Trametinib Research Articles

A Multifunctional Rocket‐Like Microneedle System with Thrusters for Self‐Promoted Deep Drug Penetration and Combination Treatment in Melanoma

AbstractPhotodynamic therapy (PDT) proves highly effective in addressing melanoma, and its synergy with targeted therapy offers a promising avenue in tumor treatment. However, the therapeutic outcome is largely impeded by the challenge of current photosensitizers and targeted drugs in reaching deep tumor tissues. Herein, a dually‐layered “microneedle rocket” termed PcNP/TRA‐HA‐Tyr/CLG‐MN is designed. The upper layer of the microneedle (MN) is composed of photodynamically active mesoporous silica nanoparticles, featuring photosensitizers covalently bonded to them. Within the mesopores of these nanoparticles lies trametinib (TRA), a compound that specifically targets the hyperactive MEK pathway present in melanoma cells. The lower layer is composed of an enzyme‐mediated hyaluronic acid‐tyramine hydrogel (HA‐Tyr/CLG) with collagenase (CLG), serving as rocket thrusters for remodeling the extracellular matrix (ECM) in tumor microenvironment. Among the three types of MNs prepared, the PcNP/TRA‐HA‐Tyr(II)/CLG‐MN exhibits the deepest penetration in tumor tissues and the longest retention time in vivo. Notably, the administrations of PcNP/TRA‐HA‐Tyr(II)/CLG‐MN alongside light irradiation significantly suppress the growth of A375‐xenografted tumors in mice. Together, the strategy of combining mesoporous silica nanoparticles, enzymatically cross‐linked hydrogels and CLG‐mediated ECM remodeling enables deep drug penetration and efficacious combination treatment against melanoma, showcasing significant potential in the realm of cancer nanomedicine.

ANP-vessel Dilator Enhances the Effect of Trametinib on Colorectal Cancer Cells via the MAPK Pathway

Background Colorectal cancer (CRC) is a common malignant disease that involves the interaction of both genetic and environmental factors. Trametinib (TNB) has been approved alone or in combination with dabrafenib to treat melanoma. Vessel dilator (VDL) is a cardiac hormone that possesses anticancer properties. This study evaluates TNB with VDL combined effects in CRC cells and explores the role of Mutagen-activated protein kinases (MAPK, ERK1/2) in this effect. Methods The HCT-15 CRC cells were treated with TNB and VDL. The MTT and ELISA assays were used to assess p-ERK, VEGF, and VEGFR levels. Results The results showed that combined treatment of TNB with VDL produced a significant and synergistic inhibition of HCT-15 cell growth with a combination index of less than 1. A combination treatment demonstrated no change in the expression of VEGF, inhibition of VEGFR2, and reduction of p-ERK1/2 in HCT-15 cells. Conclusion The findings encourage further evaluation of this combination as the combined effect is not mediated through p-ERK, VEGF, and VEGFR.

Trametinib boosts palbociclib's efficacy in breast cancer via autophagy inhibition.

Breast cancer, a predominant global health issue, requires ongoing exploration of new therapeutic strategies. Palbociclib (PAL), a well-known cyclin-dependent kinase (CDK) inhibitor, plays a critical role in breast cancer treatment. While its efficacy is recognized, the interplay between PAL and cellular autophagy, particularly in the context of the RAF/MEK/ERK signaling pathway, remains insufficiently explored. This study investigates PAL's inhibitory effects on breast cancer using both in vitro (MCF7 and MDA-MB-468 cells) and in vivo (tumor-bearing nude mice) models. Aimed at elucidating the impact of PAL on autophagic processes and exploring the potential of combining it with trametinib (TRA), an MEK inhibitor, our research seeks to address the challenge of PAL-induced drug resistance. Our findings reveal that PAL significantly decreases the viability of MCF7 and MDA-MB-468 cells and reduces tumor size in mice while showing minimal cytotoxicity in MCF10A cells. However, PAL also induces protective autophagy, potentially leading to drug resistance via the RAF/MEK/ERK pathway activation. Introducing TRA effectively neutralized this autophagy, enhancing PAL's anti-tumor efficacy. A combination of PAL and TRA synergistically reduced cell viability and proliferation, and in vivo studies showed notable tumor size reduction. In conclusion, the PAL and TRA combination emerges as a promising strategy for overcoming PAL-induced resistance, offering a new horizon in breast cancer treatment.

