Blood is the carrier of oxygen, the composition for its production and a method of obtaining a polymer modified hemoglobin

 

(57) Abstract:

The invention relates to medicine, namely to manufacture blood products. The invention is a blood - carrier of oxygen, representing an aqueous solution containing, wt.%: polymer modified hemoglobin 0,9 - 1,1, sodium chloride 1,0 - 1,2, glucose 0,68 - 0,83, ascorbic acid is 0.023 - 0,027 and water up to 100. The invention is also a composition for obtaining blood is a carrier of oxygen containing grams per 1 therapeutic dose (400 ml): polymer modified hemoglobin 3,6 - 4,4 sodium chloride 0.7 to 0.9, glucose 2,7 - 3,3, ascorbic acid 0,9 - 0,11. Polymer modified hemoglobin is produced by interaction of deoxyhemoglobin with glutaraldehyde modified at pH 6.4 to 6.6 and at a temperature of 4 - 6C with a substance from a number containing dicarboxylic acid and sodium bisulfite. Technical result: receipt of blood products, comparable in efficiency of gas transport in human blood. 3 C. p. F.-ly, 3 tables.

The present invention relates to medicines, in particular for blood substitutes, namely a blood substitutes based on polymer mo the practical solutions one of which is intended to receive the other.

The group includes, first, the blood is the carrier of oxygen, secondly, the composition for obtaining a blood substitute, thirdly, a method of obtaining a polymer modified hemoglobin, representing a substantial portion of the blood is the carrier of oxygen and composition for obtaining a blood substitute.

The invention can be widely used in medical practice for the production of blood drug comparable in efficiency of gas transport by the blood of the man.

Compared with blood polymeric products have the following advantages:

1. they are capable of long-term storage without changing its characteristics in liofilizovannyh form;

2. transfusion of them does not require a preliminary determination of blood group and additional experiments on compatibility with the blood of the patient;

3. the methods of obtaining the polymer of the blood substitute based on modified hemoglobin virtually eliminates the risk of infection in patients with viral infections in his introduction.

Known /T. M. S. Chang / Blood Substitutes: Principles, Methods, Products and Clinical Trials, 199 is a substitute because outside of the erythrocyte tetramer molecule of hemoglobin in the bloodstream falls into two subunits, which when removed cause kidney damage.

Known blood is the carrier of oxygen to the trademark "Hemolink" firm Hemosol LTD /J. C. Hsia / Biomaterials Artificial Cells and Immobilization Biotechnology, 1991. - v. 19. - P. 42/, representing a solution custom made o-raffinose hemoglobin isolated from donor blood, the lactate ringer's solution. The concentration of such a solution of 10 g/DL. O-raffinose - polyallelic obtained by the oxidation of D-raffinose. The solution of ringer's saline solution similar in composition and concentration of ions in sea water, one of the physiological fluids. Blood Hemolink contains 63 12% polyhemoglobin, less than 10% of methemoglobin has a partial pressure of oxygen at which polyhemoglobin saturated by 50% (P50), equal to 34 Torr. In phase 1 clinical trials known blood at its introduction into the body in doses up to 150 ml led to gastrointestinal discomfort: nausea, vomiting, diarrhea.

Also known /R. Przybelski et al. / Artificial Cells, Blood Substitutes and Immobilization Biotechnology, Internat. J., 1996. - v. - 24. - P. - 407./ blood is the carrier of oxygen Hemassist company Baxter Healthcare Corp., represents the Noah donor blood, in the lactate ringer's solution with a concentration of polyhemoglobin 10 g/DL. In clinical studies at doses up to 150 ml of this blood also cause gastrointestinal discomfort.

Described blood is the carrier of oxygen PolyHeme company Northfield Laboratory /L. R. Sehgal et al. / Biomaterials Artificial Cells and Artificial Organ, 1988. - v. 16 - N 1-3. P. 173/. PolyHeme is a solution preexisiting of polyhemoglobin on the basis of hemoglobin blood with a concentration of 10 g/DL of the active principle. The solution contains less than 1% of the Monomeric hemoglobin and not more than 3% methemoglobin. P50well 28-30 Torr. Clinical studies PolyHeme /S. A. Gould, G. S. Moss / World J. Surg., 1996. - v. 20. - P. 1200/ showed that infusion of known blood substitute reduced concentration of red blood cells active in the bloodstream, which is undesirable in terms of shock and great loss of blood. It was also found that while the introduction of more than 150 ml of the drug was also manifested gastrointestinal discomfort.

