PharmaGateway

Pharmaceutical Research: Metal Impurities in Food and Drugs

2010-03-09

Type: Original Paper

The major metals of potential health concern found in food, drugs (medicines), and dietary supplements are lead, cadmium, mercury, and arsenic. Other metals, such as chromium, copper, manganese, molybdenum, vanadium, nickel, osmium, rhodium, ruthenium, iridium, palladium, and platinum, may be used or introduced during manufacturing and may be controlled in the final article as impurities. Screening for metals in medicines and dietary supplements rarely indicates the presence of toxic metal impurities at levels of concern. The setting of heavy metal limits is appropriate for medicines and is appropriate for supplements when heavy metals are likely or certain to contaminate a given product. Setting reasonable health-based limits for some of these metals is challenging because of their ubiquity in the environment, limitations of current analytical procedures, and other factors. Taken together, compendial tests for metals in food and drugs present an array of issues that challenge compendial scientists.

The AAPS Journal: Role of Cannabinoids in the Development of Fatty Liver (Steatosis)

Vishnudutt Purohit; Rao Rapaka; David Shurtleff — 2010-03-05

Type: Original Paper

Emerging evidence suggests that cannabinoids play an important role in the modulation of fatty liver, which appears to be mediated via activation of cannabinoid receptors. Steatogenic agents such as ethanol and high-fat diet can upregulate the activity of cannabinoid 1 (CB1) receptors via increasing synthesis of endocannabinoids, 2-arachidonoylglycerol, and anandamide. CB1 receptors can also be upregulated by obesity. CB1 receptor activation results in upregulation of lipogenic transcription factor, sterol regulatory element-binding protein 1c and its target enzymes, acetyl-CoA carboxylase-1, and fatty acid synthase and concomitantly, downregulation of carnitine palmitoyltransferase-1. This leads to increased de novo fatty acid synthesis as well as decreased fatty acid oxidation, culminating into the development of fatty liver. High-fat diet, in addition to CB1 receptor activation, appears to activate CB2 receptors that may also contribute to fatty liver. In non-alcoholic fatty liver disease, CB2 receptor activation is associated with the development of fatty liver. Cannabis smoking can increase the severity of fatty liver in hepatitis C patients although the precise mechanism is unknown. As the mechanisms involved in endocannabinoid receptor signaling are being increasingly well understood and the biosynthetic regulatory elements elucidated, these present good opportunity for the pharmaceutical scientists to design drugs to treat liver diseases, including steatosis, based on the cannabinoids, endocannabinoids, and related templates.

Pharmaceutical Research: Classification of Annular Bed Flow Patterns and Investigation on Their Influence on the Bottom Spray Fluid Bed Coating Process

Li Wang; Paul Heng; Celine Liew — 2010-03-05

Type: Original Paper
Purpose

This study aims to classify annular bed flow patterns in the bottom spray fluid bed coating process, study their influence on coat uniformity and investigate the feasibility of developing real-time annular bed flow pattern detection as a PAT tool.

Methods

High-speed imaging and particle image velocimetry were used to visualize annular bed flow. Color coating and subsequent tristimulus colorimetry were employed to determine influence of annular bed flow pattern on coat uniformity. Feasibility of monitoring annular bed flow pattern through an observation window was tested using miniaturized particle velocity field and time series particle velocity orientation information.

Results

Three types of annular bed flow patterns were identified. Plug flow gave the best coat uniformity followed by global and localized fluidization. Plug flow may be advantageous for high spray-rate conditions, large-scale coating and prevention of particle segregation. Plug flow could be differentiated from the other flow patterns through a simulated observation window.

Conclusion

Annular bed flow patterns were classified and found to influence particle coat uniformity noticeably. Availability of annular bed flow information for large-scale coaters would enable adjustments for process optimization. This study highlights the potential of monitoring annular bed flow pattern as a PAT tool.

Pharmaceutical Research: Critical Evaluation of Nanoparticle Tracking Analysis (NTA) by NanoSight for the Measurement of Nanoparticles and Protein Aggregates

Vasco Filipe; Andrea Hawe; Wim Jiskoot — 2010-03-04

Type: Original Paper
Purpose

To evaluate the nanoparticle tracking analysis (NTA) technique, compare it with dynamic light scattering (DLS) and test its performance in characterizing drug delivery nanoparticles and protein aggregates.

Methods

Standard polystyrene beads of sizes ranging from 60 to 1,000 nm and physical mixtures thereof were analyzed with NTA and DLS. The influence of different ratios of particle populations was tested. Drug delivery nanoparticles and protein aggregates were analyzed by NTA and DLS. Live monitoring of heat-induced protein aggregation was performed with NTA.

Results

NTA was shown to accurately analyze the size distribution of monodisperse and polydisperse samples. Sample visualization and individual particle tracking are features that enable a thorough size distribution analysis. The presence of small amounts of large (1,000 nm) particles generally does not compromise the accuracy of NTA measurements, and a broad range of population ratios can easily be detected and accurately sized. NTA proved to be suitable to characterize drug delivery nanoparticles and protein aggregates, complementing DLS. Live monitoring of heat-induced protein aggregation provides information about aggregation kinetics and size of submicron aggregates.

Conclusion

NTA is a powerful characterization technique that complements DLS and is particularly valuable for analyzing polydisperse nanosized particles and protein aggregates.

AAPS PharmSciTech: Naked Plasmid DNA Formulation: Effect of Different Disaccharides on Stability after Lyophilisation

Susanne Quaak; John Haanen; Jos Beijnen; Bastiaan Nuijen — 2010-03-04

Type: Original Paper

Since plasmid DNA (pDNA) is unstable in solution, lyophilisation can be used to increase product shelf life. To prevent stress on pDNA molecules during lyophilisation, cryo- and lyoprotectants have to be added to the formulation. This study assessed the effect of disaccharides on naked pDNA stability after lyophilisation using accelerated stability studies. Naked pDNA was lyophilised with sucrose, trehalose, maltose or lactose in an excipient/DNA w/w ratio of 20. To one part of the vials extra residual moisture was introduced by placing the vials half opened in a 25°C/60% RH climate chamber, before placing all vials in climate chambers (25°C/60% RH and 40°C/75% RH) for stability studies. An ex vivo human skin model was used to assess the effect of disaccharides on transfection efficiency. Lyophilisation resulted in amorphous cakes for all disaccharides with a residual water content of 0.8% w/w. Storage at 40°C/75% RH resulted in decreasing supercoiled (SC) purity levels (sucrose and trehalose maintained approximately 80% SC purity), but not in physical collapse. The addition of residual moisture (values between 7.5% and 10% w/w) resulted in rapid collapse except for trehalose and decreasing SC purity for all formulations. In a separate experiment disaccharide formulation solutions show a slight but significant reduction (<3% with sucrose and maltose) in transfection efficiency when compared to pDNA dissolved in water. We demonstrate that disaccharides, like sucrose and trehalose, are effective lyoprotectants for naked pDNA.

AAPS PharmSciTech: Development and Characterization of Enteric-Coated Immediate-Release Pellets of Aceclofenac by Extrusion/Spheronization Technique Using κ-Carrageenan as a Pelletizing Agent

Vaishali Kilor; Nidhi Sapkal; Jasmine Awari; Bharti Shewale — 2010-03-03

Type: Original Paper

In the present study, an attempt was made to prepare immediate-release enteric-coated pellets of aceclofenac, a poorly soluble nonsteroidal anti-inflammatory drug that has a gastrointestinal intolerance as its serious side effect. Formulation of enteric-coated pellets with improved solubility of aceclofenac could address both of these problems. To achieve these goals, pellets were prepared by extrusion–spheronization method using pelletizing agents that can contribute to the faster disintegration and thereby improve the solubility of the drug. Different disintegrants like β-cyclodextrin, kollidon CL, Ac-Di-Sol, and sodium starch glycolate were tried in order to further improve disintegration time. The pellets were characterized for drug content, particle size distribution, flow properties, infrared spectroscopy, surface morphology, disintegration rate, and dissolution profile. The formulations, which showed best disintegration and dissolution profiles, were coated with Eudragit L100-55, an enteric-coated polymer which does not dissolve at gastric pH but dissolves at intestinal pH, releasing the drug immediately in the dissolution medium. The optimized enteric-coated formulation containing 20% κ-carrageenan, lactose, and sodium starch glycolate as a disintegrant did inhibit the release of the drug for 2 h in 0.1 N HCl, whereas 87% of the drug was released within 45 min. The improvement was substantial when it was compared with solubility of pure drug under the same conditions. Thus, dissolution profiles suggested that combination of κ-carrageenan and sodium starch glycolate resulted into fast-disintegrating, immediate-release pellets, overcoming the bioavailability problem of the poorly soluble drug, aceclofenac, and enteric coating of these pellets avoids the exposure of aceclofenac to ulcer-prone areas of the gastrointestinal tract.

The AAPS Journal: Near-Infrared Investigations of Novel Anti-HIV Tenofovir Liposomes

Ahmed Zidan; Crystal Spinks; Joseph Fortunak; Muhammad Habib; Mansoor Khan — 2010-03-03

Type: Original Paper

Near-infrared (NIR) approaches is considered one of the most well-studied process analyzers evolving from the process analytical technology initiatives. The objective of this study was to evaluate NIR spectroscopy and imaging to assess individual components within a novel tenofovir liposomal formulation. By varying stearylamine, as a positive charge imparting agent, five batches were prepared by the thin film method. Each formulation was characterized in terms of drug entrapment efficiency, release characteristics, particle sizing, and zeta potential. Drug excipients compatibility was tested using Fourier transform infrared spectroscopy, differential scanning calorimetry, and X-ray diffraction. The obtained results showed an increase in drug entrapment and a slower drug release by increasing the incorporated percentage of stearylamine. The compatibility testing revealed a significant interaction between the drug and some of the investigated excipients. The developed NIR calibration model was able to assess drug, phospholipid, and stearylamine levels along the batches. The calibration and prediction plots were linear with correlation coefficients of more than 0.9. The root square standard errors of calibration and prediction did not attain 5% of the measured values confirming the accuracy of the model. In contrast, NIR spectral imaging was capable of clearly distinguishing the different batches, both qualitatively and quantitatively. A linear relationship was obtained correlating the actual drug entrapped and the predicted values obtained from the partial least squares images.

The AAPS Journal: Preservation of the Immunogenicity of Dry-powder Influenza H5N1 Whole Inactivated Virus Vaccine at Elevated Storage Temperatures

2010-03-02

Type: Original Paper

Stockpiling of pre-pandemic influenza vaccines guarantees immediate vaccine availability to counteract an emerging pandemic. Generally, influenza vaccines need to be stored and handled refrigerated to prevent thermal degradation of the antigenic component. Requirement of a cold-chain, however, complicates stockpiling and the logistics of vaccine distribution. We, therefore, investigated the effect of elevated storage temperatures on the immunogenicity of a pre-pandemic influenza A H5N1 whole inactivated virus vaccine. Either suspended in liquid or kept as a freeze-dried powder, vaccines could be stored for 1 year at ambient temperature (20°C) with minimal loss of immunogenicity in mice. Elevation of the storage temperature to 40°C, however, resulted in a significant loss of immunogenic potency within 3 months if vaccines were stored in liquid suspension. In sharp contrast, freeze-dried powder formulations were stable at 40°C for at least 3 months. The presence of inulin or trehalose sugar excipients during freeze-drying of the vaccine proved to be critical to maintain its immunogenic potency during storage, and to preserve the characteristic Th1-type response to whole inactivated virus vaccine. These results indicate that whole inactivated virus vaccines may be stored and handled at room temperature in moderate climate zones for over a year with minimal decline and, if converted to dry-powder, even in hot climate zones for at least 3 months. The increased stability of dry-powder vaccine at 40°C may also point to an extended shelf-life when stored at 4°C. Use of the more stable dry-powder formulation could simplify stockpiling and thereby facilitating successful pandemic intervention.

