Note: Descriptions are shown in the official language in which they were submitted.
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SYNTHETIC WHOLE BLOOD
AND PROCESS FOR PREPARING SAME
This invention relates to a synthetic whole
blood for use as a substitute for whole natural mammalian
blood and a process for preparing same.
Authorities in the fields of physiology and
clinical medicine are in agreement that there is a need
for an acceptable substitute for whole mammalian blood
and in particular, whole human blood. Similar opinion
exists regarding hematocrit.
In the prior art, a number of compositions, i.e.
Lactated Ringer's Solution, Dextran, Modified Gelatin,
Hydroethyl Starch, Fluorocarbons and Perfluoroca~bons
are referred to as "blood substitutes". (Ref. chemical
Ab~tracts, 8th Collective Index, 1967-1971; 1972.) The
scientific literature, however, records no evidence that
any of these compositions can function as a whole blood
substitute or that they are conventionally used as such.
These substances as well as albumin are employed
principally to expand plasma volume, carry oxygen, or
enhance oxygen transport. Whole human blood is known
to possess other~important capabilities and functions.
Aside from intrinsic physiological limitations, the
available "blood substitutes" have restricted utility
by reason of known adverse reactions and incompatibilities.
The prior art contains no reference to a bIood
substitute that has both the physlcochemical charact-
eristics and the ph~siological range of whole mammalian
blood and, in particular, whole human blood. (Ref.
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Chemical Abstracts, 8th Collective Index, 1967-1971; 1972;
Chemical Abstracts, 9th Collective Index, 1977; Chemical
Abstracts, volumes, 88, 89, 90, 91, 92, 93 and 94.) More-
over, the cited prior art does not refer to a method of
5 manufacture of such a composition. Finally, the scientific
literature contains no reference to a blood substitute
which like whole mammalian blood and the claimed composition
of matter possess both polar and non polar properties.
Presently known blood substitutes are primarily either
10 polar or they are non polar.
Table 1 which follows details other fundamental
differences between the present invention and the avail
able blood substitutes described in the prior art.
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The present invention provides a process for
preparing a synthetic whole blood substitute, consisting
essentially of a non-toxic two phase liquid system,
both said phases being aqueous, comprising the steps
of: dispersing acacia in an aqueous solution; adding
to said solution a gelatin; thoroughly mixing said solution;
storing said mixture undisturbed for a period of time to
form a non-toxic two phase liquid system having physio-
logical and physicochemical properties essentially
similar to those of whole blood; one of said phases
being a relatively non-polar coacervate phase having
physiological and physicochemical properties substantially
equivalent to erythrocytes; the other of said phases
being a relatively polar liquid aqueous phase having
physiological and physicochemical properties substantially
equivalent to blood plasma; said Lelatively non-polar
coacervate phase being insoluble in and in equilibrium
with said relatively polar liquid aqueous phase.
Further, the invention provides a synthetic
whole blood substitute prepared according to said process.
:
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-- 6 --
ri`rOm d physicochemical point of view, whole
mammalian blood in the body exists and functions as a
two phase coacervate system. A coacervate system is a
polyphasic physical chemical system having two phases,
each phase, consisting chiefly of liquid water and each
phase, by reason of its thermodynamic state, heing
insoluble in the other phase. Erythrocytes, largely
composed of water comprise the relatively non-polar
coacervate phase of the coacervate system referred to
above. Plasma which also consists primarily of water
constitutes the bulk water, relatively polar aqueous
phase of the system. The two phases normally exist in
equilibrium with respect to dissolved molecules and
electrolytes.
Any change that significantly affects the
components or their concentration of the coacervate system
will disturb the normal steady state with consequent
physiological efect. Thus, as one example, alteration
of the electrolyte content of the plasma (i.e. change
in the relatively polar water state) can result in
destruction of the erythrocytes (i.e. change in the
non-polar coacervate phase) with consequent hemolysis.
In this invention, methods of manufacture are
disclosed which produce products that successfully sub-
stantially duplicate the two phase physicochemical(coacervate) system of whole mammalian blood and in
particular whole human blood~ Thus duplication enables
the claimed compositions to carry out virtually all of
the physiological functions of whole blood, with the
exception of clotting. However, introduction of the
claimed synthetic whole blood into a recipient does not
interfere with existing clotting capability. Additionally,
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the manufacturing process of the claimed products also
discloses a product which is useful as a substitute for
naturall~ occurring hematocrit.
This invention comprises safe and effective
5 substitu-tes for whole, natural blood and methods of manu-
facture thereof. The claimed invention makes use of
the concepts and process of coacervation. The claimed
process of manufacture produces a two phase coacervate
system substantially identical to the two phase physico-
10 chemical system of whole natural blood; ie: a non polarcoacervate phase insoluble in an~ in equilibrium with an
associated polar bulk water equilibrium phase. The
coacervate phase comprises from 1 to 99% of the two
phase system. The bulk water equilibrium phase con-
lS stitutes from 1 to 99~ of the coacervate system.
