Note: Descriptions are shown in the official language in which they were submitted.
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DEXTRAN-FIEMOGLOBIN CONJTJGATES
AS BLOOD SUBSTITUTES
FIELD OF THE INVENTION
This invention relates to blood substitutes, and methods of their preparation.
More
particularly, the invention relates to improved polysaccharide-hemoglobin
conjugates for use as a
blood substitute for mammals and to methods of their preparing such
conjugates.
BACKGROUND OF THE INVENTION
In recent years the quest for a safe blood substitute has accelerated rapidly.
The demand often
exceeds the supplies of blood available from human donors. In addition, in
many parts of the world,
the whole blood supply can be hazardous. Therefore, there is a need to develop
blood substitutes.
One of the most important functions of blood is to carry oxygen from lungs to
support tissue
respiration. Hemoglobin (Hb) is an attractive oxygen earner in the development
of a clinical blood
substitute, given its attributes as a respiratory pigment of extensive
solubility, uptake and release of
oxygen, and above all its capability of transporting a large quantity of
oxygen. However, one
fundamental disadvantage of free Hb itself as a blood substitute arises from
its relatively small
molecular size of 64.5 kD and consequent high renal excretion rate (EXC)
thereby leading to a rapid
clearance from the circulation. Therefore, covalent conjugation to carrier
polymers has been applied
in order to prevent renal excretion of Hb and to prolong its plasma half life.
Such conjugates are
referred to as hemoglobin based oxygen carriers (HBOC). Examples of such
polymers include:
dextran and biopolymer derivatives of dextran, inulin, hydroxyethylstarch,
polyethylene glycol,
polyvinylpyrrolidone (S.P.Tsai and J. T.-F. along, Dextran-Hemoglobin, in:
Winslow, R.N.,
Vandegriff, K.D., and Intaglietta, M. [eds.], 1997 Advances in Blood
Substitutes: Industrial
Opportunities and Medical Challenges, Birlchauser, Boston; the contents of
these references are
incorporated herein by reference).
The efficiency of oxygen delivery is determined by the total blood flow and
volume, oxygen content,
red cell or hemoglobin mass, oxygen affinity and the rate of oxygen
consumption. The relationships
between oxygen content, delivery and utilization are best exemplified by the
Fick's equation. It is
therefore apparent that a blood substitute which can carry and deliver a
maximal amount of oxygen
per unit volume, while maintaining excellent rheologic characteristics, would
be ideal.
Dextran-hemoglobin (DxHb) is one of the conjugates that has been proposed as a
blood
substitute. The combination of water solubility, availability in a wide range
of molecular sizes, and
lack of significant toxicity or tissue tropism, renders dextran an excellent
drug carrier among
biodegradable polymers. Covalent conjugation of dextran (Dx) to hemoglobin
(Hb) increases the
effective size of Hb and, therefore, reduces its excretion rate (EXC) through
the renal system.
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Methods to stabilize the viscosity of the conjugate solution have been
proposed, and exchange
transfusions with DxHb in dogs and macaques performed (Tam et al, 1976, Tam et
al, 1978, Wong
1988). However, hitherto the flow properties of the conjugate have not been
investigated.
Examples of DxHb conjugates are provided in U.S. Patents 4,064,118 and
4,650,786, the contents of
which are incorporated herein by reference. The ' 118 patent teaches a
composition useful as a blood
substitute or blood extender which is prepared by chemically coupling
hemoglobin (Hb) with dextran
(Dx) having a molecular weight of from about SkD to 2000kD. The molecular
weight of this DxHb
conjugate is in the range of 70 - 2000kD. It has however been found that, as
compared to
hemoglobin, the products according to U.S. Patent 4,064,118 tend to show a
somewhat greater affinity
for oxygen, but retain the essential oxygen transporting and releasing
capability of hemoglobin.
U.S. Patent No. 4,650,786 describes a modified dextran-hemoglobin complex
having reduced
oxygen affinity. The molecular weight of this DxHb complex is in the range of
70 - 2000kD.
One of the problems associated with DxHb complexes, or modified DxHb
complexes, is that
the viscosity of the conjugate solution increases on storage thereby rendering
the solution unsuitable
for administration. The solution to the above problem is described in U.S.
Patent No. 4,900,816, the
contents of which are incorporated herein by reference. It has been shown that
the activated sites on
the dextran moiety may be blocked, and the viscosity on storage stabililized,
without affecting the
oxygen transport properties of the hemoglobin complex. U.S. Patent No.
4,900,816 teaches a
compound having a molecular weight from about 70kD to about 2000kD, comprising
a hemoglobin
residue, an oxygen affinity reducing ligand, a polysaccharide (e.g. dextran),
covalently bonded
chemical bridging groups and a blocked activating group.
As indicated above, one of the important characteristics of a blood substitute
is its xheological
properties - in order for the substitute to be physiologically acceptable, its
viscosity must not be so
high as to hinder flow of blood. Although aggregation of red blood cells is
one of the important
causes of increased blood viscosity, especially at lower shear rates, the
actual mechanism of red cells
aggregation is still not completely elucidated. Aggregation of red cells can
be brought about by
various means. In general, both the viscosity and the RBC aggregation increase
with increasing
concentration of immunoglobulin; however, the exact relationship between the
two appears to be
quite complex. It is therefore important to characterize the possible physico-
chemical properties of
DxHb in its development as blood substitute.
As described below, in the present invention, various preparations of DxHb
were synthesized
and the rheologic properties of these solutions were examined by measuring
their viscosities and their
aggregating tendencies which were assessed based on erythrocyte sedimentation
rate (ESR) test
(Dintenfass 1985, the contents of which are incorporated herein by reference).
The presence of
macromolecules over a certain "critical concentration" in plasma could induce
red blood cell (RBC)
aggregation and in turn blood viscosity particularly at low shear rate.
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Blood flow in low shear regions, especially in the venous circulation, is
greatly reduced by
the enhancement of erythrocyte aggregation which increases blood viscosity and
impedes capillary
flow through sludge formation (Dintenfass 1981). Red blood cell, RBC,
aggregation is the result of
bridging by macromolecules between adjacent erythrocyte surfaces. When a non-
encapsulated
hemoglobin-based blood substitute is infused into the circulation, these
macromolecules could also
interact with erythrocytes and induce RBC aggregation, with ESR being one of
the foremost blood
rheological parameters to be influenced by such aggregation. Therefore, it is
of fundamental interest
to examine the ESR enhancement effects of hemoglobin-based blood substitutes
in the course of their
design and development.
It is known that molecular size is one of the critical determinants of ESR
enhancement
through the macromolecular bridging mechanism. Dextrans of 20 kD do not induce
RBC aggregation
and hence, no ESR elevation, but dextrans larger than 40 kD are entirely
capable of enhancing ESR
(Chien and Jan 1973; the contents of which are incorporated herein by
reference). Previous studies
showed that macromolecular polymerized hemoglobin larger than 220 kD would
induce RBC
aggregation, which may increase low-shear-rate blood viscosity and affect the
RBC distribution in the
circulation (Tsai and Wong 1996; the contents of which are incorporated herein
by reference).
The present invention provides a DxHb conjugate having a molecular weight
range that
results in low EXC and ESR levels and, therefore, provides an effective blood
substitute or plasma
expander.
SUMMARY OF THE INVENTION
The present invention, in one embodiment, provides an oxygen carrying compound
comprising a conjugate of hemoglobin covalently joined to a polysaccharide,
the conjugate having an
average molecular weight of from about SOkD to about SOOkD.
