Profiling of dalcetrapib metabolites in human plasma by accelerator mass spectrometry and investigation of the free phenothiol by derivatisation with methylacrylate.

Dalcetrapib, a thioester prodrug, undergoes rapid and complete conversion in vivo to its phenothiol metabolite M1 which exerts the targeted pharmacological response in human. In clinical studies, M1 has been quantified together with its dimer and mixed disulfide species that represent the 'dalcetrapib active form' in plasma. In this article, we describe the determination of the free phenothiol M1 by derivatisation with methylacrylate as a percentage of 'dalcetrapib active form'. Pharmacokinetic profiles of M1 after oral administration of dalcetrapib to humans could be established, underscoring the validity to use a composite measure of 'dalcetrapib active form' as a surrogate marker for pharmacodynamic evaluations. 'Dalcetrapib active form' and M1 made up 8.9% and 3.6% of total drug-related material, respectively. In addition, complete metabolite profiling of 14C-labeled dalcetrapib was conducted after two-dimensional HPLC using fast fractionation into 384-well plates and ultrasensitive determination of the 14C-content by accelerator mass spectrometry. M1 underwent further biotransformation to its S-methyl metabolite M3, which was further oxidized to its sulfoxide and sulfone. Another metabolic pathway was the formation of the S-glucuronide. All of these species underwent further oxidation in the ethylbutyl cyclohexyl moiety leading to a multitude of hydroxyl and keto metabolites undergoing further conjugation to O-glucuronides. More than 80 metabolites were identified, demonstrating extensive metabolism. However, it was unambiguously demonstrated that none of these metabolites were major according to the MIST guideline (exceeding 10% of drug related material in circulation). The combination of accelerator mass spectrometry with HPLC together with high resolution mass spectrometry allowed for structural characterization of the most relevant human metabolites.

Identification of GalNAc-Conjugated Antisense Oligonucleotide Metabolites Using an Untargeted and Generic Approach Based on High Resolution Mass Spectrometry.

Antisense oligonucleotides linked by phosphorothioates are an important class of therapeutics under investigation in various pharmaceutical companies. Antisense oligonucleotides may be coupled to high-affinity ligands (triantennary N-acetyl galactosamine = GalNAc) for hepatocyte-specific asialoglycoprotein receptors (ASGPR) to enhance uptake to hepatocytes and to increase potency. Since disposition and biotransformation of GalNAc-conjugated oligonucleotides is different from unconjugated oligonucleotides, appropriate analytical methods are required to identify main cleavage sites and degradation products of GalNAc conjugated and unconjugated oligonucleotides in target cells. A highly sensitive method was developed to identify metabolites of oligonucleotides using capillary flow liquid chromatography with column switching coupled to a high resolution Orbitrap Fusion mass spectrometer. Detection of GalNAc-conjugated oligonucleotides and their metabolites was achieved by combining full scan MS with two parallel MS2 experiments, one data-dependent scan and an untargeted MS2 experiment (all ion fragmentation) applying high collision energy. In the all ion fragmentation scan, a diagnostic fragment originating from the phosphorothioate backbone (O2PS-: m/z 94.936) was formed efficiently upon collisional activation. Based on this fragment an accurate determination of metabolites of oligonucleotides was achieved, independent of their sequence or conjugation in an untargeted but highly selective manner. The method was effectively applied to investigate uptake and metabolism of GalNAc-conjugated oligonucleotides in incubations of primary rat hepatocytes; the elucidation of expected and unexpected degradation products was achieved in subnanomolar range.

A double-tracer technique to characterize absorption, distribution, metabolism and excretion (ADME) of [14C]-basimglurant and absolute bioavailability after oral administration and concomitant intravenous microdose administration of [13C6]-labeled basimglurant in humans.

