Ritesh M. Pabari

Application of face centred central composite design to optimise compression force and tablet diameter for the formulation of mechanically strong and fast disintegrating orodispersible tablets

A two factor, three level (3(2)) face centred, central composite design (CCD) was applied to investigate the main and interaction effects of tablet diameter and compression force (CF) on hardness, disintegration time (DT) and porosity of mannitol based orodispersible tablets (ODTs). Tablet diameters of 10, 13 and 15 mm, and CF of 10, 15 and 20 kN were studied. Results of multiple linear regression analysis show that both the tablet diameter and CF influence tablet characteristics. A negative value of regression coefficient for tablet diameter showed an inverse relationship with hardness and DT. A positive value of regression coefficient for CF indicated an increase in hardness and DT with increasing CF as a result of the decrease in tablet porosity. Interestingly, at the larger tablet diameter of 15 mm, while hardness increased and porosity decreased with an increase in CF, the DT was resistant to change. The optimised combination was a tablet of 15 mm diameter compressed at 15 kN showing a rapid DT of 37.7s and high hardness of 71.4N. Using these parameters, ODTs containing ibuprofen showed no significant change in DT (ANOVA; p>0.05) irrespective of the hydrophobicity of the ibuprofen.

Comparative evaluation of novel biodegradable nanoparticles for the drug targeting to breast cancer cells.

Nanomedicine formulations such as biodegradable nanoparticles (nps) and liposomes offer several advantages over traditional routes of administration: due to their small size, nanocarriers are able to selectively accumulate inside tumours or inflammatory tissues, resulting in improved drug efficacy and reduced side effects. To further augment targeting ability of nanoparticles towards tumour cells, specific ligands or antibodies that selectively recognise biomarkers over-expressed on cancer cells, can be attached to the surface either by chemical bond or by hydrophilic/hydrophobic interactions. In the present work, Herceptin (HER), a monoclonal antibody (mAb) able to selectively recognise HER-2 over-expressing tumour cells (such as breast and ovarian cancer cells), was absorbed on the surface of nanoparticles through hydrophilic/hydrophobic interactions. Nps were prepared by a modified single emulsion solvent evaporation method with five different polymers: three commercial polyesters (poly(ε-caprolactone) (PCL), poly (D,L-lactide) (PLA) and poly (D,L-lactide-co-.glycolide) (PLGA)) and two novel biodegradable polyesterurethanes (PURs) based on Poly(ε-caprolactone) blocks, synthesised with different chain extenders (1,4-cyclohexane dimethanol (CDM) and N-Boc-serinol). Polyurethanes were introduced as matrix-forming materials for nanoparticles due to their high chemical versatility, which allows tailoring of the materials final properties by properly selecting the reagents. All nps exhibited a small size and negative surface charge, suitable for surface functionalisation with mAb through hydrophilic/hydrophobic interactions. The extent of cellular internalisation was tested on two different cell lines: MCF-7 and SK-BR-3 breast cancer cells showing a normal and a high expression of the HER-2 receptor, respectively. Paclitaxel, a model anti-neoplastic drug, was encapsulated inside all nps, and release profiles and cytotoxicity on SK-BR-3 cells were also assessed. Interestingly, PUR nps were superior to commercial polyester-based nps in terms of higher cellular internalisation and cytotoxic activity on the tested cell lines. Results obtained warrants further investigation on the application of these PUR nps for controlled drug delivery and targeting.

Investigation of a novel 3-fluid nozzle spray drying technology for the engineering of multifunctional layered microparticles

