Sulodexide applicable in treating pathologies involving metalloproteinases

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to using dermatan sulphate recovered from sulodexide for treating diseases involving metalloproteinase MMP-9: varicose veins and vascular malformations accompanied by a risk of thrombosis.

EFFECT: reducing and/or inhibiting individual's blood serum and saphena segments MMP-9 has been shown.

4 cl, 3 dwg, 13 tbl

 

In the present invention is described sulodexide or at least one of its components for use in reducing the level of circulating matrix metalloproteinases (MMP), particularly MMP-9.

Sulodexide and its composition useful for the treatment of pathologies involving MMP, such as cardiovascular disease, cardiovascular disease caused by diabetes, varicose veins, chronic venous insufficiency (CVI), gastrointestinal ulcers, pulmonary disease, and neoplastic pathology.

Prior art

Recent studies have demonstrated the participation of a group of zinc-dependent endopeptidases called matrix by metalloproteinases (MMP) belonging to matrixinfo family, vascular changes in cardiovascular pathological conditions.

Venous hypertension is the basis of several pathologies associated with disorders of the macro - and microcirculation and causing infiltration of leukocytes to the endothelium, the destruction of the heart valves and, finally, providing remodeling/rebuilding of the venous wall, which can lead to reflux or the formation of varicose veins and to the pathologies of the dermis, as described in Bergam J. et al. Ann. Vasc score. Surg. 21 (2007), 260-266. Raffetto JD. etc. in Thromb. Res. 123 (2009) S66-S71 report that the modifications observed in the in�free development of chronic venous pathology, may be associated with a change in the balance between matrix metalloproteinases (MMP) and their tissue inhibitors (TIMP) in blood.

As MMP and TIMP, participating in the physiological remodeling of the extracellular matrix, play an important role in cellular communication.

High concentrations of MMP were found in different pathologies, as reported by F. Mannello, etc. in Curr. Cancer Drug Targets 5 (2005), 285-298. Among MMP, MMP-9 (gelatinase, KF 3.4.24.35 92, 130 and 225 kDa) is involved in several pathologies, including neoplastic pathology, which suggests that their presence in the form of circulating enzyme and an increase associated with the development of various types of cancer, such as breast cancer, lung, ovarian, prostate and melanoma, as reported by E. Rahko, etc. in Tumor Biol., 30 (5-6) (2009), 574-64.

Pathogenic role of MMP clearly shown when the vascular pathological conditions caused by diabetes. Galis Z. S., etc. in Circ. Res. 90 (2002), 251-262, N. P. Kadoglou, etc. in Angiology 56 (2005), 173-189, Derosa and others in Diabetes Metab. 33 (2007), 129-134 and M. H. Tayebjee et in Diabetes Care 27 (2004), 2049-2051 describe elevated levels of circulating MMP in patients with diabetes.

Raffetto JD. etc. in Biochem. Pharmacol. 75 (2008), 346-359, demonstrate the importance of MMP in vascular diseases, indicating that the increased degradation of extracellular matrix (ECM) in the venous wall is involved in the early stage of relaxation of the veins, obrazovanakarakterna dilatation, dermal modification and, finally, leads to the formation of venous ulcers.

Among several pathological mechanisms associated with increased expression of MMP that are important and are a possible object of pharmacological treatment of the interaction of leukocytes with the endothelium. Mannello F. et reported in Arterioscler. Thromb, Vase. Biol. 28 (2008), 611-614, leukocytes and platelets contain higher amounts of MMP that may be released into the extracellular environment after activation/degranulation of leukocytes or during platelet aggregation. Moreover, MMP-9 is released from platelets and leukocytes after such a stimulus, as a response induced by the coagulation process.

It is known that plasmin, activated during the phase of coagulation, in turn, is an activator of MMP.

Among the proteolytic enzymes present in the human walls and potentially associated with cancer invasion, MMP, as demonstrated involved in the ability to virtually destroy the proteins of the interstitial matrix and basement membranes, enabling the cells to diffuse and penetrate into the adjacent wall.

Several compounds, such as receptors of growth factors and cell adhesion molecules, chemokines, cigarini, apoptotic ligands and angiogenic factors, can interact with MMP, and modifying their expression, � their activity.

These proteolytic enzymes act on many bioactive substrates involved in such stages of neoplastic development, as the growth of the primary tumor, angiogenesis, extravasation and intravasation neoplastic cells, migration and invasion of metastatic cells into the secondary organ, the beginning and the maintenance of tumor growth.

Several clinical studies have been conducted on animals, and human tumor models to assess the importance of reducing the level of circulating matrix metalloproteinases. The results obtained have led to the development of synthetic inhibitors, as described Ramnath N., etc., in Curr. Oncol. Rep.6 (2004), 96-102.

The main investigated inhibitors include peptide mimetics and peptides-semimatte collagen, tetracycline and biphosphonate derivatives.

Clinical trials conducted with existing compounds, showed that prolonged treatment regimens cause musculoskeletal pain and inflammation. Although it is shown that these events are reversible, and patients resumed treatment after a short period of cancellation, unexpected adverse events limited the injected dosage.

Moreover, when clinical trials for the treatment of neoplastic diseases have been expanded on more patients, in addition to serial Toxicological problems due to cyto�teticheskikh effects no significant therapeutic benefit was not observed, as reported by F. Mannello, etc. in Curr. Cancer Drug Targets 5 (2005), 285-298.

Some MMP inhibitors tested in clinical trials represent batimastat, marimastat, prinomastat, BAY 12-9566, CGS27023A and derivatives of tetracyclines.

Batimastat, BB-94 (WO 90057191), is a derivative of hydroxamic acid, which mimics the natural structure of the peptide substrates. Batimastat is a potent inhibitor of MMP, and was the first MMP inhibitor used in clinical trials, but have been shown, it has a low selectivity, caused by low solubility and absorption. Moreover, treatment with batimastat poorly tolerated by patients because should be introduced by injection into the pleural and abdominal space. Clinical Phase III trials were stopped almost immediately because of serious side effects, such as strong local inflammatory tissue reactions, nausea, abdominal pain, as reported by G. Tu, etc. in Curr. Med. Chem. 15 (2008), 1388-1395.

Marimastat, BB-2516 (WO 9402447), is a derivative of hydroxamic acids with the same structure as that of batimastat, but with a higher solubility and subsequent easier absorption after oral administration. Marimastat as batimastat, has low specificity and was t�xicnys approximately 30% of all treated patients, causing musculoskeletal pain and stiffness, starting with the small joints of the fingers and apply on the forearms and shoulders, especially near the places of joining of tendons, fibrosis and necrosis of the walls of periarticular elbow and knee and a gastric disorder associated with loss of body weight, as reported by Vihinen et in Int. J. Cancer 99 (2002), 157-166.

Prinomastat, AG3340 (WO 90720824) is a derivative of hydroxamic acid, which is selective against MMP involved in tumor invasion and metastasis. During clinical trials, side effects were observed in the joints of shoulders, knees and hands and treatment immediately cancelled, as reported by N. Ramnath, etc. in Curr. Oncol. Rep.6 (2004), 96-102.

BAY 12-9566 (US 4705798) is a derivative of butyric acid, and in several clinical trials show toxicity, manifested in thrombocytopenia, anemia, increased levels of liver enzymes and bilirubin, nausea, fatigue and headache, as reported by Nelson A. et J. Clin. Oncol. 18 (2000), 1135-1149.

CGS27023A (US 5455258) has known toxic effects with extensive skin irritation, myalgia and arthralgia.

Derivative of tetracycline has demonstrated in clinical trials of severe unwanted effects such as fatigue, confusion, nausea, vomiting, skin phototoxic�ness, increased levels of pancreatic enzymes that limit of the input dose, as reported by Hidalgo M. et al., in J. Natl. Cancer Inst. 93 (2001), 178-193.

