Counteraction of the Rapid Tolerance to 8‐Hydroxy‐2‐(di‐n‐propylamino)tetralin‐Induced Hypothermia in Rats by Activation of the GABAA Receptor‐Chloride Channel Complex

Abstract: The effects of compounds that open the GABA_A receptor‐chloride channel complex on the rapidly developed tolerance to the 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin hydrobromide(8‐OH‐DPAT)‐induced hypothermia in rats were examined. The test compound was injected 15 min. before 1 mg/kg subcutaneous 8‐OH‐DPAT or saline and 24 hr later a challenge dose of 0.1 mg/kg subcutaneous 8‐OH‐DPAT was given. The rectal temperature was measured before the challenge dose and 30, 60, 90 and 120 min. thereafter. The hypothermic effect was calculated as the area under the curve. It was found that all the GABAergic compounds examined significantly counteracted the 8‐OH‐DPAT‐induced tolerance to the hypothermic response: muscimol at 3 mg/kg subcutaneous, diazepam at 1 – 3 mg/kg subcutaneous, pentobarbitone sodium at 20 mg/kg subcutaneous, and chlormethiazole at 40 mg/kg subcutaneous. Combined treatment of the rats with the GABA_A receptor antagonist, bicucculine, or the GABA_A receptor‐chloride channel blocker, picrotoxin and diazepam, pentoparbitone sodium or chlormethiazole significantly antagonised this counteraction of the 8‐OH‐DPAT‐induced tolerance. Depletion of 5‐HT by pretreatment of the rats with the tryptophan hydroxylase inhibitor p‐chlorophenylalanine did not counteract the 8‐OH‐DPAT‐induced tolerance to the hypothermic response. Pretreatment of the rats with dexamethazone did not change the development of the tolerance to 8‐OH‐DPAT‐induced hypothermic effect which seems to exclude the involvement of the hypothalamo‐pituitary‐adrenocortical axis in the tolerance development. It is concluded that the results support the hypothesis that GABA neurones beyond the 5‐HT neurones are involved in the mechanism causing tolerance to the 5‐HT_1A receptor‐mediated hypothermia in rats.     

Potencies of vitamin D analogs, 1α‐hydroxyvitamin D3, 1α‐hydroxyvitamin D2 and 25‐hydroxyvitamin D3, in lowering cholesterol in hypercholesterolemic mice in vivo

Vitamin D₃ and the synthetic vitamin D analogs, 1α‐hydroxyvitamin D₃ [1α(OH)D₃], 1α‐hydroxyvitamin D₂ [1α(OH)D₂] and 25‐hydroxyvitamin D₃ [25(OH)D₃] were appraised for their vitamin D receptor (VDR) associated‐potencies as cholesterol lowering agents in mice in vivo. These precursors are activated in vivo: 1α(OH)D₃ and 1α(OH)D₂ are transformed by liver CYP2R1 and CYP27A1 to active VDR ligands, 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] and 1α,25‐dihydroxyvitamin D₂ [1,25(OH)₂D₂]_, respectively. 1α(OH)D₂ may also be activated by CYP24A1 to 1α,24‐dihydroxyvitamin D₂ [1,24(OH)₂D₂], another active VDR ligand. 25(OH)D₃, the metabolite formed via CYP2R1 and or CYP27A1 in liver from vitamin D₃, is activated by CYP27B1 in the kidney to 1,25(OH)₂D₃. In C57BL/6 mice fed the high fat/high cholesterol Western diet for 3 weeks, vitamin D analogs were administered every other day intraperitoneally during the last week of the diet. The rank order for cholesterol lowering, achieved via mouse liver small heterodimer partner (Shp) inhibition and increased cholesterol 7α‐hydroxylase (Cyp7a1) expression, was: 1.75 nmol/kg 1α(OH)D₃ > 1248 nmol/kg 25(OH)D₃ (dose ratio of 0.0014) > > 1625 nmol/kg vitamin D₃. Except for 1.21 nmol/kg 1α(OH)D₂ that failed to lower liver and plasma cholesterol contents, a significant negative correlation was observed between the liver concentration of 1,25(OH)₂D₃ formed from the precursors and liver cholesterol levels. The composite results show that vitamin D analogs 1α(OH)D₃ and 25(OH)D₃ exhibit cholesterol lowering properties upon activation to 1,25(OH)₂D₃: 1α(OH)D₃ is rapidly activated by liver enzymes and 25(OH)D₃ is slowly activated by renal Cyp27b1 in mouse.     

