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Aim: To report the development and validation of an LC-MS/MS method for the simultaneous determination of unconjugated payload DM4 and its metabolite S-methyl-DM4 in human plasma. Methodology: A workflow of protein precipitation followed by reduction and solid phase extraction was employed to remove antibody-maytansinoid conjugates from plasma matrix, release DM4 from endogenous conjugates, and generate a clean sample extract for analysis, respectively. Sodium adduct species of both analytes were selected for multiple reaction monitoring to meet the assay sensitivity requirement in liquid chromatography with tandem mass spectrometry. Conclusion: The method was fully validated for a dynamic range of 0.100-50.0 ng/ml for both analytes along with desired stability and acceptable incurred sample reanalysis.
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Inmunoconjugados/sangre , Maitansina/sangre , Cromatografía Liquida , Humanos , Inmunoconjugados/química , Inmunoconjugados/metabolismo , Maitansina/análogos & derivados , Maitansina/metabolismo , Espectrometría de Masas en TándemRESUMEN
A high resolution accurate mass LC-MS method was developed to facilitate the characterization of a subset of antibody drug conjugate (ADC) biotherapeutics, where the payload is linked to the antibody by a thioether bond. Desulfuration of the thioether linker was optimized for release of the payload to take advantage of the high resolution and high mass accuracy of the Orbitrap to characterize metabolism of the payload. Two model ADCs, trastuzumab emtansine (T-DM1) and SigmaMAb dansyl-cadavarine-SMCC (SigmaMAb ADC mimic) were selected for optimization of the desulfuration reaction as a function of reaction time, pH, organic solvent, and chaotropic reagents (urea, guanidine HCl) by monitoring the yield of released desulfurated DM1 from T-DM1 and desulfurated dansyl-cadaverine-SMCC from SigmaMAb ADC mimic, respectively. The optimized desulfuration technique was successfully applied to enable characterization of the ADC following its incubation in hepatocytes, liver microsomes, and buffers, as illustrated by the identification of a hydrolyzed thiosuccinimide ring of SigmaMAb ADC mimic following incubation in buffer for 48 h. The results from this study demonstrate that the chemical cleavage of thioether bond by desulfuration is simple, efficient, and specific. This technique is useful in characterization of metabolism on the payload of ADC to provide guidance for improvement of its biopharmaceutical profile. This is the first report on characterization of modification to payload of ADC following desulfuration.
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Ado-Trastuzumab Emtansina/química , Cadaverina/análogos & derivados , Inmunoconjugados/química , Maitansina/sangre , Ado-Trastuzumab Emtansina/sangre , Animales , Boranos/química , Cadaverina/sangre , Cromatografía Liquida , Liberación de Fármacos , Estabilidad de Medicamentos , Hepatocitos/metabolismo , Humanos , Inmunoconjugados/sangre , Microsomas Hepáticos/metabolismo , Níquel/química , Ratas , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
INTRODUCTION: Our objective was to determine correlations between the tumor uptake and T/B ratios for 89Zr-labeled T-DM1 (89Zr-DFO-T-DM1) in mice with human BC xenografts by microPET/CT and biodistribution studies with HER2 expression and response to treatment with trastuzumab-DM1 (T-DM1). METHODS: The tumor and normal tissue uptake and T/B ratios for 89Zr-DFO-T-DM1 (10⯵g; 7.0â¯MBq) incorporated into a therapeutic dose (60⯵g) were determined by microPET/CT and biodistribution studies at 96â¯h p.i. in NOD/SCID mice with s.c. MDA-MB-231 (5â¯×â¯104 HER2/cell), MDA-MB-361 (5â¯×â¯105 HER2/cell) and BT-474 (2â¯×â¯106 HER2/cell) human BC xenografts. Mice bearing these tumors were treated with T-DM1 (3.6â¯mg/kg every 3â¯weeks) and the tumor doubling time estimated by fitting of tumor volume vs. time curves. A tumor doubling time ratio (TDR) was calculated by dividing the doubling time for T-DM1 and normal saline treated control mice. The clonogenic survival (CS) of BC cells with increasing HER2 expression treated for 72â¯h in vitro with T-DM1 or trastuzumab (0-100⯵g/mL) was compared. Correlations were determined between the T/B ratios for 89Zr-DFO-T-DM1 and HER2 expression, TDR and CS, and between CS and TDR. RESULTS: Uptake of 89Zr-DFO-T-DM1 in MDA-MB-231, MDA-MB-361 and BT-474 tumors was 2.4⯱â¯0.4%ID/g, 6.9⯱â¯2.2%ID/g and 9.8⯱â¯1.1%ID/g, respectively. There was a non-linear but direct correlation between the T/B ratios for 89Zr-DFO-T-DM1 and HER2 expression with the T/B ratio ranging from 4.5⯱â¯0.7 for MDA-MB-231 to 18.2⯱â¯1.8 for MDA-MB-361 and 35.9⯱â¯5.1 for BT-474 xenografts. Tumor intensity on microPET/CT images was proportional to HER2 expression. The standard uptake value (SUV) for the tumors on the images was strongly correlated with the T/B ratio in biodistribution studies. There was a direct linear correlation between the T/B ratio for 89Zr-DFO-T-DM1 and TDR, with TDR ranging from 0.9 for MDA-MB-231 to 1.6 for MDA-MB-361 and 2.1 for BT-474 tumors. The cytotoxicity of T-DM1 in vitro on BC cells was dependent on HER2 expression but T-DM1 was more potent than trastuzumab. There was an inverse correlation between the TDR for mice treated with T-DM1 and CS of BC cells exposed in vitro to T-DM1. CONCLUSIONS: Based on the direct correlations between the T/B ratio for 89Zr-DFO-T-DM1 by PET and HER2 expression and response to T-DM1, our results suggest that PET with 89Zr-DFO-T-DM1 may predict response of HER2-positive BC to treatment with T-DM1. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Our results suggest that PET with 89Zr-DFO-T-DM1 may predict response to treatment with T-DM1 in HER-positive BC.
