Inflammation therapy

Abstract

A method is provided for treating a patient in need of therapy for central nervous system inflammation comprising administering to that patient a therapeutically effective amount of a cannabinoid agonist having efficacy at the CB.sub.2 receptor but having substantially no efficacy at the CB.sub.1 receptor at that amount.

Claims

1. A method of treating cerebral ischemia/reperfusion injury or spinal cord injury in a patient in need thereof or preventing spinal cord injury in a patient, comprising administering to the patient a therapeutically effective amount of a cannabinoid CB.sub.2 receptor agonist, wherein the agonist is O-1966.

2. The method as claimed in claim 1, wherein the patient suffers from a disease selected from the group consisting of head trauma, stroke, cerebral bleeds, Alzheimer's and Parkinson's diseases and CNS inflammation in multiple sclerosis.

3. The method as claimed in claim 1, wherein the agonist is administered in combination with a CB.sub.1 receptor antagonist.

4. A pharmaceutical composition for cerebral ischemia/reperfusion injury or spinal cord injury in a patient in need thereof after the cerebral ischemia/reperfusion injury or spinal cord injury, comprising a therapeutically effective amount of a cannabinoid CB.sub.2 receptor agonist, wherein the agonist is O-1966.

Description

FIGURES

(1) FIG. 1. Experimental designs for pre-ischemic treatment test (A) and post-reperfusion treatment test (B).

(2) FIG. 2. Typical closed cranial window video images (A)-(D) and infarct areas (E) of MCAO mouse.

(3) FIG. 3. Graph of rCBF with time for CB.sub.2 agonists (O-3853 or O-1966) compared with vehicle treated control group.

(4) FIG. 4. Graph of rCBF with time for model CB.sub.1 and CB.sub.2 antagonists alone, together and with O-1966.

(5) FIG. 5. Histograms of infarct vol mm3 and % of the ipsilateral hemisphere with time with and without CB.sub.2 agonists (O-3853 or O-1966) for either 1 hour before MCAO or 10 minutes after reperfusion significantly.

(6) FIG. 6. Histogram of infarct volume in mm3 and % ipsilateal hemisphere with and without O-1966 treatment at 1 hr pre, 1 hr post and 3 hours post ischemia

(7) FIG. 7. Histogram of Neurological score at 23 hours after insult with and without CB.sub.2 agonists (O-3853 or O-1966) 1 hour before MCAO and 10 minutes after reperfusion.

(8) FIG. 8. Histograms of leukocyte rolling after MCAO and adhesion following reperfusion with and without CB.sub.2 agonists (O-3853 or O-1966).

(9) FIG. 9. Histograms of Leukocyte rolling after administration of either CB.sub.2 agonist (O-3853 or O-1966) either 1 hour before MCAO attenuated leukocyte/endothelial interactions.

(10) FIG. 10. Infarct areas and histograms for effect of SR141716 and SR144528 administered 1 hour before MCAO at 20 mg/kg i.p. O-1966 was injected 1 hour before MCAO at 1 mg/kg i.v. n=5-7 per group.

(11) FIG. 11. Histogram of CB.sub.2 mRNA expression for sham and spinal cord injury (SCI) contusion model mice.

(12) FIG. 12. Plots of BMS and BBB score with time following SCI with vehicle and CB.sub.2 agonist O-1966.

(13) FIG. 13. Plots of % animals recovering bladder function with time after SCI with vehicle and with O-1966.

(14) FIG. 14. Histogram of TNF-α expression in sham, SCI and SCI with O-1966.

EXAMPLES

(15) Materials and Methods

(16) Animals

(17) The cerebral ischemia/reperfusion studies were carried out on 8 week old male C57BL/6 mice (weighing 23 to 27 g; Taconic N.Y.) and conducted in accordance with the guidelines approved by Institute for Animal Care and Use Committee at Temple University. The behavioural evaluation of the cannabinoid analogs was carried out in 8-week old ICR male mice weighing 23-27 g in accordance with the guidelines approved by the Institute for Animal Care and Use Committee at Virginia Commonwealth University.