Nanoparticles targeting Sialyl-Tn for efficient tyrosine kinase inhibitor delivery in gastric cancer

Gastric cancer (GC) is the fourth leading cause of cancer-related deaths worldwide and, therefore, it is urgent to develop new and more efficient therapeutic approaches. Foretinib (FRT) is an oral multikinase inhibitor targeting MET (hepatocyte growth factor receptor) and RON (recepteur d'origine nantais) receptor tyrosine kinases (RTKs) that has been used in clinical trials for several solid tumors. Targeted uptake of therapeutic polymeric nanoparticles (NPs) represents a powerful approach in cancer cell drug delivery. Previously, a nanodelivery system composed of polymeric NPs functionalized with B72.3 antibody, which targets the tumor-associated antigen Sialyl-Tn (STn), has been developed. Herein, these NPs were loaded with FRT to evaluate its capacity in delivering the drug to multicellular tumors spheroids (MCTS) and mouse models. The data indicated that B72.3 functionalized FRT-loaded PLGA-PEG-COOH NPs (NFB72.3) specifically target gastric MCTS expressing the STn glycan (MKN45 SimpleCell (SC) cells), leading to a decrease in phospho-RTKs activation and reduced cell viability. In vivo evaluation using MKN45 SC xenograft mice revealed that NFB72.3 were able to decrease tumor growth, reduce cell proliferation and tumor necrosis. NFB72.3-treated tumors also showed inactivation of phospho-MET and phospho-RON. This study demonstrates the value of using NPs targeting STn for FRT delivery, highlighting its potential as a therapeutic application in GC. Statement of significanceDespite the advances in gastric cancer therapeutics, it remains one of the diseases with the highest incidence and mortality in the world. Combining targeted therapies with a controlled drug release is an attractive strategy to reduce drug cytotoxic effects and improve specific drug delivery efficiency to the cancer cells. Thus, we developed nanoparticles loaded with a tyrosine kinase inhibitor and targeting a specific tumor glycan exclusive of cancer cells. In in vivo gastric cancer xenograft mice models, these nanoparticles efficiently reduced tumor growth, cell proliferation and tumor necrosis area and inactivated phosphorylation of targeting receptors. This approach represents an innovative therapeutic strategy with high impact in gastric cancer.

Efficacy of adjuvant therapy in patients (pts) with AJCC v8 stage IIIA cutaneous melanoma.

9518 Background: Pts with resected AJCC v8 stage IIIA melanoma have been under-represented in clinical trials of adjuvant drug therapy. The benefit of adjuvant targeted and immunotherapy in this population is unclear. Methods: In this multicenter, retrospective study, pts with stage IIIA melanoma (AJCC v8) who received adjuvant pembrolizumab or nivolumab (PD1), BRAF/MEK-targeted therapy dabrafenib + trametinib (TT), or no adjuvant treatment (OBS) were included. Recurrence free survival (RFS), distant metastasis free survival (DMFS), and toxicity rates were examined. Results: 613 pts from 34 centers across Australia, Europe and USA were included. The median follow-up was 2.6 yrs (IQR, 1.6-3.4 yrs). Pt characteristics and follow-up were similar across the cohorts (Table). Completion rates of 12-month PD1 and TT therapy were 57.0% and 69.2% respectively. 1-yr RFS was 93.3% (95% CI, 90.3-96.4) in PD1, 100% in TT and 91.3% (95% CI, 88.1-94.7) in OBS cohorts. 2-yr RFS was 79.3% (95% CI, 74.1-84.8) for PD1, 100% for TT and 84.3% (95% CI, 79.9-89.0) in OBS cohorts. 2-yr DMFS was 88.4% (95% CI, 84.3-92.8) in PD1, 100% in TT and 91.1% (95% CI, 87.7-94.7) in OBS cohorts. Risk of recurrence was associated with higher breslow thickness (HR 1.73, 95% CI 1.36-2.20), higher mitotic rate (HR 1.07, 95% CI 1.02-1.12) and neck as the site of nodal metastases (HR 2.71, 95% CI 1.45-5.06) in the overall cohort (p<0.05). Neck nodal metastases were associated with higher risk of recurrence in OBS cohort (HR 3.82, 95% CI 1.91-7.66; p<0.05) but not in the PD1 cohort. Rates of ≥ Grade 3 toxicities were 10.9% with PD1 and 20.0% with TT; discontinuation due to toxicity occurred in 25.0% and 30.0%, respectively. No new safety signals were observed. Rates of unresolved toxicity at last follow-up were 26.9% in PD1 and 7.7% in TT cohorts. Conclusions: In this large international study, adjuvant PD1 or TT did not significantly improve RFS or DMFS compared to OBS in pts with resected stage IIIA melanoma. Prognosis in stage IIIA melanoma is favourable and outcomes after adjuvant therapy in this population needs further study in prospective randomised trials. [Table: see text]

