Poster Absracts, 2008

ISPE Boston Area Chapter - Undergraduate

Directed Evolution of the RhlI Protein
Jason Lajoie, Undergraduate Student - University of Massachusetts Amherst

Dawn Eriksen, Jason Lajoie, Pavan Kambam, Lianhong Sun
University of Massachusetts Amherst Department of Chemical Engineering

Abstract: In protein engineering, techniques are used to modify the functionalities of proteins, leading to insight into the design principles of the enzymes. A specific protein of interest in this project is the RhlI protein found in the RhlI-RhlR quorum-sensing system of Pseudomonas aeruginosa. The quorum-sensing system of the bacteria is prominent in chronic infections that afflict patients with Cystic Fibrosis. By identifying crucial residues in the RhlI protein, it is possible to identify improved treatments for Cystic Fibrosis patients. The project’s objective was to alter the functionality of the RhlI protein to produce the signaling molecule 3-oxo-hexanoyl-HSL (OHHL) which is novel to the system. Through directed evolution and rational design, structure-activity relationships can be established to identify the crucial amino acid residues for the protein’s function. Active mutants that produced OHHL were determined through a genetic selection method that coupled RhlI with the LuxR protein. Initial results are positive: two mutants with increased activity compared to the wild-type have been identified.

ISPE Boston Area Chapter - Graduate

Epidermal Growth Factor Receptor-Targeted Engineered Gelatin Nanovectors for Gene Delivery and Transfection in Pancreatic Cancer Cells
Padmaja Magadala, Graduate Student - Northeastern University

Padmaja Magadala and Mansoor Amiji*
Department of Pharmaceutical Sciences, School of Pharmacy, Northeastern University

Purpose: Over 90% of human pancreatic cancer expresses epidermal growth factor family of receptors on the cell surface. In this study, we have examined the potential of EGFR-targeted gelatin-based engineered nanovectors (GENS) for plasmid DNA delivery and transfection in pancreatic cancer cells.

Methods: EGFP-N1 plasmid coding for green fluorescent protein, was encapsulated in gelatin nanoparticles. Nanoparticle surface-modification with 15-mer EGF receptor binding peptide via a poly(ethylene glycol) (PEG) spacer was achieved. Panc-1 human pancreatic adenocarcinoma cells were established in culture. EGFR expression in Panc-1 cells was analyzed by western blot and immunocytochemistry techniques. GFP transfection efficiency was evaluated by flow cytometry, ELISA and fluorescence microscopy.

Results: Surface-modified gelatin nanoparticles of ~200 nm in diameter were reproducibly synthesized. The relative cell viability of Panc-1 cells treated with control and targeted NPs was found to be 99% and 80% respectively. The DNA loading efficiency of GENS was >95%. Up to 9%, 30% and 55% transfection efficiency was achieved with unmodified, PEGylated and EGFR targeted gelatin nanoparticles respectively. These results were confirmed with ELISA.

Conclusion: The results of this study showed that EGFR-targeted gelatin-based nanovectors can serve as a safe and efficiency carrier for potential in vivo gene therapy in pancreatic cancer.

Acknowledgements: Padmaja Magadala is an NSF-IGERT Fellow in Nanomedical Science and Technology program at Northeastern University (Boston, MA). This doctoral training program is financially co-sponsored by the NCI and NSF.

*Corresponding author: Dr. Mansoor M. Amiji
Address: 360 Huntington Avenue, 110 Mugar Building
Boston, MA 02115
Phone: (617) 373-3137
Email: m.amiji@neu.edu

ISPE Boston Area Chapter - Graduate

Role of Microenvironment Stiffness in Directing Mouse Embryonic Stem Cell Differentiation
Akshay Navaladi, Graduate Student - Boston University

Akshay Navaladi1,2,4, Bong Geun Chung3,4, Ali Khademhosseini1,3,4

1) Harvard Stem Cell Institute, Cambridge, MA 02138 2) Department of Biomedical Engineering, Boston University, Boston, MA 02215 3) Harvard-MIT division of Health Sciences and Technology, MIT, Cambridge, MA 02139 4) Centre for Biomedical Engineering, Brigham and Women’s Hospital, Harvard Medical School. Boston, MA 02115

The mechanical aspects of the cellular microenvironment have been shown to play an important role in controlling adult stem cell self-renewal and differentiation. However, not much has been studied with respect to the effects of these forces on embryonic stem cells (ES cells). Being able to control stem cell differentiation is essential to the fields of tissue engineering and regenerative medicine and therefore, the development of technology that can be used to study the effects of mechanical forces on ES cells and thus direct ES cell differentiation in a controlled manner is of great importance. We present here, a platform to encapsulate Embryoid Bodies (EBs) within poly(ethylene glycol) (PEG) hydrogels of varying, but controlled mechanical properties, specifically molecular weight and stiffness. Furthermore we have performed preliminary analysis of the effects that these variations in mechanical properties have on the differentiation of encapsulated mouse EBs. It was found that cells can be encapsulated as cell aggregates within PEG hydrogels of various molecular weights and stiffnesses without losing viability for at least 7 days. Also, it was found that which germ layers the encapsulated EBs give rise to, depends on the molecular weight and stiffness of PEG hydrogel within which they were encapsulated.

