International Journal of Pharmaceutics (v.326, #1-2)

Synergistic effect of anionic lipid enhancer and electroosmosis for transcutaneous delivery of insulin by S. Narasimha Murthy; Ya-Li Zhao; Sek-Wen Hui; Arindam Sen (1-6).
A lipid formulation consisting of 1,2-dimyristoyl-sn3-phosphatidylserine (DMPS) in a 0.2% sodium dodecylsulfate (SDS) solution was tested as an in vivo enhancer for the transcutaneous delivery of insulin. The formulation when applied to for 15 min was found to permeabilize porcine epidermis and prolong the permeable state as evidenced by electric resistance measurement. The formulation enhanced the transport of insulin through the epidermis by 40- to 100-fold, as compared to epidermis that was treated with SDS or DMPS alone. Application of electroosmosis across the formulation-treated epidermis enhanced the transport of insulin by an additional 10-fold. Pharmacokinetic studies were carried out in Sprague-Dawley rats. Transcutaneous delivery of insulin with formulation treatment and electroosmosis increased the plasma level of insulin by ∼10-fold over delivery by formulation treatment alone. With the above protocol plasma insulin concentration remained relatively constant for up to 4 h. The synergistic application of anionic lipid formulation and electroosmosis offers a promising non-invasive technique to deliver insulin transcutaneously.
Keywords: Anionic lipid; Insulin; Porcine epidermis; Rat skin; Transcutaneous delivery; Electroosmosis; Pharmacokinetics;

Monitoring small molecule diffusion into hydrogels at various temperatures by fluorescence technique by Gülşen Akın Evingür; Kadir Karslı; Önder Pekcan (7-12).
Steady state fluorescence technique was used to study small molecule diffusion into polyacrylamide (PAAm) gels at various temperatures. Pyranine (Py), dissolved in water was introduced as a probe and fluorescence emission (I p) from Py was monitored during diffusion. Scattered light intensities, I sc from PAAm gel was also monitored to observe structural variations during diffusion process. Increase in I p intensity was attributed to Py diffusion into PAAm gel. On the other hand decrease in I sc intensity was interpreted as the variation of the spatial heterogeneities in the system. Li-Tanaka and Fickian models were used to quantify the swelling and diffusion experiments and diffusion coefficients were produced in both cases. Related activation energies were also calculated from the corresponding physical processes.
Keywords: Diffusion; Hydrogel; Fluorescence; Temperatures;

Effect of electroporation and pH on the iontophoretic transdermal delivery of human insulin by Seiji Tokumoto; Naruhito Higo; Kenji Sugibayashi (13-19).
The synergistic effect of electroporation (EP) and iontophoresis (IP) on the in vivo percutaneous absorption of human insulin was evaluated in rats. Passive diffusion and IP alone (0.4 mA/cm2) resulted in almost no skin permeation of insulin at pH 7, whereas EP treatment (150 or 300 V, 10 ms, and 10 pulses) resulted in a high plasma level of insulin and the combined use of EP and IP led to a further increase of the plasma level of insulin compared with that measured after EP alone. Interestingly, a much higher plasma level was observed when the pH of the insulin solution at 7 was increased to 10. One of the reasons was the different aggregation properties of insulin at pH 7 and pH 10. The nonassociation ratio of insulin was significantly higher at pH 10 than at pH 7. Insulin monomers and dimers were observed in addition to the normal form of insulin, hexamer, albeit in low percentages, at pH 10, whereas most of the insulin was in the hexamer form at pH 7. To confirm the influence of the aggregation properties of insulin, the commercially available human insulin analogue insulin lispro was then evaluated. Its skin permeation was found to be extremely high compared to that of conventional human insulin without increasing the solution pH. Marked decreases in blood glucose levels reflecting the increases in the plasma concentration of insulin were also observed after EP/IP treatment.The present study suggests that percutaneous absorption of insulin is synergistically enhanced by a combined use of EP and IP and that altering the aggregation properties of insulin is important to enhance the percutaneous absorption of insulin by IP and/or EP.
Keywords: Insulin; Percutaneous absorption; Electroporation; Iontophoresis; Insulin lispro;