1. The emerging technique of employing intravenous microdose administration of an isotope tracer concomitantly with an [14C]-labeled oral dose was used to characterize the disposition and absolute bioavailability of a novel metabotropic glutamate 5 (mGlu5) receptor antagonist under clinical development for major depressive disorder (MDD). 2. Six healthy volunteers received a single 1 mg [12C/14C]-basimglurant (2.22 MBq) oral dose and a concomitant i.v. tracer dose of 100 µg of [13C6]-basimglurant. Concentrations of [12C]-basimglurant and the stable isotope [13C6]-basimglurant were determined in plasma by a specific LC/MS-MS method. Total [14C] radioactivity was determined in whole blood, plasma, urine and feces by liquid scintillation counting. Metabolic profiling was conducted in plasma, urine, blood cell pellet and feces samples. 3. The mean absolute bioavailability after oral administration (F) of basimglurant was ∼67% (range 45.7-77.7%). The major route of [14C]-radioactivity excretion, primarily in form of metabolites, was in urine (mean recovery 73.4%), with the remainder excreted in feces (mean recovery 26.5%). The median tmax for [12C]-basimglurant after the oral administration was 0.71 h (range 0.58-1.00) and the mean terminal half-life was 77.2 ± 38.5 h. Terminal half-life for the [14C]-basimglurant was 178 h indicating presence of metabolites with a longer terminal half-life. Five metabolites were identified with M1-Glucuronide as major and the others in trace amounts. There was minimal binding of drug to RBCs. IV pharmacokinetics was characterized with a mean ± SD CL of 11.8 ± 7.4 mL/h and a Vss of 677 ± 229 L. 4. The double-tracer technique used in this study allowed to simultaneously characterize the absolute bioavailability and disposition characteristics of the new oral molecular entity in a single study.

In Vivo Biotransformation of the Fusion Protein Tetranectin-Apolipoprotein A1 Analyzed by Ligand-Binding Mass Spectrometry Combined with Quantitation by ELISA.

The in vivo biotransformation of a novel fusion protein tetranectin/apolipoprotein A1 (TN-ApoA1) was investigated by ligand-binding mass spectrometry (LB-MS) in support of enzyme-linked immunosorbent assays (ELISA). The main focus was on catabolites formed by proteolysis of the fusion protein in rabbit following intravenous administration of lipidated TN-ApoA1. The drug and its catabolites were isolated from rabbit plasma by immunocapture with a monoclonal antibody (mAb) binding to the fusion region of TN-ApoA1. The captured drug and catabolites were released from the streptavidin-coated magnetic beads, separated by monolithic RP capillary HPLC, and online detected by high-resolution mass spectrometry. In addition, the same extract was digested with LysN to confirm or further narrow down the structure of the found catabolites. Two pharmacologically active catabolites were identified with conserved fusion region. The major catabolite [3-285] was formed by truncation of AP at the N-terminus and the minor catabolite [29-270] by truncations of either side of the TN-ApoA1 sequence. Since the ELISA determined the sum of TN-ApoA1, along with its two main catabolites, the individual PK profiles of all three components could be derived by applying their mass peak composition for each sampling point. Parent drug accounted for 25% of drug-related material, whereas that of the catabolites [3-285] and [29-270] accounted for 66% and 9%, respectively. This result could be obtained without catabolite specific ELISAs or quantitative LC-MS assays. It was also confirmed that all relevant functional molecules of TN-ApoA1 in the plasma samples were quantified by the ELISA, which provided a good relationship for pharmacokinetic/pharmacodynamic evaluations.

Quantitative profiling of prostaglandins as oxidative stress biomarkers in vitro and in vivo by negative ion online solid phase extraction - Liquid chromatography-tandem mass spectrometry.

Free radical-mediated oxidation of arachidonic acid to prostanoids has been implicated in a variety of pathophysiological conditions such as oxidative stress. Here, we report on the development of a liquid chromatography-mass spectrometry method to measure several classes of prostaglandin derivatives based on regioisomer-specific mass transitions down to levels of 20 pg/ml applied to the measurement of prostaglandin biomarkers in primary hepatocytes. The quantitative profiling of prostaglandin derivatives in rat and human hepatocytes revealed the increase of several isomers on stress response. In addition to the well-established markers for oxidative stress such as 8-iso-prostaglandin F2α and the prostaglandin isomers PE2 and PD2, this method revealed a significant increase of 15R-prostaglandin D2 from 236.1 ± 138.0 pg/1E6 cells in untreated rat hepatocytes to 2001 ± 577.1 pg/1E6 cells on treatment with ferric NTA (an Fe(3+) chelate with nitrilotriacetic acid causing oxidative stress in vitro as well as in vivo). Like 15R-prostaglandin D2, an unassigned isomer that revealed a more significant increase than commonly analyzed prostaglandin derivatives was identified. Mass spectrometric detection on a high-resolution instrument enabled high-quality quantitative analysis of analytes in plasma levels from rat experiments, where increased concentrations up to 23-fold change treatment with Fe(III)NTA were observed.

