Composition for treating tissue lesions

Abstract

Some embodiments are directed to a pharmaceutical composition including a biocompatible polymer in association with a eukaryotic cell, a platelet extract and/or lysate, or a growth factor, to be used as a drug for the prevention and/or treatment of tissue lesions. Some other embodiments are also directed to a pharmaceutical kit which includes a biocompatible polymer in association with a eukaryotic cell for the prevention and/or treatment of tissue lesions. Some other embodiments are also directed to the use of a pharmaceutical composition including a biocompatible polymer in association with a eukaryotic cell, a platelet extract and/or lysate, or a growth factor, for manufacturing a drug for the treatment of tissue lesions. Some other embodiments can be used in particular in the veterinary and pharmaceutical fields.

Claims

1. A method of using a pharmaceutical or dermatological composition as a medicament for at least one selected from the group consisting of gastric or digestive ulcers, skin wound lesions, diabetic ulcers, venous ulcers, ischemic ulcers, pressure sores and burns, joint and tendon lesions and digestive system tissue lesions, comprising: administering a biocompatible polymer of general formula (I) below
AaXxYy  (I) in which: A represents a glucose monomer, X represents an R.sub.1COOR.sub.2 group, Y represents an R.sub.7SO.sub.3R.sub.8 group, in which: R.sub.1 independently represents an aliphatic hydrocarbon-based chain which is optionally branched and/or unsaturated and which optionally contains one or more aromatic rings, R.sub.2 and R.sub.8 independently represent a hydrogen atom or a cation, and R.sub.7 independently represents a bond, or an aliphatic hydrocarbon-based chain which is optionally branched and/or unsaturated, when R.sub.8 is a cation, then the cation is at least one selected from the group consisting of lithium, sodium, potassium, rubidium and cesium, and the administering including administering the biocompatible polymer to a patient in need of treatment for at least one selected from the group consisting of gastric or digestive ulcers, skin wound lesions, diabetic ulcers, venous ulcers, ischemic ulcers, pressure sores and burns, joint and tendon lesions and digestive system tissue lesions; a represents the number of monomers, x represents the degree of substitution of the monomers A by groups X, y represents the degree of substitution of the monomers A by groups Y, and; administering an eukaryotic cell selected from the group consisting of mesenchymal cells, adipocyte cells and bone marrow cells, the administering the eukaryotic cell is performed 12-24 hours after the administering the biocompatible polymer; and the administering the biocompatible polymer is performed in a manner directed to treating at least one selected from the group consisting of: gastric or digestive ulcers, skin wound lesions, diabetic ulcers, venous ulcers, ischemic ulcers, pressure sores and burns, joint and tendon lesions and digestive system tissue lesions; and wherein the number of monomers “a” is such that the weight of the polymers of formula (I) is greater than 2000 daltons; wherein the degree of substitution “x” is between 20% and 150%; and wherein the degree of substitution “y” is between 30% and 150%.

2. The method as claimed in claim 1, wherein the biocompatible polymer also includes functional chemical groups Z, different than X and Y, capable of conferring additional biological or physicochemical properties on the polymer.

3. The method as claimed in claim 2, wherein the degree of substitution “z” of all of the monomers A by groups Z is from 0% to 50%.

4. The method as claimed in claim 2, wherein the group Z is a substance capable of conferring better solubility or lipophilicity on the polymers.

5. The method as claimed in claim 4, wherein the groups Z are identical or different and are chosen from the group including amino acids, fatty acids, fatty alcohols, ceramides, or derivatives thereof, or targeting nucleotide sequences.

6. The method as claimed in claim 1, wherein the eukaryotic cell is chosen from at least one selected from the group consisting of adult or embryonic eukaryotic cells, bone marrow cells and adipose tissue cells.

7. The method as claimed in claim 1, wherein the biocompatible polymer is administered in the treatment of at least one selected from the group consisting of gastric or digestive ulcers, skin wound lesions, diabetic ulcers, venous ulcers, ischemic ulcers, pressure sores and burns, joint and tendon lesions and digestive system tissue lesions, and: the biocompatible polymer is administered by at least one selected from the group consisting of: intravenously or intramuscularly at a dose of from 0.1 to 5 mg/kg of body weight, by local injection at a dose of from 1 to 100 micrograms per milliliter, orally in 2 to 5 equal intakes per day, in an amount of a daily total of from 10 microg to 5 mg/kg of body weight, sublingually before eating with a concentrated aqueous solution of from 1 to 100 mg/ml, and by administration of a solution as a nasal aerosol or spray, and wherein the eukaryotic cell is administered in the treatment of at least one selected from the group consisting of gastric or digestive ulcers, skin wound lesions, diabetic ulcers, venous ulcers, ischemic ulcers, pressure sores and burns, joint and tendon lesions and digestive system tissue lesions by injection.

