International Journal of Pharmaceutics (v.249, #1-2)

Title page (iii).

The hypoglycemic effect of orally given insulin was studied on rabbits, using different bile salts as absorption promoters, in two different carriers to form an artificial chyloform system ready to be absorbed by intestinal mucosa. The rank order of enhancement by bile salts in the presence of 1% ethanol was deoxycholate>cholate>glycocholate>glycodeoxycholate>taurodeoxycholate>no bile salts. The dose response studies with increased insulin loaded in the chyle showed a greater corresponding hypoglycemic effect with the system of cholate–palmitic-α-tocopherol dispersions than the cholate–palmitic acid dispersions. A more effective hypoglycemic effect was achieved using lower doses of the deoxycholate–palmitate–tocopherol–chyle dispersions.
Keywords: Insulin; Fatty acids; Tocopherol; Bile salts; Chyles;

Floating (F) microcapsules containing melatonin (MT) were prepared by the ionic interaction of chitosan and a negatively charged surfactant, sodium dioctyl sulfosuccinate (DOS). The DOS/chitosan complex formation was confirmed employing infrared spectroscopy, differential scanning calorimetry (DSC), solubility and X-ray diffraction analysis. The characteristics of the F microcapsules generated compared with the conventional non-floating (NF) microspheres manufactured from chitosan and sodium tripolyphosphate (TPP) were also investigated. The effect of various factors (crosslinking time, DOS and chitosan concentrations, as well as drug/polymer ratio) on microcapsule properties were evaluated. The use of DOS solution in coagulation of chitosan produced well-formed microcapsules with round hollow core and 31.2–59.74% incorporation efficiencies. Chitosan concentration and drug/polymer ratio had a remarkable effect on drug entrapment in DOS/chitosan microcapsules. The dissolution profiles of most of microcapsules showed near zero order kinetics in simulated gastric fluid (S.G.F: pH 1.2). Moreover, release of the drug from these microcapsules was greatly retarded with release lasting for several hours (t 50% (S.G.F.): 1.75–6.7 h, depending on processing factors), compared with NF microspheres where drug release was almost instant. Most of the hollow microcapsules developed tended to float over simulated biofluids for more than 12 h. Swelling studies conducted on various drug-free formulations, clearly indicated that DOS/chitosan microcapsules showed less swelling and no dissolution in S.G.F. for more than 3 days, whereas, TPP/chitosan microspheres were markedly swollen and lost their integrity in S.G.F. within 5 h. Therefore, data obtained suggest that the F hollow microcapsules produced would be an interesting gastroretentive controlled-release delivery system for drugs.
Keywords: Floating chitosan microcapsules; Non-floating chitosan microspheres; Zero order release; Melatonin;

The aim of the present study was to develop a single unit, site-specific drug formulation allowing targeted drug release in the colon. Tablets were prepared using polysaccharides or synthetic polymer as binders. These included xanthan gum, guar gum, chitosan and Eudragit E. Indomethacin was used as a model drug. The prepared tablets were enteric coated with Eudragit-L 100 to give protection in the stomach. The coated tablets were tested in-vitro for their suitability as colon specific drug delivery systems. The drug release studies were carried out in simulated stomach environment (pH 1.2) for 2 h followed by small intestinal environment at pH 6.8. The dissolution data obtained from tablets demonstrates that the dissolution rate of the tablet is dependent upon the type and concentration of polysaccharide/polymer used as binder. The results demonstrate that enteric coated tablets containing 3% chitosan as a binder, showed only 12.5% drug release in the first 5 h, which is the usual upper gastrointestinal transit time, whereas, tablets prepared using guar gum as binder, were unable to protect drug release under similar conditions. Preparations with xanthan gum as a binder formed time-dependent release formulations. When used in a concentration of 5.92% in the tablets, 28% drug release was observed in the usual upper gastrointestinal tract conditions. It was also found that enteric coated preparation formulated with 8.88% of Eudragit E as binder could be used to carry water insoluble drug molecules to the colon especially in IBD. The above study shows that chitosan could be successfully used as a binder, for colon targeting of water insoluble drugs in preference to guar gum when used in the same concentration. Additionally, formulations developed with chitosan and Eudragit E would be highly site specific since drug release would be at a retarded rate till microbial degradation or polymer solubilization takes place in the colon.
Keywords: Colon specific drug delivery; Colon targeting; Chitosan; Guar gum; Xanthan gum; Eudragit E;

