International Journal of Pharmaceutics (v.367, #1-2)

A database of human dermatopharmacokinetic parameters of 12 transdermal patches is established. The effect of system design, application site, and metabolism on pharmacokinetic data is discussed, and interindividual variability of data and its possible sources evaluated. Using multiple regression analysis, two equations based on drugs physicochemical characteristics are suggested for partial prediction of peak plasma concentration (C max) after patch application. Patch application presumably decreases variance as rub-off, wash and exfoliation steps are diminished.The results showed that interindividual variation, in terms of coefficient of variation (CV) of C max, is inversely correlated with drugs molecular weight and lipophilicity in the range of 200 < MW < 400 and 1.6 < log  K oct  < 4.3. Multiple regression analysis of C max against physichochemical parameters demonstrated the prominent contribution of hydrogen bonding acceptability of the molecules on their maximal plasma concentration after patch administration.The findings suggest that the serum concentration profile for transdermal therapeutic systems (TTS) is a net result of the system performance, drug absorption and elimination. Thus, the variability in serum concentration is a function of variability of each process involved. This should be noted in explanation of effect of molecular features of drugs on their plasma concentration profile.
Keywords: Transdermal drug delivery; Pharmacokinetics; Interindividual variation; QSPR; Skin absorption; Transdermal patch;

The purpose of this research is to investigate the response of crystalline griseofulvin to mechanically induced stress through cryogenic milling. Crystalline griseofulvin was subjected to cryogenic milling for two different lengths of time. Following cryo-milling, the samples were immediately analyzed by differential scanning calorimetry (DSC) and X-ray powder diffraction (XRPD). The DSC thermograms of cryo-milled griseofulvin showed a complex exothermic event at around 65 °C for the 30 min cryo-milled sample and around 75 °C for the 60 min cryo-milled sample. A glass transition event was not observed for the cryo-milled samples. This is in direct contrast to the X-ray amorphous griseofulvin sample prepared through the quench melt method The XRPD patterns of cryo-milled griseofulvin show a loss of the crystalline Bragg peaks and a corresponding increase in diffuse scattering (halos). The disordered griseofulvin material produced through cryo-milling appears X-ray amorphous, yet different from the amorphous phase produced using the quench melt method. Both X-ray amorphous materials have distinctive DSC thermograms and X-ray powder patterns. These findings suggest that the evolution of the griseofulvin crystal structure during cryo-milling is not simply a crystal-to-amorphous transition but a transition to an intermediate mesophase.
Keywords: Milling; Griseofulvin; Amorphous; Defects; Mesophase;

Influence of polymeric subcoats on the drug release properties of tablets powder-coated with pre-plasticized Eudragit® L 100-55 by Dorothea Sauer; Alan B. Watts; Lonique B. Coots; Weijia C. Zheng; James W. McGinity (20-28).
The aim of the study was to investigate the properties of sodium valproate tablets that were dry powder-coated with pre-plasticized Eudragit® L 100-55. Polyethylene glycol 3350 (PEG 3350) was used as primer to facilitate initial coating powder adhesion. Solubility parameters were employed to determine the wetting properties of the PEG 3350 primer. Additional PEG 3350 within the powder coating formulation was required to enable powder adhesion to the tablet cores. The application of a subcoat of either Eudragit® E PO or Eudragit® RL PO facilitated adhesion of the enteric polymer to the tablet cores and reduced the amount PEG 3350 required in the coating formulation. Since reduction of the PEG 3350 content produced less water-vapor permeable films, the enteric coating level necessary to control the drug release was decreased. PEG 3350 and Methocel® K4M were incorporated in both Eudragit® E PO and Eudragit® RL PO subcoating formulations as pore forming agents. The influence of the pore forming excipients on physicochemical properties of free powder-cast films was investigated. The miscibility of the PEG 3350 and Methocel® K4M in the film coating was correlated with their ability to function as pore forming agent.
Keywords: Dry powder coating; Sodium valproate; Enteric; Eudragit; Subcoat; Pore forming agent;

The aim of the current study is the evaluation of a recently optimized SEDDS, composed of Solutol® HS15 and medium chain glycerides, and self-emulsifying pellets by means of ESR.Tempol-benzoate (TB)-loaded SEDDS were produced and electron spin resonance (ESR) spectroscopy was used to evaluate the diluted self-emulsifying mixtures. Moreover, ESR in vitro digestion experiments were carried out to have an insight on the characteristics of the different phases formed during the digestion process and to evaluate the distribution and the localization of TB in these phases. In addition, self-emulsifying pellets were produced using nitroxide-loaded SEDDS and the microenvironment within the pellets during release process was monitored in an online process using ESR spectroscopy.After dilution of nitroxide-loaded SEDDS, the percent of TB localized in the lipophilic compartment was decreasing with increasing the surfactant fraction in the mixture. Moreover, it was found that different phases with variable viscosity and polarity were produced as a result of the enzymatic digestion of SEDDS in physiologically relevant media. This change in lipid composition has largely affected the distribution and the localization of the spin probe during the digestion process. A rapid increase in the mobility of the spin probe inside the pellets was noticed after exposure to the release media. Additionally, TB was localized within the self-emulsifying mixture environment for the time of the experiment.ESR is considered a powerful non-invasive tool to assess the microenvironment of the diluted SEDDS and to monitor in vitro digestion process. Digestion induces a change in lipid composition which can affect the solubilization capacity of the administered drug. Therefore, monitoring in vitro digestion process using ESR spectroscopy will help in providing greater understanding of the interaction between the administered drug and the digested lipid vehicles.
Keywords: Self-emulsifying drug delivery systems; ESR; In vitro digestion; Poorly water soluble drugs; Pellets; Oral delivery system;

