Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
  • A61K31/727—Heparin; Heparan
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like

Definitions

• the invention relates to a composition of glycosaminoglycans for the treatment of chronic ulcers, in particular diabetic foot ulcers, and pressure ulcers. More particularly, it refers to compositions of low molecular weight heparins (LMWH) and very low molecular weight heparins (HMBPM) in the treatment of chronic ulcers, and more specifically in the manufacture of a medicament for the treatment of chronic ulcers .
• LMWH low molecular weight heparins
• HMBPM very low molecular weight heparins
• DM Diabetes Mellitus
• Spain could be between 2% and 6%; 10% would correspond to type 1 DM, with 50% of patients with type 2 DM undiagnosed. Taking into account that the published studies of prevalence of DM in different regions of Spain are prior to 2002 and the tendency to increase the incidence, currently in Spain it is estimated that there would be more than 2.5 million people with diabetes.
• diabetic foot proposed by the Consensus Group on Diabetic Foot of the Spanish Society of Angiology and Vascular Surgery is: "Clinical alteration of neuropathic etiopathogenic base induced by sustained hyperglycemia, in which with or without coexistence of ischemia, and prior traumatic trigger, the injury and / or ulceration of the foot occurs. "
• Foot ulceration is a significant complication of diseases such as diabetes with an annual incidence slightly higher than 2% (Abbott CA, et al (2002) The North-West Diabetes Foot Care Study: incidence of, and risk factors for, new diabetic foot ulceration in a community-based patient cohort. Diabet. Med. 19 (5): 377-84). It is estimated that 15% of patients with diabetes will develop ulcers at some time in their lives (Reiber GE (1996) The epidemiology of diabetic foot problems. Diabet. Med. 13 Suppl 1: S6-11) and that about 10 % - 30% of those with ulcers will progress with the amputation of the limb (Lipsky BA (2004) Medical treatment of diabetic foot infections. Clin. Infeci Dis. 39 Suppl 2: S104-14). In addition, this is complicated in the case in which an ischemia occurs in the lower limb in which the ulcer appears, being produced in most cases due to insufficient blood supply due to a high incidence of thrombosis.
• Dermagraft® is produced by sowing fibroblasts of human dermis on a synthetic scaffold of bioabsorbable material that has proven effective in low-grade ulcers with a greater proportion of healing in a shorter time (Marston WA, et al. (2003) Dermagraft Diabetic Foot Ulcer Study Group. The efficacy and safety of Dermagraft® in improving the healing of chronic diabetic foot ulcers: results of a prospective randomized trial. Diabetes Care 26: 1701-5).
• Apligraf® consists of a dermis layer composed of human fibroblasts in a matrix of bovine type I collagen and an epidermis layer formed of human keratinocytes.
• PDGF Platelet Derived Growth Factor
• a method of administering a healing agent such as Epidermal Growth Factor (EGF) has been published a few years ago, which consists of the infiltration of a solution of the biomolecule into the lesion by several injections (WO 03053458).
• EGF Epidermal Growth Factor
• This treatment has been shown to be effective in the prevention of amputation of the diabetic foot but has the disadvantage that it is traumatic for the patient since the application of injections in the lesion is very painful and in each treatment several injections must be applied for several weeks.
• WO2007087759 refers to a pharmaceutical composition containing microspheres with epidermis growth factors for parenteral administration for patients who have chronic skin conditions, such as diabetic foot ulcers.
• Many other patents have focused on other methods of accelerating the healing range. However, none of these methods have proven to be widely effective.
• LMWH low molecular weight heparin
• dalteparin improves the evolution of diabetic foot ulcers in patients with peripheral occlusive arterial disease.
• acetylsalicylic acid that is, it can be seen how the association of two active principles with anticoagulant effect synergistically favors the evolution of diabetic foot ulcers in patients with peripheral occlusive arterial disease .
• Low molecular weight heparin seem to improve local capillary circulation and healing of chronic foot ulcers in diabetic patients", VASA, Brand 22, 1993, FET 2, the realization of double-blind, placebo-controlled clinical trials is disclosed.
• dalteparin could have a beneficial effect on the prevention of this type of wounds, although always in prophylaxis doses, that is, at no time are doses of dalteparin greater than 2500 Ul / day used, since that hemorrhages are anticipated during treatment at higher doses, without this increase being more effective in the case of patients with diabetic foot ulcer.
• This is due to the general belief in the state of the art that diabetic patients have a higher risk of bleeding than patients who do not have the disease (Adverse impact of bleeding on prognosis in patients with acute coronary syndromes, Eikelboom JW et al. Circulation 2006 Aug 22; 114 (8) 774-82).
• glycosaminoglycans in the treatment of chronic ulcers, specifically diabetic foot ulcers, and in particular the use of some low molecular weight heparins for this purpose, has been roughly described for patients who have Severe circulatory conditions since these low molecular weight heparins have antithrombotic and anticoagulant activity, expressed as an activated anti-factor X effect (Xa) and anti-Na factor effect.
• Xa activated anti-factor X effect
• anti-Na factor effect anti-Na factor effect
• the antithrombotic activity of fondaparinux is a consequence of the selective inhibition of factor Xa, mediated by antithrombin III (ATIII).
• ATIII antithrombin III
• the fondaparinux power approximately 300 times the innate neutralization of factor Xa by the ATIII.
• the neutralization of factor Xa interrupts the blood coagulation cascade and inhibits thrombin formation and thrombus development (see Figure I).
• the researchers of the present invention have been able to determine experimentally that the beneficial healing effect of the administration of glycosaminoglycans with less amount of pentasaccharide in any type of ulcer that is considered chronic - not only in diabetic foot ulcers - and that said Administration is effective through three main routes of administration: parenteral, oral and topical.
• the proportion of pentasaccharide is limited to a maximum of 20% of the characteristic disaccharide of pentasaccharide (D-Glucuronic acid linked to N-sulfo-3-sulfo-D-Glucosamine) in the structure of the glycosaminoglycan which, according to the state of the art, is responsible for the anti FXa activity and that, therefore, according to it, should be in greater proportion.
