Extended siponimod release via low-porosity PLGA fibres: a comprehensive three-month in vitro evaluation for neovascular ocular diseases.

Neovascular ocular diseases are among the most common causes of preventable or treatable vision loss. Their management involves lifelong, intravitreal injections of anti-vascular endothelial growth factor (VEGF) therapeutics to inhibit neovascularization, the key pathological step in these diseases. Anti-VEGF products approved for ocular administration are expensive biological agents with limited stability and short half-life. Additionally, their therapeutic advantages are hindered by high treatment resistance, poor patient compliance and the need for frequent, invasive administration. Herein, we used electrospinning to develop a unique, non-porous, PLGA implant for the ocular delivery of siponimod to improve ocular neovascular disease management. Siponimod is an FDA-approved drug for multiple sclerosis with a novel indication as a potential ocular angiogenesis inhibitor. The electrospinning conditions were optimised to produce a microfibrous, PLGA matte that was cut and rolled into the desired implant size. Physical characterisation techniques (Raman, PXRD, DSC and FTIR) indicated siponimod was distributed uniformly within the electrospun fibres as a stabilised, amorphous, solid dispersion with a character modifying drug-polymer interaction. Siponimod dispersion and drug-polymer interactions contributed to the formation of smooth fibres, with reduced porous structures. The apparent reduced porosity, coupled with the drug's hydrophobic dispersion, afforded resistance to water penetration. This led to a slow, controlled, Higuchi-type drug diffusion, with ∼30% of the siponimod load released over 90 days. The released drug inhibited human retinal microvascular endothelial cell migration and did not affect the cells' metabolic activity at different time points. The electrospun implant was physically stable after incubation under stress conditions for three months. This novel siponimod intravitreal implant broadens the therapeutic possibilities for neovascular ocular diseases, representing a potential alternative to biological, anti-VEGF treatments due to lower financial and stability burdens. Additionally, siponimod interaction with PLGA provides a unique opportunity to sustain the drug release from the electrospun fibres, thereby reducing the frequency of intravitreal injection and improving patient adherence.

Nano-Hydroxyapatite/PLGA Mixed Scaffolds as a Tool for Drug Development and to Study Metastatic Prostate Cancer in the Bone.

(1) Background: Three-dimensional (3D) in vitro, biorelevant culture models that recapitulate cancer progression can help elucidate physio-pathological disease cues and enhance the screening of more effective therapies. Insufficient research has been conducted to generate in vitro 3D models to replicate the spread of prostate cancer to the bone, a key metastatic site of the disease, and to understand the interplay between the key cell players. In this study, we aim to investigate PLGA and nano-hydroxyapatite (nHA)/PLGA mixed scaffolds as a predictive preclinical tool to study metastatic prostate cancer (mPC) in the bone and reduce the gap that exists with traditional 2D cultures. (2) Methods: nHA/PLGA mixed scaffolds were produced by electrospraying, compacting, and foaming PLGA polymer microparticles, +/- nano-hydroxyapatite (nHA), and a salt porogen to produce 3D, porous scaffolds. Physicochemical scaffold characterisation together with an evaluation of osteoblastic (hFOB 1.19) and mPC (PC-3) cell behaviour (RT-qPCR, viability, and differentiation) in mono- and co-culture, was undertaken. (3) Results: The results show that the addition of nHA, particularly at the higher-level impacted scaffolds in terms of mechanical and degradation behaviour. The nHA 4 mg resulted in weaker scaffolds, but cell viability increased. Qualitatively, fluorescent imaging of cultures showed an increase in PC-3 cells compared to osteoblasts despite lower initial PC-3 seeding densities. Osteoblast monocultures, in general, caused an upregulation (or at least equivalent to controls) in gene production, which was highest in plain scaffolds and decreased with increases in nHA. Additionally, the genes were downregulated in PC3 and co-cultures. Further, drug toxicity tests demonstrated a significant effect in 2D and 3D co-cultures. (4) Conclusions: The results demonstrate that culture conditions and environment (2D versus 3D, monoculture versus co-culture) and scaffold composition all impact cell behaviour and model development.

In-Vial Detection of Protein Denaturation Using Intrinsic Fluorescence Anisotropy.

The conventional quality control techniques for identifying the denaturation of biopharmaceuticals includes sodium dodecyl sulfate-polyacrylamide gel electrophoresis for identifying fragmentation, ion exchange chromatography and isoelectric focusing for identifying deamidation, reverse-phase high-performance liquid chromatography (HPLC) for identifying oxidation, and size-exclusion HPLC for identifying aggregation. These stability assessments require essential processes that are destructive to the product tested. All these techniques are lab based and require sample removal from a sealed storage vial, which can breach the sterility. In this work, we investigate the heat- and surfactant-induced denaturation of an in-vial-stored model protein, bovine serum albumin (BSA), by analyzing its intrinsic fluorescence without removing the sample from the vial. A lab-based bespoke setup which can do the measurement in vial is used to demonstrate the change in fluorescence polarization of the protein to determine the denaturation level. The results obtained are compared to circular dichroism and size-exclusion HPLC measurements. The results prove that in-vial fluorescence measurements can be performed to monitor protein denaturation. A cost-effective portable solution to provide a top-level overview of biopharmaceutical product stability from manufacture to the point of patient administration can be further developed using the same technique.

Fluorescence spectroscopy for the determination of reconstitution time of an in-vial lyophilised product.

Lyophilisation is a prominent technique used to create stabilised, dried forms of biopharmaceutical formulations. Reconstitution of lyophilised parenteral formulations is a key step prior to patient administration. The accurate determination of reconstitution time is a necessity to aid formulation development and support product quality control. Traditional methods for quantifying reconstitution time involve the visual identification of the endpoint, which has led to variable values reported across studies. In this work, the use of ultra-violet (UV) excited fluorescence spectroscopy as an alternative to the visual quantification of the reconstitution time was investigated. Spectrographic information was collected via a bespoke setup that allowed the measurement of the reconstitution time in a standard sealed lyophilisation vial. The spectra were analysed via principal component analysis (PCA) to obtain a time-based representation of the changes in a reconstituting formulation. The analysis was followed by the identification of an endpoint using three techniques ranging from fully automated to manual with regards to the required level of user input. At high protein concentration, the variability of the reconstitution time measurements was reduced from 80.4% relative standard deviation obtained via the traditional method to 8.2% for the instrumental method presented in.

Deep UV Laser-Induced Fluorescence for Pharmaceutical Cleaning Validation.

Cleaning verification and validation is a requirement in the pharmaceutical industry. Due to the limited number of mobile devices that do effective and accurate onsite cleaning verification, it is mostly done via lab-based quality control techniques. These techniques, such as high-performance liquid chromatography (HPLC) or total organic carbon, often lead to extending the validation of cleaning by days. The void of more sensitive, accurate, and portable instruments to verify cleaning onsite has to be filled. The article discusses the use of deep ultra violet (DUV) laser-induced fluorescence for detecting carryover of active pharmaceutical ingredients (APIs) and detergents onsite. A modified spectrometer was used as an offsite bench type prototype for analyzing trace samples of API and cleaning detergents with various substrates. Even if the API to be detected has a low fluorescence efficiency, the specificity of the technique allows API traces having concentrations as low as ≈0.20 µg/cm2 to be identified. The work also shows the possibility of using a probe for validating cleaning of hard to reach areas using DUV laser-induced fluorescence. DUV laser-induced fluorescence of trace API over any polymer/glass substrate has better signal to background ratio (SBR) compared to FTIR absorption techniques. Processing times of DUV laser-induced fluorescence trace detection are shown to be much less than swab based methods.