Does selective digestive decontamination (SDD) increase antibiotic resistance? Long-term comparison of two intensive care units (with and without SDD) of the same tertiary hospital.

PURPOSE: The aim of this study was to to compare the antimicrobial resistance rate and its relationship with the antibiotic consumption in two separate Intensive Care Units (ICUs) of the same hospital, one with and other without selective decontamination of the digestive tract (SDD). METHODS: We performed a retrospective study in the two ICUs of the Araba University Hospital. Trauma and neurosurgical patients are admitted to the SDD-ICU, and general digestive surgery patients go to the no SDD-ICU. From 2014 to 2018 we analyzed the number of isolates, and the bacterial resistance trends of 47 antimicrobial-microorganism combinations. Additionally, antimicrobial consumption was estimated in both ICUs. Resistance rates were also compared with those reported in ENVIN-HELICS Spanish national registry. RESULTS: In the ICU with SDD protocol, there was a significant decrease in the resistance of E. coli to amoxicillin/clavulanic acid and in the resistance of E. faecalis to high concentration of gentamycin and high concentration of streptomycin. A significant increase of resistance of Staphylococcus coagulasa negative (CoNS) to linezolid in the no SDD-ICU was also detected. Overall, the level of resistance in the SDD-ICU was lower or of the same order than in the ICU without SDD and that reported in the Spanish national registry. CONCLUSIONS: SDD had neither a clinically relevant impact on emergence and spread of resistance, nor in the overall systemic antimicrobial use. The patient type rather than the SDD protocol showed to condition the ecology and therefore, the resistance rate in the ICUs.

Ceftaroline and Avibactam Removal by Continuous Renal Replacement Therapies: An in vitro Study.

INTRODUCTION: Continuous renal replacement therapies (CRRTs) are frequently used in critically ill patients; however, there are scarce in vitro and in vivo studies showing the extracorporeal elimination of ceftaroline and avibactam. The aim of this study was to assess, through an in vitro model, the extracorporeal elimination of ceftaroline and avibactam by continuous veno-venous hemofiltration (CVVH), continuous veno-venous hemodiafiltration (CVVHDF), and continuous veno-venous hemodialysis (CVVHD), using a polysulfone hemofilter. METHODS: Simulated in vitro experiments were performed using a multiFiltrate machine with a 1.4 m2 Ultraflux® AV600S polysulfone hemofilter. Isofundin® without or with bovine serum albumin was circulated as vehicle for ceftaroline or avibactam. Pre-filter, post-filter, and effluent samples were taken over a period of 60 min, and they were immediately stored at 4°C until processed in the same day. The quantification of ceftaroline and avibactam in the samples was performed by high-performance liquid chromatography with ultraviolet detection. Protein binding, extraction coefficient (EC), and extracorporeal clearance (CLCRRT) were calculated. RESULTS: The elimination of both ceftaroline and avibactam during the three extracorporeal modalities followed first-order pharmacokinetics. Regardless of the CRRT technique, EC values for both molecules were around 1, similar to the unbound fraction of avibactam (0.96) and higher than the unbound fraction of ceftaroline (0.79). CLCRRT of ceftaroline ranged from 15.63 to 17.66 mL/min when CVVH and CVVHD were used with a flow rate of 1,000 mL/h, and from 29.25 to 32.95 mL/min for the CVVHDF modality with a flow rate of 2,000 mL/h. For avibactam, CLCRRT ranged from 15.07 to 18.82 mL/min for CVVH and CVVHD, and from 33.74 to 34.13 mL/min for CVVHDF. DISCUSSION: Avibactam and ceftaroline are extensively removed through the polysulfone membrane, and a dose adjustment may be recommended for patients under CRRT to ensure pharmacodynamic target achievement.

Pharmacokinetic/pharmacodynamic evaluation of the antimicrobial therapy of pneumococcal invasive disease in adults in post-PCV13 vaccine period in Madrid, Spain.

