Deciphering pharmacokinetics and pharmacodynamics of fosfomycin.

Fosfomycin, a low molecular weight and hydrophilic drug with negligible protein binding, is eliminated almost exclusively by glomerular filtration, whose clearance is subject to patient renal function. The volume of distribution approximates to the extracellular body water (about 0.3 L/Kg) in healthy volunteers, but it is increased in critically ill patients with bacterial infections. Fosfomycin presents a high ability to distribute into many tissues, including inflamed tissues and abscess fluids. Based on PK/PD analysis and Monte Carlo simulations, we have evaluated different fosfomycin dosing regimen to optimize the treatment of septic patients due to Enterobacterales and Pseudomonas aeruginosa. As PK/PD targets, we selected %T>MIC > 70% for all pathogens, and AUC24/MIC > 24 and AUC24/MIC > 15 for net stasis of Enterobacterales and P. aeruginosa, respectively. Pharmacokinetic parameters in critically ill patients were obtained from the literature. Several dosing regimens were studied in patients with normal renal function: fosfomycin 2-8 g given every 6-12 hours, infused over 30 minutes- 24 hours. At the susceptibility EUCAST breakpoint for Enterobacterales and Staphylococcus spp. (MIC ≤ 32 mg/L), fosfomycin 4 g/8h or higher infused over 30 minutes achieved a probability of target attainment (PTA) > 90%, based in both %T>MIC and AUC24/MIC. For MIC of 64 mg/L, fosfomycin 6 g/6h in 30-minute infusion and 8 g/ 8h in 30-minute and 6 hours infusions also achieved PTA values higher than 90%. No fosfomycin monotherapy regimen was able to achieve PK/PD targets related to antimicrobial efficacy for P. aeruginosa with MICs of 256-512 mg/L.

Application of pharmacokinetic/pharmacodynamic analysis to evaluate the adequacy of antimicrobial therapy for pediatric acute otitis media in Spain before and after the introduction of the PCV7 vaccine.

OBJECTIVE: To evaluate, by applying pharmacokinetic/pharmacodynamic (PK/PD) analysis, if the change in antibiotic susceptibility after the introduction of the 7-valent pneumococcal conjugate vaccine (PCV7) in Spain had any influence on the usefulness of the antimicrobials more frequently used as empirical treatment of pediatric acute otitis media (AOM). METHODS: PK parameters and susceptibility of Streptococcus pneumoniae and Haemophilus influenzae were obtained from bibliography. Monte Carlo simulation was used to estimate the cumulative fraction of response (CFR), understood as the expected probability of therapy success. For amoxicillin and amoxicillin/clavulanate, the target was free antibiotic concentration remaining above the minimum inhibitory concentration (MIC) for ≥50% of the dosing interval (fT>MIC≥50%), whereas for cefuroxime axetil and cefotaxime, the target was fT>MIC≥60%. CFR values ≥90% were considered successful. RESULTS: When all serotypes of S. pneumoniae are considered, amoxicillin and cefotaxime turned out to reach a high probability of success, and difference before and after vaccination was scarce. For H. influenzae, CFR values were higher with amoxicillin/clavulanate than with amoxicillin. For both microorganisms, cefuroxime axetil resulted in low probability of success in the two periods of study. CONCLUSIONS: We have shown that the introduction of the PCV7 vaccination did not lead to changes in the probability of success of the current empiric treatments of the AOM. Integrated PK/PD analysis has demonstrated to be a useful tool to identify changes in antimicrobial activity after the implantation of a vaccination program, providing complementary information to the simple assessment of MIC values.

Population pharmacokinetics of daptomycin in critically ill patients.

Daptomycin has shown activity against a wide range of Gram-positive bacteria; however, the approved dosages usually seem insufficient for critically ill patients. The aim of this study was to develop a population pharmacokinetic model for daptomycin in critically ill patients and to estimate the success of the therapy by applying pharmacokinetic/pharmacodynamic (PK/PD) criteria. Sixteen intensive care unit patients were included, four of whom underwent continuous renal replacement therapies (CRRT). Blood and, when necessary, effluent samples were drawn after daptomycin administration at previously defined time points. A population approach using NONMEM 7.3 was performed to analyse data. Monte Carlo simulations were executed to evaluate the suitability of different dosage regimens. The probabilities of achieving the PK/PD target value associated with treatment success (ratio of the area under the plasma concentration-time curve over 24 h divided by the minimum inhibitory concentration (AUC24/MIC ≥ 666)) and to reach daptomycin concentrations linked to toxicity (minimum concentration at steady-state (Cminss) ≥ 24.3 mg/L) were calculated. The pharmacokinetics of daptomycin was best described by a one-compartment model. Elimination was conditioned by the creatinine clearance (Clcr) and also by the extra-corporeal clearance when patients were subjected to continuous renal replacement therapy (CRRT). The PK/PD analysis confirmed that 280- and 420-mg/d dosages would not be enough to achieve high probabilities of target attainment for MIC values ≥ 1 mg/L in patients with Clcr ≥ 60 mL/min or in subjects with lower Clcrs but receiving CRRT. In these patients, higher dosages (560-840 mg/d) should be needed. When treating infections due to MIC values ≥ 4 mg/L, even the highest dose would be insufficient.