ISPE Carolina-South Atlantic Chapter - Graduate

Regulation of Adrenergic Receptor β2 by C/EBP and cAMP Pathways

T. Higgins, I. Tchivileva, C. Downey, W. Maixner, L. Diatchenko
Thomas Higgins, University of North Carolina, Chapel Hill

Neurosensory Disorders Unit, School of Dentistry, University of North Carolina
Chapel Hill, NC 27599

Adrenergic receptor β2 (ADRB2) is a primary target for epinephrine. It plays a critical role in mediating physiological responses to environmental stressors and affects a complex array of psychological and physiological characteristics: including psychological distress, blood pressure, and pain sensitivity. However, on immune cells ADRB2 functions as an inflammatory mediator. Increased pain sensitivity is one of the characteristics of both acute and chronic inflammation. We thus hypothesized that regulation of ADRB2 expression under inflammatory conditions would mediate inflammation-induced pain. We first searched the ADRB2 promoter region for putative binding sites of pro-inflammatory mediators, which revealed CREB (cAMP response element binding protein) and C/EBP (CCAAT/enhancer binding protein) binding sites. We constructed a luciferase reporter vector containing three major polymorphic variants haplotypes) of a 1.9kb human ADRB2 promoter region, and transiently transected the constructs into neuronal and immune cell lines. These variants showed different basal activity in all assessed cell lines. We measured promoter response to various treatments; modulating inflammatory state. Cells were stimulated with epinephrine and forskolin and co-transfected with an expression vector containing C/EBPα or C/EBPβ. In the neuronal cells, reporter activity decreased significantly below baseline upon stimulation, and increased upon co-transfection with C/EBPα and C/EBPβ. Stimulation with forskolin in the immune cells increased reporter activity. In summary, we demonstrated; i) different levels of basal promoter activity related to three major human haplotypes; ii) tissue specific regulation of ADRB2 promoter at the basal level and upon treatment; iii) haplotypes exhibited similar response patterns, demonstrating haplotype-independent transcription regulation by C/EBP and cAMP. These results are the first to demonstrate regulation of ADRB2 by C/EBP and cAMP pathways, and suggest a mechanism by which inflammation modulates pain sensitivity. Supported by NIH grants DE016558 and NS045685.

ISPE Carolina-South Atlantic Chapter

The Release Profile and Diffusion of Morphine Sulfate From PLO and Lipoderm Gels through Polycarbonate Membranes

Ishwin Dembla
Campbell University

Objective: The main objective of the study was to compare the diffusion profiles of Morphine Sulfate from two gel systems, namely Lipoderm and PLO through polycarbonate membranes. Narcotic, Analgesic used to cure mild to severe pain. It binds to brain and spinal cord receptors thereby decreasing the feeling of pain. PLO gel and LPO gel were used as vehicles for the study. Pluronic lecithin organogel (PLO) is a polymeric surfactant which allows the formation of drug micelles in a gel matrix. PLO contains lecithin that acts as a permeation enhancer. Lipoderm is used for delivering hydrophilic and lipophilic drugs. Lipoderm is less greasy than PLO and also contains lecithin.

Method: Lipoderm gels and PLO gels (0.2 ml) were placed on the surface (0.67 cm-square) of a polycarbonate membrane (0.2 um; 25 mm) in a vertical Franz diffusion cell (Temperature = 37 C). The receiver compartment contained 5-ml of phosphate buffer (pH = 7.4). Samples (0.3 ml) were collected at designated time periods over 24 hours, and replaced with an equal volume of fresh buffer. Samples were stored in the refrigerator until they were analyzed by HPLC for their content of morphine sulfate. The HPLC mobile phase consisted of 60:40 aqueous hexane sulfonic acid:methanol.

Conclusion: The HPLC results indicated that morphine sulfate was linear over the range of concentrations used in this study. The diffusion experiments showed that diffusion of morphine sulfate from a PLO gel followed Higuchi?s model for diffusion. The Higuchi?s constant (k) was estimated to be equal to 1.65 ± 0.69 (Mean ± S.E.) (n = 6). The diffusion of morphine sulfate from a lipoderm gel showed zero-order kinetics with a characteristic zero-order constant of 0.0251 ± 0.00155 %/minute (n = 6) (p < 0.0001) and a t1/2 of 33.2 hours. The cumulative percent morphine sulfate release from PLO gel and Lipoderm gel after 24 hours was not statistically different (46.55% vs. 34.70%, p = 0.4508).

ISPE Carolina-South Atlantic Chapter - Undergraduate

2000L Bioreactor Design

Abigail Lithgo, Michael Perkins, Kevin Brown, Tracy Sapp
Undergraduate Student, North Carolina State University

As the biopharmaceutical industry continues to optimize manufacturing performance and flexibility, facilitating innovative bioreactor technologies is critical to operation. To aid in the development of current bioreactors, Biogen Idec, in conjunction with BTEC, has requested that the Biogen Senior Design Group design a 2000 liter bioreactor that would support the growth and recombinant protein expression of animal cells. The purpose of this project then became the proposal of two designs: a conventional stirred tank bioreactor and a non-conventional disposable airlift reactor that could promote Biogen Idec’s current processes and offer flexibility for future products. To accomplish this task, the group performed experimentation on bench-scale STR and airlift reactors as well as literature research on these two reactors along with the disposable bioreactor design. The experiment goals were to evaluate oxygen transfer in both a stirred tank and airlift reactor by finding kLα values that corresponded to appropriate flow rates found in literature and to explore foam reduction using a hydrophobic net. The experimental results were used along with the literature findings to design operational parameter limits with respect to aeration and mixing and mechanical components of each reactor. After evaluating the economic feasibility of the two designs and comparing the flexibility and production benefits from both reactors, a recommendation will be made as to which bioreactor or bioreactor component are most efficient for manufacturing performance at Biogen Idec.

