Mechanistic and quantitative insight into cell surface targeted molecular imaging agent design.

Molecular imaging agent design involves simultaneously optimizing multiple probe properties. While several desired characteristics are straightforward, including high affinity and low non-specific background signal, in practice there are quantitative trade-offs between these properties. These include plasma clearance, where fast clearance lowers background signal but can reduce target uptake, and binding, where high affinity compounds sometimes suffer from lower stability or increased non-specific interactions. Further complicating probe development, many of the optimal parameters vary depending on both target tissue and imaging agent properties, making empirical approaches or previous experience difficult to translate. Here, we focus on low molecular weight compounds targeting extracellular receptors, which have some of the highest contrast values for imaging agents. We use a mechanistic approach to provide a quantitative framework for weighing trade-offs between molecules. Our results show that specific target uptake is well-described by quantitative simulations for a variety of targeting agents, whereas non-specific background signal is more difficult to predict. Two in vitro experimental methods for estimating background signal in vivo are compared - non-specific cellular uptake and plasma protein binding. Together, these data provide a quantitative method to guide probe design and focus animal work for more cost-effective and time-efficient development of molecular imaging agents.

Self-assembled gold nanoparticle molecular probes for detecting proteolytic activity in vivo.

Target-activatable fluorogenic probes based on gold nanoparticles (AuNPs) functionalized with self-assembled heterogeneous monolayers of dye-labeled peptides and poly(ethylene glycol) have been developed to visualize proteolytic activity in vivo. A one-step synthesis strategy that allows simple generation of surface-defined AuNP probe libraries is presented as a means of tailoring and evaluating probe characteristics for maximal fluorescence enhancement after protease activation. Optimal AuNP probes targeted to trypsin and urokinase-type plasminogen activator required the incorporation of a dark quencher to achieve 5- to 8-fold signal amplification. These probes exhibited extended circulation time in vivo and high image contrast in a mouse tumor model.

Carbohydrate absorption by blackcap warblers (Sylvia atricapilla) changes during migratory refuelling stopovers.

Passerine birds migrating long distances arrive at stopover sites to refuel having lost as much as 50% of their initial body mass (mb), including significant losses to digestive organs that may serve as a reservoir of protein catabolised for fuel during flight. Birds newly arrived at a stopover show slow or no mb gain during the initial 2-3 days of a stopover, which suggests that energy assimilation may be limited by reduced digestive organs. Measurements of migrants and captive birds subjected to simulated migratory fasts have shown reductions in intestine mass, morphological changes to the mucosal epithelium, and reductions in food intake and assimilation rate upon initial refeeding. We found that blackcaps (Sylvia atricapilla, Linnaeus) newly arrived at a migratory stopover after crossing the Sahara and Sinai deserts had significantly increased paracellular nutrient absorption (non-carrier mediated uptake occurring across tight junctions between enterocytes) that may provide partial compensation for reduced digestive capacity resulting from changes to intestinal tissues. Indeed, newly arrived birds also had a slightly reduced capacity for absorption of a glucose analogue (3-O-methyl-D-glucose) transported simultaneously by both carrier-mediated and non-mediated mechanisms. Increased paracellular absorption coupled with extended digesta retention time may thus allow migratory blackcaps to maintain high digestive efficiency during initial stages of refuelling while digestive organs are rebuilt.

CYP2C8 activity recovers within 96 hours after gemfibrozil dosing: estimation of CYP2C8 half-life using repaglinide as an in vivo probe.

Gemfibrozil 1-O-beta-glucuronide is a mechanism-based inhibitor of cytochrome P450 2C8. We studied the recovery of CYP2C8 activity after discontinuation of gemfibrozil treatment using repaglinide as a probe drug, to estimate the in vivo turnover half-life of CYP2C8. In a randomized five-phase crossover study, nine healthy volunteers ingested 0.25 mg of repaglinide alone or after different time intervals after a 3-day treatment with 600 mg of gemfibrozil twice daily. The area under the plasma concentration-time curve (AUC) from time 0 to infinity of repaglinide was 7.6-, 2.9-, 1.4- and 1.0-fold compared with the control phase when it was administered 1, 24, 48, or 96 h after the last gemfibrozil dose, respectively (P < 0.001 versus control for 1, 24, and 48 h after gemfibrozil). Thus, a strong CYP2C8 inhibitory effect persisted even after gemfibrozil and gemfibrozil 1-O-beta-glucuronide concentrations had decreased to less than 1% of their maximum (24-h dosing interval). In addition, the metabolite to repaglinide AUC ratios indicated that significant (P < 0.05) inhibition of repaglinide metabolism continued up to 48 h after gemfibrozil administration. Based on the recovery of repaglinide oral clearance, the in vivo turnover half-life of CYP2C8 was estimated to average 22 +/- 6 h (mean +/- S.D.). In summary, CYP2C8 activity is recovered gradually during days 1 to 4 after gemfibrozil discontinuation, which should be considered when CYP2C8 substrate dosing is planned. The estimated CYP2C8 half-life will be useful for in vitro-in vivo extrapolations of drug-drug interactions involving induction or mechanism-based inhibition of CYP2C8.

