CA1236014A

CA1236014A - Polysaccharide haemoglobin complexes

Info

Publication number: CA1236014A
Application number: CA000466410A
Authority: CA
Inventors: Jeffrey T. Wong
Original assignee: Fisons Ltd
Current assignee: Fisons Ltd
Priority date: 1983-10-28
Filing date: 1984-10-26
Publication date: 1988-05-03
Also published as: ATE51629T1; EP0140640A2; FI74979C; NO165709B; EP0140640A3; US4710488A; JPH0714961B2; GB8328917D0; JPS60123503A; AU567593B2; NO165709C; JPH06298802A; DE3481844D1; IL73334A; US4650786A; AU3475384A; JPH0643444B2; IL73334A0; DK509884A; FI844204L

Abstract

COMPOUNDS
ABSTRACT
There is described a water soluble compound having a molecular weight of from about 70,000 to about 2,000,000, and having the formula I, (PS)-X-(HB)-Z I

where PS represents a physiologically acceptable polysaccharide of molecular weight from about 2,000 to about 2,000,000, X represents a covalently bonded chemical bridging group, HB represents a haemoglobin residue, and Z represents an oxygen affinity reducing ligand, containing 2 or more phosphate groups.
There is also described haemoglobin linked to an oxygen affinity reducing ligand derived from inositol phosphate, and polymers of such linked haemoglobin.
Processes for making the compounds, and their formulation and use as oxygen transporting agents, are also described.

Description

6~

This invention relates to blood sub~titutes and me~hods for their preparation.
US Patent No. 4,064,118 describes a composition useul as a blood substitu~e or blood extender composition which comprises the water soluble product of covalently coupling haemoglobin with dextran or hydroxyethyl starch having a molecular weight of from about 5,000 to about

2,000,000. This US patent also describes a process for preparing such a composition, which comprises chemically coupling haemoglobin with dextran or hydroxyethyl starch of a molecular weight from about S,000 to about 2,000,000.
It has however been found that, as compared with haemoglobin, the products according to US Patent No.
4,064,118 tend to show a somewhat greater affinity for oxygen, but retain the essential oxygen transporting and releasing capability of haemoglobin. As measured by the hal~-saturation oxygen tension, the dextran-haemoglobin complex prepared by method I of US Patent No. 4,064,118 shows approximately 2.5-fold greater affinity for oxygen than does ree haemoglobin.
Pyridoxal-5~phosphate is known to bind to haemo~lobin ir. a reversible manner, and it is also known that this binding can be made irreversible by reduction, both the reversibly and the irreversibly bound products having less ~æ3.~0~

strong oxygen binding characteristics than the haemoglobin itself~ However, it has been found ~hat covalent derivatization with pyridoxal-5'-phosphate fails to reduce the oxygen affinity of dextran-haemoglobin to approach that of haemoglobin derivatiæed with pyridoxal-5'-phosphate ~PLP-Hb). An oxygen affinity close to or below that of PLP-~b is considered desirable for a satisfactory haemoglobin-based blood substitute.
We have now found that physiologically acceptable polysaccharide haemoglobin complexes, for example as described in US Patent No. 4,064,118, can be made in modified form having a lower oxygen affinity than hitherto.
According to the invention we provide a water soluble compound having a molecular weight of from about 70,000 to about 2,000,000, and having the formula I, (PS)-X-tHB)-Z

where PS represents a physiologically acceptable polysaccharide of molecular weight from about 2,000 to about 2,000,000, X represents a covalently bonded chemical bridging group, HB represents a haemoglobin residue, and Z represents an oxygen affinity reducing ligand, ~æ36031 ~

containing 2 or more phosphate groups.
We prefer Z to be a polyol, two or more of the hydroxy groups of which are esterified with phosphoric acid. We particularly prefer Z to contain from 2 to 6, and more preferably 2 to 4, phosphate groups. When Z is a phosphate ester of a polyol, at least 2, and preferably all, of the hydroxy groups have been esterified with phosphoric acid. We also prefer Z to contain from 4 to 8 carbon atoms and to be a straight chain ~roup. We 10 further prefer 2 to comprise a polyol in which each of the carbon atoms (other than at least one of the term;nal carbon atoms) carries an optionally phosphoric acid esterified hydro~y group.
The Z group may ke linked to the haemoglobin in a wide variety of ways which will be readily recognised by those skilled in the art.
Thus according to the invention we also provide a process for the production of a compound of formula I t which comprises (a) linking a compound o formula II, (PS)-X-(HB) II