Zymography and reverse zymography are methods currently used for analysis of MMP and TIMP in biological samples. All types of substrates for sonografii take the gelatin out of the way of sonografii. Methods are the same, except that the substrate differs depending on the type of MMP or TIMP. In these methods, proteins are separated by electrophoresis in denaturing, Sevostyanova conditions in the presence of sodium dodecyl sulfate (SDS). Gelatin zymography is a technique used mainly for the determination of MMP representing gelatinase, and she is extremely sensitive for the detection of MMP-2 and MMP-9 in a concentration of a few picograms. Note that other MMP, such as MMP-1, MMP-8 and MMP-13 can also degrade this substrate.

This method is based on separation by electrophoresis in polyacrylamide gel with a specific substrate; SDS denatures MMP, which become inactive and after migration in electrophoresis, SDS was removed from the gel in a suitable buffer that restores the structure and function of MMP. After that, if the gel was incubated, then activated gelatinase digest gelatin, which turns into n�scimolecular peptides, which are removed by washing, as described Mannello F., etc. in Clin. Chem. 49 (2003), 339-34.

Gels are placed in a developing solution, for example containing Kumassi, and then decolorized solutions containing methyl or ethyl alcohol and acetic acid. Coloured areas of the gel are the result of the presence of undigested gelatin, whereas the unpainted areas are the result of the enzymatic activity of MMP. Enzymatic activity is proportional to the width of the transparent stripes, so it can be analyzed and quantified using a densitometer equipped with image analyzer.

Zymogram give specific and clear information about MMP and, in particular MMP-2 and MMP-9, as these two enzymes can be separated on the basis of different electrophoretic mobilities, which depend on their molecular masses. To this end a suitable molecular weight standards corresponding to 72 kDa Pro-MMP-2 and 92, 130 and 225 kDa for the complex of Pro-MMP-9 and MMP-9, is applied for comparison on the same gel.

Mannello F., etc. in Clin. Biochem. 41 (2008), 1466-73 investigated the effect of adding a high-molecular-weight heparin (HMWH) lithium in samples of peripheral blood to assess the impact of HMWH on the expression of MMP and TIMP in the blood. The described effect was that HMWH has had direct and indirect influence by changing the amount of C�kulinowski of MMP and TIMP, and increasing the release of TIMP-2. The purpose of this article was essentially in the description of the influence of high-molecular-weight heparin (HMWH) on the expression of MMP when handling blood in order to standardize procedures for handling samples of blood in way of measuring MMP. In this publication there are no links and/or suggestions related to the pharmacological uses of heparin. Moreover, not reported concentrations of lithium heparin, in which there is an effect. Consequently, it is impossible to know whether the concentration of lithium heparin, which provides the observed effect, to be therapeutically useful, as is well known the high risk of bleeding associated with HMWH. Nevertheless, since it is known that heparin is usually administered through injection, you should assume that any use of heparin for the purpose of concentration changes in plasma MMP should follow the same path.

Effect of heparin on the expression of mRNA of MMP-2 and TIMP-2 are also described in Caenazzo I. and others in Nephrol. Dial. Transplant 2001, 16, 1769-75.

Therefore, there is still a need for active ingredients for medicines, useful in reducing circulating concentrations of MMP in the blood for the treatment of all pathologies involving DFID, such as the pathogenesis of inflammatory, infectious, or neoplastic or cardiovascular disease. Among et�x abnormalities have for example, pathologies caused by diabetes, varicose veins, chronic venous insufficiency (CVI), atherosclerosis, cardiac rupture after myocardial infarction, abdominal aortic aneurysm, pulmonary pathology, the increase and development of the tumor, the primary tumor ingrowth, abnormal angiogenesis, extravasation and intravasation neoplastic cells.

Vascular diseases selected from the group of varicose veins, atherosclerosis in humans, cardiac rupture after myocardial infarction, abdominal aortic aneurysm.

Moreover, there is a need in getting medicines without any side or toxic effects at therapeutically effective dosage, acceptable to patients and administered by oral, intramuscular or intravenous injection for the treatment of all pathologies involving MMP.

Suddenly discovered, and it is the subject of the present invention that the sulodexide or one of its components, in particular dermatan sulfate, obtained by using sulodexide (SDX-DS), or heparin (SDX-HEP), obtained by using sulodexide inhibits and/or reduces the level of circulating MMP.

Sulodexide is a natural blend of natural glycosaminoglycans (GAG), extracted from intestinal mucosa of mammals, as described in US 3936351, cataractogenic mixture of mastrodicasa heparin (SDX-HEP) with an average molecular weight of 7 kDa and dermatan sulfate (SDX-DS) with an average molecular weight of 25 kDa. Other possible components contained in the sulodexide, are chondroitin sulfate and heparin. Sulodexide should be considered as a unique active ingredient that differs from other glycosaminoglycans containing about 80% by weight SDX-HEP and about 20% by weight SDX-DS. These components are still not highlighted and components of sulodexide is determined by an analytical method such as gel filtration and electrophoresis.

It is known that sulodexide shows a high affinity to antithrombin III (AT III) and the cofactor II heparin inhibits factor XA and thrombin, activates tissue plasminogen and reduces the level of fibrinogen, as described Ofosu F. A. in Semin. Thromb. Hemost. 24 (1998), 127-38 and Ceriello A. Diab and others in, Nutr. Metab., 6 (1993), 1-4.

Since it is known that the activation of plasmin from plasminogen is one of the local activation mechanisms of MMP, known properties of sulodexide lead to the conclusion that this compound should increase the concentration of circulating MMP.

In the present invention is described sulodexide or one of its components, in particular SDX-DS and SDX-HEP, for inhibiting and/or reducing the level of circulating MMP and, in particular MMP-9, also known as gelatinase In that are involved in many diseases, in particular, infection or neoplastic disease, vascular disease, product�door frames with a high level of MMP, gastrointestinal ulcers, and increase the development of the tumor, the growth of the primary tumor, altered angiogenesis, extravasation and intravasation neoplastic cells, and combinations thereof. With regard to vascular diseases characterized by high levels of MMP, the present invention in particular relates to chronic venous insufficiency (CVI), varicose veins, rupture of the heart after myocardial infarction, aneurysm of the abdominal aorta, the relaxation of the veins, pulmonary pathology and neoplastic pathologies.

Another unexpected and important aspect of the present invention is a dermatan sulfate (SDX-DS) and low molecular weight heparin (SDX-HEP), isolated from sulodexide, a process for their preparation and their use for inhibiting and/or reducing the release of MMP in vitro and in vivo, alone or in mixtures thereof.

This result is really unexpected, because in the literature, for example, in Cell Biol. Int. 2003, 27, 779-84, N. Isnard and others report that dermatan sulfate activates the expression of MMP-2 and MMP-9.

Unexpected features of SDX-DS were demonstrated when compared with commercial dermatan sulfate, and the results are shown in Examples 2-9.

These results really do sulodexide unique connection that is different from any other glycosaminoglycans, which can be obtained by using niskama�molecular heparins and dermatosurgical, available on the market for the treatment of all pathologies involving MMP.

Another aspect of the present invention relates to a SDX-DS, isolated from sulodexide.

Another aspect of the present invention relates to a SDX-HEP, isolated from sulodexide.

Another particular aspect the present invention relates to dermatan sulfate derived from sulodexide the method of purification, such as affinity chromatography with AT III-functionalized environment.

Another particular aspect the present invention relates to low molecular weight heparin obtained from sulodexide the method of purification, such as affinity chromatography with AT III-functionalized environment.

Another aspect of the present invention relates to pharmaceutical compositions containing SDX-DS and SDX-HEP, and their use as a medicine.

