Continuous Drug Exposure Research Articles

5-Fluorouracil induces apoptosis in nutritional deprived hepatocellular carcinoma through mitochondrial damage.

5-Fluorouracil (5-FU) is the leading chemotherapeutic drug used to treat hepatocellular carcinoma, one of the major cancer diseases after atherosclerosis. Because of chemo-resistance, the success rate of treatment declines with time due to continuous drug exposure. Though autophagy induction is majorly responsible for acquired resistance, the exact role of this evolutionary conserved mechanism is unknown in cancer cell survival and suppression. The usual practice involves the combinatorial use of chemotherapeutic drugs with autophagy inhibitors like Chloroquine and Bafilomycin A, while neglecting the side effects caused by autophagy impairment in healthy cells. Starvation is a well-known physiological inducer of autophagy. In this study, by caloric modulation, we tried to circumvent the resistance imposed by prolonged drug exposure and investigated the effect of 5-FU in nutrient-sufficient and deficient conditions. Our findings show a substantial correlation between autophagy and increased cancer cell death in the presence of 5-FU, with negligible effects on normal cells. Experimental data revealed that nutritional deprivation augmented cell death in the presence of 5-FU through mitochondrial membrane damage and excessive reactive oxygen species (ROS) production, initiating apoptosis. Lipidation study also unveiled that under such combinatorial treatment cellular metabolism shifts from glucose to lipid biosynthesis. Overall, our experimental findings suggest that nutritional deprivation in combination with chemotherapeutic medication can be a new effective strategy to control hepatocellular carcinoma.

Abstract PO4-23-11: Resistance Mechanisms to CDK4/6 Inhibitors and/or Tamoxifen Using Comprehensive Thermostable Group II Intron Reverse Transcriptase Sequencing

Abstract Background Understanding the mechanisms of resistance to CDK4/6 inhibitors (CDK4/6i) and endocrine therapy (ET) is pivotal in exploring new therapeutic strategies for hormone receptor-positive (HR+), HER2-negative metastatic breast cancer. To decipher these resistance mechanisms, we analyzed the alterations in comprehensive RNA-seq (coding and non-coding RNAs) of HR+ HER2-negative BC cell lines via thermostable group II intron reverse transcriptase sequencing (TGIRT-seq). Methods We established Tamoxifen-resistant (TMR), Abemaciclib-resistant (ACR), Palbociclib-resistant (PCR), Tamoxifen/Abemaciclib double-resistant (TMR-ACR), and Tamoxifen/Palbociclib double-resistant (TMR-PCR) BC cell lines from MCF7 and T47D HR+/HER2- BC cell lines through stepwise dose-escalation continuous drug exposure. We performed a TGIRT-seq transcriptomic analysis using a protocol that allows the sequencing of both long and short non-coding and protein-coding RNAs in a single library. All libraries were sequenced using paired-end 150 bp on the Novaseq platform, resulting in an average of 50 million reads per library. For analysis, raw reads underwent adapter trimming, small RNA mapping, whole genome mapping, and the generation of annotated genes' read count. We used raw counts to detect differentially expressed genes (DEGs) with DESeq2 in R using the cut-off (log2 [fold change] > 1, FDR < 1e-3). We then used the selected DEGs for Gene Set Enrichment Analysis (GSEA) and Kaplan-Meier survival analysis from the TCGA database. Results The TGIRT-seq analysis identified 1171 to 3472 DEGs in different drug resistance and cell line combinations. Most DEGs in resistant cells were consistently downregulated compared to parent cells. Seventy-five percent of DEGs were protein-coding genes, with the rest being non-coding RNAs, such as small nucleolar RNAs, microRNAs, long non-coding RNAs, and transposable element RNAs, which conventional RNA-seq poorly detects. In principal component analysis (PCA) plots, replicates per drug resistance clustered clearly in both cell line backgrounds. PCA also indicated distinctive pathways for acquiring drug resistance in MCF7 and T47D cells. The estrogen receptor (ER) expression decreased, while HER2 increased in resistant cell lines. DEGs identified were enriched in ESR1-related pathways in all MCF7 and T47D cell lines resistant models. Several ER-regulating genes like IL-1R1 and RET were upregulated, while ADCY1 was consistently downregulated across different resistance types. A significant overlap with single-resistance DEGs was observed among the DEGs in double-resistance cell lines, but 20-50% of up/down-regulated DEGs in double-resistance cell lines were uniquely altered. Several previously identified DEGs (e.g., SALL4, TOP2A) and 21 novel candidate genes correlated with poor survival outcomes. Conclusion The analysis identified unique DEGs in double resistance cell lines, suggesting that double resistance mechanisms may not merely be a cumulative effect of single resistance mechanisms, necessitating further investigation and validation. CDK4/6i and/or ET-resistant BC cell lines displayed significant transcriptome reprogramming during the development of drug resistance. In previously published research, ESR1-related pathway alterations were proposed in tamoxifen resistance cell lines. Here we find that CDK4/6i-resistant cells also modify these ESR1-related pathways. Additionally, we identified several targetable genes, such as IL-1R1 and RET, involved in ESR1-related pathways that could pave the way for developing new treatment strategies. Citation Format: Toshiaki Iwase, Hengyi Xu, Nakyung Oh, Jangsoon Lee, Alan M Lambowitz, Naoto Ueno. Resistance Mechanisms to CDK4/6 Inhibitors and/or Tamoxifen Using Comprehensive Thermostable Group II Intron Reverse Transcriptase Sequencing [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-23-11.

