Multiple Tyrosine Kinase Inhibitor Research Articles

Hepatocellular carcinoma: Advances in systemic therapies

Advanced hepatocellular carcinoma (HCC) is traditionally associated with limited treatment options and a poor prognosis. Sorafenib, a multiple tyrosine kinase inhibitor, was introduced in 2007 as a first-in-class systemic agent for advanced HCC. After sorafenib, a range of targeted therapies and immunotherapies have demonstrated survival benefits in the past 5 years, revolutionizing the treatment landscape of advanced HCC. More recently, evidence of novel combinations of systemic agents with distinct mechanisms has emerged. In particular, combination trials on atezolizumab plus bevacizumab and durvalumab plus tremelimumab have shown encouraging efficacy. Hence, international societies have revamped their guidelines to incorporate new recommendations for these novel systemic agents. Aside from treatment in advanced HCC, the indications for systemic therapy are expanding. For example, the combination of systemic therapeutics with locoregional therapy (trans-arterial chemoembolization or stereotactic body radiation therapy) has demonstrated promising early results in downstaging HCC. Recent trials have also explored the role of systemic therapy as neoadjuvant treatment for borderline-resectable HCC or as adjuvant treatment to reduce recurrence risk after curative resection. Despite encouraging results from clinical trials, the real-world efficacy of systemic agents in specific patient subgroups (such as patients with advanced cirrhosis, high bleeding risk, renal impairment, or cardiometabolic diseases) remains uncertain. The effect of liver disease etiology on systemic treatment efficacy warrants further research. With an increased understanding of the pathophysiological pathways and accumulation of clinical data, personalized treatment decisions will be possible, and the field of systemic treatment for HCC will continue to evolve.

Cabozantinib plus pembrolizumab as first-line therapy for cisplatin-ineligible advanced urothelial carcinoma (PemCab).

539 Background: Pembrolizumab (pem) was initially approved in the first line setting for cisplatin-ineligible metastatic urothelial carcinoma (mUC) patients (pts) with high PD-L1 expression and platinum ineligible pts regardless of PD-L1 status; this later changed to only platinum ineligible pts. There is a high disease progression (PD) rate with pem monotherapy. Cabozantinib (cabo) is a multiple receptor tyrosine kinase inhibitor targeting MET and VEGFR2 approved in multiple other malignancies as monotherapy or in combination with PD-1 inhibition. We hypothesized that combination of pem and cabo therapy will be safe and efficacious in patients with treatment naïve mUC. Methods: This is a phase II, open label, multi-center, single arm trial evaluating the tolerability and activity of pem 200 mg every 3 weeks and cabo 40 mg daily as first-line treatment for patients with mUC. Key inclusion criteria were histologically proven locally advanced or mUC, ECOG-PS 0-2, cisplatin-ineligible (including patient refusal of cisplatin), treatment naïve, and no prior PD-1/L1 therapy. The primary endpoint was objective response rate (ORR). Key secondary endpoints included progression-free survival (PFS) and overall survival (OS). There was a lead-in safety cohort of 6 pts. We hypothesized that combination therapy would improve ORR to > 32%. With 35 evaluable subjects, the lower bound of the 95% confidence interval would extend no more than 26% from the observed proportion. With ≥17 objective responses, the confidence interval excludes 32%. Results: Between December 26, 2018, and April 19, 2023, 36 pts were enrolled of which 35 were evaluable for response with the median follow up of 14.4 months (95% CI 12.2 – 16.2). The median age was 72.5y [range, 47-82]; ECOG PS 0-1 in 89% pts. Responses were observed in 15 pts (ORR= 42.8%) including complete responses in 5 pts (14.3%). Stable disease was seen in 10 pts (28.6%). The disease control rate was 68.5% (25/35). Median PFS was 7.7 months (95% CI 4.2 – 11.2). Fifty-two percent of pts developed any grade treatment-related adverse events (TRAE) attributable to either cabozantinib or pembrolizumab. The most common grade 1/2 TRAE were diarrhea, anorexia, dysgeusia, weight loss, and nausea. Forty-four percent pts developed grade 3/4 TRAEs most common being hypertension, hypophosphatemia, alanine transaminase elevation, diarrhea, and fatigue. Conclusions: This novel phase II trial of pem + cabo demonstrated a manageable toxicity profile and promising efficacy as first-line therapy in mUC including patient’s ineligible for cisplatin. Further investigation with a focus on predictive biomarkers is ongoing. Clinical trial information: NCT03534804 .

A simple and sensitive liquid chromatography triple quadrupole mass spectrometry method for the determination of XL092 in monkey plasma and its application to pharmacokinetic study.

XL092 is a potent ATP-competitive inhibitor of multiple receptor tyrosine kinases that is undergoing clinical development for the treatment of lung cancer. In this study, an LC triple quadrupole mass spectrometry method was established to measure XL092 in monkey plasma. A Waters ACQUITY UPLC BEH C18 column was used for chromatographic separation. The mobile phase consisted of water containing 0.1% formic acid and acetonitrile with a gradient elution at the flow rate of 0.4 mL/min. Multiple reaction monitoring mode was used for quantitative analysis of XL092 in positive electrospray ionization. In the concentration range of 0.5-1000 ng/mL, XL092 showed excellent linearity in monkey plasma with a correlation coefficient greater than 0.995 (r > 0.995). The lowest limit of quantification was 0.5 ng/mL. The intra- and inter-day relative standard deviations were <9.99%, while the relative error ranged from -12.50% to 8.10%. The mean recovery was over 82.51%. XL092 was stable in monkey plasma after storage under certain conditions. The validated method was demonstrated to be selective, sensitive, and reliable, and has been successfully applied to the pharmacokinetic study of XL092 in monkey plasma. XL092 showed moderate short half-life (~3.81 h) and good oral bioavailability (80%).

