BTK (IC50 = 5.69 nM)

Pirtobrutinib (LOXO-305) is a next-generation Bruton's tyrosine kinase (BTK) inhibitor that is highly selective, non-covalent, and exhibits strong equilibrium binding to both WT BTK and several BTK C481 substitution mutations.It also potently inhibits the cellular activity of BTK C481S, T, and R mutations.[2]

Pirtobrutinib significantly inhibits tumor growth in human lymphoma xenografts in vivo. [3]

Pirtobrutinib activity against BTK, BTK C481S, and selected non-BTK kinases was determined by monitoring incorporation of [33P]-PO4 from [33P]-adenosine triphosphate (ATP) into polyglutamic acid–tyrosine (poly-EY) peptide substrate in the HotSpot Kinase Assay (Reaction Biology, Malvern, PA).25 Data were analyzed using standard-curve fitting methods. Pirtobrutinib (1 μM) was tested for kinase activity inhibition of 371 human kinases using the HotSpot Kinase Assay and Km ATP concentrations. Pirtobrutinib, ibrutinib, zanubrutinib, and acalabrutinib (100 nM) were tested using the HotSpot Kinase Assay at a concentration of 10 μM ATP. Percentage of control activity was calculated for each enzyme.[3]
Both LOXO-305 and ibrutinib potently inhibited IgM-induced phospho-BTK with IC50 values equal to 1.34 ± 1.23 nM for LOXO-305 (n = 7, p < 0.0001) and 1.04 ± 1.26 nM for ibrutinib (n = 7, p < 0.0001). We also found a significant reduction in phosphorylation of PLCγ2 (Y1217), the immediate downstream effector of BTK (IC50 33 nM for each agent, n = 7, p = 0.02 for LOXO-305, p = 0.0017 for ibrutinib).[1]

HEK293T cell lines that were transiently expressing WT BTK and BTK C481 substitution mutations were treated for 30 minutes with LOXO-305, ibrutinib, or acalabrutinib before orthovanadate was added. Following a 2-hour incubation period, cells were lysed, and total BTK and phosphorylated Y223 BTK were identified using MesoScale (C481R) or immunoblot (BTK WT, C481S, and C481T). Using GraphPad Prism, the bands and MSD signals were quantified and the IC50 values were computed.

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. doi: 10.1182/blood.2022018674.

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

Solid powder

3.3

125Ų

C[C@@H](C(F)(F)F)N1C(=C(C(=N1)C2=CC=C(C=C2)CNC(=O)C3=C(C=CC(=C3)F)OC)C(=O)N)N

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

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

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)

Solubility in Formulation 1: 2.75 mg/mL (5.74 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (5.74 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.21 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: ≥ 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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 (Please use freshly prepared in vivo formulations for optimal results.)