The objective of our study was to evaluate by pharmacokinetic/pharmacodynamic (PK/PD) analysis, if the antimicrobials used for the treatment of invasive pneumococcal disease (IPD) in adults, including meningitis, are adequate considering the susceptibility profile of S. pneumoniae in Spain after the implantation of PVC13 vaccine. Pharmacokinetic parameters of benzylpenicillin and cefotaxime were obtained from the literature, and susceptibility data of invasive S. pneumoniae strains recovered in 2017 (post-PCV13 vaccination period) were provided by the Public Health Regional Laboratory of Madrid. We have also studied levofloxacin because it is used to treat pneumococcal pneumonia previously to be diagnosed as bacteremic pneumonia. Monte Carlo simulation was used to estimate the probability of target attainment (PTA) and the cumulative fraction of response (CFR). All doses of benzylpenicillin except 2 mU q6h provide a high probability of treatment success for MIC values ≤ 1 mg/L; 4 mU q4h is even useful for MIC values up to 4 mg/L. This high dose, used for the treatment of meningitis, also provides high probability of treatment success for MIC ≤ 0.5 mg/L. At the susceptibility EUCAST breakpoint (≤ 0.5 mg/L), cefotaxime provides a high rate of PD target achievement, even at the lowest dose (1 g q8h). For meningitis, 2 g q6h ensures probabilities of target attainment ≥90% for MIC up to 1 mg/L. Our study confirms that after the implementation of PCV13 vaccine, the treatment with benzylpenicillin and cefotaxime provides high probability of the therapy success of IPD, including meningitis.

Nucleic Acid Delivery by Solid Lipid Nanoparticles Containing Switchable Lipids: Plasmid DNA vs. Messenger RNA.

The development of safe and effective nucleic acid delivery systems remains a challenge, with solid lipid nanoparticle (SLN)-based vectors as one of the most studied systems. In this work, different SLNs were developed, by combination of cationic and ionizable lipids, for delivery of mRNA and pDNA. The influence of formulation factors on transfection efficacy, protein expression and intracellular disposition of the nucleic acid was evaluated in human retinal pigment epithelial cells (ARPE-19) and human embryonic kidney cells (HEK-293). A long-term stability study of the vectors was also performed. The mRNA formulations induced a higher percentage of transfected cells than those containing pDNA, mainly in ARPE-19 cells; however, the pDNA formulations induced a greater protein production per cell in this cell line. Protein production was conditioned by energy-dependent or independent entry mechanisms, depending on the cell line, SLN composition and kind of nucleic acid delivered. Vectors containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) as unique cationic lipid showed better stability after seven months, which improved with the addition of a polysaccharide to the vectors. Transfection efficacy and long-term stability of mRNA vectors were more influenced by formulation-related factors than those containing pDNA; in particular, the SLNs containing only DOTAP were the most promising formulations for nucleic acid delivery.

Antibiotic susceptibility trend before and after long-term use of selective digestive decontamination: a 16 year ecological study.

OBJECTIVES: The aim of this study was to compare antimicrobial susceptibility rates in a Spanish ICU before and after the introduction of selective digestive decontamination (SDD) and also to compare these with susceptibility data from other Spanish ICUs without SDD. METHODS: We performed a retrospective study in the ICU of the University Hospital of Alava, where SDD was implemented in 2002. The SDD protocol consisted of a 2% mixture of gentamicin, colistin and amphotericin B applied on the buccal mucosa and a suspension of the same drugs in the gastrointestinal tract; additionally, for the first 3 days, systemic ceftriaxone was administered. From 1998 to 2013 we analysed the susceptibility rates for 48 antimicrobial/organism combinations. Interrupted time series using a linear dynamic model with SDD as an intervention was used. Data from other ICUs were obtained from the ENVIN-HELICS national registry. RESULTS: Only amoxicillin/clavulanic acid against Escherichia coli and Proteus mirabilis, and a high concentration of gentamicin against Enterococcus faecalis, resulted in a significant decrease in the susceptibility rate after the implementation of SDD, with a drop of 20%, 27% and 32%, respectively. Compared with other Spanish ICUs without SDD, the susceptibility rate was higher in the ICU of our hospital in most cases. When it was lower, differences were <10%, except for a high concentration of streptomycin against Enterococcus faecium, for which the difference was 19%. CONCLUSIONS: No relevant changes in the overall susceptibility rate after the implementation of SDD were detected. Susceptibility rates were not lower than those in the Spanish ICUs without SDD.

