BTK/Bruton tyrosine kinase
BGB-3111 is more selective for BTK vs. EGFR, FGR, FRK, HER2, HER4, ITK, JAK3, LCK, BLK, and TEC than ibrutinib in biochemical and cellular assays[1].
BGB-3111 potently inhibits cell proliferation in multiple MCL and DLBCL cell lines by blocking downstream PLC-γ2 signaling and inhibiting BTK autophosphorylation triggered by BCR aggregation[2].

BGB-3111 is more selective for BTK vs. EGFR, FGR, FRK, HER2, HER4, ITK, JAK3, LCK, BLK, and TEC than ibrutinib in biochemical and cellular assays[1].
BGB-3111 potently inhibits cell proliferation in multiple MCL and DLBCL cell lines by blocking downstream PLC-γ2 signaling and inhibiting BTK autophosphorylation triggered by BCR aggregation[2].

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In preclinical studies, BGB-3111 showed more restricted off-target activities against a panel of kinases, including ITK, compared to ibrutinib. Due to its weaker activity on ITK, BGB-3111 was at least 10 times weaker than ibrutinib in inhibiting rituximab-induced antibody-dependent cellular cytotoxicity (ADCC) activity. [3]

BGB-3111 exhibits better oral bioavailability than ibrutinib in preclinical animal studies, achieving higher exposure and more complete target inhibition in the tissues[1].
BGB-3111 treatment causes a dose-dependent BTK occupancy in mouse BTK occupancy assays, and it exhibits approximately three times the potency of ibrutinib in target organs, such as the spleen and PBMC[2].
BGB-3111 exhibits better efficacy than ibrutinib and induces dose-dependent anti-tumor effects in both the REC-1 MCL and ABC subtype DLBCL (TMD-8) xenograft models. According to a rat toxicity study, BGB-3111 is highly well tolerated, and even at doses of up to 250 mg/kg/day, the MTD is not reached[3].

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In the REC-1 mantle cell lymphoma (MCL) xenograft model and the ABC subtype diffuse large B-cell lymphoma (DLBCL, TMD-8) xenograft model, BGB-3111 induced dose-dependent antitumor effects and demonstrated superior efficacy in comparison with ibrutinib. [3]

Procedure for protein precipitation experiment:[4]
human BTK protein were pre-incubated with BGB-3111 (31a, Zanubrutinib) or B43 (a reversible BTK inhibitor without the reactive chemical group) to form BTK/compound complexes (with BTK kinase in excess to ensure complete binding of compounds by BTK protein). The human BTK protein was then denatured and precipitated by centrifugation. The supernatants were analyzed for quantity of the original compound by LC-MS/MS or HPLC. B43 remained in the supernatant regardless of presence or absence of BTK protein during pre- S8 incubation, consistent with the reversible nature of its BTK binding (Figure 3B). In contrast, 31a was only recovered in the supernatant when pre-incubated without BTK protein. Upon preincubation with BTK, 31a largely disappeared from the supernatant (Figure 3A), consistent with the notion that 31a bound covalently/irreversibly to BTK protein thereby was removed from supernatant with precipitated BTK protein. The data also showed that the covalent bond formation between BTK and 31a was largely completed within 5 minutes[4].

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In both biochemical and cellular assays, BGB-3111 demonstrated nanomolar BTK inhibition activity. In several MCL and DLBCL cell lines, BGB-3111 inhibited BCR aggregation-triggered BTK autophosphorylation, blocked downstream PLC-γ2 signaling, and potently inhibited cell proliferation. In comparison with ibrutinib, BGB-3111 showed much more restricted off-target activities against a panel of kinases, including ITK. While ibrutinib significantly inhibited rituximab-induced NK cell IFN-γ secretion and in vitro cytotoxicity on mantle cell lymphoma cells, BGB-3111 was at least 10-fold weaker than ibrutinib in inhibiting rituximab induced ADCC, consistent with its weak ITK inhibition activity.[2]

