Moxifloxacin-Loaded Nanoemulsions Having Tocopheryl Succinate as

Article pubs.acs.org/molecularpharmaceutics

Moxifloxacin-Loaded Nanoemulsions Having Tocopheryl Succinate as the Integral Component Improves Pharmacokinetics and Enhances Survival in E. coli-Induced Complicated Intra-Abdominal Infection Prashant Shukla,† Ajeet Kumar Verma,‡ Pankaj Dwivedi,† Arti Yadav,‡ Pramod Kumar Gupta,† Srikanta Kumar Rath,‡ and Prabhat Ranjan Mishra*,† †

Pharmaceutics Division and ‡Toxicology Division, CSIR-Central Drug Research Institute (Council of Scientific and Industrial Research), B 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow, UP 226031, India ABSTRACT: In the present work, a novel nanoemulsion laden with moxifloxacin has been developed for effective management of complicated intra-abdominal infections. Moxifloxacin nanoemulsion fabricated using high pressure homogenization was evaluated for various pharmaceutical parameters, pharmacokinetics (PK) and pharmacodynamics (PD) in rats with E. coli-induced peritonitis and sepsis. The developed nanoemulsion MONe6 (size 168 ± 28 nm and zeta potential (ZP) 24.78 ± 0.45 mV, respectively) was effective for intracellular delivery and sustaining the release of MOX. MONe6 demonstrated improved plasma (AUCMONe6/MOX = 2.38-fold) and tissue pharmacokinetics of MOX (AUCMONe6/MOX = 2.63 and 1.47 times in lung and liver, respectively). Calculated PK/PD index correlated well with a reduction in bacterial burden in plasma as well as tissues. Enhanced survival on treatment with MONe6 (65.44%) and as compared to the control group (8.22%) was a result of reduction in lipid peroxidation, neutrophil migration, and cytokine levels (TNF-α and IL6) as compared to untreated groups in the rat model of E. coli-induced sepsis. Parenteral nanoemulsions of MOX hold a promising advantage in the therapy of E. coli-induced complicated intra-abdominal infections and is helpful in the prevention of further complications like septic shock and death. KEYWORDS: complicated intra-abdominal infections, cytokines, moxifloxacin, sepsis, lipopolysaccharide, myeloperoxidase

1. INTRODUCTION

Escherichia coli remains one of the most common pathogens in cIAI infections.6 Moxifloxacin (MOX) is a broad-spectrum fluoroquinolone that has recently been positioned as first-line agent in the treatment of cIAI.7 MOX has a better therapeutic viability as it has various advantages over older fluoroquinolones such as ciprofloxacin (not a first line treatment in therapy of cIAIs) or beta-lactams. This is due to facts that MOX is having a wider antimicrobial spectrum (including Gram-positive bacteria such as streptococci and staphylococci as well as atypical pathogens, more specifically anaerobes).8 A reduced prevalence of emergence of bacterial resistance among Gram-positive pathogens in in vitro studies was observed with moxifloxacin as compared with other fluoroquinolones tested specifically in treatment of cIAIs.9−11 MOX is a rapid bactericidal agent and also has anti-inflammatory effects that might exert additional beneficial effects in hyperinflammatory conditions associated

Complicated intra-abdominal infections (cIAI) represent an intricate clinical challenge, as they differ from other types of infections. The clinical spectrum of cIAI is very broad, ranging from uncomplicated acute appendicitis to generalized peritonitis caused by a perforated ischemic bowel. The microbial etiology and diagnosis pose another challenge in treatment of cIAIs.1 With emergence of multidrug resistance, the treatment of cIAIs has become more grueling.2 Critically ill patients also have more pronounced risk profiles, as cIAI is more often associated with acute kidney injury and sepsis often leading to severe inflammatory reactions and ultimately death due to lipopolysaccharide (LPS) released by antibiotic therapy as well as over burden of infection in case of nontreated patients.3 So peritonitis and the accompanying systemic inflammatory response syndrome are important causes of death in adult intensive care units.4 The mortality of patients with peritonitis accompanied abdominal sepsis is high (up to 60%), as compared to the overall mortality (25−30%) of patients with infection-associated sepsis.5 Although different bacteria have been identified as causative organisms in abdominal sepsis, © XXXX American Chemical Society

Received: May 20, 2014 Revised: October 2, 2014 Accepted: October 15, 2014

A

dx.doi.org/10.1021/mp5003762 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Molecular Pharmaceutics

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with cIAIs.12−14 Recently it has been demonstrated that MOX was equally effective as monotherapy as compared to other antibiotic treatment options in cIAIs.15 MOX has also shown better outcomes in cIAIs due to its better tissue penetration and accumulation in peritoneal fluid as compared to other treatments such as β-lactam/lactamase inhibitors, cephalosporin, and carbapenems. MOX has demonstrated antimicrobial activity against the majority of organisms involved in cIAIs16 as the molecular target for MOX is DNA gyrase. LPS has been widely reported as a very important inflammatory mediator in development of hyperinflammatory conditions associated with cIAIs,17−20 and the conditions affecting levels of circulating LPS in plasma are usually governed by two factors, initial bacterial burden and type of antimicrobial therapy employed for treatment of cIAIs. Antibiotics used to treat cIAI may also play a role in the pathophysiological process of sepsis, mainly through their ability to liberate LPS bacterial cell wall during destruction of the microorganism. Apparent failure of these different treatment options with antibiotics having different LPS liberating potential is due to the negligence of the fact that LPS liberating potential of these antibiotics may play an important role in progression of sepsis.21 MOX has been reported to release less LPS from E. coli as compared to other antibiotics specially beta lactams22 thereby reducing the probability of fatal outcomes like sepsis as compared to carbapenems and penicillin group of antibiotics.23 Continuing improvement in the pharmacological and therapeutic properties of drugs is driving the revolution in novel drug delivery systems. In fact, a wide spectrum of therapeutic nanocarriers has been extensively investigated to address this emerging need.24,25 The design of nanoengineered drug delivery systems to improve the targeting of antibiotics as well as to reduce the undesired outcomes such as organ damage is needed in therapy of peritonitis as well as its progression to sepsis. These systems should also retain and release the drug over specific durations at a therapeutic level so as to maximize therapeutic outcomes. Drug laden nanoemulsions are gaining importance as a drug carrier due to the fact that parenteral emulsions are very popular as total parenteral nutrition in critical care units. Simultaneously parenteral emulsions can also enhance solublization and stability of incorporated drug to obtain sustained release and targeting.26,27 Experimentally parenteral nanoemulsions have demonstrated utility in redirecting LPS to liver28 and therefore enhancing their removal by hepatocytes.29 Anionic derivatives of tocopherol have been employed as lipid phase in various formulations to enhance the oil solubility of water-soluble drugs via ionic interactions.30,31 Tocopheryl succinate has also been reported to be beneficial in the condition of sepsis arising due to complication of intraabdominal infections.32−34 The present work has been designed for development of a nanoemulsion for the delivery of MOX utilizing tocopherol succinate in order to improve its pharmacokinetics, tissue distribution in organs of interest, i.e., lung, liver, and spleen, as well as its efficacy in reducing bacterial burden, oxidative damage, and cytokine levels in the rat model of E. coli-induced peritonitis as a model of complicated intra-abdominal infection.