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Regulación Neoplásica de la Expresión Génica , Neoplasias Mamarias Experimentales/diagnóstico por imagen , Maitansina/análogos & derivados , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Radioisótopos , Receptor ErbB-2/metabolismo , Trastuzumab/metabolismo , Trastuzumab/uso terapéutico , Circonio , Ado-Trastuzumab Emtansina , Animales , Transporte Biológico , Línea Celular Tumoral , Femenino , Humanos , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/terapia , Maitansina/sangre , Maitansina/metabolismo , Maitansina/farmacocinética , Maitansina/uso terapéutico , Ratones , Distribución Tisular , Trastuzumab/sangre , Trastuzumab/farmacocinética , Resultado del TratamientoRESUMEN
AIM: Compared with small molecules, LC-MS quantitation of larger biotherapeutic proteins such as antibodies and antibody-drug conjugates at the intact level presents many challenges in both LC and MS due to their higher molecular weight, bigger size, structural complexity and heterogeneity. RESULTS & CONCLUSION: In this study, quantitation of an intact lysine-linked antibody-drug conjugate, trastuzumab emtansine is presented. Trastuzumab emtansine was extracted from rat plasma using bead-based immunoaffinity capture; after elution from the beads, it was directly analyzed on a LC-HRMS system. Quantitation using both extracted ion chromatogram and deconvoluted mass peaks was evaluated. A limit of quantitation was approximately 20 ng on column with a linear dynamic range from 5 to 100 µg/ml. In addition, the reproducibility and distribution of the drug-to-antibody ratio at different trastuzumab emtansine concentrations were discussed.
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Análisis Químico de la Sangre/métodos , Cromatografía Liquida/métodos , Inmunoconjugados/sangre , Espectrometría de Masas/métodos , Maitansina/análogos & derivados , Trastuzumab/sangre , Ado-Trastuzumab Emtansina , Animales , Inmunoconjugados/química , Inmunoconjugados/aislamiento & purificación , Límite de Detección , Maitansina/sangre , Maitansina/química , Maitansina/aislamiento & purificación , Ratas , Trastuzumab/química , Trastuzumab/aislamiento & purificaciónRESUMEN
AIMS: We conducted population pharmacokinetic (PopPK) and exposure-response analyses for trastuzumab emtansine (T-DM1), to assess the need for T-DM1 dose optimization in patients with low exposure by using TH3RESA [A Study of Trastuzumab Emtansine in Comparison With Treatment of Physician's Choice in Patients With human epidermal growth factor receptor 2 (HER2)-positive Breast Cancer Who Have Received at Least Two Prior Regimens of HER2-directed Therapy] study data (NCT01419197). The randomized phase III TH3RESA study investigated T-DM1 vs. treatment of physician's choice (TPC) in patients with heavily pretreated HER2-positive advanced breast cancer. METHODS: We compared a historical T-DM1 PopPK model with T-DM1 pharmacokinetics in TH3RESA and performed exposure-response analyses using model-predicted cycle 1 maximum concentration (Cmax ), cycle 1 minimum concentration (Cmin ) and area under the concentration-time curve at steady state (AUCss ). Kaplan-Meier analyses [overall survival (OS), progression-free survival (PFS)] and logistic regression [overall response rate (ORR), safety] were stratified by T-DM1 exposure metrics. Survival hazard ratios (HRs) in the lowest exposure quartile (Q1) of cycle 1 Cmin were compared with matched TPC-treated patients. RESULTS: T-DM1 concentrations in TH3RESA were described well by the historical PopPK model. Patients with higher cycle 1 Cmin and AUCss exhibited numerically longer median OS and PFS and higher ORR than patients with lower exposure. Exposure-response relationships were less evident for cycle 1 Cmax . No relationship between exposure and safety was identified. HRs for the comparison of T-DM1-treated patients in the Q1 subgroup with matched TPC-treated patients were 0.96 [95% confidence interval (CI) 0.63, 1.47] for OS and 0.92 (95% CI 0.64, 1.32) for PFS. CONCLUSIONS: Exposure-response relationships for efficacy were inconsistent across exposure metrics. HRs for survival in patients in the lowest T-DM1 exposure quartile vs. matched TPC-treated patients suggest that, compared with TCP, the approved T-DM1 dose is unlikely to be detrimental to patients with low exposure.