Erythroid variant evolving from chronic myeloid leukemia resistant to multiple tyrosine kinase inhibitors: a rare case report

BackgroundChronic myeloid leukemia (CML) is characterized by the presence of BCR::ABL1 fusion gene resulting from a reciprocal translocation, t(9;22)(q34;q11.2), leading to prominent granulocytic proliferation. The majority of patients initially present in chronic phase (CP), which may progress to advanced CML with predominantly granulocytic phenotypes in the absence of proper treatment or response to tyrosine kinase inhibitors (TKIs). We present an exceptionally rare case in which an erythroid variant emerged from a CML patient resistant to multiple TKIs. This variant is characterized by the detection of t(9;22) BCR::ABL1 fusion in erythroid precursors at various maturation stages and the absence of granulocytic progenitor hyperplasia typically seen in classical CML.Case presentationA 33-year-old female with CP-CML had received multiple TKI therapies since her initial diagnosis in 2015. Due to intolerable side effects and inconsistent adherence, she exhibited an inadequate response and developed new-onset pancytopenia. Bone marrow (BM) biopsy specimen revealed a hypercellular marrow with significant erythroid hyperplasia (90% of marrow cellularity) and a reversed myeloid-to-erythroid (M: E) ratio of 1:10. Both erythroid and myeloid cells displayed progressive maturation without dysplasia or excess blasts. Chromosomal analysis identified t(9;22) (q34;q11.2) in 19 out of 20 metaphase cells. BCR::ABL1 fusion transcript (p210 isoform) was confirmed by real-time quantitative polymerase chain reaction (RT-qPCR) and next-generation sequencing (NGS). Notably, no additional pathogenic cytogenetic abnormalities or ABL1 kinase domain mutations were detected. Here, we report the first published case of an erythroid variant emerging in a CML patient resistant to multiple TKIs—a distinct entity from the erythroid blast crisis evolving from CML.ConclusionThe erythroid variant of CML is distinguished by the presence of t(9;22) (q34;q11.2) BCR::ABL1 in predominant erythroid precursors at different stages of maturation. In a myeloid neoplasm showing predominant erythroid hyperplasia without typical CML features, it is vital to correlate morphology and t(9;22) BCR::ABL1 cytogenetic testing for accurate diagnosis, and to prevent confusion with PEL transformation in CML.

Analysis of vascular disruption in zebrafish embryos as an endpoint to predict developmental toxicity

Inhibition of angiogenesis is an important mode of action for the teratogenic effect of chemicals and drugs. There is a gap in the availability of simple, experimental screening models for the detection of angiogenesis inhibition. The zebrafish embryo represents an alternative test system which offers the complexity of developmental differentiation of an entire organism while allowing for small-scale and high-throughput screening. Here we present a novel automated imaging-based method to detect the inhibition of angiogenesis in early life stage zebrafish. Video subtraction was used to identify the location and number of functional intersegmental vessels according to the detection of moving blood cells. By exposing embryos to multiple tyrosine kinase inhibitors including SU4312, SU5416, Sorafenib, or PTK787, we confirmed that this method can detect concentration-dependent inhibition of angiogenesis. Parallel assessment of arterial and venal aorta ruled out a potential bias by impaired heart or blood cell development. In contrast, the histone deacetylase inhibitor valproic acid did not affect ISV formation supporting the specificity of the angiogenic effects. The new test method showed higher sensitivity, i.e. lower effect concentrations, relative to a fluorescent reporter gene strain (Tg(KDR:EGFP)) exposed to the same tyrosine kinase inhibitors indicating that functional effects due to altered tubulogenesis or blood transport can be detected before structural changes of the endothelium are visible by fluorescence imaging. Comparison of exposure windows indicated higher specificity for angiogenesis when exposure started at later embryonic stages (24 h post-fertilization). One of the test compounds was showing particularly high specificity for angiogenesis effects (SU4312) and was, therefore, suggested as a model compound for the identification of molecular markers of angiogenic disruption. Our findings establish video imaging in wild-type strains as viable, non-invasive, high-throughput method for the detection of chemical-induced angiogenic disruption in zebrafish embryos.

Development and characterization of inhaled nintedanib-loaded PLGA nanoparticles using scalable high-pressure homogenization technique

Presently, non-invasive targeted drug delivery to the lungs represents a routinely investigated approach to treat challenging disorders such as pulmonary fibrosis. Nintedanib (Nint) is an FDA-approved drug for treating idiopathic pulmonary fibrosis and certain interstitial lung diseases. Nint acts as a multiple tyrosine kinase inhibitor, demonstrating anti-neoplastic effects in non-small cell lung cancer. However, limitations of oral administration of Nint include low bioavailability and high dose requirement for adequate therapeutic efficacy, resulting in adverse effects like abdominal pain, sickness, diarrhea, and vomiting. In this project, Nint was reformulated into PLGA nanoparticles (Nint-NPs) for direct-lung administration to address these deficiencies and enhance its potential. In this project, Nint-NPs were designed utilizing a high-pressure homogenization (HPH) approach with excellent reproducibility and scale-up feasibility. Selected Nint-NP formulation revealed 43.3 ± 4.0 % of entrapment efficiency and 179.8 ± 3.1 nm of size. In-vitro release study showed an initial burst release of 36.0 ± 7.5 % within 30 min, followed by sustained release up to 71.0 ± 4.3 % by 96 h. Further, Nint-NPs were found to be stable after 1-month of storage in refrigerated and accelerated conditions. Interestingly, the results of lung deposition demonstrated ideal MMAD values less than 5 μm in all three nebulizer types tested (jet, breath-actuated jet, and vibrating mesh nebulizers), indicating Nint-NPs are appropriate for deep-lung delivery. In-vitro cellular uptake analysis exhibited higher internalization of NPs in alveolar epithelial cells and lung fibroblasts while avoiding macrophage uptake. Toxicity studies on normal lung cells indicated Nint-NPs were safe for use. Taken together, inhalation therapy of Nint-NPs offers a favorable alternative to oral capsules for treating respiratory diseases.