Pharmacovigilance of nephrotoxic drugs in neonates: the Pottel method for acute kidney injury detection in ELBW neonates

BackgroundExtremely low birth weight (ELBW) neonates (birth weight ≤ 1000 g) are at high risk to develop drug-induced acute kidney injury (AKI). However, we lack a pragmatic detection tool to capture their time-dependent (patho)physiologic serum creatinine (Scr) patterns. Pottel et al. suggested rescaling Scr by dividing Scr with the mean Scr value of the age- and sex-specific reference population. We explored if this Pottel method can detect drug-related nephrotoxicity in ELBW neonates.MethodsA previously reported dataset on Scr changes in ELBW neonates exposed to ibuprofen, amikacin, or vancomycin was updated to calculate Pottel scores for every available Scr value in the first 28 postnatal days. We hereby used previously published postnatal age-specific 50th centile values in an ELBW population. Linear mixed models were applied, analyzing Pottel scores as response variable and continuous time (day), drug exposure, and interaction thereof in the explanatory model.ResultsSerum creatinine (n = 3231) observations in 201 ELBW neonates were collected. A statistically significant rise of Pottel scores was observed with ibuprofen starting from postnatal day 4. In addition, a cumulative effect of treatment with mean Pottel scores on day 0 of 1.020 and on day 3 during treatment of 1.106 (95% CI 1.068–1.145, p < 0.001) was observed, corrected for effect of antibiotics. Antibiotic administrations showed a small but statistically significant difference up to postnatal day 5.ConclusionsAs rescaled Scr biomarker, the Pottel method showed a clear association with ibuprofen-exposed ELBW neonates, suggesting its applicability as a pragmatic bedside alternative tool to assess nephrotoxicity.Graphical abstractA higher resolution version of the Graphical abstract is available as Supplementary information

Abstract 4608: Combinations of Cdc-like kinase (CLK) inhibitors with targeted oncology agents or standard chemotherapy in patient-derived multi-cell type tumor spheroids

Abstract The alternative splicing of mRNA precursors allows one gene the capacity to yield multiple protein isoforms, each with distinct functions. Although this intricate process is subject to stringent regulation under normal physiological conditions, aberrant alternative splicing can generate atypical proteins and contribute to various diseases, including cancer. By activating splicing factors, Cdc-like kinases (CLKs) serve as pivotal regulators of alternative splicing and are considered promising therapeutic targets for various tumors. In this study, we investigated the activity of two CLK inhibitors, cirtuvivint (SM08502) and CC-671, in combination with precision oncology agents or conventional chemotherapeutics. Thirty highly characterized patient-derived cancer cell lines were selected from the National Cancer Institute’s Patient-Derived Models Repository (https://pdmr.cancer.gov/models/database.htm). The cell lines were derived from a range of cancer types including head and neck, bladder, pancreas, endometrial, colon, and melanoma, and contained clinically relevant variants of KRAS (G12C, G12D). Multi-cell type tumor spheroids were grown from a ratio of 6:2.5:1.5 malignant cells, endothelial cells, and mesenchymal stem cells, respectively. Following three days of growth, the spheroids were treated with single agents or combinations at concentrations up to their clinical Cmax value, if reported. After seven days of continuous drug exposure, cell viability was assessed using the CellTiter-Glo 3D assay. As a single agent, cirtuvivint showed concentration-dependent activity in all spheroid types with 1 – 3 logs of cytotoxicity, whereas CC-671 did not show activity in all spheroid types and achieved no more than 1 log of cytotoxicity. Among the standard chemotherapeutic drugs, doxorubicin and SN-38 demonstrated additive and greater-than-additive cytotoxicity in various spheroid types when combined with either CLK inhibitor. Several of the targeted oncology drugs exhibited noteworthy additive and greater-than-additive cytotoxicity when combined with a CLK inhibitor. These agents included the XPO1 inhibitor, eltanexor, the MCL-1 inhibitor, tapotoclax, the α-isoform-specific PI3K inhibitor, inavolisib, and the pan-PI3K inhibitor, copanlisib. Notably, combinations of the CLK inhibitors with the KRAS G12D variant-specific inhibitor, MRTX-1133, showed selective activity against all tumor cell lines harboring this genetic variant. Interestingly, a strong antagonistic interaction between paclitaxel and CC-671 was observed in all thirty spheroid types, while no such interaction was observed with cirtuvivint. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Thomas Steven Dexheimer, Thomas Silvers, Nathan P. Coussens, Steven D. Gore, James H. Doroshow, Beverly A. Teicher. Combinations of Cdc-like kinase (CLK) inhibitors with targeted oncology agents or standard chemotherapy in patient-derived multi-cell type tumor spheroids [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 4608.

