International Journal of Pharmaceutics (v.257, #1-2)

Evaluation of different formulation studies on air-filled polymeric microcapsules by multivariate analysis by Kathrin Bjerknes; Jorunn Undheim Brænden; Gro Smistad; Irenè Agerkvist (1-14).
Air-filled polymeric microcapsules have been prepared by freeze-drying of emulsions containing the wall-forming polymer in the organic phase of oil in water emulsions. Echogenic air-filled microcapsules were prepared from emulsions containing either (−)-camphene, cyclohexane or cyclooctane as the solvent in the organic phase. Formulation studies have been performed to improve the yield and acoustic quality of the microcapsule suspensions. The yield was measured as particle concentration or efficacy, i.e. normalised attenuation at 3.5 MHz, related to the amount of polymer used.No overall conclusion could be made for all the variables when visually comparing the results from the different investigations. Multivariate analyses (PCA and PLS) were therefore necessary to be able to reveal any relevant systematic information from all the investigations. Different parameters describing the formulation, the production process and parameters describing the characterisation of the intermediates and the final product were set as independent X-variables.Three to four percent (w/v) of polymer was found to be the appropriate concentration of wall forming polymer. Including PEG 3000 resulted in improved freeze-dried product and suspension. Quenching of the emulsions by freezing in dry ice/methanol prior to freeze-drying was not necessary.Process parameters for homogenising and freeze-drying should be optimised with regard to the single systems, due to the different physico-chemical properties of the different solvents, especially melting point and vapour pressure.
Keywords: Polymeric microcapsules; Emulsion; PLS; PCA; Freeze-drying; Ultrasound contrast agent;

Two types of chitosan, i.e. the free amine (CS J) and the glutamate salt (CS G), were evaluated for their enhancing effect on in vivo nasal absorption of salmon calcitonin (sCT) in rats. The results were subsequently compared with β-cyclodextrins, one of the most commonly studied enhancers. Solutions containing sCT and chitosan (0–1.25% w/v) in isotonic phosphate buffers (IPB; pH 3.0–6.0) were nasally administered at the dose of 10 IU/kg. The plasma calcium lowering effect in each sCT-treated rat was determined by calculating the total percent decrease in plasma calcium (%D). CS J showed an increase in %D as the solution pH was decreased in accordance with the increased ionization and hydration of the free amine chitosan at the more acidic pH. However, CS G showed an increase in %D with increasing pH, with maximum hypocalcemic effect observed at pH 6.0. At their optimal pH (4.0 for CS J and 6.0 for CS G), the absorption enhancing effect of both chitosans was concentration dependent from 0.25 to 1.0% and leveled off at 1.25%. Using specific RIA, the absolute bioavailability of sCT after comparison with i.v. administration was determined to be 2.45, 1.91, and 1.22% for 1% CS J, 5% dimethyl-β-cyclodextrin (DM-β-CD) and control group (intranasal (in) sCT alone), respectively. Although the absolute nasal bioavailability seemed to be low when compared to the i.v. administration, the inclusion of 1% CS J resulted in two-fold increase in the AUC0–180 of plasma sCT relative to that of the control group. Addition of 5% DM-β-CD also led to 1.56-fold increase in absorption over the control group. All the enhancers showed significant absorption enhancement (P<0.05) with the highest effect observed with CS J. In conclusion, cationic polymer chitosan may have promising potential as a safe and effective nasal absorption enhancer of sCT.
Keywords: Salmon calcitonin; Nasal absorption enhancers; Free amine chitosan; Chitosan glutamate; Hydroxypropyl-β-cyclodextrin; Dimethyl-β-cyclodextrin; Nasal bioavailability;

The objective of this work was to study the in vitro characteristics as well as in vivo pharmacokinetic performance of a series nalbuphine (NA) prodrug-loaded microspheres. An oil-in-water solvent evaporation method was used to incorporate the various NA prodrugs into poly(d,l-lactide-co-glycolide) (PLGA)-based microspheres. The morphology of microspheres under the scanning electron microscopy (SEM) revealed a spherical shape with smooth surface. Drug release rates for the microspheres were found to be a function of prodrug hydrophilicity, with higher drug release rates for microspheres loaded with more hydrophilic prodrugs. The release profiles fit well to the Baker and Lonsdale’s spherical matrix model, suggesting the drug release from microspheres was consistent with a diffusion mechanism. The in vivo pharmacokinetic studies after s.c. injection of microspheres into rabbits showed sustained plasma NA-time profiles, with approximately 104.7, 67.2, and 41.0% relative bioavailability for microspheres loaded with nalbuphine propionate (NAP), nalbuphine pivalate (NPI), and nalbuphine decanoate (NDE), respectively. The in vitro release characteristics correlated well with the in vivo pharmacokinetic profiles. The results indicated that the prodrug hydrophilicity had significant effects on the in vitro as well as in vivo drug release kinetics. The present study demonstrates the feasibility of using biodegradable polymeric microspheres for controlled delivery of NA prodrugs.
Keywords: Controlled release; Nalbuphine prodrug; Microsphere; Poly(d,l-lactide-co-glycolide);