(MTT reagent), soya phosphotidylcholine (soya PC; the major phospholipids as assayed by TLC (provided by manufacturer) are typically an average of 55% (42−63%) phosphotidylcholine and 20% (10−32%) phosphatidylethanolamine), Pluronic F-68, Tocopheryl succinate (TS), cell culture media, and other routine chemicals were purchased from Sigma Chemicals (St. Louis, MO). All other chemicals and solvents were of reagent grade. 2.2. HPLC Method for Analysis of MOX. The amount of MOX was estimated by HPLC equipped with LC 10 ATVP isocratic pumps (Shimadzu), Rheodyne (Cotati, CA, USA) model 7125 injector with a 20 μL loop and RF-10A XL fluorescence detector (Shimadzu). HPLC separation was achieved on a Waters Nova Pak C18 (5 μm) column of length 150 mm. Data was acquired and processed using Shimadzu LC Solution software. Column effluent was monitored at λex 290 and λem 460 nm using mobile phase of acetonitrile (ACN)/50 mM phosphate buffer pH 2.6 (20:80 v/v) at a flow rate of 1.5 mL/min. 2.3. Formulation Development. 2.3.1. Preparation of MOX Base. MOX as HCl salt is poorly soluble in oils; therefore, to increase the lipophilicity, MOX was converted into the base form by using a reported method with slight modifications.35,36 A solution of MOX HCl was made in distilled water. The pH of the solution was adjusted to 12.0 with NaOH 30% and later to pH = 8.0−8.2 with HCl 35% v/v and is extracted with methylene chloride (3 × 125 mL). The combined organic extracts are dried over anhydrous MgSO4 and are concentrated to dryness, obtaining 6.72 g of base MOX (yield: 89.3%). 2.3.2. Solubility of MOX Base in Lipid Phase. To find appropriate oils that have good solubilizing capacity of MOX base and thus can be used as the oil phase in nanoemulsion, the solubility of MOX base in various oils was measured. Oils employed were vegetable oils (soybean oil, corn oil, sesame oil, and 1% tocopheryl succinate in soybean oil). An excess amount of MOX base was added to oil (10 mL) and shaken reciprocally at 50 °C for 6 h to aid dissolution and kept at 25 °C to allow the precipitation of excess drug. The suspension was filtered through 0.45 μm membrane filter (Nylon Minisart, Sartorius stedim biotech GmbH, Germany), and the drug concentration in the filtrate was determined using the HPLC method described above after the appropriate dilution with mobile phase. 2.3.3. Preparation of Nanoemulsions. Nanoemulsions of MOX were prepared by using a high pressure homogenizer (HPH) (MP110 Micro fluidics, Newton, Massachusetts USA) in a batch size of 50 mL. Briefly, different quantities of lipidic phase (1% TS in soybean oil) containing the drug was emulsified by using different ratios of surfactants, i.e., pluronic F 68 and soya PC using phosphate buffer saline pH 7.4 emulsified into a coarse emulsion by using high speed homogenizer (Ultra Turrax T25) at 5000 rpm for 5 min. The obtained coarse emulsion was processed by initial premilling at 50 bar for five cycles then homogenized by using HPH at 500 bar for 20 cycles. The samples were analyzed after 5, 10, 15, and 20 cycles, respectively, to monitor changes in size and zeta potential. 2.4. Characterization of Nanoemulsions. 2.4.1. Particle Size, Zeta Potential, and Surface Characterization of Nanoemulsion. The developed nanoemulsion formulations were characterized for particle size, size distribution, and zeta potential using Zetasizer Nano-ZS (Malvern Instruments, U.K.). The particle size was analyzed using the dynamic light

2. MATERIALS AND METHODS 2.1. Materials and Reagents. MOX was kindly provided by MSN pharmachem Pvt. Ltd. (Madek, Andra Pradesh India). 3-[4,5-Dimethylthiazolyl]-2,5-diphenyltetrazolium bromide B



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scattering technique at 173° angle backscattering at 25 °C temperature; the samples were suitably diluted with deionized distilled water before analysis. The numbered average oil droplet’s hydrodynamic diameter and the polydispersity index were determined. Zeta potential was measured by Laser Doppler Anemometry, by diluting nanoemulsion suitably with deionized distilled water and placing it in the electrophoretic cell, and the average surface charge was determined. The morphology of nanoemulsion was observed under scanning electron microscopy (Tecnai-F 30, Oregon, USA) after negative staining. Briefly, nanoemulsion was diluted with water and adsorbed onto a copper grid and air-dried for 1 min at room temperature after removing the excessive sample with filter paper. A drop of 2% phosphotungstic acid (w/v, pH 6.5 in distilled water) was then added, and the nanocapsules were stained for 30 s. At the end, the sample was air-dried for another 5 min at room temperature before TEM observation. 2.4.2. Drug Content in Nanoemulsion. Drug content in nanoemulsion was determined by diluting the emulsion with ACN. Briefly, 200 μL of emulsion was diluted up to 10 mL with ACN, and an aliquot of this solution was suitably diluted with phosphate buffer of pH 2.6 and analyzed by HPLC method as described above. To determine partitioning of drug between oil and aqueous phase of nanoemulsion, the formulations were centrifuged and the drug was estimated in the oil phase after suitable dilution with ACN, using the above-described method. 2.4.3. In Vitro Drug Release Studies. The release of MOX from nanoemulsion was determined by dynamic dialysis method. Drug loaded nanoemulsions (0.5 mL) were taken in a dialysis bag of 12 kDa molecular weight cut off. The bags were suspended in 250 mL of triple distilled water with dissolution medium at 37 ± 1 °C in dissolution apparatus (DISSO 2000, LABINDIA) at 100 rpm to simulate the sink conditions.37 At predetermined time intervals (0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 24 h) aliquots of 1 mL samples were withdrawn and filtered through 0.22 μm filter, and the medium was replaced with fresh dissolution medium. The concentration of MOX was estimated by HPLC method as described above. 2.5. In Vitro Safety Evaluations. 2.5.1. In Vitro Hemolytic Assay. Blood was collected from a healthy rabbit in heparinized tube and centrifuged at 3000 rpm for 5 min, and blood cells in sediment were washed thrice with PBS (pH 7.4) followed by centrifugation and were diluted 10 times with PBS. The formulations were added to RBC suspension and incubated at 37 °C in humidified 5% CO2, 95% air atmosphere for 30 min having prior treatment with formulation. Then, the samples were centrifuged at 5000 rpm for 10 min. Supernatant (50 μL) was added to the PBS (150 μL) in a 96-well plate. Finally, absorbance was checked at 540 nm. Absorbance of samples was recorded using enzyme-linked immunosorbent assay (ELISA) plate reader, Bio-TEK Power Wave XS. Blank PBS and triple distilled water was used as negative and positive control, respectively. Percentage hemolysis was calculated from the formula

