Crystalline forms of monosodium salt of d-isoglutamyl-d-tryptophan

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to crystalline modifications: 1 (polymorphous form F), 2 (polymorphous form I) and 3 (polymorphous form X) of monosodium salt of D-isoglytamyl-D-tryptophan (1:1) characterised by powder X-ray pattern peaks presented in the application materials, as well as to pharmaceutical compositions containing them. The invention describes their use for treating various diseases and body conditions of at least one autoimmune diseases specified in a group consisting of psoriasis, atopic dermatitis and rheumatoid arthritis.

EFFECT: present invention describes the methods for producing the declared crystalline modifications of monosodium salt of D-isoglytamyl-D-tryptophan (1:1).

42 cl, 4 ex, 9 dwg

 

The technical FIELD

The present invention relates to crystalline forms of the mono-sodium salt of D-isoglutamine-D-tryptophan, methods for their preparation, pharmaceutical preparations containing them and their use for the treatment of various conditions and diseases. More specifically, the present invention relates to crystal modification 1 (polymorphic form F), crystal modification 2 (polymorphic form I) and the crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan.

PRIOR art

The compound D-isoglutamine-D-tryptophan (also known as H-D-γ-Glu-D-Trp-OH or H-D-iGlu-D-Trp-OH, or iDD, or D-(iEW), or thymodepressin) is a synthetic samoreguliruemaya dipeptide having the following chemical structure:

.

Thymodepressin refers to free digitatum. Chemical abstract service (HRS) assigned registration number ® 186087-26-3. He is immunosupressants and selectively inhibits the proliferation of bone marrow cells. Its action is effective to suppress the immune system during bone marrow transplantation, organs and tissues (Semin O.V. and others (2001) Bulletin of experimental biology and medicine, 131(5), 493-495); to protect cells from bone marrow and immune system from harmful what about the actions of chemotherapy and radiation (U.S. patent No. 5736519, 6103699 and 6410515); and for the treatment of autoimmune diseases such as psoriasis and atopic dermatitis (Sipuncula YEAR, and others (may 2002), Bulletin of experimental biology and medicine, 133(5), 488-490).

The method of receiving thymodepressin described in example 1 of U.S. patent No. 5736519, 6103699 and 6410515. However, the production of thymodepressin in large industrial scale based on the experimental details of this method is not possible, because the result of this method is a mixture of D-glutamyl-D-tryptophan and D-isoglutamine-D-tryptophan, which is then required to separate and purify by ion-exchange chromatography, which results in very low (12,25%) yield of thymodepressin.

U.S. patent No. 5736519, 6103699 and 6410515 show that described in the peptides can be converted to the acid additive salts by reaction with inorganic acids including hydrochloric acid, sulfuric acid, Hydrobromic acid, phosphoric acid, etc. or organic acids including formic acid, acetic acid, propionic acid, glycolic acid, pyruvic acid, lactic acid, oxalic acid, succinic acid, malic acid, tartaric acid, citric acid, benzoic acid, salicylic acid, benzosulfimide acid and toluenesulfonate sour the s. However, in these patents are not described or basic additive salts of thymodepressin or methods for such salts.

Thymodepressin is not bioavailable as a drug for oral administration in the traditional form - in the form of tablets or capsules. Currently being a solid, it is sold in Russia in the form of a disodium salt in the form of a liquid preparation for injection and intranasal in the treatment of psoriasis, atopic dermatitis and rheumatoid arthritis. The solid form of the disodium salt of D-isoglutamine-D-tryptophan is an amorphous powder, having a high hygroscopic property, and handling very difficult. The disodium salt of D-isoglutamine-D-tryptophan has the molecular formula C16H17N3Na2O5and the following chemical structure:

The disodium salt of D-isoglutamine-D-tryptophan was not identified registration system HRS not listed in the registration file XPCSMand does not have an assigned registration number HRS®. Identification and structure determination of disodium salt of D-isoglutamine-D-tryptophan were performed by means of infrared (IR) spectroscopy (Kashirin D.M. and others (2000), Chemical and pharmaceutical journal, 34(11), 619-622). However, despite the fact that the disodium salt of D-isoglutamine-D-three the Tofana known, its receipt, allocation and further characterization of the properties are not described.

Through research in his laboratory, the inventors have determined that dried disodium salt of D-isoglutamine-D-tryptophan has extremely high hygroscopicity, becoming in the open air in the gel for a few minutes, so the handling is very difficult. Use for pharmaceutical needs powder or amorphous forms of the compounds may lead to increased production problems related to the bulk density of the output, hygroscopicity and varying water content that cannot be fixed by vacuum drying. D-isoglutamine-D-tryptophan is a dipeptide, and the drying of the amorphous form at elevated temperature, for example 80 to 100°C in vacuum is not recommended. Thus, the cleaning methods disodium salt of D-isoglutamine-D-tryptophan and producing pure disodium salt in an industrial scale is associated with serious difficulties. In addition, as mentioned above, the way of obtaining not published.

Monosodium salt of D-isoglutamine-D-tryptophan identified registration system HRS listed in the registration file XPCSMunder the registration number ® 863988-88-9, it has the following chemical structure:

However, there are no sources describing the monosodium salt of D-isoglutamine-D-tryptophan, and therefore, no publications about her, about her physical and/or chemical properties, its characterization in the solid state or the manner of its acquisition and allocation. Thus, there is no evidence of the existence of the monosodium salt of D-isoglutamine-D-tryptophan.

Bulk density of the output of lyophilised powder and monosodium Dimitrievich salts of peptide drugs may not be verifiable in the ranges of values that are required for the formulation of drugs that may require significant investment in technology dispersion based on freeze-drying.

There is therefore a need to develop pharmaceutically acceptable salts D-isoglutamine-D-tryptophan in crystalline form. Such crystalline salt in the General case can be cleaned much easier than the amorphous form, and may have other useful properties, for example, related to their specific crystalline form, and/or characteristics of solubility, and/or lack of hygroscopicity they and/or their stability characteristics, including properties of thermal stability and/or their ability to resist oxidative degradation.

A BRIEF DESCRIPTION AND THE GAIN

Previously the authors of this proposal have been invented is a method of obtaining D-isoglutamine-D-tryptophan and crystalline polymorph new stable monoammonium salt and method for producing this monoammonium salt. These questions were the subject of a patent application Canada No. 2569204, filed November 28, 2006 and incorporated into the present application by reference. The authors also previously invented calcium, magnesium, potassium and lithium salts of D-isoglutamine-D-tryptophan and methods of obtaining these salts. These questions were the subject of a patent application Canada No. 2571645, filed December 19, 2006 and incorporated into the present application by reference.

For methods of producing dipeptide pharmaceuticals is often useful to use a carboxyl group in the form of specific sodium salt with, for example, the preferred value of solubility, the preferred characteristics of absorption, better stability, the preferred pH value, or, in General, the preferred set of characteristics.

The inventors have found that D-isoglutamine-D-tryptophan, interacting with the sodium hydroxide may form a salt, for example, monosodium salt, in which the hydrogen atom in the carboxylic acid is replaced by a sodium ion, and which can be formally represented by formula III, Il is the disodium salt, in which two hydrogen atoms are replaced by two atoms of sodium, and which can be formally represented by formula II.

The inventors conducted a study of speciation, the results of which is represented by the diagram in figure 1. The authors determined that for use in pharmaceutical preparations definite advantage is monosodium salt, D-isoglutamine-D-tryptophan, which can be formally represented by formula III, and which is mainly pharmaceutical salt with a neutral pH. This advantage is determined by the fact that the amino acid is zwitter-ion. The authors found that monosodium salt represented by the formula III, is the most preferred salt for pharmaceutical drugs. Disodium salt represented by the formula II is preferably at a pH of about 12 or higher. The solution of the disodium salt in water will have a pH value approximately equal to 12, and therefore, this salt is not suitable for use in liquid preparations. Bringing the pH of the solution disodium salt to values in the range from about 7 to 7.4 actually gives monosodium salt as the preferred salt in solution.

Therefore, the aim of the present invention to provide a monosodium salt of D-isoglutamine-D-tryptophan in the form convenient for pharmace the political application, and obtained using the method suitable for implementation in large industrial scale.

Stable crystalline monosodium salt can be used in pharmaceutical preparations of the appropriate composition, and method of administration which provides a therapeutic effect that is required in this case.

Now the inventors have found that monosodium salt, D-isoglutamine-D-tryptophan can be obtained in a solid crystalline form suitable for pharmaceutical use, by the reaction of D-isoglutamine-D-tryptophan with the basic compounds of sodium, for example, with sodium hydroxide. As obvious chemical equivalents of sodium hydroxide can be considered sodium hydride, sodium carbonate, sodium bicarbonate, sodium alcoholate C1-C4. The surprise was that the solid crystalline mono-sodium salt of D-isoglutamine-D-tryptophan can be obtained in several different crystalline modifications, i.e. polymorphic forms, which have different physico-chemical properties and which can be obtained by adjusting the reaction conditions and/or crystallization. In General, these crystalline modifications can differ from each other by characteristics such as, for example, solubility, rate of dissolution or p is doing in a method of producing a pharmaceutical preparation, which allows the production of pharmaceutical preparations with different sets of properties from a single source connection.

In accordance with one aspect of the present invention presents monosodium salt, D-isoglutamine-D-tryptophan in crystalline form.

In another embodiment of the present invention, the crystalline form of the mono-sodium salt of D-isoglutamine-D-tryptophan in the analysis method x-ray diffraction gives a distinct peaks with a width at half height of less than 2º, when measuring reflection angle 2θ using CuKαthe radiation.

In another embodiment of the present invention, the crystalline form of the mono-sodium salt of D-isoglutamine-D-tryptophan is a crystal modification 1 (polymorphic form F).

In another embodiment of the present invention is a picture of the powder x-ray diffraction (powder x-ray) crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan has the form shown in figure 2.

In another embodiment of the present invention the crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan is characterized by peaks in the powder x-ray at the following 2θ values: 9,23±0,20; 9,91±0,20; 12,41±0,20; 13,76±0,20; 14,87±0,20; 15,75±0,20; 17,88±0,20; 18,78±0,20; 19,57±0,0; 19,84±0,20; 20,31±0,20; 21,32±0,20; 21,55±0,20; 22,95±0,20; 23,45±0,20; 24,34±0,20; 24,96±0,20; 27,49±0,20; 27,94±0,20; 29,27±0,20; 30,07±0,20; 30,43±0,20; 31,29±0,20; 32,25±0,20; 34,07±0,20; 34,94±0,20; 35,53±0,20; 36,08±0,20; 37,21±0,20; 38,17±0,20; 39,19±0,20; and 9,23±0,20.

In another embodiment of the present invention the crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan is characterized by peaks in the powder x-ray at the following 2θ values: 9,23±0,10; 9,91±0,10; 12,41±0,10; 13,76±0,10; 14,87±0,10; 15,75±0,10; 17,88±0,10; 18,78±0,10; 19,57±0,10; 19,84±0,10; 20,31±0,10; 21,32±0,10; 21,55±0,10; 22,95±0,10; 23,45±0,10; 24,34±0,10; 24,96±0,10; 27,49±0,10; 27,94±0,10; 29,27±0,10; 30,07±0,10; 30,43±0,10; 31,29±0,10; 32,25±0,10; 34,07±0,10; 34,94±0,10; 35,53±0,10; 36,08±0,10; 37,21±0,10; 38,17±0,10; 39,19±0,10; and 9,23±0,10.

