Protection against cocaine toxicity in mice by the dopamine D3/D2 agonist R-(+)-trans-3,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol [(+)-PD 128,907].

Cocaine abuse is a public health concern with seizures and death being one consequence of overdose. In the present study, dopamine D(3/)D(2) receptor agonists dose dependently and completely prevented the convulsant and lethal effects of cocaine. The D(3)-preferring agonists R-(+)-trans-3,4a,10b-tetrahydro-4-propyl-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol) [(+)-PD 128,907], (+)-7-hydroxy-dipropylaminotetralin, and the mixed D(3/)D(2) agonists quinpirole and quinelorane were all effective against cocaine toxicity in mice. The anticonvulsant effects of these compounds occurred at doses below those that produced motor impairment as assessed in the inverted screen test. Protection against the convulsant effects of the selective dopamine uptake inhibitor 1-[2-[bis(4-fluorophenyl)methoxy] ethyl]-4-[3-phenyl-propyl]piperazine (GBR 12909) was also conferred by (+)-PD 128,907. The possible selectivity of the effects of (+)-PD 128,907 (3 mg/kg) for these dopaminergic compounds was demonstrated by its general lack of protective efficacy against a host of convulsants acting through other neural mechanisms [pentylenetetrazol, (+)-bicuculline, and picrotoxin, 4-aminopyridine, and t-butylbiclyclophosphoorothionate, N-methyl-d-aspartate, kainate, pilocarpine, nicotine, strychnine, aminophylline, threshold electric shock, and 6-Hz electrical stimulation]. Direct and correlational evidence suggests that these effects were mediated by D(3) receptors. Protection was stereospecific and reversible by an antagonist of D(3) receptors [3-[4[1-(4-[2[4-(3-diethyamino-propoxy)-phenyl]-benzoimidazol-l-yl]-butyl)-1H-benzoimidazol-2-yl]-phenoxy]-propyl)-diethyl-amine; PD 58491] but not D(2) receptors [3[[4-(4-chlorophenyl)-4 hydroxypipeidin-1-yl]methyl-1H-indole; L-741,626]. Anticonvulsant potencies were positively associated with potencies in a functional assay of D(3) but not D(2) receptor function. Together, these findings suggest that the prevention of cocaine convulsions and lethality by (+)-PD 128,907 may be due to D(3) receptor-mediated events.

Neuropharmacological profile of a selective sigma ligand, igmesine: a potential antidepressant.

Igmesine is a selective sigma (sigma(1)) ligand that was reported to exert antidepressant action through an unknown mechanism of action. A number of neurochemical measures were taken in this study in efforts to understand its mode of action. Following 21-day drug treatments, the actions of igmesine on a number of neurochemical measures were investigated. Data obtained showed significant decreases in the densities of beta-adrenergic but not 5-HT(1A), sigma(1) and GABA(B) receptors in fluoxetine (18%), desipramine (DMI, 32%) and igmesine (20%)-treated groups when compared with control. Tyrosine hydroxylase (TH) activity was significantly (30-32%) reduced in all treated groups. Further, fluoxetine and DMI excluding the igmesine-treated groups showed 85 and 40% reductions in serotonin (5-HT) and noradrenaline (NE) neuronal uptake, respectively. Following acute treatment, igmesine lacked activity for monoamine oxidase (MAO) A or B (IC(50)>10 microM). In in vivo studies, at behaviorally active doses, igmesine showed weak effects on the NE uptake but lacked activity in altering 5-HT and DA synthesis or antagonizing selective drug-induced depletion of monoamine neuronal uptake. N-methyl-D-aspartate (NMDA)-induced increases in cGMP was blocked by igmesine indicating that igmesine may interfere with the NMDA receptor/nitric oxide synthase/cGMP pathway. Although it appears that part of the pharmacological actions of igmesine is mediated by the monoaminergic system, there is still need to explore other possible mechanisms of antidepressant action.

PD 158771, a potential antipsychotic agent with D(2)/D(3) partial agonist and 5-HT(1A) agonist actions. I. Neurochemical effects.

