Preparation and evaluation of a novel oral delivery system for low molecular weight heparin.

OBJECTIVE: The objective of the present work was to prepare and evaluate a novel oral formulation for systemic delivery of low molecular weight heparin (LMWH). The formulation consisted of Eudragit S 100-coated positively charged liposomes encapsulating LMWH and a penetration enhancer. MATERIALS AND METHODS: Positively charged liposomes were first prepared by the thin film hydration method using lipid (soy phosphotidylcholine and cholesterol) and stearyl amine (SA) in the optimum ratio of 16:1, along with cetylpyridinium chloride (CPC) as a penetration enhancer. Prepared liposomes were coated with negatively charged Eudragit S 100 (0.3% w/v). The formulations were studied for various in vitro and in vivo properties. Differential scanning calorimetry (DSC), x-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) studies, and in vitro drug release were used for in vitro characterization of the formulations. Ex vivo permeation studies were performed by using distal small intestine of rat. Oral absorption studies were conducted with the rat model. RESULTS: Coating of the liposomes was confirmed by SEM and particle size determination studies. In vitro release studies of coated liposomes have demonstrated that the release of LMWH was in the following order: Stomach < small intestine < distal small intestine < colon. Ex vivo permeation studies have shown a fivefold increase in permeation of LMWH with Eudragit S 100-coated liposomes compared to uncoated, uncharged liposomes. Oral absorption studies have showed that with Eudragit-coated liposomes, the oral bioavailability of LMWH was improved, compared to plain LMWH solution. This is revealed by a threefold increase in the area under the curve (AUC) of the plasma concentration time curve. CONCLUSION: A novel formulation for oral delivery of LMWH was thus successfully prepared and evaluated.

Development of PEG-PLGA based Intravenous Low Molecular Weight Heparin (LMWH) Nanoparticles Intended to Treat Venous Thrombosis.

BACKGROUND: Anticoagulant therapy is effective in the treatment of DVT. In this regard, LMWH demonstrated significant promise. It is widely used clinically. The goal of this study was to prepare and evaluate intravenous sustained release stealth nanoparticles encapsulating LMWH using PLGA (polylactidecoglycolide) and different grades of PEG (poly ethylene glycols). METHODS: The nanoparticles were prepared using w/o/w solvent evaporation technique. Prepared nanoparticles were evaluated for particle size, encapsulation efficiency, in-vitro drug release, anti-thrombotic activity in venous thrombosis rat model, estimation of aPTT, tissue bio-distribution studies and stability. RESULTS: Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) studies confirmed the formation of smooth spherical particles. FTIR study reveals successful coating of PEG on the nanoparticles. DSC and XRD results demonstrated that drug changed its physical form in the formulation. The encapsulation efficiency was 63-74%. In vitro drug release was 57-75% for 48 hrs. Macrophage uptake of LMWH with pegylated nanoparticles was less compared to conventional PLGA nanoparticles. In vivo drug release was sustained for 48hrs; Optimized formulation exhibited good enhancement in pharmacokinetic parameters when compared to free drug solution. In vivo sustained release was also demonstrated with antithrombotic activity as well aPTT activity. Optimized formulation demonstrated significant stability, excellent antithrombotic activity in venous thrombosis rat model, improved aPTT levels when compared to free drug solution. CONCLUSION: An effective stealth LMWH nanoparticle formulation to treat venous thrombosis was successfully developed using w/o/w solvent evaporation technique.

Development of subcutaneous sustained release nanoparticles encapsulating low molecular weight heparin.

The objective of the present research work was to prepare and evaluate sustained release subcutaneous (s.c.) nanoparticles of low molecular weight heparin (LMWH). The nanoparticles were prepared by water-in-oil in-water (w/o/w) emulsion and evaporation method using different grades of polylactide co-glycolide (50:50, 85:15), and different concentrations of polyvinyl alcohol (0.1%, 0.5%, 1%) aqueous solution as surfactant. The fabricated nanoparticles were evaluated for size, shape, zeta potential, encapsulation efficiency, in vitro drug release, and in vivo biological activity (anti-factor Xa activity) using the standard kit. The drug and excipient compatibility was analyzed by Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and X-ray diffraction (XRD) studies. The formation of nanoparticles was confirmed by scanning electron microscopy; nanoparticles were spherical in shape. The size of prepared nanoparticles was found between 195 nm and 251 nm. The encapsulation efficiency of the nanoparticles was found between 46% and 70%. In vitro drug, release was about 16-38% for 10 days. In vivo drug, release shows the sustained release of drug for 10 days in rats. FTIR studies indicated that there was no loss in chemical integrity of the drug upon fabrication into nanoparticles. DSC and XRD results demonstrated that the drug was changed from the crystalline form to the amorphous form in the formulation during the fabrication process. The results of this study revealed that the s.c. nanoparticles were suitable candidates for sustained delivery of LMWH.

Development of a novel 3-month drug releasing risperidone microspheres.

