International Journal of Pharmaceutics (v.366, #1-2)

Characterization, selection, and development of an orally dosed drug polymorph from an enantiotropically related system by Lee M. Katrincic; Yan T. Sun; Robert A. Carlton; Ann M. Diederich; Ronald L. Mueller; Frederick G. Vogt (1-13).
Solid-state characterization methods are used to study a dimorphic pharmaceutical compound and select a form for development. Polymorph screening found that {4-(4-chloro-3-fluorophenyl)-2-[4-(methyloxy)phenyl]-1,3-thiazol-5-yl} acetic acid can crystallize into two non-solvated polymorphs designated Forms 1 and 2. Physical methods including vibrational spectroscopy, X-ray powder diffraction, solid-state NMR (SSNMR), thermal analysis, and gravimetric water vapor sorption are used to fully characterize the two polymorphs. Temperature-dependent competitive ripening experiments and solubility measurements indicated that the polymorphs in this system exhibit enantiotropy with a thermodynamic transition temperature of 35 ± 3 °C. This complicates the selection of a polymorph to progress in drug development. Both forms had undesirable qualities; however, a particular drawback of Form 1 was found in its tendency to convert to Form 2 upon milling. Combining this effect and the desired formulation approach with physical property results led to a rationale for the choice of Form 2 for further development. Because this form is thermodynamically metastable at room temperature, analytical approaches were developed to ensure its exclusive presence, including a quantitative infrared spectroscopic method for drug substance and 13C and 19F solid-state NMR limit tests for the undesired form in drug product at drug loads of 8.3% (w/w).
Keywords: Polymorphism; Physical characterization; Solid-state NMR; X-ray diffraction; Solubility; Enantiotropic phases;

Intestinal first-pass glucuronidation activities of selected dihydroxyflavones by Yin Cheong Wong; Li Zhang; Ge Lin; Zhong Zuo (14-20).
Flavonoids have low bioavailabilities due to extensive intestinal first-pass metabolisms, especially glucuronidation. The present study aimed to evaluate the intestinal glucuronidation of dihydroxyflavones and provide more information on their structure–activity relationships. Seven dihydroxyflavones, namely 3,7-, 5,7-, 6,7-, 7,8-, 2′,7-, 3′,7-, and 4′,7-dihydroxyflaovne and a monohydroxyflavone, 7-hydroxyflavone, were investigated by incubating each hydroxyflavone at various concentrations with either human jejunum microsome or rat intestinal microsome. Two mono-glucuronides were identified for each dihydroxyflavone. For human jejunum microsome, most of the studied dihydroxyflavones demonstrated greater glucuronidation activities than that of 7-hydroxyflavone except for 3,7-dihydroxyflavone and 4′,7-dihydroxyflavone. 3′,7-dihydroxyflavone had the greatest intrinsic clearance which was at least seven times greater than that of all other dihydroxyflavones. In addition, species difference in glucuronidation activity was observed with human jejunum microsome higher than rat intestinal microsome for all hydroxyflavones except for 3,7-dihydroxyflavone. The results further demonstrated that the hydroxyl group positions do affect the intestinal glucuronidation activity of hydroxyflavones. Increasing the number of hydroxyl groups on A- or B-ring (except for 4′-OH) would enhance the glucuronidation activity of flavones, whereas adding a 3-OH on C-ring might not. Furthermore, existence of hydroxyl group at 3′ position may enhance the glucuronidation activity of flavonoids.
Keywords: Intestine; Glucuronidation; Dihydroxyflavones;

A series of thermoresponsive-co-biodegradable polymers, containing varying molar ratios of N-isopropylacrylamide (NIPA) and poly-lactic acid diacrylate macromer (PLAM) were prepared and characterised. Chemical structures were confirmed by nuclear magnetic resonance (NMR) and Fourier transform infrared (FTIR). The hydrogels were thermoresponsive, exhibiting an increase in the lower critical solution temperature (LCST) the higher the percent of PLAM present. Swelling properties were dependant on both temperature and PLAM content. The degradation behaviour of the three-dimensional polymeric networks formed was dependent on both structural (mesh size, molecular weight distribution, composition) and environmental parameters (temperature). Swelling and in vitro biodegradation-induced morphological structural changes were examined using scanning electron microscopy (SEM). A greater rate of degradation and disruption to the porous network could be seen with increasing lactide content. Degradation was faster below the LCST, demonstrated by FTIR, pH decrease and acid release, consistent with the increased hydrophilicity of the network. The release profiles of the model drug indomethacin (IDM), from these thermoresponsive-co-biodegradable polymers, were found to be dependant on copolymer composition, drug loading and temperature, more rapid release occurring below the LCST.
Keywords: Thermoresponsive-biodegradable hydrogels; Indomethacin; Poly-lactic acid; N-isopropylacrylamide; LCST;

Injectable hydrogels have potential biomedical applications ranging from tissue fillers to drug delivery vehicles. This study focussed on evaluating the structure of poly(vinyl alcohol) (PVA) hydrogels of variable solid content and high molecular weight model drug release from the networks formed via either conventional photo-polymerization compared with chemical initiation of polymerization using an oxidation–reduction (redox) reaction. Swelling behaviour was characterised in water to assess the structural properties. Model drugs, FITC-Dextran, 20 kDa (FD20) and 4 kDa (FD4) were loaded in the hydrogels prior to curing and drug release studies conducted. Redox-cured hydrogels were more swollen than UV-cured systems, lost ∼20% of their polymer mass compared to only 5% from UV-cured hydrogels and subsequently exhibited networks of larger mesh sizes. Also, networks of variable solid contents showed different structural properties with systems of higher polymer concentration exhibiting a smaller mesh size. The difference in structural properties of the networks affected release of FD20, being faster in redox-cured than UV-cured hydrogels, and slower from systems of higher solid content. Release of FD4 was faster than FD20 from networks of same solid content. This study suggested that PVA hydrogels can be cured by redox-initiation to function as a controlled delivery system for macromolecular drugs.
Keywords: PVA hydrogels; Redox curing; Drug delivery; Networks;

