A Novel Approach for Development of Intraocular Biodegradable Ranibizumab Implant: A Solution for Stability of Protein Activity.

Purpose: Ranibizumab is a monoclonal antibody fragment, targeting all isoforms of vascular endothelial growth factor A (VEGF-A), a protein involved in angiogenesis. It is used to treat age-related macular degeneration (AMD), retinal vein occlusion (RVO), and diabetic macular edema (DME), which are associated with blindness worldwide. However, proper treatment can decrease the loss of vision in about 90% of patients. Because of poor drug uptake in topical therapy and several adverse side effects of systemic irregularities and intravitreal injections, sustained-release drug delivery systems are more suitable for treatment. However, there are many challenges in the development of these systems due to the loss of protein activities. Methods: After drug complexation by the ion pairing method and preparation of a polymeric implant, containing the drug, the characteristics of the complexes were examined by Fourier-transform infrared spectroscopy and circular dichroism spectroscopy. The stability of antibody activity and biocompatibility of the released drug from the implant were assessed by bioassays and MTT assay, respectively. Finally, the release kinetics were investigated. Results: The bioassays showed the higher activity of the drug complex, compared to the free form, besides good biocompatibility in vitro. Also, the release data confirmed sustained and controlled release characteristics for the prepared implant. Conclusion: In this study, for the first time, we proposed a method for developing a sustained-release intraocular implant, consisting of ranibizumab by the heating method. This method allows for the industrial production of ranibizumab by extrusion and eliminates the complications related to reservoir systems.

Design and In Vitro Evaluation of a Slow-Release Intraocular Implant of Betamethasone.

Posterior eye diseases are a common cause of vision problems in developing countries, which have encouraged the development of new treatment models for these degenerative diseases. Intraocular implants are one of the drug delivery systems to the posterior region of the eye. Using these implants, the blood-eye barrier can be bypassed; the complications caused by repeated in vitro administrations can be eliminated, and smaller amounts of the drug would be used during the treatment process. Meanwhile, biodegradable implants have received more attention due to their biodegradable structure and the lack of need for re-surgery to remove the rest of the system from the eye. The aim of this study is to employ biodegradable implants composed of polyethylene glycol (PEG) and 3-hydroxybutyrate-co-3-hydroxyvalerat (PHBV) to deliver betamethasone to the back of the eye in the treatment of retinopathy. PHBV polymer has been selected as the main polymer with a certain ratio of drug to polymer for fabrication of enamel and different amounts of PEG with three molecular weights used as pore generators to control drug release over a period of time. Based on the analysis of the results of differential scanning calorimetry (DSC) and FTIR spectroscopy, none of the polymers were degraded in the temperature range of the manufacturing process, and among betamethasone derivatives, the best option for implant preparation is the use of its basic form. Drug release studies over a period of three months showed that implants containing PHBV HV2% and PEG 6000 had a more appropriate release profile.

A mechanistic study of the effect of transferrin conjugation on cytotoxicity of targeted liposomes.

This study was performed to prepare 5-fluorouracil (5FU) containing targeted liposomes for the safety and efficacy enhancement. Liposomes were prepared using thin layer method and transferrin (Tf) was employed as the targeting ligand. Morphology of 5FU-loaded liposomes was assessed by transmission electron microscopy (TEM). The in vitro cytotoxicity was investigated via MTT assay on HT-29, CT26 and fibroblast cells. Mitochondrial membrane and cell death evaluations were also investigated. Resulted showed that the encapsulation efficiency (EE%) and particle size of the liposomes were 40.12% and 130 nm, respectively. TEM image implied that liposomes were spherical in shape. In cancer cells, targeted liposomes triggered the mitochondrial apoptotic pathway by lower production of reactive oxygen species (ROS) (63.58 vs 84.95 fluorescence intensity), reduced mitochondrial membrane potential and releasing of cytochrome c (68.66 vs 51.13 ng/mL). The results of this study indicated that Tf-targeted 5FU liposomes can be employed as promising nanocarrier for the delivery of drugs to cancer cells.

Nanoparticles Prepared From N,N-Dimethyl-N-Octyl Chitosan as the Novel Approach for Oral Delivery of Insulin: Preparation, Statistical Optimization and In-vitro Characterization.

In this study, N,N-Dimethyl-N-Octyl chitosan was synthesized. Nanoparticles containing insulin were prepared using PEC method and were statistically optimized using the Box-Behnken response surface methodology. The independent factors were considered to be the insulin concentration, concentration and pH of the polymer solution, while the dependent factors were characterized as the size, zeta potential, PdI and entrapment efficiency. The optimized nanoparticles were morphologically studied using SEM. The cytotoxicity of the nanoparticles on the Caco-2 cell culture was studied using the MTT cytotoxicity assay method, while the permeation of the insulin nanoparticles across the Caco-2 cell monolayer was also determined. The optimized nanoparticles posed appropriate physicochemical properties. The SEM morphological studies showed spherical to sub-spherical nanoparticles with no sign of aggregation. The in-vitro release study showed that 95.5 ± 1.40% of the loaded insulin was released in 400 min. The permeability studies revealed significant enhancement in the insulin permeability using nanoparticles prepared from octyl chitosan at 240 min (11.3 ± 0.78%). The obtained data revealed that insulin nanoparticles prepared from N,N-Dimethyl-N-Octyl chitosan can be considered as the good candidate for oral delivery of insulin compared to nanoparticles prepared from N,N,N-trimethyl chitosan.

