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Structure–activity studies around the 4-position of 2-anilinopyrimidine corticotropin releasing factor (CRF) antagonists suggest that there is a large lipophilic cavity in the rat CRF receptor, which can accommodate a wide variety of substituents at this position in contrast to the steric constraints observed for other positions on the 2-anilinopyrimidine core. The chemical syntheses and biological activities of 2-anilinopyrimidine CRF antagonists with carbon-linked substituents at the 4-position are reported. Significant improvements in rat pharmacokinetic parameters were achieved relative to those for the lead structure. While the lead compound 1 (rCRF Ki=44 nM) afforded no detectable rat plasma levels after intraperitoneal (ip) or oral (po) dosing, compounds 3 Download : Download high-res image (104KB) Download : Download full-size image Scheme 1 . (A) HNR′R″, NaH, THF or DMF (35–90% yields); (B) BH3-THF (60–80% yields); (C) (i) LiBH4, THF, (ii) MsCl, Et3N, CH2Cl2, 0 °C, (30–70% yields); (D) R′MgBr, THF, −78°C (30–80% yields); (E) (i) 1 N NaOH, EtOH, (ii) R′Li, CeCl3, THF, −78°C (30–70% yields); (F) R′Li, THF, −78°C (30–70% yields); (G) NaBH4, EtOH (60–80% yields); (H) 1 equiv R′Li, THF, −78°C (30–70% yields); (I) R′Li, THF, −78°C (30–70% yields). - 3 (rCRF Ki=16 nM) and 3 -4 (rCRF Ki=59 nM) gave high rat plasma levels at 30 mg/kg (ip, po) (Cmax=1389 nM and 8581 nM (ip) respectively; Cmax=113 nM and 988 nM (po), respectively). Furthermore 3 - 3 and 3 -4 had superior bioavailabilities at these doses (59 and 46% (ip), respectively; 2 and 10% (po), respectively).

The Suzuki reaction has been used to synthesize a variety of aryl-substituted heterocyclic antagonists of the CRH1 receptor. Examples with several different heterocyclic cores are potent CRH receptor ligands.

A series of 4-aryl-2-(N-ethylanilino)pyrimidines has been synthesized as corticotropin-releasing hormone (CRH) inhibitors. The effect of substitution on each aromatic ring on receptor binding was investigated.

Screening of our chemical library using a rat corticotropin-releasing hormone (CRH) receptor assay led to the discovery that 2-anilinopyrimidine 15-1 weakly displaced [125I]-0-Tyr-oCRH from rat frontal cortex homogenates when compared to the known peptide antagonist alpha-helical CRH(9-41) (Ki = 5700 nM vs 1 nM). Furthermore, 15-1 weakly inhibited CRH-stimulated adenylate cyclase activity in the same tissue, but it was less potent than alpha-helical CRH(9-41) (IC50 = 20 000 nM vs 250 nM). Systematic structure-activity relationship studies, using the cloned human CRH1 receptor assay, defined the pharmacophore for optimal binding to hCRH1 receptors. Several high-affinity 2-anilinopyrimidines and -triazines were discovered, some of which had superior pharmacokinetic profiles in the rat. This paper describes the structure-activity studies which improved hCRH1 receptor binding affinity and pharmacokinetic parameters in the rat. Compound 28-17 (mean hCRH1 Ki = 32 nM) had a significantly improved pharmacokinetic profile in the rat (19% oral bioavailability at 30 mg/kg) as well as in the dog (20% oral bioavailability at 5 mg/kg) relative to the early lead structures.

The Stratospheric Observatory for Infrared Astronomy (SOFIA) has recently concluded a set of engineering flights for Observatory performance evaluation. These in-flight opportunities are viewed as a first comprehensive assessment of the Observatory's performance and are used to guide future development activities, as well as to identify additional Observatory upgrades. Pointing stability was evaluated, including the image motion due to rigid-body and flexible-body telescope modes as well as possible aero-optical image motion. We report on recent improvements in pointing stability by using an active mass damper system installed on the telescope. Measurements and characterization of the shear layer and cavity seeing, as well as image quality evaluation as a function of wavelength have also been performed. Additional tests targeted basic Observatory capabilities and requirements, including pointing accuracy, chopper evaluation and imager sensitivity. This paper reports on the data collected during these flights and presents current SOFIA Observatory performance and characterization., Comment: 13 pages, 13 figure, and 2 tables; accepted by ApJS

The Suzuki reaction has been used to synthesize a variety of aryl-substituted heterocyclic antagonists of the CRH1 receptor. Examples with several different heterocyclic cores are potent CRH receptor ligands.

