International Journal of Pharmaceutics (v.298, #1)

Effect of cholesterol and ethanol on dermal delivery from DPPC liposomes by J.M. López-Pinto; M.L. González-Rodríguez; A.M. Rabasco (1-12).
The main objective of the present work was to compare the dermal delivery of minoxidil (Mx), a lipophilic drug from ethosomes versus classic liposomes, containing different cholesterol (CHOL) concentrations. All the systems were characterized for shape, lamellarity, particle size and entrapment efficiency percentage (EE), by transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), laser diffraction and ultracentrifugation or dialysis methods, respectively. Multilamellar vesicles (MLVs) were obtained and one to six lamellae were visualized by CLSM. The presence of ethanol in the formulations affects the particle size in terms of reducing this parameter. In addition, it was possible to appreciate the influence of CHOL on the vesicle size, because it was increased, as CHOL concentration was higher. When the EE was determined by two different methods (ultracentrifugation and dialysis methods), a clear losing of entrapped drug by the ultracentrifugation method was observed, because the strong energy transmitted to the samples disrupted vesicles.Vesicles were non-occlusively applied on rat skin and the permeation pattern of the different systems, depth into the skin and the main permeation pathway were studied by using β-carotene as a fluorescent probe. CLSM studies showed that ethosomal systems were much more efficient at delivering the fluorescent substance into the skin in terms of quantity and depth, than either liposomes or hydroalcoholic solutions.
Keywords: Liposome; Ethosome; Lamellarity; Confocal laser scanning microscopy; Franz cell; Minoxidil;

The entrapment of kojic oleate in bilayer vesicles by A. Manosroi; P. Wongtrakul; J. Manosroi; U. Midorikawa; Y. Hanyu; M. Yuasa; F. Sugawara; H. Sakai; M. Abe (13-25).
The entrapment of kojic acid and its newly synthesized ester (kojic oleate) has been evaluated. Kojic oleate was synthesized by DCC (N,N′-dicyclohexylcarbodiimide, DCC)/(4-(N,N-dimethylamino)pyridine, DMAP) esterification method and identified by FAB–MS and 1H NMR. The synthesized product was mainly 7-O-kojic oleate with more than 80% yield. It was entrapped in vesicular membrane prepared from 9.5:9.5:1.0 molar ratio of amphiphiles (Span 60, Tween 61 or DPPC), cholesterol and dicetyl phosphate. Kojic acid was encapsulated in the water compartment of these vesicles in order to confirm the vesicle formation. The morphology and particle size of the vesicles were characterized by an optical microscope and transmission electron microscope (TEM). The entrapment efficiencies of kojic acid and kojic oleate in the vesicles were investigated by dialysis and column chromatography, respectively. The contents of the entrapped kojic acid and kojic oleate were assayed by HPLC. The entrapment efficiency of kojic acid was 0.01–0.04 mol, whereas kojic oleate gave higher entrapment efficiency of 0.25–0.35 mol/mol of the total compositions of amphiphile/cholesterol/dicetyl phosphate. Structural modification of kojic acid improved its entrapment in the vesicles. Tween 61 vesicles could entrap kojic oleate more than did Span 60 vesicles. The πA isotherms revealed the lower area per molecule of Span 60, which formed a more rigid pack of its molecule on air/water interface than that of Tween 61. This implied the high rigidity of vesicular membrane prepared with Span 60 led to the lower amount of kojic oleate entrapped in the vesicles. From the release study of kojic acid through the dialysis membrane, it indicated that the intercalation of kojic oleate in the vesicular membranes did not significantly affect the release of kojic acid from the vesicles.
Keywords: Kojic acid; Kojic oleate; Tween 61; Span 60; DPPC; Bilayer vesicles;

In vitro evaluation of coating polymers for enteric coating and human ileal targeting by Nathalie Huyghebaert; An Vermeire; Jean Paul Remon (26-37).
Recombinant interleukin-10 producing Lactococcus lactis is an alternative therapy for Crohn's disease. For in vivo interleukin-10 production, thymidine, the essential feed component of these recombinant bacteria should be coadministered. Different coating polymers were evaluated in vitro for enteric properties and targeting suitability to the ileum, the major site of inflammation in Crohn's disease. To guarantee ileal delivery, the polymer must dissolve from pH 6.8 and allow complete release within 40 min. Aqoat® AS-HF coated pellets (15%) showed poor enteric properties and thymidine was released below pH 6.8. Eudragit® FS30D coated pellets (15%) showed good enteric properties, but no thymidine was released within 40 min at pH 6.8. Eudragit® S coated pellets (15%) showed good enteric properties after curing at elevated temperature while no thymidine was released within 40 min at pH 6.8. In another approach to pass the proximal small intestine intact, pellets were coated with 30% Eudragit® L30D-55. At pH 6.0, they showed a lag-phase of 20 min. No influence of layer thickness was seen above pH 6.5. Alternatively, pellets were coated with a mixture of Eudragit® FS30D/L30D-55 but they showed poor enteric properties and thymidine was released below pH 6.8. In conclusion, none of the tested polymers/mixtures ensured enteric properties and ileal targeting.
Keywords: Thymidine; Ileum; Crohn; Coating; Eudragit; Aqoat;

