BTK (IC50=0.5 nM)

B-cell activity and signaling are specifically inhibited by imatinib (PCI-32765). It prevents Btk (IC50=11 nM) from autophosphorylating, Btk's physiological substrate PLCγ (IC50=29 nM) from being phosphorylated, and ERK (IC50=13 nM), a further downstream kinase, from being phosphorylated[1]. BCR-activated primary B cell growth is inhibited by imatinib (PCI-32765) (IC50=8 nM). Ibrutinib (PCI-32765) suppresses the production of TNFα, IL-1β, and IL-6 in primary monocytes after FcγR stimulation (IC50=2.6, 0.5, and 3.9 nM, respectively)[3]. Cysteine481, or C481 of BTK, is bound by imatinib, with an optimal IC50 of 0.5 nM. The hydroxyl group of serine is incompatible with imatinib, and the C481S mutation raises the IC50 against BTK-C481S phosphorylation from 2.2 nM to 1 μM

In mice with collagen-induced arthritis, ibrutinib (PCI-32765) (3.125–50 mg/kg, po) totally suppresses the disease and lowers the amount of circulating autoantibodies. In the MRL-Fas(lpr) lupus model, imatinib (PCI-32765) prevents the formation of autoantibodies and the progression of kidney disease. In MRL-Fas(lpr) mice, ibrutinib (PCI-32765) (3.125–50 mg/kg, po) ameliorates renal disease and autoantibody production[1]. When compared to T cells, Ibrutinib (PCI-32765) (0.1 μM) selectively cytotoxically affects B cells, but it modifies the production of cytokines by activated T cells. It also inhibits the proliferation of CLL cells when activated. In a therapeutic CIA model, ibrutinib (PCI-32765) with an ED50 of 2.6 mg/kg/day potently and dose-dependently reverses arthritic inflammation. Clinical arthritis is also prevented in CAIA models by ibrutinib (PCI-32765)[3].

After incubating with kinase, 33P-ATP, Ibrutinib, and substrate [0.2 mg/mL poly (EY) (4:1)] for 1 hour, the in vitro kinase IC50 values were measured using a 33P filtration binding assay.

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B-cell receptor (BCR) signaling is aberrantly activated in chronic lymphocytic leukemia (CLL). Bruton tyrosine kinase (BTK) is essential to BCR signaling and in knockout mouse models its mutation has a relatively B cell-specific phenotype. Herein, we demonstrate that BTK protein and mRNA are significantly over expressed in CLL compared with normal B cells. Although BTK is not always constitutively active in CLL cells, BCR or CD40 signaling is accompanied by effective activation of this pathway. Using the irreversible BTK inhibitor PCI-32765, we demonstrate modest apoptosis in CLL cells that is greater than that observed in normal B cells. No influence of PCI-32765 on T-cell survival is observed. Treatment of CD40 or BCR activated CLL cells with PCI-32765 results in inhibition of BTK tyrosine phosphorylation and also effectively abrogates downstream survival pathways activated by this kinase including ERK1/2, PI3K, and NF-κB. In addition, PCI-32765 inhibits activation-induced proliferation of CLL cells in vitro, and effectively blocks survival signals provided externally to CLL cells from the microenvironment including soluble factors (CD40L, BAFF, IL-6, IL-4, and TNF-α), fibronectin engagement, and stromal cell contact. Based on these collective data, future efforts targeting BTK with the irreversible inhibitor PCI-32765 in clinical trials of CLL patients is warranted.[3]

B and T Cells. CD20+ B and CD3+ T cells were purified by negative selection (RosetteSep, >90% purity) from buffy coat PBMCs and viably frozen in 10% DMSO. Cells were thawed at 37 °C and maintained in growth media (RPMI media containing 10% FCS). B cells were stimulated with goat antihuman IgM F(ab′)2 (10 μg/mL; Invitrogen) and T cells were stimulated with anti-CD3/CD28 coated beads (Dynabeads) at a 1:1 bead/cell ratio. Cells were stained with PE-CD69 (BD Biosciences) and analyzed by flow cytometry, gating on viable lymphocytes. PCI-32765 at concentrations lower than 10 μM did not decrease B- or T-cell viability during the course of the experiment, although PCI-32765 did block the modest survival benefit of anti-IgM stimulation in B cells. For washout experiments, cells were rinsed three times in 10 volumes of growth media, a protocol that was confirmed to completely wash away inhibition of BCR signaling by PCI-29732, a reversible Btk inhibitor.[1]

