International Journal of Pharmaceutics (v.396, #1-2)

Approaches to improve the stability of the antiviral agent UC781 in aqueous solutions by Festo Damian; Judit Fabian; David R. Friend; Patrick F. Kiser (1-10).
In this work, we evaluated the chemical stability profiles of UC781 based solutions to identify excipients that stabilize the microbicidal agent UC781. When different antioxidants were added to UC781 in sulfobutylether-β-cyclodextrin (SBE-β-CD) solutions and subjected to a 50 °C stability study, it was observed that EDTA was a better stabilizing agent than sodium metabisulfite, glutathione or ascorbic acid. Some antioxidants accelerated the degradation of UC781, suggesting metal-catalyzed degradation of UC781. Furthermore, we observed substantial degradation of UC781 when stored in 1% Tween 80 and 1% DMSO solutions alone or in those with 10 mM EDTA. On the other hand, improved stability of UC781 in the presence of 100 and 200 mM of EDTA was observed in these solutions. The addition of both EDTA and citric acid in the stock solutions resulted in recovery of more than 60% of UC781 after 12 weeks. Generally, 10% SBE-β-CD in the presence of EDTA and citric acid stabilized UC781 solutions: the amount of UC781 recovered approaching 95% after 12 weeks of storage at 40 °C. We also showed that the desulfuration reaction of the UC781 thioamide involves oxygen by running solution stability studies in deoxygenated media. Improved stability of UC781 in the present study indicates that the incorporation of EDTA, citric acid and SBE-β-CD and the removal of oxygen in formulations of this drug will aid in increasing the stability of UC781 where solutions of the drug are required.
Keywords: UC781; Sulfobutylether-β-cyclodextrin; Complexation; Solubility; Stability; Microbicides;

Local gene expression and immune responses of vaginal DNA vaccination using a needle-free injector by Takanori Kanazawa; Yuuki Takashima; Toshiaki Tamura; Miki Tsuchiya; Yasunori Shibata; Haruhide Udagawa; Hiroaki Okada (11-16).
The vaginal mucosa is the most common site of initiation of virus infections that are transmitted through heterosexual intercourse, including HIV and papillomavirus. Thus, in order to prevent or treat these infections, strong vaginal immunity is required as the first line of defense. In this study, to establish a less invasive, safe, convenient and effective immunization method, we examined the local (skin and vagina) gene transfection efficiency of a non-needle jet injector for daily insulin injection. In the skin experiment, the needle-free injector resulted in a marked increase in marker gene expression, compared to the conventional needle-syringe injection. In addition, intradermal DNA vaccination using the needle-free injector dramatically induced IFN-γ and antibody systemic responses in mice. Furthermore, we investigated the applicability of the needle-free injector as a vaginal vaccination tool in rabbits. Vaginal gene expression using the needle-free injector was significantly greater than that using needle-syringe injection. Moreover, intravaginal vaccination by the needle-free injector promoted vaginal IgA secretion and IFN-γ mRNA expression in the blood lymphocytes, to a degree significantly higher than that by needle-syringe injection. In conclusion, local vaginal DNA vaccination using a needle-free jet injector is a promising approach for the prevention and treatment of mucosal infectious diseases.
Keywords: Vaginal DNA vaccine; Needle-free injector; Cellular immunity; Local humoral immunity;

Effective bacterial inactivation using low temperature radio frequency plasma by A. Sureshkumar; R. Sankar; Mahitosh Mandal; Sudarsan Neogi (17-22).
Staphylococcus aureus is one of the most common pathogens responsible for hospital-acquired infections. In this study, S. aureus was exposed to 13.56 MHz radiofrequency (RF) plasma generated by two different gases namely nitrogen and nitrogen–oxygen mixture and their sterilization efficacies were compared. Nitrogen plasma had a significant effect on sterilization due to generation of ultraviolet (UV) radiation. However, the addition of 2% oxygen showed enhanced effect on the sterilization of bacteria through nitric oxide (NO) emission and various reactive species. The presence of these reactive species was confirmed by optical emission spectroscopy (OES). Scanning electron microscopy (SEM) analysis was carried out to study the morphological changes of bacteria after plasma treatment. From the SEM results, it was observed that the bacterial cells treated by N2–O2 mixture plasma were severely damaged. As a result, a log10 reduction factor of 6 was achieved using N2–O2 plasma after 5 min treatment with 100 W RF power.
Keywords: Plasma sterilization; Staphylococcus aureus; Radio frequency; Optical emission spectroscopy; Scanning electron microscopy;

A novel method to measure the glass and melting transitions of pharmaceutical powders by Mohamad G. Abiad; David C. Gonzalez; Behic Mert; Osvaldo H. Campanella; M. Teresa Carvajal (23-29).
A method to measure thermo-mechanical properties of pharmaceutical and polymeric powders was developed. The measurements are conducted by characterizing the material's response to applied acoustic waves. Measurements were performed using griseofulvin, felodipine and indomethacin as model drugs and polyethylene oxide (MW = 200,000, 900,000, 2,000,000 Da) as model polymers. The method employed measures the mechanical impedance enabled the calculation of the powder rheological and thermo-mechanical properties. Measurements attained with this new technique are compared with measurements made using differential scanning calorimetry (DSC) and dynamic mechanical analysis (DMA). The new method detects the melting and glass transitions events while providing complementary information to that provided by DSC and DMA.
Keywords: Calorimetry (DSC); Glass transition; Mechanical properties; Thermal analysis; Mobility; Dynamic mechanical analysis; Squeezing flow;

