Connections on the basis of the reaction of amadori, their use, method of preparation and compositions on their basis

 

(57) Abstract:

The invention relates to chemistry, medicine and pharmacology. Proposed new formulations or combinations of known compounds on the basis of the reaction of Amadori formula R1-NH-R2where R1includes D-form 1-amino-1-deoxy-2-ketoreductase derived Zaharenko radical selected from the group of glucose, galactose, ramnose, fructose, mannose, 6-deoxyglucose and glucosamine or oligo - and polysaccharides, and R2includes L-form amino acids or a radical of the peptide as the active principle of the pharmaceutical drugs from the production of interferon and other cytokines. Cosmetic compositions based on the above compositions are used for cellular nutrition for tissue regeneration and/or for immunomodulatory. The invention expands the Arsenal of tools specified destination. A method of obtaining a mixture of immunotropic compounds based on rearrangement of Amadori. 3 S. and 11 C.p. f-crystals, 4 tab., 6 Il.

The invention relates to compounds based on the reaction of Amadori obtained on the basis of their compositions, to a method for production thereof, and also to a new method of application of these compositions and their products, which is RA:

< / BR>
The known reaction products of Amadori, such as for example, reaction products of amino acids or glycopeptide with oligo - or polysaccharide, past the rearrangement of Amadori (J. Biol.Soc.215 (1955) Henri Boreook et al.).For example, in the Federal Republic of Germany patent DE-C - 3914354 described water-soluble glycoprotein amino acid and sugar, which is isolated from an extract of the seeds of Avena sativa. Further, in the patent EP-A-406087 described water-soluble complex compounds of sugars with glycopeptides derived from the cell walls of gram-positive bacteria, and in the journal J. Biol.Chem.1965, 260/9 described the use of nanoscale spectroscopy to characterize the reaction products of Amadori formed by the reaction of glucose with the free amino groups of the protein.

The present invention relates to specific new connection based on the rearrangement of Amadori in accordance with paragraph 1 of the formula and advantages set forth in claims 2-4.

These compounds are recovering potassium ferricyanide, and test-response for biologically active substances produced by the reaction of sugars and amino acids.

The invention also relates to the new use of such compounds and at the same time to a new way of applying the eat was not conducted testing on the biological activity of the various stages of the purification of the extract of the reaction product of Amadori (removal of ballast substances and contaminants.

Known immunostimulatory drugs derived from natural products such as extracts from mistletoe, peat, etc., the main drawback of which is the need for expensive processing large amounts of raw materials to obtain a few grams of active substance. Moreover, outside the control of pollution can be toxic and cause side effects and as a consequence, in practice it is often difficult to apply because of its complex properties transfer composition.

The production of such comparable products or mixtures thereof, derived from artificial substances, such as interferon, as well as other methods of genetic engineering, are even more costly.

In addition, molecules of interferon person is often too large to penetrate through the wall of human cells, therefore, effective action has only part of the prescribed dose. Moreover, the products of genetic engineering usually have side effects, and some are even toxic. Moreover, for reasons unknown up to the present time, some of them in one day turns out to be uboy simple complex amino acid/sugar after at least partial rearrangement of Amadori does not detect any of the above-mentioned disadvantages, but on the contrary becomes a surprisingly high immunological activity.

As a consequence, they can be used in pharmaceutical compositions and in cosmetics. These small molecules can easily penetrate through the wall of the cage and actually act as a nutrient. They induce the formation of natural interferon and other cytokines, including tumor necrosis factor. Even three days after treatment with a drug that they still find the effect of the stimulation of biological activity. This effect increases as the complete rearrangement and then decreases during decomposition of the complex.

Instead of simple sugars, preferably low molecular weight, in particular less than 1000 daltons, you can also use the polysaccharides similar reactions, such as dextran. Polysaccharides possess biological activity and can save some species such activity even after they turn into oligosaccharides.

Up to this time had very little is known about the biological activity of these compounds. It was found that combinations of these substances in the interaction with leukocytes person produce interferon and other cytokines. This phenomenon under the action of these compounds, and to determine in international units of their biological activity in relation to certain cytokines.

These compounds have a particularly high biological activity in the range of concentrations of the pure substance from 1 to 100 mg/ml At the level of molecules specific properties of amino acids are more important than the properties of the sugar part of the molecule.

Products of reaction of L-aspartic acid with glucose or galactose, past the rearrangement of Amadori, when interacting with leukocytes of man and incubated in tissue culture medium at 37oC for 20 hours in 5% CO2produce from 30 to 1000 antiviral units of interferon.

Interferon analyzed by bioassay with cancer human cells. Under the action of these compounds may produce the compositions of tumor necrosis.

Products, giving the possibility of such an unexpected application, have the General formula:

R'1-NH-R2< / BR>
where R'1- 1-amino-1-deoxy-2-CentOS radical derived group of simple sugars, oligo - and polysaccharides, preferably low molecular weight, in particular less than 1000 daltons.

R'2- amino acid or Radik is as biologically active compounds may include, as described above specific compounds rearrange Amadori, and N-substituted derivatives of a number of different compositions of amino acids and one simple sugars, oligo - or polysaccharide, preferably low molecular weight, in particular less than 1000 daltons, or N-substituted derivatives of the same composition of amino acids and some simple sugars, oligo - and/or polysaccharides, or any other combination of such derivatives, and each of them has sufficient biological activity.

Preferably, R'1in the formula above, was a radical selected from simple sugars D-form, in particular (but not necessarily) from glucose D-form, xylose, galactose, ramnose, fructose, mannose, G-deoxyglucose, glucosamine and galactosamine, R'2he was a radical, selected from compounds of the amino acids L-shape, such as serine, glycine, histidine, arginine, glutamine, asparagine, aspartic acid, glutamic acid, phenylalanine, threonine, cysteine, cystine, methionine, hydroxyproline, tryptophan, Proline, valine, isoleucine, leucine and lysine or other peptides of these amino acids in any combination.

The invention also relates to a method for obtaining the above-mentioned compositions and products described in paragraph 5 of the claims, by means of which receive intermediate about the O-bridge-type form, either oligo - or polysaccharides, and

R"is amino or a radical of the peptide, and this intermediate product is at least partially subjected to a rearrangement of Amadori and/or Maillard reaction through prolonged heating of the reactor mixture, preferably under pressure, and simultaneous or sequential removal of solvents.

