Inflammation as a Possible Trigger for Mitoxantrone-Induced Cardiotoxicity: An In Vivo Study in Adult and Infant Mice.

Mitoxantrone (MTX) is a pharmaceutical drug used in the treatment of several cancers and refractory multiple sclerosis (MS). Despite its therapeutic value, adverse effects may be severe, namely the frequently reported cardiotoxicity, whose mechanisms need further research. This work aimed to assess if inflammation or oxidative stress-related pathways participate in the cardiotoxicity of MTX, using the mouse as an animal model, at two different age periods (infant or adult mice) using two therapeutic relevant cumulative doses. Histopathology findings showed that MTX caused higher cardiac toxicity in adults. In MTX-treated adults, at the highest dose, noradrenaline cardiac levels decreased, whereas at the lowest cumulative dose, protein carbonylation increased and the expression of nuclear factor kappa B (NF-κB) p65 subunit and of M1 macrophage marker increased. Moreover, MTX-treated adult mice had enhanced expression of NF-κB p52 and tumour necrosis factor (TNF-α), while decreasing interleukin-6 (IL-6). Moreover, while catalase expression significantly increased in both adult and infant mice treated with the lowest MTX cumulative dose, the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and glutathione peroxidase only significantly increased in infant animals. Nevertheless, the ratio of GAPDH to ATP synthase subunit beta decreased in adult animals. In conclusion, clinically relevant doses of MTX caused dissimilar responses in adult and infant mice, being that inflammation may be an important trigger to MTX-induced cardiotoxicity.

Development of a Platform to Align Education and Practice: Bridging Academia and the Profession in Portugal.

Limited fitness for practice may result from a mismatch between education and practice. Aiming to meet the common interests of academics and practitioners, the Portuguese Pharmaceutical Society (PPS) developed the Education and Practice Platform (EPP). The EPP includes one representative from each pharmacy faculty, and all Councils of Speciality Boards of Practice. Brainstorming with involved parties enabled sharing of interests, concerns and identifying a common path. Aims, mission, vision and values were set. The EPP's mission is to: act as an enabler to foster the quality and adequacy of education through sharing best practices, ultimately leading to facilitate professional integration, and to foster quality development in teaching practices with recognition for autonomy in freedom to teach and to learn. Its vision is an alignment of education and practice with the PPS' statutes to ensure validation of the competences defined for each practice area, and compliance with international guidance. Key performance indicators (KPIs) were set. Activities developed include the creation of a national forum to discuss education and practice, development of workshops on teaching methods and pharmacy internships, enhanced representation in international events and response to global and national requests. Ongoing work focuses on the creation of a common training framework in hospital and community pharmacy practice adapted to Portugal. The EPP is a worldwide case study, encouraging the development of discussion contributing to an open climate of sharing best practices, indirectly leading to foster a better alignment between education and practice. Many of these results are so far intangible in scientific terms but worth describing.

Triple co-culture of human alveolar epithelium, endothelium and macrophages for studying the interaction of nanocarriers with the air-blood barrier.

