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1.  AAPS pharmSci: An advanced publication forum has matured 
AAPS PharmSci  2003;5(2):86-87.
doi:10.1208/ps050216
PMCID: PMC2751524  PMID: 12866943
2.  A photo-crosslinked poly(vinyl alcohol) hydrogel growth factor release vehicle for wound healing applications 
AAPS PharmSci  2003;5(4):101-111.
The objective of this study was to develop and evaluate a hydrogel vehicle for sustained release of growth factors for wound healing applications. Hydrogels were fabricated using ultraviolet photo-crosslinking of acrylamide-functionalized nondegradable poly(vinyl alcohol) (PVA). Protein permeability was initially assessed using trypsin inhibitor (TI), a 21 000 MW model protein drug. TI permeability was altered by changing the solids content of the gel and by adding hydrophilic PVA fillers. As the PVA content increased from 10% to 20%, protein flux decreased, with no TI permeating through 20% PVA hydrogels. Further increase in model drug release was achieved by incorporating hydrophilic PVA fillers into the hydrogel. As filler molecular weight increased, TI flux increased. The mechanism for this is most likely an alteration in protein/gel interactions and transient variations in water content. The percent protein released was also altered by varying protein loading concentration. Release studies conducted using growth factor in vehicles with hydrophilic filler showed sustained release of platelet-derived growth factor (PDGF-β,β) for up to 3 days compared with less than 24 hours in the controls. In vitro bioactivity was demonstrated by doubling of normal human dermal fibroblas numbers when exposed to growth factor-loaded vehicle compared to control. The release vehicle developed in this study uses a rapid and simple fabrication method, and protein release can be tailored by modifying solid content, incorporating biocompatible hydrophilic fillers, and varying protein loading concentration.
doi:10.1208/ps050433
PMCID: PMC2750995  PMID: 15198521
photo-crosslinkable hydrogel; poly(vinyl alcohol); platelet-derived growth factor; bioactivity; sustained release
3.  Pharmacokinetics of paclitaxel-containing liposomes in rats 
AAPS PharmSci  2003;5(4):90-100.
In animal models, liposomal formulations of paclitaxel possess lower toxicity and equal antitumor efficacy compared with the clinical formulation, Taxol. The goal of this study was to determine the formulation dependence of paclitaxel pharmacokinetics in rats, in order to test the hypothesis that altered biodistribution of paclitaxel modifies the exposure of critical normal tissues. Paclitaxel was administered intravenously in either multilamellar (MLV) liposomes composed of phosphatidylglycerol/phosphatidylcholine (L-pac) or in the Cremophor EL/ethanol vehicle used for the Taxol formulation (Cre-pac). The dose was 40 mg/kg, and the infusion time was 8 to 9 minutes. Animals were killed at various times, and pharmacokinetic parameters were determined from the blood and tissue distribution of paclitaxel. The area under the concentration vs time curve (AUC) for blood was similar for the 2 formulations (L-pac: 38.1±3.32 μg-h/mL; Cre-pac: 34.5±0.994 μg-h/mL), however, the AUC for various tissues was formulation-dependent. For bone marrow, skin, kidney, brain, adipose, and muscle tissue, the AUC was statistically higher for Cre-pac. For spleen, a tissue of the reticuloendothelial system that is important in the clearance of liposomes, the AUC was statistically higher for L-pac. Apparent tissue partition coefficients (Kp) also were calculated. For bone marrow, a tissue in which paclitaxel exerts significant toxicity, Kp was 5-fold greater for paclitaxel in Cre-pac. The data are consistent with paclitaxel release from circulating liposomes, but with efflux delayed sufficiently to retain drug to a greater extent in the central (blood) compartment and reduce penetration into peripheral tissues. These effects may contribute to the reduced toxicity of liposomal formulations of paclitaxel.
doi:10.1208/ps050432
PMCID: PMC2750994  PMID: 15198520
drug delivery; paclitaxel; liposomes; physiological modeling; cancer chemotherapy
4.  Properties of microcrystalline cellulose and powder cellulose after extrusion/spheronization as studied by fourier transform Raman spectroscopy and environmental scanning electron microscopy 
AAPS PharmSci  2003;5(4):77-89.
In this study, the effect of powder cellulose (PC) and 2 types of microcrystalline cellulose (MCC 101 and MCC 301) on pellet properties produced by an extrusion/spheronization process was investigated. The different investigated types of cellulose displayed different behavior during the extrusion/spheronization process. Pure PC was unsuitable for extrusion, because too much water was required and the added water was partly squeezed during the extrusion process. In contrast, MCC 101 and MCC 301 were extrudable at a wide range of water content, but the quality of the resulting products varied. In the extrusion/spheronization process, MCC 101 was the best substance, with easy handling and acceptable product properties. The properties of the extrudates and pellets were determined by Fourier transform (FT) Raman spectroscopy and environmental scanning electron microscopy (ESEM). FT-Raman spectroscopy was able to distinguish between the original substances and also between the wet and dried extrudates. The particle sizes of the raw material and of the extrudates were determined by ESEM without additional preparation. For MCC, the size of the resulting particles within the extrudate or pellet was smaller. However, in the extrudates of PC, changes in particle size could not be observed.
doi:10.1208/ps050431
PMCID: PMC2750993  PMID: 15198519
powder cellulose; microcrystalline cellulose; pellet; Raman spectroscopy; environmental scanning electron microscopy; extrusion/spheronization
5.  Lipid-based supramolecular systems for topical application: A preformulatory study 
AAPS PharmSci  2003;5(4):62-76.