8. A method of using a pharmaceutical composition comprising: i. administering a biocompatible polymer of general formula (I) below
AaXxYy  (I) in which: A represents a monomer, X represents an R.sub.1COOR.sub.2 group, Y represents an R.sub.7SO.sub.3R.sub.8 group, in which: R.sub.1 independently represents an aliphatic hydrocarbon-based chain which is optionally branched and/or unsaturated and which optionally contains one or more aromatic rings, R.sub.2 and R.sub.8 independently represent a hydrogen atom or a cation, and R.sub.7 independently represents a bond, or an aliphatic hydrocarbon-based chain which is optionally branched and/or unsaturated, and when R.sub.8 is a cation, then the cation is at least one selected from the group consisting of lithium, sodium, potassium, rubidium and cesium, a represents the number of monomers, x represents the degree of substitution of the monomers A by groups X, y represents the degree of substitution of the monomers A by groups Y, and; the administering including administering the biocompatible polymer to a patient in need of treatment for at least one selected from the group consisting of gastric or digestive ulcers, skin wound lesions, diabetic ulcers, venous ulcers, ischemic ulcers, pressure sores and burns, joint and tendon lesions and digestive system tissue lesions; ii. administering an eukaryotic cell to treat at least one selected from the group consisting of gastric or digestive ulcers, skin wound lesions, diabetic ulcers, venous ulcers, ischemic ulcers, pressure sores and burns, joint and tendon lesions and digestive system tissue lesions the administering the eukaryotic cell is performed 12-24 hours after the administering the biocompatible polymer; and the administering the biocompatible polymer is performed in a manner directed to treating at least one selected from the group consisting of: gastric or digestive ulcers, skin wound lesions, diabetic ulcers, venous ulcers, ischemic ulcers, pressure sores and burns, joint and tendon lesions and digestive system tissue lesions.

Description

EXAMPLES

Example 1

Preparation of RGTAs and Method of Administration

(1) The RGTA synthesis is widely described in the related art, for example in the patent entitled—“PROCEDE DE SULFONATION DE COMPOSES COMPRENANT DES GROUPEMENTS HYDROXYLES (OH) LIBRES OU DES AMINES PRIMAIRES OU SECONDARES” [“PROCESS FOR SULFONATION OF COMPOUNDS INCLUDING FREE HYDROXYL (OH) GROUPS OR PRIMARY OR SECONDARY AMINES”] and also in the literature references: Yasunori I. et al., Biomaterials 2011, 32:769e776) and Petit E. et al. Biomacromolecules. 2004 March-April; 5(2):445-52.

(2) In the examples below, several known and described RGTAs were used, of which OTR4120 (described in Khammari-Chebbi et al., J Fr Ophtalmol. 2008 May;31 (5):465-71) and OTR4131 (described in Frescaline G. et al., Tissue Eng Part A. 2013 Jul.;19(13-14):1641-53. doi: 10.1089/ten.TEA.2012.0377) which are commercially available. In addition, the compound OTR4131 is a compound including a Z radical which is a fatty acid, namely the acetic acid described in Virginie Coudry, et al. Long-Term Follow-up of Superficial Digital Flexor Tendonitis Treated by a Single Intralesional Injection of a ReGeneraTing Agent in 51 Horses Journal of Equine Veterinary Science 34 (2014) 1357-1360 and a compound in which Z is an amino acid such as phenylalanine, described in U.S. Pat. Nos. 7,998,922, 8,790,631, were also used in the examples below.

(3) In the examples below, the administration was carried out as described above. In other words, when the RGTA is not mixed in a single solution with the cells, the RGTA was administered according to the methods of administration known for this compound.

(4) Advantageously, when the RGTA is administered via an independent composition, one of the effects obtained may be a preparation of the tissue or of the organ to be treated in order to promote an implantation, colonization and expansion by selected cells which allow a more efficient and synergistic tissue repair and regeneration by virtue of the combination of RGTA and cells.

(5) The frequency of administration may be single or repeated every week or fifteen days or even monthly, rarely for a total period of several months, depending on the success of the colonization and the required number of injections/administrations of cells, the time required for the preparation of the niche before implantation.

(6) The concentrations and doses of the RGTA that are administered depended on the local or systemic administration forms, on the frequencies, on the tissues, organs or areas to be treated, and on the volumes or surface area of the lesion.

(7) In the examples below, when the administration is carried out orally, the RGTAs may be in solution in water or in the form of any other oral presentation form, but also in the form of a tablet, a gel capsule or any other form compatible with an oral intake. In addition, advantageously, the orally administered RGTAs exhibit a notable resistance to degradation by acid and by digestive juices. Advantageously, since the product has no taste and is completely soluble in water, the preferred intake is in the form of an aqueous solution in a volume of 10 to 25 ml and at a concentration of 0.1 mg.ml.sup.−1 so that the amount is from 1 to 2.5 mg per intake, two to five times a day. In the majority of the examples, these intakes were in the morning before eating and in the evening at bedtime for patient weights ranging from 50 to 100 kg, the doses and frequencies possibly themselves also varying; thus, overall, the daily intake was between 1 and 500 mg/day. The period of this administration can be from several months without adverse effects having been observed. In particular, an intake by an individual of 25 mg/day over the course of more than one year induces no impairment nor any noticeable side effects.