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Antineoplásicos Inmunológicos/farmacocinética , Neoplasias de la Mama/tratamiento farmacológico , Maitansina/análogos & derivados , Modelos Biológicos , Receptor ErbB-2/antagonistas & inhibidores , Trastuzumab/farmacocinética , Moduladores de Tubulina/farmacocinética , Ado-Trastuzumab Emtansina , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/efectos adversos , Antineoplásicos Inmunológicos/sangre , Área Bajo la Curva , Neoplasias de la Mama/sangre , Neoplasias de la Mama/enzimología , Neoplasias de la Mama/mortalidad , Supervivencia sin Enfermedad , Femenino , Humanos , Infusiones Intravenosas , Estimación de Kaplan-Meier , Modelos Logísticos , Maitansina/administración & dosificación , Maitansina/efectos adversos , Maitansina/sangre , Maitansina/farmacocinética , Tasa de Depuración Metabólica , Dinámicas no Lineales , Modelos de Riesgos Proporcionales , Receptor ErbB-2/metabolismo , Medición de Riesgo , Trastuzumab/administración & dosificación , Trastuzumab/efectos adversos , Trastuzumab/sangre , Resultado del Tratamiento , Moduladores de Tubulina/administración & dosificación , Moduladores de Tubulina/efectos adversos , Moduladores de Tubulina/sangreRESUMEN
Antibody-drug conjugates (ADCs) are complex drug platforms composed of monoclonal antibodies (mAbs) conjugated to potent cytotoxic drugs (payloads) via chemical linkers, enabling selective payload delivery to neoplastic cells, resulting in improved efficacy and reduced toxicity. Brentuximab vedotin (Adcetris®, SGN-35) and adotrastuzumab emtansine (Kadcyla®, T-DM1) are the two FDA-approved and commercially available ADCs, and both drugs exhibit ADC-related thrombocytopenia and neutropenia. A pharmacokinetic/pharmacodynamic (PK/PD) model for ADCs was developed to identify the analyte from each ADC that is most associated with the observed hematopoietic toxicities and to determine the role of the apparent in vivo payload release rate on the severity of thrombocytopenia and neutropenia. Murine xenograft experiments and data from literature were combined, and the PK of both ADCs and their analytes were described with two-compartment models, with linear elimination and first-order payload release rate constants (k rel). ADC-associated hematotoxicities were captured with a previously published PD model for myelosuppression driven by various analytes. ADC half-lives were about 5 days, and k rel values were 0.46 (T-DM1) and 0.12 h-1 (SGN-35). The lifespans of platelets following T-DM1 and neutrophils following SGN-35 were 3.73 and 4.72 days. Comparison of alternate model structures suggested that mechanisms of myelosuppression are payload-driven for SGN-35 and ADC pinocytosis-dependent for T-DM1. Model simulations suggested that a 4-fold increase (T-DM1) and 70% decrease (SGN-35) in k rel would improve hematotoxicity to grade 1. The proposed model successfully captured the PK and associated myelosuppression of both ADCs and might serve as a general PK/PD platform for assessing hematological toxicities to ADCs.
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Médula Ósea/efectos de los fármacos , Inmunoconjugados/toxicidad , Ado-Trastuzumab Emtansina , Animales , Brentuximab Vedotina , Línea Celular Tumoral , Femenino , Inmunoconjugados/sangre , Maitansina/análogos & derivados , Maitansina/sangre , Maitansina/toxicidad , Ratones , Ratones Endogámicos BALB C , Modelos Biológicos , Trastuzumab/sangre , Trastuzumab/toxicidadRESUMEN
BACKGROUND: This phase 1/2 study evaluated the dose-limiting toxicity and maximum tolerated dose of MLN2704, a humanized monoclonal antibody MLN591 targeting prostate-specific membrane antigen, linked to the maytansinoid DM1 in patients with progressive metastatic castration-resistant prostate cancer. PATIENTS AND METHODS: A total of 62 patients received MLN2704 at ascending doses on 4 schedules: weekly (60, 84, 118, and 165mg/m2; 12 patients); every 2 weeks (120, 168, 236, and 330mg/m2; 15 patients); every 3 weeks (330 and 426mg/m2; 18 patients); and on days 1 and 15 of a 6-week schedule (6-week cycle, 330mg/m2; 17 patients). The primary efficacy endpoint was a sustained ≥50% decline from baseline prostate-specific antigen (PSA) without evidence of disease progression. Toxicity, pharmacokinetics, immunogenicity, and antitumor activity were assessed. RESULTS: Neurotoxicity was dose-limiting. Overall, 44 patients (71%) exhibited peripheral neuropathy: 6 (10%) had grade 3/4. Neurotoxicity rates remained high despite increasing the dosing interval to 3 (13 of 14; one grade 3) and 6 weeks (16 of 17; three grade 3). MLN2704 pharmacokinetics were dose-linear. Rapid deconjugation of DM1 from the conjugated antibody was seen. In all, 5 patients (8%) experienced ≥50% decline in PSA; 5 (8%) had PSA stabilization lasting≥90 days. Only 2 of 35 patients on the 3- and 6-week schedules achieved a PSA decline of >50%. CONCLUSIONS: MLN2704 has limited activity in metastatic castration-resistant prostate cancer. Disulfide linker lability and rapid deconjugation lead to neurotoxicity and a narrow therapeutic window.
