L-isocorypalmine reduces behavioral sensitization and rewarding effects of cocaine in mice by acting on dopamine receptors

Abstract

Background: We previously reported isolation of l-isocorypalmine (l-ICP), a mono-demethylated analog of l-tetrahydropalmatine (l-THP), from the plant Corydalis yanhusuo. Here we characterized its in vitro pharmacological properties and examined its effects on cocaine-induced behaviors in mice.

Methods: Receptor binding, cAMP and [(35)S]GTPγS assays were used to examine pharmacological actions of l-ICP in vitro. Effects of l-ICP on cocaine-induced locomotor hyperactivity and sensitization and conditioned place preference (CPP) in mice were investigated. HPLC was employed to analyze metabolites of l-ICP in mouse serum.

Results: Among more than 40 targets screened, l-ICP and l-THP bound only to dopamine (DA) receptors. l-ICP was a high-affinity partial agonist of D1 and D5 receptors and a moderate-affinity antagonist of D2, D3 and D4 receptors, whereas l-THP bound to only D1 and D5 receptors, with lower affinities than l-ICP. At 10mg/kg (i.p.), l-ICP inhibited spontaneous locomotor activity for a shorter time than l-THP. Pretreatment with l-ICP reduced cocaine-induced locomotor hyperactivities. Administration of l-ICP before cocaine once a day for 5 days reduced cocaine-induced locomotor sensitization on days 5 and 13 after 7 days of withdrawal. Pretreatment with l-ICP before cocaine daily for 6 days blocked cocaine-induced CPP, while l-ICP itself did not cause preference or aversion. HPLC analysis showed that l-ICP was the main compound in mouse serum following i.p. injection of l-ICP.

Conclusions: l-ICP likely acts as a D1 partial agonist and a D2 antagonist to produce its in vivo effects and may be a promising agent for treatment of cocaine addiction.

Keywords: Cocaine; Conditioned place preference; Dopamine receptor; Isocorypalmine; Sensitization; Tetrahydropalmatine.

Figure 1. Chemical structures of dopamine and l-isocorypalmine (l-ICP) and l-tetrahydropalmatine (l-THP)

Note that part of the l-ICP structure resembles dopamine (DA) structure.

Figure 2. l-ICP acts as a partial agonist at (A1, A2) the dopamine D1 and (B) D5 receptors

(A1, A2) dopamine- and l-ICP-stimulated cAMP formation in HEK cells expressing a (A1) high or (A2) moderate level of the D1 receptor were performed with different doses and dose-response curves of dopamine and l-ICP were generated. Potencies (EC₅₀) and efficacies (E_max) of l-ICP were determined and shown in Table 2. (B). Dose-response curves of dopamine and l-ICP in stimulating cAMP formation in HEK cells expressing the D5R were generated. EC₅₀ and E_max of l-ICP were shown in Table 2. Each value represents mean ± s.e.m. of three experiments performed in duplicate.

Figure 3. l-ICP is an antagonist at dopamine D2, D3 and D4 receptors

Membranes of HEK293 cells stably transfected with the human dopamine D2, D3 or D4 receptor were prepared. Dose-response curves of dopamine-induced increase in [³⁵S]GTPγS binding were generated in the presence or absence of 1 μM l-ICP. Each value represents the mean ± s.e.m. of three independent determinations performed in duplicate.

Figure 4. Comparison of the effects of l-ICP and l-THP on spontaneous locomotor activity in mice

CD-1 mice were injected with vehicle, l-ICP or l-THP (10 mg/kg, i.p.), placed in activity chambers and activity counts were recorded for 120 min as described in Methods. Areas under curves (AUCs) of the total activity of l-ICP or l-THP from 0-30 and 30-100 min were shown as percentage (%) of the vehicle-treated group. Each value represents mean ± s.e.m (n=8 per group). ***P<0.001 by Student’s t test.

Figure 5. Effects of l-ICP on cocaine-induced hyperactivity in mice

CD-1 mice were habituated to activity chambers for 50 min, pretreated with vehicle or l-ICP (1, 3 and 10 mg/kg, i.p.) for 10 min followed by saline or cocaine (20 mg/kg, i.p.) as described in Methods. Areas under curves (AUCs) were calculated for (A) total activity, (B) ambulatory activity and (C) repetitive non-ambulatory activity. Each value represents mean ± s.e.m (n=16 for veh/saline, n= 10 for all other groups). **P<0.01, ***P<0.001, compared with the veh/cocaine group; ⁺⁺⁺P<0.001, ⁺P<0.05 compared with veh/saline, both by two-way ANOVA followed by Bonferroni post hoc test.

Figure 6. Effects of l-ICP on development of locomotor sensitization induced by repeated cocaine treatment in mice

(A) Timeline of the experimental procedure and drug treatment. (B) Locomotor activities on Day 1 and Day 5. (C) Locomotor activities on Day 13. From day 1 to day 5, mice were treated once/day with vehicle or l-ICP (10 mg/kg, i.p.) followed by saline or cocaine (20 mg/kg, i.p.)10 min later as described in Methods. Following withdrawal from day 6 to day 12, on day 13, mice were challenged with cocaine. Activities were monitored on days 1, 5 and 13 for 120 min. The areas under curves of total activities (0-60 minutes) are shown in the B and C. Each value represents mean ± s.e.m (N=8 for each group). *** P<0.001 and *P<0.05 compared with veh/COC; ⁺⁺P<0.01 and ⁺P<0.05, compared with day 1 veh/cocaine by two-way ANOVA followed by Bonferroni post hoc test.

Figure 7. Effects of l-ICP on acquisition of cocaine-induced conditioned place preference (CPP) in mice. (A) Timeline of the CPP paradigm and testing procedure. (B) Pretreatment with l-ICP significantly blocked acquisition of cocaine CPP, but by itself did not induce preference or aversion

An unbiased and counterbalanced procedure was used for the CPP experiments. At C (cocaine) in the timeline, mice were treated with vehicle or l-ICP (10 mg/kg, i.p.) followed by cocaine (20 mg/kg, i.p.) or saline injection 10 min later and placed in one side of CPP chambers. At S (saline) in the timeline, mice were treated with vehicle followed by saline injection 10 min later and placed in the other side of CPP chambers. Test of preference was conducted on day 7. Results are expressed as mean ± s.e.m. time spent (in seconds) on the cocaine-paired compartment minus the saline-paired compartment. Eight mice in each group were used. (a)V/S: vehicle/saline, (b)V/C: vehicle/cocaine, (c)I/S: l-ICP/saline, (d)I/C: l-ICP/cocaine. **P<0.01 compared with the Pre-test of the same group, ^#P <0.05 compared with the Post-test of the V/C group, both by one-way ANOVA followed by Newman-Keuls multiple comparison test.

Figure 8. High performance liquid chromatography (HPLC) analysis of l-ICP and its possible metabolites in mouse serum

(A) Chromatogram of serum from mice injected with vehicle; (B) Chromatogram of serum from mice injected with l-ICP (20 mg/kg, i.p); (C) Chromatogram of l-ICP standard (10 μg). Male CD-1 mice were injected with vehicle or l-ICP (20 mg/kg, i.p), sacrificed 30 min later and blood was collected. Serum was processed and HPLC was performed using a C18 reverse-phase column and methanol (25-35%) / 0.05% triethylamine water as the mobile phase as described in Methods.