A series of 4-aryl-2-(N-ethylanilino)pyrimidines has been synthesized as corticotropin-releasing hormone (CRH) inhibitors. The effect of substitution on each aromatic ring on receptor binding was investigated.

Iodine-125-labelled recombinant human interleukin-1 (IL-1) receptor antagonist ([125I]IL-1ra) was utilized to further determine the characteristics of IL-1 receptors in the brain-endocrine-immune axis. The binding of [125I]IL-1ra in homogenates of mouse hippocampus, spleen and testis was linear over a broad range of membrane protein concentrations, saturable, reversible, and of high affinity (KD, 20-30 pM). In competition studies, IL-1ra, recombinant human IL-1 alpha, IL-1 beta and a weak IL-1 beta analog inhibited [125I]IL-1ra binding to mouse tissues in parallel with their biological activities. In autoradiographic studies, [125I]IL-1ra and [125I]IL-1 alpha binding showed comparable distribution patterns with highest densities of binding sites present in the dentate gyrus of the hippocampus, choroid plexus, anterior pituitary, marginal zones and red pulp regions of the spleen, epididymis and interstitial area of the testis. The binding characteristics and distribution of [125I]IL-1ra are comparable to those of previously characterized Type I IL-1 receptors. These data provide further support for a role for IL-1 in coordinating brain-endocrine-immune responses to physiological and pharmacological stimuli.

The importance of the route of drug administration (oral vs. subcutaneous) on the neurochemical effects and pharmacokinetics of repeated d,1-fenfluramine administration in rats (1–24 mg/kg b.i.d., i.e., 2–48 mg/kg/day for 4 days) was examined. Overall, comparable dose-dependent alterations in brain monoamine markers were observed following repeated oral (PO) and subcutaneous (SC) administration of fenfluramine. Doses of 1 and 2 mg/kg fenfluramine were without significant effects on the density of 3 H-paroxetine-labeled serotonin (5-HT) uptake sites. Higher doses of fenfluramine (4, 12 and 24 mg/kg) produced dose-dependent decreases in 5-HT, 5-hydroxyindoleacetic acid and 5-HT uptake sites with maximal decreases (80–90%) occurring at the 12 mg/kg dose. Fenfluramine administration produced dose-dependent and biphasic effects on brain dopamine markers with increases in homovanillic acid (HVA) observed at 2 hours, whereas decreases in the levels of dopamine, HVA and dihydroxyphenylacetic acid were evident at 18 hours posttreatment. Norepinephrine levels were only decreased at the highest dose of fenfluramine. Significantly higher levels of brain fenfluramine were observed following SC than following PO administration of the drug. On the other hand, comparable levels of its active metabolite norfenfluramine were present in the brain following the two routes of fenfluramine administration. These data suggest the importance of norfenfluramine levels in the brain in determining the high-dose neurotoxic effects of fenfluramine on brain 5-HT neurons in rats.

The effect of the noradrenergic neurotoxin DSP-4 on high affinity transport of noradrenaline (NAT) was studied using rat brain synaptosomes. DSP-4 decreased NAT with the characteristics of a competitive inhibitor. The neurotoxin was more potent in inhibiting NAT into cortical synaptosomes (Ki = 179 +/- 39 nM) than into hypothalamic synaptosomes (Ki = 460 +/- 35 nM). Differences in NAT into cortical and hypothalamic synaptosomes were also observed with noradrenaline itself (Km = 39.5 +/- 7.5 nM and 100 +/- 12.1 nM, respectively) and with the catecholamine uptake blocker mazindol (Ki = 0.55 +/- 0.05 nM and 0.30 +/- 0.08 nM, respectively). The differences in the pharmacological properties of the noradrenaline uptake carrier in cerebral cortex and hypothalamus may account for the differential effects of DSP-4 on noradrenergic axons in these two brain regions.