Effect of polyols on the solubility of bovine serum albumin (BSA) in the presence of polyethylene glycols (PEGs) was investigated in order to strengthen the understanding of the observed effects of polyols and PEGs on protein properties in solution. Effect of polyols and/or PEGs on the thermodynamic (conformational) stability of BSA was measured using DSC and circular dichroism (CD). Glucose, sucrose, raffinose, glycerol and sorbitol, all reduced the extent of protein precipitation. Solubility of BSA in the presence of ethylene glycol increased in the case of PEG 1450 and PEG 8000, but was unaffected in the case of PEG 400. DSC studies indicated that smaller PEGs have destabilizing influence on protein structure. CD studies showed that smaller PEGs (ethylene glycol) induce subtle unfolding while stabilizing polyols induce subtle compaction. Results show that, effect of polyols on the apparent solubility of the protein correlates with their effect on the thermodynamic stability of the protein, smaller PEGs are not appropriate for estimating the activity of proteins in saturated solutions, and subtle changes in protein conformation can significantly affect protein precipitation. Though smaller PEGs have weak attractive interactions with protein molecules, perturbation of protein structure by PEGs can be balanced by utilizing appropriate stabilizing solutes.
Keywords: Polyethylene glycol (PEG); Precipitation; Polyol; Proteins; Secondary structure;

Dissolution and powder flow characterization of solid self-emulsified drug delivery system (SEDDS) by Vikas Agarwal; Akhtar Siddiqui; Hazem Ali; Sami Nazzal (44-52).
In this study, the dynamics of powder flow upon griseofulvin-self-emulsified drug delivery system (SEDDS) addition to silica and silicates and the effect of these adsorbents on drug release were investigated. SEDDS was adsorbed at SEDDS/adsorbent ratios from 0.25:1 to 3:1 on magnesium aluminum silicate [5 and 80 μm], calcium silicate [25 μm], and silicon dioxide [3.6, 20, and 300 μm]. Powder flow was evaluated using the powder rheometer and compared to angle of repose. Release of drug from a 1:1 SEDDS/adsorbent powder was determined by dissolution using USP Type 2 apparatus. Powder rheometer profiles indicated that effect of SEDDS on the flow behavior of the adsorbents could be correlated to stepwise or continuous growing behavior as observed in wet granulation process. However, due to their porous nature, adsorbents exhibited an initial lag phase during which no change in flow was observed. Dissolution of drug from adsorbed-SEDDS was found to be dependent on pore length and nucleation at the lipid/adsorbent interface. Increase in dissolution rate was observed with an increase in surface area and was independent of the chemical nature of the adsorbents. Therefore, in order to manufacture free flowing powder containing liquid SEDDS, special attention should be given to particle size, specific surface area, type and amount of adsorbent.
Keywords: Self-emulsified drug delivery systems; Solid dosage forms; Powder; Rheometry; Dissolution; Powder flow dynamics;

Azithromycin loaded on hydrogels of carbomer: Chemical stability and delivery properties by Silvia L. Esteban; Ruben H. Manzo; Fabiana L. Alovero (53-57).
Hydrogels of carbomer (C) and azithromycin (AZI) were prepared by neutralizing with AZI 50% of the carboxylic groups of 0.25% C974 and C934 dispersions. The hydrogels exhibit pH close to 8 and are physically stable. Titration with NaCl revealed a high degree of counterion condensation C–AZI. The release of AZI in a Franz cell was almost negligible when the receptor compartment was filled with water but was increased about 20 times as water is replaced by NaCl solution. Two analytical methods were used to evaluate the effect of the counterionic condensation on the chemical stability of AZI, a microbial assay and an HPLC method. Degradation of AZI in buffered aqueous solution was used as reference. The stability of AZI was significantly improved in the hydrogels retaining more than 75% of the initial concentration along a period of 18–20 months evaluated and the self life (t 90) of the drug was increased 27 and 20 times over the reference. The improvement of AZI stability could be attributed to the high degree of counterion condensation in which drug molecules remain associated to the macromolecular phase having a high negative electrokinetic potential and higher viscosity and lower kinetic energy than those in the fluid phase.
Keywords: Azithromycin; Anionic polyelectrolyte; Hydrogels; Stability; Microbial assay; HPLC;

