<Emphasis Type="BoldItalic">In Silico</Emphasis> Absorption Analysis of Valacyclovir in Wildtype and <Emphasis Type="BoldItalic">Pept1</Emphasis> Knockout Mice Following Oral Dose Escalation

Purpose

We developed simulation and modeling methods to predict the in vivo pharmacokinetic profiles of acyclovir, following escalating oral doses of valacyclovir, in wildtype and Pept1 knockout mice. We also quantitated the contribution of specific intestinal segments in the absorption of valacyclovir in these mice.

Methods

Simulations were conducted using a mechanistic advanced compartmental absorption and transit (ACAT) model implemented in GastroPlus?. Simulations were performed for 3 h post-dose in wildtype and Pept1 knockout mice following single oral doses of 10, 25, 50 and 100 nmol/g valacyclovir, and compared to experimentally observed plasma concentration-time profiles of acyclovir.

Results

Good fits were obtained in wildtype and Pept1 knockout mice. Valacyclovir was primarily absorbed from duodenum (42%) and jejunum (24%) of wildtype mice, with reduced uptake from ileum (3%) and caecum/colon (1%), for a total of 70% absorption. In contrast, the absorption of valacyclovir in Pept1 knockout mice was slow and sustained throughout the entire intestinal tract in which duodenum (4%), jejunum (14%), ileum (10%) and caecum/colon (12%) accounted for a total of 40% absorption.

Conclusion

The ACAT model bridged the gap between in situ and in vivo experimental findings, and facilitated our understanding of the complicated intestinal absorption processes of valacyclovir.

     

A Dual-Biotic System for the Concurrent Delivery of Antibiotics and Probiotics: <Emphasis Type="Italic">In Vitro</Emphasis>, <Emphasis Type="Italic">Ex Vivo</Emphasis>, <Emphasis Type="Italic">In Vivo and In Silico</Emphasis> Evaluation and Correlation

Purpose

A delayed release bio-polymeric Dual-Biotic system has been extensively evaluated in this study to overcome the therapeutic issue of probiotic killing due to incorrect administration with the antibiotic.

Methods

In vitro and ex vivo release and characterization studies have been undertaken on the Dual-Biotic system. In vivo analyses utilizing a Large White pig model were also performed with commercial products used as a comparison. Intestinal fluid for probiotic quantification was aspirated using a surgically implanted intestinal cannula with Lactobacillus acidophilus cell counts determined through luminescence and inoculation onto Lactobacilli-specific agar. Plasma amoxicillin concentrations were determined through Ultra-Performance Liquid Chromatography. The reactional profile and crosslinking mechanism of ovalbumin and genipin was elucidated using molecular mechanic energy relationships in a vacuum system by exploring the spatial disposition of different concentrations of genipin with respect to ovalbumin with ovalbumin/genipin ratios of 1:1, 1:5 and 1:10.

Results

In vivo evaluation of the Dual-Biotic system detailed maximum Lactobacillus viability (~455% baseline viability) 6 h after oral administration. Concurrent administration of the commercial products revealed a 75% decrease in bacterial viability when compared to the controls analyzed. A level A in vitro-in vivo correlation was also established with 96.9% predictability of amoxicillin release ascertained. The computational results achieved corroborated well with the experimental findings and physicochemical data.

Conclusions

Evaluation and correlation of the Dual-Biotic system has detailed the success of the formulation for the concurrent delivery of an antibiotic and probiotic.

     

<Emphasis Type="Italic">In Vivo</Emphasis> Toxicity and Immunological Characterization of Detoxified Recombinant Botulinum Neurotoxin Type A

Purpose

A double-mutant E224A/E262A full-length botulinum neurotoxin (BoNT) Type A with structural similarity to native BoNT/A but lacking the endopeptidase activity provides an ideal surrogate for testing pharmacokinetics and immunochemical characteristics of BoNT.

Methods

We determined lethality (LD₅₀) of deactivated recombinant botulinum neurotoxin (drBoNT/A) to be 24.0 μg by intraperitoneal route (i.p). The polypeptide drBoNT/A labeled with near infra-red dye 800 (NIR 800) was used to examine its distribution to different organs using whole body imaging when administered to mice via intravenous (i.v) or i.p route. Also, drBoNT/A was used to evaluate its immunogenicity in Balb/C mice model.