This article describes the production and characterization of monoglyceride-based supramolecular systems by a simple processing technique, avoiding time-consuming procedures, high energy input, and the use of organic solvents. A preformulatory study was performed to study the influence of the experimental parameters on the production of monoglyceride-based disperse systems. In particular the effects of (1) stirring speed, (2) type and concentration of monoglyceride mixture, and (3) type and concentration of surfactant were investigated on the recovery, fraction of larger particles, mean diameter, and shape of smaller particles (so called nanosomes). Dispersions were first characterized by optical microscopy and freeze-fracture electron microscopy. The mean diameter of standard nanosomes, analyzed by photon correlation spectroscopy (PCS) after elimination of larger particles by filtration, was 193.5 nm. Cryotransmission electron microscopy studies, conducted in order to investigate the structure of dispersions, showed the coexistence of vesicles and particles characterized by a cubic organization. X-ray diffraction data revealed the coexistence of 2 different cubic phases, the first being a bicontinuous cubic phase of spatial symmetry Im3m (Q229) and the second belonging to the Pn3m spatial symmetry. A study on the stability of monoglyceride-based dispersions based on macroscopical analysis of organoleptic properties and dimensional analysis by time was performed after elimination of larger particles by filtration. Organoleptic and morphological features do not change by time, appearing free from phase-separation phenomena for almost 1 year from production. PCS studies showed that nanosomes undergo an initial increase in mean diameter within the first month following production; afterwards they generally maintain their dimensions for the next 4 months.
doi:10.1208/ps050430
PMCID: PMC2750992  PMID: 15198518
monoglycerides; nanosome dispersions; photon correlation spectroscopy
6.  Pharmacogenomics of multigenic diseases: Sex-specific differences in disease and treatment outcome 
AAPS PharmSci  2003;5(4):49-61.
Numerous genetic variations have been shown to affect disease susceptibility and drug response. Pharmacogenomics aims at improving therapy on the basis of genetic information for each individual patient. Furthermore, sex chromosomes broadly determine biological differences between males and females. Consequently, substantial sex differences exist in phenotypic manifestation of disease and treatment response. This review discusses the role of sex in coronary artery disease, schizophrenia, and depression—complex multigenic disorders with considerable sex differences in frequency and presentation. Moreover, genetic factors underlying disease and drug response appear to differ between male and female patients. This appears to result at least in part from different physiological effects exerted by sex hormones such that polymorphisms in susceptibility genes may have physiological relevance only in males or females. However, few examples have been discovered to play a role in complex multigenic diseases, and the mechanistic basis of genetic variants as sex-dependent susceptibility factors has yet to be explored. Therefore, pharmacogenomic studies must consider sex differences in an effort to optimize individual drug therapy.
doi:10.1208/ps050429
PMCID: PMC2750991  PMID: 15198517
pharmacogenomics; sex differences; multigenic disease; candidate genes; coronary artery disease; depression; schizophrenia
7.  Hydroxyzine from topical phospholipid liposomal formulations: Evaluation of peripheral antihistaminic activity and systemic absorption in a rabbit model 
AAPS PharmSci  2003;5(4):41-48.
Hydroxyzine, an effective but sedating H1-antihistamine is given orally to treat allergic skin disorders. This study was performed to assess the peripheral H1-antihistaminic activity and extent of systemic absorption of hydroxyzine from liposomes applied to the skin. Using L-α-phosphatidylcholine (PC), small unilamellar vesicles (SUVs) and multilamellar vesicles (MLVs) containing hydroxyzine were prepared. Hydroxyzine in Glaxal Base (GB) was used as the control. Using a randomized, crossover design, each formulation, containing 10 mg of hydroxyzine, was applied to the shaved backs of 6 rabbits (3.08±0.05 kg). Histamine-induced wheal tests and blood sampling were performed at designated time intervals up to 24 hours. Compared with baseline, hydroxyzine from all formulations significantly suppressed histamine-induced wheal formation by 75% to 95% for up to 24 hours. Mean maximum suppression, 85% to 94%, occurred from 2 to 6 hours, with no differences among the formulations. The areas of plasma hydroxyzine concentration versus time area under the curve (AUCs) from PC-SUV and PC-MLV, 80.1±20.8 and 78.4±33.9 ng/mL/h, respectively, were lower than that from GB, 492±141 ng/mL/h (P<.05) over 24 hours. Plasma concentrations of cetirizine arising in-vivo as the active metabolite of hydroxyzine, from PC-SUV, PC-MLV, and GB, were similar with AUCs of 765±50, 1035±202, and 957±227 ng/mL/h, respectively (P<.05). Only 0.02% to 0.06% of the initial hydroxyzine dose remained on the skin after 24 hours. In this model, hydroxyzine from SUV and MLV had excellent topical H1-antihistaminic activity, and minimal systemic exposure occurred. Cetirizine formed in-vivo contributed to some of H1-antihistaminic activity.
