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In the last decades, liposomes acquired a striking success in the biomedical field thanks to their biocompatibility and drug delivery ability. Many liposomal drug formulations have been already approved by the Food and Drug Administration (FDA) and used for the treatment of a wide range of pathologies with or without further engineering. Their clinical application requires strict compliance with high standard quality rules, and it is crucial to employ storage methods that do not affect the integrity of the vesicles and preventing the leakage of their cargo. In this work, the design of a suitable formulation for freeze-drying had been investigated for two different liposomes, DOPC-DOTAP and the PEGylated counterpart, DOPC-DOTAP-DSPE-PEG. The role of various cryoprotectants was evaluated paying attention to their ability to preserve the structural integrity of liposomes. At first, the study was focused on freezing and two methodologies were investigated, quenching in liquid nitrogen and shelf-ramped freezing. This analysis showed that the disaccharides (cellobiose, glucose, lactose, sucrose, and trehalose) and the polyol (mannitol) protected successfully the integrity of liposomes, while during the process, in the presence of a surfactant, liposomes were strongly damaged and fragmented by the ice crystals. Furthermore, the choice of the rate of freezing depended on the different compositions of the lipid bilayer. Finally, the effects of lyophilization on liposomes with and without additives were studied; cellobiose, lactose and trehalose showed encouraging results for the maintenance of the morpho-functional parameters of liposomes during the entire freeze-drying process., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Genetic variants are considered risk factors for gastric cancer. To date, 61 polymorphisms have been identified as associated with this disease. The aim of the present study was to analyze the association of some of those polymorphisms with GC in Chile. We performed a case-control study including 310 gastric cancer cases and 311 controls to assess the association of 36 single-nucleotide polymorphisms genotyped by Global Screening Array (GSA). Three polymorphisms was significantly associated: PSCA rs2294008 (allele model, OR = 1.49, 95%CI 1.17-1.88, P = 1.08 × 10-3), IL-4 rs2243250 (allele model, OR = 1.28, 95%CI 1.01-1.62, P = 0.04), and MUC1 rs4072037 (allele model, OR = 0.78, 95%CI 0.61-0.99, P = 0.04).PSCA rs2294008, IL-4 rs2243250 and MUC1 rs4072037 are associated with gastric cancer in Chile. It suggests that those polymorphisms could be used as biomarkers to assess the genetic risk for this cancer outside of the previously studied populations, not only for East Asians and Caucasians populations.

The protein-osmolyte interaction has been shown experimentally to follow an additive construct, where the individual osmolyte-backbone and osmolyte-side-chain interactions contribute to the overall conformational stability of proteins. Here, we computationally reconstruct this additive relation using molecular dynamics simulations, focusing on sugars and polyols, including sucrose and sorbitol, as model osmolytes. A new set of parameters (ADD) is developed for this purpose, using the individual Kirkwood-Buff integrals for sugar-backbone and sugar-side-chain interactions as target experimental data. We show that the ADD parameters can reproduce the additivity of protein-sugar interactions and correctly predict sucrose and sorbitol self-association and their interaction with water. The accurate description of the separate osmolyte-backbone and osmolyte-side-chain contributions also automatically translates into a good prediction of preferential exclusion from the surface of ribonuclease A and α-chymotrypsinogen A. The description of sugar polarity is improved compared to previous force fields, resulting in closer agreement with the experimental data and better compatibility with charged groups, such as the guanidinium moiety. The ADD parameters are developed in combination with the CHARMM36m force field for proteins, but good compatibility is also observed with the AMBER 99SB-ILDN and the OPLS-AA force fields. Overall, exploiting the additivity of protein-osmolyte interactions is a promising approach for the development of new force fields.

Proteins are unstable molecules that may be severely injured by stresses encountered during freeze-thawing. Despite this, the selection of freeze-thaw conditions is currently empirical, and this results in reduced process control. Here we propose a mathematical model that takes into account the leading causes of protein instability during freeze-thawing, i.e. cold denaturation and surface-induced unfolding, and may guide the selection of optimal operating conditions. It is observed that a high cooling rate is beneficial for molecules that are extremely sensitive to cold denaturation, while the opposite is true when ice-induced unfolding is dominant. In all cases, a fast thawing rate is observed to be beneficial. The simulation outputs are confirmed by experimental data for myoglobin and lactate dehydrogenase, suggesting that the proposed modeling approach can reproduce the main features of protein behavior during freeze-thawing. This approach can therefore guide the selection of optimal conditions for protein-based formulations that are stored in a frozen or freeze-dried state.

The controlled modification of surface properties represents a pervasive requirement to be fulfilled when developing new technologies. In this paper, we propose an easy-to-implement protocol for the functionalization of glass with self-assembled monolayers (SAMs). The adaptivity of the synthesis route was demonstrated by the controlled anchoring of thiol, amino, glycidyloxy, and methacrylate groups onto the glass surface. The optimization of the synthetic pathway was mirrored by extremely smooth SAMs (approximately 150 pm roughness), layer thickness comparable to the theoretical molecule length, absence of silane islands along the surface, quasi-unitary degree of packing, and tailored wettability and charge. The functionalization kinetics of two model silanes, 3-mercapto- and 3-amino-propyltrimethoxysilane, was determined by cross-comparing x-ray photoelectron spectroscopy and time of flight secondary ion mass spectrometry data. Our SAMs with tailored physicochemical attributes will be implemented as supports for the crystallization of pharmaceuticals and biomolecules in upcoming studies. Here, the application to a small molecule drug model, namely aspirin, was discussed as a proof of concept.

