BTK (IC50=0.5 nM)

伊马替尼 (PCI-32765) 特异性抑制 B 细胞活性和信号传导。它可以防止 Btk (IC50=11 nM) 自身磷酸化、Btk 的生理底物 PLCγ (IC50=29 nM) 被磷酸化以及 ERK (IC50=13 nM)（更下游的激酶）被磷酸化[1]。伊马替尼 (PCI-32765) 抑制 BCR 激活的原代 B 细胞生长 (IC50=8 nM)。 Ibrutinib (PCI-32765) 在 FcγR 刺激后抑制原代单核细胞中 TNFα、IL-1β 和 IL-6 的产生（IC50 分别 = 2.6、0.5 和 3.9 nM）[3]。 Cysteine481 或 BTK 的 C481 与伊马替尼结合，最佳 IC50 为 0.5 nM。丝氨酸的羟基与伊马替尼不相容，C481S 突变将针对 BTK-C481S 磷酸化的 IC50 从 2.2 nM 提高至 1 μM[4]。

在患有胶原诱导关节炎的小鼠中，依鲁替尼 (PCI-32765)（3.125-50 mg/kg，口服）完全抑制疾病并降低循环自身抗体的量。在 MRL-Fas(lpr) 狼疮模型中，伊马替尼 (PCI-32765) 可防止自身抗体的形成和肾脏疾病的进展。在 MRL-Fas(lpr) 小鼠中，依鲁替尼 (PCI-32765)（3.125–50 mg/kg，口服）可改善肾脏疾病和自身抗体的产生[1]。与 T 细胞相比，依鲁替尼 (PCI-32765) (0.1 μM) 选择性地对 B 细胞产生细胞毒作用，但它会改变活化 T 细胞产生的细胞因子。当它被激活时，它还能抑制 CLL 细胞的增殖。在治疗性 CIA 模型中，依鲁替尼 (PCI-32765) 的 ED50 为 2.6 mg/kg/天，可有效且剂量依赖性地逆转关节炎炎症。依鲁替尼 (PCI-32765) 还可在 CAIA 模型中预防临床关节炎[3]。

将激酶, 33P-ATP, Ibrutinib, 和底物 [0.2 mg/mL 聚(EY)(4:1)]一起温育1小时后，使用33P 过滤结合实验测量体外激酶IC50值。

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b细胞受体(BCR)信号在慢性淋巴细胞白血病(CLL)中异常激活。布鲁顿酪氨酸激酶(BTK)对BCR信号传导至关重要，在敲除小鼠模型中，其突变具有相对的B细胞特异性表型。本研究表明，与正常B细胞相比，BTK蛋白和mRNA在CLL中显著过表达。尽管BTK在CLL细胞中并不总是具有组成性活性，但BCR或CD40信号传导伴随着该途径的有效激活。使用不可逆的BTK抑制剂Ibrutinib (PCI-32765)，我们发现CLL细胞的适度凋亡大于正常B细胞。未观察到Ibrutinib (PCI-32765)对t细胞存活的影响。用Ibrutinib (PCI-32765)处理CD40或BCR活化的CLL细胞可抑制BTK酪氨酸磷酸化，并有效地消除由该激酶激活的下游生存途径，包括ERK1/2、PI3K和NF-κB。此外，Ibrutinib (PCI-32765)在体外抑制活化诱导的CLL细胞增殖，并有效阻断微环境向CLL细胞提供的生存信号，包括可溶性因子(CD40L、BAFF、IL-6、IL-4和TNF-α)、纤连蛋白结合和基质细胞接触。基于这些集体数据，未来在CLL患者的临床试验中，有必要使用不可逆抑制剂Ibrutinib (PCI-32765)靶向BTK。[3]

B和T细胞。CD20+B和CD3+T细胞通过阴性选择（RosetteSep，>90%纯度）从血沉棕黄层PBMC中纯化，并可在10%DMSO中冷冻。细胞在37°C下解冻，并保持在生长培养基（含有10%FCS的RPMI培养基）中。用山羊抗人IgM F（ab′）2（10μg/mL；Invitrogen）刺激B细胞，用1∶1珠/细胞比例的抗CD3/CD28包被珠（Dynabeads）刺激T细胞。用PE-CD69（BD Biosciences）对细胞进行染色，并通过流式细胞术对活淋巴细胞进行门控分析。尽管PCI-32765确实阻断了B细胞中抗IgM刺激的适度生存益处，但在实验过程中，浓度低于10μM的PCI-327650并没有降低B细胞或T细胞的生存能力。对于冲洗实验，在10体积的生长培养基中冲洗细胞三次，该方案被证实完全冲洗了可逆Btk抑制剂PCI-29732对BCR信号传导的抑制。[1]

