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Tirabrutinib (formerly ONO-4059; GS4059; ONO-WG-307; Steboronine) is a novel, potent, highly selective, covalent/irreversible and orally bioavailable BTK (Bruton agammaglobulinemia tyrosine kinase) inhibitor with anticancer activity. It has been approved in Japan since March 2020 for the treatment of recurrent or refractory primary central nervous system lymphoma. It inhibits BTK with an IC50 of 2.2 nM. Tirabrutinib inhibits B-cell development by covalently attaching to BTK within B cells, which stops B-cell receptor signaling. This means that this substance might prevent B-cell cancers from spreading. As a cytoplasmic tyrosine kinase belonging to the Tec family, BTK is crucial for B lymphocyte activation, development, proliferation, and survival.
Physicochemical Properties
| Molecular Formula | C25H22N6O3 |
| Molecular Weight | 454.490 |
| Exact Mass | 454.175 |
| Elemental Analysis | C, 66.07; H, 4.88; N, 18.49; O, 10.56 |
| CAS # | 1351636-18-4 |
| Related CAS # | Tirabrutinib hydrochloride;1439901-97-9;ONO-4059 analog;1351635-67-0 |
| PubChem CID | 54755438 |
| Appearance | White to off white powder |
| Density | 1.4±0.1 g/cm3 |
| Boiling Point | 672.0±65.0 °C at 760 mmHg |
| Flash Point | 360.2±34.3 °C |
| Vapour Pressure | 0.0±2.1 mmHg at 25°C |
| Index of Refraction | 1.700 |
| LogP | 2.31 |
| Hydrogen Bond Donor Count | 1 |
| Hydrogen Bond Acceptor Count | 6 |
| Rotatable Bond Count | 4 |
| Heavy Atom Count | 34 |
| Complexity | 825 |
| Defined Atom Stereocenter Count | 1 |
| SMILES | O=C1N(C2C=CC(=CC=2)OC2C=CC=CC=2)C2=C(N)N=CN=C2N1C1CN(C(C#CC)=O)CC1 |
| InChi Key | SEJLPXCPMNSRAM-GOSISDBHSA-N |
| InChi Code | InChI=1S/C25H22N6O3/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)/t18-/m1/s1 |
| Chemical Name | 6-amino-9-[(3R)-1-but-2-ynoylpyrrolidin-3-yl]-7-(4-phenoxyphenyl)purin-8-one |
| Synonyms | ONO-4059; GS4059; ONO-WG-307; ONO4059; GS-4059;ONO 4059; GS-4059; Btk Kinase inhibitor; ONO-4059(Free base); Tirabrutinib [INN]; Tirabrutinib free base; ONO-4059; GS 4059; ONO WG-307 |
| HS Tariff Code | 2934.99.9001 |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets |
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) Bruton’s tyrosine kinase (BTK) inhibitor. No specific IC50, Ki, or EC50 values for BTK were provided in this study. [1] |
| ln Vitro |
Tirabrutinib (0.1-1000 nM or 0.001-100 nM; 72 h) has IC50 values of 9.127 nM and 17.10 nM, respectively, which limit the growth of OCI-L Y10 and SU-DHL-6 cells[1]. Tirabrutinib (0.5, 5, 50 μM; 24, 48 h) induces apoptosis in SU-DHL-6 cells; however, it requires a high dosage and long administration (48 hours of incubation at a concentration of up to 50 μM)[1]. Tirabrutinib (300 nM, 72 h) causes caspase-3 and PARP cleavage in TMD8 cells[2]. 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] Tirabrutinib exhibited anti-proliferative effects against diffuse large B-cell lymphoma (DLBCL) cell lines in vitro. In the SU-DHL-6 cell line, the IC50 value for tirabrutinib after 72 hours of treatment was 17.10 µM (n=2). In the OCI-LY10 cell line, the IC50 value for tirabrutinib after 72 hours of treatment was 9.127 µM (n=2). Among the three BTK inhibitors tested (ibrutinib, zanubrutinib, tirabrutinib), tirabrutinib showed the weakest anti-proliferative effect in both cell lines. [1] Tirabrutinib induced apoptosis in SU-DHL-6 cells in a dose- and time-dependent manner. When cells were treated with a high concentration of tirabrutinib (50 µM) for 48 hours, a significant increase in dead/late apoptotic cells (Annexin V+/7-AAD+) was observed. No significant apoptosis was induced at lower concentrations (0.5 µM or 5 µM) after 24 or 48 hours of treatment. [1] |
| ln Vivo |
