International Journal of Pharmaceutics (v.307, #2)

Development of photostable sunscreens is extremely important to preserve the UV protective capacity and to prevent the reactive intermediates of photounstable filter substances behaving as photo-oxidants when coming into direct contact with the skin. Thus, the objective of this study was to evaluate the photostability of four different UV filter combinations in a sunscreen by using HPLC analysis and spectrophotometry. The formulations that were investigated included four different UV filter combinations often used in SPF 15 sunscreens. The UV filter combinations were: octyl methoxycinnamate (OMC), benzophenone-3 (BP-3) and octyl salicylate (OS) (formulation 1); OMC, avobenzone (AVB) and 4-methylbenzilidene camphor (MBC) (formulation 2); OMC, BP-3 and octocrylene (OC) (formulation 3); OMC, AVB and OC (formulation 4). In the photostability studies, 40 mg of each formulation were spread onto a glass plate and left to dry before exposure to different UVA/UVB irradiation. Exposed samples were then immersed in isopropanol and the dried film dissolved ultrasonically. The filter components in the resulting solution were quantified by HPLC analysis with detection at 325 nm and by spectrophotometry. In this study, the four UV filter combinations showed different photostability profiles and the best one was formulation 3 (OMC, BP-3 and OC), followed by formulations 4, 1 and 2. In addition, OC improved the photostability of OMC, AVB and BP-3.
Keywords: Sunscreens; Photostability; Octyl methoxycinnamate; Avobenzone;

Rheological, adhesive and release characterisation of semisolid Carbopol/tetraglycol systems by Giulia Bonacucina; Marco Cespi; Monica Misici-Falzi; Giovanni F. Palmieri (129-140).
Gels dosage forms are successfully used as drug delivery systems considering their ability to control drug release and to protect medicaments from an hostile environment. This study deals with the gelation properties of Carbopol 971 e 974 polymeric systems in tetraglycol, a water-miscible cosolvent. In this paper, the attention was noted of the thickening properties of the different Carbopol in tetraglycol solvent at increasing temperatures, in order to obtain gels avoiding neutralisation and, at the same time, to make possible the dissolution in these gels of insoluble or poorly soluble water drugs. Samples were prepared by simply dispersing different Carbopols amount (0.5–4%) were added to tetraglycol and different systems were prepared at room temperature and by heating at 70 °C. All these systems were then characterised rheologically. Frequency sweep, creep-recovery, temperature sweep and time sweep analyses outlined that Carbopol 971 and 974 in tetraglycol gave rise after heating to gels with satisfactory rheological behaviour: the elastic modulus was greater than the viscous one and a remarkable elastic character was found to be present.Systems obtained by heating procedure were examined also from a mechanical point of view using a texture profile analysis. Besides, being Carbopols well known mucoadhesive polymers, gels adhesive properties were also studied using the ex vivo method. Texture and adhesion characterisation confirmed rheological results pointing out a certain greater elasticity and adhesiveness of Carbopol 974 systems. Then, the possible cutaneous irritation was also tested using the in vivo method (Draize test). No signs of cutaneous irritation were obtained for all the samples that were analysed.After rheological and mucoadhesive properties were set, paracetamol as a model drug was inserted in the composition of the gels and the release characteristics were defined. Dissolution tests pointed out the greater release control properties of tetraglycol/Carbopol 971 samples. These studies showed tetraglycol/Carbopol systems as a first-rate alternative to traditional water gels when low water-soluble drugs have to be added.
Keywords: Hydrogels; Viscoelasticity; Viscosity; Mechanical properties; Mucoadhesion; Controlled release;

Surface treatment: A potential approach for enhancement of solid-state photostability by Alyaa Ramadan; Magda El-Massik; Labiba El-Khordagui; Nabila Daabis; Youssef Hammouda (141-149).
The potentials of a simple surface treatment technique aiming at modifying solid-state properties with emphasis on photostability were investigated using methyldopa (MD), a photosensitive drug substance. MD was treated with a preselected solvent by stirring a drug suspension in the solvent in a predetermined solid/solvent ratio under controlled conditions. At the end of the solvent treatment period, MD powder was separated, dried and screened. Changes in the solid-state properties of surface-treated MD were monitored using flowability measurements, scanning electron microscopy, thermal analysis and dissolution rate. Further, photostability testing, according to the ICH guidelines, was conducted using compressed disks of MD treated with solvents in the absence and presence of antioxidants, namely ascorbic acid, butylated hydroxytoluene (BHT) and cysteine HCl. The color change (ΔE) was determined according to the CIELAB system. Surface treatment of MD drug substance with ethanol, methanol and isopropanol resulted in a marked improvement in flowability which was associated with morphological crystalline changes. Treatment of MD with methanol provided free flowing spherical agglomerates. Disks of solvent-treated MD showed improved photostability which was further potentiated by the inclusion of antioxidants, although only traces of the antioxidant were retained in the treated powder. Tablets containing MD surface treated with methanol containing a mixture of 2% ascorbic acid and 0.2% BHT were prepared by direct compression using a simple formula. The tablets conformed to official requirements.
Keywords: Surface treatment; Methyldopa; Antioxidants; Photostability; CIELAB system; Flowability;