QbD-driven RP-HPLC method for novel chemo-herbal combination, in-silico, force degradation studies, and characterization of dual drug-loaded polymeric and lipidic nanocarriers

BackgroundIn cancer therapies, chemo-herbal combinations are receiving increased attention. A multiple tyrosine kinase inhibitor, lenvatinib (LTB) is beneficial in treating thyroid, lung, endometrial, and liver cancers. An isoflavone called biochanin A (BCA) is well known for its diverse biological properties that have been studied to potentiate the anti-cancer potential and lower the normal cell toxicities of other therapeutics. LTB and BCA can be combined for cancer treatment and may increase their therapeutic potential at lower doses. In brief, the quality by design (QbD)-driven RP-HPLC method was developed, validated, and utilized for applications employing the study of forced degradants and the successful development of LTB and BCA co-loaded nanocarriers.ResultsThe RP-HPLC method employed Box–Behnken design with peak resolution 6.70 ± 0.006, tailing factor 1.06 ± 0.05 for BCA and 1.17 ± 0.021 for LTB, and theoretical plates number > 2000. RP-HPLC applications utilized the investigation of a total of 41.17% and 70.58% degradants for LTB and BCA in contrast to in-silico predicted studies using Zeneth software. The poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) were formed with particle size 185.3 ± 12.3 nm, zeta potential − 13.3 ± 0.35 mV, and percentage entrapment efficiency (%EE) for the LTB and BCA 53.64 ± 4.81% and 61.29 ± 4.67%, respectively. However, the developed Cubosomes (CBs) exhibited 182.4 ± 16.3 nm aerodynamic particle size, − 10.8 ± 0.39 mV zeta potential, and % EE for LTB and BCA 55.62 ± 7.73% and 72.88 ± 5.52%, respectively. The percentage drug loading (%DL) of LTB and BCA from PLGA NPs was found to be 3.7 ± 0.46% and 4.63 ± 0.48%, whereas CBs exhibited higher % DL for BCA (5.42 ± 1.10%) and LTB (4.43 ± 0.77%).ConclusionThe RP-HPLC method was developed and validated according to ICH and USP guidelines. In-vitro and in-silico forced degradation studies are evident to quantify the type of degradant and its exact mechanism of degradation. In-silico toxicity assessment for LTB, BCA, and their degradants explains the necessity of conducting degradation studies during drug development. Finally, the applications of the developed RP-HPLC method explain the usefulness of analytical methods in the development of chemo-herbal drug nanocarriers (polymeric and lipidic).Graphical abstract

Tern-701 (HS-10382) Is a Potent Inhibitor of BCR::ABL1 and Is Synergistic with Active Site Tyrosine Kinase Inhibitors

CONCLUSIONS Although tyrosine kinase inhibitors (TKIs) targeting the ATP-binding site of the BCR::ABL1 oncoprotein are effective therapeutics for chronic myeloid leukemia (CML), patients often develop drug resistance due to ATP-site mutations that inhibit drug binding. TERN-701 is a potent and highly selective inhibitor of BCR::ABL1 that is designed to bind the allosteric myristoyl site on the kinase, circumventing resistance to active site mutations, with potential for synergistic combination with active site TKIs. Competition binding screens were used to assess the selectivity of TERN-701 on more than 450 targets spanning the human kinome. The potency of TERN-701 against the proliferation of wild type and mutant CML cell lines, as well as a panel of 102 cancer cell lines, was assessed using CellTiter-Glo®. Synergy between TERN-701 and active site TKIs was assessed in vitro with interactions quantified using Bliss, combination, and curve-shift analyses. Prospective mutagenesis experiments were conducted by treating BaF/3 cells engineered to overexpress the BCR::ABL1 kinase with various concentrations of both TERN-701 and the TKI imatinib, followed by observation of clonal outgrowth of treated cells over time. We have previously shown that TERN-701 is a potent inhibitor of native CML cell lines such as K562. Here, we show that TERN-701 inhibited cell proliferation in various clinically relevant mutant CML cell lines (including T315I) with IC 50s ranging from 5 to 26 nM and 70 to 1200 nM against active site and myristoyl site mutations, respectively. TERN-701 was highly selective for BCR::ABL1, with no appreciable activity (&amp;gt;50%) against &amp;gt;450 purified kinase targets. Against a panel of cancer cell lines, TERN-701 was more selective than the comparator allosteric BCR::ABL1 inhibitor asciminib while maintaining similar potency. In vitro combination studies previously revealed that TERN-701 works synergistically with multiple TKIs in the non-mutant K562 cell line. Here, expanded studies using KCL22-s and BCR::ABL1-T315I mutant cell lines identified strong synergistic interactions between TERN-701 and active site TKIs, including ponatinib and dasatinib. Furthermore, prospective mutagenesis studies showed that the combination of TERN-701 and imatinib resulted in greater inhibition of the outgrowth of resistant clones relative to either compound alone. TERN-701 is a potent and highly selective allosteric inhibitor of BCR::ABL1 that can act synergistically with active site TKIs in vitro, even against the T315I gatekeeper mutation that confers resistance to all approved active site TKIs except ponatinib, which has known safety liabilities. These data support the continued development of TERN-701 for the treatment of CML using both monotherapy and combination approaches.

