Positive allosteric modulation of the human cannabinoid (CB) receptor by RTI-371, a selective inhibitor of the dopamine transporter

Abstract

Background and purpose: In our search for an indirect dopamine agonist as therapy for cocaine addiction, several selective inhibitors of the dopamine transporter (DAT), which are 3-phenyltropane analogues, were assayed for their effect on locomotor activity in mice. Interestingly, several of the compounds showed a poor correlation between stimulation of locomotion and DAT inhibition. One of the compounds, 3beta-(4-methylphenyl)-2beta-[3-(4-chlorophenyl)isoxazol-5-yl]tropane (RTI-371), was shown to cross the blood-brain barrier, by binding studies in vivo, and block cocaine-induced locomotor stimulation. As poor pharmacokinetics could not explain the behavioural effects of RTI-371, this compound was screened through our functional assays for activity at other CNS receptors. Initial screening identified RTI-371 as a positive allosteric modulator of the human CB(1) (hCB(1)) receptor.

Experimental approach: The effect of RTI-371 and other DAT-selective inhibitors on CP55940-stimulated calcium mobilization was characterized in a calcium mobilization-based functional assay for the hCB(1) receptor. Selected compounds were also characterized in a similar assay for human mu opioid receptor activation to assess the specificity of their effects.

Key results: RTI-371 and several other DAT-selective inhibitors with atypical actions on locomotor behaviour increased the efficacy of CP55940 in a concentration-dependent manner.

Conclusions and implications: These results suggest that the lack of correlation between the DAT-binding affinity and locomotor stimulation of RTI-371 could be due at least in part to its activity as a positive modulator of the hCB(1) receptor.

Figure 1

Structures for cocaine, JHW007, RTI-31, RTI-112, RTI-370, RTI-371, RTI-549 and methylphenidate.

Figure 2

Positive allosteric modulation of hCB₁ receptor activation. RTI-370, RTI-371, JHW007 and GBR12909 increased the intrinsic activity of the agonist with variable effects on potency. The data represent the mean ± SEM from at least three independent experiments per compound. The maximum net (MAX-MIN) relative fluorescence units in this assay were typically 4000–5000.

Figure 3

Effects of other 3-phenyltropanes, cocaine and methylphenidate. These compounds produced locomotor behaviour similar to those observed with cocaine and had little or no allosteric modulator activity. The data represent the mean ± SEM from at least three independent experiments per compound.

Figure 4

DAMGO-mediated calcium mobilization. The specificity of the positive allosteric effect of the two most active compounds in the hCB₁ receptor assays was evaluated in this assay as a control for non-specific effects of the compounds or solvents on calcium mobilization. In contrast to their effects at hCB₁ receptors, these compounds at 10 µmol·L⁻¹ had no effect on, or inhibited, the DAMGO-stimulated calcium mobilization. The data represent the mean ± SEM from at least two independent experiments per compound. The maximum net (MAX-MIN) relative fluorescence units in this assay were typically 9000–10 000.