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1.
Anal Chem ; 2024 Jun 17.
Article in English | MEDLINE | ID: mdl-38887018

ABSTRACT

This study investigated the added value of combining both near-infrared (NIR) and Raman spectroscopy into a single NIRaman Combi Fiber Probe for in-line blend potency determination in the feed frame of a rotary tablet press. A five-component platform formulation was used, containing acetylsalicylic acid as the Active Pharmaceutical Ingredient (API). Calibration models for the determination of 1 and 5%w/w label claim tablets were developed using NIR and Raman spectra of powder blends ranging from 0.75 to 1.25%w/w and 3.75 to 6.25%w/w API, respectively. Step-change experiments with deliberate 10% deviation steps from the label claims were performed, from which the collected spectra were used for model validation. For model development and validation, low-level data fusion was explored through concatenation of preprocessed NIR and Raman spectra. Mid-level data fusion was also evaluated, based on extracted features of the preprocessed data. Herewith, score vectors were extracted by transforming preprocessed spectra through Principal Component Analysis, followed by critical feature selection through Elastic Net Regression. Partial Least Squares regression was applied to regress singular, low-level or mid-level fused data versus blend potency. It could be concluded that irrespective of the data fusion technique, an increase in Step-Change Sensitivity (SCS) and decrease in Root Mean Squared Error (RMSE) was observed when predicting the 5%w/w step-change experiment. For the prediction of the 1%w/w step-change experiment, no added benefit with regard to SCS and RMSE was observed due to the addition of the noisy NIR spectra.

2.
Cryobiology ; 116: 104907, 2024 May 26.
Article in English | MEDLINE | ID: mdl-38768801

ABSTRACT

When cells are cryopreserved, they go through a freezing process with several distinct phases (i.e., cooling until nucleation, ice nucleation, ice crystal growth and cooling to a final temperature). Conventional cell freezing approaches often employ a single cooling rate to describe and optimize the entire freezing process, which neglects its complexity and does not provide insight into the effects of the different freezing phases. The aim of this work was to elucidate the impact of each freezing phase by varying different process parameters per phase. Hereto, spin freezing was used to freeze Jurkat T cells in either a Me2SO-based or Me2SO-free formulation. The cooling rates before ice nucleation and after total ice crystallization impacted cell viability, resulting in viability ranging from 26.7% to 52.8% for the Me2SO-free formulation, and 22.5%-42.6% for the Me2SO-based formulation. Interestingly, the degree of supercooling upon nucleation did not exhibit a significant effect on cell viability in this work. However, the rate of ice crystal formation emerged as a crucial factor, with viability ranging from 2.4% to 53.2% for the Me2SO-free formulation, and 0.3%-53.2% for the Me2SO-based formulation, depending on the freezing rate. A morphological study of the cells post-cryopreservation was performed using confocal microscopy, and it was found that cytoskeleton integrity and cell volume were impacted, depending on the formulation-process parameter combination. These findings underscore the importance of scrutinizing all cooling and freezing phases, as each phase impacted post-thaw viability in a distinct way, depending of the specific formulation used.

3.
Int J Pharm ; 658: 124137, 2024 Jun 10.
Article in English | MEDLINE | ID: mdl-38670472

ABSTRACT

The shift from batch manufacturing towards continuous manufacturing for the production of oral solid dosages requires the development and implementation of process models and process control. Previous work focused mainly on developing deterministic models for the investigated system. Furthermore, the in silico tuning and analysis of a control strategy are mostly done based on deterministic models. This deterministic approach could lead to wrong actions in diversion strategies and poor transferability of the controller performance if the system behaves differently than the deterministic model. This work introduces a framework that explicitly includes the process variability which is characteristic of powder handling processes and tests it on a novel continuous feeding-blending unit (i.e., the FE continuous processing system (CPS)), followed by a tablet press (i.e., the FE 55). It employs a stochastic model by allowing the model parameters to have a probability distribution. The performance of a model predictive control (MPC), steering the feed rate of the main excipient feeder to compensate for the feed rate deviations of the active pharmaceutical ingredient (API) feeder to keep the API concentration close to the desired value, is evaluated and the impact of process variability is assessed in a Monte Carlo (MC) analysis. Next to the process variability, a model for the prediction error of the chemometric model and realistic feed rate disturbances were included to increase the transferability of the results to the real system. The obtained results show that process variability is inherently present and that wrong conclusions can be drawn if it is not taken into account in the in silico analysis.


