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1.  Stabilized dynorphin derivatives for modulating antinociceptive activity in morphine tolerant rats: Effect of different routes of administration 
The AAPS Journal  2004;6(4):68-73.
Dynorphins, such as dynorphin A(1–13) (Dyn A(1–13)), have been shown to enhance analgesia in morphine-tolerant animals, despite their very short half-life after intravenous administration. The potential use of dynorphins in humans is therefore of interest. This laboratory has recently evaluated the metabolic fate of stabilized dynorphin derivatives. This study was conducted to evaluate whether such stabilized derivatives, ie, [N-Met-Tyr1]-Dynorphin A(1–13) (N-MT Dyn A, stabilized at the N-terminal end) and [N-Met-Tyr1]-Dynorphin A(1–13) amide (N-MT Dyn A amide, stabilized at the C-and N-terminal ends), would enhance the antinociceptive activity of morphine not only after intravenous administration but also after subcutaneous and pulmonary delivery. Intravenous administration of N-MT Dyn A (5 μmol/kg) and N-MT Dyn A amide (5 μmol/kg) to morphine-tolerant rats resulted in significantly higher tail-flick latencies than those observed for the saline group. These effects could be observed for up to 2.0±0.1 hours after intravenous administration of N-MT Dyn A and for up to 3.4±1.4 hours for N-MT Dyn A amide. The time-averaged effects of both peptides were similar. After pulmonary delivery of the same dose, derivatives remained active. The duration of the effects after pulmonary administration of the amide was 4.4±2.5 hours while that of N-MT Dyn A was slightly shorter (2.8±0.9 hours). No effect was observed after subcutaneous administration of N-MT Dyn A. These results suggest that pulmonary delivery of stabilized dynorphin derivatives represents a possible alternative to intravenous administration.
doi:10.1208/aapsj060436
PMCID: PMC2751232  PMID: 18465259
dynorphin derivatives; morphine; pulmonary delivery; tail-flick test; suppression of tolerance
2.  Fourier transform infrared spectroscopy for the analysis of neutralizer-carbomer and surfactant-carbomer interactions in aqueous, hydroalcoholic, and anhydrous gel formulations 
The AAPS Journal  2004;6(4):61-67.
The objective of the present study is to evaluate the polymer-surfactant and polymer-neutralizer interactions in topical aqueous, anhydrous, and hydroalcoholic gel formulations using Fourier transform infrared (FTIR) spectroscopy. The gels were prepared by dispersing Carbomer (Carbopol 980) in water and ethanol for aqueous and anhydrous systems, respectively. Glycerol and propylene glycol were also added to ensure that the compositions of gels closely resembled those used in typical topical gel formulations. Comparisons of the spectra of Carbopol dispersions in aqueous, anhydrous, and hydroalcoholic systems, performed for the first time, show Carbopol-neutralizer and Carbopol-surfactant interactions vary depending on the nature of the solvents used for gel formation. Analysis of the spectra of aqueous gel formulations indicates significant presence of ionized carboxyl groups only at higher pH (∼8.0). Drying of the aqueous gels causes a shift in the carbonyl stretch band toward higher energy, suggesting changes in polymer-neutralizer interaction. Anhydrous gels exhibit 2 different carbonyl stretch bands: the one at ∼1653 cm−1 is related to the carboxyl group that is hydrogen bonded and is akin to hydrous gels; the second one at ∼1717 cm−1 is indicative of free carbonyl groups. The carbonyl bands of dried gels appear at different energy levels than the solvated gels. This shift resulting from solvent evaporation, reported for the first time, indicates changes in hydrogen bond characteristics. The results show that FTIR can be a good technique compared with other more time-consuming means of analysis for topical formulations.
doi:10.1208/aapsj060435
PMCID: PMC2751231  PMID: 15760100
FTIR; spectroscopy; neutralizer; surfactant; Carbopol; Carbomer; topical gel
3.  Selective and validated spectrophotometric methods for the determination of nicorandil in pharmaceutical formulations 
The AAPS Journal  2004;6(4):53-60.
