Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/795—Porphyrin- or corrin-ring-containing peptides
  • C07K14/805—Haemoglobins; Myoglobins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
  • Y10S530/827—Proteins from mammals or birds
  • Y10S530/829—Blood

Definitions

• ABSTRACT Method for obtaining a storage-stable, infusionable and hepatitis-safe hemoglobin solution having a stabilized 2,3-diphospho-glycerate level which method comprises a. treating a starting material containing human erythrocyte with a dilute solution of B-propiolactone at temperatures between 5 and 15C. to result in a mixture containing B-propiolactone in an amount of from 4 to 12 g per liter of erythrocyte sediment, thereafter b. treating the mixture with a neutral or weakly alkaline washing solution to wash out the excess B-propiolactone and its reaction products,
• the invention relates to a method for obtaining hemoglobin solutions, particularly hemoglobin solutions which are storage-stable and infusionable and which are free of potential hepatitis causing infectants or contaminants, and to such compositions per se.
• the present invention provides a method for obtaining hepatitis-safe, storage-stable and infusionable hemoglobin solutions with a stabilized 2,3-diphosphoglycerate level, which thus substantially overcame the disadvantages of known compositions.
• the inventive method comprises a. treating a starting material containing human erythrocyte with a dilute solution of B-propiolactone at temperatures between 5 and C. to result in a mixture containing B-propiolactone in an amount of from 4 to 12 g per liter of erythrocyte sediment, thereafter b. treating the mixture with a neutral or weakly alkaline washing solution to wash out the excess B-propiolactone and its reaction products,
• step (a), above) is performed preferably with 6 to 16 g of B-propiolactone in solutions of 1.2 tp 3.2 g/lOO ml per 1.5 liter erythrocyte sediment, and for no longer than 25 minutes.
• the content of not yet completely reacted B-propiolactone in the erythrocyte supernatant is decreased to 20 to 10 mg%, whereby preferably washing solutions are used which contain sodium chloride and- /or sodium bicarbonate.
• Other suitable washing agents are solutions of tert. sodium citrate, tert. sodium phosphate and sodium carbonate.
• the remaining B-propiolactone is further diluted to l/50. Only in such a concentration does the B-propiolactone come into direct contact with the hemoglobin and the other components of the cell nucleus, and it can then completely react.
• Distilled water is used as a preferred diluent.
• physiologically suitable electrolyte solutions can be added, for example sodium chloride solutions, glucose solutions or bicarbonate solutions.
• the step of the B-propiolactone treatment is ommited, after 3 months of storage, the product has about double the content of free phosphate than has the product according to the invention
• the treatment according to the invention fi-propiolactone results in stabilization of the 2,3-diophospho-glycerate content in hemoglobin solutions at a value of about 0.4 mmole per 0.8 mmole of hemoglobin over a period of time of more than three month, as determined by direct measurements. Without this stabilization, the corresponding 2,3-diphospho-glcerate value fluctuates from charge to charge between 0 and 0.25 mmole.
• Solutions of 3.8% of sodium bicarbonate or 1.6% of sodium chloride are particularly suitable solutions for the washing treatment according to the invention. When diluting, particularly the erythrocyte volume is diluted 4.5 times.
• Particularly suitable cation exchange resins are acid polystyrene-sulfonate cation exchangers, especially the resins commercially sold by the Dow Chemical Company, Midland, Michigan, under the designation Dowex 5O WX, which have an exchange capacity of 4 to 5 milliequivalents per each gram of dry resin.
• Suitable exchangers are, for example, Amberlite lR-120 of the Rohm and Haas Company, Philadelphia "Zero-Karb 225" of the Permutit Company Ltd., London, and Lewatit" of Wegriken Bayer, Leverkusen.
• the lowering of the potassium content takes place in the method according to the invention preferably by the cation exchange 1t. against 11* and the subsequent centrifuge treatment.
• the cation exchange 1t. against 11* and the subsequent centrifuge treatment there should be present about 6.3 g of hemoglobin per 100 ml.
• the stabilized pH-value at the end of the process amounts advantageously to 7.4.
• a preferred agent for the control of isotonicity is glucose, however all other agents conventional for this purpose are also suitable.
• a product In freezing the hemoglobin solution obtained according to the invention and partial re-thawing, a product can be withdrawn which is rich in hemoglobin, which product, by repetition of this process, can be brought up to a 15% hemoglobin content (this corresponds to the total hemoglobin content of blood).
• the relative viscosity of such a solution of about 1.3 at 37C. is still within the normal range of plasma.
