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Synthesis and crystal structures of new chiral 3-amino-2H-azirines and the Pd complex of one of them.
No full text3-Amino-2H-azirines are potentially versatile building blocks in heterocyclic and peptide synthesis. Three new 3-amino-2H-azirines have been synthesized as racemates or mixtures of diastereoisomers in cases where another chiral residue is incorporated as the exocyclic amine. The crystal structures of two of them, an approximately 1:1 diastereoisomeric mixture of (2R)- and (2S)-2-ethyl-3-[(2S)-2-(1-methoxy-1,1-diphenylmethyl)pyrrolidin-1-yl]-2-methyl-2H-azirine, C23H28N2O, 11, and 2-benzyl-3-(N-methyl-N-phenylamino)-2-phenyl-2H-azirine, C22H20N2, 12, and the third as its diastereoisomeric trans-PdCl2 complex, trans-dichlorido[(2R)-2-ethyl-2-methyl-3-(X)-2H-azirine][(2S)-2-ethyl-2-methyl-3-(X)-2H-azirine]palladium(II), where X = N-{[(1S,2S,5S)-6,6-dimethylbicyclo[3.1.1]heptan-2-yl]methyl}-N-phenylamino, [PdCl2(C21H30N2)2], 14, have been determined and the geometries of the azirine rings compared with those of 11 other 3-amino-2H-azirine structures reported in the literature. Most notable is the very long formal N-C single bond, which is, with one exception, around 1.57 Å. Each compound has crystallized in a chiral space group. The Pd atom in the trans-PdCl2 complex is coordinated by one of each of the pair of diastereoisomers, while both of the diastereoisomers share the same crystallographic site in the structure of 11; this property thereby manifesting itself as disorder. The chosen crystal of 12 is either an inversion twin or composed of a pure enantiomorph, but this could not be established specifically
A common mechanism of Sec61 translocon inhibition by small molecules.
No full textThe Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural and synthetic small molecules specifically inhibit Sec61, generating cellular effects that are useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of human Sec61 inhibited by a comprehensive panel of structurally distinct small molecules-cotransin, decatransin, apratoxin, ipomoeassin, mycolactone, cyclotriazadisulfonamide and eeyarestatin. All inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of Sec61. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain in a closed state, thereby preventing the protein-translocation pore from opening. Our study provides the atomic details of Sec61-inhibitor interactions and the structural framework for further pharmacological studies and drug design
Ribociclib Dose Justification in Advanced Breast Cancer Patients With Renal Impairment by Integrating PK, Safety and Efficacy Data
No full textBackground: Renal impairment (RI) is common in cancer patients and can alter the pharmacokinetics (PK) and thus the safety and efficacy of drugs. We assessed the impact of RI during treatment with ribociclib, a cyclin-dependent kinase 4/6 inhibitor, and determined dose recommendations for advanced breast cancer (ABC) patients with RI.
Methods: A comprehensive assessment integrating PK, safety and efficacy data from a phase I RI study in non-cancer subjects and six phase I–III trials in cancer patients was performed.
Results: Ribociclib showed higher PK exposure in subjects with RI than those with normal renal function following a single 400 mg dose in the RI study. However, in the clinical trials, both single-dose and steady‑state exposure of ribociclib was comparable between cancer patients with mild/moderate RI and those with normal renal function following the recommended starting dose of 600 mg. Model-predicted steady‑state exposure in ABC patients was also similar across the renal function groups. Progression-free survival was similar and safety profiles were generally consistent across the renal cohorts (normal/mild/moderate) in ABC patients with low-grade and manageable adverse events, demonstrating a positive benefit-risk profile.
Conclusion: From the collective evidence and considering real-world clinical setting, no dose adjustment is recommended for patients with mild/moderate RI, whereas a reduced dose is recommended for patients with severe RI. This report presented a holistic strategy to determine dose in patients with RI and demonstrated the effectiveness of integrating the data of both clinical pharmacology study and patient trials to justify doses in patients with RI
Quantitative systems pharmacology model of GITR-mediated T cell dynamics in tumor microenvironment.