Quantification of low levels of amorphous content in sucrose by hyperDSC by Minna Lappalainen; Ilkka Pitkänen; Päivi Harjunen (150-155).
A method was developed for the quantification of low levels of amorphous content in sucrose with hyperDSC. The method was based on the fact that the change of specific heat at the glass transition is linearly proportional to the amorphous content. It was found out that as annealing time increased, the glass transition temperature moved to a higher temperature and the change of specific heat increased. ΔC p for annealed totally amorphous sucrose was 0.761 ± 0.012 J g−1  °C−1. Synthetic mixtures with various proportions of crystalline and amorphous sucrose were prepared. The following linear regression between ΔC p and amorphous content was obtained: ΔC p  = 0.0075x  − 0.00484 (R  = 0.999). The limit of detection (LOD) and the limit of quantification (LOQ) values were 0.062 and 0.207%, respectively. The effect of grinding time on the amorphous content of crystalline sucrose was studied and a correlation between grinding time and amorphous content of sucrose was found. It was also found that the amorphous content could only attain a value of about 80–90% by grinding in the way used in this study.
Keywords: Sucrose; Amorphous content; Glass transition; HyperDSC;

Effects of absorption promoters on insulin absorption through colon-targeted delivery by Masataka Katsuma; Shunsuke Watanabe; Hitoshi Kawai; Shigeo Takemura; Kazuhiro Sako (156-162).
The aim of this study were to investigate the effect of sodium glycocholate (GC-Na) as an absorption promoter and the effects of the co-administration of GC-Na and various absorption promoters on orally administered insulin absorption utilizing a colon-targeted delivery system. The system containing insulin and GC-Na (CDS) was administered to dogs, and plasma glucose and insulin levels were then measured at 24 h after administration. For CDS, the C max in plasma glucose level was significantly higher than a reference formulation without GC-Na. The pharmacological availability for CDS was not significantly higher than the reference formulation. In contrast, CDS with poly(ethylene oxide) as a gelling agent (CDSP) showed prolonged hypoglycemia effects. The pharmacological availability was 5.5% and significantly different from the reference formulation. The absolute bioavailability for CDS was 0.25%, and for CDSP it was 0.42%. Consequently, the results of this study demonstrated that colon-specific delivery of insulin with GC-Na was more effective in increasing hypoglycemic effects after oral administration, and the combination of GC-Na and poly(ethylene oxide) tended to prolong the colonic absorption of insulin and might be more effective for improvement of orally administered insulin absorption utilizing the colon-targeted delivery system.
Keywords: Absorption promoters; Colon-targeted delivery; Sodium glycocholate; Insulin; Lactulose; Enterobacteria;

Sorption of unoprostone isopropyl to packaging materials by Michelle Wong; Robert Marion; Ken Reed; Youmin Wang (163-167).
This study investigated the stability of an ophthalmic solution formulation of unoprostone isopropyl (UI), a prostaglandin like compound, in two types of packaging materials, polypropylene (PP) and low-density polyethylene (LDPE). We determined the concentration of UI and its degradation products as a function of time and found that the rate of disappearance of drug was faster for the formulation stored in LDPE bottles than that stored in PP bottles. Further studies indicated that the inferior stability observed with the LDPE packaging was primarily due to the sorption of UI to the packaging material and to a lesser degree, chemical degradation. The sorption was temperature dependent, lowering the temperature reduced the sorption, thus improving the shelf-life of the product.
Keywords: Sorption; Polypropylene; LDPE; Stability; Unoprostone isopropyl;

Positively charged mitoxantrone (MTO) was absorbed by negatively charged blank bovine serum albumin (BSA) and chitosan (CS) nanospheres to form MTO–BSA–NS and MTO–CS–NS, respectively. In addition to other conditions, values of pH of every step were optimized. On optimized conditions, MTO–BSA–NS of a mean size of 77 nm with an encapsulation yield (EY) of (98.86 ± 1.43)% [drug loading rate (DL) (19.82 ± 0.29)%] and MTO–CS–NS of a mean size of 75 nm with an EY of (97.57 ± 1.00)% [DL (9.78 ± 0.10)%] were obtained. After lyophilization and sterilization by 60Co, the mean size increased about 10% but no significant change was observed in EY and DL. Tests for in vitro release in physiological saline or physiological saline containing 0.5% (w/v) ascorbic acid by a dialysis bag showed sustained release and little burst effect.
Keywords: Mitoxantrone; Bovine serum albumin; Chitosan; Nanospheres; Nanoprecipitation; Ion gelation; Lyophilization;

Bovine serum albumin (BSA) and chitosan (CS) nanospheres of mitoxantrone (MTO) were comparatively evaluated in terms of tissue distribution, acute toxicity and therapeutic efficiency against breast cancer and its lymph node metastases. After local injection in rats, MTO nanospheres showed a slower elimination rate and a much higher drug concentration in lymph nodes compared with MTO solution, and a lower drug concentration in other tissues. There was no observed acute toxicity to the main tissues of Kunming mice after local injection of MTO-BSA-NS. Mild toxicity to liver and lung was observed for MTO-CS-NS, but, for MTO solution, severe toxicity to liver and lung and much lower number of white blood cells were observed. Human MCF-7 breast cancer in nude mice and animal model of P388 lymph node metastases in Kunming mice were applied to investigate the therapeutic efficiency. The inhibition rate of the nanospheres against breast cancer was much higher than that of MTO solution, and lymph node metastases were efficiently inhibited by the nanospheres, especially MTO-BSA-NS.
Keywords: Mitoxantrone; Site-specific delivery; Bovine serum albumin; Chitosan; Nanospheres; Breast cancer; Lymph node metastases;

