International Journal of Pharmaceutics (v.237, #1-2)

A 24−1 fractional factorial design was used to evaluate the effect of various process variables in fluid bed granulation, on the physico-chemical properties of granule and tablet containing a high dose, poorly water soluble, low density, and micronized drug. The process variables studied were inlet air temperature, inlet air flow, spray rate of the binder solution, and atomization air pressure. Tablets with identical composition, weight, size and hardness were also manufactured in a high shear granulator and their physical properties were determined and compared with those produced by the fluidized bed granulation method. Except for the granule size distribution, other physical properties of granulations and tablets produced in a fluid bed granulator are independent of the selected process variables within the study range. Both atomization air pressure and spray rate of the binder solution had strong impact on granule size distribution. Irrespective of the process conditions used in the fluid bed granulation, granules from this process were more porous, less dense and more compressible than the granules from the high shear granulation process. Comparable tablet dissolution rates to those prepared by the optimized high shear granulation method can be achieved by selecting the appropriate process conditions in fluid bed granulation. These results suggest that wet granulation tablets of a high dose, poorly water soluble, low density, micronized drug can be manufactured using a fluidized bed granulation method, with comparable tablet dissolution rates to those produced with an optimized high shear granulation method.
Keywords: Fluid bed granulation; High shear granulation; High dose, poorly water soluble, low density, micronized drug; Granule size; Tablet dissolution;

Application of acid-treated yeast cell wall (AYC) as a pharmaceutical additive. III. AYC aqueous coating onto granules and film formation mechanism of AYC by Hiroshi Yuasa; Junichi Kaneshige; Tetsuya Ozeki; Takahide Kasai; Takahiro Eguchi; Naomu Ishiwaki (15-22).
From the viewpoint of effective utilization of natural resources and development of new pharmaceutical materials, acid-treated yeast cell wall (AYC) was prepared via a novel approach involving acidification of brewers’ yeast cell wall. AYC aqueous dispersion containing 5% (w/v) AYC and 0.5% (w/v) glycerol was prepared. Subsequently, AYC was coated onto core granules containing acetaminophen (AAP). Spray mist size under various spray conditions and viscosity of the AYC aqueous dispersion at various AYC concentrations were measured. AYC spray mists were optically observed. The surface of AYC cast film and AYC-coated granules were observed with a confocal scanning laser microscope. We attempted to show the utility of AYC as a novel material for granule coating, following the tablet coating in our previous report. In addition, the film formation mechanism of AYC was investigated. A smooth surface of the AYC-coated granules was obtained at a coating ratio of only 5%, which generally requires approximately 15–30% coating against the core granule weight, with no aggregation. These results are attributable to the fact that the granules were coated with a large number of small mists of AYC and the coating progressed efficiently, and the thin film layer of AYC was formed on the granules by mutual tangling of the hydrogel layers of AYC polysaccharides. AAP release from AYC-coated granules was obviously rapid, suggesting the high utility of AYC as a coating material for the rapidly releasing granules.
Keywords: Acid-treated yeast cell wall; Spray mist; Granule; Film formation mechanism; Pharmaceutical additive;

The H2-receptor antagonist ranitidine has previously been shown to be a substrate for colonic bacterial metabolism. The objective of the present study was to assess the in vitro stability of the other H2-receptor antagonists, cimetidine, famotidine and nizatidine, to colonic bacteria. One hundred milligrams of each drug were introduced into individual batch culture fermenters (100 ml) consisting of buffer medium inoculated with freshly voided human faeces (10% w/v). Control experiments, equivalent drug quantities in buffer medium without the presence of faeces, were also run in parallel. Samples were removed at set time intervals over a 24 h period and were subsequently analysed by HPLC. A selection of the samples removed from the fermenters was also subjected to analysis by UV spectroscopy and mass spectrometry. Following an initial dissolution phase in the fermentation system, a marked decline in nizatidine concentration was noted over time with virtually no drug remaining after 12 h, thereby suggesting degradation and metabolism of the drug by colonic bacteria. No such decline in concentration was noted for cimetidine or famotidine or for any of the drugs in the control buffer systems. The metabolic reaction pathway for nizatidine was complex, although UV and mass spectrometry analysis indicated that metabolism was initiated via cleavage of an N-oxide bond within the molecule. These results in combination with those obtained from a previous study indicate that of the four commercially available H2-receptor antagonists, nizatidine and ranitidine are susceptible to metabolism by colonic bacteria, which in turn has ramifications for drug delivery and absorption.
Keywords: H2-receptor antagonists; Metabolism; Colon; Bacteria; Drug delivery;