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Microsome intrinsic clearance: [4]
Compounds (1 µM) were incubated in the liver microsomes at protein concentration of 0.5 mg/mL. The reactions were terminated S50 at 0, 2, 6, 10, 20, 30 min for human and 0, 5, 10, 20, 40, 60 min for dog, rat and mouse after the reactions were initiated. Compounds were analyzed using LC-MS/MS. Natural log of percentage remaining of compounds was plotted against the incubation time. The intrinsic clearance (CLint) was calculated with the following equation, where Cprotein was the microsomal protein concentration in the incubation system. CLint = -slope/Cprotein.[4]

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Cytochrome P450 Inhibition: [4]
The human CYPs inhibition potential of compounds was evaluated based on the measurement of IC50 values in pooled human liver microsomes using 8 CYP probe substrates along with sample analysis by LC-MS/MS methods. Serial concentrations of compounds were incubated with each probe substrate in pooled human liver microsomes. Selective inhibitors were used as the positive controls to validate the incubation systems. Formation rate of the probe metabolites was monitored to calculate the IC50 values.

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The BTK biochemical assay used recombinant BTK protein. Compounds were pre-incubated with the enzyme for 1 hour at room temperature in assay buffer. The reaction was initiated by adding ATP (at its Km concentration) and a biotinylated peptide substrate. After 1 hour incubation, the reaction was stopped by adding a solution containing EDTA, a europium cryptate-conjugated anti-phosphotyrosine antibody, and streptavidin-labeled XL665. After a further 1-hour incubation, time-resolved fluorescence resonance energy transfer (TR-FRET) signals were measured [4]
Assays for ITK, TEC, JAK3, EGFR, and HER2 were performed using a similar TR-FRET-based method [4]
A broad kinase selectivity panel (342 kinases) was assessed using a filter-binding assay with ³³P-ATP. Compounds were pre-incubated with kinases for 1 hour, followed by addition of ATP to a final concentration of 1 μM. Reactions proceeded for 120 minutes before being spotted onto filter paper, washed, and quantified [4]

Cellular toxicity evaluation of BGB-3111 (31a, Zanubrutinib) (HEK293 and Ramos Proliferation Assay): The growthinhibitory activity of compounds in HEK293 and Ramos was determined using CellTiter-Glo luminescent cell viability assay. The number of cells seeded per well of a 96-well plate was optimized for each cell line to ensure logarithmic growth over 6 days treatment period (1000 cells/well for HEK293 cells, 6000 cells/well for Ramos cells). Cells were treated in triplicate with a 10-point dilution series. Following a 6-day exposure to the compound, a volume of CellTiter-Glo reagent equal to the volume of cell culture medium present in each well was added. Mixture was mixed on an orbital shaker for 5 minutes to allow cell lysis, followed by 10 minutes incubation at room temperature to allow development and stabilization of luminescent signal, which corresponded to quantity of ATP and thus the quantity of metabolically active cells. Luminescent signal was measured using PHERAstar FS reader. IC50 values for cell viability were determined with GraphPad Prism software.[4]

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The BTK pY223 cellular assay was conducted in Ramos cells. Cells were serum-starved overnight, treated with compounds for 3 hours, and then stimulated with pervanadate for 20 minutes to induce phosphorylation. Cells were lysed, and lysates were mixed with an HTRF detection antibody mixture (anti-BTK-d2 and anti-pBTK-K). After overnight incubation, fluorescence emission at 665 nm and 620 nm was measured to calculate the inhibition of phosphorylation [4]
Cell viability assays for Rec-1 and OCI-LY10 cells were performed using a luminescent ATP detection assay. Cells were treated with compounds for 6 days. An equal volume of detection reagent was added to lyse cells and generate a luminescent signal proportional to viable cell number [4]
The EGFR pY1068 cellular assay was performed in A431 cells. Cells were treated with compounds for 1 hour, stimulated with EGF for 10 minutes, lysed, and then analyzed using an HTRF detection kit specific for phosphorylated EGFR [4]
The ITK p-PLCγ1 assay was performed in Jurkat cells. Cells were treated with compounds for 2 hours, stimulated with hydrogen peroxide, lysed, and analyzed by western blot using antibodies against total and phosphorylated PLCγ1 [4]
The TEC p-TEC assay used TEC-overexpressing HEK293 cells. Serum-starved cells were treated with compounds for 3 hours, stimulated with pervanadate, lysed, and analyzed using an electrochemiluminescence-based immunoassay (Meso Scale Discovery) with plates coated with an anti-TEC capture antibody and a sulfotag-labeled anti-phosphotyrosine detection antibody [4]

