Conjugates of hemoglobin with polysaccharide

 

The invention relates to biologically compatible carriers of oxygen for administration to patients as a Supplement or partial substitution of whole blood. Conjugates of hemoglobin, which can be used as carriers of oxygen on the basis of hemoglobin, is obtained through the reaction of hemoglobin with polysaccharides open in the oxidation of a ring, such as gidroxiatilkrahmal or dextran, and storing the resulting conjugate under conditions that allow it after conjugation to transform into a product with a lower molecular weight. Then the conjugate is subjected to reductive stabilization for the formation of secondary aminokwasy between hemoglobin and polysaccharide and is made in the composition as a carrier of oxygen on the basis of hemoglobin. Effect: method provides receiving a new conjugate polysaccharide-hemoglobin. 2 C. and 43 C.p. f-crystals, 12 tab., table 1.

This invention relates to biologically compatible carriers of oxygen for administration to patients as a Supplement or partial substitution of whole blood. More specifically, the invention relates to vectors kikori and to methods for their preparation.

Hemoglobin as a natural component of blood is to transport oxygen, is an obvious candidate for education foundations Deputy of blood, for example, in the form of an aqueous solution. In attempts to obtain a satisfactory solution of hemoglobin, acting as Deputy of blood, was conducted extensive research work, the results of which were published. However, the chemical properties of hemoglobin outside of erythrocytes differ significantly from its properties inside red blood cells, for example, with respect to its affinity with oxygen. For a long time recognized the need for some form of chemical modification of hemoglobin, in order to make it suitable for use as a Deputy of blood, and in this direction was conducted fairly intensive research.

It is well known that hemoglobin comprises a tetramer of four subunits, namely two-subunit, each of which has a peptide chain globin and two-subunit, each of which has a peptide chain of the globin. The tetramer has a molecular weight of approximately 64 kDa, and each subunit has approximately ="https://img.russianpatents.com/chr/945.gif">--dimers and later in the same conditions - even to-subunit monomers and-subunit monomers. Dimers and monomers have too low a molecular weight to hold in the circulatory system of the body and filtered by the kidneys for excretion in the urine. This leads to unacceptably short half-life of this product from the body. Was previously recognized beneficial effect of chemical binding between the subunits to ensure tetramer forms ("intramolecular cross-stitching). Desirable in many cases, it also recognized the linking together of two or more tetramer units for the formation of oligomers and polymers of hemoglobin with a molecular mass of more than 64 kDa ("intermolecular cross-stitching").

Accordingly, one approach to developing NVOS for clinical use was in intramolecular cross-linking of units of hemoglobin in stable tetramer with a molecular weight of about 64 kDa and not necessarily in the oligomerization of these tetramers in oligomers of 2-6 such tetramers using intermolecular cross-linking. For this purpose have been proposed raffinose (for example, U.S. patent 4857636 issued Hsia, and U.S. patent 5532352 issued Pliura et A1.), bifunctional imidate, such as diethylmalonate hydrochloride (U.S. patent 3925344 issued by Muzur), a halogenated triazine, diphenylsulfone, diisocyanates, glutaraldehyde and other dialdehyde (U.S. patent 4001200 issued by Bonsen et al.), bis-desirenature (U.S. patent 5529719 issued Tue), bis - and Tris-acylphosphate (U.S. patent 5250665 issued by Kluger et al.) and others.

Another approach to obtaining NVOS with the appropriate molecular weight for clinical use was in connection with hemoglobin biocompatible polysaccharide. Such conjugates would have the advantage in comparison with the cross stitched and oligomerization the hemoglobins, requiring smaller quantities of hemoglobin per unit NVOS, and therefore receiving them would be more economical and would reduce associated with hemoglobin toxicity. Algae colloid with hemoglobin in obtaining NVOS also provides the ability to control the liquid properties such as viscosity and colloid-osmotic pressure, by adjusting the size of the colloid, the degree of modification and the ratio between the colloid and hemoglobin. These same indicators can be used to regulate data Wond, the proposed receipt of blood or Deputy filler blood using chemical compounds of hemoglobin with a polysaccharide material selected from dextran and hydroxyethylamine with a molecular weight of approximately from 5 to 2000 kDa. However, in this patent provides only an example of the application of dextran.

In article Baldwin et al. , "Tetrahedron" 37, PP. 1723-1726 (1981), "Synthesis of Polymer-Bound Hemoglobin Samples described chemical modification of dextran and hydroxyethylamine (HES) for the formation of polymers of substituted aldehydes and their subsequent reaction with hemoglobin to form a soluble hemoglobin associated with the polymer. Although educated so the products were capable of binding oxygen, report them unsuitable for use as substituents blood, because their curves binding of oxygen were significantly shifted to the left, indicating that they have too high affinity for oxygen (too low R50).

The present invention is to provide a new NVOS.

Another object of the invention is the provision of a new conjugate polysaccharide-hemoglobin, which can be used as NVOS.

Another object of the invention is to accommodat the C.

In the method of the present invention uses a polysaccharide in the form of a ring which is open during the oxidation process. In this oxidative form at least part of the Monomeric units of the saccharide oxidized, introducing an aldehyde group. The so formed oxidized polysaccharide then reacts with the extracellular hemoglobin, the hemoglobin by the primary amine groups of the globin chains that reacts with the aldehyde groups of the oxidized polysaccharide covalently bound to the polysaccharide via a Schiff's base linkages. Original and very quickly to a large extent formed product, which includes species with high molecular mass of about 500 kDa or higher, and with a wide distribution of molecular weight (128->500 kDa).