doi:10.1208/ps050428
PMCID: PMC2750990  PMID: 15198516
hydroxyzine; L-α-phosphatidylcholine; liposomes; antihistamine; skin; rabbit
8.  AAPS/RAPS/CAPRA collaborative program: Exploring the challenges of drug regulation in a global environment: Clinical concerns 
AAPS PharmSci  2003;5(4):13-40.
Globalization of the pharmaceutical industry has led to a need to harmonize the regulatory requirements governing the marketing of medicinal products. To minimize the barriers impeding global drug product registration, the International Conference on the Harmonization of Technical Requirements of Pharmaceuticals for Human Use (ICH) was established in 1990. The ICH has developed a series of guidelines that reflect agreements reached by participating nations on aspects of the chemistry and clinical technical sections that will fulfill the regulatory requirements of these various jurisdications. Nevertheless, there continue to be points of divergent perspectives and barriers that can impede the use of foreign clinical data. Given the importance of these issues, the Regulatory Science (RS) section of the American Association of Pharmaceutical Scientists (AAPS), in conjunction with the Regulatory Affairs Professional Society (RAPS) and the Canadian Association of Professional Regulatory Affairs (CAPRA) cosponsored a public forum on this topic. This manuscript provides a summary of the speaker presentations and audience discussions regarding the design of clinical trials and the extrapolation of results from these trials to support international drug registration.
doi:10.1208/ps050427
PMCID: PMC2750989  PMID: 15198515
clinical trials; regulatory requirements; international harmonization; foreign clinical data
9.  Amphiphilic star-like macromolecules as novel carriers for topical delivery of nonsteroidal anti-inflammatory drugs 
AAPS PharmSci  2003;5(4):1-12.
The objective of this study was to evaluate amphiphilic star-like macromolecules (ASMs) as a topical drug delivery system. Indomethacin, piroxicam, and ketoprofen were individually encapsulated into the ASMs using coprecipitation. The effects of the ASMs on percutaneous permeation of nonsteroidal anti-inflammatory drugs (NSAIDs) across full thickness, hairless mouse skin were evaluated in vitro using modified Franz diffusion cells. In addition, solubility and in vitro release experiments were performed to characterize ASMs behavior in aqueous media. Poly(ethylene glycol) (PEG) and Pluronic P-85 were used as polymer controls to compare the role of PEG and amphiphilic behavior in the ASMs. In vitro release experiments indicated that ASMs can delay drug release (P⋖05), whereas solubility measurements showed that ASMs can increase NSAIDs aqueous solubility (P⋖05). Percutaneous permeation studies revealed that ASMs decreased both flux and Q24 of drugs compared with the control (P⋖10). Skin pretreatment studies with ASM-containing solution before drug application demonstrated that pretreatment similarly influenced NSAID percutaneous permeation. In conclusion, ASMs likely slow drug permeation through 2 mechanisms, delayed drug diffusion from its core and skin dehydration by its shell. Thus, ASMs may be useful for delayed dermal delivery or prevention of compound permeation through the skin (eg, sunscreens, N,N-diethyl-m-toluamide [DEET]) from aqueous formulations.
doi:10.1208/ps050426
PMCID: PMC2750988  PMID: 15198514
topical drug delivery; NSAIDs; polymeric micelle; permeation; drug release
10.  Nutraceutical-definition and introduction 
AAPS PharmSci  2003;5(3):27-28.
Dr Stephen DeFelice coined the term “Nutraceutical” from “Nutrition” and “Pharmaceutical” in 1989. The term nutraceutical is being commonly used in marketing but has no regulatory definition. An attempt to redefine nutraceuticals and functional foods is made in this article. The proposed definitions can help distinguish between functional foods, nutraceuticals, and dietary supplements. The advantages and disadvantages of nutraceuticals are also briefly discussed.
doi:10.1208/ps050325
PMCID: PMC2750935  PMID: 14621960
nutraceutical; functional food; dietary supplement
11.  High glucose concentration in isotonic media alters Caco-2 cell permeability 
AAPS PharmSci  2003;5(3):17-25.
