2025 Volume 58 Issue 2 Pages 93-100
Ibrutinib is an oral irreversible Bruton’s tyrosine kinase (BTK) inhibitor that blocks BTK activity by forming covalent bonds with the thiol group of cysteine in the ATP-binding pocket via Michael addition. However, it also reacts with a variety of off-target nonspecific proteins. In this study, we attempted to generate a specific antibody against ibrutinib and develop an immunohistochemical method to detect the ibrutinib-protein conjugates. Ibrutinib has the same amino group as the nucleobase adenine. Paraformaldehyde fixation could not fix it to the tissue via this amino group. However, ibrutinib covalently binds to proteins such as BTKs to exert its action and is therefore immobilized in tissue as ibrutinib-protein conjugates. Thus, immunohistochemistry for ibrutinib detects the location of the ibrutinib-protein conjugates, that is, the sites of covalently bound to the tissue via Michael addition. Using this immunohistochemical method, we visualized the ibrutinib-protein conjugates in the rat gastrointestinal tract (gastric body, duodenum, jejunum, ileum, and colon). This study is the first to elucidate the location of the ibrutinib-protein conjugates in the rat gastrointestinal tract and helps to clarify the mechanism of ibrutinib-induced toxicity.
Bruton’s tyrosine kinase (BTK) is a non-receptor kinase that plays a role in the signal transduction of B cell antigen receptor, which is involved in the differentiation and proliferation of B cells [1, 8]. Ibrutinib, which exerts pharmacological action by inhibiting BTK, was the first BTK inhibitor approved by the United States Food and Drug Administration as a breakthrough therapy for chronic lymphocytic leukemia in 2013 [17]. Ibrutinib possesses an α,β-unsaturated ketone moiety and is designed to inhibit BTK by forming an irreversible covalent bond with the SH group at cysteine 481, close to the ATP-binding site of BTK, via the Michael addition reaction [7]. Therefore, the activity of ibrutinib is strong and persistent, and its off-target interactions with various nonspecific proteins are well established [3]. Adverse effects of ibrutinib include gastrointestinal disorders, skin disorders, bone marrow suppression, liver dysfunction, and atrial fibrillation, which can lead to dose reduction or withdrawal [11, 13]. In addition to BTK, ErbB family kinases and TEC family kinases have a cysteine residue close to the ATP-binding site [7]. Inhibition of kinase activity by ibrutinib is thought to cause several of the adverse effects [3]. However, the cells in which kinase activity is inhibited are unclear, and elucidation of these cells would enable a more detailed investigation of the mechanisms underlying the adverse effects.
Immunohistochemistry (IHC) generally requires that antigens be fixed to tissue. The fixation is achieved by cross-linking the amino groups of an antigen with those of the tissue protein using a fixative such as formalin. Ibrutinib has the same amino group as the nucleobase adenine. Previous investigations demonstrated that formaldehyde-induced adducts, such as methylol (hydroxymethyl) groups and methylene bridge cross-links on the amine moiety of an adenine base, were reversible in model systems [4]. In addition, these formaldehyde adducts are mostly hydrolyzed by heating for 30 min at 70°C in diluted Tris, phosphate, or similar buffer (pH 8), breaking the formaldehyde cross-links [4]. A similar reaction mechanism would apply to ibrutinib paraformaldehyde fixation. In general, IHC protocols involve deparaffinization (60°C, 60 min), treatment with 2 M HCl (room temperature, 30 min) and treatment with 6% hydrogen peroxide (room temperature, 30 min), etc. Presumably, these treatments would hydrolyze the ibrutinib-paraformaldehyde adducts. Therefore, irreversible paraformaldehyde fixation of ibrutinib itself to the tissue would be impossible. However, ibrutinib is covalently bound to proteins such as BTKs to exert its action and is therefore immobilized in the tissue as ibrutinib–protein conjugates. Thus, IHC for ibrutinib detects the location of the ibrutinib–protein conjugates, that is, the sites of covalently bound to the tissue via Michael addition. We have already developed IHC for afatinib [19], dacomitinib [20], and osimertinib [21], which act by covalently binding to target kinases such as ibrutinib. Using these immunohistochemical methods, we have successfully visualized the action sites of these drugs in rat tissue. However, IHC for ibrutinib has not been developed. Detection of the ibrutinib-protein conjugates would be very useful for studies of ibrutinib action and toxicity.
