Compositions and methods for thromboembolism dissolution

Abstract

Disclosed herein are novel thrombolytic formula for safe and effective treatment of blood clots. In clinical trials, cats of various breeds and ages presented partial or full paralysis of one or more limbs, due to arterial thromboembolism (ATE). Average treatment ranged from 3 days to 2 weeks, depending on the clot size and time elapsed since clot was dislodged from the subject's heart. These are significant findings considering the lack of treatment options available in veterinary medicine for cats with HCM and ATE. Veterinarians often euthanize cats presenting ATE, as no safe and effective thrombolytic agent is currently available for veterinary use. Previous thrombolytics were shown to have an average mortality rate of 60% due to reperfusion injury and hemorrhaging. The disclosed formula, on the other hand, overcomes these shortcomings.

Claims

1. A method of dissolving a feline thrombus comprising: administering to the feline from 10 to 30 tablets of an enzymatic formula containing nattokinase and an enteric coating; wherein each tablet comprises nattokinase at 37.5 mg/750 fibrinolytic units.

2. The method of claim 1, wherein each tablet further comprises rutin bioflavonoid at 50 mg.

3. The method of claim 1, wherein each tablet comprises nattokinase at 112.5 mg/2,250 fibrinolytic units.

4. The method of claim 1, wherein the thrombus comprises a feline arterial thromboembolism.

5. The method of claim 3, wherein each tablet further comprises rutin bioflavonoid at 150 mg.

6. The method of claim 1, wherein each tablet further comprises rutin bioflavonoid at 50 mg.

7. The method of claim 6, further comprising administering a second amount of 10 to 30 tablets 12 hours after the initial amount.

8. The method of claim 3, further comprising administering a second amount of 10 to 30 tablets 12 hours after the initial amount.

9. The method of claim 7, wherein orally administering the second amount is continued with 2 tablets daily.

10. The method of claim 7, wherein orally administering the second amount is continued with 4 tablets daily.

11. The method of claim 8, wherein orally administering the second amount is continued with of 6 tablets daily.

12. The method of claim 7, wherein orally administering the second amount is continued with 5 tablets about every 12 hours, wherein each tablet comprises nattokinase at 225 mg/4500 fibrinolytic units, and rutin bioflavonoid at 300 mg.

13. A method of administering a thrombolytic formula to a feline comprising: administering to the feline a formula orally every 12 hours for a treatment duration, the formula comprising 10 to 30 tablets, wherein each tablet comprises (A) nattokinase at 112.5 mg/2250 fibrinolytic units, and rutin bioflavonoid at 150 mg, or (B) nattokinase at 225 mg/4,500 fibrinolytic units, and rutin bioflavonoid at approximately 300 mg.

Description

DESCRIPTION

(1) The present disclosure relates to compositions and methods for dissolving thromboembolisms, and in particular thromboembolisms in felines. Embodiments of the enzymatic formula include nattokinase and rutin bioflavonoid. In some embodiments, the nattokinase may be present in a single dose at approximately 37.5 mg/750 fibrinolytic units, although the concentration may vary in other embodiments. In some embodiments, the rutin bioflavonoid may be present in a single dose at approximately 50 mg, although the concentration may vary in other embodiments. Some embodiments may be in the form of a tablet, the tablet having nattokinase at approximately 37.5 mg/750 fibrinolytic units, and rutin bioflavonoid at approximately 50 mg. It should be appreciated, though, that the dose may be larger or smaller, depending on the patient.

(2) Some embodiments may also include an enteric coating to reduce dissolution of the formula in the gastric environment. In some embodiments, the enteric coating may be a polymer barrier such as, for example, a methacrylic acid copolymer and triethyl citrate coating. For example, some embodiments may be in the form of a tablet having an enteric coating.

(3) Treatment methods may include oral administration of a dose of the enzymatic formula. In some embodiments, a treatment regimen includes orally administering 10-30 tablets initially, and then orally administering 10-30 tablets after approximately 12 hours after the initial administration, each tablet having nattokinase at approximately 37.5 mg/750 fibrinolytic units, and rutin bioflavonoid at approximately 50 mg. For example, 15 tablets may be orally administered at time 0 hours, and another 15 tablets may be orally administered at time 12 hours.

