Anticancer Tyrosine Kinase Inhibitors Research Articles

Pharmacological inhibition of PDGF-C/neuropilin-1 interaction: A novel strategy to reduce melanoma metastatic potential

Activation of neuropilin-1 (NRP-1) by platelet derived growth factor (PDGF)-C sustains melanoma invasiveness. Therefore, in the search of novel agents capable of reducing melanoma spreading, PDGF-C/NRP-1 interaction was investigated as a potential druggable target. Since the PDGF-C region involved in NRP-1 binding is not yet known, based on the sequence and structural homology between PDGF-C and vascular endothelial growth factor-A (VEGF-A), we hypothesized that the NRP-1 b1 domain region involved in the interaction with VEGF-A might also be required for PDGF-C binding. Hence, this region was selected from the protein crystal structure and used as target in the molecular docking procedure. In the following virtual screening, compounds from a DrugBank database were used as query ligands to identify agents potentially capable of disrupting NRP-1/PDGF-C interaction. Among the top 45 candidates with the highest affinity, five drugs were selected based on the safety profile, lack of hormonal effects, and current availability in the market: the antipsychotic pimozide, antidiabetic gliclazide, antiallergic cromolyn sodium, anticancer tyrosine kinase inhibitor entrectinib, and antihistamine azelastine. Analysis of drug influence on PDGF-C in vitro binding to NRP-1 and PDGF-C induced migration of human melanoma cells expressing NRP-1, indicated gliclazide and entrectinib as the most specific agents that were active at clinically achievable and non-toxic concentrations. Both drugs also reverted PDGF-C ability to stimulate extracellular matrix invasion by melanoma cells resistant to BRAF inhibitors. The inhibitory effect on tumor cell motility involved a decrease of p130Cas phosphorylation, a signal transduction pathway activated by PDGF-C-mediated stimulation of NRP-1.

Synthesis of Bis-thiazoles Tethered 1,4-Dihydropyridine and Pyridine Linkers via Simple Oxidation and their Molecular Docking as VEGFR -TK Inhibitors.

In this study, a neoteric and expedient oxidation method is applied for a variety of Hantzsch 1,4-dihydropyridine derivatives such as 1,4-dihydro- 2,6-dimethyl-3,5-diacetylpyridine, 3,5-bis-hydrazono--2,6-dimethyl-1,4-dihydropyridine, and 3,5-bis-thiazoly-2,6-dimethyl-1,4-dihydro pyridine. This simple oxidation is based upon the in situ generation of nitrous acid from an aqueous sodium nitrite and acetic acid mixture and could be used to downgrade costs, sustain resources, and minimize chemical wastes. Also, a molecular modeling strategy was used to study the mechanism of action for various derivatives of bis-hydrazinylidene- thiazole as the protein Vascular Endothelial Growth Factor Receptor Tyrosine Kinase (VEGFR TK) inhibitor through evaluating their binding scores and modes compared with Sorafenib as a reference standard. The results revealed that the interaction of hydrazinylidene and thiazole as an anticancer Tyrosine Kinase inhibitor has been improved. Additionally, the compounds exhibiting the highest activity were assessed for their potential anticancer effects against HepG-2, MCF-7, and WI-38 cells, and the outcomes demonstrated encouraging activity against cancer.

Novel Phthalic-Based Anticancer Tyrosine Kinase Inhibitors: Design, Synthesis and Biological Activity.

In this work, fragments of isophthalic and terephthalic acids are proposed as a structural scaffold to develop potential inhibitors of protein kinases. Novel isophthalic and terephthalic acid derivatives were designed as type-2 protein kinase inhibitors, synthesized and subjected to physicochemical characterization. The screening of their cytotoxic actions against a panel of cell lines derived from different types of tumors (liver, renal, breast and lung carcinomas, as well as chronic myelogenous and promyelocytic leukemia) and normal human B lymphocyte, for the sake of comparison, was performed. Compound 5 showed the highest inhibitory activity against four cancer cell lines, K562, HL-60, MCF-7 and HepG2 (IC50 = 3.42, 7.04, 4.91 and 8.84 µM, respectively). Isophthalic derivative 9 revealed a high potency against EGFR and HER2, at the levels of 90% and 64%, respectively, being comparable to lapatinib at 10 µM. In general, tumor cell cultures were more sensitive to isophthalic acid derivatives than to terephthalic acid ones. In cell cycle studies, isophthalic analogue 5 showed a pronounced dose-dependent effect, and with the increase in its concentration up to 10.0 µM, the number of living cells decreased to 38.66%, while necrosis reached 16.38%. The considered isophthalic compounds had a similar docking performance to that of sorafenib against the VEGFR-2 (PDB id: 4asd, 3wze). The correct binding of compounds 11 and 14 with VEGFR-2 was validated using MD simulations and MM-GPSA calculations.