Comparison of artificial sebum with human and hamster sebum samples by Guang Wei Lu; Satyanarayana Valiveti; Julie Spence; Christine Zhuang; Lora Robosky; Kimberly Wade; Ann Love; Lain-Yen Hu; David Pole; Matt Mollan (37-43).
To understand drug delivery to the sebum filled hair and sebaceous follicles, it is essential to use an artificial sebum as a surrogate of the human sebum for the investigation of drug transport properties. Artificial sebum L was developed in-house based on the chemical similarity to human sebum. The partition and diffusion of model compounds (ethyl 4-hydroxybenzoate, butyl 4-hydroxybenzoate, and hexyl 4-hydroxybenzoate) were measured in human sebum, hamster ear and body sebum (a commonly used animal model), and four representative artificial sebum samples (N, S, F, and L) in which artificial sebums, N, S and F were selected based on the available literature. DSC and NMR studies were also conducted on all sebums to compare their melting properties and chemical compositions. In vitro studies show that the partition coefficients of the three model compounds in artificial sebum L were similar to that of human sebum, whereas the hamster ear and body sebum, and other three artificial sebum samples were different from that of human sebum. Additionally, the in vitro sebum flux (μg/(cm2  min) of three model compounds through artificial sebum L was closer to that of human sebum when compared with the other three artificial sebum (N, S and F), hamster body and hamster ear sebum. The results of this study indicate that the artificial sebum L could be used as an alternative to human sebum, as the physicochemical properties of this artificial sebum is relatively similar to human sebum.
Keywords: Artificial sebum; Human sebum; Hamster sebum; Partition; Diffusion;

Skin permeation and ex vivo skin metabolism of O-acyl haloperidol ester prodrugs by Andrew P. Morris; Keith R. Brain; Charles M. Heard (44-50).
Ethyl (HE), propyl (HP), butyl (HB), octyl (HO) and decyl (HD) O-acyl esters of haloperidol (HA) were evaluated for permeation across full-thickness human and guinea pig skin. The inclusion of 0.5 mg mL−1 cetrimide as a receptor phase solubilising agent did not significantly alter the barrier properties of the membranes. The permeation of the parent drug, HA, across guinea pig skin was found to be greater than that of its derivatives. Prodrug hydrolysis by cutaneous esterases was minimal. The permeation of HE, HP and HB across freshly excised guinea pig skin was subsequently investigated, however, prodrug hydrolysis remained low. Hydrolysis studies using a skin extract revealed only limited prodrug metabolism. However, in the presence of a liver extract, hydrolysis of all prodrugs was rapid. It was proposed that GGGX esterases, required for the hydrolysis of tertiary esters, were not present at a sufficiently high concentration within the skin for substantial prodrug hydrolysis to occur. This does not necessarily detract from the system as post-transdermal delivery liberation of HA in vivo is an equally useful mode for delivering this drug to the systemic circulation.
Keywords: Skin; Transdermal drug delivery; Haloperidol; Prodrug; Ester; Cetrimide; Receptor phase;

Characterisation and aerosolisation of mannitol particles produced via confined liquid impinging jets by P. Tang; H.-K. Chan; H. Chiou; K. Ogawa; M.D. Jones; H. Adi; G. Buckton; R.K. Prud’homme; J.A. Raper (51-57).
Mannitol particles, produced by spray drying (SD), have been used commercially (Aridol™) in bronchial provocation test. In this study, we propose an alternative method to produce inhalable mannitol powders. The elongated mannitol particles (number median length 4.0 μm, and axial ratio of 3.5) were prepared using a confined liquid impinging jets (CLIJs) followed by jet milling (JM). Spray dried and jet milled raw mannitol particles were compared in an attempt to assess the performance of the particles produced by the new method. Aerosol performance of the three different powders (CLIJ, SD, and JM) was relatively poor (fine particle fraction or FPFloaded below 15%) when dispersed by the Rotahaler®. Dispersion through the Aeroliser® led to better aerosol performance of the CLIJ mannitol (FPFloaded 20.3%), which is worse than the JM (FPFloaded 30.3%) and SD mannitol particles (FPFloaded 45.7%) at 60 L/min, but comparable (FPFloaded 40.0%) with those of the JM (FPFloaded 40.7%) and SD (FPFloaded 45.5%) powders at 100 L/min. Hence, the optimum use of these elongated mannitol particles can be achieved at increased air flow with a more efficient inhaler. In addition to crystallinity, morphology, and particle size distribution, the surface energies of these powders were measured to explain the differences in aerosol performance. A major advantage of using the CLIJ method is that it can be scaled up with a good yield as the precipitate can be largely collected and recovered on a filter, compared with spray drying which has a low collection efficiency for fine particles below 2 μm.
Keywords: Particle shape; Confined liquid impinging jet; Mannitol; Inhalation drug delivery; Inverse gas chromatography (IGC);

