U.S. patent application number 10/303777 was filed with the patent office on 2003-07-03 for hemoglobin-polysaccharide conjugates.
This patent application is currently assigned to Hemosol, Inc.. Invention is credited to Adamson, Gordon J..
Application Number | 20030125238 10/303777 |
Document ID | / |
Family ID | 4162275 |
Filed Date | 2003-07-03 |
United States Patent
Application |
20030125238 |
Kind Code |
A1 |
Adamson, Gordon J. |
July 3, 2003 |
Hemoglobin-polysaccharide conjugates
Abstract
Hemoglobin conjugates useful as a hemoglobin-based oxygen
carriers are prepared by reacting hemoglobin with oxidatively
ring-opened polysaccharides such as hydroxyethyl starch or dextran,
and storing the resultant conjugate under conditions which allow it
to transform to a lower molecular weight product, after
conjugation. The conjugate is then reductively stabilized to form
secondary amino bonds between the hemoglobin and the
polysaccharide, and formulated as an HBOC.
Inventors: |
Adamson, Gordon J.;
(Georgetown, CA) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
1100 N GLEBE ROAD
8TH FLOOR
ARLINGTON
VA
22201-4714
US
|
Assignee: |
Hemosol, Inc.
|
Family ID: |
4162275 |
Appl. No.: |
10/303777 |
Filed: |
November 26, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10303777 |
Nov 26, 2002 |
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09276686 |
Mar 26, 1999 |
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6500930 |
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Current U.S.
Class: |
530/385 ;
514/13.4 |
Current CPC
Class: |
A61K 9/0026 20130101;
A61P 7/00 20180101; A61K 47/61 20170801; A61P 7/08 20180101 |
Class at
Publication: |
514/6 ;
530/385 |
International
Class: |
A61K 038/42; C07K
014/805 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 31, 1998 |
CA |
2,233,725 |
Claims
What is claimed is:
1. A polysaccharide-hemoglobin conjugate useful as an oxygen
transporter, comprising hemoglobin covalently linked through
secondary amine linkages between amino groups on the hemoglobin and
residues of aldehyde groups on the polysaccharide produced by
oxidative saccharide ring-opening thereof, the secondary amine
linkages having been formed by reaction of said amino groups and
said aldehyde groups in a first stage to form Schiff base linkages,
and in a second, subsequent stage by reduction to effect
stabilization, the conjugate having no detectable residual unbound
hemoglobin and no detectable residual amounts of components of
molecular weight higher than about 500 kDa.
2. The conjugate of claim l wherein the polysaccharide is
hydroxyethyl starch or dextran
3. The conjugate of claim 2 wherein hemoglobin is human
hemoglobin.
4. The conjugate of claim 3 herein the hemoglobin is
deoxyhemoglobin.
5. The conjugate of claim 3 wherein the hemoglobin is
intramolecularly cross-linked.
6. The conjugate of claim 3 wherein the polysaccharide is
hydroxyethyl starch of molecular weight from about 70 to about 1000
kDa.
7. The conjugate of claim 6 wherein the hydroxyethyl starch has a
substitution ratio from about 0.5 to 0.7.
8. The conjugate of claim 6 having a P50 from 4-50 at 37.degree.
C.
9. A process of preparing a hemoglobin based oxygen carrier which
comprises reacting an oxidatively ring-opened polysaccharide
carrying aldehyde groups, with hemoglobin, to form a conjugate
thereof, maintaining the conjugate under controlled conditions of
aqueous solution with predetermined pH to effect controlled
molecular weight reduction and molecular weight re-distribution of
the conjugate, stabilizing the conjugate by reduction of the Schiff
base linkages, between the polysaccharide and the hemoglobin to
stable, secondary amine linkages, and recovering a solution of the
polysaccharide-hemoglobin conjugate so formed which has no
detectable unbound hemoglobin residue and no detectable product
residue of molecular weight greater than about 500-600 kDa.
10. The process of claim 9 wherein the polysaccharide is dextran or
hydroxyethyl starch(HES).
11. The process of claim 10 wherein the conjugate is maintained in
aqueous solution under pH conditions from about 7.2-10, at
temperatures from about 15-30.degree. C. to effect molecular weight
reduction and molecular weight re-distribution.
12. The process of claim 11 wherein the molecular weight reduction
and molecular weight redistribution is terminated at a
predetermined extent by said stabilization of the conjugate by
reduction of the aldehyde-amine bonds to secondary amine
linkages.
13. The process of claim 12 including a single stage of reduction,
to effect said stabilization and substantially simultaneously to
reduce residual aldehyde groups.
14. The process of claim 12 including two stages of reduction, the
first to effect said stabilization and the second to reduce the
aldehyde groups.
15. The process of claim 13 wherein the reduction is effected with
a boron-based reducing agent.
16. The process of claim 15 wherein the reducing agent is borane
dimethylamine.
Description
FIELD OF THE INVENTION
[0001] This invention relates to biocompatible oxygen carriers for
administration to patients as a supplement for or a partial
replacement for whole blood. More specifically, the invention
relates to hemoglobin-based oxygen carriers (HBOCs) for
administration to mammals as a blood substitute or supplement, and
processes for their preparation.
BACKGROUND OF THE INVENTION
[0002] Hemoglobin, as the natural oxygen transporter component of
blood, is an obvious candidate to form the basis of a blood
substitute, e.g. as an aqueous solution. Extensive scientific work
has been done and reported, on attempts to provide a satisfactory
hemoglobin solution to act as a blood substitute. The chemical
properties of hemoglobin outside the red blood cells are, however,
markedly different from its properties inside the red blood cells,
e.g. as regards its oxygen affinity. The need for some form of
chemical modification of hemoglobin to render it suitable for use
as a blood substitute has long been recognized and has been quite
expensively investigated.