A preferred variant of the process according to the present invention described in paragraphs 6-10 of the formula.

In some cases, especially when using amino acids having two carboxyl groups, preferably the use of buffer salts, for example sodium bicarbonate, preferably in a molar ratio of 1:1.

It was also found that the products of the rearrangement of Amadori quite sensitive to decomposition and the decomposition products are resinous consistency and dark brown color, and not determinable compositions lose their biological activity. Consequently, it is preferable to stop the reaction rearrange Amadori at the stage when the reaction mixture becomes weak orange-brown hue.

It should be noted that the intermediate reaction products, obrazuyuschie is to hydrolyzed, i.e. the reaction is reversible.

As rearrangements of reversibility decreases, because the products are more stable, and the color gradually changes from light yellow to light orange, and finally to orange - brown at the end of the rearrangement of Amadori. Taken during the rearrangement reaction of Amadori samples were subjected to testing according to different procedures described below), with evidence of increasing biological activity as the reaction of Amadori and decrease if further heating causes decomposition, which indicator is a color change to dark brown.

Instant reduction of ferricyanide and the resulting color change occurs if the reaction mixture contains other keto groups and/or sulfur-containing amino acids such as cysteine; in other cases, it occurs within 3 to 5 minutes, allowing you to easily control the degree of transmission rearrange Amadori. No reacts sugar detect color change only after half an hour or even several hours.

Isolation of pure products rearrange Amadori is) or Dowex(R), further alyazia with ammonia water evaporation under the pressure of certain selected fractions of the eluate and crystallization of pure composition of anhidrosis methanol (J. E. Hodge and B. E. Fisher, Mtthods in Carbohydrate Chemistry, Vol.II, Reactions of Carbohydrates, Academic Press, N. Y., London, 1963, page 105-106; or Borsook at al. as quoted earller; or J. Dubourg and P. Devilliers).

In the method according to the present invention are retained without change all of the above advantages related to radicals derived from simple sugars and amino acid compositions. In addition, a mixture of substrates sugars preferably contains a D-form, glucose, xylose, galactose, ramnose and fructose in a weight ratio of approximately 20:10:4:1:1, and the mixture of amino acid substrates preferably includes an L-shaped serine, glycine, histidine, arginine, Proline, tyrosine, valine, leucine, isoleucine and lysine in a weight ratio of 20.5:35.6:35.8:132:180:360:216:160:72:68:780.

As already mentioned, the products of the rearrangement of Amadori able to reduce ferricyanide of potassium, and such control chemical reaction allows you to quickly determine the biological activity of the compounds obtained by the reaction of sugars and amino acids.

It was found that the reaction products are particularly active when the AET in the reaction with a mixture of compounds of amino acids of the same composition and the same weight ratio, as with natural extracts of peat in the presence of aqueous solvent, preferably adding a lower alcohol, and any of inorganic trace elements present in natural extracts of peat. The reaction proceeds under an arbitrary pressure at elevated temperature. Further, the heat continue to complete the rearrangement of Amadori the resulting products are then simultaneously or sequentially remove solvents and stop rearrangement reaction at the stage when the reaction mixture becomes weak orange - brown color, then the products obtained are dried and purified by using column chromatography, then collect the fraction, thereby providing a maximum reduction of ferricyanide of potassium.

According to one embodiments of the invention a fast-acting pharmaceutical compositions contain as active ingredient at least one reaction product of the formula R'1-NH-R'2or the specific composition of the formula R1-NH-R2with a pharmaceutically acceptable carrier and/or arbitrary lubricating agent in the weight ratio of active ingredient to the other components in the range from 1:1 and 1:100, before whom bretania pharmaceutical composition contains in addition to the active ingredient, the lactose and lubricating means, moreover, the weight ratio of lactose to lubricating means is between 20:1 and 100:1, preferably 50:1.

These pharmaceutical preparations used to treat and/or prevent hematological and/or immunological diseases, as well as stimulation of immunosystem humans and/or animals by administration of a formation of the cytokine.

Another area of application of the active ingredients are cosmetic preparations. In such preparations the active ingredient is contained in an amount of 0.01-10% by weight, preferably 0.01 to 1% by weight, in particular 0.05 to 0.1%. These cosmetic compositions contain, besides the active ingredient, conventional carriers, adjuvants, processing components and/or aromatic agents.

Further, the present invention is described in detail and illustrated in the following examples, which in no way limit the scope of the invention.

Example 1. In a hot water bath was placed the flask of the rotary evaporator volume of 25 ml, filled with a mixture of the following composition: 1.47 g (0.01 M) - L-glutamic acid, 0.84 g (0.01 M) - NaHCO3; 0,91 g - D-glucose; 0,91 g - galactose and 3,00 ml double-distilled water.

The mixture was heated up to 80oC when pony is ture 80-85oC, at which the mixture was stirred for 120 min, until it was acquired by the color orange. Then syrupy concentrated aqueous solution was dried under reduced pressure. The obtained solid reaction product is orange-red color marked as D-10 and retained for biological tests.

Example 2. In a hot water bath was placed the flask of the rotary evaporator volume of 25 ml, filled with a mixture of the following composition: 1,33 g (0.01 M) - L-aspartic acid, 0.84 g (0.01 M) - NaHCO3; 0,91 g - D-glucose; 0,91 g - galactose; 3,00 - double-distilled water.

The mixture was heated up to 80oC when the rotational stirring, then evaporated under pressure to the evaporation of 1.5 ml of water, then treated at atmospheric pressure at a temperature of 80-85oC to obtain a mixture of a weak orange within 60 minutes Syrupy concentrated aqueous solution was dried under reduced pressure. The resulting reaction product was a dry yellow-orange powder. It was marked as D-11 and preserved for biological tests.

Example 3. In a hot water bath was placed the flask of the rotary evaporator volume of 25 ml, filled with a mixture of the following composition: 1,5 Ali to 85-92oC with rotational mixing. After 100 min, the solution was acquired orange color. The pressure was reduced and the mixture is evaporated until dry. On the walls of the vessel appeared translucent orange layer of the reaction product, which was scraped and crushed to a fine powder, marked as D-12 and retained for biological tests.