Permeation of quinine across sublingual mucosa, in vitro by Charlene M.Y. Ong; Charles M. Heard (58-64).
Quinine is the first line treatment in severe P. falciparum malaria and nocturnal leg cramps and a fast, convenient delivery method of this drug quinine is needed. The purpose of this study was to investigate in vitro the sublingual route for the delivery of quinine. Permeation studies were carried out with Franz diffusion cells containing sublingual mucosa membranes with PBS receptor phase and dosed with solutions of quinine hydrochloride or quinine/2-hydroxypropyl-β-cyclodextrin complexes. Receptor phase samples were taken 2 hourly over a 12 h period and quinine was determined by reverse-phase HPLC analysis. The ventral surface of the tongue was significantly more permeable than porcine floor of the mouth (p  < 0.05) and there was no significant effect of freezing on the ventral surface of the tongue (p 0.2444). The presence of saliva caused a decrease in the permeation of quinine across the ventral surface of the tongue by up to 68%. Inclusion complexation between quinine and 2-HP-β-CD was supported by 1H NMR spectral data, and an ethanol vehicle provided the highest quinine flux from the inclusion complex solutions compared to deionised water and PEG. Overall, the data support further investigations into the clinical use of sublingual quinine, particularly for children with falciparum malaria or patients with nocturnal leg cramps. Use of quinine/cyclodextrin inclusion complexes may circumvent compliance issues due to bitter taste.
Keywords: Quinine; Malaria; Leg cramps; Sublingual; Tongue;

A comparison of the fit of flux through hairless mouse skin from water data to three model equations by Scott C. Wasdo; J. Juntunen; H. Devarajan; K.B. Sloan (65-73).
Data for the delivery of total species containing parent drugs from water through hairless mouse skin by prodrugs, log  J MMAQ, has been fitted to the Roberts–Sloan, RS, the Kasting–Smith–Cooper, KSC, and Magnusson–Anissimov–Cross–Roberts, MACR, equations. The RS model which contains a parameter for the dependence of flux on solubility in water, S AQ, as well as solubility in the lipid isopropyl myristate, S IPM, gave the best fit: log  J MMAQ  = −2.30 + 0.575 log  S IPM  + 0.425 log  S AQ  − 0.0016 MW, r 2  = 0.903. The values for the coefficients to the parameters are quite similar to those obtained when the RS model was fit to flux of solutes from water through human skin, log  J MHAQ. There was no trend in predicting the under or over-performance of prodrugs based on their fit to the RS model and whether they were more or less soluble than their parent drugs. There was an inverse dependence of log  J MMAQ on partition coefficients or permeability coefficients similar to that observed for log  J MHAQ. The similarities in trends for results for log  J MMAQ and log  J MHAQ suggests that design directives obtained from mouse skin can be extended to design new prodrugs or select new drugs for delivery through human skin.
Keywords: Lipid solubility; Water solubility; Roberts–Sloan model; Diffusion cell experiments; Prodrugs; Mouse skin;

Quantitative determination of polymorphic impurity by X-ray powder diffractometry in an OROS® formulation by Mehdi Varasteh; Zhengyu Deng; Helen Hwang; Yoo Joong Kim; Geoffrey B. Wong (74-81).
An ALZA OROS® drug delivery system was evaluated for its potential to increase drug load and reduce side effects when used with RWJ-333369 (S-2-O-carbamoyl-1-o-chlorophenyl-ethanol), a novel neuromodulatory agent initially developed by SK Bio-Pharmaceuticals and licensed by Johnson & Johnson. RWJ-333369 was found to have two crystalline polymorphs (A and B) that are enantiotropically related. Polymorph A, used in the formulation, was thermodynamically stable at room temperature. A partial polymorphic conversion in the solid state was observed at an elevated temperature of 60 °C during a two-week stability test.The OROS® RWJ-333369 manufacturing process included milling, granulation, compression, sub-coating, membrane coating, drilling, and drying to produce a capsule-shaped OROS® tablet. The potential for polymorphic conversion during manufacturing and stability testing was evaluated using Fourier Transform Infrared Spectroscopy (FTIR), Raman Spectroscopy (Raman), and X-ray Powder Diffractometry (XRD) to detect impurities; the latter was determined to be preferred method.Pure polymorph A and polymorph B reference materials were used for method development. Mixtures with different ratios of polymorph A and polymorph B were scanned using XRD, and the peak heights and areas were used to generate a calibration curve. OROS® RWJ-333369 formulations were spiked with polymorph B reference, and the detection limit was about 2% using the 22° 2θ diffraction angle relative peak area. Samples from different OROS® manufacturing process and stability tests were analyzed. The results indicated that polymorph A was not converted to polymorph B during manufacturing process. Polymorph B impurity was, however, detected in stability samples.
Keywords: Powder X-ray diffraction; Polymorph; Quantitation; OROS; Granulation; Drying; RWJ-333369;

LC method for telithromycin in tablets: A stability-indicating assay by Lauren C. Vaucher; Clésio S. Paim; Alini D. Lange; Elfrides E.S. Schapoval (82-87).
A liquid chromatographic (LC) method for the quantitative determination of telithromycin, the first member of the ketolides, which is a new class of macrolides, was developed. Analytical parameters were studied according to International Conference on Harmonization (ICH) guidelines. An Ace RP-18 octadecyl silane column (250 mm × 4.6 mm, 5 μm) maintained at 50 °C was used as the stationary phase, and methanol and 0.067 M potassium monobasic phosphate buffer pH 4.0 (55:45, v/v) were used as the mobile phase with UV detection at 265 nm. In forced degradation studies, the effects of acid, base, oxidation, UV light and temperature were investigated showing no interference in the drug peak.The method was linear (r  = 0.9999) at concentration ranging from 10.0 to 40.0 μg/mL, precise (intra-day relative standard deviation [RSD] and inter-day RSD values < 2.0%), accurate (mean recovery = 100.76%), specific and robust. Detection and quantitation limits were 0.0027 and 0.0082 μg/mL, respectively. The results showed the proposed method is suitable for its intended use.The validated method may be used to quantify telithromycin tablets and to determine the stability of the drug. The method is able to separate telithromycin from its degradation products and tablet excipients for its sensitivity and reproducibility.These results are in accordance with a previous microbiological assay study, which used the same tested conditions showing that the methods can be interchangeable.
Keywords: Validation; Liquid chromatography; Stability-indicating; Telithromycin assay; Telithromycin tablets;

