PK Properties Research Articles

In Silico Studies of Four Compounds of Cecropia obtusifolia against Malaria Parasite.

Malaria is a disease that affects many people in the world. In Mexico, malaria remains an active disease in certain regions, particularly in the states of Chiapas and Chihuahua. While antimalarial effects have been attributed to some species of Cecropia in various countries, no such studies have been conducted in Mexico. Therefore, the objective of this study was to evaluate the in silico antimalarial activity of some active compounds identified according to the literature in the species of Cecropia obtusifolia, belonging to the Cecropiaceae family, such as ursolic acid, α-amyrin, chrysin, and isoorientin. These compounds were evaluated with specific molecular docking and molecular dynamics (MD) studies using three different malarial targets with the PDB codes 1CET, 2BL9, and 4ZL4 as well as the prediction of their pharmacokinetic (Pk) properties. Docking analysis revealed the following best binding energies (kcal/mol): isoorientin-1CET (-9.1), isoorientin-2BL9 (-8.8), and chrysin-4ZL4 (-9.6). MD simulation validated the stability of the complexes. Pharmacokinetics analysis suggested that the compounds would generally perform well if administered. Therefore, these results suggest that these compounds may be used as potential drugs for the treatment of malaria.

Development of Liposome Systems for Enhancing the PK Properties of Bivalent PROTACs.

Proteolysis-Targeting Chimeras (PROTACs) are a promising new technology in drug development. They have rapidly evolved in recent years, with several of them in clinical trials. While most of these advances have been associated with monovalent protein degraders, bivalent PROTACs have also entered clinical trials, although progression to market has been limited. One of the reasons is the complex physicochemical properties of the heterobifunctional PROTACs. A promising strategy to improve pharmacokinetics of highly lipophilic compounds, such as PROTACs, is encapsulation in liposome systems. Here we describe liposome systems for intravenous administration to enhance the PK properties of two bivalent PROTAC molecules, by reducing clearance and increasing systemic coverage. We developed and characterized a PROTAC-in-cyclodextrin liposome system where the drug was retained in the liposome core. In PK studies at 1 mg/kg for GNE-01 the PROTAC-in-cyclodextrin liposome, compared to the solution formulation, showed a 80- and a 380-fold enhancement in AUC for mouse and rat studies, respectively. We further investigated the same PROTAC-in-cyclodextrin liposome system with the second PROTAC (GNE-02), where we monitored both lipid and drug concentrations in vivo. Similarly, in a mouse PK study of GEN-02, the PROTAC-in-cyclodextrin liposome system exhibited enhancement in plasma concentration of a 23× increase over the conventional solution formulation. Importantly, the lipid CL correlated with the drug CL. Additionally, we investigated a conventional liposome approach for GNE-02, where the PROTAC resides in the lipid bilayer. Here, a 5× increase in AUC was observed, compared to the conventional solution formulation, and the drug CL was faster than the lipid CL. These results indicate that the different liposome systems can be tailored to translate across multiple PROTAC systems to modulate and improve plasma concentrations. Optimization of the liposomes could further improve tumor concentration and improve the overall therapeutic index (TI). This delivery technology may be well suited to bring novel protein targeted PROTACs into clinics.

Silver-Catalyzed Decarboxylative Acylation of Isocyanides Accesses to α-Ketoamides with Air as a Sole Oxidant.

α-Ketoamide moieties, as privileged units, may represent a valuable option to develop compounds with favorable biological activities, such as low toxicity, promising PK and drug-like properties. An efficient silver-catalyzed decarboxylative acylation of α-oxocarboxylic acids with isocyanides was developed to derivatize the α-ketoamide functional group via a multicomponent reaction (MCR) cascade sequence in one pot. A series of α-ketoamides was synthesized with three components of isocyanides, aromatic α-oxocarboxylic acid analogues and water in moderate yields. Based on the research, the silver-catalyzed decarboxylative acylation confirmed that an oxygen atom of the amide moiety was derived from the water and air as a sole oxidant for the whole process.

Pharmacokinetic and Pharmacodynamic Interactions Between Henagliflozin, a Novel Selective SGLT-2 Inhibitor, and Warfarin in Healthy Chinese Subjects

