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Pharmaceutical Research: An Official Journal of the American Association of Pharmaceutical Scientists (v.19, #7)

Biopharmaceutics Classification System: The Scientific Basis for Biowaiver Extensions by Lawrence X. Yu; Gordon L. Amidon; James E. Polli; Hong Zhao; Mehul U. Mehta; Dale P. Conner; Vinod P. Shah; Lawrence J. Lesko; Mei-Ling Chen; Vincent H. L. Lee; Ajaz S. Hussain (pp. 921-925).

Rational Design of Peptide-Based Tumor Vaccines by Wilson S. Meng; Lisa H. Butterfield (pp. 926-932).
Administration of synthetic peptides derived from proteins uniquely or overexpressed in tumor cells (tumor-associated antigens) can elicit tumor-specific immune responses in vivo. This is because cytotoxic T lymphocytes can recognize and lyse tumor cells that display peptides derived from tumor-associated antigens (TAAs) in the context of class I major histocompatibility complex (MHC) molecules. TAA peptides, in contrast to peptides of viral origin, generally bind weakly to the MHC molecule. In many cases, this explains the poor magnitude of T cell response directed at the tumor in vivo. Improving MHC binding as a strategy to upregulate antigen recognition can convert low affinity TAA peptides into useful tools in clinical trial settings. High-resolution structures of class I MHC molecules reported over the past two decades provided the framework for designing peptides that can induce optimal T cell response. This review will discuss the basic and clinical aspects of modifying native TAA peptides as tumor vaccines.

Keywords: class I MHC; antigenic peptides; tumor-associated antigens; cytotoxic T cells and tumor immunotherapy


Cationic Cholesterol Promotes Gene Transfection Using the Nuclear Localization Signal in Protamine by Ari Noguchi; Naohide Hirashima; Mamoru Nakanishi (pp. 933-938).
Purpose. The purpose of this study was to evaluate protamine-mediated gene transfection by liposomes with a novel cationic cholesterol derivative (I) compared to those with DC-Chol or DOTMA (Lipofectin). Methods. Plasmid pGL3 DNA was complexed to the cationic liposomes with the derivative (I), DC-Chol, or DOTMA in SFM101(Nissui) at room temperature for 15 min, and thereafter the complex was incubated with target cells (NIH3T3) for 4 h at 37°C. The cells then were washed and cultured for another 40 h in the growth medium at 37°C before luciferase assay. Results. The transfection efficiency by the liposomes with the derivative (I) was much higher than that by the liposomes with DC-Chol or DOTMA. In addition, its transfection efficiency was enhanced greatly by the addition of protamine. Atomic force microscopy showed clearly how the size of the DNA-liposome complex was changed by protamine. Furthermore, fluorescence microscopic images showed that Cy5-labeled antisense DNAs were transferred quicker into the nucleus of the target cells by the liposomes with the derivative I in the presence of protamine. Conclusion. Although there exist several possible mechanisms, such as improved protection of DNA intracellularly by derivative (I), one possibility is that the DNA-protamine-liposome complex with the derivative (I) promoted gene transfection more significantly into the nucleus of the target cells using the nuclear localization signal of protamine.

Keywords: cationic liposome; gene transfection; atomic force microscopy; confocal laser scanning microscopy; protamine; nuclear localization signal


Genetic Immunization Using Nanoparticles Engineered from Microemulsion Precursors by Zhengrong Cui; Russell J. Mumper (pp. 939-946).
Purpose. Genetic immunization using “naked” plasmid DNA (pDNA) has been shown to elicit broad humoral and cellular immune responses. However, more versatile and perhaps cell-targeted delivery systems are needed. To this end, a novel process to engineer cationic nanoparticles coated with pDNA for genetic immunization was explored. Methods. Cationic nanoparticles were engineered from warm oil-in-water microemulsion precursors composed of emulsifying wax as the oil phase and cetyltrimethylammonium bromide (CTAB) as the cationic surfactant. Plasmid DNA was coated on the surface of the cationic nanoparticles to produce pDNA-coated nanoparticles. An endosomolytic lipid and/or a dendritic cell-targeting ligand (mannan) were incorporated in or deposited on the nanoparticles to enhance the in vitro cell transfection efficiency and the in vivo immune responses after subcutaneous injection to Balb/C mice. The IgG titer to expressed β-galactosidase and the cytokine release from isolated splenocytes after stimulation were determined on 28 days. Results. Cationic nanoparticles (around 100 nm) were engineered within minutes. The pDNA-coated nanoparticles were stable at 37°C over 30 min in selected biologic fluids. Transmission electron microscopy showed the nanoparticles were spherical. Plasmid DNA-coated nanoparticles, especially those with both an endosomolytic lipid and dendritic cell-targeting ligand, resulted in significant enhancement in both IgG titer (over 16-fold) and T-helper type-1 (Th1-type) cytokine release (up to 300% increase) over “naked” pDNA. Conclusion. A novel method to engineer pDNA-coated nanoparticles for enhanced in vitro cell transfection and enhanced in vivo immune responses was reported.

