David J. Brayden

Advances in PEGylation of important biotech molecules: delivery aspects

Background: Although various injected peptide and protein therapeutics have been developed successfully over the past 25 years, several pharmacokinetic and immunological challenges are still encountered that can limit the efficacy of both novel and established biotech molecules. Objective and method: PEGylation is a popular technique to address such properties. PEGylated drugs exhibit prolonged half-life, higher stability, water solubility, lower immunogenicity and antigenicity, as well as potential for specific cell targeting. Although PEGylated drug conjugates have been on the market for many years, the technology has steadily developed in respect of site-specific chemistry, chain length, molecular weights and purity of conjugate. These developments have occurred in parallel to improvements in physicochemical methods of characterization. Conclusion: This review will discuss recent achievements in PEGylation processes with an emphasis on novel PEG-drugs constructs, the unrealized potential of PEGylation for non-injected routes of delivery, and also on PEGylated versions of polymeric nanoparticles, including dendrimers and liposomes.

Binding and uptake of biodegradable poly-DL-lactide micro- and nanoparticles in intestinal epithelia

The use of biodegradable particles as oral delivery vehicles for macromolecular drugs was investigated. We evaluated the binding, uptake and absorption of poly-dl-lactide (PLA) micro- and nanoparticles in Caco-2 monolayers and in ileal tissue and gut associated lymphoid tissue (GALT) of anaesthetised rats and rabbits. Using a range of experimental techniques, we found that approximately 10% of administered micro- and nanoparticles were adsorbed to the apical membranes of each of the five intestinal models. Nanoparticles were found to be absorbed better than microparticles. Overall, little discrimination in uptake patterns was evident between Peyers patch (PP) and non-PP tissue while rat ileum showed a greater uptake capacity than rabbit. Our results show that uptake of PLA particles was low capacity, size-dependent and predominantly transcellular in all systems. A low proportion of the apically-bound particles was absorbed, with uptake exclusion evident for particles >4microm. The affinity of PLA particles for intestinal epithelia and GALT needs to be greatly enhanced in order to achieve improved oral bioavailability of macromolecules.

Keynote review: intestinal Peyer's patch M cells and oral vaccine targeting.

Specialized M cells in the follicle-associated epithelium of intestinal Peyers patches serve as portals for diverse particulates. Following antigen handover to dome lymphocytes, a protective mucosal antibody secretion ensues. One approach to oral vaccine delivery is to mimic the entry pathways of pathogens via M cells. The paucity of human tissue for in vitro investigation has hampered the discovery of M-cell pathogen receptors; however an in vitro human M like-cell culture model displays many expected phenotypic features. Comparative studies using microarrays reveal several novel M-cell surface receptors that could be used to potentially target orally delivered antigens.

Safety and efficacy of sodium caprate in promoting oral drug absorption: from in vitro to the clinic.

A major challenge in oral drug delivery is the development of novel dosage forms to promote absorption of poorly permeable drugs across the intestinal epithelium. To date, no absorption promoter has been approved in a formulation specifically designed for oral delivery of Class III molecules. Promoters that are designated safe for human consumption have been licensed for use in a recently approved buccal insulin spray delivery system and also for many years as part of an ampicillin rectal suppository. Unlike buccal and rectal delivery, oral formulations containing absorption promoters have the additional technical hurdle whereby the promoter and payload must be co-released in high concentrations at the small intestinal epithelium in order to generate significant but rapidly reversible increases in permeability. An advanced promoter in the clinic is the medium chain fatty acid (MCFA), sodium caprate (C(10)), a compound already approved as a food additive. We discuss how it has evolved to a matrix tablet format suitable for administration to humans under the headings of mechanism of action at the cellular and tissue level as well as in vitro and in vivo efficacy and safety studies. In specific clinical examples, we review how C(10)-based formulations are being tested for oral delivery of bisphosphonates using Gastro Intestinal Permeation Enhancement Technology, GIPET (Merrion Pharmaceuticals, Ireland) and in a related solid dose format for antisense oligonucleotides (ISIS Pharmaceuticals, USA).

Protection against Bordetella pertussis infection following parenteral or oral immunization with antigens entrapped in biodegradable particles : Effect of formulation and route of immunization on induction of Th1 and Th2 cells

The immunogenicity and protective efficacy of systemically and orally delivered pertussis antigens entrapped in either microparticle poly-lactide-co-glycolide (PLG) or nanoparticle PLG formulations were evaluated in a murine respiratory challenge model for infection with Bordetella pertussis. The results demonstrate that immunization with two parenteral doses of 1 microg or three oral doses of 100 microg of pertussis toxoid (PTd) and filamentous haemagglutinin (FHA) encapsulated in PLG conferred a high level of protection against B. pertussis challenge. Furthermore protection could be generated with a single parenteral immunization with a combined microparticle and nanoparticle formulation. However, the route of immunization and the size of the particles affected the type of T cell response induced. Parenteral immunization with PTd and FHA entrapped in PLG microparticles elicits a potent type 1 T cell response and potent antibody response when given by the intraperitoneal (i.p.) or intramuscular (i.m.) route. In contrast, nanoparticle formulations favoured the induction of Th2 cells.

Targeting polymerised liposome vaccine carriers to intestinal M cells

Due to their transcytotic capability, intestinal M cells may represent an efficient potential route for oral vaccine delivery. We previously demonstrated that the lectin Ulex europaeus agglutinin 1 (UEA1, specific for alpha-L-fucose residues) selectively binds to mouse Peyers patch M cells and targets 0.5 microm polystyrene microparticles to these cells. Using a gut loop model we now demonstrate that covalently-membrane-bound UEA1 similarly targets polymerised liposomes (Orasomes, approximately 200 nm diameter), potential biocompatable oral vaccine delivery vehicles, to mouse M cells. Targeting was inhibited by alpha-L-fucose while the co-entrapped adjuvant, monophosphoryl Lipid A (MPL), failed to exert any detrimental effect on UEA1-mediated M cell targeting. Lectin-mediated M cell targeting may thus permit the efficacy of mucosal vaccines to be enhanced if cellular relationship between particle binding and immune outcome can be established.

