Administration Of Afatinib Research Articles

Clinical and structural insights into the rare but oncogenic HER2-activating missense mutations in non-small cell lung cancer: a retrospective ATLAS cohort study

BackgroundUnlike human epidermal growth factor receptor 2 (HER2) amplification or exon 20 insertions, missense mutations in the extracellular domain (ECD), transmembrane domain (TMD), and intracellular domain (ICD) of the HER2 protein have been implicated as oncogenic in non-small cell lung cancer (NSCLC). However, their molecular subtypes, structural disparities, and clinical responses to current medical treatments, particularly HER2-targeted tyrosine kinase inhibitors (TKIs), remain unclear in NSCLC and warrant investigation.MethodsA real-world observational ATLAS study was conducted to gather and analyze therapeutic outcomes of chemotherapy or TKIs for heterogeneous HER2 missense mutations in NSCLC. Computational models of typical ECD, TMD, and ICD mutations were utilized to explore their structural variances.ResultsWe screened 37 eligible patients with HER2-activating missense mutations, of which 35 patients who had received chemotherapy or HER2-targeted TKIs as first-line therapy were available for response assessment. The median progression-free survival (PFS) for chemotherapy was 4.43 months (95% confidence interval [CI], 3.77–5.10), with an objective response rate (ORR) of 26.1% (6/23) and a disease control rate (DCR) of 17/23 (73.9%). The administration of afatinib, dacomitinib, and pyrotinib, HER2-targeted TKIs, achieved a median PFS of 4.65 months, with an ORR of 33.3% (4/12) and a DCR of 83.3% (10/12). Molecular modeling and computational simulations of ECD, TMD, and ICD mutations revealed their distinct structural characteristics.ConclusionIn comparison to chemotherapy, HER2-targeted TKIs demonstrated similar activity and PFS benefits for HER2-activating missense mutations in NSCLC.

Abstract 7223: A triplet therapeutic strategy targeting MEK, BCL-XL, and EGFR, for KRAS-mutant pancreatic ductal adenocarcinoma

Abstract KRAS mutations drive over 90% of pancreatic cancers, but KRAS has been proven difficult to target. Downstream of KRAS, MEK inhibition in combination with BCL-XL inhibition is synthetically lethal and an effective treatment for gynecologic, but not colorectal malignancies. Like colorectal cancer, pancreatic cancer often exhibits aberrant HER/EGFR activity. We hypothesized that a triplicate regimen targeting MEK, BCL-XL, and EGFR/HER would be effective for pancreatic cancer. Trametinib (MEK inhibitor), Afatinib (pan-EGFR/HER inhibitor), and Navitoclax (BCL-XL inhibitor); or DT2216 (a BCL-XL/BCL-1 proteolysis targeting chimera, PROTAC) were explored using various combinatorial strategies. These drug combinations were tested using four primary human pancreatic cancer cell lines using live cell imaging (Incucyte) and an in vivo murine model. Group comparisons were analyzed using one-way ANOVAs, and adjusted p-values <0.05 were considered significant. We observed that no single-agent treatments were effective. MEK inhibition plus anti-BCL-XL (Trametinib + Navitoclax or Trametinib + DT2216) demonstrated efficacy in one of the four cell lines. We noted synergistic effects on reduced cell growth and increased cell death when EGFR/HER inhibition was included by the administration of Afatinib. The triplet combination resulted in synergistic effects in three of four cell lines with Navitoclax and four of four cell lines with DT2216 (q<0.05). This effect was confirmed with DT2216 inhibition of BCL-XL in an in-vivo murine model. In conclusion, our study shows that a combinatory strategy targeting MEK, BCL-XL, and pan-EGFR/HER receptor kinase shows significant synergistic effects in pancreatic cancer. Clinical trials that assess the efficacy and safety of this strategy for patients with KRAS-mutant pancreatic cancer are warranted. Our data also suggests that novel KRAS inhibitors as monotherapy are likely to fail. Citation Format: Gerik W. Tushoski-Alemán, Alexandra J. Crespin, Chibeze J. Oguejiofor, Jordan A. McKean, Kelly M. Herremans, Thomas J. George, Carmen J. Allegra, Steven J. Hughes, Song Han. A triplet therapeutic strategy targeting MEK, BCL-XL, and EGFR, for KRAS-mutant pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7223.

Feasibility of Gastric Tumor Xenograft (GTX)-derived Cell Lines for Individualized Anti-cancer Drug Screening.

