Extended Nasal Residence Time of Lysostaphin and an Anti-Staphylococcal Monoclonal Antibody by Delivery in Semisolid or Polymeric Carriers

PURPOSE: Eradication of Staphylococcus aureus nasal colonization reduces the risk of nosocomial and community acquired infections with this organism. This study describes the formulation and use of lysostaphin and BSYX-A110, an anti-lipoteichoic acid monoclonal antibody, for eradication of S. aureus nasal colonization. METHODS: Lysostaphin was formulated into a hydrophilic cream that forms an emulsion with the secretions of the nasal mucosa, and aqueous formulations of BSYX-A110 were made containing the mucoadhesive polymers polystyrene sulfonate and chitosan. Intranasal pharmacokinetics of the drugs was measured in mice and cotton rats. RESULTS: Lysostaphin formulated in the cream increased nasal retention of the drug by 10-fold at 3 h post-cream installation and 50-fold at 24 h as compared to lysostaphin in saline drops. Furthermore, the levels of lysostaphin in the nose 24 h post-cream instillation are still above the minimum bactericidal concentration for most bacterial strains. The liquid polymer formulations also resulted in prolonged retention of antibody in the nose, with 4-fold higher levels at 3 h post-instillation as compared to antibody in saline drops. CONCLUSIONS: These results demonstrate that cream and polymer delivery systems significantly decrease the clearance rate of lysostaphin and BSYX-A110 from the nose, thereby enhancing their therapeutic potential for eradicating S. aureus nasal colonization.     

Using rifapentine – hen egg lipoprotein conjugate as macrophage-targeted drug delivery carrier against intracellular Staphylococcus aureus

Abstract

Context: Hen egg low-density lipoprotein (heLDL), which is present in large quantities in egg yolk, share a high identity with human apolipoprotein B-100 precursor.

Objective: This study investigated the use of heLDL as a macrophage-targeted drug delivery carrier against intracellular Staphylococcus aureus.

Methods: Rifapentine (RPT) was incorporated into heLDL (RPT–heLDL). Staphylococcus aureus ATCC 29740 and human U937 macrophage were used as intracellular infection models.

Results and conclusion: The loading efficiency of RPT into the heLDL was 66.10?±?2.28?μg RPT/mg heLDL. Fluorescence microscopy and oil red O staining results indicated RPT–heLDL can be taken up by U937 macrophages. The cell viability (MTT assay) was increased when the concentration of heLDL was <150?μg/mL. Unloaded heLDL (100?μg/mL) can inhibit the growth of intracellular S. aureus compared with the untreated control group after 18?h incubation. RPT–heLDL (6.6?μg/mL RPT, 100?μg/mL heLDL) eliminated 94% of intracellular S. aureus, whereas the corresponding dose of free RPT (6.6?μg/mL) induced an 87% reduction. The in vitro results of the current study indicated that heLDL might be used as a suitable drug carrier for targeting human macrophages.     

In vivo efficacy of the antimicrobial peptide ranalexin in combination with the endopeptidase lysostaphin against wound and systemic meticillin-resistant Staphylococcus aureus (MRSA) infections

New treatments are urgently required for infections caused by meticillin-resistant Staphylococcus aureus (MRSA) as these strains are often resistant to multiple conventional antibiotics. Earlier studies showed that ranalexin, an antimicrobial peptide (AMP), in combination with lysostaphin, an antistaphylococcal endopeptidase, synergistically inhibits the growth of MRSA, meaning that it deserved consideration as a new anti-S. aureus therapy. Using haemolysis and Vero cell viability assays, ranalexin with lysostaphin is proven to be non-toxic at antibacterial concentrations. In human serum, ranalexin with lysostaphin is significantly more effective against MRSA than treatment with either component alone. In a rabbit model of wound infection, ranalexin with lysostaphin reduced MRSA in the wound by ca. 3.5 log₁₀ colony-forming units (CFU) compared with the untreated control. The combination is significantly more effective than treatment with ranalexin or lysostaphin alone. In a mouse model of systemic infection, ranalexin with lysostaphin reduced MRSA kidney burden by ca. 1 log₁₀ CFU/g compared with untreated controls or treatment with ranalexin or lysostaphin alone. Importantly, the combination is synergistically bactericidal against various S. aureus isolates in vitro, including those with reduced susceptibility to lysostaphin or vancomycin. Ranalexin and lysostaphin could be incorporated in wound dressings for the prevention and treatment of topical S. aureus infections. That AMPs can enhance the antibacterial effectiveness of lysostaphin in vivo highlights a new avenue of research in the fight against drug-resistant staphylococci.     