The closest to the essential features of the claimed solution is blood is the carrier of oxygen containing polymeric hemoglobin, modified pyridoxal-5-phosphate, and sodium chloride dissolved in water 3% methemoglobin, P50about 25 Torr.

For modification of hemoglobin in the prototype used a biologically active pyridoxal-5-phosphate, which is introduced into the reaction mixture in excess, and then unreacted pyridoxal-5-phosphate should be removed from the target product, because it causes unwanted side effects, once in the blood stream. In addition, as was shown in article L. R. Sehgal, etc. / Biomaterials Artificial Cells and Artificial Organ, 1988. - v. 16 - N 13. P. 173/ and according to our data, polyhemoglobin, modified pyridoxal-5-phosphate, reduces the concentration of red blood cells active in the bloodstream and does not stimulate the release of erythrocytes from the depot.

The task of the invention is to improve the portability of blood is a carrier of oxygen and promote the excretion of red blood cells from the depot.

The problem is solved by blood is the carrier of oxygen, representing an aqueous solution of polymer modified hemoglobin, which take the product of the interaction of deoxyhemoglobin modified with glutaraldehyde, sodium chloride, optionally, the blood contains glucose and ascorbic acid, all the components of the initial sodium 1,0 - 1,2

Glucose - 0,68 - 0,83

Ascorbic acid is 0.023 - 0,027

Water Up to 100

The problem is solved also anhydrous composition to obtain blood is a carrier of oxygen containing polymer modified hemoglobin, which take the product of the interaction of deoxyhemoglobin modified with glutaraldehyde; the composition further comprises a glucose and ascorbic acid in the following ratio of components in g 1 therapeutic dose:

Polymer modified hemoglobin - 3,6 - 4,4

Sodium chloride - 0,7 - 0,9

Glucose - 2,7 - 3,3

Ascorbic acid - 0,09 - 0,11

Indicated therapeutic dose to obtain blood is a carrier of oxygen dissolved in 400 ml of isotonic sodium chloride solution (Ogs. Pharmacopoeia XI, issue 1. - S. 175).

The problem is solved also claimed a method of obtaining a polymer modified hemoglobin. Polymer modified hemoglobin is produced by interaction of deoxyhemoglobin in aqueous solution with a bifunctional cross-linking agent, which take glutaric aldehyde-modified dicarboxylic acid, such as TSA is particular essential features of the solution is a method of obtaining a polymer modified hemoglobin with high oxygen transport efficiency /patent RF N 96108679/14, IPC: 5 A 61 K 35/14, the Official Newsletter of Rospatent "Inventions", 10.07.1998. - 1 o'clock. - N 19/. In accordance with the known method of polymer modified hemoglobin is produced by interaction of 1 to 10 wt.% an aqueous solution of deoxyhemoglobin with cross-linking agent is modified with glutaraldehyde at a molar ratio of hemoglobin:a crosslinking agent 1: 4-24. A crosslinking agent is modified glutaric aldehyde (HA) is produced by interaction of HA and modifier in aqueous buffer solution at a pH of 6.8 to 7.4, at concentrations of HA and a modifier is 0.1 to 5.0 wt.% and when the molar ratio of HA: modifier 1: 0,2-1,2. The reaction is carried out at 12 - 15oC. the Reaction is completed by addition of an aqueous solution of sodium borohydride. As a modifier take the substance from the series, including dicarboxylic amino acid (e.g. aspartic, glutamic) and sodium bisulfite. The main disadvantage of this method is the complexity of the regulation of the reaction modification of HA, which in the stated conditions is very rapid (10 - 15 minutes), despite a lower reaction temperature. The result is a modified HA with difficult reproducible characteristics. Further interaction of such a modified HA with detoxicate is.

The objective of the proposed method is to obtain polymer modified hemoglobin, well played by its characteristics.

This problem was solved by the method of obtaining a polymer modified hemoglobin, including the interaction of 1 - 10% aqueous solution of deoxyhemoglobin with modified HA, which is different from the known fact that the modification of HA is conducted in aqueous buffer solution at a pH of 6.4 - 6.6 and at a temperature of 4 - 6oC for 1.5 - 2 hours. The interaction of the modified HA with deoxyhemoglobin also conducted at a pH of 6.4 - 6.6 and at a temperature of 4 - 6oC.

