Abstract Introduction: Cell division cycle 7 (CDC7) is a highly conserved serine/threonine kinase that plays a critical role in initiation of DNA replication and in replication stress checkpoint. Aberrant CDC7 expression has been implicated in various malignancies, highlighting its potential as a therapeutic target in cancer treatment. Consequently, CDC7 inhibitors are being explored as a novel class of anticancer agents. However, the mechanisms underlying cancer cell death induced by CDC7 inhibition are not fully elucidated. Monzosertib (AS-0141) is a potent, selective, and orally bioavailable CDC7 inhibitor, currently undergoing Phase I clinical trials in patients with solid and hematologic malignancies. In this study, we investigated the mechanisms of cell death induced by monzosertib in human cervical cancer cell lines by comparing cell lines with differential sensitivity to CDC7 inhibition. Method: Six human cervical cancer cell lines (HeLa, CaSki, ME-180, SKG-I, SKG-IIIa, and C33-A) were used to evaluate their sensitivity to CDC7 inhibitors including monzosertib and simurosertib (TAK-931). Cell viability was assessed by the resazurin assay. Cell cycle distribution and apoptosis were analyzed by flow cytometry. Protein and gene expression levels related to CDC7 and cell cycle regulation were analyzed by Western blotting and real-time PCR, respectively. FOXM1 knockdown was performed by introducing shRNA via lentiviral transduction. Result: We evaluated the antiproliferative activity of CDC7 inhibitors in six human cervical cancer cell lines. Among tested cell lines, HeLa cells were the most sensitive to CDC7 inhibition, whereas SKG-I cells exhibited the highest resistance. Treatment with monzosertib led to an increase in the sub-G1 population in these cell lines, depending on their sensitivity. Monzosertib increased the expression of the G2 and M-phase marker, cyclin B1 and phospho-CDK1 in HeLa cells in a dose-dependent manner, but not in SKG-I cells. Cell cycle analysis revealed that monzosertib induced cyclin B1 expression during the S-phase, which is characteristic of premature mitosis. These findings indicate that monzosertib-induced cell death appears to result from impaired mitotic progression. Since cyclin B1 expression was found to be elevated, we next examined the role of FOXM1, a transcription factor which regulates cyclin B1 gene expression. Treatment with monzosertib significantly increased FOXM1 expression in HeLa cells, whereas only a mild increase was observed in SKG-I cells. Knockdown of FOXM1 in HeLa cells resulted in reduced cyclin B1 expression and decreased sensitivity to CDC7 inhibition. Conclusion: We found that the FOXM1- cyclin B1 axis contributes to cell death in cervical cancer cells induced by the selective CDC7 inhibitor, monzosertib. Citation Format: Hiroko Endo, Yu Nishioka, Mariko Hatakeyama, Youichi Tajima, Sayuri Ito, Akinori Arimura, Hisao Masai, Masaaki Sawa. Monzosertib, a selective CDC7 inhibitor, induces cell death via premature mitosis promoted by the FOXM1-cyclinB1 axis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2925.

Abstract BTK inhibitors (BTKi) have become standard of care for treatment of patients with chronic lymphocytic leukemia (CLL). Covalent BTKi (cBTKi) such as ibrutinib, acalabrutinib, and zanubrutinib are effective but alterations in the kinase domain at C481 or BTK gatekeeper residue T474 mutations result in development of resistance. Noncovalent BTK inhibitors (ncBTKi) such as pirtobrutinib are effective in patients with C481x mutations developed through use of cBTKi. However, resistance to ncBTKi can occur owing to second site aberrations in BTK, generating novel mutations such as L528x and T316x. Sometimes, CLL cells with double BTK mutations are also observed. These BTK aberrations underscore a need for new inhibitors that target pan-BTK-mutants. We evaluated the efficacy of a new ncBTKi, docirbrutinib (AS-1763), against 14 BTK mutants, including C481S, T474x, and L528x, as well as gatekeeper and kinase domain double mutants, using biochemical assays, cell-line models, and primary CLL lymphocytes. Docirbrutinib potently inhibited BTK autophosphorylation and mutant BTK–driven cell proliferation, with greater effects than ibrutinib and pirtobrutinib against certain mutants. In treatment-naïve and relapsed/refractory CLL samples, docirbrutinib disrupted B-cell receptor signaling and sensitized cells to apoptosis induced by venetoclax and AZD5991. In a dose-escalation trial (NCT05602363), docirbrutinib decreased CCL3/CCL4 biomarkers and inhibited the B-cell receptor pathway signaling in longitudinal samples from patients with relapsed/refractory CLL. These findings establish docirbrutinib as a pan-mutant ncBTKi with potential to improve outcomes for CLL patients, including those with disease resistant to cBTKi and other ncBTKi.

