International Journal of Pharmaceutics (v.278, #2)

Stability of crystallised and spray-dried lysozyme by Amal A Elkordy; Robert T Forbes; Brian W Barry (209-219).
Moisture and temperature promote protein degradation. The stabilities of commercial, crystallised and spray-dried lysozyme, a model protein, were assessed under these stresses to explore whether a crystalline protein had better storage stability than a conventionally produced one. Samples were maintained at different relative humidities (RH) and temperatures for 20 weeks and stabilities estimated in solid and aqueous states. Differential scanning calorimetry (DSC) and thermogravimetry (TGA) characterised solid samples. Fourier transform Raman (FT-Raman) spectroscopy analysed solid material and aqueous solutions. High sensitivity differential scanning calorimetry (HSDSC) and enzymatic assays were used to monitor solutions. DSC and HSDSC data revealed that crystals maintained thermal stability at high RH; spray drying appreciably changed melting characteristics. These results correlated with enzymatic assays that demonstrated good activity retention for crystals but less so for spray-dried material (e.g. 95 and 87% relative to fresh samples after 20 weeks at 40 °C/75% RH). FT-Raman analysis showed that crystallised lysozyme better-maintained protein conformational integrity compared to spray-dried samples in accelerated stability studies. Based on TGA data, spray-dried protein absorbed water on storage under humid conditions, which induced instability. Thus, crystallisation enhanced storage stability of lysozyme with negligible loss of activity.
Keywords: Lysozyme; Crystallisation; Spray-dried; Stability; Calorimetry; Raman;

Solubility of drugs in aqueous solutions by E. Ruckenstein; I. Shulgin (221-229).
As in our previous publications in this journal [Int. J. Pharm. 258 (2003a) 193; Int. J. Pharm. 260 (2003b) 283; Int. J. Pharm. 267 (2003c) 121], this paper is concerned with the solubility of poorly soluble drugs in aqueous mixed solvents. In the previous publications, the solubilities of drugs were assumed to be low enough for the so-called infinite dilution approximation to be applicable. In contrast, in the present paper, the solubilities are considered to be finite and the dilute solution approximation is employed. As before, the fluctuation theory of solutions is used to express the derivatives of the activity coefficient of a solute in a ternary solution (dilute solute concentrations in a binary solvent) with respect to the concentrations of the solvent and cosolvent. The expressions obtained are combined with a theoretical equation for the activity coefficient of the solute. As a result, the activity coefficient of the solute was expressed through the activity coefficients of the solute at infinite dilution, solute mole fraction, some properties of the binary solvent (composition, molar volume and activity coefficients of the components) and parameters reflecting the nonidealities of binary species. The expression thus obtained was used to derive an equation for the solubility of poorly soluble drugs in aqueous binary solvents which was applied in two different ways. First, the nonideality parameters were considered as adjustable parameters, determined from experimental solubility data. Second, the obtained equation was used to correct the solubilities of drugs calculated via the infinite dilution approximation. It was shown that both procedures provide accurate correlations for the drug solubility.
Keywords: Solubility; Drugs; Dilute approximation;

Cross-linked high amylose starch derivatives for drug release by Jérôme Mulhbacher; Pompilia Ispas-Szabo; Mircea Alexandru Mateescu (231-238).
Acetate (Ac-), aminoethyl (AE-), and carboxymethyl (CM-) high amylose starch cross-linked 6 (HASCL-6) derivatives were previously shown to control the release of drugs over 20 h from monolithic tablets highly loaded (up to 60% drug). This report describes the swelling characteristics, which allow a better understanding of the mechanisms involved in the control of the drug release from the said polymeric matrices. The tablet swelling of HASCL-6, Ac-HASCL-6, and AE-HASCL-6 was found to not be affected by the ionic strength and by the pH between 1.2 (gastric) and 7 (intestinal), whereas the swelling of CM-HASCL-6 was shown to depend on both ionic strength and pH of the release medium. For all the studied polymers the drug loading did not change the equilibrium swelling ratio but affected the initial swelling velocity, seemingly due to the competition between drug and polymer for water uptake, a phenomenon probably influenced by the loading and the drug solubility. It was also shown that the increase of ionic strength would slightly increase the drug release time probably by decreasing the amount of free water still available to solubilize the drug present into the matrix.
Keywords: High amylose starch derivatives; Polymeric networks; Controlled release mechanisms; Swelling properties;

