FR2667789A1 - Cicatrising composition and method for preparing it - Google Patents

Abstract

The invention relates to the method for preparing a cicatrising composition which is obtained using as its basis the supernatent of platelets of human or animal origin, the supernatent being activated in vitro, wherein this supernatent undergoes at least one step of acid treatment at a pH below 5 for a sufficient time to activate the TGF-ss, followed by recovery of the treated supernatent and removal of the precipitate obtained after neutralisation. The invention also relates to a pharmaceutical composition intended, in particular, for cicatrisation.

Description

 The present invention relates to a method for preparing a composition with healing activity.

 The healing of endothelial wounds in humans and / or animals involves a complex process involving interactions between numerous substances such as serum enzymes, growth factors, platelets, monocytes and macrophages in particular.

 It is now recognized that platelets and macrophages are the most involved cells in this healing process. Platelets initiate healing by releasing angiogenic and growth factors at the wound level that act on angiogenesis and stimulate collagen synthesis.

These factors, and more specifically the growth factor
PDGF, have already been the subject of many studies given their importance in this process of healing. (Journal of Cellular
Physiology, vol. 113, p. 261; Blood, Vol. 64, No. 2, 1984, p.458).

 It is now known that they are present in platelet alpha-granules, and that they circulate in this form until the platelets are induced, by external activation, to degranulate.

Thus, it has been demonstrated, in vitro (Stakenow et al., Exp Cell Res, 1981, 136, 321-25, Burke JM et al., Exp Cell Res, 1977, 107-387) and in vivo (Knighton D. et al., Annal of Surgery 1982, vol 196, No. 4, p.379) that platelet activation by thrombin releases a mitogen or growth factor for fibroblasts, and stimulated the synthesis of collagen by these cells.

 Healing efficiency is therefore limited by the bioavailability of these growth factors that control cell migration and proliferation.

 Today it is interesting to have compositions based on growth factors that can be administered to patients suffering from a deficiency in this type of compounds, or simply having difficulty in healing, such as diabetics and the elderly.

 WO 86 03122 discloses the use of a total supernatant of thrombin-activated platelets to enhance the healing process.

 The present invention relates in particular to a method for preparing an improved healing composition obtained from a platelet supernatant of human or animal origin, activated in vitro, characterized in that said supernatant undergoes at least one acid treatment stage at pH less than 5 for a time sufficient to obtain TGFPw, then that the treated supernatant is recovered and the precipitate obtained after neutralization is removed.

 In this method, platelet activation releases the content of granules, i.e., plasma proteins and hormonal or growth factor peptides.

 Thus, albumin, fibrinogen, vW factor, serotonin, histamine, Connective Tissue Activating Peptide III (CTAP III), thromboglobin, were detected. PTG), - Platelet Factor IV (PFIV), - Platelet Derived Growth Factor (PDGF), - Epidermal Growth Factor (EGF), - Insulin like Growth Factor I and II (IGFI and II) - Transforming Growth Factor and (TGF (ss).

 It is therefore a cocktail of biologically active products that is released. However, some growth factors, linked to a carrier protein, are inactive (Furlanetto and Marino, Meth in Enz, Vol.

146, p. 216-226).

 This is particularly the case of T.G.F, which is present in a latent form in the extract of? granules (Pircher et al., B.B.R.C., Vol 136, P. 36-37 (1986)).

 Thanks to the method according to the invention which comprises an acid treatment step, the supernatant obtained contains TGF in the active state and leads to healing results much higher than those obtained for untreated extracts. This improvement in activity is due in large part to the activation of TGFA.

 Preferably, this acid treatment is conducted at a pH of less than 3, for example 2.5, for a period of a few minutes, in particular about ten minutes. Of course, this treatment can be optimized depending on the supernatant used.

 The supernatant to be treated will preferably be obtained in the following manner - platelet separation of the plasma fraction and leukocyte depletion, - activation in a buffer solution, in the presence of an activator, - homogenization of the mixture, and - recovery of the supernatant.

 Certain operations carried out in the different stages of this process make use of techniques familiar to those skilled in the art, in particular the separation of platelets and leukocyte depletion; they will not be recalled here in detail, but will appear more clearly on reading the examples below.

 The activation is preferably carried out on a platelet suspension placed in the presence of an activator. Among the activators, it is preferred to use thrombin, but calcium ADP is an activator which has certain advantages.

 Indeed, activators of the thrombin type, inactivated collagen are of human or animal origin, and may have drawbacks related to these origins.