Drug output of unvented jet nebulizers as a function of time by H. Diederik; P.P.H. Le Brun; H.W. Frijlink; P.M.B. Vitányi; M. Weda; D.M. Barends (33-39).
Nebulizer drug output rate increases during the nebulization. For unvented jet nebulizers, a physical and mathematical model based on the efficiency of the nebulization process is presented for this phenomenon. Formulas are derived for the cumulative drug output and the drug output rate of the nebulization process. The model is compared with the model proposed by Coates et al. [J. Aerosol. Med. 11 (1998) 101]. Both models are supported by experimental literature data. Both models predict the experimental values well but the proposed model allows more easy prediction of the influence of small changes in the nebulization conditions and the calculation of the cumulative drug output for a related process. From literature data it is shown that the efficiency of an unvented jet nebulization process of diluted aqueous solutions is relatively insensitive to small changes in the concentration as well as to small changes in aspiration flow but is sensitive to the humidity of the compressor gas only.
Keywords: Jet nebulizer; Drug output; Efficiency;

Iontophoretic and chemical enhancement of drug delivery by L.M.A Nolan; J Corish; O.I Corrigan; D Fitzpatrick (41-55).
This paper reports on measurements of the release characteristics of the model drug salbutamol base from a liquid crystalline vehicle across a non-rate limiting synthetic membrane. The measured passive release rates were compared with analogous behaviour: (i) when a penetration enhancer such as oleic acid was incorporated into the vehicle; (ii) when the release was iontophoretically assisted; and (iii) when the penetration enhancer and iontophoretic assistance were used simultaneously. The effects of using isotonic phosphate buffer solution as the aqueous domain of the vehicle and in the receptor were also separately assessed. The passive release from the standard system was consistent with matrix diffusion control. The addition of oleic acid indicated association of the drug with the fatty acid so that its release into an aqueous medium was significantly retarded. With buffer ions present in the vehicle the release rate increased consistent with reduced association, and when phosphate buffer was used as a receptor medium the release rate exceeded that of the standard vehicle due to an ion exchange process. The delivery of salbutamol from the fatty acid containing systems was substantially enhanced by iontophoresis and the rates were shown to be approximately proportional to the assisting currents. The data clearly indicate the iontophoretic process to be significantly less efficient in the presence of buffer ions but with the iontophoretic delivery rates being enhanced by the presence of a fatty acid.
Keywords: Buffer; Enhancers; Iontophoresis; Liquid crystals; Oleic acid; Salbutamol; Transdermal;

The Peschl-split bed shear tester was utilised to study the formation of different liquid states during wet massing for granulation. Using lactose monohydrate as a model bulking agent the threshold between pendular and funicular state was found to be at about 6% (w/w) of liquid binder added to the wet mass, here a 5% colloidal solution of HPMC in water. The upper limit of the funicular state appeared to be at approximately 15% (w/w) of liquid binder. The threshold values obtained from the shear cell measurements did correlate with values obtained from dried granule characteristics such as granule density and compressive Young’s modulus determined by Dynamic Mechanical Analysis. The compressive Young’s modulus increased with an increasing density of the wet mass during the shear experiments and decreased with an increase in the angle of internal friction. The results suggest that stiffer granules were a result of densification, not the strength of liquid bridge bond formation.
Keywords: Split-bed shear tester; Granule growth mechanisms; Wet granulation;

Interactions of amphiphilic calix[4]arene-based Solid Lipid Nanoparticles with bovine serum albumin by Jérôme Gualbert; Patrick Shahgaldian; Anthony W Coleman (69-73).
The interaction of Solid Lipid Nanoparticles (SLN) based on amphiphilic calix[4]arene with one of the major circulatory protein, serum albumin, has been investigated by Photon Correlation Spectroscopy (PCS) and Atomic Force Microscopy (AFM). The carrier systems have shown the ability to interact with bovine serum albumin (BSA), which forms a capping layer up to 17 nm in depth. AFM imaging revealed that the SLNs are protected by this layer against flattening on surfaces.
Keywords: Solid Lipid Nanoparticles; Calixarene; Bovine serum albumin (BSA); Photon Correlation Spectroscopy (PCS); Atomic Force Microscopy (AFM);

Under ultrasound exposure, the stability of plasmid DNA protected by polymer-based gene delivery system is an important factor for achieving optimal transfection into cells. We have evaluated the effectiveness of various polymer-based plasmid DNA delivery systems, which are interactive polymers and cationic polymers, to avoid shear degradation induced by ultrasound exposure. Alternatively, it is shown that sonication of plasmid DNA for exposure time as low as 10 s resulted in total DNA fragmentation and the loss of transfection potency in NIH/3T3 cells. Among these polymer-based plasmid DNA delivery systems, only cationic polymers had the ability to provide the protection of plasmid DNA from ultrasonic degradation as indicated by the reservation in supercoiled circular (SC) and open circular (OC) forms of plasmid DNA on the agarose gel electrophoresis. The DNA stability protected by cationic polymers decreased after ultrasound exposure in 1 M sodium chloride solution. Also, higher molecular weight of cationic polymers and sufficient cationic polymer/DNA weight ratios are essential to prevent DNA from degradation under ultrasound exposure in aqueous or salt solution. These results suggest that the protective mechanism by cationic polymers is due to the attractive bonding between cationic polymer and negative plasmid DNA. Whereas, DNA condensation alone provoked by the addition of polyethylene glycols was not sufficient to resist the DNA fragmentation induced by ultrasound exposure.
Keywords: DNA; Stability; Ultrasound; Polymer-based plasmid DNA delivery systems; Gel electrophoresis;