Metabolism and mass balance of SGLT2 inhibitor tofogliflozin following oral administration to humans.

1. Tofogliflozin is a novel and selective SGLT2 inhibitor increasing glucosuria by inhibition of glucose re-absorption in the kidney for the treatment of type 2 diabetes mellitus. 2. In this study, the metabolism and the mass balance of tofogliflozin was evaluated following administration of a single oral dose of 20 mg [(14)C]-tofogliflozin to six healthy subjects. 3. Tofogliflozin underwent mainly oxidative metabolism in the ethylphenyl moiety, but also minor glucuronide conjugates of metabolites and the parent drug were formed. 4. In plasma, the parent drug and its major phenyl acetic acid metabolite M1 accounted for 42% and 52% of the total drug-related material, respectively. The hydroxyl metabolites and their successor ketone metabolite showed an exposure well below 5%, along with an acyl glucuronide of M1. 5. Tofogliflozin was completely absorbed with subsequent predominate metabolic clearance and a small contribution of direct urinary elimination. Approximately, 76% of the dose was excreted in urine and 20% in faeces within 72 h. The high absorption of tofogliflozin was exemplified by the small trace of parent drug in faeces. The phenyl acetic acid metabolite M1 was the major component excreted in urine and faeces accounting for more than half of the dose. Tofogliflozin demonstrated a high metabolic turnover.

Determination of dalcetrapib by liquid chromatography-tandem mass spectrometry.

The cholesteryl ester transfer protein modulator dalcetrapib is currently under development for the prevention of dyslipidemia and cardiovascular disease. Dalcetrapib, a thioester, is rapidly hydrolyzed in vivo to the corresponding thiophenol which in turn is further oxidized to the dimer and mixed disulfides (where the thiophenol binds to peptides, proteins and other endogenous thiols). These forms co-exist in an oxidation-reduction equilibrium via the thiol and cannot be stabilized without influencing the equilibrium, hence specific determination of individual components, i.e., in order to distinguish between the free thiol, the disulfide dimer and mixed disulfide adducts, was not pursued for routine analysis. The individual forms were quantified collectively as dalcetrapib-thiol (dal-thiol) after reduction under basic conditions with dithiothreitol to break disulfide bonds and derivatization with N-ethylmaleimide to stabilize the free thiol. The S-methyl and S-glucuronide metabolites were determined simultaneously with dal-thiol with no effect from the derivatization procedure. Column-switching liquid chromatography-tandem mass spectrometry provided a simple, fast and robust method for analysis of human and animal plasma and human urine samples. Addition of the surfactant Tween 80 to urine prevented adsorptive compound loss. The lower limits of quantitation (LLOQ) were 5 ng/mL for dal-thiol, and 5 ng/mL for the S-methyl and 50 ng/mL for the S-glucuronide metabolites. Using stable isotope-labeled internal standards, inter- and intra-assay precisions were each <15% (<20% at LLOQ) and accuracy was between 85 and 115%. Recovery was close to 100%, and no significant matrix effect was observed.

Pharmacokinetics and metabolism of the dipeptidyl peptidase IV inhibitor carmegliptin in rats, dogs, and monkeys.