Antibacterial properties of citral against Staphylococcus aureus: From membrane damage to metabolic inhibition

Citral, a natural antimicrobial agent, has been found to induce severe membrane damage resulting in bacterial death. Despite its effectiveness, there is a lack of in-depth research on its interaction with bacterial cells, especially at low concentrations. This work aimed to investigate the impact of citral concentration on its antibacterial effect against Staphylococcus aureus (S. aureus) and to analyze the relevance between membrane damage and cell death. In this study, the minimal inhibitory concentration of citral against S. aureus was measured at 0.25 mg/mL. At a concentration of 0.5 mg/mL, citral significantly disturbed the cell membrane fluidity and permeability, leading to the leakage of intracellular macromolecules and rapid cell death. However, when the concentration was decreased to 0.25 mg/mL, the membrane damage was greatly reduced, and a complex biological process between citral and membrane components was found. Under continuous drug exposure, the cell membrane underwent gradual rigidification, concomitant with the loss of membrane function, ultimately causing cell death. Despite the fact that no notable disturbance to membrane functions was discovered, the presence of 0.125 mg/mL citral could still weaken the metabolic vitality and growth of S. aureus. Therefore, it was suggested that there could be multiple mechanisms of citral action on S. aureus cells, with drug concentration being a critical factor affecting the mode of action. Overall, the findings of this study provide valuable insights into the antibacterial mechanisms of natural compounds and broaden the potential applications of citral as a natural antimicrobial.

Novel Perspectives on the Design and Development of a Long-Acting Subcutaneous Raltegravir Injection for Treatment of HIV-In Vitro and In Vivo Evaluation.

Antiretrovirals (ARVs) are a highly effective therapy for treatment and prevention of HIV infection, when administered as prescribed. However, adherence to lifelong ARV regimens poses a considerable challenge and places HIV patients at risk. Long-acting ARV injections may improve patient adherence as well as maintaining long-term continuous drug exposure, resulting in improved pharmacodynamics. In the present work, we explored the aminoalkoxycarbonyloxymethyl (amino-AOCOM) ether prodrug concept as a potential approach to long-acting ARV injections. As a proof of concept, we synthesised model compounds containing the 4-carboxy-2-methyl Tokyo Green (CTG) fluorophore and assessed their stability under pH and temperature conditions that mimic those found in the subcutaneous (SC) tissue. Among them, probe 21 displayed very slow fluorophore release under SC-like conditions (98% of the fluorophore released over 15 d). Compound 25, a prodrug of the ARV agent raltegravir (RAL), was subsequently prepared and evaluated using the same conditions. This compound showed an excellent in vitro release profile, with a half-life (t½) of 19.3 d and 82% of RAL released over 45 d. In mice, 25 extended the half-life of unmodified RAL by 4.2-fold (t½ = 3.18 h), providing initial proof of concept of the ability of amino-AOCOM prodrugs to extend drug lifetimes in vivo. Although this effect was not as pronounced as seen in vitro-presumably due to enzymatic degradation and rapid clearance of the prodrug in vivo-the present results nevertheless pave the way for development of more metabolically stable prodrugs, to facilitate long-acting delivery of ARVs.

Abstract 4905: Anticancer activity and paraptosis induction of novel bipyridine-silver(I) compounds against resistant colorectal and ovarian cancer cell lines