In another embodiment, the invention provides a method for preparing an oxygen
carrying
compound, the compound comprising a conjugate of hemoglobin covalently bound
to a
polysaccharide, the method comprising:
1) reacting the polysaccharide with a bromine compound to provide bromine
groups on the
polysaccharide, thereby providing an activated polysaccharide;
2) filtering the activated polysaccharide with a first filter;
3) reacting the activated polysaccharide with hemoglobin thereby providing a
coupled
dextran-hemoglobin molecule;
4) filtering the dextran-hemoglobin molecule with a second filter.
BRIEF DESCRIPTION OF THE DRAWINGS
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These and other features of the preferred embodiments of the invention will
become more
apparent in the following detailed description in which reference is made to
the appended drawings
wherein:
Figure 1 illustrates the size dependence of erythrocyte sedimentation rate
(ESR) and excretion
rate (EXC) for dextran-hemoglobin synthesized from dextran molecules of two
starting sizes, l OkD
(DxTlO) and 20kD (DxT20).
Figure 2 illustrates a calibration of gel filtration column, SuperdexTM 200,
26/600 with
standard proteins.
Figure 3 illustrates a molecular weight distribution of four most preferred
preparations of
DxHb as measured with FPLC and MiniDawn laser detector.
Figures 4 and 5 illustrate the results of the experiments of Example 5.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
As used herein, the term "hemoglobin" will be understood to comprise
hemoglobin derived
1 S from red blood cells of any mammal. Although primarily directed to human
hemoglobin, the
invention is equally applicable to hemoglobin derived from other animals and
includes bovine and
porcine hemoglobin.
In a preferred embodiment, the present invention provides a carrier
polysaccharide-
hemoglobin conjugate and, more preferably, an improved dextran-hemoglobin
(DxHb) conjugate for
use as a blood substitute or a HBOC. As discussed further below, the DxHb
conjugate has, according
to a preferred embodiment of the invention, a molecular weight (MW) of from
about SOkD to about
SOOkD, and, more preferably, from about 89kD to 116kD. This range of molecule
size is based
primarily on the amount of conjugation between the Hb and the Dx molecules due
to the fact that the
size of the Hb is generally constant. This range of conjugate size has been
found to provide a
molecule that is sufficiently larger than Hb in size so that the EXC rate of
such molecule is not high,
while also providing a molecule that is sufficiently small in size so that the
ESR for such molecule is
also not high. As described further below, the term "high" in relation to EXC
rate is 1 % and that for
ESR is 20 mrn/hr. The invention also provides a method for production of such
conjugate. The
examples contained herein are provided for illustrative purposes alone and are
not meant to limit the
scope of the invention as will be apparent to persons skilled in the art.
1. Preparation Of Stroma-Free Human Hemoglobin (SFH)
A pure hemoglobin solution was produced by lysing red blood cells and
releasing
hemoglobin for use in the process of the present invention. These preparations
are then processed to
remove stroma in the solution so as to avoid renal damage. A hemoglobin
solution with a high degree
of purity was prepared according to the preferred embodiment of the present
invention by standard
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techniques of filtration as described by Winslow and Chapman (Meth. Enzymol.,
1994, 231:3-16), the
contents of which are incorporated herein by reference.
2. Activation Of Dextran By The Alkylation Method
As discussed above, covalent conjugation of polysaccharides to hemoglobin
increases its
effective size thereby preventing its renal excretion. The polysaccharides of
the present invention are
of established biocompatibility and are capable of being bound to hemoglobin.
The preferred
polysaccharide of the present invention is dextran.
The dextran of the present invention may have an average molecular weight of
10 kD
(referred to as Dextran TI O) to 20 kD (referred to as Dextran T20). It should
be noted that
commercially available dextran is categorized by its average molecular weight
and a variation in the
size of the dextran molecules is inherent. It is speculated that there is
higher renal as well as non-
renal excretion of DxHb when 10 kD dextran is used. Therefore, the most
preferred starting size for
dextran is 20kD according to the preferred embodiment of the present
invention.
To modify dextran to become capable of reacting with hemoglobin it has to be
"activated",
preferably with by an alkylation reaction. Specifically, the dextran was
reacted with cyanogen
bromide (CNBr) at alkaline pH and subsequently with diaminoethane. The
resultant
aminoethylamino-dextran was dialysed. This dialysis step is used to wash away
small reactant
molecules or reacted residual substances, such as diaminoethane.
Following this, the aminoethylamino-dextran was then acylated with bromo-
acetylbromide at
neutral pH, and was subsequently subjected to dialysis against water and
lyophilization. This process
for dextran activation is described in more detail by S.C. Tam, J. Blumenstein
and J.T.F. along, Proc.
Natl.Acad.Sci. USA, 1976, 73:2128-2131; the contents of which are incorporated
herein by reference.
Two tests, ninhydrin and silver nitrate tests, were used to monitor the
completeness of the
dialysis. Specifically, these tests were conducted on the filtrate from the
dialysis step whereby the
presence of amino groups was detected by the ninhydrin test as described by S.
Moore and W.H. Stein
(J. Biol. Chem., 1948, 176:367-388; the contents of which are incorporated
herein by reference). The
silver nitrate test was conducted to detect the presence of bromo groups, as
discussed below. Both of
these tests are further described below.
3. Preparation Of Dextran-Hemoglobin Conjugates
The final step of coupling hemoglobin to dextran was performed by adding
stroma-free
hemoglobin to the activated dextran, referred to as N-bromo-acetylamino-
ethylamino-dextran (DxBr).
The coupling reaction comprises the removal of the -Br groups of the activated
dextran (DxBr) and
the removal of the -H atoms of the sulfhydryl (-SH) groups of hemoglobin,
allowing for the binding
of Dx- to the HbS- to result in DxHb. The linkage between the bromo-
aminoethylamino-dextran
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(DxBr) and Hb is mediated through the free -SH at ~3-93 cysteine, the position
of the covalent linkage.
In the course of the coupling reaction, the -Br is detached from DxBr and
becomes free Br ion, which
is later dialysed away. The silver nitrate test (described further below) can
be used to test for the
presence of the Br ion.
The conjugation of dextran to hemoglobin was performed according to S.C. Tam,
J.
Blumenstein and J.T.F. along (Proc. Natl. Acad. Sci. USA, 1976, 73:2128-2131,
the contents of
which are incorporated herein by reference). According to the preferred
embodiment of the present
invention, the conjugation reaction was performed by mixing DxBr and stroma-
free hemoglobin in an
aqueous solution comprising 0.33% DxBr and 1% stroma-free hemoglobin solution.
Such
concentrations of Hb and DxBr ensures a DxHb coupling of 3:1 by weight or 1:1
by molar mass ratio.
However, other initial concentrations of the reactants to achieve these ratios
will be apparent to
persons skilled in the art.
Experimentation with coupling (i.e. conjugation) of the bromo-dextran (DxBr)
with a solution
of higher concentration of hemoglobin resulted in a higher degree of cross-
linkage, and therefore,
higher molecular weight of the resultant conjugates. Therefore, in order to
have a higher proportion
of relatively small DxHb molecules, it is preferred that the coupling reaction
take place using lower
(i.e. 1%) concentrations of Hb. However, it will be understood that it is
possible to perform coupling
of DxBr with hemoglobin of either higher or lower than 1% concentration.