• D-Glucuronic acid linked to N-sulfo-3-sulfo-D-Glucosamine in the structure of the glycosaminoglycan
• chronic ulcers are understood as continuity solutions with loss of substance in the skin, depending on the origin of the ulcer, they can be classified as: pressure ulcers, diabetic ulcer, ischemic ulcer (arterial or venous) , post-burn ulcer, posttradiotherapy ulcers, etc.
• ischemia can be caused by external pressure on the capillaries
• the composition object of the present invention works for chronic ulcers even for patients who do not have diabetes and for whom certain types of chronic ulcers appear that do not heal easily such as pressure ulcers, understanding pressure ulcers such as those that have areas of damage to the skin and underlying tissue caused by prolonged pressure on a hard plane, not necessarily intense, and independent of the position.
• ulcer by decubitus is currently discarded because it does not refer to pressure, a determining factor in its appearance, and to exclude that which has not appeared in recumbency.
• a main aspect of the present invention is directed to a glycosaminoglycan composition that contains a proportion of certain monosaccharides and contains a proportion that does not exceed 20%, of the characteristic disaccharide of the pentasaccharide, for the treatment of chronic ulcers and in particular Ia diabetic foot ulcer.
• the inventors of the present invention have also developed a relationship between the necessary amount of the monosaccharides mentioned above and the dose of a medicament containing a pharmaceutically effective amount of glycosaminoglycan, relating the proportion of monosaccharides (Ul) with the plasma half-life of the product, to achieve a dose of "treatment" of the disease and not of prophylaxis.
• the amount of pentasaccharide responsible for the antithrombotic activity is more than enough to favor the vascularization of lower extremities in patients with diabetic foot ulcer, and at doses higher than the indicated Ia general belief is that the risk of bleeding it increases by excess of said antithrombotic effect, taking into account that diabetic patients have a greater predisposition to bleeding, without realizing that the really important thing is to administer a greater amount of other monosaccharides that are not part of the pentasaccharide in the formulation.
• glycosaminoglycan compositions such as low molecular weight heparins without being prophylactic doses
• these are not curative but preventive and do not really cure ulcers by healing with regeneration of granulation tissue, but are used because they are believed to prevent their formation and in most cases used in synergistic treatment with other drugs such as Acetylsalicylic acid (Effect of Dalteparin of healing of chronic foot ulcers in diabetic patients with peripheral arterial occlusive disease ", Diabetes Care, vol. 26 (9), September 2003; M. Kalani, A. et al. Titled Beneficial effects of dalteparin on haemostatic function and local tissue oxygenation in patients with diabetes, severe vascular disease and foot ulcers. Thrombosis Research. 120, 653-661, 2007) .
• the inventors of the present invention have managed to eliminate prejudices of the state of the art, since they have determined that in the case of certain glycosaminoglycans, such as LMWH and HMBPM, the amount of pentasaccharide for the treatment of foot ulcers Diabetic (in diabetic patients) is not significant, but what is really important is the presence of a proportion of oligosaccharide sequences rich in certain specific monosaccharides.
• This proportion of certain specific monosaccharides is responsible for the healing of this type of ulcers in diabetic patients since when this type of glycosaminoglycans is administered subcutaneously or parenterally, the healing effect It is due to the presence of certain oligosaccharide fractions that do not contain the pentasaccharide commonly linked in the state of the art with the anti-factor Xa effect in heparins of low molecular weight and very low molecular weight.
• the inventors of the present invention have determined that provided that the pentasaccharide is slightly altered (quantitatively and qualitatively) in the heparinoid structure, the anti FXa activity decreases considerably. However, an analogous decrease in healing power is not observed.
• an important problem in the treatment of diabetic foot ulcers is to achieve the effective and effective dose of a medication, which achieves the regeneration of ischemic tissue and prevents
• Another additional advantage that the treatment of choice must have is that it is not very traumatic for the patient since the diabetic foot ulcers are characterized by being extremely painful, so that the route of administration must be little traumatic in the proximity of the wound. Therefore, for the purposes of the present invention the preferred routes of administration are the parenteral route, the oral route and the non-invasive topical route or of direct application on
• the mucosa to be treated The mucosa to be treated.
• Figure 1 Represents the mechanism of action of synthetic pentasaccharide (fondaparinux).
• Figure 2A Representation of the region of the anomeric signals (H1-C1 correlations) of the 13 C- 1 H HSQC spectrum of fondaparinux (pentasaccharide responsible for the interaction with antirombine III) recorded at 298 K in deuterated water (D2O). In the spectrum we can observe five correlation peaks that correspond to the five monosaccharides that make up the pentasaccharide.
• Figure 2B Representation of the region of the anomeric signals (H1-C1 correlations) of the 13 C- 1 H HSQC spectrum of the low molecular weight heparin bemiparin, recorded at 298 K in deuterated water (D2O). The spectrum is much more complex than that obtained for fondaparinux (see figure
• the intensity of this signal has decreased compared to the fondaparinux sample due to the increase in the proportion of D-glucuronic acid linked to N-sulfo-D-glucosamine G- (ANS), this being the majority glucuronic ring.
• the signals have also been labeled: ANSred, N-sulfo-D-glucosamine of the reducing end; ANS, 3S, N-sulfo-30-sulfo-D-glucosamine.
• Figure 2C Representation of the region of the anomeric signals (H1-C1 correlations) of the 13 C- 1 H HSQC spectrum of the very low molecular weight heparin R014 ckw2_13_11 L, recorded at 298 K in deuterated water (D2O).
• the intensity of this signal has decreased compared to the fondaparinux sample due to the increase in the proportion of D-glucuronic acid linked to N-sulfo-D-glucosamine G- (ANS), this being the majority glucuronic ring.
• Figure 2D Representation of the region of the anomeric signals (H1-C1 correlations) of the 13 C- 1 H HSQC spectrum of the enoxaparin low molecular weight heparin, recorded at 298 K in deuterated water (D2O).
• the spectrum is much more complex than that obtained for fondaparinux (see Figure 2A), due to the presence of new monosaccharide units that were not present in fondaparinux (I, sulphated L-iduronic acid; ANAc, N-acetyl-D-glucosamine ) and the alteration of the proportion of the existing units, as is the case of the sulfated L-iduronic acid ring in 2, I2S, which is present in greater proportion.
