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1.
Mol Cell ; 84(6): 1139-1148.e5, 2024 Mar 21.
Artículo en Inglés | MEDLINE | ID: mdl-38452765

RESUMEN

Eukaryotic genomes are folded into DNA loops mediated by structural maintenance of chromosomes (SMC) complexes such as cohesin, condensin, and Smc5/6. This organization regulates different DNA-related processes along the cell cycle, such as transcription, recombination, segregation, and DNA repair. During the G2 stage, SMC-mediated DNA loops coexist with cohesin complexes involved in sister chromatid cohesion (SCC). However, the articulation between the establishment of SCC and the formation of SMC-mediated DNA loops along the chromatin remains unknown. Here, we show that SCC is indeed a barrier to cohesin-mediated DNA loop expansion along G2/M Saccharomyces cerevisiae chromosomes.


Asunto(s)
Proteínas Cromosómicas no Histona , Proteínas de Saccharomyces cerevisiae , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Cromátides/metabolismo , Cohesinas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ADN/genética , ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
Mol Cell ; 84(5): 867-882.e5, 2024 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-38295804

RESUMEN

The structural maintenance of chromosomes (SMC) protein complexes-cohesin, condensin, and the Smc5/6 complex (Smc5/6)-are essential for chromosome function. At the molecular level, these complexes fold DNA by loop extrusion. Accordingly, cohesin creates chromosome loops in interphase, and condensin compacts mitotic chromosomes. However, the role of Smc5/6's recently discovered DNA loop extrusion activity is unknown. Here, we uncover that Smc5/6 associates with transcription-induced positively supercoiled DNA at cohesin-dependent loop boundaries on budding yeast (Saccharomyces cerevisiae) chromosomes. Mechanistically, single-molecule imaging reveals that dimers of Smc5/6 specifically recognize the tip of positively supercoiled DNA plectonemes and efficiently initiate loop extrusion to gather the supercoiled DNA into a large plectonemic loop. Finally, Hi-C analysis shows that Smc5/6 links chromosomal regions containing transcription-induced positive supercoiling in cis. Altogether, our findings indicate that Smc5/6 controls the three-dimensional organization of chromosomes by recognizing and initiating loop extrusion on positively supercoiled DNA.


Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN Superhelicoidal/genética , Cohesinas , ADN/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromosomas/metabolismo
3.
Mol Cell ; 84(5): 883-896.e7, 2024 Mar 07.
Artículo en Inglés | MEDLINE | ID: mdl-38309275

RESUMEN

DNA loop-extruding SMC complexes play crucial roles in chromosome folding and DNA immunity. Prokaryotic SMC Wadjet (JET) complexes limit the spread of plasmids through DNA cleavage, yet the mechanisms for plasmid recognition are unresolved. We show that artificial DNA circularization renders linear DNA susceptible to JET nuclease cleavage. Unlike free DNA, JET cleaves immobilized plasmid DNA at a specific site, the plasmid-anchoring point, showing that the anchor hinders DNA extrusion but not DNA cleavage. Structures of plasmid-bound JetABC reveal two presumably stalled SMC motor units that are drastically rearranged from the resting state, together entrapping a U-shaped DNA segment, which is further converted to kinked V-shaped cleavage substrate by JetD nuclease binding. Our findings uncover mechanical bending of residual unextruded DNA as molecular signature for plasmid recognition and non-self DNA elimination. We moreover elucidate key elements of SMC loop extrusion, including the motor direction and the structure of a DNA-holding state.