Description of the invention

The object of the present invention is to sulodexide or one of its components, in particular, SDX-DS and SDX-HEP, isolated from sulodexide, and their use as a medicine when all the pathologies that involve MMP. In particular, the invention describes the use of sulodexide, or SDX-DS or SDX-HEP to reduce and/or inhibition of circulating MMP, in particular, the inducible form gelatinase MMP-9, with a subsequent decrease in the concentration�and circulating MMP in the blood. Selected components can be used for drugs either alone or in mixtures thereof.

Sulodexide is presented in the form of the drug available commercially under the trademark VESSEL 2F®, and it is administered by mouth, by intramuscular or intravenous injection for the treatment of vascular pathologies with a risk of thrombosis, such as a disorder of the peripheral arteries, postphlebitic syndrome, for the treatment of albuminuria nephropathy, as described Harenberg J. Med. Res. Rev. 18 (1998), 1-20 and Tamarin R. in Medical Praxis 8 (1997), 1.

It also provides that sulodexide and its derivatives, in particular SDX-DS and SDX-HEP, include their pharmaceutically acceptable salts, solvates, hydrates, and clathrates.

An important aspect of the present invention is the specificity of sulodexide in the inhibition/reduction of the level of MMP-9, also called gelatinase In, which is involved in relaxing the veins, varicose veins, chronic venous insufficiency (CVI) and ulcers of the lower extremities.

Sulodexide, or SDX-DS or SDX-HEP can be used as active ingredient in all the pathologies characterized by increased levels of inducible MMP-9, without any effect on constitutive MMP-2. The advantage of sulodexide or its components is the possibility of introducing oral or inject�Clennam way intramuscular or intravenous. Another advantage of sulodexide and its components is the possibility of the introduction of high dosage with no side effect, as described in EP 1292315 that is well tolerated up to 1000 mg/day and above.

The present invention describes a new use of sulodexide, or SDX-DS or SDX-HEP for reducing and/or inhibiting concentrations of MMP, particularly MMP-9 (gelatinase, KF 3.4.24.35 mol. mass. 92, 130 and 225 kDa).

In the present invention is described sulodexide or one of its components, in particular SDX-DS and SDX-HEP, for use in inhibiting or reducing the level of MMP, particularly MMP-9, more than 20%.

The present invention describes the amount of sulodexide or SDX-DS and SDX-HEP, sufficient to achieve reduction and/or inhibition of the release of MMP-9 from blood cells circulating in the plasma and subsequent reduction of the activity of circulating MMP in the venous apparatus and subsequent decrease of enzymatic activity in situ in the venous walls. This effect occurs when the concentration of sulodexide in vitro and ex vivo in the range of 8 µg/ml to 12 μg/ml. These concentrations correspond to a therapeutically acceptable dosage, which can be achieved in the blood through the introduction of sulodexide intravenous, intramuscular or oral means with dosages of 20 mg and up to 1000 mg/day. �effect of sulodexide, SDX-DS and SDX-HEP on MMP in the described concentrations was demonstrated in experiments ex vivo biological samples.

In detail, the blood samples were taken from 50 healthy volunteers, both men and women. The samples that were processed to obtain the appropriate samples of plasma and serum were analyzed with or without the addition of varying concentrations of sulodexide SDX-DS and SDX-HEP.

The invention also describes the SDX-DS contained in the sulodexide, in a concentration of about 20% by weight, SDX-HEP at a concentration of about 80%, a process for their preparation and the use as a medicament and for the treatment of all pathologies involving MMP. SDX-DS and SDX-HEP can be obtained by the method of purification and extraction.

In particular, Example 1 describes a method of producing SDX-DS and SDX-HEP from sulodexide through purification affinity chromatography, where the heparin fraction adsorb on the stationary phase functionalized by AT III, and dermatology fraction collected in neadsorbirovanne the eluate.

Example 2 describes the effect of adding in whole blood of increasing amounts of sulodexide to a final concentration of from about 10 μg/ml to about 50 μg/ml and SDX-DS, extracted from sulodexide, in a final concentration from about 0 μg/ml to about 10 μg/ml. After centrifugation, the serum from the whole blood samples were collected and anal�increased in gelatin of sonografii, as described Mannello F., etc. in Clin. Chem. 2003, 49, 339-34. To normalize the activity of MMP in different samples, sample volume was adjusted to equal amounts of total proteins. Densitometric results of gelatin sonografii shown in Table 2, where a value of 100% assigned area of MMP serum samples without the addition of sulodexide or inhibitor of MMP, and the calculated reduction in the area of MMP in the presence of sulodexide and SDX-DS. It was shown that the size of the band corresponding to MMP-9, is reduced at least about 20% when the concentration of sulodexide, corresponding to about 10 μg/ml and to less than one-tenth when the concentration of sulodexide, corresponding to about 50 μg/ml.

The addition of sulodexide does not affect the concentration of MMP-2.

It turns out that the addition of sulodexide to samples of whole blood to a final concentration in the range from about 10 μg/ml to about 50 μg/ml reduces the concentration of circulating MMP, particularly MMP-9, in the respective serum samples and proportional manner, whereas the concentration of MMP-2 remained unchanged.

Table 2 also shows the densitometric results in samples treated with SDX-DS, where the size of the bands related to MMP-9, is reduced at least about 20% at the concentration corresponding to about 2.5 μg/ml, and at measures� approximately 60% at a final concentration of about 10 μg/ml.

From these results it can be concluded that the effect of the reduction/inhibition of MMP are given preference due to the presence of SDX-DS contained in the sulodexide, and these results do sulodexide unique active ingredient among glycosaminoglycans.

These data are also confirmed by quantitative ELISA performed using the Biotrak kit® MMP Amersham, which recognizes Pro-MMP gelatinase with a limit of detection (LOD). limit of detection), corresponding to 0.37 to 0.25 μg/ml.

Table 3 confirms that sulodexide at a concentration of about 25 μg/ml reduces at least about 20% the size of the bands related to MMP-9, and at least about 60% at a concentration of about 50 μg/ml. the Addition of SDX-DS, obtained in Example 1, reduces the number of MMP of at least about 20% at a concentration of about 2.5 μg/ml and at least about 60% at a concentration of about 10 μg/ml.

The demonstration that sulodexide also acts to reduce the release of MMP platelets and leukocytes, is obvious in Example 3, in which the sulodexide is added to the plasma samples after removal of the cellular component of whole blood by centrifugation.

Example 3 describes the results related to the addition of sulodexide to blood samples obtained from 50 healthy Dobrov�law with an average age of 37 years, previously treated with 0.13 m solution of sodium citrate in amounts of between 1% and 10% by volume and then centrifuged. To samples of the supernatants were added amounts of sulodexide to a final concentration of from about 10 μg/ml to 50 μg/ml. Samples were analyzed using the gelatin sonografii as described in Example 2.

The value corresponding to 100%, attributed to the band serum sample without any addition (control) and determined the proportional decrease of the area depending on the increasing amounts of sulodexide in serum samples. Watched transparent strip of digestion of gelatin related to proteolytic activity of MMP-9 remained unchanged after the addition of sulodexide, as shown in Table 4 in Example 3, and this result demonstrates that the sulodexide not affect MMP activity when the enzyme has now been released into the plasma.

The same result you get when sulodexide is added to the serum samples, as described in Example 4, to which were added increasing amounts of sulodexide. In particular, in Example 4 describes the effect of increasing amounts of sulodexide from about 10 μg/ml to about 50 μg/ml, added to the serum samples, obtained after centrifugation of blood samples taken from 50 healthy volunteers with an average age of 37 years. Are� serum samples were analyzed by the method of sonografii, as described in Example 2.

Relative densitometric values relationalities areas correlated with Gelati politically mi bands of MMP-2 and MMP-9, as shown in Table 5, where there was no reduction within the bands of the sample without the addition of sulodexide.