• the correlation peak H1-C1 of the unit G- (ANS, 3S) characteristic of the pentasaccharide has been highlighted with a circle.
• the intensity of this signal has decreased compared to the fondaparinux sample due to the increase in the proportion of D-glucuronic acid linked to N-sulfo-D-glucosamine G- (ANS), this being the majority glucuronic ring.
• Figure 2E Representation of the region of the anomeric signals (H1-C1 correlations) of the 13 C- 1 H HSQC spectrum of the low molecular weight heparin tinzaparin, recorded at 298 K in deuterated water (D2O).
• the spectrum is much more complex than that obtained for fondaparinux (see Figure 2A), due to the presence of new monosaccharide units that were not present in fondaparinux (I, sulphated L-iduronic acid; ANAc, N-acetyl-D-glucosamine ) and the alteration of the proportion of the existing units, as is the case of the sulfated L-iduronic acid ring in 2, I2S, which is present in greater proportion.
• the correlation peak H1-C1 of the unit G- (ANS, 3S) characteristic of the pentasaccharide has been highlighted with a circle.
• the intensity of this signal has decreased compared to the fondaparinux sample due to the increase in the proportion of D-glucuronic acid linked to N-sulfo-D-glucosamine G- (ANS), this being the majority glucuronic ring.
• Figure 2F Representation of the region of the anomeric signals (H1-C1 correlations) of the 13 C- 1 H HSQC spectrum of the very low molecular weight heparin RO14_H13_96_5, recorded at 298 K in deuterated water (D2O).
• the intensity of this signal has decreased compared to the fondaparinux sample due to the increase in the proportion of D-glucuronic acid linked to N-sulfo-D-glucosamine G- (ANS), this being the majority glucuronic ring.
• Figure 2G Representation of the region of the anomeric signals (H1-C1 correlations) of the 13 C- 1 H HSQC spectrum of the low molecular weight heparin BEMI_99_4, recorded at 298 K in deuterated water (D2O).
• the spectrum is very different from that obtained for fondaparinux (see Figure 2A), due to the presence of new monosaccharide units that were not present in fondaparinux (I, sulphated L-iduronic acid; ANAc, N-acetyl-D-glucosamine) and The disappearance of some of the characteristic signals such as ANS, 3S and G- (ANS, 3S).
• Figure 2H Representation of the region of the anomeric signals (H1-C1 correlations) of the 13C-1H HSQC spectrum of sodium heparin, recorded at 298 K in deuterated water (D20).
• the spectrum is much more complex than that obtained for fondaparinux (see figure 1), due to the presence of new monosaccharide units that were not present in fondaparinux (I, sulphated L-iduronic acid; ANAc, N-acetyl-D-glucosamine ) and the alteration of the proportion of the existing units, as is the case of the sulfated L-iduronic acid ring in 2, I2S, which is present in greater proportion.
• the correlation peak H1-C1 of the unit G- (ANS, 3S) characteristic of the pentasaccharide has been highlighted with a circle.
• the intensity of this signal has decreased compared to the fondaparinux sample due to the increase in the proportion of D-glucuronic acid linked to N-sulfo-D-glucosamine G- (ANS), this being the majority glucuronic ring.
• Figure 21 Representation of the region of the anomeric signals (H1-C1 correlations) of the 13C-1H HSQC spectrum of the low molecular weight heparin BEMI_99_2, recorded at 298 K in deuterated water (D20).
• the spectrum is very different from that obtained for fondaparinux (see figure 1), due to the presence of new monosaccharide units that were not present in fondaparinux (I, sulphated L-iduronic acid; ANAc, N-acetyl-D-glucosamine; ANH2 , D-glucosamine; ANH2,3S, D-3-0-sulfo-glucosamine) and the disappearance of some of the characteristic signals such as ANS, 3S and G- (ANS.3S).
• I sulphated L-iduronic acid
• ANAc N-acetyl-D-glucosamine
• ANH2 D-glucosamine
• ANH2,3S D-3-0-sulfo-glucosamine
• Figure 2J Representation of the region of the anomeric signals (H1-C1 correlations) of the 13C-1H HSQC spectrum of calcium heparin, recorded at 298 K in deuterated water (D20).
• the spectrum is much more complex than that obtained for fondaparinux (see figure 1), due to the presence of new monosaccharide units that were not present in fondaparinux (I, sulphated L-iduronic acid; ANAc, N-acetyl-D-glucosamine ) and the alteration of the proportion of the existing units, as is the case of the sulfated L-iduronic acid ring in 2, I2S, which is present in greater proportion.
• the correlation peak H1-C1 of the unit G- (ANS, 3S) characteristic of the pentasaccharide has been highlighted with a circle.
• the intensity of this signal has decreased compared to the fondaparinux sample due to the increase in the proportion of D-glucuronic acid linked to N-sulfo-D-glucosamine G- (ANS), this being the majority glucuronic ring.
• the signals have also been labeled:
• Figure 2K Representation of the region of the anomeric signals (H1-C1 correlations) of the 13 C- 1 H HSQC spectrum of the low molecular weight heparin BP06408-66 / 27-A, recorded at 298 K in deuterated water (D2O) .
• the spectrum is very different from that obtained for fondaparinux (see figure 2A), due to the presence of new monosaccharide units that were not present in fondaparinux (I, unsaturated L-iduronic acid; ANAc, N-acetyl-D-glucosamine) and the decrease in signals corresponding to ANS, 3S and G- (ANS , 3S).
• the decrease in the signals of the pentasaccharide, G- shows that this type of low molecular weight heparin has a low concentration of the structural motive responsible for the interaction with the antithrombin III.
• Figure 2L Representation of the region of the anomeric signals (H1-C1 correlations) of the 13C-1H HSQC spectrum of the low molecular weight heparin BP06408-66 / 28-A, recorded at 298 K in deuterated water (D20).
