Psychostimulant pharmacological profile of paraxanthine, the main metabolite of caffeine in humans

Abstract

Caffeine induces locomotor activation by its ability to block adenosine receptors. Caffeine is metabolized to several methylxanthines, with paraxanthine being the main metabolite in humans. In this study we show that in rats paraxanthine has a stronger locomotor activating effect than caffeine or the two other main metabolites of caffeine, theophylline and theobromine. As previously described for caffeine, the locomotor activating doses of paraxanthine more efficiently counteract the locomotor depressant effects of an adenosine A(1) than an adenosine A(2A) receptor agonist. In drug discrimination experiments in rats trained to discriminate a maximal locomotor activating dose of caffeine, paraxanthine, unlike theophylline, generalized poorly to caffeine suggesting the existence of additional mechanisms other than adenosine antagonism in the behavioral effects of paraxanthine. Pretreatment with the nitric oxide inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) reduced the locomotor activating effects of paraxanthine, but not caffeine. On the other hand, pretreatment with the selective cGMP-preferring phosphodiesterase PDE9 inhibitor BAY 73-6691, increased locomotor activity induced by caffeine, but not paraxanthine. Ex vivo experiments demonstrated that paraxanthine, but not caffeine, can induce cGMP accumulation in the rat striatum. Finally, in vivo microdialysis experiments showed that paraxanthine, but not caffeine, significantly increases extracellular levels of dopamine in the dorsolateral striatum, which was blocked by l-NAME. These findings indicate that inhibition of cGMP-preferring PDE is involved in the locomotor activating effects of the acute administration of paraxanthine. The present results demonstrate a unique psychostimulant profile of paraxanthine, which might contribute to the reinforcing effects of caffeine in humans.

Figure 1

Locomotor activation induced by the methylxanthines caffeine (caff), theophylline (theoph), paraxanthine (parax) and theobromine (theobr) in non-habituated rats after 40 min following drug administration. Data represent means ± S.E.M. of transformed data (square root) of accumulated counts during 60 min period of observation (n = 6–8 per group). * and **: P <0.05 and P < 0.01 compared to vehicle-treated animals (VEH), respectively; one-way ANOVA with Dunnett’s post-hoc comparisons. #: P < 0.05 compared to caff 30 mg/kg (one-way ANOVA followed by Bonferroni’s post-hoc test).

Figure 2

Counteraction of adenosine receptors agonist-induced motor depression by the selective A₁R antagonist CPT and the selective A_2AR antagonist MSX-3. The possible counteracting effect of the PDE9 inhibitor BAY-73-6691 was also tested. Data represent means ± S.E.M. of transformed data (square root) of total accumulated counts during a 60 min period of observation (n = 6–8 per group). The adenosine A₁R agonist CPA (0.1 mg/kg) and the A_2AR agonist CGS 21680 (CGS; 0.5 mg/kg) were administrated 10 min before the animals were placed in the activity cages. CPT (1 mg/kg), MSX-3 (MSX, 1 mg/kg) BAY 73-6691 (BAY, 3 mg/kg) or vehicle (VEH) were administered 30 min before CPA or CGS 21680. The dashed horizontal line represents the average of locomotion in animals not treated with adenosine agonists. * and **: P <0.05 and P <0.01, respectively, compared to the group that received the same dose of CPA or CGS 21680 alone (one-way ANOVA with Dunnett’s post hoc comparisons).

Figure 3

Counteraction of adenosine receptors agonist-induced motor depression by caffeine, theophylline, paraxanthine and theobromine. Data represent means ± S.E.M. of transformed data (square root) of total accumulated counts during a 60 min period of observation (n = 6–8 per group). The adenosine A₁R agonist CPA (0.1 mg/kg) and the A_2AR agonist CGS 21680 (0.5 mg/kg) were administrated 10 min before the animals were placed in the activity cages. Caffeine (caff), theophylline (theoph), paraxanthine (parax) and theobromine (theobr), at 1, 3, 10 or 30 mg/kg, were administered 30 min before CPA or CGS 21680. * and **: P <0.05 and P <0.01, respectively, compared to the group that received the same dose of CPA or CGS 21680 alone (one-way ANOVA with Dunnett’s post hoc comparisons).

