The Role of PK/PD Analysis in the Development and Evaluation of Antimicrobials

Pharmacokinetic/pharmacodynamic (PK/PD) analysis has proved to be very useful to establish rational dosage regimens of antimicrobial agents in human and veterinary medicine. Actually, PK/PD studies are included in the European Medicines Agency (EMA) guidelines for the evaluation of medicinal products. The PK/PD approach implies the use of in vitro, ex vivo, and in vivo models, as well as mathematical models to describe the relationship between the kinetics and the dynamic to determine the optimal dosing regimens of antimicrobials, but also to establish susceptibility breakpoints, and prevention of resistance. The final goal is to optimize therapy in order to maximize efficacy and minimize side effects and emergence of resistance. In this review, we revise the PK/PD principles and the models to investigate the relationship between the PK and the PD of antibiotics. Additionally, we highlight the outstanding role of the PK/PD analysis at different levels, from the development and evaluation of new antibiotics to the optimization of the dosage regimens of currently available drugs, both for human and animal use.This research was funded by the Department of Education of the Basque Government (PIBA 2019-57) and by the University of the Basque Country UPV/EHU (GIU 17/32)

Development of nucleic acid vaccines: use of self-amplifying RNA in lipid nanoparticles

Self-amplifying RNA or RNA replicon is a form of nucleic acid-based vaccine derived from either positive-strand or negative-strand RNA viruses. The gene sequences encoding structural proteins in these RNA viruses are replaced by mRNA encoding antigens of interest as well as by RNA polymerase for replication and transcription. This kind of vaccine has been successfully assayed with many different antigens as vaccines candidates, and has been shown to be potent in several animal species, including mice, nonhuman primates, and humans. A key challenge to realizing the broad potential of self-amplifying vaccines is the need for safe and effective delivery methods. Ideally, an RNA nanocarrier should provide protection from blood nucleases and extended blood circulation, which ultimately would increase the possibility of reaching the target tissue. The delivery system must then be internalized by the target cell and, upon receptor-mediated endocytosis, must be able to escape from the endosomal compartment into the cell cytoplasm, where the RNA machinery is located, while avoiding degradation by lysosomal enzymes. Further, delivery systems for systemic administration ought to be well tolerated upon administration. They should be safe, enabling the multiadministration treatment modalities required for improved clinical outcomes and, from a developmental point of view, production of large batches with reproducible specifications is also desirable. In this review, the concept of self-amplifying RNA vaccines and the most promising lipid-based delivery systems are discussed.This work was supported by the Basque Government's Department of Education, Universities and Investigation (IT-341-10)

Pharmacokinetic/Pharmacodynamic Analysis of Tedizolid Phosphate Compared to Linezolid for the Treatment of Infections Caused by Gram-Positive Bacteria

Tedizolid and linezolid have antibacterial activity against the most important acute bacterial skin and skin-structure infection (ABSSSIs) pathogens. The objective of this work was to apply PK/PD analysis to evaluate the probability of attaining the pharmacodynamic target of these antimicrobials based on the susceptibility patterns of different clinical isolates causing ABSSSI. Pharmacokinetic and microbiological data were obtained from the literature. PK/PD breakpoints, the probability of target attainment (PTA) and the cumulative fraction of response (CFR) were calculated by Monte Carlo simulation. PTA and CFR are indicative of treatment success. PK/PD breakpoints of tedizolid and linezolid were 0.5 and 1 mg/L, respectively. Probability of treatment success of tedizolid was very high (>90%) for most staphylococci strains, including MRSA and coagulase-negative staphylococci (CoNS). Only for methicillin- and linezolid-resistant S. aureus (MLRSA) and linezolid resistant (LR) CoNS strains was the CFR of tedizolid very low. Except for LR, daptomycin-non-susceptible (DNS), and vancomycin-resistant (VRE) E. faecium isolates, tedizolid also provided a high probability of treatment success for enterococci. The probability of treatment success of both antimicrobials for streptococci was always higher than 90%. In conclusion, for empiric treatment, PK/PD analysis has shown that tedizolid would be adequate for most staphylococci, enterococci, and streptococci, even those LR whose linezolid resistance is mediated by the cfr gene.This research was funded by the University of the Basque Country UPV/EHU (GIU17/032)

Augmented Renal Clearance in Critically Ill Patients: A Systematic Review

Background Traditionally, renal function in critically ill patients has been assessed to identify renal dysfunction, and dose adjustment is generally accepted in such a context. Nevertheless, augmented renal clearance (ARC) is a less well-studied phenomenon that could lead to faster elimination of drugs, resulting in subtherapeutic concentrations and poorer clinical outcomes when standard dosage guidelines are followed. Objective The aim of this systematic review was to gather and summarise all the available evidence on ARC in critically ill patients, including its definition, underlying mechanisms, epidemiology, diagnosis and impact on both drug pharmacokinetics and clinical outcomes. Method A systematic review was conducted to include all the original studies that provided information on ARC in critically ill patients, and is reported following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. Results Augmented renal clearance, defined as a creatinine clearance (CrCl)[130 mL/min/1.73 m2, preferably measured in urine, is present in 20–65% of critically ill patients. Younger age, polytrauma and lower severity illness have been identified as risk factors. An influence of ARC on antimicrobial pharmacokinetics has been observed, with ARC consistently being associated with subtherapeutic antibiotic plasma concentrations. Conclusion ARC is a prevalent condition in critically ill patients, especially in young people, with urinary CrCl being the best diagnostic method because mathematical estimates tend to underestimate CrCl. ARC increases renal drug elimination and has a clear influence on certain antimicrobial plasma levels, but is yet to define its impact on clinical outcomes and on pharmacokinetics of other types of drugs. Research on the need to stage ARC and establish specific dosing guidelines is warranted.This study was financially supported by the University of the Basque Country (UPV/EHU) (PPG17/65)

