JP2006342184A - Reduction of infarct volume using citicoline - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide combination medicament for treatment of a subject who experienced an ischemic event. <P>SOLUTION: A method reduces the extent of infarction, particularly cerebral infarction subsequent to cerebral ischemia, by the administration of citicoline shortly after an ischemic episode and continuing daily treatment for up to about 30 days, preferably for at least about 6 weeks. The method is useful in the treatments of stroke and severe head trauma patients and maximizes the chances for a full or substantially full recovery of the patient. Combination treatment regimens are also disclosed along with compositions for use therewith. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for reducing the extent of infarct, particularly cerebral infarction following an ischemic event. Reduction of infarct volume, if combined with accelerated repair of some damaged tissue, results in the possibility of recovery after loss of critical blood flow, such as the symptoms that occur after a stroke. Can be maximized. More particularly, the present invention relates to the use of citicoline (cytidine-5′-diphosphocholine or CDP-choline) in a novel therapy for reducing cerebral infarct volume and improving the chance of complete or substantial recovery. Regarding usage.

The brain is more dependent on a relatively constant supply of oxygenated blood compared to all other organs of the body for its survival and proper functioning. The brain receives 15% of the heart's blood output and consumes 20% of the oxygen used by the body, even though it accounts for only 2% of the body weight. Furthermore, in order to supply glucose to the brain, a constant blood supply is required, which is the main energy substrate used by the brain to produce high energy phosphates such as ATP. .
Ischemia is defined as a loss of blood flow to the tissue. Cerebral ischemia is the interruption or reduction of blood flow in an artery that feeds into the brain, which is usually caused by a clot (thrombosis) or other substance (emboli) that occludes the artery. The loss of blood flow to a particular vascular region is known as focal ischemia, and the loss of blood flow to the entire brain is known as global ischemia.

Once blood (and thus oxygen and glucose) is deprived, brain tissue can undergo ischemic necrosis or infarction. Metabolic events believed to underlie such cell degeneration and death include energy loss due to ATP depletion, cellular acidosis, glutamate release, calcium ion influx, stimulation of membrane phospholipid degradation and subsequent free fatty acid accumulation, and Examples include free radical generation.
From knowledge of these underlying events, investigators investigating certain types of ischemic injury have found calcium channel blockers, glutamate and glycine antagonists, CDP-amines, free radical scavengers / antioxidants Drugs such as perfluorocarbons and thrombolytic agents to improve cerebral blood flow and / or nerve exit have been used. All were confused. In fact, some vasodilators can improve blood flow and thus can be used as anti-ischemic agents. However, nothing has been shown to reduce infarct volume, especially in patients with ischemic stroke. Conversely, although certain calcium channel blockers have been reported to reduce infarct size, these drugs also do not produce consistent results and have unwanted side effects such as reduced pulse or perfusion pressure. It has been reported to occur. For example, Kaste, M .; Stroke (1994) 25: 1348-1353 (Non-Patent Document 1).

More particularly, glutamate antagonists have been observed to reduce infarct size under certain experimental conditions. For example, Olney, J .; W. Science (1991) 254: 1515-1518 (Non-patent Document 2). However, most if not all of these compounds cause vacuolation of the brain, and most produce phencyclidine-like subjective effects in animals and humans. Ingestion of phencyclidine is associated with good mood, anxiety, mood instability and long-term psychosis.
Free radical scavengers / antioxidants are a group of heterogeneous compounds. In general, the effects of these compounds on infarct volume are inconsistent. For example, superoxide dismutase inhibitors have been found to reduce infarct volume only when injected intraventricularly. Kinouchi, H .; Proc. Natl. Acad. Sci. USA (1991) 88: 11158-11162 (Non-Patent Document 3). Other compounds such as lubelazole have clinical advantages but have been shown to have very narrow safety limits. Diener, H.C. C. Stroke (1995) 26:30 (Non-Patent Document 4).

Although perfluorocarbons show some advantage in the outcome resulting from ischemic stroke, these compounds have an extremely long half-life and need to be injected into the brain and spinal fluid. In addition, these compounds have been observed to cause gonad enlargement. Bell, R.A. D. Stroke (1991) 22: 80-83 (Non-Patent Document 5).
Thrombolytic agents such as t-PA (tissue plasminogen activator), streptokinase, and urokinase have shown some promise for the treatment of ischemia. However, these drugs have a propensity to increase intracranial hemorrhage, which can ultimately lead to increased mortality. For example, del Zoppo, G.M. T. T. et al. Seminars in Neurology (1991) 11 (4): 368-384 (Non-Patent Document 6); The Ancrod Stroke Study Investigators, Stroke (1994) 25: 1755-1759 (Non-Patent Document 7): Hacke, W., et al. Stroke (1995) 26: 167 (Non-Patent Document 8). Furthermore, the effectiveness of these agents may be limited to treatment within the first 3 hours of stroke.

Citicoline monosodium is an exogenous form of cytidine-5′-diphosphocholine (CDP-choline). Endogenous CDP-choline is a key intermediate in the biosynthesis of membrane phosphatidylcholine, which has major importance for dynamic regulation of cellular integrity. The role of phospholipids in maintaining neuronal function is very important under conditions where ischemia can induce the destruction of these membranes.
Citicoline has been extensively studied in clinical trials. The results of these trials showed improvements in various clinical signs including headache, dizziness, motor coordination and insomnia. These trials also showed improved motor function and improved stroke sequelae. However, such trials were limited to the use of citicoline during the recovery phase of patients who may have had a stroke, and thus such treatment was effectively performed after an event presumed to be ischemia. Nevertheless, such trials have shown that stroke and head trauma patients have well tolerated doses of citicoline in the range of 250 mg to 1000 mg per day for weeks.
Kaste, M.C. Stroke (1994) 25: 1348-1353. Olney, J .; W. Science (1991) 254: 1515-1518. Kinouchi, H .; Proc. Natl. Acad. Sci. USA (1991) 88: 11158-11162. Diener, H.C. C. Stroke (1995) 26:30 Bell, R.A. D. Stroke (1991) 22: 80-83. del Zoppo, G.M. T. T. et al. Et al., Seminars in Neurology (1991) 11 (4): 368-384. The Ancrod Stroke Study Investigators, Stroke (1994) 25: 1755-1759 Hacke, W.M. Stroke (1995) 26: 167.