Incorporation of hydrophobic porphyrins into liposomes: characterization and structural requirements by F Postigo; M Mora; M.A De Madariaga; S Nonell; M.L Sagristá (239-254).
The ability of photosensitisers to give reactive oxygenated products is considered decisive for photodynamic applications, but the hydrophobic nature of many porphyrins makes necessary to obtain suitable pharmaceutical formulations. This paper reports the structural photosensitiser features that allow the preparation of stable liposomal formulations. Metallated and non-metallated TPPs and TPyPs and different lipid/porphyrin ratios were considered in order to procure liposomal preparations containing porphyrin concentrations adequate to necessary doses. The results show that the incorporation of porphyrins into liposomes can be related with their ability to form aggregates in a watery media. Thus, ZnTPP, which structural properties avoid the formation of aggregates, was efficiently incorporated into stable liposomes. Moreover, the efficient generation of singlet oxygen by ZnTPP liposomal suspensions has been shown. Because of this, the synthesis of hydrophobic porphyrin derived structures or other sensitisers, which do not aggregate in a watery media and with Q-bands shifted to higher λ values than ZnTPP, will be efficiently incorporated into liposomes and useful for clinical applications.
Keywords: Liposomes; Porphyrins; Characterization; Interaction; Singlet oxygen;

In vivo gene transfection via intravitreal injection of cationic liposome/plasmid DNA complexes in rabbits by Shigeru Kawakami; Ayaka Harada; Koji Sakanaka; Koyo Nishida; Junzo Nakamura; Toshiyuki Sakaeda; Nobuhiro Ichikawa; Mikiro Nakashima; Hitoshi Sasaki (255-262).
To optimize the in vivo ocular transfection efficiency of plasmid DNA (pDNA)/cationic liposome complexes, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)/dioleoylphosphatidylethanolamine (DOPE) (1:1 molar ratio) liposomes and DOTMA/cholesterol (Chol) (1:1 molar ratio) liposomes were prepared with varying amounts of pDNA. pDNA/cationic liposome complexes were intravitreally injected (100 μL) in rabbits, and luciferase activity in the cornea, aqueous humor, iris–ciliary body, lens, vitreous body, and retina was measured. Transfection efficiency of pDNA alone did not change with pDNA ranging from 40 to 85 mg. In contrast, transfection efficiency of pDNA complexed with DOTMA/Chol liposomes significantly increased with the amount of pDNA ranging from 40 to 85 μg (P<0.05). pDNA complexed with DOTMA/DOPE liposomes could not be prepared with pDNA greater than 60 μg. Among these experiments, pDNA (85 μg) complexed with DOTMA/Chol liposomes (pDNA:cationic liposome charge ratio (−:+) = 1.0:2.0) showed the highest transfection efficiency in the ocular tissue and its transfection-mediated luciferase activity peaked at 3 days. Among the ocular tissues, the highest gene expression was observed in the aqueous humor.
Keywords: Transfection; Vitreous body; Eye; Cationic liposomes; Plasmid DNA;

Incorporation of ovalbumin into ISCOMs and related colloidal particles prepared by the lipid film hydration method by Patrick H Demana; Nigel M Davies; Bianca Berger; Thomas Rades (263-274).
The aim of this study was to investigate the incorporation of a model antigen, fluorescently labelled ovalbumin (FITC-OVA), into various colloidal particles including immune stimulating complexes (ISCOMs), liposomes, ring and worm-like micelles, lamellae and lipidic/layered structures that are formed from various combinations of the triterpene saponin Quil A, cholesterol and phosphatidylethanolamine (PE) following hydration of PE/cholesterol lipid films with aqueous solutions of Quil A. Colloidal dispersions of these three components were also prepared by the dialysis method for comparison. FITC-OVA was conjugated with palmitic acid (P) and PE to produce P-FITC-OVA and PE-FITC-OVA, respectively. Both P-FITC-OVA and PE-FITC-OVA could be incorporated in all colloidal structures whereas FITC-OVA was incorporated only into liposomes. The incorporation of PE-FITC-OVA into all colloidal structures was significantly higher than P-FITC-OVA (P<0.05). The degree of incorporation of protein was in the order: ring and worm-like micelles<liposomes and lipidic/layered structures<ISCOMs and lamellae. The incorporation of protein into the various particles prepared by the lipid film hydration method was similar to those for colloidal particles prepared by the dialysis method (provided both methods lead to the formation of the same colloidal structures). In the case of different colloidal structures arising due to the preparation method, differences in encapsulation efficiency were found (P<0.05) for formulations with the same polar lipid composition. This study demonstrates that the various colloidal particles formed as a result of hydrating PE/cholesterol lipid films with different amounts of Quil A are capable of incorporating antigen, provided it is amphipathic. Some of these colloidal particles may be used as effective vaccine delivery systems.
Keywords: Ovalbumin; Quil A; ISCOMs; Liposomes; Micelles; Vaccine delivery;