in which PS, X and HB are as defined above, with a compound capable of providing a Z ligand, (b) linking a compound of formula III, (~B)-Z III

in which HB and Z are as defined above, wi~h a polysaccharide PS, or (c) producing a reduced form of a compound of formula I, by reduction of a corresponding compound of ~ormula I containing a reducible double bond.
Thus for example the Z group may be linked to an amino group on the haemoglobin by means of an amide linkage, RCONHlHBx)-X-(PS) in which X and PS are as defined above, (HBx)NH is a haemoglobin residue, and RCO is a Z group.
Such a linkage may be made by conventional acylatlon techniques, e.g. converting a compound RCOOH to an active ester therof, e.g. an N-hydroxysuccinimide ester~ and reacting the resulting ester with the preformed polysaccharide - haemoglobin complex~ However, such acylation reactions are somewhat unspecific and may lead tc linkage of the Z group to less preferred positions on the haemo~lobin.

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We therefore prefer the Z group to be linked to the haemoglobin through a Schiff's base linkage, and preferably a reduced Schiffls base linkage, R-CH-N-~HBx)-X-(PS) or R-CH2-NH-(HBx)-X-(PS) in which X and PS are a~ defined above, (HBx)-N= and (HBx)-NH- are haemoglobin residues, and R-CH= or R-CH2- is a Z group.
Such linkages may be made by conventional techniques, e.g. reacting an aldehyde RCHO with the preformed polysaccharide - haemoglobin complex, followed if necessary by selective reduction of the resulting Schiff's base. Suitable reducing agents include diethylamine borane or sodium cyano borohydride, or more preferably dimethylamine borane or sodium borohydride.
The linkage of the Z group to the (PS)-X-(HB) moiety is efected by procedures which are not affected by the size of the ~PS)-X-~HB) moiety. Thus a wide range of sizes of product molecules can be made with differing ranges of polysaccharide to haemoglobin ratios.
We prefer the Z group to be derived from an inositol phosphate.
Inositol phosphates are known compounds and may be 25 made and isolated in a manner which is also known E~ se We particularly prefer ~he Z group to be derived from inositol tetraphosphate, e.g. from a mixture containing a major proportion of inositol tetraphosphate and a minor proportion of other inositol phosphates.
Thus the compound RCHO is preferably an inositol phosphate aldehyde, and more preferably an inositol phosphate dialdehyde of formula IV, OHC-CHRx-CHRx-CHRx-CHRx-CHO IV
in which at least 2, and preferably 4, of the groups Rx are phosphate groups and the remainder are -OH
groups. Compounds of formula IV in which less than four groups Rx are phosphate groups exist as structural isomers 15 and also as a variety of stereoisomers. Compounds of formula IV in which all four groups Rx are phosphate groups exist as a variety of stereoisomers. The co~pounds of formula IV may, if desired, be separated into ~heir various structural and/or optical isomeric forms, 20 using conventional techniques known per se.
Alternatively, and preferably, the compounds of formula IV
may be used as a mixture for further reac~ion with the haemoglobin or haemoglobin-polysaccharide complex.
The compounds of formula IV can be made by selestive 25 oxidation of an ~ppropriate inositol pl~osphate having two adjacent free hydroxy groups. The oxidation may suitably be carried out using mildly oxidising conditions, e.g. by use of a perhalo acid, such as periodic acid, The reaction may conveniently be carried out at a low p~ or an elevated temperature, particularly when an inositol tetrapho~phate is used as starting material.
The polysaccharide-haemoglobin complex may be derived from a wide variety of polysaccharides, e.g. a hydroxy alkyl starch (such as hydroxyethyl starch), inulin or preferably ~extran. More particularly we prefer the complex to be one which has been prepared by the reaction of haemoglobin with N-bromoacetylaminoethylamino dextran as described by Tam, Blumenstein and Wong (1976) Proc.
Nat. Acad. Sci. USA 73, 2128-2131 or Example 1 of British Patent Specification No 1,549,246. We prefer the polysaccharide to be of molecular weight of from about 5,000 to 2,000,000, e.g. to be of average molecular weight 10,000 to 100,000. We prefer the polysaccharide to be a dextran of average molecular weight 70,000, 40,noo or ~0,000.
A specific dextran-haemoglobin complex which may be used is a 1:1 complex between human haemoglobin and dextran of about 20,000 molecular weight (J ~ Chang and J T Wong, Canadian Journal of Biochemistry~ Vol. 55, pp ~98-403, 1977).