While maintaining this product under appropriate conditions in aqueous solution it can in a controlled extent in a relatively short period (for example, 4-48 hours, depending on conditions) to be transformed into a product with a much lower molecular weight (90-200 kDa) and with a much more narrow distribution of molecular masses. It turns out that this product after chemical recovery to restore relations core is about to oxygen, in the range P50from 4 to 50 mm RT. Art. at 37oWith, depending on ligand-state hemoglobin during conjugation, which makes it highly suitable as a candidate in carriers of oxygen on the basis of hemoglobin for clinical use in mammals. The degree of transformation can be controlled by determining the time of application of the recovery phase. In addition, the resulting product does not contain unreacted detected hemoglobin, which in the case of presence are subject to dissociation with formation-dimers, causing, as suggested, kidney damage, and does not contain detectable quantities of products with excessively high molecular weight (approximately more than 500-600 kDa).

Thus, in accordance with the first aspect of the present invention provides a conjugate, a polysaccharide-hemoglobin, which can be used as a carrier of oxygen on the basis of hemoglobin and which has affinity for oxygen, expressed as partial pressure oxygen environment required to maintain 50% oxygen saturation, R50= 4-50 mm RT. Art. at 37oWith, and does not contain molecular weight above about 500 kDa, moreover, these conjugates obtained by the reaction of hemoglobin with oxidized polysaccharide to form the complex conjugates with high molecular weight, allowing complex macromolecular conjugates to disintegrate when stored in solution at a suitable pH value, easily determined by simple experiments, and form specified conjugate polysaccharide-hemoglobin at a temperature of from 2oWith up to approximately 45oC.

Another aspect of the invention relates to a conjugate, a polysaccharide-hemoglobin, which can be used as a carrier of oxygen, including hemoglobin, covalently linked through a secondary amine linkages of amino groups on the hemoglobin with the remnants of the aldehyde groups on the polysaccharide, and these aldehyde groups formed by disclosure by the oxidation of ring sharidny Monomeric units of the polysaccharide.

In accordance with another aspect of the present invention relates to a method of obtaining a carrier of oxygen on the basis of hemoglobin, which comprises carrying out the reaction between the subject disclosure by the oxidation of ring a polysaccharide bearing aldehyde groups, and hemoglobin with the formation of konut to reduce the molecular weight of the conjugate, stabilizing the conjugate by restoring ties Schiff's base to a stable secondary amine linkages, and removing the solution formed thereby conjugate polysaccharide-hemoglobin, which has no unbound detectable residue of hemoglobin and has no detectable residue of a product with a molecular weight of approximately 500 to 600 kDa.

A brief description of the drawings Fig. 1, 2 and 3 are sets of chromatograms of products below example 2.

Fig.4 represents the analysis by gel-filtration products are presented below in example 3.

Fig.5 is a similar set of chromatograms of products below example 6.

Fig.6 is a similar set of chromatograms of products below example 7.

Fig. 7 and 8 represent the same sets of chromatograms of products below example 8.

Fig. 9 is a similar set of chromatograms illustrating the results are presented below in example 10.

Fig. 10, 11 and 12 represent the same sets of chromatograms of products below example 11.

The apparent molecular weight described in this application was received poekoelan mass, obtained by the method of gel filtration. Since the conjugates of hemoglobin-colloid, as expected, contain significant amounts of trapped water in the large component colloidal chains, the actual molecular weight of the conjugate, comprising only molecules covalently associated with hemoglobin less than the apparent molecular mass. However, it is apparent molecular mass, or excluded amount, will determine the retention time of the conjugate in vivo and, thus, the above-described molecular weight will be used to describe these conjugates.

Haemoglobin designed for use in the method of the present invention, preferably is a human hemoglobin derived from erythrocytes. However, the invention is applicable also to other types of hemoglobin for education foundations Deputy of blood, such as hemoglobin animals, especially bovine hemoglobin, porcine hemoglobin and the like, and hemoglobin, derived from cell culture. Currently, the human hemoglobin is preferred to form the basis of the Deputy of blood for the introduction of man.

Hemoglobin for use in the present invention may be allocated and cellular fragments and stroma removed from them using standard centrifugation techniques, filtering and the like. Preferably use a solution of hemoglobin concentration from 2 to 20 wt. % of hemoglobin to yield a product having the most desirable composition and combination of properties. Final purification can be performed using chromatography. Successfully use the method of pressurization adsorption chromatography, described in U.S. patent 5439591 issued Pliura et al.

Hemoglobin may naturally exist in the dense (T) conformation, which usually takes deoxyhemoglobin, or relaxed (R) conformation, which usually takes the oxyhemoglobin or monooksighenaznuu hemoglobin. Characteristics of the binding of oxygen by hemoglobin in the state of T are more desirable characteristics, because the affinity to oxygen in this conformation allows for efficient binding of oxygen in the vascular system of the lungs and delivery of oxygen to peripheral tissues. Accordingly, in the method according to the invention, it is preferable to apply deoxyhemoglobin. After conjugation with hydroxyethylcellulose (HES) with prior cross-stitching or without her deoxyhemoglobin retains the characteristics of the binding of oxygen configuration So However, if the persons in accordance with the invention stabilizes hemoglobin in the configuration R. A mixture of hemoglobins in state R and T can react with HES to obtain products with the properties of binding oxygen, intermediate between the properties of the configuration state of R, and So

In accordance with known techniques, deoxygenation hemoglobin to form deoxyhemoglobin preferably carried out by exposing a solution of hemoglobin processing deoxygenating gas, such as nitrogen. It is preferable to continue processing a stream of nitrogen, followed by the respective degassing over a long enough period of time to thus achieve a complete transformation of deoxyhemoglobin.

