Biochemical and pharmacological properties of an allosteric modulator site of the human P-glycoprotein (ABCB1).

The drug-transport function of the human P-glycoprotein (Pgp or ABCB1) is inhibited by a number of structurally unrelated compounds, known as modulators or reversing agents. Among them, the thioxanthene derivative flupentixol inhibits Pgp-mediated drug transport by an allosteric mechanism. Unlike most other Pgp modulators, the cis isomer of flupentixol [cis-(Z)-flupentixol] facilitates interaction of Pgp with its transport-substrate [125I]iodoarylazidoprazosin (or [125I]IAAP), yet inhibits transport. In this study, we show that the flupentixol site acts as a common site of interaction for the tricyclic ring-containing modulators thioxanthenes and phenothiazines. The allosteric stimulation of [125I]IAAP binding to Pgp occurs independent of the phosphorylation status of the transporter. Stimulation is retained in purified Pgp reconstituted into proteoliposomes, suggesting no involvement of any other cellular protein in the phenomenon. However, perturbation of the lipid environment of the reconstituted Pgp by nonionic detergent octylglucoside abolishes stimulation by cis-(Z)-flupentixol of [125I]IAAP binding. Extensive trypsin digestion of the [125I]IAAP-labeled Pgp generates a 5.5 kDa fragment with 80% of the stimulated level of labeling associated with it. Sensitivity to inhibition by transport-substrate vinblastine and competitive modulator cyclosporin A suggests that the elevated level of [125I]IAAP binding to the fragment represents a functionally relevant interaction with the substrate site of Pgp. In summary, we demonstrate that allosteric modulation by cis-(Z)-flupentixol is mediated through its interaction with Pgp at a site specific for tricyclic ring-containing Pgp modulators of thioxanthene and phenothiazine backbone, independent of other cellular components and the phosphorylation status of the protein.

Clinical pharmacokinetics of the depot antipsychotics.

The clinical pharmacokinetics of the 4 depot antipsychotics for which plasma level studies are available (i.e. fluphenazine enanthate and decanoate, haloperidol decanoate, clopenthixol decanoate and flupenthixol decanoate) are reviewed. The proper study of these agents has been handicapped until recently by the necessity of accurately measuring subnanomolar concentrations in plasma. Their kinetic properties, the relationship of plasma concentrations to clinical effects, and conversion from oral to injectable therapy are discussed. The depot antipsychotics are synthesised by esterification of the active drug to a long chain fatty acid and the resultant compound is then dissolved in a vegetable oil. The absorption rate constant is slower than the elimination rate constant and therefore, the depot antipsychotics exhibit 'flip-flop' kinetics where the time to steady-state is a function of the absorption rate, and the concentration at steady-state is a function of the elimination rate. Fluphenazine is available as both an enanthate and decanoate ester (both dissolved in sesame oil), although the decanoate is more commonly used clinically. The enanthate produces peak plasma concentrations on days 2 to 3 and declines with an apparent elimination half-life (i.e. the half-time of the apparent first-order decline of plasma concentrations) of 3.5 to 4 days after a single injection. The decanoate produces an early high peak which occurs during the first day and then declines with an apparent half-life ranging from 6.8 to 9.6 days following a single injection. After multiple injections of fluphenazine decanoate, however, the mean apparent half-life increases to 14.3 days, and the time to reach steady-state is 4 to 6 weeks. Withdrawal studies with fluphenazine decanoate suggest that relapsing patients have a more rapid plasma concentration decline than non-relapsing patients, and that the plasma concentrations do not decline smoothly but may exhibit 'lumps' due to residual release from previous injection sites or multicompartment redistribution. Cigarette smoking has been found to be associated with a 2.33-fold increase in the clearance of fluphenazine decanoate. In 3 different studies, fluphenazine has been proposed to have a therapeutic range from less than 0.15 to 0.5 ng/ml with an upper therapeutic range of 4.0 ng/ml. Plasma concentrations following the decanoate injection are generally lower than, but clinically equivalent to, those attained with the oral form of the drug. Haloperidol decanoate plasma concentrations peak on the seventh day following injection although, in some patients, this peak may occur on the first day.(ABSTRACT TRUNCATED AT 400 WORDS)

Further evidence that 3H-cis(Z)flupenthixol binds to the adenylate cyclase-associated dopamine receptor (D-1) in rat corpus striatum.