Subject(s)
Computer Simulation , Excipients , Monte Carlo Method , Powders , Tablets , Excipients/chemistry , Powders/chemistry , Technology, Pharmaceutical/methods , Drug Compounding/methods , Chemistry, Pharmaceutical/methods , Pharmaceutical Preparations/chemistry
4.
Int J Pharm ; 657: 124135, 2024 May 25.
Article in English | MEDLINE | ID: mdl-38643808

ABSTRACT

Pharmaceutical twin-screw wet granulation is a multifaceted and intricate process pivotal to drug product development. Accurate modeling of this process is indispensable for optimizing manufacturing parameters and ensuring product quality. The fluid bed dryer, an integral component of this granulation process, significantly influences the granular critical quality attributes. This study builds upon prior research by integrating experimental findings on granule segregation during fluid bed drying into an existing compartmental model, enhancing its predictive capabilities. An additional model layer on granule segregation behavior is composed and integrated into the existing model structure in this study. The added model compartment describes probability distributions on the vertical position of granules within each granule size class considered. To beware of overfitting, predictions of both the moisture content after drying and the granule bed temperature throughout drying are discussed in this study relative to experimental data from earlier published studies. These independent analyses demonstrated a marked improvement in prediction accuracy compared to earlier published model structures. The refined model accurately predicts the residual moisture content after drying for an untrained formulation. Moreover, it simultaneously makes accurate predictions of the granular bed temperature, which emboldens its structural correctness. This advancement makes it a powerful tool for predicting the behavior of the pharmaceutical fluid bed drying, which holds significant promise to facilitate pharmaceutical product development.


Subject(s)
Desiccation , Temperature , Desiccation/methods , Particle Size , Drug Compounding/methods , Technology, Pharmaceutical/methods , Chemistry, Pharmaceutical/methods , Models, Theoretical , Excipients/chemistry
5.
ACS Chem Biol ; 19(2): 233-242, 2024 02 16.
Article in English | MEDLINE | ID: mdl-38271588

ABSTRACT

In the field of drug discovery, understanding how small molecule drugs interact with cellular components is crucial. Our study introduces a novel methodology to uncover primary drug targets using Tandem Affinity Purification for identification of Drug-Binding Proteins (TAP-DBP). Central to our approach is the generation of a FLAG-hemagglutinin (HA)-tagged chimeric protein featuring the FKBP12(F36V) adaptor protein and the TurboID enzyme. Conjugation of drug molecules with the FKBP12(F36V) ligand allows for the coordinated recruitment of drug-binding partners effectively enabling in-cell TurboID-mediated biotinylation. By employing a tandem affinity purification protocol based on FLAG-immunoprecipitation and streptavidin pulldown, alongside mass spectrometry analysis, TAP-DBP allows for the precise identification of drug-primary binding partners. Overall, this study introduces a systematic, unbiased method for identification of drug-protein interactions, contributing a clear understanding of target engagement and drug selectivity to advance the mode of action of a drug in cells.