The pharmacokinetics and excretion of carmegliptin, a novel dipeptidyl peptidase IV inhibitor, were examined in rats, dogs, and cynomolgus monkeys. Carmegliptin exhibited a moderate clearance, extensive tissue distribution, and a variable oral bioavailability of 28-174%. Due to saturation of intestinal active secretion, the area under the plasma concentration-time curve (AUC) in dogs and monkeys increased in a more than dose-proportional manner over an oral dose range of 2.5-10 mg/kg. Following oral administration of [(14)C]carmegliptin at 3 mg/kg, > 94% of the radioactive dose was recovered in 72-h post-dose from Wistar rats and Beagle dogs. Virtually, the entire administered radioactive dose was excreted unchanged in urine, intestinal lumen, and bile. Approximately 36%, 29%, and 19% of the dose were excreted by respective routes. Consistently, in vitro, carmegliptin was highly resistant to hepatic metabolism in all species tested. Based on in vitro studies, carmegliptin is a good substrate for Mdr1/MDR1. Breast cancer resistance protein (Bcrp) is not expected to be involved in the transport of carmegliptin since in vitro carmegliptin was not significantly transported by this transporter. The very high extravascular distribution of carmegliptin in the intestinal tissues, as demonstrated in Wistar rats and Beagle dogs, could play a significant role in its therapeutic effect.

Rapid screening and characterization of drug metabolites using a new quadrupole-linear ion trap mass spectrometer.

The application of a new hybrid RF/DC quadrupole-linear ion trap mass spectrometer to support drug metabolism and pharmacokinetic studies is described. The instrument is based on a quadrupole ion path and is capable of conventional tandem mass spectrometry (MS/MS) as well as several high-sensitivity ion trap MS scans using the final quadrupole as a linear ion trap. Several pharmaceutical compounds, including trocade, remikiren and tolcapone, were used to evaluate the capabilities of the system with positive and negative turbo ionspray, using either information-dependent data acquisition (IDA) or targeted analysis for the screening, identification and quantification of metabolites. Owing to the MS/MS in-space configuration, quadrupole-like CID spectra with ion trap sensitivity can be obtained without the classical low mass cutoff of 3D ion traps. The system also has MS(3) capability which allows fragmentation cascades to be followed. The combination of constant neutral loss or precursor ion scan with the enhanced product ion scan was found to be very selective for identifying metabolites at the picogram level in very complex matrices. Owing to the very high cycle time and, depending on the mass range, up to eight different MS experiments could be performed simultaneously without compromising chromatographic performance. Targeted product ion analysis was found to be complementary to IDA, in particular for very low concentrations. Comparable sensitivity was found in enhanced product ion scan and selected reaction monitoring modes. The instrument is particularly suitable for both qualitative and quantitative analysis.

Preformulation approaches to improve the oral bioavailability of two novel piperidine renin inhibitors in dog.

Different experimental formulations based on aqueous and oily systems, water miscible solvents, and solid dispersions were investigated for their potential to increase the oral bioavailability (F) of two novel piperidine renin inhibitors (Ro-X1: (R)-1-methoxy-3-[(3S,4R,5R)-4-[4-[3-(2-methoxy-benzyloxy)-propoxy]-phenyl]- 5-(4-methoxy-naphthalen-2-ylmethoxy)-piperidin-3-yloxy]-propan-2-ol; Ro-X2: (R)-3-[(3S,4R,5R)-4-[4-[3-(2-methoxy-benzyloxy)-propoxy]-phenyl]-5-(4- methoxy-naphthalen-2-ylmethoxy)-piperidin-3-ylmethoxy]-propane-1,2-diol) in dogs compared to their administration as acidic aqueous solution. The compounds were characterized by a low solubility at pH 7 (Ro-X1: 3 micrograms/ml, Ro-X2: 24 micrograms/ml) and a high lipophilicity (Ro-X1: LogP = 5.7, Ro-X2: LogP = 3.7). For Ro-X1 oil-based vehicles resulted in an improvement in the oral bioavailability compared to the aqueous solution (F = 6 +/- 1.2%) with the best result being achieved with a solution in Capmul (F = 14.6 +/- 3.5%). By contrast, for Ro-X2 the highest bioavailability (F = 27.1 +/- 8.4%) was achieved using an aqueous solution. Computer simulations based on the physicochemical parameters of the compounds only predicted that the fraction of compound absorbed in man should be almost quantitative for Ro-X2 and only about 28% for Ro-X1. These results suggest that other factors such as extensive gut and/or hepatic metabolism as well as exclusion by intestinal transporters such as p-glycoprotein, rather than incomplete solubilization in the gut, are the major reasons for the limited oral bioavailability of both compounds.