Asunto(s)
ADN , Endonucleasas , ADN/metabolismo , Plásmidos/genética , Células Procariotas , Proteínas de Ciclo Celular/metabolismo
4.
EMBO J ; 2024 Aug 19.
Artículo en Inglés | MEDLINE | ID: mdl-39160275

RESUMEN

The ring-shaped cohesin complex topologically entraps two DNA molecules to establish sister chromatid cohesion. Cohesin also shapes the interphase chromatin landscape with wide-ranging implications for gene regulation, and cohesin is thought to achieve this by actively extruding DNA loops without topologically entrapping DNA. The 'loop extrusion' hypothesis finds motivation from in vitro observations-whether this process underlies in vivo chromatin loop formation remains untested. Here, using the budding yeast S. cerevisiae, we generate cohesin variants that have lost their ability to extrude DNA loops but retain their ability to topologically entrap DNA. Analysis of these variants suggests that in vivo chromatin loops form independently of loop extrusion. Instead, we find that transcription promotes loop formation, and acts as an extrinsic motor that expands these loops and defines their ultimate positions. Our results necessitate a re-evaluation of the loop extrusion hypothesis. We propose that cohesin, akin to sister chromatid cohesion establishment at replication forks, forms chromatin loops by DNA-DNA capture at places of transcription, thus unifying cohesin's two roles in chromosome segregation and interphase genome organisation.

5.
Development ; 151(1)2024 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-38205947

RESUMEN

Cell junctions play key roles in epithelial integrity. During development, when epithelia undergo extensive morphogenesis, these junctions must be remodeled in order to maintain mechanochemical barriers and ensure the cohesion of the tissue. In this Review, we present a comprehensive and integrated description of junctional remodeling mechanisms in epithelial cells during development, from embryonic to adult epithelia. We largely focus on Drosophila, as quantitative analyses in this organism have provided a detailed characterization of the molecular mechanisms governing cell topologies, and discuss the conservation of these mechanisms across metazoans. We consider how changes at the molecular level translate to tissue-scale irreversible deformations, exploring the composition and assembly of cellular interfaces to unveil how junctions are remodeled to preserve tissue homeostasis during cell division, intercalation, invagination, ingression and extrusion.


Asunto(s)
Drosophila , Uniones Intercelulares , Animales , División Celular , Desarrollo Embrionario , Células Epiteliales
6.
Proc Natl Acad Sci U S A ; 121(21): e2401494121, 2024 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-38753513

RESUMEN

In mammalian cells, the cohesin protein complex is believed to translocate along chromatin during interphase to form dynamic loops through a process called active loop extrusion. Chromosome conformation capture and imaging experiments have suggested that chromatin adopts a compact structure with limited interpenetration between chromosomes and between chromosomal sections. We developed a theory demonstrating that active loop extrusion causes the apparent fractal dimension of chromatin to cross-over between two and four at contour lengths on the order of 30 kilo-base pairs. The anomalously high fractal dimension [Formula: see text] is due to the inability of extruded loops to fully relax during active extrusion. Compaction on longer contour length scales extends within topologically associated domains (TADs), facilitating gene regulation by distal elements. Extrusion-induced compaction segregates TADs such that overlaps between TADs are reduced to less than 35% and increases the entanglement strand of chromatin by up to a factor of 50 to several Mega-base pairs. Furthermore, active loop extrusion couples cohesin motion to chromatin conformations formed by previously extruding cohesins and causes the mean square displacement of chromatin loci during lag times ([Formula: see text]) longer than tens of minutes to be proportional to [Formula: see text]. We validate our results with hybrid molecular dynamics-Monte Carlo simulations and show that our theory is consistent with experimental data. This work provides a theoretical basis for the compact organization of interphase chromatin, explaining the physical reason for TAD segregation and suppression of chromatin entanglements which contribute to efficient gene regulation.


Asunto(s)
Proteínas de Ciclo Celular , Cromatina , Proteínas Cromosómicas no Histona , Cohesinas , Interfase , Cromatina/metabolismo , Cromatina/química , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Humanos , Animales , Segregación Cromosómica/fisiología
7.
Proc Natl Acad Sci U S A ; 121(28): e2407077121, 2024 Jul 09.
Artículo en Inglés | MEDLINE | ID: mdl-38954553

