International Journal of Pharmaceutics (v.329, #1-2)

Cyclodextrins and their pharmaceutical applications by Thorsteinn Loftsson; Dominique Duchêne (1-11).
Cyclodextrins were first described by Villiers in 1891. Schardinger laid the foundation of the cyclodextrin chemistry in 1903–1911 and identified both α- and β-cyclodextrin. In the 1930s, Freudenberg identified γ-cyclodextrin and suggested that larger cyclodextrins could exist. Freudenberg and co-workers showed that cyclodextrins were cyclic oligosaccharides formed by glucose units and somewhat later Cramer and co-workers described their ability to form inclusion complexes. By the early 1950s the basic physicochemical characteristics of cyclodextrins had been discovered, including their ability to solubilize and stabilize drugs. The first cyclodextrin-related patent was issued in 1953 to Freudenberg, Cramer and Plieninger. However, pure cyclodextrins that were suitable for pharmaceutical applications did not come available until about 25 years later and at the same time the first cyclodextrin-containing pharmaceutical product was marketed in Japan. Later cyclodextrin-containing products appeared on the European market and in 1997 also in the US. New cyclodextrin-based technologies are constantly being developed and, thus, 100 years after their discovery cyclodextrins are still regarded as novel excipients of unexplored potential.
Keywords: Complexation; Cyclodextrin; Drug delivery; Formulation; History; Regulatory; Toxicology;

Determination of n-octanol/water partition and membrane binding of cationic porphyrins by Fábio M. Engelmann; Silvia V.O. Rocha; Henrique E. Toma; Koiti Araki; Maurício S. Baptista (12-18).
Porphyrin and their derivatives are being systematically studied as photosensitizers for photodynamic therapy. The ability to predict their membrane partition properties is of key importance to unveil their in vivo activity and applications. Several n-octanol/water partition coefficients (log  P OW) of porphyrin derivatives have been reported in the literature but large discrepancies have been observed. Reproducible and reliable log  P OW data for a series of 20 cationic meso-phenyl(pyridyl)porphyrin derivatives were determined by correlating log  P OW with the partition coefficients measured in a more adequate n-butanol/water system. Linear correlations as a function of the number of positively charged groups bound to the periphery of the porphyrin rings were found within each series. A significant effect of the stereochemistry and nature of the positively charged substituents was also observed, but diminished steadily converging to a similar value in the mono-substituted derivatives. Binding constants to liposomes were shown to be proportional to log  P OW, except for the cis-isomers of doubly charged porphyrins. The cis-isomer presented smaller log  P OW and higher membrane affinity. The effect was explained based on the amphiphilic nature of the cis-porphyrin.
Keywords: Partition coefficients; n-Octanol/water; n-Butanol/water; Cationic porphyrins; Amphiplilic porphyrins; Membrane binding;

The role of N-acetyl-methioninate as a new stabilizer for albumin products by Makoto Anraku; Yousuke Kouno; Toshiya Kai; Yasufumi Tsurusaki; Keishi Yamasaki; Masaki Otagiri (19-24).
Sodium octanoate (Oct) and N-acetyl-l-tryptophanate (N-AcTrp) are widely used as stabilizers during the pasteurization of albumin products. However, N-AcTrp has a possible side effect of intracerebral disease. To provide safe and risk-free albumin products, we validated N-acetyl-methioninate (N-AcMet) as a new stabilizer for albumin products. The effect of N-AcMet on oxidation was examined using 2,2′-azobis(2-amidino-propane) dihydrochloride (AAPH) as an oxidizing agent. Carbonyl content in the presence of N-AcMet, as well as that in the presence of N-AcTrp after 24 h (), was significantly decreased. The effect of AAPH on the oxidative status of 34-Cys on human serum albumin was also studied by HPLC. It was found that N-AcMet as well as N-AcTrp, has a large protective effect on the sulfhydryl group after 1 h. Further, N-AcMet was found to be a superior radical scavenger to N-AcTrp using 1,1′-diphenyl-2-picrylhydrazyl (DPPH) radicals. The thermal stabilizing role of N-AcMet manifested as an increase in denaturation temperature and calorimetric enthalpy, as determined by differential scanning calorimetry (DSC). In the present study, we suggest that use of N-AcMet in albumin preparation is safe and free of risk of side effects.
Keywords: Albumin; Sulfhydryl groups; Calorimetry (DSC); Antioxidant activity; N-Acetyl-l-methioninate; Oxidation;