In an attempt to prepare topical formulations of griseofulvin that can deliver the drug locally in effective concentration, various hydrogel formulations were prepared using carbomer (940 NF) as base; essential oils, propylene glycol (PG), N-methyl-2-pyrrolidone (NMP) as penetration enhancers. The in vitro skin permeation studies through Laca mouse skin were performed using vertical type cells. PG in the hydrogel formulation was found to influence drug release rate by increasing its solubility and partitioning. Further combinations of PG with varying amounts of NMP in the hydrogel formulations exhibited a significantly greater increase in the flux on comparison with the control and formulation containing PG alone. The diffusion samples obtained by in vitro permeation studies through mouse skin when subjected to microbioassay using Microsporum gypseum as tester microorganism exhibited antifungal activity. This indicates that the drug permeated through the mouse skin possess sufficient antifungal activity in vitro against the tested microorganism. The prepared hydrogels did not show any skin sensitization and histological studies were carried out to check the safety of permeation enhancers used. Further these formulations were found to be stable at three different temperatures 4, 25 and 40 °C with respect to percent drug content, release characteristics, pH, transparency, feel and viscosity.
Keywords: Griseofulvin; Topical; Gels; Permeation enhancers; N-Methyl-2-pyrrolidone;

Bioavailability of salvianolic acid B in conscious and freely moving rats by Yu-Tse Wu; Yen-Fei Chen; Yen-Ju Hsieh; Ine Jaw; Ming-Shi Shiao; Tung-Hu Tsai (25-31).
Salvianolic acid B is an herbal ingredient isolated from Salvia miltiorrhiza. The aim of this study was to apply an automated blood sampling system coupled to a simple liquid chromatographic system to determine the bioavailability of salvianolic acid B in stress-free rats. The plasma sample (25 μl) was vortex-mixed with 50 μl of internal standard solution (chloramphenicol 10 μg/ml in acetonitrile) to achieve protein precipitation. Salvianolic acid B in the rat plasma was separated using a reversed-phase C18 column (250 mm × 4.6 mm, 5 μm) with a mobile phase of acetonitrile–methanol–20 mM NaH2PO4 (adjusted to pH 3.5 with H3PO4) (20:10:70 v/v/v) containing 0.1 mM 1-octanesulfonic acid, and the flow-rate of 1 ml/min. The UV detection wavelength was 286 nm. The concentration–response relationship from the present method indicated linearity over a concentration range of 0.5–200 μg/ml. Intra- and inter-assay precision and accuracy of salvianolic acid B fell well within the predefined limits of acceptability (<15%). The plasma sample of salvianolic acid B was further identified by LC–MS/MS in the negative ion mode using mass transition m/z 358.2 to the product ion m/z 196.9. After salvianolic acid B (100 mg/kg, i.v.; 500 mg/kg, p.o.) was given in conscious and freely moving rats, the AUC were 5030 ± 565 and 582 ± 222 min μg/ml for intravenous (100 mg/kg) and oral (500 mg/kg) doses, respectively. The oral bioavailability of salvianolic acid B in freely moving rats was calculated to be 2.3%.
Keywords: Automated blood sampling; Bioavailability; Pharmacokinetics; Salvianolic acid B; Tandem mass spectrometry;

Investigation into the potential of low-frequency ultrasound facilitated topical delivery of Cyclosporin A by Hongzhuo Liu; Sanming Li; Weisan Pan; Yongjun Wang; Fei Han; Huimin Yao (32-38).
The potential for low-frequency ultrasound facilitated topical transport of Cyclosporin A was investigated using rat skin. Studies of intensity and exposure time acting on the deposition of Cyclosporin A into deeper skin of in vitro sonophoresis were performed. Low-frequency ultrasound increased the amount of Cyclosporin A retained in the skin only seven times than the passive diffusion. Furthermore, we also tested the synergistic effect of ultrasound and other approaches such as chemical enhancers and electroporation on topical drug delivery of Cyclosporin A. We found that the efficacy of low-frequency ultrasound in enhancing topical delivery could be further increased by pretreatment of skin with chemical enhancers, such as laurocapram (Azone) and sodium lauryl sulfate (SLS). Meanwhile only a small amount was seen to across the full skin into the receiver compartment. Trimodality treatment comprising of pretreatment with Azone + ultrasound in combination followed by electroporation was not effective in enhancing the topical delivery of Cyclosporin A. However, this combination strategy increased the penetration of Cyclosporin A through rat skin by order of 15. The histopathological findings revealed that there was almost no change observed in the structure of skin after ultrasound or combination with ultrasound and enhancers as compared with the control group. In general, the enhanced skin accumulation of Cyclosporin A by the combination of low-frequency ultrasound and chemical enhancers could help significantly to optimize the targeting of the drug without of a concomitant increase of the systemic side effects.
Keywords: Low-frequency ultrasound; Chemical enhancer; Eletroporation; Synergistic; Cyclosporin A;

