Trastuzumab Concentrations Research Articles

Development and validation of cell-based ELISA for the quantification of trastuzumab in human plasma

Trastuzumab is a therapeutic monoclonal antibody against the Her2 oncoprotein, which is over-expressed in approximately 30% of breast cancers, and is now used routinely in the management of early and metastatic Her2+ disease. However, not all Her2+ breast cancer patients respond to trastuzumab and the pharmacodynamic and pharmacokinetic parameters behind this variation in response are unknown. Pharmacological investigations into variable response to trastuzumab have been hampered by the lack of a published feasible method to determine trastuzumab concentration in plasma. Here we describe the development and validation of a cell-based ELISA to measure trastuzumab in human plasma. The assay specifically measures the interaction between trastuzumab and Her2 and has a dynamic range of between 10 and 120 µg/ml. The mean intra-assay and inter-assay variability of the ELISA was 9%. Trastuzumab in plasma was stable for at least 10 weeks at − 20 °C and 72 h at 4 °C, and was unaffected by 5 freeze/thaw cycles. Having validated the assay, the trough plasma trastuzumab concentrations of 30 patients being treated for metastatic or early disease were measured. The median trough concentration was 62 (range 21 to 441) µg/ml. This cell-based ELISA method has undergone appropriate validation and is suitable for quantification of trastuzumab in the plasma of patients treated with Herceptin.

Trastuzumab-induced CCL20 and interleukin-8 mRNA in human whole blood ex vivo

Heparinized human whole blood from 16 adult volunteers was stimulated with achievable blood concentrations of trastuzumab and rituximab at 37 degrees C for 4 h, then CCL20, IL8, and beta-actin mRNA were quantified. The fold increase of beta-actin was all less than 1.5, and heat aggregated IgG induced both IL8 and CCL20 mRNA in all cases, suggesting that the assay was performed appropriately. Rituximab reduced the levels of CCL20 mRNA in approximately 1/3 of subjects, whereas 50 μg/ml trastuzumab induced IL8 and CCL20 mRNA in more than half of subjects. Although the results do not directly indicate the toxicity of antibody medicines, the individual variation found under physiological ex vivo condition will be an interesting clinical research model for drug safety analysis.

Dose scheduling and pharmacokinetic (PK) study of trastuzumab in patients (pts) with HER2/neu overexpressing breast cancer?

1109 Has been demonstrated that 10–20 μg/mL is the minimum serum trough concentration (Cmin) of trastuzumab that causes maximal tumor growth inhibition. This concentration was shown to be achievable using a weekly (4 mg/kg loading dose followed by 2 mg/kg weekly) and a three-weekly (8 mg/kg loading dose followed by 6 mg/kg) schedule, but only indirect comparisons of the two schedule have been performed. We prospectively analysed the PK distribution of trastuzumab in three different schedules, as follows: weekly (cohort I), bi-weekly (trastuzumab 6 mg/kg loading dose followed by 4 mg/kg, cohort II) and three-weekly schedule (cohort III). The PK analysis was performed in 18 pts (4 pts receiving the loading dose of cohort III, 8 pts receiving the maintenance dose of cohort III, 3 pts receiving the maintenance dose of cohort II, and 3 pts receiving loading dose of cohort I). Of the 18 pts analyzed, 9 were metastatic. The median age was 54 years (range 39–76), the median weight was 61,5 kg (range 53–80). At diagnoses, 10 pts had stage I disease, 5 and 3 pts had stage II and III respectively, ER was negative in 10 pts and PgR was negative in 12 pts; Ki67 was highly expressed in 14 pts; Her/neu was overexpressed and amplified in 16 and 2 pts respectively. To evaluate preliminary interpatient variability, 3 pts receiving 6mg/kg of trastuzumab repeated the PK analysis after four cycles: the comparison between two groups was not statistically significative. Comparison of Cmax and AUC mean between groups was statistically significative (P<0.05, Bonferroni test) except for 8 mg/kg vs 6 mg/kg AUC.All the doses and schedules analysed are able to reach the minimum serum concentration of trastuzumab that causes the maximal tumor growth inhibition. [Table: see text] No significant financial relationships to disclose.