In order to find whether torasemide is metabolized via CYP isozymes in rats, torasemide at a dose of 2 mg/kg was infused in rats pretreated with SKF 525-A, a non-specific CYP isozyme inhibitor in male Sprague–Dawley rats. The total area under the plasma concentration–time curve from time zero to time infinity (AUC) of torasemide was significantly greater in rats pretreated with SKF 525-A (a non-specific CYP isozyme inhibitor in rats) than that in control rats (3570 versus 1350 μg min/ml). This indicated that torasemide is metabolized via CYP isozymes in rats. Hence, torasemide was infused in rats pretreated with various enzyme inducers and inhibitors to find what types of CYP isozymes are involved in the metabolism of torasemide in rats. The AUC values were not significantly different in rats pretreated with 3-methylcholanthrene, phenobarbital, isoniazid, quinine and troleandomycin (main inducers of CYP1A1/2, CYP2B1/2, and CYP2E1, and main inhibitors of CYP2D1 and CYP3A1/2 in rats, respectively) compared with those in respective control rats. However, in rats pretreated with dexamethasone (a main inducer of CYP3A1/2 in rats), the AUC was significantly smaller than that in control rats (1290 versus 1590 μg min/ml). Dexamethasone probably also induces rat CYP2C11; this could be due to an increase in CYP2C11 in rats pretreated with dexamethasone. It has been reported from our laboratories that in rats pretreated with sulfaphenazole (a main inhibitor of CYP2C11 in rats) the AUC was significantly greater than that in control rats (2970 versus 1610 μg min/ml). The above data suggested that torasemide could be metabolized in male rats mainly via CYP2C11.
Keywords: Torasemide; CYP2C11; Enzyme inducers or inhibitors; Pharmacokinetics; Rats;

Topical absorption of piroxicam from organogels—in vitro and in vivo correlations by T. Pénzes; G. Blazsó; Z. Aigner; G. Falkay; I. Erős (47-54).
In view of their good skin tolerability, glyceryl fatty acid esters were used as organogelators, and their effects in the topical penetration of piroxicam (Px) were investigated. The in vivo skin penetration was evaluated by measuring the anti-inflammatory effect in rats, where we found that Px incorporated into glyceryl fatty acid ester organogels exhibited a significantly greater inhibition of oedema than that of the placebo control either when applied locally (p  < 0.001), or via transdermal absorption (p  < 0.01 and <0.05, respectively). As the Px concentration was increased, the extent of oedema inhibition rose in accordance with a power law. Comparisons with traditional galenic organogels and a marketed product revealed that the relative biological availability of Px was better from glyceryl fatty acid ester organogels, except when calculated for D1 versus T2 and T3. In order to predict the extent of in vivo skin absorption, we measured the penetration coefficient and the in vitro penetration. In accordance with theory, the extent of in vivo oedema inhibition increased as P oct/w increased, and maximum inhibition was observed at log  P  = 2.0211. However, the in vitro penetration through a synthetic membrane did not correlate with the in vivo results, the reason for which might be the different natures of the model barriers.
Keywords: Organogel; Piroxicam; Penetration; Bioavailability;

Preparation and characterisation of liposomes encapsulating ketoprofen–cyclodextrin complexes for transdermal drug delivery by Francesca Maestrelli; Maria Luísa González-Rodríguez; Antonio Maria Rabasco; Paola Mura (55-67).
Multilamellar vesicle (MLV) liposomes containing ketoprofen–cyclodextrin complexes intended for drug topical delivery were prepared, with the aim of simultaneously exploiting the favourable properties of both carriers. Drug complexes with β-cyclodextrin (βCyd) and hydroxypropyl-βCyd (HPβCyd), prepared by coevaporation and sealed-heating methods, were characterised by differential scanning calorimetry, hot stage microscopy, scanning electron microscopy and tested for dissolution properties. The coevaporated system with HPβCyd was the most effective, enabling an about 11-fold increase in drug dissolution. Drug and drug-Cyd systems were incorporated in MLV liposomes prepared by the thin layer evaporation technique. All liposomal formulations were characterised for encapsulation efficiency, particle size and morphology, using dialysis, light scattering and transmission electron microscopy techniques, respectively. MLV formation was negatively influenced by the presence of Cyd; nevertheless, it was possible to prepare stable MLVs containing ketoprofen-Cyd complexes. The presence of the Cyd complex affected MLV dimensions but not their lamellar structure. The complex with HPβCyd, in virtue of its greater stability than the βCyd one, allowed higher percentages of encapsulation and gave rise to more stable MLV systems. Permeability studies of drug and drug-Cyd complexes, as such or incorporated in liposomes, performed both across artificial membranes and rat skin, highlighted a favourable effect of Cyd on drug permeation rate, due to its solubilizing action; by contrast, unexpectedly, no skin-permeation enhancer property of liposomes has been evidenced. Confocal laser scanning microscopy studies carried out with the rhodamine-Cyd complex as fluorescent marker, confirmed such results, showing that the label permeated deeper across rat skin layers when it was in solution than when entrapped in liposomes.
Keywords: Ketoprofen; Cyclodextrins; Liposomes; Skin permeation studies;