(8) In the case of treatment of the digestive tract with cells in order to repair and regenerate lesions, the amounts of RGTA can advantageously be reduced. This is because the RGTA molecules can advantageously reassemble directly with the lesioned tissue and, for example, recreate a niche, advantageously allowing better implantation of the cells and, where appropriate, making it possible to promote their proliferation. The doses used during treatment of the digestive tract may be, for example, from 1 to 50 ml at 100 microg.ml.sup.−1 per day. They may for example be doses that are identical or similar to that described in the document Meddahi et al., J Biomed Mater Res 60: 497-501 2002, for example for the treatment of gastric or digestive ulcers, doses identical or similar to that described in the document Alexakis et al., Gut 2004;53:85-90 for the treatment of Crohn's diseases, or doses identical or similar to that described in the document Alexakis et al., FASEB J. 2001, 15,1546-1554 for tissues lesioned after irradiation.

(9) In the examples below, when the administration is carried out by systemic injection, the RGTAs were preferably in solution in physiological saline of injectable quality or any other form compatible with injection, in particular solutions with glucose or other excipients that are usual with polysaccharides such as heparin, or mixed with therapeutic products having other properties, provided that the risk of interactions with other active ingredients has been evaluated. The RGTAs were used at concentrations of from 0.1 to 5 mg.kg.sup.−1, preferably from 1 to 2.5 mg.ml.sup.−1 intravenously (IV) or intramuscularly (IM). For this route of injection, the injection may be a single, daily or weekly injection.

(10) In the examples below, when it was possible, the local administration in or in the vicinity of the lesioned tissue or site was preferred. In particular, this route of administration was preferred for the mucosae, for example buccal, vaginal, urethral, digestive mucosae with endoscopic vascular or cardiac access, and/or in areas where access is more difficult but can be directly reached, such as the bone marrow, the peri-retinal area, the intraventricular area, the pulmonary airways or else during surgery that opens up direct access or else directly through other tissues or organs with catheters, needles or suitable endoscopes.

(11) The RGTA may also be administered as a spray on a tissue surface in the case of implantations of isolated cells or cells in sheets or else cells impregnated in implanted materials which are a support for existing or future cell colonization.

(12) With regard to the airways, the administration may also be envisioned by inhalation.

(13) With regard to the mucosae and/or the walls of the digestive tract or of the uterine muscles or of the ligaments, the rectal or vaginal route was preferred when accessible.

(14) For the treatment of ocular tissues, the preferred administration form was, for example, in an eyewash, for example for the treatment of the cornea, by transcorneal injection for example in the treatment of the Descemet membrane, in the treatment of tissues covering the base of the eyeball, for example for the treatment of sight defects, or else for example for the treatment of a lesion of cells associated with hearing, for example for the implantation of cochlear ciliary cells.

(15) In the examples, the local administration or injection may be single, daily or weekly, the dose then being related to the surface area or volume of the lesion. In particular, in the examples, the RGTA concentration was preferably 100 microg/ml which is the preferred concentration, and the volume used was chosen so as to cover the lesioned surface area or to impregnate the volume of the lesion. Thus, the perilesional injection into the tissue or the organ or into the site of 0.1 to 0.5 ml of RGTA made it possible to impregnate a tissue in a volume that was 5 to 100 times greater. In the surface application or application as a spray, where the RGTA penetrates by adsorption, the coverage of the lesioned area was sufficient. Thus, three to four “sprays” each of 140 ml at a distance of 5 cm from the lesion were sufficient to impregnate a surface area of 10 cm.sup.2, alternatively a few milliliters of a cream or of a gel or ointment can be spread on the lesion or in the vicinity and thus provide access to the RGTA. The galenical forms of creams, ointments, gels, milks, foams, emulsions, powder pastes, etc. are those known to those with ordinary skill in the art and preferably chosen for their hydrophilic and moisturizing properties compatible with polysaccharides, for example hyaluronic acid.

(16) In the examples, another method of administration of the RGTA used was that of impregnation of the tissues, for example for the case of transplants of a tissue or of an organ such as kidney, liver, heart, lung, skin, cornea, ear drum, muscles, nerves, tendons, ligaments, bones, vessels or intestins, colon, both in areas of anastomoses and in implants, bladder, etc., without this list being exhaustive. In this embodiment which can be used with respect to all tissues and grafts, the organ to be transplanted, which may be impregnated, for example either by immersing it in a solution of RGTA, or by perfusion, or by spraying or any other method known to those with ordinary skill in the art. Advantageously, the impregnation of the tissues, as mentioned above, advantageously makes it possible to recreate the tissue microenvironment. Indeed, RGTA advantageously binds specifically to the heparan-binding sites available after the lesion and induced by the harvesting of the graft or the preparation of the transplant recipient. Advantageously, this binding makes it possible to recreate a niche which promotes colonization by the cells. The preferred concentrations of RGTA were from 0.01 to 100 microgram (mg) per ml. The impregnation time was short since a few minutes are sufficient. Advantageously, an impregnation of a few hours or day(s) can make it possible to simplify the procedure since the RGTA can be added to the preserving solutions and can prove to be a protective and anti-apoptotic agent as is described in the related art (Barritault D., Caruelle J-P. BIP121532, “POLYMERES BIOCOMPATIBLES, LEUR PROCEDE DE PREPARATION ET LES COMPOSITIONS LES CONTENANT” [“BIOCOMPATIBLE POLYMERS, PROCESS FOR PREPARING SAME AND COMPOSITIONS CONTAINING SAME”]; and Yue X-L, Lehri S, Li P, Barbier-Chassèfiere V, Petit V, Huang Q-F, Albanese P, Barritault D, Caruelle J-P, Papy-Garcia D and Morin C. Insights on a new path of pre-mitochondrial apoptosis regulation by a glycosaminoglycan mimetic.; Yue X-L and al, Cell Death and Differentiation, 2009, 1-12). The tissues were then exposed to solutions or suspensions enriched, as appropriate, with cells of a desired specificity, this being before or after the implantation. It is possible to add growth factor-enriched platelet extracts or lysates to this technique.