Caco-2 cell permeability was evaluated in isotonic media containing high (25mM) or physiological (5.5mM) glucose concentrations. Transepithelial electrical resistance (TEER) and membrane fluidity were measured to assess glucose-induced alterations in physical barrier properties. In parallel, distribution of the actin filament (F-actin) and zonula occludens-1 (ZO-1) proteins was assessed by confocal microscopy. Transepithelial fluxes of mannitol, hydrocortisone, digoxin, and glycyl sarcosine (Gly-Sar) that permeate the intestinal mucosa by various pathways were measured to quantify the effect of glucose-induced changes on Caco-2 cell permeability. High glucose decreased maximum TEER of cell monolayers by 47%, whereas membrane fluidity at the hydrophobic core and lipid/polar head interphase was significantly increased. F-actin distribution in high glucose cells appeared more diffuse while ZO-1 was unchanged. Mannitol and hydrocortisone fluxes across Caco-2 cells cultured in high glucose increased by 65% and 24%, respectively. In addition, high glucose decreased the maximum transport capacity (Vmax) of PepT-1. P-glycoprotein activity, however, was unchanged. In conclusion, high extracellular glucose concentration in isotonic media significantly alters physical barrier properties of Caco-2 cell monolayers, which predominantly affects transepithelial transport of solutes permeating the cell barrier by paracellular and transcellular passive diffusion and facilitated transport mediated by the proton-dependent oligopeptide transporter (PepT-1).
doi:10.1208/ps050324
PMCID: PMC2750934  PMID: 14621959
Caco-2; glucose; tight junctions; TEER; membrane fluidity; solute flux
12.  Quantitation of motexafin lutetium in human plasma by liquid chromatography-tandem mass spectrometry and inductively coupled plasma-atomic emission spectroscopy 
AAPS PharmSci  2003;5(3):1-16.
Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and inductively coupled plasma-atomic emission spectroscopy (ICP-AES) methods were developed and validated for the evaluation of motexafin lutetium (MLu, lutetium texaphyrin, PCI-0123) pharmacokinetics in human plasma. The LC-MS/MS method was specific for MLu, whereas the ICP-AES method measured total elemental lutetium. Both methods were fast, simple, precise, and accurate. For the LC-MS/MS method, a closely related analogue (PCI-0353) was used as the internal standard (IS). MLu and the IS were extracted from plasma by protein precipitation and injected onto and LC-MS/MS system configured with a C18 column and an electrospray interface. The lower limit of quantitation was 0.05 μg MLu mL−1, with a signal-to-noise ratio of 15∶1. The response was linear from 0.05 to 5.0 μg MLu mL−1. For the ICP-AES method, indium was used as the IS. The sample was digested with nitric acid, diluted, filtered, and then injected onto the ICP-AES system. Two standard curve ranges were validated to meet the expected range of sample concentrations: 0.5 to 50, and 0.1 to 10 μg Lu mL−1. The LC-MS/MS and ICP-AES methods were validated to establish accuracy, precision, analyte stability, and assay robustness. Interday precision and accuracy of quality control samples were ≤6.3% coefficient of variation (CV) and within 2.2% relative error (RE) for the LC-MS/MS method, and ≤8.7% CV and within 4.9% RE for the ICP-AES method. Plasma samples from a subset of patients in a clinical study were analyzed using both methods. For a representative patient, over 90% of the elemental lutetium in plasma could be ascribed to intact MLu at early time points. This percentage decreased to 59% at 48 hours after dosing, suggesting that some degradation and/or metabolism of the drug may have occurred.
doi:10.1208/ps050323
PMCID: PMC2750933  PMID: 14621958
motexafin lutetium; LC-MS/MS; ICP-AES; pharmacokinetics; lutetium texaphyrin
13.  Evaluation of sucrose esters as alternative surfactants in microencapsulation of proteins by the solvent evaporation method 
AAPS PharmSci  2003;5(2):123-131.
Sucrose esters (SE) are surfactants with potential pharmaceutical applications because of their low toxicity, biocompatibility, and excellent biodegradability. The objective of the study was to investigate SE as alternative surfactants in stabilizing emulsions for the preparation of protein-loaded microparticles. To achieve this goal, using bovine serum albumin as model protein and 75/25 poly(d,1-lactide-co-glycolide) as polymer carrier, we have investigated the influence of the following formulation variables on particle characteristics: (1) SE concentration from 0.01% to 1% (wt/vol), (2) hydrophile-lipophile balance (HLB) value of SE from 6 to 15, and (3) the nature of emulsion stabilizer. The formulations were characterized using ATR-FTIR spectroscopy, bicinchoninic acid protein assay, optical microscopy and SDS-PAGE. Results showed that at 0.05% (wt/vol) surfactant concentration, SE with HLB of 6 to 15 provided discrete and spherical microparticles with the highest encapsulation efficiency compared with controls polyvinyl alcohol (PVA) and poloxamer 188. These results may be explained by the difference in critical micelle concentration, diffusion, and partition coefficient among the tested surfactants. HLB values were consistent with SE spectral data. The protein molecular weight was preserved after the encapsulation process. The effective SE concentration was far less (20-to 200-fold) than that is usually required for PVA in microencapsulation of proteins. However, the encapsulation efficiency was relatively lower (∼13.5%). These preliminary results suggest that it may be desirable to optimize such formulations in vitro and in vivo for SE to be eventually used as altermative surfactants in the development of microparticulate systems for parenteral delivery of protein and gene medicines.
doi:10.1208/ps050222
PMCID: PMC2751529  PMID: 12866947
proteins; microencapsulation; solvent evaporation; sucrose ester; surfactants
14.  Atenolol quantification in human plasma by high-performance liquid chromatography: Application to bioequivalence study 
AAPS PharmSci  2003;5(2):116-122.