Studies of intestinal permeability of 36 flavonoids using Caco-2 cell monolayer model by Xiao-Juan Tian; Xiu-Wei Yang; Xiaoda Yang; Kui Wang (58-64).
To investigate the structure–permeability relationship of dietary/nutriceutic flavonoids, the transepithelial transport and cellular uptake of 36 flavonoids (including flavones, flavonols, dihydroflavones, dihydroflavonols, isoflavones, chalcones, flavanes, flavanols, methylated and glycosidic derivatives) were investigated using the Caco-2 cell monolayer. The apparent permeability coefficients (P app) of the flavonoids were calculated from bilateral transport assays in the Transwell system with flavonoid determination using a high performance liquid chromatography (HPLC) coupled with a UV detector. The most flavonoids exhibited concentration-independent P app values and a ratio of 0.5–1.8 for P app AB to BL/P app BL to AB, suggesting passive diffusion pathways. However, certain flavonoids e.g. morin and some flavonoid glycosides may involve the efflux mechanisms. For isoflavones, flavones, and dihydroflavones, the oil/water partition coefficients (additionally modified by the number and position of the three hydroxyl groups) was the key determinant for Caco-2 cell permeation. However, the permeability of flavonols is more complex with their structure possibly related to their high rate of cell accumulation. Overall, the parental skeleton structure, the number and position of free hydroxyl groups, accumulation and efflux in Caco-2 cell play the key roles in the transport of flavonoids across Caco-2 cell monolayer.
Keywords: Flavonoids; Caco-2 cell monolayer; Permeability; Structure–permeability relationship;

Evaluation of a taste sensor instrument (electronic tongue) for use in formulation development by Julie K. Lorenz; Joseph P. Reo; Ondrej Hendl; Jeffrey H. Worthington; Vanik D. Petrossian (65-72).
A taste sensor instrument (electronic tongue) was evaluated to determine its utility in developing a taste-enhanced liquid formulation. To train the electronic tongue, human sensory panel data were collected for two prototype formulations, a solution of the drug in water and several marketed products. Studies using the electronic tongue were conducted to determine taste-masking effectiveness of formulations compared to a matching placebo, to establish correlation with human sensory data, and to evaluate unknown formulations and predict their bitterness scores.In the first experiment, the effectiveness of a proposed taste-masking strategy was determined by comparing formulation prototypes containing a bitter active pharmaceutical ingredient (API) against corresponding placebos (i.e. formulations without an active ingredient) using electronic tongue data. The analysis of the electronic tongue data was based on the assumption that the drug was well taste masked if the placebo matched the formulation with API. In a second set of experiments, electronic tongue data were compared to existing data from a human taste panel for several marketed products and prototype formulations. A good correlation (r 2  = 0.99) was achieved from this comparison, and the relative taste of prototype formulations not tasted by humans was predicted.
Keywords: Taste sensor; Taste masking; Human taste panel; Pharmaceutical bitterness intensity; Electronic tongue;

Concentration dependent aggregation properties of chlorhexidine salts by Pengyun Zeng; Guifang Zhang; Aruna Rao; Walter Bowles; Timothy Scott Wiedmann (73-78).
Chlorhexidine (CHX), a chemical antiseptic, is known to bind to dentin and has been shown to be effective in treating bacterial infections caused by microbes. The solubility and aggregation properties of CHX salts were determined to guide the development of a sustained release formulation for long-term disinfection.The amount of CHX in solution was determined as a function of counterion concentration (chloride, acetate (Ac) or gluconate (G)) by UV spectrophotometry at 255 nm. The weight average molecular weight was determined from the angular dependence of the scattered light. Proton NMR spectroscopy was used to investigate the dependence of the peak intensity and chemical shift on solution concentration and diffusion measurements were performed by Fourier-transform pulsed-field gradient spin-echo (PFG-SE) 1H NMR.The observed CHX concentration was highly dependent on the type and concentration of salt present in solution with the greatest CHX concentration achieved with gluconate, moderate to low with diacetate, and very low with dichloride solutions. Addition of sodium gluconate enhanced the amount of CHX-Ac2 in solution; however, only low concentrations of chlorhexidine can be achieved in the presence of chloride ions. For solutions of CHX-G2, the aggregate number appeared to range from a dimer at 40 mM to perhaps a pentamer at 150 mM. In contrast, no aggregation of CHX-Cl2 or CHX-Ac2 was detected, which was corroborated by diffusion NMR results. The change in chemical shift of protons is consistent with association of the phenyl group of one CHX with the hexamethylene chain of a second CHX. Based on the analysis of NMR peak intensities of CHX, gluconate, and acetate in saturated solutions, it appears that solubilization of the diacetate species occurs within digluconate aggregates, since the solubility product of chlorhexidine diacetate is such that the concentration of CHX will exceed the critical micelle concentration (CMC). However, no solubilization of CHX-Cl2 occurs because the solubility product falls below the CMC.The low concentration of CHX that can be achieved in physiological concentrations of chloride in the oral cavity may be problematic for dental and slow release formulations. Achieving a high concentration of CHX appears to require that the monomer be present at a concentration greater than that required to produce self-association.
Keywords: Chlorhexidine; Chlorhexidine digluconate; Chlorhexidine diacetate; Chlorhexidine dichloride; Micelle; Solubility product; Light scattering; NMR spectroscopy;

The objective of this work was to examine the physicochemical properties of spray dried budesonide, formoterol fumarate and their mixtures at two different weight ratios: 100:6 and 400:6 of budesonide and formoterol fumarate, respectively. A comparison of the thermal properties, crystalline/amorphous nature and particle size of the starting micronised as well as processed materials was carried out. The micronised drugs on their own and the physical mixtures were crystalline in contrast to the spray dried counterparts which were shown to be amorphous. The glass transition temperatures (T gs) of the processed actives were determined and appeared at 89.5 and 88 °C for budesonide and formoterol fumarate, respectively. As for the spray dried composites, an indication of miscibility and/or interactions between the components was indicated by differential scanning calorimetry and infrared analysis. The spray drying in all cases resulted in smooth, spherical microparticles of sizes suitable for inhalation.
Keywords: Budesonide; Formoterol fumarate; Spray drying; Thermal properties; Amorphous; Glass transition;