Subject(s)
Carrier Proteins , Tandem Affinity Purification , Tandem Affinity Purification/methods , Tacrolimus Binding Protein 1A/metabolism , Proteins/metabolism , Chromatography, Affinity/methods
6.
Int J Pharm ; 650: 123671, 2024 Jan 25.
Article in English | MEDLINE | ID: mdl-38065345

ABSTRACT

In the last few years, twin-screw wet granulation (TSWG) has become one of the key continuous pharmaceutical unit operations. Despite the many studies that have been performed, only little is known about the effect of the starting material properties on the stepwise granule formation along the length of the twin-screw granulator (TSG) barrel. Hence, this study obtained a detailed understanding of the effect of formulation properties (i.e., Active Pharmaceutical Ingredient (API) properties, formulation blend particle size distribution and formulation drug load) and process settings on granule formation in TSWG. An experimental set-up was used allowing the collection of granules at the different TSG compartments. Granules were characterized in terms of granule size, shape, binder liquid and API distributions. Liquid-to-solid (L/S) ratio was the only TSG process parameter impacting the granule size and shape evolution. Particle size and flow properties (e.g., flow rate index) had an important effect on the granule size and shape changes whereas water-related properties (e.g., water binding capacity and solubility) became influential at the last TSG compartments. The API solubility and L/S ratio were found to have a major impact on the distribution of binder liquid over the different granule size fractions. In the first TSG compartment (i.e., wetting compartment), the distribution of the API in the granules was influenced by its solubility in the granulation liquid.


Subject(s)
Bone Screws , Water , Solubility , Particle Size , Wettability , Drug Compounding , Technology, Pharmaceutical
7.
Int J Pharm ; 646: 123493, 2023 Nov 05.
Article in English | MEDLINE | ID: mdl-37813175

ABSTRACT

This paper presents an application case of model-based design of experiments for the continuous twin-screw wet granulation and fluid-bed drying sequence. The proposed framework consists of three previously developed models. Here, we are testing the applicability of previously published unit operation models in this specific part of the production line to a new active pharmaceutical ingredient. Firstly, a T-shaped partial least squares regression model predicts d-values of granules after wet granulation with different process settings. Then, a high-resolution full granule size distribution is computed by a hybrid population balance and partial least squares regression model. Lastly, a mechanistic model of fluid-bed drying simulates drying time and energy efficiency, using the outputs of the first two models as a part of the inputs. In the application case, good operating conditions were calculated based on material and formulation properties as well as the developed process models. The framework was validated by comparing the simulation results with three experimental results. Overall, the proposed framework enables a process designer to find appropriate process settings with a less experimental workload. The framework combined with process knowledge reduced 73.2% of material consumption and 72.3% of time, especially in the early process development phase.


Subject(s)
Bone Screws , Desiccation , Drug Compounding/methods , Particle Size , Computer Simulation , Desiccation/methods , Technology, Pharmaceutical/methods , Tablets
8.
Int J Pharm ; 646: 123481, 2023 Nov 05.
Article in English | MEDLINE | ID: mdl-37805145

ABSTRACT

This work presents a granule size prediction approach applicable to diverse formulations containing new active pharmaceutical ingredients (APIs) in continuous twin-screw wet granulation. The approach consists of a surrogate selection method to identify similar materials with new APIs and a T-shaped partial least squares (T-PLS) model for granule size prediction across varying formulations and process conditions. We devised a surrogate material selection method, employing a combination of linear pre-processing and nonlinear classification algorithms, which effectively identified suitable surrogates for new materials. Using only material properties obtained through four characterization methods, our approach demonstrated its predictive prowess. The selected surrogate methods were seamlessly integrated with our developed T-PLS model, which was meticulously validated for high-dose formulations involving three new APIs. When surrogating new APIs based on Gaussian process classification, we achieved the lowest prediction errors, signifying the method's robustness. The predicted d-values were within the range of uncertainty bounds for all cases, except for d90 of API C. Notably, the approach offers a direct and efficient solution for early-phase formulation and process development, considerably reducing the need for extensive experimental work. By relying on just four material characterization methods, it streamlines the research process while maintaining a high degree of accuracy.


