International Journal of Pharmaceutics (v.289, #1-2)

Protein aggregation is arguably the most common and troubling manifestation of protein instability, encountered in almost all stages of protein drug development. Protein aggregation, along with other physical and/or chemical instabilities of proteins, remains to be one of the major road barriers hindering rapid commercialization of potential protein drug candidates. Although a variety of methods have been used/designed to prevent/inhibit protein aggregation, the end results are often unsatisfactory for many proteins. The limited success is partly due to our lack of a clear understanding of the protein aggregation process. This article intends to discuss protein aggregation and its related mechanisms, methods characterizing protein aggregation, factors affecting protein aggregation, and possible venues in aggregation prevention/inhibition in various stages of protein drug development.
Keywords: Protein aggregation; Aggregation mechanism; Protein refolding; Protein formulation; Protein stabilization;

The objective of this study was to investigate the reasons for the difference in physical stability of two amorphous cefditoren pivoxil samples that had been prepared using spray drying at inlet-air temperatures of 40 °C (SD-A) and 100 °C (SD-B). The two samples appeared amorphous by powder X-ray diffraction and had indistinguishable glass transition temperatures. Despite the fact that glass transition is often regarded as an indicator of the stability of amorphous forms, crystallisation was observed for SD-A, but not for SD-B, during storage at 60 °C and 81% relative humidity (RH). Gravimetric water sorption data demonstrated very similar water sorption until high RH values, at which point SD-A sorbed more water than did SD-B. The values of the dispersive, acidic (K A) and basic (K D) components of surface energy of the spray-dried samples were obtained using inverse gas chromatography (IGC), in the dry state and after equilibration with different RH environments. The data showed that the two amorphous samples had different surface properties and that the effect of sorbed water on these samples was also different. It is concluded that the two samples did not have long-range order, but had differences in the orientation of molecules at the surface, which were significant enough to alter the stability when the samples were stressed with water vapour and high temperature storage. IGC proved a valuable tool with which to study changes in the surface properties of amorphous materials.
Keywords: Amorphous; Cefditoren pivoxil; Inverse gas chromatography; Molecular mobility; Physicochemical stability; Surface energy;

There are a number of situations where there is a need to determine the concentrations of components in solid-state mixtures without dissolving the sample. Diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) coupled with partial-least-squares (PLS) data analysis has been used to determine the minor component in a mixture of structurally similar solid-state compounds, in this case mixtures of ephedrine and pseudoephedrine. Factors that limit the precision and accuracy of the determinations are discussed. It is shown that when care is taken to produce homogeneous calibration samples very good results can be obtained, in this case cross-validated standard error of predictions of 0.74 wt% when the minor component spanned the concentration range of 0–50 wt%, and 0.11 wt% when the minor component spanned the concentration range of 0–5 wt%. Results are presented that indicate that the amount of data available to the PLS calibration routine relative to the range over which the calibration is performed can limit the precision and accuracy of the determinations.
Keywords: Ephedrine; Infrared spectroscopy; Partial-least-squares analysis;

Lyophilised wafers as a drug delivery system for wound healing containing methylcellulose as a viscosity modifier by K.H. Matthews; H.N.E. Stevens; A.D. Auffret; M.J. Humphrey; G.M. Eccleston (51-62).
Lyophilised wafers have potential as drug delivery systems for suppurating wounds. A dual series of wafers made from low molecular weight sodium alginate (SA) and xanthan gum (XG) respectively, modified with high molecular weight methylcellulose (MC) were produced. The swelling and flow properties of these wafers on model suppurating surfaces were both qualitatively and quantitatively investigated. The wafers instantaneously adhered to the surfaces, absorbing water and transforming from glassy, porous solids to highly viscous gels. The rate at which this occurred varied for the series studied with clear distinctions between the behaviour of SA and XG systems. For SA wafers there was a distinct relationship between the flow-rate and MC content. Increased amounts of MC decreased the rate at which the SA wafers flowed across a model gelatine surface. Flow rheometry was used to quantify the effect of increased MC content on both series of wafers and for the SA series, highlighted a substantial increase in apparent viscosity as a function of incremental increases in MC content. These results reflected those from the gelatine model. Observations of the reluctance of a swollen, unmodified XG wafer to flow compared with the relative ease of unmodified, low molecular weight SA was attributed to the yield stress characteristic of xanthan gels. XG is known to exhibit complex, loosely bound network structures in solution via the association of helical backbone structures. The inclusion of sodium fluorescein as a visible model for a soluble drug highlighted the potential of lyophilised wafers as useful drug delivery systems for suppurating wounds.
Keywords: Lyophilised wafer; Wound healing; Sodium alginate; Methylcellulose; Xanthan; Model surface;

