Steve Brocchini

Poly(2-methacryloyloxyethyl phosphorylcholine) for Protein Conjugation

The water-soluble, biocompatible polymer poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) was evaluated for protein conjugation. PMPC is a zwitterionic polymer that is able to form a more compact conformation in aqueous solution than poly(ethylene glycol) (PEG). While a terminally functionalized N-hydroxysuccinimide derivative of PMPC was not efficient for conjugation to an amine moiety on interferon-alpha2a (IFN), we found that a bis-thiol specific derivative of PMPC could be conjugated after reduction of the disulfide bonds in IFN. Utilizing PMPC that displayed a similar hydrodynamic volume to 20 kDa PEG, we evaluated the in vitro antiviral and antiproliferative activity and pharmacokinetics of a PMPC-IFN conjugate. As a hygroscopic zwitterionic polymer, PMPC is able to form a compact conformation in aqueous solution, which was found to be more compact than PEG. This suggests that PMPC protein conjugates may display different plasma elimination characteristics than PEG protein conjugates. PMPC-IFN displayed marked resistance to antibody binding in Western blot analysis with a polyclonal anti-IFN antibody while displaying comparable in vitro antiviral and antiproliferative activity to PEG-IFN. During an in vivo pharmacokinetic study, the absorption t(1/2) for PMPC-IFN was considerably extended compared to the native IFN and 20 kDa PEG analogue. This is also consistent with the SDS-PAGE result where an apparent reduction in mobility through a hydrated medium was observed. The elimination t(1/2) was also vastly extended over the native IFN and twice the value of 20 kDa PEG-IFN. This suggests that tissue migration of PMPC-IFN occurs more slowly than the 20 kDa PEG-IFN despite their similarity in hydrodynamic volume, leading to an an improved depot effect, which could explain the longer elimination t(1/2). In this study, we demonstrate a potential use of PMPCylation as a novel tool for enhancing the pharmacokinetic profile of therapeutic proteins in ways that complement PEGylation.

Cancer immunotherapy: nanodelivery approaches for immune cell targeting and tracking

Cancer is one of the most common diseases afflicting people globally. New therapeutic approaches are needed due to the complexity of cancer as a disease. Many current treatments are very toxic and have modest efficacy at best. Increased understanding of tumor biology and immunology has allowed the development of specific immunotherapies with minimal toxicity. It is important to highlight the performance of monoclonal antibodies, immune adjuvants, vaccines and cell-based treatments. Although these approaches have shown varying degrees of clinical efficacy, they illustrate the potential to develop new strategies. Targeted immunotherapy is being explored to overcome the heterogeneity of malignant cells and the immune suppression induced by both the tumor and its microenvironment. Nanodelivery strategies seek to minimize systemic exposure to target therapy to malignant tissue and cells. Intracellular penetration has been examined through the use of functionalized particulates. These nano-particulate associated medicines are being developed for use in imaging, diagnostics and cancer targeting. Although nano-particulates are inherently complex medicines, the ability to confer, at least in principle, different types of functionality allows for the plausible consideration these nanodelivery strategies can be exploited for use as combination medicines. The development of targeted nanodelivery systems in which therapeutic and imaging agents are merged into a single platform is an attractive strategy. Currently, several nanoplatform-based formulations, such as polymeric nanoparticles, micelles, liposomes and dendrimers are in preclinical and clinical stages of development. Herein, nanodelivery strategies presently investigated for cancer immunotherapy, cancer targeting mechanisms and nanocarrier functionalization methods will be described. We also intend to discuss the emerging nano-based approaches suitable to be used as imaging techniques and as cancer treatment options.