Feasibility studies were performed to develop a process for obtaining stable dry protein formulations based on in situ polyethylene glycol (PEG)-induced precipitation and vacuum drying of interferon alpha-2a (IFNα2a) solution in a vial. Using a laboratory scale freeze dryer, the process was carried out in two phases: first, protein solution containing PEG was concentrated to achieve protein precipitation, and second, remaining water was removed by further reducing the chamber pressure. Drying conditions, i.e. temperature and pressure, and solution composition were selected to ensure maximal precipitation (solubility of IFNα2a), to achieve precipitation without boiling, and to ensure stability. Dried formulations were subjected to stability studies (40 °C). Concentration and precipitation could be achieved at a fast rate by utilizing pressures slightly above the vapor pressure of water. Fluorescence and circular dichroism (CD) studies showed that precipitated IFNα2a maintained its native structure. Fourier transform infrared spectroscopy (FTIR) studies showed that IFNα2a when dried in the presence of trehalose, maintained its secondary structure. Trehalose also prevented formation of aggregates during drying. Moisture contents of 1% (w/w) were achieved within 48 h of drying. Dry formulation containing 1:20:100 (w/w) IFNα2a:trehalose:mannitol was stable against aggregation and oxidation (6% oxidized at 40 °C, 6 months). Stability profile was comparable to a similar lyophilized formulation.
Keywords: Proteins; Formulation; Polyethylene glycol (PEG); Precipitation; Vacuum drying;

Co-operative thermal inactivation of herpes simplex virus and influenza virus by arginine and NaCl by Hirotoshi Utsunomiya; Masao Ichinose; Kazuko Tsujimoto; Yukiko Katsuyama; Hisashi Yamasaki; A. Hajime Koyama; Daisuke Ejima; Tsutomu Arakawa (99-102).
Elevated temperatures have been used to inactivate viruses for plasma-derived biopharmaceuticals. This paper describes the effects of arginine and NaCl in conjunction with elevated temperature for inactivation of two enveloped viruses, i.e., herpes simplex virus type 1 (HSV-1) and influenza virus type A at neutral pH. In phosphate-buffered saline, a significant inactivation of HSV-1 occurred above 40 °C, resulting in less than 10% surviving virus (over 90% virus inactivation) at 50 °C. Arginine concentration dependently decreased the temperature required for virus inactivation, leading to temperature shift by almost 17 °C at 1.2 M. NaCl also decreased the inactivation temperature, but to a considerably lesser extent, indicating that virus inactivation effect of arginine is not simply due to ionic strength. Influenza virus was also inactivated by high temperature, but its responses to arginine and NaCl were different from those on HSV-1, suggesting that virus inactivation mechanism is different between these two viruses, i.e., the effects of these reagents are virus specific.
Keywords: Virus inactivation; Arginine; Temperature dependence; HSV-1; Influenza virus;

Serum albumin-alginate coated microspheres: Role of the inner gel in binding and release of the KRFK peptide by Maïté Callewaert; Jean-Marc Millot; Jacques Lesage; Dominique Laurent-Maquin; Florence Edwards-Lévy (103-110).
In continuation with our previous study using fluorescein-isothiocyanate (FITC)–Lys-Arg-Phe-Lys (KRFK) peptide, the aim of this work was to study the interaction of the unlabelled KRFK with calcium alginate gel microspheres coated with a serum albumin (HSA)-alginate membrane prepared using a transacylation method. Coated microspheres were prepared with two main sizes and two gel strengths. Control microspheres made of cross-linked alginate-HSA without calcium alginate gel were also prepared. A series of loading and release assays conducted with methylene blue showed the requirement of inner gel for binding the cationic molecule. Release experiments were performed in different media using unlabelled KRFK and coated microspheres. A plateau was reached within 1 h, in contrast with the slow release of the FITC-peptide observed in our previous work. This discrepancy was attributed to modified properties of the labelled peptide. Adsorption assays of KRFK on coated microspheres were performed in the presence of growing concentrations of NaCl or imidazole. The ions were able to displace the peptide from the particles, which demonstrated ionic interactions, probably involving carboxylate groups of alginate. Adsorption isotherms showed that gel strength influenced affinity (4 × 105  L/mol or 8 × 105  L/mol for gelation with 5% or 20% CaCl2, respectively). Binding site number doubled (from 2.6 × 10−7  mol/mg to more than 5 × 10−7  mol/mg) when microsphere size decreased from 450 μm to 100 μm. Binding sites were assumed to be located in the gel underneath the membrane.
Keywords: Affinity; Binding properties; Membrane; Microsphere; Propylene glycol alginate; Transacylation;