Development of a laser diffraction method for the determination of the particle size of aerosolised powder formulations by Christopher Marriott; Helen B. MacRitchie; Xian-Ming Zeng; Gary P. Martin (39-49).
Impactor data are an essential component of marketing authorisation for new dry powder aerosol formulations. However such data are time-consuming to obtain and therefore impede the rapid screening of pilot formulations. In this phase of development it would be of considerable benefit to employ a technique where data acquisition was more rapid, such as laser diffraction, to predict the fine particle fraction. It was the aim of this study to investigate whether this is a feasible premise. Five different formulations were prepared, each containing 1.5% (w/w) micronised salbutamol base (volume median diameter: 2.42 μm) blended with the sieved fraction (63–90 μm) of one of the following sugars: regular crystalline lactose, spray dried lactose “Zeparox™”, sorbitol, maltose and dextrose monohydrate. A Perspex™ box was constructed to contain particles released from a glass inhaler and allow the particles to be measured by laser diffraction at different flow rates. After being validated using monodisperse aerosols, this assembly was then employed to measure the particle size distributions of each powder formulation and its respective sugar carrier at flow rates ranging from 28.3 to 100 l min−1. Aerodynamic particle size distribution of salbutamol base from each formulation was also measured after aerosolisation at 28.3 l min−1 from the glass inhaler into an Andersen cascade impactor. The flight of monodisperse particles with diameters (2–6 μm) in the desired size range of dry powders for inhalation could be contained and the size distribution determined by laser diffraction using the assembly at all flow rates investigated. Treatment of the particle size distributions measured by laser diffraction, i.e. examining only the aerosol particles with diameter <60 μm, highlighted the fine fraction (<5 μm) and enabled the aerosolisation of different blends to be feasibly compared at a range of different flow rates. The blends containing the following excipients could be placed in the following order of increasing fine fraction: spray-dried lactose < dextrose ≪ maltose < lactose < sorbitol. At 28.3 l min−1 a significant linear correlation was found between the fine fractions measured by laser diffraction and the salbutamol fine fractions determined by inertial impaction (R 2  = 87.4%, p  = 0.02, ANOVA). Therefore, the laser diffraction technique could prove to be an important tool for particle size characterisation of dry powder aerosol formulations.
Keywords: Laser diffraction; Inertial impaction; Salbutamol; Dry powder inhaler formulations; Fine particle fraction; Aerodynamic particle size distribution;

Multifunctional polyvinylpyrrolidinone-polyacrylic acid copolymer hydrogels for biomedical applications by Declan M. Devine; Sinead M. Devery; John G. Lyons; Luke M. Geever; James E. Kennedy; Clement L. Higginbotham (50-59).
Copolymers of N-vinylpyrrolidinone and acrylic acid, crosslinked with ethylene glycol dimethacrylate and polyethylene glycol 600 dimethacrylate were prepared by UV-polymerisation. These polymers were analysed for their extractable content by Soxhlet extraction of the samples at 100 °C for 72 h. Aspirin and paracetamol were incorporated into the polymer structure at 25 wt.% during the curing process and their presence confirmed by Fourier transform infrared spectroscopy. It was found that the release rate of the drug from the polymer matrix was dependent on intermolecular bonding between the polymer and active agent with aspirin being released slower than paracetamol in all cases. Results showed that paracetamol was completely released after 24 h whereas complete release of aspirin took up to 70 h. Finally preliminary in vitro biocompatibility testing was performed for crosslinked polyvinylpyrrolidinone, by determining human hepatoma HepG2 cell viability in the MTT assay and DNA damage in the comet assay following direct contact with various concentrations of polyvinylpyrrolidinone-containing media. Cytotoxicity data suggests a dose-dependent effect for both crosslinkers, with concentrations in the range 0.025–2.5 mg ml−1 showing a marginal decrease in viability to, at most, 70% that of untreated cells. Again DNA migration in the comet assay following short-term exposure to EGDMA crosslinked hydrogels correlates with MTT data.
Keywords: Acetylsalicyclic acid; Paracetamol; Controlled release; Hydrogels; pH-responsive; Biocompatibility;