Preserving enhancement in freeze-dried contrast agent ST68: Examination of excipients by Carl Solis; Flemming Forsberg; Margaret A. Wheatley (30-38).
The perfluorcarbon (perfluorobutane) ultrasound contrast agent ST68, composed of sonicated mixtures of non-ionic surfactants, is stable in solution for only a few weeks at 4 °C. Freeze-drying critically diminished ST68's ability to reflect ultrasound (its echogenicity). A method of incorporating specific lyoprotectants before lyophilization was investigated. Reintroduction of perfluorobutane to the protected freeze-dried sample, followed by reconstituting with water preserved echogenicity. Glucose, trehalose, sucrose, and mannitol were tested at 100 mM and in vitro echogenicity data was collected from samples with dose concentrations of 50–300 μl/l. Glucose was found to be the best lyoprotectant providing an average (n  = 3) maximum peak enhancement of 23.2 ± 1.2 dB in vitro, measured at 5 MHz, 684 kPa, and a pulse repetition frequency (PRF) of 100 Hz (p  < 0.05 over freeze-dried ST68 control) and 20.8 ± 0.8 dB in vivo in New Zealand white rabbits at 5 MHz and a PRF of 6.7 kHz. Pulse inversion harmonic US images of a rabbit kidney, pre- and post-contrast injection (0.1 ml/kg), showed excellent enhancement and clear vascular delineation, similar to that of the original agent. For the first time this contrast agent can be successfully freeze-dried yielding a longer self-life without the need for refrigeration.
Keywords: Ultrasound contrast agent; Freeze-drying; Lyoprotectant; Surfactant; Sugar; Glassy matrix;

Effect of food components and dosing times on the oral pharmacokinetics of nifedipine in rats by Qing-Ri Cao; Jing-Hao Cui; Jun Bom Park; Hyo-Kyung Han; Jaehwi Lee; Kyung Taek Oh; Inchoon Park; Beom-Jin Lee (39-44).
The present study was aimed to investigate the effect of food components and dosing time on the oral exposure of nifedipine in rats. Nifedipine was given orally to rats with and without food components at 8:00 a.m. (morning time) or 4:00 p.m. (evening time) during winter periods. Food components included milk, sodium chloride, oleic acid, and sodium taurocholate. Plasma concentration profiles of nifedipine showed double peak phenomena which were generally retained regardless of food components, vehicle types and the dosing time. Sodium chloride, milk and sodium taurocholate significantly increased the AUC while oleic acid did not, when drug was dosed in the morning time. After the dosing in the evening time, milk and sodium chloride significantly increased the plasma concentrations of nifedipine but oleic acid and sodium taurocholate decreased them. Overall, the systemic in vivo exposure of nifedipine was invariably lower with the evening dosing compared to the dosing in the morning, but this circadian rhythm dependency was not reversed by the multiple dosing of food components in rats. Food components and dosing time significantly altered the oral pharmacokinetics of nifedipine in rats, implying that the altered bioavailability and higher plasma concentrations in the morning time may influence dosing regimens of nifedipine for hypertension patients.
Keywords: Nifedipine; Food components; Multiple dosing; Dosing times; Oral pharmacokinetics; Double peak phenomena; Circadian zeitgeber;

We demonstrate drug release properties from hydroethanolic formulations as a function of the drug's lipophilicity (log  P), solvent lipophilicity and drug–polymer interactions, for the first time.A hydrophilic polymer, hydroxypropyl cellulose (HPC), provides the non-Fickian slower release of the lipophilic drug, lidocaine (log  P  = 2.6) and the burst (Fickian) release of hydrophilic drug, lidocaine hydrochloride (log  P  ≤ 0). Thus, log  P of drugs helps predict the drug release properties.Hydrophobic Eudragit polymers provided the burst release of lidocaine. However, the cationic hydrophobic polymer (Eudragit E100) retained more lidocaine (∼50%) topically than other hydrophobic polymers: Eudragit S100 (anionic) and Eudragit RLPO (cationic copolymer with quaternary ammonium group) (∼25% lidocaine retention) which release lidocaine systematically. Thus, minute changes in functional groups of hydrophobic polymers help tune the lidocaine release topically or systemically.An interaction between HPC and lidocaine as determined by FTIR helps the non-Fickian slower lidocaine release from HPC formulations. However, no interactions between lidocaine and hydrophobic Eudragit polymers explain the Fickian burst release of lidocaine from their formulations.A lipophilic solvent, isostearyl alcohol which when replacing ethanol by 30%, slows the release rate and enhances the topical adsorption of lidocaine. Thus, solvent lipophilicity also modulates drug release properties.
Keywords: Polymer chemistry; Drug–polymer interactions; Lipophilicity of drugs; Lipophilicity of solvents; Hydroethanolic gels;

Roller compaction of different pseudopolymorphic forms of Theophylline: Effect on compressibility and tablet properties by Ervina Hadžović; Gabriele Betz; Šeherzada Hadžidedić; Silvia Kocova El-Arini; Hans Leuenberger (53-62).
The effect of roller compaction on disintegration time, dissolution rate and compressibility of tablets prepared from Theophylline anhydrate powder, Theophylline anhydrate fine powder and Theophylline monohydrate was studied. In addition, the influence of adding microcrystalline cellulose, a commonly used excipient, in mixtures with these materials was investigated. Theophylline anhydrate powder was used as a model drug to investigate the influence of different compaction pressures on the tablet properties. Tablets with same porosity were prepared by direct compaction and by roller compaction/re-compaction. Compressibility was characterized by Heckel and modified Heckel equations. Due to the property of polymorphic materials to change their form during milling and compression, X-ray diffraction analysis of Theophylline anhydrate powder, Theophylline anhydrate fine powder and Theophylline monohydrate powders and granules was carried out. After roller compaction the disintegration time and the dissolution rate of the tablets were significantly improved. Compressibility of Theophylline anhydrate powder and Theophylline anhydrate fine powder was decreased, while Theophylline monohydrate showed higher compressibility after roller compaction. Microcrystalline cellulose affected compressibility of Theophylline anhydrate powder, Theophylline anhydrate fine powder and Theophylline monohydrate whereby the binary mixtures showed higher compressibility than the individual materials. X-ray diffraction analyses confirmed that there were no polymorphic/pseudopolymorphic changes after roller compaction.
Keywords: Roller compaction; Theophylline; Polymorphism; Disintegration; Dissolution; Compressibility;

