MMG22 Potently Blocks Hyperalgesia in Cisplatin-treated Mice

Abstract

MMG22 is a bivalent ligand containing MOR agonist and mGluR5 antagonist pharmacophores connected by a 22-atom linker. Intrathecal (i.t.) administration of MMG22 to inflamed mice has been reported to produce fmol-range antinociception in the reversal of LPS-induced hyperalgesia. MMG22 reduced hyperalgesia in the spared nerve injury (SNI) model of neuropathic pain at 10 days after injury but not at 30 days after injury, perhaps related to the inflammation that occurs early after injury but subsequently subsides. The present study determined the efficacy of MMG22 in cisplatin-treated male mice in order to provide data relating to the efficacy of MMG22 in the treatment of neuropathic pain that is associated with inflammation. Groups of eight mice each received daily intraperitoneal (i.p.) injections of cisplatin for seven days to produce robust mechanical allodynia defined by the decrease in withdrawal threshold using an electronic von Frey applied to the plantar surface of the hind paw. Intrathecal administration of MMG22 potently reduced mechanical hyperalgesia (ED₅₀ 0.04 fmol/mouse) without tolerance, whereas MMG10 was essentially inactive. Morphine was less potent than MMG22 by >5-orders of magnitude and displayed tolerance. Subcutaneous MMG22 was effective (ED₅₀ = 2.41 mg/kg) and devoid of chronic tolerance. We propose that MMG22 induces the formation of a MOR-mGluR5 heteromer through selective interaction with the upregulated NR2B subunit of activated NMDAR, in view of the 4600-fold reduction of i.t. MMG22 antinociception by the selective NR2B antagonist, Ro25-6981. A possible explanation for the substantially reduced potency for MMG22 in the SNI model is discussed.

Keywords: MMG22; antihyperalgesia; antinociception; cisplatin; neuropathic pain.

Figure 1.. Intrathecal dose-response and acute tolerance (AT) for MMG22, MMG10 and morphine in the cisplatin model of neuropathic pain.

A) ED₅₀ values (n=8 per group) are listed as %MPE ± 95% C.I. There was no potency change for the highest dose of MMG22 when repeated after 24 hours (AT) in the same mice. Paired two-tail t-tests for both morphine and MMG10 comparing the original ED₈₀ dose from original dose response curve to the same dose measured 24 hours later both showed significant acute tolerance (t (7= 4.136) p ≤ 0.004) and (t (7= 6.467) p ≤ 0.0003). B) MMG22 and morphine (n=8 per group) are compared at 1.0 nmol/mouse (ED₈₀ of MMG10) for antinociception and acute tolerance. MMG22 and morphine were tested at their peak times and compared to the same dose re-administered 24 hours later for determination of acute tolerance. MMG22 at ~25 million times its ED₅₀ dose (0.04 fmol/mouse-i.t.) on day 1, did not exhibit 24-hour acute tolerance (t (7 =1.644) NS). This was in contrast to morphine at 50 times its ED₅₀ (0.02 nmol/mouse i.t.) dose (t (7 = 6.193) p ≤ 0.0004***) and MMG10 (t (7 = 6.467 p ≤ 0.0003***) both of which displayed acute tolerance.

Figure 2.. Dose-response relationships for subcutaneous administration of MMG22 and morphine.

A) ED₅₀ values (%MPE ± 95% C.I.) were determined for MMG22 and morphine (n=8 per group). Morphine was approximately 5-times more potent than MMG22 when given subcutaneously. B). Subcutaneous administration of MMG22 did not produce acute tolerance. The peak antihyperalgesic effect following 10.0 mg/kg MMG22 was 30 and 60 min (t (7=3.346, NS) and antihyperalgesia was reduced by 120 minutes (t (7=5.060) p ≤ 0.05*). Hyperalgesia returned by 24 hours, and a second injection of MMG22 produced the same degree of antihyperalgesia as that given 24 hours earlier.

Figure 3.. Tolerance produced by chronic administration of MMG22 and morphine.

A) Repeated administration of MMG22ndid not produce tolerance. Cisplatin produced robust hyperalgesia compared to pre-cisplatin values (t (7 = 8.288) p ≤ 0.001***). After the first injection (Day 1) of MMG22 (10.0 mg/kg, s.c., n=8) there was a significant increase in mechanical threshold to the post-cisplatin day 1 baseline (t (7 = 4.837) p ≤ 0.054). On days 3 and day 9 both the post-cisplatin baseline and treatment with MMG22 were not different from the pre-cisplatin control. However, there was a significant difference between the baselines. Post-cisplatin baseline day 1 was significantly different from both post-cisplatin days 3 and 9 (t (7 = 6.854) p ≤ 0.001+++) and (t (7 = 3.941) p ≤ 0.05+) respectively. There was no difference in mechanical thresholds among the three measured time points for MMG22. B) Repeated administration of morphine produced tolerance. Morphine (n=8) reduced antihyperalgesic efficacy of cisplatin on day-1 with the appearance of significant tolerance on days 2 and 3. There was significant difference between the pre-cisplatin and post-cisplatin baselines on day 1, 3 and 9 (t (7 = 13.21) p ≤ 0.0001****), (t (7 = 8.428) p ≤ 0.001***) and (t (7 = 13.56) p ≤ 0.0001****) respectively. On treatment day 1 (after the initial dose of morphine (5.0 mg/kg-s.c.) there was a significant increase in mechanical threshold (t (7=10.36) p ≤ 0.001$$$) between the post-cisplatin baseline and morphine. Morphine exhibited chronic tolerance on both Days 3 and 9 as there was no significant difference between the post-cisplatin baseline and morphine on either day. However, the morphine treatments on Days 3 and 9 were significantly different from the pre-cisplatin baseline (t (7 = 3.832) p ≤ 0.05#) and (t (7 = 7.863) p ≤ 0.001###) respectively. On Days 3 and 9 there were also significant reductions in potency of morphine ((t (7 = 4.086 p ≤ 0.05= and ((t (7 = 5.019) p ≤ 0.01==) as compared to morphine Day 1.

Figure 4.. Proposed mechanism for the induction of a MOR-mGluR5 heteromer with concomitant activation of MOR and inhibition of both activated mGluR5 and NMDAR.

The opioid agonist pharmacophore (triangle) of MMG22 associates with MOR homomer (A→B) which then undergoes heteromer formation with activated mGluR5 which becomes antagonized by the MPEP antagonist pharmacophore linked to MMG22-bound MOR (B→C). Activated MOR together with antagonism of the NMDAR amplifies the potency of MMG22.