Aquaphilus dolomiae extract counteracts the effects of cutaneous S. aureus secretome isolated from atopic children on CD4+ T cell activation

Context: Skin microbiota takes part in the control of cutaneous inflammation. In skin diseases such as atopic dermatitis (AD) cutaneous dysbiosis and the emergence of Staphylococcus aureus contribute to the pathophysiology of the disease. New therapeutic approaches consist in topical application of natural products able to counteract S. aureus effects through activation of resident immune cells producing anti-inflammatory cytokines such as IL-10.

Objective: This study investigates the potential immunosuppressive properties of Aquaphilus dolomiae (Neisseriaceae), a flagellated bacterium contained in Avène Thermal Spring Water used in hydrotherapy treatments of AD patients.

Materials and methods: An aqueous protein extract of Aquaphilus dolomiae (ADE, 60?μg/mL) was added to human monocyte-derived dendritic cells (moDC) for 24?h. Expression of HLA-DR, CD86 and CD83 was evaluated by flow cytometry and released cytokines (IL-10, IL-12) by cytometry bead array assay. The proliferation of allogeneic CFSE-labelled CD4^+?T cells stimulated with ADE-conditioned moDC and S. aureus secretome was analysed by flow cytometry.

Results: MoDC exposed to ADE expressed lower levels of HLA-DR and CD86 than untreated cells, no CD83 and secreted barely detectable IL-12 but high amounts of IL-10 (N?=?12, p?<?0.0002). The proliferative effect of S. aureus secretome on CD4^+?T cells was reduced (p?<?0.001) in the presence of ADE-moDC.

Conclusion: ADE counteracted the mitogenic effect of a S. aureus secretome on CD4⁺T cells. Owing to the role of S. aureus colonization in driving inflammation in AD the immunosuppressive property of the ADE might be useful to reduce disease severity.     

Antioxidant,antimicrobial and wound healing properties of Struthanthus vulgaris

Context: Struthanthus vulgaris (Vell.) Mart. (Loranthaceae) has been widely used in traditional medicine in Brazil to bathe wounds.

Objective: The objective of this study is to investigate the in vitro wound healing effects, together with the antioxidant and antimicrobial activities of S. vulgaris leaf and branch extracts.

Material and methods: Ethanol leaf and branch extracts of S. vulgaris were investigated at 1–100?µg/ml concentrations in the scratch assay after 14?h. Antioxidant activity was investigated using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay, and the antibacterial activity was tested at concentrations up to 1000?µg/ml against Gram-positive and Gram-negative bacteria by the microdilution test after 24?h. The total phenolic and flavonoid contents were determined by colorimetric methods.

Results: Struthanthus vulgaris leaf and branch extracts at 100?µg/ml concentration stimulated migration and proliferation of fibroblasts and enhanced cell numbers by 56.2% and 18.6%, respectively. Antioxidant activity exhibited IC₅₀ values of 24.3 and 18.9?µg/ml for the leaf and branch extracts, respectively. The ethanol leaf extract showed antimicrobial activity against the Gram-positive Staphylococcus mutans and Staphylococcus aureus bacteria, exhibiting minimum inhibitory concentration values of 125 and 500?µg/ml, respectively. An appreciable total phenolic content in the leaves (813.6?±?2.7?mg/g) and branches (462.8?±?9.6?mg/g), and relatively low concentration of flavonoids in the leaves (13.3?±?4.3?mg/g) and branches (1.9?±?0.2?mg/g), was detected.

Discussion and conclusion: The antioxidant and antibacterial activities, together with the strong ability to stimulate proliferation and migration of fibroblasts, provide some support for the traditional use of S. vulgaris.     

Nasal PMN response to repeated challenge with endotoxin in healthy volunteers

Rationale: We have employed nasal challenge with lipopolysaccharide (LPS) followed by nasal lavage (NL) to experimentally induce and examine upper airway inflammation in human volunteers. It is unclear however whether adaptation within individuals occurs following repeated nasal challenge. This was a pilot study to determine if repeated nasal LPS challenge yields attenuation of markers of inflammation (primarily neutrophil response) in the NL fluid of healthy humans.