[0003] It is well known that hemoglobin comprises a tetramer of
four sub-units, namely two .alpha. sub-units each having a globin
peptide chain and two .beta. sub-units each having a globin peptide
chain. The tetramer has a molecular weight of approximately 64
kilodaltons, and each sub-unit has approximately the same molecular
weight. The tetarameric hemoglobin in dilute aqueous solution
readily dissociates into .alpha.-.beta. dimers, and even further
under some conditions to .alpha.-sub-unit monomers and
.beta.-sub-unit monomers. The dimers and monomers have too low a
molecular weight for retention in the circulatory system of the
body, and are filtered by the kidneys for excretion with the urine.
This results in an unacceptably short half life of such a product
in the body. The benefit of chemical bonding between the sub-units
to ensure the maintenance of the tetrameric form ("intramolecular
cross-linking") has previously been recognized. Also, the linking
together of two or more tetrameric units to form hemoglobin
oligomers and polymers of molecular weight greater than 64
kilodaltons ("inter-molecular cross-linking") has also been
recognized as desirable in many instances.
[0004] Accordingly, one approach to developing HBOCs for clinical
use has been intramolecularly cross-linking the hemoglobin units
into stabilized tetramers, of molecular weight c. 64 kilodaltons,
and optionally oligomerizing these tetramers into oligomers of 2-6
such tetramers, by intermolecular cross-linking. A variety of
cross-linking reagents have been proposed for this purpose,
including oxidatively ring-opened saccharides such as o-raffinose
(U.S. Pat. No. 4,857,636 Hsia and U.S. Pat. No. 5,532,352 Pliura et
al., for example), bifunctional imidates such as
diethyl-malonimidate hydrochloride (U.S. Pat. No. 3,925,344 Muzur),
halogenated triazines, divinylsulphones, diisocyanates,
glutaraldehyde and other dialdehydes (U.S Pat. No. 4,001,200 Bonsen
et al.), bis-diaspirin esters (U.S Pat. No. 5,529,719 Tye), bis-
and tris-acyl phosphates (U.S. Pat. No. 5,250,665 Kluger et al.)
and others.
[0005] Another approach to the preparation of HBOCs with
appropriate molecular weight for clinical use has been the coupling
of hemoglobin to a biocompatible polysaccharide. Such conjugates
would have the advantage as compared with cross-linked and
oligomerized hemoglobins of requiring lower quantities of
hemoglobin per unit of HBOC, and hence would be more economical to
prepare, and have diminished hemoglobin-related toxicities.
Conjugation of a colloid to hemoglobin in preparing an HBOC also
permits control of fluid properties such as viscosity and colloid
osmotic pressure by adjusting the size of the colloid, its degree
of modification and the colloid-to-hemoglobin ratio. These same
parameters can be used to control the final molecular weight and
vascular retention time of the product.
[0006] U.S. Pat. No. 4,064,118 Wong proposes the preparation of a
blood substitute or blood extender by chemically coupling
hemoglobin with a polysaccharide material selected from dextran and
hydroxyethyl starch of molecular weight from about 5 kDa-2,000 kDa.
Only the use of dextran is exemplified in this patent, however.
[0007] Baldwin et al. "Tetrahedron" 37, pp 1723-1726 (1981)
"Synthesis of Polymer-Bound Hemoglobin Samples" describe the
chemical modification of dextran and hydroxyethyl starch (HES) to
form aldehyde-substituted polymers, and their subsequent reaction
with hemoglobin, to form soluble, polymer-bound hemoglobin. Whilst
the products so formed were capable of binding oxygen, they are
reported as unsuitable for use as blood substitutes, since their
oxygen-binding curves were considerably left-shifted, indicating
that they have too high an oxygen affinity (P.sub.50 too low).
SUMMARY OF THE INVENTION
[0008] It is an object of the present invention to provide a novel
HBOC.
[0009] It is a further object of the invention to provide a novel
polysaccharide-hemoglobin conjugate useful as an HBOC.
[0010] It is a further object to provide a process for preparing a
novel polysaccharide-hemoglobin conjugate useful as an HBOC.
[0011] In the process of the present invention, a polysaccharide is
used, in oxidatively ring-opened form. In this oxidative form, at
least a portion of the saccharide monomeric units are oxidized to
present aldehyde groups. The oxidized polysaccharide so formed is
then reacted with extracellular hemoglobin, so that the hemoglobin,
through primary amine groups of the globin chains reacting with the
aldehyde groups of the oxidized polysaccharide, covalently binds to
the polysaccharide through Schiff base linkages. Initially and very
rapidly there is formed a product which includes species of very
high molecular weight, of the order of 500 kDa or higher, in
substantial amounts and a wide molecular weight distribution
(128.fwdarw.500 kDa).
[0012] On maintaining this product under appropriate conditions, in
aqueous solution, it can be transformed, to a controlled extent,
over a relatively short period of time (e.g. 4-48 hours depending
upon the conditions) to a much lower molecular weight product
(90-200 kDa) with a much narrower molecular weight distribution.
This product, after chemical reduction to reduce the Schiff base
linkages between the hemoglobin and the polysaccharide to secondary
amine bonds, turns cut to have properties such as oxygen affinity
in the range P.sub.50=4 to 50 mmHg at 37.degree. C., depending on
the ligand state of the hemoglobin at the time of conjugation,
which makes it eminently suitable as a candidate for a hemoglobin
based oxygen carrier for clinical use in mammals. The degree of
transformation can be controlled by the timing of the application
of the reduction step. Moreover, the resulting product contains no
detectable unreacted hemoglobin which, if present, would dissociate
to give .alpha..beta.-diners suspected of causing renal injury, and
no detectable amounts of excessively high molecular weight products
(over about 500-600 kDa).
[0013] Thus according to the first aspect of the present invention,
there is provided a polysaccharide-hemoglobin conjugate useful as a
hemoglobin based oxygen carrier and having an oxygen affinity,
expressed as partial pressure of oxygen environment required to
maintain 50% oxygen saturation, P.sub.50 of =4-50 mmHg, at
37.degree. C., and containing no detectable residual unbound
hemoglobin and no detectable residual amounts of components of
molecular weight higher than about 500 kDa, said conjugate having
been prepared by reacting hemoglobin with oxidized polysaccharide
to form a high molecular weight conjugate complex, and allowing the
high molecular weight conjugate complex to degrade by storage in
solution at a suitable pH value, readily determinable by simple,
routine experiments, and at a temperature from 2.degree. C. to
about 45.degree.0 C. to form said polysaccharide-hemoglobin
conjugate.