Predictive in vitro models are valuable alternatives to animal experiments for evaluating the transport of molecules and (nano)particles across biological barriers. In this work, an improved triple co-culture of air-blood barrier was set-up, being exclusively constituted by human cell lines that allowed to perform experiments at air-liquid interface. Epithelial NCI-H441 cells and endothelial HPMEC-ST1.6R cells were seeded at the apical and basolateral sides of a Transwell® membrane, respectively. Differentiated THP-1 cells were also added on the top of the epithelial layer to mimetize alveolar macrophages. Translocation and permeability studies were also performed. It was observed that around 14-18% of 50-nm Fluorospheres®, but less than 1% of 1.0 µm-Fluorospheres® could pass through the triple co-culture as well as the epithelial monoculture and bi-cultures, leading to the conclusion that both in vitro models represented a significant biological barrier and could differentiate the translocation of different sized systems. The permeability of isoniazid was similar between the epithelial monoculture and bi-cultures when compared with the triple co-culture. However, when in vitro models were challenged with lipopolysaccharide, the release of interleukin-8 increased in the bi-cultures and triple co-culture, whereas the NCI-H441 monoculture did not show any proinflammatory response. Overall, this new in vitro model is a potential tool to assess the translocation of nanoparticles across the air-blood barrier both in healthy state and proinflammatory state. STATEMENT OF SIGNIFICANCE: The use of in vitro models for drug screening as an alternative to animal experiments is increasing over the last years, in particular, models to assess the permeation through biological membranes. Cell culture models are mainly constituted by one type of cells forming a confluent monolayer, but due to its oversimplicity they are being replaced by three-dimensional (3D) in vitro models, that present a higher complexity and reflect more the in vivo-like conditions. Being the pulmonary route one of the most studied approaches for drug administration, several in vitro models of alveolar epithelium have been used to assess the drug permeability and translocation and toxicity of nanocarriers. Nevertheless, there is still a lack of 3D in vitro models that mimic the morphology and the physiological behavior of the alveolar-capillary membrane. In this study, a 3D in vitro model of the air-blood barrier constituted by three different relevant cell lines was established and morphologically characterized. Different permeability/translocation studies were performed to achieve differences/similarities comparatively to each monoculture (epithelium, endothelium, and macrophages) and bi-cultures (epithelial cells either cultured with endothelial cells or macrophages). The release of pro-inflammatory cytokines (namely interleukin-8) after incubation of lipopolysaccharide, a pro-inflammatory inductor, was also evaluated in this work.

Mannose-functionalized solid lipid nanoparticles are effective in targeting alveolar macrophages.

Mannose receptor is highly expressed on alveolar macrophages, being a potential target to promote the specific local drug delivery of anti-tuberculosis agents through the use of functionalized nanocarriers. In this work, isoniazid (Isn)-loaded solid lipid nanoparticles (SLN), reinforced with stearylamine (SA) were produced by double emulsion technique and further surface-functionalized with mannose in a straightforward chemical approach. Upon pre-formulation assessment, SLN close to 500 nm average size, positively charged and with association efficiency of ISN close to 50% were obtained. Functionalization with mannose was performed after SLN production and confirmed by Fourier transform infrared spectroscopy (FTIR). Both functionalized and non-functionalized SLN demonstrated to devoid of toxicity when tested in human lung epithelial cell line (NCI-H441) and differentiated THP-1 (dTHP-1), reducing the intrinsic cytotoxicity of Isn when incorporated into SLN. Uptake studies were conducted on same macrophage-like cells and the results showed that fluorescent mannosylated SLN (M-SLN) were more efficient in be internalized comparatively to SLN. Moreover, the uptake of M-SLN was reduced when cells were pre-incubated with mannose, demonstrating the receptor-dependence internalization of functionalized SLN. These functionalized nanocarriers may represent a useful platform to target alveolar macrophages for delivering anti-infective drugs.

Nanoparticles-in-film for the combined vaginal delivery of anti-HIV microbicide drugs.

Combining two or more antiretroviral drugs in one medical product is an interesting but challenging strategy for developing topical anti-HIV microbicides. We developed a new vaginal delivery system comprising the incorporation of nanoparticles (NPs) into a polymeric film base - NPs-in-film - and tested its ability to deliver tenofovir (TFV) and efavirenz (EFV). EFV-loaded poly(lactic-co-glycolic acid) NPs were incorporated alongside free TFV into fast dissolving films during film manufacturing. The delivery system was characterized for physicochemical properties, as well as genital distribution, local and systemic 24h pharmacokinetics (PK), and safety upon intravaginal administration to mice. NPs-in-film presented suitable technological, mechanical and cytotoxicity features for vaginal use. Retention of NPs in vivo was enhanced both in vaginal lavages and tissue when associated to film. PK data evidenced that vaginal drug levels rapidly decreased after administration but NPs-in-film were still able to enhance drug concentrations of EFV. Obtained values for area-under-the-curve for EFV were around one log10 higher than those for the free drugs in aqueous vehicle (phosphate buffered saline). Film alone also contributed to higher and more prolonged local drug levels as compared to the administration of TFV and EFV in aqueous vehicle. Systemic exposure to both drugs was low. NPs-in-film was found to be safe upon once daily vaginal administration to mice, with no significant genital histological changes or major alterations in cytokine/chemokine profiles being observed. Overall, the proposed NPs-in-film system seems to be an interesting delivery platform for developing combination vaginal anti-HIV microbicides.

