Spray Dried Powders and Powder Blends of Recombinant Human Deoxyribonuclease (rhDNase) for Aerosol Delivery

Purpose. We have used rhDNase to investigate the feasibility of developing a dry protein powder aerosol for inhalation delivery. Methods. Powders of rhDNase alone and with sodium chloride were prepared by spray drying. Powder blends were obtained by mixing (tumbling and sieving) pure rhDNase powder with 'carrier' materials (lactose, mannitol or sodium chloride). The weight percent of drug in the blends was between 5 and 70%. The particle size distributions and crystallinity of the spray dried powders were obtained by laser diffraction and X-ray powder diffraction, respectively. Particle morphology was examined by scanning electron microscopy. The ability of the powders and powder blends to be dispersed into respirable aerosols was measured using a Rotahaler connected to a multistage liquid impinger operating at 60 L/min. Results. Pure rhDNase powder was quite cohesive with a fine particle fraction (FPF or 'respirable fraction': % wt. of particles < 7 m in the aerosol cloud) of about 20%. When particles also contained NaCl, the powders were dispersed better to form aerosols. A linear relationship was observed between the NaCl content and FPF for a similar primary size (~3 m volume median diameter) of particles. The particle morphology of these powders varied systematically with the salt content. For the blends, SEM revealed a monolayer-like adhesion of the fine drug particles to the carriers at drug contents 50 % wt. An overall 2-fold increase in FPF of rhDNase in the aerosol cloud was obtained for all the blends compared to the pure drug aerosols. Conclusions. The aerosol properties of spray dried rhDNase powders can be controlled by incorporation of a suitable excipient, such as NaCl, and its relative proportion. Coarse carriers can also enhance the performance of rhDNase dry powder aerosols.     

Protein Powders for Encapsulation: A Comparison of Spray-Freeze Drying and Spray Drying of Darbepoetin Alfa

Purpose. To evaluate spray-freeze drying and spray drying processes for fabricating micron-sized particles of darbepoetin alfa (NESP, Aranesp®) with uniform size distribution and retention of protein integrity, requirements for encapsulation. Methods. Darbepoetin alfa was spray-freeze dried using ultrasonic atomization at 120 kHz and 25 kHz and spray dried at bench-top and pilot scales. Reconstituted powders were evaluated by size exclusion chromatography and UV/VIS spectroscopy. Powder physical properties were also characterized. Results. Spray-freeze drying resulted in aggregation of darbepoetin alfa. Aggregates (primarily insoluble) formed on drying and/or reconstitution. Particle size distributions were broad (span 3.6) at both nozzle frequencies. Annealing before drying reduced aggregate levels slightly but increased particle size over 5-fold. Spray drying at inlet temperatures up to 135°C (and outlet temperatures up to 95°C) showed little impact on integrity. Integrity at bench-top and pilot scales was identical, with 0.2% dimer and no high molecular weight or insoluble aggregates detected. Particle size was small ( 2.3 m) with uniform distribution (span 1.4) at both process scales. Conclusions. Under the conditions tested spray drying, conducted at bench-top and pilot scales with commercially available equipment, was superior to spray-freeze drying for the fabrication of darbepoetin alfa particles for encapsulation.     

Influence of Particle Size,Air Flow,and Inhaler Device on the Dispersion of Mannitol Powders as Aerosols

Purpose. To study the effect of particle size, air flow and inhaler type on the dispersion of spray dried mannitol powders into aerosols. Methods. Mannitol powders were prepared by spray drying. The solid state properties of the powders were determined by laser diffraction, X-ray powder diffraction, scanning electron microscopy, freeze fracture, Karl Fischer titration and gas pycnometry. The powders were dispersed using Rotahaler^® and Dinkihaler^®, connected to a multistage liquid impinger at different air flows. Results. Three crystalline mannitol powders with primary particle size (MMD) 2.7, 5.0, 7.3 m and a similar polydispersity were obtained. The particles were spherical with a density of 1.5 g/cm³ and a moisture content of 0.4 wt.%. At an air flow of 30 L/min all the powders were poorly dispersed by both inhalers. With the Rotahaler^® increasing the flow (60–120 L/min) increased the fine particle fraction (FPF) in the aerosols for the 2.7 m powder, and decreased the FPF for the 7.3 m powder; whereas the FPF for 5.0 m powder was unaffected. With the Dinkihaler^®, all the powders were near complete dispersion at 60 L/min. Conclusions. The FPF in the mannitol powder aerosols was determined by an interplay of the particle size, air flow and inhaler design.     

