International Journal of Pharmaceutics (v.297, #1-2)

The aim of this study was to investigate the effect of quercetin on the bioavailability of diltiazem after administering diltiazem (15 mg/kg) orally to rabbits either co-administered or pretreated with quercetin (2, 10, 20 mg/kg). The plasma concentrations of diltiazem in the rabbits pretreated with quercetin were increased significantly (p  < 0.05, at 2 mg/kg; p  < 0.01, at 10 and 20 mg/kg) compared with the control, but the plasma concentrations of diltiazem co-administered with quercetin were not significant. The areas under the plasma concentration-time curve (AUC) and the peak concentrations (C max) of the diltiazem in the rabbits pretreated with quercetin were significantly higher (p  < 0.05, at 2 mg/kg; p  < 0.01, at 10 and 20 mg/kg) than the control. The absolute bioavailability (AB%) of diltiazem in the rabbits pretreated with quercetin was significantly (p  < 0.05 at 2 mg/kg, p  < 0.01 at 10 and 20 mg/kg) higher (9.10–12.81%) than the control (4.64%). AUC, AB% and C max of diltiazem co-administered with quercetin were higher than the control, but these were not significant.The bioavailibility of diltiazem in the rabbits pretreated with quercetin is increased significantly compared with the control, but not in the rabbits co-administered with quercetin. The increased bioavailability of diltiazem in the rabbits pretreated with quercetin might have been resulted result from the quercetin, which inhibits the efflux pump P-glycoprotein and the first-pass metabolizing enzyme CYP 3A4.
Keywords: Diltiazem; Quercetin; Bioavailability; Co-administration; Pretreatment;

Model analysis of flux enhancement across hairless mouse skin induced by chemical permeation enhancers by Ning He; Kevin S. Warner; William I. Higuchi; S. Kevin Li (9-21).
Previous permeant partitioning studies with hairless mouse skin (HMS) in the presence of several chemical skin permeation enhancers have revealed that, when such enhancers induce significant skin permeability coefficient enhancement, it is accompanied by significant enhancement in the equilibrium uptake (partitioning) of the permeant into the intercellular lipid component of the stratum corneum (SC). Particularly, it was found that the 1-alkyl-2-pyrrolidones and the 1-alkyl-2-azacycloheptanones, at aqueous solution concentrations that gave skin permeation enhancement (E) of 10 for corticosterone (CS, the permeant), enhanced the equilibrium uptake of β-estradiol (E2β, a surrogate permeant) from the aqueous phase into the intercellular lipids of HMS SC by a factor of 5–7. This finding raised the question of whether this uptake enhancement induced by the permeation enhancer under equilibrium conditions would be essentially the same as that determined kinetically from time-dependent permeation experiments utilizing appropriate SC membrane models and Fick's laws of diffusion to treat the data. HMS transport experiments were conducted with CS as the permeant and 1-octyl-2-pyrrolidone (OP) and 1-hexyl-2-azacyloheptanone (HAZ) as the enhancers. In treating the experimental data, a one-layer skin transport model (SC only) and a two-layer model (SC layer and the epidermis/dermis layer) were both investigated. Both the partition coefficient enhancement (E K ) and the diffusion coefficient enhancement (E D ) were deduced from the data treatment. The results showed that when the total transport enhancement of CS was around 11, E K was in the range of 6–8 and E D was in the range of 1.5–1.9 using both the one-layer and the two-layer models. This E K value was found to be in good agreement with the E2β partition enhancement obtained directly under equilibrium conditions in previous studies. This indicates that (a) the rate-limiting domain for the transport of the lipophilic permeants across HMS and the HMS SC intercellular lipid domain probed in the equilibrium partitioning experiments are essentially the same, and (b) the total flux enhancement (E) of lipophilic permeants across HMS was driven mainly by enhancing the partitioning of the permeant into the rate-limiting domain (E K ) and secondarily by enhancing the diffusion coefficients (E D ) of the permeant in the domain. Comparison of the one-layer and two-layer skin model results revealed that non-steady-state transport of lipophilic compounds across HMS was better described by the two-layer model because the dermis/viable epidermis played a significant role in lipophilic permeant binding.
Keywords: Chemical permeation enhancers; Enhancer mechanism; Transport model; Hairless mouse skin;

Cross-linked high amylose starch derivatives for drug release by Jérôme Mulhbacher; Mircea Alexandru Mateescu (22-29).
Keywords: Diffusion; Partition coefficients; Permeability; Hydrogels; High amylose starch derivatives; Controlled release mechanisms;

