International Journal of Pharmaceutics (v.309, #1-2)

Development of new polymer-based particulate systems for anti-glioma vaccination by A. Sapin; E. Garcion; A. Clavreul; F. Lagarce; J.P. Benoit; P. Menei (1-5).
Biodegradable and biocompatible microspheres represent a promising alternative to conventional adjuvants for anti-tumour vaccination. Focusing on glioma, we developed two poly(d,l-lactide-co-glycolide) (PLGA)-based particulate systems presenting tumour antigens associated with plasma membranes or with cell lysates. Glioma cell fractions were prepared for adsorption onto poly-d-lysine (PDL)-coated PLGA microspheres formulated using a double-emulsion procedure. Adsorption was followed by 125I-radiolabelling, Western blot and confocal laser scanning microscopy. Only a panel (34%) of the proteins isolated from both cell fractions adsorbed onto PDL-coated PLGA microspheres. The integrity of the epitopes after loading was preserved, as shown by identification of plasma membrane and cytoplasmic markers. Finally, one of the major potential advantages of those particulate systems resides in the fact they not only serve as injectable adjuvant matrices presenting tumour antigens to antigen presenting cells, but also as potential reservoirs for controlled delivery of active immunostimulant molecules.
Keywords: Antigens; Adjuvant; Anti-tumour vaccination; Glioma; Particulate systems; PLGA microspheres;

Lipid excipients are usually used for the development of sustained-release formulations. When used in relatively high quantities, Precirol® ATO 5 imparts sustained-release properties to solid oral dosage forms, by forming a lipid matrix. To control or adjust the drug release kinetics from such lipid matrix however, one must often resort to complementary ingredients or techniques. This study investigates the influence of poloxamers (Lutrol®) included in lipid matrices composed of glyceryl palmitostearate (Precirol® ATO 5) on their dissolution performance and their stability. The addition of these hydrophilic polymers in the lipid matrix increased the amount of theophylline released thanks to the swelling of the hydrophilic polymer and the creation of a porous network into the inert lipid matrix. The grade and the quantity of Lutrol® could modulate the extent of drug release. Theophylline was released mainly by the matrix erosion but also by diffusion through the pores as suggested by the Peppas’ model. Moreover, the addition of Lutrol® enhanced the stability during storage. The theophylline release was quite steady after 6 months in different conditions (temperature and humidity). Thus, the mixture of glyceryl palmitostearate and poloxamers is an approach with many advantages for the development of controlled-release formulations by capsule molding.
Keywords: Capsule molding; Controlled release; Glyceryl palmitostearate; Poloxamer; Precirol®; Swelling;

This paper presents in situ observations of the epitaxial nucleation and growth of the stable polymorph of a steroid, 7αMna, on a specific face of the metastable form at low supersaturation, using optical microscopy and in situ Raman spectroscopy. The presence of the metastable polymorph is essential for the nucleation and growth of the stable one. The order of the metastable zones of the stable and metastable polymorphs is reversed for the epitaxial growth process as compared to the case of 3D nucleation. The rate of transformation of the metastable polymorph to the stable one can be controlled by the supersaturation.
Keywords: Polymorphism; Epitaxial nucleation; Metastable zone;

Evaluation of sodium alginate as drug release modifier in matrix tablets by Celine Valeria Liew; Lai Wah Chan; Ai Ling Ching; Paul Wan Sia Heng (25-37).
Alginates are useful natural polymers suitable for use in the design of pharmaceutical dosage forms. However, the effects of particle size, viscosity and chemical composition of alginates on drug release from alginate matrix tablets are not clearly understood. Hence, 17 grades of sodium alginate with different particle size distributions, viscosities and chemical compositions were used to prepare matrix tablets at various concentrations to screen the factors influencing drug release from such matrices. Particle size was found to have an influence on drug release from these matrices. Sodium alginate was subsequently classified into several size fractions and also cryogenically milled to produce smaller particle size samples. Cryogenic milling could be successfully applied to pulverize coarse alginate particles without changing the quality through degradation or segregation. This study showed the significance of each alginate property in modulating drug release: particle size is important in initial alginic acid gel barrier formation as it affected the extent of burst release; higher alginate viscosity slowed down drug release rate in the buffer phase but enhanced release rate in the acid phase; high M-alginate might be more advantageous than high-G-alginate in sustaining drug release; and, the effect of increasing alginate concentration was greater with larger alginate particles. This can serve as a framework for formulators working with alginates. Furthermore, the results showed that sodium alginate matrices can sustain drug release for at least 8 h, even for a highly water-soluble drug in the presence of a water-soluble excipient.
Keywords: Alginate; Particle size; Viscosity; Cryogenic milling; Controlled release;

A mathematical model for interpreting in vitro rhGH release from laminar implants by A. Santoveña; J.T. García; A. Oliva; M. Llabrés; J.B. Fariña (38-43).
Recombinant human growth hormone (rhGH), used mainly for the treatment of growth hormone deficiency in children, requires daily subcutaneous injections. The use of controlled release formulations with appropriate rhGH release kinetics reduces the frequency of medication, improving patient compliance and quality of life. Biodegradable implants are a valid alternative, offering the feasibility of a regular release rate after administering a single dose, though it exists the slight disadvantage of a very minor surgical operation. Three laminar implant formulations (F 1, F 2 and F 3) were produced by different manufacture procedures using solvent-casting techniques with the same copoly(d,l-lactic) glycolic acid (PLGA) polymer (Mw = 48 kDa). A correlation in vitro between polymer matrix degradation and drug release rate from these formulations was found and a mathematical model was developed to interpret this. This model was applied to each formulation. The obtained results where explained in terms of manufacture parameters with the aim of elucidate whether drug release only occurs by diffusion or erosion, or by a combination of both mechanisms. Controlling the manufacture method and the resultant changes in polymer structure facilitates a suitable rhGH release profile for different rhGH deficiency treatments.
Keywords: PLGA; rhGH; Polymeric laminar implant; Solvent-casting; Drug release;