Growing empirical evidence reveals that traditional set-theoretic structures cannot in general be applied to cognitive phenomena. This has raised several problems, as illustrated, for example, by probability judgement errors and decision-making (DM) errors. We propose here a unified theoretical perspective which applies the mathematical formalism of quantum theory in Hilbert space to cognitive domains. In this perspective, judgements and decisions are described as intrinsically non-deterministic processes which involve a contextual interaction between a conceptual entity and the cognitive context surrounding it. When a given phenomenon is considered, the quantum-theoretic framework identifies entities, states, contexts, properties and outcome statistics, and applies the mathematical formalism of quantum theory to model the considered phenomenon. We explain how the quantum-theoretic framework works in a variety of judgement and decision situations where systematic and significant deviations from classicality occur.

The ice-water interface is commonly encountered in our life, and comes into play in a wide number of natural phenomena. Here, attention will be focused on its effects on protein stability, with specific reference to the case of pharmaceutical proteins. This field represents a fascinating, and not yet fully understood, subject of investigation. Some background information on the ice-water phase diagram, as well as to the mechanisms of nucleation and crystal growth, will be provided. We will eventually discuss the effect of ice on protein activity, reviewing the mechanisms of ice-induced denaturation that have been proposed so far and discussing the strategies that may help prevent, or minimize, undesired loss of therapeutic activity., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

During the freezing step of a typical freeze drying process, the temperature at which nucleation is induced is generally stochastically distributed, resulting in undesired within-batch heterogeneity. Controlled nucleation techniques have been developed to address this problem; these make it possible to trigger the formation of ice crystals at the same time and temperature in all the batch. Here, the controlled nucleation technique known as vacuum induced surface freezing is compared to spontaneous freezing for the freeze drying of human plasma, a highly concentrated system commonly stored in a dried state. The potency of Factor VIII (FVIII), a sensitive, labile protein present in plasma, and the reconstitution time of the dried cakes are evaluated immediately after freeze drying, and after 1, 3, 6 or 9 months storage at different degradation temperatures. We show that the application of controlled nucleation significantly reduces the reconstitution time and in addition helps to improve FVIII stability., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Crown Copyright © 2020. Published by Elsevier B.V. All rights reserved.)

The process of freezing proteins is widely used in applications ranging from processing and storage of biopharmaceuticals to cryo-EM analysis of protein complexes. The formation of an ice-water interface is a critical destabilization factor for the protein, which can be offset by the use of cryo-protectants. Using molecular dynamics simulation, we demonstrate that the presence of the ice-water interface leads to a lowering of the free-energy barrier for unfolding, resulting in rapid unfolding of the protein. The unfolding process does not require direct adsorption of the protein to the surface, but is rather mediated by nearby liquid molecules that show an increased tendency for hydrating nonpolar groups. The observed enhancement in the cold denaturation process upon ice formation can be mitigated by addition of glucose, which acts as a cryoprotectant through preferential exclusion from side chains of the protein.

In the present work, the effect of controlled nucleation on the stability of human growth hormone (hGH) during freeze-drying has been investigated. More specifically, the vacuum-induced surface freezing technique has been compared to conventional freezing, both with and without an annealing step. Size exclusion chromatography and cell-based potency assays have been used to characterize the formation of soluble aggregates and the biological activity of hGH, respectively. The results obtained indicate that controlled nucleation has a positive effect on both cycle performance and product homogeneity because of the formation of bigger ice crystals, and characterized by a narrower dimensional distribution. From the point of view of hGH stability, we observed that vacuum-induced surface freezing is not detrimental to the biological activity of the protein, or aggregate formation. In addition, the effect of 2 different formulations, including trehalose or cellobiose, on protein preservation was also considered for this study., (Crown Copyright © 2020. Published by Elsevier Inc. All rights reserved.)

Freezing is widely used during the manufacturing process of protein-based therapeutics, but it may result in undesired loss of biological activity. Many variables come into play during freezing that could adversely affect protein stability, creating a complex landscape of interrelated effects. The current approach to the selection of freezing conditions is however nonsystematic, resulting in poor process control. Here we show how mathematical models, and a design space approach, can guide the selection of the optimal freezing protocol, focusing on protein stability. Two opposite scenarios are identified, suggesting that the ice-water interface is the dominant cause of denaturation for proteins with high bulk stability, while the duration of the freezing process itself is the key parameter to be controlled for proteins that are susceptible to cold denaturation. Experimental data for lactate dehydrogenase and myoglobin as model proteins support the model results, with a slow freezing rate being optimal for lactate dehydrogenase and the opposite being true for myoglobin. A possible application of the calculated design space to the freezing and freeze-drying of biopharmaceuticals is finally described, and some considerations on process efficiency are discussed as well., (Copyright © 2020 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The association of an active principle with a nanocarrier is known to improve its stability and protect it from external factors. Nevertheless, loading of nanoparticles with highly hydrophilic substances like caffeine remains a tricky issue. In the present study, inverse miniemulsion systems were successfully coupled to UV radiation to synthesize polymeric nanohydrogels for drug delivery. The proper choice of the continuous and dispersed phase chemical composition led to the entrapment of active principle into the miniemulsion droplets. Our confinement-based strategy enabled unprecedented caffeine encapsulation efficiency inside 100-nm particles. Dimensional, thermal, and spectroscopic characterizations were carried out to investigate both unloaded and loaded nanohydrogels. Furthermore, in vitro release studies evaluated caffeine release kinetics from nanohydrogels by means of dialysis tests. It was demonstrated that controlled and sustained release occurred within the first 50 hours. Experimental data were found to fit the Higuchi model suggesting that the active principle release is diffusion controlled.