Use of γ-inulin/liposomes/Vitamin E adjuvant combination in contraceptive vaccines by P Fuentes; P.D Cooper; R Barnadas; M Sabés; C Osterhoff; P Martı́nez (85-95).
The adjuvanticity of two gamma inulin/liposomes/Vitamin E combinations was evaluated in the mouse, in contraceptive vaccines with sperm protein extracts or a synthetic HE2 peptide (“Human Epididymis gene product”; residues 15–28) as antigen. The HE2 peptide was not conjugated to a protein carrier, to ensure that the antibodies elicited were specific against the HE2 peptide. The adjuvant combinations were designed to increase adjuvanticity, as their components have complementary mechanisms, and their performance was compared to Freund’s adjuvant. Antibody production against native sperm structures was determined in sera by ELISA immunoassay and immunohistology. Toxicity of adjuvants was determined by histopathological study and treated mice were monitored for signs of pain or distress. Our results show that the gamma inulin (1–2 μm particle size)/liposomes/Vitamin E combination, with sperm protein extracts, is better than Freund’s adjuvant because it elicits good antibody titres without any toxicity. When the synthetic HE2 peptide is used as antigen, the gamma inulin (1–2 μm particle size)/liposomes/Vitamin E combination is less effective than Freund’s adjuvant; nevertheless, the anti-HE2 antibodies elicited are highly specific and recognize native structures in sperm.
Keywords: HE2 sperm antigen; Contraception; Gamma inulin; Liposomes; Vitamin E; Freund’s adjuvant;

CDRI-85/92, a new antiulcer drug, acts as a proton pump inhibitor arresting the secretion of acid in the stomach. The absorption kinetics of CDRI-85/92 was evaluated in situ using rat intestinal recirculation perfusion method. The experiment was conducted at pH 2.6 and 7.4 representing the acidic and the mild alkaline environment, which the drug experiences through the GIT during oral treatment. The rate of absorption was the same (0.12 h−1) at pH 2.6 and 7.4, thus suggesting equal absorption profile of the CDRI-85/92 throughout the GIT irrespective of the pH. Equal rates of absorption can also be correlated with the presence of acidic and basic groups in the structure of CDRI-85/92.Protein binding studies of CDRI-85/92 using ultrafiltration were conducted in vitro and in vivo. Protein binding was found to be in the range of 31.49–32.91% both in in vitro and in vivo (employing 5-min post dose samples of rat serum after 20 mg kg−1 i.v. treatment of CDRI-85/92). The binding was found to be linear in the concentration range of 156.25–2000 ng ml−1 (r 2>0.99).
Keywords: Reversed phase chromatography; CDRI-85/92; Anti-ulcer agent; In situ absorption; Protein binding;

Dual-labeled galactosylated chitosan-graft-poly(ethylene glycol) (PEG) (GCP)/DNA complexes were prepared and their hepatocyte-specific delivery and cellular distribution were investigated by confocal laser scanning microscopy (CLSM). The complexes were transfected into hepatocyte through specific interaction of galactose moiety of the GCP and asialoglycoprotein receptors (ASGPR) of the hepatocytes. The GCP/DNA complexes taken up by the hepatocytes were rapidly released into the cytoplasm, but nuclear trafficking of the released complexes was slow and rate-limiting process. The more efficient transfection of the complex occurred in the human-derived HepG2 cells than in primary hepatocytes.
Keywords: Chitosan; Galactose; PEG; Nuclear trafficking; Confocal laser scanning microscopy; Hepatocyte;

A PEGylated dendritic nanoparticulate carrier of fluorouracil by D. Bhadra; S. Bhadra; S. Jain; N.K. Jain (111-124).
The present study was aimed at developing and exploring the use of uncoated and PEGylated newer PAMAM dendrimers for delivery of anti-cancer drug 5-fluorouracil. For this study, successive Michael addition and exhaustive amidation reactions were used to synthesize 4.0G PAMAM dendrimers, using ethylenediamine as core and methylmethacrylate as propagating agent. The dendrimer was PEGylated using N-hydroxysuccinimide-activated carboxymethyl MPEG-5000. IR and NMR data proved the synthesis. Various physicochemical parameters, SEM, TEM, λ max values, hemolytic toxicity, drug entrapment, drug release and blood-level studies of both PEGylated and non-PEGylated systems were determined and compared. The PEGylation of the systems was found to have increased their drug-loading capacity, reduced their drug release rate and hemolytic toxicity. TEM study revealed surface properties of the systems. Stability studies had shown its stability at room temperature in dark. The systems were found suitable for prolonged delivery of an anti-cancer drug by in vitro and blood-level studies in albino rats, without producing any significant hematological disturbances. PEGylation has been found to be suitable for modification of PAMAM dendrimers for reduction of drug leakage and hemolytic toxicity. This, in turn, could improve drug-loading capacity and stabilize such systems in body. The study suggests use of such PEGylated dendrimeric systems as nanoparticulate depot type of system for drug administration.
Keywords: Poly(ethylene glycol); Polyamidoamine; Nanoparticle; Dendrimers; Fluorouracil; Anti-cancer;