On the use of the Weibull function for the discernment of drug release mechanisms by Vasiliki Papadopoulou; Kosmas Kosmidis; Marilena Vlachou; Panos Macheras (44-50).
Previous findings from our group based on Monte Carlo simulations indicated that Fickian drug release from Euclidian or fractal matrices can be described with the Weibull function. In this study, the entire drug release kinetics of various published data and experimental data from commercial or prepared controlled release formulations of diltiazem and diclofenac are analyzed using the Weibull function. The exponent of time b of the Weibull function is linearly related to the exponent n of the power law derived from the analysis of the first 60% of the release curves. The value of the exponent b is an indicator of the mechanism of transport of a drug through the polymer matrix. Estimates for b  ≤ 0.75 indicate Fickian diffusion in either fractal or Euclidian spaces while a combined mechanism (Fickian diffusion and Case II transport) is associated with b values in the range 0.75 <  b  < 1. For values of b higher than 1, the drug transport follows a complex release mechanism.
Keywords: Weibull function; Power law; Drug release; Mechanism; Controlled release;

Effect of milling and sieving on functionality of dry powder inhalation products by H. Steckel; P. Markefka; H. teWierik; R. Kammelar (51-59).
α-Lactose monohydrate is the standard excipient used as diluent or carrier in dry powder inhaler (DPI) formulations. Earlier studies have already revealed that raw materials for the production of inhalation grade lactose have to be carefully selected in order to avoid batch-to-batch variability. In the present study, the effect of milling and milling intensity on the flow properties and the physico-chemical characteristics of lactose crystals has been determined. The milled lactoses were then further processed by sieving to give lactose qualities with identical size distribution data, but different batch history (non-milled and milled at different conditions). These were then used to manufacture low concentration (0.25%) drug blends with the model drugs salbutamol sulphate (SBS) and beclometasonedipropionate (BDP); the blends were analysed with a Multistage Liquid Impinger (MLI) after delivery from an Easyhaler® and an Aerolizer® device. It could be shown that gentle milling already results in surface defects on the lactose crystal which are further enhanced by using a higher milling intensity. Produced fine lactose particles during the milling process strongly adhere to the lactose surface and cannot be removed by compressed air which is used for the particle sizing. By trend, a higher milling intensity resulted in higher fine particle fractions (FPF) with both devices. Also, SBS was found to generally give higher fine particle fractions than BDP, independent from the device used. In conclusion, lactose pre-treatment by gentle or strong milling affects the carrier surface and thereby the aerosolization properties of drug/lactose blends produced.
Keywords: Lactose for inhalation; Salbutamol sulphate; Beclometasonedipropionate; Carrier; Raw material; Milling;

Effect of intestinal fluid flux on ibuprofen absorption in the rat intestine by Majella E. Lane; Karl A. Levis; Owen I. Corrigan (60-66).
Previously the apparent permeability coefficient (P app) of ibuprofen was observed to vary depending on the perfusion medium employed. The present work explores the possible contributions to these differences. Studies were undertaken using an in situ single pass rat gut technique. Lumenal drug concentrations and plasma drug levels were assayed by HPLC. Absorption rate constants (k 0) were determined from fractions of drug unabsorbed from the intestineat steady state. Plasma data were fitted to a two compartment open model with zero-order input. Significant differences in net fluid flux were observed between the various buffered perfusion media, with fluxes varying from −0.044 ± 0.006 ml min−1 to +0.057 ± 0.013 ml min−1, the lower and negative values occurring for lower pH media and the larger positive values tending to occur with media of higher pH. A linear relationship was found between the P app of ibuprofen and net water flux (y  = 1.13 + 11.3x; r 2  = 0.80). Apparent zero-order rate constants for ibuprofen appearance in plasma correlated well with absorption rate constants estimated from steady state lumenal drug concentration [k 0(gut)]. From the linear relationship between P app and fluid flux a normalized P app for ibuprofen (i.e. the P app in the absence of net fluid flux) of 1.1 × 10−4  cm s−1 was determined Net lumenal fluid flux is dependent on perfusion medium composition and significantly alters ibuprofen absorption. The differences observed for P app were reflected in systemic drug absorption concentrations. The findings of these studies underline the importance of standardizing the osmolarity of experimental media used for the determination of intestinal permeability data.
Keywords: Fluid flux; Ibuprofen; Osmolarity; Permeability; Intestine;