(17) The autologous cells were added with the graft before or after the implantation, advantageously making it possible to facilitate the colonization of host-graft junction areas and to promote the transplant engraftment. This method allows transplant engraftment, prevents necrosis and increases functional recovery.

(18) In the examples, the platelet extracts were administered alone after having administered the RGTA or with the RGTA. In this case, the mixture was prepared with an RGTA/PRP extract ratio such that the lysate of platelets obtained from 10.sup.9 platelets suspended in 1 ml of physiological saline is placed in the presence of thrombin, or at 100 mg of RGTA. At the end of the platelet degranulation, the solution was centrifuged at low speed. The supernatant containing the platelet factors and the RGTA was then administered on the lesion site.

(19) In another embodiment, the RGTAs alone or mixed with platelet extracts (PRPs) or isolated factors were injected with the therapeutic cells.

(20) In the examples, the administration of the RGTA was carried out at the latest at the time of the first administration of the cells, or preferably a few hours or days before. Thus, in the case of oral intakes of RGTA, it was observed that one or more daily intakes one week before the cell therapy increased the final tissue regeneration result, the preferred dose then being to drink 25 ml of the RGTA OTR4120 at 100 mg/ml in the morning before eating and in the evening before going to bed, for at least one week. When an IV or local injection of the RGTA was given, the cell therapy could be carried out within the hour following the administration of RGTA.

(21) In the examples described below, the cells are autologous and are preferably administered on the same day as that on which they are taken.

Example 2

Example of Treatment of a Tissue Lesion with the Administration of RGTA and of Mesenchymal Stem Cells (MSCs) in a Murine Model

(22) In the present example, the mice used were 70 ten-week-old female (C57/BL6) mice from Charles River divided up into 7 groups of 10 mice, and correspond to a mouse model acknowledged in the related art.

(23) A skin wound was made on the back of the mice using a punch 6 mm in diameter (used clinically for biopsies) and the wound was then left in the open air. Four wounds were made on each animal.

(24) The polymer used was OTR4120 and it was administered by subcutaneous (SC) injection in a proportion of 25 microl (μl) of a solution at 100 μl at two (diametrically opposed) points. The mesenchymal stem cells (MSCs) originating from bone marrows (tibia) isolated according to conventional protocols such as those described in “A protocol for isolation and culture of mesenchymal stem cells from mouse bone marrow”, Soleimani M, Nadri S. Nat Protoc. 2009;4(1):102-6 was suspended in a phosphate-buffered saline PBS at a concentration of 1×10.sup.6 cells per ml and were injected in a proportion of 2 injections of 50 μl per wound at two points (symmetrical, orthogonal to those of the RGTA) at various times and the wound was then measured 3, 5, 7 and 10 days after the injury.

(25) During the experiments, for each mouse, several wounds were made and a comparison was performed between: two injections that were diametrically opposed with respect to the wound for the RGTA, two injections that were diametrically opposed with respect to the wound for the MSCs, and four injections at the 4 cardinal points coupling the RGTA and MSCs.

(26) Various groups of mice were defined according to the composition administered according to the abovementioned processes and are given in detail below: Group 1 Control (Placebo): administration of physiological saline within 15 minutes following the wound/lesion. Group 2 Administration of MSCs: 1 million MSCs per administration at 2 symmetrical points opposite to the RGTA injection sites, 24 hours after the injury after the injury. Group 3: administration of RGTA after injury. Group 4: administration of RGTA mixed with MSCs (co-injected). Group 5: administration of RGTA after the injury, followed by administration of MSCs 5 minutes later. Group 6: administration of RGTA after the injury, followed by administration of MSCs 6 hours later. Group 7: administration of RGTA after the injury, followed by administration of MSCs 12 hours later. Group 8: administration of RGTA after the injury, followed by administration of MSCs 24 hours later.

(27) The table below summarizes the results regarding the wound closure kinetics measured in % on each animal according to the treatments. At time zero, the surface area of the lesion is by definition 100% for each animal, when the wound is closed the value is equal to 0%.

(28) TABLE-US-00001 TABLE 1 Change in the wound as a function of time days Group 0 3 5 7 10 1-PBS 100  53 +/− 15  29 +/− 15  15 +/− 10 5 +/− 5 2-MSCs 100  38 +/− 10 20 +/− 5 10 +/− 5 0 3-RGTA 100 40 +/− 5 25 +/− 5 10 +/− 5 0 4-RGTA + MSCs 100 35 +/− 5 20 +/− 5  8 +/− 5 0 co-injection 5-RGTA + MSCs 5 min 100 38 +/− 5 20 +/− 5  8 +/− 5 0 6-RGTA + MSCs 6 h 100 30 +/− 5 15 +/− 5  5 +/− 5 0 7-RGTA + MSCs 12 h 100 15 +/− 5  5 +/− 5 0 0 8-RGTA + MSCs 24 h 100 10 +/− 5 0 0 0

(29) Table 1 demonstrates that the administration of MSCs and of RGTA makes it possible to improve the tissue repair and in particular makes it possible to significantly accelerate healing. In particular, the administration of the cells 24 hours after the RGTA allows closing of the wound that is much better than that obtained with the RGTA alone or the MSCs alone.