The effect of water solubility of solutes on their flux through human skin in vitro by Susruta Majumdar; Joshua Thomas; Scott Wasdo; Kenneth B. Sloan (25-36).
The Flynn database (n  = 97) for determining the effect of the physicochemical properties of solutes on their skin absorption has been edited to give a database for which the solubilities of the solutes in water, S AQ, and their maximum fluxes from water through human skin in vitro, J MAQ, are known or can be calculated (n  = 76). Data from the six major contributors to the original Flynn database have been included. Data for solutes, which were significantly ionized or for experiments using different thicknesses of skin were not excluded so that the edited database is as diverse as the original. The edited database was fit to five equations where the independent variables were solubility in octanol (S OCT) in water (S AQ) or molecular weight (MW), and combinations of those three variables; and the dependent variable was J MAQ. The best fit was obtained from the Roberts–Sloan (RS) equation: log  J MAQ  =  x  +  y  log  S OCT  + (1 −  y) log  S AQ  −  z  MW, x  = −3.00, y  = 0.73, z  = 0.0048, r 2  = 0.934, S.D. = 0.37 and F  = 274. This result is important because J (amount/area time) is the more clinically useful descriptor of permeation compared to P (distance/time); and because the identification of S AQ as a significant variable in predicting flux changes the design parameters for optimizing topical delivery of drugs from solubility in lipids (or partition coefficients between OCT and AQ, K OCT:AQ) and MW, to solubility in lipids, S OCT, and in water, S AQ, as well as MW.
Keywords: Flux; Human skin; In vitro; Water solubility; Lipid solubility;

Detection and characterization of protein aggregates by fluorescence microscopy by Barthélemy Demeule; Robert Gurny; Tudor Arvinte (37-45).
Aggregation may compromise the stability as well as the biological activity of protein drugs. Detection of protein aggregates is needed in the process of protein characterization and during optimization of pharmaceutical formulations. This paper describes a technique, which consists of analysing protein aggregates by fluorescence microscopy after staining with the hydrophobic probe Nile Red. Dilution, filtration or other modifications of the sample are not needed. Assessment of aggregation was possible in highly concentrated protein samples (193 mg/ml). Fluorescence microscopy observations allowed the detection and characterization of protein aggregates not easily detected by spectroscopic techniques. Nile Red was shown to be very sensitive for the detection and analysis of immunoglobulin aggregates. Nile Red, Congo red and Thioflavine T stainings were compared. Nile Red and Thioflavine T fluorescence were colocalized. The diameter of immunoglobulin aggregates was determined, and the number of aggregates was correlated with 90° light scattering measurements. Studies of human calcitonin aggregates brought to light new aspects of the human calcitonin aggregation mechanisms. Thus, Nile Red staining not only allows detection of very low levels of protein aggregates, but also contributes to a better understanding of the complex mechanisms governing protein aggregation.
Keywords: Immunoglobulin; Calcitonin; Aggregation; Nile Red; Microscopy; Physical stability;

Computational prediction of solubilizers’ effect on partitioning by Jan Hoest; Inge T. Christensen; Flemming S. Jørgensen; Lars Hovgaard; Sven Frokjaer (46-52).
A computational model for the prediction of solubilizers’ effect on drug partitioning has been developed. Membrane/water partitioning was evaluated by means of immobilized artificial membrane (IAM) chromatography. Four solubilizers were used to alter the partitioning in the IAM column. Two types of molecular descriptors were calculated: 2D descriptors using the MOE software and 3D descriptors using the Volsurf software. Structure–property relationships between each of the two types of descriptors and partitioning were established using partial least squares, projection to latent structures (PLS) statistics. Statistically significant relationships between the molecular descriptors and the IAM data were identified. Based on the 2D descriptors structure–property relationships R 2 Y  = 0. 99 and Q 2  = 0.82–0.83 were obtained for some of the solubilizers. The most important descriptor was related to log  P. For the Volsurf 3D descriptors models with R 2 Y  = 0.53–0.64 and Q 2  = 0.40–0.54 were obtained using five descriptors. The present study showed that it is possible to predict partitioning of substances in an artificial phospholipid membrane, with or without the use of solubilizers.
Keywords: IAM chromatography; In silico prediction; Solubilizers; Molecular descriptors; Structure–property relationship;