[0014] A further aspect of the invention provides a
polysaccharide-hemoglobin conjugate useful as an oxygen
transporter, comprising hemoglobin covalently linked through
secondary amine linkages from amino groups on the hemoglobin to
residues of aldehyde groups on the polysaccharide, said aldehyde
groups having been formed by oxidative ring-opening of saccharide
monomeric units of the polysaccharide.
[0015] According to another aspect, the present invention provides
a process of preparing a hemoglobin based oxygen carrier which
comprises reacting an oxidatively ring-opened polysaccharide
carrying aldehyde groups with hemoglobin to form a Schiff
based-linked conjugate thereof, allowing the conjugate to stand
under conditions which effect molecular weight reduction of the
conjugate, stabilizing the conjugate by reduction of the Schiff
base linkages to stable, secondary amine linkages, and recovering a
solution of the polysaccharide-hemoglobin conjugate so formed which
has no detectable unbound hemoglobin residue and no detectable
product residue of molecular weight greater than about 500-600
kDa.
BRIEF REFERENCE TO THE DRAWINGS
[0016] FIGS. 1, 2 and 3 are sets of chromatograms of products of
Example 2 below;
[0017] FIG. 4 is a size exclusion chromatographic analysis of
products of Example 3 below;
[0018] FIG. 5 is a similar set of chromatograms of products of
Example 6 below; and
[0019] FIG. 6 is a similar set of chromatograms of products of
Example 7 below.
[0020] FIGS. 7 and 8 are similar sets of chromatograms of products
of Example 9 below.
[0021] FIG. 9 is a similar set of chromatograms illustrating the
results of Example 10 below.
[0022] FIGS. 10, 11 and 12 are similar sets of chromatograms from
products of Example 11 below.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0023] Apparent molecular weights described in this application are
derived by comparison with size exclusion chromatography elution
times of o-raffinose polymerized hemoglobin (polyOR-Hb) standards
of known molecular weight. Since hemoglobin-colloid conjugates are
suspected to contain significant amounts of trapped water in the
extended colloid chain component, the actual molecular weight of
the conjugate including only molecules covalently attached to the
hemoglobin is less than the apparent molecular weight. However, it
is the apparent molecular weight, or excluded volume, that will
dictate the in vivo retention time of the conjugate, and so
molecular weight as described above will be used to describe the
conjugates reported here.
[0024] The hemoglobin for use in the process of the present
invention is preferably human hemoglobin, derived from red blood
cells. However, the invention is applicable also to other types of
hemoglobin to form the basis of a blood substitute, such as animal
hemoglobins especially bovine hemoglobin, and porcine hemoglobin
and the like, and hemoglobin derived from cell culture. Human
hemoglobin is currently the preferred choice, to form the basis of
a blood substitute for administration to human patients.
[0025] The hemoglobin can be recovered and prepared for use in the
present invention according to standard, known techniques. Thus,
red blood cells are lysed, and cellular debris and stroma are
removed therefrom by standard techniques of centrifugation,
filtration and the like. Preferably, a solution of hemoglobin with
a concentration of 2-20% by weight of hemoglobin is used, to yield
a product having the most desirable composition and combination of
properties. Final purification can suitably take place
chromatographically. The displacement chromatography process
described in U.S. Pat. No. 5,439,591 Pliura et al. is beneficially
used.
[0026] Hemoglobin can naturally exist in the tight (T) conformation
as normally assumed by deoxyhemoglobin, or in the relaxed (R)
conformation as normally assumed by oxyhemoglobin or carbon
monoxyhemoglobin. The oxygen binding characteristics of hemoglobin
in the T state are the more desirable characteristics, since the
oxygen affinity of hemoglobin in this conformation allows efficient
oxygen binding in the lung vasculature and oxygen offloading in the
peripheral tissues. It is accordingly preferred to use
deoxyhemoglobin in the process of the invention. After conjugation
to the hydroxyethyl starch (HES) with or without prior
cross-linking, the deoxy-hemoglobin retains oxygen binding
characteristics of the T-configuration, If, however, one chooses
for any reason to start with R-configuration hemoglobin, the
preferred process according to the invention stabilizes the
hemoglobin into the R-configuration throughout. Mixtures of R- and
T-state hemoglobins can be reacted with the HES to obtain products
with oxygen binding properties intermediate between those of the R-
and T-state configurations.
[0027] Deoxygenation of hemoglobin to form deoxyhemoglobin is
preferably conducted by subjecting the hemoglobin solution to
treatment with a non-oxygenating gas such as nitrogen, according to
known techniques. It is preferred to continue the treatment with a
stream of nitrogen, followed by appropriate degassing, for
sufficiently long periods of time to effect complete conversion to
deoxyhemoglobin in this manner.
[0028] Polysaccharides useful in the present invention include
those of established biocompatibility, and having saccharide
monomeric units capable of oxidative ring opening to form reactive
aldehyde groups. They include starches and starch deivatives,
dextran, inulin and the like. Preferred among the polysaccharides
for use in the present invention are hydroxethyl starch and
dextran, with HES being most preferred.
[0029] The hemoglobin can be reacted with the oxidized hydroxyethyl
starch in its native, non-cross-linked form, or in its
cross-linked, 64 kDa tetrameric stabilized form, or in its
cross-linked and oligomerized form comprising 64-<500 kDa
adducts. When used in its cross-linked form, the preferred
cross-linking reagent for preparing cross-linked and
cross-linked-oligomerized hemoglobin is a polyaldehyde derived from
the oxidative ring-opening of an oligosaccharide such as raffinose
(i.e. o-raffinose). A suitable process for preparation of
o-raffinose and for its reaction with hemoglobin is described in
the above-mentioned U.S. Pat. No. 5,532,352 Pliura et al., the
disclosure of which is incorporated herein by reference. Whilst
o-raffinose is the preferred cross-linking reagent for use in this
embodiment of the invention, it is by no means limited thereto. Any
of the other known Hb cross-linking reagents, such as those
mentioned previously, for example trimesoylmethyl phosphate (TMMP)
described in U.S. Pat. No. 5,250,665 Kluger et al. can be
satisfactorily used.
