Neratinib Research Articles

Evaluation of the Feasibility of Transdermal Delivery of Neratinib (NB) Loaded Microneedles to Treat Breast Cancer

Evaluation of the Feasibility of Transdermal Delivery of Neratinib (NB) Loaded Microneedles to Treat Breast Cancer

Abstract PO2-19-05: Randomized Phase II trial evaluating three anti-diarrhoeal prophylaxis strategies in patients with HER2+/HR+ early breast cancer treated with extended adjuvant neratinib (DIANER GEICAM/2018-06)

Abstract Background: Neratinib (NER), and irreversible pan-HER tyrosine kinase inhibitor, is approved for the extended adjuvant treatment of HER2+/HR+ early breast cancer (EBC) patients within 1 year of trastuzumab (T)-based therapy completion. The ExteNET trial showed that the extended adjuvant NER compared with placebo modifies the risk of recurrence by an absolute rate of 2.5% at 5 years in HR+/HER2+ patients (Martin et al., 2017). However, without prespecified prophylaxis, diarrhoea was the most common reason for NER discontinuation in the first three cycles in said study. The CONTROL trial evaluated effectiveness of different anti-diarrhoeal strategies showing a reduction of severe diarrhoea and early discontinuation when indirectly compared to ExteNET (Chan et al., 2022). DIANER (NCT05252988) evaluates the 3 best reported anti-diarrhoeal strategies from CONTROL in patients receiving NER as per European approved label. Trial design and patients: DIANER is an international, multicenter, controlled, randomized, phase II study. Patients with HER2+/HR+ EBC (stage IB-IIIC) who completed prior neo/adjuvant T-based therapy within 2 weeks and 1 year will be included and randomized 1:1:1 to 3 treatment arms. Arm A: NER (240 mg/day[d] x 1 year) + loperamide (12 mg/d x 14 d → 8 mg/d till end of cycle 2 → as needed [PRN]), Arm B: NER dose escalation (120 mg/d x 7 d → 160 mg/d till d 14 → 240 mg/d x 13 cycles) + loperamide PRN, and Arm C: NER (like Arm A) + loperamide (12 mg/d x 14 d → 8 mg/d till end of cycle 1 → PRN) + colesevelam (3,750 mg/d x 28 d). Patients are stratified by menopausal status and prior anti-HER2 therapy (T versus T + pertuzumab). Specific aims: Primary objective is incidence of NER discontinuations due to diarrhoea in the first 3 cycles. Secondary objectives are: incidence of NER discontinuations; AEs and hospitalizations; duration, severity, and treatments for diarrhoea; NER exposure; and health-related quality of life (HRQoL) using FACT B and EQ5D-5L. Exploratory objectives include HRQoL using STIDAT; minimal residual disease and molecular alterations. Statistical methods: A maximum of 315 pts (105 per arm) will be needed using a Simon’s optimal two-stage design with the hypotheses of a desirable early discontinuation rate of 5% (H1) versus an undesirable early discontinuation rate of 13% (H0). In the first stage, arms with ≥ 4 pts discontinuing due to diarrhoea in the first 3 cycles, will be closed for enrolment. The 2-sided 90% confidence intervals using the method described by Koyama et al. (Koyama and Chen 2008) will be provided for the primary endpoint at the end of the 2nd stage of the Simon 2-stage design. Present accrual and target accrual: DIANER will take place in 81 sites in 5 European countries. As of Jul 10/2023 the study is open in 52 sites in Spain with 80 randomized patients. Contact information for people with a specific interest in the trial: Evelia Cortazar (Geicam PM): ecortazar@geicam.org Citation Format: Miguel Gil-Gil, Miguel Martín, Eva Carrasco, Elena Galve, Noelia Martínez-Jáñez, Santiago González-Santiago, Barbara Adamo, María Valero, Clara Martínez, Angel Guerrero, Silvia Antolin Novoa, Manuel Ruíz - Borrego, Isabel Blancas, Raquel Andrés, José L Alonso-Romero, ANDREA Vethencourt, J. Ignacio Chacón, Natalia Chavarría, Enrique Espinosa, Blanca Hernando, Encarnación Adrover, Federico Rojo, Maribel Casas, Leanne McCulloch, Jean-Claude Vedovato. Randomized Phase II trial evaluating three anti-diarrhoeal prophylaxis strategies in patients with HER2+/HR+ early breast cancer treated with extended adjuvant neratinib (DIANER GEICAM/2018-06) [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-19-05.

Neratinib is a TFEB and TFE3 activator that potentiates autophagy and unbalances energy metabolism in ERBB2+ breast cancer cells

Neratinib (NE) is an irreversible pan-ERBB tyrosine kinase inhibitor used to treat breast cancers (BCa) with amplification of the ERBB2/HER2/Neu gene or overexpression of the ERBB2 receptor. However, the mechanisms behind this process are not fully understood. Here we investigated the effects of NE on critical cell survival processes in ERBB2+ cancer cells. By kinome array analysis, we showed that NE time-dependently inhibited the phosphorylation of two distinct sets of kinases. The first set, including ERBB2 downstream signaling kinases such as ERK1/2, ATK, and AKT substrates, showed inhibition after 2 h of NE treatment. The second set, which comprised kinases involved in DNA damage response, displayed inhibition after 72 h. Flow cytometry analyses showed that NE induced G0/G1 cell cycle arrest and early apoptosis. By immunoblot, light and electron microscopy, we revealed that NE also transiently induced autophagy, mediated by increased expression levels and nuclear localization of TFEB and TFE3. Altered TFEB/TFE3 expression was accompanied by dysregulation of mitochondrial energy metabolism and dynamics, leading to a decrease in ATP production, glycolytic activity, and a transient downregulation of fission proteins. Increased TFEB and TFE3 expression was also observed in ERBB2-/ERBB1 + BCa cells, supporting that NE may act through other ERBB family members and/or other kinases. Overall, this study highlights NE as a potent activator of TFEB and TFE3, leading to the suppression of cancer cell survival through autophagy induction, cell cycle arrest, apoptosis, mitochondrial dysfunction and inhibition of DNA damage response.

