Viscous composition for treating ischemia

Abstract

1. A pharmaceutical composition for treating ischemic tissue, comprising: a core component comprising a thrombolytic drug; and a matrix component comprising hyaluronan or a derivative thereof, wherein the matrix component has a viscosity of greater than 10 mPa·s. [Selected Figure] Figure 1

Description

Technical Field

[0001] Cross - reference to related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 553,269, filed on September 1, 2017, the content of which is incorporated herein by reference.

Background Art

[0002] In ischemia, the blood content of an organ or tissue decreases. Ischemia can be the result of the manifestation of systemic anemia locally or local blood circulation disorders. The types of ischemia include the following: 1) Compressive ischemia is caused, for example, by pressure on arterial vessels from tumors, tight bandages, and exudates, which can lead to narrowing or occlusion of the lumen of the blood vessels. Clinically, hemorrhoids or ulcers formed from long - term lying down are examples of tissue necrosis caused by ischemia due to compression of the outer blood vessels, which may cause muscle damage. 2) Occlusive ischemia from arterial thrombosis or embolism results in occlusion of the blood vessels, which can inhibit blood supply to, for example, limbs or the heart. 3) Ischemia of the outer limbs is caused by a sudden large flow of blood to abdominal organs, which can lead to ischemia of other organs and tissues.

[0003] Most patients with peripheral arterial disease of the lower extremities are over 60 years old, and approximately 1 / 2 of these patients have diabetes. Currently, angiogenesis treatments for ischemic lower extremities are on the market. For example, the AutoloGel system is a wound dressing material prepared by extracting the patient's autologous high - concentration platelet - rich plasma (PRP) and adding growth factors and cytokines that promote wound healing to form a gelatinous substance. However, such treatments are only used to treat chronic injuries and cannot treat the underlying ischemia. Other treatments such as bypass surgery, vasodilation, and placement of vascular stents are required to restore blood vessel occlusion.

[0004] Numerous studies on ischemic lower limbs have led to the active development of angiogenesis therapies, such as cytokines or recombinant growth factors related to angiogenesis signaling, like VEGF and FGF, which stimulate angiogenesis. Platelet-derived growth factor (PDGF) has been found to stimulate the proliferation, migration, and differentiation of mesenchymal cells in developing or adult tissues and is used to promote the release of bone marrow-derived endothelial-derived cells in patients to proliferate blood vessels. Human umbilical vein endothelial cells (HUVEC) can also be stimulated by substances indirectly related to angiogenesis signaling and can stimulate angiogenesis. Treatments using tissue plasminogen activator (tPA) and HUVEC are provided to increase the number of endothelial progenitor cells migrating from the bone marrow to the blood vessels and to promote the activation (rejuvenation) of vascular endothelium to achieve a therapeutic effect.

[0005] The pathophysiology of hyperglycemia caused by diabetes reduces the secretion of endothelial growth factors and can lead to amputation in severe vascular diseases. Most current treatment methods contain angiogenesis factors but face many difficulties in clinical use or medical effectiveness. Nevertheless, currently, there is still no effective treatment to regenerate ischemic tissue without amputating the limb. Therefore, the development of a therapeutic composition for ischemic tissue suitable for most patients is an important problem to be solved. SUMMARY OF THE INVENTION

[0006] In one aspect, described herein is a pharmaceutical composition for treating ischemic tissue, comprising a core component containing a thrombolytic agent and a matrix component containing hyaluronic acid or a derivative thereof, wherein the pharmaceutical composition has a viscosity of more than 10 mPa·s. In some embodiments, the viscosity is from 10 to 10000 mPa·s. In some embodiments, the pharmaceutical composition contains hyaluronic acid at a concentration of 1 mg / ml to 100 mg / ml.

[0007] In some embodiments, hyaluronic acid has an average molecular weight of 100 kDa to 5000 kDa. For example, hyaluronic acid can have an average molecular weight of 700 kDa to 2000 kDa.