Development and evaluation of an artificial membrane for determination of drug availability by Thorsteinn Loftsson; Fífa Konrádsdóttir; Már Másson (60-68).
Various artificial membranes (e.g. PAMPA) and cellular-based membranes (e.g. Caco-2) are used for screening during early stages of drug discovery. However, these methods are not well suited for evaluation of pharmaceutical formulations and the effects of various excipients on drug availability. When drug molecules permeate biological membranes they encounter two types of permeation resistance, a membrane resistance in the lipophilic membrane and diffusion resistance in the unstirred water layers adjacent to both surfaces of the lipophilic membrane. We have developed an artificial membrane that is cheap and simple to prepare. The unstirred water layer consists of a hydrated semi-permeable cellophane membrane with a molecular weight cutoff (MWCO) of 12,000–14,000 Da and a lipophilic membrane of pure n-octanol in a nitrocellulose matrix. In the diffusion cell the hydrated cellophane membrane (thickness 210–230 μm) is on the donor side and the lipophilic octanol membrane (thickness about 120 μm) on the receptor side. Permeation of ionizable lipophilic drug molecules was diffusion-controlled when the drug was unionized but lipophilic membrane controlled when the drug was ionized. Drug permeation patterns from cyclodextrin containing formulations through the membrane were similar to those previously observed for biological membranes such as hairless mouse skin and the eye cornea.
Keywords: Octanol membrane; Permeation; Unstirred water layer; Formulation; Cyclodextrin; Availability;

Solid state characterization of olanzapine polymorphs using vibrational spectroscopy by A.P. Ayala; H.W. Siesler; R. Boese; G.G. Hoffmann; G.I. Polla; D.R. Vega (69-79).
FT-Raman, infrared and near infrared investigations of two polymorphs of olanzapine are presented, establishing the main features that allow the discrimination of these crystalline forms using vibrational spectroscopic methods. Ab initio calculations on the basis of the density functional theory were used to determine the stable conformations. The calculated vibrational spectra were compared to the experimental ones in order to identify the conformers corresponding to each polymorph and to assign the vibrational bands to the internal vibrations of the olanzapine molecule. Our results support the hydrogen bonding pattern proposed by the reported crystalline structure and provide valuable information on the structural and thermodynamical relationship between the investigated polymorphs.
Keywords: Olanzapine; Polymorphism; Raman spectroscopy; Infrared spectroscopy; Hydrogen bonding; Ab initio; DFT;

Development of polysaccharide gel coated pellets for oral administration by Pornsak Sriamornsak; Mark A. Burton; Ross A. Kennedy (80-88).
Spherical pellets containing theophylline, calcium acetate and microcrystalline cellulose were extruded and spheronized, before being coated with six different pectins or alginates by interfacial complexation. The aim of this study was to discover the effect of the coatings on physico-mechanical properties that will be crucial in determining the pellets’ utility as sustained release systems. An insoluble, smooth and uniformly thick coat of calcium polysaccharide was formed around the core pellets. A factorial experiment was designed to investigate the effect of pellet size and polysaccharide type and concentration on the entrapment efficiency, mechanical properties and other physical characteristics. Coated pellets were observed by scanning electron microscopy and, depending on the particular polysaccharide used, the dry coats were found to be 30–80 μm thick. The size of pellet, the type and concentration of polysaccharide influenced the yield of theophylline in the coated pellets. Although the mechanical properties of the pellets were improved by applying any of the gel coats, use of an alginate with a high content of guluronic acid or an amidated pectin coating gave the best results. This is probably because both of these have significant potential to form very stable cross-links within the gel coats.
Keywords: Extrusion and spheronization; Alginate; Pectin; Polysaccharide coatings; Physico-chemical properties of coated spheres;