Biopharmaceutics of intrathecal baclofen-loaded microparticles in a goat model by Frederic Lagarce; Nathalie Faisant; Jean-Claude Desfontis; Laurent Marescaux; Freddy Gautier; Delphine Holopherne; Marie-Christine Rousselet; Philippe Menei; Jean-Pierre Benoit (68-79).
The goal of this study was to develop a goat model allowing reliable pharmacokinetic (PK) studies of intrathecal baclofen (ITB) sustained release dosage forms using an implanted silicone catheter. ITB PK parameters (clearance, volume of distribution) following intrathecal bolus injection were determined for doses ranging from 100 to 560 μg and a comparison to human data was made. Baclofen-loaded microparticles were then implanted in the intrathecal space of goats and the resulting baclofen levels were determined during 28 days. Finally, PK parameters were used to predict cerebrospinal fluid (CSF) baclofen rates from in vitro release profiles of baclofen-loaded microspheres.The catheter was well tolerated and did not interfere with behavioral testings. Baclofen CSF clearance (mean = 8.59 ± 2.43 ml/h) and volume of distribution (21.06 ± 13.32 ml) were not significantly affected by the increase of the dose (p  > 0.05). In vivo, the baclofen levels in CSF were stabilized at 200 μg/l after a period of 3 days. The predictive value of the in vitro release studies was good since the theoretical levels ranged between 128 and 257 μg/l. In conclusion, a large animal model was developed and allowed the biopharmaceutic evaluation of baclofen microparticles injected via intrathecal route.
Keywords: Drug delivery; Baclofen; Pharmacokinetics; Goat; Microspheres; Sustained release dosage form;

Application of Raman spectroscopy for on-line monitoring of low dose blend uniformity by Debra S. Hausman; R. Thomas Cambron; Adel Sakr (80-90).
On-line Raman spectroscopy was used to evaluate the effect of blending time on low dose, 1%, blend uniformity of azimilide dihydrochloride. An 8qt blender was used for the experiments and instrumented with a Raman probe through the I-bar port. The blender was slowed to 6.75 rpm to better illustrate the blending process (normal speed is 25 rpm). Uniformity was reached after 20 min of blending at 6.75 rpm (135 revolutions or 5.4 min at 25 rpm). On-line Raman analysis of blend uniformity provided more benefits than traditional thief sampling and off-line analysis. On-line Raman spectroscopy enabled generating data rich blend profiles, due to the ability to collect a large number of samples during the blending process (sampling every 20 s). In addition, the Raman blend profile was rapidly generated, compared to the lengthy time to complete a blend profile with thief sampling and off-line analysis. The on-line Raman blend uniformity results were also significantly correlated (p-value < 0.05) to the HPLC uniformity results of thief samples.
Keywords: Raman; On-line spectroscopy; Blend uniformity; Low dose; Content uniformity;

Study on the characteristics of pectin–ketoprofen for colon targeting in rats by Miao Miao Xi; San Qi Zhang; Xin Yi Wang; Kun Quan Fang; Yi Gu (91-97).
Pectin–ketoprofen (PT-KP) prodrug with the potential for colon targeted delivery has been evaluated. A sensitive HPLC method was established for the determination of concentration of ketoprofen (KP) in rats. This method was also used to evaluate the colon targeting property of PT-KP. KP or PT-KP was given to rats by oral administration at a dosage of 10 mg/kg. Plasma and the different parts of gastrointestinal (GI) tract were taken after 2, 4, 6, 8, 10 and 12 h of oral administration of KP or PT-KP to rats and the concentration of KP was measured by HPLC. Preliminary experiments show KP distributes mainly in stomach, proximal small intestine and distal small intestine. However, KP released from PT-KP mainly distributes in cecum and colon. Therefore, this approach suggests that PT-KP prodrug has a good colon targeting property.
Keywords: Pectin–ketoprofen; Prodrug; Ketoprofen; Colon targeting;