[0030] The hydroxyethyl starch starting material for use in
preferred embodiments of the present invention suitably has a
molecular weight of from about 70 to about 1000 kDa. It is
commercially available, in various types and varieties. The type
and variety for use in the present invention is not critical.
Substantially any of the currently commercially available varieties
of HES can be used as the starting material, provided that they
have a molecular weight approximately as set out above. Those with
a substitution ratio (i.e. number of hydroxyethyl groups to glucose
units) of from about 0.5 to 0.7 are particularly suitable.
[0031] To prepare the HES for use in the present invention, it is
oxidized, so as to create thereon substantial numbers of aldehyde
groups. This can be accomplished by a variety of oxidation
processes, the preferred one being reaction with a periodate
(sodium or potassium). This reaction can take place in aqueous
solution at low temperature, e.g. 0-5.degree. C., using an
appropriate quantity of sodium periodate, chosen according to the
desired degree of oxidation. The reaction is complete in about 1-4
hours. Ultrafiltration or dialysis can be used to remove
undesirable low molecular weight salts and HES components, thereby
offering a means of controlling the molecular weight range of
oxidized HES to be conjugated to the Hb. The oxidized HES can be
used directly or is suitably recovered, e.g. by lyophilization, and
redissolved in water for conjugation to the hemoglobin.
[0032] The conjugation reaction suitably takes place in aqueous
solution. The hemoglobin may optionally be cross-linked Hb and/or
oligomerized Hb. It may be liganded e.g. with carbon monoxide,
(CO-Hb). Lower P.sub.50 values in the final products are obtained
with CO-Hb, higher values with deoxy Hb. Molar ratios of
Hb:oxidized HES can range anywhere from about 0.25:1 to 5:1, but
are preferably in the 0.5:1-3:1 approximate range. The reaction
best takes place at alkaline pH, e.g. in the range 7.5-9.0, and at
room temperatures.
[0033] The reaction product formed initially, e.g. after about 1
hour is found or analysis to have a very high molecular weight,
with components of molecular weight well in excess of 500 kDa, no
matter what the molecular weight of the starting polysaccharide may
have been. This initial product also contains a broad range of
molecular weight products. One can effect a controlled reduction in
the molecular weight of the product, to a product containing no
components of molecular weight higher than about 500,000, and to a
product of narrow molecular weight distribution (e.g containing
predominantly species of 100-200 kDa molecular weight), by
maintaining the product in aqueous solution, preferably in the
approximate pH range 7.2-10, and at or close to room temperature
(15.degree. C.-30.degree. C.), for a period of time up to about 48
hours. There is very little, if any, residual 32 kDa species. The
amount of 32 kDa species is so small that no special steps for its
removal are necessary.
[0034] The conjugate so formed must be stabilized by reducing the
(reversible) Schiff base linkages between the Hb and the HES to
stable secondary amine linkages and by reducing any unreacted
aldehyde groups. The reduction can be accomplished in a single
stage, in which the Schiff base linkages and the aldehyde groups
are reduced in a single stage, or in two separate stages. Powerful
reducing agents will be effective in one stage, less powerful
reducing agents requiring a two-stage process.
[0035] This step of reduction is preferably used as the means of
control of the molecular weight and molecular weight distribution
of the final product, by appropriate timing thereof. Once the
reduction has been completed, the product is stabilized and no
further changes in molecular weight or molecular weight
distribution of any significance will occur on storage.
Accordingly, analysis of samples of reaction product at intervals
allows timing of the reduction step to stabilize the product at the
chosen characteristics.
[0036] Borane dimethylamine is the preferred choice as the reducing
agent. This is powerful enough to accomplish both reduction
reactions in a single stage. Other water soluble borane lower alkyl
amine reducing agents including but not limited to
borane-tert-butylamine, borane-ammonia; borane-dimethylamine;
borane-trimethylamine; borane triethylamine; and pyridine borane
can also be used. Other useful reducing agents are sodium
cyanoborohydride and sodium borohydride.
[0037] Reduction of the Schiff bases formed during the conjugation,
and reduction of any residual unreacted aldehyde groups, most
suitably takes place in aqueous solution at a temperature range of
2-25.degree. C., for a period of time from 10-36 hours, preferably
24 hours. The reaction mixture is suitably buffered to pH 7-10,
preferably to 8.0-9.5. The molar ratio of reducing agent to the sum
of imine and aldehyde groups is in the range 1:1 to 5:1, preferably
1.5:1 to 3.5:1 based on the stoichiometry of reducing agent to
aldehyde groups added to initiate cross-linking.
[0038] It is preferred to use a final step of diafiltration, to
remove residual low molecular weight products such as starch
degradation residues, dimethylamino borane residues, salts, buffer
residues, etc. Then the product can be mixed with a suitable
excipient, to form an HBOC.
[0039] The conjugate so prepared exhibits eminently suitable
properties for use as the basis of an HBOC. It exhibits low oxygen
affinity (P.sub.50=20-50 mmHg) along with a narrow molecular weight
distribution of product (MWD 100-200 kDa), with no detectable
product of m. wt. 32 kDa under conditions which promote
dissociation to .alpha..beta.-dimers, or m. wt. above about 500
kDa.
[0040] For storage prior to use, it is suitable to remove all
oxygen from the product to prevent autoxidation. Deoxygenated
product can be stored under conditions which prevent introduction
of oxygen, either frozen or at higher temperatures. Oxygen can be
introduced prior to administration, or the product can be allowed
to acquire oxygen in vivo. The carbonmonoxy form can be stored in a
similar manner and oxygenated prior to use. The product can be
stored frozen in the oxygenated form, or at higher temperatures
until the degree of autoxidation is deemed unacceptable.
