A Unique Automation Platform for Measuring Low Level Radioactivity in Metabolite Identification Studies

Generation and interpretation of biotransformation data on drugs, i.e. identification of physiologically relevant metabolites, defining metabolic pathways and elucidation of metabolite structures, have become increasingly important to the drug development process. Profiling using 14C or 3H radiolabel is defined as the chromatographic separation and quantification of drug-related material in a given biological sample derived from an in vitro, preclinical in vivo or clinical study. Metabolite profiling is a very time intensive activity, particularly for preclinical in vivo or clinical studies which have defined limitations on radiation burden and exposure levels. A clear gap exists for certain studies which do not require specialized high volume automation technologies, yet these studies would still clearly benefit from automation. Use of radiolabeled compounds in preclinical and clinical ADME studies, specifically for metabolite profiling and identification are a very good example. The current lack of automation for measuring low level radioactivity in metabolite profiling requires substantial capacity, personal attention and resources from laboratory scientists. To help address these challenges and improve efficiency, we have innovated, developed and implemented a novel and flexible automation platform that integrates a robotic plate handling platform, HPLC or UPLC system, mass spectrometer and an automated fraction collector

A perspective on tritium versus carbon-14: ensuring optimal label selection in pharmaceutical research and development

Tritium (3H) and carbon-14 (14C) labels applied in pharmaceutical research and development each offer their own distinctive advantages and disadvantages coupled with benefits and risks. The advantages of 3H have a higher specific activity, shorter half-life that allows more manageable waste remediation, lower material costs, and often more direct synthetic routes. The advantages of 14C offer certain analytical benefits and less potential for label loss. Although 3H labels offer several advantages, they might be overlooked as a viable option because of the concerns about its drawbacks. A main drawback often challenged is metabolic liability. These drawbacks, in some cases, might be overstated leading to underutilization of a perfectly viable option. As a consequence, label selection may automatically default to 14C, which is a more conservative approach. To challenge this ‘14C-by-default’ approach, pharmaceutical agents with strategically selected 3H-labeling positions based on non-labeled metabolism data have been successfully implemented and evaluated for 3H loss. From in-house results, the long term success of projects clearly would benefit from a thorough, objective, and balanced assessment regarding label selection (3H or 14C). This assessment should be based on available project information and scientific knowledge. Important considerations are project applicability (preclinical and clinical phases), synthetic feasibility, costs, and timelines

Design, synthesis and evaluation of novel inhibitors and fluorogenic substrates for cysteine proteases and metallo proteases

Ph.D.James C. Power

Metal Complexes of Modified Cyclen as Catalysts for Hydrolytic Restriction of Plasmid DNA

Simple and novel nuclease models have been synthesized. These involve metal-binding ligand 1,4,7,10-tetraazlcyclododecane (cyclen) tethered to an acridine ring (a DNA-binding group) by amide linkers of various lengths. Binding of these probes to DNA was studied by monitoring changes in their UV-visible spectra affected by the presence of DNA. Titration of these compounds with increasing amounts of pBR322 DNA caused hypochromic effects and shifted the acridine absorption at 360nm to a longer wavelength. Under biologically relevant conditions (37°C and pH 7.4), specific transition metal complexes of these compounds are found to be highly effective catalysts toward the hydrolysis of plasmid DNA. This is demonstrated by their ability to convert the super-coiled DNA (form I) to open-circular DNA (form II). Structure-activity correlation studies show that hydrolytic activity depends on both the structure of ligand (L1\u3eL2\u3eL3) and the nature of metal ion cofactor (Co3+\u3eZn2+\u3eCr2+\u3eNi2+\u3eCu2+\u3eFe3+)

MS565, a SPECT tracer for evaluating the brain penetration of BAF312 (siponimod)

BAF312 (siponimod) is a sphingosine 1-phosphate (S1P) receptor modulator in clinical development for the treatment of multiple sclerosis, with faster organ distribution and elimination kinetics than its precursor FTY720 (Gilenya®). Our aim was to better quantify the penetration of BAF312 in the human brain, after labeling with a radioisotope for positron emission tomography (PET) or single photon emission computer tomography (SPECT). The radioisotopes 11C and 18F, which could have been introduced in BAF312 without modifying its structure, do not have decay kinetics compatible with the time required for observing the organ distribution of the drug in patients. In contrast, we identified a longer-lived iodinated derivative of BAF312 (18, MS565), which retains the blood pharmacokinetics and organ distribution of the original molecule as well as its affinity, selectivity for S1P receptors, and overall physicochemical properties. MS565 can easily be radiolabeled with 123I for SPECT or 124I for PET imaging. [123I]MS565 is currently under development as a clinical imaging agent

Application of a deuterium replacement strategy to modulate the pharmacokinetics of NVS-CRF38, a novel CRF1 antagonist

Deuterium isotope effects were evaluated as a strategy to optimize the pharmacokinetics of NVS-CRF38, a novel CRF1 receptor antagonist. In an attempt to supress O-demethylation of NVS-CRF38 without losing activity against the CRF1 receptor, the protons at the site of metabolism were replaced with deuterium. For in vitro and in vivo studies intrinsic primary isotope effects (KH/KD) were determined by the ratio of CLint obtained from the nondeuterated and deuterated substrate. In vitro isotope effects were more pronounced when CLint values were calculated based on the rate of formation of the O-desmethyl metabolite (KH/KD ~7), compared to the substrate depletion method (KH/KD ~2). In vivo isotope effects were measured in the rat after intravenous (1 mg/kg) and oral (10 mg/kg) administration. For both administration routes isotope effects calculated from in vivo CLint corresponding to all biotransformation pathways were lower (KH/KD ~2) compared to CLint values calculated from the O-demethylation reaction alone (KH/KD ~7). Comparative metabolite identification studies were undertaken using rat and human microsomes to explore the potential for metabolic switching. As expected a marked reduction of the O-demethylated metabolite was observed for the nondeuterated substrate, however levels of NVS-CRF38’s other metabolites increased, compensating to some extent for the isotope effect

Tritium labeling of full length small interfering RNA’s

A simple procedure is described for full length single internal [3H]-radiolabeling of oligonucleotides. Previous labeling strategies have been applied to large molecular weight compounds like proteins and oligonucleotides, e.g. iodination, and 111In labeling via covalently bounded chelators. However, a procedure has not yet been reported for single internal radiolabeling of oligonucleotides that preserves the molecular structure (3H replacing a 1H). Following our strategy the radiolabel can be strategically placed within a stable and predetermined internal position of the siRNA. This placement was accomplished by placing a 5-bromouridine or 5-bromo-2´-O-methyluridine phosphoramidite building block into the middle of the antisense strand using standard phosphoramidite chemistry. The de-protected full length antisense strand was tritium labeled by bromine/tritium exchange, catalyzed by palladium on charcoal in the predetermined 5-position of either uridine or 2´-O-methyluridine. Internal placement of the building block within the oligonucleotide sequence and label placement at 5-position decreases the likelihood of the label to be readily cleaved from the oligonucleotide in vivo, and loss of the label by spontaneous tritium-hydrogen exchange. The tritiated single stranded and double stranded RNAs were also shown to both radio and chemically stable for at least 6 months at -80ºC. This allows more than sufficient time to conduct pharmaceutical formulation and pharmacokinetic studies

Reconstructed UPLC radiochromatogram of [¹⁴C]-NVP123 and its metabolites from dog urine.

<p>Note, “M#” (e.g. M13) denotes a designated metabolite number, which is not assigned based on order of chromatographic elution.</p