Biosynthetic Potential of the Endophytic Fungus Helotiales sp. BL73 Revealed via Compound Identification and Genome Mining.

Endophytic fungi have been recognized as prolific producers of chemically diverse secondary metabolites. In this work, we describe a new representative of the order Helotiales isolated from the medicinal plant Bergenia pacumbis. Several bioactive secondary metabolites were produced by this Helotiales sp. BL 73 isolate grown on rice medium, including cochlioquinones and isofusidienols. Sequencing and analysis of the approximately 59-Mb genome revealed at least 77 secondary metabolite biosynthesis gene clusters, of which several could be associated with detected compounds or linked to previously reported molecules. Four terpene synthase genes identified in the BL73 genome were codon optimized and expressed, together with farnesyl-, geranyl-, and geranylgeranyl-pyrophosphate synthases, in Streptomyces spp. An analysis of recombinant strains revealed the production of linalool and its oxidized form, terpenoids typically associated with plants, as well as a yet unidentified terpenoid. This study demonstrates the importance of a complex approach to the investigation of the biosynthetic potential of endophytic fungi using both conventional methods and genome mining. IMPORTANCE Endophytic fungi represent an as yet underexplored source of secondary metabolites, of which some may have industrial and medical applications. We isolated a slow-growing fungus belonging to the order Helotiales from the traditional medicinal plant Bergenia pacumbis and characterized its potential to biosynthesize secondary metabolites. We used cultivation of the isolate with a subsequent analysis of compounds produced, bioinformatics-based mining of the genome, and heterologous expression of several terpene synthase genes. Our study revealed that this Helotiales isolate has enormous potential to produce structurally diverse natural products, including polyketides, nonribosomally synthesized peptides, terpenoids, and ribosomally synthesized and posttranslationally modified peptides (RiPPs). Identification of meroterpenoids and xanthones, along with establishing a link between these molecules and their putative biosynthetic genes, sets the stage for investigation of the respective biosynthetic pathways. The heterologous production of terpenoids suggests that this approach can be used for the discovery of new compounds belonging to this chemical class using Streptomyces bacteria as hosts.

Triamcinolone acetonide can be detected in cerebrospinal fluid after intratympanic injection.

Intratympanically applied treatments are of increasing interest to the otologic community to treat sudden sensorineural hearing loss or vestibular disorders but also to deliver gene therapy agents, or biologics to the inner ear. Further diversion from the middle ear and perilymph to blood circulation and cerebrospinal fluid via the cochlear aqueduct are one of the limiting factors and so far not understood well enough. In this study, intratympanically applied triamcinolone acetonide was determined in cerebrospinal fluid. Additionally, perilymph was sampled through the round window membrane as well as at the lateral semicircular canal to determine drug levels. Of the twenty-one included patients, triamcinolone acetonide was quantifiable in cerebrospinal fluid in 43% at very low levels (range 0 ng/ml-6.2 ng/ml) which did not correlate with perilymph levels. Drug levels at the two different perilymph sampling sites were within a range of 13.5 ng/ml to 1180.0 ng/ml. Results suggest an equal distribution of triamcinolone acetonide to semicircular canals, which might support the use of triamcinolone acetonide as a treatment option for vestibular pathologies such as Menièrés disease. On the other hand, the distribution to cerebrospinal fluid might be limiting current approaches in gene therapy where a central distribution is unwanted.

Azithromycin concentrations during long-term regimen, a pilot study in patients with MALT lymphoma.

