Respiratory Administration of Infliximab Dry Powder for Local Suppression of Inflammation.

The airways are verified as a relevant route to improve antibody therapeutic index with superior lung concentration but limited passage into systemic blood stream. The current research aimed to process spray-dried (SD) powder of Infliximab to assess the feasibility of respiratory delivery of antibody for local suppression of lung-secreted tumor necrosis factor α (TNFα). Molecular and structural stability of powders were determined through size exclusion chromatography (SEC-HPLC) and Fourier transform infrared (FTIR) spectroscopy. Particle properties were characterized by laser light scattering, twin stage impinger (TSI), and scanning electron microscopy (SEM). In vitro biological activity was quantified applying L-929 cell line. Ovalbumin (OVA)-challenged balb/c mice were employed to evaluate the anti-TNFα activity of antibody formulation as in vivo experimental model. SD sample consisting of 36 mg trehalose, 12 mg cysteine, and 0.05% of Tween 20 was selected with minimum aggregation/fragmentation rate constants of 0.07 and 0.05 (1/month) based on 1 and 2 months of storage at 40°C and relative humidity of 75%. Fine particle fraction (FPF) value of this formulation was 67.75% with desired particle size and surface morphology for respiratory delivery. EC50 was 8.176 and 6.733 ng/ml for SD Infliximab and Remicade®, respectively. SD antibody reduced TNFα (26.56 pg/ml) secretion in mouse lung tissue, more than 2 orders of magnitudes comparing positive control group (TNFα, 68.34 pg/ml). The success of antibody inhalation mainly depended on the spray drying condition, formulation components, and stability of antibody within aerosolization. Inhaled Infliximab could be a potential drug for local inhibition of lung inflammation.

The effect of excipients on the stability and aerosol performance of salmon calcitonin dry powder inhalers prepared via the spray freeze drying process.

Spray freeze drying was developed to produce dry powders suitable for applications such as inhalation delivery. In the current study, the spray freeze drying technique was employed to produce inhalable salmon calcitonin microparticles. Effects of the carrier type, concentration of hydroxyl propyl-ß-cyclodextrin and the presence of Tween 80 on the chemical and structural stability, as well as on the aerosol performance of the particles were investigated. The results indicated that hydroxyl propyl-ß-cyclodextrin had the most important effect on the chemical stability of the powder and strongly increased its stability by increasing its concentration in the formulation. Chemically stable formulations (over 90 % recovery) were selected for further examinations. Fluorescence spectroscopy and circular dichroism suggested that the formulations were structurally stable. Aerosol performance showed that the Tween-free powders produced higher fine particle fraction values than the formulations containing Tween (53.7 vs. 41.92 % for trehalose content and 52.85 vs. 43.06 % for maltose content).

A comparative study on the physicochemical and biological stability of IgG1 and monoclonal antibodies during spray drying process.

BACKGROUND: The main concern in formulation of antibodies is the intrinsic instability of these labile compounds. To evaluate the physicochemical stability of antibody in dry powder formulations, physical stability of IgG1 and a monoclonal antibody (trastuzumab) during the spray drying process was studied in a parallel study and the efficacy of some sugar based excipients in protection of antibodies was studied. RESULTS: The SDS-PAGE analysis showed no fragmentation of antibodies after spray drying in all formulations. The secondary structure of antibodies contained 40.13 to 70.19% of ß structure in dry state. Also, CD spectroscopy showed the similar secondary structure for trastuzumab after reconstitution in water. ELISA analysis and cell culture studies were conducted in order to evaluate bioactivity of monoclonal antibody. Formulations containing combination of excipients provided maximum tendency of trastuzumab to attach to the ELISA antigen (86.46% ± 2.3) and maximum bioactivity when incubated with SKBr3 cell line (the cell viability was decreased to 65.99% ± 4.6). Incubation of formulations with L929 cell line proved the biocompatibility of the excipients and non-toxic composition of formulations. CONCLUSION: The IgG1 and trastuzumab demonstrated similar behavior in spray drying process. The combination of excipients containing trahalose, hydroxypropyl beta cyclodextrin and beta cyclodextrin with proper ratio improved the physical and chemical stability of both IgG1 and monoclonal antibody.

The effective encapsulation of a hydrophobic lipid-insoluble drug in solid lipid nanoparticles using a modified double emulsion solvent evaporation method.

