International Journal of Pharmaceutics (v.299, #1-2)

New frontiers in proteomics research: A perspective by Vikas Dhingra; Mukta Gupta; Tracy Andacht; Zhen F. Fu (1-18).
Substantial advances have been made in the fundamental understanding of human biology, ranging from DNA structure to identification of diseases associated with genetic abnormalities. Genome sequence information is becoming available in unprecedented amounts. The absence of a direct functional correlation between gene transcripts and their corresponding proteins, however, represents a significant roadblock for improving the efficiency of biological discoveries. The success of proteomics depends on the ability to identify and analyze protein products in a cell or tissue and, this is reliant on the application of several key technologies. Proteomics is in its exponential growth phase. Two-dimensional electrophoresis complemented with mass spectrometry provides a global view of the state of the proteins from the sample. Proteins identification is a requirement to understand their functional diversity. Subtle difference in protein structure and function can contribute to complexity and diversity of life. This review focuses on the progress and the applications of proteomics science with special reference to integration of the evolving technologies involved to address biological questions.
Keywords: Proteomics; Two-dimensional electrophoresis; Mass spectrometry; Post-translational modifications; Bioinformatics; Proteins;

Experiments were conducted to elucidate the relationship between risedronate sodium (RS) hydration state and the physical stability of tablets containing RS. The RS crystal lattice contains channels occupied by water, which is removed by drying processes at temperatures below the boiling point of water, causing a reversible contraction of the crystal lattice. In this study, risedronate sodium was wet granulated followed by fluid bed drying to final granulation moisture contents between 1 and 7%, and then compressed into tablets. During drying, the RS solid-state form was continuously monitored using on-line Raman spectroscopy. Raman spectra acquired in these experiments enabled direct monitoring of changes in the RS crystal lattice, due to dehydration, which provided key information relating RS solid-state form characteristics to final granulation moisture content. Final granulation moisture was found to have a significant effect on the change in RS hydration state measured by Raman spectroscopy. As the final granulation moisture decreased, the amount of RS dehydrated form increased. The largest Raman spectral changes were in the C―H stretching region and the region including the 3-picoline ring and PO2 stretches. These changes are indicative of substantial changes in the RS solid-state structure. Final granulation moisture also had a significant effect on the change in tablet thickness over time. Lower final granulation moisture caused a greater increase in tablet thickness as the RS rehydrated. In addition, the change in RS hydration state during fluid bed drying, measured by on-line Raman, was correlated to the increase in tablet thickness and subsequent loss of tablet integrity. Raman spectroscopy allowed direct RS hydration state monitoring, rather than inference from a bulk moisture measurement. Development of a Process Analytical Technology (PAT), specifically Raman, to monitor RS solid-state during drying enabled establishment of relationships between fundamental hydration dynamics associated with RS and final product performance attributes.
Keywords: Raman; On-line spectroscopy; Hydration state; Physical stability; Fluid bed drying;

Kinetic analysis of protein production after DNA transfection by Yuma Yamada; Hiroyuki Kamiya; Hideyoshi Harashima (34-40).
The production of an exogenous protein by the transfection of a plasmid DNA encoding the protein was kinetically analyzed, to determine the efficiency of the transfection. Cultured NIH3T3 or HeLa cells, and the luciferase protein were used as a model system in this experiment. The findings indicate that at least a 8 × 104- and 4 × 103-fold molar amounts of luciferase protein was produced from one copy of the plasmid DNA molecule in NIH3T3 and HeLa cells, respectively. The rate of elimination of luciferase activity upon DNA transfection was smaller than that for the luciferase protein itself (k el for DNA transfection <  k el for the luciferase protein), suggesting that a decrease in intranuclear active DNA was the main determinant of the elimination rate in this case. A preliminary pharmacokinetic model is proposed, based on the results obtained.
Keywords: DNA transfection; AUC; Intranuclear disposition;