• the spectrum is very different from that obtained for fondaparinux (see figure 2A), due to the presence of new monosaccharide units that were not present in fondaparinux (I, sulphated L-iduronic acid; ANAc, N-acetyl-D-glucosamine) and The disappearance of the signals corresponding to ANS, 3S and G- (ANS, 3S).
• the disappearance of the pentasaccharide signal, G- shows that this type of low molecular weight heparin does not show detectable amounts of the structural motive responsible for the interaction with the antithrombin III.
• Figure 3 Shows the percentage reduction of ulcerated area in the mice of Example 2 when bemiparin is administered with a ratio between the half-life and the dose of 1: 950, bemiparin with a ratio between the half-life and the dose of 1: 472 and fondaparinux at equivalent doses of treatment, ie 5mg / ml, since it has a plasma half-life of 17 hours in healthy individuals, so that in wistar rat it is 0.22 mg per rat.
• Figure 4 Shows the evolution of the diabetic control mice of Example 4 with respect to mice treated with sodium bemiparin. The area of the wound has been represented to the right of each animal.
• Figure 5 Shows the evolution of the percentage of wound healing over time in the mice of Example 4.
• FIG. 1 Photomicrographs of the wound sections of the mice of Example 4 after staining with hematoxylin-eosin.
• a diabetic control B diabetic animal treated with sodium fondaparinux, C diabetic animal treated with sodium bemiparin.
• Figure 7 The percentage of reduction in ulcerated area of the mice of Example 5 when they are administered a control composition topically, a topical composition of sodium bemiparin and a topical composition of fondaparinux sodium, a topical composition of unfractionated heparin.
• Figure 8 Shows the evolution of the non-diabetic control mice of Example 5 with respect to the mice treated with calcium heparin. The area of the wound has been represented to the right of each animal.
• Figure 9 Shows the evolution of the percentage of wound healing over time in the mice of Example 5.
• Figure 10 Photomicrographs of the wound sections of the mice of Example 5 after staining with hematoxylin-eosin. A non-diabetic control. B non-diabetic animal treated with sodium bemiparin, C non-diabetic animal treated with calcium heparin.
• Figure 11 The percentage reduction of ulcerated area in diabetic wistar rat when an oral control solution, an oral suspension of sodium bemiparin of 100 mg / kg and a suspension of sodium fondaparinux of 10 mg / kg is represented
• the problem to be solved by the present invention is, therefore, to provide a pharmaceutical composition that solves the problems of the prior art.
• the solution is based on the fact that the inventors have identified that a pharmaceutical composition of glycosaminoglycans subcutaneously or parenterally, orally and / or topically, in particular LMWH and HMBPM, for the treatment of chronic ulcers such as diabetic foot, which contains Ia following proportion of the following monosaccharides (in which all the percentages are over the total percentage of monosaccharides of the composition): a) N-sulfo-D-Glucosamine: 25-50% b) D-Glucuronic Acid: 3-25% and that it is characterized in that the proportion of the disaccharide unit D-Glucuronic Acid linked to N-sulfo-3-sulfo-D-Glucosamine is not more than 25%, and preferably 20%, manages to solve the indicated problem, since it achieves Tissue regeneration, preventing limb amputation, and is effective as a therapeutic treatment and not only in prophylaxis.
• Another aspect of the invention also refers to the use of a composition of glycosaminoglycans subcutaneously or parenterally, orally and / or topically, in particular LMWH and HMBPM, which contain a characteristic proportion of at least the monosararides selected from the N- group.
• LMWH and HMBPM which contain a characteristic proportion of at least the monosararides selected from the N- group.
• sulfo-D- Glucosamine, N-acetyl-D-Glucosamine, L-lduronic acid, L-lduronic acid 2-sulfated and D-Glucuronic acid in the manufacture of an alternative medication for the treatment of chronic ulcers such as pressure ulcers and those with diabetic foot.
• the solution is based on the fact that the inventors have identified that, contrary to the generalized belief, it is possible to administer to a patient suffering from diabetic foot ulcer a treatment consisting of a dose of low molecular weight heparin (LMWH) classified as a dose of treatment of venous thromboembolic disease without a greater risk of bleeding, and surprisingly better results are obtained than if administered at lower concentrations such as usual concentrations for prophylaxis, due to the presence of a certain proportion of certain monosaccharides in glycosaminoglycans .
• LMWH low molecular weight heparin
• the present invention provides a pharmaceutical composition for the treatment of diabetic foot ulcer that uses the usual doses of treatment for venous thromboembolic disease, understood as treatment doses, those in which the relationship between the time of Plasma half-life and the dose in LMW of the LMWH is between 1: 800 and 1: 5,000, that is, those that contain a greater amount of specific monosaccharides
• a pharmaceutical composition of glycosaminoglycans is provided subcutaneously or parenterally, orally and / or topically, in particular LMWH and HMBPM, for treatment of diabetic foot ulcer containing the following proportion of the following monosaccharides:
• a pharmaceutical composition of glycosaminoglycans is provided subcutaneously or parenterally, orally and / or topically, in particular LMWH and HMBPM, for treatment of diabetic foot ulcer containing the following proportion of the following monosaccharides:
• a pharmaceutical composition of glycosaminoglycans is provided subcutaneously or parenterally, orally and / or topically, in particular LMWH and HMBPM, for treatment of diabetic foot ulcer containing the following proportion of the following monosaccharides:
• N-sulfo-D-Glucosamine 25-50%
• N-acetyl-D-Glucosamine 0.1-8%
• the present invention is directed to a pharmaceutical composition of glycosaminoglycans subcutaneously or parenterally, orally and / or topically, in particular LMWH and HMBPM, for the treatment of chronic ulcers such as diabetic foot, which contains a proportion of the disaccharide unit D-Glucuronic acid linked to N-sulfo-3-sulfo-D-Glucosamine (ANS 3S) less than 20%, even more preferably less than 10% and more preferably less than 5% of the totality of The saccharide composition.