[0008] In some embodiments, the viscosity of the pharmaceutical composition is within the range of the viscosity of 3 to 10 mg / ml of hyaluronic acid having an average molecular weight of 700 to 2000 kDa. In some embodiments, the viscosity is the same as the viscosity of 5 mg / ml of hyaluronic acid having an average molecular weight of 1560 kDa. The average molecular weight of hyaluronic acid can be 700 to 2000 kDa, and the concentration of hyaluronic acid can be 3 to 10 mg / ml. In some embodiments, the average molecular weight of hyaluronic acid is 1560 kDa, and the concentration of hyaluronic acid is 5 mg / ml.

[0009] In some embodiments, the matrix component in the pharmaceutical composition further comprises collagen, extracellular matrix factor, protein, or polysaccharide.

[0010] The thrombolytic agent in the pharmaceutical composition can be selected from the group consisting of ticlopidine, warfarin, tissue plasminogen activator, eminase, retavase, streptase, tissue plasminogen activator, tenecteplase, abokinase, kinlytic, urokinase, prourokinase, anisoylated purified streptokinase activator complex (APSAC), fibrin, and plasmin.

[0011] In some embodiments, the pharmaceutical composition further comprises an angiogenesis compound (such as vascular endothelial growth factor).

[0012] In another aspect, provided herein is a method of treating ischemic tissue. The method includes administering directly to the ischemic tissue of a subject the pharmaceutical composition described herein, provided that the pharmaceutical composition is not administered intravenously.

[0013] In some embodiments, the ischemic tissue is an ulcer of the subject or is in the subject's heart or limb. The ischemic tissue can be muscle. In some embodiments, the subject has diabetes.

[0014] Details of one or more embodiments are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of these embodiments will be apparent from this description, the drawings, and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0015]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

[0016] Unexpectedly, it has been discovered that a pharmaceutical composition containing hyaluronic acid having a specific viscosity and a thrombolytic agent is effective in treating ischemic tissue.

[0017] Pharmaceutical Composition Therefore, in this specification, a pharmaceutical composition for treating ischemic tissue is described. This composition includes a core component containing a thrombolytic agent and a matrix component containing hyaluronic acid or its derivative. This pharmaceutical composition has a viscosity of more than 10 mPa·s. Depending on the parameters (such as spindle and rotation speed) selected for viscosity measurement, the viscosity of this composition can be in the range of 10 to 10,000 mPa·s (for example, 10 to 100, 50 to 150, 100 to 200, 150 to 250, 250 to 500, 500 to 1000, 1000 to 1500, 1500 to 2000, 2000 to 2500, 2500 to 3000, 3000 to 3500, 3500 to 5000, 5000 to 6000, 6000 to 7000, 7000 to 8000, 8000 to 9000, or 9000 to 10,000).

[0018] The viscosity of this pharmaceutical composition can be within the viscosity range of hyaluronic acid with an average molecular weight of 700 to 2000 kDa (for example, 700, 800, 900, 1000, 1500, 1600, 1700, 1800, 1900, or 2000) at 3 to 10 mg / ml (for example, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg / ml). See Tables 2 to 5 below. In some embodiments, the viscosity of this composition is the same as the viscosity of 5 mg / ml of hyaluronic acid having an average molecular weight of 1560 kDa. For example, the data described below shows that 4 mg / ml of 2000 kDa hyaluronic acid, 5 mg / ml of 1560 kDa hyaluronic acid, and 6.5 mg / ml of 700 kDa hyaluronic acid have approximately the same viscosity.

[0019] The molecular weight of hyaluronic acid in this pharmaceutical composition can be in the range of 4 kDa to 5000 kDa (for example, 4 - 20, 20 - 100, 100 - 500, 500 - 1000, 1000 - 2000, 2000 - 2500, 2500 - 5000, 5, 10, 50, 100, 200, 300, 400, 500, 750, 1000, 1500, 1800, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 kDa). The concentration of hyaluronic acid in this pharmaceutical composition can be 1 - 100 mg / ml (for example, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / ml). In particular, when using hyaluronic acid having an average molecular weight of 700 - 2000 kDa, the concentration of hyaluronic acid in this pharmaceutical composition can be 3 - 10 mg / ml (for example, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mg / ml). A person skilled in the art can select an appropriate combination of molecular weight and concentration to obtain a composition with the desired viscosity. Also, a person skilled in the art can determine the viscosity of the composition using methods known in the art and commercially available instructions.