Arthritis and Lupus Models.[1]Male DBA/1 mice were immunized with type II collagen plus Freund adjuvant and boosted 21 d later. On a rolling basis, as significant swelling appeared in at least one paw, mice were enrolled and randomized. Ibrutinib (PCI-32765) or dexamethasone (0.2 mg/kg) were administered orally once per day for 11 d. Arthritis scores (0–5) were assigned to the mice based on the degree and extent of paw swelling. Mouse anti–type II collagen antibody and total IgG levels were measured by ELISA (Chondrex and Bethyl). Female MRL/MpJ-Faslpr mice received Ibrutinib (PCI-32765) by oral gavage once per day from week 8 through week 20. Proteinuria was monitored weekly. At week 20, serum was collected and analyzed for BUN (IDEXX) and mouse anti-dsDNA antibody levels. Kidney histology was scored according to established criteria. No drug-induced weight loss was observed at any of the dose levels tested. These studies were carried out at Boulder Biopath according to approved animal care protocols. Results are presented as the mean ± SEM. Statistical significance between groups were evaluated with repeated measures one-way ANOVA or one-way ANOVA using GraphPad Prism with Tukey or Bonferroni multicomparison posttest.

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Spontaneous Canine Lymphoma. [1]Spontaneous canine lymphoma studies were conducted with approval from the Colorado State University Institutional Animal Care and Use Committee and the Colorado State University Veterinary Medical Teaching Hospital Clinical Review Board. Client-owned dogs presenting as patients to the Colorado State University Animal Cancer Center were enrolled with the following inclusion criteria: (i) confirmed histologic or cytologic diagnosis of B-cell lymphoma (immunohistochemistry or flow cytometry for CD21 and CD79a or PCR for monoclonal Ig gene rearrangement), (ii) adequate organ function as indicated by standard laboratory tests, and (iii) modified Eastern Cooperative Oncology Group performance status of 0 or 1 on d 0. Exclusion criteria were (i) T cell or null-cell immunophenotype, (ii) chemotherapy within 3 wk, (iii) radiation therapy within 6 wk, and (iv) corticosteroids within 72 h. Signed informed consent was obtained from all owners before study entry. Ibrutinib (PCI-32765) was administered daily until disease progression with 40 mg and 200 mg hard gelatin capsules prepared using standard pharmaceutically acceptable excipients. Animals were rechecked weekly for 4 wk and then biweekly thereafter. Tumor burden was defined as the sum of the longest diameters of all target lesions. Response (complete response/partial response/stable disease/progressive disease) was evaluated according to Veterinary Cooperative Oncology Group criteria for assessment of response in peripheral nodal lymphoma in dogs, an adaptation of published RECIST criteria. Adverse events were recorded and prospectively graded according to the Veterinary Cooperative Oncology Group Common Terminology for Adverse Events, version 1.0. For pharmacodynamic analysis, blood was collected in CPT tubes and PBMCs purified using standard techniques. PBMC pellets were snap-frozen and stored at −80 °C. Tumor biopsies were stored at −80 °C and subsequently pulverized in PBS solution before analysis. PBMCs or tumor cells were lysed and 50 μg of soluble protein was labeled with PCI-33880 as described earlier.

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Male DBA1/1OlaHsd mice are injected on days 0 and 21 with Freunds' Complete Adjuvant containing bovine type II collagen. On days 21 to 35, mice are randomized into treatment groups when the average clinical score of each animal is 1.5 (in a scale of 5). Ibrutinib (PCI-32765) treatment (1.56-12.5 mg/kg, p.o.) is initiated following enrollment and continues for 18 days. Clinical scores are given to each mouse daily for each paw. Clinical score assessment is made using the following criteria: 0=normal; 1=one hind paw or fore paw joint affected or minimal diffuse erythema and swelling; 2=two hind or fore paw joints affected or mild diffuse erythema and swelling; 3=three hind or fore paw joints affected or moderate diffuse erythema and swelling; 4=marked diffuse erythema and swelling or four digit joints affected; 5=severe diffuse erythema and severe swelling of entire paw, unable to flex digits.[3]

