BIOLOGICAL GLUE AND USE THEREOF AS A MEDICAMENT

Abstract

Disclosed is a thrombin-free, liquid biological glue for therapeutic use, including fibrinogen and factor VIIa. The ratio of fibrinogen concentration to FVIIa concentration is 20000:1 to 1000:1, with the concentrations being expressed in weight per volume. The fibrinogen concentration is lower than 60 mg/ml. Also disclosed are a kit for preparing such a biological glue, a method to prepare the glue, and a medicament.

Claims

1-26. (canceled)

27. Thrombin-free, liquid biological glue for therapeutic use, comprising fibrinogen and factor VIIa, wherein the ratio of fibrinogen concentration to FVIIa concentration is 20000:1 to 1000:1, with the concentrations being expressed in weight per volume, and wherein the fibrinogen concentration is lower than 60 mg/ml.

28. The biological glue according to claim 27, having a fibrinogen content of 0.1 mg to 45 mg per ml of biological glue.

29. The biological glue according to claim 27, having a FVIIa content of 0.1 μg to 10 μg per ml of biological glue.

30. The biological glue according to claim 27, comprising a source of calcium ions.

31. The biological glue according to claim 30, comprising 2 μmoles to 30 μmoles of the calcium ion source per ml of biological glue.

32. The biological glue according to claim 27, further comprising at least one gelling agent.

33. A therapeutic method comprising administering a thrombin-free, liquid biological glue comprising fibrinogen, factor VIIa and at least one gelling agent.

34. The therapeutic method according to claim 33, wherein the biological glue has a fibrinogen content lower than 60 mg/ml.

35. The therapeutic method according to claim 33, wherein the biological glue has a FVIIa content of 0.1 μg to 10 μg per ml of biological glue.

36. The therapeutic method according to claim 33 wherein the biological glue comprises a source of calcium ions.

37. The biological glue according to claim 32, wherein said at least one gelling agent is a cellulose derivative.

38. The biological glue according to claim 32, wherein said at least one gelling agent derived from cellulose is hydroxypropyl cellulose.

39. The biological glue according to claim 32, wherein said at least one gelling agent is contained in a proportion of 0.01 to 10% by weight/volume (i.e. 0.01 to 10 gram(s)/100 ml of solution).

40. The biological glue according to claim 32, wherein said at least one gelling agent has a molecular weight higher than 60 kDa.

41. The biological glue according to claim 27, further comprising factor XIII.

42. Kit for preparing the biological glue according to claim 27, comprising packaging means including a lyophilised fibrinogen factor pack, lyophilised factor FVIIa pack, an aqueous solvent and optionally a gelling agent(s) pack in powder form and calcium ion source pack in powder form.

43. A method to prepare a biological glue such as defined claim 27 comprising: providing a kit for preparing the biological glue, the kit comprising packaging means including a lyophilised fibrinogen factor pack, lyophilised factor FVIIa pack, an aqueous solvent and optionally a gelling agent(s) pack in powder form and calcium ion source pack in powder form; and reconstituting the components of the kit in a biologically compatible aqueous solvent.

44. A medicament comprising the biological glue such as defined according to claim 27.

45. A method to stimulate return to haemostasis and/or healing of lesioned biological tissues comprising administering the biological glue according to claim 27.

46. A method to stimulate return to haemostasis and/or healing of lesioned biological tissues comprising administering the biological glue according to claim 27 which is in contact with a fabric or membrane (biocompatible and/or biodegradable).

Description

FIGURES

[0091] FIG. 1 shows the influence of the gelling agent, its concentration and concentration of fibrinogen (Fbg) on the total formation time (CT+CFT) of the formed clot.

[0092] FIG. 2 shows the influence of the gelling agent, its concentration and concentration of fibrinogen on the firmness of the formed clot.

[0093] FIG. 3 shows the influence of the gelling agent, its concentration and concentration of fibrinogen on the efficacy of the formed clot (ratio of clot strength to rate of clot formation).

[0094] FIG. 4 gives studies on the stability of the glues in relation to total clot formation time (CT+CFT), without gelling agent at different concentrations of fibrinogen.

[0095] FIG. 5 gives studies on the stability of the glues in relation to clot firmness without gelling agent at different concentrations of fibrinogen.

[0096] FIG. 6 gives studies on the stability of the glues in relation to total clot formation time (CT+CFT) in the presence of 1% Klucel MF at different concentrations of fibrinogen.

[0097] FIG. 7 gives studies on the stability of the glues in relation to clot firmness in the presence of 1% Klucel MF at different concentrations of fibrinogen.

