Example 1

Initial evaluation of the hemostatic properties of a composition comprising an anion exchanger and a calcium salt was carried out in an in-vivo heparinized porcine spleen circular punch model as described above, using DEAE covalently bound to Sephadex (DEAE-SEPHADEX™) as the anion exchanger. In this experiment, the punch size was 4 mm diameter, 2 mm depth. A compression time of 30 or 60 seconds was used following application. DEAE SEPHADEX™ A-50 was tested at two concentrations. In this experiment, the Post-Application Bleeding Intensity was evaluated qualitatively.

The following compositions (see elaboration in Table 1 above) were evaluated for their hemostatic efficacy:

    • 1. DEAE SEPHADEX™ A-50, prepared as 10% w/v slurry in 20 mM CaCl2 solution, (0.5 ml contains 50 mg DEAE SEPHADEX™ A-50) (30 seconds compression time);
    • 2. DEAE SEPHADEX™ A-50, prepared as 6.6% w/v slurry in 20 mM CaCl2 solution, (0.5 ml contains 33 mg DEAE SEPHADEX™ A-50) (60 seconds compression time);
    • 3. SEPHADEX™ G-75 Superfine, prepared as 10% w/v slurry in 20 mM CaCl2 solution, (0.5 ml contains 50 mg per SEPHADEX™ G-75 Superfine) (60 seconds compression time);
    • 4. SEPHADEX™ G-50 Medium, prepared as 10% w/v slurry in 20 mM CaCl2 solution, (0.5 ml contains 50 mg SEPHADEX™ G-50 Medium) (60 seconds compression time);
    • 5. Commercial gelatin hemostat prepared as a slurry (0.5 ml contains 55 mg gelatin) (60 seconds compression time).

All four SEPHADEX™ samples comprise the same base polymer, cross-linked dextran. Compositions 1-4 were provided as powders, from which slurries were prepared as described in the Table 1 above. A commercial gelatin flowable hemostat was used as control.

It was found that SEPHADEX™ G-50 Medium, SEPHADEX™ G-75 Superfine and commercial gelatin hemostat, failed to stop the bleeding, i.e. no reduction in bleeding intensity was observed (results not shown).

Surprisingly, DEAE SEPHADEX™ A-50 reduced the bleeding at all tested compression times. Following the application of DEAE SEPHADEX™ A-50, the spleen was manually manipulated by folding the organ from both sides. No re-bleeding occurred at either of the tested concentrations and following the two different compression times (results not shown). Since hemostasis only occurred in the matrix supplemented with DEAE groups it was concluded that the hemostatic effect was due the presence of the DEAE groups.

FIG. 1 shows an exemplary result obtained using DEAE SEPHADEX™ A-50 10% (w/v) and commercial gelatin. As shown in the figure, commercial gelatin hemostat failed to stop the bleeding after a compression time of 60 seconds, whereas DEAE SEPHADEX™ A-50 10% (w/v) successfully stopped the bleeding even following a shorter compression time of 30 seconds.

Example 2

In the following Example, the effect on hemostasis of each of the components of a composition comprising an anion exchanger and a calcium salt was evaluated, separately and in combination, using an in-vivo heparinized porcine liver circular punch model, as described above. This experiment identifies which of the components of the composition are required for achieving hemostasis.

The preparation of each composition is described in Table 1 above. Compression time is listed in Table 2 below. In this experiment, the Initial Bleeding Intensity and Post-Application Bleeding Intensity were evaluated according to the 0-5 scale.

The following compositions were evaluated:

    • 1. DEAE SEPHADEX™ A-50, prepared as 8% w/v slurry in 20 mM CaCl2 solution (0.5 ml contains 40 mg DEAE SEPHADEX™ A-50);
    • 2. Commercial gelatin hemostat, prepared as a slurry (0.5 ml contains 55 mg gelatin);
    • 3. Commercial gelatin hemostat with thrombin, prepared as a slurry (0.5 ml contains 55 mg gelatin);
    • 4. SEPHADEX™ G-50 Medium, prepared as 14% w/v slurry in 20 mM CaCl2 solution (0.5 ml contains 70 mg SEPHADEX™ G-50 Medium).
    • 5. DEAE SEPHADEX™ A-50, prepared as 8% w/v slurry in 20 mM NaCl solution (0.5 ml contains 40 mg DEAE SEPHADEX™ A-50);
    • 6. SP SEPHADEX™ C-50, prepared as 8% w/v slurry in 20 mM CaCl2 solution (0.5 ml contains 40 mg SP SEPHADEX™ C-50);
    • 7. QAE SEPHADEX™, prepared as 8% w/v slurry in 20 mM CaCl2 solution (0.5 ml contains 40 mg QAE SEPHADEX™);
    • 8. DEAE SEPHACEL™, prepared as a slurry (100 mg); and
    • 9. TOYOPEARL DEAE-650M™, prepared in powder form (100 mg).