The objectives of this study were to assess the utility of near infrared (NIR) spectroscopy for simultaneous in-line quantification of the contents of drug and excipients in tablets and to monitor the tabletting process in real time. Direct compression tablet formulations comprising micronized chlorpheniramine maleate, lactose, microcrystalline cellulose and magnesium stearate were used. A custom built NIR setup was used for in-line spectral acquisition (980–1900 nm with 1 nm resolution) during the tabletting process. Calibration models using dynamic spectral acquisition were prepared and validated using design of experiment approach. During tabletting, stratified sampling of tablets was also carried out to compare the NIR prediction results and subsequent UV analysis results for drug content. The results obtained with calibration and validation statistics confirmed the accuracy of models used to predict contents of tablet components. Stratified sampling results for drug content did not exhibit any significant statistical variation. However, in-line quantification enabled the content analysis of individual tablets in the production batch and detection of content uniformity problems towards the end of the tabletting process. Furthermore, it provided the assurance of in-process content uniformity monitoring of the individual excipients during the tabletting process.
Keywords: Near infrared (NIR) spectroscopy; Stratified sampling; In-line sampling; Tabletting; Content uniformity; Continuous quality monitoring;

Establishing a method to evaluate the reliability of an optimal solution is an exciting challenge for the nonlinear response surface method. We reported previously that the bootstrap (BS) resampling technique and Kohonen's self-organizing map are promising tools for meeting this challenge. To understand the usefulness of these techniques further, we employed a formulation optimization study of photocrosslinked polyacrylic acid (PAA) hydrogel as a case study. In a series of experiments, a large number of optimal solutions were generated with BS resampling and they were classified into three distinct clusters with SOM clustering. Using analysis of Bayesian estimation, we clarified the mode of generating clusters; e.g., cluster 2 was distinguished by the difference in features between the BS optimal solutions and the original optimal solution, whereas cluster 3 was distinguished by the substantial change in the shape of the response surfaces. We concluded that cluster 1 represents the global optimal solution, and then estimated 95% confidence intervals of the optimal solutions using the BS optimal solutions. These findings prove that our method is a valid approach for evaluating nonlinear optimal solutions. This method has applications for establishing a science-based rationale for, and a design space in, pharmaceutical formulation development.
Keywords: Formulation optimization; Confidence intervals; Bootstrap resampling technique; Kohonen's self-organizing map; Bayesian estimation; Dermatological patch;

The purpose of this study was to investigate the milling effect on the polymorphic transformation of four gabapentin (GBP) Forms I–IV in the absence of additive. Four polymorphs of GBP were previously prepared and identified, in which the GBP Form I was proven to be a monohydrate, but other GBP Forms II–IV belonged to anhydrate. The GBP Form II was the most stable polymorph available in the market. The co-milling process affecting the polymorphic stability of GBP Form II with different additives was also examined. During the 120-min-milling or co-milling course, the milled sample was withdrawn at prescribed intervals for Fourier transform infrared (FTIR) microspectroscopic determination. In the absence of additive, each polymorph of GBP exhibits a different polymorphic transformation behavior in the 120-min-milling course. The results indicate that GBP Form I was previously dehydrated and transited to Form II; GBP Form II was first transformed to Form III and then changed to Form IV; GBP Form III was previously transited to Form II, then changed to Form III and transformed to Form IV at last; whereas GBP Form IV was first changed to Form II, then transited to Form III and finally to Form IV. It was clearly evidenced that if GBP Form III or IV appeared in the milled mixture a little amount of GBP-lactam was certainly detected. In the presence of additives, there was almost lack of polymorphic transition for GBP Form II by co-milling GBP Form II with Emcompress, β-cyclodextrin, mannitol, corn starch or magnesium stearate. By co-milling GBP Form II with Avicel, dextrin, hydroxypropyl β-cyclodextrin, hydroxypropyl methylcellulose, Kollidon K-30 or gelatin, GBP Form II was transformed to Form IV alone. On the other hand, the GBP Form IV and a little amount of GBP-lactam were also found in the co-milled mixture after co-milling with GBP Form II with Aerosil or talc. This reveals that the solid-state transformation of GBP Form II after co-milling was markedly dependent on the types of additive used.
Keywords: Gabapentin; Milling; Co-milling; Polymorphic transition; Additives;

Enhanced dissolution of megestrol acetate microcrystals prepared by antisolvent precipitation process using hydrophilic additives by Eunbi Cho; Wonkyung Cho; Kwang-Ho Cha; Junsung Park; Min-Soo Kim; Jeong-Soo Kim; Hee Jun Park; Sung-Joo Hwang (91-98).
Microcrystals of megestrol acetate (MA), a poorly water-soluble drug, were successfully prepared using an antisolvent precipitation technique for improving the dissolution rate. The effective hydrophilic polymers and surfactants used were screened for their abilities to produce smaller particle sizes. Raw micronized MA and processed MA microcrystals were ranked by the Student–Newman–Keuls test in order of increasing particle size and SPAN values as follows: processed MA microcrystals in the presence of polymer and surfactant (mean diameter 1048 nm) < processed MA microcrystals in the presence of polymer (1654 nm) < processed MA microcrystals in the absence of polymer and surfactant (3491 nm) < raw micronized MA (4352 nm). The order of BET surface area was reversely ranked. Processed MA microcrystals in the presence of polymer and surfactant slightly decreased crystallinity and altered crystal habit and preferred orientation without change in polymorph. In addition, the dissolution properties of the processed MA microcrystals in the presence of polymer and surfactant were significantly enhanced as compared to that of the raw micronized MA. This effect is mainly due to a reduction in particle size resulting in an increased surface area. Therefore, it was concluded that the antisolvent precipitation technique in mild conditions could be a simple and useful technique to prepare poorly water-soluble drug particles with reduction in particle size, a narrow particle size distribution and enhanced dissolution properties.
Keywords: Megetsrol acetate; Microcrystals; Antisolvent precipitation; Hydrophilic additives; Dissolution properties;

