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 foot ulcers diabetic 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 enhances 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 experimentally determine 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 It is effective by topical route or external mucosa, that is applied directly on the affected area.
• LMWH or HMBPM we have physically and chemically modified different LMWH or HMBPM (see examples) to alter the pentasaccharide responsible for the anti FXa activity in a way qualitative and quantitative, to demonstrate that in the case of ulcers, specifically those of diabetic foot, what is of interest is that in this type of glycosaminoglycans there is a greater proportion of oligosaccharide sequences rich in 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, L-lduronic acid and L-lduronic acid 2-sulphated and not so much of the pentasaccharide responsible for the anti activity FXa (the presence of high concentrations of the N-sulfo-30-sulfo-D-glucosamine unit essential for the antithrombotic activity of pentasaccharide not being necessary).
• 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 (pressure ulcers or pressure), by caloric injury (burn), or by vascular obstruction (diabetes, arteriosclerosis, etc.).
• vascular obstruction diabetes, arteriosclerosis, etc.
• 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.
• 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 the low molecular weight and very low molecular weight heparins.
• the inventors of the present invention have determined that whenever 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 amputation of the diabetic foot and which is also effective not only in prophylaxis but as a therapeutic treatment
• 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 route of administration is the topical route, since it is the most convenient route of administration that allows patients to directly apply the amount stipulated by the doctor in an easy way.
• topical route presents a series of advantages over the parenteral route, since it is much more comfortable for the patient, since in many cases these treatments require long periods of therapy, such as in some types of diabetes in which treatment is needed for life, being the frequency of daily administration.
• Another important fact is that patients who suffer from this type of condition receive a lot of medication to treat other areas (not the ulcer itself), such as diabetes, and in many cases patients have established medication guidelines that on many occasions do not they allow other medications to be administered because of the interaction that may exist with the treatments they have previously established.
• the researchers of the present invention have achieved the absorption of the different glycosaminoglycans topically without the formulations causing the exacerbated growth of the affected cells in the ulcerated area, as occurs with the topical application of growth factors about the wounds that are currently in the state of the art.
• the topical route for this type of compositions is the route of choice since, as can be seen in the state of the art, in the case of topical applications of growth factors on the affected area there is a risk of producing uncontrolled growth. of the cells in the affected area (see the case of Regranex ® ), there being a risk of cancer and its use being limited to patients suffering from malignant diseases such as cancer.
• the researchers of the present invention have not observed an uncontrolled growth of the affected cells, so that the use of the combination object of the present The invention is not limited to patients who are not at risk of malignant diseases such as cancer, but the composition of the present invention is directed to any type of chronic ulcer.
• 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
• oligosaccharide chains do not have the structural motive responsible for the interaction with antithrombin III.
• 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.
• the signals have also been labeled: ANSred, N-sulfo-D-glucosamine of the reducing end; ANS, 3S, N-sulfo-30-sulfo-D-glucosamine.
• 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 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.
• the signals have also been labeled:
• 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 spectrum
• 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, sulphated L-iduronic acid; ANAc, N-acetyl-D-glucosamine) and The decrease of the 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 spectrum
• Figure 3 The percentage of reduction of ulcerated area of the mice of Example 5 is represented when they are administered a topical control composition, a topical sodium bemiparin composition, a topical sodium fondaparinux composition and a topical unfractionated heparin composition .
• Figure 4 Digital photographs of the evolution of the wound of the treatment groups against the control group and representation of the area of the wound. As it is reflected in the images, in the control group no granulation tissue is produced at day 7 (the base of the wound is clear and bright), while it is observed in the treated groups. This denotes a slowing in the healing in the control group compared to the treated groups, which demonstrates the favorable effect of the treatment in wound healing.
• Figure 5 Global values of the evolution of the percentage of reduction of the wound of the treated group versus the control group. A greater reduction of the wound is reflected in the group of animals treated with RO-14 compared to the control, which demonstrates its favorable effect in the healing of the same.
• Figure 6 Digital photographs of the evolution of the wound in the treated group against the control and representation of the area of the wound. As can be seen, the reduction in the area of the wound is greater in the group treated with RO-14 than in the control group, which demonstrates its beneficial effect in wound healing.
• 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 topically, in particular LMWH and HMBPM, for the treatment of chronic ulcers such as diabetic foot, which contains the following proportion of the following monosaccharides (in which all percentages are over the total percentage of monosaccharides of Ia composition): a) N-sulfo-D-Glucosamine: 25-50% b) D-Glucuronic Acid: 3-25% and that 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 topical glycosaminoglycan composition, in particular LMWH and HMBPM, which contain a characteristic proportion of at least the monosarcharides selected from the N-sulfo-D-Glucosamine, N-acetyl- group.
• 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 diabetic foot ulcers.
• the composition to which it refers can be formulated in an aqueous or oily phase, with liquid or solid texture, in the form of gel, cream, ointment, spray, patch and / or quick-release implant or sustained, that is, any formulation of direct application on the ulcer with pharmaceutically acceptable vehicles that allow the absorption of the glycosaminoglycans object of the present invention.