Simultaneous estimation of neratinib and naringenin from pharmaceuticals using a stability-indicating liquid chromatographic method: Application to analytical quality by design concept and multi-tool method greenness assessment

A validated stability-indicating simultaneous liquid chromatographic method was developed to estimate neratinib (NRB) and naringenin (NRN). We used the principles of analytical quality by design (AQbD) to develop a robust analytical method. The ACN%, pH, and flow rate were the three method variables investigated and optimized using the Box-Behnken experimental design (BBD). The optimized chromatographic condition used a mobile phase of ACN: phosphate buffer pH 3.8 (70:30 v/v) flowing at 0.6 mL.min−1 on a 250 mm C-18 column. Diode array detection was performed at 277 nm. The method was robust, linear (r2 = 0.999), accurate and precise. The detection and quantitation limits were 0.275 and 1.0 µg.mL−1 for both drugs. Forced degradation revealed non-interference of degradation peaks with the analyte peaks and ensured method specificity. The LC estimation demonstrated higher solubility of NRB and NRN in Precirol ATO5 and Tween 80 as solid lipid and surfactant, respectively. In addition, the entrapment efficiencies were 78 % and 86 % for SLNs of NRB and NRN, respectively. After 24 h, the In-vitro release study indicated that 40 % of the drugs were released from the pure drug suspension and >95 % released from the SLN. Afterwards, the results from five different greenness assessment approach established the present method's green nature. Overall, a green, federally robust, and flexible liquid chromatographic method was developed and can be applied routinely to estimate NRB and NRN from the developed nano formulation.

Abstract 3962: Development of selection-free HER4-mutated CRISPR/Cas9 knock-ins in cutaneous melanoma cell lines

Abstract Background: Cutaneous melanoma showed the highest HER4 genomic alteration frequency (17.34%) across 33 different cancer types (cBioPortal). HER4 is a tyrosine kinase receptor belonging to the human epidermal growth factor receptor (HER/ErbB) family. The role of HER4 in cancer has not been fully studied, potentially due to its complex biology (four isoforms, different stability/tissue-specific expression patterns, and oncogenic/tumor suppressor roles). Using computational analysis, we identified two potentially oncogenic HER4 mutations in melanoma (HER4 R106C and R711C). We then developed CRISPR/Cas9-modified selection-free melanoma cell lines for interrogating the role of these mutations as biomarkers of response to the oral, irreversible pan-HER (EGFR, HER2, HER4) tyrosine kinase inhibitor (TKI) neratinib (NER). Methods: Two melanoma cell lines (WM115, SKMEL24) were genome-edited by using a selection-free CRISPR/Cas9 approach. Single guide RNAs (sgRNAs) were designed by using the CRISPRon (v1.0) web tool, and Homology Directed Repair (HDR) single-stranded OligoDeoxyriboNucleotides (ssODNs), containing the HER4 mutations, were designed with the SnapGene Software (v6.1.1). The RNP complex (Cas9 and sgRNAs) and the HDR_ssODNs, for each HER4 mutation, were delivered into the melanoma cells through electroporation. Upon validation of the CRISPR/Cas9-efficiency via PCR and Sanger Sequencing, the CRISPR/Cas9-modified cells were single-cell sorted via FACS, by using two singlets gating (FSC-A/FSC-H and SSC-A/SSC-H) and a live/dead (propidium iodide) gating, to perform single-cell cloning. Colonies were then amplified and screened via PCR and Sanger sequencing to isolate pure selection-free HER4 CRISPR/Cas9 knock-ins clones of melanoma cells. Results: The WM115 and SKMEL24 melanoma cell lines were found to be wild-type for the HER4 receptor in the Depmap Portal (mutation 22Q2, 2022 data release 2). R106C and R711C CRISPR/Cas9 knock-ins were created in the melanoma cell lines three days after the electroporation of the CRISPR/Cas9 complex. The efficiency of the HER4 R106C CRISPR/Cas9 knock-in was 22% and 7% in the SKMEL24 and WM115 melanoma cells, respectively, with a respective total indel percentage of 76% (SKMEL24) and 55% (WM115). The HER4 R711C CRISPR/Cas9 knock-in efficiency was 15% in the SKMEL24 (with 26% total indels) and 1% in the WM115 (with 33% total indels). Single-cell colonies of the HER4 CRISPR/Cas9-mediated knock-ins (HER4 R106C and R711C), were formed at four days (SKMEL24) and at one week (WM115) after single-cell sorting. Conclusions: CRISPR/Cas9 technology can be utilised to introduce single-point mutations in HER4 to melanoma cell lines. In vitro functional characterisation of the isolated HER4 CRISPR/Cas9-edited melanoma cell lines will be performed in order to understand the activating potential of HER4 R106C and R711C and their sensitivity to NER. Citation Format: Marta Valenti, Yonglun Luo, Neil T. Conlon, Lisa D. Eli, Alvin Wong, John Crown, Denis M. Collins. Development of selection-free HER4-mutated CRISPR/Cas9 knock-ins in cutaneous melanoma cell lines. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3962.

