RESUMO
Polyethylene glycol (PEG) was introduced into synthetic bilirubin 3α and a PEGylated bilirubin 3α nanoparticle (BX-001N, Brixelle®) was developed for the first time.An in vitro microsomal stability study, in vivo PK studies with intravenous bolus (IV) and subcutaneous injection (SC), and a semi-mass balance study of BX-001N were investigated to evaluate its pharmacokinetic (PK) properties in male Sprague-Dawley (SD) rats using developed liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-qTOF/MS).Following IV administration at 10 or 30 mg/kg, BX-001N showed very low clearance (0.33-0.67 mL/min/kg) with predominant distribution in the vascular system (Vd = 51.73-83.02 mL/kg). BX-001N was also very stable in vitro liver microsomal stability study.Following SC administration at 10 or 30 mg/kg, the bioavailability of BX-001N in plasma at 10 mg/kg was around 43% and showed the less dose-proportionality at 30 mg/kg dose.BX-001N was mainly excreted via the urinary pathway (86.59-92.99% of total amount of parent drug in excreta; urine and feces) not via the biliary one.
RESUMO
There have been many attempts in pharmaceutical industries and academia to improve the pharmacokinetic characteristics of anti-tumor small-molecule drugs by conjugating them with large molecules, such as monoclonal antibodies, called ADCs. In this context, albumin, one of the most abundant proteins in the blood, has also been proposed as a large molecule to be conjugated with anti-cancer small-molecule drugs. The half-life of albumin is 3 weeks in humans, and its distribution to tumors is higher than in normal tissues. However, few studies have been conducted for the in vivo prepared albumin-drug conjugates, possibly due to the lack of robust bioanalytical methods, which are critical for evaluating the ADME/PK properties of in vivo prepared albumin-drug conjugates. In this study, we developed a bioanalytical method of the albumin-conjugated MAC glucuronide phenol linked SN-38 ((2S,3S,4S,5R,6S)-6-(4-(((((((S)-4,11-diethyl-4-hydroxy-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano [3',4':6,7] indolizino [1,2-b] quinolin-9-yl)oxy)methyl)(2 (methylsulfonyl)ethyl)carbamoyl)oxy)methyl)-2-(2-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylpropanamido)acetamido)phenoxy)-3,4,5-trihydroxytetra-hydro-2H-pyran-2-carboxylic acid) as a proof-of-concept. This method is based on immunoprecipitation using magnetic beads and the quantification of albumin-conjugated drug concentration using LC-qTOF/MS in mouse plasma. Finally, the developed method was applied to the in vivo intravenous (IV) mouse pharmacokinetic study of MAC glucuronide phenol-linked SN-38.
Assuntos
Albuminas , Imunoprecipitação , Irinotecano , Espectrometria de Massa com Cromatografia Líquida , Animais , Humanos , Camundongos , Albuminas/química , Albuminas/farmacocinética , Glucuronidase/metabolismo , Glucuronídeos/química , Glucuronídeos/metabolismo , Imunoprecipitação/métodos , Irinotecano/sangue , Irinotecano/química , Irinotecano/metabolismo , Irinotecano/farmacocinética , Espectrometria de Massa com Cromatografia Líquida/métodos , Magnetismo , Fenol/químicaRESUMO
Preclinical models suggest anticancer activity of IM156, a novel biguanide mitochondrial protein complex 1 inhibitor of oxidative phosphorylation (OXPHOS). This first-in-human dose-escalation study enrolled patients with refractory advanced solid tumors to determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D). Eligible patients received oral IM156 every other day (QOD) or daily (QD) and were assessed for safety, dose-limiting toxicities (DLTs), pharmacokinetics, and preliminary signals of efficacy. 22 patients with advanced cancers (gastric, n = 8; colorectal, n = 3; ovarian, n = 3; other, n = 8) received IM156 100 to 1,200 mg either QOD or QD. There were no DLTs. However, 1,200 mg QD was not well tolerated due to nausea; 800 mg QD was determined as the RP2D. The most frequent treatment-related AEs (TRAEs) were nausea (n = 15; 68%), diarrhea (n = 10; 46%), emesis (n = 9; 41%), fatigue (n = 4; 18%) and abdominal pain, constipation, and blood lactate increased (n = 2 each; 9%). Grade 3 nausea (n = 3; 14%) was the only grade ≥ 3 TRAE. Plasma exposures increased dose proportionally; mean Day 27 area under the curve (AUC0-24) values were higher following QD administration compared to the respective QOD regimen. Stable disease (SD), observed in 7 (32%) patients (confirmed in 2 [9%]), was the best response. To our knowledge, this is the first phase 1 study of an OXPHOS inhibitor that established a RP2D for further clinical development in cancer. Observed AEs of IM156 were manageable and SD was the best response.