The purpose of this study was to determine the effects of ionization and penetration enhancers on the transdermal delivery of 5-fluorouracil (5-FU) through excised human stratum corneum. The in vitro transport of 5-FU was determined at three physiologically relevant pH values of 5.0, 7.4 and 8.0, and in the presence of suitable penetration enhancers, namely Azone® (AZ), lauryl alcohol (LA), and isopropyl myristate (IPM). The results showed that passive permeation of 5-FU is dependent upon the pH of the donor solution, although did not fully conform to the pH-partition hypothesis. A further analysis of data suggested an inverse relationship (i.e., negative correlation) between steady-state flux and aqueous solubility of 5-FU at these pH values (correlation coefficient = −0.4205), although correlation was not statistically significant (p  = 0.7237). In the absence of a penetration enhancer, the in vitro permeability of 5-FU was quite low (0.82 ± 0.06 × 104  cm/h). This delivery rate was enhanced by approximately by 3, 4 and 24-fold, respectively, when IPM, LA, and AZ were incorporated into the donor solution. All these enhancements were statistically significant (p  < 0.05) compared to control, and occurred regardless of the polarity (solubility parameters) of these enhancers. Out of three examined enhancers, AZ appears to be a suitable enhancer for enhancing transport of 5-FU, which merits in vivo investigation in a suitable animal model. Possible mechanisms of enhancement by these penetration enhancers are also discussed.
Keywords: 5-FU; Azone®; Lauryl alcohol; Isopropyl myristate; Transdermal; Influence of pH;

Development of a controlled release formulation based on a starch matrix system by Yongsong Cao; Lu Huang; Jiuxin Chen; Ji Liang; Shengyou Long; Yitong Lu (108-116).
Controlled release formulation (CRF) of the insecticide acetamiprid was made using tapioca starch, urea and sodium borate. The data show the recovery of this CRF process is >95.43%, there is no obvious difference with increase of sodium borate added, however, with the increase of urea in the mixture, the formulation has a decrease recovery. In stability test, the decomposition rate of acetamiprid CRF was less one tenth than that of the acetamiprid emulsifiable concentrate (EC) under UV radiation. The release kinetics of acetamiprid from granules with variation content of urea, sodium borate and granule sizes were evaluated in water under laboratory condition. The release data were fitted to the generalized model M t /M z  =  kt n , where M t /M z is the percentage of insecticide released at time t, k and n are constants, and n is constant that indicates the mechanism of release. The results indicated that the release of acetamiprid was diffusion-controlled. The time taken for 50% of the active ingredient to be released into water, T 50, was also calculated for the comparison of formulations. The results showed that the formulation with the increasing urea in formulation had the higher value of T 50, which means a slower release of the active ingredient, while that the formulation with the increasing sodium borate in formulation had the lower value of T 50, which means a faster release of the active ingredient. It was also found that as the size of this formulation decreased, the release of the active ingredient was faster.
Keywords: Acetamiprid; Controlled release formulation; Tapioca starch; Stability; Kinetics;

Release of theophylline from polymer blend hydrogels by Jianhong Liu; Shiqi Lin; Lin Li; Erjia Liu (117-125).
Polymer blending is a simple yet attractive method to obtain combined physical and mechanical properties of polymers. In this paper, three types of blend hydrogels were prepared, each by physically blending two different natural polymers, and a model drug, theophylline (TPH), was immobilized into these hydrogels for the studies of drug release. The release profiles of TPH from various types of hydrogels were determined by UV–vis absorption measurement at 272 nm. The experimental results show that the releases of TPH from these hydrogels are dependent upon the composition of the hydrogel, the type of component, the possible interactions between two component polymers, as well as external temperature. All the release profiles clearly demonstrate a temperature effect. Among the three blend hydrogels, the slowest release was observed from the blend hydrogel of gelatin and agar with a weight ratio of 1:1. The drug release patterns and release mechanisms have been discussed by considering the possible molecular interactions and gel network structures.
Keywords: Drug release; Hydrogel; Polymer blend; Theophylline;