0~

The proportion of Z groups in the polysaccharide complex (or in the compound of formula III) is preferably such that the phosphate to haemoglobin ratio is in the range 2 to 16 : 1, pre~erably 4 to 16 : 1 as determined by the method described in Example 1.
When a dialdehyde is used to produce the group Z some cross-linking between haemoglobin moieties may take place or the two aldehyde groups may react with different amino groups on the same haemoglobin molecule.
The modified haemoglobins of formula (HB)-Za in which Za is a group derived from an inositol phosphate, and in particular in which Z is derived, as described above, from an inositol phosphate aldehyde, are rew compounds and form a feature of the present invention. These new modified haemoglobins have oxygen transporting capabili~y and, in addition to their utility for linking to a polysaccharide, are useful in their own right, or in polymerised form, as oxygen transporters capability.
Thus according to the invention we further provide ~0 polymerised (HB)-Za and a process for the production of pol~merised (HB)-Za, which comprises polymerisation of ~HB)-Za.
The polymerisation of the (HB)-Za may be carried out using processes which are known ~ se for the 2~ polymerisation of haemoglobin, e.g. by reaction of the haemoglobin with glutaraldehyde, for example at a temperature of 0 to 10C in an aqueous solution. The reaction may be carried out in a buffer to maintain an approximately neutral p~. The (B)-Za need not be 5 deoxygenated before polymerisation. The polymerised product is advantageous in that its oxygen dissociation curve is right shifted compared to known polymerised haemoglobins or modified polymerised haemoglobins~ The degree of polymerisation can vary with the particular 10 purpose for which the product is desired. However, in general too high a degree of polymerisation will make the solutions of the polymer ~oo viscous while too low a degree of polymerisation will leave many free haemo~lobin molecules which may cause damage to the ~idneys.
As an alternative to linking the Z group to the preformed haemoglobin polysaccharide complex (process a) above), the Z group may first be attached to the haemoglobin, e.g. using the techniques described above or other conventional linking techniques. The modified 20 haemoglobin may then, if desired, be further linked to the polysaccharide (process b) above), e.g. using the techniques described in US Patent No 4,064~1180 The haemoglobin referred ~o in this specification may be derived from any appropriate animal, e.g. a bovine 25 animal, but is preferably human haemoglobin.

The compounds of the invention, i.~. compo~nds of formula I, compounds Hb-Za and polymerised ~b-Za, are useful in that they have oxygen transporting capability.
Thus the compounds are useful as immobilised oxygen extractants, for example in the system described in US
Patent No 4,343,715. ~he compounds are also indicated for use in blood substitute or blood expander compositions~ Thus the compounds may be used to provide enhanced oxygenation of poorly perfused ~issues. Such poorly perfused tissues may be present in cancerous growths, in cases of myocardial infarction or in cases of cerebral haemorrhage. The compounds may also be used as blood substitutes, e.g. for the victims of ac~idents or violence; where blood typing and matching is not possible or is not possible in the time available; where patients are at risk from, or refuse, normal blood transfusion;
for the purpose of delivery of oxygen to tissues or organs which are to be preserved; for priming extracorporeal ciraulatory systems; or for other situations where 20 erythrocytes are normally indicated.
The compounds may be administered to the patients concerned in admixture with a pharmaceutically acceptable exicipient, diluent or carrier, for example as an aqueous solution, which may be a buffered balanced salt 25 solution. In general the compounds will be administered ~3~