Pulmonary delivery of isoxyl may increase drug efficacy by targeting alveolar macrophages which are host cells for Mycobacteria. Isoxyl microparticles (1–2 μm) were obtained by antisolvent precipitation and simultaneous spray drying method. The controls were made by mixing isoxyl solution in DMSO with cell culture media. Depending on the drug concentration, either isoxyl solution or nanosuspension was obtained in these controls. In the study, MTT (methylthiazol tetrazolium) and LDH (lactose dehydrogenase) assays were utilized to test cytotoxicity of these particle suspensions or solutions toward macrophages. Isoxyl microparticles and controls in concentrations up to 100 μg/ml were not toxic to macrophages. Both isoxyl microparticle suspensions and controls showed bactericidal activity, as estimated by death of mycobacteria inside the macrophages, at a concentration of 5 μg/ml.
Keywords: Pulmonary delivery; Phagocytosis; Microparticles; Nanoparticles; Cell viability; Efficacy;

The most appropriate method to preserve Bufferin 81-mg tablets dispensed for unit-dose packaging in the hospital pharmacy was examined. The surface color change of the tablets was investigated over time by spectrophotometry, and the decomposition rate of aspirin was measured by high-performance liquid chromatography (HPLC). To overcome these, it was found that we can effectively prevent color changes and preserve the quality by maintaining the humidity as 55% or less, storage with drying agent in a plastic or aluminum pack. It was revealed that the color changes became greater and the decomposition rate became higher as time passed. Color changes markedly affect the patients’ compliance, and are found to be a very important factor. It was considered that the clarity of the correlation between the color change and decomposition rate may contribute to a decrease in the number of tablets discarded before the expiration date.
Keywords: Aspirin tablet; Color change; Decomposition rate; Discard; Storage method;

The objective was to synthesize prodrugs of niacin and ketoprofen that target the human apical sodium-dependent bile acid transporter (ASBT) and potentially allow for prolonged drug release. Each drug was conjugated to the naturally occurring bile acid chenodeoxycholic acid (CDCA) using lysine as a linker. Their inhibitory binding and transport properties were evaluated in stably transfected ASBT-MDCK monolayers, and the kinetic parameters K i, K t, normJ max, and P p were characterized. Enzymatic stability of the conjugates was evaluated in Caco-2 and liver homogenate. Both conjugates were potent inhibitors of ASBT. For the niacin prodrug, substrate kinetic parameter K t was 8.22 μM and normJ max was 0.0917. In 4 h, 69.4% and 26.9% of niacin was released from 1 μM and 5 μM of the conjugate in Caco-2 homogenate, respectively. For the ketoprofen prodrug, K t was 50.8 μM and normJ max was 1.58. In 4 h, 5.94% and 3.73% of ketoprofen was released from 1 μM and 5 μM of the conjugate in Caco-2 homogenate, and 24.5% and 12.2% of ketoprofen was released in liver homogenate, respectively. In vitro results showed that these bile acid conjugates are potential prolonged release prodrugs with binding affinity for ASBT.
Keywords: Bile acid transporter; MDCK cells; Prolonged release; Ketoprofen; Niacin; Stability;

Hyperbranched poly(esteramides) as solubility enhancers for poorly water-soluble drug glimepiride by Sebastjan Reven; Jože Grdadolnik; Julijana Kristl; Ema Žagar (119-126).
The aim of this work was to study the feasibility of using hyperbranched polymers with highly branched structure and a large number of functional groups as solubilization enhancers for poorly water-soluble drugs. Antidiabetic drug glimepiride was used as a model drug and commercially available hyperbranched poly(esteramide)s as drug carriers.The results of in vitro dissolution studies showed significantly enhanced aqueous-solubility of glimepiride in the form of solid dispersions with hyperbranched poly(esteramide)s as compared to pure glimepiride in crystalline or amorphous form. The results of IR spectroscopic measurements revealed that improved solubility is a consequence of a complex formation between glimepiride and hyperbranched polymer. HB poly(esteramide)s with carbonyls of ester (O)–C=O and amide (N)–C=O groups serve mainly as a source of proton acceptor groups to which NH groups of glimepiride establish hydrogen bonds. Due to complex formation, glimepiride is within solid dispersions with HB polymers amorphous up to concentration of 5% (w/w) as revealed by X-ray powder diffraction measurements. Above this limit, glimepiride crystallizes as a separate phase during solvent evaporation.
Keywords: Antidiabetic drug; Glimepiride; Hyperbranched polymer; Solid dispersion; Amorphization; H-bond interaction; Solubility; Dissolution rate;

Pharmacokinetics of free-form levobupivacaine (LB) and free-form racemic bupivacaine (BU) using microdialysis sampling technique were conducted in this study. Three microdialysis probes were implanted in the jugular vein toward the right atrium, brain striatum and bile duct of male Sprague–Dawley rats for concurrently sampling free drug. Effects of P-glycoprotein (P-gp) on the brain distribution and hepatobiliary excretion of LB and BU were examined for the first time after cyclosporine (CsA) administration. LB and BU in samples were determined by HPLC. The blood pharmacokinetics of free LB and free BU were not significantly different. For brain pharmacokinetics, the CsA pretreatment raised significantly the area under curve (AUC) of BU (24.0 ± 5.9 vs. 14.6 ± 4.4 min μg/mL, p  = 0.015). Brain regions concentrations of LB and BU were significantly higher than plasma concentrations of LB (p  < 0.001) and BU (p  < 0.001), respectively. The BU concentration of cerebral cortex was significant higher than that of striatum (6.06 ± 1.03 vs. 4.04 ± 0.90 μg/mL, p  = 0.005) and hippocampus (6.06 ± 1.03 vs. 4.45 ± 1.07 μg/mL, p  = 0.04), suggesting that BU might display an uneven brain distribution. For bile pharmacokinetics, LB and BU went through hepatobiliary excretion, and the AUC of BU was significantly higher than that of the LB group (6.6 ± 1.0 vs. 4.6 ± 0.6 min μg/mL, p  = 0.005). In addition, the pretreatment of CsA significantly reduced the hepatobiliary excretion of BU in terms of AUC (4.4 ± 0.8 vs. 6.6 ± 1.0 min μg/mL, p  = 0.003). Furthermore, the maximum concentration (C max) of BU diminished significantly as a result of the CsA pretreatment (0.10 ± 0.03 vs. 0.20 ± 0.05 μg/mL, p  = 0.002). To sum up, enantioselective brain distribution and hepatobiliary excretion of free LB and free BU were observed, and P-gp may relate to the drug transport.
Keywords: Levobupivacaine; Bupivacaine; Microdialysis; Free-form pharmacokinetics;