Studies on the poly(lactic-co-glycolic) acid microspheres of cisplatin for lung-targeting by Dongjie Huo; Shuhai Deng; Lingbing Li; Jianbo Ji (63-67).
Lung-targeting cisplatin-loaded poly(lactic-co-glycolic) acid microspheres (CDDP-PLGA-MS) were prepared by a solvent evaporation method. The uniform design was used to optimize the technology of preparation, the appearance and size distribution were examined by scanning electron microscope, and the aspects such as in vitro release characteristics, stability, drug loading, loading efficiency, pharmacokinetics and tissue distribution in rabbit were studied. The experimental results showed that the microspheres were globular in appearance and dispersed well. The average particle size was 12.8 μm with 98% of the microspheres being in the range of 5–30 μm. The drug loading and loading efficiency were 17.68 and 53.2%, respectively. The in vitro release behavior could be expressed by the following equation: 1−Q  = 0.424e−0.360t  + 0.474e−0.001t . After i.v. administration (15 min), the drug concentration of microspheres group in lung in rabbits was 212 μg/g, while that of controlled group was 1.37 μg/g. CDDP-PLGA-MS showed a combination of lung-targeting and sustained drug release in experiments on rabbits.
Keywords: Cisplatin; Poly (lactic-co-glycolic) acid; Microspheres; Lung-targeting;

Effect of adsorbents on the absorption of lansoprazole with surfactant by Yukako Ito; Harumi Arai; Kaori Uchino; Kouji Iwasaki; Nobuhito Shibata; Kanji Takada (69-77).
Lansoprazole (LPZ) is a representative drug that shows a high inter-subject variation of bioavailability (BA). Solid preparation composed of surfactant, adsorbent and LPZ were prepared to improve the dissolution and absorption of LPZ, and the BA of LPZ was measured in rats and dogs. As surfactant, Tween 80, polyoxy 60 hydrogenated caster oil derivative (HCO-60) and PEG-8 caprylic/capric glycerides (Labrasol) were used. As adsorbant, porous silicon dioxide (Sylysia 550, 320), magnesium aluminometa silicate (Neusilin S2, NS2N, US2,) and porous calcium silicate (Florite RE) were used. After small intestinal administration of LPZ, 5.0 mg/kg, solution with HCO-60 showed the highest plasma LPZ concentration versus time curve of which C max and AUC was 0.46 ± 0.01 μg/mL and 0.73 ± 0.03 μg h/mL. By comparing to that after i.v. injection of LPZ solution, 2.0 mg/kg, the BA of LPZ from HCO-60 solution was 39.0%, which was about seven times higher than that of LPZ powder. To solidify the LPZ solution with HCO-60, adsorbents were used and the obtained solid preparations were used for in vitro release experiment. Sylysia 320, Neusilin S2 and Neusilin NS2 showed the T50% of about 1 h. To evaluate the BA of these solid preparations, absorption study was performed in rats. Sylysia 550 system showed the higher AUC than other systems, showing the BA of 28.1%. Sylysia 550 system was filled in an enteric capsule and was orally administered to dogs and BA was compared with enteric tablet. The AUC of Sylysia 550 system was 2.16 ± 0.26 μg h/mL and was greater than enteric tablet and the BA of 71.7% was obtained. Solid system composed of LPZ, surfactant and adsorbent has suggested the possibility as a good tool to improve the BA of LPZ.
Keywords: Lansoprazole; Absorption; Bioavailability; Adosorbent; Surfactant; Oral administration;

