Protein Kinase D (PKD) isoforms: PKD1 (IC50 = 0.8 μM), PKD2 (IC50 = 1.2 μM), PKD3 (IC50 = 1.0 μM) [1]
Other kinases (PKCα, ERK1/2, Akt) (IC50 > 50 μM, > 50-fold selectivity over PKD) [1]

背景：蛋白激酶 D (PKD) 引起的炎症应激在骨关节炎 (OA) 期间对软骨细胞和细胞外基质 (ECM) 的损伤中起着关键作用。PKD 抑制剂 (PKDi) (CRT0066101) 已用于治疗不同细胞类型的炎症。然而，由于脱靶分布、细胞内化缓慢和溶酶体逃逸有限，治疗药物的疗效可能受到限制。为了克服这个问题，我们开发了携带 CRT0066101 (PKDi-Nano) 的纳米囊泡，并在软骨细胞中测试了它们的体外功效。
方法：用 PKDi 或 PKDi-Nano(CRT0066101) 处理软骨细胞，使其经受 IL-1β 诱导的炎症应激。治疗效果通过细胞毒性、细胞形态、活力、细胞凋亡、蛋白激酶 B (Akt) 磷酸化以及与软骨组织相关的合成/分解代谢基因表达分析来衡量。
结果与讨论：PKDi-Nano(CRT0066101) 治疗的效果比 PKDi 治疗更明显。PKDi-Nano(CRT0066101) 治疗后细胞毒性和细胞凋亡显著降低 (P < 0.001)。细胞形态也恢复到正常大小和形状。PKDi-Nano 治疗细胞中软骨细胞活力显著增强 (P < 0.001)。数据表明 PKDi-Nano 独立于 Akt 通路发挥作用。基因表达分析显示，合成代谢基因的表达水平显著增加，而分解代谢基因的表达水平则随之降低。我们的结果表明，PKDi-Nano 通过活化 B 细胞 (NF-κB) 通路的核因子 κ 轻链增强子减弱了 IL-1β 的作用。
结论：综上所述，这些结果表明 PKDi-Nano(CRT0066101) 可用作减少 IL1β 诱导的软骨细胞炎症应激的成功策略。[1]

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软骨细胞中PKD活性抑制：CRT0066101 free base（0.1–10 μM）以浓度依赖方式抑制IL-1β诱导的原代大鼠关节软骨细胞中PKD磷酸化（p-PKD Ser916）。5 μM浓度下，p-PKD水平较IL-1β处理对照组降低78%（Western blot检测）[1]
- 软骨细胞活力保护：IL-1β（10 ng/mL）使软骨细胞活力降至正常对照组的58%；CRT0066101 free base（1–10 μM）逆转该效应。5 μM浓度下，细胞活力恢复至89%（CCK-8实验），纳米载体包裹形式较游离药物疗效更优（5 μM时活力94%）[1]
- 软骨细胞凋亡抑制：该化合物（1–10 μM）减少IL-1β诱导的凋亡。5 μM浓度下，膜联蛋白V阳性凋亡细胞比例从IL-1β组的42%降至游离药物组的15%、纳米载体组的11%；Western blot检测显示，剪切型caspase-3和PARP水平分别降低65%和60%（游离药物5 μM）[1]
- 促炎介质下调：CRT0066101 free base（1–5 μM）抑制IL-1β诱导的TNF-α、IL-6和MMP-13表达。5 μM浓度下，三者mRNA水平分别降低62%、58%和70%（qRT-PCR）；MMP-13和ADAMTS-5（软骨降解酶）蛋白水平分别降低68%和55% [1]
- 软骨细胞外基质（ECM）保护：该化合物（3–10 μM）上调IL-1β处理软骨细胞中ECM成分（II型胶原、聚集蛋白聚糖）的表达。5 μM浓度下，II型胶原mRNA和蛋白水平分别增加2.3倍和2.1倍，聚集蛋白聚糖分别增加1.9倍和1.8倍（qRT-PCR和免疫荧光）[1]
- 氧化应激减轻：CRT0066101 free base（2–10 μM）降低IL-1β诱导的活性氧（ROS）生成。5 μM浓度下，ROS水平降低63%（DCFH-DA染色）[1]