Structural recovery of the retina in a retinoschisin-deficient mouse after gene replacement therapy by solid lipid nanoparticles.

X-linked juvenile retinoschisis (XLRS) is a retinal degenerative disorder caused by mutations in the RS1 gene encoding a protein termed retinoschisin. The disease is an excellent candidate for gene replacement therapy as the majority of mutations have been shown to lead to a complete deficiency of the secreted protein in the retinal structures. In this work, we have studied the ability of non-viral vectors based on solid lipid nanoparticles (SLN) to induce the expression of retinoschisin in photoreceptors (PR) after intravitreal administration to Rs1h-deficient mice. We designed two vectors prepared with SLN, protamine, and dextran (DX) or hyaluronic acid (HA), bearing a plasmid containing the human RS1 gene under the control of the murin opsin promoter (mOPS). In vitro, the nanocarriers were able to induce the expression of retinoschisin in a PR cell line. After injection into the murine vitreous, the formulation prepared with HA induced a higher transfection level in PR than the formulation prepared with DX. Moreover, the level of retinoschisin in the inner nuclear layer (INL), where bipolar cells are located, was also higher. Two weeks after vitreal administration into Rs1h-deficient mice, both formulations showed significant improvement of the retinal structure by inducing a decrease of cavities and PR loss, and an increase of retinal and outer nuclear layer (ONL) thickness. HA-SLN resulted in a significant higher increase in the thickness of both retina and ONL, which can be explained by the higher transfection level of PR. In conclusion, we have shown the structural improvement of the retina of Rs1h-deficient mice with PR specific expression of the RS1 gene driven by the specific promoter mOPS, after successful delivery via SLN-based non-viral vectors.

Meropenem dosing requirements against Enterobacteriaceae in critically ill patients: influence of renal function, geographical area and presence of extended-spectrum ß-lactamases.

The aim of this study was the evaluation of the influence of the susceptibility patterns of Escherichia coli and Klebsiella pneumoniae isolates, specifically the presence of extended-spectrum ß-lactamases and the geographical area (Europe and USA), on the meropenem dosing requirements in critically ill patients with different degrees of renal function by estimation of the probability of pharmacokinetic/pharmacodynamic (PK/PD) target attainment. Additionally, estimation of the PK/PD breakpoints according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) approach was also an objective. Six dosing regimens were evaluated: 0.5 g, 1 g and 2 g every 8 h given as 0.5-h or 3-h infusions. Pharmacokinetic data were obtained from the literature, and susceptibility data to meropenem for E. coli and K. pneumoniae were collected from the Tigecycline Evaluation and Surveillance Trial (T.E.S.T.) surveillance study. For the same dose level, the 3-h infusion provided a probability of target attainment (PTA) ≥90 % for minimum inhibitory concentration (MIC) values up to two-fold dilution higher than those obtained with the 0.5-h infusion. For E. coli, the cumulative fraction of response (CFR) was 100 % in most cases, and neither the dose nor the infusion length nor the geographical area significantly affected the probability to reach the target. With regards to K. pneumoniae, the CFR increased when increasing the dose and decreasing the creatinine clearance (CLCR). The CFR for Spanish and USA strains was higher than that calculated for European strains. Meropenem PK/PD breakpoints are dependent on the dose, infusion length and CLCR, ranging from 2 to 32 mg/L. Based on our results, meropenem administered as a extended infusion is the best option to treat infections due to E. coli and K. pneumoniae.

Solid lipid nanoparticle-based vectors intended for the treatment of X-linked juvenile retinoschisis by gene therapy: In vivo approaches in Rs1h-deficient mouse model.