Evaluation of sustained release suppositories prepared with fatty base including solid fats with high melting points by Toshihito Takatori; Norihito Shimono; Kazutaka Higaki; Toshikiro Kimura (275-282).
To prepare the sustained release suppositories, solid fats such as polyglycerol ester of fatty acids (PGEFs) or beeswax were utilized with a fatty suppository base, Witepsol H15. PGEFs such as decaglycerol heptabehenate (HB750) and hexaglycerol pentastearate (PS500), and beeswax have relatively high melting points. The addition of PGEFs or beeswax to Witepsol H15 increased the apparent viscosity of suppository bases at 37 °C without any large change in the melting point of Witepsol H15. Moreover, the apparent viscosity of a mixed base with HB750, PS500 or beeswax at 37 °C was significantly correlated with the amount of each solid fat in a mixed base. The release of acetaminophen (AAP), a model drug, from suppositories was delayed by HB750, PS500 or beeswax, and an excellent correlation was observed between the apparent viscosity of these mixed bases and Higuchi’s rate constants in each mixed base suppository, suggesting that these solid fats could regulate the drug release from the mixed base suppositories by changing their viscosity. In the in vivo absorption study in rats, several suppositories made from Witepsol H15–HB750 or Witepsol H15–beeswax mixed bases prolonged the rectal absorption of AAP without reducing AUC. In conclusion, by using solid fats such as HB750 and beeswax with relatively high melting points, it is possible to control the rate of drug release from fatty base suppositories for maintaining the plasma concentration of drugs for longer time periods.
Keywords: Fatty suppository; Polyglycerol ester of fatty acid; Beeswax; Viscosity; Solid fat with high melting point; Controlled release;

Adsorption of methotrexate and calcium leucovorin onto cholestyramine in vitro by M Merino-Sanjuán; A Carrera; E Monte; N.V Jiménez-Torres (283-291).
Methotrexate (MTX), an antimetabolite of folic acid, is a drug widely used in the treatment of different types of cancer. When high doses are administered, it is necessary to interrupt its action by administering calcium leucovorin (CaL). The main pathway of MTX and CaL elimination in humans occurs through the kidney, but about 10% is excreted in the faeces via the bile. Drugs, foods and sorbents in intestinal lumen modify MTX and CaL reabsorption. Individual and simultaneous studies on the adsorption of MTX and CaL from aqueous phosphate buffer by cholestyramine were carried out in order to calculate the adsorption process of MTX and CaL to cholestyramine, and to characterize the influence of CaL in the adsorption of MTX to cholestyramine and vice versa. The Langmuir binding isotherms determined in buffer solutions at pH 6 indicated a greater (12.58%) adsorption capacity of cholestyramine (1.43 mmol of drug/g of resin) than at pH 7 (1.25 mmol of drug/g of cholestyramine). The affinity constant of MTX to cholestyramine was a 45.27% higher (6.67 mM−1) than the affinity constant of CaL to the resin (3.65 mM−1). Results from simultaneous assays indicate that a displacement of the MTX bound to cholestyramine by CaL is not foreseeable. The results suggest that cholestyramine may be a potentially useful adjunctive therapy in the treatment of an overdose of MTX. Consequently, cholestyramine may be of clinical value in patients who develop early renal function impairment whilst undergoing MTX therapy.
Keywords: Methotrexate; Calcium leucovorin; Cholestyramine; Langmuir adsorption;

Purpose: The objective of this study is to develop indocyanine green (ICG)-loaded biodegradable nanoparticles by using biodegradable polymer, poly(dl-lactic-co-glycolic acid) (PLGA). Method: PLGA nanoparticles entrapping ICG were prepared by a modified spontaneous emulsification solvent diffusion method. To optimize the nanoparticle formulation, the influence of formulation parameters such as types of ICG, amount of ICG and the polymer were investigated. The ICG entrapment in nanoparticles, nanoparticle size and zeta potential were determined. The surface characterization was performed by atomic force microscopy (AFM) and the release of ICG from nanoparticles was determined. Results: All PLGA nanoparticle formulations were found to have the mean diameter within the range of 300–410 nm with polydispersity index (PI) within the range of 0.01–0.06. Indocyanine green showed more efficient entrapment as compared to indocyanine green sodium iodide salt. All indocyanine green-loaded nanoparticle formulations were found to have almost similar ICG content of nanoparticles and showed increase in ICG entrapment with increase in the amount of polymer. The ICG entrapment reached 74% when ICG: PLGA weight ratio in the formulation reached 1:800. AFM images indicated that the nanoparticles were almost spherical in shape and had numerous pores on their surfaces. The release pattern consisted of two phases, with initial exponential phase releasing about 78% of ICG (within 8 h) followed by a slow phase releasing about 2% of ICG (within next 16 h). Conclusions: ICG-loaded PLGA nanoparticles were prepared and the formulation was optimized. The increase in amount of polymer in formulation leads to higher ICG entrapment. Nanoparticles formed were spherical and had porous surfaces and exhibited the characteristic release pattern of a monolithic matrix based system.
Keywords: Indocyanine green; PLGA; Nanoparticles; Fluorescence; Near-infrared spectroscopy;

