7196 research outputs found
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Identification and validation of marker genes for the detection of residual iPSCs in iPSC-derived Cardiomyocytes
No full textHuman induced pluripotent stem cell (iPSC) based cell therapies represent a promising approach for regenerative medicine. However, iPSC-derived cell therapy products may contain residual undifferentiated iPSCs that could, due to their teratogenic properties and genetic instability, lead to tumor formation after implantation into patients. Hence, highly sensitive and specific methods to detect residual undifferentiated iPSCs are indispensable for safety evaluation of cell therapy products. Here, we used RNA-seq data to identify potential marker genes for iPSC impurities in iPSC-derived tissues. We demonstrated that identifying such marker genes in a tissue-specific way provides a larger and more specific set of potential marker genes than considering all tissue types in the analysis. By spiking different amount of iPSCs in iPSCs derived cardiomyocytes (iCMs), we evaluated selected candidate genes regarding their specificity as well as sensitivity in iCMs, and compared their performance to the previously suggested markers LIN28A, Nanog and TDGF1. The genes CAMKV, EPHA1, ESRG, IDO1, LECT1, LINC00678, LIN28A, LCK, L1TD1, VRTN and ZCAN10 specifically detected contaminant iPSC amongst iCMs with an LOD of 0.001-0.1%. In conclusion, we provide an approach on how to identify a set of highly specific and sensitive markers that can be used for safety assessment of iPSC-derived tissues
Accelerating Availability of Medicines to Pediatrics via Extrapolation of Efficacy from Adults
No full textImproving pediatric therapeutic development is a mission of universal importance amongst health authorities, pharmaceutical companies, academic institutions, and healthcare professionals. Following the passage of legislation in the United States and Europe, we witnessed the most significant advancement yet in pediatric data generation, resulting in added pediatric use information to almost 700 product labels. Tools to accelerate generation of data for the pediatric population are available for use today, and when utilized in accordance with current practices and laws, these tools could increase the amount and timeliness of pediatric information available for clinicians and patients. If we utilize the current laws that allow regulators to incentivize and require evidence generation, apply extrapolation and utilize modeling and simulation, as well as including adolescents in the pivotal studies alongside adults as appropriate, two strategic goals could be achieved by 2030: 1) reduce the time to pediatric approval by 50%, and 2) renew pediatric labeling information for 15 priority pediatric drugs without patent and/or exclusivity
On the Choice of Active Site Sequences for Kinase-Ligand Affinity Prediction.
No full textRecent work showed that active site rather than full-protein-sequence information improves predictive performance in kinase-ligand binding affinity prediction. To refine the notion of an "active site", we here propose and compare multiple definitions. We report significant evidence that our novel definition is superior to previous definitions and better models of ATP-noncompetitive inhibitors. Moreover, we leverage the discontiguity of the active site sequence to motivate novel protein-sequence augmentation strategies and find that combining them further improves performance
Discovery and Preclinical Pharmacology of INE963, A Potent and Fast-Acting Blood-Stage Antimalarial with a High Barrier to Resistance and Potential for Single-Dose Cure in Uncomplicated Malaria
No full textA series of 5-aryl-2-amino-imidazothiadiazole (ITD) derivatives were identified by phenotype based hit-to-lead discovery using a blood stage Plasmodium falciparum (Pf) growth inhibition assay. A lead optimization program focused on improving antimalarial potency, selectivity against human kinases, ADMET properties, and extended pharmacological profiles culmi-nated in the identification of INE963 (1), which demonstrates potent cellular activity against Pf 3D7 (EC50 = 0.006 M) and achieves ‘artemisinin-like’ kill kinetics in vitro with a parasite clearance time of <24 hours. INE963 (1) has a single dose ED90 = 11.7 mg/kg in the Pf-humanized SCID-mouse model and is fully curative without recrudescence with a single dose of 30 mg/kg. INE963 (1) also demonstrates a high barrier to resistance in drug selection studies conducted with P. falciparum blood stage cultures in vitro. In pharmacokinetic studies, INE963 (1) has low clearance (CL) with high volume of distribu-tion (Vss) across mouse, rat, dog, and thus a long half-life (T1/2) is projected in humans. Taken together, these properties craft a great potential for INE963 (1) to provide a curative therapy for uncomplicated malaria with short dosing regimens. For these reasons, INE963 (1) was progressed through GLP toxicology studies and is now undergoing Ph1 clinical trials
Quantification of accurate compositions and total abundance of homologous proteins in human tissues using conserved-plus-surrogate peptide (CPSP) approach: Application in the quantification of UDP glucuronosyltransferases
No full textCharacterization of accurate compositions and total abundance of homologous drug-metabolizing enzymes, such as UDP glucuronosyltransferases (UGTs), is important for predicting the fractional contribution of individual isoforms involved in the metabolism of a drug for applications in physiologically based pharmacokinetic (PBPK) modeling. Conventional targeted proteomics utilizes surrogate peptides, which often results in high technical and inter-laboratory variability due to peptide-specific digestion efficiency leading to data inconsistencies. To address this problem, we developed a novel universal conserved-plus-surrogate peptide (CPSP) approach for determining the accurate compositions and total or cumulative abundance of homologous UGTs in commercially available pooled human liver microsomes (HLM), human intestinal microsomes (HIM), human kidney microsomes (HKM), and human liver S9 (HLS9) fractions. The relative percent composition of UGT1A and UGT2B isoforms in human liver was 35:5:36:11:13 for UGT1A1:1A3:1A4:1A6:1A9, and 20:32:22:21:5 for UGT2B4:2B7:2B10:2B15:2B17. The human kidney and intestine also show unique compositions of UGT1As and UGT2Bs. The reproducibility of the approach was validated by assessing correlations of UGT compositions between HLM and HLS9 (R2>0.91). The analysis of conserved peptides also provided the absolute abundance for individual UGT isoforms included in this investigation as well as the total abundance (pmol/mg protein) of UGT1As and UGT2Bs across tissues, i.e., 268 and 342 (HLM), 21 and 92 (HIM), 138 and 99 (HKM), respectively. In summary, the CPSP approach could be utilized for applications in the in-vitro to in-vivo extrapolation (IVIVE) of drug metabolism and PBPK modeling