Melt granulation using a twin-screw extruder: A case study by Barbara Van Melkebeke; Brenda Vermeulen; Chris Vervaet; Jean Paul Remon (89-93).
The purpose of this study was to use a twin-screw extruder for melt granulation. Polyethylene glycols (PEG 400 and 4000) were used as binders for the development of a drinking water formulation with immediate drug release. The effect of drug content, PEG 400/4000-ratio, surfactant (type and concentration) and granulation temperature on granule properties and dissolution characteristics was determined. The granulation temperature had an important influence on the granule formation. High yield (95% of the granules <1400 μm) was obtained only at a temperature near the melting point of PEG 4000. During granulation the drug of BCS class II was finely dispersed in the PEGs, creating a micro-environment around the drug particles enhancing the dissolution rate. To obtain complete drug release within 10 min for a formulation containing 10% drug, the addition of 2% (w/w) surfactant (polysorbate 80 or Cremophor® RH40) was required. At a higher drug content (20%), the PEG 4000 concentration had to be increased to 20% to improve granule properties and 4% polysorbate 80 was required to obtain 100% drug release. X-ray diffractograms showed distinct peaks of crystalline drug, the crystallinity of the drug did not change after 50 days, independent of the storage conditions.
Keywords: Continuous granulation; Extruder; Melt agglomeration; Polyethylene glycol;

True density results for a batch of commercial aspartame are highly variable when helium pycnometry is used. Alternatively, the true density of the problematic aspartame lot was obtained by fitting tablet density versus pressure data. The fitted true density was in excellent agreement with that predicted from single crystal structure. Tablet porosity was calculated from the true density and tablet apparent density. After making the necessary measurements for calculating tablet apparent density, the breaking force of each intact tablet was measured and tensile strength was calculated. With the knowledge of compaction pressure, tablet porosity and tensile strength, powder compaction properties were characterized using tabletability (tensile strength versus pressure), compactibility (tensile strength versus porosity), compressibility (porosity versus pressure) and Heckel analysis. Thus, a wealth of additional information on the compaction properties of the powder was obtained through little added work. A total of approximately 4 g of powder was used in this study. Depending on the size of tablet tooling, tablet thickness and true density, 2–10 g of powder would be sufficient for characterizing most pharmaceutical powders.
Keywords: Aspartame; True density; Material sparing; Compaction; Porosity; Tensile strength;

Vitamin A palmitate and aciclovir biodegradable microspheres for intraocular sustained release by C. Martínez-Sancho; R. Herrero-Vanrell; S. Negro (100-106).
The aim of this study was to obtain a prolonged release of Vitamin A palmitate (RAP) and aciclovir from biodegradable microspheres for intraocular administration with an antiviral action and to be capable of preventing the inherent risks of intravitreal administration. The RAP effect on the microsphere characteristics was also studied. Poly(d,l-lactic-co-glycolic) acid microspheres were prepared by the solvent evaporation method. Different quantities of aciclovir (40–80 mg) and RAP (10–80 mg) were added to the internal phase of the emulsion. Microspheres were analysed by scanning electron microscopy, which revealed a spherical surface and a porous structure, and granulometric analysis that showed an adequate particle size for intraocular administration. The aciclovir loading efficiency increased when Vitamin A palmitate was added. Differential scanning calorimetry detected no differences in the polymer glass transition temperature and the aciclovir melting endotherm in all formulations. The release of aciclovir during the first days of the in vitro assay was improved with respect to microspheres without RAP. The microspheres showed a constant release of aciclovir and RAP for 49 days. Best results were obtained for microspheres prepared with 40 mg aciclovir, 80 mg RAP and 400 mg polymer. A dose of 4.74 mg of microspheres would be therapeutic for the herpes simplex and Epstein-Barr viruses’ treatment in an animal model and would reduce the intravitreal adverse effects. The injectability of a suspension of microspheres in isotonic saline solution resulted appropriate for its injection through a 27 G needle.
Keywords: Vitamin A palmitate; Aciclovir; PLGA microspheres; Intraocular;

Formulation of an antispasmodic drug as a topical local anesthetic by Sameh M. Abdel-Hamid; S.E. Abdel-Hady; A.A. El-Shamy; H.F. El-Dessouky (107-118).
Mebeverine hydrochloride, a spasmolytic agent on GIT smooth muscles, was reported to have a local anesthetic effect. Thus, it was desired in this study to formulate mebeverine HCl into a gel that could be used locally in the treatment of different oral painful conditions. Poloxamer 407 (P-407) was used as the base for this gel. Different additives were used to enhance drug release from the preparation while others were used to enhance the residence time for the preparation. Different formulae were characterized in terms of drug release and mucoadhesion. The formula which has shown the best compromise between the aforementioned parameters was selected for clinical evaluation in comparison to Lidocaine HCl gel® and rheologically examined. The best drug release enhancer was cetrimide (0.005%, w/w), while hydroxypropylcellulose (0.5%, w/w) as a mucoadhesive additive has shown the best compromise between fast drug release and mucoadhesion. The gel formula (G) has shown a better pain reduction efficiency (p  = 0.0078) and longer duration (p  = 0.0313) than Lidocaine HCl gel®. Histopathological examination has shown no change in the inflammatory cells count of rat oral mucosa. Therefore, it could be concluded that (G) is very promising as a local anesthetic preparation for the treatment of different oral painful conditions.
Keywords: Mebeverine HCl; Local anesthetic; Gel; Oral painful conditions; Histopathology;

