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.
nutraceutical; functional food; dietary supplement
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).
Caco-2; glucose; tight junctions; TEER; membrane fluidity; solute flux
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.
motexafin lutetium; LC-MS/MS; ICP-AES; pharmacokinetics; lutetium texaphyrin
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.
proteins; microencapsulation; solvent evaporation; sucrose ester; surfactants
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.
atenolol; bioequivalence; HPLC; plasma; determination
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.
chemiluminescence; luminol; antioxidants; flavonoids; peroxidase
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.
prescription drug coverage; Medicare; older adults
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.
Cytochrome P450; CYP2B6; fluorescent substrate; drug metabolism; high-throughput screening (HTS)
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.
nucleoside; nucleobase; cornea; SIRC; transport; antiviral agent
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.
fine particle ethylcellulose; wet granulation; ionizable drugs; nonionizable drugs; Fickian diffusion; polymer relaxation
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.
erythromycin; crystallization; solvated crystals; isostructurality; clathrate; x-ray diffraction
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.
micronization; anti-inflammatory drug; rapid expansion of supercritical solution (RESS); aerosol solvent extraction system (ASES)
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.
protein formulation; protein stability; recombinant humanized monoclonal antibody; aggregation; FT-IR spectrometry
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.
pulmonary delivery; insulin; CAP-PEG particles; pharmacokinetics; pharmacodynamics
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.
multidose formulation; preservative; experimental design; monoclonal antibody; protein
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.
multiple emulsions; stability; rheology; surfactant
The objective of this study was to determine whether FM-VP4, a novel compound derived from plant sterols, can effectively reduce cholesterol accumulation within rat intestinal epithelial crypt (IEC-6) cells. IEC-6 cells were cultured in Dulbeccos minimal essential medium (DMEM) containing 5% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.1 units/mL insulin at 37°C under a humidified 5% CO2 atmosphere and seeded at 6.4×104 cells/well in 48-well plates. Experiments were initiated 14 days postconfluence. IEC-6 cells were exposed to [3H]cholesterol micelles (containing oleic and taurcholic acids), co-incubated with FM-VP4 (0, 10, 50, and 100 μM) in Hepes Buffered Sterile Saline (HBSS). Cells were also preincubated with FM-VP4 prior to [3H]cholesterol micelle incubation to determine whether its effects are elicited intracellularly. The cellular localization of cholesterol was determined using digitonin. To determine the effects of cholesterol on the extent of FM-VP4 accumulation within IEC-6 cells, [3H]FM-VP4 was incubated with IEC-6 cells in the presence of unlabeled cholesterol micelles (0, 10, and 50 μM). The extent of [3H]cholesterol or [3H]FM-VP4 associated with cell monolayers was determined after cell lysis using liquid scintillation counting in a Beckman LS6500 Scintillation Counter. Dose-response and time course studies were performed in which control (no FM-VP4 treatment) and FM-VP4 (10–100 μM) were co-incubated with 50-μM [3H]cholesterol micelles from 1 minute to 24 hours. Incubation with only 50-μM FM-VP4 for less than 24 hours resulted in a 50% to 60% reduction (n=6, P<.05) in [3H]cholesterol associated with the monolayer compared with control (n=6). Preincubation of FM-VP4 did not elicit a significant reduction in cholesterol accumulation compared with control (n=6). Approximately 25% of the total [3H]cholesterol associated with the cells was determined to be cytosolic, while 75% was noncytosolic in the presence and/or absence of FM-VP4. [3H]FM-VP4 was also shown to associate with IEC-6 cells at similar concentrations to cholesterol with the most pronounced inhibition of FM-VP4 accumulation occurring at a cholesterol concentration of 50 μM. However, cholesterol-induced inhibition was detectable only after 1 hour of incubation. FM-VP4 inhibits cholesterol accumulation within IEC-6 cells and is most effective at equimolar concentrations with cholesterol. Our findings further suggest that the action of FM-VP4 is likely at the cell surface and not elicited intracellularly.