RESUMEN

An array of motor proteins consumes chemical energy in setting up the architectures of chromosomes. Here, we explore how the structure of ideal polymer chains is influenced by two classes of motors. The first class which we call "swimming motors" acts to propel the chromatin fiber through three-dimensional space. They represent a caricature of motors such as RNA polymerases. Previously, they have often been described by adding a persistent flow onto Brownian diffusion of the chain. The second class of motors, which we call "grappling motors" caricatures the loop extrusion processes in which segments of chromatin fibers some distance apart are brought together. We analyze these models using a self-consistent variational phonon approximation to a many-body Master equation incorporating motor activities. We show that whether the swimming motors lead to contraction or expansion depends on the susceptibility of the motors, that is, how their activity depends on the forces they must exert. Grappling motors in contrast to swimming motors lead to long-ranged correlations that resemble those first suggested for fractal globules and that are consistent with the effective interactions inferred by energy landscape analyses of Hi-C data on the interphase chromosome.


Asunto(s)
Cromosomas , Cromatina/química , Cromatina/metabolismo , Proteínas Motoras Moleculares/metabolismo , Proteínas Motoras Moleculares/química
8.
Semin Cell Dev Biol ; 156: 44-57, 2024 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-37400292

RESUMEN

Epithelial cell death is highly prevalent during development and tissue homeostasis. While we have a rather good understanding of the molecular regulators of programmed cell death, especially for apoptosis, we still fail to predict when, where, how many and which specific cells will die in a tissue. This likely relies on the much more complex picture of apoptosis regulation in a tissular and epithelial context, which entails cell autonomous but also non-cell autonomous factors, diverse feedback and multiple layers of regulation of the commitment to apoptosis. In this review, we illustrate this complexity of epithelial apoptosis regulation by describing these different layers of control, all demonstrating that local cell death probability is a complex emerging feature. We first focus on non-cell autonomous factors that can locally modulate the rate of cell death, including cell competition, mechanical input and geometry as well as systemic effects. We then describe the multiple feedback mechanisms generated by cell death itself. We also outline the multiple layers of regulation of epithelial cell death, including the coordination of extrusion and regulation occurring downstream of effector caspases. Eventually, we propose a roadmap to reach a more predictive understanding of cell death regulation in an epithelial context.


Asunto(s)
Apoptosis , Células Epiteliales , Células Epiteliales/metabolismo , Muerte Celular , Apoptosis/fisiología
9.
Bioessays ; : e2400137, 2024 Aug 02.
Artículo en Inglés | MEDLINE | ID: mdl-39093600

RESUMEN

TAD boundaries are genomic elements that separate biological processes in neighboring domains by blocking DNA loops that are formed through Cohesin-mediated loop extrusion. Most TAD boundaries consist of arrays of binding sites for the CTCF protein, whose interaction with the Cohesin complex blocks loop extrusion. TAD boundaries are not fully impermeable though and allow a limited amount of inter-TAD loop formation. Based on the reanalysis of Nano-C data, a multicontact Chromosome Conformation Capture assay, we propose a model whereby clustered CTCF binding sites promote the successive stalling of Cohesin and subsequent dissociation from the chromatin. A fraction of Cohesin nonetheless achieves boundary read-through. Due to a constant rate of Cohesin dissociation elsewhere in the genome, the maximum length of inter-TAD loops is restricted though. We speculate that the DNA-encoded organization of stalling sites regulates TAD boundary permeability and discuss implications for enhancer-promoter loop formation and other genomic processes.

10.
Mol Microbiol ; 2024 Apr 05.
Artículo en Inglés | MEDLINE | ID: mdl-38578226

RESUMEN

The interplay between bacterial chromosome organization and functions such as transcription and replication can be studied in increasing detail using novel experimental techniques. Interpreting the resulting quantitative data, however, can be theoretically challenging. In this minireview, we discuss how connecting experimental observations to biophysical theory and modeling can give rise to new insights on bacterial chromosome organization. We consider three flavors of models of increasing complexity: simple polymer models that explore how physical constraints, such as confinement or plectoneme branching, can affect bacterial chromosome organization; bottom-up mechanistic models that connect these constraints to their underlying causes, for instance, chromosome compaction to macromolecular crowding, or supercoiling to transcription; and finally, data-driven methods for inferring interpretable and quantitative models directly from complex experimental data. Using recent examples, we discuss how biophysical models can both deepen our understanding of how bacterial chromosomes are structured and give rise to novel predictions about bacterial chromosome organization.

