Transport-controlled growth decoupling for self-induced protein expression with a glycerol-repressible genetic circuit.

Decoupling cell formation from recombinant protein synthesis is a potent strategy to intensify bioprocesses. Escherichia coli strains with mutations in the glucose uptake components lack catabolite repression, display low growth rate, no overflow metabolism, and high recombinant protein yields. Fast growth rates were promoted by the simultaneous consumption of glucose and glycerol, and this was followed by a phase of slow growth, when only glucose remained in the medium. A glycerol-repressible genetic circuit was designed to autonomously induce recombinant protein expression. The engineered strain bearing the genetic circuit was cultured in 3.9 g L-1 glycerol + 18 g L-1 glucose in microbioreactors with online oxygen transfer rate monitoring. The growth was fast during the simultaneous consumption of both carbon sources (C-sources), while expression of the recombinant protein was low. When glycerol was depleted, the growth rate decreased, and the specific fluorescence reached values 17% higher than those obtained with a strong constitutive promoter. Despite the relatively high amount of C-source used, no oxygen limitation was observed. The proposed approach eliminates the need for the substrate feeding or inducers addition and is set as a simple batch culture while mimicking fed-batch performance.

Recombinant protein expression in proteome-reduced cells under aerobic and oxygen-limited regimes.

Industrial cultures are hindered by the physiological complexity of the host and the limited mass transfer capacity of conventional bioreactors. In this study, a minimal cell approach was combined with genetic devices to overcome such issues. A flavin mononucleotide-based fluorescent protein (FbFP) was expressed in a proteome-reduced Escherichia coli (PR). When FbFP was expressed from a constitutive protein generator (CPG), the PR strain produced 47% and 35% more FbFP than its wild type (WT), in aerobic or oxygen-limited regimes, respectively. Metabolic and expression models predicted more efficient biomass formation at higher fluxes to FbFP, in agreement with these results. A microaerobic protein generator (MPG) and a microaerobic transcriptional cascade (MTC) were designed to induce FbFP expression upon oxygen depletion. The FbFP fluorescence using the MTC in the PR strain was 9% higher than that of the WT bearing the CPG under oxygen limitation. To further improve the PR strain, the pyruvate dehydrogenase complex regulator gene was deleted, and the Vitreoscilla hemoglobin was expressed. Compared to oxygen-limited cultures of the WT, the engineered strains increased the FbFP expression more than 50% using the MTC. Therefore, the designed expression systems can be a valuable alternative for industrial cultivations.

Effect of Basic Amino Acids on Folic Acid Solubility.

To prevent neural tube defects and other cardiovascular diseases in newborns, folic acid (FA) is recommended in pregnant women. A daily dose of 600 µg FA consumption is widely prescribed for women during pregnancy and 400 µg for women with childbearing potential. FA is a class IV compound according to the Biopharmaceutics Classification System (BCS) due to its low permeability (1.7 × 10-6 cm/s) and low solubility (1.6 mg/L); therefore, it must be administered via a formulation that enhances its solubility. Studies reported in the literature have proved that co-amorphization and salt formation of a poorly soluble drug with amino acids (AA) can significantly increase its solubility. Although arginine has been used with FA as a supplement, there is no information on the effect of basic AA (arginine and lysine) on the physical and chemical properties of FA-AA binary formulations. The present study implemented a conductimetric titration methodology to find the effective molar ratio to maximize FA solubility. The results showed that a 1:2.5 FA:AA molar ratio maximized solubility for arginine and lysine. Binary formulations were prepared using different methods, which led to an amorphous system confirmed by the presence of a glass transition, broad FTIR bands, and the absence of an X-ray diffraction pattern. Results of FA:AA (1:2.5) solubility increased in the range of 5500-6000 times compared with pure FA. In addition to solubility enhancement, the binary systems presented morphological properties that depend on the preparation method and whose consideration could be strategic for scaling purposes.

