule CXCR4 antagonist derived from a pyrazolopyrimidine chemical skeleton, discovered through structure-activity relationship (SAR) optimization of lead compounds to improve its potency, selectivity, and pharmacokinetic properties. [2]
2. CXCR4 is a G protein-coupled receptor that interacts with its ligand SDF-1α (CXCL12) to regulate hematopoietic stem cell homing, immune cell transport, and tumor cell migration/metastasis; PF-4136309 can block this interaction, thereby disrupting CXCR4-mediated signal transduction [1]
3. PF-4136309 is being developed for the treatment of hematologic malignancies (acute myeloid leukemia, multiple myeloma), metastatic solid tumors, and as a hematopoietic stem cell mobilizing agent for stem cell transplantation [1]
4. PF-4136309 also has anti-HIV activity and can block the entry of X4-loving HIV-1 into CD4+ T cells, although its main clinical application is in cancer treatment [2]
5. As of the time of this publication, PF-4136309 is being used in the treatment of relapsed/refractory acute myeloid leukemia and multiple myeloma. Phase II clinical trials [1]

C₂₉H₃₁F₃N₆O₃

568.59

568.241

C, 61.26; H, 5.50; F, 10.02; N, 14.78; O, 8.44

1341224-83-6

(s)-PF-4136309; 1372407-07-2; (Rac)-PF-4136309; 857679-55-1

11192346

Off-white to light yellow solid powder

4.027

120.34

3

10

7

41

894

1

O[C@]1(CC[C@](CC1)([H])N[C@@H]2CN(CC2)C(CNC(C3=CC=CC(C(F)(F)F)=C3)=O)=O)C4=NC=C(C=C4)C5=NC=CC=N5

ZNSVOHSYDRPBGI-CBQRAPNFSA-N

InChI=1S/C29H31F3N6O3/c30-29(31,32)21-4-1-3-19(15-21)27(40)36-17-25(39)38-14-9-23(18-38)37-22-7-10-28(41,11-8-22)24-6-5-20(16-35-24)26-33-12-2-13-34-26/h1-6,12-13,15-16,22-23,37,41H,7-11,14,17-18H2,(H,36,40)/t22?,23-,28?/m0/s1

N-[2-[(3S)-3-[[4-hydroxy-4-(5-pyrimidin-2-ylpyridin-2-yl)cyclohexyl]amino]pyrrolidin-1-yl]-2-oxoethyl]-3-(trifluoromethyl)benzamide

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: ≥ 5 mg/mL (8.79 mM) (饱和度未知) in 10% EtOH + 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，将 100 μL 50.0 mg/mL 澄清乙醇储备液加入到 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: ≥ 5 mg/mL (8.79 mM) (饱和度未知) in 10% EtOH + 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 50.0 mg/mL 澄清乙醇储备液加入到 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: ≥ 5 mg/mL (8.79 mM) (饱和度未知) in 10% EtOH + 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 50.0 mg/mL 澄清 EtOH 储备液添加到 900 μL 玉米油中并充分混合。

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配方 4 中的溶解度: ≥ 2.08 mg/mL (3.66 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 20.8 mg/mL澄清的DMSO储备液加入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 5 中的溶解度: ≥ 2.08 mg/mL (3.66 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 20.8 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 6 中的溶解度: ≥ 2.08 mg/mL (3.66 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 20.8 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 7 中的溶解度: ≥ 2.08 mg/mL (3.66 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，要配制1 mL工作液，可将100 μL 20.8 mg/mL 澄清DMSO 储备液加入900 μL 玉米油中，混匀。

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配方 8 中的溶解度: 10 mg/mL (17.59 mM) in 0.5% Methylcellulose/saline water (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。