Assuntos
Antineoplásicos , Neoplasias , Antineoplásicos/efeitos adversos , Biguanidas/uso terapêutico , Relação Dose-Resposta a Droga , Humanos , Dose Máxima Tolerável , Náusea/induzido quimicamente , Neoplasias/metabolismo , Fosforilação OxidativaRESUMO
Daporinad (FK866) is one of the highly specific inhibitors of nicotinamide phosphoribosyl transferase (NAMPT) and known to have its unique mechanism of action that induces the tumor cell apoptosis. In this study, a simple and sensitive liquid chromatography-quadrupole-time-of-flight-mass spectrometric (LC-qTOF-MS) assay has been developed for the evaluation of drug metabolism and pharmacokinetics (DMPK) properties of Daporinad in mice. A simple protein precipitation method using acetonitrile (ACN) was used for the sample preparation and the pre-treated samples were separated by a C18 column. The calibration curve was evaluated in the range of 1.02~2220 ng/mL and the quadratic regression (weighted 1/concentration2) was used for the best fit of the curve with a correlation coefficient ≥ 0.99. The qualification run met the acceptance criteria of ±25% accuracy and precision values for QC samples. The dilution integrity was verified for 5, 10 and 30-fold dilution and the accuracy and precision of the dilution QC samples were also satisfactory within ±25% of the nominal values. The stability results indicated that Daporinad was stable for the following conditions: short-term (4 h), long-term (2 weeks), freeze/thaw (three cycles). This qualified method was successfully applied to intravenous (IV) pharmacokinetic (PK) studies of Daporinad in mice at doses of 5, 10 and 30 mg/kg. As a result, it showed a linear PK tendency in the dose range from 5 to 10 mg/kg, but a non-linear PK tendency in the dose of 30 mg/kg. In addition, in vitro and in vivo metabolite identification (Met ID) studies were conducted to understand the PK properties of Daporinad and the results showed that a total of 25 metabolites were identified as ten different types of metabolism in our experimental conditions. In conclusion, the LC-qTOF-MS assay was successfully developed for the quantification of Daporinad in mouse plasma as well as for its in vitro and in vivo metabolite identification.
Assuntos
Plasma , Espectrometria de Massas em Tandem , Animais , Calibragem , Cromatografia Líquida/métodos , Camundongos , Espectrometria de Massas em Tandem/métodosRESUMO
The purpose of this study is to investigate the difference of in vitro-in vivo correlation of α-amanitin from clearance perspectives as well as to explore the possibility of extra-hepatic metabolism of α-amanitin. First, a liquid chromatography-quadrupole-time-of-flight-mass spectrometric (LC-qTOF-MS) method for α-amanitin in rat plasma was developed and applied to evaluate the in vitro liver microsomal metabolic stability using rat and human liver microsomes and the pharmacokinetics of α-amanitin in rat. The predicted hepatic clearance of α-amanitin in rat liver microsomes was quite low (5.05 mL/min/kg), whereas its in vivo clearance in rat (14.0 mL/min/kg) was close to the borderline between low and moderate clearance. To find out the difference between in vitro and in vivo metabolism, in vitro and in vivo metabolite identification was also conducted. No significant metabolites were identified from the in vivo rat plasma and the major circulating entity in rat plasma was α-amanitin itself. No reactive metabolites such as GSH-adducts were detected either. A glucuronide metabolite was newly identified from the in vitro liver microsomes samples with a trace level. A semi-mass balance study was also conducted to understand the in vivo elimination pathway of α-amanitin and it showed that most α-amanitin was mainly eliminated in urine as intact which implies some unknown transporters in kidney might play a role in the elimination of α-amanitin in rat in vivo. Further studies with transporters in the kidney would be warranted to figure out the in vivo clearance mechanism of α-amanitin.