Raloxifene HCl (RH), a selective estrogen receptor modulator (SERM), is indicated for the prophylaxis or treatment of postmenopausal osteoporosis. RH shows extremely poor bioavailability due to limited solubility and an extensive intestinal/hepatic first-pass metabolism. Solid lipid nanoparticles (SLNs) are valuable carriers that can enhance drug bioavailability. However, in the case of RH, the encapsulation of the drug in SLNs remains a challenge because of its poor solubility in both water and lipids. In this study, a series of RH-containing SLNs (RH-SLNs) were generated using a modified double emulsion solvent evaporation (DESE) method. Briefly, RH with various drug/lipid ratios was solubilized in the inner core of a double emulsion using different water/organic solvent mixtures. Our best formulation was achieved with the formation of negatively charged nanoparticles, 180nm in diameter, with an encapsulation and loading efficiency of 85% and 4.5%, respectively. It also showed a Fickian mechanism of the drug release in the basic dissolution media. Thermal analysis revealed a distinct decrease in the crystallinity of lipids and RH in comparison with the unprocessed materials. The results of a cell viability assay also showed a better antiproliferative effect of the drug-loaded SLNs versus the free drug solution. Thus, these results indicated that the modified DESE method could be proposed for the effective encapsulation of RH in SLNs with appropriate physicochemical and biological properties.

Stability studies of chitosan-DNA-FAP-B nanoparticles for gene delivery to lung epithelial cells.

A successful gene delivery system requires efficiency and stability during storage. Stability studies are imperative for nanomedicines containing biotechnological products such as plasmids and targeting peptides. Chitosan-DNA-FAP-B nanoparticles are novel non-viral vectors for specific gene delivery to the lung epithelial cells. In this study, the storage stability of chitosan-DNA-FAP-B nanoparticles at -20, 5 and 24 °C was examined. Size, zeta potential and transfection efficiency of these nano-particles in storage were also evaluated. Stability studies showed that chitosan-DNA-FAP-B nanoparticles were stable after 1 month when stored at -20 °C and retained their initial size, zeta potential and transfection efficiency. However, their stability was not desirable at 5 and 24 °C. Based on these results, it can be concluded that chitosan-DNA-FAP-B nanoparticles can be a promising candidate for gene delivery to lung epithelial cells with good storage stability at -20 °C during 1 month.

Loading hydrophilic drug in solid lipid media as nanoparticles: statistical modeling of entrapment efficiency and particle size.

Solid lipid nanoparticle (SLN) is a very well tolerated carrier systems for dermal application due to the employment physiological and/or biodegradable lipids. The effects of five factors, two categorical and three quantitative factors, were studied on the mean diameter and entrapment efficiency of the produced SLNs using response surface method (RSM), D-optimal design. Two methods of microemulsion and solvent diffusion and two types of lipid, cetyl palmitate and stearic acid, were examined comparatively. The quantitative variables were studied in three levels; amount of original Paromomycin (60, 90 and 120 mg), fraction of surfactant (0.5, 0.75 and 1 w/v %) and drug to lipid ratio (2, 4 and 6). Mean particle size and entrapment efficiency of the loaded Paromomycin were modeled statistically and the optimal condition was determined to approach to the maximum entrapment efficiency. The drug release profile of the optimal formulated material was examined in aqueous media and 64% of the Paromomycin loaded in SLNs was gradually released during 24h, which reveals efficient prolonged release of the drug.

Preparation and evaluation of inhalable itraconazole chitosan based polymeric micelles.

BACKGROUND: This study evaluated the potential of chitosan based polymeric micelles as a nanocarrier system for pulmonary delivery of itraconazole (ITRA). METHODS: Hydrophobically modified chitosan were synthesized by conjugation of stearic acid to the hydrophilic depolymerized chitosan. FTIR and 1HNMR were used to prove the chemical structure and physical properties of the depolymerized and the stearic acid grafted chitosan. ITRA was entrapped into the micelles and physicochemical properties of the micelles were investigated. Fluorescence spectroscopy, dynamic laser light scattering and transmission electron microscopy were used to characterize the physicochemical properties of the prepared micelles. The in vitro pulmonary profile of polymeric micelles was studied by an air-jet nebulizer connected to a twin stage impinger. RESULTS: The polymeric micelles prepared in this study could entrap up to 43.2±2.27 µg of ITRA per milliliter. All micelles showed mean diameter between 120-200 nm. The critical micelle concentration of the stearic acid grafted chitosan was found to be 1.58×10-2 mg/ml. The nebulization efficiency was up to 89% and the fine particle fraction (FPF) varied from 38% to 47%. The micelles had enough stability to remain encapsulation of the drug during nebulization process. CONCLUSIONS: In vitro data showed that stearic acid grafted chitosan based polymeric micelles has a potential to be used as nanocarriers for delivery of itraconazole through inhalation.

