Haemostatic composition comprising crystalline polyphosphate

Abstract

The present invention relates to the use of polyphosphate in crystalline form for treatment of wounds, especially bleeding wounds, wherein the anion of the polyphosphate has a (numerical) average of at least 4 phosphorus atoms per polyphosphate anion. The invention additionally relates to a composition suitable for treatment of wounds, especially bleeding wounds, comprising an inventive polyphosphate and a carrier material. The invention further provides a method suitable for production of the inventive composition, which comprises the introduction of the polyphosphate and optionally further haemostatic agents into the carrier material, or into a component or into a precursor of the carrier material.

Claims

1. A method for treating bleeding wounds comprising applying a composition that comprises a polyphosphate in crystalline form, wherein an anion of the polyphosphate has on average (number average) at least 4 phosphorus atoms per polyphosphate anion, and wherein the crystalline polyphosphate is substantially water-insoluble.

2. The method of claim 1, wherein the polyphosphate has a degree of crystallinity, determined by X-ray diffractometry, of at least 90%.

3. The method of claim 2, wherein the anion of the polyphosphate is substantially linear.

4. The method of claim 1, wherein the polyphosphate anion has on average from 8 to 500 phosphorus atoms.

5. The method of claim 1, wherein cations of the polyphosphate are independently selected from Na.sup.+, K.sup.+, NH.sub.4.sup.+, Ca.sup.2+, Mg.sup.2+, Zn.sup.2+, Sn.sup.2+, Fe.sup.3+ and Al.sup.3+.

6. The method of claim 5, wherein the cations of the polyphosphate are selected from Na.sup.+, K.sup.+, NH.sub.4.sup.+ and Ca.sup.2+.

7. The method of claim 1, wherein particles of the crystalline polyphosphate have a weight-average particle size within the range of 1 m to 1 mm.

8. The method of claim 2, wherein the degree of crystallinity is at least 95%.

9. The method of claim 1, wherein the polyphosphate anion has an average 12 to 300 phosphorus atoms.

10. The method of claim 1, wherein the polyphosphate anion has an average 15 to 150 phosphorus atoms.

11. The method of claim 7, wherein the particles of the crystalline polyphosphate have a weight-average particle size within the range of 1 mm to 0.5 mm.

12. The method of claim 6, wherein the cations of the polyphosphate are selected from Na.sup.+, K.sup.+ and NH.sub.4.sup.+.

13. The method of claim 1, wherein the polyphosphate has a degree of crystallinity of at least 95% as determined by X-ray diffractometry, wherein the polyphosphate anion has on average from 15 to 150 phosphorus atoms; and wherein the cations of the polyphosphate are selected from Na.sup.+, K.sup.+, NH.sub.4.sup.+ and Ca.sup.2+.

14. The method of claim 13, wherein the cations of the polyphosphate are selected from Na.sup.+, K.sup.+ and NH.sub.4.sup.+.

Description

FIGURES

(1) FIGS. 1 to 4 show graphs depicting the thrombin concentrations in blood plasma, as determined by the herein-described assay for real-time measurement of thrombin formation, after addition of polyphosphate in relation to time (so-called thrombograms). The abbreviation w/o means in each case that no polyphosphate was added. The thrombograms measured without polyphosphate (i.e., the w/o lines) are in each case on the x-axis of the graph.

(2) FIG. 1: Thrombogram determined in the presence of 0, 1, 5, 10 and 50 g/ml crystalline ammonium polyphosphate 1 (chain length K: 38).

(3) FIG. 2: Thrombogram determined in the presence of 0, 1, 5, 10 and 50 g/ml crystalline potassium polyphosphate (chain length K: 31).

(4) FIG. 3: Thrombogram determined in the presence of 0, 1, 5, 10 and 50 g/ml crystalline sodium polyphosphate (chain length K: 44).

(5) FIG. 4: Thrombogram determined in the presence of 0, 1, 5, 10 and 50 g/ml amorphous, water-soluble sodium polyphosphate 3 (chain length K: approx. 27).