Covalent and non-covalent Bruton's tyrosine kinase inhibitors (c/ncBTKis) are approved for the treatment of chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) and B-cell non-Hodgkin lymphoma (NHL), but their long-term efficacies are limited due to the off-target toxicities and the acquired resistance mutations in BTK such as C481x, T474I and L528W. Docirbrutinib (AS-1763) is a highly selective, pan-mutant ncBTKi, which inhibits both wild-type and various c/ncBTKi-resistant mutations including C481x, T474x and L528x with IC50 values of <10 nM (Kawahata et al. J Med Chem 2021; 64,14129-14141, Timofeeva et al. ASH2024). Docirbrutinib demonstrated strong anti-tumor activities in B-cell lymphoma cell lines harboring resistant BTK mutations including a kinase-dead BTK L528W (unpublished). Here we report the results of docirbrutinib from the ongoing Phase 1b study in B-cell malignancies (NCT05602363). This is a multicenter, open-label, Phase 1b study in patients (pts) with CLL/SLL or B-cell NHL who received ≥2 prior therapies including c/ncBTKis. The study includes 3+3 dose escalation and dose expansion parts. The dose expansion part consists of three cohorts; CLL/SLL pts are allocated to Cohort 1 and NHL pts to Cohort 2, both aimed at determining the recommended Phase 2 dose (RP2D), and CLL/mantle cell lymphoma (MCL) pts previously treated with a ncBTKi, pirtobrutinib, to Cohort 3 for exploratory efficacy evaluation. Each cohort includes 2-3 dose levels. Docirbrutinib was orally administered twice daily (BID). As of the 18 July 2025 cutoff date, 31 pts (19 CLL, 1 SLL, 4 follicular lymphoma [FL], 3 MCL, 1 marginal zone lymphoma [MZL], 3 Waldenström macroglobulinemia [WM]) were assigned to 5 dose levels; 100 (n=3), 200 (n=3), 300 (n=18), 400 (n=4), and 500 mg BID (n=3). Median age was 69 years (range, 46-86). The median number of prior lines of therapy for CLL was 4 (range, 2-7) including cBTKi (20/20 [100%]) and venetoclax (13/20 [63%]), and that for NHL was 4 (range, 2-6) including cBTKi (1/4 [25%] FL, 2/3 [67%] MCL, 1/3 [33%] WM) and pirtobrutinib (1/3 [33%] MCL). For 20 CLL/SLL pts, baseline genetic features include IGHV-unmutated (19/19, 100%), del(17p)/TP53 mutated (8/19, 42%), del(11q) (3/18, 17%), and BTK C481S mutation (5/19, 26%). Median treatment duration was 6.0 (range, 0.5-23.6) months. Docirbrutinib was well tolerated across all dose levels in the dose escalation part with no dose-limiting toxicity up to the highest dose level of 500 mg BID, and the maximum tolerated dose was not reached. For the dose expansion part, 300 mg BID was selected as a low dose level in Cohorts 1 and 2 and 400 mg BID in Cohort 3. The dose expansion part is currently ongoing to determine the RP2D. Among all 31 pts, no drug-related atrial fibrillation or hypertension was reported. Drug-related ≥G3 TEAEs reported were neutrophil count decreased in 2 pts, ALT/AST increased in 1 pt, anemia, hematoma and hemorrhagic shock in 1 pt on concomitant anticoagulation. Due to drug-related AEs, 1 pt temporarily experienced dose interruption/reduction (ALT/AST elevation), and 1 pt discontinued the study (hematoma). 24 pts (16 CLL, 3 FL, 2 MCL, 1 MZL, 2 WM) who had at least one response assessment after dosing were considered evaluable for efficacy across all dose levels. Of the 16 efficacy-evaluable CLL pts, 8 pts achieved partial response (PR) or PR with lymphocytosis (overall response rate [ORR] 50%), and 7 pts achieved stable disease with 4 of them remaining on treatment and showing 10.8-49.9% reduction in tumor size, with responses continuing to deepen. The median duration of response of the 8 CLL responders has not been reached, with 2 pts achieving >18 months and 2 pts >12 months. For 8 efficacy-evaluable NHL pts, 2 of 2 MCL pts achieved tumor response (ORR 100%, 1 complete response and 1 PR) and 1 of 2 WM pts achieved PR (ORR 50%,1 PR and 1 minor response). All MCL and WM pts enrolled are still on treatment. In the ongoing Phase 1b study, docirbrutinib demonstrated encouraging anti-tumor activity with a favorable safety profile and durable responses in CLL pts heavily treated with prior therapies including cBTKi and venetoclax and promising responses in MCL and WM pts. Updated data will be presented at the annual meeting.

Cell division cycle 7 (CDC7) is a highly conserved serine-threonine kinase that plays an important role in the initiation of DNA replication and cell cycle progression. Aberrant expression of CDC7 has been reported in many cancers and is thought to promote abnormal proliferation of cancer cells. Thus, CDC7 has been recognized as a potential drug target for treating cancers.Monzosertib (AS-0141) is a potent, selective, orally bioavailable small molecule inhibitor of CDC7, currently under Phase I clinical study in patients with advanced, metastatic, relapsed or refractory malignancies. In the previous studies, we demonstrated that monzosertib exerted strong synergy effects with DNA methyltransferase inhibitor (DNMTi; azacitidine or decitabine) or BCL2 inhibitor (venetoclax) in human acute myeloid leukemia (AML) cell lines.In this study, we evaluated the antitumor efficacy of monzosertib, DNMTi and venetoclax “triplet” therapy in human AML xenograft models. Fa-CI plot (the Chou-Talalay method) was used to assess combination effects of monzosertib with DNMTi and venetoclax in human AML cell lines. Effects of drug combination on cellular signaling pathway were examined by western blotting. In vivo antitumor activity of monzosertib alone or in triplet combination with DNMTi and venetoclax were evaluated in human AML tumor-bearing mice. Triplet combination of monzosertib with DNMTi (azacitidine or decitabine) and venetoclax resulted in a synergistic effect on antiproliferative activity in human AML cell lines. Treatment of MV4-11 cells (human AML cell line) with the triplet combination significantly increased in γH2AX, a biomarker of cellular response to DNA damage (DNA double strand breaks). Combining monzosertib with DNMTi (azacitidine or decitabine) and venetoclax resulted in enhanced tumor growth inhibition in a MV4-11 xenograft mouse model without body weight loss. Monzosertib demonstrated significant synergistic activity in combination with DNMTi (azacitidine or decitabine) and venetoclax in both in vitro and in vivo human AML models. The triplet combination of monzosertib with DNMTi (azacitidine or decitabine) and venetoclax was well-tolerated in these mouse AML models, suggesting the triplet therapy of monzosertib combined with the standard of care (DNMTi plus venetoclax) for AML patients may provide a new therapeutic opportunity in AML. Hatsuo Furuichi, Hiroko Endo, Akinori Arimura, Yu Nishioka, Masaaki Sawa. Triplet combination of monzosertib, a potent CDC7 inhibitor, with DNMT and BCL2 inhibitors is highly active in human AML xenograft mouse models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6867.