Polysaccharides that can be used in the present invention include polysaccharides with established biological compatibility and having sacharine Monomeric units capable of ring opening oxidation for the formation of reactive aldehyde groups. They include starches and starch derivatives, dextran, inulin, and the like. Among the preferred polysaccharides for use in the present invention are starch and dextran, with the most preferred HES.

Hemoglobin can react with oxidized hydroxy is, Lucaya the adducts from 64 to <500 kDa. When used in its cross stitched form the preferred cross linking reagent to obtain cross stitched and cross stitched oligomerization hemoglobin is polyallelic obtained as a result of a disclosure by the oxidation of ring oligosaccharide, such as raffinose (i.e., o-raffinose). A suitable method of obtaining o-raffinose and its reaction with hemoglobin described in the aforementioned U.S. patent 5532352 issued Pliura et al., description of which is included here as a reference. Although o-raffinose is a preferred cross linking reagent for use in this embodiment of the invention, it is by no means limited to it. To successfully can use any other known reagents and cross-linking b, such as the previously mentioned reagents, such as trimethylsilylmethyl (TMR), described in U.S. patent 5250665 issued by Kluger et al.

A suitable molecular weight of the source material hydroxyethylamine for use in preferred embodiments of the present invention is from about 70 to 1000 kDa. It is available in various types and varieties. To apply this to issuingca, essentially, any of the currently available for sale varieties HES, provided that they have a molecular weight approximately equal to the above. Particularly suitable varieties with a ratio of substitution (i.e., the number expressing the ratio hydroksyetylowy groups in the glucose units), constituting approximately from 0.5 to 0.7.

To obtain HES for use in the present invention it oxidizes, so as to create therein a significant amount of aldehyde groups. This can be done using different methods of oxidation, and it is preferable reaction with periodate (sodium or potassium). This reaction can occur in aqueous solution at low temperature, for example 0 to 5oWith, using the appropriate number of periodate sodium, selected in accordance with the desired degree of oxidation. The reaction is terminated after approximately 1-4 hours To remove unwanted salts and components HES with low molecular weight can be used ultrafiltration or dialysis, thus presenting a means of adjustment of the range of molecular masses of oxidized HES, which will be pairing ties with b. Oxidized HES moeetsa in water for conjugation with hemoglobin.

The conjugation reaction can occur in aqueous solution. Hemoglobin may not necessarily be a cross stitched b and/or oligomerization b. It can be in the form of a ligand, for example, carbon monoxide (CO-Hb). Lower values of R50the final products are obtained with CO-Hb, higher value - deoxy-b. The molar ratio b: oxidized HES can be concluded approximately in the range of about 0.25:1 to 5:1, but preferably are in the approximate range from 0.5:1 to 3:1. The reaction works best at an alkaline pH, for example in the range from 7.5 to 9, and at room temperature.

If the analysis reveals that regardless of the molecular mass of the initial polysaccharide originally formed by the reaction product, i.e., after approximately 1 h, has a very high molecular weight components having a molecular weight far greater than 500 kDa. This initial product also contains products with a wide range of molecular weight. Cause effect a controlled reduction of the molecular weight of the product to the product does not contain components with a molecular weight above about 500000 and up product with a narrow distribution of molecular Azania product in aqueous solution, preferably in the approximate range of pH from 7.2 to 10 and at room temperature (15-30oC) or close to it within a period of time approximately 48 hours There is very little residual species with a molecular mass of 32 kDa, if any remain. The number of species with a molecular mass of 32 kDa was so little that there is no need for special steps to remove them.

The so formed conjugate must be stabilized by restoring the (reversible) Schiff's base linkages between b and HES in stable secondary amine of communication and with the recovery of any unreacted aldehyde groups. Recovery can be carried out in one stage, at which the links of the Schiff's base and aldehyde groups are recovered in a single step or in two separate phases. At one stage effective will be a powerful rejuvenating tools, and less powerful rejuvenating tools require a two-step process.

This recovery step is preferably used as a means of adjusting the molecular weight and molecular weight distribution of the final product by choosing for it the appropriate time. Once the restore is complete the lar mass or molecular weight distribution. Accordingly, the analysis of samples of the reaction product through the intervals of time allows you to set the time for the recovery phase to stabilize the product by selected characteristics.

The preferred choice as a reducing agent is dimethylamine. It has sufficient capacity for the implementation of both reactions recovery in a single step. Can also be used with other water-soluble reducing means from the lower alkylamino, including tert-butylamino, ammonium salt of boron; dimethylamino; trimethylamine; triethylamine and pyridinol, but are not limited to. Other recovery tools that can be used are cyanoborohydride sodium and borohydride sodium.

The most appropriate, if the recovery of Schiff bases formed during conjugation, and recover any residual unreacted aldehyde groups occurs in aqueous solution in the temperature range from 2 to 25oIn the period from 10 to 36 hours, preferably 24 hours, the Reaction mixture it is advisable to tabularity to pH 7-10, preferably up to 8.0 to 9.5. The molar ratio between the regenerating means and the amount minetree reducing agent to aldehyde groups, added to initiate cross-linking.

To remove residual products of low molecular weight, such as the remains of the decomposition of starch residues dimethyl-amibara, salts, residues buffer, and so on, it is preferable to use the final stage of diafiltration. Then the product can be mixed with a suitable filler for education NVOS.

Thus obtained conjugate exhibits extremely favorable properties for use as the basis NVOS. He exhibits a low affinity for oxygen (P50=20-50 mm RT. Art.) along with a narrow distribution of molecular mass of the product (RMP 100-200 kDa), in the absence of a detectable product with a molecular mass of 32 kDa in conditions that promote the dissociation-dimers, or with a molecular weight above about 500 kDa.