Compression coating has been found to be useful for colonic drug delivery. The aim of the present investigation was to evaluate a formulation with a considerably reduced coat weight and gum concentration for colonic drug delivery in vivo using gamma scintigraphy. In vitro studies have found this formulation to be useful for delivery of 5-fluorouracil to the colon. Rapidly disintegrating core tablets containing 99mTc-DTPA were prepared and compression coating with 150 mg of granules containing a mixture of xanthan (XG), guar gum (GG) and starch. The ratios of the two gums XG:GG in the coat was kept 10:20. In vitro dissolution studies on XG:GG::10:20 tablets containing 99mTc-DTPA were carried out in simulated upper GIT conditions and also in presence of colonic contents. Cumulative percent release of technetium in the upper GIT conditions and transit time amounted to 4%. The total amount of technetium released in the 24 h of the dissolution study was 53 ± 3.23%. Upon introduction of cecal content into the dissolution medium (4%), the release of technetium from the compression-coated tablet increased to 78.34 ± 5.34%. Gamma scintigraphy studies carried out in six healthy human volunteers showed that the tablet remained intact during its transit through the upper GIT. The anatomical site of disintegration was found to be the ascending colon/hepatic flexure and the disintegration of the tablet started between 4 and 6 h post-dose in all the volunteers with a further spread of tracer into the ascending, transverse, descending and sigmoidal colon.
Keywords: Gamma-scintigraphy; Xanthan gum; Guar gum; Starch; Colon-specific drug delivery;

The purpose of this study was to optimize the pH-dependent release of nicardipine hydrochloride extended release formulations by using simultaneously combination two hydrophilic polymers: hydroxypropylmethylcellulose (HPMC) and sodium alginate as retardant and avicel as additive. The constrained mixture experimental design was used to prepare systematic model formulations which were composed of three formulation variables: the content of HPMC (X 1), avicel (X 2), and sodium alginate (X 3). The response surface methodology (RSM) and multiple response optimization utilizing the polynomial equation were used to search for the optimal formulation with specific release rate at different time intervals and to quantify the effect of each formulation variables. The drug release percent at 3, 6 and 12 h were the target responses and were restricted to 10–30% (Y 3 h), 40–65% (Y 6 h) and not less than 80% (Y 12 h), respectively. The results showed that the effect of combination of HPMC and sodium alginate was the most influence factor on the drug release from extended-release matrix tablets. The observed results of Y 3 h, Y 6 h and Y 12 h coincided well with the predictions in the RSM optimization technique, indicating it was quite useful for optimizing pharmaceutical formulation. The mechanism of drug release from extended-release matrix tablets was dependent on the added amount of alginate. The release kinetic of drug from HPMC matrix tablets with alginate was followed the zero-order release pattern.
Keywords: Extended-release dosage form; Mixture experimental design; Nicardipine response surface methodology; Dissolution;

A new scleroglucan/borax hydrogel: swelling and drug release studies by Tommasina Coviello; Mario Grassi; Antonio Palleschi; Gianfranco Bocchinfuso; Gina Coluzzi; Fateme Banishoeib; Franco Alhaique (97-107).
The aim of the work was the characterization of a new polysaccharidic physical hydrogel, obtained from Scleroglucan (Sclg) and borax, following water uptake and dimension variations during the swelling process. Furthermore, the release of molecules of different size (Theophylline (TPH), Vitamin B12 (Vit. B12) and Myoglobin (MGB)) from the gel and from the dried system used as a matrix for tablets was studied. The increase of weight of the tablets with and without the loaded drugs was followed together with the relative variation of the dimensions. The dry matrix, in the form of tablets was capable, during the swelling process, to incorporate a relevant amount of solvent (ca. 20 g water/g dried matrix), without dissolving in the medium, leading to a surprisingly noticeable anisotropic swelling that can be correlated with a peculiar supramolecular structure of the system induced by compression. Obtained results indicate that the new hydrogel can be suitable for sustained drug release formulations. The delivery from the matrix is deeply dependent on the size of the tested model drugs. The experimental release data obtained from the gel were satisfactorily fitted by an appropriate theoretical approach and the relative drug diffusion coefficients in the hydrogel were estimated. The release profiles of TPH, Vit. B12 and MGB from the tablets have been analyzed in terms of a new mathematical approach that allows calculating of permeability values of the loaded drugs.
Keywords: Hydrogel; Scleroglucan; Anisotropic swelling; Sustained drug release; Modelling;

