Patent Application
20070254861
By way of example and to make the description more clear, reference is made to the accompanying drawing in which:
As hereunder, the present invention will be specifically illustrated by way of Examples although the present invention is not limited by those Examples at all.
In some cases, the compound of the present invention forms a salt and, so far as such a salt is pharmaceutically acceptable, that is included in the present invention. Its specific examples are salt with inorganic acid such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid and phosphoric acid, etc; an acid-addition salt with organic acid such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, aspartic acid and glutamic acid, etc. The present invention further includes various kinds of hydrate and solvate of the compound of the present invention and a pharmaceutically acceptable salt and a substance having crystal polymorphism as well.
Depending upon the type of the substituent, the compound of the present invention represented by the formula (I) may contain asymmetric carbon atom and there may be optical isomer due to that. The present invention includes all of those optical isomers both as a mixture thereof and as isolated ones. In some cases, tautomers may be present in the compound of the present invention and the present invention includes all of those tautomers both as isolated ones and as a mixture thereof.
Injection for parenteral administration includes aseptic aqueous or non-aqueous solution, it includes suspension and emulsion. Aqueous solution and suspension may contain distilled water for injection and physiological saline for example. Examples of non-aqueous solution and suspension are propylene glycol, polyethylene glycol, plant oil such as olive oil, alcohol such as EtOH and Polysolvate 80. Such a composition may further contain adjuvant such as antiseptic, moisturizer, emulsifier, dispersing agent, stabilizer and dissolving aid. They are sterilized by, for example, filtration through a bacteria-retaining filter or compounding or irradiation of bactericide. They may be also used in such a manner that an aseptic solid composition is manufactured and, before use, it is dissolved in an aseptic water or in an aseptic solvent for injection.
The dose is appropriately decided depending upon each case by taking route of administration, symptom, age, sex, etc. into consideration. In the case of a common intra-arterial (i.a.) administration, it is appropriate that the daily dose is about 0.01 to 100 mg/body weight, preferably about 0.05 to 5 mg/body weight and it is administered once or divided into two to four times in a day to administer.
Drug Preparation for Intra-Arterial Injection
The compound (I) was dissolved in 100% ethanol and adjusted to 20 mg/ml. Appropriate concentrations of compound (I) were prepared using saline at final concentrations of 0.5% ethanol.
Vasorelaxant Effect of Compound (I) on Vasoconstruction in Isolated Rat Aorta Induced Various Vasoconstruction
Four male SD rats (Japan CLEA) were used in each group of this experiment. Rats were anesthetized with pentobarbital (60 mg/kg i.p.) and euthanized via exsanguination. The aortas were isolated and all adjacent tissue was removed. Rings of aorta, approximately 2-3 minutes length, were suspended in 10-ml organ baths containing Krebs-Henseleit solution (henceforth referred to as Krebs) of the following composition (mM): NaCl, 112.0; KCl, 4.7; KH2PO4, 1.2; MgSO4, 1.2; CaCl2, 2.5; NaHCO3, 25.0; glucose, 11.0. The Krebs was maintained at 37±1° C. and aerated with a gas mixture of 95% O2:5% CO2 (pH 7.4). Experiments were conducted on aortic tissue of two types. For type 1, the endothelium was removed by gently rubbing the internal surface of the vessel. For type 2, care was taken to maintain the integrity of the endothelium. One ring was placed on a hook that was suspended from a force displacement transducer (SB-1T, Nihon Kohden, Tokyo, Japan), 1.0 g tension was applied, and changes in the force of contraction were isometrically measured. Following a 1-h equilibration period, the rings were pretreated with 1 μM phenylephrine. Compound (I) was dissolved in dimethyl sulfoxide (DMSO) and added to the baths (0.1% DMSO, v/v), after which a cumulative compound (I) (1-100 nM) concentration-response curve was constructed for each type of ring. Contractions were caused in aorta rings with endothelium using three different compounds: 10 μM serotonin (5-HT), 30 nM endothelin-1 (ET-1), and 60 mM KCl. The vasorelaxant effect of compound (I) on the contractions caused by each of these compounds was then compared. Various concentrations of compound (I) were added 30 minutes before treatment with each vasoconstrictor. For 5-HT and KCl, the concentration-inhibition rate was measured for each tissue. For ET-1, each tissue was used to measure the inhibition rate in response to a given concentration of compound (I).