Effects of intra-amygdala R(+) 7-OH-DPAT on intra-accumbens d-amphetamine-associated learning I. Pavlovian conditioning

We have previously obtained evidence that the mesoamygdaloid dopamine projection modulates the acquisition of a conditioned response (CR) elicited by presentation of a conditioned stimulus (CS) predicting the availability of a natural (sucrose) reward. This property was found to be dependent upon D₃, but not D₁ or D₂, dopamine receptor activation. The aim of the present study was to determine whether the mesoamygdaloid dopamine projection is similarly involved in the acquisition of a drug-associated CR. Thus, two groups of rats with guide cannulae aimed at the nucleus accumbens and amygdala were trained using a Pavlovian conditioning procedure in which an initially neutral CS was paired with a computer-controlled, bilateral intra-accumbens infusion of d-amphetamine (the unconditioned stimulus; US). Conditioning sessions were conducted in standard operant chambers, with each session consisting of a single CS-US trial. For one group of rats, CS presentation was positively correlated with the drug US (Paired group), while for the second group CS and US presentations were negatively correlated (Unpaired group). During training, locomotor activity was recorded and was utilised as the measure both of the unconditioned (UR) and conditioned response (CR). A within-subjects design was utilised to investigate the effect of post-session bilateral intra-amygdala administration of R(+) 7-OH-DPAT on the development of the drug-associated CR. Hence, both Paired and Unpaired groups were exposed to two different CSs which were presented on alternate sessions. Post-session bilateral intra-amygdala administration of R(+) 7-OH-DPAT (10 nmol) followed sessions in which one CS was presented, while intra-amygdala vehicle followed sessions in which the alternate CS was presented. The development of a CR occurred only in the presence of a CS that had been positively correlated with presentation of the drug US. Post-session, intra-amygdala administration of R(+) 7-OH-DPAT enhanced the acquisition of this CR. However, R(+) 7-OH-DPAT was without effect upon the unconditioned response to intra-accumbens d-amphetamine. Our previous data indicate a comparable effect of R(+) 7-OH-DPAT on conditioning to a CS associated with a non-drug, natural reward. Therefore, taken together, these findings suggest that D₃ dopamine receptors within the amygdala modulate specifically the acquisition of Pavlovian conditioned responses, regardless of whether drug or natural rewards constitute the US. Received: 28 November 1997/Final version: 9 April 1998     

Differential Sensitivity to 8‐Hydroxy‐2‐(di‐n‐propylamino)tetralin‐Induced Corticosterone Secretion and Hypothermia between Sprague‐Dawley Rats from Different Breeding Colonies

Abstract: The sensitivity of Sprague‐Dawley rats from 4 different breeding colonies (ALAB, M&B, B&K, Charles River) and one breeding colony of Wistar rat (M&B) to the 5‐hydroxytryptamine_1A (5‐HT_1A) receptor stimulatory effect of 8‐hydroxy‐2‐(di‐n‐propylamino)tetralin hydrobromide (8‐OH‐DPAT) resulting in corticosterone secretion and hypothermia was compared. The dose‐response curves of the increase in plasma corticosterone showed that ALAB and M&B rats were 3.5 times more sensitive to 8‐OH‐DPAT than B&K and Charles River rats, the Wistar rats being in between. The attenuation of the corticosterone response 24 hr after a single injection of 1 mg/kg 8‐OH‐DPAT was greater for the ALAB and M&B rats than for B&K, Charles River and Wistar rats. The comparison of the 8‐OH‐DPAT‐induced hypothermia in the various rat colonies showed a similar pattern: the sensitivity of ALAB rats was about twice that of M&B, B&K and Wistar rats, Charles River rats being 9 times less sensitive. The attenuation of the response 24 hr after 1 mg/kg 8‐OH‐DPAT measured as the shift in dose‐response showed similar shift factors (4.1 to 6.7) for all rat colonies except for the B&K rats (3.0). The hypothermic response at 0.1 mg/kg 8‐OH‐DPAT was significantly lower for the Charles River and B&K rats than for the ALAB rats. Similarly was the maximal attenuation of the hypothermic effect in these rats less than half of that of the ALAB rats. The possible cause of the observed differences in the response to 8‐OH‐DPAT between these rat colonies is discussed in terms of receptor reserves and the involvement of other transmitter systems in the responses.     

