International Journal of Pharmaceutics (v.197, #1-2)

Mechanisms by which cyclodextrins modify drug release from polymeric drug delivery systems by David C. Bibby; Nigel M. Davies; Ian G. Tucker (1-11).
For many drug candidates a modified in vivo drug release is desired to improve efficacy, sustain effect or minimise toxicity. Polymeric delivery systems, such as microspheres, nanospheres and polymeric films, have been extensively researched in an attempt to achieve modified drug release. Cyclodextrins offer an alternative approach. These cyclic oligosaccharides have the ability to form non-covalent complexes with a number of drugs and in so doing alter their physicochemical properties. In addition, the primary and secondary hydroxyl groups of the native (α, β, γ-) cyclodextrins are potential sites for chemical modification. It follows that the incorporation of these agents into polymeric drug delivery systems, as physical mixtures, covalently bound conjugates or cross-linking agents, frequently permits a greater degree of control of drug release. This paper reviews the incorporation of various cyclodextrins into polymeric formulations. The mechanisms by which cyclodextrin/polymer formulations act to modify drug release are considered.
Keywords: Cyclodextrin; Microspheres; Polymer; Modified release; Mechanism;

Carrier-enhanced human growth hormone absorption across isolated rabbit intestinal tissue by Gwen M. Mlynek; Laura J. Calvo; Joseph R. Robinson (13-21).
Small molecular weight alpha acid derivatives are able to enhance the intestinal absorption of human growth hormone through isolated rabbit intestinal tissue. The enhancement is not through the usual tissue modification associated with traditional penetration enhancers nor is it through an active transport process. Rather these small molecules associate with human growth hormone in solution to make it more transportable through intestinal tissue. It is shown that the enhancer has specificity for a particular protein and the enhancer and human growth hormone must be in solution together to be effective, i.e. pretreating the tissue with enhancer and then adding the protein does not increase tissue permeability. Moreover, the enhancer does not increase the permeability of mannitol or progesterone, thus providing additional evidence of specificity and establishing that these agents are not classical penetration enhancers.
Keywords: Intestinal tissue; Enhancers; Permeability enhancer; Human growth hormone; Oral delivery;

A comprehensive model is proposed to accurately describe drug release kinetics from a coated plane sheet when drug loading in the core is above its saturation level. The general solutions are acquired in a dimensionless form by the Laplace transform and the solution for the special case-a perfect sink condition, is derived from these general ones. On the basis of the model calculations, the effects of the diffusion ratios and thickness ratios of the coatings to the core, and drug loading entrapped in the core during the release processes have been discussed over wide range of variables. To validate the model equations proposed, the coated systems, 5-fluoracilum/ethylene-vinyl alcohol copolymer (5-Fu/EVAL) core matrix coated with various polymeric materials such as EVAL, cellulose acetate (CA), poly(2-hydroxyethyl methacrylate) (PHEMA) and poly( methyl methacrylate) (PMMA), having different diffusivities, are designed, experimentally investigated and graphically and quantitatively compared with the theoretical values. The results show a good correlation between the theory and experiment.
Keywords: Coated planar matrix; Controlled release; Mathematical analysis; In-vitro release;

Interactions during aqueous film coating of ibuprofen with Aquacoat ECD by S Schmid; C.C Müller-Goymann; P.C Schmidt (35-39).
During the development of a coated ibuprofen formulation a sticking tendency occurred when applying Aquacoat ECD. This interaction indicated the formation of a eutectic mixture. The compatibility of the components of Aquacoat ECD with ibuprofen was investigated by differential scanning calorimetry. Cetyl alcohol, a stabilizing excipient in Aquacoat, was found to form a eutectic system with ibuprofen. It was characterized by the construction of a phase diagram with 33 mol% ibuprofen and an onset temperature of 40.5°C. Wide-angle X-ray diffraction was used to identify the polymorphic forms of cetyl alcohol. The results confirmed the amorphous state in the aqueous dispersion in contrast to the β0- and γ4-polymorphs of solid cetyl alcohol.
Keywords: Ibuprofen; Cetyl alcohol; Phase diagram; Eutectic; Polymorphism;