The oral application is the application of the first choice for drug administration. A lot of drugs exhibit relatively low bioavailability. This may be caused by binding of the drug in the gastro-intestinal tract, by poor penetration of the intestinal mucose or by highly hydrophilic properties. Therefore, problem drugs were only used for i.v. administration (intravenously) or for i.m. administration (intramuscularly). In the present study, cefpirom was investigated as a model substance. Cefpirom (Cp) is a semi-synthetic amino-2-thiazolyl-methoxyimino cephalosporin. It exhibits highly hydrophilic properties (P ow  = 0.02 ± 0.01) and a very low bioavailability (AUC = 524 ± 403 μg min/ml). It was only applied i.v. or i.m. In this work, the influence of absorption enhancers (aggregation and ion-pair formation) on the bioavailability and on the hydrophilic properties of Cp was investigated. The bioavailability of cefpirom was improved through the combination with absorption enhancers (hexadecyldimethylbenzylammonium chloride, BAC; hexylsalicylic acid, HSA). The absolute bioavailability of the Cp combination with absorption enhancers was 21 times larger for BAC and 15 times larger for HSA than in the case when Cp was used alone.
Keywords: Cefpirom; Partition coefficients; Pharmacokinetics;

In this study the micronization of beclomethasone-17,21-dipropionate (BECD), used as an inhaled steroid for the treatment of asthma, was studied using the gas-antisolvent (GAS) process as a “green” alternative to pharmaceutical recrystallization. A systematic investigation of the influence of the key GAS process parameters: antisolvent addition rate (1, 50, 75 and 100 ml/min), temperature (25, 32.5, 40 and 52.5 °C), solute concentration (5, 25, 70 and 100%), and agitation rate (500, 1000, 2000 and 3000 rpm) were investigated on particle morphology, size distribution, and crystallinity. It was found using scanning electron microscopy (SEM) and laser diffraction, that increasing the antisolvent addition rate and the agitation rate, while decreasing the temperature and solute concentration, led to a decrease in the steroids mean particle diameter. These parameters could be tuned to give a mean particle diameter of 1.8 μm, and an average mass median aerodynamic diameter (MMAD) of 7.9 μm. High-performance liquid chromatography (HPLC) results showed the recrystallized BECD was purer than the non-processed material. The role of the solvent (acetone, methanol and ethanol) in the BECD crystal structure was investigated using X-ray diffraction (XRD), which showed acetone gave a more crystalline structure, hence having lower incorporation into the crystal structure. These results showed that the GAS process has the potential to produce steroid with powder properties suitable for inhalation therapy.
Keywords: Beclomethasone dipropionate; Asthma; Supercritical carbon dioxide; GAS; MMAD;

Contributions of intestinal P-glycoprotein and CYP3A to oral bioavailability of cyclosporin A in mice treated with or without dexamethasone by Mingji Jin; Tsutomu Shimada; Koichi Yokogawa; Masaaki Nomura; Yukio Kato; Akira Tsuji; Ken-ichi Miyamoto (81-86).
The contributions of P-glycoprotein (P-gp) and CYP3A to the oral bioavailability (BA) of cyclosporin A (CyA) were separately evaluated by using wild-type and mdr1a/1b knockout mice treated with dexamethasone (DEX). Mice were treated with DEX (1 or 75 mg/kg/day, i.p.) daily for 7 days, and the blood concentrations of CyA were measured after an i.v. or p.o. dose of CyA (10 mg/kg) at 1.5 h after the last DEX treatment. The BA values of CyA in wild-type and mdr1a/1b knockout mice were similar, 0.25 and 0.287, respectively. As regards expression of mdr1a and CYP3A mRNAs, expression of mdr1a mRNA was weakest in the duodenum, the main absorption site of CyA, along the whole intestine of wild-type mice, while expression of CYP3A was strongest in the duodenum of both types of mice. After treatment with 1 and 75 mg/kg DEX, the BA values decreased to 43 and 25% of the control in wild-type mice, respectively, and to 89 and 73% of the control in mdr1a/1b knockout mice, respectively. Expression of mdr1a mRNA in duodenum of wild-type mice was potently induced by DEX treatment. The expression of CYP3A mRNA in liver and duodenum of both strains was enhanced only by high-DEX treatment. These results suggest that P-glycoprotein plays only a small role in the absorption of CyA under physiological conditions, but the protein is readily induced by DEX and then functions as a more substantial absorption barrier to CyA than does CYP3A in the intestine.
Keywords: Cyclosporin A; Bioavailability; P-glycoprotein; Dexamethasone; CYP3A; mdr1a/1b knockout mice;

Long-acting interferon-α 2a modified with a trimer-structured polyethylene glycol: Preparation, in vitro bioactivity, in vivo stability and pharmacokinetics by Yeong Woo Jo; Yu Seok Youn; Sung Hee Lee; Byong Moon Kim; Soo Hyung Kang; Moohi Yoo; Eung Chil Choi; Kang Choon Lee (87-93).
The proper selection of size and shape for polyethylene glycol (PEG) is one of the most important points in PEGylation technology. Therefore, PEGs of various sizes and shapes have been widely developed to endow specific properties. In this study, a unique, trimer-structured, 43 kDa PEG was conjugated to interferon-α 2a (IFN) by forming an amide bond to improve the pharmacokinetic properties and minimize the loss of IFN bioactivity. Mono-PEGylated IFN (PEG3–IFN) prepared by utilizing this unique PEG was purified and characterized by cation-exchange chromatography and MALDI-TOF mass spectrometry. The in vitro bioactivity, in vivo stability, and pharmacokinetics of PEG3–IFN were examined and compared to those of native IFN. PEG3–IFN exhibited comparable in vitro bioactivities to native IFN and an excellent stability of the conjugation linkage in rat serum and various organs following subcutaneous injection. Furthermore, it showed slow absorption and markedly reduced clearance in rats, thereby increasing the biological half-life by about 40-fold compared to that of native IFN. This is the first report on the application of unique, trimer-structured PEG to bioactive proteins. The results suggest that unique, trimer-structured 43 kDa PEG can provide some advantages to improve the pharmacokinetic properties and to maintain the bioactivity of therapeutic proteins in clinical use.
Keywords: PEG; PEGylation; Interferon; Bioactivity; Stability; Pharmacokinetics;