Because there are ongoing efforts to identify and develop novel drugs in the treatment of refractory gastric cancer, it is necessary to develop effective preclinical studies. Here, the preclinical efficacy of gastric tumor xenograft (GTX)-derived cell line models for the personalized treatment of gastric cancer was investigated. Anti-cancer drugs were scanned with high-throughput screening (HTS) using pre-established GTX-derived cell lines. The efficacy of a selected drug (afatinib) was re-confirmed in vivo and intracellular signaling pathways were investigated in xenograft tumor cell lysates using western blotting. Validation studies, such as cell proliferation and caspase activity assays, were also conducted in vitro with GTX-derived cell lines. HTS indicated that afatinib was effective in one of the five GTX-derived cell lines (GTX-087). A xenograft mouse model was established from GTX-087, and administration of afatinib at 1 mg/20 g body weight/day per oral resulted in tumor-suppressive activity in vivo. The RAS-ERK pathway was inactivated by an increase in Bax and cleaved caspase-3 in this xenograft model. In vitro cell proliferation assay also revealed that afatinib was able to suppress the growth of the GTX-087 cell line. Caspase activity assay confirmed that afatinib had an apoptotic role on GTX-087 and showed that caspase-3/7 activity increased in a time dependent manner. The GTX-derived cell line model might be useful for estimating novel drug responses and could be an alternative to patient-derived xenograft animal models.

P30-2 Examination of improvement mechanism by minocycline application for skin disorders caused by afatinib

We have clarified that minocycline petrolatum ointment prevents and repairs skin disorders caused by afatinib in mice, but its mechanism of action is unknown. In order to make this minocycline ointment usable in clinical practice, we proposed to dilute the already commercially available dental Perioclin ointment (containing minocycline) with petrolatum and use it, and its skin disorder improving effect and stability as a preparation. (1) After continuous oral administration of afatinib (20 mg / kg BW) to ddy male mice, the expression levels of EGFR and TNFa in the skin of mice coated with minocycline (0.03 mg / 0.1 g) were measured by the ELISA method. (2) The effect of minocycline ointment obtained by diluting Perioclin ointment with petrolatum was compared with that of minocycline vaseline ointment, and the minocycline content was measured using HPLC. (1) Both epidermal EGFR and TNFa levels were significantly decreased in the afatinib-administered group and recovered to normal levels by application of minocycline. (2) The minocycline ointment prepared by diluting the Perioclin ointment was as stable as or more stable than the conventional minocycline vaseline ointment, and improved the dryness of the skin by afatinib. It was suggested that minocycline may repair skin tissue by recovering epidermal EGFR level and TNFa level in response to afatinib-induced skin damage. Sano et al. BMC Cancer (2020) 20:279.

Drastic antitumor response following administration of afatinib immediately after atezolizumab in a patient with epidermal growth factor receptor tyrosine kinase inhibitor-resistant lung cancer.

Drastic antitumor response following administration of afatinib immediately after atezolizumab in a patient with epidermal growth factor receptor tyrosine kinase inhibitor-resistant lung cancer.

A prospective phase II study of multimodal prophylactic treatment for afatinib-induced adverse events in advanced non-small cell lung cancer (Niigata Lung Cancer Treatment Group 1401).

BackgroundAfatinib has shown clinical benefits in patients with non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) mutations. Many patients treated with afatinib experience skin or gastrointestinal toxicity. However, an effective management strategy has not been established. This prospective study was conducted to evaluate the efficacy of multimodal prophylactic treatment for afatinib-induced toxicity.MethodsThis single-arm prospective study was conducted to evaluate the efficacy of multimodal prophylactic treatment for afatinib-induced toxicity in patients with EGFR mutation positive advanced NSCLC who planned to receive a 40 mg dose of afatinib. Eligible patients were treated with oral loperamide (2 mg twice per day), prophylactic minocycline (100 mg once per day), topical medium-class steroids, and gargling with sodium azulene. The primary endpoint was the ability of prophylactic loperamide to prevent severe or intolerable diarrhea during the 4 weeks after the initial administration of afatinib. The incidence, severity and time to occurrence of diarrhea, rash, oral mucositis and paronychia were evaluated based on a daily patient questionnaire.ResultsForty-six patients were enrolled. The primary endpoint analysis was performed in 35 patients as the per-protocol (PP) population. The 4-week successful prophylaxis rate for severe or intolerable diarrhea was 82.9% (90% confidence interval: 70.1–91.9%). In the total population, the incidences of grade 3 or higher rash, oral mucositis and paronychia within 4 weeks were 4%, 2% and 4%, respectively.ConclusionsProphylactic loperamide administration was not effective in preventing severe or intolerable diarrhea during afatinib treatment. Adequate dose reduction will be a better approach to manage afatinib-induced diarrhea. Multimodal prevention using minocycline, topical steroids and gargling with sodium azulene may be helpful to maintain compliance with afatinib treatment (UMIN000016167).