In view of the antineoplastic effects of the macrolide clarithromycin in mucosa associated lymphatic tissue (MALT)-lymphoma, we performed a pilot study assessing levels of azithromycin in plasma, peripheral blood mononuclear cells (PBMC) and polymorphonuclear leukocytes (PMN) of MALT-lymphoma patients to determine the pharmacokinetics and potential influences of respective concentrations on the therapeutic outcome. In total 16 patients with MALT-lymphoma received 1.5 g of oral azithromycin once-weekly over 6 months. Blood was sampled directly prior to the following dose every 4 weeks during treatment. Drug levels were analysed by high performance liquid chromatography in plasma and intracellularly in PBMC and PMN. They were correlated with patients' age, weight and body-mass-index and compared between patients responsive or unresponsive to treatment. Mean azithromycin plasma levels of all patients were 58.97 ± 30.48 ng/ml, remaining stable throughout the treatment period. Correlation analysis of plasma azithromycin showed no significance. Intracellular PBMC concentrations were 6648 ± 8479 ng/ml, without any significant difference between responders and non-responders. Mean PMN levels were 39,274 ± 25,659 ng/ml and significantly higher in patients unresponsive to treatment (t = 2.858, p = 0.017). Our drug regime led to continuously high plasma and exceedingly high intracellular concentrations of azithromycin in PBMC and PMN. Age, weight or body-mass-index had no significant influence on plasma levels and thence should not be considered in dosage finding. High AZM levels in PBMC did not lead to a better treatment response, whereas enrichment in PMN suggested a poorer outcome. The threshold for immunomodulatory effects on lymphoma cells might not have been reached. Additionally, the finding of stable plasma and intracellular concentrations over months with high-dose azithromycin administered in intervals might also be important for the further design of azithromycin-based trials against MALT-lymphoma.Trial registration: EudraCT 2016-001521-13, 14/06/2016.

Evaluation of Levels of Triamcinolone Acetonide in Human Perilymph and Plasma After Intratympanic Application in Patients Receiving Cochlear Implants: A Randomized Clinical Trial.

Importance: The use of intratympanically applied steroids is of increasing interest. Consequently, research has focused on finding an ideal drug that diffuses through the round window membrane and can be retained in the perilymph. Objective: To compare levels of triamcinolone acetonide (TAC) in perilymph and plasma after intratympanic injection. Design, Setting, and Participants: This randomized clinical trial included 40 patients receiving cochlear implants at a single tertiary care center in Vienna, Austria. Patients were randomized to 1 of 4 treatment groups receiving 1 of 2 intratympanic doses of TAC (10 mg/mL or 40 mg/mL) at 1 of 2 approximate time points (24 hours or 1 hour) before sampling the perilymph. Inclusion was carried out between November 2017 and January 2020, and data were analyzed in December 2020. Interventions: All patients underwent intratympanic injection of TAC. During cochlear implantation, perilymph and plasma were sampled for further analysis. Main Outcomes and Measures: Levels of TAC measured in perilymph and plasma. Results: Among the 37 patients (median [range] age, 57 [26-88] years; 18 [49%] men) included in the analysis, TAC was present at a median (range) level of 796.0 (46.4-7706.7) ng/mL. In the majority of patients (n = 29; 78%), no drug was detectable in the plasma after intratympanic injection. Levels above the limit of detection were less than 2.5 ng/mL. The 1-factorial analysis of variance model showed lower TAC levels in the group that received TAC, 10 mg/mL, 24 hours before surgery (median, 271 ng/mL) compared with the group that received TAC, 10 mg/mL, 1 hour before surgery (median, 2877 ng/mL), as well as in comparison with the groups receiving TAC, 40 mg/mL, 24 hours before surgery (median, 2150 ng/mL) and 1 hour before surgery (median, 939 ng/mL). The 2-factorial analysis of variance model showed lower TAC levels in the group receiving TAC, 10 mg/mL, 24 hours before surgery than the group receiving TAC, 10 mg/mL, 1 hour before surgery, and higher TAC levels in the group receiving TAC, 40 mg/mL, 24 hours before surgery compared with the group receiving TAC, 10 mg/mL, 24 hours before surgery. Patients with thickening of the middle ear had statistically significantly higher plasma levels (median, 1.4 ng/mL vs 0 ng/mL) and lower perilymph levels (median, 213.1 ng/mL vs 904 ng/mL) than individuals with unremarkable middle ear mucosa. Conclusions and Relevance: In this randomized clinical trial, TAC was shown to be a promising drug for intratympanic therapies, with similar levels in perilymph 1 hour and 24 hours after injection (distinctly in the groups receiving the 40 mg/mL dose). There was also minimal dissemination to the plasma, especially in patients with unremarkable middle ear mucosa. Trial Registration: ClinicalTrials.gov Identifier: NCT03248856.

Intratympanic application of poloxamer 407 hydrogels results in sustained N-acetylcysteine delivery to the inner ear.

