Showing papers in "Pharmaceutical Research in 2005"
TL;DR: It is suggested that a Biopharmaceutics Drug Disposition Classification System (BDDCS) using elimination criteria may expand the number of Class 1 drugs eligible for a waiver of in vivo bioequivalence studies and provide predictability of drug disposition profiles for Classes 2, 3, and 4 compounds.
Abstract: The Biopharmaceutics Classification System (BCS) was developed to allow prediction of in vivo pharmacokinetic performance of drug products from measurements of permeability (determined as the extent of oral absorption) and solubility. Here, we suggest that a modified version of such a classification system may be useful in predicting overall drug disposition, including routes of drug elimination and the effects of efflux and absorptive transporters on oral drug absorption; when transporter-enzyme interplay will yield clinically significant effects (e.g., low bioavailability and drug-drug interactions); the direction, mechanism, and importance of food effects; and transporter effects on postabsorption systemic drug concentrations following oral and intravenous dosing. These predictions are supported by a series of studies from our laboratory during the past few years investigating the effect of transporter inhibition and induction on drug metabolism. We conclude by suggesting that a Biopharmaceutics Drug Disposition Classification System (BDDCS) using elimination criteria may expand the number of Class 1 drugs eligible for a waiver of in vivo bioequivalence studies and provide predictability of drug disposition profiles for Classes 2, 3, and 4 compounds.
1,292 citations
TL;DR: A fully scalable and extrusion-free method was developed to prepare rapidly and reproducibly stabilized plasmid lipid particles (SPLP) for nonviral, systemic gene therapy, which will enable the scale-up and manufacture of SPLP required for preclinical and clinical studies.
Abstract: A fully scalable and extrusion-free method was developed to prepare rapidly and reproducibly stabilized plasmid lipid particles (SPLP) for nonviral, systemic gene therapy Liposomes encapsulating plasmid DNA were formed instantaneously by mixing lipids dissolved in ethanol with an aqueous solution of DNA in a controlled, stepwise manner Combining DNA-buffer and lipid-ethanol flow streams in a T-shaped mixing chamber resulted in instantaneous dilution of ethanol below the concentration required to support lipid solubility The resulting DNA-containing liposomes were further stabilized by a second stepwise dilution Using this method, monodisperse vesicles were prepared with particle sizes less than 200 nm and DNA encapsulation efficiencies greater than 80% In mice possessing Neuro 2a tumors, SPLP demonstrated a 13 h circulation half-life in vivo, good tumor accumulation and gene expression profiles similar to SPLP previously prepared by detergent dialysis Cryo transmission electron microscopy analysis showed that SPLP prepared by stepwise ethanol dilution were a mixed population of unilamellar, bilamellar, and oligolamellar vesicles Vesicles of similar lipid composition, prepared without DNA, were also <200 nm but were predominantly bilamellar with unusual elongate d morphologies, suggesting that the plasmid particle affects the morphology of the encapsulating liposome A similar approach was used to prepare neutral egg phosphatidylcholine:cholesterol (EPC:Chol) liposomes possessing a pH gradient, which was confirmed by the uptake of the lipophilic cation safranin O This new method will enable the scale-up and manufacture of SPLP required for preclinical and clinical studies Additionally, this method now allows for the acceleration of SPLP formulation development, enabling the rapid development and evaluation of novel carrier systems
307 citations
TL;DR: In this review, PEG copolymers as gene carriers are introduced, and their characteristics are discussed.
Abstract: Polymeric gene delivery systems have been developed as an alternative for viral gene delivery systems to overcome the problems in the use of viral gene carriers Polymeric carriers have many advantages as gene carriers such as low cytotoxicity, low immunogenicity, moderate transfection efficiency, no size-limit, low cost, and reproducibility In the efforts to develop safe and efficient polymeric gene carriers, polyethylene glycol (PEG) has widely been used because of its excellent characteristics PEG-conjugated copolymers have advantages for gene delivery: 1) The PEG-conjugated copolymers show low cytotoxicity to cells in vitro and in vivo, 2) PEG increases water-solubility of the polymer/DNA complex, 3) PEG reduces the interaction of the polymer/DNA complex with serum proteins and increases circulation time of the complex, 4) PEG can be used as a spacer between a targeting ligand and a cationic polymer A targeting ligand at the end of a PEG chain is not disturbed by the interaction of a cationic polymer with plasmid DNA, and the PEG spacer increases the accessibility of the ligand to its receptor In this review, PEG copolymers as gene carriers are introduced, and their characteristics are discussed
273 citations
TL;DR: Dog seems to be a good model for man with respect to dissolution in the small intestine after intake of a meal, whereas FeSSIF is a poorer means of determining intestinal saturation solubility in the fed state.
Abstract: This study was conducted to determine the effect of food on drug solubility and dissolution rate in simulated and real human intestinal fluids (HIF) Dissolution rate obtained via the rotating disk method and saturation solubility studies were carried out in fed and fasted state HIF, fed dog (DIF), and simulated (FeSSIF) intestinal fluid for six aprotic low solubility drugs The intestinal fluids were characterized with respect to physical–chemical characteristics and contents Fed HIF provided a 35- to 30-times higher solubility compared to fasted HIF and FeSSIF, whereas fed DIF corresponded well (difference of less than 30%) to fed HIF The increased solubility of food could mainly be attributed to dietary lipids and bile acids The dissolution rate was also 2 to 7 times higher in fed HIF than fasted HIF This was well predicted by both DIF and FeSSIF (difference of less than 30%) Intestinal solubility is higher in fed state compared to fasted state However, the dissolution rate does not increase to the same extent Dog seems to be a good model for man with respect to dissolution in the small intestine after intake of a meal, whereas FeSSIF is a poorer means of determining intestinal saturation solubility in the fed state
259 citations
TL;DR: PEO-modified PbAE nanoparticles showed considerable passive tumor targeting potential in early stages of biodistribution via the enhanced permeation and retention (EPR) mechanism, which prompts a detailed biodist distribution profiling of the nanocarrier for prolonged periods to provide conclusive evidence for superiority of the delivery system.
