BTK (IC50 = 5.69 nM)

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]
LOXO-305和伊布替尼均能有效抑制IgM诱导的磷酸化BTK，LOXO-30的IC50值为1.34±1.23 nM（n=7，p<0.0001），伊布替尼为1.04±1.26 nM（n=7，p>0.0001）。我们还发现BTK的直接下游效应物PLCγ2（Y1217）的磷酸化显著降低（每种药剂的IC50为33 nM，n=7，LOXO-305的p=0.02，伊布替尼的p=0.0017）。[1]

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]

Absorption, Distribution and Excretion
With single oral doses between 300 mg and 800 mg (1.5 to 4 times the approved recommended dose) and once daily doses between 25 mg and 300 mg (0.125 to 1.5 times the recommended dose), pirtobrutinib follows a dose-proportional pharmacokinetic profile. Within 5 days of once-daily dosing, pirtobrutinib reached steady-state concentration, with an accumulation ratio of 1.63 based on AUC after 200 mg dosages. With the recommended dose, pirtobrutinib has a steady-state AUC and Cmax of 91300 h⋅ng/mL and 6460 ng/mL, respectively. On day 8 of cycle 1, pirtobrutinib had an AUC0-24 of 81800 h⋅ng/mL and a Cmax of 3670 ng/mL. After approximately 2 hours, pirtobrutinib reaches peak plasma concentration (tmax). After a single oral dose of 200 mg, pirtobrutinib reaches an absolute bioavailability of 85.5%. The administration of a high-fat, high-calorie meal to healthy subjects did not have a clinically significant effect on the pharmacokinetics of pirtobrutinib. A high-fat meal decreased the Cmax of pirtobrutinib by 23%, delayed tmax by 1 hour and had no effects on the AUC.
Pirtobrutinib is mainly excreted in urine and feces. In healthy subjects given a single dose of 200 mg of radiolabeled pirtobrutinib, 57% of the dose was recovered in urine (10% unchanged), and 37% was recovered in feces (18% unchanged).
Pirtobrutinib has an apparent central volume of distribution of 32.8 L.
Pirtobrutinib has an apparent clearance of 2.02 L/h.

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Metabolism / Metabolites
In vitro studies suggest that pirtobrutinib is mainly metabolized by CYP3A4 and direct glucuronidation by UGT1A8 and UGT1A9.

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Biological Half-Life
Pirtobrutinib has an effective half-life of approximately 19 hours.

Hepatotoxicity
In the open label prelicensure clinical trials of pirtobrutinib in patients with various B cell related malignancies serum aminotransferase elevations occurred in up to 23% of pirtobrutinib treated subjects. The elevations were typically mild and transient, but 2% to 3% of patients developed values above 5 times the upper limit of normal (ULN). The elevations occasionally led to temporary discontinuations, but more often resolved even without dose adjustment. In prelicensure studies, there were no instances of acute liver injury with symptoms or jaundice attributed to pirtobrutinib. Since approval and more widescale availability of pirtobrutinib, there have been no published reports of hepatotoxicity associated with its use. However, drug induced liver injury including acute liver failure and deaths have been reported with other Bruton tyrosine kinase inhibitors (mainly with ibrutinib) and several have been linked to instances of reactivation of hepatitis B which can be fatal.
Likelihood score: E* (unproven but suspected rare cause of clinically apparent liver injury as well as reactivation of chronic hepatitis B).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of pirtobrutinib during breastfeeding. It is 96% bound to plasma proteins, so concentrations in milk are likely low. The manufacturer recommends that breastfeeding be discontinued during pirtobrutinib 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
The human protein binding of pirtobrutinib is 96%, independent of _in vitro_ concentration. Pirtobrutinib has a blood-to-plasma ratio of 0.79.

[1]. Blood (2019) 134 (Supplement_1): 478.

[2]. Blood (2019) 134 (Supplement_1): 4644.
[3]. Blood . 2023 Jul 6;142(1):62-72. doi: 10.1182/blood.2022018674.