Keywords: plasmid DNA; mannan; pullulan; T-helper cell; β-galactosidase


Bilayer Films for Mucosal (Genetic) Immunization via the Buccal Route in Rabbits by Zhengrong Cui; Russell J. Mumper (pp. 947-953).
Purpose. The oral buccal mucosa may be an ideal site for mucosal immunization, allowing for the needle-free administration of cost-effective vaccines. A novel mucoadhesive bilayer film was developed to test the feasibility of this route of immunization in rabbits. Methods. Bilayer films were developed using different ratios of Noveon and Eudragit S-100 as the mucoadhesive layer and a pharmaceutical wax as the impermeable backing layer. Optimal 3/8-inch films were post-loaded with 100 μg of plasmid DNA (CMV-β-gal) or β-galactosidase protein. The in vitro release rates and stability of the postloaded antigens were determined. The films were applied to the buccal pouch of rabbits on days 0, 7, and 14, and the humoral and splenocyte proliferative immune responses to β-gal were determined through day 28 and compared to those responses after conventional subcutaneous injection of adjuvanted protein. Results. The weight ratio of Noveon and Eudragit S-100 had a significant effect on adhesion time of bilayer films. Postloaded plasmid DNA and β-gal remained stable after being released from bilayer films (release of ∼60-80% in 2 h for both). Buccal immunization using novel bilayer films (109 ± 6-μm thickness) containing plasmid DNA led to comparable antigen-specific IgG titer to that of subcutaneous protein injection. All rabbits immunized with plasmid DNA via the buccal route but none by the subcutaneous route with protein antigen demonstrated splenocyte proliferative immune responses. Conclusion. The feasibility of buccal (genetic) immunization with these novel bilayer films was demonstrated.

Keywords: plasmid DNA; β-galactosidase; splenocyte; cholera toxin; vaccine


Controlled Release of Plasmid DNA from a Genetically Engineered Silk-Elastinlike Hydrogel by Zaki Megeed; Joseph Cappello; Hamidreza Ghandehari (pp. 954-959).
Purpose. The purpose of this study was to evaluate the potential of a genetically engineered silk-elastinlike polymer (SELP) as a matrix for the controlled release of plasmid DNA. Methods. The influences of SELP concentration, DNA concentration, SELP cure time, and buffer ionic strength on the release of DNA from SELP hydrogels were investigated. To calculate the average effective diffusivity of DNA within the hydrogels, the release data were fitted to a known equation. Results. DNA was released from SELP hydrogels by an ion-exchange mechanism. Under the conditions studied, the release rate was influenced by buffer ionic strength, SELP concentration, and SELP cure time but not DNA concentration. The apparent diffusivity of pRL-CMV plasmid DNA in SELP hydrogels ranged from 3.78 ± 0.37 × 10-10 cm2/s (for hydrogels containing 12% w/w SELP and cured for 4 h) to 4.69 ± 2.81 × 10-9 cm2/s (for hydrogels containing 8% w/w SELP and cured for 1 h). Conclusions. The ability to precisely customize the structure and physicochemical properties of SELPs using recombinant techniques, coupled with their ability to form injectable, in situ hydrogel depots that release DNA, renders this class of polymers an interesting candidate for further evaluation in controlled gene delivery.

Keywords: genetically engineered polymers; controlled release; gene delivery; silk-elastinlike polymers; hydrogels


The Lower-Generation Polypropylenimine Dendrimers Are Effective Gene-Transfer Agents by Bernd H. Zinselmeyer; Simon P. Mackay; Andreas G. Schatzlein; Ijeoma F. Uchegbu (pp. 960-967).
Objective. To evaluate polypropylenimine dendrimers (generations 1-5: DAB 4, DAB 8, DAB 16, DAB 32, and DAB 64) as gene delivery systems. Methods. DNA binding was evaluated by measuring the reduced fluorescence of ethidium bromide, and molecular modelling of dendrimer-DNA complexes also was performed. Cell cytotoxicity was evaluated against the A431 cell line using the MTT assay. In vitro transfection was evaluated against the A431 cell line using the β-galactosidase reporter gene and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulphate (DOTAP) served as a positive control. Results. Molecular modeling and experimental data revealed that DNA binding increased with dendrimer generation. Cell cytotoxicity was largely generation dependent, and cytotoxicity followed the trend DAB 64 > DAB 32 > DAB 16 > DOTAP > DAB 4 > DAB 8, whereas transfection efficacy followed the trend DAB 8 = DOTAP = DAB 16 > DAB 4 > DAB 32 = DAB 64. Conclusion. The generation 2 polypropylenimine dendrimer combines a sufficient level of DNA binding with a low level of cell cytoxicity to give it optimum in vitro gene transfer activity.

