International Journal of Pharmaceutics (v.351, #1-2)

Stabilization of a polypeptide in non-aqueous solvents by Wei Wang; Sheryl Martin-Moe; Clark Pan; Laszlo Musza; Y. John Wang (1-7).
A pituitary adenylate cyclase-activating peptide (PACAP) analogue (HSDAVFTDNYTRLRKQVAAKKYLQSIKNKRY, P66) was formulated in several non-aqueous solvents in anticipation of improved shelf-life stability. However, the stability of this peptide in these solvents was found to be as poor as in an aqueous solution. The major degradation reaction in non-aqueous solvents was dimer formation. The proposed mechanism for dimerization was a nucleophilic attack of a basic amino acid on cyclic imide formed by dehydration or deamidation of Asp or Asn. Two approaches were found to be effective in stabilizing the peptide in non-aqueous solvents: (1) acidification of the peptide and (2) use of zinc chloride in the formulation. Stabilization could be attributed to reduction of the nucleophilicity of the reactive groups through protonation and metal–peptide interaction through chelation. The stabilization approaches are applicable only in a non-aqueous environment for this peptide, and possibly for other peptides with similar reactive moieties.
Keywords: Peptide stability; Non-aqueous solvent; Deamidation; Zinc stabilization; Dimerization; PACAP;

Zonula occludens toxin (Zot) and its biologically active fragment, delta G, have been shown to reversibly open tight junctions (TJ) in endothelial and epithelial cells. Recently, a six-mer synthetic peptide H-FCIGRL-OH (AT1002) was identified and synthesized that retains the Zot permeating effect on intercellular TJ. The objective of this study was to evaluate the biological activity of AT1002 on enhancing the oral administration of cyclosporin A (CsA). The intestinal permeability enhancing effect of AT1002 on the transport of CsA across Caco-2 cell monolayers was examined after the following treatments, i.e., CsA, CsA/protease inhibitors (PI), CsA/PI/benzalkonium chloride (BC), CsA/AT1002, CsA/PI/AT1002, and CsA/PI/BC/AT1002 (CsA 0.5 μCi/ml, PI (bestatin 15 mM and E-64 5 mM), BC 0.005 w/v%, and AT1002 5 mM, respectively). Apparent permeability coefficients (P app) were calculated for each treatment. In addition, four treatments, i.e., CsA, CsA/PI/BC, CsA/AT1002, and CsA/PI/BC/AT1002 (CsA 120 μCi/kg, PI (bestatin 30 mg/kg and E-64 10 mg/kg), BC 0.1 w/v%, and AT1002 doses of 5, 10 or 40 mg/kg, respectively) were prepared and administered intraduodenally to male Sprague–Dawley rats (230–280 g, n  = 4–5). Blood samples were collected at 0, 20, 60, and 120 min post-dosing and CsA plasma concentrations were determined subsequently using a Beckman Liquid Scintillation Counter. No significant increases in CsA transport were observed in the Caco-2 cell culture experiments following pre-treatment with AT1002 (5 mM). Even though, AT1002 appeared to increase the P app of CsA in each treatment (CsA/AT1002, 1.54 ± 0.13 × 10−6  cm/s and CsA/PI/AT1002, 1.76 ± 0.05 × 10−6  cm/s) compared to each control (CsA and CsA/PI), respectively. The plasma concentration of CsA was significantly increased over a range of 1.55–2.50 at 10 and 40 mg/kg dose of AT1002. Also, AUC0–120 min of CsA over a range of 1.64–2.14 and the C max of CsA over a range of 1.77–2.56 was statistically and significantly increased at 10 and 40 mg/kg of AT1002 after the intraduodenal administration of CsA/PI/BC/AT1002 to Sprague–Dawley rats. AT1002 significantly increased the in vivo oral absorption of CsA in the presence of PI. This study suggests that AT1002-mediated tight junction modulation, combined with metabolic protection and stabilization, may be used to enhance the low oral bioavailability of certain drugs when administered concurrently.
Keywords: Absorption enhancer; Delta G; Zonula occludens toxin; Intestinal absorption;

Development of a self-emulsifying formulation that reduces the food effect for torcetrapib by M.E. Perlman; S.B. Murdande; M.J. Gumkowski; T.S. Shah; C.M. Rodricks; J. Thornton-Manning; D. Freel; L.C. Erhart (15-22).
Torcetrapib is a highly lipophilic (Clog  P  = 7.45) and water insoluble cholesteryl ester transfer protein (CETP) inhibitor developed for the treatment of atherosclerosis. Self-emulsifying drug delivery system (SEDDS) formulations have been developed to reduce the food effect observed in early clinical trials using medium chain triglyceride (MCT) softgels and to increase the dose per capsule. MCT/Triacetin/Polysorbate 80/Capmul MCM (20/30/20/30) (MTPC) increased fasted exposure and thus reduced the food effect from 5- to 3-fold in dogs at a dose of 90 mg. Self-emulsifying formulations also accelerated absorption and generally decreased variability. Use of the lipophilic, GRAS cosolvent triacetin allowed a 2-fold increase in the dose per capsule. For the three formulations evaluated in dogs that showed significant differences in absorption, emulsion droplet size did not appear to play a significant role. Absorption did increase with Cremophor RH40 content, and at 50% Cremophor RH40 there was no food effect (at 30 mg). High polar surfactant content also resulted in poor dose proportionality, while there was good dose proportionality for MTPC. Studies of in vitro lipolysis are being conducted to aid in understanding the in vitro/in vivo relationships for torcetrapib SEDDS absorption.
Keywords: Self-emulsifying drug delivery system; Particle size; Medium chain triglycerides; Oral bioavailability; Food effect;