Pirtobrutinib is a secondary carboxamide resulting from the formal condensation of the carboxy group of 5-fluoro-2-methoxybenzoic acid with the amino group of 5-amino-3-[4-(aminomethyl)phenyl]-1-[(2S)-1,1,1-trifluoropropan-2-yl]-1H-pyrazole-4-carboxamide. It is a BTK inhibitor used for the treatment of adult patients with chronic lymphocytic leukemia or small lymphocytic lymphoma. It has a role as an EC 2.7.10.2 (non-specific protein-tyrosine kinase) inhibitor, an antineoplastic agent and an apoptosis inducer. It is a monomethoxybenzene, a member of monofluorobenzenes, a member of benzamides, a secondary carboxamide, a primary carboxamide, an organofluorine compound, a member of pyrazoles and a primary amino compound.
Pirtobrutinib is a small molecule and a highly selective non-covalent inhibitor of Bruton’s tyrosine kinase (BTK). Its high selectivity has been associated with lower discontinuation rates due to adverse events and a lower incidence of atrial fibrillation. Unlike BTK covalent inhibitors, such as [ibrutinib], that bind to the cysteine 481 (Cys481) amino acid within the active site of BTK, the inhibitory activity of pirtobrutinib is maintained even in the presence of Cys481 mutations. Although the mechanisms of resistance to covalent BTK inhibitors have not been fully elucidated, it appears that the presence of Cys481 mutations is the most common reason for resistance to covalent BTK inhibitors. However, other mutations may confer resistance to non-covalent BTK inhibitors such as pirtobrutinib. In January 2023, the use of pirtobrutinib for the treatment of relapsed or refractory mantle cell lymphoma (MCL) after at least two lines of systemic therapy was approved under the FDA's Accelerated Approval pathway.
Pirtobrutinib is a Kinase Inhibitor. The mechanism of action of pirtobrutinib is as a Bruton's Tyrosine Kinase Inhibitor, and P-Glycoprotein Inhibitor, and Cytochrome P450 2C8 Inhibitor, and Cytochrome P450 2C19 Inhibitor, and Cytochrome P450 3A Inhibitor, and Breast Cancer Resistance Protein Inhibitor.
Pirtobrutinib is an orally available small molecule inhibitor of Bruton tyrosine kinase that is used to treat chronic lymphocytic leukemia and lymphomas. Pirtobrutinib is associated with transient and usually mild elevations in serum aminotransferase levels during therapy but has yet to be linked to cases of clinically apparent acute liver injury.
Pirtobrutinib is an orally available, selective, non-covalent Bruton's tyrosine kinase (BTK) inhibitor with potential antineoplastic activity. Upon oral administration, pirtobrutinib selectively and reversibly binds to BTK. This prevents both the activation of the B-cell antigen receptor (BCR) signaling pathway and BTK-mediated activation of downstream survival pathways, thereby inhibiting the growth of malignant B-cells that overexpress BTK. Reversible binding of LOXO-305 may preserve antitumor activity in the presence of certain acquired resistance mutations, including C481 mutated BTK, and limit toxicity associated with inhibition of other non-BTK kinases. BTK, a member of the Src-related BTK/Tec family of cytoplasmic tyrosine kinases, is overexpressed or mutated in B-cell malignancies; it plays an important role in the development, activation, signaling, proliferation and survival of B-lymphocytes.

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Drug Indication
Pirtobrutinib is indicated for the treatment of adult patients with relapsed or refractory mantle cell lymphoma (MCL) after at least two lines of systemic therapy, including a BTK inhibitor.
Treatment of mantle cell lymphoma (MCL)

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Mechanism of Action
Bruton’s tyrosine kinase (BTK) is a tyrosine kinase located in the cytoplasm that is recruited to the cytoplasm upon activation. In B-cells, BTK participates in the activation of B-cell antigen receptor (BCR) signaling and cytokine receptor pathways, both critical for B-cell development, function, adhesion and migration. Therefore, the inhibition of BTK is a valuable target for the treatment of B-cell cancers. Pirtobrutinib binds to Bruton’s tyrosine kinase (BTK) in a non-covalent manner and inhibits its activity. Unlike other BTK inhibitors that bind covalently to the active site of BTK, the inhibitory activity of pirtobrutinib is maintained even in the presence of mutations in this region, such as the presence of Cys481. In nonclinical studies, pirtobrutinib inhibited BTK-mediated B-cell CD69 expression and inhibited malignant B-cell proliferation.

C22H21F4N5O3

479.4356

479.1581

C, 55.12; H, 4.42; F, 15.85; N, 14.61; O, 10.01

2101700-15-4

(R)-Pirtobrutinib;2101700-14-3

129269915

White to yellow solid powder

1.44±0.1 g/cm3

619.2±55.0 °C

3.3

125Ų

3

9

7

34

719

1

FC([C@H](C)N1C(=C(C(N)=O)C(C2C=CC(CNC(C3C=C(C=CC=3OC)F)=O)=CC=2)=N1)N)(F)F

FWZAWAUZXYCBKZ-NSHDSACASA-N

InChI=1S/C22H21F4N5O3/c1-11(22(24,25)26)31-19(27)17(20(28)32)18(30-31)13-5-3-12(4-6-13)10-29-21(33)15-9-14(23)7-8-16(15)34-2/h3-9,11H,10,27H2,1-2H3,(H2,28,32)(H,29,33)/t11-/m0/s1

5-amino-3-[4-[[(5-fluoro-2-methoxybenzoyl)amino]methyl]phenyl]-1-[(2S)-1,1,1-trifluoropropan-2-yl]pyrazole-4-carboxamide

Pirtobrutinib; LY-3527727; Jaypirca; LOXO-305; LY 3527727; LOXO305; LY3527727; LOXO 305; JAYPIRCA; RXC-005; LY3527727; JNA39I7ZVB

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（例如氮气保护），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)

DMSO: 50~96 mg/mL (104.3~200.2 mM)
Ethanol: ~48 mg/mL (~100.1 mM)

配方 1 中的溶解度: 2.75 mg/mL (5.74 mM) 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), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 27.5 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 2 中的溶解度: ≥ 2.75 mg/mL (5.74 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 27.5 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.21 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 4 中的溶解度: ≥ 2.5 mg/mL (5.2 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + + 45% Saline
≥ 2.5 mg/mL (5.2 mM) in 10% DMSO + 90% (20% SBE-β-CD in saline)
≥ 2.5 mg/mL (5.2 mM) in 10% DMSO + 90% Corn oil

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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。