Methods: We employed a 3-day nasal LPS challenge protocol with NL using a “split nose” design. The control and LPS nares received two consecutive day saline (0.9% saline/day) and LPS (2 µg LPS/day) challenges, respectively followed by an LPS (2 µg/day) challenge to each nare on Day 3. NL was performed immediately pre Day 1 challenges and 6-h post nasal LPS challenges on both Days 1 and 3. Markers of inflammation (PMNs/mg, cytokines) were assessed in NL and the inflammatory response to LPS (measured as the difference between pre and post challenge) was evaluated in both nares on Day 3 and compared to Day 1.

Results: Significant (p?<?0.05) blunting of the LPS-induced polymorphonuclear leukocyte (PMN) response was observed in the nare that received repeated LPS challenges as compared to the control nare (67.60?±?22.39 vs. 157.8?±?76.04 PMN/mg) and initial LPS challenge on Day 1 (121?±?32 PMN/mg). Decreased soluble CD14 and significantly decreased interleukin-8 were also found in the repeat LPS-treated nare. In the LPS-treated nare, the blunted PMN response on Day 3 correlated well with the observed PMN response on Day1 (r?=?0.58, p?=?0.02).

Conclusions: We show attenuation of PMN response to repeated LPS in the nasal airways in healthy humans. Effect of repeat endotoxin exposure prior to allergen delivery on local airway inflammation in both healthy and atopic subjects can be studied.     

Antimicrobial and antioxidant activities of crude extracts of two Nigerian chewing sticks

In vitro determinations of the antimicrobial and antioxidant activities of ethanol and aqueous extracts of Disthemonanthus benthamianus Baill. (Caesalpiniaceae) and Zanthoxylum zanthoxyloides Lam. (Rutaceae), which are used as chewing sticks in Nigeria, were investigated. The extracts were screened against eight strains of Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, one methicillin-resistant strain of Staphylococcus epidermidis, twelve strains of Pseudomonas aeruginosa, five strains of Candida albicans and four strains of Bacillus cereus. The in vitro antimicrobial assay was performed by using a Mast Multipoint Inoculator based on the principles of an agar dilution technique. The aqueous extracts had no inhibitory effects on any of the tested microorganisms. The ethanol D. benthamianus extract inhibited P. aeruginosa, E. faecalis, and B. cereus with MIC ≤ 2.64?mg mL^?1 and S. aureus and S. epidermidis with MIC ≤ 0.44?mg mL^?1. Z. zanthoxyloides ethanol extract was less effective but noteworthy was the MIC ≤ 2.52?mg mL^?1 against C. albicans and 0.28?mg mL^?1 against some S. aureus strains. D. benthamianus ethanol extract had the best antioxidant activity and Z. zanthoxyloides ethanol extract second best. IC₅₀ for DPPH free radical scavenging of these extracts were 87.76 and 128.28?μg mL^?1/mL, respectively; ascorbic acid equivalents were 2.4 and 9.5?mg mg^?1 and total antioxidant capacities using the FRAP assay were 4068.06?mM Fe⁺⁺ mg^?1 and 624.86?mM Fe⁺⁺ mg^?1, respectively. The ethanol extracts of D. benthamianus and Z. zanthoxyloides showed significant antimicrobial and antioxidant activities which could be beneficial in oral hygiene.     

Antibacterial activity of aqueous and methanol extracts of selected species used in livestock health management

Context: Salvadora persica L. (Salvadoraceae), Colophospermum mopane (J.Kirk ex Benth.) J. Léonard (Leguminosae) and Dichrostachys cinerea (L.) Wight &; Arn. (Leguminosae) crude extracts are used by local farmers against many livestock infections with little or no side effects usually associated with synthetic antimicrobials. However, their efficacy has rarely been tested.

Objective: These plants were tested for potential antibacterial activity against clinical isolates of Staphylococcus aureus ATCC33862 and Escherichia coli ATCC25922. Minimal inhibitory concentrations (MIC) of the crude plant extracts were determined.