(4) In a preferred embodiment, the thrombolytic enzymatic formula includes the following active ingredients: nattokinase (37.5 mg/750 fibrinolytic units) and rutin bioflavonoid (50 mg). Nattokinase is a naturally occurring proteolytic enzyme derived from natto, a traditional Japanese food produced from the fermentation of soybeans with Bacillus subtilis natto. Bacillus subtilis are rod-shaped, gram-positive and catalase-positive bacteria. Nattokinase is an enzyme that digests fibrin both directly and indirectly. Indirectly, it activates pro-urokinase and tissue plasminogen activator (t-PA), supporting the fibrinolytic activity of plasmin. These combined actions promote healthy platelet function, circulation and blood flow. In its purified form, the enzyme shows its optimum conditions at 37° C. and a pH of 7. The enzyme activity gradually declines at temperatures beyond 70° C. The enzyme may contribute to the regular healthy function of the heart and cardiovascular system by maintaining proper blood flow, thinning the blood and preventing blood clots. For this reason, nattokinase may hydrolyze fibrin in blood clots.

(5) The inventor confirmed that oral administration of nattokinase produced a mild and frequent enhancement of the fibrinolytic activity in the plasma and the production of tissue plasminogen activator. In a Japanese study, nattokinase capsules had been administered orally to dogs that received artificially induced thrombosis, and lysis of the thrombi was observed by angiography. The enzyme was already active in circulation prior to the clot being induced, so this does not demonstrate the enzymes efficacy to dissolve clots dislodged over a period of days or weeks. Regardless, results obtained from this Japanese study suggest that nattokinase may represent a possible drug for use not only in the treatment of embolism but also in the prevention of the disease. There are no known studies that examine the efficacy of nattokinase as a thrombolytic agent in cats.

(6) Rutin, also called rutoside, quercetin-3-O-rutinoside and sophorin, is the glycoside between the flavonolquercetin and the disaccharide rutinose (α-L-rhamnopyranosyl-(1.fwdarw.6))-β-D-glucopyranose). Rutin is one of the phenolic compounds found in the invasive plant species Carpobrotus edulis and contributes to the antibacterial properties of the plant. Its name comes from the name of Ruta graveolens, a plant that also contains rutin. Rutin is a citrus flavonoid glycoside found in many plants including buckwheat, the leaves and petioles of Rheum species, and asparagus. Tartary buckwheat seeds have been found to contain more rutin (about 0.8-1.7% dry weight) than common buckwheat seeds (0.01% dry weight). Rutin is also found in the fruit of the fava d'anta tree (from Brazil), fruits and flowers of the pagoda tree, fruits and fruit rinds (especially the citrus fruits orange, grapefruit, lemon, and lime) and apple; berries such as mulberry, ash tree fruits, aronia berries and cranberries.

(7) In the fava d'anta tree, the synthesis is done via a rutin synthase activity. Rutin is one of the primary flavonols found in ‘clingstone’ peaches. It is also found in green tea infusions. Rutin inhibits platelet aggregation, as well as decreases capillary permeability, making the blood thinner and improving circulation. Recent studies show rutin could help prevent blood clots so could be used to treat patients at risk of heart attacks and strokes. Rutin may also have a veterinary use in the management of chylothorax in dogs and cats. In other words, rutin may potentially help decrease fluid buildup in the lungs during congestive heart failure. Along with blood clots, congestive heart failure is a common occurrence in cats with hypertrophic cardiomyopathy (HCM).

(8) Since both nattokinase and rutin have demonstrated thrombolytic activity based on data collected in the inventor's private research study, the inventor's private research study investigated combining the two agents to make a more powerful thrombolytic pharmaceutical. However, both agents can be dissolved in stomach acids, rendering certain dosage forms nearly useless as thrombolytic agents. This may explain why the potency of previous oral thrombolytics is decreased in comparison to injected thrombolytic agents. Enteric coatings reduce the active ingredients' rate of digestion in stomach acids, leading to enhanced efficacy of the active ingredient(s).

(9) To ensure the efficacy of these two thrombolytic agents, an enteric coating is appropriate for some orally administered embodiments. The enteric coating allows the nattokinase and rutin bioflavonoid to travel through the acidic environment of the stomach without being dissolved. Nattokinase and rutin can then be absorbed into the bloodstream or circulation via the small intestine. Once absorbed, nattokinase remains active in the bloodstream for approximately 12 hours. In one embodiment, the enteric coating is composed of methacrylic acid copolymer and triethyl citrate. Additional ingredients may include, for example, arabinoglactan, microcrystalline cellulose, maltodextrin, stearic acid, croscarmellose sodium, silicon dioxide. During this clinical trial, participants were not on any other thrombolytic or fibrinolytic (blood thinning) agents.

