Biological impact and therapeutic potential of a novel camptothecin derivative (FLQY2) in pancreatic cancer through inactivation of the PDK1/AKT/mTOR pathway.

BACKGROUND: Camptothecin (CPT), a pentacyclic alkaloid with antitumor properties, is derived from the Camptotheca acuminata. Topotecan and irinotecan (CPT derivatives) were first approved by the Food and Drug Administration for cancer treatment over 25 years ago and remain key anticancer drugs today. However, their use is often limited by clinical toxicity. Despite extensive development efforts, many of these derivatives have not succeeded clinically, particularly in their effectiveness against pancreatic cancer which remains modest. AIM OF THE STUDY: This study aimed to evaluate the therapeutic activity of FLQY2, a CPT derivative synthesized in our laboratory, against pancreatic cancer, comparing its efficacy and mechanism of action with those of established clinical drugs. METHODS: The cytotoxic effects of FLQY2 on cancer cells were assessed using an MTT assay. Patient-derived organoid (PDO) models were employed to compare the sensitivity of FLQY2 to existing clinical drugs across various cancers. The impact of FLQY2 on apoptosis and cell cycle arrest in Mia Paca-2 pancreatic cancer cells was examined through flow cytometry. Transcriptomic and proteomic analyses were conducted to explore the underlying mechanisms of FLQY2's antitumor activity. Western blotting was used to determine the levels of proteins regulated by FLQY2. Additionally, the antitumor efficacy of FLQY2 in vivo was evaluated in a pancreatic cancer xenograft model. RESULTS: FLQY2 demonstrated (1) potent cytotoxicity; (2) superior tumor-suppressive activity in PDO models compared to current clinical drugs such as gemcitabine, 5-fluorouracil, cisplatin, paclitaxel, ivosidenib, infinitinib, and lenvatinib; (3) significantly greater tumor inhibition than paclitaxel liposomes in a pancreatic cancer xenograft model; (4) robust antitumor effects, closely associated with the inhibition of the TOP I and PDK1/AKT/mTOR signaling pathways. In vitro studies revealed that FLQY2 inhibited cell proliferation, colony formation, induced apoptosis, and caused cell cycle arrest at nanomolar concentrations. Furthermore, the combination of FLQY2 and gemcitabine exhibited significant inhibitory and synergistic effects. CONCLUSION: The study confirmed the involvement of topoisomerase I and the PDK1/AKT/mTOR pathways in mediating the antitumor activity of FLQY2 in treating Mia Paca-2 pancreatic cancer. Therefore, FLQY2 has potential as a novel therapeutic option for patients with pancreatic cancer.

DNA damage response and repair gene mutations are associated with tumor mutational burden and outcomes to platinum-based chemotherapy/immunotherapy in advanced NSCLC patients.

BACKGROUND: DNA damage response and repair (DDR) genes are crucial for maintaining the integrity of the genome. This study aims to explore the correlation of DDR gene mutations with TMB, clinical characteristics, and outcomes to platinum-based chemotherapy and platinum-based chemotherapy/immunotherapy in non-small cell lung cancer (NSCLC) without EGFR and ALK alterations. METHODS: Tumor tissue from 49 patients with stage III or IV NSCLC who were without EGFR and ALK alterations were analyzed using targeted next-generation sequencing (NGS). Among them, 13 patients received first-line platinum-based chemotherapy, 32 patients received first-line platinum-based chemotherapy/immunotherapy. RESULTS: In these NSCLC patients without EGFR and ALK alterations, the frequently mutated genes included TP53, KMT2D and KRAS, the most frequently mutated DDR gene was FANCG, DDR gene mutations were detected in 20 patients. The mutation frequency of homologous recombination (HR) pathway was significantly higher in lung squamous cell carcinoma (LUSC) than that in lung adenocarcinoma (LUAD) (30.8% vs. 5.7%). Among DDR positive patients, a lower percentage exhibited metastasis. Patients with DDR gene mutations, cell-cycle checkpoint pathway mutations, and BER pathway mutations had significantly higher TMB compared to those without corresponding mutations. In the patients receiving platinum-based chemotherapy/immunotherapy, the disease control rate was significantly lower in the DDR-positive group compared with that in the DDR-negative group (55.6% vs. 100.0%). Among LUAD patients receiving platinum-based chemotherapy/immunotherapy, we observed a worse overall survival (OS) in DDR-positive group, as well as poorer progression-free survival(PFS)and OS in BER-positive and FANCG mutated group. CONCLUSIONS: DDR gene mutations are associated with tumor metastasis, TMB, and outcomes to platinum-based chemotherapy/immunotherapy in advanced NSCLC patients.