Targeting corneal inflammation by gene therapy: Emerging strategies for keratitis.

Inflammation is the underlying process of several diseases within the eye, specifically in the cornea. Current treatment options for corneal inflammation or keratitis, and related neovascularization, are restricted by limited efficacy, adverse effects, and short duration of action. Gene therapy has shown great potential for the treatment of diseases affecting the ocular surface, and major efforts are being targeted to inflammatory mediators and neovascularization, in order to develop potential treatments for corneal inflammation. Gene therapy to treat ocular disorders is still starting, and current therapies are primarily experimental, with most human clinical trials still in research state, although some of them have already shown encouraging results. In this review, we focus on the progress and challenges of gene therapy to treat corneal inflammation. After introducing the inflammation process, we present the main nucleic acid delivery systems, including viral and non-viral vectors, and the most studied strategies to address the therapy: control of neovascularization and regulation of pro- and anti-inflammatory cytokines.

Tranexamic Acid Compared with Placebo for Reducing Total Blood Loss in Hip Replacement Surgery: A Randomized Clinical Trial.

BACKGROUND: Tranexamic acid (TXA) reduces bleeding in patients undergoing hip replacement surgery, but optimal doses and timing have yet to be established. Our primary objective in this study was to assess total blood loss 48 hours after surgery with different regimens. METHODS: This was a multicenter, parallel-group, randomized, placebo-controlled clinical trial that included all ASA physical status I to III patients undergoing unilateral total hip replacement surgery who met the inclusion criteria. Patients were randomly allocated to 1 of 3 groups: a single-dose group (15 mg/kg TXA before the start of surgery and saline 3 hours later after the start of surgery), a 2-dose group (10 m/kg TXA before and 10 mg/kg of TXA 3 hours after the start of surgery), and a control group (saline before and 3 hours after the start of surgery). Total blood loss was calculated using a formula considering hematocrit values and blood transfusions received. RESULTS: We included 108 patients in the study. Total blood loss volumes up to day 2 were 1377 ± 689, 1308 ± 641, and 2215 ± 1136 mL in the single-dose, 2-dose and control groups, respectively (P < 0.001 between the placebo and the experimental groups). Blood transfusions were given to 22.9% of patients (n = 8) in the single-dose group, 11.1% (n = 4) in the 2-dose group, and 37.8% (n = 14) in the control group (P = 0.028). CONCLUSIONS: A single preoperative dose of TXA or 2 infusions of a lower dose, preoperatively and then after 3 hours after the start of surgery, resulted in lower blood loss during the first 2 days after surgery and less need for blood transfusion, with good levels of safety.

Nanostructured lipid carriers: Promising drug delivery systems for future clinics.

During the past decade, the number of studies describing nanostructured lipid carriers (NLCs)-based formulations has been dramatically increased. The raise in NLC exploitation is essentially due to defeated barriers within the technological process of lipid-based nanoparticles' formulation and increased knowledge of the underlying mechanisms of transport of NLCs via different routes of administration. This review article aims to give an overview on the current state of the art of NLC as controlled drug delivery systems for future clinics through novel NLC applications providing examples of successfull outcomes. The reported data clearly illustrate the promise of these nanoparticles for novel treatments in the near future. From the Clinical Editor: The understanding of the nanostructured lipid carriers (NLC)-based formulations has improved with continuing research recently. The result has seen an increase in the use of these in the clinical setting. In this comprehensive review, the authors discussed the current state and major challenges in the use of nanostructured lipid carriers as controlled drug delivery systems.