An in vitro method for the quantitative determination of the antimicrobial efficacy of silver-containing wound dressings by Simon Gaisford; Anthony E. Beezer; Alistair H. Bishop; Michael Walker; David Parsons (111-116).
Treatment with silver-containing wound dressings is becoming an increasingly popular strategy to eliminate growth of opportunistic wound pathogens during the healing process. However, there are concerns over the possible side-effects of silver to the patient; coupled to the cost of silver as an ingredient there is a desire to ensure that wound dressings contain the least quantity of active ingredient to ensure the minimum bactericidal concentration (MBC) of silver is maintained in the wound environment. This requires the ability to determine the efficacy of silver directly within the wound environment; an extremely complicated task that is difficult using classical (plate counting) microbiological assays because these cannot be conducted in situ. Here, we report a quantitative method for determining the efficacy of silver in wound dressings using an isothermal calorimetric method. The growth curves of P. aeruginosa (NCIMB 8628) were recorded in growth medium and in growth medium containing AQUACEL® Ag Hydrofiber® dressing. It was found that 10 mg of dressing was sufficient to ensure no detectable growth of organism in 2.5 mL of medium inoculated to 106  cfu/mL. This corresponded to a silver load of 1.1 × 10−6  moles (equivalent to 4.4 × 10−4  M, in the volume of medium used in the experiment). Experiments conducted with silver nitrate rather than dressing indicated the MBC of silver against P. aeruginosa was 1 × 10−4  M. The results suggested that not all of the silver in the dressing was bioavailable, at least over the lifetime of the experiment. One advantage of this effect would be the lack of excess availability of the silver, which allays fears of potential toxicity to the patient and may provide an extended period of time over which the dressing is bactericidal.
Keywords: Silver-containing wound dressings; AQUACEL® Ag Hydrofiber® dressing; Isothermal calorimetry; P. aeruginosa; Efficacy;

In vitro evaluation of the dissolution behaviour of itraconazole in bio-relevant media by Heba S. Ghazal; A. Mark Dyas; James L. Ford; Gillian A. Hutcheon (117-123).
Drugs in the gastrointestinal tract are exposed to a medium of partially digested food, comprising mixtures of fat, protein and carbohydrate. The dissolution behaviour of itraconazole was evaluated in bio-relevant media which were developed to take this into account. Media containing milk with different fat contents, protein (albumin, casein, gluten and gelatin), carbohydrates (glucose, lactose and starch) and amino acids (lysine, glycine, alanine and aspartic acid) to mimic a digested meal and bile components (sodium taurocholate and lecithin) to represent a key endogenous digestive material were investigated. The effect of medium composition on the intrinsic dissolution rate of itraconazole was evaluated as this drug has extremely poor solubility and its bioavailability is affected by food. Dissolution tests were carried out in simple compendial media based on dilute solutions of hydrochloric acid or neutral solutions of phosphate buffer and in more complex media containing the dietary components. The data obtained showed that most of the dietary components enhanced the solubility compared to simulated gastric fluid (SGF) but to differing extents. The greatest increase in dissolution was observed with the addition of milk and albumin although an increase was also seen with other proteins, amino acids and simulated gastrointestinal fluids.
Keywords: Dissolution; Solubility; Intrinsic dissolution rate; Bio-relevant dissolution media; Itraconazole;

To characterise the adhesive interactions between three pulmonary active pharmaceutical ingredient (API) materials and the components of pressurised metered dose inhalers (pMDIs) obtained from two commercially available products (termed ‘Prod-1’ and ‘Prod-2’). This is of potential interest, as a greater understanding of the interactions between specific APIs and surfaces may aid manufacturers in component selection during pMDI system development.The theoretical work of adhesion (ΔG 132) for each API–pMDI component interaction was calculated using the surface component analysis (SCA) approach. These results were correlated with corresponding API–pMDI component separation energy measurements determined using colloid probe AFM.Strong correlations existed between separation energy and the ΔG 132 parameters where the polar contribution was accounted for. This highlighted the adhesive influence of polar surface energy on each interaction in this study. Generally the largest adhesive interactions involved APIs and pMDI components which have a bipolar surface energy (i.e. both γ and γ + >1 mJ m−2).For each API–pMDI interaction in this study, the polar component of surface energy has the greater influence on adhesive events. The bipolar surface energetics of certain APIs and pMDI components were deemed responsible for the increased adhesive interactions observed with these materials. This study highlights that different materials can have different effects on the adhesive interactions with particulate APIs; information that could aid the manufacturer in producing more effective and efficient pMDI systems.
Keywords: Pressurised metered dose inhalers (pMDIs); Atomic force microscopy (AFM); Surface energy; Surface component analysis (SCA);

An in vitro study of liposomal curcumin: Stability, toxicity and biological activity in human lymphocytes and Epstein-Barr virus-transformed human B-cells by Changguo Chen; Thomas D. Johnston; Hoonbae Jeon; Roberto Gedaly; Patrick P. McHugh; Thomas G. Burke; Dinesh Ranjan (133-139).
Curcumin is a multi-functional and pharmacologically safe natural agent. Used as a food additive for centuries, it also has anti-inflammatory, anti-virus and anti-tumor properties. We previously found that it is a potent inhibitor of cyclosporin A (CsA)-resistant T-cell co-stimulation pathway. It inhibits mitogen-stimulated lymphocyte proliferation, NFκB activation and IL-2 signaling. In spite of its safety and efficacy, the in vivo bioavailability of curcumin is poor, and this may be a major obstacle to its utility as a therapeutic agent. Liposomes are known to be excellent carriers for drug delivery. In this in vitro study, we report the effects of different liposome formulations on curcumin stability in phosphate buffered saline (PBS), human blood, plasma and culture medium RPMI-1640 + 10% FBS (pH 7.4, 37 °C). Liposomal curcumin had higher stability than free curcumin in PBS. Liposomal and free curcumin had similar stability in human blood, plasma and RPMI-1640 + 10% FBS. We looked at the toxicity of non-drug-containing liposomes on 3H-thymidine incorporation by concanavalin A (Con A)-stimulated human lymphocytes, splenocytes and Epstein-Barr virus (EBV)-transformed human B-cell lymphoblastoid cell line (LCL). We found that dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylglycerol (DMPG) were toxic to the tested cells. However, addition of cholesterol to the lipids at DMPC:DMPG:cholesterol = 7:1:8 (molar ratio) almost completely eliminated the lipid toxicity to these cells. Liposomal curcumin had similar or even stronger inhibitory effects on Con A-stimulated human lymphocyte, splenocyte and LCL proliferation. We conclude that liposomal curcumin may be useful for intravenous administration to improve the bioavailability and efficacy, facilitating in vivo studies that could ultimately lead to clinical application of curcumin.
Keywords: Curcumin; Liposome; Human lymphocytes; Human splenocytes; EBV-transformed B-cells;