Keywords: polypropylenimine dendrimers; gene delivery; Astramol; cytotoxicity; molecular modeling


Glucose-Modulated Transgene Expression via Recombinant Adeno-Associated Virus by Ya-Wun Yang; Yuan-Chiao Hsieh; Chih-Kai Chao (pp. 968-975).
Purpose. The objective of this study was to examine glucose-modulated reporter gene expression via recombinant adeno-associated viral vectors both in vitro and in vivo. Methods. Huh7 human hepatoma cells were transduced by recombinant adeno-associated virus (rAAV) vectors containing the luciferase gene under control of the rat insulin I gene promoter and a cytomegalovirus immediate-early promoter driving-enhanced green fluorescence protein gene. The reporter gene expression was evaluated by glucose stimulation either in the absence or presence of insulin secretagogues, including phorbol-12-myristate-13-acetate, dibutyryl cyclic AMP, and forskolin. In vivo studies were performed by injecting rAAV into the livers of streptozotocin-induced diabetic C57BL/6J mice followed by measurements of blood glucose concentration and luciferase activity assays 2 weeks after rAAV injection. Results. At a multiplicity of infection of 500, approximately 66-69% of cells expressed enhanced green fluorescence protein at 48 h post-transduction. Luciferase activities, driven by the insulin gene promoter, in the rAAV-transduced hepatoma cells responded to millimolars of glucose. The addition of phorbol-12-myristate-13-acetate, dibutyryl cyclic AMP, and forskolin increased luciferase expression in the presence of either 1 mM or 25 mM glucose. The stimulation of luciferase activities by these substances was inhibited by the presence of 100 nM staurosporine. Exposure to increments of exogenous insulin up to 10-7 M inhibited luciferase gene expression in rAAV-transduced Huh7 cells. The in vivo experiments demonstrated good correlation between luciferase activities and blood glucose levels in streptozotocin-induced diabetic animals. Conclusion. rAAV is a promising vector for hepatic gene therapy for diabetes. Glucose and insulin secretagogues modulated transgene expression in rAAV-transduced hepatoma cells, suggesting that conditions affecting insulin gene promoter function in pancreatic islet beta cells also affect transgene expression in human hepatoma cells conferred with insulin gene promoter. Results obtained from in vivo experiments demonstrated that glucose modulated transgene expression can be obtained in rAAV-treated diabetic C57BL/6J mice.

Keywords: diabetes mellitus; gene delivery; glucose-responsive- ness; recombinant adeno-associated virus


Prediction of Drug Transport Through the Blood-Brain Barrier in Vivo: A Comparison Between Two in Vitro Cell Models by Stefan Lundquist; Mila Renftel; Julien Brillault; Laurence Fenart; Roméo Cecchelli; Marie-Pierre Dehouck (pp. 976-981).
Purpose. Studies were conducted to evaluate whether the use of an in vitro model of the blood-brain barrier (BBB) resulted in more accurate predictions of the in vivo transport of compounds compared to the use of a human intestinal cell line (Caco-2). Methods. The in vitro BBB model employs bovine brain capillary endothelial cells co-cultured with primary rat astrocytes. The Caco-2 cells originate from a human colorectal carcinoma. The rat was used as experimental animal for the in vivo studies. Results. Strong correlations (r = 0.93-0.95) were found between the results generated by the in vitro model of the BBB and two different methodologies to measure the permeability across the BBB in vivo. In contrast, a poor correlation (r = 0.68) was obtained between Caco-2 cell data and in vivo BBB transport. A relatively poor correlation (r = 0.74) was also found between the two in vitro models. Conclusion. The present study illustrates the limitations of the Caco-2 model to predict BBB permeability of compounds in vivo. The results emphasize the fact that the BBB and the intestinal mucosa are two fundamentally different biologic barriers, and to be able to make accurate predictions about the in vivo CNS penetration of potential drug candidates, it is important that the in vitro model possesses the main characteristics of the in vivo BBB.