• LMWH and HMBPM disaccharide unit D-Glucuronic acid linked to N-sulfo-3-sulfo-D-Glucosamine
• One of the merits of this invention is not only to have eliminated a prejudice of the state of the art, but to achieve an effective correlation between the proportion of the monosaccharides mentioned above with the dose of medicine containing the pharmaceutical composition mentioned above necessary to treat The diabetic foot ulcer as well as to promote tissue healing and regeneration in patients based on the plasma half-life of these glycosaminoglycans.
• a further aspect of the invention is directed to the use of glycosaminoglycans, in particular low molecular weight heparins, in the manufacture of a medicament for the treatment of diabetic foot ulcer that is characterized in that the relationship between the average life time Plasma and the dose in Ul of LMWH is between 1: 800 and 1: 5,000, that is, it is characterized in that a composition containing a greater amount of the monosaccharides selected from the group formed by N-sulfo-D-Glucosamine N- is administered.
• acetyl-D-Glucosamine and D-Glucuronic Acid and in preferred embodiments of the invention also L-lduronic acid and L-lduronic acid 2-sulphated, and a proportion of the disaccharide unit D-Glucuronic Acid linked to N-sulfo-3-sulfo-D-Glucosamine less than 20% of the entire saccharide composition, for treatment of diabetic foot ulcer, more preferably less than 10% and Io more preferably less than 5%
• the invention is directed to the use of a low molecular weight heparin in the manufacture of a medicament for the treatment of diabetic foot ulcer in which LMWH is a heparin of average molecular weight less than 6,000 daltons.
• the invention is directed to the use of a low molecular weight heparin in the manufacture of a medicament for the treatment of diabetic foot ulcer characterized in that the plasma half-life is between 5.2 and 5.4 and the dose daily average is 5,000 Ul, which ensures a specific proportion of the monosaccharides selected from the group formed by N-sulfo-D-
• Glucosamine and D-Glucuronic Acid and in preferred embodiments of the invention also N-acetyl-D-
• Glucosamine, L-lduronic acid and L-lduronic acid 2-sulphated and the D-Glucuronic acid disaccharide unit linked to N-sulfo-3-sulfo-D-Glucosamine less than 20% of the total saccharide composition, more preferably less than 10% and more preferably less than 5%
• the invention is directed to the use of a low molecular weight heparin in the manufacture of a medicament for the treatment of diabetic foot ulcer characterized in that the plasma half-life is between 4.0 and 4.4 and Ia Average daily dose is 7,600 Ul, which ensures a specific proportion of the monosaccharides selected from the group consisting of N-sulfo-D-Glucosamine, N-acetyl-D-Glucosamine, L-lduronic acid, L-lduronic acid 2- sulfated and D-Glucuronic Acid.
• the invention is directed to the use of a low molecular weight heparin in the manufacture of a medicament for the treatment of diabetic foot ulcer characterized in that the plasma half-life is between 2.3 and 2.8 and the average daily dose is of 10,000 Ul, which ensures having a specific proportion of the monosaccharides selected from the group consisting of N-sulfo-D-Glucosamine and D-Glucuronic Acid, and in preferred embodiments of the invention also N-acetyl-D-Glucosamine, Acid L -duronic and L-lduronic acid 2-sulfated.
• the inventors of the present invention consider the results of the examples to endorse their hypothesis, since if they use the synthetic pentasaccharide (fondaparinux) for the treatment of diabetic foot ulcer, the healing activity and / or regenerative is very diminished (when it should be the other way around) in the same way as if they modify chemically and / or enzymatically the monosaccharides selected from the group formed by N-sulfo-D-Glucosamine, N-acetyl-D-Glucosamine, Acid L -duronic, L-lduronic acid 2-sulphated and D-Glucuronic acid, the activity of the glycosaminoglycan composition decreases dramatically, therefore it is concluded that although there is a portion of pentasaccharide in the structure it is not responsible for the healing activity and / or regenerative tissue as well as if we qualitatively or quantitatively alter the aforementioned monosaccharides we do not obtain a bu
• glycosaminoglycans such as low molecular weight heparins
• the relationship between the life time Plasma mean of each LMWH and the dose in Ul of LMWH is between 1: 800 and 1: 5,000 provide the indicated advantages of the invention.
• Pentasaccharide analog with high affinity for antithrombin III obtained by chemical synthesis. Among others it has the following characteristics: Molecular Weight of 1728 Daltons and an anti-factorXa activity: 700 Ul / mg.
• Bemiparin it is a second generation heparin, of low molecular weight (average molecular weight of 3,600 daltons) and an anti-Xa / anti-lla ratio greater than 8. Bemiparin is obtained by a new method of depolymerization and fractionation -beta elimination in non-aqueous medium in order to achieve an even lower molecular weight than the previous heparins, as well as an optimal distribution of its fragments, so that the percentage of fragments of more than 6,000 Daltons resulting is much lower than the rest of HPBM, with a high proportion of chains below the critical length (PM ⁇ 5.400 D).
• - Enoxaparin Low molecular weight heparin obtained by depolymerization by a method of ⁇ -elimination in aqueous medium of the previously formed benzyl esters of heparin. Among others, it has the following characteristics (Ph. Eur. 6th Edition): Average Molecular Weight of 3500 - 5500 Daltons and an anti-factorXa activity: 90 - 125 Ul / mg - Dalteparin: Low molecular weight heparin obtained by depolymerization with nitrous acid . Among others it has the following characteristics (Ph. Eur. 6th Edition): Average Molecular Weight of 5600 - 6400 Daltons and an anti-factorXa activity: 110 - 210 Ul / mg
• Tinzaparin Low molecular weight heparin obtained by enzymatic depolymerization with heparinase I. Among others it has the following characteristics (Ph. Eur. 6th Edition): Average Molecular Weight of 5500 - 7500 Daltons and an anti-factorXa activity: 70 - 120 Ul / mg
• - RO-14 Very low molecular weight heparin obtained by depolymerization by a method of ⁇ -elimination in non-aqueous medium. Among others it has the following characteristics: Average molecular weight of 1800-3000 Daltons and has an anti-factorXa activity: 80-140 Ul / mg. - BEMI-99/4: It is a low molecular weight heparin that has altered the degree of sulphation, so that it only has sulfate groups in the N position of glucosamines. For this reason, its anti-factor Xa activity decreases considerably since the portion of the pentasaccharide is affected qualitatively and quantitatively. The modifications to obtain this heparin are based on an N, O-desulphation reaction that is carried out according to the conditions described by Nagasawa and Inoue
• the eluate is neutralized with pyridine and lyophilized, obtaining 11.2 g of Bemiparin pyridinium salt.