Development of a simple method for predicting the levels of di(2-ethylhexyl) phthalate migrated from PVC medical devices into pharmaceutical solutions by Yuji Haishima; Fumie Seshimo; Tae Higuchi; Haruko Yamazaki; Chie Hasegawa; Shun-ichiro Izumi; Tsunehisa Makino; Keisuke Nakahashi; Rie Ito; Koichi Inoue; Yoshihiro Yoshimura; Koichi Saito; Takeshi Yagami; Toshie Tsuchiya; Hiroyuki Nakazawa (126-142).
This study deals with the development of a simple method for predicting the elution levels of di-2-ethylhexyl phthalate (DEHP) from medical devices made of polyvinyl chloride (PVC) by using the physicochemical properties of pharmaceutical injections as a marker. GC-MS analysis showed that the release of DEHP from medical grade PVC product was concentration-dependently increased by extraction with two kinds of lipophilic injections (Sandimmun® and Prograf®) and three kinds of surfactants (HCO-60, Tween® 80, and SDS). The solubility of lipophilic pigments such as Sudan III, methyl yellow, and 1,4-diamino-anthraquinone against these solutions were also increased in a concentration-dependent manner, in which methyl yellow showed the highest response regarding the increase of optical density (O.D.). Further, electrical conductivity and static contact angle to the PVC sheet of the solutions were also increased or decreased in the same manner. As a result of the comparative study, significant correlation was found between DEHP release levels and these three physicochemical properties, particularly methyl yellow solubility, of the solutions tested. To evaluate the relationship in detail, DEHP release levels from PVC tubing and methyl yellow solubility of 53 injections used in gynecologic and obstetric fields were determined. None of the hydrophilic medicines showed any significant release of DEHP, and all showed low solubility of methyl yellow. On the other hand, the lipophilic medicines releasing a large amount of DEHP showed high solubility of methyl yellow (greater than O.D. 0.8). These results indicate that a significant proportional relationship exists between DEHP release potency and methyl yellow solubility of pharmaceutical solutions, and the risk of DEHP exposure to the patients administered pharmaceuticals through transfusion set could be easily predicted by the solubility test without complicated elution tests of DEHP using GC-MS or LC-MS.
Keywords: DEHP; PVC; Medical device; Prediction; Risk assessment;

Utilization of pure nuclear quadrupole resonance spectroscopy for the study of pharmaceutical crystal forms by S.C. Pérez; L. Cerioni; A.E. Wolfenson; S. Faudone; S.L. Cuffini (143-152).
Solid-state physical characterization of a pharmaceutical substance is necessary for successful development and approval of the final product. Different physical analytical techniques are available to do so: X-ray diffraction (XRD), IR, Raman, DSC, TG and NMR. Moreover, all of them detect the presence of excipients perturbing the analysis of the pure substance in low doses. In order to study polymorphism and pseudo polymorphism of drug, this paper introduces possible applications of pure nuclear quadrupole resonance, as a non-destructive technique in qualitative and quantitative approaches. Chlorpropamide and diclofenac sodium were used as examples. Unlike the mentioned techniques, the nuclear quadrupole resonance (NQR) signal of pharmaceutical compounds is not perturbed by the presence of solid excipient or other substances unless they possess resonance frequencies in the same frequency range of the compound studied.
Keywords: Pure nuclear quadrupole resonance; Pharmaceutical drugs; Quantitative analysis; Qualitative analysis; Polymorphism and pseudopolymorphism;

Aim of this study was the detection of polysaccharides with antioxidative properties as potential lipid protectors for topical administration. The effects of eight different polysaccharides on UV irradiation induced lipid peroxidation were investigated in a concentration dependent manner. An aqueous linolenic acid dispersion was used as an in vitro test system to examine the influences of acacia gum, agar agar, alginic acid, guar gum, novelose 330 and xanthan gum on the lipid peroxidation level after UV exposure. Four different samples of pectin and locust bean gum resulting from a swing mill grinding series were tested as well. Iron ions were added as transition metal catalysts. A UV irradiation device was used to create high level radiation. The amount of lipid peroxidation secondary products was quantified by the thiobarbituric acid assay detecting malondialdehyde. All of the tested polysaccharides showed antioxidative effects at least at one concentration. For acacia and xanthan gum, a concentration dependency of the protective effects was measured. The samples of agar agar, guar gum and novelose 330 acted antioxidatively without showing any concentration dependency. For alginic acid, prooxidative effects were determined. A correlation between grinding time and the effects of pectin and locust bean gum on the model lipid was not observed. The administration of lipid protective polysaccharides in cosmetic formulations or sunscreens could be helpful for the protection of the human skin against UV induced damage. In vivo experiments with the lipid protective polysaccharides found in this study should follow.
Keywords: Polysaccharides; Oxidative stress; Topical application; Antioxidants; Thiobarbituric acid assay;

Differences between eccentric and rotary tablet machines in the evaluation of powder densification behaviour by Giovanni F. Palmieri; Etienne Joiris; Giulia Bonacucina; Marco Cespi; Annalisa Mercuri (164-175).
Differences in the dynamics of powder densification between eccentric and rotary machine were pointed out by compressing at different compression pressures microcrystalline cellulose, lactose monohydrate and dicalcium phosphate dihydrate and recovering the corresponding stress/strain data in both machines equipped to monitor punches displacement and compression forces. Heckel plots were then obtained from these stress/strain data.Curves obtained in the rotary machine possess a narrower zone of linearity for the calculation of P Y and D A. The effect of the different compression mechanism of the rotary machine on the shape of the Heckel plot is more noticeable in a non-deforming material such as dicalcium phosphate. The effect of the longer dwell time of the rotary machine on the porosity reduction occurring after the maximum pressure has been reached, is more noticeable in a ductile material such as microcrystalline cellulose.Heckel parameters obtained in the rotary press are in some cases different from those recovered in the eccentric machine because of the longer dwell time, machine deflection and punch tilting occurring in the rotary machine, although theoretically they could better describe the material densification in a high speed production rotary machine.
Keywords: Compression; Compaction; Tableting; Mechanical properties; Rotary tablet machine; Densification; Heckel; Stress; Strain;