Two simple and sensitive validated spectrophotometric methods have been described for the assay of nicorandil in drug formulations. Method A is based on the reaction of the drug with phloroglucinol-sulfanilic acid reagent in sulfuric acid medium to give yellow-colored product, which absorbs maximally at 425 nm. Method B uses the oxidative coupling of 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) with DL-3,4-dihydroxyphenylalanine (DL-dopa) in the presence of nicorandil as oxidant in sulfuric acid medium to form an intensely colored product having maximum absorbance at 530 nm. Beer's law is obeyed in the concentration range 2.5 to 50.0 and 1.0 to 15.0 μg mL−1 with methods A and B, respectively. Both methods have been successfully applied for the analysis of drug in pharmaceutical formulations. The reliability and the performance of the proposed methods are established by point and interval hypothesis and through recovery studies. The experimental true bias of all samples is smaller than ±2%.
doi:10.1208/aapsj060434
PMCID: PMC2751230  PMID: 15760099
nicorandil; phloroglucinol; DL-dopa; 3-methyl-2-benzothiazolinone hydrazone hydrochloride; pharmaceutical formulations; validation parameters
4.  A novel method for the determination of biliary clearance in humans 
The AAPS Journal  2004;6(4):45-52.
Biliary excretion is an important route of elimination and the biliary tract is a potential site of toxicity for many drugs and xenobiotics. Quantification of biliary excretion in healthy human volunteers is logistically challenging and is rarely defined during drug development. The current study uses a novel oroenteric tube coupled with a specialized clinical protocol to examine the pharmacokinetics of99mTechnetium (Tc-99m) mebrofenin, a compound that undergoes rapid hepatic uptake and extensive biliary excretion. A custommade multilumen oroenteric tube was positioned in the duodenum of healthy human volunteers. Subjects were positioned under a gamma camera and 2.5 mCi of Tc-99m mebrofenin was administered intravenously. Duodenal aspirates, blood samples, and urine were collected periodically for 3 hours. Two hours after Tc-99m mebrofenin administration, the gallbladder was contracted with an intravenous infusion of cholecystokinin-8. Gamma scintigraphy was used to determine the gallbladder ejection fraction in each subject. Total systemic clearance of Tc-99m mebrofenin approximated liver blood flow (Cltotal 17.3±1.7 mL/min/kg), and 35% to 84% of the Tc-99m mebrofenin dose was recovered in bile. However, when the data were corrected for the gallbladder ejection fraction, 71% to 92% of theexcreted Tc-99m mebrofenin dose was recovered. This novel croenteric tube and clinical protocol provide a useful method to quantify biliary excretion of xenobiotics in healthy human volunteers.
doi:10.1208/aapsj060433
PMCID: PMC2751229  PMID: 18465257
oroenteric tube; gallbladder; Tc-99m mebrofenin; biliary excretion; biliary clearance
5.  The potential use of raman mapping to investigate in vitro deposition of combination pressurized metered-dose inhalers 
The AAPS Journal  2004;6(4):41-44.
Scanning near-infrared Raman microscopy has been used to map aerosol particulate deposits produced from pressurized metered-dose inhalers (pMDI). A commercially available combination asthma therapy pMDI (Ventide, Allen and Hanbury, UK), containing salbutamol and beclometasone dipropionate, was analyzed by conventional in vitro quantitative analysis and scanning Raman microscopy. Raman maps, taken from Andersen cascade impactor plate stages 3 and 5 (over 100 × 100 μm areas) suggested good correlation with chemical analysis of the respective stages. Scanning Raman microscopy allows visual differentiation between formulation components (not possible using conventional imaging techniques), while potentially allowing chemical quantification.
doi:10.1208/aapsj060432
PMCID: PMC2751228  PMID: 15760097
pMDIs; combination therapy; Raman mapping; SEM; Andersen Cascade Impactor
6.  Regional permeability of salmon calcitonin in isolated rat gastrointestinal tracts: Transport mechanism using Caco-2 cell monolayer 
The AAPS Journal  2004;6(4):36-40.
