Mai Hazekawa

Delayed Treatment With Minocycline Ameliorates Neurologic Impairment Through Activated Microglia Expressing a High-Mobility Group Box1–Inhibiting Mechanism

Background and Purpose— Minocycline, a semisynthetic tetracycline antibiotic, has been reported to ameliorate brain injury and inhibit microglial activation after focal cerebral ischemia. However, the cerebroprotective mechanism of minocycline remains unclear. In the present study, we investigated that mechanism of minocycline in a murine model of 4-hour middle cerebral artery (MCA) occlusion. Methods— One day after 4-hour MCA occlusion, minocycline was administered intraperitoneally for 14 days. Neurologic scores were measured 1, 7, and 14 days after cerebral ischemia. Motor coordination was evaluated at 14 days by the rota-rod test at 10 rpm. Activated microglia and high-mobility group box1 (HMGB1), a cytokine-like mediator, were also evaluated by immunostaining and Western blotting. In addition, terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling immunostaining was carried out 14 days after cerebral ischemia. Results— Repeated treatment with minocycline (1, 5, and 10 mg/kg) for 14 days improved neurologic score, motor coordination on the rota-rod test, and survival in a dose-dependent manner. Minocycline decreased the expression of Iba1, a marker of activated microglia, as assessed by both immunostaining and Western blotting. Moreover, minocycline decreased the activation of microglia expressing HMGB1 within the brain and also decreased both brain and plasma HMGB1 levels. Additionally, minocycline significantly decreased the number of terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling–positive cells and prevented ischemic brain atrophy 14 days after cerebral ischemia. Conclusions— Our results suggest that minocycline inhibits activated microglia expressing HMGB1 and decreases neurologic impairment induced by cerebral ischemia. Minocycline will have a palliative action and open new therapeutic possibilities for treatment of postischemic injury via an HMGB1-inhibiting mechanism.

Cannabidiol potentiates pharmacological effects of Δ9-tetrahydrocannabinol via CB1 receptor-dependent mechanism

Cannabidiol, a non-psychoactive component of cannabis, has been reported to have interactions with Delta(9)-tetrahydrocannabinol (Delta(9)-THC). However, such interactions have not sufficiently been clear and may have important implications for understanding the pharmacological effects of marijuana. In the present study, we investigated whether cannabidiol modulates the pharmacological effects of Delta(9)-THC on locomotor activity, catalepsy-like immobilisation, rectal temperature and spatial memory in the eight-arm radial maze task in mice. In addition, we measured expression level of cannabinoid CB(1) receptor at striatum, cortex, hippocampus and hypothalamus. Delta(9)-THC (1, 3, 6 and 10 mg/kg) induced hypoactivity, catalepsy-like immobilisation and hypothermia in a dose-dependent manner. In addition, Delta(9)-THC (1, 3 and 6 mg/kg) dose-dependently impaired spatial memory in eight-arm radial maze. On the other hand, cannabidiol (1, 3, 10, 25 and 50 mg/kg) did not affect locomotor activity, catalepsy-like immobilisation, rectal temperature and spatial memory on its own. However, higher dose of cannabidiol (10 or 50 mg/kg) exacerbated pharmacological effects of lower dose of Delta(9)-THC, such as hypoactivity, hypothermia and impairment of spatial memory. Moreover, cannabidiol (50 mg/kg) with Delta(9)-THC (1 mg/kg) enhanced the expression level of CB(1) receptor expression in hippocampus and hypothalamus. Cannabidiol potentiated pharmacological effects of Delta(9)-THC via CB(1) receptor-dependent mechanism. These findings may contribute in setting the basis for interaction of cannabinoids and to find a cannabinoid mechanism in central nervous system.

Cannabidiol prevents a post-ischemic injury progressively induced by cerebral ischemia via a high-mobility group box1-inhibiting mechanism.