Release behavior of diethylhexyl phthalate from the polyvinyl-chloride tubing used for intravenous administration and the plasticized PVC membrane by Hanawa Takehisa; Endoh Naoko; Suzuki Masahiko; Terada Katsuhide; Ohkuma Moriyuki; Sakuta Keizoh; Tanaka Mutsuko; Kawano Kenji; Nakajima Shin’ichiro; Oguchi Toshio (30-37).
The release behavior of diethylhexyl phthalate (DEHP) from polyvinyl-chloride (PVC) tubing, which composes materials in an intravenous administration set, was investigated using polysorbate 80 (Tween 80) aqueous solutions. When Tween 80 solution was circulated in PVC tubing, the amount of DEHP released increased with increasing circulation velocity and temperature. In order to clarify the effect of temperature on the release behavior of DEHP, PVC films containing varying amounts of DEHP were mounted on a cylindrical shaft and rotated at 5 and 40 °C. The cumulative amount of DEHP released increased with an increase in temperature, the diffusion coefficients [D  × 10−10  cm2  min−1] at 5 and 40 °C were 9.1 and 156.0, respectively. The glass transition temperature (T g) of PVC films decreased with an increase in DEHP in the PVC film, as measured by differential scanning calorimeter (DSC) and release of DEHP occurred at temperatures above T g. These results indicate that the release of DEHP from PVC tubing is closely associated with the state of the PVC and is related to diffusion of DEHP throughout the PVC.
Keywords: Diethylhexyl phthalate; PVC tubing; Tween 80; Release behavior; Glass transition temperature; Diffusion;

The effect of taste masking agents on in situ gelling pectin formulations for oral sustained delivery of paracetamol and ambroxol by Shozo Miyazaki; Wataru Kubo; Kunihiko Itoh; Yasuhiro Konno; Mariko Fujiwara; Masatake Dairaku; Mitsuo Togashi; Ryozo Mikami; David Attwood (38-49).
The aim of this study was to examine the influence of polyhydric alcohols (taste masking agents) on the rheological properties of in situ gelling pectin formulations and on the in vitro and in vivo release of paracetamol and ambroxol from these formulations. Gelation of orally administered pectin solutions containing calcium in complexed form occurred on release of calcium in the acidic environment of the stomach. Inclusion of 10% (w/v) sorbitol in 2% (w/v) pectin sols reduced the viscosity and ensured Newtonian flow properties. Xylitol and mannitol in similar concentrations were less effective in reducing viscosity; sucrose increased viscosity and caused non-Newtonian flow. The in vitro release of paracetamol from 2% (w/v) pectin gels formulated with 10% (w/v) of sorbitol, erythritol, xylitol or mannitol, and of ambroxol from 2% (w/v) pectin gels containing 10% (w/v) sorbitol, followed diffusion-controlled kinetics. Pectin gels (2%, w/v) containing sorbitol (10%, w/v) sustained the release of paracetamol in the rat stomach and bioavailabilities of approximately 90% of those from an orally administered paracetamol syrup were achieved. Sustained release of ambroxol from in situ gelling formulations was achieved with pectin concentrations of 1.5 and 1% (w/v) and a sorbitol concentration of 10% (w/v).
Keywords: In situ gelation; Oral drug delivery; Sustained release; Pectin gels; Taste masking agents; Paracetamol; Ambroxol;

Impurity formation studies with peptide-loaded polymeric microspheres by Santos B. Murty; B.C. Thanoo; Qui Wei; Patrick P. DeLuca (50-61).
The purpose of the present investigation was to assess the peptide related substances or impurities formed during incubation of drug loaded poly-(d,l-lactide-co-glycolide) (PLGA) and poly-(d,l-lactide) (PLA) microspheres under in vivo conditions. Sprague–Dawley rats were injected with separate batches of octreotide microspheres prepared by either an oil/water or oil/oil dispersion technique. At specified time points (days 14, 22, 30, and 41), animals were sacrificed and microsphere particles were recovered from the subcutaneous injection sites. The recovered particles were further extracted with 1:1 mixture of dimethylsulfoxide:dichloromethane for subsequent impurity analysis by HPLC and mass spectrometry. During incubation, the percentage purity of parent compound depended on the PLGA co-monomer ratio (e.g. 50:50, 85:15, and 100:0 glycolide:lactide ratios). After 41 days of incubation, for instance, octreotide area percentage by HPLC was determined to be ∼47% for PLGA 50:50 microspheres, ∼75% for PLGA 85:15 microspheres, and ∼87% for PLA microspheres. Spectral analysis of particle extracts revealed the presence of peptide related substances with 58 m/z and 72 m/z units higher than the parent peptide m/z value. This indicated the presence of glycoyl and lactoyl covalent substitutions on the drug compound, resulting from chemical interaction between peptide amine groups and PLGA or PLA ester groups.
Keywords: Somatostatin analogues; Octreotide acetate; PLA microspheres; PLGA microspheres; Peptide acylation; Peptide stability;