11.
J Pathol ; 263(2): 226-241, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38572612

RESUMEN

Loss of the cell-cell adhesion protein E-cadherin underlies the development of diffuse-type gastric cancer (DGC), which is characterized by the gradual accumulation of tumor cells originating from the gastric epithelium in the surrounding stroma. How E-cadherin deficiency drives DGC formation remains elusive. Therefore, we investigated the consequences of E-cadherin loss on gastric epithelial organization utilizing a human gastric organoid model and histological analyses of early-stage DGC lesions. E-cadherin depletion from gastric organoids recapitulates DGC initiation, with progressive loss of a single-layered architecture and detachment of individual cells. We found that E-cadherin deficiency in gastric epithelia does not lead to a general loss of epithelial cohesion but disrupts the spindle orientation machinery. This leads to a loss of planar cell division orientation and, consequently, daughter cells are positioned outside of the gastric epithelial layer. Although basally delaminated cells fail to detach and instead reintegrate into the epithelium, apically mispositioned daughter cells can trigger the gradual loss of the single-layered epithelial architecture. This impaired architecture hampers reintegration of mispositioned daughter cells and enables basally delaminated cells to disseminate into the surrounding matrix. Taken together, our findings describe how E-cadherin deficiency disrupts gastric epithelial architecture through displacement of dividing cells and provide new insights in the onset of DGC. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Asunto(s)
División Celular , Organoides , Neoplasias Gástricas , Células de Riñón Canino Madin Darby , Animales , Perros , Estómago/patología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Epitelio/metabolismo , Epitelio/patología , Proliferación Celular
12.
Methods ; 223: 106-117, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38295892

RESUMEN

The connection between the patterns observed in 3C-type experiments and the modeling of polymers remains unresolved. This paper presents a simulation pipeline that generates thermodynamic ensembles of 3D structures for topologically associated domain (TAD) regions by loop extrusion model (LEM). The simulations consist of two main components: a stochastic simulation phase, employing a Monte Carlo approach to simulate the binding positions of cohesins, and a dynamical simulation phase, utilizing these cohesins' positions to create 3D structures. In this approach, the system's total energy is the combined result of the Monte Carlo energy and the molecular simulation energy, which are iteratively updated. The structural maintenance of chromosomes (SMC) protein complexes are represented as loop extruders, while the CCCTC-binding factor (CTCF) locations on DNA sequence are modeled as energy minima on the Monte Carlo energy landscape. Finally, the spatial distances between DNA segments from ChIA-PET experiments are compared with the computer simulations, and we observe significant Pearson correlations between predictions and the real data. LoopSage model offers a fresh perspective on chromatin loop dynamics, allowing us to observe phase transition between sparse and condensed states in chromatin.


Asunto(s)
Cromatina , Proteínas Cromosómicas no Histona , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromosomas/metabolismo , Cohesinas
13.
Biochem Soc Trans ; 52(2): 793-802, 2024 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-38451192

RESUMEN

Eukaryotic genomes are compacted and organized into distinct three-dimensional (3D) structures, which range from small-scale nucleosome arrays to large-scale chromatin domains. These chromatin structures play an important role in the regulation of transcription and other nuclear processes. The molecular mechanisms that drive the formation of chromatin structures across scales and the relationship between chromatin structure and function remain incompletely understood. Because the processes involved are complex and interconnected, it is often challenging to dissect the underlying principles in the nuclear environment. Therefore, in vitro reconstitution systems provide a valuable approach to gain insight into the molecular mechanisms by which chromatin structures are formed and to determine the cause-consequence relationships between the processes involved. In this review, we give an overview of in vitro approaches that have been used to study chromatin structures across scales and how they have increased our understanding of the formation and function of these structures. We start by discussing in vitro studies that have given insight into the mechanisms of nucleosome positioning. Next, we discuss recent efforts to reconstitute larger-scale chromatin domains and loops and the resulting insights into the principles of genome organization. We conclude with an outlook on potential future applications of chromatin reconstitution systems and how they may contribute to answering open questions concerning chromatin architecture.