Arthritis and Lupus Models.[1] Male DBA/1 mice were immunized with type II collagen plus Freund adjuvant and boosted 21 d later. On a rolling basis, as significant swelling appeared in at least one paw, mice were enrolled and randomized. Ibrutinib (PCI-32765) or dexamethasone (0.2 mg/kg) were administered orally once per day for 11 d. Arthritis scores (0–5) were assigned to the mice based on the degree and extent of paw swelling. Mouse anti–type II collagen antibody and total IgG levels were measured by ELISA (Chondrex and Bethyl). Female MRL/MpJ-Faslpr mice received Ibrutinib (PCI-32765) by oral gavage once per day from week 8 through week 20. Proteinuria was monitored weekly. At week 20, serum was collected and analyzed for BUN (IDEXX) and mouse anti-dsDNA antibody levels. Kidney histology was scored according to established criteria. No drug-induced weight loss was observed at any of the dose levels tested. These studies were carried out at Boulder Biopath according to approved animal care protocols. Results are presented as the mean ± SEM. Statistical significance between groups were evaluated with repeated measures one-way ANOVA or one-way ANOVA using GraphPad Prism with Tukey or Bonferroni multicomparison posttest.

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Spontaneous Canine Lymphoma. [1] Spontaneous canine lymphoma studies were conducted with approval from the Colorado State University Institutional Animal Care and Use Committee and the Colorado State University Veterinary Medical Teaching Hospital Clinical Review Board. Client-owned dogs presenting as patients to the Colorado State University Animal Cancer Center were enrolled with the following inclusion criteria: (i) confirmed histologic or cytologic diagnosis of B-cell lymphoma (immunohistochemistry or flow cytometry for CD21 and CD79a or PCR for monoclonal Ig gene rearrangement), (ii) adequate organ function as indicated by standard laboratory tests, and (iii) modified Eastern Cooperative Oncology Group performance status of 0 or 1 on d 0. Exclusion criteria were (i) T cell or null-cell immunophenotype, (ii) chemotherapy within 3 wk, (iii) radiation therapy within 6 wk, and (iv) corticosteroids within 72 h. Signed informed consent was obtained from all owners before study entry. Ibrutinib (PCI-32765) was administered daily until disease progression with 40 mg and 200 mg hard gelatin capsules prepared using standard pharmaceutically acceptable excipients. Animals were rechecked weekly for 4 wk and then biweekly thereafter. Tumor burden was defined as the sum of the longest diameters of all target lesions. Response (complete response/partial response/stable disease/progressive disease) was evaluated according to Veterinary Cooperative Oncology Group criteria for assessment of response in peripheral nodal lymphoma in dogs, an adaptation of published RECIST criteria. Adverse events were recorded and prospectively graded according to the Veterinary Cooperative Oncology Group Common Terminology for Adverse Events, version 1.0. For pharmacodynamic analysis, blood was collected in CPT tubes and PBMCs purified using standard techniques. PBMC pellets were snap-frozen and stored at −80 °C. Tumor biopsies were stored at −80 °C and subsequently pulverized in PBS solution before analysis. PBMCs or tumor cells were lysed and 50 μg of soluble protein was labeled with PCI-33880 as described earlier.

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Male DBA1/1OlaHsd mice are injected on days 0 and 21 with Freunds' Complete Adjuvant containing bovine type II collagen. On days 21 to 35, mice are randomized into treatment groups when the average clinical score of each animal is 1.5 (in a scale of 5). Ibrutinib (PCI-32765) treatment (1.56-12.5 mg/kg, p.o.) is initiated following enrollment and continues for 18 days. Clinical scores are given to each mouse daily for each paw. Clinical score assessment is made using the following criteria: 0=normal; 1=one hind paw or fore paw joint affected or minimal diffuse erythema and swelling; 2=two hind or fore paw joints affected or mild diffuse erythema and swelling; 3=three hind or fore paw joints affected or moderate diffuse erythema and swelling; 4=marked diffuse erythema and swelling or four digit joints affected; 5=severe diffuse erythema and severe swelling of entire paw, unable to flex digits.[3]

Absorption
Ibrutinib is rapidly absorbed after oral administration and it presents a Cmax, tmax and AUC of approximately 35 ng/ml, 1-2 hour and 953 mg.h/ml respectively.