(18) In Vitro and In Vivo Evaluation of CB.sub.2 Receptor Selectivity

(19) Both analogs depicted below were assessed for cannabinoid properties by determining their affinities for CB.sub.1 and CB.sub.2 receptors, functional activity using .sup.35S-GTPγS binding, and assessment in cannabinoid behavioural assays in mice using methodologies described in detail in recent publications. (Martin et al. 2002; Wiley et al. 2002) CB.sub.1 and CB.sub.2 receptor affinities were determined using .sup.3H-CP 55,940 binding to rat brain membranes and to Chinese Hamster Ovary (CHO) cells stably expressing the human CB.sub.2 receptor, respectively. In vitro functional activity was determined in these same preparations using .sup.35S-GTPγS binding. For in vivo behavioural effects, mice were injected intravenously with the drugs prepared in ethanol:emulphor:saline (1:1:18). The mice were evaluated for locomotor activity, analgesia, body temperature, and catalepsy. These behaviours are collectively referred to as the tetrad test and are indicative of CB.sub.1 receptor activity.

(20) Cranial Windows

(21) On the day of cranial window implantation, the animals were anesthetized with an intraperitoneal injection of Ketamine (100 mg/ml)-Xylazine (20 mg/kg) mixture (1:1) at a dose of 1 ml/kg. The head was shaved and positioned in a stereotactic head holder. A 1 cm.sup.2 area of skin on the dorsal surface of the skull over the right cortical hemisphere was excised and the periosteum was removed. A 4 mm diameter circular craniotomy was performed using a high speed drill (Champ-Air Dental Drill Benco Dental) over the right parietal cortex extending from attachment of the temporal muscle to midpoint of sagittal suture in the coronal direction and aligned to middle of the sagittal suture, so that the window contained some terminal branches of the middle cerebral artery. Normal saline was dripped over the cranium to avoid thermal injury of the cortex. The dura was removed and exposed brain was kept moisture with 37° C. artificial cerebrospinal fluid (CSF) solution. A 5 mm diameter coverglass was then placed over the exposed brain, and an airtight seal was produced using Nexaband Quick seal. The coverglass provided adequate mechanical protection from infection or contamination. A recovery period of four days was allowed between implantation of the cranial window and the induction of transient focal ischemia. (Ni et al. 2004) A typical closed cranial window is presented in FIG. 2A.

(22) Middle Cerebral Artery Occlusion and Reperfusion (MCAO/R)

(23) The animals were anesthetized with an intraperitoneal injection of Ketamine (100 mg/ml)-Xylazine (20 mg/kg) mixture (1:1) at a dose of 1 ml/kg. Body temperature was maintained at 37±5° C. by a heating lamp and heating pad. Middle cerebral artery occlusion was achieved by the intraluminal filament methods. (Hata et al. 1998) Briefly, a midline neck incision was made under the operation microscope; the right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were isolated. The ECA was ligated with 6-0 silk suture distal from the ICA-ECA branch and then cut distal from ligated point. Another 6-0 silk suture was tied loosely around ECA at close to the origin at the CCA. A blunted 5-0 monofilament nylon suture coated with poly-L-lysine (0.1% in deionized water, Sigma) (Belayev et al. 1999) was introduced from a small incision on ECA and then advanced into the circle of Willis, and finally to the origin of the middle cerebral artery. The silk suture around the ECA stump was tied tightly to prevent bleeding and secure the nylon suture. The nylon suture was removed after 60 minutes occlusion and ECA was permanently tied. Reperfusion was confirmed when pulsations were again observed in ICA.

(24) A laserPro Blood Perfusion Monitor (TSI Inc) was used to monitor regional cerebral blood flow (rCBF) prior to ischemia, during MCAO and reperfusion. A 1 mm diameter microfiber laser-Doppler probe was attached to the skull 4 mm lateral and 2 mm posterior of bregma. The MCAO was considered adequate if rCBF showed a sharp drop to 25% of baseline (pre-ischemia) level, otherwise, animals were excluded. (Tsuchiya et al. 2003)

(25) Injection of CB.sub.2 Agonists in MCAO/R

(26) The CB.sub.2 agonists (O-1966 and O-3853: described in Wiley et al and WO 03/091189, incorporated herein by reference, and the copending application of Razdan et al)) were dissolved in a pure ethanol:emulphor:saline mixed solution at 1:1:18. The CB.sub.2 agonists (1 mg/kg) or equal volume of vehicle were administered as an intravenous injection into the jugular vein 1 hour before MCAO or 10 minutes after reperfusion. The investigator was blinded with regard to whether the animals were members of vehicle or treatment groups during all experimental procedures and measurements.