(10) During the inventor's research study, cats presented signs of ATE including hindlimb paralysis, low or absent pulse in affected limb(s), cold and darkened paw pads from low or no blood supply in affected limb(s). Hardened tissue, raw skin and hair loss on affected limb(s). Cats exhibited pain or numbness in affected limb(s) and some cats showed signs of congestive heart failure in the form of coughing and other symptoms directly related to pleural effusion. These cats were immediately put on diuretics including Furosemide (Lasix) to drain fluid from their lungs. Once the cats were stable, 15 tablets of an embodiment of the enzymatic formula were administered orally in the morning. 12 hours later, 15 tablets of an embodiment of the enzymatic formula were administered orally.

(11) For the embodiments used in the inventor's research study, it was preferred to reduce the likelihood that a subject would chew up a tablet, because the enteric coating had to remain intact for maximum efficacy. Additionally, the inventor's research study indicated that stress on the subject may be reduced by breaking up the treatment dose over the course of a day. For example, with subjects between about 7 pounds and about 21 pounds in bodyweight, dividing the initial dosage of about 1,125 mg/day into two doses, approximately 12 hours apart, appears sufficient to minimize stress on the subject and also maintain an adequately high concentration of effective ingredients to break down the clot.

(12) Of the 46 cats in the inventor's research study, 2 had dislodged clots from their heart and into circulation within an hour to 5 hours of treatment. The rest had dislodged clots from their heart within 1 day to 2 weeks of treatment. In all cases, circulation and mobility returned, with no adverse reactions to the formula embodiment. One cat had not received treatment until 2 weeks after the onset of ATE. This cat regained full circulation to his hindlimbs, but lost 1 digit on his right hindlimb, because necrosis had set into that tissue prior to treatment. After a full 3 week treatment of 15 tablets in the morning and 15 tablets at night, this cat regained function of his affected hindlimbs and was able to walk again. Damaged peripheral tissue had also regenerated during treatment.

(13) The recommended treatment dose for 1 blood clot is approximately 10-30 tablets, and in embodiments about 15 tablets, every 12 hours, and for the earlier of approximately 2 weeks or until full circulation and mobility has returned to affected limbs. If more than one clot is present, the treatment dose may be extended, e.g., to about 3 weeks instead of about 2 weeks. In some embodiments, the daily treatment dose of 30 tablet/day does not need to be increased for the treatment dose. For kittens or cats weighing less than 71bs, 15 tablets/day may be administered to determine whether the lower concentration is adequate for the subject. In some embodiments, for kittens or cats weighing less than 7 lbs, the maintenance dose of 1 tablet in the morning and 1 tablet at night, may be effective and safe. Though it is difficult to determine if more than one clot is present, the treatment may continue past 2 weeks if multiple clots are suspected. Generally, treatment should continue until full mobility, color, and temperature have returned to affected limbs. This may mean the treatment may exceed 4 weeks, though in some embodiments it may be appropriate to lower the dosage to half of the treatment dosage after the third week. In some embodiments, after recovery and full dissolution of the thromboembolism, cats are placed on a maintenance dose of 1 tablet in the morning and 1 tablet at night, 12 hours apart. Generally, the treatment should be maintained as long as the subject is at risk of developing further blood clots. For some subjects, this may be for the life of the subject. In the private research study, the maintenance dose effectively prevented new clot formation in the hearts of cats with hypertrophic cardiomyopathy (HCM). Therefore, the disclosed treatment may be used to prevent blood clot formation in the heart.

(14) In one embodiment, a method for treating thromboembolism in felines may take the form of:

(15) Nattokinase Rutin Thrombolytic Treatment Dose: 2 week treatment. 15 tablets in the morning before meals and 15 tablets in the evening before meals every day until full circulation returns, wherein each tablet has nattokinase at approximately 37.5 mg/750 fibrinolytic units, and rutin bioflavonoid at approximately 50 mg. Each tablet may also include an enteric coating.