Influence of Hydrodynamics and Particle Size on the Absorption of Felodipine in Labradors

Purpose. To study the influence of GI hydrodynamics and drug particle size on felodipine absorption in the dog. Methods. Labradors fistulated at midjejunum were used to selectively study the influence of hydrodynamics and particle size on the in vivodissolution and absorption of the poorly soluble, lipophilic drug felodipine. A combination of infusion and oral administration of either normal saline or a 5% glucose solution was used to maintain fasted and establish fed state motility patterns, respectively. The absorption characteristics of both a micronized (8 m) and a coarse fraction (125 m) of felodipine were subsequently studied under these two motility patterns. Results. A reduction in particle size led up to an approximate 22-fold increase in maximum plasma concentration and up to an approximate 14-fold increase in area under the curve, with a commensurate decrease in the time at which the maximum plasma concentration occurred. Although the absorption of felodipine from the solution and micronized suspension was not influenced by a change in the hydrodynamics, felodipine was absorbed from the coarse suspension almost twice as well in the fed state as under fasted conditions. Conclusions. Absorption from coarse suspensions of felodipine was sensitive to luminal hydrodynamics, whereas micronized suspensions were not. However, the particle size seems to have a much more important influence on the bioavailability of felodipine than the hydrodynamics per se.     

How Much Particle Surface Corrugation Is Sufficient to Improve Aerosol Performance of Powders?

No HeadingPurpose. The current study aimed to quantify the different degree of particle surface corrugation and correlate it to the aerosol performance of powders.Methods. Powders of different degree of surface corrugation were prepared by spray drying under varying conditions. The solid-state properties of the powders including particle size, morphology, crystal form, true density, and moisture content were characterized. The degree of surface corrugation was quantified by the surface fractal dimension (D_S) obtained by light scattering. The aerosol performance was studied by dispersing the powders using the Rotahaler at 60 L/min into a multi-stage liquid impinger. Fine particle fraction (FPF) was expressed as the wt% of BSA particles of size 5 m in the aerosol.Results. Four powders of increasing degree of particle surface corrugation were prepared, with D_S ranging from 2.06 for the least corrugated to 2.41 for the most corrugated. The powders had a similar size distribution (VMD 3 m, span 1.4–1.5) and solid-state properties. Increasing the surface corrugation, D_S, slightly from 2.06 to 2.18 enhanced the FPF significantly from 27% to 41%. This was explained by the reduced area of contacts and increased separation distance between the particles. Further increase of corrugation (D_S 2.18) did not improve FPF.Conclusion. Powders with varying degrees of corrugation were successfully obtained by spray drying with their surface roughness quantified by fractal analysis. It was shown that only a relatively small degree of surface corrugation was sufficient to accomplish a considerable improvement in the aerosol performance of the powder.     

Use of Solid Corrugated Particles to Enhance Powder Aerosol Performance

Purpose. To study the dispersion performance of non-porous corrugated particles, with a focus on the effect of particle surface morphology on aerosolization of bovine serum albumin (BSA) powders. Methods. The solid-state characteristics of the spray-dried BSA powders, one consisting of smooth spherical particles and another corrugated particles, were characterized by laser diffraction, X-ray powder diffraction, scanning electron microscopy, confocal microscopy, thermogravimetric analysis, surface area analyzer, and buoyancy method. The powders were dispersed using the Rotahaler® and the Dinkihaler® coupled to a four-stage liquid impinger operating at 30 to 120 L/min. Fine particle fraction (FPF) was expressed as the wt. % of BSA particles of size 5 m collected from the liquid impinger. Results. Apart from the morphology and morphology-related properties (specific surface area, envelope density), the corrugated particles and spherical particles of BSA had very similar solid-state characteristics (particle size distribution, water content, true density, amorphous nature). Using the Dinkihaler®, the FPFs of the corrugated particles were 10-20 wt. % higher than those of the smooth particles. Similar FPF differences were found for the powders dispersed by the Rotahaler®, but the relative changes were larger. In addition, the differences were inversely proportional to the air flows (17.3% at 30 L/min, 25.2% at 60 L/min, 13.8% at 90, 8.5% at 120 L/min). Depending on the inhaler, capsule and device retention and impaction loss at the impinger throat were lower for the corrugated particles. Conclusions. Enhanced aerosol performance of powders can be obtained by surface modification of the particles. The surface asperities of the corrugated particles could lower the true area of contact between the particles, and thus reduce the powder cohesiveness. A distinct advantage of using corrugated particles is that the inhaler choice and air flow become less critical for these particles.     