Abstract P6-12-09: Retinoic acid receptor (RAR) signalling plays a role in neratinib (NER) resistance in HER2+ breast cancer (BC) cell lines

Abstract Introduction: NER is a pan-HER tyrosine kinase inhibitor (TKI) approved for the treatment of HER2+ BC in the adjuvant setting following trastuzumab and in combination with capecitabine for advanced disease. Resistance to small molecule TKIs like NER can develop in the clinic. Pre-clinical studies have highlighted that retinoic acid can inhibit BC growth and modulate HER2 signalling pathways. The RAR family of nuclear transcription factors consists of RARα, RARβ and RARγ. The synthetic retinoic acid fenretinide (FN) acts as a pan-RAR agonist, while AGN194310 (AG) acts as a pan-RAR antagonist. In order to investigate the anti-proliferative potential of co-targeting RAR and HER2 pathways in sensitive and resistant BC cell line models, we examined the effect of FN and AG in combination with NER in two HER2+, estrogen receptor-negative, trastuzumab-resistant cell lines HCC1569 and HCC1954, and their NER-resistant (NR) sub-lines HCC1569-NR and HCC1954-NR. Methods: HCC1569 and HCC1954 cell lines were cultured in RPMI/10% FCS at 370C/5% CO2. NR cell lines were generated by continuous exposure to 150nM NER for 6 months. 10 mM stock solutions of FN (H7779-Sigma), AG (SML2665-Sigma) and NER (supplied by Puma Biotechnology, Inc) were made in DMSO. Proliferation was measured as percentage growth versus DMSO control using an acid phosphatase based assay after 5 days drug exposure. The half-maximal inhibitory concentration (IC50) was calculated for each drug using CalcuSyn. The combination assays were performed using fixed ratios. The combination index (CI) values were calculated at the effective dose that inhibits 50% growth (ED50) using CalcuSyn. Values < 1 represent a synergistic effect, a value of 1 is additive and values > 1 represent an antagonistic effect. All data presented as the mean of biological triplicate experiments ± standard deviation. Results: This research found that the NR cell lines were >10-fold resistant to NER (HCC1569-NR IC50 0.44 ± 0.1 μM, HCC1954-NR IC50 0.198 ± 0.019 μM) compared to the parental HCC1569 (IC50 0.018 ± 0.015 μM) and HCC1954 (IC50 0.017 ± 0.001 μM) cell lines. Pan-RAR agonism by FN had a potent anti-proliferative effect in the HCC1569 (FN IC50 0.22 ± 0.02 μM) and the HCC1569-NR cell lines (FN IC50 0.28 ± 0.13 μM), with the HCC1954 and HCC1954-NR cell lines proving less sensitive (IC50 6.47 ± 1.3 μM and 1.9 ± 0.2 μM, respectively). When combined with NER, FN produced a strong antagonistic effect in the HCC1569 cell line (CI value: 15.63 ± 9.5) and a strong synergistic effect in the HCC1954 cell line (CI value: 0.42 ± 0.06). For the NR cell line models, the NER/FN combination proved synergistic (HCC1569-NR, CI value: 0.84 ± 0.46) or additive (HCC1954-NR, CI value: 0.97 ± 0.15). Next, we wanted to assess the impact of antagonising rather than activating RAR activity in our cell line models. All four cell lines were less sensitive to antagonist AG (IC50 >8μM for all cell lines) compared to FN. The addition of AG to NER resulted in responses diametrically opposed to the FN/NER combination. The AG/NER combination produced a strong synergistic effect in the HCC1569 cell line (CI value: 0.52 ± 0.17), an antagonistic effect in the HCC1954 cell line (CI value: 2.1 ± 0.4) and an antagonistic effect in both NR cell lines (HCC1954-NR, CI value: 2.69 ± 0.6 and HCC1569-NR, CI value: 1.58 ± 0.12). Conclusions: This pre-clinical study suggests involvement of the RAR signalling pathway in response to NER and the development of NR. Results also suggest pan-RAR agonism, rather than pan-RAR antagonism, as a potential therapeutic strategy to overcome NR. Further investigation is warranted to determine how targeting the RAR signalling pathway may assist in the treatment of HER2+ BC. Citation Format: Debbie O’Reilly, Lisa D. Eli, Alvin Wong, John Crown, Denis M. Collins. Retinoic acid receptor (RAR) signalling plays a role in neratinib (NER) resistance in HER2+ breast cancer (BC) cell lines [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-12-09.

Dual targeting multiwalled carbon nanotubes for improved neratinib delivery in breast cancer.