Immunohistochemical localization of afatinib in male rat intestines and skin after its oral administration

Afatinib, a second-generation tyrosine kinase inhibitor, was designed to bind covalently to and irreversibly inhibit active ErbB family receptors. The major metabolites of afatinib in human plasma are adducts of afatinib covalently bound to plasma proteins via. the Michael addition reaction. These findings suggest that afatinib may form covalent bonds with proteins in tissue and be localized in tissue. However, there is no method for the specific detection of afatinib-protein conjugates localized in tissue. In this paper, we aimed to develop an immunohistochemical protocol to detect afatinib-protein conjugates. Immunostainings were performed with male rat intestinal tract and skin at 24 h after an oral administration of afatinib. In the intestinal tract, strong staining was observed in the ileum and colon, but only slight staining was observed in the duodenum and jejunum. In the skin, strong staining was observed in the epidermis, sebaceous glands and hair follicles. Immunohistochemistry for afatinib-protein conjugates could be a useful tool to detect the localization of such conjugates. This study is the first to elucidate the localization of afatinib-protein conjugates in the rat intestinal tract and skin and is expected to be of great use in efforts to clarify the mechanism underlying afatinib-induced diarrhoea or skin toxicities.

The clinical features of squamous cell lung carcinoma with sensitive EGFR mutations.

The process of selecting patients on the basis of epidermal growth factor receptor (EGFR) mutations would likely result in a patient population with greater sensitivity to EGFR tyrosine kinase inhibitors (EGFR-TKIs). However, EGFR mutation status is not routinely examined in patients with squamous cell lung cancer (Sq) because of the low incidence of EGFR mutations and the poor clinical response to EGFR-TKIs. We retrospectively reviewed the clinical features of patients at our hospital with Sq who carried EGFR-TKI-sensitive EGFR mutations and assessed their responses to EGFR-TKIs. EGFR mutation status was tested in 23 of 441 patients with Sq (5.2%) admitted to our hospital during the study period. An EGFR mutation (exon 19 deletion 3, L858R 2) was identified in five of the 23 patients (21.7%), all of whom were female never-smokers. Of these five patients, four (4/9; 44.4%) were in the normal lung group, one (1/12; 8.3%) was in the emphysematous lung group, and none (0/2; 0%) in the fibrotic lung group. Two of these five patients with the EGFR mutation received gefitinib and two received afatinib. Although the two patients who were treated with gefitinib did not respond well to treatment (stable disease, 1 patient; progressive disease, 1 patient), the two patients who were treated with afatinib showed a good response (partial response, 2 patients). The administration of afatinib to Sq patients after selecting patients using the EGFR mutation test based on their underlying pulmonary disease and smoking status would likely result in a population with a greater sensitivity to afatinib.

Breast cancer resistance protein (BCRP/ABCG2) and P-glycoprotein (P-gp/ABCB1) transport afatinib and restrict its oral availability and brain accumulation

Afatinib is a highly selective, irreversible inhibitor of EGFR and HER-2. It is orally administered for the treatment of patients with EGFR mutation-positive types of metastatic NSCLC. We investigated whether afatinib is a substrate for the multidrug efflux transporters ABCB1 and ABCG2 and whether these transporters influence oral availability and brain and other tissue accumulation of afatinib.We used in vitro transport assays to assess human (h)ABCB1-, hABCG2- or murine (m)Abcg2-mediated transport of afatinib. To study the single and combined roles of Abcg2 and Abcb1a/1b in oral afatinib disposition, we used appropriate knockout mouse strains.Afatinib was transported well by hABCB1, hABCG2 and mAbcg2 in vitro. Upon oral administration of afatinib, Abcg2−/−, Abcb1a/1b−/− and Abcb1a/1b−/−;Abcg2−/− mice displayed a 4.2-, 2.4- and 7-fold increased afatinib plasma AUC0-24 compared with wild-type mice. Abcg2-deficient strains also displayed decreased afatinib plasma clearance. At 2h, relative brain accumulation of afatinib was not significantly altered in the single knockout strains, but 23.8-fold increased in Abcb1a/1b−/−;Abcg2−/− mice compared to wild-type mice.Abcg2 and Abcb1a/1b restrict oral availability and brain accumulation of afatinib. Inhibition of these transporters may therefore be of clinical importance for patients with brain (micro)metastases positioned behind an intact blood-brain barrier.