Unravelling the Origin of Solvate Formation in the Anticancer Drug Trametinib: Insights from Crystal Structure Analysis and Computational Modeling

The discovery of new crystalline forms of large, flexible drug molecules is of significant interest to the pharmaceutical industry as a means of improving one or more physicochemical properties of the active pharmaceutical ingredient (API). Trametinib (TRMB) is an anticancer drug, for which no solid-state forms are yet reported. Here, we report the results of an extensive screen for cocrystals of TRMB in an effort to improve the poor aqueous solubility of this API. Contrary to expectations, this effort led to the discovery of only six isostructural solvates of TRMB, where the solvent molecules fill the voids of the crystal structure and are stabilized by various intermolecular interactions. Computational crystal structure prediction methods were used to rationalize the high propensity for solvent inclusion in TRMB as arising from the poor crystal packing in TRMB. All predicted polymorphs of TRMB within a relative lattice energy of 44 kJ mol–1 of the global minimum display solvent accessible voids ranging from 1.4 to 16.5% of the unit cell volume. The serendipitous discovery of solvates in experiments targeting cocrystals,strongly suggests that the well-established synthon approach for the discovery of cocrystals works relatively well for documented literature examples that use rigid coformers with minimal conformational flexibility. However, for large APIs such as TRMB, in-silico screening methods should be adopted to reduce the need for extensive experimental cocrystal screens as the ability to form a cocrystal for drug candidates relies on more than synthon complementarity.

Co-Treatment of Chloroquine and Trametinib Inhibits Melanoma Cell Proliferation and Decreases Immune Cell Infiltration.

Autophagy is characterized as a cytoprotective process and inhibition of autophagy with medicinally active agents, such as chloroquine (CQ) is proposed as a prospective adjuvant therapy for cancer. Here, we examined the preclinical effects of CQ combined with the MEK inhibitor trametinib (TRA) on melanoma. We found that cotreatment of CQ and TRA markedly slowed melanoma growth induced in Tyr-CreER.BrafCa.Ptenfl/fl mice. Immunostaining showed that trametinib decreased Ki-67+ proliferating cells, and increased TUNEL+ apoptotic cells. The combo treatment induced a further decrease of Ki-67+ proliferating cells. Consistent with the in vivo findings, CQ and TRA inhibited melanoma cell proliferation in vitro, which was correlated by decreased cyclin D1 expression. In addition, we found that tissues treated with CQ and TRA had significantly decreased numbers of CD4+ and CD8+ T-lymphocytes and F4/80+ macrophages. Together, these results indicate that cotreatment of CQ and TRA decreases cancer cell proliferation, but also dampens immune cell infiltration. Further study is warranted to understand whether CQ-induced immune suppression inadvertently affects therapeutic benefits.

Abstract CT197: Phase Ib study of LXH254 + trametinib (TMT) in patients (pts) with NRAS-mutant melanoma