Impurity formation studies with peptide-loaded polymeric microspheres by Santos B. Murty; Dong Hee Na; B.C. Thanoo; Patrick P. DeLuca (62-72).
Since acylated peptide impurities were isolated from octreotide microspheres following incubation in an in vivo environment, the present investigation was undertaken to determine the dosage form dynamics responsible for facilitating acylation. In particular, microsphere batches made with poly(l-lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) 85:15 were studied for in vitro drug release, mass balance relationships, mass loss behavior, hydration uptake, and solid-state stability. Furthermore, native octreotide was incubated in a varying pH stability model (heat treated lactic acid solutions 42.5%, w/w) to determine the effects of acidity on impurity formation. From a review of the experimental results, the appearance of octreotide impurities or related substances occurred with the onset of polymeric mass loss. In fact, the significant formation of acylated peptide did not appear until >90% mass loss, which was observed at 14 days. It was surmised that because of water uptake, the hydrolytic cleavage of the polymeric backbone created an acidic microenvironment to facilitate the covalent coupling of peptide with polymer. The lactic acid solution stability model corroborated with greater evidence of acylation at pH 2.25 where the presence lactoyl (+72 m/z) derivatives of octreotide were confirmed by MALDI-TOF mass spectrometry.
Keywords: Somatostatin analogues; Octreotide acetate; PLA microspheres; PLGA microspheres; Peptide acylation; Peptide stability;

Determination of lamivudine in human plasma by HPLC and its use in bioequivalence studies by Eunice Kazue Kano; Cristina Helena dos Reis Serra; Eunice Emiko Mori Koono; Simone Schramm Andrade; Valentina Porta (73-79).
A simple, accurate, precise and sensitive high-performance liquid chromatographic (HPLC) method with ultraviolet detection was developed to quantificate lamivudine (3-TC) in human plasma samples from bioequivalence studies. 3-TC and stavudine (internal standard, I.S.) were extracted from 0.5 ml of human plasma by acetonitrile protein precipitation. The method was validated over a concentration range of 0.05–3.00 μg/ml and used in a bioequivalence trial between two lamivudine formulations, to assess its usefulness in this kind of study. FURP–lamivudine (Fundação para o Remédio Popular, Brazil, as test formulation) and Epivir® (GlaxoSmithKline, Brazil, as reference formulation) were evaluated following a single 150 mg oral dose to 24 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 determined by calculating 90% confidence intervals (90% CI) for the ratio of C max, AUC0−t and AUC0−inf values for the test and reference products, using logarithmic transformed data. The 90% confidence intervals for the ratio of C max (0.86–1.06), AUC0−t (0.96–1.04) and AUC0−inf (0.97–1.05) values for the test and reference products are within the 0.80–1.25 interval proposed by FDA and EMEA. It was concluded that the two 3-TC formulations are bioequivalent in their rate and extent of absorption, and thus, may be used interchangeably.
Keywords: Lamivudine; 3-TC; Bioequivalence; HPLC; Plasma;

Immediate release direct compression tablet formulations require a strict control of the particle characteristics (i.e. particle size (distribution) and shape) of both the active pharmaceutical ingredient (API) and the excipients. In this publication, the development of a dry dispersion laser diffraction (LD) method has been outlined. With this method, the chemical development of an API meant for the manufacturing of an immediate release direct compression tablet formulation can be supported. Comparison with static image analysis (SIA) and scanning electron microscopy (SEM) data often shows laser diffraction to generate different size data. However, since LD is fast and frequently shows an adequate precision over a wide particle size range, the technique is still considered as a valuable analytical tool in the screening of the particle size distribution of API batches. In the future, automated (static) image analysis and dynamic image analysis are believed to become more and more important, since these techniques will allow the fast analysis of large amounts of particles with a minimum intervention of the operator.
Keywords: Laser diffraction; Static image analysis; Scanning electron microscopy; Particle size distribution; Direct compression;

Asymmetric membrane capsules for delivery of poorly water-soluble drugs by osmotic effects by Chun-Yu Wang; Hsiu-O Ho; Ling-Hong Lin; Ying-Ku Lin; Ming-Thau Sheu (89-97).
A non-disintegrating polymeric capsule system, in which asymmetric membrane offers an improved osmotic effect, was used to deliver poorly water-soluble drugs in a control manner. The capsule wall membrane was made by a phase inversion process, in which asymmetric membrane was formed on stainless-steel mold pins by dipping the mold pins into a coating solution containing a polymeric material followed by dipping into a quench solution. This study evaluates the influence of coating formulation that was cellulose acetate (CA), ethylcellulose (EC), and plasticizer (glycerin and triethyl citrate). Results show capsule that made by CA with glycerin (formulation A), which appear in asymmetric structure and are able to release chlorpheniramine maleate (CM) in significant percentage. Two poorly water-soluble drugs of felodipine (FL) and nifedipine (NF) were selected as the model drug to demonstrate how the controlled release characteristics can be manipulated by the design of polymeric capsules with an asymmetric membrane and core formulations. Results show that sodium lauryl sulfate (SLS) is able to promote the release of FL from polymeric capsules prepared with CA with asymmetrical membrane. The addition of solubilizer, including RH40, PVP K-17, and PEG 4000 could enhance the release of FL but with an extent not being related to its solubility. Based on these results, influence of core formulation variables, including the viscosity and added amount of hydroxypropyl methylcellulose (HPMC), the added amount of SLS, and drug loading were examined on the release of NF. It was found that HPMC of 50 cps was suitable to be a thickening agent and both added amount of HPMC and SLS showed a comparable and profoundly positive effect, whereas NF loading had no influence on the drug release percent and rate. There existed a synergistic interaction between HPMC and SLS on the release percent and rate.
Keywords: Asymmetrical membrane; Osmotic effect; Poorly water-soluble drugs; Sodium lauryl sulfate;