Inhibitory Effect of Afatinib on Platelet Activation and Apoptosis

Background/Aims: The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor afatinib is used for the treatment of several malignancies. Afatinib is at least partially effective by triggering apoptosis of tumor cells. Platelets may similarly undergo apoptosis, which is characterized by caspase 3 activation, cell shrinkage and phosphatidylserine translocation. However, an effect of afatinib on platelets has never been reported. The present study explored whether treatment of platelets with afatinib modifies platelet activation and apoptosis in the absence and presence of platelet activators thrombin or collagen related peptide (CRP). Methods: Platelets isolated from wild-type mice were exposed for 30 minutes to afatinib (18 µg/ml) without or with subsequent treatment with thrombin (0.005 U/ml or 0.01 U/ml) or CRP (2 µg/ml or 5 µg/ml). Flow cytometry was employed to estimate Orai1 abundance at the platelet surface with specific antibodies, cytosolic Ca²⁺-activity ([Ca²⁺]_i) from Fluo-3 fluorescence, platelet degranulation from P-selectin abundance, integrin activation from α_IIbβ₃ integrin abundance, caspase activity utilizing an Active Caspase-3 Staining kit, phosphatidylserine abundance from annexin-V-binding, platelet volume from forward scatter and aggregation utilizing staining with CD9-APC and CD9-PE. Results: In the absence of thrombin and CRP, the administration of afatinib (18 µg/ml) slightly, but significantly, increased [Ca²⁺]_i and annexin-V-binding, but did not significantly modify Orai1 abundance, P-selectin abundance, activated α_IIbβ₃ integrin, cell volume, caspase activity and aggregation. Exposure of platelets to 0.005 U/ml or 0.01 U/ml thrombin or 2 µg/ml or 5 µg/ ml CRP was followed by a significant increase of Orai1 abundance, increase of [Ca²⁺]_i, P-selectin abundance, α_IIbβ₃ integrin activity, annexin-V-binding, caspase activity, and aggregation, as well as a significant decrease of forward scatter, all effects significantly blunted (thrombin) or virtually abolished (CRP) by afatinib. Conclusions: Afatinib is a powerful inhibitor of platelet activation, platelet apoptosis and platelet aggregation.

COMPARATIVE PHARMACOECONOMIC EVALUATION OF THE USE OF AFATINIB AND GEFITINIB IN THERAPY FOR LUNG CANCER

Background: Preliminary results of the study of clinical effectiveness and safety of afatinib in comparison with gefitinib as the first line therapy for the patients with the metastatic non-small-cell lung cancer (NSCLC) and activating EGFR mutation have been reported in December of 2015. According to this study afatinib has significantly increased the rate of progression-free survival (PFS) (RR = 0.73; 95% CI, 0.57, 0.95; р = 0.0165), as well as the time before treatment discontinuation (TTD) (RR = 0.73; 95% CI, 0.58, 0.92; р = 0.0073) in comparison with gefitinib and the objective response rate (ORR) (70% vs 56%, р = 0.0083). The aim of the present study: Economic evaluation of afatinib in comparison with gefitinib as the first line therapy during the long-term treatment of locally advanced or metastatic NSCLC in the patients with the activating EGFR mutation. Methods: The cost-effectiveness analysis has been performed using Markov model. The model has been constructed on the basis of the results of clinical study LUX-Lung 7, taking into account the results of the study LUX-Lung 3. The direct medical costs have been taken into account: the cost of drug treatment of metastatic NSCLC during the first and second line therapy, the expenses for palliative care, correction of adverse events, as well as for the follow-up of patients in the course of the treatment. The cost/effectiveness ratio (CER) (Rubles/ Quality adjusted life year, or QALY) has been calculated for afatinib and gefitinib. In the baseline scenario (all patients with frequent activating mutations) the cost-effectiveness of afatinib for all patients with activating EGFR mutation has been evaluated; in addition the costs and outcomes in patients with Del19 and L858R mutation have been analyzed separately. The probabilistic sensitivity analysis of the analysis results to the variability of initial parameters has been performed. Results: In the baseline scenario the cost of first year treatment with afatinib as the first line therapy has been by 13.56% cheaper than the cost of treatment with gefitinib and amounted to 1.058 million Rubles for afatinib and 1.224 million Rubles for gefitinib. The general expenses for the first year of treatment have amounted to 946 726 Rubles for afatinib, and 1 024 096 Rubles for gefitinib. In the baseline scenario the modeled progression-free survival for afatinib has amounted to 13.15 months and for gefitinib to 10.65 months. The total direct medical costs for first 3 years of treatment for afatinib have amounted to 1.577 million Rubles, for gefitinib 1.585 million Rubles, the number of QALY gained - 1.341 and 1.280 correspondingly. CER for afatinib has amounted to 1.176 million, for gefitinib - 1.239 million Rubles/QALY In the additional scenarios the same results have been demonstrated: CER for afatinib was less than for gefitinib. Conclusions: The administration of afatinib leads to the better clinical results, expressed by the increase of the rate of progression-free survival and longer-term therapy on retention of life quality of the patients in comparison with the administration of gefitinib accompanying by the cost advantage. Subsequently the cost - effectiveness ratio for afatinib is better than for gefitinib. Afatinib is the dominant alternative for the treatment of NSCLC during first line therapy in comparison with gefitinib. The prescription of afatinib offers the possibility to spare funds in the health system and is appropriate from pharmacoeconomic standpoint.