Abstract Background: MAPK pathway dysregulation is a characteristic molecular abnormality of melanoma. NRAS mutations in melanoma lead to constitutive MAPK activation and subsequent MEK and ERK activation. Inhibiting the pathway with a combination of BRAF/CRAF and MEK inhibitors (LXH254 and TMT, respectively) has demonstrated synergistic activity in preclinical models of NRAS-mutant melanoma. Methods: We report data from an open-label, Phase Ib study (NCT02974725), that tested LXH254 + TMT combination in a dose-escalation (ESC) part in pts with KRAS- or BRAF-mutant NSCLC or NRAS-mutant melanoma and in a dose-expansion (EXP) part in NRAS-mutant melanoma pts. In ESC, five dose levels were explored: LXH254 200 mg twice daily (BID) + TMT (1 mg or 0.5 mg) daily (QD); LXH254 400 mg BID + TMT (1 mg or 0.5 mg) QD; LXH254 400 mg BID + TMT 1 mg (QD, 2 wk on/2 wk off). Primary objectives were to evaluate safety and tolerability, and determine the recommended dose for expansion (RDE). Secondary objectives were to characterize pharmacokinetics and pharmacodynamics of LXH254 + TMT, and evaluate antitumor activity. Results: As of Dec 9, 2021, 36 and 30 pts were enrolled in ESC and EXP, respectively; median age was 63.5 and 69 y, respectively. Pts in ESC and EXP received a median of 3 (range: 1-6) and 2 (range: 1-7) prior treatments, respectively, including IO (ESC: 88.9% pts; EXP: 96.7% pts). All pts from ESC and 24 pts from EXP discontinued, mainly due to progressive disease (61.1% and 60%, respectively); 6 pts were ongoing in EXP at data cut-off. In ESC, Grade ≥3 (G≥3) DLTs were reported in 6 pts: dermatitis acneiform (n=2), maculopapular rash (n=2), increased lipase (n=1), and Stevens-Johnson syndrome (n=1). The RDEs were LXH254 200 mg BID + TMT 1 mg QD and LXH254 400 mg BID + TMT 0.5 mg QD. Treatment-related adverse events (TRAEs) were reported in 34 pts (94.4%) in ESC and all pts in EXP. G≥3 TRAEs in ESC and EXP were experienced by 19 (52.8%) and 24 (80%) pts, respectively; the most common being rash or dermatitis acneiform (13.9% each) in ESC and rash (33.3%) in EXP. One fatal TRAE (hypovolemic shock) was reported in the LXH254 400 mg BID + TMT 0.5 mg QD group. The ORR in EXP was 46.7% (7 pts; 95% CI: 21.3-73.4) for LXH254 200 mg BID + TMT 1 mg QD and 13.3% (2 pts; 95% CI: 1.7-40.5) for LXH254 400 mg BID + TMT 0.5 mg QD; overall in EXP, 9 pts (30%) achieved PR and 13 pts (43.3%) reported SD. In EXP the median DOR was 3.8 months (95% CI: 2.6-7.4). Exposures of LXH254 (200 mg or 400 mg BID) + TMT (0.5 mg or 1 mg QD) vs LXH254 single agent were comparable, indicating no apparent LXH254-TMT interactions. Limited paired tumor biopsy data (n=8) showed substantial MAPK pathway inhibition (DUSP6). Conclusions: LXH254 in combination with TMT has shown preliminary antitumor activity in the pretreated, NRAS-mutant melanoma population. A Phase II trial of LXH254 combinations in pts with pretreated BRAF- or NRAS-mutant melanoma is currently ongoing (NCT04417621). Citation Format: Filippo de Braud, Christophe Dooms, Rebecca S. Heist, Celeste Lebbe, Martin Wermke, David Planchard, Dirk Schadendorf, Piotr Rutkowski, Jürgen Wolf, Paolo A. Ascierto, Ignacio Gil-Bazo, Shumei Kato, Maria Wolodarski, Meredith McKean, Eva Muñoz Couselo, Martin Sebastian, Armando Santoro, Vesselina Cooke, Luca Manganelli, Kitty Wan, Anil Gaur, Tanay S. Samant, Jaeyeon Kim, Giordano Caponigro, Xuân-Mai Couillebault, Michele Moschetta, Adil Daud. Phase Ib study of LXH254 + trametinib (TMT) in patients (pts) with NRAS-mutant melanoma [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 CT197.

Abstract 368: Improving glioblastoma treatment specificity and efficacy of mApoE-targeted liposome by MMP2-controlled drug releasee