In another embodiment of the present invention the crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan is characterized by the following powder x-ray, which is expressed in terms of inter-planar distance d, Bragg angles 2θ and relative intensity (expressed as percentage relative to the beam of greatest intensity):

24,34
2θ (°)D-distance (angstroms)Relative intensity (%)
9,23from KZT9, 5732
to 9.918,91741,3
12,417,12637,6
13,76to 6.430,8
14,875,95435,8
of 15.755,6227,6
17,884,9575,5
18,784,721of 58.9
19,574,53230,9
19,844,47128,1
20,314,3682,9
one-21.324,16553,5
21,554,1230,3
22, 95mm3,87367,4
23,453,7924,5
3,65419,4
24,963,565to 85.2
27,493,242100
of 27.943,1923,3
to 29.273,04919,1
30,072,9727,2
30,432,93515,2
31,292,85639,9
32,252,77413
34,072,62919,3
34,942,5667,8
35,532,5255
36,082,4878,4
37,212,41415,5
38,172,3569,1
39,192,2973,1

In another embodiment of the present invention, the crystalline form of the mono-sodium salt of D-isoglutamine-D-tryptophan is a crystal modification 2 (polymorphic form I).

In another embodiment of the present invention the powder x-ray crystal modification 2 (polymorphic form I) of the monosodium salt of D-isoglutamine-D-tryptophan has the form shown in figure 3.

In another embodiment of the present invention is a crystal modification 2 (polymorphic form I) monosodium salt of D-isoglutamine-D-tryptophan is characterized by peaks in the powder x-ray at the following 2θ values: 9,65±0,20; 10,41±0,20; 11,2±0,20; 11,71±0,20; 13,45±0,20; 13,93±0,20; 14,44±0,20; 15,61±0,20; 17,01±0,20; 18,18±0,20; 18,65±0,20; 20,02±0,20; 20,85±0,20; 21,39±0,20; 21,73±0,20; 22,52±0,20; 23,27±0,20; 24,3±0,20; 25,84±0,20; 26,82±0,20; 28,49±0,20; 30,18±0,20; 30,76±0,20; 31,49±0,20; 33,03±0,20; 34,55±0,20; 34,97±0,20; 35,74±0,20; 37,25±0,20; 37,71±0,20; and 38,79±0,20.

In another embodiment of the present invention is a crystal modification 2 (polymorphic form I) monosodium salt of D-isoglutamine-D-tryptophan is characterized by peaks in the powder x-ray at the following 2θ values: 9,65±0,10; 10,41±0,10; 11,2±0,10; 11,71±0,10; 13,45±0,10; 13,93±0,10; 14,44±0,10; 15,61±0,10; 17,01±0,10; 18,18±0,10; 18,65±0,1; 20,02±0,10; 20,85±0,10; 21,39±0,10; 21,73±0,10; 22,52±0,10; 23,27±0,10; 24,3±0,10; 25,84±0,10; 26,82±0,10; 28,49±0,10; 30,18±0,10; 30,76±0,10; 31,49±0,10; 33,03±0,10; 34,55±0,10; 34,97±0,10; 35,74±0,10; 37,25±0,10; 37,71±0,10; and 38,79±0,10.

In another embodiment of the present invention is a crystal modification 2 (polymorphic form I) monosodium salt of D-isoglutamine-D-tryptophan is characterized by the following powder x-ray, which is expressed in terms of inter-planar distance d, Bragg angles 2θ and relative intensity (expressed as percentage relative to the beam of greatest intensity):

td align="center"> 28,49
2θ (°)D-distance (angstroms)Relative intensity (%)
9,659,1615,3
10,418,49223,7
11,27,89740,4
11,717,5494,5
13,45to 6.5890,2
13,936,35115,9
6,1283,7
15,615,67232,4
17,015,2079,9
18,184,87611,7
18,654,75547,8
20,024,43259,2
20,854,25735,9
21,394,1524,1
21,734,08627,3
22,523,945100
23,273,81913,7
24,33,6632,4
25,843,44569,5
26,823,32282,5
3,1330,1
30,182,95958,8
30,762,90486,9
31,492,83935,3
33,032,718,7
34,55at 2,59417,8
34,972,56443,4
35,74of 2.518,5
37,252,41228,1
37,712,38328,5
38,792,31916,9

In another embodiment of the present invention, the crystalline form of the mono-sodium salt of D-isoglutamine-D-tryptophan is a crystal modification 3 (polymorphic form X).

In another embodiment of the present invention the powder x-ray crystal the modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan has the form, shown in figure 4.

In another embodiment of the present invention is a crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan is characterized by peaks in the powder x-ray at the following 2θ values: 9,187±0,200; 11,058±0,200; 11,713±0,200; 12,239±0,200; 13,785±0,200; 14,806±0,200; 15,763±0,200; 17,126±0,200; 17,693±0,200; 18,268±0,200; 18,562±0,200; 19,261±0,200; 20,033±0,200; 20,63±0,200; 21,006±0,200; 21,778±0,200; 22,268±0,200; 23,054±0,200; 23,361±0,200; 23,851±0,200; 24,626±0,200; 24,981±0,200; 25,507±0,200; 26,257±0,200; 26,963±0,200; 27,329±0,200; 27,807±0,200; 28,243±0,200; 28,975±0,200; 29,264±0,200; 29,687±0,200; 30,409±0,200; 30,798±0,200; 31,193±0,200; 31,724±0,200; 32,505±0,200; 32,985±0,200; 33,645±0,200; 34,249±0,200; 34,587±0,200; 35,048±0,200; 35,41±0,200; 35,933±0,200; 36,833±0,200; 37,276±0,200; 37,937±0,200; 38,467±0,200; and 39±0,200.

In another embodiment of the present invention is a crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan is characterized by peaks in the powder x-ray at the following 2θ values: 9,187±0,100; 11,058±0,100; 11,713±0,100; 12,239±0,100; 13,785±0,100; 14,806±0,100; 15,763±0,100; 17,126±0,100; 17,693±0,100; 18,268±0,100; 18,562±0,100; 19,261±0,100; 20,033±0,100; 20,63±0,100; 21,006±0,100; 21,778±0,100; 22,268±0,100; 23,054±0,100; 23,361±0,100; 23,851±0,100; 24,626±0,100; 24,981±0,100; 25,507±0,100; 26,257±0,100; 26,963±0,100; 27,329±0,100; 27,807±0,100; 28,243±0,100; 28,975±0,100; 29,264±0,100; 29,687±0,100; 30,409±0,100; 30,798±0,100; 31,193±0,100; 31,724±0,100; 32,505±0,100; 32,985±0,100; 33,645±0,100; 34,249±0,100; 34,587±0,100; 35,048±0,100; 35,41±0,100; 35,933±0,100; 36,833±0,100; 37,276±0,100; 37,937±0,100; 38,467±0,100; and 39±0,100.

In another embodiment of the present isoramaria.castilla modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan is characterized by the following powder x-ray, expressed in terms of inter-planar distance d, Bragg angles 2θ and relative intensity (expressed as percentage relative to the beam of greatest intensity):

td align="center"> 2,662
2Θ (°)D-distance (angstroms)Relative intensity (%)
9,1879,61825,4
11,0587,9952,3
11,7137,54918,7
12,2397,22634,2
13,7856,41923,5
14,8065,97813
15,7635,6185
17,1265,17329,3
17,6935,0098,4
18,2684,852 48,2
18,5624,77628,2
19,2614,60414,3
20,0334,42914,5
20,634,30217,2
21,0064,22612
21,7784,0782,4
22,2683,989100
23,0543,8556,4
23,3613,8057,4
23,8513,7281,8
24,6263,61214,9
24,9813,56214,7
25,5073,48911,1
26,2573,391 34,3
26,9633,30411,1
27,3293,26120,6
27,8073,20635
28,2433,15725,6
28,9753,0791,1
29,2643,0492,3
29,6873,0079,5
30,4092,93720,9
30,7982,9016,1
31,1932,8656,9
31,7242,81824,7
32,5052,7528
32,9852,71312,1
33,64526,5
34,2492,61615,2
34,5872,5918,6
35,0482,5584,6
35,412,5333,3
35,9332,49715,3
36,8332,4388
37,2762,4110,8
37,9372,37a 12.7
38,4672,33814,7
392,3082,6

In another embodiment of the present invention, the crystalline form of the mono-sodium salt of D-isoglutamine-D-tryptophan is a mixture of crystal modification 1 (polymorphic form F) and crystal modification 2 (polymorphic form I) monosodium salt of D-isoglutamine-D-tryptophan.

In another embodied the attachment of the present invention, the crystalline form of the mono-sodium salt of D-isoglutamine-D-tryptophan is a mixture of crystal modification 1 (polymorphic form F) and crystal modification 3 (polymorphic form X) monosodium salt, D-isoglutamine-D-tryptophan.

In another embodiment of the present invention x ray powder mixture of crystal modification 1 (polymorphic form F) and 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan has the form shown in Fig.9.

In accordance with another aspect of the present invention presents a pharmaceutical composition comprising any of the above-described crystalline forms of the mono-sodium salt of D-isoglutamine-D-tryptophan, and at least one pharmaceutically acceptable carrier.

In accordance with another aspect of the present invention presents a method of obtaining a pharmaceutical composition containing any of the above crystalline forms of the mono-sodium salt of D-isoglutamine-D-tryptophan, comprising combining any of the above crystalline forms of the mono-sodium salt of D-isoglutamine-D-tryptophan with at least one pharmaceutically acceptable carrier.

In accordance with another aspect of the present invention features the use of any of the above crystalline forms of the mono-sodium salt of D-isoglutamine-D-tryptophan without chromatographic purification in obtaining pharmaceutical compositions.

In accordance with another aspect of the present invention features the use of any of the described crystalline forms of the mono-sodium salt of D-isoglutamine-D-tryptophan as a tool against psoriasis.

In accordance with another aspect of the present invention proposes a method of obtaining mono-sodium salt of D-isoglutamine-D-tryptophan in crystalline form, which includes stages:

(a) obtaining a solution of D-isoglutamine-D-tryptophan and sodium hydroxide in water at a pH of from about 6.5 to about 7.2;

(b) filtering the solution to remove solid particles;

(C) evaporation of water for the purpose of concentrating the filtrate; and

(g) adding isopropanol order of deposition of monosodium salt of D-isoglutamine-D-tryptophan,

or

(d) mixing the solids obtained in stage (a), (b), (C) and (d) ways with ethyl acetate; and

(e) filtering solids

or

after the above steps (a) and (b) the following stages:

(W) evaporation of the filtrate obtained in stage (b), with the aim of obtaining solids;

(C) adding water to obtain a solution of the monosodium salt of D-isoglutamine-D-tryptophan; and

(I) evaporation of water over a period of time exceeding 5 hours to obtain the mono-sodium salt of D-isoglutamine-D-tryptophan in crystalline form,

or

(K) obtaining a solution of the monosodium salt of D-isoglutamine-D-tryptophan in methanol;

(l) filtering the solution to remove solid particles; and

(m) adding isopropanol with the aim of deposition Mononitrate the salt D-isoglutamine-D-tryptophan,

or

(h) obtaining the solution monoammonium salt D-isoglutamine-D-tryptophan and sodium hydroxide in water

then perform the above stage (b), (C) and (d).

In one of the embodiments of the present invention, when the method includes a stage (a), (b), (C) and (d), the duration of mixing on stage (d) is from about 1.5 to 16 hours.

In another embodiment of the present invention, when the method includes a stage (a), (b), (C) and (d), the duration of mixing on stage (d) is approximately 1 hour.

In another embodiment of the present invention, when the method includes a stage (a), (b), (C), (d), (e) and (f), the duration of mixing on stage (d) is approximately 1 hour and duration of stirring in ethyl acetate at stage (e) is approximately 2.5 hours.

In another embodiment of the present invention, when the method includes a stage (a), (b), (g), (h) and (I)at the stage (C) add water rate of approximately 18 to 22 ml of water for every gram of monosodium salt of D-isoglutamine-D-tryptophan, and the duration of the evaporation stage (s) is approximately 5 to 6 hours at the temperature of evaporation in the range from approximately 30 to 35°C.

In another embodiment of the present invention, when the method includes a stage (K), (l) and (m), on stage (K) is taken from about 11 is about 13 ml of methanol per 1 g of monosodium salt of D-isoglutamine-D-tryptophan, while the ratio of isopropanol to methanol at stage (m) is approximately 0.4 to 0.6 ml to 1 ml

In another embodiment of the present invention, when the method includes a stage (h), (b), (C) and (d), the duration of mixing on stage (g) is approximately 12 to 16 hours.