Drug delivery to inflamed colon by nanoparticles: Comparison of different strategies

For inflammatory bowel disease (IBD) treatment, local delivery of molecules loaded in nanoparticles to the inflamed colon could be a promising strategy. The aim of this study was to investigate how drug-loaded polymeric nanoparticles target the site of inflammation and to analyse the influence of different colon-specific delivery strategies. Three different polymeric nanoparticles were formulated using ovalbumin (OVA) as a model drug. pH-sensitive nanoparticles were made with Eudragit(®) S100. Mucoadhesive nanoparticles were created with trimethylchitosan (TMC). A mix of polymers, PLGA, PEG-PLGA and PEG-PCL, were used to obtain a sustained drug delivery. Furthermore, ligands targeting immune cells (i.e. mannose) or the inflamed colon (i.e. a specific peptide) were grafted on the PEG chain of PCL. Interaction of nanoparticles with the intestinal epithelium was explored using Caco-2 monolayers designed to mimic an inflamed epithelium and then visualized using confocal laser microscopy. TMC nanoparticles had the highest apparent permeability for OVA in the untreated model. However, in the inflamed model, there were no difference between TMC, PLGA-based and Eudragit(®) nanoparticles. The uptake of nanoparticles in the inflamed mouse colon was assessed in a horizontal diffusion chamber. Mannose-grafted PLGA nanoparticles showed the highest accumulation of OVA in inflamed colon. Based on these results, active targeting of macrophages and dendritic cells may be a promising approach for targeting the colon in IBD.

Conjugation of salmon calcitonin to a combed-shaped end functionalized poly(poly(ethylene glycol) methyl ether methacrylate) yields a bioactive stable conjugate

Salmon calcitonin (sCT) was conjugated via its N-terminal cysteine to a comb-shaped end-functionalized poly(poly(ethylene glycol) methyl ether methacrylate) (PolyPEG, 6.5 kDa), and to linear PEG (5 kDa). Conjugate molecular weight and purity was assessed by SEC-HPLC and MALDI-TOF MS. Bioactivity of conjugates was measured by cyclic AMP assay in T47D cells. Calcium and calcitonin levels were measured in rats following intravenous injections. Stability of conjugates was tested against serine proteases, intestinal and liver homogenates and serum. Cytotoxicity of conjugates was assessed by lactate dehydrogenase (LDH) assay and by haemolytic assay of rat red blood cells. Results showed that the two conjugates were of high purity with molecular weights similar to predictions. Both conjugates retained more than 85% bioactivity in vitro and had nanomolar EC(50) values similar to sCT. While both sCT-PolyPEG(6.5 K) and sCT-PEG(5 K) were resistant to metabolism by serine proteases, homogenates and serum, PolyPEG (6.5 K) was more so. Although both conjugates reduced serum calcium to levels similar to those achieved with sCT, PolyPEG(6.5 K) extended the T(1/2) and AUC of serum sCT over values achieved with sCT-PEG and sCT itself. None of PolyPEG, PEG or methacrylic acid displayed significant cytotoxicity. PolyPEG may therefore have potential to improve pharmacokinetic profiles of injected peptides.

Direct Peptide Bioconjugation/PEGylation at Tyrosine with Linear and Branched Polymeric Diazonium Salts

Direct polymer conjugation at peptide tyrosine residues is described. In this study Tyr residues of both leucine enkephalin and salmon calcitonin (sCT) were targeted using appropriate diazonium salt-terminated linear monomethoxy poly(ethylene glycol)s (mPEGs) and poly(mPEG) methacrylate prepared by atom transfer radical polymerization. Judicious choice of the reaction conditions-pH, stoichiometry, and chemical structure of diazonium salt-led to a high degree of site-specificity in the conjugation reaction, even in the presence of competitive peptide amino acid targets such as histidine, lysines, and N-terminal amine. In vitro studies showed that conjugation of mPEG(2000) to sCT did not affect the peptides ability to increase intracellular cAMP induced in T47D human breast cancer cells bearing sCT receptors. Preliminary in vivo investigation showed preserved ability to reduce [Ca(2+)] plasma levels by mPEG(2000)-sCT conjugate in rat animal models.

Oral vaccination in man using antigens in particles: current status

Over 30 publications suggest that antigens given orally to mice in biocompatible microspheres stimulate an immune response and, in some cases, can give rise to protective immunity. Of those responses in mice that have been reproduced, confirmation in large animal models and in Phase 1 studies has not resulted. Particles containing antigens given orally and mixed with soluble adjuvants like cholera toxin have generally not produced any better data in mice than that seen with mixed solutions of unprotected antigens and adjuvants. Peyers patch M cell targeting of antigens in particles remains however a relatively untested hypothesis. While binding and uptake of M cell-targeted latex particles and stable liposomes by mouse M cells has been clearly shown using the mouse M cell-specific lectin, Ulex europaeus 1 (UEA-1), a direct relationship between M cell particle uptake and immune outcome remains illusive. Some studies have produced increased serum antibodies from UEA-1- and cholera toxin B (CTB)-coated liposomes containing antigens. Other groups are currently working on developing novel human M cell ligands for attachment to stable particles for oral delivery. Use of untargeted antigen-containing particles with adjuvants administered by the nasal route remains an alternative option.