ISPE Carolina-South Atlantic Chapter - Graduate

A Role for a Cardiac-specific MicroRNA in Hypertrophy
Tom Callis, Graduate Student - University of North Carolina-Chapel Hill

The major response of the heart to biomechanical stress and pathological stimuli is to undergo extensive cardiac remodeling known as hypertrophic growth. Cardiac hypertrophy is defined by an increase in myocyte size without a change in myocyte number and helps to sustain cardiac output in the face of such stress. Although hypertrophy induced by pathological stimuli is an adaptive mechanism that is beneficial in the short term, prolonged hypertrophy has adverse consequences associated with heart failure and sudden death. MicroRNAs (miRNAs) are recently discovered class of ~22-nucleotide long noncoding RNA molecules that regulate gene expression through complementary base pairing with the 3’ untranslated region (UTR) of targeted messenger RNAs (mRNAs). Over 500 human miRNA genes have been identified, though relatively few have been critically studied and assigned specific biological functions. Here, using gain- and loss-of-function approaches in genetically engineered mice, we find that a single cardiac-specific miRNA, miR-208a, is sufficient to induce hypertrophic growth. This previously unrecognized relationship sheds new light onto the regulatory mechanisms underlying heart pathology, and suggests the potential of miRNAs as diagnostic markers and therapeutic targets for cardiovascular disease.

ISPE Central Canada Chapter - Undergraduate

Comparison of hybridoma cells culture in suspension and attached to geotextile membrane

R-M. Poulin, M. Jolicoeur
Rose-Marie Poulin, Undergraduate Student, École Polytechnique de Montréal

For the production of monoclonal antibodies in bioreactors, it is advantageous to own mathematical models of cellular metabolism-producing cells. The technique of nuclear magnetic resonance is used to produce these models. However, this technique requires to attached cells on a geotextile membrane. To achieve a better comprehension of hybridoma cells behavior, a comparative study on the growth rate and antibodies production by hybridoma cells in suspension and attached to geotextile membrane was made. The concentration of glucose, lactate and ammonium show a highest growth rate of hybridoma cells when attached to geotextile membrane treated with the PPE:N.

The production of anti-PSMA antibodies was evaluated by western blot and by surface plasmon resonance (SPR). Tests such as the quantification of glutamine and the evaluation of cell density on membrane should be made to draw reliable conclusions.

ISPE Central Canada Chapter - Graduate

Enabling Real-Time release using Near Infrared Spectroscopy technique as a PAT tool for quality control of multi-component pharmaceutical intact tablets
Antoine Cournoyer, Graduate Student - Université de Montréal

A. Cournoyera,b, J.-S. Simardb, L. Cartiliera, N. Abatzoglouc

a Université de Montréal, Faculty of Pharmacy, Montreal, Que., Canada H3C 3J7
b Process Analytical Techniques & Technologies, Product and Process Development, Technology, Wyeth Canada, Montreal, Que., Canada H4R 1J6
c Université de Sherbrooke, Department of Chemical Engineering, Sherbrooke, Que., Canada J1K 2R1

Continuous development and implementation of improved Process Analytical Technologies (PAT) and Quality by Design (QbD) tools are a priority of the contemporary industrial production of pharmaceutics. These tools, when proved sufficiently performing, allow for a more efficient and timely Quality Control and decrease the associated costs while keeping or even improving the level of quality of such produced pharmaceutical goods.

This study was aimed at developing a rapid, robust and versatile Near Infra Red Spectroscopy (NIRS) analysis protocol for the quality control of intact tablets containing 2 active pharmaceutical ingredients (API) and three excipients. Reference samples were prepared and used to build a calibration model. Three different FT-NIR apparatuses using transmission measurements have been tested and quantitative prediction models using Multivariate Data Analysis (MVDA) have been built and statistically validated. Calibration was carried out using a partial least square (PLS) regression method; signal pre-processing techniques were applied to optimize the efficiency of the models. High Performance Liquid Chromatography (HPLC) was used as reference analytical method. Samples prepared for statistical validation were used to confirm the efficiency of the MVDA models built.

Four out of five ingredients were precisely quantified with the three MVDA models developed, allowing a quantification of at least 99% of the formulation. The results show that a versatile in-line or at-line NIR spectroscopic method can be developed without sample preparation for real-time pharmaceutical tablets quality control.

Key-Words: NIRS Analysis, Pharmaceutical tablets, Chemometrics, Process Analytical technologies (PAT), Quality Control

ISPE Central Canada Chapter - Graduate

Bioengineering scaffold-stabilized blood clots for regenerative medicine applications
Catherine Marchand, Graduate Student - École Polytechnique de Montréal

C. Marchand 1, G.-E. Rivard 2, J. Sun 3 C. Hoemann 1,4

1Institute of Biomeical Engineering & 4Dept Chemical Engineering, École Polytechnique, Montréal, Québec, Canada.
2Hematology-Oncology, Hôpital Sainte-Justine, Montréal, Québec, Canada. 3BioSyntech, Laval, Québec, Canada.

We recently developed a novel implant for cartilage repair that is generated by mixing autologous patient whole blood with chitosan-glycerol phosphate, to generate a scaffold-stabilized clot in the cartilage defect. As a faster in situ solidification of this hybrid clot could improve clinical ease-of-use, we investigated implant solidification mechanisms using thromboelastography, and evaluated the effect of adding clotting factors on the solidification rate. Chitosan-GP/blood clots solidified in an atypical biphasic manner, with a higher initial viscosity followed by a similar final clot tensile strength as normal blood clots. Chitosan-GP/blood clots showed a more sustained thrombin generation, platelet activation, and greater resistance to lysis. Thrombin and Tissue Factor with recombinant human Factor VIIa shortened implant solidification in vivo in a rabbit cartilage repair model. Our data indicate that chitosan-GP/blood implants solidify through thrombin-dependent coagulation mechanisms and that clotting factors can be used to shorten in situ solidification time in cartilage defects.

