Simultaneous Determination of Dexpanthenol, Lidocaine Hydrochloride, Mepyramine Maleate and their Related Substances by a RP-HPLC Method in Topical Dosage Forms.

The pharmaceutical combination of dexpanthenol (DPA), lidocaine hydrochloride (LIH) and mepyramine maleate (MAM) is used for their anti-allergic, anti-inflammatory, anti-pruritic, anesthetic and antiseptic properties. The present study was aimed to develop and validate a new, first and rapid high performance liquid chromatographic method for simultaneous determination of DPA, LIH and MAM in the presence of their stress-induced degradation products in pharmaceutical gel/fluigel formulations. The chromatographic separation was performed on an Inertsil ODS-3 V, 250 × 4.6 mm (5 µm) column using a gradient mobile phase of an aqueous solution of ammonium acetate (0.01 M) and methanol mixture at gradient flow rates of 1.3 mL/min and 1.5 mL/min with detection at 230 nm. The retention times for DPA, LIH and MAM were ~3.28 min, 11.67 min and 12.99 min, respectively. The method was validated in accordance with International Conference on Harmonisation guidelines. Calibration curves were linear in the ranges of 9-54 µg/mL for MAM and LIH and 30-180 µg/mL for DPA with satisfactory correlation coefficients (R2 > 0.999). The mean % recoveries obtained were found to be 99.9% for MAM, 100.3% for LIH and 99.3% for DPA. Precision % RSD was <2. Robustness results were uniform, there were no marked changes, so method is highly validated. All drugs were subjected to stress conditions and degradation products were separated with acceptable peak tailing (T ≤ 2) and good resolution (Rs > 2). The validated method therefore can be adapted for quality control procedures of the drugs in pharmaceutical dosage forms and their stability studies.

Characterization of bevacizumab by dynamic light scattering while maintaining its native structure.

Bevacizumab, is a humanized monoclonal antibody and patents on Avastin® (Bevacizumab, Roche) will expire in the US in 2019 and in Europe in 2022. Therefore, bevacizumab is a popular target for biosimilar developers. One of the most common problems in the formulation of antibody drugs is protein aggregation. Dynamic light scattering (DLS) is a well-established method for the determination of hydrodynamic dimensions, aggregates, and aggregation points of proteins. In contradistinction to other techniques that require diluted samples or specific conditions, proteins and aggregates can maintain their native structure during DLS measurements. In recent studies, bevacizumab was characterized by DLS using diluted samples. In this study, we aimed at investigating the hydrodynamic dimensions, aggregates, and aggregation onset of bevacizumab (Altuzan®, Turkey, Roche) by DLS, while maintaining its native structure. The intensity, volume, and number-based particle size distribution profiles of the test samples were evaluated and the aggregation onset of the formulation was successfully determined against increasing temperature. It is shown that the preservation of the native structure of commercial formulations in DLS measurements provides an opportunity to the characterization of commercial products and development of biosimilars.

Development and validation of a LC-FL method for the simultaneous determination of doxorubicin and celecoxib in nanoparticulate fixed dose combination (NanoFDC).

An isocratic reversed phase HPLC method for the simultaneous determination of doxorubicine (DOX) and celecoxib (CXB) out of a nanoparticulate fixed dose combination (NanoFDC) was developed and validated. Linearity of the results was demonstrated from 1-11 µg/mL for both components. Lower limits of detection were determined as 7 ng/mL for DOX and 13 ng/mL for CXB. Total run time was approximately 15 min.

Using PVA and TPGS as combined emulsifier in nanoprecipitation method improves characteristics and anticancer activity of ibuprofen loaded PLGA nanoparticles.