We examined the cerebroprotective mechanism of cannabidiol, the non-psychoactive component of marijuana, against infarction in a 4-h mouse middle cerebral artery (MCA) occlusion model. Cannabidiol was intraperitoneally administrated immediately before and 3h after cerebral ischemia. Infarct size and myeloperoxidase (MPO) activity, a marker of neutrophil, monocyte/macropharge, were measured at 24h after cerebral ischemia. Activated microglia and astrocytes were evaluated by immunostaining. Moreover, high-mobility group box1 (HMGB1) was also evaluated at 1 and 3 days after MCA occlusion. In addition, neurological score and motor coordination on the rota-rod test were assessed at 1 and 3 days after cerebral ischemia. Cannabidiol significantly prevented infarction and MPO activity at 20h after reperfusion. These effects of cannabidiol were not inhibited by either SR141716 or AM630. Cannabidiol inhibited the MPO-positive cells expressing HMGB1 and also decreased the expression level of HMGB1 in plasma. In addition, cannabidiol decreased the number of Iba1- and GFAP-positive cells at 3 days after cerebral ischemia. Moreover, cannabidiol improved neurological score and motor coordination on the rota-rod test. Our results suggest that cannabidiol inhibits monocyte/macropharge expressing HMGB1 followed by preventing glial activation and neurological impairment induced by cerebral ischemia. Cannabidiol will open new therapeutic possibilities for post-ischemic injury via HMGB1-inhibiting mechanism.

Repeated treatment with cannabidiol but not Δ9-tetrahydrocannabinol has a neuroprotective effect without the development of tolerance

Both Delta(9)-tetrahydrocannabinol (Delta(9)-THC) and cannabidiol are known to have a neuroprotective effect against cerebral ischemia. We examined whether repeated treatment with both drugs led to tolerance of their neuroprotective effects in mice subjected to 4h-middle cerebral artery (MCA) occlusion. The neuroprotective effect of Delta(9)-THC but not cannabidiol was inhibited by SR141716, cannabinoid CB(1) receptor antagonist. Fourteen-day repeated treatment with Delta(9)-THC, but not cannabidiol, led to tolerance of the neuroprotective and hypothermic effects. In addition, repeated treatment with Delta(9)-THC reversed the increase in cerebral blood flow (CBF), while cannabidiol did not reverse that effect. Repeated treatment with Delta(9)-THC caused CB(1) receptor desensitization and down-regulation in MCA occluded mice. On the contrary, cannabidiol did not influence these effects. Moreover, the neuroprotective effect and an increase in CBF induced by repeated treatment with cannabidiol were in part inhibited by WAY100135, serotonin 5-HT(1A) receptor antagonist. Cannabidiol exhibited stronger antioxidative power than Delta(9)-THC in an in vitro study using the 1,1-diphenyl-2-picryhydrazyl (DPPH) radical. Thus, cannabidiol is a potent antioxidant agent without developing tolerance to its neuroprotective effect, acting through a CB(1) receptor-independent mechanism. It is to be hoped that cannabidiol will have a palliative action and open new therapeutic possibilities for treating cerebrovascular disorders.

Delayed treatment with cannabidiol has a cerebroprotective action via a cannabinoid receptor-independent myeloperoxidase- inhibiting mechanism

We examined the neuroprotective mechanism of cannabidiol, non‐psychoactive component of marijuana, on the infarction in a 4 h mouse middle cerebral artery (MCA) occlusion model in comparison with Δ9‐tetrahydrocannabinol (Δ9‐THC). Release of glutamate in the cortex was measured at 2 h after MCA occlusion. Myeloperoxidase (MPO) and cerebral blood flow were measured at 1 h after reperfusion. In addition, infarct size and MPO were determined at 24 and 72 h after MCA occlusion. The neuroprotective effect of cannabidiol was not inhibited by either SR141716 or AM630. Both pre‐ and post‐ischemic treatment with cannabidiol resulted in potent and long‐lasting neuroprotection, whereas only pre‐ischemic treatment with Δ9‐THC reduced the infarction. Unlike Δ9‐THC, cannabidiol did not affect the excess release of glutamate in the cortex after occlusion. Cannabidiol suppressed the decrease in cerebral blood flow by the failure of cerebral microcirculation after reperfusion and inhibited MPO activity in neutrophils. Furthermore, the number of MPO‐immunopositive cells was reduced in the ipsilateral hemisphere in cannabidiol‐treated group. Cannbidiol provides potent and long‐lasting neuroprotection through an anti‐inflammatory CB1 receptor‐independent mechanism, suggesting that cannabidiol will have a palliative action and open new therapeutic possibilities for treating cerebrovascular disorders.