X-linked juvenile retinoschisis (XLRS), which results from mutations in the gene RS1 that encodes the protein retinoschisin, is a retinal degenerative disease affecting between 1/5000 and 1/25,000 people worldwide. Currently, there is no cure for this disease and the treatment is based on the application of low-vision aids. The aim of the present work was the in vitro and in vivo evaluation of two different non-viral vectors based on solid lipid nanoparticles (SLNs), protamine and two anionic polysaccharides, hyaluronic acid (HA) or dextran (DX), for the treatment of XLRS. First, the vectors containing a plasmid which encodes both the reporter green fluorescent protein (GFP) and the therapeutic protein retinoschisin, under the control of CMV promoters, were characterized in vitro. Then, the vectors were subretinally or intravitreally administrated to C57BL/6 wild type mice. One week later, GFP was detected in all treated mice and in all retinal layers except in the Outer Nuclear Layer (ONL) and the Inner Nuclear Layer (INL), regardless of the administration route and the vector employed. Finally, two weeks after subretinal or intravitreal injection to Rs1h-deficient mice, GFP and retinoschisin expression was detected in all retinal layers, except in the ONL, which was maintained for at least two months after subretinal administration. The structural analysis of the treated Rs1h-deficient eyes showed a partial recovery of the retina related to the production of retinoschisin. This work shows for the first time a successful RS1 gene transfer to Rs1h-deficient animals using non-viral nanocarriers, with promising results that point to non-viral gene therapy as a feasible future therapeutic tool for retinal disorders.

Solid Lipid Nanoparticles as Non-Viral Vectors for Gene Transfection in a Cell Model of Fabry Disease.

Here, we demonstrate the ability of solid lipid nanoparticle-based non-viral vectors to increase the α-galactosidase A levels of the IMFE1 cell line, an in vitro model for target cells in Fabry disease. For this purpose, vectors containing the pR-M10-αGal A plasmid, which encodes the α-galactosidase A enzyme, were prepared; the in vitro transfection efficacy was studied in IMFE1 cells, and the results were confirmed by RT-PCR. The cellular uptake of the vectors, intracellular disposition of the plasmid, and probable endocytosis pathways of the nanoparticles were also analyzed. The vectors used for the studies carried protamine (P-DNA-SLN), dextran and protamine (D-P-DNA-SLN), or hyaluronic acid of two different molecular weights and protamine (HA150-P-DNA-SLN or HA500-P-DNA-SLN). The new formulations, which presented a particle size in the range of nanometers (from 218 nm to 348 nm) and a positive superficial charge, were able to increase α-galactosidase A activity up to 4-fold in comparison to non treated IMFE1 cells. The most efficient vectors were those that included HA, and no differences due to changes in the molecular weight of HA were detected. The observed lack of colocalization with each of the four different Nile Red-labeled vectors and transferrin or cholera toxin appears to indicate that clathrin- and caveolae-independent pathways may be involved in their cellular uptake. Additionally, colocalization with LysoTracker indicated that the formulations were exposed to lysosomal activity, which may be responsible for the release of the plasmid from the vector. In conclusion, we reveal the potential of SLN-based vectors to efficiently transfect an immortalized Fabry patient cell line.

Pharmacokinetic/pharmacodynamic analysis to evaluate ceftaroline fosamil dosing regimens for the treatment of community-acquired bacterial pneumonia and complicated skin and skin-structure infections in patients with normal and impaired renal function.

In this study, the probability of pharmacokinetic/pharmacodynamic target attainment (PTA) of ceftaroline against clinical isolates causing community-acquired bacterial pneumonia (CABP) and complicated skin and skin-structure infection (cSSSI) in Europe was evaluated. Three dosing regimens were assessed: 600 mg every 12 h (q12 h) as a 1-h infusion (standard dose) or 600 mg every 8 h (q8 h) as a 2-h infusion in virtual patients with normal renal function; and 400 mg q12 h as a 1-h infusion in patients with moderate renal impairment. Pharmacokinetic and microbiological data were obtained from the literature. The PTA and the cumulative fraction of response (CFR) were calculated by Monte Carlo simulation. In patients with normal renal function, the ceftaroline standard dose (600 mg q12 h as a 1-h infusion) can be sufficient to treat CABP due to ceftazidime-susceptible (CAZ-S) Escherichia coli, CAZ-S Klebsiella pneumoniae, meticillin-susceptible Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis (CFR>90%). However, against meticillin-resistant S. aureus (MRSA), the CFR was 72%. In cSSSI, the CFR was also <80% for MRSA. Administration of ceftaroline 600 mg q8 h as a 2-h infusion or 400 mg q12 h as a 1-h infusion in patients with moderate renal insufficiency provided a high probability of treatment success (CFR ca. 100%) for most micro-organisms causing CABP and cSSSI, including MRSA and penicillin-non-susceptible S. pneumoniae. These results suggest that in patients with normal renal function, ceftaroline 600 mg q8 h as a 2-h infusion may be a better option than the standard dose, especially if the MRSA rate is high.