Role of the mucous/glycocalyx layers in insulin permeation across the rat ileal membrane by Yoshinobu Aoki; Mariko Morishita; Kozo Takayama (98-109).
The contribution of mucous/glycocalyx layers, as a diffusional or enzymatic barrier, to the absorption of insulin was investigated in situ and in vitro studies using rats. To remove the mucous/glycocalyx layers, ileal segments were exposed to a hyaluronidase solution in situ. The removal of the layers was confirmed by transmission electron microscopy, and the safety of the hyaluronidase pretreatment was established based on light microscopy, a constant mucosal membrane electrical resistance and the absence of lactate dehydrogenase leakage. In the in situ loop absorption experiment, hyaluronidase pretreatment significantly increased the plasma insulin level accompanied by an obvious hypoglycemic response. In the in vitro transport experiment, the apparent permeability coefficient of insulin was significantly increased by the hyaluronidase pretreatment, whereas that of 4.4 kDa fluorescein isothiocyanate-labeled dextran and of antipyrine, respective markers for passive para- and transcellular permeation, was unaffected. In the insulin degradation experiment in vitro, a significant amount of insulin was degraded in the compartment removed by hyaluronidase pretreatment. Thus, the mucous/glycocalyx layers functioned in insulin absorption as an enzymatic barrier and insignificantly affected diffusive absorption. In addition, co-administration of aprotinin, a protease inhibitor, further increased insulin absorption from ileum pretreated with hyaluronidase, implying the existence of another enzymatic barrier that influences insulin mucosal absorption.
Keywords: Insulin; Hyaluronidase; Mucous; Glycocalyx; Macromolecular drug delivery; Intestinal metabolism; Aprotinin;

Development and application of high throughput plasma stability assay for drug discovery by Li Di; Edward H. Kerns; Yan Hong; Hong Chen (110-119).
Plasma stability plays an important role in drug discovery and development. Unstable compounds tend to have rapid clearance and short half-life, resulting in poor in vivo performance. This paper examines the variables that affect the plasma stability assay results, including substrate concentration, %DMSO, plasma concentration, enzyme activity upon incubation and batch variation. The results show that plasma stability can accommodate a wide range of experimental conditions. Relatively minor differences in results are produced with major differences in conditions. Significant batch-to-batch variations were observed for rat plasma. We selected the following conditions: 1 μM substrate concentration, 2.5% DMSO, and 50% dilution of plasma in pH 7.4 buffer. Plasma stability can be used as a diagnostic assay when compounds are unexpectedly rapidly cleared, as a special assay when structural classes contain groups that may be susceptible to plasma enzyme hydrolysis, or as general screen for compounds if resources are available. Plasma stability assay has many applications in drug discovery: to alert teams to labile structural motifs, to prioritize compounds for in vivo studies and to screen prodrugs and antedrugs.
Keywords: Plasma stability; Stability; Hydrolysis; Prodrugs; Enzymes; HPLC; LC–MS; High throughput;

Accumulation of extractables in buffer solutions from a polyolefin plastic container by Dennis R. Jenke; Jill M. Jene; Mitchell Poss; James Story; Tom Tsilipetros; Alex Odufu; William Terbush (120-133).
Plastic materials are widely used in medical items, such as solution containers, transfusion sets, transfer tubing and devices. An emerging trend in the biotechnology industry is the utilization of large plastic containers to prepare, transport and store an assortment of solutions including buffers, media and in-process and finished products. The direct contact of such containers with the product at one or more points in its lifetime raises the possibility that container extractables may end up in the finished product. The interaction between a polyolefin container material and several test solutions representative of buffers and media used in biopharmaceutical applications was investigated. This manuscript summarizes the identification of the major extractables associated with the polyolefin container and documents the levels to which targeted extractables accumulate in the test solutions under several storage regimes.
Keywords: Container extractables; Material/solution compatibility; Polyolefin containers;