Prednisolone (PDS) beads were coated sequentially with (i) innermost hydrophobic layer of Eudragit® RS/RL, (ii) middle drug release-triggering layer of chitosan, organic acid and Eudragit® RS/RL, and (iii) outermost enteric coating layer. Continuous dissolution studies were carried out in artificial gastric fluid (pH 1.2), followed by intestinal fluid (pH 6.8), and finally in colonic fluid (pH 4 and 6) with and without β-glucosidase. While drug release was prevented in the gastric and small-intestinal fluids, a continuous release was observed in the colonic fluid. Succinic acid provided the fastest rate of release in the colonic fluid compared to citric, tartaric or malic acid. A combined mechanism of drug release is proposed, which considers the swelling of chitosan and Eudragit® RS/RL in the presence of succinic acid possibly via electrostatic interaction between the amine groups of chitosan/quaternary ammonium groups of Eudragit® RS/RL and the carboxyl groups of succinic acid in aqueous medium. The results of plasma pharmacokinetic studies in Sprague–Dawley rats showed that the developed system provided a significant delay (T max 9.3 h) in the absorption profile of PDS compared with simple enteric-coated (T max 4 h) or powder (T max 1 h) formulation that was taken as proof for the colon-targeted delivery.
Keywords: Colon-targeted delivery; Chitosan; Succinic acid; Eudragit®; Electrostatic interaction;

Bioequivalence assay between orally disintegrating and conventional tablet formulations in healthy volunteers by Yara Popst Armando; Simone Grigoleto Schramm; Marina de Freitas Silva; Eunice Kazue Kano; Eunice Emiko Mori Koono; Valentina Porta; Cristina Helena dos Reis Serra (149-153).
The purpose of this study was to evaluate bioequivalence of two commercial 8 mg tablet formulations of ondansetrona available in the Brazilian market. In this study, a simple, rapid, sensitive and selective liquid chromatography–tandem mass spectrometry method is described for the determination of ondansetron in human plasma samples. The method was validated over a concentration range of 2.5–60 ng/ml and used in a bioequivalence trial between orally disintegrating and conventional tablet ondansetron formulations, to assess its usefulness in this kind of study. Vonau flash® (Biolab Sanus Farmacêutica, Brazil, as test formulations) and Zofran® (GlaxoSmithKline, Brazil, as reference formulation) were evaluated following a single 8 mg dose to 23 healthy volunteers of both genders. The dose was administered after an overnight fast according to a two-way crossover design. Bioequivalence between the products was determinated by calculating 90% confidence interval (90% CI) for the ratio of C max, AUC0–t and AUC0–∞ values for the test and reference products, using logarithmically transformed data. The 90% confidence interval for the ratio of C max (87.5–103.8%), AUC0–t (89.3–107.2%) and AUC0–∞ (89.7–106.0%) values for the test and reference products is within the 80–125% interval, proposed by FDA, EMEA and ANVISA. It was concluded that two ondansetron formulations are bioequivalent in their rate and extent of absorption.
Keywords: Bioequivalence; Ondansetron; LC–MS; Plasma;

Preparation of polysaccharide glassy microparticles with stabilization of proteins by Weien Yuan; Yan Geng; Fei Wu; Yajun Liu; Meiyan Guo; Hao Zhao; Tuo Jin (154-159).
This study investigates a method of preparing hazard-resistant protein-loaded polysaccharide glassy microparticles using freezing-induced phase separation method without exposure to water/oil, water/air interface and cross-linking reagents. Model protein (such as bovine serum albumin, myoglobin and β-galactosidase (β-Gal)) was dissolved in water together with dextran and polyethylene glycol (PEG), followed by a freezing process to form a temperature-stabilized aqueous–aqueous emulsion wherein dextran separated out as the dispersed phase with protein partitioned in preferentially. The frozen sample was freeze-dried and washed with dichloromethane (DCM) to remove the PEG continuous phase, after which protein-loaded polysaccharide particles, 1–4 μm in diameter, were harvested. Differential scanning calorimetry (DSC) and X-ray diffraction (XRD) patterns showed that the particles were in glassy state. These glassy polysaccharide microparticles can well protect the delicate structure of proteins and preserve their bioactivities under deleterious environment interacting with organic solvents, vortex and centrifugation processes that often involve during the formulation processes leading to polymer-based sustained-release systems. Therefore, this freezing-induced phase separation method is a mild and effective way to encapsulate protein into hazard-resistant polysaccharide glassy particles, which ensure its stability in subsequent formulating processes that leads to polymer-based sustained-release system.
Keywords: Protein delivery; Polysaccharide microparticles; Freezing-induced phase separation; Protein aggregation;