[0098] FIG. 8 illustrates the adhesive power at 15 minutes in the carotid region of eight glue compositions.

[0099] FIG. 9 illustrates the adhesive power at 15 minutes in the hepatic region of seven glue compositions.

[0100] FIG. 10 illustrates the adhesive power at 15 minutes in the spleen region of five glue compositions.

[0101] FIG. 11 illustrates hepatic bleeding in grams at 15 minutes after incisions and application of one of the eight tested glue compositions.

[0102] FIG. 12 illustrates spleen bleeding in grams at 15 minutes after incisions and application of one of the five tested glue compositions.

[0103] FIG. 13 illustrates kidney bleeding in grams at 15 minutes after incisions and application of one of the five tested glue compositions.

EXAMPLES

Example 1: Study on Type of Gelling Agent

[0104] Four compounds were tested for their glue gelling capability: Klucel HF (MW: 1 150 kDa), Klucel MF (MW: 850 kDa), PlasdoneC30 (MW: 58 kDa) and PlasdoneC17 (MW: 10 kDa).

[0105] Strong concentrations of the gelling agents PlasdoneC30 and PlasdoneC17 (concentrations higher than 30% in g/mL of solution) were needed to obtain satisfactory viscosity, contrary to Klucel HF and Klucel MF of which concentrations of 0.01 to 10% provided major thickening of the glue preparation. Therefore, the properties of the gelled glue were researched in the presence of Klucel HF and Klucel MF.

Example 2: Influence of the Gelling Agent and Fibrinogen Concentration on Total Clot Formation Time

[0106] Gelling of the biological glue was measured by ROTEM. ROTEM is a measurement of thromboelastometry which systematically provides several parameters: coagulation time (CT) and clot formation time (CFT), two kinetic parameters characterizing the rapidity of coagulation of the solution. For greater simplicity, the value corresponding to the sum of these two parameters (CT+CFT) is given, corresponding to the total clot formation time. The system also provides a value reflecting maximum clot firmness (MCF in mm).

[0107] In brief, a solution of gelling agent was prepared by reconstituting the powder gelling agent in 25 mM HEPES buffer, 175 mM NaCl pH 7.4 for 16 h at ambient temperature. A 2× concentrated solution of gelling agent was thus prepared (1 or 2 g/100 ml). The next day, a volume of this gelling solution (or buffer for negative control) was added to a volume of 2× concentrated fibrinogen solution previously dialysed against the same HEPES buffer (fibrinogen solution of 6 to 90 mg/ml before dilution). FVIIa was then added in the desired concentration (2-5 μg/ml) in minimum volume (<1% of total volume). FVIIa was previously diluted against HEPES buffer. The experiment was initiated through the addition of 0.5 pM tissue factor, 4 μM phospholipids, 5% human plasma and 5 μM CaCl.sub.2 in a small ROTEM dish (500 μl). Fibrin formation was recorded by increase in the density of the solution.

[0108] The sum of clot formation time and coagulation time was measured under each of the conditions (FIG. 1).

[0109] Without gelling agent, the increase in fibrinogen concentration leads to a regular decrease in clot onset time from 1164 to 300 s. In the presence of 0.5% Klucel HF, the times with 10 mg/ml fibrinogen remain higher than those obtained in the absence of gelling agent, this difference not being significant. In the presence of 0.5% or 1% Klucel MF, the clot formation times are similar to those without gelling agent, even lower with 20 mg/ml fibrinogen and 0.5% Klucel MF. As a result, the presence of Klucel MF or Klucel HF at between 0.5 and 1% does not significantly modify clot formation time.

Example 3: Influence of Gelling Agent Concentration and Fibrinogen Concentration on Clot Strength

[0110] Clot firmness (MCF in mm) was measured by ROTEM as described in Example 2 (FIG. 2). Without gelling agent clot firmness increases regularly with fibrinogen concentration, increasing from 27 mm with 3 mg/ml fibrinogen to 93 mm with 20 mg/ml fibrinogen. The presence of 0.5% Klucel MF or Klucel HF does not significantly modify firmness, in particular on and after 10 mg/ml de fibrinogen.

Example 4: Influence of Gelling Agent Concentration and Fibrinogen Concentration on the Efficacy of Clot Formation

[0111] The ratio of clot firmness (in mm) to rate of clot formation (in sec) allows evaluation of the overall quality of clot formation in a single parameter. This ratio was calculated and is given as a function of different parameters of the study (FIG. 3). Without gelling agent, the ratio increases regularly as a function of fibrinogen concentration, reaching a maximum at 20 mg/ml (ratio=0.31). In the presence of 0.5% Klucel HF, this ratio is reduced with 10 mg/ml de fibrinogen. In the presence of 1% Klucel MF, the ratio is better with low concentrations of fibrinogen (3 and 5 mg/ml) than when not present. In the presence of 0.5% Klucel MF, the ratios are globally maintained relative to a solution without gelling agent. An optimum is seen in the region of 20 mg/ml fibrinogen.