The objective of this study was to design an all-trans retinoic acid (RA) topical release system that modifies drug diffusion parameters in the vehicle and the skin in order to reduce systemic absorption and the side-effects associated with topical application of the drug to skin. Three cases of application of hydrogels containing RA either in free form or encapsulated in stratum corneum lipid liposomes (SCLLs) have been considered. For this purpose, we have evaluated the RA in formulations with combinations of Carbopol® Ultrez™ 10 (U10) and hyaluronic acid (HA) for percutaneous absorption. In vitro permeability experiments with [3H]-t-RA were carried out using a Franz-type diffusion cell in abdominal rat skin samples. Accumulation of the drug in the surface and skin layers was evaluated by both the tape stripping method and a dissection technique, and subsequently, all the radiolabelled samples were analyzed by liquid scintillation counting. The results show that RA encapsulation not only prolongs drug release but also promotes drug retention by the viable skin. At the same time, interaction between RA and HA has an obstructive effect on diffusion, which contributes to the formation of a reservoir of the latter.
Keywords: Stratum corneum lipid liposomes (SCLLs); All-trans retinoic acid (RA); Skin permeation; Skin penetration; Drug reservoir;

Buccal penetration enhancement properties of N-trimethyl chitosan: Influence of quaternization degree on absorption of a high molecular weight molecule by Giuseppina Sandri; Silvia Rossi; Maria Cristina Bonferoni; Franca Ferrari; Ylenia Zambito; Giacomo Di Colo; Carla Caramella (146-155).
The aim was to evaluate the influence of the degree of quaternization of N-trimethyl chitosans (TMCs) on the mucoadhesive and penetration enhancement properties towards buccal mucosa. Fluorescein isothiocyanate dextran (MW 4400 Da) (FD4) was used as model molecule.TMCs, obtained from chitosans of different MW (1460 and 580 kDa, respectively), were hydrated in distilled water and in pH 6.4 phosphate buffer (simulating the buccal fluid).The polymer solutions were subjected to mucoadhesion measurements towards bovine submaxillary mucin dispersion and porcine buccal mucosa and to FD4 permeation tests through porcine cheek epithelium.The trimethylation of chitosan allows maintenance or improvement of the mucoadhesive properties of the starting chitosans dependently on quaternization degree. In particular, the mucoadhesive properties increase on increasing degree of quaternization. The trimethylation does not produce any change in chitosan penetration enhancement properties when the medium is distilled water while if pH 6.4 buffer is used, the trimethylation produces an improvement in chitosan penetration enhancing effect.TMC derived from the lower MW chitosan and characterized by the highest degree of quaternization shows the best mucoadhesive and penetration enhancement properties and is the most promising TMC to improve the bioavailability of hydrophilic and large MW molecules (like peptides and proteins) when administered via buccal route.
Keywords: Trimethyl chitosan; Quaternization degree; Mucoadhesive properties; Penetration enhancement properties;

Enhanced bioavailability of piroxicam via salt formation with ethanolamines by Hye-Sun Gwak; Jun-Shik Choi; Hoo-Kyun Choi (156-161).
Piroxicam can be ionized as a zwitterion that has two pKa values (pKa1  = 1.86 and pKa2  = 5.46). Consequently, piroxicam has a low solubility in both polar and nonpolar media, and a low lipophilicity, which results in a low permeability. Three piroxicam-ethanolamine salts were prepared, which had a higher area under the curve (AUC) than piroxicam. There were minimal differences in the AUC among the salt forms. It was reported that the piroxicam triethanolamine salt had a lower permeability across the skin than piroxicam but it had a higher oral bioavailability. Piroxicam monoethanolamine showed the highest C max followed by piroxicam diethanolamine and piroxicam triethanolamine. The dissolution rates of piroxicam and its salts were similar at pH 1.2. Piroxicam monoethanolamine showed the highest dissolution rate at pH 6.8, which was followed by the piroxicam diethanolamine and piroxicam triethanolamine salts. The order of dissolution rate at pH 6.8 matched the order of C max or the AUC after oral administration.
Keywords: Piroxicam; Ethanolamine; Salt; Dissolution; Bioavailability;