ISPE Central Canada Chapter - Graduate

Bioreactor Culture Optimization for Cartilage Cells
Anne Gigout, Graduate Student - École Polytechnique de Montréal

Anne Gigout‡, Michael Buschmann*, Mario Jolicoeur‡

‡Chemical engineering department, *Biomedical engineering department, École Polytechnique de Montréal.

Research on cartilage and its unique cell type, the chondrocyte, leads to a better understanding of initiation and progression of joint diseases, such as arthritis and enables to propose new therapeutic strategies. We developed a new culture system consisting in a scaffold-free suspension culture in a defined serum-free medium (SFM). In this system, chondrocytes are stable and condense to form aggregates. They also re-create a cartilage-like microtissue maintaining them in a physiological environment. However, these aggregates can be several mm large, what impede nutrient accessibility to all cells. To circumvent this problem, we investigated the use of an agitated bioreactor to simultaneously improve nutrient distribution, control aggregate size and homogeneously distribute aggregates in the culture volume. The previously optimized SFM was used as a control. In addition, two modified media were tested, one containing Pluronic F-68 and the other one containing Pluronic F-68 and a 10-fold increase in growth factor concentration. This culture system enabled to obtain i) smaller aggregates (200-300μm versus >1mm in static conditions) with an excellent viability, ii) aggregates which structurally and biochemically resemble cartilage iii) a cell proliferation in presence of Pluronic-F68 accompanied with a good to excellent phenotype maintenance, depending on the medium used. This latter result is of high importance as cell growth is usually associated with loss of the chondrocyte phenotype in other types of culture. Agitated suspension culture thus enables to engineer cartilaginous aggregates composed of stable chondrocytes that have a therapeutic interest and could possibly used as transplants for cartilage repair.

ISPE Delaware Valley Chapter - Graduate

Optimization of alginate gel fabrication for maximal neurite growth in vitro

Nicola Francis and Dr. Margaret A. Wheatley
Nicola Francis, Graduate Student, Drexel University

Transplantation of cells genetically modified to produce neurotrophic agents is a promising strategy for spinal cord repair. We have previously shown that fibroblasts genetically engineered to produce brain-derived neurotrophic factor (Fb/BDNF) encapsulated in coated alginate microcapsules survive in vitro, continue to grow for at least one month, and express bioactive BDNF. To further study the properties of alginate-encapsulated Fb/BDNF, we compared the effects of different alginate cross-linking solutions on neurotrophin release and evaluated the effects of a BDNF concentration gradient on neurite growth using dorsal root ganglia (DRGs) as a model.

Alginate that was coated with PLO and laminin was found to have neurotrophic properties by itself, yet the addition of Fb/BDNF to the alginate stimulated significantly more neurite growth. Alginate gels cross-linked with calcium carbonate and D-glucono-δ-lactone were found to be excellent for DRG growth in vitro because of their pH buffering and gelation properties. Neurotrophin concentration gradients (BDNF) produced by alginate-encapsulated fibroblasts were shown to direct the growth of DRG neurites. This positive neurotrophic effect of alginate-encapsulated BDNF-producing fibroblasts on the growth and guidance of neurites shows promise for enhanced regeneration and guidance of injured axons towards a specific target in the injured spinal cord.

ISPE Delaware Valley Chapter - Undergraduate

A Bioactive Ultrasound Contrast Agent: Ovarian Cancer and Melanoma Cell Apoptosis Induced by TRAIL-Conjugated Microbubbles
Kelleny Oum, Undergraduate Student - Drexel University

Kelleny Oum, Dr. Margaret A. Wheatley
School of Biomedical Engineering, Science & Health Systems, Drexel University, Philadelphia, PA

Tumor necrosis (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF family of cytokines that promotes apoptosis. TRAIL induces apoptosis via death receptors (DR4 and DR5) in tumor cells but not in normal cells. We have developed a novel ultrasound contrast agent to induce death in cancer cells by the surface ligation of TRAIL to microbubbles via a thoil-conjugation procedure. The biodegradable polymeric, hard-shelled microbubbles are produced using a double emulsion method that results in a hollow gas-filled sphere which reflects impinging ultrasound beams to enhance ultrasound images. When exposed to the TRAIL-bound microbubbles, significant apoptosis was observed in both OVCAR-3 human ovarian cancer and A2058 human melanoma cancer cell models over a 24 hour period. Apoptosis was markedly lower in cells incubated with unmodified microbubbles. The apoptotic abilities of TRAIL were not hindered by its conjugation to the microbubbles. Potential applications of this approach may include the simultaneous ultrasound imaging and treatment of malignant tumors, especially if further adapted to a nano-scale contrast agent platform.