EXAMPLES

(6) Determination of Solubility:

(7) The polyphosphates to be investigated were each weighed out in a 250 ml Erlenmeyer flask (5 g0.01 g) and admixed with distilled water (50 ml, 20-22 C.). The mixture thus obtained was shaken at 200 revolutions per minute (rpm, rotary movement) for 20 min. Thereafter, the mixture was transferred to a centrifuge tube and centrifuged at 5000 rpm for 20 min. 10 ml of the supernatant solution were pipetted into a predried aluminum bowl (diameter of 60 mm, height of 15 mm), which was then dried at 105 C. in a forced-air oven for 150 min. The solubility in g per 100 g of water was then obtained from the formula TR10, where TR represents the determined dry residue in grams.

(8) Determination of the Chain Length of Polyphosphates:

(9) For the polyphosphates used, a .sup.31P solid-state NMR spectrum was recorded and the integrals of the signals of the centrally located phosphate groups (a) as well as the integrals of the signals of the terminal phosphate groups (b) were determined. The number-average chain length K could then be determined in each case with the aid of the formula K=2(a/b+1). For some of the water-soluble polyphosphates serving as comparison, the number-average chain length K was accordingly also determined in solution (D.sub.2O) by means of .sup.31P NMR. The values obtained for the chain lengths K of the investigated polyphosphates are listed in Table 1.

(10) Determination of the Degree of Crystallinity of Polyphosphates

(11) The degrees of crystallinity of the investigated polyphosphates were determined on the basis of their X-ray powder diffractograms. This involved using the full widths at half maximum of the recorded signals of the diffractograms in a manner known per se to quantify the crystallinity.

(12) The following polyphosphates were used: (1) sodium triphosphate: sodium tripolyphosphate 1331 (BK Giulini); (2) sodium oligophosphate: sodium polyphosphate P60 (BK Giulini); (3) sodium polyphosphate A: sodium polyphosphate P64 (BK Giulini); (4) sodium polyphosphate B: sodium polyphosphate P68 hexametaphosphate (BK Giulini); (5) sodium polyphosphate C: sodium polyphosphate P70 (BK Giulini); (6) sodium polyphosphate D: acidic sodium polyphosphate P70.5 (BK Giulini); (7) sodium polyphosphate E: Maddrell's salt (BK Giulini); (8) ammonium polyphosphate A: Phos-Chek P30 (BK Giulini); (9) ammonium polyphosphate B: Phos-Chek P42 (BK Giulini); (10) calcium polyphosphate: laboratory product from BK Giulini; (11) potassium polyphosphate: potassium metaphosphate (BK Giulini); (12) sodium trimetaphosphate: sodium trimetaphosphate (BK Giulini); (13) calcium hydrogen phosphate: DCP-D, dicalcium phosphate dihydrate (BK Giulini);

(13) Assay for Measuring Thrombin Formation in Real-Time

(14) This method was used to determine the formation of thrombin in blood plasma, triggered in each case by the polyphosphates, in relation to time and to the amounts of polyphosphate used. With the aid of the so-called thrombograms thus obtained, it is possible to draw conclusions concerning the response rate and the extent of thrombin formation. The measurements were carried out in accordance with the instructions from Thrombinoscope BV (manufacturer of the below-mentioned fluorogenic substrate) in a fluorometer (Fluoroscan Ascent, Thermo Scientific) equipped with a dispenser (see F. Mller et al., Cell 2009, 139, 1143-1156 and Supplemental Data). In said measurements, the thrombin formation occurred in a total volume of 120 l which contained low-thrombocyte plasma (80 l), tissue factor (0.5 pM or 0.0 pM), factor VIIa inhibited at the active site, phospholipid (4 M) consisting of phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the ratio of 20 mol %/20 mol %/60 mol %, Ca.sup.2+ (20 mM) and the fluorogenic substrate Z-Gly-Gly-Arg-7-amino-4-methylcoumarin (2.5 mM, ZGGR-AMC, Thrombinoscope BV). Trypsin inhibitor from maize, monoclonal antibodies against human tissue factor pathway inhibitor (TFPI) or thrombin activatable fibrinolysis inhibitor (TAFI) and carboxypeptidase inhibitor (all from Sigma-Aldrich GmbH) were added up to final concentrations of 40 g/ml, 1 g/ml or 10 g/ml. Inner filter effects and the substrate consumption were corrected by calibrating each measurement against a fluorescence curve which was determined by means of a mixture of the plasma (80 l) with a fixed amount of thrombin/2-macroglobulin complex (Thrombin Calibrator, Thrombinoscope BV). All measurements were each left to run for 60 min and carried out in duplicate. The values obtained relating to thrombin formation were calibrated using the software from Thrombinoscope (version 3.0.0.29). The data determined for ammonium polyphosphate 1 (chain length K: 38), potassium polyphosphate (chain length K: 31), sodium polyphosphate (chain length K: 44) and water-soluble sodium polyphosphate 3 (chain length K: approx. 27) as comparative example are summarized in the thrombograms in FIGS. 1 to 4.