Development of optimized pyrimido-thiazole scaffold derivatives as anticancer and multitargeting tyrosine kinase inhibitors using computational studies

Cancer is one of the frontier causes of leading death ratesglobally and one of the most researched domains. Despite many drugs available in the market currently, their effectiveness towards resistance, randomly mutating cancer targets, and side effects initiate the necessity of developing target selective and effective anti-cancer drugs.This study aimed to develop a congeneric series of fused pyrimidine and thiazole-containing hybrid series and evaluate its anticancer potential using in-silico tools. Molecular modeling strategies were used to develop various derivatives of fused thiazole-pyrimidines as anticancer agents for target-based drug discovery. Strategically, based on Structural Activity Relationship (SAR), of the synthesized or studied compounds, a combinatorial library was generated using the lead grow tool to develop a possible combination of various derivatives. Using Swiss ADME and Osiris; the ADMETproperties were predicted and screened molecules were docked using Auto-dock vina and Swiss Dockto obtain possible interactions with 5 listed targets of Tyrosine Kinase Inhibitors (TKI's) namely BCR-ABL kinase (PDB- 2GQG), c-ABL Kinase (PDB-3PYY), EGFR(PDB-1M17), VEGFR(PDB- 4ASD), and BRAF inhibitors (PDB- 2FB8). SAR of the pyrimido-thiazole molecules was derived from the literature survey and substitutions that enhance the anticancer activity were incorporated into the Combilib to generate a library of 221 compounds. This library of compounds was subjected to a Lipinski Filter. About 45 compounds were screened and tested for their drug-likeliness parameters. The 24 compounds that qualified with good absorption, drug-likeliness, and ADME were tested for their toxicity for developing relatively safe drugs with low side effects. These molecules were docked intofivedifferent PDB's and their binding affinity scores along with prominent interactions were noted. Compound 33 has shown comparable binding affinity and key interactions with all the five PDB's and thus can be used as a lead for further investigations. These studies have helped to optimize the relationship between fused pyrimidine and thiazoles as anticancer Tyrosine Kinase Inhibitors.

Adrenal Insufficiency with Anticancer Tyrosine Kinase Inhibitors Targeting Vascular Endothelial Growth Factor Receptor: Analysis of the FDA Adverse Event Reporting System.

Simple SummaryThis real-world post-marketing research described clinical features of adrenal insufficiency with anticancer drugs targeting vascular endothelial growth factor receptor reported to the Food and Drug Administration pharmacovigilance database. A robust signal emerged for multi-targeted tyrosine kinase inhibitors, especially in combination with immunotherapy, namely checkpoint inhibitors in renal cancer. These signals should promote both prospective research and a multidisciplinary proactive monitoring by healthcare professionals. These findings strengthen the role of timely pharmacovigilance to detect and characterize post-marketing adverse events of special interest, thus supporting patient care.Background: We described clinical features of adrenal insufficiency (AI) reported with tyrosine kinase inhibitors (TKIs) targeting vascular endothelial growth factor receptor (VEGFR) in the Food and Drug Administration Adverse Event Reporting System (FAERS). Methods: Reports of AI recorded in FAERS (January 2004–March 2022) were identified through the high-level term “adrenal cortical hypofunctions”. Demographic and clinical features were inspected, and disproportionality signals were detected through the Reporting Odds Ratio (ROR) and Information Component (IC) with relevant 95% confidence/credibility interval (CI), using different comparators and adjusting the ROR for co-reported corticosteroids and immune checkpoint inhibitors (ICIs). Results: Out of 147,153 reports with VEGFR-TKIs, 314 cases of AI were retained, mostly of which were serious (97.1%; hospitalization recorded in 44.9%). In a combination regimen with ICIs (43% of cases), VEGFR-TKIs were discontinued in 52.2% of the cases (26% as monotherapy). The median time to onset was 72 days (IQR = 14–201; calculated for 189 cases). A robust disproportionality signal emerged, also in comparison with other anticancer drugs (ROR = 2.71, 95%CI = 2.42–3.04; IC = 0.25, 95%CI = 0.07–0.39). Cabozantinib, sunitinib and axitinib generated robust disproportionality even after ROR adjustment. Conclusions: We call pharmacologists, internists, oncologists and endocrinologists to raise awareness of serious AI with VEGFR-TKIs, and to develop dedicated guidelines, especially for combination regimens with immunotherapy.