Other and further advantages and features of the present invention will become clear to experts in the industry from the subsequent detailed description of the invention, filed in conjunction with the accompanying drawings.

BRIEF DESCRIPTION of DRAWINGS

The accompanying figures are:

Figure 1 - chart of speciation of the dipeptide H-D-iGlu-D-Trp-OH and its salts, calculated based on the experimentally derived values of pKa of the acid and amine groups.

Figure 2 is a characteristic powder x-ray crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan.

Figure 3 is a characteristic powder x-ray crystal modification 2 (polymorphic form I) of the monosodium salt of D-isoglutamine-D-tryptophan.

4 is a characteristic powder x-ray crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan.

5 is a characteristic infrared spectrum (IR) absorption of the crystal modification 1 (p is limarino F) monosodium salt of D-isoglutamine-D-tryptophan.

6 is a characteristic infrared spectrum (IR) absorption of the crystal modification 2 (polymorphic form I) of the monosodium salt of D-isoglutamine-D-tryptophan.

7 is a characteristic infrared spectrum (IR) absorption of the crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan.

Fig represents the comparison of powder x-ray crystal modification 1 (polymorphic form F), crystal modification 2 (polymorphic form I) and the crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan.

Fig.9 is a characteristic powder x-ray of a mixture of crystal modification 1 (polymorphic form F) and crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan.

The INVENTION

In the present description D-isoglutamine-D-tryptophan is presented in the form of the dipeptide

Chemistry of amino acids or simple dipeptides complicated by the fact that the group-NH2represents the base, and the group-CO2H is an acid. Therefore, in aqueous solution ion H+moving from one end of the molecule to another, forming zwitter-ions.

Zwitter ions are simultaneously and e is chicheste charged, and electrically neutral. They contain positive and negative charges, but the total charge of the molecule is equal to zero. Although not in full compliance with theoretical rationale, the amino acid group iGlu molecules H-D-iGlu-D-Trp-OH exists in the form of zwitter-ion battery, and therefore for the formation of salt remains only one group-CO2H, when only one equivalent of a monovalent sodium hydroxide is used to bring the pH to neutral values. If the monosodium salt of H-D-iGlu-D-Trp-OH according to the formula III present in the above format, then only one group of CO2H is able to take one atom of metal sodium, which results in a salt according to formula III. In the compound of formula (III) cation sodium replaces one hydrogen atom in the compounds of formula (I), which is a carboxylic acid.

Figure 1 represents a diagram of the speciation of the dipeptide H-D-iGlu-D-Trp-OH and its salts, calculated using the program Hyperquad Simulation & Speciation using obtained on the basis of experimental pKa values of the acid and amine groups. LH2=H-D-iGlu-D-Trp-OH in dikilitas form, represented by a compound of formula I, LH=salt of monocarboxylic acid such as monosodium salt represented by the compound of formula III, L=salt of dicarboxylic acid, such as disodium salt, presents the the group of formula II, LH3=acid additive salt of the compound H-D-iGlu-D-Trp-OH, such as monohydrochloride salt of the compounds of formula I, where the group of NH2added proton. On the X-axis represents the values of the pH of the solution. On the Y-axis are determined by the molar fraction of the types of compounds present at specific pH values. Note: the share connection type in % with respect to L is the terminology of the program adopted by default. Concentration of 0.5 M is used to reflect the equivalence of 1 g of thymodepressin in 6 ml of water during the allocation method. This figure 1 shows that almost 100% of thymodepressin is in the form of the monosodium salt in water at a pH in the range of about 7.0 to 7.4.

The present invention is directed to a new mono-sodium salt of D-isoglutamine-D-tryptophan according to the formula (III), a new crystalline form of the mono-sodium salt of D-isoglutamine-D-tryptophan, including the new crystal modification 1 (polymorphic form F), a new crystal modification 2 (polymorphic form I) and a new crystal modification 3 (polymorphic form X).

The present invention is also directed to a method of obtaining a new crystal modification 1 (polymorphic form F), new crystal modification 2 (polymorphic form I), the new crystal modification 3 (polymorphic Faure is s X), and also a mixture of new crystalline modifications 1 and 3, monosodium salt, D-isoglutamine-D-tryptophan, and this method includes the following stages:

(a) obtaining a solution of D-isoglutamine-D-tryptophan and sodium hydroxide in water at a pH of approximately 6.5 to 7.0;

(b) filtering the solution to remove solid particles;

(C) evaporation of water for the purpose of concentrating the filtrate;

(g) adding isopropanol order of deposition of monosodium salt of D-isoglutamine-D-tryptophan,

or

the above stage (a), (b), (C) and (d), followed by stage:

(d) mixing the solids obtained in stage (a), (b), (C) and (d) ways with ethyl acetate; and

(e) filtering solids

or

the above stage (a) and (b), after which stage are:

(g) the process of evaporation of water from the filtrate obtained in stage (b) to obtain a solid substance;

(C) adding water to obtain a solution of the monosodium salt of D-isoglutamine-D-tryptophan; and

(I) evaporation of water over a period of time exceeding 5 hours, to obtain the mono-sodium salt of D-isoglutamine-D-tryptophan in crystalline form,

or

(K) obtaining a solution of the monosodium salt of D-isoglutamine-D-tryptophan in methanol;

(l) filtering the solution to remove solid particles;

(m) adding isopro is Enola order of deposition of monosodium salt of D-isoglutamine-D-tryptophan,

or

(h) obtaining the solution monoammonium salt D-isoglutamine-D-tryptophan and sodium hydroxide in water

then perform the above stage (b), (C) and (d).

In a preferred variant of the method, including the above stage (a), (b), (C) and (d), is used for obtaining the crystal modification 1 (polymorphic form F) and crystal modification 2 (polymorphic form I) of the monosodium salt of D-isoglutamine-D-tryptophan. This method includes the following stages:

(a) obtaining a solution of D-isoglutamine-D-tryptophan and sodium hydroxide in water;

(b) filtering the solution to remove solid particles;

(C) evaporation of water for the purpose of concentrating the filtrate;

(g) adding isopropanol order of deposition of monosodium salt of D-isoglutamine-D-tryptophan.

Depending on the ratio of the isopropanol by volume solution of monosodium salt of D-isoglutamine-D-tryptophan, as well as on the concentration of the solution and mixing time, as a result of this method can be obtained in pure crystalline modification 1 or pure crystalline modification of 2. On the basis of the calculated data for the chart of speciation, shown in figure 1, it is seen that the pH value of the solution at the stage of (a) to phase (b) must be in the range of about 6.5 to 7.2, it would be optimal value is 7,0.

A solution of D-isoglutamine-D-tryptophan and sodium hydroxide in water is obtained by adding a solid D-isoglutamine-D-tryptophan in solution of sodium hydroxide. D-isoglutamine-D-tryptophan has limited solubility in water (<20 mg / 1 ml water), however, the sodium salt dissolves in water perfectly. Sodium hydroxide is selected, on the basis of ease of obtaining sodium hydroxide solution, however, can be applied to other sodium bases, such as sodium hydride, sodium carbonate, sodium bicarbonate. These chemicals are obvious chemical equivalents of sodium hydroxide to complete the stage (a) of the method. At the stage (b) the solution is filtered to remove any solid particles to move to stage (b).

In the production method of the crystal modification 1 concentration of the filtrate obtained in stage (b), increase in stage (C) to remove water and achieve concentrations from about 1.3 to 3 mmol/ml of the dissolved substance in the solution. Soluto is a monosodium salt of D-isoglutamine-D-tryptophan. For this deposition monosodium salt is applied antibacterial. In our case, antibacterial is a solvent, which leads to the precipitation of the solute. Examples of antibacterially suitable for the applications in the present invention, are isopropanol and alkanol C1-C4. In a preferred variant embodiment of the present invention as antibacterial for deposition of monosodium salt is used isopropanol. At stage (g) for every 1 ml of sodium salt of D-isoglutamine-D-tryptophan in water, add approximately 30 to 40 ml of isopropanol in order to initiate the precipitation of the crystalline modification 1 of monosodium salt of D-isoglutamine-D-tryptophan. The duration of mixing is from about 1.5 to 16 hours, more preferred range is from about 12 to 16 hours. The solid is separated by filtration and dried in a vacuum oven under high vacuum. The preferred drying temperature is from about 40 to 45°C, and the preferred value of vacuum is lower than 8 mm Hg Even more preferably, the value of the applied vacuum was below 5 mm Hg

In the method of obtaining a crystalline modification of 2 the filtrate obtained in stage (b), then undergoes an increase in its concentration at the stage (C) to remove water and achieve the values of the concentration of the solute in the solution is from about 3 to 18 mmol/ml Dissolved substance is a monosodium salt of D-isoglutamine-D-tryptophan. For deposition of monosodium salt of PR is changing isopropanol as antibacterial. For the deposition of crystalline modification of 2 monosodium salt, D-isoglutamine-D-tryptophan add approximately 40 ml of isopropanol per 1 ml of monosodium salt of D-isoglutamine-D-tryptophan in water. The duration of mixing is approximately 1 hour. The solid is separated by filtration and dried in a vacuum oven under high vacuum. The preferred drying temperature is from about 40 to 45°C, and the preferred value of vacuum is lower than 8 mm Hg Even more preferably, the value of the vacuum was below 5 mm Hg

Not limited to theoretical information, the inventors have however found that, in the deposition method using isopropanol at stage (g) an important role in determining what kind of crystal modification 1 or 2 will be the reaction product, plays a duration of mixing. The result of a short mixing (within one hour or less than one hour) is crystal modification 2 (polymorphic form I), and the result of mixing a duration of about 16 hours is crystal modification 1 (polymorphic form F). The output of the crystalline modification also depends on the concentration of monosodium salt in solution and the amount of isopropanol added to qualities is antibacterial. Details about the experiment can be found in examples 1 and 2 described below.

The inventors also found that if the deposition of monosodium salt of formula III at a concentration of approximately 3 mmol/ml solution in water is carried out using isopropanol, the result can be obtained a mixture of crystal modification 1 (polymorphic form F) and crystal modification 3 (polymorphic form X). If this mixture is mixed with ethyl acetate and then filtered, the resulting crystalline form is represented mainly by the crystal modification 1, and more detailed information on the experiment are given in the following example 4B.

Thus, in a preferred embodiment, a method of obtaining a crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan includes the following stages:

(a) obtaining a solution of D-isoglutamine-D-tryptophan and sodium hydroxide in water at a pH of approximately 6.5 to 7.0;

(b) filtering the solution to remove solid impurities;

(C) evaporation of water for the purpose of concentrating the filtrate; and

(g) adding isopropanol order of deposition of monosodium salt of D-isoglutamine-D-tryptophan;

(d) mixing the solids obtained in stage (g)with ethyl acetate; and

(e) filter the Finance solids.

The presence of crystal modification 3 (polymorphic form X) in a mixture with crystal modification 1 (polymorphic form F) demanded further research that led to the invention of two ways to get crystal modification 3.

In a preferred embodiment, a method of obtaining a crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan includes the above stage (a), (b), (g), (h) and (I). More specifically, this method includes the following stages:

(a) obtaining a solution of D-isoglutamine-D-tryptophan and sodium hydroxide in water at a pH of from about 6.5 to about 7.2;

(b) filtering the solution to remove solid particles;

(W) evaporation of the filtrate obtained in stage (b) to obtain a solid substance;

(C) adding water to obtain a solution of the monosodium salt of D-isoglutamine-D-tryptophan;

(I) evaporation of water over a period of time exceeding 5 hours, to obtain the mono-sodium salt of D-isoglutamine-D-tryptophan in crystalline form.