For storage prior to use, it is advisable to remove all the oxygen from the product to prevent oxidation. Deoxygenating the product can be stored under conditions that prevent the introduction of oxygen, or in a frozen state, or at higher temperatures. Oxygen can be introduced before the introduction or product, you can give the change before using. The product can be stored in a frozen state in the oxygenated form or at higher temperatures as long as the degree of oxidation will not be considered unacceptable.

Hereinafter the invention is described only for purposes of illustration, in the following specific, non-limiting examples.

Example 1. The production of oxidized hydroxyethylamine.

9.0 g of hydroxyethylamine with an average molecular mass (MM) 450 kDa, having a degree of substitution of hydroxyethyl 0,7, was dissolved in 90 ml of water. 0,49, and 0.98 and 1.96 g of metaperiodate sodium, representing respectively about 0.3, 0.6 and 1.2 EQ. periodate 1 mol adjacent diol present in HES, were added to separate aliquots of 30 ml of this solution. These quantities sufficient to provide approximately 30%, 60% and 100% oxidation available dolovich groups. After 4 h of reaction in the dark at 4oThe solutions were subjected to extensive dialysis against the chilled water using cut-off membrane, inhibiting compound with a molecular mass of 15 kDa. End detainees products liofilizirovanny in the white powder was stored at room temperature. An alternative solution is subjected to dialysis oxidized HES can be used f the th HES also prepared by direct oxidation HES, prepared in 0.9% NaCl. Measure consumption periodate and final content of aldehyde show that the range used periodate led to oxidation, from partial to full, all available dolovich groups and that the degree of oxidation is easily adjustable by changing the number of periodate.

Example 2. Obtaining conjugates with different oxidized HES and CO-hemoglobin.

Studied reaction-b with different relative ratios of oxidized HES (HES-CHO). Based on the expected content of the neighboring diol in HES was calculated equivalents of periodate for oxidation. In one case of 0.54 g of oxidized HES 450 kDa, obtained using 1.2 EQ. periodate as described in example 1, was dissolved in 3.0 ml of 100 mm HEPES buffer at pH 8.1. This solution HES-CHO was added in monooksighenaznuu hemoglobin (b, 200 mg/ml in water) in the following proportions: 0,76 ml of HES-CHO:0,041 ml b, 0,73: 0,078 and 0.61: of € 0.195, receiving a final concentration b respectively about 10, 20 and 50 mg/ml Reactions were carried out at 22-25oSince at pH 8, and samples were removed at different times to determine mm using a Pharmacia column Superdex 200 (1 x 30 cm), elyuirovaniya 0.5 M gl2+25 mm Tris with a pH of 7.2 at a speed of 0.4 ml/min In all three soothes the elution, compared with controls b MM with a maximum of 128 kDa and in the range exceeding the limit of the excluded volume of the column (b >500 kDa). In Fig. 1 shows the chromatogram obtained for a product with a ratio of Hb:HES of 0.5: 1 (50 mg Hb/ml), taken at different periods of time and in comparison with control b (lower curve). The dotted vertical line represents the elution unmodified-dimer 32 kDa. In eluruumid fractions was observed spectrum absorption at 414 nm, characteristic of hemoglobin. Over the next 30 h elution of the product was decreased, and were obtained species having the time of elution, comparable to the controls b with MM 128 kDa, which were not identified nor unmodified b, neither species that exceed the limit column. The type of evolution of MM and MM ranges of the final product were similar in all three ratios HES:Hb as and when HES oxidized 0.6 EQ. periodate. The conjugates obtained with the use of HES, oxidized 0.3 EQ. periodate on diol, usually contained a considerable quantity of material together aliremove with unmodifiedthe dimer. Therefore, to generate conjugates, free, obrazovannyh within the first few hours was lower when using fewer b. Similar reactions and results were obtained when using oxidized 200 kDa HES as described in example 1. Average MM of the final products was higher when using the HES 450 kDa, compared to the HES 200 kDa. In Fig.2 shows the chromatogram of the final products Hb+HES-CHO to HES 200/0,5 (broken line) and HES 450/0,7 (solid lines) when these various oxidation States, all at the ratios of Hb:HES-CHO 1:1.

Conjugates Hb-HES obtained using oxidized periodata HES 70 kDa (of 0.3, 0.6 and 1.2 EQ. periodate in comparison with the calculated diola) also formed a species with a higher MM during the early phase conjugation with subsequent transformation into a lower MM (Fig.3). Average MM conjugates early phase, and the time required to turn in species with lower MM, depended on the degree of oxidation HES 70. After 48 h of conjugation certain amount of material, together aliremove with unmodified component b 32 kDa control R-b remained in the conjugate obtained from the lowest degree of oxidation HES (0.3 EQ. periodate on the calculated diol). After 48 h was still a significant amount of material, elwira is certain HES 70 (1.2 EQ. periodate on the calculated diol). Within 48 h of reaction with HES, oxidized 0.6 EQ. periodate on the calculated diol was obtained a product that does not contain unmodified hemoglobin, and material eluruumis at the limit of the excluded volume of the column.

Example 3. Simultaneous large-scale obtaining conjugates Hb-HES high and low MM.

Two conjugate Hb-HES with different MM were obtained from a single reaction, which was isolated and stabilized portion of the product early conjugation with high MM, allowing the remaining product conjugation before stabilization to undergo transformation into a product with a lower MM. Comparison of physical properties and in vivo two products is carried out so as to demonstrate the favorable properties of one compared to the others.

