[11C]NS8880, a promising PET radiotracer targeting the norepinephrine transporter.

INTRODUCTION: Positron emission tomography (PET) imaging of the norepinephrine transporter (NET) is still hindered by the availability of useful PET imaging probes. The present study describes the radiosynthesis and pre-clinical evaluation of a new compound, exo-3-(6-methoxypyridin-2-yloxy)-8-H-8-azabicyclo[3.2.1]octane (NS8880), targeting NET. NS8880 has an in vitro binding profile comparable to desipramine and is structurally not related to reboxetine. METHODS: Labeling of NS8880 with [(11)C] was achieved by a non-conventional technique: substitution of pyridinyl fluorine with [(11)C]methanolate in a Boc-protected precursor. The isolated [(11)C]NS8880 was evaluated pre-clinically both in a pig model (PET scanning) and in a rat model (µPET scanning) and compared to (S,S)-[(11)C]-O-methylreboxetine ([(11)C]MeNER). RESULTS: The radiolabeling technique yielded [(11)C]NS8880 in low (<10%) but still useful yields with high purity. The PET in vivo evaluation in pig and rat revealed a rapid brain uptake of [(11)C]NS8880 and fast obtaining of equilibrium. Highest binding was observed in thalamic and hypothalamic regions. Pretreatment with desipramine efficiently reduced binding of [(11)C]NS8880. CONCLUSION: Based on the pre-clinical results obtained so far [(11)C]NS8880 displays promising properties for PET imaging of NET.

Development of an inhalation physiologically based pharmacokinetic (PBPK) model for 2,2, 4-trimethylpentane (TMP) in male Long-Evans rats using gas uptake experiments.

2,2,4-Trimethylpentane (TMP) is a volatile colorless liquid used primarily to increase the octane rating of combustible fuels. TMP is released in the environment through the manufacture, use, and disposal of products associated with the gasoline and petroleum industry. Short-term inhalation exposure to TMP (< 4 h; > 1000 ppm) caused sensory and motor irritations in rats and mice. Like many volatile hydrocarbons, acute exposure to TMP may also be expected to alter neurological functions. To estimate in vivo metabolic kinetics of TMP and to predict its target tissue dosimetry during inhalation exposures, a physiologically based pharmacokinetic (PBPK) model was developed for the chemical in Long-Evans male rats using closed-chamber gas-uptake experiments. Gas-uptake experiments were conducted in which rats (80-90 days old) were exposed to targeted initial TMP concentrations of 50, 100, 500, and 1000 ppm. The model consisted of compartments for the closed uptake chamber, lung, fat, kidney, liver, brain, and rapidly and slowly perfused tissues. Physiological parameters were obtained from literature. Partition coefficients for the model were experimentally determined for air/blood, fat, liver, kidney, muscle, and brain using vial equilibration methods. Common to other hydrocarbons, metabolism of TMP via oxidative reactions is assumed to mainly occur in the liver. The PBPK model simulations of the closed chamber data were used to estimate in vivo metabolic parameters for TMP in male Long-Evans rats.

Synthesis and evaluation of a novel 2-azabicyclo[2.2.2]octane class of long chain fatty acid elongase 6 (ELOVL6) inhibitors.

A series of novel 2-azabicyclo[2.2.2]octane derivatives was synthesized and evaluated as long chain fatty acid elongase 6 (ELOVL6) inhibitors. Screening of our corporate chemical collections against ELOVL6 resulted in the identification of lead 1. Exploratory chemistry efforts were applied to lead 1 to identify the orally available, potent, and selective ELOVL6 inhibitor 28a.

Synthesis and biological evaluation of a novel 3-sulfonyl-8-azabicyclo[3.2.1]octane class of long chain fatty acid elongase 6 (ELOVL6) inhibitors.

Long chain fatty acid elongase 6 (ELOVL6) catalyzes the elongation of long chain fatty acyl-CoAs and is a potential target for the treatment of metabolic disorders. The ultrahigh throughput screen of our corporate chemical collections resulted in the identification of a novel 3-sulfonyl-8-azabicyclo[3.2.1]octane class of ELOVL6 inhibitor 1a. Optimization of lead 1a led to the identification of the potent, selective, and orally available ELOVL6 inhibitor 1w.