[0020] The term "hyaluronan" refers to a naturally occurring anionic non-sulfated glycosaminoglycan containing repeating disaccharide units of N-acetylglucosamine and D-glucuronic acid, and its derivatives. Naturally occurring hyaluronan (also known as hyaluronic acid or hyaluronate) can be isolated via conventional methods from its natural sources, such as the capsules of streptococcus, the combs of roosters, cartilage tissue, synovial joints fluid, umbilical cords, skin tissue, and the vitreous humor of the eye. See, for example, Guillermo Lago et al. Carbohydrate Polymers 62(4): 321-326, 2005; and Ichika Amagai et al. Fisheries Science 75(3): 805-810, 2009. Alternatively, this can be purchased from commercial vendors, such as Genzyme Corporation, Lifecore Biomedical, LLC, and Hyaluron Contract Manufacturing. Derivatives of naturally occurring hyaluronan include, but are not limited to, hyaluronan esters, hyaluronan modified with adipic dihydrazide, hyaluronan amide products, cross-linked hyaluronic acid, hemiesters of succinic acid or its heavy metal salts with hyaluronic acid, partial esters or total esters of hyaluronic acid, sulfated hyaluronic acid, N-sulfated hyaluronic acid, and hyaluronic acid modified with amides or diamines. These can be obtained by chemically modifying one or more of its functional groups (e.g., carboxylic acid groups, hydroxyl groups, reducing end groups, N-acetyl groups). Carboxyl groups can be modified via esterification or reactions mediated by carbodiimide and bishydrazide. Modifications of hydroxyl groups include, but are not limited to, sulfation, esterification, isourea coupling, activation with cyanogen bromide, and oxidation with periodate. Reducing end groups can be modified by reductive amination. This can also bind to phospholipids, dyes (e.g., fluorophores or chromophores), or agents suitable for the preparation of affinity matrices.Derivatives of naturally occurring hyaluronan can also be obtained by crosslinking using a crosslinking agent (e.g., bisepoxide, divinyl sulfone, biscarbodiimide, small molecule homo-bifunctional linker, formaldehyde, cyclohexyl isoimide, lysine ethyl ester, metal cation, hydrazide, or a mixture thereof), or can be obtained via internal esterification, photo-crosslinking, or surface plasma treatment. To prepare a hyaluronan solution, hyaluronan can be dissolved in a phosphate buffer (e.g., <0.05 M at pH 7 ± 1) and / or NaCl (e.g., ≤ 0.9%).

[0021] The matrix component can include one or more other matrix molecules as long as the viscosity of the composition remains within the desired range. These matrix molecules can include gelatin, collagen, hyaluronan, fibronectin, elastin, tenacin, laminin, vitronectin, polypeptide, heparan sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, dermatan sulfate, carrageenan, heparin, chitin, chitosan, alginate, agarose, agar, cellulose, methylcellulose, carboxymethylcellulose, glycogen, and derivatives thereof. Further, the matrix component can include fibrin, fibrinogen, thrombin, polyglutamic acid, synthetic polymers (e.g., acrylate, polylactic acid, polyglycolic acid, or poly(lactic-co-glycolic acid)), or a crosslinking agent (e.g., genipin, glutaraldehyde, formaldehyde, or epoxide).

[0022] Thrombolytic agents can be ticlopidine, warfarin, tissue plasminogen activator (t-PA), eminase (anisoylated plasminogen streptokinase activator complex: APSAC), reteplase (retavase), streptase (streptokinase, kabikinase), activase, tenecteplase (TNKase), abokinase, kinlytic (lokinase), urokinase, prourokinase, fibrin, plasmin. The pharmaceutical composition may contain one or more thrombolytic agents. The pharmaceutical composition may contain a thrombolytic agent in a dose similar to or less than the recommended clinical dose.

[0023] The pharmaceutical composition may further contain an angiogenesis compound such as vascular endothelial growth factor ( VEGF ).

[0024] Treatment method An effective amount of the pharmaceutical composition can be administered to a patient for ischemic treatment. This can be administered directly to or near the ischemic tissue (such as muscle), for example, by injection or application. The composition is gelatinous or viscous in consistency and is not administered intravenously.