The coupling reaction was conducted in a solution having a pH of 9.5 with
sodium
bicarbonate buffer. The solution mixture was first sterilized with a 0.22 p,m
filter and the coupling
reaction was allowed to proceed at 4°C overnight, according to the
method described by H. Xue and
J.T.F. along (Meth. Enzymol, 1994, 231:308-322, the contents of which are
incorporated herein by
reference). The coupling reaction was allowed to proceed for about 10 to 16
hours, and most
preferably for 16 hours. The longer coupling time resulted in DxHb of higher
average molecular
weight due to higher degree of cross-linking.
~3 -mercaptopropionic acid was then added to react with any residual bromo
groups on the
DxBr thereby stopping the coupling reaction. Further, the cross linking
reaction was also stopped
with this addition thereby preventing any further elevation in the solution
viscosity (S.P. Tsai and
J.T.F. along, In: Winslow, R.W., Vandegriff, K.D. and Intaglietta, M.(Eds.),
Advances in Blood
Substitutes, 1997, Birkhauser, Boston, the contents of which are incorporated
herein by reference).
The solution was then subjected to dialysis against phosphate buffered saline
to clear any residual
reactants and reaction by-products.
Thus, the alkylation method resulting in the above conjugation can be
summarized as follows
(as shown in Blumenstein et al., Experimental Transfusion of Dextran-
Hemoglobin, (1978); the
contents of which are incorporated herein by reference):
1) Dx + CNBr -~ activated Dx
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2) Activated Dx + diaminoethane ~ aminoethylamino-dextran
3) Aminoethylamino-dextran + bromoacetylbromide ~ N-bromo-
acetylaminoethylamino-
dextran
4) N-bromo-acetylaminoethylamino- dextran + hemoglobin-SH -~ hemoglobin-S-
acetylaminoethylamino- dextran
4. DxHb Fractionation and Correlation Between ESR, EXC and MW
Fractionation steps were introduced after a discovery of the ESR enhancement
by excessively
large DxHb molecules. In order to determine the correlation between molecule
size and ESR and
EXC, the DxHb was synthesized preferably from dextran molecules of at least
two starting average
sizes, IOkD (DxTlO) and 20kD (DxT20). The resultant conjugates, DxTI~OHb and
DxT20Hb, were
fractionated using the Waters 650E Advanced Protein Purification System. More
specifically, the
fractionation was carried out on a Hiload~ 26/60 Superdex~ 200 prep grade gel
filtration column
(Pharmacia). A solution of l OmM phosphate buffered saline, pH 7.4, was used
as the elution buffer
and the elution flow rate was 0.5 ml/min. The sample used was 8 ml of 8% DxHb.
The fractions
were collected using an ISCO Retriever II (fractions were collected for 8
min., i.e. 4 ml per fraction).
The fractions were tested for ESR and EXC as discussed below. However, since
the amount of
sample that is obtained from the column fractionation step is insufficient for
both ESR and EXC tests,
the fractions from several runs were pooled and concentrated with a Centriprep
30 (Amicon) in order
to have enough sample (of each size) to conduct both tests. The average MW of
each fraction was
also determined. Although the above mentioned fractionation step is described
in relation to DxHb, it
will be appreciated by persons skilled in the art that such fractionation step
may also be performed on
the DxBr precursor molecule. In this case, the DxBr fractionate is conjugated
to Hb using the above
mentioned process to result in DxHb fractions of the desired size range. The
details of the
measurements performed according to the preferred embodiment of the present
invention are
described below.
4.1. ESR Measurements
The erythrocyte sedimentation rate (ESR) is a means of quantifying the
aggregation of red
blood cells. Such aggregation is generally caused by the presence of macro-
molecules. For this
measurement, the sample is preferably prepared by mixing a 6.0% test solution
with an equal volume
of freshly withdrawn citrated rat whole blood. ESR measurement should be
conducted within 3-4
hours after withdrawing fresh blood due to the potential changes in the
suspension stability and
erythrocyte deformability of red blood cells over prolonged standing. This
fording was made when
using blood extracted from the rat. ESR measurements were conducted in
accordance with
instructions provided by the manufacturer, Clay Adams, Division of Becton
Dickison and Company,
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Parisippany, N.J., 07054 USA . Solutions were kept in a vertical position in
reusable Clay Adams
Wintrobe Blood sedimentation tubes (115 mm long, with a 3.Omm bore) fox 60
minutes on a universal
ESR rack (Chase Jnstruments) at room temperature (about 20-22° C). This
method is commonly
referred to herein as "Wintrobe's method" and a description of this method is
provided in
"Introduction to Medical Laboratory Technology", Baker & Silverstein (eds.),
5t'' ed., pp. 605-606,
Butterworths, the contents of which are incorporated herein by reference.
The sedimentation tubes were scaled at 1 mm intervals. For some preparations,
there was no
clear boundary between the sediment and supernatant, but rather a gradual
color change was observed
in the upper part of the ESR tube. For such preparations, ESR was recorded in
a range format (e.g. 1-
l8mm/hr, Table 2). An acceptable ESR was taken to be 20 mxn/hr.
It should be noted that other methods for measuring ESR will be apparent to
those skilled in
the art. For example, instead of the Wintrobe's method mentioned above, it
would also be possible to
use the Westergren method as described in "Introduction to Medical Laboratory
Technology", Baker
& Silverstein (eds.), 5~' ed., pp. 606-607, Butterworths, the contents of
which are incorporated herein
by reference. . These methods will result in different ESR values; however,
the same conclusions will
be drawn as in the present case.
4.2. EXC Measurements
Excretion rate (EXC) experiments were conducted ~:o determine the extent of
renal excretion
of the DxHb conjugate of the present invention. For determining the EXC, a
test solution of DxHb
conjugates was infused into the jugular vein of an anesthetized male Sprague-
Dawley rat of an
average weight of about 300-350g. The excretion rate was estimated by
determining the hemoglobin
concentration in urine sample of a rat by the method described by D.L. Drabkin
and J.H. Austin, (J.
Biol. Chem.,1935, 112:51-65), the contents of which are incorporated herein by
reference. Although
the EXC value is preferably 0, an acceptable value can be taken to be less
than 1 %. Above this level,
the urine of the rat was found to be reddish in colour.
4.3. Molecular Weight Measurements
The molecular weights of the DxHb conjugates were measured using gel
filtration
chromatography wherein molecules elute from the column in order of decreasing
molecular weight.
As DxHb molecules get fractionated over the gel filtration.column, a detector
monitors their retention
time. Fast Protein Liquid Chromatography (FPLC) was employed for molecular
weight determination
of DxHb conjugates of the present invention, however High Performance Liquid
Chromatography
(HPLC) could be employed as well. For the purposes of illustrating the
invention, molecular weights
were determined using the Waters 650E Advanced Protein Purification System.
The following two
columns were used in series in this system: 1) the TSK-Gel GMPWXL (7.8x300);
and, 2) the '
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Pharmacia Superose 6 HR10/30. The elution buffer consisted of lOmM Tris-IICI,
0.05% sodium
azide, pH 8.0, which was used with a flow rate of 0.4 ml/min. In the preferred
embodiment, the
composition of the eluent is verified using detectors that can be used for MW
determination of DxHb.
For example, a Wyatt Technology MiniDAWN laser detector or a UV detector could
be used for this
purpose. In the preferred embodiment, the following three detectors were used
in series: a Waters 440
LJV Detector A280; a Wyatt Technology MiniDAWN detector; and, a Waters 410
Differential
Refractometer. Table 1, below, lists the peak molecular weight values of the
various DxHb
manufactured. Further, the following molecular weight data was obtained using
the above described
system:
Sample Calculated Average
Mol.Wt.