In the preparation of nanoparticles (NPs) by the nanoprecipitation method, emulsifiers play a key role for NPs' characteristics. The present study aimed to investigate the combined emulsifier effect on ibuprofen loaded poly(lactic-co-glycolic acid) (PLGA) NPs' characteristics and anticancer activity. Ibuprofen loaded PLGA NPs were prepared by nanoprecipitation using different concentrations of PVA (poly(vinyl alcohol)) or PVA-TPGS (d-α-tocopherol polyethylene glycol 1000 succinate) combination as emulsifier. It was found that encapsulation efficiencies of NPs varied between 17.9 and 41.9 % and the highest encapsulation efficiency was obtained with 0.5% PVA + 0.1% TPGS (coded as PLGA PVA/TPGS NPs). PLGA PVA/TPGS NPs were characterized and compared with PLGA PVA NPs, which was obtained by 0.5% PVA alone. Polydispersity index of PLGA PVA/TPGS and PLGA PVA NPs were found to be 0.08 and 0.15, respectively. Incorporation of TPGS with PVA slightly decreased the initial ibuprofen release. Transmission electron microscopy analyses demonstrated a nearly uniform particle size distribution and spherical particle shape of the PLGA PVA/TPGS NPs. Additionally, PLGA PVA/TPGS NPs were significantly more cytotoxic than PLGA PVA NPs on the MCF-7 (human breast adenocarcinoma cells) and Caco-2 (human epithelial colorectal adenocarcinoma) cells (p<0.05). Also PLGA PVA/TPGS NPs were not cytotoxic on normal cells (L929, mouse healthy fibroblast cells) (p>0.05). In conclusion, these results indicated that using a combination of TPGS and PVA as an emulsifier in nanoprecipitation could be a promising approach for preparing ibuprofen loaded PLGA NPs because of their improved characteristics and anticancer activity.

An aquaporin 4 antisense oligonucleotide loaded, brain targeted nanoparticulate system design.

Aquaporins (AQPs), members of the water-channel protein family, are highly expressed in brain tissue especially in astrocytic end-feet. They are important players for water hemostasis during development of cytotoxic as well as vasogenic edema. Increased expression of AQPs is important in pathophysiology of neurological diseases such as neuroinflammation and ischemia. Unfortunately, there are a few pharmacological inhibitors of AQP4 with several side effects limiting their translation as a drug for use in clinical conditions. Another therapeutic approach is using antisense oligonucleotides (ASOs) to block AQP4 activity. These are short, synthetic, modified nucleic acids that bind RNA to modulate its function. However, they cannot pass the blood brain barrier (BBB). To overcome this obstacle we designed a nanoparticulate system made up of chitosan nanoparticles surface modified with PEG and conjugated with monoclonal anti transferrin receptor-1 antibody via streptavidin-biotin binding. The nanocarrier system could be targeted to the transferrin receptor-1 at the brain endothelial capillaries through monoclonal antibodies. It is hypothesized that the nanoparticles could pass the BBB via receptor mediated transcytosis and reach brain parenchyma. Particle size, zeta potential, loading capacity and release profiles of nanoparticles were investigated. It was observed that all types of chitosau (CS) nanoparticles had positive zeta potential values and nanoparticle particle size distribution varied between 100 and 800 nm. The association efficiency of ASOs into the nanoparticles was between 80-97% and the release profiles of the nanoparticles exhibited an initial burst effect followed by a controlled release. The results showed that the designed chitosan based nanocarriers could be a promising carrier system to transport nucleic acid based drugs to brain parenchyma.

Formulation and evaluation of clozapine orally disintegrating tablets prepared by direct compression.

In this study, clozapine orally disintegrating tablets (ODTs) were prepared by direct compression method. Disintegration time, resistance to crushing of tablets, porosity, friability, dissolution tests were performed and dissolution profiles of ODTs were investigated. Morphological and interaction studies were also performed. Friability values were found to be less than 1%. All tablet formulations disintegrated within 1 min and fulfilled the 3 min disintegration time required for ODTs given in the European Pharmacopoeia. More than 85% of the labeled amount of clozapine was dissolved in 15 min from the ODTs. No interaction or changes were found between active substance and excipients. As a result of the studies, ODT formulations developed in this study can be suggested as promising formulations, which assist development and manufacturing a generic product of clozapine.

The influence of technological parameters on the physicochemical properties of blank PLGA nanoparticles.

