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Time matters – in vitro cellular disposition kinetics help rationalizing cellular potency disconnects
No full textLoss in 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.
We developed a novel cellular disposition assay in MDCK cells to determine passive uptake clearance (PSinf), cell-to-medium ratios at steady-state (Kp) and the time to reach 90% steady-state (TTSS90) from a single experiment in a high-throughput format.
The assay was validated using 40 marketed drugs, showing a wide distribution of PSinf and Kp values. The parameters generally correlated with transcellular permeability and lipophilicity, while PSinf data revealed better resolution in the high and low permeability ranges compared to traditional permeability data. A linear relationship between the Kp/PSinf ratio and TTSS90 was mathematically derived and experimentally validated, demonstrating the dependency of TTSS90 on the rate and extent of cellular accumulation.
Cellular disposition parameters could explain potency (IC50) disconnects noted for seven Bruton’s tyrosine kinase degrader compounds in a cellular potency assay. In contrast to transcellular permeability, PSinf data enabled identification of the compounds with IC50 disconnects based on their time to reach equilibrium. Overall, the novel assay offers the possibility to address potency disconnects in early drug discovery
People of TM: Video of Franziska Saxer
No full textThe video will be used for an external social media engagement campaign on platforms like linked-in, facebook, etc. featuring stories of people in TM. No IP related content
Defining the substrate envelope of SARS-CoV-2 main protease to predict and avoid drug resistance.
No full textCoronaviruses can evolve and spread rapidly to cause severe disease morbidity and mortality, as exemplified by SARS-CoV-2 variants of the COVID-19 pandemic. Although currently available vaccines remain mostly effective against SARS-CoV-2 variants, additional treatment strategies are needed. Inhibitors that target essential viral enzymes, such as proteases and polymerases, represent key classes of antivirals. However, clinical use of antiviral therapies inevitably leads to emergence of drug resistance. In this study we implemented a strategy to pre-emptively address drug resistance to protease inhibitors targeting the main protease (Mpro) of SARS-CoV-2, an essential enzyme that promotes viral maturation. We solved nine high-resolution cocrystal structures of SARS-CoV-2 Mpro bound to substrate peptides and six structures with cleavage products. These structures enabled us to define the substrate envelope of Mpro, map the critical recognition elements, and identify evolutionarily vulnerable sites that may be susceptible to resistance mutations that would compromise binding of the newly developed Mpro inhibitors. Our results suggest strategies for developing robust inhibitors against SARS-CoV-2 that will retain longer-lasting efficacy against this evolving viral pathogen
Tolerance Interval Approach for the Determination of Overfill of Liquid Parenteral Drug Products.
No full textLiquid parenteral products contain an overfill to ensure withdrawal of the declared volume. The overfill must be sufficiently high to compensate for the expected loss during product preparation and administration, but it should also be minimized to prevent accidental overdosing and unforeseen dose splitting of single-dose products. Finding the right balance between too much and too little overfill with an acceptable risk of product failure is challenging and requires consideration of the relevant sources of variability of the extractable volume. This article provides a novel approach for the calculation of the required overfill based on tolerance interval methodology. In a first step, a tolerance interval multiplier from the literature is proposed, and a simulation study is conducted to assess the appropriateness of its use for overfill determination. In a second step, this multiplier is adapted to cover operator-to-operator variability in the loss data and compared with other multipliers via a second simulation study. Use of a tolerance interval multiplier enables adaptation of the overfill such that the risk of not reaching the minimum extractable volume fulfills predefined acceptance criteria. By this, the scientific justification of the selected overfill is strengthened and control over a critical quality attribute is improved
Model-informed drug development for immuno-oncology agonistic anti-GITR antibody GWN323: Dose selection based on MABEL and biologically active dose.