No full textT cell interaction in the tumor microenvironment is a key component of immuno-oncology therapy. Glucocorticoid-induced tumor necrosis factor receptor (TNFR)-related protein (GITR) is expressed on immune cells including regulatory T cells (Tregs) and effector T cells (Teffs). Preclinical data suggest that agonism of GITR in combination with Fc-γ receptor-mediated depletion of Tregs results in increased intratumoral Teff:Treg ratio and tumor shrinkage. A novel quantitative systems pharmacology (QSP) model was developed for the murine anti-GITR agonist antibody, DTA-1.mIgG2a, to describe the kinetics of intratumoral Tregs and Teffs in Colon26 and A20 syngeneic mouse tumor models. It adequately captured the time profiles of intratumoral Treg and Teff and serum DTA-1.mIgG2a and soluble GITR concentrations in both mouse models, and described the response differences between the two models. The QSP model provides a quantitative understanding of the trade-off between maximizing Treg depletion versus Teff agonism, and offers insights to optimize drug design and dose regimen
Screening for Differences in Early Exposure in the Fasted State with in Vitro Methodologies can be Challenging: Experience with the BioGIT System.
No full textThe Biorelevant Gastrointestinal Transfer (BioGIT) system is a useful screening tool for assessing the impact of dose and/or formulation on early exposure after administration of immediate release or enabling drug products with a glass of water in the fasted state. The objective of this study was to investigate potential limitations. BioGIT experiments were performed with five low solubility active pharmaceutical ingredients with weakly alkaline characteristics: mebendazole (tablet and chewable tablet), Compound E (aqueous solutions, three doses), pazopanib-HCl (Votrient™ tablet, crushed Votrient™ tablet and aqueous suspension), Compound B-diHCl (hard gelatin capsule, three doses) and Compound C (hard gelatin capsule containing nanosized drug and hard gelatin capsule containing micronized drug). For all formulation or dose comparisons the ratio of mean BioGIT AUC0-50 min values was not predictive of the ratio of mean plasma AUC0-60 min values which became available after completion of BioGIT experiments. BioGIT experimental conditions have not been designed to simulate the gastrointestinal drug transfer process after administration of chewable tablets or aqueous solutions, therefore, BioGIT may not be useful for the assessment of intraluminal performance early after administration of such drug products. Also, based on this study, BioGIT may not be useful in investigating the impact of dose and/or formulation on early exposure when the dose is not administered with a glass of water to fasted healthy individuals or when BioGIT data are highly variable. Finally, the rapid dissolution of nanocrystals after administration of low solubility weak bases may require adjustment of the pH in the gastric compartment of BioGIT to slightly higher pH values. Limitations identified in this study for the BioGIT system may be also relevant to other in vitro systems proposed for similar evaluations
Closed System Transfer Devices (CSTDs): understanding potential over- and under- dosing of liquid vial drug products and how to generally mitigate.
No full textClosed system transfer devices (CSTDs) are a major challenge for drug manufacturers to assess and assure drug compatibility and acceptable dosing accuracy for a range of clinical administration strategies. In this article, we systematically investigate parameters affecting the loss of product during transfer by CSTDs from vials to infusion bags. We show that liquid volume loss increases with vial size, vial neck diameter, and solution viscosity - while dependent on stopper design. We further compared CSTDs' performance with a traditional syringe transfer and learned that loss is larger for CSTDs than for syringe transfer. Based on experimental data, a statistical model was developed to predict drug loss upon transfer by CSTDs. The model predicted that, for single dose vials with USP conforming overfill, a complete extraction and transfer of the full dose can be assured for a broad range of CSTDs, product viscosities, and vial types (2R, 6R, 10R, 20R) if a flush (of syringe, syringe adaptor, bag spike) is performed. The model also predicted that complete transfer cannot be achieved for fill volumes ≤ 2.0 mL. For multi-dose vials and pooling of several vials, respectively, the effective dose transfer (i.e., ≥ 95%) for all CSTDs tested was predicted to be achieved if a minimum of 5.0 mL is transferred
Drug Development Through the Prism of Biomarkers: Current State and Future Outlook
No full textno abstrac
Improved Repeat Protein Stability by Combined Consensus and Computational Protein Design.