Sustained release of protein from poly(ethylene glycol) incorporated amphiphilic comb like polymers by M. Srividhya; S. Preethi; A. Gnanamani; B.S.R. Reddy (119-127).
Amphiphilic comb like macromonomer containing hydrophilic poly(ethylene glycol) groups covalently linked to poly(hydromethyl siloxane) (PHMS) were prepared by hydrosilylation reaction. The epoxy reacting sites were introduced to this amphiphilic system by the reaction with allyl epoxy propyl ether (AEPE). Bovine serum albumin (BSA), a model protein drug was loaded to the PEG–PDMS system and very thin membranes were made from this macromonomer adopting solution casting technique. The in vitro protein release studies at various pH conditions showed a controlled release profile without exhibiting any initial burst. The control of the initial burst might be due to the strong linkages of the protein with the membrane and the aggregation of the protein at the surface. The morphology of the membrane before and after the protein release, and the mechanical strength were evaluated. The surface properties of the membrane were studied using the contact angle measurements.
Keywords: Controlled drug release; Drug delivery; Siloxane; Surface energy;

Enteric microparticles were prepared by a novel microencapsulation method in order to improve the oral bioavailability of lipophilic drugs. This method involved the addition of an aqueous polymer solution to an organic enteric polymer solution containing lipophilic drugs. In contrast to classical coacervation microencapsulation methods, the drugs were initially also dissolved and not dispersed in the organic polymer solution. The hydrophilic polymer (hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC) and Poloxamer 407) was dissolved in the aqueous phase and acted as a stabilizer for the coacervate droplets, preventing their coalescence and leading to the formation of enteric microparticles. The size of the enteric microparticles decreased with higher concentrations of the hydrophilic polymers, a higher pH of the aqueous polymer solution, a higher content of carboxyl groups of the enteric polymer and with better polymer solvents. Amide-containing lipophilic drugs, such as carbamazepine, lidocaine and cyclosporine A, were successfully encapsulated in the enteric microparticles in a non-crystalline state and were physically stable for 5 months. The high solubility of carbamazepine in the enteric polymer (>30%, w/w), a high partition coefficient between polymer-rich/-poor regions and strong drug/polymer interactions contributed to the high drug encapsulation efficiency (90%, w/w). In contrast, carboxyl-containing drugs (indomethacin, ibuprofen) and hydroxyl-containing drug (17β-estradiol hemihydrate) crystallized inside or outside the polymeric matrix due to their low solubility in the enteric polymer.
Keywords: Coacervation; Enteric polymer; Lipophilic drugs; Microencapsulation;

Degradation behaviour of microspheres prepared by spray-drying poly(d,l-lactide) and poly(d,l-lactide-co-glycolide) polymers by M. Dolores Blanco; Roberto L. Sastre; César Teijón; Rosa Olmo; José M. Teijón (139-147).
Polymeric microsphere degradation must be taken into account in the design of drug delivery systems to be injected in in vivo systems, thus a prior analysis of in vitro degradation behaviour of microspheres appears to be necessary. In this study degradation characteristics of poly(lactide-co-glycolide) (PLGA) and poly(d,l-lactide) (PLA) microspheres prepared by the spray-drying technique have been examined. It was found that a slow decrease in molecular weight took place during the first stage of degradation, and the value of the rate constant decreased with the increase of the percentage of lactic acid of the polymer in a linear way. Thus, the period of time of this first stage decreased with the increase of content of glycolidyl units of the polymer, and it was the unique stage observed in PLA microspheres after 5 months of study. During this period of time, significant mass loss was not observed in the microspheres. The second stage of degradation of PLGA microspheres showed a larger rate constant, whose value increased with the content of glycolidyl units of the polymer. Mass loss was observed from number-average molecular weight about 6000. A sharp decrease of glass transition temperature (T g) was observed coinciding with the start of mass loss. This fact was accompanied by a physical change of the samples, fusion of microspheres to form large particles, which also fusion to form a unique mass of polymer; moment from that the degradation process was quicker.
Keywords: Poly(d,l-lactide); Poly(lactide-co-glycolide); Spray-dryer; Microspheres; Degradation;