Hemoglobin receive osmotic hemolysis washed erythrocytes blood /S. E. Rabiner / J. Exp. Med., 1967. - v. 126. - N-6. - P. 1127/. For this RBC mass, isolated from donor blood with retention periods of 5 to 36 days, washed six times from plasma proteins and antigens solution of sodium chloride. The washed mass is subjected to hemolysis pyrogen-free water. Stroma (erythrocyte membrane) is separated by centrifugation. A solution of hemoglobin is filtered through a filter Cuno to remove residual stroma. The filtrate is treated with dry sodium chloride for planting agamoglanov proteins. the native GB and the products of its chemical modification determined by spectrophotometric cyanmethemoglobin derived, wavelength = 540 nm /M C. Kushakovskij / Clinical forms of damage to the hemoglobin, L-d: Medicine, 1968. - N-23/. Use sodium chloride brand CHP ("Chemically pure", GOST 42-2572-88), glucose (FS 42-2419-86), ascorbic acid (GF X, S - 6), isotonic (GF XI, vol. 1, page 175). Use 25% aqueous solution of glutaraldehyde firm Reanal (Hungary). The concentration of HA is determined by the method of differential pH-metry with hydroxylamine /10. H. R. Roe, G. Mitchel / Analyt. Chem., 1951. - v. 23. - N 12. - P. 1758-1760/.

Sodium bisulfite (NaHSO3) get immediately prior to use /Y. C. Karyakin, I. I. Angels / Pure chemical reagents, Moscow. GNTI. Chem. l-RA, 1955. - S. 397 - 398/. Its concentration determined iodometrically.

The concentration of amino acids is determined by the reaction with 2,4,6-trinitrobenzenesulfonic /R. Fields / Biochern. J., 1971. - v. 124. - P. 581/.

The molecular mass (MM) of the target polymer modified hemoglobin is determined on the basis of the molecular mass distribution (MMD) method gel chromatography on sepharose 6B in phosphate buffer at neutral pH. The calculation is carried out by the original method /N. P. Kuznetsov, G. C. Samsonov / Vysokomolecul. Conn. , ser. But, 1985. - so 27. - N 12. - S. 2611/

Curves of dissociation of oxyhemoglobin get to a recording device "of Hem-O-SC is the oxygen from 0 to 150 Torr, achieved by blowing nitrogen or mixtures of nitrogen - oxygen (75:25 v/v). Based on the curves of oxyhemoglobin dissociation determines the amount of polonesian of hemoglobin with oxygen (P50). The error in the determination of P50is 10 - 15%.

Distinctive from the solution of the prototype signs of blood is a carrier of oxygen and composition to obtain are: compounds in General, and characterization of polymer modified hemoglobin.

Analysis of the known prior art did not allow to find solutions that exactly match the set of essential characteristics of the claimed compositions. This confirms the compliance of decisions on PP 1 and 2 of the patent formula, the condition of "novelty."

The analysis also showed that the prototype used polymer modified hemoglobin obtained by merging pre-modified hemoglobin. In the proposed solution the effect achieved by pre-modification cross-linking agent is glutaraldehyde.

Distinctive from the solution of the prototype (method for producing a polymer modified hemoglobin - p. 3 patent formula) symptoms are:

1. the pH of the water booth - ,6;

2. the reaction temperature modification and interaction of the modified HA with deoxyhemoglobin equal to 4 to 6oC.

The analysis of the prior art showed the lack of publicly disclosed information on how completely the same set of features with the stated solution. This confirms the relevance of the proposed invention, the eligibility condition of "novelty". The experiment showed that the distinctive features of the proposed method under item 3 patent formula provided a solution to the above problem is getting the playing characteristics of the blood substitute.

A new and unexpected feature of the proposed blood is the carrier of oxygen were increased its stability during long-term storage to autocycling with preservation of functional properties, as well as the ability to initiate allocation of erythrocytes from the depot of the body intravenously. This ability was discovered when conducting clinical trials and noted in the final reports of these studies.

