International Journal of Pharmaceutics (v.203, #1-2)

Developing recombinant protein pharmaceuticals has proved to be very challenging because of both the complexity of protein production and purification, and the limited physical and chemical stability of proteins. To overcome the instability barrier, proteins often have to be made into solid forms to achieve an acceptable shelf life as pharmaceutical products. The most commonly used method for preparing solid protein pharmaceuticals is lyophilization (freeze-drying). Unfortunately, the lyophilization process generates both freezing and drying stresses, which can denature proteins to various degrees. Even after successful lyophilization with a protein stabilizer(s), proteins in solid state may still have limited long-term storage stability. In the past two decades, numerous studies have been conducted in the area of protein lyophilization technology, and instability/stabilization during lyophilization and long-term storage. Many critical issues have been identified. To have an up-to-date perspective of the lyophilization process and more importantly, its application in formulating solid protein pharmaceuticals, this article reviews the recent investigations and achievements in these exciting areas, especially in the past 10 years. Four interrelated topics are discussed: lyophilization and its denaturation stresses, cryo- and lyo-protection of proteins by excipients, design of a robust lyophilization cycle, and with emphasis, instability, stabilization, and formulation of solid protein pharmaceuticals.
Keywords: Aggregation; Cryoprotection; Denaturation; Excipient; Formulation; Freeze-drying; Glass transition; Stability; Lyoprotection; Residual moisture;

In vivo evaluation of doxorubicin carried with long circulating and remote loading proliposome by Wang Junping; Yoshie Maitani; Kozo Takayama; Tsuneji Nagai (61-69).
Long circulating and remote loading proliposome (LRP-L) was a kind of transparent solution and composed of soybean phosphatidylcholine (SPC), cholesterol, polyethylene glycol derivative of distearoylphosphatidyl ethanolamine (PEG-DSPE) and oleic acid sodium salt. When LRP-L was mixed with 0.9% NaCl aqueous solution containing doxorubicin (DXR), liposomes formed and automatically loaded DXR, in which sonication and extruders were not needed. The average diameter of the liposomal DXR in saline was 129.0±1.9 nm and the encapsulation efficiency was 98.1±0.6%. The pharmacokinetics, biodistribution, acute toxicity and anticancer effect of DXR carried with LRP-L (LRP-L-DXR) were studied. The plasma concentration–time curves of DXR were best fitted to the triexponential decay curves. The area under the plasma concentration-time curve (AUC) of LRP-L-DXR was 22 and five times of free DXR (F-DXR) and conventional cardiolipin liposomal DXR (CL-DXR), respectively. Following i.v. administration, the biodistribution of LRP-L-DXR in the heart and the liver, unlike that of CL-DXR, was not greater than that of F-DXR. However, the biodistribution of LRP-L-DXR in the spleen was less than that of CL-DXR and greater than that of F-DXR. The acute toxicity of LRP-L-DXR was decreased compared with that of F-DXR. The anticancer effect of LRP-L-DXR was significantly increased compared with that of F-DXR in the ascitic M5076 tumor model of C57BL/6 mice and had no significant difference compared with that of doxorubicin HCl liposome injection (Doxil).
Keywords: Proliposome; Long circulation; Remote loading; Doxorubicin; Pharmacokinetics; Biodistribution; Anticancer action;

Tensile strength of tablets containing two materials with a different compaction behaviour by B. van Veen; K. van der Voort Maarschalk; G.K. Bolhuis; K. Zuurman; H.W. Frijlink (71-79).
The tensile strength of tablets compressed from binary mixtures is in general not linearly related to the strength of tablets prepared from single materials; in many cases it shows a decreased tensile strength relative to interpolation. The materials used in this study, sodium chloride and pregelatinised starch, are both plastically deforming materials, but have a different densification and relaxation behaviour. The yield pressure of the binary mixtures shows an almost linear relationship. As an effect of their lower yield pressure, starch particles yield earlier than sodium chloride particles. The following enclosure prevents some sodium chloride particles to yield or crack. The relaxation of the tablets is higher than the relaxation calculated by linear interpolation of the relaxation behaviour of the two pure materials. The difference between the measured porosity expansion and the data obtained by linear interpolation can be considered as a measure for the reduced interparticle bonding. SEM-photographs indicate that the reduced interparticle bonding is caused by the low adhesive forces. The measured decrease of the tensile strength of the tablets is also considered to be the result of reduced interparticle bonding. In this paper it is shown that there exists a similar relationship between the tensile strength reduction and the percentage of starch on the one hand and the extra porosity expansion and the starch percentage on the other hand.
Keywords: Tablet strength; Binary mixture; Densification; Interparticle bonding; Relaxation;