Studies on lactulose formulations for colon-specific drug delivery by Masataka Katsuma; Shunsuke Watanabe; Hitoshi Kawai; Shigeo Takemura; Yoshinori Masuda; Muneo Fukui (33-43).
A novel, colon-targeted delivery system (CODES™), which uses lactulose, was investigated in this study. Lactulose is not absorbed in the upper GI tract, but degraded to organic acids by enterobacteria in the lower gastrointestinal tract, especially the colon. A CODES™ consists of three components: a core containing lactulose and the drug, an inner acid-soluble material layer, and an outer layer of an enterosoluble material. When a CODES™ containing a pigment was introduced into the rat cecum directly after shaking in JP 2nd fluid for 3 h, pigment release was observed 1 h after introduction. A CODES™ containing 5-aminosalicylic acid (5-ASA) was orally administered to fasting and fed dogs to evaluate its pharmacokinetic profiles. 5-ASA was first detected in plasma after 3 h, which is the reported colon arrival time for indigestible solids, after dosing to fasting dogs. The T max in fed dogs was delayed by 9 h when compared to fasting dogs. This corresponds to the gastric emptying time. However, the C max and AUC under fed conditions were almost as same as those under fasting conditions. The results of this study show that lactulose can act as a trigger for drug release in the colon, utilizing the action of enterobacteria.
Keywords: Oral drug delivery; Colon targeting; Lactulose; Enterobacteria;

Solid dispersion literature, describing the mechanism of dissolution of drug–polyethylene glycol dispersions, still shows some gaps; (A) only few studies include experiments evaluating solid solution formation and the particle size of the drug in the dispersion particles, two factors that can have a profound effect on the dissolution. (B) Solid dispersion preparation involves a recrystallisation process (which is known to be highly sensitive to the recrystallisation conditions) of polyethylene glycol and possibly also of the drug. Therefore, it is of extreme importance that all experiments are performed on dispersion aliquots, which can be believed to be physico-chemical identical. This is not always the case. (C) Polyethylene glycol 6000 (PEG6000) crystallises forming lamellae with chains either fully extended or folded once or twice depending on the crystallisation conditions. Recently, a high resolution differential scanning calorimetry (DSC)-method, capable of evaluating qualitatively and quantitatively the polymorphic behaviour of PEG6000, has been reported. Unraveling the relationship between the polymorphic behavior of PEG6000 in a solid dispersion and the dissolution characteristics of that dispersion, is a real gain to our knowledge of solid dispersions, since this has never been thoroughly investigated. The aim of the present study was to fill up the three above mentioned gaps in solid dispersion literature. Therefore, physical mixtures and solid dispersions were prepared and in order to unravel the relationship between their physico-chemical properties and dissolution characteristics, pure drugs (diazepam, temazepam), polymer (PEG6000), solid dispersions and physical mixtures were characterised by DSC, X-ray powder diffraction (Guinier and Bragg-Brentano method), FT-IR spectroscopy, dissolution and solubility experiments and the particle size of the drug in the dispersion particles was estimated using a newly developed method. Addition of PEG6000 improves the dissolution rate of both drugs. Mechanisms involved are solubilisation and improved wetting of the drug in the polyethylene glycol rich micro-environment formed at the surface of drug crystals after dissolution of the polymer. Formulation of solid dispersions did not further improve the dissolution rate compared with physical mixtures. X-ray spectra show that both drugs are in a highly crystalline state in the solid dispersions, while no significant changes in the lattice spacings of PEG6000 indicate the absence of solid solution formation. IR spectra show the absence of a hydrogen bonding interaction between the benzodiazepines and PEG6000. Furthermore, it was concluded that the reduction of the mean drug particle size by preparing solid dispersions with PEG6000 is limited and that the influence of the polymorphic behavior of PEG6000 (as observed by DSC) on the dissolution was negligible.
Keywords: Solid dispersion; PEG6000; Benzodiazepines; Dissolution; Differential scanning calorimetry; X-ray powder diffraction; FT-IR spectroscopy; Solubility; GPC; Particle size determination;

Investigations into the stabilisation of drugs by sugar glasses: I. Tablets prepared from stabilised alkaline phosphatase by H.J.C Eriksson; W.L.J Hinrichs; B van Veen; G.W Somsen; G.J de Jong; H.W Frijlink (59-70).
The aim of this study was to investigate the formulation of sugar glass stabilised alkaline phosphatase from bovine intestine (BIAP) into tablets. Two major subjects of tablet formulation were investigated. First, the compaction behaviour of the inulin sugar glass was investigated. Secondly, the effect of the compaction process on the physical stability of sugar glass stabilised BIAP was investigated, comparing inulin and trehalose glass. The tabletting properties of freeze-dried inulin without BIAP were studied first. Freeze-dried inulin conditioned at either 20 °C/0% relative humidity (RH) or 20 °C/45% RH was compacted at various pressures. As expected, the yield pressure of the material conditioned at 0% RH was higher (68 MPa) than after conditioning at 45% RH (39 MPa). Tablets made of the material stored at 0% RH showed severe capping tendency, especially at high compaction pressures. In contrast, material conditioned at 45% RH gave tablets without any capping tendency and a friability of less than 1%. Sugar glasses of BIAP and either inulin or trehalose were prepared by freeze-drying (BIAP/sugar 1/19 (w/w)). The material was subsequently compacted. Tablets and powders were stored at 60 °C/0% RH. The activity of the incorporated BIAP was measured at various time intervals. It was found that inulin was by far superior to trehalose as stabiliser of BIAP in tablets. The poor stabilising capacities of trehalose after compaction are explained by crystallisation of trehalose induced by the compaction process and moisture in the material. The results clearly show that inulin is an excellent stabiliser for BIAP. The tabletting properties are adequate, showing sufficient tablet strengths and low friability. Furthermore, the good (physical) stability of inulin glass with respect to exposure to high relative humidities makes it practical to work with.
Keywords: Stabilisation; Proteins; Sugar glass; Tablets; Compaction; Crystallisation;