In this work, PLGA nanoparticles were prepared by an emulsification-diffusion technique. The main objective was to optimize the preparation of formulations by evaluating the influence of the technological parameters on the physicochemical properties of PLGA nanoparticles. The effects of variations in polymer and emulsifier concentrations, and homogenization duration, rate and type on the particle size distribution, surface charge and morphology of nanoparticles were assessed. The smallest nanoparticles (177.53 +/- 2.78 nm) were obtained with a 2% PLGA (w/v) concentration in the organic phase and 3% PVA (w/v) in the aqueous phase and were prepared by an emulsification-diffusion method via ultrasonic homogenization at a power of 80 W applied for 30 s. It was observed that nanoparticles prepared by Ultra Turrax were more spherical but larger. In addition, increasing the PVA concentration in the aqueous phase, increasing the PLGA concentration in the organic phase and increasing the homogenization rate decreased the zeta potential values of PLGA nanoparticles.

Comparative evaluation of in vitro parameters of tamoxifen citrate loaded poly(lactide-co-glycolide), poly(epsilon-caprolactone) and chitosan nanoparticles.

Tamoxifen (TAM), the clinical choice for the antiestrogen treatment of advanced or metastatic breast cancer, was formulated in nanoparticulate carrier systems in the form of poly(lactide-co-glycolide) (PLGA), poly-epsilon-caprolactone (PCL) and chitosan (CS) nanoparticles. The PLGA and PCL nanoparticles were prepared by a nanoprecipitation technique whereas the CS nanoparticles were prepared by the ionic gelation method. Mean particle sizes were under 260 nm for PLGA and PCL nanoparticles and around 400 nm for CS nanoparticles. Polydispersity indices were less than 0.4 for all formulations. Zeta potential values were positive for TAM loaded nanoparticles because of the positive charge of the drug. Drug loading values were significantly higher for PCL nanoparticles when compared to PLGA and CS nanoparticles. All nanoparticle formulations exhibited controlled release properties. These results indicate that TAM loaded PLGA, PCL and CS nanoparticles may provide promising carrier systems for tumor targeting.

Preparation and evaluation of alpha-phenyl-n-tert-butyl nitrone (PBN)-encapsulated chitosan and PEGylated chitosan nanoparticles.

Alpha-phenyl-n-tert-butyl nitrone (PBN) shows its major effect by scavenging free radicals formed in the ischemia and it has the ability to penetrate through the blood brain barrier easily. The in vivo stability of PBN is very low and when administered systemically, it has a mean plasma half life of about three hours. Therefore, formulations which are able to prolong the plasma residence time of PBN are of major interest, because oxygen radicals are usually continuously formed under pathological conditions. In this study, PBN, a nitrone compound having neuroprotective properties, was encapsulated in chitosan (CS) and chitosan-poly(ethylene glycol) (CS-PEG) nanoparticles for treatment of diseases such as stroke, in which sustained free radical production is reported. The nanoparticles were characterized through particle size determination, zeta potential, encapsulation efficiency, surface morphology determinations and in vitro release studies. The surface morphologies were evaluated by transmission electron microscopy (TEM) and nanoparticles having spherical shapes were characterized. The particle size distribution was between approximately 97 nm and approximately 322 nm; and the zeta potentials varied between approximately 9 mV and approximately 33 mV. Size of the nanoparticle formulations was important for the release of PBN from nanoparticles. The quantitative determination of PBN has been evaluated by a validated analytical HPLC method. The presented chitosan-based nanotechnology opens new perspectives for testing antioxidant activity in vivo.

Preparation and characterization of PLGA nanospheres for the targeted delivery of NR2B-specific antisense oligonucleotides to the NMDA receptors in the brain.

Treatment of central nervous system (CNS) diseases with potentially useful pharmaceuticals is prevented by the blood-brain barrier (BBB). The BBB is a unique protective barrier in the body. It is formed by epithelial-like tight junctions, which are expressed by the brain capillary endothelial cells. Although most molecules are potentially active in the CNS, they cannot readily enter the brain because of their properties. Antisense oligonucleotides (ODNs) have a great potential as neuropharmaceuticals; however, the large size and polar nature of nucleic acid drugs prevent these molecules from bypassing the BBB and readily entering the CNS following systemic administration. One approach to improve both the pharmacokinetics and the pharmacodynamics of ODNs involves the use of sustained-release polymer formulations, such as poly(lactide-co-glycolide) (PLGA) nanoparticulate systems. In this study, nanospheres were prepared by the emulsification diffusion technique and characterized in terms of particle size, surface morphology, encapsulation efficiency, in vitro release profiles and ODN stability. The nanospheres produced were spherical with homogenous size distribution. Nanospheres were prepared with different encapsulation efficiency. Release profiles of formulations were also evaluated. The results show that formulations with different ODN content exhibited different release profiles. Moreover, the chemical integrity of ODN during the processes was conserved. These results demonstrate that a stable ODN formulation could be prepared utilizing PLGA nanospheres as a potential delivery system for the treatment of CNS diseases.