2-hydroxypropyl-β-cyclodextrin extracts 2-phenylphenol from silicone tubing by Monique W.J den Brok; Sabien C.van der Schoot; Bastiaan Nuijen; Michel J.X Hillebrand; Jos H Beijnen (303-309).
Cyclodextrins are capable to solubilise lipophilic drugs via (partial) inclusion in their lipophilic cavity. This, however, also provides the potential for the extraction of small molecules from production materials. In the present study, the potency of the commercially available and used cyclodextrin, 2-hydroxypropyl-β-cyclodextrin (HPβCD) to extract the preservative 2-phenylphenol (2-PP) from platinum cured silicone tubing was tested. The presence of 2-PP was structurally confirmed with HPLC–UV and LC/MS/MS in HPβCD solutions after incubation with platinum cured silicone tubing. HPβCD concentration and prior tubing sterilisation were found not to influence the levels of 2-PP extracted. Interestingly, extraction to ethanol was 15-fold higher than observed for HPβCD solutions.2-PP was extracted from silicone tubing during routine manufacture of a blank dosage form formulated with only HPβCD, resulting in detectable levels of 2-PP in the final product. In a freeze-dried dosage form containing HPβCD and an active pharmaceutical ingredient (exhibiting a stability constant for HPβCD/drug of 1045 L/mol), on the other hand, 2-PP was undetectable.
Keywords: 2-Hydroxypropyl-β-cyclodextrin; 2-Phenylphenol; Silicone tubing; Extraction; Formulation;

Intravenous liposomal benznidazole as trypanocidal agent: increasing drug delivery to liver is not enough by M.J Morilla; J.A Montanari; M.J Prieto; M.O Lopez; P.B Petray; E.L Romero (311-318).
With the aim of investigating if delivery of benznidazole (BNZ) to liver could be increased by incorporating the drug in multilamellar liposomes, single bolus of free BNZ or liposomal BNZ formulations (MLV-BNZ) composed of HSPC:DSPG:Chol 2:1:2 (mol/mol/mol) at 0.7% (w/w) drug/total lipid ratio, were injected by intramuscular (i.m.), subcutaneous (s.c.) and intravenous (i.v.) routes, at 0.2 mg BNZ/kg, in rats. The resulting blood concentrations were followed along 9 h post-injection (p.i.) and drug accumulation in liver was determined after 4 and 9 h p.i. Only upon i.v. injection of MLV-BNZ, a threefold higher BNZ accumulation in liver was obtained, together with blood BNZ concentrations of 1.1 μg/ml (30% lower than the blood BNZ concentration achieved upon i.v. administration of free drug) occurred 4 h p.i. However, such increased liver uptake of BNZ, raised twice a week had no effect on parasitaemia levels of mice infected with the RA strain of Trypanosoma cruzi. Our results indicate that the relationship between increased selectivity for an infected tissue and therapeutic effect is not always straightforward, at least for the MLV-BNZ regimen used in the present study.
Keywords: Mutilamellar liposomes; Benznidazole;

The purpose of this investigation was to evaluate the effect of matrix crosslinking and solute size on release of a random coil macromolecular solute from crosslinked gelatin matrices. Gelatin hydrogel matrices crosslinked with different molar ratios of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC):ε-amino groups on gelatin (1:1, 4:1, and 10:1) were prepared containing dextran of molecular weights 12, 20, and 77 kDa, and hydrodynamic diameters 54, 74, and 133 Å, respectively. The extent of matrix crosslinking was determined quantitatively and used to calculate the molecular weight between crosslinks (M c). The M c parameter and equilibrium swelling ratio (Q m) were used to calculate an estimated matrix mesh size (ξ). The in vitro release of incorporated dextran was evaluated at 37 °C in PBS at pH 7.4 for approximately 80 h. The one-, four- and 10-fold molar ratios of crosslinking agent EDC yielded 24, 41, and 78% of gelatin matrix crosslinking, respectively. The calculated average matrix mesh size ranged from 338 to 90 Å. The effect of matrix crosslinking varied with solute size, from retarding diffusional release of the dextran to completely entrapping it inside the crosslinked matrices. These results support the threshold concept of solute size relative to matrix mesh size for release of a flexible, random coil macromolecular solute from a hydrogel.
Keywords: Crosslinked gelatin matrices; Matrix mesh size; Physical and chemical crosslinks; Random coil macromolecular solute release; Hindered diffusion; Dextran;

