A High Throughput Protein Formulation Platform: Case Study of Salmon Calcitonin

Purpose: The feasibility of using high throughput spectroscopy for characterization and selection of physically stable protein formulations was studied. Materials and Methods: A hundred aqueous formulations of salmon calcitonin (sCT) were prepared using 20 buffer compositions. The solutions had pH values between 2.5 and 10.5. The stability of the sCT formulations was analyzed over 1week by the following assays: (1) protein concentration, (2) volume control by measuring pathlength, (3) turbidity (absorbance at 350nm), (4) intrinsic tyrosine fluorescence, (5) 1-anilino-naphthalene-8-sulfonate (ANS) fluorescence, (6) Nile Red fluorescence. Addition of the dyes (Nile Red and ANS) was used to study protein conformational changes. Results: After 1day, 27 out of the 100 formulations of salmon calcitonin were stable. After 7days, 12 stable sCT formulations remained. The best salmon calcitonin formulation was in 10mM sodium acetate buffer with pH values between 3.5 and 5.5. Conclusions: The findings are in accordance with the sCT formulations that were patented and used commercially. This can be considered as a proof of concept for the high throughput protein formulation platfor

Breaking the Aggregation of the Monoclonal Antibody Bevacizumab (Avastin®) by Dexamethasone Phosphate: Insights from Molecular Modelling and Asymmetrical Flow Field-Flow Fractionation

ABSTRACT: Purpose: To investigate the mechanism behind the aggregation breaking properties of dexamethasone phosphate and related corticosteroids on the IgG1 antibody bevacizumab (Avastin®). Methods: An in silico 3D dimer model is developed to identify the bevacizumab-bevacizumab interface, and different corticosteroids are docked onto the model to distinguish preferred binding sites. In silico predictions are validated by in vitro stability studies, where the antibody is stressed in presence or absence of each corticosteroid and formed aggregates are quantified by asymmetrical flow field-flow fractionation. Results: The dimer model features one close crystal contact area: Lys445 on the Fc region interacts with one Fab arm of the second bevacizumab. Docking reveals an interaction between the phosphate group of dexamethasone phosphate and Lys445, while the rest of the molecule is hindering dimer formation. Predictions are confirmed in vitro, demonstrating that dexamethasone phosphate and betamethasone phosphate partly prevent antibody aggregation, whereas triamcinolone acetonide phosphate does not. Conclusions: Results suggest that bevacizumab monomers follow a specific mechanism to form dimers in which a protein-protein interaction hotspot can be distinguished. The dimer formation can be hindered by corticosteroids in a specific way. This approach allows a simple way to stabilize IgG1 antibodie

Preparation of surfactant-free nanoparticles of methacrylic acid copolymers used for film coating

The aim of the present study was to prepare surfactant-free pseudolatexes of various methacrylic acid copolymers. These aqueous colloidal dispersions of polymeric materials for oral administration are intended for film coating of solid dosage forms or for direct manufacturing of manoparticles. Nanoparticulate dispersions were produced by an emulsification-diffusion method involving the use of partially water-miscible solvents and the mutual saturation of the aqueous and organic phases prior to the emulsification in order to reduce the initial thermodynamic instability of the emulsion. Because of the self-emulsifying properties of the methacrylic acid copolymers, it was possible to prepare aqueous dispersions of colloidal size containing up to 30% wt/vol of Eudragit RL, RS, and E using 2-butanone or methyl acetate as partially water-miscible solvents, but without any surfactant. However, in the case of the cationic Eudragit E, protonation of the tertiary amine groups by acidification of the aqueous phase was necessary to improve the emulsion stability in the absence of surfactant and subsequently to prevent droplet coalescence during evaporation. In addition, a pseudolatex of Eudragit E was used to validate the coating properties of the formulation for solid dosage forms. Film-coated tablets of quinidine sulfate showed a transparent glossy continuous film that was firmly attached to the tablet. The dissolution profile of quinidine sulfate from the tablets coated with the Eudragit E pseudolatex was comparable to that of tablets coated with an acetonic solution of Eudragit E. Furthermore, both types of coating ensured similar taste masking. The emulsification-evaporation method used was shown to be appropriate for the preparation of surfactant-free colloidal dispersions of the 3 types of preformed methacrylic acid copolymers; the dispersions can subsequently be used for film coating of solid dosage form