• the pyridinium salt of bemiparin (3 g) is dissolved in 75 ml of dimethylsulfoxide containing 10% methanol, and the solution is maintained for 24 h at 105 0 C. After this time, 75 ml of water are added, the pH at 9.0-9.5 with sodium hydroxide and stir for 15 minutes.
• the solution is neutralized with hydrochloric acid, 0.5 g of sodium chloride is dissolved and precipitated with the addition of 3 volumes of methanol, obtaining the N, O-desulphated derivative.
• the N, O-desulphated derivative (1 g) is N-sulfata, according to the conditions of Lloyd et al.
• the product is dissolved in 75 ml of a saturated sodium bicarbonate solution and the pH is adjusted to 9.
• the solution is heated to 55 0 C and 3 g of sulfur trioxide are added - trimethylamine.
• the reaction is maintained 3 h at 55 0 C.
• 3 g of sulfur trioxide trimethylamine complex is added again, leaving the reaction at 3 0 more for 3 hours.
• the solution is cooled and the pH is adjusted to 7.
• With hydrochloric acid 1 g of sodium chloride is added and precipitated with three volumes of methanol. 0.88 g of BEMI-99/4 are obtained.
• the product obtained has the following characteristics: Average molecular weight of 3468 Daltons and an anti-factor Xa activity: 4 Ul / mg.
• Sepharose 4B previously activated with ATIII Human
• ATIII Human
• HóóK et al. Hóók M, Bjórk I, Hopwood J, Lindahl U. FEBS Lett. 1976; 66,: 90-93.
• the column is eluted at 4 0 C with a pH 7.40 buffer solution of 1mM Tris-HCI + 0.4M NaCI, thus eluting the low affinity fraction.
• This fraction is purified by a Biogel P2 column, eluting with water.
• the solution containing the product is lyophilized, finally obtaining 7.6 mg of product.
• the product obtained has the following characteristics: Average molecular weight of 3567 Daltons and an anti-factor Xa activity: 32 Ul / mg.
• This fraction is purified by a Biogel P2 column, eluting with water.
• the solution containing the product is lyophilized, finally obtaining 3.6 mg of product.
• the product obtained has the following characteristics: Average molecular weight of 3611 Daltons and an anti-factorXa activity: 5.4 Ul / mg.
• the experiments carried out have generally been the following: preparation and quantification and identification of fractions of glycosaminoglycan samples by NMR and administration in previously diabetized wistar rats and in genetically modified mice to assess the healing of said ulcers.
• the inventors of the present invention have analyzed commercial samples and samples of their own research, to determine the monosaccharide proportions of those monosaccharides responsible for the healing of the diabetic foot ulcer as well as the different saccharide fractions present in the pentasaccharide.
• the average content of monosaccharides in the samples of glycosaminoglycans has been determined by the Nuclear Magnetic Resonance (NMR) technique, using quantitative 13 C- 1 H HSQC (heteronuclear single quantum coherence) experiments, according to the method described by Marco Guerrini et al.
• NMR Nuclear Magnetic Resonance
• HSQC heteroclear single quantum coherence
• the amount of the unit of D-glucuronic acid linked to N-sulfo-3-sulfo-D-glucosamine G- (ANS, 3S) present in GAGs obtained from natural heparin, can be directly related to the anti-Xa activity of them, as described by M. Guerrini et al.
• This disaccharide belongs to the pentasaccharide responsible for the interaction with the antithrombin III and is only detected in the active sequences.
• the correlation signal of the anomeric carbon of this type of glucuronic acid with the directly bound hydrogen appears in a characteristic and free region of overlap in the HSQC spectrum and therefore it can be used to quantify the proportion of the pentasac on the GAG.
• the GAGs studied present new monosaccharide units, such as the rings of unsaturated iduromic acid (I) or N-acetylated glusamines (ANAc).
• the proportion of the sulfated iduromic acid unit in 2 is much greater than in the pentasaccating. Therefore, the samples analyzed mostly contain oligosacan chains other than fondaparmux.
• Wistar rat diabetization Wistar rat diabetization.
• streptozocin (STZ) were administered intramuscularly of 45 mg / kg of streptozocin to make a batch of 8 rats diabetic (Andrades Ja et al. Engineering, expression, and renaturation of a collagen-targeted human bFGF fusion protein Growth Factors 2001; 18 (4): 261-75)
• Table 1 Wistar rats weight-glycemia measure
• the selection of rats included in each group was randomized.
• Bemiparin administrations were performed using a solution of bemiparin subcutaneously (100 ⁇ L) for the treatment of ulcers.
• the diameter of the ulcer was quantified by tracing the ulcer in transparency to see the progress of the healing.
• Table 2 shows the calculation of percentage of decrease in area of day 9 with respect to day 1:
• streptozocin (STZ) were administered intramuscularly of 45 mg / kg of streptozocin to make a batch of 8 rats diabetic (Andrades Ja et al. Engineering, expression, and renaturation of a collagen- targeted human bFGF fusion protein Growth Factors 2001; 18 (4): 261-75).
• the selection of rats included in each group was randomized.
• the diameter of the ulcer was quantified by tracing the ulcer in transparency to see the progress of the healing.
• Table 4 shows the calculation of percentage of decrease in area of day 9 with respect to day 1:
• streptozocin (STZ) was administered intramuscularly of 45 mg / kg of streptozocin to make a batch of 10 rats diabetic (Andrades Ja et al. Engineering, expression, and renaturation of a collagen- targeted human bFGF fusion protein Growth Factors 2001; 18 (4): 261-75)
• Diabetic rats were given sugar water (350 mg / dL) for three days and glucose measurements were taken with a glucometer every day to verify that the rats were indeed diabetized. Therefore, before the administration and on time 4 days after the administration of streptozocin, a drop of blood was drawn from the vein of the tail of the rat for the subsequent quantification of the glycemia and see if they were diabetic to begin with the ulceration test .