Materials and methods: Aqueous and methanol extraction of 100?g each of the bark of C. mopane, roots of D. cinerea and leaves of S. persica was done by placing the samples in 250?mL of either water or methanol. Nutrient broth was used as growth medium for the bacteria, and McFarland standard for bacterial standardization. 2,3,5-Triphenyltetrazoliumchloride (TTC) was the indicator salt. Each of the aqueous and methanol extracts (100?μL) was tested. Gentamycin and ampicillin were the controls.

Results: MIC of aqueous extracts ranged from 1.03–14.6?mg/mL against S. aureus, and from 12.1–34.3?mg/mL against E. coli. Methanol extracts ranged between 5.31 and 9.64?mg/mL against S. aureus, and between 7.86 and 13.6?mg/mL against E. coli. Aqueous and methanol extracts of S. persica were significantly higher (p?C. mopane and D. cinerea.

Discussion and conclusion: Colophospermum mopane, S. persica and D. cinerea exhibited antibacterial activity, with methanol extracts performing better than aqueous extracts, justifying use as ethnoveterinary medicine. Further study to isolate the active components should be pursued.     

Mometasone furoate-loaded cold processed oil-in-water emulsions: in vitro and in vivo studies

Abstract

Over the years, research has focused on strategies to increase benefit/risk ratio of corticoids. However, vehicles intended for topical glucocorticoids delivery with an improved benefit/risk ratio are still on demand. The aim of this work was the in vitro and in vivo characterization of cold processed oil-in-water (o/w) emulsions intended for mometasone furoate (MF) delivery to induce drug targeting to upper skin strata, decreasing adverse effects. Two o/w emulsions, containing 0.1% of MF, were developed differing in the glycol used (2-methyl-2,4-pentanediol – PT and ethoxydiglycol – TC emulsions). In vitro permeation studies revealed that these emulsions are suitable vehicles for the delivery of MF containing ingredients which are responsible for a drastically increased on the permeability coefficients of MF from a theoretical value of 1.18?×?10^?4?cm/h to 5.20?×?10^?4?±?2.05?×?10^?4?cm/h and 6.30?×?10^?4?±?2.94?×?10^?4?cm/h, for PT and TC, respectively. The tape stripping results showed that the amount of drug that reached the viable skin layers was very low (1.99 %) and the amount that remained in the stratum corneum (SC) was 10.61%. The in vivo studies showed that the developed formulations decreased the edema and erythema in mice skin in more that 90%, assuring, at least, the same anti-inflammatory effect compared with the commercial cream. PT placebo demonstrated to contribute to restore the skin barrier by increasing the amount of lipids within the human skin.     

Effect of pramlintide as an adjunct to basal insulin on markers of cardiovascular risk in patients with type 2 diabetes

ABSTRACT

Objective: Intensification of insulin therapy in patients with type 2 diabetes, while improving glycemic control, often leads to an increase in body weight and other markers of cardiovascular risk. The effects of pramlintide as an adjunct to basal insulin titration (without mealtime insulin) on glycemia and cardiovascular risk markers were examined.

Research design and methods: This was a post hoc analysis of a 16-week, double-blind, placebo-controlled study in patients with type 2 diabetes (N?=?211) using insulin glargine (without mealtime insulin)?±?oral agents. Patients were randomized to treatment with placebo or pramlintide (60 or 120?µg with major meals), and insulin glargine was titrated to target a fasting plasma glucose concentration of ≥70 to <100?mg/dL.

Main outcome measures: Endpoints included the change from baseline to Week 16 in body weight, high sensitivity C-reactive protein (hsCRP), triglycerides, HDL, LDL, and blood pressure.

Results: Pramlintide-treated patients lost weight and placebo-treated patients gained weight during 16 weeks of treatment (?1.6?±?0.3?kg vs. +0.7?±?0.3?kg, p?<?0.001; mean?±?SE). hsCRP was reduced in pramlintide-treated versus placebo-treated patients (?0.8?±?0.2?mg/L vs. 0.1?±?0.2?mg/L, p?<?0.01; mean?±?SE). Patients with baseline hsCRP?>?3?mg/L (high cardiovascular risk) demonstrated greater hsCRP reductions with pramlintide versus placebo treatment at Week 16 (p?<?0.05). Patients with baseline triglycerides ≥150?mg/dL or ≥200?mg/dL (high cardiovascular risk) showed significant reductions from baseline in triglyceride concentrations with pramlintide (?43?±?14?mg/dL or ?59?±?19?mg/dL; p?<?0.05; mean?±?SE) but not with placebo (1?±?29?mg/dLor ?3?±?54?mg/dL; mean?±?SE). No significant differences between pramlintide and placebo were observed for changes in HDL, LDL, or blood pressure. Pramlintide treatment was generally well tolerated. The most frequent adverse event related to pramlintide was mild-to-moderate nausea (31% pramlintide vs. 10% placebo). Pramlintide added to basal insulin did not increase the incidence of hypoglycemia. A limitation of the study was its relatively short duration.