We investigated various chitosan types and batches successfully with a combination of asymmetrical flow field-flow fractionation and multi-angle light scattering using a customized separation method. Advantageous were the separation capability of a broad applicable molar mass range, the injection of unpurified solutions and the determination of absolute molar masses. Most of the measured samples followed molar mass distributions of monomodal logarithmic Gaussian type. Only one sample showed a very broad distribution caused by either high molar mass amounts or aggregate formation. It was also found that batch-to-batch variations were quite high, which is a common problem for nature-derived products. Thus, especially pharmaceutical products and semi-synthetic derivates should benefit from the development of products with a predictable average molar mass and a narrow distribution. On the basis of received data for each slice eluting from the channel, molar mass–gyration radius relationships could be generated for every single measurement. The hereof received molecule structure parameters were verified to be molar mass dependent, ranging from open structures of rinsed thoroughly molecules to theta coil conditions or an even more compact conformation.
Keywords: Chitosan; Field-flow fractionation; Multi-angle light scattering (MALS); Molecular weight; Molar mass distribution; Molecular structure;

Transport characteristics of ginkgolide B by Caco-2 cells and examination of ginkgolide B oral absorption potential using rat in situ intestinal loop method by Hua Lv; Guangji Wang; Xiaolan Wu; Lin Xie; Chenrong Huang; Hao Li; Yan Liang; Haiping Hao; Jie Sun (31-35).
The intestinal absorptive characteristics of ginkgolide B were investigated. The Caco-2 cells and the in situ closed loop were used as models of the intestinal mucosa to assess transepithelial transport of ginkgolide B. The determination of ginkgolide B was performed by HPLC–MS. In the Caco-2 cells, the absorptive transepithelial transport of ginkgolide B was pH dependent and the transport was enhanced at weakly acidic pH on the apical side. No concentration dependence and saturation were observed for the absorptive transport of ginkgolide B at concentrations up to 50 μM. In the in situ closed loop, the absorption of ginkgolide B was intestinal segment-selective. The results indicate that the intestinal absorption of ginkgolide B in the upper intestine was significantly higher than that in the lower intestine.
Keywords: Ginkgolide B; Intestinal absorption; Caco-2 cells; In situ closed intestinal loop;

The taste sensory evaluation of medicinal plants and Chinese medicines by Masumi Kataoka; Emi Tokuyama; Yohko Miyanaga; Takahiro Uchida (36-44).
The purpose of this study was to investigate the use of the artificial taste sensor in the evaluation of 11 medicinal plants and 10 Chinese medicines with bitter and/or astringent tastes, and to assess the possible application of the sensor in the evaluation of taste and quality control of medicinal products.Aqueous extracts of the six bitter medicinal plants could be classified into three types, and those of the five astringent medicinal plants into two types, on the basis of sensor output pattern profiles. These differences seem to derive from the different structures of the main components.In the principal component analysis of the taste sensor output of 10 Chinese medicines, a new measure developed, the ‘Euclidean distance’, defined as the distance between a control and the targeted substance on the principal component map. This measure offers a possibility for indicating the different tastes of Chinese medicines.Lastly, we confirmed that berberine adsorption on the surface of the artificial membrane of the taste sensor was of the Langmuir type. The berberine content in extracts of medicinal plants could be evaluated by the taste sensor, and it was shown to be possible to use the taste sensor for the quality control of medicinal plants.
Keywords: Taste sensor; Medicinal plants; Chinese medicine; Bitterness; Astringency; Berberine;

Particle design of naproxen-disintegrant agglomerates for direct compression by a crystallo-co-agglomeration technique by Maryam Maghsoodi; Omid Taghizadeh; Gary P. Martin; Ali Nokhodchi (45-54).
The purpose of the present research was to obtain directly compactible agglomerates of naproxen containing disintegrant by a novel crystallo-co-agglomeration (CCA) technique. Acetone–water containing hydroxypropylcellulose (HPC) was used as the crystallization medium. Acetone acted as a good solvent for naproxen as well as a bridging liquid for agglomeration of crystallized drug with disintegrant and aqueous phase as non-solvent. The agglomerates were characterized by differential scanning calorimetry (DSC), powder X-ray diffraction (XRPD) and scanning electron microscopy. The agglomerates were compressed at different compression pressures and dissolution studies were carried out for the tablets produced at lowest compression force. The growth of particle size and the spherical form of the agglomerates resulted in formation of products with good flow and packing properties. The improved compaction properties of the agglomerated crystals were due to the fragmentation which occurred during compression. DSC and XRPD studies showed that naproxen particles, crystallized in the presence of HPC and disintegrant did not undergo structural modifications. The dissolution rate of naproxen from the agglomerates could be controlled by the amount of included disintegrant, being enhanced as the latter was increased. Moreover, the results showed that when the disintegrants were included both intragranularly and extragranularly during agglomeration of naproxen particles, tablets containing these agglomerates dissolved at a faster rate than the tablets containing crystallized naproxen with the same amount of disintegrant incorporated only extragranularly by physical mixing. In conclusion, the properties of agglomerated crystals, such as flowability, compactibility and dissolution rate were improved profoundly using the developed technique resulting in successful direct tableting without need to additional process of physical blending of agglomerates and disintegrants.
Keywords: Disintegrant; Crystallo-co-agglomeration; Direct tableting; Tensile strength; Disintegration time; Dissolution;

Interaction between azelnidipine and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy and circular dichroism (CD). Azelnidipine effectively quenched the intrinsic fluorescence of BSA via a combination of static and dynamic quenching, forming azelnidipine–BSA complex with binding constant (K a) of the order of 105. The thermodynamic parameters obtained from van’t Hoff equation revealed that both ΔH° and ΔS° were negative, that is, −49.77 kJ mol−1 and −64.47 J mol−1  K−1, respectively, suggesting that the binding is mainly driven by the enthalpy and hydrogen bonding plays major role in stabilizing azelnidipine–BSA complex. The binding of azelnidipine to BSA leads to changes in the conformation of BSA according to synchronous fluorescence spectra and CD data. The presence of metal ion decreases the binding constant of azelnidipine–BSA complex.
Keywords: Azelnidipine; Bovine serum albumin (BSA); Fluorescence quenching; Synchronous fluorescence; Circular dichroism (CD);