OBJECTIVE: The purpose of this study was to develop an ideal microsphere formulation of risperidone that would prolong the drug release for 3 months in vivo and avoid the need for co-administration of oral tablets. MATERIALS AND METHODS: Polycaprolactones (PCL) were used as polymers to prepare microspheres. The research included screening and optimizing of suitable commercial polymers of variable molecular weights: PCL-14000, PCL-45000, PCL-80000 or the blends of these polymers to prepare microspheres with zero-order drug-releasing properties without the lag phase. In the present study, the sustained release risperidone microspheres were prepared by o/w emulsion solvent evaporation technique and the yield was determined. Microspheres were evaluated for their drug content and in vitro drug release. Microspheres prepared using a blend of PCL-45000 and PCL-80000 at a ratio of 1:1 resulted in the release of the drug in a time frame of 90 days, demonstrated zero-order drug release without lag time and burst release. This formulation was considered optimized formulation. Optimized formulation was characterized for solid state of the drug using differential scanning calorimetry, surface morphology using scanning electron microscopy and in vivo drug release in rats. RESULTS: The surface of the optimized formulation was smooth, and the drug changed its physical form in the presence of blends of polymers and upon fabrication of microspheres. The optimized formulation also released the drug in vivo for a period of 90 days. CONCLUSIONS: From our study, it was concluded that these optimized microspheres showed great potential for a better depot preparation than the marketed Risperdal Consta™ and, therefore, could further improve patient compliance.

Preparation and characterization of amorphous ezetimibe nanosuspensions intended for enhancement of oral bioavailability.

OBJECTIVE: The objective of this study was to prepare and investigate better and stable amorphous ezetimibe nanosuspensions for oral bioavailability enhancement. MATERIALS AND METHODS: Nanosuspensions of ezetimibe were prepared by solvent-antisolvent precipitation technique using the surfactant, Tween 80 as stabilizer. The nanosuspension preparation was optimized for particle size by investigating two factors that is, solvent:antisolvent ratio and surfactant concentration, at three levels. The formulations were characterized for particle size, surface morphology, crystallinity, zeta potential, saturation solubility, in vitro drug release and in vivo drug absorption. RESULTS: The nanosuspensions of ezetimibe were successfully prepared using solvent-antisolvent precipitation. The two factors solvent:antisolvent ratio and surfactant concentration influenced the particle size of the nanosuspensions prepared. Nanosuspensions were smooth and spherical. The X-ray powdered diffraction and differential scanning calorimetry results indicated that the antisolvent-solvent method led to the amorphization of ezetimibe. Under storage, the amorphous ezetimibe nanosuspensions demonstrated significant physical stability. Ezetimibe nanosuspensions increased the saturation solubility to an extent of 4-times. Ezetimibe nanosuspensions completely dissolved in the dissolution medium within 1 h, while pure drug was dissolved up to 42% during same time. The Cmax with ezetimibe nanosuspension was approximately 3-fold higher when compared with that of ezetimibe conventional suspensions administered orally. CONCLUSIONS: Stable amorphous ezetimibe nanosuspensions were successfully prepared and these nanosuspensions demonstrated dramatic improvement in oral bioavailability of the active.

Development of novel risperidone implants using blends of polycaprolactones and in vitro in vivo correlation studies.

The objective of this study was to develop a novel implant containing risperidone intended for long-term treatment in Schizophrenia utilizing in vitro in vivo correlation (IVIVC) studies. Different implants (F1-F8) containing an antipsychotic drug, risperidone, were prepared using a hot melt extrusion technique by taking polycaprolactones of different molecular weights (Mwt. 15000, 45000, 80000) either alone or as their blends, and PLGA (75:25). The implants contained 40% of the drug. After fabrication, the implants were characterized for various in vitro properties such as drug release and physical strength. Prior to conducting drug release studies, optimum drug release method was developed based on IVIVC studies. An optimized formulation based on drug release and physical strength at the end of fabrication was selected from the various implants fabricated. The bioactivity, reversibility, and IVIVC of optimized formulation were determined using pharmacokinetic studies in rats. Short-term stability studies were conducted with optimized formulation. Drug release depended on polymer molecular weight. Implant fabricated using 50:50 polycaprolactone 45,000 and polycaprolactone 80,000 was considered optimized implant. Optimized formulation selected released the drug for 3-months in vitro and was physically rigid. The optimized implant was able to release the drug in vivo for a period of 3 months, the implants are reversible throughout the delivery interval and, a 100% IVIVC was achieved with optimized implant, suggesting the development of 3-month drug-releasing implant for risperidone. The optimized implant was stable for 6 months at room temperature (25°C) and 45°C. A novel implant for risperidone was successfully prepared and evaluated.

Development and evaluation of a novel biodegradable sustained release microsphere formulation of paclitaxel intended to treat breast cancer.