• 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 chronic ulcer a treatment consisting of a dose of low molecular weight heparin (LMWH) classified as a treatment dose of venous thromboembolic disease without a greater risk of bleeding, and surprisingly better results are obtained than if administered at lower concentrations such as the 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 The 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 topically, in particular LMWH and HMBPM, for the treatment of diabetic foot ulcer containing the following proportion of the following monosaccharides: - N-sulfo-D-Glucosamine: 25-50%
• a pharmaceutical composition of glycosaminoglycans is provided 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 topically, in particular LMWH and HMBPM, for treatment of diabetic foot ulcer containing the following proportion of the following monosaccharides:
• the present invention is directed to a pharmaceutical composition of glycosaminoglycans by topical route, in particular LMWH and HMBPM, for the treatment of chronic ulcers such as those of diabetic foot, which contains a proportion of the linked D-Glucuronic Acid disaccharide unit.
• 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 entire saccharide composition.
• One of the merits of this invention is not only to have eliminated a prejudice of the state of Ia technique, but to achieve an effective correlation between the proportion of the monosaccharides mentioned above with the dose of medication containing the pharmaceutical composition mentioned above necessary to treat the diabetic foot ulcer as well as to promote the healing and regeneration of tissue in patients in function of 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 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 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 , L-lduronic acid and L-lduronic acid 2-sulfated, 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 lower 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 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-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 removal 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, such that the percentage of fragments of more than 6,000
• 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 -
• - 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. Sodium Heparin: Unfractionated Heparin. Among others it has the following characteristics (Ph.
• 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.
• 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 presents the following characteristics: Average molecular weight of 3468 Daltons and an anti-factor Xa activity: 4 Ul / mg.
• BP06408-66 / 27-A It is a low molecular weight heparin of reduced anti factor Xa activity, obtained by fractionation of Bemiparin by affinity chromatography on ATIII. To obtain this derivative, 10 mg of Bemiparin is passed through a CNBr-activated column
• Sepharose 4B previously activated with ATIII Human
• ATIII Human
• 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 dissolution that Contains 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-factorXa activity: 32 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-glucuromic acid linked to N-sulfo-3-sulfo-D-glucosamine G- (ANS, 3S) present in GAGs obtained from natural heparma, can be directly related to the anti-Xa activity of them, as described by M. Guer ⁇ ni et al. This disacá ⁇ do belongs to the pentasacando 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 glucuric acid with the directly bound hydrogen appears in a characteristic region and free of overlap in the HSQC spectrum and therefore 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.
• Example 1 C57BL / KS BKS diabetic mouse ulceration. Cg-m + Leprdb / + Leprdb / J, measurement of the degree of healing and histopathological evaluation. Study of the healing effect of Bemiparina, Fondaparinux and HNF in semi-solid forms.
• the C57BL / KsJ-db / db diabetic mouse has been used as a model of type II diabetes that presents an altered scarring, unlike the model of the wistar rat with streptozotocin-induced diabetes used in the previous examples, which is a model of type diabetes I ⁇ 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 C57BL / KsJ-db / db is a model that develops insulin resistance and hyperglycemia similar to those observed in the diabetes of an adult patient. Regarding the behavior in what refers to the healing, the wounds made in the back of these animals take longer to form the granulation tissue and to 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 spine was shaved, the area was cleaned with disinfectant and the situation of the wound was marked by 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.
• FIG 4 shows the evolution of the diabetic control mice with respect to the treated diabetic mice.
• the area of the wound has been represented to the right of each animal.
• 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.
• Figure 3 shows the global values of the evolution of the percentage of reduction of the wound of the treatment groups against the control group. As can be seen in the graph, the best wound healing was observed with the bemiparin, obtaining a reduction value of the area greater than 60% at the end of the treatment.
• Untreated diabetic animals are those that have a slower healing rate, reaching wound reduction values close to 20% in time 15 days. In all treatments, a continuous healing of the wounds has been observed, reaching healing values higher than those obtained with the control from the formation of the same. The best evaluation of the wounds was observed with the bemiparin, obtaining a reduction value of the area greater than 60% at the end of the treatment, followed by the HNF and the fondaparinux. It can be concluded that the three treatments have a favorable effect on wound healing compared to the untreated diabetic group.
• control Bemiparin, Fondaparinux and unfractionated calcium heparin (HNF)
• HNF unfractionated calcium heparin
• Fine and immature granulation tissue dominated by inflammatory cells but with few fibroblasts, capillaries or collagen deposition; minimal epithelial migration
• Moderately thick granulation tissue It can vary from being dominated by
• 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. This indicates that, in the case of the control group, the healing process is at an earlier stage, thus explaining the slower healing speed revealed in the studies of reduction of the area of the wound.
• the skin samples belonging to the groups treated with bemiparin and HNF present 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.
• Example 2 C57BUKS BKS diabetic mouse ulceration. Cg-m + Lepr db Z + Lep ⁇ / J, measurement of the degree of healing and histopathological evaluation. Study of the healing effect of RQ-H in semi-solid forms.
• the C57BL / KsJ-db / db diabetic mouse has been used again as a model of type II diabetes that presents an altered healing.
• the animals were anesthetized on the first day of the test (Ketamine + Xylazine, 100 + 10 mg / kg, Lm.). The spine was shaved, the area was cleaned with disinfectant and the situation of the wound was marked by 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.
• Diabetic control Diabetic animal treated with RO-14 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 6 shows the evolution of the diabetic control mice with respect to the treated diabetic mice.
• the area of the wound has been represented to the right of each animal.
• Figure 5 shows the evolution of the percentage reduction of the wound over time.

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