Background: Bruton's tyrosine kinase inhibitors are a standard therapy for chronic lymphocytic leukemia (CLL). Despite their efficacy, covalent BTK inhibitors suffer from drug resistance driven by mutation at C481 in BTK. To address this, noncovalent BTK inhibitors (ncBTKi) have been developed, however, resistance can still emerge through mutations such as T474I and L528W. These resistant mutations in BTK emphasize the importance of developing novel inhibitors capable of targeting multiple resistant variants. Previously, we demonstrated that docirbrutinib (AS-1763) is a potent and highly selective ncBTKi, orally available and effective against both wild-type (WT) and multiple resistant BTK mutants, including those resistances to covalent BTKi and ncBTKi. This work extends our previous findings by examining the efficacy of docirbrutinib in human diffuse large B-cell lymphoma (DLBCL) cell lines harboring BTK mutations, both alone and in combination with a BCL2 inhibitor. Study Design and Methods: We investigated the effects of docirbrutinib on BCR signaling using HEK293 or OCI-Ly10 cells expressing various BTK mutants. For L528W kinase dead mutant, we generated OCI-Ly10 cells overexpressing L528W mutant to hijack the BCR signaling. The off-rate of docirbrutinib was measured using the rapid dilution assay. Apoptosis was assessed in OCI-Ly10 cells harboring mutant BTK using a Annexin V Apoptosis Detection Kit, and data were acquired by flow cytometry. Synergistic effects between docirbrutinib and venetoclax in OCI-Ly10 cells were evaluated using isobologram analysis. Results: The enzymatic activity of BTK is regulated by upstream kinases, which phosphorylate Tyr551 and induce a conformational change of BTK. Compounds that bind to a Src-like inactive conformation of BTK can sequester Tyr551 from these kinases, thereby allowing Tyr551 to remain unphosphorylated. Docirbrutinib potently inhibited phosphorylation at Tyr551 as well as Tyr223, an autophosphorylation site, suggesting that docirbrutinib binds to a Src-like inactive conformation of BTK, as predicted by our docking model. Inhibitors that bind to the inactive conformation of kinases have previously been reported to exhibit slow off-rates. To investigate this property for docirbrutinib, we performed a rapid dilution assay. As anticipated, the rate of product formation following docirbrutinib treatment was markedly reduced, demonstrating that docirbrutinib acts as a slow off-rate inhibitor. The inhibitory potency of docirbrutinib against BTK mutants was evaluated in OCI-Ly10 cells harboring resistance mutations. Docirbrutinib demonstrated strong antiproliferative activity against BTK WT, C481S, and T474I mutants. Overexpression of the L528W mutant in OCI-Ly10 cells conferred resistance to both ibrutinib and pirtobrutinib; however, docirbrutinib retained its antiproliferative activity in these cells. This effect is likely attributable to the tight binding of docirbrutinib to the Src-like inactive conformation of BTK L528W, which may disrupt its scaffold function and thereby inhibit cell survival signaling. Given the known clinical synergy between BTK inhibitors and venetoclax, we investigated the synergistic antiproliferative effects of docirbrutinib and venetoclax in OCI-Ly10 cells using isobologram analysis. The results demonstrated a synergistic effect between docirbrutinib and venetoclax. We also assessed apoptosis induced by the combination of docirbrutinib and venetoclax in BTK-mutant OCI-Ly10 cells. The combination treatment resulted in a significant increase in apoptosis compared to either agent alone. In ex vivo treatment of three L528W-mutated CLL samples, the combination of docirbrutinib and venetoclax reduced cell mean viability more effectively than venetoclax alone at 72 hours (35.5% vs 50.3%). Conclusions: Docirbrutinib is a novel ncBTKi characterized by a slow off-rate that enables sustained BTK inhibition, potentially leading to prolonged therapeutic effects similar to those of cBTKi. Profiling across a panel of BTK mutants demonstrated that docirbrutinib is effective against both wild-type and multiple resistant BTK variants, including the kinase-dead L528W mutant. In combination with venetoclax, docirbrutinib induced significant cell death in BTK-mutant OCI-Ly10 cells and primary CLL. The detailed mechanism underlying these effects will be presented. Ongoing clinical trial is testing efficacy of docirbrutinib in CLL patients with WT or mutant BTK.

AbstractBruton's tyrosine kinase (BTK) is a potential therapeutic target for allergic and autoimmune diseases. This first‐in‐human phase I study evaluated safety, pharmacokinetic, and pharmacodynamic profiles of sofnobrutinib (formerly AS‐0871), a highly selective, orally available, non‐covalent BTK inhibitor, in healthy adult subjects. Single ascending doses (SAD; 5–900 mg) and multiple ascending doses (MAD; 50–300 mg twice daily [b.i.d.] for 14 days [morning dose only on Day 14]) of sofnobrutinib were tested. In the entire study, all adverse events (AEs) were mild or moderate, and no apparent dose‐proportional trend in severity or frequency was observed. No serious treatment‐emergent AEs, cardiac arrythmias, or bleeding‐related AEs were reported. In the SAD part, sofnobrutinib exhibited approximately dose‐dependent systemic exposures up to 900 mg with rapid absorption (median time to maximum concentration of 2.50–4.00 h) and gradual decline (mean half‐lives of 3.7–9.0 h). In the MAD part, sofnobrutinib showed low accumulation after multiple dosing (mean accumulation ratios of ≤1.54) and reached a steady state on ≤Day 7. Single dosing of sofnobrutinib rapidly and dose‐dependently suppressed basophil and B‐cell activations in ex vivo whole blood assays. Multiple dosing of sofnobrutinib achieved 50.8%–79.4%, 67.6%–93.6%, and 90.1%–98.0% inhibition of basophil activation during the dosing interval of 50, 150, and 300 mg b.i.d., respectively. Based on pharmacokinetic‐pharmacodynamic analysis, half‐maximal inhibitory concentration (IC50) of sofnobrutinib for basophil activation was 54.06 and 57.01 ng/mL in the SAD and MAD parts, respectively. Similarly, IC50 for B‐cell activation was 187.21 ng/mL. These data support further investigation of sofnobrutinib in allergic and autoimmune diseases.