In the present study, we developed an immunohistochemical method in which a specific antibody against ibrutinib is used to detect the ibrutinib-protein conjugates. Using this method, we visualized the ibrutinib-protein conjugates in the rat gastrointestinal tract (gastric body, duodenum, jejunum, ileum, and colon).
Ibrutinib was purchased from AvaChem Scientific (San Antonio, TX). (R)-3-(4-Phenoxyphenyl)-1-(piperidin-3-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PPPA) was obtained from BLD Pharmatech (Cincinnati, OH). Tirabrutinib and zanubrutinib were purchased from Selleck Chemicals (Tokyo, Japan). 3,3',5,5'-Tetramethylbenzidine (TMB) was bought from Sigma-Aldrich (Ingelheim, Germany). 3,3'-Diaminobenzidine tetrahydrochloride (DAB) was purchased from Jun Seikagaku (Tokyo, Japan). As the enzyme-labeled secondary antibody, Histofine Simple Stain MAX PO (M) was obtained from Nichirei Bioscience (Tokyo, Japan). Bovine serum albumin (BSA) and 1-ethyl-3,3-dimethyl-aminopropyl carbodiimide hydrochloride (EDC) were purchased from Fujifilm Wako Pure Chemicals (Osaka, Japan).
Preparation of antigen for ibrutinibThe ibrutinib antigen was prepared using PPPA, a substructure of ibrutinib (Fig. 1). PPPA (10 mg, 25.9 μmol) was dissolved in 200 μL of pyridine. Succinic anhydride (2.5 mg, 25.0 μmol) was then dissolved in the solution and incubated overnight at 60°C. The pyridine solution was volatilized with nitrogen, and the remaining reaction product was dissolved in 200 μL of 95% 1,4-dioxane. Next, N-hydroxysuccinimide (3.0 mg, 26.0 μmol) and EDC (25 mg, 130.4 μmol) were added and allowed to react for 2 hr at room temperature. To the reaction solution, 1 mL of 0.1 M phosphate buffer (pH 7.5) in which BSA (10 mg, 0.15 μmol) was dissolved was added and the reaction was incubated for 2 hr at room temperature with stirring. Ibrutinib antigen was obtained by dialysis four times with 1 L of 0.01 M phosphate buffer (pH 7.5). The number of moles of PPPA bound to BSA was calculated by the 2,4,6-trinitrobenzenesulfonic acid method [6]. Based on the decrease in the primary amine, about 38 ibrutinib moles were coupled to each mole of BSA.
Preparation of the antigen for ibrutinib.
For primary immunization, 0.1 mg of ibrutinib antigen emulsified with Freund’s complete adjuvant was intraperitoneally administered to 5-week-old female BALB/c mice (Kyudo Exp. Animals, Kumamoto, Japan). Subsequently, 0.05 mg of the complex emulsified with Freund’s incomplete adjuvant was administered three times every 2 weeks. Seven days after the last dose, blood was drawn from the axillary vein of the mice. The blood was centrifuged at 1,048 × g for 10 min at 4°C to obtain antiserum. The antiserum was deactivated at 55°C for 30 min. Sera obtained by blood sampling were stored at −20°C, and the antibody titer and specificity were confirmed by enzyme-linked immunosorbent assay (ELISA).