The formulation of botanical dietary supplements is challenging due to their complex activity–composition relationship, as well as physical and chemical stability issues. As excipient compatibility testing is a major component of sound formulation development, the objectives of this work were: (1) explore excipient compatibility storage paradigms; (2) determine interactions between phytochemicals of interest in Saint John's Wort (SJW) with several excipients; and (3) explore the application of similarity metrics to the data. Modifications to conventional isothermal stress testing paradigms included additional storage conditions of heat and moisture (5, 50 °C, 5 and 0% added water), as well as more rigorous controls. Binary blends of SJW and ten commonly used excipients were prepared and neat SJW was used as control. After 3 weeks, the percentage remaining of each phytochemical was determined by HPLC. Several similarity metrics were applied to the data. Common storage paradigms were suitable for excipient compatibility testing when controls of neat material are stored under similar conditions and the percentage of phytochemicals remaining in excipient:SJW blends and neat SJW are compared. Excipient incompatibilities were determined for SJW phytochemicals of interest. Similarity metrics applied to the phytochemical profiles conveniently summarized the data. This work allows logical decisions to be made regarding the formulation of SJW.
Keywords: Hypericum perforatum extract; Excipient compatibility testing; Botanical formulation; Similarity metrics;

Studies on mechanism of 8-methoxypsoralen–DNA interaction in the dark by A Arabzadeh; S.Z Bathaie; H Farsam; M Amanlou; A.A Saboury; A Shockravi; A.A Moosavi-Movahedi (47-55).
The interaction of 8-methoxypsoralen (8-MOP) with calf thymus DNA was studied in darkness at 25 °C and pH 7.4. The enthalpy curve for 8-MOP–DNA interaction was obtained by isothermal titration calorimetry and showed a two-step process for the interaction. According to the spectrophotometric data, it was suggested that some compaction may occur in the DNA structure at higher [8-MOP]t/[DNA] ratio. Using the fluorescence quenching data, the Scatchard analysis was performed for 8-MOP–DNA interaction at the extended ranges of drug concentration. The results indicated that the first set of binding sites was occupied by 1 mol of drug bound per near eight base pairs of DNA. Also 8-MOP caused the quenching of the fluorescence emission of DNA–ethidium bromide complex. The Scatchard analysis of these data indicated the non-competitive manner for quenching. A non-displacement based quenching mechanism has been suggested for this behavior. The circular dichroism spectra also confirmed the non-intercalative binding of 8-MOP at higher concentrations accompanied by some conformational changes in DNA structure. It has been suggested that at low drug load, 8-MOP binds to DNA as an intercalator, which is an endothermic process, whereas at higher ratios of [8-MOP]t/[DNA], it binds to the outside of DNA, probably in the minor groove and causes some compaction in DNA, which is the exothermic process.
Keywords: DNA; 8-MOP; Intercalation; Microcalorimetry; Binding sites;

Protection mechanism of Tween 80 during freeze–thawing of a model protein, LDH by Anna Hillgren; Jan Lindgren; Maggie Aldén (57-69).
The purpose of the study was to investigate the protective mechanism of a non-ionic surfactant, Tween 80, at freeze–thawing with controlled temperature history of a model protein, lactate dehydrogenase (LDH). The system was examined by differential scanning calorimetry (DSC) and infrared spectroscopy (IR). LDH activity assays were performed spectrophotometrically. In all samples, independent of temperature history and addition of surfactant, all water was crystallized to polycrystalline ice at temperatures below −20 °C. The size and perfection of the ice crystals could be varied by a range of cooling rates giving different degrees of undercooling. At Tween concentrations below the cmc at crystallization, lower concentrations were required at low cooling rates compared to higher cooling rates to protect LDH. Concentrations above cmc of Tween reduced the protection at a cooling rate of 5 °C min−1 and at quenching in N2(l). The amount of Tween needed for complete protection correlated to the surface area of the ice crystals at a certain temperature history. Tween 80 protects LDH from denaturation at freeze–thawing by hindering its destructive interaction with the ice crystals. The protective effect might be obtained when Tween molecules compete with the protein for sites on the ice surface. The optimum concentration of Tween needed for complete protection is dependent on the temperature history.
Keywords: Tween 80; LDH; Freeze–thawing; Temperature history; DSC; IR spectroscopy;