Mechanical Activation by Ball Milling as a Strategy to Prepare Highly Soluble Pharmaceutical Formulations in the Form of Co-Amorphous, Co-Crystals, or Polymorphs.

Almost half of orally administered active pharmaceutical ingredients (APIs) have low solubility, which affects their bioavailability. In the last two decades, several alternatives have been proposed to modify the crystalline structure of APIs to improve their solubility; these strategies consist of inducing supramolecular structural changes in the active pharmaceutical ingredients, such as the amorphization and preparation of co-crystals or polymorphs. Since many APIs are thermosensitive, non-thermal emerging alternative techniques, such as mechanical activation by milling, have become increasingly common as a preparation method for drug formulations. This review summarizes the recent research in preparing pharmaceutical formulations (co-amorphous, co-crystals, and polymorphs) through ball milling to enhance the physicochemical properties of active pharmaceutical ingredients. This report includes detailed experimental milling conditions (instrumentation, temperature, time, solvent, etc.), as well as solubility, bioavailability, structural, and thermal stability data. The results and description of characterization techniques to determine the structural modifications resulting from transforming a pure crystalline API into a co-crystal, polymorph, or co-amorphous system are presented. Additionally, the characterization methodologies and results of intermolecular interactions induced by mechanical activation are discussed to explain the properties of the pharmaceutical formulations obtained after the ball milling process.

Glucose transport engineering allows mimicking fed-batch performance in batch mode and selection of superior producer strains.

BACKGROUND: Fed-batch mode is the standard culture technology for industrial bioprocesses. Nevertheless, most of the early-stage cell and process development is carried out in batch cultures, which can bias the initial selection of expression systems. Cell engineering can provide an alternative to fed-batch cultures for high-throughput screening and host selection. We have previously reported a library of Escherichia coli strains with single and multiple deletions of genes involved in glucose transport. Compared to their wild type (W3110), the mutant strains displayed lower glucose uptake, growth and aerobic acetate production rates. Therefore, when cultured in batch mode, such mutants may perform similar to W3110 cultured in fed-batch mode. To test that hypothesis, we evaluated the constitutive expression of the green fluorescence protein (GFP) in batch cultures in microbioreactors using a semi defined medium supplemented with 10 or 20 g/L glucose + 0.4 g yeast extract/g glucose. RESULTS: The mutant strains cultured in batch mode displayed a fast-growth phase (growth rate between 0.40 and 0.60 h-1) followed by a slow-growth phase (growth rate between 0.05 and 0.15 h-1), similar to typical fed-batch cultures. The phase of slow growth is most probably caused by depletion of key amino acids. Three mutants attained the highest GFP fluorescence. Particularly, a mutant named WHIC (ΔptsHIcrr, ΔmglABC), reached a GFP fluorescence up to 14-fold greater than that of W3110. Strain WHIC was cultured in 2 L bioreactors in batch mode with 100 g/L glucose + 50 g/L yeast extract. These cultures were compared with exponentially fed-batch cultures of W3110 maintaining the same slow-growth of WHIC (0.05 h-1) and using the same total amount of glucose and yeast extract than in WHIC cultures. The WHIC strain produced approx. 450 mg/L GFP, while W3110 only 220 mg/L. CONCLUSION: The combination of cell engineering and high throughput screening allowed the selection of a particular mutant that mimics fed-batch behavior in batch cultures. Moreover, the amount of GFP produced by the strain WHIC was substantially higher than that of W3110 under both, batch and fed-batch schemes. Therefore, our results represent a valuable technology for accelerated bioprocess development.

Global transcriptomic response of Escherichia coli to p-coumaric acid.