1) purified Hb (66 +/- 7) kD
2) dextran T20P (DxT20P - Pharmacia) 16.0 kD
3) activated dextran (DxBr) after filtering 24.4 kD
to obtain a filtrate with
components lower than 300kD
4) DxHb after filtering to obtain a filtrate115.6 kD
with components less than
SOOkD and then filtering to obtain a retentate
with components
greater than 80 kD
Molecular weight determinations of various fractions were made by comparing
the ratio of
Ve/V° for the molecule in question to the Ve/Vo of protein standards of
known molecular weight (Ve is
the elution volume and V~ is the void volume). The void volume of a given
column is based on the
volume of effluent required for the elution of a large molecule such as Blue
Dextran or the like. A
calibration curve can then be prepared by plotting the logarithms of the known
molecular weights of
protein standards versus their respective Ve/Vo values.
It should be noted that measured molecular weight values can vary depending
upon the
equipment/methods used. For example, S.P. Tsai and J.F.T. along (Artificial
Cells Blood Subst.
Immob. Biotech., 1996, 24:513-523) reported an anomaly in measuring the
molecular weight of
hemoglobin. According to such report, the Hb molecule, because of its compact,
round molecular
structure and shape, was found to have a MW measurement of just 46kD, as
determined by FPLC
system, whereas its theoretical value is 64.SkD. As indicated above, the
molecular weight
measurement of Hb using the MiniDAWN laser method was found to be 66+/- 7 kD
giving a range of
58 - 73 kD, which is very near the theoretical value. Furthermore, dextran,
because of its linear
structure, results in a molecular weight reading using gel filtration, that is
higher than its theoretical
value. This fact should be taken into consideration when determining MW limits
for DxHb
conjugates of the present invention. For this reason, the above mentioned
laser detector was used in
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order to obtain more accurate results. For example, using such laser detector,
the measured molecular
weight of H6 was found to be close to its theoretical value.
4.4. The Size Dependence of ESR and EXC for DxHb Conjugates
Data for ESR, EXC and MW as measured at the peak of the elution volume as
mentioned
above, are provided below in Table 1 and are illustrated in Figure 1.
Table 1. The size dependence of ESR and EXC fox DxHb conju abates synthesized
from dextran
molecules of two startin sg izes (lOkD and 20kD)
Dx(T10)-Hb Dx(T20)-Hh
Elution Vol MW (1cD)ESR (mm/hr)EXC ESR (mm/hr)EXC
(mL) (%) (%)
124 1217 75
128 1067 74 73
132 935 73
136 820 72 73
140 719 71
144 630 67 70
148 553 53
152 484 43 46
156 425 13 28
160 372 8 0.00 16
164 326 2 5
168 286 1 1
172 25I 0.5
I76 220 0.00 0.00
180 193 0.2 0.2
184 169 0.09
188 148 0.2 0.00
192 I30 0.2 0.00
196 114 0.00
200 100 0.2 0.56
204 88 1.44 I .34
208 77 0.2
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Dx(T10)-Hb Dx(T20)-Hb
Elution Vol MW (1cD)ESR (mm/hr)EXC (%) ESR (mm/hr)EXC
(mL) (%)
212 67 2.85 1.03
216 59 0.2
220 52 7.77
224 45 0.2
228 40 16.49
As discussed above, the problem of rapid excretion of hemoglobin by itself
appears to be a
consequence of its relatively low molecular weight. In order to increase the
molecular weight of
hemoglobin to allow for adequate retention, it is coupled to a polysaccharide
such as dextran or the
like. Ideally, no renal excretion of hemoglobin should be observed if such
hemoglobin is
administered in the form of DxHb conjugates as a blood substitute. However, an
excretion rate lower
than about 1%, and more preferably lower than about 0.2%, is preferred (S.C.
Tam and J.T.F. along,
Impairment of Renal Function by Stroma-Free Hemoglobin in Rats, J. Lab. Clin.
Med, 1988,
111:189-193; the contents of which are incorporated herein by reference).
As also discussed above, although a higher molecular weight leads to reduced
EXC, such
larger molecules result in an increased ESR. According to the preferred
embodiment of the present
invention, ESR should be less than about 20mm/hr, and, more preferably, the
ESR should be less than
about 1 mm/hr.
After the DxHb conjugates of the present invention were fractionated on the
basis of
molecular weight using gel filtration chromatography and the collected
fractions were tested for their
effect on the ESR, it was found that DxHb fractions with peak molecular
weights less than about
500kD did not enhance ESR over the acceptable limit of up to 20rnm/hr. On the
other hand, DxHb
conjugates greater than peak molecular weight of about 501cD resulted in an
EXC value within the
acceptable range of between 0 and 1 %. According to the preferred embodiment
of the present
invention, the results define an acceptable range of molecular weight for DxHb
conjugates of about
50kD to about 500kD that results in the desired levels of EXC and ESR.
5. Selection For The Optimal Fractionation Procedure To Obtain Preferred Dxhb
Conjugates
Once the above desired molecular weight range of DxHb conjugates was
determined, the
search for the optimal fractionation procedure was launched. One factor to
consider in determining an
optimal fractionation procedure is the maximization of the yield of the DxHb
product. A high yield of
the final product avoids any unnecessary waste of the DxHb.
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Several methods were employed to achieve the goal of obtaining the final DxHb
products of
the optimal molecular size. These methods included ethanol precipitation,
choice of the starting
dextran, and filtering. Fractionation of Dx, DxBr and DxHb was done before and
after the activation
step, as well as before and after the conjugation step to screen for a better
production scheme. The
S procedures employed, and the resulting ESR and EXC, for the various DxHb
preparations are listed in
Table 2 below.
Table 2. Erythrocyte Sedimentation Rate and Excretion Rate of DxHb Conjugates
Obtained by
Various Procedures.