Dry powder formulations for inhalation usually comprise a mixture of coarse lactose (CL), employed as a carrier, and micronized drug. It was the aim of this study to determine the effects of fine lactose (FL), blended as a tertiary component on the mixing homogeneity and dispersibility of a model hydrophobic drug, beclomethasone dipropionate (BDP). BDP particles (volume median diameter (VMD) 4.6 μm) existed mainly as agglomerates, the majority of which were not dispersed into primary particles after aerosolization at a high shear force (4.7 psi). The resultant particle size distribution of BDP was multi-modal with VMD varying between 4.7 and 30.2 μm. Ternary interactive mixtures were prepared to consist of CL, FL and BDP with a fixed ratio of lactose to BDP of 67.5:1 w/w, but two concentrations of FL, i.e. 2.5 and 5%, w/w. The mixing was carried out using different sequences of adding the three components for two mixing times (15 and 60 min). Binary mixtures composed of CL and BDP were prepared for both mixing times as the controls, and these exhibited a coefficient of variation (COV) in BDP content≤5%. Addition of FL to the binary formulations greatly reduced the content uniformity of BDP if the final powder were prepared by first mixing CL with FL before mixing with the drug (COV>20%, after mixing for 15 min). However, the mixtures, prepared using other mixing sequences, had a similar uniformity of BDP content to the binary mixtures. All ternary mixtures containing 2.5% FL consistently produced a significantly higher (ANOVA P<0.01) fine particle fraction (FPF, 3.1–6.1%) and fine particle dose (FPD, 13.6–30.1 μg) of BDP than the binary mixtures (FPF, 0.3–0.4%; FPD, 1.6–2.1 μg) after aerosolization at 60 l min−1 via a Rotahaler into a twin stage liquid impinger. The mixing sequences exerted a significant (P<0.05) effect on the dispersion and deaggregation of BDP from the formulations prepared using a mixing time of 15 min but such an effect disappeared when the mixing time was lengthened to 60 min. The dispersibility of BDP was always higher from the ternary mixtures than from the binary mixtures. BDP delivery from dry powder inhalers was improved markedly by adding FL to the formulation, without substantial reduction in the content uniformity of the drug.
Keywords: Dry powder inhalers; Lactose; Beclomethasone dipropionate; BDP; Ternary mixtures; Mixing homogeneity; Aerodynamic particle size; Dry powder aerosols;

Chitosans as nasal absorption enhancers of peptides: comparison between free amine chitosans and soluble salts by Parkpoom Tengamnuay; Amorn Sahamethapat; Achariya Sailasuta; Ashim K Mitra (53-67).
A total of three free amine chitosans (CS J, CS L and CS H) and two soluble chitosan salts (CS G and CS HCl) were evaluated for their efficacy and safety as nasal absorption enhancers of peptides based on in situ nasal perfusion and subacute histological evaluation in rat. At 0.5% w/v, all chitosans were effective in enhancing the nasal absorption of [d-Arg2]-Kyotorphin, an enzymatically stable opioid dipeptide. The enhancing effect of the free amine chitosans increased as the pH was decreased from 6.0 to 4.0 (P<0.05). However, the pH effect was not significant for the two chitosan salts (P>0.05), suggesting that their adjuvant activity may be less pH-dependent than the free amine form. CS J and CS G were subsequently selected for further studies. At only 0.02% w/v, their enhancing effect was already significant and comparable to that of 5% w/v hydroxypropyl-β-cyclodextrin (HP-β-CD). Both chitosans at 0.1% caused minimal release of total protein and phosphorus from the rat nasal mucosa, with the values similar to that of 5% HP-β-CD. At 0.5% the two chitosans also stimulated smaller release of lactate dehydrogenase, an intracellular enzyme used as marker of nasal membrane damage, than 1.25% dimethyl-β-cyclodextrin. Morphological evaluation of the rat nasal mucosa following 2-week daily administration indicated that the two chitosans (1.0%) produced only mild to moderate irritation. In conclusion, both the free amine and the acid salt forms of chitosans are effective in enhancing the nasal absorption of [d-Arg2]-Kyotorphin and have potential for further studies as a safe and effective nasal absorption enhancer of peptide drugs.
Keywords: Free amine chitosans; Chitosan salts; Nasal absorption enhancers; Hydroxypropyl-β-cyclodextrin; Dimethyl-β-cyclodextrin; Protein release; Phosphorus release; Subacute histological evaluation;