Design, synthesis and biological evaluation of novel phenylfuran-bisamide derivatives as P-glycoprotein inhibitors against multidrug resistance in MCF-7/ADR cell.

The co-administration of anticancer drugs and P-glycoprotein (P-gp) inhibitors was a treatment strategy to surmount multidrug resistance (MDR) in anticancer chemotherapy. In this study, novel phenylfuran-bisamide derivatives were designed as P-gp inhibitors based on target-based drug design, and 31 novel compounds were synthesized and screened on MCF-7/ADR cells. The result of bioassay revealed that compound y12d exhibited low cytotoxicity and promising MDR reversal activity (IC50 = 0.0320 µM, reversal fold = 1163.0), 3.64-fold better than third-generation P-gp inhibitor tariquidar (IC50 = 0.1165 µM, reversal fold = 319.3). The results of Western blot and rhodamine 123 accumulation verified that compound y12d exhibited excellent MDR reversal activity by inhibiting the efflux function of P-gp but not expression. Furthermore, molecular docking showed that compound y12d bound to target P-gp by forming the double H-bond interactions with residue Gln 725. These results suggest that compound y12d might be a potential MDR reveal agent acting as a P-gp inhibitor in clinical therapeutics, and provide insight into design strategy and skeleton optimization for the development of P-gp inhibitors.

Preparation of a camptothecin analog FLQY2 self-micelle solid dispersion with improved solubility and bioavailability.

BACKGROUND: 7-p-trifluoromethylphenyl-FL118 (FLQY2) is a camptothecin analog with excellent antitumor efficacy against various solid tumors. However, its poor solubility and low bioavailability limited the development of the drug. Polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (Soluplus®), an emerging carrier for preparing solid dispersion (SD), encapsulated FLQY2 to circumvent the above limitations. RESULTS: In this project, FLQY2-SD was prepared by solvent evaporation method and self-assembled into micelles in aqueous solutions owing to the amphiphilic nature of Soluplus®. The physicochemical characterizations demonstrated that FLQY2 existed in a homogeneous amorphous form in SD and was rapidly dissolved. The micelles did not affect cytotoxicity or cellular uptake of FLQY2 in vitro, and the oral bioavailability was increased by 12.3-fold compared to the FLQY2 cyclodextrin suspension. The pharmacokinetics of FLQY2-SD showed rapid absorption, accumulation in the intestine, and slow elimination via fecal. Metabolite identification studies showed 14 novel metabolites were identified, including 12 phase I metabolites (M1-M12) and 2 phase II metabolites (M13-M14), of which M2 (oxidation after decarboxylation) and M7 (dioxolane ring cleavage) were the primary metabolites in the positive mode and negative mode, respectively. The tumor growth inhibition rate (TGI, 81.1%) of FLQY2-SD (1.5 mpk, p.o./QW) in tumor-bearing mice after oral administration was higher than that of albumin-bound Paclitaxel (15 mpk, i.v./Q4D) and Irinotecan hydrochloride (100 mpk, i.p./QW). CONCLUSIONS: The successful preparation, pharmacokinetics, and pharmacodynamics studies of FLQY2-SD showed that the solubility and bioavailability of FLQY2 were improved, which facilitated the further druggability development of FLQY2.

