Investigating discovery strategies and pharmacological properties of stereodefined phosphorodithioate LNA gapmers.

The introduction of sulfur into the phosphate linkage of chemically synthesized oligonucleotides creates the stereocenters on phosphorus atoms. Researchers have valued the nature of backbone stereochemistry and early on investigated drug properties for the individual stereocenters in dimers or short oligomers. Only very recently, it has become possible to synthesize fully stereodefined antisense oligonucleotides in good yield and purity. Non-bridging phosphorodithioate (PS2) introduces second sulfur into the phosphorothioate linkage to remove the chirality of phosphorus atom. Here, we describe the application of symmetrical non-bridging PS2 linkages in the context of stereodefined locked nucleic acids (LNAs) antisense oligonucleotides with the goal of reducing chiral complexity and, ultimately, resulting in single molecules. In addition, we propose a rather simple strategy to rapidly identify stereodefined gapmers, combining PS2 and a preferred stereochemistry motif (RSSR), which supports RNase-H-mediated target knockdown. Pharmacological efficacy and metabolic stability are investigated systematically using ApoB as a target sequence, where in vivo data correlate well to what is observed in vitro.

Addressing Today's Absorption, Distribution, Metabolism, and Excretion (ADME) Challenges in the Translation of In Vitro ADME Characteristics to Humans: A Case Study of the SMN2 mRNA Splicing Modifier Risdiplam.

Small molecules that present complex absorption, distribution, metabolism, and elimination (ADME) properties can be challenging to investigate as potential therapeutics. Acquiring data through standard methods can yield results that are insufficient to describe the in vivo situation, which can affect downstream development decisions. Implementing in vitro-in vivo-in silico strategies throughout the drug development process is effective in identifying and mitigating risks while speeding up their development. Risdiplam (Evrysdi)-an orally bioavailable, small molecule approved by the US Food and Drug Administration and more recently by the European Medicines Agency for the treatment of patients ≥2 months of age with spinal muscular atrophy-is presented here as a case study. Risdiplam is a low-turnover compound whose metabolism is mediated through a non-cytochrome P450 enzymatic pathway. Four main challenges of risdiplam are discussed: predicting in vivo hepatic clearance, determining in vitro metabolites with regard to metabolites in safety testing guidelines, elucidating enzymes responsible for clearance, and estimating potential drug-drug interactions. A combination of in vitro and in vivo results was successfully extrapolated and used to develop a robust physiologically based pharmacokinetic model of risdiplam. These results were verified through early clinical studies, further strengthening the understanding of the ADME properties of risdiplam in humans. These approaches can be applied to other compounds with similar ADME profiles, which may be difficult to investigate using standard methods. SIGNIFICANCE STATEMENT: Risdiplam is the first approved, small-molecule, survival of motor neuron 2 mRNA splicing modifier for the treatment of spinal muscular atrophy. The approach taken to characterize the absorption, distribution, metabolism, and excretion (ADME) properties of risdiplam during clinical development incorporated in vitro-in vivo-in silico techniques, which may be applicable to other small molecules with challenging ADME. These strategies may be useful in improving the speed at which future drug molecules can be developed.

Safety, Tissue Distribution, and Metabolism of LNA-Containing Antisense Oligonucleotides in Rats.

Antisense oligonucleotides (ASOs) are chemically modified nucleic acids with therapeutic potential, some of which have been approved for marketing. We performed a study in rats to investigate mechanisms of toxicity after administration of 3 tool locked nucleic acid (LNA)-containing ASOs with differing established safety profiles. Four male rats per group were dosed once, 3, or 6 times subcutaneously, with 7 days between dosing, and sacrificed 3 days after the last dose. These ASOs were either unconjugated (naked) or conjugated with N-acetylgalactosamine for hepatocyte-targeted delivery. The main readouts were in-life monitoring, clinical and anatomic pathology, exposure assessment and metabolite identification in liver and kidney by liquid chromatography coupled to tandem mass spectrometry, ASO detection in liver and kidney by immunohistochemistry, in situ hybridization, immune electron microscopy, and matrix-assisted laser desorption/ionization mass spectrometry imaging. The highly toxic compounds showed the greatest amount of metabolites and a low degree of tissue accumulation. This study reveals different patterns of cell death associated with toxicity in liver (apoptosis and necrosis) and kidney (necrosis only) and provides new ultrastructural insights on the tissue accumulation of ASOs. We observed that the immunostimulatory properties of ASOs can be either primary from sequence-dependent properties or secondary to cell necrosis.