Comparative effects of 1α‐hydroxyvitamin D3 and 1,25‐dihydroxyvitamin D3 on transporters and enzymes in fxr(+/+) and fxr(‐/‐) mice

Previous studies have shown that 1α,25‐dihydroxyvitamin D₃ [1,25(OH)₂D₃] treatment in mice resulted in induction of intestinal and renal Cyp24a1 and Trpv6 expression, increased hepatic Cyp7a1 expression and activity, as well as higher renal Mdr1/P‐gp expression. The present study compared the equimolar efficacies of 1α‐hydroxyvitamin D₃ [1α(OH)D₃] (6 nmol/kg i.p. q2d × 4), a lipophilic precursor with a longer plasma half‐life that is converted to 1,25(OH)₂D₃, and 1,25(OH)₂D₃ on vitamin D receptor (VDR) target genes. To clarify whether changes in VDR genes was due to VDR and not secondary, farnesoid X receptor (FXR)‐directed effects, namely, lower Cyp7a1 expression in rat liver due to increased bile acid absorption, wildtype [fxr(+/+)] and FXR knockout [fxr(‐/‐)] mice were used to distinguish between VDR and FXR effects. With the exception that hepatic Sult2a1 mRNA was increased equally well by 1α(OH)D₃ and 1,25(OH)₂D₃, 1α(OH)D₃ treatment led to higher increases in hepatic Cyp7a1, renal Cyp24a1, VDR, Mdr1 and Mrp4, and intestinal Cyp24a1 and Trpv6 mRNA expression in both fxr(+/+) and fxr(‐/‐) mice compared to 1,25(OH)₂D₃ treatment. A similar induction in protein expression and microsomal activity of hepatic Cyp7a1 and renal P‐gp and Mrp4 protein expression was noted for both compounds. A higher intestinal induction of Trpv6 was observed, resulting in greater hypercalcemic effect following 1α(OH)D₃ treatment. The higher activity of 1α(OH)D₃ was explained by its rapid conversion to 1,25(OH)₂D₃ in tissue sites, furnishing higher plasma and tissue 1,25(OH)₂D₃ levels compared to following 1,25(OH)₂D₃‐treatment. In conclusion, 1α(OH)D₃ exerts a greater effect on VDR gene induction than equimolar doses of 1,25(OH)₂D₃ in mice. Copyright © 2013 John Wiley & Sons, Ltd.     

Pharmacological evidence that 5‐HT1D activation induces renal vasodilation by NO pathway in rats

5‐HT is a powerful vasoconstrictor substance in renal vasculature (mainly by 5‐HT₂ activation). Nevertheless, 5‐HT is notable for its dual cardiovascular effects, producing both vasodilator and vasoconstrictor actions. This study aimed to investigate whether, behind the predominant serotonergic vasoconstrictor action, THE 5‐HT system may exert renal vasodilator actions, and, if so, characterize the 5‐HT receptors and possible indirect pathways. Renal perfusion pressure (PP), systemic blood pressure (SBP) and heart rate (HR) measurement in in situ autoperfused rat kidney was determined in phenylephrine infused rats. Intra arterial (i.a.) bolus administration of 5‐HT (0.00000125–0.1 μg/kg) decreased renal PP in the presence of a phenylephrine continuous infusion (phenylephrine‐infusion group), without modifying SBP or HR. These vasodilator responses were potentiated by 5‐HT₂ antagonism (ritanserin, 1 mg/kg i.v.), whereas the responses were abolished by 5‐HT_1/7 antagonist (methiothepin, 100 μg/kg i.v.) or 5‐HT_1D antagonist (LY310762, 1 mg/kg i.v.). The i.a. administration (0.00000125 to 0.1 μg/kg) of 5‐CT or L‐694,247 (5‐HT_1D agonist) mimicked 5‐HT vasodilator effect, while other agonists (1‐PBG, α‐methyl‐5‐HT, AS‐19 (5‐HT₇), 8‐OH‐DPAT (5‐HT_1A) or CGS‐12066B (5‐HT_1B)) did not alter baseline haemodynamic variables. L‐694,247 vasodilation was abolished by i.v. bolus of antagonists LY310762 (5‐HT_1D, 1 mg/kg) or L‐NAME (nitric oxide, 10 mg/kg), but not by i.v. bolus of indomethacin (cyclooxygenase, 2 mg/kg) or glibenclamide (ATP‐dependent K⁺ channel, 20 mg/kg). These outcomes suggest that 5‐HT_1D activation produces a vasodilator effect in the in situ autoperfused kidney of phenylephrine‐infusion rats mediated by the NO pathway.     