Abstract: This study was carried out to determine the biodistribution profiles and tumor localization potential of poly(ethylene oxide) (PEO)-modified poly(β-amino ester) (PbAE) as a novel, pH-sensitive biodegradable polymeric nanoparticulate system for tumor-targeted drug delivery. The biodistribution studies of PEO-modified PbAE and PEO-modified poly(ɛ-caprolactone) (PCL), a non-pH-sensitive polymer, nanoparticle systems were carried out in normal mice using 111indium-oxine [111In] as a lipophilic radiolabel encapsulated within the polymeric matrix, and the distribution of the nanoparticles was studied in plasma and all the vital organs following intravenous administration. Solid tumors were developed on nude mice using human ovarian carcinoma xenograft (SKOV-3) and the change in concentrations of tritium [3H]-labeled paclitaxel encapsulated in polymeric nanoparticles was examined in blood, tumor mass, and liver. Study in normal mice with a gamma-emitting isotope [111In] provided a thorough biodistribution analysis of the PEO-modified nanoparticulate carrier systems, whereas 3H-paclitaxel was useful to understand the change in concentration and tumor localization of anticancer compound directly in major sites of distribution. Both PEO-PbAE and PEO-PCL nanoparticles showed long systemic circulating properties by virtue of surface modification with PEO-containing triblock block copolymer (Pluronic®) stabilizer. Although the PCL nanoparticles showed higher uptake by the reticuloendothelial system, the PbAE nanoparticles effectively delivered the encapsulated payload into the tumor mass. PEO-modified PbAE nanoparticles showed considerable passive tumor targeting potential in early stages of biodistribution via the enhanced permeation and retention (EPR) mechanism. This prompts a detailed biodistribution profiling of the nanocarrier for prolonged periods to provide conclusive evidence for superiority of the delivery system.
254 citations
TL;DR: Cross-linking of small PEIs with judiciously designed amide- and ester-bearing linkers boosts their gene delivery efficiency both in vitro and in vivo without increasing the cytotoxicity.
Abstract: Purpose.
Polyethylenimine (PEI) is among the most efficient nonviral gene delivery vectors. Its efficiency and cytotoxicity depend on molecular weight, with the 25-kDa PEI being most efficient but cytotoxic. Smaller PEIs are noncytotoxic but less efficient. Enhancement in gene delivery efficiency with minimal cytotoxicity by cross-linking of small PEIs via potentially biodegradable linkages was explored herein. The hypothesis was that cross-linking would raise the polycation’s effective molecular weight and hence the transfection efficiency, while biodegradable linkages would undergo the intracellular breakdown after DNA delivery and hence not lead to cytotoxicity. Toward this goal, we carried out cross-linking of branched 2-kDa PEI and its 1:1 (w/w) mixture with a linear 423-Da PEI via ester- and/or amide-bearing linkages; the in vitro and in vivo gene delivery efficiency, as well as toxicity to mammalian cells, of the resultant cross-linked polycations were investigated.
250 citations
TL;DR: Overall, the empirical SF method and the drug-specific SF method appear to be the best methods; they show lower bias than the physiologic SF and better precision than allometric approaches.
Abstract: Purpose.
The aim of this study is to compare the accuracy of five methods for predicting in vivo intrinsic clearance (CLint) and seven for predicting hepatic clearance (CLh) in humans using in vitro microsomal data and/or preclinical animal data.
230 citations
TL;DR: Reflectance spectroscopy demonstrated that indomethacin incorporated into MO dispersions can be released in a prolonged fashion and can be proposed as nanoparticulate systems able to control the percutaneous absorption of indometHacin.
Abstract: The present study concerns the production and characterization of monooleine (MO) dispersions as drug delivery systems for indomethacin, taken as model anti-inflammatory drug. Dispersions were produced by emulsification and homogenization of MO and poloxamer in water. Morphology and dimensional distribution of the disperse phase have been characterized by cryo-transmission electron microscopy and photon correlation spectroscopy, respectively. X-ray diffraction has been performed to determine the structural organization of the disperse phase. Sedimentation field flow fractionation (SdFFF) has been performed to investigate drug distribution in the dispersion. An in vitro diffusion study was conducted by Franz cell associated to stratum corneum epidermis membrane on cubosome dispersions viscosized by carbomer. In vivo studies based on skin reflectance spectrophotometry and tape stripping were performed to better investigate the performance of cubosome as indomethacin delivery system. Microscopy studies showed the coexistence of vesicles and cubosomes. X-ray diffraction revealed the presence of a bicontinuous cubic phase of spatial symmetry Im3m (Q229). SdFFF demonstrated that no free drug was present in the dispersion. Indomethacin incorporated in viscosized MO dispersions exhibited a lower flux with respect to the analogous formulation containing the free drug in the aqueous phase and to the control formulation based on carbomer gel. Reflectance spectroscopy demonstrated that indomethacin incorporated into MO dispersions can be released in a prolonged fashion. Tape-stripping experiments corroborated this finding. MO dispersions can be proposed as nanoparticulate systems able to control the percutaneous absorption of indomethacin.
228 citations
TL;DR: Dendrimer–oligonucleotide complexes were moderately effective for delivery of antisense and only poorly effective for Delivery of siRNA.