Absolute Bioavailability of Vemurafenib in Patients With BRAFV600 Mutation-Positive Malignancies.

Vemurafenib is a BRAF kinase inhibitor indicated for the treatment of patients with BRAFV600 mutation-positive unresectable or metastatic melanoma and Erdheim-Chester disease. This phase 1, open-label, single-arm study was designed to estimate absolute bioavailability of oral vemurafenib at steady state and to characterize the pharmacokinetics of a single intravenous microdose of 14 C-labeled vemurafenib in patients with BRAFV600 mutation-positive malignancies. Patients received oral vemurafenib 960 mg twice daily on days 1 through 28, with a single intravenous infusion of 14 C-labeled vemurafenib solution (3 mL, corresponding to a radioactive dose of 18.5 kBq and a vemurafenib dose of 20 µg) given on the morning of day 21, immediately following the morning dose of oral vemurafenib. A total of 6 patients were enrolled. Four patients who received 14 C-labeled vemurafenib infusion were included in the pharmacokinetic and bioavailability analyses. Geometric mean absolute bioavailability of oral vemurafenib at steady state, calculated as the ratio of dose-normalized area under the curve during the dosing interval (AUCτ ) following oral vemurafenib dose to dose-normalized AUC from time 0 extrapolated to infinity (AUC0-inf ) following vemurafenib intravenous dose, was 57.8%. The majority of radioactivity (geometric mean 41%) was recovered in feces, and a small proportion (geometric mean 1.4%) was recovered in urine. Treatment-emergent adverse events occurred in 5 of 6 (83%) patients and were all grade 1/2 in severity, except for 1 grade-4 anaphylactic reaction occurring during infusion of 14 C-labeled vemurafenib, which was thought to be related to the excipient polysorbate 80 in the intravenous formulation.

Studying the Biotransformation of Phosphorothioate-Containing Oligonucleotide Drugs by LC-MS.

Across the pharmaceutical industry, there is increasing interest and need to investigate the biotransformation of oligonucleotide drugs. The method of choice is high-resolution mass spectrometry due to its unmet sensitivity and specificity.Here, we describe a method developed and applied in our laboratory studying the biotransformation of phosphorothioate-containing oligonucleotide drugs. This method is based on capillary flow liquid chromatography with column switching coupled to high-resolution mass spectrometry.

The In Vitro Biotransformation of the Fusion Protein Tetranectin-Apolipoprotein A1.

As more and more protein biotherapeutics enter the drug discovery pipelines, there is an increasing interest in tools for mechanistic drug metabolism investigations of biologics in order to identify and prioritize the most promising candidates. Understanding or even predicting the in vivo clearance of biologics and to support translational pharmacokinetic modeling activities is essential, however there is a lack of effective and validated in vitro cellular tools. Although different mechanisms have to be adressed in the context of biologics disposition, the scope is not comparable to the nowadays widely established tools for early characterization of small molecule disposition. Here, we describe a biotransformation study of the fusion protein tetranectin apolipoprotein A1 by cellular systems. The in vivo biotransformation of tetranectin apolipoprotein A1 has been described previously, and the same major biotransformation product could also be detected in vitro, by a targeted and highly sensitive detection method based on chymotrypsin digest. In addition, the protease responsible for the formation of this biotransformation product could be elucidated to be DPP4. To our knowledge, this is one of the first reports of an in vitro biotransformation study by cells of a therapeutic protein.

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.

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.