Timothy John Norman

Hepato-biliary and renal excretion in mice of charged and neutral gadolinium complexes of cyclic tetra-aza-phosphinic and carboxylic acids

Tissue distribution of 21 new 157/153Gd complexes was measured at 5 min and 24 hr after an intravenous injection into mice. A complex was judged to be stable in vivo when the percentage of 153Gd retained in the liver and skeleton at 24 hr was comparable with that of 153Gd(DOTA)-. Complexes varied in net charge and lipophilicity and 20 were phosphinic or carboxylic acid derivatives of tetra-aza-cyclo-dodecane. Three anionic, lipophilic complexes were cleared predominantly by the hepato-biliary pathway and were stable in vivo. The remaining 18 complexes were cleared mainly by the kidneys. Of these 18, 1 anionic, 8 neutral, and 3 cationic complexes were stable in vivo. These findings augur well for the future of hepato-biliary and general purpose Gd contrast enhancing agents for MRI.

Synthesis of charged and uncharged complexes of gadolinium and yttrium with cyclic polyazaphosphinic acid ligands for in vivo applications

The synthesis of 18 new macrocyclic complexing agents incorporating phosphinic acid (and carboxylic acid) groups is reported, based on the 1,4,7,10-tetraazacyclododecane ring. Through selective functionalisation of one ring nitrogen or by changing the nature of the P-substituent, anionic, neutral and cationic complexes of yttrium and gadolinium may be prepared of varying lipophilicity. Diamagnetic complexes have been characterised by 1H, 31P and 89Y NMR spectroscopy, and the rate of uptake of 90Y of selected ligands compared. The kinetics of dissociation of nine gadolinium complexes has been measured in the pH range 1–2 using 153Gd-labelled complexes. Charge-neutral complexes dissociate more slowly than their anionic analogues, and the phosphinate complexes, although slightly less stable than their carboxylate analogues, are nevertheless sufficiently kinetically inert for in vivo applications.

Nuclear magnetic resonance, luminescence and structural studies of lanthanide complexes with octadentate macrocyclic ligands bearing benzylphosphinate groups

The solution and solid-state structures of lanthanide complexes of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetryltetramethylenetetra(benzylphosphinate), L1a, and of its o-, m- and p-methoxybenzyl analogues (L2, L3, L4) have been investigated by NMR, relaxometry, crystallographic and photophysical methods. It has been shown that the number of proximate water molecules is dependent on the size of the bound lanthanide ion: the complex [LaL1a]– is nine-co-ordinate and adopts a twisted square-antiprismatic structure with one bound water molecule. The analogous [YL1a]–, [YbL1a]–, [GdL1a]– and [EuL1a]– complexes also adopt a twisted square-antiprismatic geometry, but are eight-co-ordinate, with no metal-bound water. Relaxivity studies with the gadolinium complexes showed that they are purely ‘outer-sphere’ contrast agents, but they associate strongly with proteins leading to a pronounced relaxivity enhancement. Detailed biodistribution studies with [GdL1a]– revealed avid biliary uptake at low doses and a well defined tendency for the complex to be cleared more slowly from tumour tissue, allowing tissue differentiation.

Towards selective DNA targeting: synthesis of an antibody–macrocycle–intercalator conjugate

The synthesis is reported of a trivalent conjugate incorporating a macrocyclic bifunctional complexing agent, an engineered human monoclonal antibody fragment and an established DNA intercalator.

Synthesis and biodistribution of 111In, 67Ga and 153Gd-radiolabelled conjugates of nitroimidazoles with bifunctional complexing agents: imaging agents for hypoxic tissue?

Abstract Conjugates of the putative hypoxia-selective agent 2-nitroimidazole with bifunctional complexing agents have been prepared and radiolabelled with 111In, 67Ga or 153Gd. Although some evidence for tumour localisation was found control experiments revealed that the gallium and indium labelled complexes of the simple ligand 1,4,7-triazacyclononane-triacetate are themselves promising tumour-imaging agents.

Improved tumour targeting with recombinant antibody–macrocycle conjugates

Linkage of a macrocyclic complexing agent to a spaced tri-maleimide allows formation of a recombinant trivalent antibody by reaction with a Δ-Cys Fab fragment of an engineered human antibody.

Stable anionic, neutral and cationic complexes of gadolinium with functionalised amino-phosphinic acid macrocyclic ligands

The synthesis and stability of anionic, neutral and cationic gadolinium complexes based on tetraazaphosphinic acid ligands is compared: lipophilic anionic complexes show biliary rather than renal clearance.

Insight into small molecule binding to the neonatal Fc receptor by X-ray crystallography and 100 kHz magic-angle-spinning NMR.

Aiming at the design of an allosteric modulator of the neonatal Fc receptor (FcRn)–Immunoglobulin G (IgG) interaction, we developed a new methodology including NMR fragment screening, X-ray crystallography, and magic-angle-spinning (MAS) NMR at 100 kHz after sedimentation, exploiting very fast spinning of the nondeuterated soluble 42 kDa receptor construct to obtain resolved proton-detected 2D and 3D NMR spectra. FcRn plays a crucial role in regulation of IgG and serum albumin catabolism. It is a clinically validated drug target for the treatment of autoimmune diseases caused by pathogenic antibodies via the inhibition of its interaction with IgG. We herein present the discovery of a small molecule that binds into a conserved cavity of the heterodimeric, extracellular domain composed of an α-chain and β2-microglobulin (β2m) (FcRnECD, 373 residues). X-ray crystallography was used alongside NMR at 100 kHz MAS with sedimented soluble protein to explore possibilities for refining the compound as an allosteric modulator. Proton-detected MAS NMR experiments on fully protonated [13C,15N]-labeled FcRnECD yielded ligand-induced chemical-shift perturbations (CSPs) for residues in the binding pocket and allosteric changes close to the interface of the two receptor heterodimers present in the asymmetric unit as well as potentially in the albumin interaction site. X-ray structures with and without ligand suggest the need for an optimized ligand to displace the α-chain with respect to β2m, both of which participate in the FcRnECD–IgG interaction site. Our investigation establishes a method to characterize structurally small molecule binding to nondeuterated large proteins by NMR, even in their glycosylated form, which may prove highly valuable for structure-based drug discovery campaigns.