Efficacy of afatinib, an irreversible ErbB family blocker, in the treatment of intracerebral metastases of non-small cell lung cancer in mice.

Few effective therapeutic options are currently available for the treatment of non-small cell lung cancer (NSCLC) with brain metastases (BM). Recent evidence shows that NSCLC patients with BMs respond well to afatinib, but little is known about the underlying mechanisms. In this study, we evaluated the efficacy of afatinib in treatment of BMs in mice and investigated whether afatinib could actively penetrate the brain-blood barrier and bind to its target. NSCLC BM model was established in nude mice by intracerebral injection of PC-9.luc cells. The tumors were measured weekly using in vivo quantitative bioluminescence. The mice are administrated afatinib (15, 30 mg·kg-1·d-1, ig) for 14 d. The antitumor efficacy of afatinib was determined by tumor growth inhibition (TGI), which was calculated as [1-(change of tumor volume in treatment group/control group)×100]. Pharmacokinetic characteristics were measure in mice receiving a single dose of afatinib (30 mg/kg, ig). Pharmacodynamics of afatinib was also assessed by detecting the expression of pEGFR (Tyr1068) in brain tumor foci using immunohistochemistry. Administration of afatinib (15, 30 mg·kg-1·d-1) dose-dependently inhibited PC-9 tumor growth in the brain with a TGI of 90.2% and 105%, respectively, on d 14. After administration of afatinib (30 mg/kg), the plasma concentration of afatinib was 91.4±31.2 nmol/L at 0.5 h, reached a peak (417.1±119.9 nmol/L) at 1 h, and was still detected after 24 h. The cerebrospinal fluid (CSF) concentrations followed a similar pattern. The T1/2 values of afatinib in plasma and CSF were 5.0 and 3.7 h, respectively. The AUC(0-24 h) values for plasma and CSF were 2375.5 and 29.1 nmol/h, respectively. The plasma and CSF concentrations were correlated (r=0.844, P<0.01). Pharmacodynamics study showed that the expression levels of pEGFR were reduced by 90% 1 h after afatinib administration. The Emax was 86.5%, and the EC50 was 0.26 nmol/L. A positive correlation between CSF concentrations and pEGFR modulation was revealed. Afatinib penetrates the BBB in NSCLC BM mice and contributes to the brain tumor response. The CSF exposure level is correlated with the plasma level, which in turn is correlated with the modulation of pEGFR in the tumor tissues. The results support for the potential application of afatinib in NSCLC patients with BMs.

In vitro and in vivo efficacy of afatinib as a single agent or in combination with gemcitabine for the treatment of nasopharyngeal carcinoma.

PurposeEpidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC). We tested the antitumor effects of irreversible ErbB family inhibitor afatinib on human NPC using in vitro and in vivo models.Materials and methodsThe effect of afatinib on NPC cells was evaluated using the Cell Counting Kit 8 (CCK8) assay, flow cytometry, and Western blot analyses. The effect of afatinib, as either a single agent or in combination with gemcitabine (GEM), on tumor growth was determined using NPC tumor xenografts in mice.ResultsAfatinib inhibited cell growth in all three NPC cell lines tested in a dose-dependent manner. Afatinib promoted cell cycle arrest at the S and G2/M phases, and it significantly inhibited epidermal growth factor (EGF)-induced activation of EGFR and its downstream signaling factors. Co-treatment with afatinib and GEM more effectively inhibited tumor growth than either drug alone but was associated with increased toxicity.ConclusionAfatinib induced cell cycle arrest and inhibited the proliferation of NPC cell lines. Afatinib in combination with GEM demonstrated significant antitumor effect in an NPC xenograft model. The administration of afatinib with GEM in NPC needs to be modified in order to be effective and tolerable.