Keywords: blood-brain barrier; drug delivery; in vitro models; in vivo studies; intestinal barrier


Role of Ascorbic Acid in Stratum Corneum Lipid Models Exposed to UV Irradiation by Hagen Trommer; Rolf Böttcher; Andreas Pöppl; Joachim Hoentsch; Siegfried Wartewig; Reinhard H. H. Neubert (pp. 982-990).
Purpose. The effects of ascorbic acid on Stratum corneum lipid models following ultraviolet irradiation were studied adding iron ions as transition metal catalysts. Methods. Lipid peroxidation was quantified by the thiobarbituric acid assay. The qualitative changes were studied on a molecular level by mass spectrometry. To elucidate the nature of free radical involvement we carried out electron paramagnetic resonance studies. The influence of ascorbic acid on the concentration of hydroxyl radicals was examined using the spin trapping technique. Moreover, we checked the vitamin's ability to react with stable radicals. Results. Ascorbic acid was found to have prooxidative effects in all lipid systems in a concentration dependent manner. The degradation products of ascorbic acid after its prooxidative action were detected. The concentration of the hydroxyl radicals in the Fenton assay was decreased by ascorbic acid. The quantification assay of 2,2-diphenyl-1-picrylhydrazyl hydrate showed reduced concentration levels of the stable radical caused by ascorbic acid. Conclusions. Considering human skin and its constant exposure to UV light and oxygen, an increased pool of iron ions in irradiated skin and the depletion of co-antioxidants, the administration of ascorbic acid in cosmetic formulations or in sunscreens could unfold adverse effects among the Stratum corneum lipids.

Keywords: ascorbic acid; thiobarbituric acid assay; mass spectrometry; EPR, oxidative stress


Transdermal Delivery of Pergolide from Surfactant-Based Elastic and Rigid Vesicles: Characterization and in Vitro Transport Studies by P. Loan Honeywell-Nguyen; Peter M. Frederik; Paul H. H. Bomans; Hans E. Junginger; Joke A. Bouwstra (pp. 991-997).
Purpose. The aim of this study was to investigate the effect of elastic and rigid vesicles on the penetration of pergolide across human skin. Methods. Vesicles used consisted of the bilayer-forming surfactant L-595 (sucrose laurate ester) and the micelle-forming surfactant PEG-8-L (octaoxyethylene laurate ester), together with the stabilizer sulfosuccinate. A series of L-595/PEG-8-L/sulfosuccinate vesicles were investigated, ranging from very rigid to very elastic. Pergolide-loaded elastic and rigid vesicles were visualized using Cryo-TEM and characterized for size and stability. Transdermal penetration of pergolide from different vesicle compositions was studied in vitro using flow-through Franz diffusion cells. A saturated buffer solution served as the control. Results. Vesicle composition had a major effect on the physico-chemical characteristics, morphology and drug solubility of the vesicular system. L-595/PEG-8-L/sulfosuccinate (70/30/5) elastic vesicles gave the best balance between vesicle stability and elasticity, as well as the highest drug solubility. Transport studies clearly showed that elastic vesicles were superior to rigid vesicles. Elastic vesicles enhanced the drug transport compared to the buffer control, although rigid vesicles decreased the drug transport. The best drug transport was achieved from L-595/PEG-8-L/sulfosuccinate (70/30/5) elastic vesicles, resulting in a steady-state flux of 13.6 ± 2.3 ng/(h*cm2). This was a 6.2-fold increase compared to the most rigid vesicles. Conclusions. This study supports the hypothesis that elastic vesicles are superior to rigid vesicles as vehicles for transdermal drug delivery.

Keywords: transdermal delivery; elastic vesicles; pergolide


Nasal Delivery of Insulin Using Novel Chitosan Based Formulations: A Comparative Study in Two Animal Models Between Simple Chitosan Formulations and Chitosan Nanoparticles by A. M. Dyer; M. Hinchcliffe; P. Watts; J. Castile; I. Jabbal-Gill; R. Nankervis; A. Smith; L. Illum (pp. 998-1008).
Purpose. To investigate whether the widely accepted advantages associated with the use of chitosan as a nasal drug delivery system, might be further improved by application of chitosan formulated as nanoparticles. Methods. Insulin-chitosan nanoparticles were prepared by the ionotropic gelation of chitosan glutamate and tripolyphosphate pentasodium and by simple complexation of insulin and chitosan. The nasal absorption of insulin after administration in chitosan nanoparticle formulations and in chitosan solution and powder formulations was evaluated in anaesthetised rats and/or in conscious sheep. Results. Insulin-chitosan nanoparticle formulations produced a pharmacological response in the two animal models, although in both cases the response in terms of lowering the blood glucose levels was less (to 52.9 or 59.7% of basal level in the rat, 72.6% in the sheep) than that of the nasal insulin chitosan solution formulation (40.1% in the rat, 53.0% in the sheep). The insulin-chitosan solution formulation was found to be significantly more effective than the complex and nanoparticle formulations. The hypoglycaemic response of the rat to the administration of post-loaded insulin-chitosan nanoparticles and insulin-loaded chitosan nanoparticles was comparable. As shown in the sheep model, the most effective chitosan formulation for nasal insulin absorption was a chitosan powder delivery system with a bioavailability of 17.0% as compared to 1.3% and 3.6% for the chitosan nanoparticles and chitosan solution formulations, respectively. Conclusion. It was shown conclusively that chitosan nanoparticles did not improve the absorption enhancing effect of chitosan in solution or powder form and that chitosan powder was the most effective formulation for nasal delivery of insulin in the sheep model.