The AERX™ Aerosol Delivery System

Purpose. We describe the AER_X aerosol delivery system, a new, bolus inhalation device that is actuated at preprogrammed values of inspiratory flow rate and inhaled volume. We report on its in vitro characterization using a particular set of conditions used in pharmacokinetic and scintigraphic studies. Methods. Multiple doses of aerosol were delivered from single use collapsible plastic containers containing liquid formulation. The aerosol was generated by forcing the formulation under pressure through an array of 2.5 micron holes. Air was drawn through the device at 70 LPM, and the aerosol was collected onto a filter or Andersen cascade impactor. The emitted dose was quantified from the filter collection data, and the particle size distribution was obtained from the best fit log-normal distribution to the impactor data. Results. 57.0 ± 5.9% of the dose of drug placed as an aqueous solution in the 45 L collapsible container was delivered as an aerosol (n = 40). The best fit size distribution had an MMAD = (2.95 ± 0.06) m and a geometric standard deviation _g = 1.24 ± 0.01 (n = 6). Conclusions. The AER_X aerosol delivery system generates a nearly monodisperse aerosol with the properties required for efficient and repeatable drug delivery to the lung.     

Artificial Neural Network Prediction of Aerosol Deposition in Human Lungs

Purpose. To develop a rapid and reliable method for predicting the pattern of aerosol particle deposition within the human lungs, using artificial neural networks (ANNs). Methods. Experimental data from the literature were used to train multi-layer perceptron (MLP) networks to allow for prediction of regional and total aerosol particle deposition patterns in human lungs. These data covered particle sizes in the range 0.05-15 m and three different breathing patterns (ranging from quiet breathing to breathing under physical work conditions). Three different MLPs were trained, to provide separate predictions of aerosol particle deposition in the laryngeal, bronchial, and alveolar regions. The total deposition fraction for a given set of breathing conditions was computed simply as the sum of the outputs produced from the corresponding regional deposition MLPs. Results. The ANNs developed are shown to give highly accurate predictions for both regional and total aerosol deposition patterns for all particle sizes and breathing conditions (with errors typically less than 0.04%). Conclusions. We conclude that the current set of ANNs can be used to give good predictions of particle deposition from polydisperse pharmaceutical aerosols generated from breath-actuated dry powder inhalers, nebulizers, and metered dose inhalers with spacers.     

Adhesion of Powders for Inhalation: An Evaluation of Drug Detachment from Surfaces Following Deposition from Aerosol Streams

Purpose. To evaluate micronized powder retention and detachment from inhaler surfaces following reproducible deposition by impaction, coupled with centrifugal particle detachment (CPD). Methods. Micronized albuterol sulfate (AS) and beclomethasone dipropionate (BDP) were aerosolized as dry powders and deposited by cascade impaction onto different contact surfaces. Drug detachment from the surfaces was characterized using CPD, coupled with HPLC assay and scanning electron microscopy. Results. Drugs which accumulated as aggregates on model surfaces detached with distinctive profiles for % remaining vs. applied centrifugal force; each profile showed reproducible values for the minimum force required to initiate drug detachment, F_yield. While differences occurred in the observed detachment profiles for different drugs and contact surfaces (polyacetal vs. aluminum), the deposited drug particle size had the most significant effect on these profiles, e.g., F_yield for AS (2.1-3.3 m) was 383 12.7 N compared with 18 13.8 N for AS (4.7-5.8 m). Conclusions. A technique was developed which enabled the experimental review, and subsequent data analysis, of the adhesive properties between different DPI construction materials and drug substances deposited from aerosol clouds. The technique appears to be of greater relevance to inhaler design decisions than earlier studies in the literature claiming to show differences in the adhesion of single drug particles to surfaces.     