The procedure of stages (a) and (b) is the same as described above. At stage (g) per 1 g of monosodium salt of D-isoglutamine-D-tryptophan add approximately 18 to 22 ml of water to obtain a solution. Slow evaporation of the solvent under reduced pressure, which gives the possibility of formation of crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan. At the stage (C) is the evaporation of the solvent in a round bottom flask using a rotary evaporator under vacuum. The preferred temperature of an external water bath for evaporation of the solvent is from about 30°to 35 ° C, the preferred value of vacuum from about 14 to 20 mm RT. Art. and the preferred duration of evaporation is approximately 5 to 7 hours.

In the alternative, but also the preferred method for obtaining a crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan is used a process comprising a stage (K), (l) and (m). More specifically, this method includes the following stages:

(K) obtaining a solution of the monosodium salt of D-isoglutamine-D-tryptophan in methanol;

(l) filtering the solution to remove solid particles; and

(m) adding isopropanol order of deposition of monosodium salt of D-isoglutamine-D-tryptophan.

Phase (K) is a solution of the monosodium salt of D-isoglutamine-D-tryptophan in methanol by dissolving solid monosodium salt (in any polymorphic form) in methanol. To effect the dissolution of the applied heating the resulting suspension in methanol. The preferred concentration of the dissolved substance (monosodium salt) is 1 g is storeimage matter about 11-13 ml of methanol. Then on stage (l) any insoluble particles filtered. At stage (m) add isopropanol rate of approximately from 0.4 to 0.6 ml isopropanol per 1 ml solution in methanol. Undissolved material is highlighted by filtering by suction and is a crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan.

In another variant embodiment of the present invention monosodium salt, D-isoglutamine-D-tryptophan can be obtained by exchange reactions monoammonium salt D-isoglutamine-D-tryptophan with sodium hydroxide. This method includes the above stage (h), (b), (C) and (d) and is as follows:

(h) obtaining the solution monoammonium salt D-isoglutamine-D-tryptophan with sodium hydroxide in water;

(b) filtering the solution to remove solid impurities;

(C) evaporation of water for the purpose of concentrating the filtrate;

(g) adding isopropanol order of deposition of monosodium salt of D-isoglutamine-D-tryptophan.

At stage (n) monoammonium Sol D-isoglutamine-D-tryptophan is mixed with sodium hydroxide in a ratio of approximately 1:1 in water. At the stage (b) the resulting solution is filtered. It should be noted that at the stage (n) results in the release of ammonium hydroxide. Therefore, the pH level is greater than the value of 7.5. No neo is the need to adjust pH. Further concentration of the filtrate increases to reach values in the range from approximately 0.25 to 0.5 g dissolved substances (monosodium salt) in 1 ml of water. At stage (g) to precipitate the crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan in the solution obtained in stage (C), add isopropanol rate of approximately 15-30 ml isopropanol per 1 ml of solution.

As described in example 4 of the present invention, by varying the reaction conditions D-isoglutamine-D-tryptophan with sodium hydroxide can get mixed polymorph monosodium salt containing crystal modifications 1 and 3. As an integral part of the present invention the inventors have illustrated in example 4 conversion of a mixture of crystalline modifications 1 and 3 mainly in the crystal modification 1 by stirring the solids in ethyl acetate.

As generally described above for the method of obtaining the monosodium salt of D-isoglutamine-D-tryptophan, when executing this method, and other methods described here, the benefits of additional add isopropanol to the aqueous solution of sodium salt, to isolate the sodium salt in the form of a solid crystalline substance.

Installed, but without theoretical justification that paramesh is improving with isopropanol in a short period of time (usually no more than an hour) results in a crystal modification 2 (polymorphic form I) monosodium salt of formula III. Stirring for a long time (16 hours) results in a crystal modification 1 (polymorphic form F). In those cases, when the method are a mixture of crystal modification 1 (polymorphic form F) and crystal modification 3 (polymorphic form X), pure crystalline modification 1 can be obtained by stirring the mixture in ethyl acetate.

If the solution of the monosodium salt get in methanol, the use of isopropanol as antibacterial provides the ability to get the crystal modification 3 of the present invention.

Pharmacological properties of monosodium salt of D-isoglutamine-D-tryptophan and crystal modifications 1, 2 and 3, as well as the possibility of their use for the treatment and prevention of disorders in the body, match, provided that these substances are present in the target organ or target cell in the dissolved form, regardless of the original form of solids, the properties described for thymodepressin and its disodium salt, is described in many sources, in particular, in U.S. patent No. 5736519, 6103699 and 6410515l, in the works Semina O.V. and others (2001), Bulletin of experimental biology and medicine, 131(5), 493-495); and Sapunova YEAR, and others (may 2002), Bulletin of experimental biology and medicine, 133(5), 488-490).

Action Chris is allicesia modifications 1, 2 and 3 can be investigated, for example, by using pharmacological models, described in particular in U.S. patent No. 5736519, 6103699 and 6410515, in the works Semina O.V. and others (2001)Bulletin of experimental biology and medicine, 131(5), 493-495); and Sapunova YEAR, and others (may 2002), Bulletin of experimental biology and medicine, 133(5), 488-490), which are incorporated here by reference, and the relevant sections of which are part of the present description.

Crystalline modification of the monosodium salt of D-isoglutamine-D-tryptophan of the present invention can be used as pharmaceuticals for animals, preferably to mammals, and in particular, for people, both in pure form and as mixtures of these modifications with each other or in the form of pharmaceutical preparations (or pharmaceutical compositions). Therefore, the present invention relates to crystalline mono-sodium salt of D-isoglutamine-D-tryptophan and crystalline modifications of monosodium salt of D-isoglutamine-D-tryptophan for use as pharmaceuticals, their use as anti-psoriasis and, in particular, to their use as immunosuppressants, as well as to their use for the production of medicines based on them. In addition, the present invention relates to Pharma is autocheckin drugs, containing as active ingredients an effective dose of the crystalline mono-sodium salt of D-isoglutamine-D-tryptophan, in particular, monosodium salt, D-isoglutamine-D-tryptophan in the form of one or several crystal modifications 1, 2 and 3 and at least one pharmaceutical acceptable carrier, i.e. one or more substances that contribute to the delivery to any point of the body, and/or excipients. These pharmaceutical preparations contain, for example, monosodium salt, D-isoglutamine-D-tryptophan in the form of crystal modification 1, and at least one pharmaceutical acceptable carrier or monosodium salt, D-isoglutamine-D-tryptophan in the form of a crystalline modification of 2, and at least one pharmaceutical acceptable carrier or monosodium salt, D-isoglutamine-D-tryptophan in the form a crystal modification 3, and at least one pharmaceutical acceptable carrier, or a combination of the two crystalline modifications according to the present invention, such as crystal modifications 1 and 2 or crystal modifications 1 and 3 or crystalline modification of 2 and 3, in each case together with at least one pharmaceutically acceptable carrier.

APPLICATION AND INTRODUCTION

The disodium salt of D-isoglutamine-D-tripto the Ana has found application in the treatment of psoriasis, atopic dermatitis and rheumatoid arthritis. Therefore, monosodium salt, D-isoglutamine-D-tryptophan of the present invention can be used in pharmaceutical compositions for administration to patients in amounts effective from a therapeutic point of view and in a biologically acceptable form for administration in vivo, i.e. in the form of peptides, therapeutic effects from the use of which greatly exceeds the resulting toxicity.

In accordance with the speciation diagram shown in figure 1, the dominant species at neutral pH is a monocarboxylic form of thymodepressin, that is, the monosodium salt of the dipeptide D-isoglutamine-D-tryptophan in that case, if the counterion is sodium. The disodium salt of D-isoglutamine-D-tryptophan has an extremely high hygroscopicity, and it is very difficult to dose. Depending on the concentration of the disodium salt in the solution pH of the solution exceeds the value of 8.2. The pH of a solution containing 100% disodium salt, exceed 11,5 as shown in the diagram of speciation (figure 1). A solution with a high pH is not suitable for introduction into the human body. Adjusting pH value of about 7.2 to 7.4 for using inorganic acids means the introduction in the preparation of additional salts, such as sodium chloride.

Crystalline form of the monosodium salt of t is Auda ideal candidates for replacement of the disodium salt upon receipt of various drugs. The introduction of these described here new crystalline salts of the present invention may be any of the acceptable methods of administering drugs for systemic effects. These methods include oral, parenteral or other system introduction in the organism as a whole, introduction to aerosol or local form.

Depending on the intended route of administration of these compositions can be applied in the form of solid, semi-solid or liquid dosage forms, such as tablets, sublingual tablets, suppositories, pills, capsules, powders, liquids, aerosols, suspensions and the like, preferably in unit dosage forms, for the convenience of one introduction accurately assigned doses. The composition of these compositions will include at least one pharmaceutical acceptable carrier or excipient and crystalline D-isoglutamine-D-tryptophan of moonacre, in addition, they can also include other medicinal agents, pharmaceutical agents, carriers, adjuvants, etc.

To conventional non-toxic solid carriers which can be used in the composition of solid compositions include, for example, corresponding pharmaceutical standards mannitol, lactose, starch, magnesium stearate, saccharin sodium, talc, cellulose, glucose, sucrose, magnesium carbonate, and others. As from what was echalas above, the active substance may be in a dosage form that represents suppositories, including as a carrier, for example, polyalkylene glycols or propylene glycol. Liquid pharmaceutical compositions intended for insertion into the body, can be obtained, for example, by dissolving, dispersing, etc. described above active substances and optional pharmaceutical adjuvants in a carrier, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like, resulting in a solution or suspension. Optionally, the pharmaceutical compositions intended for insertion, can also contain minor amounts of nontoxic auxiliary substances, such as moisturizing or emulsifying agents, buffering agents to control pH and the like, which include, for example, sodium acetate, sorbitanoleat, triethanolamine sodium acetate, triethanolamine oleate, and other Practical methods for such dosage forms are known to specialists in this field, they are also described, for example, in Remington: The Science and Practice of Pharmacy, 21stEdition, 2006, Part 5, Pharmaceutical Manufacturing, Chapters 37, 39, 41-47 and 50, pp. 702-719, 745-775, 802-938, and 1000-1017 (formerly Remington''s Pharmaceutical Sciences), David B. Troy (Ed.), Lipincott Williams & Wilkins, Baltimore, Maryland. In any case, part of this is oppozitsii or drug intended for introduction into the body, will be composed of active substance (substances) in a quantity sufficient to affect the symptoms in the subject's treatment.

Parenteral administration in the General case is an injection that can be subcutaneous, intramuscular or intravenous. Preparations for injection can be obtained in a convenient form - it can be liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, and emulsions. To an acceptable excipients include, for example, water, saline, dextrose, glycerol, ethanol, and others. In addition, if necessary, the pharmaceutical composition intended for insertion, can also contain minor amounts of nontoxic auxiliary substances, such as moisturizing or emulsifying agents, buffering agents to control pH and the like, which include, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate, and other.

Preferred routes of administration monosodium salt, D-isoglutamine-D-tryptophan are either oral or nasal (bronchial), depending on the nature of the violation which is subject to treatment.

For oral administration is made of non-toxic pharmaceutically acceptable composition is, including any of the traditionally used excipients, such as appropriate pharmaceutical standards mannitol, lactose, starch, magnesium stearate, saccharin sodium, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. Such compositions can be obtained in the form of solutions, suspensions, tablets, pills, capsules, powders, drugs with a slow release, etc. of the Active ingredient in such compositions may comprise from about 1 to 95%, in the preferred embodiment, this range is approximately from 25 to 70%.

For administration to the lungs by oral or nasal application can also be used in aerosol form of delivery. For the introduction of a spraying method, it is desirable that the active ingredient was in fine form, together with the surface-active agent and a propellant. Typical values of the content of the active ingredients are in the range of from about 0.01 to 20 wt.%, preferably 0.04 to 1.0 wt.%.