(30) A comparable effect was obtained with cells originating from adipocytes of mice from the same litter.

Examples of Some Embodiments Observed in Clinical Practice

(31) Many examples carried out in clinical practice, mentioned below, illustrate the effects of some embodiments. The RGTA product in its commercial form OTR4120 or CACIPLIQ® is readily accessible. On the other hand, objectification is difficult and only clinical observations document the effects of some embodiments.

Example 3

Effects of the Combined Treatment with the RGTA and The Autologous Mesenchymal Stem Cells (MSCs) in Chronic Wound Healing

(32) In these examples, patients having various types of chronic wounds of varied etiology, such as diabetic ulcers, venous ulcers, ischemic ulcers, pressure sores, burns and transplant engraftments have been experiencing therapeutic failure for months. Several treatments had been tried without success, including a local treatment with CACIPLIQ® (RGTA OTR4120) according to the manufacturers' recommendations.

(33) This clinician was also used to combining RGTA technology with platelet extracts or carrying out cell therapies using autologous MSCs obtained by sampling 5 ml of bone marrow from the sternum of the consenting patient. The sample, having been filtered and enriched by centrifugation according to the techniques described for MSCs, were suspended in 1.5 ml of physiological saline and the cell solution was injected into the edges of the wound subcutaneously and also in the center of the wound. In all, about twelve injections of 0.1 ml around the outline of the ulcer were given (and according to the image likening the ulcer to the quarter of a clock and injecting at each position of the hours). According to this clinician, the use either of RGTA alone or of cell therapy alone did not allow wound healing.

(34) A combined administration according to some embodiments of RGTA and cells was also carried out. CACIPLIQ® was applied locally according to the protocol recommended for the CACIPLIQ product: the solution of 5 ml containing 100 μl of CACIPLIQ® (RGTA OTR4120) was poured on to a 15×15 cm compress and the impregnated compress was applied to the well-cleaned wound for 5 minutes and then removed. The MSCs were then injected as described above (example 1) within an hour after application of the RGTA. The CACIPLIQ® was then administered locally twice a week. For a few patients, a second bone marrow sample was taken 3 weeks later, and the patients were again treated as previously with an MSC injection. The result was very rapid since closure of all the ulcers was observed in less than 6 weeks.

(35) Another series of clinical trials was also carried out on other patients, by injection of CACIPLIQ® (RGTA) and then injection of the cells within an hour following the sampling of the cells (the time to prepare the cells). The result was then even more astonishing since all the patients were able to completely heal in less than one month. Furthermore, he even noted that some of these patients experienced closure of their wound in 15 days, even though the wound had persisted for months without any sign of improvement. Such rapidity had never been observed by this clinician, regardless of the treatment applied, in particular during treatment with CACIPLIQ® alone or the MSCs alone.

(36) This example therefore clearly demonstrates that the composition according to some embodiments advantageously and surprisingly make it possible to effectively treat tissue lesions, in particular chronic wounds, and advantageously allows a considerable acceleration of healing.

(37) In addition, this example clearly demonstrates that the composition according to some embodiments provide a novel solution to a problem for which no solution existed in the related art.

(38) In addition, a comparable if not even better effect was observed on patients using co-administration of RGTA, of PRP and of cells. In one preferred embodiment, the administration of RGTA was followed by an injection of PRP and then of cells, the administration, for regulatory reasons, having been carried out during the same operating period. The RGTA was administered alone (1 ml at 100 mg/ml of OTR4120), with or sequentially to the PRP (originating from a lysate of platelets from a blood sample from the patient, for example 50 ml), with or sequentially to the mesenchymal autologous cells, for example, but without this being limited, from various tissue sources.

(39) In another embodiment, a sample of mesenchymal stem cells was taken from the adipose tissues of the patient to be treated, by liposuction. The method for isolating the MSCs was identical to the method described in example 1 above.

(40) In another example of treatment using the composition according to some embodiments, a patient presented with a deep wound covering part of the underneath of the foot including the large toe and including a visible area of necrosis on at least half of the underneath of the toe. There was a strong odor of decomposition in the consultation room. A first treatment by cell therapy using injections of 0.1 ml at several points around the wound for a total of 1 ml of mesenchymal cells originating directly from the bone marrow from the patient's sternum was carried out unsuccessfully, and in addition the condition of the wound had worsened. A second treatment using the administration of only CACIPLIQ (registered trademark) locally also did not make it possible to improve the wound and only made it possible to prevent a greater degradation and “to maintain” a condition of status quo regarding the progression and aggravation of the wound, thus avoiding immediate amputation. Following these failures, an injection of 0.5 ml at 5 points of 0.1 microg.ml.sup.−1 to 100 microg.ml.sup.−1 of the RGTA (CACIPLIQ) at the junction between the still healthy areas and the necrotic area of the toe, followed one hour later by 5 injections, each of 0.1 ml, of cells originating directly from a further sample of 0.5 ml of cells by puncture of the sternum, were given. Following the administration of the composition according to the some embodiments a regression of the wound was observed, making it possible to avoid any amputation, and going as far as complete recovery of the wound. In particular, the necrotic area was totally eliminated and then replaced with budding originating from the still healthy tissues contiguous to the wound until there was complete coverage of the wound. In this example, a single injection sequence (RGTA and then mesenchymal autologous cells) was sufficient to treat this patient and allowed closure of the patient's wound.