Development of pH-sensitive liposomes that efficiently retain encapsulated doxorubicin (DXR) in blood by Tatsuhiro Ishida; Yurie Okada; Tomotaka Kobayashi; Hiroshi Kiwada (94-100).
We have reported that targeted, pH-sensitive sterically stabilized liposomes are able to increase the cytotoxicity of DXR in vitro against B lymphoma cells, but the rate of release of DXR in plasma was too rapid to permit the results to be extended to in vivo applications. The purpose of the study reported here is two-fold. First, to understand the mechanism of the rapid release of DXR from pH-sensitive sterically stabilized liposomes (PSL) in human plasma. Second, to reformulate the above liposomes to improve their drug retention, while retaining their pH sensitivity. The stability of the PSL formulations in human plasma was evaluated by comparing the rate of release of encapsulated DXR with that of HPTS, a water-soluble fluorescent marker. Since DXR, but not HPTS, a water soluble-less membrane permeable fluorescence marker, was rapidly released from liposomes in the presence of plasma, the rapid release of DXR is likely caused by the diffusion of DXR molecules through the lipid bilayer, not by the disruption of the membrane. In order to develop more stable PSL formulations, various molar ratios of the membrane rigidifying lipid, hydrogenated soy HSPC and/or CHOL, were added to the lipid composition and the rate of release of encapsulated solutes and pH-sensitivity were evaluated. The compositions that showed the best drug retention and pH-sensitivity were a mixture of DOPE/HSPC/CHEMS/CHOL/mPEG2000-DSPE at a molar ratio of 4:2:2:2:0.3 and DOPE/HSPC/CHEMS/CHOL at a molar ratio of 4:2:2:2. Our formulations, if targeted to internalizing antigens on cancer cells, may increase intracellular drug release rates within acidic compartment, resulting in a further increase in the therapeutic efficacy of targeted anticancer drug-containing liposomes.
Keywords: pH-sensitive liposomes; Encapsulation; Controlled drug release; Doxorubicin; Triggered drug release;

Stabilization and encapsulation of a staphylokinase variant (K35R) into poly(lactic-co-glycolic acid) microspheres by Jin-Tian He; Hua-Bo Su; Guo-Ping Li; Xian-Mei Tao; Wei Mo; Hou-Yan Song (101-108).
The aim of this study is to prepare poly(lactic-co-glycolic acid) (PLGA) microspheres containing a staphylokinase variant K35R (DGR) with purpose of preserving the protein stability during both encapsulation and drug release. DGR-loaded microspheres are fabricated using a double-emulsion solvent extraction technique. Prior to encapsulation, the effect of ultrasonication emulsification of DGR solutions with methylene chloride on protein recovery was investigated. Moderate ultrasonic treatment of aqueous DGR/dichloromethane mixtures caused approximately 84% DGR aggregation. Polyvinyl alcohol (PVA) added into aqueous DGR solutions significantly improved DGR recovery to >90%. The effects of co-encapsulated PVA and NaCl in the external aqueous phase on the characteristics of the microspheres were investigated. When 2% PVA was co-encapsulated and 2.5% NaCl was added to the external water phase, DGR encapsulation efficiency was significantly increased from 7.1% to 78.1% and DGR was distributed uniformly throughout the microspheres. In vitro release test showed that DGR was released from PLGA microspheres in a sustained manner over 15 days. A large amount of released DGR was inactive in the absence of co-encapsulated PVA. On the contrary, when 2% PVA was co-encapsulated, the released DGR was almost completely intact within 9 days. In conclusion, PLGA microspheres can be an effective carrier for DGR and form a promising depot system.
Keywords: Staphylokinase; Microspheres; Protein delivery; Polyvinyl alcohol; Protein stability;

A simplified model to predict P-glycoprotein interacting drugs from 3D molecular interaction field by Xiao-Mei Zhuang; Jun-Hai Xiao; Jin-Tong Li; Zhen-Qing Zhang; Jin-Xiu Ruan (109-114).
A new two components partial least squares discriminant analysis (PLS) model for the prediction of P-glycoprotein-associated ATPase activity of drugs by using VolSurf compute theoretical molecular descriptors derived from 3D molecular interaction field was reported in the present study. By using 27 diverse drugs from literature, two models were constructed (R 2  = 0.9003, 0.8150; Q 2  = 0.7165, 0.7630) in this paper, which were similar to models that utilized MolSurf parametrization (R 2  = 0.7760, 0.7180; Q 2  = 0.7420, 0.6950) by using 22 drugs reported in the same literature. The results investigated VolSurf software was superior to MolSurf in its simplicity. Properties associated with the volume, polarizability, and hydrogen bond could have important impact on the P-glycoprotein-associated ATPase activity.
Keywords: P-glycoprotein; VolSurf; QSAR; Molecular interaction field;