Naturally, surfactants must be non-toxic and preferably soluble in aerosol media. Representatives of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as Caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenate is, alasaarela and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride such as ethylene glycol, glycerin, aritra, arabitol, mannitol, sorbitol anhydrides of exit derived from sorbitol (esters sorbitan, commercially available under the trademark SPANS®), as well as polyoxyethylene and polyoxypropylene derivatives of these esters. Can also be used mixed esters, for example, mixed or natural glycerides. Of surface-active agents, the most preferred are the oleates or sorbitan, for example, commercially available under the trademarks ARLACEL® C (sorbitan poletariat), SPAN® 80 (sorbitan monooleate) and SPAN® 85 (sorbitan trioleate). Mass fraction of surfactant in the composition may range from 0.1 to 20 wt.%, preferred is a range of values from about 0.25 to 5 wt.%.

The rest of the song is usually aerosol media. Liquefied aerosol media in environmental conditions are usually gases and condensed under pressure. Examples of acceptable liquefied aerosol carriers are the lower alkanes containing up to five carbon atoms, such as butane and propane, and preferably fluorinated or ferroresonance alkanes, for example, commercially available n the d a trademark FREON®. Can also be used mixtures of the aforementioned substances.

In the manufacture of aerosol spray with a suitable valve is filled with the corresponding aerosol media containing finely dispersed active ingredient and a surfactant. Thus, the ingredients of the composition will be under high pressure to the pushing of the valve.

For the local introduction of these compositions include an effective amount of the active substances of this class in a mixture with acceptable non-toxic pharmaceutical carrier. The acceptable range of values of the proportion of active ingredient in the composition is from about 0.1 to 10%, and the rest is media, the preferred value of a share of active ingredient from about 1%to 2%. The concentration of active ingredient in pharmaceutical compositions suitable for topical application, will vary depending on the specific activity of the active substances in combination with the patient's condition, which introduces the song. Acceptable carriers or means of drug delivery for local injection of these substances are creams, ointments, lotions, emulsions, solutions, etc.

For example, acceptable ointment for local application of the compounds of the present invention contains from about 15 to 45 percent is in saturated fatty alcohol, containing from 16 to 24 carbon atoms in the molecule, for example, cetyl alcohol, stearyl alcohol or BeginInvoke alcohol, and from 45 to 85 wt.% glycol solvent such as propylene glycol, polyethylene glycol, dipropyleneglycol, and mixtures thereof. The ointment may also contain from 0 to 15 wt. percent plasticizer, such as polyethylene glycol, 1,2,6-hexanetriol, sorbitol, glycerol and the like; from about 0 to 15 wt. percent binder, for example, a saturated fatty acid containing from 16 to 24 carbon atoms, such as stearic acid, palmitic acid, Bekenova acid, fatty acid amide, such as oleamide, palmitate, stearate, beginnig, and the complex ester of a fatty acid containing from 16 to 24 carbon atoms, such as sorbitol monostearate, polyethylene glycol monostearate, polypropylenglycol or appropriate monoether other fatty acids, such as oleic acid and palmitic acid; and from about 0 to 20 wt.% substances promoting the penetration of, for example, dimethyl sulfoxide or dimethylacetamide.

Therapeutically active amount of the crystalline mono-sodium salt of D-isoglutamine-D-tryptophan may vary depending on factors such as stage of disease, age, sex and weight of the patient. The dosed mode which I can modify to obtain the optimal therapeutic effect. In General, the dose of daily intake should be approximately from 1 to 200 mg of peptide.

Below are illustrative examples of the compositions, which in no way limit the scope of the invention in obtaining various pharmaceutical compositions.

IngredientsThe number one tablet mg
The active ingredient25
Lactose, spray dried20
Corn starch153
Magnesium stearate2

The above ingredients are thoroughly mixed and pressed into a tablet with a single notch.

IngredientsThe number one tablet mg
The active ingredient100
Lactose, spray dried148
Magnesium stearate2

Presented above in redient mixed and are enclosed in a capsule shell made of hard gelatin

IngredientsThe number one tablet mg
The active ingredient200
Lactose145
Corn starch50
Magnesium stearate5

The above ingredients are thoroughly mixed and pressed into a tablet with one notch

IngredientsThe number one tablet mg
The active ingredient108
Lactose15
Corn starch25
Magnesium stearate2

The above ingredients are mixed and are enclosed in a capsule shell made of hard gelatin

IngredientsThe number one tablet mg
The active ingredient150
Lactose92

The above ingredients are mixed and are enclosed in a capsule shell made of hard gelatin.

The preparation for injection with pH adjusted with buffer to a value of 7, obtained in the following composition:

Ingredients
The active ingredient0.2 g
KH2PO42 ml
KOH (1 BC)amount to obtain a pH of 7
Water (distilled, sterile)number to obtain 20 ml

The preparation for injection with pH adjusted with buffer to a value of 7, obtained in the following composition:

Ingredients
The active ingredient0.01 g
Water (distilled, sterile) number to obtain 1 ml
NaOH (0,2 ad)amount to obtain a pH of 7

Suspension for oral administration prepared in the following composition:

Ingredients
The active ingredient0.1 g
Fumarola acid0.5 g
Methyl paraben2.0 g
Granulated sugar0.1 g
sorbitol (70% solution)25,5 g
Veegum K (Vanderbilt Co.)is 12.85 g
Flavouring substance1.0 g
Dyesa 0.035 ml
Distilled waternumber to obtain 100 ml

Form for local use

IngredientsGrams
Active is emesto 0,2-2
Span 602
Tween 602
Mineral oil5
Petrolatum10
Methyl paraben0,15
Propyl paraben0,05
BGA (bottled hydroxyanisol)0,01
Distilled waternumber to obtain 100 ml

All of the above ingredients, except water, are mixed and heated to 45 degrees Celsius with stirring. Then add the required amount of heated to 45 degrees Celsius water with vigorous stirring to obtain emulsion of the ingredients, then water is added to obtain 100 g of the emulsion.

More preferred embodiments of the invention are illustrated in the following examples, which should not be considered as limitations to the following appended claims.

EXAMPLES

Example 1

Obtaining crystalline option is the ratification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan (1:1)

Method A: From monoammonium salt D-isoglutamine-D-tryptophan (1:1) and sodium hydroxide

A solution of H-D-iGlu-D-Trp-OH, monoammonium salt (1:1), (496 mg, of 1.34 mmol) and 1N sodium hydroxide (1.4 ml of 1.40 mmol) in water (15 ml) was stirred at room temperature for 30 minutes. The reacted mixture was subjected to evaporation under reduced pressure to a residual amount of the solvent 1-2 ml After cooling to room temperature was added isopropanol (30 ml) prior to the deposition of sediment. The resulting suspension was stirred over night at room temperature, after which the solid is separated by filtering with suction. This solid was washed with isopropyl alcohol (2×40 ml), then dried overnight in an oven at a temperature of 44°C. was obtained off-white powder (462 mg, yield 97%). This material is called the crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan (1:1).

The water content of this material as specified by the test Karl-Fischer, was 3.4%.

MS (m/z): 356,0 [M]+, 334,1 [C16H20N3O5]+, 187,9 (100%).

Powder x-ray for this material is shown in figure 2. This powder x-ray can also be expressed in the parameters of the interplanar distance d, Bragg's angle 2θ and relative the Oh intensity (expressed in percentage relative to the maximum beam intensity), as follows:

2Θ (°)D-distance (angstroms)Relative intensity (%)
9,23from KZT9, 5732
to 9.918,91741,3
12,417,12637,6
13,76to 6.430,8
14,875,95435,8
of 15.755,6227,6
17,884,9575,5
18,784,721of 58.9
19,574,53230,9
19,844,47128,1
20,314,3682,9
one-21.324,165 53,5
21,554,1230,3
22, 95mm3,87367,4
23,453,7924,5
24,343,65419,4
24,963,565to 85.2
27,493,242100
of 27.943,1923,3
to 29.273,04919,1
30,072,9727,2
30,432,93515,2
31,292,85639,9
32,252,77413
34,072,62919,3
34,94 2,5667,8
35,532,5255
36,082,4878,4
37,212,41415,5
38,172,3569,1

The samples in powder form were prepared using conventional technology front packing and tested the system diffraction D8 Discovery source Cu-kα mode 45 kV/45 mA. The system is equipped with a proportional 2D - detector (GADDS). Research data were collected on two frames with exposures of 600 s for each, the result covers a range 3-35° (2-θ). Then the received 2D image diffraction were combined and processed to produce the standard of pictures diffraction, showing the relationship between I and 2θ. These data were then processed using various data processing programs Bruker AXS, including: EVA™ 8.0 and TOPAS™ v. 2.1 (for analysis matches with the application, if necessary). All powder x-ray presented in the present description, were obtained using the above-described technology, equipment and modes.

Spectrum of this material obtained is output by way of spectroscopy PF-IR (KBr)-, shown in figure 5.

Method B: From D-isoglutamine-D-tryptophan and sodium hydroxide.

In a round bottom flask with a volume of 100 ml with a magnetic rod agitator placed 2,91 ml of sodium hydroxide (1,000 N, only 2.91 mmol) and only 2.91 ml of deionized water. The solution was cooled to 0°C in an ice bath, then one portion was added H-D-iGlu-D-Trp-OH (1,00 g, 3.00 mmol) as a solid. Added 3 ml of deionized water, and the resulting solution was stirred for another 15 minutes. The pH of this solution was approximately 5.5 to 6.0. The pH is brought up to a value of 6.5 by adding 40 μm solution 0,500 N. NaOH. The mixture was filtered to remove any solid particles, and then the filtrate was concentrated in vacuo to obtain the value of the solution volume of 0.5 ml at a bath temperature of 30°C. the Obtained residue was diluted with deionized water to a volume of 0.6 ml) and isopropanol (10 ml) with vigorous stirring, resulting in a was obtained a solid residue. In several minutes the mixture was affected by ultrasound. Then added 30 ml of isopropanol. After stirring for 90 minutes, the mixture was divided into two parts (a and b).

A. the Part a mixture was filtered, and the solid was washed with isopropanol (2×10 ml). The solid was air dried for 1 hour and then dried overnight under vacuum in an oven at 40°C. the Powder roentgenol the Amma and the IR-spectrum (KBr) of this part A of the material was the same, as presented in figure 2 and figure 5 for the polymorphic form F obtained according to the method As described above.

B. Part B was stirred over night. Then the mixture was filtered, and the solid was washed with isopropanol (2×10 ml), dried with air for 15 minutes, and then during the night was dried in vacuum at 40°C. Powder x-ray and IR-spectrum (KBr) of this part In the material was the same as presented in figure 2 and figure 5 for the polymorphic form F obtained according to the method As described above.

Example 2

Getting crystal modification 2 (polymorphic form I) of the monosodium salt of D-isoglutamine-D-tryptophan (1:1)

In a round bottom flask with a volume of 100 ml with a magnetic rod agitator placed 2,97 ml of sodium hydroxide (1,000 H of 2.97 mmol) and 3.0 ml of deionized water. The solution was cooled to 0°C in an ice bath, then one portion was added H-D-iGlu-D-Trp-OH (1,00 g, 3.00 mmol) in solid form, resulting in a transparent slightly pink solution. The pH of this solution was approximately 7,0. The mixture was filtered to remove any remaining solid particles, and then the filtrate was concentrated in vacuo to obtain oil. The residue was diluted with deionized water to a volume of 0.6 ml and with vigorous stirring was added isopropanol (40 ml) prior to the deposition of sediment. After paramasivan what I'm in for 1 hour the mixture was filtered, and the solid was washed with isopropanol (2×15 ml), dried with air for about 15 minutes, then dried overnight under vacuum at 36°C. there Was obtained a white crystalline solid (1,00 g, yield 94%). This material is called crystal modification 2 (polymorphic form I) monosodium salt of D-isoglutamine-D-tryptophan (1:1).

The purity of this material is determined by the HPLC method (proportion of peak area percent), amounted to 98.5%. method HPLC; Column: XTerra MS C18; 5 μm, a 4.6×250 mm; mobile phase: A=water phase: 4 mm hydroxyethylaminomethyl, 2 mm add, pH 7,4; B=organic phase: CH3CN; gradient: B%: 0 min 5%, 15 minutes and 55%, 30 minutes and 55%, 32 minutes, 5%, 35 minutes, 5%a; flow Rate: 1 ml/min; volume of injection: 5 ál; λ: 222, 254, 280, 450 nm; retention time of product: 6,39 minutes

The water content of this material, as determined by the test of Karl-Fischer, was 6.0%.