PurposeWhile controlling blood glucose, patients with diabetes and abnormal coagulation should be treated with positive anticoagulation because the hypercoagulable state of their blood is the primary cause of macroangiopathy. The goal of this study was to evaluate the pharmacokinetic and pharmacodynamic (PK/PD) interactions between henagliflozin, a novel selective sodium-glucose cotransporter 2 inhibitor, and warfarin in healthy subjects. MethodsThis single-center, open-label, single-arm clinical study was conducted in 16 healthy male Chinese subjects. According to the study protocol, the PK properties of henagliflozin 10 mg/d and warfarin 5 mg/d were collected and tabulated in accordance with sampling time. All study drugs were given with once-daily administration. Subjects were monitored for adverse reactions and their severity, outcomes, and relationship to study drug. This influences of warfarin on the PK properties of henagliflozin (Cmax,ss and AUCτ,ss), the effects of henagliflozin on the PK properties of warfarin (Cmax, AUC0–t, and AUC0–∞), and the influences of henagliflozin on the PD properties of warfarin (PTmax, PTAUC, INRmax, and INRAUC) were evaluated. FindingsThe geometric mean ratios (GMRs; 90% CIs) of henagliflozin Cmax,ss and AUCτ,ss were 101.75% (96.11%–107.72%) and 102.21% (100.04%–104.42%), respectively. The GMRs (90% CIs) of S- and R-warfarin Cmax, AUC0–t, and AUC0–∞ were as follows: Cmax, 114.31% (106.30%–122.91%) and 115.09% (109.46%–121.01%), respectively; AUC0–t, 120.15% (116.71%–123.69%) and 119.01% (116.32%–121.76%); and AUC0–∞, 120.81% (117.17%–124.58%) and 121.94% (118.90%–125.05%). The GMRs (90% CIs) of warfarin PTmax and PTAUC were 92.73% (91.25%–94.22%) and 97.42% (96.61%–98.24%). The GMRs (90% CIs) of warfarin INRmax and INRAUC were 92.66% (91.17%–94.17%) and 97.36% (96.52%–98.21%). A total of 32 cases of mild adverse events were reported, and were recovered/resolved. There were no serious adverse events reported. ImplicationsNo significant clinically relevant effects on the PK/PD properties of henagliflozin or warfarin were found with coadministration of the two drugs in these healthy male Chinese subjects. Based on these findings, it is expected that henagliflozin and warfarin can be used in combination without dose adjustment. Chinadrugtrials.org.cn identifier: CTR20190240.

Discovery and Structural Optimization of Novel Quinolone Derivatives as Potent Irreversible Pan-Fibroblast Growth Factor Receptor Inhibitors for Treating Solid Tumors.

Aberrant activation of fibroblast growth factor receptors (FGFRs) has been identified as an oncogenic driver force for multiple cancer types, making FGFRs a compelling target for anticancer therapy. Because of the renewed interest in irreversible inhibitors, considerable efforts have been made to find irreversible FGFR inhibitors. Herein, we discovered a series of novel quinolone-based covalent pan-FGFR inhibitors by further optimizing the lead compound (lenvatinib) under the guidance of molecular docking. The representative pan-FGFR inhibitor I-5 exhibited significant inhibitory potency against FGFR1-4 with nanomolar activity and effectively suppressed the proliferation of Huh-7 and Hep3B HCC cells. I-5 displayed high selectivity against a panel of 369 kinases at 1 μM. The irreversible binding to target proteins was characterized by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Moreover, I-5 exhibited favorable PK properties in vivo and induced significant TGI in the Huh-7 and NCI-H1581 xenograft mouse models.

Assessing Boron-Pleuromutilin AN11251 for the Development of Antibacterial Agents.

Pleuromutilins are a group of antibiotics derived from the naturally occurring compound. The recent approval of lefamulin for both intravenous and oral doses in humans to treat community-acquired bacterial pneumonia has prompted investigations in modifying the structure to broaden the antibacterial spectrum, enhance the activity, and improve the pharmacokinetic properties. AN11251 is a C(14)-functionalized pleuromutilin with a boron-containing heterocycle substructure. It was demonstrated to be an anti-Wolbachia agent with therapeutic potential for Onchocerciasis and lymphatic filariasis. Here, the in vitro and in vivo PK parameters of AN11251 were measured including PPB, intrinsic clearance, half-life, systemic clearance, and volume of distribution. The results indicate that the benzoxaborole-modified pleuromutilin possesses good ADME and PK properties. AN11251 has potent activities against the Gram-positive bacterial pathogens tested, including various drug-resistant strains, and against the slow-growing mycobacterial species. Finally, we employed PK/PD modeling to predict the human dose for treatment of disease caused by Wolbachia, Gram-positive bacteria, or Mycobacterium tuberculosis, which might facilitate the further development of AN11251.

Phase I clinical study of orally-dosed HBW-3220 in patients with relapsed/refractory B-cell lymphoma.