DMEM medium (Sigma, USA), and HepG2 in low glucose medium DMEM (Sigma, USA). All were supplemented with 10% fetal bovine serum and 1× antibiotic antimycotic solution (Sigma, USA). Cell cultures were maintained in a humidified 95% O2/5% CO2 atmosphere at 37 °C. For evaluation of in vitro cytotoxicity, cells (THP1, J774A.1 macrophages, HepG2, and HEK) were seeded at density 1 × 103 cells per well in a 96-well cell culture plate and allowed to grow for 24 h. Different dilutions of formulations were made using PBS, while plain PBS was taken as untreated control. Two hundred microliters of diluted sample were added in each well and then culture plates were further incubated for 24 h in a humidified atmosphere of 5% CO2 at 37 °C. Then 10 μL of MTT solution (5 mg/mL MTT in PBS) was added to each well and incubated for 4 h at 37 °C. Growth of cells was assessed by the ability of living cells to reduce the yellow dye MTT to a blue formazan product. After incubation, the MTT reagent was removed before adding 200 μL of DMSO to each well, gently shaken and incubated for 15 min. The optical density (OD) of the formazan solution was measured at 540 nm by ELISA plate reader (Biotek, USA). Each experiment was performed using three replicate wells for each concentration. Cytotoxicity was calculated according to the following equation: cytotoxicity(%) = (abs treatment/abs blank) × 100

2.6. Uptake Studies by Confocal Microscopy. Cells (J774) were seeded on glass coverslips (poly-L-lysine coated) in six-well microplate and incubated with 20 μL Nile red-loaded nanoemulsions (MONe6) diluted with 2 mL of media per well and nucleus were stained with Hoechst 33342 dye. After 6 h of incubation, cells were washed and coverslips were fixed onto glass slides. The fluorescence images of cells were obtained using Confocal Laser Scanning Microscope (Carl Zeiss CLSM 510 META). 2.7. In vivo Evaluations. 2.7.1. Pharmacokinetic Studies and Tissue Distribution Studies. The experimental protocol was approved by the Institutional Animal Ethics Committee (CSIR-Central Drug Research Institute, Lucknow, and study number IAEC 2012/38). Male Wistar rats of approximately 4− 6 weeks of age weighing between 150 and 180 g were taken for the study. The rats had a free access to potable water and standard animal diet. Throughout the study period, room temperature and relative humidity were maintained at 25 °C ± 2 °C and 30% and 70% RH, respectively. Illumination was controlled to give 12 h dark cycles during the 24 h period. Overnight fasted rats were used for the study. Prior to the initiation of the study rats were weighed for the body weights. Twenty-four rats were randomized based on their body weights and distributed equally into four groups. Each group of rats received MOX solution and MOX nanoemulsion. Both the formulations were administered intraperitoneally at the dose equivalent to 10 mg/kg of MOX. Following i.p. administration, approximately 500 μL of blood sample was collected after anesthetizing with ketamine (30 mg/kg) from a group of 3 animals per time point from the respective group at different time intervals, i.e., 0.5, 1, 2, 4, 6, 8, and 24 h postdosing from the retro-orbital plexus in prelabeled eppendorf tubes. In the case of tissue, homogenates (100 μg/mL) were prepared and 200 μL of standard as well as test samples were processed as described for plasma samples. Analysis of blood and tissue samples: A volume (200 μL) of the study sample and calibration curve samples and quality

%hemolysis =

[(abs treatment − negative control) × 100] positive control

2.5.2. Cytotoxicity of Nanoemulsions. Human monocytic cell line THP1 was grown in RPMI-1640 medium (Sigma, USA), murine macrophage cell line J774 in high glucose C



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Table 1. Composition and Characterization of Developed Nanoemulsion Formulations in Terms of Globule Size, Polydispersity Index (PDI), and Zeta Potential

a

batch code

lipidic phasea (%v/v)

PF-68 (% w/v)

phoshotidylcholine (% w/v)

MONe1 MONe2 MONe3 MONe4 MONe5 MONe6

10 10 10 10 20 20

1 1 2 2 1 2

0.5 1 0.5 1 1 1

globule size (nm) (mean ± SD) 364 234 216 148 254 168

± ± ± ± ± ±

55 36 18 26 46 28

PDI (mean ± SD) 0.184 0.168 0.112 0.128 0.128 0.138

± ± ± ± ± ±

0.085 0.108 0.048 0.016 0.086 0.036

zeta potential (mV) (mean ± SD) −26.56 −29.62 −22.24 −27.78 −29.46 −24.67

± ± ± ± ± ±

0.46 0.62 0.52 0.28 0.87 0.45

Lipidic phase consisted of 1% TS in soy bean oil containing 10 mg/mL of MOX.

rat cytokines (R&D system, USA). Cytokine concentrations in plasma have been expressed as pg/mL. 2.7.4.2. Effect on Lipid Peroxidation in Various Tissues on Treatment with MOX Emulsion. Oxygen derived free radicals are believed to be important mediators of cellular injury contributing to the acute-phase response in sepsis. Free radical mediated lipid peroxidation is a indicator of oxidative burden in sepsis.40 Lipid peroxidation level was estimated by using the method reported by Ohkawa et al. with slight modifications.41 Tissue homogenates (10%) in SDS (10% w/v) was taken and incubated with glacial acetic acid (20%) for 2 min. TBA (0.8%) was added, and the reaction mixture was incubated in a boiling water bath for 1 h. After centrifugation at 10 000g for 5 min at 4 °C, the supernatant was collected; absorbance was recorded at 532 nm against the control blank. 2.7.4.3. Leukocyte Myeloperoxidase (MPO) Assay. Neutrophils are the first cells to be activated in the host immune response to infection or injury and are critical cellular effectors in both humoral and innate immunity, central to the pathogenesis of sepsis and multiorgan dysfunction. Myeloperoxidase activity is widely employed to assess neutrophil accumulation and often correlates with tissue injury. In order to access tissue injury, MPO assay was performed briefly; tissues were homogenized in a solution containing 0.5% hexadecyltrimethyl-ammonium bromide dissolved in 10 mM potassium phosphate buffer (pH 7) and centrifuged for 30 min at 20 000g at 4 °C. An aliquot of the supernatant was then allowed to react with a solution of tetra-methylbenzidine (1.6 mM) and 0.1 mM H2O2. The rate of change in absorbance was measured with a spectrophotometer at 650 nm. MPO activity, an index of PMN accumulation, was defined as the quantity of enzyme degrading 1 pmol of H2O2 per min at 37 °C and was expressed in units per gram of wet tissue.42