Abstract Background: Platinum-based anticancer drugs (e.g. Carboplatin and Oxaliplatin) are frequently applied in chemotherapy regimens against several types of cancer including ovarian carcinoma (OC) and colorectal carcinoma (CRC). However, intrinsic or acquired tumor resistance severely limit their clinical application. Here, we investigated the anticancer activity of several novel silver(I) 2,2’-bipyridine derivatives, containing either triphenylphosphane (PPh3) or 1,2-bis(diphenylphosphino)ethane (dppe) ligands and tested their potential to overcome platinum resistance. Methods: Cytotoxicity and cross resistance profiles of the newly synthesized compounds were tested against two OC models (SKOV-3 and MES-OV), a CRC model (HCT-116) and their corresponding sublines resistant to carboplatin or oxaliplatin, as well as non-malignant fibroblasts (F331) via an MTT-based cell viability assay after 72 h continuous drug exposure. Moreover, the intracellular levels of silver were measured via ICP-MS. In addition, thioredoxin reductase (TrxR) inhibition properties, impact on cell cycle distribution and apoptosis induction potential were investigated via multiple molecular biological methods. Morphological changes induced by the silver compounds were characterized via spinning disk confocal microscopy. Results: All tested compounds displayed pronounced anticancer activity and cancer cell selectivity. Moreover, cell intrinsic resistance to carboplatin or oxaliplatin was not impacting on the cytotoxic activity of the compounds. Noteworthy, MES-OV and HCT-116 cells showed exceptional sensitivity to several silver(I) 2,2’-bipyridine derivatives with a 1,2-bis(diphenylphosphino)ethane (dppe) (AP-compounds). This sensitivity was not based on enhanced drug uptake, pronounced TrxR inhibition nor apoptosis induction or cell cycle arrest. In contrast, hypersensitive cells displayed pronounced endoplasmic reticulum (ER)-derived vesicles together with multiple others hallmarks of paraptotic cell death. Conclusion: Our findings did not only demonstrate that the new silver compounds were unaffected by cancer cell intrinsic resistance mechanisms to platinum-based chemotherapy, but also indicated a high vulnerability of tumor (sub)types towards these bipyridine silver complexes. This hypersensitivity is based on the induction of paraptosis, a novel form of programmed cell death. To summarize, these silver compounds represent a new class of promising anticancer drugs, which demand further preclinical development. Citation Format: Alessia Stefanelli, Ricardo G. Teixeira, Adhan Pilon, Rebecca Warmers, Ingo Ott, Christian R. Kowol, Anamaria Brozovic, Ana Isabel Tomaz, Andreia Valente, Petra Heffeter. Anticancer activity and paraptosis induction of novel bipyridine-silver(I) compounds against resistant colorectal and ovarian cancer cell lines. [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 4905.

Data quality considerations for evaluating COVID-19 treatments using real world data: learnings from the National COVID Cohort Collaborative (N3C)

BackgroundMulti-institution electronic health records (EHR) are a rich source of real world data (RWD) for generating real world evidence (RWE) regarding the utilization, benefits and harms of medical interventions. They provide access to clinical data from large pooled patient populations in addition to laboratory measurements unavailable in insurance claims-based data. However, secondary use of these data for research requires specialized knowledge and careful evaluation of data quality and completeness. We discuss data quality assessments undertaken during the conduct of prep-to-research, focusing on the investigation of treatment safety and effectiveness.MethodsUsing the National COVID Cohort Collaborative (N3C) enclave, we defined a patient population using criteria typical in non-interventional inpatient drug effectiveness studies. We present the challenges encountered when constructing this dataset, beginning with an examination of data quality across data partners. We then discuss the methods and best practices used to operationalize several important study elements: exposure to treatment, baseline health comorbidities, and key outcomes of interest.ResultsWe share our experiences and lessons learned when working with heterogeneous EHR data from over 65 healthcare institutions and 4 common data models. We discuss six key areas of data variability and quality. (1) The specific EHR data elements captured from a site can vary depending on source data model and practice. (2) Data missingness remains a significant issue. (3) Drug exposures can be recorded at different levels and may not contain route of administration or dosage information. (4) Reconstruction of continuous drug exposure intervals may not always be possible. (5) EHR discontinuity is a major concern for capturing history of prior treatment and comorbidities. Lastly, (6) access to EHR data alone limits the potential outcomes which can be used in studies.ConclusionsThe creation of large scale centralized multi-site EHR databases such as N3C enables a wide range of research aimed at better understanding treatments and health impacts of many conditions including COVID-19. As with all observational research, it is important that research teams engage with appropriate domain experts to understand the data in order to define research questions that are both clinically important and feasible to address using these real world data.

Continuous dosing of nikkomycin Z against systemic candidiasis, in vivo and in vitro correlates.

Candidiasis is the most common cause of fungal sepsis, and new agents are of interest to ameliorate current deficiencies in therapy. Nikkomycin Z (NIKZ) is an inhibitor of chitin synthase, interfering with fungal cell wall development. We studied NIKZ therapy of disseminated murine candidiasis, via continuous drug exposure, in drinking water, to compensate for rapid clearance of the drug. Drinking, and thus drug intake in the NIKZ groups, as well as body weight, was affected by the degree of illness. NIKZ effect on survival, despite reduced drinking initially after infection, was highly efficacious and dose-related, and comparable to fluconazole, though neither were curative with the regimens employed. The challenge was rapidly lethal to all untreated animals, whereas NIKZ groups achieved >50% survival. Assays of residual fungal infection were consistent with impressions of efficacy based on survival. Although NIKZ MIC for Candida albicans appeared unpromising, mycelial formation assays more closely correlated with in vivo observations. In vitro-in vivo disparity may be explained by NIKZ tissue concentration in the target tissue and/or by enhanced NIKZ action on mycelial formation, a morphological change in vivo wherein chitin synthesis is more critical, compared to NIKZ activity in inhibiting planktonic growth. A sustained release oral form of NIKZ in drug development for humans could hold promise, possibly also in future exploring previously demonstrated synergy in vitro with other antifungals.

