BMX (IC50 = 6 nM); BTK (IC50 = 6.8 nM); TEC (IC50 = 48 nM); TXK (IC50 = 92 nM); BLK (IC50 = 0.3 μM); ERBB4 (IC50 = 0.77 μM); EGFR (IC50 = 3.02 μM); JAK3 (IC50 = 5.52 μM); ERBB2 (IC50 = 7.31 μM)

OCI-L Y10 and SU-DHL-6 cell growth is inhibited by tirabrutinib hydrochloride (0.1-1000 nM or 0.001-100 nM; 72 h), with IC50 values of 9.127 nM and 17.10 nM, respectively [1]. Tibrutinib hydrochloride (0.5, 5, 50 μM; 24, 48 h) causes SU-DHL-6 cells to undergo apoptosis; this requires large doses and prolonged administration (48 h of incubation at concentrations up to 50 μM) [1]. In TMD8 cells, tirabotinib hydrochloride (300 nM, 72 hours) causes caspase-3 and PARP cleavage [2].

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Tirabrutinib irreversibly and covalently binds to BTK Cys-481. The inactivation efficiency kinact/Ki was measured and used to calculate selectivity among different kinases for each of the four inhibitors studied. Tirabrutinib showed a kinact/Ki value of 2.4 ± 0.6 × 104 M-1 s-1 for BTK with selectivity against important off-targets. Conclusions: For the BTK inhibitors tested in this study, analysis of the inactivation kinetics yielded a more accurate measurement of potency and selectivity than conventional single-time point inhibition measurements. Subtle but clear differences were identified between clinically tested BTK inhibitors which may translate into differentiated clinical efficacy and safety. General significance: This is the first study that offers a detailed side-by-side comparison of four clinically-relevant BTK inhibitors with respect to their inactivation of BTK and related kinases.[3]

The oral single-dose dose of tiprunitinib hydrochloride (10 mg/kg) is rapidly absorbed into the brain and plasma, reaching its maximum concentration two hours after administration (blood Cmax = 339.53 ng/mL, brain Cmax = 28.9 ng/mL) [1]. In vivo, tibrutinib hydrochloride (6, 20 mg/kg; oral; once daily for 3 weeks) has demonstrated growth-inhibiting properties for tumors [2].

Determination of covalent binding[3]
Protein labeling experiments were performed using BTK at a final concentration of 2 μM in a buffer solution containing 10 mM HEPES, pH 7.5, 150 mM sodium chloride, 10 mM magnesium chloride, 2 mM Tris(2-carboxyethyl)phosphine (TCEP), and 1% glycerol. Inhibitors were added to a final concentration of 10 μM, with a final concentration of 1% DMSO in all samples. Four conditions were tested, each run in triplicate: BTK + tirabrutinib, BTK + staurosporine, BTK + ibrutinib, and BTK + DMSO control. After compound addition, samples were incubated overnight at 4 °C in a rotating shaker (1200 rpm). After an 18-h incubation, aliquots were collected from each condition for analysis and this time point was termed t = pre-chase. A chase step was then performed with the remaining sample by addition of ibrutinib into the BTK + tirabrutinib and BTK + staurosporine samples to a final concentration of 100 μM. An equivalent amount of DMSO was added to the BTK + ibrutinib and BTK + DMSO control samples to maintain the same volume. After incubating for 6 h at 4 °C, the remaining sample was collected at the final time point, termed t = post-chase. Aliquots taken at both time points were analyzed at the time of collection using mass spectrometry and enzyme activity assays.
Mass spectrometry analysis was performed on an Agilent 6210 Time of Flight Mass Spectrometer with an Agilent 1200 Rapid Resolution HPLC using Masshunter B.05 Acquisition software. Samples were run on an Agilent Zorbax 300 Extend C18 rapid resolution column at 70 °C, using reverse phase chromatography with a gradient from 20% to 90% acetonitrile containing 0.1% formic acid. Data were processed using Agilent MassHunter Qualitative Analysis B.06, with a BioConfirm workflow allowing for protein deconvolution to obtain neutral mass values.