Transdermal iontophoretic delivery of timolol maleate in albino rabbits by N. Kanikkannan; J. Singh; P. Ramarao (69-76).
The use of transdermal iontophoresis is a promising technique for the systemic delivery of water soluble and ionic drugs of relatively large molecular size. The present study investigates the skin pre-treatment with Azone® (laurocapram) and iontophoresis on the pharmacodynamic effect of timolol maleate (TM) in vivo in albino rabbits. The pharmacodynamic effect of TM was evaluated by transdermal delivery and compared with an intravenous (i.v.) administration. Iontophoresis of TM (0.1 mg/ml) produced a significant inhibition in the isoprenaline (ISP)-induced tachycardia. Iontophoresis with higher concentration of TM (1 mg/ml) produced a 100% inhibition of the ISP induced tachycardia. Pre-treatment of skin with Azone® (3% v/v emulsion) eliminated the lag time and prolonged the duration of action of iontophoresis from 4 to 6 h. The AUC of Azone® treated group was significantly higher than that of the untreated group (P<0.05). Further, the AUC with iontophoretic delivery and pre-treatment of Azone® was comparable to that of intravenous TM (30 μg/kg). In conclusion, iontophoresis in combination with Azone® can increase the transdermal delivery of TM, whereby the required transport rate can be achieved with a lower drug concentration.
Keywords: Transdermal; Azone®; Iontophoresis; Timolol maleate; Rabbits;

The dermal delivery of lignocaine: influence of ion pairing by C. Valenta; U. Siman; M. Kratzel; J. Hadgraft (77-85).
The purpose of the present study was to determine the significance of ion pairing on the permeation of lignocaine. Results of diffusion studies through polydimethylsiloxane (PDMS) at different pH values 4.0, 6.0, 7.0, 8.0 indicated that lignocaine hydrochloride (L-HCl) flux significantly increased with the amount of unionized base. In order to see if similar results could be obtained using human skin, permeation runs were performed with human skin at pH of 4.0, 5.5 and 7.0. These values were chosen to simulate an appropriate range of physiological conditions. Results of the experiments with human epidermis showed increasing L-HCl flux with increasing pH, confirming the trends seen with PDMS membranes. A linear relationship was found between the apparent partition coefficient and the steady state flux. Further experiments were conducted at donor pH 4.0 to minimise the contribution of the unionized species. Although an excess of different ions such as nitrate, mesylate and bromide increased the apparent partition coefficient, the steady state flux was not significantly increased. The steady state lignocaine flux was increased up to 2.45-fold using different counter ions. The highest flux was measured from lignocaine morpholinopropane sulfonate (L-mps). It is possible to enhance the flux of salts across lipophilic membranes by using an ion pair approach. The degree to which this is possible depends on the lipophilicity of the counter ion, the medium in which the ion pair forms, and the ionic strength.
Keywords: Lignocaine; Percutaneous absorption; Silicone membrane; Human epidermis; pH effect; Ion pairing;