Example 4

Effect of the Combined Treatment with the RGTA and Cell Therapy in Bone Tissue Healing

(41) In this example, the treatment of a non union fracture placing was carried out using the composition according to some embodiments.

(42) Likewise, the related art describes in namely an ineffective and dissuasive combination of the administration of RGTA with a cell therapy was carried out in the related art but did not make it possible to obtain any improvement in the fractures, this being after several months. The results on open wounds made it possible to obtain a notable acceleration of the healing process.

(43) Patients with a non union bone fracture of the tibia who had not successfully improved either through application of the RGTA alone or after the administration of cells from bone marrow according to the protocols of the art described above or that described by Hernigou P, Homma Y, Flouzat-Lachaniette C H, Poignard A, Chevallier N, Rouard H. Cancer risk is not increased in patients treated for orthopaedic diseases with autologous bone marrow cell concentrate. J Bone Joint Surg Am. 2013;95:2215-21.

(44) Patients having responded neither to the treatment with the RGTA alone nor to the treatment with autologous cells taken directly from the bone marrow were treated with the composition according to some embodiments. Surprisingly and unexpectedly, in particular in the light of the teaching of the related art, the use of the composition according to some embodiments, in particular the combination of RGTA with the cells from the bone marrow, made it possible to trigger the healing process in these patients and to obtain what no treatment had successfully obtained alone, thus showing the advantage of some embodiments. In particular, three patients experiencing therapeutic failure were treated with the composition according to the some embodiments and were able to benefit from this double treatment. In these cases, the cells from bone marrow were administered 1 h after administration of 1 ml of RGTA by local injection in the area of non union and of 1 ml of mesenchymal cells, taken by puncture of the patient's sternum and reinjected into the area of the non union bone fracture without any other treatment step.

(45) A combination additionally including platelet extracts was also performed and gives excellent results when it is used between the administration of the RGTA and of the cells. In particular, very positive results were obtained with the administration of the RGTA, followed one hour later by the PRP and then the cells.

(46) In this other example, the effect of the composition according to some embodiments on the bone preservation and action on the ischemic necrosis of the femur head were also studied. A treatment using the RGTA and the cells or the RGTA, PRP and cells was carried out. The patients presented with cases of early-stage femur head ischemia before deep necrosis causing considerable pain had set in. A treatment using the RGTA and the cells according to some embodiments made it possible to prevent the destruction of the femur head and the need to perform surgical procedures in order to implant a prosthesis. The treatment included or consisted of the local injection of 1 ml of RGTA OTR4131 at 10 microg.ml.sup.−1 in the periosteal ischemic area close to the lesion, followed 30 minutes later by the local injection of 5 ml of autologous cells from bone marrow taken from the iliac fossa on the same day.

(47) In another case, a cotreatment with PRP was carried out; the PRP was prepared from a sample of 10 ml of peripheral blood according to the usual methods and injected locally a few minutes before the cells of the sample. The results obtained via these two treatments showed a preservation of the femur head. It should be noted that, in several of these patients presenting with tissue necrosis that had already set in, there was complete disappearance of the necrosis, suggesting a process of regeneration and replacement of the necrotic tissues. The combination of the RGTA and of the autologous cells advantageously makes it possible to obtain this regeneration with a synergistic and surprising effect, improved by the injection of the PRP. In this example, the effect of the composition according to some embodiments in the treatment of bone destructions in the case of a post-infection osteonecrosis was studied.

(48) Bone tissue destruction is a frequent consequence and a complication of the treatment of chronic wounds subsequent to osteomyelitis of infectious bacterial origin normally treated over a long period with antibiotics. The composition/method according to fee some embodiments was used and made it possible to save and to reform bone tissue. The synergistic action of the RGTA supplemented by the cell therapy enabled a regeneration of the lesioned bone tissue such that has never been observed with RGTA alone or the cell therapy alone. Several patients thus treated with several local injections of 0.1 ml of CACIPLIQ (OTR4120 at 100 microg.ml.sup.−1) in the healthy tissue part around the necrotic area, followed by injection of the same volume of mesenchymal cells taken from the patient's sternum without any other preparation were thus able to recover bone tissue before it was completely destroyed, which would have been difficult without this therapy. It should be noted that this treatment was given while maintaining the antibiotic treatment and that, however, no integral bone regrowth was observed, the treatment making it possible to save or even to recover, but not enabling ex nihilo regrowth of an entirely destroyed bone.