Strategies for maintaining the particle size of peptide DNA condensates following freeze-drying by Kai Y Kwok; Roger C Adami; Kelly C Hester; Youmie Park; Steve Thomas; Kevin G Rice (81-88).
The particle size of peptide DNA condensates were studied after freeze-drying and rehydration as a function of sugar excipient, concentration, pH, DNA concentration, and peptide condensing agent. In the absence of an excipient, freeze-dried 50 μg/ml AlkCWK18 (iodoacetic acid alkylated Cys-Typ-Lys18) DNA condensates formed large fibrous flocculates on rehydration. Of the sugars tested as lyoprotectants, sucrose proved most effective at preserving particle size during rehydration. The addition of 5 wt/vol% sucrose preserved a mean particle diameter of less than 50 nm during rehydration of AlkCWK18 DNA condensates prepared at DNA concentrations up to 200 μg/ml; however, higher DNA concentrations led to the formation of insoluble fibrous flocculates. Substitution of polyethylene glycol (PEG)-CWK18 as a DNA condensing peptide eliminated the need for sucrose, resulting in peptide DNA condensates that retained particle size when rehydrated in water or normal saline at concentrations up to 5 mg/ml. The results suggest that sucrose functions primarily as a bulking agent during freeze-drying that only preserves the particle size of AlkCWK18 DNA condensates up to a maximum concentration of 200 μg/ml. Alternatively, the steric layer created on the surface of PEG-CWK18 DNA condensates provides far more efficient lyoprotection, preserving their particle size at a concentration of 5 mg/ml without a bulking agent.
Keywords: Peptide DNA condensates; Freeze-drying; Lyoprotectant; Polyethylene glycol; Particle size; Solubility; Aggregation prevention;

Characterization of thermosensitive chitosan gels for the sustained delivery of drugs by E Ruel-Gariépy; A Chenite; C Chaput; S Guirguis; J.-C Leroux (89-98).
The aim of this study was to investigate the physical properties of a chitosan/glycerophosphate (GP) thermosensitive solution which gels at 37°C and evaluate the in vitro release profiles of different model compounds. The gelation rate was dependent on the temperature and on the chitosan deacetylation degree. The solution containing 84%-deacetylated chitosan could be stored 3 months at 4°C without apparent change in viscosity. The in vitro release profiles of the model compounds depended on the presence of GP in the chitosan solution, on their molecular weight and on the presence of lysozyme in the release media. They were not affected by the electrostatic charge of the model compound when present at low concentrations. During the first 4 h, the release was accompanied by a substantial loss of the gel weight which was mainly attributed to the leaching of water and excess GP. Scanning electron micrographs revealed that the solutions yield gels with a highly porous structure after 24 h of exposure to a continuous flow of phosphate buffered saline. These results indicate that the chitosan/GP thermosensitive solutions gel rapidly at body temperature, can remain in the sol state at 4°C and can sustain the delivery of macromolecules.
Keywords: Chitosan; Thermosensitivity; Hydrogel; Sustained-delivery; Macromolecule;