UV (water, c=22,4 μm, λmaxnm): 221 (ε 30528), 280 (ε 4958).

MS (m/z): 356,0 [M]+, 334,2 [C16H20N3O5]+, 187,9 (100%).

Powder x-ray for this material is shown in figure 3. This powder x-ray can also be expressed in the parameters of the interplanar distance d, Bragg angle 2θ and relative intensity (expressed as percentage relative to the maximum beam intensity), as follows:

34,97
2Θ (°)D-distance (angstroms)Relative intensity (%)
9,659,1615,3
10,418,49223,7
11,27,89740,4
11,717,5494,5
13,45to 6.5890,2
13,936,35115,9
14,446,1283,7
15,615,67232,4
17,015,2079,9
18,184,87611,7
18,654,75547,8
20,024,43259,2
20,854,25735,9
21,394,1524,1
21,734,08627,3
22,523,945100
23,273,81913,7
24,33,6632,4
25,843,44569,5
26,823,32282,5
28,493,1330,1
30,182,95958,8
30,762,90486,9
31,492,83935,3
33,032,718,7
34,55at 2,59417,8
2,56443,4
35,74of 2.518,5
37,252,41228,1
37,712,38328,5
38,792,31916,9

Range of PF-IR (KBr) for this material is shown in Fig.6.

This material was prepared by the method described below.

Deionized water (18 ml) and a solution of 1 N. NaOH in (18.0 ml, 18 mmol) were mixed in a flask of 250 ml, round bottom and cooled to 0°C. Then added H-D-iGlu-D-Trp-OH (6.0 g 18 mmol) in solid form and slowly dissolved. After 1 hour the solution was painted a pale peach color. Part of this solution (6 ml) were collected and subjected to evaporation in vacuo to an oil. Then this oil was diluted with deionized water to a volume of 0.6 ml) and dropwise added 40 ml of isopropyl alcohol (IPA). The mixture was vigorously stirred for 1 hour and then was filtered. The solid was air dried and then dried overnight in a vacuum oven, resulting in a received 450 mg of white solid, MP: 186,9-189,2°C. Powder x-ray and IR spectrum of this material kazaliste presented respectively in figure 3 and 6, as explained above.

Example 3

Getting crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan (1:1)

Method A: monosodium salt of D-isoglutamine-D-tryptophan (800 mg) in solid form obtained as described above in example 1, method B, was suspended in methanol (10 ml). The mixture is gently heated to dissolve the solid. The solution was filtered twice, passing through a funnel with a sintered glass filter, and was placed in a round bottom flask with a volume of 100 ml Flask was equipped with a stirrer, and 4 ml of isopropyl alcohol was slowly added before the formation of solids. Within 4 hours, the suspension was stirred and then filtered. Then the solid is washed with isopropyl alcohol (3×10 ml). The solid was dried air (solid colored in a pale peach color), and then dried overnight in a vacuum oven. There was obtained a white solid substance (480 mg, yield 60%), TPL: 182,3-186,1°C. This material is called crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan (1:1).

X ray powder of this material is shown in figure 4. This powder x-ray can also be expressed in terms of inter-planar distance d, Bragg angle 2θ and relative intensities (expr is authorized in percentage relative to the maximum beam intensity), as follows:

34,587
2Θ (°)D-distance (angstroms)Relative intensity (%)
9,1879,61825,4
11,0587,9952,3
11,7137,54918,7
12,2397,22634,2
13,7856,41923,5
14,8065,97813
15,7635,6185
17,1265,17329,3
17,6935,0098,4
18,2684,85248,2
18,5624,77628,2
19,261 4,60414,3
20,0334,42914,5
20,634,30217,2
21,0064,22612
21,7784,0782,4
22,2683,989100
23,0543,8556,4
23,3613,8057,4
23,8513,7281,8
24,6263,61214,9
24,9813,56214,7
25,5073,48911,1
26,2573,39134,3
26,9633,30411,1
7,329 3,26120,6
27,8073,20635
28,2433,15725,6
28,9753,0791,1
29,2643,0492,3
29,6873,0079,5
30,4092,93720,9
30,7982,9016,1
31,1932,8656,9
31,7242,81824,7
32,5052,7528
32,9852,71312,1
33,6452,66226,5
34,2492,61615,2
2,5918,6
35,0482,5584,6
35,412,5333,3
35,9332,49715,3
36,8332,4388
37,2762,4110,8
37,9372,37a 12.7
38,4672,33814,7
392,3082,6

The water content of this material as specified by the test Karl-Fischer, amounted to 9.3%.

MS (m/z): 356,0 [M]+, 334,2 [C16H20N3O5]+, 187,9 (100%).

Range of PF-IR (KBr) presented on Fig.7.

Method B: Deionized water (6 ml) and a solution of 1 N. NaOH (6.0 ml, 6 mmol) were mixed in a round bottom flask of 100 ml and cooled to 0°C, then added H-D-iGlu-D-Trp-OH (2.0 g, 6 mmol)and the mixture was affected by ultrasound for 2 minutes to dissolve all the solid part of the s. The pH value was approximately 6,0. Part of this solution (6 ml) was collected and filtered to remove any remaining solid particles. Then the filtrate is evaporated in vacuum to obtain a solid substance (1,02 g). A portion of this solid (0.5 g) was dissolved in deionized water (10 ml), resulting in a clear colorless solution. Removal of volatiles was performed in vacuum using a rotary evaporator with cooling of the condenser with ice water and with a bath temperature of 30°C for 6 hours, the resulting solid substance. Powder x-ray and IR spectrum of this material was similar to the one shown respectively in figure 4 and 7, as described above in method A.

Example 4

The mixture of crystal modification 1 (polymorphic form F) and crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan (1:1)

In a round bottom flask of 50 ml volume, equipped with a magnetic rod-stirrer, was placed 3.0 ml of NaOH (1,0 M, 3 mmol) and 3 ml of distilled water. Then the solution was cooled to 0°C using a bath of ice water, and added H-D-iGlu-D-Trp-OH (1,00 g, 3.00 mmol), which resulted in a clear solution pink. The solution is allowed to mix for 1 hour at the temperature of ice water, and then heated to room so the temperature. The solution was filtered, and then concentrated until the volume of solvent was less than 1 ml. of isopropyl alcohol in the amount of 38 ml) was added before the formation of a solid precipitate. The solution was strongly stirred for 1 hour. Half of this solution was filtered and washed IPA (2×15 ml). The solid was air dried and then dried overnight in a vacuum oven at 35°C, resulting in a received 379 mg of a white crystalline solid. This material is a mixture of crystal modification 1 (polymorphic form F) and crystal modification 3 (polymorphic form X) of sodium salt of D-isoglutamine-D-tryptophan (1:1). Powder x-ray of the material presented on Fig.9.

Part of a solid (50 mg), obtained as described above, suspended in 3 ml of ethyl acetate at room temperature and was stirred. After 2.5 hours the solution was filtered, the solid was air dried and then dried overnight in a vacuum oven, resulting in a received 38 mg (yield 78%) of white crystalline solid. Analysis of the powder x-ray showed that this material mainly represents the crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan (1:1), described above in example 1, powder x-ray which is presented in figure 2.

Powder x-ray for the above-described crystalline modifications 1, 2 and 3, monosodium salt, D-isoglutamine-D-tryptophan are presented respectively in figure 2, 3 and 4. Specialists in this field will understand that the values of angle 2θ for powder x-ray for crystal modifications 1, 2 and 3, monosodium salt, D-isoglutamine-D-tryptophan may slightly vary when using different devices and/or from sample to sample, so these values should not be considered absolute. In a typical case, the values of angles 2θ shall be as specified with a tolerance of about ±0.2 degrees, preferably about ±0.1 degrees. Specialists in this field will understand that the value of the relative intensity of peaks in the powder x-ray for crystal modifications 1, 2 and 3, monosodium salt, D-isoglutamine-D-tryptophan can vary when using different devices and/or from sample to sample, so these values should not be considered absolute.

Although this description presents preferred embodiments of the present invention, the experts in this field will understand that these options may be amended, does not violate the essence of the invention and within the scope of the claims.

1. Crystal m the modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan (1:1), characterized by peaks in the powder x-ray with the following 2θ values: 18,78±0,20, 21,32±0,20, 22,95±0,20, 24,96±0,20 and 27,49±0,20.

2. Crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan (1:1) according to claim 1, characterized by peaks in the powder x-ray with the following 2θ values: 18,78±0,10, 21,32±0,10, 22,95±0,10, 24,96±0,10 and 27,49±0,10.

3. Crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan (1:1) according to claim 1, having a powder x-ray as shown in figure 2.

4. Crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan according to claim 1, characterized by peaks in the powder x-ray with the following 2θ values: 9,23±0,20; 9,91±0,20; 12,41±0,20; 13,76±0,20; 14,87±0,20; 15,75±0,20; 17,88±0,20; 18,78±0,20; 19,57±0,20; 19,84±0,20; 20,31±0,20; 21,32±0,20; 21,55±0,20; 22,95±0,20; 23,45±0,20; 24,34±0,20; 24,96±0,20; 27,49±0,20; 27,94±0,20; 29,27±0,20; 30,07±0,20; 30,43±0,20; 31,29±0,20; 32,25±0,20; 34,07±0,20; 34,94±0,20; 35,53±0,20; 36,08±0,20; 37,21±0,20; 38,17±0,20; and 39,19±0,20.

5. Crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan of claim 2, characterized by peaks in the powder x-ray with the following 2θ values: 9,23±0,10; 9,91±0,10; 12,41±0,10; 13,76±0,10; 14,87±0,10; 15,75±0,10; 17,88±0,10; 18,78±0,10; 19,57±0,10; 19,84±0,10; 20,31±0,10; 21,32±0,10; 21,55±0,10; 22,95±0,10; 23,45±0,10; 24,34±0,10; 24,96±0,10; 27,49±0,10; 27,94±0,10; 29,27±0,10; 30,07±0,10; 30,43±0,10; 31,29�0,10; 32,25±0,10; 34,07±0,10; 34,94±0,10; 35,53±0,10; 36,08±0,10; 37,21±0,10; 38,17±0,10; and 39,19±0,10.

6. Crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan according to claim 4 or 5, characterised by the following powder x-ray obtained using a diffractometer (copper cathode) and expressed in the parameters of the interplanar distance d, Bragg angle 2θ and relative intensity (expressed as percentage relative to the beam of greatest intensity):

2θ (°)D-distance (angstroms)Relative intensity (%)
9,23from KZT9, 5732
to 9.918,91741,3
12,417,12637,6
13,76to 6.430,8
14,875,95435,8
of 15.755,6227,6
17,884,9575,5
18,784,721of 58.9
19,574,53230,9
19,844,47128,1
20,314,3682,9
one-21.324,16553,5
21,554,1230,3
22, 95mm3,87367,4
23,453,7924,5
24,343,65419,4
24,963,565to 85.2
27,493,242100
of 27.943,1923,3
to 29.273,04919,1
30,072,97 27,2
30,432,93515,2
31,292,85639,9
32,252,77413
34,072,62919,3
34,942,5667,8
35,532,5255
36,082,4878,4
37,212,41415,5
38,172,3569,1
39,192,2973,1

7. Crystal modification 2 (polymorphic form I) monosodium salt of D-isoglutamine-D-tryptophan (1:1), characterized by peaks in the powder x-ray with the following 2θ values: 3,45±0,20, 20,02±0,20, 22,52±0,20, 25,84±0,20, 26,82±0,20, 30,18±0,20 and 30,76±0,20.

8. Crystal modification 2 (polymorphic form I) monosodium salt of D-isoglutamine-D-tryptoph is at (1:1) according to claim 7, characterized by peaks in the powder x-ray with the following 2θ values: 3,45±0,10, 20,02±0,10, 22,52±0,10, 25,84±0,10, 26,82±0,10, 30,18±0,10 and 30,76±0,10.