 In contrast, calcium ADP is a product that can be synthesized chemically, which ensures its safety.

 After activation, the resulting supernatant undergoes viral inactivation, preferably by pasteurization. This pasteurization step makes it possible to get rid of any trace of bacterial or viral contamination possibly present in the supernatant. It also makes it possible to inactivate the activator containing the thrombin that may be present.

 Pasteurization is preferably carried out by heating the supernatant for about 10 hours at a temperature of about 60 ° C. The pH is a neutral or slightly acidic pH.

 It is this supernatant obtained after viral inactivation, possibly without having been separated from the precipitate formed by the heating step, which is subjected to acid treatment.

 The treated supernatant obtained by carrying out the process according to the invention has the advantage, given its protein purity, of being stable and of being able to be stored under good conditions.

 It is also possible to provide that the healing composition is prepared extemporaneously from the platelets of the subject to be treated, which eliminates any biological risk, reaction or contamination.

 The present invention also relates to healing compositions obtained from this supernatant.

 These include such compositions containing more than 1 μg / ml of activated TGF, preferably 3 μg / ml.

 Given its good stability, the supernatant can be stored in lyophilized form for example, available for extemporaneous mixing at the time of its application to the wound with a carrier agent, or for the preparation of healing compositions.

 The compositions according to the invention may of course contain other therapeutically active principles, which may be chosen from antiseptic, antibiotic and / or anesthetic compounds for example, or else other active substances known for their activity in the healing of wounds.

 It is also possible to provide compositions containing biological glues based on an extemporaneous mixture of fibrinogen / fibronectin and thrombin, in particular the biological glue described in French patent applications 90 07505 and 89 10355.

 These compositions may also comprise acceptable pharmaceutical excipients, chosen from compounds having good compatibility with the active ingredients and the supernatant, contained in said composition.

 These pharmaceutically acceptable excipients may thus be chosen from water-soluble polymers of the natural polymer type, such as polysaccharides (xanthan gum, locust bean gum, peptin, etc.) or polypeptides, cellulosic derivatives such as methylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose or else synthetic polymers. , polaxamer, carbomer, PVA or PVP.

 Of course, such compositions should have a pH as well as an osmolarity within the limits of physiology.

 Finally, it is within the scope of any person skilled in the art to add in these compositions various cosolvent type excipients such as ethanol, glycerol, benzyl alcohol, humectants (glycerol), diffusion-promoting agents (transcurol , urea), or anti-bacterial preservatives.

 These compositions may be in the form of liquid, milk, powder, spray, and preferably in the form of a hydrogel.

 The examples presented below in a non-limiting way will make it possible to demonstrate other characteristics of the present invention.

EXAMPLE 1
The platelets are extracted from the plasma by double centrifugation. They are leukylated by filtration on a PALL filter and washed in 0.04 M Tris buffer, pH 7.4.

 Approximately 3 x 10 10 platelets are recovered per standard blood donation, suspended in 5 to 10 ml of buffer.

To this suspension is added thrombin 5
U / ml and allowed to act for 10 minutes; the resulting supernatant is adjusted to pH 7.0 and heated for 10 h at 60 ° C in a water bath.

 This produces a product that can be treated by the process of the invention.

EXAMPLE 2: ACIDIFICATION
The extract according to Example 1 is brought to pH 2.5 by addition of HCl, maintained for 10 min. with gentle stirring and then neutralized to pH 7.0 by the addition of NaOH.

 The precipitate formed during pasteurization and the acidic pH treatment step are centrifuged and the supernatant is filtered if necessary.

EXAMPLE 3
The platelets are extracted from the plasma by double centrifugation.

Platelets are depleted by filter filtration
PALL and washed in 0.02 M TRISS buffer pH 7.4.

 About 3.10 exp 10 platelets are thus recovered per standard blood donation, suspended in 5 to 10 ml of buffer.

 ADP is added to this suspension at a level of 2.5 micromoles per liter and 19 millimoles / liter of calcium. The suspension is homogenized and then left standing for 20 minutes. The platelet aggregate is then centrifuged.

 The platelet extract is heated to pH 7.0 for 10 hours at 60 ° C in a water bath.

 The large precipitate that occurs during heating is centrifuged and the supernatant recovered.

 This supernatant can be treated as in Example 2.

EXAMPLE 4
The extract according to Example 2 is diluted to 1 / 5th in a hydroxymethylcellulose solution at 2 g / l or in a solution of collagen at 20 g / l.