Example 4

Treatment of Joint and Tendon Lesions with an Example of a Composition According to Some Embodiments

(49) The related art describes the effects of the injection of RGTA alone, in particular OTR4131, in the treatment of tendon and joint lesions in sport and race horses (Coudry V et al. Journal of Equine Veterinary Science, 2014, 34 pages 1357-1360, David Carnicer, research report “Preliminary report: ultrasonographic evolution of tendon lesions treated with RGTA in horses” école nationale vétérinaire de [national veterinary school of] Maison Alfort); the same is true for the use of cell therapy, in particular of mesenchymal cells taken from horses at the iliac fossa or more commonly the sternum (Pechayre M. and Betizeau C. S0704, AVAC Conference, 2-4 Dec. 2011. Lyon annual conference).

(50) The PRP was also used in the same indications (http://fr.slideshare.net/dvmfun/platelet-rich-plasma-prp-therapy).

(51) A comparative study was carried out using horses treated by combining either RGTA and the cells according to the some embodiments, the RGTA and PRP, or RGTA, PRP and cell therapy, compared with a treatment with only the cells.

(52) In this example, the horses presented with either tendon lesions (at the level of the SDF tendon) or joint lesions (osteochondrosis dessicans, subchondral bone cyst, lesions of the meniscus). In all cases, the combined RGTA and mesenchymal cell treatment allowed a much faster recovery of the race horses (return in approximately 5-6 months, that is to say a gain of one to two months compared with the recovery time after treatment with the RGTA alone or a cell treatment alone). This recovery was evaluated in terms of the recovery from limping; a faster return to training and to prior performances for almost all the horses treated. For these horses, the RGTA was injected firstly within a week following the tendon lesion under echography. The injection of cells was carried out, as appropriate, on the same day but after that of the RGTA (most commonly approximately 30 minutes to one hour corresponding to the time required to prepare the cells). The RGTA injection was sometimes followed a few minutes later by the injection of PRP and then by the injection of the autologous cells.

(53) The amounts of RGTA, of cells and of PRP were respectively 1 ml of OTR4131, 1.5 ml of PRP lysate (from 10 ml of blood taken) and 1 ml of autologous cells, the administration of the PRP and of the cells being carried out one hour after the first injection of RGTA.

(54) The best results were obtained in the case of joint lesions when the cells were injected mixed with a biomaterial, such as collagen or hyaluronic acid or a combination of the two. In these cases, these products are of injectable quality in solution (used in plastic surgery for wrinkles or joints in human beings) and were directly added to the solution of cells before injection. The cell solution was then diluted approximately two-fold: 1 ml of cells and 1 ml of solution of biomaterials, preferably between 0.1 to 3 mg.ml.sup.−1.

(55) In another embodiment, an administration as a bolus was carried out, including either RGTA and cells, or RGTA, PRP and cells. The results obtained showed an efficacious and significantly improved treatment compared with a treatment including the administration of only one of the abovementioned elements. Moreover, even more surprising results were obtained by spacing out the administration of RGTA and of cells by a period of 30 to 60 minutes to a few hours (preferably the same).

(56) Currently, the treatment of joint lesions and suffering in patients is performed using cell therapies and in particular by local injection of autologous mesenchymal cells originating from bone marrow or from adipose tissue.

(57) However, this treatment does not make it possible to obtain an effective treatment and has failures. Thus, a treatment using the composition according to some embodiments via in particular the administration (injection) of RGTA into the synovial fluid, followed by the injection of autologous mesenchymal cells or of adipocytes, was carried out in several patients experiencing failure of a cell therapy alone. To do this, the amounts of RGTA and of cells were respectively 1 ml at 100 microg.ml.sup.−1, from 1 to 5 ml of autologous cells enriched with adipocyte mesenchymal cells by centrifugation and injected on the same day, generally 30 minutes to one hour after the injection of RGTA. The timing of administration by injection of the cells was carried out within the hour following the injection of RGTA or an injection of two solutions, one of RGTA immediately followed by an injection of the cells, or the simultaneous administration of the RGTA and of the cells via a single solution.

(58) The addition of hyaluronic acid after the injection of RGTA but before or with the injection of cells and also the combination with other biomaterials such as type 2 collagens gave even better results in certain patients.

(59) Following this treatment, and surprisingly, the lesions and associated suffering were resorbed, advantageously enabling the patients to gradually walk again without requiring the implantation of a knee prosthesis and without pain.

(60) In particular, in about twelve patients suffering from osteochondral lesions, very disabled in their movements and by the pain, who had previously been unsuccessfully treated with cells nor with the RGTA alone; following the abovementioned treatment, eight of them were able to recover a motor capacity by virtue of the double treatment of RGTA followed by autologous cells, clearly demonstrating the surprising and unexpected advantages of the composition according to some embodiments.

Example 5

Treatment of Digestive System Tissue Lesions with a Composition According to Some Embodiments

(61) The related art describes the properties of the RGTA on the digestive tract mucosae, buccal mucosae (Morvan et al., Am J Pathol. 2004 Feb; 164(2):739-46), gingival mucosae (Escartin et al., FASEB J. 2003 Apr; 17(6):644-51) and on ulcerations in the stomach or intestins (Meddahi et al., J Biomed Mater Res. 2002, 60(3):497-501); and also their capacity to reduce fibroses by acting on collagen synthesis, on normal or irradiated isolated cells (Alexakis C. et al., FASEB J. 2001 Jul; 15(9):1546-54) and also on biopsies from tissues from patients suffering from Crohn's disease (Alexakis C. et al., Gut. 2004 Jan; 53(1):85-90). However, no effect of RGTAs on digestive system tissue lesions, in particular regarding a possible lesion repair, has been observed or identified.