The aim of this study was to develop biotinylated chitosan (Bio-Chi) decorated multi-walled carbon nanotubes (MWCNTs) for breast cancer therapy with the tyrosine kinase inhibitor, neratinib (NT). For achieving such a purpose, carboxylic acid functionalized multiwalled carbon nanotubes (c-MWCNTs) were initially decorated non-covalently with biotin-chitosan (Bio-Chi) coating for achieving a dual targeting mode; pH-dependent release with chitosan and biotin-receptor mediated active targeting with biotin. Afterwards, Bio-Chi decorated c-MWCNTs were loaded with the tyrosine kinase inhibitor, neratinib (NT). The formulation was then characterized by dynamic light scattering, FTIR and EDX. The drug loading efficiency was estimated to be 95.6 ± 1.2%. In vitro drug release studies revealed a pH-dependent release of NT from Bio-Chi decorated c-MWCNTs, with a higher drug release under acidic pH conditions. Sulforhodamine B (SRB) cytotoxicity assay of different NT formulations disclosed dose-dependent cytotoxicities against SkBr3 cell line, with a superior cytotoxicity observed with NT-loaded Bio-Chi-coated c-MWCNTs, compared to either free NT or NT-loaded naked c-MWCNTs. The IC50 values for free NT, NT-loaded c-MWCNTs and NT-loaded Bio-Chi-coated c-MWCNTs were 548.43 ± 23.1 μg mL-1, 319.55 ± 17.9 μg mL-1, and 257.75 ± 24.5 μg mL-1, respectively. Interestingly, competitive cellular uptake studies revealed that surface decoration of drug-loaded c-MWCNTs with Bio-Chi permitted an enhanced uptake of c-MWCNTs by breast cancer cells, presumably, via biotin receptors-mediated endocytosis. To sum up, Bio-Chi-decorated c-MWCNTs might be a promising delivery vehicle for mediating cell-specific drug delivery to breast cancer cells.

Development and Characterization of Oral Raft Forming In Situ Gelling System of Neratinib Anticancer Drug Using 32 Factorial Design.

Neratinib (NTB) is an irreversible inhibitor of pan-human epidermal growth factor receptor (HER-2) tyrosine kinase and is used in the treatment of breast cancer. It is a poorly aqueous soluble drug and exhibits extremely low oral bioavailability at higher pH, leading to a diminishing of the therapeutic effects in the GIT. The main objective of the research was to formulate an oral raft-forming in situ gelling system of NTB to improve gastric retention and drug release in a controlled manner and remain floating in the stomach for a prolonged time. In this study, NTB solubility was enhanced by polyethylene glycol (PEG)-based solid dispersions (SDs), and an in situ gelling system was developed and optimized by a two-factor at three-level (32) factorial design. It was analyzed to study the impact of two independent variables viz sodium alginate [A] and HPMC K4M [B] on the responses, such as floating lag time, percentage (%) water uptake at 2 h, and % drug release at 6 h and 12 h. Among various SDs prepared using PEG 6000, formulation 1:3 showed the highest drug solubility. FT-IR spectra revealed no interactions between the drug and the polymer. The percentage of drug content in NTB SDs ranged from 96.22 ± 1.67% to 97.70 ± 1.89%. The developed in situ gel formulations exhibited a pH value of approximately 7. An in vitro gelation study of the in situ gel formulation showed immediate gelation and was retained for a longer period. From the obtained results of 32 factorial designs, it was observed that all the selected factors had a significant effect on the chosen response, supporting the precision of design employed for optimization. Thus, the developed oral raft-forming in situ gelling system of NTB can be a promising and alternate approach to enhance retention in the stomach and to attain sustained release of drug by floating, thereby augmenting the therapeutic efficacy of NTB.

Abstract 3333: Pre-clinical investigation of neratinib plus dasatinib in breast cancer

Abstract Background: HER2-targeted therapies have revolutionized the prognosis of HER2-positive (HER2+) breast cancer (BC). However, the emergence of acquired resistance or de novo resistance are persistent clinical problems. The objective of this study was to examine neratinib (NER), an irreversible pan-HER inhibitor approved for early-stage and metastatic HER2+ BC, and dasatinib (DAS), a multi-kinase inhibitor used to treat certain leukemias, as a new treatment for HER2-targeted therapy resistant HER2+ BC and triple negative BC (TNBC). Methods: The anti-proliferative effect of NER plus DAS was screened in a panel of 20 breast cancer cell lines using a 5-day acid phosphatase assay. This panel included 12 HER2+ (3 trastuzumab (TRAS) sensitive (SKBR3, BT474, EFM192A), 2 acquired TRAS resistant (BT474-T, SKBR3-T), 3 innately TRAS resistant (HCC1954, HCC1569, JIMT1), and 4 acquired neratinib resistant cell lines (HCC1954-N, HCC1569-N, SKBR3-N, BT474-N), 6 triple negative (BT20, HCC1143, HCC1937, MDA-MB- 157/231/468), and 2 estrogen receptor positive (ER+) cell lines (MCF7, ZR-75-1). Drug synergy was examined using Combenefit and Calcusyn software. Combination Index (CI) values < 1 indicated synergy, > 1 antagonism. Neratinib IC50 values < 150nM were considered physiologically relevant. Prolonged exposure in vitro efficacy was tested in these cell lines by treating twice weekly with NER +/- DAS until resistance emerged. Cell confluence was then quantified using a crystal violet-based method. The safety and efficacy of NER (10 mg/kg) plus DAS (15 mg/kg) was investigated in vivo in HER2+ HCC1954 xenografts in BALB/c nude mice. Results: NER was highly effective against HER2+ BC cell lines regardless of TRAS sensitivity (IC50 values range 1-172 nM), showed moderate activity in TNBC (IC50 value range 31 nM - >1 μM), and did not achieve <1 μM activity in ER+ BC cell lines. NER resistant cell lines had IC50 values greater than 150 nM DAS alone displayed IC50 values from 8-135 nM in 4/6 TNBC cell lines, with all 6 TNBC cell lines sensitive to either the single agents or the combination at concentrations less than 100 nM. DAS enhanced the anti-proliferative activity of NER in the HCC1954, HCC1569, JIMT1 cell lines (CI values = 0.27-0.62). The NER/DAS combination overcame NER resistance in all acquired NER-resistant cell line models and no antagonism was observed in any cell line tested. Matrix assays confirmed synergy at low nanomolar concentrations of both drugs. NER plus DAS had a greater long term in vitro anti-proliferative effect over NER in HCC1954, HCC1569, and JIMT1 cells, preventing or delaying the development of NER resistance. NER plus DAS was well tolerated in mice for over 60 days. The combination was better than single agents and showed prolonged anti-tumor effect against HCC1954 xenografts in vivo (p=0.02). Conclusions: This study suggests that the addition of DAS to NER may have potential as a new treatment strategy for BC, particularly for HER2+ BC. Citation Format: Neil T. Conlon, Sandra Roche, Fiona O'Neill, Justine Meiller, John Crown, Denis M. Collins. Pre-clinical investigation of neratinib plus dasatinib in breast cancer [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 3333.