Abstract Recent evidence from our laboratory provided proof-of-concept for therapeutic potential for glioblastoma (GBM) of a combination strategy based on radiation and adjuvant doxorubicin-loaded liposomes (LIPs) conjugated with a modified Apolipoprotein E-derived peptide (mApoE), known to facilitate Blood Brain Barrier (BBB)-crossing. Significant glioma stem cell (GSC) apoptosis, tumor growth inhibition and increased overall survival were observed in vivo upon combined treatment offering attractive and innovative therapeutic possibilities for GBM. To strengthen therapeutic efficacy and lower off-target effects, we implemented mApoE-LIPs with a matrix metalloproteinases (MMP)-activable element that allows controlled payload release only in the MMPs rich tumor microenvironment, thus concurring to reduce unspecific interaction in healthy tissue where MMPs are low or absent Given the MMP2 overexpression in GBM, a MMP2-activable block (M2AB) was included in the phospholipid bilayer of mApoE-LIPs. The M2AB efficacy was evaluated on patient-derived GSCs displaying different MMP2 enzymatic activities by means of calcein-loaded M2AB/mApoE-LIPs. Human endothelial cells (hCMEC/d3), not expressing MMP2, were included to validate the targeted strategy as well as to assay the cytotoxicity on non-tumoral cells. Intracellular calcein quantification showed that: 1) calcein uptake correlates with MMP2 activity level; 2) M2AB/mApoE functionalization augmented calcein internalization into GSCs compared to mApoE alone. The MEK/ERK pathway, known to supports GBM cell survival, migration, and radio-resistance was considered as target strategy against GBM. A survey of the MEK/ERK inhibitors Trametinib (TRAM) and Pimasertib (PIMA) in several GSC lines indicated significant induction of GSC apoptosis associated to reduction of ERK phosphorylation. TRAM and PIMA were then encapsulated into mApoE-LIPs and their anti-GSC activity was investigated. Indeed, a dose dependent inhibition of GSC survival and induction of apoptosis combined to a significant lower level of phospho-ERK was observed upon 72h treatment. In conclusion, M2AB/mApoE-LIPs demonstrate: 1) a MMP2-dependent payload release; 2) stability when MMP2 lacks in the cellular milieu; 3) a synergic effect of the double M2AB/mApoE functionalization. TRAM and PIMA encapsulation into LIPs does not alter their anti-GSC activity. Funding by FRRB grant NEVERMIND (CP2_16/2018) Citation Format: Milena Mattioli, Marco Pizzocri, Elisabetta Stanzani, Valentino Ribecco, Simone Olei, Maria Pia Tropeano, Sabrina Giofrè, Antonio Renda, Sara Pellegrino, Pierfausto Seneci, Francesca Re, Federico Pessina, Michela Matteoli, Lorena Passoni. Improving glioblastoma treatment specificity and efficacy of mApoE-targeted liposome by MMP2-controlled drug releasee [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 368.

Tumor suppressor miR-193a-3p enhances efficacy of BRAF/MEK inhibitors in BRAF-mutated colorectal cancer.

Patients with BRAF‐mutated colorectal cancer (CRC) have a poor prognosis despite recent therapeutic advances such as combination therapy with BRAF, MEK, and epidermal growth factor receptor (EGFR) inhibitors. To identify microRNAs (miRNAs) that can improve the efficacy of BRAF inhibitor dabrafenib (DAB) and MEK inhibitor trametinib (TRA), we screened 240 miRNAs in BRAF‐mutated CRC cells and identified five candidate miRNAs. Overexpression of miR‐193a‐3p, one of the five screened miRNAs, in CRC cells inhibited cell proliferation by inducing apoptosis. Reverse‐phase protein array analysis revealed that proteins with altered phosphorylation induced by miR‐193a‐3p were involved in several oncogenic pathways including MAPK‐related pathways. Furthermore, overexpression of miR‐193a‐3p in BRAF‐mutated cells enhanced the efficacy of DAB and TRA through inhibiting reactivation of MAPK signaling and inducing inhibition of Mcl1. Inhibition of Mcl1 by siRNA or by Mcl1 inhibitor increased the antiproliferative effect of combination therapy with DAB, TRA, and anti‐EGFR antibody cetuximab. Collectively, our study demonstrated the possibility that miR‐193a‐3p acts as a tumor suppressor through regulating multiple proteins involved in oncogenesis and affects cellular sensitivity to MAPK‐related pathway inhibitors such as BRAF inhibitors, MEK inhibitors, and/or anti‐EGFR antibodies. Addition of miR‐193a‐3p and/or modulation of proteins involved in the miR‐193a‐3p–mediated pathway, such as Mcl1, to EGFR/BRAF/MEK inhibition may be a potential therapeutic strategy against BRAF‐mutated CRC.