The aromatic compound p-coumaric acid (p-CA) is a secondary metabolite produced by plants. This aromatic acid and derived compounds have positive effects on human health, so there is interest in producing them in biotechnological processes with recombinant Escherichia coli strains. To determine the physiologic response of E. coli W3110 to p-CA, dynamic expression analysis of selected genes fused to a fluorescent protein reporter as well as RNA-seq and RT-qPCR were performed. The observed transcriptional profile revealed the induction of genes involved in functions related to p-CA active export, synthesis of cell wall and membrane components, synthesis of amino acids, detoxification of formaldehyde, phosphate limitation, acid stress, protein folding and degradation. Downregulation of genes encoding proteins involved in energy production, carbohydrate import and metabolism, as well as several outer and plasma membrane proteins was detected. This response is indicative of cell envelope damage causing the leakage of intracellular components including amino acids and phosphate-containing compounds. The cellular functions responding to p-CA that were identified in this study will help in defining targets for production strains improvement.

[The impact of COVID-19 on sick leave of the persons working in nursing homes in Spain. Usefulness of sick leave for the surveillance of the pandemic evolution.] / El impacto de la Covid-19 en la incapacidad temporal de las personas que trabajan en centros socio-sanitarios en España. Utilidad de la incapacidad temporal para el seguimiento de la evolución de la pandemia.

OBJECTIVE: The nursing homes represented high-risk settings for SARS-CoV-2 infection, both for residents and for the employees. The COVID-19 impact on long-term care facilities (LTCFs) is evaluated, measured through the employees sick leave (SL). The pandemic evolution in the general population aged between 16 and 65 years was analyzed together with the sick leave to assess the latter as a complementary indicator of the SARS-CoV-2 surveillance. METHODS: A descriptive study of all sick leave processes due to COVID-19 recorded between February 15th 2020 and May 1st 2021 in nursing homes was carried out. The close contact sick leave/infection sick leave ratios, the 100,000 affiliated/occupied sick leave rates were computed and compared with the COVID-19 cases cumulative incidence notified to the National Network of epidemiological Surveillance (RENAVE). RESULTS: 261.892 SL processes were recorded. The close contact sick leave/infection sick leave median ratio in nursing homes was 1.8 (Interquartile range, ICR: 1.1-3.3), with values lower than 1 at certain periods. The infection sick leaves were higher in number and ratio and prior to the cases recorded in RENAVE. The sick leave ratio ranged between 81.679/100.000 occupied in nursing homes with medical care and 4.895/100.000 in other residential facilities. CONCLUSIONS: The results confirmed the dramatic impact of COVID-19 in nursing homes and the inequalities characterizing this impact. They also confirmed the potential use of sick leave as an alternative source for epidemiological and public health surveillance, especially now, when the transition of the COVID-19 surveillance to a system not including universal individual surveillance is being discussed.

OBJETIVO: Los centros sociosanitarios representaron entornos de alto riesgo de contagio por SARS-CoV-2, tanto para los residentes como para las personas trabajadoras. Se evaluó el impacto en términos de incapacidad temporal (IT) por COVID-19 en las personas que trabajan en centros sociosanitarios y se comparó con la evolución de la pandemia en la población general de 16 a 65 años, para valorar la utilidad de la IT como indicador complementario de la epidemia por SARS-CoV-2. METODOS: Se realizó un estudio descriptivo de todos los procesos de incapacidad temporal por COVID-19 registrados entre el 15 de febrero de 2020 y el 1 de mayo de 2021 en establecimientos residenciales. Se obtuvieron las ratios de incapacidad temporal por contacto estrecho /incapacidad temporal por infección, las tasas de incapacidad temporal por 100.000 afiliados/ocupados y se compararon con la incidencia acumulada de casos COVID-19 notificados a la Red Nacional de Vigilancia Epidemiológica (RENAVE). RESULTADOS: Se registraron 261.892 procesos de incapacidad temporal. La mediana de la ratio de incapacidad temporal por contacto estrecho /incapacidad temporal por infección en residencias fue de 1,8 (Rango intercuartílico, RIC: 1,1-3,3), con valores menores a 1 en periodos. Las IT por infección fueron superiores en número, tasa y anteriores en el tiempo a los casos registrados en RENAVE. Por tipo de residencia, la tasa de incapacidad temporal osciló entre 81.679/100.000 ocupados en asistencia en establecimientos residenciales con cuidados sanitaros y 4.895/100.000 en otros establecimientos residenciales. CONCLUSIONES: Los resultados confirmaron el enorme impacto que tuvo la COVID-19 en los centros sociosanitarios y la desigualdad que ha caracterizado este impacto. Apoyan también la posible utilización de la incapacidad temporal como fuente de información alternativa para la vigilancia epidemiológica y de salud pública, lo cual resulta de especial interés en este momento en el que se está planteando una transición en la vigilancia del COVID-19 hacia un sistema que ya no incluya una vigilancia individualizada universal.