A novel system for the delivery of drugs to bacterial biofilms has been developed. The system is based on the use of anionic and cationic liposomes as drug carriers adsorbed on the surface of zinc citrate particles. The adsorption process results in the formation of solid supported vesicles (SSVs) which aids the stabilisation of the liposomes. Anionic liposomes have been prepared by incorporation of phosphatidylinositol (PI) into dipalmitoylphosphatidylcholine (DPPC) liposomes and cationic liposomes have been prepared by incorporation of dioctadecyldimethylammonium bromide (DDAB) into DPPC plus cholesterol liposomes. The liposomes were adsorbed onto zinc citrate particle and targeted to immobilised biofilms of the oral bacterium Streptococcus oralis. The liposomes were used to carry the bactericides, Triclosan®, a lipid-soluble agent, and the aqueous-soluble penicillin-G, and their ability to inhibit bacterial growth from immobilised biofilms was accessed. Zinc citrate is itself a bactericide and is used in the formulation of toothpastes. The SSVs carrying the drugs have therapeutic properties. To trace the origin of these properties, each component of the SSV was investigated alone and in combination in binary systems. Some combinations showed synergistic (or additive) antibacterial effects while others showed regressive effects compared with their components.
Keywords: Solid supported liposomes (or vesicles) (SSVs); Zinc citrate particles; Triclosan®; Penicillin-G; Streptococcus oralis biofilms; Antibacterial properties;

The in vitro uptake and release behaviour of cromolyn sodium, ketotifen fumarate, ketorolac tromethamine and dexamethasone sodium phosphate with silicon-containing (lotrafilcon and balafilcon) and p-HEMA-containing (etafilcon, alphafilcon, polymacon, vifilcon and omafilcon) hydrogel contact lenses indicated that both drug and material affected the uptake and release behaviour. Rapid uptake and release (within 50 min) was observed for all drugs except ketotifen fumarate which was more gradual taking approximately 5 h. Furthermore, the maximum uptake differed significantly between drugs and materials. The highest average uptake (7879±684 μg/lens) was cromolyn sodium and the lowest average uptake (67±13 μg/lens) was dexamethasone sodium phosphate. Partial release of the drug taken up was observed for all drugs except dexamethasone sodium phosphate where no release was detected. Sustained release was demonstrated only by ketotifen fumarate. Drug uptake/release appeared to be a function of lens material ionicity, water and silicon content. The silicon-containing materials released less drug than the p-HEMA-containing materials. The lotrafilcon material demonstrated less interactions with the drugs than the balafilcon material which can be explained by their different bulk composition and surface treatment.
Keywords: Silicon; p-HEMA; Hydrogel; Contact lens; Drug; Drug delivery;

Preparation of solid lipid nanoparticles by a solvent emulsification–diffusion technique by Michele Trotta; Francesca Debernardi; Otto Caputo (153-160).
A preparation method for nanoparticles based on the emulsification of a butyl lactate or benzyl alcohol solution of a solid lipid in an aqueous solution of different emulsifiers, followed by dilution of the emulsion with water, was used to prepare glyceryl monostearate nanodispersions with narrow size distribution. To increase the lipid load the process was conducted at 47±2 °C and in order to reach submicron size a high-shear homogenizer was used. Particle size of the solid lipid nanoparticles (SLN) was affected by using different emulsifiers and different lipid loads. By using lecithin and taurodeoxycholic acid sodium salt, on increasing the GMS percentage from 2.5 to 10% an increase of the mean diameter from 205 to 695 nm and from 320 to 368 nm was observed for the SLN prepared using benzyl alcohol and butyl lactate, respectively. Transmission electron micrographs of SLN reveal nanospheres with a smooth surface.
Keywords: Solid lipid nanoparticles; Glyceryl monostearate; Solvent diffusion;

The 16HBE14o− cell line, which forms polarised cell layers in vitro, provides a promising opportunity to develop a convenient epithelial cell culture model in which respiratory drug transport can be evaluated in vitro. This study investigated the effect of cell seeding density, collagen substratum and time in culture on the development of barrier properties in this cell line, after which the permeability of the 16HBE14o− cell layers to a series of solutes was studied. Seeding cells at a density of 2.5×105 cells per cm2 on a monofibrillar Vitrogen-100 collagen substratum, followed by culture at an air–liquid interface for 6 days resulted in cell layers with a transepithelial electrical resistance (TER) of 247±47 Ω cm2 and an apparent permeability coefficient of 2.5×10−6  cm s−1 for mannitol. The permeability of the 16HBE14o− cells to hydrophilic molecules (log  P<1.9) was of an order of magnitude greater than that of typical alveolar cell cultures, possibly reflecting barrier properties more representative of the airways. More lipophilic drugs showed higher permeabilities indicating a sigmoidal relationship between permeability and lipophilicity similar to that observed for solute transport across primary cultured epithelial cell layers. These results indicate that under appropriate culture conditions, 16HBE14o− cell layers provide a discriminatory barrier to solute transport.
Keywords: Biopharmaceutics; Cell culture; Air-interface; Permeability; Drug absorption; Lung;

