In vitro and in vivo efficacy of a potent opioid receptor agonist, biphalin, compared to subtype-selective opioid receptor agonists for stroke treatment

Abstract

To meet the challenge of identification of new treatments for stroke, this study was designed to evaluate a potent, nonselective opioid receptor (OR) agonist, biphalin, in comparison to subtype selective OR agonists, as a potential neuroprotective drug candidate using in vitro and in vivo models of ischemic stroke. Our in vitro approach included mouse primary neuronal cells that were challenged with glutamate and hypoxic/aglycemic (H/A) conditions. We observed that 10nM biphalin, exerted a statistically significant neuroprotective effect after glutamate challenge, compared to all selective opioid agonists, according to lactate dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Moreover, 10nM biphalin provided superior neuroprotection after H/A-reoxygenation compared to selective opioid agonists in all cases. Our in vitro investigations were supported by in vivo studies which indicate that the nonselective opioid agonist, biphalin, achieves enhanced neuroprotective potency compared to any of the selective opioid agonists, evidenced by reduced edema and infarct ratios. Reduction of edema and infarction was accompanied by neurological improvement of the animals in two independent behavioral tests. Collectively these data strongly suggest that concurrent agonist stimulation of mu, kappa and delta ORs with biphalin is neuroprotective and superior to neuroprotection by activation of any single OR subtype.

Keywords: Blood–brain barrier; Neuropeptide; Neuroprotection; Opioid receptors; Stroke.

Figure 1

Structures of biphalin and selective OR agonists. A. Non-selective OR agonist biphalin; B. DOR selective agonist DPDPE (Tyr-D-Pen-Gly-Phe-D-Pen); C. MOR selective agonist DAMGO (Tyr-D-Ala-Gly-NMe-Phe4, Gly-ol5-enkephalin); D. KOR selective agonist U 50488.

Figure 2

Effect of biphalin and selective OR agonists on glutamate challenge. For both graphs; *P<0.05; **P<0.01; ***P<0.001; data from 3 - 4 independent primary neuronal isolations with 2 - 3 replicate treatments per isolation. Selective agonists, DPDPE, DAMGO and U50,488 did not show any significant effect compared to no drug treatment group. All experimental groups were significantly different compared to normoxia (P<0.0001). A. LDH assay; effect of biphalin and selective OR agonists (10 nM) on glutamate exposure (25 uM) for 24 h. Compared to no drug treated group biphalin significantly decreased neuronal death (P<0.001) and effect of biphalin on neuronal death is statistically significant compared to DPDPE (P<0.01), DAMGO (P<0.01) and U50,488 (P<0.05). B. MTT assay; effect of biphalin and selective OR agonists (10 nM) on glutamate exposure (25 uM) for 24 h. Biphalin is significantly improving neuronal survival (P<0.01) compared to no drug treated group. In comparison to DPDPE (P<0.05), DAMGO (P<0.05) and U50,488 (P<0.05), biphalin showed statistical significant improvement in terms of neuronal survival. Naltrexone reversed the effect (P<0.001) of biphalin.

Figure 3

Effects of biphalin, morphine and selective OR agonists on H/A and reoxygenation challenge. For both graphs; ‘#’ compared to no drug group; ‘*’ compared to biphalin; ^#P<0.05; ^##P<0.01; ^###P<0.001;*P<0.05; **P<0.01; ***P<0.001; ****p<0.0001; data from 3 - 4 independent primary neuronal isolations with 2 - 3 replicate treatments per isolation. Compared to normoxia or 0.1% triton X, all experimental groups were significantly different (P<0.0001). A. LDH assay; effect of biphalin and selective OR agonists (10 nM) on 3 h H/A and 24 h reoxygenation. Compared to no drug treated group biphalin (P<0.0001), DPDPE (P<0.001), DAMGO (P<0.001), U50,488 (P<0.001) and Morphine (P<0.01) significantly decreased neuronal cell death. Again biphalin in comparison to DPDPE (P<0.05), DAMGO (P<0.05), U50,488 (P<0.05) and morphine (P<0.0001) showed better neuroprotection in terms of decreased neuronal death. NTX (P<0.0001) reversed the effect of biphalin. B. MTT assay; effect of biphalin, morphine and selective OR agonists (10 nM) on 3 h H/A and 24 h reoxygenation. Biphalin (P<0.0001), morphine (P<0.05), DPDPE (P<0.05), DAMGO (P<0.001) and U50.488 (P<0.001) significantly improved neuronal cell survival compared to no drug treated group. In comparison to morphine (P<0.01), DPDPE (P<0.001), DAMGO (P<0.05) and U50,488 (P<0.05), biphalin showed statistically significant improvement in terms of neuronal survival. NTX (P<0.0001) reversed the effect of biphalin.

Figure 3

Effects of biphalin, morphine and selective OR agonists on H/A and reoxygenation challenge. For both graphs; ‘#’ compared to no drug group; ‘*’ compared to biphalin; ^#P<0.05; ^##P<0.01; ^###P<0.001;*P<0.05; **P<0.01; ***P<0.001; ****p<0.0001; data from 3 - 4 independent primary neuronal isolations with 2 - 3 replicate treatments per isolation. Compared to normoxia or 0.1% triton X, all experimental groups were significantly different (P<0.0001). A. LDH assay; effect of biphalin and selective OR agonists (10 nM) on 3 h H/A and 24 h reoxygenation. Compared to no drug treated group biphalin (P<0.0001), DPDPE (P<0.001), DAMGO (P<0.001), U50,488 (P<0.001) and Morphine (P<0.01) significantly decreased neuronal cell death. Again biphalin in comparison to DPDPE (P<0.05), DAMGO (P<0.05), U50,488 (P<0.05) and morphine (P<0.0001) showed better neuroprotection in terms of decreased neuronal death. NTX (P<0.0001) reversed the effect of biphalin. B. MTT assay; effect of biphalin, morphine and selective OR agonists (10 nM) on 3 h H/A and 24 h reoxygenation. Biphalin (P<0.0001), morphine (P<0.05), DPDPE (P<0.05), DAMGO (P<0.001) and U50.488 (P<0.001) significantly improved neuronal cell survival compared to no drug treated group. In comparison to morphine (P<0.01), DPDPE (P<0.001), DAMGO (P<0.05) and U50,488 (P<0.05), biphalin showed statistically significant improvement in terms of neuronal survival. NTX (P<0.0001) reversed the effect of biphalin.