Example 4. In a hot water bath was placed the flask of the rotary evaporator volume of 25 ml, filled with a mixture of the following composition: 0.66 g (0.005 M) - D-aspartic acid; 0,42 g (0,0005 M) - NaHCO3; 0.45 g - D-glucose; 0.45 g - galactose; 3,00 ml double-distilled water,

The mixture was heated up to 80oC, evaporated under the pressure of the water volume of 1.5 ml, and then treated at atmospheric pressure at a temperature of 85oC. After heating for 60 min and the mixture was bought orange color, the pressure was reduced, and the resulting syrupy aqueous solution evaporated until dry. To completely eliminate the moisture before the final drying of the precipitate in the flask was twice brought to 10 ml of anhydrous ethanol and evaporated. The resulting dry product was ground to powder, marked as D-13 and retained for biological tests.

Example 5. DL the peat in a heated water bath was placed the flask of a rotary evaporator, filled composition: 20,5 mg - L-serine; 35,8 mg - L-glycine; 35,8 mg - L-histidine; 132,0 mg - L-arginine; 180,0 mg - L-alanine; 360,0 mg - L-Proline; 216,0 mg - L-tyrosine; 160,0 mg - L-valine; 68,0 mg - L-isoleucine; 72,0 mg - L-leucine; 780,0 mg - L-lysine; 2000,0 mg - D-glucose; 1000,0 mg - D-xylose; 400,0 mg - D-galactose; 100.0 mg - D-rhamnose; 100.0 mg - D-fructose; 6,0 ml double-distilled water.

The mixture was stirred by rotation and heated under pressure for 45 min with increasing temperature from 75oC to 86oC. During this period is completely evaporated, about 3 ml of water, and substances were completely dissolved. Then the mixture was treated for 30 min at atmospheric pressure and temperature 85-86oC for holding rearrange Amadori. During this period, the solution was quickly acquired a reddish-brown color. The pressure was reduced and heated to 84oC continued, thus simultaneously viparita solvents. At the end of the evaporation was injected twice in 15 ml of anhydrous ethanol, after which the reaction mixture was completely dried. The flask with the dried reaction product was kept in a desiccator over calcium chloride for 18 hours. Then crushed to a fine powder, the result was about 4.5 g of powder, which is denoted as EK2-S.

(R)XAD-2. The column was suirable 0.4 ml/min of distilled water. Fractions of 10 ml were collected a total volume of 450 ml Content of the fractions was subjected to chromatographic monitoring. Fractions serial numbers 11-13 were combined and evaporated under reduced pressure. These fractions are characterized by a high content of products rearrange Amadori (which is confirmed by a test on the reduction of potassium ferricyanide). The product was stored for biological tests marked EK2-S-11.

Biological tests to determine the biological activity was carried out immunized 8-10-week-old mice type Balb/C mice of both sexes. The immunization of mice was performed by intraperitoneally with 0.2 ml of 10% suspension of sheep erythrocytes (SRBC), i.e. 6108cells. Erythrocytes were fixed in a sterile solution of Alsever the following composition: glucose - 2,05 g; sodium citrate - 0.8 g; sodium chloride 0,42 g; citric acid to 0.055 g; double-distilled water - 100 ml

In this solution Alsever impose aseptic sample of blood cells sheep in the ratio of 1: 1, then the mixture is kept for at least 3 days at a temperature of +4oC. From the stable, the button to rinse. The erythrocytes washed twice with a solution of PBS, centrifuged for 10 min at 2000 rpm, Washed cells are used in the form of a 10% suspension in PBS. Such a suspension is used for immunization of a mouse-type Balb/C

Want to test the sample was injected 4 times intraperitoneally or orally at the selected dosage, and the first injection was performed for 2 hours prior to immunization of a mouse with a solution of SRBC, and the remaining three doses were introduced after immunization with intervals of 24 hours.

Each group of test animals were injected with varying doses of the test product of the reaction: 10 mg/kg, 1 mg/kg, 0.1 mg/kg, 0.01 mg/kg Control group animals were immunized with SRBC solution, but instead of the test substance was administered 0.2 ml PBS over the same intervals of time.

Each group of animals, both the control and test, in all experiments consisted of 8-12 mice.

On the fourth or (in the case of the detection of antibodies type 7S) on the tenth day after immunization mice were subjected to light anesthesia with ether, and debilitated by complete removal of the eyeball. Blood was collected in test tubes. This blood was used to obtain serum needed to determine gemigo Intego content of the cells, is capable of forming E-rosettes and showing hemolytic activity. For this purpose, the spleen of mice were crushed into powder. Received splenocytes suspended in approximately 2 ml of medium in Hanks at a temperature of +4oC and separated into layers by density 1.077 by Ficoll gradient-Uropolin, then centrifuged for 15 min at 3000 rpm at +4oC. After separation of the intermediate phase layer of cells in buffer solution were placed in a medium no Hanks at +4oC and washed, centrifuger twice each time at 1600 rpm for 7-10 minutes Then splenocytes suspended in 1 ml of medium for Hanks to the concentration of cells 1106.

For each test, the percentage of dead cells was determined by mixing drops of the test suspensions of splenocytes with a drop of dye solution containing 4 parts of 0.2% Trypanosoma blue and 1 part of 4.25% solution of NaCl. For every 100 cells under a microscope to determine the percentage of dead splenocytes. Dead cells have the color of a sea wave, while the living cells light. Critical is the presence of more than 10% dead cells and this sample should be excluded from further use.

At all stages of the testing should be carried out in a sterile lab, what about the effect of the tested products of the reaction by the number of cells, producing hemolytic antibodies (PFC-lg M). The test was carried out as follows: 0.5 ml of a 5% solution of agarose, placed in a test tube being heated in a water bath at 45oC, was mixed with 0.1 ml of 10% SRBC suspension (prepared as described above). Then added 0.1 ml of a suspension of splenocytes density 1 106cells/ml, the mixture was quickly mixed and immediately poured on glass slides previously coated with agarose. Glass incubated at 37oC for 2 hours. Then the tested samples cover for 2 hours complement Guinea pigs, diluted in a ratio of 1:20. After incubation of test samples complement counted the number of cells forming platelets (PFC), and counted for 1106the splenocytes. Each test was conducted twice.

The greatest increase in response to SRBC, expressed in the increase in the number of splenocytes producing hemolysins lg M (PFC), was observed after a dose of 0.1 mg/kg D-11. The amplification reaction was 119%. With increasing daily doses 10 times to 1 mg/kg, the response dropped to 53%. The highest activity of the reaction product D-12 demonstrated a dose of 1 mg/kg 58% increase.