Objective: To examine the potential of a novel 3-fluid nozzle spray drying technology to formulate differentiated layered microparticles (MPs) of diclofenac sodium (DFS)/ethyl cellulose (EC). Methods: DFS/EC MPs were formulated using the inner and/or outer nozzles of a novel 3-fluid nozzle and compared with MPs formed using conventional (2-fluid) spray drying. MPs were characterised for particle size and for morphology by TEM and SEM. Distribution of DFS and EC of MPs was analysed by FT-IR and DSC. A two-factor, three-level (32) factorial design was applied to investigate the effect and interaction of total feed solid content (TSC) and feed flow rate (FFR) on MP size, D50% and D90%, bulk density and MP yield. Results: Interestingly, TEM demonstrated that MPs formed by 3-fluid nozzle spray drying showed a heterogeneous internal morphology consisting of a core and coat, characteristic of a microcapsule. In comparison, MPs from conventional spray drying showed a homogeneous internal morphology, characteristics of a matrix system. This differential distribution of DFS/EC was supported by FT-IR and DSC. Results of multiple linear regression analysis showed a linear relationship for the effect of TSC and FFR on all responses except for D50% where a quadratric model was valid. The effect of TSC/FFR on MP size and yield was similar to conventional spray drying. Conclusion: The novel 3-fluid nozzle spray drying offers a new method of designing layered microparticles or microcapsules which can have wide applications from drug stabilisation to controlled drug delivery and targeting.

Effect of Microencapsulation Shear Stress on the Structural Integrity and Biological Activity of a Model Monoclonal Antibody, Trastuzumab

The aim of the present study was to investigate the influence of process shear stressors on the stability of a model monoclonal antibody, trastuzumab. Trastuzumab, at concentrations of 0.4–4.0 mg/mL, was subjected to sonication, freeze-thaw, lyophilisation, spray drying and was encapsulated into micro- and nanoparticles. The stressed samples were analysed for structural integrity by gel electrophoresis, SDS-PAGE, and size exclusion chromatography (SEC), while the conformational integrity was analysed by circular dichroism (CD). Biological activity of the stressed trastuzumab was investigated by measuring the inhibition of cell proliferation of HER-2 expressing cell lines. Results show that trastuzumab was resistant to the process shear stresses applied and to microencapsulation processes. At the lowest concentration of 0.4 mg/mL, a low percent (<9%) of soluble/reversible aggregates may have been formed. No loss of structural integrity, conformation was observed and no significant change in the biological activity of trastuzumab was observed (ANOVA; p > 0.05). The results of this study conclude that trastuzumab may be resistant to various processing stresses. These findings have important implications with respect to pharmaceutical processing of monoclonal antibodies.

Intranasal and intravenous administration of octa‐arginine modified poly(lactic‐co‐glycolic acid) nanoparticles facilitates central nervous system delivery of loperamide

The potential of poly(lactic‐co‐glycolic acid) (PLGA) nanoparticles (NPs) surface modified with octa‐arginine (R8) for central nervous system (CNS) delivery was investigated.

Influence of Parathyroid Hormone-Loaded PLGA Nanoparticles in Porous Scaffolds for Bone Regeneration

Biodegradable poly(lactide-co-glycolide) (PLGA) nanoparticles, containing human parathyroid hormone (PTH (1–34)), prepared by a modified double emulsion-solvent diffusion-evaporation method, were incorporated in porous freeze-dried chitosan-gelatin (CH-G) scaffolds. The PTH-loaded nanoparticles (NPTH) were characterised in terms of morphology, size, protein loading, release kinetics and in vitro assessment of biological activity of released PTH and cytocompatibility studies against clonal human osteoblast (hFOB) cells. Structural integrity of incorporated and released PTH from nanoparticles was found to be intact by using Tris-tricine SDS-PAGE. In vitro PTH release kinetics from PLGA nanoparticles were characterised by a burst release followed by a slow release phase for 3–4 weeks. The released PTH was biologically active as evidenced by the stimulated release of cyclic AMP from hFOB cells as well as increased mineralisation studies. Both in vitro and cell studies demonstrated that the PTH bioactivity was maintained during the fabrication of PLGA nanoparticles and upon release. Finally, a content of 33.3% w/w NPTHs was incorporated in CH-G scaffolds, showing an intermittent release during the first 10 days and, followed by a controlled release over 28 days of observation time. The increased expression of Alkaline Phosphatase levels on hFOB cells further confirmed the activity of intermittently released PTH from scaffolds.

Comparative evaluation of the degree of pegylation of poly(lactic-co-glycolic acid) nanoparticles in enhancing central nervous system delivery of loperamide.

In this study, we examined the relative cellular uptake of nanoparticles (NPs) formulated using poly(lactic‐co‐glycolic acid) (PLGA) polymers with increasing degree of pegylation (PLGA‐PEG) and their potential to deliver loperamide to the brain of a mouse.