Layered double hydroxides (LDHs) were investigated as cordycepin delivery nanocarriers for the first time in this study. Negatively charged biomolecule-cordycepin was intercalated in the gallery spaces of [Mg–Al–NO3] as the charge-compensating species, which was confirmed by the results of XRD, FT-IR, TEM, CZE and electrophoretic mobility. Cell experiment suggested that the new bio-LDH nanohybrid could prevent cordycepin decomposition by adenosine deaminase. This new formulation could possibly be used as a novel form cordycepin intravenous injection.
Keywords: Layered double hydroxide; Cordycepin; Nanohybrid; Intercalate;

The aim of this study was to investigate the influence of the proteolytic enzyme bromelain on the permeation of heparin across the gastrointestinal epithelial barrier. Stability of the complex and effect of heparin on the enzymatic activity of bromelain was analysed photometrically by measuring bromelain enzymatic activity in complex with the heparin. In vitro permeation studies were performed with Caco-2 cell monolayer and rat small intestinal mucosa in Ussing-type chambers, respectively. Results revealed that enzymatic activity of bromelain remained uninfluenced by the immobilization of heparin on it. Transport studies across Caco-2 cell monolayer and rat small intestine showed that the permeation of heparin could be significantly increased in presence of bromelain. In the study with Caco-2 cells, the most effective molar ratio of bromelain to heparin was 2:1, leading to 6.7-fold improvement in uptake, whereas the molar ratio 1:1 showed the highest permeation enhancing effect in the study on intestinal mucosa. This study provides evidence that heparin and bromelain form stable complexes leading to a significantly improved uptake of heparin.
Keywords: Bromelain; Heparin; Permeation; Caco-2 cell monolayer;

Nanomolar cationic dendrimeric sulfadiazine as potential antitoxoplasmic agent by M.J. Prieto; D. Bacigalupe; O. Pardini; J.I. Amalvy; C. Venturini; M.J. Morilla; E.L. Romero (160-168).
The high doses of sulfadiazine (SDZ), used in synergistic combination with pyrimethamine, are mainly responsible for severe side effects and discontinuation of toxoplasmosis treatments. In the search for new strategies that improve the efficacy of treatments with reduced doses of SDZ, we have determined the performance of cationic G4 (DG4) and anionic G4.5 (DG4.5) poly(amidoamine) (PAMAM) dendrimers to act as SDZ nanocarriers. Both dendrimers could efficiently load SDZ (SDZ–DG4 and SDZ–DG4.5) up to a ratio of 30 molecules SDZ per dendrimer molecule. The MTT assay on Vero and J774 cells showed no cytotoxicity for DG4.5 and its SDZ complex incubated between 0.03 and 33 μM of dendrimer concentration. On the other hand, DG4 and its SDZ complex resulted cytotoxic when incubated at dendrimer concentrations higher than 3.3 μM. Finally, complexes and empty dendrimers were in vitro tested against Vero cells infected with RH strain of Toxoplasma gondii along 4 h of treatment. For SDZ–DG4.5 and DG4.5 to cause an infection decrease between 25 and 40%, respectively, a dendrimer concentration of 33 μM was required; however, SDZ–DG4 produced the highest infection decrease of 60% at 0.03 μM. These preliminary results, achieved with nanomolar doses of SDZ–DG4 as unique active principle, point to this complex as a suitable potential candidate for antitoxoplasmic therapy.
Keywords: Dendrimers; Sulfadiazine; Toxoplasmosis;