Population pharmacokinetics of trastuzumab in patients With HER2+ metastatic breast cancer

To characterize the population pharmacokinetics of trastuzumab in patients with metastatic breast cancer. A nonlinear mixed effect model was based on pharmacokinetic data from phase I, II, and III studies of 476 patients. The phase I study enrolled patients with advanced solid tumors. The phase II and III studies enrolled patients with HER2-positive metastatic breast cancer. Patients in the pivotal phase II and III studies were treated with a 4 mg/kg loading dose of trastuzumab followed by 2 mg/kg weekly for up to 840 days. The model adequately predicted observed trastuzumab concentrations. Model stability and performance were verified using bootstrap simulations. Percentiles, mean, and standard deviation of observed levels were compared with their distributions from 100 replicates of datasets simulated under the model. A two-compartment linear pharmacokinetic model best described the data and accounted for the long-term accumulation observed following weekly administration of trastuzumab. Population estimates from the base model for clearance (CL) and volume of distribution of the central compartment (V1) of trastuzumab were 0.225 L/day, and 2.95 L, respectively. Estimated terminal halflife (t1/2) based on the population estimate was 28.5 days. Interpatient variabilities in clearance and volume were 43 and 29%, respectively. The number of metastatic sites, plasma level of extracellular domain of the HER2 receptor, and patient weight were significant baseline covariates for clearance, volume, or both (P<0.005). However, these covariate effects on trastuzumab exposure were modest and not clinically important in comparison with the large inter-patient variability of CL. Concomitant chemotherapy (anthracycline plus cyclophosphamide, or paclitaxel) did not appear to influence clearance. This population pharmacokinetic model can predict trastuzumab exposure in the long-term treatment of patients with metastatic breast cancer and provide comparison of alternative dosage regimens via simulation.

Phase II Study of Efficacy, Safety, and Pharmacokinetics of Trastuzumab Monotherapy Administered on a 3-Weekly Schedule

This phase II study investigated the efficacy, safety, and pharmacokinetics of trastuzumab monotherapy given as first-line treatment once every 3 weeks (3-weekly) in women with human epidermal growth factor receptor 2 (HER2) -positive metastatic breast cancer (MBC). Patients with previously untreated HER2-positive MBC received a loading dose of trastuzumab, 8 mg/kg intravenously (IV) and then 6 mg/kg IV at 3-week intervals until disease progression or patient withdrawal. In total, 105 patients received a median of five cycles of therapy (range, 1 to 35+). The overall response rate was 19% (23% in patients with measurable centrally confirmed immunohistochemistry [IHC] 3+ and/or fluorescence in situ hybridization [FISH] -positive disease) and clinical benefit rate (complete and partial responses plus stable disease for at least 6 months) was 33% (36% in patients with measurable centrally confirmed IHC 3+ and/or FISH-positive disease). Median time to progression was 3.4 months (range, 0.6 to 23.6 months). The most common treatment-related adverse events were rigors, pyrexia, headache, nausea, and fatigue. Median baseline left ventricular ejection fraction was 63%; this did not significantly change over the course of the study. The average exposure to trastuzumab observed in this study was similar to that in previous studies of the weekly regimen. However, as expected, mean trough trastuzumab concentrations were lower and peak levels were higher with 3-weekly trastuzumab compared with weekly treatments. Administering higher doses on a 3-weekly schedule did not compromise the efficacy and safety of trastuzumab in women with HER2-positive MBC, and average exposure was similar to that observed with weekly therapy. Three-weekly trastuzumab may represent a convenient alternative to weekly administration.

Cellular uptake of radioiodine delivered by trastuzumab can be modified by the addition of epidermal growth factor

The purpose of this study was to analyse whether non-radiolabelled epidermal growth factor (EGF) can modify the cellular uptake of 125I when delivered as [125I]trastuzumab. 125I was used as a marker for the diagnostically and therapeutically more interesting isotopes 123I (SPECT), 124I (PET) and 131I (therapy). The cell-associated radioactivity was measured in squamous carcinoma A431 cells following addition of [125I]trastuzumab. Different concentrations of [125I]trastuzumab and unlabelled EGF were used, and the total, membrane-bound and internalised radioactivity was measured. We also analysed how EGF and trastuzumab affected the cell growth. It was generally found that the cellular 125I uptake was decreased by the addition of EGF when [125I]trastuzumab was added for short incubation times. However, if the incubation times were longer, EGF increased the 125I uptake. This shift came earlier when higher [125I]trastuzumab concentrations were applied. The addition of EGF also influenced cell proliferation, and concentrations above 10 ng/ml reduced cell growth by approximately 20% after 24 h of incubation. By adding unlabelled EGF, it was possible to modify the cellular uptake of [125I]trastuzumab. This points towards new approaches for the modification of radionuclide uptake in EGFR- and HER2-positive tumours.