using types of formulations, packages and forms of administration which are conventional for the administration of blood plasma expanders. The compounds may also be freeze dried, optionally with a cry~protective agent, and subsequently be reconstituted for use~
The amount of the compound which is administered will vary with the size, condition of the patient and treatment desired. However, in certain severe instances substantially all the patient's blood may be replaced by a formulation containin~ a compound of the invention.
~ he compounds of formula I, and the other compounds of the invention, are advantageous in that they possess more desirable oxygen absorption and release properties than similar known compounds. Thus certain compounds of the invention have oxygen dissociation curves (cf Example

3) which are more right shifted than phosphopyridoxylated haemoglobin. The compounds of the invention are also advantaqeous in that they may be prepared easily and in relatively high yield.
~ Molecular weights in this specification are expressed as Mw rather than Mn The invPntion is illustrated, but in no way limited by the following Examples.
Example 1 (a) Preparation of Inosi~ol Tetrakisphosphate Sodium phytate (inositol hexaphosphate) was dephosphorylated at with soluble wheat phytase as described by R V Tomlinson and C E Ballou, Biochemistry 1, 166-171(1962). Alternatively wheat bran washed twice with 50% ethanol and once with 95% ethanol may be used as the source of phytase. Dephosphorylation was allowed to proceed until no significant amounts of inositol hexaphosphate or inositol pentaphosphate remained in solution. The amount of phytase employed was chosen so that this procedure required about 72 hours. The reaction mixture was clarified by filtration followed by centrifugation. IM FeC13 was added to the resulting solution to give a 3:1 molar ratio of FeC13: original phytate. The yellow precipitate was collected by lS centrifugation and resuspended in distilled water. Solid NaOH was added to the suspension to give pH 12, and the mixture was stirred and maintained at p~ 12 for 3 hours.
Af~er filtration, the filtrate was neutralised with acetic acid to pH 5.2. The filtrate obtained from 5 litres of digest (containing originally 50mM inositol hexaphosphate) was loaded on a 1 litre column of DowexPl resin equilibra~ed with distilled water. After loading, the column was washed with 1 litre distilled water, and eluted with 7 litres of 0.3 N HCl and then 4 litres of 1 ~ HCl.
25 Fractions containing the inositol tetraphosphate were ~ r~

precipitated with 3 volumes of 95% ethanol. After standing overnight at 4C, the supernatant was poured off, leaving a viscous precipitate. Absolute ethanol was added until the precipitate turned into a white powder.
S The powder was washed with absolute ethanol, 1:1 ethanol:ether and finally ether to yield the desired product.
~b) Oxidation of Inositol Tetrakisphosphate A solution of 119 of inositol tetrakisphosphate (prepared as in step a)) in 322ml of 0.6 M ~I04.2H20, was incubated in the dark at 22C for eight hours. The reaction mixture was neutralised to p~ 5.2 with SN KOH, kept at 0C for 10 minutes and then centrifuged to remove KI04. Ascorbic acid (56.7 g) was added to the lS supernatant. After 10 minutes 3 volumes of ethanol were added and the mixture was kept at 4C for 1 houx.
clear viscous precipitate was collected by centrifugation. The precipitate was added to 100ml of absolute ethanol. Upon trituration the precipitate became 20 more solid, and eventually broke up into a powder as the ethanolic supernatant was replaced by 4 changes, 50 ml each, of absolute ethanol. The resulting white powder was washed with ethanol, 1:1 ethanol:ether and ether successively, and air dried. ~he yield was 7.79 o the 25 dialdehyde derivative of inositol tetrakisphosphate (FPA).