N-acetylcysteine is a thiol-containing antioxidant, which has shown otoprotective effects in in vitro as well as in vivo models of cisplatin-induced hearing loss. Systemic administration of antioxidants, however, is associated with the major potential drawback of interference with the tumoricidal effect of cisplatin. This therapeutic limitation can be overcome by local intratympanic injection of the antioxidant N-acetylcysteine, which results in very restricted systemic uptake of the drug, whilst intracochlear drug levels are substantially higher. Furthermore, osmolality and pH properties of formulations for intratympanic injection need to be controlled, as they impact the fraction of drug crossing the barriers of the inner ear and could potentially damage middle and inner ear structures. This study focused on (i) the evaluation of concentration-time profiles of N-acetylcysteine in perilymph, cerebrospinal fluid and plasma after intratympanic administration, (ii) the influence of the dosage form, i.e. a thermoreversible poloxamer 407 hydrogel versus a solution, on N-acetylcysteine pharmacokinetics, and (iii) the development of a pH- and osmolality-adjusted formulation for intratympanic N-acetylcysteine delivery. 49 female albino guinea pigs were randomized into two treatment groups, receiving either a single intratympanic injection of a 4% N-acetylcysteine poloxamer 407 hydrogel or a 4% N-acetylcysteine solution. 8 animals served as untreated controls. N-acetylcysteine levels in perilymph, cerebrospinal fluid and plasma were monitored over a period of 24 h. Samples were taken at 1, 3, 6, 12 and 24 h (poloxamer 407 hydrogel group) and 1, 6 and 24 h (solution group) post injection, and analysed by high performance liquid chromatography-tandem mass spectrometry. Intratympanic application of the 4% N-acetylcysteine poloxamer 407 hydrogel resulted in a 4-fold larger perilymph area under the concentration-time curve (0-24 h) than topical administration of the equally concentrated N-acetylcysteine solution but in similar plasma N-acetylcysteine levels. N-acetylcysteine concentrations in the cerebrospinal fluid were below the level of detection (5 ng/ml) in both treatment groups. N-acetylcysteine-containing formulations applied to the middle ear were isohydric and osmolality was reduced by up to 200 mosmol/kg compared to equally concentrated formulations used in previous studies. In summary, we were able to demonstrate that the intratympanic injection of thermoreversible poloxamer 407 hydrogels increases and sustains N-acetylcysteine delivery to the inner ear. Given the low plasma N-acetylcysteine levels after topical application and the physiological pH and osmolality of the hydrogel, the risk of compromising the antineoplastic effects of cisplatin therapy and of local side effects is minimal.

Compatibility of aztreonam in four commercial peritoneal dialysis fluids.

The preferable route for treatment of peritoneal dialysis related peritonitis remains the intraperitoneal administration of antibiotics admixed to peritoneal dialysis fluids. It is important to know whether the administered drug is compatible with the PD fluids and its container. In the present study the compatibility of aztreonam with four commercial PDFs at storing temperatures and duration representative for storing conditions in the clinical settings was investigated. Aztreonam concentrations were determined using high-performance liquid chromatography. The antimicrobial activity of aztreonam was evaluated using an E. coli diffusion disk inhibition assay and P. aeruginosa time-kill curves. In Extraneal evaluated at 6 °C, 25 °C and 37 °C aztreonam was stable over the whole study period of 14 days and 24 hours, respectively. In Physioneal and Nutrineal aztreonam was stable at 6 °C for up to 14 days. Antimicrobial activity was retained in all PD fluids over the whole study period. Aztreonam remained stable and was compatible with the PD fluids, particularly with Extraneal or Nutrineal, and no compensatory dose adjustment is needed when stored for up to 14 days at refrigeration temperature before use.

Short Chain (≤C4) Esterification Increases Bioavailability of Rosmarinic Acid and Its Potency to Inhibit Vascular Smooth Muscle Cell Proliferation.