El impacto de la COVID-19 en la incapacidad temporal de las personas que trabajan en centro / The impact of COVID-19 on sick leave of the persons working in nursing homes in Spain. Usefulness of sick leave for the surveillance of the pandemic evolution

Fundamentos: Los centros sociosanitarios representaron entornos de alto riesgo de contagio por SARS-CoV-2, tanto para los residentes como para las personas trabajadoras. Se evaluó el impacto en términos de incapacidad temporal (IT) por COVID-19 en las personas que trabajan en centros sociosanitarios y se comparó con la evolución de la pandemia en la población general de 16 a 65 años, para valorar la utilidad de la IT como indicador complementario de la epidemia por SARS-CoV-2. Métodos: Se realizó un estudio descriptivo de todos los procesos de incapacidad temporal por COVID-19 registrados entre el 15 de febrero de 2020 y el 1 de mayo de 2021 en establecimientos residenciales. Se obtuvieron las ratios de incapacidad temporal por contacto estrecho /incapacidad temporal por infección, las tasas de incapacidad temporal por 100.000 afiliados/ocupados y se compararon con la incidencia acumulada de casos COVID-19 notificados a la Red Nacional de Vigilancia Epidemiológica (RENAVE). Resultados: Se registraron 261.892 procesos de incapacidad temporal. La mediana de la ratio de incapacidad temporal por contacto estrecho /incapacidad temporal por infección en residencias fue de 1,8 (Rango intercuartílico, RIC: 1,13,3), con valores menores a 1 en periodos. Las IT por infección fueron superiores en número, tasa y anteriores en el tiempo a los casos registrados en RENAVE. Por tipo de residencia, la tasa de incapacidad temporal osciló entre 81.679/100.000 ocupados en asistencia en establecimientos residenciales con cuidados sanitaros y 4.895/100.000 en otros establecimientos residenciales. Conclusiones: Los resultados confirmaron el enorme impacto que tuvo la COVID-19 en los centros sociosanitarios y la desigualdad que ha caracterizado este impacto.(AU)

Background: The nursing homes represented highrisk settings for SARS-CoV-2 infection, both for residents and for the employees. The COVID-19 impact on longterm care facilities (LTCFs) is evaluated, measured through the emplo yees sick leave (SL). The pandemic evolution in the general population aged between 16 and 65 years was analyzed together with the sick leave to assess the latter as a complementary indicator of the SARS-CoV-2 surveillance. Methods: A descriptive study of all sick leave processes due to COVID-19 recorded between February 15th 2020 and May 1st 2021 in nursing homes was carried out. The close contact sick leave/infection sick leave ratios, the 100,000 affiliated/occupied sick leave rates were computed and compared with the COVID-19 cases cumulative incidence notified to the National Network of epidemiological Surveillance (RENAVE). Results: 261.892 SL processes were recorded. The close contact sick leave/infection sick leave median ratio in nursing homes was 1.8 (Interquartile range, ICR: 1.1-3.3), with values lower than 1 at certain periods. The infection sick leaves were higher in number and ratio and prior to the cases recorded in RENAVE. The sick leave ratio ranged between 81.679/100.000 occupied in nursing homes with medical care and 4.895/100.000 in other residential facilities. Conclusions: The results confirmed the dramatic impact of COVID-19 in nursing homes and the inequalities characterizing this impact. They also confirmed the potential useof sick leave as an alternative source for epidemiological and public health surveillance, especially now, when the transition of the COVID-19 surveillance to a system not including universal individual surveillance is being discussed.(AU)

High Hydrostatic Pressure to Increase the Biosynthesis and Extraction of Phenolic Compounds in Food: A Review.