A New Reagent for Stable Thiol-Specific Conjugation

Many clinically used protein therapeutics are modified to increase their efficacy. Example modifications include the conjugation of cytotoxic drugs to monoclonal antibodies or poly(ethylene glycol) (PEG) to proteins and peptides. Monothiol-specific conjugation can be efficient and is often accomplished using maleimide-based reagents. However, maleimide derived conjugates are known to be susceptible to exchange reactions with endogenous proteins. To address this limitation in stability, we have developed PEG-mono-sulfone 3, which is a latently reactive, monothiol selective conjugation reagent. Comparative reactions with PEG-maleimide and other common thiol-selective PEGylation reagents including vinyl sulfone, acrylate, and halo-acetamides show that PEG-mono-sulfone 3 undergoes more efficient conjugation under mild reaction conditions. Due to the latent reactivity of PEG-mono-sulfone 3, its reactivity can be tailored and, once conjugated, the electron-withdrawing ketone is easily reduced under mild conditions to prevent undesirable deconjugation and exchange reactions from occurring. We describe a comparative stability study demonstrating a PEG-maleimide conjugate to be more labile to deconjugation than the corresponding conjugate obtained using PEG-mono-sulfone 3.

Molecular Modeling to Study Dendrimers for Biomedical Applications

Molecular modeling techniques provide a powerful tool to study the properties of molecules and their interactions at the molecular level. The use of computational techniques to predict interaction patterns and molecular properties can inform the design of drug delivery systems and therapeutic agents. Dendrimers are hyperbranched macromolecular structures that comprise repetitive building blocks and have defined architecture and functionality. Their unique structural features can be exploited to design novel carriers for both therapeutic and diagnostic agents. Many studies have been performed to iteratively optimise the properties of dendrimers in solution as well as their interaction with drugs, nucleic acids, proteins and lipid membranes. Key features including dendrimer size and surface have been revealed that can be modified to increase their performance as drug carriers. Computational studies have supported experimental work by providing valuable insights about dendrimer structure and possible molecular interactions at the molecular level. The progress in computational simulation techniques and models provides a basis to improve our ability to better predict and understand the biological activities and interactions of dendrimers. This review will focus on the use of molecular modeling tools for the study and design of dendrimers, with particular emphasis on the efforts that have been made to improve the efficacy of this class of molecules in biomedical applications.

The PK-Eye: A Novel In Vitro Ocular Flow Model for Use in Preclinical Drug Development

A 2-compartment in vitro eye flow model has been developed to estimate ocular drug clearance by the anterior aqueous outflow pathway. The model is designed to accelerate the development of longer-acting ophthalmic therapeutics. Dye studies show aqueous flow is necessary for a molecule injected into the vitreous cavity to clear from the model. The clearance times of proteins can be estimated by collecting the aqueous outflow, which was first conducted with bevacizumab using phosphate-buffered saline in the vitreous cavity. A simulated vitreous solution was then used and ranibizumab (0.5 mg) displayed a clearance time of 8.1 ± 3.1 days, which is comparable to that observed in humans. The model can estimate drug release from implants or the dissolution of suspensions as a first step in their clearance mechanism, which will be the rate-limiting step for the overall resident time of a candidate dosage form in the vitreous. A suspension of triamcinolone acetonide (Kenalog®) (4.0 mg) displayed clearance times spanning 26-28 days. These results indicate that the model can be used to determine in vitro-in vivo correlations in preclinical studies to develop long-lasting therapeutics to treat blinding diseases at the back of the eye.

Expression of soluble and active interferon consensus in SUMO fusion expression system in E. coli

Protein production can be improved if methods for soluble protein expression are developed. Interferon consensus (IFN-con) is used to treat hepatitis C. IFN-con has superior activity compared to other clinically used interferon α subtypes. However IFN-con is a challenging protein to produce in a soluble form using an Escherichia coli expression system. Here we describe the expression of soluble and active recombinant IFN-con in E. coli. The IFN-con gene sequence was optimised for expression in E. coli, which was then cloned into the Champion™ pET SUMO expression vector downstream of the SUMO fusion protein and under strong T7lac promoter. The SUMO-IFN-con fusion protein was efficiently expressed using the SHuffle™ E. coli strain and existed in soluble form as 86-88% of the total IFN-con. After removal of the SUMO fusion partner, approximately 50mg of recombinant IFN-con of at least 98% purity (by RP-HPLC) was obtained from a 1L fermentation culture. Using an A549/EMCV antiviral assay, the specific activity of the recombinant IFN-con was determined to be 960×10(6) IU/mg as calculated to NIBSC standard for IFN-con (3×10(5)pfu/mL virus titre). Comparison of the antiviral activity of the produced IFN-con to IFN α-2a showed that IFN-con displays 2.8 times greater activity, which is in good agreement with what has been reported in the literature for pure protein. IFN-con expression in a soluble form from E. coli allowed us to use a simple, two-step purification process to yield highly pure and active IFN-con which is more efficient than obtaining IFN-con from inclusion bodies.