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Covalent binding to BTK Cys-481[3]
Samples were prepared as follows: 25 μg (2 μM) of in-house recombinant BTK protein was incubated in 50 mM ammonium bicarbonate and 10 μM tirabrutinib for 1 h at 37 °C. Samples were then reduced using 5 mM DTT at 55 °C for 45 min, followed by alkylation with 10 mM iodoacetamide at 25 °C for 1 h. Samples were then digested using a 50:1 ratio of GluC endoproteinase for 12 h at 37 °C, followed by addition of trypsin at a 50:1 ratio and another 4-h digestion at 37 °C to yield the expected peptide target of BTK with sequence YMANGCLLNYLR. Digested samples were then lyophilized and re-suspended in 3% acetonitrile, 0.1% formic acid and submitted for mass spectrometry analysis.
Mass spectrometry analysis was conducted as follows: Samples were injected using a ThermoFisher UltiMate 3000 RSLCnano System. Separation was performed using a ThermoFisher Scientific ES800 Easy Spray LC column (150 mm × 75 μm) at a flow rate of 300 nL/min on a 60-min gradient using 1% acetonitrile, 0.1% formic acid as solvent A and 90% acetonitrile, 0.1% formic acid as solvent B [3% B - 35% B (45 min), 35% B - 90% B (15 min), 90% B (5 min), re-equilibration (20 min)]. Mass spectrometry analysis was performed on a ThermoFisher Q-Exactive HF using a top 20 data dependent acquisition. Automatic gain control settings used 50 ms fill time and 3E6 ion counts for MS scans (60 K resolution) and 100 ms file times and 1E5 ion counts for MSMS scans (15 K resolution). Data were searched using Proteome Discoverer 2.2 against a Swissprot human database using variable chemical modification by tirabrutinib.

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IC50 determination against BTK and other tyrosine kinases by tirabrutinib, ibrutinib, acalabrutinib, and spebrutinib in Z'-LYTE™ and LanthaScreen™ assays[3]
The IC50 values of tirabrutinib, ibrutinib, acalabrutinib, and spebrutinib were first studied in a standard Z'-LYTE™ kinase or LanthaScreen™ binding assay against BTK and other TEC family kinases at Life Technologies/ThermoFisher Scientific (Waltham, MA). Various enzyme concentrations were used in each biochemical assays. The concentration of each enzyme was as follows: 0.26 nM BLK, 6.2 nM BMX, 3.1 nM BTK, 8.3 nM EGFR, 17.5 nM ERBB2, 16.2 nM ERBB4, 30.0 nM ITK, 2.7 nM JAK3, 8.1 nM TXK, and 1 nM TEC. The reactions were carried out at room temperature for 1 h. Detailed assay conditions can be found in the Supplementary Data.

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BTK enzyme activity and IC50 evaluation[3]
BTK activity was quantified by determining phosphorylation of a fluorescein-labeled substrate using a LanthaScreen™ Assay Kit. The final reaction mixture contained kinase Buffer A [50 mM HEPES (pH 7.5), 10 mM MgCl2, 0.01% brij-35, 1 mM EGTA, and 0.5 mg/mL BSA], 200–300 pM of BTK, 0.2 μM of fluorescein-Poly GT substrate, and 180 μM of ATP (2× Km). All pre-incubations and reactions were carried out in black, 96-well nonbinding surface (NBS™) assay plates at room temperature. To evaluate the enzyme activity in the samples used in the mass spectrometry analysis, an aliquot of each of the samples was diluted to 300 pM BTK and 1.5 nM inhibitor in kinase reaction Buffer A. For IC50 determinations, compound dilutions were prepared using an HP D300 liquid dispenser and the final DMSO concentration in the reactions was kept at 1%. After a 30-min pre-incubation of inhibitors and BTK in Buffer A, the kinase reaction was initiated by addition of an equal volume of 2× fluorescein-Poly GT substrate and ATP in Buffer A to reach a final 100 μL reaction mixture containing 200 pM BTK. The kinase reaction was allowed to proceed for 30 min and was terminated with 100 μL of 2× EDTA/LanthaScreen™ Tb-PY20 antibody mixture in Life Technologies' TR-FRET Dilution Buffer to reach final concentrations of 10 mM EDTA and 2 nM antibody. Reactions were then incubated for at least 90 min at room temperature before fluorescence intensity (λ ex 332 nm/λ em 486/515 nm) was read on a TECAN Infinite M1000 Pro Multimode reader. The ratio of fluorescence at 515 nm to that at 486 nm was the measure of product formation. The TR-FRET ratio was plotted against the inhibitor concentration and normalized to enzyme/no enzyme controls. IC50 values were calculated with a four-parameter logistic fit using GraphPad Prism.