(27) Intravital Microscopy

(28) The animals were anesthetized and immobilized on a plexiglass stage and secured on the microscopic stage. Intravital microscopy was performed with an epi-illuminiscence microscope (BHI Water Immersion, Olympus, Japan). A 20× water-immersion objective (WI 20, 0.4; Olympus, Tokyo, Japan), an image intensifier (Ceniisys Image Intensifier, Dage-MTI) and a monitor (12VM968; Audiotronics) were used to gain a final total magnification of 660×. Leukocytes were stained in vivo by a bolus injection of 0.05 ml of a 0.01% solution of the fluorescent dye Rhodamine 6G (Sigma, Inc) into the jugular vein. The light leaving the lamp housing was filtered to allow light with a peak wavelength of 605 nm to be transmitted to tissue. Excitation of fluorescent dyes in the leukocytes caused a shift in the wavelength of the emitted light. Selective filtering allowed visualization of the fluorescent cells on a dark background. A non-intensified black-and-white charge-coupled device (CCD) camera (CCD72, Dage-MTI) was used for visualization of the microscopic image. The image from the CCD72 camera was then displayed on the monitor, captured and recorded by a computer controlled real time TV Tuner (ATI-TV WONDER, ATI Technologies Inc) at a video frame rate of 36 frames/sec. (FIG. 2B-D) The interactions between leukocyte and endothelium were investigated offline. The investigator was blinded to drug treatment of the experimental animals when making measurement of leukocyte/endothelial interactions.

(29) Measurement of Leukocyte/Endothelial Interactions

(30) Leukocyte/endothelial interactions were evaluated before MCAO, 1 hour after MCAO and 24 hours after MCAO. Each vessel was exposed to the light of the microscope for 30 seconds at each viewing to minimize phototoxicity (Saetzler et al. 1997). Three venules (with diameter 30-50 μm) and three arterioles (with diameter 20-40 μm) in each animal were assessed. The number of rolling leukocytes was considered to be the total number of leukocytes moving along the endothelial cells at substantially slower velocity compared with the midstream blood cell velocity. They were counted when they passed an arbitrary line perpendicular to the longitudinal axis of the vessel over a period of 30 seconds. Adhering leukocytes were defined as the total number of the leukocytes firmly attached to the microvascular endothelium that did not change their location during the entire 30 seconds of observation period. Adhering leukocytes were scored as the number of cells per mm.sup.2 of the vascular surface area, calculated from the diameter and standardized length (100 μm) of the vessel segment under investigation.

(31) Infarct Volume Assessment

(32) Animals were euthanized with an overdose of pentobarbital (200 mg/kg i.p) 24 hours after MCAO and then the brains were removed. The brains were chilled in ice for 10 minutes to slightly harden the tissue. Five 2 mm coronal sections were cut using a mouse brain matrix (Zivic lab).The brain sections were placed in 2% triphenyltetrazolium chloride (TTC) (Sigma, Inc) dissolved in saline and stained for 20 minutes at 37° C. in the dark. The brain sections were then fixed in 4% paraformaldehyde at 4° C. for 24 hours and the anterior and caudal face of each section was scanned by a flatbed color scanner (Microtek Inc). The resulting images were captured as JPEG files (FIG. 2E) and analyzed with NIH image software. The infarct volumes were expressed as mm.sup.3 as well as the percentage of the ipsilateral hemisphere.

(33) Neurological Evaluation

(34) The severity of neurological deficits was evaluated 24 hours after ischemic insult using a five-point deficit score (0=normal motor function; 1=flexion of torso and of contralateral forelimb upon lifting of the animal by tail; 2=circling to the contralateral side but normal posture at rest; 3=leaning to contralateral side at rest; and 4=no spontaneous motor activity) (Hata et al. 1998).

(35) Statistical Analysis

(36) Numbers of leukocyte rolling on and adhering to venules or arterioles were analyzed by one-way (times) analysis of variance (ANOVA) in control group or two-way (treatments, times) ANOVA with repeated measurements followed by Bonferroni's test in treated groups. Bonferroni's test after one way ANOVA was used for analyzing differences in average of rCBF, infarct volume or neurological score. Data were presented as means±SEM. A statistically significant difference was assumed at P<0.05.

Results

(37) In Vitro and In Vivo Characterization of CB.sub.2 Cannabinoid Selectivity

(38) The affinity of O-1966 for CB.sub.1 and CB.sub.2 cannabinoid receptors was reported previously to be 5,055±984 and 23±2.1 nM, respectively(Wiley et al. 2002). We report herein that it stimulated .sup.35S-GTPγS binding with a EC50 of 70±14 nM and an Emax of 74±5 (percent of maximal stimulation produced by the full agonist CP 55,940). O-3853 binds to CB.sub.1 and CB.sub.2 receptors with respective affinities of 815 f 127 and 17.3±2.5 nM. It was also effective in stimulating .sup.35S-GTPγS binding with an EC50 of 6.0±2.5 nM and an Emax of 87±5%, whereas its ability for stimulating CB.sub.1 .sup.35S-GTPγS was very low (EC50=1509±148 and Emax of 43±3%). I.v. administration of O-1966 to mice failed to produce effects in the tetrad test (the measurements for locomotor activity, analgesia, body temperature, and catalepsy) in doses up to 30 mg/kg, consistent with its very low CB.sub.1 receptor affinity.