Abstract 2361: The functional roles of miRNA-193a-3p in colorectal cancer

Abstract Background: Patients with BRAF-mutated colorectal cancer (CRC) have a poor prognosis despite recent therapeutic advances such as combination therapy with BRAF, MEK, and EGFR inhibitors. Aim: Our aim was to clarify the functions of the microRNAs (miRNAs) involved in oncogenesis and sensitivity to BRAF inhibitors and MEK inhibitors in BRAF-mutated CRC. Methods and Results: To identify miRNAs that sensitize a BRAF inhibitor dabrafenib (DAB) and a MEK inhibitor trametinib (TRA), we screened 240 miRNAs in BRAF-mutated CRC cell line RKO. Among the screened miRNAs, we tried to elucidate the novel functional roles of miR-193a-3p in CRC cell lines. We found that miR-193a-3p overexpression inhibited cell proliferation in CRC cell lines. The protein array analysis revealed that protein phosphorylation levels were altered by miR-193a-3p overexpression in various pathways such as cell-cycle-related pathways and PI3K-AKT pathways. The enhanced cytotoxicity by miR-193a-3p in addition to DAB and TRA was validated in multiple BRAF-mutated cell lines. Furthermore, the enhanced cytotoxicity by miR-193a-3p in addition to DAB and TRA was shown to be through inhibiting reactivation of MAPK signaling. This enhanced cytotoxicity by miR-193a-3p was even observed under treatment of DAB, TRA, and an anti-EGFR antibody. Conclusions: Our study shows that miR-193a-3p regulates multiple proteins involved in oncogenesis, and proposes that the combination therapy of miR-193a-3p and BRAF/MEK inhibitors could be a promising treatment strategy in BRAF-mutated CRC. Citation Format: Sakura Hiraide, Masanobu Takahashi, Yuya Yoshida, Keigo Komine, Chikashi Ishioka. The functional roles of miRNA-193a-3p in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2361.

Primary Malignant Melanoma of the Esophagus: Prognosis and Therapeutic Challenges

Primary malignant melanoma of the esophagus (PMME) is a very rare and highly aggressive tumor, representing <1% of esophageal malignancies. We are reporting a case of PMME in a 54-year-old White, Non-Hispanic male, who presented to the hospital with dysphagia and weight loss. Computed tomography (CT) of the chest revealed a large mid-to-distal esophageal mass with mass effect on adjacent structures, with no evidence of invasion. The patient subsequently underwent esophagogastroduodenoscopy (EGD) with guided biopsy of the esophageal mass. Histopathology and immunohistochemistry of the biopsied specimen were performed and consistent with malignant melanoma. Tumor staging by positron emission tomography and computed tomography (PET/CT) scans revealed extensive, locally advanced disease with lymph node involvement. Subsequently, esophageal ultrasound (EUS) with biopsy of the involved lymph node was performed and was positive for malignant melanoma. The mass was deemed unresectable due to the extensive degree of locally advanced disease. To assist in our therapeutic decision making, the patient was screened for the presence of a BRAF V600 mutation. He was then started on a combination of the BRAF inhibitor (dabrafenib) in combination with the mitogen-activated extracellular kinase (MEK) Inhibitor (trametinib) given the presence of a BRAF V600 mutation.

Response surface methodology for optimization of micellar-enhanced spectrofluorimetric method for assay of foretinib in bulk powder and human urine

This work investigates a sensitive and precise enhanced spectrofluorimetric assay for assay of foretinib (FTB); a tyrosine kinase inhibitor drug used for treatment of breast cancer, in tablets and urine through response surface optimization by micelle mediated protocol. The basis of the described method is the enhancement of the fluorescence behavior of FTB in Cremophor RH 40 (Cr RH 40) micellar medium and measuring the fluorescence of FTB at 344 nm after excitation at 245 nm. Optimization was performed through evaluation of diluting solvent, types of organized media, buffer type and its relevant pH. Response surface methodology was applied to obtain the optimized values of variables that mostly affect interaction of Cr RH 40 with FTB using Box–Behnken design. ICH guidelines were adhered for the validation of merit figures. Acceptable linear relationship was obtained between relative fluorescence intensity (RFI) and FTB concentrations in the range of 50 – 1000 µg L−1, with correlation coefficient of 0.998. Accuracy was ≥ 99.82% and calculated limit of detection (LOD) was 10.60 µg L−1. Method applications included FTB assaying in pure bulk powder. Furthermore, applications on urine samples were performed with accuracy of 100.59 ± 3.40%. The method represents echo-friendly approach and effective alternating methodology to the relevant analytical ones for FTB assaying.