Identification of Multivariable microRNA and Clinical Biomarkers Panels to Predict Imatinib Response in Chronic Myeloid Leukemia at Diagnosis

Imatinib Mesylate (IM) was once hailed as the magic bullet for chronic myeloid leukemia (CML) and remains a front-line therapy for CML to this day alongside other tyrosine kinase inhibitors (TKIs). However, TKI treatments are rarely curative and while they demonstrate efficacy in managing the disease, patients are often required to receive life-long treatment or risk experiencing relapse. Patients also frequently harbor innate TKI-resistance mechanisms to front-line IM therapy and without prior screening, patients can spend precious months on non-optimal treatment before transitioning to other therapeutic strategies. Thus, there is a growing interest in studying biomarkers in patients which can predict TKI response upon diagnosis. CML has unique microRNA (miRNA) expression profiles at different stages of the disease and in response to TKI-treatment. We previously generated global transcriptome profiles on treatment-naïve CD34 + CML cells with known subsequent imatinib (IM) responses and identified several differentially expressed miRNAs in IM-nonresponders as compared to IM-responders. We also reported that IM response could be predicted in treatment-naïve CD34 + cells by an in vitro colony forming cell (CFC) assay. Additionally, we previously analyzed patient data from the ENESTxtnd clinical trial to show that miRNA expression combined with CFC output data could accurately predict Nilotinib response in patients. In this study, we used a Cox Proportional Hazard (CoxPH) analysis to identify 22 out of a pool of 35 pre-validated miRNAs which were associated with IM nonresponse in CD34 + CML cells of 80 patients at diagnosis. These patients were later classified as IM-responders or IM-nonresponders based on the latest European LeukemiaNet (ELN) guidelines. Interestingly, a Welch t-test revealed 16 of these 22 IM-response associated miRNAs to be differentially expressed between IM-responders and IM-nonresponders. Between IM-responders and IM-nonresponders we also found 7 clinical parameters that were associated with IM-response of which 3 matched parameters had significantly different values including CFC assay outputs ( p=0.0015), Sokal scores ( p=0.0070) and white blood cell counts (WBC, p=0.0028). We then trained a machine learning model employing the random-forest (RF), gradient-boosting (GBM) and naïve-bayes (NB) algorithms with different combinations of the 16 miRNAs with and without the clinical parameters of these patients to identify panels with high predictive performance based on area-under-curve (AUC) values of receiver-operating-characteristic (ROC) and precision-recall (PR) curves. Notably, the multivariable panel consisting of both miRNAs and clinical features (AUC-ROC RF=0.83, GBM=0.83, NB=0.84, AUC-PR RF=0.68, GBM=0.67, NB=0.70) performed better than either miRNA (AUC-ROC RF=0.72, GBM=0.73, NB=0.78, AUC-PR RF=0.49, GBM=0.51, NB=0.4) or clinical (AUC-ROC RF=0.82, GBM=0.84, NB=0.84, AUC-PR RF=0.64, GBM=0.67, NB=0.64) panels alone. Interestingly, 2 miRNAs in this panel, miR-185 and miR-145, were also significant classifiers for our Nilotinib predictive study suggesting that expression patterns of these miRNAs may have predictive properties for multiple TKI responses. Thus, we show that predictive accuracy of biomarkers may be supplemented by inclusion of multivariable parameters, and our findings may inform future studies on developing predictive panels for more optimized treatment plans in the clinic.

Tyrosine Kinase Inhibitor (TKI) Treatment Patterns in 5,261 Chronic Phase (CP) Chronic Myeloid Leukemia (CML) Patients in “Real-Life” Settings: A Population-Based Study Using a Nationwide Claim Database

Introduction The introduction of TKI in CP-CML enabled responders to reach a similar survival to that of the general population. However, recent small-sized real-life studies showed that 1 in 5 CP-CML patients (pts) received at least 3 lines of TKI, mainly due to resistance or the onset of adverse events (AE). The objective of this study is to document real-life TKI treatment patterns nationwide in CML pts with a long-term follow-up, with a focus on pts who initiated at least a 3 rd line and associated disease burden. Methods This real-life non-interventional study was conducted using the French nationwide claim database (SNDS, Système National des Données de Santé), covering over 99% of French beneficiaries. Pts who initiated their first TKI with an indication for CML (imatinib, dasatinib, nilotinib, bosutinib, ponatinib) between January 2010 and December 2018, aged 18 years old or older at TKI initiation (index date T 0), and with healthcare resource use (HCRU) markers of CML - i.e., reasons for hospitalization and reported Long-Term Disease - without markers of confounding disease for the use of TKIs, were included. Treatment patterns were assessed from T 0 to December 2020 or death, through the description of TKI treatment sequences (TKI lines), adherence - discontinuations (≥ 3 months) during a TKI line and Medication Possession Ratio (MPR) -, and line duration (Time to Next Treatment (TTNT) estimates). TTNT was calculated as cumulative incidence of TKI switches, with death as competing risk; switches were defined as discontinuation of a TKI followed by the start of a different TKI. Results A total of 5,261 CML pts initiated their 1 st line TKI, with a median age at T 0 of 59.5 years (IQR: 23.2) and a M/F ratio of 1.2. The median follow-up from T 0 was 5.6 years (IQR: 4.6). Respectively 2,299, 1,017 and 426 pts initiated 2 nd, 3 rd and 4 th TKI line. Most frequent TKIs used as 1 st and 2 nd lines were imatinib (73.1%), and dasatinib (60.4%; Figure 1), respectively. From the 3 rd line, TKI patterns changed following the reimbursement of bosutinib in 2015 and ponatinib in 2016, and subsequently stabilized; shares in pts treated in 3 rd line and more at the beginning of 2020 were roughly equally distributed between marketed TKIs. TKI discontinuations rates during a line varied from 76.2% for 1 st line to 56.2% for 4 th lines. The median cumulative duration of discontinuations tended to decrease across lines (2.9, 2.5, 2.3 and 2.1 months for 1 st, 2 nd, 3 rd, and 4 th line, respectively); IQRs across lines were similar. The proportion of pts with a good medication adherence (MPR ≥ 80%) was around 80% for all TKI lines. TTNT was similar across all TKIs and all lines; 10-year TTNT rates were 48% (95CI: [47%; 50%]), 56% [53%; 59%], 56% [51%; 61%], 48% [42%; 54%] for 1 st, 2 nd, 3 rd, and 4 th lines. For pts who switched, most of switches occurred during the first year following line initiation (51% of switches observed in the first year for both 1 st, 2 nd, 3 rd, and 4 th lines), 24% occurred during the second year, and 10% during the third year. The 10-year cumulative incidences of death were 12% (95CI: [11%; 14%]), 9% [8%; 11%], 12% [9%; 16%], 14% [6%; 25%] for 1 st, 2 nd, 3 rd, and 4 th lines, respectively. The proportion of CML pts who initiated at least 3 lines of TKIs was 19.3% from raw estimates (n=1,017/5,261) and would be 27% based on TTNT estimates. Their characteristics were similar to pts who initiated 1 st line TKI (sociodemographic, comorbidities). Regarding HCRU, respectively 37.2% and 29.8% of pts had at least one hospitalization during 1 st and 6 th semesters following 3 rd line initiation; 7.9% to 5.8% visited emergency room, and around 10% had at least one sick leave per semester. Conclusion Using one of the largest cohorts of CML pts ever studied, with a follow-up period up to 10 years (median 5.6 years), this real-life study confirmed that multiple TKI switches are now common. We confirmed that at least 1 out of 5 CML patient fails at least two TKI lines. From TTNT estimates, surrogate marker for the duration of clinical benefit, lines of TKI would be effective over the long-term for around half of pts, and half of the switches occur within the first two years, regardless of line position. Finally, this real-world study illustrates the need for permanent care of these pts and alternative treatment options to avoid resistance or intolerance and induce a similar survival as the general population of the same age.