Evaluation of ibuprofen-loaded microspheres prepared from novel copolyesters by C.J. Thompson; D. Hansford; S. Higgins; C. Rostron; G.A. Hutcheon; D.L. Munday (53-61).
The utility of two novel linear random copolyesters to encapsulate and control the release of ibuprofen, via microspheres, was investigated. Various manufacturing parameters, including temperature, disperse phase volume and polymer:ibuprofen ratios were altered during the microsphere production. The effects of these changes on the morphological characteristics of the microspheres, yield, drug loading, encapsulation efficiency and drug release rates were examined. The diameter of the microspheres ranged from 36 to 89 μm and showed both smooth and ridged surfaces. Microsphere diameter was probably determined by the internal phase volume, while surface morphology was controlled by manufacturing temperature. Greater encapsulation efficiency was obtained by increasing the polymer:ibuprofen ratio and by reducing the internal phase volume. For all batches there was an initial burst drug release into phosphate buffer (pH 7.4) over the first 2–4 h, which was followed by a much slower release rate over the remaining time period. Drug release rates during both these phases were dependent upon the amount and nature of the polymer in the microspheres, noting that the more hydrophilic polymer provided faster release rates. Ibuprofen solubility appeared to play a dominant role in controlling release, although both encapsulation efficiency and microsphere morphology were also contributing factors.
Keywords: Polyesters; Microspheres; Emulsion solvent evaporation; Manufacturing parameters;

Preparation of monolithic matrices for oral drug delivery using a supercritical fluid assisted hot melt extrusion process by John G. Lyons; Mark Hallinan; James E. Kennedy; Declan M. Devine; Luke M. Geever; Paul Blackie; Clement L. Higginbotham (62-71).
The use of supercritical fluids as plasticisers in polymer processing has been well documented. The body of work described in this research paper outlines the use of a supercritical CO2 assisted extrusion process in the preparation of a hot melt extruded monolithic polymer matrix for oral drug delivery. Several batches of matrix material were prepared with Carvedilol used as the active pharmaceutical ingredient (API). These batches were subsequently extruded both with and without supercritical CO2 incorporation. The resultant matrices were characterised using steady-state parallel plate rheometry, differential scanning calorimetry (DSC), atomic force microscopy (AFM), micro-thermal analysis (μTA) and dissolution testing. Dissolution analysis showed that the use of supercritical CO2 during the extrusion process resulted in a faster dissolution of API when compared with unassisted extrusion. The supercritical CO2 incorporation also resulted in reduced viscosity during processing, therefore allowing for quicker throughput and productivity. The results detailed within this paper indicate that supercritical fluid assisted hot melt extrusion is a viable enhancement to conventional hot melt extrusion for the production of monolithic dosage forms.
Keywords: Supercritical fluid; Extrusion; Polymer; Drug release; Monolithic matrix;

Effect of the melt granulation technique on the dissolution characteristics of griseofulvin by Dong Yang; Rajesh Kulkarni; Robert J. Behme; Pramila N. Kotiyan (72-80).
This work describes a melt granulation technique to improve the dissolution characteristics of a poorly water-soluble drug, griseofulvin. Melt granulation technique is a process by which pharmaceutical powders are efficiently agglomerated by a meltable binder. The advantage of this technique compared to a conventional granulation is that no water or organic solvents is needed. Because there is no drying step, the process is less time consuming and uses less energy than wet granulation. Granules were prepared in a lab scale high shear mixer, using a jacket temperature of 60 °C and an impeller speed of approximately 20,000 rpm. The effect of drug loading (2.5/5%), binder (PEG 3350/Gelucire 44/14), filler (starch/lactose), and HPMC on the dissolution of griseofulvin was investigated using a half two level-four factor factorial design. The granules were characterized using powder XRD, DSC and SEM techniques. A significant enhancement in the in vitro dissolution profiles of the granules was observed compared to the pure drug and drug excipient physical mixtures. The factorial design results indicated that higher drug loading and the presence of HPMC reduced the extent of dissolution of the drug, whereas, the presence of starch enhanced the dissolution rate. XRD data confirmed crystalline drug in formulation matrices. DSC results indicated monotectic mixtures of griseofulvin with PEG in the granulated formulations. In conclusion, the results of this work suggest that melt granulation is a useful technique to enhance the dissolution rate of poorly water-soluble drugs, such as, griseofulvin.
Keywords: Griseofulvin; Melt granulation; PEG 3350; Gelucire 44/14; HPMC; Dissolution enhancement;

Granule breakage during drying processes by Florentine J.S. Nieuwmeyer; Kees van der Voort Maarschalk; Herman Vromans (81-87).
The drying of wet granules often involves an unwanted and uncontrolled size reduction. Current FDA PAT guidance stresses importance of process control and understanding. The aim of this study is to determine and understand the breakage phenomena during drying processes in order to control these processes. High shear granulated lactose granules with water as binding liquid were dried during variable periods. Subsequently the (partially) dried granules were exposed to agitation by the impeller and chopper in the granulator. Granule characterization revealed that the change in granule size of (partially) dried granules is dependent on water content and follows a three phase system characterized by a growth, plateau and breakage phase. The derived yield stress of the granules is a function of velocity. From this it is concluded that in the plateau phase above minimum water content, stress behavior of granules can be described with Rumpfs’ dynamic granule strength, whereas below minimum water content (breakage phase) granule strength is determined by the solid bridges. The extent and velocity of stress and water content of the granules during the process determine the size reduction phenomena.
Keywords: Drying; Granule; Breakage; Attrition; Water;