Preparation and dissolution characteristics of griseofulvin solid dispersions with saccharides by Masataka Saito; Takashi Ugajin; Yasuo Nozawa; Yasuyuki Sadzuka; Atsuo Miyagishima; Takashi Sonobe (71-79).
To improve the solubility of poorly water-soluble drugs, we studied physical characteristics of griseofulvin (GF) solid dispersions with saccharides as the dispersion carrier using a roll mixing method. In all carriers tested, roll mixtures of GF and saccharides gradually became amorphous, and the solubility of GF increased. The solubility of GF was higher in the mixtures with higher molecular weight carriers such as corn starch and processed starch. The dissolution of GF was markedly improved by the GF–Britishgum roll mixture. The initial dissolution rate of these mixtures was 170-fold higher than GF alone. The surface tension of carrier aqueous solutions was low in the processed starch with branched sugar chains. The initial dissolution rate of GF in physical mixtures was correlated with the surface tension of carrier aqueous solutions. The stability of the amorphous state of GF at a high humidity was maintained in the mixtures with carriers with a high molecular weight. These results indicated that the solubility of GF was markedly improved in the roll mixtures. It was suggested that the saccharides with a high molecular weight are useful carriers for solid dispersions.
Keywords: Solid dispersion; Roll mixing; Saccharides; Corn starch; Processed starch; Griseofulvin;

Enhancement of transdermal absorption by switching iontophoresis by Osamu Ishikawa; Yoshinori Kato; Hiraku Onishi; Tsuneji Nagai; Yoshiharu Machida (81-88).
The enhancing effect of switching iontophoresis on transdermal absorption of phthalic acid (PA), benzoic acid (BA), salicylic acid (SA), p-phenylenediamine (PD), aniline (AN) and verapamil (VR) and its mechanism were examined. An electric current with pulsed waveform (4 kHz, 50% duty) was passed through the skin for 2 h at 10 V. Iontophoretic application was carried out with switching at intervals of 5, 10 and 20 min, or without switching. Each drug solution was injected into the donor side of the cell, and phosphate buffer (pH 7.4) was injected into the receiver side. Transport of PA, BA and VR was affected by switching the polarity of electrodes but no effect was observed on that of SA, PD and AN. Cumulative amount permeated and apparent permeability coefficients were apparently high at switching intervals with a short period. The partition coefficient suggested that there was no interrelation between the affinity for skin and the permeability of each drug. The resistance values of PA and glucose were low at intervals of 5 min suggesting the participation of enhanced hydration of the skin. These results suggested that enhancement of skin hydration plays an important role in the enhancing effect of switching iontophoresis on skin permeation.
Keywords: Switching iontophoresis; Transdermal absorption; Mechanism; Skin hydration; Skin resistance;

Development and in vitro characterisation of a benznidazole liposomal formulation by M.J Morilla; P Benavidez; M.O Lopez; L Bakas; E.L Romero (89-99).
The purpose of this study was to find a multilamellar liposomal formulation for the antichagasic drug Benznidazole (BNZ). Different lipid matrices and organic solvents for BNZ were tested in order to obtain the liposomes with the highest g BNZ/100 g total lipid (D/TL) ratio. The best lipid matrices resulted from hydrogenated phosphatidylcholine from soybean (HSPC): Cholesterol (Chol): distearoyl-phosphatidylglycerol (DSPG) (molar ratio 2:2:1) prepared with BNZ dissolved in DMSO. Drug loading of 2 g BNZ/100 g total lipids at a total lipid concentration of 20–30 mM was obtained. Two in vitro assays on the HSPC:Chol:DSPG formulation to predict its in vivo behaviour were performed. In the first experiments, after 60 min at 1–450-fold dilution in buffer at 37 °C, the amount of drug associated to liposomes was reduced from 2 to 0.25 g BNZ/100 g total lipids at a rate of 65% (drug lost) min−1 at the first minute followed by 0.4% (drug lost) min−1 during the next hour. When incubated in plasma at 37 °C, the HSPC:Chol:DSPG formulations bounded a high amount of plasma proteins: r=2400 μg plasma protein per μmol total lipid.
Keywords: Multilamellar liposomes; Benznidazole;

