Safety and efficacy evaluation of carnosine, an endogenous neuroprotective agent for ischemic stroke

Abstract

Background and purpose: An urgent need exists to develop therapies for stroke that have high efficacy, long therapeutic time windows, and acceptable toxicity. We undertook preclinical investigations of a novel therapeutic approach involving supplementation with carnosine, an endogenous pleiotropic dipeptide.

Methods: Efficacy and safety of carnosine treatment was evaluated in rat models of permanent or transient middle cerebral artery occlusion. Mechanistic studies used primary neuronal/astrocytic cultures and ex vivo brain homogenates.

Results: Intravenous treatment with carnosine exhibited robust cerebroprotection in a dose-dependent manner, with long clinically relevant therapeutic time windows of 6 hours and 9 hours in transient and permanent models, respectively. Histological outcomes and functional improvements including motor and sensory deficits were sustained on 14th day poststroke onset. In safety and tolerability assessments, carnosine did not exhibit any evidence of adverse effects or toxicity. Moreover, histological evaluation of organs, complete blood count, coagulation tests, and the serum chemistry did not reveal any abnormalities. In primary neuronal cell cultures and ex vivo brain homogenates, carnosine exhibited robust antiexcitotoxic, antioxidant, and mitochondria protecting activity.

Conclusions: In both permanent and transient ischemic models, carnosine treatment exhibited significant cerebroprotection against histological and functional damage, with wide therapeutic and clinically relevant time windows. Carnosine was well tolerated and exhibited no toxicity. Mechanistic data show that it influences multiple deleterious processes. Taken together, our data suggest that this endogenous pleiotropic dipeptide is a strong candidate for further development as a stroke treatment.

Figure 1. Effects of carnosine on infarct size and neurological function in rats at 24 h after 3 h tMCAO

A. Cerebral blood flow (CBF) was monitored during the surgical procedure. B. Effect of carnosine on neurological deficits. C. Left, representative TTC-stained sections; Right, quantification of the infarct volume. *p<0.05 and **p<0.01 vs. saline-treated group. A:n=20, B:n=14-15, C:n=14-15. All values are means ± SEM and analyzed by ANOVA tests.

Figure 2. Therapeutic time window of carnosine neuroprotection against ischemic stroke in rats

Carnosine treatment was initiated at indicated time in transient- (A) or permanent-(B) ischemic rat models. *p<0.05 and **p<0.01 vs. saline-treated group. A:n=7-15, B:13-15. All values are means ± SEM and analyzed by Student's t-test.

Figure 3. Protective effects of carnosine in rats with 3 h-tMCAO through 14 d survival duration

A. The distribution of infarct (left) and infarct volume (right) at 14 d after ischemia are shown. B to D. Functional outcome was evaluated by adhesive tape removal test (B), rotarod test (C), and neurological scoring (D). Sham rats underwent the same surgical procedure apart from MCAO. *p<0.05 and **p<0.01 vs. saline-treated group. A:n=17-18, B:n=13-14, C:n=17, D=17-18 (Sham animal;n=5 for B to D). All values are means ± SEM and analyzed by Student's t-test.

Figure 4. Evaluation of safety and tolerability of carnosine after intravenous administration

A and B, After single intravenous administration of saline or carnosine to rats, body weight (A) and food consumption (B) were monitored for 14 d. C, Histopathological evaluation of toxicity in various organs was performed using HE staining. A: n=7, B: n=7, C: n=4. All values are means ± SEM and analyzed by Student's t-test. Representative photos are shown.

Figure 5. The effect of carnosine on clot lysis by tPA

The amount of clot lysis was obtained after addition of tPA or carnosine (CAR) using spectrophotometry. A, Control: No treatment, tPA: tissue plasminogen activator (8.3 μg/mL), PAI: plasminogen activator inhibitor (8.3 μg/mL), CAR: carnosine (30 μg/mL) B, The thrombolytic activity of tPA (8.3 μg/mL) was not affected by carnosine (10, 20, and 30 μg/mL). A: n=5, B: n=5. All values are means ± SEM.

Figure 6. Underlying mechanisms for carnosine neuroprotection

A to C, Primary cortical neurons or astrocytes were isolated from neonatal mice, and were exposed to ischemic stimulus of NMDA or oxygen-glucose deprivation (OGD). Cell death (A), generation of reactive oxygen species (B), and transition of mitochondrial membrane potential (C) were decreased by carnosine. *p<0.05 and **p<0.01 vs. ischemic stimulus. D. Effect of carnosine on respiratory control ratio was examined in ex vivo rat brain homogenates after pMCAO. *p<0.05 and **p<0.01. P/M, Pyruvate and malate; A, ADP; O, Oligomycine A; C, CCCP; S, Succinate. A: n=4, B: n=3,C: n=3, D: n=4. All values are means ± SEM and analyzed by ANOVA tests or by Student's t-test. Representative tracings of oxygen consumption are shown.