In particular, the results in Example 2 show that sulodexide and SDX-DS contained in the sulodexide, prevent the increase and the decrease of MMP in the blood through a mechanism of action that selectively acts on the inducible form of MMP-9, identifying possible therapeutic use for the treatment of all pathologies, where the presence of MMP-9 directly or indirectly correlated with the disease progression.

Example 5 describes the comparison between sulodexide, parnaparin and SDX-DS, extracted from sulodexide, MMP present in the serum. Samples of whole blood taken from healthy volunteers were processed as in Example 2, the group of samples treated with sulodexide to a final concentration of about 25 ug/ml group with dermatan sulfate to a final concentration of approximately 5 μg/ml and the group with low molecular weight heparin (parnaparin) to a final concentration of about 20 μg/ml In Table 8 and Table 9 describes the results obtained when sonografii and ELISA analysis, respectively, g�e sulodexide demonstrates a decrease in the level of MMP-9 in the range from 50% to 70%, parnaparin in the range from about 20% to 30%, and SDX-DS in the range from about 50% to 70% relative to the control sample without any addition.

The unique property of sulodexide to release MMP should be attributed to natural mixtures of GAG (glycosaminoglycans), which represent the sulodexide.

Example 6 confirms the characteristics of SDX-DS in reducing the level of MMP compared with two different dermatosurgical, commercially available on the market. The same concentration of 5 μg/ml SDX-DS induces a decrease in the level of MMP-9 by more than 50%, whereas the two commercial dermatan sulfate (Sigma and Calbiochem) induce a reduction of less than 10%.

Found that the SDX-DS reduces the level of MMP, particularly MMP-9, at least three times more than commercial preparations DS.

Example 7 demonstrates that SDX-HEP also has unique properties, in fact, a mixture of low molecular weight heparin and dermatan sulfate contained in the sulodexide, act differently than synthetic mixture obtained by adding parnaparin to commercial dermatan sulfate and SDX-DS.

In more detail, sulodexide compared with mixtures of low molecular weight heparin and dermatosurgical, mixed approximately in the same ratio, which takes place in the sulodexide, about 80% parnaparin and about 20% dermatan sulfate. The data presented in Table 11, SVID�suggests, that sulodexide demonstrates a significantly higher percentage of inhibition relative to compounds parnaparin/dermatan sulfate and parnaparin/SDX-DS.

In particular, the observation that sulodexide has excellent activity against a mixture of heparin/SDX-DS, allows to demonstrate that the heparin component contained in the sulodexide, also has unique properties that influence the level of circulating MMP.

It was also demonstrated by the ability of sulodexide in relation to MMP on such biological sample, as the inner wall of the veins.

The inner walls of the veins are particularly important because they are the site of action of MMP, causing some pathology related to proteolytic remodeling of the extracellular matrix.

In comparative Example 8 illustrates the effect of sulodexide, SDX-DS and parnaparin at MMP on the venous walls in immunohistochemical experiments. This experiment confirms the effect of sulodexide and SDX-DS in all pathologies involving DFID, and, in particular, in the reduction and/or inhibition concentration and expression of MMP. Added sulodexide, SDX-DS and parnaparin in a concentration of, respectively, about 25 μg/ml, 5 μg/ml and 20 μg/ml to segments of the saphenous vein of the leg of a person, surgically obtained from 10 healthy women (mean age 42 years), subjected to saphenectomy in �the elations varicose veins.

After proper treatment, the tissue serial sections with a thickness of about 5 μm were incubated with a monoclonal antibody against MMP-9, and after incubation with biotinylated secondary antibody complex was detected using peroxidase reaction in accordance with standard methods. Complex after incubation were detected using avidin-peroxidase in accordance with the standard methods.

Fabric samples treated with sulodexide showed a small area of the painted area that demonstrates a lower local concentration of MMP compared to the control and treated heparin samples.

Table 12 shows that, if we ascribe a value corresponding to 100, the sample without any additives, the sulodexide at a concentration of about 25 μg/ml reduces the intensity of the colored region of MMP-9, respectively, to approximately 60%; SDX-DS at a concentration of about 5 μg/ml reduces the intensity to about 70%, and parnaparin in a concentration of about 20 μg/ml reduces the intensity of MMP-9. These in vitro findings in respect of sulodexide and SDX-DS can be transferred to the use of in vivo and can be used to reduce the level of MMP for all pathologies involving MMP.

Example 9 demonstrates the effect of sulodexide, SDX-DS and parnaparin on MMP in the venous walls using sonografii in stu (cytochemistry).

Sulodexide, SDX-DS and parnaparin in a concentration of, respectively, about 25 μg/ml, 5 μg/ml and 20 μg/ml was added to segments of the saphenous vein of the leg of a person, surgically obtained from 10 healthy women (mean age 42 years), subjected to saphenectomy against varicose veins.

After processing, the sections of the veins were incubated with the substrate, which is fluorogenic gelatin, and proteolytic activity was observed, comparing the microscopic image areas of the veins at 530 nm.

In these experiments, the level of fluorescence is proportional to the activity of MMP in tissue, and the results of sonografii in situ are presented in Table 13.

Table 13 shows that, if we ascribe a value corresponding to 100, the sample without any additives, the sulodexide at a concentration of about 25 μg/ml reduces the intensity of MMP-9 to about 50%; SDX-DS at a concentration of about 5 μg/ml reduces the intensity of MMP-9 to about 80%, and parnaparin in a concentration of about 20 μg/ml reduces the intensity of MMP-9 to about 40%.

The effect of sulodexide, SDX-DS and SDX-HEP on the fabric of Vienna in terms of reduction and/or inhibition can be distributed to all tissues, where MMP involved.

For example, sulodexide, SDX-DS and SDX-HEP can also be used in the treatment of intestinal ulcers and other lesions related to MMP, without limitation.

Order received�of effective concentrations in vivo to reduce the level of MMP and, in particular, MMP-9, sulodexide can be administered intravenously by, inyazura of 1-4 bottles of solution containing about 60 mg/ml of sulodexide with pharmaceutically acceptable excipients. Alternative, sulodexide can be administered in a therapeutically equivalent dose of intramuscular or oral route. In this latter case, you can enter up to 1000 mg/day and a higher dosage without any adverse events, as described in EP 1292315.

Property of sulodexide to reduce the level of circulating MMP and especially selective activity against MMP-9 is a unknown and unexpected effect, in particular, useful for all pathologies involving DFID, and, in particular, MMP-9, such as vascular disease, chronic venous insufficiency (CVI), varicose veins, venous ulcers, lung disease, and increase the development of the tumor, the primary tumor ingrowth, angiogenesis, extravasation and intravasation neoplastic cells.

Described and non-limiting experiments demonstrate a new use of sulodexide, SDX-DS and heparin fractions of sulodexide for the reduction/inhibition of the release of MMP, particularly MMP-9 released by white blood cells and platelets, and inhibition values of MMP in the inner walls of the veins. Results ex-vivo can be fully transferred to the application�s of sulodexide, SDX-DS and heparin fractions of sulodexide in vivo using pharmaceutical compositions containing the sulodexide in an amount of from about 20 to 1000 mg and above; SDX-DS in an amount of from about 4 mg to about 200 mg and above; SDX-HEP in an amount of from about 16 to about 700 and above.

Furthermore, the introduction of sulodexide, SDX-DS and SDX-HEP, can be done by injection, intramuscular and/or intravenous or by mouth, without limitation.

Dosage of active ingredient: sulodexide, SDX-DS and SDX-HEP, can vary greatly depending on the method of administration, patient's age, weight and General condition of the patient, and the severity of the disease, and can be determined by standard methods. In addition, for the identification of possible ranges of dosages you can use the in vitro assays and analysis on animals. Preferably, the pharmaceutical composition is in a form acceptable for oral administration. Sulodexide, SDX-DS and heparin fraction of sulodexide, the present invention may be contained in the composition is in the form of tablets, capsules, granulates, suspensions, solutions or aerosols.