Abstract 3988: Identification of HER4 mutations with potential as biomarkers of response to neratinib

Abstract Background: HER4 is a member of the human epidermal growth factor receptor (HER/ErbB) family of receptor tyrosine kinases. HER4 is activated through ligand binding, transmitting intracellular signals by homodimerizing, or by heterodimerizing with other family members (EGFR, HER2, HER3). The role of HER4 in cancer or normal tissue has not been studied as comprehensively as EGFR and HER2, potentially due to a more complex biology that includes four different isoforms and different stability/tissue-specific expression patterns, and oncogenic or oncosuppressor roles. The oral, irreversible pan-HER (EGFR, HER2, HER4) tyrosine kinase inhibitor (TKI) neratinib (NER) is clinically active against HER2-mutant and HER2-amplified cancers. Using publicly available datasets including cBioPortal and COSMIC and predicted structural and functional impacts, this study aimed to identify HER4 mutations with the greatest oncogenic potential that may have value as potential biomarkers of response to NER. Methods: The frequency of HER4 mutations across 33 cancer types was determined by using the clinical database cBioPortal and TCGA PanCancer Atlas datasets (n=10967). A shortlist of potentially oncogenic HER4 mutations was created based on the occurrence in cBioPortal and COSMIC databases, annotation as putative driver and cancer hotspot, structural and functional impacts according to OncoKB, Mutation Assessor, SIFT and Polyphen-2 software, and HER4 protein folding studies. Analysis was carried out using the R-studio statistical environment. Results: HER4 amplification is rare in cancer, with the majority of genetic changes reported as point mutations; particularly missense (31.93%), synonymous (6.43%) and nonsense (3%) substitutions. Melanoma and esophagogastric (EG) cancers showed the highest HER4 alteration frequency, 17.34% and 13.42%, respectively, across different cancer types in the cBioPortal database. Melanoma and EG cancers showed the second and third highest absolute count of HER4 genomic alterations (n=77 and n=69, respectively). Based on the criteria outlined in methods, a priority list of HER4 mutations with greatest oncogenic potential was curated for melanoma (HER4 R106C, R306C, R711C, G936E, F1003I), stomach (HER4 C217R, S774N, K1002R, Y1150C) and esophageal (HER4 R488W, S449Y) adenocarcinomas. Mutations at these sites, located across ligand-binding, dimerization and intracellular signaling domains, have the potential to impact HER4 activity and thus possibly act as biomarkers of response to an irreversible pan-HER TKI like NER. Conclusions: These data suggest that melanoma and EG cancers are the cancer types with the highest rate of HER4 mutations. In vitro functional characterization of the identified HER4 mutations is now required to further explore their value as biomarkers of response to NER. Citation Format: Marta Valenti, Neil T. Conlon, Lisa D. Eli, Irmina Diala, John Crown, Denis M. Collins. Identification of HER4 mutations with potential as biomarkers of response to neratinib [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 3988.

Safety and efficacy of monoclonal antibodies and tyrosine kinase inhibitors in advanced breast carcinoma.

e13017 Background: Novel drugs that target HER2 receptors have shown promise in the treatment of Advanced breast carcinoma (BC). We study here the safety and efficacy of select monoclonal drugs (Mab) and tyrosine kinase inhibitors (TKIs) in the treatment of advanced BC. Methods: Following the PRISMA guidelines, we searched the literature on PubMed, Cochrane, Embase & clinicaltrials.gov. A total of 1054 articles were screened, and 6 studies were included. Results: In a phase III trial comparing Margetuximab (MAR) vs Trastuzumab (TRA), the overall survival (OS) and Progression free survival (PFS) was superior for MAR (21.6m Vs 19.8m & 5.7m Vs 4.4m respectively). However, the side effect profile is comparable for both drugs.Neutropenia (50 %) was a key problem in an ongoing trial including Zenocutuzumab (ZENO), resulting in dosage reduction. Similarly, phase III trials incorporating TKIs such as Neratinib (NER) and Tucatinib (TUC), there was improved OS (21m Vs 18.7, 24.7m Vs 19.2m respectively) as compared to Standard of care and chemotherapy. Regarding safety profile, these therapies are generally well tolerated with manageable side effects as listed in the table. Conclusions: Both monoclonal antibodies and tyrosine kinase inhibitors show clinically significant benefits in advanced HER2 positive breast carcinoma. However, more evidence and randomized clinical trials are needed to further establish their role in this rapidly evolving field.[Table: see text]