Applications of the pharmacokinetic/pharmacodynamic (PK/PD) analysis of antimicrobial agents.

The alarming increase of resistance against multiple currently available antibiotics is leading to a rapid lose of treatment options against infectious diseases. Since the antibiotic resistance is partially due to a misuse or abuse of the antibiotics, this situation can be reverted when improving their use. One strategy is the optimization of the antimicrobial dosing regimens. In fact, inappropriate drug choice and suboptimal dosing are two major factors that should be considered because they lead to the emergence of drug resistance and consequently, poorer clinical outcomes. Pharmacokinetic/pharmacodynamic (PK/PD) analysis in combination with Monte Carlo simulation allows to optimize dosing regimens of the antibiotic agents in order to conserve their therapeutic value. Therefore, the aim of this review is to explain the basis of the PK/PD analysis and associated techniques, and provide a brief revision of the applications of PK/PD analysis from a therapeutic point-of-view. The establishment and reevaluation of clinical breakpoints is the sticking point in antibiotic therapy as the clinical use of the antibiotics depends on them. Two methodologies are described to establish the PK/PD breakpoints, which are a big part of the clinical breakpoint setting machine. Furthermore, the main subpopulations of patients with altered characteristics that can condition the PK/PD behavior (such as critically ill, elderly, pediatric or obese patients) and therefore, the outcome of the antibiotic therapy, are reviewed. Finally, some recommendations are provided from a PK/PD point of view to enhance the efficacy of prophylaxis protocols used in surgery.

Development and validation of a LC-MS assay for the quantification of ikh12 a novel anti-tumor candidate in rat plasma and tissues and its application in a pharmacokinetic study.

IKH12 is a novel histone deacetylase 6 selective inhibitor. A rapid and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for the quantification of IKH12 in rat plasma and tissue with kendine 91 as internal standard (IS). The samples were prepared by liquid-liquid extraction with tert-butyl methyl ether. The chromatographic separation was accomplished by using a Zorbax Extend C18 4.6 × 150 mm, 5 µm column, with a mobile phase consisting of methanol and 0.1% formic acid (75:25 v/v). Multiple reaction monitoring, using electrospray ionization in positive ion mode, was employed to quantitatively detect IKH12 and IS. The monitored transitions were set at m/z 418 → 252 and 444 → 169 for IKH12 and kendine 91, respectively. The calibration curve was linear over the concentration range 2-1000 ng mL(-1) . The intra- and inter-assay precision and accuracy of the quality controls and the limit of quantification were satisfactory in all cases (according to European Medicines Agency guidelines). Stability studies showed that plasma samples were stable in the chromatography rack for 24 h and at -80°C for 2 months and also after three freeze-thaw cycles. This method was successfully applied to a pharmacokinetic study of IKH12 in rat.

[Pharmacokinetic/pharmacodynamic analysis in microbiology: a tool for the evaluation of the antimicrobial treatment]. / Análisis farmacocinético-farmacodinámico en microbiología: herramienta para evaluar el tratamiento antimicrobiano.

The selection of multiresistant microorganisms, as a side-effect of the use of antimicrobials, together with the lack of new therapeutic drugs expected in the near future, forces to a rational use of antibiotics. The optimisation of antibacterial treatments based on pharmacokinetic/pharmacodynamic analysis (PK/PD) may contribute to prolong the life of antibiotics and to contain the bacterial resistance to them. A review is made of the importance of the appropriateness of the dose regimen selected, the application of PK/PD analysis of antimicrobials, the Monte Carlo simulation, PK/PD indices for efficacy, and PK/PD cut-off points. PK/PD analysis is also applicable to the prevention of bacterial resistance. Different methods have been used to study the factors that lead to its emergence and spread, such as in vitro and animal models, and resistance prevention studies (mutant selection window). Although the PK/PD analysis is a very useful tool for the selection of the most appropriate dose regimen of antibiotics, several problems limit its use in clinical practice.