Drug carrier systems based on water-soluble cationic β-cyclodextrin polymers by Jianshu Li; Huining Xiao; Jiehua Li; YinPing Zhong (329-342).
This study was designed to synthesize, characterize and investigate the drug inclusion property of a series of novel cationic β-cyclodextrin polymers (CPβCDs). Proposed water-soluble polymers were synthesized from β-cyclodextrin (β-CD), epichlorohydrin (EP) and choline chloride (CC) through a one-step polymerization procedure by varying molar ratio of EP and CC to β-CD. Physicochemical properties of the polymers were characterized with colloidal titration, nuclear magnetic resonance spectroscopy (NMR), gel permeation chromatography (GPC) and aqueous solubility determination. The formation of naproxen/CPβCDs inclusion complexes was confirmed by NMR and fourier transform infrared spectroscopy (FT-IR). Cationic β-CD polymers showed better hemolytic activities than parent β-CD and neutral β-CD polymer in hemolysis test. The morphological study of erythrocytes revealed a cell membrane invagination induced by the cationic groups. The effects of molecular weight and charge density of the polymers on their inclusion and release performance of naproxen were also investigated through phase-solubility and dissolution studies. It was found that the cationic β-CD polymers with high molecular weight or low charge density exhibited better drug inclusion and dissolution abilities.
Keywords: Cationic β-cyclodextrin polymer; Physicochemical analysis; Inclusion complexes; Human erythrocytes; Hemolysis; Morphological changes;

Preparation of poly(N-isopropylacrylamide/itaconic acid) copolymeric hydrogels and their drug release behavior by Betül Taşdelen; Nilhan Kayaman-Apohan; Olgun Güven; Bahattin M Baysal (343-351).
N-isopropylacrylamide/itaconic acid copolymeric hydrogels were prepared by irradiation of the ternary mixtures of N-isopropylacrylamide/itaconic acid/water by γ-rays at ambient temperature. The effect of comonomer concentration, irradiation dose and pH on the swelling equilibria were studied. Lidocaine was used as a model drug for the investigation of drug release behaviour of hydrogels. Lidocaine adsorption capacity of the hydrogels were found to increase from 3.6 to 862.1 (mg lidocaine/g dry gel) with increasing amount of itaconic acid in the gel structure. Adsorption and release processes were followed at 4 and 37 °C, respectively. The release studies showed that the basic parameters affecting the drug release behaviour of the hydrogels were pH and temperature of the solution and cross-link density of the gels.
Keywords: Hydrogel; N-isopropylacrylamide; Itaconic acid; Lidocaine; Drug release;

Comparative evaluation of cytotoxicity of a glucosamine-TBA conjugate and a chitosan-TBA conjugate by Davide Guggi; Nina Langoth; Martin H Hoffer; Michael Wirth; Andreas Bernkop-Schnürch (353-360).
d-Glucosamine and chitosan were modified by the immobilization of thiol groups utilizing 2-iminothiolane. The toxicity profile of the resulting d-glucosamine-TBA (4-thiobutylamidine) conjugate, of chitosan-TBA conjugate and of the corresponding unmodified controls was evaluated in vitro. On the one hand, the cell membrane damaging effect of 0.025% solutions of the test compounds was investigated via red blood cell lysis test. On the other hand, the cytotoxity of 0.025, 0.25 and 0.5% solutions of the test compounds was evaluated on L-929 mouse fibroblast cells utilizing two different bioassays: the MTT assay (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide), which asses the mitochondrial metabolic activity of the cells, and the BrdU-based enzyme-linked immunosorbent assay, which measures the incorporation in the DNA of 5-bromo-2′-deoxyuridine and consequently the cell proliferation.Results of the red blood cell lysis test showed that both thiolated compounds displayed a lower membrane damaging effect causing a significantly lower haemoglobine release than the unmodified compounds. Data obtained by the MTT assay and the BrdU assay revealed a concentration dependent relative cytotoxicity for all tested compounds. The covalent linkage of the TBA-substructure to d-glucosamine did not cause a significant increase in cytotoxicity, whereas at higher concentrations a slightly enhanced cytotoxic effect was caused by the derivatisation of chitosan.In conclusion, the –TBA derivatives show a comparable toxicity profile to the corresponding unmodified compounds, which should not compromise their future use as save pharmaceutical excipients.
Keywords: Glucosamine; Chitosan; Thiomers; Red blood cells lysis; Cytotoxicity; L-929 cells;