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(c~ Covalent ~inkaqe of FPA to dextran haemoqlobin with dimethylamine-borane (DM~B~
To 0.25ml 6% w/w (measured on the haemoglobin alone) dextran haemoglobin (Dx-Hb) produced by the method of Example 1 of BP 1,549,246, save that de~tran of molecular weight 20,000 was used, in 0.05M (bis~[2-hydroxyethyl]
amino-tris-~hydroxymethyl])methane (bis-tris) p~ 7.4, was added 1.25 mg of FPA in 0.17 ml 0.05 M sodium acetate buffer, pH 5.2 (about 7.3 FPA:l Dx-Hb). The pH of the mixture was adjusted to 7.4 with dilute NaOH, 0.025 ml of 0.5 M dimethylamine borane (DMAB~ was added, and the solution was incubated for 2 hours a~ 0C. The resulting mixture was applied to a 30ml Sephadex G25 column equilibrated in 0.lM 2-amino-2-(hydroxymethyl)-1,3-propandiol (tris) lN NaCl pH 8.5 buffer, and run at4C. The first two-thirds of the peak was collected, dialysed against bis-tris buffer, 0.05M, pH7.4 overnight and ~hen used for oxygen dissociation determination. The use of the Sephadex column may, if desired, be omitted.
~d) Covalent Linkage_of FPA to Dx-Hb with NaBH4 To 0.25ml 6~ Dx-Hb (as used in c) above) in 0.05M
bis-tris pH 7.4 was added 3.41mg of FPA in a 20mg/ml solution (20FPA:lDx-Hb). The final p~ of the solution was 7Ø After a 20 minute incubation at 0C, 0.02$ml of 0.8M NaBH4 was added. The solution was incubated ~23~

for a further 2 hours a~ 0C. The reaction mixture was then applied to a 30ml Sephadex~G25 column equilibrated in 0.lM tris lN NaCl pH 8.5 and run at 4C. The first two-thirds of the peak was collected, dialysed as in 5 paragraph c) above, and used for oxygen dissociation determination. Again the use of the Sephadex column, may, if desired, be omitted, In an alternative method 0.2ml of 6~ Dx-Hb (as used in c) above) in 0.05M bis-tris p~ 7,4 was added 0.68mg of 10 FPA in a 5mg/ml solution (SFPA:lDx-Hb). The mixture was incubated at 0C for 20 minutes, and 0.02ml of 0.5M
NaBH4 was added at 0C, over a period of 2 hours with constant stirring.
(e) Covalent Linkage of FPA to Hb with Dimethylamine-15 Borane or NaBH4 To 0.2Sml 6% w/w haemoglobin dissolved in 0~05M bistris at p~ 7.4, was added 1.25mg of FPA in 0.17ml 0.05M
sodium acetate buffer, p~ 5.2. The pH of the mixture was adjusted to 7.4 with dilute NaO~, 0.025ml of 0.5M
~0 dimethylamine borane was added, and the solution was incubated for 2 hours at 0C. The reaction mixture was applied to a 30ml Sephadex G25 column equilibrated in 0.lM
tris buffer, pH 805, containing 1 N NaCl, and run at 4C. The first two-thirds of the peak was collected, ~5 dialysed against bis-tris buffer~ 0.05M, pH 7.4 overnight ~23~

and then used for oxygen determination. (The use of theSephadex column may, if desired, be omitted.) The FPA-~b so obtained has an oxygen dissociation curve that is right-shifted as compared to free Hb, and therefore may be employed as oxygen carrier where a low affinity for oxygen is of importance, either for the purpose of blood substitution or ~or incorporation into an immobilised oxygen extractant. NaBH4 may be used in place of the DMAB. The FPA-Hb so produced is also useful in parts (f) and (9) below.
(f) Covalent Linka~e of Cyanoqen Rromide-~ctivated Dextran to FPA-Hb 29 of dextran with molecular weight of 70,000 was dissolved in a mixture of 80% formamide-~0% water v~v, and lS the solution was chilled to -15C. 1.69 of CNBr in 3.2ml of 80~ formamide-20% water v/v at 4C was then added with stirring. Further addition of 15.lml of 1.5M
triethylamine in dimethyl formamide at -15C was carried out dropwise with stirring. After incubation for 10 minutes at -15C, one volume of acetone (at -20C) was added to the mixture to precipitat~ the activated dextran. The precipitate was washed twice with acetone (-20C) by centrifugation, once with ether, and air-dried to yield an activated dextran. 35mg of the activated dextran was added to lml of FPA-Hb (3~ with Z~6~