(15) Characteristic of the thrombograms are, in particular, the delay time and the endogenous thrombin potential (ETP). The delay time is the period of time from time 0 up to the start of the explosive rise in thrombin concentration. ETP refers to the integral of the measured curve of the thrombin concentration. The delay time corresponds in a first approximation to the time until onset of blood coagulation, whereas the ETP is a good indicator of the extent of blood coagulation (cf. Hemker et al., Thromb. Haemost. 2006, 96 (55), 553-561).

(16) The comparison of the thrombograms for the crystalline polyphosphates according to the invention with the thrombogram of the water-soluble sodium polyphosphate 3 shows that the polyphosphates according to the invention have a lag time that is about 3 to 5 times shorter and additionally have ETPs that are many times larger. It can be concluded therefrom that, with respect to the water-soluble sodium polyphosphate 3, the water-insoluble, crystalline polyphosphates of the invention not only result in a drastically shortened period of time until the onset of blood coagulation, but also lead to an enormous rise in thrombin concentration and thus to a very much more intense blood coagulation.

(17) Table 1 below characterizes the investigated phosphates in terms of their water solubility, their chain length, their solid structure and their hemostatic action. Here, water-soluble refers to phosphates having a water solubility of at least 1 g per 100 g of water, as determined by the above-described technique. Here, hemostatic refers to phosphates where the thrombograms, as determined by the above-described technique, have a maximum thrombin concentration of at least 50 nM. As can be clearly read off from Table 1, all the crystalline, water-insoluble polyphosphates according to the invention are notable for a hemostatic action, whereas amorphous, water-soluble polyphosphates and also crystalline mono-, tri- and metaphosphates are not hemostatic.

(18) TABLE-US-00001 TABLE 1 Hemostatic action of the polyphosphates according to the invention as per Examples 1 to 5 and of the phosphates or polyphosphates not in accordance with the invention as per Examples V1 to V8. Chain length K as per .sup.31P NMR in: Phosphates solid Water- Example investigated Structure phase solution soluble Hemostatic V1 Sodium Crystalline 3 3 Yes No triphosphate V2 Sodium oligo- Amorphous 5 Yes No phosphate V3 Sodium poly- Amorphous 7 Yes No phosphate A V4 Sodium poly- Amorphous 15 Yes No phosphate B V5 Sodium poly- Amorphous Approx. 31 99% No phosphate C 27 V6 Sodium poly- Amorphous 98% No phosphate D 1 Sodium poly- Crystalline 44 No Yes phosphate E 2 Ammonium poly- Crystalline 38 No Yes phosphate A 3 Ammonium poly- Crystalline 110 No Yes phosphate B 4 Calcium poly- Crystalline 16 No Yes phosphate 5 Potassium poly- Crystalline 31 No Yes phosphate V7 Sodium trimeta- Crystalline Three- Yes No phosphate membered ring V8 Calcium Crystalline 1 No No hydrogen phosphate