This paper provides an organic overview of the most interesting continuous freeze-drying concepts that have been proposed over the years. Attention has mainly been focused on the field of pharmaceuticals, but some background has also been given on the food industry. This work aims at providing a solid starting point for future research on continuous manufacturing for the freeze-drying of pharmaceuticals., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Background: Gray scale ultrasound (US), Doppler and Contrast Enhanced Ultrasound (CEUS) represent important surveillance tools in the early post-operative period after pancreas transplantation (PTx), when complications are more common. This review summarizes the available evidence on their clinical application in this setting., Methods: We searched the Pub-Med database from inception to October 2018 for English literature on the clinical use of US, Doppler and CEUS in the post-PTx surveillance. Article selection was carried out according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses criteria (PRISMA)., Results: Twenty-nine articles concerning the clinical applications of US, Doppler and CEUS were identified, 13 of which, involving 264 patients, were focused on the sonographic findings in immunologic rejection, whereas 11 studies reporting on 887 patients were focused on post-PTx vascular complications. The remaining five articles, involving a total of 196 patients, described US or CEUS applied in the study of pancreatic morphology and texture to diagnose peri-graft fluids collections or to obtain experimental data on allograft endocrine function., Conclusions: US, Doppler and CEUS have proven to be valuable assets in post-PTx follow up, thanks to the combination of their non-invasiveness with a high accuracy in the detection of early abnormalities, in particular regarding vascular complications. Preliminary experiences are directing towards functional research; however, future prospective trials are necessary to precisely correlate organ perfusion, early abnormalities and allograft function., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Patients with pancreatic cystic neoplasms (PCN), without surgical indication at the time of diagnosis according to current guidelines, require lifetime image-based surveillance follow-up. In these patients, the current European evidenced-based guidelines advise magnetic resonance imaging (MRI) scanning every 6 mo in the first year, then annually for the next five years, without reference to any role for trans-abdominal ultrasound (US). In this study, we report on our clinical experience of a follow-up strategy of image-based surveillance with US, and restricted use of MRI every two years and for urgent evaluation whenever suspicious changes are detected by US., Aim: To report the results and cost-efficacy of a US-based surveillance follow-up for known PCNs, with restricted use of MRI., Methods: We retrospectively evaluated the records of all the patients treated in our institution with non-surgical PCN who received follow-up abdominal US and restricted MRI from the time of diagnosis, between January 2012 and January 2017. After US diagnosis and MRI confirmation, all patients underwent US surveillance every 6 mo for the first year, and then annually. A MRI scan was routinely performed every 2 years, or at any stage for all suspicious US findings. In this communication, we reported the clinical results of this alternative follow-up, and the results of a comparative cost-analysis between our surveillance protocol (abdominal US and restricted MRI) and the same patient cohort that has been followed-up in strict accordance with the European guidelines recommended for an exclusive MRI-based surveillance protocol., Results: In the 5-year period, 200 patients entered the prescribed US-restricted MRI surveillance follow-up. Mean follow-up period was 25.1 ± 18.2 mo. Surgery was required in two patients (1%) because of the appearance of suspicious features at imaging (with complete concordance between the US scan and the on-demand MRI). During the follow-up, US revealed changes in PCN appearance in 28 patients (14%). These comprised main pancreatic duct dilatation ( n = 1), increased size of the main cyst ( n = 14) and increased number of PNC ( n = 13). In all of these patients, MRI confirmed US findings, without adding more information. The bi-annual MRI identified evolution of the lesions not identified by US in only 11 patients with intraductal papillary mucinous neoplasms (5.5%), largely consisting of an increased number of very small PCN ( P = 0.14). The overall mean cost of surveillance, based on a theoretical use of the European evidenced-based exclusive MRI surveillance in the same group of patients, would have been 1158.9 ± 798.6 € per patient, in contrast with a significantly lower cost of 366.4 ± 348.7 € ( P < 0.0001) incurred by the US-restricted MRI surveillance used at our institution., Conclusion: In patients with non-surgical PCN at the time of diagnosis, US surveillance could be a safe complementary approach to MRI, delaying and reducing the numbers of second level examinations and therefore reducing the costs., Competing Interests: Conflict-of-interest statement: No conflict of interest.

Biopharmaceuticals are often stored in a lyophilized form. However, stresses due to both the freezing and the drying steps of the lyophilization process can be harmful to protein stability, and appropriate excipients must be added to minimize detrimental effects. In this work, molecular dynamics was used to provide insight into the mechanisms of protein stabilization by different osmolytes, using lactate dehydrogenase as model protein. Our simulations indicate that good cryoprotectants are not always equally good as lyoprotectants, suggesting that synergistic effects may arise when different excipients are combined. This observation is in accordance with the experimental results. In fact, the enzymatic activity of lactate dehydrogenase after freeze-drying was investigated for various formulations, and the trend predicted by molecular dynamics was confirmed. More specifically, we found that the most effective stabilization of the protein structure is achieved when a good cryoprotectant is coupled with an efficient lyoprotectant. Ultimately, we propose a new approach to the design of formulations for protein-based therapeutics to be lyophilized, which combines simulations and experiments. In this new concept, the computational investigation allows a more knowledge-driven and targeted experimental campaign for the selection of the optimal excipients, making the whole process extremely time- and cost-effective., (Copyright © 2019 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The RAS/RAF/MEK/ERK pathway regulates certain cellular functions, including cell proliferation, differentiation, survival, and apoptosis. Dysregulation of this pathway leads to the occurrence and progression of cancers mainly by somatic mutations. This study aimed to assess if polymorphisms of the RAS/RAF/MEK/ERK pathway are associated with gastric cancer. A case-control study of 242 gastric cancer patients and 242 controls was performed to assess the association of 27 single nucleotide polymorphisms (SNPs) in the RAS/RAF/MEK/ERK pathway genes with gastric cancer. Analyses performed under the additive model (allele) showed four significantly associated SNPs: RAF1 rs3729931 (Odds ratio (OR) = 1.54, 95%, confidence interval (CI): 1.20⁻1.98, p -value = 7.95 × 10 -4 ), HRAS rs45604736 (OR = 1.60, 95% CI: 1.16⁻2.22, p -value = 4.68 × 10 -3 ), MAPK1 rs2283792 (OR = 1.45, 95% CI: 1.12⁻1.87, p -value = 4.91 × 10 -3 ), and MAPK1 rs9610417 (OR = 0.60, 95% CI: 0.42⁻0.87, p -value = 6.64 × 10 -3 ). Functional annotation suggested that those variants or their proxy variants may have a functional effect. In conclusion, this study suggests that RAF1 rs3729931, HRAS rs45604736, MAPK1 rs2283792, and MAPK1 rs9610417 are associated with gastric cancer.