Conclusions: Pramlintide, as an adjunct to basal insulin, was associated with improvements in several cardiovascular risk markers, warranting long-term clinical studies to determine its potential effects on cardiovascular risk.     

Chemical composition,antioxidant and antibacterial activities of two Spondias species from Northeastern Brazil

Context: The leaves of Spondias tuberosa Arr. Cam. (Anacardiaceae) and Spondias mombin L. have been traditionally used for medicinal purposes. Some studies reveal their antibacterial, antimicrobial, and antiviral properties.

Objective: Determine the chemical composition, antioxidant, and antimicrobial activities of Spondias species to justify its ethnopharmacological use.

Materials and methods: Spondias species extracts were prepared with methanol:water 80:20 and analyzed by silica gel column chromatography and reversed phase liquid chromatography (HPLC). The antioxidant activity was evaluated by scavenging the radicals 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS?+) and measuring antimicrobial activity (agar well diffusion method, minimum inhibitory concentration and minimum bactericidal concentrations).

Results: The HPLC analysis of Spondias extracts demonstrated the occurrence of high yield of flavonoids. Found in S. mombin were quercetin (2.36?±?0.01?mg/g) and ellagic acid (41.56?±?0.01?mg/g) and in S. tuberosa species rutin (53.38?±?1.71?mg/g), quercetin (24.46?±?0.87?mg/g), and ellagic acid (169.76?±?0.17?mg/g). The antibacterial activity of the extracts against the various bacteria strains varied from 8.8 to 20.1?mm. MIC values from 62.5 to 125 µg/mL were satisfactory when compared with other plant products. Medium DPPH scavenging activity IC₅₀ for Spondias extracts varied from 0.042 to 0.558?mg/mL and for ABTS from 0.089 to 0.465?mg/mL. DPPH scavenging activity for constituent ellagic acid IC₅₀?=?0.042?mg/mL and for quercetin IC₅₀?=?0.081?mg/mL.

Discussion and conclusion: The chemical study of Spondias leaf extracts showed the occurrence of quercetin, rutin and ellagic acid, substances with relevant antioxidant and antimicrobial activities.     

Chemical composition and evaluation of modulatory of the antibiotic activity from extract and essential oil of Myracrodruon urundeuva

Context: The combination of antibiotics with natural products has demonstrated promising synergistic effects in several therapeutic studies.

Objective: The aim of this study was to determine the effect of a combination of an ethanol extract of Myracrodruon urundeuva Fr. All. (Anacardiaceae) (aroeira plant) and its essential oil with six antimicrobial drugs against multiresistant strains of Staphylococcus aureus and Escherichia coli from clinical isolates.

Materials and methods: After identification of the chemical components by GC-MS, the antibacterial activity of the natural products and antibiotics was assessed by determining the minimal inhibitory concentration (MIC) using the microdilution method and concentrations ranging 8–512?μg/mL and 0.0012–2.5?mg/mL, respectively. Assays were performed to test for a possible synergistic action between the plant products and the antimicrobials, using the extract and the oil at a sub-inhibitory concentration (128?μg/mL) and antibiotic at concentrations varying between 8 and 512?μg/mL.

Results: The GC-MS analysis identified the main compound as δ-carene (80.41%). The MIC of the natural products was >1024?μg/mL, except against S. aureus ATCC25923. Only the combinations of the natural products with gentamicin, amikacin and clindamycin were effective against S. aureus 358, enhancing the antibiotic activity by reducing the MIC.

Conclusions: The extract from aroeira showed a higher antibacterial activity and the oil was more effective in potentiating the activity of conventional antibiotics.     