The objective of the study was to determine the region of maximum permeation of salmon calcitonin (sCT) through the gastrointestinal tract and to investigate the mechanism of permeation. For regional permeability determination, male Sprague-Dawley rats (250–300 g) were anesthetized and the gastrointestinal tissues were isolated. Stomach, duodenum, jejunum, ileum, or colon tissues were mounted on Navicyte side-by-side diffusion apparatus. Salmon calcitonin solutions (50 μM in phosphate-buffered saline, pH 7.4, 37°C) were added to the donor side, and the samples were removed from the receiver compartment and analyzed by competitive radioimmunoassay (RIA). For mechanistic studies, Caco-2 cells were grown on Transwell inserts (0.4-μm pore size, 0.33 cm2 area) in a humidified 37°C incubator (with 5% CO2). Transport experiments were conducted for sCT solutions (50 μM in Dulbecco's modified eagle's medium [DMEM], pH 7.4) from the apical-to-basolateral (A-to-B) direction and B-to-A direction at 37°C and from the A-to-B direction at 4°C. Cell monolayer integrity was monitored by mannitol permeability and transepithelial electrical resistance (TEER) measurements. The permeability coefficients (× 10−9, cm/sec) for sCT through rat stomach, duodenum, jejunum, ileum, and colon tissues were 0.482±0.086, 0.718±0.025, 0.830±0.053, 1.537±0.32, and 0.934±0.15, respectively. The region of maximum sCT permeability is ileum followed by colon, jejunum, duodenum, and stomach. The permeability coefficients (× 10−6, cm/sec) for sCT through Caco-2 cell monolayer were 8.57±2.34 (A-to-B, 37°C), 8.01±1.22 (A-to-B, 4°C), and 6.15±1.97 (B-to-A, 37°C). The mechanism of its permeation is passive diffusion through the mucosa as determined from the Caco-2 monolayer permeability of sCT.
doi:10.1208/aapsj060431
PMCID: PMC2751227  PMID: 15760096
salmon calcitonin; regional permeability; stomach; duodenum; jejunum; ileum; colon; Caco-2
7.  The role of halogen substitution in classical cannabinoids: A CB1 pharmacophore model 
The AAPS Journal  2004;6(4):23-35.
The presence of halogens within the classical cannabinoid structure leads to large variations in the compounds' potencies and affinities for the CB1 receptors. To explore the structure activity relationships within this class of analogs we have used a series of halogen-substituted (−)-Δ8-tetrahydrocannabinol analogs and compared their affinities for the CB1 cannabinoid receptor. Our results indicate that halogen substitution at the end-carbon of the side chain leads to an enhancement in affinity with the bulkier halogens (Br, I) producing the largest effects. Conversely, 2-iodo substitution on the phenolic ring leads to a 2-fold reduction in affinity while iodo-substitution in the C1'-position of the side chain lowers the compound's affinity for CB1 by more than 8-fold. The pharmacophoric requirements resulting from halogen-substitution are explored using computer modeling methods.
doi:10.1208/aapsj060430
PMCID: PMC2751226  PMID: 15760095
tetrahydrocannabinol; halogen substitution; CB1 cannabinoid receptors
8.  Anionic liposomal delivery system for DNA transfection 
The AAPS Journal  2004;6(4):13-22.
The present study investigates the use of novel anionic lipoplexes composed of physiological components for plasmid DNA delivery into mammalian cells in vitro. Liposomes were prepared from mixtures of endogenously occurring anionic and zwitterionic lipids, 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), respectively, at a molar ratio of 17∶83 (DOPG:DOPE). Anionic lipoplexes were formed by complexation between anionic liposomes and plasmid DNA molecules encoding green fluorescence protein (GFP) using Ca2+ ions. Transfection and toxicity were evaluated in CHO-K1 cells using flow cytometry and propidium iodide staining, respectively. Controls included Ca2+-DNA complexes (without lipids), anionic liposomes (no Ca2+), and a cationic liposomal formulation. Efficient delivery of plasmid DNA and subsequent GFP expression was achieved using anionic lipoplexes. Transfection efficiency increased with Ca2+ concentration up to 14 mM Ca2+, where transfection efficiency was 7-fold higher than in untreated cells, with minimum toxicity. Further increase in Ca2+ decreased transfection. Transfection efficiency of anionic lipoplexes was similar to that of cationic liposomes (lipofect Amine), whereas their toxicity was significantly lower. Ca2+-DNA complexes exhibited minimal and irregular transfection with relatively high cytotoxicity. A model was developed to explain the basis of anionic lipoplex uptake and transfection efficacy. Effective transfection is explained on the formation of nonbilayer hexagonal lipid phases. Efficient and relatively safe DNA transfection using anionic lipoplexes makes them an appealing alternative to be explored for gene delivery.