The neurochemical effects of a novel dopamine (DA) D(2)-like and serotonin (5-HT) 5-HT(1A) agonist, PD 158771, are described. PD 158771 exhibited affinities for human D(2L), D(3) and D(4.2) receptors expressed in Chinese hamster ovary (CHO)-K1 cells with K(i) (nM) values of 5.2, 13.7 and 34.8 respectively. PD 158771 showed high affinity for cloned human 5-HT(1A) (K(i) = 2.6 nM) and rat hippocampal 5-HT(1A) receptors (K(i) = 3.5 nM). Weaker affinities were observed at alpha 1-adrenergic (K(i) = 43 nM), histamine H(1) (IC(50) = 30 nM), 5-HT(2A) (K(i) = 24.5 nM) and sigma (sigma) -1 binding sites (K(i) = 24.5 nM). In measures of in vitro functional activity, PD 158771 stimulated [(3)H]thymidine uptake in CHO p-5 cells transfected with hD(3) receptors with a maximal effect of 23% relative to quinpirole. In hD(2)L, the corresponding value was 60% with an EC(50) of 29 nM, again indicating partial DA agonist action of PD 158771. In vivo, PD 158771 produced a dose-related decrease in DA synthesis in the striatum and mesolimbic regions of rat brain treated with gamma-butyrolactone (GBL), indicating a DA autoreceptor agonist action. In animals not treated with GBL, PD 158771 produced a dose-related decrease in DA synthesis and extracellular DA. A decrease in 5-HT synthesis in several brain areas was observed consistent with an agonist response. Further support for DA autoreceptor agonist action is that PD 158771 produced a partial inhibition of the firing of substantia nigra zona compacta DA neurons, an effect reversed by haloperidol. In conclusion, PD 158771 exhibited affinities for DA and 5-HT receptors, appears to possess DA and 5-HT agonist actions; and it could provide improved antipsychotic profile with minimal side effects.

A series of 6- and 7-piperazinyl- and -piperidinylmethylbenzoxazinones with dopamine D4 antagonist activity: discovery of a potential atypical antipsychotic agent.

As part of a program to develop dopamine D4 antagonists for the treatment of schizophrenia, we discovered a series of 6- and 7-(phenylpiperazinyl)- and -(phenylpiperidinyl)methylbenzoxazinones through mass screening of our compound library. A structure-activity relationship SAR study was carried out involving substituents on the phenyl ring, and several selective D4 antagonists were identified. The 7-substituted benzoxazinones showed more activity in neurochemical and behavioral tests than the 6-substituted series. One of the most potent and selective compounds (26) was found to have potent activity in animal tests predictive of antipsychotic activity in humans after oral administration. This paper describes the SAR of the benzoxazinone series and the preclinical characterization of 26.

Pharmacological characterization of PD 152255, a novel dimeric benzimidazole dopamine D3 antagonist.

152255 (E-1,1'-(2-butene-1,4-diyl)bis[2-[4-[3-(1-piperidinyl)propoxy]-phe nyl]-1H-benzimidazole]) exhibited high affinity (Ki = 12.7 nM) for human dopamine (DA) D3 receptors expressed in CHO K1 cells but not for DA D2L receptors (Ki = 565 nM), DA D42 or DA D1 receptors (Ki > 3 microM) and a number of other neurotransmitter receptors. Affinity for human muscarinic receptors was seen in vitro but no functional muscarinic agonist and/or antagonist action was observed in vivo. Antagonist activity at DA D3 receptors was demonstrated by blockade of quinpirole-stimulated [3H]-thymidine uptake in D3 transfected cells, an effect that was 28-fold more potent than in D2-transfected cells. Unlike classical DA D2 antagonists, PD 152255 did not increase rat brain DA synthesis and it increased locomotion in habituated rats. However, like antipsychotics, PD 152255 reduced locomotor activity in mice and reduced spontaneous and amphetamine-stimulated locomotion in nonhabituated rats. These results demonstrate that PD 152255 is a DA D3 antagonist that may have antipsychotic activity.

Effects of the dopamine D3 antagonist PD 58491 and its interaction with the dopamine D3 agonist PD 128907 on brain dopamine synthesis in rat.