[0025] The composition can be administered to a subject as needed, for example, 1 to 5 times a day, 1 to 5 times a week, 1 to 5 times a month, over an appropriate treatment period, for example, 1 to 4 weeks, 1 to 12 months, or 1 to 3 years. It is preferably administered as soon as possible (for example, within 0 to 48 hours or within 1 to 7 days) after ischemia or ischemic injury has occurred.

[0026] The amount of the pharmaceutical composition administered should be sufficient to provide an effective amount of the therapeutic compound, such as a thrombolytic agent. The effective amount can be, for example, 0.00001 to 10 μg (such as 0.00001 to 0.001, 0.001 to 0.005, 0.005 to 0.01, 0.05 to 0.1, 0.1 to 0.5, 0.5 to 1, 0.00001, 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μg) per gram of the body weight of the subject, depending on, for example, the potency of the thrombolytic agent.

[0027] "Treating" means administering a pharmaceutical composition to a subject suffering from a disorder or at risk of developing the disorder for the purpose of curing, reducing, alleviating, remedying, delaying the onset, preventing, or remission of the disorder, the symptoms of the disorder, the disease state secondary to the disorder, or the tendency to become the disorder. "Effective amount" means the amount of the composition that can bring about a medically desirable result in the subject being treated. This treatment method can be carried out alone or in combination with other pharmaceuticals or treatments. The subject to be treated can be a human, or a research or domestic animal.

[0028] The following specific examples should be construed as merely illustrative and not limiting the rest of the disclosure in any way. Without further elaboration, it is believed that one of ordinary skill in the art can utilize the present disclosure to its fullest extent based on the description herein. All publications cited herein are hereby incorporated by reference in their entirety.

[0029] Example 1: Mouse Model of Diabetic Lower Limb Ischemia Male C57BL / 6 mice were procured from the Research Animal Center of National Cheng Kung University (NCKU) or BioLASCO Taiwan Co., Ltd. These mice were housed in the animal facility of the Institute of Biotechnology at NCKU for at least one week to adapt to the environment before the experiments were conducted. All the experiments performed were pre-approved by the IACUC (Institutional Animal Care and Use Committee) at NCKU.

[0030] The animal model for this experiment was established to test therapeutic treatments for lower limb ischemia. Mice older than 6 months were treated with a 50 mg / kg body weight streptozotocin (STZ) solution to induce type I diabetes to exhibit the characteristics of aged and slowly healing wounded diabetic tissues. To prevent this result from the hypoglycemic values of the mice, mice with blood glucose values in the range of 400 mg / dl to 550 mg / dl were used in this experiment, and a small amount of insulin was used in the mice to avoid life-threatening hyperglycemia. To avoid the possibility of self-renewing angiogenesis, the femoral artery and its peripheral blood vessels in the lower limbs of the mice were severed. This model minimized the possibility of angiogenesis and enabled a more accurate assessment of the angiogenic ability of the test pharmaceutical.

[0031] To induce lower limb ischemia, shaved diabetic mice were placed in an anesthesia gas chamber with ventilating gas containing 1 - 3% isoflurane per liter of gas per minute. After the mice lost consciousness, they were transferred to the operating table and maintained under anesthesia gas. After fixing the limbs using breathable tape, the body temperature of the mice was kept constant using a heating pad at 37°C. After disinfecting the lower abdomen and limbs of the mice, the skin of the limb was incised from a small opening at the left ankle to the thigh. Both ends of two lateral blood vessels on the dorsal side of the gastrocnemius muscle of the mice were ligated with suture threads, and the blood vessels were removed to block the blood flow in the dorsal blood vessels. Next, the collateral branches and main blood vessels of the ventral femoral artery were blocked. The arterial end near the ankle and the surrounding blood vessels were ligated with suture threads to ensure complete blockage of the femoral artery and peripheral blood flow.