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Adenocarcinoma/secundario , Antineoplásicos Inmunológicos/uso terapéutico , Inmunoconjugados/uso terapéutico , Maitansina/uso terapéutico , Neoplasias de la Próstata Resistentes a la Castración/tratamiento farmacológico , Adenocarcinoma/sangre , Adenocarcinoma/tratamiento farmacológico , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/farmacocinética , Supervivencia sin Enfermedad , Relación Dosis-Respuesta Inmunológica , Monitoreo de Drogas , Enfermedades Gastrointestinales/inducido químicamente , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/farmacocinética , Masculino , Dosis Máxima Tolerada , Maitansina/administración & dosificación , Maitansina/efectos adversos , Maitansina/sangre , Persona de Mediana Edad , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Antígeno Prostático Específico/sangre , Neoplasias de la Próstata Resistentes a la Castración/sangreRESUMEN
For antibody drug conjugates (ADCs), the fate of the cytotoxic payload in vivo needs to be well understood to mitigate toxicity risks and properly design the first in-patient studies. Therefore, a distribution, metabolism, and excretion (DME) study with a radiolabeled rat cross-reactive ADC ([(3)H]DM1-LNL897) targeting the P-cadherin receptor was conducted in female tumor-bearing nude rats. Although multiple components [total radioactivity, conjugated ADC, total ADC, emtansine (DM1) payload, and catabolites] needed to be monitored with different technologies (liquid scintillation counting, liquid chromatography/mass spectrometry, enzyme-linked immunosorbent assay, and size exclusion chromatography), the pharmacokinetic data were nearly superimposable with the various techniques. [(3)H]DM1-LNL897 was cleared with half-lives of 51-62 hours and LNL897-related radioactivity showed a minor extent of tissue distribution. The highest tissue concentrations of [(3)H]DM1-LNL897-related radioactivity were measured in tumor. Complimentary liquid extraction surface analysis coupled to micro-liquid chromatography-tandem mass spectrometry data proved that the lysine (LYS)-4(maleimidylmethyl) cyclohexane-1-carboxylate-DM1 (LYS-MCC-DM1) catabolite was the only detectable component distributed evenly in the tumor and liver tissue. The mass balance was complete with up to 13.8% ± 0.482% of the administered radioactivity remaining in carcass 168 hours postdose. LNL897-derived radioactivity was mainly excreted via feces (84.5% ± 3.12%) and through urine only to a minor extent (4.15% ± 0.462%). In serum, the major part of radioactivity could be attributed to ADC, while small molecule disposition products were the predominant species in excreta. We show that there is a difference in metabolite profiles depending on which derivatization methods for DM1 were applied. Besides previously published results on LYS-MCC-DM1 and MCC-DM1, maysine and a cysteine conjugate of DM1 could be identified in serum and excreta.
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Anticuerpos/metabolismo , Antineoplásicos Fitogénicos/farmacocinética , Neoplasias de la Mama/tratamiento farmacológico , Inmunoconjugados/farmacocinética , Maitansina/farmacocinética , Administración Intravenosa , Animales , Anticuerpos/administración & dosificación , Anticuerpos/sangre , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/sangre , Área Bajo la Curva , Disponibilidad Biológica , Biotransformación , Neoplasias de la Mama/sangre , Neoplasias de la Mama/patología , Cadherinas/inmunología , Cadherinas/metabolismo , Línea Celular Tumoral , Heces/química , Femenino , Semivida , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/sangre , Maitansina/administración & dosificación , Maitansina/sangre , Tasa de Depuración Metabólica , Ratas Desnudas , Distribución TisularRESUMEN
A sensitive and specific method was developed and validated for the quantitation of maytansinoid (DM1) in human serum using on-line solid phase extraction (SPE)-liquid chromatography-tandem mass spectrometry (LC-MS/MS). Because DM1 contains a free thiol moiety, likely to readily dimerize or react with other thiol-containing molecules in serum, samples were pre-treated with a reducing agent [tris (2-carboxyethyl) phosphine] (TCEP) and further blocked with N-ethylmaleimide (NEM). The resulting samples were diluted with acetonitrile prior to the on-line solid phase extraction (SPE) on a C18 cartridge. A C18 (150×4.6mm ID 3µm particle size) column was used for chromatographic separation with a 10.0min HPLC gradient and DM1-NEM was detected in the selected reaction monitoring mode of a triple quadrupole mass spectrometer. DM1 concentrations were back-calculated from DM1-NEM amount found in the human serum samples. The quantitation range of the method was 0.200-200ng/mL when using 0.25mL serum. Within-run day precisions (n=6) were 0.9-4.4% and between-run day (3 days runs; n=18) precisions 2.5-5.6%. Method biases were between 3.5-14.5% across the whole calibration range. DM1-NEM exhibited sufficiently stability under all relevant analytical conditions and no DM1 losses from the ADC were observed. Finally, the assay was used for DM1 determination in human serum concentration after the intravenous administration of an investigational antibody drug conjugate (ADC) containing DM1 as payload.