Thermodynamic considerations of solvent/enhancer uptake into a model membrane by W.J. McAuley; G. Oliveira; D. Mohammed; A.E. Beezer; J. Hadgraft; M.E. Lane (134-139).
The aim of this study was to conduct a thermodynamic analysis of the uptake of solvents into a model membrane as a precursor to skin transport studies. The investigation was designed so that the methodology may be applied to analyse data produced from measurement of the uptake of enhancers into skin. The uptake of a series of alcohols into polydimethylsiloxane (silicone) membranes in the temperature range 5–45 °C was examined. A thermodynamic analysis of the data was performed to provide fundamental insight into the uptake process. A simple structure activity relationship was found to exist for the uptake of alcohols with a carbon chain length greater than four, with additional methylene groups exponentially decreasing the equilibrium uptake. Two separate straight lines were observed in the van’t Hoff plot for the equilibrium solvent uptake above and below 16 °C.The two separate straight lines in the van’t Hoff plot suggest a change in the mechanism of solvent uptake and solvent structure in the membrane above and below 16 °C. This is likely to have implications for the effect of the solvents on the partitioning of drugs into the membrane and will be used to provide insight into dynamic measurements of the effect of temperature on the transport of drug molecules in the same vehicles, across the membrane. The analysis described here should provide a useful methodology for investigating the uptake of solvents into model membranes.
Keywords: Solvent uptake; Membrane partitioning; Temperature effects; Thermodynamics; Polydimethylsiloxane;

An in vitro study of epoetin β intravenous injection site at the end of hemodialysis by Fabien Xuereb; Valérie de Précigout; Christian Combe; Gilles Sinnasse-Raymond; Marie-Claude Saux; Dominique Breilh (140-142).
The aim of this study (SITEPO™) is to evaluate the influence of the intravenous injection site (drip chamber injection site, venous injection site or venous fistula needle) on plasma concentration of epoetin β (Neorecormon®, Roche), a recombinant Human Erythropoietin (rHuEPO), at the end of in vitro hemodialysis sessions. No practical administration guidelines are available.Twenty 1-h dialysis sessions are performed. Before each dialysis, the circuit is filled with 270 ml, of heparinized total human blood whose hematocrit is adjusted to 35%. A common dosage of epoetin β in clinical practice (3000 IU) is studied for the three injection sites and for reference experiments in which rHuEPO is not injected into the dialysis circuit. Plasma concentrations of erythropoietin are measured by ELISA. The physiologically endogenous erythropoietin concentration is systematically determined and removed from the total epoetin β concentration.Average epoetin β plasma levels returned are not significantly different between the three injection sites and no significant rHuEPO loss is observed after injection into the drip chamber, the venous injection site and the venous fistula needle compared with reference experiments.The three intravenous injection sites of rHuEPO can be used at the end of dialysis without significant epoetin β loss.
Keywords: Anaemia; Epoetin β; Hemodialysis; Injection site; In vitro prediction; PVC tubing;

In this study, a dual-ligand liposomal system comprised of a specific ligand and a cell penetrating peptide (CPP) is described to enhance selectivity and cellular uptake. Dual-ligand PEGylated liposomes were prepared by modifying the end of the PEG with an NGR motif peptide, followed by a surface coating of the liposomes with stearylated oligoarginine (STR-RX). The NGR motif recognizes CD13, a marker protein located on tumor endothelial cells. A suitable number of RX units was determined to be R4, since it can be masked by the PEG aqueous layer. Although no enhanced cellular uptake was observed when a single modification of PEGylated liposomes with either NGR- or STR-R4 was used, the dual-modification with NGR and STR-R4 stimulated uptake of PEGylated liposomes by CD13 positive cells, and this uptake was superior to that obtained by PEG-unmodified liposomes modified with STR-R4. The dual-ligand system shows a synergistic effect on cellular uptake. Collectively, the dual-ligand system promises to be useful in the development efficient and specific drug delivery systems.
Keywords: Dual-ligand; Active targeting; Cell penetrating peptide; NGR; Tumor endothelial cells;

Nanosized particles of orlistat with enhanced in vitro dissolution rate and lipase inhibition by Andrej Dolenc; Biljana Govedarica; Rok Dreu; Petra Kocbek; Stane Srčič; Julijana Kristl (149-155).
Orlistat is locally acting inhibitor of gastrointestinal lipases which has been developed for the treatment of obesity. The present study was designed with the intent to formulate orlistat in a different way compared to the current practice and investigate its inhibition of gastrointestinal lipases. Orlistat is considered as a technologically problematic and unmanageable substance because of waxy nature, low melting point and low chemical stability. The manuscript presents the critical issues regarding engineering of its nanosuspension with controlled particle size by melt emulsification and high pressure homogenization. In order to formulate dry product, lactose was dissolved in nanosuspension as filler and spray drying has been performed for obtaining the final powder product. Laser diffraction, scanning electron microscopy and atomic force microscopy have been used for orlistat nanosuspension characterization, dissolution studies and lipase inhibition studies were performed to characterize the in vitro efficacy of formulated orlistat. The advantage of selected technological procedures is nanosized orlistat with elevated in vitro dissolution rate in comparison to raw drug, physical mixture and marketed product. Furthermore, nanosuspension demonstrated significantly higher in vitro lipase inhibition in comparison to references. To conclude, the results show new technological solution and remarkable increase of pharmacological effect which could potentially lead to decreasing the dose and consequently dose dependent side effects.
Keywords: Orlistat; Obesity; Nanosuspension; Dissolution; In vitro lipase inhibition; Melt emulsification; High pressure homogenization;

