Drug delivery of aminolevulinic acid from topical formulations intended for photodynamic therapy

Topical photodynamic therapy is used for a variety of malignant and pre-malignant skin disorders, including Bowen's Disease and Superficial Basal Cell Carcinoma. A haem precursor, typically 5-aminolevulinic acid (ALA), acting as a prodrug, is absorbed and converted by the haem biosynthetic pathway to photoactive protoprophyrin IX (PpIX), which accumulates preferentially in rapidly dividing cells. Cell destruction occurs when PpIX is activated by an intense light source of appropriate wavelength. Topical delivery of ALA avoids the prolonged photosensitivity reactions associated with systemic administration of photosensitisers but its clinical utility is influenced by the tissue penetration characteristics of the drug, its ease of application and the stability of the active agent in the applied dose. This review, therefore, focuses on drug delivery applications for topical, ALA-based PDT. Issues considered in detail include physical and chemical enhancement strategies for tissue penetration of ALA and subsequent intracellular accumulation of PpIX, together with formulation strategies and drug delivery design solutions appropriate to various clinical applications. The fundamental aspects of drug diffusion in relation to the physicochemical properties of ALA are reviewed and specific consideration is given to the degradation pathways of ALA in formulated systems that, in turn, influence the design of stable topical formulations.     

A phase I, randomized study of combined IL-2 and therapeutic immunisation with antiretroviral therapy

Background  

Fully functional HIV-1-specific CD8 and CD4 effector T-cell responses are vital to the containment of viral activity and disease progression. These responses are lacking in HIV-1-infected patients with progressive disease. We attempted to augment fully functional HIV-1-specific CD8 and CD4 effector T-cell responses in patients with advanced chronic HIV-1 infection.     

ZiCo: a peptide designed to switch folded state upon binding zinc

We describe a novel approach to the design of a metal-triggered conformational switch. Specifically, two distinct protein-folding motifs were merged into one polypeptide sequence. The target structures were an alpha-helical coiled-coil trimer and zinc-bound monomer. Solution-phase spectroscopic, sedimentation, and binding studies confirmed the key aspects of the design. Both forms of the peptide were cooperatively folded, and the switch between them was reversible. This design process potentially presents a novel route to peptide-based biosensors.     

Electrostatic control of thickness and stiffness in a designed protein fiber

Attempts to design peptide-based fibers from first principles test our understanding of protein folding and assembly, and potentially provide routes to new biomaterials. Several groups have presented such designs based on alpha-helical and beta-strand building blocks. A key issue is this area now is engineering and controlling fiber morphology and related properties. Previously, we have reported the design and characterization of a self-assembling peptide fiber (SAF) system based on alpha-helical coiled-coil building blocks. With preceding designs, the SAFs are thickened, highly ordered structures in which many coiled coils are tightly bundled. As a result, the fibers behave as rigid rods. Here we report successful attempts to design new fibers that are thinner and more flexible by further programming at the amino-acid sequence level. This was done by introducing extended, or "smeared", electrostatic networks of arginine and glutamate residues to the surfaces of the coiled-coil building blocks. Furthermore, using arginine--rather than lysine--in these networks plays a major role in the fiber assembly, presumably by facilitating multidentate intra and intercoiled-coil salt bridges.     

Controlling the Assembly of Coiled–Coil Peptide Nanotubes

An ability to control the assembly of peptide nanotubes (PNTs) would provide biomaterials for applications in nanotechnology and synthetic biology. Recently, we presented a modular design for PNTs using α‐helical barrels with tunable internal cavities as building blocks. These first‐generation designs thicken beyond single PNTs. Herein we describe strategies for controlling this lateral association, and also for the longitudinal assembly. We show that PNT thickening is pH sensitive, and can be reversed under acidic conditions. Based on this, repulsive charge interactions are engineered into the building blocks leading to the assembly of single PNTs at neutral pH. The building blocks are modified further to produce covalently linked PNTs via native chemical ligation, rendering ca. 100 nm‐long nanotubes. Finally, we show that small molecules can be sequestered within the interior lumens of single PNTs.     

Coiled coils 9-to-5: rational de novo design of α-helical barrels with tunable oligomeric states

The rational design of linear peptides that assemble controllably and predictably in water is challenging. Short sequences must encode unique target structures and avoid alternative states. However, the non-covalent forces that stabilize and discriminate between states are weak. Nonetheless, for α-helical coiled-coil assemblies considerable progress has been made in rational de novo design. In these, sequence repeats of nominally hydrophobic (h) and polar (p) residues, hpphppp, direct the assembly of amphipathic helices into dimeric to tetrameric bundles. Expanding this pattern to hpphhph can produce larger α-helical barrels. Here, we show that pentameric to nonameric barrels are accessed by varying the residue at one of the h sites. In peptides with four L/I–K–E–I–A–x–Z repeats, decreasing the size of Z from threonine to serine to alanine to glycine gives progressively larger oligomers. X-ray crystal structures of the resulting α-helical barrels rationalize this: side chains at Z point directly into the helical interfaces, and smaller residues allow closer helix contacts and larger assemblies.

Systematic de novo design of peptides that form α-helical barrels with functionalisable central channels with a range of internal diameters.     

The d'--d--d' vertical triad is less discriminating than the a'--a--a' vertical triad in the antiparallel coiled-coil dimer motif

Elucidating relationships between the amino-acid sequences of proteins and their three-dimensional structures, and uncovering non-covalent interactions that underlie polypeptide folding, are major goals in protein science. One approach toward these goals is to study interactions between selected residues, or among constellations of residues, in small folding motifs. The α-helical coiled coil has served as a platform for such studies because this folding unit is relatively simple in terms of both sequence and structure. Amino acid side chains at the helix-helix interface of a coiled coil participate in so-called "knobs-into-holes" (KIH) packing whereby a side chain (the knob) on one helix inserts into a space (the hole) generated by four side chains on a partner helix. The vast majority of sequence-stability studies on coiled-coil dimers have focused on lateral interactions within these KIH arrangements, for example, between an a position on one helix and an a' position of the partner in a parallel coiled-coil dimer, or between a--d' pairs in an antiparallel dimer. More recently, it has been shown that vertical triads (specifically, a'--a--a' triads) in antiparallel dimers exert a significant impact on pairing preferences. This observation provides impetus for analysis of other complex networks of side-chain interactions at the helix-helix interface. Here, we describe a combination of experimental and bioinformatics studies that show that d'--d--d' triads have much less impact on pairing preference than do a'--a--a' triads in a small, designed antiparallel coiled-coil dimer. However, the influence of the d'--d--d' triad depends on the lateral a'--d interaction. Taken together, these results strengthen the emerging understanding that simple pairwise interactions are not sufficient to describe side-chain interactions and overall stability in antiparallel coiled-coil dimers; higher-order interactions must be considered as well.