[0041] The invention is further described or illustrative purposes
only, in the following specific, non-limiting examples.
EXAMPLE 1--PREPARATION OF OXIDIZED HYDROXYETHYL STARCH
[0042] 9.0 g hydroxyethyl starch with weight average molecular
weight (MW) of 450 kDa, having a degree of hydroxyethyl
substitution of 0.7, was dissolved in 90 mL water. 0.49, 0.98 and
1.96 g sodium meta-periodate, representing approximately 0.3, 0.6
and 1.2 eq, respectively, of periodate per mol of vicinal diol
present in the HES, were added to separate 30 mL aliquots of this
solution. These amounts are sufficient to provide approximately
30%, 60% and 100% oxidation of available diol groups. After 4 hours
reaction in the dark at 4.degree. C., the solutions were dialyzed
extensively against chilled water using a 15 kDa molecular weight
cutoff membrane. Final retentates were lyophilized to white powders
and stored at room temperature. Alternatively, the dialyzed
oxidized HES solution could be used directly for conjugation of Hb.
HES with MW of 200 kDa and substitution of 0.5 was oxidized and
prepared in a similar manner. Oxidized HES was also prepared by
direct oxidation of HES formulated in 0.9% NaCl. Measurements of
periodate consumption and final aldehyde content indicated that the
range or periodate used resulted in partial to complete oxidation
of all available diol groups, and that the degree of oxidation was
readily controlled by varying the amount of periodate used.
EXAMPLE 2--PREPARATION OF CONJUGATES WITH VARIOUS OXIDIZED HES and
CO-HEMOGLOBIN
[0043] The reaction of CO-Hb with various relative ratios or
oxidized HES (HES-CHO) was studied. Periodate equivalents for
oxidation were calculated based on the expected vicinal diol
content of the HES. In one case, 0.54 g oxidized 450 kDa HES,
prepared using 1.2 eq periodate as described in example 1, was
dissolved in 3.0 mL 100 mM HEPES buffer pH 8.1. This HES-CHO
solution was added to carbonmonoxylated hemoglobin (COHb, 200 mg/L
in water) in the following ratios: 0.76 mL HES-CHO:0.041 mL COHb,
0.73:0.0.078, and 0.61:0.195, giving final Hb concentrations of
approximately 10, 20 and 50 mg/mL, respectively. The reactions were
allowed to proceed at 22-25.degree. C., at pH 8, and samples were
withdrawn at various times for MW determination using a Pharmacia
Superdex 200 column (1.times.30 cm) eluted with 0.5 M MgCl2+25 mM
Tris pH 7.2 at 0.4 mL/min. At all three HES:COHb ratios, Hb was
completely modified in the fist several hours to give species
having elution times comparable to polyHb controls with MW greater
than 128 kDa and ranging to above the exclusion limit of the column
(>500 kDa polyHb). FIG. 1 of the accompanying drawings shows the
chromatogram derived from the 0.5:1 Hb:HES product (50 mg Hb/ml),
taken at various times and compared with (bottom curve) the poly Hb
control. The dashed vertical line represents the elution time of 32
kDa unmodified .alpha.-.beta. dimer. The absorbance at 414 nm
characteristic of hemoglobin is tracked in the eluting fractions.
During the next 30 hours, the elution times of the product
decreased to give species having elution times comparable to polyHb
controls with MW of 128 kDa, with no unmodified Hb detectable and
no species above the exclusion limit of the column. The pattern of
MW evolution and final product MW ranges were similar at all three
HES:Hb ratios, as with HES oxidized with 0.6 eq periodate.
Conjugates prepared using HES oxidized with 0.3 eq periodate per
diol typically contained significant material co-eluting with
unmodified a-b dimer. Higher levels of oxidation were therefore
preferable for generating conjugate free of unmodified dimer.
[0044] The average MW of conjugates formed during the first several
hours was lower when less Hb was used. Similar reactions and
results were obtained using oxidized 200 kDa HES as described in
Example 1. Average MW of final products were higher when 450 kDa
HES was used in comparison to 200 kDa HES. FIG. 2 of the
accompanying drawings shows chromatograms of the final products of
Hb+HES-CHO for HES 200/0.5 (broken lines) and HES 450/0.7 (solid
lines), at different degrees of oxidation as indicated, all at 1:1
Hb:HES-CHO ratios.
[0045] Hb-HES conjugates obtained using periodate oxidized 70 kDa
HES (0.3, 0.6 and 1.2 eq. periodate vs. calculated diol) also
formed higher MW species during the early phase of conjugation,
followed by transformation to lower MW (FIG. 3). Average MW of
early phase conjugates, as well as the time required to convert to
lower MW species, was dependent on the degree of oxidation of the
HES 70. After 48 hours conjugation, some material coeluting with
the 32 kDa unmodified Hb component of the polyOR-Hb control
remained in the conjugate derived from the lowest degree of HES
oxidation (0.3 eq periodate per calculated diol). Significant
material eluting at the analytical column exclusion limit remained
after 48 hour in the reaction using the most highly oxidized HES 70
(1.2 eq periodate per calculated diol). Product free of unmodified
hemoglobin and material eluting at the column exclusion limit was
obtained within 48 hours of reaction with HES oxidized by 0.6 eq.
periodate per calculated diol.
EXAMPLE 3: SIMULTANEOUS LARGE SCALE PREPARATION OF HIGH AND LOW MW
Hb-HES CONJUGATES
[0046] Two Hb-HES conjugates of different MW were prepared from a
single reaction, in which a portion of the early conjugation
product having high MW was isolated and stabilized, allowing the
remaining conjugation product to undergo transformation to a lower
MW product before stabilization. A comparison of physical and in
vivo properties of the two products was made so that the beneficial
properties of one over the other could be demonstrated.
