Pirtobrutinib (LOXO-305) HCl   中文名称: 盐酸吡托布替尼（LOXO-305）

BTK (Bruton's tyrosine kinase), non-covalent inhibitor

Pirtobrutinib（LOXO-305）是下一代布鲁顿酪氨酸激酶（BTK）抑制剂，具有高度选择性、非共价性，并与WT BTK和几个BTK C481取代突变表现出强平衡结合。它还有效地抑制BTK C481S、T和R突变的细胞活性。[2]

吡妥布替尼在体内显著抑制人淋巴瘤异种移植物的肿瘤生长。[3]

在HotSpot激酶测定（反应生物学，Malvern，PA）中，通过监测[33P]-三磷酸腺苷（ATP）中[33P]-PO4掺入聚谷氨酸-酪氨酸（poly-EY）肽底物来测定Pirtobrutinib对BTK、BTK C481S和选定的非BTK激酶的活性。25使用标准曲线拟合方法分析数据。使用HotSpot激酶测定和Km-ATP浓度测试了Pirtobrutinib（1μM）对371种人类激酶的激酶活性抑制。使用HotSpot激酶测定法在10μM ATP的浓度下测试Pirtobrutinib、ibrutinib，zanubrutinib和acalabrutinib（100 nM）。计算每种酶的对照活性百分比。[3]

在加入原钒酸盐之前，用LOXO-305、伊布替尼或阿克拉布替尼处理瞬时表达WT BTK和BTK C481取代突变的HEK293T细胞系30分钟。在2小时的孵育期后，裂解细胞，并使用MesoScale（C481R）或免疫印迹（BTK-WT、C481S和C481T）鉴定总BTK和磷酸化的Y223-BTK。使用GraphPad Prism，对频带和MSD信号进行量化，并计算IC50值。

OCI-Ly10 cells were implanted subcutaneously into male nonobese diabetic/severe combined immunodeficiency mice and tumors were allowed to grow to a volume of between ∼150 and 200 mm3. Mice were randomized by tumor size across dose groups and dosed orally twice-daily (BID) with 10 or 50 mg/kg pirtobrutinib (12 mice per group) or vehicle (11 mice per group) for 28 days. Tumor volumes were measured thrice weekly during the study and for an additional 35 days after dosing (Axis Bioservices, Coleraine, United Kingdom). In the TMD8 study, cells were injected subcutaneously into Balb/c SCID mice and allowed to grow for 19 days until a mean tumor volume of 400 mm3 was reached. Mice were randomized by tumor size across dose groups and orally dosed BID with 15 or 30 mg/kg pirtobrutinib (10 mice per group) for 18 days or vehicle (10 mice per group) for 14 days. Tumor volumes were measured thrice weekly. REC-1 cells were injected subcutaneously into female athymic nude mice and allowed to grow for 18 days when a mean tumor volume of 150 mm3 was reached. Mice were randomized by tumor volume across dose groups and orally dosed BID with pirtobrutinib at 10, 30, or 50 mg/kg (6 mice per group) or vehicle (10 mice per group) for 21 days. Tumor volumes were measured twice weekly. TMD8 cells expressing BTK C481S were injected into female Balb/c SCID mice and allowed to grow for 12 days when a mean tumor volume of 150 mm3 was reached. Mice were randomized by tumor size and orally dosed BID with pirtobrutinib at 3, 10, and 30 mg/kg (10 mice per group) or vehicle (14 mice per group) for 14 days. Tumor volumes were measured 2 or 3 times per week. Animal procedures for the OCI-Ly10 xenograft tumor studies were performed under the guidance of the United Kingdom Animal (Scientific Procedures) Act 1986. Mice used in the TMD8 and TMD8 BTK C481S studies were treated in accordance with guidelines by the Association for Assessment and Accreditation of Laboratory Animal Care International, and protocols were authorized by the French Ministry of Education, Advanced Studies and Research. All procedures used in the REC-1 xenograft study were compliant with the United States Department of Agriculture’s Animal Welfare Act (9 CFR Parts 1, 2, and 3) and the Guide for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Research, The National Academies Press, Washington, DC).[3]

[1]. Blood (2019) 134 (Supplement_1): 478.
[2]. Blood (2019) 134 (Supplement_1): 4644.
[3]. Blood . 2023 Jul 6;142(1):62-72.
[4]. J Clin Oncol. 2015; 35:1437-1443.
[5]. Haematologica. 2018; 103(5):874-879.

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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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。