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Maitansina/análogos & derivados , Extracción en Fase Sólida/métodos , Extracción en Fase Sólida/normas , Espectrometría de Masas en Tándem/métodos , Espectrometría de Masas en Tándem/normas , Cromatografía Liquida/métodos , Cromatografía Liquida/normas , Humanos , Infusiones Intravenosas , Maitansina/administración & dosificación , Maitansina/sangre , Neoplasias/sangre , Neoplasias/tratamiento farmacológico , Reproducibilidad de los ResultadosRESUMEN
DM1, a derivative of maytansine, is the cytotoxic component of the antibody-drug conjugate trastuzumab emtansine (T-DM1). Understanding the disposition and metabolism of DM1 would help to assess (1) any tissue-specific distribution and risk for potential drug-drug interactions and (2) the need for special patient population studies. To this end, the current study determined the disposition and metabolism of DM1 following single intravenous administration of [(3)H]-DM1 in Sprague Dawley rats. Blood, tissues, urine, bile, and feces were collected up to 5 days after dose administration and analyzed for total radioactivity and metabolites. Results showed that radioactivity cleared rapidly from the blood and quickly distributed to the lungs, liver, kidneys, spleen, heart, gastrointestinal tract, adrenal glands, and other tissues without significant accumulation or persistence. The majority of dosed radioactivity was recovered in feces (~100% of the injected dose over 5 days) with biliary elimination being the predominant route (~46% of the injected dose over 3 days). Excretion in urine was minimal (~5% of the injected dose over 5 days). Mass balance was achieved over 5 days. An analysis of bile samples revealed a small fraction of intact DM1 and a predominance of DM1 metabolites formed through oxidation, hydrolysis, S-methylation, and glutathione and its related conjugates. Collectively, these data demonstrate that DM1 is extensively distributed and quickly cleared from blood, and undergoes extensive metabolism to form multiple metabolites, which are mainly eliminated through the hepatic-biliary route, suggesting that hepatic function (but not renal function) plays an important role in DM1 elimination.
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Anticuerpos Monoclonales Humanizados/farmacocinética , Antineoplásicos/farmacocinética , Hígado/metabolismo , Maitansina/análogos & derivados , Ado-Trastuzumab Emtansina , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/sangre , Antineoplásicos/administración & dosificación , Antineoplásicos/sangre , Bilis/metabolismo , Biotransformación , Heces/química , Femenino , Glutatión/metabolismo , Eliminación Hepatobiliar , Hidrólisis , Inyecciones Intravenosas , Maitansina/administración & dosificación , Maitansina/sangre , Maitansina/farmacocinética , Metilación , Estructura Molecular , Oxidación-Reducción , Ratas Sprague-Dawley , Eliminación Renal , Distribución Tisular , TrastuzumabRESUMEN
OBJECTIVE: Trastuzumab emtansine (T-DM1), an antibody-drug conjugate composed of the cytotoxic agent DM1 conjugated to trastuzumab via a stable thioether linker, has shown clinical activity in human epidermal growth factor receptor 2-positive metastatic breast cancer patients. This study evaluated the maximum tolerated dose, toxicity and pharmacokinetics of trastuzumab emtansine in Japanese breast cancer patients. METHODS: Inoperable advanced or recurrent human epidermal growth factor receptor 2-positive breast cancer patients were administered trastuzumab emtansine intravenously at a dose of 1.8, 2.4 or 3.6 mg/kg every 3 weeks. The maximum tolerated dose was estimated using the continual reassessment method. RESULTS: This study enrolled 10 patients who were administered trastuzumab emtansine for a median of seven cycles. The dose-limiting toxicity was Grade 3 elevation of aspartate aminotransferase/alanine aminotransferase at the 2.4 mg/kg dose level. The maximum tolerated dose was estimated to be 3.6 mg/kg because at the point when dose-limiting toxicity was evaluable in 10 patients, the probability of dose-limiting toxicity estimated using the continual reassessment method was closest to 25% at a dose of 3.6 mg/kg and this was unchanged by the results for patients enrolled after that. The most frequent adverse events were nausea, arthralgia, fever, fatigue and decreased appetite. Adverse events were generally tolerable. The maximum concentration and area under the concentration-time curve increased linearly with the dose. CONCLUSIONS: Trastuzumab emtansine up to 3.6 mg/kg was well tolerated by Japanese breast cancer patients. Although thrombocytopenia and hepatotoxicity tended to be more severe than was seen in Western patients in previous trastuzumab emtansine trials, those adverse events recovered without special supportive treatment.