Assuntos
Alfa-Amanitina , Microssomos Hepáticos , Ratos , Humanos , Animais , Alfa-Amanitina/metabolismo , Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Microssomos Hepáticos/metabolismo , Plasma , Cromatografia Líquida de Alta Pressão/métodosRESUMO
Sulfasalazine (SAS), an anti-inflammatory drug with potent cysteine/glutamate antiporter system xc-(SXC) inhibition has recently shown beneficial effects in brain-related diseases. Despite many reports related to central nervous system (CNS) effect of SAS, pharmacokinetics (PK) and metabolite identification studies in the brain for SAS were quite limited. The aim of this study was to investigate the pharmacokinetics and metabolite identification of SAS and their distributions in mouse brain. Using in vivo brain exposure studies (neuro PK), the PK parameters of SAS was calculated for plasma as well as brain following intravenous and oral administration at 10 mg/kg and 50 mg/kg in mouse, respectively. In addition, in vivo metabolite identification (MetID) studies of SAS in plasma and brain were also conducted. The concentration of SAS in brain was much lower than that in plasma and only 1.26% of SAS was detected in mouse brain when compared to the SAS concentration in plasma (brain to plasma ratio (%): 1.26). In the MetID study, sulfapyridine (SP), hydroxy-sulfapyridine (SP-OH), and N-acetyl sulfapyridine (Ac-SP) were identified in plasma, whereas only SP and Ac-SP were identified as significant metabolites in brain. As a conclusion, our results suggest that the metabolites of SAS such as SP and Ac-SP might be responsible for the pharmacological effect in brain, not the SAS itself.
Assuntos
Encéfalo/metabolismo , Sulfassalazina/análise , Sulfassalazina/metabolismo , Animais , Masculino , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos ICR , Sulfassalazina/farmacocinética , Fatores de TempoRESUMO
Ticagrelor (TCG) has been used as an antiplatelet agent for acute coronary syndrome patients. The aim of this research was to establish a population pharmacokinetic/pharmacodynamic (PK/PD) model of TCG and to apply the model for predicting the PD response of the TCG-loaded self-microemulsifying drug delivery system (TCG-SME) in rats. Pure TCG and TCG-SME (2, 5, and 10 mg/kg of TCG) were orally administered to male Sprague-Dawley rats. Plasma samples were collected at scheduled time-points and then analyzed for TCG plasma concentrations and antiplatelet effects. The inhibition of platelet aggregation of TCG was measured as a PD response. The PK profiles of pure TCG and TCG-SME could be well-explained with a two-compartment PK model. The accuracy of the PK model was assessed with a goodness-of-fit plot and conditional weight residual error (CWRES). Also, the visual predictive check was investigated based on the predictions. A population PK/PD model for pure TCG was established as an indirect response Emax model linked to the two-compartment PK model of pure TCG. The PK/PD model proposed a suitable fitting to link the plasma concentration of TCG simultaneously with platelet aggregation. Based on the PK data of TCG-SME, as well as the established PK/PD model of pure TCG, the PD profiles of TCG-SME were simulated. TCG-SME was more effective in inducing the antiplatelet effect than pure TCG at equivalent doses of TCG. The accuracy of the simulation was verified by comparing the simulated PD profile with the profile observed in rats. The observations were close to the model simulations. In addition, the values of CWRES were almost within ±2. In conclusion, the PK/PD modeling approach can provide a way for predicting mathematically the PD responses from PK profiles of other TCG formulations and a conceptual prediction for future clinical assessment.