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Determination of inactivation kinetics for BTK[3]
The rate of enzyme inactivation was studied as a function of inhibitor concentration using a Sox-based fluorescence assay that allows real-time measurement of enzyme activity. In this assay, kinase activity is measured by an increase in fluorescence as a result of phosphorylation of a Sox-labeled substrate. Briefly, a Master Mix containing 1.3× Sox-labeled substrate, ATP, and DTT was prepared in reaction Buffer B [20 mM Tris-HCl (pH 7.5), 5 mM β-glycerophosphate, 1 mM EGTA, 5 mM MgCl2, and 5% glycerol]. 75 μL of the Master Mix was added to the assay plate containing 1 μL compound solution in DMSO. Reactions were initiated with the addition of 25 μL of 4× in-house recombinant BTK protein in reaction Buffer B, except for the no-enzyme control in which only reaction Buffer B was used. The final assay mixtures contained 5 nM BTK, 10 μM Sox-labeled substrate AQT0101 (AQT0104 for ibrutinib), 300 μM ATP (2× Km), and 200 μM DTT in reaction Buffer B. Fluorescence intensity readings (λ ex 360 nm/λ em 485 nm) were collected every 30 s for 4 h at room temperature using a TECAN Infinite M1000 Pro plate reader.

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Determination of inactivation kinetics for EGFR, BMX, ITK, and TEC[3]
Enzymatic inhibition by tirabrutinib, ibrutinib, acalabrutinib, and spebrutinib were tested against EGFR and the TEC family kinases BMX, ITK, and TEC. Inactivation kinetics for the inhibitors were studied in a similar manner as previously described for BTK: EGFR at 2.5 nM with 70 mM ATP (2× Km), BMX at 1.25 nM with 100 mM ATP (2× Km), ITK at 5 nM with 50 mM ATP (2× Km), and TEC at 2.5 nM with 80 mM ATP (2× Km). The Sox substrates used were AQT0001 for EGFR, AQT0025 for BMX and ITK, and AQT0102 for TEC. Selectivity is defined as the ratio of the kinetic parameter kinact/Ki for compound binding to BTK to kinact/Ki for compound binding to EGFR, ITK, BMX, or TEC.

Cell proliferation assay [1]
Cell Types: SU-DHL-6 and OCI-LY Y10 Cell
Tested Concentrations: 0.1-1000 nM; 0.001nM-100nM.
Incubation Duration: 72 hrs (hours)
Experimental Results: demonstrated good anti-proliferative activity with IC50 of 9.127 nM and 17.10 nM for OCI-L Y10 and SU-DHL-6 cells respectively.

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Cell apoptosis analysis [1]
Cell Types: SU-DHL-6 Cell
Tested Concentrations: 0.5, 5, 50 μM
Incubation Duration: 24, 48 h
Experimental Results: Cell apoptosis was induced when the concentration reached 50 μM and incubated for 48 h.

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Western Blot Analysis[2]
Cell Types: TMD8 Cell
Tested Concentrations: 300 nM
Incubation Duration: 72 hrs (hours)
Experimental Results: Induction of caspase-3 and PARP cleavage.