We examined the incorporation of [3H]methylenedioxyamphetamine ([3H]MDA) and [3H]amphetamine into rat brain synaptosomes. Saturation studies, using increasing concentrations of nonradioactive ligand, revealed that [3H]-MDA interacted with two saturable sites that were sensitive to boiling of the tissue. Eadee-Scatchard plots of [3H]MDA saturation data were curvilinear; nonlinear curve-fitting analysis of these data suggested the presence of high- and low-affinity [3H]MDA sites of association: KD high = 295 nM, Bmax high = 32 pmol/mg of protein; KD low = 45 microM, Bmax low = 5.2 nmol/mg of protein. Association of [3H]MDA to the low-affinity site was dependent on the presence of isotonic sucrose in the incubation medium. The high capacities of these sites argue against a bimolecular interaction of [3H]MDA with monovalent protein binding sites. [3H]MDA incorporation was reduced under conditions that disrupt the integrity of plasma membranes, such as sonication, incubation in hypotonic media, and incubation in the presence of the detergent digitonin. These data indicate that [3H]MDA incorporation into synaptosomes may represent an internalization and sequestration phenomenon. [3H]MDA incorporation was also reduced by preincubation of the synaptosomal preparation at 37 degrees C or in hypotonic buffer at 4 degrees C, a result suggesting that this sequestration is maintained by an intrasynaptosomal component that is lost under the preincubation conditions described above. [3H]MDA incorporation was pH dependent (maximal at pH 7.5) and temperature sensitive (maximal incorporation occurred at 21 degrees C and was substantially reduced at 37 degrees C). [3H]Amphetamine was also incorporated into synaptosomes, and this incorporation was sensitive to the same physical manipulations of the tissue preparation as [3H]MDA incorporation. The synaptosomal sequestration of both [3H]MDA and [3H]amphetamine was inhibited by permeant cations, such as sodium and potassium, a result suggesting that the proposed intrasynaptosomal component that maintains the sequestration is anionic. Preliminary pharmacological profiles of [3H]MDA and [3H]amphetamine sequestration were identical. The rank order of inhibitor potencies for the incorporation of both ligands was desipramine greater than amphetamine greater than MDA greater than methylphenidate. This order of potency does not correspond to the lipophilicity of the test drugs. The synaptosomal incorporation and sequestration of [3H]MDA, [3H]methylenedioxymethamphetamine, and [3H]amphetamine described in the present report may be important in the molecular mechanism of action of monoamine release induced by the amphetamines.

An ELISA assay was developed for murine IL-1 beta (mIL-1 beta) using a polyclonal antibody generated in rabbits. The antibody was purified by affinity chromatography on protein A coupled to Sepharose followed by chromatography on mIL-1 beta coupled to Sepharose. The protein A and affinity purified populations were compared using radiolabeled mIL-1 beta and the results used to develop the conditions for the ELISA. The assay developed is sensitive to pg/ml concentrations of mIL-1 beta, is comparable in sensitivity to one which uses a hamster monoclonal antibody as the capture antibody, and can be used to detect IL-1 beta in peritoneal washings or tissue lysates from either mouse or rat. There is no cross reaction with any cytokine tested. The use of ELISA enhancement kits can increase the resolution at the lower concentration ranges without affecting assay sensitivity. This assay should prove useful for defining the presence and potential role for IL-1 beta in animal models of disease.

The cytokine interleukin-1 (IL-1) alters a variety of immune, central nervous system, and neuroendocrine activities characteristic of an integrator of the brain-endocrine-immune response to stress. In an attempt to define the regulation of IL-1 and IL-1 receptors in the mouse brain-endocrine-immune axis, we measured tissue levels of IL-1 beta using an enzyme-linked immunosorbent assay and iodine-125-labeled recombinant human IL-1 alpha ([125I]IL-1 alpha) binding in hippocampus, hypothalamus, pituitary, epididymus, testis, and spleen after ip injection of the bacterial endotoxin lipopolysaccharide (LPS). Basal IL-1 beta levels were detectable in all of the tissues examined. IL-1 beta levels were dramatically increased in the peripheral tissues (pituitary, testis, and spleen) 2-6 h after a single LPS injection; however, no significant changes were observed in the brain (hippocampus and hypothalamus). [125I]IL-1 alpha binding was decreased in the spleen, but was unchanged in the hippocampus and testis after a single LPS injection. To determine whether activation of IL-1 in brain may require more sustained exposure to endotoxin, we examined the effects of two injections of LPS at 0 and 12 h. After two LPS injections (0 and 12 h), significant increases in IL-1 beta concentrations were noted in the hippocampus, hypothalamus, spleen, testis and epididymus; [125I]IL-1 alpha binding using quantitative autoradiography was significantly decreased in all tissues, including the pituitary gland. Saturation studies in whole testis homogenates demonstrated that the LPS-induced decrease in [125I]IL-1 alpha binding was primarily due to a decrease in the density of IL-1 receptors. These data demonstrate that LPS treatment results in elevated circulating and/or tissue levels of IL-1, which, in turn, down-regulates IL-1 receptors in the brain-endocrine-immune axis.