Population pharmacokinetics of piperacillin and tazobactam in critically ill patients undergoing continuous renal replacement therapy: application to pharmacokinetic/pharmacodynamic analysis.

OBJECTIVES: To evaluate the pharmacokinetics of piperacillin/tazobactam in critically ill patients undergoing continuous renal replacement therapy (CRRT) and to assess the success of the therapy against susceptible bacteria. PATIENTS AND METHODS: Sixteen patients undergoing CRRT with different degrees of renal function were included in the study. Blood and ultrafiltrate samples were drawn after administration of piperacillin/tazobactam (4/0.5 g) every 4, 6 or 8 h. The data were analysed by a population approach using NONMEM 7.2. The probability of target attainment (PTA) of maintaining free piperacillin levels above the MIC during the entire dosing interval was estimated by simulation of intermittent and continuous infusions. RESULTS: The pharmacokinetics of piperacillin and tazobactam were best described by two-compartment models where the elimination of both drugs was conditioned by renal [dependent on creatinine clearance (CLCR)], non-renal and extracorporeal clearances. A 20 min infusion of piperacillin/tazobactam administered every 6 h provided high PTAs against MICs ≤ 32 mg/L in patients with severe renal failure. In patients with normal or moderate renal function PTAs ≥ 90% were only obtained up to MICs ≤ 8 mg/L with short infusions. However, simulating continuous infusion, higher probabilities of success were obtained against MICs of 32 and 16 mg/L when CLCR was 50 and 100 mL/min, respectively. CONCLUSIONS: Population pharmacokinetic models have been developed and validated for piperacillin and tazobactam. Based on the pharmacokinetic/pharmacodynamic analysis, dosing recommendations are given considering the residual renal function of the patient and the MIC for the isolated bacteria.

Fate of nanostructured lipid carriers (NLCs) following the oral route: design, pharmacokinetics and biodistribution.

The aim of this study was to develop a nanostructured lipid carriers (NLC) formulation containing spironolactone (SPN-NLCs), and to investigate its potential for the oral delivery of poorly water-soluble compounds. SPN-NLCs were orally administered to rabbits and the pharmacokinetics of spironolactone and its metabolites was evaluated. As reference formulation, we administered syrup. Spironolactone was only detected in a few plasma samples; hence, metabolite levels were employed for the pharmacokinetic analysis. The absolute bioavailability of 7α-TMS was significantly higher with the syrup than those obtained with the SPN-NLCs (0.7 versus 0.4, p < 0.05). However, no significant differences were observed in the bioavailability of canrenone, revealing a different canrenone/7α-TMS ratio depending on the administered formulation. Orally administered (99m)Tc-radiolabeled SPN-NLCs were mainly detected in the small intestine. These results suggest the retention of the nanocarriers in the underlying epithelium and further uptake by the epithelial cells.

Targeting strategies with lipid vectors for nucleic acid supplementation therapy in Fabry disease: a systematic review.

Fabry disease (FD) results from a lack of activity of the lysosomal enzyme α-Galactosidase A (α-Gal A), leading to the accumulation of glycosphingolipids in several different cell types. Protein supplementation by pDNA or mRNA delivery presents a promising strategy to tackle the underlying genetic defect in FD. Protein-coding nucleic acids in FD can be either delivered to the most affected sites by the disease, including heart, kidney and brain, or to specialized organs that can act as a production factory of the enzyme, such as the liver. Lipid-based systems are currently at the top of the ranking of non-viral nucleic acid delivery systems, and their versatility allows the linking to the surface of a wide range of molecules to control their biodistribution after intravenous administration. This systematic review follows the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement guidelines and provides an overview and discussion of the targeting ligands that have been employed so far to actively vectorize intravenously administered non-viral vectors based on lipid carriers to clinically relevant organs in the treatment of FD, for protein-coding nucleic acid (pDNA and mRNA) supplementation. Among the thirty-two studies included, the majority focus on targeting the liver and brain. The targeting of the heart has been reported to a lesser degree, whereas no articles addressing kidney-targeting have been recorded. Although a great effort has been made to develop organ-specific nucleic acid delivery systems, the design of active-targeted carriers with high quality, good clinical translation, and large-scale manufacturing capacity is still challenging.