The present inventors have not achieved sufficient potential therapeutic effects of citicoline in previous studies that would be associated with membrane stabilization through increased phospholipid synthesis in the ischemic brain, I think. The reason is that conventional studies include administering citicoline well after the onset of ischemia, typically only during a therapeutic effort to promote patient recovery. Moreover, such conventional treatment may not have progressed for a sufficiently long period.
Stroke is a serious and potentially devastating disease that affects approximately 500,000 people annually in the United States. Clinicians had to rely on treatments that were effective in maintaining patient fitness and nonspecific drugs such as steroids and mannitol to reduce brain swelling. As 25-50% of stroke victims become incapacitated, improved treatment of these patients is needed.
Intravenous thrombolytic therapy has been shown to be somewhat promising when given within 3 hours of stroke, but started within 24 hours after stroke and 3 months after stroke It is possible that oral medications that would have a positive impact on the neurological outcomes of this would be a new and important weapon in the fight against this disease.

The invention relates to an ischemic event (eg, cerebral ischemia) caused by any of a number of disorders, such as ischemic stroke, comprising administering an effective amount of citicoline or a pharmaceutically acceptable salt thereof. The present invention relates to a method for reducing an infarct volume that occurs, particularly a cerebral infarct volume. Such disorders include thromboembolic or hemorrhagic stroke, cerebral vasospasm, hypoglycemia, cardiac arrest, and epilepsy, and also schizophrenia, epilepsy, neurodegenerative disorders, Alzheimer's disease, and hunting Toncho is also included, but is not limited to them.
The present invention further relates to the use of citicoline for the manufacture of a medicament for reducing infarct volume, particularly cerebral infarct size, comprising administering an effective amount of citicoline together with a pharmaceutically acceptable carrier. Related.

  In the method of reducing the infarct volume or size, an effective amount of citicoline needs to be administered within a short period of time after onset of ischemic symptoms, preferably within 24 hours. Subsequent citicoline administration is then performed for a specific period, typically at least about 1 week (about 5-10 days, but preferably at least about 7 days), most preferably at least about 2-3 weeks to several weeks. (Eg 3 to 8 weeks, preferably at least about 6 weeks). The dose may vary within certain limits. Typically, about 100 mg to 2000 mg of citicoline can be administered one or more times per day, preferably once a day for the duration of the treatment. A preferred daily dose includes about 500 mg to 1000 mg of citicoline, most preferably about 500 mg. Again, however, this single dose may be administered one or more times per day, for example twice a day, if desired.

Compared to other drugs developed for the reduction of infarct volume after an ischemic event, citicoline could be expected to have a number of advantages. Citicoline is intrinsically safe because it is an endogenous compound. Citicoline is very toxic and has an extremely broad therapeutic index.
The various potential effects of citicoline will also prove advantageous. Although the relative contribution of each potential mode of action to reducing infarct size is not known, citicoline and its hydrolysates cytidine and choline produce phospholipids involved in membrane formation and repair It is thought to play an important role. These compounds are also believed to contribute to the synthesis of important metabolic functions such as nucleic acid and protein formation and the neurotransmitter acetylcholine. Ulus, I. et al. H. See Brain Research (1989) 484: 217-227. Thus, under ischemic conditions, citicoline (1) stabilizes the membrane by providing a substrate for membrane maintenance, (2) repairs the damaged membrane by providing a substrate important for membrane formation And (3) providing a substrate for acetylcholine formation to restore neuronal function. Moreover, unlike other proposed therapeutic agents, citicoline not only reduces the size of the initial infarct, but also has the potential to contribute to the repair of the damaged area.

  Accordingly, one object of the present invention is to provide a method for improving the recovery of victims who have suffered strokes and head injuries. Therefore, it is another object of the present invention to administer citicoline to a patient very early after the onset of ischemia, preferably within 24 hours after the ischemic event. Most preferably, the initial dose of citicoline is administered within about 12 hours to about 15 hours of the ischemic event.

Yet another object of the present invention is a method of reducing the infarct volume of a patient who is injured by any condition that can result, for example, ischemic stroke, head trauma, or impaired blood flow in the affected organ or tissue. Is to provide.
These and other objects of the present invention will be apparent to those skilled in the art in view of the foregoing discussion and the further detailed description set forth below with respect to preferred embodiments of the present invention.

The present invention relates to citicoline or a pharmaceutically acceptable salt thereof at a dose of about 100 mg to about 2000 mg once to 4 times a day, starting very early after an ischemic event for at least about 1 week (eg about 7 days). ) To several weeks, preferably 4 to 8 weeks, more preferably 5 to 8 weeks, and most preferably at least about 6 weeks.
Without being limited by theory, citicoline is believed to have at least two different mechanisms of action. That is, to limit brain damage caused by injuries such as strokes or severe head trauma, and to help repair damaged neuronal tissue.
Administration of citicoline is believed to limit the extent of infarction or tissue damage by preventing the accumulation of toxic free fatty acids. Furthermore, following its administration, citicoline is thought to be broken down into two components, cytidine and choline, which are substrates required for the formation of neuronal cell membranes. In addition, it is postulated that nerve cells damaged by stroke must create new membrane elements in order to normalize brain function. As described below, in the animal model prior to the onset of stroke, it is shown that administration of citicoline significantly reduces infarct size. This result is confirmed by a clinical test described further below in which a selected group of patients undergoing early intervention with citicoline treatment is examined in humans by magnetic resonance imaging techniques.

The present invention relates to a new and important use of citicoline (reduction of infarct size following cerebral ischemia). Although membrane stabilization is believed to be beneficial in ischemic symptoms, it has not been clearly demonstrated that membrane stabilization would result in a reduction in infarct volume. The inventors have surprisingly found that administration of citicoline for just 7 days significantly reduces infarct volume, possibly due to alterations in phosphatidylcholine synthesis and membrane formation.
The cerebral protective effect of citicoline was shown in one case in a transient forebrain ischemia model in rats where the middle cerebral artery (MCA) was closed with sutures. Treatment with 500 mg / Kg of citicoline significantly reduced the mean volume of the infarct compared to the control.