944 g HES (200 kDa, degree of substitution of 0.5) was dissolved in 8 liters of water for injection (VDI), cooled at 4oWith, then added 370 g NaIO4and the mixture was stirred in the dark for 5,3 including All NaIO4dissolved in less than 1 h the Mixture was filtered (0.2 μm), and then subjected diafiltration against 12 volumes of FDI at room temperature using a membrane of regenerated cellulose with MM 30 kDa. the th the samples was determined, that the final concentration was 128 mg HES-SNO/ml 1.2 l b (23,2 g/DL, VDI) was combined with 2.0 l of 200 mm HEPES buffer at pH 8.1, then oxygenerator and desoxyribose by contact with O2then N2through the membrane of the hollow fibers. 4.5 kg of the solution of the HES-CHO (128 g/l) combined with deoksigenirovanii b (3,2 l at 9.0 g/DL) and the mixture is maintained under conditions of deoxygenation. RMP formed conjugates was monitored by gel-filtration.

After 3 h of conjugation half of the volume of the reaction mixture was transferred under the N2in a separate vessel and added 56 ml of 3M NaOAc and 196 g DMB, dissolved in 1.7 l VDI. The final ratio of DMB: initial aldehyde was 1.5:1. Before loading WITH and diafiltrate the mixture was kept under N2at ambient temperature for 23 hours After 29 h after initiation of the reaction conjugation Hb-HES the other half of the mixture in the course of 17.5 h similarly processed NaOAc and DMB. Then both restored DMB reaction mixture was loaded and subjected diafiltration against VDI, and then lactate ringer (approximately 10 volumes for each solution). Using Hcl 0.1 N. the pH was brought to 7.5 and 7.6. Both solution was concentrated to colloidal osmoticheskoe pressure was 80-100 mm RT. senior hydroxy Products is RME. The remaining quantity of each half was desoxyribose and Packed in deoksigenirovanii form. Oxygendemand products were stored at -80oWith, desoxyribose products - at 4oC. the Product with higher MM, obtained by using product recovery early stages of conjugation, hereinafter referred to as here HIMW HES-Hb. Product with lower MM, obtained by using product recovery late stage of conjugation, hereinafter referred to as here HIMW HES-Hb. The distribution of MM, estimated by gel filtration, as shown in Fig.4. Within 4 months of storage either at 4 or at -80oWith the distribution of MM did not change.

Colloid osmotic pressure (CODE) HIMW HES-Hb investigated at different concentrations was firmly higher than LOMW HES-Hb for both products (see table at the end of the description). The viscosity at the hemoglobin content of 6.5 and 9.0 g b/DL amounted to HIMW HES-Hb and LOMW HES-Hb, respectively, 8,9 and 3.0 cSt. So HIMW HES-Hb, which is comparable to the distribution of MM conjugates HES-Hb and dextran-b obtained by other authors, had colloidal properties, which, as would be expected, could lead to more change fluid and blood rheological properties than LOMW HES-Hb. Were described adverse vozdeistvia erythrocytes, and on blood clotting (Treib et al. Thrombosis and Haemostasis 74: 1452-6 (1995)).

Example 4. The influence of ligand States at finite P50.

b (55 mg/ml in water) oxygenerator and desoxyribose respectively through contact with oxygen and then nitrogen. HES 200 kDa oxygenerator using 0.6 EQ. periodate as described in example 1 was brought to a concentration of 60 mg/ml in 100 mm HEPES at pH 8.1, was degirolami and was purged with nitrogen. 2.5 ml of this oxidized HES solution was added to 0.8 ml deoxygenating solution b, providing 1 EQ. b on 1 mol of the original non-oxidized 200 kDa HES. After 48 h at 22-25oWith under nitrogen, the reaction mixture was brought to 0.3 M in sodium acetate was then added 3 EQ. dimethylamino on 1 mol of the initial aldehyde. After 24 h, the solution was loaded WITH gaseous and subjected to the full dialysis against a solution of lactate ringer. Carried out a similar procedure in which b without removal of the ligand WITH reacts with HES 200 kDa, oxidized 0.6 EQ. periodate. Properties of binding oxygen was measured for both products using a Hemox-Analyzer (TCS Instruments, Southhampton, Pennsylvania, U. S. A.) at 37oC. Conjugation deoxygenating b led to the end5026 mm RT. senior Conjugation b Pref the imposition of the transverse cross-linked hemoglobin.

HES 200 kDa oxygenerator with 0.3 EQ. and 0.6 EQ. periodate in separate reactions as described in example 1 and brought to a concentration of 125 mg/ml 270 mm sodium bicarbonate at pH 8.1. 3.0 ml of each oxygendemanding of HES solution was added to separate aliquots of 1.0 ml trimethyl-Tris-(methylphosphate) (TMR)-cross stitched b (cross stitched b 64 kDa, U.S. patent 5250665 issued by Kluger et al., 125 mg/ml in water) and similarly in aliquots of 1.0 ml b, polymerized o-raffinose (polymers b from 64 to <500 kDa, U.S. patent 5532352 issued Pliura et ai., 117 mg/l) for 4 reactions, providing in all cases, 1 EQ. Hb 1 mol of the original non-oxidized 200 kDa HES. Both products of hemoglobin presents in the form. After 30 h of reaction at 22-25oWith under gas was added sodium acetate to a final concentration of 0.3 M was Then added 3 EQ. dimethylaminoborane on 1 mol of the initial aldehyde. After 24 h the reaction mixture was subjected to dialysis (MWCO) against water and then the solution of lactate ringer's solution at pH 7.4. The properties of the bonding of oxygen was detected using Namah-Analyzer at 37oC.