In vitro percutaneous absorption studies and in vivo evaluation of anti-inflammatory activity of essential fatty acids (EFA) from fish oil extracts by Carmelo Puglia; Salvatore Tropea; Luisa Rizza; Natale Alfredo Santagati; Francesco Bonina (41-48).
The aim of the present study was to evaluate the in vitro percutaneous absorption and the in vivo anti-inflammatory activity of EPA and DHA fatty acids from three oily extracts, obtained by acetonic extractions from the entrails of different varieties of Mediterranean fishes such as mackerel (Scomber scombrus), sardine (Sardina pilchardus) and horse mackerel (Trachurus mediterraneus).In the first part of our research, we focused our attention on the characterization of the oily extracts to determine their ω-3 polyunsaturated fatty acid content, then, we evaluated the in vitro percutaneous absorption through excised human skin (stratum corneum/epidermis membranes; SCE) of EPA and DHA contained in the extracts. In the second part, the fish oil which guaranteed the best in vitro permeation profile of these ω-3 fatty acids was studied in order to evaluate its inhibiting ability towards the in vivo UVB-induced skin erythema.From the results obtained, all the fish oils tested in this study presented significant amounts of ω-3 fatty acids EPA and DHA, and particularly sardine oil extract showed higher concentrations of these substances compared to the other two fish oils. The in vitro experiments revealed interesting fluxes of these compounds from sardine extract through the stratum corneum/epidermis membranes and an appreciable anti-inflammatory activity against UVB-induced erythema in human volunteers was also observed.
Keywords: EPA; DHA; Anti-inflammatory activity; In vitro and in vivo studies; Skin;

The study aimed to demonstrate satisfactory inter-UK transit of cold storage clinical trial material. The product environment had to be maintained between 0 and 8 °C throughout transit until delivery. Straightforward, low cost and simplified shipping arrangements were sought that would be appropriate for small-scale Phase I clinical trial activities. A laboratory test defined an optimal three frozen gel pack configuration to maintain refrigerated environmental conditions for dummy product packs in a single type and size of insulated shipper. The internal environment was temperature monitored at 30-min intervals in all tests. Twelve Glasgow to London transits were then studied over 2 years to include all seasonal temperature variations. A configuration using three frozen gel packs and 4 h pre-chill of the transit container maintained the internal environment at 0–8 °C for up to 48 h during autumn, winter and spring. A modified four frozen gel pack configuration was suitable for summer transit. Thus cold shipment verification was successfully carried out for a small-scale distribution operation. It was proven that refrigerated shipping conditions could be maintained using a straightforward and cost effective ‘passive’ type system consisting of frozen gel packs and insulated transit containers.
Keywords: Phase I; Cold chain; Distribution;

A polyglycolised glyceride carrier, Gelucire 50/13, was incorporated with paracetamol as a model drug, filled into hard gelatin capsules and stored at three different temperatures for various lengths of time. The resultant solidified matrix within the capsule was subjected to thermal analysis using differential scanning calorimetry (DSC) to ascertain its supramolecular structure. Polymorphic transformations towards more stable gelucire forms were observed upon aging the matrices, with samples stored at a temperature near the melting range of the lower temperature gelucire melting fraction showing the most profound changes. The increase in the rate of drug release from aged samples could be correlated to the alterations to the supramolecular structure of the gelucire. Accelerated drug release from aged samples could also be seen from in vivo studies using healthy human volunteers, although the extent of absorption was not affected. Therefore, even though the sustainability of release may be compromised by aging the gelucire matrices, the bioavailability of the incorporated drug is unlikely to be affected.
Keywords: Lipid matrices; Gelucire®; Aging; In vivo; Sustained release; DSC;