It is generally preferred that citicoline be administered orally as a pharmaceutically acceptable salt. A preferred salt is the monosodium salt of citicoline. This is because this form is readily available in pharmaceutically acceptable purity.
As noted above, treatment under the present invention should be initiated within at least about 24 hours, preferably within about 12-15 hours, and most preferably as soon as possible after the onset of initial ischemic symptoms. In embodiments of the invention, treatment is continued from its start for at least about 7 days, preferably for at least about 14 days, and most preferably for at least about 30 days.
Thus, according to one aspect of the present invention, an initial effective amount of citicoline or a pharmaceutically acceptable salt thereof is administered shortly after the onset of an ischemic event but within 24 hours, followed by citicoline. Or disclosed is a method of reducing infarct volume in a patient experiencing an ischemic event comprising administering an effective amount of a subsequent effective amount of a pharmaceutically acceptable salt thereof over a period of at least about 1 week. Preferably, the initial amount is administered within about 12 to about 15 hours after the occurrence of the ischemic event, and the initial amount is followed by a subsequent amount of citicoline or a pharmaceutically acceptable salt thereof for at least about 7 days, preferably Administer for 14 days, most preferably 30 days.
A preferred dose of about 500 to about 1000 mg of citicoline or a pharmaceutically acceptable salt thereof may be administered once or more daily, preferably once or twice daily.

The methods of the present invention can be most advantageously used in human patients who have experienced ischemic events that typically occurred in the brain, typically from cerebral ischemia, head trauma, and the like. However, it cannot be fully emphasized that the initial dose of an effective amount of citicoline or its pharmaceutically acceptable salt must be administered as soon as possible after the ischemic event, but within 24 hours after such occurrence.
Various dosage ranges are suitable. The citicoline dose in the present invention is about 100 to about 1000 mg and 1
It can be 1 to about 4 times per day. For example, if once daily administration is desired, citicoline is about 100 to about 4000 mg per day, preferably about 500 to about 2000 per day.
Administered in mg. In one embodiment of the invention, the dose is 1000 mg per day.

  In medical applications, the amount of citicoline or a pharmacologically acceptable salt thereof (“active ingredient”) to achieve a therapeutic effect will vary depending on the route of administration and the particular disorder or disease to be treated. Suitable systemic dosages of the active ingredient for mammals suffering from or potentially having any of the symptoms described herein range from 100 to 4000 mg per day, with a preferred amount of 1 Administer 500 mg twice a day at 1000 mg per day. A daily dose of 1000 mg of citicoline will produce a plasma choline concentration of 1.5 ng / ml. This is the same as occurs by administering 500 mg / kg citicoline per day to rats as further described in the examples. However, 500 mg per day has been shown to obtain most of the benefits of citicoline treatment while minimizing all side effects such as dizziness that some patients may experience.

In the trial, 259 ischemic stroke patients were enrolled within 24 hours after onset of symptoms. Patients were randomly assigned to one of three oral doses of placebo or citicoline (500 mg, 1000 mg or 2000 mg / day) over 6 weeks and monitored for an additional 6 weeks. The primary efficacy outcome measure was an improvement in neurological function at 12 weeks post-stroke as assessed by the Barthel Index.
Patients given 500 mg or 2000 mg citicoline per day showed a significantly higher (p <0.05) improvement in Basel index at week 12 compared to placebo-treated patients. Efficacy outcome measures for the 1000 mg per day group did not reach statistical significance, but demographic imbalance and confused variables between patients in the group could fully explain this finding .
Furthermore, the global neurological status assessed by the other well-known measure, Rankin Scale, was significantly improved with piticholine treatment compared to placebo (p <0.04).
The Barcel Index uses a 100-point evaluation scale.
A score of 95 or higher is an indicator of complete or almost complete recovery from stroke. Overall, 33% of placebo-treated patients reached a score higher than 95 at 12 weeks post-stroke, compared to 53% of patients receiving 500 mg citicoline daily (p <0.04).

Another outcome measure, the NIH Stroke Scale, is 34% of citicoline treated patients as indicated by a score below or equal to 1 at 12 weeks after stroke (p <0.04). Was shown to achieve full or almost complete normalization of function in 16% of placebo-treated patients.
There was no significant difference in the number of deaths within the four treatment groups. Preliminary analysis of adverse events and laboratory findings indicated that all doses of citicoline were well tolerated. The only statistically significant difference between citicoline-treated patients and placebo-treated patients was an increase in dizziness and accidental injury (eg, falls). However, the 500 mg citicoline dose group was not significantly different from the placebo group in these parameters.

Due to the degree of effectiveness of this daily dose of 500 mg and the absence of significant differences in adverse events between this dose level and placebo, a daily dose of 500 mg is the optimal dose derived from this study. It seems to be.
While it is possible for the active ingredient to be administered alone, it may be preferable to present the active ingredient as a formulation.
Formulations of the active ingredients suitable for oral administration are in the form of discrete units such as capsules, cachets, tablets or lozenges; powders or granules for reconstitution; solutions or suspensions in aqueous or non-aqueous liquids It can be in the form of a suspension; or in the form of an oil-in-water emulsion or a water-in-oil emulsion. The active ingredient may also be in the form of a bolus, electuary or paste.

Active ingredient formulations suitable for parenteral administration include sterile aqueous preparations of the active ingredients. The formulation can be provided in unit dosage form and can be prepared by any method well known in the pharmacological industry.
All of the above formulations may contain other therapeutically active substances in addition to standard and well known pharmaceutical carriers and / or excipients. Accordingly, the present invention also contemplates a combination therapy relating to the co-administration of citicoline and at least one second therapeutic agent or respective pharmaceutically acceptable salt thereof.

  A broad category of this at least one second therapeutic agent is contemplated. These drugs include antiplatelet agents (eg, Alboagregin A), BB-2113, BN-50726, BN-50737, “Corsevin M”, C68-22, Integrelin, KB-3022, Linotroban, platelet factor 4, Staurosporine, S-1452, ticlopidine, TP-9201, etc.), anticoagulants (eg, alpha-1 antitrypsin, anti Thrombin III, antithrombin polypeptide, Argatroban, coagulation factor Xa, CTC-110, CTC-111 and other protein C products, CX-397, dalteparin (Dalt) parin), Danaproid sodium, Enoxaparin, Factor XIIa inhibitor, Fraxiparine, heparin, hirudin, hirugen, Hoe-023, HV-1, ITF-300 and ITF-1300, monoclonal antibodies, ONO-3307, persulfated LMW heparin, raviparin sodium, rTAP, R-020, SC-597, thrombomodulin, TMD1-105, etc., thrombolytic agents and related agents (eg, mold-2161 (Kabi-2161) ), Kunitz protease inhibitor, plasminogen activator, plasminogen activator inhibitor, tissue plasminogen activator, etc.), anti-ischemic agent and “nerve Neuroprotectives "(e.g., inhibitors of excitatory action, amino acids, ACEA-1021, ACPC, Aptiganel, BW-619C, CNS-1145, CNS-1505, CPC-71 and CPC-702, Dextst Lorphan and dextromethorphan, eliprodil, ES-242-1, FPL-15896, FR-115427, GP-1-4688, L-687414, L-689560, L-695902, LY-104658, LY-235959, LY-274614, LY-293558, Memantine, NNC-07-9202, NS-257, NPC 17742, “Protara”, Remacemide, Riluzole, SDZ EAA 494, Selfel, SYM-1010, SYM-1207, YM-90K, MK-801, etc. It is not limited to these.