Oral pharmaceutical composition can provide controlled release.

Oral pharmaceutical composition can provide bioadhesive properties to increase mucoadhesive�STI.

Excipient for oral drug is selected from one or more than one coupling agent, a sliding agent, a disintegrant, a diluent, a natural agent.

Excipient for oral use may contain disintegrants, diluents and combinations thereof. Leavening agents can be selected from polyplasdone, sodium starch glycolate, corn starch, cellulose derivatives, and combinations thereof. The diluents may be selected from lactose, mannitol, xylitol, microcrystalline cellulose, sugar, dextrin, hydrophilic carbohydrate, and combinations thereof. Oral pharmaceutical forms can contain bioadhesive excipient.

The tablets may optionally contain a membrane, which contains hydroxypropyl methylcellulose, hydroxymethylcellulose, polyvinylpyrrolidone, polyethylene glycol, dextrin, maltodextrin, compounds based on polyvinyl acetate, hydroxypropyl cellulose, cellulose acetate, cellulose phthalate, derivative or combination thereof.

The oral form may also contain flavors, colors or sweeteners.

Pharmaceutical compositions containing sulodexide, or SDX-DS and SDX-HEP, in comparison with known and studied compounds in reducing/inhibiting the controlled release of MMP, provide numerous advantages � solve many problems, like specific selectivity against MMP-9, tolerability, safety and acceptability.

The present invention relates to a new therapeutic use, other than those for which the sulodexide is available in the prevention of vascular pathologies with a risk of thrombosis.

In pharmaceutical practice, sulodexide, SDX-DS and SDX-HEP can be combined with acceptable excipients, using conventional methods known to the expert.

The composition can be administered in single or multiple doses, once or more than once a day in the composition of controlled release.

The prior art describes the use of sulodexide in the pathologies related to diabetes, such as diabetic nephropathy, while only now, after the present invention, it is possible to have a new medical use of sulodexide.

The object of the present invention are also pharmaceutical compositions containing SDX-DS and SDX-HEP, useful in the treatment of all pathologies involving MMP, particularly MMP-9, without restriction.

A particular aspect of the present invention relates to the above application for the treatment of pathologies involving the relaxation of the veins, for example, the formation of varicose veins, chronic venous insufficiency, gastro-intestinal ulcers pulmonary abnormalities and neoplastic pathologies.

Another specific and important aspect of the present invention relates to a SDX-DS, isolated from sulodexide and its use for inhibiting and/or reducing the release of MMP in vitro and in vivo.

Another aspect of this invention relates to SDX-DS, obtained by using sulodexide, using the cleaning process.

Another particular aspect relates to the preparation of SDX-DS by affinity chromatography with the use of AT III-functionalized column.

A method of separating SDX-DS includes the following stages:

a) preparation of affinity chromatography columns with ATIII in a concentration of from 5 to 10 mg/ml of gel;

b) loading of sulodexide in the specified column (from step a) in a concentration of from 0.1 to 5 mg/ml of gel in a buffer containing 50 mm TRIS and 50 mm NaCl at pH 7.4;

b) Association-eluted fractions of dermatan sulfate in unabsorbed fractions and lyophilization may specified United dermatan sulfate;

g) possible transformation specified dermatan sulfate in its pharmaceutically acceptable salt, solvate, hydrate, clathrate.

Another aspect of this invention relates to SDX-HEP, obtained by using sulodexide, using the cleaning process.

Another particular aspect relates to the preparation of SDX-HEP by affinity chromatography with the use of AT III-functionalized column.

Ability� selection SDX-HEP comprising the following steps:

a) preparation of affinity chromatography columns with ATIII in a concentration of from 5 to 10 mg/ml of gel;

b) loading of sulodexide at a concentration of from 0.1 to 5 mg/ml of gel in a buffer solution containing 50 mm TRIS and 50 mm NaCl at pH 7.4;

C) elution of low molecular weight heparin with the specified chromatographic column with a buffer solution containing 50 mm TRIS and 3 M NaCl at pH 7.4;

g) the Association-eluted fraction of low molecular weight heparin and possibly lyophilization of the specified combined heparin;

d) you can transform the specified low molecular weight heparin in its pharmaceutically acceptable salt, solvate, hydrate, clathrate.

Another specific and important aspect of the present invention relates to a SDX-HEP and its use for inhibiting and/or reducing the release of MMP in vitro and in vivo.

Another aspect relates to pharmaceutical compositions containing sulodexide, in solid form, suspension, granule, aerosol for the treatment of pathologies involving DFID and the level of which should be reduced.

Another aspect relates to kits containing a pharmaceutical composition comprising sulodexide, SDX-DS and SDX-HEP.

Another aspect relates to use of pharmaceutical compositions containing the sulodexide or one of its components, in particular SDX-DS and SDX-HEP, or a mixture thereof, in a dosage that is appropriate for the entered�I at least about 5 mg/day. Examples of daily dosages are from 5 to 1000 mg/day, from 15 to 800 mg/day, 50 to 200 mg/day, 20 to 1000 mg/day or more for one or more introductions, without restriction.

The preferred daily dosage for SDX-DS is from 1 mg to 300 mg or more than, in particular, from 4 mg to 300 mg, in one or more introductions, without restriction.

The preferred daily dosage for SDX-HEP ranges from 16 mg to 900 mg or more, in particular from 19 mg to 800 mg, in one or more introductions, without restriction.

Pharmaceutical composition containing sulodexide, SDX-DS and SDX-HEP, you can type in patients receiving concomitant therapy without any negative effect.

The use of sulodexide, SDX-DS and SDX-HEP, described in the present invention, should be considered the new medical use for the treatment of all pathologies involving DFID, and will require the reduction and/or inhibition. The use of sulodexide, SDX-DS and heparin fractions of sulodexide compared to compounds previously investigated in the prior art overcomes several important issues, such as specific selectivity against MMP-9, patient compliance with treatment and ease of administration.

This invention is described in detail in the following examples, which should not be construed as limiting this and�the acquisition.

Graphic materials

Fig.1: Gel filtration profile of sulodexide;

Fig.2: Gel filtration profile of dermatan sulfate, obtained by using sulodexide;

Fig.3: Gel filtration profile of heparin, obtained by using sulodexide.

Example 1

A method of producing dermatosurgical fraction of sulodexide

The number of sulodexide extracted from intestinal mucosa of the animal, corresponding to 8 mg, was dissolved in the buffer for binding, containing 50 mm TRIS and 50 mm NaCl at pH 7.4, and applied to affinity column obtained by combining 7.5 mg ATIII/ml CNBr-activated Safronova gel (GE).

The column was washed with the same buffer solution, and the absorbed substance was suirable buffer solution consisting of 50 mm TRIS and 3 M NaCl at pH 7.4.

The dermatan sulfate fraction was collected in the unabsorbed substance present in the eluate, whereas heparin fraction is collected in-eluted fractions.

The fraction of dermatan sulfate and heparin can be stored at -20° or can be liofilizirovanny.

The combined fractions of heparin and the United dermatan sulfate fractions were analyzed by gel-filtration chromatography (GPC) and in agarose gel.

Chromatographic profiles were obtained in the HPLC system HP 1090 equipped with a GPC column with TSK gel, G2000 SWXL (of 7.8×300 mm), elwira using 0.2 M N 2SO4, pH 5, at a flow rate corresponding to 0.2 ml/min, and determining with a detector refractive index (RI, Refraction Index).

Fig.1 shows the Chromatographic profiles of sulodexide applied to affinity chromatography, with a broad chromatographic profile of GPC with a sharp maximum peak at RT (retention time), corresponding to about 30 min.