Differential vascular endothelial cell toxicity of established and novel BCR-ABL tyrosine kinase inhibitors.

BCR-ABL tyrosine kinase inhibitors (TKIs) have dramatically improved survival in Philadelphia chromosome-positive leukemias. Newer BCR-ABL TKIs provide superior cancer outcomes but with increased risk of acute arterial thrombosis, which further increases in patients with cardiovascular comorbidities and mitigates survival benefits compared to imatinib. Recent studies implicate endothelial cell (EC) damage in this toxicity by unknown mechanisms with few side-by-side comparisons of multiple TKIs and with no available data on endothelial impact of recently approved TKIs or novels TKIs being tested in clinical trials. To characterize BCR-ABL TKI induced EC dysfunction we exposed primary human umbilical vein ECs in 2D and 3D culture to clinically relevant concentrations of seven BCR-ABL TKIs and quantified their impact on EC scratch-wound healing, viability, inflammation, and permeability mechanisms. Dasatinib, ponatinib, and nilotinib, the TKIs associated with thrombosis in patients, all significantly impaired EC wound healing, survival, and proliferation compared to imatinib, but only dasatinib and ponatinib impaired cell migration and only nilotinib enhanced EC necrosis. Dasatinib and ponatinib increased leukocyte adhesion to ECs with upregulation of adhesion molecule expression in ECs (ICAM1, VCAM1, and P-selectin) and leukocytes (PSGL1). Dasatinib increased permeability and impaired cell junctional integrity in human engineered microvessels, consistent with its unique association with pleural effusions. Of the new agents, bafetinib decreased EC viability and increased microvessel permeability while asciminib and radotinib did not impact any EC function tested. In summary, the vasculotoxic TKIs (dasatinib, ponatinib, nilotinib) cause EC toxicity but with mechanistic differences, supporting the potential need for drug-specific vasculoprotective strategies. Asciminib and radotinib do not induce EC toxicity at clinically relevant concentrations suggesting a better safety profile.

Lenvatinib activates anti-tumor immunity by suppressing immunoinhibitory infiltrates in the tumor microenvironment of advanced hepatocellular carcinoma

BackgroundLenvatinib, a multiple receptor tyrosine kinase inhibitor, might exert antitumor effects via tumor immune modulation. However, changes in the tumor immune microenvironment induced by lenvatinib are poorly understood. We investigated the effect of lenvatinib on immune features in clinical samples from patients with hepatocellular carcinoma.MethodsFifty-one patients with advanced hepatocellular carcinoma who received lenvatinib monotherapy as first-line treatment were enrolled. We collected blood sample (n = 51) and tumor tissue (n, baseline/four weeks after treatment initiation/post-progression = 50/8/12). DNA, RNA, and proteins extracted from the tissues were subjected to multi-omics analysis, and patients were classified into two groups according to baseline immune status. Each group was investigated in terms of the dynamics of tumor signaling. We also longitudinally analyzed circulating immune proteins and chemokines in peripheral blood.ResultsHere we show that lenvatinib has similar anti-tumor efficacy with objective response rate and progression-free survival in both Immune-Hot and Immune-Cold subtypes. Immune signatures associated with T-cell functions and interferon responses are enriched in the early phase of treatment, while signatures associated with immunoinhibitory cells are downregulated along with efficient vascular endothelial growth factor receptor and fibroblast growth factor receptor blockades. These findings are supported by imaging mass cytometry, T-cell receptor repertoire analysis and kinetics of circulating proteins. We also identify interleukin-8 and angiopoietin-2 as possible targets of intervention to overcome resistance to existing immunotherapies.ConclusionsOur findings show the ability of lenvatinib to modulate tumor immunity in clinical samples of hepatocellular carcinoma.

