7196 research outputs found
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Combined Free-Energy Calculation and Machine Learning Methods for Understanding Ligand Unbinding Kinetics.
No full textThe determination of drug residence times, which define the time an inhibitor is in complex with its target, is a fundamental part of the drug discovery process. Synthesis and experimental measurements of kinetic rate constants are, however, expensive and time consuming. In this work, we aimed to obtain drug residence times computationally. Furthermore, we propose a novel algorithm to identify molecular design objectives based on ligand unbinding kinetics. We designed an enhanced sampling technique to accurately predict the free-energy profiles of the ligand unbinding process, focusing on the free-energy barrier for unbinding. Our method first identifies unbinding paths determining a corresponding set of internal coordinates (ICs) that form contacts between the protein and the ligand; it then iteratively updates these interactions during a series of biased molecular dynamics (MD) simulations to reveal the ICs that are important for the whole of the unbinding process. Subsequently, we performed finite-temperature string simulations to obtain the free-energy barrier for unbinding using the set of ICs as a complex reaction coordinate. Importantly, we also aimed to enable the further design of drugs focusing on improved residence times. To this end, we developed a supervised machine learning (ML) approach with inputs from unbiased "downhill" trajectories initiated near the transition state (TS) ensemble of the string unbinding path. We demonstrate that our ML method can identify key ligand-protein interactions driving the system through the TS. Some of the most important drugs for cancer treatment are kinase inhibitors. One of these kinase targets is cyclin-dependent kinase 2 (CDK2), an appealing target for anticancer drug development. Here, we tested our method using two different CDK2 inhibitors for the potential further development of these compounds. We compared the free-energy barriers obtained from our calculations with those observed in available experimental data. We highlighted important interactions at the distal ends of the ligands that can be targeted for improved residence times. Our method provides a new tool to determine unbinding rates and to identify key structural features of the inhibitors that can be used as starting points for novel design strategies in drug discovery
Proteomic analysis identifies circulating proteins associated with plasma amyloid βeta and incident dementia
No full textBackground
Plasma amyloid beta (Aβ42, Aβ40, and Aβ42/Aβ40), biomarkers of the Alzheimer’s form of dementia, are under consideration for clinical use. The associations of these peptides with circulating proteins may identify novel plasma biomarkers of dementia and inform peripheral factors influencing the levels of these peptides.
Methods
We analyzed the association of these three plasma Aβ measures with 4,638 circulating proteins among a subset of the participants of the Atherosclerosis Risk in Communities (ARIC) study (midlife: n=1,955; late-life: n=2,082), related the Aβ-associated proteins with incident dementia in the overall ARIC cohort (midlife: n=11,069, late-life: n=4,110) with external replication in the Age, Gene/Environment Susceptibility (AGES) - Reykjavik Study (n=4,973), estimated the proportion of Aβ variance explained, and conducted enrichment analyses to characterize the proteins associated with the plasma Aβ peptides.
Results
At midlife, of the 296 Aβ-associated proteins, 8 were associated with incident dementia from midlife and late-life in ARIC, and NPPB, IBSP, and THBS2 were replicated in AGES. At late-life, of the 34 Aβ-associated proteins, none were associated with incident dementia at midlife, and kidney function explained 10%, 12%, 0.2% of the variance of Aβ42, Aβ40, and Aβ42/Aβ40, respectively. Aβ42-associated proteins at midlife were found to be enriched in the liver and those at late-life were found to be enriched in the spleen.
Conclusions
This study identifies circulating proteins associated with plasma Aβ levels and incident dementia and informs peripheral factors associated with plasma Aβ levels
Immunosuppression Profile of CFZ533 (Iscalimab), a Non-Depleting Anti-CD40 Antibody, and the Presence of Opportunistic Infections in a Rhesus Monkey Toxicology Study
No full textImmunosuppression Profile of CFZ533 (Iscalimab), a Non-Depleting Anti-CD40 Antibody, and the Presence of Opportunistic Infections in a Rhesus Monkey Toxicology Study
miR-579-3p Controls Hepatocellular Carcinoma Formation by Regulating the Phosphoinositide 3-Kinase-Protein Kinase B Pathway in Chronically Inflamed Liver.