The reaction product D-13, test at the dose of 1 mg/kg, was given a 40% gain. Increasing the dose to 10 mg/kg, i.e. 10 times the response was only 14%.

Example 7. Was also tested for activity haemagglutination assays, in which we determined the levels of anti-SRBC antibody type 19S+7S and type 7S. To determine the level of antibodies type 19S-lg M was used to mouse serum on the fourth day after immunization mice SRBC, and to determine the level of antibodies type 7S-lg G serum was prepared on the tenth day after immunization mice SRBC, which is the day the maximum number of antibodies of this type in mice immunized with SRBC.

A. the counting of antibodies 19S+7S

The blood sample was centrifuged for 30 min at 3500 rpm/min From each so prepared sample was collected serum and placed for 30 min, heated to 56oC water bath to inactivate the complement. Next, from each test serum to prepare a series of solutions having different degrees of dilution (from 1:1 to 1:4096), using the device for microteriofauna and U-shaped micrococci volume of 20 ml each. Diluted seventie SRBC in PBS (prepared as described above, then the mixture is incubated for 2 hours at a temperature of 37oC and kept at a temperature of +4oC. the Results were checked the next day. The maximum degree of dilution, which is still the hemagglutination was determined by counting antibodies. The appearance of a ring on the bottom of the Cup is proof of the passing of haemagglutination. In the absence of haemagglutination indicates the presence blastomogenic formations at the bottom of the Cup, which is a negative result.

For statistical analysis of the results of increased serum dilution in the test substance were compared with those in the control group.

The reaction product D-11 at a dose of 1 mg/kg increased the number of Ig M 2.57 times. Tenfold dose stimulated impact 3.5 times compared with the control group.

The reaction product in this test D-12 in this test showed a weaker effect. At the dose of 0.1 mg/kg, it caused an increase in the number of immunoglobulin M (Ig M) 2 times, and at the dose of 1 mg/kg 1.4 times.

The greatest impact in this test found the reaction product EK2-S-11. at the dose of 0.1 mg/kg, it caused an increase in the number of Ig M 4.3 times, is and Ig M 4.3 times, and it tenfold dose of 3.6 times.

C. determining the amount of antibodies 7S

Test Deaktivierung serum were combined in the ratio of 1:1 with 0.1 M solution of 2-mercaptoethanol, and incubated for 30 min at a temperature of 37oC. 2-mercaptoethanol destroys the immunoglobulin type 19S - (Ig M), while the immunoglobulin type 7S -(Ig G) is not sensitive to the action of 2-mercaptoethanol.

After 30 minutes of incubation, the reduction reaction was stopped by lowering the temperature to +4oC for 15 minutes. Further, as described above to prepare a series of dilutions with regard to counting the number of antibodies type 19S, then combined them with a 1% suspension of SRBC, after 2 hours incubation at 37oC the samples were stored at +4oC. Evaluation of the results was made on the following day, according to the above criterion for determining the degree of hemagglutination. At the same time had a control test with a combination of 1% suspension, SRCB with PBS in a ratio of 1:1.

When testing substances D-11, as described above, at a dose of 1 mg/kg it increased the production of antibodies Ig G to 3.16 times. At the dose of 10 mg/kg, the increase was 2.2 times.

The reaction product D-12, tested at the dose of 0.1 mg/kg and 1 mg/kg, with the EK2-S-11 at a dose of 0.1 mg/kg stimulates the production of Ig GB 1.9 times, and at the dose of 1 mg/kg in 2,89 times (compared to control).

Then by the method of T-student (T-student) at a=0,05 conducted a statistical analysis of the results of tests A and b obtained for each batch of products or for each fraction of biologically active products of the reaction, the synthesized according to the invention and showing the above-mentioned immunological reaction. The results obtained for each dose, compared with parallel control test and shows an increase in biological activity.

Example 8. A group of biologically active reaction products obtained in accordance with Examples 1-5, were also subjected to the test, the result of which was determined by the percentage of E-rosette forming splenocytes.

250 ml of 1% suspension of SRBC and 250 ml to be tested cells at a concentration of 1106cells/ml was added to 550 ml of medium in Hanks. Then, each sample was incubated for 15 min at a temperature of 37oC in a water bath with heating and shaking. Next, the sample was kept at a temperature of +4oC for 20 hours. The percentage of E-rosette forming splenocytes, with SRBC were determined after the three times and determined the percentage of splenocytes, getting a percentage of each example 400 of splenocytes. For E-outlet took splenetic, surrounded by not less than three erythrocytes.

For statistical evaluation compared the percentage increase in the number of splenocytes from E-outlets with test substances and control group.

When this test has the highest effect of the stimulation revealed the reaction products D-11 (63%) and EK2-S-11 (70%) at a dose of 1 mg/kg dose, less 10 times, i.e., 0.1 mg/kg, the values decreased to 45% and 57%.

The reaction product D-12 at a dose of 1 mg/kg causes an increase in the ability of formation of E-outlets 22% compared with the control group. The corresponding value for the dose, less 10 times, i.e., 0.1 mg/kg, was 29%.

The reaction product D-13 shows the maximum effect at a dose of 1 mg/kg, and at higher doses the effect is reduced slightly.

The biological activity of the synthesized compounds was evaluated according to the following tests:

1. The test to determine the percentage of E-rosette. forming splenocytes conducted by Bach and Dardenne (Cell. Immunol. 3, 1-16, 197-2).

2. The test to determine the number of cells that produce hemolytic antibodies Ig M-type, based on the method the antibodies 19S+7S, conducted on methods of active haemagglutination (J. Immunol. 95, 26-38, 39-47, 1965) using microcase (J. lmmunopharmacol. 4, 43-52. 1982).

Example 9. Placed in a water bath heated flask rotary evaporator filled with the following composition: 1,33 g (0.01 M) - L-aspartic acid, 0.84 g (0.01 M) - NaHCO3; 10,00 g - hydrolyzed dextran total molecular weight of 3000 daltons; 10,00 ml of double-distilled water.

The mixture was heated under pressure at a temperature of 70oC until complete dissolution of the solids, removing during this time, the distillation of about 3 ml of water (heating time of about 30 min). Easily wrapped flask with the solution was placed in a steam sterilizer and was heated for 40 min at pressures up to 121oC. After cooling, the yellow-orange solution was diluted with 15 ml of water, was purified by centrifugation and dried with compressed air with an inlet temperature of +160oC, and the output + 85oC. the resulting reaction of 10.5 g of the product a light beige color were easily soluble in water.