Abstract: PAMAM G5 dendrimer (P) was conjugated to Tat peptide (T), a cell penetrating peptide, in search of an efficient cellular delivery vehicle for antisense and siRNA oligonucleotides. PAMAM G5 dendrimer was reacted with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid, sulfosuccinimidyl ester, sodium salt (BODIPY) for visualization to yield the conjugate BP. Bifunctional sulfosuccinimidyl 6-[α-methyl-α-(2-pyridyldithio)toluamido]hexanoate (sulfo-LC-SMPT) was then used to conjugate primary amino groups of BP to cysteine derivatized Tat peptide to give the designed conjugate, BPT. This conjugate was complexed with antisense and siRNA oligonucleotides designed to inhibit MDR1 gene expression. NIH 3T3 MDR cells were used for the evaluation of biological activity of the conjugate. Both antisense and siRNA readily formed complexes with the synthesized BPT, introduced into NIH 3T3 MDR cells, and primarily accumulated in intracellular vesicles. MDR1 gene expression was partially inhibited by the antisense–BPT complex and weakly inhibited by the siRNA–BPT complex when both were tested at nontoxic levels of dendrimer. Conjugation with Tat peptide did not improve the delivery efficiency of the dendrimer. Dendrimer–oligonucleotide complexes were moderately effective for delivery of antisense and only poorly effective for delivery of siRNA. Conjugation of the dendrimer with the Tat cell penetrating peptide failed to further enhance the effectiveness of the dendrimer.
214 citations
TL;DR: High potential of spray drying technique for obtaining stable amorphous SDs of low Tg drugs is demonstrated and in vivo study in rats justified the improvement in therapeutic efficacy of SD 1:2:2 over pure SIM.
Abstract: To obtain free flowing, stable, amorphous solid dispersions (SDs) of simvastatin (SIM), a drug with relatively lower glass transition temperature (Tg) by spray drying technique, and to perform comparative in vivo study in rats, which could justify the improvement in rate and extent of in vitro drug release. Dichloromethane suspensions of SIM either alone or in combination with PVP (1:1 or 1:2 parts by weight) were spray dried with proposed quantity of Aerosil 200 (1:1, 1:1:1, 1:2:2 parts by weight of SIM, Aerosil 200 and PVP, respectively). SDs were characterized initially in comparison with pure drug and corresponding physical mixtures in same ratios by drug content, saturation solubility, SEM, DSC, XRPD, IR, and in vitro drug release. SD 1:2:2 was further subjected to accelerated stability testing and checked for in vitro drug release and presence of crystallinity using DSC and XRPD. In addition, improvement in rate and extent of in vitro drug release from SD 1:2:2 was justified by in vivo study in rats. Combination of SD and surface adsorption techniques has been attempted to overcome the limitations of spray drying technique for amorphization of low Tg drugs. Based on powder characteristics, drug content, saturation solubility, and feasibility of processing into tablets; SD 1:2:2 was selected as the optimized formulation. During initial characterization, SEM, DSC, and XRPD analyses confirmed the presence of amorphous form in SD 1:2:2. IR spectroscopy revealed possibility of hydrogen bonding interaction between SIM and PVP in SDs. Also, there was dramatical improvement in rate and extent of in vitro drug release of SD 1:2:2. Insignificant decrease in dissolution was observed with no evidence of crystallinity during accelerated stability studies of SD 1:2:2. Moreover in vivo study in rats also justified the improvement in therapeutic efficacy of SD 1:2:2 over pure SIM. Thus, present study demonstrates high potential of spray drying technique for obtaining stable amorphous SDs of low Tg drugs.
214 citations
TL;DR: 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.
Abstract: Purpose.
The current study aimed to quantify the different degree of particle surface corrugation and correlate it to the aerosol performance of powders.
TL;DR: The in vitro and in vivo results of this study clearly show that a long-circulating, biocompatible and biodegradable, DNA-encapsulating nanoparticulate system would be highly desirable for systemic delivery of genetic constructs to solid tumors.
Abstract: To develop safe and effective systemically administered nonviral gene therapy vectors for solid tumors, DNA-containing poly(ethylene glycol)-modified (PEGylated) gelatin nanoparticles were fabricated and evaluated in vitro and in vivo. Reporter plasmid DNA encoding for β-galactosidase (pCMV-β) was encapsulated in gelatin and PEGylated gelatin nanoparticles using a water-ethanol solvent displacement method under controlled pH and temperature. Lewis lung carcinoma (LLC) cells in culture were transfected with the pCMV-β in the control and nanoparticle formulations. Periodically, the expression of β-galactosidase in the cells was measured quantitatively using an enzymatic assay for the conversion of o-nitrophenyl-β-d-galactopyranoside (ONPG) to o-nitrophenol (ONP). Qualitative expression of β-galactosidase in LLC cells was observed by staining with 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal). Additionally, the plasmid DNA-encapsulated gelatin and PEGylated gelatin nanoparticles were administered intravenously (i.v.) and intratumorally (i.t.) to LLC-bearing female C57BL/6J mice. At various time points postadministration, the animals were sacrificed and transgene expression in the tumor and liver was determined quantitatively by the ONPG to ONP enzymatic conversion assay and qualitatively by X-gal staining. Almost 100% of the pCMV-β was encapsulated in gelatin and PEGylated gelatin nanoparticles (mean diameter 200 nm) at 0.5% (w/w) concentration. PEGylated gelatin nanoparticles efficiently transfected the LLC cells and the β-galactosidase expression, as measured by the ONPG to ONP enzymatic conversion assay at 420 nm absorbance, increased starting from 12 h until 96 h post-transfection. The efficient expression of LLC cells was also evident by the X-gal staining method that shows blue color formation. The in vivo studies showed significant expression of β-galactosidase in the tumor following administration of DNA-containing PEGylated gelatin nanoparticles to LLC-bearing mice by both i.v. and i.t. routes. Following i.v. administration of pCMV-β in PEGylated gelatin nanoparticles, for instance, the absorbance at 420 nm per gram of tumor increased from 0.60 after 12 h to 0.85 after 96 h of transfection. After i.t. administration, the absorbance values increased from 0.90 after 12 h to almost 1.4 after 96 h. The in vitro and in vivo results of this study clearly show that a long-circulating, biocompatible and biodegradable, DNA-encapsulating nanoparticulate system would be highly desirable for systemic delivery of genetic constructs to solid tumors.