(62) In this example, a treatment using the composition according to some embodiments, namely an RGTA and cells, was applied to a patient suffering from Crohn's disease and suffering from a perineal fistula for several years. In this example, 0.5 ml of the RGTA, used at a concentration of 100 microg.ml.sup.−1, was injected in proximity to the fistula (0.1 ml/per injection and at 3 sites) followed, within 30 minutes of the injection, by the fraction enriched with cells originating from a 10 ml liposuction sample) (0.1 ml per injection, at two or three sites, of cells).

(63) The administration of the composition according to some embodiments made it possible to close the perianal fistula, whereas neither the RGTA alone nor the administration of these same cells had made it possible to obtain this closure and recovery.

(64) A second patient having undergone a surgical ablation of an epidermoid carcinoma very close to the sphincter, followed by chemoradiotherapy, who had lost the function of his sphincter was able to benefit from a local injection of RGTA OTR4120 (0.1 ml at 100 microg.ml.sup.−1) at several sites, followed, after 30 minutes, by a local injection of autologous adipocyte cells (100 microl/injection originating from an initial liposuction volume of 10 ml). Following this administration, rapid colonization of the cells of the lesional site was observed, allowing the patient to experience a functional recovery of the sphincter, which did not seem to be difficult.

Example 6

Treatment of Tissue Lesions Including the Administration of a Composition According to Some Embodiments

(65) In these examples, there was no objectification of the additive effect combining RGTA and cell therapy, because each case was isolated and unique, which did not allow an objectification of the effect(s). The only criterion observed and capable of being studied is whether or not the patient improved after treatment.

(66) In this example, the effect of the composition according to some embodiments in pulmonary regeneration was studied. Patients suffering from lesions of the pulmonary mucosae caused by exposure to toxic vapors from fires inhaled RGTA OTR4120 placed in a vaporizer and the autologous adipocyte cells were injected on the very same day, one hour after the inhalation. The vaporizer contained a solution of OTR4120 at 100 microg.ml.sup.−1, and the inhalation was for 10 minutes enabling the inhalation of approximately 5 ml. Within the following half an hour, autologous cells taken on the very same day by liposuction (without concentration) were injected (5 ml, IV). This administration allowed, surprisingly and unexpectedly, a rapid and functional recovery of the patient, namely improved breathing, whereas the patient treated had been experiencing therapeutic failure for several months.

(67) In this example, the effect of the composition according to some embodiments on lesioned tissues following the implanting of a stent was observed. To do this, an oral administration of RGTA was performed on the day of the implantation of the stent and followed, after 24 h, by the IV administration of autologous adipocyte cells (originating from a 50 ml liposuction). The oral intake of RGTA OTR4120 was in an amount of 50 ml of a solution at 100 microg.ml.sup.−1 and was maintained for 1 month. The injection of cells was repeated after 10 days and 20 days.

(68) Recolonization after the area lesioned after implantation of the stent was observed but not documented, showing, surprisingly, a re-endothelialization of the area of implantation of the stent, which had never been observed with the RGTA alone or the cells alone.

(69) In this example, the effect of the composition according to some embodiments on lesioned tissues, in this case a muscle accidentally irradiated and having suffered a functional loss its function. To do this, an oral administration of RGTA was carried out, namely 25 ml at 100 mg/ml for two days followed by the administration, in the irradiated area of the muscle, of cells, namely 5 injections of 1 ml of a solution of cells originating from 50 ml of bone marrow sample taken from the iliac fossa on the same day and then enriched by centrifugation and suspended in 5 ml of physiological saline. Following the administration, an evaluation of its functional activity was carried out. Surprisingly and unexpectedly, the treatment allowed a functional motor recovery, which appeared to be difficult.

(70) In this example, the effect of the composition according to some embodiments on the re-epithelialization of the cornea was studied. To do this, the sample of cells taken from the patient's buccal mucosae was seeded, on the same day, on to corneas locally pretreated with two drops of RGTA per cornea. This treatment made it possible to obtain repair of the lesioned tissue and in particular the effect was considerably increased by the administration of CACICOL alone.

(71) Finally, an evaluation of the effect of an example of a composition according to some embodiments on a spinal cord lesion was also carried out. A 20-year-old patient presented with degradation of motor function following a recent lesion of the spinal cord, giving him paralysis of the lower limbs. A treatment over the course of three days using an example of a composition according to some embodiments for which the amounts of RGTA and of cells were respectively 25 ml/day of RGTA orally of a solution at 100 mg/ml of OTR4120 and local injection (in the area peripheral to the spinal cord lesion) of 1 ml, at 4 points, of cells originating from a 50 ml puncture of bone marrow from the iliac fossa. This injection was repeated at 10 and 20 days, while the daily oral intake of RGTA was maintained for a period of two months.

(72) Unexpectedly and very positively, the patient began to recover a small amount of neuromotor function after the first three days of days of treatment and gradually a noticeable improvement as far as motor recovery.