IL87706A - Hemoglobin-based non-pyridoxylated blood substitute and method for the preparation thereof - Google Patents

Description

The present invention relates to blood substitute material. More particularly, the invention relates to a hemoglobin-based blood substitute material and to a method for the preparation thereof.

BACKGROUND OF THE INVENTION As known, blood transfusion has become a universally accepted, life-saving procedure used in modern clinical medicine. Blood losses at substantial amounts are very dangerous, since they decrease the blood volume and pressure, limit the oxygen supply to vital tissues and slow the metabolic processes. However, the collection of blood for transfusion, its storage and administration is a complex subject which involves serious problems such as limited resources, compatibility and risk of transmitting certain diseases (e.g. hepatitis, AIDS, malaria, etc.). Moreover, when considering performing the blood transfusion in the field, even more complicated problems are encountered not only of medical origin but also logistic, such as availability at site, transport and storage conditions , - etc .

In view of the above problems, the search for blood substitutes was seriously considered and a number of related papers and patents can be found in the respective literature.

Two main groups of blood substitutes are mentioned in the literature! (a) perfluorocarbon-based , and (b) hemoglobin-based, Perfluorocarbons-based substitutes are cyclic -branched or straight-chain molecules, in which hydrogen atoms have been replaced with fluorine. Due to the strength of the carbon-fluorine bond, these compounds are generally considered as very inert, both chemically and biologically. They are capable of absorbing oxygen by physical dissolution, the amount absorbed being linearly dependent on the composition of the equilibrating gas phase. However, recent studies have shown that perfluoro-carbons have several disadvantages such as adverse reactions on the immune system,hematological disturbance, alterations in prostaglandin metabolism, impairment of the coagulation process, etc.

The present invention relates to a hemoglobin-based substitute, and therefore some more discussion will be presented on this subject. As known, hemoglobin binds oxygen in a process having the characteristic of non-linearity and a chemical species known as oxyhemoglobin is thus formed. An ideal hemoglobin-based blood tetramer, having a molecular weight of about 65,000. Xn mammals, hemoglobin is present in erythrocytes, the membrane of which contains stroma which consists of phospholipids, cholesterol, and proteins.

In the followings, the terms stroma-free hemoglobin and stroma-depleted hemoglobin can be used interchangeably, and both mean a product from which essentially all the stromal components were removed.

Attempts have been made to use stroma-free hemoglobin solutions as blood substitute, exhibiting oxygen transport capabilities. These solutions endure storage at room temperature for limited periods of time, without changes in their properties. However, oxygen binds to hemoglobin very strongly, so that only a small part of the bound oxygen is released to the cells at the oxygen tension normally prevailing in tissues. Also, not all of the important characteristics of these solutions are equal to those of human blood. Thus for instance, at the required concentration, the corresponding oncotic pressure is far from that of normal blood. Further investigations have also shown that stroma is a toxic factor, probably by causing thrombosis of the small renal vasculature.

In view of some favorable properties possessed by stroma-free hemoglobin, intensive work was done to improve the disadvantages encountered with its use as blood substitute. One approach was to modify its structure i order to decrease its oxygen affinity. For this purpose, it was suggested by Benesch et al [Biochemistry 11, 3576-3582 (1972)3 to treat the stroma-free hemoglobin with pyridoxal-5 · -phosphate (hereafter referred to as PLP). The product obtained pyridoxal-5 ' -phosphate hemoglobin (hereafter referred to as PLP-hemoglobin) , indeed has a satisfactory oxygen affinity, but the intravascular life time is too short compared to what is required for a resuscitation material. - 4 a- 87706/2 ■According to U.S. Patent No. 4,053,590 a blood substitute composition based on a macromolecul ar "hemoglobin is described. The composition is obtained b an inter- molecular erosslinking with a reagent selected from heterocyclic triazines, dialdehydes , sulfones, diisocya- nates , diepoxides and halogenated aromatic cycloalkanes. Some improvements are claimed to be obtained for certain properties but still the product resulted differs from that of the normal blood.

In a paper by DeVenuto et al (C.A. 98.221668) it is described a method for the preparation of pyridoxy 1 ated- pclymer ized human hemoglobin. It is mentioned that the product obtained did not showed adverse coagulant activity.