We present results on the effect of two anti-inflammatory agents, naproxen and indomethacin, on the structure, assembly and gelation transitions of Pluronic® F127 micelles. Small-angle neutron scattering experiments on micellar solutions indicate that the micelle aggregation number decreases significantly in the presence of drug solutes, causing the number density of micelles to increase. However, only slight changes were observed in the critical micelle concentration of F127 in the presence of these drugs. Both anti-inflammatory agents were found to shift the liquid-to-gel and gel-to-liquid transitions of the copolymer to lower temperatures. This may be the result of an increase in the micellar volume fraction, caused by the presence of the hydrophobic drugs. Using an ultraviolet spectroscopy technique, we have also measured the solubilities and micelle–water partition coefficients (K mw) of naproxen and indomethacin in water and F127 solutions. The values of K mw for naproxen and indomethacin are 355±64 and 474±33, respectively. They are larger than previously reported lipid–water partition coefficients, indicating that F127 micelles may be a better choice for drug loading than lipid vesicles. The slightly greater effects of indomethacin on the gelation boundary as compared with naproxen may be attributable to a higher value of K mw.
Keywords: PEO–PPO–PEO; SANS; TIM; Liquid–gel transition; Partition coefficient; DLS;

Cholera toxin B subunit conjugated bile salt stabilized vesicles (bilosomes) for oral immunization by Paramjit Singh; D Prabakaran; Sanyog Jain; Vivek Mishra; K.S Jaganathan; Suresh P Vyas (379-390).
Bile salt stabilized vesicles, bilosomes appear to be a promising and potential carrier system for oral delivery of peptides and proteins. Bilosomes containing bovine serum albumin (BSA), a model antigen, were prepared and conjugated with cholera toxin B subunit (CTB) in order to enhance their affinity towards M cells of Peyer’s patches. Stability studies were undertaken to ascertain the effect of simulated gastric fluid (SGF, pH 1.2), simulated intestinal fluid (SIF, pH 7.5) and different concentrations of bile salts. Intactness and biological activity of CTB were checked by hemagglutination test. A single oral dose of CTB-conjugated bilosomes produced almost equivalent response compared to parenteral administration of antigen with Freund’s complete adjuvant (FCA). However, in contrast to FCA, oral administration of bilosomes is convenient and devoid of any adverse effects that are observed with parenteral administration of FCA. Serum IgG titers after single administration were significantly better (P<0.05) than oral administration of antigen with other systems for 3 consecutive days, suggesting an effective stimulation of systemic immune response. Mucosal IgA titers obtained advocated a possible application of CTB-conjugated bilosomes as oral vaccine delivery system.
Keywords: Bilosomes; Bile salts; CTB; BSA; Oral immunization; M cell targeting;

Xerostomia is commonly known as ‘dry mouth’ and is characterised by a reduction or loss in salivary production. A bioadhesive gel for its localised treatment was formulated to help enhance the residence time of the product, based on the polymer Carbopol 974P. The bioadhesion of various formulations was evaluated on different mucosal substrates, as simulations of the oral mucosa of xerostomic patients. Depending on the type of model substrate used, the mechanism of bioadhesion could alter. When the rheology of various formulations was examined, changes in bioadhesion were more easily interpreted, as the presence of other excipients caused an alteration in the rheological profile, with a change from a fully expanded and partially cross-linked system to an entangled system. Improving the lubricity of the product was considered important, with optimum incorporation of vegetable oil causing a desirable lowering of the observed friction of the product. The final complex formulation developed also contained salivary levels of electrolytes to help remineralisation of teeth, fluoride to prevent caries, zinc to enhance taste sensation, triclosan as the main anti-microbial/anti-inflammatory agent and non-cariogenic sweeteners with lemon flavour to increase the palatability of the product while stimulating any residual salivary function.
Keywords: Xerostomia; Saliva substitute; Bioadhesion; Rheology; Lubricity; Carbopol 974P; Excipients;