• the selection of rats included in each group was randomized.
• Table 6 shows the calculation of the percentage of healing on day 9 with respect to day 1:
• the rats in a 9-day study process, have a healing percentage of approximately 50%, that is to say that in 9 days the wound is cut in half.
• an oligosaccharide composition with a higher percentage of monosaccharides a) N-sulfo-D-Glucosamine: 25-50% b) D-Glucuronic Acid: 3-25 % in which the proportion of the disaccharide unit D-Glucuronic Acid linked to N-sulfo-3-sulfo-D-Glucosamine is not higher than 20%, it is observed that the healing increases to approximately 70%, and even more so if The dose is higher (greater amount in relation to all monosaccharides).
• non-diabetic Bemiparin 217 Ul / rat
• the rats of Group 1 it is observed that the healing is substantially reduced by 71.82% compared to its non-diabetic control, so that it is concluded that in the case of chronic ulcers of non-diabetic patients (such as pressure ulcers) the administration of an oligosaccharide composition with a higher percentage of monosaccharides: a) N-sulfo-D-Glucosamine: 25-50% b) D-Glucuronic Acid: 3-25% in which the proportion of the disaccharide unit D-Glucuronic Acid bound to N-sulfo-3-sulfo-D-Glucosamine is not higher than 20%, it is observed that the healing of 70 % approximately.
• the objective of this study is to evaluate the healing activity of the test products: fondaparinux sodium and bemiparin after oral administration in the model of wounds in diabetic rats.
• the control group received only the vehicle used to prepare the test product.
• the vehicle for the preparation of the test product is a 1.5% (w / v) bicarbonate solution in water for injections.
• the pH of the prepared formulations was found to be above 8.00.
• diabetes mellitus was induced in all animals. For this, they received a single intraperitoneal streptozotocin injection (60 mg / kg, 10 mL / kg). 72 hours later, the establishment of diabetes was checked by analyzing blood glucose levels. To do this, the animals were anesthetized with isoflurane and 0.8 mL of retrorbicular sinus blood was drawn. The animals had to having glucose values greater than 15 mmol / L One week after the induction of diabetes, the wound was made.
• the animals were anesthetized (Ketamine + Xylazine, 90 + 10 mg / kg, Lm.).
• the loin was shaved and cleaned with disinfectant (povidone iodine) and the area where the wound would be made was marked, using a template to homogenize its position in all animals.
• the wound was made through a circular incision of approximately 1.5 cm in diameter, removing the skin from the dorsal middle area. It was administered, via Lp., 1 mL / animal of physiological serum after the intervention. During the 7 days after the intervention analgesia (paracetamol, 1 mg / mL) was supplied in the drinking water.
• the area of the wounds was calculated as the percentage of reduction of the same to evaluate the degree of healing of the same, by means of the standardized formula:
• the measurement made immediately after the intervention or incision was used as the basal area.
• the animals were administered with 10 mL / kg orally.
• the animals were distributed homogeneously, according to body weight, in the following treatment groups, with at least 5 animals per group:
• the animals will be monitored, macroscopically assessing the evolution of the wounds twice a week, by digital photograph of the wound. The body weight of the animals was also recorded twice a week.
• rats treated with sodium Bemiparin reduced the area of ulceration more or less to the same extent as the control animals.
• Each cut was given a histological score in the range grade I to grade V, where I means uncured wound and V completely epithelized wound. The score is based on the degree of cell invasion, tissue formation of granulation, vascularization and epithelialization.
• Diabetic animal treated with fondaparinux sodium 9.5 III Diabetic animal treated with sodium bemiparin 9.0 III
• scab is essential in the wound healing process, since it meets the objective of limiting the loss of transdermal water and acting as a barrier against bacteria and external pathogens, preventing infection of The wound
• the rats that formed scab in previous days show in the histopathological analysis of the scar that the pattern of the dermis is closer to normal, compared with the wounds of the animals where it has not been formed or the scab It is smaller.
• the area of scarring is greater and therefore the measure is not conclusive against animals that have not formed a scab. Therefore, the formation of scab is a good indicator of the natural healing process, which in this particular case is only observed macroscopically in the group treated with bemiparin.
• the animals were monitored, macroscopically assessing the evolution of the wounds twice a week by digital photograph of the wound.
• the body weight of the animals was also recorded twice a week.
• Figure 11 shows the evolution of diabetic control rats with respect to diabetized rats treated with sodium bemiparin and fondaparinux. This graph shows how rats treated with bemiparin heal much better than those treated with fomdaparinux. In relation to the areas of the ulcers, when we compare all the products, it is observed how glycosaminoglycans that have a higher proportion of monosaccharides in the regular region have a beneficial effect on wound healing.
• Each cut was given a histological score in the range grade I to grade V, where I means uncured wound and V completely epithelized wound. The score is based on the degree of cell invasion, tissue formation of granulation, vascularization and epithelialization.
• Example 4 Ulceration of the diabetic mouse C57BUKS BKS.Cp-m + Lepr db / + LeDWJ, measurement of the degree of healing and histopathological evaluation.
• the C57BL / KsJ-db / db diabetic mouse has been used as a model of type II diabetes that presents an altered scarring, unlike the mouse with streptozotocin-induced diabetes used in the previous examples, which is a type I diabetes model (Michaels , J., et al., Db / db mice exhibit severe wound-healing impairments compared with other murine diabetic strains in a silicone-splinted excisional wound model. Wound Repair Regen, 2007. 15 (5): p. 665-70) .
• the genetically diabetic mouse develops resistance to insulin and hyperglycemia similar to those observed in adult diabetes.
• the full thickness wounds made on the back of these animals take longer to form the granulation tissue and close than the same wounds produced in non-diabetic animals.
• the animals were anesthetized on the first day of the test (Ketamine + Xylazine, 100 + 10 mg / kg, Lm.). The animal's back was shaved, the area was cleaned with disinfectant and the wound situation was marked, by means of a template to match its position in all animals.