In another paper by Stabilini et al (C.A. 100, 180066) pyridoxal 5-phosphate was covalently bound to stroma-free hemoglobin prior to the polyme ization with glutaralde- hyde. The product obtained is characterized by a high oxygen affinity of stroma-free hemoglobin, thus being able to decrease the oxygen delivery to tissues. 4 b' 87706/2 In the French Patent No. 2,600,255 (cf . C'.A. 110, 121365) an acellular erythrocite substituent is described. The main feature of the method is to polymerize the treated product by a dialysis filter followed by a purification through ultraf i Itrat i on , gel fi ltration and affinity chromatography on agarose gel -bound heptoglobin.

A general characteristic of all these methods is that only some of the properties of the blood substitute are improved and the blood product obtained does not possess all the properties substantially the same as these of the natura 1 blood .

Based on the same approach of modifying the structure of stroma-free hemoglobin, it is described in the U«S. Patent Number 4,061,736 a method for obtaining an intramolecularly crossllnked stroma-free hemoglobin, by reacting the latter with a reagent selected from aldehydes, dialdehydes, activated ureas, derivative of carboxylic acids, heterocyclic triazines and halogenated aromatic cycloalkanes .

According to U.S. Patent No, 4,598,064, stroma-free hemoglobin is modified by intramolecular crosslinking between the alpha chains of the tetramer, using an acylating agent. It is claimed that the product obtained is an effective blood substitute having suitable oxygen binding characteristics, such as low oxygen affinity, being particularly useful in the treatment of ischemia. Another approach, which was also investigated, consists in the modification of hemoglobin in an attempt to improve both the oxygen binding characteristics and the intravascular retention. Thus according to PCT Patent Application No. 84/04248, hemoglobin is first covalently crossllnked intramolecularly between the two beta chains, and then reacted with PLP. It is claimed that the product thus obtained is a stable, oxygen carrying material, capable of delivering oxygen to perfused tissue, and advantageously remaining in the Intravascular space* However, one of the main disadvantages of the blood substitute obtained according to the method described therein is its limited oxygen carrying capacity.

Still another approach involves the polymerization of PLP-hemoglobin [G.S. Moss et al. Surgery 95, 249-255 (1984)3. However, the product thus obtained has, again, a limited oxygen carrying capacity.

Summing-up the above short review of the prior art , it should be concluded that there are, indeed, some improvements towards the use of hemoglobin-based blood substitutes beyond the use of stroma-free hemoglobin. Blood substitutes containing one or more characteristics of normal blood can thus be encountered, but still there is a long felt need for a blood substitute which will possess most of the characteristics of the normal blood. It is an object of the present invention to provide a blood substitute which possesses most of the characteristics of the normal blood. It is another object of the present invention to provide a hemoglobin- based blood substitute which possesses most of the characteristics of the normal blood. It is yet another - 7 - object of the present invention to provide a hemoglobin-based blood substitute to be useful for clinical use. A further object of the present invention is to provide a simple method for obtaining a hemoglobin-based blood substitute useful for clinical use - 8 - 87706/2 BRIEF DESCRIPTION OF THE INVENTION The invention relates to a hemoglobin-based blood substitute, obtained from stroma-free hemoglobin, intra-molecularly (i.e. tetramer ca 1 ly) stabilized, with di-functional reagents selected from haloesters of . dicarbo-xylic acids, said stabi lized stroma-free hemoglobin being subsequently polymerized at an extent of above 40% under anaerobic conditions using polymerization reagents selected from glutaraldehyde or precursors thereof capable of reacting with linkable groups on the hemoglobin molecule. The product obtained, when reconstituted with an appropriate solvent, y i e l d s a blood substitute solution which is characterized by properties which are substantially equal to those of whole human blood, and which are highly improved when compared to those of solutions of unmodified hemoglobin. Table 1 below summarizes the main properties of whole human blood under normal physiological conditions, of a solution of unmodified hemoglobin £the values for unmodified hemoglobin solution were taken from Lakshman e aj_, Journal of Surgical Research 30.» 14-20 (19810], and of a solution of the hemoglobin-based blood substitute obtained according to the present invention . - 9 - TABLE 1.

Main properties of whole human blood, of aolution of unmodified hemoglobin and of solution of the blood substitute material according to the present invention.

