GB1563490A - Hemostatic formulation - Google Patents

Abstract

Haemostatic preparation containing at least two of the following components: phytonadione, adrenochrome monosemicarbazone and ascorbic acid or its salts in combination with an excipient. The preparation can be administered orally or parenterally and shows an unexpected synergistic activity in the treatment of haemorrhages in comparison to the action of any individual component.

Description

(54) HEMOSTATIC FORMULATION (71) We, SHIRAIMATSU SHIN YAKU COMPANY LTD., a Japanese Body Corporate, of 37-1, Aza-Inaba, O'aza Ukawa, Minakuchi-cho, Koga County, Shiga Prefecture, Japan, do hereby declare the invention, for which we pray that a patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement:- The present invention relates to hemostatic formulations.

Phytonadione, adrenochrome monosemi- carbazone and ascorbic acid or a salt thereof are each known hemostatics. However, no description or investigation has been reported with respect to the administration of a combination of two or three of these hemostatics for hemostatic purposes.

Accordingly, it is of particular interest to develop combined use of two or three of these hemostatics whch would be effective over an expanded area of human and animal syndromes accompanied by bleeding.

We have discovered that the formulations of these known hemostatics, when administered through an oral or parenteral route, unexpectedly produce a synergistic effectiveness in the treatment of diseases accompanied by bleeding, with a significant difference in effectiveness being shown in comparison with that which each active component produces by itself.

The present invention accordingly provides a hemostatic formulation which comprises at least two of (a) phytonadione, (b) adrenochrome monosemicarbazone, and (c) ascorbic acid and/or a salt thereof.

Phytonadione is the name adopted by the U.S. Pharmacopoeia and is another name for Vitamin K1, hereinafter referred to as K1 throughout the present specification. Adrenochrome monosemicarbazone has the formula

and is hereinafter referred to as ACS, and ascorbic acid is Vitamin C and is hereinafter referred to as AA.

The expression "a salt thereof" utilized throughout the present specification denotes any pharmaceutically or veterinary acceptable (non-toxic) salt of AA, these salts being well known to those skilled in the art and encompasses alkali metal (e.g. sodium) salts.

K1 is known to be a blood coagulant and to play a necessary part in synthesizing, in human liver cells, Factor V, Factor IX, Factor X and prothrombin, which are necessarily required for coagulation of blood. Average daily doses of from 10 mg to 50 mg are taken by adult humans under treatment.

ACS is known to have effectiveness for vasoconstriction, for enhancing resistibility of capillaries and for inhibiting elevation of capillary permeability, and is said to develop its hematostatic function without exhibiting any influence on clotting time or contractile force of a curor of blood platelets. Average daily doses of from 6 mg to 15 mg are taken by adult humans under treatment.

AA is known to fortify the walls of blood vessels, promote production of blood platelets and activate the function of thrombin, and is said to play a part in preventing bleeding or arresting hemorrhage. Average daily oral doses of from 50 mg to 500 mg are taken by adult humans under treatment.

In the preferred formulations of the present invention Kl when present, is present in an amount of about 1 part by weight, ACS, when present, is present in an amount of from 1.7 to 2.3 parts, and AA and/or a salt thereof, when present, is present in a total amount of from 17 to 23 parts by weight, the latter amount being calculated on the basis of AA itself. All the parts are based on the total weight of the formulation.

The formulations of the present invention can be administered in unit dosage forms.

Preferred unit dosage forms contain 5 mg of K1, from 8.5 mg to 11.5 mg, more preferably 10 mg, of ACS and from 85 mg to 115 mg, more preferably 100 mg, of AA or an equiva lent amount (calculated on the basis of AA itself) of a salt thereof.

The formulations of this invention may be formulated for oral administration in the form of powders, tablets, troches, solutions, syrups or capsules, for rectal administration in the form of suppositories, and for parenteral administration in the form of injectable fluids with any of the conventional and pharmaceutically or veterinarily acceptable (nontoxic) diluents, carriers, or excipients. Thus, in the formulation of solid preparations, the active components may be combined predominantly with solid and/or tabletting adjuvants such as lactose, starch, calcium carbonate or magnesium stearate. In a similar manner, solutions and injections may be formulated with any or a combination of solubilizing agents.

Whilst the formulations may be in the form of mixtures of the active components, the active components may also be in a non-mixed form e.g. in a capsule having two or three compartments each containing an active component.

The active components may also be taken separately and may be presented to the patient in an easily dispensed form such as on cards carrying measured amounts, e.g. tablets, of the two or three active components arranged in a particular order, and together with instructions, said instructions and arrangement being such as to provide guidance in relation to and to facilitate the taking of said measured amounts such that at least two of the active components are taken to inhibit bleeding.