The dopamine (DA) D3 receptor antagonist PD 58491 [3-[4-[1-[4-[2-[4-(3-diethylaminopropoxy)phenyl]benzoimidazol++ +-1-yl-butyl]-1H-benzoimidazol-2-yl]phenoxy]propyl]diethylamine] bound with high affinity and selectivity to recombinant human DA D3 versus D2L and D4.2 receptors transfected into Chinese hamster ovary cells: Ki values of 19.5 nM versus 2,362 and >3,000 nM, respectively. In contrast, the putative DA D3 receptor antagonist (+)-AJ76 displayed low affinity and selectivity for D3 versus D2L and D4.2 receptors (91 nM vs. 253 and 193 nM, respectively). In vitro, PD 58491 (1 nM-1 microM) exhibited D3 receptor antagonist activity, reversing the quinpirole (10 nM)-induced stimulation of [3H]thymidine uptake in D3 CHOpro-5 cells, but did not have any significant intrinsic activity by itself in this assay. PD 58491 did not decrease the gamma-butyrolactone-induced increase in DA synthesis (L-3,4-dihydroxyphenylalanine accumulation) in rat striatum, indicating that the compound possessed no in vivo DA D2/D3 receptor agonist action at DA autoreceptors. PD 58491 (3-30 mg/kg, i.p.) generally did not alter DA or serotonin synthesis in either the striatum or mesolimbic region of rat brain. The D3-preferring agonist PD 128907 decreased DA synthesis in striatum and mesolimbic regions, and this effect was attenuated by pretreatment with PD 58491. These findings support the hypothesis that DA D3 autoreceptors may in part modulate the synthesis and release of DA in striatum and mesolimbic regions.

Behavioral and neurochemical effects of acute and repeated administration of triadimefon in the male rat.

The effects of triadimefon (TDF) were examined in male Sprague-Dawley rats. In this study, the acute administration of TDF (100 mg/kg) was found to significantly increase locomotor activity and induce stereotyped behavior. Acute administration of TDF was also found to significantly increase dopamine (DA) and homovanillic acid (HVA) levels while the dihydroxyphenylacetic acid (DOPAC) level remained unchanged in both the nucleus accumbens (NA) and striatal (ST) tissues when compared to control. Furthermore, DOPAC:DA ratios were significantly reduced in both brain regions suggesting an increase in DA turn overrate. On the other hand, in animals receiving repeated TDF administration, only the HVA level was significantly increased in both the ST and NA. TDF neither competed for binding to D2, D3 or D4 DA receptors nor altered the Kd or the Bmax of [3H] SCH 23390 and [3H] spiperone recognition sites associated with striatal D1 and D2 receptors, respectively. Meanwhile, TDF competed with [3H] GBR 12935 for binding to DA transporter sites with strong affinity, but repeated treatment with TDF had no sustained or cumulative effect on the DA transporter system. These results clearly show that acute TDF-induced behavioral effects may not be via binding to DA receptors, but through the interaction with DA transporter binding sites. Also, TDF does not appear to produce cumulative effects in the parameters evaluated.

The pharmacology of the novel and selective sigma ligand, PD 144418.

The pharmacology of PD 144418 (1-propyl-5-(3-p-tolyl-isoxazol-5-yl)-1,2,3,6-tetrahydropyridine) was characterized using neurochemical, biochemical and behavioral techniques. For sigma (sigma 1 and sigma 2 respectively) sites, PD 144418 affinities were determined using whole guinea pig brain membranes with [3H](+)-pentazocine and neuroblastoma x glioma cell membranes using [3H]1,3,di-O-tolylguanidine (DTG) in the presence of 200 nM (+)-pentazocine. PD 144418 exhibited an affinity for sigma 1 of 0.08 nM (Ki) versus a K1 of 1377 nM for sigma 2 site. Additional receptor binding studies indicated that PD 144418 lacked affinity for dopaminergic, adrenergic, muscarinic and a variety of other receptors. In vitro studies indicated that PD 144418 reversed the N-methyl-D-aspartate (NMDA)-induced increase in cyclic GMP (cGMP) in rat cerebellar slices without affecting the basal levels, suggesting that sigma 1 sites may be important in the regulation of glutamine-induced actions. PD 144418 potentiated the decrease in 5-hydroxytryptophan caused by haloperidol in the mesolimbic region, but by itself had no effect in 5-hydroxytrypamine (5-HT) and dopamine (DA) synthesis. Behaviorally, similar to other sigma ligands, PD 144418 antagonized mescaline-induced scratching at doses that did not alter spontaneous motor activity. This action is suggestive of potential antipsychotic property. It exhibited no anxiolytic and antidepressant properties in the models used. These results show that PD 144418 is a very selective sigma 1 agent, devoid of any significant affinity for other receptors and that sigma 1 site may modulate actions in the CNS.