# Preparation ESR EXC(%) YieldYield Yield
(mm/hr) DxBr DxHb Overall
Hb Stroma-free Human Hemoglobin <1 30-40
DxHb Preparations Based on DxT20P**
1 DxT20P, activate, <100kD (Millipore),60 ND* 57 100 57
couple
2 DxT20P, activate, >100kD (Millipore),78 ND 30 100 30
couple
3 DxT20P, activate, <100kD (A/G)***,32 ND 38 100 38
couple
4 DxT20P, activate, >100kD (A/G), 50 ND 53 100 53
couple
DxT20P, activate, <300kD (A/G), 30 ND 35 100 35
couple
6 DxT20P, activate, >300kD (A/G), 78 ND 60 100 60
couple
7 DxT20P, activate, <SOOkD (A/G), 57 ND 49 100 49
couple
8 DxT20P, activate, >SOOkD (A/G), 64 ND 42 100 42
couple
Different Filters to Fractionate
DxHb After the
Coupling
9 DxT20P, activate, couple, <SOOkD1-20 ND 100 49.5 49.5
>70kD (A/G)
DxT20P, activate, couple, >SOOkD76 ND 100 33 33
(A/G)
11 DxT20P, activate, couple, <750kD51 ND 100 54.8 54.8
>70kD (A/G)
12 DxT20P, activate, couple, >750kD73 ND 100 23.7 23.7
(A/G)
Selected Filters Used Before
and After the
Coupling
13 DxT20P, activate, <300kD, couple,1 ND 35 100.2 35
<SOOkD
>80kD (A/G)
14 DxT20P, activate, <300kD, couple,1 ND 35 99.2 34.7
<SOOkD (A/G) i i i i i i
i
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# Preparation ESR EXC(%) YieldYield Yield
(mm/hr) DxBr DxHb Overall
15 DxT20P, activate, <300kD, couple,ND ND 35 0.8 0.3
>SOOkD (A/G)
16 DxT20P, activate, <SOOkD, couple,1-18 ND 49 91.1 44.6
<SOOkD (A/G)
17 DxT20P, activate, <SOOkD, couple,77 ND 49 8.9 4.4
>SOOkD (A/G)
18 DxT20P, activate, <SOOkD, couple,25 ND 49 86.3 42.3
<750kD
>70kD (AJG)
19 DxT20P, activate, <SOOkD, couple,80 ND 49 13.7 6.7
>750kD (A/G)
Filters of Different Brands to
Fractionate DxBr
Made from DxT20P
20 T20, activate, couple, >lOkD 76 0.24 I00 100 100
(A/G)
21 T20, activate, <30kD (AJG), couple<1 1.60 8 100 8
22 T20, activate, >30kD (AJG), couple76 0.30 85 100 85
24 T20, activate, <SOkD (A/G), couple1-18 ND 4 100 4
25 T20, activate, >SOIeD (A/G), 76 ND 91 100 91
couple
26 T20, activate, <SOkD (Microgon),1-9 ND 14 100 14
couple
27 T20, activate, >SOkD (Microgon),74 ND 85 100 85
couple
28 T20, activate, <SOkD (Paul Filtron),45 ND 14 100 14
couple
29 T20, activate, >SOkD (Paul Filtron),74 ND 8I 100 81
couple
30 T20, activate, <60kD (A/G), couple1-36 ND 18 100 18
31 T20, activate, >60kD (A/G), couple77 ND 82 100 82
32 T20, activate, <70kD (Paul Filtron),32 ND 17 100 17
couple
33 T20, activate, >70kD (Paul Filtron),74 ND 82 100 82
couple
34 T20, activate, <100kD (A/G), 53 ND 22 100 22
couple
35 T20, activate, >100kD (A/G), 74 ND 73 100 73
couple
DxHb Preparations Based on DxTlOP
36 T20, activate, couple, >IOkD 76 0.08 100 100 100
(A/G)
37'T20, activate, couple, >30kD ND 0.07 100 99.9 99.9
(A/G)
38 T20, activate, couple, >SOkD ND 0.27 100 99.4 99.4
(A/G)
39 T20, activate, couple, >70kD ND 0.04 100 98.4 98.4
(AlG)
40 T20, activate, couple, >100kD ND 0.00 100 65.7 65.7
(A/G)
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# Preparation ESR EXC(%) YieldYield Yield
(mm/hr) DxBr DxHb Overall
41 T10, activate, couple, >lOkD 1-15 1.35 100 100 100
(A/G)
42 T10, activate, couple, >30kD ND 0.88 100 99.8 99.8
(A/G)
43 T10, activate, couple, >SOkD ND 0.37 100 97.6 97.6
(A/G)
44 T10, activate, couple, >70kD ND 0.23 100 95.2 95.2
(A/G)
45 T10, activate, couple, >100kD ND 0.06 100 9 9
(A/G)
46 T10, activate, <30kD >lOkD (A/G),1 2.60 39 100 39
couple,
>lOkD (A/G)
47 T10, activate, <30kD >lOkD (A/G),ND 1.56 39 99.7 38.9
couple,
>301eD (A/G)
48 T10, activate, <30kD >IOkD (A/G),ND 0.19 39 97.9 38.2
couple,
>SOkD (AJG)
49 T10, activate, <30kD >IOkD (A/G),ND 0.21 39 90.3 35.2
couple,
>70kD (A/G)
50 T10, activate, <SOkD (M) >SkD <0.5 2.53 5 I00 5
(M), couple
51 T10, activate, >SOkD (M), couple10 0.47 66 100 66
52 T10, activate, <60kD (A/G) >SkD <1 4.70 25 100 25
(M), couple
53 T10, activate, >60kD (A/G), couple35 ND 67 100 67
54 T10, >SOkD (M), activate, couple1 0.18 66 100 66
Ethanol Used for Fractionating
DxTlO
55 T10, 0-55% ethanol ppt*'~**, <1 0.4I 45 100 45
activate, <30kD
>IOkD (A/G), couple, >lOkD (AJG)
56 T10, 0-55% ethanol ppt, activate,ND 0.51 45 99.8 44.9
<30kD >IOkD
(A/G), couple, >30kD (A/G)
57 T10, 0-55% ethanol ppt, activate,ND 0.47 45 97.9 44.1
<30kD >IOkD
(A/G), couple, >SOkD (A/G)
58 T10, 0-55% ethanol ppt, activate,ND 0.13 45 96.1 43.2
<301cD >lOkD
(A/G), couple, >70kD (AlG)
59 T10, 0-55% ethanol ppt, activate,ND 0.00 45 40.2 18.1
<30kD >lOkD
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# Preparation ESR EXC(%) YieldYield Yield
(mm/hr) DxBr DxAb Overall
(A/G), couple, >100kD (AlG)
60 T10, 0-49% ethanol ppt, activate,62 ND 19 100 19
couple
61 T10, 49-55% ethanol ppt, activate,15 ND 17 100 17
couple
62 T10, 55-80% ethanol ppt, activate,<1 ND 11 100 11
couple
63 T20, 0-46% ethanol ppt, activate,73 ND 40 100 40
couple
64 T20, 46-75% ethanol ppt, activate,23 ND 54 100 54
couple
Where:
1) "DxTlO" and "DxT20" refers to the starting size of the dextran (Dx)
molecule; i.e. an average
molecular weight of 10 or 20kD, respectively.
2) "activate" means activation of Dx by alkylation as described above.
3) "couple" refers to the conjugation, or coupling, of DxBr to Hb as described
above.
4) "< 100kD", or "> SOOkD", or the like, refers to the cut off value of the
filters used during the
filtering step of DxBr or DxHb. Further, the symbols "<" and ">" refer to the
values of the filtrate
and retantate, respectively. For example, "<100kD" indicates that the filter
provides a filtrate
containing only those molecules having a molecular weight less than 100kD.
Similarly, ">SOOkD"
indicates that the filter provides a retantate having molecules with molecular
weights greater than
SOOkD.
* ND = not determined
IS ** all Dx samples (i.e. T10, T20, T20P) were obtained from Pharmacia
Biotech AB.
*** A/G = A/G Technology Corp., Needham, MA, USA
**** ethanol ppt = ethanol precipitation (discussed further below)
Filters of larger cut-off sizes (e.g. SOOkD) applied after the coupling
reaction assisted in
eliminating the excessively large DxHb conjugates (those that are larger than
about SOOkD - the upper
limit for the preferred products). Intermediate filters (e.g. SOkD, 70kD)
applied after conjugation
were used to remove molecules smaller than the lower limit for preferred
products (about SOkD). The
actual function of small filters (e.g.lOkD) is the same as that of the
dialysis process that is carried out
after the conjugation reaction, that is to remove any residual reactants (e.g.
mercaptopropionic acid
etc.).
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The function of the ethanol precipitation is to eliminate the excessively
large dextran
molecules before the activation step. Ethanol was added in a stepwise manner.
For example, in
preparations # 55 - 59 (Table 2) ethanol was added slowly starting from an
initial concentration of 0%
up to a final concentration of 55%. As a result, some excessively large
dextran molecules were
precipitated, pelleted and re-dissolved for later activation.