Lipid nanocarriers as drug delivery system for ibuprofen in pain treatment by Alf Lamprecht; Jean-Louis Saumet; Jérôme Roux; Jean-Pierre Benoit (407-414).
Due to their small size, lipid nanocapsules (LNC) might be promising for an injectable as well as for an oral drug delivery system, providing both sufficient drug solubility avoiding vessel embolisation for the intravenous injection and a positive effect of drug absorption after oral administration. Biocompatible ibuprofen LNC were developed in a size range of around 50 nm with a new preparation method. Drug incorporation into LNC was successful to a high degree in all formulations tested (94–98%) and the in vitro drug release in phosphate buffer occurred within 24 h. Pharmacokinetic data were recorded in vivo from rats after intravenous or oral administration, while the antinociceptive efficiency of the LNC formulation was compared with ibuprofen solution by the tail flick test. The AUC and half-life of intravenously injected ibuprofen LNC were found to be 16 and 19%, respectively, higher than a simple drug solution, while the mean residence time was not changed. Oral administration of LNC showed an 18% increase of AUC and a 27% higher mean residence time. The antinociceptive effect was similar for oral administration, drug solution, and LNC at 30 min after administration, and was prolonged up to 4 h in the LNC group. The pain relief after intravenous administration was prolonged when administering LNC formulation for at least 2 h. A drug delivery system for intravenous administration of ibuprofen has been developed which exhibits sustained release properties by either oral or intravenous route and may be interesting in the treatment of postoperative pain.
Keywords: Nanocapsules; Nanoparticles; Pharmacokinetics; Biocompatibility; Antinociceptive effect; Ibuprofen;

Transport of leuprolide across rat intestine, rabbit intestine and Caco-2 cell monolayer by J Guo; Q Ping; G Jiang; J Dong; S Qi; L Feng; Z Li; C Li (415-422).
The purpose of this study was to investigate the transport mechanisms and causes of low bioavailability of leuprolide. The everted gut sac technique and Caco-2 cell monolayer were used to examine: (1) transport properties, enzyme degradation and apparent permeation coefficient (P app); (2) the influence of trypsin inhibitor, EDTA, chitosan and alginate on drug transport; and (3) the effect of animal species on the intestinal transport. Results showed flux increased with increasing concentration of drug, showing a passive diffusion pathway. The enzyme degradation in rabbit gut was the highest. The P app of (4.19 ± 1.33) × 10−5  cm/s in rat gut was the largest and the P app of (5.20 ± 0.20) × 10−7  cm/s in Caco-2 cell the smallest. At a low concentration of drug, trypsin inhibitor had strong enhancement effect on the P app by protecting enough drug for permeation. Chitosan had no effect on the activity of α-chymotrypsin. The increase in P app was due to opening of the tight junctions and interaction with cells. In conclusion, both inhibition of proteolytic enzymes and opening the tight junctions to allow for paracellular transport improved the intestinal absorption. At low drug concentration, reduction of enzyme degradation is the most important factor.
Keywords: Leuprolide; Transport; Trypsin inhibitor; Chitosan; Everted gut sac technique; Caco-2 cell;

The preparation of orally disintegrating tablets using a hydrophilic waxy binder by G Abdelbary; P Prinderre; C Eouani; J Joachim; J.P Reynier; Ph Piccerelle (423-433).
The demand for rapidly disintegrating tablets (RDT) has been growing during the last decade especially for elderly and children who have swallowing difficulties. The problem of certain RDT is their low physical resistance and high friability. This work describes a new approach to prepare RDT with sufficient mechanical integrity, involving the use of a hydrophilic waxy binder (Superpolystate©, PEG-6-stearate). Superpolystate© is a waxy material with a melting point of 33–37 °C and an HLB value of 9. So it will not only act as a binder and increase the physical resistance of tablets but will also help the disintegration of the tablets as it melts in the mouth and solublises rapidly leaving no residues. The incorporation of Superpolystate® in the formulation of RDT was realised by means of two different granulation methods: wet granulation by using an emulsion of this waxy binder as granulating liquid and melt granulation where the molten form of the binder was used. Granule size distributions of both wet and melt granules of crystallised Paracetamol and d-mannitol were compared using laser light diffractometer. Scanning electron microscopy (SEM) was used to examine their morphological characteristics. The potential of the intragranular addition of croscarmellose sodium as a disintegrating agent was also evaluated. The subsequent step encompassed the preparation and the evaluation of the tablets, including the effect of the extragranular introduction of croscarmellose sodium. An improvement in tablet hardness and friability was observed with both granulation methods where we were able to obtain RDT with a disintegration time of 40±2 s and a hardness of 47.9±2.5N.
Keywords: Rapidly disintegrating tablets; Hydrophilic waxy binder; Wet granulation; Melt granulation; Croscarmellose sodium;