Background : Covalent Bruton's tyrosine kinase inhibitors (cBTKis) are approved for the treatment of B-cell malignancies, but their long-term efficacies are limited due to the off-target toxicities and the acquired resistance mutations in BTK such as C481x. A non-covalent BTKi (ncBTKi), pirtobrutinib, was recently approved for chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL) after at least 2 lines of systemic therapies including cBTKis, but acquired resistance via BTK T474I and L528W mutations has been reported. Docirbrutinib (AS-1763) is a highly selective ncBTKi which inhibits both wild-type and various c/ncBTKi-resistant mutations including C481x, T474x and L528x (pan-mutant BTKi) with IC50 values of <10 nM in in vitro assays and demonstrated strong anti-tumor activities in cellular and animal studies (Kawahata et al. J Med Chem 2021; 64,14129-14141, Tantawy et al. ASH2023). Here we report the preliminary results of docirbrutinib monotherapy from the ongoing Phase 1b study in B-cell malignancies (NCT05602363). Methods: This is a multicenter, open-label, Phase 1b study in patients (pts) with B-cell malignancies who received ≥2 prior therapies, including cBTKis. Recently, the clinical protocol was amended to allow pts with prior ncBTKi. The study has 2 parts: 3+3 dose escalation and dose expansion. Docirbrutinib oral tablets were administered twice daily (BID). Dose-limiting toxicities (DLTs) were assessed during the first cycle (28 days). Results: As of 19 July 2024, 14 pts (9 CLL, 3 follicular lymphoma [FL], 1 MCL, 1 marginal zone lymphoma [MZL]) were enrolled to 5 dose levels in the dose escalation part: 100 mg BID (n=3; 3 CLL), 200 mg BID (n=3; 2 CLL, 1 FL), 300 mg BID (n=3; 2 CLL, 1 FL), 400 mg BID (n=3; 2 CLL, 1 MCL) and 500 mg BID (n=2; 1 FL, 1 MZL). Median age was 67 years (range, 46-77). Median lines of prior therapies were 4 (range, 2-5), including chemotherapy + anti-CD20 antibody (all pts [100%]), cBTKi (9/9 CLL [100%], 1/2 FL [50%]), and venetoclax (6/9 CLL [67%]). For the 9 pts with CLL, baseline genetic features include IgHV unmutated (9/9, 100%), deletion 17p (3/9, 33%), TP53 mutation (4/8, 50%), and BTK C481S (2/8, 25%). No DLTs were observed at doses up to 500 mg BID, and the maximum tolerated dose has not been reached yet. To date, among all 14 pts, no drug-related atrial fibrillation or bleeding was reported; neutropenia (2/14, 14%) and ALT/AST elevations (1/14, 7%) were the only drug-related ≥G3 adverse events (AEs). 1 pt experienced dose interruption due to asymptomatic liver enzyme elevations (G3). No treatment discontinuation due to drug-related AE occurred. Among 9 pts with CLL, 5 pts achieved partial response (PR) or PR with lymphocytosis (PR-L) and 3 pts showed stable disease (SD) with 16-45% reduction in tumor size. Among 3 efficacy-evaluable pts with NHL (2 FL and 1 MCL), 2 pts (1 FL, 1 MCL) showed SD. 2 pts with FL and MZL at 500 mg BID are awaiting the initial radiologic assessment at the end of Cycle 2. Currently available pharmacokinetic data up to 500 mg BID indicated the increase in exposures with doses, and the exposure exceeded the calculated IC90 for BTK at doses ≥300 mg BID throughout the dosing interval. Among 4 pts with CLL receiving ≥300 mg BID, 3 of 4 pts (75%) achieved PR or PR-L, and 1 pt showed SD with 45% tumor reduction. 11 of 15 pts continue on the study treatment with a median treatment duration of 4.9 months. Conclusion: The preliminary safety and efficacy data from the ongoing study of docirbrutinib in heavily pretreated pts with B-cell malignancies are encouraging. We plan to move forward to the expansion part to determine recommended phase 2 dose. Updated data will be presented at the annual meeting.

Abstract Introduction: Cell division cycle 7 (CDC7) is a highly conserved serine-threonine kinase that plays an important role in the initiation of DNA replication and cell cycle progression. Aberrant expression of CDC7 have been reported to cause uncontrolled proliferation of many cancer types, suggesting that CDC7 inhibitors may provide a great potential for the development of novel therapy for cancers. Monzosertib (AS-0141) is a potent, selective, orally bioavailable small molecule inhibitor of CDC7, and is currently being evaluated in Phase I study for the treatment of solid tumors. In a preclinical cancer cell panel study, monzosertib showed strong antiproliferative activities against a variety of cancer types, and acute myeloid lymphoma (AML) cell lines were found to be the most sensitive to monzosertib. As a single agent, treatment with monzosertib accumulates DNA damage in cancer cells and induces cell death. In this study, we aimed to investigate the antitumor effects of monzosertib alone and in combination with other anticancer drugs in AML models. Method: Antiproliferative activity of monzosertib was examined against a panel of 35 human cancer cell lines of various cancer types. DNA metyltransferase (DNMT) inhibitors (azacitidine or decitabine) and BCL2 inhibitor (venetoclax) were evaluated for their synergistic/antagonistic effects in combination with monzosertib against human AML cell lines (THP-1, HL-60, MV4-11, MOLM-14, TF-1, U-937 and NOMO-1). The combination index (CI) was calculated using the Chou-Talalay method. Flow cytometry assay was used to analyze apoptosis. To evaluate the in vivo efficacy, tumor-bearing mice were treated with monzosertib alone or in combination with venetoclax. Results: The combination of monzosertib with azacitidine, decitabine, or venetoclax resulted in a significant synergistic antiproliferative effects against AML cell lines in vitro. The flow cytometry assay indicated that azacitidine combination induced apoptosis and increased cell death in THP-1 cells. In vivo, oral administration of monzosertib demonstrated robust in vivo antitumor efficacy in a MV4-11 tumor bearing xenograft mouse model, both as a single agent and in combination with venetoclax. Conclusions: Monzosertib, a selective CDC7 inhibitor, demonstrated strong antiproliferative activity against human AML cell lines, both as a single agent and in combination with standard therapies. Monzosertib exerts synergistic antitumor effect with venetoclax in a human AML xenograft mouse model. These results suggest that monzosertib has a potential to enhance the antitumor efficacy of standard of care agents for AML patients. Citation Format: Hiroko Endo, Hatsuo Furuichi, Akinori Arimura, Yu Nishioka, Masaaki Sawa. Synergistic effect of the CDC7 inhibitor, monzosertib (AS-0141) with current therapies in AML models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5714.