Optimizing Antibiotic Therapy for Stenotrophomonas maltophilia Infections in Critically Ill Patients: A Pharmacokinetic/Pharmacodynamic Approach.

Stenotrophomonas maltophilia is an opportunistic, multidrug-resistant non-fermentative Gram-negative bacillus, posing a significant challenge in clinical treatment due to its numerous intrinsic and acquired resistance mechanisms. This study aimed to evaluate the adequacy of antibiotics used for the treatment of S. maltophilia infections in critically ill patients using a pharmacokinetic/pharmacodynamic (PK/PD) approach. The antibiotics studied included cotrimoxazole, levofloxacin, minocycline, tigecycline, cefiderocol, and the new combination aztreonam/avibactam, which is not yet approved. By Monte Carlo simulations, the probability of target attainment (PTA), the PK/PD breakpoints, and the cumulative fraction of response (CFR) were estimated. PK parameters and MIC distributions were sourced from the literature, the European Committee on Antimicrobial Susceptibility Testing (EUCAST), and the SENTRY Antimicrobial Surveillance Program collection. Cefiderocol 2 g q8h, minocycline 200 mg q12h, tigecycline 100 mg q12h, and aztreonam/avibactam 1500/500 mg q6h were the best options to treat empirically infections due to S. maltophilia. Cotrimoxazole provided a higher probability of treatment success for the U.S. isolates than for European isolates. For all antibiotics, discrepancies between the PK/PD breakpoints and the clinical breakpoints defined by EUCAST (or the ECOFF) and CLSI were detected.

Oral linezolid compared with benzathine penicillin G for treatment of early syphilis in adults (Trep-AB Study) in Spain: a prospective, open-label, non-inferiority, randomised controlled trial.

BACKGROUND: Management of syphilis, a sexually transmitted infection (STI) with increasing incidence, is challenged by drug shortages, scarcity of randomised trial data, an absence of non-penicillin alternatives for pregnant women with penicillin allergy (other than desensitisation), extended parenteral administration for neurosyphilis and congenital syphilis, and macrolide resistance. Linezolid was shown to be active against Treponema pallidum, the causative agent of syphilis, in vitro and in the rabbit model. We aimed to assess the efficacy of linezolid for treating early syphilis in adults compared with the standard of care benzathine penicillin G (BPG). METHODS: We did a multicentre, open-label, non-inferiority, randomised controlled trial to assess the efficacy of linezolid for treating early syphilis compared with BPG. We recruited participants with serological or molecular confirmation of syphilis (either primary, secondary, or early latent) at one STI unit in a public hospital and two STI community clinics in Catalonia (Spain). Participants were randomly allocated in a 1:1 ratio using a computer-generated block randomisation list with six participants per block, to receive either oral linezolid (600 mg once per day for 5 days) or intramuscular BPG (single dose of 2·4 million international units) and were assessed for signs and symptoms (once per week until week 6 and at week 12, week 24, and week 48) and reagin titres of non-treponemal antibodies (week 12, week 24, and week 48). The primary endpoint was treatment response, assessed using a composite endpoint that included clinical response, serological response, and absence of relapse. Clinical response was assessed at 2 weeks for primary syphilis and at 6 weeks for secondary syphilis following treatment initiation. Serological cure was defined as a four-fold decline in rapid plasma reagin titre or seroreversion at any of the 12-week, 24-week, or 48-week timepoints. The absence of relapse was defined as the presence of different molecular sequence types of T pallidum in recurrent syphilis. Non-inferiority was shown if the lower limit of the two-sided 95% CI for the difference in rates of treatment response was higher than -10%. The primary analysis was done in the per-protocol population. The trial is registered at ClinicalTrials.gov (NCT05069974) and was stopped for futility after interim analysis. FINDINGS: Between Oct 20, 2021, and Sept 15, 2022, 62 patients were assessed for eligibility, and 59 were randomly assigned to linezolid (n=29) or BPG (n=30). In the per-protocol population, after 48 weeks' follow-up, 19 (70%) of 27 participants (95% CI 49·8 to 86·2) in the linezolid group had responded to treatment and 28 (100%) of 28 participants (87·7 to 100·0) in the BPG group (treatment difference -29·6, 95% CI -50·5 to -8·8), which did not meet the non-inferiority criterion. The number of drug-related adverse events (all mild or moderate) was similar in both treatment groups (five [17%] of 29, 95% CI 5·8 to 35·8 in the linezolid group vs five [17%] of 30, 5·6 to 34·7, in the BPG group). No serious adverse events were reported during follow-up. INTERPRETATION: The efficacy of linezolid at a daily dose of 600 mg for 5 days did not meet the non-inferiority criteria compared with BPG and, as a result, this treatment regimen should not be used to treat patients with early syphilis. FUNDING: European Research Council and Fondo de Investigaciones Sanitarias.