[0032] After cutting the blood vessels, the pharmaceutical composition to be tested for its therapeutic effect on ischemia was applied directly to the tissue or injected into the gastrocnemius muscle at eight sites, and the surgical opening was sutured. The mice were subcutaneously injected with 1 mg / kg body weight of ketorolac analgesic and lidocaine - HCl local anesthetic, and 1 ml of physiological saline aqueous solution was administered to relieve pain and provide hydration. A glucose solution was administered whenever necessary to maintain physical strength.

[0033] On days 0, 1, 2, 3, 4, 5, 6, 7, 14, 21, and 28 after surgery, the appearance of the mice and the blood flow in the lower limbs were evaluated using the scoring system shown in Table 1 and a laser Doppler flowmeter, respectively. The ROI was calculated as the ratio of the blood flow signal in the untreated right lower limb to the blood flow signal in the left lower limb after surgery, and the ratio in percentage was normalized based on the blood flow signal performed before surgery.

Table 1

[0034] Example 2: Viscosity Test of Hyaluronic Acid A plurality of compositions containing hyaluronic acid with various molecular weights and different concentrations were generated.

[0035] The viscosities of these pharmaceutical compositions were tested using a DV2TRV viscometer (Brookfield, USA) according to the manual. Appropriate spindles (CPE40 or CPE52) were selected according to the viscosity. Before the test, this machine was calibrated and set to operate for 1 minute at 25 °C and 20 rpm. 500 μl of each sample was transferred to the sample plate with a viscosity pipette, and the operation button was pressed to start the determination of the viscosity of the sample. The viscosities of hyaluronic acid with average molecular weights of 1,560 kDa, 700 kDa, and 2,000 kDa at 5 mg / ml were determined, and these results are shown in Table 2. The viscosity of hyaluronic acid with an average molecular weight of 1,560 kDa at 5 mg / ml was used as a reference, and the viscosities of hyaluronic acid at various concentrations with average molecular weights of 700 kDa and 2,000 kDa were measured as shown in Tables 3 and 4. Next, the concentrations of hyaluronic acid with average molecular weights of 700 kDa and 2,000 kDa having viscosities close to the reference viscosity value were calculated and adjusted to 6.5 mg / ml and 4 mg / ml, respectively.

[0036] As shown in Table 5, it is noteworthy that the viscosities of hyaluronic acid having the same concentration and molecular weight range changed when the measurement parameters were changed.

Table 2

Table 3

Table 4

Table 5

[0037] Example 3: Pharmaceutical Composition Containing Vascular Endothelial Growth Factor (VEGF) A composition (DIV) containing hyaluronic acid at 5 mg / ml with an average molecular weight of 1560 kDa and VEGF was administered to mice, and the effects on their lower limbs and blood flow were evaluated as described in Example 1 above. Diabetic mice not treated with this composition after surgery were used as controls. VEGF pharmaceuticals have been described in the literature as having an angiogenesis effect. The maximum and minimum effective amounts of VEGF in humans were converted to mouse dosages based on body weight.

[0038] The appearance scores are shown in Figure 1. Administration of 100 μl of DIV provided 3.125 ng / g body weight of VEGF to the mice. Administering 3.125 ng / g of VEGF (DIV2) to the mice resulted in a higher appearance score compared to the control group. When the dosage of VEGF was lowered to 0.3 ng / g (DIV1), it was observed that the appearance score was lower than the value of the control group. When the dosage of VEGF was increased to 15 ng / g (DIV3), it was observed that ischemia and necrosis of the lower limbs worsened.

[0039] The results of the blood flow measurements are shown in Figure 2. Compared to the control group, only 3.125 ng / g of VEGF significantly increased blood flow on the 14th day and after subsequent surgery. This result indicated that VEGF was effective only at specific dosages from 0.3 ng / g to 15 ng / g.

[0040] Example 4: Pharmaceutical Composition Containing Ticlopidine A composition (DIT) containing 5 mg / ml of hyaluronic acid with a molecular weight in the range of 1000 - 1800 kDa and ticlopidine was administered to mice, and the effects on their lower limbs and blood flow were evaluated as described in Example 1 above. Diabetic mice not treated with this composition after surgery were used as controls. The maximum and minimum effective amounts of human ticlopidine were converted to mouse dosages based on body weight.