Solution of Solution of Property Whole human Unmodified Blood blood Hemoglobin Substitute Hemoglobin (g/100 ml) 12-18 7-8 10-18 PKft (torr) 26-29 12-14 20-23 COP (torr) 25-29 20-25 24-30 Methemoglobin content (% of total hemoglobin )up to 5 3-8 3-5 P50 B the Partial pressure of oxygen at which 50% of hemoglobin sites are saturated with oxygen; measured at normal physiological conditions t pH a 7.4, PCO^ 40 torr, temperature » 37.0°C. COP a colloid osmotic (i.e. oncotic) pressure.

As appears also from the above Table 1, a solution of the blood substitute material . prepared according to the present invention is characterized by its proper oxygen affinity, as well as by its adequate COP at normal physiological concentrations of hemoglobin. Moreover, it should be pointed out that the above mentioned improved properties (of the blood substiture material prepared according to the present invention) are obtained without utilizing the treatment with pyridoxal-5 · -phosphate generally used in most of the prior methods. As mentioned in some scientific papers this treatment with pyridoxal-51 -phosphate does not reduce the abnormally high (Ca.60 torr) oncotic pressure of physiological hemoglobin concentrations (12-18g/100 ml), of the stroma- free hemoglobin solution, and the obtained product has the disadvantage of a too short half-life in circulation.

The term stroma-free hemoglobin, as utilized in the specification includes also the substantially stroma-free hemoglobin.

The blood substitute material prepared according to the present invention is characterized by the following advantageous properties: a. Is not toxic. - 11 - b. Possesses appropriate lifetime In circulation. c. The solution obtained upon reconstitution in an appropriate solvent posseeses good oxygen binding and delivery characteristics. d. The solution obtained upon reconstitution in an appropriate solvent possesses normal oncotic pressure at physiological concentrations of hemoglobin, and correspondingly appropriate oxygen transport capacity.

In view of the above, transfusion with this blood substitute solution is much more convenient to perform than transfusion with whole blood, because there is no need to "type and crossmatch" prior to transfusion and thus lives may be saved by saving precious time.

One of the important properties required for a blood substitute is to have an appropriate oxygen dissociation curve. In Figure 1 there is a diagram illustrating in a clear manner this property for whole human blood (II), for the solution of the blood substituted prepared according to the present invention (III), and for the solution of stroraa-free hemoglobin (I), which is the starting material for said blood substitute material. As appears from Figure 1, the Ρ.Ω of the blood substitute - 12 solution (21 torr) is much closer to that of whole human blood (26 torr) than is the Pg0 of stroma-free hemoglobin solution (14 torr).

Another important property required for a blood substitute is to have the appropriate capacity for carrying oxygen from the lungs to the tissues* This characteristic is presented schematically in Figure 2, for whole human blood (A), for a solution of the blood-substitute prepared according to the present invention (B) , for the blood-substitute solution described in the prior art and obtained from polymerized PLP-hemoglobin (C) [G.S. Moss et al., Surgery 95, 249-255 (1984)3 and from intramolecularly crosslinked pyridoxylated hemoglobin (D) (PCT Patent Application No. 84/04248) , for fluosol DA fluorocarbon emulsion (E) [H. Ohyanagi and Y. Saitoh, Int. J. Art. Organs !J, 363-368 (1986)] and for stroma-free hemoglobin solution exhibiting the required oncotic pressure (F). As appears from this Figure, the oxygen carrying capacity - measured relatively to whole human blood at normal physiological conditions - is 90% for the blood substitute prepared according to the present invention, compared with 65% or 60% for the prior - 13 art blood substlte solutions, 856 for fluorocarbons , and 656 for the stroma-free hemoglobin.

The hemoglobin - based blood substitute material prepared according to the present invention is much superior to the perfluoro- chemicals suggested as blood substitutes in casualties suffering from hypovolemic shocks, due to its ability to carry appropriate amounts of oxygen when the victim is ventilated with room air. It may also be used to treat acute myocardial infarcts, and cerebral vascular accidents* The improved properties of the blood-substitute prepared according to the present invention result from the polymerization of the tetramerically stabilized etroma-free hemoglobin. It was found that this polymerization should be carried out only in absence of oxygen, and under conditions in which the percentage of polymerization is above 50%.