[0047] 944 g HES (200 kDa, degree of substitution=0.5) was
dissolved in 8 L WFI, cooled to 4.degree. C., then 370 g NaIO.sub.4
added and the mixture stirred in the dark for 5.3 hours. All
NaIO.sub.4 dissolved in less than 1 hour. The mixture was filtered
(0.2 um) then diafiltered against 12 volumes room temperature WFI
(water for injection) using a 30 kDa regenerated cellulose
membrane. It was then deoxygenated by contact with N.sub.2 through
a hollow fibre membrane. Lyophilized samples indicated a final
concentration of 128 mg HES-CHO/mL. 1.2 L of COHb (23.2 g/dL in
WFI) was combined with 2.0 L 200 mM HEPES pH 8.1 buffer, then
oxygenated and deoxygenated by contact with O.sub.2 then N.sub.2
through a hollow fibre membrane. 4.5 kg of the HES-CHO solution
(128 g/L) was combined with the deoxygenated Hb (3.2 L at 9.0 g/dL)
and the mixture maintained under deoxy conditions. The MWD of the
conjugates forming was monitored by size exclusion
chromatography.
[0048] After 3 hours conjugation, half of the reaction volume was
transferred under N2 to a separate vessel and 56 mL 3 M NaOAc and
196 g DMB, dissolved in 1.7 L WFI, were added. The final DMB
initial aldehyde ratio was 1.5:1. The mixture was kept under N2 at
ambient temperature for 23 hours before CO charging and
diafiltration. At 29 hours after initiation of the Hb-HES
conjugation reaction, the other half of the mixture was treated
similarly with NaOAc and DMB for 17.5 hours. Both DMB-reduced
reactions were then CO charged and diafiltered vs. WFI then
Ringer's lactate (approximately 10 volumes for each solution). The
pH was adjusted to 7.5-7.6 with 0.1 N HCl. Both solutions were
concentrated such that colloid osmotic pressure was 80-100 mm Hg.
Products were oxygenated and approximately three-quarters removed
for sterile filtration and packaging in the oxy form. The remaining
amounts of each were deoxygenated and packaged in the deoxy form.
Oxygenated products were stored at -80.degree. , deoxygenated
products at 4.degree. C. The higher MW product, obtained by
reduction of early stage conjugation product, is hereafter referred
to as HIMW HES-Hb. The lower MW product, obtained by reduction of
the late stage conjugation product, is hereafter referred to as
LOMW HES-Hb. MW distributions assessed by size exclusion
chromatography are shown in FIG. 4. MW distribution did not change
over four months storage at either 4 or -80.degree. C.
[0049] The colloid osmotic pressure (COP) of HIMW HES-Hb was
consistently higher than LOMW HES-Hb at the various concentrations
tested for both products (Table 1). Viscosities were 8.9 and 3.0
cSt at 6.5 and 9.0 g Hb/dL for HIMW HES-Hb and LOMW HES-Hb,
respectively. HIMW HES-Hb, which is comparable in MW distribution
to HES- and dextran-Hb conjugates prepared by others, therefore has
colloidal properties which would be expected to result in a greater
change in blood fluid and rheological properties than LOMW HES-Hb.
The deleterious effects of higher MW HES plasma components on
rheological factors such as viscosity and erythrocyte aggregation,
and on blood clotting, have been described (Treib et al.,
Thrombosis and Haemostasis 74:1452-6 (1995)).
1TABLE 1 Concentration dependence of COP for HIMW and LOMW HES-Hb
HIMW HES-Hb LOMW HES-Hb Conc (g Hb/dL) COP (mm Hg) Conc (g Hb/dL)
COP (mm Hg) 6.5 103.5 9.0 85.4 4.2 35.7 6.1 36.6 2.1 8.6 3.1
11.3
EXAMPLE 4--EFFECT OF LIGAND STATE ON FINAL P.sub.50
[0050] COHb (55 mg/mL in water) was oxygenated and deoxygenated by
exposure to oxygen then nitrogen, respectively. 200 kDa HES was
oxidized using 0.6 eq periodate as described in Example 1, and made
up to 60 mg/mL in 100 mM HEPES pH 8.1, and degassed and purged with
nitrogen. 2.5 mL of this oxidized HES solution was added to 0.8 mL
of the deoxygenated Hb solution, providing 1 eq Hb per mol of
initial unoxidized 200 kDa HES. After 48 hours at 22-25.degree. C.
under nitrogen, the reaction mixture was made 0.3 M in sodium
acetate, then 3 eq dimethylamine borane per mole of initial
aldehyde were added. After 24 hours, the solution was charged with
CO gas, and exhaustively dialyzed against lactated Ringer's
solution. A similar procedure was conducted in which COHb, without
removal of the CO ligand, was reacted with 200 kDa HES oxidized by
0.6 eq periodate. Oxygen binding properties were measured for both
products using a Hemox-Analyzer (TCS Instruments, Southhampton,
Pa., U.S.A.) at 37.degree. C. Conjugation of deoxygenated Hb
resulted in a final P.sub.50 of 26 mm Hg. Conjugation of COHb
resulted in a final P.sub.50 of 4 mm Hg. Both products were
non-cooperative,
EXAMPLE 5--USE OF CROSS-LINKED HEMOGLOBIN
[0051] 200 kDa HES was oxidized by 0.3 eq and 0.6 eq periodate in
separate reactions as described in Example 1, and made up to 125
mg/mL in 270 mM sodium bicarbonate pH 8.1. 3.0 mL of each oxidized
HES solution was added to separate 1.0 mL aliquots of trimesoyl
tris(methyl phosphate) (TMMP)-cross-linked Hb (64 kDa cross-linked
Hb, U.S. Pat. No. 5,250,665 Kluger et al., 125 mg/mL in water), and
likewise to 1.0 mL aliquots of o-raffinose polymerized Hb
(64-<500 kDa Hb polymers, U.S. Pat. No. 5,532,352 Pliura et al.,
117 mg/mL), for a total of four reactions, in all cases providing 1
eq Hb per mol of initial unoxidized 200-kDa HES. Both hemoglobin
products were in the CO form. After 30 hours reaction at
22-25.degree. C. under CO gas, sodium acetate was added to a final
concentration of 0.3 M. 3 eq dimethylamine borane per mol of
initial aldehyde was then added. After 24 hours, the reactions were
dialyzed (10 kDa MWCO) against water then lactated Ringer's
solution at pH 7.4. Oxygen binding properties were then recorded
using a Hemox-Analyzer at 37.degree. C.