The effects of electrically assisted methods on transdermal delivery of nalbuphine benzoate and sebacoyl dinalbuphine ester from solutions and hydrogels by Jeng-Fen Huang; K.C. Sung; Oliver Yoa-Pu Hu; Jhi-Joung Wang; Yi-Hsin Lin; Jia-You Fang (162-171).
The aim of this study was to assess the effects of iontophoresis and electroporation on transdermal delivery of nalbuphine (NA) and its two novel prodrugs: nalbuphine benzoate (NAB) and sebacoyl dinalbuphine ester (SDN) from solutions as well as from hydrogels. Hydroxypropyl cellulose (HPC) and carboxymethyl cellulose (CMC) were used in hydrogel formulations to evaluate their feasibility for delivery of NA and its prodrugs. Application of iontophoresis or electroporation significantly enhanced the in vitro permeation of NA and its prodrugs. The enhancement effect was more pronounced after applying iontophoresis. The combination of two electrically assisted methods enhanced the delivery of NA; however, no such enhancement was observed for the permeation of NAB and SDN. Hydrogels containing low concentration HPC did not affect the passive as well as electrically assisted permeation of NA and its prodrugs. The increase of hydrogel concentration as well as molecular weight significantly decreased the electrically assisted permeation of NA, whereas the permeation of NAB and SDN remained unchanged. For the electrically assisted permeation from CMC-based hydrogels, the reduced permeation from higher percentage of CMC hydrogels may be attributed the viscosity effect as well as the ion competition effect. The above results demonstrated that lipophilicity and molecular size, as well as hydrogel compositions had significant effects on skin permeation of NA, NAB and SDN via passive diffusion or under the electric field.
Keywords: Nalbuphine; Prodrugs; Transdermal delivery; Iontophoresis; Electroporation; Hydrogels;

Mucoadhesive microspheres containing either amoxicillin or clarithromycin were prepared via the interpolymer complexation of poly(acrylic acid) (PAA) with poly(vinyl pyrrolidone) (PVP) and solvent diffusion method. The complexation between the PAA and PVP in an ethanol/water mixture was confirmed by the change in the transmittance of the solution as a function of repeating PAA and PVP unit ratio. The loading efficiency of clarithromycin in the complex microspheres was higher than that of amoxicillin due to the stronger interaction of clarithromycin with the PAA. The microspheres had a spherical shape with a smooth surface and the inside of the microspheres was completely filled. The dissolution rate of the complex microspheres was significantly slower than that of the PVP microspheres, particularly at pH 2.0. Amoxicillin and clarithromycin degraded significantly during the release study at pH 2.0. Therefore, their release rates were corrected using first order degradation rate constants. The amoxicillin release rates were similar regardless of the pH of the medium, while those of clarithromycin differed depending on the pH. The release mechanism of amoxicillin was mainly by a diffusion process and that of clarithromycin was via a dissolution process. The drug release rate from the complex microspheres was significantly lower than that from the PVP microspheres.
Keywords: Poly(acrylic acid); Poly(vinyl pyrrolidone); Mucoadhesive microsphere; Gastric residence time; Amoxicillin; Clarithromycin;

In vitro and in vivo methotrexate disposition in alveolar macrophages: Comparison of pharmacokinetic parameters of two formulations by Christophe Doddoli; Olivier Ghez; Fabrice Barlési; Benoit D’journo; Stéphane Robitail; Pascal Thomas; Thierry Clerc (180-189).
MTX-liposomes, prepared with a polymerised core (LSP), were administered in anaesthetised rats by pulmonary instillation versus free drug. No toxicological effects were macroscopically observed. After each time point: 15, 30, 60 and 90 min, animals were humanely killed and analyses of radio-signal were done. This approach allowed recovery of MTX or breakdown products within biological samples. Previously, kinetics of MTX cellular uptake was performed to identify the cytotoxic concentration of drug formulation for human macrophage. Flow cytometry was set-up to characterise liposomal uptake by ex vivo pulmonary macrophage. Cells were isolated by bronchioloalveolar washes from animals. Results have shown clear different pharmacokinetic parameters between free MTX and the liposomal form of MTX. Unlike classical liposomes, which are mainly taken up by the reticulo-endothelial system, LSP-MTX was not targeted to spleen or kidney. The route of administration could be an explanation of this phenomenon. In addition, LSP-MTX was more retained by the lung tissue. Moreover, free form of the drug reaches easily lymph node. This latest result should be taken into consideration for neoplasic disease and more specifically when lymph nodes are a way for pulmonary metastasis. Finally, LSP-MTX should be tested in physio-pathological model of lung cancer to evaluate the influence of the variation of liposomal formulation pharmacokinetic parameters on the drug efficacy.
Keywords: Methotrexate; Lungs; Pharmacokinetic; Macrophages;