respect to~ Hb)in 0.05 M bis-tris, pH 7.4, and the solution was held at 0C for 16 hours.
0.15ml of the FPA-Dx-Hb obtained by this method (8%
with respect to ~b) was separated ~rom the uncoupled S FPA-Hb by chromato~raphy on a 118ml Sephadex ~150 column to produce thè optical density trace at 576nm shown in Fig. 1. The arrow indicates the position of uncoupled FPA-Hb.
(g) Preparation of Poly-FPA-~b Usin~ Glutaraldehyde as 10 Polymerisin~ A~ent To 6.5 ml of FPA-Hb (6~ wi~h respect to ~b) in 0.1 M
sodium phosphate buffer, pH 7.5, at 4 C, was added 0.195 ml of 53 glutaraldehyde in the same buffer. The mixture was kept at 4C for 16 hours and 0.15ml of the mixture 15 separated by chrQmatography on 118ml of a 5ephadex G150 column to produce the optical density trace at 576nm shown in Fig. 2. The arrow indicates the position of free Hb.
Phosphat Determination Up to 0.5ml aliquots of the FPA-Dx-Hb or FPA-Hb 20 solutions were digested by addition of 1.2ml of 70%
HC104, and then boiling until clear. 8ml ~2~ 0.05ml 5~ ammonium molybdate and 0.05ml 0.25%
l-amino-2-naphthol-4-sulphonic acid were then added. The resulting solutions were boiled for 10 minutes and the 25 absorbance or optical density at 820 nm was read to ~3~

determine phosphate concentration.
The haemoglobin concentration was determined by the method of D L Drabkin and J H Austin, J Biological Chemistry, Vol. 112, pp51-65 (1935).
S Preparation Reduction Phosphate/
Method ~b4 FPA-Dx-Hb made from bromodextran MW 20,000 DMAB 8.4:1 and human Hb FPA-Dx-Hb made from bromodextran MW 20,000 NaBH4 418:1 and human Hb FPA-Dx-Hb made from bromodextran MW 20,000 DMAB 10.9:1 and bovine ~b FPA-Hb made from human ~b DMAB 10.4:1 15 FPA-~b made from bovine Hb DMAB 7.6:1 ~Note: FPA-Dx-~b obtained by linking dextran to FPA-Hb and polyFPA-Hb both start from FPA-Hb, therefore their ph~sphate/~b4 ratio will be identical to that of the parent FPA-Hb).
Example 2 a) 0.5 Mmole of 2,3-diphosphoglycerate was dissolved in lOml water and passed through pyridine-form of Dowex~or AG)-50 column. The effluent was collected and concentrated under redu_ed pressure~ Water was removed by means of adding anhydrous pyridine and concentrating.

tr~l~km~K

:~3~

Remaining water and pyridine were removed by washing withanhydrous dimethyl formamide (twice) until there was no smell of pyridine. 6 Mmole of aminoacetaldehyde diethylacetal was added using anhydrous dimethyl sulphoxide as solvent, and mixed by shaking. 6 Mmole of dicyclohexylcarbodiimide was added and the mixture stirred ~or 3-4 hours at room tempera~ure. A small amount of water was then added and the precipitate was filtered off. The filtrate was extracted (3 X) with anhydrous ether in order to remove residual reactants and the solution concentrated to dryness to yield a white powder product.
b) The crude product of step a) (equivalent to about 0.5 Mmole of original 2,3-diphophroglycerate) was dissolved in a small quantity of water The solution was passed through Dowex-50 H+-form to remove free amine.
The acidic portion of effluent was collected, concentrated to about 2ml, and 0.5ml 1 N ~Cl added. The resulting solution was left at 4C for 30-60 minutes and an 20 equivalent amount of NH4HC03 was added to adjust the p~ to about 7.
c) 3~ ~oles of 6% dextran-haemoglobin (either oxygenated or deoxygenated), produced by the method of Example 1 of BP 1,549,246, was dissolved in 0 05M tris at p~ 7.4~ The 25 product of step b) (15 ~ mole) and sodium cyanoboro-~36~