Absorption
Ibrutinib is rapidly absorbed after oral administration, with peak plasma concentration (Cmax), time to peak concentration (tmax), and area under the curve (AUC) of approximately 35 ng/ml, 1–2 hours, and 953 mg·h/ml, respectively.
Elimination Routes
Approximately 7.8% of the administered dose is excreted cumulatively in the urine, with the majority of excretion occurring within 24 hours after administration. Approximately 80% of the administered dose is excreted cumulatively in the feces, primarily within 48 hours after the first administration. Within 168 hours after the first administration, approximately 88.5% of the administered dose is excreted.
Volume of Distribution
The steady-state volume of distribution of ibrutinib is approximately 10,000 liters.
Clearance
In patients with normal renal function, clearance ranges from 112 to 159 mL/min.
Metabolisms/Metabolites
Three metabolic pathways have been identified based on possible metabolites. These pathways include phenyl hydroxylation (M35), piperidine ring-opening and reduction of the primary alcohol (M34), and oxidation to a carboxylic acid and ethylene epoxidation, followed by hydrolysis to a dihydrodiol (PCI-45227). The latter metabolite also showed a 15-fold reduction in BTK inhibitory activity. Ibrutinib is primarily metabolized by CYP3A5 and CYP3A4, with a smaller role in CYP2D6 metabolism. Ibrutinib has been marketed as a new drug for the treatment of leukemia since 2014. Ibrutinib (trade name: Imbruvica) is primarily metabolized in the liver by the CYP3A4 isoenzyme, with a small amount metabolized by CYP2D6. Concomitant use of Imbruvica and consumption of foods containing secondary metabolites that strongly inhibit the CYP3A4 isoenzyme may significantly affect the drug's toxicity. This article lists relevant foods. Biological Half-Life: The elimination half-life of ibrutinib is approximately 4-6 hours.

Toxicity Overview
Identification and Use: Ibrutinib is an oral tyrosine kinase inhibitor that irreversibly binds to and inhibits tyrosine protein kinase BTK (Bruton's tyrosine kinase). BTK plays a crucial role in B-cell receptor signaling function and is therefore key in maintaining and expanding various B-cell malignancies. Targeting BTK with ibrutinib is an effective strategy for treating these malignancies. Ibrutinib (trade name: ibrutinib) is indicated for the treatment of patients with mantle cell lymphoma (MCL) or chronic lymphocytic leukemia (CLL) who have received at least one prior therapy. It is also indicated for the treatment of patients with CLL with 17p deletion and patients with Waldenström macroglobulinemia (WM). Human Exposure and Toxicity: Human studies have shown that ibrutinib enhances the efficacy of chemotherapy and immunotherapy without increasing toxicity. Ibrutinib is cytotoxic to malignant plasma cells in patients with multiple myeloma (MM). Furthermore, ibrutinib treatment significantly enhances the cytotoxic activity of bortezomib and lenalidomide chemotherapy. Reported hypersensitivity reactions include anaphylactic shock (fatal), urticaria, and angioedema. Patients with mantle cell lymphoma (MCL) whose disease progresses after ibrutinib treatment have a poor prognosis, with low response rates and short durations of remission with salvage therapy. Ibrutinib inhibits the proliferation of germinal center B-cell-like diffuse large B-cell lymphoma (GCB-DLBCL) cell lines and induces apoptosis by inhibiting the BCR signaling pathway and activating caspase-3. In addition, studies have found that ibrutinib treatment attenuates the production of chemokines CCL3 and CCL4 in tumor cells. Different cell lines exhibit varying sensitivities to ibrutinib. Interestingly, the decrease in p-ERK levels (rather than basal Btk expression levels) after ibrutinib treatment is associated with different drug sensitivities. Ibrutinib may be a potential treatment for GCB-DLBCL, and a decrease in p-ERK levels could be a potent biomarker for predicting treatment response. Ibrutinib was well-tolerated in combination with the R-CHOP regimen (chemotherapy regimens named alphabetically by the drugs used: rituximab, cyclophosphamide, doxorubicin (hydroxydaunorubicin), vincristine (vincristine), prednisolone) and improved treatment response in patients with B-cell non-Hodgkin lymphoma. Animal studies: In rats treated with 560 mg and 420 mg of ibrutinib daily, malformations occurred when ibrutinib exposure was 14 times the reported dose for patients with mantle cell lymphoma (MCL) and 20 times the reported dose for patients with chronic lymphocytic leukemia (CLL) or Waldenström macroglobulinemia (WM), respectively. Reduced fetal weight was observed at lower exposures.