Figure 4

Effects of the adenosine receptor antagonists paraxanthine (parax), theophylline (theoph) and theobromine (theobr) in rats trained to discriminate 30 mg/kg caffeine (caff) from vehicle (VEH). Data represent mean ± S.E.M. (n=10). Ordinates percentage of responses on the lever associated with caffeine administration (left panel) and overall rate of lever pressing expressed as responses per second averaged over the session (right panel). Drug doses are expressed in mg/kg (log scale). Complete generalization to the caffeine-training stimulus (80% caffeine-lever selection) is represented with dashed horizontal line. * and **: P < 0.05 and P < 0.01 compared with VEH, respectively (one-ANOVA followed by Bonferroni’s post-hoc test).

Figure 5

Effect of the NO synthase inhibitor L-NAME treatment on locomotor activation induced by paraxanthine and caffeine. Non-habituated animals were placed in the motility cages 40 min after vehicle (VEH), paraxanthine (parax, 30 mg/kg) or caffeine (caff, 30 mg/kg) administration and 30 min after VEH or L-NAME (L-NAME, 30 mg/kg). (a) and (c) represent time course for locomotion of transformed data (square root) of accumulated counts per 10-min period (mean ± S.E.M.; n = 6–8 per group). (b) and (d) represent the average (mean ± S.E.M.) of the 10-min period transformed values during the first 60 min of recording (n = 6–8 per group). ** and ***: P < 0.01 and P <0.001, compared to VEH-treated animals, respectively; #: P < 0.05 compared to paraxanthine alone (one-way ANOVA followed by Bonferroni’s post-hoc test).

Figure 6

Effect of the PDE9 inhibitor BAY 73-6691 treatment on locomotor activation induced by caffeine, paraxanthine, the A_2AR antagonist KW-6002 and the A₁R antagonist CPT. Animals were placed in the motility cages 40 min after vehicle (VEH), caffeine (caff, 30 mg/kg), paraxanthine (parax, 30 mg/kg), KW-6002 (KW, 0.1 mg/kg) or CPT (CPT, 4.8 mg/kg) administration and 30 min after VEH or BAY 73-6691 (BAY, 3 mg/kg). Data represent the average (mean ± S.E.M.) of the 10-min period transformed values during the first 60 min of recording (n = 6–8 per group). *, ** and ***: P < 0.05, P < 0.01 and P <0.001, compared to VEH-treated animals, respectively. #: P < 0.05 compared to caffeine alone (b) or to CPT alone (d) (one-way ANOVA with Bonferroni’s post-hoc comparisons).

Figure 7

cGMP accumulation in the rat striatum after the systemic administration of paraxanthine, caffeine or the PDE9 inhibitor BAY 73-6691. Data represent means ± S.E.M. of cGMP accumulation in striatal homogenates from animals sacrificed 30 min after the systemic administration of BAY 73-6691 (3 mg/kg; BAY 3), paraxanthine (10 or 30 mg/kg; parax 10 or parax 30) or caffeine (10 or 30 mg/kg; caff 10 or caff 30). **: P < 0.01 compared to vehicle-treated animals (VEH); no significant differences were observed between the groups treated with BAY, parax 30 and BAY 3 + caff 30 (one-way ANOVA with Newman-Keuls’ post hoc comparisons).

Figure 8

Effect of systemic administration of paraxanthine, caffeine and L-NAME on the increase of dopamine extracellular levels in the lateral striatum. Results express the mean ± S.E.M. in percentage of the average of the three stable values before the pretreatment (n = 6–7 per group). The arrows indicate the time of systemic administration of caffeine (30 mg/kg; caff 30), paraxanthine (30 mg/kg; parax 30) or L-NAME (30 mg/kg; L-NAME 30). * and **: P < 0.05, and P < 0.01, respectively, compared to the average of the three basal level values before drug administration (one-way ANOVA for repeated measurement with Dunnett’s post-hoc comparisons).