Pharmacokinetics (PK) study in rats: [4]
Sprague-Dawley rats were housed in temperature (20-25°C) and humidity (40-70%) controlled facility with 12-hour light/dark cycle. The rats were sterilized water. The animals were fasted overnight prior to dosing until 4 hours post dosing. Water was not restricted during the study. Blood samples (0.15 mL) were collected via jugular vein cannula into dry heparinized (coated with 0.25% heparin in saline) tubes. For the IV administered groups, the sampling time points were pre-dose, 5, 15, 30, 45 min, 1, 2, 3, 4 and 6 h post-dose. For the oral administered groups, the sampling time points were pre-dose 15, 30 min, 1, 2, 3, 4, 6, 8, 12, and 24 h post-dose. Plasma samples were separated via centrifugation at 5500 rpm for 10 min, and stored in -30°C freezer until analysis. The plasma samples were analysis using LC-MS/MS with low limit of quantification (LLOQ) at 1 ng/mL. All PK parameters were calculated with non-compartment analysis using Phoenix WinNonlin 6.3.

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Plasma protein binding: [4]
The plasma protein binding of compounds was measured using equilibrium dialysis with HTDialysis equilibrium dialysis chamber apparatus. In brief, an aliquot of compounds spiked plasma was added to the donor side of each designated well. An equal volume of phosphate buffer (0.002% Tween 80) was added to the receiver side. The plate was covered with adhesive sealing film to prevent evaporation and placed in a water bath at 37ºC for 5-8 h with a shaking speed at 80 rpm to reach protein binding equilibrium. Samples were taken from both sides, and analyzed using LC-MS/MS. The value of unbound fraction (fu) was determined by dividing the compounds concentration on the buffer side of the equilibrium dialysis chamber by the compounds concentration on the plasma side.[4]

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In mouse BTK occupancy assays, treatment with BGB-3111 resulted in a dose-dependent BTK occupancy and showed about 3-fold more potency than ibrutinib in target organs, including PBMC and spleen. BGB-3111 induced dose-dependent anti-tumor effects against REC-1 MCL xenografts engrafted either subcutaneously or systemically via tail vein injection in mice. In the subcutaneous xenografts, BGB-3111 at 2.5 mg/kg BID showed similar activity as ibrutinib at 50 mg/kg QD, its clinical relevant dose. In the systemic model, the median survival of BGB-3111 25 mg/kg BID group was significantly longer than those of both ibrutinib 50 mg/kg QD and BID groups. In an ABC-subtype DLBCL (TMD-8) subcutaneous xenograft model, BGB-3111 also demonstrated better anti-tumor activity than ibrutinib. Preliminary 14-day toxicity study in rats showed that BGB-3111 was very well tolerated and maximal tolerate dose (MTD) was not reached when it was dosed up to 250mg/kg/day.[2]