The stability of [Arg8]-vasopressin (AVP) as a function of buffer pH, buffer concentration, salt concentration, temperature, and skin with and without enzyme inhibitors was investigated. AVP was analyzed by reverse-phase high-performance liquid chromatography. The results indicated that the buffer’s pH affected the degradation rate of AVP. Buffer ions (H2PO4 and HPO4 2−) and salt concentrations had no effect on the degradation of AVP. Maximum stability was achieved at pH 3.35 among pH values tested. The activation energy for the overall reaction was 21.5 kcal mol−1 at pH 3.35. From the Arrhenius equation, the shelf-life of AVP at 25°C and pH 3.35 was calculated to be 1.38 years. The degradation rate of AVP in the skin (area: 9 cm2, thickness: 0.5 mm) was 0.22 h−1. Bestatin (an aminopeptidase inhibitor) had the best stabilizing effect on the degradation of AVP by skin among the three enzyme inhibitors (i.e. aprotinin, bestatin, and leupeptin) studied. The degradation rate of AVP in the skin was reduced to 0.059 h−1 in the presence of bestatin in comparison with no inhibitor (0.22 h−1).
Keywords: Stability; Vasopressin; pH; Buffer concentration; Skin; Enzyme inhibitors;

Improved compression properties of propyphenazone spherical crystals by Piera Di Martino; Roberta Di Cristofaro; Christine Barthélémy; Etienne Joiris; Giovanni Palmieri Filippo; Martelli Sante (95-106).
Spherical propyphenazone crystals were produced by an agglomeration technique using a three solvents system. After selecting the best propyphenazone solvent (ethyl alcohol), non-solvent (demineralized water) and bridging liquid (isopropyl acetate), several of their ratios were tested by a Sheffé ternary diagram. Micromeritic properties of agglomerates such as flowability, were improved and their compression behavior was investigated and compared to that of raw crystals. By compression and densification studies, along with tablet SEM analysis, we have been able to explain the compression mechanism of propyphenazone spherical crystals and have shown that their better tablet/ability can be due to the small size of individual particles in the agglomerates
Keywords: Propyphenazone; Spherical crystallization; Compression behavior; Brittle fracture;

Evaluation of low-substituted hydroxypropylcelluloses (L-HPCs) as filler-binders for direct compression by C. Alvarez-Lorenzo; J.L. Gómez-Amoza; R. Martı́nez-Pacheco; C. Souto; A. Concheiro (107-116).
The aims of this study were to assess the potential value of low-substituted hydroxypropylcelluloses (L-HPCs) as excipients of direct compression, and to investigate relationships between the chemical and physical properties of the polymers and (a) the powder rheological behavior and (b) drug release profiles from direct compressed tablets elaborated with (1:1) theophylline:L-HPC mixtures. Experiments were performed with five L-HPC varieties of different nominal particle sizes and degree of substitution. The products were characterized with regard to the moisture content, density, IR and Raman spectroscopy, hydroxypropyloxy content, heat of hydration, particle size, specific surface and porosity, and important differences were found in relation with all these properties. The differences in specific surface principally determine the flow and compaction properties of the powders, and the mechanical and microstructural properties of the tablets. The control of the hydroxypropyloxy content and the particle size of the L-HPCs allow the theophylline release profile to be regulated.
Keywords: Low-substituted hydroxypropylcellulose (L-HPC); Theophylline; Tablet excipients; Direct compression; Porosimetry;

Rapidly cooled materials are often unstable as a result of changes in their physical properties due to imperfect crystallization. In the process of spray-congealing, melted material is atomized into droplets which very quickly solidify. This increases the possibility of the material crystallizing in different metastable forms. In this study it is shown that isothermal microcalorimetry can be used to observe the change in the thermodynamic state of spray-congealed carnauba wax during storage. In order to accelerate the thermodynamic change in the spray-congealed wax, three annealing procedures have been developed and compared using isothermal microcalorimetry. By means of annealing, a spray-congealed product closer to a thermodynamically stable state has been achieved.
Keywords: Microcalorimetry; Solid dispersion; Aging; Stabilization; Annealing; Spray-congealing;