Antisticking power varies according to the talc considered. It is difficult to define the physical properties of talc implicated in its antisticking power. In this work, different talcs were characterized and an evaluation made of their performance in reducing sticking in tablet manufacturing. Determination of the specific surface area was made by permeametry, morphogranulometric analysis by laser diffractometry using a method, which made it possible to assess the mean thickness of talc particles, and measurement of water absorption kinetics was taken to assess hydrophobicity. The relationship between the characteristics of talcs and their antisticking power was then considered. There is a correlation between the particle size of talc and surface hydrophobicity. The detaching force of tablets appears to be dependent on the basal dimension of talc.
Keywords: Talc; Morphogranulometry; Hydrophobicity; Antisticking power; Functionality assay;

The purpose of this paper is to show how the utilization of Fourier Transform Infrared (FTIR) spectroscopy can be interesting in stability studying of cosmetic or pharmaceutical “oil in water” (O/W) emulsions. In this study temperature storage tests were performed to accelerate the aging process and evaluate the stability of five emulsions. Emulsions were analyzed by FTIR and classical methods (conductivity, viscosity, pH, texture analysis) in order to determine a method that would enable predicting the emulsion's stability. During the aging process, modifications of chemical functions are measured by FTIR (using spectrometric indices), such modifications included: a decrease of unsaturation index, an increase of carbonyl index and a broadening of the carbonyl band. This band was deconvoluted to evaluate the contribution of different species in the broadening phenomenon, which seems to be caused by the appearance of free fatty acids. Conductimetry seems to be the most sensitive technique to assess physical modifications during emulsion's aging. Concerning the most unstable emulsions, a progressive increasing of conductivity was observed several months before the emulsion destabilizes. Consequently, FTIR and conductimetry are two complementary techniques. Conductimetry is a useful technique to predict emulsion destabilization while FTIR allows the measurement of chemical modifications and helps to understand the chemical mechanisms which occur during the oxidation.
Keywords: Emulsion; Aging; Oxidation; FTIR; Deconvolution; Conductivity;

Studies on the reactions between daptomycin and glyceraldehyde by Walaisiri Muangsiri; William R. Kearney; Lynn M. Teesch; Lee E. Kirsch (133-150).
The objectives of this project were to determine the reaction pathways of daptomycin in the presence of glyceraldehyde in acidic solutions, and to quantitate the kinetics of the major pathways. In the presence of glyceraldehyde (pH range 1–7 at 25 to 60 °C), daptomycin formed two major products separable by RP-HPLC. The products were identified using UV spectroscopy, fluorimetry, mass spectrometry, and 2D-1H NMR. The reaction scheme involved the reversible formation of imine and anilide derivatives. Carbinolamine was believed to be a common intermediate in formation pathways of both products. The carbinolamine intermediate underwent either acid catalyzed dehydration resulting in imine formation or intramolecular hydrogen bonding and bond cleavage giving rise to anilide formation. In mild acid conditions, both products reversed to daptomycin. The reaction between daptomycin and glyceraldehyde was first-order with respect to both reactants. In a pH range of 1–7, the imine formation rate was pH dependent with a maximum rate at approximate pH values of 3–4. The observed pH dependence was consistent with the pH dependence of typical amine–aldehyde reactions.
Keywords: NMR; Proteins; Kinetics; Aldehyde–amine reaction; Anilide formation; pH dependence;

In vivo distribution of arsenic after i.p. injection of arsonoliposomes in balb-c mice by Sophia G. Antimisiaris; Pavlos Klepetsanis; Venetia Zachariou; Eleftheria Giannopoulou; Panagiotis V. Ioannou (151-158).
We recently showed that arsonoliposomes (novel arsenic containg liposomes) demonstrate differential toxicity towards various types of cancer and normal cells, in cell culture studies, as well as anti-parasitic activity. In this study, the in-vivo distribution of the active moiety of these vesicles, As, is evaluated.Sonicated arsonoliposomes were prepared using the arsonolipid with palmitic acid acyl chain (C16) mixed with egg-phosphatidyl choline (PC) and cholesterol (Chol) [C16/PC/Chol at 8:12:10 mol/mol/mol]. A dose of arsonoliposomes, corresponding to 5 mg arsenate/kg was administered by intraperitoneal injection in balb-c mice. At various time points post-injection the mice were sacrificed and the distribution of As in the organs was measured, by atomic absorption spectroscopy.Results demonstrate that a high portion of the dose administered is rapidly excreted; since 1-h post-injection only about 30% of the dose administered was detected cumulatively in the animal tissues. After this the elimination of arsenic was a slow process with a total body elimination rate constant of 0.023 h−1, corresponding to a half-life of 30 h. Tissues with the highest arsenic concentration during the study period are: spleen–kidneys–stomach, followed by lung, liver, intestines–heart, carcass + skin and finally blood. No acute toxicity, or effect on the body or organ weight of the mice was observed.
Keywords: Liposome; Arsenate; Arsenic; Distribution; Tissue; Mice; In vivo; Toxicity;