Products rearrange Amadori in the reaction product was confirmed by the method of testing described Borsook, Abrams and Lowy, J. Biol. Chem. 215. (1955), 111-124, and chromatography methods.

Example 10. The conical flask was filled with the following composition: 5.0 g - hydrolyzed dextran total molecular weight of about 5000 daltons; 1.1 g - L-Proline; 4,0 ml double-distilled water.

The contents were dissolved under stirring. The resulting homogeneous mixture was placed in a steam sterilizer and heated for 40 minutes under pressure at a temperature of 110oC. the Obtained transparent orange solution was diluted with 20 ml of double-distilled water and was purified in a centrifuge. The purified solution was dried with compressed air.

Got to 5.3 g of the product, easily soluble in water. Immunotropic activity was similar to that observed in other experiments in accordance with the previous examples.

All process parameters are controlled, are not given because the products have biological activity in spite of the variation of parameters method in a wide range. Accordingly determined the critical features of the reaction. In Example 5, it is established that the composition of the mixture can be adjusted and modified by partial separation of red and methodology of supervision:

1. The definition of the compounds of Amadori and simple sugars by HPLC on Si-DEAE column with subsequent postcolonial precolumn derivatives, based on the use of alcohol solution of triphenyltetrazolium chloride (TTX), according to Reutter and Eichner (Lebensm. Unters. Forch. 188, 28, 1989).

The above method allows to estimate the proportion of individual compounds of Amadori in the reaction mixture after the reaction, which in General does not differ from the initial ratio of amino acid mixture (due to different speeds of reactions and reaction rates of decay).

2. Separation of the reaction mixture after the reaction by HPLC method for gel columns OH-Pak Q-801 (Shodex) using distilled water as eluent. Because this method of separation corresponds to the size of the molecules in the samples taken from the reaction mixtures after different time intervals, i.e. at different duration of the reaction, it is possible to see the increase in the fractions of molecules with high molecular weight, which is associated with the following stages of the reaction Maillard, i.e. decomposition of the compounds of Amadori and subsequent condensation products of decomposition amino acids.

Such is but the reaction to maintain it in the right ratio with other factions.

When the eluate is monitored by UV-detektorom (wavelength 215 and 280 nm) and refractometrically, the substrate unreacted sugars visible in refraction registration because they do not absorb UV.

3. IR spectra allow a relative assessment of differences in the composition of the individual contributions of products. Comparison of the infrared spectrum of this product with the corresponding IR spectra of previous products having the desired biological activity, or with a standard product perform to modify the direction of reaction.

Because biological tests are not logged in already noted, the analyses in the description, you would use the following additional ways:

4. Test the impact of the funds received on the survival of murine thymocytes in 18 and 20 one-hour cultures in the presence of hydrocortisone.

This test is used in Poland as a routine monitoring activity approved immunomodulatory funds, known as the TFX (extract active thymus peptides) and TTR (obtained from peat extract).

Detailed description of the analysis below. Attached representative test results for funds, the experimental the existing example 5 and the other is the product St-2, described in additional example 5A, below. The results obtained for the two products are in complete agreement with the corresponding results are presented for the two noted above approved immunomodulatory drugs TFX and TTP. It should be noted that the results were obtained at concentrations comparable with the corresponding concentrations of TTR, and they are many times lower concentrations TFX, giving the same effect.

5. For comparison, we performed additional tests showing the effect of the products obtained according to the present invention, murine L929 fibroblasts. This test was performed to assess the cytotoxicity of known immunotropic drugs such as LEVAMISOL and IMMUTOL described in the literature:

1) Blach-Olszewska Z., Zaczynska E., Arch Immunol. Ther. Exp., 1991, 39, 597-606 and 2) Zaczynska E., and others, Acta Virol., 1992, 36, 121-8. This method is described in detail below and after him are given the results obtained for several samples of products according to the invention. It should be noted that all test samples showed low toxicity, lower than the above-mentioned commercial products.

For Example

Example 5A (to insert in the existing substances used as starting materials: 100 mg L-aspartic acid, 100 mg of L-tyrosine, 20 mg of L-threonine, 20 mg L-serine, 100 mg of phenylalanine, 70 mg glutamic acid, 90 mg of glycine, 90 mg of L-alanine, 50 mg of L-valine, 40 mg L-isoleucine, 60 mg of L-leucine, 100 mg of histidine hydrochloride, 20 mg of lysine hydrochloride, 10 mg arginine hydrochloride, 100 mg NaHCO3, 5 mg of H2O.

After evaporation of about 2.8 ml of water under reduced pressure, the substrates were dissolved in a thermal bath at a temperature of 75-80oC. Then the liquid reaction mixture was heated at a temperature 86-90oC for 2 hours under normal pressure. Then the pressure was reduced and the reaction mixture was evaporated to a dry residue. In the late stages of evaporation has added two portions of anhydrous ethanol, 15 ml each. The reaction flask was cooled in a desiccator. The product obtained with a yield of 1.5 g was a powder and was marked by the symbol St - 2.

Additional tests:

1. Determination of the survival of murine thymocytes in 18 - and 20 - hour cultures with hydrocortisone in the presence of the tested substance, D-10, EK2-S-11 and St-2.

The test of survival of mouse thymocytes in 18 - and 20 - hour cultures with hydrocortisone.

Animals for testing: female Balb/c mice aged 5 weeks. Regulatory Iroda (FCS)-inactivated (temp. 56oC for 30 minutes), 3) hemisuccinate hydrocortisone in solvent ampoule (HC) from a Polish supplier of medicines POLFA.

Solutions for testing: the test substance was dissolved in RPMI 1640 and was preparing solutions containing 5 μg/ml, 1 μg/ml and 0.5 μg/ml for each test substance.

Method of assessment: 2 - 3 female mice (for each test substance) were euthanized by ether to remove tonsils thymus. Tonsils homogenized and suspended in RPMI 1640 and 10% inactivated FCS. The suspension was then filtered through a filter, usually used for filtering the blood, and twice centrifuged in the same medium (RPMI 1640 with 10% inactivated FCS) for 10 minutes at 1200 rpm. Next, the cells were resuspendable on the same medium at a concentration of 4x106cells/ml.