The hypoglycemic effect of Eudragit S100 enteric-coated capsules containing sodium salicylate as an absorption promoter formulated with insulin in various ways: as physical mixture, by wet granulation or in suppository bases (polyethylene glycol 4000 or Witepsol W35) was studied in hyperglycemic beagle dogs. The capsules containing insulin formulated with sodium salicylate (50 mg) and prepared by either physical mixing or wet granulation using 10% polyvinyl pyrollidone gave almost the same results producing a maximum reduction in plasma glucose level (C max) of 81.53±8.21 and 79.59±5.75%, T max of 6 and 5 h, area under the curve (AUC) of 69.37±48.64 and 57.98±23.15% reduction hour (% red. h) and resulting in relative hypoglycemia (RH) of 8.73±6.12 and 7.29±2.91%, respectively. Formulation of insulin with sodium salicylate in PEG 4000 produced a lower AUC of 37.30±10.36% red. h and RH of 4.69±1.3%. While, formulation in Witepsol W35 (0.5, 1.0 and 2.0 g) that was sieved to produce particle size of 180–315 μm and filled in enteric-coated capsules showed that formulating insulin and sodium salicylate in 1 g base is the best formulation. It produced 25% reduction in plasma glucose levels of the hyperglycemic beagle dogs at T max of 4 h and the largest AUC of 100.10±25.72% red. h, resulting in the highest RH of 12.59±3.23%. In conclusion, 25–30% reduction in plasma glucose levels and RH of about 12.5% relative to subcutaneous injection of regular soluble insulin can be achieved by formulating insulin in Witepsol W35 (1 g) with sodium salicylate (50 mg) as an absorption promoter, reducing the resulting mass into particle size 180–315 μm, packing into hard gelatin capsules and coating with Eudragit S100.
Keywords: Oral insulin capsules; Sodium salicylate; Diabetic beagle dogs;

An ethyl laurate-based microemulsion system with Tween 80 as surfactant, propylene glycol and ethanol as cosolvents was developed for intranasal delivery of diazepam. Phase behavior and solubilization capacity of the microemulsion system were characterized and in vivo nasal absorption of diazepam from microemulsion formulations was investigated in rabbits. A single isotropic region, which is considered as a bicontinuous microemulsion, was found in the pseudo-ternary phase diagrams developed at various Tween 80: propylene glycol: ethanol ratios. With the increase of Tween 80 concentration, the microemulsion region area, microemulsion viscosity, and the amount of H2O and ethyl laurate solubilized into the microemulsion system increased; however, the increase of ethanol percentage produced opposite effects. Diazepam, a practically water-insoluble drug, displayed a high solubility of 41 mg/ml in a microemulsion consisting of 15% ethyl laurate, 15% H2O, and 70% (w/w) surfactant/cosurfactant (Tween 80:propylene glycol:ethanol at 1:1:1 weight ratio). Nasal absorption of diazepam from this microemulsion was found to be fairly rapid. At 2 mg/kg dose, the maximum drug plasma concentration was arrived within 2–3 min, and the bioavailability (0–2 h) after nasal spray compared with intravenous injection was about 50%. These results suggest that this ethyl laurate-based microemulsion may be a useful approach for the rapid-onset delivery of diazepam during the emergency treatment of status epilepticus.
Keywords: Diazepam; Microemulsion; Solubilization; Intranasal absorption; Status epilepticus;