e19532 Background: BTK (Bruton's tyrosine kinase), a central regulator of the B cell receptor signaling pathway, has been targeted for treating a variety of B cell malignancies and autoimmune diseases. The use of the first generation covalent BTK inhibitors (BTKis) have often resulted in the development of resistance, commonly associated with the mutation at the cysteine 481 where the inhibitor binds covalently; therefore, a non-covalent BTKi will offer greater therapeutic benefits. HBW-3220 is a novel reversible BTKi binding to BTK non-covalently. HBW-3220 has potent inhibitory effects on BTK wild-type and BTK mutants (C481S, C481R, T474I, T316A, etc.). HBW-3220 also has a superior preclinical profile, including better anti-tumor efficacy, PK properties and safety, validating its clinical study in cancer patients. Methods: This is a phase I dose-escalation study in patients with r/r B-cell non-Hodgkin's lymphoma. The dose escalation was performed with the accelerated titration and the "3+3" design. Adverse events (AEs) were assigned according to NCI Common Terminology Criteria for Adverse Events (CTCAE) v5.0. Response assessment for chronic lymphocytic leukemia (CLL) was based on the iwCLL 2018 guidelines, for Waldenström macroglobulinemia (WM) was based on the IWWM- 7 consensus, and for all other lymphomas (such as small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), marginal zone lymphoma (MZL), follicular lymphoma (FL), etc.) was based on the Lugano 2014 guidelines. Results: A total of 11 patients were enrolled, and the doses were 15, 30, 60, 90 or 120 mg QD. No dose-limiting toxicity (DLT) occurred in any patient, implicating good tolerability. AEs were similar to other BTKis. Common AEs (≥2 cases, grade 1-3) were neutropenia (n = 2), reduced platelet count (n = 3) and reduced white blood cell count (n = 2). Drug-related grade 3 AEs were neutropenia (n = 1) and pulmonary infection (n = 1). There was no AE leading to discontinuation or dose reduction. 8 patients underwent at least one response assessment, with 1 complete remission (CR) and 5 stable disease (SD). 1 patient (30 mg) with MZL achieved CR at the first response assessment, and her extranodal lesion (2cm*1cm) in the nasopharynx completely disappeared; she remains CR after 20 weeks of medication, and no obvious AEs has been observed! 3 patients in the 60 mg dose group (2 FL and 1 DLBCL) were assessed as SD during the first assessment, and all had tumor shrinkages! 1 MCL patient in the 90 mg dose group has had his lesion reduced by 43%. After oral administration of HBW-3220, the drug exposures show dose-dependent increases, and the time to maximum plasma concentration (Tmax) is 1-4 hours, and elimination half-life (T1/2) is 11-25 hours. Conclusions: The current data show that HBW-3220 is well tolerated and of efficacy in patients with B-cell lymphoma. Its clinical PK characteristics support once-a-day administration. Clinical trial information: ChiCTR20221130 .

Development of BLX-3030, a potent and selective inhibitor of CDK9 for the treatment of NMYC-driven NEPCS.

e17067 Background: Cell lineage reprogramming in cancer cells is a major mechanism for cancers to develop drug resistance and escape targeted therapy. The use of new potent targeted therapies in prostate cancers has led to the development of highly aggressive cancers, including neuroendocrine prostate cancer (NEPC).Gene amplification of N-MYC and C-MYC oncogenes leads to its overexpression in NEPC and mCRPC patients. This overexpression is highly prevalent in these lethal subtypes of cancers, detected in 2% of all Prostate Cancers (PCa) and over 10-17% of mCRPC patients. N-MYC is a transcriptional target of CDK9, which acts as an oncogenic driver, sufficient to transform human-derived prostate cancer cells to take on NEPC phenotypic changes. This results in a more aggressive disease identified in late-stage human PCa patients. Additionally, N-MYC and C-MYC are required for tumor drug resistance and its downregulation, through CDK9 inhibition, lowers tumor growth. Our efforts have demonstrated that targeting both N-MYC and C-MYC as a driver of NEPC and mCRPC with a highly selective CDK9 inhibitor (BLX-3030) is a viable approach for therapeutic intervention of mCRPC and NEPC. Methods: We employed our AI/ML driven FIELDS fragment-based design strategies, in vitro including FACS/apoptosis, cell migration, live cell imaging, immunofluorescent and in vivo tumor models, 7-day tox, PK, selectivity, and ADME-Tox and in vitro safety and secondary pharmacological assays. Results: We discovered and synthesized a series of novel small molecule CDK9 agents that reversibly bind to CDK9, competitively block the ATP site with an IC50 of < 5 nM, and elicit pharmacological responses in N-MYC, C-MYC, and other prostate cancer cells in vitro and in vivo. We detected the expression of CDK9 and N-MYC in PC-3, DU-145, LNCaP, LASCPC-01, NCI-H660, and 22RV1 cell lines. BLX-3030 exhibited potent cellular efficacy in PC cells (IC50 of 22 to 590 nM) and in N-MYC expressed cells (IC50 of 76, 470, and 132 nM), respectively. BLX-3030 reduced pCDK9, C-MYC, Mcl-1, N-MYC and its downstream RNA Pol II (Ser-2) and pAR (Ser-81) in a dose dependent manner. Additionally, we performed FACS/apoptosis, cell migration, and live cell imaging, including immunofluorescence. We also conducted three separate in vivo efficacy mouse models (LASCPC-01, 22RV1, and C4-2) which demonstrated > 70% TGI. Several in vitro ADME-Tox, hERG, P450, safety panel screen, in vivo MTD and dose range finding experiments in SD rats and beagle dogs will also be presented. Conclusions: In summary, a series of BLX-3030 and its analogs display a potency in MYC-driven PCa, with ideal PK properties in rodent, canine species ranging 28-82 % F, with low clearance, and > 3.0 hr half-life. Within this profile, BLX-3030 was selected as a candidate that had the appropriate developable properties. Our plans for IND enabling studies and future clinical trials will also be discussed.