The coacervate phase substantially possessesthe physiological and physicochemical properties of
naturally occurring hematocrit while the bulk water
equilibrium phase is substantialIy the physiological
20 and physicochemical equivalent of naturally occurring
blood plasma. Upon emulsification, the two phase co-
acervate system can be inused into a recipient and will
function as whole blood. In the preferred method,
however, the two phase coacervate system is brought into
25 even closer functional equivalence with whole, natural
blood during the process of manufacture, by the addition
of appropriate proteins, electrolytes, a sterol, and
if desired, stroma f~ee hemoglobin.
Upon separation of the phases, the coacervate
30 phase can be safely introduced intravenously to either
carry out or enhance the physiological functions of
hematocrit. It can transport and trclnsrer oxygen and
carbon dioxide much as naturally occurring erythr~ ytes
do. The introduced hematocrit substitute (ie; coacer-
35 vate phase of the two phase coacervate system~ will notadversely affect the per cent of the recipient's hematocrit~
.'
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Moreover, when it is infused, it will disperse in the
blood plasma of the recipient, thereby contributing to
the two phase physicochemical system of the whole blood
to which it has been added. In addition, the physico-
5 chemical characteristics of the coacervate phase renderit sensitive and reacti~e to both the physioloyical
state and physiological changes in the recipient~s blood.
Finally, the claimed hematocrit substitute can readily
enter and pass through the major blood vessels, capil-
10 laries and the microci~rculation.
If the intended purpose is to transfuse theequivalent of whole blood, then the emulsified form of
the two phase coacervate system is infused intravenously.
If the preferred method is followed, the infused syn-
15 thetic whole blood will be comprised of the two phasecoacervate system and appropriate additives, the com-
bination of wh;~ch will haye been emulsified prior to use.
In this form, the claimed composition can not only
carry out vital functions o~ normal whole blood but in
20 addition can be safely used in a wide variety of treat-
ment procedures including the establishment and main-
tenance of extra corporeal circulation. By reason of
its physicochemical procedures, the claimed synthetic
whole blood will circulate readily within the entire
25 vascular system.
Upon transfusion, the claimed composition can
establish, re-establish and/or maintain normal osmotic
pressures, transport and transfer physiological gases,
can carry nutrients, druq dosage forms and various
30 physiological entities over extended periods of time
without loss of stahility. The transport character-
istics of the claimed compositions of matter enable them
to serve as a ~safe and reliable Yehicle in hyperalimenta-
tion procedures. When it is desirable to introduce
35 enzyme systems into the body, such systems can be added
, .
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- 9 ~ zz~
to the coacervate phase of this invention and infused
through conYentional intrayenous methods. Enzy~e systems
introduced through these compositions of matter will
perform their nor~al physiological functions.
When it is desirable to enhance the intrinsic
oxygen carrying capacity of the claimed compositions,
stroma free hemoglobin may be added to the coacervate
phase or to the emulsif;ed coacer~ate system. Such aa-
diticn does not affect the stability or the physiological
10 capabilities o~ the claimed compositions.
The disclosed synthetic whole blood can be
rendered free of forei~n proteins and other elements
which contribute to the adverse reactions associated with
transfusion of whole human blood. Further, because this
15 invention possesses universal donor characteristics, no
blood typing is necessary priar to infusion of this com-
position of matter. An additional important advantage
of the claimed whole blood substitute over whole human
blood is that it can be readily modified to meet many of
20 the s~ecific requirements of sPecific medical and sur-
qical treatment procedures.
The guidelines which govern the quantities of
the claimed synthetic whole blood that may be safely
infused, are unlike those of the available blood sub-
25 stitutes, but are substantially identical to those thatgovern the use of whole human blood.
The Process of manufacture must be carried out
under aseptic conditions. Except for cooling steps that
are necessary to the method of pre~aration, all Pro-
30cedures are carried out at ambient temperatures and con-
ventional pressures. ~en infused into humans, the composi~ions
may be at ambient te~Perature and PreferablY should be at
a temperature that approximates9~.6 deqrees F (37C).
Either of two groups of component ingredients
35can be used in the ~re~aration of the claimed comPo-
sitions of matter. The groups are of substantially equal
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-- 10 --
utility in producing the necessary two phase coacervate
system. Irrespective of the grollp of ingredients sel-
ected for use, the fundamental manufaeturing step re-
mains the same; i.e. a process of coacervation constitutes
the method by which the component ingredients are eom-
bined.
If the two phase coacervate system is to be
prepared from the group of ingredients herein referred to
as Group A, the preferred ingredients will include al-
bumin, sodium chloride, urea, lecithin, and distilledwater. If the coaeervate system is to be manufactured
using ingredients eomprising the group herein referred to
as Group B, the preferred ingredients will eonsist of
aeaeia USP, gelatin solution, sterile water and lN
hydroehlorie aeid. If the eoacervate system is to be
manufactured using ingredients herein referred to as
Group C, the preferred ingredients will be those of
Group A and additionally a non-polar solvent or a semi-
non-polar solvent.
Regardless of the group of components employed
to produee the two phase coaeervate system, the additives
used to bring the composition into eloser physiologieal
approximation with whole mammalian blood are the same.