(16) 30 tablets per day=1,125 mg/day [of nattokinase, and 1,500 mg/day rutin bioflavonoid]

(17) 30 tablets×14 days=420 tablets for an average treatment of 1 clot

(18) Total of 37.5 mg×420 tablets=15,750 mg [of nattokinase, and 21,000 mg rutin bioflavonoid] for 1 full treatment of 1 clot

(19) Nattokinase-Rutin Maintenance Dose (for prevention of clot formation): 2 tablets per day (1 tablet in the morning and 1 tablet before bedtime) given 12 hours apart

(20) 75 mg/day for daily maintenance dose

(21) Table 1 shows data for all 46 subjects in a private research study. Data includes subject number, age, gender, weight, date of treatment, location of blood clot, length of treatment and if clot formation occurred while subject was on maintenance dose.

(22) TABLE-US-00001 TABLE 1 New Clot Formation Clot While on Subject Age Weight Location of Dislodged Length of Treatment Maintenance Number (years) Breed (lbs) Gender Clot from Heart Treatment Dose (mg) Dose 1 3 Domestic 12 Male Right 10 minutes 20 minutes 825 No Shorthair Forelimb Polydactyl 2 9 Domestic 10 Male Both 3 days 2 days 2,250 No Shorthair Hindlimbs 3 18 Domestic 13.5 Male Both 2 days 5 days 5,625 No Shorthair Hindlimbs 4 8 Domestic 7 Male Both 2 days 4 days 4,500 No Shorthair Hindlimbs 5 10 Domestic 11 Male Both 4 days 1 week 7,875 No Shorthair Hindlimbs 6 17 Domestic 9.4 Male Both 5 days 5 days 5,625 No Shorthair Hindlimbs 7 14 Domestic 9 Male Right 3 days 2 days 2,250 No Shorthair Hindlimb 8 12 Domestic 12 Male Both 1 week 3 days 3,375 No Shorthair Hindlimbs 9 8 Domestic 10 Male Right 2 weeks 6 days 6,750 No Shorthair Hindlimb Tuxedo 10 19 Domestic 12 Male Both 1 day 4 days 4,500 No Shorthair Hindlimbs 11 9 Domestic 13 Male Right 1 day 2 days 2,250 No Shorthair Forelimb 12 8 Domestic 9 Female Both 1 week 10 days 1,875 No Shorthair Hindlimbs 13 12 Domestic 11 Male Both 9 days 2 weeks 15,750 No Shorthair Hindlimbs 14 12 Domestic 9 Male Both 3 days 1 day 1,125 No Shorthair Hindlimbs Tuxedo 15 9 Domestic 8.5 Female Both 1 day 9 days 10,125 No Shorthair Hindlimbs 16 11 Domestic 12 Male Left 2 days 10 days 11,250 No Shorthair Forelimb 17 9 Siamese 12 Male Both 1 week 5 days 5,625 No Himalayan Hindlimbs 18 5 Domestic 8.5 Male Both 2 days 3 days 3,375 No Longhair Hindlimbs 19 3 Domestic 9.4 Male Both 4 days 1 day 1,125 No Shorthair Hindlimbs 20 9 Domestic 8 Male Both 1 day 8 days 9,000 No Shorthair Hindlimbs 21 14 Domestic 21 Male Both 3 days 5 days 5,625 No Longhair Hindlimbs 22 13 Domestic 16 Male Both 5 days 5 days 5,625 No Mediumhair Hindlimbs 23 9 Domestic 15.4 Female Right 3 days 1 week 7,875 No Shorthair Hindlimb 24 16 Domestic 13 Male Partial 3 weeks 3 days 3,375 No Shorthair Hindlimb 25 11 Domestic 14 Female Both 5 days 4 days 4,500 No Shorthair Hindlimbs 26 10 Domestic 9 Female Both 3 days 4 days 4,500 No Shorthair Hindlimbs 27 13 Domestic 11 Male Both 1 day 3 days 3,375 No Shorthair Hindlimbs 28 5 Domestic 14 Male Both 2 days 3 days 3,375 No Shorthair Hindlimbs 29 4 Domestic 15.4 Male Both 5 days 2 weeks 15,750 No Shorthair Hindlimbs 30 10 Domestic 10 Male Both 6 days 1 week 7,875 No Mediumhair Hindlimbs 31 4 Domestic 15 Female Both 1 week 2 weeks 15,750 No Shorthair Hindlimbs 32 2 Domestic 7.5 Female Both 5 days 4 days 4,500 No Shorthair Hindlimbs 33 1 Domestic 11 Male Left 4 days 3 days 3,375 No Shorthair Hindlimb 34 14 Domestic 13 Male Both 4 days 1 week 7,875 No Shorthair Hindlimbs 35 4 Domestic 12 Male Both 1 week 2 weeks 15,750 No Shorthair Hindlimbs 36 14 Maine Coon 8.2 Female Left 4 days 1 week 7,875 No Forelimb 37 10 Domestic 9 Female Right 6 days 2 weeks 15,750 No Shorthair Hindlimb 38 12 Persian 12 Male Both 2 weeks 2 weeks 15,750 No Hindlimbs 39 14 Domestic 11 Male Both 2 weeks 2.5 weeks 19,125 No Shorthair Hindlimbs 40 9 Domestic 16.8 Female Both 3 days 4 days 4,500 No Shorthair Hindlimbs 41 17 Persian 9 Male Both 1 week 1 day 1,125 No Hindlimbs 42 16 Domestic 14 Male Both 4 days 2 weeks 15,750 No Shorthair Hindlimbs 43 8 Domestic 12 Male Left 2 weeks 2 weeks 15,750 No Shorthair Hindlimb 44 3 Domestic 11 Female Both 4 days 1 week 7,875 No Shorthair Hindlimbs 45 1 Domestic 12 Male Right 1 week 9 days 10,125 No Shorthair Hindlimb 46 9 Bengal 14.8 Male Both 8 days 1 week 7,875 No Hindlimbs