Rosmarinic acid is a natural phenolic acid and active compound found in many culinary plants, such as rosemary, mint, basil and perilla. Aiming to improve the pharmacokinetic profile of rosmarinic acid and its activity on vascular smooth muscle cell proliferation, we generated a series of rosmarinic acid esters with increasing alkyl chain length ranging from C1 to C12. UHPLC-MS/MS analysis of rat blood samples revealed the highest increase in bioavailability of rosmarinic acid, up to 10.52%, after oral administration of its butyl ester, compared to only 1.57% after rosmarinic acid had been administered in its original form. When added to vascular smooth muscle cells in vitro, all rosmarinic acid esters were taken up, remained esterified and inhibited vascular smooth muscle cell proliferation with IC50 values declining as the length of alkyl chains increased up to C4, with an IC50 of 2.84 µM for rosmarinic acid butyl ester, as evident in a resazurin assay. Vascular smooth muscle cells were arrested in the G0/G1 phase of the cell cycle and the retinoblastoma protein phosphorylation was blocked. Esterification with longer alkyl chains did not improve absorption and resulted in cytotoxicity in in vitro settings. In this study, we proved that esterification with proper length of alkyl chains (C1-C4) is a promising way to improve in vivo bioavailability of rosmarinic acid in rats and in vitro biological activity in rat vascular smooth muscle cells.

Compatibility of ciprofloxacin with commercial peritoneal dialysis solutions.

Intraperitoneal administration of antibiotics together with peritoneal dialysis fluids (PDFs) remains the preferable route for treatment of peritoneal dialysis-related peritonitis. For home based therapy, antibiotic-containing PDFs are stored for up to two weeks and warmed up to body-temperature before administration. The present study investigated the compatibility of ciprofloxacin with five commercial PDFs at refrigeration-temperature, room-temperature and body-temperature. Ciprofloxacin concentrations were determined using high-performance liquid chromatography. Drug-diluent stability was evaluated by measurement of pH-values and visual inspection at each sampling point. The antimicrobial activity of ciprofloxacin was assessed by an E. coli disk diffusion method. Ciprofloxacin was stable at refrigeration-temperature and body-temperature in all PDFs evaluated over the whole study period of 14 days and 24 hours, respectively. At room-temperature, in contrast, ciprofloxacin demonstrated only limited stability in particular when tested in mixed Physioneal. Except for Physioneal 1.36%, no relevant drug adsorption was observed and the antimicrobial activity of ciprofloxacin was found to be preserved in each PDF at each storage condition investigated. Intraperitoneal ciprofloxacin might be used for inpatient and home based therapy of peritoneal dialysis-related peritonitis and no compensatory dose adjustment is needed when stored for up to two weeks at refrigeration-temperature before use.

Compatibility of linezolid with commercial peritoneal dialysis solutions.

PURPOSE: Results of a compatibility and stability study of linezolid admixed in commercial peritoneal dialysis (PD) solutions stored at various temperatures are reported. METHODS: Test samples were prepared by adding linezolid i.v. injection (2 mg/mL) to infusion bags of 4 PD solutions (Extraneal, Nutrineal, Physioneal 40 Glucose 1.36%, and Physioneal 40 Glucose 2.27%, all from Baxter Healthcare Corporation). Assessments were conducted at various time points during storage of test samples at refrigeration temperature (6 °C) or room temperature (25 °C) for 14 days and at body temperature (37 °C) for 24 hours. Linezolid concentrations over time were determined by high-performance liquid chromatography, physical compatibility was determined by pH measurement and visual inspection, and antimicrobial activity was monitored by a disk diffusion method. The influence of solution warming by heating plate on drug stability was investigated. RESULTS: Linezolid was stable in all tested solutions for 14 days at refrigeration and room temperatures and for 24 hours at body temperature. No linezolid adsorption to container material was detected. There were only minor variations in pH values, and visual inspection revealed no diluent abnormalities. With 1 exception, antimicrobial activity of >90% was retained in all PD solution samples for the duration of the study under all temperature conditions. CONCLUSION: Linezolid injection 2 mg/mL remained stable and was compatible with the PD solutions studied for up to 2 weeks at refrigeration or room temperature and up to 24 hours at body temperature.

Influence of different peritoneal dialysis fluids on the in vitro activity of fosfomycin against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa.