Tirabrutinib (10 mg/kg; p.o.; single) enters the brain and plasma quickly, reaching its Cmax two hours after administration (blood Cmax = 339.53 ng/mL, brain Cmax = 28.9 ng/mL)[1]. Tirabrutinib (6, 20 mg/kg; p.o.; single daily for 3 weeks) inhibits the growth of tumors in vivo[2]. In a subcutaneous xenograft model using severe combined immunodeficiency (SCID) mice implanted with TMD8 cells, oral administration of tirabrutinib via diet at doses equivalent to 6, 20, and 60 mg/kg/day for three weeks caused dose-dependent tumor regression. Treatment with 0.012% in diet (~20 mg/kg/day) led to tumor remission in all animals (10/10). No decrease in body weight was observed in any treatment group. In contrast, the same treatment (0.012% and 0.037% in diet) had no effect on tumor growth in mice implanted with tirabrutinib-resistant TMD8R cells. [2] |
| Enzyme Assay |
Determination of covalent binding[3] \nProtein 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. \n\nMass 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. \n\nCovalent binding to BTK Cys-481[3] \nSamples 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. \n\nMass 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. \n\n\n\n \n \n\nView More\n\nIC50 determination against BTK and other tyrosine kinases by tirabrutinib, ibrutinib, acalabrutinib, and spebrutinib in Z'-LYTE™ and LanthaScreen™ assays[3] \n\nBTK enzyme activity and IC50 evaluation[3] \nBTK 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.\n\nThe 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. \n\nDetermination of inactivation kinetics for BTK[3] \nThe 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. \n\nDetermination of inactivation kinetics for EGFR, BMX, ITK, and TEC[3] \nEnzymatic 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.\n\n Covalent Binding Verification by Mass Spectrometry: Recombinant BTK protein (2 µM) was incubated with 10 µM tirabrutinib (or controls: ibrutinib, staurosporine, DMSO) overnight at 4°C. For a subset of samples, a "chase" step was performed by adding 100 µM ibrutinib and incubating for an additional 6 hours. Aliquots were taken pre- and post-chase. The intact mass of BTK was analyzed using LC-TOF mass spectrometry to detect mass shifts corresponding to compound binding. To confirm the specific binding site, BTK samples incubated with tirabrutinib were reduced, alkylated, and digested with Glu-C and trypsin. The resulting peptides were analyzed by LC-MS/MS, searching for variable modifications by tirabrutinib to identify the modified peptide. [3] IC50 Determination (LanthaScreen Assay): BTK activity was measured via phosphorylation of a fluorescein-labeled peptide substrate detected by time-resolved fluorescence resonance energy transfer (TR-FRET). Reactions contained 0.2-0.3 nM BTK, 0.2 µM fluorescein-poly(GT) substrate, and 180 µM ATP in assay buffer. Serial dilutions of tirabrutinib were pre-incubated with BTK for 30 minutes at room temperature before initiating the reaction by adding substrate/ATP. After a 30-minute reaction, it was stopped with an EDTA/Tb-antibody mixture. Following a 90-minute incubation, fluorescence was read (ex 332 nm, em 486/515 nm). The ratio of emissions (515/486 nm) was used to calculate percent inhibition, and IC50 values were determined by fitting the data to a four-parameter logistic curve. [3] Inactivation Kinetics (Sox-based Fluorescence Assay): The rate of BTK inactivation by tirabrutinib was measured in real-time using a Sox-labeled peptide substrate, whose phosphorylation increases fluorescence. Assays contained 5 nM BTK, 10 µM Sox-substrate, 300 µM ATP, and varying concentrations of tirabrutinib in reaction buffer. Reactions were initiated by adding BTK to the mixture containing inhibitor and substrate. Fluorescence (ex 360 