(+)-3-[2-(Benzo[b]thiophen-2-yl)-2-oxoethyl]-1-azabicyclo[2.2.2]octane as potent agonists for the alpha7 nicotinic acetylcholine receptor.

A series of 3-substituted 1-azabicyclo[2.2.2]octanes was discovered as the alpha7 nicotinic acetylcholine (alpha7) receptor agonists. It was found that (+)-3-[2-(benzo[b]thiophen-2-yl)-2-oxoethyl]-1-azabicyclo[2.2.2]octane (+)-15b has potent agonistic activity for the alpha7 receptor.

Biotransformation of octane by E. coli HB101[pGEc47] on defined medium: octanoate production and product inhibition.

E. coli HB101[pGEc47], which is able to convert octane to octanoate, but cannot oxidize octanoate further, was grown on defined medium with glucose as carbon source in batch and continuous culture. The biomass yield on glucose decreased from 0.32 +/- 0.02 g g-1 in aqueous cultivations to 0.25 +/- 0.02 g g-1 in the presence of octane. Maximal octanoate productivities of 0.6 g L-1 h-1 were the same as found in cultivations on complex medium. The glucose-based carbon recovery in these experiments was 99 +/- 4% (in extreme, between 90% and 105%). An increase of the octane feed from 1% to 2% (v/v) or more led to washout of cells. This effect was reversible when the octane feed was decreased to its initial value of 1%. Analysis of experimental data by model simulation strongly suggested that washout was due to inhibition by octanoate only. Pulses of octanoate to a continuous culture grown on aqueous media were applied to analyze the inhibition further. Inhibition by acetate was not significant, but its presence in the medium reflected a physiological state that made the cells more sensitive to octanoate inhibition. Model simulation with linear inhibition kinetics could perfectly predict glucose consumption and the resulting glucose concentration. The linear type of inhibition was confirmed by a variety of batch experiments in the presence of different concentrations of octanoate. The glucose-based specific growth rate, mu, decreased linearly with increasing concentrations of octanoate and became zero at a threshold concentration pmax of 5.25 +/- 0.25 g L-1.

The fate of inhaled octane and the nephrotoxicant, isooctane, in rats.

To determine if inhaled nephrotoxic branched and nonnephrotoxic straight chain alkanes differ substantially in their biological fate, male F344 rats were exposed to 14C-labeled isooctane and octane vapors at approximately 1 and 350 ppm by the nose-only mode for 2 hr. Radioactivity in exhalant, urine, and feces was determined for 70 hr post exposure, after which residual radioactivity in the rat carcasses was determined. Absorbed [14C]isooctane equivalents were eliminated almost exclusively via the kidneys, while absorbed [14C]octane equivalents were excreted about equally via the kidneys and as 14CO2. Kidney excretion of isooctane-introduced 14C was protracted over the entire 70 hr postexposure observation period whereas for octane-introduced 14C, kidney excretion was essentially complete after 10-20 hr. About 5% of the [14C]octane equivalents inhaled at 1 ppm remained in the carcass 70 hr after inhalation exposure. Two percent of the [14C]octane equivalents inhaled at 350 ppm and 1-2% of the [14C]isooctane equivalents inhaled at either 1 or 350 ppm remained in the carcass 70 hr after inhalation exposure. The different patterns of excretion of metabolites of isooctane compared to octane may be a factor affecting the differences in nephrotoxicity between these two compounds.

2,2,4-Trimethylpentane-induced nephrotoxicity. I. Metabolic disposition of TMP in male and female Fischer 344 rats.