Phenolic compounds from fruits and vegetables have shown antioxidant, anticancer, anti-inflammatory, among other beneficial properties for human health. All these benefits have motivated multiple studies about preserving, extracting, and even increasing the concentration of these compounds in foods. A diverse group of vegetable products treated with High Hydrostatic Pressure (HHP) at different pressure and time have shown higher phenolic content than their untreated counterparts. The increments have been associated with an improvement in their extraction from cellular tissues and even with the activation of the biosynthetic pathway for their production. The application of HHP from 500 to 600 MPa, has been shown to cause cell wall disruption facilitating the release of phenolic compounds from cell compartments. HPP treatments ranging from 15 to 100 MPa during 10-20 min at room temperature have produced changes in phenolic biosynthesis with increments up to 155%. This review analyzes the use of HHP as a method to increase the phenolic content in vegetable systems. Phenolic content changes are associated with either an immediate stress response, with a consequent improvement in their extraction from cellular tissues, or a late stress response that activates the biosynthetic pathways of phenolics in plants.

Induced Changes in Aroma Compounds of Foods Treated with High Hydrostatic Pressure: A Review.

Since conventional thermal processing can have detrimental consequences on aroma compounds, non-thermal technologies such as high hydrostatic pressure (HHP) have been explored. HHP may alter the weak chemical bonds of enzymes. These changes can modify the secondary, tertiary, and quaternary structures of key enzymes in the production of aroma compounds. This can result in either an increase or decrease in their content, along with reactions or physical processes associated with a reduction of molecular volume. This article provides a comprehensive review of HHP treatment's effects on the content of lipid-derived aroma compounds, aldehydes, alcohols, ketones, esters, lactones, terpenes, and phenols, on various food matrices of vegetable and animal origin. The content of aldehydes and ketones in food samples increased when subjected to HHP, while the content of alcohols and phenols decreased, probably due to oxidative processes. Both ester and lactone concentrations appeared to decline due to hydrolysis reactions. There is no clear tendency regarding terpenes concentration when subjected to HHP treatments. Because of the various effects of HHP on aroma compounds, an area of opportunity arises to carry out future studies that allow optimizing and controlling the effect.

Methods for the Modification and Evaluation of Cereal Proteins for the Substitution of Wheat Gluten in Dough Systems.

The substitution of wheat gluten in the food industry is a relevant research area because the only known treatment for celiac disease is abstinence from this protein complex. The use of gluten-free cereals in dough systems has demonstrated that the viscoelastic properties of gluten cannot be achieved without the modification of the protein fraction. The quality of the final product is determined by the ability of the modification to form a matrix similar to that of gluten and to reach this, different methods have been proposed and tested. These procedures can be classified into four main types: chemical, enzymatic, physical, and genetic. This article provides a comprehensive review of the most recent research done in protein modification of cereal and pseudocereals for gluten substitution. The reported effects and methodologies for studying the changes made with each type of modification are described; also, some opportunity areas for future works regarding the study of the effect of protein modifications on gluten-free products are presented.

Infarto hemorrágico espontáneo de nódulo tiroideo / Spontaneous hemorrhagic infarction of thyroid nodule

No disponible

Production of Melanins With Recombinant Microorganisms.