Pharmaceutical Research: In Vivo Distribution of Polymeric Nanoparticles at the Whole-Body, Tumor, and Cellular Levels

Helen Lee; Bryan Hoang; Humphrey Fonge; Raymond Reilly; Christine Allen — 2010-03-02

Type: Original Paper
Purpose

Block copolymer micelles (BCMs) were functionalized with indium-111 and/or epidermal growth factor (EGF), which enabled investigation of the in vivo transport of passively and actively targeted BCMs. The integration of conventional and image-based techniques afforded novel quantitative means to achieve an in-depth insight into the fate of polymeric nanoparticles in vivo.

Methods

Pharmacokinetics and biodistribution studies were performed in athymic mice bearing human breast xenografts to evaluate the whole-body transport of NT-BCMs (non-targeted, EGF-) and T-BCMs (targeted, EGF+). The intratumoral distribution of BCMs was investigated using MicroSPECT/CT and autoradiographic imaging, complemented with quantitative MATLAB® analyses. Tumors were fractionated for quantifying intracellular uptake of BCMs via γ-counting.

Results

The intratumoral distribution of NT-BCMs and T-BCMs were found to be heterogeneous, and positively correlated with tumor vascularization (r > 0.68 ± 0.04). The enhanced in vivo cell uptake and cell membrane binding of T-BCMs were found to delay their clearance from tumors overexpressing EGFR, and therefore resulted in enhanced tumor accumulation for the T-BCMs in comparison to the NT-BCMs.

Conclusions

Adequate passive targeting is required in order to achieve effective active targeting. Tumor physiology has a significant impact on the transvascular and intratumoral transport of passively and actively targeted BCMs.

Pharmaceutical Research: Influence of Preparation Methods on Solid State Supersaturation of Amorphous Solid Dispersions: A Case Study with Itraconazole and Eudragit E100

2010-03-02

Type: Original Paper
Purpose

The present study aims to determine the drug / polymer miscibility level as a function of the preparation method for an amorphous solid dispersion model system containing itraconazole and eudragit E100. This value was compared to the theoretical crystalline drug solubility in the amorphous polymer and the miscibility of the amorphous drug in the amorphous polymer.

Methods

The amorphous solid dispersions were prepared via spray drying and film casting in order to evaluate the influence of the solvent drying rate. The experimental miscibility level was estimated using XRPD, MDSC, FT-IR, HPLC and TGA. The solubility and miscibility were estimated using the Flory-Huggins mixing theory and experimental drug in monomer solubility data.

Results

The experimental miscibility level was found to be 27.5% w/w for spray-dried and 15% for film-casted solid dispersions. FT-IR measurements confirmed the absence of saturable interactions like hydrogen bonds, and analysis of the mixed glass transition temperatures suggested low adhesion forces in the amorphous mixture. The solubility analysis rendered a positive FH interaction parameter, a crystalline solubility of approximately 0.012% w/w and an amorphous drug-polymer miscibility of approximately 7.07% w/w.

Conclusion

The solid dispersions are significantly supersaturated with respect to both crystalline solubility and amorphous miscibility demonstrating the influence of manufacturing methodology.

Pharmaceutical Research: Cell-Based Multiscale Computational Modeling of Small Molecule Absorption and Retention in the Lungs

Jing-yu Yu; Gus Rosania — 2010-03-01

Type: Original Paper
Purpose

For optimizing the local, pulmonary targeting of inhaled medications, it is important to analyze the relationship between the physicochemical properties of small molecules and their absorption, retention and distribution in the various cell types of the airways and alveoli.

Methods

A computational, multiscale, cell-based model was constructed to facilitate analysis of pulmonary drug transport and distribution. The relationship between the physicochemical properties and pharmacokinetic profile of monobasic molecules was explored. Experimental absorption data of compounds with diverse structures were used to validate this model. Simulations were performed to evaluate the effect of active transport and organelle sequestration on the absorption kinetics of compounds.

Results

Relating the physicochemical properties to the pharmacokinetic profiles of small molecules reveals how the absorption half-life and distribution of compounds are expected to vary in different cell types and anatomical regions of the lung. Based on logP, pK_a and molecular radius, the absorption rate constants (K_a) calculated with the model were consistent with experimental measurements of pulmonary drug absorption.

Conclusions

The cell-based mechanistic model developed herein is an important step towards the rational design of local, lung-targeted medications, facilitating the design and interpretation of experiments aimed at optimizing drug transport properties in lung.

The AAPS Journal: Comparison of Two Pharmacodynamic Transduction Models for the Analysis of Tumor Therapeutic Responses in Model Systems

Jun Yang; Donald Mager; Robert Straubinger — 2010-03-01

Type: Original Paper

Semi-mechanistic pharmacodynamic (PD) models that capture tumor responses to anticancer agents with fidelity can provide valuable insights that could aid in the optimization of dosing regimens and the development of drug delivery strategies. This study evaluated the utility and potential interchangeability of two transduction-type PD models: a cell distribution model (CDM) and a signal distribution model (SDM). The evaluation was performed by simulating dense and sparse tumor response data with one model and analyzing it using the other. Performance was scored by visual inspection and precision of parameter estimation. Capture of tumor response data was also evaluated for a liposomal formulation of paclitaxel in the paclitaxel-resistant murine Colon-26 model. A suitable PK model was developed by simultaneous fitting of literature data for paclitaxel formulations in mice. Analysis of the simulated tumor response data revealed that the SDM was more flexible in describing delayed drug effects upon tumor volume progression. Dense and sparse data simulated using the CDM were fit very well by the SDM, but under some conditions, data simulated using the SDM were fitted poorly by the CDM. Although both models described the dose-dependent therapeutic responses of Colon-26 tumors, the fit by the SDM contained less bias. The CDM and SDM are both useful transduction models that recapitulate, with fidelity, delayed drug effects upon tumor growth. However, they are mechanistically distinct and not interchangeable. Both fit some types of tumor growth data well, but the SDM appeared more robust, particularly where experimental data are sparse.

The AAPS Journal: Points to Consider when Establishing Drug Product Specifications for Parenteral Microspheres

Rajesh Kumar; Michael Palmieri — 2010-03-01

Type: Original Paper

Drug product specifications are a critical element of a good control strategy. Parenteral microsphere products are complex dosage forms, requiring careful development of test methods and acceptance criteria for the specifications. In particular, the in vitro release test method and acceptance criteria require rigorous scientific consideration and should be developed with an eye toward understanding the mechanisms of drug release. The final specifications need to ensure the safety, identity, strength, performance, and quality of the drug product at release and during storage through the end of its shelf-life. The specification limits are typically established based upon regulatory guidance, available data from the manufacturing process (process capability), from non-clinical, clinical, and stability studies.

The AAPS Journal: Autoradiography, MALDI-MS, and SIMS-MS Imaging in Pharmaceutical Discovery and Development

Eric Solon; Alain Schweitzer; Markus Stoeckli; Brendan Prideaux — 2010-03-01

Type: Original Paper

Whole-body autoradiography ((WBA) or quantitative WBA (QWBA)), microautoradiography (MARG), matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI), and secondary ion mass spectrometric imaging (SIMS-MSI) are high-resolution, molecular imaging techniques used to study the tissue distribution of radiolabeled and nonlabeled compounds in ex vivo, in situ biological samples. WBA, which is the imaging of the whole-body of lab animals, and/or their organ systems; and MARG, which provides information on the localization of radioactivity in histological preparations and at the cellular level, are used to support drug discovery and development efforts. These studies enable the conduct of human radiolabeled metabolite studies and have provided pharmaceutical scientists with a high resolution and quantitative method of accessing tissue distribution. MALDI-MSI is a mass spectrometric imaging technique capable of label-free and simultaneous determination of the identity and distribution of xenobiotics and their metabolites as well as endogenous substances in biological samples. This makes it an interesting extension to WBA and MARG, eliminating the need for radiochemistry and providing molecular specific information. SIMS-MSI offers a complementary method to MALDI-MSI for the acquisition of images with higher spatial resolution directly from biological specimens. Although traditionally used for the analysis of surface films and polymers, SIMS has been used successfully for the study of biological tissues and cell types, thus enabling the acquisition of images at submicrometer resolution with a minimum of samples preparation.

The AAPS Journal: Biodistribution Mechanisms of Therapeutic Monoclonal Antibodies in Health and Disease

Mohammad Tabrizi; Gadi Bornstein; Hamza Suria — 2010-03-01

Type: Original Paper

The monoclonal antibody market continues to witness an impressive rate of growth and has become the leading source of expansion in the biologic segment within the pharmaceutical industry. Currently marketed monoclonal antibodies target a diverse array of antigens. These antigens are distributed in a variety of tissues such as tumors, lungs, synovial fluid, psoriatic plaques, and lymph nodes. As the concentration of drug at the proximity of the biological receptor determines the magnitude of the observed pharmacological responses, a significant consideration in effective therapeutic application of monoclonal antibodies is a thorough understanding of the processes that regulate antibody biodistribution. Monoclonal antibody distribution is affected by factors such as molecular weight, blood flow, tissue and tumor heterogeneity, structure and porosity, target antigen density, turnover rate, and the target antigen expression profile.

The AAPS Journal: A History of Biopharmaceutics in the Food and Drug Administration 1968–1993

Jerome Skelly — 2010-03-01

Type: Original Paper

The history of biopharmaceutics is reviewed, beginning with its origin out of the Division of Clinical Research in The Bureau of Medicine. The reason for the creation of the Division of Biopharmaceutics, the certification of Food and Drug Administration authority over the functions it was to have, and the implementation of that authority are described. The determination of bioequivalence, the bioavailability decision rules, pharmacokinetics, and drug metabolism are explained. The reason for the development of the Scale-Up and Post Approval Regulations and how they were developed are also explained.

The AAPS Journal: Small Molecule Modifiers of the microRNA and RNA Interference Pathway

Alexander Deiters — 2010-03-01

Type: Original Paper

Recently, the RNA interference (RNAi) pathway has become the target of small molecule inhibitors and activators. RNAi has been well established as a research tool in the sequence-specific silencing of genes in eukaryotic cells and organisms by using exogenous, small, double-stranded RNA molecules of approximately 20 nucleotides. Moreover, a recently discovered post-transcriptional gene regulatory mechanism employs microRNAs (miRNAs), a class of endogenously expressed small RNA molecules, which are processed via the RNAi pathway. The chemical modulation of RNAi has important therapeutic relevance, because a wide range of miRNAs has been linked to a variety of human diseases, especially cancer. Thus, the activation of tumor-suppressive miRNAs and the inhibition of oncogenic miRNAs by small molecules have the potential to provide a fundamentally new approach for the development of cancer therapeutics.