An accurate, precise, and sensitive high-performance liquid chromatography (HPLC) assay was developed for the determination of atenolol in human plasma samples to compare the bioavailability of 2 atenolol tablet (50 mg) formulations in 24 volunteers of both sexes. The study had an open, randomized, 2-period crossover design with a 1-week washout period. Plasma samples were obtained over a 24-hour interval. Atenolol concentrations were analyzed by combined reversed phase liquid chromatography and fluorescence detection (λEX = 258 nm, λEM = 300 nm). From the atenolol plasma concentration versus time curves, the following pharmacokinetic parameters were obtained: AUC0–24h, AUC0–∞, and Cmax. The geometric mean of test/reference 50-mg tablets individual percent ratio was 102.2% for AUC0–24h, and 101.6% for Cmax. The 90% confidence intervals (CI) were 100.2% to 105.4% and 100.9% to 103.5%, respectively. Since the 90% CI for both Cmax and AUC0–24h were within the 80% to 125% interval proposed by the Food and Drug Administration, it was concluded that atenolol (50-mg tablets) test formulation was bioequivalent to the reference formulation, with regard to both the rate and extent of absorption.
doi:10.1208/ps050221
PMCID: PMC2751528  PMID: 12866946
atenolol; bioequivalence; HPLC; plasma; determination
15.  Evaluation of the antioxidant activity of different flavonoids by the chemiluminescence method 
AAPS PharmSci  2003;5(2):111-115.
The objective of the present investigation was to study the antioxidant action of different flavonoids (quercetin, glabridin, red clover, and Isoflavin Beta, an isoflavones mixture) in order to determine if they could be added to a topical formulation used to treat damage caused by free radicals. Samples of 10 μL of the test compounds at different concentrations were mixed with 0.1 M phosphate buffer, pH 7.4, and a luminol solution was added to yield a final concentration of 0.113 mM. Hydrogen peroxide was then added at a final concentration of 0.05 mM. The reaction was started by introducing the horse-radish peroxidase enzyme at a final concentration of 0.2 IU/mL, in a final volume of 1.0 mL. Chemiluminescence was measured for 10 minutes at room temperature, and dimethylsulfoxide (DMSO) was used as a control. All samples showed marked inhibition of oxidative stress, with a concentration-dependent action for quercetin and Isoflavin Beta. The highest inhibition was observed with glabridin and the dry red clover extract. All flavonoids proved to be adequate for addition to topical formulations because of their high antioxidant activity.
doi:10.1208/ps050220
PMCID: PMC2751527
chemiluminescence; luminol; antioxidants; flavonoids; peroxidase
16.  Exploring the demand for a voluntary medicare prescription drug benefit 
AAPS PharmSci  2003;5(2):99-110.
The purposes of this study were (1) to assess the utility of the economic theory of demand for insurance for modeling voluntary Medicare drug benefit enrollment decisions and (2) to explore the degree of adverse selection and crowd-out that might occur under a voluntary enrollment Medicare prescription benefit. Data were collected using a cross-sectional, mail survey of 2,100 community-dwelling adults aged 65 and older in Wisconsin. Respondents were asked to evaluate their likelihood of enrollment in any of 4 hypothetical drug benefit plans under the assumption that they could enroll in one of the hypothetical plans or maintain their current coverage. Data analyses included bivariate comparisons across enrollment likelihood categories and logit analysis of enrollment likelihood as a function of respondent characteristics. 1041 usable survey forms were returned for an adjusted response rate of 51.5%. Older adults with 4 or more chronic conditions were most likely to report that they were “very likely” to enroll in one of the hypothetical drug plans, as were those with the highest out-of-pocket drug spending in the previous 30 days. Respondents with no or self-purchased drug benefits were more likely than those with employer-based plans to express a higher likelihood of enrollment in one of the hypothetical plans. Adverse selection may be problematic for a voluntary enrollment Medicare drug benefit. Given that high out-of-pocket drug spending (secondary to drug coverage source) was a consistent predictor of enrollment likelihood, demand-side factors affecting the crowding out of employer-based drug coverage sources by a voluntary enrollment drug benefit appear minimal. However, the availability of a Medicare prescription benefit may still lead to crowd-out through employer incentives.
doi:10.1208/ps050219
PMCID: PMC2751526  PMID: 12866945
prescription drug coverage; Medicare; older adults
17.  High-Throughput screeening assays for CYP2B6 metabolism and inhibition usuing fluorogenic vivid substrates 
AAPS PharmSci  2003;5(2):88-98.
