Discovery of novel selective norepinephrine reuptake inhibitors: 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ols (WYE-103231).

Structural modification of a virtual screening hit led to the identification of a new series of 4-[3-aryl-2,2-dioxido-2,1,3-benzothiadiazol-1(3H)-yl]-1-(methylamino)butan-2-ols which are potent and selective inhibitors of the norepinephrine transporter over both the serotonin and dopamine transporters. One representative compound S-17b (WYE-103231) had low nanomolar hNET potency (IC(50) = 1.2 nM) and excellent selectivity for hNET over hSERT (>1600-fold) and hDAT (>600-fold). S-17b additionally had a good pharmacokinetic profile and demonstrated oral efficacy in rat models of ovariectomized-induced thermoregulatory dysfunction and morphine dependent flush as well as the hot plate and spinal nerve ligation (SNL) models of acute and neuropathic pain.

Discovery of WAY-260022, a Potent and Selective Inhibitor of the Norepinephrine Transporter.

The potency and selectivity of a series of 1-{(1S)-2-[amino]-1-[3-(trifluoromethoxy)phenyl]ethyl}cyclohexanol analogues are described. These compounds were prepared to improve in vitro metabolic stability and achieve brain penetration. Compound 13 (WAY-260022, NRI-022) was found to be a potent inhibitor of norepinephrine reuptake and demonstrated excellent selectivity over the serotonin and dopamine transporters. Additionally, 13 exhibited oral efficacy in a rat model of thermoregulatory dysfunction.

Dual acting norepinephrine reuptake inhibitors and 5-HT(2A) receptor antagonists: Identification, synthesis and activity of novel 4-aminoethyl-3-(phenylsulfonyl)-1H-indoles.

The discovery of a series of 4-aminoethyl-3-(phenylsulfonyl)-1H-indoles, dual acting norepinephrine reuptake inhibitors (NRIs) and 5-HT(2A) receptor antagonists, is described. The synthesis and structure-activity relationship (SAR) of this novel series of compounds is also presented.

Estradiol increases catecholamine levels in the hypothalamus of ovariectomized rats during the dark-phase.

Estrogens modulate critical homeostatic functions of the hypothalamus such as temperature regulation, sexual behavior and sleep with the most pronounced effects in rats occurring during the dark-phase. The neurochemical signals underlying estrogenic regulation of these hypothalamic functions have not been clearly identified, possibly due to the fact that previous studies have not explored the effects of estrogen treatments on neuronal signaling during the dark-phase. In the present study, ovariectomized rats received estradiol benzoate (5 microg/rat for 7 days, s.c.) and norepinephrine and dopamine levels were measured in the preoptic area of the hypothalamus across the light/dark cycle using in vivo microdialysis. Estradiol benzoate treatment increased extracellular norepinephrine and dopamine levels relative to vehicle treatment during the dark-phase. Increases in norepinephrine and dopamine were first detected by 30 min and 5.5h after lights-off, respectively. Subsequent increases in norepinephrine and dopamine were also noted throughout the 9.5-h collection period. The effect of estradiol benzoate on catecholamine release did not correlate with increases in either tyrosine hydroxylase (TH) protein expression or activity levels in the anterior hypothalamus, although a marked decrease in TH activity correlated with a rise in extracellular norepinephrine at the beginning of the dark-phase. We conclude that subchronic estradiol benzoate treatment increases extracellular catecholamine levels in the preoptic area of the hypothalamus during the dark-phase without a concomitant increase in neurotransmitter biosynthesis. The estradiol benzoate-induced increases in norepinephrine and dopamine levels in the preoptic area during the dark-phase may play an important role in modulating critical hypothalamic functions.

Enrichment and analysis of Alzheimer's Abeta1-42 peptide in human plasma and whole blood.

The Abeta1-42 fragment from the Amyloid Precursor Protein (APP) has presented considerable challenges from an analytical perspective. It is present at low levels in the circulation and can bind to proteins which mask its presence in assays. A number of therapeutic strategies target the lowering of this peptide, necessitating more robust and sensitive methods for its measurement. In this study, conditions for extracting and enriching Abeta1-42 using solid-phase extraction (SPE) and reverse-phase HPLC (RP-HPLC) were optimized. The new process provided reproducible recovery of Abeta1-42 of about 80% and allowed for concentration of the peptide prior to assay. Radiolabeled Abeta1-42 and ELISA for Abeta1-42 were used to determine the recovery and distribution of the peptide from whole blood collected in the presence of potassium-EDTA. Endogenous Abeta1-42 yielded a cell pellet:plasma ratio near 40:60 while exogenously added peptide distributed with a ratio of about 27:73. Additionally, the Abeta1-42 in the plasma and cell pellet fractions maintained stability over many hours. Comparing the measurement of Abeta1-42 using a commercial ELISA before and after enrichment demonstrated noticeable improvement of signal in samples enriched for the peptide. The current study also showed that conspicuous amounts of Abeta1-42 partition to the cell pellet but that this fraction can be robustly recovered and measured with SPE and HPLC. The process utilized established chromatographic techniques and is suitable for automation. It is also compatible with other detection methods including mass spectrometry.

Simultaneous telemetric monitoring of tail-skin and core body temperature in a rat model of thermoregulatory dysfunction.

