Development and validation of a liquid chromatography-triple quadrupole mass spectrometry method for the determination of isopeptide ε-(γ-glutamyl) lysine in human urine as biomarker for transglutaminase 2 cross-linked proteins.

Determination of the levels of protein cross-linking catalysed by the activity of transglutaminase 2 in various disease states has remained a significant challenge. The ability to quantify the isopeptide ε-(γ-glutamyl) lysine, which can form as a heterogeneous bond within or between proteins has significant analytical and clinical potential as a biomarker in biofluids such as human urine. Increased transglutaminase 2 activity is associated with a number of diseases, such as fibrosis. Previously published methods have been based on classical amino acid analysis, however they require a complex multi-enzyme digestion in order to achieve complete protein digestion, whilst leaving the isopeptide cross link intact. These methods require high levels of enzymes, which contaminate the analysis and alter the dynamics of digestion. The amino acid analysis detection also lacked selectivity, especially where the levels of crosslink are expected to be low relative to the background protein levels. We have systematically addressed these challenges, by optimising the precipitation of the protein in urine, the use of innovative immobilised enzyme technology, which allows for efficient digestion without enzyme contamination and LC-MS/MS detection based on multiple reaction monitoring. This method was validated for its analytical performance characteristics, showing the method has a sensitivity of 0.1 ng/mL of ε-(γ-glutamyl) lysine in human urine with precision of less than 20% CV, and is selective as no interferences were observed that may adversely affect the analysis. As such this approach represents a significant advance in the ability to detect and quantify ε-(γ-glutamyl) lysine.

IQ consortium perspective: complementary LBA and LC-MS in protein therapeutics bioanalysis and biotransformation assessment.

Increasingly diverse large molecule modalities have driven the need for complex bioanalysis and biotransformation assessment involving both traditional ligand-binding assays (LBA) and more recent hybrid immunoaffinity LC-MS platforms. Given the scientific expertise in LBA and LC-MS typically resides in different functions within the industry, this has presented operational challenges for an integrated approach for bioanalysis and biotransformation assessment. Encouragingly, over time, the industry has recognized the complementary value of the two platforms. This has not been an easy transition as organizational structures vary widely within the industry. However, there are tremendous benefits in adopting fully integrated strategies for biopharma. This IQ consortium paper presents current perspectives across the biopharma industry. It highlights the technical and operational challenges in current large molecule bioanalysis, the value of collaborations across LBA and LC-MS, and scientific expertise for fully integrated strategies for bioanalysis and biotransformation.

Discovery of a Potent, Orally Bioavailable PI4KIIIß Inhibitor (UCB9608) Able To Significantly Prolong Allogeneic Organ Engraftment in Vivo.

The primary target of a novel series of immunosuppressive 7-piperazin-1-ylthiazolo[5,4- d]pyrimidin-5-amines was identified as the lipid kinase, PI4KIIIß. Evaluation of the series highlighted their poor solubility and unwanted off-target activities. A medicinal chemistry strategy was put in place to optimize physicochemical properties within the series, while maintaining potency and improving selectivity over other lipid kinases. Compound 22 was initially identified and profiled in vivo, before further modifications led to the discovery of 44 (UCB9608), a vastly more soluble, selective compound with improved metabolic stability and excellent pharmacokinetic profile. A co-crystal structure of 44 with PI4KIIIß was solved, confirming the binding mode of this class of inhibitor. The much-improved in vivo profile of 44 positions it as an ideal tool compound to further establish the link between PI4KIIIß inhibition and prolonged allogeneic organ engraftment, and suppression of immune responses in vivo.

Fab-dsFv: A bispecific antibody format with extended serum half-life through albumin binding.

An antibody format, termed Fab-dsFv, has been designed for clinical indications that require monovalent target binding in the absence of direct Fc receptor (FcR) binding while retaining substantial serum presence. The variable fragment (Fv) domain of a humanized albumin-binding antibody was fused to the C-termini of Fab constant domains, such that the VL and VH domains were individually connected to the Cκ and CH1 domains by peptide linkers, respectively. The anti-albumin Fv was selected for properties thought to be desirable to ensure a durable serum half-life mediated via FcRn. The Fv domain was further stabilized by an inter-domain disulfide bond. The bispecific format was shown to be thermodynamically and biophysically stable, and retained good affinity and efficacy to both antigens simultaneously. In in vivo studies, the serum half-life of Fab-dsFv, 2.6 d in mice and 7.9 d in cynomolgus monkeys, was equivalent to Fab'-PEG.

Extending the half-life of a fab fragment through generation of a humanized anti-human serum albumin Fv domain: An investigation into the correlation between affinity and serum half-life.