Biodistribution of Nanostructured Lipid Carriers (NLCs) after intravenous administration to rats: influence of technological factors.

Nanoparticles for medical applications are frequently administered via parenteral administration. In this study, the tissue distribution of three lipid formulations based on Nanostructured Lipid Carriers (NLCs) after intravenous administration to rats was evaluated. NLCs were prepared by a high pressure homogenization method and varied in terms of particle size, surface charge, and surfactant content. The (99m)Tc radiolabeled NLCs were intravenously administered to rats, and radioactivity levels in blood and tissues were measured. Cmax, AUC0-24, and MRT0-24 were obtained from the radioactivity level versus time profiles. The radiolabeled nanocarriers exhibited a long circulation time since radioactivity was detected in blood even 24 h post-injection. No differences on the MRT values in blood among the NLCs were observed, in spite of the different particle size and surface charge. The highest radioactivity levels were measured in the kidney, followed by the bone marrow, the liver, and the spleen. In the kidney, there was a higher accumulation of the positive nanoparticles, and in the liver, uptake of negative nanoparticles was higher than positive ones. NLCs with the largest particle size showed a higher uptake in the lung and lower accumulation in liver and bone marrow, in comparison with the smaller ones.

Impact of acute otitis media pathogen shifts on the clinical efficacy of several antibiotics: a therapeutic outcomes model.

The aim of this study was to predict the clinical efficacy of different antimicrobials in the treatment of patients with acute otitis media (AOM), before and after the change in the proportion of middle ear pathogens observed after the introduction of the new conjugated heptavalent penumococcal vaccine (pPCV-7). The therapeutic Outcomes model was used to predict the likelihood of clinical success. According to this mathematical model the obtained rank order of predicted clinical efficacy was similar in the pre-PVC7 period and the post-PVC period. The results suggest that ceftriaxone and amoxicillin/clavulanate are the antibiotics with the highest predicted clinical efficacy, whereas cefaclor, azithromycin, erythromycin and clarithromycin are those with the lowest predicted clinical efficacy. The differences between antibiotics with good and those with low antibacterial activity were greater when only cases of bacterial AOM were considered. Antibiotics for which the highest clinical efficacy was predicted should maximize the likelihood of cure in outpatient antibiotic treatment of AOM.

The multidrug resistance modulator valspodar (PSC 833) is metabolized by human cytochrome P450 3A. Implications for drug-drug interactions and pharmacological activity of the main metabolite.

The metabolism of valspodar (PSC 833; PSC), which is developed as a multidrug resistance-reversing agent, was investigated to assess the potential for drug-drug interactions and the pharmacological activity of major metabolites. The primary metabolites of PSC produced by human liver microsomes were monohydroxylated, as revealed by LC/MS. The major site of hydroxylation was at amino acid 9, resulting in M9, as determined by cochromatography with synthetic M9. Dihydroxylated and N-demethylated metabolites were also detected. PSC metabolism in two human livers exhibited KM values of 1.3-2.8 microM. The intrinsic clearance was 9-36 ml/min/kg of body weight. PSC biotransformation was cytochrome P450 (CYP or P450) 3A dependent, based on chemical inhibition and on metabolism by Chinese hamster ovary cells expressing CYP3A. Ketoconazole was a competitive inhibitor (Ki = 0.01-0.04 microM). The inhibition by 27 compounds, including four antineoplastic agents, corresponded to the inhibitory potentials of these compounds toward CYP3A. For vinblastine, paclitaxel, doxorubicin, and etoposide, the IC50 values were 5, 12, 20, and 150 microM, respectively. M9 was also an inhibitor, with a lower apparent affinity for CYP3A (IC50 = 21 microM), compared with that of PSC. M9 was also less active as a multidrug resistance-reversing agent. M9 demonstrated low potency in sensitizing resistant cells to paclitaxel and was a poor inhibitor of rhodamine-123 efflux from paclitaxel-resistant cells. In addition, compared with PSC, a higher concentration of M9 was needed to compete with the photoaffinity labeling of P-glycoprotein. Conversely, PSC inhibited only reactions catalyzed by CYP3A, including cyclosporine A metabolism (IC50 = 6.5 microM) and p-hydroxyphenyl-C3'-paclitaxel formation (Ki = 1.2 microM). Thus, PSC behaves in a manner very similar to that of other cyclosporines, and a comparable drug-drug interaction profile is expected.