Subject(s)
Bone Screws , Technology, Pharmaceutical , Least-Squares Analysis , Particle Size , Pharmaceutical Preparations , Drug Compounding , Tablets
9.
Int J Pharm ; 645: 123391, 2023 Oct 15.
Article in English | MEDLINE | ID: mdl-37696346

ABSTRACT

Twin-screw wet granulation (TSWG) stands out as a promising continuous alternative to conventional batch fluid bed- and high shear wet granulation techniques. Despite its potential, the impact of raw material properties on TSWG processability remains inadequately explored. Furthermore, the absence of supportive models for TSWG process development with new active pharmaceutical ingredients (APIs) adds to the challenge. This study tackles these gaps by introducing four partial least squares (PLS) models that approximate both the applicable liquid-to-solid (L/S) ratio range and resulting granule attributes (i.e., granule size and friability) based on initial material properties. The first two PLS models link the lowest and highest applicable L/S ratio for TSWG, respectively, with the formulation blend properties. The third and fourth PLS models predict the granule size and friability, respectively, from the starting API properties and applied L/S ratio for twin-screw wet granulation. By analysing the developed PLS models, water-related material properties (e.g., solubility, wettability, dissolution rate), as well as density and flow-related properties (e.g., flow function coefficient), were found to be impacting the TSWG processability. In addition, the applicability of the developed PLS models was evaluated by using them to propose suitable L/S ratio ranges (i.e., resulting in granules with the desired properties) for three new APIs and related formulations followed by an experimental validation thereof. Overall, this study helped to better understand the effect of raw material properties upon TSWG processability. Moreover, the developed PLS models can be used to propose suitable TSWG process settings for new APIs and hence reduce the experimental effort during process development.


Subject(s)
Bone Screws , Technology, Pharmaceutical , Particle Size , Solubility , Wettability , Drug Compounding/methods , Technology, Pharmaceutical/methods , Tablets
10.
BMJ Open ; 13(9): e075664, 2023 09 20.
Article in English | MEDLINE | ID: mdl-37730385

ABSTRACT

INTRODUCTION: Individuals with dementia spend most of the day without care, without encounters, and usually without activity. Although this has been proven in studies, there is a knowledge gap on how individuals with dementia experience these periods of time. Such knowledge would be highly relevant for health professionals and relatives to develop adequate strategies for dealing with these periods of time. The FreiZeit study aims to reconstruct periods of time without care and encounters from the perspective of individuals with dementia and formal and informal carers. The specific objective of this review is to provide a continuously updated overview of the topical evidence that may be used to guide data synthesis and interpretation within the FreiZeit study. METHODS AND ANALYSIS: We conduct a living evidence map, based on a comprehensive systematic literature search in MEDLINE/PubMed, CINAHL, PsycINFO/Ovid and Web of Science Core Collection, citation-based searches and web searches. We include studies on times without care and encounters of individuals with dementia from the perspective of individuals with dementia themselves and formal or informal caregivers of any observational study design that were conducted in the institutional and domestic long-term care setting and published as journal article in English, French or German language without any restriction of the publication year. One reviewer screens titles, abstracts and full texts and extracts data. Key characteristics and results of the included studies are charted in a tabular format. The searches will be run and continuously updated throughout the duration of the overarching FreiZeit study (every 6 months for 2 years from 2023 to 2025). ETHICS AND DISSEMINATION: Ethics approval is not required for this evidence map. We disseminate our findings via journal articles and conference proceedings as well as other formats. REGISTRATION DETAILS: This review protocol is uploaded on Open Science Framework (OSF; DOI 10.17605/OSF.IO/GDYZ9).