Investigation of the Uptake and Transport of Two Novel Camptothecin Derivatives in Caco-2 Cell Monolayers.

Irinotecan and Topotecan are two Camptothecin derivatives (CPTs) whose resistance is associated with the high expression of breast cancer resistance protein (BCRP) and P-glycoprotein (P-gp). To reverse this resistance, two novel CPTs, FL77-28 (7-(3-Fluoro-4-methylphenyl)-10,11-methylenedioxy-20(S)-CPT) and FL77-29 (7-(4-Fluoro-3-methylphenyl)-10,11-methylenedioxy-20(S)-CPT), were synthesized by our group. In this study, the anti-tumor activities of FL77-28, FL77-29, and their parent, FL118 (10,11-methylenedioxy-20(S)-CPT), were evaluated and the results showed that FL77-28 and FL77-29 had stronger anti-tumor activities than FL118. The transport and uptake of FL118, FL77-28, and FL77-29 were investigated in Caco-2 cells for the preliminary prediction of intestinal absorption. The apparent permeability coefficient from apical to basolateral (Papp AP-BL) values of FL77-28 and FL77-29 were (2.32 ± 0.04) × 10-6 cm/s and (2.48 ± 0.18) × 10-6 cm/s, respectively, suggesting that the compounds had moderate absorption. Since the transport property of FL77-28 was passive diffusion and the efflux ratio (ER) was less than 2, two chemical inhibitors were added to further confirm the involvement of efflux proteins. The results showed that FL77-28 was not a substrate of P-gp or BCRP, but FL77-29 was mediated by P-gp. In conclusion, FL77-28 might be a promising candidate to overcome drug resistance induced by multiple efflux proteins.

Cellular Uptake and Transport Characteristics of FL118 Derivatives in Caco-2 Cell Monolayers.

In the evaluation of the druggability of candidate compounds, it was vital to predict the oral bioavailability of compounds from apparent permeability (Papp) across Caco-2 cell-culture model of intestinal epithelium cultured on commercial transwell plate inserts. The study was to investigate the transport characteristics and permeability of FL118 (10, 11-Methylenedioxy-20(S)-camptothecin) derivatives 7-Q6 (7-(4-Ethylphenyl)-10, 11-methylenedioxy-20(S)-camptothecin) and 7-Q20 (7-(4-Trifluoromethylphenyl)-10, 11-methylenedioxy-20(S)-camptothecin). Transport characteristics and permeability of the tested compounds to the small intestine were assessed at different concentrations (0.5, 1 µM) via Caco-2 cell monolayers model in vitro. Uptake studies based on Caco-2 cells, including temperatures, concentrations, and the influence of efflux transporters, were combined to confirm the transport characteristics of the tested compounds. Furthermore, cytotoxicity results showed that the concentrations used in the experiments were non-toxic and harmless to cells. In addition, The Papp of 7-Q6 was (3.69 ± 1.07) × 10-6 cm/s with efflux ratio (ER) 0.98, while the Papp of 7-Q20 was (7.78 ± 0.89) × 10-6 cm/s with ER 1.05 for apical-to-basolateral (AP→BL) at 0.5 µM, suggesting that 7-Q20 might possess higher oral bioavailability in vivo. Furthermore, P-glycoprotein (P-gp) was proved to slightly affect the accumulations of 7-Q20, while the absorption of 7-Q6 was irrelevant with P-gp and breast cancer resistant protein (BCRP) based on the cellular uptake assays. Accordingly, 7-Q6 was completely absorbed by passive diffusion, and 7-Q20 was mainly dependent on passive diffusion with being effluxed by P-gp slightly. Meanwhile, both 7-Q6 and 7-Q20 were potential antitumor drugs that might exhibit high oral bioavailability in the body.