In this study, cyclosporin-A (Cy-A) a highly lipophilic, poorly absorbable drug can be prepared easily and reproducibly as positively and negatively charged nanoparticles with the aim of improving its bioavailability and reducing its inter- and intra-individual variability. The nanoparticles were prepared by emulsification solvent diffusion method, using lecithin and poloxamer 188 as emulsifiers, and chitosan HCl, gelatin-A or sodium glycocholate (SGC) as charge inducing agents. The prepared nanoparticles were evaluated with respect to particle size, zeta potential, drug content and encapsulation efficiency. The bioavailability Cy-A from nanoparticles in comparison with the currently available Cy-A microemulsion (Neoral®) were assessed in beagle dogs. The results obtained revealed that, it was possible to prepare Cy-A as nanoparticles with size range of 104–148 nm. Chitosan HCl and gelatin-A nanoparticles exhibited +31.2 and +23.1 mV zeta potential, respectively; while SGC-nanoparticles exhibited −41.6 mV zeta potential. The in vivo results showed that, chitosan-nanoparticles gave the highest C max (2762.8 ng/ml) of Cy-A after 2.17 h (T max), while SGC-nanoparticles gave the lowest one (1202.4 ng/ml after 4.0 h). Furthermore, AUC0–24 of Cy-A from chitosan-nanoparticles was markedly increased by about 2.6-fold when compared with SGC-nanoparticles and increased by about 1.8-fold when compared with the reference Neoral® microemulsion. However, in case of gelatin-nanoparticles the AUC0–24 of Cy-A increased by about 1.8 and 1.2-fold when compared with SGC-nanoparticles and the reference Neoral® microemulsion, respectively. The relative bioavailability of Cy-A from chitosan-nanoparticles was increased by about 73%, and by about 18% from gelatin nanoparticles, while it was decreased by about 36% from SGC-nanoparticles.
Keywords: Bioavailability; Chitosan; Cyclosporin; Nanoparticles; Positively charged;

Evaluation of percutaneous absorption of 4-nerolidylcathecol from four topical formulations by Cristina D Röpke; Telma M Kaneko; Renata M Rodrigues; Vanessa V da Silva; Sonia Barros; Tânia C.H Sawada; Massuo J Kato; Silvia B.M Barros (109-116).
Antioxidants such as vitamins E and C are known to play a significant role in ameliorating or preventing oxidative damage to the skin. However, to provide a satisfactory protection they must first permeate the skin, which serves as a permeation barrier. In this study we evaluated the influence of three different formulations (gel, gel–emulsion and emulsion) on the percutaneous absorption of 4-nerolidylcathecol, an antioxidant compound isolated from Pothomorphe umbellata root extracts. Also, the absorption of the isolated 4-nerolidylcathecol was compared with its absorption when dried P. umbellata root ethanolic extract was incorporated into a gel formulation. The ‘lag time method’ was employed for the analysis of the in vitro permeation data. All formulations showed satisfactory percutaneous penetration with the 4-nerolidylcathecol–gel presenting a higher rate of penetration leading to higher dry drug levels in the tissue.
Keywords: 4-Nerolidylcathecol; Antioxidant; Skin permeation;

This work presents a simple and fast method for the simultaneous determination of amiloride and furosemide by digital derivative spectrophotometry. HCl 1×10−2 mol/l dissolved in ethanol was used as solvent and to extract drugs from formulations. Subsequently the samples were evaluated directly by first digital derivative spectrophotometry, using a smoothing factor of 8 and scale factor of 1×10−4. The simultaneous determination of furosemide and amiloride can be carried out at 241.4 and 343.6 nm, respectively. In both cases, the zero crossing approach was used. When both compounds are present together in a sample, it is possible to quantify one in the presence of the other, without mutual interference. The determination range was found to be of 6.9×10−8 to 16×10−5 and 6.8×10−8 to 8×10−5 mol/l, for amiloride and furosemide, respectively. A good level of repeatability (RSD) of 0.9 and 0.6% was observed for amiloride and furosemide, respectively. The ingredients commonly found in commercial pharmaceutical formulations do not interfere. The proposed method was applied to the determination of these drugs in pharmaceutical formulations.
Keywords: Amiloride; Furosemide; Pharmaceutical formulation; Simultaneous determination and first digital derivative spectrophotometry;