Fenfluramine is an amphetamine derivative which is used as a weight-reducing agent in the treatment of obesity. It has been postulated that fenfluramine affects brain serotonin (5HT) neurons resulting in decreased food intake and altered autonomic outflow which, in turn, increases metabolism. CRF decreases food intake and, in addition, has been demonstrated to reduce body weight in genetically obese rats through selective activation of sympathetic and inhibition of parasympathetic outflows. Because 5HT is a potent CRF secretagogue, we tested the hypothesis that the weight-reducing effects of fenfluramine administration may be mediated, in part, through altered CRF secretion. Chronic fenfluramine treatment (1-24 mg/kg sc, twice daily, 4 days) resulted in a dose-dependent decrease in hypothalamic CRF concentration at 30 min after the final drug injection and was accompanied by a significant reciprocal increase in plasma corticosterone concentration. These data suggest that the decrease in hypothalamic CRF was a consequence of increased CRF secretion. These changes in hypothalamic CRF and plasma corticosterone correlated with brain fenfluramine levels. In contrast, high dose fenfluramine treatment significantly increased hippocampus, midbrain, and spinal cord CRF concentrations whereas levels in cerebral cortex, caudate putamen, thalamus, pons/medulla, and cerebellum were unaffected. There was no effect of this fenfluramine treatment protocol on regional brain TRH or neurotensin concentrations. In keeping with the well known development of tolerance to the weight-reducing effects of fenfluramine, chronic fenfluramine treatment resulted in lesser increases in corticosterone secretion than after acute treatment. Whereas weight loss observed after chronic fenfluramine treatment was associated with stimulation of hypothalamic-pituitary-adrenocortical hormone secretion, the weight-recovery phase after cessation of drug treatment was associated with decreased levels of plasma corticosterone. These data, demonstrating fenfluramine-induced alterations in brain CRF and plasma corticosterone, suggest that CRF may represent an important endogenous transmitter which mediates the weight-reducing effects of the drug.

The rate of development of environment-independent tolerance was assessed in seven inbred strains of rats, in randomly bred Sprague-Dawley rats, and in the heterogeneous stock (HS) animals developed from the inbred strains. The results demonstrated significant differences in rates of tolerance development between the various groups of inbred animals. A relationship between the initial sensitivity of an animal to the incoordinating effects of ethanol and the rate of tolerance development was also demonstrated. Animals displaying the greatest initial sensitivity to ethanol were found to develop tolerance at a faster rate than those displaying greater resistance to the incoordinating effects of ethanol. The HS animals developed from the inbred strains were demonstrated to embody the full range of rates of tolerance development exemplified by the parental inbred strains.

The effect of in vitro addition of ethanol (0.02-1.0 M) on the binding of 35S-TBPS was examined in brain membranes from cerebellum and cortex of naive or chronically ethanol-treated C57B1 mice. In brain membranes of untreated animals, increasing concentrations of ethanol produced a dose-related inhibition of 35S-TBPS binding in the brain areas investigated. Additional studies showed that this effect of ethanol was due to a decreased affinity of 35S-TBPS for its binding sites. Chronic treatment of the animals with ethanol, which produced tolerance to and dependence on ethanol, did not alter ethanol's ability to inhibit the binding of 35S-TBPS. In naive animals, the in vitro addition of GABA or pentobarbital produced a pronounced inhibition of 35S-TBPS, both drugs being more potent in the cerebellum than in the cortex. Picrotoxin also produced a dose-dependent inhibition at 35S-TBPS, but was equally potent in the brain areas investigated. The inhibition by GABA or pentobarbital was not influenced by in vitro addition of a physiologically relevant concentration of ethanol (100 mM), whereas ethanol produced a significant increase in the IC50 values for picrotoxin both in the cortex and in the cerebellum. Furthermore, the inhibitory effects of GABA or pentobarbital on 35S-TBPS binding remained unchanged in animals chronically treated with ethanol for 7 days. Our data indicate that ethanol may affect the GABA receptor system through a rather specific interaction with the 35S-TBPS recognition site, but that this action of ethanol is not altered by the development of tolerance to and dependence on ethanol.