Monitoring the ingestion of anti-tuberculosis drugs by simple non-invasive methods by F.A. Sirgel; J.S. Maritz; A. Venter; G. Langdon; P.J. Smith; P.R. Donald (182-187).
This investigation retrospectively assessed inexpensive non-invasive qualitative methods to monitor the ingestion of anti-tuberculosis drugs isoniazid, rifampicin and rifapentine. Results showed that commercial test strips detected the isoniazid metabolites isonicotinic acid and isonicotinylglycine as efficiently as the isonicotinic acid method in 150 urine samples. The presence of rifamycins in urine samples (n  = 1085) was detected by microbiological assay techniques and the sensitivity compared to the n-butanol extraction colour test in 91 of these specimens. The proportions detected by the two methods were significantly different and the sensitivity of the n-butanol procedure was only 63.8% (95% CL 51.2–76.4%) as compared to that of the superior microbiological method. Final validation (n  = 691) showed that qualitative assays measure isoniazid and rifamycin ingestion with an efficiency similar to high-performance liquid chromatography. The qualitative procedures may therefore be valuable in clinical trials and in tuberculosis clinics to confirm drug ingestion.
Keywords: Anti-tuberculosis drugs; Monitoring adherence; Urine testing;

Epidermal cell culture model with tight stratum corneum as a tool for dermal gene delivery studies by Lauri Paasonen; Maarit Korhonen; Marjo Yliperttula; Arto Urtti (188-193).
The purpose of this study was to evaluate the feasibility of organotypic cultures of rat epidermal cells as a tool to study non-invasive dermal gene delivery. Also, a novel transfection method employing liposomal pre-treatment of stratum corneum (SC) was evaluated. Rat epidermal cells were cultured on Transwell tissue culture inserts and formation of stratum corneum barrier was evaluated in permeability studies with two model compounds. Transfections were performed with naked pCMV-SEAP2 plasmid and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)/dioleyl-phosphatidylethanolamine (DOPE)/DNA lipoplexes. Naked DNA was administered on the stratum corneum of the cell culture model with or without prior treatment of the stratum corneum with DOTAP/DOPE liposomes. Transfection was evaluated non-invasively by monitoring concentrations of secreted alkaline phosphatase (SEAP) in the culture medium of the basolateral compartment at 24-h intervals. Transfection with lipoplexes produced significant gene expression in rat epidermal keratinocyte (REK) epidermal culture model. Likewise, delivery of naked DNA on stratum corneum after DOTAP/DOPE liposome pre-treatment produced gene expression. Naked DNA alone did not result in detectable gene expression. In dermal gene delivery studies REK epidermal culture model is a suitable tool that includes tight stratum corneum and allows transgene expression in viable epidermis and non-invasive sampling of secreted gene product in the basolateral compartment. Liposomal pre-treatment of the stratum corneum augments transfection of viable epidermis.
Keywords: Gene delivery; Dermal; Cell culture model; Liposome;

Hydroxyethylstarch microcapsules: A preliminary study for tumor immunotherapy application by J. Devy; E. Balasse; H. Kaplan; C. Madoulet; M.-C. Andry (194-200).
The objective of this work was to prepare microcapsules which would allow protection and slow release of antigens used for melanoma immunotherapy treatment. Hydroxyethylstarch (HES) microcapsules were prepared using interfacial cross-linking with terephthaloyl chloride (TC). They were characterized with respect to morphology (microscopy) and size (in the 4–15 μm range). Bovine serum albumin (BSA) was used as model protein for loading and release studies. Microcapsules were loaded with solutions at different protein concentrations (0.5–5%). The maximum loading efficiency (20%) was observed with the concentration of 2.5%, which allowed a loading capacity near 100%. Confocal laser scanning microscopy (CLSM) visualization showed that BSA was entrapped within the microcapsules and not only associated to their outer surface. BSA-release studies showed a 20% BSA release within 30 min while 80% remained entrapped in the microcapsules for 4 days. Microcapsules were degraded by α-amylase and addition of esterase to α-amylase enhanced slightly their degradation. In vitro studies on melanoma cells showed that HES microcapsules were non-toxic. Preliminary in vivo studies demonstrated that microcapsules were biodegradable after intraperitoneal injection (i.p.). The observation of peritoneal wash showed a complete degradation within 7 days, indicating a possible application as an in vivo drug delivery system especially to enhance the presentation of antigens.
Keywords: Hydroxyethylstarch; Biodegradable microcapsules; Confocal microscopy; Loading efficiency; Cytotoxicity; Enzymatic degradation;