• the wound was made through a circular incision of approximately 1.5 cm in diameter, removing the skin from the dorsal middle area. Subsequently, a semipermeable dressing was placed on the wound. It was administered via i.p. 1 mL / animal of physiological serum after the intervention.
• Analgesia (Paracetamol, 1 mg / mL) was administered in the drinking water during the 7 days after the intervention.
• the area of the wounds was calculated as the percentage reduction of the same to assess the degree of healing of the mimes, using the standardized formula:
• the measurement made immediately after the intervention or incision was used as the basal area. The next day and for a total of 14 days, the animals were administered with the assigned treatments.
• Diabetic animal treated with fondaparinux sodium 1 mg / kg
• Diabetic animal treated with sodium bemiparin 1000 Ul Xa / kg
• the animals were followed up to a period of 21 days (average time required for wound healing in this animal model).
• the assessment was performed by digital photography of the wound and subsequent analysis of the image.
• Figure 4 shows the evolution of diabetic control mice with respect to mice treated with sodium bemiparin. The area of the wound has been represented to the right of each animal.
• Figure 5 shows the evolution of the percentage of wound healing over time.
• Untreated diabetic animals are those that have a slower healing rate, since they do not reach 90% healing on day 21, followed by animals treated with fondaparinux that do not reach full healing, which reach 96% in on day 21. Animals treated with bemiparin reach full healing on time 21 days and have healing values of 98% from day 18.
• Moderately thick granulation tissue It can vary from being dominated by
• IV 10-12 extensive collagen, the epithelium covers the wound partially or totally.
• V 13-15 traces of the wound.
• B Complete reepithelialization. Moderate inflammatory infiltrate, polymorphonuclear neutrophils in the dermis. Moderate presence of fibroblasts in dermis. Moderate neovascularization in the dermis. C Fully regenerated cell structure.
• topical formulations were prepared to be administered directly on ulcers whose vehicles are formed by Carbopol, Phenonip®, Span 80, Glycerol and water.
• the ulceration and measurement of the area was performed as detailed in the subcutaneous administration trials. The day after the incision and for a total of 14 days, the animals were administered with the assigned treatments, 0.15 ml / animal topically on the back of the animal, on top of the wound.
• Diabetic animal treated with Heparin caldca 10000 Ul / kg The animals were followed up to a period of 14 days (average time required for wound healing in this animal model).
• the assessment was performed by digital photography of the wound and subsequent analysis of the image.
• Figure 7 shows the global values of the evolution of the percentage of wound reduction of the treatment groups versus the control group. As seen in the graph, the best of the wounds was observed with the bemiparin, obtaining a reduction value of the area greater than 60% at the end of the treatment.
• the histopathological scores obtained for the different groups are shown in the table below.
• the three treated groups obtain a better histological score than the control group, at least two points above it.
• the scarring in all groups is characterized by the presence of moderately thick granulation tissue, the difference between them is in the greater presence of inflammatory cells, as in the case of the control group, versus to the greater presence of fibroblasts and deposition of collagen fibers, as is the case of the treated groups.
• the skin samples belonging to the groups treated with bemiparin and HNF have a somewhat more evolved scar repair index, with a more evolved granulation tissue, greater presence of fibroblasts and dermal neovascularization.
• the group treated with HNF differs in histological score within the framework of grade IV, which indicates a better quality in the healing with respect to the other groups.
• the granulation tissue formed is thicker and is dominated by fibroblasts, with extensive deposition of collagen fibers.
• the epithelium covers the wound partially or totally.
• Bemiparin has a faster healing rate, while in the case of HNF the healing is slower but with higher quality in the tissue formed.
• the C57BL / KS.Cg-m Lepr * + / + m mouse is the non-diabetic analog of the mouse used in the previous example.
• the animals were anesthetized on the first day of the test (Ketamine + Xylazine, 100 + 10 mg / kg, Lm). The animal's back was shaved, the area was cleaned with disinfectant and the wound situation was marked, by means of a template to match its position in all animals.
• the wound was made through a circular incision of approximately 1.5 cm in diameter, removing the skin from the dorsal middle area. Subsequently, a semipermeable dressing was placed on the wound. It was administered via i.p. 1 mL / animal of physiological serum after the intervention.
• Analgesia (Paracetamol, 1 mg / mL) was administered in the drinking water during the 7 days after the intervention.
• the area of the wounds was calculated as the percentage reduction of the same to assess the degree of healing of the mimes, using the standardized formula:
• the measurement made immediately after the intervention or incision was used as the basal area. The next day and for a total of 14 days, the animals were administered with the assigned treatments.
• the animals were followed up to a period of 21 days.
• the assessment was performed by digital photography of the wound and subsequent analysis of the image.
• Figure 8 shows the evolution of non-diabetic control mice with respect to mice treated with calcium heparin. The area of the wound has been represented to the right of each animal.
• Figure 9 shows the evolution of the percentage of wound healing over time.
• Untreated non-diabetic animals reach wound reduction values greater than 90% as of day 14. Treatment with sodium bemiparin does not improve healing speed. However, animals treated with calcium heparin do improve with respect to control, reaching 90% healing on day 11.
• the three groups of animals have a normal healing process, with the wounds healed on day 21. However, it seems that the group of mice treated with calcium heparin the process has accelerated since the day 11 the wound is dry and closed. , and the 21st day covered with hair.
• Each cut was given a histological score in the range grade I to grade V, where I means uncured wound and V completely epithelized wound. The score is based on the degree of cell invasion, tissue formation of granulation, vascularization and epithelialization.
• Figure 10 shows photomicrographs of the wound sections after staining with hematoxylin-eosin: A non-diabetic control. B non-diabetic animal treated with sodium bemiparin, C non-diabetic animal treated with calcium heparin. The results obtained have been the following:
• the glycosaminoglycans object of the present invention can be used to treat other chronic ulcers other than those of the diabetic foot.
• the present invention is very likely to work for other polysaccharides, branched or not, such as polysulphated pentosan, betaglucans. , chondroitin sulfates, dermatan sulfates, carrageenans, alginates, arabinoxylans, galactomannans and glucomannans, among others.

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