Background: Non-covalent BTK inhibitors (ncBTKi), such as pirtobrutinib, have emerged as critical therapeutic options for patients (pts) with chronic lymphocytic leukemia (CLL). These ncBTKi are highly relevant as they provide an alternative mechanism of action, bypassing the common resistance associated with covalent BTK inhibitors (cBTKi) that target the C481 residue. However, non-C481 mutations have been identified as contributors to pirtobrutinib resistance, further emphasizing the need for effective ncBTKi therapies. At ASH 2023 our group presented data introducing a potent, highly selective, orally available ncBTKi, AS-1763 (docirbrutinib). Building on these foundational results, our current work expands the study by preclinical and clinical investigations. Study Design and Methods: We used cell free assay systems to evaluate selectivity and potency of docirbrutinib against 15 BTK-mutants including predicted potential double mutants. We further validated potency of docirbrutinib in cells by measuring inhibition of BTK autophosphorylation in BTK-mutant-transfected HEK293 cell lines. Biological and biochemical effects were tested in treatment-naïve (TN with BTK-wild type) and relapsed/refractory (R/R with BTK and/or Bcl-2 mutant) CLL cells during in vitro investigations with 0.01, 0.1, and 1 µM docirbrutinib alone or with Bcl-2i (venetoclax) or Mcl-1i (AZD5991). Finally, we investigated biomarkers in longitudinal samples obtained from pts during dose-escalation docirbrutinib clinical trial in R/R CLL (NCT05602363 Clinical Trials.gov). Results: We have generated a total of 15 recombinant BTK mutant proteins (C481, T474, L528, T316 variants and double mutants). Docirbrutinib showed potent inhibitory activities for those BTK mutants while inhibitory potencies of ibrutinib or pirtobrutinib were diminished against some BTK mutants such as C481 or T474 and L528, respectively. Docirbrutinib inhibited BTK autophosphorylation (pY223) in HEK293 cells transfected with those BTK mutants. In proliferation assays, docirbrutinib inhibited cell growth of c/ncBTKi-resistant OCI-Ly10 cells harboring mutant BTKs (C481S, T474I or L528V). In TN CLL lymphocytes from 11 CLL pts, 72-hr incubation with docirbrutinib induced modest yet significant apoptosis which was comparable to ibrutinib or pirtobrutinib. In parallel, B-cell activation, as measured by surface expression of CD86 in cells (n=14 CLL pts), was inhibited with 10 and 100 nM docirbrutinib (p=<0.01). Docirbrutinib effectively inhibited calcium release (n= 10 pts) even at 10 and 100 nM (p=<0.0001). At 1 µM, inhibition was similar to pirtobrutinib (p=0.47) but lower than ibrutinib (p=0.016). In concert, spontaneous (p=0.04) and CXCL12-induced (p=0.007) migration (n=9 pts) was lessened by docirbrutinib yielding results comparable to ibrutinib and greater than pirtobrutinib. Docirbrutinib mitigated phosphorylation of BTKTyr223 and PLCγ2Tyr1217 while reducing levels of MCL-1 and BCL-XL proteins. In R/R patient samples, docirbrutinib inhibited BCR pathway and inhibited cell viability in CLL lymphocytes containing C481S, C481R, L528W, or T474I mutants. Notably, in vitro incubation of R/R CLL cells (n=5) harboring either BTK and/or BCL-2 (G101V, D103E, A113G) mutations were sensitized by docirbrutinib to venetoclax- and AZD5991-induced cell death. Cell death was caspase-dependent and was blocked by QVD. Longitudinal analysis of samples from 3 pts enrolled in the docirbrutinib clinical trial revealed a decrease in plasma concentrations of CCL3/CCL4 during treatment, as well as reduced BTKTyr223 and PLCγ2Tyr1217 phosphorylation and decreased MCL-1 protein levels (Figure 1). One patient exhibited disease progression, accompanied by a resurgence of CCL3/CCL4 levels. Conclusions: Docirbrutinib, pan-mutant ncBTKi demonstrated equipotent activity against both wild-type and multiple BTK mutants in biochemical and cellular assays, as well as its capacity to disrupt BCR signaling pathway in BTK mutant cell lines. In both TN and R/R primary CLL cells, docirbrutinib effectively blocked BCR pathway signaling and increased sensitivity to Bcl-2 and MCL-1 inhibitors even with diverse BTK-mutant and Bcl-2 mutant backgrounds. In the dose-escalation trial, docirbrutinib treatment showed decreased BCR pathway biomarkers such as CCL3/CCL4 and phospho-BTK and PLCγ2. Updated data will be presented.

Background: Covalent Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib are approved for treating patients with B-cell malignancies, but their long-term efficacy is limited by toxicity related to off-target kinase inhibition and acquired resistance due to BTK C481 mutation. AS-1763 is a potent, highly selective, orally available, and non-covalent BTK inhibitor, equipotent against both wild-type and C481S-mutated BTK with sub-nanomolar IC50 values (Kawahata et al. J Med Chem 2021; 64,14129-14141). In a Phase 1 single-ascending dose study in healthy volunteers, AS-1763 was well tolerated and safe at doses of 5-600 mg and achieved maximum inhibition of B cell activation at doses of 100 mg and above at 1-2 h post-dose and the duration of inhibitory effect increased with dose [Arimura et al. AACR 2022; Cancer Res 2022;82(12_Suppl)]. In preclinical studies, AS-1763 demonstrated a broad spectrum of inhibition against BTK mutations that confer resistance to other non-covalent BTK inhibitors in addition to the C481 mutation. Study Design and Methods: This study is an open-label, multi-center, Phase 1b study of oral AS-1763 in patients with advanced chronic lymphocytic leukemia (CLL)/small lymphocytic leukemia (SLL) and other B-cell non-Hodgkin lymphomas (NHLs) who have failed or are intolerant to at least two prior lines of systemic therapy. Prior therapy with a covalent BTK inhibitor is permitted; prior use of a noncovalent BTK inhibitor is excluded. This study consists of two parts, i.e., dose escalation and expansion parts. The dose escalation part will follow a 3+3 design with a starting dose of 100 mg BID. Each cycle will be 28 days. Three dose levels will be selected for dose expansion part based on a comprehensive review of data from dose escalation. The dose expansion part consists of 2 cohorts, i.e., Cohort 1 for CLL/SLL and Cohort 2 for B-cell NHL. In each cohort, the first 30 patients will be allocated to the 3 dose levels (10 patients for each), then the same provisional recommended Phase 2 Dose (RP2D) will be selected for Cohort 1 and Cohort 2 after 30 patients have enrolled in either cohort. After the provisional RP2D is selected, up to 28 patients in Cohort 1 and up to 15 patients in Cohort 2 may be treated at the provisional RP2D. In this study, up to 110 patients will be enrolled. Treatment will continue for 24 cycles or until disease progression, occurrence of unacceptable toxicity or discontinuation because of other reasons. Eligible patients include CLL/SLL, Waldenstrӧm macroglobulinemia (WM), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), and follicular lymphoma (FL). Patients with transformed disease (e.g., Richter's transformation) prior to or during screening are not eligible. Key exclusion criteria include CNS involvement by systemic lymphoma, stem cell transplant or CAR-T therapy <30 days, and clinically significant cardiovascular disease. The primary objective of the dose escalation part is to determine the maximum tolerated dose/dose-limiting toxicity. Key secondary endpoints include the evaluation of safety profile and tolerability, pharmacokinetics properties, and preliminary anti-tumor activity based on overall response rate (ORR) by investigator according to iwCLL 2018 for CLL, IWW6 for WM, and Lugano Treatment Response Criteria for MCL, MZL, SLL and FL. The primary objective of the dose expansion part is to assess the preliminary anti-tumor activity based on ORR by the Safety Monitoring Committee (SMC). Key secondary objectives are to investigate safety, tolerability, and pharmacokinetic profiles and to assess the preliminary antitumor activity based on ORR by investigator and best overall response, duration of response, progression-free survival and overall survival by investigator and the SMC. This trial will evaluate several key exploratory objectives including evaluation of minimal residual disease negativity, characterization of patient subsets defined by mutation status of BTK and phospholipase C gamma 2 (PLCG2), and characterize BTK and PLGC2 gene mutation before and after disease progression. Changes in the level of biomarkers such as CCL3 and CCL4 chemokines and B-cell receptor pathway signaling will be evaluated prior to, during therapy, and at time of disease progression. The RP2D will be determined based on all the data generated in the study. The study has been registered on ClinicalTrials.gov (NCT05602363).