The AAPS Journal: Compartmental Analysis and its Manifold Applications to Pharmacokinetics

Aldo Rescigno — 2010-03-01

Type: Original Paper

In this paper, I show how the concept of compartment evolved from the simple dilution of a substance in a physiological volume to its distribution in a network of interconnected spaces. The differential equations describing the fate of a substance in a living being can be solved, qualitatively and quantitatively, with the help of a number of mathematical techniques. A number of parameters of pharmacokinetic interest can be computed from the experimental data; often, the data available are not sufficient to determine some parameters, but it is possible to determine their range.

The AAPS Journal: How Minimally Invasive is Microdialysis Sampling? A Cautionary Note for Cytokine Collection in Human Skin and other Clinical Studies

Julie Stenken; Martin Church; Carolyn Gill; Geraldine Clough — 2010-03-01

Type: Original Paper

It is common to refer to microdialysis as a minimally invasive procedure, likening it to insertion of an artificial capillary. While a comparison of this type allows the process to be easily visualized by those outside the field, it tends to provide a false impression of the localized perturbation of the tissue space that is caused by catheter insertion. With the increased acceptance of microdialysis sampling as a viable in vivo sampling method, many researchers have begun to use the technique to explore inflammatory and immune-mediated diseases in the skin and other organs. Unfortunately, many of the molecules of interest, particularly chemokines and cytokines, are known to be generated during the inflammatory response to wounding and the subsequent cellular events leading to wound repair. With more than 11,000 reports citing the use of microdialysis sampling, only a few researchers have sought to assess the tissue damage that is incurred by probe insertion. For this reason, caution is warranted when collecting these molecules and inferring a role for them in clinical disease states. This commentary seeks to remind the research community of the confounding effects that signaling molecules related to the wounding response will have on clinical studies. Proper controls must be incorporated into all studies in order to assess whether or not particular molecules are truly related to the disease state under investigation or have been generated as part of the tissue response to the wound incurred by microdialysis catheter implantation.

Pharmaceutical Research: Pseudovirions as Vehicles for the Delivery of siRNA

Paul Lund; Ryan Hunt; Michael Gottesman; Chava Kimchi-Sarfaty — 2010-03-01

Type: Review Paper

Over the last two decades, small interfering RNA (siRNA)-mediated gene silencing has quickly become one of the most powerful techniques used to study gene function in vitro and a promising area for new therapeutics. Delivery remains a significant impediment to realizing the therapeutic potential of siRNA, a problem that is also tied to immunogenicity and toxicity. Numerous delivery vehicles have been developed, including some that can be categorized as pseudovirions: these are vectors that are directly derived from viruses but whose viral coding sequences have been eliminated, preventing their classification as viral vectors. Characteristics of the pseudovirions discussed in this review, namely phagemids, HSV amplicons, SV40 in vitro-packaged vectors, influenza virosomes, and HVJ-Envelope vectors, make them attractive for the delivery of siRNA-based therapeutics. Pseudovirions were shown to deliver siRNA effector molecules and bring about RNA interference (RNAi) in various cell types in vitro, and in vivo using immune-deficient and immune-competent mouse models. Levels of silencing were not always determined directly, but the duration of siRNA-induced knockdown lasted at least 3 days. We present examples of the use of pseudovirions for the delivery of synthetic siRNA as well as the delivery and expression of DNA-directed siRNA.

The AAPS Journal: Comparing Exponentially Weighted Moving Average and Run Rules in Process Control of Semiquantitative Immunogenicity Immunoassays

2010-03-01

Type: Original Paper

The immunogenicity immunoassay validation process ensures development of a robust, reproducible method. However, no matter how well developed, validated, and maintained a method is, in the course of running a large number of samples over time, it is not uncommon to see bad reagents, poorly calibrated equipment, personnel errors, or other unknown and unpredictable factors that have an impact in the performance of the method and quality of the sample results. The immunogenicity immunoassay thus needs to be closely monitored with an internal statistical quality control process overtime to ensure a consistent and reliable output. The statistical process control has been widely applied to monitor manufacturing processes and in clinical laboratories. Its application to immunogenicity immunoassays is relatively novel. Limited guidance is available to implement the process to monitor semiquantitative immunogenicity immunoassay performance. Here, we have performed a suitability evaluation for process control charts with actual laboratory data from three immunogenicity immunoassay methods each utilizing a different technology platform. Additionally, a panel of prepared samples designed to assess long-term method performance were periodically evaluated for over a year. Finally, we make recommendations for an internal quality control process based on the results of these evaluations.

The AAPS Journal: Molecular Targets of Dietary Phenethyl Isothiocyanate and Sulforaphane for Cancer Chemoprevention

Ka Cheung; Ah-Ng Kong — 2010-03-01

Type: Review Paper

Development of cancer is a long-term and multistep process which comprises initiation, progression, and promotion stages of carcinogenesis. Conceivably, it can be targeted and interrupted along these different stages. In this context, many naturally occurring dietary compounds from our daily consumption of fruits and vegetables have been shown to possess cancer preventive effects. Phenethyl isothiocyanate (PEITC) and sulforaphane (SFN) are two of the most widely investigated isothiocyanates from the crucifers. Both have been found to be very potent chemopreventive agents in numerous animal carcinogenesis models as well as cell culture models. They exert their chemopreventive effects through regulation of diverse molecular mechanisms. In this review, we will discuss the molecular targets of PEITC and SFN potentially involved in cancer chemoprevention. These include the regulation of drug-metabolizing enzymes phase I cytochrome P450s and phase II metabolizing enzymes. In addition, the signaling pathways including Nrf2–Keap 1, anti-inflammatory NFκB, apoptosis, and cell cycle arrest as well as some receptors will also be discussed. Furthermore, we will also discuss the similarities and their potential differences in the regulation of these molecular targets by PEITC and SFN.

The AAPS Journal: Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum

Li Xue; Rasheeda Johnson; Boris Gorovits — 2010-03-01

Type: Original Paper

Host cell-derived protein impurities may be present at low levels in biopharmaceutical products. Antibodies to host cell proteins are present in individuals with no known exposure to these products. In this study, antibodies to drug process-specific Chinese hamster ovary host cell-derived proteins (CHO-HCP) were measured in unexposed individuals using a validated enzyme-linked immunosorbent assay. Samples that tested positive for anti-CHO-HCP reactivity were further characterized to determine the isotypes and IgG subclasses expressed in each positive individual. The specificity of the detected anti-CHO-HCP antibody isotypes was confirmed by the competitive inhibition assay and the uncoated plate specificity testing. These antibody characterization experiments revealed that the prevalent anti-CHO-HCP antibody subclasses were predominantly IgG1 (present in 66.6% of individuals) and IgG2 (60%), with 33.3% positive for IgG3 while IgG4 was detected in low abundance. Forty percent (40%) of the individuals with IgG type reactivity were also identified as IgM-positive. Anti-CHO-HCP-specific IgE was not detected in the assays used in this study. Some individuals exhibited single isotype anti-CHO-HCP reactivity; others contained a combination of multiple antibody isotypes. Data presented in this study demonstrate the isotypic complexity and the high prevalence of anti-CHO-HCP antibodies in human serum with no known exposure to CHO cell-derived therapeutic biologics.

The AAPS Journal: Erratum to: Autoradiography, MALDI-MS, and SIMS-MS Imaging in Pharmaceutical Discovery and Development

Eric Solon; Alain Schweitzer; Markus Stoeckli; Brendan Prideaux — 2010-03-01

Type: Erratum
No abstract available.

Pharmaceutical Research: Toxicological Study and Efficacy of Blank and Paclitaxel-Loaded Lipid Nanocapsules After i.v. Administration in Mice

2010-03-01

Type: Original Paper
Purpose

Lipid nanocapsules (LNCs) are solvent-free drug nanocarriers permitting entrapment of paclitaxel and increasing its antitumoural effect in animal models after i.v. injection. The tolerance and efficacy of LNCs after repeated dose i.v. administration were assessed in mice. The maximum tolerated dose (MTD) and 50% lethal dose (LD50) were studied.

Methods

Paclitaxel-loaded LNC formulation was given i.v. at the dose of 12 mg/kg per day for 5 consecutive days in comparison with blank LNCs and saline. Histological examination, complete blood counts and biochemical quantification were performed after a recovery of 7 days. Growth of NCI-H460 subcutaneous xenografts in nude mice receiving one of the aforementioned schedules was assessed. MTD and LD50 were determined by Irwin test.

Results

No mortality was observed in repeated injections studies. Histological studies revealed no lesions and no accumulation of lipids. Blood studies were normal. The tumoural growth was significantly reduced in the group treated by paclitaxel-loaded LNCs. The MTDs/LD50s of Taxol®, paclitaxel-loaded LNCs and blank LNCs were 12/19.5, 96/216 and above 288/288 mg/kg, respectively.

Conclusions

This study demonstrates that a five-day i.v. injection schedule of paclitaxel-loaded LNC dispersions induces no histological or biochemical abnormalities in mice and improves paclitaxel efficacy and therapeutic index in comparison with Taxol®.

Pharmaceutical Research: A Plant-Derived Hydrolysable Tannin Inhibits CFTR Chloride Channel: A Potential Treatment of Diarrhea

Nisa Wongsamitkul; Lalida Sirianant; Chatchai Muanprasat; Varanuj Chatsudthipong — 2010-03-01

Type: Original Paper
Purpose

The present study examined the effects and mechanisms of actions of penta-m-digalloyl-glucose (PDG), a hydrolysable tannin extracted from Chinese gallnut, on cystic fibrosis transmembrane conductance regulator protein (CFTR).

Materials and Methods

Fisher rat thyroid cells stably expressing human CFTR (FRT cells) and human intestinal T84 cells were used as cell models to investigate the effects of PDG on chloride secretion using short-circuit current analysis. The mechanisms by which PDG affected chloride secretion were also examined. Finally, in vivo antidiarrheal efficacy and effects of PDG on intestinal fluid absorption were evaluated in mouse closed-loop models.

Results

In FRT cells, apical chloride current induced by forskolin, CPT-cAMP and apigenin were reversibly inhibited by PDG (IC50 ∼ 10 µM) without effects on intracellular cAMP content and cell viability. Similarly, in T84 cells, PDG effectively inhibited chloride secretion induced by forskolin and cholera toxin. However, it had no effect on calcium-induced chloride secretion. In mice, a single intraluminal injection of PDG (0.6 mg/kg) reduced cholera toxin-induced intestinal fluid secretion by 75% with no effect on intestinal fluid absorption.

Conclusions

PDG represents a new class of CFTR inhibitors. Further development of this class of compounds may provide a new therapeutic intervention for diarrhea.

Pharmaceutical Research: Leakiness and Size Exclusion of Paracellular Channels in Cultured Epithelial Cell Monolayers–Interlaboratory Comparison

Alex Avdeef — 2010-03-01

Type: Original Paper
Purpose

To determine and compare the paracellular characteristics of permeability (P_app) of Caco-2, MDCK, and 2/4/A1 cell lines.

Methods

The P_app data from 14 studies were analyzed by weighted nonlinear regression in terms of the paracellular parameters: porosity-pathlength (ε/δ), pore radius (R), and electrostatic potential drop (Δφ). Aqueous diffusivities, D_aq, for the analysis, were empirically determined. The required hydrodynamic radii, r_HYD, were estimated without knowledge of compound density. Mannitol iso-paracellular profiles allowed comparisons of “leakiness” across labs.