Moving toward personalized medicine in rheumatoid arthritis: SNPs in methotrexate intracellular pathways are associated with methotrexate therapeutic outcome.

AIM: Evaluate the potential of selected SNPs as predictors of methotrexate (MTX) therapeutic outcome. PATIENTS & METHODS: In total, 35 SNPs in 14 genes involved in MTX intracellular pathways and Phase II reactions were genotyped in 233 rheumatoid arthritis (RA) patients treated with MTX. Binary logistic regressions were performed by genotype/haplotype-based approaches. Non-Response- and Toxicity-Genetic Risk Indexes (Non-RespGRI and ToxGRI) were created. RESULTS: MTX nonresponse was associated to eight genotypes and three haplotypes: MTHFR rs1801131 AA and rs1801133 TT; MS rs1805087 AA; MTRR rs1801394 A carriers; ATIC rs2372536 C carriers, rs4673993 T carriers, rs7563206 T carriers and rs12995526 T carriers; CC for GGH rs3758149 and rs12681874; CGTTT for ATIC combination 1; and CTTTC for ATIC combination 2. From overall Non-RespGRI patients with indexes 6-8 had more than sixfold increased risk for MTX nonresponse than those patients with indexes 0-5. MTX-related toxicity was associated to five genotypes and two haplotypes: ATIC rs2372536 G carriers, rs3821353 T carriers, rs7563206 CC and rs12995526 CC; ADORA2A rs2267076 T; CTTCC for ATIC combination 1; and TC for ADORA2A rs2267076 and rs2298383. From overall ToxGRI, patients with indexes 3-4 had more than sevenfold increased risk for MTX-related toxicity than those patients with indexes 1-2. CONCLUSION: Genotyping may be helpful to identify which RA patients will not benefit from MTX treatment and, consequently, important to personalized medicine in RA. Nevertheless, further studies are required to validate these findings.

Synthesis and characterization of non-toxic and thermo-sensitive poly(N-isopropylacrylamide)-grafted cashew gum nanoparticles as a potential epirubicin delivery matrix.

Cashew gum (CG) was grafted with N-isopropylacrylamide (NIPA) by radical polymerization to originate a stimuli-sensitive copolymer for drug delivery purposes. NMR and IR spectroscopy confirmed the insertion of NIPA onto the cashew gum chains. The graft copolymer (CG:NIPA) demonstrated thermal responsiveness. The critical aggregation concentration of the copolymers at 25°C was higher than at 50°C. At temperatures lower than the LCST, the nanoparticle size ranged from 12 to 21nm, depending on the CG:NIPA ratio, but above the LCST the particles aggregated, increasing the particle size. Regarding the potential for future oral application, the nanoparticles showed no cytotoxic activity against the Caco-2 and HT29-MTX intestine cell lines. Epirubicin was encapsulated into nanoparticles of CG-NIPA (1:1), resulting in a 64% association efficiency and 22% loading capacity. Thus, the CG:NIPA graft copolymer demonstrates good potential for used in controlled drug delivery systems.

Development and in vivo safety assessment of tenofovir-loaded nanoparticles-in-film as a novel vaginal microbicide delivery system.