In vitro cytotoxicity of mitoxantrone-incorporated albumin microspheres on acute promyelocytic leukaemia cells.

In the present study, the preparation and characterization of bovine serum albumin (BSA) microspheres and the evaluation of the in vitro cytotoxicity of these microspheres on acute promyelocytic leukaemia (HL-60) cells were described. Mitoxantrone (MTZ)-incorporated microspheres were evaluated for particle size, drug loading, release characteristics and surface morphology. The biological effect of MTZ released from BSA microspheres was determined on an in vitro cultured HL-60 cell line, showing that, after encapsulation, MTZ still retains cytotoxic activity. For this purpose, methyl-thiazol-tetrazolium (MTT) assay was used to evaluate the in vitro cytotoxicity of MTZ-loaded microspheres. Particle size of BSA microspheres was determined between 17.61-20.38 microm and they were smooth and spherical in shape. Encapsulation efficiency of the drug-loaded microspheres was between 22.26-60.50%. For MTZ-containing microspheres, the cell death ratios were greater than 80% for all formulations. This study demonstrate that BSA microspheres were well suited for the controlled release of MTZ and were promising for anti-cancer chemotherapy.

In vitro/in vivo studies on a buccal bioadhesive tablet formulation of carbamazepine.

A buccoadhesive controlled-release system for delivery of carbamazepine (CBZ) was prepared by compression of hydroxypropyl methylcellulose (HPMC) and carbomer, incorporating a penetration enhancer, sodium glycodeoxycholate (GDC). The release behaviour of systems containing CBZ and various amounts of the two polymers with and without GDC was found to be non-Fickian. Formation of an interpolymer complex between HPMC and carbomer was confirmed in acidic medium by turbidity, viscosity and FT-IR measurements. Addition of the drug to the buccoadhesive formulation reduced the adhesion force significantly (p < 0.1). GDC did not have any effect on bioadhesion. Permeability of bovine buccal mucosa to CBZ was determined using Ussing diffusion chambers [1]. In vivo interaction between the tablet and tissue was examined histologically as well as by scoring mucosal irritation. Histological changes observed in the buccal epithelium after 4 h contact with the tablets containing GDC recovered completely within 24 h after removal. No measurable plasma level of CBZ was obtained either in the absence or presence of GDC.

Stability of poly(L-lysine)-complexed plasmid DNA during mechanical stress and DNase I treatment.

The aim of this study was to investigate the formation and stability of complexes between plasmid DNA (pDNA) and poly(L-lysine) (PLL). Formation of pDNA/PLL complexes with various ratios was determined by a fluorescence spectrophotometric method using fluorescamine. The effects of sonication, vortexing, and exposure to DNase I on the stability of free pDNA and pDNA/PLL complexes are discussed. A linear correlation between PLL added and PLL bound was obtained with overall reaction efficiency of 84.2-92.6%. Sonication degraded both free and PLL-complexed pDNA within 15 sec of vortexing. However, vortexing did not alter the stability of free and complexed pDNA. Dramatic increase in the protection of pDNA in pDNA/PLL complexes was observed in the DNase I digestion experiment; 68.1-89.0% of total pDNA in the pDNA/PLL complexes was protected from DNase I digestion compared to only 19.2% of total pDNA that remained undegraded after DNase I treatment of free pDNA. An increase in the PLL/pDNA ratio led to an increase in the protection of supercoiled pDNA; 15.5-38.2% of supercoiled pDNA pin PLL/pDNA complexes was protected after DNase I treatment. The results show that complexation of pDNA with PLL can stabilize the supercoiled structure of pDNA for the development of biodegradable microspheres as a delivery system for pDNA. Stability of pDNA/PLL complex can be monitored by PicoGreen dye and fluorescence densitometric assay methods.

Influence of formulation parameters on the characteristics of poly(D, L-lactide-co-glycolide) microspheres containing poly(L-lysine) complexed plasmid DNA.