A calibration strategy for the continuous monitoring of solvent-mediated phase transition was developed using in situ Raman spectroscopy. Citric acid which exhibits enantiotropy during its anhydrous/monohydrate phase transition was selected as a model organic product. Using 25 samples in suspension, specific calibration of the spectral data was obtained for estimating in-line both the overall solid concentration in suspension and the composition of the solid phase. In addition to such key-measurements, reliable estimates of supersaturation were computed in-line from the mass balance of the solute. In order to validate the technique, anhydrous to monohydrate phase transition experiments were performed in suspensions at 15 °C and the kinetic process involved was monitored. Despite the use of various solid concentrations and reactor configurations, both the reproducibility and the reliability of the in situ Raman measurements are shown to be satisfactory.
Keywords: Raman spectroscopy; Crystallization; Quantitative analysis; Solubility; Pseudo-polymorphism; Solvate; Transition kinetics;

Studies were conducted to develop oral leuprolide microemulsions using oleic acid as an absorption enhancer and to evaluate its absorption and pharmacological responses in rats. Oral administration of leuprolide microemulsion at a dose of 3 mg/kg showed a greater in vivo exposure level (C max and AUC) than its saline solution. When male rats were orally given a microemulsion formulation of leuprolide acetate at 0.25, 0.5, and 1 mg/day for 14 consecutive days, a significant decrease in testis, prostate and seminal vesicle weights was observed. In a 35-day study, the reduction of the male genital organ weights by once a day treatment (2 mg/rat, qd) was similar to that by twice a day treatment (1 mg/rat, bid) at the same dose level. From both 14- and 35-day studies, plasma testosterone levels were sharply increased at the beginning of the treatment, and then significantly decreased to below normal control level which was also maintained during the treatment. In female rats, similar reduction of uterus and ovary weights was obtained following oral administration of leuprolide microemulsion for 35 days. These antagonistic activities from oral leuprolide microemulsion were similar to a single subcutaneous injection of Lupron® depot (3.75 mg/rat), a commercial leuprolide product. The results indicated that leuprolide absorbed into systemic blood circulation from the oral microemulsion containing oleic acid reached the plasma level which can exert its pharmacological effects. Increasing oral absorption of leuprolide observed in this study could be mediated by improved membrane permeation from oleic acid and reduced enzymatic degradation from microemulsions. These findings suggest that systemic absorption of highly water-soluble protein or peptide drugs could be enhanced by oral microemulsions containing oleic acid.
Keywords: Leuprolide acetate; Oleic acid; Permeation enhancer; Peptide; Microemulsion; Testosterone; Absorption; Rat; formulation; Peptide delivery;

Interactions of water with the surfaces of crystal polymorphs by M. Teresa Carvajal; John N. Staniforth (216-224).
The purpose of this study is to investigate the interactions of water adsorption on the surfaces of different crystal forms of the same drug. The energy of interaction between water vapor and the surfaces of the two crystal polymorphs has been investigated as a function of temperature and water activity. One of the adsorbents, the metastable form of the monotropically related pair used here, showed greater adsorptive capacity in terms of both the amount of water uptake as well the integral heat of adsorption. However, the specific heat of adsorption values revealed that even though the surface of the thermodynamically stable crystal adsorbs less water, water molecules are actually more strongly bound when adsorbed on the surface of the stable crystal form. This means that the metastable crystal form adsorbs a greater amount of more weakly bound water. Conversely, the thermodynamically stable form, presents on its surface a smaller number of stronger adsorption sites for water. This study also shows that the crystalline character of the surfaces of the two polymorphs, shown as quantifiable differences in their surface interactions, is maintained despite the presence of any crystal defects incorporated upon milling.
Keywords: Water activity; Water uptake; Water interactions; Moisture sorption; Isothermal microcalorimetry; Vapor sorption; Crystal surface; Polymorphs; Water–solid interactions; Surface energetics;

Interactions of oleic acid and model stratum corneum membranes as seen by 2H NMR by Amy C. Rowat; Neil Kitson; Jenifer L. Thewalt (225-231).
We have investigated the mechanism through which the penetration enhancer oleic acid acts on stratum corneum (SC) model membranes (bovine brain ceramide:cholesterol:palmitic acid, 1:1:1 molar ratio). We used solid state deuterium nuclear magnetic resonance to monitor such multilamellar SC dispersions containing either cholesterol-d6, palmitic acid-d31, or oleic acid-d2 as a function of both fatty acid concentration (2:2:1:1 and 1:1:1:1 bovine brain ceramide:cholesterol:palmitic acid:oleic acid) and temperature (18–75 °C). Our results show that below 40 °C, oleic acid (OA) is in an ‘isotropic’ phase, indicating that it has not incorporated into the lamellar membrane phase. At and above the SC model membrane's crystalline to liquid crystalline melting temperature, T m  = 40–42 °C, OA interacts with lamellar SC membranes with a slight dependence on OA concentration. T m does not change upon the exposure of the SC model membrane to OA, nor do we see any significant change in membrane chain disorder as monitored by the labelled PA. However, the spectra of both the palmitic acid (PA) and cholesterol SC model membrane components contain an isotropic peak that grows with increasing temperature. Our results thus indicate that oleic acid extracts a fraction of the endogenous SC membrane components, promoting phase separation in the SC membrane system. Reducing the proportion of crystalline lipids and creating more permeable OA-rich domains is a plausible mechanism that explains how OA enhances transdermal penetration.
Keywords: Penetration enhancer; Transdermal drug delivery; Deuterium NMR; Fatty acids;