Cytotoxicity assessment of heparin nanoparticles in NR8383 macrophages by H. Eidi; O. Joubert; G. Attik; R.E. Duval; M.C. Bottin; A. Hamouia; P. Maincent; B.H. Rihn (156-165).
The bioavailability of low molecular weight heparin (LMWH) has been increased by encapsulation in nanoparticles. As a complement to these results, the cytotoxicity and apoptosis induced by LMWH nanoparticles prepared by two methods [nanoprecipitation (NP) and double emulsion (DE)] using Eudragit® RS (RS) and poly-ɛ-caprolactone (PCL) have been analysed. Particle sizes varied from 54 to 400 nm with zeta potential values between −65 and +63 mV. Our results showed that the method of nanoparticle preparation affects their properties, especially in terms of drug incorporation and cell tolerance. Cell viability ranged from 6% to 100% depending on the preparation method and physicochemical properties of the particles and the type of toxicity assay. Particle diameter and zeta potential seemed to be the most valuable cytotoxicity markers when cell viability was measured by Trypan blue exclusion and MTT respectively. Nanoparticles prepared by DE were better tolerated than those of NP. LMWH encapsulation into the cationic nanoparticles reduces remarkably their toxicity. Apoptosis evaluation showed activated caspases in exposed cells. However, no nuclear fragmentation was detected in NR8383 cells whatever the tested nanoparticles. DE nanoparticles of RS and PCL can be proposed as a good LMWH delivery system due to their low toxicity (IC50  ∼ 2.33 and 0.96 mg/mL, respectively).
Keywords: Nanoparticles; Nanoprecipitation; Double emulsion; Cytotoxicity; Macrophages; Apoptosis;

Cutanvoa Nanorepair Q10 cream, the first NLC containing cosmetical product introduced to the market in October 2005, was compared to an identical o/w cream without NLC with regards to particle size, melting behaviour, rheological properties and the in vivo effect on skin hydration. The consistency, the spreadability on the skin and the subjective feeling of increase in skin hydration were evaluated using a standardized questionnaire, and compared to hydration data measured. Furthermore, it was shown by epicutaneous patch test that Cutanova Nanorepair Q10 cream has no irritating effects on the skin. By laser diffraction (LD) and differential scanning calorimetry (DSC) measurements it could be shown that NLC are physically stable in Cutanova Nanorepair Q10 cream. After 7 days application of Cutanova Nanorepair Q10 cream and NLC negative control cream an increase in skin hydration could be objectively confirmed by measurements in vivo. From day 28 on the skin hydration measured in the test areas of Cutanova Nanorepair Q10 cream was significantly higher than the skin hydration in the test areas of the NLC negative control cream (p  = 0.05). The subjective feeling of increase in skin hydration was also rated from the volunteers as superior for Cutanova Nanorepair Q10 cream. The rheological properties of Cutanova Nanorepair Q10 cream contributed to a better subjective impression of consistency and spreadability on the skin than found for NLC negative control cream.
Keywords: Nanostructured lipid carriers (NLC); Dermal application; Coenzyme Q10; Cosmetic product; Skin hydration; Epicutaneous patch test;

A gene-delivery system specific for hepatoma cells and an intracellular kinase signal based on human liver-specific bionanocapsules and signal-responsive artificial polymer by Jun Oishi; Joohee Jung; Akira Tsuchiya; Riki Toita; Jeong-Hun Kang; Takeshi Mori; Takuro Niidome; Katsuyuki Tanizawa; Shun’ichi Kuroda; Yoshiki Katayama (174-178).
Recently, our group has proposed a novel gene-regulation system responding to cAMP-dependent protein kinase (PKA) that has been applied to living cells. In this study, human liver-specific bionanocapsules (BNCs) are used as a gene-delivery system to increase transfection efficiency and to target specific cell types. BNCs can efficiently deliver a target gene to human hepatocytes and hepatoma cells in vitro or in vivo. The combination of a signal-responsive gene-delivery system with BNCs led to an increase in the transfection efficiency and selectivity for hepatoma cells. Expression from the delivered gene was identified from PKA-activated hepatoma cells (HepG2), but not from colon tumor cells (WiDr). These results show that the combination of a gene-regulation system responding to an intracellular signal with BNC can be used for the selective treatment of human hepatoma cells.
Keywords: Gene therapy; Intracellular signal; Protein kinase; Tumor-targeted gene delivery; Hepatocellular carcinoma;

The object of this study was to design novel self-assembled liquid crystalline nanoparticles (cubosomes) as an ophthalmic delivery system for dexamethasone (DEX) to improve its preocular retention and ocular bioavailability. DEX cubosome particles were produced by fragmenting a cubic crystalline phase of monoolein and water in the presence of stabilizer Poloxamer 407. Small angle X-ray diffraction (SAXR) profiles revealed its internal structure as Pn3m space group, indicating the diamond cubic phase. In vitro, the apparent permeability coefficient of DEX administered in cubosomes exhibited a 4.5-fold (F1) and 3.5-fold (F2) increase compared to that of Dex-Na phosphate eye drops. Preocular retention studies revealed that the retention of cubosomes was significantly longer than that of solution and carbopol gel, with AUC0→180 min of Rh B cubosomes being 2–3-fold higher than that of the other two formulations. In vivo pharmacokinetics in aqueous humor was evaluated by microdialysis, which indicated a 1.8-fold (F1) increase in AUC0→240 min of DEX administered in cubosomes relative to that of Dex-Na phosphate eye drops, with about an 8-fold increase compared to that of DEX suspension. Corneal cross-sections after incubation with DEX cubosomes demonstrated an unaffected corneal structure and tissue integrity, which indicated the good biocompatibility of DEX cubosomes. In conclusion, self-assembled liquid crystalline nanoparticles might represent a promising vehicle for effective ocular drug delivery.
Keywords: Cubosome; Dexamethasone; Ocular bioavailability; Ophthalmic delivery; Preocular retention;