Ocular implant containing bevacizumab-loaded chitosan nanoparticles intended for choroidal neovascularization treatment.

Choroidal neovascularization (CNV) is among the leading causes of blindness worldwide. Bevacizumab has demonstrated promising effects on CNV treatment; however, frequent intravitreal injection is its major drawback. Current study aimed to address this issue by developing a sustained release formulation through nanoparticles of bevacizumab imbedded in an ocular implant. Bevacizumab-loaded chitosan nanoparticles were prepared by ionic gelation method and inserted in the matrix of hyaluronic acid and zinc sulfate. Despite the common approaches in using ultraviolet (UV)-spectrophotometry, microprotein-Bradford, and bicinchoninic acid (BCA), assay for protein assessment, our results revealed a remarkable UV-Vis absorption overlap of protein and chitosan during these analysis and thus enzyme-linked immunosorbent assay was employed for the antibody concentration assay. The size of optimized nanoparticles obtained through statistical analysis based on design of experiments was 78.5 ± 1.9 nm with polydispersity index of 0.13 ± 0.05 and the entrapment-efficiency and loading-efficiency were 67.6 ± 6.7 and 15.7 ± 5.7%, respectively. The scanning electron microscopy and confocal microscopy images revealed a homogenous distribution of nanoparticles in the implant matrix and the release test results indicated an appropriate extended release of bevacizumab from the carrier over two months. In conclusion, the prepared system provided a sustained release bevacizumab delivery formulation which can introduce a promising ocular drug delivery system intended for posterior segment disease. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2261-2271, 2018.

Preparation, characterization and in vivo evaluation of a combination delivery system based on hyaluronic acid/jeffamine hydrogel loaded with PHBV/PLGA blend nanoparticles for prolonged delivery of Teriparatide.

In the current study, biodegradable PHBV/PLGA blend nanoparticles (NPs) containing Teriparatide were loaded in hyaluronic acid/jeffamine (HA-JEF ED-600) hydrogel to prepare a combination delivery system (CDS) for prolonged delivery of Teriparatide. The principal purpose of the present study was to formulate an effective and prolonged Teriparatide delivery system in order to reduce the frequency of injection and thus enhance patient's compliance. Morphological properties, swelling behaviour, crosslinking efficiency and rheological characterization of HA-JEF ED-600 hydrogel were evaluated. The CDS was acquired by adding PHBV/PLGA NPs to HA-JEF ED-600 hydrogel simultaneously with crosslinking reaction. The percentage of NPs incorporation within the hydrogel as well as the loading capacity and morphology of Teriparatide loaded CDS were examined. Intrinsic fluorescence and circular dichroism spectroscopy proved that Teriparatide remains stable after processing. The release profile represented 63% Teriparatide release from CDS within 50days with lower burst release compared to NPs and hydrogel. MTT assay was conducted by using NIH3T3 cell line and no sign of reduction in cell viability was observed. Based on Miller and Tainter method, LD50 of Teriparatide loaded CDS was 131.8mg/kg. In vivo studies demonstrated that Teriparatide loaded CDS could effectively increase serum calcium level after subcutaneous injection in mice. Favourable results in the current study introduced CDS as a promising candidate for controlled delivery of Teriparatide and pave the way for future investigations in the field of designing prolonged delivery systems for other peptides and proteins.

Efficient gene delivery to primary human retinal pigment epithelial cells: The innate and acquired properties of vectors.

The purpose of this study is designing non-viral gene delivery vectors for transfection of the primary human retinal pigment epithelial cells (RPE). In the design process of gene delivery vectors, considering physicochemical properties of vectors alone does not seem to be enough since they interact with constituents of the surrounding environment and hence gain new characteristics. Moreover, due to these interactions, their cargo can be released untimely or undergo degradation before reaching to the target cells. Further, the characteristics of cells itself can also influence the transfection efficacy. For example, the non-dividing property of RPE cells can impede the transfection efficiency which in most studies was ignored by using immortal cell lines. In this study, vectors with different characteristics differing in mixing orders of pDNA, PEI polymer, and PLGA/PEI or PLGA nanoparticles were prepared and characterized. Then, their characteristics and efficacy in gene delivery to RPE cells in the presence of vitreous or fetal bovine serum (FBS) were evaluated. All formulations showed no cytotoxicity and were able to protect pDNA from premature release and degradation in extracellular media. Also, the adsorption of vitreous or serum proteins onto the surface of vectors changed their properties and hence cellular uptake and transfection efficacy.

An Estimation of the Potential Utilization in Iranian Pharmaceutical Industry Involved in the Stock Exchange, 2008-2012.