LC/MS Assessment of Glycoform Clearance of A Biotherapeutic MAb in Rabbit Ocular Tissues

Many biotherapeutics such as monoclonal antibodies (mAbs) consist of various glycoforms, which can have different PK properties upon administration to animals and human. As a result, it is necessary to monitor the abundance of glycoforms and limit lot-to-lot variability during the manufacturing process. However, limited information is known about the clearance of mAb glycoforms from ocular space upon intravitreal injection. We present here an assessment of glycoform clearance of a biotherapeutic mAb (IgG1) from rabbit vitreous humor, aqueous humor and retina tissue using LC/MS. The results show that G0, G0F and G1F have similar T1/2, while mannose-5 has a longer T1/2 and is cleared slower in rabbit ocular space, which contradicted with what has been reported in the literature in which Mann5 was cleared faster systematically.

Abstract 490: Discovery of novel, potent and orally available MAT2A inhibitors

Abstract The methylthioadenosine phosphorylase (MTAP) gene is co-deleted with CDKN2A in approximately 15% of all cancers, but targeting a loss of function mutant is not trivial due to the lack of a target protein. The metabolic enzyme methionine adenosyltransferase 2A (MAT2A) was identified as a synthetic lethal target in MTAP-deleted cancers. We describe here potent and orally bioavailable MAT2A inhibitors (“Euregen MAT2Ais”) with superior potency and selectivity compared to AG-270. Euregen MAT2Ais bind at the interface of two MAT2A subunits and exhibit low nanomolar IC50s in biochemical assay. These compounds demonstrated more than 20-fold selective for MTAP−/− cell lines over isogenic MTAP WT. Oral administration of Euregen MAT2Ais drived dose-dependent antitumor activity without meaningful body weight loss in HCT116 MTAP−/− xenograft model. Furthermore, Euregen MAT2Ais revealed excellent oral bioavailability and PK properties in pre-clinical species. We are optimizing multiple MAT2A inhibitors that exhibit activity across a wide range of cancers with MTAP-deletion. Citation Format: Xuxing Chen, Jing Li, Yanhong Chen. Discovery of novel, potent and orally available MAT2A inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 490.

Abstract 3088: Identification of a novel series of MTAP-selective PRMT5 inhibitors, and first disclosure of AZ-PRMT5i-1

Abstract Background: PRMT5, a type 2 arginine methyltransferase, has a critical role in regulating cell growth and survival in cancer. Despite this therapeutic potential, there is growing evidence that the first generation of clinical PRMT5 inhibitors may not be able to achieve high clinical efficacy due to PRMT5-driven (e.g. hematological) toxicities. Aims: In order to pharmacologically exploit the MTAP ‘collateral vulnerability’ mechanism for the potential of increased therapeutic index and clinical efficacy, next-generation PRMT5 inhibitors are required to bind PRMT5 in a cooperative manner with natural metabolite methylthioadenosine (MTA). Whilst MTAP-selective PRMT5 inhibitors have recently been reported,1,2,3 there is continued interest in identifying new and structurally diverse in vivo-quality compounds to further investigate this therapeutic opportunity. Results: To identify novel inhibitors of PRMT5 suitable for the MTAP line of sight, our hit finding strategy used a ‘biased’ biochemical high throughput screen of a 2.4M compounds collection. MTA-cooperative hits were validated using techniques including SPR and Xray crystallography. One series identified from this screen was extensively optimized using techniques including structure-based design to improve potency, MTAP selectivity and DMPK properties. Through these optimizations, we have identified AZ-PRMT5i-1, which shows potent in vitro cellular inhibition of PRMT5, over 50-fold MTAP selectivity margin, excellent selectivity against a broad panel of methyltransferases, and suitable oral PK properties. Conclusion: AZ-PRMT5i-1 is a structurally novel and potent PRMT5 inhibitor with high MTAP-selectivity suitable for use in in vitro and in vivo PRMT5-driven models of cancer’. Note: the structure of AZ-PRMT5i-1 will be disclosed at this meeting PRMT5 Cell SDMA: IC50 nM (HCT116 MTAP KO/WT) SDMA MTAP selectivity (fold) LogD/Rat heps CLint/Caco2 intr. A-B HTS hit A 660/4300 6.5 2.1/34/1.6 AZ-PRMT5i-1 5.4/290 54 2.8/5.0/48

Abstract 1615: NMS-812, a novel potent PERK inhibitor that also inhibits GCN2, exhibits strong anti-tumor activity as single agent and in combination in preclinical models

