Effects of the novel glycopeptide opioid agonist MMP-2200 in preclinical models of Parkinson's disease

Abstract

In Parkinson's disease (PD), the consequence of dopaminergic denervation is an imbalance in the activity of the direct and indirect striatofugal pathways, which include potentially important changes in opioid peptide expression and/or activity. The systemic administration of a novel glycosylated opioid peptide MMP-2200 (a.k.a. lactomorphin) was shown to have potent effects in two standard models of PD: 1) amphetamine-induced rotations in the hemi-Parkinsonian 6-hydroxydopamine (6-OHDA)-treated rat and 2) locomotion in the reserpine-treated rat. MMP-2200, an opioid mu and delta receptor agonist, reduced amphetamine-induced rotations in severely-lesioned hemi-Parkinsonian rats; this effect was fully blocked by naloxone, an opioid receptor antagonist. The selective δ-opioid receptor antagonist naltrindole only partially blocked the effect of MMP-2200. MMP-2200 alone did not induce rotations. This effect was also observed in a mild progressive rat 6-OHDA-lesion model. In animals treated with reserpine, profound akinesia was induced that was reversed with apomorphine. There was a prominent overshoot in animals that received apomorphine compared to non-reserpine treated animals, reflecting the well described phenomenon of dopamine supersensitivity indicating that apomorphine not only reversed akinesia but also induced hyper-kinesia. The opioid peptide MMP-2200 blocked the apomorphine-induced hyper-kinesia. This effect of MMP-2200 was prevented by pre-administration of naloxone. MMP-2200 had no effect in preventing the reserpine-induced akinesia, nor did it affect locomotion in control animals. Taken together, the results from these two models are consistent with the glycopeptide opioid agonist MMP-2200 having a potent effect on movements related to dopaminergic hyper-stimulation following striatal dopamine depletion that are best explained by a reduction in the downstream effects of dopamine agonists in these models.

Figure 1

Structure of MMP-2200.

Figure 2. Unilateral injections of 6-OHDA into the striatum creates a severe lesion in the substantia nigra

The presence of the unilateral lesion is verified using tyrosine hydroxylase (TH) antibody staining as a marker of dopaminergic neurons. The loss of cell bodies in the substantia nigra (A) and the loss of dopaminergic terminals in the striatum (B) is visible. For visualization DAB staining was performed after binding of the anti-TH antibody.

Figure 3. MMP-2200 decreased the rotations induced by amphetamine in severely 6-OHDA lesioned rats

(A) Amphetamine injection induced turning ipsiversive to the lesion in a standard hemi-Parkinsonian model that utilized a unilateral injection of 6-OHDA (more than 90% loss of dopaminergic cell bodies in the substantia nigra). Amphetamine-induced rotation (▲) reached a plateau at 30–40 min, and then gradually decreased from 40 min onwards. Co-injection of MMP-2200 markedly reduced the amphetamine-induced rotation over the entire time-course (●). This effect was most pronounced 20–45 minutes after injection (* P < 0.05 & ** P < 0.01 compared A+M with A respectively, n=8, t-test). MMP-2200 alone (■) did not induce rotation. (B) The total number of amphetamine-induced rotations over a 1 hr period was significantly reduced by co-injection with MMP-2200 (*P < 0.05, n=8, t-test).

Figure 4. Dose-response of the effect of MMP-2200 in severely 6-OHDA lesioned rats

The total number of amphetamine-induced rotations over a 1 hr period was counted. The graph depicts the effects of 3 different MMP-2200 doses plotted as % of amphetamine-only control. For statistical analysis the raw rotation data before normalization (n=10) was subjected to a repeated measures ANOVA with a Fisher LSD post-hoc test revealing that the 5 mg (*P < 0.05) and the 10 mg (**P < 0.005) MMP-2200 doses reduced amphetamine-induced rotations significantly. The 1 mg MMP-2200 dose showed no significant effect.

Figure 5. The effect of MMP-2200 on amphetamine-induced rotation is blocked by naloxone

Baseline rotations were established in severely-lesioned hemi-Parkinsonian rats challenged with amphetamine to establish the control level of 100%. The data are plotted as % of amphetamine-only control. MMP-2200 co-injections led to a marked decrease in amphetamine-induced rotation (A+M, ^#P < 0.05, one way ANOVA with a Bonferroni post-hoc test of log-transformed rotation data before normalization to % control, n=16) compared to the amphetamine-only control baseline. This effect was completely reversed by 3 mg/kg naloxone (Nal+A+M, *P < 0.05 compared with A+M group, n=16). Naloxone alone did not affect amphetamine-mediated rotations (Nal+A, P > 0.05 compared with control; n=16).

Figure 6. The effect of MMP-2200 on amphetamine-induced rotations is partially reversed by the selective δ-opioid antagonist naltrindole

Baseline rotations were measured in severely-lesioned hemi-Parkinsonian rats challenged with amphetamine to establish the 100% level. The data are plotted as % of amphetamine-only control. MMP-2200 significantly reduced the amphetamine-induced rotations (A+M, ^###P < 0.005, n=15, one way ANOVA with a Fisher LSD post-hoc test of the raw rotation data before normalization to % control) compared to this baseline. Naltrindole partially reversed this effect (A+M vs NTL+A+M, n=7, *P < 0.05; NTL+A+AM vs NTL+A, *P < 0.05), confirming that MMP-2200 exerts some of its effect through the δ opioid receptor. Naltrindole alone had no effect on amphetamine-induced rotations (NTL+A; n=7) nor did it directly induce rotations (NTI; n=5).

Figure 7. MMP-2200 decreased the rotations induced by amphetamine in a mild 6-OHDA lesioned rat model

Amphetamine injection induced turning ipsiversive to the lesion in this mild progressive unilateral 6-OHDA lesion model (less than 50% loss of dopaminergic cell bodies in the substantia nigra). The total number of amphetamine-induced rotations over a 1 hr period was significantly reduced by co-injection with MMP-2200 when compared to amphetamine alone (**P < 0.01, n=11, paired t-test).

Figure 8. MMP-2200 alone did not induce changes in locomotion in control or reserpinized rats

MMP-2200 induced a small but insignificant decrease in locomotion in control rats compared with vehicle control. Treatment with reserpine induced severe akinesia (***) that could be reversed with apomorphine with the expected overshoot due to dopamine super-sensitivity (**). Unlike apomorphine, MMP-2200 was not effective to reverse akinesia in reserpinized rats.

Figure 9. The reduction of apomorphine-induced hyper-active locomotion by MMP-2200 was blocked by the non-selective opioid receptor antagonist naloxone in the reserpinized rat model

Dopamine depletion was accomplished by systemic administration of reserpine to induce akinesia (^###). Apomorphine was administered 18 hr later to induce hyper-kinesia in the reserpinized group, as shown by the overshoot of the R+A group compared to control (***). This overshoot was abolished by MMP-2200 (R+A+M, *P < 0.05, t-test). The MMP-2200-effect was totally blocked by naloxone (^# P < 0.05, t-test). Naloxone alone had no effect on the apomorphine-induced hyper-kinesia (R+Nal+A).

Figure 10. Schematic of MMP-2200's action on the basal ganglia circuitry

This schematic depicts part of the classical model of basal ganglia pathways and includes the potential side of action for MMP-2200 in the direct and indirect pathways. SNc = substantia nigra pars compacta; GP_e = external segment of the globus pallidus; GP_i = internal segment of the globus pallidus.