Results

D_aq (37°C) was predicted as 9.9 × 10⁻⁵ MW^−0.453; r_HYD = (0.92 + 21.8 MW⁻¹)·r_SE, where r_SE is the Stokes-Einstein radius. Values of pore radius ranged from 4.0(±0.1) to 18(±3) Å, with the 2/4/A1 indicating the largest pores. The ε/δ capacity factor ranged from 0.2 (±0.1) to 69 (±5) cm⁻¹, with most values <1.5 cm⁻¹. The average potential drop for Caco-2 models was Δφ_{wt avg}^Caco-2 = −43 ± 20 mV. The paracellular model predicted measured log P_app values with pooled r²= 0.93 and s = 0.17 (n = 108).

Conclusion

R and ε/δ are negatively correlated to a large extent. P_app can be rate-limited by either factor, with a wide range of possible combinations still indicating nearly constant leakiness for a given marker.

Pharmaceutical Research: Neuropilin-1 Targeting Photosensitization-Induced Early Stages of Thrombosis via Tissue Factor Release

2010-03-01

Type: Original Paper
Purpose

This article characterizes the vascular effects following vascular-targeted photodynamic therapy with a photosensitizer which actively targets endothelial cells.

Methods

This strategy was considered by coupling a chlorin to a heptapeptide targeting neuropilin-1 in human malignant glioma-bearing nude mice. A laser Doppler microvascular perfusion monitor was used to monitor microvascular blood perfusion in tumor tissue. Endothelial cells’ ultra structural integrity was observed by transmission electron microscopy. The consequences of photosensitization on tumor vessels, tissue factor expression, fibrinogen consumption, and thrombogenic effects were studied by immunohistochemical staining.

Results

Treatment of glioma-bearing mice with the conjugate showed a statistically significant tumor growth delay. Vascular effect was characterized by a decrease in tumor tissue blood flow at about 50% baseline during treatment not related to variations in temperature. This vascular shutdown was mediated by tumor blood vessels’ congestion. A pro-thrombotic behavior of targeted endothelial cells in the absence of ultra structural changes led to the induction of tissue factor expression from the earliest times post-treatment. Expression of tissue factor-initiated thrombi formation was also related to an increase in fibrinogen consumption.

Conclusion

Using a peptide-conjugated photosensitizer targeting neuropilin-1, induction of tissue factor expression immediately post-treatment, led to the establishment of thrombogenic effects within the vessel lumen.

Pharmaceutical Research: New Core-Shell Nanoparticules for the Intravenous Delivery of siRNA to Experimental Thyroid Papillary Carcinoma

2010-03-01

Type: Original Paper
Purpose

Development of efficient in vivo delivery nanodevices remains a major challenge to achieve clinical application of siRNA. The present study refers to the conception of core-shell nanoparticles aiming to make possible intravenous administration of chemically unmodified siRNA oriented towards the junction oncogene of the papillary thyroid carcinoma.

Methods

Nanoparticles were prepared by redox radical emulsion polymerization of isobutylcyanoacrylate and isohexylcyanoacrylate with chitosan. The loading of the nanoparticles with siRNA was achieved by adsorption. The biological activity of the siRNA-loaded nanoparticles was assessed on mice bearing a papillary thyroid carcinoma after intratumoral and intravenous administration.

Results

Chitosan-coated nanoparticles with a diameter of 60 nm were obtained by adding 3% pluronic in the preparation medium. siRNA were associated with the nanoparticles by surface adsorption. In vivo, the antisense siRNA associated with the nanoparticles lead to a strong antitumoral activity. The tumor growth was almost stopped after intravenous injection of the antisense siRNA-loaded nanoparticles, while in all control experiments, the tumor size was increased by at least 10 times.

Conclusion

This work showed that poly(alkylcyanoacrylate) nanoparticles coated with chitosan are suitable carriers to achieve in vivo delivery of active siRNA to tumor including after systemic administration.

Pharmaceutical Research: A Semi-physiological-Based Pharmacokinetic/Pharmacodynamic Model to Describe the Effects of Topotecan on B-Lymphocyte Lineage Cells

2010-03-01

Type: Original Paper
Purpose

To develop a semi-physiological-based model describing simultaneously the time course of immature and mature B-lymphocytes after topotecan (TPT) administration to tumor-bearing rats.

Methods

Twenty-four tumor-bearing BDIX male rats received a single 6 mg/kg intra-peritoneal dose of TPT or saline. Mature and immature B-cell levels were measured every two days during three weeks and showed a very different temporal pattern. Both B-cell populations declined rapidly, reaching the nadir at 3–4 days after TPT administration; however, mature cells returned to baseline at day 8, while immature B-cells stayed at nadir until day 9 instead. Data were modeled using the population approach with NONMEM VI.

Results

The model developed maintains the proliferation, maturation and degradation elements of previous published models for myelosuppresion. In order to describe the rapid recovery of mature cells, it includes a peripheral compartment providing a constant supply of mature cells to the bloodstream.

Conclusions

The major contribution of the model is its new structure and the dynamical consequences, demonstrating an independent behavior between mature and immature B-cells during recovery. The final model could represent a good basis for the optimization of cytotoxic drugs oriented to attain a maximum antitumor efficacy while minimizing hematological toxicity.

Pharmaceutical Research: The Pharmaceutical Sciences in 2020: Report of a Conference Organized by the Board of Pharmaceutical Sciences of the International Pharmaceutical Federation (FIP)

Vinod Shah; Luc Besancon; Pieter Stolk; Geoffrey Tucker; Daan Crommelin — 2010-03-01

Type: Report

The Board of Pharmaceutical Sciences (BPS) of the International Pharmaceutical Federation (FIP) has developed a view on the future of pharmaceutical sciences in 2020. This followed an international conference with invited participants from various fields (academicians, scientists, regulators, industrialists, venture capitalists) who shared their views on the forces that might determine how the pharmaceutical sciences will look in 2020. The commentary here provides a summary of major research activities that will drive drug discovery and development, enabling technologies for pharmaceutical sciences, paradigm shifts in drug discovery, development and regulations, and changes in education to meet the demands of academia, industry and regulatory institutions for pharmaceutical sciences in 2020.

Pharmaceutical Research: Reaching Out to Collaborators: Crowdsourcing for Pharmaceutical Research

Sean Ekins; Antony Williams — 2010-03-01

Type: Editorial Notes
No abstract available.

Pharmaceutical Research: Erratum to: Movement of Heparins Across Rat Gastric Mucosa is Dependent on Molecular Weight and pH

Bita Moazed; Linda Hiebert — 2010-03-01

Type: Erratum
No abstract available.

Pharmaceutical Research: Oxidation of Free L-histidine by tert-Butylhydroperoxide

Bruce Mason; Melissa McCracken; Edward Bures; Bruce Kerwin — 2010-03-01

Type: Original Paper
Purpose

L-histidine, a commonly used buffer for protein formulations, has the potential to oxidize and form multiple byproducts. Previous studies were performed using metal catalyzed oxidation with Fe²⁺ or Cu²⁺. We re-examined the oxidation of L-histidine under conditions more appropriate to protein formulations.

Methods

Solutions of free L-histidine, protected from light, were initially reacted with tert-butylhydroperoxide and the products analyzed by UV absorption spectroscopy, reversed phase HPLC and mass spectrometric analysis and NMR. Experimental parameters investigated were oxidizing agent, pH, temperature, metal ion and metal chelator content.

Results

The initial reaction produced a number of known products, along with an unknown product that was identified as 4(5)-imidazolecarboxaldehyde. The reaction was highly pH and oxidizing-agent specific. The product was not observed at pH 5.0 or below, while there was a dramatic increase for reactions carried out at pH 6.0 or above. Addition of FeSO₄ to the reaction dramatically increased the amount of 4(5)-imidazolecarboxaldehyde produced, while addition of the metal chelators EDTA or DTPA completely inhibited the reaction.

Conclusions

The presence of oxidants and trace concentrations of metal ions in high purity L-histidine solutions results in the formation of 4(5)-imidazolecarboxaldehyde which has the potential to covalently modify proteins.

Pharmaceutical Research: AAPS Connection

2010-03-01

Type: News
No abstract available.

Pharmaceutical Research: DNA Methylation Profiles of Organic Anion Transporting Polypeptide 1B3 in Cancer Cell Lines

2010-03-01

Type: Original Paper
Purpose

Multispecific organic anion transporter, OATP1B3/SLCO1B3, is expressed in several cancer cell lines as well as tumor tissues, and its expression sensitizes the cells to some anti-cancer agents. The present study was aimed to characterize the DNA methylation profiles around the transcriptional start site (TSS) of OATP1B3 and correlate them with the mRNA expression in cancer and immortalized cell lines.

Methods

The mRNA expression and DNA methylation profiles of OATP1B3 were determined by RT-PCR and bisulfite sequencing, respectively.

Results

The expression of OATP1B3 mRNA was detected in DLD-1, TFK-1, PK-8, and PK-45P cells, but below the limit of detection in HepG2, Caco-2, and HEK293 cells. Bisulfite sequencing demonstrated that CpG dinucleotides around the TSS are differentially methylated among cell lines and partly associated with the mRNA expression profile of OATP1B3. Furthermore, treatment with 5-aza-2′-deoxycytidine, an inhibitor of DNA methyltransferase, significantly increased the mRNA expression of OATP1B3 in HepG2 and Caco-2 cells by 18- and 14-fold, respectively, but not in DLD-1 and TFK-1 cells.

Conclusion

DNA methylation-dependent gene silencing is at least partly involved in the regulation of OATP1B3 expression in cancer/immortalized cell lines.

Pharmaceutical Research: Model Analysis of the Concentration-Dependent Permeability of P-gp Substrates

2010-03-01

Type: Original Paper
Purpose

Recently, it was reported that the apparent Michaelis-Menten constant (Km_(app)) of a P-glycoprotein (P-gp) substrate, defined for the extracellular substrate concentration, increases as the P-gp expression level in the cell increases. By its nature, the Km value should not depend on the level of P-gp expression. The purpose of this study is to establish a model which can estimate the Km value independent of the P-gp expression level in cells.

Methods

The previously reported concentration-dependent permeability of verapamil, quinidine, and vinblastine in MDR1-MDCKII, P-gp-highly induced Caco-2, P-gp-induced Caco-2, normal Caco-2, and MDR1-knockdown Caco-2 cells data were analyzed using a model in which the Km value was defined for the intracellular substrate concentration.

Results

The estimated Km values defined for the substrate concentration inside the cells were almost the same among various cells with different levels of P-gp expression. The estimated Vmax values were approximately proportional to the P-gp expression level.

Conclusion

The established kinetic model was found to be rational based on the results that the Km values of P-gp substrates were about the same for cells expressing various levels of P-gp, while the Vmax values were proportional to the expression levels of P-gp.

Pharmaceutical Research: AAPS Connection

2010-02-26

Type: News
No abstract available.

The AAPS Journal: Assessment of a Spectrophotometric Assay for Monoacylglycerol Lipase Activity

Nadine Ulloa; Dale Deutsch — 2010-02-26

Type: Brief Communication
No abstract available.