The distribution of the MM all forms of Hb was shifted toward higher values. Using Hb and Hb, cross stitched TMR can be yousie conditions). Conjugates R-b contain significant amounts of material by volume of voids. P50(37o(C) amounted to: HES+CO-TM-b 5-7 mm RT. Art.; HES+CO-R-b 5-7 mm RT. Art. Both products showed the property cooperatively.

Example 6. Change the time and temperature of reaction.

The impact of shorter reaction time and lower temperature (12 versus 22o(C) the distribution of MM b-HES studied on a small scale. Used oxidized form HES 200 and 450 kDa.

Used deoxygenating Hb. b (50 mg/ml in 75 mm HEPES buffer with pH 8.1) oxygenerator and desoxyribose use impacts, respectively, oxygen, then nitrogen. Oxygenated HES derived from the HES 200 or 450 kDa using 0.6 or 1.2 EQ. periodate 1 mol adjacent diol was dissolved in 100 mm HEPES buffer with a pH of 8.1 to a final concentration of 60 mg/ml and the solutions were then degirolami and was purged with nitrogen. 0,253 ml b combined with 1.6 ml of the oxidized solution HES 200 kDa and 0,498 ml b combined with 1.4 ml of the oxidized solution HES 450 kDa, in both cases providing 1 EQ. b on 1 mol of the original non-oxidized HES 200 or 450 kDa. This solution was allowed to react at 22oWith under nitrogen, was prepared identisch time points, as described in example 2. Chromatographic profiles shown in Fig.5.

The final distribution of MM was narrower at the 22oWith both oxidized and HES at a longer reaction time for both temperature regimes. Average MM product HES 450 kDa (solid line) was greater than that derived 200 kDa (dashed line), and difference MM was greater at lower temperatures. The reaction proceeded more slowly at lower temperatures, leading to greater average MM and to a wider range of molecular masses, in comparison with the same reaction time at higher temperatures.

Example 7. The increased conjugation.

The conjugation of oxidized HES with b was strengthened to assess in vivo.

The concentration b in the buffer 100 mm HEPES at pH 8,1 brought up to 125 mg/ml and were released from the ligand through contact with oxygen and then nitrogen when using gazoobmenna with hollow fibers. 47 g of oxidized 200 kDa HES obtained as in example 1, using 0.6 EQ. periodate, was dissolved in 280 ml of buffer 100 mm HEPES at pH 8.1, then degirolami and was purged with nitrogen. Then the solution is oxidized HES added in b and maintained under nitrogen at 22-25oWith periodic measurement was repraisals (Fig.6). The lowest curve is presented for comparison purposes, received from b, cross stitched with raffinose with a ring which is open at oxidation (R-b). The reaction was carried out at a concentration of basic chemicals of 0.4 M sodium acetate was added 36 g dimethylamine, representing approximately 3 EQ. boron on 1 mol of the initial aldehyde. After 21 h, the reaction mixture oxygenerator, diafiltrate (with MM 10 kDa) against the solution of lactate ringer and the pH was brought to 7.4. The product had a P50(37oC) 26 mm RT. Art. and showed the property cooperatively. Analysis of the dispersion nizkoposhibnogo laser radiation arising after gel filtration of the solution indicated in MM from 90 to 210 kDa. The free aldehyde was not detected.

Analysis of half-life in vivo showed that the product Hb-HES held for extended periods of time. Volume of product in concentrations increased to 3.0 g/DL solution of lactate ringer equivalent to 10% of the total blood volume, poured awake rats and determined the retention time in the vascular system. The half-life rate of 6.0 h in comparison with 5.1 h, defined for the equivalent volume R-b with a concentration of 10.0 g/DL solution of lactate ringer.

Example 8. The conjugation of H by using either of 0.45, or 1,36 EQ. periodate on diol (diol 2 to monomer chain dextran). A solution of 2.0 g of dextran (260 kDa) was dissolved in 40 ml of water at 4oWith processed or 2,39 or 7,18 g periodate sodium (respectively of 0.45 and 1.36 equiv.). After 4 h of stirring in the dark at 4oWith the solution dialyzed (cut-off MM 10 kDa) and liofilizirovanny in the white powder. 50 mg of oxidized dextran in 1 ml of buffer 80 mm HEPES with pH 8.1 combined with 0,062 ml b (200 mg/ml) and the conjugation reaction was monitored by gel-filtration dissociable, adenocarinoma conditions (0.5 M MgCl2+25 mm Tris with a pH of 7.4). The results are shown in Fig.7 for the product in which the dextran was oxidized with the use of 0.45 EQ. periodate on diol, and Fig.8 - 1,36 EQ. periodate on diology experiment. Both reactions showed the initial formation of species with high MM, eluruumid mainly in the excluded volume of the column. The conjugate obtained from highly oxidized dextran, quickly evolved into a species with low MM than when using less oxidized dextran. The similarity of profile MM control R-b and highly oxidized dextran conjugate, except in General higher MM latter suggests that the conjugate composed of PoE it is also anticipated to oxidized HES conjugates, obtained under the same conditions (Fig.3).

Example 9. The half-life of the conjugates Hb-HES low and high MM.