Abstract PERK and GCN2 are eIF-2α serine-threonine kinases activated by endoplasmic reticulum (ER) stress and amino acid deprivation respectively. These kinases are key effectors of the unfolded protein response (UPR) and amino acid Response (AAR) branches of the integrated stress response (ISR). ISR downregulates protein synthesis while upregulating specific genes in response to various cellular stresses, thus allowing adaptation and survival. PERK and GCN2 cooperate to modulate survival under stress conditions in cancer cells, with GCN2 activation compensating when PERK is inhibited, therefore suggesting a certain degree of redundancy between the two. Multiple myeloma (MM) and other protein-secreting tumors are highly dependent on UPR for survival, due to their high protein load. Therefore, PERK inhibition represents a promising anticancer approach in these tumor types. Notably, in these contexts, dual PERK/GCN2 inhibition may be more effective than inhibiting the single kinase.NMS-03597812 is a novel, very potent, ATP-competitive PERK/GCN2 inhibitor, NMS-812 is preferentially active in vitro on multiple myeloma cell lines; activity has however been observed also on lymphoma and selected solid tumor cell lines, suggesting a broader but still specific role of PERK and GCN2 inhibition depending on cell context. In assays revealing the mechanism of action, as expected, a transient dose-dependent eIF2α pathway activation is observed, suggesting the attempt of GCN2 to compensate for PERK inhibition. However, at higher doses, NMS-812 strongly induces pathway downmodulation and apoptosis compared to prototype PERK selective inhibitors. NMS-812 treatment of a MM xenograft model significantly prolongs the median survival time as single agent and demonstrates synergistic activity with MM standard of care (SOC) agents. In lymphoma, NMS-812 shows activity both as single agent and in combination with SOC.NMS-812 shows a favorable in vitro ADME profile and good PK properties with oral bioavailability in preclinical species and an acceptable toxicity profile. NMS-812 is a novel dual, PERK/GCN2 inhibitor with potential for first-in-class, that validates dual PERK and GCN2 inhibition as a therapeutic approach for MM and other PERK/GCN2-dependent settings. Based on these promising results, a Ph1 study has been started in Multiple Myeloma with plans to expand to other malignancies in the near future. Citation Format: Claudia Perrera, Maurizio Pulici, Patrizia Banfi, Nilla Avanzi, Davide Carenzi, Daniele Casero, Antonella Ciavolella, Laura Gianellini, Fabio Gasparri, Grazia Saturno, Barbara Valsasina, Elena Ardini, Alessia Montagnoli, Arturo P. Galvani, Antonella Isacchi. NMS-812, a novel potent PERK inhibitor that also inhibits GCN2, exhibits strong anti-tumor activity as single agent and in combination in preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1615.

Abstract 4027: TSN084, a multi-kinase inhibitor, overcomes acquired drug-resistant mutations of cMet, Trks, and Flt3, and displays broad activities against kinase oncotargets Axl, DDRs, and CDK8/19

Abstract Background: Up to date, over 80 small molecule kinase inhibitors have been approved for the treatment of cancers worldwide. This class of targeted therapy benefits millions of patients by improving their disease management and quality of life. However, acquired drug resistance can develop quickly after treatment in most of the patients. Herein, we disclose TSN084, a unique small molecule multi-kinase inhibitor, which can overcome many common acquired drug-resistant mutations of cMet, Trks, and Flt3, etc. It also inhibits other oncotargets including Axl, DDRs, and CDK8/19. Method: The primary screen of TSN084 against 468 kinases at a single concentration was carried out by Eurofins DiscoverX Corporation. Its inhibitory activity on interested kinases and relevant cellular proliferation were determined separately. A series of cell-derived xenograft (CDX) models were used to evaluate its in vivo antitumor effect. Both in vitro and in vivo PK study was performed in mouse, rat, dog, and monkey. An in vitro safety panel and in vivo GLP animal studies were carried out to assess its preliminary toxicity profile in preparation for human clinical trials. Results: Kinase activity profiling assay revealed that TSN084 is a multi-kinase inhibitor. Among the 468 kinases tested, 33 showed >50% inhibition at 0.1 uM, including many oncotargets and their mutants such as Met, Met-Y1235D, Met-M1250T, Flt3, Flt3-N841I, Flt3-ITD, Flt3-K663Q, Flt3-D835Y, Flt3-D835V, Axl, TrkA, DDR1, and CDK8/19. It demonstrated potent anti-proliferation activity against many tumor cell lines, e.g. KM12, MKN45, MV-4-11, and HS746T. It also effectively inhibited tumor growth in several CDX drug-resistant models like NTRK-G667C and NTRK3-G696C and showed outstanding survival advantage. TSN084 is an orally bioavailable small molecule agent in mouse, rat, dog, and monkey. It is clean in a safety panel assessment of 44 concerning targets and considered safe enough to advance into clinical study. Conclusion: TSN084 targets several oncogenic kinases and their drug-resistant mutants. It demonstrated potent antitumor effects in both in vitro and in vivo studies. It showed excellent PK properties and acceptable preclinical safety profile. A phase I clinical trial of TSN084 in patients with advanced or metastatic malignancies is currently ongoing (NCT05300438). As of Oct 30, 2022, 6 patients have been enrolled into the trial, and no DLTs or treatment-related AEs were observed. It is a promising agent for the potential treatment of resistant cancers in the clinic. Citation Format: Boyu Zhong, Tony Zhang, Chunlan Dong, Shengtang Mark Ma, Ziyang Jia, Guangming Chen, Renjuan Zheng, Jing Cindy Li, Han Fu. TSN084, a multi-kinase inhibitor, overcomes acquired drug-resistant mutations of cMet, Trks, and Flt3, and displays broad activities against kinase oncotargets Axl, DDRs, and CDK8/19. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4027.

