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Unambiguous identification of α-Gal epitopes in intact monoclonal antibodies by NMR spectroscopy.
No full textThe α-Gal epitope consisting of the terminal trisaccharide Galα1,3Galβ1,4GlcNAc exposed on cell or protein surfaces can cause severe immune reactions, such as hypersensitivity reactions, in humans. This epitope is also called the xenotransplantation epitope because it is one of the main reasons for the rejection of non-human organ transplants by the human innate immune response. Recombinant therapeutic proteins expressed in murine cell lines may contain α-Gal epitopes, and therefore their absence or presence needs to be tightly monitored to minimize any undesired adverse effects. The analytical identification of α-Gal epitopes in glycoproteins using the common standard techniques based on liquid chromatography and mass spectrometry is challenging, mainly due to the isobaricity of hexose stereoisomers. Here, we present a straightforward NMR approach to detect the presence of α-Gal in biotherapeutics based on a quick screen with sensitive 1H-1H TOCSY spectra followed by a confirmation using 1H-13C HSQC spectra.Abbreviations: α-Gal: α1,3-linked galactose; AGC: automatic gain control; CHO: Chinese hamster ovary; CE: capillary electrophoreses coupled to mass spectrometry; COSY: correlation spectroscopy; DSS: 2,2-dimethyl-2-silapentane-5-sulfonate; DTT: dithiothreitol; GlcNAc: N-acetyl glusomamine; HCD: higher-energy collisional dissociation; HMBC: heteronuclear multiple-bond correlation; HPLC: high-performance liquid chromatography; HSQC: heteronuclear single-quantum corre; LacNAc: N-acetyl lactosamine; mAb: monoclonal antibody; MS: mass spectrometry; NMR: nuclear magnetic resonance; NOESY: 2D) nuclear Overhauser spectroscopy; PEG: polyethylenglycol; pH*: observed pH meter reading without correction for isotope effects; PTM: post-translational modification; TCEP: tris(2-carboxyethyl) phosphine hydrochloride; TOCSY: total correlation spectroscopy; xCGE-LIF: multiplex capillary gel electrophoresis with laser-induced fluorescence detection
Novel Bruton's tyrosine kinase inhibitor remibrutinib: Assessment of drug-drug interaction potential as a perpetrator of cytochrome P450 enzymes and drug transporters and the impact of covalent binding on possible drug interactions.
No full textPharmacokinetic drug-drug interactions (DDIs) are investigated to ensure safety for patients receiving concomitant medications. Here, we present a strategy to characterise the DDI potential of remibrutinib, as an inhibitor of drug-metabolising enzymes and drug transporters, and as an inducer. Initial in vitro studies were performed, followed by a biomarker-based assessment of induction in a first in human study, concluded by a clinical study to verify initial results. Remibrutinib is a covalent inhibitor of Bruton's Tyrosine kinase (BTKi) carrying a reactive acrylamide moiety (warhead), thus the potential contribution of covalent binding (off-target) to observed interactions was investigated as this could lead to prolonged and more potent drug interactions.DDI assessment was focused on the putative inhibition of key metabolic enzymes (Cytochrome P450, CYP), drug transporters and a potential effect on oral contraceptives (OC) by induction of enzymes that are involved in their clearance (CYP3A4). The impact of covalent binding was assessed by synthesising an identical reference molecule but with an inactivated warhead.An interaction potential of limited clinical relevance was revealed for remibrutinib for CYP enzymes and drug transporters. The reactive warhead of remibrutinib had no impact on CYP enzyme and transporter inhibition, including time-dependent inhibition of CYP3A4, but may increase the induction potential of remibrutinib.Observed inhibition of metabolic enzymes indicated that remibrutinib is a weak inhibitor of CYP3A4 and CYP2C9 and is not a clinically relevant inhibitor of uptake and efflux transporters, except for intestinal P-glycoprotein and breast cancer resistance protein inhibition. OC may be safely administered and are effective when given with pharmacologically relevant doses of remibrutinib
Publication of results from DFV890D12201 study: DFV890, a new oral NLRP3 inhibitor, tested for the treatment of SARS-CoV-2 infected patients with COVID-19 pneumonia and impaired respiratory function: Results from a randomised Phase 2 trial
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A practical approach to the optimization of positron emission tomography imaging agents for the central nervous system.
No full textThe discovery of novel imaging agents for positron emission tomography (PET) relies on medicinal chemistry best practices, including a good understanding of molecular and pharmacological properties required for the acquisition of relevant, high-quality images. This short note reviews the characteristics of a series of clinically successful imaging agents, providing guidance for the optimization of such molecular tools. PET imaging plays an important role in staging disease and in helping clinical dose selection, which is critical for the efficient development of drug candidates
Scratch that itch: Farrowing crate scratching enrichment for sows
No full textDeveloping effective enrichments is important for improving pig (Sus scrofa) welfare as it increases species-specific behaviours, decreases abnormal behaviours, and increases time active. However, few enrichments are available for sows in farrowing crates. Pigs are often observed to scratch, or rub against objects, however enrichments designed to provide a scratching outlet have never been tested in sows. We examined the behaviour and welfare of sows in farrowing crates when they were presented with one of two types of scratchpad enrichment. Sows (n = 18) of parities two (P2) and three (P3) were housed for 25 days and assigned no enrichment (Control) or a scratch-pad made of plastic mats (Plastic) or coir fibre mats (Fibre). Parity two Plastic sows scratched for a longer total duration than P2 and P3 Fibre sows, P3 Plastic sows, and P2 Control sows. Parity two Plastic sows also displayed scratching bouts more frequently than all except P3 Control sows. There were no body lesion differences between treatments. Abnormal behaviour and proportion of time spent in different postures also did not differ between treatments. Plastic scratch-pads may be a suitable enrichment for farrowing crates as they increased the natural behaviour of scratching. More research is needed to refine the scratch-pad design and measure motivation before it can be concluded that scratch-pads are a successful enrichment that should be implemented on-farm
Time matters – In vitro cellular disposition kinetics help identifying disconnects in cell-based potency assays
No full text1. Decreasing potency is commonly observed in early drug discovery when moving from biochemical to more complex cellular systems. Among other factors low permeability is often considered to cause such potency disconnects.