The proposal that 3H-cis(Z)flupenthixol (3H-FPT) preferentially binds to striatal dopamine receptors associated with adenylate cyclase activity (D-1), distinct from dopamine receptors to which 3H-haloperidol (3H-hal) binds (D-2), has been investigated further. The dopamine agonists bromocriptine and ergotamine were 60-times more potent as displacers of 3H-hal than 3H-FPT binding. Other dopamine agonists (ergometrine, dopamine, apomorphine, 2-amino-6,7-dihydroxy-tetralin (ADTN), and ergocornine) also shared this profile, although a smaller ratio was found for these compounds. Substituted benzamides (clebopride greater than sultopride greater than sulpiride greater than metoclorpramide greater than tiapride) displace 3H-hal but have only a very slight displacing effect towards 3H-FPT, and did not inhibit dopamine-stimulated adenylate cyclase activity. The same pattern is shared by oxiperomide and molindone. Together, these results support the idea that 3H-FPT labels another class of dopamine receptors than does 3H-hal, and that the former class most likely is associated with adenylate cyclase.

3H-Flupenthixol binding in post-mortem brains of schizophrenics: evidence for a selective increase in dopamine D2 receptors.

The binding of 3H-cis flupenthixol (3H-FPT) to dopamine receptors in membrane preparations from control subjects and schizophrenics was studied. Using a fixed concentration of 3H-FPT, no differences were observed between controls and all schizophrenics, although 3H-FPT binding was increased in schizophrenics who apparently were drug-free at the time of death. Scatchard analysis of 3H-FPT binding revealed that in drug-treated schizophrenics both the number of binding sites (BM) and the dissociation constant (KD) were increased, whilst in drug-free schizophrenics only the BM was increased. Using domperidone to differentiate 3H-FPT binding to D1 and D2 dopamine sites, it was found that only D2 sites were increased in drug-free schizophrenics. The results are discussed with reference to previous studies on dopamine receptors in schizophrenia, and the effects of neuroleptic treatment. It is suggested that a selective increase in D2 receptors may be associated with the disease process in schizophrenia.

A comprehensive method for the quantitative determination of dopamine receptor subtypes.

We have demonstrated that three subtypes of dopamine receptors can be characterized using several radioligand binding techniques. Indirect binding assays in which several competing ligands were used to inhibit the binding of the nonselective radioligand spiroperidol resulted in shallow displacement curves with Hill coefficients less than 1. Nonlinear regression analysis of these curves also indicated that there were two subtypes of the D-2 receptor present in a ratio of approximately 3 to 1. Direct binding assays with [3H]alpha-flupenthixol showed that this radioligand nonselectively labeled D-2A, D-2B, and D-1 receptors. Inhibition of the binding of [3H]alpha-flupenthixol by spiroperidol revealed that spiroperidol had a much higher affinity for D-2A and D-2B receptors than for D-1 receptors. Masking D-2 receptors with nanomolar concentrations of spiroperidol permitted characterization of D-1 receptors with the radioligand [3H]alpha-flupenthixol. Indirect binding assays of D-1 receptors with numerous competing ligands resulted in steep displacement curves with Hill coefficients of 1. This is consistent with the existence of a single, homogeneous population of D-1 receptors.

Autoradiographic comparison of D1-specific binding of [3H]SCH39166 and [3H]SCH23390 in the primate cerebral cortex.

Quantitative autoradiography was used to compare the binding of the novel dopamine D1 receptor antagonist, [3H]SCH39166, with that of the widely used radioligand, [3H]SCH23390 (in the presence of ritanserin), in the primate cerebral cortex. Specific binding of both radioligands, determined using SCH23390 or cis-flupentixol as displacing agents, had very similar densities and distributions throughout the cortex. However, the specific binding of [3H]SCH39166 obtained with SCH39166 as a blank was significantly higher than that obtained using SCH23390 or cis-flupentixol as displacing agents in some layers of motor, somatosensory and occipital cortices. In addition, the non-specific binding of [3H]SCH39166 obtained in the presence of an excess of SCH23390 of cis-flupentixol displayed a complex laminar pattern very different from that of the specific binding. These observations suggest that [3H]SCH39166 may have a high affinity to more than the D1 receptor subtype bound by SCH23390 or cis-flupentixol. Also, these additional sites are likely to be different from 5-HT2 or 5-HT1C receptors since the latter sites were not displaced by 1 microM SCH23390.

Muscarinic and dopaminergic receptors in the aging human brain.