Fig.2 shows the fraction of dermatan sulfate, which is present in the eluate of affinity chromatography, with a shorter peak with a maximum at RT for about 27 min.

Fig.3 shows the fraction of heparin-eluted by affinity chromatography, which is characterized by a broad peak.

The analysis in agarose gel was made by dissolving agarose in a concentration of 0.6% (wt./about.) in the barium-acetate buffer 0.04 M, pH of 5.8. The warm solution was poured onto a glass plate with dimensions of about 7.5 cm×4.5 cm and sulodexide, SDX-DS and SDX-HEP at a concentration of about 10 mg/ml, containing a small amount of dye cresol red, were applied to the agarose gel. Electrophoretic separation was carried out in two stages: the first stage in the barium-acetate buffer 0.04 M, pH of 5.8 At 100 V for about 25 min and second phase 1.3-diaminopropane 0.05 M, pH 9 at 110 V for about 30-40 minutes. The plate was then immersed in a fixative solution consisting of 0.1% (wt./about.) CET�pyridine chloride in water for about 3 hours. After drying the plate in warm air, the plate was immersed in a staining solution composed of 0.1% (wt./about.) toluidine blue in a mixture of ethanol/water/acetic acid 50:50:1 (vol./about./vol.), they bleached in the same solution without the dye and dried with warm air.

The relative mobility (Rf) of sulodexide, a dedicated component of SDX-DS and SDX-HEP and commercial dermatan sulfate are presented in Table 1.

Table 1
Component (mcg/ml)Rf
Sulodexide0,86
0,75
Fraction of dermatan sulfate (SDX-DS)0,84
The fraction of heparin (SDX-DS)0,75
Commercial dermatan sulfate (Sigma)0,86

Example 2

Effect of sulodexide on circulating MMP in blood samples

Blood samples taken from 50 healthy volunteers, 25 women and 25 men, aged between 25 and 58 years, mean age 37 years, was placed in a test-tube and received two sets of samples; the first set was added a varying amount�of sulodexide to obtain final concentration, corresponding to 0, 12, 24, or 48 mg/ml, in the second set were added varying amounts of dermatan obtained as in Example 1, to a final concentration corresponding to 0, 2,5, 5, or 10 μg/ml. the Samples without the addition of sulodexide and dermatan used as a control, and the bands of MMP these samples was ascribed an arbitrary value intensity of 100%. The intensity of the bands of MMP-treated samples were expressed in percentage relative to untreated samples.

The final samples were centrifuged at 1550 g for 10 minutes at 4°C, the number of supernatants of serum samples (S) corresponding to 150 μg of total protein determined by Bradford, was analyzed with the help of gelatin sonografii. Samples were applied to gel sonografii, and separation in the electrophoresis, compared with the standard MMP obtained by diluting the capillary blood from healthy volunteers with 15 volumes of buffer Laemmli U. K (Nature, 227 (1970), 680-685) in Sevostyanova conditions.

Zymogram received in the gelatin 7.5% (wt./about.) polyacrylamide containing 0.2% (wt./about.) gelatin extracted from porcine skin (type a 90 Bloom, Sigma).

Electrophoresis was carried out at 35 mV for 75 minutes, the gels were washed with solutions of Triton X-100 at a concentration of 2.5% (about./about.) and were incubated for 24 hours at 37°C in a solution containing 50 mm TRIS-HCl, pH 7.5, 5 mm CaCl , 100 mm NaCl, 1 mm ZnCl2, 0.3 mm NaN3, 0,02% (mass./about.) detergent Brij®-35 and 0.25% (vol./about.) Triton X-100. Detection gelatinase activity is carried out in situ by staining the gels with Coomassie Brilliant blue R-250 in a concentration of 0.2% (wt./about.) and then bleaching with a solution of methanol-acetic acid in a volume ratio of 80/20.

Sulodexide and SDX-DS, obtained as in Example 1, is valid only on the band related to MMP-9, whereas the intensity of the band of MMP-2 remained unchanged relative to control, and she is credited with the value of 100%. The calculation of the area was carried out using a densitometer with image analyzer. Table 1 shows the area percentage gelatinolytic digestion associated with MMP-9 in serum samples with or without sulodexide, a fraction of dermatan or without it.

Table 2
Gelatin zymography: serum Concentrations of MMP-9 after treatment with sulodexide and dermatan sulfate, obtained as in Example 1
Sulodexide (µg/ml)Area, % MMP-9Dermatan sulfate obtained as in Example 1 (µg/ ml)Area, % MMP-9
0 100±70100±7
1257±42,546±3
2441±5530±7
487±1104±7

The concentration of MMP-9 in the samples are also determined in an ELISA for the measurement of the enzymatic activity of metalloproteinases, using analysis of MMP Biotrack (Amersham), which recognizes forms of Pro-MMP gelatinase with LOD (limit of detection, from the English. limit of detection) from 0.37 to 0.25 µg/l for MMP-9 and MMP-2. The results are presented in Table 2. These ELISA results did not show statistically significant differences from gelatinolytic activity discovered sonografii.

Table 3
ELISA analysis: the Concentration of serum MMP after treatment with sulodexide and dermatan sulfate, obtained as in Example 1
Sulodexide (µg/ml)% MMP-9Dermatan sulfate obtained as Example 1 % MMP-9
0100±40100±4
1261±62,547±4
2443±8531±5
486±3105±1

Example 3

Effect of sulodexide on the number of circulating plasma MMP

Blood samples were taken from 50 healthy volunteers, 25 women and 25 men, aged between 25 and 58 years, mean age 37 years, and 1 ml of each sample was added to 38 μl of 0.13 M sodium citrate solution and centrifuged at 1550 g for 10 minutes at 4°C.

Increasing amounts of sulodexide added to the samples to obtain the final concentration corresponding to 0, 12, 24 and 48 μg/ml.

The amount corresponding to 150 μg of total protein from supernatants of plasma samples (P), obtained by centrifugation, measured by the Bradford analyzed by gelatin of sonografii. The gel is applied standard gelatinase obtained by diluting the capillary blood of healthy volunteers 15 volumes of the buffet�and Laemmli, in Sevostyanova conditions.

Zymogram received in the gelatin and electrophoresis was performed as described in Example 2.

Square streaks zymogram was determined using a densitometer with image analyzer, calculating the increase in the area of the bands related to the metalloproteinases, compared with stripes for a plasma without the addition of sulodexide. Area in percent gelatinolytic digestion related to MMP-9, in the blood sample with or without sulodexide is presented in Table 4.

Table 4
Gelatin zymography: Concentration of MMP-9 in plasma after treatment with sulodexide
The concentration of sulodexide (µg/ml)Area of MMP-9, %
0100±5
1298±4
24101±4
4899±4

Densitometric analysis of MMP-9 on zymograms confirmed by quantitative ELISA analysis whose data is presented in Table 4 do not show any significant differences in comparison with the densitometric Ana�Isom of zymogram.

Table 5
ELISA analysis: the Concentration of MMP in plasma after treatment with sulodexide
The concentration of sulodexide (µg/ml)The concentration of MMP, %
0100±7
1299±5
24100±3
4898±4

Example 4

Effect of sulodexide on the number of circulating MMP in serum after the formation of whey

Blood samples taken from 50 healthy volunteers, 25 women and 25 men, aged between 25 and 58 years, mean age 37 years, is placed in a test tube and centrifuged at 1500 g for ten minutes at 4°C.

The increasing number of sulodexide added to 150 μg of protein of serum, measured according to Bradford, to obtain a final concentration of sulodexide corresponding to 0, 12, 24, 48 µg/ml. the resulting samples were analyzed by gelatin of sonografii in comparison with the standard gelatinase obtained by diluting the capillary blood from healthy volunteers 15 volumes of Laemmli buffer, Sevostyanova�their terms.

Getting zymogram, electrophoresis and staining/discoloration is carried out as described in Example 2.