The aim of this study was to develop a microemulsion formulation providing an improved efficacy of orally administered insulin. The microemulsions were prepared using Labrafil M 1944 CS, Phospholipon 90 G (lecithin), absolute alcohol and bi-distilled water. The microemulsions of recombinant human (rh)-insulin and aqueous solution (200 IU/kg) were administered intragastrically by a canulla to diabetic and non-diabetic rats. Aprotinin (2500 KIU/g) was added as the enzyme inhibitor to the formulation. Upon the administration of intragastric rh-insulin solution (IS) to non-diabetic rats, the plasma glucose and insulin levels were not changed significantly. Therefore, the hypoglycemic effect caused by subcutaneous rh-insulin solution (SC), microemulsion containing rh-insulin (IME) and microemulsion containing rh-insulin and aprotinin (IMEA) were analyzed in diabetic rats. The area above the plasma glucose levels time curves (AAC), minimum glucose concentration (C min) and time to C min (t min) were derived from the plasma glucose profiles. IME and IMEA caused approximately 30% decrease in plasma glucose levels. The decrease in the plasma glucose levels continued after the 90th min. The highest AAC value was obtained when IMEA was administered to rats. The maximum plasma insulin concentration (C max), time to reach C max (t max), terminal half-life (t 1/2), area under the plasma concentration–time curve (AUC), mean residence time (MRT) and elimination rate constant (k el) values were also calculated. It was observed that t 1/2 values varied between 0.53 and 1.31 h. No significant difference could be found between the pharmacokinetic parameters of the IME and IMEA administered groups. Addition of aprotinin to the microemulsion containing rh-insulin increased bioavailability when compared to those not containing it, although the difference is not significant.
Keywords: Insulin; Microemulsion; Oral administration; Lecithin; Hypoglycemic effect;

Benzocaine (BZC), butambene (BTN) and isobutambene (BTI) are basic local anaesthetic agents of the ester type, preferentially used for surgery and dental procedures. The compounds, official in the USP (BZC and BTN) and Ph. Eur. (BZC), were each found to exist in two polymorphic crystal forms and their solid state characteristics have been determined by thermomicroscopy, differential scanning calorimetry (DSC), FTIR-, FT-Raman-spectroscopy as well as X-ray powder diffractometry. This work further emphasizes the comparison of solid state characteristics of three compounds with closely related structural features on molecular level, leading to opportunities for the investigation of structure-property relationships. Mod. I0 is the particular thermodynamically stable form at room temperature in all of the three systems. This form is present in commercial products and can be crystallized from solvents at room conditions. Mod. II can be obtained by annealing the supercooled melt or fast cooling of a saturated solution, respectively. The endothermic transformation of mod. II to mod. I0 upon heating confirms that mod. I0 is thermodynamically stable at ambient conditions (heat of transition rule) whereas mod. II is enantiotropically related to mod. I0, i.e. is metastable at temperatures above the transition temperature. The metastable forms show different kinetic stabilities at room temperature.
Keywords: Local anaesthetics; Crystal forms; Crystal polymorphism; Thermal analysis; Hot-stage microscopy; Solid state properties;

The microencapsulation vesicle (MCV) method is a liposome preparation technique that reproducibly produces liposomes with homogeneous particle sizes with a high encapsulation efficiency. Liposomes encapsulating water-soluble drugs, lipophilic drugs and an amphiphilic drug were prepared by the MCV method and the encapsulation efficiency of the drugs was examined. Three kinds of egg yolk lecithin with different iodine values, i.e., purified egg yolk lecithin (PEL), partially hydrogenated purified egg yolk lecithin (R-20) and completely hydrogenated purified egg yolk lecithin (R-5), were used for membrane materials in order to explore the possible effects of membrane rigidity or surface area on the encapsulation efficiency of the drug. Water-soluble 5-fluorouracil showed 12–15% encapsulation efficiency, which was higher than those reported in the literature (less than 10%). With the MCV method, theoretically the initial drug-containing water phase was always separated from the dispersion medium by the lecithin-containing oil phase, which was advantageous to maintaining a higher encapsulation efficiency of the water-soluble drug. The encapsulation efficiency of lipophilic ibuprofen and flurbiprofen was around 90%. As for ketoprofen and liposomes were not formed when using hydrogenated egg yolk lecithin R-5, while the encapsulation efficiency using PEL or R-20 was around 80%. Amphiphilic amitriptyline hydrochloride resulted in a slightly higher encapsulation efficiency when dissolved in the water than the chloroform. Among the three kinds of lecithin, the most unsaturated PEL tended to show a higher encapsulation efficiency, probably due to differences in the packing geometry of the hydrophobic carbon chains in the membrane bilayer. The encapsulation efficiency of these drugs strongly correlated to the log  P octanol/water and also tended to correlate to the log  P chloroform/water for the order of the log  P chloroform/water was almost the same as the order of the log  P octanol/water in the drugs examined. As far as the results of this study, the log  P octanol/water was considered to be a better indicator of the encapsulation efficiency of a drug in the MCV method.
Keywords: Liposome; Encapsulation efficiency; Partition coefficient; Hydrogenated egg yolk lecithin;