AAPS PharmSciTech: In Vitro Release Kinetics and Bioavailability of Gastroretentive Cinnarizine Hydrochloride Tablet

Ramesh Nagarwal; Devendra Ridhurkar; J. Pandit — 2010-02-25

Type: Original Paper

An oral sustained release dosage form of cinnarizine HCl (CNZ) based on gastric floating matrix tablets was studied. The release of CNZ from different floating matrix formulations containing four viscosity grades of hydroxypropyl methylcellulose, sodium alginate or polyethylene oxide, and gas-forming agent (sodium bicarbonate or calcium carbonate) was studied in simulated gastric fluid (pH 1.2). CNZ release data from the matrix tablets were analyzed kinetically using Higuchi, Peppas, Weibull, and Vergnaud models. From water uptake, matrix erosion studies, and drug release data, the overall release mechanism can be explained as a result of rapid hydration of polymer on the surface of the floating tablet and formation of a gel layer surrounding the matrix that controls water penetration into its center. On the basis of in vitro release data, batch HP1 (CNZ, HPMC-K100LV, SBC, LTS, and MgS) was subjected to bioavailability studies in rabbits and was compared with CNZ suspension. It was concluded that the greater bioavailability of HP1 was due to its longer retention in the gastric environment of the test animal. Batch no. HP1 of floating tablet in rabbits demonstrated that the floating tablet CNZ could be a 24-h sustained release formulation.

AAPS PharmSciTech: Freeze Thaw: A Simple Approach for Prediction of Optimal Cryoprotectant for Freeze Drying

Praveen Date; Abdul Samad; Padma Devarajan — 2010-02-25

Type: Original Paper

The present study evaluates freeze thaw as a simple approach for screening the most appropriate cryoprotectant. Freeze–thaw study is based on the principle that an excipient, which protects nanoparticles during the first step of freezing, is likely to be an effective cryoprotectant. Nanoparticles of rifampicin with high entrapment efficiency were prepared by the emulsion-solvent diffusion method using dioctyl sodium sulfosuccinate (AOT) as complexing agent and Gantrez AN-119 as polymer. Freeze–thaw study was carried out using trehalose and fructose as cryoprotectants. The concentration of cryoprotectant, concentration of nanoparticles in the dispersion, and the freezing temperature were varied during the freeze–thaw study. Cryoprotection increased with increase in cryoprotectant concentration. Further, trehalose was superior to fructose at equivalent concentrations and moreover permitted use of more concentrated nanosuspensions for freeze drying. Freezing temperature did not influence the freeze–thaw study. Freeze-dried nanoparticles revealed good redispersibility with a size increase that correlated well with the freeze–thaw study at 20% w/v trehalose and fructose. Transmission electron microscopy revealed round particles with a size ∼400 nm, which correlated with photon correlation spectroscopic measurements. Differential scanning calorimetry and X-ray diffraction suggested amorphization of rifampicin. Fourier transfer infrared spectroscopy could not confirm interaction of drug with AOT. Nanoparticles exhibited sustained release of rifampicin, which followed diffusion kinetics. Nanoparticles of rifampicin were found to be stable for 12 months. The good correlation between freeze thaw and freeze drying suggests freeze–thaw study as a simple and quick approach for screening optimal cryoprotectant for freeze drying.

AAPS PharmSciTech: Preparation and Bioavailability Assessment of SMEDDS Containing Valsartan

Adhvait Dixit; Sadhana Rajput; Samir Patel — 2010-02-25

Type: Original Paper

A self-microemulsifying drug delivery system (SMEDDS) has been developed to enhance diffusion rate and oral bioavailability of valsartan. The solubility of valsartan was checked in different oils, surfactants, and cosurfactants and ternary phase diagrams were constructed to evaluate the microemulsion domain. The valsartan SMEDDS was prepared using Capmul MCM (oil), Tween 80 (surfactant), and polyethylene glycol 400 (cosurfactant). The particle size distribution, zeta potential, and polydispersity index were determined and were found to be 12.3 nm, −0.746, and 0.138, respectively. Diffusion rate of valsartan was measured by in vitro dialysis bag method using phosphate buffer pH 6.8 as diffusion media. Developed high-performance liquid chromatography method was used to determine drug content in diffusion media. Oral bioavailability of valsartan SMEDDS was checked by using rabbit model. Results of diffusion rate and oral bioavailability of valsartan SMEDDS were compared with those of pure drug solution and of marketed formulation. Diffusion of valsartan SMEDDS showed maximum drug release when compared to pure drug solution and marketed formulation. The area under curve and time showed significant improvement as the values obtained were 607 ng h/mL and 1 h for SMEDDS in comparison to 445.36 and 1.36 h for market formulation suggesting significant increase (p < 0.01) in oral bioavailability of valsartan SMEDDS.

AAPS PharmSciTech: Dual-Drug Delivery System Based on In Situ Gel-Forming Nanosuspension of Forskolin to Enhance Antiglaucoma Efficacy

Saurabh Gupta; Malay Samanta; Ashok Raichur — 2010-02-25

Type: Original Paper

The present study was designed to improve the bioavailability of forskolin by the influence of precorneal residence time and dissolution characteristics. Nanosizing is an advanced approach to overcome the issue of poor aqueous solubility of active pharmaceutical ingredients. Forskolin nanocrystals have been successfully manufactured and stabilized by poloxamer 407. These nanocrystals have been characterized in terms of particle size by scanning electron microscopy and dynamic light scattering. By formulating Noveon AA-1 polycarbophil/poloxamer 407 platforms, at specific concentrations, it was possible to obtain a pH and thermoreversible gel with a pH_gel/T_gel close to eye pH/temperature. The addition of forskolin nanocrystals did not alter the gelation properties of Noveon AA-1 polycarbophil/poloxamer 407 and nanocrystal properties of forskolin. The formulation was stable over a period of 6 months at room temperature. In vitro release experiments indicated that the optimized platform was able to prolong and control forskolin release for more than 5 h. The in vivo studies on dexamethasone-induced glaucomatous rabbits indicated that the intraocular pressure lowering efficacy for nanosuspension/hydrogel systems was 31% and lasted for 12 h, which is significantly better than the effect of traditional eye suspension (18%, 4–6 h). Hence, our investigations successfully prove that the pH and thermoreversible polymeric in situ gel-forming nanosuspension with ability of controlled drug release exhibits a greater potential for glaucoma therapy.

Pharmaceutical Research: Measurement and Modeling of Diffusion Kinetics of a Lipophilic Molecule Across Rabbit Cornea

Chhavi Gupta; Anuj Chauhan; Raj Mutharasan; Sangly Srinivas — 2010-02-25

Type: Original Paper
Purpose

To develop a kinetic model for representing the diffusion and partitioning of Rhodamine B (RhB), a fluorescent lipophilic molecule, across the cornea for gaining insights into pharmacokinetics of topical drugs to the eye.

Methods

Rabbit corneas mounted underneath a custom-built scanning microfluorometer were perfused with Ringers on both sides of the tissue. After a step change in RhB on the tear side, transients of trans-corneal fluorescence of RhB were measured at a depth resolution ∼ 8 μm.

Results

RhB distribution exhibited discontinuities at the interface between epithelium and stroma, and between stroma and endothelium. In each of the layers, fluorescence was non-uniform. Fluorescence was elevated in the epithelium and endothelium relative to the stroma. Modeling of RhB transport by diffusion in each layer and stipulation of partitioning of RhB at the cellular interfaces were required to account for trans-corneal penetration kinetics of RhB. The model parameters, estimated using the unsteady state trans-corneal RhB profiles, were found to be sensitive, and the model predicted the experimental profiles accurately.

Conclusions

Conventional pharmacokinetic models that depict cornea as a single compartment do not predict the depth-dependent kinetics of RhB penetration. The proposed model incorporates realistic transport mechanisms and thereby highlights the influence of physicochemical properties of drugs on trans-corneal kinetics.

Pharmaceutical Research: Prostate Cancer-Targeted Imaging Using Magnetofluorescent Polymeric Nanoparticles Functionalized with Bombesin

2010-02-25

Type: Original Paper
Purpose

In this work, the aim was to prepare and characterize a magnetofluorescent polymeric nanoparticle for prostate cancer imaging in vivo.

Methods

Glycol chitosan (GC) was chemically modified with N-acetyl histidine (NAHis) as a hydrophobic moiety, and bombesin (BBN) was conjugated to the hydrophobically modified GC for use in targeting gastric-releasing peptide receptors (GRPR) overexpressed in prostate cancer cells. NAHis-GC conjugates were labeled with the near-infrared (NIR) fluorophore Cy5.5 (C-NAHis-GC conjugate).

Results

BBN-conjugated C-NAHis-GC nanoparticles (BC-NAHis-GC nanoparticles) showed significantly higher binding to the PC3 cell surface than nanoparticles without BBN, and the cellular binding was clearly inhibited by BBN. The tumor-to-muscle ratios of C- and BC-NAHis-GC nanoparticles were 2.26 ± 0.66 and 5.37 ± 0.43, respectively. The tumor accumulation of BC-NAHis-GC nanoparticles was clearly reduced by co-injection of BBN. Further, iron oxide nanoparticles (IO) were loaded into BC-NAHis-GC nanoparticles to investigate the possibility of use as a probe for MRI. IO-BC-NAHis-GC nanoparticles were well observed in the PC3 cells, and the blocking with BBN significantly reduced the cellular binding of the nanoparticles.

Conclusion

These results demonstrate that the BBN conjugation to NAHis-GC nanoparticles improves their tumor accumulation in PC3-bearing mice in comparison to nanoparticles without BBN, suggesting that BC-NAHis-GC nanoparticles may be useful for prostate cancer imaging.

Pharmaceutical Research: Phenethyl Isothiocyanate Sensitizes Androgen-Independent Human Prostate Cancer Cells to Docetaxel-Induced Apoptosis In Vitro and In Vivo

Dong Xiao; Shivendra Singh — 2010-02-25

Type: Original Paper
Purpose

The present study was undertaken to determine efficacy of phenethyl isothiocyanate (PEITC) for sensitization of androgen-independent human prostate cancer cells (AIPC) to Docetaxel-induced apoptosis using cellular and xenograft models.

Methods

Cell viability was determined by trypan blue dye exclusion assay. Microscopy and DNA fragmentation assay were performed to quantify apoptotic cell death in cultured cells. Protein levels were determined by immunoblotting. PC-3 prostate cancer xenograft model was utilized to determine in vivo efficacy of the PEITC and/or Docetaxel treatments.

Results

Pharmacologic concentrations of PEITC augmented Docetaxel-induced apoptosis in PC-3 and DU145 cells in association with suppression of Bcl-2 and XIAP protein levels and induction of Bax and Bak. The PEITC-Docetaxel combination was markedly more efficacious against PC-3 xenograft in vivo compared with PEITC or Docetaxel alone. Significantly higher counts of apoptotic bodies were also observed in tumor sections from mice treated with the PEITC-Docetaxel combination compared with PEITC or Docetaxel alone. The PEITC and/or Docetaxel-mediated changes in the levels of apoptosis regulating proteins in the tumor were generally consistent with the molecular alterations observed in cultured cells.

Conclusion

These results offer obligatory impetus to test PEITC-Docetaxel combination for the treatment of AIPC in a clinical setting.