Affinity for dopamine D2, D3, and D4 receptors of 2-aminotetralins. Relevance of D2 agonist binding for determination of receptor subtype selectivity.

A series of 2-aminotetralins, substituted with a methoxy or a hydroxy group on the 5- or 7-position, and with varying N-alkyl or N-arylalkyl substituents, were prepared and evaluated in binding assays for human dopamine (DA) D2, D3, and D4 receptors. Some members of this series were prepared in former studies, but were never tested in vitro with single receptor subtypes, and these were examined again. None of the tested 2-aminotetralins showed high affinity for the dopamine D4 receptor. However, a number of the 2-aminotetralins showed high affinity for both the D2 and the D3 DA receptors, as exemplified by compounds 11-15 and 21-26, while some had a reasonable selectivity for the DA D3 receptors. The affinities of the 2-aminotetralins for the D21, receptor depended on the type of radioligand (agonist or antagonist) used. The agonist affinity data, obtained by using the agonist ligand [3H]N-0437, are thought to be more relevant for calculating DA receptor subtype selectivity.

Aryl 1-but-3-ynyl-4-phenyl-1,2,3,6-tetrahydropyridines as potential antipsychotic agents: synthesis and structure-activity relationships.

A novel series of aryl 1-but-3-ynyl-4-phenyl-1,2,3,6-tetrahydropyridines with dopaminergic activity is described. The structure-activity relationships of this series were studied by synthesis of analogs and evaluation of their affinities for the dopamine (DA) D2 receptor and inhibition of locomotor activity (LMA) in rodents. The basic amine, alkyne chain length, and aryl groups were varied. Compounds having a 4-phenyl-1,2,3,6-tetrahydropyridine and an aryl group with hydrogen-bonding substituents separated by a butynyl chain were found to have the most potent dopaminergic activity. Several compounds that were found to have exceptional in vivo activity in LMA inhibition in rodents were evaluated for additional pharmacological activity including binding affinities for other DA receptor subtypes as well as effects on brain DA synthesis, DA neuronal firing, and conditioned avoidance responding in squirrel monkeys.

Functional activity of new C-terminal cyclic-neurotensin fragment analogs.

Neurotensin (NT, pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) is a tridecapeptide that displays a wide spectrum of biological actions. Cyclic derivatives of a hexapeptide NT [(8-13)] (N alpha MeArg-Lys-Pro-Trp-Tle-Leu, Tle = tert-leucine) were designed and prepared by a combination of solution and solid-phase peptide synthetic methodologies. As reported previously, several analogs possessed nanomolar binding affinities for NT receptors in newborn (10-day-old) mouse brain membrane preparations. In this study, we determined the functional ability of these analogs to mobilize intracellular free calcium, [Ca2+]i, in HT-29 cells (human colonic adenocarcinoma). Of greatest interest were the cyclic compounds 2, 6 and 9 that had Ki values of 0.19, 3.50 and 4.18 microM for [3H]NT labeled receptors in the HT-29 cell membrane assay, respectively. In the functional assay, compounds 2 and 6 mobilized [Ca2+] with EC50 values of 0.13 and 20 microM, respectively. In comparison, Compound 9 blocked the NT-induced mobilization of [Ca2+]i, with an IC50 of 1.70 microM. The present findings indicate that small molecule cyclic analogs, that possess functional activity, can be designed and may have therapeutic utility in the treatment of schizophrenia and possibly other neurological disorders.

Neurochemical and functional characterization of the preferentially selective dopamine D3 agonist PD 128907.