UNLABELLED: Topical pre-exposure prophylaxis (PrEP) with antiretroviral drugs holds promise in preventing vaginal transmission of HIV. However, significant biomedical and social issues found in multiple past clinical trials still need to be addressed in order to optimize protection and users' adherence. One approach may be the development of improved microbicide products. A novel delivery platform comprising drug-loaded nanoparticles (NPs) incorporated into a thin polymeric film base (NPs-in-film) was developed in order to allow the vaginal administration of the microbicide drug candidate tenofovir. The system was optimized for relevant physicochemical features and characterized for biological properties, namely cytotoxicity and safety in a mouse model. Tenofovir-loaded poly(lactic-co-glycolic acid) (PLGA)/stearylamine (SA) composite NPs with mean diameter of 127nm were obtained with drug association efficiency above 50%, and further incorporated into an approximately 115µm thick, hydroxypropyl methylcellulose/poly(vinyl alcohol)-based film. The system was shown to possess suitable mechanical properties for vaginal administration and to quickly disintegrate in approximately 9min upon contact with a simulated vaginal fluid (SVF). The original osmolarity and pH of SVF was not affected by the film. Tenofovir was also released in a biphasic fashion (around 30% of the drug in 15min, followed by sustained release up to 24h). The incorporation of NPs further improved the adhesive potential of the film to ex vivo pig vaginal mucosa. Cytotoxicity of NPs and film was significantly increased by the incorporation of SA, but remained at levels considered tolerable for vaginal delivery of tenofovir. Moreover, histological analysis of genital tissues and cytokine/chemokine levels in vaginal lavages upon 14days of daily vaginal administration to mice confirmed that tenofovir-loaded NPs-in-film was safe and did not induce any apparent histological changes or pro-inflammatory response. Overall, obtained data support that the proposed delivery system combining the use of polymeric NPs and a film base may constitute an exciting alternative for the vaginal administration of microbicide drugs in the context of topical PrEP. STATEMENT OF SIGNIFICANCE: The development of nanotechnology-based microbicides is a recent but promising research field seeking for new strategies to circumvent HIV sexual transmission. Different reports detail on the multiple potential advantages of using drug nanocarriers for such purpose. However, one important issue being frequently neglected regards the development of vehicles for the administration of microbicide nanosystems. In this study, we propose and detail on the development of a nanoparticle-in-film system for the vaginal delivery of the microbicide drug candidate tenofovir. This is an innovative approach that, to our best knowledge, had never been tested for tenofovir. Results, including those from in vivo testing, sustain that the proposed system is safe and holds potential for further development as a vaginal microbicide product.

Effect of the Freezing Step in the Stability and Bioactivity of Protein-Loaded PLGA Nanoparticles Upon Lyophilization.

PURPOSE: The freezing step in lyophilization is the most determinant for the quality of biopharmaceutics. Using insulin as model of therapeutic protein, our aim was to evaluate the freezing effect in the stability and bioactivity of insulin-loaded PLGA nanoparticles. The performance of trehalose, sucrose and sorbitol as cryoprotectants was evaluated. METHODS: Cryoprotectants were co-encapsulated with insulin into PLGA nanoparticles and lyophilized using an optimized cycle with freezing at -80°C, in liquid nitrogen, or ramped cooling at -40°C. Upon lyophilization, the stability of protein structure and in vivo bioactivity were assessed. RESULTS: Insulin was co-encapsulated with cryoprotectants resulting in particles of 243-394 nm, zeta potential of -32 to -35 mV, and an association efficiency above 90%. The cryoprotectants were crucial to mitigate the freezing stresses and better stabilize the protein. The insulin structure maintenance was evident and close to 90%. Trehalose co-encapsulated insulin-loaded PLGA nanoparticles demonstrated enhanced hypoglycemic effect, comparatively to nanoparticles without cryoprotectant and added with trehalose, due to a superior insulin stabilization and bioactivity. CONCLUSIONS: The freezing process may be detrimental to the structure of protein loaded into nanoparticles, with negative consequences to bioactivity. The co-encapsulation of cryoprotectants mitigated the freezing stresses with benefits to protein bioactivity.

The formulation of nanomedicines for treating tuberculosis.

Recent estimates indicate that tuberculosis (TB) is the leading cause of death worldwide, alongside the human immunodeficiency virus (HIV) infection. The current treatment is effective, but is associated with severe adverse-effects and noncompliance to prescribed regimens. An alternative route of drug delivery may improve the performance of existing drugs, which may have a key importance in TB control and eradication. Recent advances and emerging technologies in nanoscale systems, particularly nanoparticles (NPs), have the potential to transform such approach to human health and disease. Until now, several nanodelivery systems for the pulmonary administration of anti-TB drugs have been intensively studied and their utility as an alternative to the classical TB treatment has been suggested. In this context, this review provides a comprehensive analysis of recent progress in nanodelivery systems for pulmonary administration of anti-TB drugs. Additionally, more convenient and cost-effective alternatives for the lung delivery, different types of NPs for oral and topical are also being considered, and summarized in this review. Lastly, the future of this growing field and its potential impact will be discussed.