• the final adjustment of the isotonicity is advantageously carried out only after the initial concentration, so that the obtained V hyperoncotic concentrate, i.e., concentrate whose colloidal osmotic pressure exceeds that of normal plasma is not also hypertonic.
• the method according to the invention is illustrated by the following examples.
• the products obtained in accordance with these examples have already been tested on mammals such as mice, rats, rabbits, dogs, pigs, and humans, and have been found compatible as well as oxygen-transport effective.
• EXAMPLE 1 Erythrocyte sediments from 6 blood donors, donating 500 ml of blood each, were separated from the plasma, combined, diluted with 1.6% sodium chloride solution to a volume of 2 liters and at C. mixed with 5 g of freshly distilled B-propiolactone for minutes at 10 to 12C. The mixture was subjected for minutes to a cooling centrifugation, the supernatant was sucked off and the erythrocyte sediments were washed with fresh, 1.6% sodium chloride solution four times in succession in proportions of about 1.1 liters, each, on the centrifuge and then placed into 5.2 liters of sterile, demineralized and distilled water.
• the oxygen binding curve of a product prepared in accordance with this example showed that after 2 months of storage at 10C., a P 50 value of 19 mm Hg and a pH-value of 7.4. Calculated on an intraerythrocytic pH-value of 7.2, the P 50 value of the hemoglobin of said preparation would be 23 mm Hg.
• the P 50 value is the oxygen partial pressure under which the hemoglobin preparation is saturated to 50% of its maximum oxygen binding capacity.
• Example 1 was repeated but 16 g of ,B-propiolactone was added to 2 liters of erythrocyte suspension. After centrifugation and sucking off the supernatant, the erythrocyte sediment was washed with about 1.1 liters of each of the following solutions:
• Example 2 The sediment was then further processed as in Example 1 through the stroma separation. Prior to the sterile filtration the pH-value was adjusted with n-NaOl-l to 7.0, 33 g per liter of glucose were added, then 2.5 g per liter sodium bicarbonate were added and adjusted with n-NaOH to a final pH-value of 7.4.
• Example 1 was repeated through the stroma separation. Thereafter, the mixture was concentrated to blood-analogous hemoglobin content of 15 g/ ml by way of the following steps: the hemoglobin solution was frozen in portions of 500 ml each, in 1000 ml bottles lying on their side, and thereafter thawed at room temperature with the bottles placed in an upside down position in such a manner that by perforation of the stopper closure with a sterile blood-taking-device the deepred melt could be continuously removed from the remaining white ice stick. A repetition of the process resulted in a solution containing about g/liter of hemoglobin, which was then worked-up in accordance with Example 1 to an infusionable product.
• Method for obtaining a hemoglobin solution having a stabilized 2,3-diophospho-glycerate level comprises a. treating a starting material containing human erythrocytes with a diluted solution of B-propiolactone at temperatures between 5 and 15C. to result in a mixture containing B-propiolactone in an amount of from 4 to 12 g per liter of erythrocyte sediment, thereafter treating the mixture with a neutral or weakly alkaline washing solution to wash out the excess B-propiolactone and its reaction products,
• step (a) of the method from 6 to 16 g. of ,B-propiolactone are used in solutions of 1.2 to 3.2 g/lOO ml per 1.5 liter erythrocyte sediment.
• washing step (b) is effected with a solution of sodium bicarbonate.
• washing step (b) is effected with a solution of sodium chloride.
• step (c) is a polystyrenesulfonate resin in H-form.
• Method as claimed in claim 1 including the additional step between steps (d) and (e) of adjusting the isotonicity by adding glucose, sodium acetate or sodium bicarbonate.
• Method as claimed in claim 1 including the additional step between steps (d) and (e) of adjusting the pl-l-value to about 7.4 with caustic soda.
• step (e) is effected by use of celluloseasbestos filters or cellulose acetate discs.
• a hemoglobin solution having a stabilized 2,3-diphospho-glycerate level prepared by the method claimed in claim 1.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• Life Sciences & Earth Sciences (AREA)
• Genetics & Genomics (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Gastroenterology & Hepatology (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicinal Preparation (AREA)

Applications Claiming Priority (1)

Publications (1)

Family

ID=5858076

Family Applications (1)

Country Status (5)

Cited By (16)

Families Citing this family (4)

Citations (2)

• 1972
  • 1972-10-03 DE DE2248475A patent/DE2248475C3/de not_active Expired
• 1973
  • 1973-10-02 FR FR7335228A patent/FR2201102B1/fr not_active Expired
  • 1973-10-03 GB GB4615773A patent/GB1430217A/en not_active Expired
  • 1973-10-03 JP JP48110633A patent/JPS6025411B2/ja not_active Expired
  • 1973-10-03 US US403141A patent/US3864478A/en not_active Expired - Lifetime

Patent Citations (2)

Also Published As

Similar Documents

Legal Events