Staying the Course: Toward Strong HQIM Implementation in Delaware

With the implementation of high-quality instructional materials (HQIM) and curriculum-based professional learning, Delaware educators, students, and families have ventured into promising, challenging new territory. HQIM ask a great deal of their users. Educators are called upon to abandon traditional approaches to instruction, allowing kids to loudly drive classroom discourse rather than passively taking notes on teacher lectures. Students are asked to grapple with rigorous, problem-based subject matter that offers no easy answers and requires deep analytical thinking and collaboration. Families are asked to support their children’s learning when the materials and resources that come home may feel unfamiliar and overwhelming. For all stakeholders, implementation can, at times, feel like an arduous journey with no clear destination

Tolerance of high and low amounts of PLGA microspheres loaded with mineralocorticoid receptor antagonist in retinal target site

Mineralocorticoid receptor (MR)contributes to retinal/choroidal homeostasis. Excess MR activation has been shown to be involved in pathogenesis of central serous chorioretinopathy (CSCR). Systemic administration of MR antagonist (MRA) reduces subretinal fluid and choroidal vasodilation, and improves the visual acuity in CSCR patients. To achieve long term beneficial effects in the eye while avoiding systemic side-effects, we propose the use ofbiodegradable spironolactone-loaded polylactic-co-glycolic acid (PLGA)microspheres (MSs). In this work we have evaluated the ocular tolerance of MSs containing spironolactone in rats’ eyes. As previous step, we have also studied the tolerance of the commercial solution of canrenoate salt, active metabolite of spironolactone. PLGA MSs allowed in vitro sustained release of spironolactone for 30 days. Rat eyes injected with high intravitreous concentration of PLGA MSs (10 mg/mL) unloaded and loaded with spironolactone maintained intact retinal lamination at 1 month. However enhanced glial fibrillary acidic protein immunostaining and activated microglia/macrophages witness retinal stress were observed. ERG also showed impaired photoreceptor function. Intravitreous PLGA MSs concentration of 2 mg/mL unloaded and loaded with spironolactone resulted well tolerated. We observed reduced microglial/macrophage activation in rat retina compared to high concentration of MSs with normal retinal function according to ERG. Spironolactone released from low concentration of MSs was active in the rat retina. Low concentration of spironolactone-loaded PLGA MSs could be a safe therapeutic choice for chorioretinal disorders in which illicit MR activation could be pathogenic

Patent Literature Review of Ophthalmic Inserts

In the area of topical ocular administration, important efforts concern the design and the conception of new ophthalmic drug delivery systems able to prolong the residence time. The use of inserts, which are solid devices to be placed in the cul-the-sac or on the cornea represents one of the possibilities to reach increased residence time. These solid ophthalmic devices present the advantage of avoiding a pulsed release due to multiple applications. In the scientific literature, patents are often not mentioned resource and this remark is particularly true in the case of ophthalmic inserts: there is no review yet describing patented ophthalmic inserts. Thus the review presented herein essentially takes into account patented ophthalmic inserts. The solid ophthalmic devices are classified into three major categories based upon their solubility behavior: the insoluble, the soluble and the bioerodible inserts. When necessary, these groups are subdivided into more specific sub-groups. The discussion essentially takes into account the design, the conception, the release mechanisms, the in vivo and in vitro assays, the limitations, and the therapeutic rationale for their use

Chemical and physical parameters of tears relevant for the design of ocular drug delivery formulations

This paper provides a summary of the most important chemical and physical parameters of tears that can help the formulator in the development of new ocular formulations and in the conception of innovative ophthalmic delivery approaches. For each physiological parameter, the relevance in ocular drug delivery is discussed in detail and the analytical tools that are used for the determination of these parameters are described and summarized. The aim of this review is also to give a description of the main analytical techniques available in ophthalmology that can be used for pharmacokinetic studies of active compounds. The importance of tear sampling techniques used in the determination of the parameters is also discussed