Interactions between liposomes and hydroxypropylmethylcellulose by Concepción Gutiérrez de Rubalcava; José L Rodriguez; Roberto Duro; Carmen Alvarez-Lorenzo; Angel Concheiro; Begoña Seijo (99-108).
The characteristics of the adsorption process of hydroxypropylmethylcellulose (HPMC) of molecular weight 35 400 Da and nominal viscosity 100 cps onto liposomes prepared with different egg lecithin–cholesterol molar ratios were examined. Adsorption isotherms were constructed and analysed to investigate the mechanisms implicated in the incorporation of the polymer to the interface. Only the isotherms obtained with cholesterol-free liposomes were fitted with Langmuir model. When cholesterol is present in the composition they present a sigmoidal slope. The mechanism of adsorption depends on liposome composition being the main force that drives polymer adsorption of hydrophobic nature. The apparent volumes of HPMC indicate that the conformation of the adsorbed macromolecules depends on liposome composition. Hydration enthalpy values show that adsorbed polymers do not give more hydrophilic systems after freeze-drying as expected with the hydrophilic characteristics of the HPMC.
Keywords: Liposomes; Hydroxypropylmethylcellulose; Interfacial adsorption;

Microbiological assay for terbinafine hydrochloride in tablets and creams by Simone Gonçalves Cardoso; Elfrides E.S Schapoval (109-113).
The optimization of a microbiological assay, applying the cylinder-plate method, for the determination of the antifungal terbinafine hydrochloride is described. Using a strain of Aspergillus flavus ATCC 15546 as the test organism, terbinafine hydrochloride at concentrations ranging from 0.125 to 0.5 μg ml−1 could be measured in tablets and creams. A prospective validation of the method showed that the method was linear (r=0.9999), precise (intra-day: CV=0.48%-tablets and 0.43%-creams; inter-day: CV=0.98%-tablets and 0.64%-creams) and accurate (it measured the added quantities). The method shows results that confirm its precision, not differing significantly the others methods described in the literature. We conclude that the microbiological assay is satisfactory for quantitation of in vitro antifungal activity of terbinafine.
Keywords: Aspergillus flavus; Microbiological assay; Cylinder-plate method; Terbinafine;

The degradation pathways of glucagon in acidic solutions by Anjali B Joshi; Elena Rus; Lee E Kirsch (115-125).
Objective: Glucagon is a 29 amino acid peptide hormone that exhibits degradation via both chemical and physical pathways. The objective of the studies reported herein was to identify the degradation products and scheme for glucagon hydrolysis in acidic solutions. Methods: Solutions of glucagon in 0.01 N HCl (pH 2.5) were degraded at 60°C for 70 h. One isocratic and two gradient RP-HPLC methods were developed to separate the degradation products. Structure elucidation of the separated peaks was achieved using amino acid sequencing, amino acid analysis, and mass spectrometry. Degradation was carried out in the pH range 1.5–5 to check for changes in degradation scheme with pH. Authentic samples of degradation products were degraded under similar acidic conditions to confirm precursor successor relationships in the degradation scheme. Results: Sixteen major degradation products were isolated and identified. The major pathways of degradation were found to be aspartic acid cleavage at positions 9, 15, and 21 and glutaminyl deamidation at positions 3, 20, and 24. Cleavage occurred on both sides of Asp-15 but only on the C-terminal side of Asp-9 and Asp-21. Deamidation of the Asn residue at position 28 was not detected.
Keywords: Degradation pathways; Deamidation; Glucagon; Glutaminyl; Hydrolysis; Peptide cleavage;

Semisolid liquid paraffin-in-water emulsions (aqueous creams) prepared from cetrimide/fatty alcohol mixed emulsifiers, and ternary systems formed by dispersing the mixed emulsifier in controlled percentages of water were examined as they aged using a combination of low and high angle X-ray diffraction measurements (Daresbury Laboratory Synchrotron Radiation Source). The results were correlated with the rheological properties measured in earlier studies. The cationic emulsifying wax showed phenomenal swelling in water. The reflection that incorporates interlamellar water increased continuously from 74 Å at 28% water to over 500 Å at 93% water. The trend was not influenced by the method of incorporation of the components and swollen lamellar phase was also identified in the corresponding emulsion. The swelling, which was due to electrostatic repulsion, was suppressed by salt and was reduced when the surfactant counterion was changed from Br to Cl. Changes in rheological properties on storage and in the presence of salt were correlated with changes in water layer thickness. High angle diffraction confirmed that the hydrocarbon bilayers were in the hexagonal α-crystalline mode of packing. Ternary systems and creams prepared from pure alcohols, although initially semisolid, were rheologically unstable and broke down. Low angle X-ray study into the kinetics of structure breakdown showed that the swollen lamellar gel phase formed initially swells even further on storage before separating.
Keywords: Pharmaceutical cream; Cetrimide/cetostearyl alcohol ternary system; Lamellar phase; Emulsifying wax; Synchrotron radiation; X-ray diffraction;