No full textGWN323, an agonistic human anti-GITR (glucocorticoid-induced TNFR-related protein) IgG1 antibody, was studied clinically as an immuno-oncology therapeutic agent. A model-based minimum anticipated biological effect level (MABEL) approach integrating in vitro and in vivo data informed dose selection for the first-in-human (FIH) study. Data evaluated included pharmacokinetics (PK) of DTA-1.mIgG2a (mouse surrogate GITR antibody for GWN323), target-engagement pharmacodynamic (PD) marker soluble GITR (sGITR), tumor shrinkage in Colon26 syngeneic mice administered with DTA-1.mIgG2a, cytokine release of GWN323 in human peripheral blood mononuclear cells, and GITR binding affinity. A PK model was developed to describe DTA-1.mIgG2a PK, and its relationship with sGITR was also modeled. Human GWN323 PK was predicted by allometric scaling of mouse PK. Based on the totality of PK/PD modeling and in vitro and in vivo pharmacology and toxicology data, MABEL was estimated to be 3-10 mg once every 3 weeks (Q3W), which informed the starting dose selection of the FIH study. Based on tumor kinetic PK/PD modeling of tumor inhibition by DTA-1.mIgG2a in Colon26 mice and the predicted human PK of GWN323, the biologically active dose of GWN323 was predicted to be 350 mg Q3W, which informed the dose escalation of the FIH study. GWN323 PK from the FIH study was described by a population PK model; the relationship with ex vivo interleukin-2 release, a target-engagement marker, was also modeled. The clinical PK/PD modeling data supported the biological active dose projected from the translational PK/PD modeling in a "learn and confirm" paradigm of model-informed drug development of GWN323
Refractive Index to Monitor Solid-Phase Oligonucleotide Synthesis
No full textSolid-phase oligonucleotide synthesis is a mature and efficient automated process, which is used in academia and industry since decades. However, some steps are still run in a black box modus and only a few process analytical technology tools for in-process control are available, namely UV-Vis spectroscopy and conductivity. In order to ensure high yields in each of the numerous steps, the process is usually performed using an excess of starting materials, reagents, solvents. The reaction times are defined according to standard procedures and not the real need of the reaction.. In consequence, Solid-phase oligonucleotide synthesis suffers from generating large amounts of toxic waste and it is a challenge from the sustainability perspective.. One approach to make more sustainable that process is to use add more complementary monitoring tools that provide online information about the progress of the process. Herein, we disclose our promising preliminary results in the use of refractive index to monitor solid phase oligonucleotide synthesis
Discovery of Small Molecules that Target a Tertiary-Structured RNA
No full textThere is growing interest in therapeutic intervention that targets disease-relevant RNAs using
small molecules. While there have been some successes in RNA-targeted small molecule
discovery, a deeper understanding of structure-activity relationships in pursuing these targets has
remained elusive. One of the best studied tertiary-structured RNAs is the theophylline aptamer,
which binds theophylline with high affinity and selectivity. Although not a drug target, this
aptamer has had many applications, especially pertaining to genetic control circuits. Heretofore,
no compound has been shown to bind the theophylline aptamer with greater affinity than
theophylline itself. However, by carrying out a high-throughput screen of low molecular weight
compounds, several unique hits were identified that are chemically distinct from theophylline
and bind with up to 340-fold greater affinity. Multiple atomic-resolution X-ray crystal structures
were determined to investigate the binding mode of theophylline and four of the best hits. These
structures reveal both the rigidity of the theophylline aptamer binding pocket and the opportunity
for other ligands to bind more tightly in this pocket by forming additional hydrogen bonding
interactions. These results give encouragement that the same approaches to drug discovery that
have been applied so successfully to proteins can also be applied to RNAs
A behavior and physiology-based decision support tool to predict thermal comfort and stress in non-pregnant, mid-gestation, and late-gestation sows
No full textBackground
Although thermal indices have been proposed for swine, none to our knowledge differentiate by reproductive stage or predict thermal comfort using behavioral and physiological data. The study objective was to develop a behavior and physiology-based decision support tool to predict thermal comfort and stress in multiparous (3.28 ± 0.81) non-pregnant (n = 11), mid-gestation (n = 13), and late-gestation (n = 12) sows.