The aim of this paper is to establish a novel method to calculate the extent and amount of drug transported to brain after administration. The cerebrospinal fluid (CSF) was chosen as the target region. The intranasal administration of meptazinol hydrochloride (MEP) was chosen as the model administration and intravenous administration was selected as reference. According to formula transform, the extent was measured by the equation of (X A)CSF, ∞/X 0  = ClCSF (AUC0→∞)CSF/X 0 and the drug amount was calculated by multiplying the dose with the extent. The drug clearance in CSF (ClCSF) was calculated by a method, in which a certain volume of MEP solution was injected directly into rat cistern magna and then clearance was assessed as the reciprocal of the zeroth moment of a CSF level-time curve normalized for dose. In order to testify the accurateness of the method, 14C-sucrose was chosen as reference because of its impermeable characteristic across blood-brain barrier (BBB). It was found out that the MEP concentrations in plasma and CSF after intranasal administration did not show significant difference with those after intravenous administration. However, the extent and amount of MEP transported to CSF was significantly lower compared with those to plasma after these two administrations. In conclusion, the method can be applied to measure the extent and amount of drug transported to CSF, which would be useful to evaluate brain-targeting drug delivery.
Keywords: Brain-targeting; Extent; Intranasal administration; Clearance; Cerebrospinal fluid; Meptazinol hydrochloride;

Skin permeation of propranolol from polymeric film containing terpene enhancers for transdermal use by Chomchan Amnuaikit; Itsue Ikeuchi; Ken-ichi Ogawara; Kazutaka Higaki; Toshikiro Kimura (167-178).
To develop the suitable film formulations of propranolol hydrochloride (PPL) containing enhancers for transdermal use, polymeric film formulations were prepared by employing ethyl cellulose (EC) and polyvinyl pyrrolidone (PVP) as a film former, and dibutyl phthalate (DBP) as a plasticizer. Terpenes such as menthol and cineole, and propylene glycol (PG) were also employed as a chemical enhancer to improve the skin penetration of PPL. The film preparations were characterized in physical properties such as uniformity of drug content, thickness and moisture uptake capacity. Release and skin permeation kinetics of PPL from film preparations were examined in the in vitro studies using a Franz-type diffusion cell. The uniformity of drug content was evidenced by the low S.D. values for each film preparation. The moisture uptake capacity and drug release rate increased with the increase of PVP in each preparation. Enhancers examined in the present study also increased the moisture uptake capacity and release rate of PPL from the film preparations. Increasing the concentration of PPL from 1 to 2 mg/cm2 in the film enhanced the release rate of PPL, while no effect of enhancer concentrations on the release rate from the film preparations was observed. In vitro skin permeation study showed that cineole was the most promising enhancer among the enhancers examined in the present study and suggested that the suitable compositions of film preparation would be EC:PVP:PPL = 6:3:4 with 10% (w/w) cineole and 7:2:4 with 10% (w/w) PG and cineole, which provided high skin permeation rates at 93.81 ± 11.56 and 54.51 ± 0.52 μg/cm2/h, respectively.
Keywords: Propranolol hydrochloride; Terpene; Transdermal absorption; Polymeric film; Cineole; Propylene glycol;