As shown in
Effect of Compound (I) on Ex Vivo Platelet Aggregation Inhibition After Single Administration in the Rat Femoral Artery
Four male SD rats (Japan CLEA) were used in each group of this experiment. All animals were fasted overnight before the experiment. After being anesthetized with pentobarbital (60 mg/kg i.p.), vehicle (5% ethanol) or compound (I) was injected into either the left femoral artery or the right jugular vein via a catheter. The rats were anesthetized with pentobarbital (60 mg/kg i.p.) before blood sampling. Blood was collected from the vena cava in syringes containing 3.8% sodium citrate at the following time points: 5 minutes after an intra-arterial (i.a.) single bolus injection of compound (I) (1-10 μg/kg). Platelet-rich plasma was obtained by centrifuging the blood at 200×g for 5 minutes at ambient temperature. This residue was further centrifuged at 2,000×g for 10 minutes in order to obtain platelet-poor plasma. Platelet counts were measured with an automatic cell counter (MEK-6258, Nihon Kohden, Tokyo, Japan), and adjusted to 3×105/μl with platelet-poor plasma. Platelet aggregation in platelet-rich plasma was measured using an aggregometer (MCM Hema Tracer 212, MC Medical, Tokyo, Japan) to record the increase in light transmission detected through a stirred suspension maintained at 37° C. for 5 minutes. Platelet aggregation was induced in 90 μl of platelet-rich plasma (3×105/μl) by adding 10 μl of 50 μM ADP. The inhibitory rate was calculated by dividing the absorbance area for the mixture containing the test sample by the absorbance area obtained for the vehicle-treated group.
As shown in
Inhibitory Effect of Compound (I) on Lesion Progression in a Rat Peripheral Arterial Disease Model Induced by Lauric Acid
A rat peripheral arterial disease model was produced by using a lauric acid injection method modified from that of Ashida et al. (Thrombosis research, 18, 55, 1980). Six to 24 animals were used in each group of this experiment. After being fasted overnight, each rat was anesthetized with pentobarbital (60 mg/kg i.p.), and the left femoral artery was freed from the surrounding tissue. The femoral artery was cannulated with a polyethylene catheter for i.a. injection of the lauric acid and the test drug. For the i.v. infusion of PGE1, the jugular vein was cannulated with a polyethylene catheter. To induce peripheral arterial vascular injury, 0.33 ml/kg of lauric acid (7.5 mg/ml in distilled water) was injected into the distal side of the arterial. Compound (I) was administered in a single i.a. bolus injection 15 minutes after the lauric acid injection. PGE1 was administered either intravenously for 30 minutes at 1 μg/kg/minutes starting 5 minutes before the lauric acid injection or intra-arterially for 15 minutes at 0.2 μg/kg/minutes starting 5 minutes after the lauric acid injection. Clopidogrel was administered orally either at 3 and 30 mg/kg 4 hours before the lauric acid injection, or at 30 mg/kg 2 hours after the lauric acid injection. It was also administered orally once a day for 3 days after the lauric acid injection. The treated hindlimb was examined macroscopically on days 3 after the lauric acid injection. The progress of the lesion was assessed using the following 5-point graded scoring system: grade 0: normal appearance, grade 1: the affected region was limited to the nails, grade 2: the affected region was limited to the fingers, grade 3: either the fingers are beginning to fall off or lesions appear on the paw, grade 4: either the paw is beginning to fall off or lesions appear on the leg. The condition of each toe was assessed and scored, and the sum of the scores for the five toes was used as the lesion index. If a lesion developed on the sole of the foot, 5 more points were added.