Optimization of the preparation process for human serum albumin (HSA) nanoparticles by K. Langer; S. Balthasar; V. Vogel; N. Dinauer; H. von Briesen; D. Schubert (169-180).
Nanoparticles prepared by desolvation and subsequent crosslinking of human serum albumin (HSA) represent promising carriers for drug delivery. Particle size is a crucial parameter, in particular for the in vivo behaviour of nanoparticles after intravenous injection. The objective of the present study is the development of a desolvation procedure for the preparation of HSA-based nanoparticles under the aspect of a controllable particle size between 100 and 300 nm in combination with a narrow size distribution. A pump-controlled preparation method was established which enabled particle preparation under defined conditions. Several factors of the preparation process, such as the rate of addition of the desolvating agent, the pH value and the ionic composition of the HSA solution, the protein concentration, and the conditions of particle purification were evaluated. The pH value of the HSA solution prior to the desolvation procedure was identified as the major factor determining particle size. Varying this parameter, (mean) particle diameters could be adjusted between 150 and 280 nm, higher pH values leading to smaller nanoparticles. Washing the particles by differential centrifugation led to significantly narrower size distributions. The reproducibility of the particle size and particle size distribution under the proposed preparation conditions was demonstrated by sedimentation velocity analysis in the analytical ultracentrifuge and the cellular uptake of those nanoparticles was studied by confocal microscope imaging and FACS analysis. The stability of the resulting nanoparticles was evaluated by pH and buffer titration experiments. Only pH values distinctly outside the isoelectric pH range of HSA and low salt concentrations were able to prevent nanoparticle agglomeration.
Keywords: Nanoparticles; Human serum albumin (HSA); Particle size distribution; Cellular uptake;

The effect of formulation variables on the stability of nebulized aviscumine by Hartwig Steckel; Fadi Eskandar; Klaus Witthohn (181-194).
The pulmonary drug delivery of proteins present an alternative to parenteral and oral administration. Nebulization of aqueous protein solutions is an ideal method for pulmonary application of therapeutic proteins considering the difficulties of their formulation as MDIs or DPIs. This research presents the effect of variable excipients on the stability of freeze-dried aviscumine after reconstitution and nebulization. Formulations containing different lyoprotectants have been lyophilized and reconstituted with isotonic salt solution. The loss of aviscumine activity in the nebulizer reservoir and after nebulization with a PariBoy® air-jet nebulizer, a Multisonic® ultrasonic nebulizer and a Systam® ultrasonic nebulizer was determined by a binding assay. The effect of variable lyoprotectants such as 8% (w/v) Dextran T1, HES130, HES450, HP-β-CD and 6% (w/v) HES450 plus 2% (w/v) mannitol on the stability of aviscumine to air-jet and ultrasonic nebulization has been evaluated. Only 50% of aviscumine activity was retained after 20 min nebulization, where 8% (w/v) HES450 was shown to be the best stabilizer. Stabilization of aviscumine by the addition of variable surfactants as 0.01 and 0.1% (w/v) Poloxamer 188, 0.03 and 0.1% (w/v) PEG 8000, 0.03 and 0.1% (w/v) Solutol HS15 and 0.03 and 0.1% (w/v) octanoyl-N-methyl-glucamide to the reconstitution solution has also been studied. By the addition of 0.03% (w/v) octanoyl-N-methyl-glucamide, 70% of the activity was retained after 20 min nebulization.
Keywords: Nebulizer; Octanoyl-N-methyl-glucamide; Stability of proteins; Lyoprotectants; Aviscumine;

Liver specific polymeric nanoparticles were designed and synthesized from biotinylated poly(ethylene glycol) conjugated with lactobionic acid containing a galactose moiety (abbreviated as BEL). Synthesized BEL conjugate was identified by Fourier transform-infrared (FT-IR) and 1 H -nuclear magnetic resonance (NMR) spectroscopy. The fluorescence spectroscopy data showed that BEL conjugate was self-assembled in water to form core-shell structure nanoparticles, and the critical association concentration (CAC) value was estimated as 0.028 g/l. From the transmission electron microscope (TEM) observation, the BEL nanoparticles were spherically shaped and ranged in size between 30 and 60 nm. The particle size distribution was measured by photon correlation spectroscopy (PCS), and the result was 41.2±11.7 nm. Anti-cancer drug all-trans-retinoic acid (ATRA) was loaded into the BEL nanoparticles for evaluating its efficacy as a drug delivery carrier. The crystallinities of ATRA and ATRA-loaded nanoparticles were examined by X-ray diffraction (XRD) patterns. The ATRA release kinetics from the BEL nanoparticles showed a pseudo zero-order pattern during 1-month period.
Keywords: Polymeric nanoparticle; Biotin; Poly(ethylene glycol); Lactobionic acid; All-trans-retinoic acid;