Physicochemical studies on Ciclopirox olamine complexes with divalent metal ions by Ruba T. Tarawneh; Imad I. Hamdan; Ahmad Bani-Jaber; Rula M. Darwish (179-187).
Ciclopirox olamine (CPO) metal complexes have been prepared and characterized using elemental analysis, infra red (IR), melting point and differential scanning calorimetry (DSC). Spectroscopic titration using molar ratio method indicated the occurrence of 1:1 complexes for CPO with almost all the examined metals. Physicochemical properties were also studied including aqueous solubility and apparent partition coefficient. Results showed that generally complex formation dramatically decreased the solubility and increased apparent partition coefficient. However, some metal complexes exhibited opposite effect. It could be concluded that complex formation can modify the solubility and apparent partition coefficient, which may suggest the use of complexes to manipulate the physicochemical properties of the drug.
Keywords: Ciclopirox olamine; Metal ion complexes; Partition coefficient;

The interference of colloidal tin oxides on the biodistribution of 99mTechnetium radiolabeled chitosan nanoparticles has been overcome by using sodium borohydride instead of commonly used stannous salts as reducing agent for the reduction of 99mTc (VII) to lower valency states. Biodistribution of radiolabeled chitosan nanoparticles prepared by using stannous chloride method revealed localization of the radioactivity mainly in the liver and spleen while that of radiolabeled chitosan nanoparticles prepared by using sodium borohydride method manifested the presence of radioactivity in blood up to an extent of 10% even after 2 h. Interestingly, the reduction of radioactivity in the latter case with the progress of time was not manifested through an increase in activity in the liver. Rather, a time dependent increased accumulation of radioactive materials was observed in the stomach. From the results it has been concluded that the biodistribution is strongly influenced by the presence of colloidal particles of tin oxides and 99mTc labeled chitosan nanoparticles are RES evading and long circulating in blood when Tc (VII) is reduced by sodium borohydride and not by stannous chloride during radiolabeling process.
Keywords: 99mTc; Chitosan nanoparticles; Gamma scintigraphy; Hydrogels;

Physicochemical characterization and gene transfection efficiency of lipid emulsions with various co-emulsifiers by Chi-Feng Hung; Tsong-Long Hwang; Chia-Chun Chang; Jia-You Fang (197-208).
Transfection systems based on complexes of DNA and lipid emulsions were evaluated with respect to their effectiveness, toxicity, physicochemical characteristics, and cell-type dependence. The potential of a series of co-emulsifiers to serve as vectors was investigated. The co-emulsifiers examined included 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), Tween, cholesterol, stearylamine, and polyethylenimine (PEI). Squalane and 1,2-dioleoyl-sn-glycero-3-trimethylammonium-propane (DOTAP), respectively, were the main oil phase and cationic lipid added to the lipid emulsions. Cell viability was reduced after inclusion of either of the two cationic components of stearylamine and PEI. DOPE and cholesterol showed both higher transfection activity and cell viability as compared to the other co-emulsifiers. The incorporation of DOPE and cholesterol also prevented droplet aggregation of the emulsions after long-term storage. Results of the transfection of COS-1, A549, or HaCat cell lines with lipid emulsions indicated differences in transfection activities of each formulation for the different cell lines. It is concluded that DOPE and cholesterol as co-emulsifiers for DOTAP were preferable for stability and DNA transfection of emulsions.
Keywords: Lipid emulsion; Transfection; Gene therapy; DOTAP; Squalane;

Protective properties of melatonin-loaded nanoparticles against lipid peroxidation by S.R. Schaffazick; A.R. Pohlmann; C.A.S. de Cordova; T.B. Creczynski-Pasa; S.S. Guterres (209-213).
The aim of this study was to prepare melatonin-loaded nanoparticles (nanocapsules and nanospheres) by nanoprecipitation, using Eudragit S100® as polymer. The potential of these systems to protect lipids against peroxidation was evaluated in comparison to melatonin in aqueous solution and nanoemulsion. Liposomes and microsomes were used as model of a lipid membrane and lipid peroxidation was induced by free radical ascorbyl. Nanocapsule and nanosphere suspensions presented total recoveries of melatonin near 100% and associated drug around 55%. The zeta potential values were negative and the hydrodynamic diameter of particles were lower than 255 nm. The results demonstrate that the lipids were protected against peroxidation from 8 to 51% due to the presence of the melatonin and that this effect depended on the drug dose, the type of the lipid substrate and the type of colloid, in which melatonin was incorporated. Nanocapsules and nanospheres provided an important increase in the antioxidant effect of melatonin against lipid peroxidation.
Keywords: Melatonin; Nanoparticles; Lipid peroxidation;

Noticeboard (215-219).