Asunto(s)
Ensamble y Desensamble de Cromatina , Cromatina , Genoma , Nucleosomas , Nucleosomas/metabolismo , Cromatina/metabolismo , Cromatina/genética , Cromatina/química , Humanos , Animales
14.
Crit Rev Biotechnol ; : 1-21, 2024 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-38797671

RESUMEN

Global food security has recently been under serious threat from the rapid rise in the world's population, the problems brought on by climate change, and the appearance of new pandemics. As a result, the need for novel and innovative solutions to solve the existing problems and improve food sustainability has become crucial. 3D printing is expected to play a significant role in providing tangible contributions to the food industry in achieving sustainable development goals. The 3D food printing holds the potential to produce highly customized food in terms of shape, texture, flavor, structure and nutritional value and enable us to create new unique formulations and edible alternatives. The problem of whether the cost of the printed meal and 3D printing itself can be sustainably produced is becoming more and more important due to global concerns. This review intends to provide a comprehensive overview of 3D printed foods with an overview of the current printing methodologies, illustrating the technology's influencing factors, and its applications in personalized nutrition, packaging, value addition, and valorization aspects to fully integrate sustainability concerns thus exploring the potential of 3D food printing.

15.
Chemistry ; 30(17): e202304060, 2024 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-38206188

RESUMEN

This work establishes the first direct mechanocatalytic reaction protocol within an extruder, focusing on the Suzuki-Miyaura reaction. Through the coating of either the extruder screws or barrel with Pd, we executed the cross-coupling reaction without the reliance on molecular catalyst compounds or powders, and solvents continuously. We identified the influence and interplay of crucial reaction parameters such as temperature, mechanical energy input, residence time, rheology, and catalyst contact time and finally obtained 36 % and 75 % of the reaction product after one and four reactor passes respectively.

16.
J Nutr ; 154(7): 2053-2064, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38797481

RESUMEN

BACKGROUND: Industrial processing can alter the structural complexity of dietary proteins and, potentially, their digestion and absorption upon ingestion. High-moisture extrusion (HME), a common processing method used to produce meat alternative products, affects in vitro digestion, but human data are lacking. We hypothesized that HME of a mycoprotein/pea protein blend would impair in vitro digestion and in vivo postprandial plasma amino acid availability. METHODS: In Study A, 9 healthy volunteers completed 2 experimental trials in a randomized, double-blind, crossover design. Participants consumed a beverage containing 25 g protein from a "dry" blend (CON) of mycoprotein/pea protein (39%/61%) or an HME content-matched blend (EXT). Arterialized venous blood samples were collected in the postabsorptive state and regularly over a 5-h postprandial period to assess plasma amino acid concentrations. In Study B, in vitro digestibility of the 2 beverages were assessed using bicinchoninic acid assay and optical fluorescence microscopy at baseline and during and following gastric and intestinal digestion using the INFOGEST model of digestion. RESULTS: Protein ingestion increased plasma total, essential (EAA), and branched-chain amino acid (BCAA) concentrations (time effect, P < 0.0001) but more rapidly and to a greater magnitude in the CON compared with the EXT condition (condition × time interaction, P < 0.0001). This resulted in greater plasma availability of EAA and BCAA concentrations during the early postprandial period (0-150 min). These data were corroborated by the in vitro approach, which showed greater protein availability in the CON (2150 ± 129 mg/mL) compared with the EXT (590 ± 41 mg/mL) condition during the gastric phase. Fluorescence microscopy revealed clear structural differences between the 2 conditions. CONCLUSIONS: These data demonstrate that HME delays in vivo plasma amino acid availability following ingestion of a mycoprotein/pea protein blend. This is likely due to impaired gastric phase digestion as a result of HME-induced aggregate formation in the pea protein. This trial was registered at clinicaltrials.gov as NCT05584358.