(39) Intravenous administration of O-3853 to mice resulted in weak activity in two of the tetrad measures. It depressed spontaneous activity and blocked tail-flick response with ED50's (confidence limits) of 10.3 (6.2-17.0) and 11.4 (8.2-16.0) mg/kg. It failed to either alter body temperature or produce catalepsy up to doses of 30 mg/kg. The ED50's of the CB.sub.1/CB.sub.2 cannabinoid receptor agonist Δ.sup.9-tetrahydrocannabinol are approximately 1-2 mg/kg in these four measures.

(40) CB.sub.2 Agonists Did not Change the rCBF During MCAO

(41) During MCAO, rCBF decreased to approximately 25% of baseline value. Administration of the CB.sub.2 agonists (O-3853 or O-1966) 1 hour prior to occlusion at a dose of 1 mg/kg had no effect on rCBF during the 1 hour occlusion period when compared with the vehicle-treated group (FIG. 3).

(42) Effects of CB.sub.2 Agonists on Cerebral Infarction

(43) Administration of CB.sub.2 agonists (O-3853 or O-1966) at either 1 hour before MCAO (pre-ischemic treatment) or 10 minutes after reperfusion (post-reperfusion treatment) significantly reduced the cerebral infarction compared with vehicle-treated group. Infarct volumes were similar in pre-ischemic treated controls (99.2±6.9 mm.sup.3, 34±2.3%) and in post-reperfusion treated controls (99.8±4.6 mm.sup.3, 38±1.6%). Administration of O-3853 prior to ischemia reduced infarct size to 68.2±5.0 mm.sup.3 and 24±2.4%. Furthermore, administration of O-3853 after reperfusion reduced infarct size to 71.9±6.1 mm.sup.3 and 28±1.3%. Likewise, O-1966 reduced infarct size to 65.6±4.0 mm.sup.3, 25±2.5%; and 71.3±5.5 mm.sup.3, 27±2% when administered either before ischemia (FIG. 5A) or after reperfusion (FIG. 5B), respectively.

(44) Effects of CB.sub.2 Agonists on Neurological Function

(45) Administration of the CB.sub.2 agonists (O-3853 or O-1966) at either 1 hour before MCAO (pretreatment) or during reperfusion significantly improved the motor function at 24 hours post-ischemia. (FIG. 7) Motor function score in animals receiving vehicle prior to MCAO was 3.25±0.20 which was reduced to 2.04±0.18 with O-3853 pretreatment and 1.69±0.30 with O-1966 pretreatment.

(46) MCAO Enhanced Leukocyte/Endothelial Interactions

(47) Leukocyte rolling and adhesion on both venules and arterioles were significantly enhanced during 1 hour MCAO followed by 23 hours reperfusion. Leukocyte rolling on venules increased from 3±0.2 (before MCAO) to 6.9±0.8 (1 hour after MCAO) and 8.9±0.8 (24 hours after MCAO); leukocyte adhesion on venules increased from 18±8.5 (before MCAO) to 134.7±30.9 (1 hour after MCAO) and 163±25 (24 hours after MCAO); leukocyte rolling on arterioles increased from 0.2±0.2 (before MCAO) to 1.7±0.6 (24 hours after MCAO); leukocyte adhesion on arterioles increased from 0 (before MCAO) to 196±54.7 (1 hour after MCAO) and 160±21.9 (24 hours after MCAO) (FIG. 8).

(48) Effects of CB.sub.2 Agonists on Leukocyte/Endothelial Interactions During Cerebral Ischemia/Reperfusion Injury

(49) Treatment with either of the CB.sub.2 agonists 1 hour prior to MCAO attenuated leukocyte/endothelial interactions during cerebral ischemia/reperfusion injury. Both CB.sub.2 agonists significantly decreased leukocyte rolling and adhesion on venules 1 hour after ischemia and following 23 hours of reperfusion. Leukocyte adhesion to arterioles was also attenuated by both agonists during both measurement periods following ischemia. Although both agents reduced leukocyte rolling along arterioles 24 hours after MCAO, there was no difference in leukocyte rolling as a result of treatment 1 hour after MCAO. (FIG. 9).