Results

drBoNT/A was found to be highly immunogenic when tested under various in vivo conditions in Balb/C mice model. For the first time we have demonstrated that a full length 150 kDa drBoNT/A, by administering via inhalation route in mice model, has evoked both circulating immunoglobulin levels of IgG and secretory IgA at the mucosal surface. The immunoglobulin levels were sufficient enough to protect against the challenge dose of native BoNT toxin in mice model. Tissue distribution of drBoNT/A seems to be similar to that of native toxin.

Conclusions

Based on the characteristics described in this report this nontoxic holotoxin protein will assist us to explore the window of opportunity available for therapeutic treatment in case of unnatural poisoning, and also it can be an effective vaccine candidate.

     

A Study on Toxicity of Chemically Synthesised Silver Nanoparticle on <Emphasis Type="Italic">Eudrilus eugeniae</Emphasis>

Objective

To evaluate the toxicity of the silver nanoparticle against earthworms - Eudrilus eugeniae, a model for soil organism.

Methods

Silver nanoparticles were synthesised by chemical reduction and further characterised by UV Visible Spectroscopy and FeSEM. Earthworms were allowed to interact with different concentrations of the synthesized silver nanoparticles. After exposure period, histology and inductively coupled plasma optical emission spectrometry (ICP-OES) were done to determine the accumulation and toxic effects exhibited by the nanoparticle on earthworms.

Results

The synthesized nanoparticle was found to be between the size of 180 and 200 nm. Histology studies revealed that silver nanoparticles to cause fibrosis, lipofuscin-like deposits and also gut disruption in earthworms.

Conclusion

Silver nanoparticles were found to be toxic to Eudrilus eugeniae, which was evidenced by histology.

     

Predicting Pulmonary Pharmacokinetics from <Emphasis Type="Italic">In Vitro</Emphasis> Properties of Dry Powder Inhalers

Purpose

The ability of two semi-mechanistic simulation approaches to predict the systemic pharmacokinetics (PK) of inhaled corticosteroids (ICSs) delivered via dry powder inhalers (DPIs) was assessed for mometasone furoate, budesonide and fluticasone propionate.

Methods

Both approaches derived the total lung doses and the central to peripheral lung deposition ratios from clinically relevant cascade impactor studies, but differed in the way the pulmonary absorption rate was derived. In approach 1, the rate of in vivo drug dissolution/absorption was predicted for the included ICSs from in vitro aerodynamic particle size distribution and in vitro drug solubility estimates measured in an in vivo predictive dissolution medium. Approach 2 derived a first order absorption rate from the mean dissolution time (MDT), determined for the test formulations in an in vitro Transwell^® based dissolution system.

Results

Approach 1 suggested PK profiles which agreed well with the published pharmacokinetic profiles. Similarly, within approach 2, input parameters for the pulmonary absorption rate constant derived from dissolution rate experiments were able to reasonably predict the pharmacokinetic profiles published in literature.

Conclusion

Approach 1 utilizes more complex strategies for predicting the dissolution/absorption process without providing a significant advantage over approach 2 with regard to accuracy of in vivo predictions.

     

The <Emphasis Type="BoldItalic">In vitro</Emphasis> Sub-cellular Localization and <Emphasis Type="BoldItalic">In vivo</Emphasis> Efficacy of Novel Chitosan/GMO Nanostructures containing Paclitaxel

Purpose

To determine the in vitro sub-cellular localization and in vivo efficacy of chitosan/GMO nanostructures containing paclitaxel (PTX) compared to a conventional PTX treatment (Taxol^®).

Methods

The sub-cellular localization of coumarin-6 labeled chitosan/GMO nanostructures was determined by confocal microscopy in MDA-MB-231 cells. The antitumor efficacy was evaluated in two separate studies using FOX-Chase (CB17) SCID Female-Mice MDA-MB-231 xenograph model. Treatments consisted of intravenous Taxol^® or chitosan/GMO nanostructures with or without PTX, local intra-tumor bolus of Taxol^® or chitosan/GMO nanostructures with or without PTX. The tumor diameter and animal weight was monitored at various intervals. Histopathological changes were evaluated in end-point tumors.

Results

The tumor diameter increased at a constant rate for all the groups between days 7-14. After a single intratumoral bolus dose of chitosan/GMO containing PTX showed significant reduction in tumor diameter on day 15 when compared to control, placebo and intravenous PTX administration. The tumor diameter reached a maximal decrease (4-fold) by day 18, and the difference was reduced to approximately 2-fold by day 21. Qualitatively similar results were observed in a separate study containing PTX when administered intravenously.