Dry skin and other skin disorders such as atopic dermatitis are characterized by impaired stratum corneum (SC) barrier function and by an increase in transepidermal water loss (TEWL) leading to a decrease in skin hydration. The possibility that dermatological and cosmetic products containing SC lipids could play a part in the restoration of disturbed skin barrier function is of great interest in the field of dermatology and cosmetics. The aim of the present study was to evaluate the effect of positively charged oil/water nanoemulsions (PN) containing ceramide 3B and naturally found SC lipids (PNSC) such as ceramide 3, cholesterol, and palmitic acid on skin hydration, elasticity, and erythema. Creams of PNSC were compared to PN creams, to creams with negatively charged o/w nanoemulsion and SC lipids (NNSC) and to Physiogel® cream, a SC lipid containing formulation, which is already on the market. The formulations (PN, PNSC, and NNSC) were prepared by high-pressure homogenization. After adding Carbopol 940 as thickener, particle size and stability of the creams were not significantly changed compared to the nanoemulsions. The studies were carried out on three groups, each with 14 healthy female test subjects between 25 and 50 years of age, using Corneometer® 825, Cutometer® SEM 575 and Mexameter® 18 for measurements of skin hydration, elasticity, and erythema of the skin, respectively. The creams were applied regularly and well tolerated throughout the study. All formulations increased skin hydration and elasticity. There was no significant difference between PNSC and Physiogel®. However, PNSC was significantly more effective in increasing skin hydration and elasticity than PN and NNSC indicating that phytosphingosine inducing the positive charge, SC lipids and ceramide 3B are crucial for the enhanced effect on skin hydration and viscoelasticity.
Keywords: Ceramide; Phytosphingosine; Positively charged nanoemulsion; Skin elasticity; Skin hydration; Skin measurement in vivo;

Development of parenteral formulations and evaluation of the biological activity of the trypanocide drug benznidazole by María C. Lamas; Luciano Villaggi; Isabel Nocito; Georgina Bassani; Darío Leonardi; Fernanda Pascutti; Esteban Serra; Claudio J. Salomón (239-243).
Chagas disease, caused by Trypanosoma cruzi, is a major public health problem in Latin America. According to the World Health Organization, around 20 million people are infected and another 40 million are at risk of acquiring the disease. One of the drugs most frequently used for the treatment of Chagas disease is benznidazole (BZL). It is practically insoluble in water (0.4 mg/ml), which precludes the preparation of liquid dosage forms, in particular, parenteral formulations. Thus, the aim of this work was to investigate the solubilization of BZL at two pH values using various cosolvents such as ethyl alcohol, propylene glycol, polyethylene glycol 400, benzyl alcohol, diethylene glycol monoethyl ether (Transcutol) and surfactants such as polysorbates (Tween) 40 and 80, and sodium dioctyl sulfosuccinate (AOT). Solvent systems based on PEG 400, with the addition ethyl alcohol and/or potassium biphthalate buffer solution, increased the BZL solubility up to 10 mg/ml. These alcoholic vehicles showed no toxicity against parasite when assayed at 1%. Physical and chemical stability studies showed that the formulations were stable for at least 1.5 years. In agreement with the biological activity results, the selected formulations are suitable for further clinical studies. Moreover, increasing the aqueous solubility of BZL reduced the problems in vitro testing techniques and bioassays leading to more reliable results and/or reproducibility.
Keywords: Chagas disease; Benznidazole; Water solubility; Cosolvent systems; Parenteral formulation;

Enhanced activity of liposomes-encapsulated antibiotics against clinical isolates of Pseudomonas aeruginosa has been documented with liposomes of low encapsulation efficiency. We sough to construct liposomes with high yield entrapment of aminoglycoside and macrolide antibiotics as well as favorable stability in storage and physiological conditions. Liposome-entrapped aminoglycosides (amikacin, gentamicin, tobramycin) and a macrolide (erythromycin) were prepared by a modified dehydration–rehydration vesicles (DRVs) method, and their particle size and entrapment efficiency were determined. We studied in vitro stability of these vesicles over a 48 h period at 4 and 37 °C in phosphate-buffered saline (PBS) and in plasma at 37 °C. The mean particle size of DRVs loaded with antibiotics varied from 163.37 ± 38.44 to 259.83 ± 11.80 nm with no significant difference in regard with the type of the antibiotics encapsulated. Encapsulation efficiency of DRVs loaded with amikacin, gentamicin, tobramycin, and erythromycin were 29.27 ± 1.17, 33 ± 0.76, 22.33 ± 1.48 and 32.06 ± 0.82% of initial amount of the drug, respectively. These vesicles were stable regardless of the experimental temperature. Indeed, the liposomes retained more than 75% of the initially encapsulated drugs for the study period of 48 h. DRVs incubated in plasma however, released more antibiotics than those incubated in PBS. In conclusion, using this modified DRV method, we obtained small sized vesicles with high yield entrapment for aminoglycoside and macrolide antibiotics. The technique may be utilized to overcome the low encapsulation efficiency associated with aminoglycoside and macrolide antibiotics.
Keywords: DRVs; Size; Encapsulation efficiency; Stability;