The melanins constitute a diverse group of natural products found in most organisms, having functions related to protection against chemical and physical stresses. These products originate from the enzyme-catalyzed oxidation of phenolic and indolic substrates that polymerize to yield melanins, which include eumelanin, pheomelanin, pyomelanin, and the allomelanins. The enzymes involved in melanin formation belong mainly to the tyrosinase and laccase protein families. The melanins are polymeric materials having applications in the pharmaceutical, cosmetic, optical, and electronic industries. The biotechnological production of these polymers is an attractive alternative to obtaining them by extraction from plant or animal material, where they are present at low concentrations. Several species of microorganisms have been identified as having a natural melanogenic capacity. The development and optimization of culture conditions with these organisms has resulted in processes for generating melanins. These processes are based on the conversion of melanin precursors present in the culture medium to the corresponding polymers. With the application of genetic engineering techniques, it has become possible to overexpress genes encoding enzymes involved in melanin formation, mostly tyrosinases, leading to an improvement in the productivity of melanogenic organisms, as well as allowing the generation of novel recombinant microbial strains that can produce diverse types of melanins. Furthermore, the metabolic engineering of microbial hosts by modifying pathways related to the supply of melanogenic precursors has resulted in strains with the capacity of performing the total synthesis of melanins from simple carbon sources in the scale of grams. In this review, the latest advances toward the generation of recombinant melanin production strains and production processes are summarized and discussed.

Highly Soluble Glimepiride and Irbesartan Co-amorphous Formulation with Potential Application in Combination Therapy.

One-third of the population of the USA suffers from metabolic syndrome (MetS). Treatment of patients with MetS regularly includes drugs prescribed simultaneously to treat diabetes and cardiovascular diseases. Therefore, the development of novel multidrug formulations is recommended. However, the main problem with these drugs is their low solubility. The use of binary co-amorphous systems emerges as a promising strategy to increase drug solubility. In the present study, irbesartan (IBS) and glimepiride (GMP), class II active pharmaceutical ingredients (API), widely used in the treatment of arterial hypertension and diabetes, were selected to develop a novel binary co-amorphous system with remarkable enhancement in the dissolution of both APIs. The phase diagram of IBS-GMP was constructed and co-amorphous systems were prepared by melt-quench, in a wide range of compositions. Dissolution profile (studied at pH 1.2 and 37°C for mole fractions 0.01, 0.1, and 0.5) demonstrated that the xGMP = 0.01 formulation presents the highest enhancement in its dissolution. GMP went from being practically insoluble to reach 3.9 ± 0.9 µg/mL, and IBS showed a 12-fold increment with respect to the dissolution of its crystalline form. Infrared studies showed that the increase in the dissolution profile is related to the intermolecular interactions (hydrogen bonds), which were dependent of composition. Results of structural and thermal characterization performed by XRD and DSC showed that samples have remained in amorphous state for more than 10 months of storage. This work contributes to the development of a highly soluble co-amorphous drugs with potential used in the treatment of MetS.

Co-Amorphous Simvastatin-Nifedipine with Enhanced Solubility for Possible Use in Combination Therapy of Hypertension and Hypercholesterolemia.

The high index of simultaneous incidence of hypertension and hypercholesterolemia in the population of many countries demands the preparation of more efficient drugs. Therefore, there is a significant area of opportunity to provide as many alternatives as possible to treat these illnesses. Taking advantage of the solubility enhancement that can be achieved when an active pharmaceutical ingredient (API) is obtained and stabilized in its amorphous state, in the present work, new drug-drug co-amorphous formulations (Simvastatin SIM- Nifedipine NIF) with enhanced solubility and stability were prepared and characterized. Results show that the co-amorphous system (molar ratio 1:1) is more soluble than the pure commercial APIs studied separately. Aqueous dissolution profiles showed increments of solubility of 3.7 and 1.7 times for SIM and NIF, correspondingly, in the co-amorphous system. The new co-amorphous formulations, monitored in time, (molar fractions 0.3, 0.5 and 0.7 of SIM) remained stable in the amorphous state for more than one year when stored at room temperature and did not show any signs of crystallization when re-heating. Inspection on the remainder of a sample after six hours of dissolution showed no recrystallization, confirming the stability of co-amorphous system. The enhanced solubility of the co-amorphous formulations makes them promising for simultaneously targeting of hypertension and hypercholesterolemia through combination therapy.