It should be understood that the amounts indicated above for each component take into account the allowable range of deviation which is usually recognizable from a medical point of view.

Results of the pharmacological tests conducted on the formulations of this invention will now be given, in comparison with controls, with reference to the accompanying drawings in which: Fig. 1 is a chart showing the effect of formulations of this invention on bleeding time in CCl4-treated mice.

Fig. 2 is a chart showing the effect of formulations of this invention on bleeding time in dicumarol-treated mice.

Fig. 3 is a chart showing the effect of formulations of this invention on pulmomary bleeding in mice under negative pressure.

I. Experimental Procedure.

K1 was utilized in a solubilizing form with a non-toxic surface active agent such as HCO 60. ACS was utilized in a suspension form of 2% by weight gum arabic, and AA was utilized in an aqueous form. They were admixed with each other just before administration to animals.

The test animals were ICR-line and SDline rats, and they were given water and solid feed ad libitum for a week before commenc ing the experiments, under the conditions of 22 t 20C and 65% humidity; those animals which seemed systemically sound were selected for the experiments.

II. Experimental Results.

(1) Time of Bleeeding from Tail Vein of CCl4-injected Mice: Each group consisting of 10 ICR-line mice, each weighing 20 + 2 g, was separately ad- ministered through an oral route with (a) Kl at the rate of 5 mg/Kg of animal weight, hereinafter referred to as Group K1, (b) ACS at the rate of 10 mg/Kg of animal weight, hereinafter referred to as Group ACS, (c) AA at the rate of 100 mg/Kg of animal weight, hereinafter referred to as Group AA, (d) a combination of 5 mg of K1 and 10 mg of ACS, referred to as Group Kl + ACS, (e) a combination of 5 mg of K1 and 100 mg of AA, referred to as Group Kl + AA, (f) a combination of 10 mg of ACS and 100 mg of AA, referred to as Group ACS + AA, or (g) a combination of 5 mg of Kl, 10 mg of ACS and 100 mg of AA, referred to as Group K1 + ACS + AA.

Each test mouse was subcutaneously injected with CC14 at the rate of 0.05 ml per Kg of animal weight 2 hours after the administration of each test medicament Thus treated mice were fixed in fixing boxes, and 48 hours after the CC14 injection, the right-side vein at a point 3 cm in length frost the end of their tail was cut to a certain depth by means of a 1/2 mm injection needle under a non anesthetic condition. The time of bleeding was determined by successively applying pieces of filter paper to the wound at intervals of 5 seconds until no stain of blood was found on the filter paper.

The results of this bleeding test are illus trated in graphic form in Fig. 1 of the draw ings, wherein symbol A represents a group -(control group) to which neither CCI4 nor a medicament was administered, symbol B repre sents a group which was subcutaneously in jected only with CCl4 at the rate of 0.05 ml per Kg of animal weight, and symbols C, D, E, F, G, H and I represent Group Kl, Group ACS, Group AA, Group K1 + ACS, Group K1 + AA, Group ACS + AA and Group K1 + ACS + AA, respectively. P represents the level of difference, and the mark ** repre sents P to be less than 0.01. From Fig. 1, it is apparent that Groups Kl, ACS and AA show reduced bleeding time in comparison with that of the symbol B group; however, no significant difference is found. On the other hand, Groups K1 + ACS, Kit + AA, ACS + AA and K1 + ACS + AA have a significant reduction in bleeding time (P < 0. 01) as compared to the symbol B group, and still further, Group K1 + ACS + AA shows a significant reduction in the bleeding time (P < 0.05) as compared to those of each single medication group.

(2) Action against Kallikrein: Male rats of SD-line weighing 200 + 10 g were divided into the groups indicated in Table 1, and each rat of each group (except the control group) was separately administered with the test medicament in the same manner as explained in the foregoing Experiment (1).

Two hours after administration, each rat of each group was intravenously injected with 0.5% Evan's blue solution at the rate of 5ml/ Kg of animal weight, immediately after which each rat was intracutaneously injected, in its abdomen, with 0.1 ml of Kallikrein solution (concentration: 1 unit/ml). Two hours after the Kallikrein injection, each rat was venesected to death and its blue-dyed skin was flayed and extended on a glass plate, after which the area of the blue-dyed area was determined.

Results are shown in Table 1.