Solubilization and quantification of lycopene in aqueous media in the form of cyclodextrin binary systems by Maria Vertzoni; Theodora Kartezini; Christos Reppas; Helen Archontaki; Georgia Valsami (115-122).
An optimized kneading method for the preparation of lycopene–cyclodextrin binary systems was developed leading to solubilization of lycopene in water and 5% (w/v) dextrose solution. Lycopene quantification in the prepared binary systems was performed by a developed spectrometric method that followed a successful single-step extraction with dichloromethane. Storage stability characteristics of the binary systems were studied at 4 °C in solution and at −20 °C in the lyophilized products. Lycopene content was monitored at λ max  = 482 nm, the limit of detection was 0.41 μg/ml and relative standard deviation was less than 3.1%. The results obtained with the spectrometric method were confirmed by a HPLC method. In the presence of cyclodextrins, lycopene concentration in water was 8.0 ± 1.0, 27.1 ± 3.2 and 16.0 ± 2.2 μg/ml for β-CD, HP-β-CD and Me-β-CD, respectively. In 5% (w/v) aqueous dextrose solutions the corresponding values were 16.0 ± 1.8, 48.0 ± 5.1 and 4.0 ± 0.5 μg/ml, respectively. At 4 °C, storage stability of lycopene–cyclodextrin binary systems in water or 5% (w/v) aqueous dextrose solutions, was limited (t 1/2  = 1–4 days). Addition of the antioxidant sodium metabisulfite increased the stability of lycopene–HP-β-CD binary system in water. At −20 °C, the lyophilized lycopene–cyclodextrin binary systems were stable for at least 2 weeks.
Keywords: Lycopene solubilization; Lycopene quantification; Spectrometric method; Kneading technique; Lycopene–cyclodextrin binary systems; Lyophilization;

The present study aimed to investigate whether β-cyclodetxrin (β-CD) and its water-soluble derivatives, hydroxypropyl-β-cyclodextrin (HP-β-CD) and sulfobutyl ether β-cyclodextrin (SBE-β-CD), exert any effects on the permeation of two drug transport markers (propranolol and lucifer yellow) across rat intestinal epithelium. Rat ileum was stripped of its serosa and mounted inside an Ussing Chamber. Apparent permeability coefficients (P app) of the markers from the mucosal to serosal side of the tissue were determined at 37 °C in the presence and absence of the β-cyclodextrins on the mucosal side. Potential difference (PD) was constantly monitored during each experiment to ensure maintenance of the viability and integrity of the tissue. Pre-incubation with 1% β-CD, 1% HP-β-CD or 1.48% SBE-β-CD on the mucosal side for 30 min did not significantly alter the PD and the propranolol permeability (p  > 0.05). Co-incubation with 1% β-CD or 1% HP-β-CD exerted no significant effect on the P app of both propranolol and lucifer yellow (p  > 0.05), but co-incubation with 1.48% SBE-β-CD lowered the P app of propranolol from (1.71 ± 0.44) × 10−5 to (0.19 ± 0.04) × 10−5  cm/s, which may be ascribed to the molecular complexation of propranolol with SBE-β-CD. All three β-cyclodextrins exert no apparent impact on both (passive) transcellar and paracellular drug transports.
Keywords: β-Cyclodextrins; Drug transport; Intestinal permeability; Ussing Chamber;

Formulation and biological evaluation of glimepiride–cyclodextrin–polymer systems by H.O. Ammar; H.A. Salama; M. Ghorab; A.A. Mahmoud (129-138).
Glimepiride is one of the third generation sulfonylureas used for treatment of type 2 diabetes. Poor aqueous solubility and slow dissolution rate of the drug lead to irreproducible clinical response or therapeutic failure in some cases due to subtherapeutic plasma drug levels. Consequently, the rationale of this study was to improve the biological performance of this drug through enhancing its solubility and dissolution rate. Inclusion complexes of glimepiride in β-cyclodextrin (β-CyD), hydroxypropyl-β-cyclodextrin (HP-β-CyD) and sulfobutylether-β-cyclodextrin (SBE-β-CyD), with or without water soluble polymers were prepared by the kneading method. Binary systems were characterized by thermogravimetric analysis, IR spectroscopy and X-ray diffractometry. Phase solubility diagrams revealed increase in solubility of the drug upon cyclodextrin addition, showing Ap type plot indicating high order complexation. All the ternary systems containing β-CyD or HP-β-CyD showed higher dissolution efficiency compared to the corresponding binary systems. The hypoglycemic effect of the most rapidly dissolving ternary system of glimepiride–HP-β-CyD–PEG 4000 was evaluated after oral administration in diabetic rats by measuring blood glucose levels. The results indicated that this ternary system improves significantly the therapeutic efficacy of the drug. In conclusion, the association of water soluble polymers with glimepiride–CyD systems leads to great enhancement in dissolution rate, increased duration of action and improvement of therapeutic efficacy of the drug.
Keywords: Glimepiride; Cyclodextrins; Water soluble polymers; Complexation;

To develop an efficient female controlled drug delivery system (FcDDS) against sexually transmitted diseases (STDs), the polymeric films containing sodium dodecyl sulfate (SDS) were prepared with various compositions of Carbopol 934P, hydroxypropyl methylcellulose (HPMC) and polyethylene glycol (PEG). The physicochemical properties of mucoadhesive polymeric films, such as tensile strength, contact angle, swelling ratio and erosion rate in a vaginal fluid stimulant (VFS), were characterized. In addition, the drug release profile of SDS from the films and mucosal residence time were evaluated using a simulated dynamic vaginal system. It was demonstrated that the films made of Carbopol, HPMC and PEG were colorless, thin and soft and had proper physicodynamic properties for FcDDS. An increase in Carbopol content elevated tensile strength and swelling ratio but decreased the contact angle, erosion rate and the SDS release rate from the films. The films containing 0.25% (w/v) PEG as well as 0.75% (w/v) of combining Carbopol and HPMC remained on the vaginal tissue for up to 6 h. The films containing the ratio of Carbopol:HPMC:PEG = 1.5:1.5:1 and 1:2:1 seem to be optimal compositions for FcDDS, as they showed good peelability, relatively high swelling index and moderate tensile strength, and achieved the target release rate of SDS for 6 h.
Keywords: Mucoadhesive polymeric films; Female controlled drug delivery system; Carbopol; Hydroxypropyl methylcellulose; Physicochemical properties;