[3H]1-Quinuclidinyl(phenyl)-4-benzilate ([3H]QNB), [3H]spiroperidol and [3H]flupenthixol were used to label brain muscarinic, dopamine D2 and D1 receptors, respectively, in altogether 78 patients aged from 4 to 93 years. The binding of [3H]QNB declined with age in the frontal cortex, hippocampus, caudate nucleus and putamen. Scatchard analysis showed that the reduced binding was due to a decline in the number of receptors. The binding of the dopaminergic ligands was determined in the caudate nucleus, putamen, pallidum and substantia nigra. [3H]Spiroperidol binding showed age-dependent decline in all the brain areas examined, while no change was seen in [3H]flupenthixol binding in any brain area studied. Also the decrease in [3H]spiroperidol binding was due to the reduced number of receptors. The results of this study suggest that aging is more likely to affect certain neurotransmitter receptor systems than certain brain areas.

Characteristics of 3H-cis-flupenthixol binding to calf brain membranes.

The binding of 3H-cis flupenthixol (3H-FPT) to calf brain membranes was investigated in vitro. Highest levels of 3H-FPT binding were observed in striatum and nucleus accumbens, whilst no significant binding was observed in pituitary. The potencies of neuroleptics, dopaminergic ergot alkaloids and dopamine (DA) agonists in inhibiting 3H-FPT binding to calf striatal membranes were compared with their potencies in inhibiting 3H-spiperone (3H-SPIP) binding and DA-stimulated adenylate cyclase (AC). A high correlation was observed between drug potencies in inhibiting 3H-FPT binding and DA stimulated AC. However, potencies of the drugs inhibiting 3H-SPIP binding were only weakly correlated with their potencies in inhibiting either 3H-FPT binding or DA stimulated AC. A detailed analysis of the inhibition of 3H-FPT binding by butyrophenones revealed two classes of 3H-FPT binding sites. It is suggested that 3H-FPT binds predominantly to a class of dopaminergic sites associated with DA-stimulated AC.

Dopamine receptors in pancreatic acinar cells from dog.

Dispersed acini from dog pancreas were used to characterize dopamine receptors in a study on the stimulation of cellular cyclic AMP formation and the binding of [3H]dopamine. Dopamine elicited concentration-dependent stimulation of cellular cyclic AMP with a maximal increase occurring at a concentration of 0.1 mM (EC50 = 1 microM). Epinine produced a potent stimulation similar to that by dopamine; the alpha-adrenoceptor agonists (amidephrine and noradrenaline) were less potent. Isoproterenol and apomorphine were ineffective. The effect of dopamine was potently blocked by dopamine receptor antagonists such as cis-(Z)-flupenthixol, haloperidol, fluphenazine, whereas spiperone, sulpiride and domperidone were weakly active. Apomorphine acted as an antagonist to inhibit dopamine-stimulated cellular cyclic AMP formation as well as phenoxybenzamine. Yohimbine, prazosin or clonidine were poorly or not active. The affinity of agents to stimulate cellular cyclic AMP formation or to inhibit dopamine-stimulated cellular cyclic AMP formation was closely related to their affinity to inhibit the binding of [3H]dopamine to pancreatic acini, providing evidence that dopamine binding sites are receptors that mediate the action of dopamine on cAMP accumulation. This was further substantiated by the demonstration of specific binding sites for [3H]cis-(Z)-flupenthixol.

Chronic neuroleptic treatment increases D-2 but not D-1 receptors in rat striatum.

In vitro binding of [3H]cis(Z)-flupentixol (FPT) to rat striatal membranes was evaluated as an assay for D-1 receptors. It was found that under the appropriate assay conditions [3H]FPT bound to a single saturable site which was most abundant in striatum and bound dopaminergic agonists in the potency order that these drugs demonstrate for adenylate cyclase. These data support previous work suggesting that [3H]FPT labels the D-1 receptor. Next, rats received haloperidol or fluphenazine for 54 days and striatal dopamine receptors were assayed 72 h later. The drug treatments increased the density of D-2 receptors as measured by [3H]spiperone binding by 40% and 25% respectively. However, no change was observed in D-1 receptor density. We conclude that effects of chronic neuroleptic treatment that depend upon increased dopamine receptor density are mediated via the D-2 receptor subtype.

Kainate lesion dissociates striatal dopamine receptor radioligand binding sites.