[0052] MW distributions of all Hbs were shifted to higher values.
With Hb and TMMP-cross-linked-Hb, it was possible to modify all
starting Hb and there was no detectable void volume material
(Superose 12, dissociating conditions) within 48 hr. PolyOR-Hb
conjugates contained significant void volume material. P.sub.50s
(37.degree.) were: HES+CO-TM-Hb, 5-7 mmHg; HES+CO-polyOR-Hb, 5-7
mmHg. All products were non-cooperative.
EXAMPLE 6--VARIATION IN REACTION TIME AND TEMPERATURE
[0053] The effect Of shorter reaction times and lower temperature
(12 vs. 22.degree. C.) on Hb-HES MWD was studied on a small scale.
Oxidized forms of 200 kDa and 450 kDa HES were used.
[0054] Deoxygenated Hb was used. COHb (50 mg/mL in 75 mM HEPES
buffer pH 8.1) was oxygenated and deoxygenated by exposure to
oxygen then nitrogen, respectively. Oxidized HES, derived from
either 200 or 450 kDa HES using 0.6 or 1.2 eq periodate per mol of
vicinal diol, respectively, was dissolved in 100 mM HEPES buffer pH
8.1 to a final concentration of 60 mg/mL and the solutions were
then degassed and purged with nitrogen. 0.253 mL of Hb was combined
with 1.6 mL of oxidized 200 kDa HES solution, and 0.498 mL of Hb
was combined with 1.4 mL of oxidized 450 kDa HES solution, in both
cases providing 1 eq Hb per mol of initial unoxidized 200 kDa or
450 kDa HES. These solutions were allowed to react at 22.degree. C.
under nitrogen, and identical solutions were prepared and allowed
to react at 12.degree. C. MWD were determined at various time
points as described in Example 2. Chromatographic profiles are
shown in FIG. 5.
[0055] Final MWD was narrower at 22.degree. C. for both oxidized
HES's, and at longer reaction times for both-temperatures. Average
MW of the 450 kDa YES product (solid lines) was greater than for
the 200 kDa derivative (dashed lines), with the MW difference being
larger at the lower temperature. Reactions proceeded more slowly at
lower temperature, resulting in greater average MW and wider
molecular weight range compared to similar reaction times at higher
temperature.
EXAMPLE 7--SCALE-UP
[0056] Conjugation of oxidized HES to deoxy Hb was scaled up for in
vivo evaluation.
[0057] COHb was made up to 125 mg/mL in 100 mM HEPES buffer pH 8.1
and rendered ligand-free by contact with oxygen then nitrogen using
a hollow fibre gas exchanger. 47 g of oxidized 200 kDa HES,
prepared as in Example 1 using 0.6 eq periodate, was dissolved in
280 mL 100 mM HEPES buffer pH 8.1, then degassed and purged with
nitrogen. The oxidized HES solution was then added to the deoxyHb
and maintained under nitrogen at 22-25.degree. C. with periodic
measurement of MWD. Within 16 hours, all Hb was modified and no
product eluted at the exclusion limit of the column (FIG. 6). The
lowermost curve, presented for comparison purposes, is derived from
Hb cross-linked with oxidatively ring-opened raffinose (polyOR-Hb)
The reaction was made 0.4 M in sodium acetate, and 36 g
dimethylamine borane was added, representing approximately 3 eq
borane per mole of initial aldehyde. After 21 hours, the reaction
mixture was oxygenated, diafiltered (10 kDa MWCO) against lactated
Ringer's solution and adjusted to pH 7.4. The product had a P50
(37.degree. C.)=26 mmHg and was non-cooperative. Low angle laser
light scattering analysis of size exclusion chromatographic
effluent indicated a MW of 90-210 kDa. No free aldehyde was
detectable.
[0058] Analysis of in vivo halflife shows that the Hb-HES product
is retained for extensive periods. A volume of the product,
adjusted to 3.0 g Hb/dL in lactated Ringer's solution, equivalent
to 10% of total blood volume was infused into conscious rats and
the vascular retention time determined. The half-life was 6.0
hours, compared to 5.1 hours determined for an equivalent volume of
polyOR-Hb, adjusted to 10.0 g/dL in lactated Ringer's solution.
EXAMPLE 8: CONJUGATION OF Hb WITH OXIDIZED DEXTRAN AND
TRANSFORMATION TO LOWER MW
[0059] Two oxidized dextrans were prepared using either 0.45 or
1.36 eq periodate per diol (2 diols per dextran chain monomer).
Solutions of 2.0 g dextran (260 kDa) dissolved in 40 mL 4.degree.
C. water were treated with either 2.39 or 7.18 g sod-um periodate
(0.45 and 1.36 eq, respectively). After 4 hours stirring in the
dark at 4.degree. C., the solutions were dialyzed (10 kDa MW
cutoff) and lyophilized to white powders. 50 mg of oxidized dextran
in 1 mL 80 mM HEPES pH 8.1 buffer was combined with 0.062 mL COHb
(200 mg/mL) and the conjugation reaction was monitored by size
exclusion chromatography under dissociating, non-denaturing
conditions (0.5 M MgCl2+25 mM Tris pH 7.4). The results are shown
on FIG. 7, for the product where dextran was oxidized using 0.45
equivalents of periodate per diol, and on FIG. 8 for the 1.36
equivalents periodate per diol experiment. Both reactions showed
initial formation of high MW species eluting largely in the column
exclusion volume. Conjugate derived from the highly oxidized
dextran was more rapidly transformed to low MW species than when
using less oxidized dextran. The similarity in MW profiles for the
polyOR-Hb control and the highly oxidized dextran conjugate, with
the exception of overall higher MW for the latter, suggests the
conjugate is made up of polymerized, cross-linked Hb species which
are decorated with polysaccharide fragments. This configuration is
also suggested for oxidized HES conjugates obtained under some
conditions (FIG. 3).