Assuntos
Plaquetas/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Inibidores da Agregação Plaquetária/farmacocinética , Ticagrelor/farmacologia , Ticagrelor/farmacocinética , Animais , Sistemas de Liberação de Medicamentos/métodos , Masculino , Modelos Biológicos , Agregação Plaquetária/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
MMAE is a potent antimitotic drug used as payload of an antibody-drug conjugate which shows potent activity in preclinical and clinical studies against a range of lymphomas, leukemia and solid tumors. Liquid chromatography-high resolution mass spectrometric method was developed for the quantification of MMAE and its preclinical pharmacokinetics. The method consisted of protein precipitation using acetonitrile (ACN) for sample preparation and liquid chromatography - quadrupole - time-of-flight - tandem mass spectrometry (LC-qTOF-MS/MS) analysis in the positive ion mode. A quadratic regression (weighted 1/concentration2 ), with an equation y = ax2 + bx + c, was used to fit calibration curves over the concentration range of 1.01-2,200 ng/mL for MMAE. The qualification run met the acceptance criteria of ±25% accuracy and precision values for QC samples. Recovery was 42.84%. The dilution integrity was determined for 5-fold dilution and the accuracy and precision ranged within ±25%. The stability results indicated that MMAE was stable for the following conditions: short-term (4 h), long-term (4 weeks), freeze/thaw (3 cycles) and post-preparative stability (12 h). This qualified method was successfully applied to a pharmacokinetic study of MMAE in rat as a preclinical animal model. The PK results suggest that MMAE has moderate CL and low BA.Also, these results would be helpful in having a comprehensive understanding of the PK characteristics of MMAE and developing ADC in future.
Assuntos
Cromatografia Líquida/métodos , Espectrometria de Massas/métodos , Oligopeptídeos/sangue , Oligopeptídeos/farmacocinética , Animais , Modelos Animais de Doenças , Imunoconjugados , Modelos Lineares , Masculino , Oligopeptídeos/administração & dosagem , Oligopeptídeos/química , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
5-Amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo(4,3-e)-1,2,4-triazolo(1,5-c) pyrimidine (SCH 58261) is one of the new chemical entities that has been developed as an adenosine A2A receptor antagonist. Although SCH 58261 has been reported to be beneficial, there is little information about SCH 58261 from a drug metabolism or pharmacokinetics perspective. This study describes the metabolism and pharmacokinetic properties of SCH 58261 in order to understand its behaviors in vivo. Rats were used as the in vivo model species. First, an LC-MS/MS method was developed for the determination of SCH 58261 in rat plasma. A GastroPlus™ simulation, in vitro microsomal metabolic stability, and bile duct-cannulated studies were also performed to understand its pharmacokinetic profile. The parameter sensitivity analysis of GastroPlus™ was used to examine the factors that influence exposure when the drug is orally administered. The factors are as follows: permeability, systemic clearance, renal clearance, and liver first-pass effect. In vitro microsomal metabolic stability indicates how much the drug is metabolized. The extrapolated hepatic clearance value of SCH 58261 was 39.97 mL/min/kg, indicating that the drug is greatly affected by hepatic metabolism. In vitro microsomal metabolite identification studies revealed that metabolites produce oxidized and ketone-formed metabolites via metabolic enzymes in the liver. The bile duct-cannulated rat study, after oral administration of SCH 58261, showed that a significant amount of the drug was excreted in feces. These results imply that the drug is not absorbed well in the body after oral administration. Taken together, SCH 58261 showed quite a low bioavailability when administered orally and this was likely due to significantly limited absorption, as well as high metabolism in vivo.