Asunto(s)
Aminoácidos , Estudios Cruzados , Proteínas en la Dieta , Digestión , Periodo Posprandial , Humanos , Aminoácidos/sangre , Aminoácidos/metabolismo , Adulto , Masculino , Proteínas en la Dieta/administración & dosificación , Femenino , Método Doble Ciego , Adulto Joven , Disponibilidad Biológica , Manipulación de Alimentos , Proteínas de Guisantes
17.
Mol Pharm ; 21(3): 1309-1320, 2024 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-38345459

RESUMEN

Producing amorphous solid dispersions (ASDs) by hot-melt extrusion (HME) is favorable from an economic and ecological perspective but also limited to thermostable active pharmaceutical ingredients (APIs). A potential technology shift from spray-drying to hot-melt extrusion at later stages of drug product development is a desirable goal, however bearing the risk of insufficient comparability of the in vitro and in vivo performance of the final dosage form. Hot-melt extrusion was performed using API/polymer/surfactant mixtures with hydroxypropyl methylcellulose acetate succinate (HPMCAS) as the polymer and evaluated regarding the extrudability of binary and ternary amorphous solid dispersions (ASDs). Additionally, spray-dried ASDs were produced, and solid-state properties were compared to the melt-extruded ASDs. Tablets were manufactured of a ternary ASD lead candidate comparing their in vitro dissolution and in vivo performance. The extrudability of HPMCAS was improved by adding a surfactant as plasticizer, thereby lowering the high melt-viscosity. d-α-Tocopheryl polyethylene glycol succinate (TPGS) as surfactant showed the most similar solid-state properties between spray-dried and extruded ASDs compared to those of poloxamer 188 and sodium dodecyl sulfate. The addition of TPGS, however, barely affected API/polymer interactions. The in vitro dissolution experiment and in vivo dog study revealed a higher drug release of tablets manufactured from the spray-dried ASD compared to the melt-extruded ASD; this was attributed to the different particle size. We could further demonstrate that the drug release can be controlled by adjusting the particle size of melt-extruded ASDs leading to a similar release profile compared to tablets containing the spray-dried dispersion, which confirmed the feasibility of a technology shift from spray-drying to HME upon drug product development.


Asunto(s)
Polietilenglicoles , Polímeros , Animales , Perros , Composición de Medicamentos , Solubilidad , Tensoactivos
18.
Mol Pharm ; 2024 Aug 13.
Artículo en Inglés | MEDLINE | ID: mdl-39135353

RESUMEN

There has been a significant volume of work investigating the design and synthesis of new crystalline multicomponent systems via examining complementary functional groups that can reliably interact through the formation of noncovalent bonds, such as hydrogen bonds (H-bonds). Crystalline multicomponent molecular adducts formed using this approach, such as cocrystals, salts, and eutectics, have emerged as drug product intermediates that can lead to effective drug property modifications. Recent advancement in the production for these multicomponent molecular adducts has moved from batch techniques that rely upon intensive solvent use to those that are solvent-free, continuous, and industry-ready, such as reactive extrusion. In this study, a novel eutectic system was found when processing albendazole and maleic acid at a 1:2 molar ratio and successfully prepared using mechanochemical methods including liquid-assisted grinding and hot-melt reactive extrusion. The produced eutectic was characterized to exhibit a 100 °C reduction in melting temperature and enhanced dissolution performance (>12-fold increase at 2 h point), when compared to the native drug compound. To remove handling of the eutectic as a formulation intermediate, an end-to-end continuous-manufacturing-ready process enables feeding of the raw parent reagents in their respective natural forms along with a chosen polymeric excipient, Eudragit EPO. The formation of the eutectic was confirmed to have taken place in situ in the presence of the polymer, with the reaction yield determined using a multivariate calibration model constructed by combining spectroscopic analysis with partial least-squares regression modeling. The ternary extrudates exhibited a dissolution profile similar to that of the 1:2 prepared eutectic, suggesting a physical distribution (or suspension) of the in situ synthesized eutectic contents within the polymeric matrix.