To increase the local concentration of tamoxifen in estrogen receptor (ER) positive breast cancer, we have developed and characterized nanoparticle formulation using poly(ε-caprolactone) (PCL). The nanoparticles were prepared by solvent displacement method using acetone–water system. Particle size analysis, scanning electron microscopy, zeta potential measurements, and differential scanning calorimetry (DSC) were used for nanoparticle characterization. Biodegradation studies were performed in the presence and absence of Pseudomonas lipase in phosphate-buffered saline (PBS, pH 7.4) at 37 °C. Tamoxifen loading over different concentrations was analyzed by high-performance liquid chromatography (HPLC) and the optimum loading concentration was determined. In vitro release studies were performed in 0.5% (w/v) sodium lauryl sulfate (SLS) containing PBS at 37 °C. Cellular uptake and distribution of fluorescent-labeled nanoparticles was examined in MCF-7 breast cancer cells. SEM micrographs and Coulter analysis showed nanoparticles with spherical shape and uniform size distribution (250–300 nm), respectively. Zeta potential analysis revealed a positive surface charge of +25 mV on the tamoxifen-loaded formulation. Being hydrophobic crystalline polyester, PCL did not degrade in PBS alone, but the degradation was enhanced by the presence of lipase. The maximum tamoxifen loading efficiency was 64%. Initial burst release of tamoxifen was observed, probably due to significant surface presence of the drug on the nanoparticles. A large fraction of the administered nanoparticle dose was taken up by MCF-7 cells through non-specific endocytosis. The nanoparticles were found in the perinuclear region after 1 h. Results of the study suggest that nanoparticle formulations of selective ER modulators, like tamoxifen, would provide increased therapeutic benefit by delivering the drug in the vicinity of the ER.
Keywords: Targeted delivery system; Poly(ε-caprolactone); Tamoxifen; Nanoparticles; MCF-7 cells;

Bioadhesive polysaccharide in protein delivery system: chitosan nanoparticles improve the intestinal absorption of insulin in vivo by Yan Pan; Ying-jian Li; Hui-ying Zhao; Jun-min Zheng; Hui Xu; Gang Wei; Jin-song Hao; Fu-de Cui (139-147).
There are many ongoing investigations to improve the oral bioavailability of peptide and protein formulations. Bioadhesive polysaccharide chitosan nanoparticles (CS-NPs) would seem to further enhance intestinal absorption of them. In this study, Insulin-loaded CS-NPs were prepared by ionotropic gelation of CS with tripolyphosphate anions. Its particle size distribution and zeta potential were determined by photon correction spectroscopy and laser Dopper anemometry. The ability of CS-NPs to enhance intestinal absorption of insulin and increase the relative pharmacological bioavailability of insulin was investigated by monitoring the plasma glucose level of alloxan-induced diabetic rats after oral administration of various doses of insulin-loaded CS-NPs. CS-NPs had a particle size in the range of 250–400 nm and its polydispersity index was smaller than 0.1, positively charged, stable. Insulin association was found up to 80% and its in vitro release showed a great initial burst with a pH-sensitivity property. CS-NPs enhanced the intestinal absorption of insulin to a greater extent than the aqueous solution of CS in vivo. Above all, after administration of 21 I.U./kg insulin in the CS-NPs, the hypoglycemia was prolonged over 15 h and the average pharmacological bioavailability relative to SC injection of insulin solution was up to 14.9%.
Keywords: Insulin; Chitosan-nanoparticles; Oral administration; Relative pharmacological bioavailability;

Pharmacokinetic and pharmacodynamic evaluation of cyclosporin A O/W-emulsion in rats by Soo-Jin Kim; Hoo-Kyun Choi; Yong-Bok Lee (149-156).
The pharmacokinetics and pharmacodynamics of the cyclosporin A (CSA) O/W-emulsion were studied after intravenous and oral administration to Sprague–Dawley rats. Two commercial products, CIPOL Inj.® and Sandimmun Neoral®, were used as the reference formulations. CSA concentration and lymphocyte populations in whole blood were measured by TDxFLx® and Coulter STKS®, respectively. The pharmacokinetic and pharmacodynamic parameters were obtained by fitting experimental data to two-compartment model and to indirect pharmacodynamic model, respectively, using winnonlin. The area under the concentration–time curve (AUC), terminal half-lives (T 1/2), total clearance (CLt) and relative bioavailability (F) after intravenous administration of CSA O/W-emulsion were not significantly different from those of intravenous administration of CIPOL Inj.® (P>0.05). In oral administration, AUC and C max of CSA O/W-emulsion were significantly decreased (P<0.05), while T 1/2, MRT, T max and F were not significantly different (P>0.05) from those of Sandimmun Neoral®. However, the area between the baseline and effect curves (ABEC) and pharmacodynamic efficiency (EFF) of CSA O/W-emulsion were significantly greater than those of references regardless of routes of administration (P<0.05). The pharmacodynamic availability (FPD) of CSA O/W-emulsion was 1.79- and 2.13-fold higher than that of CIPOL Inj.® and Sandimmun Neroal® (P<0.05), respectively.
Keywords: Cyclosporin A; O/W-emulsion; Pharmacokinetics; Pharmacodynamics;