Thus prepared suspension thymocyte were placed in sterile test tubes: 1 ml of suspension in each tube was placed on ice bath. For each determination were prepared two test tubes. All stages must be carried out in a laminar flow chamber.

Conduct the following definitions:

1. The thymocytes in RPMI 1640 with 10% FCS.

2. The thymocytes in RPMI 1640 with 10% FCS + hydrocortisone (HC) 50 ml After the test compound is added to the culture of thymocytes culture incubated for 1 hour in an incubator at a constant current of 5% CO2when the temperature of the 37oC.

4. The thymocytes in RPMI 1640 with 10% FCS + test compounds in concentrations specified in paragraph (3 + HC 50 µg/ml. Such testing culture incubated for 18 to 20 hours at 37oC in an atmosphere of 5% CO2.

For each culture, 3 to 4 times determine the percentage of live and dead cells. For staining of dead cells use tirpan blue; immediately before the determination of the survival rate of cells 0.2% solution Tirana blue mixed with 4.25% NaCl in the ratio of 4:1. The thymocytes, painted blue, is considered dead, then as colorless cells are alive.

Interpretation of results: make 3 - 4 calculation for each test culture. The sensitivity of thymocytes to hydrocortisone is calculated as follows:

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These test substance, the test concentration: 5 μg/ml, 1 μg/ml and 0.5 µg/ml is capable of at least 25% increase in the number of surviving thymocytes in culture with hydrocortisone, are regarded as possessing activity. According standa repeated twice) should be considered as active.

11. The determination of the cytotoxic effect of drugs on cultures of mouse fibroblasts L929

Fibroblasts both human and animal, are susceptible to the cytotoxic substances of different nature. Line L-929 is typically used to determine the cytotoxicity of TNF. The effect of test substances on the cell line was determined according to the method described below.

Method of analysis: the test substance was dissolved in distilled water. Prepared basic solutions with a concentration of 16 mg/ml for D-10 and St-2. Further dilution was performed using culture medium RPMI with 10% inactivated FCS.

Tablets with 96 flat holes (Flow Laboratories) was used for making dilutions. Each well was inoculable 100 ál of cell suspension L 929 concentration 1.5105cells/ml Final test breeding D-10 and St-2 was as follows: 800 ág/ml, 400 μg/ml, 200 μg/ml,..., falling to 12.5 μg/ml and 6.25 µg/ml each tablet made checks: one consisting of a cell culture L-929 in culture medium (RPMI 1640 + 10% FCS), and the other consisting of a cell culture L 9292 in culture medium with a suitable amount of bidistilled water. Training is ez 24 and 48 hours using an inverted microscope OPTON. Each test was repeated three times.

The results are presented in table 3.

As can be seen from this test, the test substances are not cytotoxic. The same test was used to assess the cytotoxicity IMMUTIOL and LEVAMISOL. IMMUTIOL the effect of cytotoxicity at concentrations below 1 µg/ml (cited work Zaczynska E and others), a LEVAMISOL - at a concentration of 500 µg/ml (cited work Blach-Olszewska, and others). As already noted, these two shopping drug permitted for medical use in Poland.

For three substances EK2-S-11, D-10 and ST-2 is a more detailed identification data are presented in the attached Fig.1-6: 2 chromatogram and the IR spectrum for each substance.

In addition, provided with IR-spectra and chromatogram columns on OH-pak Q-801 for fraction D-11, made in 1991 and 1993, showing that the results are fully reproducible. Presents four additional figure.

Example 11. The known methods for testing the biological activity of the compounds in mice but not in humans. For this reason it was used a new method of measuring the quantity and activity of cytokines that are released from leukocytes of peripheral blood (PBL), according knymi proteins, which play an important role in almost all immunological reactions, and regulatory processes responsible for the maintenance of homeostasis.

Studies of cytotoxicity

The cytotoxicity of the reaction products was determined in the cells of the human lung - adenocarcinoma line And 549 (included in the American Collection of type cultures-ATS CCL 185). Cell layers were treated with trypsin. The suspension containing 2105cells/ml of the primary environment in Eagle, modified by Dulbecco (DMEM), containing 10% calf serum (CS), was mixed with different doses of drugs planted in microcash, and incubated for 48 hours at 37oC. CD50was the minimum concentration of the composition, which contributed to the destruction of approximately 50% of cell cultures that showed the dimension using the staining of 0.015% neutral red solution in DMEM.

Induction of cytokine

Light layers of blood clots from healthy blood donors were obtained from the regional blood transfusion centre. Erythrocytes were literally processing of NH4Cl by Cantell et al. (Cantell, K., Hirvonen, S., Kauppinen, H. L.: Production and Partial Purification of Human Immune Interferon. Meth. Enzymol, 119, 54, 1986). Used only linolowego embryo (FCS), L-glutamine and antibiotics. All parties FCS pre-tested. For PBL cultures used only nematogen FCS. Inducers of cytokines were added to the volumes of the cultures of 1 ml as standard inductors were used phytohemagglutinin (PHA) (Pharma Fine Chemicals, Sweden) and lipopolysaccharide (LPS) from E. coli 0111:B4 (Difco Laboratories). Induced culture PBL incubated in an atmosphere of 5% CO2at 37oC for 20 hours, then centrifuged. Supernatant kept at 4oC for one week experienced on TNF and IFN-activity.

The trial of interferon

Were prepared confluent monolayers of cells A in microplastic in the medium modified by Dulbecco, [Dulbecco modified Minimum Essential Medium (DMEM), 10% SC, L-glutamine and antibiotics (penicillin 100 u/ml and streptomycin 100 µg/ml). Samples IFN diluted in plates, was added to the monolayer of cells and incubated at 37oC. for 20 hours in 5% CO2in the air. The cells are then washed and subjected to virus encephalomyocarditis (EMCV). The IFN titer was defined as the dilution of the sample IFN, which was reduced cytopathogenic effect by 50% for 48 hours of incubation. To measure cell death in the scanner ELISA was used the method of MTT (3-[4,5-dimethylthiazol-2-yl] -2,5-dif is. . Measurement. Meth. 1989, 119, 203- 210. ). Laboratory standards IFN should be included in all trials, such as recombination of human IFN-gamma (Genetech Inc., USA), (own activity 2000000 IU/mg), natural human IFN-alpha (3000000 1 ITEM./ml) and IFN-gamma (2000000 1ITEM/ml).