Evaluation of the anti-ibrutinib antibody titerThe anti-ibrutinib antibody titer was confirmed by dilution ELISA [5]. Microtiter plate wells were coated with 100 μL of ibrutinib antigen (10 μg/mL) for 60 min at 37°C. The wells were then blocked with 1% skimmed milk for 30 min at room temperature. A five-fold dilution series of anti-ibrutinib serum was prepared using 10 mM PBS containing 1% BSA (PBS-BSA), and 100 μL was added to each well and incubated at 37°C for 90 min. The wells were washed three times with PBS-BSA, and Simple Stain Rat MAX PO (M) (1:1000; 100 μL) was then added and further incubated at 37°C for 60 min. The wells were again washed three times with PBS-BSA and the activity of the enzyme was measured by the addition of 100 μL of 0.42 mM TMB in 0.05 M acetate-citric acid buffer (pH 5.5) containing 0.01% H2O2 and incubation of the wells for 30 min at 37°C. The reaction was stopped by adding 50 μL of 1 M H2SO4, and the absorbance at 450 nm was measured using a MultiskanTM FC ELISA reader (Thermo Fisher Scientific, Inc., Waltham, MA).
Anti-ibrutinib antibody cross-reactivity by inhibition ELISAThe specificity of the anti-ibrutinib antibody was confirmed by inhibition ELISA [5]. Microtiter plate wells were coated with 100 μL of the ibrutinib antigen (10 μg/mL) for 60 min at 37°C. The wells were then blocked with 1% skimmed milk for 30 min at room temperature. After three washes with PBS-BSA, 50 μL of anti-ibrutinib antiserum diluted 1:10000 in PBS-BSA and 50 μL of step-diluted ibrutinib analogues were added to the wells and subjected to a competitive reaction for 3 hr at 37°C. The wells were washed three times with PBS-BSA, and Simple Stain Rat MAX PO (M) (1:1000; 100 μL) was then added and further incubated at 37°C for 60 min. The wells were washed three times with PBS-BSA and the activity of the enzyme was measured by the addition of 100 μL of 0.42 mM TMB in 0.05 M acetate-citric acid buffer (pH 5.5) containing 0.01% H2O2 and incubation of the wells for 30 min at 37°C. The reaction was stopped by adding 50 μL of 1 M H2SO4, and the absorbance at 450 nm was measured using an ELISA reader. Cross-reactivity (%) was calculated from the 50% inhibitory concentration (IC50) of ibrutinib and each analogous compound by inhibition ELISA.
Tissue samplesNormal adult male Wistar rats (6–8 weeks old; Japan SLC, Inc., Shizuoka), weighing 180–200 g, were used in this study. Three rats were administered a single oral dose of 100 mg ibrutinib/kg body weight. The drug was suspended in methylcellulose (0.5%, m/v water) at a concentration of 100 mg/mL. No abnormalities were observed in these rats after drug administration. After 24 hr, the three rats were anesthetized with sodium pentobarbital (60 mg/kg; Abbott Laboratories, North Chicago, IL) and transcardially perfused with 2.5% heparin followed by a fresh preparation with 4% paraformaldehyde (PFA) in 10 mM phosphate buffer (pH 7.4). The gastric body, duodenum, jejunum, ileum, and colon were removed and fixed overnight in 4% PFA in 10 mM phosphate buffer (pH 7.4). Tissues were dehydrated with ethanol or xylene and embedded in paraffin. Three rats administered with saline and killed 24 hr after administration were used as controls.
Cross-linking of ibrutinib and BSA by paraformaldehydeWhether paraformaldehyde fixation of ibrutinib to the tissue via its amino group is possible was evaluated using BSA as a tissue protein substitute. Briefly, ibrutinib (1 mg, about 2.3 μmol) dissolved in DMF (100 μL) was mixed with BSA (1 mg, about 15 nmol) dissolved in 400 μL of 10 mM phosphate buffer (pH 7.0). The solution was added 500 μL of 4% PFA in 10 mM phosphate buffer (pH 7.0), and the entire mixture was incubated for 12 hr with slow stirring at room temperature. The reaction mixture was chromatographed on a Sephadex G-25 column (PD-10) from Cytiva (Tokyo, Japan) equilibrated with 0.1 M phosphate buffer (pH 7.0) to remove any small molecular compounds remaining. The ibrutinib-BSA fraction obtained was incubated for 1 hr at two different temperatures (at 60°C and at room temperature). Each treated ibrutinib-BSA fraction was used as a solid-phase antigen for dilution ELISA [5].