The objective of this work was to identify, using a statistical experimental design, the critical processing variables that affect content uniformity and loading of active agent coated on tablets in a 24″ Accela-Cota. United States Pharmacopeia (USP) specifies that the % relative standard deviation (RSD) of drug content within a batch should be less than 6%. A Plackett–Burman experimental design was used to identify the process variables that influence the content uniformity and loading efficiency of the drug in the aqueous-based film coat of the tablets. The process variables investigated were inlet airflow, pan speed, inlet air temperature, coating time, atomization pressure, and fan pressure. Atomization pressure was identified as a major variable with respect to content uniformity (P<0.01). Pan speed and coating duration were also identified as variables significantly affecting content uniformity (P<0.05). Fan pressure was identified as a critical variable affecting recovery (P≪0.01). Temperature also significantly affected recovery (P<0.05). A good correlation was obtained between observed and predicted values for content uniformity (r 2=0.85) and recovery (r 2=0.95). It was possible to achieve % RSD less than 6% while maintaining the recovery at 80% or higher.
Keywords: Accela-cota; Coating; Experimental design;

Prediction of Caco-2 cell permeability using a combination of MO-calculation and neural network by Shin-ichi Fujiwara; Fumiyoshi Yamashita; Mitsuru Hashida (95-105).
In the present study, we developed an approach involving a combination of molecular orbital (MO) calculation and neural network to predict Caco-2 cell permeability (log  P app) from the molecular structure of compounds. For a total of 87 compounds with log  P app values obtained from the literature, three-dimensional molecular structures were determined by MO-calculation, and then five molecular descriptors were obtained, namely, the dipole moment, polarizability, sum of charges of nitrogen atoms (sum(N)), oxygen atoms (sum(O)), and hydrogen atoms bonding to nitrogen or oxygen atoms (sum(H)). The correlation between these five molecular descriptors and log  P app was analyzed using a feed-forward back-propagation neural network with a configuration of 5-4-1 for input, hidden, and output layers found suitable for predicting Caco-2 cell permeability. A leave-one-out cross-validation procedure revealed that the neural network model possesses a fairly good predictability as far as Caco-2 cell permeability is concerned (predictive root mean square error (RMSE)=0.507), and better than the simple and quadratic regression model (predictive RMSE=0.584 and 0.568, respectively).
Keywords: Intestinal absorption; Caco-2 cells; Permeability; Molecular structure; Neural networks;

Didanosine extended-release matrix tablets: optimization of formulation variables using statistical experimental design by Carla Sánchez-Lafuente; Sandra Furlanetto; Mercedes Fernández-Arévalo; Josefa Alvarez-Fuentes; Antonio M. Rabasco; M.Teresa Faucci; Sergio Pinzauti; Paola Mura (107-118).
Statistical experimental design was applied to evaluate the influence of some process and formulation variables and possible interactions among such variables, on didanosine release from directly-compressed matrix tablets based on blends of two insoluble polymers, Eudragit RS-PM and Ethocel 100, with the final goal of drug release behavior optimization. The considered responses were the percent of drug released at three determined times, the dissolution efficiency at 6 h and the time to dissolve 10% of drug. Four independent variables were considered: tablet compression force, ratio between the polymers and their particle size, and drug content. The preliminary screening step, carried out by means of a 12-run asymmetric screening matrix according to a D-optimal design strategy, allowed evaluation of the effects of different levels of each variable. The drug content and the polymers ratio had the most important effect on drug release, which, moreover, was favored by greater polymers particle size; on the contrary the compression force did not have a significant effect. The Doehlert design was then applied for a response-surface study, in order to study in depth the effects of the most important variables. The desirability function was used to simultaneously optimize the five considered responses, each having a different target. This procedure allowed selection, in the studied experimental domain, of the best formulation conditions to optimize drug release rate. The experimental values obtained from the optimized formulation highly agreed with the predicted values. The results demonstrated the reliability of the model in the preparation of extended-release matrix tablets with predictable drug release profiles.
Keywords: Statistical experimental design; Response surface methodology; Multiple response optimization; Didanosine; Extended-release inert matrix tablets;