Biochemical profile of anseculin (KA‐672) at different brain receptors

Anseculin (KA‐672) is a multifunctional antidementia agent under development that may be of therapeutic value in the treatment of progressive neuronal and cognitive decline, which are common in Alzheimer disease. In vitro and ex vivo experiments have characterized anseculin as a potent functional antagonist of central α₁‐adrenoceptors. In comparison to prazosin, the in vitro potency of anseculin for α₁‐adrenoceptors is about 8 times less. Like prazosin, anseculin shows an inhibition of phenylephrine‐ (norepinephrine‐) induced accumulation of inositol phosphates. Oral treatment with 1 mg/kg anseculin or 1 mg/kg prazosin show similar effects on α₁‐adrenergic receptor binding ex vivo, indicating a greater ex vivo potency of anseculin on central α₁‐adrenergic receptors. Anseculin has only weak affinity for central muscarinic cholinergic receptors. Anseculin inhibited adenylate cyclase and reduced intracelluar cAMP levels of about 30%, similar to the 5‐HT_1A agonist, 8‐OH‐DPAT. Together with its high affinity for 5‐HT_1A receptors (IC₅₀ for ³H‐8‐OH‐DPAT binding is about 8 nM), these data suggest that anseculin also exhibits 5‐HT_1A agonistic properties. Drug Dev. Res. 55:187–196, 2002. © 2002 Wiley‐Liss, Inc.     

Dopaminergic Involvement in Improving Effects of Dynorphin A-(1-13) on Galanin-Induced Impairment of Passive Avoidance Learning in Mice

The present study was designed to clarify whether dopaminergic systems are involved in the effects of dynorphin A-(1-13), an endogenous κ-opioid receptor agonist, on the galanin-induced impairment of passive avoidance learning in mice. Galanin (0·3 μg, i.c.v.) shortened step-down latency of passive avoidance learning, while the dopamine D₁ receptor agonist SKF 38393 (3 and 10 mg/kg, s.c.), the dopamine D₂ receptor agonist RU 24213 (0·3 and 1 mg/kg, s.c.), the dopamine D₁ receptor antagonist SCH 23390 (0·01 and 0·03 mg/kg, i.p.) or the dopamine D₂ receptor antagonist S(−)-sulpiride (10 and 30 mg/kg, i.p.) failed to influence it. Dynorphin A-(1-13) (3 μg, i.c.v.) and SKF 38393 (10 mg/kg, s.c.) markedly improved the galanin (0·3 μg, i.c.v.)-induced shortening of step-down latency. However, RU 24213 (0·3 and 1 mg/kg, s.c.), SCH 23390 (0·01 and 0·03 mg/kg, i.p.) or S(−)-sulpiride (10 and 30 mg/kg, i.p.) did not affect the galanin (0·3 μg, i.c.v.)-induced shortening of step-down latency. In contrast, SCH 23390 (0·3 mg/kg, i.p.) significantly reversed the improving effects of dynorphin A-(1-13) (3 μg, i.c.v.) on the galanin (0·3 μg, i.c.v.)-induced dysfunction of passive avoidance learning, although SKF 38393 (1 and 3 mg/kg, s.c.), RU 24213 (0·3 and 1 mg/kg, s.c.) or S(−)-sulpiride (10 and 30 mg/kg, i.p.) did not influence the effects of dynorphin A-(1-13) (3 μg, i.c.v.). These results suggest that dynorphin A-(1-13) improves the galanin-induced amnesia resulting from indirect stimulation of dopamine D₁ receptors. © 1997 John Wiley & Sons, Ltd.     

Optimization and pharmacological characterization of receptor‐mediated Gi/o activation in postmortem human prefrontal cortex

The biochemical abnormalities in transmembrane signal transduction mediated through G protein‐coupled receptors (GPCRs) have been postulated as underlying pathophysiology of psychiatric diseases such as schizophrenia and mood disorders. In the present study, the experimental conditions of agonist‐induced [³⁵S]GTPγS binding in postmortem human brain membranes were optimized, and the responses induced by a series of agonists were pharmacologically characterized. The [³⁵S]GTPγS binding assay was performed in postmortem human prefrontal cortical membranes by means of filtration techniques, and standardized as to GDP concentration, membrane protein content, MgCl₂ and NaCl concentrations in assay buffer, incubation period and effect of white matter contamination. Under the standard assay conditions, the specific [³⁵S]GTPγS binding was stimulated by the addition of 15 compounds in a concentration‐dependent manner. Of these agonists, R(+)‐8‐OH‐DPAT, UK‐14,304, DAMGO and DPDPE showed apparently biphasic concentration‐response curves. As for these four responses, only higher‐potency site was pharmacologically characterized. The receptors involved in the responses investigated were 5‐HT_1A receptor (probed with R(+)‐8‐OH‐DPAT or 5‐HT), α_2A‐adrenoceptor (UK‐14,304 or (?)‐epinephrine), M₂/M₄ mAChRs (carbachol), adenosine A₁ receptor (adenosine), histamine H₃ receptor (histamine), group II mGlu (l ‐glutamate), GABA_B receptor (baclofen), μ‐opioid receptor (DAMGO or endomophin‐1), δ‐opioid receptor (DPDPE or SNC‐80) and NOP (nociceptin). Although dopamine also activated specific [³⁵S]GTPγS binding, this response was likely mediated via α_2A‐adrenoceptor, but not dopamine receptor subtypes. The present study provides us with fundamental aspects of the strategy for elucidation of probable abnormalities of neural signalling mediated by G proteins activated through multiple GPCRs in the brain of psychiatric patients.     