CYP2B6 is a highly polymorphic P450 isozyme involved in the metabolism of endo-and xenobiotics with known implications for the activation of many procarcinogens resulting in carcinogenesis. However, lack of validated high-throughput screening (HTS) CYP2B6 assays has limited the current understanding and full characterization of this isozyme’s involvement in human drug metabolism. Here, we have developed and characterized a fluorescence-based HTS assay employing recombinant human CYP2B6 and 2 novel fluorogenic substrates (the Vivid CYP2B6 Blue and Cyan Substrates). Assay validation included testing the inhibitory potency of a panel of drugs and compounds known to be metabolized by this isozyme, including CYP2B6 substrates, inhibitors, and known inducers. Compound rankings based on inhibitory potency in the Vivid CYP2B6 Blue and Cyan Assays matched compound rankings based on relative affinity measurements from previously published data (Ki, Kd, or Km values) for the CYP2B6 isozyme. In conclusion, these assays are proven to be robust and sensitive, with broad dynamic ranges and kinetic parameters allowing screening in HTS mode of a large panel of compounds for CYP2B6 metabolism and inhibition, and are a valuable new tool for CYP2B6 studies.
doi:10.1208/ps050218
PMCID: PMC2751525  PMID: 12866948
Cytochrome P450; CYP2B6; fluorescent substrate; drug metabolism; high-throughput screening (HTS)
18.  Functional differences in nucleoside and nucleobase transporters expressed on the rabbit corneal epithelial cell line (SIRC) and isolated rabbit cornea 
AAPS PharmSci  2003;5(2):72-85.
The purpose of this study was to investigate the expression of nucleoside/nucleobase transporters on the Statens Seruminstitut rabbit corneal (SIRC) epithelial cell line and to evaluate SIRC as an in vitro screening tool for delineating the mechanism of corneal permeation of nucleoside analogs. SIRC cells (passages 410–425) were used to study uptake of [3H]thymidine, [3H]adenine, and [3H]ganciclovir. Transport of [3H]adenine and [3H]ganciclovir was studied across isolated rabbit cornea. Uptake and transport studies were performed for 2 minutes and 120 minutes, respectively, at 34°C. Thymidine uptake by SIRC displayed saturable kinetics (Km=595.9±80.4μM, and Vmax=289.5±17.2 pmol/min/mg protein). Uptake was inhibited by both purine and pyrimidine nucleosides but not by nucleobases. [3H]thymidine uptake was sodium and energy independent but was inhibited by nitrobenzylthioinosine at nanomolar concentrations. Adenine uptake by SIRC consisted of a saturable component (Km=14.4±2.3μM, Vmax=0.4±0.04 nmol/min/mg protein) and a nonsaturable component. Uptake of adenine was inhibited by purine nucleobases but not by the nucleosides or pyrimidine nucleobases and was independent of sodium, energy, and nitrobenzylthioinosine. [3H]ganciclovir uptake involved a carrier-mediated component and was inhibited by the purine nucleobases but not by the nucleosides or pyrimidine nucleobases. However, transport of [3H]adenine across the isolated rabbit cornea was not inhibited by unlabeled adenine. Further, corneal permeability of ganciclovir across a 100-fold concentration range remained constant, indicating that ganciclovir permeates the cornea primarily by passive diffusion. Nucleoside and nucleobase transporters on rabbit cornea and corneal epithelial cell line, SIRC, are functionally different, undermining the utility of the SIRC cell line as an in vitro screening tool for elucidating the corneal permeation mechanism of nucleoside analogs.
doi:10.1208/ps050215
PMCID: PMC2751523  PMID: 12866942
nucleoside; nucleobase; cornea; SIRC; transport; antiviral agent
19.  Wet granulation fine particle ethylcellulose tablets: Effect of production variables and mathematical modeling of drug release 
AAPS PharmSci  2003;5(2):48-60.
In the present study, the applicability of fine particle ethylcellulose (FPEC) to produce matrix tablets by a wet granulation technique was evaluated. The effect of various formulation and process variables, such as FPEC content, hardness of the tablet, and solubility of the drug, on the release of drug from these tablets was examined. Tablets were prepared by wet granulation of drug and FPEC in an appropriate mass ratio. Theophylline, caffeine, and dyphylline were selected as nonionizable model drugs with solubilities from 8.3 to 330 mg/mL at 25°C. Ibuprofen, phenylpropanolamine hydrochloride, and pseudoephedrine hydrochloride were selected as ionizable drugs with solubilities from 0.1 to 2000 mg/mL at 25°C. Drug release studies were conducted in 37°C water with UV detection. As the FPEC content and the hardness of the tablets increased, the release rate of the drug decreased. The drug release rate increased with an increase in the solubility of the drug. Model equations, intended to elucidate the drug release mechanism, were fitted to the release data. Parameters were generated and data presented by SAS software. The Akaike Information Criterion was also considered to ascertain the best-fit equation. Fickian diffusion and polymer relaxation were the release mechanisms for nonionizable and ionizable drugs.
doi:10.1208/ps050213
PMCID: PMC2751521  PMID: 12866940
fine particle ethylcellulose; wet granulation; ionizable drugs; nonionizable drugs; Fickian diffusion; polymer relaxation
20.  Influence of solvents on the variety of crystalline forms of erythromycin 
AAPS PharmSci  2003;5(2):39-47.