The aim of this paper is the modification of the release behaviour of hydrophilic HPMC-based matrices of different viscosity grade by the introduction of a new inert polymeric excipient hydroxypropylcellulose-methyl methacrylate (HCMMA). The drug released could be control by both mechanisms, the swelling rate from the hydrophilic matrices, and the porosity, tortuosity and water uptake capacity from inert matrices. The effects of drying methods, presence or absence of viscosity (HCMMA in relation with HPMC), proportion of two polymers and different viscosity grade of HPMC were studied. It was observed that the mixtures with FD-HCMMA needed less pressure, presented higher plasticity and their tablets were easier to obtain compared with OD-HCMMA mixtures. Only FD-HCMMA:K100M mixtures did not show any differences in the percentage of theophylline released when FD-HCMMA proportion changed (f 2  > 95). All mixtures show double release mechanism, diffusion and erosion from the gel layer, but with higher contribution of the relaxation factor than on HPMC tablets. For the different mixtures HCMMA–HPMC, it is possible to see fronts movement profiles similar to swellable matrices. The results demonstrate that the use of high viscosity differences of HPMC or 50% HCMMA or above was required to produce modifications on theophylline monoaxial release modulation.
Keywords: Hydroxypropyl methylcellulose; Hydroxypropylcellulose-methyl methacrylate; Viscosity; Release modulation; Drug delivery system; Theophylline;

Solubility of molecular crystals: Polymorphism in the light of solubility theory by P. Bennema; J. van Eupen; B.M.A. van der Wolf; J.H. Los; H. Meekes (74-91).
The thermodynamic theory of solubility of molecular crystals in solvents is reviewed with an emphasis on solutes showing polymorphism as in case of many pharmaceuticals. The relation between solubility and binary phase diagrams of the solute solvent system is treated. The astonishing variety of possible solubility curves as a function of temperature is explained using simple models for the solution thermodynamics assuming no mixing between the solvent and solute in the solid phase, though including the case of solvates or pseudo polymorphs. In passing a new method is introduced that allows to estimate the transition temperature of enantiotropically related polymorphs from melting temperatures and enthalpies of the polymorphs.
Keywords: Solubility; Pharmaceuticals; Regular solution; Polymorphism; Phase diagrams;

A database (n  = 50) consisting of values of solubility in water, S AQ, solubility in octanol, S OCT, molecular weight, MW, and maximum flux based on the delivery of total species containing a parent drug by its prodrugs through human skin in vitro from water has been integrated into the extended Flynn database (n  = 114). In addition, data for two more recent contributions (n  = 8) and one (n  = 7) contribution that was overlooked for inclusion in the extended Flynn database were added to the prodrug database, as well as the data for the parent drugs (n  = 6), to give n  = 71 and n  = 185 for the total integrated database. This integrated database was fit to five equations where the independent variable was S AQ, S OCT or MW alone or were combinations of S OCT and MW (Kasting–Smith–Cooper, KSC model) or S OCT, S AQ and MW (Roberts–Sloan, RS model). The RS equation gave the best fit: log  J MAQ  = −2.506 + 0.538 log  S OCT  + 0.462 log  S AQ  − 0.00402MW, r 2  = 0.839, S.D. = 0.423 and the residual (Δlog  J MAQ) was 0.474 log units. Integration of a substantial number of prodrugs into the extended Flynn database did not change the dependence of J MAQ on a balance of S AQ and S OCT. No trend in the effect of the prodrug being more or less water soluble than its parent drug on over- or underpredicting flux (±Δ′log  J MAQ) by the RS model was found. Thus optimization of the S AQ of a prodrug in its design, as well as the design of new drugs, is indicated.
Keywords: Prodrugs; Water solubility; Octanol solubility; Roberts–Sloan equation; Hydrolysis;

Fabrication of novel core-shell hybrid alginate hydrogel beads by Hongxia Liu; Chaoyang Wang; Quanxing Gao; Xinxing Liu; Zhen Tong (104-112).
Novel hybrid alginate hydrogel beads with shells of porous CaCO3 microparticles were fabricated by templating water-in-oil emulsion and subsequent in situ gelation. Porous CaCO3 microparticles were self-assembled at interfaces of water-in-oil emulsion. Water droplets containing alginate in the emulsion were subsequently in situ gelated by Ca2+ released from CaCO3 through decreasing pH with slow hydrolysis of d-glucono-δ-lactone (GDL). The resulting hybrid beads with alginate gel cores and shells of porous CaCO3 microparticles were called colloidosomes. The packed density of CaCO3 microparticles in the shell increased with increasing the ratio of the CaCO3 microparticle weight to the water phase volume M p/V w and decreased with addition of NaCl into water. The size of the produced colloidosome beads was independent of M p/V w. Increasing the volume fraction of water Φ w to 0.5, some colloidosome beads deformed to nonspheral shape and even broken. Brilliant blue (BB) as a drug model was loaded into the colloidosome beads by being dissolved in the alginate aqueous solution before gelation. The BB release from the colloidosome beads was slowed down because of the formation of the shells of CaCO3 microparticles. The colloidosome beads may find applications as delivery vehicles for drugs, cosmetics, food supplements and living cell.
Keywords: Alginate hydrogel bead; Porous CaCO3 microparticle; Self-assembly; Water-in-oil emulsion; Controlled release;