Keywords: chitosan; nanoparticles; complex; nasal delivery; insulin


Evaluation of the AERx Pulmonary Delivery System for Systemic Delivery of a Poorly Soluble Selective D-1 Agonist, ABT-431 by Franklin W. Okumu; Rai-Yun Lee; James D. Blanchard; Anthony Queirolo; Christine M. Woods; Peter M. Lloyd; Jerry Okikawa; Igor Gonda; Stephen J. Farr; Reid Rubsamen; Akwete L. Adjei; Richard J. Bertz (pp. 1009-1012).
Purpose. ABT-431 is a chemically stable, poorly soluble prodrug that rapidly converts in vivo to A-86929, a selective dopamine D-1 receptor agonist. This study was designed to evaluate the ability of the AERx™ pulmonary delivery system to deliver ABT-431 to the systemic circulation via the lung. Methods. A 60% ethanol formulation of 50 mg/mL ABT-431 was used to prepare unit dosage forms containing 40 μL of formulation. The AERx system was used to generate a fine aerosol bolus from each unit dose that was collected either onto a filter assembly to chemically assay for the emitted dose or in an Andersen cascade impactor for particle size analysis. Plasma samples were obtained for pharmacokinetic analysis after pulmonary delivery and IV dosing of ABT-431 to nine healthy male volunteers. Doses from the AERx system were delivered as a bolus inhalation(s) (1, 2, 4, and 8 mg) and intravenous infusions were given over 1hr (5 mg). Pharmacokinetic parameters of A-86929 were estimated using noncompartmental analysis. Results. The emitted dose was 1.02 mg (%RSD = 11.0, n = 48). The mass median aerodynamic diameter of the aerosol was 2.9 ± 0.1 μm with a geometric standard deviation of 1.3 ± 0.1 (n = 15). Tmax (mean ± SD) after inhalation ranged from 0.9 ± 0.6 to 11.5 ± 2.5. The mean absolute pulmonary bioavailibility (as A-86929) based on emitted dose ranged from 81.9% to 107.4%. Conclusions. This study demonstrated that the AERx pulmonary delivery system is capable of reproducibly generating fine nearly monodisperse aerosols of a small organic molecule. Aerosol inhalation utilizing the AERx pulmonary delivery system may be an efficient means for systemic delivery of small organic molecules such as ABT-431.

Keywords: AERx; dopamine D-1; ABT-431; DAS-431; pulmonary delivery; non-invasive delivery


Characterization of Mefenamic Acid-Guaiacol Ester: Stability and Transport Across Caco-2 Cell Monolayers by Vimon Tantishaiyakul; Kamonthip Wiwattanawongsa; Sirirat Pinsuwan; Srirat Kasiwong; Narubodee Phadoongsombut; Sanae Kaewnopparat; Nattha Kaewnopparat; Yon Rojanasakul (pp. 1013-1018).
Purpose. Prodrug of non-steroidal anti-inflammatory drugs (NSAIDs) or NSAIDs linked with guaiacol have been reported to suppress gastrointestinal (GI) toxicity or induce GI protective effect. In this study, mefenamic-guaiacol ester was synthesized and its physicochemical properties, stability, and transport across Caco-2 monolayers were investigated. Methods. Synthesis of the ester was carried out using mefenamic acid, guaiacol, N, N′-dimethylaminopyridine, and N, N′dicyclohexylcarbodiimide. The hydrolysis of the ester was investigated in aqueous buffer solutions pH 1-12 as well as in Caco-2 homogenate, human plasma, and porcine liver esterase. Caco-2 cell monolayers were utilized for transport studies. Due to the high lipophilicity of the ester with a calculated logP of 6.15, bovine serum albumin (BSA, 4%) was included in the receiver compartment to obtain a good in vitro-in vivo correlation. Permeation of the ester was assessed with or without the exposure of cells to PMSF, an inhibitor of esterase. Results. The ester was stable at a wide pH range from 1-10. However, it was hydrolyzed by enzymes from porcine liver esterase and Caco-2 homogenate. With the PMSF exposure on the apical (AP) side and in the presence of 4% BSA on the basolateral (BL) side, the transported amount of the ester from AP-to-BL direction was 14.63% after 3 hr with a lag time of 23 min. The Papp for the ester was 4.72 × 10-6 cm s-1. Conclusion. The results from hydrolysis studies indicate that this ester is a prodrug. The Papp value suggests the moderate absorption characteristic of the compound. The accumulation of this highly lipophilic ester in Caco-2 cells is reduced in the presence of BSA.

