79 research outputs found
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Recombinant adenoviral-meditated alterations of cytochrome P450 3A2 and 2C11
No full texttextRecombinant adenovirus serotype 5 (Ad) is a key vector extensively employed in gene therapy clinical trials and vaccine development protocols. Although this vector has a natural tropism for the liver, there is limited understanding of how Ad infection affects one of the primary hepatic processes, drug metabolism. Preliminary data investigating the effect of a single dose of 5.7 x 1012 adenovirus particles/kilogram on the hepatic cytochrome P450 enzyme system (CYP) revealed not only that significant suppression occurs following systemic administration, but also that enzymatic activity remains suppressed for 14 days. In addition to the vector dose, various components of the virus (transgene, virus gene expression and capsid-receptor interactions)
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could be responsible for the observed suppression in hepatic CYP. Investigation of treatment (5.7 x 1010-5.7 x 1012 vp/kg) of a recombinant vector expressing a non-therapeutic transgene significantly suppressed CYP3A2 expression (mean value of 39%) and function (mean value of 41%) four days after administration. Doses in the range of 5.7 x 106-5.7 x 109 vp/kg did not alter CYP3A2 expression or function, but significantly increased CYP2C11 one day after administration. Expression levels (mean value of 88%) and activity levels (mean value of 93%) were markedly increased. Doses of vectors expressing self transgenes and no transgene revealed that CYP is altered regardless of the transgene cassette used. Treatment with the Null vector, a vector without a transgene cassette, significantly altered CYP3A2 activity throughout the duration of the study (14 days) and CYP2C11 activity at early time points (6 and 24 hours). In addition, modifications at the molecular and macromolecular level do not eliminate aberrations in CYP following Ad administration. Treatment with an adenoviral vector lacking all viral genes markedly suppressed both CYP isoforms, at the transcriptional and translational level, for 14 days. The data presented here suggest that the binding of Ad to cell surface receptors and subsequent internalization of the virion significantly alters posttranslational CYP function, possibly through altered signal transduction pathways. In addition these studies show that shifts in cellular machinery to support the production of the transgene product, and not the transcription of viral genes, also repress CYP transcription.Pharmaceutical Science
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Enhancing intratumoral drug transport by optimizing the surface physicochemical properties of nanotherapeutics
No full textCurrent therapies against solid cancers fail in part due to their inability to penetrate through the extracellular matrix (ECM) of tumors and reach the cancer cells at therapeutic doses. Increasing penetration promotes tumor regression and improves the therapeutic index of compounds. To achieve distribution and penetration through tumors, molecules must achieve unhindered diffusion through the size and interaction barriers presented by the heterogeneous ECM network. The interactions between the molecules and the ECM are driven by the surface physicochemical properties of the therapeutics. To address the challenges presented by current surface chemistries, we used peptide-presenting phage libraries as a high-throughput approach to screen and identify peptide coatings with physicochemical properties able to facilitate improved diffusive transport through the tumor microenvironment. Through iterative screening of these phage libraries against tumor ECM and identification through next-generation DNA sequencing and analysis, we identified a peptide that enhanced the diffusivity of both biological and synthetic nanoparticles. Interestingly, in contrast to most studies, we found that a positively charged peptide “surface” enhanced penetration, uptake, and the retention of particles in tumor tissue when compared to neutrally charged peptides. Mechanistically, we demonstrated that the positively charged particle partitioned into the net negative tumor tissue due to electrostatic interactions. Further, weak and reversible binding of the particles with the tumor bed allowed for deep penetration within the tissue. Additionally, the particle had a high number of intra-tissue binding sites in the tumor ECM that enabled longer retention in the tissue. Conjugating these peptides to immune checkpoint inhibitor antibodies improved the binding of the antibodies in a murine melanoma tumor environment. The longer retention of peptide-antibody conjugates recruited a higher number of activated tumor-infiltrating T-cells, resulting in a delayed tumor growth. This study indicates that net negative tumor ECM acts as a drug delivery depot for positively charged solutes toward improved intratumoral drug penetration and retention. Leveraging favorable interactions between the tumor ECM with therapeutics results in improved drug accumulation and retention for improved antitumor efficacy. As a result, this work can upend the current dogma of designing inert drug delivery systems and exploit electrostatic interactions to significantly improve therapeutic index of drugs and in the long-term, improve therapeutic outcomes in solid cancers.Pharmaceutical Science