Stabilization and sustained-release effect of Misoprostol with Methacrylate copolymer by David Chen; Rong-Jer Tsay; Hue-In Lin; Huilan Chen; Shou-Chung Chao; Hao Ku (141-148).
The use of ammonio methacrylate copolymer (Eudrgit RS, RL) to form a sustained-release solid dispersion of Misoprostol can improve and enhance two important physical and chemical properties of Misoprostol. First, the solid dispersion matrix formed by the copolymer can protect Misoprostol from being degraded by water so that its stability is improved. Second, Misoprostol can be slowly released by diffusion from the copolymer matrix. Accelerated stability studies of Misoprostol–Eudragit solid dispersion after storing at various temperatures for different time periods were carried out. According to high performance liquid chromatography (HPLC) analyses, the stability of Misoprostol in a series of Eudragit appeared significantly improved at different ratios. The Misoprostol–Eudragit dispersion can be used in a powder form, filled in capsules, or compressed into tablets. The dissolution profiles of Misoprostol–Eudragit solid dispersion and its tablets in water, pH 1.2, 4.5 and 6.8, dissolution media show that this stable solid dispersion is a sustained-release type.
Keywords: Misoprostol; Eudragit; Stability; Sustained-release effect; Dissolution profile;

Adsorption and controlled release of terbinafine hydrochloride (TER-HCl) to and from pH sensitive poly(acrylamide/maleic acid) (P(AAm/MA)) hydrogels were investigated. P(AAm/MA) hydrogels were prepared by irradiating the ternary mixtures of AAm/MA/and water by γ-rays at ambient temperature. Antifungal drug, TER-HCl containing hydrogels, at different drug to polymer ratios, was prepared by direct adsorption method. The influence of MA content in the gel on the adsorption capacities of hydrogel and the effect of pH on the releasing behavior of TER-HCl from gel matrix were investigated. Terbinafine adsorption capacity of hydrogels are found to increase from 2 to 38 mg TER-HCl per g dry gel with increasing amount of MA in the gel system. In vitro drug release studies in different buffer solutions show that the basic parameters affecting the drug release behavior of hydrogel are the pH of the solution and MA content of hydrogel.
Keywords: Terbinafine hydrochloride; Poly(acrylamide/maleic acid); Hydrogel; pH sensitive;

Development of spray-dried acetaminophen microparticles using experimental designs by A Billon; B Bataille; G Cassanas; M Jacob (159-168).
Experimental factorial designs were built to investigate the effects of five parameters on production yields and moisture contents of spray-dried products. These factors concerned both the solution feed (drug concentration, colloidal silica concentration and polymer/drug ratio) and the spray dryer (inlet temperature and feed rate). Three formulations containing cellulose derivatives and acetaminophen were tested. The aim of the study was to optimize the operating conditions to maximize production yields while minimizing moisture contents. First screening experiments consisting of fractional factorial designs revealed the most significant factors to be inlet temperature, feed rate and their interaction for both formulations containing sodium carboxymethylcellulose and feed rate and colloidal silica concentration for the formulation containing microcrystalline cellulose. Then, the optimal operating conditions were estimated by response surface methodology. Central rotational composite designs showed quadratic models were adequate. New assays were carried out using these last conditions to evaluate both the repeatability and reproducibility of the spray-drying technique. Yields above 80% and moisture content of ∼1% were reached. The characterization of microparticles revealed the poor flowability of the spray-dried products due to significant cohesiveness and very small size (less than 55 μm).
Keywords: Spray-drying; Acetaminophen; Central composite designs; Optimization; Microparticle characterization;