Background: Covalent Bruton's tyrosine kinase (BTK) inhibitors (cBTKi) have transformed treatment landscape of chronic lymphocytic leukemia (CLL). These inhibitors bind to C481 residue in the kinase domain of BTK. This is also the site for the most common mutations rendering cells resistant to cBTKi. To circumvent this limitation of the cBTKi, non-covalent BTKi (ncBTKi) such as pirtobrutinib have been developed. Recently, non-C481 BTK mutations have been reported in patients with CLL at the time of disease progression during pirtobrutinib treatment. These observations underscore the need for ncBTKi that can target C481 as well as non-C481 mutations of BTK. AS-1763 is a potent, highly selective, orally available, and ncBTKi, equipotent against both wild-type and C481S-mutated BTK when tested in biochemical assays. In vivo, AS-1763 demonstrated significant antitumor effects in OCI-LY10 tumor xenograft models harboring wild-type or C481S mutant BTK (Kawahata et al. J Med Chem 64:14129, 2021). Study Design and Methods: In the present project, first we used cell free assay systems to evaluate selectivity and potency of AS-1763 by a kinome-wide profiling and inhibitory effect of AS-1763 in enzyme assays using recombinant mutant BTK. Second, we examined dose- and time-dependent inhibition of mutant BTK that is expressed in HEK293 cell line. Third, we tested biological, biochemical, and molecular impact of AS-1763 in primary CLL cells. Fourth, we determined sensitivity of CLL cells to AS-1763 when combined with other targeted agents. Results: AS-1763 showed a highly selective profile for BTK in a panel of 291 kinase assays with >260-fold selectivity except 3 Tec family kinases (BMX, ITK, and TEC). We have generated a total of 17 recombinant BTK mutant proteins (C481, T474, L528 variants and other BTK mutants; Table 1) reported in the literature or predicted by single nucleotide change in the codon, and established assay methods to measure inhibitory potency of BTKi for those BTK mutants (Table 1). AS-1763 showed potent inhibitory activities for those BTK mutants while inhibitory potencies of other cBTKi and ncBTKi were diminished against some BTK mutants such as T474 and/or L528 mutations. AS-1763 exhibited dose-dependent and slow-off rate inhibitions of BTK autophosphorylation (pY223) in HEK293 cells transfected with various BTK mutants. Furthermore, the observed inhibitory effects of AS-1763 on the BTK autophosphorylation (pY223) in HEK293 cells were continued up to 24 h after washing out of AS-1763. In primary CLL samples, AS-1763, pirtobrutinib or ibrutinib induced a modest apoptosis. AS-1763 effectively inhibited the BCR signaling pathway in a dose dependent manner as evidenced by downregulation of pY223-BTK expression which was also observed in cBTKi and ncBTKi relapsed/refractory CLL samples. In vitro incubations with AS-1763 inhibited CLL cell spontaneous migration, decreased CCL3/CCL4 levels in culture supernatant and was accompanied with the inhibition of intracellular calcium release and B-cell activation, as measured by surface CD86 expression. Besides, AS-1763 incubation for 24 hours was shown to modulate the expression of BCL-2 family proteins with the downregulation of MCL-1 and BCL-xl. Interestingly, in vitro treatment of CLL patient samples with AS-1763 demonstrated a notable elevation in cellular ROS and mitochondrial superoxide levels starting at 1 µM, concurrently impacting SOD1 expression in CLL patient samples. Evaluation of drug interaction models utilizing Compusyn and Synergy Finder applications predominantly indicated additive effects between AS-1763 with BCL-2 inhibitor, venetoclax as well as p53 activator APR-246. Consistent with this data, AS-1763 and venetoclax combinations showed high-rates of apoptosis in samples that were relapsed/refractory to cBTKi and ncBTKi. Conclusions: AS-1763 is a selective ncBTKi that inhibits both wild-type and mutant BTKs listed in Table 1. In CLL cells, AS-1763 was effective in inhibiting BCR pathway signaling and sensitized cells to other agents such as venetoclax. Based on these encouraging data we have initiated a clinical trial to test AS-1763 in patients with CLL and other B cell malignancies who have failed or are intolerant to at least two prior lines of systemic therapy, including cBTKi (NCT05602363 Clinical Trials.gov).