Determination of dabrafenib and trametinib in serum by dispersive solid phase extraction with multi-walled carbon nanotubes and capillary electrophoresis coupled to ultraviolet/visible detection

Dispersive solid phase extraction (DSPE) prior to capillary electrophoresis with ultraviolet and visible detection was used for the first time for the determination of dabrafenib (DB) and trametinib (TT), the main drugs used for the treatment of metastatic melanoma, in serum. Six different magnetic and non-magnetic nanomaterials were evaluated as DSPE sorbents for the extraction and preconcentration of the analytes. The best results were obtained with 4 mg of pristine multi-walled carbon nanotubes in phosphate buffer (pH 12.5; 10 mM). The electrophoretic separation was carried out in a fused silica capillary (30 cm × 75 µm i.d.) at 22 °C in a voltage ramp (8–12 kV) with a background electrolyte consisting of borate buffer (pH 9.2; 25 mM), 37 mM sodium dodecylsulfate, 22% (v/v) acetonitrile and 3% (v/v) methanol.The preconcentration factor achieved was 4, and the limits of detection were 0.012 and 0.008 mg L−1 for DB and TT in serum, respectively. Linearity was observed up to 5 mg/L and precision (n = 7) was below 3% in migration time and 7.8% in peak areas. The present method was applied to six patients under treatment with DB and TT for metastatic melanoma. It requires a less sophisticated instrumentation and lower volume of harmful organic solvents than the methods reported up to present.

NLRP1 Functions Downstream of the MAPK/ERK Signaling via ATF4 and Contributes to Acquired Targeted Therapy Resistance in Human Metastatic Melanoma.

The BRAF V600E mutation leads to constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway and its downstream effector responses. Uncovering the hidden downstream effectors can aid in understanding melanoma biology and improve targeted therapy efficacy. The inflammasome sensor, NACHT, LRR, and PYD domains-containing protein 1 (NLRP1), is responsible for IL-1β maturation and itself is a melanoma tumor promoter. Here, we report that NLRP1 is a downstream effector of MAPK/ERK signaling through the activating transcription factor 4 (ATF4), creating regulation in metastatic melanoma cells. We confirmed that the NLRP1 gene is a target of ATF4. Interestingly, ATF4/NLRP1 regulation by the MAPK/ERK pathway uses distinct mechanisms in melanoma cells before and after the acquired resistance to targeted therapy. In parental cells, ATF4/NLRP1 is regulated by the MAPK/ERK pathway through the ribosomal S6 kinase 2 (RSK2). However, vemurafenib (VEM) and trametinib (TRA)-resistant cells lose the signaling via RSK2 and activate the cAMP/protein kinase A (PKA) pathway to redirect ATF4/NLRP1. Therefore, NLRP1 expression and IL-1β secretion were downregulated in response to VEM and TRA in parental cells but enhanced in drug-resistant cells. Lastly, silencing NLRP1 in drug-resistant cells reduced their cell growth and inhibited colony formation. In summary, we demonstrated that NLRP1 functions downstream of the MAPK/ERK signaling via ATF4 and is a player of targeted therapy resistance in melanoma. Targeting NLRP1 may improve the therapeutic efficacy of targeted therapy in melanoma.

Simultaneous quantification of dabrafenib, hydroxy-dabrafenib and trametinib in human plasma by liquid chromatography-tandem mass spectrometry

A new liquid chromatography-tandem mass spectrometry (LC–MS/MS) method for the simultaneous quantification of dabrafenib (DAB), its main metabolite hydroxy-dabrafenib (OHD) and trametinib (TRA) in human plasma has been developed and validated. After addition of internal standard (dabrafenib-d9), extraction was achieved after protein precipitation with acetonitrile containing 1 % (v/v) formic acid. Chromatographic separation was performed on an Accucore® C18 (2.1 × 50 mm; 2.6 μm) column using a gradient elution of water acidified with 0.1 % (v/v) formic acid (A) and acetonitrile containing 0.1 % (v/v) formic acid (B) at a flow rate of 500 μL/min. The calibration ranged from 10 to 2000 ng/mL for DAB and OHD and from 5 to 50 ng/mL for TRA. This method was validated with satisfactory results including good precision (intra- and inter-assay coefficient of variation from 2.0 %–14.9 %) and good accuracy (inter- and intra-day bias between -1.2 % and 10.9 %), as well as long term stability in unprocessed plasma at −20 °C. This newly proposed method is useful for clinical research purposes as well as therapeutic drug monitoring for patients with a Rapidly Accelerated Fibrosarcoma kinase B (BRAF)-mutated cancer.