Polysaccharide coated liposomes were prepared, characterized and evaluated for their potential use in oral immunization. Liposomes were prepared by reverse phase evaporation method. Bovine serum albumin (BSA) was chosen as the model antigen. Pulluan, a naturally occurring polysaccharide produced by a yeast like fungus, was chemically modified into its palmitoyl derivative (O-palmitoylpullulan; OPP) and was used for coating of the liposomes. The synthesized OPP was characterized by IR and NMR spectroscopy. The liposomes prepared were characterized for their size, shape, surface charge, encapsulation efficiency and stability in simulated gastric fluid. The immune stimulating activity was studied by measuring the serum IgA and IgG following oral administration of the prepared polysaccharide coated liposomes. Similarly, other formulations were studied and the results were compared. BSA loaded liposomes coated with OPP and plain polysaccharide could produce better IgG and IgA titre levels as compared to plain alum adsorbed BSA. The plain liposomes containing BSA could however produce significantly higher IgG and IgA levels as compared to equivalent BSA–alum based oral immunization. The results indicate that chemically modified polysaccharide coated liposomes can be used as a potential adjuvants for effective oral immunization.
Keywords: Liposomes; Oral immunization; O-palmitoylpullulan; Polysaccharide;

Directly compressed matrices were produced containing either xanthan gum or karaya gum as a release-controlling agent. These swellable hydrophilic natural gums were used to control the release of varying proportions of two model drugs, caffeine and diclofenac sodium, which have different solubilities in aqueous medium. Gum erosion, hydration and drug release studies were carried out using a dissolution apparatus (basket method) at two agitation speeds. Xanthan gum displayed a high degree of swelling due to water uptake and a small degree of erosion due to polymer relaxation. Neither agitation speed nor drug solubility had any significant effect on water uptake, but matrices with the lower proportion of gum produced a lesser degree of hydration. In contrast, karaya gum displayed a much lower hydration capacity and a higher rate of erosion, both markedly affected by agitation speed. Drug release from xanthan and karaya gum matrices depended on agitation speed, solubility and proportion of drug. Both xanthan and karaya gums produced near zero order drug release with the erosion mechanism playing a dominant role, especially in karaya gum matrices.
Keywords: Xanthan gum; Karaya gum; Hydration; Matrices; Drug release; Erosion;

Leuprolide acetate, an analogue of luteinizing hormone-releasing hormone (LH-RH), was hydrophobically ion paired with a long chain fatty acid, sodium oleate, in an aqueous solution. Solution behaviors of the complex formed between leuprolide and sodium oleate were investigated in terms of aqueous solubility, turbidity, particle size, and zeta potential as a function of molar ratio between the two species. It was found that with increasing the stoichiometric molar amounts of sodium oleate to leuprolide approached up to 2.5–3, the solution became gradually turbid with increasing particle sizes, indicating leuprolide precipitation as a result of hydrophobic ion pairing. On the other hand, beyond that critical molar ratio range, the solution turned into clear with much reduced particle size, indicative of micelle formation. The hydrophobically modified leuprolide–oleate complex was lyophilized and directly encapsulated within biodegradable poly(d,l-lactic-co-glycolic acid) (PLGA) microspheres via a single oil-in-water (O/W) emulsion method. Microsphere morphology, leuprolide release behavior, and polymer mass erosion profiles were examined in comparison to the PLGA microspheres prepared with free leuprolide.
Keywords: Leuprolide acetate; PLGA; Microspheres; Hydrophobic ion pairing;