Physicochemical and pharmacological characterization of α-tocopherol-loaded nano-emulsion system by Junya Hatanaka; Hina Chikamori; Hideyuki Sato; Shinya Uchida; Kazuhiro Debari; Satomi Onoue; Shizuo Yamada (188-193).
The main purpose of the present study is to develop a novel nano-emulsion (NE) formulation of α-tocopherol (α-TC) with enhanced oral bioavailability and pharmacological effects. Three NE formulations of α-TC at different loading amounts (10%, 30% and 50%) were prepared by a mechanochemical method. Physicochemical properties of NE formulations were characterized with a focus on the morphology by transmission electron microscopy (TEM), droplet size distribution and zeta-potential by dynamic light scattering (DLS), and long-term stability. According to the TEM images and DLS data, mean diameters of NE droplets ranged from 80 to 400 nm, in proportion to the amount of loaded α-TC. Although all NE formulations of α-TC were found to be negatively charged with the zeta-potential of ca −40 mV, NE formulations at α-TC content of 30% or higher exhibited severe aggregation of droplets in NE formulations during long-term storage. After oral administration of 10% α-TC-loaded NE formulation (30 mg α-TC/kg) in rats, higher α-TC exposure was observed with a 2.6-fold increase of bioavailability as compared to the control mixture of oil and α-TC. In streptozotocin-induced diabetic rats, oral administration of the α-TC-loaded NE formulation (30 mg α-TC/kg) exhibited a significant reduction of lipoperoxidant in several organs, especially the liver; however, the control mixture was less effective. With these findings, the NE approach might be efficacious to improve the oral bioavailability and anti-oxidative activities of α-TC.
Keywords: α-Tocopherol; Nano-emulsion; Absorption; Oxidative stress; Lipid peroxidation;

In vitro characterization and in vivo toxicity study of repaglinide loaded poly (methyl methacrylate) nanoparticles by U.M. Dhana lekshmi; G. Poovi; Narra Kishore; P. Neelakanta Reddy (194-203).
With the objective to achieve prolonged drug release, especially for the treatment of diabetes mellitus, and thereby to reduce the side effects of administration of conventional dosage form, repaglinide loaded PMMA nanoparticles have been formulated. These nanoparticles have been developed by solvent evaporation method and were subjected to various studies for characterization including photon correlation spectroscopy (PCS), scanning electron microscopy (SEM) and X-ray diffraction (XRD). These studies favorably revealed that the mean particle diameter of optimized formulation was 108.3 nm and had spherical morphology with amorphous nature.Moreover, these particles were also subjected to Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and thermo gravimetric analysis (TGA) for compatibility analysis between drug and polymer. The results were positive and showed that, there were no interaction between drug and polymer. The optimized formulation demonstrated favorable in vitro prolonged release characteristics. Experimental in vitro release data were substituted with available mathematical models to establish the mechanism of release of repaglinide and was found to follow zero order, diffusion and erosion mechanisms.The in vivo toxicity study in albino rats showed no significant change in biochemical and pathological examinations. Hence, the designed system could possibly be advantageous in terms of prolonged release, to achieve reduced dose frequency and improve patient compliance of repaglinide.
Keywords: Repaglinide; Anti-diabetic; PMMA; Nanoparticles;

Post-insertion into Lipid NanoCapsules (LNCs): From experimental aspects to mechanisms by Thomas Perrier; Patrick Saulnier; Florian Fouchet; Nolwenn Lautram; Jean-Pierre Benoît (204-209).
Over the last decade, Lipid NanoCapsules (LNCs) have been intensively used as effective drug delivery systems; they are classically prepared using a phase-inversion method. Following formulation of the LNCs, the molecular insertion of commercially-available disteraoylphosphatidylethanolamine-peg amphiphiles is performed into the LNC shell, using a post-insertion method, more classically applied with liposomes. The subsequent LNC interfacial modifications are investigated by using size and electrokinetic measurements. More particularly, the length and the nature of the hydrophilic part of the post-inserted surfactant are modified. The results are discussed in order to improve our understanding of post-insertion mechanisms.
Keywords: Lipid NanoCapsules; Post-insertion; Amphiphilic phospholipids; Soft-particle analysis; Electrokinetic measurements;

New drug substances from early development are often poorly water-soluble, which causes poor bioavailability upon peroral administration and hampers drug administration through other routes such as the parenteral or ocular routes. One approach to improve drug solubility and administration flexibility is by wet milling to nanosize. Particle size reduction increases the surface energy which requires adequate stabilization by excipients. In this study, the practically water-insoluble miconazole was nanoground, and a variety of surface active and polymeric excipients were tested for their stabilizing effects. For efficient milling, two preformulation criteria had to be fulfilled: a relatively low contact angle (<70°) and high dispersibility of the native drug particles in the milling medium. Hydroxypropylcellulose (HPC-LF) in combination with sodium dodecyl sulfate (SDS) stabilized best the miconazole nanosuspensions. A design of experiments was used to achieve drug particle mean sizes of 140–170 nm by varying the concentrations of miconazole (5 and 20%, w/w), SDS (0.05 and 0.2%, w/w), and HPC-LF (1.25 and 5%, w/w). Further experiments revealed that minimal 0.0125% SDS and 3.125% HPC-LF were required for miconazole nanogrinding and nanosuspension stabilisation. Storage of the nanosuspensions at 5 °C for up to 6 months caused only minor changes, whereas storage at 25 °C resulted in particle agglomeration and single crystal growth. Altogether the study showed that excellent wetting of drug particles as well as their electrostatic and steric stabilization by excipients is necessary to produce stable nanosuspensions by nanogrinding.
Keywords: Nanosuspension; Nanogrinding; Nanonization; Suspension stability; Miconazole; Drug solubility;