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Intrinsically Disordered Proteins & Regions in Viruses and Possibly Pharmaceuticals
No full textThis review examines the significance of intrinsically disordered proteins (IDPs) and regions to viruses and their prospects as vaccine components. Using a content analysis of scientific articles, reviews, interviews, industry updates and magazines, it demonstrates how intrinsically disordered proteins support virus infections allowing them to subvert the host immune response, facilitate virus evolution, conserve genomic space, and enhance viral stability. It also shows that protein biologics are being challenged in the pharmaceutical industry with the rise of mRNA technology in therapeutics and vaccines. While some findings additionally indicate strong potential for targeting IDPs particularly in the treatment of cancer and neurological disorders, this study proposes the possible function of IDPs as excipients for the stabilization of other biologics.Cellular and Molecular Biolog
Optimized Adenovirus-Antibody Complexes Stimulate Strong Cellular and Humoral Immune Responses Against an Encoded Antigen in Naive Mice and Those with Preexisting Immunity
No full textThe immune response to recombinant adenoviruses is the most significant impediment to their clinical use for immunization. We test the hypothesis that specific virus-antibody combinations dictate the type of immune response generated against the adenovirus and its transgene cassette under certain physiological conditions while minimizing vector-induced toxicity. In vitro and in vivo assays were used to characterize the transduction efficiency, the T and B cell responses to the encoded transgene, and the toxicity of 1 x 10(11) adenovirus particles mixed with different concentrations of neutralizing antibodies. Complexes formed at concentrations of 500 to 0.05 times the 50% neutralizing dose (ND(50)) elicited strong virus-and transgene-specific T cell responses. The 0.05-ND(50) formulation elicited measurable anti-transgene antibodies that were similar to those of virus alone (P = 0.07). This preparation also elicited very strong transgene-specific memory T cell responses (28.6 +/- 5.2% proliferation versus 7.7 +/- 1.4% for virus alone). Preexisting immunity significantly reduced all responses elicited by these formulations. Although lower concentrations (0.005 and 0.0005 ND(50)) of antibody did not improve cellular and humoral responses in naive animals, they did promote strong cellular (0.005 ND(50)) and humoral (0.0005 ND(50)) responses in mice with preexisting immunity. Some virus-antibody complexes may improve the potency of adenovirus-based vaccines in naive individuals, while others can sway the immune response in those with preexisting immunity. Additional studies with these and other virus-antibody ratios may be useful to predict and model the type of immune responses generated against a transgene in those with different levels of exposure to adenovirus.Immunobiosciences, Inc.Cellular and Molecular Biolog
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Development and optimization of pH-responsive oral delivery systems for protein therapeutics
No full textA pH-responsive anionic linear polymer was synthesized via controlled radical polymerization using reversible addition-fragmentation chain transfer (RAFT) polymerization. The linear polymer was used to form pH-responsive self-assembled nanoparticles via the nanoprecipitation technique capable of loading antibodies with high efficiency at high weight loading percentages. The linear polymer system and the nanoprecipitation process were optimized using a Design of Experiments approach, resulting in a system capable of maintaining high antibody loading while exhibiting favorable release characteristics in simulated gastric, intestinal, and circulatory conditions. The optimized polymer and nanoparticle system was able to achieve a loading efficiency of 94% with a final weight loading of 32.9% protein compared to polymer. This optimized system showed minimal release in simulated gastric and intestinal conditions, while exhibiting high release at physiological pH, which is ideal for a nanocarrier designed to transit the intestinal epithelium before releasing its payload.