Increasing pinosylvin production in Escherichia coli by reducing the expression level of the gene fabI-encoded enoyl-acyl carrier protein reductase

Background: The plant secondary metabolite pinosylvin is a polyphenol from the stilbene family, which have positive effects on human health. Biotechnological production is an attractive alternative for obtaining this stilbene. In Escherichia coli, malonyl-CoA is the precursor for both stilbene and fatty acid syntheses. In this study, with the aim of increasing pinosylvin production, we evaluated a novel approach that is based on reducing the expression of the gene fabI, which encodes the enzyme enoyl-acyl carrier protein reductase that is involved in fatty acid synthesis. Results: A recombineering method was employed to eliminate the chromosomal -35 promoter sequence and the upstream region of the gene fabI in E. coli strain W3110. Analysis, employing RT-qPCR, showed that such modification caused a 60% reduction in the fabI transcript level in the mutant strain W3110Δ-35fabI::Cm compared to the wild type W3110. Synthetic genes encoding a mutant version of 4-coumaroyl-CoA ligase from Streptomyces coelicolor A3 with improved catalytic activity employing cinnamic acid as substrate and a stilbene synthase from Vitis vinifera were cloned to generate the plasmid pTrc-Sc4CL(M)-VvSTS. The production performance of strains W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS and W3110/pTrc-Sc4CL(M)- VvSTS was determined in shake flask cultures with Luria-Bertani medium supplemented with 10 g/L glycerol and 3 mM cinnamic acid. Under these conditions, the strain W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS produced 52.67 mg/L pinosylvin, a level 1.5-fold higher than that observed with W3110/pTrc-Sc4CL(M)-VvSTS. Conclusion: A reduction in the transcript level of fabI caused by the elimination of the -35 and upstream promoter sequences is a successful strategy to improve pinosylvin production in E. coli.

Two-phase amorphous-amorphous solid drug dispersion with enhanced stability, solubility and bioavailability resulting from ultrasonic dispersion of an immiscible system.

Amorphization of active pharmaceutical ingredients (APIs) and the preparation of solid dispersions are strategies that can be synergized to improve the solubility of oral drugs. Immiscibility between an API and a carrier in the molten state that could be perceived as a problem in the preparation of solid dispersions, may actually introduce an advantage. In the present work, a two-phase amorphous-amorphous solid dispersion (AASD) was prepared by ultrasonicating a molten immiscible mixture of indomethacin (IND) and glucose (GLU) prior quenching. By introducing this novel ultrasound assisted method, the immiscible API particles were uniformly dispersed as microscopic glassy clusters of the drug in the solid amorphous GLU matrix; particle sizes of IND in the AASD range from 600nm to 1.4µm. As a result of the amorphization and particle size reduction of IND, its aqueous solubility increased to reach almost 40ppm (8 times more soluble compared to indomethacin in its crystalline state). In addition, the oral bioavailability and its resistance against crystallization were also enhanced; AASD samples have remained amorphous for more than two years of storage.

Long-Term Stability of New Co-Amorphous Drug Binary Systems: Study of Glass Transitions as a Function of Composition and Shelf Time.

The amorphous state is of particular interest in the pharmaceutical industry due to the higher solubility that amorphous active pharmaceutical ingredients show compared to their respective crystalline forms. Due to their thermodynamic instability, drugs in the amorphous state tend to recrystallize; in order to avoid crystallization, it has been a common strategy to add a second component to hinder the crystalline state and form a thermally stable co-amorphous system, that is to say, an amorphous binary system which retains its amorphous structure. The second component can be a small molecule excipient (such as a sugar or an aminoacid) or a second drug, with the advantage that a second active pharmaceutical ingredient could be used for complementary or combined therapeutic purposes. In most cases, the compositions studied are limited to 1:1, 2:1 and 1:2 molar ratios, leaving a gap of information about phase transitions and stability on the amorphous state in a wider range of compositions. In the present work, a study of novel co-amorphous formulations in which the selection of the active pharmaceutical ingredients was made according to the therapeutic effect is presented. Resistance against crystallization and behavior of glass transition temperature ( T g were studied through calorimetric measurements as a function of composition and shelf time. It was found that binary formulations with T g temperatures higher than those of pure components presented long-term thermal stability. In addition, significant increments of T g values, of as much as 15 ∘ C, were detected as a result of glass relaxation at room temperature during storage time; this behavior of glass transition has not been previously reported for co-amorphous drugs. Based on these results, it can be concluded that monitoring behavior of T g and relaxation processes during the first weeks of storage leads to a more objective evaluation of the thermomechanical stability of an amorphous formulation.