Major adverse cardiovascular events associated with VEGF-targeted anticancer tyrosine kinase inhibitors: a real-life study and proposed algorithm for proactive management

BackgroundVascular endothelial growth factor receptor (VEGFR)-targeted tyrosine kinase inhibitors (TKIs) are widely used in cancer treatment and burdened by cardiovascular toxicity. The majority of data come from clinical trials, thus in selected populations. The aim of our study is to evaluate the cardiotoxicity profile of VEGFR-targeted TKIs and the impact of cardiovascular risk factors in a real-life population.Patients and methodsIn this cohort, population-based study, patients treated with VEGFR-targeted TKIs, bevacizumab and trastuzumab between 2009 and 2014 were analyzed. A multi-source strategy for data retrieval through hospital, pharmaceutical and administrative databases of the Lombardy region, Italy, has been adopted. The primary endpoint was to determine the incidence and type of major adverse cardiovascular events (MACEs) along with their temporal trend. The secondary endpoint was to define the impact of cardiovascular risk factors in the occurrence of MACEs.ResultsA total of 829 patients were treated with VEGFR-targeted TKIs. Eighty-one MACEs occurred in the first year of follow-up [crude cumulative incidence (CCI): 9.79%] mainly consisting of arterial thrombotic events (ATEs, 31 events, CCI: 3.99%), followed by rhythm disorders (22 events, CCI: 2.66%), pulmonary embolisms and heart failures (13 events each, CCI: 1.57%). While the incidence of most MACEs showed a plateau after 6 months, ATEs kept increasing along the year of follow-up. Hypertension and dyslipidemia were associated with an increase in risk of ATEs [relative risk difference (RRD) +209.8% and +156.2%, respectively], while the presence of previous MACEs correlated with a higher risk of all MACEs in multivariate analysis (RRD 151.1%, 95% confidence interval 53.6% to 310.3%, P < 0.001).ConclusionsMACEs occur in a clinically significant proportion of patients treated with VEGFR-targeted TKIs, with ATEs being predominant, mainly associated with hypertension and dyslipidemia. A clinical algorithm for effective proactive management of these patients is warranted.

Abstract 365: Ponatinib Triggers Cardiac Inflammation by STAT-3 Dependent Immune Checkpoint Blockade

Background: Ponatinib is a potent anticancer tyrosine kinase inhibitor (TKI) with considerable cardiotoxicity. The manifestation of cardiovascular adverse events, including fatal myocardial infarction and congestive heart failure, has hampered its clinical use. Therefore, a better understanding of the mechanism by which ponatinib exerts cardiotoxicity is urgently warranted to efficiently counteract treatment-related adversities. Methods: Wild type C57BL/6, comorbidity mouse model ApoE -/- , and pressure overload (PO) mouse model were used to investigate the cardiotoxic mechanism of ponatinib. Echocardiography was performed to assess cardiac function. Flow cytometry analysis was performed to assess the dynamics of inflammation. Results: We observed that high-fat diet (HFD) fed ApoE -/- mice develop cardiac dysfunction within 2 weeks of ponatinib treatment. An unbiased RNA-Seq analysis revealed significant upregulation of inflammatory genes (CCR1, CCR5, CCL6, CCL8, CCL9, CXCL4) in ponatinib treated hearts. Since ApoE -/- background, and HFD are known confounder of inflammation signal, we validated this observation in naïve C57BL/6 mice. Despite the lack of cardiac dysfunction in ponatinib treated naïve C57BL/6 mice, comprehensive immune profiling depicted upregulation of myocardial inflammation as evident by infiltrated immune cells (CD45 + TNFα + , CD45 + CD11b + F4/80 + Ly6C + CCR2 + , CD45 + Gal1 + ). Interestingly, we also demonstrated the downregulation of immune checkpoints over T cells (TCRαβ + CTLA4 + , TCRαβ + PD1 + ) in ponatinib treated hearts. Next, in the PO mouse model, ponatinib treated mice showed significant cardiac dysfunction with myocardial inflammation as reflected by increased frequencies of inflammatory parameters (TNFα + , IL1β + , IL6 + , MCP1 + , CXCL9 + ). Mechanistically, we demonstrated that ponatinib potentially suppresses PD-L1 expression over cardiomyocytes via inhibition of STAT3, subsequently leading to immune cells mediated myocardial inflammation. Conclusions: These findings uncover a novel mechanism of ponatinib induced cardiac inflammation leading to adverse cardiac function. It also suggests that strategies to attenuate inflammation may be an effective therapy to prevent ponatinib induced cardiac adverse events.