Assuntos
Antagonistas de Receptores Purinérgicos P1 , Pirimidinas , Espectrometria de Massas em Tandem , Triazóis , Animais , Disponibilidade Biológica , Cromatografia Líquida , Fígado/metabolismo , Masculino , Microssomos Hepáticos/metabolismo , Antagonistas de Receptores Purinérgicos P1/química , Antagonistas de Receptores Purinérgicos P1/farmacocinética , Antagonistas de Receptores Purinérgicos P1/farmacologia , Pirimidinas/química , Pirimidinas/farmacocinética , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Triazóis/química , Triazóis/farmacocinética , Triazóis/farmacologiaRESUMO
The novel prenyl transferase-mediated, site-specific, antibody-drug conjugate LCB14-0110 is comprised of a proprietary beta-glucuronide linker and a payload (Monomethyl auristatin F, MMAF, an inhibitor for tubulin polymerization) attached to human epidermal growth factor receptor 2 (HER2)-targeting trastuzumab. A LC-MS/MS method was developed to quantify the antibody-conjugated drug (acDrug) for in vitro linker stability and preclinical pharmacokinetic studies. The method consisted of affinity capture, enzymatic cleavage of acDrug, and LC-MS/MS analysis in the positive ion mode. A quadratic regression (weighted 1/concentration2), with the equation y = ax2 + bx + c, was used to fit calibration curves over the concentration range of 19.17~958.67 ng/mL for acDrug. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control (QC) samples. The overall recovery was 42.61%. The dilution integrity was for a series of 5-fold dilutions with accuracy and precision values ranging within ±25%. The stability results indicated that acDrug was stable at all stability test conditions (short-term: 1 day, long-term: 10 months, Freeze/Thaw (F/T): 3 cycles). This qualified method was successfully applied to in vitro linker stability and pharmacokinetic case studies of acDrug in rats.
Assuntos
Cromatografia Líquida , Imunoconjugados/química , Imunoconjugados/farmacocinética , Neopreno , Espectrometria de Massas em Tandem , Transferases , Animais , Monitoramento de Medicamentos , Estabilidade de Medicamentos , Humanos , Estrutura Molecular , Neopreno/química , Ratos , Transferases/química , Trastuzumab/química , Trastuzumab/farmacocinéticaRESUMO
Parkinson's disease is one of the most common neurodegenerative diseases. Adenosine regulates the response to other neurotransmitters in the brain regions related to motor function. In the several subtypes of adenosine receptors, especially, adenosine 2A receptors (A2ARs) are involved in neurodegenerative conditions. ZM241385 is one of the selective non-xanthine A2AR antagonists with high affinity in the nanomolar range. This study describes the in vitro and in vivo pharmacokinetic properties of ZM241385 in rats. A liquid chromatography-quadrupole time-of-flight mass spectrometric (LC-qToF MS) method was developed for the determination of ZM241385 in rat plasma. In vivo IV administration studies showed that ZM241385 was rapidly eliminated in rats. However, the result of in vitro metabolic stability studies showed that ZM241385 had moderate clearance, suggesting that there is an extra clearance pathway in addition to hepatic clearance. In addition, in vivo PO administration studies demonstrated that ZM241385 had low exposure in rats. The results of semi-mass balance studies and the in silico PBPK modeling studies suggested that the low bioavailability of ZM241385 after oral administration in rats was due to the metabolism and by liver, kidney, and gut.
Assuntos
Antagonistas do Receptor A2 de Adenosina , Simulação por Computador , Triazinas , Triazóis , Antagonistas do Receptor A2 de Adenosina/farmacocinética , Antagonistas do Receptor A2 de Adenosina/farmacologia , Animais , Masculino , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Ratos , Ratos Sprague-Dawley , Receptor A2A de Adenosina/metabolismo , Triazinas/farmacocinética , Triazinas/farmacologia , Triazóis/farmacocinética , Triazóis/farmacologiaRESUMO
A simple liquid chromatography-quadrupole-time-of-flight-mass spectrometric assay (LC-TOF-MS/MS) has been developed for the evaluation of metabolism and pharmacokinetic (PK) characteristics of monomethyl auristatin F (MMAF) in rat, which is being used as a payload for antibody-drug conjugates. LC-TOF-MS/MS method was qualified for the quantification of MMAF in rat plasma. The calibration curves were acceptable over the concentration range from 3.02 to 2200 ng/mL using quadratic regression. MMAF was stable in various conditions. There were no significant matrix effects between rat and other preclinical species. The PK studies showed that the bioavailability of MMAF was 0% with high clearance. Additionally, the metabolite profiling studies, in vitro/in vivo, were performed. Seven metabolites for MMAF were tentatively identified in liver microsome. The major metabolic pathway was demethylation, which was one of the metabolic pathways predicted by MedChem Designer. Therefore, these results will be helpful to understand the PK, catabolism, and metabolism behavior of MMAF comprehensively when developing antibody-drug conjugates (ADCs) in the future.