Evaluation of bioactivity and pharmacokinetic characteristics of PEGylated P.pastoris-expressed erythropoietin.

High costs of production and relatively short serum half-life of mammalian cell-derived recombinant human erythropoietin (rHuEpo) necessitate finding and developing superior hosts/technologies for more efficient production of longer-acting erythropoietic agents. With these aims, we provide the first report on reductive alkylation of low-cost P.pastoris-expressed rHuEpo (PPEpo) with PEG-aldehyde. The PCR-amplified cDNA of native rHuEpo was cloned into the pPICZαA vector and transformed into the yeast Pichia pastoris. The best expressing transformant was selected and employed for secreted-expression of PPEpo using the standard protocols. Purified PPEpo was N-terminally PEGylated with 20-kDa mPEG-propionaldehyde in a low pH (5) condition. The in vitro and in vivo biological activities of purified mono-PEGylated PPEpo was evaluated by the UT-7 cells proliferation assay and normocythaemic mice assay, respectively. Pharmacokinetic parameters were determined following intravenous administration of Epo proteins in rabbits. While PPEpo showed a higher in vitro bioactivity compared to rHuEpo, no in vivo efficiency was determined for PPEpo. However, the in vivo activity of PEG-PPEpo conjugate was comparable to that of rHuEpo. Pharmacokinetic studies showed that the terminal half-life and mean residence time of PEG-PPEpo were increased approximately 4-fold and 6.5-fold respectively, compared with those of PPEpo. The results indicate that N-terminal PEGylation of Pichia-expressed Epo could be considered as a promising approach for generating cost-effective and long-acting erythropoiesis-stimulating agents.

Delivery of a cocktail DNA vaccine encoding cysteine proteinases type I, II and III with solid lipid nanoparticles potentiate protective immunity against Leishmania major infection.

Earlier generations of Leishmania vaccines have reached the third-phase of clinical trials, however none of them have shown adequate efficacy due to lack of an appropriate adjuvant. In this study, cationic solid lipid nanoparticles (cSLNs) were used to formulate three pDNAs encoding L. major cysteine proteinase type I (cpa), II (cpb) and III (cpc). BALB/c mice were immunized twice with a 3-week interval, with SLN-pcDNA-cpa/b/c, pcDNA-cpa/b/c, SLN, SLN-pcDNA and PBS. Footpad assessments, parasite burden, cytokine and antibody responses were evaluated. Mice vaccinated with SLN-pcDNA-cpa/b/c significantly (p<0.05) showed higher protection levels with specific Th1 immune response development compared to other groups. This is the first report demonstrating cSLNs as a nanoscale vehicle boosting immune response quality and quantity; in a designable trend. The nanomedical feature of this novel formulation can be applied for wide-spread use in genetic vaccination against leishmaniasis, which is currently managed only through relatively ineffectual therapeutic regimens.

Entrapment of 5-fluorouracil into PLGA matrices using supercritical antisolvent processes.

OBJECTIVES: Two different supercritical antisolvent processes were performed to co-precipitate 5-fluorouracil (5-FU) and poly(lactide-co-glycolide) simultaneously. 5-FU is a hydrophilic antitumor agent, and is more effective when administered at a lower dose for a longer period of time. METHODS: Controlled-release polymeric systems of 5-FU were produced, and morphology, thermal behavior, in-vitro release and cytotoxicity of microparticles were analysed. KEY FINDINGS: Dissolution studies showed that 33% of drug was released in 21 days, which represents a long-lasting profile. To evaluate the efficacy of the released drug on cancer cells, the MTT assay cytotoxicity test was performed using human lung carcinoma A549 cell lines. There was no significant difference between the cell inhibition rates of the released drug and unprocessed 5-FU at the same drug concentration level. IC50 values were 69.12 mg/ml for unprocessed 5-FU and 68.71 mg/ml for the released drug. CONCLUSIONS: Application of supercritical processing for co-precipitation of 5-FU and PLGA provided mild and non-aqueous conditions, so the hydrophilic drug incorporated in the polymer had good stability during the process.