The purpose of this paper is to prepare a new formulation of clarithromycin emulsion (ClaE) with the clarithromycin–phospholipid complex which was analyzed by DSC. High-pressure homogenization, Nicomp™ 380 Particle Sizing system, and HPLC were used to prepare and investigate ClaE, while UPLC/MS/MS for pharmacokinetic study. Clarithromycin and soybean lecithin were reacted in dehydrated alcohol at a ratio of 1:10 for 3 h at 65 °C to prepare the complex. The ClaE formulation consisted of, according to quality percentage, the complex (clarithromycin 0.25% in ClaE), LCT 4%, MCT 16%, soybean lecithin 1.0%, F68 0.2%, Tween80 0.2%, glycerol 2.5%, sodium oleate 0.1% and l-cysteine 0.02%. ClaE was sterilized in a 100 °C revolving water bath for 30 min. The drug content, particle size distribution and entrapment efficiency of ClaE before and after sterilization and over 6 months storage at 10 °C were almost unchanged, while ζ-potential increased from −20.32 mV to −23.71 mV. These results show that ClaE has enough physicochemical stability to undergo sterilization and storage. The pharmacokinetic study showed that both ClaE and ClaS fitted a three-compartment model, their pharmacokinetic curves were similar and the main parameters showed no significant difference except Vss. ClaE has a great potential for clinical applications and industrial-scale production.
Keywords: Clarithromycin–phospholipids complex; Formulation; Pharmacokinetic; Emulsion; Intravenous;

Lipid nanoparticles (SLN, NLC) in cosmetic and pharmaceutical dermal products by Jana Pardeike; Aiman Hommoss; Rainer H. Müller (170-184).
Solid lipid nanoparticles (SLN) are distinguishable from nanostructured lipid carriers (NLC) by the composition of the solid particle matrix. Both are an alternative carrier system to liposomes and emulsions. This review paper focuses on lipid nanoparticles for dermal application. Production of lipid nanoparticles and final products containing lipid nanoparticles is feasible by well-established production methods. SLN and NLC exhibit many features for dermal application of cosmetics and pharmaceutics, i.e. controlled release of actives, drug targeting, occlusion and associated with it penetration enhancement and increase of skin hydration. Due to the production of lipid nanoparticles from physiological and/or biodegradable lipids, this carrier system exhibits an excellent tolerability. The lipid nanoparticles are a “nanosafe” carrier. Furthermore, an overview of the cosmetic products currently on the market is given and the improvement of the benefit/risk ratio of the topical therapy is shown.
Keywords: Solid lipid nanoparticles (SLN); Nanostructured lipid carriers (NLC); Dermal application; Cosmetic use; Pharmaceutical use; Dermal safety;

Physical chemistry behavior of enteric polymer in drug release systems by H.P. de Oliveira; J.J.F. Albuquerque; C. Nogueiras; J. Rieumont (185-189).
We report an analysis based on the electrical impedance (EI) spectrum of the samples of enteric random copolymer poly-methacrylic acid-co-methyl methacrylate as a function of pH of media. Important aspects of the charge transport and conformational processes in enteric polymer can be identified by mapping the complex impedance as a function of the frequency, which allows that some parallelism between titration and EI measurements can be obtained. However, the latter technique reveals details of this complex equilibrium that not appear using common titration methods. The relaxation frequency observed in the impedance spectrum act as a probe for the detection of phase transitions and conformational changes of the polymeric chains, once the distribution of size of particles can be related with this parameter. The progressive introduction of the alkali and the variation of pH between 4 and 10 are associated with a three steps process, related to the equilibrium shift from a precipitated solid or suspension, to a colloidal-like dispersion and to a complete solubilization of the copolymer. All those experimental features were reflected simultaneously as a turning point in plots of impedance, relaxation frequency and visible absorption with alkali addition giving a better and detailed insight to these processes.
Keywords: Impedance spectroscopy; Enteric polymer; Dielectric relaxation;

Microneedle mediated delivery of nanoparticles into human skin by Sion A. Coulman; Alexander Anstey; Chris Gateley; Anthony Morrissey; Peter McLoughlin; Chris Allender; James C. Birchall (190-200).
The development of novel cutaneous delivery technologies that can produce micron-sized channels within the outermost skin layers has stimulated interest in the skin as an interface for localised and systemic delivery of macromolecular and nanoparticulate therapeutics. This investigation assesses the contribution of physicochemical factors to the rate and extent of nanoparticle delivery through microchannels created in a biological tissue, the skin, by novel delivery technologies such as the microneedle array.The hydrodynamic diameter, zeta potential and surface morphology of a representative fluorescent nanoparticle formulation were characterised. Permeation studies using static Franz-type diffusion cells assessed (i) the diffusion of nanoparticle formulations through a model membrane containing uniform cylindrical microchannels of variable diameter and (ii) nanoparticle penetration across microneedle treated human skin.Wet-etch microneedle array devices can be used to significantly enhance the intra/transdermal delivery of nanoparticle formulations. However the physicochemical factors, microchannel size and particle surface charge, have a significant influence on the permeation and subsequent distribution of a nanoparticle formulation within the skin. Further work is required to understand the behaviour of nanoparticle formulations within the biological environment and their interaction with the skin layers following disruption of the skin barrier with novel delivery devices such as the microneedle array.
Keywords: Microneedle; Nanoparticles; Transdermal; Skin; Skin permeation; Vaccination;