5‐HT modulates the rat mesenteric vasopressor outflow by 5‐HT1D sympatholytic receptors

5‐hydroxytryptamine (5‐HT) modulates noradrenergic activity in different cardiovascular territories, but its effect on the mesenteric vasopressor outflow has not yet been clarified. This study investigated the in vivo serotonergic influence, characterizing 5‐HT receptors implicated, in sympathetic innervation of mesenteric vasculature. Wistar rats were anaesthetised and prepared for the in situ autoperfused rat mesentery, monitoring systemic blood pressure (SBP), heart rate (HR) and mesenteric perfusion pressure (MPP). Electrical stimulation of mesenteric sympathetic nerves resulted in frequency‐dependent increases in MPP (9 ± 1.6, 25.7 ± 3.9 and 60.2 ± 5 mmHg for 2, 4 and 8 Hz, respectively), without altering SBP or HR. 5‐HT (1‐25 μg/kg), 5‐carboxamidotryptamine (5‐HT_1/7 agonist; 25 μg/kg) or L‐694,247 (5‐HT_1D agonist; 1‐25 μg/kg) i.a. bolus inhibited vasopressor responses by mesenteric nerves electrical stimulation, unlike i.a. bolus of agonists 8‐OH‐DPAT (5‐HT_1A), CGS‐12066B (5‐HT_1B), BRL 54443 (5‐HT_1e/1F), α‐methyl‐5‐HT (5‐HT₂), 1‐PBG (5‐HT₃), cisapride (5‐HT₄) or AS‐19 (5‐HT₇) (25 μg/kg each). Interestingly, i.a. L‐694,247 (25 μg/kg) also reduced the exogenous norepinephrine‐induced vasoconstrictions. Pretreatment with selective 5‐HT_1D receptor antagonist, LY310762 (1 mg/kg, i.v.), completely abolished L‐694,247‐ and 5‐HT‐induced mesenteric sympathoinhibition. Furthermore, ELISA analysis confirmed 5‐HT_1D receptors expression in mesenteric artery. These findings suggest that serotonergic mechanisms‐induced sympathoinhibition of mesenteric noradrenergic outflow is mediated by pre and/or postjunctional 5‐HT_1D receptors.     

Effects of venlafaxine given repeatedly on α1‐adrenergic,dopaminergic and serotonergic receptors in rat brain

Venlafaxine (VEN), a representative of a new class of antidepressants (serotonin and noradrenaline reuptake inhibitors, SNRI), administered repeatedly affects—as was demonstrated by us previously—the behavioural responsiveness of α₁‐adrenergic, dopaminergic (D₂ and D₃) and serotonergic systems to their agonists. In the present study we aimed to find out whether parallel changes in the binding to the respective receptors also occurred. The experiment was carried out on male Wistar rats. VEN was administered in a dose of 10 mg/kg once or repeatedly (14 days, twice daily). The obtained results showed that VEN did not change the binding (B_max and K_D) of α₁‐adrenergic receptors to [³H]‐prazosin in the cerebral cortex, having increased only its displacement by phenylephrine. The binding (B_max and K_D) to D₁ and D₂ receptors in the limbic forebrain and the striatum was not affected by repeated venlafaxine when [³H];‐SCH 23390 and [³H]‐spiperone, respectively, were used as ligands. When [³H]‐quinpirole was used as a ligand, the binding was enhanced in the striatum, the nucleus accumbens (shell and core) and islands of Calleja. VEN also increased the binding of [³H]‐7‐OH‐DPAT to D₃ receptors in islands of Calleja and the nucleus accumbens (shell). In the serotonergic system, a decrease in the density of 5‐HT_1A receptors was observed in the hippocampus, whereas no changes occurred in the binding of 5‐HT₂ receptors in the cortex. Thus VEN given repeatedly enhanced the binding (of the ligands that are agonists) to dopamine D₂ and D₃ receptors. Weaker effects were observed in the α₁‐adrenergic and the serotonergic systems. Copyright © 1999 John Wiley & Sons, Ltd.     