This work investigated the simultaneous permeation of ketoprofen and propylene glycol (PG) across pig ear skin from simple gel formulations administered under simulated in-use conditions. The aims were to quantify rates of permeation of both solvent and active, probe the effects of formulation drying and gain insight into drag/complexation interactions. Simple 3-component gels were formulated using a fixed amount of ketoprofen and hydroxypropyl cellulose thickener with decreasing content of solvent propylene glycol. Multiple finite (5 mg × 15 mg) doses were massaged over 24 h into full thickness pig ear skin in vertical Franz-type diffusion cells. The permeation of ketoprofen was inversely proportional to the content of PG, whereas the permeation of PG was directly proportional, although the amount of PG permeated was always greater than ketoprofen, even from the driest gel practically achievable. In this state, the molar ratio of PG/ketoprofen was ∼12, suggesting that this number of PG molecules constitutes the solvation cage of ketoprofen. Dragging/pulling effect extends throughout the skin and into the receptor compartment and probably the system, in an in vivo situation. Although PG may represent a worse case scenario given its well-documented skin permeation enhancement properties, it is probable that other solvents exert a similar effect on solutes across skin. A drying film will behave in different ways depending on the nature of both the thickener and solvent, where the outcomes are not readily predictable. It is important to account for the fate of all species administered from a topical formulation.
Keywords: Ketoprofen; Propylene glycol; Topical; Permeation; Drag effect; Solvated complex; Drying; Skin;

Folate-mediated targeting of polymeric conjugates of gemcitabine by Gennara Cavallaro; Licciardi Mariano; Stefano Salmaso; Paolo Caliceti; Giammona Gaetano (258-269).
The synthesis of two new macromolecular prodrugs for active tumor targeting was set up. Gemcitabine (2′-deoxy-2′,2′-difluorocytidine) was conjugated to α,β-poly(N-2-hydroxyethyl)-dl-aspartamide (PHEA) through succinyl or diglycolyl hydrolysable spacers. The targeting agent folic acid was attached to the macromolecular backbone through the aminocaproic spacer. The two conjugates [PHEA-(5′-succinylgemcitabine)-1′-carboxypentyl-folamide and PHEA-(5′-diglycolyl-gemcitabine)-1′-carboxypentyl-folamide], were purified and extensively characterised by spectroscopic (UV, IR and NMR) and chromatographic analyses to determine the correct chemical structure, the purity degree and the reaction yield. In vitro studies demonstrated that the drug release depends on the spacer arm (diglycolyil or succinyl) and incubation pH. After 30 h incubation at pH 7.4, mimicking the plasma and extracellular compartments, the gemcitabine release from the succinyl and diglycolyl derivatives was 28 and 31%, respectively. After 30 h incubation at pH 5.5, mimicking the lisosomial compartment, the drug released from both bioconjugates was lower than 13%. In plasma, the polymer conjugation increased the drug stability and provided for a sustained drug release. In vitro citotoxicity studies performed using human nasopharyngeal epidermal carcinoma KB cells demonstrated that PHEA-(5′-succinylgemcitabine)-1′-carboxypentyl-folamide displays an higher dose dependent cytotoxic effect with respect to PHEA-(5′-diglycolyl-gemcitabine)-1′-carboxypentyl-folamide.
Keywords: Polymeric conjugates; Gemcitabine; Polyaspartamide; Folic acid; Folate-mediated targeting;

Thiolated chitosan microparticles: A vehicle for nasal peptide drug delivery by Alexander H. Krauland; Davide Guggi; Andreas Bernkop-Schnürch (270-277).
The goal of this study was to develop a microparticulate delivery system based on a thiolated chitosan conjugate for the nasal application of peptides. Insulin was used as model peptide. For thiolation of chitosan 2-iminothiolane was covalently linked to chitosan. The resulting chitosan–TBA (chitosan-4-thiobutylamidine) conjugate featured 304.89 ± 63.45 μmol thiol groups per gram polymer. 6.5% of these thiol groups were oxidised. A mixture of the chitosan–TBA conjugate, insulin and the permeation mediator reduced glutathione were formulated to microparticles. Control microparticles comprised unmodified chitosan and insulin. As second control served mannitol–insulin microparticles. All microparticulate systems were prepared via the emulsification solvent evaporation technique. In 100 mM phosphate buffer pH 6.8 chitosan–TBA–insulin microparticles swelled 4.39 ± 0.52-fold in size, whereas chitosan based microparticles did not swell at all. Chitosan–TBA microparticles showed a controlled release of fluorescein isothiocyanate (FITC)-labelled insulin over 6 h. Nasal administered chitosan–TBA–insulin microparticles led to an absolute bioavailability of 7.24 ± 0.76% (means ± S.D.; n  = 3) in conscious rats. In contrast, chitosan–insulin microparticles and mannitol–insulin microparticles exhibited an absolute bioavailability of 2.04 ± 1.33% and 1.04 ± 0.27%, respectively (means ± S.D.; n  = 4).Because of these results microparticles comprising chitosan–TBA and reduced glutathione seem to represent a useful formulation for the nasal administration of peptides.
Keywords: Thiomer; Nasal drug delivery; Microparticles; Peptides; Glutathione; Bioavailability; Controlled release/delivery;

