Quantification and Metabolite Identification of Sulfasalazine in Mouse Brain and Plasma Using Quadrupole-Time-of-Flight Mass Spectrometry.

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.

Qualification and application of liquid chromatography-quadrupole time-of-flight mass spectrometric method for the determination of carisbamate in rat plasma and prediction of its human pharmacokinetics using physiologically based pharmacokinetic modeling.

Carisbamate is an antiepileptic drug and it also has broad neuroprotective activity and anticonvulsant reaction. In this study, a liquid chromatography-quadrupole time-of-flight mass spectrometric (LC-qTOF-MS) method was developed and applied for the determination of carisbamate in rat plasma to support in vitro and in vivo studies. A quadratic regression (weighted 1/concentration2), with an equation y = ax2 + bx + c, was used to fit calibration curves over the concentration range from 9.05 to 6,600 ng/mL for carisbamate in rat plasma. Preclinical in vitro and in vivo studies of carisbamate have been studied through the developed bioanalytical method. Based on these study results, human pharmacokinetic (PK) profile has been predicted using physiologically based pharmacokinetic (PBPK) modeling. The PBPK model was optimized and validated by using the in vitro and in vivo data. The human PK of carisbamate after oral dosing of 750 mg was simulated by using this validated PBPK model. The human PK parameters and profiles predicted from the validated PBPK model were similar to the clinical data. This PBPK model developed from the preclinical data for carisbamate would be useful for predicting the PK of carisbamate in various clinical settings.

Profiling and Identification of Omeprazole Metabolites in Mouse Brain and Plasma by Isotope Ratio-Monitoring Liquid Chromatography-Mass Spectrometric Method.

Neuro-inflammation is known to be one of the pathogenesis for the degenerative central nervous system (CNS) disease. Recently various approaches for the treatment of brain diseases by controlling neuro-inflammation in the brain have been introduced. In this respect, there is a continuous demand for CNS drugs, which could be safer and more effective. Omeprazole, a well-known proton-pump inhibitor (PPI) is generally prescribed for the treatment of peptic ulcer. In addition to the anti-gastric acid secretion mechanism, recent studies showed that omeprazole or PPIs would likely have anti-inflammation effects in vitro and in vivo, but their effects on anti-inflammation in brain are still unknown. In this study, omeprazole and its metabolites in a mouse's brain after various routes of administration have been explored by stable isotope ratio-patterning liquid chromatography-mass spectrometric method. First, a simple liquid chromatography-mass spectrometric (LC-MS) method was established for the quantification of omeprazole in mouse plasma and brain. After that, omeprazole and its stable isotope (D3-omeprazole) were concomitantly administered through various routes to mice in order to identify novel metabolites characteristically observed in the mouse brain and were analyzed using a different LC-MS method with information-dependent analysis (IDA) scan. With this unique approach, several new metabolites of omeprazole were identified by the mass difference between omeprazole and stable isotope in both brain and plasma samples. A total of seventeen metabolites were observed, and the observed metabolites were different from each administration route or each matrix (brain or plasma). The brain pharmacokinetic profiles and brain-to-plasma partition coefficient (Kp) were also evaluated in a satellite study. Overall, these results provide better insights to understand the CNS-related biological effects of omeprazole and its metabolites in vivo.

Assessment of Pharmacokinetics and Metabolism Profiles of SCH 58261 in Rats Using Liquid Chromatography-Mass Spectrometric Method.

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.

Liquid chromatography-high resolution mass spectrometric method for the quantification of monomethyl auristatin E (MMAE) and its preclinical pharmacokinetics.

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.

Quantification of an Antibody-Conjugated Drug in Fat Plasma by an Affinity Capture LC-MS/MS Method for a Novel Prenyl Transferase-Mediated Site-Specific Antibody-Drug Conjugate.

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.

Development and evaluation of TPGS/PVA-based nanosuspension for enhancing dissolution and oral bioavailability of ticagrelor.

In this study, we developed ticagrelor-dispersed nanosuspension (TCG-NSP) to enhance the dissolution and oral bioavailability of ticagrelor (TCG) through a statistical design approach. TCG, a reversible P2Y12 receptor antagonist, is classified as a biopharmaceutics classification system (BCS) class IV drug with low solubility and permeability, resulting in low oral bioavailability. Nanosuspension (NSP) is an efficient pharmaceutical technique for overcoming the disadvantages. First, we optimized TCG-NSP consisting of D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) and polyvinyl alcohol (PVA), which exhibited homogeneously dispersed TCG particle (233 nm) and low precipitation (3%). Characterization studies demonstrated that TCG-NSP provided amorphous TCG particles and supersaturation effect, resulting in higher dissolution than a commercial product. In addition, everted gut sac and pharmacokinetic studies confirmed that TCG-NSP improved the gastrointestinal permeation of TCG by 2.8-fold compared to commercial product, thereby enhancing the oral bioavailability (2.2-fold). These results suggested that TCG-NSP could be successfully used as an efficient pharmaceutical formulation to achieve the enhanced dissolution and oral bioavailability of TCG.