Immunohistochemical staining for ibrutinibImmunohistochemical staining for ibrutinib was performed according to our previously established immunohistochemical protocol for afatinib [19]. Briefly, paraffin-embedded samples of the gastric body, duodenum, jejunum, ileum, and colon were cut into 5-μm-thick sections. The paraffin sections were melted at 60°C for 60 min and the sections were treated with xylene and ethanol (100%, 95%, 90%, and 70%) and washed with 10 mM PBS. To inactivate endogenous peroxidase activity and reduce background staining, the sections were treated with 6% H2O2 at room temperature for 30 min. The slides were then washed three times with 10 mM PBS for 5 min each time. To restore the antigenicity of the drug in the nuclei of the cells [10], the slides were treated with 2 N HCl at room temperature for 30 min. After the slides were washed three times for 5 min with 10 mM PBS, 70 μL of blocking buffer (10% normal goat serum, 1% BSA, and 0.1% saponin in 50 mM Tris-buffered saline [TBS]) was applied to the sections and the slides were incubated at room temperature for 1 hr. Anti-ibrutinib serum was diluted 1:1000 in 10% normal goat serum, 0.25% BSA, and 0.1% saponin in 50 mM TBS, and 70 μL was applied to the slides and incubated overnight at 4°C. The absorbance control comprised 30 μg/mL ibrutinib added to the ibrutinib antibody solution. After an overnight reaction, the slides were washed three times with 50 mM TBS-0.5% Triton X-100 (TBST) and further washed twice with 50 mM TBS for 5 min. Then, 60 μL of Histofine Simple Stain was added to the slides and incubated for 2 hr at room temperature. After the reaction, the slides were washed three times with 50 mM TBST and 50 mM TBS for 5 min each. They were then immersed in 100 mL of DAB substrate (50 mM TBS containing 0.05% DAB and 0.012% H2O2) for 10 min at room temperature to develop the color and washed several times with distilled water. The slides were then dehydrated through an ethanol series (70%, 90%, 95%, and 100%) and immersed three times in xylene for 5 min each time. Finally, the sections were sealed in Marinol and observed under an optical microscope.
Ibrutinib forms a covalent bond with tissue proteins through its α,β-unsaturated ketone moiety. To recognize tissue protein-bound ibrutinib, it is necessary to create antibodies that recognize the 4-phenoxyphenyl moiety end of the ibrutinib structure. Thus, an ibrutinib antigen was bound to BSA carrier protein through the piperidine moiety of PPPA, part of the ibrutinib structure (Fig. 1). The resulting PPPA–BSA conjugate (ibrutinib immunogen) induced the formation of specific antibodies in each of five immunized mice. Dilution ELISA tested the binding activity of the anti-ibrutinib antibody. The results showed that the antibody exhibited strong binding activity for ibrutinib antigen even at a 300,000-fold dilution and little binding activity for BSA (Fig. 2). Thus, the anti-ibrutinib antiserum had a sufficient antibody titer.
Evaluation of the anti-ibrutinib serum titer. ELISA measurements of the binding of serially diluted anti-ibrutinib serum to a solid phase coated with PPPA–BSA (●) or BSA (○). Error bars represent the standard deviation with n = 3.
Inhibition ELISA tested the cross-reactivity of the anti-ibrutinib antibody. The cross-reactivity values were set as the ratio of each compound to ibrutinib at the concentration needed for 50% inhibition of anti-ibrutinib antibody binding to the coating ibrutinib antigen. The anti-ibrutinib antibody showed 100% cross-reactivity to PPPA as the hapten antigen, 3.6% to zanubrutinib, 2.4% to tirabrutinib, and 0.85% to 4-phenoxyaniline (Table 1). These results suggest that the anti-ibrutinib antibody recognizes at least the 4-phenoxyphenyl moiety to the 1H-pyrazolo[3,4-d]-pyrimidin-4-amine moiety in the ibrutinib structure. In other words, the anti-ibrutinib antibody was sufficiently specific for the structure of ibrutinib. Ibrutinib is mainly metabolized to dihydrodiol-ibrutinib by CYP3A4 [12], which is formed via epoxidation of the ethylene in the acryloyl moiety followed by hydrolysis to dihydrodiol. The cross-reactivity of dihydrodiol-ibrutinib has not yet been confirmed. Judging from the specificity of the anti-ibrutinib antibody, dihydrodiol-ibrutinib would be expected to show a similar cross-reaction to ibrutinib. However, dihydrodiol-ibrutinib does not have α,β-unsaturated carbonyl groups that can covalently bind to proteins. Therefore, the major metabolite dihydrodiol-ibrutinib is not fixed in tissue. These findings suggest that our immunohistochemical method is not affected by dihydrodiol-ibrutinib. Therefore, the anti-ibrutinib antibody was shown to have sufficient specificity to detect the target site of ibrutinib.