Assuntos
Cromatografia Líquida , Metabolômica , Oligopeptídeos/farmacocinética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Animais , Biomarcadores , Cromatografia Líquida/métodos , Monitoramento de Medicamentos , Humanos , Masculino , Redes e Vias Metabólicas , Metabolômica/métodos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Ratos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
Tozadenant is one of the selective adenosine A2a receptor antagonists with a potential to be a new Parkinson's disease (PD) therapeutic drug. In this study, a liquid chromatography-mass spectrometry based bioanalytical method was qualified and applied for the quantitative analysis of tozadenant in rat plasma. A good calibration curve was observed in the range from 1.01 to 2200 ng/mL for tozadenant using a quadratic regression. In vitro and preclinical in vivo pharmacokinetic (PK) properties of tozadenant were studied through the developed bioanalytical methods, and human PK profiles were predicted using physiologically based pharmacokinetic (PBPK) modeling based on these values. The PBPK model was initially optimized using in vitro and in vivo PK data obtained by intravenous administration at a dose of 1 mg/kg in rats. Other in vivo PK data in rats were used to validate the PBPK model. The human PK of tozadenant after oral administration at a dose of 240 mg was simulated by using an optimized and validated PBPK model. The predicted human PK parameters and profiles were similar to the observed clinical data. As a result, optimized PBPK model could reasonably predict the PK in human.
Assuntos
Benzotiazóis/sangue , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Antagonistas do Receptor A2 de Adenosina , Animais , Benzotiazóis/farmacocinética , Ratos , Verapamil/sangue , Verapamil/farmacocinéticaRESUMO
RATIONALE: The cassette-dosing technique is a technique that administers various drugs to a single animal at once and quantitated simultaneously. The purpose of this study was to evaluate the feasibility of cassette-dosing as a means of increasing throughput and decreasing animal usage for pharmacokinetic studies of biopharmaceuticals using liquid chromatography/time-of-flight mass spectrometric (LC/TOF-MS) analysis. METHODS: Brentuximab, trastuzumab, cetuximab and adalimumab were used as model biopharmaceuticals. The method consisted of immunoprecipitation followed by tryptic digestion for sample preparation and LC/TOF-MS analysis of specific signature peptides in the positive ion mode using electrospray ionization. The specific signature peptides used for quantification were from the complementarity-determining regions of each mAb. All rats received a single intravenous bolus injection containing either a single mAb or a mixture of four mAbs. RESULTS: The proposed method has been qualified in linearity range of 1-100 µg/mL with correlation coefficients higher than 0.990. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control (QC) samples. This qualified LC/TOF-MS method was successfully applied to a pharmacokinetic study in the rat. The PK properties of mAbs administered as a cassette-dosage were similar to the pharmacokinetics of each antibody drug when administered as a single entity. CONCLUSIONS: These findings suggest that the cassette-dosing approach could be used to evaluate the PK properties of biopharmaceuticals in the early drug discovery stage. Also, this method would be useful for other preclinical sample analysis without developing new reagents for sample preparation.
Assuntos
Anticorpos Monoclonais Humanizados/sangue , Cromatografia Líquida/métodos , Espectrometria de Massas em Tandem/métodos , Adalimumab/administração & dosagem , Adalimumab/sangue , Animais , Anticorpos Monoclonais Humanizados/administração & dosagem , Brentuximab Vedotin , Calibragem , Cetuximab/administração & dosagem , Cetuximab/sangue , Imunoconjugados/administração & dosagem , Imunoconjugados/sangue , Limite de Detecção , Camundongos , Controle de Qualidade , Ratos Sprague-Dawley , Sensibilidade e Especificidade , Trastuzumab/administração & dosagem , Trastuzumab/sangueRESUMO
A liquid chromatographic-electrospray ionization-time-of-flight/mass spectrometric (LC-ESI-TOF/MS) method was developed and applied for the determination of WKYMVm peptide in rat plasma to support preclinical pharmacokinetics studies. The method consisted of micro-elution solid-phase extraction (SPE) for sample preparation and LC-ESI-TOF/MS in the positive ion mode for analysis. Phenanthroline (10 mg/mL) was added to rat blood immediately for plasma preparation followed by addition of trace amount of 2 m hydrogen chloride to plasma before SPE for stability of WKYMVm peptide. Then sample preparation using micro-elution SPE was performed with verapamil as an internal standard. A quadratic regression (weighted 1/concentration2 ), with the equation y = ax2 + bx + c was used to fit calibration curves over the concentration range of 3.02-2200 ng/mL for WKYMVm peptide. The quantification run met the acceptance criteria of ±25% accuracy and precision values. For quality control samples at 15, 165 and 1820 ng/mL from the quantification experiment, the within-run and the between-run accuracy ranged from 92.5 to 123.4% with precision values ≤15.1% for WKYMVm peptide from the nominal values. This novel LC-ESI-TOF/MS method was successfully applied to evaluate the pharmacokinetics of WKYMVm peptide in rat plasma.