NSCLC Cells Resistance to PI3K/mTOR Inhibitors Is Mediated by Delta-6 Fatty Acid Desaturase (FADS2)

Hyperactivation of the phosphatidylinositol-3-kinase (PI3K) pathway is one of the most common events in human cancers. Several efforts have been made toward the identification of selective PI3K pathway inhibitors. However, the success of these molecules has been partially limited due to unexpected toxicities, the selection of potentially responsive patients, and intrinsic resistance to treatments. Metabolic alterations are intimately linked to drug resistance; altered metabolic pathways can help cancer cells adapt to continuous drug exposure and develop resistant phenotypes. Here we report the metabolic alterations underlying the non-small cell lung cancer (NSCLC) cell lines resistant to the usual PI3K-mTOR inhibitor BEZ235. In this study, we identified that an increased unsaturation degree of lipid species is associated with increased plasma membrane fluidity in cells with the resistant phenotype and that fatty acid desaturase FADS2 mediates the acquisition of chemoresistance. Therefore, new studies focused on reversing drug resistance based on membrane lipid modifications should consider the contribution of desaturase activity.

Should Patients with Waldenström Macroglobulinemia Receive a BTK Inhibitor as Frontline Therapy?

Waldenström Macroglobulinemia (WM) is a rare indolent lymphoma with heterogeneous clinical presentation. As there are no randomised trials suggesting the best treatment option in treatment-naive patients, guidelines suggest either rituximab-combining regimens or BTK-inhibitors (BTKi) as feasible alternatives. Several factors play in the decision-making process: patients’ age and fitness, disease characteristics and genotype. Chemoimmunotherapy (CIT) represents a fixed-duration, less expensive and effective option, able to achieve prolonged time-to-next treatment even in patients with unfavourable genotypes. Immunosuppression and treatment-related second cancers may represent serious concerns. Proteasome-inhibitor-based regimens are effective with rapid disease control, although bortezomib-related neuropathy discourages the choice of these agents and treatment schedules may not be easily manageable in the elderly. BTKi have demonstrated high rates of response and prolonged survival together with the convenience of an oral administration and limited cytopenias. However, outcomes are impacted by genotype and some concerns remain, in particular the continuous drug exposure that may result in extra-haematological complications and drug resistance. Although next-generation BTKi have improved treatment tolerance, the question whether BTKi should be offered as frontline therapy to every patient is still debated. Giving fixed-duration schedule, prolonged time-to-next treatment and outcomes independent of genotype, CIT is still our preferred choice in WM. However, BTKi remain a valuable option in frail patients unsuitable for CIT.

Continuous antioxidant drug exposure: a bridge from ideal world to real world of therapy for amyotrophic lateral sclerosis

Continuous antioxidant drug exposure: a bridge from ideal world to real world of therapy for amyotrophic lateral sclerosis

Abstract 5443: BPI-421286: A highly potent small molecule inhibitor targeting KRASG12C mutation

Abstract KRAS is a GTP-binding protein that links receptor tyrosine kinase activation to intracellular signaling. Mutation of KRAS favors the GTP-bound active state and constitutive activation of downstream effects. KRASG12C mutation has been identified as an oncogenic driver of tumorigenesis, which is found in approximately 14% of lung cancer, 4% of colorectal (CRC), and 1-3% of other solid tumors. Herein, we report BPI-421286, a highly potent and selective covalent small molecular KRASG12C inhibitor. In vitro, BPI-421286 showed a high Kinact/Ki value (256,000 M-1 s-1), and inhibited KRAS-GTP/GDP exchange with a lower IC50 (55 nM). In cell-based assays, BPI-421286 suppressed proliferation of KRASG12C mutant cell lines with sub-nanomolar potency, but did not affect cell lines with wide type or non-G12C KRAS mutations. BPI-421286 also inhibited phospho-ERK in MIA PaCa-2 cell with a low IC50 value (2.9 nM). In vivo, daily oral administration of 3-100 mg/kg BPI-421286 led to tumor growth suppression or regression in multiple KRASG12C mutant xenograft or PDX models of NSCLC, CRC and PDAC. The in vivo PKPD in MIA PaCa-2 xenograft model showed that durable pERK inhibition can be achieved without continuous drug exposure, featuring BPI-421286 as a covalent, non-reversible KRAS inhibitor. Combining BPI-421286 with several targeted agents, including a SHP2 inhibitor BPI-442096 and a CDK4/6 inhibitor BPI-16350, demonstrated enhanced tumor growth inhibition compared to either agent alone. Notably, for the acquired resistance mutations that are clinically observed after KRASG12C inhibitor treatment, such as G12C/H95D, G12C/H95Q, G12C/R68S and G12C/Y96C, BPI-421286 also exhibited strong inhibitory activities, suggesting that BPI-421286 could overcome the acquired resistance of currently approved KRASG12C inhibitors. Taken together, BPI-421286 is a highly potent and selective KRASG12C inhibitor, with favorable ADME properties and wide therapeutic index in pre-clinical toxicology studies. BPI-421286 is currently in Phase 1 clinical study. Citation Format: Xiaoguan Zhu, Yuan Lu, Jing Guo, Dan Yan, Boyan Li, Xiaoping Chen, Haibo Chen, Hong Lan, Hao Wu, Lieming Ding, Jiabing Wang. BPI-421286: A highly potent small molecule inhibitor targeting KRASG12C mutation [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 5443.