Keywords: mefenamic acid; guaiacol; prodrug; caco-2 cells; hydrolysis; transport


Modifying the Bitterness of Selected Oral Pharmaceuticals with Cation and Anion Series of Salts by Russell S. J. Keast; Paul A. S. Breslin (pp. 1019-1026).
Purpose. NaCl has proven to be an effective bitterness inhibitor, but the reason remains unclear. The purpose of this study was to examine the influence of a variety of cations and anions on the bitterness of selected oral pharmaceuticals and bitter taste stimuli: pseudoephedrine, ranitidine, acetaminophen, quinine, and urea. Method. Human psychophysical taste evaluation using a whole mouth exposure procedure was used. Results. The cations (all associated with the acetate anion) inhibited bitterness when mixed with pharmaceutical solutions to varying degrees. The sodium cation significantly (P < 0.003) inhibited bitterness of the pharmaceuticals more than the other cations. The anions (all associated with the sodium cation) also inhibited bitterness to varying degrees. With the exception of salicylate, the glutamate and adenosine monophosphate anions significantly (P < 0.001) inhibited bitterness of the pharmaceuticals more than the other anions. Also, there were several specific inhibitory interactions between ammonium, sodium and salicylate and certain pharmaceuticals. Conclusions We conclude that sodium was the most successful cation and glutamate and AMP were the most successful anions at inhibiting bitterness. Structure forming and breaking properties of ions, as predicted by the Hofmeister series, and other physical-chemical ion properties failed to significantly predict bitterness inhibition.

Keywords: bitter taste; bitterness blocking; salts; taste psychophysics; pseudoephedrine; ranitidine; acetaminophen


Influence of Structural Variations in Peptidomimetic 4-Amidinophenylalanine-Derived Thrombin Inhibitors on Plasma Clearance and Biliary Excretion in Rats by Jörg Hauptmann; Torsten Steinmetzer; Helmut Vieweg; Peter Wikström; Jörg Stürzebecher (pp. 1027-1033).
Purpose. Systemic and hepato-biliary clearance of peptidomimetic thrombin inhibitors of the 4-amidinophenylalanine amide-type, derived from NAPAP (Nα-[2-naphthylsulfonyl-glycyl]-4-amidinophenylalanine-piperidide) by substituting Gly in P2 for natural and unnatural amino acids or by varying the C- and N-terminal moieties, resp., were investigated. Methods. Concentrations of the compounds administered as intravenous bolus injection at a dose of 1 mg/kg to bile duct-cannulated rats were determined in plasma and bile samples collected over 4 hours using reversed-phase HPLC. Results. NAPAP and the derivatives with additional charged groups are comparatively hydrophilic compounds. For NAPAP and most of the derivatives the biliary clearance accounted for a high percentage of the rapid systemic plasma clearance. Derivatives 2a-c with a second basic group in P2 position showed lower systemic and biliary clearance compared to NAPAP, whereas their cumulative biliary excretion after a period of 120 min was less affected. Bis-benzamidine derivatives 4a and 5 with the second amidino group in the N-terminal moiety had the lowest biliary clearance. Additional carboxylic groups reduced the systemic and biliary clearance only as free amidinophenylalanine carboxyl in 3a and 5. No influence compared to NAPAP was observed for 2d with a free carboxyl group in P2 position. Conclusions. The weak correlation of the log P values of the compounds with the clearance parameters indicates the influence of structural variations, especially of charged groups, in this series of compounds rather than overall lipophilicity on hepato-biliary elimination mediated by hepatocellular transporters.