Conclusion

Chitosan/GMO nanostructures containing PTX are safe and effective administered locally or intravenously. Partially supported by DOD Award BC045664

     

<Emphasis Type="BoldItalic">In Vitro</Emphasis> and <Emphasis Type="BoldItalic">In Vivo</Emphasis> Modeling of Hydroxypropyl Methylcellulose (HPMC) Matrix Tablet Erosion Under Fasting and Postprandial Status

Purpose

To develop a model linking in vitro and in vivo erosion of extended release tablets under fasting and postprandial status.

Methods

A nonlinear mixed-effects model was developed from the in vitro erosion profiles of four hydroxypropyl methylcellulose (HPMC) matrix tablets studied under a range of experimental conditions. The model was used to predict in vivo erosion of the HPMC matrix tablets in different locations of the gastrointestinal tract, determined by magnetic marker monitoring. In each gastrointestinal segment the pH was set to physiological values and mechanical stress was estimated in USP2 apparatus rotation speed equivalent.

Results

Erosion was best described by a Michaelis–Menten type model. The maximal HPMC release rate (V_MAX) was affected by pH, mechanical stress, HPMC and calcium hydrogen phosphate content. The amount of HPMC left at which the release rate is half of V_MAX depended on pH and calcium hydrogen phosphate. Mechanical stress was estimated for stomach (39.5 rpm), proximal (93.3 rpm) and distal (31.1 rpm) small intestine and colon (9.99 rpm).

Conclusions

The in silico model accurately predicted the erosion profiles of HPMC matrix tablets under fasting and postprandial status and can be used to facilitate future development of extended release tablets.

     

<Emphasis Type="BoldItalic">In Silico</Emphasis> Modelling of Transdermal and Systemic Kinetics of Topically Applied Solutes: Model Development and Initial Validation for Transdermal Nicotine

Purpose

The purpose was to develop a mechanistic mathematical model for predicting the pharmacokinetics of topically applied solutes penetrating through the skin and into the blood circulation. The model could be used to support the design of transdermal drug delivery systems and skin care products, and risk assessment of occupational or consumer exposure.

Methods

A recently reported skin penetration model [Pharm Res 32 (2015) 1779] was integrated with the kinetic equations for dermis-to-capillary transport and systemic circulation. All model parameters were determined separately from the molecular, microscopic and physiological bases, without fitting to the in vivo data to be predicted. Published clinical studies of nicotine were used for model demonstration.

Results

The predicted plasma kinetics is in good agreement with observed clinical data. The simulated two-dimensional concentration profile in the stratum corneum vividly illustrates the local sub-cellular disposition kinetics, including tortuous lipid pathway for diffusion and the “reservoir” effect of the corneocytes.

Conclusions

A mechanistic model for predicting transdermal and systemic kinetics was developed and demonstrated with published clinical data. The integrated mechanistic approach has significantly extended the applicability of a recently reported microscopic skin penetration model by providing prediction of solute concentration in the blood.

     

<Emphasis Type="Italic">In Vivo</Emphasis> and <Emphasis Type="Italic">In Vitro</Emphasis> Evidence for Brain Uptake of 4-Phenylbutyrate by the Monocarboxylate Transporter 1 (MCT1)

Purpose

4-Phenylbutyrate (4-PBA) is expected to be a potential therapeutic for several neurodegenerative diseases. These activities require 4-PBA transport into the brain across the blood-brain barrier (BBB). The objective of the present study was to characterize the brain transport mechanism of 4-PBA through the BBB.

Methods

The brain transport of 4-PBA across the BBB was investigated following intravenous (IV) injection and internal carotid artery perfusion (ICAP) in vivo. The mechanism of transport was examined using TR-BBB cells, an in vitro model of the BBB.