New self-assembling polyaspartylhydrazide copolymer micelles for anticancer drug delivery by Mariano Licciardi; Gennara Cavallaro; Mauro Di Stefano; Giovanna Pitarresi; Calogero Fiorica; Gaetano Giammona (219-228).
A new amphiphilic copolymer have been synthesized starting from the hydrosoluble polyaspartylhydrazide (PAHy) polymer, by grafting both hydrophilic PEG2000 chains and hydrophobic palmitic acid (C16) moieties on polymer backbone, and the structure of obtained PAHy–PEG2000–C16 copolymer have been characterized by 2D 1H/13C NMR experiments. PAHy–PEG2000–C16 copolymer showed the ability of self-assembling in aqueous media giving a core–shell structure and resulted potentially useful for encapsulating and dissolving hydrophobic drug. The formation of micellar core–shell structure has been investigated by 2D 1H NMR NOESY experiments. The presence of cross-peaks for protons of C16 and PAHy portions, indicated that the two domains are in close proximity forming micelle core. The critical aggregation concentration (CAC) values of PAHy–PEG2000–C16 amphiphilic graft copolymer was determined in water by fluorescence technique, and it was demonstrated that PAHy–PEG2000–C16 micelles are well suited to be micellar vehicle of highly hydrophobic molecules. Therefore, anticancer drug tamoxifen, used as a model hydrophobic molecule, was loaded into PAHy–PEG2000–C16 micelles obtaining an increase of drug solubility of about 3000 times. Transmission electron microscopy (TEM) observations showed the spherical morphology of micelles formed by PAHy–PEG2000–C16 copolymer with a mean diameter of about 30 nm, as confirmed also by dynamic light scattering (DLS) studies. Finally, in vitro cell viability studies were carried out on human breast cancer cells (MCF-7) testing the pharmacological activity of tamoxifen-loaded PAHy–PEG2000–C16 micelles, in comparison with free tamoxifen at different drug concentrations, demonstrating that tamoxifen-loaded PAHy–PEG2000–C16 micelles exhibited a concentration-dependent cytotoxic activity.
Keywords: Polyaspartylhydrazide (PAHy); Self-assembling copolymer; Polymeric micelles; Tamoxifen;

Development of cell-penetrating peptide-modified MPEG-PCL diblock copolymeric nanoparticles for systemic gene delivery by Ko Tanaka; Takanori Kanazawa; Yasunori Shibata; Yumiko Suda; Tsunehiko Fukuda; Yuuki Takashima; Hiroaki Okada (229-238).
To develop a safe and efficient systemic non-viral gene vector, methoxy poly(ethylene glycol) (MPEG)/poly(ɛ-caprolactone) (PCL) diblock copolymers conjugated with a Tat analog through the ester or disulfide linkage were synthesized and their suitability as a systemic non-viral gene carrier evaluated. The physicochemical properties of the MPEG-PCL diblock copolymers were determined by GPC, 1H NMR and FT-IR spectroscopy. The particle sizes and in vitro (COS7 and S-180 cells) transfection efficiencies and cytotoxicity were evaluated. Furthermore, the luciferase activity was then determined in various tissues after intravenous injection of MPEG-PCL-SS-Tat/pCMV-Luc complex into mice bearing S-180 cells. The particle sizes of the MPEG-PCL-Tat copolymers with or without pDNA were about 40 and 60 nm, respectively. The luciferase activity in COS7 cells transfected with pCMV-Luc with MPEG-PCL-ester-Tat or MPEG-PCL-SS-Tat was higher than that with pDNA only. MPEG-PCL-SS-Tat greatly increased the transfection efficiency compared to MPEG-PCL-ester-Tat in COS7 and S-180 cells. In an in vitro cytotoxicity test MPEG-PCL-SS-Tat did not induce any remarkable cytotoxicity. In an in vivo experiment, the synthesized MPEG-PCL-SS-Tat copolymers promoted the delivery and expression of pDNA into tumor tissue in tumor-bearing mice. In conclusion, this vector might be applicable as a tumor-targeting non-viral systemic gene carrier in the clinical setting.
Keywords: Block copolymeric nanoparticles; Tat analog; Disulfide linkage; Polymer micelles; Systemic non-viral gene carrier;

Preparation and pharmaceutical characterization of amorphous cefdinir using spray-drying and SAS-process by Junsung Park; Hee Jun Park; Wonkyung Cho; Kwang-Ho Cha; Young-Shin Kang; Sung-Joo Hwang (239-245).
The aim of this study was to investigate the effects of micronization and amorphorization of cefdinir on solubility and dissolution rate. The amorphous samples were prepared by spray-drying (SD) and supercritical anti-solvent (SAS) process, respectively and their amorphous natures were confirmed by DSC, PXRD and FT-IR. Thermal gravimetric analysis was performed by TGA. SEM was used to investigate the morphology of particles and the processed particle had a spherical shape, while the unprocessed crystalline particle had a needle-like shape. The mean particle size and specific surface area were measured by dynamic light scattering (DLS) and BET, respectively. The DLS result showed that the SAS-processed particle was the smallest, followed by SD and the unprocessed cefdinir. The BET result was the same as DLS result in that the SAS-processed particle had the largest surface area. Therefore, the processed cefdinir, especially the SAS-processed particle, appeared to have enhanced apparent solubility, improved intrinsic dissolution rate and better drug release when compared with SD-processed and unprocessed crystalline cefdinir due not only to its amorphous nature, but also its reduced particle size. Conclusions were that the solubility and dissolution rate of crystalline cefdinir could be improved by physically modifying the particles using SD and SAS-process. Furthermore, SAS-process was a powerful methodology for improving the solubility and dissolution rate of cefdinir.
Keywords: Cefdinir; Amorphous; Anhydrous; Micronization; Supercritical anti-solvent (SAS);