(50) The closed cranial window technique was utilized to evaluate the effect of selective CB.sub.2 agonists on endothelial/leukocyte interactions. Before ischemia, baseline leukocyte rolling and adhesion on venules was very low and there were almost no leukocyte/endothelial interactions on arterioles because of high shear stress. One hour after ischemia, there was a significant increase in leukocytes rolling on venules.

(51) The lack of increase in leukocyte rolling in arterioles during the first hour of reperfusion was probably the result of an increase in shear rate resulting from the ischemia induced reactive hyperemia. The increased leukocyte/endothelial interactions that resulted from ischemia/reperfusion injury were dramatically diminished by both CB.sub.2 agonists. Since leukocytes rolling and adhesion on endothelial cells are critical steps for their full activation and extravasation into brain tissue to participate in the inflammatory response (Heinel et al. 1994), it is possible that CB.sub.2 activation exerts at least part of its neuroprotective effects via modulation of white cell contributions to inflammatory reactions during ischemia/reperfusion injury.

(52) While it is likely that the attenuation of leukocyte rolling and adhesion following stroke is directly caused by CB.sub.2 receptor activation on these cells, it is also possible that the attenuation of rolling and adhesion is not a direct contributory mechanism but rather a reflection of a decrease in damage due to CB.sub.2 receptor activation in other cells such as microglia.

(53) In a previous study using selective CB.sub.1 and CB.sub.2 antagonists, we found WIN55212-2, exerted its neuroprotective effects in a mouse EAE model via CB.sub.2 not CB.sub.1 activation, and that this neuroprotective effect was also associated with an attenuation of leukocyte/endothelial cell interactions. (Ni et al. 2004)

(54) In addition to modulating inflammatory responses through inhibition of leukocyte/endothelial adhesion, a number of laboratories have also reported that the CB.sub.2 receptors also exist on microglial cells and that CB.sub.2 receptors were highly up-regulated by inflammatory stimulation in microglia (Maresz et al. 2005; Nunez et al. 2004). Activated microglia play an active role in cerebral ischemia/reperfusion injury, through phagocytic activity, inflammatory cytokine production and the release of destructive proteolytic enzymes as well as neurotoxin secretion (Mabuchi et al. 2000; Schilling et al. 2005). It is therefore possible that the beneficial effects of CB.sub.2 agonist treatment in stroke may result in part from inhibition of microglial activation.

(55) Another possible mechanism through which CB.sub.2 receptor agonists could exert a protective effect is by modulating cerebral blood flow through alterations in cerebral vascular resistance. However rCBF during ischemia was not changed by the agonists in the model used in this investigation. Therefore it seems unlikely that CB.sub.2 activation exerts its protective effects by influencing cerebral vascular resistance during ischemia. Since transient MCAO should result in maximal vasodilation early in the reperfusion period, it is also unlikely that vasodilation caused by the CB.sub.2 agonists during this time period is a contributing factor.

(56) Effect of CB.sub.2 Agonists in Spinal Cord Injury.

(57) A thoracic spinal cord injury model was induced in female 6-8 week old C57BL/6 mice. Spinal cord CB.sub.2 mRNA expression was evaluated in sham animals and SCI animals 24 hours after injury by real time RT-PCR. Animals were randomized into two groups: the experimental group received intraperitoneal injections of a selective CB.sub.2 agonist (O-1966; 1 mg/kg) one hour before injury, 24 and 48 hours after injury; the control group received equal volumes of vehicle. Both groups were tested for motor function by using the Basso Mouse Scale for Locomotion (BMS) and the Basso, Beattie, Bresnahan Locomotor Rating Scale (BBB) on post-procedure days 1, 7, and 14. Urine mass was recorded twice daily to assess bladder recovery. The inflammatory cytokine TNF-α was measured by real time RT-PCR in both groups 24 hours after SCI.

(58) Results:

(59) CB.sub.2 expression in spinal cord increased dramatically after injury (see FIG. 11). Animals treated with the received CB.sub.2 agonist treatment demonstrated better motor function recovery than the control group at all evaluation times after injury (See FIG. 12). The CB.sub.2 agonist treated group also had significantly improved bladder function recovery rate (45%) than the control group (14%) 14.sup.th days after SCI (FIG. 13). Administration of the CB.sub.2 agonist also decreased TNF-α expression in the injured spinal cord compared to control group (FIG. 14).

(60) These results indicate that CB.sub.2 agonist administration improved motor and autonomic function in a mouse model of SCI. The improvement may be mediated by the attenuation of inflammation in spinal cord after injury.

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