Peritonitis is still the main infectious complication among patients on peritoneal dialysis. For treatment of peritoneal dialysis-related peritonitis, the intraperitoneal administration of antibiotics admixed to peritoneal dialysis fluids (PDFs) should be preferred. However, the influence of diverse PDFs on the activity of frequently used antibiotics has been investigated insufficiently. Thus, the present study set out to investigate the in vitro activity of fosfomycin against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus epidermidis, and Staphylococcus aureus in commercially available PDFs. Time-kill curves in four different PDFs (Dianeal®, Extraneal®, Nutrineal®, and Physioneal®) were performed over 24 h with two different concentrations of fosfomycin (150 and 400 mg/L) and without antibiotics as control. Cation-adjusted Mueller Hinton broth (CA-MHB) was used as a comparator solution. In blank PDFs, bacterial growth of each organism evaluated was reduced when compared to CA-MHB. For S. aureus in blank Physioneal®, a reduction under the limit of detection was observed within 24 h. The activity of fosfomycin was reduced in all PDFs when compared to CA-MHB except for P. aeruginosa in Nutrineal® where the activity of fosfomycin was increased when investigated at 400 mg/L. Against E.coli, bactericidal activity was demonstrated in Extraneal®, Nutrineal®, and Physioneal®. Fosfomycin resistance (MIC > 1024 mg/L) was observed for P. aeruginosa in CA-MHB at both concentrations and in Nutrineal® at 150 mg/L. Fosfomycin is active in PDFs particularly against the frequently isolated enterobacterium E. coli. The choice of the respective PDF considerably influences the microbiological outcome in vitro. Further studies are warranted to investigate the clinical relevance of these findings.

Quality Control of Valerianae Radix by Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) Spectroscopy.

(Acetoxy-)valerenic acid and total essential oil content are important quality attributes of pharmacy grade valerian root (Valerianae radix). Traditional analysis of these quantities is time-consuming and necessitates (harmful) solvents. Here we investigated an application of attenuated total reflection Fourier transform infrared spectroscopy for extractionless analysis of these quality attributes on a representative sample comprising 260 wild-crafted individuals covering the Central European taxonomic diversity of the Valeriana officinalis L. s. l. species aggregate with its three major ploidy cytotypes (i.e., di-, tetra- and octoploid). Calibration models were built by orthogonal partial least squares regression for quantitative analysis of (acetoxy-)valerenic acid and total essential oil content. For the latter, we propose a simplistic protocol involving apolar extraction followed by gas chromatography as a reference method for multivariate calibration in order to handle the analysis of samples taken from individual plants. We found good predictive ability of chemometric models for quantification of valerenic acid, acetoxyvalerenic acid, total sesquiterpenoid acid, and essential oil content with a root mean squared error of cross-validation of 0.064, 0.043, and 0.09 and root mean squared error of prediction of 0.066, 0.057, and 0.09 (% content), respectively. Orthogonal partial least squares discriminant analysis revealed good discriminability between the most productive phenotype (i.e., the octoploid cytotype) in terms of sesquiterpenoid acids, and the less productive ones (i.e., di- and tetraploid). All in all, our results demonstrate the application of attenuated total reflection Fourier transform infrared spectroscopy for rapid, extractionless estimation of the most important quality attributes of valerian root and minimally invasive identification of the most productive phenotype in terms of sesquiterpenoid acids.

Compatibility of Meropenem with Different Commercial Peritoneal Dialysis Solutions.

♦ BACKGROUND: Intraperitoneal administration of antimicrobial agents is recommended for the treatment of peritoneal dialysis (PD)-related peritonitis. For home-based antimicrobial therapy it is common to supply patients with PD fluid bags with admixed antibiotic. Thus, the compatibility of meropenem with different PD fluids (PDFs), namely Extraneal, Physioneal 1.36% and Physioneal 2.27% (all Baxter Healthcare Corp., Deerfield, IL, USA), was investigated under varying storage conditions. ♦ METHODS: Meropenem (Venus Pharma, Werne, Germany) was stored at 6°C and 25°C over 14 days and at 37°C over 24 hours. Drug concentration over time was determined using high performance liquid chromatography, drug activity by a diffusion disk method, diluent stability by visual inspection and drug adsorption was calculated. Blank PD fluids and deionized water were used as comparator solutions. ♦ RESULTS: Compared to water, the stability of meropenem was minimally lower in Extraneal but markedly reduced in both Physioneal solutions. No significant drug adsorption was detected for any PDF investigated. ♦ CONCLUSIONS: Meropenem is stable and compatible with Extraneal and might be stored for up to a week at refrigeration temperature (6°C). A loss of ~20% of meropenem after 2 days at room temperature should be considered. Mixed Physioneal appears not suitable for storage at any temperature after meropenem has been admixed. A considerable drug degradation due to the warming up to body temperature through heating plates should further be taken into account in clinical practice.