Augmentative effect of spinosin on pentobarbital-induced loss of righting reflex in mice associated with presynaptic 5-HT1A receptor

Objectives This study investigated whether spinosin potentiates pentobarbital‐induced loss of righting reflex (LORR) in mice via 5‐HT_1A receptors. Methods Our primary endpoint for sedation was LORR. In addition, the basal rectal temperature was measured. Key findings The results demonstrated that the 5‐HT_1A agonist 8‐OH‐DPAT (s.c.) induced reductions in duration of LORR at 0.1, 0.5 and 1.0 mg/kg (P < 0.01), and prolongation of LORR latency at 0.5 and 1.0 mg/kg (s.c., P < 0.01) in pentobarbital (45 mg/kg, i.p.)‐treated mice. This effect of 8‐OH‐DPAT was antagonized either by 5‐HT_1A antagonist p‐MPPI (5 mg/kg, i.p.) or by spinosin (15 mg/kg, i.g.) with significance, respectively. Co‐administration of spinosin and p‐MPPI both at ineffective doses (spinosin at 5.0 mg/kg, i.g. and p‐MPPI at 1.0 mg/kg, i.p.) showed significant augmentative effects in reducing latency to LORR, and increasing LORR duration (P < 0.01) in pentobarbital‐treated mice. On the other hand, spinosin inhibited 8‐OH‐DPAT‐induced hypothermia, which has been generally attributed to the activation of somatodendritic 5‐HT_1A autoreceptors in mice. Conclusions Based on our previous results and the present data, it should be presumed that presynaptic 5‐HT_1A autoreceptor mechanisms may be involved in the inhibitory effect of spinosin on 8‐OH‐DPAT‐induced hypothermia and also in the potentiating effect of spinosin on pentobarbital‐induced LORR in mice.     

Synthesis of [2‐13C, 4‐13C]‐(2R,3S)‐catechin and [2‐13C, 4‐13C]‐(2R,3R)‐epicatechin

The first synthesis of doubly labeled, [2‐¹³C, 4‐¹³C]‐(2R,3S)‐catechin 15 and [2‐¹³C, 4‐¹³C]‐(2R,3R)‐epicatechin 18 starting from labeled 2‐hydroxy‐4, 6‐bis(benzyloxy)acetophenone 3 and labeled 3, 4‐bis(benzyloxy)‐benzaldehyde 7 are described. Copyright © 2010 John Wiley & Sons, Ltd.     

Functional reversal of (−)‐Stepholidine analogues by replacement of benzazepine substructure using the ring‐expansion strategy

(?)‐Stepholidine is an active ingredient of the Chinese herb Stephania and naturally occurring tetrahydroprotoberberine alkaloid with mixed dopamine receptor D₁ agonistic and dopamine receptor D₂ antagonistic activities. In this work, a series of novel hexahydrobenzo[4,5]azepino [2,1‐a]isoquinolines were designed and synthesized as ring‐expanded analogues of (?)‐Stepholidine. Initial pharmacological assays demonstrated that a benzazepine replacement was associated with significant increase in selectivity and functional reversal at dopamine receptor D₁. Compound‐(?)‐ 15e (K_i = 5.32 ± 0.01 nm ) is more potent than (?)‐Stepholidine (K_i = 13 nm ) and was identified as a selective dopamine receptor D₁ antagonist (IC₅₀ = 0.14 μm ). Moreover, molecular modeling suggested that (?)‐ 15e might exert its dopamine receptor D₁ antagonistic activities through interacting with the transmembrane helix 7 of dopamine receptor D₁.     

Synthesis and Evaluation of a Series of 3,4,5‐Trimethoxycinnamic Acid Derivatives as Potential Antinarcotic Agents