The objective of this study was to evaluate the feasibility of using T ZERO modulated temperature differential scanning calorimetry (MDSC) as a novel technique to characterize protein solutions using lysozyme as a model protein and IgG as a model monoclonal antibody. MDSC involves the application of modulated heating program, along with the standard heating program that enables the separation of overlapping thermal transitions. Although characterization of unfolding transitions for protein solutions requires the application of high sensitive DSC, separation of overlapping transitions like aggregation and other exothermic events may be possible only by use of MDSC. A newer T ZERO calibrated MDSC model from TA instruments that has improved sensitivity than previous models was used. MDSC analysis showed total, reversing and non-reversing heat flow signals. Total heat flow signals showed a combination of melting endotherms and overlapping exothermic events. Under the operating conditions used, the melting endotherms were seen in reversing heat flow signal while the exothermic events were seen in non-reversing heat flow signal. This enabled the separation of overlapping thermal transitions, improved data analysis and decreased baseline noise. MDSC was used here for characterization of lysozyme solutions, but its feasibility for characterizing therapeutic protein solutions needs further assessment.
Keywords: T ZERO modulated DSC; Circular dichroism; Thermal stability; Thermal analysis; Lysozyme; Protein unfolding;

Pore size distributions of biodegradable polymer microparticles in aqueous environments measured by NMR cryoporometry by Oleg Petrov; István Furó; Michael Schuleit; Rainer Domanig; Mark Plunkett; John Daicic (157-162).
NMR cryoporometry is a unique method permitting the investigation of pores in the microporous and mesoporous regimes for samples in aqueous environments. Here, we apply the technique to porous biodegradable polymer microparticles designed as devices for drug delivery in depot formulations. The results indicate that structural features too small to be captured in surface and fracture images obtained by SEM are able to be accessed using the technique, and that the evolution of pore structure can be studied for several days as the particles swell and degrade in the aqueous environment.
Keywords: Microparticles; Drug release; Pore size distribution; Swelling; Degradation; NMR cryoporometry; SEM;

The aim of the present study was to screen the effect of seven factors – POLYOX molecular weight (X 1) and amount (X 2); carbopol (X 3), lactose (X 4), sodium chloride (X 5), citric acid (X 6); compression pressure (X 7) – on (1) the release of theophylline from hydrophilic matrices, demonstrated by changes in dissolution rate, and (2) their impact on the release exponent [n] indicative of the drug transport mechanism through the diffusion matrix. This objective was accomplished utilizing the Placket–Burman screening design. Theophylline tablets were prepared according to a 7-factor–12-run statistical model and subjected to a 24-h dissolution study in phosphate buffer at pH 7.2. The primary response variable, Y 4, was the cumulative percent of theophylline dissolved in 12 h. The regression equation for the response was Y 4  = 66.2167 − 17.5833X 1  − 3.3833X 2  − 9.366X 3  − 1.1166X 4  − 0.6166X 5  + 2.6X 6  − 2.783X 7. This polynomial model was validated by the ANOVA and residual analysis. The results showed that only two factors (X 2 and X 3) had significant effect (p-value < 0.10) on theophylline release from the hydrophilic polymer matrix. Factors (X 2 and X 7) had significant effect (p-value < 0.10) on [n], the exponent.
Keywords: Theophylline; Hydrophilic polymer matrix; Placket–Burman; Screening; Controlled release; Dissolution;

Multiple W/O/W emulsions with high content of inner phase (Φ1 = Φ2 = 0.8) were prepared using relatively low concentrations of lipophilic polymeric primary emulsifier, PEG 30-dipolyhydroxystearate, and diclofenac diethylamine (DDA) as a model drug. The investigated formulations were characterized and their stability over the time was evaluated by dynamic and oscillatory rheological measurements, microscopic analysis and in vitro drug release study. In vitro release profiles of the selected model drug were evaluated in terms of the effective diffusion coefficients and flux of the released drug. The multiple emulsion samples exhibited good stability during the ageing time. Concentration of the lipophilic primary emulsifier markedly affected rheological behaviour as well as the droplet size and in vitro drug release kinetics of the investigated systems. The multiple emulsion systems with highest concentration (2.4%, w/w) of the primary emulsifier had the lowest droplet size and the highest apparent viscosity and highest elastic characteristics. Drug release data indicated predominately diffusional drug release mechanism with sustained and prolonged drug release accomplished with 2.4% (w/w) of lipophilic emulsifier employed.
Keywords: W/O/W emulsions; Rheology; Droplet size; In vitro release; Diclofenac diethylamine;