The inhibitory effects of fluoroquinolones on l-carnitine transport in placental cell line BeWo by Takeshi Hirano; Satoru Yasuda; Yuki Osaka; Masaru Asari; Masaki Kobayashi; Shirou Itagaki; Ken Iseki (113-118).
l-Carnitine plays an important role in lipid metabolism by facilitating the transport of long-chain fatty acids across the mitochondrial inner membrane followed by fatty acid beta-oxidation. It is known that members of the OCTN family play an important role in l-carnitine transport in the placenta. Investigation of drug–drug or drug–nutrient interaction in the placenta is important for establishment of safety drug medication during pregnancy. The aim of this study was to determine the effects of fluoroquinolones, inhibitors of OCTN2, on l-carnitine transport in the placenta which is known to have a high expression level of OCTN2.We investigated the inhibitory effect of five fluoroquinolones, ciprofloxacin (CPFX), gatifloxacin (GFLX), ofloxacin (OFLX), levofloxacin (LVFX) and grepafloxacin (GPFX), on l-carnitine transport mediated by OCTN2 in placental cell line BeWo cells. We found that all of the fluoroquinolones inhibited l-carnitine transport, GPFX being the strongest inhibitor. We also found that the inhibitory effects of LVFX and GPFX depended on their existence ratio of zwitterionic forms as, we reported previously.Furthermore, we elucidated the LVFX transport mechanism in BeWo cells. LVFX was transported actively by transporters. However, we found that LVFX transport was Na+-independent and l-carnitine had no inhibitory effect on LVFX transport, suggesting that LVFX acts as inhibitor of OCTN2, not as a substrate for OCTN2.
Keywords: l-Carnitine; BeWo; Levofloxacin; Fluoroquinolone;

This study is aimed to elucidate the physicodynamic phenomena governing diffusion coefficient (D) of the loaded drugs in a female controlled drug delivery system (FcDDS) and to find the most influencing variable on the diffusivity using artificial neural networks (ANN). The release profiles of sodium dodecyl sulphate (SDS), a topical microbicide used as a model drug, from FcDDS were obtained using in vitro apparatus, the Simulant Vaginal System (SVS), under various conditions. The effects of formulation and intrinsic/extrinsic variables on the diffusivity of SDS were assessed using artificial neural networks (ANN). The release profiles of SDS from FcDDS revealed a non-linear relationship between the diffusivity and formulation/physiological variables. Intrinsic variables (vaginal fluid pH, vaginal fluid secretion rate) have a more prominent role in defining the diffusion coefficient of SDS from FcDDS than formulation variables (formulation loading weight and loaded doses in the formulation) or extrinsic variables (inserting position). Among 5 variables, pH of vagina fluids is the most influencing factor in defining the diffusion coefficient (maximum value of 0.95 ± 0.04) of SDS from FcDDS. The external exposure conditions clearly outweighed the effects of the formulation variables on the diffusion coefficient of SDS. A model-based approach can be used to assess the diffusion coefficient of loaded drugs in FcDDS under the given conditions, leading to a parameter-specific prevention strategy against sexually transmitted diseases (STD) with a high degree of confidence.
Keywords: Diffusion coefficient; Mucoadhesive barrier devices; Intrinsic/extrinsic variables; Artificial neural network;

A continuous dissolution/absorption system using a hexadecane membrane (HDM) as the permeation measurement has been examined for three distinct formulations of metformin hydrochloride. This system was used to correlate the absorption rate of metformin through the membrane after release from the dosage form to rate of appearance of metformin in the plasma from the same formulations. These correlations were then used to make predictions of the in vivo plasma profile for each formulation. Successful predictions of AUC were accomplished for both immediate release and extended release formulations of metformin hydrochloride.
Keywords: Dissolution; Absorption; Metformin hydrochloride;

The aim of this study was to evaluate the effects of docosahexaenoic acid (DHA) on the intestinal cytochrome P450 isoenzyme (CYP3A) and P-glycoprotein (P-gp) functions using midazolam and rhodamine-123 as specific substrates of CYP3A and P-gp, respectively. Perfused everted intestinal segments from rats were employed to determine the effects of DHA on midazolam metabolism and rhodamine-123 transport. In addition, the effects of DHA on in vitro midazolam metabolism in rat intestinal microsomes and on midazolam bioavailability in rats were examined. The intestinal extraction ratio (ERG) of midazolam was determined to be 0.43 and decreased significantly to 0.12, 0.07, and 0.06 in the presence of 50, 100, and 200 μM DHA, respectively, in a concentration-dependent manner. The results from an in vitro study using rat intestinal microsomes demonstrated that DHA competitively inhibited the intestinal CYP3A activity with K i of 15.7 and 27.1 μM for the formations of 1′-OH midazolam and 4-OH midazolam, respectively. Moreover, the oral administration of DHA (100 mg/kg) increased the AUC, C max, and oral bioavailability (F) of midazolam by about 50% in rats, without affecting the T 1/2, V dss/F, or CLtot/F. In contrast, DHA did not change the serosal-to-mucosal transport of rhodamine-123 in the perfused everted intestine and oral administration of DHA (100 mg/kg) had no influence on the pharmacokinetics of intravenously administered midazolam in rats, thus suggesting that DHA has little effect on the intestinal P-gp activity and hepatic clearance of midazolam. This study provided the first direct evidence to show that DHA has an inhibitory effect on the intestinal pre-systemic metabolism of a CYP3A substrate and that DHA has little, if any, effect on the P-gp activity in the gut.
Keywords: Docosahexeanoic acid (DHA); Midazolam; Rhodamine-123; CYP3A; P-glycoprotein (P-gp); Perfused everted intestinal segment;

Polymorphism of linezolid: A combined single-crystal, powder diffraction and NMR study by Elisabetta Maccaroni; Enrica Alberti; Luciana Malpezzi; Norberto Masciocchi; Chiara Vladiskovic (144-151).
Linezolid (S)-N-[[3-(3-fluoro-4-morpholinylphenyl)-2-oxo-5-oxazolidinyl]methyl] acetamide is one of the first commercially available (and most widely used) oxazolidinone antibiotics. It was selectively prepared as two anhydrous polymorphic forms, labelled form II and IV in accordance with preliminary reports in the patent literature. Form II has been characterized by single-crystal X-ray diffraction methods (orthorhombic, P212121, a  = 6.536(1), b  = 9.949(1), c  = 24.807(3) Å, V  = 1613.1(3) Å3, Z  = 4, Z′ = 1), while powders of form IV could be fully characterized by employing ab initio powder diffraction methods (triclinic, P1, a  = 6.5952(7) Å, b  = 10.9875(10) Å, c  = 12.9189(14) Å, α  = 110.683(4)°, β  = 88.186(6)°, γ  = 105.826(6)°, V  = 840.5(2) Å3, Z  =  Z′ = 2). The interconversion of form II into form IV was studied by TG, DSC and thermodiffractometry, which indicated a quantitative (endothermic and irreversible) transformation (in air) just above 160 °C. On cooling from the melt, linezolid gives an oily material, stable at RT, which can be crystallized into form IV by controlled heating near 100 °C. These materials were further characterized by high-resolution 1H and 13C NMR studies, as well as by 13C solid-state NMR.
Keywords: Linezolid; Crystal and molecular structure; Powder diffraction; Phase transition;