The administration of drugs by subcutaneous infusion is routinely practiced in palliative medicine for the management of patients who are no longer able to take oral medication. It is common for two or more drugs to be combined in subcutaneous solutions. The combination of an opioid with other drugs (haloperiol lactate and hyoscine N-butyl bromide) can be very valuable. Unfortunately, the compatibility and stability of morphine hydrochloride, haloperidol lactate and hyoscine N-butyl bromide combined in the same solution has not yet been determined. Therefore, this study examined the stability of ternary solutions containing morphine HCl, haloperidol lactate and hyoscine N-butyl bromide at different dose ranges. Twelve different solutions were assessed for 15 days after preparation in polypropylene syringes using 0.9% saline as diluent. Triplicate syringes were stored at 25 °C. HPLC was the analytical technique used to measure morphine HCl, haloperidol lactate and hyoscine N-butyl bromide. Initial concentration ranges were 1.67–10.0 mg/ml for morphine HCl, 0.417–0.625 mg/ml for haloperidol lactate and, 5.0–6.67 mg/ml for hyoscine N-butyl bromide. All three drugs were very stable (>92.5%) when stored at 25 °C. The clinical performance of the admixture was retrospectively assessed in 21 terminal oncology patients. Total symptom control was achieved in 17 out of 21 patients with very good local tolerance.
Keywords: Subcutaneous; Stability; Morphine hydrochloride; Hyoscine N-butyl bromide; Haloperidol lactate; Palliative care;

Are chitosan formulations mucoadhesive in the human small intestine? by Mia Säkkinen; Janne Marvola; Hanna Kanerva; Kai Lindevall; Aapo Ahonen; Martti Marvola (285-291).
Rapid passage through the proximal intestine can result in the low bioavailability of a drug substance with site-specific absorption characteristics in the upper gastrointestinal tract. To overcome this, there is increasing interest in developing gastro-retentive formulations and/or formulations that linger in the proximal parts of the small intestine, e.g. by using mucoadhesive polymers as excipients in formulations. In our recent study, we used neutron activation-based gamma scintigraphy to evaluate the gastro-retentive properties of formulations containing chitosan (Mw 150 kDa) in man. At the same time, we had an opportunity to monitor the transit of the formulations (40 or 95% of chitosan) in the small intestine. Gamma scintigraphic investigations revealed that although the chitosan studied had exhibited marked mucoadhesive capacities in vitro, retention of the chitosan formulations in the upper gastrointestinal tract was not sufficiently reproducible and the duration of retention was relatively short. In 3 volunteers out of 10, the formulation adhered to the gastric mucosa (retention times varied from 1.25 to 2.5 h) and in two volunteers to the upper small intestine (approximate retention time 45 min). In one case, the formulation adhered to the oesophagus. The system failed to increase the bioavailability of furosemide, a drug site-specifically absorbed in the upper gastrointestinal tract. As far as the kind of formulation studied is concerned, preparation of a system that is site-specific to the stomach and/or the upper small intestine seems difficult if the proposed mechanism of action is mucoadhesion. The results suggest that other mechanisms of action should also be studied.
Keywords: Mucoadhesion; Small intestine; Chitosan; Gamma scintigraphy;

LC/MS/MS quantitation assay for pharmacokinetics of naringenin and double peaks phenomenon in rats plasma by Yan Ma; Peibo Li; Dawei Chen; Tiezheng Fang; Haitian Li; Weiwei Su (292-299).
A highly sensitive and specific electrospray ionization (ESI) liquid chromatography–tandem mass spectrometry (LC/MS/MS) method for quantitation of naringenin (NAR) and an explanation for the double peaks phenomenon was developed and validated. NAR was extracted from rat plasma and tissues along with the internal standard (IS), hesperidin, with ethyl acetate. The analytes were analyzed in the multiple-reaction-monitoring (MRM) mode as the precursor/product ion pair of m/z 273.4/151.3 for NAR and m/z 611.5/303.3 for the IS. The assay was linear over the concentration range of 5–2500 ng/mL. The lower limit quantification was 5 ng/mL, available for plasma pharmacokinetics of NAR in rats. Accuracy in within- and between-run precisions showed good reproducibility. When NAR was administered orally, only little and predominantly its glucuronidation were into circulation in the plasma. There existed double peaks phenomenon in plasma concentration–time curve leading to the relatively slow elimination of NAR in plasma. The results showed that there was a linear relationship between the AUC of total NAR and dosages. And the double peaks are mainly due to enterohepatic circulation.
Keywords: LC/MS/MS; Pharmacokinetics; Double peaks; Naringenin; Enterohepatic circulation;