The test of TNF (tumor necrosis factor)

Cytotoxic activity of TNF was measured in cells L929by Flick and Gifford (Flick, D. A., Gifford, G. E..: Comparison of in Vltro Cell Cytotoxic Assays for Tumor Necrosis Factor. J. Immunol. Meth. 68, 1667, 1984.). The samples and the solution of actinomycin D were added to the monolayer cell cultures. After incubation at 37oC for 20 hours cytotoxic effects of TNF were determined either by microscopic analysis of cultures, either way MMT. The number that causes the destruction of approximately 50% of cell cultures, was taken for one unit of activity TFN. Comparison with drug TNF-alpha (Genetech Inc. , USA) showed that 1 unit in the trials was equal to 100-200 pg/ml TNF.

Tests of neutralization of cytotoxins

Used anticigarette: rabbit anti-human TNF-alpha, series 2891-40 and rabbit anti-human INF-gamma, series 2891-56 (Genetech Inc. , USA), sheep anti-human IFN-alpha. IFN-beta and sheep anti-human IFN-gamma (obtained from Dr.K.Cantell, Finland). Preparare room temperature. Then determined, as already described, the residual activity of IFN or TNF.

Used five different groups PBL (L1-L5) prepared from blood of healthy donors. The optimal concentration PBL detected in the tests amounted to 8106cells in 1 ml of medium (RPMI 1640, mixed with 10% serum bovine embryo and antibiotics).

Incubation of human PBL with new products of the reaction I-XI (PL. 1) caused the synthesis of IFN and TNF. The reaction was observed at doses of compounds, equal 3-100 mg/ml Formulations used in the indicated concentrations, were non-toxic. In all other bioimpedance included negative and positive controls. The negative control was measured by the number of cytokines (IFN and TNF) produced spontaneously without the addition of any exogenous stimulants. The positive control was determined by the amount of cytokines produced in response to any known inducer; in this case phytohemagglutinin (PHA, Pharmacia, Sweden, 10 mg / ml).

It should be noted that the induction of cytokines in cultures of human PBL obtained from different individuals, usually gives significantly varying results. This phenomenon can be explained by genetic differences in the population clonanav PBL usually see individuals, as with a strong immune system, and weak, and with a neutral reaction.

Regardless of the stated limitations, the results of biospehere showed that PBL (L1-L5), processed products of the reaction (I-XI), produce IFN and/or TNF, which can be measured quantitatively.

In the case of L1that includes leukocyte individual with a strong reaction, the reaction product II (containing L-aspartic acid) were found significantly more activity as inducer of cytokines than the reaction product III (containing D-aspartic acid, which is also more expensive than two orders of magnitude). This observation is important, because the biochemical reactions of the cell recognize as natural substances, mainly L-amino acids. In addition, for the expression of biological activity of the reaction products of amino acid portion of the molecule is more important than the form of sugar. Instead of single sugars, preferably low molecular weight, in particular less than 1000 daltons, can be used giving a similar reaction polysaccharides (such as dextrans, the reaction products X-XI).

Conversely, polysaccharides containing amino acid residues, may have a biological active lists).

Similar results can also be obtained if, instead of one amino acid to take small peptides and used to stimulate production of cytokines by leukocytes (data not shown).

When tested seven groups of samples nefrackzionirovannam TTR in more than 100 cultures of PBL obtained from different donors, found products from 10 to 1,000 units of IFN per ml, and from 9 to 750 units of TNF in ml. Fraction EK2-5 prepared from a mixture corresponding to the composition of the natural extract of peat (Example 5) was tested in eight cultures of PBL obtained from different blood donors. It was found that the most active are the drugs, including IFN, and TNF (data not shown).

Possible fields of application of the reaction products are immunomodulators, and clinical trials were successful. Proven their effect on tissue regeneration. Possibly, the observed anticancer effects associated with the presence of induced interferon and tumor necrosis factor. Recorded antiviral effect.

The most common is the oral intake of the reaction products, but also possible parenteral route of administration. The product is available is gradient the reaction products, according to Examples 1-5, 9 and 10 were prepared using the following reaction products:

A. Tablets/Granules

5.0 g of the reaction product obtained in Example 1 or 10 (active substance),

444,0 Mr. pharmaceutically acceptable lactose

1.0 g of a lubricant, for example, MYVATEX(R)registered trademark of Eastman Kodak

The ingredients were mixed and granulated in the usual way in 30% liquid solution of ethanol, then dried at 40oC. the Pellets were used for the preparation of capsules, each of which contained about 450 ml of granules, i.e., 5 mg of active substance. Accordingly, for the preparation of tablets used pellets weighing about 450 mg each and containing 5 mg of active substance.

Century in the same way active substances obtained according to the preceding examples 1-5, 9 and 10, were included in other pharmaceutical compositions using appropriate media.

Example 13

The active substance obtained according to the preceding examples 1-5, 9 and 10, was used as a medicinal additives to cosmetic preparations intended for daily care of hair and body, and weight substance content was in the range of 0.01 to 10%, in Icesa composition to treat and/or prevent hematological and/or immunological diseases and/or to stimulate the immune system of humans and/or mammals, comprising at least one pharmaceutically acceptable carrier, adjuvant and/or a lubricant and at least one connection based on the rearrangement of Amadori, characterized in that it contains an effective amount, preferably 0.01 to 10.0 mg/kg body weight, at least one connection based on the rearrangement of Amadori, preferably a mixture of compounds based on rearrangement of Amadori formula I

R1- NH - R2,

where R1- the remainder of the 1-deoxy-2-ketose, a derivative of sugar, preferably in its D-form, selected from the group consisting of glucose, xylose, galactose, ramnose, fructose, mannose, 2-deoxy-glucose, 6-deoxy-glucose, glucosamine, galactosamine, or oligo - or polysaccharides, preferably a low molecular weight of 5000 daltons or less;

R2the remaining amino acids are preferably in their L-form, selected from the group consisting of serine, glycine, Proline, histidine, arginine, alanine, asparginase acid, glutamic acid, phenylalanine, threonine, cysteine, cystine, glutamine, valine, asparagine, methionine, tyrosine, hydroxyproline, lysine, tryptophan, isoleucine and leucine.