The direct solidification process of a melt into a particulate solid is studied to achieve the desired size and shape of the product and to predict the required crystallization time. For an example, a Bisacodyl melt is chosen. The role of the contact angle of the droplet is investigated as a function of Reynolds number, degree of subcooling and characteristic of used cooled surface (substrate). The static contact angle increases with increasing degree of surface roughness of the substrate. The contact angle, however, decreases with increasing Reynolds number and degree of subcooling. The phenomena of spreading and rebouncing of droplets are used in the discussion of the deformation process. By the model of Madejski the degree of deformation is found to be proportional to the Reynolds number to the power of 0.2. On the basis of a simple droplet solidification model and experimental data, a numerical study is presented. The equations allow to estimate the normalized deformation and crystallization times, which are proportional to the Reynolds number to the power of 1.23 and help the design of solidification processes.
Keywords: Contact angle; Degree of deformation; Crystallization time; Direct solidification process;

The migration of the antioxidant additives pentaerythrityl tetrakis(3,5-di-tert-butyl-4-hydroxyphenyl)propionate (Irganox 1010) and tris(2,4-di-tert-butylphenyl)phosphite (Irgafos 168) from polyolefinic packaging into oily vehicles was investigated. The polyolefins included in the study were from the following classes: isotactic polypropylene homopolymer (PP), ethylene-co-propylene random copolymer (RACO), ethylene-propylene heterophasic copolymer and ethylene-propylene amorphous copolymer blend (EP) and high-density polyethylene (HDPE). Each polymer was additioned with Irganox 1010 (0.15%, w/w) and Irgafos 168 (0.15%, w/w) and processed into blown bottles. To study the antioxidant release process, plastic sheets were cut from the bottles and dipped for various time intervals into a mixture of five oils (caprylic/capric triglyceride, cyclomethicone, dicaprylyl ether, isohexadecane and C12–15 alkyl benzoate) representative of lipophilic excipients used in pharmaceutical and cosmetic formulations. After exposure to the oil medium, the non-migrated Irganox 1010 and Irgafos 168 were recovered from the polymeric matrices using microwave-assisted extraction with ethyl acetate-hexane and assayed by HPLC. The leaching of the two antioxidants varied remarkably depending on the polyolefin crystallinity and structure. The amount of Irganox 1010 transferred into the contact medium at 25 °C decreased in the order EP>RACO>PP>HDPE. The same polyolefin ranking was observed in the case of Irgafos 168, except for PP and HDPE which exhibited similar depletion of this additive. Migration of Irgafos 168 was greater than that of Irganox 1010 and the release of both antioxidants increased at higher temperature (50 °C). The obtained data are useful for the selection of polyolefinic matrices as raw-materials for the production of pharmaceutical and cosmetic containers.
Keywords: Polyolefins; Antioxidant additives; Containers; Migration; Oily vehicles;

The powder flow and compact mechanical properties of sucrose and three high-intensity sweeteners used in chewable tablets by Matthew P Mullarney; Bruno C Hancock; Glenn T Carlson; Dauda D Ladipo; Beth A Langdon (227-236).
The physical, flow, and mechanical properties of four common pharmaceutical sweeteners were measured to assess their relative manufacturability in solid dosage formulations. Sucrose, acesulfame potassium (Sunett®), saccharin sodium, and aspartame were evaluated to determine significant differences in particle shape, size distribution, and true density. Powder flow and cohesivity as well as compact mechanical properties such as ductility, elasticity, and tensile strength were measured and found to be noticeably different. Among these sweeteners, sucrose and acesulfame potassium demonstrated excellent flowability and marginal mechanical property performance relative to over 100 commonly used pharmaceutical excipients evaluated in the authors’ laboratory. Saccharin sodium and aspartame demonstrated poor flowability and superior compact strength relative to sucrose and acesulfame, despite their noticeably higher brittleness. These data suggest that careful selection of an appropriate sweetener is warranted in obtaining desirable process and tableting robustness, particularly if sweetener loading is high. Detailed descriptions of each material property and recommendations for sweetener selection in formulation development are included.
Keywords: Pharmaceutical sweeteners; Tableting indices; Flow; Mechanical; Tensile strength; Indentation hardness;

Binding of primaquine to epidermal membranes and keratin by C.M Heard; B.V Monk; A.J Modley (237-244).
The localisation of primaquine was studied within epidermal membranes following the application of a topical dose. A depth profile was constructed by tape-stripping human epidermis following permeation of a 70 mg ml−1 solution of primaquine in Miglyol 840. Comparative binding studies of primaquine were carried out on isolated human stratum corneum and whole epidermis, using normal and delipidised tissue. An additional study was undertaken using bovine keratin powder as a model of human keratin. The depth profile showed that primaquine decreased with depth and decreasing keratin content, and the total primaquine recovered (15.5 mg cm−2) was 300× the amount of extractable lipid. Binding to delipidised skin was saturable, whereas binding to normal skin was unsaturable, reflecting the high miscibility of drug in the lipid domains as opposed to a finite, but large number of binding sites on the corneocytes. Binding was greater for stratum corneum than stratum corneum plus viable epidermis, probably due to greater accessibility of corneocytes keratin. Binding was dose dependent, although binding to delipidised skin was far greater than to normal skin, demonstrating that primaquine had an affinity for lipoidal regions and an even higher affinity for the proteinaceous domains of the stratum corneum. This was supported by high saturable levels of primaquine binding to bovine horn keratin. The results indicated extensive binding to corneocyte keratin has a significant effect on reservoir formation and the permeability of primaquine across human skin. It is speculated that the large amount of keratin presented at the skin surface may be an evolutionary protective process for the sequestration of ingressing molecules.
Keywords: Keratin; Skin; Stratum corneum; Primaquine;

