Targeting organic cation transporters at the blood-brain barrier to treat ischemic stroke in rats

Abstract

Drug discovery and development for stroke is challenging as evidenced by few drugs that have advanced beyond a Phase III clinical trial. Memantine is a N-methyl-d-aspartate (NMDA) receptor antagonist that has been shown to be neuroprotective in various preclinical studies. We have identified an endogenous BBB uptake transport system for memantine: organic cation transporters 1 and 2 (Oct1/Oct2). Our goal was to evaluate Oct1/Oct2 as a required BBB mechanism for memantine neuroprotective effects. Male Sprague-Dawley rats (200-250 g) were subjected to middle cerebral artery occlusion (MCAO) for 90 min followed by reperfusion. Memantine (5 mg/kg, i.v.) was administered 2 h following intraluminal suture removal. Specificity of Oct-mediated transport was evaluated using cimetidine (15 mg/kg, i.v.), a competitive Oct1/Oct2 inhibitor. At 2 h post-MCAO, [³H]memantine uptake was increased in ischemic brain tissue. Cimetidine inhibited blood-to-brain uptake of [³H]memantine, which confirmed involvement of an Oct-mediated transport mechanism. Memantine reduced post-MCAO infarction and brain edema progression as well as improved neurological outcomes during post-stroke recovery. All positive effects of memantine were attenuated by co-administration of cimetidine, which demonstrates that Oct1/Oct2 transport is required for memantine to exert neuroprotective effects in ischemic stroke. Furthermore, Oct1/Oct2-mediated transport was shown to be the dominant mechanism for memantine brain uptake in the MCAO model despite a concurrent increase in paracellular "leak." These novel and translational findings provide mechanistic evidence for the critical role of BBB transporters in CNS delivery of stroke therapeutics, information that can help such drugs advance in clinical trials.

Keywords: Blood-brain barrier; Drug delivery; Endothelial cell; Ischemic stroke; Memantine; Organic cation transporters.

Figure 1:. Functional Expression of Oct1 and Oct2 at the BBB in Male Sprague-Dawley Rats.

A: Oct1 and Oct2 protein expression was measured by western blot analysis of brain microvessels isolated from control (i.e., untreated) male Sprague-Dawley rats. Isolated microvessels (10 μg) were resolved on a 4-12% SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and analyzed for expression of Oct1, Oct2, or α-tubulin (i.e., the loading control). Each lane triplet on the depicted western blot corresponds to a microvessel sample obtained from a single experimental animal. The image depicts a representative blot showing transporter expression data from six experimental animals (n = 6). B: Chemical structure of memantine. C: Uptake of [³H]memantine (0.5 μCi/ml) in brain tissue isolated from control (i.e., untreated) male Sprague-Dawley rats. Inhibition experiments were conducted in the presence and absence of cimetidine (25 μM), a competitive Oct transport inhibitor, that was perfused for 10 min prior to administration of [³H]memantine. Results are expressed as mean ± SD of six animals per time point (n = 6). Asterisks represent statistical significance between animals perfused with [³H]memantine and animals perfused with [³H]memantine in the presence of cimetidine (*** p < 0.001).

Figure 2:. Protein Expression of Oct1 in Ipsilateral and Contralateral Cerebral Cortical Microvessels after MCAO.

A: Oct1 protein expression was measured by western blot analysis of brain microvessels isolated from ipsilateral and contralateral cerebral cortex following transient MCAO (90 min) with reperfusion (2 h). Isolated microvessels (10 μg) were resolved on a 4-12% SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and analyzed for expression of Oct1. Each lane doublet on the depicted western blot corresponds to a microvessel sample obtained from a single experimental animal. The image depicts a representative blot from six individual animals (n = 6). B: Relative levels of Oct1 protein expression in ipsilateral and contralateral microvessels were determined by densitometric analysis and normalized to total protein. Quantitative results are expressed as mean ± SD from six individual animals (n = 6).

Figure 3:. Protein Expression of Oct2 in Ipsilateral and Contralateral Cerebral Cortical Microvessels after MCAO.