Effect of occlusion on the percutaneous penetration of linoleic acid and glycerol by Louise J Taylor; Robert S Lee; Mark Long; Anthony V Rawlings; Joseph Tubek; Lynne Whitehead; Gary P Moss (157-164).
The effect of occlusion on the in vitro percutaneous absorption of linoleic acid was investigated. A greater skin concentration of linoleic acid from an ethanolic vehicle was observed in non-occluded experiments compared with occluded experiments (P<0.05). Such changes were not observed as consistently when ethanol was replaced with a less volatile organic solvent (cyclomethicone). These observations were attributed to the increase in the concentration gradient due to the unimpeded evaporation of volatile solvents, which provided a greater driving force and enhanced non-occluded delivery in these systems, compared with occluded systems. Conversely, the percutaneous absorption of a polar material (glycerol) from an aqueous solution did not yield any such differences. While more conclusive comparisons between volatile and non-volatile solvents and penetrants would be required to substantiate fully these comparisons, it is apparent that non-occlusion of volatile solvents may enhance percutaneous absorption. The physicochemical properties of the penetrant, for example its natural state at skin temperature (i.e. solid or liquid) may further determine the degree of enhanced percutaneous absorption compared with occluded environments.
Keywords: Occlusion; Percutaneous absorption; Linoleic acid; Glycerol; Porcine skin;

Chitosan microparticles were prepared with tripolyphosphate (TPP) by ionic crosslinking. The particle sizes of TPP-chitosan microparticles were in range from 500 to 710 μm and encapsulation efficiencies of drug were more than 90%. The morphologies of TPP-chitosan microparticles were examined with scanning electron microscopy. As pH of TPP solution decreased and molecular weight (MW) of chitosan increased, microparticles had more spherical shape and smooth surface. Release behaviors of felodipine as a model drug were affected by various preparation processes. Chitosan microparticles prepared with lower pH or higher concentration of TPP solution resulted in slower felodipine release from microparticles. With decreasing MW and concentration of chitosan solution, release behavior was increased. The release of drug from TPP-chitosan microparticles decreased when cross-linking time increased. These results indicate that TPP-chitosan microparticles may become a potential delivery system to control the release of drug.
Keywords: Chitosan; Microparticle; Tripolyphosphate; Drug delivery system;

Polymerized rosin: novel film forming polymer for drug delivery by S.V Fulzele; P.M Satturwar; A.K Dorle (175-184).
Polymerized rosin (PR) a novel film forming polymer is characterized and investigated in the present study for its application in drug delivery. Films were produced by a casting/solvent evaporation method from plasticizer free and plasticizer containing solutions. Films prepared from different formulations were studied for their mechanical (tensile strength, percent elongation and Young's modulus), water vapour transmission and moisture absorption characteristics. Neat PR films were slightly brittle and posed the problem of breaking during handling. Hydrophobic plasticizers, dibutyl sebacate and tributyl citrate, improved the mechanical properties of free films with both the plasticizers showing significant effects on film elongation. Release of diclofenac sodium (model drug) from coated pellets was sustained with high coating levels. Concentration of plasticizer was found to affect the release profile. PR films plasticized with hydrophobic plasticizers could therefore be used in coating processes for the design of oral sustained delivery dosage forms.
Keywords: Polymerized rosin; Hydrophobic plasticizers; Films; Mechanical properties; Pellet coating; Sustained release;

This study was performed in order to evaluate the effects of binder droplet size and powder particle size on agglomerate formation and growth in fluid bed spray agglomeration using a meltable binder. Three different lactose grades, 100, 125 or 350 mesh, were agglomerated using polyethylene glycol (PEG) 3000 at two different concentrations, 11.5 or 22% (volume/mass), and three spray droplet sizes, 30, 60 or 90 μm were applied. The ratio of droplet size/particle size was found to determine whether the mechanism of nucleation was distribution or immersion. Distribution was promoted by a low ratio, whereas immersion was promoted by a high ratio. Distribution as nucleation mechanism led to a more open agglomerate structure and immersion to a denser structure. When the nucleation phase was terminated, coalescence between rewetted nuclei or agglomerates was the growth mechanism with both preceding mechanisms of nucleation. A larger particle size of the lactose led to larger agglomerates. The difference in the effect on growth between the 30 and 60 μm droplets was generally low. The 90 μm droplets at 22% binder concentration offered a potential for uncontrollable growth giving rise to markedly larger agglomerates and a lower reproducibility than 30 and 60 μm droplets.
Keywords: Binder droplet size; Fluid bed agglomeration; Growth mechanism; Melt agglomeration; Nucleation mechanism; Powder particle size;