control samples were transferred to the prelabeled eppendorf tubes. Samples were mixed with 50 μL of 7% perchloric acid, and the contents were vortexed vigorously and centrifuged at 10,000 rpm for 10 min. Twenty microliters of the clear supernatant was directly injected into the HPLC column using the method described in the previous section. 2.7.2. Effect of Formulation on E. coli-Induced Peritonitis in Rats. Wistar rats with 12 animals in each group (no. of groups = 3) in a set were anesthetized with an intramuscular injection of ketamine (30 mg/kg of body weight), and rats received an i.p. inoculum of 1 mL of saline containing 2 × 1010 CFU of E. coli. Immediately after bacterial challenge, animals received treatment comprising MOX solution and MOX nanoemulsion, containing MOX equivalent to 10 mg/kg. The control group received isotonic sodium chloride solution through i.p. route. Animals (n = 3) in each group were sacrificed after 2, 4, and 8 h, MOX concentration in different tissues, i.e., liver, spleen, lung, and kidney were determined along with simultaneous bacterial counts in the blood and tissue homogenate, and the concentration of endotoxin in plasma was estimated at 8 h. Blood samples of the formulation treated and untreated groups from infected rats were obtained prior to sacrificing of animals by aseptic percutaneous transthoracic cardiac puncture in sterile heparinized evacuated blood collection tubes. Plasma was separated by centrifugation collected, frozen, and kept at −70 °C until further analysis. Similarly different tissues were collected in aseptic condition using sterilized equipment after sacrificing animals. 2.7.3. Survival Studies. Survival of rats (n = 12) was observed after bacterial challenge (as described previously) and treatment comprising MOX solution and MOX nanoemulsion, containing MOX equivalent to 10 mg/kg. The control group received isotonic sodium chloride solution through i.p. route, 2 times a day (at 12 h interval) for 7 days. Survival was observed daily for 7 days after injection of E. coli (i.p.), and treatment and deaths were recorded as they occurred. The end point for this experiment was death. Survivors were euthanized at the end of the experimental period. 2.7.4. Estimation of Pharmacodynamic Parameters. 2.7.4.1. Release of Free LPS and Cytokines TNF-α and IL-6 in Plasma. In severe cases E. coli-induced peritonitis may lead to bacteremia and septic shock due to release of endotoxins (LPS) in the circulation. To evaluate the possibility of septic shock, the plasma levels of LPS were determined at different time intervals. Plasma endotoxin (LPS) concentrations were measured using commercially available LAL test (E-TOXATE; Sigma-Aldrich). The endotoxin content was determined as described previously.38 Plasma TNF-α levels were determined by ELISA as described previously,39 using commercial kits that are selective

3. RESULTS 3.1. Solubility of MOX Base in Different Oils. Solubility studies indicated that MOX base was soluble in highest amounts in 1% w/w tocopheryl succinate in soy bean oil (10.28 ± 0.46 mg/mL) after slight heating to aid dissolution as compared to oils like corn oil (1.66 ± 0.62 mg/mL), sesame oil (1.08 ± 0.64 mg/mL), and soybean oil (1.26 ± 0.45 mg/mL). 3.2. Nanoemulsion Formulation and Characterization. 3.2.1. Size, Zeta Potential, and Drug Content. In a series of initial experiments, the optimal composition of nanoemulsion was evaluated with respect to oil and emulsifier concentration, ratio of emulsifiers, droplet size, and drug loading. MOX containing nanoemulsion formulations was prepared using HPH (Microfluidics 110P). The processing conditions were optimized to achieve globule size less than 400 nm. Homogenization of nanoemulsion at 500 mPa for 20 cycles D



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Figure 1. (a) In vitro release studies of nanoemulsion formulations. (b) Transmission electron micrograph of optimized nanoemulsion MONe6. (c) In vitro safety assessment of the formulation on THP1, HepG2, and J774 cell lines. (d) In vitro uptake studies by confocal microscopy in J774 MΦ of Nile red-loaded optimized formulation (MONe6).

triple distilled water as well as PBS was observed in our experimental conditions. The solubility of MOX free base was found to be 4.18 ± 0.56 and 4.48 ± 0.32 mg/mL in water. The mean cumulative % MOX released versus time plot for nanoemulsion and drug solution inside the dialysis bag has been presented in Figure1a. As can be observed, nanoemulsions were able to control release of MOX (38% to 55% at 24 h). The release kinetics of MOX from the nanoemulsions is slower and hence release is prolonged compared to that of the free drug (immediate release). During the first 4 h (initial burstrelease phase), the amount of MOX released from the emulsions was in the order of MONe6 < MONe5 < MONe4. The highest burst release (38.82 ± 2.26%) was observed with MONe4 due to a less rigid interfacial film in the case of MONe4 as compared to other formulations. The release of drug entrapped in the oily core as well as at the interface was hindered by the presence of phospholipid and a layer of polymeric surfactant PF-68 acting as a barrier in drug release. The in vitro release pattern may not mimic actual in vivo release behavior due to the factors such as phagocytosis of nanoemulsion droplets. 3.2.3. TEM Analysis. TEM analysis of optimized nanoemulsion (MONe6) revealed a spherical globule size of emulsion, and a dense region on the interphase indicated the presence of a tough film comprising PF-68 and PC. The size of nanoemulsion droplets is in accordance to the data obtained previously (Figure 1b). 3.2.4. Stability Studies. Stability studies of developed formulations were conducted in terms of pH, zeta potential, drug content, and globule size at 37 °C. Reduction in pH values was observed for all formulations with a variation range of 7.1 to 7.4 after 3 months. Increase in zeta potential was observed after storage for three months, but the changes were not significant. Changes in pH and zeta potential can be attributed

was found to be optimum to achieve monodisperse nanoemulsion. The main parameter affecting the size was found to be number of homogenization cycles and concentrations of PF-68 and PC. In the case of MONe4 having 2 and 0.5% (w/v) of PF68 and PC, respectively, globule size was reduced to 148 ± 36 nm as compared to MONe1 (364 ± 55 nm) containing 1 and 0.5% of PF-68 and PC, respectively. However, as the oil content was increased from 10 to 20% v/v, globule size was increased from 234 ± 36 nm (MONe2) to 254 ± 46 nm (MONe5) when nanoemulsion was prepared containing 1% w/ v each of PF-68 and PC. A similar phenomenon was observed with MONe4 (148 ± 36 nm) and MONe6 (168 ± 28 nm) nanoemulsion formulations (Table 1). Zeta potential of all batches of nanoemulsion formed by HPH was found to be in the range of −20 to −30 mV. Zeta potential was decreased when the PF-68/PC ratio was increased from 1:1 to 2:1 at both 10% (MONe2 and MONe4; −29.62 ± 0.62 and −27.78 ± 0.28 mV, respectively) and 20% oil phase (MONe5 and MONe6; −29.46 ± 0.87 and −24.78 ± 0.45 mV, respectively). Increase in oil/water phase ratio resulted in minimal change in zeta potential as zeta potential is mainly dependent upon the type of emulsifier present on the surface of the emulsion. Total amount of MOX present in the emulsion MONe6 was found to be 1.9834 mg/mL (99.17%). MOX encapsulated in oil globule was found to be 1.3844 mg/mL of emulsion (69.80%). 3.2.2. In Vitro Release Profile. In vitro dissolution studies of most of the fluoroquinolones has been widely reported in triple distilled water as official methods described in pharmacopoeias. This may be due to the fact that most of the fluoroquinolones have isoelectric points near to pH 7.4 and may lead to erroneous conclusions in PBS, and therefore, it was avoided. Moreover, no significant difference in solubility of MOX base in E



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Table 2. Stability of Various Nanoemulsion Formulations in Terms of pH, Globule Size, Drug Content, and Zeta Potential (at 25 °C) after Storage up to 3 Months batch

time

pH value

globule size (nm)

MONe1

initial 30 days 90 days initial 30 days 90 days initial 30 days 90 days initial 30 days 90 days initial 30 days 90 days initial 30 days 90 days

7.32 7.24 7.18 7.28 7.18 7.12 7.24 7.21 7.16 7.36 7.32 7.28 7.42 7.4 7.38 7.38 7.34 7.32

364.5 ± 55.4 386.1 ± 61.2 450.6 ± 76.3 234.7 ± 36.3 258.3 ± 45.4 286.8 ± 65.7 216.8 ± 18.6 236.6 ± 38.3 248.4 ± 46.5 148.1 ± 26.3 152.3 ± 30.2 156.4 ± 36.9 254.6 ± 46.2 258.7 ± 54.6 261.5 ± 58.8 168.8 ± 28.4 170.7 ± 32.4 175 ± 49.8