Comparative study and optimisation of the administration mode of three proton pump inhibitors by nasogastric tube by D. Messaouik; V. Sautou-Miranda; S. Bagel-Boithias; J. Chopineau (65-72).
Patients in intensive care often develop stress-induced ulcers. As a preventive measure, proton pump inhibitors (PPIs) are administered by nasogastric tube. However, some PPIs can block the tube. The aim of this study was to compare the behaviour of three PPIs (omeprazole, lanzoprazole and esomeprazole) during the transit of the granules through the tube and to optimise their modes of administration. For each IPP, the experiment was designed to study the influence of four variables: the tube material (silicone or polyurethane), the solvent used to dilute the granules (water or apple juice), the mode of administration (in two or three doses) and the rinse volume (10 or 20 ml). We counted the granules before transit and at the tube outlet, and assayed the active drug ingredient by UV spectrometry. The assay showed complete transit of esomeprazole through the tube, but average losses of omeprazole and lanzoprazole of 39 and 33%, respectively, were observed. No significant improvement was obtained by the variables ‘diluent’ and ‘mode of administration’. The variable ‘rinse’ had a significant influence. For lanzoprazole, a polyurethane tube allowed recovery of on average 86% of the active ingredient. Esomeprazole is thus the choice PPI for the treatment of patients by nasogastric tube. Using a polyurethane tube and a rinse volume of 20 ml, the administration of lanzoprazole by tube can be considered. Use of omeprazole is not recommended because none of the modes of administration tested ensured that a sufficient concentration of active ingredient reached the stomach.
Keywords: Omeprazole; Lansoprazole; Esomeprazole; Nasogastric tube; Granule;

A comparison of chemical reference materials for solution calorimeters by Rita Ramos; Simon Gaisford; Graham Buckton; Paul G. Royall; Barbara T.S. Yff; Michael A.A. O’Neill (73-83).
Solution calorimeters are based on semi-adiabatic or isothermal heat-conduction principles and differ in the way they record data. They also have different measuring sensitivities and require different quantities of solute and solvent. As such, the choice of chemical test substance is not straightforward. Usually the dilution of KCl is recommended; it is possible to purchase a reference sample of KCl that has a certified enthalpy of solution and this standard material is usually used to test semi-adiabatic instruments. Here, we review the suitability of a range of chemical test substances (KCl, sucrose and Tris) for an isothermal heat-conduction solution calorimeter. It was found that KCl was not the best test material because its relatively high enthalpy of solution (Δsol H) necessitated the use of small samples (2 mg), resulting in a relatively large standard deviation (σ n  − 1) in the values recorded (Δsol H  = 17.14 ± 0.49 kJ mol−1); furthermore, KCl data must be corrected to account for the effect of dilution, although the correction was found to be small (0.07 kJ mol−1) under the experimental conditions employed here. Sucrose appears to be a much more robust test material for isothermal heat-conduction instruments because its lower enthalpy of solution allows the use of much larger samples (20 mg), which minimises experimental errors. The Δsol H value returned (6.14 ± 0.08 kJ mol−1) is in excellent agreement with the literature. It is also cheap, readily available and requires minimal preparation although its widespread use would require the preparation of a certified reference sample.
Keywords: Solution calorimetry; Calibration; Chemical test material; Potassium chloride; Sucrose; Tris;

ZK 811 752, a potent candidate for the treatment of autoimmune diseases, demonstrated pH-dependent solubility. The resulting release from conventional mini matrix tablets decreased with increasing pH-values of the dissolution medium. The aim of this study was to overcome this problem and to achieve pH-independent drug release. Mini matrix tablets were prepared by direct compression of drug, matrix former (polyvinylacetate/polyvinylpyrrolidone; Kollidon® SR) and excipients (lactose, calcium phosphate or maize starch). To solve the problem of pH-dependent solubility fumaric acid was added to the drug–polymer excipient system. The addition of fumaric acid was found to maintain low pH-values within the mini tablets during release of ZK 811 752 in phosphate buffer pH 6.8. Thus, micro environmental conditions for the dissolution of the weakly basic drug were kept constant and drug release was demonstrated to be pH-independent. Incorporation of water-soluble (lactose) or highly swellable (maize starch) excipients accelerated drug release in a more pronounced manner compared to the water-insoluble excipient calcium phosphate. Stability studies demonstrated no degradation of the drug substance and reproducible drug release patterns for mini matrix tablets stored at 25 °C/60% RH and 30 °C/70% RH for up to 6 months.
Keywords: pH-Independence; Weakly basic drug; Controlled release; Fumaric acid; Mini matrix tablets;