A stability indicating method development and validation of a rapid and sensitive RP-HPLC method for Nintedanib and its application in quantification of nanostructured lipid carriers

Background: Nintedanib (NTB) is a multiple tyrosine kinase inhibitor, been investigated for many disease conditions like idiopathic pulmonary fibrosis (IPF), systemic sclerosis interstitial lung disease (SSc-ILD) and non-small cell lung cancer (NSCLC). NTB is available as oral capsule formulation, but its ability to detect degradants formed through oxidative, photolytic and hydrolytic processes makes it difficult to quantify. In the current work, a novel reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and validated. Methods: The developed method is simple, precise, reproducible, stable and accurate. The inherent stability of NTB was evaluated using the proposed analytical method approach and force degradation studies were carried out. NTB was separated chromatographically on the Shimadzu C18 column as stationary phase (250 ×4.6 mm, 5 µm) using an isocratic elution method with 0.1% v/v triethyl amine (TEA) in HPLC grade water and acetonitrile (ACN) in the ratio 35:65% v/v. The mobile phase was pumped at a constant flow rate of 1.0 ml/min, and the eluent was detected at 390 nm wavelength. Results: NTB was eluted at 6.77±0.00 min of retention time (tR) with a correlation coefficient of 0.999, the developed method was linear in the concentration range of 0.5 µg/ml to 4.5 µg/ml. The recovery rate was found to be in the range of 99.391±0.468% for 1.5 µg/ml concentration. Six replicate standards were determined to have an % RSD of 0.04. Conclusion: The formulation excipients didn't interfere with the determination of NTB, demonstrating the specificity of the developed method. The proposed approach of the analytical method developed can be used to quantify the amount of NTB present in bulk drugs and pharmaceutical formulations.

Differential network analysis of ROS1 inhibitors reveals lorlatinib polypharmacology through co-targeting PYK2

Multiple tyrosine kinase inhibitors (TKIs) are often developed for the same indication. However, their relative overall efficacy is frequently incompletely understood and they may harbor unrecognized targets that cooperate with the intended target. We compared several ROS1 TKIs for inhibition of ROS1-fusion-positive lung cancer cell viability, ROS1 autophosphorylation and kinase activity, which indicated disproportionately higher cellular potency of one TKI, lorlatinib. Quantitative chemical and phosphoproteomics across four ROS1 TKIs and differential network analysis revealed that lorlatinib uniquely impacted focal adhesion signaling. Functional validation using pharmacological probes, RNA interference, and CRISPR-Cas9 knockout uncovered a polypharmacology mechanism of lorlatinib by dual targeting ROS1 and PYK2, which form a multiprotein complex with SRC. Rational multi-targeting of this complex by combining lorlatinib with SRC inhibitors exhibited pronounced synergy. Taken together, we show that systems pharmacology-based differential network analysis can dissect mixed canonical/non-canonical polypharmacology mechanisms across multiple TKIs enabling the design of rational drug combinations.

Assessing the safety and activity of cabozantinib combined with lanreotide in gastroenteropancreatic and thoracic neuroendocrine tumors: rationale and protocol of the phase II LOLA trial

BackgroundWell-differentiated (WD) neuroendocrine tumors (NETs) are a group of rare neoplasms with limited therapeutic options. Cabozantinib is an inhibitor of multiple tyrosine kinases with a pivotal role in NET pathogenesis, including c-MET and Vascular Endothelial Growth Factor Receptor 2 (VEGFR2). LOLA is the first prospective phase II trial aiming to assess the safety and activity of cabozantinib combined with lanreotide in WD NETs of gastroenteropancreatic (GEP), thoracic and of unknown origin.MethodsThis is a multicenter, open-label, double-cohort, non comparative, non-randomized, three-stage phase II trial. Eligible patients have to meet the following inclusion criteria: diagnosis of advanced or metastatic, progressive, non-functioning WD thoracic NETs, GEP-NETs or NETs of unknown origin with Ki67 ≥ 10%; positive 68 Ga-PET uptake or somatostatin receptor 2 immunohistochemical (IHC) stain; maximum 1 prior systemic regimen for metastatic disease. Two cohorts will be considered: pNETs and carcinoids (typical or atypical lung and thymus NETs, gastro-intestinal NETs or NETs of unknown origin). In stage I, the primary objective is to find the optimal dose of cabozantinib in combination with lanreotide and to evaluate the safety of the combination (percentage of patients experiencing grade 3–5 toxicities according to NCI-CTCAE version 5.0). Starting dose of cabozantinib is 60 mg/day continuously, plus lanreotide 120 mg every 28 days. In stage II and III, co-primary endpoints are safety and overall response rate (ORR) according to RECIST version 1.1. The uninteresting antitumor activity is fixed in ORR ≤ 5%. Secondary endpoints are progression-free survival and overall survival. Exploratory objectives include the assessment of c-MET, AXL and VEGFR2 IHC expression, to identify predictive or prognostic tissue biomarkers. Enrolment started in July 2020, with an expected trial duration of 42 months comprehensive of accrual, treatment and follow-up. Considering a drop-out rate of 5%, the maximum number of enrolled patients will be 69.DiscussionSupported by a solid rationale, the trial has the potential to generate milestone data about the synergistic effects of cabozantinib plus lanreotide in a group of NET patients with relatively aggressive disease and limited therapeutic options.Trial registrationLOLA is registered at ClinicalTrials.gov (NCT04427787) and EudraCT (2019–004506-10).