Subject(s)
Dementia , Health Facilities , Humans , Health Personnel , Knowledge , Language , Observational Studies as Topic , Review Literature as Topic
11.
Virology ; 587: 109866, 2023 10.
Article in English | MEDLINE | ID: mdl-37741199

ABSTRACT

We previously reported the discovery and characterization of two novel proteins (ORF1 and ORF2) generated by the alternative splicing of the JC virus (JCV) late coding region. Here, we report the discovery and partial characterization of three additional novel ORFs from the same coding region, ORF3, ORF4 and ORF5, which potentially encode 70, 173 and 265 amino acid long proteins respectively. While ORF3 protein exhibits a uniform distribution pattern throughout the cells, we were unable to detect ORF5 expression. Surprisingly, ORF4 protein was determined to be the only JCV protein specifically targeting the promyelocytic leukemia nuclear bodies (PML-NBs) and inducing their reorganization in nucleus. Although ORF4 protein has a modest effect on JCV replication, it is implicated to play major roles during the JCV life cycle, perhaps by regulating the antiviral response of PML-NBs against JCV infections and thus facilitating the progression of the JCV-induced disease in infected individuals.


Subject(s)
JC Virus , Leukoencephalopathy, Progressive Multifocal , Polyomavirus , Humans , JC Virus/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Open Reading Frames , Promyelocytic Leukemia Nuclear Bodies
12.
Int J Pharm ; 646: 123447, 2023 Nov 05.
Article in English | MEDLINE | ID: mdl-37770009

ABSTRACT

In this work, a mechanistic fluidized bed drying model computing the granule moisture content in function of granule size, drying time, process settings and formulation properties is developed. Modeling the moisture content distribution concerning the granule size is essential for tabletability and drug product quality. This work combines a mechanistic bulk model and a single-particle drying kinetics model in a semicontinuous mode. The added model complexity allows physical approximations of drying phenomena at both the drying system level and the granular level. This includes quantifying the variations in moisture content by taking into account the specific dryer design and the variations in granule size. The model performance was quantified through industrially relevant case studies. It was revealed that the proposed model structure accurately predicts the drying behavior of the yield fraction. However, systematic model biases were observed for the fine and coarse fractions of the granule size distribution. In addition, discrepancies in the predicted outgoing air properties (relative air humidity and air temperature) were obtained. Further enhancement of the model complexity, e.g. complete incorporation of fluidization and segregation phenomena, is likely to improve the model performance. Notwithstanding, the developed model forms a step towards a formulation-generic fluidized bed drying model as interacting mechanisms on different levels of the drying system are considered.

13.
Pharmaceuticals (Basel) ; 16(8)2023 Jul 26.
Article in English | MEDLINE | ID: mdl-37630976

ABSTRACT

The present study aimed to develop 3D printed dosage forms, using custom-made filaments loaded with diclofenac sodium (DS). The printed tablets were developed by implementing a quality by design (QbD) approach. Filaments with adequate FDM 3D printing characteristics were produced via hot melt extrusion (HME). Their formulation included DS as active substance, polyvinyl alcohol (PVA) as a polymer, different types of plasticisers (mannitol, erythritol, isomalt, maltodextrin and PEG) and superdisintegrants (crospovidone and croscarmellose sodium). The physicochemical and mechanical properties of the extruded filaments were investigated through differential scanning calorimetry (DSC), X-ray diffraction (XRD) and tensile measurements. In addition, cylindrical-shaped and tubular-shaped 3D dosage forms were printed, and their dissolution behaviour was assessed via various drug release kinetic models. DSC and XRD results demonstrated the amorphous dispersion of DS into the polymeric filaments. Moreover, the 3D printed tablets, regardless of their composition, exhibited a DS release of nearly 90% after 45 min at pH 6.8, while their release behaviour was effectively described by the Korsmeyer-Peppas model. Notably, the novel tube design, which was anticipated to increase the drug release rate, proved the opposite based on the in vitro dissolution study results. Additionally, the use of crospovidone increased DS release rate, whereas croscarmellose sodium decreased it.