Formulation and in vitro evaluation of ciprofloxacin containing niosomes for pulmonary delivery.

In order to develop a niosome-encapsulated ciprofloxacin (CPFX) HCl formulation for pulmonary delivery, the feasibility of encapsulation of CPFX in niosomes, its stability and nebulization capability was evaluated. Various combinations of nonionic surfactants with cholesterol were used to prepare the formulations. The in vitro deposition data of the niosomal formulations were examined using an Andersen cascade impactor. Formulations composed of Span 60 and Tween 60 in combination with 40 mol% of cholesterol exhibited high encapsulation efficacy and stability and also had fine particle fraction and nebulization efficiency of about 61.9% ± 1.0 and 77.9 ± 2.8, respectively. Minimal inhibitory concentration of the niosomal CPFX against some pulmonary pathogens were lower than free CPFX. Using the MTT assay in human lung carcinoma cell line (A549), niosome-entrapped CPFX showed significantly lower cytotoxicity in comparison to the free drug. These results indicate that niosome can be used as a carrier for pulmonary delivery of CPFX via nebulization.

High Expression of Methylotrophic Yeast-Derived Recombinant Human Erythropoietin in a pH-Controlled Batch System.

To accomplish the worldwide demand for recombinant human erythropoietin (rHuEpo) as a therapeutic, application of cost-efficient expression system of methylotrophic yeast Pichia pastoris (P. pastoris) rather than mammalian cells is indispensable. Herein, a report on high levels secreted-expression of Pichia-derived rHuEpo by batch fermentation in a pH stabilized format is presented. The full length cDNA of rHuEpo was inserted into pPICZαA vector under control of AOX1 promoter, downstream of the secretion-α-factor and electroporated into P. pastoris strain X33. The highest expression transformant was selected by screening among the colonies surviving high concentration of Zeocin (1.0 mg/ml), followed by comparative small scale expression analysis by ELISA. Stabilization of pH around 6.0 by adding phosphoric acid into the culture media during induction period, improved the yield of expression to 150 mg/l of the media. Single-step Nickel-affinity chromatography was employed for purification of rHuEpo-6xHis to 80% purity. Analyses by SDS-PAGE, Western blot and N-terminal protein sequencing confirmed the authenticity of the 33 kDa expressed rHuEpo with a native N-terminal indicating the proper cleavage of secretion-signal. Results of this study, further confirmed the possibility of employing methylotrophic yeast for scaled up production aims of rHuEpo as a cost-efficient expression system when provided evidence for higher expression yields through application of pH-controlled systems.

The effect of formulation variables on the characteristics of insulin-loaded poly(lactic-co-glycolic acid) microspheres prepared by a single phase oil in oil solvent evaporation method.

Biodegradable polymeric microspheres are ideal vehicles for controlled delivery applications of drugs, peptides and proteins. Amongst them, poly(lactic-co-glycolic acid) (PLGA) has generated enormous interest due to their favorable properties and also has been approved by FDA for drug delivery. Insulin-loaded PLGA microparticles were prepared by our developed single phase oil in oil (o/o) emulsion solvent evaporation technique. Insulin, a model protein, was successfully loaded into microparticles by changing experimental variables such as polymer molecular weight, polymer concentration, surfactant concentration and stirring speed in order to optimize process variables on drug encapsulation efficiency, release rates, size and size distribution. A 2(4) full factorial design was employed to evaluate systematically the combined effect of variables on responses. Scanning electron microscope (SEM) confirmed spherical shapes, smooth surface morphology and microsphere structure without aggregation. FTIR and DSC results showed drug-polymer interaction. The encapsulation efficiency of insulin was mainly influenced by surfactant concentration. Moreover, polymer concentration and polymer molecular weight affected burst release of drug and size characteristics of microspheres, respectively. It was concluded that using PLGA with higher molecular weight, high surfactant and polymer concentrations led to a more appropriate encapsulation efficiency of insulin with low burst effect and desirable release pattern.