Whisker growth of l -menthol in coexistence with various excipients by Hiroshi Yuasa; Michi Ooi; Yuki Takashima; Yoshio Kanaya (203-210).
The purpose of the present study was to clarify the mechanism for l -menthol whisker growth. l -Menthol was mixed with an excipient, and the interaction was examined by IR measurement, thermal analysis and powder X-ray diffraction. Then we examined the involvement of the capillary condensation using the pore size distribution measurement. By mixing l -menthol with an excipient with whisker growth, the hydroxyl group stretching band of l -menthol was shifted to the higher wavenumber in the IR spectrum, the melting point and heat of fusion of l -menthol became lower in the thermal analysis, and the diffraction intensity of l -menthol became lower in the powder X-ray diffraction. The excipients with whisker growth showed the tendency to have the meso-pore involved in the capillary condensation in the pore size distribution measurement. From the above results, the whisker growth mechanism is considered as follows. When l -menthol was mixed with an excipient with whisker growth, the crystallinity of l -menthol was lowered and the vapor pressure was increased by the interaction mainly consisting of the hydrogen bond. The generated l -menthol vapor entered meso-pore, the saturated vapor pressure was lowered by the capillary condensation, and the nucleation occurred. The vapor was further supplied, generating the growth of whisker.
Keywords: Whisker; l -Menthol; Excipient; Hydrogen bond; Capillary condensation;

Lipid and ultrastructural characterization of reconstructed skin models by Maria Ponec; Esther Boelsma; Arij Weerheim; Aat Mulder; Joke Bouwstra; Mieke Mommaas (211-225).
The study aimed at evaluating tissue architecture and quality of the permeability barrier in commercially available reconstructed human skin models; EpiDerm™, SkinEthic™ and Episkin™ in comparison to native tissue. For this purpose, tissue architecture was examined by electron microscopy and epidermal lipid composition was analyzed by HPTLC. Stratum corneum lipid organization was investigated by electron microscopy in combination with RuO4 post-fixation and by SAXD. Ultrastructurally, the overall tissue architecture showed high similarities with native epidermis. In the stratum corneum extracellular space, lipid lamellae consisting of multiple alternating electron-dense and electron-lucent bands were present. This regular pattern was not seen throughout the whole stratum corneum probably due to the observed irregular lamellar body extrusion in some areas. Lipid analyses revealed the presence of all major epidermal lipid classes. Compared with native epidermis the content of polar ceramides 5 and 6 was lower, ceramide 7 was absent, and the content of free fatty acids was very low. These differences in lipid composition may account for differences observed in SAXD pattern of Episkin and EpiDerm penetration models. In the latter only the long-distance periodicity unit of about 12 nm was observed and the short periodicity unit was missing. In conclusion, all three skin models provide a promising means for studying the effects of topically applied chemicals, although the observed deviations in tissue homeostasis and barrier properties need to be optimized.
Keywords: Reconstructed epidermis; Skin barrier function; Epidermal lipids; Morphology;

Spinal biopharmaceutics of bupivacaine and lidocaine by microdialysis after their simultaneous administration in rabbits by Rozenn Clément; Jean-Marc Malinovsky; Pascal Le Corre; Gilles Dollo; Francois Chevanne; Roger Le Verge (227-234).
The aim of the present study was to determine the intrathecal bioavailability of a mixture of lidocaine and bupivacaine in a rabbit model of spinal anesthesia by using the microdialysis technique. Catheter and microdialysis probe were inserted under control of the view either in the epidural or in the intrathecal space. First, the epidural disposition of the mixture of bupivacaine and lidocaine was studied after epidural administration. Then, the intrathecal and plasma dispositions of bupivacaine and lidocaine were investigated following intrathecal or epidural administration. The epidural clearance of bupivacaine was higher than that of lidocaine, suggesting a more significant uptake of bupivacaine into the systemic circulation and/or into the CSF. The intrathecal bioavailability of bupivacaine and lidocaine was 12.3 and 17.9%, respectively, while it was 5.5 and 17.7% following the separate administration of each agent [Clément, R., Malinovsky, J.M., Le Corre, P., Dollo, G., Chevanne, F., Le Verge, R., 1999. Cerebrospinal fluid bioavailability and pharmacokinetics of bupivacaine and lidocaine following intrathecal and epidural administrations in rabbits using microdialysis. J. Pharmacol. Exp. Ther. 289, 1015–21]. After intrathecal administration, a decrease in C max and AUC values was observed for bupivacaine in comparison with the separate administration. Moreover, after epidural administration, the systemic resorption was slower and lower, especially for bupivacaine. Such a reduction in the systemic absorption of bupivacaine might increase its intrathecal bioavailability, resulting from a vasoconstrictor effect of lidocaine reducing the systemic absorption of bupivacaine from the epidural space leading to an increase of its extent of absorption through meninges into CSF although its absorption rate was not modified.
Keywords: Microdialysis; Lidocaine–bupivacaine; Intrathecal administration; Epidural administration; Intrathecal bioavailability;