Several preparations of DxHb were selected from the list in Table 2 as the
most preferred.
These preparations are identified in Table 2 as numbers: 13, 14, and 16. The
choice of the selected
preparations was based on the desired ESR and EXC values as well as the yield
of the product.
Peak molecular weights for these preparations were determined as described
above and are
0 illustrated in Figure 3. As shown in Figure 3, the peak MW of the preferred
preparations, two batches
of preparation #14 and two batches of preparation #I6, was found to be 102,
96, 93, 89kD. In "'
addition, although not, shown in Figure 3, the average MW of preparation #13
was found to be 1 I6kD.
These numbers define the most preferred range for the average MW of the most
preferred DxHb
preparations of the present invention.
Thus, according to the present invention, the preferred size range of the DxHb
conjugates is
from about 50kD to about SOOkD, with the most preferred range of about 89kD to
about 116kD. The
optimal procedure for synthesizing these preparations comprises the activation
of dextran having a
starting size of 20kD, filtrating the activated DxBr through a 500kD or 300kD
filter, coupling the
products of filtration with stroma-free hemoglobin, filtrating the resulting
DxHb through a 500kD
'0 filter to eliminate any excessively large conjugates. The most preferred
procedure for producing the
DxHb conjugates of the present invention also includes a final step of
filtrating the resultant
conjugates through a 80kD filter to eliminate any excessively small conjugates
that might potentially
increase the EXC value.
Table 3 below presents data for the preparation of DxHb conjugates that is
similar to that of
?5 Table 2. However, Table 3 includes additional further preparation and
analytical information
concerning the various conjugates. The data in Table 3 also indicates the
molecular weight
distribution for the various batches.
Table 3: Preparation Procedures for Dx-Hb and Analytical Results:
Batch Procedures Compo Lower UpperAve ESR
&
Code Coupling vent Limit LimitMW mm/ >200 >300 >400 >50
Conditions Yield hr 1cD 1cD kD 0
(1)
2 kD
P5.1 DxT20P, activate,23.0 1300 6300 too 100 100 100 100
couple at viscou
6%,
>500kD s
P5.5 DxT20P, activate,25.9 500 6000 930 77 100 I00 100 97
couple at
1%,
>500kD
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P5.3 DxT20P, activate,6.6 300 3000 S72 76. 100 100 74 58
couple at 1%,
>SOOkD .
P4.1 DxT20P, activate,30.2 220 3000 490 73 I00 82 65 S3
coupleat 6%,
>750kD
P4.3 DxT20P, activate,14.9 350 3000 778 73 . 100 95 84
100
coupleat 6%,
>750kD
P DxT20P, activate,83.3 100 2000 207 70 53 34.5 22.5 15
1.
I
S
couple at 6%,
<750kD
P3.2 DxT20P, activate,46.5 80 1800 160 63 39 23 14 8
couple at 6%,
<500kD,>70kD
P5.4 DxT20P, activate,93.4 57 1000 105 62 21 10 -5 3
couple at 6%,
<SOOkD, >70kD
P4.4 DxT20P, activate,85.1 60 1300 140 60 ~ 21 13 7
36
couple at 6%,
<750kD,>70kD
P4.2 DxT20P, activate,69.8 60 1000 127 51 30 14 5.5 3
couple at 6%,
<750kD,>70kD
P1.3SDxT20P, activate,74.4 60 700 l 3S 25 10 3.5 1
I7
<300kD, couple
at
2%, <750kD
P DxT20P, activate,83.6 70 1300 130 25 24 10 4 2
1..25
couple at 2%,
<750kD
Samples
with
acceptable
ESR
values
(3):
P5.2 DxT20P, activate,77.0 53 S00 107 I-3215 5 1 0.5
couple at 1%,
<SOOkD, >70kD
P3.4 DxT20P, activate,60.0 64 700 110 1-2019 6 2 1
couple at 6%,
<SOOkD, >70kD
P8.1 DxT20P, activate,91.1 53 500 104 1-1815 5 1.5 1
couple at 1%,
<500kD
P5.6 DxT20P, activate,74.1 52 400 90 1-9 7 2 1 0
couple at I
%,
<SOOIeD, >70kD
P102 DxT20P, activate,97.7 62 400 121 1 18 7 0.5 0
<300kD, couple
at
1%, <SOOkD,
>80kD
P6.4 DxT20P, activate,93.2 52 700 IOZ 2 17 6 2.5 1
<SOOkD, couple
at
1%, <SOOkD
P6.2 DxT20P, activate,99.3 50 500 96 1 9 2 0.5 0
<300kD, cou
1e at
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1%, <500kD
P6.8 DxT20P, activate,80,1 50 700 93 1 11 3 l 0.5
<SOOkD, couple
at
1%, <500kD
P8.3 DxT20P, activate,83.2 50 450 91 1 5 1 0 0
couple at 1
%,
<500kD
P6.6 DxT20P, activate,99.1 49 S00 89 1 9 2 O.S 0
<300kD, couple
at
1%, <SOOkD
Notes:
- (1): The coupling of Dx Hb indicates a % value which corresponds to the
concentration of
Hb. For example, for batch S.S, the DxHb coupling was effected with 1% Hb and
0.33% DxBr. --.
- {2): Normalized yield of the final DxHb fractionation step, showing the %
component of the
Dx-Hb produced.
- (3): Table 3 includes data sorted by ESR values and indicates a "cut-off '
where ESR values
are acceptable as defined above.
The following examples serve to illustrate the preferred embodiments of the
invention and are
not intended to limit the invention in any way.
Example 1: Preparation of Stroma-Free Human Hemoglobin SFH)
SFH of the present invention was prepareii according to the method described
above.
Outdated human blood was supplied by the Hong Kong Red Cross Blood Transfusion
Service. Fifteen
units (about 4.S liters) of whole blood, type A, AB, or O, were pooled and
mixed with 4.SL of 10 mM
PBS (phosphate buffered saline - a mixture of lOmM sodium phosphate buffer and
154mM Natal ),
pH 7.4 in a 10-liter glass bottle. The red blood cells (RBC) were washed with
7 volumes of chilled
buffer by diafiltration with a hollow fiber filter of 0.65 ~Cm (CFP-6-D-6A)
mounted an FlexStrand~
(A/G Technology Corp., Needkam, MA) at a constant volume of about 10 L. RBC
were then lysed
slowly with hypotonic 10 mM phosphate buffer at the same pH with a 0.1 pin
membrane cartridge
(CFP-I-E-6A, A/G Technology Corp). The volume of RBC corpuscle was washed
thoroughly with
up to 5 volume of the buffer. The filtrate was then diafiltrated through a
S001cD filter (UFP-500-E-
SA, A/G Technology Corp.) to assure stroma-free and then concentrated to 20
g/dL by circulating
through a 10 kD membrane cartridge (UFP-10-E-9A, A/G Technology Corp.). The
solution was
diafiltrated with 10 rnM PBS, pH 7.4, which was the final storage buffer. The
sterility of the final
hemoglobin solution was further ascertained by passing through a pre-filter
and a 0.22 ~m filter (293
mm, Millipore) in series. The stroma-free hemoglobin solution was bottled and
stored at 4°C.