In Figure 3 are presented two graphs which correlate the percentage of oxygen saturation and the partial pressure of oxygen (expressed in torr). Graph I is the oxygen dissociation curve of a solution of the blood substitute prepared according to the present invention, particularly in case that the said polymerization is carried out in presence or absence of oxygen / Product obtained Product obtained in the absence of in the presence of oxygen - Hemoglobin concentration (g/100 ml) 14.0 12.8 - Methemoglobin content (% of total hemoglobin) 2.9 4.7 - Pso (at pH-7.4; pCO2«-40 torr and 137 deg.C) 21 14 - 15. - From the above data it clearly appears that the polymerization in the presence of oxygen, leads to a product with a P50 of 14 torr, which is significantly lower than that of the corresponding product obtained by polymerization in the absence of oxygen (p5Q = 21 torr) . Also, the methemoglobin, content in the product is much higher and increases from 2.956 to 4.7¾ when the polymerization is carried out in the presence of oxygen rather than in its absence (4.756 and 2.9% respectively).

Polymerization reagents for this step should be selected from various compounds such as glutaraldehyde _ or precursors thereof such as 2-ethoxy-3 , 4-dihydro-1, 2-pyran, 2,5-diethoxy tetrahydrofuran etc.

Generally, the polymerization reagent is added to a buffered solution of the stabilizd stroma-free hemoglobin having a pH between 6.5 and 8.5, and preferably of 7.5.

According to a most preferred embodiment, the polymerized product obtained is quenched in an amine solution in order to avoid that moieties containing active groups of the polymerization reagent remain in the final product. Accordingly, the amine should be added in an amount - 16 - sufficient to quench all the unreacted groups of the polymerization reagent. Although amines such as ethanol-amine and lysine are preferred reagents, other reagents may be utilized, such as bisulfites, diols alcohols, isocyanates and mercaptans.

An additional optional step which, if carried out anaeroblcally, would avoid an increase in the methemoglobin content, is reduction with sodium borohydride. This step ensures the saturation of the double bonds formed by the polymerization reagent, thus giving extra stability to the polymerized hemoglobin. Since, at the introduction of sodium borohydride, apart from the reduction of the double bonds, the residual carbonyl groups introduced by the polymerization reagent are also reduced, the addition of a quenching agent is generally superfluous. In most cases, only one additional step after the polymerization, i.e. quenching or reduction with borohydride will be indicated, but for specific cases such as when extra stability of the polymerized product is needed together with precise regulation of the polymerization step, the two additional steps might be required. A person skilled in the art, - 17 - will select the proper step according to the specific requirement for the blood-substitute.

One o the advantages of the process for obtaining the hemoglobin-based substitute according to the present invention, is the use of common reagents, commercially available or easily synthesized, which are generally utilized for this purpose. The product obtained may be lyophilized in the presence of 3% glucose or other protective agents as known in the art , without changing its oxygen- transporting properties as a potential resuscitation fluid upon reconstltutlon.

In summary, the blood-substitute hemoglobin-based material prepared according to the present invention is a superior product for the rapid treatment of hypovolemic shock, because it can be used by paramedical personnel without delay. It is easy to prepare, has a long storage life and delivers oxygen to perfused tissues when the victim is inhaling room air. It may be used in many critical situations other than combat casualty and hypovolemic shock. It should be an ideal substitute of whole blood for use as priming fluid in extracorporeal pumps employed in cardiac surgery, or in extracorporeal devices employed in blood treatments such as hemoperfu- - le ¬ sion and hemodialysis. It also could be used to treat Ischemic episodes including sludging and pain in sickle cell crises. It could be used as an organ perfusant to maintain organ viability until transplanted.

DETAILED DESCRIPTION OF THE INVENTION The first step requires the obtaining of stroma-free hemoglobin. This is carried out starting with packed red blood cells or whole blood from various mammalian species. There are several known methods for obtaining the stroma-free hemoglobin, most of them producing a suitable starting material for the present invention. As known, when stroma is present in hemoglobin preparations in large amounts, it interferes with renal function. Investigations in the past had shown that stroma caused thrombosis of the small renal vasculature. It was already described in many references that, by removal of most of the stroma from hemoglobin solution, this disadvantage is eliminated.