Liquid chromatography-tandem mass spectrometric assay for the tyrosine kinase inhibitor afatinib in mouse plasma using salting-out liquid-liquid extraction.

A quantitative bioanalytical liquid chromatography-tandem mass spectrometric (LC-MS/MS) assay for afatinib, an irreversible inhibitor of the ErbB (erythroblastic leukemia viral oncogene homolog) tyrosine kinase family, was developed and validated. Plasma samples were pre-treated using salting-out assisted liquid-liquid extraction (SALLE) with acetonitrile, magnesium chloride and a stable isotopically labeled internal standard. After dilution, the extract was directly injected into the reversed-phase liquid chromatographic system. The eluate was transferred into the electrospray interface with positive ionization and compounds were detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. The assay was completely validated for plasma in a 0.5-500ng/ml calibration range with r(2)=0.995±0.002 (n=6) using linear regression with the inverse square of the concentration as the weighting factor for the calibration. Within-run precisions (n=18) were 2.7-11.7% and between-run (3 runs; n=18) precisions 3.0-14.5%. Accuracies were between 96-109% for the whole calibration range. The drug was sufficiently stable under all relevant analytical conditions. Finally, the assay was successfully applied to determine plasma drug levels and study pharmacokinetics after oral administration of afatinib to female FVB mice.

Simultaneous and rapid determination of gefitinib, erlotinib and afatinib plasma levels using liquid chromatography/tandem mass spectrometry in patients with non-small-cell lung cancer.

A simultaneous, selective, sensitive and rapid liquid chromatography/tandem mass spectrometry method was developed and validated for the quantification of gefitinib, erlotinib and afatinib in 250 μL samples of human blood plasma. Diluted plasma samples were extracted using a liquid-phase extraction procedure with tert-butyl methyl ether. The three drugs were separated by high-performance liquid chromatography using a C18 column and an isocratic mobile phase running at a flow rate of 0.2 mL/min for 5 min. The drugs were detected using a tandem mass spectrometer with electrospray ionization using imatinib as an internal standard. Calibration curves were generated over the linear concentration range of 0.05-100 nm in plasma with a lower limit of quantification of 0.01 or 0.05 nm for all compounds. Finally, the validated method was applied to a clinical pharmacokinetic study in patients with nonsmall-cell lung cancer (NSCLC) following the oral administration of afatinib. These results indicate that this method is suitable for assessing the risks and benefits of chemotherapy in patients with NSCLC and is useful for therapeutic drug monitoring for NSCLC treatment. As far as we know, this is the first report on LC-MS/MS method for the simultaneous quantification of NSCLC tyrosine kinase inhibitor plasma concentrations including afatinib. Copyright © 2015 John Wiley & Sons, Ltd.

Abstract 2591: Activity of MSC2156119J in non-small cell lung cancer models with activating EGFR mutation