No full textHigh protein stability is an important feature for proteins used as therapeutics, as diagnostics, and in basic research. We have previously employed consensus design to engineer optimized Armadillo repeat proteins (ArmRPs) for sequence-specific recognition of linear epitopes with a modular binding mode. These designed ArmRPs (dArmRPs) feature high stability and are composed of M-type internal repeats that are flanked by N- and C-terminal capping repeats that protect the hydrophobic core from solvent exposure. While the overall stability of the designed ArmRPs is remarkably high, subsequent biochemical and biophysical experiments revealed that the N-capping repeat assumes a partially unfolded, solvent-accessible conformation for a small fraction of time that renders it vulnerable to proteolysis and aggregation. To overcome this problem, we have designed new N-caps starting from an M-type internal repeat using the Rosetta software. The superior stability of the computationally refined models was experimentally verified by circular dichroism and nuclear magnetic resonance spectroscopy. A crystal structure of a dArmRP containing the novel N-cap revealed that the enhanced stability correlates with an improved packing of this N-cap onto the hydrophobic core of the dArmRP. Hydrogen exchange experiments further show that the level of local unfolding of the N-cap is reduced by several orders of magnitude, resulting in increased resistance to proteolysis and weakened aggregation. As a first application of the novel N-cap, we determined the solution structure of a dArmRP with four internal repeats, which was previously impeded by the instability of the original N-cap
Complex binding and elution behavior of therapeutic proteins under column overloading conditions
No full textThe binding and elution behavior of two therapeutic bispecific monoclonal antibodies (bsAbs) on the strong cation exchange resin POROS™ XS is investigated and modeled over broad ranges of pH, salt concentrations, and column loadings. One of the two bsAbs exhibits common Langmuir elution behavior under high loading and column overloading conditions, whilst the other bsAb exhibits uncommon anti-Langmuir elution behavior as a consequence of multi-layer binding on the stationary phase surface. The frequently used Steric Mass Action (SMA) model modified with an activity coefficient for the salt in solution is used to simulate the Langmuirian elution behavior. A Self-Association Steric Mass Action (SAS-SMA) model extended with two activity coefficients for the protein and salt in solution is applied to describe the anti-Langmuir elution behavior. The SAS-SMA model is able to describe self-dimerization on the resin surface and thus can predict anti-Langmuir elution behavior. The binding models are each combined with a lumped rate model to describe mass transfer inside the chromatography column. To apply these models for describing protein elution over wide ranges of pH, the pH-dependences of all model parameters, including the linear and especially the non-linear model parameters, are investigated, described, and implemented into the binding models. Therefore, extensive data sets were generated that consist of linear gradient elution experiments comprising a pH range from pH 4.5 to 8.9 and column loadings from 0.5 to 90.0 mgbsAb/mLresin. The modeling results of both antibodies show that the pH of the mobile phase has a strong influence on the non-linear model parameters, thus valuable process insights can be gained by interpretation of these results. An increasing buffer pH leads to an increase in binding sites shielded by the antibodies, whilst self-dimerization on the resin surface becomes less with increasing pH. Empirical correlations describing the non-linear model parameters as functions of pH are established and implemented into the SMA and SAS-SMA formalisms. The functionality of these modified pH-dependent binding models is verified with linear salt, pH and dual gradient elution experiments using single-component simulations. Most of these experiments can be accurately predicted under high loading and overloading conditions, whereby especially the peak shapes are well-described. Slight discrepancies between the simulated and experimental data can be observed for some of these experiments, especially when they were performed under overloading conditions. In this dissertation, it is clearly shown that these discrepancies are not primarily a consequence of limitations of the SMA and SAS-SMA models. At lower pH values (pH ≤ 5.3), overloading phenomena such as protein breakthrough during the loading phase, additional peaks, and peak-shoulders occur. The outcomes of additional experiments in which the antibodies were loaded onto the column with different counterion concentrations and loading times show that intraparticle diffusion effects and conformational changes of the bsAbs are responsible for these overloading phenomena at low pH. The applied lumped rate mass transfer model is not adequate here since it cannot describe hindered intraparticle transport and should be extended to consider these effects. Additional peaks and peak shoulders due to bsAb conformations can only be predicted by describing multi-state binding, which is shown in this dissertation for one case by a simple extension to a multi-component simulation. Furthermore, it is shown that the description of complex peak shapes arising due to competitive binding and multi-component elution of the antibodies' charge variants cannot be adequately predicted using single-component simulations. However, an extension of the model to a simple multi-component system consisting of two charge variants enables accurate prediction of some of these complex elution profiles
Activation of human STING by a molecular glue-like compound
No full textStimulator of interferon genes (STING) is a dimeric transmembrane adaptor protein that plays an important role in the human innate immune response to infection and has been therapeutically exploited for its antitumor activity. The activation of STING requires its high-order oligomerization, which could be induced by binding of the endogenous ligand cGAMP to the cytosolic ligand-binding domain or binding of an artificial compound, C53, to a transmembrane domain pocket. Here we report the discovery through functional screens of a new class of compounds, named NVS-STGs, that activate human STING using an alternative mechanism. Our cryo-electron microscopic (Cryo-EM) structures show that NVS-STG2 induces the high-order oligomerization of human STING by binding to a pocket between the transmembrane domains of the neighboring STING dimers, effectively acting as a molecular glue. Our functional assays showed that NVS-STG2 could elicit potent STING-mediated immune responses in cells and antitumor activities in animal models. These findings establish a distinct class of agonists that can act synergistically with both cGAMP and C53 in activating STING for cancer therapy