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Hepatotoxicity
In premarketing clinical trials of ibrutinib in patients with CLL and mantle cell lymphoma, the incidence of elevated serum enzymes during treatment was 20% to 30%, similar to the control group, and the elevation was usually mild (less than 5 times the upper limit of normal) and resolved spontaneously. No clinically significant liver injury or premature discontinuation due to hepatotoxicity was reported in multiple controlled trials. The main toxicities of ibrutinib are similar to those of tyrosine kinase receptor inhibitors, including bleeding and myelosuppression. While ibrutinib reduces peripheral blood lymphocyte counts and causes lymphopenia and neutropenia, it has minimal effect on serum immunoglobulin levels and was not found to be associated with tuberculosis relapse or opportunistic infections in premarketing studies. However, with the approval and widespread use of ibrutinib, rare cases of acute liver injury have been reported, including acute liver failure and severe hepatitis B relapse. The latency period for liver injury ranges from several weeks to 9 months. The injury pattern is hepatocellular, but its course differs from typical acute hepatitis-like injury, resembling more acute hepatic necrosis with early liver failure.
Probability Score: D (Possibly a rare cause of clinically significant liver injury).

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Effects during Pregnancy and Lactation
◉ Overview of Use During Lactation
There is currently no information on the clinical use of ibrutinib during lactation. Because ibrutinib binds to plasma proteins at a rate exceeding 97%, its concentration in breast milk may be low. The manufacturer recommends discontinuing breastfeeding during ibrutinib treatment and for one week after the last dose.
◉ Effects on Breastfed Infants
No published information found as of the revision date.
◉ Effects on Lactation and Breast Milk
No published information found as of the revision date.

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Protein Binding
The irreversible binding rate of ibrutinib to plasma proteins gradually increases over time, reaching 25% of the administered dose 8 hours after the first dose. Studies have shown that ibrutinib primarily binds to albumin and also to α1-Acid glycoprotein (α1-AGP). Irreversible protein binding of ibrutinib to plasma proteins accounts for up to 97.3% of the administered dose.

[1]. Honigberg LA, et al. The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc Natl Acad Sci U S A. 2010 Jul 20;107(29):13075-80.
[2]. Herman SE, et al. Bruton tyrosine kinase represents a promising therapeutic target for treatment of chronic lymphocytic leukemia and is effectively targeted by PCI-32765. Blood. 2011 Jun 9;117(23):6287-96.
[3]. Chang BY, et al. The Bruton tyrosine kinase inhibitor PCI-32765 ameliorates autoimmune arthritis by inhibition of multiple effector cells. Arthritis Res Ther. 2011 Jul 13;13(4):R115.
[4]. Sun Y, et al. PROTAC-induced BTK degradation as a novel therapy for mutated BTK C481S induced ibrutinib-resistant B-cell malignancies. Cell Res. 2018 Jul;28(7):779-781

1-[3-[4-amino-3-(4-phenoxyphenyl)-1-pyrazolo[3,4-d]pyrimidinyl]-1-piperidinyl]-2-propen-1-one is an aromatic ether.
See also: ibrutinib (note moved to).

C₂₅H₂₄N₆O₂

440.50

440.196

936563-87-0

Ibrutinib;936563-96-1;Ibrutinib-d5;1553977-17-5

16126651

White to off-white solid powder

4.736

1

6

5

33

678

0

C=CC(=O)N1CCCC(C1)N2C3=NC=NC(=C3C(=N2)C4=CC=C(C=C4)OC5=CC=CC=C5)N

XYFPWWZEPKGCCK-UHFFFAOYSA-N

InChI=1S/C25H24N6O2/c1-2-21(32)30-14-6-7-18(15-30)31-25-22(24(26)27-16-28-25)23(29-31)17-10-12-20(13-11-17)33-19-8-4-3-5-9-19/h2-5,8-13,16,18H,1,6-7,14-15H2,(H2,26,27,28)

1-[3-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one

PCI-32765 Racemate; PCI32765 Racemate; 936563-87-0; PCI-32765 Racemate; Ibrutinib Racemate; PCI-32765 (Racemate); Ibrutinib (Racemate); 1-[3-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl]prop-2-en-1-one; PCI-32765 (Ibrutinib); 1-{3-[4-amino-3-(4-phenoxyphenyl)pyrazolo[3,4-d]pyrimidin-1-yl]piperidin-1-yl}prop-2-en-1-one; PCI 32765 Racemate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~25 mg/mL (~56.75 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.68 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 2: ≥ 2.5 mg/mL (5.68 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (5.68 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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 (Please use freshly prepared in vivo formulations for optimal results.)