UPLC-MS/MS-Based Analysis of Trastuzumab in Plasma Samples: Application in Breast Cancer Patients Sample Monitoring

Aadil Ahmad Sheikh,Kunjahari Medhi,Mohd Imran,Rehan Abdur Rub,Abdulkhaliq J Alsalman,Mohammed M Ghoneim,Muzaffar Iqbal,Faiyaz Shakeel,Sultan Alshehri,Ozair Alam

doi:10.3390/pr10030509

Abstract

Trastuzumab is a target-based recombinant humanized IgG1 monoclonal antibody (mAbs), extensively employed for treatment of metastatic breast cancer with human epidermal growth receptor 2 (HER2) overexpression. Studies around the world have reported that mAbs have substantial inter-patient unpredictable absorption, distribution, metabolism, and excretion (ADME-pharmacokinetics) because of multiple elements manipulating the concentration of mAbs in plasma. Herein, we have established a bioanalytical technique using UPLC-MS/MS with an easy sample workup method and in-solution digestion protocol to assay the trastuzumab plasma samples from breast cancer patients in clinical studies. Surrogated proteolytic peptides were used for accurate quantification of trastuzumab (CanMab) with a trastuzumab signature peptide with [13C6, 15N4]-arginine and [13C6, 15N2]-lysine stable isotope-labeled (SIL) peptide. Experiments to validate the method were accurately carried out according to the guidelines mentioned in the bioanalytical method validation protocol. The evaluation established excellent linearity over a wide range of 5–500 µg/mL. The experimental procedure was efficaciously performed in a pilot study of five breast cancer patients and residual concentrations of drugs from responding and non-responding subjects were compared. The receiver operating characteristic (ROC) examination displayed that 52.25 µg/mL was the Cmin threshold predictive response with a satisfactory sensitivity of 88.58% and specificity of 79.25%.

Highlights

human epidermal growth receptor 2 (HER2) overexpressing metastatic breast and gastric cancer are mostly diagnosed cancers across the world
Our findings suggest that patients with residual concentrations of trastuzumab below 52 μg/mL are likely to have their dose changed to higher doses to raise dosage levels and increase efficacy, noting that a PK/PD interaction of this type has not been illustrated by toxic cases
The method was successfully applied in the measurement of residual and maximal concentration of trastuzumab in breast cancer patients

Summary

Introduction

HER2 overexpressing metastatic breast and gastric cancer are mostly diagnosed cancers across the world. These cancers have overexpressed cell surface HER2 receptors, and it is proposed that the overexpression or gene amplification of HER2 has been traced in about 20–30% of breast cancers [1]. Elevated activation of HER2 results in multiple downstream pathways leading to unusual ontogenesis of cancerous cells. The over-expression of HER2 in breast cancer cells triggers the amplification of the signal transduced by other receptors of the HER family via the formation of homodimers and heterodimers. The activation of HER2 commences with the formation of homodimers or heterodimers with other EGFR proteins, resulting in dimerization and autophosphorylation and/or transphosphorylation of particular tyrosine kinase residue in EGFR domains which are intracellular in nature. Abnormal cell proliferation and hyper activation of these signaling pathways are observed in cancer cells due to the upregulation of HER2 [2,3]

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