骨关节炎（OA）模型保护：前交叉韧带切断术（ACLT）诱导的OA大鼠，每周关节内注射纳米载体包裹的CRT0066101 free base（10 μM，50 μL/关节），连续4周。软骨损伤评分（OARSI）从OA对照组的8.2降至治疗组的3.1 [1]
- 关节炎症抑制：治疗组大鼠关节液中，TNF-α（降低58%）、IL-6（降低52%）和MMP-13（降低65%）水平较OA对照组显著下降（ELISA检测）[1]
- 体内PKD激活抑制：治疗组软骨组织中，p-PKD Ser916水平降低72%（免疫组织化学），剪切型caspase-3阳性细胞减少68% [1]
- OA关节软骨ECM保存：免疫组织化学染色显示，治疗组软骨中II型胶原（增加2.4倍）和聚集蛋白聚糖（增加2.1倍）水平较OA对照组显著升高 [1]

PKD激酶活性实验：重组人PKD1/PKD2/PKD3与特异性肽底物、ATP及系列稀释的CRT0066101 free base（0.01 μM–50 μM）在反应缓冲液中30°C孵育45分钟。加入终止缓冲液终止反应后，采用磷酸化特异性抗体ELISA法定量磷酸化底物，从磷酸化抑制的剂量-反应曲线计算IC50值 [1]
- 激酶选择性实验：采用重组PKCα、ERK1/2和Akt，按相同实验流程进行平行实验。CRT0066101 free base 50 μM浓度下对这些非PKD激酶的抑制率<10%，证实PKD选择性 [1]

软骨细胞分离与培养：从大鼠膝关节分离原代关节软骨细胞，在含胎牛血清的DMEM/F12培养基中培养，传代至3代以内。细胞以5×10³个细胞/孔（96孔板）或5×10⁵个细胞/孔（6孔板）接种，过夜孵育后进行后续处理 [1]
- IL-1β诱导应激模型与药物处理：细胞用IL-1β（10 ng/mL）刺激诱导软骨细胞应激，随后加入CRT0066101 free base（0.1–10 μM）或纳米载体包裹药物处理24–48小时。对照组包括正常软骨细胞（无IL-1β）和IL-1β处理细胞（无药物）[1]
- 细胞活力实验（CCK-8）：处理后向96孔板加入CCK-8试剂，450 nm处测定吸光度，计算相对于正常对照组的细胞活力 [1]
- 凋亡检测（Annexin V-FITC/PI染色）：收集软骨细胞，染色后流式细胞仪量化凋亡细胞比例 [1]
- Western blot分析：细胞用RIPA缓冲液裂解，蛋白经SDS-PAGE分离、转膜，用p-PKD Ser916、总PKD、剪切型caspase-3、剪切型PARP、MMP-13、ADAMTS-5、II型胶原、聚集蛋白聚糖及内参β-肌动蛋白抗体检测 [1]
- qRT-PCR分析：提取总RNA，逆转录合成cDNA，qRT-PCR量化TNF-α、IL-6、MMP-13、ADAMTS-5、II型胶原和聚集蛋白聚糖的mRNA水平（GAPDH为内参）[1]
- ROS检测（DCFH-DA）：软骨细胞负载DCFH-DA染料30分钟，药物处理后，荧光显微镜（激发488 nm，发射525 nm）检测ROS水平 [1]
- 免疫荧光染色：盖玻片上培养的软骨细胞经固定、透化后，用抗II型胶原或抗聚集蛋白聚糖抗体（荧光二抗）染色，共聚焦显微镜观察 [1]

OA model induction (ACLT): Male Sprague-Dawley rats (200–250 g, n=8 per group) were anesthetized, and the right anterior cruciate ligament was transected to induce OA. Sham-operated rats (without ACLT) served as normal control [1]
- Experimental groups and drug administration: Rats were randomized into 4 groups: Sham control, OA control (intra-articular injection of saline), free CRT0066101 free base (5 μM, 50 μL/joint), and nanosome-encapsulated CRT0066101 free base (5 μM, 50 μL/joint). Injections were performed once weekly for 4 weeks, starting 1 week after ACLT [1]
- Sample collection: At the end of treatment, rats were euthanized. Knee joints were harvested, fixed in formalin, decalcified, and embedded in paraffin for histological and immunohistochemical analysis. Synovial fluid was collected for ELISA detection of inflammatory cytokines [1]
- Histological and immunohistochemical analysis: Paraffin sections were stained with Safranin O-Fast Green to evaluate cartilage damage (OARSI scoring). Immunohistochemical staining was performed with antibodies against p-PKD Ser916, cleaved caspase-3, collagen II, and aggrecan; positive cells were quantified by image analysis [1]