pH-dependent association of SN-38 with lipid bilayers of a novel liposomal formulation by Viktor Peikov; Sydney Ugwu; Manjeet Parmar; Allen Zhang; Imran Ahmad (92-99).
The aim of this study was to determine the location of SN-38 molecules in a liposomal formulation as a function of pH. Steady-state fluorescence polarization anisotropy and gel filtration studies of blank (placebo) liposomes, liposomes containing SN-38 and SN-38 solutions (in some cases suspensions) were conducted before lyophilization and after re-hydration at different pH conditions. SN-38, l-(4-trimethylammoniumphenyl)-6-phenyl-l,3,5-hexatriene p-toluenesulfonate (TMA–DPH), N-((4-(6-phenyl-l,3,5-hexatrienyl)phenyl)propyl)trimethylammonium p-toluenesulfonate (TMAP–DPH) and l,6-diphenyl-l,3,5-hexatriene (DPH) were used as fluoroprobes in the polarization anisotropy measurements. The localization of SN-38 was governed by the degree of hydrophobicity of the drug molecules. At high pH, SN-38 is in its inactive, hydrophilic form and partitioned into the water phase of the liposome suspensions. In lyophilized LE-SN38 liposomes re-hydrated with low pH buffer, SN-38 was found at the water–lipid interface of the bilayer.
Keywords: SN-38; Liposomes; Lyophilization; Bilayer; Interaction; Fluorescence; Anisotropy;

Self-association properties of CS-088, an antiglaucoma ophthalmic agent, were investigated. Various analytical methods, such as surface tension measurement, demonstrated that CS-088 is a self-associating compound with critical micellar concentration (CMC) of approximately 10 mg/mL. Light scattering analysis revealed that the micellar molecular weight (MMW) of CS-088 aggregates well above the CMC was approximately 2260, corresponding to a pentamer. In addition, the MMW corresponding to a dimer was detected by NMR spectroscopy, indicating that self-association of monomers to pentamers is via the formation of dimers. According to the Stokes–Einstein equation, hydrodynamic radii of the dimer and pentamer were calculated to be 0.87 and 1.16 nm, respectively. The concentration-dependent change in the NMR chemical shift indicated that hydrophobic interaction between biphenyl groups is an important factor in the self-association of CS-088 molecules. Furthermore, measurement of particle size distribution using a Nicomp Submicron Particle-Sizer revealed that the addition of either n-propanol or urea to CS-088 solution led to monomerization of the dimers and pentamers, suggesting that not only hydrophobic interaction but also hydrogen bonding is involved in stabilizing CS-088 aggregates. No bigger aggregate than a pentamer was formed in the absence of NaCl, whereas further aggregation was observed with increasing concentrations of NaCl.
Keywords: CS-088; Self-association; Light-scattering; Particle sizing; Aggregation number; NMR;

This study was conducted to clarify the penetration properties of 4-[1-hydroxy-1-methylethyl]-2-propyl-1-[4-[2-[tetrazole-5-yl]phenyl]phenyl]methylimidazole-5-carboxylic acid monohydrate (CS-088), an ophthalmic agent, and the mechanism of the permeability-enhancing effect of EDTA and boric acid (EDTA/boric acid) on the corneal penetration of CS-088. In the absence of additives, corneal permeability decreased with increasing concentration of CS-088 as CS-088 monomers self-associate to form dimers. Presence of EDTA/boric acid caused no significant changes in the physicochemical properties of CS-088, the apparent partition coefficient or the mean particle size of CS-088. EDTA/boric acid induced only a slight change in the zeta potential of liposomes used as a model of the biological membrane.On the other hand, EDTA/boric acid significantly increased membrane fluidity of liposomes, whereas other buffering agents tested did not. This effect was synergistic and concentration-dependent for both EDTA and boric acid as was observed in in vitro corneal penetration of CS-088. In accordance with the result, the rate of CS-088 permeation into the liposomes significantly increased by the addition of EDTA/boric acid. Therefore, it was demonstrated that EDTA/boric acid promotes corneal penetration of CS-088 through the transcellular pathway by increasing membrane fluidity. Conversely, other buffering agents decreased corneal permeability of CS-088 by inducing further self-association of CS-088 aggregates.
Keywords: CS-088; Penetration enhancer; EDTA; Boric acid; Membrane fluidity; Ophthalmology;