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Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/farmacocinética , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/sangre , Neoplasias de la Mama/tratamiento farmacológico , Maitansina/análogos & derivados , Receptor ErbB-2/análisis , Ado-Trastuzumab Emtansina , Adulto , Anciano , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales Humanizados/sangre , Antineoplásicos/efectos adversos , Antineoplásicos/sangre , Área Bajo la Curva , Pueblo Asiatico , Neoplasias de la Mama/química , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Femenino , Humanos , Hipopotasemia/inducido químicamente , Japón , Dosis Máxima Tolerada , Maitansina/administración & dosificación , Maitansina/efectos adversos , Maitansina/sangre , Maitansina/farmacocinética , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Trombocitopenia/inducido químicamente , TrastuzumabRESUMEN
PURPOSE: Trastuzumab emtansine (T-DM1), an antibody-drug conjugate (ADC) comprised of trastuzumab linked to the antimitotic agent DM1, has shown promising results in patients with human epidermal growth factor receptor 2-positive metastatic breast cancer. Investigations of the mechanisms of the action of ADCs, including T-DM1, have been primarily descriptive or semiquantitative. However, quantitative pharmacokinetic/pharmacodynamic (PK/PD) analysis may provide insights into their complex behavior. The analyses described herein applied PK/PD modeling to nonclinical studies of maytansinoid conjugates. METHODS: The maytansinoid conjugates T-DM1 and T-SPP-DM1, with thioether and disulfide linkers, respectively, were tested in mouse efficacy, PK, and tumor uptake studies. (3)[H]DM1-bearing ADCs were used to facilitate the quantitation of the ADCs in plasma, as well as ADC and ADC catabolites in tumors. Three mechanistic PK/PD models were used to characterize plasma ADC, tumor ADC, and tumor catabolite concentrations. Tumor catabolite concentrations were used to fit tumor response. Model parameters were estimated using R software and nonlinear least squares regression. RESULTS: Plasma ADC-associated DM1 concentrations of T-DM1 decreased more slowly than those of T-SPP-DM1, likely due to slower DM1 release. A comparison of the mechanistic models found that the best model allowed catabolism and catabolite exit rates to differ between ADCs, that T-DM1 exhibited both faster tumor catabolism and catabolite exit rate from tumors than T-SPP-DM1; findings inconsistent with expected behavior based on the physicochemical nature of the respective catabolites. Tumor catabolite concentrations adequately described tumor response with both ADCs showing similar potency. CONCLUSION: Mechanistic PK/PD studies described herein provided results that confirmed and challenged current hypotheses, and suggested new areas of investigation.
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Anticuerpos Monoclonales Humanizados/farmacocinética , Maitansina/análogos & derivados , Modelos Biológicos , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Ado-Trastuzumab Emtansina , Algoritmos , Animales , Anticuerpos Monoclonales Humanizados/sangre , Área Bajo la Curva , Línea Celular Tumoral , Femenino , Humanos , Maitansina/sangre , Maitansina/farmacocinética , Ratones Desnudos , Trastuzumab , Resultado del Tratamiento , TritioRESUMEN
PURPOSE: Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate comprising the humanized monoclonal antibody trastuzumab linked to DM1, a highly potent cytotoxic agent. A population pharmacokinetic (PK) analysis was performed to estimate typical values and interindividual variability of T-DM1 PK parameters and the effects of clinically relevant covariates. METHODS: Serum samples were collected from 671 patients with human epidermal growth factor receptor 2-positive locally advanced or metastatic breast cancer (MBC) who received single-agent T-DM1 in five phase I to phase III studies. Nonlinear mixed-effects modeling with the first-order conditional estimation method was used. RESULTS: A linear two-compartment model with first-order elimination from the central compartment described T-DM1 PKs in the clinical dose range. T-DM1 elimination clearance was 0.676 L/day, volume of distribution in the central compartment (V c) was 3.127 L, and terminal elimination half-life was 3.94 days. Age, race, region, and renal function did not influence T-DM1 PK. Given the low-to-moderate effect of all statistically significant covariates on T-DM1 exposure, none of these covariates is expected to result in a clinically meaningful change in T-DM1 exposure. CONCLUSIONS: T-DM1 PK properties are consistent and predictable in patients. A further refinement of dose based on baseline covariates other than body weight for the current 3.6 mg/kg regimen would not yield clinically meaningful reductions in interindividual PK variability in patients with MBC.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacocinética , Antineoplásicos/farmacocinética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Maitansina/análogos & derivados , Receptor ErbB-2/antagonistas & inhibidores , Ado-Trastuzumab Emtansina , Anticuerpos Monoclonales Humanizados/sangre , Antineoplásicos/sangre , Disponibilidad Biológica , Neoplasias de la Mama/epidemiología , Neoplasias de la Mama/secundario , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Modelos Lineales , Maitansina/sangre , Maitansina/farmacocinética , Pronóstico , Receptor ErbB-2/metabolismo , Distribución Tisular , Trastuzumab , Estados Unidos/epidemiologíaRESUMEN
Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) therapeutic for treatment of human epidermal growth factor receptor 2 (HER2)-positive cancers. The T-DM1 dose product contains a mixture of drug-to-antibody ratio (DAR) moieties whereby the small molecule DM1 is chemically conjugated to trastuzumab antibody. The pharmacokinetics (PK) underlying this system and other ADCs are complex and have not been elucidated. Accordingly, we have developed two PK modeling approaches from preclinical data to conceptualize and understand T-DM1 PK, to quantify rates of DM1 deconjugation, and to elucidate the link between trastuzumab, T-DM1, and DAR measurements. Preclinical data included PK studies in rats (n = 34) and cynomolgus monkeys (n = 18) at doses ranging from 0.3 to 30 mg/kg and in vitro plasma stability. T-DM1 and total trastuzumab (TT) plasma concentrations were measured by enzyme-linked immunosorbent assay. Individual DAR moieties were measured by affinity capture liquid chromatography-mass spectrophotometry. Two PK modeling approaches were developed for T-DM1 using NONMEM 7.2 software: a mechanistic model fit simultaneously to TT and DAR concentrations and a reduced model fit simultaneously to TT and T-DM1 concentrations. DAR moieties were well described with a three-compartmental model and DM1 deconjugation in the central compartment. DM1 deconjugated fastest from the more highly loaded trastuzumab molecules (i.e., DAR moieties that are ≥3 DM1 per trastuzumab). T-DM1 clearance (CL) was 2-fold faster than TT CL due to deconjugation. The two modeling approaches provide flexibility based on available analytical measurements for T-DM1 and a framework for designing ADC studies and PK-pharmacodynamic modeling of ADC efficacy- and toxicity-related endpoints.