Differences in the pH values of 6.4 and 6.6 (for the claimed process) and 6.8 to 7.4 (for the prototype method) significant, because it was always considered that the hemoglobin is claimed conditions hemoglobin remains relatively stable to the formation of metal-form, and the reaction products are more stable during storage.

Thus, the claimed integrated solution ensures the achievement of the unobvious and unexpected features of the target blood - increased resistance to autocycling during prolonged storage in liofilizovannyh form and the ability to initiate allocation of erythrocytes from the depot when administered intravenously.

New and unexpected functions gives the claimed integrated solution in General compliance with this important condition of patentability as "inventive step".

To confirm the compliance of the claimed solution to the condition of "industrial applicability" and to better understand the essence of the proposals provide examples of specific implementations of the invention, which do not exhaust the essence of the proposals.

Example 1. Obtaining polymer modified hemoglobin blended with the product of the interaction of glutaraldehyde dicarboxylic amino acid.

40 ml of an aqueous solution of hemoglobin (GB), obtained from blood, with a concentration of 5.0 wt.% (pH 6,6) are placed in a flask with a capacity of 200 ml and spend deoxygenation in a stream of nitrogen with stirring at 4oC. To Rast is going (HA) and 1.47 wt.% by glutamic acid in 0.1 M phosphate buffer solution with a pH of 6.6. The reaction is carried out at a stirring for 2 hours, then add 2.4 ml of 1 wt.% aqueous solution of sodium borohydride with a pH of 9.0. Stirring is continued for another 30 minutes. Take 1 ml of the reaction mixture and spend the day dialysis against 0.05 M solution of Tris-buffer with a pH of 7.4, containing 0.15 M NaCl. In the thus prepared sample is measured dissociation curve of oxyhemoglobin.

For comparison, the measured dissociation curve of oxyhemoglobin in the original solution GB, otdelitelnogo in the same conditions. To the resulting polymer modified hemoglobin P50is 30 Torr, for the original GB 22 Torr; MM (Mw) the resulting polymer modified hemoglobin 230 thousand Days

The main part of the resulting solution of polymer modified hemoglobin cialiswhat against 2 l of 1 wt.% solution of sodium chloride during the day and add ascorbic acid and glucose. The resulting solution was dried by lyophilization.

The final product contains the following components g 1 dose:

Polymer modified hemoglobin - 4,4

Sodium chloride at 0.9

Glucose - 3,3

Ascorbic acid - 0,11

To obtain a dosage form liofilizovannye, wt.%:

Polymer modified hemoglobin - 1,1

Sodium chloride - 1,2

Glucose - 0,83

Ascorbic acid - 0,027

Water Up to 100

Polymer modified hemoglobin in example 1 has a high resistance to autocycling, which is caused both by the production method, and the claimed composition of the dosage form. Long preparation is stored in liofilizovannyh form without loss of functional activity. After two years of storage the value of P50changed from 30 mm to 27 mm, the content of met-forms increased from 4% to 12%.

Blood products based on polymer modified hemoglobin obtained in accordance with the method of the prototype in similar conditions, during two years of storage in liofilizovannyh form changed the value of P50from 22 mm to 17 mm, the content of met-forms increased from 5% to 19%.

Example 2. Obtaining polymer modified hemoglobin blended with the product of the interaction of glutaraldehyde sodium bisulfite.

In a flask of 50 ml was placed 10 ml of an aqueous solution GB with a concentration of 4.7 wt. % (pH 6.4) and are deoxygenation by blowing nitrogen at a 6oC. To a solution of deoxygenating GB if the W is on the sodium bisulfite to 0.73 wt.%. The mixture is stirred for 1.5 hours. The reaction is stopped by adding 0.5 ml of 1 wt.% aqueous solution of sodium borohydride with pH 8.5, followed by stirring for 30 minutes. In the conditions of example 1 to produce the target product and determine its characteristics.

To the resulting polymer modified hemoglobin P50is 23 Torr, for the original GB 17 Torr; MM (Mw) the resulting polymer modified hemoglobin 180 thousand Days

The main part of the resulting solution of polymer modified hemoglobin cialiswhat against 2 l of 1 wt.% solution of sodium chloride during the day and add ascorbic acid and glucose. The resulting solution was dried by lyophilization.