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For pharmacokinetic (PK) studies in rats, Zanubrutinib was administered intravenously as a 1 mg/kg solution or orally as a 5 mg/kg suspension in methylcellulose. Blood samples were collected at various time points for plasma concentration analysis [4]
For pharmacodynamic (PD) studies in ICR mice, Zanubrutinib was administered orally via gavage as a solution or suspension in a volume of 10 mL/kg body weight. Mice were euthanized at specified times post-dose (e.g., 4 hours for dose-response, multiple time points for kinetics) to collect blood (for PBMCs and plasma) and spleen for BTK occupancy analysis [4]
For the OCI-LY10 xenograft efficacy study, female NCG mice were inoculated subcutaneously with tumor cells. When tumors were palpable, mice were randomized into groups and treated orally with Zanubrutinib or vehicle, twice daily (BID) at doses of 2.5 or 7.5 mg/kg for 28 days. Tumor dimensions and body weights were measured regularly [4]

Absorption, Distribution and Excretion
Following oral administration of zanubrutinib 160 mg twice daily and 320 mg once daily, the mean (%CV) steady-state concentrations of zanubrutinib were 2295 (37%) ng·h/mL and 2180 (41%) ng·h/mL, respectively. The mean Cmax (%CV) after 160 mg twice daily was 314 (46%) ng/mL, and the mean Cmax after 320 mg once daily was 543 (51%) ng/mL. The Cmax and AUC of zanubrutinib increased proportionally to the dose, with minimal systemic accumulation after repeated dosing. The median time to peak concentration (Tmax) was 2 hours. After oral administration of 320 mg of radiolabeled zanubrutinib, approximately 87% of the dose was excreted in feces, and approximately 8% was recovered in urine, of which less than 1% was unmetabolized parent drug. The geometric mean (%CV) of the apparent steady-state volume of distribution (Vd) was 881 (95%) L. The plasma concentration-to-dose ratio was approximately 0.7 to 0.8. The mean (%CV) of the apparent oral clearance (CL/F) of zanubrutinib was 182 (37%) L/h. Metabolites/Metabolites: Zanubrutinib is primarily metabolized via CYP3A4. Its metabolites are not yet known. Biological Half-Life: Following a single oral dose of 160 mg or 320 mg of zanubrutinib, the mean half-life is approximately 2 to 4 hours. In rats, after intravenous injection of 1 mg/kg zanubrutinib, the terminal half-life (T₁/₂) was 0.53 hours, the steady-state volume of distribution (Vss) was 2.32 L/kg, and the clearance (CL) was 76.8 mL/min/kg.
After oral administration of 5 mg/kg zanubrutinib, the time to peak concentration (Tmax) was 0.33 hours, the maximum concentration (Cmax) was 235 ng/mL, the area under the curve (AUC) was 257 h·ng/mL, and the oral bioavailability (F) was 23.6%.
The plasma protein-bound free fraction (fu) was species-dependent: 5.8% in humans, 6.7% in dogs, 3.3% in rats, and 5.1% in mice.
The intrinsic clearance of microsomes was high in humans (109 μL/min/mg) and rats (148 μL/min/mg), and moderate in dogs (25.6 μL/min/mg) and mice (67.0 μL/min/mg)[4].

Hepatotoxicity
In premarket clinical trials of zanubrutinib for mantle cell lymphoma, liver dysfunction was common but usually mild. Elevated alanine aminotransferase (ALT) levels were observed in 28% of participants, and elevated bilirubin levels were observed in 24%, but less than 1% of participants had ALT levels exceeding five times the upper limit of normal (ULN). In these trials involving over 600 patients, no clinically significant liver injury, premature discontinuation of treatment due to liver injury, or liver-related death were reported. However, other Bruton's kinase inhibitors (such as ibrutinib and acalabrutinib) have been associated with rare cases of acute liver injury, including acute liver failure. The latency period for liver injury caused by these drugs ranges from several weeks to nine months. Liver injury is typically hepatocellular and immunologically uncommon. Although the injury pattern is hepatocellular, the course is not typical of acute hepatitis-like injury, but rather resembles acute liver necrosis with early liver failure. Other Bruton's kinase inhibitors have also been associated with multiple cases of hepatitis B virus reactivation, which can be severe and even fatal. Probability score: E (Unproven, but suspected cause of clinically significant liver damage (including hepatitis B virus reactivation) in susceptible patients). Pregnancy and Lactation Effects ◉ Overview of Use During Lactation There is currently no information regarding the clinical use of zanubrutinib during lactation. Because zanubrutinib binds to plasma proteins at a rate of 94%, its concentration in breast milk may be low. The manufacturer recommends discontinuing breastfeeding during zanubrutinib treatment and for at least 2 weeks after the last dose. ◉ Effects on Breastfed Infants No published information was found as of the revision date. ◉ Effects on Lactation and Breast Milk No published information was found as of the revision date. Protein Binding The plasma protein binding rate of zanubrutinib is approximately 94%.