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Example 2: Activation of Dextran by the Alkvlation Method
Description of dextran activation by the alkylation method according to the
preferred
embodiment of the present invention includes small-scale and pilot-scale
activation. Small-scale
dextran activation may result in the production of DxHb only in limited
amounts that is enough for
research purposes only, while pilot-scale dextran activation procedure can be
used in the industrial
setting for the subsequent conjugation of DxHb in larger amounts.
a) Small Scale Activation
0.3 g cyanogen bromide (CNBr) (Riedel-de Haen) dissolved in 3 mL of
acetonitrile was
added to 95 mL of 2% dextran (mean MW 20kD, Pharmacia), and the activation was
allowed to
proceed for 5 minutes. During the activation, pH is maintained at I0.8 by
continuous addition of 1 M
NaOH. Afterwards, it was lowered to 2.0-2.5 with 2 M HCI. Then, 2mL of
diaminoethane (Sigma)
was added along with additional HCl to prevent the pH from exceeding 9.5.
After stirring at 4°C
overnight, the mixture was thoroughly dialyzed against distilled water.
Completeness of dialysis was
preferably confirmed by testing the dialysate with the ninhydrin method.
Aminoethylamino-dextran
was thus obtained. Solid NaZHP04 was then added into the mixture to a
concentration of O.I M and
pH 7.0, and 3 mL of bromoacetyl bromide (Fluka) was added over a period of 2
hours, accomplished
with vigorous stirring and maintenance of pH at 7.0 by addition of 1 M NaOH.
Then, the mixture was
again dialyzed against distilled water. Completeness of dialysis was
preferably confirmed by testing
the dialysate with silver nitrate solution. After dialysis, the product N-
bromoacetyl-aminoethylamino-
dextran (DxBr) was lyophilized and stored in freezer, ready for use.
b) Pilot Scale Activation
Twenty-eight grams of CNBr (Riedel-de Haen) were dissolved in 50 mL of
acetonitrile, which was then added to 4.0 L of 3.5% dextran (Pharmacia). The
pH of the solution was
maintained at 10.8 by continuous addition of 6 M NaOH (about 50 mL) for 5-10
minutes.
Afterwards, about 50 mL of 6 N HCl was added to lower the pH to around 2Ø
Then, 210 mL of
diaminoethane (Sigma) was added along with 6 N HC1 (about 500 mL) to keep the
pH below 9.5.
After stirring at 4°C overnight, the mixture was thoroughly
diafiltrated against 40 L of distilled water
with Millipore Pellicon Cassette Filter Acrylic Holder and three stacking PTGC
0005 cassettes
(Millipore) at a flow rate of around 8 L per minute. The circulation of the
dextran solution was
maintained by the use of a Cole-Parmer peristaltic pump (model 7549-40) with
an Easy-load pump
head (Model 7529-80, MasterFlex). The circulating volume was kept at 3-4 L. To
monitor the
completeness of the dialysis, the filtrate was then subjected to the ninhydrin
test as described below.
Later, 180 mL of bromo-acetyl bromide (Fluka) was added slowly accompanied
with
vigorous stirring over a period of two hours, during which the solution was
maintained at neutral pH
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by adding 6 M NaOH. Then, the mixture was thoroughly dialyzed with the
Pellicon cassette against
distilled water (about 50 L). Completeness of dialysis was confirmed by
subjecting the filtrate to the
silver nitrate test as described below. The activated bromodextran was
lyophilized and stored at -
20°C.
c) Ninhydrin Test
Firstly, ninhydrin solution was prepared as previously described by Moore, S.
and Stein,
W.H. (J. Biol. Chem., 1948, 176:367-388; incorporated herein by reference). In
short, ninhydrin
solution was prepared by mixing 1.0 L of 4.0 M sodium acetate, pH 5.5 with 3.0
L of ethylene glycol
monomethyl ether (Sigma). The mixture was bubbled with nitrogen for an hour.
Then, 80 g of
ninhydrin (Sigma) and 7.5 mL of 21 % titanous chloride solution (Sigma) were
added. The ninhydrin
solution was kept under nitrogen.
To carry out the test, 1.0 mL of ninhydrin solution was mixed with 1.0 mL of
the DxBr
solution and the mixture was allowed to react at 100°C for 15 minutes.
A blue coloration which could
be stabilized by adding 2.0 mL of 50% ethanol indicated a positive result.
Quantitative analysis was
obtained spectrophotornetrically at 570 nm. Ethylamine was employed for
calibration. Absence of
amino group which would result in colorless solution indicates the
thoroughness of the reaction.
A simpler ninhydrin test could be performed to obtain qualitative results,
where 1.0 g
ninhydrin (Sigma) is dissolved in 50 mL distilled water. One half mL of this
ninhydrin solution is
mixed with an equal volume of the DxBr solution. The mixture would turn yellow
if there were any
residual amino groups.
d) Silver Nitrate Test
The test was performed by using a silver nitrate (AgN03) (Nalcalai Tesque)
solution. The
bromo groups were first released by alkaline hydrolysis. Three drops of 1 M
NaOH were added to
each of 0.5 mL DxBr samples and the solutions were incubated at 37°C
for 30 minutes.
Subsequently, three drops of concentrated nitric acid were added, followed by
another three drops of
1 % AgN03 solution. A white precipitate of silver bromide would result if
bromide is present, and the
solution would turn milky. In the actual experiment described above, there was
no white precipitate,
which indicated the absence of the bromo group in DxBr solution.
Examine 3: Preparation of Dextran-Hemoglobin Conjugates
According to the present invention, the conjugation reaction was preferably
performed by
dissolving 16.7 g of DxBr in 5 L (0.33% DxBr) of 1% stroma-free hemoglobin
solution. Sodium
bicarbonate buffer was added to a final concentration of 0.1 M and the pH
adjusted to 9.5 with I M
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NaOH. The solution mixture was first sterilized by passing through a 0.22 ,u m
filter (Millipore),
stirred and the coupling reaction was allowed to proceed at 4°C for up
to 16 hours.
l6mM ~ -mercaptopropionic acid (Sigma, pH adjusted to 0.5 with NaOH) were
added to react with
any residual bromo groups and to stop the coupling reaction. The solution was
subjected to dialysis
against 10 mM phosphate buffered saline, pH 7.4, 60 minutes later to clear any
residual reactants such
as ~ -mercaptopropionic acid, bromo groups, etc. A conventional dialysis bag
was employed in
small test tube scale, while a 10 kD filter cartridge (UFP-10-E-9A, A/G
Technol.) was used for the
diafiltration in pilot-scale
Examule 4: Molecular Weisht Measurement: Calibration of the Filtration Column
The preferred procedure for determining molecular weights of DxHb preparations
of the
present invention using gel filtration chromatography is outlined in Technical
Bulletin No. GF-3,
Sigma Chemical Company, October 1987. SuperdexTM 200, 26/600 gel filtration
column (Pharmacia)
was employed. The column was calibrated with standard proteins before the MW
was determined for
I S DxHb conjugates of the present invention. The standard proteins employed
were as follows:
cytochrome c (MW 12.4kD), carbonic anhydrase (MW 29kD), albumin (MW 66kD),
alcohol
dehydrogenase (MW 150kD), (3 -amylase (MW 200kD), ferntin (MW 440kD). The
calibration curve
is shown in Figure 2, where molecular weight is plotted versus Ve/V°
for each respective protein
standard. The average molecular weight was determined for each fraction since
each fraction may
contain DxHb of various MW values.
Example 5: Develoument of Hemorrha~ic Model in Guinea Pig
Method
Six normal, healthy Guinea pigs were used in the optimization of bleeding
conditions to
develop a useful hemorrhagic model to study the effect on survival and
assessment of delivery of
oxygen to tissues. Guinea pigs was housed fox at least 1 week before
experimentations. Food and
water were supplied et libido.