Prediction of human intestinal permeability using artificial membrane permeability by Kiyohiko Sugano; Yoshiaki Nabuchi; Minoru Machida; Yoshinori Aso (245-251).
The purpose of the present study was to examine a correlation between the human intestinal permeability (P eff) and the bio-mimetic artificial membrane permeability corrected by the paracellular pathway model based on the Renkin function (P PAMPA-PP-RF) and to construct a prediction scheme. The effect of the unstirred water layer was incorporated to the prediction scheme. Eighteen P eff values of passively absorbed drugs were employed for the analysis. The correlation coefficient (CC) between the predicted and observed log  P eff was 0.91. P eff of furosemide, hydrochlorothiazide and creatinine were underestimated by P PAMPA-PP-RF. When these compounds were excluded, CC was 0.97. Without the correction for the paracellular pathway, P eff of small, cationic and hydrophilic compounds were underestimated. Therefore, P PAMPA-PP-RF was found to be an adequate in vitro surrogate for P eff.
Keywords: Permeability; Artificial membrane; Paracellular; Unstirred water layer; PAMPA;

Among different technological variables that influence drug release from hydrophilic matrices, different proportions of the polymer and a water-soluble excipient have been used to control the drug release properties. These variables were used to modify the drug release rate and to examine its effect on the mechanism controlling the process. Tablets of the model drug 4-aminopyridine (4-AP) were prepared varying the matrix proportion of hydroxypropyl methylcellulose (HPMC) and citric acid (CA). The matrices release behavior (USP apparatus 2, paddle, at 50 rpm) was examined using 0.1N HCl and 0.2 M phosphate buffer as dissolution media. Dissolution curves were described by M t/M inf=kt n , applied separately for each dissolution medium. The increase of the HPMC matrix content reduced the release rate of the drug. The release mechanism showed a linear trend toward higher n values with a continuous reduction of drug release. The addition of increasing proportions of CA produced the opposite. An increasing drug release rate produced logarithmic decreasing n values. The results demonstrate, as a general rule, that every restriction of the drug release rate is associated with increasing values of the mechanism-indicating exponent n. This relationship means a logarithmic movement away from a release mechanism controlled by diffusion toward a mechanism controlled by relaxation, erosion and dissolution of the polymeric matrix as the drug release rate is restricted. These results are attributed to an increasing hydration and dissolution of the polymeric matrix, as the drug release is subject to limitation.
Keywords: Sustained release; 4-Aminopyridine; Dissolution restriction; HPMC; Release mechanism;

The development of isothermal microcalorimetry to a study of the kinetic and thermodynamics of polymorphic transitions in seratrodast ((±)-7-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-7-phenylheptanoic acid) Form II is reported. Sieved samples of Form II were allowed to convert to Form I, in a reaction vessel of an isothermal microcalorimeter, under 13, 31, 63 and 93% relative humidity (RH) between 48 and 65 °C. The power (Φ, in Watts) versus time curves from the microcalorimeter were integrated into the heat output (q, in Joules) versus time curves to yield fractional extent of Form I converted versus time curves. The change in enthalpy (−5.70 kJ mol−1) agreed very closely with that obtained by differential scanning calorimetry and solution calorimetry, which indicated that the power measured by the microcalorimeter was due only to the Form II-to-Form I transition. Application of the theoretical kinetic method [J. Am. Ceram. Soc. 55 (1972) 74] revealed that the transition took place via a two-dimensional growth of nuclei mechanism at all the studied relative humidities and temperatures. The rate constant increased with increasing RH and temperature, and with decreasing the particle size of sample. The activation energies obtained from Arrhenius plots were 292, 290, 280 and 284 kJ mol−1, and the extrapolated rate constants at 25 °C were also 3.01×10−10, 3.11×10−10, 9.65×10−10 and 3.84×10−9  s−1 for 13, 31, 63 and 93% RH, respectively.
Keywords: Isothermal microcalorimetry; Kinetics; Thermodynamics; Polymorphic transition; Seratrodast;

Investigations into the stabilisation of drugs by sugar glasses: II by H.J.C Eriksson; W.R Verweij; K Poelstra; W.L.J Hinrichs; G.J de Jong; G.W Somsen; H.W Frijlink (273-281).
In this study the possibility to deliver the acid-sensitive enzyme alkaline phosphatase (AP) from calf intestine (CIAP) to the intestinal system by oral administration was investigated. Tablets were prepared and in vitro evaluated. Final proof of concept studies were performed in rats. This acid labile enzyme is potentially useful in the treatment of sepsis, a serious condition during which endotoxins can migrate into the blood stream. The CIAP was freeze-dried with inulin and subsequently compacted into round biconvex tablets with a diameter of 4 mm and a weight of 25–30 mg per tablet. The tablets were coated with an enteric coating in order to ensure their survival in the stomach.In vitro evaluation of tablets containing alkaline phosphatase from bovine intestine (BIAP) was the first step in the development. It was found that tablets without enteric coating dissolved rapidly in 0.10 M HCl with total loss of enzymatic activity of the alkaline phosphatase. Tablets that were coated were stable for at least 2 h in 0.10 M HCl, but dissolved rapidly when the pH was increased to 6.8. Furthermore, it was shown that the enzymatic activity of the released BIAP was fully preserved.The in vivo test clearly showed that the oral administration of enteric coated tablets resulted in the release of enzymatically active CIAP in the intestinal lumen of rats. The location of the enhanced enzymatic activity of AP in the intestines varied with the time that had passed between the administration of the tablets and the sacrificing of the rats. Also, the level of enzymatic activity increased with an increasing number of tablets that were administered.
Keywords: Sugar glass; Proteins; Oral administration;