In vivo drug release from solid lipid nanoparticles (SLN) takes place by diffusion and degradation of the lipid matrix. SLN with different degree of crystallinity were prepared to study the effect of crystallinity on the degradation velocity. These SLN were produced by using glycerides with different length of fatty acid chains and known differences in crystallisation velocity (Dynasan 114 and 116), and using stabilisers interfering differently with the crystallisation process of the lipid matrix (cholic acid sodium salt (NaCh), Poloxamer 407 (Plx 407)). NaCh disturbs the crystallisation process, Poloxamer shows little interference. The particles were characterised by photon correlation spectroscopy (PCS) and differential scanning calorimetry (DSC), degradation velocity was determined directly after production and during storage up to 4 weeks under different storage conditions using an especially developed assay based on the NEFA Test kit. After production, SLN with a lower crystallinity matrix (Dynasan 114 and 116, NaCh) degraded faster than higher crystalline particles (all SLN with Plx 407), and showed a decrease in degradation velocity with increasing crystallinity during storage. Fast crystallising particles made from Dynasan 116 stabilised with the non-interfering Plx 407 showed no change in the degradation velocity during storage. SLN produced with a higher crystalline lipid in combination with the crystallisation-disturbing NaCh (Dynasan 116, NaCh) required a ‘ripening time’ to reach sufficient crystallinity.
Keywords: SLN; Nanoparticles; Pancreatic lipase; Crystallinity; Dynasan;

Targetability and intracellular delivery of anti-BCG antibody-modified, pH-sensitive fusogenic immunoliposomes to tumor cells by Toshiro Mizoue; Toshiya Horibe; Kazuo Maruyama; Tomoko Takizawa; Motoharu Iwatsuru; Kenji Kono; Hironobu Yanagie; Fuminori Moriyasu (129-137).
We prepared tumor-specific immunoliposomes by coupling anti-BCG monoclonal antibodies to pH-sensitive fusogenic liposomes modified with succinylated polyglycidol (sucPG), in order to obtain efficient binding to, and endocytotic internalization into, the tumor cells. Mouse colon carcinoma 26 cells, which are known to share a common antigen with BCG, were used in in vitro experiments. BCG-sucPG immunoliposomes showed fusion ability under acidic conditions. Fluorescence microscopic observation indicated that BCG-sucPG immunoliposomes bound to colon 26 tumor cells and induced receptor-mediated endocytosis at 37 °C. Fusion assay by resonance energy transfer using N-(7-nitro-2-1,3-benzoxadiazol-4-yl) diacyl phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl) diacyl phosphatidylethanolamine suggested that fusion between BCG-sucPG immunoliposomes and endosomal and/or lysozomal membrane did occur. These results imply that the BCG-sucPG immunoliposomes transfer their content into the cytoplasm by fusing with the endosomal and/or lysozomal membrane after recognition of target cells and subsequent internalization into the cells by endocytosis.
Keywords: Liposome; pH-sensitive liposome; Immunoliposome; Drug delivery system; BCG;

Comparison of the mechanical destructive force in the small intestine of dog and human by Masaharu Kamba; Yasuo Seta; Akira Kusai; Kenji Nishimura (139-149).
The purpose of this study was to evaluate the destructive force that oral solid dosage forms receive in the small intestine of dogs and humans. Information on the mechanical destructive forces of the gastrointestinal tract (GI) helps formulation design research in the following way: (1) to improve the predictability of the dissolution test since in vivo drug release is affected by not only agitation intensity but also mechanical stress; (2) to design safe and robust products by avoiding dose-dumping or unintended drug release at an inadequate site; (3) to better understand the species difference in bioavailability by comparing the destructive forces against dosage forms in the GI of dogs with those of humans. “Destructive force Dependent Release System” (DDRS) was developed to measure the mechanical destructive forces of the GI tract by using highly hydrophobic Teflon powder. In a DDRS, a marker drug contained in the core tablet is released only when the DDRS receives a force larger than its pre-determined crushing strength. DDRS-Small Intestine (DDRS-SI), a modified DDRS, was prepared for targeting the small intestine. DDRS-SI was encapsulated in starch capsules (Capill®) and then the capsules were coated with an enteric film (DDRS-SI-Ecap). The capsules were administered to six dogs and nine human volunteers. Both dogs and human volunteers crushed a DDRS-SI having a crushing strength of 1.2 N. Therefore, these controlled-release formulations should withstand a destructive force of 1.2 N when they pass through the small intestine.
Keywords: Stomach; Small intestine; Destructive force; Teflon; Gastrointestinal transit;

