Enhancing pharmaceutical formulations to improve efficacy and delivery of drug molecules

Thesis (Ph. D. in Biological Chemistry)--Massachusetts Institute of Technology, Dept. of Chemistry, 2013.Cataloged from PDF version of thesis.Includes bibliographical references.Major impediments to the full utility of current and potential drugs include issues of resistance and delivery. To address these challenges, in this thesis two directions of research were pursued: (1) the use of multivalent polymeric inhibitors to overcome drug resistance in human and avian influenza and (2) low-viscosity, high-concentration protein suspensions for therapeutic antibody, in particular monoclonal antibody (MAb), delivery. (1) Influenza resistance to small molecule neuraminidase (NA) inhibitors is spreading. Little emphasis, however, has been placed on alternative formulations of inhibitors. We investigated the design of multivalent antivirals, wherein small molecule ligands of viral proteins are conjugated via a linker to a linear polymeric backbone. Unexpectedly, we found that a poly-L-glutamine bearing pendant zanamivir (ZA) groups is at least as potent as those containing both ZA and sialic acid (SA). By examining the structure-activity relationship of such monofunctional conjugates, we show that the most potent one has 10% ZA attached to a neutral, high molecular weight backbone through a short alkyl linker. Importantly, we also demonstrate that such a polymer conjugate entirely compensates for weakened binding in and has 2,000-fold enhanced anti-viral potency against, ZA-resistant strains. We further evaluated this optimized inhibitor in vivo and observed that it is an effective therapeutic of established infection in ferrets and reduces viral titers up to 190-fold when used as a combined prophylactic/therapeutic in mice. Additionally, we see no evidence that the conjugate stimulates an immune response in mice upon repeat administration. (2) Typically, high doses of MAb therapeutics are required for clinical effect. Ideally, these MAbs would be delivered by subcutaneous injection of a small liquid volume. Such highly concentrated MAb solutions, however, are far more viscous than the 50 centipose (cP) permitted by the FDA. We evaluated approaches to reduce formulation viscosity by forming protein suspensions. Aqueous suspensions induced by poly(ethylene glycol), precipitating salts, or ethanol actually increased viscosity. However, non-aqueous suspensions of amorphous antibody powders in organic solvents that have s 1 hydrogen atom available for hydrogen-bonding, exhibited up to a 38-fold decrease in viscosity.by Alisha K. Weight.Ph.D.in Biological Chemistr

Attaching zanamivir to a polymer markedly enhances its activity against drug-resistant strains of influenza a virus

Effects of the commercial drug zanamivir (Relenza™) covalently attached to poly-l-glutamine on the infectivity of influenza A viruses are examined using the plaque reduction assay and binding affinity to viral neuraminidase (NA). These multivalent drug conjugates exhibit (i) up to a 20,000-fold improvement in anti-influenza potency compared with the zanamivir parent against human and avian viral strains, including both wild-type and drug-resistant mutants, and (ii) superior neuraminidase (NA) inhibition constants, especially for the mutants. These findings provide a basis for exploring polymer-attached inhibitors as more efficacious therapeutics, particularly against drug-resistant influenza strains.National Institutes of Health (U.S.) (Grant Number U01-AI074443)Fundación Ramón Areces. Postdoctoral Fellowshi

Non-Aqueous Suspensions of Antibodies are Much Less Viscous Than Equally Concentrated Aqueous Solutions

Purpose: The aim of this study was to markedly lower the viscosities of highly concentrated protein, in particular antibody, formulations. An effective approach elaborated herein for γ-globulin and a monoclonal antibody is to replace aqueous solutions with equimolar suspensions in neat organic solvents. Methods: Viscosities of aqueous solutions and non-aqueous suspensions of the model protein bovine γ-globulin and a murine monoclonal antibody were examined under a variety of experimental conditions. In addition, protein particle sizes were measured using dynamic light scattering and light microscopy. Results: Concentrated suspensions of amorphous γ-globulin powders (up to 300 mg/mL, composed of multi-micron-sized particles) in absolute ethanol and a number of other organic solvents were found to have viscosities up to 38 times lower than the corresponding aqueous solutions. Monoclonal antibody follows the same general trend. Additionally, the higher the protein concentration and lower the temperature, the greater the viscosity benefit of a suspension over a solution. Conclusions: The viscosities of concentrated γ-globulin and monoclonal antibody suspensions in organic solvents are drastically reduced compared to the corresponding aqueous solutions; the magnitude of this reduction depends on the solvent, particularly its hydrogen-bonding properties.Sanofi Aventis (Firm)Massachusetts Institute of Technology. Biophysical Instrumentation Facility (Study of Complex Macromolecular Systems (NSF-0070319))Massachusetts Institute of Technology. Biophysical Instrumentation Facility (Study of Complex Macromolecular Systems (NIH GM68762 instrumentation grant