AAPS PharmSciTech: Preparation and In Vitro Evaluation of a New Fentanyl Patch Based on Functional and Non-functional Pressure Sensitive Adhesives

Seyed Taghizadeh; Arezou Soroushnia; Fatemeh Mohamadnia — 2010-02-25

Type: Original Paper

In this study, some single-layer and double-layer transdermal drug delivery systems (TDDSs) with different functional and non-functional acrylic pressure-sensitive adhesives (PSAs) were prepared. For this purpose, fentanyl as a drug was used. The effects of PSAs type, single-layer and double-layer TDDSs on skin permeation and in vitro drug release from devices were evaluated using a hydrodynamically well-characterized Chien permeation system fitted with excised rat abdominal skin. The adhesion properties of devices such as peel strength and tack values were obtained as well. It was found that TDDS with –COOH functional PSA showed the lowest steady-state flux. Double-layer TDDS displayed a constant flux up to 72 h. In double- and single-layer devices after 1 and 3 h, respectively, drug release followed Higuchi’s kinetic model. Formulations with the highest percentage of –COOH functional PSA have displayed the lowest flux. The double-layer TDDSs with non-functional PSA demonstrated the suitable skin permeation rate close to Duragesic® TDDS and suitable adhesion properties.

AAPS PharmSciTech: Composition Optimization and Stability Testing of a Parenteral Antifungal Solution based on a Ternary Solvent System

Kristóf Kovács; István Antal; György Stampf; Imre Klebovich; Krisztina Ludányi — 2010-02-24

Type: Original Paper

An intravenous solution is a dosage forms intended for administration into the bloodstream. This route is the most rapid and the most bioavailable method of getting drugs into systemic circulation, and therefore it is also the most liable to cause adverse effects. In order to reduce the possibility of side effects and to ensure adequate clinical dosage of the formulation, the primarily formulated composition should be optimized. It is also important that the composition should retain its therapeutic effectiveness and safety throughout the shelf-life of the product. This paper focuses on the optimization and stability testing of a parenteral solution containing miconazole and ketoconazole solubilized with a ternary solvent system as model drugs. Optimization of the solvent system was performed based on assessing the risk/benefit ratio of the composition and its properties upon dilution. Stability tests were conducted based on the EMEA (European Medicines Agency) “guideline on stability testing: stability testing of existing active substances and related finished products”. Experiments show that both the amount of co-solvent and surface active agent of the solvent system could substantially be reduced, while still maintaining adequate solubilizing power. It is also shown that the choice of various containers affects the stability of the compositions. It was concluded that by assessing the risk/benefit ratio of solubilizing power versus toxicity, the concentration of excipients could be considerably decreased while still showing a powerful solubilizing effect. It was also shown that a pharmaceutically acceptable shelf-life could be assigned to the composition, indicating good long-term stability.

AAPS PharmSciTech: Stomach-Specific Controlled Release Gellan Beads of Acid-Soluble Drug Prepared by Ionotropic Gelation Method

Mrunalini Narkar; Praveen Sher; Atmaram Pawar — 2010-02-24

Type: Original Paper

The purpose of the present work was the development and evaluation of stomach-specific controlled release mucoadhesive drug delivery system prepared by ionotropic gelation of gellan beads, containing acid-soluble drug amoxicillin trihydrate, using 3² factorial design with concentration of gellan gum and quantity of drug as variables. The study showed that beads prepared in alkaline cross-linking medium have higher entrapment efficiency than the acidic cross-linking medium. The entrapment efficiency was in the range of 32% to 46% w/w in acidic medium, which increased up to 60% to 90% w/w in alkaline medium. Batches with lowest, medium, and highest drug entrapment were subjected to chitosan coating to form a polyelectrolyte complex film. As polymer concentration increases, entrapment efficiency and particle size increases. Scanning electron microscopy revealed spherical but rough surface due to leaching of drug in acidic cross-linking solution, dense spherical structure in alkaline cross-linking solution, and rough surface of chitosan-coated beads with minor wrinkles. The in vitro drug release up to 7 h in a controlled manner following the Peppas model (r = 0.9998). In vitro and in vivo mucoadhesivity study showed that beads have good mucoadhesivity and more than 85% beads remained adhered to stomach mucosa of albino rat even after 7 h. In vitro growth inhibition study showed complete eradication of Helicobacter pylori. These results indicate that stomach-specific controlled release mucoadhesive system of amoxicillin gellan beads may be useful in H. pylori treatment.

AAPS PharmSciTech: Effects of Thermal Curing Conditions on Drug Release from Polyvinyl Acetate–Polyvinyl Pyrrolidone Matrices

2010-02-20

Type: Original Paper

This study aimed to investigate the effects of dry and humid heat curing on the physical and drug release properties of polyvinyl acetate–polyvinyl pyrrolidone matrices. Both conditions resulted in increased tablet hardness; tablets stored under humid conditions showed high plasticity and deformed during hardness testing. Release from the matrices was dependent on the filler's type and level. Release profiles showed significant changes, as a result of exposure to thermal stress, none of the fillers used stabilized matrices against these changes. Density of neat polymeric compacts increased upon exposure to heat; the effect of humid heat was more evident than dry heat. Thermograms of samples cured under dry heat did not show changes, while those of samples stored under high humidity showed significant enlargement of the dehydration endotherm masking the glass transition of polyvinyl acetate. The change of the physical and release properties of matrices could be explained by the hygroscopic nature of polyvinyl pyrrolidone causing water uptake; absorbed water then acts as a plasticizer of polyvinyl acetate promoting plastic flow, deformation, and coalescence of particles, and altering the matrices internal structure. Results suggest that humid heat is more effective as a curing environment than dry heat for polyvinyl acetate–polyvinyl pyrrolidone matrices.

Pharmaceutical Research: Influence of Gallate Esterification on the Activity of Procyanidin B2 in Androgen-Dependent Human Prostate Carcinoma LNCaP Cells

Shen-Chieh Chou; Manjinder Kaur; John Thompson; Rajesh Agarwal; Chapla Agarwal — 2010-02-17

Type: Original Paper
Purpose

Present study assessed the influence of gallate esterification on the anti-cancer activity of procyanidin B2 (B2) in androgen-dependent human prostate carcinoma LNCaP cells employing B2-3,3′-di-O-gallate (B2-G₂), two mono-gallate esters B2-3-O-gallate (B2-3G) and B2-3′-O-gallate (B2-3′G) and the parent compound B2, all isolated from grape seed extract (GSE).

Materials and Methods

Study compounds were isolated from GSE by several chromatographic steps and structures determined by a combination of enzymatic hydrolysis, mass spectrometry and comparisons with standards. Cells, treated with these compounds, were assessed for viability and apoptosis and examined by western blotting.

Results

Gallate esters B2-G₂, B2-3G and B2-3′G significantly decreased LNCaP cell viability; however, B2 and gallic acid were ineffective. Furthermore, only B2-G₂ also significantly decreased cell growth. Decreases in cell viability were largely due to apoptosis induction with B2-G₂ and B2-3′G exhibiting comparable effects, whereas B2-3G was less effective. In mechanistic studies, B2-G₂ and B2-3′G treatments caused caspases-9 and -3 and PARP cleavage, and down-regulated Bcl-2, Bcl-Xl and androgen receptor levels.

Conclusion

Together, our findings demonstrate anti-PCA efficacy of B2-G₂ and suggest that a gallate ester moiety at 3′ position of procyanidin B2 contributes more extensively toward the biological activity of the di-gallate ester than esterification of position 3.

Pharmaceutical Research: Identification of Peptide Ligands for Targeting to the Blood-Brain Barrier

2010-02-17

Type: Original Paper
Purpose

Transport of drugs to the brain is limited by the blood-brain barrier. New, specific brain endothelium ligands can facilitate brain-specific delivery of drugs.

Methods

We used phage display in an in situ brain perfusion model to screen for new brain endothelium peptide ligands.

Results

Two phage clones, displaying 15 amino acid-peptides (GLA and GYR) that were selected for brain binding in the mouse model, showed significant binding to human brain endothelium (hCMEC/D3), compared to a random control phage. This binding was not seen for other human endothelial cells (HUVEC). Binding to hCMEC/D3 cells was dose dependent. When phage GLA and GYR were individually perfused through the murine brain, their ability to bind to the brain was 6-fold (GLA) and 5-fold (GYR) higher than the control phage. When compared to lung perfusion, phage showed an 8.5-fold (GYR) and 48-fold (GLA) preference for brain over lung compared to the control.

Conclusions

These results indicate that two new peptide ligands have been identified that may be used for specific targeting of drugs to the blood-brain barrier.

Pharmaceutical Research: Dendrimeric Alkylated Polyethylenimine Nano-carriers with Acid-Cleavable Outer Cationic Shells Mediate Improved Transfection Efficiency Without Increasing Toxicity

Terry Steele; Wayne Shier — 2010-02-17

Type: Original Paper
Purpose

Improved polycation-based non-viral DNA vectors were sought by preparing dendrimers with polyethylenimine cores surrounded by various shells incorporating structural features intended to facilitate steps in transfection mechanisms. Dendrimeric vectors were designed with (a) an outer oligocation shell, intended to facilitate DNA release inside cells, (b) a hydrophobic C-16 alkyl shell, and (c) a polycationic core, the latter two intended to combine lipid-depletion and osmotic burst endosome escape mechanisms, respectively, and were (d) attached through an a acid-cleavable linker reported to hydrolyze at endosomal pH values.

Methods

Vectors and DNA complexes were characterized by dynamic and static light scattering. Flow cytometry was used to quantitate transfection activity and cytotoxicity in CHO–K1 cells.

Results

About 5-fold increased transfection activity was obtained for a vector constructed with an outer shell of oligocations attached through acid-cleavable linkers, relative to a control dendrimer with an acid-stable linker. The most effective oligocation component of outer shells tested was spermine. Neither modification was associated with increased cytotoxicity. This vector design did not permit an evaluation of the benefit of combining endosome release mechanisms.

Conclusion

Using acid-cleavable linkers to attach an outer shell of short, highly-charged oligocations to a PEI-based dendrimeric vector substantially increased transfection efficiency without increasing cytotoxicity.

Pharmaceutical Research: Characteristics of rhVEGF Release from Topical Hydrogel Formulations

2010-02-13

Type: Original Paper
Purpose

To study recombinant human vascular endothelial growth factor (rhVEGF), the release characteristics from topical gel formulations, and its interaction with the gelling agents.

Methods

The release kinetics were followed by quantifying rhVEGF that diffused into the receptor chamber of Franz cells. Analytical ultracentrifuge (AUC) was used to characterize the sedimentation velocity of rhVEGF experienced in the gel. The interactions were characterized by isothermal calorimetry (ITC), and rhVEGF conformation was assessed by circular dichroism (CD).

Results

The fraction of protein released was linear with the square root of time. The release rate constants did not show significant change within a wide range of bulk viscosities created by different concentrations of hydroxypropyl methylcellulose (HPMC) or MC gels. Sedimentation velocity determined by AUC generated comparable sedimentation coefficients of protein in these gels. AUC and ITC revealed no significant interaction between rhVEGF and HPMC and some change on secondary structure of the protein by Far UV CD, which was not the case with carboxymethyl cellulose (CMC).

Conclusions

Microviscosity, not bulk viscosity, was the key factor for the release of rhVEGF from cellulosic gels such as HPMC. Interaction between rhVEGF and CMC resulted in slower, and reduced amount of, release from the gel.

Pharmaceutical Research: New Techniques for Drug Delivery to the Posterior Eye Segment

Esther Eljarrat-Binstock; Jacob Pe’er; Abraham Domb — 2010-02-13

Type: Review Paper

Ocular drug delivery has become an increasingly important field of research especially when treating posterior segment diseases of the eye, such as age-related macular degeneration, diabetic retinopathy, posterior uveitis and retinitis. These diseases are the leading causes of vision loss in developed countries which require repeated long-term administration of therapeutic agents. New drugs for the medication of the posterior ocular segment have emerged, but most drugs are delivered by repeated intravitreal injections associated with ocular complications. Advances in ocular drug delivery system research are expected to provide new tools for the treatment of the posterior segment diseases, providing improved drug penetration, prolonged action, higher efficacy, improved safety and less invasive administration, resulting in higher patient compliance. This review provides an insight into the recent progress and trends in ocular drug delivery systems for treating posterior eye segment diseases, with an emphasis on transscleral iontophoresis.