Lipid droplet-mediated scavenging as novel intrinsic and adaptive resistance factor against the multikinase inhibitor ponatinib.

Ponatinib is a small molecule multi‐tyrosine kinase inhibitor clinically approved for anticancer therapy. Molecular mechanisms by which cancer cells develop resistance against ponatinib are currently poorly understood. Likewise, intracellular drug dynamics, as well as potential microenvironmental factors affecting the activity of this compound are unknown. Cell/molecular biological and analytical chemistry methods were applied to investigate uptake kinetics/subcellular distribution, the role of lipid droplets (LDs) and lipoid microenvironment compartments in responsiveness of FGFR1‐driven lung cancer cells toward ponatinib. Selection of lung cancer cells for acquired ponatinib resistance resulted in elevated intracellular lipid levels. Uncovering intrinsic ponatinib fluorescence enabled dissection of drug uptake/retention kinetics in vitro as well as in mouse tissue cryosections, and revealed selective drug accumulation in LDs of cancer cells. Pharmacological LD upmodulation or downmodulation indicated that the extent of LD formation and consequent ponatinib incorporation negatively correlated with anticancer drug efficacy. Co‐culturing with adipocytes decreased ponatinib levels and fostered survival of cancer cells. Ponatinib‐selected cancer cells exhibited increased LD levels and enhanced ponatinib deposition into this organelle. Our findings demonstrate intracellular deposition of the clinically approved anticancer compound ponatinib into LDs. Furthermore, increased LD biogenesis was identified as adaptive cancer cell‐defense mechanism via direct drug scavenging. Together, this suggests that LDs represent an underestimated organelle influencing intracellular pharmacokinetics and activity of anticancer tyrosine kinase inhibitors. Targeting LD integrity might constitute a strategy to enhance the activity not only of ponatinib, but also other clinically approved, lipophilic anticancer therapeutics.

Anti-cancer tyrosine kinase inhibitors increase oxidative stress in primary cardiac fibroblasts

Anti-cancer tyrosine kinase inhibitors (TKI) are known to exert cardiotoxicity that can result in heart failure. Here we have used primary adult rat cardiac fibroblasts (CFs) to examine whether imatinib and sunitinib (both TKIs) increase oxidative stress and cause increased oxidation and activation of calcium/calmodulin dependent protein kinase II (CaMKII). Activation of CaMKII is known to be important for cellular function but excessive activation is linked to cardiac pathology. CFs, isolated from adult Wistar rat hearts, were maintained in culture and treated over 24 hour with the TKIs sunitinib and imatinib (0.1–20 µM) in serum-free medium (DMEM). Cell phenotype and growth was monitored using a Nikon Eclipse (TE300) inverted microscope and ROS assays performed using a specially designed assay kit (Abcam, UK) which uses DCFDA to measure hydroxyl, peroxyl and other ROS activity. Quantitative immunoblotting was used to assess ox-CaMKII protein levels. Treatment with both TKIs caused a dose-dependent decrease in cell viability. Sunitinib exerted more toxic effects at lower concentrations than imatinib. Decreased cell viability was evident at concentrations as low as 1 µM for sunitinib with dramatic cell loss (>50%) evident at 10–20 µM. Significantly increased ROS levels (using the DFCDA-based assay) were observed in the presence of both imatinib (4956±102 vs 1984±358 (imatinib vs vehicle (a.u.), n=3, p In conclusion, both imatinib and sunitinib cause increased oxidative stress in adult primary CFs with resultant activation of CaMKII. These effects are evident at lower concentrations of sunitinib than for imatinib and reflect the increased cardiotoxicity of this TKI reported in the patient.