Background: Covalent Bruton’s tyrosine kinase (BTK) inhibitors such as ibrutinib are approved for treating patients with B-cell malignancies, but their long-term efficacy is limited due to their toxicity (i.e., on-target and off-target inhibition of kinases) and acquired resistance (i.e., BTK C481 mutation). AS-1763 is a potent, highly selective, orally active, non-covalent inhibitor of both wild-type and C481S-mutant BTK. AS-1763 strongly inhibits the proliferation of B cell lymphoma cell line OCI-LY10 carrying wild-type and C481S-mutatant BTK in vitro and in vivo. AS-1763, therefore, is expected to be a promising BTK inhibitor for patients who have failed or are intolerant to a covalent BTK inhibitor. This study reports the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) in healthy volunteers who received single, ascending doses of AS-1763 in an alternating cohort design. Methods: This is a first-in-human, double-blind, placebo-controlled, randomized, single-center, single-ascending dose Phase I study of AS-1763. Two cohorts of 8 healthy male and female subjects were alternately dosed with AS-1763 (5, 25, 100, 300, 500, and 600 mg) or placebo orally under fasted condition. Intensive blood sampling for PK (up to 72 h) and PD (up to 24 h) was conducted before and post-dosing to allow a successful PK/PD correlation. For the PD assessment, whole blood was stimulated ex vivo with an anti-IgD antibody, and CD69 upregulation on B cells was assessed by flow cytometry. Results: Single doses of up to 600 mg AS-1763 were well-tolerated. No serious adverse events (AE) were reported during the trial. All treatment-emerged AEs reported were of mild intensity and showed no apparent dose-relationship in frequency. No clinically relevant changes from baseline were observed in all other safety parameters assessed (clinical laboratory, ECGs, vital signs, or physical examinations). AS-1763 was rapidly absorbed with a median time to maximum plasma concentrations (tmax) of 0.5-2.0 h post-dose and thereafter gradually decreased with mean elimination half-time (t1/2) of 8.5-12 h. Overall, mean plasma concentrations of AS-1763 increased with dose up to 500 mg. AS-1763 at 100 mg or higher induced >80% inhibition of B cell activation (%CD69 upregulation in naïve B cells normalized to baseline) at 1-2 h post-dose and the duration of inhibitory effect increased with dose. PK/PD analysis demonstrated that the IC50 and IC90 (90% CI) values for inhibition of CD69 upregulation in naïve B cells were 10.5 ng/mL (9.7-11.2 ng/mL) and 34.6 ng/mL (28.8-40.4 ng/mL), respectively. Conclusions: Single doses of AS-1763 up to 600 mg were well-tolerated and showed encouraging pharmacodynamics (inhibition of CD69 upregulation) with a favorable safety profile. The data support continued development of AS-1763 for patients with B-cell malignancies. Citation Format: Akinori Arimura, Kyoko Miyamoto, Maria Velinova, Marieke van den Dobbelsteen, Katsuhiro Mihara, Robert M. Miller, Masaaki Sawa. Safety, pharmacokinetics, and pharmacodynamics of AS-1763, a highly selective, orally bioavailable, non-covalent BTK inhibitor, in healthy volunteers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT137.

Abstract Background Epertinib (S-222611) is a potent reversible inhibitor of HER2, EGFR and HER4. This trial evaluated the safety, tolerability, pharmacokinetics and antitumour activity of daily oral epertinib combined with trastuzumab (arm A), with trastuzumab plus vinorelbine (arm B) or with trastuzumab plus capecitabine (arm C), in patients with HER2-positive metastatic breast cancer (MBC). Methods Eligible patients, with or without brain metastases, had received prior HER2-directed therapy. A dose-escalation phase determined the tolerability of each combination and established a dose for further study. Further, patients were recruited to expansion cohorts in each of the 3 arms to further explore efficacy and safety. Results The recommended doses of epertinib were 600 mg, 200 mg and 400 mg in arms A, B and C, respectively. The most frequent grade 3/4 adverse event (AE) was diarrhoea in all arms, which was manageable with medical intervention and dose modification. The objective response rate (complete response [CR] plus partial response [PR]) in heavily pre-treated HER2-positive MBC patients at the recommended doses of epertinib combined with trastuzumab was 67% (N = 9), with trastuzumab plus vinorelbine was 0% (N = 5) and with trastuzumab plus capecitabine was 56% (N = 9). Notably, 4 of 6 patients previously treated with T-DM1 responded in the arm A expansion cohort (epertinib plus trastuzumab). In the arm C expansion cohort (epertinib plus trastuzumab plus capecitabine), 4 of 7 patients responded despite previous exposure to capecitabine. Measurable regression of brain metastases was observed in patients with CNS target lesions treated in both arms A and C. Conclusion We observed safety, tolerability and encouraging antitumour activity of epertinib combined with trastuzumab, or with trastuzumab plus capecitabine. This supports further evaluation of these combinations in patients with pre-treated HER2-positive MBC, with or without brain metastases. Trial registration EudraCT Number: 2013-003894-87; registered 09-September-2013.

W. Artibani, Verona M. Babjuk, Prague G. Baretton, Dresden P. Bassi, Rome N.N.P. Buchholz, Ilford M. Burger, Regensburg E. Costantini, Perugia A.K. Ebert, Ulm M. Fisch, Hamburg P. Fornara, Halle B. Frea, Udine M. Froehner, Dresden S. Füssel, Dresden J.P. Gearhart, Baltimore T. Hanus, Prague J. Heesakkers, Nijmegen W. Horninger, Innsbruck G. Janetschek, Salzburg S. Kliesch, Münster S.F. Krause, Linz J.N. Krieger, Seattle F. Kunath, Erlangen M. Lazzeri, Rozzano (Milan) A. Manseck, Ingolstadt V.R. Marshall, Mintaro M. Mas, La Laguna, Tenerife M. May, Straubing L. Mearini, Perugia S. Minhas, London E. Montanari, Milan G. Morgia, Catania G. Nicita, Florence R.T.D. Oliver, London S. Propping, Dresden C. Protzel, Rostock P. Radziszewski, Warsaw P.N. Schlegel, New York S. Schmidt, Berlin B.J. Schmitz-Dräger, Fürth S. Siracusano, Verona N. Sofikitis, Ioannina W.L. Strohmaier, Coburg H. Taubert, Erlangen H.-G. Tiselius, Stockholm L. Türkeri, Istanbul F. Verrey, Zurich F. Wagenlehner, Giessen A. Zucchi, Perugia Founded 1955 by Peter A. Narath and Heinrich Heusser Successors: G. Mayor (1968–1987); D. Hauri (1988–2005) Official Scientific Journal of the Deutsche Gesellschaft für Urologie/ German Society of Urology