[0041] The postoperative appearance scores are shown in Figure 3. Administration of 100 μl of DIT provided mice with 0.7 μg / g body weight of dichloropyridine (DIT2). At this dose, the appearance scores from day 2 to day 28 were significantly different from those of the control group (P < 0.05). Also, the appearance scores were significantly different from those of the control group even when 0.07 μg / g of dichloropyridine (DIT1) was administered.

[0042] However, when the dose was increased to 7 μg / g body weight (DIT3) or 110 μg / g body weight (DIT4), the appearance scores were not significantly different from those of the control group during the observation period. These results also indicated that dichloropyridine could effectively reduce the gangrene caused by ischemia at low doses, but this effect decreased when the dose was below a specific threshold.

[0043] The results of blood flow measurement are shown in Figure 4. These results showed that with 0.07 μg / g body weight (DIT1) and 0.7 μg / g body weight (DIT2) of dichloropyridine, the blood flow signal in the ischemic lower limb increased significantly compared to the control from day 7 after surgery (P < 0.05). However, at higher doses (DIT3 and DIT4) than this, there was no significant difference in blood flow compared to the control.

[0044] Example 5: Pharmaceutical Composition Containing Warfarin A composition (DIW) containing hyaluronic acid with a concentration of 5 mg / ml and an average molecular weight of 1,560 kDa and warfarin was administered to mice, and its effects on the lower limbs and blood flow were evaluated as described in Example 1 above. Diabetic mice that were not treated with this composition after surgery were used as controls. The maximum effective dose and minimum effective dose of human warfarin were converted to the mouse dose according to body weight.

[0045] The postoperative appearance scores are shown in Fig. 5. Administration of 100 μl of DIW provided mice with 70 ng / g body weight of warfarin. At this dose (DIW2), the appearance scores from day 2 to day 28 were most significantly different compared to the scores of the control group (P<0.05). Furthermore, when this dose was doubled up to 140 ng / g body weight (DIW4), the appearance scores from day 5 to day 28 were also significantly different from the scores of the control group (P<0.001 - 0.05). On the other hand, when the dose was increased to 3 times, 210 ng / g body weight (DIW5), the appearance scores were not significantly different from the scores of the control group. These results suggested that the optimal dose of DIW was 70 ng / g body weight of warfarin, which maintained the integrity of the lower limb appearance and avoided tissue gangrene in cases of ischemic diseases.

[0046] The results of blood flow measurement are shown in Fig. 6. These results showed that when the dose of warfarin was 70 ng / g body weight (DIW2), the blood flow signal in the lower limb was significantly different from the signal of the control group from day 7 after surgery. When the doses were 35 ng / g (DIW1), 105 ng / g (DIW3), 140 ng / g (DIW4), or 210 ng / g (DIW5), the blood flow signals in the lower limb were significantly different from the signal of the control group from day 14 after surgery. However, with 210 ng / g of warfarin, there was no significant difference compared to the control group on day 28 after surgery.

[0047] Comparing the results obtained with DIV (Example 3), DIT (Example 4), and DIW, administration of DIW with 70 ng / g body weight of warfarin (DIW2) seemed to be the most effective. In the DIW2 group, only the distal ends of the toes of the lower limb were slightly darkened during the postoperative observation period. In contrast, in the control group, the left lower limb had a blackened appearance on day 3 after surgery. Also, some tissue shedding may be observed on day 7 after surgery, and gangrene of the lower limb may be observed on day 14 after surgery. Therefore, the appearance of gangrene induced by ischemia could be significantly reduced by administration of DIW2.

[0048] Furthermore, the distribution of blood flow signals detected by laser Doppler indicated that the blood flow signals in the DIW2 group gradually increased after the 14th day after surgery. Conversely, in the control group, no increase in blood flow signals was observed. Additionally, due to lower limb gangrene, the laser Doppler imager was unable to detect blood flow in the lower limbs of the control group. These results further indicated that the appearance of lower limb gangrene was significantly reduced in the DIW2 group compared to the control group.