Pharmaceutical Research: Understanding the Behavior of Amorphous Pharmaceutical Systems during Dissolution

David Alonzo; Geoff Zhang; Deliang Zhou; Yi Gao; Lynne Taylor — 2010-02-12

Type: Original Paper
Purpose

To investigate the underlying physical processes taking place during dissolution of amorphous pharmaceuticals and correlate them to the observed solution concentration-time profiles. Felodipine and indomethacin were used as model hydrophobic compounds.

Methods

Concentration-time profiles were monitored during dissolution of the model amorphous compounds using in situ fiber-optic ultraviolet spectroscopy. Crystallization of the solid exposed to an aqueous environment was monitored using Raman spectroscopy and/or powder X-ray diffraction. Polarized light microscopy was used to provide qualitative information about crystallization processes.

Results

For felodipine, a small extent of supersaturation was generated via dissolution at 25°C but not at 37°C. The amorphous solid was found to crystallize rapidly at both temperatures upon exposure to the dissolution medium. Addition of low concentrations of polymers to the dissolution medium was found to delay crystallization of the amorphous solid, leading to the generation of supersaturated solutions. Amorphous indomethacin did not crystallize as readily in an aqueous environment; hence, dissolution resulted in supersaturated solutions. However, crystallization from these supersaturated solutions was rapid. Polymeric additives were able to retard crystallization from supersaturated solutions of both indomethacin and felodipine for up to 4 h.

Conclusions

The dissolution advantage of amorphous solids can be negated either by crystallization of the amorphous solid on contact with the dissolution medium or through rapid crystallization of the supersaturated solution. Polymeric additives can potentially retard both of these crystallization routes, leading to the generation of supersaturated solutions that can persist for biologically relevant timeframes.

Pharmaceutical Research: Micellar Nanocarriers: Potential Nose-to-Brain Delivery of Zolmitriptan as Novel Migraine Therapy

Ratnesh Jain; Swapna Nabar; Prajakta Dandekar; Vandana Patravale — 2010-02-12

Type: Original Paper
Purpose

The investigation was aimed at developing micellar nanocarriers for nose-to-brain delivery of zolmitriptan with the objective to investigate the pathway involved in the drug transport.

Methods

The micellar nanocarrier was successfully formulated and characterized for particle size and shape by multi-angle dynamic light scattering, small angle neutron scattering and cryo-transmission electron microscopy. Toxicity and biodistribution studies were carried out in rat. The distribution of the nasally administered labeled micellar nanocarrier in various regions of the rat brain was determined using the brain localization and autoradiography studies.

Results

Micellar nanocarrier of zolmitriptan, with size of around 23 nm, was successfully formulated. The spherical nature of the nanocarrier was confirmed using DLS, SANS and cryo-TEM. Toxicity studies indicated the safety for administration in the nasal cavity. In vivo biodistribution studies indicated the superiority of the developed nanocarrier for brain targeting when compared with the intravenous and nasal solutions of the drug. Brain localization and autoradiography studies illustrated the distribution of the drug in various regions of the brain and revealed a possible nose-to-brain transport pathway for the labeled drug.

Conclusion

The investigation indicated the potential of the developed nanocarrier as an effective new-generation vehicle for brain targeting of zolmitriptan.

AAPS PharmSciTech: Ciprofloxacin as Ocular Liposomal Hydrogel

Khaled Hosny — 2010-02-12

Type: Original Paper

The purpose of this study was to prepare and characterize an ocular effective prolonged-release liposomal hydrogel formulation containing ciprofloxacin. Reverse-phase evaporation was used for preparation of liposomes consisting of soybean phosphatidylcholine (PC) and cholesterol (CH). The effect of PC/CH molar ratio on the percentage drug encapsulation was investigated. The effect of additives such as stearylamine (SA) or dicetyl phosphate (DP) as positive and negative charge inducers, respectively, were studied. Morphology, mean size, encapsulation efficiency, and in vitro release of ciprofloxacin from liposomes were evaluated. For hydrogel preparation, Carbopol 940 was applied. In vitro transcorneal permeation through excised albino rabbit cornea was also determined. Optimal encapsulation efficiency of 73.04 ± 3.06% was obtained from liposomes formulated with PC/CH at molar ratio of 5:3 and by increasing CH content above this limit, the encapsulation decreased. Positively charged liposomes showed superior entrapment efficiency (82.01 ± 0.52) over the negatively charged and the neutral liposomes. Hydrogel containing liposomes with lipid content PC, CH, and SA in molar ratio 5:3:1, respectively, showed the best release and transcorneal permeation with the percentage permeation of 30.6%. These results suggest that the degree of encapsulation of ciprofloxacin into liposomes and prolonged in vitro release depend on composition of the vesicles. In addition, the polymer hydrogel used in preparation ensure steady and prolonged transcorneal permeation. In conclusion, ciprofloxacin liposomal hydrogel is a suitable delivery system for improving the ocular bioavailability of ciprofloxacin.

AAPS PharmSciTech: Stability of 5-Fluoro-2′-deoxycytidine and Tetrahydrouridine in Combination

2010-02-12

Type: Original Paper

In vivo, the DNA methyltransferase inhibitor, 5-fluoro-2′-deoxycytidine (FdCyd, NSC-48006), is rapidly converted to its unwanted metabolites. Tetrahydrouridine (THU, NSC-112907), a cytidine deaminase inhibitor can block the first metabolic step in FdCyd catabolism. Clinical studies have shown that co-administration with THU can inhibit the metabolism of FdCyd. The National Cancer Institute is particularly interested in a 1:5 FdCyd/THU formulation. The purpose of this study was to investigate the in vitro pH stability of FdCyd and THU individually and in combination. A stability-indicating high-performance liquid chromatography method for the quantification of both compounds and their degradants was developed using a ZIC®-HILIC column. The effect of THU and FdCyd on the in vitro degradation of each other was studied as a function of pH from 1.0 to 7.4 in aqueous solutions at 37°C. The degradation of FdCyd appears to be first-order and acid-catalyzed. THU equilibrates with at least one of its degradants. The combination of FdCyd and THU in solution does not affect the stability of either compound. The stability and compatibility of FdCyd and THU in the solid state at increased relative humidity and at various temperatures are also evaluated.

Pharmaceutical Research: Formulation and In Vitro-In Vivo Evaluation of Black Raspberry Extract-Loaded PLGA/PLA Injectable Millicylindrical Implants for Sustained Delivery of Chemopreventive Anthocyanins

Kashappa Desai; Karl Olsen; Susan Mallery; Gary Stoner; Steven Schwendeman — 2010-02-11

Type: Original Paper
Purpose

The objective of this study was to formulate and evaluate freeze-dried black raspberry (FBR) ethanol extract (RE) loaded poly(DL-lactic-co-glycolic acid) (PLGA) and poly(DL-lactic acid) (PLA) injectable millicylindrical implants for sustained delivery of chemopreventive FBR anthocyanins (cyanidin-3-sambubioside (CS), cyanidin-3-glucoside (CG) and cyanidin-3-rutinoside (CR)).

Methods

Identification and quantitation of CS, CG, and CR in RE was performed by mass spectroscopy and HPLC. RE:triacetyl-β-cyclodextrin (TA-β-CD) inclusion complex (IC) was prepared by a kneading method and characterized by X-ray diffraction (XRD), nuclear magnetic resonance spectroscopy (NMR) and UV-visible spectroscopy. RE or RE:TA-β-CD IC-loaded PLGA or PLA implants were prepared by a solvent extrusion method. In vitro and in vivo controlled release studies were conducted in phosphate-buffered saline Tween-80 (pH 7.4, 37°C) and after subcutaneous administration in male Sprague-Dawley rats, respectively. Anthocyanins were quantified by HPLC at 520 nm.

Results

The content of CS, CG, and CR in RE was 0.2, 1.5, and 3.5 wt%, respectively. The chemical stability of anthocyanins in solution was determined to be pH-dependent, and their degradation rate increased with an increase in pH from 2.4 to 7.4. PLGA/PLA millicylindrical implants loaded with 5 or 10 wt% RE exhibited a high initial burst and short release duration of anthocyanins (35–52 and 80–100% CG + CR release after 1 and 14 days, respectively). The cause for rapid anthocyanins release was linked to higher polymer water uptake and porosity associated with the high osmolytic components of large non-anthocyanin fraction of RE. XRD, ¹H NMR and UV-visible spectroscopy indicated that the non-anthocyanin fraction molecules of RE formed an IC with TA-β-CD, decreasing the hydrophilicity of RE. Formation of an IC with hydrophobic carrier, TA-β-CD, provided better in vitro/in vivo sustained release of FBR anthocyanins (16–24 and 97–99% CG + CR release, respectively, after 1 and 28 days from 20 wt% RE:TA-β-CD IC/PLA implants) over 1 month, owing to reduced polymer water uptake and porosity.

Conclusion

PLA injectable millicylindrical implants loaded with RE:TA-β-CD IC are optimal dosage forms for 1-month slow and continuous delivery of chemopreventive FBR anthocyanins.

Pharmaceutical Research: Basolateral Efflux Mediated by Multidrug Resistance-Associated Protein 3 (Mrp3/Abcc3) Facilitates Intestinal Absorption of Folates in Mouse

Yoshiaki Kitamura; Hiroyuki Kusuhara; Yuichi Sugiyama — 2010-02-11

Type: Original Paper
Purpose

This study investigated the role of an ABC transporter, Mrp3/Abcc3 in intestinal folate absorption.

Methods

Plasma concentrations of folic acid and leucovorin, given orally, were determined in wild-type and Mrp3^−/− mice. Mucosal-to-serosal transport was determined in the everted intestinal sacs. The plasma concentrations of endogenous 5-methyltetrahydrofolic acid, homocysteine and vitamin B₁₂, and mRNA levels of hepatic and intestinal folate metabolizing enzymes were compared between wild-type and Mrp3^−/− mice.

Results

C_max and area-under plasma concentration–time curve of folic acid were 3.0- and 2.3-fold lower in Mrp3^−/− mice compared with wild-type mice, whereas the total body clearance was unchanged. Absorption of leucovorin was significantly delayed in Mrp3^−/− mice. Mucosal-to-serosal transport of folic acid and leucovorin was significantly decreased in the duodenum of Mrp3^−/− mice, where their PS_serosal was decreased to 6.3 and 22% of that in wild-type mice, respectively. PS_serosal of 5-methyltetrahydrofolic acid was moderately decreased in Mrp3^−/− mice. There was no obvious abnormality in folate homeostasis in Mrp3^−/− mice.

Conclusions

Mrp3 accounts for the serosal efflux of folic acid and leucovorin, while it makes a moderate contribution to the serosal efflux of 5-methyltetrahydrofolic acid in mice. Mrp3 dysfunction does not disrupt folate homeostasis in mouse.

