Lawrence D. Wise

Autoradiographic localisation of D3-dopamine receptors in the human brain using the selective D3-dopamine receptor agonist (+)-[3H]PD 128907

Abstract The selective D3-dopamine receptor agonist 4aR,10bR-(+)-trans-3,4,4a,10b-tetrahydro-4-[N-propyl-2,3-3H]-2H,5H-[1]benzopyrano[4,3-b]-1,4-oxazin-9-ol ([3H]PD 128907) was used to visualise D3-dopamine receptors in whole hemisphere cryosections from post-mortem human brain. [3H]PD 128907 has an 18- to 40-fold selectivity for D3- over D2-dopamine receptors as compared to a 7- to 24-fold selectivity of the more commonly used ligand [3H]7-OH-DPAT. [3H]PD 128907 accumulated markedly in the nucleus accumbens and in the ventral parts of caudate nucleus and putamen, with a slightly heterogeneous (patch-matrix like) distribution. The binding in the lateral parts of caudate nucleus and putamen was much less dense. No binding was obtained in any other regions. A very high proportion of [3H]PD 128907 was specifically bound, as judged from the low binding remaining in the presence of the D2/D3-dopamine receptor antagonist raclopride. This gives the ligand a potential for the detection of low density D3-dopamine receptors in the human brain. The binding obtained with [3H]PD 128907 was qualitatively similar to that using [3H]7-OH-DPAT in the presence of GTP. However, [3H]7-OH-DPAT labelled, in contrast to [3H]PD 128907, also D3-dopamine receptors in neocortex. The new compound [3H]PD 128907 appears to be a suitable radioligand for autoradiographic examination of the D3-dopamine receptor localisation in the human brain, and should also be useful for pharmacological studies of this receptor subtype.

Isoindolinone enantiomers having affinity for the dopamine D4 receptor.

PD 108635 (1) was identified as a potent dopamine D4 ligand and we wanted to replace the benzylic alcohol with a metabolically more stable moiety. Investigations led to the discovery of a series of isoindolinones having D4 affinity.

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.

Discovery of selective dopamine D4 receptor antagonists: 1-Aryloxy-3-(4-aryloxypiperidinyl)-2-propanols

Abstract High volume screening identified 3-(4-benzylpiperidinyl)-1-naphthoxy-2-propanol as a selective dopamine D4 receptor ligand. A systematic structure-activity study revealed that the benzyl group could be replaced with phenoxy and the naphthalene with phenyl to improve potency almost tenfold. The (R) enantiomer of this compound had a D4 affinity of 2 nM and was over 100-fold weaker at dopamine D2 and D3 receptors.

4-bromo-1-methoxy-N-[2-(4-aryl-1-piperazinyl)ethyl]-2-naphthalenecarboxamides: Selective dopamine D3 receptor partial agonists

Abstract A series of 4-bromo-1-methoxy-N-[2-(4-aryl-1-piperazinyl)ethyl]-2-naphthalenecarboxamide dopamine (DA) D 3 receptor agonists has been identified. These compounds were found to be selective for DA D 3 over D 2 receptors and were shown to be partial to full agonists as measured by stimulation of mitogenesis in D 3 -transfected CHO p-5 cells.

8-amino-6-(arylsulphonyl)-5-nitroquinolines: novel nonpeptide neuropeptide Y1 receptor antagonists

A novel series of 8-amino-6-(arylsulphonyl)-5-nitroquinoline neuropeptide Y1 (NPY) receptor antagonists is reported. The 8-amino and 5-nitro groups were important for NPY1 binding affinity as changes caused large drops in potency. The 6-arylsulphonyl group was necessary; however, substitution on the phenyl was tolerated. The 2-isopropyl analog 21 was a moderately potent, highly selective NPY1 receptor antagonist.

Novel cyclohexyl amides as potent and selective D3 dopamine receptor ligands

Abstract The dopamine D3 receptor is an attractive target for the treatment of schizophrenia. We identified PD137557 (V) as a ligand for the D2 receptor and desired to prepare a selective D3 compound. SAR studies involving different amides and different phenyl piperazines have led to the discovery of 8a and 8c as selective D3 receptor ligands.

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.

Agonist properties of a stable hexapeptide analog of neurotensin, NαMeArg-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.