 A series of 6 circular dorsal punches with a diameter of 6 mm and a depth of approximately 2 mm which are symmetrical with respect to the spine is produced on a 200 g female Wistar rat.

 A series of 3 wells is filled with 10 ul of diluted extract, the other series by 3 wells of placebo.

 Three batches of rats are thus made - 1 batch using the extract according to Example 1, - 1 batch using the extract according to Example 2, - 1 lot comparing the acidified extract versus the non acidified extract ( 2 VS 3).

 The holes are then covered with a plastic film, itself covered with tulle gras, all held in place by a self-adhesive tape.

 The rats are sacrificed on days 4 and 7 after the treatment, the wound is then removed and fixed for 48 hours in 4% paraformaldehyde containing saccharin (0.4M), 1% CaCl 2 and NacI (0.15M). then washed under running water.

For histology, fixed parts are embedded in tissue
Teak, cut or cryotome and colored with a trichome of Masson.

RESULTS
Here are the results obtained from a suspension at 5 × platelets / ml.

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<SEP> Preview <SEP> Next <SEP> Preview <SEP> Next <SEP> Preview <SEP> Next
<tb><SEP> Example <SEP> 1 <SEP> * <SEP> Example <SEP> 1 <SEP> Example <SEP> 2
<tb><SEP> PDGF <SEP> 350 <SEP> ng / ml <SEP> 300 <SEP> ng / ml <SEP> 280 <SEP> ng / ml <SEP>
<tb> TGFssactif <SEP> 30 <SEP> ng / ml <SEP> 50 <SEP> ng / ml <SEP> 2,8 <SEP> g / ml <SEP>
<tb> TGFsstotal <SEP> 3.0 <SEP> g / ml <SEP> 3.0 <SEP> g / ml <SEP> 3.0 <SEP> g / ml <SEP>
<tb> * before pasteurization
The assay of TGFss is done according to the agar cloning technique of Pircher et al. (BBRC, Vol 136, P. 30-37)
Active TGFss is the amount spontaneously measured in the sample.

 T.G.F. ss total is the quantity measured after complete activation of the sample.

 The difference between the two corresponds to the latent T.G.F.ss.

 It is therefore found that the activation obtained persists despite the neutralization according to Example 2.

Results on the animal: effects observed after 7 days
The scar tissue of the placebo control is organized into a fibrillated, loose, weakly cellularized weft.

 In the presence of extracts according to Example 2 or 3, the regeneration tissue is differentiated to several fibers - the outer density of the cell much higher, - the collagen matrix is better defined, its general organization is dense. It is expressed in the form of fibrillar deposits announcing the classical organization of the dermis, the stratum corneum is better differentiated for the extract according to Example 3 than for the extract according to Example 1 and the maturation is started as in attests the differentiation of hair follicle.

 It is therefore found that the acidified fractions are more effective than the non-acidified fractions, in particular by an earlier cell differentiation.

 The effects on day 4 are similar, but less pronounced.

Claims (10)

 1. Process for the preparation of a healing composition obtained from the supernatant of platelets of human or animal origin, activated in vitro, characterized in that the said supernatant undergoes at least one acid treatment step at a pH of less than 5 for a period of time. sufficient to activate the TGF, then recovering the treated supernatant and removing the precipitate obtained after neutralization.  2. Method according to claim 1, characterized in that the acid treatment is carried out at a pH of less than 3.  3. Method according to claim 2, characterized in that the acidic treatment lasts approximately 10 min.  4. Method according to one of claims 1 to 3, characterized in that before the acid treatment the supernatant is pasteurized.  5. Preparation process according to one of claims 1 to 4, characterized in that the supernatant to be treated is obtained from insulated platelets suspended and subjected to activation.  6. Method according to claim 5, characterized in that the activation is carried out using thrombin or calcium ADP.  7. Method according to one of claims 1 to 6, characterized in that the healing composition is prepared extemporaneously from the platelets of the subject to be treated.  8. Method according to one of claims 1 to 7, characterized in that the concentration of activated platelets from 109 to 101 platelets / ml.  9. Pharmaceutical composition intended in particular for wound healing, characterized in that it comprises a healing composition obtainable by the process of one of claims 1 to 8.  10. Pharmaceutical composition characterized in that it comprises an activated platelet supernatant freed of platelets and containing at least 1 .mu.g / ml of active TGFO.