ISPE Delaware Valley Chapter - Undergraduate

Large Scale Production of Peptides for Biotechnology
Jennifer Nugent, Undergraduate Student - Widener University

Jennifer Nugent, Caitlin Feeser, Jonathan Hurst,
Dr. Thomas Twardowski, Dr. Shirley Fischer-Drowos, Dr. Alexis Nagengast,
Dr. James San Antonio

The objective of this project is to develop techniques for synthesis of a heparin binding peptide that can be scaled to economical levels, resulting in production costs consistent with commercial supplies. The approaches considered for production of the peptide were solid phase peptide synthesis, biosynthesis via E. coli, and synthesis via tissue degradation. The stepwise synthesis approach is currently used to produce the peptide on a small sale; a large scale production plant based on this approach was designed on paper. Laboratory work was performed to assess the feasibility of the latter approaches. Based on the work in this project, the most effective source of the target peptide would be a solid phase synthesis plant. This approach is well-known and reliable, and proceeds to high yields with cost estimated at 150 $/g. The biosynthesis approach is shown to be 20 $/g less expensive than the solid phase plant after accounting for five years of future research and development; however, the technical feasibility is not yet entirely established. The tissue degradation approach has been shown to be feasible, yet the methodology is sub-optimized.

ISPE Great Lakes Chapter - Undergraduate

Development of a Swine Model for the Evaluation of Novel Compounds in the Prevention of Pelvic Adhesions
Maureen Chung, Undergraduate Student - University of Akron

Maureen Cheung 1,2; Bradford W. Fenton, MD, PhD 1; Michelle Chapman, BS 1;
Mark Kovacik, BS 1; Don Noe, BSE 1; Nicholas Ree, MD 1;
Stephanie Lopina, PhD 2; James Fanning, DO 1

1: Summa Health System, Akron, Ohio, USA
2: The University of Akron, Akron, Ohio, USA

Introduction: Adhesions following surgery represent a significant problem often resulting in pain, disability, and additional surgeries. There are compounds available for the prevention of post-operative adhesions, but their effectiveness is difficult to assess. Currently accepted models of adhesion comparison are limited to qualitative methods with much potential bias and little comparability.

Objective: The purpose of this study was to develop a standard injury technique and quantitative assessment method to evaluate adhesion prevention by novel compounds.

Development: The primary focus of this research was the creation of an adhesion complex that was suitable to quantitative testing using the Material Testing System (MTS) and histological assessment.

Result:The MTS measures force by displacement which can be quantitatively analyzed and interpreted, while the histology confirmed that adhesion was present and being quantified as well as providing a comprehensive description of the vascularity, density, collagen content, and organization of the adhesion. This model is unique and advantageous in its ability to quantitatively assess the strength of the adhesion complex. Our assessments showed that previous visual grading methods were neither consistent nor accurate. Our model minimizes the potential for bias by using quantitative testing and histological assessment. This method of assessing adhesion strength can now be used to test current industry standard barriers and newly developed novel compounds for adhesion prevention.

ISPE Great Lakes Chapter - Graduate

Nanoparticle “Paint-Brush” Conjugates for Targeted Cancer Nanotherapy: Synthesis & In-Vitro Activity
Vinu Krishnan, Graduate Student - University of Akron

Vinu Krishnan1*, Dr. Stephanie T. Lopina2, 1 PhD

1The Department of Biomedical Engineering, The University of Akron, Ohio
2The Department of Chemical & Biomolecular Engineering, The University of Akron, Ohio

Statistical figures outline the five-year survival rate for all cancers diagnosed between 1996 and 2002 as 66%, which depicts a marked rise from the 51% that survived in 1975-19771. However, cancer still remains the second leading cause of death in the United States, following heart disease. An American Cancer Society report estimated that in 2007, there will be over 1.4 million new cancer cases and over half a million cancer deaths in the United States1. The National Institutes of Health had estimated the overall costs of cancer treatment in 2006 to be at $206.3 billion even with a declining death rate in the last decade1. Moreover, any use of traditional biomolecules as a treatment strategy would only elevate the treatment costs to higher levels. Though significant oncology drug discoveries have been made during the past 30 years, conventional chemotherapeutic agents exhibit poor specificity in reaching the tumor site and are often restricted by toxicity factors. The lack of a uniform biodistribution leads to harmful side-effects to healthy tissues and the need for administration of a larger than necessary drug dosage; with a higher repetitive rate so as to elicit a satisfactory pharmacological response.

Wide interest in cancer nanotherapy has led to the development of nanoparticle based “smart drugs” that have not only improved pharmacological and therapeutic properties of anticancer drugs, but also offer a less invasive alternative enhancing the patient’s life expectancy and quality of life as well. Dendrimers, due to their unique architecture and macromolecular characteristics are currently used extensively in research of nanoparticles for targeted and controlled drug delivery. The research objective is to design, synthesize and characterize a novel nanoparticle based “Paint-Brush” like multi-hydroxyl capped poly (ethylene glycol) (PEG) conjugate using the dendron – bishomotris that may have a potential use in targeted cancer nanotherapy.

Characterization of the conjugates suggested that the synthesis was successful; resulting in the formation of nanoparticle “Paint-Brush” conjugates. It was also found that these conjugates would remain stable during transport inside the body but would activate in response to an acidic pH (a characteristic trait of target cancer cells) so as to release the anticancer drug. The research also presents the relative cell viability of human epithelial carcinoma cell line (HeLa S3) with respect to in-vitro characterization of the conjugate to determine its suitability as a drug delivery vector. The uniqueness in the model envisioned lies in the fact that the structure can be exploited in more than one way to develop novel hybrids that may have the ability to carry antibodies; enzymes or even additional PEG linkages that may enhance the system’s biocompatibility along with its purpose as a targeted drug delivery system for malignancies.