Specificity of anti-ibrutinib
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Whether paraformaldehyde fixation of ibrutinib to the tissue via its amino group is possible was evaluated using BSA as a tissue protein substitute. ibrutinib-BSA binding was evaluated by dilution ELISA to measure immunoreactivity. Fixation time in paraformaldehyde was 12 hr, as in actual tissue fixation. As shown in Fig. 3, mixing the ibrutinib solution with BSA and paraformaldehyde showed moderate immunoreactivity. However, the mixture showed hardly any immunoreactivity after heating at 60°C for 60 min in phosphate buffer. These results show that ibrutinib is temporarily cross-linked to BSA by paraformaldehyde via its amino group, which is hydrolyzed almost completely by heating at 60°C for 60 min, dissociating ibrutinib from BSA. This reaction is probably similar to the previously reported reaction mechanism of adenine [4]. This IHC protocols involve deparaffinization (60°C, 60 min), treatment with 2 M HCl (room temperature, 30 min) and treatment with 6% hydrogen peroxide (room temperature, 30 min), etc. As these treatments dissociate the amino group-mediated paraformaldehyde adducts of ibrutinib, our IHC likely detected only ibrutinib that was covalently bound to the tissue via Michael addition.
Evaluation of cross-linking of ibrutinib and BSA by paraformaldehyde. ELISA assay of the binding of serially diluted anti-ibrutinib serum to a solid-phase antigen coated with a PFA-treated ibrutinib-BSA fraction reacted at two different temperatures (open circles: 60°C, 1 hr. closed circles: room temperature, 1 hr). Each point represents the mean ± SD of three replicates.
Immunostaining of the rat gastrointestinal tract (gastric body, duodenum, jejunum, ileum, and colon) was performed 24 hr after the administration of a single oral dose of 100 mg ibrutinib/kg body weight. Ibrutinib was administered at approximately ten times the clinical dose. To demonstrate the specificity of the immunostaining method, two types of negative control experiments were performed. One comprised immunostaining using rat tissues that had not been administered ibrutinib. The second negative control was an absorption control, in which an excessive amount of ibrutinib (30 μg/mL) was added to the antibody solution before the antibody was added to the sections. These negative control experiments were all negative (Figs. 4C, 4D, 5C–F, 6D, 6E, and 7D, 7C), demonstrating that the immunostaining was specific for ibrutinib.
Immunohistochemistry for the sites of ibrutinib action in the rat gastric body. A: Immunostaining was performed in the rat gastric body collected 24 hr after a single oral dose of ibrutinib (100 mg/kg). B: High-magnification images of the boxed area of A. C: Untreated (not administered ibrutinib) rat gastric body. D: Absorption control for rat gastric body. Bars = 100 μm.
Immunohistochemistry for the sites of ibrutinib action in the rat duodenum and jejunum. A, B: Immunostaining was performed in the rat duodenum and jejunum collected 24 hr after a single oral dose of ibrutinib (100 mg/kg). C, D: Untreated (not administered ibrutinib) rat duodenum and jejunum. E, F: Absorption control for rat duodenum and jejunum. Bars = 100 μm.
Immunohistochemistry for the sites of ibrutinib action in the rat ileum. A: Immunostaining was performed with rat ileum collected 24 hr after a single oral dose of ibrutinib (100 mg/kg). B, C: High-magnification images of the boxed area of A. D: Untreated (not administered ibrutinib) rat ileum. E: Absorption control for rat ileum. Auerbach’s plexus (open arrow head). A, D, E: Bars = 100 μm; B, C: Bars = 50 μm.