Results

The volume of distribution (V_D) of 4-PBA by rat brain was about 7-fold greater than that of sucrose, a BBB impermeable vascular space marker, suggesting the blood-to-brain transport of 4-PBA through the BBB in the physiological state. [¹⁴C]4-PBA uptake by TR-BBB cells showed time-, pH- and concentration-dependence with a K _m of 13.4 mM at pH 7.4 and 3.22 mM at pH 6.0. The uptake was Na⁺ independent, and was significantly inhibited by alpha-cyano-4-hydroxycinnamate (a typical inhibitor for monocarboxylate transport), endogenous monocarboxylate compounds and monocarboxylic drugs. Lactate and valproate competitively inhibited [¹⁴C]4-PBA uptake with K _i value of 13.5 mM and 7.47 mM, respectively. These results indicate the role of monocarboxylate transporters (MCTs) in 4-PBA transport into the brain at the BBB. TR-BBB cells expressed mRNA of rMCT1, 2, and 4, especially, rMCT1 showed high mRNA expression level. In addition, [¹⁴C]4-PBA uptake was inhibited by rMCT1 specific small interfering RNA.

Conclusion

The transport mechanism of 4-PBA from blood to brain across the BBB likely involves MCT1.

     

Mutagenicities of 2-Propylaniline and 4-Propylaniline Determined by a Bacterial Reverse Mutation (<Emphasis Type="Italic">Ames</Emphasis>) Assay

Objective

Although the organic compounds, 2-propylaniline and 4-propylaniline are frequently used in many industrial sectors, and have little information about the potential genetic toxicity, and it is covered by the Occupational Safety & Health Act (OSHAct) in Korea.

Methods

The mutation test of 2-propylaniline and 4-propylaniline was evaluated in five different doses for each chemical through a well-known Ames bacterial mutation test. This test was performed regardless of metabolic activation.

Results

In this assay, we obtained positive results under all tested conditions, indicating that these two chemicals have mutagenic and potentially carcinogenic properties.

Conclusion

Both 2-propylaniline and 4-propylaniline were mutagenic under the conditions of these tests. This result means that all of these chemicals exhibit mutations and potential carcinogenicity.

     

<Emphasis Type="BoldItalic">In Vitro</Emphasis> Investigation of Influences of Chitosan Nanoparticles on Fluorescein Permeation into Alveolar Macrophages

Purpose

Pulmonary infection namely tuberculosis is characterized by alveolar macrophages harboring a large microbe population. The chitosan nanoparticles exhibit fast extracellular drug release in aqueous biological milieu. This study investigated the matrix effects of chitosan nanoparticles on extracellular drug diffusion into macrophages.

Methods

Oligo, low, medium and high molecular weight chitosan nanoparticles were prepared by nanospray drying technique. These nanoparticles were incubated with alveolar macrophages in vitro and had model drug sodium fluorescein added into the same cell culture. The diffusion characteristics of sodium fluorescein and nanoparticle behavior were investigated using fluorescence microscopy, scanning electron microscopy, differential scanning calorimetry and Fourier transform infrared spectroscopy techniques.

Results

The oligochitosan nanoparticles enabled macrophage membrane fluidization with the extent of sodium fluorescein entry into macrophages being directly governed by the nanoparticle loading. Using nanoparticles made of higher molecular weight chitosan, sodium fluorescein permeation into macrophages was delayed due to viscous chitosan diffusion barrier at membrane boundary.

Conclusion

Macrophage-chitosan nanoparticle interaction at membrane interface dictates drug migration into cellular domains.

     

Estimating Maximal <Emphasis Type="BoldItalic">In Vitro</Emphasis> Skin Permeation Flux from Studies Using Non-sink Receptor Phase Conditions

Purpose

This study explored the impact of non-sink receptor conditions on the in vitro skin permeation test (IVPT) and sought to estimate equivalent sink condition IVPT data.

Methods

Simulated diffusion model and experimental IVPT data were generated for ethyl salicylate across human epidermal membranes in Franz diffusion cells using six different receptor phases, with a 10 fold variation in ethyl salicylate solubility.

Results

Both simulated and experimental IVPT – time profiles were markedly affected by receptor phase solubility and receptor sampling rates. Similar sink condition equivalent estimated maximum fluxes were obtained by nonlinear regression and adjustment of linear regression estimates of steady state flux for relative saturation of the receptor phase over time for the four receptor phases in which the ethyl salicylate was relatively soluble. The markedly lower steady - state fluxes found for the other two phases in which ethyl salicylate was less soluble was attributed to an aqueous solution boundary layer effect.

Conclusions

Non-sink receptor phase IVPT data can be used to derive equivalent sink receptor phase IVPT data provided the receptor phase solubility and hydrodynamics are sufficient to minimise the impact of aqueous diffusion layers on IVPT data.