IEC-6 cells; phytostanols; cholesterol accumulation; cytotoxicity
The development of vaginal medications, especially antifungal medications, requires that the drug is solubilized as well as retained at or near the mucosa for sufficient periods of time to ensure adequate bioavailability. Itraconazole is a broad-spectrum antifungal agent, which has been used for some time orally and intravenously but for which a vaginal formulation has not yet been developed. We present here a novel itraconazole formulation intended for vaginal use based on hydroxypropyl-β-cyclodextrin (HPβCD), a functional excipient that increases drug solubility and generates a mucoadhesive system in the presence of other ingredients. An aqueous phase was prepared by solubilizing itraconazole with HCl in the presence of propylene glycol and then adding an aqueous solution of HPβCD. After pH adjustment, the itraconazole/HPβCD solution was added to the oil phase (paraffin oil, trihydroxystearate, and cetyl dimethicon copolyol) and the desired cream containing 1%, 2%, and 2.5% drug obtained by homogenization. Primary irritation studies and subchronic toxicity studies using a rabbit vaginal model indicated that the formulation was safe, well tolerated, and retained in the vaginal space. Clinical investigations indicated that application of 5 g of a 2% cream was very well tolerated and itraconazole was not systemically absorbed. Additional studies in women found that the itraconazole cream was highly effective in reducing or eliminating fungal cultures with few adverse effects. These studies suggested that an HPβCD-based, emulsified wax cream formulation was a useful and effective dosage form for treating vaginal candidiasis.
Itraconazole; vaginal; cyclodextrin; mucoadhesive; toxicity; clinical investigation; candidiasis
The purpose of this study was to examine absorption of basic drugs as a function of the composite solubility curve and intestinally relevant pH by using a gastrointestinal tract (GIT) absorption simulation based on the advanced compartmental absorption and transit model. Absorption simulations were carried out for virtual monobasic drugs having a range of pKa, log D, and dose values as a function of presumed solubility and permeability. Results were normally expressed as the combination that resulted in 25% absorption. Absorption of basic drugs was found to be a function of the whole solubility/pH relationship rather than a single solubility value at pH 7. In addition, the parameter spaces of greatest sensitivity were identified. We compared 3 theoretical scenarios: the GIT pH range overlapping (1) only the salt solubility curve, (2) the salt and base solubility curves, or (3) only the base curve. Experimental solubilities of 32 compounds were determined at pHs of 2.2 and 7.4, and they nearly all fitted into 2 of the postulated scenarios. Typically, base solubilities can be simulated in silico, but salt solubilities at low pH can only be measured. We concluded that quality absorption simulations of candidate drugs in most cases require experimental solubility determination at 2 pHs, to permit calculation of the whole solubility/pH profile.
GIT; absorption simulation; pH solubility curve; BCS; solid-state properties; solubility screening
The transdermal delivery of buspirone hydrochloride across hairless mouse skin and the combined effect of iontophoresis and terpene enhancers were evaluated in vitro using Franz diffusion cells. Iontophoretic delivery was optimized by evaluating the effect of drug concentration, current density, and pH of the vehicle solution. Increasing the current density from 0.05 to 0.1 mA/cm2 resulted in doubling of the iontophoretic flux of buspirone hydrochloride, while increasing drug concentration from 1% to 2% had no effect on flux. Using phosphate buffer to adjust the pH of the drug solution decreased the buspirone hydrochloride iontophoretic flux relative to water solutions. Incorporating buspirone hydrochloride into ethanol:water (50:50 vol/vol) based gel formulations using carboxymethylcellulose and hydroxypropylmethylcellulose had no effect on iontophoretic delivery. Incorporation of three terpene enhancers (menthol, cineole, and terpineol) into the gel and when combined with iontophoresis it was possible to deliver 10 mg/cm2/day of buspirone hydrochloride.
iontophoresis; terpene; buspirone hydrochloride; gel; transdermal
Purpose: In an attempt to increase the local concentration of tamoxifen in estrogen receptor positive breast cancer cells, we have prepared and characterized poly (ε-caprolactone) (PCL) nanoparticle formulation. Methods: PCL (mol wt 14,800 daltons) nanoparticles were prepared by the solvent displacement method in acetonewater system in the presence of Pluronic F-68. PCL nanoparticles, labeled with rhodamine 123, were incubated with MCF-7 estrogen receptor positive breast cancer cells to determine uptake, intracellular distribution, and localization as a function of time. Intracellular drug concentrations over a specified period of time using different initial doses were examined using tritiated [3H]-tamoxifen. Results: A significant fraction of the administered rhodamine 123-loaded PCL nanoparticles was found in the perinuclear region of the MCF-7 cells, where estrogen receptors are also localized, after 1 hour of incubation. Measurements of the intracellular concentrations revealed that most of the administered nanoparticle dose was internalized within the first 30 minutes of incubation, and the uptake followed saturable transport kinetics. Conclusion: Results of this study show that PCL nanoparticles were rapidly internalized in MCF-7 cells and intracellular tamoxifen concentrations followed a saturable process. This approach may provide better therapeutic benefit by delivering the drug locally, near the tumor cells, for a longer period of time.