A series of 3,4,5‐trimethoxycinnamic acid derivatives was prepared and evaluated for antinarcotic effects on morphine dependence in mice and binding affinities on serotonergic receptors. The key synthetic strategies involve generation of ketones 6–7 , esters 9–12 through condensation reaction, and amides 13–19 via coupling reaction using 1‐hydroxybenzotriazole/ethyl(dimethylaminopropryl)carbodiimide system in high yield. We found that the naloxone‐induced morphine withdrawal syndrome was significantly suppressed by new synthetic 3,4,5‐trimethoxycinnamic acid derivatives (20 mg/kg/day). Most of 3,4,5‐trimethoxycinnamic acid derivatives were found to have high affinity to 5‐HT_1A receptor. The naloxone‐induced morphine withdrawal syndrome was attenuated by (+)8‐OH‐DPAT (0.1 mg/kg/day, i.p.), a 5‐HT_1A receptor agonist. In cortical neuronal cells, (+)8‐OH‐DPAT (1 μm ) produced an elevation of the pERK 1/2 expression, and the elevated pERK levels were inhibited by WAY 100635, a 5‐HT_1A receptor‐specific antagonist. Interestingly, the pERK levels were increased by the 3,4,5‐trimethoxycinnamic acid derivatives and the derivatives‐mediated changes in pERK levels were blocked by the WAY 100635. These results suggested that new synthetic 3,4,5‐trimethoxycinnamic acid derivatives have a potential antinarcotic effect through acting as a 5‐HT_1A receptor agonist in mice.     

Synthesis of C‐14 and C‐13, H‐2‐labeled IKK inhibitor: [14C] and [13C4,D3]‐N‐(6‐chloro‐7‐methoxy‐9H‐pyrido[3,4‐b]indol‐8‐yl)‐2‐methyl‐3‐pyridinecarboxamide

[¹⁴C]‐N‐(6‐Chloro‐7‐methoxy‐9H‐pyrido [3,4‐b]indol‐8‐yl)‐2‐methyl‐3‐pyridinecarboxamide (5B ), an IKK inhibitor, was synthesized from [¹⁴C]‐barium carbonate in two steps in an overall radiochemical yield of 41%. The intermediate, [carboxyl‐¹⁴C]‐2‐methylnicotinic acid, was prepared by the lithiation and carbonation of 3‐bromo‐2‐methylpyridine. [¹³C₄,D₃]‐N‐(6‐chloro‐7‐methoxy‐9H‐pyrido [3,4‐b]indol‐8‐yl)‐2‐methyl‐3‐pyridinecarboxamide (5C ) was synthesized from [1,2,3,4‐¹³C₄]‐ethyl acetoacetate and [D₄]‐methanol in six steps in an overall yield of 2%. [¹³C₄]‐2‐methylnicotic acid, was prepared by condensation of [¹³C₄]‐ethyl 3‐aminocrotonate and acrolein, followed by hydrolysis with lithium hydroxide. Copyright © 2006 John Wiley & Sons, Ltd.     

Synthesis and Anticonvulsant Properties of New Mannich Bases Derived from 3‐Aryl‐pyrrolidine‐2,5‐diones. Part 1

A series of new Mannich bases of N‐[(4‐arylpiperazin‐1‐yl)‐methyl]‐3‐(chlorophenyl)‐pyrrolidine‐2,5‐diones 10–23 have been synthesized and evaluated for their anticonvulsant activity in maximum electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure threshold tests. Their neurotoxicity was determined using a rotorod screen. Several molecules showed a promising anticonvulsant profile especially in the MES‐test. In this model of seizures, the most active were N‐[{4‐(4‐chlorophenyl)‐piperazin‐1‐yl}‐methyl]‐3‐(3‐chlorophenyl)‐pyrrolidine‐2,5‐dione 16 and N‐[{4‐(3‐trifluoromethylphenyl)‐piperazin‐1‐yl}‐methyl]‐3‐(3‐chlorophenyl)‐pyrrolidine‐2,5‐dione 17 with ED₅₀ values of 21.4 mg/kg and 28.83 mg/kg, respectively. Selected derivatives 10 , 14 , and 16 were tested in the psychomotor seizure 6‐Hz test from which N‐[{4‐(2‐chlorophenyl)‐piperazin‐1‐yl}‐methyl]‐3‐(2‐chlorophenyl)‐pyrrolidine‐2,5‐dione 10 revealed the highest protection with an ED₅₀ of 78 mg/kg. Compounds 10 , 12 , and 17 were also tested in the pilocarpine‐induced status PIPS test. Furthermore, 17 was examined in the hippocampal kindling screen after i. p. administration to rats.     