Effervescent floating matrix tablets of a novel anti-cancer drug neratinib for breast cancer treatment

The main purpose of this study was to develop a novel gastro-retentive drug delivery system via effervescent technique for the treatment of breast cancer. Neratinib (NTB) is a novel anticancer drug, poorly soluble at higher pH, was been used as a model drug and an attempt was made to develop a floating tablet using hydroxypropyl methylcellulose-90SH 15000 (HPMC-90SH 15000), microcrystalline cellulose, sodium bicarbonate, talc and lactose using direct compression method. The particulate materials were studied for the pre-compression parameters before compression. After compression, the floating matrix tablets of NTB were developed and studied in terms of post-compression parameters, floating lag time, buoyancy evaluation, water uptake/swelling studies, in vitro release, release kinetics and cytotoxicity studies. The parameters of pre- and post-compression evaluation were within the limits of United States Pharmacopoeia. Fourier transform infra-red evaluation indicated no interaction between the NTB and excipients used. The developed floating matrix tablets of NTB showed good swelling/floating properties for more than 24 h, and floating lag time was within 120 s. The NTB release from an optimized formulation G2 (100% release) offered a sustained release profile for up to 12 h. The release mechanism of NTB from formulation G2 was non-Fickian release mechanism. Cytotoxicity evaluation on MCF-7 human breast cancer cells suggested that NTB in an optimized formulation G2 (cytotoxicity = 98.60% at 800 nM) was more efficacious than free NTB (cytotoxicity = 91.29% at 800 nM). All these results and observations suggested that developed floating matrix tablets of NTB could be effectively applied for the treatment of breast cancer.

Abstract CT026: A phase II trial of neratinib (NER) or NER plus fulvestrant (FUL) (N+F) in HER2 mutant, non-amplified (HER2mut) metastatic breast cancer (MBC): Part II of MutHER

Abstract Introduction: The irreversible pan-HER inhibitor NER showed modest single agent activity for HER2mut MBC in Part I of MutHER trial. In Part II, we hypothesized that (1) N+F would improve activity in estrogen receptor positive (ER+) HER2mut MBC due to ER-HER2 crosstalk and (2) dual HER2 blockade by adding trastuzumab at disease progression (PD) could overcome resistance. Methods: Pts with ER+HER2mut MBC were enrolled to 2 cohorts (FUL treated or naive) to receive N+F with diarrhea prophylaxis. ER- pts received NER in an exploratory ER- cohort. Trastuzumab was added at PD if approved by insurance. Simon's Minimax 2-stage phase II design with the primary endpoint of clinical benefit rate (CBR: rates of complete/partial response [CR/PR] plus stable disease [SD] >24 weeks [wks]), with anticipated vs null hypothesis being CBR of 55% vs 35% (FUL treated) or 65% vs 40% (FUL naïve) with 80% power, 1 sided 0.05 alpha, was used. Secondary endpoints included progression free survival (PFS) and adverse events (AEs). Serial blood samples were analyzed for circulating tumor DNA (ctDNA) by Guardant360 for concomitant mutations, HER2mut variant allele frequency (VAF) dynamics, and resistance mechanisms. Results: Between Sep. 2015 and Oct. 2020, 40 pts with HER2mut MBC were enrolled, completing the 1st stage of each ER+ cohort. 35 pts (21 FUL treated, 10 FUL naïve, 4 ER-) were evaluable for response, with median age 63 (35-82) years, 3 (0-12) prior MBC regimen, lobular BC in 13 (37%) and visceral mets in 32 (91%) pts. 21 (68%) ER+ pts had prior CDK4/6 inhibitor. All but 1 pt has come off study due to PD. Table 1 shows the efficacy by cohort. Further enrollment is closed per protocol. Adding trastuzumab at PD induced CB in 4 (3 PR, 1 SD≥24 wks) of 5 pts (1 ER-, 4 ER+), with PFS 28 (95% CI 18~NA) wks. Common AEs across cohorts were diarrhea (G3 21%) and fatigue (G3 5%). No G4 AEs. ctDNA HER2mut was detected in 72% (23/32) baseline (BL) samples tested. In pts with paired samples, HER2mut VAF decreased at C1D15/C2D1 from BL in 75% (15/20) and rose in 89% (16/18) at PD. Acquired HER2mut, including the T798I gatekeeper mutation, were detected in 2 pts at PD. Mutations in TP53 (53%), PIK3CA (43%), and CDH1 (35%) were common, but none significantly associated with PFS in all or ER+ pts. Conclusions: NER, or N+F, is active for HER2mut MBC with good tolerability. Adding trastuzumab at PD induced further response, supporting dual HER2 blockade for HER2mut MBC. Table 1.EfficacyCohortFUL treatedFUL naïveER-Best Response, n evaluablen = 21n = 10n = 4CR, n100PR, n431SD (≥ 24 wks), n300SD (< 24 wks), n1030PD, n343CBR, n with CB/total n evaluable, % (95% CI)8 of 20*, 40% (19~64%)3 of 10, 30% (7~65%)1 of 4, 25% (0.6~81%)mPFS (95% CI), wks, ITT (n)24 (16~31) wks, (n = 24)20 (8~NA) wks, (n = 11)8.5 (8~NA) wks, (n = 5)*20 of 21 pts are evaluable for CBR in the FUL treated Cohort as 1 pt had SD as best response and treatment is still ongoing. ITT (intent to treat) population is used for mPFS estimate. Citation Format: Cynthia X. Ma, Jingqin Luo, Rachel A. Freedman, Timothy Pluard, Julie Nangia, Janice Lu, Frances Valdez-Albini, Melody Cobleigh, Jason Jones, Nancy U. Lin, Eric Winer, P. Kelly Marcom, Shana Thomas, Jill Anderson, Brittney Haas, Kimberly M. Hamann, Richard Bryce, Alshad S. Lalani, Lisa Carey, Matthew Goetz, Feng Gao, Gretchen Kimmick, Mark Pegram, Matthew J. Ellis, Ron Bose. A phase II trial of neratinib (NER) or NER plus fulvestrant (FUL) (N+F) in HER2 mutant, non-amplified (HER2mut) metastatic breast cancer (MBC): Part II of MutHER [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 CT026.