Piperacillin, a potent β-lactam antibiotic, is effective in a large variety of Gram+ and Gram− bacterial infections but its administration is limited to the parenteral route as it is not absorbed when given orally. In an attempt to overcome this problem, we have synthesized a novel series of charged liposaccharide complexes of piperacillin comprising a sugar moiety derived from d-glucose conjugated to a lipoamino acid residue with varying side-chain length (cationic entity) and the piperacillin anion. A complete multiple reaction monitoring LC–MS/MS method was developed to detect and characterize the synthesized complexes. The same method was then successfully applied to assess the in vitro apparent permeability values of the charged liposaccharide complexes in Caco-2 monolayers.
Keywords: Piperacillin; Charged liposaccharide; Penetration enhancer; LC–MS/MS; Caco-2; Permeability;

Pulmonary absorption of aerosolized fluorescent markers in the isolated rabbit lung by Kerstin Lahnstein; Thomas Schmehl; Ute Rüsch; Monika Rieger; Werner Seeger; Tobias Gessler (158-164).
For the development of aerosolized controlled release formulations such as liposomes or nanoparticles, the use of suitable model drugs is necessary. This study compared the pulmonary absorption of the three structurally diverse fluorescent markers 5(6)-carboxyfluorescein (CF), 8-methoxypyrene-1,3,6-trisulfonic acid trisodium salt (MPTS) and rhodamine 6G (R6G) after nebulization in an isolated, perfused and ventilated rabbit lung. Aerosol particle size and lung deposition as well as lipophilicity of the fluorescent markers were determined. Dye concentrations were measured in the recirculating buffer and in the bronchoalveolar lavage.The MMAD of the dye aerosols ranged between 4.70 and 4.88 μm, total lung deposition was 0.40 ± 0.05 ml. The 1-octanol/water partition coefficient as measure for lipophilicity was −3.45 ± 0.16 for CF, −4.95 ± 0.21 for MPTS and 2.69 ± 0.18 for R6G. The perfusate concentration showed an increase to ∼400 ng/ml (53.4 ± 6.8% of the intrapulmonary deposited dye) for CF and ∼230 ng/ml (29.1 ± 2.0%) for MPTS, respectively; R6G concentration increased in the first 30 min to ∼38 ng/ml followed by a gradual decrease to ∼26 ng/ml (3.3 ± 0.7%).In conclusion, these data suggest that the hydrophilic dye CF is suitable to study drug transport from aerosolized controlled release formulations across the lung barrier. In contrast, the highly water-soluble fluorescent probe MPTS demonstrates insufficient recovery and the lipophilic R6G high accumulation in lung tissue.
Keywords: Isolated lung; Nebulization; Pulmonary absorption; Fluorescent markers; Controlled release formulations;

Drug tablet thickness estimations using air-coupled acoustics by Ilgaz Akseli; Cetin Cetinkaya (165-173).
A non-contact/non-destructive acoustic technique for predicting the coating layer thickness of a drug tablet is presented. Quality of tablet coatings can play a major role in the effectiveness of drug delivery. Many pharmaceutical tablets consist of a tablet core and a coated outer cover. Variations in the tablet coating can be indicative of various process problems and, therefore, is of a concern for quality assurance. In the current non-contact measurement system, an air-coupled excitation and laser interferometric detection for predicting the coating layer thickness of a drug tablet is introduced. Drug tablets with different coating thicknesses are vibrated via an acoustic field generated by an air-coupled transducer in a frequency range sufficiently high to excite their several vibrational modes. The tablet surface vibrational responses are acquired at a number of measurement points by a laser interferometer in a non-contact manner. An iterative computational procedure, based on the FE method and Newton's method, was developed and demonstrated to extract the coating layer thicknesses of the tablets from a subset of the measured resonance frequencies.
Keywords: Tablet coating thickness; Thickness measurements; Drug tablet coating; Acoustic process monitoring; Process Analytical Technology; PAT;

Screening poly(ethyleneglycol) micro- and nanogels for drug delivery purposes by T.G. Van Thienen; J. Demeester; S.C. De Smedt (174-185).
This study investigates poly(ethyleneglycol) (PEG)-based micro- and nanogels, with or without lipid coating, with the aim to slowly deliver encapsulated molecules. Hydroxyethylmethacrylated PEG (PEG-HEMA), PEG-HEMA with an oligo lactate spacer (PEG-lac-HEMA), and eight-armed PEG end capped with HEMA (▪-PEG-HEMA) were used. PEG-lac-HEMA matrices degraded very fast (in terms of days), while it took about 1 month for linear PEG-HEMA and several months for ▪-PEG-HEMA hydrogels to become degraded. PEG-based microgels were made by use of an all aqueous technique and could be lipid-coated by mixing the microgels (made positively or negatively charged through copolymerization with respectively methacrylic acid and dimethyl aminoethyl methacrylate with a suspension of oppositely charged liposomes. The release of fluorescently labeled molecules incorporated in the PEG-based microgels could be clearly governed by the type of molecules used (lasting from hours to months). PEG-based nanogels could be made using liposomes as a nanoscopic mold, resulting in particles with a PEG gel core surrounded by a lipid coating. BSA could be easily encapsulated in the PEG nanogels which released the BSA over a period of about 1 week.
Keywords: Microgels; Nanogels; PEG; Drug delivery systems; Biodegradable; Controlled release;