Clemizole and trazodone are effective antiseizure treatments in a zebrafish model of STXBP1 disorder.

CRISPR‐Cas9–generated zebrafish carrying a 12 base‐pair deletion in stxbpb1b, a paralog sharing 79% amino acid sequence identity with human, exhibit spontaneous electrographic seizures during larval stages of development. Zebrafish stxbp1b mutants provide an efficient preclinical platform to test antiseizure therapeutics. The present study was designed to test antiseizure medications approved for clinical use and two recently identified repurposed drugs with antiseizure activity. Larval homozygous stxbp1b zebrafish (4 days postfertilization (dpf)) were agarose‐embedded and monitored for electrographic seizure activity using a local field recording electrode placed in midbrain. Frequency of ictal‐like events was evaluated at baseline and following 45 min of continuous drug exposure (1 mM, bath application). Analysis was performed on coded files by an experimenter blinded to drug treatment and genotype. Phenytoin (PHT), valproate (VPA), ethosuximide (ESX), levetiracetam (LEV), and diazepam (DZP) had no effect on the ictal‐like event frequency in stxbp1b mutant zebrafish. Clemizole and trazodone decreased ictal‐like event frequency in stxbp1b mutant zebrafish by 80% and 83%, respectively. These results suggest that repurposed drugs with serotonin receptor–binding affinities could be effective antiseizure treatments. Clemizole and trazodone were previously identified in a larval zebrafish model for Dravet syndrome. Based primarily on these preclinical zebrafish studies, compassionate‐use and double‐blind clinical trials with both drugs have progressed. The present study extends this approach to a preclinical zebrafish model representing STXBP1 (syntaxin‐binding protein 1)‐related disorders and suggests that future clinical studies may be warranted.

DIPG Harbors Alterations Targetable by MEK Inhibitors, with Acquired Resistance Mechanisms Overcome by Combinatorial Inhibition.

We report alterations in the MAPK pathway in DIPGs to confer initial sensitivity to targeted MEK inhibition. We further identify for the first time the mechanism of resistance to single-agent targeted therapy in these tumors and suggest a novel combinatorial treatment strategy to overcome it in the clinic. This article is highlighted in the In This Issue feature, p. 587.

Prolonged Exposure to Oxaliplatin during HIPEC Improves Effectiveness in a Preclinical Micrometastasis Model.

Simple SummaryAbsence of survival benefits when adding hyperthermic intraperitoneal chemotherapy (HIPEC) with oxaliplatin to cytoreductive surgery in peritoneal metastasis from colorectal cancer has recently been shown in the randomized controlled PRODIGE 7 trial. We therefore aimed to investigate the effects of this treatment modality in a preclinical micrometastasis model. Cancer cells were incubated with either patient samples obtained during HIPEC procedures or with defined oxaliplatin-containing solutions prepared according to clinically established HIPEC protocols. Our results demonstrate a limited effectiveness of short-term HIPEC in simulations with oxaliplatin to eliminate micrometastases, although we used platinum-sensitive cell lines for our model. Since these results are in line with findings from current research, our studies might offer further convincing evidence and potential explanations for HIPEC futility observed in clinical application.Cytoreductive surgery combined with hyperthermic intraperitoneal chemotherapy (HIPEC) was considered a promising treatment for patients with peritoneal metastasis from colorectal cancer. However, the recently published randomized controlled PRODIGE 7 trial failed to demonstrate survival benefits through the addition of short-term oxaliplatin-based HIPEC. Constituting a complex multifactorial treatment, we investigated HIPEC in a preclinical model concerning the elimination of minimal tumor residues, thereby aiming to better understand the size of effects and respective clinical trial results. Patient samples of peritoneal perfusates obtained during HIPEC treatments and oxaliplatin-containing solutions at clinically relevant dosages, conforming with established HIPEC protocols, were assessed regarding their ability to eliminate modelled ~100 µm thickness cancer cell layers. Impedance-based real-time cell analysis and classical end-point assays were used. Flow cytometry was employed to determine the effect of different HIPEC drug solvents on tumor cell properties. Effectiveness of peritoneal perfusate patient samples and defined oxaliplatin-containing solutions proved limited but reproducible. HIPEC simulations for 30 min reduced the normalized cell index below 50% with peritoneal perfusates from merely 3 out of 9 patients within 72 h, indicating full-thickness cytotoxic effects. Instead, prolonging HIPEC to 1 h enhanced these effects and comprised 7 patients’ samples, while continuous drug exposure invariably resulted in complete cell death. Further, frequently used drug diluents caused approximately 25% cell size reduction within 30 min. Prolonging oxaliplatin exposure improved effectiveness of HIPEC to eliminate micrometastases in our preclinical model. Accordingly, insufficient penetration depth, short exposure time, and the physicochemical impact of drug solvents may constitute critical factors.