The aim of this study was to investigate the effect of changing spray drying parameters on the production of a budesonide/formoterol fumarate 100:6 (w/w) composite. The systems were spray dried as solutions from 95% ethanol/5% water (v/v) using a Büchi 191-Mini Spray Dryer. A 25−1 factorial design study was undertaken to assess the consequence of altering spray drying processing variables on particle characteristics. The processing parameters that were studied were inlet temperature, spray drier airflow rate, pump rate, aspirator setting and feed concentration. Each batch of the resulting powder was characterised in terms of thermal and micromeritic properties as well as an in vitro deposition by twin impinger analysis. Overall, the parameter that had the greatest influence on each response investigated was production yield – airflow (higher airflow giving greater yields), median particle size – airflow (higher airflow giving smaller particle sizes) and Carr's compressibility index – feed concentration (lower feed concentration giving smaller Carr's indices). A six- to seven-fold difference in respirable fraction can be observed by changing the spray drying process parameters. The co-spray dried composite system which displayed best in vitro deposition characteristics, showed a 2.6-fold increase in respirable fraction in the twin impinger experiments and better dose uniformity compared with the physical mix of micronised powders.
Keywords: Budesonide; Formoterol fumarate; Spray drying; Factorial design; Inhalation; Amorphous;

Influence of pH on the dissolution of folic acid supplements by Islam R. Younis; Mary K. Stamatakis; Patrick S. Callery; Paula Jo. Meyer-Stout (97-102).
The vitamin folic acid has received considerable attention because of its role in decreasing the risk of neural tube birth defects, and its potential role in reducing the risks of cardiovascular and psychiatric diseases. A significant concern is the quality of commercially available folic acid products. We evaluated the pharmaceutical performance of 15 currently available folic acid products in terms of meeting the USP standards for disintegration and dissolution, and showed that there has been significant improvement in the past decade in the quality of these products. However, at least one product failed to meet the requirement of each test performed. Since folic acid absorption is maximal at the proximal jejunum, dissolution was further evaluated in simulated gastric fluid. All the products failed to release more than 75% of the active ingredient in 60 min. While some excipient-related factors were preliminarily considered, it was ultimately proposed that the failure may be related to the pH-dependency of the solubility of folic acid, a premise supported by faster dissolution of laboratory prepared buffered folic acid tablets. The more limited solubility of folic acid in acidic medium should be taken into consideration in the required dissolution testing methods, as well as in product formulation to optimize release.
Keywords: Folic acid; Nutraceuticals; Disintegration; Dissolution;

Novel oral absorption system containing polyamines and bile salts enhances drug transport via both transcellular and paracellular pathways across Caco-2 cell monolayers by Fuyuki Mukaizawa; Koji Taniguchi; Masateru Miyake; Ken-ichi Ogawara; Masaaki Odomi; Kazutaka Higaki; Toshikiro Kimura (103-108).
The combinatorial use of spermine (SPM), a typical polyamine, and sodium taurocholate (STC), a typical bile salt, was found to be a promising safe preparation for improving the oral absorption of poorly water-soluble and/or poorly absorbable drug in our previous studies utilizing rats and dogs. To clarify the mechanisms behind the synergistic enhancement effect of the polyamine and bile salt, the transport of rebamipide, which is classified into Biopharmaceutics Classification System Class IV, was investigated in Caco-2 cell monolayers. The synergistic enhancement of rebamipide transport by SPM and STC was certainly observed in Caco-2 cells as well, while the separate use of either SPM or STC did not significantly improve the transport of rebamipide. The combinatorial use of SPM and STC significantly decreased the transepithelial electrical resistance (TEER) in Caco-2 cell monolayers, suggesting that the opening of paracellular pathway. On the other hand, it was also confirmed that the decrease in TEER was transient and reversible after removal of SPM and STC and that cell viability was maintained. Voltage-clamp study clearly showed that their combinatorial use improved rebamipide transport via both paracellular and transcellular pathways, and that the contribution of transcellular route could be larger than paracellular route.
Keywords: Spermine; Sodium taurocholate; Transport pathways; Electrophysiological study; Caco-2 cells;

The purpose of this work was to explore the feasibility of preparing itraconazole hydrochloride to improve the solubility and dissolution rate. Itraconazole dihydrochloride was synthesized by bubbling anhydrous hydrogen chloride gas into the acetone suspension of itraconazole. Results of the elementary analysis gave the molecular formula of C35H38Cl2N8O4·2HCl and its structure was confirmed by Fourier transform infrared (FTIR), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Powder X-Ray diffraction (PXRD) suggested that a new crystalline form of the salt was formed. The morphology and mean size distribution study by scanning electron microscopy (SEM) and dynamic light scattering (DLS) confirmed that the salt was dispersable nanoparticle aggregation. Aqueous solubility measurements showed that the solubility of the salt, its 1:1, 1:2 and 1:3 (w/w) physical mixtures with beta-cyclodextrin (β-CD) was 6, 99, 236 and 388 times greater than itraconazole. More than 94% of itraconazole was dissolved out of the salt/β-CD 1/3 physical mixture after 60 min. The stability studies indicated that the physical mixture remained stable for 24 months in assay, the related substances and dissolution. Based on the present results, it is concluded that hydrochloride formation can significantly increase solubility and dissolution rate of itraconazole, and the formulation of itraconazole dihydrochloride/β-CD (1/3) would be an environment-friendly, economic and practical alternative to the commercially available itraconazole capsules (Sporanox®).
Keywords: Itraconazole; Itraconazole dihydrochloride; Nanocrystals; Solubility; Dissolution rate;