Exploring Structural Conformations of Human P450 1A1 with Diverse Ligands

The human P450 enzyme 1A1 (CYP1A1) is an extrahepatic P450 often expressed in tumor tissues where it may be clinically significant in procarcinogens and toxin bioactivation. Prototypical CYP1A1 ligands are planar and polycyclic aromatic compounds, such as the procarcinogen benzo[a]pyrene found in environmental combustion and tobacco smoke. The single crystal structure of CYP1A1 is with the polycyclic, planar molecule alpha‐napthoflavone (αNF) and reveals a narrow active site that is consistent with planar CYP1A1 ligands. However, a number of known CYP1A1 substrates and inhibitors do not adhere to these typical ligand features, including promising pro‐drugs in cancer therapy based on a duocarmycin scaffold and clinically used tyrosine kinase inhibitors. Such CYP1A1 ligands could not be accommodated in the active site of the CYP1A1/αNF structure. This study aims to determine how CYP1A1 binds atypical CYP1A1 substrates and inhibitors using co‐crystallization.Potential ligands for co‐crystallization trials with CYP1A1 were selected based on structural diversity, focusing on those that are not compatible with the 1A1/αNF crystal structure active site dimensions. Refinement of these ligands was performed by ranking based on their ability to inhibit CYP1A1 metabolism of a pro‐luciferin substrate. Anticancer tyrosine kinase inhibitors (erlotinib, imatinib, and ponatinib) displayed nanomolar to low micromolar IC50 values. A series of antifungal azoles, which are generally potent P450 inhibitors, resulted in a range of inhibitory responses. Compact, lower MW clotrimazole, miconazole, and tioconazole exhibited sub‐nanomolar potency, but the much larger extended azoles ketoconazole and posaconazole were also potent nanomolar inhibitors. The azoles fluconazole and letrozole were significantly less potent inhibitors with micromolar IC50 values. The pro‐duocarmycin drug ICT2700 and antitumor benzothiazole GW 610 inhibited CYP1A1 in the sub‐micromolar to nanomolar range. Compounds displaying potent inhibition are being employed in cocrystallization and structure determination. The structural results with atypical CYP1A1 ligands should provide a more robust framework for both prediction of procarcinogen and toxin activation and the development of novel CYP1A1‐activated cancer therapies.Support or Funding InformationThis work was supported by National Institutes of Health Grants F37 GM076343 (to EES).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exploring the crizotinib resistance mechanism of NSCLC with the L1196M mutation using molecular dynamics simulation.

Crizotinib is an anticancer tyrosine kinase inhibitor that is approved for use as a first-line treatment for some non-small-cell lung cancers. L1196M is the most frequently observed mutation in NSCLC patients. This mutation, known as the gatekeeper mutation in the ALK kinase domain, confers resistance to crizotinib by sterically blocking the binding of the drug. However, the molecular mechanism of crizotinib resistance caused by the L1196M mutation is still unclear. Molecular dynamics simulation was therefore utilized in this study to investigate the mechanism by which the L1196M mutation may affect crizotinib resistance. Our results suggest that larger fluctuations in some important regions of the mutant complex compared to the wild-type complex may contribute to the resistance of the mutant complex to crizotinib. Also, mutation-induced alterations to the secondary structure of the complex as well as unstable hydrogen-bonding patterns in the A-loop and P-loop regions decrease the total binding energy of the complex. This study therefore provides a molecular explanation for the resistance to crizotinib caused by the L1196M mutation, which could aid the design of more efficient and selective drugs.

Attenuation of Sunitinib-induced cardiotoxicity through the A3 adenosine receptor activation

Sunitinib is an anti-cancer tyrosine kinase inhibitor associated with severe cardiotoxic adverse effects. Using rat Langendorff heart model and human acute myeloid leukaemia 60 (HL60) cell line we detected the involvement of protein kinase C (PKC) α during Sunitinib-induced cardiotoxicity and the effect of Sunitinib on cancer progression. The cardioprotective and anti-cancer properties of the A3 adenosine receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (IB-MECA) were investigated.The cardiac effect of Sunitinib (1µM) and IB-MECA (1nM) treatment was measured through haemodynamic and infarct size assessment. The cytotoxic effect of Sunitinib (0.1 – 10μM) and IB-MECA (10nM – 10μM) on HL60 cells was assessed using the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay technique. Myocardial injury associated microRNAs (miR-1, miR-27a, miR-133a and miR-133b) and cancer associated microRNAs (miR-15a, miR-16-1 and miR-155) were profiled by qRT-PCR in the cardiac tissue and HL60 cells, while phosphorylated PKCα levels were measured by Western Blot analysis.Sunitinib treatment increased infarct size and decreased left ventricular developed pressure and heart rate. Co-treatment of IB-MECA reversed the myocardial injury produced by Sunitinib administration. IB-MECA did not jeopardize the anti-cancer effect of Sunitinib in HL60 cells. The expression signature of the specific microRNAs in cardiac tissue and HL60 cells showed an altered expression profile when treated with Sunitinib and IB-MECA. pPKCα levels were increased by Sunitinib treatment in cardiac tissue and HL60 cells and co-administration of IB-MECA attenuated this increase in the cardiac tissue.This study reveals that A3 adenosine receptor activation by IB-MECA attenuates Sunitinib-induced cardiotoxicity through the involvement of PKCα.

