Optimizing the bioavailability of small molecular optical imaging probes by conjugation to an albumin affinity tag.

Small molecular imaging probes are often found to be rapidly cleared from the circulation. In order to improve signal to noise ratio (SNR) by high probe accumulation in the target tissue we intended to prolong the presence of the probes in the circulation by exploiting inherent transport mechanisms. Human serum albumin (HSA) is playing an increasingly important role as a drug carrier in clinical settings and drugs directly bound to albumin or attached to albumin binding moieties have been successfully developed for treatment approaches. To optimize the bioavailability of existing fluorescent probes, a hydrophobic affinity tag is installed, which enhances albumin binding. In a first experiment an endothelin-A receptor (ETAR) probe is modified by inserting a trivalent linker, attaching an albumin affinity tag and labeling the conjugate with the fluorescent dye Cy 5.5. The spectroscopic properties of the conjugate are examined by photometer- and fluorometer measurements in comparison to a probe without albumin binding tag. Albumin binding was proven by agarose gel electrophoresis. The affinity towards ETAR was confirmed in vitro by cell binding assays on human fibrosarcoma cells (HT-1080) and in vivo by murine xenograft imaging studies. In vitro, the modified probe retains high target binding in the absence and presence of albumin. Binding could be blocked by predosing with ETAR antagonist atrasentan, proving specificity. The in vivo examinations in comparison to the established probe showed a reduced renal elimination and a prolonged circulation of the tracer resulting in significantly higher signal intensity (SI) at the target and a higher signal-to-noise ratio (SNR) between 3h and 96 h after injection. In summary, we designed a small molecular, non-peptidic fluorescent probe which targets ETAR and reversibly binds to serum albumins. The reversible binding to albumin enhances the biological half-life of the probe substantially and enables near infrared optical imaging of subcutaneous tumors for several days. This approach of reversibly attaching probes to serum albumin may serve as a tool to optimize tracer distribution for more precise target characterization in molecular imaging experiments.

Characterisation of the contribution of the GABA-benzodiazepine α1 receptor subtype to [(11)C]Ro15-4513 PET images.

This positron emission tomography (PET) study aimed to further define selectivity of [(11)C]Ro15-4513 binding to the GABARα5 relative to the GABARα1 benzodiazepine receptor subtype. The impact of zolpidem, a GABARα1-selective agonist, on [(11)C]Ro15-4513, which shows selectivity for GABARα5, and the nonselective benzodiazepine ligand [(11)C]flumazenil binding was assessed in humans. Compartmental modelling of the kinetics of [(11)C]Ro15-4513 time-activity curves was used to describe distribution volume (V(T)) differences in regions populated by different GABA receptor subtypes. Those with low α5 were best fitted by one-tissue compartment models; and those with high α5 required a more complex model. The heterogeneity between brain regions suggested spectral analysis as a more appropriate method to quantify binding as it does not a priori specify compartments. Spectral analysis revealed that zolpidem caused a significant V(T) decrease (~10%) in [(11)C]flumazenil, but no decrease in [(11)C]Ro15-4513 binding. Further analysis of [(11)C]Ro15-4513 kinetics revealed additional frequency components present in regions containing both α1 and α5 subtypes compared with those containing only α1. Zolpidem reduced one component (mean±s.d.: 71%±41%), presumed to reflect α1-subtype binding, but not another (13%±22%), presumed to reflect α5. The proposed method for [(11)C]Ro15-4513 analysis may allow more accurate selective binding assays and estimation of drug occupancy for other nonselective ligands.

Chronic administration of 2-(2-benzofuranyl)-2-imidazoline (2-BFI) induces region-specific increases in [3H]2-BFI binding to rat central imidazoline I2 sites.

Chronic administration of I(2) ligands increases the density of central I(2) sites as measured in brain homogenates. Here, we have used autoradiography to examine whether the increase in I(2) site density induced by chronic administration of 2-(2-benzofuranyl)-2-imidazoline (2-BFI) is uniform across brain regions. We dosed rats with 2-BFI 7 mg/kg or with saline vehicle i.p. over 96 days. Compared with vehicle-treated rats, this treatment significantly increased specific [(3)H]2-BFI binding only in the arcuate nucleus and area postrema, by 63% and 67% respectively. There were no significant effects in the pineal gland or interpeduncular nucleus which, like the arcuate nucleus and area postrema, are rich in I(2) sites. These data indicate that chronic administration of 2-BFI selectively alters radioligand binding in two I(2) rich brain ideas, namely the arcuate nucleus and area postrema, suggesting there may be more than one population of I(2) sites in the rat brain.

Isolation and culture of rat sensory neurons having distinct sensory modalities.

We have recently published papers in which sensory neurons that innervate either the tooth pulp or masseter muscle spindles were labelled in vivo and later identified and studied in primary tissue culture (Taddese et al., 1995; Cook et al., 1997). Here, we provide detailed descriptions of cell labelling and tissue culture methods that we used. The purpose of the preparations is to compare nociceptive and non-nociceptive sensory neurons in vitro. The spindles in mastication muscles are the only muscle afferents whose cell bodies reside in the mesencephalic nucleus (MeN5) of the fifth nerve (Corbin and Harrison, J Neurophysiol, 1940; Cody et al., J Physiol, 1972). Thus, labelling neurons projecting to the masseter muscle and dissecting the MeN5 isolates muscle spindle afferents. Pain is the only conscious sensation elicited by physiological stimulus of tooth pulp (Anderson and Matthews, 1967; Edwall and Olgart, 1977; Ahlquist et al., 1984; Narhi et al., 1994); there may be unconscious sensations that arise from the pulp, but these have never been demonstrated. Thus, tooth pulp afferents represent at least a highly enriched, and possibly a pure, population of nociceptors. In broad outline, the methods of labelling and tissue culture are standard, but we have honed many details in order to obtain practical yields.

Site-directed disulfide reduction using an affinity reagent: application on the nicotinic acetylcholine receptor.

The aim of this study was to present a new concept of site-directed reduction of disulfide bonds based upon the use of an affinity ligand harbouring a readily oxidizable dithiol. The cysteine bond involved in the acetylcholine binding site of the AChoR was specifically reduced by a carbamylcholine analogue. The ligand, in its oxidized form, was characterized by an affinity constant of 20 microM for the agonist binding site. In its dithiol form, it specifically reduced the disulfide between Cys-192 and Cys-193 on the alpha-subunits of the nicotinic acetylcholine receptor. This reduction needed 10 times lower concentration when carried out with site-directed reducing agent (ARA) than with DTT, and was highly specific for the alpha-subunits. The contribution of the carbamylcholine moiety of the site-directed reducing agent was clearly demonstrated in kinetic studies where reduction abilities of ARA, DTT and the methylated analogue of ARA (MeRA) were compared. At the same concentration (20 microM), DTT and MeRA had a 25 times lower initial rate of reduction than ARA. With 200 microM of DTT this initial reduction was still 4 times lower. Furthermore, the use of a maleimido undecagold cluster which specifically labeled the reduced nicotinic receptor opens the way to structural analysis of the agonist binding site by electron microscopy. These results demonstrate the potency of this kind of site-directed reducing agent for structural study of receptors or enzymes involving a disulfide bond in their active site.