A Randomized Phase II/III Study to Assess the Efficacy of Trametinib in Patients with Recurrent or Progressive Low-Grade Serous Ovarian or Peritoneal Cancer

Objective: Low-grade serous carcinoma of the ovary/peritoneum (LGSOC) is a rare subtype, accounting for 5%–10% of all serous cancers and is characterized by alterations in the MAPK pathway, relative chemoresistance, and prolonged overall survival (OS) compared to high-grade serous carcinoma. NRG Oncology in the United States and the National Cancer Research Network (NCRN) in the United Kingdom collaborated on a phase II–III trial to assess the efficacy of a MEK inhibitor trametinib (TRAM) compared to physician’s choice standard of care (SOC) in recurrent LGSOC.

MEK blockade overcomes the limited activity of palbociclib in head and neck cancer.

Head and neck cancer (HNC) is characterized with multiple aberrations in cell cycle pathways, including amplification of cyclin D1. Palbociclib (PAL), a cyclin-dependent kinase 4/6 (CDK4/6) inhibitor, has been reported to regulate cell cycle progression in HNC. However, recent studies have revealed the acquired resistance of certain cells to PAL through activation of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. Therefore, we investigated whether the inhibition of MEK/ERK pathway by trametinib (TRA) may overcome the limited efficacy of PAL in HNC. We evaluated the effect of PAL alone and in combination with TRA on the viability of HNC cells, and found that the combination treatment synergistically inhibited the proliferation of HNC cells. The combination treatment induced G0/G1 cell cycle arrest and apoptotic cell death. In particular, apoptosis mediated by the combination treatment was accompanied with an increase in caspase-3 activity and the number of TUNEL-positive apoptotic cells. These results were consistent with the decrease in cell cycle progression and mitogen-activated protein kinase (MAPK) pathway activation. In a xenograft mouse model of HNC, PAL and TRA synergistically inhibited tumor growth and enhanced tumor cell apoptosis, consistent with the increase in the number of TUNEL-positive cells. The anti-proliferative effects were evident in tumor tissues subjected to the combination treatment as compared with those treated with single drug. Taken together, our study demonstrates that the combination of PAL and TRA exerts synergistic anticancer effects and inhibits cell cycle check points and MEK/ERK pathway in HNC, suggestive of their potential application for HNC treatment.

Exploring the effect of khat (Catha edulis) chewing on the pharmacokinetics of the antiplatelet drug clopidogrel in rats using the newly developed LC-MS/MS technique

AbstractClopidogrel (CLOP) is widely used worldwide for cardiovascular complications. CLOP is highly metabolized in the liver to its active metabolite by cytochrome P450 enzymes. Studies have shown that khat, an addictive substance, is a powerful inhibitor of cytochrome P450 enzymes and can influence the metabolism of drugs that are concomitantly used. Therefore, this study was designed to evaluate the effects of khat on the pharmacokinetics of CLOP in rats. In this study, rats were administered either CLOP alone or CLOP combined with khat and their plasma were obtained at different time intervals and analyzed using the newly developed and validated liquid chromatography with tandem mass spectrometry (LC-MS/MS) method using foretinib (FTB) as the internal standard. The corresponding peak area of the analyte versus FTB was used for calculating the peak ratio. The validated LC-MS/MS method resulted in the separation of the well-defined quantifiable peaks of CLOP, FTB, and CLOP metabolite within 7 min. Results showed a significant influence of khat on the peak ratio of CLOP metabolite, which was found to be significantly decreased (P &lt; 0.05) in comparison to CLOP alone, suggesting significant decrease in the conversion of CLOP to its active metabolite due to the inhibition of CYP450 enzymes by khat. Therefore, there might be a need for dose adjustment for regular khat chewers using CLOP.

Monocyte-derived multipotent cell delivered programmed therapeutics to reverse idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a highly heterogeneous and fatal disease. However, IPF treatment has been limited by the low drug delivery efficiency to lungs and dysfunctional "injured" type II alveolar epithelial cell (AEC II). Here, we present surface-engineered nanoparticles (PER NPs) loading astaxanthin (AST) and trametinib (TRA) adhered to monocyte-derived multipotent cell (MOMC) forming programmed therapeutics (MOMC/PER). Specifically, the cell surface is designed to backpack plenty of PER NPs that reach directly to the lungs due to the homing characteristic of the MOMC and released PER NPs retarget injured AEC II after responding to the matrix metalloproteinase-2 (MMP-2) in IPF tissues. Then, released AST can enhance synergetic effect of TRA for inhibiting myofibroblast activation, and MOMC can also repair injured AEC II to promote damaged lung regeneration. Our findings provide proof of concept for developing a strategy for cell-mediated lung-targeted delivery platform carrying dual combined therapies to reverse IPF.