In vivo iontophoretic delivery and pharmacokinetics of salmon calcitonin by Ayyappa Chaturvedula; Dipty P. Joshi; Carter Anderson; Russell L. Morris; Walter L. Sembrowich; Ajay K. Banga (190-196).
In vivo iontophoretic delivery of salmon calcitonin (SCT) in hairless rats using a self-contained wearable and disposable iontophoretic patch was investigated. Iontophoretic patches with built-in proprietary Zn/AgCl electrodes were used. SCT was formulated in citrate buffer (50 mM, pH 4.0) to impart a positive charge for anodal iontophoresis. SCT was delivered intravenously to determine primary pharmacokinetic parameters. Pharmacokinetics of iontophoretic delivery of SCT was compared with subcutaneous route of administration. Blood samples were collected through tail vein and analyzed for serum SCT and calcium levels. Pharmacokinetic parameters were calculated by non-compartmental analysis. An average current of 0.43 ± 0.01 mA was maintained during patch application. Iontophoretic patches delivered SCT at an average infusion rate of 177.9 ± 58.7 ng/(min kg) and an average steady state concentration of 7.58 ± 1.35 ng/ml was achieved. There was no difference between the calcium lowering effect of iontophoretic patch and subcutaneous injection (p  > 0.05). Clearance and half-life of SCT after IV administration were found to be 16.8 ± 0.9 ml/(min kg) and 33.5 ± 3.3 min, respectively. The iontophoretic delivery of SCT was well defined by a one-compartment model with zero-order infusion. Iontophoretic patch delivered therapeutically relevant concentrations of SCT in hairless rats and delivery was comparable to conventional routes.
Keywords: Salmon calcitonin; Iontophoresis; Pharmacokinetics; Calcaemia;

The effect of packaging materials on the stability of sunscreen emulsions by Maria Inês R.M. Santoro; Daniella Almança Gonçalves Da Costa E. Oliveira; Erika R.M. Kedor-Hackmann; Anil K. Singh (197-203).
The purpose of this research was to study the stability of a emulsion containing UVA, UVB and infrared sunscreens after storage in different types of packaging materials (glass and plastic flasks; plastic and metallic tubes). The samples, emulsions containing benzophenone-3 (B-3), octyl methoxycinnamate (OM) and Phycocorail®, were stored at 10, 25, 35 and 45 °C and representative samples were analyzed after 2, 7, 30, 60 and 90 days period. The stability studies were conducted by analyzing samples at pre-determined intervals by high performance liquid chromatography (HPLC) along with periodic rheological measurements.
Keywords: Cosmetic products; Emulsions; Sunscreens; Packaging materials; HPLC; Rheology;

Skin permeation enhancement by sucrose esters: A pH-dependent phenomenon by J. Cázares-Delgadillo; A. Naik; Y.N. Kalia; D. Quintanar-Guerrero; A. Ganem-Quintanar (204-212).
The purpose of the present study was to evaluate the effect of sucrose esters (particularly, sucrose laureate and sucrose oleate in Transcutol®) on the percutaneous penetration of a charged molecule as a function of ionization. We have investigated the influence of these sucrose esters on the in vitro diffusion profiles of lidocaine hydrochloride, a weak ionizable base (pK a  = 7.9), at different pH values, using porcine ear skin as the barrier membrane. As expected, lidocaine flux in the absence of an enhancer, increased from pH 5 to 9 with a corrresponding increase in the level of the unionized base. However, when skin was pretreated with 2% laureate in Transcutol (2% L-TC), drug permeation was higher at pH 5.0 and 7.0 than at 9.0. A different trend was observed in experiments with 2% oleate in Transcutol (2% O-TC), where skin flux was maximal at a more basic pH, when the degree of ionization is low. The results suggest that sucrose laureate enhances the penetration of the ionized form of the drug (12-fold greater flux relative to control), whereas sucrose oleate is more effective in promoting permeation of the unionized species. The structural properties of the sucrose esters as well as the degree of ionization of the drug are important characteristics affecting the transdermal flux of lidocaine.
Keywords: Lidocaine hydrochloride; Sucrose esters; Enhancer; Percutaneous absorption;

Cyclodextrin solubilization of the antibacterial agents triclosan and triclocarban: Formation of aggregates and higher-order complexes by Matt S. Duan; Nelson Zhao; Ína B. Össurardóttir; Thorsteinn Thorsteinsson; Thorsteinn Loftsson (213-222).
It is well known that water-soluble cyclodextrins form inclusion complexes with many lipophilic water-insoluble drugs and that such complexation frequently enhances the aqueous solubility of drugs. It is also well known that various excipients, such as water-soluble polymers, organic acids and bases and metal ions can enhance the solubilizing effects of cyclodextrins. However, it is not clear how these excipients enhance the effects. The effects of cyclodextrins, 2-hydroxypropyl-β-cyclodextrin (HPβCD) and randomly methylated β-cyclodextrin (RMβCD) on the aqueous solubility of triclosan and triclocarban were investigated. The phase-solubility profiles were all of type AP indicating formation of higher-order complexes or complex aggregates. Addition of lysine and other excipients enhanced the RMβCD solubilization of triclocarban. NMR spectroscopic studies, including 2D ROESY and 1D gROESY techniques, indicated that HPβCD and RMβCD, as well as their complexes, form aggregates of two to three cyclodextrin molecules. The critical concentration for the aggregate formation was determined to be 5.4% (w/v). Lysine, polyvinylpyrrolidone and magnesium ions formed non-inclusion complexes resulting in formation of multiple-component cyclodextrin complexes in aqueous solutions with triclocarban.
Keywords: Cyclodextrins; Inclusion complexes; Antibacterial agents;