     

Population <Emphasis Type="BoldItalic">In Vitro-In Vivo</Emphasis> Correlation Model Linking Gastrointestinal Transit Time,pH, and Pharmacokinetics: Itraconazole as a Model Drug

Purpose

To establish an in vitro-in vivo correlation (IVIVC) model for Sporanox and SUBA-itraconazole formulations and to understand the impact of gastrointestinal (GI) pH and transit times on itraconazole dissolution and absorption.

Methods

IVIVC was developed based on fed/fasted pharmacokinetic data from randomized cross-over trials, in vitro dissolution studies, and prior information about typical and between subject variability of GI pH and transit times. Data were analysed using the population modelling approach as implemented in NONMEM.

Results

Dissolution kinetics were described using first order models. The in vivo pharmacokinetics of itraconazole was described with a 2-compartment model with 4-transit absorption compartments. Pharmacokinetic profiles for fasted itraconazole periods were described based on the in vitro dissolution model, in vivo disposition model, and the prior information on GI pH and transit times. The IVIVC model indicated that drug dissolution in the fed state required an additional pH-independent dissolution pathway. The IVIVC models were presented in a ‘Shiny’ application.

Conclusion

An IVIVC model was established and internally evaluated for the two itraconazole formulations. The IVIVC model provides more insight into the observed variability of itraconazole pharmacokinetics and indicated that GI pH and transit times influence in vivo dissolution and exposure.

     

Benchmarking of Human Dose Prediction for Inhaled Medicines from Preclinical <Emphasis Type="Italic">In Vivo</Emphasis> Data

Purpose

A scientifically robust prediction of human dose is important in determining whether to progress a candidate drug into clinical development. A particular challenge for inhaled medicines is that unbound drug concentrations at the pharmacological target site cannot be easily measured or predicted. In the absence of such data, alternative empirical methods can be useful. This work is a post hoc analysis based on preclinical in vivo pharmacokinetic/pharmacodynamic (PK/PD) data with the aim to evaluate such approaches and provide guidance on clinically effective dose prediction for inhaled medicines.

Methods

Five empirically based methodologies were applied on a diverse set of marketed inhaled therapeutics (inhaled corticosteroids and bronchodilators). The approaches include scaling of dose based on body weight or body surface area and variants of PK/PD approaches aiming to predict the therapeutic dose based on having efficacious concentrations of drug in the lung over the dosing interval.

Results

The most robust predictions of dose were made by body weight adjustment (90% within 3-fold) and by a specific PK/PD approach aiming for an average predicted 75% effect level during the dosing interval (80% within 3-fold). Scaling of dose based on body surface area consistently under predicted the therapeutic dose.

Conclusions

Preclinical in vivo data and empirical scaling to man can be used as a baseline method for clinical dose predictions of inhaled medicines. The development of more sophisticated translational models utilizing free drug concentration and target engagement data is a desirable build.

     

Antitumour Efficacy of Two Paclitaxel Formulations for Hyperthermic Intraperitoneal Chemotherapy (HIPEC) in an <Emphasis Type="Italic">In Vivo</Emphasis> Rat Model

Purpose

To evaluate the tumour growth delay of a peritoneal carcinomatosis (PC) of colorectal origin after intraperitoneal chemotherapy with paclitaxel/randomly-methylated-β-cyclodextrin (Pac/RAME-β-CD) versus Taxol® at normo- and hyperthermic conditions in rats.

Methods

Hyperthermic intraperitoneal chemotherapy (HIPEC) was performed 7 days post implantation of the tumour with both formulations at a Pac concentration of 0.24 mg/ml. Tumour evaluation was performed via positron emission tomography (PET) and magnetic resonance imaging (MRI) imaging, measuring tumour activity and tumour volume, respectively. Scans were taken at 2 and 7 days post treatment.

Results

PET and MRI data showed a significant reduction in tumour activity and tumour volume for rats treated with Pac/RAME-β-CD (at normo- and hyperthermic conditions), compared to the control group. Treatment with Taxol® did not result in a significant reduction of tumour activity and tumour volume. No significant differences between the normo- and hyperthermic conditions were observed for both formulations, indicating that hyperthermia and paclitaxel were not synergistic despite the direct cytotoxic effect of hyperthermia.