Doxorubicin skin penetration from monoolein-containing propylene glycol formulations by Haluka Herai; Taís Gratieri; José Antônio Thomazine; Maria Vitória Lopes Badra Bentley; Renata Fonseca Vianna Lopez (88-93).
Topical chemotherapy with the antineoplastic doxorubicin (DXR) could be an alternative to treat skin cancer, however its poor skin penetration often limits the efficacy of topical formulations. The aim of this work was to study the effect of monoolein (MO), a penetration enhancer, on the in vitro skin permeation and retention of DXR. DXR was incorporated in a propylene glycol preparation containing 0–20% of MO. DXR release rate and topical delivery were evaluated in vitro using acetate cellulose membrane and porcine skin, respectively, mounted in a Franz diffusion cell. At 5%, MO did not significantly change DXR release rate, but MO concentrations larger than 10% decreased almost twice its release. In vitro skin penetration studies showed that the presence of MO in the propylene glycol formulations markedly increased DXR presence in the stratum corneum (SC). At 5%, MO significantly increased the amount of DXR in the SC already in the first hours, attained a maximum in 6 h. Comparing propylene glycol formulations containing more than 10% MO with that containing 5%, the former took the double of the time (12 h) to reach the same amount of DXR in the skin, result that is in agreement with in vitro release studies. Interesting, despite the fact that MO significantly increased the amount of DXR in the SC, drug transdermal delivery did not change. These findings suggest a cutaneous delivery of DXR that is an important condition for topical treatment of skin tumors. Further in vivo experiments can show DXR delivery to deeper skin layers.
Keywords: Topical delivery; Monoolein; Doxorubicin; Skin penetration; Topical tumor therapy;

Effect of transferrin receptor-targeted liposomal doxorubicin in P-glycoprotein-mediated drug resistant tumor cells by Tomotaka Kobayashi; Tatsuhiro Ishida; Yurie Okada; Saori Ise; Hideyoshi Harashima; Hiroshi Kiwada (94-102).
The over-expression of P-glycoprotein (P-gp) has been associated with the development of multidrug resistance (MDR) in cancer cells. In this study, we examined whether transferrin receptor (Tf-R) targeted liposomes can efficiently deliver encapsulated doxorubicin (DXR) into MDR cells (SBC-3/ADM) via Tf-R-mediated endocytosis thus overcoming MDR by by-passing P-gp-mediated drug efflux. We prepared four types of liposome, i.e. untargeted and Tf-R-targeted, made of either egg-PC/cholesterol or hydrogenated egg PC/cholesterol. Only with the targeted EPC-liposome we achieved significant delivery of encapsulated DXR and increased cytotoxicity of encapsulated DXR on the MDR cells (3.5-fold higher than free DXR). Confocal microscopy and an intracellular drug-accumulation assay indicated that the targeted liposomes efficiently delivered DXR into cells where it readily accumulated in the nucleus, in both drug-sensitive and MDR cells. These findings suggest that the targeted liposomes are rapidly internalized via Tf-R-mediated endocytosis followed by release of their contents into the cytoplasm. The rapid internalization and content release, most likely facilitated by the higher fluidity of the EPC-based liposomes, may explain why only targeted EPC-liposomes were able to prevent drug efflux by P-gp and to consequently circumvent MDR. Our results indicate that in order to achieve MDR circumvention by means of liposomal encapsulation of DXR the liposomes not only need to be targeted, but also to have the proper physicochemical properties for adequate release of the drug. Furthermore, these in vitro results suggest that Tf-R targeted EPC-liposomes are a potentially useful drug delivery system to circumvent P-gp-mediated MDR of tumors.
Keywords: Liposome; Targeting; Multidrug resistance (MDR); P-glycoprotein (P-gp); Transferrin;