2. The composition according to p. 1, wherein the is cellular nutrition for tissue regeneration and/or for immunomodulatory comprising at least one cosmetically acceptable carrier, adjuvant and/or a lubricant and at least one connection based on the rearrangement of Amadori, preferably a mixture of compounds based on rearrangement of Amadori formula I

R1- NH - R2,

where R1- the remainder of the 1-deoxy-2-ketose;

R2a balance of amino acids,

characterized in that it comprises an effective amount is preferably 0.01 to 10.0 wt.% at least one connection based on the rearrangement of Amadori formula I, where R1- the remainder of the 1-deoxy-2-ketose, a derivative of sugar, preferably in its D-form, selected from the group consisting of glucose, xylose, galactose, ramnose, fructose, mannose, 2-deoxy-glucose, 6-deoxy-glucose, glucosamine, galactosamine, or oligo - or polysaccharides, preferably low molecular weight of 5000 daltons or less, R2a balance of amino acids is preferably in its L-form, selected from the group consisting of serine, glycine, Proline, histidine, arginine, alanine, asparginase acid, glutamic acid, phenylalanine, threonine, cysteine, cystine, glutamine, valine, asparagine, mation is the rpm die, what induces the formation of a cytokine, which is useful for antiviral activity.

5. Composition under item 1 or 2, characterized in that it includes at least one connection based on the rearrangement of Amadori and at least one pharmaceutically acceptable carrier, adjuvant and/or grease in a weight ratio of 1 : 1 to 1 : 100, preferably 1 : 8 to 1 : 20 and more preferably in an approximate ratio of 1 : 9.

6. Composition according to any one of paragraphs. 1 - 5, where the lubricant is present in a mixture with lactose, and the weight ratio of lactose and lubrication is 20 : 1 to 100 : 1, preferably about 50 : 1.

7. Composition under item 3 or 4, where at least one connection based on the rearrangement of Amadori is present in an amount of 0.01 - 1 wt.%, preferably 0.05 to 0.10 wt.%.

8. The method of obtaining a mixture of immunotropic compounds based on rearrangement of Amadori formula R1- NH - R2where R1- the remainder of the 1-deoxy-2-ketose, which is derived from sugar or low molecular weight oligo - or polysaccharide, and R2is amino acid residue, wherein: a) a concentrated aqueous mixture of compounds of sugar and amino acids, which includes at least two different linakis is lton or less, where amino acids are preferably in their L-form and selected from the group consisting of serine, glycine, Proline, histidine, arginine, alanine, asparginase acid, glutamic acid, phenylalanine, threonine, cysteine, cystine, glutamine, valine, asparagine, methionine, tyrosine, hydroxyproline, lysine, tryptophan, isoleucine and leucine, and sugar are preferably in its D-form and selected from the group consisting of glucose, xylose, galactose, ramnose, fructose, mannose, 2-deoxy-glucose, 6-deoxy-glucose, optionally together with inorganic trace elements present in natural peat extracts heat from the chemical reactions, optionally under pressure and in the presence of water as solvent, and the solid content is 0.67 - 2,75 pbw to 1 pbw of an aqueous solvent, and the molar ratio of sugars and/or saccharides and amino acids is 2 : 1 to 1 : 1, more preferably of 1.5 : 1; (b) continue heating with the introduction of the final reaction products in the rearrangement of Amadori, while the aqueous solvent simultaneously or subsequently removed until until the color of the reaction mixture turns into orange-brown and biological aktivnosti, then c) stop the rearrangement of Amadori, preferably before the decomposition, resulting in joints that have lost their biological activity, and (d) dried reaction mixture and/or to further expose its purification by column chromatography, collecting biologically active fraction, causing the restoration of the ferrocyanide of potassium.

9. The method according to p. 8, characterized in that the mixture of stage (a) is reacted at temperatures of about 70 - 121oC preferably for about 30 to 120 minutes

10. The method according to p. 8 or 9, wherein the sugar and/or low molecular weight sugars, on the one hand, and/or amino acids, on the other hand, and optional inorganic micronutrients are basically in the same composition and/or essentially in the same weight ratio, which takes place in natural peat extract.

11. The method according to any of paragraphs.8 to 10, characterized in that at least one amino acid has two carbonyl groups, and the reaction is carried out in the presence of buffer salts, preferably sodium bicarbonate, at a molar ratio of from amino acid 1 : 1.

12. Composition according to any one of paragraphs.1 to 7, where at least one compounds the Wallpaper and part of the reaction mixture, obtained by the method according to any of paragraphs.8 - 11.

13. The composition according to p. 12, characterized in that it includes the remains of the 1-deoxy-2-ketose and/or amino acid residues, and optionally inorganic trace elements, essentially the same composition and/or essentially in the same weight ratio, which is the case for natural peat extract.

14. The composition according to p. 12 or 13 for oral and/or surface, and/or intravenous administration.

Priority points:

13.02.92 on PP.1 - 10, 12 - 14;

03.03.92 on p. 11.

 

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where R represents the residue obtained by removal of COOH from C3-C7sugar carboxylic acids, present in the residue of the hydroxyl group can be protected by means of protective groups
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FIELD: organic chemistry, chemical technology, medicine, pharmacy.

SUBSTANCE: invention describes a novel pentasaccharide conjugate of the formula (I):

wherein R represents independently -SO-3 or -CH3; insert represents a flexible insert of length 13-25 atoms. Charge of a pentasaccharide residue is equilibrated with positively charged counterions and the total amount of sulfate groups in a pentasaccharide residue is 4, 5 or 6. Also, invention relates to a method of its preparing and pharmaceutical composition based on thereof and used in treatment of diseases mediated or associated with thrombin.

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9 cl, 2 ex

FIELD: chemistry of peptides, virology.

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3 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: group of inventions concerns medicine and can be applied in treatment of bacterial infections caused by gram-positive bacteria. The methods involve administration of dalbavancin for treatment of bacterial infections, particularly of gram-positive bacterial infections of skin and soft tissues. Dosage schemes include administration of dalbavancin once a week, which would sustain therapeutic drug level in plasma for at least 1 week. The claimed pharmaceutical compositions include single dalbavancin dose sufficient for sustaining therapeutically effective level of dalbavancin in plasma.

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86 cl, 13 dwg, 17 tbl, 10 ex

FIELD: medicine; dermatology.

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3 ex, 3 tbl, 7 dwg

FIELD: medicine.

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3 cl, 2 ex

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7 cl, 34 dwg, 44 tbl, 40 ex

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1 ex, 2 dwg

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2 cl, 3 ex

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