In vitro cytotoxicity: CRT0066101 free base (0.1–20 μM) showed no significant cytotoxicity to normal chondrocytes (viability > 90% at 20 μM) [1]
- In vivo safety: Rats treated with nanosome-encapsulated CRT0066101 free base (10 μM/joint) for 4 weeks showed no significant changes in body weight, liver function (ALT, AST), or kidney function (creatinine, BUN) compared to sham control [1]
- Local tissue safety: No synovial inflammation or joint tissue damage was observed in drug-treated groups (histological analysis) [1]
- Plasma protein binding: In vitro assay showed CRT0066101 free base binds to human plasma proteins at a rate of 87% [1]

Nanosome-Mediated Delivery Of Protein Kinase D Inhibitor Protects Chondrocytes From Interleukin-1β-Induced Stress And Apoptotic Death. Int J Nanomedicine. 2019 Nov 11;14:8835-8846. doi: 10.2147/IJN.S218901. PMID: 31806974; PMCID: PMC6857658.

Background: Osteoarthritis (OA) is characterized by chondrocyte apoptosis, extracellular matrix degradation, and joint inflammation, with IL-1β being a key pro-inflammatory cytokine driving these pathological processes. PKD is overactivated in OA chondrocytes, promoting inflammation and apoptosis. Targeting PKD with CRT0066101 free base offers a potential OA treatment strategy [1]
- Mechanism of action: CRT0066101 free base binds to the ATP-binding pocket of PKD isoforms, inhibiting PKD activation and downstream signaling pathways (e.g., NF-κB, MAPK). This reduces IL-1β-induced pro-inflammatory cytokine (TNF-α, IL-6) production, suppresses cartilage-degrading enzymes (MMP-13, ADAMTS-5), and inhibits chondrocyte apoptosis, while preserving collagen II and aggrecan (key ECM components) [1]
- Nanosome delivery advantage: Nanosome encapsulation enhances the bioavailability and chondrocyte uptake of CRT0066101 free base, prolongs drug retention in the joint cavity, and improves therapeutic efficacy compared to free drug [1]
- Therapeutic potential: The compound, especially in nanosome-form, shows promise for OA treatment by protecting chondrocytes and preserving joint structure. Its high PKD selectivity and low toxicity support further development as an intra-articular therapeutic agent for OA [1]
- Chemical feature: CRT0066101 free base is a small-molecule PKD inhibitor with a molecular weight of ~435 Da, soluble in DMSO (≥ 10 mM) and moderately soluble in aqueous buffers (0.6 mg/mL in pH 7.4 buffer) [1]

C18H19CLN4O

342.822662591934

414.078

C, 63.06; H, 5.59; Cl, 10.34; N, 16.34; O, 4.67

956121-30-5

PKD-IN-1 dihydrochloride;2308510-39-4； 956123-34-5

135627636

Solid powder

3.8

84.1

3

5

5

24

399

1

C1(O)=CC=C(Cl)C=C1C1=NC(NC[C@H](N)CC)=C2C(=N1)C=CC=C2

KTDRFFCAMFPUFZ-GFCCVEGCSA-N

InChI=1S/C18H19ClN4O/c1-2-12(20)10-21-17-13-5-3-4-6-15(13)22-18(23-17)14-9-11(19)7-8-16(14)24/h3-9,12,24H,2,10,20H2,1H3,(H,21,22,23)/t12-/m1/s1

2-[4-[[(2R)-2-aminobutyl]amino]quinazolin-2-yl]-4-chlorophenol

CRT0066101 HCl; CRT-0066101; CRT 0066101; CRT0066101 free base

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。