Still other therapeutic agents useful in combination with citicoline include calcium channel blockers (eg, AJ-394, AK-275, calpain inhibitors, CD-349, Clentiaze, CNS-1237, CNS -2103, CPC-304 and CPC-317, Dazodipine, Diperdinine, Emopamil, Fasudil, Lacidipine, Rifarizine MD, Larizine MD, L 28170, NB-818, Nilvadipine, Nimodipine, NS-626 and Reaming compounds, SM-6586, SNX-111, S-312-d, U-92032, UK-74505, US-35, etc.), drugs targeted to nitric oxide, and various others agents, neurotransmitters (e.g., alpha ² - receptor therapeutics, CV-5197, dopamine receptors, enadoline (enadoline), lazabemide (lazabemide), milnacipran (milnacipran), nalmefene (nalmefene), RP-60180, SR-57746A, synaptic uptake blockers, etc.), cytokines, hormones and related products (eg, AN-10025 and AN-100226, brain-derived neurotrophic factor, calcitonin gene related peptide, CEP-075 and related compounds, ciliary Somatotrophic factor, endothelial cell factor Endothelin inhibitor, FR-139317 interleukin-1 receptor antagonist (lipocortin), JTP-2942, macrophage regulatory compound, motor neuron trophic factor NBI-117, nerve growth factor, neural stem cell, neutrophil inhibitor NS-506, NT-3, Posatirelin, Schwann cell promoter, sCR1, somatomedin-1 (Somatomedin-1), etc.), free radical scavengers (eg, EPC-K1, MCI-186, Nicaraven, Phenazoviridine, Resorstatin, Rambrin, Superoxide Dismutase, Tirilazad ( irilazad) mesylate, U-88999E, Ysumum project P-000619, YM-737, etc.), gangliosides and related products (eg LIGA4, LIGA4, monosialoganglioside (GM1), ND-37, siad) (Siagoside) etc.).

  Yet another class of second therapeutic agents include various specific enzyme modulators (eg, CEP-217, CEP-245, CEP-392, CNS-1531, Ebselen, Epalrestat, JTP-4819, K-7259, protease nexin-1 (SKin-827, tyrosine kinase modulator, Z-312, etc.), memory enhancers or “notropics” (eg, Aloracetam) ), Choline-L-alfoscerate, DN-2574, Idebenone, Oxiracetam, Piracetam, Pramiracet m), tacrine and analogs thereof, vinconate, neuroprotective agents having "various" action (eg, ademethionine sulfate tosylate, ancrod, apocandine) CPC-111, CPC-211, HSV vector, KF17329 and KF-19863, LY-178002, MS-153, Nicorandil, N-3393 and N-3398, SUN4757, TJ-8007, VA-045, etc.) , Hemorheological drugs and blood substitutes (eg, Drotaverine acephylinate, “RheothRx” substitute) Blood) and contrast agents, but are not limited thereto.

  Accordingly, a method of treating a patient suffering from ischemia, comprising: an effective amount of citicoline and a first dose of at least one second therapeutic agent, or each pharmaceutically acceptable salt thereof, ischemic There is provided a method comprising co-administration in a short period after onset of, but preferably within about 24 hours. After this first dose, an effective amount of citicoline alone, at least one second therapeutic agent alone, or one or more subsequent doses of each of their pharmaceutically acceptable salts, or a subsequent combination thereof May be administered simultaneously. As with the other methods disclosed herein, this first dose can be co-administered within about 12 to about 15 hours after the onset of ischemia. The term “co-administration” means that citicoline and at least one second therapeutic agent, or their respective pharmaceutically acceptable salts, are administered together or sequentially. To do.

Methods using the intended combination therapy include administration of subsequent doses or co-administration preferably over a period of at least about 30 days. In certain aspects of the invention, the subsequent administration of subsequent doses occurs for at least about 4-8 weeks, preferably for at least about 6 months to about 1 year. Further, the first dose or subsequent doses are co-administered one or more times daily for a predetermined period of time. It is expected that the patients who will benefit most from this combination therapy are those suffering from head injury or stroke. Thus, the combination therapy may include a second therapeutic agent, which is t-PA, streptokinase, urokinase, and may also be aspirin or dipyridamole.
Accordingly, for the treatment of patients suffering from ischemia comprising an effective amount of citicoline and at least one second therapeutic agent, or a pharmaceutically acceptable salt thereof, respectively, in a pharmaceutically acceptable carrier. There is further provided a composition for. In such compositions, the effective amount of active ingredient may vary depending on the particular need. However, a typical range is about 100 mg to about 1000 mg of citicoline and about 10 mg to about 500 mg of at least one second therapeutic agent.
The present invention is illustrated by the following examples, but it will be understood that the invention is not limited to the specific details of these examples.

Example 1 Animal Test 1
The cerebral protective effect of citicoline was demonstrated in a transient forebrain ischemia model in rats where the middle cerebral artery (MCA) is occluded with sutures. Treatment with 500 mg / kg citicoline significantly reduced the mean volume of infarcts compared to controls.
Thirty male Sprague-Dawley rats weighing 280-350 grams were randomly divided into three groups of 10 rats each: 10 animals treated with 500 mg / kg citicoline; 100 mg / kg Ten animals treated with kg citicoline; and 10 control animals treated with saline, a vehicle of citicoline.
All animals were anesthetized by intraperitoneal administration of 400 mg / kg chloral hydrate. A PE-50 polyethylene tube was cannulated into the left femoral artery to sample blood for continuous monitoring of arterial blood pressure and analysis of arterial blood gases. Prior to reperfusion, measurements were recorded before surgery, 1 hour after ischemia and 2 hours after ischemia. During surgery and MCA occlusion, the rectal temperature was maintained at 37 ° C. with a thermostatically controlled heating lamp.