The optimized polymer and nanoparticle system were then evaluated in a series of in vitro models to determine their cytocompatibility and transport capabilities. The linear polymer showed no cytotoxicity at concentrations up to 1 mg/mL in two model cell lines using two different assays, which is higher than any concentration likely to be achieved in vivo. Nanoparticles were conjugated with Fc ligands to evaluate if targeting the neonatal Fc receptor (FcRn) on intestinal enterocytes could enhance transport of nanoparticles across the epithelial cell barrier. The nanoparticles were then evaluated in a transepithelial transport model using Caco-2 and HT29-MTX model cell lines. Transepithelial transport was analyzed using an ELISA assay as well as confocal microscopy of fluorescently labeled antibodies. Nanoparticles conjugated with Fc ligands resulted in transepithelial protein transport up to 25x higher than unconjugated nanoparticles or unencapsulated protein based on ELISA results. Confocal microscopy proved inconclusive in differentiating transport between groups, likely due to fluorescence quenching of the FITC probe in acidic intracellular vesicles combined with high levels of background fluorescence.
Finally, cell penetrating peptides (CPPs) were evaluated as an alternative means of increasing transepithelial transport of proteins. CPPs were evaluated in a pH-responsive complexation hydrogel system delivering insulin. The crosslinking density and crosslinker type were found to have a strong effect on the loading and release characteristics of CPP-insulin dual loaded microparticles. In an in situ closed-loop intestinal model in rats, the CPP L-PenetraMax showed a promising ability to increase transepithelial transport of insulin, resulting in a decrease in blood glucose levels compared to microparticles loaded with insulin alone. Future work will likely benefit from a combination of CPPs with Fc-conjugated nanoparticle delivery systems.Chemical Engineerin
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Itaconic acid-based pH-responsive nanoscale hydrogels for the oral delivery of high isoelectric point proteins
No full textThe field of protein-based therapeutics is rapidly expanding and will be of high importance in the years to come due to their high specificity and ability to target pathological markers. This class of therapeutic agents is currently only available to patients via injection, which is not considered to be a patient-friendly method of administration. Oral delivery offers multiple advantages over parenteral administration, but there are multiple obstacles that these systems have to overcome in order to deliver active protein across the intestinal epithelium into the bloodstream. Environmentally responsive polymer carriers have been explored for the development of an oral delivery platform for protein drugs. These pH-responsive carriers rely on charge interactions for the complexation behavior that facilitates protein loading and release. This characteristic presents a difficulty with proteins that exhibit a high isoelectric point, as the protein is likely to remain bound to the anionic hydrogel carrier at the pH of the small intestine instead of being repelled. Thus, the goal of this project was to develop a platform that will facilitate the delivery of high isoelectric point protein drugs, such as interferon beta, which is used to treat multiple sclerosis. This work was approached in three main aims: first, the optimization of itaconic acid-based copolymeric nanogels for the loading and release of high isoelectric point proteins. Second, protein loading and release from these optimized nanogels was evaluated. Third, the copolymeric nanoparticle system was assessed in vitro to determine cytocompatibility and protein transport was measured using intestinal epithelial models grown on Transwell supports. The development of these technologies will help in the future design of the new-generation of delivery vehicles for high isoelectric point protein therapeutics. The work combined fundamental research comprised of the rational design of novel copolymeric nanoparticles with the practical platform application in the oral delivery of therapeutic proteins. Due to the lack of progress made thus far in the oral delivery of these proteins, this research has the potential to greatly impact the scientific community.Chemical Engineerin
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A mechanistic study of how hepatic cytochrome P450 3A4 is regulated during infectious and non-infectious conditions