Characterization of chondroitin sulfate and its interpenetrating polymer network hydrogels for sustained-drug release by Shiu-Chih Wang; Bing-Hung Chen; Li-Fang Wang; Jenn-Shing Chen (103-109).
The goal of this work was to utilize the chondroitin sulfate (CS) based hydrogels for a drug delivery matrix. CS is a good structure/disease-modifying anti-osteroarthritis drug (S/DMOAD). However, the readily water-soluble nature limits its application as a solid-state drug-delivery vehicle. In this study, two methods were used to prepare CS hydrogels: directly crosslinking CS with poly(ethylene glycol) diglycidyl ether (EX-810) abbreviated as CS-EX or forming an interpenetrating polymer network named CS-EX-IPN. The CS-EX-IPN hydrogel was carried out by sequentially crosslinking reaction between CS and EX-810 in one phase and acrylic acid and di(ethylene glycol) diacrylate (DEGDA) as a counter phase. The swelling percent, cross-section morphology, and effective crosslinking density of hydrogels were characterized. The values of compression modulus and effective crosslinking density of CS-EX-IPN were ∼3.6-fold higher than CS-EX. We also characterized the release of a model drug, diclofenac sodium (DS) and a model protein, bovine serum albumin (BSA), from CS-EX and CS-EX-IPN. The similar release profiles of DS were observed in the both hydrogels but slower release rate of BSA occurred in CS-EX-IPN. The release profiles of the two model drugs fit in a diffusion-controlled mechanism. The D eff values are in the order of 10−5 for DS and 10−7 for BSA.
Keywords: Diclofenac sodium; Bovine serum albumin; Chondroitin sulfate; Hydrogels; Interpenetrating polymer network;

Effect of coupling of albumin onto surface of PEG liposome on its in vivo disposition by Kentaro Furumoto; Jun-Ichi Yokoe; Ken-ichi Ogawara; Sayuri Amano; Maki Takaguchi; Kazutaka Higaki; Toshiya Kai; Toshikiro Kimura (110-116).
To evaluate the effect of coupling of albumin onto the surface of poly(ethylene glycol)-modified liposome (PEG liposome) on the in vivo disposition of liposome, pharmacokinetics and tissue distribution were examined after intravenous administration of rat serum albumin-modified PEG (RSA/PEG) liposome into rats. RSA/PEG liposome showed longer blood-circulating property than PEG liposome and the hepatic clearance for RSA/PEG liposome was significantly smaller than that for PEG liposome. Single-pass liver perfusion experiments also showed that the hepatic disposition of RSA/PEG liposome was much less than that of PEG liposome and that pre-treatment of liver with trypsin did not significantly reduce the hepatic disposition of RSA/PEG liposome, suggesting that RSA/PEG liposome could avoid the hepatic uptake via the receptor-mediated endocytosis. To unravel the mechanism behind the less affinity of RSA/PEG liposome to the liver, serum proteins associated on their surface were quantitatively and qualitatively assessed. The results showed that the coupling of albumin onto PEG liposome significantly reduced the total amount of serum proteins associated onto the surface, and SDS-PAGE revealed that the decrease in the association with liposomes for several serum proteins, which might have opsonic activity. From these findings, introduction of serum albumin onto PEG liposome could be useful to develop a new nanoparticulate formulation with a better pharmacokinetic property.
Keywords: Liposomes; Poly(ethylene glycol); Rat serum albumin; Hepatic disposition; Serum opsonins;

Enhanced immune responses induced by vaccine using Sendai virosomes as carrier by Qiang Li; Jian-Qing Gao; Li-Yan Qiu; Fu-De Cui; Yi Jin (117-121).
Sendai virosomes can deliver encapsulated contents into the cytoplasm directly in a virus fusion-dependent manner. In this paper, Sendai virosomes-formulated melanoma vaccine was constructed and its anti-tumor effects were investigated. The melanoma vaccine was prepared by encapsulating mixture antigen into the Sendai virosomes. The antigen, mixture proteins were extracted from B16 melanoma cells. The cytotoxic T lymphocyte (CTL) response level was evaluated by 51Cr release method, and the change of CD4+ and CD8+ expression as well as the concentration of IgG in serum of immunized mice was measured. The results showed that Sendai virosomes-formulated melanoma vaccine can effectively elicit not only systemic immune response but also strong CTL response. Sendai virosomes can be used as an effective vector for use in anti-tumor vaccine therapy.
Keywords: Vaccine; Melanoma; Sendai virosomes;