Influence of Different Peritoneal Dialysis Fluids on the In Vitro Activity of Cefepime, Ciprofloxacin, Ertapenem, Meropenem and Tobramycin Against Escherichia Coli.

♦ BACKGROUND: Peritonitis is a major problem among patients on peritoneal dialysis (PD). The influence of diverse PD fluids on the activity of frequently used antibiotics has been insufficiently investigated. Thus, the present study set out to investigate the impact of different PD fluids on the activity of cefepime, ciprofloxacin, ertapenem, meropenem, and tobramycin against Escherichia coli. ♦ METHODS: Time-kill curves in 4 different PD fluids (Dianeal PDG4, Extraneal, Nutrineal PD4 and Physioneal 40, all Baxter Healthcare Corp., Deerfield, IL, USA) were performed over 24 hours with 4 different concentrations (1 × minimum inhibitory concentration [MIC], 4 × MIC, 8 × MIC, 30 × MIC) of each antibiotic evaluated and without antibiotics as control. Cation-adjusted Mueller Hinton broth (CA-MHB) was used as comparator solution. ♦ RESULTS: In all PD fluids investigated, bacterial growth and antimicrobial activity of all antibiotics tested was significantly reduced compared with the CA-MHB comparator solution. Except at high concentrations of 30 × MIC, cefepime, ertapenem and meropenem demonstrated a strongly reduced activity in all PD fluids investigated. Ciprofloxacin and tobramycin were highly active and bactericidal in all PD fluids and demonstrated dose-dependent activity. ♦ CONCLUSION: The antimicrobial activity of cefepime, ertapenem and meropenem is limited or even nullified in certain PD fluids in vitro, whereas ciprofloxacin and tobramycin show excellent activity. The choice of PD fluids can impact the activity of antimicrobial agents and might influence microbiological outcome. Further studies are required to verify the clinical relevance of our findings.

An Open, Randomized, Single-Center, Crossover Pharmacokinetic Study of Meropenem after Intraperitoneal and Intravenous Administration in Patients Receiving Automated Peritoneal Dialysis.

The objective of this study was to determine the pharmacokinetic profile of meropenem in automated peritoneal dialysis (APD) patients. In 6 patients without peritonitis, a single dose of 0.5 g of meropenem was applied intraperitoneally (i.p.) or intravenously (i.v.) and concentrations in serum and dialysate were measured at specified intervals over 24 h with high-performance liquid chromatography-mass spectrometry. The mean maximum concentrations of meropenem in serum (Cmax) were 27.2 mg/liter (standard deviation [SD], ±6.9) and 10.1 mg/liter (SD, ±2.5) and in dialysate were 3.6 mg/liter (SD, ±2.3) and 185.8 mg/liter (SD, ±18.7) after i.v. and i.p. administrations, respectively. The mean areas under the curve from 0 to 24 (AUC0-24) of meropenem in serum were 173.5 mg · h/liter (SD, ±29.7) and 141.4 mg · h/liter (SD, ±37.5) (P = 0.046) and in dialysate were 42.6 mg · h/liter (SD, ±20.0) and 623.4 mg · h/liter (SD, ±84.1) (P = 0.028) after i.v. and i.p. administrations, respectively. The ratios for dialysate exposure over plasma exposure after i.v. and i.p. treatments were 0.2 (SD, ±0.1) and 4.6 (SD, ±0.9), respectively (P = 0.031). A mean target value of 40% T>MIC (time for which the free meropenem concentration exceeds the MIC) for clinically relevant pathogens with EUCAST susceptibility breakpoints of 2 mg/liter was reached in serum after i.p. and i.v. administrations and in dialysate after i.p. but not after i.v. administration. The present data indicate that low i.p. exposure limits the i.v. use of meropenem for PD-associated peritonitis. In contrast, i.p. administration not only results in superior concentrations in dialysate but also might be used to treat systemic infections.