Respirable PLGA Microspheres Containing Rifampicin for the Treatment of Tuberculosis: Manufacture and Characterization

Purpose. Particles with aerodynamic diameters of 1–5m deposit in the periphery of the lungs and are phagocytized by alveolarmacrophages, the primary site of Mycobacterium tuberculosisinfection. Aerosols of biodegradable polymeric microspheres containingantitubercular agents may be delivered to the lungs to improve the treatmentof tuberculosis. Methods. Poly(lactide-co-glycolide) (PLGA) microspherescontaining rifampicin were prepared using solvent evaporation and spraydrying methods. The solvent evaporation process was optimized usingfactorial experimental design and surface response methodology. Themorphology, particle size, drug loading, and dissolution of microspheres wasevaluated. Results. The spray dried rifampicin loaded PLGAmicroparticles were shriveled, unlike the spherical particles produced bysolvent evaporation. Drug loadings of 20;pc and 30;pc were achieved forsolvent evaporation and spray dried products, respectively. The particlesprepared by solvent evaporation and spray drying had 3.45 m and 2.76m median diameters by volume, respectively. Conclusions. Respirable rifampicin loaded PLGAmicrospheres were produced by both solvent evaporation and spray dryingmethods. These particles are being evaluated in an animal model oftuberculosis.     

One-Dimensional and Two-Dimensional 1H- and 13C-Nuclear Magnetic Resonance (NMR) Analysis of Vitamin E Raw Materials or Analytical Reference Standards

Two-dimensional spectral analysis (COSY, HETCOR) was utilized to make the complete ¹³C- and ¹H-NMR assignments for -, -, --, and -tocopherol as well as for the acetate and succinate esters of -tocopherol. ¹³C-NMR was found to be especially useful in distinguishing between the various tocopherols and distinguishing between the d-isomer and the d,l-racemic mixture. HETCOR spectra were also found to be useful for the qualitative identification of mixtures of the tocopherols and sesame oil. Using a procedure designed to minimize errors arising from spin relaxation and nuclear Overhauser effects, ¹³C-NMR peak integrals were used to quantitate -tocopherol and -tocopherol in the presence of sesame oil using benzoic acid as the standard for calibration of the quantitation. The NMR results were compared to a capillary column gas chromatographic analysis of the individual -tocopherol and -tocopherol reference materials.Chris W. Myers: Recipient of the Analysis and Pharmaceutical Quality Section Undergraduate Award in Pharmaceutical Analysis from the APQ Section of the American Association of Pharmaceutical Scientists.     

Biliary Excretion of 17β-Estradiol 17β-d-Glucuronide Is Predominantly Mediated by cMOAT/MRP2

Purpose. The mechanism for the biliary excretion of 17-estradiol170-d-glucuronide (E₂17G), a cholestatic metabolite of estradiol, isstill controversial. The purpose of the present study is to examine thetransport of E₂17G across the bile canalicular membrane. Methods. We examined the uptake of [³H]E₂17G by isolatedcanalicular membrane vesicles (CMVs) prepared from Sprague-Dawley (SD)rats and Eisai Hyperbilirubinemic rats (EHBR) whose canalicularmultispecific organic anion transporter/multidrug resistance associatedprotein 2 (cMOAT/MRP2) function is hereditarily defective. Also,in vivo biliary excretion of intravenously administered [³H]E₂17Gwas examined. Results. In CMVs prepared from SD rats, but not from EHBR, amarked ATP-dependent uptake of [³H]E₂17G was observed.Moreover, E₂17G competitively inhibited the ATP-dependent uptake of[³H]2,4-dinitrophenyl-S-glutathione (DNP-SG). In addition, nosignificant inhibitory effect of verapamil (100 M) and PSC-833 (5 M) onthe uptake of [³H]E₂17G was observed. In vivo, the biliary excretionof intravenously administered [³H]E₂17G was severely impaired inEHBR while the biliary excretion of [³H]E₂17G in SD rats wasreduced by administering a cholestatic dose (10 mol/kg) unlabeledE₂17G, but not by PSC-833 (3 mg/kg). Conclusions. The transport of E₂17G across the bile canalicularmembrane is predominantly mediated by cMOAT/MRP2.     