MONe2

MONe3

MONe4

MONe5

MONe6

ζ-potential (mV) −26.56 −27.96 −29.42 −29.62 −31.34 −33.02 −22.24 −23.36 −25.81 −27.78 −28.24 −30.21 −29.46 −30.57 −32.08 −24.67 −25.19 −27.38

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.46 0.56 0.23 0.62 0.43 0.18 0.52 0.34 0.22 0.28 0.32 0.18 0.87 0.68 0.92 0.45 0.58 0.14

total drug content (mg/mL) 0.9764 ± 0.0086 0.9783 ± 0.0097 0.9842 ± 0.081 0.9856 ± 0.0068 0.9732 ± 0.0094 0.9702 ± 0.0086 0.9816 ± 0.0056 0.9791 ± 0.0042 1.9789 ± 0.0038 1.9763 ± 0.0062 1.9834 ± 0.032 1.9734 ± 0.062

Figure 2. (a) Plasma concentration time profile of intraperitoneally injected MOX base solution (10 mg/kg) and a solution of MONe6 lipid nanoemulsion. (b) Tissue distribution of intraperitoneally injected MOX base solution (10 mg/kg) and MONe6 lipid nanoemulsion at 2, 4, and 8 h.

formulation (i.e., 20, 40, and 80 μg of lipid/mL of media). In the case of J774 Mφ cells, MONe6 was found to be least toxic where 96.46 ± 1.32% of the cells were viable as compared to MONe4 and MONe5 with 92.34 ± 2.32 and 93.6 ± 2.64% viability, respectively. In the case of THP1 and HepG2, the viability following treatment was better than J744 cells as uptake of emulsion was more in J774 Mφ and the viability ranged from 97 to 99.5% for the formulation at all treatment levels (Figure 1c). Nanoemulsion (MONe6) was selected to study uptake by confocal microscopy and for pharmacokinetic studies as it demonstrated better stability, less hemolysis, and less cytotoxicity in different cell lines. To investigate the distribution of emulsions in J774 macrophages, we observed the cells after incubation with MONe6 using confocal fluorescence microscopy. The emulsions were labeled by Nile red (red). Confocal microscopy (Figure 1d) clearly demonstrates intracellular uptake of MONe6 in J774 macrophages via pinocytosis in phagosomes suggesting that the nanoemulsion efficiently delivered the payload to the subcellular sites in the cell.

to liberation of free fatty acid from phospholipids leading to a decrease in pH and an increase in zeta potential. There was a drastic change in globule size of MONe1, MONe2, and MONe3 after three months (Table 2) of incubation demonstrating the instability of nanoemulsions. In the case of MONe4, MONe5, and MONe6, the change in globule size was not significant indicating stable nanoemulsions. As drug stability of emulsions was concerned, no significant decrease in drug content was observed (Table 2). 3.2.5. Hemolytic Study. The hemolytic potential of various formulations was evaluated in the concentration range of 10 to 40 μL of nanoemulsion/mL diluted in PBS. The percent hemolysis for all formulations was found to be in between 10 and 20% and were regarded as safe43 in comparison to other formulations as hemolysis for MONe6 was less than 10% (i.e., 8.82 ± 0.24%); therefore, it was carried forward for pharmacokinetic studies and in vivo evaluations. 3.2.6. Toxicity Studies on J774, HepG2, and THP1 Cell Lines and Uptake in J774 Cells. Toxicity on the cell lines were conducted at three lipid concentrations of each nanoemulsion F



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3.3. Pharmacokinetics and Tissue Distribution. Parenteral nanoemulsions containing MOX base was formulated to fulfill the objective to alter the biodistribution so as to improve tissue penetration thereby enhancing in vivo antibacterial efficacy. Nanoemulsion (MONe6) and MOX solution were administered in rat via i.p. route at the dose of 10 mg/kg of MOX. Encapsulation of MOX into nanoemulsion significantly improved the pharmacokinetics as compared to MOX solution. Maximum blood concentration (Cmax) of MOX released from MONe6 was 3 times that as compared to MOX solution (Figure 2a), indicating slower clearance of MONe6 than MOX solution. Nanoemulsion increased the Cmax (0.34 ± 0.05 μg/ mL) and AUC (4.07 ± 0.46 μg/mL) indicating improvement in therapeutic efficacy as an increased ratio of AUC/MIC was observed (Figure 3). A significant difference in distribution of

MONe6. The reduction correlated well with the AUC/MIC ratio in the tissue except in the case of spleen where the bacterial burden increased significantly as compared to control group in both treatment groups at 6 h (P < 0.05), but at 24 h, the burden was reduced significantly, indicating an overall improvement in efficacy of MOX by formulating it in a nanoemulsion (Figure 4a). 3.4.2. Reduction in Lipopolysaccharide (LPS) and Cytokine (TNF-α and IL-6) Levels in Plasma. LPS is an important component of the outer cell wall of Gram-negative bacteria, which plays a crucial role as initiator of septic shock. They activate the host effector cells through stimulation of receptors on their surface and induce production of macrophage-derived cytokines. LPS levels in blood are an important predisposition factor for progression of E. coli-induced peritonitis to Gramnegative sepsis and septic shock and would contribute significantly to the mortality.44 There was a significant reduction in LPS levels in blood after treatment with MONe6 (12.26 ± 2.362 pg/mL) treated groups as compared to MOX solution (17.05 ± 0.46 pg/mL) and untreated group (39.86 ± 1.06 pg/mL) at 24 h, indicating that treatments with both MOX and MONe6 have a low propensity of progression of peritonitis to sepsis and septic shock as compared to untreated group (E. coli) (Figure 4b). In addition to that, overproduction of cytokines, specifically IL-6 and TNF-α, are key mediators in LPS-induced sepsis and septic shock after E. coli-induced peritonitis and increase the risk of mortality in the subjects; therefore, cytokine levels can effectively indicate the risk of mortality in sepsis. The mean peak levels of endotoxin, TNF-α, and IL-6 at 6 h in plasma were 28 ± 2.11, 1053.61 ± 110.68, and 456.20 ± 28.34 pg/mL, respectively, in the positive control group. MOX (i.p.) treated group showed 2-fold reduction in plasma endotoxin level (12.46 ± 2.13 pg/mL), TNF-α (441.66 ± 35.96 pg/mL), and IL-6 (280.78 ± 12.76 pg/mL). Treatment with MONe6 and septic mice resulted in the highest reduction in plasma LPS (7.46 ± 1.08 pg/mL), TNF-α (354.24 ± 67.42 pg/mL), and IL-6 (118.70 ± 14.56pg/mL). The administration of MOX in lipid-based emulsion by parenteral route not only produced high antimicrobial activities but also strongest reduction in plasma endotoxin and TNF-α levels, resulting in higher survival rates28 (more than 80% after 72 h). 3.4.3. Reduction in Bacterial Count in Blood. Bacteremia in blood was reduced significantly at all-time points (1, 4, and 8 h) in the treatment groups as compared to the nontreatment group. When both treatment groups (MONe6 and MOX solution) were compared, there was a significant reduction of bactermia in blood at each time point when treated with MONe6; this can be attributed to increased accumulation of MOX rendered by MONe6 (Figure 4c). 3.4.4. Effect of MOX Nanoemulsion on Lipid Peroxidation. Lipid peroxidation is a crucial indicator for oxidative damage in tissues, sepsis and septic shock, determining the crucial outcomes, which are multiple organ failure and death.45 There was a significant reduction in lipid peroxide (LPO) levels in tissues of both treatment groups (MOX solution and MONe6) as compared to control group (E. coli) (Figure 4d). It has been observed that the LPO levels in liver, lung, and kidney was lower when treated with MONe6 as compared to MOX solution, whereas in spleen LPO levels were higher at both time points, i.e., at 6 h, it was 71.64 ± 1.2 μM/mg of protein for MOX solution, while it was 72.46 ± 2.1 μM/mg of protein for MONe6. Similarly, 19.14 ± 0.9 μM/mg of protein was found