An efficient biosurfactant-producing bacterium Pseudomonas aeruginosa MR01, isolated from oil excavation areas in south of Iran.

A bacterial strain was isolated and cultured from the oil excavation areas in tropical zone in southern Iran. It was affiliated with Pseudomonas. The biochemical characteristics and partial sequenced 16S rRNA gene of isolate, MR01, was identical to those of cultured representatives of the species Pseudomonas aeruginosa. This bacterium was able to produce a type of biosurfactant with excessive foam-forming properties. Compositional analysis revealed that the extracted biosurfactant was composed of high percentages lipid ( approximately 65%, w/w) and carbohydrate ( approximately 30%, w/w) in addition to a minor fraction of protein ( approximately 4%, w/w). The best production of 2.1g/l was obtained when the cells were grown on minimal salt medium containing 1.2% (w/v) glucose and 0.1% (w/v) ammonium sulfate supplemented with 0.1% (w/v) isoleucine at 37 degrees C and 180rpm after 2 days. The optimum biosurfactant production pH value was found to be 8.0. The MR01 could reduce surface tension to 28mN/m and emulsified hexadecane up to E24 approximately 70. The results obtained from time course study indicated that the surface tension reduction and emulsification potential was increased in the same way to cell growth. However, maximum biosurfactant production occurred and established in the stationary growth phase (after 84h). Fourier Transform Infrared spectrum of extracted biosurfactant indicates the presence of carboxyl, amine, hydroxyl and methoxyl functional groups. Thermogram of biosurfactant demonstrated three sharp endothermic peaks placing between 200 and 280 degrees C. The core holder flooding experiments demonstrated that the oil recovery efficiencies varied from 23.7% to 27.1% of residual oil.

Intranasal bioavailability of insulin from carbopol-based gel spray in rabbits.

The purpose of this study was to investigate the nasal absorption of insulin from a carbopol-based nasal gel spray in rabbits. An insulin nasal gel was prepared by dispersing carbopol in distilled water, followed by the addition of insulin solution, then neutralization and viscosity adjustment. The nasal absorption of insulin from the gel, in conscious rabbits, was evaluated in comparison with absorption from an insulin solution. The absolute bioavailability of insulin from the nasal gel was studied using blood glucose level in comparison to intravenous injection. The insulin gel formulation produced a significant hypoglycemic response in rabbits, whereas no response was seen following administration of the insulin solution formulation. The bioavailability of insulin from the nasal gel formulation was 20.6% compared with the intravenous injection. The results of the present study suggest that the carbopol gel promotes the nasal absorption of insulin in rabbit model and due to its sprayability with commercially available spray pumps, could be considered as a preferred platform in nasal drug administration.

Development and physical characterization of sorbitan monoester niosomes for insulin oral delivery.

Niosomes of sorbitan monoesters (Span 20, 40, 60, and 80) were prepared using the film hydration method without sonication. Unlike the other surfactants, Span 80 did not form niosomes in the absence of a sufficient amount of cholesterol. The size of vesicles depended on the cholesterol molar ratio or charge incorporation. The amount of insulin released in simulated intestinal fluid from Span 40 and 60 was lower than Span 20 and 80 vesicles. Vesicles containing Span 60 showed the highest protection of insulin against proteolytic enzymes and good stability in the presence of sodium desoxycholate and storage temperatures.

A rapid and sensitive method for simultaneous determination of insulin and A21-desamido insulin by high-performance liquid chromatography.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV-detection at room temperature has been developed for the analysis of insulin and its main degradation product, A21-desamido insulin. Octadecylsilica was used as stationary phase and a mixture of water and acetonitrile containing tetrametylammonium hydroxide as eluent. The method produced linear response over the concentration range of 10-100 microg/ml, with an average accuracy of 97.35+/-1.36% as well as average intra- and inter-day variations of 1.29 and 5.24%, respectively. The limits of detection and quantitation of the method were 0.25 and 0.75 microg/ml, respectively. Considering the analysis specifications, the system is suitable for direct analysis of routine formulations and stability studies. By this method human insulin can be separated from its principal degradation product, A21-desamido insulin. Also there is no need for any particular requirement and it is easily available in most of laboratories.