Short-term delayed-release microcapsules spraycoated with acrylic terpolymers by Dongchun Liu; Hideki Ichikawa; Fude Cui; Yoshinobu Fukumori (300-307).
A series of poly(ethyl acrylate (EA)/methyl methacrylate (MMA)/2-hydroxyethyl methacrylate (HEMA)) lattices were synthesized to prepare short-term delayed-release microcapsules by employing the Wurster coating process. Latex with a HEMA molar fraction exceeding 60% could not be synthesized as an aqueous suspension due to latex particle precipitation. The effects of monomer composition on the particle size of latex and the water-uptake and glass transition temperature (T g) of cast films were investigated. Lattices whose T g ranged from 40 to 80 °C were used to prepare the microcapsules. Most of the lattices exhibited excellent process performance while coating particles that were smaller than 100 μm: the product yields were 85.1–90.6% and the mean particle sizes were 82–85 μm. However, since the lattices with high molar ratios of EA and HEMA were highly hydrophilic and strongly adhesive, the core particles in the coating were severely agglomerated. The microcapsules coated with lattices whose HEMA molar fractions were higher than 50% were unable to retard the release of carbazochrome sodium sulfonate, a water-soluble model drug, during the initial 0.5 min. Poly(EA/MMA/HEMA) with a molar ratio of 9:9:10 appeared to be suitable for the preparation of short-term delayed-release microcapsules by the Wurster coating process.
Keywords: Microcapsule; Controlled release; Acrylic polymer; Latex; Fluidized bed; Coating;

Cutaneous metabolism of a dipeptide influences the iontophoretic flux of a concomitant uncharged permeant by Melanie Altenbach; Nathalie Schnyder; Christine Zimmermann; Georgios Imanidis (308-317).
Passive and iontophoretic transport of the model dipeptide tyrosine-phenylalanine (TyrPhe) that is subject to cutaneous metabolism and the uncharged glucose derivative benzyl-2-acetamido-2-deoxy-α-d-glucopyranoside (BAd-α-Glc) used as electroosmosis marker through heat-separated human epidermis was investigated in vitro. TyrPhe and BAd-α-Glc were used separately and in combination in order to determine their interaction in terms of permeability and the influence of skin metabolism of TyrPhe on permeation rate and tissue retention of itself and of BAd-α-Glc. TyrPhe was chemically and electrochemically stable but underwent considerable degradation in the epidermis under reflection boundary conditions with generation of degradation products tyrosine (Tyr) and phenylalanine (Phe) confirming cutaneous metabolism of TyrPhe in heat-separated human epidermis, which was more pronounced at pH 4.5 than at pH 3.0. As a result, no reproducible epidermis permeation of TyrPhe at pH 3 and no permeation at all at pH 4.5 was measured regardless of the presence of BAd-α-Glc, accompanied by increased levels of Tyr and Phe compared to blank runs. Low temperature (4 °C) at both pH values and addition of o-phenanthroline at pH 3 but not at pH 4.5 yielded reproducible TyrPhe permeation and blank, i.e., endogenous levels of Tyr and Phe evidencing inhibition of degradation. Constant voltage anodal iontophoresis marginally reduced BAd-α-Glc flux at pH 3 and 4.5 compared to the passive flux. In combination with TyrPhe, iontophoretic flux of BAd-α-Glc was increased markedly compared to the passive one when TyrPhe was metabolized in the tissue, while no such increase was observed when TyrPhe metabolism was inhibited. The increase of BAd-α-Glc iontophoretic flux was accompanied by a considerable decrease of the BAd-α-Glc amount retained in the epidermis. The presence of the generated Tyr and Phe, therefore, appears to be related to a decrease of the BAd-α-Glc amount retained in the epidermis upon application of an electrical voltage and an enhancement of its iontophoretic flux. Thus, an interaction between the concurrent permeants at the level of tissue retention induced by metabolism can influence the apparent iontophoretic permeation.
Keywords: Iontophoresis; Cutaneous; Electroosmosis; Peptides; Amino acid; Skin; Metabolism; Transdermal drug delivery; Permeability; Transport; Tissue retention;

Design and physicochemical characterisation of a bioadhesive patch for dose-controlled topical delivery of imiquimod by Ryan F. Donnelly; Paul A. McCarron; Agnieszka A. Zawislak; A. David Woolfson (318-325).
Clinical use of the imidazoquinoline immunomodulator imiquimod for the topical treatment of dysplastic and neoplastic lesions has increased markedly in recent years. However, despite guidance from the manufacturer of the proprietary imiquimod cream, there seems to be little consensus between clinicians as to the topically applied dose. Given that patients often apply the cream themselves at home, further dosing variability is expected and, consequently, accurate comparison of the results of different published studies is difficult. This paper describes, for the first time, the formulation and physicochemical characterisation of a bioadhesive patch for dose-controlled topical delivery of imiquimod as well as a new HPLC method for sensitive fluorescence determination of imiquimod released from such systems. Patches containing imiquimod loadings of 4.75, 9.50 and 12.50 mg cm−2 all released significantly more drug across a model membrane than the proprietary cream over a period of 6 h. Inclusion of imiquimod in patches did not adversely affect their physicochemical properties. Of major importance, patches contained defined drug loadings per unit area; therefore, their use could reduce inter-clinician variability. This would make critical comparison of clinical studies and determination of an appropriate imiquimod dose for successful treatment much simpler. Since bioadhesive formulations are capable of adhering to body tissues in moist environments, the use of a bioadhesive patch system may allow extension of the clinical uses of imiquimod to the treatment of neoplastic conditions of the oral cavity and cervix, as well as the vulva.
Keywords: Imiquimod; Bioadhesive; Neoplastic; Drug delivery;

Noticeboard (326-328).