nm, em 485 nm) was monitored every 30 seconds for 4 hours at room temperature. Progress curves were fitted to a single-exponential equation to obtain the observed rate constant (kobs) at each inhibitor concentration. A plot of kobs versus [inhibitor] was then fitted to a hyperbolic equation to derive the maximal inactivation rate (kinact) and the inhibitor concentration for half-maximal inactivation (Ki). When individual kinact and Ki values could not be determined precisely, the second-order inactivation efficiency constant (kinact/Ki) was reported as the slope from a linear fit of kobs vs. [I] at low inhibitor concentrations. This assay format was also used to determine kinact/Ki for off-target kinases (EGFR, BMX, ITK, TEC) under their respective optimized conditions (enzyme concentration, ATP Km). [3] Kinase Selectivity Panel (Z’-LYTE/LanthaScreen Assay): IC50 values against a panel of ten tyrosine kinases were determined using commercial Z’-LYTE kinase activity or LanthaScreen binding assays. Enzyme concentrations varied per kinase (e.g., 3.1 nM BTK, 8.3 nM EGFR, 30.0 nM ITK). Tirabrutinib was serially diluted and incubated with the enzyme for 1 hour at room temperature before starting the kinase reaction according to the manufacturer's protocol. Dose-response curves were generated to calculate IC50 values. [3] |
| Cell Assay |
Cell Line: SU-DHL-6 and OCI-L Y10 cells Concentration: 0.1-1000 nM; 0.001 nM-100 nM Incubation Time: 72 h Result: Showed good anti-proliferative activity with IC50s of 9.127 nM, and 17.10 nM for OCI-L Y10 and SU-DHL-6 cells, respectively. 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. Inhibition Assay: DLBCL cell lines (SU-DHL-6 and OCI-LY10) were cultured in complete medium. Cells in the logarithmic growth phase were collected, resuspended, and seeded into 96-well plates. After 24 hours of incubation, cells were treated with tirabrutinib at six or ten different concentrations for 72 hours. Dimethyl sulfoxide (DMSO) was used as the vehicle control. After the treatment period, cell viability was assessed using a Cell Counting Kit-8 (CCK-8). The absorbance was measured at 450 nm using a microplate reader. The half-maximal inhibitory concentration (IC50) was calculated using nonlinear regression analysis with appropriate software. Each experiment was repeated twice. [1] Apoptosis Assay: SU-DHL-6 cells were seeded in 96-well plates. After 24 hours, cells were treated with tirabrutinib at three different concentrations (0.5 µM, 5 µM, 50 µM) for either 24 or 48 hours. DMSO served as the control. After incubation, cells were harvested and stained with Annexin V and 7-aminoactinomycin D (7-AAD) according to the kit protocol. The stained cells were then analyzed by flow cytometry. Cells were categorized as living (Annexin V-/7-AAD-), early apoptotic (Annexin V+/7-AAD-), or late apoptotic/dead (Annexin V+/7-AAD+). [1] |
| Animal Protocol |
Male SD rats (219.0–260.5g) 10 mg/kg Oral administration; single. Mouse Xenograft Model[2] To assess in vivo efficacy of tirabrutinib, severe combined immunodeficiency (SCID) mice were injected with 1 × 107 cells in Matrigel, subcutaneously. Randomization and treatment were initiated when the mean tumor volumes reached 400 mm3 for TMD8 and 200 mm3 for tirabrutinib resistant cells. Groups of mice were then dosed via diet containing tirabrutinib at concentrations of 0.0037%, 0.012% and 0.037%. The daily dosage was found to be comparable to the doses, 6, 20, and 60 mg/kg/day, respectively. Tumor growth was assessed using a caliper. Pharmacokinetic Study in Rats: Male Sprague-Dawley (SD) rats were randomly divided into groups. One group received a single oral administration of tirabrutinib at a dose of 10 mg/kg. The dosage was calculated based on the clinical equivalent dose using body surface area conversion factors. Blood samples were collected from the jugular vein at six designated time points (0.25, 0.5, 1, 2, 4, and 24 hours) after dosing. Plasma was obtained by centrifuging the blood samples. At each corresponding time point, rats were euthanized via decapitation, and whole brains were collected. Brain tissues were homogenized in saline. Both plasma and brain homogenate samples were stored frozen until analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine drug concentrations. [1] |