Male rats Sprague Dawley stood for adaptation during the week with free access to food and tap water. On the day of experiment, rats were narcoticyou drugs Ketaset (ketamine hydrochloride, 60 mg/kg, intramuscularly) and Atravet (acepromazine maleate, 2.0 mg/kg, intramuscularly). In the right femoral artery and vein were introduced cannula with tubing made of polyethylene brand RE length 2.5-3.5 cm, which is connected with a tube of polyethylene brand RE, saline-filled with heparin (50 units of heparin on the USP/ml). A tube of polyethylene brand RE length of 2.0-3.5 cm was introduced into the lower abdominal aorta via the femoral artery and Vena cava via the femoral vein. Both cannulas were placed in a subcutaneous tunnel and moved outward in the neck. At the end of surgery surgical wound was sutured with surgical thread. At the end of the procedure, both the cannula was filled with saline solution with heparin (500 units of heparin on the USP/ml). Then the animals were placed in a fixing device of rodents with miniature hinge devices for feeding and were placed individually in metabolic cages. Animals davallia the whole experiment. After a period of recovery venous cannula was connected with an automatic infusion pump. Waking animals were subjected to injection control solutions (10 g/DL OR-b in the solution of lactate ringer, and the same solution, diluted to a concentration of 4 g/DL of plasma) or investigational products (the products of example 2, HES-Hb with low molecular weight (MMOs) and high molecular weight (AMM), respectively at a concentration of 5.0 and 3.5 g b/DL solution of lactate ringer), which is equivalent to 10% of the total blood volume, when the feed rate of 0.2 ml/min blood Samples were taken after 20 min after injection (time 0,33 h after injection) and then through 1, 3, 6, 10, 22, 28 and 34 including the Plasma was separated by centrifugation and stored at -80oTo analysis by gel-filtration. The total hemoglobin content was calculated according to spectrum absorption at 414 nm, with the correction for the background and put it on the schedule depending on the time of blood collection, the periods of half-life in plasma was obtained on the basis of a single exponential approximations. Periods of half-life in plasma was 5.1, and 5.5, 8,9 and 15.6 hours for the 4 and 10 g/DL OR-b and solutions HES-Hb respectively with MMOs and TIM.

When compared with the half-life obtained for the product of example 7, which had raspredeny, that the longer the half-life obtained for the latter product, which is obtained from the higher oxygendemanding HES.

Example 10. The stability of Hb-HES in plasma in vitro.

Hb-HES obtained in example 6 was diluted 10 times in the plasma of rats and incubated at 37oWith imitating the introduction of excess, amounting to 5-10% (about. /about.). The mixture was analyzed by gel-filtration dissociable, adenocarinoma conditions (0.5 M MgCl2+25 mm Tris with pH 7.4) for 49 hours the Results are shown in Fig.9. At this time not identified the species with low molecular weight, indicating the degradation of the product. Species with high molecular weight, eluruume at ultimate excluded volume analytical column that appeared within the first hour of incubation. These types correspond to complexes of modified hemoglobins with high molecular weight and the rat haptoglobin, which is consistent with observations made using several other products polymerized hemoglobin incubated in plasma.

Example 11. Stabilization of species with different MM in the process of conjugation Hb-HES.

To complete the change of molecular weight occurring during conjugation b with ocil kombinirovannogo with 490 μl of 80 mm HEPES with pH 8.1. 674 mg loaded N2oxidized HES (obtained from HES 450/0,7, 1,10 EQ. periodate on the calculated diol) was dissolved in 6.8 ml of degassed 80 mm HEPES with pH 8.1. 950 μl of solution b was added to the oxidized solution HES getting end ratio Hb:HES (450 kDa) 1:1. After 3 h, 2 ml of this reaction mixture was added a freshly prepared solution of 152 mg of the loaded N2DMB (providing approximately 3 EQ. DMB on initial aldehyde, calculated for the oxidized HES) dissolved in 1.6 ml of degassed water in 350 μl added 4 M NaOAc. Aliquots of 2 ml of the reaction mixture Hb-HES similarly processed within 6 hours After 22 h the reaction mixture was loaded and subjected to extensive dialysis against water for 72 h at 4oC. Distribution of MM during conjugation, recovery and after dialysis was determined by the method of gel filtration in dissociable, adenocarinoma conditions (0.5 M MgCl2+25 mm Tris with a pH of 7.4). The results are shown in Fig. 10 for products subjected to recovery after 3 h of Fig.11 - for products subjected to recovery after 6 h, and in Fig.12 - for unrecovered products. The distribution of MM products observed after 3 and 6 h (both conjugate with high MM), significant changes neither in the about in the samples, which were not restored (Fig.10), the recovery of 3 EQ. DMB on initial aldehyde prevented the transformation of the conjugate Hb-HES in such lower MM. As described in example 3 stabilization of species with high MM is observed also when using fewer equivalents DMB on the original aldehyde. This recovery method can be used to stabilize any distribution MM, which develops during the conjugation reaction.

Claims

1. The method of obtaining the composition of the recovered conjugate hemoglobin-polysaccharide, which implies to carry out the following steps: (a) to expose the original polysaccharide reaction ring opening oxidation with obtaining the polysaccharide with oxidative open ring, bearing aldehyde groups; (b) to ensure the specified reaction of the polysaccharide with oxidative open ring with hemoglobin under conditions suitable for the formation of a Schiff's base linkages between hemoglobin and polysaccharide with oxidative open ring, with the formation of the composition of the original conjugate hemoglobin-polysaccharide containing oxidized polysaccharides; (C) to expose the degradation of the polysaccharide with oxidative open ring is an eye will not achieve the necessary reduction in the average molecular weight of the composition hemoglobin-polysaccharide compared with the original composition of the hemoglobin-polysaccharide, with the formation of the detectable composition further degraded conjugate hemoglobin-polysaccharide with a reduced average molecular weight and a reduced amount of unreacted hemoglobin compared with the composition of the original conjugate hemoglobin-polysaccharide; (g) to ensure recovery of the specified composition further degraded conjugate hemoglobin-polysaccharide, when it will be necessary to decrease the average molecular weight of the composition hemoglobin-polysaccharide compared with the original composition of the hemoglobin-polysaccharide, to obtain the composition of the recovered conjugate hemoglobin-polysaccharide; (d) removing the specified composition of the recovered conjugate hemoglobin-polysaccharide.