Synergistic tumor suppression by a Perilla frutescens-derived methoxyflavanone and anti-cancer tyrosine kinase inhibitors in A549 human lung adenocarcinoma.

Anti-cancer tyrosine kinase inhibitors (TKIs) are effective in many types of cancers including non-small cell lung cancer, while appearance of TKI-resistant tumors suggests a need for the development of their potentiation strategies. We have previously shown that a methoxyflavanone derivative from the Asian medicinal herb Perilla frutescens (Perilla-derived methoxyflavanone; PDMF) shows a prominent anti-tumor activity against A549 human lung adenocarcinoma. Here we show that PDMF and anti-cancer TKIs (nilotinib, bosutinib, dasatinib, and ponatinib) synergistically suppress proliferation of A549 cells. Flow cytometric analysis indicated that co-stimulation with nilotinib (4μM) and PDMF induced G2/M cell cycle arrest in low PDMF doses (10-50μM), whereas this combination triggered de novo G1 arrest in higher PDMF dosages (50-125μM). We also found that co-administration with nilotinib and PDMF significantly suppressed in vivo tumorigenicity of A549 cells in athymic nude mice.

High-throughput screening of tyrosine kinase inhibitor cardiotoxicity with human induced pluripotent stem cells.

Tyrosine kinase inhibitors (TKIs), despite their efficacy as anticancer therapeutics, are associated with cardiovascular side effects ranging from induced arrhythmias to heart failure. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), generated from 11 healthy individuals and 2 patients receiving cancer treatment, to screen U.S. Food and Drug Administration-approved TKIs for cardiotoxicities by measuring alterations in cardiomyocyte viability, contractility, electrophysiology, calcium handling, and signaling. With these data, we generated a "cardiac safety index" to reflect the cardiotoxicities of existing TKIs. TKIs with low cardiac safety indices exhibit cardiotoxicity in patients. We also derived endothelial cells (hiPSC-ECs) and cardiac fibroblasts (hiPSC-CFs) to examine cell type-specific cardiotoxicities. Using high-throughput screening, we determined that vascular endothelial growth factor receptor 2 (VEGFR2)/platelet-derived growth factor receptor (PDGFR)-inhibiting TKIs caused cardiotoxicity in hiPSC-CMs, hiPSC-ECs, and hiPSC-CFs. With phosphoprotein analysis, we determined that VEGFR2/PDGFR-inhibiting TKIs led to a compensatory increase in cardioprotective insulin and insulin-like growth factor (IGF) signaling in hiPSC-CMs. Up-regulating cardioprotective signaling with exogenous insulin or IGF1 improved hiPSC-CM viability during cotreatment with cardiotoxic VEGFR2/PDGFR-inhibiting TKIs. Thus, hiPSC-CMs can be used to screen for cardiovascular toxicities associated with anticancer TKIs, and the results correlate with clinical phenotypes. This approach provides unexpected insights, as illustrated by our finding that toxicity can be alleviated via cardioprotective insulin/IGF signaling.

Development of a Pharmacokinetic Model to Describe the Complex Pharmacokinetics of Pazopanib in Cancer Patients.

Pazopanib is a multi-targeted anticancer tyrosine kinase inhibitor. This study was conducted to develop a population pharmacokinetic (popPK) model describing the complex pharmacokinetics of pazopanib in cancer patients. Pharmacokinetic data were available from 96 patients from three clinical studies. A multi-compartment model including (i) a complex absorption profile, (ii) the potential non-linear dose-concentration relationship and (iii) the potential long-term decrease in exposure was developed. A two-compartment model best described pazopanib pharmacokinetics. The absorption phase was modelled by two first-order processes: 36% (relative standard error [RSE] 34%) of the administered dose was absorbed with a relatively fast rate (0.4h-1 [RSE 31%]); after a lag time of 1.0h (RSE 6%), the remaining dose was absorbed at a slower rate (0.1h-1 [RSE 28%]). The relative bioavailability (rF) at a dose of 200mg was fixed to 1. With an increasing dose, the rF was strongly reduced, which was modelled with an E max (maximum effect) model (E max was fixed to 1, the dose at half of maximum effect was estimated as 480mg [RSE 23%]). Interestingly, the plasma exposure to pazopanib also decreased over time, modelled on rF with a maximum magnitude of 50% (RSE 27%) and a first-order decay constant of 0.15day-1 (RSE 43%). The inter-patient and intra-patient variability on rF were estimated as 36% (RSE 16%) and 75% (RSE 22%), respectively. A popPK model for pazopanib was developed that illustrated the complex absorption process, the non-linear dose-concentration relationship, the high inter-patient and intra-patient variability, and the first-order decay of pazopanib concentration over time. The developed popPK model can be used in clinical practice to screen covariates and guide therapeutic drug monitoring.