No full textChronic liver inflammation causes continuous liver damage with progressive liver fibrosis and cirrhosis, which may eventually lead to hepatocellular carcinoma (HCC). Whereas the 10-year incidence for HCC in patients with cirrhosis is approximately 20%, many of these patients remain tumor free for their entire lives. Clarifying the mechanisms that define the various outcomes of chronic liver inflammation is a key aspect in HCC research. In addition to a wide variety of contributing factors, microRNAs (miRNAs) have also been shown to be engaged in promoting liver cancer. Therefore, we wanted to characterize miRNAs that are involved in the development of HCC, and we designed a longitudinal study with formalin-fixed and paraffin-embedded liver biopsy samples from several pathology institutes from Switzerland. We examined the miRNA expression by nCounterNanostring technology in matched nontumoral liver tissue from patients developing HCC (n = 23) before and after HCC formation in the same patient. Patients with cirrhosis (n = 26) remaining tumor free within a similar time frame served as a control cohort. Comparison of the two cohorts revealed that liver tissue from patients developing HCC displayed a down-regulation of miR-579-3p as an early step in HCC development, which was further confirmed in a validation cohort. Correlation with messenger RNA expression profiles further revealed that miR-579-3p directly attenuated phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) expression and consequently protein kinase B (AKT) and phosphorylated AKT. In vitro experiments and the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology confirmed that miR-579-3p controlled cell proliferation and cell migration of liver cancer cell lines. Conclusion: Liver tissues from patients developing HCC revealed changes in miRNA expression. miR-579-3p was identified as a novel tumor suppressor regulating phosphoinositide 3-kinase-AKT signaling at the early stages of HCC development
2021 White Paper on Recent Issues in Bioanalysis: TAb/NAb, Viral Vector CDx, Shedding Assays; CRISPR/Cas9 & CAR-T Immunogenicity; PCR & Vaccine Assay Performance; ADA Assay Comparability & Cut Point Appropriateness
No full textNo Abstract.
Gene Therapy, Cell Therapy and Vaccines (9 Topics)
1. TAb/NAb & Anti-Viral Vector Antibody Companion Diagnostic Assays
2. Viral Vector Shedding Assays
3. Viral Vector Gene Therapy Immunogenicity & Pre-Existing Immunity
4. CRISPR/Cas9 Immunogenicity & Bioanalytical Challenges
5. CAR-T Immunogenicity & Cellular Kinetics
6. qPCR/ddPCR Assay Performance
7. Analyte Stability in Vaccine Serology Assays
8. Vaccine Critical Reagent Management and Bridging
9. Vaccine Bioanalytical Assays & Immune Monitoring
Immunogenicity of Biotherapeutics (8 Topics)
1. NAb Assays – Drug and Target Interference
2. ADA Cut Points – Appropriateness and False Positive Rates
3. Circulating Immune Complexes – ADA/Drug Complexes
4. ADA Assay Comparability
5. Integrated Summary of Immunogenicity Harmonization
6. China NMPA Immunogenicity Guidance
7. Multi-Domain Biotherapeutics & Bispecific Antibody Immunogenicity
8. Biosimilar ADA Assay Validation & Harmonizatio
Sample Preparation Matters for Peptide Mapping to Evaluate Deamidation of Adeno-Associated Virus Capsid Proteins Using Liquid Chromatography–Tandem Mass Spectrometry
No full textAdeno-associated viral capsid proteins (AAV VP) are the major components that determine the tissue specificity and
immunogenicity, and in vivo transduction performance of the vector. It was reported that asparagine deamidation of AAV
capsid proteins leads to charge variants/heterogeneity and altered vector function, reduction of stability, and potency of
AAV gene therapy products. Deamidation of asparagine residue is a common post-translational modification of proteins
and is mostly detected and quantified by liquid chromatography–tandem mass spectrometry (LC-MS/MS)-based peptide
mapping. However, deamidation can be spontaneously introduced during sample preparation before LC-MS/MS analysis.
So far, no optimal sample preparations, instead, traditional sample preparation has been used for AAV VP peptide
mapping, resulting in exaggerating the original deamidation levels. It is important to accurately monitor and provide true
value of asparagine deamidation for development of AAV gene therapy products. In this study, we evaluated denaturation
temperatures, digestion durations, and digestion temperatures using three different sample preparation formats for
LC-MS/MS-based assessment of deamidation of AAV9 capsid proteins. The results demonstrated that the optimal
sample preparation method for AAV9 VP peptide mapping minimized asparagine deamidation artifacts. Although AAV9
was used for method optimization, this study may also provide a guidance on how to control deamidation artifacts for
other AAV serotypes
Distribution and Efficacy of Ofatumumab and Ocrelizumab in Humanized CD20 Mice Following Subcutaneous or Intravenous Administration
No full textApproval of B-cell-depleting therapies, such as ofatumumab and ocrelizumab, signifies an important advance in the treatment of multiple sclerosis (MS). However, it is unclear whether the route of administration of these anti-CD20 monoclonal antibodies (mAbs) impacts treatment outcome. This study aimed to investigate the distribution and efficacy of radiolabeled ofatumumab and ocrelizumab in humanized-CD20 (huCD20) transgenic mice following subcutaneous (SC) and intravenous (IV) administration. For distribution analysis, huCD20 and wildtype mice (n = 5 per group) were imaged by single-photon emission computed tomography (SPECT/CT) 72 h after SC or IV administration of ofatumumab or ocrelizumab, radiolabeled with Indium-111 (111In-ofatumumab or 111In-ocrelizumab; 5 µg, 5 MBq). For efficacy analysis, huCD20 mice with focal delayed-type hypersensitivity lesions and associated tertiary lymphoid structures (DTH-TLS) were administered SC or IV ofatumumab or ocrelizumab (7.5 mg/kg, n = 10 per group) on Days 63, 70 and 75 post lesion