Figure 3. Comparison of PK/PD parameter (AUC/MIC) of MOX solution as well as MONe6 in blood and in different tissues.

drug (denoted by AUC0−8 h) from MOX solution and MONe6 was observed with a greater distribution in spleen, kidney, lungs, and liver in the case of MONe6. MONe6 improved tissue distribution in kidney, lungs, and liver in a significant manner as compared to MOX solution, whereas in the case of spleen, there was no significant difference (at P < 0.05) (Figure 2b). The concentration of MOX in the case of spleen at 8 h was increased significantly (at P < 0.05) (MONe6 vs MOX soln; 6.64 ± 0.46 and 7.96 ± 0.12 μg/g, respectively); this observation was also true for kidney (MONe6 vs MOX soln; 2.68 ± 0.11 and 0.87 ± 0.098 μg/g, respectively), lungs (MONe6 vs MOX soln; 3.88 ± 0.098 and 0.68 ± 0.14 μg/g, respectively), and liver (MONe6 vs MOX soln; 1.84 ± 0.098 and 0.236 ± 0.078 μg/g, respectively) (Figure 2b) (at P < 0.05). 3.4. Pharmacodynamic Studies in Rats with E. coliInduced Peritonitis. 3.4.1. Effect on Bacterial Burden in Tissues and Plasma. Significant reduction in bacterial burden was observed in both treatment groups (MONe6 and MOX solution) as compared to untreated groups (E. coli) at 6 h as well as at 24 h (P < 0.05). However, higher bacterial burden was found in spleen at 6 h when treated with MOX solution and MONe6 as compared to control group, whereas at 24 h bacterial burden reduced significantly (p < 0.05). In the case of liver, the reduction in bacterial burden was insignificant in the case of MOX solution at 6 h as compared to control group (E. coli). On comparing both treatment groups, i.e., MONe6 and MOX solution, significant reduction in bacterial burden at both time points (i.e., 6 and 24 h) was observed in the case of G



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Figure 4. (a) CFU counts in various tissues in control (E. coli), MOX solution, and MONe6 at 6 and 24 h (n = 3). (b) Level of lipopolysaccharide (LPS) at various time points in control (E. coli), MOX solution, and MONe6. (c) Bacterial burden in plasma and concentration of MOX in plasma at various time points in control (E. coli), MOX solution, and MONe6. (d) Lipid peroxidation in various tissues at 6 and 24 h (n = 3). (e) Myeloperoxidase activity after treatment with MOX solution and optimized nanoemulsion at 2 and 8 h (n = 3). Control group (E. coli) = 100% MPO activity.

for MOX solution after 24 h, while it was 31.34 ± 1.2 μM/mg of protein for MONe6. 3.4.5. Effect of MOX Nanoemulsion on Neutrophil Migration in Tissues. Myeloperoxidase (MPO) activity frequently employed as a sensitive, quantitative measure of neutrophil sequestration was performed to permit quantitation of neutrophil-induced inflammation in tissues.46,47 There was a significant reduction in MPO levels in both treatment groups as compared to the untreated group (E. coli). When both treatment groups were compared at 2 and 8 h, significant reduction in MPO levels was found in liver and lung when treated with MONe6 as compared to MOX solution. In the case of spleen, there was a reduction in MPO levels in MONe6

as compared to MOX solution, but the reduction was not significant at 8 h, whereas in the case of kidney, MPO levels increased at 8 h significantly when treated with MONe6 (P < 0.05) (Figure 4e). 3.4.6. Histopathological Evaluations. Histopathology of lung, liver, kidney, and spleen revealed a difference in neutrophil infiltration as an indicator of oxidative tissue damage. The following histological changes were observed in different groups. In the untreated group (E. coli), hepatocytes showed a disordered arrangement, extensive fatty degeneration, swelling, and abundant lymphocytes infiltrated the fibrous tissue with proliferation of bile ducts. Lung tissue exhibited characteristic H



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Figure 5. (a) Histopathological evaluation of various tissues after 24 h in treated and untreated groups. (b) Cumulative percent survival after 8 days of MOX treatment with MOX solution and MOX containing nanoemulsion (MONe6) in E. coli-induced peritonitis in rats. MOX was administered at doses of 10 mg/kg two times a day.

subsequent complications such as sepsis. Tocopherol succinate can sustain release of cationic drugs from lipidic phase via ionic interaction and have a beneficial effect on counteracting the complication of infections, i.e., endotoxic shock by reducing cytokine secretion and oxidative damage to tissue in response to endotoxin.32−34 MOX-based nanoemulsions incorporating tocopheryl succinate demonstrated and enhanced plasma pharmacokinetics and tissue distribution of drug and improved various pharmacodynamic parameters like in vivo reduction in antimicrobial efficacy as well as reduction in cytokine storm, LPS levels, oxidative stress, neutrophil migration leading to reduction in organ damage, and enhancement of survival in E. coli-induced peritonitis in rats. MOX base was incorporated into lipid phase with higher efficiency than MOX HCl due to increased lipophilicity and also due to the presence of tocopheryl succinate, which enhanced the solublization of MOX in soybean oil as well as inhibit the diffusion of MOX from lipidic phase leading to reduced partitioning of drug into aqueous phase and controlling the release behavior of drug in in vitro dissolution studies. The idea of taking tocopherol succinate is based on preformulation studies since it has a free −COOH group, which leads to enhanced solubility of MOX base (containing free −NH2 group) and may also reduce partitioning into aqueous solution thereby effectively delivering MOX to tissues. In addition, tocopherol succinate has been reported to reduce LPS-induced inflammation (via antioxidant effect and other molecular mechanisms) both in in vitro cell line studies51−57 and in animal models. Similar observation has also been reported with tocopherol succinate51−53 and tocopherol acetate.58,59 Nanoemulsion was optimized in order to maximize drug loading and minimize droplet size as well as to enhance the stability of nanoemulsion. The droplet size of nanoemulsion was increased as the fraction of oil phase was increased. Stability was increased as the percentage of PF-68 and phospholipid was increased due to the formation of a rigid layer on the interphase preventing the coalescence of the droplets.60 Because of the ethylene oxide groups, stronger steric stabilization can be achieved. Therefore, mean particle size and zeta potential values containing 2% w/v PF-68 nanoemulsions were physically stable over 10 weeks showing no flocculation, creaming, coalescence, and Ostwald ripening. In vitro release studies indicated that nanoemulsion formulation of MOX was able to sustain release of MOX up to 24 h and can be useful for enhancing the tissue concentration as well as retention of drug in plasma.