Temperature dysfunction, clinically described as hot flashes/flushes and night sweats, commonly occur in women transitioning through menopause. Research in this field has yet to fully elucidate the biological underpinnings explaining this dysfunction. The need to develop animal models that can be used to study hormone-dependent temperature regulation is essential to advancing this scientific area. Development of telemetric transmitters for monitoring tail-skin (TST) and core body (CBT) temperatures for animal research has increased the accuracy of data by reducing extraneous factors associated with previous methods. However, until recently, TST and CBT could not be simultaneously measured telemetrically within the same animal. In this report, new dual temperature monitoring transmitters were validated by simultaneously evaluating them with the single measurement transmitters using the ovariectomized (OVX) rat thermoregulatory dysfunction model. A major advantage of measuring TST and CBT in the same animal is the ability to relate temporal changes on these two temperature parameters. Comparative experimentation was performed by single administration of clonidine (alpha(2) adrenergic agonist), MDL-100907 (5-HT(2a) antagonist), or a 7-day treatment of 17alpha-ethinyl estradiol (EE). Clonidine caused decreases in TST and CBT, MDL-100907 caused increases in TST while decreasing CBT, and EE caused decreases in TST with minor CBT decreases only at the higher dose. Data from either probe type showed similar results on temperature parameters regardless of transmitter used. These findings support the use of the new dual temperature transmitters and should enhance the quality and interpretation of data being generated in thermoregulation studies.

Synthesis and activity of 1-(3-amino-1-phenylpropyl)indolin-2-ones: a new class of selective norepinephrine reuptake inhibitors.

Norepinephrine and serotonin play an important role in a wide variety of biological processes and are implicated in a number of neurological disorders. A novel class of 1-(3-amino-1-phenylpropyl)indolin-2-ones was designed and synthesized that displays potent norepinephrine reuptake inhibition while maintaining high selectivity (>100-fold) against the human serotonin and dopamine transporters.

Effect of 17alpha-ethinyl estradiol on active phase rapid eye movement sleep microarchitecture.

Estrogen treatment decreases active phase rapid eye movement (REM) sleep in ovariectomized rats. Here we explored further the effect of 17alpha-ethinyl estradiol (17alpha-EE) on active phase REM sleep in ovariectomized rats by analyzing spectral properties and the number and length of REM sleep bouts. The greatest suppression of REM sleep occurred on day 4 of 17alpha-EE treatment, was due to decreases in bout length, and was accompanied by decreased EEG theta power. These results further elucidate 17alpha-EE's effects on REM sleep and provide greater understanding of the mechanisms by which estrogens alter sleep-wakefulness patterns.

ICI 182,780 penetrates brain and hypothalamic tissue and has functional effects in the brain after systemic dosing.

Previous reports suggest the antiestrogen ICI 182,780 (ICI) does not cross the blood-brain barrier (BBB). However, this hypothesis has never been directly tested. In the present study, we tested whether ICI crosses the BBB, penetrates into brain and hypothalamic tissues, and affects known neuroendocrine functions in ovariectomized rats. Using HPLC with mass spectrometry, ICI (1.0 mg/kg.d, 3 d) was detected in plasma and brain and hypothalamic tissues for up to 24 h with maximum concentrations of 43.1 ng/ml, and 31.6 and 38.8 ng/g, respectively. To evaluate antiestrogenic effects of ICI in the brain after systemic dosing, we tested its ability to block the effect of 17 alpha-ethinyl estradiol (EE) (0.3 mg/kg, 8 d) on tail-skin temperature abatement in the morphine-dependent model of hot flush and on body weight change. In the morphine-dependent model, EE abated 64% of the naloxone-induced tail-skin temperature increase. ICI pretreatment (1.0, 3.0 mg/kg.d) dose dependently inhibited this effect. ICI (3.0 mg/kg.d) alone showed estrogenic-like actions, abating 30% the naloxone-induced flush. In body weight studies, EE-treated rats weighed 58.5 g less than vehicle-treated rats after 8 d dosing. This effect was partially blocked by ICI (3.0 mg/kg.d) pretreatment. Similar to EE treatment, rats receiving 1.0 or 3.0 mg/kg.d ICI alone showed little weight gain compared with vehicle-treated controls. Thus, ICI crosses the BBB, penetrates into brain and hypothalamic tissues, and has both antiestrogenic and estrogenic-like actions on neuroendocrine-related functions.

Subchronic 17alpha-ethinyl estradiol differentially affects subtypes of sleep and wakefulness in ovariectomized rats.

In ovariectomized (OVX) Sprague-Dawley rats, estradiol benzoate (EB) has been reported to decrease rapid eye movement (REM) and non-REM (NREM) sleep during the dark phase for up to 3 days. It is unknown, however, if estrogenic effects on sleep extend beyond 3 days or if other estrogens could induce the same changes. Furthermore, it is unclear whether the increased wakefulness in the dark phase was due to changes in active or quiet wakefulness. Therefore, we examined the effects of daily injections of 17alpha-ethinyl estradiol (EE) for 6 days on sleep and wakefulness in the OVX rat. After 3 days of baseline recording using a telemetric system, rats were administered sesame oil (sc) for 3 days followed by injection with EE (20 mug/rat/day, sc) for 6 days. After treatment, sleep was recorded during hormone withdrawal for an additional 5 days. A few sporadic but statistically significant increases in light phase sleep occurred during the last 3 days of EE treatment. Starting on day 2 of the study, EE caused statistically significant decreases in dark phase REM sleep that were maintained throughout the treatment period and persisted until the 3rd day of hormone withdrawal. During the dark phase, statistically significant decreases in NREM sleep and increases in active wakefulness started on the second day of treatment and abated by the end of treatment. This study demonstrated that EE had similar effects on sleep-wakefulness to EB and demonstrates the utility of telemetric polysomnographic recording of the female OVX rat as a model for understanding the estrogen-induced changes on sleep-wakefulness.