Abstract 486: Characterization of a novel series of highly selective PARP7 inhibitors

Abstract PARP7 is a cellular stress-induced enzyme that adds mono-ADP-ribose groups to a variety of substrate proteins thereby regulating their function. One such substrate is the kinase TBK1 which regulates activity of cGAS-STING and RIG-I nucleic acid sensing pathways. Up-regulation of PARP7 expression in cancers applies a brake to cytosolic nucleic acid sensing and the Type I interferon response. This creates an immunosuppressive tumor microenvironment (TME) leading to faster tumor growth. In addition, inhibition of PARP7 has also been shown to directly arrest growth in a subset of cancer cells via the promotion of a senescence phenotype, inhibition of autophagy and regulation of metabolism. Through the use of structure-based drug design we describe the characterization of potent and selective inhibitors of PARP7. These molecules block viability of a subset of immortalized and primary cancer cells in 2D culture. We have observed potent cGAS-STING independent growth arrest in a number of cancer cell lines harboring a KRAS G12C mutation, and studies combining PARP7 inhibitors with commercially available KRAS G12C inhibitors yield additional in vitro efficacy. Recent studies have shown that inhibitors targeting oncogenic RAS pathway signaling can activate ULK1/2-mediated autophagy as an adaptive treatment resistance mechanism. Hence, our data support further studies investigating the use of PARP7 inhibitors in KRAS-driven cancers either as single agents or in combination with mutant KRAS inhibitors. Our molecules also stimulate robust induction of type I interferon (IFNβ) and downstream chemokines such as CXCL10 in a cGAS-STING pathway dependent manner in a range of cancer cells and 3D organoids. This induction is augmented by combining agonists of cGAS-STING and RIG-I pathways with PARP7 inhibitors. In addition, PARP7 inhibitors work in concert with exogenous DNA-damaging agents such as chemotherapeutics and X-ray radiation to induce the type I interferon response in cancer cells. Overall, these data highlight the opportunity to utilize PARP7 inhibitors in cancers where high genomic instability leads to aberrant cytosolic nucleic acid levels or in concert with exogenous DNA-damaging agents. Our PARP7 inhibitors exhibit highly desirable physico-chemical and in vitro ADME properties coupled with an exceptional in vitro safety profile which translates to best-in-class rodent PK. In a KRAS G12C lung cancer xenograft model (NCI-H1373), twice daily oral administration of an exemplar PARP7 inhibitor drives rapid and full tumor regression in a dose-dependent manner. The anti-tumor effects correlate with robust tumor PK/PD upon measurement of tumor IFNβ and CXCL10. In summary, we describe the characterization of a lead series of highly potent and selective PARP7 inhibitors which demonstrate excellent in vitro ADMET and in vivo PK properties leading to best-in-class anti-tumor efficacy in a KRAS-driven lung cancer xenograft model. Citation Format: Phillip M. Cowley, Barry E. McGuinness, Gillian M. Campbell, Alan Wise. Characterization of a novel series of highly selective PARP7 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 486.

Abstract 5049: Pb-203 image guided Pb-212 receptor targeted alpha particle therapy for cancer - a powerful emerging paradigm