Two new plate nozzles for the production of alginate microspheres by Fan Yang; Kang Wang; Zhimin He (206-210).
Combining the Rayleigh-type jet break-up and two new plate nozzles, the alginate microsphere was produced. Spray generators made of syringe needle and laser-drilling nozzle plate and synthetic red stone nozzle plate were fabricated and contrasted. The above two plate nozzles provided lower liquid resistance and yield well. Furthermore, the more uniform microsphere was produced within a wider range of frequency by plate nozzles. Experiments using multiple-nozzle synthetic red stone plate was easy to feasible.
Keywords: Laser-drilling nozzle plate; Synthetic red stone nozzle plate; Alginate microsphere;

The objectives of this study were to determine the effects of heat-treatment on Fungizone® (FZ)-induced cytotoxicity in human kidney (HK-2) cells and fungal isolates of Aspergillus fumigatus, and to determine the possible role of phospholipases (PLA2 and PLC) on heat-treated FZ (HFZ)-associated renal cell toxicity. HK-2 cells were grown at 37 °C in T75 flasks and seeded in 96-well plates at 20,000 cells/well. FZ and HFZ concentrations of 10, 25 and 50 μg/mL of AmpB were prepared. Snake venom PLA2 and PLC (2.15 U/mL) were pre-incubated with HFZ for 1 h prior to addition to the cells. After 18 h of incubation, an MTS assay was performed to assess cell viability through mitochondrial respiration. A spore suspension of A. fumigatus was prepared and 96-well plates were seeded at 500,000 spores/well. HFZ and FZ were prepared as above and incubated with the fungi at 35 °C. After 72 h, the minimum inhibitory concentration (MIC) was determined as the lowest concentration of drug that inhibited visible growth. Student–Newman–Keuls multiple comparisons tests were conducted to determine statistical significance. FZ-induced cytotoxicity was significantly greater than for HFZ in HK-2 cells at amphotericin B (AmpB) concentrations between 10 and 50 μg AmpB/mL (n  = 5–9, p  < 0.05). HFZ and FZ were found to have similar minimum inhibitory concentration (MIC) ranges for A. fumigatus (0.225–0.25 μg) AmpB/mL; (n  = 6). The addition of PLA2 and PLC to 50 μg heat-treated AmpB/mL significantly enhanced the cytotoxicity compared to controls (n  = 6, p  <  0.05). The presence of the phospholipases did not alter FZ-associated renal cell toxicity. Taken together, these findings suggest heat-treatment significantly decreased FZ-induced cytotoxicity in HK-2 cells without altering toxicity against a reference strain of A. fumigatus. In addition, PLA2 and PLC enhanced the renal toxicity associated with HFZ, but not that of FZ.
Keywords: Heat-treated amphotericin B; Renal toxicity; Human kidney cells; Aspergillus fumigatus; Phospholipases; Fungizone®;

Micelle-like nanoparticles of PLA–PEG–PLA triblock copolymer as chemotherapeutic carrier by Subbu S. Venkatraman; Pan Jie; Feng Min; Boey Yin Chiang Freddy; Gan Leong-Huat (219-232).
Triblock copolymer PLA–PEG–PLA were synthesized using ring opening polymerization with different LA/EG ratio. Micellar aggregates were prepared from these block copolymers and characterized. The degradation characteristics of selected copolymers were assessed in both micellar and film forms. Surface segregation of PEG was also quantified as a function of copolymer composition. Anti-cancer drugs 5-FU and paclitaxel were loaded into the micellar nanospheres with good efficiency. The drug release profile showed good control over the release of paclitaxel from these polymers.
Keywords: PLA; PEG; Drug carrier; 5-FU; Paclitaxel;