Keywords: thrombin inhibitors; structures; charged groups; systemic clearance; biliary clearance


The N-Demethylation of the Doxepin Isomers Is Mainly Catalyzed by the Polymorphic CYP2C19 by Sebastian Härtter; Gunnel Tybring; Thomas Friedberg; Harald Weigmann; Christoph Hiemke (pp. 1034-1037).
Purpose. This study was conducted to identify the cytochrome P450s (CYPs) responsible for the metabolism of the cis- and trans-isomers of the tricyclic antidepressant doxepin to its pharmacologically active N-desmethylmetabolite by in vitro techniques. Methods. The doxepin N-demethylation was studied by means of pooled human liver microsomes and chemical inhibitors, recombinant human (rh)-CYPs, and geno- and phenotyped human liver microsomes. Results. The N-demethylation of both isomers was inhibited most prominently by tranylcypromine (CYP2C19) to more than 50%. Furafylline (CYP1A2) and sulfaphenazole (CYP2C9) inhibited the N-demethylation to a lesser extent while quinidine (CYP2D6) or troleandomycine (CYP3A4) had no effect. Rh-CYP2C19, -CYP1A2, and -CYP2C9 were able to N-demethylate cis- and trans-doxepin. Only traces of trans-desmethyldoxepin were detectable when CYP3A4 was used. The maximum velocity in the cis- and trans-doxepin N-demethylation was significantly (P < 0.05) lower in microsomes with low CYP2C19 activity (345 ± 44 and 508 ± 75 pmol/min/mg protein, respectively) compared to those with high CYP2C19 activity (779 ± 132 and 1189 ± 134 pmol/min/mg). Conclusion. The present study demonstrates a significant contribution of the polymorphic CYP2C19 to the N-demethylation of doxepin. CYP2C9 and CYP1A2 play a minor role and CYP3A4 does not contribute substantially.

Keywords: doxepin; cis-/trans-isomers; in vitro ; metabolism; tricyclic antidepressants


Differential Modulation of P-Glycoprotein Expression and Activity by Non-Nucleoside HIV-1 Reverse Transcriptase Inhibitors in Cell Culture by Elke Störmer; Lisa L. von Moltke; Michael D. Perloff; David J. Greenblatt (pp. 1038-1045).
Purpose. This study investigated the effects of the non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTI) nevirapine (NVR), efavirenz (EFV), and delavirdine (DLV) on P-glycoprotein (P-gp) activity and expression to anticipate P-gp related drug-drug interactions associated with combination therapy. Methods. NNRTIs were evaluated as P-gp substrates by measuring differential transport across Caco-2 cell monolayers. Inhibition of P-gp mediated rhodamine123 (Rh123) transport in Caco-2 cells was used to assess P-gp inhibition by NNRTIs. Induction of P-gp expression and activity in LS180V cells following 3-day exposure to NNRTIs was measured by western blot analysis and cellular Rh123 uptake, respectively. Results. The NNRTIs showed no differential transport between the basolateral to apical and apical to basolateral direction. NNRTI transport in either direction was not affected by the P-gp inhibitor verapamil. DLV inhibited Rh123 transport, causing a reduction to 15% of control at 100 μM (IC50 = 30 μM). NVR caused a concentration-dependent induction of P-gp expression in LS180V cells resulting in a 3.5-fold increase in immunoreactive P-gp at 100 μM NVR. Induction attributable to EFV and DLV was quantitatively smaller. NVR significantly reduced cellular uptake of Rh123 into LS180V cells, indicating increased drug efflux due to induced P-gp activity; effects of EFV and DLV were smaller. Acute DLV treatment of LS180V cells previously induced with NVR or ritonavir did not reverse the decreased Rh123 cell accumulation. Conclusions. NNRTIs show differential effects on P-gp activity and expression in vitro. Clinical studies are required to elucidate the clinical importance of potential drug interactions.

Keywords: P-glycoprotein; induction; inhibition; non-nucleoside reverse transcriptase inhibitors; Caco-2; LS180


A Novel in Vitro Delivery System for Assessing the Biological Integrity of Protein upon Release from PLGA Microspheres by Anne Aubert-Pouëssel; David C. Bibby; Marie-Claire Venier-Julienne; François Hindré; Jean-Pierre Benoît (pp. 1046-1051).
Purpose. The development of a novel in vitro system is required to assess the stability and release kinetics of a protein microsphere formulation used for drug delivery to the brain. Methods. Microspheres containing lysozyme as model protein were prepared using a (w/o/w) emulsion-solvent evaporation process. Both the active and total (active + inactive) encapsulation efficiencies and release profiles were determined. The biologic activity of lysozyme was measured using bacterial cell lysis; total protein content was measured using a 125I-radiolabel. A novel in vitro apparatus was developed to determine kinetics over a sustained time period (>30 days). Results. The microencapsulation technique allowed an entrapment of active lysozyme at 80 ± 4% and a sustained (>42 days) in vitro release. The kinetics study showed that the novel in vitro system was able to detect the release of low amounts (ng) of protein. To improve the stability of the protein within microspheres and allow the release of biologically active lysozyme, a basic additive ( Mg(OH)2 ) was successfully encapsulated. Conclusions. This novel in vitro system was appropriate to study protein microsphere release kinetics. In addition, the model is cost-effective and mimes brain physiological conditions more closely than previous models.