Fast disintegrating crystalline solid dispersions of simvastatin for incorporation into orodispersible tablets

Aim: Spray dried solid dispersion (SDP) of crystalline simvastatin (SIM) in a fast disintegrating matrix of superdisintegrants was studied as a method to enhance SIM dispersibility, rheology, compactibility and compressibility for incorporation into orodispersible tablets (ODTs). Materials and Methods: The superdisintegrants investigated were crospovidone (CP), sodium starch glycollate (SSG) and calcium silicate (CS) were spray dried with simvastatin to form SDPs. Results: The SDPs were characterized and the median particle size of SDPs was similar or greater than the SIM, contributing to good rheology of SDPs, while the low bulk density of SDPs indicated a high compactibility. Interestingly electron micrographs for SDPs showed a CP or CS carrier coating of the SIM crystals, contributing to its rheology. Thermal analysis and X-ray diffraction confirmed that SIM was crystalline in the SDPs and no interaction between SIM and any of the carrier(s) was shown by Fourier transform-infra red. Drug content analysis showed a SIM content of 90-95% in SDPs containing CP or CS, while a higher SIM content of 143% was found in SDP containing SSG. When formulated as ODTs, blend containing SIM SDPs in CP showed ease of tableting, regardless of the turret speed. In comparison, tablet blend consisting of a physical mix (PM) of SIM and CP could only be tableted at the lower turret speed of 7 rpm. ODTs formulated using SIM SDPs in CP showed a higher extent of dissolution, compared to the ODTs containing corresponding PM or the commercially available SIM Zocor® tablets (ANOVA, P < 0.05). Conclusion: SDP using disintegrants as carriers may offer an alternative formulation approach for ODTs of poorly soluble drugs.

Physical and structural stability of the monoclonal antibody, trastuzumab (Herceptin®), intravenous solutions.

A major limitation of biological therapeutics is their propensity for degradation particularly in aqueous solutions hence resulting in their short shelf-life. In this study, the stability of trastuzumab (Herceptin®) intravenous (i.v.) solutions, an IgG1 monoclonal antibody (mAb), indicated for the treatment of HER2 positive breast cancer, stored under refrigerated conditions, was evaluated over 28 days. No change in visual appearance or average particle size was observed. The pH values of the trastuzumab i.v. solutions remained stable over time. Interestingly, no change in trastuzumab monomer concentration was observed throughout the 28-day study, as determined by SEC-HPLC. SDSPAGE showed only a monomer band corresponding to the molecular weight of trastuzumab. Circular dichroism spectra obtained following 28-day storage demonstrated integrity of the secondary structural conformation of trastuzumab. Results from this study show that trastuzumab i.v. solutions remain physically and structurally stable on storage at 2-8°C for 28 days. These findings suggest that trastuzumab in solution may not be as sensitive to degradation as expected for a mAb and therefore may have important implications in extending trastuzumab shelf life for clinical use and reducing associated healthcare cost.

Targeting the 19S proteasomal subunit, Rpt4, for the treatment of colon cancer

Deregulation of the ubiquitin-proteasome pathway has been frequently observed in a number of malignancies. Using quantitative Western blotting of normal and matched tumour tissue, we here identified a significant increase in the 19S proteasome subunit Rpt4 in response to chemoradiation in locally advanced rectal cancer patients with unfavourable outcome. We therefore explored the potential of Rpt4 reduction as a therapeutic strategy in colorectal cancer (CRC). Utilizing siRNA to down regulate Rpt4 expression, we show that silencing of Rpt4 reduced proteasomal activity and induced endoplasmic reticulum stress. Gene silencing of Rpt4 also inhibited cell proliferation, reduced clonogenic survival and induced apoptosis in HCT-116 colon cancer cells. We next developed a cell penetrating peptide-based nanoparticle delivery system to achieve in vivo gene silencing of Rpt4. Administration of Rpt4 siRNA nanoparticles reduced tumour growth and improved survival in a HCT-116 colon cancer xenograft tumour model in vivo. Collectively, our data suggest that inhibition of Rpt4 represents a novel strategy for the treatment of CRC.