The basic characteristics and the biodistribution properties of nanoparticles prepared from mixtures of poly(lactide-co-glycolide) (PLGA) with poly(lactide-co-glycolide)–poly(ethylene glycol) (PLGA–PEG) copolymers were investigated. A PLGA(45)–PEG(5) copolymer of relatively low PEG content and a PLGA(5)–PEG(5) copolymer of relatively high PEG content were included in the study. Increasing the PLGA–PEG content of the PLGA/PLGA–PEG mixture, or when PLGA(45)–PEG(5) was replaced by PLGA(5)–PEG(5), a decrease in the size of the nanoparticles and an increase in the rate of PEG loss from the nanoparticles were observed. The blood residence of the PLGA/PLGA(45)–PEG(5) nanoparticles increased as their PLGA–PEG content was increased, reaching maximum blood longevity at 100% PLGA(45)–PEG(5). On the contrary, the blood residence of PLGA/PLGA(5)–PEG(5) nanoparticles exhibited a plateau maximum in the range of 80–100% PLGA(5)–PEG(5). At PLGA–PEG proportions lower than 80%, the PLGA/PLGA(45)–PEG(5) nanoparticles exhibited lower blood residence than the PLGA/PLGA(5)–PEG(5) nanoparticles, whereas at PLGA–PEG proportions higher than 80%, the PLGA/PLGA(45)–PEG(5) nanoparticles exhibited higher blood residence than the PLGA/PLGA(5)–PEG(5) nanoparticles. These findings indicate that apart from the surface PEG content, the biodistribution properties of the PLGA/PLGA–PEG nanoparticles are also influenced by the size of the nanoparticles and the rate of PEG loss from the nanoparticles.
Keywords: Poly(lactide-co-glycolide)/poly(lactide-co-glycolide)–poly(ethylene glycol) nanoparticles; Biodistribution; Physicochemical characteristics;

Lipid nanoparticles (LNP) based on triglycerides containing high amounts of the amphiphilic lipid lecithin have been proposed as a promising alternative drug delivery system with regard to drug loading capacity. Aim of the present study is to evaluate the influence of lecithin within the lipid matrix (LM) on the crystallization behavior by thermoanalysis and wide angle X-ray diffraction (WAXD). The crystallinity of LM and LNP is mainly determined by the triglyceride content. However, lecithin influences the crystallization behavior significantly. WAXD shows an accelerated polymorphic transition of the LM to the β-modification upon storage with increasing lecithin content. Both, the melting point and the crystallization temperature are not affected by the lecithin concentration and are comparable to recrystallized triglyceride bulk. However, the crystallinity indices (CI) of LM show a general decrease by 10% suggesting an incomplete crystallization. For the formation of LNP at least 10% lecithin is necessary and all systems are present in the stable β-modification. In comparison to the undispersed LM, the crystallization temperature of LNP is significantly decreased by about 20 °C whereas the melting point is reduced by about 5 °C only. Melting enthalpy is comparable to the untreated triglyceride bulk and elevated in comparison to the undispersed LM. Isothermal heat-conduction microcalorimetry (IMC) enables the determination of crystallization kinetics after fitting of the heat flow volume according to the Avrami equation.
Keywords: Crystallization; Lipid nanoparticles; Thermal analysis; X-ray diffraction;

Physicochemical characterization of poly(l-lactic acid) and poly(d,l-lactide-co-glycolide) nanoparticles with polyethylenimine as gene delivery carrier by In-Sook Kim; Soo-Kyung Lee; Yu-Mi Park; Yong-Bok Lee; Sang-Chul Shin; Kang Choon Lee; In-Joon Oh (255-262).
Polymer nanoparticles have been used as non-viral gene delivery systems and drug delivery systems. In this study, biodegradable poly(l-lactic acid) (PLA)/polyethylenimine (PEI) and poly(d,l-lactide-co-glycolide) (PLGA)/PEI nanoparticles were prepared and characterized as gene delivery systems. The PLA/PEI and PLGA/PEI nanoparticles, which were prepared by a diafiltration method, had spherical shapes and smooth surface characteristics. The size of nanoparticles was controlled by the amount of PEI, which acted as a hydrophilic moiety, which effectively reduced the interfacial energy between the particle surface and the aqueous media. The nanoparticles showed an excellent dispersive stability under storage in a phosphate-buffered saline solution for 12 days. The positive zeta-potentials for the nanoparticles decreased and changed to negative values with increasing plasmid DNA (pDNA) content. Agarose gel electrophoresis showed that the complex formation between the nanoparticles and the pDNA coincided with the zeta-potential results. The results of in vitro transfection and cell viability on HEK 293 cells indicated that the nanoparticles could be used as gene delivery carriers.
Keywords: Poly(l-lactic acid); Poly(d,l-lactide-co-glycolide); Polyethylenimine; Nanoparticles; Gene delivery;

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