  The right MCA was occluded by the previously described transvascular approach. Minematsu, K .; et al. Neurology (1992) 42: 235-240: Zea Longa, E .; et al. See Stroke (1989) 20: 84-91. Briefly, the right common carotid artery and right external carotid artery were exposed by a midline cervical incision. Peripheral CCA and external carotid artery were ligated with 3-0 silk suture. A 4-0 monofilament nylon suture (40 mm long) (Bayer, Leverkusen, Germany), rounded by heating the tip near the flame and then coated with silicone, was inserted through the CCA arteriotomy and gently inserted. Advance to the carotid artery. When reaching approximately 17 mm from the carotid bifurcation, the suture tip unilaterally blocks the proximal anterior cerebral artery, the origin of the MCA, and the posterior traffic artery. To prevent bleeding, the CCA was loosely ligated with 3-0 silk suture distal to the arteriotomy.

110 minutes after ischemia, these animals were treated with 500 mg / kg citicoline; 100 mg / kg citicoline; or 0.3 ml saline vehicle (control) administered intraperitoneally. The MCA ligature and femoral artery catheter were then removed and the tissue was reperfused after a 2 hour total ischemia period. These animals were awakened from anesthesia and allowed to eat and drink freely. This citicoline treatment procedure was repeated for another 6 days.
On the seventh day of treatment, these animals were anesthetized again by intraperitoneal administration of 400 mg / kg chloral hydrate and decapitated. The brain was quickly removed and examined to confirm that no subarachnoid hemorrhage had occurred, and was coronally cut into 6 2 mm slices. These brain slices were fixed in a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) for 30 minutes at 37 ° C. and immersed in a 10% buffered formalin solution. TTC stains normal brain tissue (intact cell membrane) in red; ischemic tissue in pink; and necrotic tissue in white. Six brain slices per animal were TTC stained, photographed using a charge coupled device camera (EDC-1000HR computer camera, Electrim Corporation, Princeton, NJ) and the images were stored in a microcomputer.

Since brain edema is known to affect infarct size measurement, an image processing software package (Bio Scan OPTIMAS, Edmonds, Washington) was used to calculate corrected infarct volume. . The corrected infarct area was calculated using the equation: corrected infarct area = left hemisphere area− (right hemisphere area−infarct area). The corrected infarct volume was calculated by multiplying this corrected infarct area and slice thickness.
Five of the 10 animals in the control group died 24-48 hours after MCA occlusion. Five of the 10 animals in the 100 mg / kg group died, 4 of which were 24-48 hours and 1 died on day 5. Three of the ten animals in the 500 mg / kg group died, two of which died at 24-48 hours and one died on day 6.
As shown in FIG. 1, the mean volume of infarct in the control group is 243.5 ± 88.6 mm ³ (mean ± SD); 200.2 ± 19.9 mm ³ in the 100 mg / kg group; in the 500 mg / kg group Was 125.5 ± 45.2 mm ³ . The difference in mean value of infarct volume was significant in the control versus 500 mg / kg group (p <0.01, Scheffe test). There was no significant difference between the control and the 100 mg / kg group, but there was a trend towards smaller infarct volumes in the 100 mg / kg group.

2 Animal Test 2-Behavior Spontaneously hypertensive rats (SHR) weighing approximately 250-300 g were subjected to reversible middle cerebral aorta (MCA) and common carotid artery (CCA) occlusions of various durations. Briefly, animals that were anesthetized with chloral hydrate (anesthesia persisted for at least 2 hours with a 0.5 g / kg intraperitoneal (ip) bolus in 1 ml of saline). Using a 0.005 inch diameter stainless steel wire (Small Parts Inc., Miami, FL) from the left MCA rostral lower nasal cleft, proximal to the main branch of the MCA, and lens Placed distal to the striatum artery. The artery was then lifted and the wire was rotated clockwise. The two atraumatic Heifetz aneurysm clips were then occluded. As a result, the blood flow in the nucleus of the infarct (4 mm dorsal to MCA occlusion) was measured with a Vasamedics laser blood flow perfusion monitor throughout the ischemic period, and the baseline value before ischemia was measured. Reduced to 4-8%. After a CCA / MCA occlusion for a predetermined period ranging from 0 to 120 minutes, the aneurysm clip is first removed from the CCA and then reperfused by rotating the wire counterclockwise and removing it from the bottom of the MCA Established.

Temporal muscle temperature was maintained at 36.5 ± 0.3 ° C. using a heating lamp and warming blanket. This study was designed to analyze exercise performance, thus eliminating damage that could be caused by femoral vessel cannulation and could interfere with behavioral performance. Therefore, changes in blood pressure, pH, PO ₂ and pCO ₂ during surgery were not recorded. Brain temperature was maintained at 36.2 ± 0.4 ° C. during the entire duration of ischemia and the first 2 hours of reperfusion.

15 minutes after induction of ischemia, CPD- using a 0.5 g / kg ip bolus in 0.5-0.6 ml 0.9% NaCl followed by the same volume daily for 14 days. Choline was administered. Fresh CDP-choline solution was prepared daily. Control rats were injected with saline alone instead of CDP-choline solution.
An outline analysis was performed 14 days after ischemia and after all behavioral tests were completed. This takes into account the direct correlation between behavioral and histological outcomes in the same animal. After 14 days of MCA / CCA occlusion, the ischemic brain is mainly characterized by atrophy of infarcted cortical tissue. Thus, the amount of ischemic damage can be calculated as the difference between the contralateral and ipsilateral cortical volumes.
Fourteen days after ischemia, rats were sacrificed under chloral hydrate anesthesia. The brain was cooled and cut into 2 mm slices. Profile determination of ipsilateral and contralateral cortical areas of each section was performed using a computer-based Drexel University (DUMAS) image analyzer that was calibrated to display measurements in mm ² . The cortical area (mm ³ ) was calculated by summing the cortical areas of the serial sections and multiplying by the interval thickness between the sections. Finally, the volume of atrophy was calculated by subtracting the ipsilateral cortex volume from the contralateral cortex volume of the same rat. The investigator who performed this profile analysis was not informed of the treatment resume.

3 Behavioral tests The following series of tests were used to evaluate functional results after ischemia. These have been validated and standardized in this model in previous studies.