The method selected in the present invention fo preparing the stroma-free hemoglobin is fully described in a paper by De Venuto et al (J. Lab. Clinical Med. 89, 509-16, 1977), 19 The second step involves the stabilization of the stroma-free hemoglobin. This is carried out by a chemical reaction with a compound selected from halo esters of dlcarboxylie acids, such as bie(3fS-dibromo-sallcyl) glutarate; bl0(3,S-dibromoeallcyl) adipate, bis (3,S~dibromosallcyl) fumarate; bis(3,5-dlchlorosallcyl) fumarate; bls(3#5->diiodoealicyl) fumarate; bis(3,5~ dibromosalicyl) succinate* . The reaction introduces a covalently bound glutarate (or adipate, succinate, fumarate etc) bridge between the two beta chains of the hemoglobin molecule. This modification brings about an Improved intravascular retention and oxygen transport capability compared to the original stroma~free hemoglobin solution. The g0 at normal physiological conditions of the solution after this lntramolecularly stabilization increases to 23 torr, a value approaching the normal 50 of whole human blood. There are several variations for carrying out this reaction but in particular suitable for the present invention was the route described by R.W.Tye et al (Advances in Blood Substitute Research, Alan R. Lies Inc. 1983» p. 41-49). The third step is the crucial one for obtaining the blood substitute with the improved life time in circulation and - 20 - adequate oncotic pressure. This step involves the polymerization under anaerobic conditions of the stabilized stroma-free hemoglobin obtained in the previous step. This polymerization is carried out using polyfunctional agents having groups that are capable of reacting with amino groups and other linkable sites on the hemoglobin molecule. In particular useful is the reaction with bi- or polyfunctional carbonyl containing . compounds. Within this group of materials glutaraldehyde was found to produce excellent results. It was found that the preferred molar ratio of polymerizing reagent - to hemoglobin ranges between 111 to 30)1. The oxygen-free environment, required in this step is maintained by continuously bubbling a stream of inert gas such as nitrogen. Preferably, the reaction is carried out on a stabilized stroma-free hemoglobin solution having a hemoglobin concentration of between 7 to 16% w/v. In order to prevent foaming during the reaction addition of an antifoamlng reagent is suggested.

Optionally, at the end of this step, the product may be quenched and/or reduced with sodium borohydride in order to Improve its qualities.

EXAMPLE.

STEP 1.

A solution of washed dissolved crystals of stroma-free hemoglobin was prepared as per the method of De Venuto et al. (J. Lab. Clin. Med. 89, 509-516, 1977) and were dialyzed at 4°C, first twice against distilled water (It 24 by volume) in order to reduce the potassium and phosphate content and then three times more (1x16 by volume) against Tris-HCl buffer (0.1 M, pH 7.4 also containing 0.2 g/1 ascorbic acid and 1 g/1 glucose). At the end of this procedure the pH of the reteriate solution was 7.4.

In order to ensure sterility to the resulted solution, sodium penicillin G, gentamycin and streptomycin sulfate were added to final concentrations of: - 50,000 units/1 sodium penicillin G. - 50 mg/1 gentamycin; and - 50 mg/1 streptomycin sulfate.

STEP 2.

An amount of 200 ml of the stroma-free hemoglobin solution obtained in step 1, with a concentration of 1556 W-V was introduced into a 500 ml closed Pyrex container together with 1.0 ml of caprylic alcohol as antifoaming - 22 - 1' agent. An oxygen-free environment was achieved by bubbling continuousl a stream of nitrogen, and a deoxygenated solution was added, consisting o 0*67 g bis-(3,5-dibromosalicyl) fumarate dissolved in 20 ml Tris-HCl buffer (0.5 M, pH 8,0). The reaction was carried out in the absence of oxygen, nitrogen gas being bubbled continuously for about two hours, at 35°C. The resulting mixture was twice centrifuged, each time for 30 minutes at 4°C and 12,000xg,, in order to eliminate particulate matter. The clear solution thus obtained was subsequently dialyzed three times at 4°C each time against 24 volumes of sodium phosphate buffer, (0.05 M, pH 7.5) also containing 0.2 g/1 ascorbic acid. At the end of this procedure, the reagents for ensuring sterility were added to the retenate to reach the concentrations as mentioned in step 1 for the final stroma-free hemoglobin solution. STEP 3.