EXAMPLE 9: PLASMA HALF-LIFE OF LOMW AND HIMW Hb-HES CONJUGATES
[0060] Male Sprague Dawley rats were acclimatized for one week with
free access to food and tap water. On the day of the experiment,
rats were anesthetized with Ketaset (ketamin hydrochloride, 60
mg/kg, i.m.) and Atravet (acepromazine maleate, 2.0 mg/kg, i.m.).
The right femoral artery and vein were cannulated using a 2.5-3.5
cm PE10 tubing connected to a PE50 tubing filled with
heparin-saline solution (50 USP units heparin/mL). Two to 3.5 cm of
PE10 were inserted into the lower abdominal aorta via the femoral
artery and vena cava via the femoral vein. Both cannulas were
tunneled subcutaneously to the nape and exteriorized. At the end of
the surgery the surgical site was closed using surgical thread.
Both cannulas were filled with heparin-saline solution (500 USP
unit/mL) at the end of the procedure. Animals were then outfitted
with a rodent tethering harness and miniature feed-through swivels
and placed individually in metabolic cages. Animals were allowed to
recover from the surgery 0.5 to 1.5 hours and resided in the
metabolic cage throughout the entire experiment. After the recovery
period, the venous cannula was connected to an automatic infusion
pump. Conscious animals were subjected to infusion of the control
solutions (10 g/dL polyOR-Hb in lactated Ringer's solution, and the
same diluted to 4 g/dL in plasma) or test articles (the products of
Example 2, low molecular weight (LOMW) and high molecular weight
(HIMW) HES-Hb, 5.0 and 3.5 g Hb/dL in lactated Ringer's solution),
respectively equivalent to 10% of total blood volume, delivered at
0.2 mL/min. Blood samples were collected 20 min after the end of
infusion (time=0.33 hr post-infusion) and at time=1, 3, 6, 10, 22,
28 and 34 hours. Plasma was separated by centrifugation and stored
at -80.degree. C. until analyzed by size exclusion chromatography.
Total hemoglobin was calculated from background-corrected
absorbances recorded at 414 nm, and plotted against time of blood
collection, and plasma half-lives were derived from single
exponential fits. Plasma half-lives were 5.1, 5.5, 8.9 and 15.6
hours for the 4 and 10 g/dL polyOR-Hb, and the LOMW and HIMW HES-Hb
solutions, respectively.
[0061] When compared with the half-life obtained for the product of
Example 7, which had a similar molecular weight distribution to
that of LOMW, it appears from the limited experimental data that a
longer half-life is obtained for the latter product which is
derived from the more highly oxidized HES.
EXAMPLE 10: IN VITRO STABILITY OF Hb-HES IN PLASMA
[0062] Hb-HES prepared in Example 6 was diluted 10-fold in rat
plasma and incubated at 37.degree. C., simulating a 5-10% (vol/vol)
topload administration. The mixture was analyzed by size exclusion
chromatography under dissociating, non-denaturing conditions (0.5 M
MgCl2+25 mM Tris pH 7.4), over a 49 hour period. The results are
shown on FIG. 9. No low molecular weight species, indicative of
product degradation, were detected during this time. High molecular
weight species, eluting at the exclusion limit of the analytical
column, appeared within the first hour of incubation. These species
correspond to high molecular weight complexes of the modified
hemoglobins and rat haptoglobin, in agreement with observations
made using several other polymerized hemoglobin products incubated
in plasma.
EXAMPLE 11--STABILIZATION OF VARIOUS MW SPECIES DURING Hb-HES
CONJUGATION
[0063] Dimethylamine borane (DMB) reduction was used to terminate
molecular weight changes occurring during conjugation of Hb to
oxidized HES. 2.0 mL COHb (200 mg/mL in H2O) combined with 490 uL
80 mM HEPES pH 8.1 was deoxygenated. 674 mg N.sub.2 charged
oxidized HES (prepared previously from HES 450/0.7, 1.10 eq
periodate per calculated diol) was dissolved in 6.8 mL of degassed
80 mM HEPES pH 8.1. 950 uL of the Hb solution was added to the
oxidized HES solution, giving a final Hb:HES(450 kDa) ratio of 1:1.
After 3 hours, 2 mL of this reaction mixture were added to a
freshly prepared solution of 152 mg N.sub.2-charged DMB (providing
approximately 3 eq DMB per initial aldehyde calculated for the
oxidized HES), dissolved in 1.6 mL degassed water with 350 uL 4 M
NaOAc added. A 2 mL aliquot of the Hb-HES reaction was similarly
treated after 6 hours. After 22 hours, the reactions were charged
with CO and dialyzed extensively vs. water over 72 hours at
4.degree. C. MW distributions during conjugation, reduction and
after dialysis were measured by size exclusion chromatography under
dissociating, non-denaturing conditions (0.5 M MgCl2+25 mM Tris pH
7.4). The results are shown on FIG. 10, for the products subject to
reduction at 3 hours, FIG. 11 for the products subject to reduction
at 6 hours, and FIG. 12 for the non-reduced products. The MW
distribution of products observed at 3 hours and 6 hours (both high
MW conjugates) did not change significantly during 22 hours of
reduction at room temperature, nor during 72 hours dialysis at
4.degree. C. Therefore, reduction with 3 eq. DMB per initial
aldehyde prevented transformation of the Hb-HES conjugate to lower
MW, as seen in samples which were not reduced (FIG. 10).
Stabilization of high MW species was also seen when using fewer
equivalents of DMB per initial aldehyde, as described in Example 3.
This reduction method can be used to stabilize any MW distribution
that develops during the conjugation reaction.
* * * * *