No full textIt has been established that adenovirus infection alters the expression and function of hepatic cytochrome P450 3A (CYP3A) for 14 days in several animal models. To determine the mechanism behind adenovirus-mediated alterations in CYP3A4 and if this effect was relevant to humans, we characterized a human hepatocyte cell line, HC-04, as an in vitro model to study virus-mediated changes in human CYP3A4. Similar to in vivo observations, infection with a first-generation recombinant adenovirus (AdlacZ) significantly suppressed CYP3A4 catalytic activity in an isoform-specific manner in this cell line. HC-04 cells were subsequently used to determine how vaccines to be used in pandemic outbreaks impact CYP3A4 expression and function. Infection with a recombinant adenovirus-based Ebola vaccine or influenza A virus subtypes H1N1 and H3N2 significantly suppressed human hepatic CYP3A4 catalytic activity by 34%, 91%, and 90%, respectively. This effect is important to understand as these vaccines are given to people of all ages. Mechanistic study of adenovirus infection in HC-04 cells indicated that engagement of integrin receptors is key in the initiation of processes responsible for changes in CYP3A4 during infection. Mice infected with AdlacZ experienced a 70% reduction in CYP3A activity 24 hours after infection. While infection with a mutant virus with integrin-binding arginine-glycine-aspartic acid (RGD) sequences deleted from the viral capsid (AdΔRGD) did not alter CYP3A activity at the same timepoint. CYP3A mRNA and protein levels in AdlacZ-treated animals were also suppressed, whereas those of mice given AdΔRGD were similar to uninfected control mice. Silencing of the integrin β-subunit reverted adenovirus-mediated CYP3A4 suppression in vitro. Silencing of the α-subunit did not. This led us to believe that interactions with the -tail of the integrin receptor is important for virus-mediated suppression of CYP3A4. Talin-1, a cytoplasmic protein that specifically binds to the -tail of integrins therefore became the focus of remaining studies. Knockdown of talin-1 increased CYP3A4 catalytic activity by 190%. Genes associated with post-translational modifications (PTMs) were downregulated in talin-silenced samples and analysis during viral infection revealed a significant increase in PTMs of CYP3A4. These initial experiments indicate a role of talin-1 and PTMs in the regulation of CYP3A4, however further experiments are needed to confirm these findings. Understanding changes in CYP3A4 during wild-type infection and immunization is clinically important, especially for low metabolizers. In this population, suppression of CYP3A4 could lead to accumulation of drugs that normally would be metabolized and cleared via this pathway, leading to serious therapeutic failure or adverse effects.Pharmaceutical Science
Software Design and Implementation of a UNIX Based Graphics System
No full textiv, 206 p.The author describes his internship as a Programmer/Analyst at u.s. Windpower Inc., the nation’s largest windmill producer and his work on the design, implementation, and testing of a UNIX-based graphics system named AUTOGRAPH.Power Plant Systems Development. U.S. Windpower, Inc. Burlington, Massachusetts.Analytic Studies. U.S. Windpower, Inc. Burlington, Massachusetts
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Development and characterization of thermostable thin films as a novel vaccine dosage form
No full textThermostabilization of vaccines can significantly simplify vaccine storage and distribution processes, eliminating the need for cold-chain maintenance, and resulting in global access to life-saving vaccines. Despite this benefit, all approved vaccines for use by the Food and Drug Administration must be refrigerated for long term storage in order to guarantee potency. The first study described in this thesis demonstrated that formulation of live adenovirus in the novel thin film matrix protects the virus from degradation at 4°C and 20°C for a minimum of three months, as well as 14 days at 37°C and 5 days at 40°C. The film matrix protected virus through 16 freeze-thaw cycles as well. As formulations prepared with surfactant outperformed those without it, the second study was designed to characterize and evaluate the intermolecular interactions between the surfactant and adenovirus capsids. in order to better understand the surfactants contribution to stability. The data suggested that surfactant stabilizes adenovirus by preventing aggregation of capsids via electrostatic and hydrophobic interactions. Additionally, the other formulation components in our multi-component preparation mitigates the interactions between adenovirus and the surfactant without interfering with stability. Lastly, the principles of surfactant stabilization were applied to the identification of alternative excipients for stabilization
of a virus with different properties from adenovirus, H1N1 influenza. The third study evaluated the ability of the thin film platform to induce an immune response and the impact of a natural adjuvants on the cytokine response and bioavailability of the vaccine dose. A preliminary screen demonstrated that vaccination with the thin film platform resulted in a stronger humoral response following mucosal vaccination than with traditional intramuscular vaccination. Additionally, the optimized formulation improved bioavailability of the viral dose across human buccal explants. Further characterization of the immune response also revealed that sublingual routes induced a strong TH1 polarized immune response which resulted in greater protective efficacy than intramuscular immunization. Taken together, these studies identified a novel thin film platform capable of stabilizing adenovirus at ambient temperatures, provide key insights into viral stabilization in the novel thin film platform, and illustrate the utility of the thin film as mucosal vaccine dosage form.Pharmaceutical Science
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