doi:10.1208/aapsj060429
PMCID: PMC2751225  PMID: 15760094
anionic liposomes; gene delivery; transfection; nonviral vector; lipoplex; flow cytometry
9.  Exposure-response relationships and drug interactions of sirolimus 
The AAPS Journal  2004;6(4):1-12.
Sirolimus (rapamycin, RAPAMUNE, RAPA) is an immunosuppressive agent used for the prophylaxis of renal allograft rejection and exhibits an immunosuppressive mechanism that is distinct from that for cyclosporine and tacrolimus. The purpose of this manuscript is to discuss the exposure-response relationships and drug interactiosn of sirolimus. The various factors affecting sirolimus whole blood exposure included first-pass extraction, formulation, food, demographics, liver disease, assay method, and interacting drugs. Clinically significant effects caused by food, pediatric age, hepatic impairment, and interacting drugs require recommendations for the safe and efficacious use of sirolimus in renal allograft patients. An exposure-response model based on multivariate logistic regression was developed using the interstudy data from 1832 renal allograft patients. The analysis revealed an increased probability of acute rejection for sirolimus troughs <5 ng/mL, cyclosporine troughs <150 ng/mL, human leukocyte antigen (HLA) mismatches ≥4, and females. The outcomes suggested that individualization of sirolimus doses immediately after transplantation, based on HLA mismatch and sex, would likely decrease the probability of acute rejections in renal allograft recipients who receive concomitant sirolimus, cyclosporine (full-dose), and corticosteroid therapy. Sirolimus is a substrate for both Cytochrome P450 3A (CYP3A) and P-glycoprotein (P-gp) and undergoes extensive first-pass extraction. Drugs that are known to inhibit or induce these proteins may potentially affect sirolimus whole blood exposure. In healthy volunteers, cyclosporine, diltiazem, erythromycin, ketoconazole, and verapamil significantly increased sirolimus whole blood exposure, and rifampin significantly decreased sirolimus exposure. However, sirolimus whole blood exposure was not affected by acyclovir, atorvastatin, digoxin, ethinyl estradiol/norgestrel, glyburide, nifedipine, or tacrolimus. Among the 15 drugs studied, sirolimus significantly increased the exposures of only erythromycin and S-(−)verapamil.
doi:10.1208/aapsj060428
PMCID: PMC2751224  PMID: 15760093
sirolimus; exposure-response relationship; drug interactions
10.  Development and characterization of biodegradable chitosan films for local delivery of paclitaxel 
The AAPS Journal  2004;6(3):88-99.
Intratumoral and local drug delivery strategies have gained momentum recently as a promising modality in cancer therapy. In order to deliver paclitaxel at the tumor site in therapeutically relevant concentrations, chitosan films were fabricated. Paclitaxel could be loaded at 31% wt/wt in films, which were translucent and flexible. Physicochemical characterization of paclitaxel via thermal, spectroscopic, x-ray diffraction, and electron microscopy techniques revealed information on solid-state properties of paclitaxel as well as chitosan in films. While chitosan was in amorphous form, paclitaxel seemed to be present in both amorphous and crystalline forms in film. The polymeric dispersion of paclitaxel in poloxamer formed fibrous structures generating discontinuities in the film matrix, thereby leading to the introduction of perturbations in the packing arrangement of polymer chains. These films released only 10% to 15% of loaded paclitaxel by a burst effect under in vitro testing conditions, with lysozyme having no effect on the release. However, films softened after implantation in mice and lost integrity over time. The implantable delivery system is not only biodegradable but also well tolerated in vivo and hence, biocompatible as revealed by histological studies. The lack of formulation-induced local inflammatory responses of paclitaxel chitosan films suggests a new paradigm for localized chemotherapy based on implantable systems.
doi:10.1208/aapsj060327
PMCID: PMC2751252  PMID: 15760112
paclitaxel; local delivery; film; solid-state; histology; mice
11.  Optimization of chlorphenesin emulgel formulation 
The AAPS Journal  2004;6(3):81-87.