We generated an anti-albumin antibody, CA645, to link its Fv domain to an antigen-binding fragment (Fab), thereby extending the serum half-life of the Fab. CA645 was demonstrated to bind human, cynomolgus, and mouse serum albumin with similar affinity (1-7 nM), and to bind human serum albumin (HSA) when it is in complex with common known ligands. Importantly for half-life extension, CA645 binds HSA with similar affinity within the physiologically relevant range of pH 5.0 - pH 7.4, and does not have a deleterious effect on the binding of HSA to neonatal Fc receptor (FcRn). A crystal structure of humanized CA645 Fab in complex with HSA was solved and showed that CA645 Fab binds to domain II of HSA. Superimposition with the crystal structure of FcRn bound to HSA confirmed that CA645 does not block HSA binding to FcRn. In mice, the serum half-life of humanized CA645 Fab is 84.2 h. This is a significant extension in comparison with < 1 h for a non-HSA binding CA645 Fab variant. The Fab-HSA structure was used to design a series of mutants with reduced affinity to investigate the correlation between the affinity for albumin and serum half-life. Reduction in the affinity for MSA by 144-fold from 2.2 nM to 316 nM had no effect on serum half-life. Strikingly, despite a reduction in affinity to 62 µM, an extension in serum half-life of 26.4 h was still obtained. CA645 Fab and the CA645 Fab-HSA complex have been deposited in the Protein Data Bank (PDB) with accession codes, 5FUZ and 5FUO, respectively.

Introduction of a routine quan/qual approach into research DMPK: experiences and evolving strategies.

After graduating with an Oceanography degree from Swansea University, Lloyd has spent over 20 years in the field of bioanalysis and metabolite profiling. He started his career in large pharma at Wyeth UK, where he was involved in setting up the first GC and LC-MS/MS systems for both QC and early DMPK assays, employing EI/CI, thermospray, and the then new electrospray ionization techniques. Lloyd then joined Celltech, now UCB, where he is primarily tasked with metabolite profiling by LC-MS and NMR to support both early research projects and late-stage clinical studies. The application of liquid chromatography high-resolution mass spectrometry for simultaneous quantitative and qualitative (quan/qual) analysis has gained momentum across a range of different scientific arenas in recent years. The ability to acquire high quality quantitative data, whilst also capturing qualitative data for either parallel or retrospective analysis, is a powerful resource, especially in view of ever-reducing cycle times, laboratory space and budgets. The quan/qual approach employing a Q-Exactive™ Orbitrap high-resolution mass spectrometer has been successfully introduced into UCB's research DMPK department. This article describes our experiences in introducing quan/qual, issues that we discovered in establishing this new working paradigm, the evolution of the strategy and its future potential.

Design, synthesis and biological evaluation of peptide derivatives of L-dopa as anti-parkinsonian agents.

A series of dipeptide derivatives of L-dopa were synthesized and investigated for their pharmacological activity using the unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rat as an experimental model of Parkinson's disease. Among them, (S)-isopropyl 2-(2-amino-2-methylpropanamido)-3-(3,4-dihydroxyphenyl)propanoate (4 g) was found to be the most active compound, with 106% AUC activity and 149% peak activity of L-dopa after oral administration.

A mixture of functionally oligoclonal humanized monoclonal antibodies that neutralize Clostridium difficile TcdA and TcdB with high levels of in vitro potency shows in vivo protection in a hamster infection model.

Clostridium difficile infections are a major cause of antibiotic-associated diarrhea in hospital and care facility patients. In spite of the availability of effective antibiotic treatments, C. difficile infection (CDI) is still a major cause of patient suffering, death, and substantial health care costs. Clostridium difficile exerts its major pathological effects through the actions of two protein exotoxins, TcdA and TcdB, which bind to and disrupt gut tissue. Antibiotics target the infecting bacteria but not the exotoxins. Administering neutralizing antibodies against TcdA and TcdB to patients receiving antibiotic treatment might modulate the effects of the exotoxins directly. We have developed a mixture of three humanized IgG1 monoclonal antibodies (MAbs) which neutralize TcdA and TcdB to address three clinical needs: reduction of the severity and duration of diarrhea, reduction of death rates, and reduction of the rate of recurrence. The UCB MAb mixture showed higher potency in a variety of in vitro binding and neutralization assays (∼10-fold improvements), higher levels of protection in a hamster model of CDI (82% versus 18% at 28 days), and higher valencies of toxin binding (12 versus 2 for TcdA and 3 versus 2 for TcdB) than other agents in clinical development. Comparisons of the MAb properties also offered some insight into the potential relative importance of TcdA and TcdB in the disease process.

Benserazide dosing regimen affects the response to L-3,4-dihydroxyphenylalanine in the 6-hydroxydopamine-lesioned rat.