Oleic acid, the main monounsaturated fatty acid of olive oil, suppresses Her-2/neu (erbB-2) expression and synergistically enhances the growth inhibitory effects of trastuzumab (Herceptin™) in breast cancer cells with Her-2/neu oncogene amplification

The relationship between the intake of olive oil, the richest dietary source of the monounsaturated fatty acid oleic acid (OA; 18:1n-9), and breast cancer risk and progression has become a controversial issue. Moreover, it has been suggested that the protective effects of olive oil against breast cancer may be due to some other components of the oil rather than to a direct effect of OA. Using flow cytometry, western blotting, immunofluorescence microscopy, metabolic status (MTT), soft-agar colony formation, enzymatic in situ labeling of apoptosis-induced DNA double-strand breaks (TUNEL assay analyses), and caspase-3-dependent poly-ADP ribose polymerase (PARP) cleavage assays, we characterized the effects of exogenous supplementation with OA on the expression of Her-2/neu oncogene, which plays an active role in breast cancer etiology and progression. In addition, we investigated the effects of OA on the efficacy of trastuzumab (Herceptin), a humanized monoclonal antibody binding with high affinity to the ectodomain of the Her-2/neu-coded p185(Her-2/neu) oncoprotein. To study these issues we used BT-474 and SKBr-3 breast cancer cells, which naturally exhibit amplification of the Her-2/neu oncogene. Flow cytometric analyses demonstrated a dramatic (up to 46%) reduction of cell surface-associated p185(Her-2/neu) following treatment of the Her-2/neu-overexpressors BT-474 and SK-Br3 with OA. Indeed, this effect was comparable to that found following exposure to optimal concentrations of trastuzumab (up to 48% reduction with 20 microg/ml trastuzumab). Remarkably, the concurrent exposure to OA and suboptimal concentrations of trastuzumab (5 microg/ml) synergistically down-regulated Her-2/neu expression, as determined by flow cytometry (up to 70% reduction), immunoblotting, and immunofluorescence microscopy studies. The nature of the cytotoxic interaction between OA and trastuzumab revealed a strong synergism, as assessed by MTT-based cell viability and anchorage-independent soft-agar colony formation assays. Moreover, OA co-exposure synergistically enhanced trastuzumab efficacy towards Her-2/neu overexpressors by promoting DNA fragmentation associated with apoptotic cell death, as confirmed by TUNEL and caspase-3-dependent PARP cleavage. In addition, treatment with OA and trastuzumab dramatically increased both the expression and the nuclear accumulation of p27(Kip1), a cyclin-dependent kinase inhibitor playing a key role in the onset and progression of Her-2/neu-related breast cancer. Finally, OA co-exposure significantly enhanced the ability of trastuzumab to inhibit signaling pathways downstream of Her-2/neu, including phosphoproteins such as AKT and MAPK. These findings demonstrate that OA, the main monounsaturated fatty acid of olive oil, suppresses Her-2/neu overexpression, which, in turn, interacts synergistically with anti-Her-2/neu immunotherapy by promoting apoptotic cell death of breast cancer cells with Her-2/neu oncogene amplification. This previously unrecognized property of OA offers a novel molecular mechanism by which individual fatty acids may regulate the malignant behavior of breast cancer cells and therefore be helpful in the design of future epidemiological studies and, eventually, dietary counseling.

Randomized phase II trial of gemcitabine–cisplatin with or without trastuzumab in HER2-positive non-small-cell lung cancer

BackgroundTrastuzumab provides significant clinical benefits in HER2-positive metastatic breast cancer patients when administered in combination with chemotherapy. Chemotherapy has also been shown to be beneficial in some patients with advanced non-small-cell lung cancer (NSCLC). The present randomized phase II trial examined the effect of adding trastuzumab to a standard chemotherapeutic combination (gemcitabine–cisplatin) in patients with HER2-positive NSCLC. Patients and methodsPatients with untreated stage IIIB/IV HER2-positive NSCLC received up to six 21-day cycles of gemcitabine 1250 mg/m2 (days 1 and 8) and cisplatin 75 mg/m2 (day 1). Patients in the trastuzumab arm received trastuzumab 4 mg/kg intravenously (i.v.) followed by 2 mg/kg/week i.v. until progression. ResultsOf 619 patients screened, 103 were eligible. Fifty-one patients were treated with trastuzumab plus gemcitabine–cisplatin and 50 with gemcitabine–cisplatin alone. Efficacy was similar in the trastuzumab and control arms: response rate 36% versus 41%; median time to progression 6.3 versus 7.2 months; and median progression-free survival (PFS) 6.1 versus 7 months. Response rate (83%) and median PFS (8.5 months) appeared relatively good in the six trastuzumab-treated patients with HER2 3+ or fluorescence in situ hybridization (FISH)-positive NSCLC. Addition of trastuzumab to gemcitabine–cisplatin was well tolerated, side-effects were as expected, and trastuzumab did not exacerbate the known toxicity of gemcitabine and cisplatin. Symptomatic cardiotoxicity was observed in one trastuzumab-treated patient. Serum trastuzumab concentrations in the presence of gemcitabine–cisplatin were comparable to those of trastuzumab alone. ConclusionsTrastuzumab plus gemcitabine–cisplatin is well tolerated. Clinical benefit was not observed. Although HER2 3+/FISH-positive patients may benefit from trastuzumab, the subgroup is too small to provide definitive information. No significant effect of gemcitabine–cisplatin on trastuzumab pharmacokinetics was observed.