TL;DR: An equilibrium model of TMDD is developed that recapitulates the essential features of the full general model and eliminates the need for estimating drug-binding microconstants that are often difficult or impossible to identify from typical in vivo pharmacokinetic data.
Abstract: The aim of this study is to derive and evaluate an equilibrium model of a previously developed general pharmacokinetic model for drugs exhibiting target-mediated drug disposition (TMDD). A quasi-equilibrium solution to the system of ordinary differential equations that describe the kinetics of TMDD was obtained. Computer simulations of the equilibrium model were carried out to generate plasma concentration-time profiles resulting from a large range of intravenous bolus doses. Additionally, the final model was fitted to previously published pharmacokinetic profiles of leukemia inhibitory factor (LIF), a cytokine that seems to exhibit TMDD, following intravenous administration of 12.5, 25, 100, 250, 500, or 750 μg/kg in sheep. Simulations show that pharmacokinetic profiles display steeper distribution phases for lower doses and similar terminal disposition phases, but with slight underestimation at early time points as theoretically expected. The final model well-described LIF pharmacokinetics, and the final parameters, which were estimated with relatively good precision, were in good agreement with literature values. An equilibrium model of TMDD is developed that recapitulates the essential features of the full general model and eliminates the need for estimating drug-binding microconstants that are often difficult or impossible to identify from typical in vivo pharmacokinetic data.
TL;DR: This summary report of the workshop focuses on the major issues discussed during presentations and open forums and noted consensus achieved among the participants on topics from nomenclature to best practices.
Abstract: Biomarkers are increasingly used in drug development to aid scientific and clinical decisions regarding the progress of candidate and marketed therapeutics. Biomarkers can improve the understanding of diseases as well as therapeutic and off-target effects of drugs. Early implementation of biomarker strategies thus promises to reduce costs and time-to-market as drugs proceed through increasingly costly and complex clinical development programs. The 2003 American Association of Pharmaceutical Sciences/Clinical Ligand Assay Society Biomarkers Workshop (Salt Lake City, UT, USA, October 24-25, 2003) addressed key issues in biomarker research, with an emphasis on the validation and implementation of biochemical biomarker assays, covering from preclinical discovery of efficacy and toxicity biomarkers through clinical and postmarketing implementation. This summary report of the workshop focuses on the major issues discussed during presentations and open forums and noted consensus achieved among the participants on topics from nomenclature to best practices. For example, it was agreed that because reliable and accurate data provide the basis for sound decision making, biomarker assays must be validated in a manner that enables the creation of such data. The nature of biomarker measurements often precludes direct application of regulatory guidelines established for clinical diagnostics or drug bioanalysis, and future guidance on biomarker assay validation should therefore be adaptable enough that validation criteria do not stifle creative biomarker solutions.
TL;DR: Metformin is transported by the basolateral organic cation transporter hOCT2 in the human kidney and could play a role in the drug interactions between metformin and some cationic drugs.
Abstract: Purpose.
Metformin, an antihyperglycemic agent, is eliminated by tubular secretion in addition to glomerular filtration in the human kidney. This study was performed to characterize metformin transport by human organic cation transporter 2 (hOCT2), the most abundant organic cation transporter in the basolateral membranes of the human kidney.
TL;DR: The use of CFD has provided further insight into the effect of air flow on the performance of the Aerolizer® and the approach of using CFD coupled with powder dispersion is readily applicable to other dry powder inhalers to help better understand their performance optimization.
Abstract: The aims of the study are to analyze the influence of air flow on the overall performance of a dry powder inhaler (Aerolizer®) and to provide an initial quantification of the flow turbulence levels and particle impaction velocities that maximized the inhaler dispersion performance. Computational fluid dynamics (CFD) analysis of the flowfield in the Aerolizer®, in conjunction with experimental dispersions of mannitol powder using a multistage liquid impinger, was used to determine how the inhaler dispersion performance varied as the device flow rate was increased. Both the powder dispersion and throat deposition were increased with air flow. The capsule retention was decreased with flow, whereas the device retention first increased then decreased with flow. The optimal inhaler performance was found at 65 l min−1 showing a high fine particle fraction (FPF) of 63 wt.% with low throat deposition (9.0 wt.%) and capsule retention (4.3 wt.%). Computational fluid dynamics analysis showed that at the critical flow rate of 65 l min−1, the volume-averaged integral scale strain rate (ISSR) was 5,400 s−1, and the average particle impaction velocities were 12.7 and 19.0 m s−1 at the inhaler base and grid, respectively. Correlations between the device flow rate and (a) the amount of throat deposition and (b) the capsule emptying times were also developed. The use of CFD has provided further insight into the effect of air flow on the performance of the Aerolizer®. The approach of using CFD coupled with powder dispersion is readily applicable to other dry powder inhalers (DPIs) to help better understand their performance optimization.
TL;DR: In this review article, the asymmetrical transporter distribution and altered surface expression in polarized tissues are discussed.