Figure 4

Effects of biphalin and selective OR agonists (10 nM) on total ROS production following 3 h H/A and 24 h reoxygenation in primary cortical neurons. All experimental groups were significantly different compared to normoxia (P<0.0001) or H₂O₂. Compared to no drug treated group, biphalin (P<0.0001), DPDPE (P<0.01), DAMGO (P<0.0001) and U50,488 (P<0.0001) decreased total ROS production. Biphalin effects on total ROS production was statistically significant in comparison to DPDPE (P<0.01). (‘#’ compared to no drug group; ‘*’ compared to biphalin; ^##P<0.01; ^####P<0.0001; **P<0.01; **** P<0.0001; data from 3 - 4 independent primary neuronal isolations with 2 - 3 replicate treatments per isolation).

Figure 5

Effect of intraperitoneal injection of biphalin, selective OR agonists (5 mg/kg, I.P. administrated 10 min after reperfusion), non-selective OR antagonist, naltrexone, (1 mg/kg, I.P. administrated 10 min before the surgery) or vehicle on edema and infarct formation in transient MCAO (60 min occlusion and 24 h reperfusion). A. Representative TTC staining of brains from vehicle and drug -treated mice. B. Brain edema formation in vehicle and drug -treated mice. Biphalin significantly decreased edema ratio (P<0.01) compared to saline treated group. NTX reversed (P<0.05) the effect of biphalin. Compared to selective agonists DPDPE (P<0.001) and DAMGO (P<0.001), biphalin significantly reduced edema ratio. Effect of DPDPE, DAMGO and U50,488 was not statistically significant compared to saline treated group. C. Brain infarct ratio in vehicle and drug-treated mice. Biphalin significantly reduced infarct ration (P<0.01) compared to 0.9% saline treated group. Biphalin showed better neuroprotection in terms of infarct ratio reduction compared to DPDPE (P<0.01), DAMGO (P<0.05) and NTX (P<0.05). NTX reversed (P<0.01) the effect of biphalin. In terms of infarct ration reduction, DPDPE, DAMGO and U50,488 did not show a statistically significant effect compared to vehicle treated group. (*P<0.05; **P<0.01; ***P<0.001; numbers indicated in parenthesis in the figure columns donate to the number of experimental animals per group). Mean cerebral blood flow reductions ± SEM in ischemic brain for saline group 80.7 ± 1.24%, biphalin 78.5 ± 0.96%, BIP+NTX 79.7 ± 2.02 %, DPDPE 81.3 ± 2.05 %, DAMGO 79.7 ± 0.83%, U50,488 78.8 ± 1.72% and NTX 76.9 ± 2.01%.

Figure 5

Effect of intraperitoneal injection of biphalin, selective OR agonists (5 mg/kg, I.P. administrated 10 min after reperfusion), non-selective OR antagonist, naltrexone, (1 mg/kg, I.P. administrated 10 min before the surgery) or vehicle on edema and infarct formation in transient MCAO (60 min occlusion and 24 h reperfusion). A. Representative TTC staining of brains from vehicle and drug -treated mice. B. Brain edema formation in vehicle and drug -treated mice. Biphalin significantly decreased edema ratio (P<0.01) compared to saline treated group. NTX reversed (P<0.05) the effect of biphalin. Compared to selective agonists DPDPE (P<0.001) and DAMGO (P<0.001), biphalin significantly reduced edema ratio. Effect of DPDPE, DAMGO and U50,488 was not statistically significant compared to saline treated group. C. Brain infarct ratio in vehicle and drug-treated mice. Biphalin significantly reduced infarct ration (P<0.01) compared to 0.9% saline treated group. Biphalin showed better neuroprotection in terms of infarct ratio reduction compared to DPDPE (P<0.01), DAMGO (P<0.05) and NTX (P<0.05). NTX reversed (P<0.01) the effect of biphalin. In terms of infarct ration reduction, DPDPE, DAMGO and U50,488 did not show a statistically significant effect compared to vehicle treated group. (*P<0.05; **P<0.01; ***P<0.001; numbers indicated in parenthesis in the figure columns donate to the number of experimental animals per group). Mean cerebral blood flow reductions ± SEM in ischemic brain for saline group 80.7 ± 1.24%, biphalin 78.5 ± 0.96%, BIP+NTX 79.7 ± 2.02 %, DPDPE 81.3 ± 2.05 %, DAMGO 79.7 ± 0.83%, U50,488 78.8 ± 1.72% and NTX 76.9 ± 2.01%.

Figure 6

Neurological score evaluation of mice 24 h after stroke and drug treatments. Biphalin showed reduced neurological deficit in terms of neurological score compared to saline treated group (P<0.01), DPDPE (P<0.05) and DAMGO (P<0.05). NTX reversed the effect (P<0.05) of biphalin. Selective agonists DPDPE, DAMGO and U50,488 did not show a statistically significant effect when compared to saline treated group. (*: P<0.05, **P<0.01; numbers indicated in parenthesis in the figure columns donate to the number of experimental animals per group).