Protective effects of phosphatidylcholine against mechanisms of ischemia and reperfusion-induced arrhythmias in isolated guinea pig ventricular tissues

Summary The effects of exogenous phosphatidylcholine (PC) on some potential mechanisms of ischemia and reperfusion-induced arrhythmias were tested using the superfused right ventricular free wall of the guinea pig. Exposure of the preparation to simulated ischemia (hypoxia, acidosis, glucose deprivation and hyperkalemia) resulted in several electrophysiological derangements, including a marked depolarization of the maximum diastolic potential (MDP) in both endocardium and epicardium, shortening of the action potential duration (APD), and prolongation of the transmural conduction time followed by transmural conduction block. In a few preparations, coupled beats were also observed. Reperfusion was associated with arrhythmic activity in all preparations. Both the characteristics and the severity of reperfusion-associated arrhythmias were dependent upon the duration of the preceding ischemia. In hearts exposed to ischemic conditions for 40 min, transmural conduction block persisted until 45 min of reperfusion and no electrical activity was present in the epicardium during this time. However, both coupled beats as well as abnormal automaticity were observed in the endocardium. When the period of ischemia was reduced to 20 min, recovery from transmural conduction block occurred sooner and coupled beats and abnormal automaticity were detected in both epicardial and endocardial layers. Superfusion with PC during both ischemia and reperfusion (PC1 group), or during reperfusion only (PC2 group), significantly altered the response of the preparations to reperfusion. Following 40 min ischemia, preparations treated with PC recovered from transmural conduction block more rapidly (PC1 group, 4 min, P < 0.05; PC2 group, 23 min, ns), compared to control. In addition, both PC treated groups exhibited improved recovery of MDP in the epicardium early in reperfusion. Neither PC treatment altered the incidence of coupled beats but reperfusion-associated abnormal automaticity was abolished in both groups. In preparations exposed to 20 min of ischemia, PC reduced the duration of coupled beats from 5 ± 2 min in the control to 1 ± 0 min in both PC treated groups (P < 0.05). In addition, PC treatment decreased the incidence of abnormal automaticity from 7/10 in control preparations to 1/10 in both treated groups (P < 0.05). Severe ventricular tachycardia was observed in 3/10 control preparations, whereas it was completely absent in the PC treated groups. The present study demonstrates that exogenous PC can antagonize three important cellular mechanisms which may underlie reperfusion arrhythmias: transmural conduction block, coupled beats and abnormal automaticity.Abbreviations APD action potential duration - MDP maximum diastolic potential - PC phosphatidylcholine - PC1 PC included during both ischemia and reperfusion - P2 PC included during reperfusion only Send offprint requests to M. P. Moffat at the above address     

Synthesis and β-Adrenergic Activities of R-Fluoronaphthyloxypropanolamine

Purpose. Many biogenic amines where an aromatic proton is substituted with fluorine have exhibited pharmacological properties that are dependent on the position of fluorine on the aromatic ring. For example, 6-fluoroepinephrine is selective for -adrenergic receptors whereas the 2-fluoroisomer is selective for -receptors. Aryloxypropanolamines are -receptor agonists or antagonists, depending on the aryl group and its substituents. We therefore hypothesized that fluorine substitution on the aromatic ring could lead to significant biological effects in this class. A target with fluorine on naphthyl group of a known -antagonist was chosen for investigation. Methods. Synthesis of the target compound began with fluoronaphthalene and involved introduction of 4-hydroxy group by Friedel-Crafts acylation followed by Baeyer Villiger oxidation. The side chain was introduced stereoselectively using the chiral synthon (2R)-glycidyl 3-nitrobenzenesulfonate, a Sharpless epoxidation technique. The epoxide was opened with t-butyl amine. HPLC methods were used to characterize %ee of the enantiomer. Results. The target compound was synthesized in several hundred milligram quantity, and in good yield and high enantiomeric excess, showing practicality of the synthetic scheme. It exhibited potent binding activities on -adrenergic receptors, and was found to be two times selective for ₂-receptors over ₁. Conclusions. The current report demonstrates that aromatic fluorine substitution on -adrenergic ligands can be achieved, and that such can be used to obtain binding selectivity between receptors.     