The animals were anaesthetized with intraperitoneal (i.p.) injection of
pentobarbital, as it is
known that Guinea pigs have narrower respiratory tracts and ether is to be
avoided. The jugular vein
and carotid artery were cannulated for infusion of control or testing solution
and blood pressure was
measured with a pressure transducer connected to an electronic amplifier and a
l OmV recorder
respectively. The animal was bled at a rate of 30%, 50%, 70%, 90%, and 100% of
Maximum Bled
Out for the first 10 minutes, then at smaller volumes of about 0.2 - O.Sml
occasionally to keep the
blood pressure low. The blood volume bled was calculated as follows:
Total blood volume (TBV) - body weight (g) x 8%
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Maximum Bled Out (MBO) - TBV x 60%
10-minute Bled Out (LOBO) - MBO x various percentage
After bleeding and maintaining at a low blood pressure of about 25 mmHg for 90
minutes,
0.2m1 of blood was drawn for lactate determination with a serum lactate kit
(Sigma). Procedures
indicated on the Sigma diagnostic kits were followed. Briefly, blood was mixed
with equal volume of
TCA solution, centrifuged after standing for several minutes. The supernatant
stored for later lactate
assay. Blood vessels were tied, and the wound was rinsed with ampicillin and
sutured. The animals
were then monitored and survival and body weight data were recorded. Animals
that lived longer
than 7 days were classified as "survivors".
Results and Conclusion
The following results of survival and 90-minute blood lactate levels were
obtained:
lOBO > 7-Day 90-min Serum Standard Error
%MBO Survival % Lactate m /dl
0 100 6.2 1.3
30 83.3 32.2 10.0
50 58.3 44.8 26.4
70 27.3 62.1 16.3
90 8.3 77.5 26.3
100 0.0 115.1 32.5
Figures 4 and 5 illustrate the effect of 10-minute bled out volume on the long-
term survival of
the hypovolemic shocked animals and the relationship between the survival
outcome and the 90-
minute lactate levels. Generally speaking, the faster the bleeding, the higher
the lactate level,
suggesting anaerobic respiration of the animal, and that the animal was in a
hypovolemic shock state.
Furthermore, the higher the lactate level, the lower the long-term survival.
Survivors can live for
longer than 2 more months after experimentation and gradual weight gain was
observed. On the
contrary, if the animal did not recover, weight loss continued and eventually
it would die within the
first few days.
Example 6: Resuscitation with Dextran-Hemoglobin Solution after an otherwise
Fatal
Hemorrhage in Guinea Pig
Method
Commercially available Guinea pigs of body weights between 350 and 550 gm were
fasted
overnight (16 hrs) and then anesthetized with pentobarbital (Sigma) by
intraperitoneal injection. The
level of anesthesia was assessed by the response to hind toe pinch and a
sufficient response was
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WO 02/24751 PCT/CA01/01329
continuously maintained by further doses of pentobarbital inj ection. The
carotid artery was
cannulated for bleeding and arterial blood pressure measurement with a
pressure transducer connected
to an electronic amplifier and a lOmV chart recorder. The jugular vein was
cannulated for fluid
infusion.
A volume equivalent to 70% of MBO was bled through the carotid artery during
the first 10
minutes, during which time the blood pressure changed from 85rnmHg to 30 mmHg.
Subsequently,
0.2 - 0.5m1 was bled occasionally for the following 80 minutes to keep the
blood pressure at 25-
30mmHg. At 90 minutes, a bled volume of control buffer, hemoglobin (Hb) or
fractionated dextran-
hemoglobin (Dx-Hb) in kidney dialysis fluid was infused over 60 minutes. Only
those with a 90-
minute lactate level between 50-90 mgldl were included, because too low or
excessively high lactate
level might be brought about by inconsistence of bleeding skill during the
experimentation.
Results and Conclusion
Test Solution n Survival
Kidney dial sis fluid (KDF) 7 75.0
5% Hb (#29) in KDF 6 50.0
5% Dx(ZHB)-Hb >70kD 6 83.3
5% Dx ZGB)-Hb >70kD 6 66.7
5% Dx(DBB)-Hb >70kD 5 60.0
5% Dx(DBB -Hb >70kD 6 16.7
5% Dx(DBB)-Hb 70-300kD in 9 77.8
KDF
5% Dx(DDB2 -Hb 70-500kD in 9 100.0
KDF
5% Dx(DBB)-Hb 70-500kD in 8 87.5
KDF
5% Dx(DBB)-Hb 70-750kD in 4 50.0
KDF
5% Dx(DBB)-Hb 70-1,OOOkD in 9 66.7
KDF
5% Dx(DBB)-Hb 70-1,OOOkD in 5 20.0
KDF
5% Dx(DDB)-Hb >1,OOOkD in 3 0.0
KDF
Description of DxHb conjugates used:
DextranDextranVolumeDextranCNBr Diamino-BromoacythylYield
Brand Size WeightWeightethane -bromide (%)
(MW) ( )
ZGB Fisons 20,000 5L 120g 24g 180rn1 180m1 63%
ZHB Fisons 20,000 5L 120g 24g 180rn1 180m1 100%
DBB Phamacia20,000 5L*7 143g*728g*7 210m1*7210m1*7 94%
DDB Phamacia20,000 SL* 66g* l Og* 100m1* 125m1* 82%
15 15 15 15 10
DDB2 75%
DDB2: derived from DDB with a <500K A/G cartridge.
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It will be understood by persons skilled in the art that the DxHb conjugates
of the present can
be used for a variety purposes and in a variety of manners. Primarily, the
conjugates of the present
invention can be used as blood substitute or blood expander. By way of
example, the DxHb
conjugates can be used as a blood substitute to prevent hemorrhagic shock (in
trauma wards) or for
hemodialysis.
Although the invention has been described with reference to certain specific
embodiments,
various modifications thereof will be apparent to those skilled in the art
without departing from the
spirit and scope of the invention as outlined in the claims appended hereto.
References
The following is a listing of some of the references that have been discussed
above. The
contents of the following are incorporated herein by reference.
1) Chien, S., and K-M. Jan. (1973) Ultrastructural basis of the mechanism of
rouleaux formation.
Microvasc. Res. 5:155-166.
2) Chien, S., S. A. Luse, K-M. Jan, S. Usami, L. H. Miller, and H. Fremount.
(1971) Effects of
macromolecules on the rheology and ultrastructure of red cell suspensions.
6th Europ. Coyaf. Microcirc. pp.29-34.
3) Dintenfass, L. eds. (1985) Blood viscosity, hyperviscosity &
hyperviscosaemia. Boston:MTP Press,
pp.45-112.
4) Dintenfass, L. eds. (1981) Hyperviscosity in Hypertension. Sydney:Pergamon
Press, pp.l-40.
5) Fabry, T. L. (1987) Mechanism of erythrocyte aggregation and sedimentation.
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Bridging by
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7) Jan, K-M. (1979) Red cell interactions in macromolecular suspension.
Biorlaeology 16:137-148.
8) Tam, S-C., J. Blumenstein, and J. T. along. (1978) Blood replacement in
dogs by dextran-
hemoglobin. Cafa. J. Biochem. 56:981-984.
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9) Tam, S-C., J. Blumenstein, and J. T, along. (1976) Soluble dextran-
hemoglobin complex as a
potential blood substitute. Proc. Natl. Acad. Sci. USA 73:2128-2131.
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