Identification of a Mitochondrial DNA Polymerase Affecting Cardiotoxicity of Sunitinib Using a Genome-Wide Screening on S. pombe Deletion Library.

Drug toxicity is a key issue for drug R&D, a fundamental challenge of which is to screen for the targets genome-wide. The anticancer tyrosine kinase inhibitor sunitinib is known to induce cardiotoxicity. Here, to understand the molecular insights of cardiotoxicity by sunitinib at the genome level, we used a genome-wide drug target screening technology (GPScreen) that measures drug-induced haploinsufficiency (DIH) in the fission yeast Schizosaccharomyces pombe genome-wide deletion library and found a mitochondrial DNA polymerase (POG1). In the results, sunitinib induced more severe cytotoxicity and mitochondrial damage in POG1-deleted heterozygous mutants compared to wild type (WT) of S. pombe. Furthermore, knockdown of the human ortholog POLG of S. pombe POG1 in human cells significantly increased the cytotoxicity of sunitinib. Notably, sunitinib dramatically decreased the levels of POLG mRNAs and proteins, of which downregulation was already known to induce mitochondrial damage of cardiomyocytes, causing cardiotoxicity. These results indicate that POLG might play a crucial role in mitochondrial damage as a gene of which expressional pathway is targeted by sunitinib for cardiotoxicity, and that genome-wide drug target screening with GPScreen can be applied to drug toxicity target discovery to understand the molecular insights regarding drug toxicity.

Ultra High Performance Liquid Chromatography Method for the Determination of Two Recently FDA Approved TKIs in Human Plasma Using Diode Array Detection.

Generally, tyrosine kinase inhibitors have narrow therapeutic window and large interpatient variability compared to intrapatient variability. In order to support its therapeutic drug monitoring, two fast and accurate methods were developed for the determination of recently FDA approved anticancer tyrosine kinase inhibitors, afatinib and ibrutinib, in human plasma using ultra high performance liquid chromatography coupled to PDA detection. Diclofenac sodium was used as internal standard. The chromatographic separation was achieved on an Acquity UPLC BEH C18 analytical column using a mobile phase combining ammonium formate buffer and acetonitrile at a constant flow rate of 0.4 mL/min using gradient elution mode. A µSPE (solid phase extraction) procedure, using Oasis MCX µElution plates, was processed and it gave satisfying and reproducible results in terms of extraction yields. Additionally, the methods were successfully validated using the accuracy profiles approach (β = 95% and acceptance limits = ±15%) over the ranges 5–250 ng/mL for afatinib and from 5 to 400 ng/mL for ibrutinib in human plasma.

Multi-agent chemotherapy overcomes glucocorticoid resistance conferred by a BIM deletion polymorphism in pediatric acute lymphoblastic leukemia.

A broad range of anti-cancer agents, including glucocorticoids (GCs) and tyrosine kinase inhibitors (TKIs), kill cells by upregulating the pro-apoptotic BCL2 family member, BIM. A common germline deletion in the BIM gene was recently shown to favor the production of non-apoptotic BIM isoforms, and to predict inferior responses in TKI-treated chronic myeloid leukemia (CML) and EGFR-driven lung cancer patients. Given that both in vitro and in vivo GC resistance are predictive of adverse outcomes in acute lymphoblastic leukemia (ALL), we hypothesized that this polymorphism would mediate GC resistance, and serve as a biomarker of poor response in ALL. Accordingly, we used zinc finger nucleases to generate ALL cell lines with the BIM deletion, and confirmed the ability of the deletion to mediate GC resistance in vitro. In contrast to CML and lung cancer, the BIM deletion did not predict for poorer clinical outcome in a retrospective analysis of 411 pediatric ALL patients who were uniformly treated with GCs and chemotherapy. Underlying the lack of prognostic significance, we found that the chemotherapy agents used in our cohort (vincristine, L-asparaginase, and methotrexate) were each able to induce ALL cell death in a BIM-independent fashion, and resensitize BIM deletion-containing cells to GCs. Together, our work demonstrates how effective therapy can overcome intrinsic resistance in ALL patients, and suggests the potential of using combinations of drugs that work via divergent mechanisms of cell killing to surmount BIM deletion-mediated drug resistance in other cancers.