Dexamethasone nano-aggregate was prepared for the treatment of intimal hyperplasia caused by abnormal proliferation of smooth muscle cells. Triblock copolymers composed of poly(ethylene glycol) [PEG] and poly(d,l-lactic acid) [PLA] were synthesized with different chain lengths of PEG. Triblock copolymers in organic phase were mixed with dexamethasone and dexamethasone nano-aggregates was prepared by dispersing the organic phase into water. The average diameter of the nano-aggregates ranged from 200 to 300 nm. Dexamethasone was released out from the nano-aggregates and the release profile was dependent on PEG chain lengths. The dexamethasone nano-aggregates showed superior anti-proliferation effects on smooth muscle cells compared to dexamethasone. Flow cytometry showed that smooth muscle cells treated with dexamethasone nano-aggregates was arrested at a dormant phase in a dose-dependent manner. The dexamethasone nano-aggregates are expected to be a potent candidate for anti-proliferating smooth muscle tissues after a balloon-catheter treatment.
Keywords: Dexamethasone; Nano-aggregate; Smooth muscle cell; Anti-proliferation;

Hypericin-loaded nanoparticles for the photodynamic treatment of ovarian cancer by Magali Zeisser-Labouèbe; Norbert Lange; Robert Gurny; Florence Delie (174-181).
A photodynamic approach has been suggested to improve diagnosis and therapy of ovarian cancer. As Hypericin (Hy), a natural photosensitizer (PS) extracted from Hypericum perforatum, has been shown to be efficient in vitro and in vivo for the detection or treatment of other cancers, Hy could also be a potent tool for the treatment and detection of ovarian cancer. Due to its hydrophobicity, systemic administration of Hy is problematic. Thus, polymeric nanoparticles (NPs) of polylactic acid (PLA) or polylactic-co-glycolic acid (PLGA) were used as a drug delivery system. Hy-loaded NPs were produced with the following characteristics: (i) size in the 200–300 nm range, (ii) negative zeta potential, (iii) low residual PVAL and (iv) drug loading from 0.03 to 0.15% (w/w). Their in vitro photoactivity was investigated on the NuTu-19 ovarian cancer cell model derived from Fischer 344 rats and compared to free drug. Hy-loaded PLA NPs exhibited a higher photoactivity than free drug. Increasing light dose or incubation time with cells induced an enhanced activity of Hy-loaded PLA NPs. Increased NP drug loading had a negative effect on their photoactivity on NuTu-19 cells: at the same Hy concentration, the higher was the drug loading, the lower was the phototoxic effect. The influence of NP drug loading on the Hy release from NPs was also investigated.
Keywords: Photodynamic therapy; Biodegradable nanoparticles; Hypericin; Ovarian cancer; In vitro; Drug delivery system;

Mass-balanced blend uniformity analysis of pharmaceutical powders by at-line near-infrared spectroscopy with a fiber-optic probe by Weiyong Li; Mark C. Johnson; Rick Bruce; Steven Ulrich; Henrik Rasmussen; Gregory D. Worosila (182-185).
A near-infrared (NIR) fiber-optic probe was used at-line to study the blending dynamics of pharmaceutical powder blends on a mass-balanced basis. The probe with a flexible fiber-optic cable can be inserted directly into a 10-L bin blender to get quantitative readings. NIR calibration models were developed and validated for the probe based on a designed 50-sample calibration set. A model formulation containing acetaminophen, mannitol, Avicel, magnesium stearate and AcDiSol was used in the study. The blending study was conducted at 18 rpm for 20 min. NIR probe scans were performed at 1 min intervals and five different locations in the bin. Thief samples were collected and later analyzed by a bench-top NIR instrument to confirm the results from the probe. Complete blending profiles were constructed and compared based on the assay results from both instruments.
Keywords: Near-infrared spectroscopy; Diffusion reflection; Fiber-optic probe; Blend uniformity; Mass balance; Pharmaceutical;

The effect of N-methyl pyrrolidone (NMP) on the phase behavior of two ternary systems, viz. PEG-35-castor oil (Cremophore®EL)–glyceryl caprylate/caprate (Capmul® MCM)–water and PEG-35-castor oil (Cremophore®EL)–isopropyl myristate–water was studied. The study indicated that NMP has considerable influence on the phase behavior of both the systems. NMP increased the area of microemulsion formation in both the systems. Moreover, it also led to reduction/disappearance in the gelling region of the Cremophore®EL–isopropyl myristate–water system. These observations allowed us to conclude that NMP can be considered as a cosurfactant for the development of biocompatible microemulsions.
Keywords: N-Methyl pyrrolidone; Microemulsions; Cosurfactant; Phase behavior; Microemulsion region; Gelling region;

Noticeboard (190).