Annealing as a tool for the optimization of lyophilization and ensuring of the stability of protein-loaded PLGA nanoparticles.

The aim of this work was to use annealing as a tool to optimize the lyophilization cycle by decreasing its duration time, and simultaneously preserve the stability of poly(lactic-co-glycolic acid) nanoparticles and with upmost importance, maintain the structural stability of loaded insulin, used as model therapeutic protein. The contribution of a cryoprotectant in this preservation process was also evaluated. Insulin-loaded nanoparticles co-encapsulated with and without trehalose as cryoprotectant were produced, resulting in a particle size of about 250-300 nm, a PdI around 0.25 and a zeta potential in the range of -20 to -24 mV. The insulin association efficiency was higher than 90%, and the loading capacity was of 11-12%. The use of annealing allowed the decrease of duration time of primary drying in about 38%, representing a global decrease of lyophilization time of around 26%. The residual moisture content of all lyophilizates was around 1%, and the reconstitution of lyophilizates obtained using annealing was even faster than those without annealing. The co-encapsulated trehalose better preserved the nanoparticle size throughout the lyophilization process using annealing, compared to formulation containing no cryoprotectant. Fourier-transform infrared spectroscopy showed that the trehalose-containing nanoparticles presented higher insulin structural maintenance, compared to nanoparticles without cryoprotectant, presenting an insulin structural maintenance of 85.3 ± 0.7% and 86.0 ± 1.0% for annealing and no annealing, respectively. This formulation also presented the closest structural similarity with native insulin. Interestingly, the structural features of insulin loaded into nanoparticles upon lyophilization with and without annealing were practically identical, showing that annealing had no detrimental effect in insulin structure. Circular dichroism and fluorescence spectroscopy confirmed these results. Overall, this work gave rise to the importance of annealing in decreasing the duration time of lyophilization of protein-loaded poly(lactic-co-glycolic acid) nanoparticles, and simultaneously ensuring the stability of the carrier and loaded protein.

Co-encapsulation of lyoprotectants improves the stability of protein-loaded PLGA nanoparticles upon lyophilization.

The purpose of this work was to evaluate the influence of the co-encapsulation of lyoprotectants with insulin into PLGA nanoparticles, on the stability of the protein and nanoparticles upon lyophilization. Different lyoprotectants were used, namely trehalose, glucose, sucrose, fructose and sorbitol at 10% (w/v). Insulin-loaded PLGA nanoparticles with co-encapsulated lyoprotectants achieved a mean particle size of 386-466nm, and a zeta potential ranging between -34 and -38mV, dependent on the lyoprotectant used. Formulations had association efficiencies and loading capacities of 85-91% and 10-12%, respectively. The lyophilization process increased the colloidal stability of nanoparticles, and maintained their spherical shape and smooth surface, particularly in presence of lyoprotectants. XRPD revealed that the lyophilizates of nanoparticles with co-encapsulated lyoprotectants were amorphous, whereas formulations with externally added lyoprotectants, except trehalose, showed crystallinity. FTIR assessment showed that co-encapsulating lyoprotectants better preserved insulin structure upon lyophilization with a spectral area overlap of 82-87%, compared to only 72% in lyoprotectant absence. These results were confirmed by circular dichroism spectroscopy. Surprisingly, the simultaneous co-encapsulation and addition of lyoprotectants was detrimental to protein stabilization. The insulin in vitro release studies demonstrated that formulations with co-encapsulated trehalose, glucose, sucrose, fructose and sorbitol achieved 83%, 69%, 70%, 77% and 74%, respectively after 48h. In contrast, formulations added with those lyoprotectants prior lyophilization showed a lower release rate not higher than 60% after 48h. This work gives rise to a different promising strategy of co-encapsulating lyoprotectants and therapeutic proteins, to better stabilize protein structure upon lyophilization.