In a previous study we demonstrated the dependency of cyclosporine (CyA) pharmacokinetics on the age and gender of Wistar rats given 10 mg/kg intravenously. The present study has been conducted under the same experimental conditions (10 mg/kg as a single intravenous dose) to identify the mechanisms behind such differences. On the one hand, drug distribution was studied by measuring the CyA levels in blood, liver, kidney, spleen, adipose tissue, skin and muscle at 48 h post-treatment by using a specific fluorescence polarization immunoassay (m-FPIA, Abbott Laboratories). Drug blood and tissue levels in male rats were significantly higher than the female counterparts except for adipose tissue where the concentrations were 2-fold higher in females. In males, the highest CyA concentrations were observed in the liver, followed in rank order by kidney and spleen, fat, skin, muscle, then blood. On the contrary, females showed the highest drug levels in fat, followed by liver, kidney, spleen, skin, muscle and blood. Age exerted a significant influence on CyA tissue levels in males but no effect was observed in females. The potential differences in drug metabolism were established by measuring (HPLC) the amounts of CyA and its metabolites accumulated in faeces after hepatic biotransformation and biliary excretion. The amounts of circulating metabolites in blood as well as those accumulated and excreted in the liver and urine were also estimated by using specific (m-FPIA) and non-specific fluorescence polarization immunoassay (p-FPIA, Abbott Laboratories), respectively. The analysis of faeces revealed that AM9 was the major identified metabolite with females excreting lower amounts of unchanged CyA than males. In addition, the comparison of the AUC values corresponding to parent CyA and total CyA derivatives suggested that blood concentrations of CyA metabolites were higher in females indicating higher biotransformation rates. Therefore, both CyA distribution and metabolism are responsible for the sex-associated differences in drug pharmacokinetics previously found in rats.
Keywords: CyA; Metabolism; Pharmacokinetics; FPIA; Rats;

Determination of optimal combination of surfactants in creams using rheology measurements by Mirka Korhonen; Heikki Niskanen; Juha Kiesvaara; Jouko Yliruusi (143-151).
The effect of surfactant on the rheological properties of some cream formulations was studied. Two surfactants from two different series were combined to determine the combination which yielded the most viscoelastic structure for creams. The surfactants were the soybean derivatives soya sterol, polyethylene glycol 10 soya sterol and polyethylene glycol 25 soya sterol and the sorbitol derivatives sorbitan monooleate and sorbitan trioleate. The rheological properties of the creams were studied using oscillation stress sweep, oscillation frequency sweep and viscosity tests. Droplet size distribution and conductivity of the creams were also determined. The combination polyethylene glycol 10 soya sterol and sorbitan trioleate yielded the most viscoelastic structure with linearly viscoelastic behaviour.
Keywords: Rheology of creams; Surfactants; Storage modulus; Loss tangent; Hydrophile–lipophile balance;

A combination of 2% erythromycin and 0.05% tretinoin in an alcohol–isopropanol lotion was prepared. Two parameters were investigated for their influence on the stability of erythromycin and/or tretinoin, namely pH and the concentration of butylhydroxytoluene (BHT) as antioxidant. To investigate these two parameters, an optimization technique was used with two factors (pH and concentration of BHT) at two levels. Accelerated stability analysis was performed at 45°C in the dark to exclude isomerization of tretinoin. To analyse erythromycin and tretinoin in the combination preparation, a TLC method, previously developed in the laboratory, was used. The degradation of erythromycin seemed to be much faster than the tretinoin degradation. Optimal stability is shown in the pH range of 8.2–8.6 for erythromycin and 7.2–8.2 for tretinoin while the concentration of BHT had no significant influence.
Keywords: Erythromycin–tretinoin topical solution; Chemical stability; Influence of pH and concentration antioxidant; Composite rotative design;

In vitro release studies of methylmethacrylate liberation from acrylic cement powder by A. Bettencourt; A. Calado; J. Amaral; F.M. Vale; J.M.T. Rico; J. Monteiro; A. Lopes; L. Pereira; M. Castro (161-168).
Bone cement or polymethylmethacrylate (PMMA) is commonly used for anchoring cemented prosthesis to the bone. Cytotoxic effect of culture media exposed to PMMA powder may be related with long term problems associated with acrylic cement application, being the monomer (methylmethacrylate) one of the cement’s component partly responsible for the cytotoxic effect. The present work reports the studies of monomer release from acrylic bone cement powder under different experimental conditions: setting time of PMMA (in solution and air) and different culture media composition. High-performance liquid chromatography was used for the determination of residual monomer. Mathematical models were applied to experimental dissolution data revealing that monomer release is lightly affected by the studied variables. The monomer release seems to be a surface phenomena, suggesting that the possible actions of monomer will mainly be due to the initial loss of non polymerized monomer rather than to further depolymerization of the already polymerized cement.
Keywords: Poly(methylmethacrylate); Methylmethacrylate; Biomaterial; Bone cement; Release model;