The present study determined the biochemical and pharmacological effects of PD 128907 [R-(+)-trans-3,4,4a,10b-tetrahydro-4-propyl-2H,5H- [1]benzopyrano[4,3-b]-1,4-oxazin-9-ol], a dopamine (DA) receptor agonist that shows a preference for the human D3 receptor. In transfected Chinese hamster ovary cells (CHO K1), PD 128907 displaced [3H]spiperone in a biphasic fashion which fit best to a two-site model, generating Ki values of 20 and 6964 nM for the high- and low-affinity sites for the D2L receptors and 1.43 and 413 nM for the corresponding sites for the D3 receptors. Addition of sodium and the GTP analog Gpp(NH)p to both the D2L and D3 caused a modest reduction in the affinity of the compound suggestive of an agonist type action. In agonist binding ([3H]N-0437), PD 128907 exhibited an 18-fold selectivity for D3 versus D2L, a selectivity similar to that found with antagonist binding to the high-affinity sites. PD 128907 exhibited only weak affinity for D4.2 receptors (Ki = 169 nM). No significant affinity for a variety of other receptors was observed. PD 128907 stimulated cell division (measured by [3H]thymidine uptake) in CHO p-5 cells transfected with either D2L or D3 receptors exhibiting about a 6.3-fold greater potency in activating D3 as compared to D2L receptors. In vivo the compound was active in reducing DA synthesis both in normal and gamma-butyrolactone (GBL) treated rats; in the GBL model, the decrease was greater in the higher D3-expressing mesolimbic region as compared with striatum which has a lower expression of D3 receptors. PD 128907 decreased DA release (as measured by brain microdialysis) both in rat striatum, nucleus accumbens and medial frontal cortex, as well as in monkey putamen. Behaviorally PD 128907 decreased spontaneous locomotor activity (LMA) in rats at low doses, whereas at higher doses stimulatory effects were observed. PD 128907 at high doses reversed the reserpine-induced decrease in LMA and induced stereotypy in combination with the D1 agonist SKF 38393 indicating postsynaptic DA agonist actions. It is unclear which of the subtypes of DA receptors might be mediating the pharmacological effects of PD 128907. However, the present findings indicating that PD 128907 shows a preference for DA D3 over D2L and D4.2 receptors indicates that its action at low doses may be due to interaction with D3 receptors and at higher doses, with both D2 and D3 receptors.

CI-1007, a dopamine partial agonist and potential antipsychotic agent. I. Neurochemical effects.

The receptor binding and biochemical effects of the putative dopamine (DA) partial agonist CI-1007 ([R(+)-1,2,3,6-tetrahydro-4-phenyl- 1-[(3-phenyl-3-cyclohexen-1-yl)methyl]pyridine] maleate) and potential antipsychotic were evaluated with a variety of biochemical methods. In receptor binding studies, CI-1007 bound to rat striatal DA receptors exhibiting a Ki of 3 nM as assessed by inhibition of [3H]N-propylnorapomorphine binding. CI-1007 also exhibited high affinity for cloned human D2L (Ki = 25.5 nM) and D3 (Ki = 16.6 nM) receptors with less affinity for D4.2 receptors (Ki = 90.9 nM). The affinity for serotonin-1A (5-HT-1A), alpha-2 adrenergic and 5-HT-2 receptors was moderate (submicromolar range) and slight or negligible for alpha-1, DA D1 and various other receptors. Unlike dopamine, the inhibition of [3H]spiperone binding was monophasic for CI-1007 and only slightly affected by the addition of Gpp-(NH)p. In vitro CI-1007 antagonized the forskolin-induced increases in cyclic AMP levels in GH4C1 cells expressing the human D2L receptor, having an intrinsic activity of 53% of that seen with the full agonist quinpirole. In vivo CI-1007 antagonized the gamma-butyrolactone (GBL)-induced accumulation of L-3,4-dihydroxyphenylalanine in striatum and mesolimbic regions of rat brain, causing a maximal 64% reversal in striatum, consistent with a partial agonist profile. In microdialysis studies it decreased DA overflow in both striatum and nucleus accumbens, indicating decreased release of DA. CI-1007 also reduced brain DA synthesis (DOPA accumulation), metabolism (DOPAC and HVA) and utilization (after tyrosine hydroxylase inhibition with alpha-methyl-p-tyrosine). CI-1007 did not affect striatal acetylcholine levels indicating lack of potent postsynaptic DA actions. CI-1007 seemed to be selective for DA neurons as it did not alter rat brain norepinephrine (NE) synthesis in the NE-enriched brainstem or NE utilization in the mesolimbic region. In addition, it did not affect in general 5-HT synthesis and metabolism in striatum and mesolimbic regions. These neurochemical results demonstrate that CI-1007 is a selective potent brain dopamine partial agonist with limited agonist activity at postsynaptic DA receptors.

Behavioral and neurochemical changes in the dopaminergic system after repeated cocaine administration.