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Cell Death Assessment[2]
The cell lines used in this study have been previously described and were obtained either from the originators or from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ). The identity of cell lines were confirmed by both metaphase cytogenetics and short tandem repeat assessment. Cell lines were grown in RPMI 1640 supplemented with 10% fetal calf serum. Sensitivity to Tirabrutinib in cell lines as well as combination treatments was performed using a CellTiterGlo® viability assay or AnnexinV-FITC staining. The calculation of EC50 was done using GraphPadPrism. Tirabrutinib resistant TMD8 (TMD8R) was generated by continuous exposure over nine months at concentrations of tirabrutinib ranging from 3 nM to 1000 nM until stable resistance to tirabrutinib was established. Specifically, the concentration of tirabrutinib was gradually increased from 3, 6, 12, 25, 40, 60, 80, 100, 200, 400, 800, and 1000 nM. Cell passage was performed twice a week. When the growth was fine, cells were re-suspended with fresh media containing the same concentration of tirabrutinib (final cell density was 100,000 cells/mL). Sensitivity of cells to tirabrutinib was tested by CellTiterGlo® at every step of passage. The mutational status of TMD8R was determined by Sanger sequencing. The combination index was calculated using CalcuSyn based on the multiple drug-effect equation of Chou-Talalay.

Animal/Disease Models: Male SD rat (219.0–260.5g) [1].
Doses: 10 mg/kg
Route of Administration: Oral; single.
Experimental Results: 1.19 pharmacokinetic/PK/PK parameters of tilatinib in male SD rats [1]. Plasma, Cmax (ng/mL) Brain, Cmax (ng/mL) Permeability (%, Cmax, brain/Cmax, plasma) PO (10 mg/kg) 339.53 28.9 8.5

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Animal/Disease Models: Immunodeficient (SCID) mice ( Mouse xenograft model) [2].
Doses: 6, 20 mg/kg
Route of Administration: po (po (oral gavage)) one time/day for 3 weeks.
Experimental Results: Inhibited tumor growth. When the dose reached 20 mg/kg, the tumor was completely inhibited on the 14th day.

Ibrutinib and Tirabrutinib might be more suitable for brain-confined diseases than zanubrutinib[1]
As indicated by the inhibition and apoptosis assay, high level of drug concentration in a prolonged period is required for the complete inhibition of tumor cells. Therefore, to determine the feasibility of BTK inhibitors in treating PCNSL, we tested if they were able to maintain the concentration in brain efficient for tumor inhibition. SD rats were orally administered with BTK inhibitors once. Plasma and brain tissue were sampled at designated time points to test the drug concentration (Figure 4). As indicated by Figure 4, all three BTK inhibitors were rapidly absorbed into plasma and brain. Both unbound ibrutinib and Tirabrutinib reached Cmax 2 hours post administration, and maintained at a relatively stable level, both in blood and brain tissue (ibrutinib: blood Cmax =412.7 ng/mL, brain Cmax =40.4 ng/mL, n=3; Tirabrutinib: blood Cmax =339.53 ng/mL, brain Cmax =28.9 ng/mL, n=3). As for unbound zanubrutinib, it rapidly reached maximum concentration in blood and brain at 0.5-hour post administration, and exhibited a decrease in blood concentration slightly faster than the other BTK inhibitors. In brain, on the other hand, unbound zanubrutinib concentration slumped, and was below the limit of detection 4 hours after administration.
Because Cmax in blood and brain were simultaneously reached by each BTK inhibitor (ibrutinib and Tirabrutinib: 2 hour post oral administration; zanubrutinib: 0.5 hour post oral administration), unbound brain-to-plasma concentration ratio was calculated at the time they reached Cmax. Unbound brain-to-plasma concentration ratio of zanubrutinib, Tirabrutinib and ibrutinib were 3.5%, 8.5% and 9.8%, respectively (Table 2). This ratio of ibrutinib is slightly higher than Tirabrutinib. This ratio of zanubrutinib, however, was much lower than ibrutinib and Tirabrutinib, indicating its inferior ability to pass through BBB compared to ibrutinib and Tirabrutinib.
Together, these data indicate that ibrutinib, Tirabrutinib and zanubrutinib can be rapidly absorbed into blood and distributed into brain after oral administration. Compared with zanubrutinib, ibrutinib and Tirabrutinib exerted better ability in passing through BBB and maintaining a high and stable concentration in brain, facilitating these inhibitors to exert their anti-tumoral effect in brain, making them more promising candidates for the treatment of PCNSL.