This work presents an evaluation of the performance of jet spouted bed with inert particles for production of dried extracts of Maytenus ilicifolia leaves. The development of the extraction procedure was carried-out with the aid of three factors and three levels Box–Behnken design. The effects of the extraction variables, temperature (T ext); stirring time (θ); and the ratio of the plant to solvent mass (m p/m s) on the extraction yield were investigated. The drying performance and product properties were evaluated through the measurement of the product size distribution, loss on drying (U p), flavonoid degradation (D) and, process thermal efficiency (η). These parameters were measured as a function of the inlet temperature of the spouting gas (T gi), the feed mass flow rate of the concentrated extract relative to mass flow rate of the spouting gas (W s/W g), the ratio between the feed flow rate of spouting gas relative to feed flow rate at a minimum spouting condition (Q/Q ms) and the static bed height (H 0). A powder product with a low degradation of active substances and good physical properties were obtained for selected operating conditions. These results indicate the feasibility of this drying equipment for the production of dried extracts of M. ilicifolia Martius ex Reiss leaves.
Keywords: Drying; Jet spouted bed; Dried extract; Maytenus ilicifolia; Medicinal plants; Experimental design;

Controlled release of furosemide from the ethylene-vinyl acetate matrix by Cheong-Weon Cho; Jun-Shik Choi; Sang-Chul Shin (127-133).
The ethylene-vinyl acetate (EVA) matrix containing furosemide was prepared by the casting method and the release patterns were observed. The solubility of furosemide was determined as a function of volume fraction of polyethylene glycol 400. The release of drug from the matrix was studied as a function of temperature and drug concentration. Plasticizers such as the citrates and the phthalates were added for preparing the membrane to increase the flexibility of the EVA matrix. The solubility of furosemide was the highest when the concentration of PEG 400 was 40% (v/v). The release rate of drug from the EVA matrix increased with increasing temperature and drug loading doses. A linear relationship was found between the release rate and the square root of the loading dose. The activation energy (E a), which was measured from the slope of the log  P versus 1000/T plots, was 12.33 kcal/mol for the 0.5% loading dose, and 11.58 kcal/mol for the 1.0% loading dose, and 11.00 kcal/mol for the 1.5% loading dose. Among the plasticizers used such as the citrates and the phthalates groups, diethyl phthalate showed the best enhancing effects in drug release. In conclusion, the application of an EVA matrix containing a plasticizer might be useful in the development of a controlled drug delivery system.
Keywords: Furosemide; Ethylene-vinyl acetate; Controlled release; Plasticizer; Matrix;

Cross-linking of dried paracetamol alginate granules by D. Mukhopadhyay; M. Reid; D. Saville; I.G. Tucker (134-145).
This paper reports on the cross-linking of dried (<5% moisture) paracetamol alginate granules with calcium chloride solutions. The effect of calcium concentration, temperature of the treatment solution, stirring speed and time used during cross-linking of granules on water uptake by the granules during cross-linking and physical properties of the cross-linked and dried granules were studied. A full factorial study of these factors each at two levels was used (CaCl2·2H2O: 20, 100 mg/ml; temperature: 25, 45° C; stirrer speed: 25, 240 rpm; time: 1.5 and 5.5 min) to treat dried stock granules (size: 0.8–1.0 mm) containing the model drug paracetamol and sodium alginate powder (1:1) which were prepared using conventional aqueous granulation under low shear. In addition to SEM and photomicrography, the physical properties studied were water uptake during cross-linking, yield, aggregation behaviour, moisture content, drug content, early stage drug release [over 10 s (R10) and the next 50 s (R50)] and calcium and sodium content of the unwashed cross-linked granules. Dry granules were successfully cross-linked. The treatment factors significantly affected most of the response variables. The variables most affected were water uptake (78–254%), drug entrapment (58–86%), early release (R10: 1.2–6.4% and R50: 3.0–12.2%), granule aggregation (0–70%), calcium (6.02–12.4%) and sodium content (1.2–6.44%). SEM photographs suggest that low calcium treated granules were less porous in nature compared to high calcium treated granules. Low shear drug alginate granules can be cross-linked in dried state. The properties of the cross-linked granules can be modified by altering the treatment process.
Keywords: Factorial study; Treatment conditions; Reduced swollen state early stage drug release;