Molecular interactions in reverse hexagonal mesophase in the presence of Cyclosporin A by Dima Libster; Paul Ben Ishai; Abraham Aserin; Gil Shoham; Nissim Garti (115-126).
The present work investigates the detailed molecular structure of the HII mesophase of GMO/tricaprylin/phosphatidylcholine/water system in the presence of hydrophobic model peptide Cyclosporin A (CSA) via ATR-FTIR analysis. The conformation of the peptide in the hexagonal mesophase, as well as its location and specific interactions with the components of the carrier, were studied. Incorporation of phosphatidylcholine to the ternary GMO/tricaprylin/water system caused competition for water binding between the hydroxyl groups of GMO and the phosphate groups of the phosphatidylcholine (PC) leading to dehydration of the GMO hydroxyls in favor of phospholipid hydration. Analysis of CSA solubilization effect on the HII mesophase revealed a significant increase in the strength of hydrogen bonding with surfactant hydrogen-bonded carbonyls, indicating interaction of the peptide with the C=O groups of the surfactants. The peptide probably caused partial replacement of the intramolecular hydrogen bonds of the mesophase carbonyl groups with intermolecular hydrogen bonds of these carbonyl groups with the peptide. Furthermore, analysis of the Amide I’ peak in the FTIR spectra of the peptide demonstrated that two pairs of its internal hydrogen bonds are disrupted when it is incorporated. The partial disruption of the internal hydrogen bonds seems to cause an outward rotation of the peptide amide groups involved, resulting in more efficient intermolecular hydrogen-bonding ability. Apparently, this conformational change increased the hydrophilic properties of CSA, even making it susceptible to a weak interaction with the GMO hydroxyl groups in the interfacial region.
Keywords: Liquid crystals; Reverse hexagonal phase; Glycerol monooleate; Phosphatidylcholine; Cyclosporin A; ATR-FTIR;

Oral administration of the nonsteroidal anti-estrogen tamoxifen (TMX) is the treatment of choice for metastatic estrogen receptor-positive breast cancer. With the aim to improve TMX oral bioavailability and decrease its side effects, crosslinked alginate microparticles for the targeting to the lymphatic system by Peyer's patch (PP) uptake were developed and in vitro characterized. TMX was molecularly dispersed inside the microparticles and an electrostatic interaction involving the TMX tertiary amine was detected by rheological and FT-IR assays. Microparticles showed a size less than 3 μm, then suitability to be taken up by M cells in PP and a positive surface charge. Moreover, TMX loading level as well as in vitro release behaviour was affected by the polymer network connected with the mannuronic/guluronic ratio of the alginate chains.
Keywords: Lymphatic system; Microparticles; Tamoxifen; Alginate; Chitosan;

Complexation with β-cyclodextrin confers oral activity on the flavonoid dioclein by Bruno A. Rezende; Steyner F. Cortes; Frederico B. De Sousa; Ivana S. Lula; Martine Schmitt; Rubén D. Sinisterra; Virginia S. Lemos (133-139).
Dioclein is a flavonoid reported to have many beneficial effects on the cardiovascular system such as vasorelaxant, hypotensive, antioxidant and antiarrythmogenic activities. However, use as pharmaceuticals is limited due to the lack of oral activity and low water solubility. In this work, intending to improve its oral activity, we performed a 1:1 inclusion complex (IC) between dioclein and β-cyclodextrin (β-CD). The IC was characterized by nuclear magnetic resonance and infrared spectroscopy and its vasodilator and hypotensive effects were evaluated in mice. The inclusion of dioclein in β-CD increased the water solubility 44% compared to free dioclein. The IC (2.5 mg kg−1) produced a higher and long lasting change in systolic blood pressure (SBP) after intraperitoneal administration compared to free dioclein. When given orally, free dioclein (10 mg kg−1) showed no hypotensive effect while the IC induced a pronounced decrease in SBP. The in vitro vasodilator effect of dioclein was unchanged by its inclusion in β-CD showing that the IC does not change the interaction between dioclein and its cellular targets. In conclusion, our results show that the new complex prepared by inclusion of dioclein in β-CD improves the hypotensive effect of the flavonoid by increasing its bioavailability and enables dioclein to be effective after oral administration. The mechanism underling the increase in bioavailability is probably a consequence of a protective effect of β-CD against in vivo biodegradation by enzymes and possibly increased water solubility.
Keywords: Cyclodextrins; Inclusion complex; Flavonoids; Dioclein; Dioclein/β-cyclodextrin complexation; Drug effects; Oral therapeutic efficacy;