OBJECTIVE: The objective of this study was to develop a novel 1 month depot paclitaxel (PTX) microspheres that give a sustained and complete drug release. MATERIALS AND METHODS: PTX loaded microspheres were prepared by o/w emulsion solvent evaporation technique using the blends of poly(lactic-co-glycolic acid) (PLGA) 75/25, polycaprolactone 14,000 and polycaprolactone 80,000. Fourier transform infrared spectroscopy was used to investigate drug excipient compatibility. Compatible blends were used to prepare F1-F6 microspheres, the process was characterised and the optimum formulation was selected based on the release. Optimised formulation was characterised for solid state of the drug using the differential scanning calorimetry (DSC) studies, surface morphology using the scanning electron microscopy (SEM), in vivo drug release, in vitro in vivo correlation (IVIVC) and anticancer activity. Anticancer activity of release medium was determined using the cell viability assay in Michigan Cancer Foundation (MCF-7) cell line. RESULTS: Blend of PLGA with polycaprolactone (Mwt 14,000) at a ratio of 1:1 (F5) resulted in complete release of the drug in a time frame of 30 days. F5 was considered as the optimised formulation. Incomplete release of the drug resulted from other formulations. The surface of the optimised formulation was smooth and the drug changed its solid state upon fabrication. The formulation also resulted in 1-month drug release in vivo. The released drug from F5 demonstrated anticancer activity for 1-month. Cell viability was reduced drastically with the release medium from F5 formulation. A 100% IVIVC was obtained with F5 formulation suggesting the authenticity of in vitro release, in vivo release and the use of the formulation in breast cancer. CONCLUSIONS: From our study, it was concluded that with careful selection of different polymers and their combinations, PTX 1 month depot formulation with 100% drug release and that can be used in breast cancer was developed.

Nanoparticle formulation enhances the delivery and activity of a vascular endothelial growth factor antisense oligonucleotide in human retinal pigment epithelial cells.

The objective of this study was to investigate the delivery and activity of a vascular endothelial growth factor (VEGF) antisense oligonucleotide in a human retinal pigment epithelial cell line (ARPE-19) using a biodegradable nanoparticulate delivery system. A 19-mer antisense phosphorothioate oligonucleotide (PS-ODN) complementary to bases 6-24 relative to the translational start site of the VEGF mRNA, a sense PS-ODN and a mismatch PS-ODN were examined for the inhibition of secretion and mRNA expression of VEGF using an enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction, respectively. Nanoparticles of the antisense oligonucleotides were formulated using a poly(lactide-co-glycolide) (50:50) copolymer using a double emulsion solvent evaporation method. After preparing nanoparticles, drug loading, encapsulation efficiency and particle size were determined. The cells were exposed to either plain solution of oligonucleotide or nanoparticles of oligonucleotide from Day 3 through Day 6. Alternatively, the cells were incubated with PS-ODNs and lipofectin for 4 h on Day 4. In all studies, VEGF secretion and mRNA expression were determined on Day 6. The particle size, drug loading and encapsulation efficiency were 252 nm, 5.5% and 16.5%, respectively. The antisense PS-ODN inhibited VEGF mRNA and protein secretion when delivered using nanoparticles or lipofectin but not in its free form. This was consistent with the ability of nanoparticles and lipofectin to elevate the cellular uptake of the oligonucleotide by 4-fold and 13-fold, respectively. Neither mismatch nor sense oligonucleotides inhibited VEGF secretion. In conclusion, biodegradable nanoparticles enhance cellular delivery of a VEGF antisense oligonucleotide and inhibit VEGF secretion and mRNA expression in a human retinal pigment epithelial cell line.

A biodegradable injectable implant sustains systemic and ocular delivery of an aldose reductase inhibitor and ameliorates biochemical changes in a galactose-fed rat model for diabetic complications.

PURPOSE: To fabricate and characterize in vitro and in vivo performance of a sustained release biodegradable implant for N-4-(benzoylaminophenylsulfonyl glycine) (BAPSG), a novel aldose reductase inhibitor. METHODS: The ability of BAPSG to inhibit aldose reductase activity and glucose-induced vascular endothelial growth factor (VEGF) expression was assessed in a retinal pigment epithelial cell line (ARPE-19). A poly (DL-lactic-co-glycolic acid) implant containing 50% w/w BAPSG was fabricated and characterized for drug loading, in vitro drug release, and the thermal behavior of the drug and the polymer. Implants were injected subcutaneously into a galactose-fed diabetic rat model and cataract scores, plasma and tissue drug levels, galactitol levels in the lens and the retina, glutathione levels in the plasma, lens, cornea and retina and VEGF expression in the retina were determined on or until 18 days. RESULTS: BAPSG inhibited aldose reductase activity and reduced VEGF expression in ARPE-19 cells. Implants (1 x 4 mm), with a loading efficiency of 106 +/- 7% for BAPSG, were fabricated. Upon implant fabrication, while the glass transition temperature of the polymer decreased, the melting point of the drug was not affected. In vivo drug release correlated well with in vitro release, with approximately 44% drug release occurring in vivo by the end of 18 days. The implant reduced galactitol accumulation, glutathione depletion, cataract scores, and VEGF expression in galactose-fed rats. CONCLUSIONS: An injectable biodegradable implant of BAPSG sustained drug release in vitro and in vivo, and reduced galactitol accumulation, glutathione depletion, cataract scores, and VEGF expression in galactose-fed rats.