| ADME/Pharmacokinetics |
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. In SD rats following a single oral dose of 10 mg/kg tirabrutinib, the drug was rapidly absorbed and distributed. The maximum plasma concentration (Cmax, plasma) was 339.53 ng/mL. The maximum brain concentration (Cmax, brain) was 28.9 ng/mL. The time to reach Cmax (Tmax) in both plasma and brain was 2 hours post-administration. The unbound brain-to-plasma concentration ratio (calculated as Cmax, brain / Cmax, plasma at Tmax) was 8.5%. This ratio indicates the ability of tirabrutinib to cross the blood-brain barrier and was higher than that of zanubrutinib (3.5%) but slightly lower than that of ibrutinib (9.8%) in this study. [1] |
| Toxicity/Toxicokinetics |
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. In the SCID mouse xenograft study, no decreases in body weight were observed in any group treated with tirabrutinib at dietary concentrations up to 0.037% (equivalent to ~60 mg/kg/day) over a 2-3 week period, suggesting good tolerability at these efficacious doses in this model. [2] |
| References |
[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]. Tirabrutinib: First Approval. Drugs. 2020 Jun;80(8):835-840. |
| Additional Infomation |
Tirabrutinib is under investigation in clinical trial NCT02626026 (Safety and Pharmacokinetics of GS-4059 in Healthy Volunteers and Subjects With Rheumatoid Arthritis (RA)). Tirabrutinib is 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. Tirabrutinib is a second-generation Bruton's tyrosine kinase (BTK) inhibitor. Primary central nervous system lymphoma (PCNSL) is an aggressive lymphoma confined to the CNS, and most cases are of the diffuse large B-cell lymphoma (DLBCL) subtype, which often involves constitutive activation of the B-cell receptor pathway and NF-κB signaling. BTK is a crucial kinase in this pathway. This study compared tirabrutinib with two other BTK inhibitors (ibrutinib and zanubrutinib) for potential use in PCNSL. While tirabrutinib showed weaker in vitro anti-proliferative activity compared to ibrutinib and zanubrutinib, it demonstrated favorable pharmacokinetic properties for targeting brain tumors. Specifically, it achieved sustained and measurable concentrations in the brain parenchyma of rats with a brain-to-plasma ratio supportive of blood-brain barrier penetration. The study suggests that despite its lower in vitro potency, tirabrutinib's distribution profile may make it a promising candidate for treating PCNSL, where achieving effective drug levels in the brain is critical. The study also mentions that tirabrutinib has been evaluated in clinical trials for B-cell malignancies and cites a phase I/II trial in relapsed/refractory PCNSL patients reporting an objective response rate of 64%. [1] |
Solubility Data
| Solubility (In Vitro) | DMSO: ≥ 100 mg/mL (~220.0 mM) |
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (5.50 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (5.50 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (5.50 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.2003 mL | 11.0013 mL | 22.0027 mL | |
| 5 mM | 0.4401 mL | 2.2003 mL | 4.4005 mL | |
| 10 mM | 0.2200 mL | 1.1001 mL | 2.2003 mL |