Pharmacokinetic Characteristics of Long-Acting Injectable Antipsychotics for Schizophrenia: An Overview.

The availability of long-acting injectable (LAI) antipsychotics for the treatment of schizophrenia provides clinicians with options that deliver continuous drug exposure and may improve adherence compared with daily oral antipsychotics. However, all LAI antipsychotics have unique formulations and pharmacokinetic characteristics that have implications for medication selection, administration interval, and injection site. This review outlines key differences in drug formulations and pharmacokinetics among LAI antipsychotics. A systematic search of the PubMed database was conducted to identify physical and formulation properties and pharmacokinetic data of commercially available LAI antipsychotics, including flupentixol decanoate, fluphenazine decanoate, haloperidol decanoate, zuclopenthixol decanoate, aripiprazole monohydrate, aripiprazole lauroxil, olanzapine pamoate, paliperidone palmitate, risperidone microspheres, and risperidone polymeric microspheres. Additional information was obtained from package inserts and product monographs. Relevant data on drug properties, administration details, pharmacokinetic parameters, and oral dose equivalencies of LAI antipsychotics are summarized. Based on our analysis, formulation characteristics (e.g., vehicle medium) and administration characteristics (e.g., injection site) can affect rate of absorption and adverse effects and may factor into whether oral supplementation or an additional injection is needed. Dose adjustments may be necessary based on potential drug–drug interactions, and approximate dose equivalence with oral formulations can help inform titration when switching from oral to LAI formulations. Clinicians administering LAI antipsychotics should consider these formulation and pharmacokinetic factors to maximize clinical impact and to adjust to an individual patient’s needs and treatment goals.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40263-020-00779-5.

MODL-19. DIPG HARBOUR ALTERATIONS TARGETABLE BY MEK INHIBITORS, WITH ACQUIRED RESISTANCE MECHANISMS OVERCOME BY COMBINATORIAL UP- OR DOWN-STREAM INHIBITION

The survival of children with DIPG remains dismal, with new treatments desperately needed. In the era of precision medicine, targeted therapies represent an exciting treatment opportunity, yet resistance can rapidly emerge, playing an important role in treatment failure. In a prospective biopsy-stratified clinical trial (BIOMEDE), we combined detailed molecular profiling (methylation BeadArray, exome, RNAseq, phospho-proteomics) linked to drug screening in newly-established patient-derived models of DIPG in vitro and in vivo. We identified a high degree of in vitro sensitivity to the MEK inhibitor trametinib (GI50 16-50nM) in samples which harboured genetic alterations targeting the MAPK pathway, including the non-canonical BRAF_G469V mutation, and those affecting PIK3R1. Treatment of PDX models and the patient with trametinib at relapse, however, failed to elicit a significant response. We generated trametinib-resistant clones (62-188-fold, GI50 2.4–5.2µM) in the BRAF_G469V model through continuous drug exposure, and identified acquired mutations in MEK1/2 (MEK1_K57N, MEK1_I141S and MEK2_I115N) with sustained pathway up-regulation. These cells showed the hallmarks of mesenchymal transition, with overexpression of key proteins involved in invasion/migration, such as collagen-family proteins, integrins, MMPs and AHNAK2, amongst others. Resistant clones were conversely sensitive to the upstream receptor tyrosine kinase inhibitor dasatinib (GI50 36-93nM), and combinations of trametinib with dasatinib and the downstream ERK inhibitor ulixertinib showed synergistic effects in vitro. These data highlight the MAPK pathway as a therapeutic target in DIPG, and show the importance of parallel resistance modelling and rational combinatorial treatments likely to be required for meaningful clinical translation.