Role of human epidermal growth factor receptor 3 in treatment resistance of anaplastic lymphoma kinase translocated non-small cell lung cancer

ABSTRACT Background ALK tyrosine kinase inhibitors (TKI) have revolutionized the treatment of ALK+ non-small cell lung cancer (NSCLC), and therapy resistance occurs in virtually all patients. Multiple TKI resistance mechanisms have been characterized, including ERBB receptor coactivation. In this study, we investigated the role of HER3 in ALK TKI resistance. Methods In vitro studies were carried out using ALK+ NSCLC cell lines H3122, H2228, and DFCI032. Pharmacological co-targeting of ALK and HER3 was investigated with ALK and ERBB TKIs, and HER3 knockdown was achieved using the CRISPR-Cas9 system. Co-localization of ALK and HER3 was investigated by immunoprecipitation (IP) and proximity ligation assay (PLA) in vitro and in vivo using six ALK+ NSCLC tumor samples. Results In all tested cell lines, combined targeting with ALK and pan-ERBB TKI resulted in marked inhibition of colony formation and long-term (72 h) downregulation of pAKT levels. HER3 knockdown resulted in multiple effects on ALK+ cell lines, including the downregulation of ALK expression and visible morphological changes (H2228). Co-immunoprecipitation (IP) and proximation ligation assay (PLA) experiments provided evidence that both ALK and HER3 could interact in vitro, and this finding was verified by PLA using ALK+ NSCLC tumors. Conclusions This study provides evidence that HER3 may mediate TKI resistance in ALK+ NSCLC. Interestingly, we were able to show that both translocated ALK and HER3 could interact. Joint targeting of ALK and HER3 could be further investigate in ALK+ NSCLC.

Preparation of a Sunitinib loaded microemulsion for ocular delivery and evaluation for the treatment of corneal neovascularization in vitro and in vivo.

Background: Corneal neovascularization (CNV) is a pathological condition that can disrupt corneal transparency, thus harming visual acuity. However, there is no effective drug to treat CNV. Sunitinib (STB), a small-molecule multiple receptor tyrosine kinase inhibitor, was shown to have an effect on CNV. The purpose of this study was to develop an STB microemulsion (STB-ME) eye drop to inhibit CNV by topical application. Methods: We successfully prepared an STB-ME by the phase inversion emulsification method, and the physicochemical properties of STB-MEs were investigated. The short-term storage stability, cytotoxicity to human corneal epithelial cells, drug release, ocular irritation, ocular pharmacokinetics and the inhibitory effect on CNV were evaluated in vitro and in vivo. Results: The optimal formulation of STB-ME is composed of oleic acid, CRH 40, Transcutol P, water and sodium hyaluronate (SH). It is a uniform spherical particle with a mean droplet size of 18.74 ± 0.09nm and a polydispersity index of 0.196 ± 0.004. In the in vitro drug release results, STB-ME showed sustained release and was best fitted by a Korsmeyer-Peppas model (R 2 = 0.9960). The results of the ocular pharmacokinetics in rabbits showed that the formulation containing SH increased the bioavailability in the cornea (2.47-fold) and conjunctiva (2.14-fold). STB-ME (0.05% and 0.1%), administered topically, suppressed alkali burn-induced CNV in mice more effectively than saline, and high-dose (0.1%) STB-ME had similar efficacy to dexamethasone (0.025%). Conclusion: This study provides a promising formulation of STB-ME for the inhibition of CNV by topical administration, which has the excellent characteristics of effectiveness, sustained release and high ocular bioavailability.

Longitudinal, prospective cardiovascular and metabolic risk in treatment-naive patients with chronic myeloid leukemia in chronic phase (CML-CP) starting tyrosine kinase inhibitor (TKI) therapy in a real-world setting.

7053 Background: Multiple TKIs used in the treatment (tx) of CML have been associated with the development of cardiovascular/metabolic (CV/Met) comorbidities, yet no study to date has prospectively and systematically assessed CV/Met parameters after TKI initiation. CA180-653 (NCT03045120) is a non-interventional, prospective, real-world study characterizing the effects of TKIs (dasatinib [DAS], imatinib [IM], nilotinib [NIL], and bosutinib [BOS]) on CV/Met risk factors in patients (pts) with CML-CP in routine care. Baseline CV/Met risks were previously reported (Mauro, GCOS 2019). Here, we report data from the final 2-year follow-up. Methods: Adult pts with newly diagnosed CML-CP due to start tx with DAS, IM, NIL, or BOS were enrolled; the BOS cohort was not analyzed independently due to small sample size. Pts were followed for 24 mo. Blood/urine samples, CV/Met assessments, coronary calcium scoring, echocardiography, and cytogenetic/molecular response assessments were collected at routine office visits at predetermined timepoints. The primary endpoint was change in CV/Met risk from baseline. Results: At the end of follow-up, 118 pts were enrolled; 49 received DAS, 47 IM, 16 NIL, and 6 BOS. Of these, 83 (70.3%) pts completed the study and 35 (29.7%) discontinued; the most common reason for discontinuation was withdrawal of consent (n = 19). No pts died from CML. At baseline, median age was 57 (range, 20–97) y; 59.3% of pts were male and 83.9% were White. Sixty-two (52.5%) pts had CV/Met risk factors at baseline, including hypertension (n = 45/62; 72.6%), hyperlipidemia (n = 33/62; 53.2%), diabetes (n = 19/62; 30.6%), and arrhythmia (n = 10/62; 16.1%). Over the study period, 1 (3.4%) patient receiving DAS and 2 (6.9%) receiving IM developed coronary artery calcification; 2 (9.1%) pts receiving IM developed pericardial effusion. Median left ventricular ejection fraction on 2D echocardiogram was normal for all TKIs at baseline (65.4% [range, 27–81]) and end of study (65.4% [range, 40–80]). Systolic and diastolic blood pressure increased from baseline only in pts who received NIL. Blood glucose levels increased from baseline for DAS, IM, and NIL (median: 1.9, 4.9, and 5.8 mg/dL, respectively), and low-density lipoprotein levels decreased from baseline in all 3 cohorts (median: 72.7, 52.1, and 56.3 U/L, respectively); data from the NIL group were limited by the small sample size. There was 1 death from myocardial infarction related to NIL. Conclusions: These findings suggest more than half of pts with CML-CP have CV/Met risk factors at baseline. At end of study, there was indication of CV/Met effects based on TKI selection. This highlights the importance of appropriate TKI selection based on pt comorbidity characteristics, along with close monitoring and management of CV/Met risks throughout tx. Clinical trial information: NCT03045120 .