Superantigens Modulate Bacterial Density during Staphylococcus aureus Nasal Colonization

Superantigens (SAgs) are potent microbial toxins that function to activate large numbers of T cells in a T cell receptor (TCR) Vβ-specific manner, resulting in excessive immune system activation. Staphylococcus aureus possesses a large repertoire of distinct SAgs, and in the context of host-pathogen interactions, staphylococcal SAg research has focused primarily on the role of these toxins in severe and invasive diseases. However, the contribution of SAgs to colonization by S. aureus remains unclear. We developed a two-week nasal colonization model using SAg-sensitive transgenic mice expressing HLA-DR4, and evaluated the role of SAgs using two well-studied stains of S. aureus. S. aureus Newman produces relatively low levels of staphylococcal enterotoxin A (SEA), and although we did not detect significant TCR-Vβ specific changes during wild-type S. aureus Newman colonization, S. aureus Newman Δsea established transiently higher bacterial loads in the nose. S. aureus COL produces relatively high levels of staphylococcal enterotoxin B (SEB), and colonization with wild-type S. aureus COL resulted in clear Vβ8-specific T cell skewing responses. S. aureus COL Δseb established consistently higher bacterial loads in the nose. These data suggest that staphylococcal SAgs may be involved in regulating bacterial densities during nasal colonization.     

Treatment of Helicobacter pylori infected mice with Bryophyllum pinnatum,a medicinal plant with antioxidant and antimicrobial properties,reduces bacterial load

Context: Bryophyllum pinnatum (Lam.) Kurz (Crassulaceae) is a plant known for its antiulcer properties.

Objective: This study evaluates the anti-Helicobacter pylori activity of Bryophyllum pinnutum methanol extract with a mouse model and its antioxidant properties.

Materials and methods: Dried leaves of Bryophyllum pinnutum were extracted with methanol and ethyl acetate. Broth microdilution method was used to evaluate the anti-Helicobacter activity of extract samples in vitro. Swiss mice were inoculated with a suspension of Helicobacter pylori and divided into control group and four others that received 125, 250, 500?mg/kg of methanol extract or ciprofloxacin (500?mg/kg), respectively, for 7 days. Helicobacter pylori colonization and bacterial load of mouse stomach was assessed on day 1 and 7 post-treatment. The antioxidant activity of Bryophyllum pinnutum was evaluated through DPPH radical, hydroxyl radical and reducing power assay.

Results: Methanol extract showed a significant anti-Helicobacter activity with MIC and MBC values of 32 and 256?μg/mL, respectively. Bryophyllum pinnatum and ciprofloxacin reduced H. pylori colonization of gastric tissue from 100% to 17%. Bryophyllum pinnatum extract (85.91?±?52.91 CFU) and standard (25.74?±?16.15 CFU) also reduced significantly (p?50 values of 25.31?±?0.34, 55.94?±?0.68 and 11.18?±?0.74?μg/mL, respectively.

Discussion and conclusion: The data suggest that the methanol extract of Bryophyllum pinnatum could inhibit Helicobacter pylori growth, and may also acts as an antioxidant to protect gastric mucosa against reactive oxygen species.     

Calculation of AQCmax: comparison of five ophthalmic fluoroquinolone solutions

ABSTRACT

Objective: Ocular tissue penetration of five different ophthalmic fluoroquinolone solutions in the rabbit eye was measured and evaluated by an index of the maximum aqueous concentration (AQCmax).

Methods: Moxifloxacin 0.5% (MFLX), levofloxacin 0.5% (LVFX), gatifloxacin 0.3% (GFLX), ofloxacin 0.3% (OFLX), or tosufloxacin tosilate 0.3% (TFLX) were instilled into the eyes of white rabbits every 15?min for a total of three doses. Aqueous humor, cornea, iris/ciliary body and vitreous body were collected 10 to 240?min after instillation and drug concentrations were measured by high-performance liquid chromatography.

Results: The concentration of MFLX was the highest in each tissue, with maximum concentrations of MFLX in the aqueous humor (10.16?±?1.59?µg/mL) at 30?min after instillation, cornea (156.07?±?95.97?µg/g) and iris/ciliary body (11.92?±?4.00?µg/g) at 10?min after instillation, and vitreous body (0.099?±?0.033?µg/mL) at 30?min after instillation. The concentration of TFLX was the lowest in each tissue, with LVFX, GFLX, and OFLX sharing the mid-ranks. AQCmax?:?MIC₉₀ ratio for S.?aureus was 150.67 for MFLX, 10.6 for LVFX, 9.69 for GFLX, 3.48 for OFLX, and could not be determined for TFLX.