A multi-scale approach can be used to simulate the drying behavior of microparticles in packed-bed. Data outcomes from discrete element method (DEM) and computational fluid dynamics (CFD) simulations can be used to estimate some relevant product characteristics, such as the porosity, tortuosity, voids in the bed and permeability which are required by the multi scale model. Data from DEM simulations are presented, with a particular focus on the influence of the model parameters, packing characteristics and inhomogeneities (wall effect and particles segregation); computational costs and scala bility are also considered. Data on the properties of packings as modeled at the macroscale are presented with regard to the thermal conductivity of gases in the Knudsen regime and effective properties of packed-beds modeled as a pseudo-homogeneous medium. A mathematical model of the freeze-drying of single microparticles and its outcomes are first presented. Data outcomes from the mathematical model at the macroscale concerning the drying behavior of microparticles in a tray and in a vial are then presented and can be used for process design. Some further data, with detailed interpretation and discussion of the presented data, can be found in the related research data article, "A multi-scale computational framework for modelling the freeze-drying of microparticles in packed-beds" (Capozzi et al., 2019).

When proteins bind to interfaces, the resulting changes in protein structure can lead to loss of protein function. We investigate the mechanism by which surfactant molecules can counteract surface-induced protein denaturation through a detailed study of the stability of the GB1 peptide at the air-water, ice-water, and silica-water interfaces using molecular dynamics simulations coupled with metadynamics. Our simulations reveal that the air-water interface and, to a lesser extent, the ice-water interface destabilize the protein by direct interactions between the protein and surface that disrupt the hydrophobic core of the protein, while the weakly interacting silica surface stabilizes the protein through confinement effects. Addition of the surfactant Tween 80 leads to stabilization of the protein at the air-water and ice-water surfaces and mild destabilization at the water-silica interface. We show that the amphiphilic nature of the surfactant is key to its stabilizing/destabilizing effect, with an orientation-dependent mechanism in which the protein is stabilized when the hydrophilic heads of the surfactant point toward the protein.

Background/aim: Inflammation is a key process in gastric carcinogenesis. Cytokines are mediators of inflammation and are involved in metastasis and tumorigenicity. We previously assessed the role of cytokine gene polymorphisms in gastric cancer risk in Chile. In the present study, we aimed to analyze whether these polymorphisms are associated with overall survival (OS) in gastric cancer (GC) patients., Patients and Methods: A total of 153 individuals with GC diagnosis were followed-up for at least 2 years. Hazard ratios (HR) were estimated from Cox regression models using SNPs as predictor variables. The following SNPs were genotyped for study using a TaqMan assay: rs16944 (IL1B -511C>T); rs4073 (IL8 -251 T>A); rs2275913 (IL-17 -197G>A); rs1800872 (IL10 -592 C>A); rs1800896 (IL10 -1082A>G); rs28372698 (IL32)., Results: Interleukin-8 rs4073 (IL-8 -251T>A) showed association with OS under the dominant model (TA + AA) only when adjusted by clinicopathological variables (HR=1.64, 95%CI=1.05-2.55, p=0.030, q-value=0.18), but not with the univariate model (HR=1.51, 95%CI=0.98-2.31, p=0.062, q-value=0.37). No significant differences were observed after adjusting for population stratification (PC1 and PC2 from Principal Component Analysis using genotypes from Infinium Global Screening Array). After stratification by clinicopathological variables, the association with shorter overall survival was higher among patients with diffuse-type tumors (HR=2.24, 95%CI=1.16-4.45) and patients with tumor size >5 cm (HR=1.79, 95%CI=1.08-2.97)., Conclusion: These results suggest a role of IL-8 rs4073 in cancer prognosis. Its use as a prognostic marker of GC survival warrants further investigation., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Despite the wide occurrence of crystallization in the pharmaceutical industry, deep understanding and fine control of the process remain a tricky issue. Nevertheless, the successful manufacturing of finished pharmaceutical products, as well as the structural determination of biopharmaceuticals, depend on the size, form, shape and purity of the crystals. The ability of substrates with precise chemistry and topological features to induce nucleation has been thoroughly assessed during the recent years. This paper reviews the major advances and discoveries in controlling small molecule drug and protein crystallization by means of engineered surfaces. By designing superficial properties and morphology, it has been possible to tune the polymorph outcome, shorten the nucleation induction time, impose specific crystal shapes, control the crystal size and carry out crystallization at very low supersaturation levels. Such achievements underline the potential of surface-induced crystallization to provide an ideal platform for the study of the nucleation process and gain control over its stochastic nature., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The freezing step plays a central role in reaching the most stringent requirements of quality, homogeneity and standardization of freeze-dried products. In this paper, a systematic procedure has been proposed to obtain a quantitative estimation of the pore-size variability of lyophilized products resulting from uncontrollable variations of the nucleation temperature. This procedure consisted in collecting the nucleation temperature from a statistically significant number of samples and correlating each nucleation temperature to the corresponding product morphology, using a mathematical model, to obtain a statistical description of the lyophilized product structure. This approach can also be used to obtain an estimation of the variability of the mass transfer resistance to vapor flow and, finally, of the drying time. Two different freezing configurations, i.e., conventional and suspended-vial freezing, have been used as case studies since they can produce significantly different freezing rates., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Purpose: Surfactants are common stabilizers, often added to biopharmaceuticals formulations, but the mechanisms at the basis of their activity are unclear. The aim of this work is to provide insight into the molecular factors underlying surfactant effectiveness as protectants., Methods: Molecular Dynamics simulations of human growth hormone (hGH) in the presence of Tween 20 were performed. The effect of Tween 20 was compared with the activity of commonly used protectants, such as the disaccharides., Results: We found that Tween 20 could prevent the self-association of hGH, leading to the formation of a protein-surfactant complex. In the case of unfolded hGH, surfactant molecules were oriented with their hydrophilic head in the direction of the protein hydrophobic patches. This created a highly unstable situation, fostering refolding. In the case of native hGH, Tween 20 molecules oriented with their lipophilic groups in the direction of the protein surface. This thermodynamically stabilized the native conformation, preventing unfolding., Conclusions: We found that the ability of surfactants to foster protein refolding is related to their amphiphilic nature, and, more specifically, to their specific orientation with respect to the protein surface. A molecular mechanism explaining surfactant activity is proposed, which could provide direction for improvements in biopreservation., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The stochastic nature of nucleation makes it difficult to control batch homogeneity in conventional freezing, and this lack of control is in contrast with the current emphasis on Quality by Design. Among the techniques which have been developed to overcome this problem, Vacuum Induced Surface Freezing is probably the most promising for application in manufacturing, because it does not require additional equipment and can be scaled-up more easily than other proposed approaches. In this work, we summarize the impact of Vacuum Induced Surface Freezing on product morphology, and the efficiency of the subsequent drying steps as well. We will show that this controlled freezing approach is extremely beneficial for both the efficiency of the freeze-drying process, and the quality and homogeneity of the final product. The hope is that this work could contribute to the commercial implementation of controlled nucleation technology, overcoming the final resistance to its widespread use. It is our opinion that this would be a substantial improvement, beneficial for both the pharmaceutical industry and the end users., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background/aim: Epithelial-mesenchymal transition (EMT) program has been linked as a driver of metastatic dissemination by conferring migratory and invasive capacity to cancer cells. Gastric cancer (GC) patients with tumors expressing altered levels of EMT markers have low survival. This study aimed to assess if polymorphisms of CDH1, TWIST1, SNAIL2, ZEB1 and ZEB2 genes are associated with survival in GC patients., Patients and Methods: A total of 153 individuals with diagnosis of GC were recruited in Santiago, Chile. All patients were genotyped using Infinium Global Screening Array (GSA). Twenty Tag SNPs of the studied genes were retrieved., Results: Three SNPs were associated with survival: rs2526614 (TWIST1) (genotype CA + AA, adjusted HR=0.58, 95%CI=0.37-0.93), rs6953766 (TWIST1) (genotype GG, crude HR=2.02, 95%CI=1.06-3.82, adjusted HR=2.14, 95%CI=1.07-4.25), and rs431073 (ZEB1) (genotype AC + CC, crude HR=1.62, 95%CI=1.01-2.59, adjusted HR=1.96, 95%CI=1.18-3.25)., Conclusion: To the best of our knowledge, this is the first study proposing a role of these SNPs in cancer prognosis. Their use as prognostic markers of GC survival warrants further investigation., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