Square streaks shimogamo determined using a densitometer with image analyzer, calculating the reduction in the area of the bands related to the metalloproteinases, compared with those for serum without the addition of sulodexide. Area in percent gelatinolytic the digestion relating to DFID, in a sample of serum with or without sulodexide presented in Table 6.

Table 6
Gelatin zymography: Concentrations of MMP-9 in serum in the presence of increasing concentrations of sulodexide
The concentration of sulodexide (µg/ml)Area (%) MMP-9
0100±6
1298±4
24101±3
4899±4

The data obtained by demographia not show significant changes in comparison with the data obtained by ELISA, as shown in Table 7.

Table 7
ELISA: the Concentration of MMP in plasma with different concentrations of sulodexide
The concentration of sulodexide (µg/ml)The concentration of MMP-9, %
0100±5
1299±2
24101±4
4897±6

Example 5: Comparative example

Evaluation of sulodexide, dermatan sulfate, obtained as in Example 1, and parnaparin

Blood samples, obtained as described in Example 2 were divided into three groups, where one group was treated with sulodexide at a final concentration corresponding to 24 mg/ml, one group was treated with parnaparin in a final concentration corresponding to 5 μg/ml, and the last group was treated dermatology fraction obtained as in Example 1 at a concentration of 5 μg/ml, and compared sonografii and ELISA.

The results are presented in Table 8 and Table 9.

Table 8
Sravneniyu serum reviewed by demographia
Sulodexide (µg/ml)% MMP-9Parnaparin (µg/ml)% MMP-9Dermatan sulfate Sample 1 (ág/ml)Area of MMP-9, %
0100±70100±70100±7
2441±51969±7530±6
Table 9
Comparison of serum samples analyzed ELISA
Sulodexide (µg/ml)% MMP-9Parnaparin (µg/ml)% MMP-9Dermatan sulfate Sample 1 (ág/ml)Area of MMP-9, %
0/td> 100±70100±70100±7
2443±81967±9532±8

These Examples confirm the effect of sulodexide and dermatosurgical fraction present in the sulodexide, in the reduction and/or inhibition of MMP, particularly MMP-9.

Example 6: Comparative example

Comparison between commercial dermatan sulfate and dermatan sulfate, obtained as in Example 1

Blood samples, obtained as described in Example 2 were divided into three groups where the first group was treated with a commercial dermatan sulfate from Sigma Aldrich (Italy); the second group was treated with dermatan sulfate from Calbiochem (Italy), and the third was treated with DS, extracted from sulodexide, as in Example 1, where all the final concentration corresponding to 5 μg/ml. Samples were analyzed in ELISA as in Example 2, and the final results are presented in Table 10.

Table 10
Dermatan sulfate (igma Aldrich) (µg/ml) % MMP-9Dermatan sulfate Calbiochem (µg/ml)% MMP-9Dermatan sulfate from Example 1 (µg/ml)Area of MMP-9, %
0100±50100±50100±7
589±8595±7532±8

Example 7: Comparative example

Comparison between commercial dermatan sulfate and dermatan sulfate, obtained as in Example 1

Blood samples, obtained as described in Example 2 were divided into five groups:

Group 1: a sample of blood from commercial parnaparin at a concentration of 19 µg/ml;

Group 2: blood sample with sulodexide at a concentration of 24 μg/ml;

Group 3: a sample of blood from DS, obtained by using sulodexide as in Example 1 at a concentration of 5 µg/ml;

Group 4: blood sample with a mixture parnaparin at a concentration of 19 μg/ml and DS obtained with the help of sulodexide as in Example 1 at a concentration of 5 µg/ml;

Group 5: a blood sample with a mixture parnaparin at a concentration of 19 μg/ml and DS in commercial con�entrale 5 μg/ml.

The samples were analyzed in ELISA as described in Example 2 and the results are presented in Table 11.

The decrease in the level of MMP-9 was calculated in relation to the samples without any additives, which were attributed to the size of the area corresponding to 100%.

TABLE 11
CompositionConcentration (µg/ml)Area of MMP-9, %
Sulodexide2443±8
SDX-DS531±6
Parnaparin1973±9
Parnaparin + SDX-DS19±555±4
Parnaparin + DS-Sigma19±587±6

Commercial DS from Sigma and Calbiochem showed the same results.

Example 8

Evaluation of sulodexide, dermatan sulfate (SDX-DS) and parnaparin invenous walls ex vivo by immunohistochemistry

Segments of the saphenous veins of the legs of a man, surgically obtained from 10 healthy women (mean age 42 years) subjected to Safina�thomy against varicose veins maintained under sterile conditions in CO2incubator (5% CO2at 37°C for 24 hours) in 0.9% NaCl and divided into three parts. The first part was treated with sulodexide at a concentration of 24 μg/ml; the second part was treated with parnaparin at a concentration of 19 μg/ml and the third part was treated with SDX-DS, obtained as in Example 1 at a concentration of 5 μg/ml. vein Segments were fixed in 10% formalin solution, processed and placed in paraffin. Prepared paraffin blocks are oriented perpendicularly to the original direction of flow, then sliced into transverse sections with a thickness of 5 mm and placed on a cover glass coated with 3-aminopropyltriethoxysilane. The plot of the venous wall was cut into serial sections with a thickness of 5 μm. Serial sections were investigated by staining for elastin by Miller and stained with hematoxylin and eosin according to van Gisone and Harris. The sections were subjected to protein blocking normal goat serum and then overnight incubated at 4°C with primary monoclonal antibody against MMP-9 (Santa Cruz), diluted 1:50 with phosphate buffered saline (PBS).

Then slices the veins were incubated with secondary antibody (biotinylated goat antibody against mouse IgG as the standard methods, and used a complex of avidin-Biotin-peroxidase.

After washing in PBS CPE�s veins were stained with DAB solution (diaminobenzidine) and H 2O2the standard way.

Then the sections were subjected to contrast staining with hematoxylin according to Mayer, dehydrated, clarified and recorded. In each of the painted party has always included positive and negative controls to ensure consistency between successive series.

Images were obtained using an electronic microscope (40×) and the results are summarized in Table 12. The color intensity was measured using an optical control, attributing a value of 100 tissue without any additives (control), and were evaluated by visual weakening of black color, as defined under the influence of SDX-DS and SDX-HEP and parnaparin. The results are presented in Table 12.

Table 12
Concentration (µg/ml)The decrease in intensity, %
Sulodexide2460
SDX-DS570
Parnaparin190

Example 9

The ex vivo evaluation of sulodexide, dermatan sulfate and parnaparin in the venous walls of demografie� in situ (cytochemistry)

Segments of the saphenous veins of the legs of a man, surgically obtained from 10 healthy women (mean age 42 years), subjected to saphenectomy in relation to varicose veins, maintained under sterile conditions in CO2incubator (5% CO2at 37°C for 24 hours) in 0.9% NaCl and divided into three parts. The first part was treated with sulodexide at a concentration of 24 μg/ml; the second part was treated with parnaparin at a concentration of 19 μg/ml and the third part was treated with SDX-DS, obtained as in Example 1 at a concentration of 5 μg/ml. vein Segments were fixed in 10% formalin solution, processed and placed in paraffin. Prepared paraffin blocks are oriented perpendicularly to the original direction of flow, sliced into transverse sections with a thickness of 5 mm and placed on a cover glass coated with 3-aminopropyltriethoxysilane. Segments of the venous wall was cut into serial sections with a thickness of 5 μm.

Sections of the vessels were incubated at 37°C for 5 hours with fluorogenic gelatin substrate (DQ Gelatin, Molecular probes) at a concentration of 25 μg/ml in buffer containing 50 mm TR1S-HCl, pH 7.5; 5 mm CaCl2; 100 mm NaCl; 1 mm ZnCl2; 0.3 mm NaN3.