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacocinética , Antineoplásicos/farmacocinética , Neoplasias de la Mama/tratamiento farmacológico , Maitansina/análogos & derivados , Modelos Biológicos , Ado-Trastuzumab Emtansina , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/sangre , Antineoplásicos/administración & dosificación , Antineoplásicos/sangre , Biotransformación , Neoplasias de la Mama/sangre , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Cromatografía Liquida , Estabilidad de Medicamentos , Femenino , Humanos , Inyecciones Intravenosas , Macaca fascicularis , Masculino , Espectrometría de Masas , Maitansina/administración & dosificación , Maitansina/sangre , Maitansina/farmacocinética , Metástasis de la Neoplasia , Ratas , Ratas Sprague-Dawley , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/metabolismo , Reproducibilidad de los Resultados , TrastuzumabRESUMEN
Trastuzumab emtansine (Kadcyla) is a recently approved antibody-drug conjugate produced by attachment of the anti-tubulin drug, DM1, to lysine amines via the SMCC linker. The resulting product exhibits a drug load distribution from 0 to 8 drugs per antibody that can be quantified using mass spectrometry. Different statistical models were tested against the experimental data derived from samples produced during process characterization studies to determine best fit. The Poisson distribution gives the best correlation for samples manufactured using the target process conditions (yielding the target average drug to antibody ratio (DAR) of 3.5) as well as those produced under conditions that exceed the allowed manufacturing ranges and yield products with average DAR values that are significantly different from the target (i.e., ≤3.0 or ≥4.0). The Poisson distribution establishes a link between average DAR values and drug load distributions, implying that measurement and control of the former (i.e., via a simple UV spectrophotometric method) could be used to indirectly control the latter in trastuzumab emtansine.
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Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/farmacocinética , Neoplasias de la Mama/tratamiento farmacológico , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Lisina/química , Maitansina/análogos & derivados , Modelos Estadísticos , Receptor ErbB-2/antagonistas & inhibidores , Ado-Trastuzumab Emtansina , Anticuerpos Monoclonales Humanizados/sangre , Antineoplásicos/sangre , Antineoplásicos/química , Antineoplásicos/farmacocinética , Neoplasias de la Mama/metabolismo , Cromatografía Liquida , Femenino , Humanos , Inmunoconjugados/sangre , Lisina/metabolismo , Maitansina/sangre , Maitansina/química , Maitansina/farmacocinética , Estructura Molecular , Relación Estructura-Actividad , Espectrometría de Masas en Tándem , Distribución Tisular , Trastuzumab , Células Tumorales CultivadasRESUMEN
BACKGROUND AND OBJECTIVE: Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate recently approved by the US Food and Drug Administration for the treatment of human epidermal growth factor receptor 2 (HER2)-positive metastatic breast cancer previously treated with trastuzumab and taxane chemotherapy. It comprises the microtubule inhibitory cytotoxic agent DM1 conjugated to the HER2-targeted humanized monoclonal antibody trastuzumab via a stable linker. To characterize the pharmacokinetics of T-DM1 in patients with metastatic breast cancer, concentrations of multiple analytes were quantified, including serum concentrations of T-DM1 conjugate and total trastuzumab (the sum of conjugated and unconjugated trastuzumab), as well as plasma concentrations of DM1. The clearance of T-DM1 conjugate is approximately 2 to 3 times faster than its parent antibody, trastuzumab. However, the clearance pathways accounting for this faster clearance rate are unclear. An integrated population pharmacokinetic model that simultaneously fits the pharmacokinetics of T-DM1 conjugate and total trastuzumab can help to elucidate the clearance pathways of T-DM1. The model can also be used to predict total trastuzumab pharmacokinetic profiles based on T-DM1 conjugate pharmacokinetic data and sparse total trastuzumab pharmacokinetic data, thereby reducing the frequency of pharmacokinetic sampling. METHODS: T-DM1 conjugate and total trastuzumab serum concentration data, including baseline trastuzumab concentrations prior to T-DM1 treatment, from phase I and II studies were used to develop this integrated population pharmacokinetic model. Based on a hypothetical T-DM1 catabolism scheme, two-compartment models for T-DM1 conjugate and trastuzumab were integrated by assuming a one-step deconjugation clearance from T-DM1 conjugate to trastuzumab. The ability of the model to predict the total trastuzumab pharmacokinetic profile based on T-DM1 conjugate pharmacokinetics and various sampling schemes of total trastuzumab pharmacokinetics was assessed to evaluate total trastuzumab sampling schemes. RESULTS: The final model reflects a simplified catabolism scheme of T-DM1, suggesting that T-DM1 clearance pathways include both deconjugation and proteolytic degradation. The model fits T-DM1 conjugate and total trastuzumab pharmacokinetic data simultaneously. The deconjugation clearance of T-DM1 was estimated to be ~0.4 L/day. Proteolytic degradation clearances for T-DM1 and trastuzumab were similar (~0.3 L/day). This model accurately predicts total trastuzumab pharmacokinetic profiles based on T-DM1 conjugate pharmacokinetic data and sparse total trastuzumab pharmacokinetic data sampled at preinfusion and end of infusion in cycle 1, and in one additional steady state cycle. CONCLUSIONS: This semi-mechanistic integrated model links T-DM1 conjugate and total trastuzumab pharmacokinetic data, and supports the inclusion of both proteolytic degradation and deconjugation as clearance pathways in the hypothetical T-DM1 catabolism scheme. The model attributes a faster T-DM1 conjugate clearance versus that of trastuzumab to the presence of a deconjugation process and suggests a similar proteolytic clearance of T-DM1 and trastuzumab. Based on the model and T-DM1 conjugate pharmacokinetic data, a sparse pharmacokinetic sampling scheme for total trastuzumab provides an entire pharmacokinetic profile with similar predictive accuracy to that of a dense pharmacokinetic sampling scheme.