9. Crystal modification 2 (polymorphic form I) monosodium salt of D-isoglutamine-D-tryptophan (1:1) according to claim 7, having a powder x-ray as shown in figure 3.

10. Crystal modification 2 (polymorphic form I) monosodium salt of D-isoglutamine-D-tryptophan according to claim 7, characterized by peaks in the powder x-ray with the following 2θ values: 9,65±0,20; 10,41±0,20; 11,2±0,20; 11,71±0,20; 13,45±0,20; 13,93±0,20; 14,44±0,20; 15,61±0,20; 17,01±0,20; 18,18±0,20; 18,65±0,20; 20,02±0,20; 20,85±0,20; 21,39±0,20; 21,73±0,20; 22,52±0,20; 23,27±0,20; 24,3±0,20; 25,84±0,20; 26,82±0,20; 28,49±0,20; 30,18±0,20; 30,76±0,20; 31,49±0,20; 33,03±0,20; 34,55±0,20; 34,97±0,20; 35,74±0,20; 37,25±0,20; 37,71±0,20; and 38,79±0,20.

11. Crystal modification 2 (polymorphic form I) monosodium salt of D-isoglutamine-D-tryptophan of claim 8, characterized by peaks in the powder x-ray with the following 2θ values: 9,65±0,10; 10,41±0,10; 11,2±0,10; 11,71±0,10; 13,45±0,10; 13,93±0,10; 14,44±0,10; 15,61±0,10; 17,01±0,10; 18,18±0,10; 18,65±0,10; 20,02±0,10; 20,85±0,10; 21,39±0,10; 21,73±0,10; 22,52±0,10; 23,27±0,10; 24,3±0,10; 25,84±0,10; 26,82±0,10; 28,49±0,10; 30,18±0,10; 30,76±0,10; 31,49±0,10; 33,03±0,10; 34,55±0,10; 34,97±0,10; 35,74±0,10; 37,25±0,10; 37,71±0,10; and 38,79±0,10.

12. Crystal modification 2 (polymorphic form I) monosodium salt of D-isoglutamine-D-tryptophan of claim 10 or 11, characterized by a powder x-ray is th, obtained using a diffractometer (copper cathode) and expressed in the parameters of the interplanar distance d, Bragg angle 2θ and relative intensity (expressed as percentage relative to the beam of greatest intensity):

4,876 2,839
2θ (°)D-distance (angstroms)Relative intensity (%)
9,659,1615,3
10,418,49223,7
11,207,89740,4
11,717,5494,5
13,45to 6.5890,2
13,936,35115,9
14,446,1283,7
15,615,67232,4
17,015,2079,9
18,1811,7
18,654,75547,8
20,024,43259,2
20,854,25735,9
21,394,1524,1
21,734,08627,3
22,523,945100
23,273,81913,7
24,303,6632,4
25,843,44569,5
26,823,32282,5
28,493,1330,1
30,182,95958,8
30,762,90486,9
31,4935,3
33,032,718,7
34,55at 2,59417,8
34,972,56443,4
35,74of 2.518,5
37,252,41228,1
37,712,38328,5
38,792,31916,9

13. Crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan (1:1), characterized by peaks in the powder x-ray with the following 2θ values: 12,239±0,200, 18,268±0,200, 22,268±0,200, 26,257±0,200 and 27,807±0,200.

14. Crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan (1:1) in item 13, characterized by peaks in the powder x-ray with the following 2θ values: 12,239±0,100, 18,268±0,100, 22,268±0,100, 26,257±0,100 and 27,807±0,100.

15. Crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan (1:1) in item 13, the ima is based on powder x-ray, as shown in figure 4.

16. Crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan indicated in paragraph 13, characterized by peaks in the powder x-ray with the following 2θ values: 9,187±0,200; 11,058±0,200; 11,713±0,200; 12,239±0,200; 13,785±0,200; 14,806±0,200; 15,763±0,200; 17,126±0,200; 17,693±0,200; 18,268±0,200; 18,562±0,200; 19,261±0,200; 20,033±0,200; 20,63±0,200; 21,006±0,200; 21,778±0,200; 22,268±0,200; 23,054±0,200; 23,361±0,200; 23,851±0,200; 24,626±0,200; 24,981±0,200; 25,507±0,200; 26,257±0,200; 26,963±0,200; 27,329±0,200; 27,807±0,200; 28,243±0,200; 28,975±0,200; 29,264±0,200; 29,687±0,200; 30,409±0,200; 30,798±0,200; 31,193±0,200; 31,724±0,200; 32,505±0,200; 32,985±0,200; 33,645±0,200; 34,249±0,200; 34,587±0,200; 35,048±0,200; 35,41±0,200; 35,933±0,200; 36,833±0,200; 37,276±0,200; 37,937±0,200; 38,467±0,200; and 39±0,200.

17. Crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan through 14, characterized by peaks in the powder x-ray with the following 2θ values: 9,187±0,100; 11,058±0,100; 11,713±0,100; 12,239±0,100; 13,785±0,100; 14,806±0,100; 15,763±0,100; 17,126±0,100, 17,693±0,100; 18,268±0,100; 18,562±0,100; 19,261±0,100; 20,033±0,100; 20,63±0,100; 21,006±0,100; 21,778±0,100; 22,268±0,100; 23,054±0,100; 23,361±0,100; 23,851±0,100; 24,626±0,100; 24,981±0,100; 25,507±0,100; 26,257±0,100; 26,963±0,100; 27,329±0,100; 27,807±0,100; 28,243±0,100; 28,975±0,100; 29,264±0,100; 29,687±0,100; 30,409±0,100; 30,798±0,100; 31,193±0,100; 31,724±0,100; 32,505±0,100; 32,985±0,100; 33,645±0,100; 34,249±0,100; 34,587±0,100; 35,048±0,100; 35,41±0,100; 35,933±0,100; 36,833±0,100; 37,276±0,100; 37,937±0,100; 38,467±0,100; and 39±0,100.

18. Crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan p the item 16 or 17, characterized by powder x-ray obtained using a diffractometer (copper cathode) and expressed in the parameters of the interplanar distance d, Bragg angle 2θ and relative intensity (expressed as percentage relative to the beam of greatest intensity):

td align="center"> 2,713
2θ (°)D-distance (angstroms)Relative intensity (%)
9,1879,61825,4
11,0587,9952,3
11,7137,54918,7
12,2397,22634,2
13,7856,41923,5
14,8065,97813
15,7635,6185
17,1265,17329,3
17,6935,009 8,4
18,2684,85248,2
18,5624,77628,2
19,2614,60414,3
20,0334,42914,5
20,6304,30217,2
21,0064,22612
21,7784,0782,4
22,2683,989100
23,0543,8556,4
23,3613,8057,4
23,8513,7281,8
24,6263,61214,9
24,9813,56214,7
25,5073,489 11,1
26,2573,39134,3
26,9633,30411,1
27,3293,26120,6
27,8073,20635
28,2433,15725,6
28,9753,0791,1
29,2643,0492,3
29,6873,0079,5
30,4092,93720,9
30,7982,9016,1
31,1932,8656,9
31,7242,81824,7
32,5052,7528
32,98512,1
33,6452,66226,5
34,2492,61615,2
34,5872,5918,6
35,0482,5584,6
35,4102,5333,3
35,9332,49715,3
36,8332,4388
37,2762,4110,8
37,9372,37a 12.7
38,4672,33814,7
39,0002,3082,6

19. A mixture of crystal modification 1 (polymorphic form F) and crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan (1:1), where the mixture is characterized by peaks on oroscopogratisonline, has the following 2θ values: 18,79±0,20, 21,30±0,20, 22,33±0,20, 22,90±0,20, 24,96±0,20 and 27,48±0,20.

20. A mixture of crystal modification 1 (polymorphic form F) and crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan (1:1) according to claim 19, where the mixture is characterized by peaks in the powder x-ray with the following 2θ values: 18,79±0,10, 21,30±0,10, 22,33±0,10, 22,90±0,10, 24,96±0,10 and 27,48±0,10.

21. A mixture of crystal modification 1 (polymorphic form F) and crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan (1:1) according to claim 19, having a powder x-ray as shown in Fig.9.

22. The crystalline mono-sodium salt of D-isoglutamine-D-tryptophan of claim 1 to 21, in which the crystals when measured by x-ray diffraction give distinct peaks whose width at half height, measured at an angle of 2θ reflection and using CuK radiationαis less than 2°.

23. A method of obtaining a crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan (1:1), comprising the following stages:
(a) obtaining a solution of D-isoglutamine-D-tryptophan and sodium hydroxide in water with a pH from about 6.5 to about 7.2;
(b) filtering the solution to remove solid particles;
(C) evaporation of water for concentration of the filtrate and
(g) adding antipasto is Italia for deposition of monosodium salt of D-isoglutamine-D-tryptophan, and
(d) a solid substance obtained after stage (g), stirred with ethyl acetate to obtain the crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan.

24. A method of obtaining a crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan (1:1), comprising the following stages:
(a) obtaining a solution of D-isoglutamine-D-tryptophan and sodium hydroxide in water with a pH from about 6.5 to about 7.2;
(b) filtering the solution to remove solid particles;
(C) evaporation of water for concentration of the filtrate and
(d) adding about 30 ml of antibacterial 1 ml of sodium salt of D-isoglutamine-D-tryptophan in water to precipitate a crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan.

25. A method of obtaining a crystal modification 2 (polymorphic form I) of the monosodium salt of D-isoglutamine-D-tryptophan (1:1), comprising the following stages:
(a) obtaining a solution of D-isoglutamine-D-tryptophan and sodium hydroxide in water with a pH from about 6.5 to about 7.2;
(b) filtering the solution to remove solid particles;
(C) evaporation of water for concentration of the filtrate and
(g) add approximately 40 ml of antibacterial 1 ml of sodium salt of D-isoglutamine-D-tryptophan in water to precipitate crystalline mo is eficacia 2 (polymorphic form III) of the monosodium salt of D-isoglutamine-D-tryptophan.

26. A method of obtaining a crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan (1:1), comprising the following stages:
(a) obtaining a solution of D-isoglutamine-D-tryptophan and sodium hydroxide in water with a pH from about 6.5 to about 7.2;
(b) filtering the solution to remove solid particles;
(C) evaporation of the filtrate from step (b) obtaining a solid substance;
(g) adding water to obtain a solution of the monosodium salt of D-isoglutamine-D-tryptophan and
(d) evaporation of water for more than 5 h to obtain the crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan.

27. A method of obtaining a crystal modification 3 (polymorphic form X) of the monosodium salt of D-isoglutamine-D-tryptophan (1:1), comprising the following stages:
(a) obtaining a solution of D-isoglutamine-D-tryptophan in methanol;
(b) filtering the solution to remove solid particles and
(C) adding antibacterial for deposition of monosodium salt of D-isoglutamine-D-tryptophan.

28. A method of obtaining a crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan (1:1), comprising the following stages:
(a) obtaining a solution of the monosodium salt of D-isoglutamine-D-tryptophan and sodium hydroxide in water;
(b) filtering the solution to remove solid the x particles;
(C) evaporation of water for concentration of the filtrate and
(g) adding antibacterial for deposition of crystal modification 1 (polymorphic form F) monosodium salt of D-isoglutamine-D-tryptophan.

29. The method according to paragraph 24, where the duration of mixing on stage (d) is from about 1.5 to 16 hours

30. The method according A.25, where the duration of mixing on stage (g) is approximately 1 hour

31. The method according to item 23, where the duration of mixing on stage (d) is approximately 1 h, and duration of stirring in ethyl acetate at stage (d) is approximately 2.5 hours

32. The method according to p, where at stage (g) add approximately 18 to 22 ml of water per 1 g of monosodium salt of D-isoglutamine-D-tryptophan, and the duration of the evaporation stage (d) is approximately 5 to 6 hours at a temperature of evaporation from approximately 30 to 35°C.