DDRE-07. DIPG HARBOUR ALTERATIONS TARGETABLE BY MEK INHIBITORS, WITH ACQUIRED RESISTANCE MECHANISMS OVERCOME BY COMBINATORIAL INHIBITION

Abstract The survival of children with diffuse intrinsic pontine glioma (DIPG) remains dismal, with new treatments desperately needed. In the era of precision medicine, targeted therapies represent an exciting treatment opportunity, yet resistance can rapidly emerge, playing an important role in treatment failure. In a prospective biopsy-stratified clinical trial, we combined detailed molecular profiling (methylation BeadArray, exome, RNAseq, phospho-proteomics) linked to drug screening in newly-established patient-derived models of DIPG in vitro and in vivo. We identified a high degree of in vitro sensitivity to the MEK inhibitor trametinib (GI50 16-50nM) in samples, which harboured genetic alterations targeting the MAPK pathway, including the non-canonical BRAF_G469V mutation, and those affecting PIK3R1 and NF1. However, treatment of PDX models and of a patient with trametinib at relapse failed to elicit a significant response. We generated trametinib-resistant clones (62-188-fold, GI50 2.4–5.2µM) in the BRAF_G469V model through continuous drug exposure, and identified acquired mutations in MEK1/2 (MEK1_K57N, MEK1_I141S and MEK2_I115N) with sustained pathway up-regulation. These cells showed the hallmarks of mesenchymal transition, and expression signatures overlapping with inherently trametinib-insensitive primary patient-derived cells that predicted an observed sensitivity to dasatinib. Combinations of trametinib with dasatinib and the downstream ERK inhibitor ulixertinib showed highly synergistic effects in vitro. These data highlight the MAPK pathway as a therapeutic target in DIPG, and show the importance of parallel resistance modelling and rational combinatorial treatments likely to be required for meaningful clinical translation.

Abstract 312: Multicellular spheroids of A549 cells: A clinically relevant model of lung cancer

Abstract Purpose: This project aims to test the hypothesis that multicellular spheroids (MCS) of human lung cancer cells A549 can better mimic non-small cell lung cancer's (NSCLC) sensitivity to anticancer drugs in clinic than the corresponding monolayer cells. Methods: 2D monolayers and MCS of A549 cells were treated with anticancer drugs cisplatin, carboplatin, and doxorubicin. The ability of the drugs to suppress cell growth was compared. A549 cells were seeded in 96 well plates (monolayer) and 96 well ultra-low attachment plates (MCS) at 5000 cells/well. The drugs were given in a series of concentrations 24 hours or 5 days after seeding. IC50 values were obtained from cell viability assays after 72 h drug exposure. MCS of fluorescently labeled A549 cells (A549-iRFP) were treated with select anticancer drugs by a cycled dosing schedule based on guidelines for NSCLC from National Comprehensive Cancer Network with modifications to accommodate for the life span of MCS. MCS were treated by 4 cycles, 7 days/cycle. In each cycle, carboplatin was dosed on day 1, and gemcitabine on day 1 and day 4. Drug concentration was reduced by media change according to the blood half-life of the drug to mimic its pharmacokinetics (PK). Drug dosage was based on the target clinical AUC or 1/10 of the peak plasma concentration. IC50 values were obtained from MCS fluorescence and viability. Results: IC50 values of cisplatin in A549 monolayer and MCS were 9.73 μM and 20.71 μM, respectively; those of carboplatin 131.80 μM and 188.90 μM; those of doxorubicin 0.61 μM and 6.57 μM; those of gemcitabine 0.027 μM and &amp;gt;250 μM. Ratios of IC50 value in MCS to monolayer were calculated for each drug, which were 2.13 in cisplatin, 1.43 in carboplatin and 10.74 in doxorubicin. Cisplatin and Carboplatin are indicated in NSCLC but require much higher concentration to inhibit 50% of monolayer cell growth than Doxorubicin, which is not indicated in NSCLC cancer. Although IC50 values in the monolayer cell model would suggest doxorubicin as a more promising drug than platinum drugs against lung cancer, the ratio of doxorubicin's IC50 values in MCS over monolayers is as high as 10.74 folds, which is 3 to 7 times as high as the two platinum drugs. The two different drug dosages based on target clinical AUC and on 1/10 of peak plasma concentration yielded similar suppression of MCS growth after the cycled drug treatment. However, at the same drug dosage for the cycled drug treatment, PK-mimetic media exchange substantially decreased the MCS growth suppression compared to 48 h continuous drug exposure, which is commonly used in current cell culture assays on anticancer drugs. Conclusion: MCS of A549 cells better correlated with the efficacy of an anticancer drug in clinic than the monolayer. MCS can also evaluate anticancer drugs by pulsed drug exposure based on its pharmacokinetics, and by cycled dosing regiments as commonly used in clinic. Citation Format: Xinyu Pei, Henry Ling, Yifan Lu, Mallika Vadlamudi, Yingbo Huang, Ruiqi Huang, Shen Zhao, Zizhao Xu, Zhongyue Yuan, Yong Zhu, Myo-Kyoung Kim, Xin Guo. Multicellular spheroids of A549 cells: A clinically relevant model of lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 312.