Abstract Objective: Pb-203 and Pb-212 have emerged as a promising elementally-matched theranostic pair for imaging-guided alpha-particle radiotherapy for cancer. A somatostatin receptor 2 (SSTR2)-targeted peptide coupled with novel Pb specific chelator (PSC) has been developed (PSC-PEG2-TOC). Here, preclinical in vitro and in vivo evaluation of 203Pb/212Pb-labeled PSC-PEG2-TOC was conducted including head to head therapy of SSTR2+ tumors in mice vs standard of care beta particle therapy and first in humans clinical imaging of SSTR2+ tumors. Method: [203Pb]PSC-PEG2-TOC and [212Pb]PSC-PEG2-TOC was prepared by published methods. Radiochemical stability of [203Pb]PSC-PEG2-TOC, [212Pb]PSC-PEG2-TOC and [212Bi]PSC-PEG2-TOC in human serum was determined. Binding affinity of [203Pb]PSC-PEG2-TOC was determined by binding assays in AR42J cells. In vivo SSTR2-mediated tumor targeting of [203Pb]PSC-PEG2-TOC was determined by SPECT imaging in athymic nude mice bearing AR42J xenografts. In vivo biodistribution of [212Pb]PSC-PEG2-TOC in normal organs and potential redistribution of 212Bi daughter were determined in CD-1 Elite mice. Clinical imaging was conducted under appropriate regulations to determine the biodistribution of the agent in patients bearing SSTR2+ tumors as determined by gold standard 68Ga-DOTATOC PET imaging. Results: Rapid incorporation of 203Pb and 212Pb in PSC-PEG2-TOC was observed after reactions at temperatures as low as 4°C within 15 min. Greater than 95% radiochemical stability was observed for both [203Pb]PSC-PEG2-TOC and [212Pb]PSC-PEG2-TOC after incubation in human serum for up to 55 hours. Stable decay product [212Bi]PSC-PEG2-TOC with minimal free 212Bi was observed. Agents demonstrated superior binding affinity to SSTR2 with Kd=0.59 nM. In in vivo studies, rapid tumor uptake and renal clearance of [203Pb]PSC-PEG2-TOC were observed in athymic nude mice bearing AR42J xenografts. In the biodistribution study, nearly identical biodistribution profiles of [212Pb]PSC-PEG2-TOC and progeny [212Bi]PSC-PEG2-TOC were found. No redistribution of 212Bi activity was identified, indicating that 212Bi remained co-localized with parent [212Pb]PSC-PEG2-TOC in vivo. Therapeutic studies in this same mouse model resulted in 100% complete responses for both a single dose 4.4 MBq administration of [212Pb]PSC-PEG2-TOC or a fractionated regimen of 4 × 1.1 MBq. First in humans clinical imaging showed PK properties that included rapid tumor accumulation and retention at over 20 h post administration, coupled with fast renal clearance of residual agent. Conclusion: [203Pb]PSC-PEG2-TOC and [212Pb]PSC-PEG2-TOC have promising potential for image-guide alpha-particle therapy for SSTR2 positive tumors. Agents have received a Fastrack designation by the US FDA and a safe to proceed for a Phase 1 trial. Citation Format: Michael King Schultz, Yusuf Menda, Dijie Liu, Mengshi Li, Frances Johnson, Brianna Cagle, Nicholas Baumhover, Ivy Vance. Pb-203 image guided Pb-212 receptor targeted alpha particle therapy for cancer - a powerful emerging paradigm. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5049.

Design, synthesis and evaluation of novel monoamine oxidase B (MAO-B) inhibitors with improved pharmacokinetic properties for Parkinson's disease

A series of novel ((benzofuran-5-yl)methyl)pyrrolidine-2-carboxamide derivatives were designed, synthesized and evaluated as MAO-B inhibitors. SAR studies indicated that cyclizing benzyl ether into benzofuran ring resulted in the most potent MAO-B inhibitor (IC50 = 0.037 μM), (2S,4S)-4-fluoro-1-((2-(4-fluorophenyl) benzofuran-5-yl)methyl)pyrrolidine-2-carboxamide (C14). PK properties of C14 in rats and mice were significantly improved compared to our previous candidate and safinamide, indicating that benzofuran moiety is essential for improving PK properties. Moreover, C14 displayed good metabolic stability and brain-blood barrier permeability, as well as favorable in vitro properties. Finally, C14 significantly inhibited MAO-B in the mouse brain. C14 exhibited a potential efficacy for DA deficits in the MPTP-induced mouse model and significantly increased DA concentration in the striatum. Thus, we identified that C14 may be a promising drug candidate for PD treatment.

Albumin-encapsulated HSP90-PROTAC BP3 nanoparticles not only retain protein degradation ability but also enhance the antitumour activity of BP3 in vivo

Proteolysis-targeting chimaera (PROTAC) has received extensive attention in industry. However, there are still some limitations that hinder its further development. In a previous study, our group first demonstrated that the HSP90 degrader BP3 synthesised by the principle of PROTACs showed therapeutic potential for cancer. However, its application was hindered by its high molecular weight and water insolubility. Herein, we aimed to improve these properties of HSP90-PROTAC BP3 by encapsulating it into human serum albumin nanoparticles (BP3@HSA NPs). The results demonstrated that BP3@HSA NPs showed a uniform spherical shape with a size of 141.01 ± 1.07 nm and polydispersity index < 0.2; moreover, BP3@HSA NPs were more readily taken up by breast cancer cells and had a stronger inhibitory effect in vitro than free BP3. BP3@HSA NPs also demonstrated the ability to degrade HSP90. Mechanistically, the improved inhibitory effect of BP3@HSA NPs on breast cancer cells was related to its stronger ability to induce cell cycle arrest and apoptosis. Furthermore, BP3@HSA NPs improved PK properties and showed stronger tumour suppression in mice. Taken together, this study demonstrated that hydrophobic HSP90-PROTAC BP3 nanoparticles encapsulated by human serum albumin could improve the safety and antitumour efficacy of BP3.

CS27109, A Selective Thyroid Hormone Receptor-β Agonist Alleviates Metabolic-Associated Fatty Liver Disease in Murine Models.