Results
Regression analyses were performed using PROC MIXED in SAS 9.4 to determine the optimal environmental indicator [dry bulb temperature (TDB) and dew point] of heat stress (HS) in non-pregnant, mid-gestation, and late-gestation sows with respiration rate (RR) and body temperature (TB) successively used as the dependent variable in a cubic function. A linear relationship was observed for skin temperature (TS) indicating that TDB rather than the sow HS response impacted TS and so TS was excluded from further analyses. Reproductive stage was significant for all analyses (P < 0.05). Heat stress thresholds for each reproductive stage were calculated using the inflections points of RR for mild HS and TB for moderate and severe HS. Mild HS inflection points differed for non-pregnant, mid-gestation, and late gestation sows and occurred at 25.5, 25.1, and 24.0 °C, respectively. Moderate HS inflection points differed for non-pregnant, mid-gestation, and late gestation sows and occurred at 28.1, 27.8, and 25.5 °C, respectively. Severe HS inflection points were similar for non-pregnant and mid-gestation sows (32.9 °C) but differed for late-gestation sows (30.8 °C). These data were integrated with previously collected behavioral thermal preference data to estimate the TDB that non-pregnant, mid-gestation, and late-gestation sows found to be cool (TDB < TDB preference range), comfortable (TDB = TDB preference range), and warm (TDB preference range < TDB < mild HS).
Conclusions
The results of this study provide valuable information about thermal comfort and thermal stress thresholds in sows at three reproductive stages. The development of a behavior and physiology-based decision support tool to predict thermal comfort and stress in non-pregnant, mid-gestation, and late-gestation sows is expected to provide swine producers with a more accurate means of managing sow environments
CLK1/CLK2-driven signalling at the Leishmania kinetochore is captured by spatially referenced proximity phosphoproteomics.
No full textKinetochores in the parasite Leishmania and related kinetoplastids appear to be unique amongst eukaryotes and contain protein kinases as core components. Using the kinetochore kinases KKT2, KKT3 and CLK2 as baits, we developed a BirA* proximity biotinylation methodology optimised for sensitivity, XL-BioID, to investigate the composition and function of the Leishmania kinetochore. We could detect many of the predicted components and also discovered two novel kinetochore proteins, KKT24 and KKT26. Using KKT3 tagged with a fast-acting promiscuous biotin ligase variant, we took proximity biotinylation snapshots of the kinetochore in synchronised parasites. To quantify proximal phosphosites at the kinetochore as the parasite progressed through the cell cycle, we further developed a spatially referenced proximity phosphoproteomics approach. This revealed a group of phosphosites at the kinetochore that were highly dynamic during kinetochore assembly. We show that the kinase inhibitor AB1 targets CLK1/CLK2 (KKT10/KKT19) in Leishmania leading to defective cytokinesis. Using AB1 to uncover CLK1/CLK2 driven signalling pathways important for kinetochore function at G2/M, we found a set of 16 inhibitor responsive kinetochore-proximal phosphosites. Our results exploit new proximity labelling approaches to provide a direct analysis of the Leishmania kinetochore, which is emerging as a promising drug target
The bioequivalence between pediatric-centric valsartan oral solution and extemporaneous suspension formulations
No full textThe bioequivalence of valsartan 160 mg oral solution compared to extemporaneous suspension was assessed in a single-dose, open-label, randomized, two-period, two-way crossover study in 82 healthy adults. The participants were randomized (1:1) to receive a single dose of the solution or suspension formulation in each of the two treatment periods. Serial blood samples for pharmacokinetic (PK) evaluation were collected up to 48 hours post-dose. The PK parameters were estimated by noncompartmental methods and analyzed as per bioequivalence criteria of statistical analysis. The peak plasma concentration (Cmax) of valsartan was reached with median time (Tmax) of 1 and 3 hours with solution and suspension formulation, respectively. Compared to suspension formulation, the mean Cmax with solution formulation was higher by 32% (90% CI:1.27–1.38) while the geometric mean ratios (1.09) and the associated 90% CIs (1.05–1.13) of both the areas under the concentration time-curves [AUC (0-t) and AUC (0-∞)] were contained in the required range of 0.80–1.25. No new safety signals were observed with either of the formulations