References: 1) Cancer Facts and Figures 2007, American Cancer Society, Atlanta, 2007
* Corresponding Author Email: vk11@uakron.edu

ISPE New Jersey Chapter - Graduate

High Shear De-Agglomeration of Nano Particle Agglomerates in Stirred Tanks

Micaela Caramellino1 and Piero M. Armenante1
Micaela Caramellino

Graduate Student, New Jersey Institute of Technology
1Otto H. York Department of Chemical Engineering
323 King Blvd., Newark, NJ 07102-1982

The film strip manufacturing process has been analyzed in this work. In particular, Active Pharmaceutical Ingredients (APIs) with low solubility, and therefore low bioavailability, have been considered. An homogeneous dispersion of nano-particles in an aqueous phase is necessary prior to the addition of a gelling agent and the formation of the film.

The possibility of the de-agglomeration of nano-particles agglomerates has been investigated in order to obtain final aggregates with dimension compatible with the film thickness. It has been demonstrated that the formation of submicron agglomerates can be achieved by means of a high shear device.

The results show a dependence of the final agglomerates size on the local energy input and a rate of breakage that is a function of the amount of material to be processed.

ISPE New Jersey Chapter - Graduate

Using Physically-Based Dissolution Simulations to Develop Ideal Particle Distributions in Tablets

Dan Braido and Alberto Cuitino, PhD
Graduate Student, Rutgers University

Purpose: The purpose of this investigation is to develop modeling and simulation tools to conduct in-silico design of delivery systems for pharmaceuticals, in particular tablets. A basic component is the systematic correlation of end product quantities (e.g. dissolution profile) with the physical characteristics of the tablet (e.g. spatial distribution of particles). In this paper we describe a methodology to determine optimal active distribution for a prescribed dissolution profile. This methodology integrates physically-based solvers that track the effects of fluid penetration with nonlinear optimization techniques. This methodology opens the possibility of aiding in the design process of tablets with desired or required performance.

Methods: The computational tool initially simulates tablets with a predefined distribution of active particles on a three dimensional fixed grid. The steady-state concentration is used to develop a localized flux for each of the particles, and determine the amount of time it would take for each of the particles to dissolve completely. All of the particle volumes are reduced accordingly and particles below a critical volume are removed from the simulation, until all of the particles have dissolved completely. The outputs of these simulations are then compared to the desired output and the differences in composition to determine a rate of change. This rate of change is then used to develop a new particle distribution whose output is closer to the desired output. This process is repeated until the desired output is reached to within an acceptable tolerance.

Results: The simulation produces profiles for concentration and surface fluxes at every time step. It also provides a total time of dissolution for the entire tablet. The program is easily adaptable to multiple grid sizes, active particle distribution concentrations, and diffusion properties through several global constants. The backsolving algorithm is capable of dealing with particle distribution profiles of varying complexity. A series of physical experiments were performed in order to provide relevant data for comparison with simulation output. Simulation output was compared to dissolution curves from this model physical system with reasonable correlation. The release profiles were nearly identical for the first part of the simulation, but differed after the physical profile developed a sharp hump. This hump is most likely the result of tablet swelling and fracture, phenomenon which have not yet been incorporated into the numerical model.

 

Conclusions: This program provides a good basis for future extensions and demonstrates feasibility of the method. The process can be adapted into larger simulations incorporating a moving fluid boundary layer, variable tablet geometry, and composite particle systems with multiple active ingredients. The backsolving algorithm is removable from the dissolution program and is easily adapted to future dissolution simulations, and conversely, it is easy to add a new tablet design algorithm which focuses on different tablet characteristics.

ISPE New Jersey Chapter - Graduate

Regulation of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and DF508 Mutant by Rab GTPases
Simarna Kaur, Graduate Student - Stevens Institute of Technology

Cystic fibrosis (CF), an autosomal recessive disorder, is caused by the dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR). The predominant mutation found in CF patients involves the loss of phenylalanine at position 508 (DF508) resulting in a mislocalized channel, which is unable to reach the plasma membrane. CFTR regulatory mechanisms include channel transport to plasma membrane, and regulation by protein–protein interactions. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. The regulation of CFTR channel by Rab GTPases-4 and 27a was investigated in multiple cell systems comprising of the colorectal HT-29 cells, and the bronchial epithelial IB3-1 cells obtained from a CF patient expressing the mutant DF508. Over-expression of Rabs-4 and 27a were successful in inhibiting CFTR channel activity by causing a decline in the amount of cell surface CFTR. However, the total CFTR pool remained unaffected under these conditions. GDP-locked and GTPase-deficient mutants of these Rab proteins were found to regulate CFTR at varying degrees. Both the GTPases showed strong protein-protein interactions with CFTR. Native interaction of the DF508 mutant with Rab27a was shown under physiological conditions. Certain high-affinity Rab27a-binding effector proteins led to the reversal of CFTR inhibition that was caused by Rab27a over-expression. Possible models showcase a potential role for Rab4 in mediating the recycling of the CFTR or its mutant form DF508 from the plasma membrane to the microsomes inside the cell. Rab27a-related experiments reveal that this GTPase can potentially act as a key player in regulating the trafficking of CFTR or DF508 towards the lysosomes for degradation. Rab27a effector proteins might be able to rescue the channel by preferentially binding to Rab27a.