Complex regulation of Hsf1-Skn7 activities by the catalytic subunits of PKA in Saccharomyces cerevisiae: experimental and computational evidences.

BACKGROUND: The cAMP-dependent protein kinase regulatory network (PKA-RN) regulates metabolism, memory, learning, development, and response to stress. Previous models of this network considered the catalytic subunits (CS) as a single entity, overlooking their functional individualities. Furthermore, PKA-RN dynamics are often measured through cAMP levels in nutrient-depleted cells shortly after being fed with glucose, dismissing downstream physiological processes. RESULTS: Here we show that temperature stress, along with deletion of PKA-RN genes, significantly affected HSE-dependent gene expression and the dynamics of the PKA-RN in cells growing in exponential phase. Our genetic analysis revealed complex regulatory interactions between the CS that influenced the inhibition of Hsf1/Skn7 transcription factors. Accordingly, we found new roles in growth control and stress response for Hsf1/Skn7 when PKA activity was low (cdc25Δ cells). Experimental results were used to propose an interaction scheme for the PKA-RN and to build an extension of a classic synchronous discrete modeling framework. Our computational model reproduced the experimental data and predicted complex interactions between the CS and the existence of a repressor of Hsf1/Skn7 that is activated by the CS. Additional genetic analysis identified Ssa1 and Ssa2 chaperones as such repressors. Further modeling of the new data foresaw a third repressor of Hsf1/Skn7, active only in the absence of Tpk2. By averaging the network state over all its attractors, a good quantitative agreement between computational and experimental results was obtained, as the averages reflected more accurately the population measurements. CONCLUSIONS: The assumption of PKA being one molecular entity has hindered the study of a wide range of behaviors. Additionally, the dynamics of HSE-dependent gene expression cannot be simulated accurately by considering the activity of single PKA-RN components (i.e., cAMP, individual CS, Bcy1, etc.). We show that the differential roles of the CS are essential to understand the dynamics of the PKA-RN and its targets. Our systems level approach, which combined experimental results with theoretical modeling, unveils the relevance of the interaction scheme for the CS and offers quantitative predictions for several scenarios (WT vs. mutants in PKA-RN genes and growth at optimal temperature vs. heat shock).

Application of ATR-FTIR spectroscopy to the study of thermally induced changes in secondary structure of protein molecules in solid state.

FTIR spectroscopy in combination with ATR sampling technique is the most accessible analytical technique to study secondary structure of proteins both in solid and aqueous solution. Although several studies have demonstrated the applications of ATR-FTIR to study conformational changes of solid dried proteins due to dehydration, there are no reports that demonstrate the application of ATR-FTIR in the study of thermally induced changes of secondary structure of biomolecules directly on the solid state. In this study, four biomolecules of pharmaceutical interest, lysozyme, myoglobine, chymotripsin and human growth hormone (hGH), were studied on the solid state before and after different thermal treatments in order to relate changes of secondary structure to partial or total thermal denaturation processes. The results obtained provide experimental evidence that protein thermal denaturation in the solid state can be detected by displacement of carbonyl bands which correspond to conformational transformations between α-helix to ß-sheet or intermolecular ß-sheet; the molecules studied undergo this transformation when exposed to a temperature close to their denaturation temperature which may become irreversible depending on the extent of the heating treatment. These findings demonstrate that ATR-FTIR is an effective and time efficient technique that allows the monitoring of the protein thermal denaturation process of solid samples without further reconstitution or prior sample preparation.