6-O-α-(4-O-α-d-glucuronyl)-d-glucosyl-β-cyclodextrin: solubilizing ability and some cellular effects by Sumitra Tavornvipas; Fumitoshi Hirayama; Hidetoshi Arima; Kaneto Uekama; Toshihiro Ishiguro; Masahide Oka; Kenichi Hamayasu; Hitoshi Hashimoto (199-209).
Some physicochemical and biopharmaceutical properties of a new branched cyclodextrin, 6-O-α-(4-O-α-d-glucuronyl)-d-glucosyl-β-cyclodextrin (GUG-β-CyD), were investigated. The interaction of GUG-β-CyD with drugs was studied by spectroscopic and solubility methods, and compared with those of parent β-CyD and 6-O-α-maltosyl-β-CyD (G2-β-CyD). The hemolytic activity of GUG-β-CyD on rabbit erythrocytes was lower than those of β-CyD and G2-β-CyD. GUG-β-CyD and G2-β-CyD showed negligible cytotoxicity on Caco-2 cells up to at least 0.1 M. The inclusion ability of GUG-β-CyD to neutral and acidic drugs was comparable to or slightly smaller than those of β-CyD and G2-β-CyD, probably because of a steric hindrance of the branched sugar. On the other hand, GUG-β-CyD showed greater affinity for the basic drugs, compared with β-CyD and G2-β-CyD, owing to an electrostatic interaction of its carboxylate anion with positive charge of basic drugs. Thus, GUG-β-CyD may be useful as a safe solubilizing agent particularly for basic drugs.
Keywords: Branched cyclodextrin; Inclusion ability; Stability constant; Electrostatic interaction; Hemolysis; Cytotoxicity;

This study investigated the cosolubilization phenomenon of three non-polar drugs (hydrocortisone, β-estradiol, and ethynylestradiol) in polysorbate 80 solutions. It was found that the solubility of any drug decreased in the presence of other steroidal compounds. In an attempt to understand the observation, the author proposed a model to describe and to predict the drug solubility in the presence of other non-polar drugs in a non-ionic surfactant. The model indicates that, in a non-ionic surfactant solution that contains both drugs D a and D b, the total solubility [D a tot] is related not only to the physical chemical properties of D a (micellar equilibrium constant K a, the intrinsic solubility [D a]), as well as the total surfactant concentration [S tot], it is also related to the physical chemical properties of the D b. Mathematically, the [D a tot] decreases as the product of the micellar equilibrium constant K b and the intrinsic solubility [D b] increases. The model was also put to the test by comparing the cosolubilization data obtained experimentally with the data calculated from the proposed model. The fact that these two sets of data were in good agreement lent strong support to the newly proposed model.
Keywords: Solubilization; Cosolubilization; Surfactant; Polysorbate 80; Hydrocortisone; β-estradiol; Ethynylestradiol;

Cascade impactor analysis is the standard technique for in vitro characterization of aerosol clouds generated by medical aerosol generators. One important reason for using this inertial separation principle is that drug fractions are classified into aerodynamic size ranges that are relevant to the deposition in the respiratory tract. Measurement of these fractions with chemical detection methods enables establishment of the particle size distribution of the drug in the presence of excipients. However, the technique is laborious and time consuming and most of the devices used for inhaler evaluation lack sufficient possibilities for automation. In addition to that, impactors often have to be operated under conditions for which they were not designed and calibrated. Particularly, flow rates through impactors are increased to values at which the flow through the nozzles is highly turbulent. This has an uncontrolled influence on the collection efficiencies and cut-off curves of these nozzles. Moreover, the cut-off value varies with the flow rate through an impactor nozzle. On the other hand, the high air flow resistances of most impactors are rather restricting to the attainable (fixed) inspiratory flow curves through these devices. Especially for breath actuated dry powder inhalers, higher flow rates and flow increase rates may be desirable than can be achieved in combination with a particular type of impactor. In this paper, the applicability of laser diffraction technology is evaluated as a very fast and highly reliable alternative for cascade impactor analysis. With this technique, aerodynamic diameters cannot be measured, but for comparative evaluation and development, comprising most in vitro applications, this is not necessary. Laser diffraction has excellent possibilities for automated recording of data and testing conditions, and the size classes are independent of the flow rate. Practical limitations can be overcome by using a special inhaler adapter which enables control of the inspiratory flow curve through the inhaler, analysis of the emitted fine particle mass fraction and pre-separation of large particles during testing of dry powder inhalers containing adhesive mixtures.
Keywords: Inhalation; Aerosol cloud; Particle size analysis; Aerodynamic diameter; Laser diffraction; Cascade impactor;