The present work shows a rational method for the development of the freezing step of a freeze-drying cycle. The current approach to the selection of freezing conditions is still empirical and nonsystematic, thus resulting in poor robustness of control strategy. The final aim of this work is to fill this gap, describing a rational procedure, based on mathematical modeling, for properly choosing the freezing conditions. Mechanistic models are used for the prediction of temperature profiles during freezing and dimension of ice crystals being formed. Mathematical description of the drying phase of freeze-drying is also coupled with the results obtained by freezing models, thus providing a comprehensive characterization of the lyophilization process. In this framework, deep understanding of the phenomena involved is required, and according to the Quality by Design approach, this knowledge can be used to build the design space. The step-by-step procedure for building the design space for freezing is thus described, and examples of applications are provided. The calculated design space is validated upon experimental data, and we show that it allows easy control of the freezing process and fast selection of appropriate operating conditions., (Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Purpose: One of the most common classes of excipients used in protein formulations are buffers. The aim of this work is to investigate the effect of buffers on protein stabilization given by sugars during freeze drying., Methods: Molecular Dynamics simulations of human growth hormone (hGH) in the presence of sucrose and trehalose were performed, and the impact of phosphate and citrate buffers on their protective action was analyzed., Results: We found that buffers broke the hydrogen bonding network formed by excipients, and the consequences of this disruption of structure ordering were different in sucrose-based or trehalose-based formulations. More specifically, we observed that buffers increased protein recovery in the presence of excipients, such as sucrose, that exert their action mainly by preferential exclusion. By contrast, the opposite effect was sometimes noted in the case of excipients, such as trehalose, whose protective action is related to the formation of a highly structured matrix., Conclusions: We found that buffers have important properties, other than the control of pH, that can contribute to the overall stability of proteins. Some of these properties are related to their interaction with the other components of the formulation.

Biopharmaceuticals are frequently stored in the frozen state to avoid rapid degradation. Moreover, therapeutic proteins are frequently made into a dried form to provide long-term storage. However, both freezing and drying stresses can result in protein unfolding and aggregation. Thus, a proper formulation, containing suitable excipients, must be used to avoid loss of activity. Here, the conformational stability of a model protein, human growth hormone, is studied during freezing, and in the dried state as well, using molecular dynamics. The impact of the ice-water interface and of water removal is deeply investigated, and the role of protectants in preventing denaturation phenomena is addressed. We found that good cryo-protectants not always are equally effective as lyo-protectants, and experimental data confirmed simulation results. From this analysis, we also discovered that the interaction of stabilizers with specific amino acid sequences of the protein, rather than with the molecule as a whole, seems to be a crucial issue in the preservation of protein structure. This finding was confirmed for another protein, i.e., lactate dehydrogenase, thus suggesting that it is a generally applicable result. Remarkably, those sequences which unfolded during freezing and drying, generally coincided with the aggregation prone regions of the protein.