In Vitro, In Silico, and In Vivo Assessments of Pharmacokinetic Properties of ZM241385.

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.

Pharmacokinetic/Pharmacodynamic Modeling To Predict the Antiplatelet Effect of the Ticagrelor-Loaded Self-Microemulsifying Drug Delivery System in Rats.

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.

Development and evaluation of a film-forming system hybridized with econazole-loaded nanostructured lipid carriers for enhanced antifungal activity against dermatophytes.

Treatment of skin infection by dermatophytes is still limited, and the application of conventional topical formulations (ointments, creams, etc.) cause patient discomfort due to repeated administration and low efficacy. This study describes the film-forming system (FFS) hybridized with econazole (ECO)-loaded nanostructured lipid carriers (NLC) for enhanced antifungal activity against dermatophytes. We assumed that the application of NLC could effectively increase the skin permeability of ECO, thereby suppressing the growth of dermatophytes in stratum corneum as well as in epidermis. Meanwhile, ECO-NLC hybrid FFS (ECO-NLC@FFS) could increase the adhesion of ECO-NLC to the skin and prolong the antifungal activity of ECO. First, we optimized ECO-NLC, which shows nanosized particle (199 nm), high encapsulation efficiency (92.5%), and biocompatibility. ECO-NLC@FFS formed a transparent, homogeneous, and hard-to-remove film after topical application. In vitro skin permeation and deposition studies demonstrated that ECO-NLC@FFS showed 1.5-fold higher skin permeation and 3-fold higher ECO deposition in the epidermis layer than a commercial product, which resulted from the nanosized particle and its occlusion effect. And, ex vivo and in vivo antifungal activity studies confirmed that ECO-NLC@FFS improved the skin adhesion of ECO-NLC, thereby allowing ECO to be continuously exposed to the infection sited and reducing the number of applications with a single dose. These results showed that this hybrid system could be a potential for effectively improving the efficacy of antifungal agents and the patient compliance in the treatment of dermatophytes. STATEMENT OF SIGNIFICANCE: Treatment of skin infection by dermatophytes is difficult due to the inconvenience and low efficacy of conventional topical formulations. Here, we demonstrated the potential of a film-forming system (FFS) hybridized with nanostructured lipid carriers (NLC). First, we confirmed that the enhanced skin permeability of drug was improved by NLC. In addition, the hybridization of NLC with FFS improved the skin adhesion of NLC, allowing the drug to exhibit a sustained release profile and prolong antifungal activity. Given the maximized antifungal activity, this hybrid system can be used as a potential pharmaceutical technique to improve patient convenience and achieve complete treatment of skin infection.

Statistical approach for solidifying ticagrelor loaded self-microemulsifying drug delivery system with enhanced dissolution and oral bioavailability.

The aim of this study was to solidify a ticagrelor loaded self-microemulsifying drug delivery system (TCG-SM) with enhanced dissolution and bioavailability of ticagrelor (TCG) for developing TCG-SM granules and tablets. TCG was dissolved in the self-microemulsifying drug delivery system (SMEDDS) and TCG-SM was solidified by adsorption to the optimized adsorbent through statistical design. In order to select an appropriate adsorbent, the physical properties (bulk density, tapped density, angle of repose, and liquid adsorption capacity) of silica-based adsorbents (Neusilin US2, Florite R, Aerosil 200, and Florite PS-10) and non silica-based adsorbents (Avicel PH102, Pharmatose 100M, Pearlitol 200, LH-11, and Emcompress) were investigated. Neusilin US2 and Florite R were selected as suitable adsorbents and their mixing ratios were optimized using statistical experimental design. The predicted values of physical properties by statistical design showed the error percentage of <10% compared to actual values. As a result of the statistical approach, TCG-SM (490 mg) was successfully solidified with Nesulin US2 (167.8 mg) and Florite R (82.2 mg), which showed good powder properties and improved dissolution of TCG. The solidified TCG-SM (Sol-TCG-SM), disintegrant (croscarmellose sodium), diluent (microcrystalline cellulose), binder (polyvinylpyrrolidone), and lubricant (magnesium stearate) were mixed to prepare granules. And, the granules with total weight of 900 mg were tableted using 16 mm oval-shape punch. The prepared Sol-TCG-SM tablet showed good tablet properties and maintained self-microemulsifying ability, such as microemulsion formation and enhanced dissolution of TCG. In vivo pharmacokinetic study, the relative bioavailability of Sol-TCG-SM exhibited 108.1% and 632.7% compared to TCG-SM and raw TCG powder, respectively. In conclusion, we successfully solidified SMEDDS with improved oral bioavailability of insoluble drugs such as TCG through a statistical design. This suggests a new approach that can be utilized in the production of solidified SMEDDS.