A common limitation of using polymeric nanoparticles in aqueous suspension is due to their poor chemical and physical stability when conserved for a long time. Therefore, freeze drying of these colloidal systems is an alternative method to achieve long-term stability. Nanocapsules have thin and fragile shell structure, which may not resist to the stress of such process. The aim of this study is to investigate the formulation and process parameters in order to ensure the stability of polycaprolactone nanocapsules (PCL NC) by freeze drying.In this paper, we studied the freeze drying of PCL NC prepared by the emulsion–diffusion method and stabilized by poly(vinyl alcohol) (PVA). Different parameters have been tested throughout the freeze–thawing study including PVA and PCL concentration, cooling rate, cryoprotectant concentrations, nature of encapsulated oil and NC purification. On the other hand, nanocapsules have been freeze dried both before and after purification. Freeze dried purified PCL NC were characterized by particle size measurement, collapse temperature, T ′ g determination, scanning electron microscope observation, environmental scanning electron microscope imaging and residual humidity quantification. Finally, the effect of annealing on the NC stability and the sublimation rate has been well explored.The results suggest that PCL NC could be freeze dried without a cryoprotectant if the concentration of PVA stabilizer is sufficient (5%), while for the purified NC the addition of 5% of cryoprotectant seems to be necessary to ensure the stability of NC. The type of cryoprotectants had practically negligible effects on the size and the rehydration of freeze dried nanocapsules. The annealing process could accelerate the sublimation with the conservation of nanocapsules size.
Keywords: Emulsification–diffusion technique; Nanocapsules; Freeze drying; Collapse; Annealing; Environmental scanning electron microscope;

The synthesis and transfection potential of a novel cationic cholesterol cytofectin with a dimethylamino head group and a long 12 atom, 15 Å spacer incorporating relatively polar amido and dicarbonyl hydrazine linkages are reported. Thus N,N-dimethylaminopropylamidosuccinylcholesterylformylhydrazide (MS09) in equimolar admixture with dioleoylphosphatidylethanolamine (DOPE) forms stable unilamellar liposomes (80–150 nm) which cluster into very effective transfecting, serum nuclease-resistant, lipoplexes with DNA (180–200 nm) at a liposome+/DNA− molar charge ratio of 2.8:1 (12:1, w/w). Gel retardation and ethidium displacement assays confirmed that DNA was fully liposome-associated and maximally compacted at this ratio. Transfection levels in three human transformed epithelial cell lines, as established by luciferase transgene activity, was found to be optimal at this charge ratio and in the following order: cervical carcinoma (HeLa) > oesophageal carcinoma (SNO) > hepatoblastoma (HepG2). Activity in the murine fibroblast line NIH-3T3 was comparable to that in HepG2 cells. MS09 lipoplexes achieved approximately three-times and two-times greater activity than Lipofectin® complexes in HeLa and SNO cells, respectively, whilst comparable levels were recorded in HepG2 and NIH-3T3 cells. MS09 lipoplexes were well tolerated by HepG2, HeLa and SNO cells with cell numbers found to be 80, 85 and 75% of untreated cultures, respectively, at the optimal transfection concentration. These lipoplexes also exhibited high activity in the presence of 10% foetal bovine serum (FBS) in HeLa (17% inhibition) and HepG2 (33% inhibition) cells.
Keywords: Cationic liposome; Transfection; Transformed epithelial cells;

Self-assembled drug delivery systems by Yiguang Jin; Li Tong; Ping Ai; Miao Li; Xinpu Hou (199-207).
Self-assembled drug delivery systems (SADDS) were designed in the paper. They can be prepared from the amphiphilic conjugates of hydrophilic drugs and lipids through self-assembling into small-scale aggregates in aqueous media. The outstanding characteristic of SADDS is that they are nearly wholly composed of amphiphilic prodrugs. The self-assembled nanoparticles (SAN) as one of SADDS had been prepared from the lipid derivative of acyclovir (SGSA) in the previous paper. They were further studied on the properties and the in vitro/in vivo behavior in this paper. The SAN kept the physical state stable upon centrifugation or some additives including some inorganic salts, alkaline solutions, surfactants and liposomes except for HCl solution, CaCl2 solution and animal plasma. Autoclave and bath heat for sterilization hardly influenced the SAN. However, gamma-irradiation strongly destroyed the structure of SAN and SGSA was degraded. SGSA in SAN showed good stability in weak acidic or neutral buffers although it was very sensitive to alkaline solutions and carboxylester enzymes, the half-lives (t 1/2) of which in the buffer at pH 7.4, the alkaline solution at pH 12.0, pig liver carboxylester enzyme solution, rabbit plasma, and rabbit liver tissue homogenate were 495, 21, 4.7, 25 and 8.7 h, respectively. Compared with SGSA in a disordered state, the specific bilayer structures of SAN could protect SGSA from hydrolysis through hiding the sensitive ester bonds. The SAN showed hemolytic action because the amphiphilic SGSA could insert into rabbit erythrocyte membranes. Both the high concentration of SGSA in samples and the long incubation time improved hemolysis. No hemolysis was observed if the additional volume of the SAN was less than 10% of rabbit whole blood in spite of the high concentration of SGSA. Plasma proteins could interfere the interaction between the SAN and erythrocytes by binding the SAN. The in vitro antiviral activity of acyclovir SAN was limited possibly because of the weak hydrolysis of SGSA in Vero cells, and the SAN showed a little cell toxicity possible due to the amphiphilicity of SGSA. A macrophage cell line of QXMSC1 cells showed uptake of the SAN but not significantly. The SAN were rapidly removed from blood circulation after bolus iv administration to rabbits with the very short distribution t 1/2 (1.5 min) and the elimination t 1/2 (47 min). The SAN were mainly distributed in liver, spleen and lung after iv administration, and SGSA was eliminated slowly in these tissues (t 1/2, about 7 h). It would appear that the nanosized SAN were trapped by the mononuclear phagocyte system. SADDS including SAN combine prodrugs, molecular self-assembly with nanotechnology, and hopefully become novel drug delivery approaches.
Keywords: Prodrugs; Molecular self-assembly; Nanotechnology; Amphiphiles; Acyclovir; Mononuclear phagocyte system;