Peripheral aromatic amino acid decarboxylase (AADC) inhibitors, such as benserazide, are routinely used to potentiate the effects of L-3,4-dihydroxyphenylalanine (L-DOPA) in Parkinson's disease (PD) and in experimental models of PD. However, there is little information available on the optimal dose or the timing of administration relative to L-DOPA treatment. We now assess the effect of dose, timing, and supplemental administration of benserazide on the rotational response induced by L-DOPA in unilateral 6-hydroxydopamine-lesioned rats. L-DOPA (12.5 mg/kg, p.o.) concomitant with benserazide (3.125-15 mg/kg, p.o.) produced a dose-dependent increase in contraversive rotation compared with the effects of L-DOPA alone. The optimal L-DOPA response was achieved with 10 mg/kg of benserazide and this dose was used in subsequent experiments. When L-DOPA treatment was delayed for 1, 2, or 3 h after benserazide, the rotational response declined suggesting loss of AADC inhibition. Unexpectedly, there was also a progressive decline in response when benserazide and L-DOPA were given together but at increasingly later time points of 08.00, 09.00, 10.00, and 11.00 h. To assess supplemental administration of benserazide, an additional dose was given 2 h after the initial benserazide/L-DOPA treatment. This produced a further increase in the number of contralateral rotations indicating that the effect of benserazide declines while plasma levels of L-DOPA are maintained. Therefore, optimization of the dose and timing of benserazide administration is essential to achieve a consistent L-DOPA response in 6-hydroxydopamine-lesioned rats. These findings may have implications for the way in which peripheral AADC inhibitors are used in the treatment of PD.

Design, synthesis and biological evaluation of L-dopa amide derivatives as potential prodrugs for the treatment of Parkinson's disease.

A range of amide derivatives of L-dopa were synthesized and investigated for their pharmacological activity and their ability to be converted to L-dopa using the unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rat, as an experimental model of Parkinson's disease. The diacetyl derivative of L-dopa amide (11b) was found to be more active than L-dopa after its oral administration and generated plasma levels of L-dopa in the therapeutic range for an antiparkinsonian effect in man.

Development of carboxylic acid replacements in indole-N-acetamide inhibitors of hepatitis C virus NS5B polymerase.

Allosteric inhibition of the hepatitis C virus (HCV) NS5B RNA-dependent RNA polymerase enzyme has recently emerged as a viable strategy toward blocking replication of viral RNA in cell-based systems. We report here 2 series of indole-N-acetamides, bearing physicochemically diverse carboxylic acid replacements, which show potent affinity for the NS5B enzyme with reduced potential for formation of glucuronide conjugates. Preliminary optimization of these series furnished compounds that are potent in the blockade of subgenomic HCV RNA replication in HUH-7 cells.

Direct determination of unbound intrinsic drug clearance in the microsomal stability assay.

The microsomal stability assay is commonly used to rank compounds according to their metabolic stability. Determination of the unbound intrinsic clearance (CL(in,u)) is essential for the accurate comparison of compounds, since nonspecific binding to microsomes can lead to an underestimation of the microsomal clearance. In this study, a new method (linear extrapolation in the stability assay, LESA) was established, which allows direct calculation of CL(in,u) from microsomal stability data, without the need to independently determine the fraction of free (unbound) drug. The method was validated using nine drugs with different chemical structures and physicochemical properties. The CL(in,u) of these compounds was extrapolated from the intrinsic clearance values obtained at different concentrations of human liver microsomes and compared with that calculated by the conventional method, using microsomal intrinsic clearance values and the free fraction of drug determined by equilibrium dialysis, ultracentrifugation, or ultrafiltration. A good agreement was observed between the data generated by the LESA method and those determined by conventional procedures. The method was further evaluated using a published dataset for 10 additional drugs and found to yield intrinsic clearance data comparable to the previously reported values. LESA provides a convenient and rapid method to determine the influence of microsome binding on intrinsic clearance in a single assay.

The toxicity of opiates and their metabolites in HepG2 cells.

The toxicity of codeine (C), codeinone (CO), morphine (M), oxycodone (OC), pholcodine (P) and pholcodine-N-oxide (P-NOX) was assessed in HepG2 cells by determining cell viability via the measurement of lactate dehydrogenase (LDH) leakage through the membrane, depletion of reduced glutathione (GSH) and measurement of total protein content. Incubation of C, M, OC, P or P-NOX with HepG2 cells resulted in no significant loss of cell viability, depletion of GSH or decreased total protein content. In contrast, with CO there was a marked depletion of GSH with significant differences from control cells (P<0.05) being detected after as little as 5 min. This effect preceded the loss of cell viability and the decrease in total protein content. To identify the cause of GSH depletion during incubations with CO, the incubation solutions were analysed by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Analysis showed that a codeinone-glutathione conjugate (CO-SG) had been formed. This adduct was synthesised and characterised by LC/MS/MS and by nuclear magnetic resonance spectroscopy (NMR). CO-SG was quantified in the incubation solutions using the synthesised standard substance. Results obtained in this study support the hypothesis that the toxicity of CO may be partly due to GSH depletion. The absence of LDH leakage and GSH depletion in the incubations containing C or OC suggests, that the presence of both a double bond at Delta 7 and an adjoining keto-group in the 6-position are necessary to elicit the toxicity of M analogues with regard to GSH depletion.