The influence of carrier and drug morphology on drug delivery from dry powder formulations by Hassan Larhrib; Gary Peter Martin; Christopher Marriott; David Prime (283-296).
Lactose was crystallised either from neutralised Carbopol 934 gel or from water–ethanol solution without stirring, with a view to obtaining lactose α-monohydrate of favourable shape and smooth surface, suitable for use as carriers in formulations for dry powder inhalers (DPIs).Crystallisation of salbutamol sulphate was carried out in the presence of water, lecithin and ethanol to form salbutamol crystals with defined shape and smooth surface. The crystals formed were needle-shaped, with a length of less than 6 μm and a width between 0.5 and 1 μm.DSC and TGA showed that lactose crystals produced from Carbopol gel or from water–ethanol solution existed as α-lactose monohydrate. The DSC thermograms of micronised and crystallised salbutamol sulphate showed two similar endothermic transitions at 200 and 290 °C, respectively. The first transition was initially thought to correspond to the melting of salbutamol sulphate. However, the shape of the particles as observed by optical microscopy was not altered after heating the sample to 250 °C, suggesting that no transition from solid to liquid state occurred at 200 °C. This was confirmed by observations made using hot stage microscopy. The two endothermic transitions are suggested to correspond to the decomposition of the salbutamol sulphate molecule.The elongation ratio of the commercial lactose crystals, lactose crystallised from Carbopol and from water–ethanol were 1.69±0.05, 2.01±0.13 and 6.25±0.17, respectively. As the elongation ratio increased the flow properties of the carrier were affected detrimentally and this consequently reduced the content uniformity of salbutamol sulphate and drug emission from the inhaler device. Whereas, increasing the elongation ratio of the carrier or drug improved the deposition profiles of salbutamol sulphate, suggesting that the more elongated particles would be more aerodynamic and favour deep lung penetration.
Keywords: Dry powder inhalers; Lactose; Salbutamol sulphate; Decomposition; Crystallisation; Elongation ratio; Surface smoothness; Flow properties; Fine particle fraction;

This study investigated the site-specific absorption of oseltamivir using targeted delivery and gamma scintigraphy. On four separate occasions, nine healthy male subjects each received a single 150 mg of oseltamivir administered via the Enterion™ capsule to the stomach, proximal small bowel, distal small bowel and the ascending colon. Pharmacokinetic parameters of oseltamivir and its carboxylate metabolite show that absorption was similar in the proximal and distal small bowel compared to stomach delivery, but reduced from the ascending colon, demonstrating that absorption-rate limited disposition occurred only for the ascending colon. The metabolite-to-parent ratios were minimally reduced. The results support the feasibility of modified-release formulation development whilst confirming the high and consistent oral bioavailability of oseltamivir.
Keywords: Oseltamivir; Pharmacokinetics; Prodrug; Absorption; Controlled delivery; Gamma scintigraphy;

Note on the measurement of flowability according to the European Pharmacopoeia by Andrea Schüssele; Annette Bauer-Brandl (301-304).
Flowabilities of six commercially available, direct compression excipients, Emcompress™, Fujicalin™, Starch 1500™, Avicel™ PH-102, Tablettose™ 80, and Tablettose™ 100 were examined according to the technical procedure described in the current European Pharmacopoeia, and with a Sotax Flow Tester.Results revealed unfavourable properties for Fujicalin compared to other substances. Fujicalin, however, appeared macroscopically to be of extremely pronounced free-flowing properties. This contradiction was studied in more detail, proposing to express powder flow in terms of volume per time unit rather than mass per time unit (volume-flowability).
Keywords: Flowability; Bulk powder properties; Volume-flowability; Carr index; Hausner ratio; Direct compression excipients; Uniformity of mass of tablets;

A water-in-oil microemulsion based on ingredients acceptable for oral administration of substances to human was investigated. The microemulsion was studied with and without biologically active ingredients by dynamic light scattering, turbidity, diffusion-NMR, and conductivity. Also, a technology based on mixing of appropriate microemulsions was investigated as a means to produce particles of nanometer size of a heparin/chitosan complex.It was found that the microemulsion existed up to about 15% (w/w) of water with or without active ingredients. Formation of nanosized heparin/chitosan particles inside the water droplets was confirmed. It was concluded that the investigated microemulsion could be an interesting system for the oral administration of a heparin/chitosan complex.
Keywords: Microemulsions; Heparin; Chitosan; Pharmaceutical; Drug delivery;

Erratum to “Physicochemical characterization and percutaneous delivery of 2,3,5,6-tetramethylpyrazine” by Xiaohong Qi; Chrisita Ackermann; Duxin Sun; Minli Sheng; Huimin Hou (311).

NOTICEBOARD (313-315).