Conclusion: AQCmax is a useful pharmacokinetic parameter for determining the therapeutic efficacy of an ophthalmic antibiotic, especially when combined with MIC₉₀ values for intraocular pathogens. C_max of MFLX ophthalmic solution was superior in all tissues (cornea, aqueous humor, iris/ciliary body and vitreous body) among the five ophthalmic solutions studied, exceeding the MIC₉₀ of S.?aureus in all tissues, and MIC₉₀s of S.?epidermidis, B.?cereus, and P.?acnes in aqueous humor, cornea, and iris/ciliary body. AQCmax was approximately proportional to C_max in iris/ciliary body and vitreous, and may be used in combination with MIC₉₀s as an index to predict the most appropriate dose and frequency of ophthalmic antibiotics in conjunction with other PK/PD parameters. This study may provide the groundwork for calculation of AQCmax in humans.     

Nanoparticle-based delivery enhances anti-inflammatory effect of low molecular weight heparin in experimental ulcerative colitis

Epithelial administration of low molecular weight heparin (LMWH) has proven its therapeutic efficiency in ulcerative colitis (UC) but still lacks of a sufficiently selective drug delivery system. Polymeric nanoparticles were used here not only to protect LMWH from intestinal degradation but also to provide targeted delivery to inflamed tissue in experimental colitis mice. LMWH was associated with polymethacrylate nanoparticles (NP) type A (PEMT-A) or type B (PEMT-B) of a size: 150?nm resulting in a maximum drug loading: 0.1?mg/mg. In a lipopolysaccharide-stimulated macrophages both, free LMWH and LMWH-NP have significantly reduced the cytokines secretion independently from cellular uptake. The in-vivo therapeutic efficiency was dose dependent as at low doses (100?IU/kg) only minor differences between free LMWH and LMWH-NP were found and the superiority of LMWH-NP became prominent with dose increase (500?IU/kg). Administration of LMWH-NP at 500?IU/kg has markedly improved the clinical activity as compared to LMWH while similarly pathophysiological indicators revealed increased therapeutic outcome in presence of NP compared to LMWH alone: Myeloperoxidase (Colitis control: 10 480?±?5335, LMWH-PEMT-A NP: 1507?±?2165, LMWH-PEMT-B NP: 382?±?143, LMWH: 8549?±?5021 units/g) and tumor necrosis factor: (Colitis control: 1636?±?544, LMWH-PEMT-A NP: 511?±?506, LMWH-PEMT-B NP: 435?±?473, LMWH: 1110?±?309?pg/g). Associating LMWH with NP is improving the anti-inflammatory efficiency of LMWH in-vivo by its protection against degradation in luminal environment and selective drug delivery. Such a combination holds promise for a highly specific therapy by its double selectivity towards the inflamed intestinal tissue. LMWH-PEMT NP have significantly improved the clinical activity in-vivo in comparison to free LMWH.     

Research on the comparison of the demethylvancomycin’s diffusion–deposition characteristics in the ocular solid tissues of sustained subtenon drug delivery with subconjunctival injection

Purpose: To compare the demethylvancomycin’s diffusion–deposition characteristics in the ocular solid tissues of sustained subtenon drug delivery with subconjunctival injection.

Method: Sixty adult white rabbits were randomly assigned to the subtenon drug delivery group and the subconjunctival injection group. The subtenon drug delivery group was continuously infused demethylvancomycin to the subtenon of rabbits. The subconjunctival injection group was injected demethylvancomycin to the subconjunctival of rabbits. Cornea, iris and sclera were collected for high-performance liquid chromatography analyses to determine drug concentrations at one hour, three hours, six hours, 12?h and 24?h of drug administration. WinNonlin 6.3 was used to calculate the parameters of cumulative area under the curve (AUC_cum) of demethylvancomycin.

Results: The peak levels of demethylvancomycin concentration of the subtenon drug delivery group and the subconjunctival injection group were 92.406?±?21.555 and 51.778?±?14.001?μg/g in cornea, 28.451?±?10.229?μg/g and 42.271?±?27.291?μg/g in iris, 153.166?±?51.738?μg/g and 57.423?±?18.480?μg/g in sclera. The differences of concentrations between the two groups in cornea and sclera were statistically significant (F?=?487.775, p?F?=?132.748, p?F?=?4.848, p?=?0.064). The maximum of AUC_cum of the subtenon drug delivery group and the subconjunctival injection group was 1808.23?h?*?μg/g and 273.73?h?*?μg/g in cornea, 489.12?h?*?μg/g and 216.16?h?*?μg/g in iris and 2166.34?h?*?μg/g and 392.57?h?*?μg/g in sclera at 24?h of drug administration.