(23) Because blood clots are diagnosed or determined to exist through the following symptoms/observations, these factors were used to determine if they no longer existed, as well. Length of treatment was dependent on whether or not signs remained that a clot still existed. These factors or signs of a clot are: decreased mobility, partial paralysis or full paralysis of one or more limbs, decreased temperature in affected limbs, decreased color of paw pad in affected limbs. Unaffected limbs were used as a reference point to compare affected limbs. So, length of treatment was dependent on the length of time it took for the blood clot to be dissolved and these symptoms to no longer appear. With increased temperature, mobility, and the return of color to the cat's affected limb, it could be determined that the clot was in the process of being dissolved or was already dissolved. Stiffness from dehydration and lack of blood flow through affected tissue is also a symptom of a blood clot, so this can also be used to determine if the clot is still present and blocking circulation. Hence, when these symptoms subsided, the treatment dose could be changed to a maintenance dose.

(24) Dosage in the clinical trial was determined in a similar way. The first cat in this study received the treatment only minutes after having a clot dislodged into circulation from his heart. The inventor initially administered 5 tablets, noticed no change, and administered 5 more, noticed no change, but once the inventor reached 15 tablets, she started to see a slight improvement. She could feel the temperature in his affected limbs increase and, after administering 15 more tablets within the next 10 minutes, the inventor could see a full recovery occur. Since the inventor did not want to risk internal bleeding by administering more than 30 tablets, she decided to cut off the treatment at this point. The inventor concluded 30 tablets was sufficient to dissolve one clot safely, and she decided to make this the cut off point, since it had shown to be an effective dose in other cats with various sized clots. The inventor still had no way to measure clot size, but can only assume they vary in size in each case.

(25) Due to the preference for a high concentration of the nattokinase-rutin formula in order to effectively dissolve blood clots, the enzymatic formula is expected to have increased efficacy at high concentrations, e.g., as highly concentrated tablets. In embodiments used for the inventor's research study, each tablet contained about 37.5 mg of nattokinase and 50 mg of rutin, in tablet form having an enteric coating. Length of treatment would be reduced and the concentration would still be considered safe at 225 mg of nattokinase and 300 mg of rutin per tablet. This is based on the previous data where a 1 day treatment to dissolve ATE consisted of 1,125 mg nattokinase and 1,500 mg of rutin. Increasing the concentration of the current formula in tablet, capsule or gelcap form, would decrease the time it takes to dissolve clots and decrease the stress on the cat, owner and/or participating medical staff. It should be appreciated that alternative concentrations of each active ingredient, relative concentrations, and treatment regimens, other than those employed in connection with the clinical trial, may be employed without departing from the principles described herein.

(26) The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the claims of the application rather than by the description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

(27) The present embodiment may be used in dogs, horses, and other animals for clot dissolution or clot prevention. Treatment and maintenance regimens will vary based on the weight of the animal and size of the clot.