Pharmacogenomics of Methotrexate Membrane Transport Pathway: Can Clinical Response to Methotrexate in Rheumatoid Arthritis Be Predicted?

BACKGROUND: Methotrexate (MTX) is widely used for rheumatoid arthritis (RA) treatment. Single nucleotide polymorphisms (SNPs) could be used as predictors of patients' therapeutic outcome variability. Therefore, this study aims to evaluate the influence of SNPs in genes encoding for MTX membrane transport proteins in order to predict clinical response to MTX. METHODS: Clinicopathological data from 233 RA patients treated with MTX were collected, clinical response defined, and patients genotyped for 23 SNPs. Genotype and haplotype analyses were performed using multivariate methods and a genetic risk index (GRI) for non-response was created. RESULTS: Increased risk for non-response was associated to SLC22A11 rs11231809 T carriers; ABCC1 rs246240 G carriers; ABCC1 rs3784864 G carriers; CGG haplotype for ABCC1 rs35592, rs2074087 and rs3784864; and CGG haplotype for ABCC1 rs35592, rs246240 and rs3784864. GRI demonstrated that patients with Index 3 were 16-fold more likely to be non-responders than those with Index 1. CONCLUSIONS: This study revealed that SLC22A11 and ABCC1 may be important to identify those patients who will not benefit from MTX treatment, highlighting the relevance in translating these results to clinical practice. However, further validation by independent studies is needed to develop the field of personalized medicine to predict clinical response to MTX treatment.

The age factor for mitoxantrone's cardiotoxicity: multiple doses render the adult mouse heart more susceptible to injury.

Age is a known susceptibility factor for the cardiotoxicity of several anticancer drugs, including mitoxantrone (MTX). The impact of anticancer drugs in young patients is underestimated, thus we aimed to evaluate the cardiotoxicity of MTX in juvenile and adult animals. Juvenile (3 week-old) and adult (8-10 week-old) male CD-1 mice were used. Each group was treated with a 9.0mg/kg cumulative dose of MTX or saline; they were maintained in a drug-free period for 3-weeks after the last administration to allow the development of late toxicity (protocol 1), or sacrificed 24h after the last MTX administration to evaluate early cardiotoxicity (protocol 2). In protocol 1, no adult mice survived, while 2 of the juveniles reached the end of the protocol. High plasma aspartate aminotransferase/alanine aminotransferase ratio and a high cardiac reduced/oxidized glutathione ratio were found in the surviving MTX-treated juvenile mice. In protocol 2, a significant decrease in plasma creatine-kinase MB in juveniles was found 24h after the last MTX-administration. Cardiac histology showed that both MTX-treated populations had significant damage, although higher in adults. However, MTX-treated juveniles had a significant increase in fibrotic tissue. The MTX-treated adults had higher values of cardiac GSSG and protein carbonylation, but lower cardiac noradrenaline levels. For the first time, mature adult animals were shown to be more susceptible to MTX as evidenced by several biomarkers, while young animals appear to better adjust to the MTX-induced cardiac injury. Even so, the higher level of fibrotic tissue and the histological damage showed that MTX also causes cardiac damage in the juvenile population.

Targeted Drug Delivery Systems for Lung Macrophages.

Lung macrophages present an effective role in innate and immune response through specific and non-specific mechanisms, namely phagocytosis, antigen processing and presentation through major histocompatibility complex, activation of T cells and inflammatory cytokines release. Despite their protective role against injury in normal condition, they can cause several lung conditions, since they can mediate several processes, through recruitment of other inflammatory cells for alveolar space, release of proinflammatory cytokines or stimulation of collagen deposition. Lung macrophages constitute a good therapeutic target. With the development of nanotechnology, new carriers have been designed to target drugs towards these cells. One of the commonly used approaches is the attachment of ligands with affinity to receptors presented at lung macrophage surface, to the nanocarriers. This review will focus on the importance of lung macrophages in host-defense, their influence on different pulmonary diseases and different strategies for alveolar macrophage targeting.

Genetic polymorphisms in low-dose methotrexate transporters: current relevance as methotrexate therapeutic outcome biomarkers.