Conclusion: The sustained subtenon drug delivery had a better drug permeability and accumulation in the intraocular solid tissue compared to subconjunctival injection, which demonstrated it was probably a promising and effective approach for treating posterior segment diseases and endophthalmitis.     

Transdermal iontophoretic delivery of selegiline hydrochloride,in vitro

Transdermal iontophoretic delivery of selegiline hydrochloride (SH) across dermatomed human skin was studied. Electrochemical stability and various factors affecting the skin permeation were investigated. SH was stable under the influence of an electrical field. The permeation of SH was very low by passive delivery (2.29?±?0.05 μg/cm²/h) as compared to iontophoresis at 0.5 mA/cm² (65.10?±?5.04 μg/cm²/h). An increase in drug concentration from 1 to 20?mg/mL increased the iontophoretic flux by 13-fold. Optimal pH and salt (NaCl) concentration for iontophoretic delivery of SH were found to be pH 5 and 100?mM, respectively. Overall, with 20?mg/mL SH and a current density of 0.4 mA/cm², a maximum flux of 305.5?μg/cm²/h was obtained. Based on reported pharmacokinetic parameters, input target delivery rate to achieve effective plasma concentration of SH (2.2?ng/mL) was calculated. With a surface area of 40?cm², iontophoretic delivery can provide six to seven times higher levels of SH than the target delivery rate, which enables lowering of the dose and/or patch surface area. Further in vivo studies will be required to prove the efficacy of ionophoresis for enhanced delivery of SH.     

Note: A new triterpene from Luculia pinciana Hook.

A new triterpene named luculiaoic acid A (1), showing inhibitory activity of a leukaemia cell line, along with eleven known compounds, has been isolated from the ethyl acetate extract of the stems of Luculia pinciana Hook. All the structures were elucidated on the basis of NMR, MS, and IR methods. The activity to inhibit Staphylococcus aureus and Candida albicans of all compounds showed that ursolic acid inhibits the growth of Staphylococcus aureus with an MIC of 0.5?mg?ml^?1 and an MBC of 10?mg?ml^?1, and scopletin inhibits Candida albicans with an MIC of 1?mg?ml^?1 and an MBC of 5?mg?ml^?1.     

Antimicrobial,modulatory and chemical analysis of the oil of Croton limae

Context: Croton sp. are plants with a well-reported antimicrobial activity. Croton limae A.P. Gomes, M.F. Sales P.E. Berry (Euphorbiaceae), known as ‘marmeleiro-prateado’, is commonly used to manage abdominal pain in Brazil.

Objective: This work evaluates the phytochemical composition, antimicrobial and modulatory activities of the essential oil of C. limae leaves (EOCL).

Materials and methods: The minimum inhibitory concentration (MIC) and the modulation of the antibiotic activity were determined using a microdilution method. The concentration of EOCL ranged between 512 and 8?μg/mL. Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Candida tropicalis, C. krusei and C. albicans strains were used in the MIC and modulation assays. The antibiotics, amikacin, gentamicin and neomycin, and the antifungals, amphotericin B, benzoylmetronidazole and nystatin, were used in concentrations ranging between 2500 and 2.5?μg/mL. The phytochemical analysis of the EOCL was performed through gas chromatography coupled to a mass spectrometer (GC/MS).

Results: Only Staphylococcus aureus was inhibited by a clinically relevant concentration of EOCL (MIC 512?μg/mL). Synergism between the EOCL and amikacin against S. aureus (9.76?μg/mL) and E. coli (39.062?μg/mL); neomycin against E. coli (2.44?μg/mL); and benzoylmetronidazole against C. krusei (256?μg/mL) were observed. The GC/MS analysis identified cedrol, eucalyptol and α-pinene as the main compounds of EOCL.

Conclusion: EOCL inhibited the growth of S. aureus and potentiated the antibiotic and antifungal effects of drugs against all bacterial and Candida strains, respectively.