Theophylline pellets were coated with cellulosic (Aquacoat® ECD 30, Surelease® clear) or acrylic (Eudragit® NE30D, RS30D) polymer aqueous dispersions, containing 10% (related to the insoluble polymer content) of pectin HM or calcium pectinate, using a Uni-Glatt fluidized-bed coating apparatus. When commercial pectinolytic enzymes were added to the dissolution media (0.05 M acetate — phosphate buffer, pH 6.0), the release of theophylline from the coated pellets was generally slower than that observed in the media without enzymes. The enzymatic slowing down of the drug release, depending on the type of the aqueous polymer dispersion used, is more important with mixed Eudragit® NE/calcium pectinate coated pellets. The results obtained have been examined with regard to the validity of the approach based on the combination of pectins and the insoluble polymer aqueous dispersions intended for specific-delivery of drugs to the colon. The mechanism of the hydrophilic drug release from pellets coated with insoluble polymer aqueous dispersions containing an aqueous gel-forming polymer has been also discussed.
Keywords: Colonic drug delivery; Pectins; Insoluble polymers; Polymer aqueous dispersions; Coated pellets; Pectinolytic enzymes; Mechanism of drug release;

Studies of pectin HM/Eudragit® RL/Eudragit® NE film-coating formulations intended for colonic drug delivery by Rasmane Semdé; Karim Amighi; Michel J Devleeschouwer; André J Moës (181-192).
Theophylline pellets were coated with Eudragit® NE30D aqueous dispersions, containing various pectin HM/Eudragit® RL30D ionic complexes, using an Uni-Glatt fluidized-bed apparatus. Dissolution studies were then carried out on the coated pellets at pH 6.0, in absence and in presence of commercial pectinolytic enzymes. The theophylline release from the coated pellets, after an initial latency phase, occurred linearly as a function of time. The theophylline release rate was dependent on the pectin HM content of the complexes incorporated in the coatings. The lowest theophylline release from the coated pellets was obtained when the pectin HM content of the complexes was 20.0% w/w (related to Eudragit® RL), i.e. when the complexation between pectin HM and Eudragit® RL is optimal. The theophylline release from the coated pellets was slower in presence of the pectinolytic enzymes when the pectin content of complexes is higher than 20% w/w. On the other hand, the effect of the enzymes induced an increase of the theophylline release when the pectin HM content of the coatings ranged between 10.0 and 15.0% w/w (related to Eudragit® RL).
Keywords: Colonic drug delivery; Pectin; Eudragits®; Ionic complex; Enzymatic degradation; Film-coating;

Effect of surfactants on human stratum corneum: electron paramagnetic resonance study by Junichi Mizushima; Yoshiaki Kawasaki; Tatsuru Tabohashi; Takeshi Kitano; Kazutami Sakamoto; Makoto Kawashima; Roger Cooke; Howard I. Maibach (193-202).
Electron paramagnetic resonance (EPR) spectra of nitroxide spin probes are useful for studying biological membranes and chemical-membrane interactions. Recently, we established a stripping method to remove stratum corneum (SC) for this purpose. To assess this stripping method with EPR and correlate with standard methods, we quantified the irritant effects of three types of surfactants by measurements of visual score and transepidermal water loss (TEWL), SC hydration and chromametry and studied EPR spectra measurements of surfactant-treated cadaver SC (C-SC) and stripped off SC (S-SC) on patch tested sites. 5-Doxyl stearic acid was the spin label. The order parameter S obtained from the spectra of S-SC correlated with those of C-SC and TEWL values. The results suggest that this method is capable of evaluating the fluidity of SC and correlates with the above bioengineering parameters.
Keywords: Electron paramagnetic resonance spectroscopy; Human stratum corneum; 5-Doxyl stearic acid; Sodium lauryl sulfate; N-3-alkyl oxyhydroxypropyl-l-arginine;