TABLE 1

Change of Dose Blue-dyed Capillary Group (mg/Kg, per os) No. of Rats Area (mm2) Permeability (So) Control MCO-60, 3mg/Kg 6 146.0+ 27.9 9 K, 5 6 119.0 + 26.1 18.3 ACS 10 6 107.5 + 14.9* 26.4 AA 100 6 117.0 +19.1 19.7 K, + ACS 5+10 6 103.0 +9.9 29.5 K1+AA 5+100 6 61.5 i 13.4** 57.9 ACS+AA 10+100 6 82.0+ 25.4** 43.8 K1+ACS + AA 5+10+100 6 40.6 * 9.5** 72.2 * : P < 0.05, ** : P < 0.01 (3) Time of Bleeding in Dicumaroladministered Mice: Male mice of ICR-line were divided into groups and administered the test medicaments in the same manner as explained in Experiment (1). Two hours after the administration, dicumarol was orally administered at the rate of 50 mg/Kg of animal weight to each mouse 24 hours after the dicumarol administration, bleeding times were determined in the same manner as explained in Experiment (1).

Results of the test are shown in Fig. 2 of the drawings, in which symbols from A to P are defined in the same manner as indicated for Fig. 1, with the exception that symbol B represents the group orally administered with dicumarol at the rate of 50 mg/Kg of animal weight, and the mark * represents P < 0.ops.

It is apparent from Fig. 2 that every group which was medicated by a single test medicament shows less bleeding time, with a significant difference, than that of the symbol B group; still further, every combined medication group, especially Group K1 + ACS + AA (P < 0.01), shows a further reduced bleeding time as compared to those of the single medication groups.

(4) Action against Pulmonary Bleeding in Mice under Negative Pressure: Male mice of ICR-line, each weighing 20 + 2 g, were divided into groups consisting of 10 mice each, and the mice were administered with the test medicaments in the manner explained in Experiment (1). Two hours after administration, according to the procedure of G. J. Majuski et al each mouse was put into a wide-mouthed bottle, and the atmospheric pressure was rapidly reduced to 65 to 70 mmHg. After one minute, the mouse was taken out from the bottle, its cerebrum was dissected, and its lung was taken out. Then, a grade of bleeding from the lung was determined on the basis of the standard that an extracted lung which floated in 99% alcohol was assigned a mark of 0 (zero), one which floated in 70% alcohol was assigned a mark of 1, one which floated in 50% alcohol was assigned a mark of 2, one which floated in 30% alcohol was assigned a mark of 3, one which floated in water was assigned a mark of 4, and one which sunk in water was assigned a mark of 5. The mean value of marks for each group was calculated from the total of the marks for the mice within each group.

Results of the test are illustrated in Fig. 3 of the drawings, in which every symbol represents the same as those indicated in Figs. 1 and 2. From Fig. 3, it can be seen that the control group had a mean mark value of 4.4 + 0.3; bleeding was observed to spread over the entire lung surface. On the other hand, every single medication group (symbols C, D and E) showed inhibition of bleeding in degrees similar to each other, and every combined medication group (symbols F, G, H and I), especially Group K1 + ACS + AA (P < 0.01), showed an inhibition of bleeding with significant difference.

Acute Toxicity: LDso (mg/Kg of animal weight) obtained when each test medicament was administered per os to mice and rats is shown in Tables 2 and 3. TABLE 2: LD50 in Mice

Medicament Male Female Kl > 83. 333 > 83.333 ACS > 35.832 > 35.832 AA 9.030 7.990 K1+ACS > 31.104 > 31.104 K1+AA 8.200 7. 480 ACS + AA 9.600 > 8.500 K, + ACS + AA 9. 300 9.350 TABLE 3: LD50 in Rats

Medicament Male Female Kl > 33.487 > 33.487 ACS > 17.280 > 17.280 AA > 5.787 > 5.787 Kl + ACS > 18.000 > 18.000 K, + AA > 6.076 8.000 ACS + AA > 7.638 > 7.638 K, + ACS + AA > 7.985 > 6.654 Based on the above-indicated experimental results, it is apparent that the compositions of this invention unexpectedly exhibit a significant difference in effectiveness, in comparison with any one of the three active components when employed individually, for the prevention and treatment of human and animal syndromes accompanied by bleeding, for example, in internal treatment, bleeding from a lung, gastroenteric tract or kidney, bleeding caused by hypertension, and purpura; in surgical treatment, bleeding during an operation; in gynecological treatment, bleeding from the uterus, and menorrhagia; in otorhinolaryngological treatment, nasal bleeding, and bleeding from the throat; in ophthalmological treatment, bleeding from the retina and vitreous body, and bleeding caused by purpura,; and bleeding from the urinary tract.

The effective dose of the formulations of this invention will vary, of course, depending upon the type and seriousness of the disease, sex distinction and age of the patient. However, in the case of a tablet which contains 5 mg of K1, 10 mg of ACS and 100 mg of AA, as one of the unit dosage forms of the compositions of this invention, it is preferable to administer from 1 to 3 tablets each of 1 to 3 times per day to an adult human bleeding from its kidney; from 1 to 2 tablets each of 1 to 3 times per day to an adult human suffering from purpura; and from 1 to 4 tablets each of 2 to 3 times per day to an adult human bleeding from its vitreous body.