The influence of the organic solvents widely used in the pharmaceutical industry (acetone, methylethylketone, ethanol, and isopropanol) both in the presence and in the absence of water on the crystallization behavior of erythromycin (Em), a clinically relevant antibiotic of the macrolide group, was investigated. It was observed that despite a high preference for water as a guest molecule, Em rather easily forms solvates with the organic solvents studied. Consequently, 4 distinct solvates of Em have been isolated by recrystallization: acetonate, methylethylketonate, ethanolate, and isopropanolate. It was established that in a pure organic solvent, or 1∶9 or 1∶1 water-organic solvent mixtures, the corresponding solvate is always crystallized. However, the recrystallization of erythromycin from 2∶1 water-organic solvent (excluding methylethylketone) mixture results in the formation of a crystal hydrate form. X-ray powder diffraction revealed the isostructurality of the solvates with acetone and methylethylketone. Thermogravimetric analysis showed that the loss of volatiles by all of the solvated crystals is nonstoichiometric. The desolvation behavior of the solvates with the organic solvents studied by means of variable-temperature x-ray powder diffraction indicates that in contrast to erythromycin dihydrate, they belong to a different class of solvates—those that produce an amorphous material upon desolvation.
doi:10.1208/ps050212
PMCID: PMC2751520  PMID: 12866939
erythromycin; crystallization; solvated crystals; isostructurality; clathrate; x-ray diffraction
21.  Application of dense gas techniques for the production of fine particles 
AAPS PharmSci  2003;5(2):32-38.
The feasibility of using dense gas techniques such as rapid expansion of supercritical solutions (RESS) and aerosol solvent extraction system (ASES) for micronization of pharmaceutical compounds is demonstrated. The chiral nonsteroidal anti-inflammatory racemic ibuprofen is soluble in carbon dioxide at 35°C and pressures above 90 bar. The particle size decreased to less than 2 μm while the degree of crystallinity was slightly decreased when processed by RESS. The dissolution rate of the ibuprofen (a poorly water-soluble compound) was significantly enhanced after processing by RESS. The nonsteroidal anti-inflammatory drug Cu2(indomethacin)4L2(Cu-Indo); (L=dimethylformamide [DMF]), which possessed very low solubility in supercritical CO2, was successfully micronized by ASES at 25°C and 68.9 bar using DMF as the solvent and CO2 as the antisolvent. The concentration of solute dramatically influenced the precipitate characteristics. The particles obtained from the ASES process were changed from bipyramidal to spherical, with particle size less than 5 μm, as the concentration increased from 5 to 100 mg/g. A further increase in solute concentration to 200 mg/g resulted in large porous spheres, between 20 and 50 μ, when processing Cu-Indo by the ASES method. The dissolution rate of the micronized Cu-Indo was significantly higher than the commercial product.
doi:10.1208/ps050211
PMCID: PMC2751519  PMID: 12866938
micronization; anti-inflammatory drug; rapid expansion of supercritical solution (RESS); aerosol solvent extraction system (ASES)
22.  Mechanisms of aggregate formation and carbohydrate excipient stabilization of lyophilized humanized monoclonal antibody formulations 
AAPS PharmSci  2003;5(2):21-31.
The purpose of this study was to evaluate the mechanisms of aggregate formation and excipient stabilization in freeze-dried formulations of a recombinant humanized monoclonal antibody. Protein degradation was measured using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and native size exclusion chromatography, and protein structure was studied using Fourier transform-infrared spectrometry and circular dichroism. The results showed that protein aggregates present following reconstitution were composed of native antibody structure and a reduced amount of free thiol when compared to protein monomer, which implied that intermolecular disulfides were involved in the aggregation mechanism. An excipient-free formulation resulted in reversible solid-state protein structural alteration and increased aggregation during storage. This correlated with dehydration to an extent that the amount of water was less than the estimated number of surface-accessible hydrogen-bonding sites on the protein. Improved native-like solid-state protein structure and reduced aggregation were obtained by formulation with enough carbohydrate to fulfill the hydrogen-bonding sites on the surface of the protein. Carbohydrate in excess of this concentration has less of an influence on protein aggregation. Reduced aggregation during storage was obtained by the addition of sufficient excipient to both stabilized solid-state protein structure and provide an environment that consisted of an amorphous glassy state matrix.
doi:10.1208/ps050210
PMCID: PMC2751518  PMID: 12866937
protein formulation; protein stability; recombinant humanized monoclonal antibody; aggregation; FT-IR spectrometry
23.  Evaluation of novel particles as pulmonary delivery systems for insulin in rats 
AAPS PharmSci  2003;5(2):10-20.