Abstract The therapeutic success of EGFR tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC) is hampered in a subset of patients by the development of drug resistance through the EGFR T790M ‘gate keeper’ mutation. Recent investigations suggest that aberrant activation of the c-Met receptor through c-Met amplification and/or HGF upregulation may be an additional mechanism for acquired resistance. Combination of an EGFR TKI with a c-Met inhibitor could, therefore, delay and/or prevent the emergence of c-Met-driven resistance. The aim of our study was to evaluate the efficacy of MSC2156119J, a highly selective small molecule c-Met inhibitor, as monotherapy or in combination with EGFR TKIs (erlotinib, gefitinib and afatinib) in human xenograft models transplanted with two different primary NSCLC explants, LU342 and LU0858. While both tumors express the activated EGFR mutant form, LU342 expresses low levels of c-Met, while LU0858 expresses high levels of c-Met and carries an amplified c-Met locus. Single-agent MSC2156119J was inactive against the low c-Met-expressing LU342 explant and tumors progressed on treatment (median tumor volume [TV] change &amp;gt;73%). Erlotinib treatment resulted in tumor regression, with median TV change of -93% and tumor growth delay (TGD) of 56 days. The combination of MSC2156119J and erlotinib was more active, delaying tumor regrowth by 73 days compared to erlotinib monotherapy. Administration of afatinib at 5mg/kg was highly active in this model, inducing complete tumor regression (median TV change -100%), similar to the combination of afatinib and MSC2156119J. In contrast, the LU0858 explant, which carries an amplified c-Met locus, was sensitive to MSC2156119J at a dose of 100mg/kg 5 days on and 2 days off (5 on/2 off), inducing tumor stasis with a median TV change of 19% and TGD of 34 days. While gefitinib and afatinib alone at doses of 150mg/kg 5 on/2 off and 5mg/kg 5 on/2 off, respectively, were inactive (median TV changes &amp;gt;73%), in combination with MSC2156119J tumor growth inhibition was enhanced, resulting in tumor regression (median TV changes of -64% and -85%, respectively). In comparison, tumors treated with platinum-based doublet chemotherapy (cisplatin 5mg/kg QW/pemetrexed 200mg/kg QW) progressed, with TGD of 16 days. When MSC2156119J was combined with cisplatin/pemetrexed, antitumor activity was not significantly enhanced compared to single-agent MSC2156119J (median TV change -32%, TGD 30 days). Together, these findings are compatible with the hypothesis that inhibition of the c-Met pathway prevents and/or delays the emergence of c-Met-driven resistance in EGFR-mutant NSCLC. Furthermore, the enhancement of antitumor activity in the low c-Met-expressing LU342 model when MSC2156119J was combined with erlotinib suggests that the combination of an EGFR TKI + MSC2156119J may also be active in tumors with low levels of c-Met expression. Citation Format: Friedhelm Bladt, Manja Friese-Hamim, Andree Blaukat. Activity of MSC2156119J in non-small cell lung cancer models with activating EGFR mutation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2591. doi:10.1158/1538-7445.AM2015-2591

The role of extrahepatic metabolism in the pharmacokinetics of the targeted covalent inhibitors afatinib, ibrutinib, and neratinib.

Despite the fact that much progress has been made recently in the development of targeted covalent inhibitors (TCIs), their pharmacokinetics (PK) have not been well characterized in the light of extrahepatic clearance (CLextH) by glutathione (GSH)/glutathione S-transferase (GST)-dependent conjugation attributable to the unique electrophilic structure (e.g., acrylamide moiety) of TCI compounds. In the present study, CLextH values were examined in rat, dog, and monkey to predict the contribution of CLextH to the PK of the TCIs afatinib, ibrutinib, and neratinib in humans. Afatinib and neratinib both underwent extensive conjugation with GSH in buffer and cytosol fractions of liver and kidney, whereas ibrutinib showed much lower reactivity/susceptibility to GSH/GST-dependent conjugation. The CLextH in each species was calculated from the difference between observed total body clearance and predicted hepatic clearance (CLH) in cryopreserved hepatocytes suspended in 100% serum of the corresponding species. The power-based simple allometry relating the CLextH for the unbound compound to animal body weight was applicable across species for afatinib and neratinib (R(2) ≥ 0.9) but not for ibrutinib (R(2) = 0.04). The predicted AUC after oral administration of afatinib and neratinib agreed reasonably closely with reported values in phase I dose-escalation studies. Comparisons of CLextH and CLH predicted that CLextH largely determined the PK of afatinib (>90% as a proportion of total body clearance) and neratinib (∼34%) in humans. The present method can serve as one of the tools for the optimization of PK in humans at the discovery stage for the development of TCI candidates.

Adverse events of afatinib as first-line treatment for five cases of advanced lung adenocarcinoma and review of literature

背景与目的阿法替尼是一种新型的低分子量人类表皮生长因子受体（human epidermal growth factor receptor, HER）家族抑制剂，它属于第二代表皮生长因子受体-酪氨酸激酶抑制剂（epidermal growth factor receptor-tyrosine kinase inhibitors, EGFR-TKIs），在临床前和临床研究中显示了该药对具有表皮生长因子受体（epidermal growth factor receptor, EGFR）活性突变的非小细胞肺癌（non-small cell lung cancer, NSCLC）的疗效。本研究关注阿法替尼治疗晚期肺腺癌患者的安全性。方法确诊为Ⅲb期或Ⅳ期肺腺癌、具有EGFR突变的患者，一线给予阿法替尼每日40 mg口服，直至疾病进展。观察不良反应、疗效及生存情况。结果最常见不良反应为腹泻（n=5, 100%）、皮疹（n=4, 80%）、粘膜炎/口腔炎（n=4, 80%）。总体不良反应程度较轻，均≤Ⅲ级，相对最严重的副反应为粘膜炎/口腔炎。腹泻虽发生于所有患者，但程度较轻。共有3例患者因不良反应暂停药、减量。在4例可评价疗效的患者中，部分缓解（partial response, PR）2例（50%），疾病稳定（stable disease, SD）1例（25%），疾病进展（progressive disease, PD）1例（25%）。中位无进展生存期（progression-free survival, PFS）9.7个月，中位总生存期（overall survival, OS）18.4个月。结论阿法替尼一线治疗晚期肺腺癌患者疗效确切，常见不良反应除腹泻、皮疹外，还应关注粘膜炎/口腔炎的发生。由于本研究入组人数较少，此结论尚需得到研究者的进一步关注。