Principal components analysis (PCA) and multivariate regression analysis (MRA) are used to assess the predictors of permeant diffusion across human stratum corneum. Log (D/h), was estimated from log  k p+0.024−0.59 log  K oct, where D=diffusion coefficient (cm2/h), h=path length (cm), k p permeability coefficient (cm/h), K oct=partition coefficient (octanol/water). Molecular weight (MW) with (1) scaled H-bonding parameters α and β, or (2) summed modulus of partial charge from molecular modelling were tested as predictors of (D/h). Charge may be computed for any molecule, whilst α and β values are generally unavailable for molecules of biological interest. PCA suggests a dominant permeation pathway since 93% of data variation is in PC1 of log (D/h), MW and charge and 82% in PC1 of log (D/h), MW, α and β. MRA using MW, α and β is unsatisfactory because of collinearity amongst predictors. The best predictor was the product MW*charge. Similarity of the eigenvectors in PCA and normalised coefficients in MRA indicates that charge and MW are equally important predictors of diffusion.
Keywords: Principal component analysis; Multivariate regression analysis; Human stratum corneum; Partial charge; Molecular modelling; Diffusion;

Pharmacokinetics and in-situ absorption studies of a new anti-allergic compound 73/602 in rats by J.K. Paliwa; A.K. Dwivedi; S. Singh; R.C. Gutpa (213-220).
Compound 73/602 (AA) is a structural analogue of vasicinone, an alkaloid present in the leaves and roots of Adhatoda vasica (Acanthaceae). It possesses potent antiallergic activity in mice, rats and guinea pigs. The pK a of AA was determined to be 2.87±0.19 by UV spectrophotometry. The absorption kinetics of this compound were studied in-situ using a rat gut technique at pH 2.6 and 7.4. The rate of absorption at pH 2.6 (0.0288±0.004 min−1) was slightly less than at pH 7.4 (0.035±0.0008 min−1). This characteristic behavior was attributed to the low pK a of AA, a weekly basic compound, where nearly 35% of the compound remained in the unionized form at pH 2.6. Also, the return of compound into the mucosal lumen from the blood capillaries over a period of 2 h after administering a 2 mg dose in tail vein was less than 0.3%. Hence it was concluded that entero-enteric circulation of AA did not contribute significantly to the in-situ absorption rates. Pharmacokinetic parameters of AA were determined in male rats after administering a single 10 mg/kg intravenous dose (i.v.) and 50 mg/kg oral bolus dose. Following i.v. administration the initial decline in serum concentration was rapid with half-life of 20.2 min. After a single oral dose the concentration-time data of AA in rats was best described by a one-compartment model with equal first order absorption and apparent elimination rate constants. The half-life of the decline in serum concentration of AA following oral administration was 50.6 min, indicating absorption rate limiting disposition at the high dose given. Comparison of AUC of oral and i.v. data indicates that only about 60% of the oral dose reach the systemic circulation.
Keywords: Compound 73/602; Anti-allergic; Pharmacokinetics; Absorption;