A biotin-protein bond with stability in plasma.

A nonradioactive label for peptide hormones would be useful for pharmacokinetic studies in infants, children, and pregnant women. Because the binding affinity between biotin and avidin is large (Ka=10(15) M(-1)), biotin could also serve as a covalent label for subsequent detection using a variety of avidin conjugates. However, biotin labels produced by most commercially available biotinylating reagents are rapidly cleaved from protein in plasma. We sought to synthesize a stable biotin label for protein. With the use of immunoglobulin G (IgG) as a model protein, biotin was conjugated through a cysteine residue; a carboxylate group was positioned alpha to the biotinamide bond. Stability of the bond in the presence of plasma and buffer control was assessed by release of biotin. Released biotin was separated from biotinylated IgG by ultrafiltration and was quantitated by an avidin-binding assay. In plasma, less than 0.6% of bound biotin was released. This release rate is not significantly different from buffer and is less than 7% of the release rate for IgG biotinylated by N-hydroxysuccinimide-LC-biotin. We conclude that this biotin-protein bond is stable in plasma. We speculate that many uses of avidin-biotin technology could be improved by using this method for protein labeling.

Evaluation of the protective effects of Schisandra chinensis on Phase I drug metabolism using a CCl4 intoxication model.

To evaluate the potential activity of Schisandra chinensis in restoring hepatic drug metabolism in CCl4 damaged liver, antipyrine was employed as a probe for the possible effects of the herb on Phase I oxidative metabolism in rats. Schisandra lignan fraction (160 mg/kg) was given orally to male Sprague-Dawley rats (220-240 g) 30 min or 6 h before CCl4 intoxication (4 ml/kg, s.c.). Following a single oral dose of antipyrine (80 mg/kg) to the rats with damaged liver, the pharmacokinetics of antipyrine in whole blood were determined and levels of liver enzymes, e.g. SGPT, SGOT, and cytochrome P450 were measured. Pharmacokinetic parameters for antipyrine were estimated using noncompartmental analysis. Results indicated that CCl4 significantly increased the elimination half-life (t(1/2)) of antipyrine from 2.59 +/- 1.04 to 11.25 +/- 3.91 h (P < 0.001) and decreased its clearance (CL) from 65.94 to 10.84 ml/h as compared to control. Pretreatment with the Schisandra lignan fraction 30 min or 6 h before intoxication significantly (P < 0.001) improved antipyrine elimination by reducing its t(1/2) to 3.30 +/- 0.52 and 3.58 +/- 1.05 h, respectively. The corresponding improvements observed for CL, i.e. 49.06 +/- 21.75 ml/h (P < 0.01); 21.10 +/- 10.42 ml/h (P < 0.05), were also substantial. Moreover, normalization of SGPT, SGOT and P450 levels was observed with the two Schisandra pretreatment schedules. In conclusion, Schisandra lignans exhibited strong protective effect on Phase I oxidative metabolism in the liver damaged by CCl4. Furthermore, pretreatment of Schisandra 30 min before intoxication showed a more pronounced effect than that of the 6 h pretreatment. The current pharmacokinetic approach allowed the protective effects of Schisandra on oxidative drug metabolism in damaged liver to be systemically examined and will certainly help in the evaluation of hepato-protectants obtained from natural sources.

Development and validation of a solid-phase extraction and high-performance liquid chromatographic assay for a novel fluorinated 2-nitroimidazole hypoxia probe (SR-4554) in Balb/c mouse plasma.

N-(2-Hydroxy-3,3,3-trifluoropropyl)-2-(2-nitro-1-imidazolyl) acetamide, a novel 2-nitroimidazole, is currently being developed as a non-invasive probe for tumour hypoxia. A sensitive (minimum quantifiable level = 25 ng/ml; C.V. = 6.01%) and selective assay has, therefore, been developed for the analysis of this compound in mouse plasma. The assay employed a solid-phase extraction followed by a rapid (10 min) HPLC analysis with UV-photodiode-array detection. No drug-related metabolites were observed in plasma when mice were treated with 180 mg/kg of the drug. The assay has proved to be suitable for studying the plasma pharmacokinetics of this fluorinated 2-nitroimidazole in mice.