Validity of in Vitro Tests on Aqueous Spray Pumps as Surrogates for Nasal Deposition

Purpose. To determine whether deposition pattern is related to in vitromeasurements of droplet size, plume geometry, and spray pattern between two different nasal spray pumps believed to have different performance characteristics. Methods. Ten healthy volunteers inhaled radiolabeled saline from two different spray pumps (pump A and pump B). Deposition pattern was quantified from lateral views of the nose by gamma scintigraphy, expressed as the ratio of anterior to posterior (I:O) and superior to inferior (U:L) deposition. Droplet size was determined by Malvern Mastersizer S. Spray patterns were determined at 2.5 and 5 cm from the tip of the spray nozzle. Two-dimensional images of the emitted plume were captured by high-speed still photography. Results. There were no significant differences in I:O or U:L ratios for pump A compared to pump B, indicating no significant differences in deposition pattern. The volume diameters, D_v10 and D_v50, were not statistically different for pump A compared to pump B. There was a significant difference in D_v90 between pump A and pump B, (86.9 ± 5.8 m and 77.4 ± 2.4 m, respectively; P < 0.001). The ratio of the longest to shortest diameter for the spray pattern with pump A was 1.26 ± 0.06 at 2.5 cm and 1.44 ± 0.08 at 5 cm. The ratio for pump B was 1.13 ± 0.03 at 2.5 cm and 1.19 ± 0.05 at 5 cm. Ratios at both heights were statistically different for pump A compared to pump B (P < 0.00002 and P < 0.000001, respectively) Plume geometry analysis indicated statistical differences in both the width (17.0 ± 0.97 vs. 18.5 ± 0.56 cm, respectively; p<0.001) and the maximum length of the plumes (46.0 ± 1.83 vs. 53.1 ± 4.88 cm, respectively; p < .002). The differences in velocity of the plume and spray angle between the two pumps were not statistically different. Conclusions. Certain in vitrotests detected performance differences between the two pumps. However, these differences did not translate into different deposition patterns in vivo.     

Studies on the constituents of Scutellaria species (XXI): constituents of the leaves of Scutellaria strigillosa Hemsley

From the leaves of Scutellaria strigillosa, 14 compounds, chrysin, apigenin, 5,7,2-trihydroxyflavone, norwogonin, ursolic acid, 6-hydroxy-4-stigmasten-3-one, 6-hydroxy-4,22-stigmastadien-3-one, 2 R,4 R,8 R--tocopherol, (S)-5,5 -bi--tocopheryl, (R)-5,5 -bi--tocopheryl, solanachromene, tocopherylquinone, jodrellin T, and 14,15-dihydrojodrellin T were isolated. The structures were determined on the basis of chemical and spectral data.     

The Mechanism of Uptake of Biodegradable Microparticles in Caco-2 Cells Is Size Dependent

Purpose. To study the uptake of biodegradable microparticles in Caco-2 cells. Methods. Biodegradable microparticles of polylactic polyglycolic acid co-polymer (PLGA 50:50) of mean diameters 0.1 m, 1 m, and 10 m containing bovine serum albumin as a model protein and 6-coumarin as a fluorescent marker were formulated by a multiple emulsion technique. The Caco-2 cell monolayers were incubated with each diameter microparticles (100 g/ml) for two hours. The microparticle uptake in Caco-2 cells was studied by confocal microscopy and also by quantitating the 6-coumarin content of the microparticles taken up by the cells. The effects of microparticle concentration, and incubation time and temperature on microparticle cell uptake were also studied. Results. The study demonstrated that the Caco-2 cell microparticle uptake significantly depends upon the microparticle diameter. The 0.1 m diameter microparticles had 2.5 fold greater uptake on the weight basis than the 1 m and 6 fold greater than the 10 m diameter microparticles. Similarly in terms of number the uptake of 0.1 m diameter microparticles was 2.7 × 10³ fold greater than the 1 m and 6.7 × 10⁶ greater than the 10 m diameter microparticles. The efficiency of uptake of 0.1 m diameter microparticles at 100 g/ml concentration was 41% compared to 15% and 6% for the 1 m and the 10 m diameter microparticles, respectively. The Caco-2 cell microparticle (0.1 m) uptake increased with concentration in the range of 100 g/ml to 500 g/ml which then reached a plateau at higher concentration. The uptake of microparticles increased with incubation time, reaching a steady state at two hours. The uptake was greater at an incubation temperature of 37°C compared to at 4°C. Conclusions. The Caco-2 cell microparticle uptake was microparticle diameter, concentration, and incubation time and temperature dependent. The small diameter microparticles (0.1 m) had significantly greater uptake compared to larger diameter microparticles. The results thus suggest that the mechanism of uptake of microparticles in Caco-2 cell is particle diameter dependent. Caco-2 cells are used as an in vitro model for gastrointestinal uptake, and therefore the results obtained in these studies could be of significant importance in optimizing the microparticle-based oral drug delivery systems.     