Role of endocytic uptake in transfection efficiency of solid lipid nanoparticles-based nonviral vectors.

BACKGROUND: As has been shown for different vector systems, the entry pathway(s) impacts upon the transfection efficiency. The present study aimed to explore the cellular uptake mechanisms of three different vectors based on solid lipid nanoparticles (SLN) in HeLa cells. The use of endocytosis inhibitors that affect specific internalization pathways provides a tool for the study of these routes. METHODS: We prepared three vectors based on solid lipid nanoparticles: without protamine, with protamine, and with protamine and dextran. Uptake, percentage of transfected HeLa cells and enhanced green fluorescent protein (EGFP) production were all analyzed in the presence or absence of different endocytosis inhibitors. In addition, co-localization studies using lysosomal markers were carried out to determine the influence of the trafficking to late endosomal compartments on the transfection capacity of the vectors. RESULTS: Uptake and transfection of each vector was affected differently by each endocytosis inhibitor. Ethylisopropylamiloride (EIPA) did not affect uptake of the DNA-SLN vector, whereas all of the inhibitors affected transfection. In the case of protamine-DNA-SLN and dextran-protamine-DNA-SLN vectors, EIPA affected uptake and dynasore did not decrease transfection. CONCLUSIONS: DNA-SLN vector appear to enter productively by multiple pathways in HeLa cells. By contrast, dynamin does not appear to be essential in the productive entry of protamine-containing vectors. In addition, enhancement of the macropinocytic route increases EGFP production when dextran is added to the vector.

Novel Golden Lipid Nanoparticles with Small Interference Ribonucleic Acid for Substrate Reduction Therapy in Fabry Disease.

Substrate reduction therapy (SRT) has been proposed as a new gene therapy for Fabry disease (FD) to prevent the formation of globotriaosylceramide (Gb3). Nanomedicines containing different siRNA targeted to Gb3 synthase (Gb3S) were designed. Formulation factors, such as the composition, solid lipid nanoparticles (SLNs) preparation method and the incorporation of different ligands, such as gold nanoparticles (GNs), protamine (P) and polysaccharides, were evaluated. The new siRNA-golden LNPs were efficiently internalized in an FD cell model (IMFE-1), with GNs detected in the cytoplasm and in the nucleus. Silencing efficacy (measured by RT-qPCR) depended on the final composition and method of preparation, with silencing rates up to 90% (expressed as the reduction in Gb3S-mRNA). GNs conferred a higher system efficacy and stability without compromising cell viability and hemocompatibility. Immunocytochemistry assays confirmed Gb3S silencing for at least 15 days with the most effective formulations. Overall, these results highlight the potential of the new siRNA-golden LNP system as a promising nanomedicine to address FD by specific SRT.