Conclusion

Monitoring tumour growth via PET and MRI indicated that Pac/RAME-β-CD inclusion complexes had a significantly higher efficacy compared to Taxol® in a rat model for peritoneal carcinomatosis.

     

<Emphasis Type="BoldItalic">In Vitro</Emphasis> Transport Activity and Trafficking of MRP2/<Emphasis Type="BoldItalic">ABCC2</Emphasis> Polymorphic Variants

Purpose

Multidrug resistance-associated protein 2 (MRP2/ABCC2) is an efflux pump that removes drugs and chemicals from cells. We sought to characterize the expression, trafficking and in vitro activity of seven single nucleotide polymorphisms (SNPs) in the ABCC2 gene.

Methods

ABCC2 SNPs were generated using site-directed mutagenesis and stably expressed in Flp-In HEK293 cells, which allows targeted insertion of transgenes within the genome. Total and cell surface expression of MRP2 as well as accumulation of substrates (calcein AM and 5(6)-carboxy-2′,7′-dichlorofluorescein diacetate, CDCF) were quantified in cells or inverted membrane vesicles expressing wild-type (WT) or variant forms.

Results

The cell surface expression of the C-24T-, G1249A-, G3542T-, T3563A-, C3972T- and G4544A-MRP2 variants was similar to WT-MRP2. While expression was similar, transport of calcein AM was enhanced in cells expressing the G3542T-, T3563A-, C3972T-, and G4544A-MRP2 variants. By comparison, cells expressing the C2366T-MRP2 variant had 40–50% lower surface expression, which increased the accumulation of calcein AM up to 3-fold. Accumulation of CDCF in inverted membrane vesicles expressing the C2366T-MRP2 variant was also reduced by 50%. In addition, the G1249A-MRP2 variant had decreased transport of CDCF.

Conclusions

Taken together, these data demonstrate that genetic variability in the ABCC2 gene influences the in vitro expression, trafficking, and transport activity of MRP2.

     

Effect of Chemical Binding of Doxorubicin Hydrochloride to Gold Nanoparticles, <Emphasis Type="Italic">Versus</Emphasis> Electrostatic Adsorption,on the <Emphasis Type="Italic">In Vitro</Emphasis> Drug Release and Cytotoxicity to Breast Cancer Cells

Purpose

The selective delivery of chemotherapeutic agent to the affected area is mainly dependent on the mode of drug loading within the delivery system. This study aims to compare the physical method to the chemical method on the efficiency of loading DOX.HCl to GNPs and the specific release of the loaded drug at certain tissue.

Method

Bifunctional polyethylene glycol with two different functionalities, the alkanethiol and the carboxyl group terminals, was synthesized. Then, DOX·HCl was covalently linked via hydrazone bond, a pH sensitive bond, to the carboxyl functional group and the produced polymer was used to prepare drug functionalized nanoparticles. Another group of GNPs was coated with carboxyl containing polymer; loading the drug into this system by the means of electrostatic adsorption. Finally, the prepared system were characterized with respect to size, shape and drug release in acetate buffer pH 5 and PBS pH 7.4 Also, the effect of DOX.HCl loaded systems on cell viability was assessed using MCF-7 breast cancer cell line.

Results

The prepared nanoparticles were spherical in shape, small in size and monodisperse. The release rate of the chemically bound drug in the acidic pH was higher than the electrostatically adsorbed one. Moreover, both systems show little release at pH 7.4. Finally, cytotoxicity profiles against human breast adenocarcinoma cell line (MCF-7) exhibited greater cytotoxicity of the chemically bound drug over the electrostatically adsorbed one.

Conclusion

Chemical binding of DOX·HCl to the carboxyl group of PEG coating GNPs selectively delivers high amount of drug to tumour-affected tissue which leads to reducing the unwanted effects of the drug in the non-affected ones.

     

Co-Delivery of Ciprofloxacin and Colistin in Liposomal Formulations with Enhanced <Emphasis Type="BoldItalic">In Vitro</Emphasis> Antimicrobial Activities against Multidrug Resistant <Emphasis Type="BoldItalic">Pseudomonas aeruginosa</Emphasis>

Purpose

This study aims to develop liposomal formulations containing synergistic antibiotics of colistin and ciprofloxacin for the treatment of infections caused by multidrug-resistant Pseudomonas aeruginosa.