The purpose of the present study was to develop implantable poly(d,l-lactide-co-glycolide) (PLGA) microparticles for continuous delivery of intact 3,4-dihydro-3-methyl-4-oxoimidazo[5,1-d]-as-tetrazine-8-carboxamide (temozolomide, TM) for about a 1-month period and to evaluate its cytotoxicity against Glioma C6 cancer cells. The emulsifying-solvent evaporation process has been used to form TM-loaded PLGA microparticles. The influences of several preparation parameters, such as initial drug loading, polymer concentration, and stirring rate were investigated. Scanning electron microscopy (SEM) showed that such microparticles had a smooth surface and a spherical geometry, i.e. microspheres. The differential scanning calorimetry (DSC) and powder X-ray diffraction (XRD) results indicated that TM trapped in the microparticles existed in an amorphous or disordered-crystalline status in the polymer matrix. The release profiles of TM from microparticles resulted in biphasic patterns. After an initial burst, a continuous drug release was observed for up to 1 month. Finally, a cytotoxicity test was performed using Glioma C6 cancer cells to investigate the cytotoxicity of TM delivered from PLGA microparticles. It has been found that the cytotoxicity of TM to Glioma C6 cancer cells is enhanced when TM is delivered from PLGA polymeric carrier and, PLGA only did not affect the growth of the cells. Meanwhile, the cytotoxic activity of TM powder disappeared within 12 h.
Keywords: Temozolomide (TM); Poly(d,l-lactide-co-glycolide) (PLGA); Emulsifying-solvent evaporation; Microparticles; Cytotoxicity;

Data on distribution coefficients from blood or plasma to rat skin and rabbit skin have been compiled. From previous work on blood/plasma to brain or to muscle it is apparent that distributions from blood and plasma can be combined, and we show that it is possible to combine data on distribution to rat skin and rabbit skin. The combined set of blood/plasma distribution to rat and rabbit skin for 59 compounds, as log Pskin, can be correlated through a linear free energy equation with a correlation coefficient of 0.856 and a standard deviation of 0.26 log units. The predictive capability of the equation has been assessed through training and test sets, and it is shown that the S.D. value of 0.26 log units is a good estimate of the predictive ability. The equation for log Pskin has been compared to equations for a large number of possible model processes, using two mathematical methods. It is shown that there is no process amongst those we have examined that has any advantage over the present in silico linear free energy equation for the estimation of further values of log Pskin.
Keywords: Blood; Plasma; Skin; Rat; Rabbit; Distribution; Partition; Solvation equation; Hydrogen bonding; Linear free energy equation; In silico;

EO-9 bladder instillations: Formulation selection based on stability characteristics and in vitro simulation studies by S.C. van der Schoot; L.D. Vainchtein; J.H. Beijnen; A. Gore; D. Mirejovsky; L. Lenaz; B. Nuijen (135-141).
A bladder instillation of EO-9 (EOquin™) is currently used in phase II clinical trials for the treatment of superficial bladder cancer. Three alternative formulations were developed to improve its pharmaceutical properties and clinical acceptability. Freeze-dried products composed of EO-9, 2-hydroxypropyl-β-cyclodextrin (HPβCD), tri(hydroxymethyl) aminomethane (Tris), and sodium bicarbonate (NaHCO3) were tested. Selection of one formulation for further development was based on stability studies. These studies comprised stability of the freeze-dried products, stability after reconstitution and dilution and stability during bladder instillation in an experimental set-up. The stability study of the freeze-dried products showed that the formulation composed of EO-9/HPβCD/Tris (4/600/1 mg/vial) was most stable. After reconstitution and dilution all products were stable for at least 8 h. The product composed of EO9/HPβCD/NaHCO3 (4/600/20 mg/vial) was the least stable product both as freeze-dried formulation and after reconstitution and dilution. The bladder instillation simulation experiment showed that all products were stable when mixed with urine of pH 8 and unstable in urine of pH 4 and 6. The degradation products formed in urine were EO-5a and EO-9-Cl.Based on these results, the product composed of EO-9/HPβCD/Tris (4/600/1 mg/vial) was selected for further pharmaceutical development.
Keywords: EO-9; Cyclodextrin; Stability; Urine;

A new nanoparticulate delivery system for amphotericin B (AmB) has been developed by means of the polyelectrolyte complexation technique. Two opposite charged polymers were used to form nanoparticles through electrostatic interaction, chitosan (CH) as a positively charged polymer and dextran sulfate (DS) as a polymer with a negative charge, together with zinc sulfate as a crosslinking and hardening agent. The AmB nanoparticles obtained possessed a mean particle size of 600–800 nm with a polydispersity index of 0.2, indicating a narrow size distribution. The measured zeta potential of the nanoparticle surface was approximately −32 mV indicating a strong negative charge at the particle's surface. Scanning electron microscopy revealed spherical particles with a smooth surface. Drug association efficacy of up to 65% was achieved. Dissolution studies demonstrated a fast release behavior suggesting that AmB exhibits only moderate interaction with the weakly crosslinked polymers of the nanoparticles. Although, electronic absorbance spectra showed that the aggregation state of AmB was modified within the nanoparticles, a reduction of nephrotoxicity was observed in an in vivo renal toxicity study.
Keywords: Amophotericin B; Chitosan; Dextran sulfate; Nanoparticles;