Methotrexate (MTX) is used in low doses to treat a variety of diseases. Although the mechanism responsible for its therapeutic action is unknown, MTX membrane transport proteins (influx and/or efflux) can be major determinants of pharmacokinetics, adverse drug reactions and clinical response profiles. With progess in pharmacogenomics, the improvement of the prediction of patients' therapeutic outcome treated with low doses of MTX will offer a powerful tool for the translation of transporter SNPs into clinical practice and will be essential to sustain a breakthrough in the field of personalized medicine. Therefore, this paper provides an update on the current data on SNPs in genes encoding low-dose MTX membrane transport proteins and their relevance as possible biomarkers of MTX therapeutic outcome.

Role of key TYMS polymorphisms on methotrexate therapeutic outcome in portuguese rheumatoid arthritis patients.

BACKGROUND: Therapeutic outcome of rheumatoid arthritis (RA) patients treated with methotrexate (MTX) can be modulated by thymidylate synthase (TS) levels, which may be altered by genetic polymorphisms in TS gene (TYMS). This study aims to elucidate the influence of TYMS polymorphisms in MTX therapeutic outcome (regarding both clinical response and toxicity) in Portuguese RA patients. METHODS: Clinicopathological data from 233 Caucasian RA patients treated with MTX were collected, outcomes were defined and patients were genotyped for the following TYMS polymorphisms: 1) 28 base pairs (bp) variable number tandem repeat (rs34743033); 2) single nucleotide polymorphism C>G (rs2853542); and 3) 6 bp sequence deletion (1494del6, rs34489327). Chi-square and binary logistic regression analyses were performed, using genotype and haplotype-based approaches. RESULTS: Considering TYMS genotypes, 3R3R (p = 0.005, OR = 2.34), 3RC3RG (p = 0.016, OR = 3.52) and 6bp- carriers (p = 0.011, OR = 1.96) were associated with non-response to MTX. Multivariate analysis confirmed the increased risk for non-response to MTX in 6bp- carriers (p = 0.016, OR = 2.74). Data demonstrated that TYMS polymorphisms were in linkage disequilibrium (p<0.00001). Haplotype multivariate analysis revealed that haplotypes harboring both 3R and 6bp- alleles were associated with non-response to MTX. Regarding MTX-related toxicity, no statistically significant differences were observed in relation to TYMS genotypes and haplotypes. CONCLUSION: Our study reveals that TYMS polymorphisms could be important to help predicting clinical response to MTX in RA patients. Despite the potential of these findings, translation into clinical practice needs larger studies to confirm these evidences.

Stability study perspective of the effect of freeze-drying using cryoprotectants on the structure of insulin loaded into PLGA nanoparticles.

This work aimed to evaluate the influence of a freeze-drying process using different cryoprotectants on the structure of insulin loaded into poly(lactic-co-glycolic acid) (PLGA) nanoparticles and to assess the stability of these nanoparticles upon 6 months of storage following ICH guidelines. Insulin-loaded PLGA nanoparticles with a size around 450 nm were dehydrated using a standard freeze-drying cycle, using trehalose, glucose, sucrose, fructose, and sorbitol at 10% (w/v) as cryoprotectants. All formulations, except those nonadded of cryoprotectant and added with trehalose, collapsed after freeze-drying. The addition of cryoprotectants increased the nanoparticles stability upon storage. FTIR results showed that insulin maintained its structure after encapsulation in about 88%, decreasing to 71% after freeze-drying. The addition of cryoprotectants prior to freeze-drying increased insulin structural stability an average of up to 79%. Formulations collapsed after freeze-drying showed better protein stabilization upon storage, in special sorbitol added formulation, preserving 76, 80, and 78% of insulin structure at 4 °C, 25 °C/60% RH, and 40 °C/75% RH, respectively. Principal component analysis also showed that the sorbitol-added formulation showed the most similar insulin structural modifications among the tested storage conditions. These findings suggested that regarding nanoparticles stability, cryoprotectants are versatile to be used in a standard freeze-drying, however they present different performances on the stabilization of the loaded protein. Thus, on the freeze-drying of the nanoparticles field, this work gives rise to the importance of the process of optimization, searching for a balance between a good obtainable cake with an optimal structural stabilization of the loaded protein.