2. The method according to p. 1, where the detectable composition further degraded conjugate hemoglobin-polysaccharide with a reduced average molecular weight and a reduced amount of unreacted hemoglobin also has a narrower distribution of molecular masses in comparison with the specified composition of the original conjugate hemoglobin-polysaccharide.

3. The method according to p. 1, where in the specified composition of the recovered conjugate hemoglobin-polysaccharide not detected namaseb under item 1, where at the stage of (a) oxidized from 30 to 100% of the available dolovich groups on the polysaccharide with oxidative open ring.

5. The method according to p. 4, where oxidized from 60 to 100% of the available dolovich groups on the polysaccharide with oxidative open ring.

6. The method according to p. 5, where the oxidized 100% of the available dolovich groups on the polysaccharide with oxidative open ring.

7. The method according to any of paragraphs.1-6, where the original polysaccharide has a molecular weight from 70 to 1000 kDa.

8. The method according to p. 7, where the original polysaccharide has a molecular weight from 70 to 450 kDa.

9. The method according to p. 8, where the original polysaccharide has a molecular weight from 70 to 260 kDa.

10. The method according to p. 9, where the original polysaccharide has a molecular mass of 70 kDa.

11. The method according to any of paragraphs.1-10, where the original polysaccharide is a dextran or gidroxiatilkrahmal.

12. The method according to p. 11, where the original polysaccharide represents gidroxiatilkrahmal.

13. The method according to p. 12, where gidroxiatilkrahmal is the ratio of substitution of about 0.5 to 0.7.

14. The method according to any of paragraphs.1-13, where the mass ratio of hemoglobin and oxidized polysaccharide composition of the recovered conjugate hemoglobin-polysaccharide ranges from 0.25:1 to 5:1.

15. The method according to p. 14, where the mass ratio g is nyugat supported on the stage (in) in aqueous solution at pH from about 7.2 to 10 and at a temperature of from 15 to 30With to reduce the average molecular weight of the initial composition of the conjugate.

17. The method according to p. 16, where the original conjugate supported in aqueous solution within 4-48 hours

18. The method according to any of paragraphs.1-17, where the apparent molecular mass of recovered conjugates in the resultant composition is preferably from about 90 to 200 kDa.

19. The method according to any of paragraphs.1-18, where this reduction step (g) is the only stage of recovery to achieve recovery of these Schiff bases and essentially simultaneous recovery of the aldehyde groups.

20. The method according to p. 19, where recovery is achieved using a reducing agent on the basis of boron.

21. The method according to p. 20, where the reducing agent on the basis of boron is dimethylamino.

22. The method according to any of paragraphs.1-18, where this reduction step (g) is carried out in two successive stages of recovery, the first is to achieve recovery of the Schiff bases and the other for recovery of the aldehyde groups.

23. The method according to any of paragraphs.1-22, where the hemoglobin is hemoglobin a mammal.

24. The method according to p. 23, where the hemoglobin is non-human hemoglobin.

lobin is deoxyhemoglobin.

27. The method according to any of paragraphs.23-25, where the hemoglobin is carboxyhemoglobin.

28. The method according to any of paragraphs.23-27, where the hemoglobin is intramolecular and/or intermolecular cross stitched.

29. The method according to any of paragraphs.1-28, where the composition of the recovered conjugate hemoglobin-polysaccharide has rate R50from 4 to 50 at 37C.

30. The method according to any of paragraphs.1-28, where the composition of the recovered conjugate hemoglobin-polysaccharide has rate R50concluded between R - and T-state hemoglobin.

31. The composition of the recovered conjugate hemoglobin-polysaccharide obtained by the method according to any of paragraphs.1-30.

32. The composition of the recovered conjugate hemoglobin-polysaccharide obtained by the method according to p. 1.

33. The composition of the recovered conjugate hemoglobin-polysaccharide obtained by the method according to p. 6 or 12.

34. The composition of the recovered conjugate hemoglobin-polysaccharide comprising degradiruem alkali polysaccharide open during the oxidation of the ring.

35. The composition of the recovered conjugate hemoglobin-polysaccharide on p. 34, obtained using hydroxyethylamine or dextran as a polysaccharide.

36. Composition Vosstania>

37. The composition of the recovered conjugate hemoglobin-polysaccharide on p. 36, where the hemoglobin is hemoglobin a mammal.

38. The composition of the recovered conjugate hemoglobin-polysaccharide on p. 37, where the hemoglobin is human hemoglobin.

39. The composition of the recovered conjugate hemoglobin-polysaccharide on p. 37, where the hemoglobin is deoxyhemoglobin.

40. The composition of the recovered conjugate hemoglobin-polysaccharide on p. 37, where the hemoglobin is carboxyhemoglobin.

41. The composition of the recovered conjugate hemoglobin-polysaccharide according to any one of paragraphs.37-40, where the hemoglobin is intramolecular and/or intermolecular cross stitched.

42. The composition of the recovered conjugate hemoglobin-polysaccharide according to any one of paragraphs.37-41, where the polysaccharide is gidroxiatilkrahmal with a molecular weight of from about 70 to 1000 kDa.

43. The composition of the recovered conjugate hemoglobin-polysaccharide on p. 42, where gidroxiatilkrahmal is the ratio of substitution of about 0.5 to 0.7.

44. The composition of the recovered conjugate hemoglobin-polysaccharide according to any one of paragraphs.34-41, with P50from 4 to 50 at 37C.

45. Composition R - and T-state hemoglobin.

 

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