Pharmacokinetic/Pharmacodynamic Analysis of Oral Calcium Fosfomycin: Are Urine Levels Sufficient to Ensure Efficacy for Urinary Tract Infections?

Urinary tract infections (UTIs) are extremely common and a major driver for the use of antimicrobials. Calcium fosfomycin is an old antibiotic indicated for the treatment of UTIs; however, data about its urine pharmacokinetic profile are scarce. In this work, we have evaluated the pharmacokinetics of fosfomycin from urine concentrations after oral administration of calcium fosfomycin to healthy women. Moreover, we have assessed, by pharmacokinetic/pharmacodynamic (PK/PD) analysis and Monte Carlo simulations, its effectiveness considering the susceptibility profile of Escherichia coli, the main pathogen involved in UTIs. The accumulated fraction of fosfomycin excreted in urine was around 18%, consistent with its low oral bioavailability and its almost exclusively renal clearance by glomerular filtration as unchanged drug. PK/PD breakpoints resulted to be 8, 16, and 32 mg/L for a single dose of 500 mg, a single dose of 1000 mg, and 1000 mg q8h for 3 days, respectively. For empiric treatment, the estimated probability of treatment success was very high (>95%) with the three dose regimens, considering the susceptibility profile of E. coli reported by EUCAST. Our results show that oral calcium fosfomycin at a dose level of 1000 mg every 8 h provides urine concentrations sufficient to ensure efficacy for the treatment of UTIs in women.

Antimicrobial susceptibility of Treponema pallidum subspecies pallidum: an in-vitro study.

BACKGROUND: The increasing incidence of syphilis and the limitations of first-line treatment with penicillin, particularly in neurosyphilis, neonatal syphilis, and pregnancy, highlight the need to expand the therapeutic repertoire for effective management of this disease. We assessed the in-vitro efficacy of 18 antibiotics from several classes on Treponema pallidum subspecies pallidum (T pallidum), the syphilis bacteria. METHODS: Using the in-vitro culture system for T pallidum, we exposed the pathogen to a concentration range of each tested antibiotic. After a 7-day incubation, the treponemal burden was evaluated by quantitative PCR targeting the T pallidum tp0574 gene. The primary outcome was the minimum inhibitory concentration (MIC) at which the quantitative PCR values were not significantly higher than the inoculum wells. We also investigated the susceptibility of macrolide-resistant strains to high concentrations of azithromycin, and the possibility of developing resistance to linezolid, a proposed candidate for syphilis treatment. FINDINGS: Amoxicillin, ceftriaxone, several oral cephalosporins, tedizolid, and dalbavancin exhibited anti-treponemal activity at concentrations achievable in human plasma following regular dosing regimens. The experiments revealed a MIC for amoxicillin at 0·02 mg/L, ceftriaxone at 0·0025 mg/L, cephalexin at 0·25 mg/L, cefetamet and cefixime at 0·0313 mg/L, cefuroxime at 0·0156 mg/L, tedizolid at 0·0625 mg/L, spectinomycin at 0·1 mg/L, and dalbavancin at 0·125 mg/L. The MIC for zoliflodacin and balofloxacin was 2 mg/L. Ertapenem, isoniazid, pyrazinamide, and metronidazole had either a poor or no effect. Azithromycin concentrations up to 2 mg/L (64 times the MIC) were ineffective against strains carrying mutations associated to macrolide resistance. Exposure to subtherapeutic doses of linezolid for 10 weeks did not induce phenotypic or genotypic resistance. INTERPRETATION: Cephalosporins and oxazolidinones are potential candidates for expanding the current therapeutic repertoire for syphilis. Our findings warrant testing efficacy in animal models and, if successful, clinical assessment of efficacy. FUNDING: European Research Council.