Kainic acid lesion of rat striata reduces the specific dopamine receptor binding of the butyrophenone antagonist [3H]spiperone and the butyrophenone-like antagonist [3H]domperidone by 56% and 59% respectively. Significantly greater decreases in binding were observed with the agonist [3H]N-propylnorapomorphine (NPA) and the antagonist [3H]flupentixol which showed 79% and 73% losses of high affinity binding respectively. These data indicate that, in part, [3H]spiperone and [3H]domperidone label distinct dopamine receptors with different neuronal localizations from those labeled by [3H]flupentixol and [3H]NPA. Our data is consistent with the hypothesis that [3H]flupentixol and [3H]NPA bind preferentially to adenylate cyclase-linked dopamine (D1) receptors.

Kainic acid lesions dissociate [3H] spiperone and [3H]cis-flupenthixol binding sites in rat striatum.

The effects of striatal kainic acid lesions on [3H] cis-flupenthixol ([3H]FPT) and [3H]spiperone (3H-SPIP) binding to dopamine (DA) receptors in control and lesioned striata were examined. Significant reductions in both binding parameters were observed, [3H] FPT binding was depleted to a greater extent than [3H] SPIP binding. Reductions in both [3H] FPT and [3H] SPIP binding were significantly correlated with reductions in glutamic acid decarboxylase activity. After complete loss of GAD activity, 30-40% of [3H] SPIP binding sites remained in the lesioned striata, whereas all [3H] FPT binding sites were destroyed. The results are discussed in relation to the presence of distinct types of DA receptors in rat striatum, and ligand binding to these receptors.

Evidence for the existence of a dopamine receptor of the D-1 type in the rat median eminence.

By means of receptor autoradiography using the dopamine (DA) receptor radioligands [3H]cis(z)-flupenthixol ([3H]FLU), [3H]spiperone, [3H]N-propyl-norapomorphine and amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphtalene-2-(5,8[3H]) in combination with a microdensitometrical analysis indications have been obtained for the existence of a DA receptor of the D-1 type in the median eminence of the male rat. Thus, only [3H]FLU (10-20 nM) strongly labeled both the nuc. caudatus putamen and the median eminence and the labeling was markedly prevented by (+)-butaclamol in both regions. Furthermore, a DA receptor agonist of the D-1 type preferentially displaced [3H]FLU from the median eminence. Thus, a DA receptor of the low affinity type may regulate the secretion of hypothalamic hormones from the median eminence.

Characteristics of D-1 dopamine receptors labelled by [3H]fluphenazine in homogenates of rat neostriatum.

The properties of the site labelled by [3H]fluphenazine in membranes prepared from the rat neostriatum were examined using radioligand binding methodology. Binding of [3H]fluphenazine was rapid, saturable and of high affinity (K4 = 0.4 nM). Drug displacement experiments demonstrated that the site labelled by [3H]fluphenazine possessed pharmacological characteristics consistent with those of a D-1 dopamine receptor.

Binding sites for [3H]dopamine and dopamine-antagonists on cultured astrocytes of rat striatum and spinal cord: an autoradiographic study.

The cellular localization of binding sites for [3H]dopamine, and dopamine-antagonists (D1 and D2) was studied in organotypic cultures of rat striatum and spinal cord by means of autoradiography. In both types of cultures, many astrocytes were labelled by [3H]dopamine, the D1-antagonist [3H]cis-flupenthixol and the D2-antagonists [3H]domperidone and [3H]spiperone (10(-9) to 10(-8) M). Addition of unlabelled dopamine and antagonists at high concentrations (10(-6) to 10(-4) M) inhibited or markedly reduced binding of the radioligands indicating 'specific' binding of the compounds. Our autoradiographic studies are consistent with biochemical investigations by other authors, suggesting that astrocytes possess receptors for dopamine.

Biochemical and pharmacological differentiation of neuroleptic effect on dopamine D-1 and D-2 receptors.

In dopamine (DA) receptor binding tests two different receptor populations can be measured. By using 3H-haloperidol or 3H-spiroperidol as ligands D-2 receptors are detected. When the thioxanthene neuroleptics 3H-cis (Z)-flupenthixol or 3H-piflutixol are used as ligands D-1 receptors coupled to adenylate cyclase can be detected. In displacement experiments butyrophenones (haloperidol and spiroperidol) and diphenylbutylpiperidines (pimozide) interact with D-2 receptors only, whereas thioxanthenes (cis [Z]-flupenthixol, cis [Z]-piflutixol and cis [Z]-clopenthixol) have equal affinity for D-1 and D-2 receptors. A similar differentiation is also seen in behavioral studies. The methylphenidate antagonistic effect of butyrophenones and diphenylbutylpiperidines is dramatically attenuated by concomitant treatment with an anticholinergic agent or a GABA agonist. The effect of thioxanthenes is almost unchanged. When mice are treated for 12 days with neuroleptics, supersensitivity to methylphenidate is induced. The supersensitivity is reversed by thioxanthenes, whereas butyrophenones and diphenylbutylpiperidines have no effect at all. These differences do not appear to be due to a change in the affinity for DA receptors because the ability of cis (Z)-flupenthixol, haloperidol, DA or apomorphine to displace 3H-piflutixol or 3H-spiroperidol is the same in supersensitive and control mice. The results clearly demonstrate that neuroleptics acting on both D-1 and D-2 receptors have a behavioral profile different from that of neuroleptics acting exclusively on D-2 receptors.