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Anticuerpos Monoclonales Humanizados/farmacocinética , Antineoplásicos/farmacocinética , Neoplasias de la Mama/metabolismo , Maitansina/análogos & derivados , Modelos Biológicos , Ado-Trastuzumab Emtansina , Anticuerpos Monoclonales Humanizados/sangre , Antineoplásicos/sangre , Femenino , Humanos , Maitansina/sangre , Maitansina/farmacocinética , Receptor ErbB-2 , Reproducibilidad de los Resultados , TrastuzumabRESUMEN
In this report, we describe the synthesis of a panel of disulfide-linked huC242 (anti-CanAg) antibody maytansinoid conjugates (AMCs), which have varying levels of steric hindrance around the disulfide bond, in order to investigate the relationship between stability to reduction of the disulfide linker and antitumor activity of the conjugate in vivo. The conjugates were first tested for stability to reduction by dithiothreitol in vitro and for plasma stability in CD1 mice. It was found that the conjugates having the more sterically hindered disulfide linkages were more stable to reductive cleavage of the maytansinoid in both settings. When the panel of conjugates was tested for in vivo efficacy in two human colon cancer xenograft models in SCID mice, it was found that the conjugate with intermediate disulfide bond stability having two methyl groups on the maytansinoid side of the disulfide bond and no methyl groups on the linker side of the disulfide bond (huC242-SPDB-DM4) displayed the best efficacy. The ranking of in vivo efficacies of the conjugates was not predicted by their in vitro potencies, since all conjugates were highly active in vitro, including a huC242-SMCC-DM1 conjugate with a noncleavable linkage which showed only marginal activity in vivo. These data suggest that factors in addition to intrinsic conjugate potency and conjugate half-life in plasma influence the magnitude of antitumor activity observed for an AMC in vivo. We provide evidence that bystander killing of neighboring nontargeted tumor cells by diffusible cytotoxic metabolites produced from target cell processing of disulfide-linked antibody-maytansinoid conjugates may be one additional factor contributing to the activity of these conjugates in vivo.
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Anticuerpos/química , Antineoplásicos/química , Carbono/química , Neoplasias del Colon/tratamiento farmacológico , Disulfuros/química , Maitansina/química , Animales , Anticuerpos/sangre , Anticuerpos/farmacología , Antineoplásicos/sangre , Antineoplásicos/farmacología , Neoplasias del Colon/metabolismo , Disulfuros/sangre , Disulfuros/farmacología , Humanos , Maitansina/sangre , Maitansina/farmacología , Ratones , Ratones Endogámicos , Ratones SCID , Conformación Molecular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Maytansine, a potent clinically evaluated plant-derived anti-tumor drug, and its microbial counterpart, ansamitocin P-3, showed a substantially higher cytoxicity than many other anti-tumor drugs. Owing to a shortage of material and lack of sufficiently sensitive analytical methods at the time, no metabolism studies were apparently carried out in conjunction with the initial preclinical and clinical studies on maytansine, but some products of decomposition during the period of storage of the formulated drug were reported. In the current study, the in vitro metabolism of maytansine and ansamitocin P-3 was studied after incubation with rat and human liver microsomes in the presence of NADPH, and with rat and human plasma and whole blood, using liquid chromatography/multi-stage mass spectrometry. Unchanged ansamitocin P-3 and 11 metabolites and unchanged maytansine and seven metabolites were profiled and the structures of some metabolites were tentatively assigned based on their multi-stage electrospray ion-trap mass fragmentation data and in some cases accurate mass measurement. The major pathway of ansamitocin P-3 metabolism in human liver microsomes appears to be demethylation at C-10. Oxidation and sequential oxidation/demethylation also occurred, although to a lesser extent. However, the major pathway of maytansine metabolism in human liver microsomes is N-demethylation of the methylamide of the ester moiety. Several minor pathways including O/N-demethylation, oxidation and hydrolysis of the ester bond were also observed. There were no differences in maytansine metabolism between rat and human liver microsomes; however, the rate of metabolism of ansamitocin P-3 was different in rat and human liver microsomes. About 20% of ansamitocin P-3 was converted to its metabolites in rat liver microsomes and about 70% in human liver microsomes under the same conditions. Additionally, 10-O-demethylated ansamitocin P-3 was also detected in the urine after i.v. bolus administration of ansamitocin P-3 to Sprague-Dawley male rats. No metabolites were detected following incubation of maytansine and ansamitocin P-3 with human and rat whole blood and plasma.