Afamin is an 87 kDa glycoprotein with five predicted N-glycosylation sites. Afamin's glycan abundance contributes to conformational and chemical inhomogeneity presenting great challenges for molecular structure determination. For the purpose of studying the structure of afamin, various forms of recombinantly expressed human afamin (rhAFM) with different glycosylation patterns were thus created. Wild-type rhAFM and various hypoglycosylated forms were expressed in CHO, CHO-Lec1, and HEK293T cells. Fully nonglycosylated rhAFM was obtained by transfection of point-mutated cDNA to delete all N-glycosylation sites of afamin. Wild-type and hypo/nonglycosylated rhAFM were purified from cell culture supernatants by immobilized metal ion affinity and size exclusion chromatography. Glycan analysis of purified proteins demonstrated differences in micro- and macro-heterogeneity of glycosylation enabling the comparison between hypoglycosylated, wild-type rhAFM, and native plasma afamin. Because antibody fragments can work as artificial chaperones by stabilizing the structure of proteins and consequently enhance the chance for successful crystallization, we incubated a Fab fragment of the monoclonal anti-afamin antibody N14 with human afamin and obtained a stoichiometric complex. Subsequent results showed sufficient expression of various partially or nonglycosylated forms of rhAFM in HEK293T and CHO cells and revealed that glycosylation is not necessary for expression and secretion.

Molecular dynamics is here used to elucidate the mechanism of protein stabilization by carbohydrates and other additives during freezing. More specifically, we used molecular dynamics simulations to obtain a quantitative estimation of the capability of various cryoprotectants to preserve a model protein, the human growth hormone, against freezing stresses. Three mechanisms were investigated, preferential exclusion, water replacement, and vitrification. Model simulations were finally validated upon experimental data in terms of the ability of excipients to prevent protein aggregation. Overall, we found that the preferential exclusion and vitrification mechanisms are important during the whole freezing process, while water replacement becomes dominant only toward the end of the cryoconcentration phase. The disaccharides were found to be the most efficient excipients, in regard to both preferential exclusion and water replacement. Moreover, sugars were in general more efficient than other excipients, such as glycine or sorbitol.

Vacuum Induced Nucleation is often discussed in the context of primary drying performances and its tunability, with the potential to tailor the nucleation temperature to the desired porous structure. Instead, here we investigate its influence on secondary drying dynamics and, in particular, on rate of desorption and vial-to-vial inhomogeneity. So as to track the evolution of residual moisture during secondary drying, vials were regularly collected through a vacuum-tight sampling device; the residual moisture and the morphology of the porous cake was then determined by Karl Fischer titration and Scanning Electron Microscopy, respectively. The control of freezing promotes the formation of larger ice crystals and, as a result, accelerates the sublimation of ice and slows down the desorption process. Overall, we found that it reduces the total (primary and secondary) drying time and produces much more uniform batches than those obtained by the conventional freezing, and this positive effect was observed since the end of primary drying. In conclusion, the control of freezing was beneficial to the total drying time reduction, vial-to-vial homogeneity and allowed a better control of product inhomogeneity., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The STAR System (Ekymed SpA) is a novel multipurpose sponge developed for conventional manual laparoscopic surgery., Materials and Methods: Between December 2012 and December 2014, we successfully used the sponge in ten robot-assisted and ten direct manual laparoscopic operations to achieve haemostasis, for blunt dissections, for atraumatic lifting of solid organs, to check for bile leaks, for cleaning the surgical field thus avoiding frequent use of suction or the application of haemostatic agents. The reason of the insertion (RI), the main use (MU) and any further use (FU), once inserted, were registered for each operation and compared between the two groups., Results: The principal RI was haemostasis for minor bleeding, without differences between the two groups (P = not significant). Regard to MU, in the robotic group cleaning the surgical field was utilised more than laparoscopic group (100% vs. 60%; P = 0.03). About FU, atraumatic solid organs lifting was more frequent during robotically assisted surgery than with laparoscopy (50% vs. 0%; P = 0.01). A statistically more frequent use of the sponge was registered during standard laparoscopy for the blunt dissection (30% vs. 80%; P = 0.03)., Conclusions: The STAR System was beneficial in both approaches, but it imparts added benefit during robotically-assisted laparoscopic surgery organs because of the lack of tactile feedback and because the operating surgeon is remote from the patient, and has to rely on the assisting surgeon in the sterile field for dealing with bleeding episodes, cleansing/mopping the operative field when necessary, who may not be experienced or completely proficient.

This paper aimed to study the impact of freezing on both within-batch (inter-vial) and within-product (intra-vial) heterogeneity. This analysis has been carried out using two freezing protocols, the conventional shelf-ramped method and the Vacuum Induced Surface Freezing, and placebo formulations containing both crystallizing (mannitol) and amorphous (lactose and sucrose) excipients. The freezing conditions (i.e., the temperature of freezing, the temperature and time of the equilibration phase, and the filling volume) were found to have a dramatic impact on both the within-batch and the within-product homogeneity. Overall, we observed that the control of freezing can effectively minimize the variability in product characteristics, and moisture content, within the same batch. In addition to more uniform production, the control of freezing was found to be fundamental to achieve a more uniform product than that produced by the shelf-ramped freezing method. The influence of the freezing protocol on the crystallization process of mannitol was also investigated, showing that the temperature of freezing plays a key role in the formation of the mannitol polymorphs., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Robot-assisted partial nephrectomy has been proposed as a technique to overcome technical challenges of laparoscopic partial nephrectomy. We prospectively collected and analyzed data from 31 patients who underwent robotic partial nephrectomy with systematic use of hemostatic agents, between February 2009 and October 2014. Thirty-three renal tumors were treated in 31 patients. There were no conversions to open surgery, intraoperative complications, or blood transfusions. The mean size of the resected tumors was 27 mm (median 20 mm, range 5-40 mm). Twenty-seven of 33 lesions (82%) did not require vascular clamping and therefore were treated in the absence of ischemia. All margins were negative. The high partial nephrectomy success rate without vascular clamping suggests that robotic nephron-sparing surgery with systematic use of hemostatic agents may be a safe, effective method to completely avoid ischemia in the treatment of selected renal masses.