Proteolytic activity was observed in situ by comparing venous segments at 530 nm and identifying them by green fluorescence. Data of densitometric analysis using sonografii in stu in the tissue of the vein by means of cytochemical staining of MMP-9 fluorogenic gelatin in the presence of SDX-DS, SDX-HEP and parnaparin relative to control is presented in Table 13. A value of 100 was attributed to tissue without any additives and measuring the decrease in fluorescence after addition of SDX-DS, SDS-HEP and parnaparin.

Table 13
Concentration (µg/ml)The decrease in intensity, %
Sulodexide2450
SDX-DS580
Parnaparin1950

1. The use of dermatan sulfate, isolated from sulodexide, in a daily dose from 4 mg to 300 mg for the treatment of diseases involving metalloproteinase-9 MMP-9, selected from varicose veins and vascular pathologies with a risk of thrombosis.

2. The use according to claim 1, wherein the dermatan sulfate is highlighted through the use of affinity chromatography and has a molecular weight of approximately 25 kDa.

3. The use according to claim 1, wherein the vascular disease is selected from chronic venous insufficiency, venous ulcers, ulcers of the lower limbs and pulmonary pathologies.

4. The use according to claim 1, where d�metasulfite is in combination with sulodexide or with low molecular weight heparin, selected from sulodexide.



 

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4 dwg, 20 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: before operation analysis of patient's haemostasis by means of thrombodynamics is carried out, and 12 hours before beginning operation anticoagulant prophylaxis with enoxaparin in dose 40 mg s/c 1 time per day performed. Analysis of thrombodynamics and coagulogram are repeated one day after operation, In case of detection of hypercoagulation (increase of one or some indices of thrombodynamics - initial speed of clot growth, stationary speed of clot growth, clot density, appearance of spontaneous clots) dose of enoxaparin is increased to 60 mg one time per day, and in case of detection of hypocoagulation (reduction of one or several indices of thrombodynamics - initial speed of clot growth, stationary speed of clot growth, clot density, delay of clot growth) dose of anticoagulant is reduced twice - 20 mg of enoxaparin per day.

EFFECT: method makes it possible to prevent development of post-operative venous thromboembolic complications in patients with colorectal cancer; said regimen of enoxaparin introduction provides prevention of both thromboembolic and hemorrhagic complications in said group of patients.

2 ex

FIELD: chemistry.

SUBSTANCE: thrombocyte aggregation-inhibiting heteromeric peptides based on imidazo[4,5-e]benzo[1,2-c;3,4-c']difuroxane are disclosed: , where R=Phe-Ile-Ala-Asp-Thr; Arg-Tyr-Gly-Asp-Arg; Lys-Ile-Ala-Asp-Asp; His-Ile-Gly-Asp-Asp.

EFFECT: improved properties.

1 dwg, 2 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to N-carb(arginyl)oxymethylimidazo[4,5-e]benzo[1,2-c; 3,4-c']difuroxane of formula .

EFFECT: improved properties of the compound.

1 dwg, 1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to the field of pharmaceutics and represents a medication, possessing venomotor and anticoagulant action, which includes a dry extract of vine leaves, a base, preservatives, solvents, and is characterised by the fact that it additionally contains heparin in the form of a pharmaceutically acceptable salt, as the base it contains a hydrophilic gel-generating agent, as the solvents it contains purified water, propyleneglycol, ethyl alcohol rectified 95%, as the preservatives it contains nipagin, nipasol, with the following component ratio, wt %: dry extract of vine leaves with the content of the sum of flavonoides counter per rutin no less than 8% - 0.1-30.0; heparin in the form of a pharmaceutically acceptable salt - 100-1000 Units, hydrophilic gel-generating agent - 0.2-20.0; nipagin - 0.01-0.2; nipasol - 0.01-0.2, propyleneglycol - 3.0-70.0; ethyl alcohol rectified 95% - 3.0-70.0; purified water - the remaining part.

EFFECT: invention provides the creation of the medication in the form of a gel with good absorption, fast soaking, storage stability and acceptable organoleptic properties.

4 cl, 8 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to a citrate of a compound described by formula (II) below, and a pharmaceutical composition containing said citrate.

EFFECT: experimental results of the present inventions prove that said citrate can inhibit activity of phosphodiesterase type 5 and can be used for treating erectile dysfunction, for inhibiting thrombocyte aggregation and treating thrombosis, for reducing pulmonary hypertension and treating cardiovascular diseases, asthma and diabetic gastroparesis.

2 cl

FIELD: medicine.

SUBSTANCE: invention concerns preparing systemic and topical solid and soft dosage forms for external application in the form of a 1.5% hydrophilic gel and rectal capsules containing a 7.5% hydrophilic gel 1.9 g (in terms of the amount of the active substance of 0.14 g/capsule), for preventing and treating chronic venous insufficiency possessing anticoagulant, antithrombotic, anti-inflammatory, antiexudative and antitranssudative, capillary protective activities and improving haemorheology. An active pharmacologically active substance is presented by a substance of a potassium salt of sulphated arabinogalactan; a hydrophilic base is Aerosil, glycerol and pure water; the ingredients of the agent are taken in certain proportions, wt %.

EFFECT: invention provides the effective prevention and treatment of chronic venous insufficiency, has a broad spectrum of therapeutic action, improves haemorheology, tones the vessels and can be used in particular for treating varicose veins, thrombosis and posttraumatic oedema.

5 cl, 7 ex, 8 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine, particularly to pharmaceutical industry, and describes a dosage form of Clopidogrel presented in the form of a solid gelatine capsule. The dosage form contains Clopidogrel hydrogen sulphate, lactose anhydride, microcrystalline cellulose, sodium croscarmellose, colloidal silicon dioxide and magnesium stearate.

EFFECT: according to the invention, the dosage form of Clopidogrel contains a high amount of the active ingredient; it is prepared without the use of a wet granulation technique, and provides the more accurate dosage of the ingredients and the stability of the substances used.

9 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of immunology and biotechnology. Claimed is monoclonal antibody or its functional fragment, where said antibody and fragment bind with activated protein C and inhibit anticoagulant activity, but do not bind and do not inhibit activation of inactivated protein C, where said antibody is obtained by immunisation of mammal by APC and screening of binding ability of said antibody with APC, but not with protein C. Also described is pharmaceutical composition for treating diseases associated with anticoagulation activity of APC, including said antibody in effective amount and pharmaceutically acceptable carrier. Claimed are: method of inhibiting anticoagulation activity of activated protein C in subject, method of inhibiting amidolytic activity of activated protein C in subject, method of treating subject, requiring blood coagulation; method of treating subject with haemophilia; method of modulating haemostasis in subject; as well as method of modulating thrombogenesis in subject, which include introduction of effective quality of said antibody to subject. In addition, described is method of treating subject with sepsis, including introduction of effective quality of said antibody and activated protein C.

EFFECT: invention makes it possible to obtain monoclonal antibody or its functional fragment, where said antibody and fragment bind with activated protein C and inhibit anticoagulation activity, but do not bind and do not inhibit activation of inactivated protein C.

17 cl, 11 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: early postoperative period involves administering low-molecular heparins and anti-inflammatory agents. Anti-inflammatory therapy requires administering cycloferon according to the schedule: 2 tablets of cycloferon 0.15 mg on the first preoperative day, 2 tablets on the first postoperative day, 2 tablets in the morning on the 2nd, 4th, 6th postoperative day, and further, every third day throughout 1 postoperative month (on the 9th, 12th, 15th, 18th, 21st, 24th, 27th, 30th day).

EFFECT: method provides the effective prevention of postpericardiotomy syndrome with a lower risk of side effects by administering cycloferon according to the developed schedule requiring non-steroidal anti-inflammatory agents and glucocorticosteroids.

2 ex, 3 tbl

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