14.
Eur J Pharm Biopharm ; 189: 251-263, 2023 Aug.
Article in English | MEDLINE | ID: mdl-37356638

ABSTRACT

The use of in-line near-infrared (NIR) measurements for tablet potency monitoring and diversion was studied. First, the optimal sample size for in-line NIR measurements inside the feed chute and the dosing and filling chamber of the tablet press feed frame was determined to allow proper comparison between these different measurement positions. Because of the considerably longer measurement time needed to obtain the same sample size inside the feed chute compared to the feed frame, the possibility of powder segregation inside the feed chute and the additional powder mixing inside the feed frame, the latter is preferred over the feed chute for in-line blend potency monitoring. Next, a design of experiments (DoE) was performed to evaluate the effect of paddle speed, turret speed, overfill level and formulation properties upon the lead-lag and the time it takes before the powder blend that is expelled at the dosing station is measured by the NIR inside the dosing chamber. Lead-lag is defined as the difference in time and API concentration between the measured in-line NIR response inside the filling chamber of the feed frame and the off-line NIR tablet response. Paddle speed and turret speed were the only compression parameters affecting lead-lag. Lead-lag decreased with increasing paddle speed for the first formulation. For the second formulation, lead-lag decreased with decreasing paddle speed and/or increasing turret speed. Formulation properties did not have an effect on the lead-lag. The in-line NIR response inside the dosing chamber of the feed frame was found to be closely following the tablet NIR response. Therefore, the dosing chamber could be used as an additional in-line NIR position for tablet potency monitoring and diversion. It can provide an extra layer of confidence about the final tablet quality. To demonstrate this potential benefit of simultaneous in-line NIR measurements inside the filling and dosing chamber of the feed frame, a tableting experiment was performed where a surrogate API spike was introduced into the product stream to mimic a potential process disturbance. The in-line NIR measurements inside the filling chamber allow diverting tablets in-time when the blend potency crosses the predefined control limits. And because the NIR response inside the dosing chamber closely follows the tablet NIR response, tablet diversion can discontinue when the blend potency inside the dosing chamber is again within the control limits. This could increase the yield of the tableting process by avoiding a longer than needed wash-out period and rejecting tablets that meet the release limits.


Subject(s)
Technology, Pharmaceutical , Powders , Tablets , Time Factors , Pressure , Drug Compounding
15.
Int J Pharm ; 641: 123010, 2023 Jun 25.
Article in English | MEDLINE | ID: mdl-37169104

ABSTRACT

In recent years, continuous twin-screw wet granulation (TSWG) is gaining increasing interest from the pharmaceutical industry. Despite the many publications on TSWG, only a limited number of studies focused on granule porosity, which was found to be an important granule property affecting the final tablet quality attributes, e.g. dissolution. In current study, the granule porosity along the length of the twin-screw granulator (TSG) barrel was evaluated. An experimental set-up was used allowing the collection of granules at the different TSG compartments. The effect of active pharmaceutical ingredient (API) properties on granule porosity was evaluated by using six formulations with a fixed composition but containing APIs with different physical-chemical properties. Furthermore, the importance of TSWG process parameters liquid-to-solid (L/S) ratio, mass feed rate and screw speed for the granule porosity was evaluated. Several water-related properties as well as particle size, density and flow properties of the API were found to have an important effect on granule porosity. While the L/S ratio was confirmed to be the dictating TSWG process parameter, granulator screw speed was also found to be an important process variable affecting granule porosity. This study obtained crucial information on the effect of material properties and process parameters on granule porosity (and granule formation) which can be used to accelerate TSWG process and formulation development.