signs of lung injury, which include interstitial edema, alveolar thickening, and severe leukocyte infiltration in interstitium and alveoli. Renal histology revealed infiltration of neutrophils in the Bowman’s space and renal tubules leading to damage in the normal architecture of the kidney (Figure 5a). In the MOX-treated group the inflammatory process was less as compared to the control with a lower number of neutrophils in liver tissue. In the case of the MONe6-treated group, inflammation is almost absent in the portal space, few mononuclear cells are present, and tissue appeared to be normal due to less oxidative stress. A similar pattern of neutrophil infiltration was observed in the spleen with reduced neutrophil infiltrations in the tissue for both treatment groups as compared with the control group. Lung histology revealed a reduction in alveolar thickness and neutrophil infiltration, with maximal effect observed in the case of the MONe6 group, demonstrating reduction in thickness of alveolar walls. Kidney histology in MOX solution treated group demonstrated reduced infiltration of neutrophils in Bowman’s space and renal tubules, whereas histomorphological features in kidney were found to be normal in the MONe6 groups (Figure 5a). 3.4.7. Survival Analysis. Survival studies clearly demonstrated improved survival in the case of treatment with MONe6 (65.44%) and MOX (38.35%) solution as compared to the control group (8.22%) (Figure 5b). Comparison of survival curves was done by log rank (Mantel Cox) test with a calculated p value of 0.0253; the survival was significantly different at 95% confidence interval (using Graph pad Prism Software).

4. DISCUSSIONS Antimicrobial agents are a mainstay in the therapy of cIAIs. As compared to older fluoroquinolones (second generation agents like ciprofloxacin), MOX also has the advantage that its emergence of resistance has been stable and low due to its dual mechanism action (DNA gyrase and topoisomerase IV) and rapid killing, and its effectiveness against MDR pathogens has not been effected.48 Apart from these facts MOX has also been reported to play a dual role in infections, an antimicrobial role and an immunomodulatory role by inhibiting lipopolysaccharide-stimulated secretion of IL-1α, IL-1β, and TNF-α by monocytes and also causing a very low amount of LPS release in blood as compared to other antibiotic therapies.49 MOX has also been reported to stimulate white blood cell production in murine models.50 So it is a good choice for the therapeutic intervention of intra-abdominal infections in order to minimize I



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antibiotics having different LPS liberating potential is due to the negligence of the fact that LPS liberating potential of these antibiotics may play an important role in the progression of sepsis.21 LPS is a potent inducer of the pro-inflammatory cytokine response in individuals suffering from Gram-negative sepsis. This is further strengthened by the fact that the only clinically viable approach for reduction of mortality in Gramnegative sepsis is via removal of LPS by hemodiafiltration through Polymyxin B immobilized cartridge. MOX has been reported to release less LPS from E. coli as compared to other antibiotics especially beta lactams.22 The role of reactive oxygen species (ROS) produced by activated phagocytes as defense mechanism to kill bacteria is a vital part of the immune system, but excessive ROS can inadvertently cause tissue damage and, along with other inflammatory mediators (NO,TNF-α, and IL-1β), can lead to organ damage, failure, and death.20 Konukoglu et al. reported an increase in tissue oxidative stress during peritonitis, which was relieved by α-tocopherol and taurolin.66 MONe6 was effective in decreasing lipid peroxides (LPO) levels in tissues as compared to MOX base solution and control (no treatment) group. This observation can be attributed to the fact that nanoemulsion was able to increase the concentration of MOX as well as due to the presence of tocopheryl succinate, which is a potent antioxidant leading to reduced levels of LPO as reported previously.67 This observation was also in agreement with previous studies describing the suppression of ROS by MOX so that lipid peroxidation and tissue destruction due to the infection process is suppressed.68 Since, it has been reported that an increase in cytokine levels, i.e., TNF-α and IL-6,69−72 in-directly leads to an increase in tissue inflammatory conditions. Reduction of levels of inflammatory cytokines (TNF-α and IL-6) in plasma by MONe6 was beneficial in hyperinflammatory conditions associated with cIAIs as both tocopheryl succinate and MOX (via NFκB inhibition)51,73−75 has been reported to reduce cytokine levels73,76 via reducing NFκB expression from macrophages and THP1 monocytes.50,68 In addition, there may be multiple mechanisms playing a role in attenuation of tissue inflammatory infiltration and decreasing circulating LPS levels by both immunomodulation and better bacterial killing due to improved pharmacokinetics of MOX. Tissue injury is a major outcome on infection-induced sepsis, which may lead to multiple organ failure and death. It is largely mediated through neutrophil accumulation as a result of both LPS release and E. coli present at the site of infection, which triggers purging of inflammatory mediators in tissues by massive apoptosis of neutrophils. MPO content in the respective tissues are directly correlated with the accumulation of neutrophils and believed to be a major mediator in tissue inflammation.77 MONe6 was found to be effective in reducing neutrophil accumulation (as demonstrated by histopathological evaluations of tissues) indicating protection from tissue damage as well as reducing bacterial burden in tissues by reducing MPO content, due to increased concentration of MOX and tocopheryl succinate in tissues. So in the case of the present formulation, tocopheryl succinate served dual purpose by increasing loading of MOX in oil phase as well reduction of neutrophil migration in tissues as reported previously,34 leading to improved survival in rat model of E. coli-induced peritonitis on treatment with MONe6 as compared to plain drug solution.

Suitability of the developed formulation for parenteral administration was evaluated by in vitro hemolytic assays and cytotoxicity assays. Hemolysis is a very important phenomenon in safety of parenteral formulations as it may lead to vascular irritation, phlebitis, anemia, jaundice, kernicterus, acute renal failure, and, in some cases, death.61 Optimized nanoemulsion (MONe6) was found to be suitable for parenteral administration as indicated by in vitro hemolysis (100 verifies the reduced possibility of emergence of resistance, whereas values ∼35 are good predictors of bactericidal and clinical efficacy.48 The values obtained with MOX containing nanoemulsion was better as compared to MOX solution in both blood and tissues, which were far above the levels required for emergence of resistance. Formulation of MOX in the form of nanoemulsion resulted in increased AUC in blood (enhanced AUC/MIC ratio) as well as enhanced tissue distribution especially in liver and lungs leading to a better pharmacodynamic outcome in tissues. This may be attributed to prevention of rapid distribution of MOX to tissues (specifically to non-RES organs and adipose tissues) surrounding the intraperitoneal cavity resulting in low plasma concentration of MOX in plasma when administered as solution. These data are in agreement with the previous studies by Tilney,63 who reported enhancement in plasma levels of drug on i.p. administration. Nanoemulsions (as reported previously in the case of liposomal delivery system64) may bypass entrapment by lymph nodes and can be drained into blood via thoracic lymph channels from lymphatic circulations, ultimately leading to blood, thereby increasing plasma concentration of drug by preventing metabolism and distribution of drug to non-RES organs (having less blood flow). These formulations resulted in facilitated distribution in the organs of RES specifically liver, kidney, and lungs (organs most affected in the case of intra-abdominal sepsis developed after cIAIs) as demonstrated in pharmacokinetic studies. AUC/ MIC ratios in tissues correlated well the reduction of bacterial burden after 24 h. It was also demonstrated that optimized emulsion on treatment reduced the level of circulating LPS as compared to MOX solution alone. This could be an important observation as circulating LPS plays an important role in the development of sepsis in patients with cIAI, and reduced circulatory levels of LPS may be an indication of prohibition of conversion of cIAI into sepsis.65 There are numerous experimental and clinical studies suggesting that reducing LPS levels by neutralizing or removing LPS would be an effective adjunctive approach to the management of Gram-negative sepsis.18 LPS is an important mediator in the development of Gram-negative sepsis.17−19 Antibiotics used to treat cIAI may also play a role in the pathophysiological conversion of cIAIs to sepsis, mainly through their ability to liberate LPS from bacterial cell wall during destruction of the microorganisms. Apparent failure of these different treatment options with J