Design and application of a new modular adapter for laser diffraction characterization of inhalation aerosols by A.H de Boer; D Gjaltema; P Hagedoorn; M Schaller; W Witt; H.W Frijlink (233-245).
An inhaler adapter has been designed for the characterization of the aerosol clouds from medical aerosol generators such as nebulizers, dry powder inhalers (dpis) and metered dose inhalers (mdis) with laser diffraction technology. The adapter has a pre-separator, for separation of large particles (i.e. carrier crystals) from the aerosol cloud before it is exposed to the laser beam. It also has a fine particle collector for measuring the emitted mass fraction of fines by chemical detection methods after laser diffraction sizing. The closed system enables flow control through the aerosol generators and all test conditions, including ambient temperature and relative humidity, are automatically recorded. Counter flows minimize particle deposition onto the two windows for the laser beam, which make successive measurements without cleaning of these windows possible. The adapter has successfully been tested for nebulizers, mdis and dpis. In a comparative study with ten nebulizers it was found that these devices differ considerably in droplet size (distribution) of the aerosol cloud for the same 10% aqueous tobramycin solution (volume median diameters ranging from 1.25 to 3.25 μm) when they are used under the conditions recommended by the manufacturers. The droplet size distribution generated by the Sidestream (with PortaNeb compressor) is very constant during nebulization until dry running of the device. Comparative testing of dpis containing spherical pellet type of formulations for the drug (e.g. the AstraZeneca Turbuhaler) with the adapter is fast and simple. But also formulations containing larger carrier material could successfully be measured. Disintegration efficiency of a test inhaler with carrier retainment (acting as a pre-separator) could be measured quite accurately both for a colistin sulfate formulation with 16.7% of a lactose fraction 106–150 μm and for a budesonide formulation with a carrier mixture of Pharmatose 325 and 150 M. Therefore, it is concluded that, with this special adapter, laser diffraction may be a valuable tool for comparative inhaler evaluation, device development, powder formulation and quality control. Compared to cascade impactor analysis, laser diffraction is much faster. In addition to that, more detailed and also different information about the aerosol cloud is obtained.
Keywords: Inhalation; Aerosol cloud; Particle size analysis; Laser diffraction; Modular inhaler adapter; INHALER 2000™;

Equivalence testing of salbutamol dry powder inhalers: in vitro impaction results versus in vivo efficacy by M Weda; P Zanen; A.H de Boer; D Gjaltema; A Ajaoud; D.M Barends; H.W Frijlink (247-255).
The aim of the study was to evaluate several impactors for in vitro equivalence testing of salbutamol with respect to efficacy and to define in vitro equivalence limits validated with in vivo efficacy data. The four impactors described in Supplement 2000 of the European Pharmacopoeia were used: Glass Impinger (GI), Metal Impinger (MI), Multistage Liquid Impinger (MSLI) and Andersen Cascade Impactor (ACI). Three salbutamol dry powder formulations with different fine particle doses (FPDs) were prepared and the aerodynamic particle size distribution was measured. For each impactor also the recovery was determined. The same three preparations were administered to 12 asthmatic patients in a randomized, placebo-controlled, four-way crossover study. Cumulative doses from 50 μg up to 400 μg were given. The FEV1 was measured at baseline and 15 min after each dose. The in vitro results showed large differences between the FPDs of the three preparations with all impactors, whereas only small differences were observed between the four impactors. Since the recoveries of the MI and GI were low, in vitro equivalence testing should only be performed with the MSLI or ACI. The in vivo measurements did not show significant differences in efficacy between the three active preparations, even at the most discriminatory dose of 50 μg. It is concluded that in case there are no relevant differences between delivered dose, inhalation device and excipients, for salbutamol dry powder inhalers equivalence can be assumed when the 90% confidence interval for the FPD ratio of the test product and reference product is within 0.50–1.20 and each of the two products has a FPD (particles <6 μm) of at least 10 μg.
Keywords: Equivalence; Salbutamol; Dry powder inhaler; Impactor; Fine particle dose; Efficacy;

Stabilization and preformulation of anticancer drug—SarCNU by Nina Ni; Tapan Sanghvi; Samuel H Yalkowsky (257-264).
The stability of SarCNU (NSC364432), 1-(2-chloroethyl)-3-sarcosinamide-1-nitrosourea in several pharmaceutically acceptable solvents was investigated by high pressure liquid chromatography (HPLC). The influences of light, ionic strength, pH, buffer concentration, and the following excipients: benzyl alcohol, ascorbic acid, sodium bisulfite, and disodium EDTA were studied at room temperature. The stability of the drug was also determined in water, EtOH, PG, Capmul PG, DMSO, and in different combinations of these cosolvents at four different temperatures. The degradation of the drug, which is catalyzed not only by general but also by specific acid and base, follows first order kinetics. Antioxidants, EDTA, and light have no effect on the degradation rate, suggesting oxidation is not a major degradation pathway. The t 90 in pure cosolvent is 25–50 times higher than that in water or semi-aqueous vehicles. Neat EtOH can be used to store the drug in a nonaqueous concentrate that is diluted with aqueous solvent prior to injection.
Keywords: Stability; 1-(2-Chloroethyl)-3-sarcosinamide-1-nitrosourea; Double syringe;

Noticeboard (265-267).