This study was conducted to develop an emulgel formulation of chlorphenesin (CHL) using 2 types of gelling agents: hydroxypropylmethyl cellulose (HPMC) and Carbopol 934. The influence of the type of the gelling agent and the concentration of both the oil phase and emulsifying agent on the drug release from the prepared emulgels was investigated using a 23 factorial design. The prepared emulgels were evaluated for their physical appearance, rheological behavior, drug release, antifungal activity, and stability. Commercially available CHL topical powder was used for comparison. All the prepared emulgels showed acceptable physical properties concerning color, homogeneity, consistency, spreadability, and pH value. They also exhibited higher drug release and antifungal activity than the CHL powder. It was found that the emulsifying agent concentration had the most pronounced effect on the drug release from the emulgels followed by the oil phase concentration and finally the type of the gelling agent. The drug release from all the emulgels was found to follow diffusion-controlled mechanism. Rheological studies revealed that the CHL emulgels exhibited a shear-thinning behavior with thixotropy. Stability studies showed that the physical appearance, rheological properties, drug release, and antifungal activity in all the prepared emulgels remained unchanged upon storage for 3 months. As a general conclusion, it was suggested that the CHL emulgel formulation prepared with HPMC with the oil phase concentration in its low level and emulsifying agent concentration in its high level was the formula of choice since it showed the highest drug release and antifungal activity.
doi:10.1208/aapsj060326
PMCID: PMC2751251  PMID: 15760111
chlorphenesin; emulgel; factorial design
12.  Neural retina limits the nonviral gene transfer to retinal pigment epithelium in an in vitro bovine eye model 
The AAPS Journal  2004;6(3):72-80.
We investigated the permeation of liposomal and polymeric gene delivery systems through neural retina into retinal pigment epithelium (RPE) and determined the roles of various factors in permeation and subsequent uptake of the delivery systems by RPE. Anterior parts and vitreous of fresh bovine eyes were removed. Retina was left intact or peeled away. Complexes of ethidium monoazide (EMA)-labeled plasmid DNA and cationic carriers (polyethyleneimine, poly-L-lysine, DOTAP liposomes) were pipetted on the retina or RPE. Two hours later the neural retina was removed, if present, and the RPE cells were detached. Contaminants were removed by sucrose centrifugation, and the RPE cells were analyzed for DNA uptake by flow cytometry. Cellular uptake of FITC-dextrans (molecular weight [mw] 20 000, 500 000 and 2 000 000), FITC-poly-L-lysine (mw 20 000), FITC-labeled oligonucleotide (15-mer), and naked EMA-labeled plasmid DNA was determined after pipetting the solutions on the RPE or neural retina. Location of the fluorescent materials in the retina was visualized with fluorescence microscopy. Neural retina decreased the cellular uptake of DNA complexes by an order of magnitude, the uptake of FITC-dextrans slightly, whereas delivery of polycationic FITC-poly-L-lysine to RPE was almost completely inhibibited. Neural retina decreased the cellular uptake of FITC-oligonucleotides, while the uptake of uncomplexed plasmid was always negligible. conclusions from FACS and fluorescence microscopy were similar: delivery of polymeric and liposomal DNA complexes into RPE are limited by the neural retina. This is due to the size and positive charge of the complexes.
doi:10.1208/aapsj060325
PMCID: PMC2751250  PMID: 15760110
gene delivery; intravitreal; retina; liposome; polymer
13.  Morphology and buoyancy of oil-entrapped calcium pectinate gel beads 
The AAPS Journal  2004;6(3):65-71.
