Investigation of pharmacokinetic properties of a PEGylated bilirubin nanoparticle in male Sprague-Dawley rats using liquid chromatography-quadrupole time-of-flight mass spectrometry.

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.

Evaluation of In Vivo Prepared Albumin-Drug Conjugate Using Immunoprecipitation Linked LC-MS Assay and Its Application to Mouse Pharmacokinetic Study.

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.

Investigation of In Vitro and In Vivo Metabolism of α-Amanitin in Rats Using Liquid Chromatography-Quadrupole Time-of-Flight Mass Spectrometric Method.

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.