We investigated the effects of S-777469 (1-[[6-Ethyl-1-[4-fluorobenzyl]-5-methyl-2-oxo-1, 2-dihydropyridine-3-carbonyl]amino]-cyclohexanecarboxylic acid), a novel cannabinoid type 2 receptor (CB2) agonist, on 1-fluoro-2,4-dinitrobenzene (DNFB)-induced ear inflammation and mite antigen-induced dermatitis in mice. The oral administration of S-777469 significantly suppressed DNFB-induced ear swelling in a dose-dependent manner. In addition, S-777469 significantly alleviated mite antigen-induced atopic dermatitis-like skin lesions in NC/Nga mice. A histological analysis revealed that S-777469 significantly reduced the epidermal thickness and the number of mast cells infiltrating skin lesions. We demonstrated that S-777469 inhibited mite antigen-induced eosinophil accumulation in skin lesions and an endogenous CB2 ligand, 2-arachidonoylglycerol (2-AG)-induced eosinophil migration in vitro. Moreover, we confirmed that 2-AG levels significantly increased in skin lesions of mite antigen-induced dermatitis model. Together, these results suggest that S-777469 inhibits skin inflammation in mice by blocking the activities of 2-AG.

Prostaglandin (PG) D2 elicits responses through either the DP1 and/or DP2 receptor. Experimental evidence suggests that stimulation of the DP1 receptor contributes to allergic responses, such that antagonists are considered to be directed therapies for allergic diseases. In this study, we demonstrate the activity of a novel synthetic DP1 receptor antagonist termed asapiprant (S-555739) for the DP1 receptor and other receptors in vitro, and assess the efficacy of asapiprant in several animal models of allergic diseases. We determined the affinity and selectivity of asapiprant for the DP1 receptor in binding assays. In the animal models of allergic rhinitis, changes in nasal resistance, nasal secretion, and cell infiltration in nasal mucosa were assessed after antigen challenge with and without asapiprant. Similarly, in the animal models of asthma, the effect of antigen challenge with and without asapiprant on antigen-induced bronchoconstriction, airway hyper-responsiveness, mucin production, and cell infiltration in lung were assessed. In binding studies, asapiprant exhibited high affinity and selectivity for the DP1 receptor. Significant suppression of antigen-induced nasal resistance, nasal secretion, and cell infiltration in nasal mucosa was observed with asapiprant treatment. In addition, treatment with asapiprant suppressed antigen-induced asthmatic responses, airway hyper-responsiveness, and cell infiltration and mucin production in lung. These results show that asapiprant is a potent and selective DP1 receptor antagonist, and exerts suppressive effects in the animal models of allergic diseases. Thus, asapiprant has potential as a novel therapy for allergic airway diseases.

2511 Background: S-222611 is an oral, reversible ErbB tyrosine kinase inhibitor of EGFR and HER2 with potent pre-clinical activity. MTD was not reached during dose-escalation, even at the maximum dose of 1600mg QD. PK and efficacy data supported a daily dose of 800mg. A further cohort has been treated to further explore safety and efficacy. Methods: Subjects with advanced solid tumors expressing EGFR and/or overexpressing HER2 were enrolled. S-222611 was administered until disease progression or unacceptable toxicity. Results: 76 patients (33 male/43 female; median age 62 years [range 31-81], ECOG PS 0/1/2: 24/51/1), were included in this expansion phase, including breast (27), esophago-gastric (30), head & neck (12) and renal (7). Dose reduction was required because of adverse events in 11 patients (14%); the most frequent of such being diarrhea and elevated bilirubin. Only 2 patients discontinued treatment due to drug-related adverse events. Of the 41 patients with HER2-positive cancers (26 breast, 13 esophago-gastric, 2 head & neck), 1 complete response (gastric-esophageal junction cancer) and 5 partial responses (4 breast cancer, 1 gastric cancer) were observed; all these patients had received prior HER2-directed therapy. Prolonged stable disease ( ≥ 6 months) was observed in 3 additional patients with breast cancer. 6 of the 25 breast patients had brain metastases, in whom 1 intracranial response and 2 prolonged SD ( ≥ 6mo) were observed. In the EGFR+ve/HER2-ve cohort (n = 35), no RECIST responses were seen, however 4 patients had prolonged stable disease ( ≥ 6mo). Conclusions: S-222621 was well tolerated at a dose of 800mg once daily. Anti-tumour activity, including shrinkage of brain metastases, was evident in a heavily pre-treated population of patients with HER2-positive breast and esophago-gastric cancers. Clinical trial information: 2009-017817-31.

We have previously reported that S-777469 [1-([6-ethyl-1-(4-fluorobenzyl)-5-methyl-2-oxo-1,2-dihydropyridine-3-carbonyl]amino)-cyclohexanecarboxylic acid], a novel cannabinoid type 2 receptor (CB2) agonist, significantly suppressed compound 48/80-induced scratching behavior in mice in a dose-dependent manner when it was administered orally. Here, we demonstrated that the inhibitory effects of S-777469 on compound 48/80-induced scratching behavior are reversed by pretreatment with SR144528, a CB2-selective antagonist. In addition, we investigated the effects of S-777469 on itch-associated scratching behavior induced by several pruritogenic agents in mice and rats. S-777469 significantly suppressed scratching behavior induced by histamine or substance P in mice or by serotonin in rats. In contrast, the H1-antihistamine fexofenadine clearly inhibited histamine-induced scratching behavior but did not affect scratching behavior induced by substance P or serotonin. Moreover, S-777469 significantly inhibited histamine-induced peripheral nerve firing in mice. In conclusion, these results suggest that S-777469 produces its antipruritic effects by inhibiting itch signal transmission through CB2 agonism.

Our lead compound 1 showed high affinity for both CB1 and CB2 receptors, suggesting the possibility of inducing psychoactive side effects through the CB1 receptor in the brain. To solve this issue, polar functional groups were introduced at the 3-position of the pyridone core of compound 1 to find CB1/2 dual agonists such as 17 and 20 which did not show any CNS side effects.