Influence of composition and preparation parameters on the properties of aqueous monoolein dispersions by G. Wörle; M. Drechsler; M.H.J. Koch; B. Siekmann; K. Westesen; H. Bunjes (150-157).
Colloidal cubic phase particles formed in the monoolein/poloxamer/water system are being investigated as potential drug carriers for, e.g., intravenous administration. Preparation methods must, however, still be further developed to reliably yield monoolein dispersions with cubic particles in a size range acceptable for i.v. administration and adequate long-term stability. In this context, the influence of different composition and preparation parameters on the properties of monoolein dispersions prepared by high-pressure homogenization was studied. High pressure homogenization of coarse poloxamer 407-stabilized monoolein/water mixtures leads to dispersions with a large fraction of micrometer-sized particles at low poloxamer concentrations. Higher poloxamer concentrations lead to lower mean particle sizes but the fraction of cubic particles becomes smaller and vesicular particles are observed instead. A study of the characteristics of a dispersion with a standard composition indicated that the homogenization temperature has a much stronger influence on the dispersion properties than the homogenization pressure or the type of homogenizer used. Temperatures around 40–60 °C lead to the most favorable dispersion properties. The high temperature sensitivity of the preparation process appears to be at least partly correlated with the phase behavior of the dispersed particles determined by temperature-dependent X-ray diffraction.
Keywords: Dispersions of cubic phase; Nanoparticles; X-ray diffraction; Phase behavior; Monoolein;

Encapsulation of hydrophobic drugs in polymeric micelles through co-solvent evaporation: The effect of solvent composition on micellar properties and drug loading by Hamidreza Montazeri Aliabadi; Sara Elhasi; Abdullah Mahmud; Rashida Gulamhusein; Parvin Mahdipoor; Afsaneh Lavasanifar (158-165).
This study was designed to develop an optimized co-solvent evaporation procedure for the efficient encapsulation of hydrophobic drugs in polymeric micelles of methoxy poly(ethylene oxide)-block-poly(ɛ-caprolactone) (MePEO-b-PCL). MePEO-b-PCL block copolymers having varied MePEO and PCL molecular weights were synthesized, assembled to polymeric micelles, and used for the encapsulation of cyclosporine A (CyA) by a co-solvent evaporation method. The co-solvent composition was varied by changing the type of organic co-solvent (using acetone, acetonitrile and tetrahydrofuran), the ratio of organic to aqueous phase, and their order of addition. Carrier size, morphology and encapsulated CyA levels were defined by dynamic light scattering (DLS), transmission electron microscopy (TEM) and HPLC, respectively, and the effect of co-solvent composition on micellar properties and loaded CyA levels was evaluated. Application of acetone and acetonitrile as the selective co-solvent for the core-forming block led to a decrease in the average diameter of self-assembled structures. When acetone was added to water, a decrease in the ratio of organic to aqueous phase led to an increase in the loading efficiency of CyA in MePEO-b-PCL micelles. A similar trend in CyA loading was observed for MePEO-b-PCL micelles of varied MePEO and PCL block lengths. The ratio of organic to aqueous phase did not affect CyA loading when water was added to acetone. Irrespective of the order of addition, the decrease in the organic to aqueous phase ratio caused a reduction in the average diameter of the empty and CyA loaded micelles. We conclude that the co-solvent evaporation method may be optimized to improve the efficiency of drug encapsulation in polymeric micelles. For CyA encapsulation in MePEO-b-PCL micelles, addition of acetone to water at lower organic to aqueous phase ratio is shown to be the optimum procedure leading to higher drug encapsulation and smaller average diameter for the self-assembled structures.
Keywords: Polymeric micelles; Cyclosporine A; Poly(ethylene oxide); Poly(ɛ-caprolactone); Solubilization;

Self-nanoemulsifying drug delivery systems (SNEDDS) were developed with the objective to overcome problems associated with the delivery of cefpodoxime proxetil (CFP), a poorly bioavailable high dose antibiotic having pH dependant solubility. Solubility of CFP in oily phases and surfactants was determined to identify components of SNEDDS. Various surfactants and co-surfactants were screened for their ability to emulsify selected oily phases. Ternary phase diagrams were constructed to identify area of nanoemulsification for the selected systems. The influence of CFP and the pH of dilution medium on the phase behavior of selected system were assessed. The globule size of optimized CFP SNEDDS in various dissolution media was determined to check the effect of pH on its behavior. The optimized CFP SNEDDS needed surfactant content less than 40% and yielded nanoemulsion of mean globule size 170 nm, which was not affected by the pH of dilution medium. The optimized SNEDDS released CFP completely within 20 min irrespective of the pH of dissolution medium.
Keywords: Cefpodoxime proxetil; SNEDDS; pH dependant solubility;