Abstract: It is well known that transporter proteins play a key role in governing drug absorption, distribution, and elimination in the body, and, accordingly, they are now considered as causes of drug-drug interactions and interindividual differences in pharmacokinetic profiles. Polarized tissues directly involved in drug disposition (intestine, kidney, and liver) and restricted distribution to naive sanctuaries (blood-tissue barriers) asymmetrically express a variety of drug transporters on the apical and basolateral sides, resulting in vectorial drug transport. For example, the organic anion transporting polypeptide (OATP) family on the sinusoidal (basolateral) membrane and multidrug resistance-associated protein 2 (MRP2/ABCC2) on the apical bile canalicular membrane of hepatocytes take up and excrete organic anionic compounds from blood to bile. Such vectorial transcellular transport is fundamentally attributable to the asymmetrical distribution of transporter molecules in polarized cells. Besides the apical/basolateral sorting direction, distribution of the transporter protein between the membrane surface (active site) and the intracellular fraction (inactive site) is of practical importance for the quantitative evaluation of drug transport processes. The most characterized drug transporter associated with this issue is MRP2 on the hepatocyte canalicular (apical) membrane, and it is linked to a genetic disease. Dubin-Johnson syndrome is sometimes caused by impaired canalicular surface expression of MRP2 by a single amino acid substitution. Moreover, single nucleotide polymorphisms in OATP-C/SLC21A6 (SLCO1B1) also affect membrane surface expression, and actually lead to the altered pharmacokinetic profile of pravastatin in healthy subjects. In this review article, the asymmetrical transporter distribution and altered surface expression in polarized tissues are discussed.
TL;DR: The pharmacokinetic model appears to be suitable for describing the absorption and disposition of the quercetin and its conjugates and may be applicable to other flavonoids that undergo similar pharmacokinetics pathways.
Abstract: To determine the pharmacokinetics of quercetin and its glucuronide/sulfate conjugates and to develop a pharmacokinetic model to simultaneously describe their disposition after intravenous and oral administration in rats. After oral, intraportal, and intravenous administration of quercetin, serial plasma, urine, and fecal concentrations of quercetin and its conjugates were determined by an HPLC method. Enterohepatic recirculation was evaluated in a linked-rat model as well as after oral administration of bile containing quercetin and its metabolites. Based on the experimental data, a specific compartmental model was developed and validated to describe and predict the plasma concentration-time profiles of quercetin and its conjugates after oral and intravenous administration. Only 5.3% of unchanged quercetin was bioavailable, although the total quercetin absorbed was as high as 59.1%. After oral administration, about 93.3% of quercetin was metabolized in the gut, with only 3.1% metabolized in the liver. No significant enterohepatic recirculation was observed for both quercetin and its conjugated metabolites. The pharmacokinetic model fitted well the observed data of quercetin and its conjugates. Our study clarifies the relative importance of the gut, liver, and bile in the metabolism and excretion of quercetin and its conjugates. The pharmacokinetic model appears to be suitable for describing the absorption and disposition of the quercetin and its conjugates and may be applicable to other flavonoids that undergo similar pharmacokinetic pathways.
TL;DR: Manometric temperature measurement and the expert system for good practices in freeze drying does allow development of an optimized freeze-drying process during a single laboratory freeze-Drying experiment and the rate of water desorption during secondary drying may be accurately measured by a variation of the basic MTM procedure.
Abstract: To develop a procedure based on manometric temperature measurement (MTM) and an “expert system” for good practices in freeze drying that will allow development of an optimized freeze-drying process during a single laboratory freeze-drying experiment. Freeze drying was performed with a FTS Dura-Stop/Dura-Top freeze dryer with the manometric temperature measurement software installed. Five percent solutions of glycine, sucrose, or mannitol with 2 ml to 4 ml fill in 5 ml vials were used, with all vials loaded on one shelf. Details of freezing, optimization of chamber pressure, target product temperature, and some aspects of secondary drying are determined by the expert system algorithms. MTM measurements were used to select the optimum shelf temperature, to determine drying end points, and to evaluate residual moisture content in real-time. MTM measurements were made at 1 hour or half-hour intervals during primary drying and secondary drying, with a data collection frequency of 4 points per second. The improved MTM equations were fit to pressure-time data generated by the MTM procedure using Microcal Origin software to obtain product temperature and dry layer resistance. Using heat and mass transfer theory, the MTM results were used to evaluate mass and heat transfer rates and to estimate the shelf temperature required to maintain the target product temperature. MTM product dry layer resistance is accurate until about two-thirds of total primary drying time is over, and the MTM product temperature is normally accurate almost to the end of primary drying provided that effective thermal shielding is used in the freeze-drying process. The primary drying times can be accurately estimated from mass transfer rates calculated very early in the run, and we find the target product temperature can be achieved and maintained with only a few adjustments of shelf temperature. The freeze-dryer overload conditions can be estimated by calculation of heat/mass flow at the target product temperature. It was found that the MTM results serve as an excellent indicator of the end point of primary drying. Further, we find that the rate of water desorption during secondary drying may be accurately measured by a variation of the basic MTM procedure. Thus, both the end point of secondary drying and real-time residual moisture may be obtained during secondary drying. Manometric temperature measurement and the expert system for good practices in freeze drying does allow development of an optimized freeze-drying process during a single laboratory freeze-drying experiment.
TL;DR: The use of the new biomarker classification is discussed in the context of the application of mechanism-based PK/PD analysis in drug discovery and development.
Abstract: In recent years, pharmacokinetic/pharmacodynamic (PK/PD) modeling has developed from an empirical descriptive discipline into a mechanistic science that can be applied at all stages of drug development. Mechanism-based PK/PD models differ from empirical descriptive models in that they contain specific expressions to characterize processes on the causal path between drug administration and effect. Mechanism-based PK/PD models have much improved properties for extrapolation and prediction. As such, they constitute a scientific basis for rational drug discovery and development. In this report, a novel classification of biomarkers is proposed. Within the context of mechanism-based PK/PD modeling, a biomarker is defined as a measure that characterizes, in a strictly quantitative manner, a process, which is on the causal path between drug administration and effect. The new classification system distinguishes seven types of biomarkers: type 0, genotype/phenotype determining drug response; type 1, concentration of drug or drug metabolite; type 2, molecular target occupancy; type 3, molecular target activation; type 4, physiological measures; type 5, pathophysiological measures; and type 6, clinical ratings. In this paper, the use of the new biomarker classification is discussed in the context of the application of mechanism-based PK/PD analysis in drug discovery and development.