Thyroid hormone receptor-β (THR-β) agonists play crucial roles in dyslipidemia and metabolic associated fatty liver disease (MAFLD). We developed a novel oral and liver-targeted THR-β agonist, CS27109, and evaluated its efficacy in the treatment of metabolic disorders. We evaluated in vitro and in vivo efficacy and/or safety of CS27109 along with MGL3196 (a phase III THR-β agonist). CS27109 showed pronounced activity and selectivity to THR-β and favorable PK properties, which was equivalent to MGL3196. In the hamster model, animals treated with a high dose of CS27109 showed equivalent reductions in serum TC and LDL-c with groups treated with MGL3196. In the rat model, CS27109 and MGL3196 reduced serum ALT, TC, TG, LDL-c, liver weight ratio, and liver steatosis. CS27109 simultaneously decreased liver TG and TC, and MGL3196 additionally reduced AST. In the mouse model, CS27109 dose-dependently reduced serum AST, ALT, liver inflammation, and NAS score, and also downregulated TC, LDL-c, liver steatosis, and fibrosis, but not in a dose-dependent manner. MGL3196 revealed an equivalent effect with CS27109 in that model. CS27109 also exhibited tolerable toxicity to the heart. CS27109 shows comparative in vitro and in vivo efficacy with MGL3196, suggesting its potential therapeutic application in the treatment of MAFLD such as dyslipidemia and steatohepatitis.

A pH-dependent anti-CD47 antibody that selectively targets solid tumors and improves therapeutic efficacy and safety

BackgroundThe antiphagocytic molecule CD47 is overexpressed in a wide variety of cancer cells, and antibodies targeting CD47 for cancer therapies are currently under intensive investigation. However, owing to the ubiquitous expression of CD47 on healthy cells, anti-CD47 therapies often achieve only weak therapeutic benefits and can induce severe side effects. Here, we report the generation of a pH-dependent anti-CD47 antibody (BC31M4) which selectively binds to tumors under the acidic solid tumor microenvironment.MethodsBC31M4 was generated using antibody phage display and a pH-dependent selection strategy. The pH-dependent binding and blocking activities of BC31M4 were verified using in vitro assays, and the structural basis of the pH-dependent binding property was characterized. BC31M4’s antitumor effect was confirmed by both phagocytosis assays and studies in xenograft models. The tumor selectivity, mechanism of action, PK properties, side effects, and therapeutic efficacy were further evaluated in humanized (hCD47 and its receptor hSIRPα) immunocompetent syngeneic mouse models.ResultsThe crystal structure reveals that two histidines locate within the CDRs of the light chain directly contribute to the pH-dependent binding of BC31M4. BC31M4 promotes macrophage phagocytosis of tumor cells more potently at acidic-pH than at physiological-pH. Our hCD47/hSIRPα humanized syngeneic mouse model results demonstrated that BC31M4 selectively accumulates in tumors but not in normal tissues. BC31M4 causes minimal side effects and exhibits superior PK properties as compared to the other examined anti-CD47 antibodies. When combined with adoptive T cell transfer, BC31M4 efficiently promotes adaptive immune responses against tumors and also induces immune memory. Moreover, we show that BC31M4’s antitumor effects rely on an Fc that mediates strong effector functions.ConclusionsOur study illustrates that the development of a tumor-selective, pH-dependent anti-CD47 antibody safely confers strong therapeutic effects against solid tumors, thus providing a promising therapeutic strategy to overcome the challenges of anti-CD47 therapy.

Design, synthesis of novel benzimidazole derivatives as ENL inhibitors suppressing leukemia cells viability via downregulating the expression of MYC

Eleven-Nineteen-Leukemia Protein (ENL) containing YEATS domain, a potential drug target, has emerged as a reader of lysine acetylation. SGC-iMLLT bearing with benzimidazole scaffold was identified as an effective ENL inhibitor, but with weak activity against mixed-lineage leukemia (MLL)-rearranged cells proliferation. In this study, a series of compounds were designed and synthesized by structural optimization on SGC-iMLLT. All the compounds have been evaluated for their ENL inhibitory activities. The results showed that compounds 13, 23 and 28 are the most potential ones with the IC50 values of 14.5 ± 3.0 nM, 10.7 ± 5.3 nM, and 15.4 ± 2.2 nM, respectively, similar with that of SGC-iMLLT. They could interact with ENL protein and strengthen its thermal stability in vitro. Among them, compound 28 with methyl phenanthridinone moiety replacement of indazole in SGC-iMLLT, exhibited significantly inhibitory activities towards MV4-11 and MOLM-13 cell lines with IC50 values of 4.8 μM and 8.3 μM, respectively, exhibiting ∼7 folds and ∼9 folds more potent inhibition of cell growth than SGC-iMLLT. It could also increase the ENL thermal stability while SGC-iMLLT had no obvious effect on leukemia cells. Moreover, compound 28 could downregulate the expression of target gene MYC either alone or in combination with JQ-1 in cells, which was more effective than SGC-iMLLT. Besides, in vivo pharmacokinetic studies showed that the PK properties for compound 28 was much improved over that of SGC-iMLLT. These observations suggested compound 28 was a potential ligand for ENL-related MLL chemotherapy.