3.1 Rotating motion Use a rat running wheel with a 12-inch diameter and a crosspiece spacing of 2 cm. The side of the wheel was closed using a poster board, except for an openable and closeable flap that allows the animal to enter and remain in the wheel. Rats were trapped inside a wheel, which was itself placed in a home cage and recorded on videotape as they ran. The 100 steps taken by the forelimb on the opposite side of the infarct hemisphere were observed, and the number of matches that the limb stepped between the side rails of the wheel was counted. The same observation was made on the ipsilateral forelimb and hindlimb, and the value of stepping out (error) per step taken was calculated for each limb. The number of errors was then obtained by subtracting the ipsilateral error from the contralateral error. Each of the animals was tested once a day on days 4, 7, and 10 after surgery.

3.2 Arm flexion Raise the rats with their tails for 10 seconds so that their abdominal surfaces are exposed to the observer. The duration of asymmetric arm flexion is measured using a stopwatch. The animals were tested once daily (2 trials per day) on days 1-14 after surgery.

3.3 Tape Test This test for asymmetry and recovery from asymmetry has been previously described by Schallert and Whishow and has been characterized in detail. Briefly, an Avery self-adhesive label (1 cm diameter circle) was placed in the lower rib area of the ankle of each forelimb of the rat. The time it took for the animal to touch each label and remove it and the order in which it occurred (body side vs. ipsilateral) was determined and used to approximate the ipsilateral asymmetry. It is possible to offset the bias by applying a larger label on the less preferred ankle and a correspondingly smaller label on the other ankle, while at the same time being quantified by the size of the label required. The higher the ratio of the surface of the ipsilateral patch to the contralateral patch used for bias cancellation (1: 1 to 1/8: 15/8), the higher the score (1 to 7) assigned to the rat, Reflected more extensive damage. There was no prior training other than extensive handling for at least 7 days prior to surgery. Each animal was tested 2-3 days after surgery (one test per day).
Before and after surgery, all animals were placed in separate cages in an isolated room that provided a minimal stress environment. Applied daily (at least 10 minutes per day for each animal) for at least 2 weeks. During this time, animals were handled by the same investigator performing all behavioral tests. This adaptation was introduced to reduce the stress response to contact and handling and was particularly critical to the proper performance of this tape test. Investigators conducting behavioral tests were not informed of the treatment plan.

4 Composite behavior anomaly assessment Represents a composite of three individual tests, including the number of errors (stepping off / steps; rotational motion), time (arm flexion), or notch (surface area required to offset the bias ; Abnormal behavior evaluation using the tape test) for quantifying behavioral functional abnormality was calculated.
First, the average value of the behavior of each rat over the entire period in each test was calculated. The two lowest values in each of the behavior tests were averaged and subtracted from the average of the two highest values. Then, this range was equally divided into 5 and evaluated by 0-4. Next, a composite evaluation of the three upset tests was determined with equal weighting of each of them. Therefore, each rat was evaluated for three separate tests and given a combined assessment of behavior with a maximum score (defect) of 3 × 4 = 12.

5 Correlation between ischemic persistence and infarct volume or behavioral abnormalities Behavioral abnormalities evaluation and calculated atrophy volume (mm ³ ) were used to evaluate ischemic duration and abnormal behavioral evaluation or atrophic volume in untreated and CDP-choline treated animals. It was used to obtain the correlation.
The combined evaluation value or atrophy volume is input to a computer and analyzed by the curve fitting computer program ALLFIT, and in addition to the curve shape and slope, the maximum behavioral dysfunction / maximal atrophy volume (BD _max \ Vol _max ), the time required to induce half-maximal behavioral dysfunction / atrophy volume (BD ₅₀ \ T ₅₀ ), generated a curve describing the correlation between behavioral abnormality / atrophy value and duration of ischemia . The computer program (ALLFIT) used to perform this analysis used the symbolic logic function y = (ad) / [1+ (xXc) b] + d. Where y is the behavioral dysfunction evaluation, x is the duration of ischemia, a is the response when x = 0, d is BD _max -Vol _max , b is the slope factor that determines the slope of the curve, and c Is BD ₅₀ / T ₅₀ . This program was developed to simultaneously fit a set of sigmoidal dose response curves and was obtained from NIH's Laboratory of Physical and Physical Biology.

6 Statistical analysis Statistical differences in BD ₅₀ / T ₅₀ and BD _max -Vol _max between groups were calculated using the logarithm of the mean and standard error of the comparison values obtained by ALLFIT, and the student t -Evaluated using a test.

7 Results 7.1 Histology-Atrophy Volume Analysis 18 and 23 rats were analyzed in saline control and CDP-choline treated groups, respectively. 14 days of CDP-choline administration significantly (p <0.05) prolonged the duration of ischemia prior to reperfusion resulting in half-maximal atrophy (T ₅₀ ).
The T ₅₀ calculated by the ALLFIT program was 38.3 ± 5.9 minutes for control (untreated) animals and 60.5 ± 4.3 minutes for CDP-choline treated animals. In this model, never CDP- choline reduces the maximum infarct _{volume; Vol max} for control and CDP- choline treated groups, respectively, 103.3 ± 13.6 mm ³ vs. 101.6 ± 11.4 mm ³ Met.
The above results suggest that CDP-choline can primarily reduce morphological damage after a relatively short duration of ischemia that produces almost maximal damage. These results also underscore the importance of administering citicoline in a short period after ischemia has occurred, ie as soon as possible.

7.2 Behavioral analysis Sixteen untreated rats and 21 CDP-choline treated rats were analyzed. CDP-choline treatment significantly extended BD ₅₀ in the same manner as observed in histological analysis. The BD ₅₀ in control rats was 41.9 ± 4.6 minutes. In contrast, CDP-choline treatment extended BD ₅₀ by approximately 30 minutes to 72.9 ± 24.5 minutes.
CDP-choline treatment had no significant effect on BD _max ; the values were 8.5 ± 0.7 and 10.1 ± 4.0 for control and CDP-choline treated animals, respectively. there were.
These data indicate that long-term CDP-choline treatment significantly improves both histological and functional outcome by stretching T ₅₀ and BD ₅₀ . However, in this case CDP-choline had no effect on Vol _max and BD _max . These results suggest that the effectiveness of CDP-choline is greater in animals showing almost maximal ischemic damage caused by 30-75 minutes of ischemia in this model.