The lower respiratory tract provides a number of disease targets for gene therapy. Nebulisation is the most practical system for the aerosolisation of non-viral gene delivery systems. The aerosolisation process represents a significant challenge to the maintenance of the physical stability and biological activity of the gene vector. In this study we investigate the role of a condensing polycationic peptide on the stability and efficiency of nebulised lipid–DNA complexes. Complexes prepared from the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and plasmid DNA (pDNA) at mass (w/w) ratios of 12:1, 6:1 and 3:1, and complexes prepared from DOTAP, the polycationic peptide, protamine, and pDNA (LPD) at 3:2:1 w/w ratio were nebulised using a Pari LC Plus jet nebuliser. Samples from the nebuliser reservoir (pre- and post-nebulisation) and from the aerosol mist were collected and investigated for changes, including: particle diameter, retention of in-vitro transfection activity and the relative concentration and nature of the complexed pDNA remaining after the nebulisation procedure. The process of jet nebulisation adversely affected the physical stability of lipid:pDNA complexes with only those formulated at 12:1 w/w DOTAP:pDNA able to maintain their pre-nebulisation particle size distribution (145±3 nm pre-nebulisation vs. 142±2 nm aerosol mist) and preserve significant pDNA integrity in the reservoir (35% of pre-nebulisation pDNA band intensity). The LPD complexes were smaller (102±1 nm pre-nebulisation vs. 113±2 nm aerosol mist) with considerably greater retention of pDNA integrity in the reservoir (90% of pre-nebulisation pDNA band intensity). In contrast the concentration of pDNA in the aerosol mist for both the 12:1 w/w DOTAP:pDNA and LPD complexes were significantly reduced (10 and 12% of pre-nebulised values, respectively). Despite reduced pDNA concentration the transfection (% cells transfected) mediated by aerosol mist for the nebulised complexes was comparatively efficient (LPD aerosol mist 26 vs. 40% for pre-nebulised complex; the respective values for 12: 1 w/w DOTAP:pDNA were 12 vs. 28%). The physical stability and biological activity of nebulised lipid:pDNA complexes can be improved by inclusion of a condensing polycationic peptide such as protamine. The incorporation of the peptide precludes the use of potentially toxic excesses of lipid and charge and may act as a platform for the covalent attachment of peptide signals mediating sub-cellular targetting.
Keywords: Gene therapy; Cationic lipid; Plasmid DNA; Polycationic peptide; Protamine; Nebulisation; Aerosolisation; Lung;

Gene expression in an intact ex-vivo skin tissue model following percutaneous delivery of cationic liposome–plasmid DNA complexes by James C Birchall; Claire Marichal; Lee Campbell; Ashraf Alwan; Jonathan Hadgraft; Mark Gumbleton (233-238).
The skin represents an attractive site for the localised gene therapy of dermatological pathologies and as a potential antigen bioreactor following transdermal delivery. Potential also exists for the gene therapy of skin as a cosmetic intervention. The most exploited non-viral gene delivery system involves the complexation of cationic liposomes with plasmid DNA (pDNA) to form lipid:pDNA vectors that protect the DNA from nuclease-mediated degradation and improve transgene-cell interactions. Despite numerous studies examining the potential for these vectors in delivering genes to a variety of keratinocyte models, investigations into the topical application of such complexes to intact skin tissue is limited. This ex-vivo study, conducted with intact skin tissue derived from hairless mice, provides quantitative confirmation that topical administration of cationic lipid:pDNA complexes can mediate uptake and expression of reporter pDNA (33-fold higher compared with control) in viable epidermal tissue. The ex-vivo study design provides for intact skin tissue that has not been subjected to depilatory procedures of potential detriment to stratum corneum barrier function, and can be utilised for the quantitative and efficient examination of a potentially wide range of non-viral gene vectors designed for epidermal expression.
Keywords: Skin; Gene therapy; Ex vivo model; Cationic liposomes; Epidermis;

Polyamidoamine Starburst® dendrimers as solubility enhancers by O.M Milhem; C Myles; N.B McKeown; D Attwood; A D’Emanuele (239-241).
The solubility of the hydrophobic drug ibuprofen has been compared in an aqueous solution of polyamidoamine (PAMAM) G4 dendrimer and sodium dodecyl sulphate (SDS). The PAMAM G4 dendrimer solution significantly enhanced the solubility of ibuprofen compared to 2% SDS solution. It was found that the solubility of ibuprofen in dendrimer solution was directly proportional to dendrimer concentration and inversely proportional to temperature. The influence of dendrimer solution pH on the solubility enhancement of ibuprofen suggests that it involves an electrostatic interaction between the carboxyl group of the ibuprofen molecule and the amine groups of the dendrimer molecule.
Keywords: Dendrimers; Solubility enhancement; PAMAM G4 dendrimer; Ibuprofen;

Index (247-248).