TL;DR: Drug-polymer hydrogen bonding in indoprofen-PVP dispersions favors solid solubility and phase separation rate of drug from the solid dispersions depends on the initial drug content and the nature of drug- polymer interactions.
Abstract: Purpose.
To determine the factors influencing “solid solubility” and phase separation kinetics of drugs from amorphous solid dispersions.
TL;DR: Small, stable insulin nanocomplexes were formed using PEGylated trimethyl chitosan copolymers, which significantly enhanced the uptake of insulin in Caco-2 cells by adsorptive endocytosis, but nanocom Complexation did not seem to enhance transcellular insulin transport across cell monolayers, which is in line with animal data in rats.
Abstract: The effect of chitosan and polyethylene glycol (PEG)ylated trimethyl chitosan copolymer structure on the uptake and transport of insulin nanocomplexes was evaluated and transport mechanisms were investigated. Insulin nanocomplexes were prepared from chitosan and its copolymers by self-assembly. Complex uptake in Caco-2 cells was quantified by measuring the cell-associated fluorescence and cellular localization was visualized by confocal laser scanning microscopy (CLSM) using tetra-methyl-rhodamine isothiocyanate-labeled insulin. The transport of selected insulin complexes through Caco-2 monolayers was then investigated and compared with in vivo uptake by nasal epithelium in diabetic rats. All complexes were 200–400 nm in diameter, positively charged, and displayed an insulin loading efficiency of approximately 90%. In vitro release of insulin from the complexes was dependent on the medium pH. Insulin uptake was enhanced by nanocomplex formation, and was dependent on incubation time, temperature, and concentration. Complex uptake in Caco-2 cells was inhibited by 25.2 ± 1.3%, 13.0 ± 1.0%, and 16.6 ± 0.7% in the presence of cytochalasin D, sodium azide, and 2,4-dinitrophenol, respectively. The uptake mechanism was assumed to be adsorptive endocytosis. Additionally, cell uptake efficiency was shown to be influenced by a combination of polymer molecular weight, viscosity, and positive charge density. However, none of the nanocomplexes displayed improved transport properties when compared to insulin transport data after 2 h incubation with Caco-2 monolayers. This result was further confirmed with animal experiments. Small, stable insulin nanocomplexes were formed using PEGylated trimethyl chitosan copolymers, which significantly enhanced the uptake of insulin in Caco-2 cells by adsorptive endocytosis. However, nanocomplexation did not seem to enhance transcellular insulin transport across cell monolayers, which is in line with animal data in rats. This implies that PEGylated trimethyl chitosan complexes with insulin need further optimization and the Caco-2 cell line is a predictable in vitro cell culture model for drug absorption.
TL;DR: The identification of genes related to detoxification, transport, cell growth and apoptosis, cell adhesion, kinase, and transcription regulated by EGCG not only provide potential novel insight into the effect of E GCG on global gene expression and chemopreventive effects, but also point to the potential role of Nrf2 in these processes.
Abstract: This study was conducted to study global gene expression profiles elicited by (−)-epigallocatechin-3-gallate (EGCG) in mouse liver and small intestine, as well as to identify EGCG-regulated Nrf2-dependent genes. C57BL/6J and C57BL/6J/Nrf2(−/−) mice were given an oral dose of EGCG at 200 mg/kg or treated with vehicle. Both liver and small intestine were collected 3 h and 12 h after treatment. Total RNA was extracted from the tissues and gene expression profiles were analyzed using Affymetrix mouse genome 430 2.0 array and GeneSpring 6.1 software. Microarray data were validated using quantitative real-time reverse transcription-PCR chain reaction analysis. Genes that were either induced or suppressed more than two fold by EGCG treatment compared with vehicle treatment in the same genotype group were filtered using the GeneSpring software. Among these well-defined genes, 671 EGCG-regulated Nrf2-dependent genes and 256 EGCG-regulated Nrf2-independent genes were identified in liver, whereas 228 EGCG-regulated Nrf2-dependent genes and 98 EGCG-regulated Nrf2-independent genes were identified in the small intestine. Based on their biological functions, these genes mainly fall into the category of ubiquitination and proteolysis, electron transport, detoxification, transport, cell growth and apoptosis, cell adhesion, kinase and phosphatases, and transcription factors. Genes expressed in mouse liver are more responsive to oral treatment of EGCG than those expressed in small intestine. EGCG could regulate many genes in both organs in an Nrf2-dependent manner. The identification of genes related to detoxification, transport, cell growth and apoptosis, cell adhesion, kinase, and transcription regulated by EGCG not only provide potential novel insight into the effect of EGCG on global gene expression and chemopreventive effects, but also point to the potential role of Nrf2 in these processes.
TL;DR: Using pH-solubility and pH-dissolution rate interrelationships, it has been established that diffusion layer pH could be used to explain the observed rank order in dissolution rates for different salt forms.