Pharmaceutical Research: Crystalline vs. Ionic Liquid Salt Forms of Active Pharmaceutical Ingredients: A Position Paper

Jelena Stoimenovski; Douglas MacFarlane; Katharina Bica; Robin Rogers — 2010-02-09

Type: Original Paper

Why not consider liquid salt forms of active pharmaceutical ingredients (APIs) as an alternative versatile tool in the pharmaceutical industry? Recent developments have shown that known APIs can be readily converted into ionic liquids and that these novel phases often possess different properties (e.g., improved solubilities and dissolution rates), which may have a direct impact on the pharmacokinetics and pharmacodynamics of the drug. They may also offer the potential of novel and more efficient delivery modes, as well as patent protection for each of the new forms of the drug. Since these pharmaceutically active ionic liquids represent a thermodynamically stable phase, they avoid the troublesome issues surrounding polymorphism and “polymorphic transformation.” In some cases, an active cation and an active anion can be combined to produce a liquid possessing dual functionality. Here we examine and challenge the current industry reliance on crystalline APIs by discussing the breadth and potential impact of liquid salts as a possible approach to phase control.

Pharmaceutical Research: Stability of Protein Pharmaceuticals: An Update

Mark Manning; Danny Chou; Brian Murphy; Robert Payne; Derrick Katayama — 2010-02-09

Type: Review Paper

In 1989, Manning, Patel, and Borchardt wrote a review of protein stability (Manning et al., Pharm. Res. 6:903–918, 1989), which has been widely referenced ever since. At the time, recombinant protein therapy was still in its infancy. This review summarizes the advances that have been made since then regarding protein stabilization and formulation. In addition to a discussion of the current understanding of chemical and physical instability, sections are included on stabilization in aqueous solution and the dried state, the use of chemical modification and mutagenesis to improve stability, and the interrelationship between chemical and physical instability.

Pharmaceutical Research: Dietary Grape Seed Proanthocyanidins Inhibit UVB-Induced Cyclooxygenase-2 Expression and Other Inflammatory Mediators in UVB-Exposed Skin and Skin Tumors of SKH-1 Hairless Mice

Som Sharma; Santosh Katiyar — 2010-02-09

Type: Original Paper
Purpose

The purpose of this study was to determine the chemopreventive mechanism of dietary grape seed proanthocyanidins (GSPs) against ultraviolet (UV) radiation-induced skin tumor development in mice.

Methods

Six-to-seven-week-old SKH-1 hairless mice were subjected to photocarcinogenesis protocol, and exposed to UVB radiation (180 mJ/cm²) three times/week for 24 weeks. Mice were fed a standard AIN76A control diet with or without supplementation with grape seed proanthocyanidins (GSPs; 0.2% or 0.5%, w/w). At the termination of the experiment, mice were sacrificed, and skin and skin tumor samples were harvested and subjected to the analysis of biomarkers related to inflammation using immunostaining, western blot analysis, ELISA and real-time PCR.

Results

Dietary GSPs inhibited UVB-induced infiltration of proinflammatory leukocytes and the levels of myeloperoxidase, cyclooxygenase-2 (COX-2), prostaglandin (PG) E₂, cyclin D1 and proliferating cell nuclear antigen (PCNA) in the skin and skin tumors compared to non-GSPs-treated UVB irradiated mouse skin and skin tumors. GSPs also significantly inhibited the levels of proinflammatory cytokines, tumor necrosis factor-α (P < 0.01), IL-1β (P < 0.001) and IL-6 (P < 0.001), in UVB-exposed skin and skin tumors.

Conclusion

The results from this study clearly suggest that dietary GSPs inhibit photocarcinogenesis in mice through the inhibition of UVB-induced inflammation and mediators of inflammation in mouse skin.

Pharmaceutical Research: Diallyl Trisulfide-Induced G2/M Phase Cell Cycle Arrest in DU145 Cells Is Associated with Delayed Nuclear Translocation of Cyclin-Dependent Kinase 1

Anna Herman-Antosiewicz; Young-Ae Kim; Su-Hyeong Kim; Dong Xiao; Shivendra Singh — 2010-02-09

Type: Original Paper
Purpose

The present study was undertaken to gain insight into the molecular mechanism of G2/M phase cell cycle arrest resulting from treatment of DU145 cells with diallyl trisulfide (DATS), a promising cancer chemopreventive constituent of garlic.

Methods

Cell cycle distribution was determined by flow cytometry. Immunoblotting was performed to determine protein expression. Overexpression of wild-type or mutant Cdc25C was achieved by transient transfection. Nuclear and cytoplasmic localization of cyclin B1 and cyclin-dependent kinase 1 (cdk1) was studied by immunoblotting.

Results

Exposure of DU145 human prostate cancer cells to DATS resulted in concentration- and time-dependent accumulation of G2/M phase cells, which correlated with down-regulation as well as increased S216 phosphorylation of Cdc25C. Ectopic expression of wild-type or redox-insensitive mutants (C330S and C330S/C377S) or S216A mutant of Cdc25C failed to confer protection against DATS-induced G2/M phase arrest. The DATS-mediated G2/M phase cell cycle arrest was also independent of reduced complex formation between cdk1 and cyclin B1, but correlated with delayed nuclear translocation of cdk1.

Conclusion

The present study indicates that the DATS-mediated G2/M phase cell cycle arrest in DU145 cells results from differential kinetics of nuclear localization of cdk1 and cyclin B1.

Pharmaceutical Research: Electrospun Nanofibers in Oral Drug Delivery

Francis Ignatious; Linghong Sun; Chao-Pin Lee; John Baldoni — 2010-02-09

Type: Review Paper

In order to enhance the delivery of drugs with limited absorption due to poor solubility/dissolution, approaches are being developed to improve the dissolution rates and solubility of drug molecules. These approaches include identification of water-soluble salts of parent drugs, preparation of stable amorphous drug formulations, inclusion of solubility-enhancing agents in the dosage form, and particle size reduction. Technologies to reduce drug particle size to sub-micrometer range are being applied to product development more frequently. Electrospinning is being considered as one of the technologies which can produce nanosized drugs incorporated in polymeric nanofibers. In vitro and in vivo studies have demonstrated that the release rates of drugs from these nanofiber formulations are enhanced compared to those from original drug substance. This technology has the potential to be used for enhancing the oral delivery of poorly soluble drugs.

The AAPS Journal: Liposomal Simvastatin Attenuates Neointimal Hyperplasia in Rats

2010-02-09

Type: Original Paper

Monocytes, macrophages, and inflammation play a key role in the process of neointimal proliferation and restenosis. The present study evaluated whether systemic and transient depletion of monocytes could be obtained by a single intravenous (IV) injection of simvastatin liposomes, for the inhibition of neointima formation. Balloon-injured carotid artery rats (n = 30) were randomly assigned to treatment groups of free simvastatin, simvastatin in liposomes (3 mg/kg), and saline (control). Stenosis and neointima to media ratio (N/M) were determined 14 days following single IV injection at the time of injury by morphometric analysis. Depletion of circulating monocytes was determined by flow cytometry analyzes of blood specimens. Inhibition of RAW264.7, J774, and THP-1 proliferation by simvastatin-loaded liposomes and free simvastatin was determined by the 3-(4, 5-dimethylthiazolyl-2)-2, 5- diphenyltetrazolium bromide assay. Simvastatin liposomes were successfully formulated and were found to be 1.5-2 times more potent than the free drug in suppressing the proliferation of monocytes/macrophages in cell cultures of RAW 264.7, J774, and THP-1. IV injection of liposomal simvastatin to carotid-injured rats (3 mg/kg, n = 4) resulted in a transient depletion of circulating monocytes, significantly more prolonged than that observed following treatment with free simvastatin. Administration to balloon-injured rats suppressed neointimal growth. N/M at 14 days was 1.56 ± 0.16 and 0.90 ± 0.12, control and simvastatin liposomes, respectively. One single systemic administration of liposomal simvastatin at the time of injury significantly suppresses neointimal formation in the rat model of restenosis, mediated via a partial and transient depletion of circulating monocytes.

The AAPS Journal: Augmentation of Therapeutic Efficacy in Drug-Resistant Tumor Models Using Ceramide Coadministration in Temporal-Controlled Polymer-Blend Nanoparticle Delivery Systems

Lilian Vlerken; Zhenfeng Duan; Steven Little; Michael Seiden; Mansoor Amiji — 2010-02-09

Type: Original Paper

The development of multidrug resistance (MDR) is a major hindrance to cancer eradication as it renders tumors unresponsive to most chemotherapeutic treatments and is associated with cancer resurgence. This study describes a novel mechanism to overcome MDR through a polymer-blend nanoparticle platform that delivers a combination therapy of C6-ceramide (CER), a synthetic analog of an endogenously occurring apoptotic modulator, together with the chemotherapeutic drug paclitaxel (PTX), in a single formulation. The PTX/CER combination therapy circumvents another cellular mechanism whereby MDR develops, by lowering the threshold for apoptotic signaling. In vivo studies in a resistant subcutaneous SKOV3 human ovarian and in an orthotopic MCF7 human breast adenocarcinoma xenograft showed that the PTX and CER nanoparticle combination therapy reduced the final tumor volume at least twofold over treatment with the standard PTX therapy alone. The study also revealed that the cotherapy accomplished this enhanced efficacy by generating an enhancement in apoptotic signaling in both tumor types. Additionally, acute evaluation of safety with the combination therapy did not show significant changes in body weight, white blood cell counts, or liver enzyme levels. The temporal-controlled nanoparticle delivery system presented in this study allows for a simultaneous delivery of PTX + CER in breast and ovarian tumor model drug, leading to a modulation of the apoptotic threshold. This strategy has tremendous potential for effective treatment of refractory disease in cancer patients.

The AAPS Journal: Biomaterials/Tissue Interactions: Possible Solutions to Overcome Foreign Body Response

Jacqueline Morais; Fotios Papadimitrakopoulos; Diane Burgess — 2010-02-09

Type: Original Paper

In recent years, a variety of biomaterial implantable devices has been developed. Of particular significance to pharmaceutical sciences is the progress made on the development of drug/implantable device combination products. However, the clinical application of these devices is still a critical issue due to the host response, which results from both the tissue trauma during implantation and the presence of the device in the body. Accordingly, the in vivo functionality and durability of any implantable device can be compromised by the body response to the foreign material. Numerous strategies to overcome negative body reactions have been reported. The aim of this review is to outline some key issues of biomaterial/tissue interactions such as foreign body response and biocompatibility and biocompatibility assessment. In addition, general approaches used to overcome the in vivo instability of implantable devices are presented, including (a) biocompatible material coatings, (b) steroidal and nonsteroidal anti-inflammatory drugs, and (c) angiogenic drugs. In particular, strategies to overcome host response to glucose biosensors are summarized.

Alert: 2010 AAPS National Biotechnology Conference

2009-12-04

Type: Alert

May 16 - 19, 2010

Hilton San Francisco

San Francisco, CA, USA

For more info visit 2010 AAPS National Biotechnology Conference

Alert: FIP Pharmaceutical Sciences 2010 World Congress in Association with AAPS Annual Meeting and Exposition

2009-05-14

Type: Alert
November 14-18, 2010
New Orleans Convention Center
New Orleans, Louisiana, USA
For more info visit www.pswc2010.org

Alert: The Official AAPS Journals Continue to Increase Their Impact on the Pharmaceutical Sciences!

2008-10-03

Type: Alert
Pharmaceutical Research: 4.024; The AAPS Journal: 5.529; AAPS PharmSciTech: 1.445.