Transdermal penetration of zalcitabine, lamivudine and synthesised N-acyl lamivudine esters by Minja Gerber; Jaco C. Breytenbach; Jeanetta du Plessis (186-193).
The objective of this study was to determine the in vitro transdermal permeation through human epidermis of zalcitabine, lamivudine and the synthesised N-acyl lamivudine esters, with and without the use of Pheroid™ as delivery system and to establish a correlation, if any, with selected physicochemical properties. Six N-acyl lamivudine esters were prepared by acylation of lamivudine with six different acid chlorides. The experimental aqueous solubility, log  D and in vitro transdermal flux values were determined for these compounds. There was an inverse correlation between the aqueous solubility and the log  D values. The median flux of zalcitabine (0.442 μmol/cm2  h) in PBS was lower than that of lamivudine (4.289 μmol/cm2  h), but in Pheroid™, lamivudine (0.011 μmol/cm2  h) had a slightly lower median flux than zalcitabine (0.015 μmol/cm2  h). Entrapment of compounds in Pheroid was confirmed by confocal laser scanning microscopy.
Keywords: Zalcitabine; Lamivudine; N-acyl lamivudine esters; Skin penetration; Transdermal delivery;

Modelling drug dissolution from controlled release products using genetic programming by Duong Q. Do; Raymond C. Rowe; Peter York (194-200).
This study has investigated and compared genetic programming (GP) – a method of automatically generating equations that describe the cause-and-effect relationships in a system – and statistical methods for modelling two controlled release formulations—a matrix tablet and microspheres. With the improved GP models exhibiting comparable predictive power, as well as simpler equations in some cases, the results obtained indicate that GP can be considered as an effective and efficient method for modelling controlled release formulations.
Keywords: Genetic programming; Statistical methods; Modelling; Controlled release; Formulation;

The body of work described in this research paper outlines the use of PEO/PCL blends in the production of monolithic matrices for oral drug delivery. Several batches of matrix material were prepared with carvedilol used as the active pharmaceutical ingredient. The matrices were prepared using various extrusion parameters to investigate the effect of screw speed and barrel temperature on the properties of the drug delivery devices. The resultant extrudate was characterised using steady state parallel plate rheometry, differential scanning calorimetry (DSC) and dissolution testing. Higher screw speeds were observed to result in slightly lower matrix melt viscosity when compared with matrices compounded using lower screw speeds. Dissolution testing showed that the incorporation of the hydrophobic PCL polymer into a PEO matrix results in a retarded drug release profile.
Keywords: Extrusion; PEO; PCL; Drug delivery; Matrix; Temperature;

Method for screening of solid dispersion formulations of low-solubility compounds—Miniaturization and automation of solvent casting and dissolution testing by Anant Shanbhag; Shelley Rabel; Ewa Nauka; Gemma Casadevall; Padmaja Shivanand; Gary Eichenbaum; Paul Mansky (209-218).
An efficient method has been developed for screening solid dispersion formulations that are intended to enhance the dissolution of poorly soluble compounds. The method is based on miniaturization and automation of sample preparation by solvent casting, and dissolution testing, in a 96-well plate format, using less than 0.1 mg of compound per well. To illustrate the method, six polymers and eight surfactants were screened, individually and in combination, for their ability to dissolve a compound with aqueous solubility of <1 μg/ml in simulated intestinal fluid. Screening was performed at an excipient/compound ratio of 10:1, and a polymer/surfactant ratio of 3:1 for ternary formulations. Sixteen of the 48 ternary formulations dissolved the compound to a level >100 μg/ml, i.e. at least a 100-fold increase over the aqueous solubility. A number of synergies were observed wherein the performance of a ternary formulation greatly exceeded that of either of the corresponding binary formulations. Thirteen ‘hits’ from screening were scaled up with melt methods, and ∼2/3 of these showed comparable dissolution enhancement when tested at larger scale. Five of these were administered to rats, and the absolute oral bioavailability ranged from 10 to 23%, versus less than 1% for the unformulated compound.
Keywords: Solid dispersions; Solubility; High throughput; Bioavailability; Surfactants; Dissolution;

Pingyangmycin loaded bovine serum albumin microspheres for chemoembolization therapy—in vitro and in vivo studies by Changguang Wang; Jie Liu; Weisan Pan; Xinchun Wang; Qinghong Gao; Shixiang Hou (219-226).
Chemoembolization based on microspheres have been emerged as a novel and promising way for interventional therapy, however, the exact effect and probable mechanism have not been revealed. The purpose of our study was to evaluate the potential of Pingyangmycin loaded bovine serum albumin microspheres (PYM-BSA-MSs) for chemoembolization therapy both in vitro and in vivo. The effect of PYM-BSA-MSs on cell growth curves and changes of cell morphology and activities measured by MTT assay were carried out in human umbilical vein endothelial ECV-304 cells. The in vivo occlusion effect was evaluated in 24 healthy rabbits. Macroscopic examinations and Hematoxylin-Eosin (H-E) staining of cross-section of rabbits’ central auricular arteries were employed to observe the apparent and histological changes of arterioles. The results show that the PYM-BSA-MSs could inhibit the proliferation and induce the apoptosis of ECV-304 cells in a time-dependent manner. In vivo studies demonstrated that 21 days after artery embolization with the PYM-BSA-MSs, neointimal thickening of arterioles and significant hyperplasia of endothelial cells could be detected, but without completely interruption of blood flow. Compared with plain PYM aqueous solution or BSA-MSs oily suspension, PYM-BSA-MSs showed excellent potential as an alternative to interventional embolization materials.
Keywords: Chemoembolization; Pingyangmycin; Microspheres; ECV-304 cell line; Hematoxylin-Eosin staining;