Biological properties of low molecular mass peptide dendrimers by B. Klajnert; J. Janiszewska; Z. Urbanczyk-Lipkowska; M. Bryszewska; D. Shcharbin; M. Labieniec (208-217).
A series of new, low molecular mass, lysine-based peptide dendrimers with varying distribution of cationic and aromatic groups in the structure were synthesized. They expressed antimicrobial activity against Gram-positive (Staphylococcus aureus) and Gram-negative (Escherichia coli) bacteria as well as against fungal pathogens (Candida albicans). Their cytotoxic, haematotoxic, and genotoxic effects were studied. It appears that degree of branching and steric distribution and types of hydrophobic (aromatic) groups and cationic centres are important components of dendrimeric structure and influence both antimicrobial potency and toxicity. Such 3D structure of our dendrimers mimics that of the natural antimicrobial peptides and can be achieved by application of dendrimer chemistry.
Keywords: Antimicrobial dendrimers; Cytotoxicity; Dendrimer; Genotoxicity; Haemolysis; Peptide; Dendrimeric effect;

Lyotropic liquid crystalline phases formed from glycerate surfactants as sustained release drug delivery systems by Ben J. Boyd; Darryl V. Whittaker; Shui-Mei Khoo; Greg Davey (218-226).
A new class of surfactants with glycerate headgroups, that form viscous lyotropic liquid crystalline phases in excess water, have been investigated for their potential to provide sustained release matrices for depot drug delivery. Oleyl glycerate and phytanyl glycerate were used as representative surfactants of this new class, and their behaviour compared with that of glyceryl monooleate (GMO). The surfactants were found to form reverse hexagonal phase (HII) in excess water, and the matrices were loaded with a series of model hydrophobic and hydrophilic drugs, (paclitaxel, irinotecan, glucose, histidine and octreotide), and the release kinetics determined. In all cases, the release behaviour obeyed Higuchi kinetics, with linear drug release versus square root of time. The HII phases released model drugs slower than the GMO cubic phase matrix. The oleyl glycerate matrix was found to consistently release drug faster than the phytanyl glycerate matrix, despite both matrices being based on HII phase. To further demonstrate the potential utility of these materials as drug depot delivery systems, an injectable precursor formulation for octreotide was also prepared and demonstrated to provide controlled release for the peptide. The stability of the HII phase to likely in vivo breakdown products was also assessed.
Keywords: Parenteral depot formulations; Cubic phase; Hexagonal phase; Glycerate surfactant; In vitro release;

Nanodecoy system: A novel approach to design hepatitis B vaccine for immunopotentiation by Amit K. Goyal; Amit Rawat; Sunil Mahor; Prem N. Gupta; Kapil Khatri; Suresh P. Vyas (227-233).
The progress toward subunit vaccines has been limited by their poor immunogenicity and limited stability. To enhance the immune response, subunit vaccines universally require improved adjuvants and delivery vehicles. In the present study, we propose the use of ceramic core based nanodecoy systems for effective immunization, which seems to exhibit a broad range of surface properties. Nanodecoy systems were prepared by self-assembling of hydroxyapatite core and cellobiose and finally the hepatitis B surface antigen (HBsAg) was adsorbed over the preformed nanodecoy systems. HBsAg loaded nanodecoy systems were characterized for size, shape and antigen loading efficiency. The effect of processing steps on the stability and integrity of HBsAg was assessed by in vitro antigenicity and SDS-PAGE experiments. Nanodecoy preparations were nanometric in size range and almost spherical in shape. SDS-PAGE studies confirmed the integrity of HBsAg protein in the formulation. Vaccine efficacy was determined in female Balb/c mice and results indicated that specific anti-HBsAg antibody titers in mice receiving nanodecoy system were more efficient than the conventional adjuvant alum followed by subcutaneous immunization. Studies also indicated that nanodecoy formulations could elicit combined Th1 and Th2 immune response. It is inferred that nanodecoy systems are a class of novel carriers and hold potential as an alternative adjuvant in vaccine technology.
Keywords: Hydroxyapatite; Calcium phosphate; Nanodecoy; Antigen delivery; Hepatitis B vaccine;

The aim of this work was to evaluate the potential of self-assembling poly(ethyleneglycol)750-block-poly(ɛ-caprolactone-co-trimethylenecarbonate)4500 50/50 copolymers (PEG-p(CL-co-TMC)) to solubilize amphotericin B in polymeric micelles and to disaggregate the drug to the less toxic monomeric form. Amphotericin B was encapsulated in the micelles upon dilution of a mixture of the liquid polymer and the drug in water. Its solubility was increased by two orders of magnitude depending on polymer concentration. The aggregation state of amphotericin B was decreased by PEG-p(CL-co-TMC). The preparation method and the loading of the polymeric micelles influenced it. The antifungal activity of the drug was reduced by encapsulation in the polymeric micelles whereas the onset of amphotericin B-induced hemolysis was delayed. PEG-p(CL-co-TMC) micelles could be an easy method for amphotericin B encapsulation.
Keywords: Polymeric micelles; Amphotericin B; mmePEG750-block-poly(ɛ-caprolactone-co-trimethylenecarbonate);

Noticeboard (241-243).