hydride (60~ mole) were added and the reaction mixturemaintained at room temperature or 2 hours. Excess product of step b) was then removed either (i) by dialysis against lM NaCl/0.05M tris at pH 8.5, or (ii) by passage through Sephadex C25 equilibrated wi~h lM NaC1/0.05M tris at pH 8.5, and collection of the front end peak.
ExamPle 3 The oxygen dissociation curves of the products of Example 1, and of the starting haemoglobin dextran were determined using the method of Benesch, MacDuff and Benesch (1965) Anal. Biochem. 11 81-87 (1965), but using a temperature of 24C and bis-tris buffer, 0.05M, at a pH
of 7.4.
The results are shown in the attached figures 3 and 4.
Fig. 3 shows the result for the product of ~xample lg) before ~eparation on the Sephadex column.
Fig. 4 shows at 1 the result for the dextran haemoglobin starting material for Example lc), at 2 the result ~or the product of Example ld), at 3 the result for the product of Example lc) and at 4 the result for ~he product of Example (e) using DMAB.

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Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A process for the production of a water soluble compound having a molecular weight of from about 70,000 to about 2,000,000, and having the formula I, (PS)-X-(HB)-Z I

where PS represents a physiologically acceptable polysaccharide of molecular weight from about 2,000 to about 2,000,000, X represents a covalently bonded chemical bridging group, HB represents a haemoglobin residue, and Z represents an oxygen affinity reducing ligand, containing 2 or more phosphate groups, which comprises (a) linking a compound for formula II, (PS)-X-(HB) II

in which PS, X and HB are as defined above, with a compound capable of providing a Z ligand, (b) linking a compound of formula III, (HB)-Z III

in which HB and 2 are as defined above, with a polysaccharide PS, or (c) producing a reduced form of a compound of formula I, by reduction of a corresponding compound of formula I containing a reducible double bond.

2. A process according to Claim 1, wherein z is a polyol, two or more of the hydroxy groups of which are esterified with phosphoric acid.

3. A process according to Claim 1, wherein z contains from 2 to 6 phosphate groups.

4. A process according to any one of Claims 1, 2 or 3 wherein Z is derived from a compound of formula IV, OHC-CHRx-CHRx-CHRx-CHRx-CHO IV

in which at least 2 of the groups Rx are phosphate groups an the remainder are -OH groups.

5. A process according to any one of Claims 1, 2 or 3, wherein the polysaccharide is a dextran of average molecular weight of from 10,000 to 100,000.

6. A process according to any one of Claims 1, 2 or 3, wherein the phosphate to haemoglobin ratio is in the range 2 to 16:1.

25819H/jaa

7. A water soluble compound having a molecular weight of from about 70,000 to about 2,000,000, and having the formula I, (PS)-X-(HB)-Z I

where PS represents a physiologically acceptable poly-saccharide of molecular weight from about 2,000 to about 2,000,000, X represents a covalently bonded chemical bridging group, HB represents a haemoglobin residue, and Z represents an oxygen affinity reducing ligand, containing 2 or more phosphate groups.

8. A compound according to Claim 7, wherein Z is a polyol, two or more of the hydroxy groups of which are esterified with phosphoric acid.

9. A compound according to Claim 7, wherein Z contains from 2 to 6 phosphate groups.

10. A compound according to any one of Claims 7, 8 or 9, wherein Z is derived from a compound of formula IV, OHC-CHRx-CHRx-CHRx-CHRx-CHO IV

in which at least 2 of the groups Rx are phosphate groups and the remainder are -OH groups.

11. A compound according to any one of Claims 7, 8 or 9, wherein the polysaccharide is a dextran of average molecular weight from 10,000 to 100,000.

12. A compound according to any one of Claims 7, 8 or 9, wherein the phosphate to haemoglobin ratio is in the range 2 to 16:1.

13. A pharmaceutical formulation comprising a compound of formula I, as defined in Claim 7, in admixture with a pharmaceutically acceptable excipient, diluent or carrier.