Chitosan–magnesium aluminum silicate composite dispersions: Characterization of rheology, flocculate size and zeta potential by Wanwisa Khunawattanakul; Satit Puttipipatkhachorn; Thomas Rades; Thaned Pongjanyakul (227-235).
Composite dispersions of chitosan (CS), a positively charged polymer, and magnesium aluminum silicate (MAS), a negatively charged clay, were prepared and rheology, flocculate size and zeta potential of the CS–MAS dispersions were investigated. High and low molecular weights of CS (HCS and LCS, respectively) were used in this study. Moreover, the effects of heat treatment at 60 °C on the characteristics of the CS–MAS dispersions and the zeta potential of MAS upon addition of CS at different pHs were examined. Incorporation of MAS into CS dispersions caused an increase in viscosity and a shift of CS flow type from Newtonian to pseudoplastic flow with thixotropic properties. Heat treatment brought about a significant decrease in viscosity and hysteresis area of the composite dispersions. Microscopic studies showed that flocculation of MAS occurred after mixing with CS. The size and polydispersity index of the HCS–MAS flocculate were greater than those of the LCS–MAS flocculate. However, a narrower size distribution and the smaller size of the HCS–MAS flocculate were found after heating at 60 °C. Zeta potentials of the CS–MAS flocculates were positive and slightly increased with increasing MAS content. In the zeta potential studies, the negative charge of the MAS could be neutralized by the addition of CS. Increasing the pH and molecular weight of CS resulted in higher CS concentrations required to neutralize the charge of MAS. These findings suggest that the electrostatic interaction between CS and MAS caused a change in flow behavior and flocculation of the composite dispersions, depending on the molecular weight of CS. Heat treatment affected the rheological properties and the flocculate size of the composite dispersions. Moreover, pH of medium and molecular weight of CS influence the zeta potential of MAS.
Keywords: Chitosan; Magnesium aluminum silicate; Rheology; Flocculation; Zeta potential; Heat treatment;

Nanoparticle dispersion has demonstrated its effectiveness in improving the dissolution rate and oral bioavailability of poorly water-soluble compounds. When we studied the interactions of drug and polymeric stabilizers and milling parameters of a poorly water-soluble compound, Compound A, the relaxation behavior that occurred repeatedly in our nanoparticle dispersion formulation was observed. Nanomill® was used to generate the nanoparticle dispersion, and milling parameters such as time, speed, and stabilizer loading were altered to investigate their effects on relaxation. The particle size and morphology of milled products were studied using a light-scattering particle analyzer and a scanning electron microscope (SEM). X-ray powder diffraction (XRD) was employed to characterize particle crystallinity and the crystallite size. The results indicated that, after milling, nanoparticles agglomerated constantly to form clusters and reached maximum apparent size within the first 24 h. Thereafter, the clusters relaxed spontaneously and, within a few days, dissociated into individual primary particles and the suspension were stable at sub 100-nm levels near equilibrium. Milling crystalline drug substance resulted in an XRD pattern in which the peaks were observed to broaden, suggesting formation of disordered nanocrystalline. Altering milling conditions and stabilizer ratios influences the relaxation behavior and certainly led to optimization of the process and performance of nanoparticle suspension formulations.
Keywords: Poorly water-soluble compounds; Nanoparticle; Drug delivery; X-ray powder diffraction (XRD) and Scanning electron microscope (SEM);

Bupivacaine-loaded biodegradable poly(lactic-co-glycolic) acid microspheres by He Zhang; Ying Lu; Guoqing Zhang; Shen Gao; Duxin Sun; Yanqiang Zhong (244-249).
Bupivacaine has been encapsulated by solvent evaporation method based on O/W emulsion, using poly(dl-lactic-co-glycolic) acid (PLGA) 50:50. The particle size can be controlled by changing stirring rate and polymer concentration. The encapsulation efficiency was affected by polymer concentration and burst effect of bupivacaine released from particles was affected by drug/polymer mass ratio. Orthogonal design was used to optimize the formulation according to drug content, encapsulation efficiency and burst effect. The dissolution profile and release model were evaluated with two different bupivacaine microspheres (bupi-MS) groups including low drug loading (6.41%) and high drug loading (28.92%). It was observed that drug release was affected by drug loading especially the amount of drug crystal attached on surface of bupi-MS. The drug release profile of low drug loaded bupi-MS agreed with Higuchi equation and that of high drug loaded bupi-MS agreed with first order equation.
Keywords: Bupivacaine; Microspheres; Poly(dl-lactic-co-glycolic) acid (PLGA); In vitro release;

Controlled release of a protein kinase inhibitor UCN-01 from liposomes influenced by the particle size by Masahiro Yamauchi; Hiroko Kusano; Etsuko Saito; Masayuki Abe; Kyoko Tsutsumi; Yoichi Uosaki; Masashi Nakakura; Yasuki Kato; Noboru Aoki (250-258).
A protein kinase inhibitor UCN-01 binds with high affinity to human α1-acid glycoprotein (hAGP) which may compromise the drugs therapeutic effectiveness. Liposomal formulations of UCN-01 have been evaluated as a means of reducing the impact of binding to hAGP. However, in an initial study, UCN-01 was released rapidly from liposomes added to rat plasma containing hAGP. The purpose of this study was to develop a liposomal formulation of UCN-01 that only slowly released drug. Liposomes composed of lipids with a high phase transition temperature and having an average particle size of 120 nm and above reduced leaking of UCN-01 when the formulations were evaluated by adding to rat plasma containing hAGP. Furthermore, formulations composed of larger liposomes were also more effective in vivo; in tests in which liposomal preparations were injected together with hAGP into rats, more UCN-01 was retained in liposomes for 24 h after administration of 155 nm liposomes as compared to 112 nm liposomes.
Keywords: α1-Acid glycoprotein; Release; Liposomes; Pharmacokinetics; Lamellarity;