Evaluation of Eudragit-coated chitosan microparticles as an oral immune delivery system by Mika Hori; Hiraku Onishi; Yoshiharu Machida (223-234).
Chitosan microparticles containing ovalbumin (OVA), OVA-containing chitosan microparticles (Chi-OVA), were prepared, coated with Eudragit L100 (ER), and evaluated as oral vaccine. Chi-OVA with an OVA content of 34.4% (w/w) and a mean particle size of 2.3 μm were used for experiments in vitro and in vivo. ER-coated Chi-OVA (ER-Chi-OVA) contained 3.6–20.5% (w/w) OVA and had a particle size of 47.9–161.1 μm. Chi-OVA dissolved readily in JP 14 first fluid, but not in JP 14 second fluid. The release of OVA from Chi-OVA was suppressed extensively in JP 14 second fluid. ER-Chi-OVA did not dissolve in JP 14 first fluid, and the release of OVA was suppressed greatly in JP 14 first and second fluids. OVA solution, Chi-OVA and ER-Chi-OVA (200 and 800 μg OVA/mouse) were administered to Balb/C mice twice at a 1-week interval. At 7 d after the second administration, plasma OVA-specific IgG and fecal OVA-specific IgA levels were measured. OVA-specific IgG tended to be enhanced in Chi-OVA and ER-Chi-OVA, but was the highest in OVA solution. OVA-specific IgA was induced significantly more efficiently by ER-Chi-OVA than the others. These suggested that ER-Chi-OVA should be possibly useful to induce an intestinal mucosal immune response.
Keywords: Chitosan microparticle; Oral vaccine; Ovalbumin; Eudragit L100; Immune response;

Validation of the 96 well Caco-2 cell culture model for high throughput permeability assessment of discovery compounds by Anthony M. Marino; Melissa Yarde; Hetal Patel; Saeho Chong; Praveen V. Balimane (235-241).
The use of Caco-2 cells for permeability screening of discovery compounds is quite well established and serves as the “method-of-choice” across the pharmaceutical industries worldwide. The typical permeability-screening model involves growing cells on a 12 well or 24 well transwell format. In this manuscript, we report the use of Caco-2 cells grown on 96 well transwell plates for screening of discovery compounds to assess their permeability characteristics. A set of standard compounds (marketed compounds) belonging to the various class of Biopharmaceutics Classification System (BCS) were used to assess the utility of the 96 well Caco-2 cells. Extensive validations were also performed with ∼160 Bristol-Myers Squibb (BMS) discovery compounds by comparing the permeability values in the 96 well Caco-2 cells with the in-house 24 well Caco-2 cells. Functional Caco-2 cells with intact monolayers could be established in the 96 well format using optimized seeding and culturing conditions. The permeability of BCS compounds in the 96 well format was found to be comparable to the permeability in 24 well format. Similarly, there was very good correlation (R 2  = 0.93) between the two formats for the extensive validation performed with in-house discovery compounds. The validated 96 well Caco-2 cell system presents a very attractive permeability screening tool that can perform much more efficiently than the conventional 12 well or 24 well systems while providing the same high quality permeability screening data.
Keywords: Permeability; Caco-2 cells; High throughput screening; Absorption; Transport; Drug discovery;

A novel functionalised copolymer with three polymeric components, poly(ethylene glycol)-block-poly(aspartic acid-stat-phenylalanine), PEG-P(asp-phe), was synthesised and investigated for its potential to form micelles via ionic interactions with a model water-soluble drug, diminazene aceturate. Drug-free solutions of structurally related PEG-P(asp-phe) 5:6:4 and PEG-P(asp-phe) 5:4:6 copolymers indicated polymeric aggregation into micellar-type constructs. The size of PEG-P(asp-phe) 5:6:4 micelles was found to be pH and drug content-dependent. The drug-loaded systems existed as discreet units and were fairly uniform in size and shape. More drug could be included in the PEG-P(asp-phe) 5:6:4 micelles as compared to if only interaction with carboxyl groups from aspartic acid units was responsible for micelle formation, indicating the augmentative role of phenylalanine moieties in drug-incorporation. The slower in vitro drug release from PEG-P(asp-phe) 5:6:4 micelles as compared to PEG-Pasp (AB) micelles indicated the role of the phenylalanine moiety in controlling drug release. This study, therefore, confirmed the potential of a novel tri-component copolymer structure, PEG-P(asp-phe), for the formation of polyionic micelles for drug delivery.
Keywords: Drug–polymer interactions; Polyionic micelles; Drug delivery; Block copolymers;

Noticeboard (254-257).