Benznidazole (BZL) is one of the two therapeutic agents used for the treatment of Chagas’ disease. However, the use of BZL in most pharmaceutical preparations and research experiments is still limited due to its low water solubility (0.4 mg/mL). To overcome the dissolution rate-limiting step in oral absorption, chitosan microparticles prepared by the coacervation method were chosen, owing to non-toxicity of the polymer and mild conditions of the method. The influence of process parameters such as encapsulation efficiency, size, yield, and dissolution rate was optimized by using artificial neural networks (ANNs). The optimal conditions were found to be 1.5% (w/v) for the polymer concentration, 6.0% (w/v) for the coacervation agent concentration, 1400.0 rpm for the stirring rate, and 5.0 mL/min for the spraying rate. Confirmation experiments showed good agreement between predicted and experimental values of the optimized properties. These results indicate that ANNs is a valuable tool for the development of optimized BZL chitosan microparticles. To our knowledge it is the first report based on the development of optimized BZL microparticles.
Keywords: Benznidazole; Chagas’ disease; Chitosan; Microparticles; Optimization; Artificial neural networks;

In vitro cytotoxicity of paclitaxel/β-cyclodextrin complexes for HIPEC by W. Bouquet; T. Boterberg; W. Ceelen; P. Pattyn; M. Peeters; M. Bracke; J.P. Remon; C. Vervaet (148-154).
Hyperthermic intraperitoneal chemotherapy (HIPEC) is a promising strategy in the treatment of peritoneal carcinomatosis. To perform HIPEC, a tensioactive- and solvent-free paclitaxel formulation consisting of water-soluble paclitaxel/randomly methylated-β-cyclodextrin (Pac/RAMEB) complexes was developed previously. Using MTT and SRB assays the cytotoxic activity of this formulation versus Taxol®, was evaluated as well as the cytotoxicity of the different formulation excipients (RAMEB and Cremophor EL®). The possible synergistic effect of heat and paclitaxel-based chemotherapy during HIPEC was also evaluated in vitro. The cytotoxicity assays revealed differences in viability between Cremophor EL® and RAMEB treated cells of 40 and 50% for the CaCo-2 human and the CC531s rat colon cancer line, respectively, in favour of RAMEB. Despite the higher cytotoxicity of Cremophor EL®, Pac/RAMEB complexes and Taxol® were equipotent. Using the MTT and SRB assays the average difference in viability between both cell lines was below 10% and IC50 values showed no significant difference. Hyperthermia after drug administration (41 °C during 1 h) had no effect on cell viability. These results indicated that it was possible to reformulate paclitaxel with a less cytotoxic vehicle while maintaining the cytotoxic activity of the formulation and that there is no synergism between paclitaxel and heat for in vitro cytotoxicity.
Keywords: Paclitaxel; Hyperthermic intraperitoneal chemotherapy (HIPEC); Cytotoxicity; MTT; SRB; β-Cyclodextrin;

Studies on antibacterial dressings obtained by fluorinated post-discharge plasma by C. Canal; F. Gaboriau; S. Villeger; U. Cvelbar; A. Ricard (155-161).
Surface modification of wool, polyamide 6 and cotton fabrics was investigated with an Ar–CF4 post-discharge plasma. The radical F, as determined by optical emission spectroscopy, is considered to be the main active species acting on the fabrics and producing different effects as a function of the textile substrate. Fluorination of the surface is achieved on the three materials studied, but only wool and polyamide 6 fluorinated surfaces become hydrophobic at long treatment times, and show antibacterial properties. The treatment conditions used are mild enough so as not to alter surface topography, as confirmed by scanning electron and atomic force microscopy.
Keywords: Antibacterial; Wound dressings; Post-discharge plasma; Fluorine;

The effect of short-term current application (0.4 mA for 10 min) on the epidermal transport of two model penetrants (butyl paraben, BP; caffeine, CF) of differing lipohilicity was investigated and compared to that produced by employing an established method of skin penetration enhancement (delipidisation). The aim was to investigate the mechanism of enhancement and route of skin permeation associated with each penetrant and mode of treatment. Franz cell diffusion experiments were conducted using human epidermal sheets and a saturated buffer solution (pH 7.4) of the respective penetrant, at a pseudo-finite dose. The effects of electrode type (anodal or cathodal) and current treatment protocol (iontophoresis or post-iontophoresis) on solute permeation was found not to be significantly different (p  > 0.05). However, in contrast to BP, a significant increase in CF transport (3–5-fold) relative to untreated skin was observed when iontophoretic/post-iontophoretic treatment protocols were employed. The use of delipidised skin was found to enhance the permeation of both model penetrants to an extent greater than iontophoresis (BP: 3-fold; CF: 24-fold). Results from this study suggest that the permeation of the more hydrophilic CF across the skin, unlike BP, may involve multiple pathways. Electroperturbation of the epidermis was confirmed as the mechanism responsible for enhancing CF transport when electrical current was applied. Iontophoretic and post-iontophoretic enhancement may serve as a potential approach to enhance the topical delivery of CF in cosmetic or dermatological treatments (anti-cellulite, viral infections and psoriasis).
Keywords: Iontophoresis; Electroperturbation; Paraben; Caffeine; Skin permeability; Topical delivery;