The poorly soluble drug buparvaquone is used in experimental clinics against the gastrointestinal persisting parasite Cryptosporidium parvum. It was produced as nanosuspension by high pressure homogenisation. Main advantages of nanosuspensions (amongst others) are their increase of saturation solubility and dissolution velocity, improving the bioavailability of drugs. The buparvaquone nanosuspension had a bulk population of about 600 nm (analysed by photon correlation spectroscopy (PCS)). The additional analysis performed with laser diffraction showed that only a very small content of microparticles occurred, which is, for the special features of nanosuspensions, negligible because they were still below 3 μm. Another feature of nanosuspensions is the adhesion properties to surfaces, e.g. mucosa. To further increase the adhesion time of the buparvaquone nanosuspension to C. parvum, the nanosuspension was formulated with hydrogels made from mucoadhesive polymers, e.g. different types of Carbopol® and chitosan. Only a small increase of the particle size of the bulk population occurred directly after the incorporation of buparvaquone nanosuspension into the hydrogels. The nanosuspension/hydrogel systems were physically long-term stable over a period of 6 months as indicated by the unchanged particle sizes.
Keywords: Buparvaquone; High pressure homogenisation; Nanosuspensions; Hydrogels; Cryptosporidium parvum; Mucoadhesion;

Effects of polyethylene glycol attachment on physicochemical and biological stability of E. coli l-asparaginase by Alexandre Learth Soares; Gledson Manso Guimarães; Bronislaw Polakiewicz; Ronaldo Nogueira de Moraes Pitombo; José Abrahão-Neto (163-170).
l-asparaginase obtained from E. coli strains is an important enzyme widely used in leukemia treatment. However, hypersensitivity reactions must be considered a relevant adverse effect of asparaginase therapy. One approach to reduce the hypersensitivity reactions caused by this enzyme is to change its physicochemical and biological properties by means of polyethylene glycol (PEG) conjugation, resulting in a less immunogenic enzyme with much longer half-time of plasmatic life. This work investigated the factors that could interfere in PEG–enzyme's stability. The complexation did not affect the range of pH activity and stability was improved in acid medium remaining stable during 1 h at pH 3.5. The PEG–enzyme exhibited activity restoration capacity (32% after 60 min) when subjected to temperatures of 65 °C in physiological solution. The PEG–enzyme in vitro assays showed a very high stability in a human serum sample, keeping its activity practically unchanged during 40 min (strength to non-specific antibodies or proteases in serum). An increase of PEG–enzyme catalytic activity during the lyophilization was observed. The process of modification of l-asparaginase with PEG improved both physicochemical and biological stability.
Keywords: PEG–asparaginase; Leukemia; Stabilization; Storage;

The crystallisation of amorphous salbutamol sulphate prepared by spray drying was monitored using a humidity controlled microbalance (Dynamic Vapour Sorption apparatus, Surface Measurement Systems) combined with a near-infrared probe. Amorphous salbutamol sulphate was prepared by spray drying from a solution in water. The particles were then analysed using scanning electron microscopy, thermogravimetric analysis, differential scanning calorimetry, powder X-ray diffraction, isothermal microcalorimetry and water vapour sorption analysis combined with near-infrared spectroscopy (NIR). Isothermal microcalorimetry and water vapour sorption combined with NIR spectroscopy were able to detect the transition from the amorphous to crystalline state. However while the isothermal microcalorimeter showed only a classic crystallisation exotherm when the material was exposed at 75% RH, the DVS-NIR results at the same humidity highlighted a more complex process. When exposed at 75% RH, the uptake of water was followed by crystallisation that was detected using NIR. The expulsion of water after crystallisation was very slow and at a constant rate whether the material was exposed to 75 or 0% RH. The NIR and DVS studies indicated that the material had crystallised very soon after exposure to high RH. The water that was expelled during crystallisation was not displaced from the particles and remained associated with the particles for many days. This study showed that the use of gravimetric analysis together with NIR spectroscopy provided valuable information on the dynamics of the crystallisation of salbutamol sulphate. The retention of water within recently crystallised salbutamol is potentially important to the behaviour of dosage forms containing the amorphous (or partially amorphous) form of this drug.
Keywords: Salbutamol sulphate; Amorphous; Crystallisation; Calorimetry; Water sorption; Near-infrared spectroscopy;