Effect of nintedanib, a triple angiokinase inhibitor, on EGFR and HER2 in colorectal cancer (CRC) models, and rational for its combinations with the ErbB family blocker afatinib.

485 Background: We have recently shown that combinations of afatinib, a pan-HER/ErbB blocker, and nintedanib, a triple angiokinase (VEGFR, FGFR, PDGFR) inhibitor show synergistic activity in CRC models (Poindessous et al., Clin Cancer Res. 17:6522, 2011). However, the mechanistic basis for the synergistic effects of the combination is incompletely understood. EGFR is activated following exposure to a wide variety of therapeutic modalities including ionizing irradiation and irinotecan. We speculated that nintedanib exposure could also activate EGFR signaling which might explain the synergistic activity of the combination. Methods: Mice with human CRC xenografts were treated with nintedanib and afatinib alone or in combination and the influence on tumor growth, viability and the presence of phosphorylated HER family members was determined. Different scheduling regimens were explored to identify an administration schedule which combined optimal antitumor activity with minimal toxic side effects. Results: We here show that nintedanib treatment results in activation of EGFR and HER2 in multiple CRC xenograft models in a dose-dependent manner. Among the different regimens tested, continuous nintedanib with administration of afatinib every second week proved almost as efficient as continuous administration of the two agents together and was less toxic. Finally, nintedanib plus afatinib was superior to nintedanib alone in three different tumor xenografts with mutant KRAS. Conclusions: We here report that prolonged exposure to nintedanib, a small molecule angiogenesis inhibitor, is accompanied by activation of EGFR and HER2. Accordingly, afatinib, an ErbB family blocker, was synergistic with nintedanib. We subsequently identified a novel regimen for optimizing the antitumor effects of the combination with limited toxic side effects and showed that this regimen is active in four different CRC tumor models including three with mutant KRAS. These findings provide a rationale for clinical trials of the two small molecules, even in patients with mutant KRAS.

Pharmacokinetic Drug Interactions of Afatinib with Rifampicin and Ritonavir

Afatinib is a potent, irreversible, ErbB family blocker in clinical development for the treatment of advanced non-small cell lung cancer, metastatic head and neck cancer, and other solid tumours. As afatinib is a substrate for the P-glycoprotein (P-gp) pump transporter the three studies presented here investigated the pharmacokinetics of afatinib in the presence of a potent inhibitor (ritonavir) or inducer [rifampicin (rifampin)] of P-gp. We conducted phase I, open-label, single-centre studies in healthy male volunteers who received a single once-daily oral dose of afatinib (20 or 40 mg) together with either ritonavir or rifampicin; two studies had a randomised, two- and three-way crossover design and the third was a non-randomised, two-period sequential study. When afatinib 20 mg was administered 1 h after ritonavir, afatinib geometric mean (gMean) maximum plasma concentration (C max) and area under the plasma concentration-time curve from time zero to infinity (AUC∞) increased by 38.5 and 47.6 %, respectively. Coadministration of ritonavir either simultaneously or 6 h later than afatinib 40 mg resulted in minimal increase in the afatinib gMean C max and AUC∞ (4.1 and 18.6 % for simultaneous administration with AUC∞ not completely within the bioequivalence limits; 5.1 and 10.8 % for timed administration within the bioequivalence limits). Administration of afatinib 40 mg in the presence of rifampicin led to reduction in C max and AUC∞ by 21.6 and 33.8 %, respectively. In all studies, P-gp modulation mainly affected the extent of absorption of afatinib; there was no change in the terminal elimination half-life. The overall safety profile of afatinib was acceptable. Coadministration of potent P-gp modulators had no clinically relevant effect on afatinib exposure. Effects of potent P-gp inhibitors were minimal at higher afatinib doses and can be readily managed by the timing of concomitant therapy. As afatinib is not a relevant modulator or substrate of cytochrome P450 enzymes, the drug-drug interaction potential is considered to be low.