A phase 2 study of neoadjuvant lenvatinib in locally advanced invasive thyroid cancer.

TPS6105 Background: Lenvatinib is a multiple receptor tyrosine kinase (RTK) inhibitor that has demonstrated meaningful benefit in radioiodine (RAI)-refractory differentiated thyroid cancer (DTC). In SELECT, lenvatinib significantly prolonged progression-free survival (PFS) and was associated with an overall response rate (ORR) of 65% in patients with RAI-refractory DTC, with the greatest degree of tumor shrinkage occurring in the first 2 cycles. The robust activity of lenvatinib in DTC suggests that neoadjuvant treatment in patients presenting with bulky neck disease may make surgery more feasible, allows for optimal surgical outcomes and less aggressive surgical management. Neoadjuvant lenvatinib to improve surgical outcomes in locally advanced DTC has not been studied to date. This phase 2 study will examine the efficacy and safety of lenvatinib prior to thyroidectomy, with the goal of achieving more favorable surgical outcomes. Methods: This is a Simon two-stage Phase 2 open-label multicenter clinical trial for adult patients who meet the following criteria: have been diagnosed with locally advanced or persistent/recurrent thyroid and/or cervical nodal DTC, deemed at risk for R2 resection (surgical margins with macroscopic tumor) (as evident by vocal cord paralysis by laryngoscopic exam, imaging documenting extrathyroidal/extranodal extension, or major vessels involvement), have measurable disease per RECIST v1.1, have an ECOG performance status ≤ 1, and no contraindication to surgery. A total of 28 patients will be enrolled from three centers (Massachusetts General Hospital/Massachusetts Eye &amp; Ear Infirmary (MGH/MEEI), Memorial Sloan-Kettering Cancer Center (MSKCC), and MD Anderson Cancer Center (MDACC)). Participants will receive up to 6 cycles of lenvatinib prior to surgery depending on response. Participants will undergo restaging at the end of cycle 2, 4, and 6. Participants with sufficient response to treatment who projected to achieve an R0 or R1 resection will stop lenvatinib and proceed to surgery within 7-10 days from the last dose. Participants who do not have a sufficient response who are tolerating treatment, will receive an additional 2-4 cycles of lenvatinib, followed by surgery. The primary objective is the R0 (surgical margins free from tumor)/R1 (surgical margins free from macroscopic tumor) resection rate. Secondary outcome measures include R0/R1 resection rates in each of 5 pre-specified anatomic target interfaces (perithyroid muscles, cartilage, esophagus, recurrent laryngeal nerve, major vessels), evaluation of the change in surgical morbidity complexity MGH/MEEI-MSKCC-MDACC (MMM) score and the Invasive Thyroid Class, ORR prior to surgery per RECIST v1.1, and the safety of lenvatinib in this patient population. The study is currently accruing with 11 patients enrolled at time of submission. Clinical trial information: NCT04321954 .

Mechanisms of resistance to tyrosine kinase inhibitor treatments in patients with ROS1 fusion-positive non-small cell lung cancer.

9062 Background: ROS1 rearrangement was found in about 1% non-small cell lung cancer (NSCLC), and the fusion proteins created during this process will result in the successive activation of the ROS1 kinase domain. NSCLC patients with ROS1 gene fusions were considered a unique subtype highly sensitive to related tyrosine kinase inhibitor (TKI) treatments. However, the acquired TKI resistance was the major hurdle preventing patients from getting prolonged treatment benefits. Methods: 107 patients diagnosed with advanced or metastatic NSCLC harboring ROS1 fusion were retrospectively recruited. All patients were treated with crizotinib as first-line treatment, and 21 patients received lorlatinib after crizotinib progression. Samples were collected at baseline, after crizotinib progression, and after lorlatinib progression, which all underwent targeted DNA sequencing. TKIs binding to mutated ROS1 fusion proteins was simulated using molecular dynamics simulations. Results: The most witnessed fusion partner of ROS1 was CD74 (58%), followed by SDC4 (14%), EZR (11%), and SLC34A2 (9%). The median progression-free survival was 12.9 months for crizotinib and 6.4 months for lorlatinib. Patients with CD74-ROS1 and SLC34A2-ROS1 had significantly longer PFS than those with other ROS1 fusion types when treated with crizotinib. Patients with baseline TP53 mutations showed worse PFS compared to TP53 wild-type (WT) patients (P &lt; 0.001, HR: 0.19, 95%CI: 0.09-0.39) under crizotinib treatment. An accumulation of both on-target (baseline vs. post-crizotinib vs. post-lorlatinib: 0% vs. 43% vs. 62%) and off-target resistant mutations (baseline vs. post-crizotinib vs. post-lorlatinib: 22% vs. 26% vs. 43%) after multiple TKI treatments was observed. A total of ten on-target resistance mutations were detected after TKI therapies, with 4 first-reported mutations (ROS1 L2010M, G1957A, D1988N, L1982V). According to the molecular dynamics simulation results, ROS1 L2010M mutations (happening in the ROS1 kinase ATP binding cassette) maybe resistant to lorlatinib, entrectinib, cabozantinib, and crizotinib. ROS1 G1957A may lead to resistance to cabozantinib. Moreover, all four novel on-target mutations may lead to resistance to crizotinib. Conclusions: In summary, CD74- and SLC34A2-ROS1 patients showed better crizotinib efficacy with different resistance mutation patterns. Patients of these subtypes are potential beneficiaries of molecular testing after crizotinib progression, directing future treatment strategies. Multiple TKI treatments may lead to the accumulation of both on-target and off-target resistance mutations. In addition, 4 novel ROS1 on-target mutations identified underwent molecular dynamics simulations, unveiling potential resistance to different TKIs, which can provide vital information for future ROS1 fusion-positive NSCLC patient treatment selections.