33. The method according to item 27, where the ratio of the mono-sodium salt of D-isoglutamine-D-tryptophan to methanol at the stage (a) is 1 g of salt per 11-13 ml of methanol, and antibacterial to methanol at the stage (b) is about 0.4-0.6 ml 1 ml

34. The method according to p, where the duration of mixing on stage (g) is approximately 12 to 16 hours

35. The method according to any of PP-34, where antibacterial represents a C1-C4 alkanol.

36. The method according to p, where C1-C4alkanol represents isopropanol.

37. The pharmaceutical composition used as immunosuppressant containing an effective amount of the crystalline mono-sodium salt of D-isoglutamine-D-tryptophan according to any one of claims 1 to 22 and at least one pharmaceutically acceptable carrier.

38. A method of obtaining a pharmaceutical composition that is used as immunosuppressant, comprising combining an effective amount of the crystalline mono-sodium salt of D-isoglutamine-D-tryptophan according to any one of claims 1 to 22 with at least one pharmaceutically acceptable carrier.

39. The use of crystalline mono-sodium salt of D-isoglutamine-D-tryptophan according to any one of claims 1 to 22 without chromatographic purification in the pharmaceutical compositions.

40. The use of crystalline mono-sodium salt of D-isoglutamine-D-tryptophan according to any one of claims 1 to 22 as a tool against psoriasis.

41. The use of crystalline mono-sodium salt of D-isoglutamine-D-tryptophan according to any one of claims 1 to 22 for the manufacture of a medicinal product for treating at least one autoimmune diseases.

42. The application of paragraph 41, where at least one autoimmune disease selected from the group consisting of psoriasis, atopic dermatitis and rheumatoid arthritis is one.



 

Same patents:

FIELD: chemistry.

SUBSTANCE: disclosed is a method of producing lipodipeptides based on L-glutamic acid or L-glutamine and L-ornithine, L-lysine or L-arginine. L-glutamic acid or L-glutamine derivatives esterified with fatty alcohol residues are obtained by fusing an amino acid with a corresponding alcohol in the presence of a strongly acidic ion-exchange resin in H+ form. Amino groups of L-ornithine, L-lysine or L-arginine are protected and then activated with carboxyl groups. Further, a reaction takes place between esterified derivatives of L-glutamic acid or L-glutamine and the protected derivatives of L-ornithine, L-lysine or L-arginine to form lipodipeptides. The protective groups are then removed.

EFFECT: invention simplifies the process at the esterification step, reduces reaction temperature and reaction time to 2 hours.

3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to novel polyfunctional fullerene C60 amino acid derivatives of formula (1) , wherein R is H, mono- or dinitroxyC1-6alkyl, maleinimide; N-Z denotes a α, β, γ, ω-amino acid fragment of general formula where m=2-5 and M is a nitroxyC1-6alkyl group, a C1-6alkyl group or an alkali metal salt, having biological activity, as well as methods for production thereof and a method for covalent bonding of fullerene derivatives with SH-containing proteins. The invention also relates to the use of nitroxyalkyl-N-(fullerenyl)amino acids as nitrogen monoxide donors and to use of nitroxyalkyl-N-(fullerenyl)amino acids as quick-acting vasodilatators for antihypertensive therapy. The invention also relates to a method of inhibiting a metastasis process and a method of enhancing antileukemic activity of cyclophosphamide. Disclosed nitroxyalkyl-N-fullerenyl amino acid derivatives have an effect on coronary, contractile and pumping ability of the isolated heart of Vistar rats and are quick-acting vasodilatators which reduce arterial pressure and heart rate and cause relaxation of coronary vessel with less depressive effect on myocardial function compared to nitroglycerine.

EFFECT: disclosed compounds considerably intensify antileukemic activity of cyclophosphamide, increase chemosensitising activity when combined with cyclophosphamide.

9 cl, 8 ex, 3 tbl, 3 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to a 1-(1-adamantyl)ethylamine derivative (remantadin) of general formula: wherein R is a functional group of the amino acid residue (I-IV) or the lipoic acid residue (V). (I), (II), (III), (IV), (V) which possess selective antiviral activity in relation to the influenza A strains, including the viral strains resistant to action of remantadin. The compounds wherein R means the groups (l),(ll),(lll) and (V) are novel.

EFFECT: prepared compounds may be promising as substances as a part of the antiviral drugs and applicable for creating new antiviral drugs.

2 cl, 2 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: in claim described are organic compounds of formula I where radicals are given in description, which are applicable for elimination, prevention or alleviation of one or more symptoms, associated with HCV disorders.

EFFECT: obtaining pharmaceutical composition which possesses inhibiting activity with respect to NS3-4 HCV serinprotease, including formula I compound and pharmaceutically acceptable carrier.

30 cl, 25 ex, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to a compound of formula I

and pharmaceutical compositions containing them and used for IAP-function modulation by inhibiting the binding of BIR-binding protein and BIR domain of IAP and for treating proliferative diseases such as cancer. What is also disclosed is a compound of formula I marked by a detectable tag and an affinity tag which is applicable as a probe for identifying the compounds bound with BIR domain of IAP.

EFFECT: preparing the compounds used for IAP-function modulation.

33 cl, 4 tbl, 1 ex

FIELD: chemistry.

SUBSTANCE: invention relates to methods for synthesis of nonapeptide ethylamide, having strong LH-RH/FSH-RH activity, of formula pGlu-His-Trp-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NH-C2H5·2AcOH (I), and intermediate compounds for synthesis thereof. The nonapeptide ethylamide is obtained via condensation of a C-terminal tetrapeptide of formula H-D-Ser(But)-Leu-Arg-Pro-NH-C2H5·HCl (II) with a dipeptide of formula: X-Ser-Tyr-OH (IV), where X is a protective group. The obtained N-substituted hexapeptide ethylamide of formula X-Ser-Tyr-D-Ser(But)-Leu-Arg-Pro-NH-C2H5·HCl (III) is treated with an unblocking agent to remove the N-protective group, and then condensed with a tripeptide of formula pGlu-His-Trp-OH·HCl (V) and the end product is purified through chromatography and extracted in form of a monoacetate salt.

EFFECT: high output.

4 cl, 1 ex

FIELD: chemistry.

SUBSTANCE: synthesis is carried out using a liquid-phase technique via condensation of pentafluorophenyl ether of Nα-benzloxycarbonyl-Nε-tert-butoxycarbonyl-L-lysine with methyl ether of O-tert-butyl-L-threonine in the presence of N-methylmorpholine in ethyl acetate medium and followed by hydrogenation of the obtained compound with hydrogen in methyl alcohol using palladium hydroxide as a catalyst. Purification of the desired protected dipeptide is carried out using a salt with oxalic acid. The initial pentafluorophenyl ether is obtained from reaction of Nα-benzyloxycarbonyl-Nε-tert-butoxycarbonyl-L-lysine with pentafluorophenol and is then used without extraction. The disclosed method enables to obtain the desired product with output of over 96% and high degree of purity.

EFFECT: obtained protected dipeptide can be used to produce octreotide or analogues thereof, or other peptides containing a L-lysine-L-threonine fragment.

2 ex

FIELD: chemistry.

SUBSTANCE: invention describes a method of producing cyclic depsipeptides of formula (I) via intramolecular cyclisation.

EFFECT: improved method.

14 cl, 31 ex

Iap inhibitors // 2425838

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula

, which can inhibit binding of protein Smac with apoptosis protein inhibitor (IAP).

EFFECT: improved properties of the inhibitor.

4 cl, 198 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to compounds of formula

, as well as to application of such compounds for preparing a drug which is administered for treatment, relief or prevention of a condition associated with canal activating protease, such as prostasin or trypsin.

-J-(R10)p = or .

EFFECT: preparing the pharmaceutical compositions which exhibit prostasin or trypsin inhibiting activity.

13 cl, 2 tbl, 53 ex

The invention relates to products derived from histamine and, in particular, the condensation products of histamine or methylsiloxanes histamine and amino acids, the method of their preparation and use as active principle in areas such as therapy and cosmetology, as well as the factor (agent), improving the stability of compositions used in therapy, cosmetology, agriculture and food industry (region)

The invention relates to medicine, namely to new peptide structures with immunomodulatory properties, and preparations on their basis

The invention relates to medicine, namely to compounds having immunomodulatory properties

FIELD: chemistry.

SUBSTANCE: present invention relates to a method of purifying mono-pegylated erythropoietin using two cation-exchange chromatography steps, where the same type of cationite is used at both cation-exchange chromatography steps.

EFFECT: method of producing mono-pegylated erythropoietin in a substantially homogeneous form.

17 cl, 3 dwg, 2 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of purifying antibodies by adding a negatively charged polyelectrolyte such as polyvinyl sulphonic acid, polyvinyl sulphonate, polystyrene sulphonic acid or polyacrylic acid, to a mixture containing an antibody and extracting the obtained precipitate containing the antibody. The invention can further be used in extracting antibodies from body fluids and purification thereof.

EFFECT: improved method.

13 cl, 13 ex, 13 tbl, 38 dwg

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, particularly a method for production and purification of human recombinant growth hormone (rHGH). The presented method for production and purification of rHGH involves dissolution of inclusion bodies in 2 M urea at pH=11.0 followed by renaturation in a buffer solution 20 mM "Трис"-HCl at pH=8.0. Hydrogen peroxide is used as an rHGH sulphhydryl group oxidiser. N-terminal methionine is split from leucinic aminopeptidase. Chromatographic purification of rHGH is enabled with two-stage ion-exchange chromatography on the sorbent Q Sepharose FF and the stage of purification by hydrophobic chromatography on the sorbent Butyl Sepharose 4 FF. Gel filtration on Sephadex G-25 and ion-exchange chromatography on Q Sepharose FF, pH=6.5. It is followed by ion-exchange chromatography on Sephacryl S-100HR.

EFFECT: invention enables producing a preparation of high-purity rHGH protein of high compendial grade applicable for preparing drug preparations.

3 dwg, 2 tbl, 4 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention concerns biochemistry and medicine area. What is presented is a visualisation agent representing a conjugate of structure I as described in the patent claim. The visualisation agent refers to marked cMet-binding peptides. These peptides include a mark with an optical reporter group applicable for visualisation in vivo with the use of light within the range of wave length in the spectrum of 600-1200 nm. There are also presented a pharmaceutical composition for optical visualisation, containing the visualisation agent, a kit for preparing it and a method for optical visualization of a mammalian body in vivo. What is also presented is a method for managing the patients suffering colorectal cancer, involving the stage of optical visualisation in vivo.

EFFECT: presented visualisation agent possess higher cMet-binding affinity and selective cell targeting in vivo.

28 cl, 2 tbl, 7 ex

FIELD: chemistry.

SUBSTANCE: invention discloses versions of a method of cleaning a liquid composition which contains recombinant factor VII from viruses. The composition to be cleaned contains at least 5% recombinant polypeptide of factor VII in activated form. Cleaning is carried out using a nanofilter with pore size of at most 80 nm.

EFFECT: methods enable to obtain a clean recombinant polypeptide of factor VII without breakdown of its active form.

34 cl, 1 dwg, 6 ex

FIELD: medicine.

SUBSTANCE: what is presented is a method of preparative recovery of basic proteins from supramolecular structures of Escherichia coli growing population. The Escherichia coli cells are preserved in buffered 80-90% glycerol at -25°C. Then the sediment cells are washed in 3% triton X-100. It is followed by sediment extraction in salts of increasing concentrations: 0.14 M, 0.35 M; 2 M NaCl, 6 M in guanidine hydrochloride with 0.1% β-mercaptoethanol. Basic proteins are recovered from the prepared fractions by means of ion-exchange chromatography with amberlite resin IRC-50 of discontinuous gradient of guanidine hydrochloride: 6%, 8.9%, 10.6%, 13% on 0.1 M potassium phosphate buffer pH 6.8.

EFFECT: method enables producing fractions enriched by basic proteins with the use of a microamount of protein of Escherichia coli cell suprastructures.

7 dwg, 1 ex

Up!