Novel poloxamer-based nanoemulsions to enhance the intestinal absorption of active compounds by Carsten Brüsewitz; Andreas Schendler; Adrian Funke; Torsten Wagner; Ralph Lipp (173-181).
On the basis of Pluronic® P104 as primary emulsifier and Lauroglycol® 90 as amphiphilic oil phase, two nanoemulsion systems were developed with Pluronic® L62 or L81 as secondary emulsifiers. The possible nanoemulsion region of combinations of these excipients was described in ternary phase diagrams. Three formulations were selected from the nanoemulsion region and their potential impact on oral absorption was examined in the Caco-2 monolayer model of the small intestine. The apparent permeability of the BCS class III compound Atenolol was enhanced 2.5-fold, of BCS class II compound Danazol 3.2-fold and of BCS class I compound Metoprolol 1.4-fold. The three formulations were very well tolerated by the Caco-2 cells, which was confirmed by TEER measurements, a MTT test and a LDH release test.
Keywords: Absorption enhancer; Nanoemulsion; Poloxamer;

Distribution, transition, adhesion and release of insulin loaded nanoparticles in the gut of rats by Ming-Guang Li; Wan-Liang Lu; Jian-Cheng Wang; Xuan Zhang; Xue-Qing Wang; Ai-Ping Zheng; Qiang Zhang (182-191).
The purpose of this work was to investigate distribution, transition, bioadhesion and release behaviors of insulin loaded pH-sensitive nanoparticles in the gut of rats, as well as the effects of viscosity agent on them. Insulin was labeled with fluorescein isothiocyanate (FITC). The FITC-insulin solution and FITC-insulin nanoparticle aqueous dispersions with or without hydropropylmethylcellulose (HPMC, 0.2%, 0.4%, or 0.8% (w/v)) were orally administered to rats, respectively. The amounts of FITC-insulin in both the lumen content and the intestinal mucosa were quantified by a spectrofluorimeter. The release profiles in the gut were plotted by the percentages of FITC-insulin released versus time. FITC-insulin nanoparticle aqueous dispersion showed similar stomach but lower intestine empty rates, and enhanced intestinal mucosa adhesion in comparison with FITC-insulin solution. Addition of the HPMC reduced the stomach and intestine empty rates, enhanced the adhesion of FITC-insulin to the intestine mucosa. The release of FITC-insulin from nanoparticles in the gut showed an S-shape profile, and addition of HPMC prolonged the release half-life from 0.77 to 1.51 h. It was concluded that the behaviors of pH-sensitive nanoparticles tested in gastrointestinal tract of rats and the addition of HPMC were favorable to the absorption of the drug loaded.
Keywords: Insulin; Nanoparticles; Fluorescein isothiocyanate; Hydropropylmethylcellulose; Gastrointestinal distribution;

Fetuin mediates hepatic uptake of negatively charged nanoparticles via scavenger receptor by Susumu Nagayama; Ken-ichi Ogawara; Keiko Minato; Yoshiko Fukuoka; Yoshinobu Takakura; Mitsuru Hashida; Kazutaka Higaki; Toshikiro Kimura (192-198).
We tried to evaluate the possible involvement of fetuin in the scavenger receptors (SRs)-mediated hepatic uptake of polystyrene nanospheres with the size of 50 nm (NS-50), which has surface negative charge (zeta potential = −21.8 ± 2.3 mV). The liver perfusion studies in rats revealed that the hepatic uptake of NS-50 pre-coated with fetuin (NS-50-fetuin) was significantly inhibited by poly inosinic acid (poly I), a typical inhibitor of SRs, whereas that of plain NS-50 or NS-50 pre-coated with BSA (NS-50-BSA) was not. The uptake of NS-50-fetuin by cultured Kupffer cells was also significantly inhibited by poly I, and anti-class A scavenger receptors (SR-A) antibody, suggesting that fetuin on NS-50 mediated the recognition and internalization of NS-50 by Kupffer cells and at least SR-A would be responsible for the uptake. Taken that Western blot analysis confirmed that fetuin certainly adsorbed on the surface of NS-50 after the incubation of NS-50 with serum, the results obtained in the present study indicate that fetuin would be one of the serum proteins that were substantially involved in the hepatic uptake of NS-50 via SRs.
Keywords: Polystyrene nanosphere; Fetuin; Scavenger receptors; Hepatic uptake; Receptor-mediated phagocytosis;

Noticeboard (199).