Subject(s)
Drug Industry , Technology, Pharmaceutical , Porosity , Particle Size , Bone Screws , Tablets , Drug Compounding
16.
Int J Pharm ; 641: 123062, 2023 Jun 25.
Article in English | MEDLINE | ID: mdl-37209792

ABSTRACT

Continuous spin freeze-drying provides a range of opportunities regarding the implementation of several in-line process analytical technologies (PAT) to control and optimize the freeze-drying process at the individual vial level. In this work, two methods were developed to (1) control the freezing phase by separately controlling the cooling and freezing rate and (2) control the drying phase by controlling the vial temperature (and hence the product temperature) to setpoint values and monitoring the residual moisture content. During the freezing phase, the vial temperature closely followed the decreasing setpoint temperature during the cooling phases, and the crystallization phase was reproducibly controlled by regulating the freezing rate. During both primary and secondary drying, vial temperature could be maintained on the setpoint temperature which resulted in an elegant cake structure after every run. By being able to accurately control the freezing rate and the vial temperature, a homogeneous drying time (SD = 0.07-0.09 h) between replicates was obtained. Applying a higher freezing rate significantly increased primary drying time. On the other hand, fast freezing rates increased the desorption rate. Finally, the residual moisture of the freeze-dried formulation could be monitored in-line with a high accuracy providing insight on the required length of the secondary drying phase.


Subject(s)
Desiccation , Technology, Pharmaceutical , Freeze Drying/methods , Temperature , Freezing
17.
Int J Pharm ; 640: 123040, 2023 Jun 10.
Article in English | MEDLINE | ID: mdl-37172629

ABSTRACT

In the pharmaceutical industry, twin-screw wet granulation has become a realistic option for the continuous manufacturing of solid drug products. Towards the efficient design, population balance models (PBMs) have been recognized as a tool to compute granule size distribution and understand physical phenomena. However, the missing link between material properties and the model parameters limits the swift applicability and generalization of new active pharmaceutical ingredients (APIs). This paper proposes partial least squares (PLS) regression models to assess the impact of material properties on PBM parameters. The parameters of the compartmental one-dimensional PBMs were derived for ten formulations with varying liquid-to-solid ratios and connected with material properties and liquid-to-solid ratios by PLS models. As a result, key material properties were identified in order to calculate it with the necessary accuracy. Size- and moisture-related properties were influential in the wetting zone whereas density-related properties were more dominant in the kneading zones.


Subject(s)
Drug Compounding , Drug Industry , Drug Compounding/methods , Least-Squares Analysis , Particle Size , Technology, Pharmaceutical/methods
18.
Blood ; 142(11): 973-988, 2023 09 14.
Article in English | MEDLINE | ID: mdl-37235754

ABSTRACT

Diffuse large B-cell lymphoma (DLBCL) is the most common subtype of non-Hodgkin lymphoma. Up to 40% of patients with DLBCL display refractory disease or relapse after standard chemotherapy treatment (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone [R-CHOP]), leading to significant morbidity and mortality. The molecular mechanisms of chemoresistance in DLBCL remain incompletely understood. Using a cullin-really interesting new gene (RING) ligase-based CRISPR-Cas9 library, we identify that inactivation of the E3 ubiquitin ligase KLHL6 promotes DLBCL chemoresistance. Furthermore, proteomic approaches helped identify KLHL6 as a novel master regulator of plasma membrane-associated NOTCH2 via proteasome-dependent degradation. In CHOP-resistant DLBCL tumors, mutations of NOTCH2 result in a protein that escapes the mechanism of ubiquitin-dependent proteolysis, leading to protein stabilization and activation of the oncogenic RAS signaling pathway. Targeting CHOP-resistant DLBCL tumors with the phase 3 clinical trial molecules nirogacestat, a selective γ-secretase inhibitor, and ipatasertib, a pan-AKT inhibitor, synergistically promotes DLBCL destruction. These findings establish the rationale for therapeutic strategies aimed at targeting the oncogenic pathway activated in KLHL6- or NOTCH2-mutated DLBCL.