Enhanced antitumor activity of combinations of free and HPMA copolymer-bound drugs by J. Hongrapipat; P. Kopečková; S. Prakongpan; J. Kopeček (259-270).
The synergism in anticancer effect toward human renal carcinoma A498 cells by binary combinations of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound anticancer drugs, SOS thiophene (SOS), doxorubicin (DOX), and mesochlorin e6 monoethylenediamine (Mce6), was evaluated. The combination index (CI) analysis was used to quantify the synergism, antagonism, and additive effects. Both free drugs and HPMA copolymer conjugates, when used as single agents or in combination, exhibited cytotoxic activities against A498 cells, as determined using a modified MTT assay. As single agents, SOS and P-GFLG-SOS (HPMA copolymer conjugates containing SOS bound via glycylphenylalanylleucylglycine [GFLG] spacer) were significantly more effective than the other agents evaluated. The synergistic effects ranked in the order SOS + DOX > P-GFLG-DOX + P-GFLG-Mce6  ≈ DOX + Mce6  > P-GFLG-SOS + P-GFLG-DOX ≈ SOS + Mce6  > P-GFLG-SOS + P-GFLG-Mce6. The combination of SOS + DOX proved to be synergistic over all cell growth inhibition levels. All other combinations exhibited synergism in a wide range of drug effect levels. The SOS + Mce6 and P-GFLG-SOS + P-GFLG-Mce6 combinations displayed synergism up to drug affected fraction (f a) values of about 0.8 and reached slight antagonism and nearly additivity at f a  = 0.95, respectively. However, all other combinations were synergistic up to f a  < 0.9 and were additive at higher f a values. The observations that most combinations produced synergistic effects will be important for clinical translation.
Keywords: N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer; 2,5-bis(6-hydroxymethyl-2-thienyl)furan; Doxorubicin; Mesochlorin e6 monoethylenediamine; Combination index; Renal cancer;

Transport of antiretroviral agents across the blood–brain barrier (BBB) is of key importance to the treatment for the acquired immunodeficiency syndrome (AIDS). In this study, impact of exposure to electromagnetic field (EMF) on the permeability of saquinavir (SQV) across BBB was investigated. The in vitro BBB model was based on human brain-microvascular endothelial cells (HBMEC), and the concentration of SQV in receiver chamber of the transport system was evaluated. Polybutylcyanoacrylate (PBCA), methylmethacrylate-sulfopropylmethacrylate (MMA-SPM), and solid lipid nanoparticle (SLN) were employed as carriers for the delivery systems. Cytotoxicity of SLN decreased as content of cacao butter increased. Power of 5 mV was apposite for the study on HBMEC without obvious apoptosis. Square wave produced greater permeability than sine and triangle waves. The carrier order on permeability of SQV across HBMEC monolayer under exposure to EMF was SLN > PBCA > MMA-SPM. Also, a larger frequency, modulation or depth of amplitude modulation (AM), or modulation or deviation of frequency modulation (FM) yielded a greater permeability. Besides, enhancement of permeability by AM wave was more significant than that by FM wave. Transport behavior of SQV across BBB was strongly influenced by the combination of nanoparticulate PBCA, MMA-SPM, and SLN with EMF exposure. This combination would be beneficial to the clinical application to the therapy of AIDS and other brain-related diseases.
Keywords: Blood–brain barrier; Electromagnetic field; Polybutylcyanoacrylate; Methylmethacrylate-sulfopropylmethacrylate; Solid lipid nanoparticle; Saquinavir;

The objective of the present study is to formulate Naproxen nanosuspensions at high drug concentrations of up to 300 mg/ml using ball milling and is to investigate the additive effect between hydroxypropyl cellulose (HPC) and arginine hydrochloride as stabilizers. The nanosuspensions were obtained at different arginine hydrochloride/polymer weight ratios. Stability of Naproxen suspensions at 100 and 300 mg/ml was determined over a period of 14 days by measuring the particle size. The control, which contained only drug and buffers without the stabilizers agglomerated immediately after preparation. The study of the effect of arginine hydrochloride as a primary stabilizer indicated that arginine hydrochloride levels of up to 0.8% (w/v) were not able to help reduce particle size below one micron, and were also not able to provide stabilization to the suspensions on storage. Therefore, HPC was also added to the system to increase suspensions stability, presumably by a steric repulsion mechanism. When the Naproxen concentration was increased to 300 mg/ml, 1% (w/v) HPC was not able to provide good stabilization and it was found that arginine hydrochloride increased the stabilization efficiency of 1% (w/v) HPC by preventing flocculation. When HPC level was increased to 4% (w/v), HPC was high enough to sufficiently stabilize the nanosuspensions for 2 weeks and thereby could maintain the mean size diameter of the suspensions without the presence of arginine hydrochloride. Furthermore, stable nanosuspensions were successfully lyophilized without the use of additional cryoprotectants.
Keywords: Naproxen; Poorly soluble drug; Hydroxypropyl cellulose; Nanosuspensions; Arginine hydrochloride; Stabilizer;

Sulfated and non-sulfated amphiphilic-β-cyclodextrins: Impact of their structural properties on the physicochemical properties of nanoparticles by Wassim Abdelwahed; Ghania Degobert; Alix Dubes; Hélène Parrot-Lopez; Hatem Fessi (289-295).
The aim of this work was to study the preparation of nanospheres from amphiphilic β-cyclodextrins formed (a) by different acylation degrees (DA) at the secondary hydroxyl face (DA = 14 and 21) followed by varying (b) the sulfatation degrees (DS) at the primary hydroxyl face (DS = 0, 4 and 7).The physicochemical properties of the synthesized compounds such as molecular weights, the theoretical HLB values and the critical micellar concentration values and their surface area were presented. The nanoparticles prepared from amphiphilic β-cyclodextrins were characterized by mean size, zeta potential and their morphology.The compounds presented hydrophile–lipophile balance values ranging from 5.6 to 10. For sulfated amphiphilic β-cyclodextrins having HLB values higher than 8, were able to self-organize in water to form nanoparticles. However, for the amphiphilic β-cyclodextrins that HLB values lower than 6.6 are insoluble in water but soluble in organic solvents rendering possible the preparation of nanoparticles by nanoprecipitation technique.An interesting correlation between the amphiphilic-β-cyclodextrin structures and their ability to form nanospheres has been established. The association of sulfated amphiphilic-β-CDs to the peracylated amphiphilic-β-CDs was interesting, it led to improve the stability of nanospheres size and probably confer them a biological activity.
Keywords: Sulfated amphiphilic β-cyclodextrins; Nanoparticles; Chemical structure; Stability;

Notice Board (296).