The invention will further be described in reference to the following examples, which are given for the purpose of illustration, and are not meant to limit the invention.

Example 1: Powder formulation.

K1 10 Kg ACS 17 Kg AA 200 Kg Lactose 100 Kg Starch 170 Kg These components were thoroughly mixed and the mixture was passed through a suitable sieve.

Example 2: Tablet formulation.

Kl 15 Kg ACS 30 Kg AA 300 Kg Lactose 240 Kg Starch 240 Kg Gum arabic powders 15 Kg Talcum 15 Kg Gelatin 9 Kg White sugar 270 Kg Precipitated calcium carbonate 45 Kg These components were mixed, granulated and compressed into tablets, which were then sugar coated, by conventional procedures known in the art.

Example 3: Liquid formulation.

K1 2 Kg ACS 3.6 Kg AA 42 Kg Polysolvate-80 4 Kg Simple syrup 240 Kg Sterilized water ad. 800 ml Example 4: Troche formulation.

Kl 15 Kg ACS 35 Kg AA 280 Kg White sugar 1060 Kg Starch 13 Kg Sterilized water 59 Kg These components were mixed, passed through a sieve, granulated and compressed into troches by conventional procedures in the art.

Example 5: Injection formulation.

K1 5 Kg ACS 13 Kg AA 30 Kg HCO-60 25 Kg Sodium benzoate 40 Kg Propylene glycol 30 Kg Phosphate buffer solution ad. 1000 liter Thus prepared solution was formulated in a form suitable for injection by conventional procedures in the art.

Example 6: Capsule formulation.

K1 10 Kg ACS 20 Kg AA 210 Kg Lactose 132 Kg Starch 334 Kg Hydroxypropyl cellulose 3.5 Kg These components were formulated into capsules by conventional procedures in the art.

Claims (16)

WHAT WE CLAIM IS:

1. A hemostatic formulation which com prises at least two of (a) phytonadione, (b) adrenochrome monosemicarbazone, and (c) ascorbic acid and/or a salt thereof.

2. A formulation as claimed in daim 1 which further comprises a pharmaceutically or veterinarily acceptable diluent, carrier, or excipient.

3. A formulation as claimed in claim 1 or claim 2 which comprises about 1 part by weight of phytonadione and from 1.7 to 2.3 parts by weight of adrenochrome monosemicarbazone, by total weight of the formulation.

4. A formulation as claimed in claim 1 or claim 2 which comprises from 1.7 to 2.3 parts by weight of adrenochrome monosemicarbazone and from 17 to 23 parts by weight of ascorbic acid, by total weight of the formulation.

5. A formulation as claimed in claim 1 or claim 2 which comprises about 1 part by weight of phytonadione and from 17 to 23 parts by weight of ascorbic acid, by total weight of the formulation.

6. A formulation as claimed in claim 1 or claim 2 which comprises about 1 part by weight of phytonadione, from 1.7 to 2.3 parts by weight of adrenochrome monosemicarbazone, and from 17 to 23 parts by weight of ascorbic acid, by total weight of the formulation.

7. A formulation as claimed in any one of the preceding claims and in the form of a powder, tablet, troche, solution, syrup, capsule, suppository, or an injectable fluid.

8. A formulation as claimed in any one of claims 1 to 7 in unit dosage form.

9. A hemostatic composition in unit dosage form, which comprises about 5 mg of phytonadione, about 10 mg of adrenochrome mono semicarbazone and about 100 mg of ascorbic acid, together with a pharmaceutically or veterinarily acceptable carrier.

10. A composition as claimed in claim 9 in the form of a powder, tablet, liquid or troche.

11. A pharmaceutically acceptable capsule containing the composition as claimed in claim 9.

12. A composition as claimed in claim 9 in a form suitable for therapeutic injection.

13. A formulation as claimed in claim 1 substantially as hereinbefore described in any one of the Examples.

14. A method of inhibiting bleeding in a non-human animal which comprises administering to the non-human animal an effective amount of a formulation as claimed in any one of claims 1 to 8, and 13 or a composition as claimed in any one of claims 9, 10 and 12.

15. A card carrying measured amounts of at least two of (a) phytonadione, (b) adrenochrome monosemicarbazone, and (c) ascorbic acid and/or a salt thereof arranged in a particular order, and together with instructions, said instructions and arrangement being such as to provide guidance in relation to and to facilitate the taking of said measured amounts such that at least two of (a), (b) and (c) are taken to inhibit bleeding.

16. A card as claimed in claim 15, wherein said measured amounts are in the form of tablets.