Keywords: protein release; lysozyme; microspheres; poly(lactic-co-glycolic acid); stabilization


Sustained Release Chemotherapeutic Microspheres Provide Superior Efficacy over Systemic Therapy and Local Bolus Infusions by Dwaine F. Emerich; Pamela Snodgrass; Denise Lafreniere; Reginald L. Dean; Heather Salzberg; Joanne Marsh; Brigido Perdomo; Mahin Arastu; Shelley R. Winn; Raymond T. Bartus (pp. 1052-1060).
Purpose. The present studies evaluated the ability of injectable, biodegradable microspheres releasing carboplatin, doxorubicin, or 5-fluorouracil to suppress the growth of solid tumors implanted subcutaneously or intramuscularly. Methods. Seven to 10 days after implantation of MATB-III cells, rats received systemic chemotherapy, intratumoral bolus chemotherapy, or injections of chemotherapeutic microspheres into the tumor center or multiple sites along the outer perimeter of the tumor. Results. A single treatment with carboplatin, doxorubicin, or 5-fluorouracil microspheres along the perimeter of the tumors produced a significant, dose-related suppression in tumor growth, relative to injections directly into the tumor center. Moreover, five temporally-spaced microsphere treatments along the tumor perimeter (with either doxorubicin or 5-fluorouracil microspheres) completely eradicated 100% of the subcutaneous tumors and 40-53% of the intramuscular tumors. Polypharmacy, accomplished by blending doxorubicin- and 5-fluorouracil-loaded microspheres and injecting them into the tumors was even more efficacious than sustained delivery of either drug alone. Comparable doses of systemic chemotherapy or intratumoral bolus chemotherapy were ineffective. Conclusions. Injectable microspheres might be ideal for local, sustained delivery of chemotherapeutic agents to solid tumors. However, attention must be paid to the placement of the microspheres, for injections around the tumor perimeter may be required for efficacy.

Keywords: sustained release; microspheres; peripheral tumors; carboplatin; doxorubicin; 5-FU


Long-Circulating Poly(Ethylene Glycol)-Modified Gelatin Nanoparticles for Intracellular Delivery by Goldie Kaul; Mansoor Amiji (pp. 1061-1067).
Purpose. The objective of this study was to develop and characterize long-circulating, biodegradable, and biocompatible nanoparticulate formulation as an intracellular delivery vehicle. Methods. Poly(ethylene glycol) (PEG)-modified gelatin was synthesized by reacting Type-B gelatin with PEG-epoxide. The nanoparticles, prepared by pH and temperature controlled ethanol-water solvent displacement technique, were characterized for mean size, size distribution, and surface morphology. Electron spectroscopy for chemical analysis (ESCA) was used to confirm the surface presence of PEG chains. In vitro release of tetramethylrhodamine-labeled dextran (TMR-dextran, Mol. wt. 10,000 daltons) from the nanoparticle formulations was examined in PBS, with and without 0.2-mg/ml protease, at 37°C. Relative cytotoxicity profile of control and PEGylated gelatin was evaluated in BT-20 a human breast cancer cell line. The nanoparticles were incubated with BT-20 cells to determine uptake and cellular distribution using confocal microscopy. Results. Gelatin and PEGylated gelatin nanoparticles were found to be spherical in shape with a smooth surface in a size range of 200-500 nm and a unimodal size distribution. ESCA results showed an increase in the ether carbon (-C-O-) peak in the PEGylated gelatin nanoparticles due to the presence of PEG chains. The presence of PEG chains decreased the percent release of TMR-dextran in the presence of proteolytic enzyme due to steric repulsion. Cytotoxicity assays indicated that both gelatin and PEGylated gelatin were completely non-toxic to the cells. A large fraction of the administered control gelatin and PEGylated gelatin nanoparticles were found to be concentrated in the perinuclear region of the BT-20 cells after 12 hours indicating possible vesicular transport through initial uptake by endocytosis and endosomal processing. Conclusion. The results of this study show that PEGylation of gelatin may prove beneficial as long-circulating delivery system in vivo. Additionally, the nanoparticles could encapsulate hydrophilic macromolecules and are internalized by tumor cells.

Keywords: intracellular delivery vehicle; PEGylated gelatin nanoparticles; rhodamine-labeled dextran (TMR-dextran); BT-20 cells; endocytosis

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