Syntheses and radiofluorination of two derivatives of 5‐cyano‐indole as selective ligands for the dopamine subtype‐4 receptor

Two fluoroethoxy substituted derivatives, namely 2‐[4‐(2‐(2‐fluoroethoxy)phenyl)‐piperazin‐1‐ylmethyl]indole‐5‐carbonitrile ( 5a ) and 2‐[4‐(4‐(2‐fluoroethoxy)‐phenyl)piperazin‐1‐ylmethyl]indole‐5‐carbonitrile ( 5b ) were synthesized as analogs of the selective D₄ receptor ligand 2‐[4‐(4‐fluorophenyl)piperazin‐1‐ylmethyl]indole‐5‐carbonitrile (FAUC 316). In vitro characterization using CHO‐cells expressing different dopamine receptor subtypes gave K_i values of 2.1 ( 5a ) and 9.9 nM ( 5b ) for the dopamine D₄ subtype and displayed a 420‐fold D₄‐selectivity over D₂ receptors for 5b . The para‐fluoroethoxy substituted candidate 5b revealed substantially reduced α₁ and serotoninergic binding affinities in comparison to the ortho‐fluoroethoxy substituted compound. In order to provide potential positron emission tomography (PET) imaging probes for the dopamine D₄ receptor, ¹⁸F‐labelling conditions using [¹⁸F]fluoroethyl tosylate were optimized and led to radiochemical yields of 81 ± 5% ( [¹⁸F]5a ) and 47 ± 4% ( [¹⁸F]5b ) (n = 3, decay‐corrected and referred to labelling agent), respectively. Thus, ¹⁸F‐fluoroethylation favourably at the para position of the phenylpiperazine moiety of the 5‐cyano‐indole framework proved to be tolerated by D₄ receptors and could also be applied to alternative scaffolds in order to develop D₄ radioligand candidates for PET with improved D₄ receptor affinity and selectivity. Copyright © 2005 John Wiley & Sons, Ltd.     

1α,25‐dihydroxyvitamin D3 triggered vitamin D receptor and farnesoid X receptor‐like effects in rat intestine and liver in vivo

1α,25‐Dihydroxyvitamin D₃ (1,25(OH)₂D₃), a natural ligand of the vitamin D receptor (VDR), was found to increase the rat ileal Asbt and bile acid absorption. The effects of VDR, whose expression is low in liver, on hepatic transporters and enzymes are unknown. Protein and mRNA levels of target genes in the small intestine, colon and liver after intraperitoneal dosing of 1,25(OH)₂D₃ (0–2.56 nmol/kg/day for 4 days) to the rat were determined by Western blotting and qPCR, respectively. The 1,25(OH)₂D₃ treatment increased total Cyp3a protein and Cyp3a1 mRNA expressions in the proximal small intestine, and the short heterodimer partner (SHP), the fibroblast growth factor 15 (FGF15), organic solute transporter (Ostα and Ostβ) mRNA and Asbt protein expressions in the ileum. About 50% higher portal bile acid concentration (65.1±14.9 vs 41.9±7.8 µm , p<0.05) and elevated expressions of the hepatic farnesoid X receptor (FXR) and SHP mRNA resulted with 1,25(OH)₂D₃ treatment. Increased Bsep and Ostα mRNA expressions in liver and a>50% reduction in the Cyp7a1 protein level (p<0.05) and cholesterol metabolism in rat liver microsomes (p=0.002), likely consequences of the bile acid‐FXR‐SHP cascade and activation of the signaling pathway for Cyp7a1 inhibition by FGF15, were found. Increased hepatic multidrug resistance‐associated protein (Mrp3) and multidrug resistance protein 1a (Mdr1a) mRNA and P‐gp protein were also observed. It was concluded that the changes in hepatic transporters and enzymes in the rat were indirect, secondary effects of the liver FXR‐SHP cascade due to increased intestinal absorption of bile acids and elevated levels of FGF15, events that led to the activation of FXR. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of [123I]‐3‐(4‐iodobenzyl)‐1,2,3,4‐tetrahydro‐8‐hydroxychromeno[3,4‐c]pyridin‐5‐one,a potential dopamine D4 ligand for SPECT studies

Schizophrenia is a devastating mental disorder characterized by relapsing psychotic episodes accompanied with emotional, professional and social decline. The classical dopamine hypothesis of schizophrenia postulates that hyperactivity of dopaminergic neurotransmission is responsible for the positive symptoms of the disorder, more exactly hyperactivity of the dopamine D₂‐like receptors. One of these receptors is the D₄ receptor which is thought to be involved in the motor side‐effects caused antipsychotics. However, research into the specific role of this receptor has been hampered by the lack of specific ligands. Therefore, a new ¹²³I‐labelled compound was developed which may allow in vivo visualization of the D₄ receptor by SPECT. [¹²³I]‐3‐(4‐iodobenzyl)‐1,2,3,4‐tetrahydro‐8‐hydroxychromeno[3,4‐c]pyridin‐5‐one was prepared by electrophillic aromatic substitution of the tributylstannyl derivative. The radiochemical yield was 68±3% (n=5) and the specific activity was >2.96 Ci/µmol. Copyright © 2005 John Wiley & Sons, Ltd.