Antitumor activity of Cratylia mollis lectin encapsulated into liposomes by Cesar A.S Andrade; Maria T.S Correia; Luana C.B.B Coelho; Silene C Nascimento; Nereide S Santos-Magalhães (435-445).
The hemagglutinating (HA) activity of Cratylia mollis lectin (Cra) was evaluated and the influence of ultrasound and mechanical agitation on its activity examined. The antitumor activity of Cra-loaded liposomes was also investigated. Liposomes were obtained by the lipid thin film method. Physicochemical characterization was carried out and long-term stability of Cra-loaded liposomes assessed. Antitumor activity of Cra-loaded liposomes was investigated against Sarcoma 180 in Swiss mice. The treatment was performed intraperitoneally (7 mg/kg body weight per day) for 7 days. Histopathological analyses of tumor, liver, spleen and kidneys were carried out after treatment of the animals. The results showed that Cra–HA activity is affected under ultrasound exposure. However, Cra was successfully encapsulated into liposomes and the activity of the lectin was preserved despite the use of ultrasound in the liposome preparation. Cra-loaded liposomes were produced with an 84% encapsulation ratio (700 μg/ml) and a tumor inhibition of 71% was achieved. The encapsulation of Cra produced a decrease in its tissue toxicity and improved its antitumor activity. In particular, histopathological analysis revealed that treatment with Cra-loaded liposomes prevented Cra cytotoxicity in the liver and kidney of animals.
Keywords: Lectin; Cratylia mollis; Liposomes; Cytotoxicity; Antitumor activity;

The purpose of this study was to clarify the characteristics of erythritol and to develop the optimum basic formulation of a novel coating with erythritol termed thin-layer sugarless coating. Characteristics of erythritol were investigated compared with maltitol, mannitol, sorbitol, xylitol, and sucrose. Furthermore, the optimum basic formulation of thin-layer sugarless coating with erythritol was determined by coating glass beads. We selected a continuous spray mist method for thin-layer sugarless coating due to the formation of a thin sugarless coating layer by a simple method. We demonstrated that erythritol is a suitable coating material for thin-layer sugarless coating compared with maltitol, mannitol, sorbitol, xylitol, and sucrose because of its high water solubility, low hygroscopicity, instant crystallization, and low tackiness. We also demonstrated that thin-layer sugarless coating with erythritol can reduce coating time compared with the coating with maltitol or sucrose due to its characteristics. We developed the optimum basic formulation of thin-layer sugarless coating consists of erythritol, powdered acacia, and talc. We confirmed that a smooth coating layer and high coating efficiency were achieved using the formulation.
Keywords: Erythritol; Sugarless coating; Crystallization; Tackiness; Powdered acacia; Talc;

The purpose of this study was to develop and evaluate the thin-layer sugarless coated tablets containing Vitamin C, Vitamin E, Vitamin B2, calcium pantothenate, and l-cysteine. As a result of the formulation study, three coating layers, 2% under coating (UC), 38% build-up coating (BC), and 5% syrup coating (SC) were necessary for sufficient impact toughness, elegant appearance, and improvement of appearance stability after storage at 25 °C/75% RH for 6 months under open conditions. We demonstrated that the thin-layer sugarless coated tablets are superior to the sugar-coated tablets in terms of small tablet size and stability of calcium pantothenate. It was due to the coating method, the continuous spray mist method, which can minimize the thicknesses of coating layers and the moisture content in the tablets. We also demonstrated that the thin-layer sugarless coated tablets are superior to the film-coated tablets in terms of masking ability of the unpleasant odor and the appearance, stability of the appearance, and low hygroscopicity. It was due to the dense, opaque, and stable coating layers mainly consist of erythritol. We revealed that thin-layer sugarless coated tablets have both advantages of film-coated tablets and sugar-coated tablets.
Keywords: Erythritol; Thin-layer sugarless coated tablets; Masking; Stability;

Novel high pressure extraction technology by Zhang Shouqin; Zhu Junjie; Wang Changzhen (471-474).
This note describes a novel application of the food processing technology, known as high pressure processing (HPP), to the extraction of essential components of herbs. Herbal extracts may be used as drugs, as well as ingredients in food and cosmetics. We have run some pilot studies in our laboratories to demonstrate the applicability of high pressure extraction.
Keywords: High pressure extraction; Essential components; Epimedium;

Erratum to “Tuning the MR properties of blood-stable pH-responsive paramagnetic liposomes” [Int. J. Pharm. 274 (2004) 75–83] by Knut-Egil Løkling; Roald Skurtveit; Knut Dyrstad; Jo Klaveness; Sigrid L Fossheim (477).

Noticeboard (479-481).