Practical Guidelines for Therapeutic Drug Monitoring of Anticancer Tyrosine Kinase Inhibitors: Focus on the Pharmacokinetic Targets

There is accumulating evidence for potential benefits of therapeutic drug monitoring (TDM) in the treatment of cancer with tyrosine kinase inhibitors (TKIs). Relationships between exposure and response (efficacy/toxicity) have been established for several TKIs. For example, the pharmacokinetic targets for efficacy of imatinib, sunitinib and pazopanib have been defined as trough plasma concentrations (Ctrough) of >1,000, >50 and >20,000 ng/mL for selected indications, respectively. Dose adjustment based on pharmacokinetic targets could therefore increase response rates and duration. Furthermore, with appropriate target concentrations defined, excessive side effects in patients using the current fixed dosing strategy may be prevented. This review provides a practical guideline for TDM for the currently approved TKIs at 28 February 2013. The focus of this article is on the elaboration of exposure and response relationships of TKIs with proposed pharmacokinetic targets, mainly Ctrough, and further on the interpretation of the pharmacokinetic targets with recommendations for dose titrations.

Abstract 2040: Indication switching of the FDA-approved anticancer tyrosine kinase inhibitors to treat metabolic diseases.

Abstract Drug switching is a unique way to identify and extend new indications for approved drugs in drug discovery &amp; development. Tyrosine kinase inhibitors (TKIs) are used in biologically targeted cancer therapy and so far 12 TKIs have been approved by FDA. We hypothesize that these TKIs act on other molecular targets in addition to tyrosine kinases and may manage metabolic diseases given that there is a complex network of kinases that work together to regulate a number of important cellular processes. Employing a comprehensive docking method with our established chemical-protein interactome (CPI) and 11 FDA-approved TKIs, we have discovered301 PDB-deposited proteins corresponding to 353 ligand binding pockets among a total of 1,780 PDB-deposited human protein entries. Notably, sorafenib, dasatinib and crizotinib had a CPI binding score (ZZ_score) of -1.2903, -1.0278 and -1.5384 against histone deacetylase 7A, respectively. In addition, those TKIs achieved high ZZ scores against B-Raf, PPAR and VDR, suggesting a high binding affinity of sorafenib, dasatinib and crizotinib with these proteins. Our preliminary studies have showed that the acetylated-lysine in α-tublin and oncogenic Akt and Raf-signaling was inhibited significantly in human multiple melanoma cells by these TKIs. Interestingly, dasatinib increased VDRE and HDLR luciferase reporter activity in the human lung adenocarcinoma A-549 cells, and dasatinib and crizotinib induced autophage by activated LC3 in vitro. Furthermore, the erlotinib and sorafenib decreased glucose levels in Wistar STZ-D rats. These TKIs are predicted to act on a series of therapeutics targets associated with metabolic diseases (such as PPAR and Sirt1). Collectively, FDA-approved TKIs may be switched to become a “magic bullet” concurrently targeting tyrosine kinase, HDAC, PPAR, VDR and B-Raf, shedding a light for future anti-metabolic disorder and anticancer drug discovery &amp; development. Further validation of additional “hot targets” besides tyrosine kinase such as HDAC, B-Raf, PPAR, Sirt1, Akt and VDR, and in vivo evaluation of anti-metabolic disorder by TKIs are undergoing at our laboratory. These findings suggest that drug switching may represent a new and effective approach to expanding the application of existing drugs. Citation Format: Jiazhi Sun, Lun Yang, Minghua Li, Zhixin Wang, Amy Haynes, Qian Wang, Isaac Raplee, Jean Melchisedek, Anand Prasad, Kevin B. Sneed, Lin He, Shufeng Zhou. Indication switching of the FDA-approved anticancer tyrosine kinase inhibitors to treat metabolic diseases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2040. doi:10.1158/1538-7445.AM2013-2040