A new emulsion-gelation method to prepare oil-entrapped calcium pectinate gel (CaPG) beads capable of floating in the gastric condition was designed and tested. The gel beads containing edible oil were prepared by either being gently mixed or homogenized an oil phase and a water phase containing pectin, and then extruded into calcium chloride solution with gentle agitation at room temperature. The gel beads formed were then separated, washed with distilled water, and dried at 37°C for 12 hours. A model of the emulsion-gelation process to illustrate the formation of oil-entrapped CaPG beads was proposed. The effect of selected factors, such as type of oil, percentage of oil, and type of pectin on morphology and floating properties was investigated. The oil-entrapped calcium pectinate gel beads floated if a sufficient amount of oil was used. Scanning electron photomicrographs demonstrated very small pores, ranging between 5 and 40 μm, dispersed all over the beads. The type and percentage of oil play an important role in controlling the floating of oil-entrapped CaPG beads. The results suggested that oil-entrapped CaPG beads were promising as a carrier for intragastric floating drug delivery.
doi:10.1208/aapsj060324
PMCID: PMC2751249  PMID: 15760109
calcium pectinate; pectin; oil; emulsion; gel beads; floating; gastro-retentive
14.  Etoposide-incorporated tripalmitin nanoparticles with different surface charge: Formulation, characterization, radiolabeling, and biodistribution studies 
The AAPS Journal  2004;6(3):55-64.
Etoposide-incorporated tripalmitin nanoparticles with negative (ETN) and positive charge (ETP) were prepared by melt emulsification and high-pressure homogenization techniques. Spray drying of nanoparticles led to free flowing powder with excellent redispersibility. The nanoparticles were characterized by size analysis, zeta potential measurements, and scanning electron microscopy. The mean diameter of ETN and ETP nanoparticles was 391 nm and 362 nm, respectively, and the entrapment efficiency was more than 96%. Radiolabeling of etoposide and nanoparticles was performed with Technetium-99m (99mTc) with high labeling efficiency and in vitro stability. The determination of binding affinity of99mTc-labeled complexes by diethylene triamine penta acetic acid (DTPA) and cysteine challenge test confirmed low transchelation of99mTc-labeled complexes and high in vitro stability. Pharmacokinetic data of radiolabeled etoposide, ETN, and ETP nanoparticles in rats reveal that positively charged nanoparticles had high blood concentrations and prolonged blood residence time. Biodistribution studies of99mTc-labeled complexes were performed after intravenous administration in mice. Both ETN and ETP nanoparticles showed significantly lower uptake by organs of the reticuloendothelial system such as liver and spleen (P<.001) compared with etoposide. The ETP nanoparticles showed a relatively high distribution to bone and brain (14-fold higher than etoposide and ETN at 4 hours postinjection) than ETN nanoparticles. The ETP nanoparticles with long circulating property could be a beneficial delivery system for targeting to tumors by Enhanced Permeability and Retention effect and to brain.
doi:10.1208/aapsj060323
PMCID: PMC2751248  PMID: 15760108
etoposide; tripalmitin; Technetium-99m; long circulating nanoparticles; radiolabeling; biodistribution
15.  Cryoprotection mechanisms of polyethylene glycols on lactate dehydrogenase during freeze-thawing 
The AAPS Journal  2004;6(3):45-54.
The purpose of this study was to explore the cryoprotection mechanisms of high molecular weight polyethylene glycols (PEGs) (eg, PEG 4000 and PEG 8000) on lactate dehydrogenase (LDH). Ultraviolet activity assays, circular dichroism (CD) spectroscopy, gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE),14C-PEG 4000 labeling and binding, and cryostage microscopic study were conducted. Different molecular weights and concentrations of PEGs in LDH formulations were treated by freeze-thawing. Higher molecular weights and concentrations of PEGs in LDH-PEG formulations obtained better activity and secondary structure recoveries of LDH after freeze-thawing. Insoluble aggregation of LDH was not observed in gel filtration studies. SDS-PAGE results suggested surface characteristic modifications of LDH by the larger molecular weight PEGs. The14C-PEG 4000 labeling and binding study showed extensive nonspecific interactions between the PEG 4000 and LDH molecules in a concentration-dependent manner. The bound LDH-PEG 4000/free PEG 4000 ratio increased when LDH or PEG 4000 concentrations increased. Cryostage microscopic study showed that PEG 8000 delayed the ice crystallization and eutectic transition of LDH formulation. It appeared that multiple mechanisms were at work during PEGs' cryoprotection of LDH. It was unclear whether the delayed eutectic characteristics of PEGs contributed to LDH cryoprotection. The favorable interaction, rather than preferential exclusion, between LDH and PEGs (eg, 4000) cryoprotected LDH.