[0049] Further analysis of the postoperative blood flow changes in the lower limbs in the control group and the DIW2 group was performed using an oximeter. As shown in Figure 7, the blood flow in the lower limbs in the control group and the DIW2 group began to decrease immediately after surgery. The DIW2 group began to recover blood flow on the 7th day after surgery, and this blood flow reached approximately 100 - 200 AU during the observation period. The control group showed no recovery of blood flow during the 28-day observation period and actually showed a slight decrease.

[0050] Example 6: Evaluation of the function of mice treated with a pharmaceutical composition containing warfarin The mice in the DWI2 group and the control group were further evaluated for function. This evaluation was performed on the 35th day after surgery.

[0051] Each mouse was placed on the platform, its tail was pulled at a fixed height of about 5 cm, and its standing grip pose was observed. In the upright posture, it was observed that the mice in the DIW2 group still could not grip as well as normal mice. However, it was found that the step length and sway length of the DIW group were significantly increased (P<0.001) compared to the values of the control group and were comparable to those of normal mice. Please refer to Figures 8(A) and (B). These results indicated that although the lower limbs of the mice in the DIW2 group began to atrophy after surgery and their stride could not fully return to normal, they were still significantly better compared to the control group.

[0052] Furthermore, the mice were placed on a running track and their gait was analyzed according to the shape of their feet. At 5 rpm, the mice in the DIW2 group and normal mice were able to stay on the track without falling for a similar period, but it was observed that the mice in the control group fell after a significantly shorter period. Please refer to Figure 8(C). When the rotation speed was increased to 10 rpm, the mice in the DIW2 group fell much earlier than normal mice, but it was observed that they stayed on the track for a significantly longer period than the control mice. Please refer to Figure 8(D).

[0053] Example 7: Effect of Treatment Timing Diabetic mice with lower limb ischemia were prepared as described in Example 1. These mice were treated with a composition containing warfarin and hyaluronic acid with a molecular weight in the range of 1000 - 1800 kDa at a concentration of 5 mg / ml, at a dose of 70 ng / g body weight of warfarin, similar to the mice in the DIW2 group described above, but at different time points after surgery. As shown in Figure 9, when the treatment was delayed after surgery, it was observed that the appearance score of the lower limb decreased according to the length of the delay. Nevertheless, even when the treatment was delayed by about 48 hours after surgery, the appearance score was still about 9 points on the 28th day after surgery, indicating that only a part of the limb remained with necrosis of the toes. When the treatment was delayed by 72 hours, necrosis of the lower limb could not be recovered.

[0054] Example 8: Effect of Hyaluronic Acid with Different Molecular Weights on the Efficacy of Treatment It was studied whether hyaluronic acid with different molecular weights in this pharmaceutical composition affects the therapeutic effect.

[0055] A plurality of compositions containing warfarin and hyaluronic acid with different average molecular weights, namely 74 kDa, 357 kDa, 700 kDa, 1560 kDa, 2000 kDa, and 2590 kDa, at a concentration of 5 mg / ml were generated. These compositions were administered to diabetic mice with lower limb ischemia at a dose of 70 ng / g body weight of warfarin and evaluated as described in Example 1.

[0056] As shown in Figure 10, the composition containing hyaluronic acid with an average molecular weight of 1,560 kDa showed the best therapeutic effect. When the average molecular weight was increased to 2,000 kDa, the appearance score was better than that of the control group, but lower than that of 1,560 kDa hyaluronic acid. Furthermore, hyaluronic acid with an average molecular weight less than 1,560 kDa tended to lower the appearance score. The appearance scores of the 357 kDa and 74 kDa groups on the 28th day after surgery were deteriorated compared to the control group.

[0057] As shown in FIG. 11, the blood flow signals of the 2,000 kDa and 1,560 kDa groups were significantly increased after 14 days post-surgery compared to the control group.

[0058] Example 9: Effect of Viscosity on the Efficacy of Treatment It was investigated whether viscosity affects the therapeutic effect of this pharmaceutical composition.

[0059] Compositions containing 4 mg / ml of hyaluronic acid with an average of 2,000 kDa, 5 mg / ml of hyaluronic acid with an average of 1,560 kDa, or 6.5 mg / ml of hyaluronic acid with an average of 700 kDa were produced. The concentrations of hyaluronic acid were selected so that all three had similar viscosities. See Tables 2, 3, and 4 above. Each was mixed with warfarin to produce a gelatinous composition. These compositions were administered to diabetic mice with hindlimb ischemia at a dose of 70 ng / g body weight of warfarin and evaluated as described in Example 1.