Recent studies have demonstrated the presence of immunoreactive oxytocin (OT) and vasopressin (VP), OT and VP receptors and physiological functions for these two hormones in a variety of peripheral tissues, including anterior pituitary gland. The objectives of this study were to determine if (i) OT and VP genes are expressed in rat testis and anterior pituitary gland and (ii) if osmotic stimulation known to modify the regulation of OT and VP genes in hypothalamus, would modify the expression of these genes in rat testis and anterior pituitary gland. Using oligonucleotide probes (courtesy of Drs. M. Brownstein and W. Scott Young, NIMH) corresponding to the VP gene or OT gene and specific fractions of human OT and VP genes (courtesy of Dr. J. Battey, NCI) subcloned in the pGEM-3 riboprobe system, and Northern blot and slot blot techniques, OT and VP mRNAs were found in rat testis and anterior pituitary gland. When adult male rats (SD) were either deprived of drinking water or offered 2% salt solution as a sole source of drinking fluid for 72 hrs, both OT and VP mRNA levels were increased in hypothalamus, anterior pituitary gland and testis. Our data suggest that testis and anterior pituitary gland could also be sites of synthesis of OT and VP and that the same stimulus may regulate these genes in various tissues.

The effect of in vitro and in vivo administration of ethanol on the binding of 35S-t-butyl-bicyclophosphorothionate (35S-TBPS) to cortical brain membranes of C57Bl mice was investigated using KCl (100 mM) containing assay media. The in vitro addition of ethanol produced a dose-dependent inhibition of basal 35S-TBPS binding. In the presence of chloride ions, GABA and pentobarbital had a biphasic action (stimulation followed by inhibition) on 35S-TBPS binding, whereas diazepam only stimulated the binding. Ethanol reduced the stimulatory effects of GABA and pentobarbital in a dose-dependent manner, but had no effect on the enhancement of 35S-TBPS binding produced by diazepam. 35S-TBPS binding to cortical brain membranes was inhibited by the putative Cl- channel blocking agent DIDS. This inhibitory action of DIDS was significantly, and dose-dependently reduced by ethanol (greater than or equal to 100 mM ethanol). Chronic ethanol ingestion in vivo, which produced tolerance to and physical dependence on ethanol in the animals, did not alter the stimulatory and inhibitory effects of GABA and pentobarbital on 35S-TBPS binding. The enhancement of 35S-TBPS binding produced by diazepam was slightly, but significantly, enhanced in brain membranes from animals which had undergone 24 hours of ethanol withdrawal. Chronic ethanol treatment did not change the potency of picrotoxin and of the peripheral BDZ-receptor ligand RO 5-4864 to competitively inhibit 35S-TBPS binding. Our results suggest that in vitro addition of ethanol alters the activity of the GABA/benzodiazepine (BDZ) receptor complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Methyl-phenidate (Ritalin) and pemoline (Cylert) are central nervous system stimulants which are widely prescribed for attention deficit and other psychiatric disorders. Several other related stimulants, including amphetamine and methamphetamine, have been shown to cause long lasting decreases in monoamine markers in rat brain, characteristic of axonal degeneration. To assess the neurotoxic potential of methylphenidate and pemoline, we compared the effects of multiple injections (sc, bid for up to 4 days) of methylphenidate (21 and 50 mg/kg) and pemoline (20 and 70 mg/kg) with methamphetamine (5 and 15 mg/kg) on rat brain norepinephrine, dopamine, and serotonin levels and transport sites. While decreases were observed in all brain monoamine markers measured in rats treated with methamphetamine, no changes were observed in animals treated with methylphenidate as compared to saline-treated controls. Pemoline failed to induce significant changes in the level of monoamine transport sites; however, a wide array of changes were observed in the levels of 5-hydroxyindoleacetic acid, dopamine, and norepinephrine in various brain areas after a 3-day treatment regimen with a high dose (70 mg/kg) of pemoline. The lack of changes in monoamine transport sites following the repeated administration of high doses of methylphenidate and pemoline suggests that these drugs do not affect axonal integrity. However, the pattern of changes observed in the levels of monoamines after pemoline treatment may have relevance to the self-injurious behavior seen in these animals.