Nasal absorption enhancement strategies for therapeutic peptides: an in vitro study using cultured human nasal epithelium by Remigius Uchenna Agu; Hoang Vu Dang; Mark Jorissen; Tom Willems; Renaat Kinget; Norbert Verbeke (179-191).
This study examined the potential usefulness of cultured human nasal epithelium as a model to investigate nasal absorption enhancement strategies for therapeutic peptides. The transport of leucine enkephalin (Leu-Enk) in the presence of bestatin and puromycin, respectively and various combinations of these protease inhibitors with absorption enhancers capable of inhibiting proteases or protecting peptides against protease degradation (glycocholate, dimethyl-β-cyclodextrin (DMβCD)) was studied. Epithelial membrane perturbation, protein leakage, bestatin/puromycin absorption and rebound aminopeptidase activity were used as toxicological end-points. The combination of puromycin with glycocholate or DMβCD resulted in a higher absorption enhancement of Leu-Enk (9–14%) than when the absorption enhancers were combined with bestatin (1–3%) or when the inhibitors were used alone (2–4%). The higher absorption enhancement resulting from the combination of protease inhibitors with absorption enhancers caused a significant reduction of epithelial resistance and increased sodium fluorescein transport. Although only puromycin permeated the human nasal epithelium, both protease inhibitors induced a significant rebound aminopeptidase activity (25–61%), which can be associated with protein leakage (21–46%). This study highlighted (i) the potential usefulness of cultured human nasal epithelium as a model to study nasal absorption enhancement of therapeutic peptides; (ii) further studies using in vivo nasal models are required to ascertain whether the membrane perturbation and cytotoxicity observed with various combinations of the protease inhibitors and absorption enhancers really raise safety concerns.
Keywords: Nasal cell culture; Peptides; Metabolism; Absorption enhancement; Toxicity;

Twenty two salts of benzylamine and p-substituted benzoic acids were prepared and characterized. The p-substituent was varied with regard to electronic, hydrophobic, and steric effects as well as hydrogen bonding potential. A multivariate data analysis was used to describe the relationship between the aqueous solubility of the salts and experimentally determined physicochemical parameters and theoretically derived molecular descriptors. The model, based on all descriptors, gave R 2=0.86 and Q 2=0.72. The most significant descriptors exhibiting VIP (variance of importance) values above 1.0 were intrinsic dissolution rate, intrinsic solubility of the unionized acids (S 0), Hansch's hydrophobic parameter, Charton's steric parameter and molecular weight (MW). Statistically good models for predicting solubility of a selected test set were obtained by using simple models consisting of a few descriptors only: (i) Charton, Hansch and MW (R 2=0.73; Q 2=0.70), and (ii) Charton and S 0 (R 2=0.74; Q 2=0.72).
Keywords: Salt; Solubility; Crystal lattice energy; Free energy of hydration; Hydrogen bonding; Hydrophobicity; Steric effects;

Effect of different terpene-containing essential oils on permeation of estradiol through hairless mouse skin by D. Monti; P. Chetoni; S. Burgalassi; M. Najarro; M.Fabrizio Saettone; E. Boldrini (209-214).
Purpose of the present investigation was to evaluate six terpene-containing essential oils for their capacity to promote permeation of estradiol (ES) through hairless mouse skin in vitro. Tests on cajuput, cardamom, melissa, myrtle, niaouli and orange oil, all used at the 10% w/w concentration in propylene glycol (PG), evidenced niaouli oil (NIA) as the best permeation promoter for ES. Tests on the main terpene components of NIA (1,8 cineole, α-pinene, α-terpineol and d-limonene), evaluated neat (10% w/w in PG) or in admixture, confirmed the better promoting activity of whole NIA. The present data point to the validity of complex terpene mixtures, such as that composing NIA, as transdermal penetration enhancers for moderately lipophilic drugs like ES.
Keywords: Estradiol; Penetration enhancers; Hairless mouse skin; Terpenes; Essential oils; Niaouli essential oil;