[0060] As shown in FIG. 12, the appearance scores of the hindlimbs in mice treated with compositions containing hyaluronic acid of different molecular weights were not very different during the observation period. Thus, the viscosity of hyaluronic acid was considered to be more important than the molecular weight. It should be noted that, as shown in FIGS. 10 and 11, 5 mg / ml of hyaluronic acid with an average of 1,560 kDa and 5 mg / ml of hyaluronic acid with an average of 2,000 kDa, although at the same concentration, were significantly more effective than hyaluronic acid with a larger or smaller molecular weight. These results also suggest that a specific range of viscosities is optimal.

[0061] Other Embodiments All of the characteristics disclosed herein can be combined in any combination. Each characteristic disclosed herein can be replaced by another characteristic that serves the same purpose, an equivalent purpose, or a similar purpose. Thus, unless otherwise specified, each characteristic disclosed is merely an example of a general series of equivalent or similar characteristics.

[0062] From the above description, those skilled in the art can easily reach the essential features of the described embodiments and can make various changes and modifications to these embodiments in order to adapt to various uses and conditions without departing from the spirit and scope of the present invention. Therefore, other embodiments are also within the scope of the claims.

Claims

1. A pharmaceutical composition for treating ischemic tissue, comprising a core component containing a thrombolytic agent and a matrix component containing hyaluronic acid or a derivative thereof, wherein the thrombolytic agent is ticlopidine or warfarin, the derivative of hyaluronic acid is selected from the group consisting of hyaluronic acid esters, hyaluronic acid modified with adipic acid dihydrazide, hyaluronic acid amide products, cross-linked hyaluronic acid, hemiesters of succinic acid or its heavy metal salts and hyaluronic acid, partial esters or total esters of hyaluronic acid, sulfated hyaluronic acid, N-sulfated hyaluronic acid, and hyaluronic acid modified with amides or diamines, the pharmaceutical composition has a viscosity of 10 to 10,000 mPa·s, the viscosity is measured at 25°C and a rotational speed of 20 rpm, a pharmaceutical composition that is directly administered to the ischemic tissue but not intravenously.

2. The pharmaceutical composition according to claim 1, wherein the hyaluronic acid has an average molecular weight of 100 kDa to 5000 kDa.

3. The pharmaceutical composition according to claim 2, wherein the hyaluronic acid has an average molecular weight of 700 kDa to 2000 kDa.

4. The pharmaceutical composition according to claim 2, containing 1 mg / ml to 100 mg / ml of the hyaluronic acid.

5. The pharmaceutical composition according to claim 1, wherein the viscosity is within the range of the viscosity of 3 to 10 mg / ml of hyaluronic acid having an average molecular weight of 700 to 2000 kDa.

6. The pharmaceutical composition according to claim 1, wherein the viscosity is the same as the viscosity of 5 mg / ml of hyaluronic acid having an average molecular weight of 1560 kDa.

7. The pharmaceutical composition according to claim 5, wherein the average molecular weight of the hyaluronic acid is 700 to 2000 kDa and the concentration of the hyaluronic acid is 3 to 10 mg / ml.

8. The pharmaceutical composition according to claim 6, wherein the average molecular weight of the hyaluronic acid is 1560 kDa and the concentration of the hyaluronic acid is 5 mg / ml.

9. The pharmaceutical composition according to claim 1, wherein the matrix component further contains collagen, extracellular matrix factors, proteins, or polysaccharides.

10. The pharmaceutical composition according to any one of claims 1 to 9, further containing an angiogenesis compound.

11. The pharmaceutical composition according to claim 10, wherein the angiogenesis compound is vascular endothelial growth factor (VEGF).

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein the ischemic tissue is an ulcer of a subject, or is in the heart or limb of the subject.

13. The pharmaceutical composition according to claim 1, wherein the ischemic tissue is muscle.

14. The pharmaceutical composition according to claim 1, wherein the ischemic tissue is in a subject having diabetes.

Images