Purpose: Few studies have reported minimally invasive total proctocolectomy with ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC) and familial adenomatous polyposis (FAP). We herein report a novel hand-assisted hybrid laparoscopic-robotic technique for patients with FAP and UC., Methods: Between February 2010 and March 2014, six patients underwent hand-assisted hybrid laparoscopic-robotic total proctocolectomy with IPAA. The abdominal colectomy was performed laparoscopically with hand assistance through a transverse suprapubic incision, also used to fashion the ileal pouch. The proctectomy was carried out with the da Vinci Surgical System. The IPAA was hand-sewn through a trans-anal approach. The procedure was complemented by a temporary diverting loop ileostomy., Results: The mean hand-assisted laparoscopic surgery (HALS) time was 154.6 (±12.8) min whereas the mean robotic time was 93.6 (±8.1) min. In all cases, a nerve-sparing proctectomy was performed, and no conversion to traditional laparotomy was required. The mean postoperative hospital stay was 13.2 (±7.4) days. No anastomotic leakage was observed. To date, no autonomic neurological disorders have been observed with a mean of 5.8 (±1.3) bowel movements per day., Conclusions: The hand-assisted hybrid laparoscopic-robotic approach to total proctocolectomy with IPAA has not been previously described. Our report shows the feasibility of this hybrid approach, which surpasses most of the limitations of pure laparoscopic and robotic techniques. Further experience is necessary to refine the technique and fully assess its potential advantages.

Background: While conventional laparoscopic repair for giant hiatal hernias is considered difficult, robotic technology is likely to result in an improved postoperative course., Methods: We prospectively analysed patients with giant hiatal hernias who underwent robotic repair during a 3 year period. Preoperative data, operative variables, complications, clinical outcomes and anatomical recurrence after 1 year were evaluated., Results: Six patients with giant hiatal hernias underwent robotic repair using the Da Vinci surgical system. The mean operative time was 182 min. The mean hospital stay was 6 days. No patients required reoperation for disease recurrence, and all claimed the absence of postoperative symptoms., Conclusions: Robotic approaches can minimize surgical trauma in patients with giant hiatal hernias and result in favourable outcomes in terms of anatomical recurrence and quality of life. With the availability of the da Vinci System, all patients with giant hiatal hernias can be offered a minimally invasive surgical option., (Copyright © 2014 John Wiley & Sons, Ltd.)

An expert panel analyzed the available evidence and reached a consensus to release 24 recommendations for primary and secondary prevention of gastric cancer (CG) in symptomatic patients, with indication for upper GI endoscopy. The main recommendations include (1) Search for and eradicate H. pylori infection in all cases. (2) Systematic gastric biopsies (Sydney protocol) in all patients over 40 years of age or first grade relatives of patient with CG, to detect gastric atrophy, intestinal metaplasia or dysplasia. (3) Incorporate the OLGA system (Operative Link on Gastritis Assessment) to the pathological report, to categorize the individual risk of CG. (4) Schedule endoscopic follow-up according to the estimated risk of CG, namely annual for OLGA III- IV, every 3 years for OLGA I- II or persistent H. pylori infection, every 5 years for CG relatives without other risk factors and no follow-up for OLGA 0, H. pylori (-). (4) Establish basic human and material resources for endoscopic follow-up programs, including some essential administrative processes, and (5) Suggest the early CG/total CG diagnosis ratio of each institution and the proportion of systematic recording of endoscopic images, as quality indicators. These measures are applicable using currently available resources, they can complement any future screening programs for asymptomatic population and may contribute to improve the prognosis of CG in high-risk populations.

This paper deals with the problem of monitoring the freeze-drying process of thermo-labile products and particularly tracking the temperature and the position of the interface of sublimation. A novel and affordable way is proposed to estimate the vapor flow rate and, thus, retrieve the parameters of interest. This method is referred to as pressure decrease test (PDT) and is based on the shutoff of the controlled leakage valve and on the subsequent model-based analysis of the response of the process. In this paper, a detailed description of the PDT algorithm is given; furthermore, the method was tested upon two placebo formulations, that is, sucrose- and mannitol-based solutions, showing that the tool can give an accurate, complete, and noninvasive estimation of all the parameters of interest. These results also show that the method is a valid solution to various technical problems that arise when attempting to apply other techniques proposed in the literature, for example, the pressure rise test and the tunable diode laser absorption spectroscopy technology., (© 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.)

Enzymes typically have a critical instability, which dominates both formulation and process development. In this paper, the ability to preserve the enzyme activity during freeze-drying was investigated for both water-binding and non-water-binding substrates. For this purpose, acid phosphatase was used as model protein. In addition, a procedure for the fast development of a freeze-drying cycle is shown. For the secondary drying part, the effect of processing temperature and time on enzyme activity was investigated. The enzyme activity decreased continuously during secondary drying, with a dramatic drop associated with processing temperatures higher than 293 K. Besides product temperature, the residual moisture level and water mobility are also important. As the residual moisture level and water mobility depend on the product formulation, the stabilizing effect against the enzyme deactivation was studied for a number of formulations which contain different additives, that is, sucrose, lactose, mannitol, and poly-vinylpyrrolidone, with a dramatic activity loss associated with crystallizing excipients. This study also confirmed that not all water-binding substrates can successfully protect the enzyme against deactivation. In fact, water-binding substrates containing reducing sugars (lactose) showed the highest loss of activity., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This paper shows the application of mathematical modeling to scale-up a cycle developed with lab-scale equipment on two different production units. The above method is based on a simplified model of the process parameterized with experimentally determined heat and mass transfer coefficients. In this study, the overall heat transfer coefficient between product and shelf was determined by using the gravimetric procedure, while the dried product resistance to vapor flow was determined through the pressure rise test technique. Once model parameters were determined, the freeze-drying cycle of a parenteral product was developed via dynamic design space for a lab-scale unit. Then, mathematical modeling was used to scale-up the above cycle in the production equipment. In this way, appropriate values were determined for processing conditions, which allow the replication, in the industrial unit, of the product dynamics observed in the small scale freeze-dryer. This study also showed how inter-vial variability, as well as model parameter uncertainty, can be taken into account during scale-up calculations.