doi:10.1208/aapsj060322
PMCID: PMC2751247  PMID: 15760107
lactate dehydrogenase (LDH); freeze-thaw; circular dichroism (CD); SDS-PAGE; 14C-PEG 4000 binding
16.  Transporter and ion channel gene expression after caco-2 cell differentiation using 2 different microarray technologies 
The AAPS Journal  2004;6(3):35-44.
mRNA expression profiles had previously been measured in Caco-2 cells (human colonic carcinoma cells) using either custom-designed spotted oligonucleotide arrays or Affymetrix GeneChip oligonucleotide arrays. The Caco-2 cells used were from different clones and were examined under slightly different culture conditions commonly encountered when Caco-2 cells are used as a model tissue for studying intestinal transport and metabolism in different laboratories. In this study, we compared gene expression profiles of Caco-2 cells generated with different arrays to assess the validity of conclusions derived from the 2 independent studies, with a focus on changes in transporter and ion channel mRNA expression levels on Caco-2 cell differentiation. Significant changes in expression levels upon differentiation were observed with 78 genes, with probes common to both arrays. Of these, 18 genes were upregulated and 36 genes were downregulated. The 2 arrays yielded discrepant results for 24 genes, showing significant changes upon differentiation. The results from the 2 arrays correlated well for genes expressed above average levels (r=0.75,P<0.01, n=25) and poorly for genes expressed at low levels (r=0.08,P>0.05, n=25). Overall correlation across the 2 platforms wasr=0.45 (P<0.01) for the 78 genes, with similar results from both arrays. Despite differences in experimental conditions and array technology, similar results were obtained for most genes.
doi:10.1208/aapsj060321
PMCID: PMC2751246  PMID: 15760106
microarrays; Caco-2 cells; transporter; ion channel; platform comparison
17.  Particle size analysis: AAPS workshop report, cosponsored by the Food and Drug Administration and the United States Pharmacopeia 
The AAPS Journal  2004;6(3):23-34.
The concepts of particle engineering and dosage form design have become dominant themes in pharmaceutical manufacturing. This trend is not simply a reflection of the development of new, more sophisticated manufacturing methods of particles or dispersed systems but also recognition of the importance of quality control even in more traditional manufacturing processes. However, the diversity of particle treatments, methods of particle size analysis, expression and interpretation of data, and process applications results in complicated and sometimes confusing criteria for selection, adoption, or relevance of the available techniques.
doi:10.1208/aapsj060320
PMCID: PMC2751245  PMID: 15760105
18.  The back-step method—Method for obtaining unbiased population parameter estimates for ordered categorical data 
The AAPS Journal  2004;6(3):13-22.
A significant bias in parameters, estimated with the proportional odds model using the software NONMEM, has been reported. Typically, this bias occurs with ordered categorical data, when most of the observations are found at one extreme of the possible outcomes. The aim of this study was to assess, through simulations, the performance of the Back-Step Method (BSM), a novel approach for obtaining unbiased estimates when the standard approach provides biased estimates. BSM is an iterative method involving sequential simulation-estimation steps. BSM was compared with the standard approach in the analysis of a 4-category ordered variable using the Laplacian method in NONMEM. The bias in parameter estimates and the accuracy of model predictions were determined for the 2 methods on 3 conditions: (1) a nonskewed distribution of the response with low interindividual variability (IIV), (2) a skewed distribution with low IIV, and (3) a skewed distribution with high IIV. An increase in bias with increasing skewness and IIV was shown in parameters estimated using the standard approach in NON-MEM. BSM performed without appreciable bias in the estimates under the 3 conditions, and the model predictions were in good agreement with the original data. Each BSM estimation represents a random sample of the population; hence, repeating the BSM estimation reduces the imprecision of the parameter estimates. The BSM is an accurate estimation method when the standard modeling approach in NONMEM gives biased estimates.
doi:10.1208/aapsj060319
PMCID: PMC2751244  PMID: 15760104
ordered categorical; proportional odds model; bias in parameter estimates; NONMEM; Laplacian; pharmacodynamics
19.  Cetirizine from topical phosphatidylcholine-hydrogenated liposomes: Evaluation of peripheral antihistaminic activity and systemic absorption in a rabbit model 
The AAPS Journal  2004;6(3):7-12.