induction. Treatment impact on the number of CD19+ cells in select tissues and the evolution of DTH-TLS lesions in the brain were assessed. Uptake of an 111In-labelled anti-CD19 antibody in cervical and axillary lymph nodes was also assessed before and 18 days after treatment initiation as a measure of B-cell depletion. SPECT/CT image quantification revealed similar tissue distribution, with large differences in blood signal, of 111In-ofatumumab and 111In-ocrelizumab following SC and IV administration; however, an increase in both mAbs was observed in the axillary and inguinal lymph nodes following SC versus IV administration. In the DTH-TLS model of MS, both treatments significantly reduced the 111In-anti-CD19 signal and number of CD19+ cells in select tissues, but no difference between the route of administration or mAb was observed. Both treatments significantly decreased the extent of glial activation, as well as the number of B- and T-cells in the lesion following SC and IV administration, although this was achieved to a greater extent with ofatumumab versus ocrelizumab. These findings suggest that there may be more direct access to the lymph nodes through the lymphatic system with SC versus IV administration. Furthermore, preliminary findings suggest that ofatumumab may be more effective than ocrelizumab at controlling MS-like pathology in the brain
Utilization of in silico tools to investigate the in vivo performance of vildagliptin modified release (MR) 100 mg tablets in healthy subjects using physiologically based biopharmaceutics modeling (PBBM) and evaluate an approach to establish clinically relevant dissolution specifications
No full textThe objective of the study was to predict pharmacokinetic (PK) and pharmacodynamic (PD) parameters of matrix based modified release (MR) drug product of vildagliptin. Physiologically based biopharmaceutics modeling (PBBM) was developed using GastroPlus™ based on the available data including immediate-release (IR) drug product of vildagliptin.
In vitro-in vivo correlation (IVIVC) was developed using mechanistic deconvolution to predict plasma concentration-time profile and PK parameters for a MR drug product planned for clinical use. Both methods i.e., PBBM and IVIVC were compared for the predicted PK parameters.
Integration of DDDPlus™ and GastroPlus™ modeling was performed to explore clinically relevant dissolution specifications for vildagliptin MR tablets. The bioequivalence (BE) between batches with different dissolution specifications was evaluated using virtual clinical trials. The PD effect of Dipeptidyl peptidase-IV (DPP-IV) inhibition was simulated utilizing PDPlus™ model in GastroPlus™. The results indicated that IVIVC best correlated the simulated PK parameters with those observed with the clinical study. The outcomes highlight the importance of integration of in vitro and in silico tools towards predictability of PK and PD parameters for a MR drug product. However, the post absorptive phase was found to be more dependent on the demographics of the healthy subjects
Identification of NVP-CLR457 as an Orally Bioavailable Non-CNS-Penetrant pan-Class IA Phosphoinositol-3-Kinase Inhibitor.
No full textBalanced pan-class I phosphoinositide 3-kinase inhibition as an approach to cancer treatment offers the prospect of treating a broad range of tumor types and/or a way to achieve greater efficacy with a single inhibitor. Taking buparlisib as the starting point, the balanced pan-class I PI3K inhibitor 40 (NVP-CLR457) was identified with what was considered to be a best-in-class profile. Key to the optimization to achieve this profile was eliminating a microtubule stabilizing off-target activity, balancing the pan-class I PI3K inhibition profile, minimizing CNS penetration, and developing an amorphous solid dispersion formulation. A rationale for the poor tolerability profile of 40 in a clinical study is discussed
Population pharmacokinetics of asciminib in tyrosine kinase inhibitor-treated patients with Philadelphia chromosome-positive chronic myeloid leukemia in chronic and acute phases
No full textBackground: Asciminib, a first-in-class highly potent BCR-ABL1 inhibitor has shown superior efficacy compared to the existing tyrosine kinase inhibitor TKI in patients with Philadelphia chromosome-positive chronic myeloid leukemia in chronic phase, treated with ≥2 TKIs. This study aimed to describe pharmacokinetic properties of asciminib and to identify clinically relevant covariates impacting its exposure.
Methods: A population pharmacokinetics (PopPK) model was developed using a two-compartment model with delayed first-order absorption and elimination. The analysis included pharmacokinetics data from two clinical studies (Phases 1 and 3) involving 353 patients, with total daily asciminib doses range of 20-400 mg.
Results: The nominal total daily dose was incorporated as a structural covariate on clearance (CL), and body weight (BW) was included as a structural covariate via allometric scaling on CL and central volume. Renal function and formulation were included as statistically significant covariates on CL and absorption (ka), respectively. The simulation results revealed a modest but clinically non-significant effect of baseline BW and renal function on ka. Covariates such as baseline demographic and disease characteristics, hepatic function, and T315I mutation were not statistically significant and were not incorporated in the final model. Additionally, the final model-based simulations demonstrated comparable exposure and CL for asciminib 40 mg bid and 80 mg qd (an alternative regimen not studied in the Phase 3 trial), as well as similar PK properties in patients with and without T315I mutation.
Conclusions: The final PopPK model adequately characterized the pharmacokinetics properties of asciminib and assessed the impact of key covariates on its exposure. The model not only corroborates the use of the recommended asciminib dose of 40 mg bid, but also substantiates the use of 80 mg qd to facilitate patient’s compliance