Targeting colon cancer cells using PEGylated liposomes modified with a fibronectin-mimetic peptide by Ashish Garg; Alison W. Tisdale; Eman Haidari; Efrosini Kokkoli (201-210).
Integrin α5β1 is expressed on several types of cancer cells, including colon cancer, and plays an important role in tumor growth and metastasis. The ability to target the integrin α5β1 using an appropriate drug delivery nano-vector can significantly help in inhibiting tumor growth, reducing tumor metastasis, and decreasing deleterious side effects associated with different cancer therapies. Liposomes are nano-sized phospholipid bilayer vesicles that have been extensively studied as drug delivery carriers. The goal of this study is to design stealth liposomes (liposomes covered with polyethylene glycol (PEG)) that will target colon cancer cells that express the integrin α5β1. The PEG provides a steric barrier allowing the liposomes to circulate in the blood and the functionalizing moiety, PR_b peptide, will specifically recognize and bind to α5β1 expressing cells. PR_b is a novel peptide sequence that mimics the cell adhesion domain of fibronectin, and includes four building blocks, RGDSP (the primary recognition site for α5β1), PHSRN (the synergy site for α5β1), a (SG)5 linker, and a KSS spacer. In this study we have demonstrated that by varying the amount of PEG (PEG750 or PEG2000) and PR_b on the liposomal interface we can engineer nano-vectors that bind to CT26.WT, HCT116, and RKO colon cancer cells in a specific manner and are internalized through most likely α5β1-mediated endocytosis. GRGDSP-targeted stealth liposomes bind to colon cancer cells and internalize, but they have much lesser efficiency than PR_b-targeted stealth liposomes, and more importantly they are not as specific since many integrins bind to RGD peptides. PR_b-targeted stealth liposomes are as cytotoxic as free 5-Fluorouracil (5-FU) and exert the highest cytotoxicity on CT26.WT cells compared to GRGDSP-targeted stealth liposomes and non-targeted stealth liposomes. Thus, the proposed targeted delivery system has the great potential to deliver a therapeutic load directly to colon cancer cells, in an efficient and specific manner.
Keywords: Drug targeting; α5β1integrin; Fibronectin; RGD; PHSRN; CT26; Nanoparticles; Liposomes; 5-FU; PEG750; PEG2000;

Preparation and characterization of magnetic cationic liposome in gene delivery by Xiaoli Zheng; Jianping Lu; Li Deng; Yang Xiong; Jianming Chen (211-217).
Low transfection efficiency in vivo and failure to deliver therapeutic nucleic acids to the target organs limit the use of cationic liposomes (CLs) in gene therapy. Magnetic drug targeting (MDT) was applied in this study to improve the transfection efficiency and overcome the limitations. Magnetic cationic liposomes (MCLs) were prepared by incorporating MAG-T (magnetite) into CLs. The inclusion of relatively high concentration of MAG-T significantly increased the size of liposomes/lipoplexes, reduced the zeta potential, and decreased the cell viability. The transfection efficiency of MCLs in gene delivery was evaluated by using plasmid DNA (pDNA) containing a luciferase reporter gene in THLE-3 cells. Results suggested that the transfection efficiency of MCLs/pDNA complexes with a relatively lower content of MAG-T (0.75 mg/ml) was the same as that of CLs/pDNA complexes without a magnetic field but was higher (about 2.6-fold) with magnetic induction. Finally, using MCLs/pDNA complexes and a static magnetic field placed over the liver of rats, luciferase reporter gene expression in the liver increased as compared to MCLs/pDNA complexes and CLs/pDNA complexes in the absence of an external magnetic field.
Keywords: Cationic liposome; Gene transfection; Magnetic drug targeting; Magnetic cationic liposome;

Pulmonary delivery of a GLP-1 receptor agonist, BMS-686117 by Feng Qian; Neil Mathias; Paul Moench; Cecilia Chi; Sridhar Desikan; Munir Hussain; Ronald L. Smith (218-220).
Alternate delivery route of therapeutic peptides is an attractive non-invasive option to patients who must chronically self-administer their medication through injections. In recent years, much attention has centered on pulmonary peptide delivery of peptide drugs such as insulin and GLP-1 mimetic peptides in the treatment of type II diabetes. In this study, we assessed the feasibility of delivering BMS-686117, an 11-mer GLP-1 receptor peptide agonist, to the lung in rats via intratracheal administration. The pharmacokinetic profiles of three spray-dried, prototype inhaled powder formulations, 80/20 BMS-686117/trehalose (I), 100% BMS-686117 (II), and 20/80 BMS-686117/mannitol (III), as well as a lyophilized BMS-686117 powder, were compared with intravenously and subcutaneously administered peptide. The spray-dried formulations were mostly spherical particles with narrow particle size distribution between 2 to 10 μm, which are better suited for inhalation delivery than the lyophilized, irregular shape powder with a wide particle size distribution between 2 to 100 μm. Prototype III exhibited the best physical characteristics and in vivo performance, with bioavailability of 45% relative to subcutaneous administration. The T max for lung delivered peptide formulations were almost twice as fast as subcutaneous injection, suggesting potential for rapid absorption and onset of action. This study demonstrated that pulmonary delivery is a promising, non-invasive route for the administration of BMS-686117.
Keywords: Type II diabetes mellitus; Glucagon-like peptide (GLP-1); Pulmonary delivery; Spray drying; Pharmacokinetics;

Noticeboard (221).