Subject(s)
Drug Resistance, Neoplasm , Lymphoma, Large B-Cell, Diffuse , Humans , Drug Resistance, Neoplasm/genetics , Ubiquitin , Proteomics , Neoplasm Recurrence, Local/drug therapy , Rituximab/therapeutic use , Vincristine , Cyclophosphamide , Lymphoma, Large B-Cell, Diffuse/drug therapy , Lymphoma, Large B-Cell, Diffuse/genetics , Doxorubicin/pharmacology , Doxorubicin/therapeutic use , Prednisone , Mutation , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Receptor, Notch2/genetics
19.
Biomater Sci ; 11(12): 4327-4334, 2023 Jun 13.
Article in English | MEDLINE | ID: mdl-37073472

ABSTRACT

The limited thermostability and need for ultracold storage conditions are the major drawbacks of the currently used nucleoside-modified lipid nanoparticle (LNP)-formulated messenger RNA (mRNA) vaccines, which hamper the distribution of these vaccines in low-resource regions. The LNP core contains, besides mRNA and lipids, a large fraction of water. Therefore, encapsulated mRNA, or at least a part of it, is subjected to hydrolysis mechanisms similar to unformulated mRNA in an aqueous solution. It is likely that the hydrolysis of mRNA and colloidal destabilization are critical factors that decrease the biological activity of mRNA LNPs upon storage under ambient conditions. Hence, lyophilization as a drying technique is a logical and appealing method to improve the thermostability of these vaccines. In this study, we demonstrate that mRNA LNP formulations comprising a reduction-sensitive ionizable lipid can be successfully lyophilized, in the presence of 20% w/v sucrose, both by conventional batch freeze-drying and by an innovative continuous spin lyophilization process. While the chemical structure of the ionizable lipid did not affect the colloidal stability of the LNP after lyophilization and redispersion in an aqueous medium, we found that the ability of LNPs to retain the mRNA payload stably encapsulated, and mediate in vivo and in vitro mRNA translation into protein, post lyophilization strongly depended on the ionizable lipid in the LNP formulation.


Subject(s)
Lipids , Nanoparticles , Lipids/chemistry , RNA, Messenger/genetics , Cryoprotective Agents/chemistry , Drug Compounding , Nanoparticles/chemistry , Freeze Drying , RNA, Small Interfering/genetics
20.
J Control Release ; 357: 149-160, 2023 05.
Article in English | MEDLINE | ID: mdl-36958400

ABSTRACT

Messenger RNA (mRNA) lipid nanoparticles (LNPs) have emerged at the forefront during the COVID-19 vaccination campaign. Despite their tremendous success, mRNA vaccines currently require storage at deep freeze temperatures which complicates their storage and distribution, and ultimately leads to lower accessibility to low- and middle-income countries. To elaborate on this challenge, we investigated freeze-drying as a method to enable storage of mRNA LNPs at room- and even higher temperatures. More specifically, we explored a novel continuous freeze-drying technique based on spin-freezing, which has several advantages compared to classical batch freeze-drying including a much shorter drying time and improved process and product quality controlling. Here, we give insight into the variables that play a role during freeze-drying by evaluating the impact of the buffer and mRNA LNP formulation (ionizable lipid to mRNA weight ratio) on properties such as size, morphology and mRNA encapsulation. We found that a sufficiently high ionizable lipid to mRNA weight ratio was necessary to prevent leakage of mRNA during freeze-drying and that phosphate and Tris, but not PBS, were appropriate buffers for lyophilization of mRNA LNPs. We also studied the stability of optimally lyophilized mRNA LNPs at 4 °C, 22 °C, and 37 °C and found that transfection properties of lyophilized mRNA LNPs were maintained during at least 12 weeks. To our knowledge, this is the first study that demonstrates that optimally lyophilized mRNA LNPs can be safely stored at higher temperatures for months without losing their transfection properties.


Subject(s)
COVID-19 , Nanoparticles , Humans , Temperature , RNA, Messenger , COVID-19 Vaccines , Freeze Drying/methods , Lipids
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