The final product contains the following components g 1 dose:

Polymer modified hemoglobin - 3,6

Sodium chloride - 0,7

Glucose - 2,7

Ascorbic acid - 0,09

To obtain a pharmaceutical form of liofilizovannye product (1 dose) dissolved in 400 ml of isotonic sodium chloride to obtain a preparation containing, in wt.%:

Polymer modified hemoglobin - 0,9

Sodium chloride - 1,0

Glucose - 0,68

Ascorbic acid - 0,0 composition of the dosage form according to example 2, also has a high resistance to autocycling. During storage of blood products in liofilizovannyh form within two years, the value of P50changed from 23 mm to 20 mm, the concentration of met-forms has increased from 3.5% to 12.2%.

Blood products based on polymer modified hemoglobin obtained by the method prototype in similar conditions, during two years of storage in liofilizovannyh form has reduced the value of P50from 22 mm to 18 mm, the content of met-forms increased from 4.5% to 18%.

In clinical studies claimed the blood was applied during surgical operations about the various diseases of the gastrointestinal tract more than 50 patients of different ages for the correction of intraoperative blood loss, as well as in patients with posthemorrhagic anemia. Diseases which were produced by intravenous infusion of the proposed blood products during surgical intervention. It is a chronic stomach ulcer - 17 cases, stomach cancer, 9 cases, acute gastric ulcer complicated by bleeding - 2 cases, chronic duodenal ulcer - 3 cases, cancer of the colon - 4 cases, rectal cancer 7 cases, state polnych neoplastic lesions of the stomach or colon infusion claimed the blood was performed for the correction of anemia in the preoperative period, and during surgical intervention. The inventive blood substitute has also been used to assist patients with hemorrhagic and traumatic shock. In 9 cases had severe blood loss (from 1300 to 2000 ml), which was accompanied by hemorrhagic shock. In 6 cases there was a hemorrhage and traumatic shock II and III. Infusion of the proposed blood substitute was made to replenish blood loss with blood, with gas transport function (oxygen transport) and was 400 - 1600 ml.

In tables 1 - 3 presents data characterizing the blood after infusion of the proposed blood substitute.

The data presented show that the infusion of the proposed blood is a carrier of oxygen provides fast and long enough hemodynamic effect, which is manifested, first, in the stabilization of both systolic and diastolic blood pressure, and secondly, in the steady increase of the blood, third, the increased partial pressure of oxygen in arterial blood.

Clinical trials have confirmed the good tolerability and high treatment efficiency Appl is when the bleeding is severe and very severe.

Found a stimulating effect of the claimed blood is the carrier of oxygen in haematopoiesis, which is confirmed by table 2. Side effects of the claimed blood is a carrier of oxygen was not detected in any case, even when the jet introduction during hemorrhagic shock. It was also noted that the excretory function of the kidneys in the period of introduction and steps stated blood is the carrier of oxygen is not violated.

1. Blood is the carrier of oxygen, representing an aqueous solution containing polymer modified hemoglobin and sodium chloride, characterized in that it additionally contains glucose and ascorbic acid, as well as polymer modified hemoglobin contains the product of the interaction of deoxyhemoglobin modified with glutaraldehyde in the following ratio, wt.%:

Polymer modified hemoglobin - 0,9 - 1,1

Sodium chloride - 1,0 - 1,2

Glucose - 0,68 - 0,83

Ascorbic acid is 0.023 - 0,027

Water Up to 100

2. Composition to obtain blood is a carrier of oxygen, including polymer modified hemoglobin and sodium chloride, oficialbnogo hemoglobin contains the product of the interaction of deoxyhemoglobin modified with glutaraldehyde in the following ratio of components in g 1 dose (400 ml):

Polymer modified hemoglobin - 3,6 - 4,4

Sodium chloride - 0,7 - 0,9

Glucose - 2,7 - 3,3

Ascorbic acid - 0,09 - 0,11

3. A method of obtaining a polymer modified hemoglobin by successive operations deoxygenation hemoglobin blood in aqueous solution, modification of glutaraldehyde substance from a number containing dicarboxylic acid and sodium bisulfite, interaction deoxyhemoglobin modified with glutaraldehyde and with the completion of the reaction by the addition of substances covering the functional group of the crosslinking agent, characterized in that the glutaric aldehyde modify at pH 6.4 to 6.6 and at a temperature of 4 - 6oC, interaction of deoxyhemoglobin modified with glutaric aldehyde is carried out at the same pH and temperature.

 

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