High-cholesterol feeding aggravates cerebral infarction via decreasing the CB1 receptor.

We examined how feeding conditions affect the CB1 receptor and cerebral infarction caused by cerebral ischemia. Mice were divided into the following three groups: normal diet (ND), caloric restriction (CR) and high-cholesterol-enriched diet (HCD), and were kept for 6 weeks. After 6 weeks, we measured both serum and brain cholesterol and the expression level of cannabinoid CB1 receptor within the brain in intact mice. In addition, middle cerebral artery (MCA) was occluded for 2 h following reperfusion. Serum cholesterol significantly increased in the HCD group in comparison with both the ND and CR groups. However, brain cholesterol decreased in the HCD group. Then, the expression level of CB1 receptor significantly decreased in the HCD group, while that of the CR group clearly increased in comparison with the ND group in intact mice. In MCA-occluded mice, The HCD group produced the most severe cerebral infarction, while cerebral infarction was significantly decreased in the CR group. These results suggest that CR prevents infarction by increasing CB1 receptor expression, while high-cholesterol feeding aggravates cerebral infarction both by hypercholesterolemia in serum and by decreasing CB1 receptor expression modulated by hypocholesterolemia within the brain.

Evaluation of palatability of 10 commercial amlodipine orally disintegrating tablets by gustatory sensation testing, OD‐mate as a new disintegration apparatus and the artificial taste sensor

The purpose of this study was to evaluate and compare the palatability of 10 formulations (the original manufacturers formulation and nine generics) of amlodipine orally disintegrating tablets (ODTs) by means of human gustatory sensation testing, disintegration/dissolution testing and the evaluation of bitterness intensity using a taste sensor.

Single injection of ONO-1301-loaded PLGA microspheres directly after ischaemia reduces ischaemic damage in rats subjected to middle cerebral artery occlusion

Objectives  ONO‐1301 was developed as a novel long‐acting prostacyclin agonist with thromboxane synthase inhibitory activity. In this study, we investigated the therapeutic time window of oral ONO‐1301 and the effect of a single subcutaneous injection of ONO‐1301‐loaded poly(lactide‐co‐glycolide) (PLGA) microspheres (ONO‐1301 PLGA MS) on infarction volume, functional deficits and plasma ONO‐1301 levels following a 1 h middle cerebral artery occlusion (MCAO) in rats.

Preparation of ONO-1301-loaded poly(lactide-co-glycolide) microspheres and their effect on nerve conduction velocity

Objectives  The aim of this study was to prepare poly(lactide‐co‐glycolide) (PLGA) microspheres containing ONO‐1301, a novel long‐acting prostacyclin agonist with thromboxane synthase inhibitory activity, with 10% of drug released in the initial burst and a sustained‐release period of about 3 weeks after administration. The effect of PLGA type (molecular weight and the lactide/glycolide (L/G) ratio in PLGA), the preparative conditions and the particle size on the in‐vitro release profile were examined. The effect of optimized ONO‐1301‐loaded PLGA microspheres on delayed nerve condition velocity (NCV) was investigated in streptozotocin (STZ) induced diabetic rats.

Effect of antioxidants on the stability of ONO-1301, a novel long-acting prostacyclin agonist, loaded in PLGA microspheres.

The purpose of this study was to investigate the physicochemical stability of ONO-1301 in poly(lactide-co-glycolide) microspheres (PLGA MS) under storage for 28 days in the absence or presence of butylated hydroxytoluene (BHT) or α-tocopherol as antioxidant. First, we observed the hydrolysed product: (i) in acidic solution and oxidized product and (ii) in PLGA MS under storage in HPLC study, each structure was determined by liquid chromatography–nuclear magnetic resonance/mass spectrometry. Second, ONO-1301-loaded PLGA MS containing 10% BHT was shown to be superior to ONO-1301-loaded PLGA MS without BHT, in the standpoint of the stability under storage or in vitro drug-release test, and AUC0–28 following subcutaneous injection in rats. Finally, ONO-1301-loaded PLGA MS with 10% BHT were demonstrated to be significantly more effective than ONO-1301-loaded PLGA MS without BHT in a murine sponge model of angiogenesis. In conclusion, BHT is an effective antioxidant on the stability of ONO-1301 in PLGA MS under storage.