A: Oct2 protein expression was measured by western blot analysis of brain microvessels isolated from ipsilateral and contralateral cerebral cortex following transient MCAO (90 min) with reperfusion (2 h). Isolated microvessels (10 μg) were resolved on a 4-12% SDS-polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and analyzed for expression of Oct2. Each lane doublet on the depicted western blot corresponds to a microvessel sample obtained from a single experimental animal. The image depicts a representative blot from six individual animals (n = 6). B: Relative levels of Oct2 protein expression in ipsilateral and contralateral microvessels were determined by densitometric analysis and normalized to total protein. Quantitative results are expressed as mean ± SD from six individual animals (n = 6).

Figure 4:. CNS Delivery of Memantine by Oct-Mediated Transport and Paracellular Sucrose “Leak” in Male Sprague-Dawley Rats Subjected to MCAO.

A: Uptake of [³H]memantine (0.5 μCi/ml) in whole brain tissue from male Sprague-Dawley rats subjected to transient MCAO (90 min) with reperfusion (2 h) and Sham-operated controls. Inhibition experiments were conducted in the presence and absence of cimetidine (25 μM), a competitive Oct transport inhibitor, that was perfused for 10 min prior to administration of [³H]memantine. B: Uptake of [³H]memantine (0.5 μCi/ml) in ipsilateral and contralateral cortical brain tissue from experimental animals subjected to transient MCAO (90 min) with reperfusion (2 h) and Sham-operated controls. Inhibition experiments were conducted in the presence and absence of cimetidine (25 μM). C: Uptake of [³H]sucrose (0.5 μCi/ml) in whole brain tissue from male Sprague-Dawley rats subjected to transient MCAO (90 min) with reperfusion (2 h) and Sham-operated controls. D: Uptake of [³H]sucrose (0.3 μCi/ml) in ipsilateral and contralateral cortical brain tissue from experimental animals subjected to transient MCAO (90 min) with reperfusion (2 h) and Sham-operated controls. Results are expressed as mean ± SD of six animals per time point (n = 6). Asterisks represent data points that were significantly different from control animals (* p < 0.05; ** p < 0.01; **** p < 0.0001; ns = not significant).

Figure 5:. Memantine Improves Neurological Scoring via Oct1/Oct2 in Male Sprague-Dawley Rats Subjected to MCAO.

Neurological deficit score assessment in male Sprague-Dawley rats at 24 h post-MCAO (A) or 72 h post-MCAO (B). MCAO animals were administered vehicle, memantine (MEM, 5 mg/kg, i.v.) or memantine/cimetidine (MEM, 5 mg/kg, i.v.; cimetidine, 15 mg/kg, i.v.). Sham-operated animals were used as experimental controls. Results are expressed as mean ± SD of six animals per treatment group (n = 6). Asterisks represent data points that were significantly different from control animals (* p < 0.05; **** p < 0.0001; ns = not significant).

Figure 6:. Memantine Improves Motor Performance via Oct1/Oct2 in Male Sprague-Dawley Rats Subjected to MCAO.

Motor performance was assessed by rotarod performance testing in animals at 24 h post-MCAO (A) and 72 h post-MCAO (B). MCAO animals were administered vehicle, memantine (MEM, 5 mg/kg, i.v.) or memantine/cimetidine (MEM, 5 mg/kg, i.v.; cimetidine, 15 mg/kg, i.v.). Sham-operated animals were used as experimental controls. Results are expressed as mean ± SD of six animals per treatment group (n = 6). Asterisks represent data points that were significantly different from control animals (** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant).

Figure 7:. Memantine Attenuates Cerebral Infarction Volume and Edema Ratio via Oct1/Oct2 in Male Sprague-Dawley Rats Subjected to MCAO.

A: Representative TTC staining of brain tissue collected from experimental animals subjected to transient MCAO (90 min) with reperfusion (70.5 h) (i.e., 72 h post-MCAO). Animals were administered vehicle, memantine (MEM, 5 mg/kg, i.v.) or memantine/cimetidine (MEM, 5 mg/kg, i.v.; cimetidine, 15 mg/kg, i.v.). Sham-operated animals were used as experimental controls. Arrows indicate regions of infarction in individual brain slides as detected by TTC staining. B: Measurement of cerebral infarction volume in TTC-stained brain tissue. C: Measurement of brain edema ratio in TTC-stained brain tissue. Results are expressed as mean ± SD of six animals per treatment group (n = 6). Asterisks represent data points that were significantly different from control animals (** p < 0.01; *** p < 0.001; **** p < 0.0001; ns = not significant).