Assuntos
Cromatografia Líquida/métodos , Oligopeptídeos/sangue , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Modelos Lineares , Oligopeptídeos/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
A single hybrid affinity-captured-LC-TOF-MS/MS method was developed and applied for the quantification of total antibody, antibody conjugated drug and free payload of antibody drug conjugate (ADC). Adcetris®, a valine-citrulline monomethyl auristatin E conjugated ADC, was used as a model ADC compound. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 30.65-613.00 ng/mL with an equation y = ax2 + bx + c for the antibody-conjugated drug of Adcetris®. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control samples. For the analysis of total antibody, a signature peptide (TTPPVLDSDGSFFLYSK, molecular weight 1874) was used after affinity capture using magnetic beads and on-bead trypsin digestion. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 5.00-100.00 µg/mL with an equation y = ax2 + bx + c for total antibody. For free payload analysis of monomethyl auristatin E, a protein precipitation method followed by LC-TOF-MS/MS analysis was used. A quadratic regression (weighted 1/concentration) was used to fit calibration curves over the concentration range 1.01-2200 ng/mL with an equation y = ax2 + bx + c for free payload. Pharmacokinetic study samples and in vitro stability samples in rat were successfully analyzed by this a hybrid affinity-captured-LC-TOF-MS/MS method. This single platform method is a useful complementary method for the pharmacokinetics study of ADC with valine-citrulline linker at the early drug discovery stage.
Assuntos
Cromatografia Líquida/métodos , Imunoconjugados/análise , Imunoconjugados/química , Espectrometria de Massas em Tandem/métodos , Animais , Brentuximab Vedotin , Estabilidade Proteica , Ratos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Over the last decade, physiologically based pharmacokinetics (PBPK) application has been extended significantly not only to predicting preclinical/human PK but also to evaluating the drug-drug interaction (DDI) liability at the drug discovery or development stage. Herein, we describe a case study to illustrate the use of PBPK approach in predicting human PK as well as DDI using in silico, in vivo and in vitro derived parameters. This case was composed of five steps such as: simulation, verification, understanding of parameter sensitivity, optimization of the parameter and final evaluation. Caffeine and ciprofloxacin were used as tool compounds to demonstrate the "fit for purpose" application of PBPK modeling and simulation for this study. Compared to caffeine, the PBPK modeling for ciprofloxacin was challenging due to several factors including solubility, permeability, clearance and tissue distribution etc. Therefore, intensive parameter sensitivity analysis (PSA) was conducted to optimize the PBPK model for ciprofloxacin. Overall, the increase in Cmax of caffeine by ciprofloxacin was not significant. However, the increase in AUC was observed and was proportional to the administered dose of ciprofloxacin. The predicted DDI and PK results were comparable to observed clinical data published in the literatures. This approach would be helpful in identifying potential key factors that could lead to significant impact on PBPK modeling and simulation for challenging compounds.