tamoxifen; poly (ε-caprolactone); biodegradable nanoparticles; cellular uptake; intracellular concentrations
New and improved drug delivery systems are the important subject of much scientific research. The development of formulations that increase skin oxygenation and of methods for measuring oxygen levels in skin are important for dealing with healing processes affected by the level of oxygen. We have use EPR oximetry in vivo to compare the influence of liposomal formulations of different size and composition with that of hydrogel with respect to the action of the entrapped benzyl nicotinate (BN). Following the topical application of BN onto the skin of mice, pO2 increase was measured by low-frequency EPR as a function of time. The effect of BN was evaluated by 3 different parameters: lag-time, time needed for maximum pO2 increase, and overall effectiveness expressed by the area under the response-time curve. An increase in skin oxygenation was observed after BN application. The results show that the effect of BN incorporated in liposomes is achieved more rapidly than the effect from hydrophilic gel. The composition of the liposomes significantly affects the time at which BN starts to act and, to a lesser extent, the maximum increase of pO2 in skin and the effectiveness of BN action. However, the size of the liposomes influences both the effectiveness of BN action and the time at which BN starts to act. After repeated application of liposomes, the pO2 baseline increased and the response of the skin tissue was faster. Our results demonstrate that EPR oximetry is a useful method for evaluating oxygen changes after drug application and for following the time course of their action.
skin; liposome; benzyl nicotinate; oxygenation; EPR in vivo
This article describes the production and characterization of cationic submicron particles constituted with Eudragit RS 100, plus different cationic surfactants, such as dioctadecyl-dimethyl-ammonium bromide (DDAB18) and diisobutyphenoxyethyl-dimethylbenzyl ammonium chloride (DEBDA), as a transport and delivery system for DNA/DNA and DNA/peptide nucleic acid (PNA) hybrids and PNA-DNA chimeras. Submicron particles could offer advantages over other delivery systems because they maintain unaltered physicochemical properties for long time periods, allowing long-term storage, and are suitable for industrial production. Submicron particles were characterized in terms of size, size distribution, morphology, and zeta potential. Moreover, the in vitro activity and ability of submicron particles to complex different types of nucleic acids were described. Finally, the ability of submicron particles to deliver functional genes to cells cultured in vitro was determined by a luciferase activity assay, demonstrating that submicron particles possess superior transfection efficiency with respect to commercially available, liposome-based transfection kits.
peptide nucleic acids; delivery; submicron particles
Identification and functional characterization of the genes in the human genome remain a major challenge. A principal source of publicly available information used for this purpose is the National Center for Biotechnology Information database of expressed sequence tags (dbEST), which contains over 4 million human ESTs. To extract the information buried in this data more effectively, we have developed a semiautomated method to mine dbEST for uncharacterized human genes. Starting with a single protein input sequence, a family of related proteins from all species is compiled. This entire family is then used to mine the human EST database for new gene candidates. Evaluation of putative new gene candidates in the context of a family of characterized proteins provides a framework for inference of the structure and function of the new genes. When applied to a test data set of 28 families within the major facilitator superfamily (MFS) of membrane transporters, our protocol found 73 previously characterized human MFS genes and 43 new MFS gene candidates. Development of this approach provided insights into the problems and pitfalls of automated data mining using public databases.
Major facilitator superfamily; transporters; superfamily analysis; expressed sequence tags; data mining
The purpose of this study was to construct a pharmacokinetic (PK) model and to determine PK parameters of 2,3,5,6-tetramethylpyrazine (TMP) after application of TMP transdermal delivery system. Data were obtained in Sprague-Dawley (SD) rats following a single dose of TMP transdermal delivery system. Blood samples were obtained at 0, 0.25, 0.5, 1, 2, 4, 6, 16, and 24 hours after the transdermal application. In the brain level study, 18 SD rats were divided into 6 groups. Three SD rats before and after transdermal application were culled and sacrificed at each of the following time intervals: 2, 4, 6, 16, and 24 hours after the TMP-TTS application. TMP concentrations in plasma and brain tissues were determined using high performance liquid chromatography and data were fitted using a zero-order absorption and a firstorder-elimination 3-compartment PK model. Fitted parameters included 2 volumes of distribution (V1, V2) and 2 elimination rate constants (k10, k20). The elimination half-life for TMP in plasma and brain was 26.5 and 31.2 minutes, respectively. The proposed PK model fit observed concentrations of TMP very well. This model is useful for predicting drug concentrations in plasma and brain and for assisting in the development of transdermal systems.
tetramethylpyrazine; percutaneous absorption; transdermal drug delivery system; in vitro/in vivo; pharmacokinetic model