[0112] These data show that the presence of the Klucel MF thickener in the solution of biological glue does not affect formation of the fibrin clot whilst increasing the viscosity of the solution.

Example 5: Study on the Stability of Different Glues in Respect of Maximum Clot Firmness (MCF), Coagulation Time (CT) and Clot Formation Time (CFT)

[0113] The stability of several glue formulations was evaluated by ROTEM analysis. First, formulations of biological glues not containing any gelling agent were prepared and analysed. These were mixtures of 10 or 20 mg/ml fibrinogen incubated in the presence of FVIIa for 0 to 10 days or in which FVIIa was added extemporaneously. Polymerisation of the glue was triggered through the addition of an inducing mixture (0.5 pM tissue factor, 2 μM phospholipids, 3 mM calcium) in the presence of 5% human plasma.

[0114] Incubation of the solutions for up to 7 days does not affect the rate of clot onset (CT+CFT) whether FVIIa was incubated in the presence of (Fbg-FVIIa) or added at the time of measurement (Fbg) (FIG. 4). After 7 days, the clot formation time increased regularly under all examined conditions. Clot firmness was optimal with 20 mg/ml fibrinogen, whether or not FVIIa was incubated in the presence of fibrinogen (FIG. 5, .square-solid. and x). There was no variation in firmness for the 7 first days after which it decreased regularly over time under all examined conditions. The clots formed from mixtures containing 20 mg/ml fibrinogen remained more resistant than those formed from 10 mg/ml solutions.

[0115] A similar experiment was repeated by adding 1% (1 g/100 mL solution) of Klucel MF gelling agent to the fibrinogen and incubating them together for different times (FIGS. 6 and 7). The presence of gelling agent did not cause any major modification in the quality of the formed clots. It even tended to limit loss of efficacy between days 7 and 10. As previously, the mixtures formed from 20 mg/ml fibrinogen gave clots that were more resistant and formed more rapidly.

To conclude, the preparation of biological glue can be incubated up to 7 days at 25° C. without losing or reducing its coagulation function. The addition of 1% Klucel MF improves the performance of the solution over longer time periods.

Example 6: In Vivo Study on Eight Formulations of Biological Glues

[0116] In this study, several glue formulations were tested in a rabbit organ bleeding model. The eight tested formulations were denoted: Glue A, Glue B, Glue C (corresponding to 3 glue formulations with different fibrinogen concentrations) and Solution 1, Solution 2, Solution 3, Solution 4, Solution 5 (glue formulation with added gelling agent), all these solutions being compared with a placebo (buffer solution). [0117] Glue A: 3 mg/ml Fibrinogen+2 μg/ml FVIIa [0118] Glue B: 20 mg/ml Fibrinogen+2 μg/ml FVIIa [0119] Glue C: 80 mg/ml Fibrinogen+2 μg/ml FVIIa [0120] Solution 1: 3 mg/ml Fibrinogen+2 μg/ml FVIIa+1% (w/v) Klucel MF [0121] Solution 2: 20 mg/ml Fibrinogen+2 μg/ml FVIIa+1% (w/v) Klucel MF [0122] Solution 3: 10 mg/ml Fibrinogen (plasmatic origin)+2 μg/ml FVIIa+1% (w/v) Klucel MF [0123] Solution 4: 10 mg/ml Fibrinogen (transgenic origin)+2 μg/ml FVIIa+1% (w/v) Klucel MF [0124] Solution 5: 10 mg/ml Fibrinogen (transgenic origin)+2 μg/ml FVIIa+1% (w/v) Klucel MF+33 μg/ml FXIII

[0125] The primary evaluation criterion was organ bleeding, evaluated via the amount of blood lost after organ lesion. The secondary evaluation criterion was the adhesive power observed at the carotid vessels (without induced wound) and at the edges of the organ lesions, graded from 0 to 2 (0=no adhesive power, 1=low adhesion, 2=strong adhesion).

[0126] This blind, randomised, controlled study included a total of forty-eight female New Zealand rabbits weighing about 3 kg-4 kg, divided into 9 groups of 5 animals (with 3 pilot animals): Placebo group, glue A group, glue B group, glue C group, Solution 1 group, Solution 2 group, Solution 3 group, Solution 4 group and Solution 5 group. An organ bleeding model was applied to evaluate haemostatic capacity (capacity to limit bleeding) and adhesive power in rabbits with surgical wounds of organs. The entire experiment was conducted under general anaesthesia. The order of treatment (glue or placebo) was conducted by drawing lots.