Evaluation of An Aerosolized Selective COX-2 Inhibitor as a Potentiator of Doxorubicin in a Non-Small-Cell Lung Cancer Cell Line

Purpose. To evaluate the in vitro effects of an aerosolized cyclooxygenase-2 (COX-2) inhibitor, nimesulide, on the cytotoxicity and apoptotic response of doxorubicin against the human lung adenocarcinoma cell line A549. Methods. Nimesulide was formulated into a metered dose inhaler (MDI) formulation and characterized for aerodynamic particle size and medication delivery. The in vitro cytotoxicity of nimesulide-MDI in the presence or absence of doxorubicin was assessed by using the six-stage viable impactor by an already standardized method. Induction of apoptosis in A549 cells by nimesulide (nonaerosolized or aerosolized) in combination with doxorubicin was evaluated by established techniques such as caspase-3 estimation and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) staining. Finally, to understand the mechanism of action, the influence of different treatments on the expression of COX-2 and peroxisome proliferator-activated receptor- (PPAR-) in A549 cells was studied by immunoblotting. Results. The nimesulide-MDI formulation had a mass median aerodynamic diameter (MMAD) of 1.1 m, (GSD = 2.8) and a medication delivery of 51 g/shot. Nimesulide-MDI (40 shots) in combination with doxorubicin (0.01 g/ml) had a cell kill of more than 60% as determined by in vitro cytotoxicity assay. The specific caspase-3 activity in A549 cells treated with nimesulide (40 g/ml) and doxorubicin (0.25 g/ml) in combination was 3 and 5 times higher than doxorubicin and nimesulide, respectively. Further, TUNEL staining showed apoptosis in over 30% of A549 cells treated with aerosolized nimesulide and doxorubicin combination vs. negligible as seen in cells treated individually. The expression of COX-2 was not altered in control or treatments, whereas PPAR- was expressed only in the combination treatment. Conclusion. Our results indicate that aerosolized nimesulide significantly enhances doxorubicin activity against A549 cells, and the enhanced cytotoxicity was probably mediated via a COX-2-independent mechanism.     

Novel Direct Curve Comparison Metrics for Bioequivalence

Purpose. The object of this work was to devise four new direct curve comparison (DCC) metrics and examine each metric's distribution properties and performance characteristics. Methods. DCC metrics, Cmax, and AUCi were calculated from two bioequivalence studies of three sustained release carbamazepine formulations, where a range of profile similarity was observed. DCC metric values and their confidence intervals were compared to Cmax and AUCi. Results. The DCC metrics , _m, _a, and _s exhibited more favorable distributions than Cmax and AUCi ratios, which were frequently skewed. The DCC metrics performed differently than Cmax and AUCi ratios in profile comparisons due to the nature of the DCC metrics. Unlike Cmax and AUCi, the DCC metrics utilize all data points to directly compare entire profiles. Each DCC metric appears to measure exposure in a single assessment. Possible bioequivalence acceptance criteria are: 1.40, _m 0.35, _a 0.27, and _s 0.102. Conclusions. These DCC metrics, particularly p_m, are promising bioequivalence metrics for exposure.     

Subclassification of presynaptic 5-HT autoreceptors in the human cerebral cortex as 5-HT1Dβ receptors

Human cerebral cortical synaptosomes were used to determine the 5-hydroxytryptamine (5-HT) receptor subtype to which the inhibitory presynaptic 5-HT autoreceptor belongs. The synaptosomes preincubated with [³H]5-HT were superfused and tritium overflow was stimulated by high K⁺. The K⁺-evoked tritium overflow, which was Ca²⁺-dependent but tetrodotoxin-resistant, was concentration-dependently inhibited by the nonselective 5-HTlD_1D/1D receptor agonist, 5-carboxamidotryptamine. Ketanserin at a concentration which should block the 5-HT_1D but not the 5-HT_1D receptor failed to antagonize the inhibitory effect of 5-carboxamidotryptamine. In contrast, the non-selective 5-HT_1D/1D receptor antagonist, methiothepin, at a concentration which should block both the 5-HT_1D and the 5-HT_1D receptor abolished the effect of 5-carboxamidotryptamine. It is concluded that the presynaptic 5-HT autoreceptor, which has previously been classified as 5-HT_1D, belongs to the 5-HT_1D subtype.