Pulmonary drug delivery with aerosolizable nanoparticles in an ex vivo lung model by Moritz Beck-Broichsitter; Julia Gauss; Claudia B. Packhaeuser; Kerstin Lahnstein; Thomas Schmehl; Werner Seeger; Thomas Kissel; Tobias Gessler (169-178).
The use of colloidal carrier systems for pulmonary drug delivery is an emerging field of interest in nanomedicine. The objective of this study was to compare the pulmonary absorption and distribution characteristics of the hydrophilic model drug 5(6)-carboxyfluorescein (CF) after aerosolization as solution or entrapped into nanoparticles in an isolated rabbit lung model (IPL). CF-nanoparticles were prepared from a new class of biocompatible, fast degrading, branched polyesters by a modified solvent displacement method. Physicochemical properties, morphology, encapsulation efficiency, in vitro drug release, stability of nanoparticles to nebulization, aerosol characteristics as well as pulmonary dye absorption and distribution profiles after nebulization in an IPL were investigated.CF-nanoparticles were spherical in shape with a mean particle size of 195.3 ± 7.1 nm, a polydispersity index of 0.225 ± 0.017 and a ζ-potential of −28.3 ± 0.3 mV. Encapsulation efficiencies of CF were as high as about 60% (drug loading of 3% (w/w)); 90% of the entrapped CF were released during the first 50 min in vitro. Nanoparticle characteristics were not significantly affected by the aerosolization process utilizing a vibrating mesh nebulizer. After deposition of equal amounts of CF in the IPL, less CF was detected in the perfusate for CF-nanoparticles (plateau concentration 9.2 ± 2.4 ng/ml) when compared to CF aerosolized from solution (17.7 ± 0.8 ng/ml).In conclusion, the data suggest that inhalative delivery of biodegradable nanoparticles may be a viable approach for pulmonary drug delivery. Moreover, a targeting effect to the lung tissue is claimed.
Keywords: Nanoparticles; Biodegradable branched polyesters; Nebulization; Isolated lung; Pulmonary drug delivery; Targeting;

Formulation and pharmacokinetic evaluation of an asulacrine nanocrystalline suspension for intravenous delivery by Srinivas Ganta; James W. Paxton; Bruce C. Baguley; Sanjay Garg (179-186).
Asulacrine (ASL) is an inhibitor of topoisomerase II, which has shown potential against breast and lung cancer. It is a poorly water soluble drug. To allow intravenous (i.v.) administration, ASL was formulated as a nanocrystalline suspension by high pressure homogenization. The nanosuspension was lyophilized to obtain the dry ASL nanoparticles (average size, 133 ± 20 nm), which enhanced both the physical and chemical stability of the ASL nanoparticles. ASL dissolution and saturation solubility were enhanced by the nanosuspension. Differential scanning calorimetry and X-ray diffraction analysis showed that the crystallinity of the ASL was preserved during the high pressure homogenization process. The pharmacokinetics and tissue distribution of ASL administered either as a nanosuspension or as a solution were compared after i.v. administration to mice. In plasma, ASL nanosuspension exhibited a significantly (P  < 0.01) reduced Cmax (12.2 ± 1.3 μg ml−1 vs 18.3 ± 1.0 μg ml−1) and AUC0–∞ (18.7 ± 0.5 μg ml−1  h vs 46.4 ± 2.6 μg ml−1  h), and a significantly (P  < 0.01) greater volume of distribution (15.5 ± 0.6 l kg−1 vs 2.5 ± 0.1 l kg−1), clearance (1.6 ± 0.04 l h−1  kg−1 vs 0.6 ± 0.04 l h−1  kg−1) and elimination half-life (6.1 ± 0.1 h vs 2.7 ± 0.2 h) compared to the ASL solution. In contrast, the ASL nanosuspension resulted in a significantly greater AUC0–∞ in liver, lung and kidney (all P  < 0.01), but not in heart.
Keywords: Asulacrine; Nanosuspension; High pressure homogenization; Dissolution; Pharmacokinetics; Tissue distribution;

Lipid nanoparticles of the cancer drug Chlorambucil (CLB) were prepared by ultrasonication, using stearic acid as the core lipid. Four types of lipid nanoparticle formulations were studied: (i) stearic acid solid lipid nanoparticles (SLN); (ii) sterically stabilized SLN with pegylated phospholipids as stabilizer; (iii) nanostructured lipid complexes with oleic acid as adjunct lipid; (iv) lipid nanocomplexes with dimethyl dioctadecyl ammonium bromide (DDAB) as surface modifier (LN). Lipid nanoparticles were characterized for particle size, assay and encapsulation efficiency, particle morphology and physico-chemical stability over 90 days. All of the formulations were physically stable, with an average particle size of 147 (±10) nm. The drug encapsulation efficiency (DEE) of all the formulations except LN decreased significantly over time (p  < 0.05), probably due to the expulsion of CLB upon crystallization. This indicated that the presence of DDAB in stearic acid nanoparticles increases DEE, preventing CLB degradation in the aqueous disperse phase. Pharmacokinetic studies of the intravenous LN formulation revealed plasma clearance kinetics were comparable to that of CLB solution (p  > 0.01), indicating electrostatic charge mediated clearance, as reported earlier. In tissue and tumor distribution studies, lower AUC values of CLB were observed for LN compared to CLB solution in liver, kidneys, heart and lungs. However, higher AUC values of LN formulation as compared to CLB solution (p  < 0.01) in tumors suggested that the presence of DDAB on the lipid nanoparticles resulted in greater accumulation of the drug in tumors.
Keywords: Chlorambucil; Solid lipid nanoparticles; Lipid nanoparticles; Dimethyl dioctadecyl ammonium bromide; Pharmacokinetics; Stability;