In order to determine whether repeated cocaine administration produced persistent changes in dopamine (DA) receptor binding and release consistent with behavioral sensitization, rats were treated with either cocaine (25 mg/kg ip) or saline twice daily for 14 consecutive days followed by a 3-d withdrawal period. The DA transporter site was assayed using [3H]GBR 12935, whereas D1 and D2 sites were assayed using [3H]SCH 23390 and [3H]spiperone, respectively. The density (Bmax) of the DA transporter binding sites in the ST of the cocaine-treated group increased significantly (p < 0.05) over controls 3 d after the last injection, whereas the density of striatal D1 and D2 binding sites remained unchanged. The DA transporter in the nucleus accumbens (NA) was also studied with [3H]GBR 12935 and was unchanged following drug treatment. D1 and D2 binding parameters for the NA were not determined in this study. Furthermore, cocaine administration did not affect the affinities (Kd) of the radioligands used to label the transporter, D1, or D2 sites in any of the studies performed. In addition, striatal DA release was measured using in vivo microdialysis in anesthetized rats. Linear regression analysis on maximal decreases in DA release after apomorphine (0.02, 0.2, and 2.0 mg/kg sc) injection showed no difference in the functional capacity of the ST to modulate DA transmission between control and treated groups. Moreover, animals pretreated with cocaine showed a significant (p < 0.01) decrease in locomotor activity (LA) after a presynaptic, autoregulating dose of apomorphine (0.03 mg/kg sc) was given. These results suggests that the effects seen after repeated exposure to cocaine may be regulated, in part, by changes in striatal DA transporter binding site densities and not necessarily by DA-releasing mechanisms or D1 and D2 receptor modification.

Agonist properties of a stable hexapeptide analog of neurotensin, N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1).

The major signal transduction pathway for neurotensin (NT) receptors is the G-protein-dependent stimulation of phospholipase C, leading to the mobilization of intracellular free Ca2+ ([Ca2+]i) and the stimulation of cyclic GMP. We investigated the functional actions of an analog of NT(8-13), N alpha MeArg-Lys-Pro-Trp-tLeu-Leu (NT1), and other NT related analogs by quantitative measurement of the cytosolic free Ca2+ concentration in HT-29 (human colonic adenocarcinoma) cells using the Ca(2+)-sensitive dye fura-2/AM and by effects on cyclic GMP levels in rat cerebellar slices. The NT receptor binding affinities for these analogs to HT-29 cell membranes and newborn (10-day-old) mouse brain membranes were also investigated. Data obtained from HT-29 cell and mouse brain membrane preparations showed saturable single high-affinity sites and binding densities (Bmax) of 130.2 and 87.5 fmol/mg protein, respectively. The respective KD values were 0.47 and 0.39 nM, and the Hill coefficients were 0.99 and 0.92. The low-affinity levocabastine-sensitive site was not present (K1 > 10,000) in either membrane preparation. Although the correlation of binding between HT-29 cell membranes and mouse brain membranes was quite significant (r = 0.92), some of the reference agents had lower binding affinities in the HT-29 cell membranes. The metabolically stable compound NT1 plus other NT analogs and related peptides [NT, NT(8-13), xenopsin, neuromedin N, NT(9-13), kinetensin and (D-Trp11)-NT] increased intracellular Ca2+ levels in HT-29 cells, indicating NT receptor agonist properties. The effect of NT1 in mobilizing [Ca2+]i blocked by SR 48692, a non-peptide NT antagonist. Receptor binding affinities of NT analogs to HT-29 cell membranes were positively correlated with potencies for mobilizing intracellular calcium in the same cells. In addition, NT1 increased cyclic GMP levels in rat cerebellar slices, confirming the latter findings of its NT agonist action. These results substantiate the in vitro NT agonist properties of the hexapeptide NT analog NT1.

Design and structure-activity relationships of C-terminal cyclic neurotensin fragment analogues.

Neurotensin (NT) is a linear tridecapeptide with a broad range of central and peripheral pharmacological effects. The C-terminal hexapeptide of NT (NT8-13) has been shown to possess similar properties to NT itself, and in fact, an analogue of NT8-13 (N alpha MeArg8-Lys-Pro-Trp-Tle-Leu13, Tle = tert-leucine) has been reported to possess central activity after peripheral administration. Cyclic derivatives of this hexapeptide were synthesized by a combination of solution and solid-phase peptide synthetic methodologies, and several analogues had low nanomolar binding affinity for the NT receptor. In particular, cyclo[Arg-Lys-Pro-Trp-Glu]-Leu (cyclized between the alpha amine of Arg and the gamma carboxylate of Glu) possessed 16 nM NT receptor affinity and was determined to be an agonist in vitro. 1H-NMR and 13C-edited 1H-NMR spectroscopy were performed on this and related cyclic analogues to help identify structural properties which may be important for receptor recognition. These cyclic peptides represent novel molecular probes to further investigate NT receptor pharmacology, as well as to advance our understanding of the structure-conformation relationships of NT and to help establish a working basis for additional pharmacophore mapping studies.