ONO-4059 was found to be well tolerated, with no dose limiting toxicities (DLTs). A total of 18 ONO-4059-related adverse events were reported in 6 out of 14 patients; CTCAE-V4.0 G1 (n=10 [n=6 in 1 patient]) and G2 (n=5). Three ONO-4059-related G3 haematological toxicities were reported in 2 patients; thrombocytopenia (x2) and anemia. No ONO-4059-related G4 events, or related SAEs or infections were reported. The pharmacokinetics of ONO-4059 reflects rapid absorption and elimination, a half-life of ∼6 hours, a dose dependent increase in exposure with no accumulation of ONO-4059 exposure and low inter- or intra-patient variability; with Btk occupancy in peripheral blood (as measured by phosphorylated Btk) being maintained for at least 24 hours across all dose levels.

[1]. Bruton's tyrosine kinase inhibitors in primary central nervous system lymphoma-evaluation of anti-tumor efficacy and brain distribution. Transl Cancer Res. 2021 May;10(5):1975-1983.

[2]. Responses to the Selective Bruton's Tyrosine Kinase (BTK) Inhibitor Tirabrutinib (ONO/GS-4059) in Diffuse Large B-cell Lymphoma Cell Lines. Cancers (Basel). 2018 Apr 23;10(4):127.

[3]. Biochemical characterization of tirabrutinib and other irreversible inhibitors of Bruton's tyrosine kinase reveals differences in on - and off - target inhibition. Biochim Biophys Acta Gen Subj. 2020 Apr;1864(4):129531.

[4]. Dhillon S. Tirabrutinib: First Approval. Drugs. 2020 Jun;80(8):835-840.

Tirabrutinib Hydrochloride is the hydrochloride salt form of tirabrutinib, an orally available formulation containing an inhibitor of Bruton agammaglobulinemia tyrosine kinase (BTK), with potential antineoplastic activity. Upon administration, tirabrutinib covalently binds to BTK within B cells, thereby preventing B cell receptor signaling and impeding B cell development. As a result, this agent may inhibit the proliferation of B cell malignancies. BTK, a cytoplasmic tyrosine kinase and member of the Tec family of kinases, plays an important role in B lymphocyte development, activation, signaling, proliferation and survival.

C25H23CLN6O3

490.948

490.152

C, 61.16; H, 4.72; Cl, 7.22; N, 17.12; O, 9.78

1439901-97-9

Tirabrutinib;1351636-18-4;ONO-4059 analog;1351635-67-0

71571562

White to off-white solid powder

2.31

2

6

4

35

825

1

CC#CC(=O)N1CC[C@H](C1)N2C3=NC=NC(=C3N(C2=O)C4=CC=C(C=C4)OC5=CC=CC=C5)N.Cl

UQYDCIJFACDXSG-GMUIIQOCSA-N

InChI=1S/C25H22N6O3.ClH/c1-2-6-21(32)29-14-13-18(15-29)31-24-22(23(26)27-16-28-24)30(25(31)33)17-9-11-20(12-10-17)34-19-7-4-3-5-8-19/h3-5,7-12,16,18H,13-15H2,1H3,(H2,26,27,28)1H/t18-/m1./s1

(R)-6-amino-9-(1-(but-2-ynoyl)pyrrolidin-3-yl)-7-(4-phenoxyphenyl)-7,9-dihydro-8H-purin-8-one hydrochloride

GS4059; ONO-WG-307; ONO-4059 HCl; GS 4059; ONO4059; Tirabrutinib hydrochloride; ONO-4059 Hydrochloride; Tirabrutinib HCl; Tirabrutinib (hydrochloride); ONO-4059 (hydrochloride); GS-4059 hydrochloride; Ono-4059(HCl salt); ONO 4059; ONO-4059; GS-4059; Tirabrutinib HCl

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, 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 : ~100 mg/mL (~203.69 mM)

Solubility in Formulation 1: 2.5 mg/mL (5.09 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 25.0 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: ≥ 2.5 mg/mL (5.09 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: ≥ 1.79 mg/mL (3.65 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 17.9 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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 (Please use freshly prepared in vivo formulations for optimal results.)