RESUMO
An liquid chromatography-quadrupole time-of-flight (QqTOF) mass spectrometric method was developed for the determination of humanized or human monoclonal antibodies in rat plasma at the early drug discovery stage. Trastuzumab was used as a model monoclonal antibody. The method consisted of immunoprecipitation followed by tryptic digestion for sample preparation and LC-TOF-MS/MS analysis of specific signature peptides in the positive ion mode using electrospray ionization for analysis. A stable isotope-labeled signature peptide was also used as internal standard. A quadratic regression (weighted 1/concentration(2) ), with an equation y = ax(2) + bx + c, was used to fit calibration curves over the concentration range of 0.500-100 µg/mL for trastuzumab. Samples from a pharmacokinetic study in rat were analyzed by this qualified LC-TOF-MS/MS method and concentrations were compared with those generated by enzyme linked immunosorbent assays method. The LC-TOF-MS/MS method was accurate and precise, with quantitative results comparable with those of ELISA. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control samples. Within-run accuracy ranged from 1.53 to 9.20% with precision values ≤10.29%. This LC-TOF-MS/MS method approach could be used as a complementary method for humanized or human monoclonal antibodies at the early drug discovery stage.
Assuntos
Antineoplásicos/sangue , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas em Tandem/métodos , Trastuzumab/sangue , Animais , Calibragem , Cães , Humanos , Limite de Detecção , Macaca fascicularis , Camundongos , Ratos Sprague-DawleyRESUMO
A liquid chromatography-triple quadrupole mass spectrometric (LC-MS/MS) method was developed and validated for the determination of 5-nitro-5'-hydroxy-indirubin-3'-oxime (AGM-130) in human plasma to support a microdose clinical trial. The method consisted of a liquid-liquid extraction for sample preparation and LC-MS/MS analysis in the positive ion mode using TurboIonSpray(TM) for analysis. d3 -AGM-130 was used as the internal standard. A linear regression (weighted 1/concentration) was used to fit calibration curves over the concentration range of 10-2000 pg/mL for AGM-130. There were no endogenous interference components in the blank human plasma tested. The accuracy at the lower limit of quantitation was 96.6% with a precision (coefficient of variation, CV) of 4.4%. For quality control samples at 30, 160 and 1600 pg/mL, the between run CV was ≤5.0 %. Between-run accuracy ranged from 98.1 to 101.0%. AGM-130 was stable in 50% acetonitrile for 168 h at 4°C and 6 h at room temperature. AGM-130 was also stable in human plasma at room temperature for 6 h and through three freeze-thaw cycles. The variability of selected samples for the incurred sample reanalysis was ≤12.7% when compared with the original sample concentrations. This validated LC-MS/MS method for determination of AGM-130 was used to support a phase 0 microdose clinical trial.
Assuntos
Indóis/administração & dosagem , Indóis/sangue , Oximas/administração & dosagem , Oximas/sangue , Cromatografia Líquida/métodos , Quinases Ciclina-Dependentes/antagonistas & inibidores , Feminino , Humanos , Indóis/farmacocinética , Modelos Lineares , Extração Líquido-Líquido , Masculino , Oximas/farmacocinética , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/sangue , Inibidores de Proteínas Quinases/farmacocinética , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/métodosRESUMO
Characterization and prediction of the pharmacokinetics (PK) and pharmacodynamics (PD) of Antibody-Drug Conjugates (ADCs) is challenging, since it requires simultaneous quantitative understanding about the PK-PD properties of three different molecular species i.e., the monoclonal antibody, the drug, and the conjugate. Mathematical modeling and simulation provides an excellent tool to overcome these challenges, as it can simultaneously integrate the PK-PD of ADCs and their components in a quantitative manner. Additionally, the computational PK-PD models can also serve as a cornerstone for the model-based drug development and preclinical-to-clinical translation of ADCs. To provide an overview of this subject matter, this manuscript reviews the PK-PD models applicable to ADCs. Additionally, the usage of these models during different drug development stages (i.e., discovery, preclinical development, and clinical development) is also emphasized. The importance of PK-PD modeling and simulation in making rationale go/no-go decisions throughout the drug development process is also highlighted. There is an array of PK-PD models available, ranging from the systems models specifically developed for ADCs to the empirical models applicable to all chemotherapeutic agents, which one can employ for ADCs. The decision about which model to choose depends on the questions to be answered, time at hand, and resources available.