Characterization of binding of [3H]PD 128907, a selective dopamine D3 receptor agonist ligand, to CHO-K1 cells.

PD 128907 [4a R, 10 b R-(+)-trans-3, 4, 4a, 10 b - tetrahydro - 4- n -propyl2 H,5H-[1]benzop-yrano[4,3-b]1,4-oxazin-9-ol.], a selective dopamine (DA) D3 receptor agonist ligand exhibits about a 1000-fold selectivity for human D3 receptors (Ki, 1 nM) versus human D2 receptors (Ki, 1183 nM) and a 10000-fold selectivity versus human D4 receptors (Ki, 7000 nM) using [3H]spiperone as the radioligand in CHO-K1-cells. Studies with [3H]PD 128907, showed saturable, high affinity binding to human D3 receptors expressed in CHO-K1 cells (CHO-K1-D3) with an equilibrium dissociation constant (Kd) of 0.99 nM and a binding density (Bmax) of 475 fmol/mg protein. Under the same conditions, there was no significant specific binding in CHO-K1-cells expressing human D2 receptors (CHO-K1-D2). The rank order of potency for inhibition of [3H]PD 128907 binding with reference DA agents was consistent with reported values for D3 receptors. These results indicate that [3H]PD 128907 is a new, highly selective D3 receptor ligand with high specific activity, high specific binding and low non-specific binding and therefore should be useful for further characterizing the DA D3 receptors.

Differential effects of the nonpeptide neurotensin antagonist, SR 48692, on the pharmacological effects of neurotensin agonists.

In in vitro studies, SR 48692, a nonpeptide neurotensin receptor antagonist, inhibited the binding of [3H] or [125I]neurotensin to membrane preparations from 10-day-old mouse brains and from HT-29 cells with Ki values of 3.9 and 8.6 nM, respectively. SR 48692 also antagonized the neurotensin-induced mobilization of intracellular calcium in HT-29 cells, in agreement with previous findings. In rat cerebellar slices SR 48692 blocked the increase in cyclic GMP levels evoked by neurotensin in a dose-dependent manner. In vivo, SR 48692 antagonized the increase in rat brain mesolimbic dopamine turnover induced by the systemically active neurotensin peptide, EI [(N-Me)Arg-Lys-Pro-Trp-tert-Leu-Leu]. No effects on dopamine turnover of either EI or SR 48692 were observed in the striatum. SR 48692 did not antagonize the EI-induced decreases in mouse body temperature and spontaneous locomotor activity (LMA) or the decreases in LMA induced by ICV-administered neurotensin. Although other explanations are possible, these findings support the hypothesis that a subtype of the NT receptor may mediate the locomotor and hypothermic actions of this peptide and that it is different from the NT receptor that is involved in dopamine turnover.

Structure-activity and conformational studies of a series of modified C-terminal hexapeptide neurotensin analogues.

Neurotensin (NT), is a linear tetradecapeptide (pGlu1-Leu2-Tyr3-Glu4-Asn5- Lys6-Pro7-Arg8-Arg9-Pro10-Tyr11-Ile12-Leu13) that has been found in the central nervous system and peripheral tissues and appears to have a variety of physiological properties. A C-terminal hexapeptide analogue [N alpha Me-Arg-Lys-Pro-Trp-Tle-Leu, (1) Tle = tert-leucine] has recently been reported to have high affinity for the NT receptor and appears to possess central activity after systemic administration. In an effort to probe the structure-activity and conformational properties of the dipeptide, Pro-Trp for binding and functional activity, these residues have been substituted with several natural and unnatural amino acids. Some of these analogues have binding affinities similar to compound 1, while in other cases, such as D-amino acid substitutions, the peptides had negligible binding affinity. In general, the Pro10 position seems more tolerant of substitution by amino acids that favor a reverse turn, rather than those that favor an extended conformation. The Trp11 position accepted extra steric bulk more readily than conformational constraints.