Immunohistochemistry for the sites of ibrutinib action in the rat colon. A: Immunostaining was performed with rat colon collected 24 hr after a single oral dose of ibrutinib (100 mg/kg). B, C: High-magnification images of the boxed area of A. D: Untreated (not administered ibrutinib) rat colon. E: Absorption control for rat colon. A, D, E: Bars = 100 μm; B, C: Bars = 50 μm.
Immunostaining of gastric bodies revealed strong staining of the cytoplasm of superficial epithelial cells in the gastric gland (Fig. 4). The sites of ibrutinib-protein conjugates in the gastric corpus were concentrated in surface mucosal cells. ErbB family kinases are reported to be localized to surface mucosal cells, suggesting that ErbB family kinases are highly likely to be the target proteins of ibrutinib in the gastric corpus [2, 18]. Ibrutinib has been reported to cause adverse effects such as indigestion, abdominal pain, nausea, and vomiting [11, 13]. These results suggested that the influence of ibrutinib on surface mucosal cells is involved in the mechanisms underlying such adverse effects.
Immunostaining of the duodenum, jejunum, ileum, and colon demonstrated strong positive reactions in the cytoplasm of absorptive epithelial cells and lamina propria mucosa in all tissues (Figs. 5–7). Strong staining was also observed in the nuclei of absorptive epithelial cells in the ileum and colon (Figs. 6B, 7B). Furthermore, in all intestinal villi, positive reactions were concentrated at the tips. Leblond and Stevens (1948) reported that replacement of the epithelium in rats took 1.57 days in the duodenum and 1.35 days in the ileum [9]. Since our IHC results represent the time point 24 hr after drug administration, we assume that the immunoreactivity was concentrated at the villi tips due to intestinal epithelial turnover. In the ileum and colon, positive reactions were also evident in Auerbach’s plexus of the muscularis propria. The sites of ibrutinib action in the intestinal tract were concentrated in absorptive epithelial cells and lamina propria mucosa. ErbB family kinases are reported to be localized to absorptive epithelial cells [2, 15, 18], suggesting that ErbB family kinases are highly likely to be target proteins of ibrutinib in the intestinal tract. Diarrhea is reported by between 3% and 65% of ibrutinib-treated patients (0%–10% ≥ grade 3) [14, 16]. Thus, these results suggest that the effects of ibrutinib on absorptive epithelial cells and lamina propria mucosa are involved in the mechanisms underlying such adverse effects. All results mentioned above in this study were highly reproducible in the three rats.
Our developed immunohistochemical method successfully visualized sites of ibrutinib-protein conjugates in the rat gastrointestinal tract. Taken together, these findings suggest that ErbB family kinases are highly likely to be the target proteins of ibrutinib in the rat digestive tract. To investigate the mechanisms underlying these adverse effects in greater detail, it is necessary to identify the target proteins. Our immunohistochemical method can be used for tissues beyond the gastrointestinal tract, and it is anticipated to be useful for further investigating the mechanisms underlying the toxicity of ibrutinib, including skin disorders, liver dysfunction, and atrial fibrillation.
We succeeded in producing a specific polyclonal antibody with strong binding activity for ibrutinib and in using this antibody to develop an immunohistochemical method that is useful for detecting the sites of ibrutinib-protein conjugates in rat tissue. Using the developed immunohistochemical method for ibrutinib, we visualized sites of ibrutinib-protein conjugates in the rat gastrointestinal tract (gastric body, duodenum, jejunum, ileum, and colon). This work is the first to elucidate the localization of sites of ibrutinib-protein conjugates in the rat gastrointestinal tract and is expected to help to clarify the mechanism of ibrutinib-induced toxicity.
The authors declare no potential conflicts of interest.
All animal care and handling procedures were performed under the approved guidelines of the Animal Care and Ethical Review Committee of Sojo University (2024-L-006), Japan. All efforts were made to minimize animal suffering during the experiments.