This study describes the influence of polymer type, surfactant type/concentration, and target drug loading on the particle size, plasmid DNA (pDNA) structure, drug loading efficiency, in vitro release, and protection from DNase I degradation of poly(D, L-lactide-co-glycolide) (PLGA) microspheres containing poly(L-lysine) (PLL) complexed pDNA. PLGA microspheres containing pDNA-PLL were prepared using the water-in-oil-in-water (w-o-w) technique with poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) as surfactants in the external aqueous phase. A complex ratio of 1:0.33 (pDNA-PLL, w/w) enhanced the stability of pDNA during microsphere preparation. Higher pDNA-PLL loading efficiency (46.2%) and supercoiled structure (64.9%) of pDNA were obtained from hydrophobic PLGA (M(w) 31000) microspheres compared with hydrophilic PLGA or low-molecular-weight PLGA microspheres. The particle size decreased from 6.6 to 2.2 microm when the concentration of PVA was increased from 1 to 7%. At the same concentration of surfactant, PVA stabilized microspheres showed higher pDNA-PLL loading efficiency (46.2%) than PVP stabilized microspheres (24.1%). Encapsulated pDNA in PLGA microspheres was protected from enzymatic degradation and maintained in the supercoiled form. The pDNA-PLL microspheres showed in vitro release of 95.9 and 84.9% within 38 days from the low-molecular-weight PLGA and hydrophilic PLGA microspheres, respectively, compared to 54.2% release from the hydrophobic, higher-molecular-weight PLGA microspheres. The results suggest loading and release of pDNA-PLL complex can be influenced by surfactant concentration and polymer type.

Preparation and characterization of poly (D,L-lactide-co-glycolide) microspheres for controlled release of poly(L-lysine) complexed plasmid DNA.

PURPOSE: To produce and characterize controlled release formulations of plasmid DNA (pDNA) loaded in poly (D,L-lactide-co-glycolide) (PLGA) microspheres both in free form and as a complex with poly (L-lysine). METHODS: Poly (L-lysine) (PLL) was used to form pDNA/PLL complexes with complexation ratio of 1:0.125 and 1:0.333 w/w to enhance the stability of pDNA during microsphere preparation and protect pDNA from nuclease attack. pDNA structure, particle size, zeta potential, drug loading, in vitro release properties, and protection from DNase I were studied. RESULTS: The microspheres were found to be spherical with average particle size of 3.1-3.5 microm. Drug loading of 0.6% was targeted. Incorporation efficiencies of 35.1% and 29.4-30.6% were obtained for pDNA and pDNA/PLL loaded microspheres respectively. Overall, pDNA release kinetics following the initial burst did not correlate with blank microsphere polymer degradation profile suggesting that pDNA release is convective diffusion controlled. The percentage of supercoiled pDNA in the pDNA and pDNA/PLL loaded microspheres was 16.6 % and 76.7-85.6% respectively. Unencapsulated pDNA and pDNA/PLL degraded completely within 30 minutes upon the addition of DNase I. Encapsulation of DNA/PLL in PLGA microspheres protected pDNA from enzymatic degradation. CONCLUSIONS: The results show that using a novel process, pDNA can be stabilized and encapsulated in PLGA microspheres to protect pDNA from enzymatic degradation.

Formulation and in vitro/in vivo investigation of carbamazepine controlled-release matrix tablets.

Carbamazepine controlled-release tablet formulations containing hydroxypropyl methylcellulose (HPMC) as matrix material at different concentrations were developed and evaluated in vitro and in vivo. The formulation containing 10% HPMC (HPMC-10) showed a controlled-release profile comparable to that of a commercially available, controlled-release carbamazepine preparation (Tegretol CR 200). The kinetics of controlled-release carbamazepine tablets was examined in eight healthy volunteers. The peak plasma concentration of 1.99 +/- 0.56 micrograms.ml-1 was obtained for HPMC-10 at 15.0 +/- 9.0 h, and 1.33 +/- 0.35 micrograms.ml-1 for Tegretol CR 200 at 15.2 +/- 8.9 h, and AUC0-infinity values of 85.2 +/- 30.8 micrograms.h.ml-1 and 76.9 +/- 20.7 micrograms.h.ml-1, respectively. Developed formulation (HPMC-10) was found to be bioequivalent to Tegretol CR 200 and, controlled release was obtained with smoother concentration-time curve resulting in less fluctuations.