2. We developed a novel cellular disposition assays in MDCK cells to determine initial uptake rate (PSinf), cell-to-medium ratios under steady-state (Kp) and the time to reach equilibrium (TTSS90) from a single experiment in a high throughput assay.
3. The assay was validated using 40 marketed drugs, showing a wide distribution of PSinf and Kp data. The parameters generally correlated with transwell permeability and lipophilicity (logD), while PSinf data revealed better resolution in the high and low permeability ranges. A linear correlation between the Kp/PSinf ratio and TTSS90 was found, demonstrating the dependency of TTSS90 on the rate and extent of a compound to accumulate in cells.
4. Cellular disposition parameters could explain potency (IC50) disconnects for eight BTK degrader compounds in a cellular potency assay. In contrast to transwell permeability, PSinf data enabled differentiation of the compounds with and without IC50 disconnects based on their time to reach equilibrium. Overall, the novel assay offers the possibililty to address potency disconnects during an early drug discovery phase
Near infrared spectroscopy for blend uniformity monitoring: an innovative qualitative application based on the coefficient of determination
No full textBlending process is a critical unit operation in the pharmaceutical industry during the solid dosage form production. Near infrared spectroscopy (NIRS) is a powerful analytical tool to assess the blend homogeneity in real-time. In this paper, a new methodology for blending process monitoring and for end point confirmation is proposed. Quantitative procedure validation and maintenance of NIR procedure are time-consuming activities that can prevent the adoption of PAT tools in the pharmaceutical industry. Clearly, there is a need in the industry for simpler and more intuitive qualitative blend monitoring analytical procedure that are easy to build, validate and maintain. The method we are introducing herein consist of tracking the trend of CD between a mean reference spectrum from a homogeneous batch and the NIRS spectra that are recorded during the blending operation. Four formulations of commercial products were selected from different scales –including low dosage solid form- to show the usefulness of the method. In addition, this analytical procedure is tested with data from two different types of spectrometers (diode array instruments). Method calibration model was performed with five batches (representing expected process variability) for each product: one for the computation of the homogeneous batch target spectrum and four to compute the limit of the CD (CD) values related to anticipated and acceptable homogeneity. Method validation was performed with homogeneous batches and with challenge spectra for assessing the specificity of the method. Real-world examples (e.g. technical, validation batches and clinical batches) were presented in order to demonstrate that this method is able to detect inhomogeneous batches. The new qualitative method presented in this paper is useful for determination of the blending end-point, in assessing the blend uniformity in real-time and in increasing process understanding during development and troubleshooting
Pharmacokinetics of Tropifexor, a Potent Farnesoid X Receptor Agonist, in Participants With Varying Degrees of Hepatic Impairment.
No full textTropifexor, a non-bile acid farnesoid X receptor (FXR) agonist, has dose-proportional pharmacokinetics and no obvious major enterohepatic circulation. This open-label study investigated the effect of hepatic impairment (HI), as determined by Child-Pugh grade, on tropifexor's pharmacokinetics, safety, and tolerability following a 200-μg dose in the fasted state. Blood samples were collected through 168 hours after dosing for quantification and plasma protein-binding determination. Total tropifexor exposure was comparable across participants with HI vs those with normal hepatic function. Tropifexor was highly protein bound (>99%) in human plasma across participants of all groups. The average unbound fractions (percentage free) were 0.14% in participants with normal hepatic function and mild HI, which increased to 0.17% and 0.24% in participants with moderate and severe HI, respectively. Similar unbound drug exposure was noted in participants with mild HI and normal hepatic function. Participants with moderate HI (N = 8) had a 1.6-fold increase in unbound exposure (area under the plasma concentration-time curve from time 0 to infinity [AUCinf,u ]) and a 1.3-fold increase in maximal exposure (Cmax,u ) vs those with normal hepatic function (geometric mean ratio: AUCinf,u , 1.64 [90%CI, 1.25-2.16]; Cmax,u , 1.30 [90%CI, 0.96-1.76]). Participants with severe HI (N = 8) had a 1.6-fold increase in AUCinf,u (1.61 [90%CI, 1.04-2.49]) and comparable Cmax,u (1.02 [90%CI, 0.60-1.72]) compared to participants with normal hepatic function. Tropifexor was well tolerated. The relative insensitivity of tropifexor to HI offers the potential to treat patients with severe liver disease without dose adjustment
Discovery solubility measurement and assessment with drug development in mind
No full textSolubility is a key physicochemical property for the success of any drug candidate. While the methods used and their rationales for determining solubility are subject to project needs and stages along the drug discovery-drug development pipeline, an artificial boundary may exist at the discovery- development interface. This boundary results in less effective solubility knowledge sharing and data integration among scientists in both drug discovery and drug development. Herein, we present a refreshed perspective on solubility. Solubility experimentation is not a one size fits all measurement; instead, we stress the importance of constructing a seamless solubility understanding of a molecule as it progresses from a new chemical entity into a drug product. Expect, welcome, and brace yourself for an evolution thereof