Simultaneous determination of active component and vehicle penetration from F-DPPC liposomes into porcine skin layers.

Liposomes have been used as innovative delivery vehicles on skin for a number of years due to their positive influence on skin penetration. However, until now it is not entirely clear how and by which mechanism enhancement is achieved. In the present study, the skin permeation of a model substance incorporated into liposomes and a control formulation was compared to study the influence of the vehicle in Franz-type diffusion cell experiments. Furthermore, the penetration depths of both components were studied by simultaneous determination of the active substance and the vehicle component during tape stripping studies and horizontal sectioning. For these purposes we prepared liposomes with 1-palmitoyl-2-(16-fluoropalmitoyl)-sn-glycero-3-phosphocholine (F-DPPC), the monofluorinated analogue of dipalmitoylphosphaditylcholine (DPPC) loaded with sodium fluorescein (SoFl). A sodium-fluorescein solution was used as control formulation. While the semi-solid F-DPPC liposomes and the SoFl-solution performed equally well with similar permeation profiles during skin diffusion experiments, superior penetrated amounts of SoFl into the stratum corneum (SC) from F-DPPC liposomes compared to the SoFl-solution were observed possibly due to a "push" exerted by the vehicle F-DPPC. We also showed that SoFl penetrated through SC into the viable epidermis.

Semi-solid fluorinated-DPPC liposomes: Morphological, rheological and thermic properties as well as examination of the influence of a model drug on their skin permeation.

The goal of this study was to investigate the influence of an incorporated model drug on the skin permeation of the vehicle itself as it may affect the microstructure and properties of the applied formulation via molecular interactions. For this purpose, we performed skin permeation studies using liposomes prepared with F-DPPC, a monofluorinated analog of dipalmitoylphosphatidylcholine (DPPC), with and without sodium fluorescein (SoFl) serving as model drug. Interestingly, the liposome preparation with F-DPPC yielded semi-solid opalescent systems. Hence, a thorough characterization was accomplished beforehand by electron microscopy imaging, rheological and thermoanalytical experiments. Freeze-fracture electron microscopy images confirmed the existence of globular shaped vesicles in the F-DPPC preparations and oscillatory rheological measurements proved the viscoelastic properties of F-DPPC and F-DPPC+SoFl liposomes in contrast to the viscous characteristics of DPPC liposomes. Thermoanalytical measurements revealed an increased phase transition temperature Tm of about 50 °C for F-DPPC and F-DPPC+SoFl liposomes compared to pure DPPC liposomes with a Tm of about 43° C. The similar Tm of F-DPPC+SoFl and F-DPPC liposomes as well as the similar skin permeation of the vehicle compound F-DPPC compared to its drug-free counterpart suggest an incorporation of sodium fluorescein into the aqueous core of F-DPPC liposomes.

Topical delivery of acetyl hexapeptide-8 from different emulsions: influence of emulsion composition and internal structure.

Acetyl hexapeptide-8 (AH-8) is a well-known component of anti-aging products and was recently explored as a promising topical treatment of blepharospasm. Although AH-8 appears in a variety of cosmetic products, its skin penetration is sparsely studied and controversially discussed. Therefore, the aim of the present study was to investigate the influence of the vehicle type on the AH-8 delivery to the skin. Besides skin permeation experiments with Franz type diffusion cells, the spatial distribution of AH-8 in the stratum corneum after a real in-use application was investigated by in vitro tape stripping on porcine ear skin. By applying LC-MS/MS for quantification of AH-8, we demonstrated that a multiple water-in-oil-in-water (W/O/W) emulsion can significantly increase penetration of AH-8 into porcine skin compared to simple O/W and W/O emulsions. The internal structure of the developed multiple emulsion was confirmed by electron microscopic investigations and NMR self diffusion studies. In general, a clear superiority of water-rich W/O/W and O/W emulsions over an oil-rich W/O emulsion in terms of dermal delivery of AH-8 was found. This enhanced delivery of AH-8 could be explained by an increased absorption of the water-rich emulsions into the skin, confirmed by combined ATR-FTIR and tape stripping experiments.