As shown in
Effect of Compound (I) on Decreased Blood Flow in the Rat Femoral Artery after the Lauric Acid Injection
Three male SD rats (Japan CLEA) were used in each group of this experiment. A laser Doppler blood flow meter (Laser Doppler Perfusion Imager System, Lisca) was used to evaluate the perfusion in the left (ischemic) and right (non-ischemic) rat hindlimbs. Before and during scanning, animals were placed on a heating plate set at 37° C. to minimize variations in temperature. The femoral artery was cannulated with a polyethylene catheter for i.a. injection of the lauric acid and test drug. For i.v. PGE1 infusion, the jugular vein was cannulated with a polyethylene catheter. Either compound (I) (10 μg/kg) or vehicle was administered in an i.a. bolus injection 15 minutes after the lauric acid injection (0.33 ml/kg, 7.5 mg/ml in distilled water). The laser Doppler images were recorded just before and 10 minutes after the lauric acid injection, as well as 10 minutes after the compound (I) (or vehicle) injection. PGE1 was administered by i.v. infusion at a rate of 1 μg/kg/minutes for 30 minutes beginning 5 minutes before the lauric acid injection. The laser Doppler images were recorded just before and 5 minutes after the start of infusion (just before the lauric acid injection), and then again 10 minutes after the lauric acid injection. Either vehicle (0.5% methylcellulose solution) or clopidogrel (30 mg/kg) was orally administered 4 hours before the experiment. For the oral studies, the laser Doppler images were recorded just before and 10 minutes after the lauric acid injection.
Effect of compound (I) on decreased blood flow in the rat femoral artery after the lauric acid injection was judged by perfusion images before and after the lauric acid injection in each treatment group. The perfusion signal was subdivided into 6 separate intervals, with each displayed as a different color. The perfusion values upon which the color-coded pixels are based are stored and remain available for further data analysis. In the vehicle (i.a.) group, dermal blood flow decreased markedly (center panel) immediately after the lauric acid injection, and did not improve at all after vehicle injection. Compound (I) (10 μg/kg), when administered intra-arterially in a bolus 15 minutes after the lauric acid injection, improved the lauric acid-induced reduction in dermal blood flow somewhat, but did not correct it completely. In the PGE1 group, a marked increase in dermal blood flow was observed in both hindlimbs 5 minutes after the start of the infusion; however, there was no improvement in the blood flow despite continuous infusion of the drug. Oral administration of clopidogrel improved the blood flow only slightly.
Effect of Compound (I) on Blood Pressure and Heart Rate after Single Bolus Injection in Femoral Artery of Rats
Five male SD rats (Japan CLEA) were used in each group of this experiment. The rats, which were fasted overnight, were anesthetized with urethane (1.4 g/kg i.p.) and then secured on an operating table. The left carotid artery was cannulated with polyethylene catheters in order to monitor systemic blood pressure and heart rate. The left femoral artery was likewise cannulated with polyethylene catheters for the injection of compound (I). Systemic blood pressure was measured with a pressure transducer (AP-200T, Nihon Kohden) and heart rate was measured with a tachometer (AT-600G, Nihon Kohden) that was triggered by the arterial pulse wave. Changes in these parameters were continuously monitored by a thermal recorder (WT-685G, Nihon Kohden). Compound (I) was administered in an i.a. bolus injection at the rate of 5 mL/kg. The dosage (10 to 100 μg/kg) was increased every 10 to 20 minutes. The inhibitory rate was calculated by dividing the heart rate and mean blood pressure levels obtained after the compound (I) injection by them obtained after the vehicle injection.
As shown in
The compound (I) or its salt exerts potent platelet aggregation inhibition and vasodilative effect. The local administration of this compound (I) can provide safer and more effective treatment for patients with PAD/severe PAD than its systemic administration. This invention produces decrease of pain at rest, prevention of ulcer/necrosis development, decrease of amputation rate. The compound (I) is expected to be effective in the treatment for patients with ASO and Buerger Disease.
While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the scope thereof.
This application is based on Japanese patent applications No. 2006-124797 filed Apr. 28, 2006 and No. 2006-213356 filed Aug. 4, 2006, the entire contents thereof being hereby incorporated by reference.
The patents, patent applications and publications cited herein are incorporated by reference.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2006-124797 | Apr 2006 | JP | national |
| 2006-213356 | Aug 2006 | JP | national |