SLC19A1, SLC46A1 and SLCO1B1 polymorphisms as predictors of methotrexate-related toxicity in Portuguese rheumatoid arthritis patients.

Methotrexate (MTX) is used for rheumatoid arthritis (RA) treatment showing a wide toxicity profile. This study aimed to evaluate the influence of single nucleotide polymorphisms (SNPs) in genes encoding for MTX transporters with the occurrence of MTX-related toxicity (overall and gastrointestinal). A total of 233 Portuguese RA patients were genotyped for 23 SNPs. Haplotype analyses were performed and a toxicogenetic risk index (TRI) was created for SNPs that revealed to be statistically significant. Regarding MTX overall toxicity, an increased risk was associated to SLC19A1 rs7499 G carriers (p = 0.017), SLC46A1 rs2239907 GG (p = 0.030) and, SLCO1B1 rs4149056 T carriers (p = 0.040) and TT (p = 0.019). TRI revealed that patients with Index 3 were 18-fold more likely to present an adverse drug reaction when compared to those with Index 1 (p = 0.001). For MTX gastrointestinal toxicity, results demonstrated an increased risk associated with SLC19A1 rs7499 G carriers (p = 0.012) and GG (p = 0.045), SLC19A1 rs1051266 G carriers (p = 0.034), SLC19A1 rs2838956 A carriers (p = 0.049) and, SLCO1B1 rs4149056 T carriers (p = 0.042) and TT (p = 0.025). Haplotype analyses showed association between GGAG haplotype for SLC19A1 rs7499, rs1051266, rs2838956 and rs3788200 with MTX gastrointestinal toxicity (p = 0.029). TRI revealed that patients with Index 4 were 9-fold more likely to present a gastrointestinal disorder when compared to those with Index 1 (p = 0.020). This study demonstrated that SLC19A1, SLC46A1 and SLCO1B1 genotypes may help to identify patients with increased risk of MTX-related overall toxicity and that SLC19A1 and SLCO1B1 genotypes, and SLC19A1 haplotypes may help to identify patients with increased risk of MTX-related gastrointestinal toxicity.

Prediction of methotrexate clinical response in Portuguese rheumatoid arthritis patients: implication of MTHFR rs1801133 and ATIC rs4673993 polymorphisms.

OBJECTIVE: Methotrexate (MTX), the most used drug in rheumatoid arthritis (RA) treatment, showing variability in clinical response, is often associated with genetic polymorphisms. This study aimed to elucidate the role of methylenetetrahydrofolate reductase (MTHFR) C677T and aminoimidazole carboxamide adenosine ribonucleotide transformylase (ATIC) T675C polymorphisms and clinicopathological variables in clinical response to MTX in Portuguese RA patients. METHODS: Study included 233 RA patients treated with MTX for at least six months. MTHFR C677T and ATIC T675C polymorphisms were genotyped and clinicopathological variables were collected. Statistical analyses were performed and binary logistic regression method adjusted to possible confounding variables. RESULTS: Multivariate analyses demonstrated that MTHFR 677TT (OR = 4.63; P = 0.013) and ATIC 675T carriers (OR = 5.16; P = 0.013) were associated with over 4-fold increased risk for nonresponse. For clinicopathological variables, noncurrent smokers (OR = 7.98; P = 0.001), patients positive to anti-cyclic citrullinated peptide (OR = 3.53; P = 0.004) and antinuclear antibodies (OR = 2.28; P = 0.045), with higher health assessment questionnaire score (OR = 2.42; P = 0.007), and nonsteroidal anti-inflammatory drug users (OR = 2.77; P = 0.018) were also associated with nonresponse. Contrarily, subcutaneous administration route (OR = 0.11; P < 0.001) was associated with response. CONCLUSION: Our study suggests that MTHFR C677T and ATIC T675C genotyping combined with clinicopathological data may help to identify patients whom will not benefit from MTX treatment and, therefore, assist clinicians in personalizing RA treatment.