8 Double-blind, placebo-controlled clinical trial Based on a review of two clinical trials and 17 trials reported in the literature, citicoline (250-1000 mg / kg dose range) was administered for 5 days to 8 weeks. It seems consistently well tolerated among patients with stroke and head injury. Based on a review of over 50 studies reported in the literature, citicoline appears consistently well tolerated among other populations. Side effects were generally not clinically significant except in cases of death as a result of ischemic severity (which is not unexpected in the population study). No clinically significant changes in experimental parameters, vital signs, or ECG have been reported.
This study was designed to evaluate the effect of three doses of citicoline (500 mg, 1000 mg and 2000 mg) on placebo in patients with acute ischemic stroke.

8.1 Dosing Instructions / Schedule Patients will receive 4 tablets on the day of participation, 2 tablets at the time of participation in the study and 2 tablets at the next meal time if they were randomized before 3:00 pm It was. Otherwise, patients were given 2 tablets (morning dose) at the time of participation, and another 2 tablets (evening dose) on day 1 were returned without use. For the remainder of the study, patients were given 2 tablets in the morning and 2 tablets in the evening (see treatment plan below). In patients who cannot take the entire study tablet, acceptable routes of administration include nasogastric administration, or crushing the tablet and mixing with food or beverage.

8.2 Treatment Phase Study drugs were administered twice a day (2 tablets in the morning and 2 tablets in the evening) for 6 weeks.
The effectiveness of various dose levels of citicoline was assessed by the Barthel Index.
Secondary efficacy assessments collected during this study included the Basel Index at Week 12 (successful with a score of at least 61), the modified Rankin Score, the NIH scale grade total , NIH stroke grade exercise items, days to discharge, mortality, and other neuropsychological battery scores. The NIH stroke grade and the Barcel index are standard measures of functional performance related to stroke symptoms and daily life.
This double-blind placebo-controlled trial showed that patients who received 500 milligrams of citicoline daily had a disability at 12 weeks post-stroke that was more than twice that of patients who received placebo as measured by the NIH stroke grade. It was shown that it seemed to reveal that there was minimal or no, and that the Barcel index seemed to reveal that there was minimal or no capacity impairment 1.6 times.

9 Diffusion Weighted MRI (DWI) Study DWI detects areas of ischemic injury within minutes after the occurrence of ischemia, and animal models show that the utility of DWI is to monitor neuroprotective treatment Show. Placebo-controlled trials suggest that citicoline reduces infarct size in the rat temporary occlusion model described above.
In this study, 12 patients were studied from a phase III double-blind placebo controlled trial of citicoline (500/1000/2000 mg daily for 6 weeks) in acute stroke (MCA area, within 24 hours of onset). The overall study design and results are shown above. Multi-slice echo planar DWI and T2-weighted MRI were performed at acute and chronic time points. That is, DWI scanning at baseline (i.e., 8-24 hours after onset, estimated stroke time) and at least one imaging during treatment were performed. The average time to do again was 9.3 weeks (range 4.1-26.3 weeks; median 6.6 weeks). Lesion volume was measured twice by three observers who were unaware of the treatment and clinical information about the patient. Thereafter, the percent change in infarct volume between the baseline scan and the second scan after treatment was determined.

Three of the four placebo patients showed lesion growth. In contrast, 7 out of 8 patients treated with citicoline showed a reduction in lesion volume (4/5 at 500 mg; 3/3 at 2000 mg). The dose-based trend chi-square test was significant at p = 0.031. Fisher exact test yielded a value of p = 0.067. These determined values were compared to a series of histological control patients (n = 31) collected in the same manner. Of the 31 patients, 22 had increased infarct volume, 7 had decreased infarct volume, and 2 did not change. Of the 4 placebo patients in this study, 3 had increased infarct volume and 1 had decreased infarct volume, just the proportion of histological controls. Of the 8 patients treated under the practice of the present invention (5 at 500 mg qd, 3 at 2000 mg bid), 7 decreased infarct volume and 1 increased infarct volume.
The percentage of patients with reduced infarct volume under the practice of the invention was tested against the percentage in the histological control (ie 22/7 vs. 1/7) using the Fisher exact test. A difference was obtained in support of being found to be significant (p = 0.0021). Thus, as assessed by DWI, these trials are insignificant in infarct volume due to treatment of ischemia with citicoline when treatment is initiated as soon as possible after ischemia and continued for a period of up to several weeks. Indicates a decrease.

10 Combination treatment 40-60 non-fasted male Sprague-Dawley rats weighing 280-365 g are each anesthetized by intraperitoneal administration of chloral hydrate (400 mg / kg body weight). To maintain anesthesia during the surgical procedure, 100 mg / kg chloral hydrate is administered periodically after the first injection. This anesthesia is maintained for only 90 minutes after arterial occlusion. A catheter is introduced into the inferior vena cava via the left femoral vein for drug administration. During the entire anesthesia, the body temperature of the rat is monitored and maintained at 37 ° C.
The luminal structure MCA occlusion model described in detail elsewhere is used. Minematsu, K .; et al. Neurology (1991) 42: 235-240. Briefly, a luminal occlusion body, 4-0 monofilament nylon suture, whose tip is rounded by flame heating, is introduced into the internal carotid artery via a ligated right CCA, and then approximately approximately from the CCA branch. Slowly advance through the 17 mm skull. This suture unilaterally occludes the proximal anterior cerebral artery, the distal internal carotid artery and the origin of the MCA and the posterior traffic artery.

  The animals are divided into 4 groups: (i) NMDA antagonist treatment, (ii) saline + citicoline treatment, (iii) NMDA antagonist + citicoline treatment and (iv) saline control group. 10-15 rats are slowly intravenously injected with 0.5 mg / kg body weight of MK-801 (NMDA antagonist) in 1 ml / kg of saline 5 minutes after MCA occlusion. A maintenance dose of 0.5 mg / kg MK-801 is administered intraperitoneally (ip) 8 and 20 hours after MCA occlusion. A second group of another 10-15 rats is administered an equal volume of saline at the same time as the NMDA antagonist group, which is administered intraperitoneally at 5 minutes, 8 hours and 20 hours after MCA occlusion. Accompanying 500 mg / kg citicoline administered. The above NMDA antagonist treatment regimen is repeated except that another group of 10-15 rats is further administered 500 mg / kg citicoline intraperitoneally at 5 minutes, 8 hours and 20 hours after MCA occlusion. A fourth final group of another 10-15 rats is administered an equal volume of saline at the same time as the NMDA antagonist group.