Abstract: To evaluate the effect of pH on solubility and dissolution rates of a model weak base, haloperidol, and two different salt forms, hydrochloride and mesylate. pH-solubility profiles were determined by using haloperidol base, haloperidol hydrochloride, and haloperidol mesylate as starting materials; concentrated or diluted HCl or NaOH solutions were added to aqueous suspensions of solids to adjust pH to desired values. Intrinsic dissolution rates were determined using intrinsic dissolution apparatus under various pH-stat conditions. Further, approximation of diffusion layer pH was estimated from that of 10% w/w slurries of drug substances in dissolution media, which were used to correlate with intrinsic dissolution rates of haloperidol and its salt forms under different pHs. pH-solubility profiles of haloperidol base and its HCl salt were similar, while when the mesylate salt was used as starting material, it exhibited a higher solubility between pH 2 and 5. The higher solubility of the mesylate salt at pH 2–5 is attributed to its higher solubility product (Ksp) than that of the hydrochloride salt. The pH-solubility profiles indicated a pHmax (pH of maximum solubility) of ∼5, indicating that the free base would exist as the solid phase above this pH and a salt would be formed below this pH. Below pH 1.5, all solubilities were comparable due to a conversion of haloperidol base or the mesylate salt to the HCl salt form when HCl was used as the acidifying agent. These were confirmed by monitoring the solid phase by differential scanning calorimeter. When their dissolution rates are tested, dissolution rates of the mesylate salt were much higher than those of the free base or the HCl salt, except at very low pH (<2). Dissolution rates of free base and HCl salt also differed from each other, where that of HCl salt exhibits higher dissolution rates at higher pHs. A direct correlation of dissolution rate with solubility at diffusion layer pH at the surface of dissolving solid was established for haloperidol, its hydrochloride, and mesylate salts. Using pH-solubility and pH-dissolution rate interrelationships, it has been established that diffusion layer pH could be used to explain the observed rank order in dissolution rates for different salt forms. A non-hydrochloride salt, such as a mesylate salt, may provide advantages over a hydrochloride salt due to its high solubility and lack of common ion effect unless at very low pH.
TL;DR: The data suggest that rutin acted as a quercetin deliverer to the large intestine and its anti-inflammatory action in TNBS-induced colitis rats may be through quercETin-mediated inhibition of TNF-α-induced NFκB activation.
Abstract: Orally administered rutin reportedly ameliorates 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis of rats. We investigated the metabolic and pharmacological properties of rutin underlying the rutin-mediated amelioration of the rat colitis. Apparent partition coefficients of rutin and its aglycone quercetin were compared. The biochemical/chemical stability of rutin was examined in the contents of various segments of gastrointestinal tracts of rats. Inflammatory indices were determined in the colitis rats after oral administration of rutin or rectal administration of quercetin. In human colon epithelial cells, the effect of quercetin on tumor necrosis factor-alpha (TNF-α)-induced nuclear factor kappa B (NFκB) activation was examined. The sugar residue in rutin greatly lowered the apparent partition coefficient and was rapidly deglycosylated to liberate quercetin in the cecal contents, whereas it was stable in the contents of the upper intestine. Not only oral administration of rutin but also rectal administration of quercetin remarkably ameliorated TNBS-induced colitis rats, indicating that quercetin liberated from rutin is therapeutically active. Furthermore, quercetin dose-dependently inhibited an inflammatory signal TNF-α-dependent NFκB activation. Our data suggest that rutin acted as a quercetin deliverer to the large intestine and its anti-inflammatory action in TNBS-induced colitis rats may be through quercetin-mediated inhibition of TNF-α-induced NFκB activation.
TL;DR: The experimental data let us conclude that NLC lipid nanoparticles are not spherical solid lipid particles with embedded liquid droplets, but they are rather solid platelets with oil present between the solid platelet and the surfactant layer.
Abstract: Recently, colloidal dispersions made of mixtures from solid and liquid lipids have been described to overcome the poor drug loading capacity of solid lipid nanoparticles (SLN). It has been proposed that these nanostructured lipid carriers (NLC) are composed of oily droplets, which are embedded in a solid lipid matrix. High loading capacities and controlled release characteristics have been claimed. It is the objective of the present paper to investigate these new NLC particles in more detail to obtain insights into their structure. Colloidal lipid dispersions were produced by high-pressure homogenization. Particle sizes were estimated by laser diffraction and photon correlation spectroscopy. The hydrophobic fluorescent marker nile red (NR) was used as model drug, and by fluorometric spectroscopy, the molecular environment (polarity) was elucidated because of solvatochromism of NR. The packaging of the lipid nanoparticles was investigated by Raman spectroscopy and by densimetry. The light propagation in lipid nanodispersions was examined by refractometry to obtain further insights into the nanostructural compositions of the carriers. Fluorometric spectroscopy clearly demonstrates that NLC nanoparticles offer two nanocompartments of different polarity to accommodate NR. Nevertheless, in both compartments, NR experiences less protection from the outer water phase than in a nanoemulsion. In conventional SLN, lipid crystallization leads to the expulsion of the lipophilic NR from the solid lipid. Measurements performed by densimetry and Raman spectroscopy confirm the idea of intact glyceryl behenate lattices in spite of oil loading. The lipid crystals are not disturbed in their structure as it could be suggested in case of oil incorporation. Refractometric data reveal the idea of light protection because of incorporation of sensitive drug molecules in NLC. Neither SLN nor NLC lipid nanoparticles did show any advantage with respect to incorporation rate compared to conventional nanoemulsions. The experimental data let us conclude that NLC lipid nanoparticles are not spherical solid lipid particles with embedded liquid droplets, but they are rather solid platelets with oil present between the solid platelet and the surfactant layer.
TL;DR: The sustained-release PLG polymer vehicle is a promising delivery system for therapeutic neovascularization applications and resulted in sustained VEGF delivery, improved tissue perfusion, greater capillary density, and more mature vasculature compared to the controls.
Abstract: We hypothesized that sustained delivery of vascular endothelial growth factor (VEGF) using a polymer [85:15 poly(lactide-co-glycolide) (PLG)] would enhance angiogenesis and improve perfusion of ischemic tissue. C57BL/6J mice (n = 20/group) underwent unilateral hind limb ischemia surgery and were randomized to groups of no scaffold implantation (∅-Implant), unloaded scaffold implantation (Empty-PLG), or implantation of scaffolds incorporating 3 μg of VEGF165 (PLG-VEGF). Endpoints included laser Doppler perfusion imaging (LDPI, ischemic/nonischemic limb, %), local vessel counts, immunohistochemistry for CD31, and α-smooth muscle actin. In vitro release kinetics of VEGF from PLG was also measured. PLG-VEGF resulted in improved lower extremity perfusion vs. controls as measured by LDPI% at 7, 14, 21, and 28 days (p < 0.05). PLG-VEGF was associated with significantly greater percentage of vessels staining for CD31 and α-smooth muscle actin compared to the Empty-PLG or ∅-Implant (p < 0.05 for both). The PLG-VEGF scaffolds resulted in sustained VEGF delivery, improved tissue perfusion, greater capillary density, and more mature vasculature compared to the controls. The sustained-release PLG polymer vehicle is a promising delivery system for therapeutic neovascularization applications.