Polymeric nanoparticles for siRNA delivery and gene silencing by Yogesh Patil; Jayanth Panyam (195-203).
Gene silencing using small interfering RNA (siRNA) has several potential therapeutic applications. In the present study, we investigated nanoparticles formulated using the biodegradable polymer, poly(d,l-lactide-co-glycolide) (PLGA) for siRNA delivery. A cationic polymer, polyethylenimine (PEI), was incorporated in the PLGA matrix to improve siRNA encapsulation in PLGA nanoparticles. PLGA-PEI nanoparticles were formulated using double emulsion-solvent evaporation technique and characterized for siRNA encapsulation and in vitro release. The effectiveness of siRNA-loaded PLGA-PEI nanoparticles in silencing a model gene, fire-fly luciferase, was investigated in cell culture. Presence of PEI in PLGA nanoparticle matrix increased siRNA encapsulation by about 2-fold and also improved the siRNA release profile. PLGA-PEI nanoparticles carrying luciferase-targeted siRNA enabled effective silencing of the gene in cells stably expressing luciferase as well as in cells that could be induced to overexpress the gene. Quantitative studies indicated that presence of PEI in PLGA nanoparticles resulted in 2-fold higher cellular uptake of nanoparticles while fluorescence microscopy studies showed that PLGA-PEI nanoparticles delivered the encapsulated siRNA in the cellular cytoplasm; both higher uptake and greater cytosolic delivery could have contributed to the gene silencing effectiveness of PLGA-PEI nanoparticles. Serum stability and lack of cytotoxicity further add to the potential of PLGA-PEI nanoparticles in gene silencing-based therapeutic applications.
Keywords: RNA interference; siRNA; poly(d,l-lactide-co-glycolide); Luciferase; Sustained release;

Nanocomposites coated with xyloglucan for drug delivery: In vitro studies by C. Ribeiro; G.G.C. Arizaga; F. Wypych; M.-R. Sierakowski (204-210).
Enalaprilate (Enal), an active pharmaceutical component, was intercalated into a layered double hydroxide (Mg/Al-LDH) by an ion exchange reaction. The use of a layered double hydroxide (LDH) to release active drugs is limited by the low pH of the stomach (pH ∼1.2), in whose condition it is readily dissolved. To overcome this limitation, xyloglucan (XG) extracted from Hymenaea courbaril (jatobá) seeds, Brazilian species, was used to protect the LDH and allow the drug to pass through the gastrointestinal tract. All the materials were characterized by X-ray diffraction, Fourier transform infrared spectroscopy, elemental analyses, transmission electronic microscopy, thermal analyses, and a kinetic study of the in vitro release was monitored by ultraviolet spectroscopy. The resulting hybrid system containing HDL–Enal–XG(3) slowly released the Enal. In an 8-h of test, the system protected 40% (w/v) of the drug. The kinetic profile showed that the drug release was a co-effect behavior, involving dissolution of inorganic material and ion exchange between the intercalated anions in the lamella and those of phosphate in the buffer solution. The nanocomposite coated protection with XG was therefore efficient in obtaining a slow release of Enal.
Keywords: Layered double hydroxide; Enalaprilate; Xyloglucan coating; Nanocomposite; Drug release;

The interaction between Pseudomonas aeruginosa cells and cationic PC:Chol:DOTAP liposomal vesicles versus outer-membrane structure and envelope properties of bacterial cell by Zuzanna Drulis-Kawa; Agata Dorotkiewicz-Jach; Jerzy Gubernator; Grzegorz Gula; Tomasz Bocer; Wlodzimierz Doroszkiewicz (211-219).
The interactions between cationic liposomal formulations (PC:Chol:DOTAP 3:4:3) and 23 Pseudomonas aeruginosa strains were tested. The study was undertaken because different antimicrobial results had been obtained by the authors for Pseudomonas aeruginosa strains and liposomal antibiotics (Drulis-Kawa, Z., Gubernator, J., Dorotkiewicz-Jach, A., Doroszkiewicz, W., Kozubek, A., 2006. The comparison of in vitro antimicrobial activity of liposomes containing meropenem and gentamicin. Cell. Mol. Biol. Lett., 11, 360–375; Drulis-Kawa, Z., Gubernator, J., Dorotkiewicz-Jach, A., Doroszkiewicz W., Kozubek, A., 2006. In vitro antimicrobial activity of liposomal meropenem against Pseudomonas aeruginosa strains. Int. J. Pharm., 315, 59–66). The experiments evaluate the roles of the bacterial outer-membrane structure, especially outer-membrane proteins and LPS, and envelope properties (hydrophobicity and electrostatic potential) in the interactions/fusion process between cells and lipid vesicles. The interactions were examined by fluorescent microscopy using PE-rhodamine-labelled liposomes. Some of the strains exhibited red-light emission (fusion with vesicles or vesicles surrounding the cell) and some showed negative reaction (no red-light emission). The main aim of the study was to determine what kinds of bacterial structure or envelope properties have a major influence on the fusion process. Negatively charged cells and hydrophobic properties promote interaction with cationic lipid vesicles, but no specific correlation was noted for the tested strains. A similar situation concerned LPS structure, where parent strains and their mutants possessing identical ladder-like band patterns in SDS-PAGE analysis exhibited totally different results with fluorescent microscopy. Outer-membrane protein analysis showed that an 18-kDA protein occurred in the isolates showing fusion with rhodamine-labelled vesicles and, conversely, strains lacking the 18-kDA protein exhibited no positive reaction (red emission). This suggests that even one protein may be responsible for favouring stronger interactions between Pseudomonas aeruginosa cells and cationic liposomal formulations (PC:Chol:DOTAP 3:4:3).
Keywords: Pseudomonas aeruginosa; Outer membrane; Liposomes; Interactions;

Noticeboard (220).