Cytochrome P450 3A-mediated metabolism of the topoisomerase I inhibitor 9-aminocamptothecin: impact on cancer therapy.

The metabolism of 9-aminocamptothecin (9-AC) was investigated in human and rat liver microsomes. In both species 9-AC was almost exclusively biotransformed to dihydroxy-9-AC (M1) and monohydroxy-9-AC (M2). The enzymatic efficiencies of the formation of M1 and M2 (V(max)/K(m)) were 1.7- and 2.7­fold higher in rat than in human liver microsomes indicating species-related differences in 9-AC hydroxylation. Incubation in the presence of human recombinant cytochrome P450 (CYP) enzymes demonstrated that the formation of M1 and M2 is mainly catalyzed by CYP3A4 and only to a minor extent by extrahepatic CYP1A1. The predominant role of CYP3A4 was further supported by a dramatic inhibition of metabolite formation in the presence of the CYP3A4 substrates troleandomycin and ketoconazole. Experiments conducted in isolated perfused rat livers further demonstrated that biliary excretion of 9-AC, M1 and M2 during 60 min of perfusion was pronounced and accounted for 17.7±2.59, 0.05±0.01 and 2.75±0.14% of total 9-AC applied to the liver, respectively. In summary, this study established that CYP3A-dependent hydroxylation is the main metabolic pathway for 9-AC in rat and human liver, which have to be taken into consideration during cancer therapy of patients.

Efficacy of fosfomycin compared to vancomycin in treatment of implant-associated chronic methicillin-resistant Staphylococcus aureus osteomyelitis in rats.

Fosfomycin monotherapy was compared to therapy with vancomycin for the treatment of implant-associated methicillin-resistant Staphylococcus aureus (MRSA) osteomyelitis in an experimental rat model. The proximal tibiae were inoculated with 15 µl of a suspension containing 1×10(8) to 5×10(8) CFU/ml of a clinical isolate of MRSA with simultaneous insertion of a titanium wire. Four weeks later, treatment was started for 28 days with either 50 mg/kg of body weight vancomycin intraperitoneally twice daily (n=11) or 75 mg/kg fosfomycin intraperitoneally once daily (n=10). Eleven animals were left untreated. After treatment, quantitative cultures from bone were found to be positive for MRSA in all animals in the untreated group (median, 3.29×10(6) CFU/g of bone) and the vancomycin group (median, 3.03×10(5) CFU/g of bone). In the fosfomycin group, MRSA was detectable in 2 out of 10 (20%) animals (3.42×10(2) and 1.51×10(3) CFU/g of bone). Vancomycin was superior to the no-drug control (P=0.002), and fosfomycin was superior to the no-drug control and vancomycin (P<0.001). The cultures from the wires were positive in all untreated animals (median, 2.5×10(3) CFU/implant), in 10 animals in the vancomycin group (median, 1.15×10(3) CFU/implant), and negative in all animals in the fosfomycin group. Based on the bacterial counts from the implants, vancomycin was not superior to the no-drug control (P=0.324), and fosfomycin was superior to the no-drug control and vancomycin (P<0.001). No emergence of resistance was observed. In conclusion, it was demonstrated that fosfomycin monotherapy is highly effective for the treatment of experimental implant-associated MRSA osteomyelitis.

Sustained release of triamcinolone acetonide from an intratympanically applied hydrogel designed for the delivery of high glucocorticoid doses.

The pharmacokinetic properties and tolerability of a triamcinolone acetonide poloxamer 407 hydrogel for intratympanic application were investigated in a guinea pig model. Evaluation of in vivo release kinetics showed very high initial perilymph drug levels, with clinically relevant levels present for a minimum of 10 days. Assessment of auditory brainstem response thresholds showed a minimal, delayed and transient threshold shift, which was apparent on day 3 and resolved by day 10. No relevant histological changes of the middle and inner ear structures were noted, and hair cell counts showed no significant differences between treated and untreated ears. Thus, the triamcinolone-acetonide-loaded poloxamer 407 hydrogel is an effective vehicle for sustained high-dose inner ear glucocorticoid delivery.