Lipid Nanoparticles as Carriers for RNAi against Viral Infections: Current Status and Future Perspectives

The efforts made to develop RNAi-based therapies have led to productive research in the field of infections in humans, such as hepatitis C virus (HCV), hepatitis B virus (HBV), human immunodeficiency virus (HIV), human cytomegalovirus (HCMV), herpetic keratitis, human papillomavirus, or influenza virus. Naked RNAi molecules are rapidly digested by nucleases in the serum, and due to their negative surface charge, entry into the cell cytoplasm is also hampered, which makes necessary the use of delivery systems to exploit the full potential of RNAi therapeutics. Lipid nanoparticles (LNP) represent one of the most widely used delivery systems for in vivo application of RNAi due to their relative safety and simplicity of production, joint with the enhanced payload and protection of encapsulated RNAs. Moreover, LNP may be functionalized to reach target cells, and they may be used to combine RNAi molecules with conventional drug substances to reduce resistance or improve efficiency. This review features the current application of LNP in RNAi mediated therapy against viral infections and aims to explore possible future lines of action in this field.This work was supported by the Basque Government's Department of Education, Universities and Investigation (IT-341-10)

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.This research was funded by MCIU/AEI/FEDER, UE, grant number RTI2018-098672-B-I00; by the UNIVERSITY OF THE BASQUE COUNTRY UPV/EHU, grant number GIU20/048; and by the BASQUE GOVERNMENT, grant number IT1587-22, GIC21/34

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.This research was funded by the MCIU/AEI/FEDER, UE (RTI2018-098672-B-I00) and by the UPV/EHU (GIU17/032)

MMP-9 Downregulation with Lipid Nanoparticles for Inhibiting Corneal Neovascularization by Gene Silencing

Gene silencing targeting proangiogenic factors have been shown to be a useful strategy in the treatment of corneal neovascularization (CNV). Among interference RNA (RNAi) molecules, short-hairpin RNA (shRNA) is a plasmid-coded RNA able to down-regulate the expression of the desired gene. It is continuously produced in the host cell, inducing a durable gene silencing effect. The aim of this work was to develop a solid lipid nanoparticle (SLN)-based shRNA delivery system to downregulate metalloproteinase 9 (MMP-9), a proangiogenic factor, in corneal cells for the treatment of CNV associated with inflammation. The nanovectors were prepared using a solvent emulsification-evaporation technique, and after physicochemical evaluation, they were evaluated in different culture cell models. Transfection efficacy, cell internalization, cell viability, the effect on MMP-9 expression, and cell migration were evaluated in human corneal epithelial cells (HCE-2). The inhibition of tube formation using human umbilical vein endothelial cells (HUVEC) was also assayed. The non-viral vectors based on SLN were able to downregulate the MMP-9 expression in HCE-2 cells via gene silencing, and, consequently, to inhibit cell migration and tube formation. These results demonstrate the potential of lipid nanoparticles as gene delivery systems for the treatment of CNV-associated inflammation by RNAi technology.This research was funded by the Ministerio de Economía y Competitividad (SAF2014-53092-R), by FEDER funds from the EU, and by the UPV/EHU (PPG17/65, GIU17/032). J Torrecilla and I Gómez-Aguado thank UPV/EHU for their research grants

Galactomannan-Decorated Lipidic Nanocarrier for Gene Supplementation Therapy in Fabry Disease

Gene supplementation therapy with plasmid DNA (pDNA) represents one of the most promising strategies for the treatment of monogenic diseases such as Fabry disease (FD). In the present work, we developed a solid lipid nanoparticles (SLN)-based non-viral vector with a size below 100 nm, and decorated with galactomannan (GM) to target the liver as an α-Galactosidase A (α-Gal A) production factory. After the physicochemical characterization of the GM-SLN vector, cellular uptake, transfection efficacy and capacity to increase α-Gal A activity were evaluated in vitro in a liver cell line (Hep G2) and in vivo in an animal model of FD. The vector showed efficient internalization and it was highly efficient in promoting protein synthesis in Hep G2 cells. Additionally, the vector did not show relevant agglutination of erythrocytes and lacked hemolytic activity. After the systemic administration to Fabry mice, it achieved clinically relevant α-Gal A activity levels in plasma, liver, and other organs, importantly in heart and kidneys, two of the most damaged organs in FD. This work shows the potential application of GM-decorated lipidic nanocarries for the treatment of FD by pDNA-based gene augmentation.This research was funded by MCIU/AEI/FEDER, UE (RTI2018-098672-B-I00) and by the University of the Basque Country UPV/EHU (GIU20/048)

Quantification of Ceftaroline in Human Plasma Using High-Performance Liquid Chromatography with Ultraviolet Detection: Application to Pharmacokinetic Studies

This study was conducted to develop a rapid, simple and reproducible method for the quantification of ceftaroline in plasma samples by high-performance liquid chromatography with ultraviolet detection (HPLC-UV). Sample processing consisted of methanol precipitation and then, after centrifugation, the supernatant was injected into the HPLC system, working in isocratic mode. Ceftaroline was detected at 238 nm at a short acquisition time (less than 5 min). The calibration curve was linear over the concentration range from 0.25 to 40 µg/mL, and the method appeared to be selective, precise and accurate. Ceftaroline in plasma samples was stable at −80 °C for at least 3 months. The method was successfully applied to characterize the pharmacokinetic profile of ceftaroline in two critically ill patients and to evaluate whether the pharmacokinetic/pharmacodynamic (PK/PD) target was reached or not with the dose regimen administered.This research was funded by Department of Education of the Basque Government (PIBA 2019-57) and by the University of the Basque Country UPV/EHU (GIU 17/32)