Pharmacokinetic and Metabolism Studies of Monomethyl Auristatin F via Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry.

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.

Effect of Ticagrelor, a Cytochrome P450 3A4 Inhibitor, on the Pharmacokinetics of Tadalafil in Rats.

Tadalafil is a cytochrome P450 (CYP) 3A4 substrate. Because there are few data on drug-drug interactions, it is advisable to take sufficient consideration when co-administering tadalafil with CYP3A4 inducers or inhibitors. This study was conducted to assess the effect of ticagrelor, a CYP3A4 inhibitor, on the pharmacokinetic properties of tadalafil after oral administration to rats. A total of 20 Sprague-Dawley male rats were randomly divided into the non-pretreated group and ticagrelor-pretreated group, and tadalafil was orally administered to each group after pretreatment with or without ticagrelor. Blood samples were collected at predetermined time points after oral administration of tadalafil. As a result, systemic exposure of tadalafil in the ticagrelor-pretreated group was significantly increased compared to the non-pretreated group (1.61-fold), and the clearance of tadalafil in the ticagrelor-pretreated group was significantly reduced than the non-pretreated group (37%). The prediction of the drug profile through the one-compartment model could explain the differences of pharmacokinetic properties of tadalafil in the non-pretreated and ticagrelor-pretreated groups. This study suggests that ticagrelor reduces a CYP3A-mediated tadalafil metabolism and that tadalafil and a combination regimen with tadalafil and ticagrelor requires dose control and specific pharmacotherapy.

Systemic Design and Evaluation of Ticagrelor-Loaded Nanostructured Lipid Carriers for Enhancing Bioavailability and Antiplatelet Activity.

Ticagrelor (TGL), a P2Y12 receptor antagonist, is classified as biopharmaceutics classification system (BCS) class IV drug due to its poor solubility and permeability, resulting in low oral bioavailability. Nanostructured lipid carriers (NLC) are an efficient delivery system for the improvement of bioavailability of BCS class IV drugs. Hence, we prepared TGL-loaded NLC (TGL-NLC) to enhance the oral bioavailability and antiplatelet activity of TGL with a systemic design approach. The optimized TGL-NLC with Box-Behnken design showed a small particle size of 87.6 nm and high encapsulation efficiency of 92.1%. Scanning electron microscope (SEM), differential scanning calorimetry (DSC), and powder X-ray diffraction (PXRD) were performed to investigate the characteristics of TGL-NLC. Furthermore, TGL-NLC exhibited biocompatible cytotoxicity against Caco-2 cells. Cellular uptake of TGL-NLC was 1.56-fold higher than that of raw TGL on Caco-2 cells. In pharmacokinetic study, the oral bioavailability of TGL-NLC was 254.99% higher than that of raw TGL. In addition, pharmacodynamic study demonstrated that the antiplatelet activity of TGL-NLC was superior to that of raw TGL, based on enhanced bioavailability of TGL-NLC. These results suggest that TGL-NLC can be applied for efficient oral absorption and antiplatelet activity of TGL.

Quantitative Analysis of Tozadenant Using Liquid Chromatography-Mass Spectrometric Method in Rat Plasma and Its Human Pharmacokinetics Prediction Using Physiologically Based Pharmacokinetic Modeling.

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.

Strategic approach to developing a self-microemulsifying drug delivery system to enhance antiplatelet activity and bioavailability of ticagrelor.