Characteristics of 3H-cis-flupenthixol binding in rat striatum.

Characteristics of membrane receptor binding by 3H-cis-flupenthixol were examined in rat striatum. Using modifications of standard dopamine receptor binding techniques, it was possible to obtain 70% specific binding with 3H-cis-flupenthixol. Association and dissociation rates were very rapid, with equilibrium reached in 2 min and half-time of dissociation being 1 min. Analysis of saturation and competition studies using cis-flupenthixol and spiroperidol indicated that cis-flupenthixol binds to two striatal receptors with apparent KD's of 0.7 and 4.8 nM. It is suggested these represent D1 and D2 dopamine receptors respectively. The further characterization of the properties of cis-flupenthixol binding presented here should enable more detailed studies of multiple dopamine receptors to be designed.

Interactions of novel dopaminergic ligands with D-1 and D-2 dopamine receptors.

The interactions of three novel dopaminergic ligands, SKF38393, SKF82526 and SKF83742, with D-1 and D-2 dopamine (DA) receptors have been investigated using radioligand binding techniques and computer modeling procedures. Using the bovine anterior pituitary D-2 DA receptor system, SKF38393 and SKF82526 behave as agonists demonstrating biphasic agonist/3H-antagonist competition curves. For both drugs, the high affinity phase comprised 30% of the total displacement curve. Such findings are atypical as previously tested classical dopamine agonists demonstrated high and low affinity displacement phases in equal proportions. Such behavior exhibited by the SKF agonists may be related to their activity as partial agonists. In contrast, SKF83742 behaves as an antagonist exhibiting homogeneous monophasic competition curves. Similar results are obtained in the rat striatal membrane D-2 DA receptor system. Both SKF38393 and SKF82526 also demonstrate shallow biphasic displacement curves on rat striatal D-1 receptors labeled with 3H-flupentixol whereas SKF83742/3H-flupentixol curves are uniphasic. Of all the ligands, only SKF38393 clearly demonstrates higher affinity for 3H-flupentixol labeled D-1 receptors.

Binding of a novel dopaminergic agonist radioligand [3H]-fenoldopam (SKF 82526) to D-1 receptors in rat striatum.

Fenoldopam (SKF 82526), a dopamine agonist which exhibits D-1 receptor subtype selectivity, was evaluated as a radioligand for this receptor subtype. In saturation studies in rat striatal membrane preparations, [3H]-fenoldopam appeared to label a single binding site with a KD of 2.3 +/- 0.1 nM and a Bmax of 590 +/- 40 fmoles/mg protein. In competition binding experiments, binding was shown to be stereoselective, and rank ordering of affinities of dopaminergic and non-dopaminergic compounds closely correlated with potencies of these compounds in stimulating or inhibiting dopamine-sensitive adenylate cyclase (D-1) and in binding to D-1 sites labelled with the antagonist [3H]-cis-flupenthixol. The most potent competitors were the recently identified D-1 selective antagonists, SCH 23390 and SKF R-83566. [3H]-Fenoldopam was also used to assess agonist/D-1 receptor interactions. The results suggest that [3H]-fenoldopam is a useful and selective agonist radioligand for the D-1 receptor.

Irreversible interaction of beta-haloalkylamine derivatives with dopamine D1 and D2 receptors.

The interaction of beta-haloalkylamine derivatives of dopamine agonists and antagonists with 3H-spiperone binding (D2 sites) and 3H-flupenthixol binding (D1 sites) was studied. N-chloroethyl derivatives of phenothiazines and thioxanthenes were potent inhibitors of the binding of both ligands. The in vitro inhibition of binding produced by these compounds was irreversible. The drugs were however only weakly active in vivo. The results suggest that beta-haloalkylamine derivatives of neuroleptics may be useful compounds for studying dopamine receptors in vitro.