ISPE New Jersey Chapter

Mechanical analysis of an expandable interbody cage

S. Gadol, F. Hardenbrook, W. Kowalski, M. Trapani, A. Quiray

Stevens Institute of Technology
1 Castle Point on the Hudson
Hoboken, NJ 07030

Improper spinal fusions result in patient discomfort and revision surgery serves as an option for relief. These revi-sions are time consuming and costly for clinicians and patients. An expandable intervertebral cage (Back Up Cage) has been designed to improve positioning during surgery, providing solid support for fusion. The Back Up Cage was subjected to static testing, with an axial load applied at a rate of 25mm/min. Load displacement curves were plotted with maximum load and com-pressive stiffness extracted. Fatigue testing was performed, with axial loading applied for 5 million cycles. The net displacement and stiffness were plotted. Results of the static test signify that the cage is able to withstand intervertebral compressive forces. The fatigue test results show the cage’s ability to remain de-ployed with minimal subsidence under normal cyclic loading. The Back Up Cage successfully endured intervertebral forces under dynamic and static loading, proving the strength and sta-bility of the design.

ISPE New Jersey Chapter

Precipitation of Griseofulvin by Impinging Jets in Stirred-Tank Reactors

Giuseppe Di Benedetto1, Ewa Sukcik1, Piero M. Armenante1, and Somenath Mitra2

1 Otto H. York Department of Chemical Engineering, New Jersey Institute of Technology,
323 King Blvd., Newark, NJ 07102-1982
2 Department of Chemistry and Environmental Science, New Jersey Institute of Technology
323 King Blvd., Newark, NJ 07102-1982

An impinging jet system placed within a stirred-tank reactor was utilized in this work to precipitate an active pharmaceutical ingredient (API), namely griseofulvin, using a solvent/anti-solvent approach. The API was dissolved in an acetone solution. The antisolvent solution was water added with a polymer (HPMC) and a surfactant (SDS) combination in order to enhance particle stability.

The impinging jet mixer consisted of two jet nozzles arranged diametrically opposed to each other with the outlet tips of the nozzles directed to face each other. The jet nozzles were organized in a submerged arrangement within the stirred-tank reactor. Ultrasonics were also introduced at the point of impingement by way of a sonication probe. The effect of sonication power and angle of impingement of the two jets on the resulting particle size distribution and crystal structure was observed. The particle size distribution was measured for each experiment using a laser diffraction particle size analyzer. The precipitated griseofulvin crystals were also analyzed using Scanning Electron Microscopy (SEM) and X-ray Diffraction (XRD) to obtain an image of the crystal shape and structure.

The impinging jets system produced crystals with smaller mean particle sizes when the two jets were oriented 180 degrees apart and pointed directly at each other. The introduction of ultrasonic power at the impingement point resulted in markedly smaller mean particle size and a tighter particle size distribution. In general, the results were highly reproducible. X-Ray diffraction results showed that the crystal structure was unaffected by different operating conditions.

ISPE San Francisco/Bay Area Chapter - Graduate

MRI/PET Probes for the Integrated Assessment of Activated Microglia in Relation to Alzheimer Plaques
Erica Andreozzi, Graduate Student - University of California Davis

Authors: Erica Andreozzi, Benjamin Jarrett, Angelique Y. Louie

Organizations: University of California, Davis Department of Biomedical Engineering

Activated microglia, a macrophage-lineage cell located in the central nervous system (CNS), have been implicated to be important in the neuro- inflammatory response associated with Alzheimer’s disease (AD). They are activated in the presence of senile plaques, current pathological hallmarks of AD, and this activation appears to be mediated by macrophage scavenger receptors. Since microglia have been identified as a target for anti-neuroinflammatory drugs as a possible treatment for AD, there is great interest for non-invasive in vivo methods to identify senile plaques as a way to confirm diagnoses of AD.

We plan to target novel imaging probes to a type of scavenger receptor (SR-A) that is highly expressed by microglia in AD but not in normal microglia, allowing in vivo visualization of activated microglia associated with senile plaques. We have developed two types of SR-A probes: 1) Iron oxide nanoparticles coated with dextran sulfate ligands (SR-A)and coupled to 64Cu chelates; and 2) Maleylated BSA molecules (SR-A ligand), coupled to gadolinium (Gd+3) and 64Cu chelates. These probes are capable of both magnetic resonance imaging (MRI) and positron emission tomography (PET). While PET is utilized to assess general probe biodistribution, Magnetic Resonance imaging (MRI) will be investigated for labeling plaques too small to resolve by PET. This multimodal imaging approach will allow for the integrated assessment of activated microglia in relation to plaques in mouse models of AD using MRI and PET. In this way, we can attempt to correlate activated microglia with both senile plaque location and degree of neurotoxicity to more effectively monitor its role during AD disease progression as well as during therapeutic intervention.

ISPE San Francisco/Bay Area Chapter - Graduate

Layered Biodegradable Star Polymer Complexes for Versatile and Controlled Surface-Based Drug Delivery
Cecile S. Bonifacio, Graduate Student - San Jose State University

Authors: Cecile S. Bonifacio, Eric A. Appel, Lilian Chang, Melanie McNeil, Jane Frommer, Joseph Sly, and Robert D. Miller

Organizations: San Jose State University, California Polytechnic State University, IBM Almaden Research Center