In vitro studies on enhancing effect of sodium glycocholate on transbuccal permeation of morphine hydrochloride.

During the perioperative period, gastric emptying rate and first-pass metabolism limit the use of peroral morphine. Buccal mucosa appears to be a potential site for delivery of morphine as it provides direct entry into the system circulation thereby avoiding the hepatic first-pass effect. However, the low permeability of the buccal epithelium results in a low flux of the drug. The use of a penetration enhancer is required to improve the bioavailability of the drug via buccal route. In this study, the enhancing effect of sodium glycocholate (GC) used at 10 mM and 100 mM concentrations on permeation of morphine hydrochloride (MPH) across the porcine buccal mucosa was studied in vitro. Furthermore, in conjunction with its permeation, accumulation of GC in the tissue with time was also studied in order to elucidate the relationship between GC and enhanced mucosal permeation of the drug. Franz diffusion cells were used in the experiments. Permeation of MPH was increased in the presence of 100 mM GC with an enhancement factor of 9.3 whereas no enhancement was obtained with 10 mM GC. The calculated permeability coefficient for MPH in the presence of 100 mM GC was 2.35 x 10(-5) cm/s. Accumulation of GC at 100 mM in the tissue appears to be more significant at 100 mM concentration which correlated well with the increased permeation of the drug. GC was diffused through the buccal epithelium significantly at 100 mM concentration. Interaction of GC with the tissue appears to be more significant at 100 mM concentration compared to 10 mM concentration, thus resulting in a significant enhancing effect.

Formulation and in vitro-in vivo evaluation of buccoadhesive morphine sulfate tablets.

Buccoadhesive controlled-release systems for the delivery of morphine sulfate were prepared by compression of hydroxypropyl methylcellulose (HPMC) with carbomer (CP), which served as the bioactive adhesive compound. The release behavior of systems containing 30 mg of morphine sulfate and various amounts of the two polymers was found to be non-Fickian. The adhesion force was significantly affected by the mixing ratio of HPMC and CP in the tablet, and the weakest adhesion force was observed at a ratio of 1:1 (HPMC:CP). Interpolymer complex formation was confirmed between HPMC and CP in acidic medium by turbidity, viscosity, and FT-IR measurements. The amount absorbed (percentage of the drug loaded) of the controlled-release buccoadhesive tablets in six healthy volunteers and was 30 +/- 5%.

Physico-chemical and bioadhesive properties of polyacrylic acid polymers.

In this study, influence of several physico-chemical properties of bioadhesive polyacrylic acid poylmers (Carbopol 910, Polycarbophil, Carbopol 940 and Carbopol 934 PH) are investigated. The effect of pH on the swelling of polyacrylic acid polymers was studied at constant temperature, ionic strength and osmolality. The polymers showed the pH dependence of equilibrium swelling. The bioadhesive strength of the bond formed between surface-preswollen systems and the sublingual bovine mucus was determined by tensile experiments. It was found that the least substituted polymers Polycarbophil and Carbopol 910 with low molecular weight showed the same degree of bioadhesion while highly branched polyacrylic acid polymers Carbopol 940 and 934 PH showed reduced bioadhesion.

Factors affecting the formulation of sustained release potassium chloride tablets.

In this study, influence of several formulation factors on the release kinetics of potassium chloride from directly compressed matrices are investigated. Formulations containing hydrophilic (methylcellulose, carbomer), plastic (polyvinyl chloride), and wax (glycerol palmitostearate) matrix materials at concentrations of 10, 15 and 20%, incorporated with potassium chloride as active ingredient and insoluble excipients were prepared and studied in vitro using the USP XXI/NF XVI rotating paddle method. Hardness had no markedly effect on the release characteristics of formulations except for wax matrix material formulation. Results of goodness of fit analysis applied to release data had shown that the release mechanism was described by the Higuchi diffusion controlled model. Positive deviations from Higuchi equation might be due to the erosion of gel layer. Analysis of in vitro release mechanisms indicated that the most suitable results were obtained by methylcellulose and glycerol palmitostearate.