Competitive Reactions During Amine Addition to cis-Aconityl Anhydride

Amine addition to cis-aconityl anhydride has been used to prepare monomers needed to make water-soluble polymers that are being developed for biomedical applications. Unfortunately competitive decarboxylation, double bond isomerization, and hydrolysis reactions of cis-aconityl anhydride have been observed during monomer synthesis. Since cis-aconityl anhydride has also been used for bioconjugation applications, these deleterious side reactions should be accounted for. Reactions and product profiles of cis-aconityl anhydride with amines and amino acids in aqueous and organic media are described. The side reactions could be avoided with weakly nucleophilic aromatic amines and by the interfacial reaction of glycine with cis-aconityl anhydride.

Characterisation of Ilomastat for Prolonged Ocular Drug Release

We are developing tablet dosage forms for implantation directly into the subconjunctival space of the eye. The matrix metalloproteinase inhibitor, ilomastat, has previously been shown to be efficacious at suppressing scarring following glaucoma filtration surgery (GFS). We report on the physical characterisation of ilomastat which is being developed for ocular implantation. Since ilomastat is being considered for implantation it is necessary to examine its polymorphs and their influence on aspects of the in vitro drug release profile. X-ray powder diffraction identified two polymorphs of ilomastat from different commercial batches of the compound. Tablets were prepared from the two different polymorphs. Isothermal perfusion calorimetry was used to show that amorphous content is not increased during tablet formulation. The melting points of the two polymorphs are 188 and 208°C as determined by differential scanning calorimetry. Utilising single crystal X-ray diffraction, the structural conformations and packing arrangements of the different polymorphs were determined. The orthorhombic crystal crystallised as a monohydrate while the second monoclinic crystal form is non-solvated. Ilomastat tablets prepared from the two different solid forms exhibited similar drug release profiles in vitro under conditions mimicking the aqueous composition, volume and flow of the subconjunctival space after GFS. This suggests that a reproducible dose at each time point during release after implantation should be achievable in vivo with ilomastat tablets prepared from the two polymorphs identified.

Solid-state protein formulations.

When formulated as liquid dosage forms, therapeutic proteins and peptides often show instability during handling as a result of chemical degradation. Solid formulations are frequently required to maintain protein stability during storage, transport and upon administration. Herein we highlight current strategies used to formulate pharmaceutical proteins in the solid form. An overview of the physical instabilities which can arise with proteins is first described. The key solidification techniques of crystallization, freeze-drying and particle forming technologies are then discussed. Examples of current commercial products that are formulated in the solid state are provided and include neutral protamine Hagedorn - insulin crystal suspensions, freeze-dried monoclonal antibodies and leuproride polylactide-co-glycolide microparticles. Finally, future perspectives in solid-state protein formulation are described.

Poly(methacrylic acid) complexation of amphotericin B to treat neglected diseases

Amphotericin B (AmB) is used to treat a neglected disease called visceral leishmaniasis (VL). We hypothesised that direct non-covalent association of AmB with poly(methacrylic acid) (PMAA) would replicate many of the properties of liposomal AmB (AmB-L), which is more efficacious than micellar AmB (AmB-D). Water-soluble AmB–PMAA complexes with AmB loadings ranging from ∼20 to 45% were reproducibly prepared. The AmB in the PMAA complex displayed similar aggregation properties to the AmB within AmB-L. The AmB–PMAA complex displayed low heamolytic properties while maintaining in vitro activity against Leishmania donovani amastigotes with no macrophage toxicity observed at an IC50 of 0.043 (±0.003) μM. AmB–PMAA complexes were well tolerated in vivo at a total dose of 6 mg kg−1 and both the complex (2.2 mg kg−1 AmB) and AmB-L (2.5 mg kg−1 AmB) achieved greater than 90% parasite inhibition in vivo after a single dose against L. donovani in HU3 infected BALB/c mice.