One of the great challenges in modern pharmacy is the engineering of platforms for the slow and controlled delivery of hydrophobic therapeutics within the body. The use of thin film surface coatings for this purpose is receiving increased attention; however, there currently remain many significant problems with this approach, such as controlling the film structure, incorporating the drug within the film and then controlling the release of the drug from the film. To address these key issues we are examining the combination of the “layer-by-layer” (LBL) self-assembly process with three dimensional, hyperbranched, star polymers as building blocks for nanostructured, composite, thin film formation. Using model star polymer materials and surface plasmon resonance (SPR) to monitor thin film formation it was shown that successive layers of the star polymers could be rapidly deposited (18 seconds) to form reproducible, uniform, and robust organic multilayer star polymer thin films. By atomic force microscopy (AFM), the star polymer thin films were characterized as being stable and contiguous surfaces in the dry state. These LBL techniques were then adapted for thin film formation with more structurally complex core/shell (hydrophobic/hydrophilic) biodegradable star polymers materials. These materials can be “preloaded” with hydrophobic materials prior to thin film deposition to form a versatile and controlled composite building block for use in multilayered star polymer thin film formation. Polyethyleneoxide/polycaprolactone based star polymer occlusion complexes with hydrophobic materials, for example, have been shown be us to be indefinitely stable in polar organic solvents but slowly degrade under physiological conditions releasing their cargo to the environment over time. Investigations into both the formation and degradation/release profiles of multilayered thin film structures formed from these star polymer composite materials will be presented. The ability to “preload” the star polymer building blocks with different materials for sequential multilayer build up within the thin film structure will then be discussed as an avenue for the engineering of complimentary, sequential controlled drug delivery platforms.

ISPE Singapore Affiliate - Undergraduate

Screening of novel actinomycetes for secondary metabolites

Siti Hajar Ab Halim
Undergraduate Student, Ngee Ann Polytechnic University

Actinomycetes were isolated from several terrestrial sources in Singapore using various selective media. The amplified 16S rRNA gene fragments of each isolate were sequenced and matched against the NCBI nucleotide database for identification. Majority of the isolates identified belonged to the Streptomyces genera, while the remaining belonged to other genera such as Kitasatospora, Amycolatopsis, Lechevalieria and Nocardia. They were then screened for their ability to produce bioactive secondary metabolites. 8 isolates with anti-microbial activity against at least 1 out of 5 test cultures have been obtained. Future work would include separation and identification of compounds that successfully inhibited microbial activity.

ISPE Turkey Affiliate

Transforming Drugs Which Are Used Topically for External Hemorrhoids to Spray Form

Gülþah Gökdemir, Özlem Akbal, Ata Cömert
Ýstanbul University, Faculty of Pharmacy

Hemorrhoids are one of the important disorders of the anus and rectum. Hemorrhoids are swollen and inflamed veins in anus and rectum, enlarged prolapsed anal cushions and its pathophysiology involves degeneration of the supporting fibro elastic tissue and smooth muscle with enlargement and protrusion of the cushions.

In this study we focus on external hemorrhoids. Symptoms of external hemorrhoids are; these hemorrhoids tend to be painful. Sometimes blood may pool in it, causing severe pain, swelling and inflammation. An acutely thrombosed external hemorrhoid is a purple black edematous, subcutaneous perianal skin becomes quite tender. The thrombus occasionally causes ischemia and necrosis of the overlying skin resulting in bleeding.

Irritated external hemorrhoids can itch or bleed after a bowel movement. Less commonly, hemorrhoids may discharge mucus (oozing) and create a feeling that rectum isn?t completely emptied.

Generally, we can classify treatments as medical treatments

1. Advice for daily life and drugs (orally and topically)
2. Surgical or other procedures (rubber band ligation, sclerotherapy, infrared light, stapling)

Patients can use topical drugs like hemorrhoid creams, suppositories and pomades. If hemorrhoids are producing mild discomfort, topical drugs are more helpful for patients. Drugs which are used topically classify as four parts:

1. Anti-inflammatory drugs contain steroids
2. Topical local anesthetics
3. Oily pomades (for skin protection and healing)
4. Vasculotropic drugs. ( usually oral drugs)

Also they may combine with analgesics and antiseptics.

The aim of this study is giving idea for transforming topical drugs to spray form. We think spray form would help patients to manage symptoms of external hemorrhoids by themselves more easily.

Using spray form would be more hygienic because they don?t need to touch or feel the swallowed, bleeding skin. They can carry and apply the drug easily. They won?t touch cream, suppository or pomades by hands and fingers. They just need to push the head of the spray. When they are using topical dosage forms like creams to internal hemorrhoids, they push the cream inside the anal region, anus. And they need to wait for suppositories to melt and be absorbed by mucosa because if they move before it melts, it can dirty their clothes and underwear, leaving stains. They don?t need to wait for these; they just push the head of the spray and maybe wait a little bit for the spray to dry. Some patients need somebody to help them use suppositories; by using spray form they won?t need anybody to apply the drug. Transforming topical drugs to spray form would increase patient compliance.

We chose some compounds used for hemorrhoids in dosage forms and some compounds that have the potency to be an active agent for treatment of external hemorrhoids. These compounds have different effects on hemorrhoids. And some of them have same or similar effects. By using only one therapeutic agent we can treat symptoms or make some symptoms better. They have lots of characteristic properties; they need to be analyzed and researched.

1. Anti-inflammatory drugs (not containing steroids)
   • Hippocastani semen extract: Escin
   • Hamamelis cortex extract : Proanthociyanidins (catechin, epicatechin, epigallocatechin)
   • Matricaria flower extract: Flavones (apigenin) , Sesquiterpenes (bisabolol, matricin, chamazulen)

2. Anti-inflammatory drugs: (containing steroids)
   • Hydrocortisone acetate

3. Local anesthetics
   • Tetracaine hydrochloride
   • Lidocaine

4. Skin protectants
   • Hippocastani semen extract: Esculin
   • Hamamelis leaf and cortex extract: Hamamelitannin and Gallic acid
   • Matricaria flower extract: Chamazulen, Azulene

By using these agents we can form a dosage form as spray. Spray form may bring lots of advantages and comforts to the patients? life and may increase life quality.