19.
Mol Pharm ; 21(4): 1933-1941, 2024 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-38502549

RESUMEN

Islatravir, a highly potent nucleoside reverse transcriptase translocation inhibitor (NRTTI) for the treatment of HIV, has great potential to be formulated as ethylene-vinyl acetate (EVA) copolymer-based implants via hot melt extrusion. The crystallinity of EVA determines its physical and rheological properties and may impact the drug-eluting implant performance. Herein, we describe the systematic analysis of factors affecting the EVA crystallinity in islatravir implants. Differential scanning calorimetry (DSC) on EVA and solid-state NMR revealed drug loading promoted EVA crystallization, whereas BaSO4 loading had negligible impact on EVA crystallinity. The sterilization through γ-irradiation appeared to significantly impact the EVA crystallinity and surface characteristics of the implants. Furthermore, DSC analysis of thin implant slices prepared with an ultramicrotome indicated that the surface layer of the implant was more crystalline than the core. These findings provide critical insights into factors affecting the crystallinity, mechanical properties, and physicochemical properties of the EVA polymer matrix of extruded islatravir implants.


Asunto(s)
Desoxiadenosinas , Etilenos , Polivinilos , Compuestos de Vinilo , Polivinilos/química
20.
Pharm Res ; 41(6): 1233-1245, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38744732

RESUMEN

PURPOSE: This study was designed to develop ibuprofen (IBU) sustained-release amorphous solid dispersion (ASD) using polymer composites matrix with drug release plateaus for stable release and to further reveal intrinsic links between polymer' matrix ratios and drug release behaviors. METHODS: Hydrophilic polymers and hydrophobic polymers were combined to form different composite matrices in developing IBU ASD formulations by hot melt extrusion technique. The intrinsic links between the mixed polymer matrix ratio and drug dissolution behaviors was deeply clarified from the dissolution curves of hydrophilic polymers and swelling curves of composite matrices, and intermolecular forces among the components in ASDs. RESULTS: IBU + ammonio methacrylate copolymer type B (RSPO) + poly(1-vinylpyrrolidone-co-vinyl acetate) (PVP VA64) physical mixtures presented unstable release behaviors with large error bars due to inhomogeneities at the micrometer level. However, IBU-RSPO-PVP VA64 ASDs showed a "dissolution plateau phenomenon", i.e., release behaviors of IBU in ASDs were unaffected by polymer ratios when PVP VA64 content was 35% ~ 50%, which could reduce risks of variations in release behaviors due to fluctuations in prescriptions/processes. The release of IBU in ASDs was simultaneously regulated by the PVP VA64-mediated "dissolution" and RSPO-PVP VA64 assembly-mediated "swelling". Radial distribution function suggested that similar intermolecular forces between RSPO and PVP VA64 were key mechanisms for the "dissolution plateau phenomenon" in ASDs at 35% ~ 50% of PVP VA64. CONCLUSIONS: This study provided ideas for developing ASD sustained-release formulations with stable release plateau modulated by polymer combinations, taking full advantages of simple process/prescription, ease of scale-up and favorable release behavior of ASD formulations.


Asunto(s)
Preparaciones de Acción Retardada , Composición de Medicamentos , Liberación de Fármacos , Ibuprofeno , Polímeros , Preparaciones de Acción Retardada/química , Ibuprofeno/química , Ibuprofeno/administración & dosificación , Polímeros/química , Composición de Medicamentos/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Solubilidad , Tecnología de Extrusión de Fusión en Caliente/métodos , Compuestos de Vinilo/química , Pirrolidinas/química , Química Farmacéutica/métodos , Povidona/química
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