TL;DR: RhIFNα2b immunogenicity is related to its structural integrity and depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size.
Abstract: This study was conducted to study the influence of protein structure on the immunogenicity in wild-type and immune tolerant mice of well-characterized degradation products of recombinant human interferon alpha2b (rhIFNα2b). RhIFNα2b was degraded by metal-catalyzed oxidation (M), cross-linking with glutaraldehyde (G), oxidation with hydrogen peroxide (H), and incubation in a boiling water bath (B). The products were characterized with UV absorption, circular dichroism and fluorescence spectroscopy, gel permeation chromatography, reverse-phase high-pressure liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and mass spectrometry. The immunogenicity of the products was evaluated in wild-type mice and in transgenic mice immune tolerant for hIFNα2. Serum antibodies were detected by enzyme-linked immunosorbent assay or surface plasmon resonance. M-rhIFNα2b contained covalently aggregated rhIFNα2b with three methionines partly oxidized to methionine sulfoxides. G-rhIFNα2b contained covalent aggregates and did not show changes in secondary structure. H-rhIFNα2b was only chemically changed with four partly oxidized methionines. B-rhIFNα2b was largely unfolded and heavily aggregated. Nontreated (N) rhIFNα2b was immunogenic in the wild-type mice but not in the transgenic mice, showing that the latter were immune tolerant for rhIFNα2b. The anti-rhIFNα2b antibody levels in the wild-type mice depended on the degradation product: M-rhIFNα2b > H-rhIFNα2b ∼ N-rhIFNα2b ≫ B-rhIFNα2b; G-rhIFNα2b did not induce anti-rhIFNα2b antibodies. In the transgenic mice, only M-rhIFNα2b could break the immune tolerance. RhIFNα2b immunogenicity is related to its structural integrity. Moreover, the immunogenicity of aggregated rhIFNα2b depends on the structure and orientation of the constituent protein molecules and/or on the aggregate size.
TL;DR: This review recapitulates how pharmacologic agents administered into the CSF system prevent disease or enhance the brain’s ability to recover from chemical and physical insults and discusses the BCSFB interaction with the blood–brain barrier.
Abstract: The choroid plexus (CP), i.e., the blood-cerebrospinal fluid barrier (BCSFB) interface, is an epithelial boundary exploitable for drug delivery to brain. Agents transported from blood to lateral ventricles are convected by CSF volume transmission (bulk flow) to many periventricular targets. These include the caudate, hippocampus, specialized circumventricular organs, hypothalamus, and the downstream pia-glia and arachnoid membranes. The CSF circulatory system normally provides micronutrients, neurotrophins, hormones, neuropeptides, and growth factors extensively to neuronal networks. Therefore, drugs directed to CSF can modulate a variety of endocrine, immunologic, and behavioral phenomema; and can help to restore brain interstitial and cellular homeostasis disrupted by disease and trauma. This review integrates information from animal models that demonstrates marked physiologic effects of substances introduced into the ventricular system. It also recapitulates how pharmacologic agents administered into the CSF system prevent disease or enhance the brain's ability to recover from chemical and physical insults. In regard to drug distribution in the CNS, the BCSFB interaction with the blood-brain barrier is discussed. With a view toward translational CSF pharmacotherapy, there are several promising innovations in progress: bone marrow cell infusions, CP encapsulation and transplants, neural stem cell augmentation, phage display of peptide ligands for CP epithelium, CSF gene transfer, regulation of leukocyte and cytokine trafficking at the BCSFB, and the purification of neurotoxic CSF in degenerative states. The progressively increasing pharmacological significance of the CP-CSF nexus is analyzed in light of treating AIDS, multiple sclerosis, stroke, hydrocephalus, and Alzheimer's disease.
TL;DR: Pegylated and nonpegylated E78 NPs have potential blood compatibility at clinically relevant doses and very rapidly inhibited agonist-induced platelet activation and aggregation in a dose-dependent manner.
Abstract: Pegylated and nonpegylated cetyl alcohol/polysorbate nanoparticles (E78 NPs) are being tested as drug carriers for specific tumor and brain targeting. Because these nanoparticle formulations are designed for systemic administration, it is important to test the compatibility of these lipid-based NPs with blood and blood cells. The hemocompatibility of E78 NPs was evaluated with a particular focus on hemolytic activity, platelet function, and blood coagulation. Human red blood cell lysis was determined by measuring hemoglobin release. Activation and aggregation of human platelets were determined using flow cytometry and aggregometry, respectively. Finally, the whole blood clotting time was measured using human blood. E78 NPs did not cause in vitro red blood cell lysis at concentrations up to 1 mg/mL. In addition, under conditions tested, E78 and polyethylene glycol (PEG)-coated E78 NPs (PEG-E78 NPs) did not activate platelets. In fact, both NP formulations very rapidly inhibited agonist-induced platelet activation and aggregation in a dose-dependent manner. Additionally, E78 NPs significantly prolonged in vitro whole blood clotting time at a concentration of 500 μg/mL or greater. It was concluded that PEG-coated and nonpegylated E78 NPs have potential blood compatibility at clinically relevant doses. Based on the calculated nanoparticle-to-platelet ratio, the concentration at which E78 NPs could potentially affect platelet function in vivo was approximately 1 mg/mL.