Imaging of Endocytic Trafficking and Extracellular Vesicles Released Under Neratinib Treatment in ERBB2+ Breast Cancer Cells.

Breast cancers (BCa) with ERBB2 amplification show rapid tumor growth, increased disease progression, and lower survival rate. Deregulated intracellular trafficking and extracellular vesicle (EVs) release are mechanisms that support cancer progression and resistance to treatments. Neratinib (NE) is a Food and Drug Administration-approved pan-ERBB inhibitor employed for the treatment of ERBB2+ BCa that blocks signaling and causes survival inhibition. However, the effects of NE on ERBB2 internalization, its trafficking to multivesicular bodies (MVBs), and the release of EVs that originate from these organelles remain poorly studied. By confocal and electron microscopy, we observed that low nanomolar doses of NE induced a modest ERBB2 internalization along with an increase of clathrin-mediated endocytosis and of the CD63+ MVB compartment in SKBR-3 cells. Furthermore, we showed in the culture supernatant two distinct EV subsets, based on their size and ERBB2 positivity: small (30-100 nm) ERBB2- EVs and large (>100 nm) ERBB2+ EVs. In particular, we found that NE increased the overall release of EVs, which displayed a reduced ERBB2 positivity compared with controls. Taken together, these results provide novel insight into the effects of NE on ERBB2+ BCa cells that may lead to a reduction of ERBB2 potentially transferred to distant target cells by EVs.

Blocking c-MET/ERBB1 Axis Prevents Brain Metastasis in ERBB2+ Breast Cancer.

Simple SummaryTargeted monotherapies are ineffective in the treatment of brain metastasis of ERBB2+ breast cancer (BC) underscoring the need for combination therapies. The lack of robust preclinical models has further hampered the assessment of treatment modalities. We report here a clinically relevant orthotopic mouse model of ERBB2+ BC that spontaneously metastasizes to brain and demonstrates that targeting the c-MET/ERBB1 axis with a combination of cabozantinib and neratinib decreases primary tumor growth and prevents brain metastasis in ERBB2+ BC.Brain metastasis (BrM) remains a significant cause of cancer-related mortality in epidermal growth factor receptor 2-positive (ERBB2+) breast cancer (BC) patients. We proposed here that a combination treatment of irreversible tyrosine kinase inhibitor neratinib (NER) and the c-MET inhibitor cabozantinib (CBZ) could prevent brain metastasis. To address this, we first tested the combination treatment of NER and CBZ in the brain-seeking ERBB2+ cell lines SKBrM3 and JIMT-1-BR3, and in ERBB2+ organoids that expressed the c-MET/ERBB1 axis. Next, we developed and characterized an orthotopic mouse model of spontaneous BrM and evaluated the therapeutic effect of CBZ and NER in vivo. The combination treatment of NER and CBZ significantly inhibited proliferation and migration in ERBB2+ cell lines and reduced the organoid growth in vitro. Mechanistically, the combination treatment of NER and CBZ substantially inhibited ERK activation downstream of the c-MET/ERBB1 axis. Orthotopically implanted SKBrM3+ cells formed primary tumor in the mammary fat pad and spontaneously metastasized to the brain and other distant organs. Combination treatment with NER and CBZ inhibited primary tumor growth and predominantly prevented BrM. In conclusion, the orthotopic model of spontaneous BrM is clinically relevant, and the combination therapy of NER and CBZ might be a useful approach to prevent BrM in BC.

Influence of antioxidant flavonoids quercetin and rutin on the in-vitro binding of neratinib to human serum albumin.

This study was designed to examine the interaction of neratinib (NRB) with human serum albumin (HSA) in presence of flavonoids quercetin and rutin. Both quercetin and rutin can compete with NRB to bind to HSA and displace NRB from its binding site. The interaction mechanism was studied with several spectroscopic techniques and molecular docking. Static fluorescence quenching mechanism was observed on interaction of HSA with NRB. van der Waals force and hydrogen bond were involved in the HSA-NRB interaction as per the results of thermodynamic parameters. Further, the conformational changes were observed in the HSA on its interaction with NRB. Interaction of NRB with HSA in presence of quercetin and rutin resulted in changes in the binding constants of HSA-NRB suggesting some impact on the binding of NRB in the presence of flavonoids.