These animals are awakened from anesthesia and are allowed to eat and drink freely. After any neurological evaluation, the animals are anesthetized again by intraperitoneal administration of 300 mg / kg chloral hydrate and immediately decapitated. The brain was quickly removed, a test to confirm MCA occlusion with luminal sutures was performed, and the coronal shape was cut at intervals of 2 mm, and using a 2% solution of 2,3,5-triphenyltetrazolium chloride (TTC) at 37 ° C. Stain for 30 minutes and fix by dipping in 10% buffered formalin solution. In order to measure the size of the infarction, a brain section of each animal stained with TTC is photographed. Evaluate the enlarged photo blindly. It is considered that an infarct is formed in a region not stained red with TTC. Infarct volume (mm ³ ) is calculated by numerically integrating the infarct area of all TTC sections and the distance between them for each animal.
Standard tests are performed to assess the significance of monitored physiological variables between groups and differences in infarct area and volume. A two-sided probability value of less than 0.05 is considered significant.

  It is found that the three groups of rats receiving a particular type of treatment show a statistically significant reduction in infarct volume over the control group. In fact, the two groups of rats receiving either NMDA antagonist alone or citicoline alone show a reduction in infarct volume compared to the control group, whereas the third group receiving the combination of NMDA antagonist and citicoline. This group of rats is found to be better than any of the other groups. In fact, a surprising trend towards synergy may be shown. The third group of rats is also better evaluated than the other groups for neurological evaluation. Similar gains are observed in citicoline combination formulations with other presenting therapeutic agents selected from among those described above. The presented therapeutic agents include, but are not limited to, tissue plasminogen activator (t-PA), streptokinase and urokinase. The initial dose of the combined treatment regime may optionally be followed by one or more subsequent doses of citicoline alone, at least one second therapeutic agent alone, or both.

10.1 Citicoline and Aspirin The present invention also contemplates the co-administration of citicoline and aspirin, where citicoline and aspirin are administered together or sequentially for a short period after the onset of an ischemic event, as part of a combination therapy. is doing. (In practice, the determination that this “event” is a non-hemorrhagic stroke and therefore the stroke can be ischemic, thromboembolic, or otherwise is sufficient to initiate a combination therapy. Yes.) The amount of aspirin and citicoline administered may vary depending on the needs of the individual patient. However, typically about 50 to about 500 mg (preferably about 70 to 300 mg) of aspirin is administered per dose, and about 100 mg to about 1000 mg (preferably about 300 to 700 mg) of citicoline or a salt thereof per dose. Is administered. In a preferred embodiment, the composition comprising about 70-90 mg aspirin and about 400-600 citicoline or a salt thereof in a pharmaceutically acceptable carrier is administered once daily for a short period after the onset of stroke or It is administered twice. This combination therapy regimen can last at least 30 days, preferably up to several weeks, more preferably months. Most preferably, the prescription is continued for 6 months to 1 year to minimize damage to the tissue, maximize patient recovery, and reduce the occurrence of secondary symptoms such as stroke.

Accordingly, the present invention contemplates a combination therapy that includes co-administering citicoline and at least one second therapeutic agent for a short period after ischemia has occurred. This simultaneous administration may be performed simultaneously or sequentially. When sequentially co-administered, it is preferred to administer citicoline or a pharmaceutically acceptable salt thereof and at least one second therapeutic agent by the first 24 hours of the onset of ischemia.
Other aspects should be apparent to those of ordinary skill in the art in light of the detailed disclosure presented herein. Nevertheless, these embodiments are within the scope and spirit of the present invention. For example, dipyridamole or the like can be used instead of aspirin. Accordingly, the preferred embodiments described above should not be construed as limiting the invention in any way.

FIG. 1 shows a bar graph of infarct volume in a group of animals given vehicle control, citicoline 100 mg / kg, and citicoline 500 mg / kg. Values are mean ± standard deviation (SD).

Claims (19)

  A combination drug for treating a subject experiencing an ischemic event, comprising a first effective amount of citicoline and at least one second therapeutic agent or a pharmaceutically acceptable salt thereof respectively, The combination drug administered within about 24 hours after the occurrence of the ischemic event.   After the initial dose administration, an effective amount of citicoline or a pharmaceutically acceptable salt thereof is administered one or more times, or an effective amount of the at least one second therapeutic agent or a pharmaceutically acceptable salt thereof. The combination according to claim 1, wherein the combination is administered one or more times, or an effective amount of citicoline and at least one second therapeutic agent or their respective pharmaceutically acceptable salts are administered simultaneously one or more times. Drug.   The combination drug of claim 1, wherein the initial dose is administered within about 12 to about 15 hours after the occurrence of the ischemic event.   The combination drug of claim 1, wherein an effective amount of the citicoline and the at least one second therapeutic agent or their respective pharmaceutically acceptable salts are administered together or sequentially.   An effective amount of the citicoline and the at least one second therapeutic agent or their respective pharmaceutically acceptable salts are administered sequentially within about 12 to about 15 hours after the occurrence of the ischemic event. Item 5. A combination drug according to Item 4.   3. The combination drug of claim 2, wherein the subsequent administration or co-administration is performed over at least about 30 days.   3. The combination agent of claim 2, wherein the subsequent administration or co-administration is performed for at least about 4-8 weeks.   The combination agent according to claim 2, wherein the subsequent administration or simultaneous administration is performed for at least about 6 months to about 1 year.   The combination drug according to claim 2, wherein the initial or subsequent administration or simultaneous administration is performed once or more per day.   The combination drug according to claim 9, wherein the first or subsequent administration or simultaneous administration is performed twice a day.   The combination drug of claim 1, wherein the ischemic event occurs in the brain.   The combination drug according to claim 1, wherein the subject is a human.   The combination drug of claim 1, wherein the subject is suffering from cerebral ischemia, head trauma, or stroke.   The combination drug according to claim 1, wherein the at least one second therapeutic agent is t-PA, streptokinase or urokinase.   The combination drug according to claim 2, wherein the at least one second therapeutic agent is aspirin or dipyridamole.   A therapeutic composition for a subject experiencing an ischemic event comprising an effective amount of citicoline and at least one second therapeutic agent or their respective pharmaceutically acceptable salt in a pharmaceutically acceptable carrier. object.   17. The composition of claim 16, wherein the effective amount ranges from about 100 to about 1000 mg of citicoline and from about 10 to about 500 mg of at least one second therapeutic agent.   17. The composition of claim 16, wherein the at least one second therapeutic agent is t-PA, streptokinase or urokinase.   17. The composition of claim 16, wherein the at least one second therapeutic agent is aspirin or dipyridamole.

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