Molecular and Structural Stability of Infliximab: Spray-Dried Powder versus Freeze-Dried Cake.

he current study compared the stability of Infliximab powder following 2 drying procedures namely spray drying and freeze drying. Introduction: Infliximab as a chimeric anti-TNFα monoclonal antibody was approved for the treatment of inflammatory diseases namely Crohn's disease and rheumatoid arthritis. In prospect of preparing stable formulation of Infliximab, freeze drying and spray drying were compared as processing method. Methods and Results: Spray-dried formulation was prepared in the presence of Trehalose and Sucrose besides Cysteine. Powders were characterized via SEC-HPLC to quantify the level of induced aggregates/fragments after process along with upon 1 and 3 months of storage at 45̊C. Kinetic of aggregation and fragmentation was calculated for each sample. FTIR-spectroscopic assessments were employed to determine the secondary structure of antibody. Trehalose generated more stable particle within spray drying, with least aggregation and fragmentation rate constants of 0.22 and 0.27 (1/month). Combination of Cysteine and Trehalose significantly reduced aggregation upto 0.87, 1.07 and 2.26 % after process, up on 1 and 3 months of storage (rate constant of aggregation of 0.14,(1/month)). Fragmentation was 0.31, 0.38 and 0.98 % respectively with 0.17 (1/month) rate constant of fragmentation. The induced aggregates in Remicade were 0.11, 0.18 and 0.32% (aggregation rate constant:0.15 (1/month)) and fragments were 0.1, 0.14 and 0.29% after process, 1 month and 3 months at 45̊C (fragmentation rate constant of 0.15 (1/month)). The conformation of antibody was shown to be composed of 66.68% and 69.43% beta-sheet in spray-dried powder and freeze-dried cake (Remicade®) respectively. Conclusions: This study demonstrated that, both spray drying and freeze drying may be efficient for powder production of Infliximab with regards to molecular and structural stability after storage at high temperatures

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 β 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

C-Terminal Domain Deletion Enhances the Protective Activity of cpa/cpb Loaded Solid Lipid Nanoparticles against Leishmania major in BALB/c Mice

Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis with an annual incidence of approximately 2 million cases and is endemic in 88 countries, including Iran. CL's continued spread, along with rather ineffectual treatments and drug-resistant variants emergence has increased the need for advanced preventive strategies. We studied Type II cysteine proteinase (CPA) and Type I (CPB) with its C-terminal extension (CTE) as cocktail DNA vaccine against murine and canine leishmaniasis. However, adjuvants' success in enhancing immune responses to selected antigens led us to refocus our vaccine development programs. Herein, we discuss cationic solid lipid nanoparticles' (cSLN) ability to improve vaccine-induced protective efficacy against CL and subsequent lesion size and parasite load reduction in BALB/c mice. For this work, we evaluated five different conventional as well as novel parasite detection techniques, i.e., footpad imaging, footpad flowcytometry and lymph node flowcytometry for disease progression assessments. Vaccination with cSLN-cpa/cpb-CTE formulation showed highest parasite inhibition at 3-month post vaccination. Immunized mice showed reduced IL-5 level and significant IFN-ã increase, compared to control groups. We think our study represents a potential future and a major step forward in vaccine development against leishmaniasis

FORMATION OF SALBUTAMOL SULPHATE MICROPARTICLES USING SOLUTION ENHANCED DISPERSION BY SUPERCRITICAL CARBON DIOXIDE

ABSTRACT Salbutamol sulphate (SS) was precipitated by supercritical carbon dioxide (SC-CO 2 ) using a homemade system at two different pressures. This process is characterized by spraying a methanolic solution of the drug into the supercritical fluid (SCF), extraction of the solvent by SC-CO 2 and formation of drug particles. The morphology and size distribution of precipitated SS particles were characterized using scanning electron microscope and laser diffraction particle size analyzer respectively. FTIR spectra were used before and after processing to asses crystal modifications. Depending on the processing conditions, needle-like and flake-like particles with different size distributions were observed. The average size of the flake like particles was less than needle-like particles and the span parameter showed a narrower size distribution of the processed in comparison with the unprocessed materials. Analysis by FTIR showed that there was no significant effect on the structure of the drug under these processing conditions

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-β-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-β-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)

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 24 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. © 2009 Elsevier B.V. All rights reserved

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 andtransmission 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

Ispitivanje stabilnosti kitozanskih nanočestica s DNA-FAP-B za gensku isporuku u epitelne stanice pluća

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 ºC was examined. Size, zeta potential and transfection efficiency of these nanoparticles 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 ºC. 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 ºC during 1 month.Dobar sustav za gensku isporuku mora biti učinkovit i stabilan prilikom skladištenja. Stoga su ispitivanja stabilnosti nužna za nonosustave s biotehnološkim produktima kao što su plazmidi i ciljni peptidi. Kitozanske nanočestice s DNA-FAP-B su novi nevirusni vektori za specifičnu gensku isporuku u epitelne stanice pluća. U radu je ispitana njihova stabilnost na –20, 5 i 24 ºC, veličina čestica, zeta potencijal i transfekcijska učinkovitost. Dobiveni rezultati pokazuju da su kitozanske nanočestice s DNA-FAP-B stabilne nakon mjesec dana skladištenja na –20 °C i da zadržavaju početnu veličinu, zeta potencijal i sposobnost transfekcije, a nisu stabilne skladištenjem na 5 i 24 ºC. Može se zaključiti da su kitozanske nanočestice s DNA-FAP-B potencijalni kandidati za gensku isporuku u epitelne stanice pluća te da su dovoljno stabilne ako se skladište mjesec dana na –20 ºC