An amount of 20 ml solution of the stabilized stroma-free hemoglobin, obtained in step 2 and containing 12% (w/v) hemoglobin obtained in step 2, was introduced into a 100 ml closed pyrex container together with 0.1 ml of caprylic alcohol (as antifoamlng agent). A continuous stream of nitrogen gas was bubbled through the solution - 23 ■ - for about 1.5 hours. To the resulted deoxygenated solution, 0.75 ml of deoxygenated solution of glutaral-aldehyde [2,5% w/v) in sodium phosphate buffer (0.05 M pH 7.5) was slowly added, while the stream of nitrogen gas continued. The reaction was continued for about three hours at 25°C still under nitrogen streaming.

In order to ensure the elimination of the residual active groups, the product obtained above was quenched with 2.0 ml deoxygenated solution of 1.0 M ethanolamine in sodium phosphate buffer (0.05 M), the pH of which was adjusted to 7.5 with phosphoric acid. After 1 hour quenching at 25°C, 0.6 ml of sodium borohydride solution (20 moles/ mole of hemoglobin)in 1 mM NaOH were added anaerobically, and nitrogen bubbling into the solution was continued. After 2 hours, the reaction was terminated by dialysis, at 4°C, four times (1:20 V/V each time) against a standard kidney dialysis fluid [for composition see: De Venuto et al., J. Lab. Clin. Med. 89, 509-516 (1977)3 also containing 0.2 g/1 ascorbic acid, the pH being adjusted to 7.4 with lactic acid.

The hemoglobin-based blood substitute material was further treated (sterilization, etc.) to obtain the transfusion solution as known in the art. - 24 Preliminary toxicity tests were performed on six white mice with transfusion solution of the blood substitute material obtained according to the present Invention. Thus, an amount of 0.1 ml of the blood substitute solution containing 14g/100 ml hemoglobin was injected through the tail of each mouse. Normal behaviour of the mice was noticed until they were sacrificed fifteen days after the transfusion.

Claims (12)

- 25 - 87706/3

1. C L A I M S :- 1 . Hemcglobin-based. blood substitute, resulted from stroma-free hemoglobin intramolecularly stabilized with difunctional reagents selected from haloesters of dicar-boxylic acids, and subsequently polymerized using polymerization reagents selected from, glutaraldehyde and precursors thereof capable of reacting with linkable groups on the hemoglobin molecule under anaerobic conditions, being characterized by the following properties: - possessing a hemoglobin concentration in the range of 10 to 18 g/100 ml; - possessing a P5Q in the range of 20 to 23 torr; - possessing a colloid osmotic pressure in the range of 24 to 30 torr, and - possessing a methemoglobin content in. the range of 3% to 5% of the total hemoglobin.

2. Hemoglobin-based blood substitute according to Claim 1 , wherein the hemoglobin is derived from whole human blood, or packed human red blood cells.

3. Hemoglobin-based blood substitute according to Claim 1 , wherein the extent of polymerization is in the range of 50% to 80% . C - 26 - 87706/3

4. A method for obtaining hemoglobin-based blood substitute which comprises the steps of: (a) producing stroma-free hemoglobin; (b) intramolecular stabilization of the stroma-free hemoglobin by a reaction with a halo-ester of dicarboxy- lic acid; and (c) polymerization at an extent of above 50%, under anaerobic conditions, of the i ntramolecu 1 a ly stabilized hemoglobin using a polymerization reagent selected from gl utaraldehyde and precursors thereof.

5. The method according to Claim 4, wherein the molar ratio between the polymerizing reagent and hemoglobin is between 1:1 to 30:1.

6. The method according to Claims 4 to 6, wherein the stabilized, stroma-free hemoglobin solution used in the polymerization has a hemoglobin concentration in the range of between 7-16% (w/v).

7. The method according to Claims 4 to 6, wherein the polymerized, stabilized, stroma-free hemoglobin is quenched in a quencher solution.

8. The method according to Claim 7, wherein said quencher is selected from the group consisting of amines, bisulfites, alcohols, isocyanates and mercaptans. - 27 - 87706/3

9. The method according to Claim 8, wherein said amine is selected from the group consisting of ethanol-amine and lysine.

10. The method according to Claims 4 to 9, wherein the polymerized ,stabi 1 ized , stroma-free hemoglobin is reduced by a solution of sodium borohydride.

11. Hemoglobin-based blood substitute, substantially as described in the specification and claimed in any one of Claims 1 to 4.

12. A method for obtaining hemoglobin-based blood substitute, substantially as described in the specification and claimedin any one of Claims 4 to 10. For the Appl icants , Simon Lavie / Patent Attorney