Preparation in high-shear mixer of sustained-release pellets by melt pelletisation by D. Voinovich; M. Moneghini; B. Perissutti; J. Filipovic-Grcic; I. Grabnar (235-244).
The preparation of sustained-release pellets by melt pelletisation was investigated in a 10-l high shear mixer and ternary mixtures containing stearic acid as a melting binder, anhydrous lactose as a filler and theophylline as a model drug. A translated Doehlert matrix was applied for the optimisation of process variables and quality control of pellets characteristics. After determination of size distribution, the pellets were characterised with scanning electron microscopy, X-ray photoelectron spectroscopy and porosimetric analysis. Finally, the in vitro release from every single size fraction was evaluated and the release mechanism was analysed. Since the drug release rate decreased when enhancing the pellet size fraction, the 2000-μm fraction, exhibiting a substantially zero-order release, was selected for further in vivo biovailability studies. These data demonstrated that pellets based on the combination of stearic acid and lactose can be used to formulate sustained release pellets for theophylline.
Keywords: Doehlert design; Melt pelletisation; High shear mixer; Sustained release;

The influence of Span®20 on stratum corneum lipids in Langmuir monolayers: comparison with Azone® by A López-Castellano; C Cortell-Ivars; G López-Carballo; M Herráez-Domı́nguez (245-253).
Recently we have proved that Span 20 has the same enhancer effect as Azone on in vitro percutaneous penetration of lipophilic compounds (log  P oct from 1.34 to 2.33). The purpose of this work is to study the interactions of Span 20 with stratum corneum lipids monolayers and to compare them with Azone. The surface pressure–area characteristics of Span 20 in mixed monolayers with different model lipids (ceramides, cholesterol, free fatty acids and two mixtures of ceramides+cholesterol, and ceramides+cholesterol+free fatty acids) in similar proportions to that which exists in human stratum corneum lipids were recorded as compression isotherms at 25°C. Azone was also investigated on monomolecular films of some of these lipids. The results indicate that the effect exerted upon lipid packing by the Span 20 correspond, as in the case of Azone, to increased fluidity within monolayers. To quantify and compare the effect of Span 20 and Azone, the compressibility of enhancer–lipid model mixed monolayers was calculated, and expressed as a function of mole fraction of enhancer present on the films. Statistical comparison of the results obtained from both enhancers shows that they are equally potent in their interaction with the lipid models assayed. These models, if restricted, seem to be good for predict the activity and potency of percutaneous enhancers on the fluididity of the lipidic structure of the stratum corneum.
Keywords: Percutaneous enhancers; Azone; Span 20; Monolayer; Stratum corneum lipids;

We describe a method for determining incorporated amounts of poly(ethylene glycol) (PEG)-derivatized lipids in liposomes for the physicochemical characterization of PEG-coated liposomes. This method is based on the spectrophotometric determination of complexes of polyethers with sodium ions after their extraction as picrates into 1,2-dichloroethane, developed by Favretto for measuring levels of polyoxyethylene alkylphenyl-ether non-ionic surfactants in waste water. The same assay was applied to the estimation of PEG-derivatized lipids in liposomes and percent incorporation of PEG-derivatized lipids into liposomes was successfully determined. To prevent the interference from liposomal lipids other than PEG-derivatized lipids in this assay, liposomal samples were diluted at least to a concentration of less than 0.2 mM. The percent incorporation of PEG-lipids varied, depending on the molecular weight of PEG and anchor acyl chain length in PEG-lipids and it was suggested that the percent incorporation of PEG-lipids into liposomes would be a good parameter of quality control of PEG-liposomes in manufacturing facility and the picrate method used in the present study allows for the determination of this parameter without the need for hazardous radioisotopes.
Keywords: Liposomes; Poly(ethylene glycol); Physicochemical characterization;

Index (271-272).