Bilayer nicotine mucoadhesive tablets were prepared and evaluated to determine the suitability of the formulation as a nicotine replacement product to aid in smoking cessation. A range of formulations containing 0–50% w/w Carbopol 934® and 0–50% w/w hydroxypropylcellulose (HPC) were prepared and tested for adhesive properties and drug release. Mucoadhesion was assessed using bovine buccal mucosa. Peak detachment force of the tablets was found to reach a maximum at 20% w/w Carbopol 934®, whilst work of adhesion continued to increase with Carbopol 934® concentration. HPC concentrations of 20–30% w/w were found to provide nicotine hydrogen tartrate (NHT) release approaching zero order kinetics over a 4 h test period. A combination of 20% w/w Carbopol 934® and 20% w/w HPC was thus found to provide suitable adhesion and controlled drug release. The formulation of a bilayer tablet containing the adhesive controlled release layer (CRL) and a fast releasing layer provided an initial burst release of NHT followed by the controlled release for a period of up to 4 h. The same biphasic type of release was identified during an in vivo assessment using human volunteers This biphasic drug release could represent an improvement over current methods of nicotine replacement.
Keywords: Nicotine; Nicotine replacement therapy; Buccal tablets; Bioadhesion; Controlled release;

Lectin-mediated drug delivery: binding and uptake of BSA-WGA conjugates using the Caco-2 model by Franz Gabor; Andrea Schwarzbauer; Michael Wirth (227-239).
To examine whether the dietary lectin wheat germ agglutinin (WGA) can facilitate binding and uptake of protein drugs due to its cytoadhesive and cytoinvasive properties, conjugates were prepared by covalent coupling of fluorescein-labeled bovine serum albumin (F-BSA) to WGA using divinylsulfone for crosslinking. Increasing the molar ratio of F-BSA/WGA resulted in 2.6–8.7 times higher Caco-2 binding as compared with glycyl-F-BSA. About 75% of F-BSA-WGA were bound specifically to Caco-2 cells according to inhibition studies in presence of the complementary carbohydrate. The Caco-2 association of F-BSA-WGA was temperature-dependent indicating active uptake of membrane bound conjugate, which was confirmed by confocal microscopy. The conjugate accumulated within lysosomal compartments followed by proteolytic degradation of F-BSA-WGA 1–4 h after conjugate loading as observed by equilibrating the intracellular pH with monensin. Finally low molecular weight degradation products of the proteinaceous prodrug appear in the extracellular medium. Contrary to Caco-2 single cells, a minor part of the conjugate is degraded by brush border proteases already 30 min after exposure to Caco-2 monolayers. But most of the conjugate is taken up into differentiated cells and processed as in single cells. Though the enzymic barrier remains to be surmounted, WGA-mediated drug delivery is a promising strategy for peroral delivery of even high molecular weight drugs to overcome the mucosal barrier.
Keywords: Active transport; Caco-2; Lectin; Mucosal barrier; Monensin; Protein drug delivery; Wheat germ agglutinin;

Determination of the required HLB values of some essential oils by Lara O. Orafidiya; F.A. Oladimeji (241-249).
The required HLB values of eucalyptus, lippia and peppermint essential oils were determined using droplet size analysis and turbidimetric method on emulsions prepared with emulsifier blends of varying HLB values. The percentage increase in mean droplet diameter and the degree of dispersion of droplet sizes were determined before and after centrifugation of the emulsions. The HLB value of the emulsion with the least dispersion ratio or the least percentage increase in mean droplet diameter was taken as the required HLB of the respective essential oil. The turbidimetric method was validated by the existence of correlation (r=−0.958) between the mean droplet diameter and the turbidity of the emulsions. The turbidity curve went through a maximum at the HLB value where the mean droplet diameter was least. Based on these methods, the required HLB values of eucalyptus, lippia and peppermint oils were determined as 9.8, 12.1 and 12.3, respectively (P<0.05). Liquid paraffin was used as a reference standard and its required HLB fell within literature value.
Keywords: Required HLB; Essential oils; Degree of dispersion of droplets; Turbidimetric method;

Corrigendum to: “Effect of dendrimer on entrapment and release of bioactive from liposomes” by Ajay J. Khopade; Frank Caruso; Pushpendra Tripathi; Surekha Nagaich; Narendra K. Jain (251-253).

Noticeboard (255-257).