Methods

Colistin (Col) and ciprofloxacin (Cip) were co-encapsulated in anionic liposomes by ammonium sulfate gradient. Particle size, encapsulation efficiency, in vitro drug release and in vitro antibiotic activities were evaluated.

Results

The optimized liposomal formulation has uniform sizes of approximately 100 nm, with encapsulation efficiency of 67.0% (for colistin) and 85.2% (for ciprofloxacin). Incorporation of anionic lipid (DMPG) markedly increased encapsulation efficiency of colistin (from 5.4 to 67.0%); however, the encapsulation efficiency of ciprofloxacin was independent of DMPG ratio. Incorporation of colistin significantly accelerated the release of ciprofloxacin from the DMPG anionic liposomes. In vitro release of ciprofloxacin and colistin in the bovine serum for 2 h were above 70 and 50%. The cytotoxicity study using A549 cells showed the liposomal formulation is as non-toxic as the drug solutions. Liposomal formulations of combinations had enhanced in vitro antimicrobial activities against multidrug resistant P. aeruginosa than the monotherapies.

Conclusions

Liposomal formulations of two synergistic antibiotics was promising against multidrug resistant P. aeruginosa infections.

     

Lipid-based Formulations for Danazol Containing a Digestible Surfactant,Labrafil M2125CS: <Emphasis Type="BoldItalic">In Vivo</Emphasis> Bioavailability and Dynamic <Emphasis Type="BoldItalic">In Vitro</Emphasis> Lipolysis

Purpose

To evaluate the use of Labrafil® M2125CS as a lipid vehicle for danazol. Further, the possibility of predicting the in vivo behavior with a dynamic in vitro lipolysis model was evaluated.

Methods

Danazol (28 mg/kg) was administered orally to rats in four formulations: an aqueous suspension, two suspensions in Labrafil® M2125CS (1 and 2 ml/kg) and a solution in Labrafil® M2125CS (4 ml/kg).

Results

The obtained absolute bioavailabilities of danazol were 1.5?±?0.8%; 7.1?±?0.6%; 13.6?±?1.4% and 13.3?±?3.4% for the aqueous suspension, 1, 2 and 4 ml Labrafil® M2125CS per kg respectively. Thus administration of danazol with Labrafil® M2125CS resulted in up to a ninefold increase in the bioavailability, and the bioavailability was dependent on the Labrafil® M2125CS dose. In vitro lipolysis of the formulations was able to predict the rank order of the bioavailability from the formulations, but not the absorption profile of the in vivo study.

Conclusions

The bioavailability of danazol increased when Labrafil® M2125CS was used as a vehicle, both when danazol was suspended and solubilized in the vehicle. The dynamic in vitro lipolysis model could be used to rank the bioavailabilities of the in vivo data.

     

Physical Characterization and Innate Immunogenicity of Aggregated Intravenous Immunoglobulin (IGIV) in an <Emphasis Type="Italic">In Vitro</Emphasis> Cell-Based Model

Purpose

To investigate in vitro the innate immune response to accelerated stress-induced aggregates of intravenous immunoglobulin (IGIV) using a well-defined human cell-line model, and to correlate the innate response to physical properties of the aggregates.

Methods

IGIV aggregates were prepared by applying various accelerated stress methods, and particle size, count and structure were characterized. Immune cell activation as tracked by inflammatory cytokines released in response to aggregates was evaluated in vitro using peripheral blood mononuclear cells (PBMC), primary monocytes and immortalized human monocyte-like cell lines.

Results

IGIV aggregates produced by mechanical stress induced higher cytokine release by PBMC and primary monocytes than aggregates formed by other stresses. Results with the monocytic cell line THP-1 paralleled trends in PBMC and primary monocytes. Effects were dose-dependent, enhanced by complement opsonization, and partially inhibited by blocking toll-like receptors (TLR2 and TLR4) and to a lesser extent by blocking Fc gamma receptors (FcγRs).

Conclusions

Stress-induced IGIV aggregates stimulate a dose-dependent cytokine response in human monocytes and THP-1 cells, mediated in part by TLRs, FcγRs and complement opsonization. THP-1 cells resemble primary monocytes in many respects with regard to tracking the innate response to IgG aggregates. Accordingly, the measurement of inflammatory cytokines released by THP-1 cells provides a readily accessible assay system to screen for the potential innate immunogenicity of IgG aggregates. The results also highlight the role of aggregate structure in interacting with the different receptors mediating innate immunity.