Numerical modelling of the dissolution of drug nanocrystals and its application to industrial product development
No full textThe apparent solubility of drug nanocrystals in equilibrium was experimentally determined for a drug-stabilizer system with different particle size distributions. True supersaturation was identified for ultrafine drug nanocrystals with an almost 2-fold increase compared to the thermodynamic solubility of related coarse drug crystals, highlighting their enabling potential to enhance bioavailability. The experimental results were applied to investigate in silico the associated dissolution behaviour in a closed system by numerical modelling according to the Ostwald-Freundlich and Noyes-Whitney / Nernst-Brunner equations. Calculated results were found to be in agreement with the experimental results only when the entire particle size distribution of drug nanocrystals was considered. In silico dissolution studies were conducted to simulate the complex interplay between drug nanocrystals, dissolution conditions and resulting temporal progression during dissolution up to the equilibrium state. Calculations were performed for selected in vivo and in vitro scenarios considering different drug nanocrystal particle size distributions, drug amount, dissolution media and volume. The achieved results demonstrated the importance of ultrafine drug nanocrystals for potential bioavailability improvement and the simplicity to investigate their dissolution behaviour for configurable formulation variables in product development in terms of in vivo and in vitro relevant conditions
Pegylated insulin-like growth factor-1 biotherapeutic delivery promotes rotator cuff regeneration in a rat model.
No full textTears in the rotator cuff are challenging to repair because of the complex, hypocellular, hypovascular, and movement-active nature of the tendon and its enthesis. Insulin-like Growth Factor-1 (IGF-1) is a promising therapeutic for this repair. However, its unstable nature, short half-life, and ability to disrupt homeostasis has limited its clinical translation. Pegylation has been shown to improve the stability and sustain IGF-1 levels in the systemic circulation without disrupting homeostasis. To provide localized delivery of IGF-1 in the repaired tendons, we encapsulated pegylated IGF-1 mimic and its controls (unpegylated IGF-1 mimic and recombinant human IGF-1) in polycaprolactone-based matrices and evaluated them in a pre-clinical rodent model of rotator cuff repair. Pegylated-IGF-1 mimic delivery reestablished the characteristic tendon-to-bone enthesis structure and improved tendon tensile properties within 8 weeks of repair compared to controls, signifying the importance of pegylation in this complex tissue regeneration. These results demonstrate a simple and scalable biologic delivery technology alternative to tissue-derived grafts for soft tissue repair
Enhanced tendon healing by a tough hydrogel with an adhesive side and high drug-loading capacity
No full textHydrogels that provide mechanical support and sustainably release therapeutics have been used to treat tendon injuries. However, most hydrogels are insufficiently tough, release drugs in bursts, and require cell infiltration or suturing to integrate with surrounding tissue. Here we report that a hydrogel serving as a high-capacity drug depot and combining a dissipative tough matrix on one side and a chitosan adhesive surface on the other side supports tendon gliding and strong adhesion (larger than 1,000 J m−2) to tendon on opposite surfaces of the hydrogel, as we show with porcine and human tendon preparations during cyclic-friction loadings. The hydrogel is biocompatible, strongly adheres to patellar, supraspinatus and Achilles tendons of live rats, boosted healing and reduced scar formation in a rat model of Achilles-tendon rupture, and sustainably released the corticosteroid triamcinolone acetonide in a rat model of patellar tendon injury, reducing inflammation, modulating chemokine
secretion, recruiting tendon stem and progenitor cells, and promoting macrophage polarization to the M2 phenotype. Hydrogels with ‘Janus’ surfaces and sustained-drug-release functionality could be designed for a range of biomedical applications
Photo-Brook rearrangement of acyl silanes as a new tool for photoaffinity probes design
No full textPhotoaffinity labeling is a powerful tool for the identification of small molecule-protein interactions important to drug dis-covery and medicinal chemistry, but only a handful of robust photochemical warheads exist that are capable of capturing such transient interactions. Acyl silanes are an alternative car-bene precursors to diazirines, which upon irradiation, generate an α-siloxy carbene capable of inserting into polar X–H bonds, but have remained unexplored for photoaffinity labeling appli-cations. Herein, we report the synthesis of a new class of acyl silane photochemical warheads with long-UV wavelength ab-sorption and fast rates of reaction. Small molecule (+)-JQ1 and rapamycin derived acyl silanes selectively label BRD4-BD1 and FKBP12, respectively, with minimal background. Further, acyl silane (+)-JQ1 probe 16 was used in live cell proteome profiling experiments for identification of potential off-target binders and confirmation of endogenous target engagement. Site-of-modification studies showed (+)-JQ1 probe 16 inserted into T134 and K141, consistent with known previously report-ed binding data. Together, these data highlight the untapped potential of acyl silanes as a photoaffinity probe amenable to modern workflows. Further development of this class of modu-lar warheads could lead to advances in target identification and binding site mapping