The aim of this study was to measure the potential of production and the capacity used in the pharmaceutical industry. Capacity use is the actual production rate to the potential output, which reflects the gap between actual production and production capacity. Through econometric methods, translog cost function in the short run along with functions of share cost of production factors is estimated through seemingly unrelated repeated regression (SURE) as a multivariate regression analysis provided by zeller. During the study the capacity used is decreasing. The capacity used, which calculated by weighted average, also decreased and the amount during the study period is much less than the simple average of the industry. Average capacity utilization in the industry over five years of study is equal to 57% while the average capacity used calculated by the weighted of industry average is 37%. To enhance the economic potential requires a proper use of resources, creation of favorable economic structure and productivity of the industry. Due to the large amount of unused capacity in the pharmaceutical industry there is no need to invest anymore unless in new grounds and it is obvious that more investment will change using capacity.

Preparation, statistical optimisation and in vitro characterisation of poly (3-hydroxybutyrate-co-3-hydroxyvalerate)/poly (lactic-co-glycolic acid) blend nanoparticles for prolonged delivery of teriparatide.

The purpose of this study was the preparation, optimisation and in vitro characterisation of PHBV and PLGA blend nanoparticles (NPs) for prolonged delivery of Teriparatide. Double emulsion solvent evaporation technique was employed for the fabrication of NPs. The nanoformulation was optimised using the Box-Behnken methodology. The morphological properties of NPs were assessed by both SEM and transmission electron microscopy (TEM). Encapsulation of Teriparatide within the NPs and lacking of chemical bonds between drug and copolymers were proved by XRPD, FTIR and DSC. The structural stability of Teriparatide after processing was confirmed by fluorescence spectrometry. The average size of optimised NPs was 250.0 nm with entrapment efficiency (EE) of 89.5% and drug loading (DL) of 5.0%. Teriparatide release from optimised NPs led to 64.4% release over 30 days and it showed a diffusion-based mechanism. Based on the favourable results, PHBV/PLGA blend NPs could be a promising candidate for designing a controlled release formulation of Teriparatide.

Economic burden of hepatitis B virus-related diseases: evidence from iran.

BACKGROUND: Hepatitis B infection is still the main cause of chronic liver disease in Iran, which is associated with significant economic and social costs. OBJECTIVES: This study aimed to estimate the financial burden caused by CHB infection and its complications in Iran. PATIENTS AND METHODS: Prevalence-based and bottom-up approaches were used to collect the data. Data on direct medical costs were extracted from outpatient medical records in a referral gastroenterology and hepatology research center, inpatient medical records in several major hospitals in Tehran and Shiraz in 2013, and the self-reports of specialists. Data on direct non-medical and indirect costs were collected based on the patients' self-reports through face-to-face interviews performed in the mentioned centers. To calculate the indirect costs, friction cost approach was used. To calculate the total cost-of-illness in Iran, the total cost per patient at each stage of the disease was estimated and multiplied by the total number of patients. RESULTS: The total annual cost for the activate population of CHB patients and for those receiving treatment at various disease stages were respectively 450 million and 226 million dollars, with 64% and 70% of which allocated to direct costs respectively, and 36% and 30% to indirect costs respectively. The total direct costs alone for each group were respectively 1.17% and 0.6% of the total health expenditure. Furthermore, the cost spent on drugs encompasses the largest proportion of the direct medical cost for all stages of the disease. CONCLUSIONS: According to the perspectives of payers, patients, and community, CHB infection can be considered as one of the diseases with a substantial economic burden; the disease, specifically in extreme cases, can be too expensive and costly for patients. Therefore, patients should be protected against more severe stages of the disease through proper treatment and early diagnosis.

Development, characterizations and biocompatibility evaluations of intravitreal lipid implants.

BACKGROUND: The treatment of posterior eye diseases is always challenging mainly due to inaccessibility of the region. Many drugs are currently delivered by repeated intraocular injections. OBJECTIVES: The purpose of this study was to investigate the potential applications of natural triglycerides as alternative carriers to synthetic polymers in terms of drug release profile and also biocompatibility for intraocular use. MATERIALS AND METHODS: In vitro/in vivo evaluations of intravitreal implants fabricated from the physiological lipid, glyceride tripalmitate containing clindamycin phosphate as a model drug was performed. The micro-implants with average diameter of 0.4 mm were fabricated via a hot melt extrusion method. The extrudates were analyzed using scanning electron microscopy, differential scanning calorimetry, and in vitro drug dissolution studies. For biocompatibility, the implants were implanted into rabbit eyes. Clinical investigations including fundus observations, electroretinography as well as histological evaluations were performed. RESULTS: In vitro tests guaranteed usefulness of the production method for preparing the homogenous mixture of the drug and lipid without affecting thermal and crystalinity characteristics of the components. In vitro releases indicated a bi-phasic pattern for lower lipid ratios, which were completed by the end of day three. With higher lipid ratios, more controlled release profiles were achieved until about ten days for a lipid ratio of 95%. Clinical observations did not show any abnormalities up to two months after implantation into the rabbit eye. CONCLUSIONS: These results suggest that although the implant could not adequately retard release of the present drug model yet, due to good physical characteristics and in vivo biocompatibility, it can represent a suitable device for loading wide ranges of therapeutics in treatment of many kinds of retinochoroidal disorders.