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A rat toxicity study showed that BGB-3111 was well tolerated, and the maximum tolerated dose (MTD) was not reached at doses up to 250 mg/kg/day. [3]
In an ongoing Phase I first-in-human trial in patients with advanced B-cell malignancies, no drug-related adverse events (AEs) or dose-limiting toxicities (DLTs) were reported as of the time of reporting. The maximum tolerated dose (MTD) has not been reached. [3]

[1]. Cancer Cell. 2015 Aug 10;28(2):225-39.

[2]. Cancer Res (2015) 75 (15_Supplement): 2597.

[3]. J Hematol Oncol . 2016 Sep 2;9(1):80.

[4]. J Med Chem . 2019 Sep 12;62(17):7923-7940.

Pharmacodynamics
Zanubrutinib is an immunomodulator that reduces the survival rate of malignant B cells. It inhibits BTK by binding to the active site of BTK. Its mechanism of action is to inhibit the proliferation and survival of malignant B cells, thereby reducing the tumor volume of mantle cell lymphoma. BGB-3111 is a second-generation, highly selective, and potent BTK inhibitor currently under clinical development. [3] BGB-3111 is being developed as a potential treatment for B-cell malignancies. [3] A first-in-human, open-label, phase I clinical trial of BGB-3111 in patients with advanced B-cell malignancies is underway, using a modified 3+3 dose escalation design (40, 80, 160, 320 mg orally once daily; 160 mg orally twice daily). Preliminary results showed an objective response rate of 64% (16/25) in 25 patients, including 1 complete response (CR) and 6 stable disease (SD). [3]

C27H29N5O3

471.56

471.227

C, 68.77; H, 6.20; N, 14.85; O, 10.18

1691249-45-2

(±)-Zanubrutinib;1633350-06-7;(R)-Zanubrutinib;1691249-44-1;Zanubrutinib-d5

135565884

White to off-white solid powder

1.3±0.1 g/cm3

713.4±60.0 °C at 760 mmHg

385.2±32.9 °C

0.0±2.3 mmHg at 25°C

1.680

3.64

2

5

6

35

756

1

O=C(C=C)N1CCC(CC1)[C@]1([H])CCNC2=C(C(N)=O)C(C3C=CC(=CC=3)OC3C=CC=CC=3)=NN12

RNOAOAWBMHREKO-QFIPXVFZSA-N

InChI=1S/C27H29N5O3/c1-2-23(33)31-16-13-18(14-17-31)22-12-15-29-27-24(26(28)34)25(30-32(22)27)19-8-10-21(11-9-19)35-20-6-4-3-5-7-20/h2-11,18,22,29H,1,12-17H2,(H2,28,34)/t22-/m0/s1

(7S)-2-(4-phenoxyphenyl)-7-(1-prop-2-enoylpiperidin-4-yl)-4,5,6,7-tetrahydropyrazolo[1,5-a]pyrimidine-3-carboxamide

2934.99.9001

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (4.41 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 20.8 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 2: ≥ 2.08 mg/mL (4.41 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 20.8 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 3: ≥ 2.08 mg/mL (4.41 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)