BACKGROUND: Ticagrelor (TCG) is used to inhibit platelet aggregation in patients with acute coronary syndrome, but its poor solubility and low bioavailability limit its in vivo efficacy. The purpose of this study was to manufacture an optimized TCG-loaded self-microemulsifying drug delivery system (SMEDDS) to enhance the oral bioavailability and antiplatelet activity of TCG. MATERIALS AND METHODS: Solubility and emulsification tests were conducted to determine the most suitable oils, surfactants, and cosurfactants. Scheffé's mixture design was applied to optimize the percentage of each component applied in the SMEDDS formulation to achieve optimal physical characteristics, ie, high solubility of TCG in SMEDDS, small droplet size, low precipitation, and high transmittance. RESULTS: The optimized TCG-loaded SMEDDS (TCG-SM) formulation composed of 10.0% Capmul MCM (oil), 53.8% Cremophor EL (surfactant), and 36.2% Transcutol P (cosurfactant) significantly improving the dissolution of TCG in various media compared with TCG in Brilinta® (commercial product). TCG-SM exhibited higher cellular uptake and permeability in Caco-2 cells than raw TCG suspension. In pharmacokinetic studies in rats, TCG-SM exhibited higher oral bioavailability with 5.7 and 6.4 times higher area under the concentration-time curve and maximum plasma concentration, respectively, than a raw TCG suspension. Antiplatelet activity studies exhibited that the TCG-SM formulation showed significantly improved inhibition of platelet aggregation compared with raw TCG at the same dose of TCG. And, a 10 mg/kg dose of raw TCG suspension and a 5 mg/kg dose of TCG-SM had a similar area under the inhibitory curve (907.0%±408.8% and 907.8%±200.5%⋅hours, respectively) for antiplatelet activity. CONCLUSION: These results suggest that the developed TCG-SM could be successfully used as an efficient method to achieve the enhanced antiplatelet activity and bioavailability of TCG.

Analysis of Vipadenant and Its In Vitro and In Vivo Metabolites via Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry.

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 (PK) properties of vipadenant in rat, a selective A2a receptor antagonist as one of the novel immune checkpoint inhibitors. A simple protein precipitation method using acetonitrile was used for the sample preparation and the pre-treated samples were separated by a reverse-phase C18 column. The calibration curve was evaluated in the range of 3.02 ~ 2200 ng/mL and the quadratic regression (weighted 1/concentration) was used for the best fit of the curve with a correlation coefficient ≥0.997. The in vivo PK studies in rats showed that vipadenant bioavailability was 30.4 ± 8.9% with a low to moderate drug clearance. In addition, in vitro/in vivo metabolite profiles in rat were also explored. Five different metabolites were observed in our experimental conditions and the major metabolites were different between in vitro and in vivo conditions. As far as we know, there has been no report on the development of quantitative methods for its PK samples nor the identification of its metabolites since vipadenant was developed. Therefore, this paper would be very useful to better understand the pharmacokinetic and drug metabolism properties of vipadenant in rat as well as other species.

Qualification and Application of a Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometric Method for the Determination of Adalimumab in Rat Plasma.

A liquid chromatography⁻quadrupole time-of-flight (Q-TOF) mass spectrometric method was developed for early-stage research on adalimumab in rats. The method consisted of immunoprecipitation followed by tryptic digestion for sample preparation and LC-QTOF-MS/MS analysis of specific signature peptides of adalimumab in the positive ion mode using electrospray ionization. This specific signature peptide is derived from the complementarity-determining region (CDR) of adalimumab. A quadratic regression (weighted 1/concentration), with an equation y = ax² + bx + c, was used to fit calibration curves over the concentration range of 1⁻100 μg/mL for adalimumab. The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control (QC) samples. This qualified LC-QTOF-MS/MS method was successfully applied to a pharmacokinetic study of adalimumab in rats as a case study. This LC-QTOF-MS/MS approach would be useful as a complementary method for adalimumab or its biosimilars at an early stage of research.

A Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometric Assay for the Quantification of Fabry Disease Biomarker Globotriaosylceramide (GB3) in Fabry Model Mouse.

Fabry disease is a rare lysosomal storage disorder resulting from the lack of α-Gal A gene activity. Globotriaosylceramide (GB3, ceramide trihexoside) is a novel endogenous biomarker which predicts the incidence of Fabry disease. At the early stage efficacy/biomarker study, a rapid method to determine this biomarker in plasma and in all relevant tissues related to this disease simultaneously is required. However, the limited sample volume, as well as the various levels of GB3 in different matrices makes the GB3 quantitation very challenging. Hereby we developed a rapid method to identify GB3 in mouse plasma and various tissues. Preliminary stability tests were also performed in three different conditions: short-term, freeze-thaw, long-term. The calibration curve was well fitted over the concentration range of 0.042⁻10 µg/mL for GB3 in plasma and 0.082⁻20 µg/g for GB3 in various tissues. This method was successfully applied for the comparison of GB3 levels in Fabry model mice (B6;129-Glatm1Kul/J), which has not been performed previously to the best of our knowledge.

Rapid and simultaneous quantification of a mixture of biopharmaceuticals by a liquid chromatography/quadrupole time-of-flight mass spectrometric method in rat plasma following cassette-dosing.

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.