The purpose of the study was to evaluate the influence of calcium phosphate (CAP) and polyethylene glycol (PEG) particles on the systemic delivery of insulin administered by the pulmonary route. Two methods of pulmonary delivery were employed: intratracheal instillation and spray instillation. Insulin-CAP-PEG particles in suspension (1.2 U/kg, 110–140 μL) were administered to the lungs of fasted rats by intratracheal instillation (INCAPEG) or spray instillation (SINCAPEG). Control treatments consisted of insulin solution (1.2 U/kg) by intratracheal instillation, spray instillation, and subcutaneous administration (SC). Plasma concentrations of insulin and glucose were determined by chemiluminescence and colorimetric methods, respectively. Data were analyzed by compartmental and non-compartmental methods, and pharmacokinetic (PK) and pharmacodynamic (PD) parameters of insulin disposition were determined. PK analysis suggested that insulin administered in particles had a longer half-life, a longer mean residence time, and a smaller rate of elimination than insulin in solution. In addition, insulin bioavailability after SINCAPEG was 1.8-fold that of insulin solution administered SC. PD analysis showed that smaller areas under the effect curve and, conversely, larger areas above the effect curve were obtained after INCAPEG in comparison to insulin solution. The magnitude of this effect was increased after SINCAPEG. The presence of CAP-PEG particles appears to positively influence the disposition of insulin administered to the lungs of Sprague-Dawley rats. Spray instillation appears to be a more efficient method of delivering insulin to the lungs of rats than intratracheal instillation.
doi:10.1208/ps050209
PMCID: PMC2751517  PMID: 12866936
pulmonary delivery; insulin; CAP-PEG particles; pharmacokinetics; pharmacodynamics
24.  Development of a multidose formulation for a humanized monoclonal antibody using experimental design techniques 
AAPS PharmSci  2003;5(2):1-9.
The purpose of this study was to identify optimal preservatives for a multidose formulation of a humanized monoclonal antibody using experimental design techniques. The effect of antimicrobial parenteral preservatives (benzyl alcohol, chlorobutanol, methyl paraben, propylparaben, phenol, and m-cresol) on protein stability was assessed using size-exclusion chromatography, differential scanning calorimetry, right-angle light scattering, UV spectroscopy, and potency testing using a cell-based fluorescence-activated cell sorting method. A quick, cost-effective preservative screening test was designed. Combinations of preservatives were examined using an I-optimal experimental design. The protein was most stable in the presence of methylparaben and propylparaben, and was compatible with benzyl alcohol and chlorobutanol at low concentrations. Phenol and m-cresol were not compatible with the protein. The I-optimal experimental design indicated that as an individual preservative, benzyl alcohol was promising. The model also indicated several effective combinations of preservatives that satisfied the antimicrobial efficacy and physical stability constraints. The preservative screening test and the experimental design approach were effective in identifying optimal concentrations of antimicrobial preservatives for a multidose protein formulation; (1) benzyl alcohol, and (2) the combination of methylparaben and chlorobutanol were screened as potential candidates to satisfy the regulatory requirements of various preservative efficacy tests.
doi:10.1208/ps050208
PMCID: PMC2751516  PMID: 12866935
multidose formulation; preservative; experimental design; monoclonal antibody; protein
25.  Rheology and stability of water-in-oil-in-water multiple emulsions containing Span 83 and Tween 80 
AAPS PharmSci  2003;5(1):62-73.
Multiple emulsions are often stabilized using a combination of hydrophilic and hydrophobic surfactants. The ratio of these surfactants is important in achieving stable multiple emulsions. The objective of this study was to evaluate the long-term stability of water-in-oil-in-water (W/O/W) multiple emulsions with respect to the concentrations of Span 83 and Tween 80. In addition, the effect of surfactant and electrolyte concentration on emulsion bulk rheological properties was investigated. Light microscopy, creaming volume, and rheological properties were used to assess emulsion stability. It was observed that the optimal surfactant concentrations for W/O/W emulsion long-term stability were 20% wt/vol Span 83 in the oil phase and 0.1% wt/vol Tween 80 in the continuous phase. Higher concentrations of Tween 80 had a destructive effect on W/O/W emulsion stability, which correlated with the observation that interfacial film strength at the oil/water interface decreased as the Tween 80 concentration increased. High Span 83 concentrations increased the storage modulus G′ (solidlike) values and hence enhanced multiple emulsion stability. However, when 30% wt/vol Span 83 was incorporated, the viscosity of the primary W/O emulsion increased considerably and the emulsion droplets lost their shape. Salt added to the inner aqueous phase exerted an osmotic pressure that caused diffusion of water into the inner aqueous phase and increased W/O/W emulsion viscosity through an increase in the volume fraction of the primary W/O emulsion. This type of viscosity increase imposed a destabilizing effect because of the likelihood of rupture of the inner and multiple droplets.
doi:10.1208/ps050107
PMCID: PMC2751475  PMID: 12713279
multiple emulsions; stability; rheology; surfactant

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