[0127] After induced anaesthesia, the rabbits were tracheotomised and mechanically ventilated. First, the adhesive power was examined in the carotid region (without induced bleeding). At 5 and 15 minutes, the adhesive power was evaluated using a score of 0 to 2 (0=no adhesive power, 1=low adhesion, 2=strong adhesion).

[0128] Organ bleeding was then examined after median xyphopubic laparotomy and hepatic, splenic then renal lesions with blood collection over 15 minutes for each organ. The lost, collected blood was weighed. In more detail, first 6 standardised incisions were made perpendicular to the free edge of the liver and away from the vascular pedicle. The haemostatic product was applied to the lesions. Bleeding was evaluated after 15 minutes by weighing the compresses placed underneath and around the liver. The adhesive power at 5 and 15 minutes was graded between 0 and 2. Similarly, spleen bleeding was obtained after 4 incisions perpendicular to the convexity, then right and left renal bleeding after 3 transfixing wounds on each kidney. For each organ, after application of the haemostatic product, bleeding was measured at 15 minutes, and the adhesive power graded at 5 and 15 minutes for the liver and spleen.

On completion of the experiment, the animals were sacrificed by injection of a lethal dose of pentobarbital.

[0129] Three pilot animals allowed adaption and standardisation of the type and number of lesions for each organ.

TABLE-US-00001 TABLE 1 Study on the adhesive power of different formulations Placebo Glue A Glue B Glue C Carotid score 5 min* 0/20 0/20 10/20 3/20 median 5 min 0 (0-0) 0 (0-0) 1 (0-2) 0 (0-2) score 15 min* 0/20 9/20 11/20 6/20 median 15 min 0 (0-0) 1 (0-2) 1 (0-2) 0 (0-2) p score vs placebo 0.005 <0.001 0.03 Liver score 5 min ** 0/10 3/10  4/10 3/10 median 5 min 0 (0-0) 1 (0-1) 1 (0-1) 0 (0-2) score 15 min ** 0/10 3/10  6/10 4/10 median 15 min 0 (0-0) 1 (0-1) 1 (0-2) 1 (0-2) p score vs placebo 0.05   0.018 NS .sub.(0.053) Spleen score 5 min ** 0/10 1/10  3/10 1/10 median 5 min 0 (0-0) 0 (0-1) 0 (0-2) 0 (0-1) score 15 min ** 0/10 3/10  5/10 1/10 median 15 min 0 (0-0) 0 (0-2) 1 (0-2) 0 (0-1) p score vs placebo NS NS .sub.(0.053) NS N° of animals 5 5   5 5

[0130] Glues A et B have stronger adhesive power than the placebo in the carotid and liver regions. Glue C, having the highest fibrinogen content, only has a significantly stronger adhesive power than the placebo in the carotid region. Therefore, the use of glues having fibrinogen concentrations lower than 60 mg/ml generally allows an increase in the adhesive power of the glue.

TABLE-US-00002 TABLE 1bis Study on the adhesive power of different formulations Placebo Solution 1 Solution 2 Solution 3 Solution 4 Solution 5 Carotid score 5 min* 0/20 8/20 10/20  26/52 20/52  7/20 median 5 min 0 (0-0) 1 (0-2) 1 (0-2) 1 (0-2) 1 (0-2) 1 (0-2) score 15 min* 0/20 15/20 15/20  40/52 46/52 15/20 median 15 min 0 (0-0) 2 (0-2) 1.5 (1-2)   2 (0-2) 2 (1-2) 2 (0-2) p score vs placebo <0.001  <0.001  <0.001  <0.001  Liver score 5 min ** 0/10 8/10 3/10 11/26 13/26 median 5 min 0 (0-0) 2 (0-2) 1 (0-1) 1 (0-2) 1 (0-2) score 15 min ** 0/10 8/10 7/10 12/26 15/26 median 15 min 0 (0-0) 2 (1-2) 1 (1-2) 1 (0-2) 1 (0-2) p score vs placebo 0.005 0.005 0.015 0.004 Spleen score 5 min ** 0/10 1/10 2/10 median 5 min 0 (0-0) 0 (0-1) 0 (0-2) score 15 min ** 0/10 4/10 3/10 median 15 min 0 (0-0) 1 (0-1) 1 (0-1) p score vs placebo 0.014 0.05  No of animals 5 5    5    13    13    5 *sum of scores (of both carotids), at 5 and 15 minutes after application ** sum of scores, at 5 and 15 minutes after application median (minimum-maximum) NS: non-significant

[0131] Solutions 1 to 5 have stronger adhesive power than the placebo on the tested regions (FIGS. 8, 9, 10). In addition, the use of gelling agent most advantageously allows an increase in the adhesive power of the glue (comparison of glues A and B and of Solutions 1 and 2).