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(7) Blot, S.; De Waele, J. J.; Vogelaers, D. Essentials for selecting antimicrobial therapy for intra-abdominal infections. Drugs 2012, 72 (6), e17−e32. (8) Goldstein, E. J. C.; Solomkin, J. S.; Citron, D. M.; Alder, J. D. Clinical efficacy and correlation of clinical outcomes with in vitro susceptibility for anaerobic bacteria in patients with complicated intraabdominal infections treated with moxifloxacin. Clin. Infect. Dis. 2011, 53 (11), 1074−1080. (9) Lemaire, S.; Kosowska-Shick, K.; Appelbaum, P. C.; Glupczynski, Y.; Van Bambeke, F.; Tulkens, P. M. Activity of moxifloxacin against intracellular community-acquired methicillin-resistant Staphylococcus aureus: comparison with clindamycin, linezolid and co-trimoxazole and attempt at defining an intracellular susceptibility breakpoint. J. Antimicrob. Chemother. 2011, 66 (3), 596−607. (10) Balfour, J. B.; Lamb, H. Moxifloxacin. Drugs 2000, 59 (1), 115− 139. (11) Fraimow, H.; Nahra, R. Resistant Gram-negative infections. Crit. Care Clin. 2013, 29 (4), 895−921. (12) Brunkhorst, F. M.; Oppert, M.; Marx, G.; et al. Effect of empirical treatment with moxifloxacin and Meropenem vs Meropenem on sepsis-related organ dysfunction in patients with severe sepsis: A randomized trial. JAMA 2012, 307 (22), 2390−2399. (13) Weiss, T.; Shalit, I.; Blau, H.; Werber, S.; Halperin, D.; Levitov, A.; Fabian, I. Anti-inflammatory effects of moxifloxacin on activated human monocytic cells: inhibition of NF-κB and mitogen-activated protein kinase activation and of synthesis of proinflammatory cytokines. Antimicrob. Agents Chemother. 2004, 48 (6), 1974−1982. (14) Blau, H.; Klein, K.; Shalit, I.; Halperin, D. Moxifloxacin but not ciprofloxacin or azithromycin selectively inhibits IL-8, IL-6, ERK1/2, JNK, and NF-κB activation in a cystic fibrosis epithelial cell line. Am. J. Physiol., Lung Cell. Mol. Physiol. 2007, 292 (1), L343−L352. (15) Mu, Y. P.; Liu, R. L.; Wang, L. Q.; Deng, X.; Zhu, N.; Wei, M. D.; Wang, Y. Moxifloxacin monotherapy for treatment of complicated intra-abdominal infections: a meta-analysis of randomised controlled trials. Int. J. Clin. Pract. 2012, 66 (2), 210−217. (16) Goldstein, E. J.; Solomkin, J. S.; Citron, D. M.; Alder, J. D. Clinical efficacy and correlation of clinical outcomes with in vitro susceptibility for anaerobic bacteria in patients with complicated intraabdominal infections treated with moxifloxacin. Clin. Infect. Dis. 2011, 53 (11), 1074−1080. (17) Ma, C.-Y.; Shi, G.-Y.; Shi, C.-S.; Kao, Y.-C.; Lin, S.-W.; Wu, H.L. Monocytic thrombomodulin triggers LPS- and Gram-negative bacteria-induced inflammatory response. J. Immunol. 2012, 188 (12), 6328−6337. (18) Davies, B.; Cohen, J. Endotoxin removal devices for the treatment of sepsis and septic shock. Lancet Infect. Dis. 2011, 11 (1), 65−71. (19) Mignon, F.; Piagnerelli, M.; Van Nuffelen, M.; Vincent, J. Effect of empiric antibiotic treatment on plasma endotoxin activity in septic patients. Infection 2014, 42 (3), 521−528. (20) Shukla, P.; Rao, G. M.; Pandey, G.; Sharma, S.; Mittapelly, N.; Shegokar, R.; Mishra, P. R. Therapeutic intervention of sepsis: Current and anticipated pharmacological agents. Br. J. Pharmacol. 2014, 171 (22), 5011−5031. (21) Lepper, P.; Held, T.; Schneider, E.; Bölke, E.; Gerlach, H.; Trautmann, M. Clinical implications of antibiotic-induced endotoxin release in septic shock. Intensive Care Med. 2002, 28 (7), 824−833. (22) Trautmann, M.; Scheibe, C.; Wellinghausen, N.; Holst, O.; Lepper, P. M. Low endotoxin release from Escherichia coli and Bacteroides f ragilis during exposure to moxifloxacin. Chemotherapy 2010, 56 (5), 364−370. (23) Nau, R.; Eiffert, H. Minimizing the release of proinflammatory and toxic bacterial products within the host: A promising approach to improve outcome in life-threatening infections. FEMS Immunol. Med. Microbiol. 2005, 44 (1), 1−16. (24) Shukla, P.; Gupta, G.; Singodia, D.; Shukla, R.; Verma, A. K.; Dwivedi, P.; Kansal, S.; Mishra, P. R. Emerging trend in nanoengineered polyelectrolyte-based surrogate carriers for delivery of bioactives. Expert Opin. Drug Delivery 2010, 7 (9), 993−1011.

5. CONCLUSIONS In the present study, focus was mainly on improved antibacterial activity and immunomodulation using MOXloaded nanoemulsion leading to improvement in conditions of intra-abdominal sepsis via improving tissue penetration. In the present studies, multiple mechanisms might be playing a role in the attenuation of sepsis. As we have observed, improved conditions of intra-abdominal sepsis may be due to improved pharmacokinetics of MOX because of a new preparation in the form of nanoemulsion, and improved antibacterial activity and immunomodulation may be due to the presence of tocopheryl succinate as well as the reduced bacterial burden and low LPS release. Nanoemulsions also reduced the oxidative stress and tissue damage in E. coli-induced peritonitis by reducing the levels of LPO and MPO in tissues specifically in lungs and liver with low endotoxin release and cytokine levels in plasma compared to MOX solution and control groups. Therefore, parenteral nanoemulsions of MOX may hold a promising advantage in the therapy of E. coli-induced peritonitis and may be helpful in the prevention of further complication like sepsis and septic shock.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

Authors acknowledge the funding received from Indian Council of Medical Research (ICMR) from the GAP project entitled “Delivery system for the management of septic shock; rational approach towards lipopolysaccharide neutralization and detoxification” and senior research fellowship provided by CSIR New Delhi. Authors also acknowledge MSN pharmachem Pvt. Ltd. (Medhak Andra Pradesh India) for providing moxifloxacin HCl as a gift sample. This is CDRI communication number 8830.

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