Antioxidant activity of gamma-oryzanol: Mechanism of action and its effect on oxidative stability of pharmaceutical oils by Claudia Juliano; Massimo Cossu; Maria Cristina Alamanni; Luisella Piu (146-154).
Gamma-oryzanol, a phytosteryl ferulate mixture extracted from rice bran oil, has a wide spectrum of biological activities; in particular, it has antioxidant properties and is often used in cosmetic formulations as a sunscreen. The first objective of the present investigation was to elucidate the molecular mechanism(s) of the antioxidant activity of gamma-oryzanol by utilising different in vitro model systems, such as scavenging of stable DPPH• radical, OH• and O2 radicals scavenging, and azocompound AMVN-initiated lipid peroxidation. The effect of gamma-oryzanol on the oxidative stability of vegetable oils of pharmaceutical and cosmetic interest was then evaluated in a oxidation accelerate test and compared with the effect of the well-known antioxidants BHA and BHT. Our results demonstrate that gamma-oryzanol is an organic radical scavenger able to prevent AMVN-triggered lipoperoxidation. Moreover, when added to oils at concentrations ranging between 2.5 and 10 mmol/kg, gamma-oryzanol shows a dose-dependent increase of the induction times; in particular, it improved the oxidative stability of oils very prone to lipoperoxidation because of their high content of polyunsaturated fatty acids. On the ground of our results, we can conclude that gamma-oryzanol may have a potential application for the stabilization of lipidic raw materials.
Keywords: Gamma-oryzanol; Antioxidant activity; Oxidative stability; Pharmaceutical and cosmetic oils;

Preparation of coated nanoparticles for a new mucosal vaccine delivery system by Olga Borges; Gerrit Borchard; J. Coos Verhoef; Adriano de Sousa; Hans E. Junginger (155-166).
It has been found that the adsorption of antigens onto chitosan particles is an easy and unique mild loading process suitable to be used with vaccines. In order to increase the stability of this particles and to prevent an immediate desorption in gastrointestinal fluids, a coating process with sodium alginate was developed. One of the challenges of this developing process was to keep the particles in the nanosized range in order to be taken up by M-cells of the Peyer's patches. The observed inversion of the particles’ zeta potential values after coating suggested the presence of an alginate coating layer. These results were confirmed by FTIR and DSC techniques. Additionally, in vitro release studies showed that the presence of the alginate layer around the particles was able to prevent a burst release of loaded ovalbumin and to improve the stability of the nanoparticles in simulated intestinal fluid at 37 °C. The optimisation of the coating process resulted in 35% (w/w) for the loading capacity of the coated particles. SEM investigations confirmed a suitable size of the coated nanoparticles for the uptake by M-cells.
Keywords: Chitosan; Sodium alginate; Ovalbumin adsorption; Coated nanoparticles; Mucosal vaccination;

Poorly water-soluble drugs such as nifedipine (NIF) (∼20 μg/ml) offer challenging problems in drug formulation as poor solubility is generally associated to poor dissolution characteristics and thus to poor oral bioavailability. In order to enhance these characteristics, preparation of nifedipine nanoparticles has been achieved using high pressure homogenization. The homogenization procedure has first been optimized in regard to particle size and size distribution. Nanoparticles were characterized in terms of size, morphology and redispersion characteristics following water-removal. Saturation solubility and dissolution characteristics were investigated and compared to the un-milled commercial NIF to verify the theoretical hypothesis on the benefit of increased surface area. Crystalline state evaluation before and following particle size reduction was also conducted through differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD) to denote eventual transformation to amorphous state during the homogenization process. Through this study, it has been shown that initial crystalline state is maintained following particle size reduction and that the dissolution characteristics of nifedipine nanoparticles were significantly increased in regards to the commercial product. The method being simple and easily scaled up, this approach should have a general applicability to many poorly water-soluble drug entities.
Keywords: Nanoparticles; Drugs; High pressure homogenization; Dissolution; Crystalline state; Nifedipine;

Noticeboard (179-181).