TABLE-US-00003 TABLE 2 Study on blood loss Placebo Glue A Glue B Glue C Liver Loss in g 4.40 1 2.11 2.91 (2.73-8.57)  (0.62-4.62)  (1.38-6.60)  (1.38-5.45)  p vs placebo 0.028 NS NS Spleen Loss in g 19.20 11.47 11.41 14.39 (5.86-23.43) (5.11-28.15) (6.47-21.47) (4.86-27.98) p vs placebo NS NS NS Kidneys Loss in g 3.22 4.21 3.14 4.0 (1.67-4.09)  (3.75-9.79)  (2.36-8.32)  (1.9-9.0)   p vs placebo 0.028 NS NS N° of animals 5 5 5 5

TABLE-US-00004 TABLE 2bis Study on blood loss Placebo Solution 1 Solution 2 Solution 3 Solution 4 Solution 5 Liver Loss in g 4.40 1.99 2.83 1.08 0.94 0.83 (2.73-8.57) (1.05-7.60) (0.90-6.64) (0.53-2.18) (0.46-5.81) (0.53-2.18) p vs placebo NS (0.076) NS 0.001 0.005 Spleen Loss in g 19.20  7.8  6.21  (5.86-23.43)  (5.58-15.30)  (4.13-12.65) p vs placebo NS NS Kidneys Loss in g 3.22 1.29 0.74 (1.67-4.09) (1.28-4.06) (0.62-2.33) p vs placebo NS (0.075)  0.016 No of animals 5   5   5   13 13 5
Blood losses are expressed as a median (minimum-maximum).

[0132] Hepatic bleeding in the <<Glue A>> group is statistically lower than in the placebo group. On account of an extreme value in the Solution 1 group, there is no significant difference between the a <<Solution 1>> group and placebo group (FIG. 11). However, a significant trend is observed towards a reduction in hepatic bleeding in the Solution 3 and 4 groups.

[0133] Also, there is no significant difference between the different groups regarding spleen bleeding on account of a wide dispersion of values (FIG. 12). However, a non-significant trend is observed towards reduced spleen and liver bleeding in the Solution 1 and 2 groups.

[0134] It appears that Solutions 1 and 2 allow a reduction in renal bleeding, although for Solution 1 this effect is not significant (FIG. 13).

[0135] In general, the use of fibrinogen concentrations lower than 60 mg/ml appears to be preferable to limit bleeding. The use of gelling agent in the glue formulation may also allow a reduction in bleeding, in particular in the spleen and kidney regions.

Example 7: In Vivo Study on Healing Property

[0136] Two glues were tested in pigs after skin sampling. The grafts were bandaged in algostéril® alone or in the presence of glues comprising 45 mg/ml fibrinogen.

[0137] Four skin samples (7 cm×7 cm×4, i.e. about 15% of body surface) were taken using a dermatome from the back of Large White Landras pigs under general anaesthesia. Glue 1 comprised 20 mg/mL fibrinogen, 4 μg/mL FVIIa and 1% Klucel MF, Glue 2 comprised 45 mg/mL fibrinogen, 4 μg/mL FVIIa and 1% Klucel MF. Both glues were tested in association with a Jelonet® membrane (Smith & Nephew, London, UK) and the clinical progress of healing was compared with application of algostéril® at D2, 5 and 14.

[0138] Algostéril® containing calcium alginate is a haemostatic dressing conventionally employed in surgery for wound management. The Jelonet® was only used here as substrate for the glue of the invention, since it is advantageously employed as non-adhesive dressing and does not intrinsically contain any healing active ingredient.

[0139] <<100%>> healing denotes complete healing. The percentage represents the healed surface of the total lesioned surface area (49 cm.sup.2).

[0140] Table 3 gives the results obtained.

TABLE-US-00005 TABLE 3 Progress of healing as a function of treatment Glue 1 (n = 4) Glue 2 (n = 4) Algostéril (n = 3) Day 2   0%   0%  3% Day 5   82% 52.5%  5% Day 14 100% 82.5% 70%

[0141] These results show that the glues used allowed a very signification acceleration of the healing process compared with the standard dressing method.