Evaluation of competitive binding interaction of neratinib and tamoxifen to serum albumin in multidrug therapy.

Co-administration of two drugs to obtain a therapeutic goal is a common practice clinically and for effective use of drug therapy. However, the co-administration can sometimes cause adverse effects due to pharmacokinetic drug interactions. Breast Cancer treatment regimen include tyrosine kinase inhibitor neratinib (NRB) and/or tamoxifen (TMX). In this study neratinib and tamoxifen interaction with bovine serum albumin (BSA) and human serum albumin (HSA) individually and in combination using fluorescence spectroscopy was studied. The aim of this study was to find out whether there is a possibility of either of the two drugs interfering in the plasma protein binding of the other drug. Subdomain IIA of both the BSA and HSA was found to bind tamoxifen and neratinib. The λex = 280 nm and 295 nm were used for the analysis of neratinib-SA, tamoxifen-SA, neratinib: SA in presence of constant concentration of tamoxifen and similarly tamoxifen-SA in presence of constant concentration of neratinib. The interaction study of the binary and the ternary systems suggest that neratinib doesn't affect the interaction between SA and tamoxifen. In contrast, the interaction between neratinib and SA was affected by tamoxifen. The binding constant and quenching constant values suggest that tamoxifen dislodges neratinib from its serum albumin complex whereas neratinib doesn't affect the interaction between SA and tamoxifen. Thus, it was concluded from the results the study that during simultaneous administration of neratinib and tamoxifen, their competition for the SA binding sites should be taken into account.

Neratinib for HER2-Positive Breast Cancer: 5-Year Outcomes

The most recent agent to be approved for women with HER2-positive, early-stage breast cancer is the oral, irreversible, pan-HER tyrosine kinase

Comparative pharmacokinetic profiles of selected irreversible tyrosine kinase inhibitors, neratinib and pelitinib, with apigenin in rat plasma by UPLC–MS/MS

Neratinib (NER) and pelitinib (PEL) are irreversible tyrosine kinase inhibitors (TKIs) that have been recently employed in cancer treatment. Apigenin (API), among other flavonoids, is known to have antioxidant, anti-proliferative, and carcinogenic effect. API can potentiate the antitumor effect of chemotherapeutic agents and/or alleviate the side effects of many anticancer agents. Since TKIs are mostly metabolized by CYP3A4 enzymes and that API could alter the enzymatic activity, potential drug interactions could be expected following their co-aministration. In the present study, a bioanalytical UPLC–MS/MS method has been developed and validated for the quantification of NER and PEL in rat plasma, using domperidone (DOM) as an internal standard. Sample preparation was carried out using solid phase extraction (SPE) with C18 cartridges with good extraction recovery of not less than 92.42% (NER) and 89.73% (PEL). Chromatographic analysis was performed on a Waters BEH C18 column with a mobile phase composed of acetonitrile and water, (70:30, v/v), each with 0.1% formic acid. Quantitation was performed using multiple reaction monitoring (MRM) of the transitions from protonated precursor ions [M+H]+, at m/z 557.30 (NER), m/z 468.21 (PEL), and at m/z 426.27 (DOM), to selected product ions at m/z 112.05 (NER), m/z 395.22 (PEL), and at m/z 175.18 (DOM). The method was fully validated as per the FDA guidelines over the concentration range of 0.5–200ng/mL with very low lower limit of quantification (LLOQ) of 0.5ng/mL for both NER and PEL. The intra- and inter-day assay precision and accuracy were evaluated for both drugs and the calculated values of percentage relative standards deviations (%RSD) and relative errors (%Er) were within the acceptable limits (<15%) for concentrations other than LLOQ and 20% for LLOQ. The applicability of the method was extended to study the possibility of drug interactions following the oral co-administration of NER/PEL with API. Thus, this study could be readily applied in therapeutic drug monitoring (TDM) of cancerous patients receiving such drug combinations.

An eco-friendly direct spectrofluorimetric method for the determination of irreversible tyrosine kinase inhibitors, neratinib and pelitinib: application to stability studies.

A new rapid and simple stability-indicating spectrofluorimetric method has been developed for the determination of two irreversible tyrosine kinase inhibitors (TKIs), neratinib (NER) and pelitinib (PEL). The method is based upon measurement of the native fluorescence intensity of both drugs at λex 270nm in aqueous borate buffer solutions (pH 10.5). The fluorescence intensity recorded at 545nm (NER) and 465nm (PEL) were rectilinear over the concentration range of 0.1-10μg/mL for both drugs with a high correlation coefficient (r>0.999). The proposed method provided low limits of detection and of quantitation of 0.07, 0.11μg/mL (NER) and 0.02, 0.05μg/mL (PEL), respectively. The method was successfully applied for the determination of NER and PEL in bulk powder. The proposed methods were fully validated as per the International Conference on Harmonisation (ICH) guidelines. The application of the method was extended to stability studies of both NER and PEL under different forced-degradation conditions (acidic-induced, base-induced, oxidative, wet heat, and photolytic degradation). Moreover, the kinetics of the base-induced and oxidative degradation of both drugs was investigated and the pseudo-first-order rate constants and half-lives were estimated at different temperatures. Also, an Arrhenius plot was applied to predict the stability behaviour of the two drugs at room temperature. Copyright © 2016 John Wiley & Sons, Ltd.