Route of Elimination
The cumulative excretion of ibrutinib in urine is of about 7.8% of the administered dose and most of this excretion is found during the first 24 hours after administration. In feces, the cumulative excretion accounts for 80% of the administered dose and the excretion occurs within 48 hours of the initial administration. The total excretion of ibrutinib during the first 168 hours after initial administration accounts for 88.5% of the administered dose.

Volume of Distribution
The volume of distribution at steady-state of ibrutinib is in approximately 10,000 L.

Clearance
In patients with normal renal function, the clearance rate is in the range of 112-159 ml/min.

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Metabolism / Metabolites
Three metabolic pathways have been identified according to the possible metabolites. These pathways are the hydroxylation of the phenyl group (M35), the opening of the piperidine with a reduction of the primary alcohol (M34) and the oxidation to a carboxylic acid and epoxidation of the ethylene followed by a hydrolysis to the formation of dihydrodiol (PCI-45227). The latter metabolite presents also 15 times lower inhibitory activity against BTK. The metabolism of ibrutinib is mainly performed by CYP3A5 and CYP3A4. and in a minor extent it is seen to be performed by CYP2D6.

Since 2014, Ibrutinib has been available as a new drug for the treatment of leukemic diseases. Ibrutinib (Imbruvica) is metabolized in the liver mainly by the isoenzyme CYP3A4 and to a minor extent by CYP2D6. Simultaneous application of Imbruvica and consumption of foods containing secondary metabolites strongly inhibiting the CYP3A4 isoform, could significantly influence the toxicity of this drug. This article references the respective foods.

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Biological Half-Life
The elimination half-life of ibrutinib is of approximately 4-6 hours.

Toxicity Summary
IDENTIFICATION AND USE: Ibrutinib is an oral tyrosine kinase inhibitor that irreversibly binds and inhibits tyrosine-protein kinase BTK (Bruton tyrosine kinase). BTK is important in the function of B-cell receptor signaling and therefore in the maintenance and expansion of various B-cell malignancies. Targeting BTK with ibrutinib is an effective strategy in treating these malignancies. Ibrutinib, marketed as Imbruvica, is indicated for the treatment of patients with mantle cell lymphoma (MCL) or chronic lymphocytic leukemia (CLL) who have received at least one prior therapy. It is also indicated for the treatment of patients with chronic lymphocytic leukemia (CLL) with 17p deletion and patients with Waldenstrom's macroglobulinemia (WM). HUMAN EXPOSURE AND TOXICITY: Studies in humans have shown that ibrutinib may enhance chemoimmunotherapy efficacy without additive toxicities. Ibrutinib is cytotoxic to malignant plasma cells from patients with multiple myeloma (MM) and furthermore treatment with ibrutinib significantly augments the cytotoxic activity of bortezomib and lenalidomide chemotherapies. Hypersensitivity reactions including anaphylactic shock (fatal), urticaria, and angioedema have been reported. The outcome of patients with MCL who experience disease progression following ibrutinib therapy is poor, with both low response rates to salvage therapy and short duration of responses. ibrutinib inhibited the proliferation and induced apoptosis of Germinal center B-cell like diffuse large B-cell lymphoma (GCB-DLBCL) cell lines through suppression of BCR signaling pathway and activation of caspase-3. Furthermore, the chemokines CCL3 and CCL4 production from tumor cells were also found to be attenuated by ibrutinib treatment. Different cell lines exhibited distinct sensitivity after ibrutinib treatment. Interestingly, the decreasing level of p-ERK after ibrutinib treatment, but not the basal expression level of Btk, correlated with different drug sensitivity. Ibrutinib could be a potentially useful therapy for GCB-DLBCL and the decreasing level of p-ERK could become a useful biomarker to predict related therapeutic response. Ibrutinib is well tolerated when added to R-CHOP (chemotherapy named after the initials of the drugs used: rituximab, cyclophosphamide, doxorubicin (hydroxydaunomycin), vincristine (Oncovin ), prednisolone), and could improve responses in patients with B-cell non-Hodgkin lymphoma. ANIMAL STUDIES: Ibrutinib caused malformations in rats at exposures 14 times those reported in patients with MCL and 20 times those reported in patients with CLL or WM, receiving the ibrutinib dose of 560 mg per day and 420 mg per day, respectively. Reduced fetal weights were observed at lower exposures.