The compression time following the application of each tested composition, and the bleeding intensity results are shown in Table 2. Bleeding Intensity Reduction was calculated by subtracting the Post-Application Bleeding Intensity from the Initial Bleeding Intensity.

TABLE 2
Effect of tested compositions in reduction of
bleeding intensity (liver bleeding model)
Compres-Bleeding Intensity
Calciumsion timeIni-PostReduc-
Tested CompositionSalt(seconds)tialApplicationtion*
DEAE+60505
SEPHADEX ™+30303
A-50+10413
(8% w/v)+0211
*Calculated by subtracting the Post Application Bleeding Intensity from the Initial Bleeding Intensity.

TABLE 3
Effect of tested compositions in reduction of
bleeding intensity (liver bleeding model)
Bleeding Intensity
CalciumIni-PostReduc-
Tested CompositionSalttialApplicationtion*
DEAE SEPHACEL ™+303
(100 g slurry)
TOYOPEARL DEAE-650M ™+404
(100 mg powder)
QAE SEPHADEX ™+431
(8% w/v)
DEAE SEPHADEX ™ A-50−330
(8% w/v)
with 20 mM NaCl
SEPHADEX ™ G50+330
(14% w/v)
SP SEPHADEX ™ C-50+330
(8% w/v)
Commercial gelatin+303
hemostat with thrombin
Commercial−330
gelatin hemostat
*Calculated by subtracting the Post Application Bleeding Intensity from the Initial Bleeding Intensity.

In general, it can be seen, that a composition comprising an anion exchanger, such as DEAE bound to a matrix, together with a calcium salt, lead to complete hemostasis (see Table 2 for DEAE SEPHADEX™ A-50, DEAE SEPHACEL™, and TOYOPEARL DEAE-650M™, all containing a calcium salt). These compositions substantially lead to complete hemostasis regardless of the specific matrix used. For example, matrices such as SEPHADEX™, SEPHACEL™ and TOYOPEARL™ (dextran, cellulose and hydroxylated methacrylic polymer, respectively) had a similar effect in reducing the bleeding intensity.

More particularly, DEAE SEPHADEX™ A-50 8% w/v in CaCl2 was able cease bleeding after 60, and 30 seconds of compression. The results also showed DEAE SEPHADEX™ A-50 (8% w/v) application could be used, without compression, to reduce bleeding intensity (Table 2).

The hemostatic capabilities of a composition comprising DEAE SEPHADEX™ A-50 and a calcium salt exhibited similar efficacy to that of commercial gelatin hemostat with thrombin, when using the same compression time (30 seconds), and even with only 10 seconds of compression. However, the hemostatic capability of a hemostat based on an anion exchanger comprising DEAE bound to a matrix, and a calcium salt, was substantially superior to that of commercial gelatin hemostat in the absence of a biologically active component, such as thrombin.

As shown in Table 3, a composition comprising DEAE SEPHADEX™ prepared with NaCl, and lacking a calcium salt produced no reduction in bleeding intensity, such that it can be concluded that the sample was not effective in stopping the bleeding.

When evaluating the impact of the ion exchange group on the hemostatic capability, it was shown that SP SEPHADEX™ containing an anionic group, sulfopropyl (SP) with a calcium salt, did not reduce the bleeding intensity. In other words, a material with a negative (SP) group, instead of a positive (DEAE) group was not effective as a hemostat.

It was further shown that a quaternary aminoethyl, QAE SEPHADEX™ with a calcium salt was able to reduce bleeding intensity.

It was also shown, as in Example 1, that the matrix alone, in the absence of a functional group (SEPHADEX™ G-50 with a calcium salt but without DEAE groups) had no hemostatic efficacy.

The results show that use of a composition comprising DEAE groups bound to a matrix in presence of a calcium salt effectively achieved hemostasis.

The results also showed that compression time of 30 and 60 seconds following DEAE SEPHADEX™ A-50 (8% w/v) application resulted in complete hemostasis and therefore the TTH was defined as 30 seconds.

It was thus shown that an anion exchanger such as DEAE bound to a matrix together with a calcium salt provided complete hemostasis. This result was obtained regardless of the matrix used. The results are comparable to those obtained when using a commercial hemostat such as gelatin with thrombin.

It was found that QAE SEPHADEX™ together with a calcium salt reduced bleeding.

A composition devoid of a calcium salt and/or a matrix had no effect on the bleeding intensity.

These results suggest that a composition comprising an anion exchanger and a calcium salt is effective as a hemostat.

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