2,2,4-Trimethylpentane (TMP), a component of unleaded gasoline, causes nephrotoxicity in male, but not in female, rats. In the present study, male and female Fischer 344 rats were treated with a single oral dose of [14C]TMP (4.4 mmol/kg; 2 microCi/mmol). Radiolabeled material in kidney, liver, and plasma was determined at 4, 8, 12, 24, and 48 hr after dosing. Maximum concentration of TMP-derived radioactivity in kidney, liver, and plasma of male rats was found after 12 hr (1252, 1000, and 403 nmol eq/g, respectively), whereas those measured in females were found after 8 hr (577, 1163, and 317 nmol eq/g, respectively). A selective retention of the TMP-derived radiolabel in the kidneys of male rats was noted when peak tissue concentration was expressed as a percentage of administered dose. Kidney concentrations of TMP-derived radiolabel increased in a nonlinear, but dose-dependent, manner; the kidney to plasma ratio was greater at low doses than at higher doses. Increased retention of radiolabel material in the kidney was associated with a significant increase in renal concentration of the male-rat-specific protein, alpha 2u-globulin, 24 and 48 hr after TMP administration. Total radioactivity collected in urine 48 hr after TMP administration was similar in males and females (32 and 31% of dose). Identification and quantitation of the urinary metabolites of TMP showed that both male and female rats metabolize TMP via the same pathway and at a similar rate. Female rats, however, excreted more conjugates of 2,4,4-trimethyl-2-pentanol in urine than males. 2,4,4-Trimethyl-2-pentanol was the major metabolite present in the male rat kidney, but was absent in the female rat kidney. The renal retention of 2,4,4-trimethyl-2-pentanol appears to account for the delayed clearance observed in the disposition of [14C]TMP-derived radiolabel. Based on the concomitant accumulations in renal alpha 2u-globulin concentration and renal 2,4,4-trimethyl-2-pentanol concentration, an association is speculated between these two components. The male-rat-specific accumulation of 2,4,4-trimethyl-2-pentanol may therefore reflect the accumulation of a "metabolite-alpha 2u-globulin" complex. This may be relevant to the male-rat-specific nephrotoxicity produced by TMP.

2,2,4-Trimethylpentane-induced nephrotoxicity. II. The reversible binding of a TMP metabolite to a renal protein fraction containing alpha 2u-globulin.

Trimethylpentane (TMP) produces nephrotoxicity in male but not in female rats. The toxicity is characterized by an increase in protein droplets in proximal convoluted tubular cells and an increase in the renal concentration of the male-rat-specific protein alpha 2u-globulin. Subcellular fractionation of the kidneys from male rats 24 hr after [3H]TMP administration showed that about 60% of the radiolabeled material was localized in the 116,000g supernatant. Column chromatography of this supernatant resolved the radioactivity into two components; one, which contained about 26% of the radiolabel, coeluted with alpha 2u-globulin and cross-reacted with an antibody specific for alpha 2u-globulin. The remaining component eluted in the low-molecular-weight range (less than 1000 Da) and was assumed to be TMP metabolites. Radiolabel from [3H]TMP in male rat urine also resolved into two components with about 0.1% of the radiolabel in urine coeluting with the alpha 2u-globulin-containing fraction. Radiolabel from TMP in male rat liver 116,000g supernatant and plasma and in female rat kidney 116,000g supernatant eluted as a single component in the low-molecular-weight range. Dialysis (1000-Da cutoff) of male kidney 116,000g supernatant led to a loss of the low-molecular-weight components, but nondialyzable radiolabel (about 20%) still coeluted with the alpha 2u-globulin after gel chromatography. Dialysis against 0.1% sodium dodecyl sulfate led to a loss of both the low- and high-molecular-weight radioactive material. These results suggested that the high-molecular-weight radioactive material was formed by the reversible binding of a radioactive component of TMP to a male-rat-specific protein. Gas chromatography-mass spectrometry of an ethyl acetate extract of the alpha 2u-globulin-containing fractions of TMP-treated male rat kidney 116,000g supernatant identified 2,4,4-trimethyl-2-pentanol as the only bound metabolite to alpha 2u-globulin. These studies provide the first evidence for a reversible binding between a metabolite of TMP and a male-rat-specific protein in the kidney and thus provide important insight delineating a potential mechanism of hydrocarbon-induced hyaline-droplet nephropathy.