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Hepatotoxicity
In the prelicensure clinical trials of ibrutinib in patients with CLL and mantle cell lymphoma, the rates of serum enzyme elevations during therapy were 20% to 30% but were similar to comparator arms, and elevations were generally mild (less than 5 times ULN) and self limited. In multiple controlled trials there were no reports of clinically apparent liver injury or need for early discontinuation because of hepatotoxicity. The major toxicities of ibrutinib resembled those of the tyrosine kinase receptor inhibitors and included hemorrhage and myelosuppression. While ibrutinib depressed peripheral lymphocyte counts and caused both lymphopenia and neutropenia, it has little effect on serum immunoglobulin levels and was not associated with reactivation of tuberculosis or opportunistic infections in prelicensure studies. Nevertheless, with approval and more widespread use of ibrutinib, rare cases of acute liver injury including acute liver failure and severe instances of reactivation of hepatitis B have been reported. The latency to onset of liver injury varied from several weeks to 9 months. The pattern of injury was hepatocellular, but the course was atypical of an acute hepatitis-like injury and more similar to acute hepatic necrosis with early onset of hepatic failure.
Likelihood score: D (possible rare cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of ibrutinib during breastfeeding. Because ibrutinib is more than 97% bound to plasma proteins, the amount in milk is likely to be low. The manufacturer recommends that breastfeeding be discontinued during ibrutinib therapy and for 1 week after the last dose.

◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.

◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Irreversible plasma protein binding increases gradually over time and reaches 25% of the administered dose 8 hours after initial administration. From the plasma proteins, ibrutinib has been shown to be mainly bound to albumin and to bind to α1 AGP. The irreversible protein binding of ibrutinib to plasma proteins can account for 97.3% of the administered dose.

[1]. Honigberg LA, et al. The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc Natl Acad Sci U S A. 2010 Jul 20;107(29):13075-80.
[2]. Herman SE, et al. Bruton tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765. Blood. 2011 Jun 9;117(23):6287-96.
[3]. Chang BY, et al. The Bruton tyrosine kinase inhibitor PCI-32765 ameliorates autoimmune arthritis by inhibition of multiple effector cells. Arthritis Res Ther. 2011 Jul 13;13(4):R115.
[4]. Sun Y, et al. PROTAC-induced BTK degradation as a novel therapy for mutated BTK C481S induced ibrutinib-resistant B-cell malignancies. Cell Res. 2018 Jul;28(7):779-781

1-[3-[4-amino-3-(4-phenoxyphenyl)-1-pyrazolo[3,4-d]pyrimidinyl]-1-piperidinyl]-2-propen-1-one is an aromatic ether.
See also: Ibrutinib (annotation moved to).

C₂₅H₂₄N₆O₂

440.50

440.196

936563-87-0

Ibrutinib;936563-96-1;Ibrutinib-d5;1553977-17-5

16126651

White to off-white solid powder

4.736

99.16

1

6

5

33

678

0

XYFPWWZEPKGCCK-UHFFFAOYSA-N

InChI=1S/C25H24N6O2/c1-2-21(32)30-14-6-7-18(15-30)31-25-22(24(26)27-16-28-25)23(29-31)17-10-12-20(13-11-17)33-19-8-4-3-5-9-19/h2-5,8-13,16,18H,1,6-7,14-15H2,(H2,26,27,28)

1-[3-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one

PCI-32765 Racemate; PCI32765 Racemate; 936563-87-0; PCI-32765 Racemate; Ibrutinib Racemate; PCI-32765 (Racemate); Ibrutinib (Racemate); 1-[3-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one; PCI-32765 (Ibrutinib); 1-{3-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl}prop-2-en-1-one; PCI 32765 Racemate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

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

DMSO : ~25 mg/mL (~56.75 mM)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.68 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 25.0 mg/mL澄清DMSO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: ≥ 2.5 mg/mL (5.68 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 25.0 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: ≥ 2.5 mg/mL (5.68 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 25.0 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。