Purpose: This paper shows how to optimize the primary drying phase, for both product quality and drying time, of a parenteral formulation via design space., Methods: A non-steady state model, parameterized with experimentally determined heat and mass transfer coefficients, is used to define the design space when the heat transfer coefficient varies with the position of the vial in the array. The calculations recognize both equipment and product constraints, and also take into account model parameter uncertainty., Results: Examples are given of cycles designed for the same formulation, but varying the freezing conditions and the freeze-dryer scale. These are then compared in terms of drying time. Furthermore, the impact of inter-vial variability on design space, and therefore on the optimized cycle, is addressed. With this regard, a simplified method is presented for the cycle design, which reduces the experimental effort required for the system qualification., Conclusions: The use of mathematical modeling is demonstrated to be very effective not only for cycle development, but also for solving problem of process transfer. This study showed that inter-vial variability remains significant when vials are loaded on plastic trays, and how inter-vial variability can be taken into account during process design.

Background: Collapsing glomerulopathy is a cause of nephrotic syndrome with massive proteinuria secondary to podocyte proliferation and glomerular collapse. It is characterized by an almost inevitable progression to end stage renal failure, poor response to treatment and high post-transplant recurrence. Its frequency has increased in recent years due to its common association with Human Immunodeficiency Virus (HIV) infection and the growing recognition of new etiologic agents such as drugs and parvovirus B19. Therefore, it is a disease of growing interest for clinicians. The aim of this review is to update the clinical presentation, diagnosis, pathogenesis and therapeutic alternatives of this disease.

This paper deals with the design space of a pharmaceutical freeze-drying process. Mathematical modeling is used to investigate the effect of the operating conditions [shelf temperature (T(shelf)) and chamber pressure (P(c))] on product temperature (that has to remain below a limit value) and sublimation flux (that has to be lower than a level that would cause choked flow). The algorithm takes into account the variation of the design space with time due to the increase in the dried layer thickness. Besides T(shelf) and P(c), the dried layer thickness is used as the third coordinate of the diagram, thus resulting in just one graph that can be used to build recipes with variable operating conditions, as well as to analyze the effect of process failures. Such results are compared with those obtained when the variation of the design space with time is not accounted for; in this case, the design space comprises those operating conditions that fulfill the operation constraints throughout primary drying, thus giving a much more conservative recipe when designing the process or potentially misleading results when analyzing process failures. Finally, the proposed method has been used to design, and experimentally validate, a recipe for a pharmaceutical formulation., (Copyright © 2011 Wiley-Liss, Inc.)

This paper is focused on the in-line monitoring of the secondary drying phase of a lyophilization process. An innovative software sensor is presented to estimate reliably the residual moisture in the product and the time required to complete secondary drying, that is, to reach the target value of the residual moisture or of the desorption rate. Such results are obtained by coupling a mathematical model of the process and the in-line measurement of the solvent desorption rate and by means of the pressure rise test or another sensors (e.g., windmills, laser sensors) that can measure the vapor flux in the drying chamber. The proposed method does not require extracting any vial during the operation or using expensive sensors to measure off-line the residual moisture. Moreover, it does not require any preliminary experiment to determine the relationship between the desorption rate and residual moisture in the product. The effectiveness of the proposed approach is demonstrated by means of experiments carried out in a pilot-scale apparatus: in this case, some vials were extracted from the drying chamber and the moisture content was measured to validate the estimations provided by the soft-sensor., (Copyright © 2010 Wiley-Liss, Inc.)

This paper deals with the in-line optimization and control of the freeze-drying process of pharmaceuticals in vials. The proposed control system, named LyoDriver, uses a mathematical model of the process to calculate the values of the temperature of the heating fluid: the goal is to minimize the time required to get the desired amount of residual water in the dried product, and to maintain product temperature below the maximum allowed value, thus preserving product quality. The values of product temperature and residual ice content, as well as other parameters, are required to perform the calculations: these variables are estimated in-line by means of the Dynamic Parameters Estimation algorithm, an advanced tool based on the pressure rise test, but also other monitoring systems can be used. Two different control algorithms are presented and investigated by means of mathematical simulation and experiments carried out in a small industrial-type apparatus (LyoBeta 25 by Telstar). Results show the effectiveness of LyoDriver in a wide range of operating conditions, even when the process becomes mass-transfer controlled, or when the operating pressure is changed., (© 2010 Wiley-Liss, Inc. and the American Pharmacists Association)

Newborn babies admitted into the neonatal intensive care unit (NICU) often require many supportive invasive devices and frequently receive antimicrobial therapy. We investigated the microbial flora in NICU patients reporting the distribution of infections in different catheter sites. Results showed that 97% of samples were positive; in particular 11% were positive for two or more microbial agents. Coagulase negative Staphylococci were the most commonly isolated. The detection of Gram-negative bacteria and yeasts suggested that these microorganisms are also involved in infections of hospitalized infants. Finally, no correlation between a specific microbial agent and a particular catheter type was found.