Cetirizine, an effective, minimally sedating, second-generation H1-antihistamine is widely used orally to treat allergic skin disorders. This study was performed to assess the peripheral H1-antihistaminic activity and extent of systemic absorption of cetirizine from liposomes applied to the skin. Cetirizine was incorporated into small unilamellar vesicles (SUV) and multilamellar vesicles (MLV) prepared using L-α-phosphatidylcholine hydrogenated (HPC), and into Glaxal Base (GB) as the control. In a randomized, crossover study, each formulation, containing 10 mg of cetirizine, was applied to the depilated backs of 6 rabbits (3.08±0.05 kg). Histamine-induced wheal tests and blood sampling were performed before cetirizine application and at designated times for up to 24 hours afterwards. Compared with baseline, histamine-induced wheal formation was suppressed by cetirizine in SUV only at 24 hours, in MLV from 0.5 to 24 hours, and in GB from 0.5 to 8 hours (P<.05). Wheal suppression by cetirizine in SUV at 24 hours (91.7%±5.2%) and in MLV from 1 to 24 hours (93.8%±2.2% to 76.2%±6.5%) was greater than in GB (36.5%±7.4% to 60.6%±14.2%) from 1 to 24 hours (P<.05). Faster onset, as well as greater and more persistent suppression was obtained from cetirizine in MLV. Plasma cetirizine concentrations from MLV (area under the curve [AUC] of 221.2±42.3 ng.hr/mL) were lower than from GB (AUC of 248.3±34.6 ng.hr/mL). In this model, cetirizine from MLV had excellent topical H1-antihistamine activity, while systemic exposure was reduced, compared with cetirizine from GB.
doi:10.1208/aapsj060318
PMCID: PMC2751243  PMID: 18465266
cetirizine; L-α-phosphatidylcholine hydrogenated; liposomes; antihistamine; rabbit's skin
20.  Formulation design of double-layer in the outer shell of dry-coated tablet to modulate lag time and time-controlled dissolution function: Studies on micronized ethylcellulose for dosage form design (VII) 
The AAPS Journal  2004;6(3):1-6.
The dry-coated tablet with optimal lag time was designed to simulate the dosing time of drug administration according to the physiological needs. Different compositions of ethylcellulose (EC) powder with a coarse particle (167.5 μm) and several fine particles (<6 μm), respectively, were mixed to formulate the whole layer of the outer shell of dry-coated tablets. The formulations containing different weight ratios of coarse/fine particles of EC powders or 167.5 μm EC powder/excipient in the upper layer of the outer shell to influence the release behavior of sodium diclofenac from dry-coated tablet were also explored. The results indicate that sodium diclofenac released from all the dry-coated tablets exhibited an initial lag period, followed by a stage of rapid drug release. When the mixture of the coarse/fine particles of EC powders was incorporated into the whole layer, the lag time was almost the same. The outer shell broke into 2 halves to make a rapid drug release after the lag time, which belonged to the time-controlled disruption of release mechanism. When the lower layer in the outer shell was composed of 167.5 μm EC powder and the upper layer was formulated by mixing different weight ratios of 167.5 μm and 6 μm of EC powders, the drug release also exhibited a time-controlled disruption behavior. Its lag time might be freely modulated, depending on the amount of 6 μm EC powder added. Once different excipients were respectively incorporated into the upper layer of the outer shell, different release mechanisms were observed as follows: time-controlled explosion for Explotab, disruption for Avicel and spray-dried lactose, erosion for dibasic calcium phosphate anhydrate, and sigmoidal profile for hydroxypropyl methylcellulose.
doi:10.1208/aapsj060317
PMCID: PMC2751242  PMID: 15760102
micronized ethylcellulose; dry-coated tablet; outer layer; time-controlled dissolution; lag time

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