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 and it presents a Cmax, tmax and AUC of approximately 35 ng/ml, 1-2 hour and 953 mg.h/ml respectively.

Route of Elimination
The cumulative excretion of ibrutinib in urine is of about 7.8% of the administered dose and most of this excretion is found during the first 24 hours after administration. In feces, the cumulative excretion accounts for 80% of the administered dose and the excretion occurs within 48 hours of the initial administration. The total excretion of ibrutinib during the first 168 hours after initial administration accounts for 88.5% of the administered dose.

Volume of Distribution
The volume of distribution at steady-state of ibrutinib is in approximately 10,000 L.

Clearance
In patients with normal renal function, the clearance rate is in the range of 112-159 ml/min.

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Metabolism / Metabolites
Three metabolic pathways have been identified according to the possible metabolites. These pathways are the hydroxylation of the phenyl group (M35), the opening of the piperidine with a reduction of the primary alcohol (M34) and the oxidation to a carboxylic acid and epoxidation of the ethylene followed by a hydrolysis to the formation of dihydrodiol (PCI-45227). The latter metabolite presents also 15 times lower inhibitory activity against BTK. The metabolism of ibrutinib is mainly performed by CYP3A5 and CYP3A4. and in a minor extent it is seen to be performed by CYP2D6.

Since 2014, Ibrutinib has been available as a new drug for the treatment of leukemic diseases. Ibrutinib (Imbruvica) is metabolized in the liver mainly by the isoenzyme CYP3A4 and to a minor extent by CYP2D6. Simultaneous application of Imbruvica and consumption of foods containing secondary metabolites strongly inhibiting the CYP3A4 isoform, could significantly influence the toxicity of this drug. This article references the respective foods.

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Biological Half-Life
The elimination half-life of ibrutinib is of approximately 4-6 hours.

Toxicity Summary
IDENTIFICATION AND USE: Ibrutinib is an oral tyrosine kinase inhibitor that irreversibly binds and inhibits tyrosine-protein kinase BTK (Bruton tyrosine kinase). BTK is important in the function of B-cell receptor signaling and therefore in the maintenance and expansion of various B-cell malignancies. Targeting BTK with ibrutinib is an effective strategy in treating these malignancies. Ibrutinib, marketed as Imbruvica, 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 chronic lymphocytic leukemia (CLL) with 17p deletion and patients with Waldenstrom's macroglobulinemia (WM). HUMAN EXPOSURE AND TOXICITY: Studies in humans have shown that ibrutinib may enhance chemoimmunotherapy efficacy without additive toxicities. Ibrutinib is cytotoxic to malignant plasma cells from patients with multiple myeloma (MM) and furthermore treatment with ibrutinib significantly augments the cytotoxic activity of bortezomib and lenalidomide chemotherapies. Hypersensitivity reactions including anaphylactic shock (fatal), urticaria, and angioedema have been reported. The outcome of patients with MCL who experience disease progression following ibrutinib therapy is poor, with both low response rates to salvage therapy and short duration of responses. ibrutinib inhibited the proliferation and induced apoptosis of Germinal center B-cell like diffuse large B-cell lymphoma (GCB-DLBCL) cell lines through suppression of BCR signaling pathway and activation of caspase-3. Furthermore, the chemokines CCL3 and CCL4 production from tumor cells were also found to be attenuated by ibrutinib treatment. Different cell lines exhibited distinct sensitivity after ibrutinib treatment. Interestingly, the decreasing level of p-ERK after ibrutinib treatment, but not the basal expression level of Btk, correlated with different drug sensitivity. Ibrutinib could be a potentially useful therapy for GCB-DLBCL and the decreasing level of p-ERK could become a useful biomarker to predict related therapeutic response. Ibrutinib is well tolerated when added to R-CHOP (chemotherapy named after the initials of the drugs used: rituximab, cyclophosphamide, doxorubicin (hydroxydaunomycin), vincristine (Oncovin ), prednisolone), and could improve responses in patients with B-cell non-Hodgkin lymphoma. ANIMAL STUDIES: Ibrutinib caused malformations in rats at exposures 14 times those reported in patients with MCL and 20 times those reported in patients with CLL or WM, receiving the ibrutinib dose of 560 mg per day and 420 mg per day, respectively. Reduced fetal weights were observed at lower exposures.

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Hepatotoxicity
In the prelicensure clinical trials of ibrutinib in patients with CLL and mantle cell lymphoma, the rates of serum enzyme elevations during therapy were 20% to 30% but were similar to comparator arms, and elevations were generally mild (less than 5 times ULN) and self limited. In multiple controlled trials there were no reports of clinically apparent liver injury or need for early discontinuation because of hepatotoxicity. The major toxicities of ibrutinib resembled those of the tyrosine kinase receptor inhibitors and included hemorrhage and myelosuppression. While ibrutinib depressed peripheral lymphocyte counts and caused both lymphopenia and neutropenia, it has little effect on serum immunoglobulin levels and was not associated with reactivation of tuberculosis or opportunistic infections in prelicensure studies. Nevertheless, with approval and more widespread use of ibrutinib, rare cases of acute liver injury including acute liver failure and severe instances of reactivation of hepatitis B have been reported. The latency to onset of liver injury varied from several weeks to 9 months. The pattern of injury was hepatocellular, but the course was atypical of an acute hepatitis-like injury and more similar to acute hepatic necrosis with early onset of hepatic failure.
Likelihood score: D (possible rare cause of clinically apparent liver injury).

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Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
No information is available on the clinical use of ibrutinib during breastfeeding. Because ibrutinib is more than 97% bound to plasma proteins, the amount in milk is likely to be low. The manufacturer recommends that breastfeeding be discontinued during ibrutinib therapy and for 1 week after the last dose.

◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.

◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
Irreversible plasma protein binding increases gradually over time and reaches 25% of the administered dose 8 hours after initial administration. From the plasma proteins, ibrutinib has been shown to be mainly bound to albumin and to bind to α1 AGP. The irreversible protein binding of ibrutinib to plasma proteins can account for 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 (annotation 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

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.)