Exeluding the substances used in titration and establish-
ment of isotonicity, the additives comprise the follow-
ing: cholesterol, caleium ehloride, potassium chloride,
electrolytes and stromafree hemoglobin.
Certain treatment regimens may make the ad-
dition of mueopolysaccarides, glycoproteins, proteins,
enzymes and other molecules such as heparin desirable.
These substanees ean be added to the elaimed eompositions;
they will perform their eonventional funetions without
altering the composition.
During the manufaeutre of the deseribed eo-
acervate system, eoaeervated struetured, water-insoluble
droplets are formed. Under the eonditions of the manu-
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facture of this invention, these droplets coalesce toform the coacervate phase of the two phase coacervate
system. This system can be readily emulsified to form
droplets of any desired size. ~n this invention, the
preferred size can range from 2 to 9 microns.
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The prior art contains no reference which sug-
gests or hints at a method of manufacture of a synthetic
whole blood based upon the process of coacervation; nor
does it hint at or suggest the composition of a two
5 phase coacer~ate system which will function virtuall~
as whole blood. Further, the prior art makes no mention
of a substitute for hematocrit comprised of the coacervate
phase of the claimea coaceP~ate system.
In ordeE to explain the invention more fully,
lO the following are descriptions of the preferred methods
of preparing the claimed compositions. Specific ex-
amples of the practice of this invention are detailed
in the subsequent sections of this application.
The preferred process used to manufacture the
15 claimed synthetic whole blood and the claimed substitute
fore hematocrit, using the ingredients described in this
document as comprising Group A is briefly presented
below:
At the point that manufacture oE the two phase
20 coacervate system is co~pleted, the two phases may be
separated. In the pre~erred procedure, the two com-
ponent phases are separated after the ingredients de-
scribed below ha~e been mixed but before e~ulsification
of the coacervate system and addition of the incorpora-ted
25 additives.
Disperse from 5 to 25% weiqht to volume o~ pow-
dered albumin in distilled water containinq 0.9% weight
to volu~e sodium chloride, 1 to 5% weight to volume urea
and 0.1 to 10% weight to ~olume of lecithin. Store the
30 resulting solution, undisturbed at 4 degrees C for twelve
hours. Remove the resulting two phase coacervate s~stem
from refrigeration. Then add such amount of distilled
water at ambient temperature to the coacervate system as
will result in a 5 to 25~ weight to volume of albumin;
35 add such quantity of sodium chloride to the coacervate
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system as wil~ render said system isotonic with whole
mammalian blood. The coacerYate system may now be
separated into its two component phases, or alternatively,
emulsified to produce droplets ranging in size from 2 to
5 9 microns. In its emulsified form, the composition can
be used as a syntKetic whole blood. The separated co-
acervate phase of the two phase system can be used as a
substitute for hematocrit. Storage of the coacervate
phase, if desired, should be at 4 to lO degrees C.
In the preferred-method of manufacture, emul-
sification of the two phase coacerYate system is delayed
until the procedures described immediately below, are
completed; aad such amount of cholesterol as will result
in a 0.1 to 2~ weight to yolume concentration of cholesterol
15 in the said system. Add calcium chloride powder to a
concentration of l to 5 mg.%. Next, add potassium
chloride to a concentration of l to 3 mg.%. The prep-
aration is then titrated using sodium bicarbonate until
a pH in the range of 7.3 to 7.45 is reached. Next,
20 add such quantity of distilled water as will render the
coacervate system isotonic with whole mammalian blood.
Mix the prepar~tion ~igorously for one hour; follow the
mixing step by storing the preparation at lO degrees C.
for 24 to 148 hours. (Longer periods of storage yiéld
25 greater volumes of the coacerYate phase.) Remo~e the
preparation from storage and emulsify it at ambient
temperature to produce droplets ranging in size from 2 to
9 microns. The resulting composition comprises the
claimed synthetic whole blood. For infusion into a
30 human recipient, the claimed synthetic whole blood should
be at temperatures approximating normal human temperatures
~i.e. 98.7F which is 37C.). Storage of the claimed
synthetic whole blood, if necessary, should be at 4 to
lO degrees- C.
If desired, the two phases of the coacervate
~2~
- 14 -
system may be separated, for instance, by mear~s of a
separatory funnel prior to emulsification. The separated
coacervate phase is useful as a substitute for hemato-
crit. Five to ten per cent weight to volume of stroma
5 free hemoglobin optionally may be added to the separated
coacervate phase, or optionally may be added, prior to
the emulsification step, to the two phase coacervate
system. ~lso, stroma free he~oglob;n optionally may be
added, prior to the emulsiication step, to the two phase
10 coacervate system containin~ the additive ingredients
described immediately abo~e. ~ddition of stroma free
hemoglobin will enhance the intrinsic oxygen transport
capability of both compositions of matter.
When the invention is prepared with the in-
15 gredients of the previously described Group A, a phos-
pholipid is included as a necessary component. Lecithin
is the preferred phospholipid. Any of the following or
mixtures thereof can be used in combination with or in
place of lecithin: cephalin, isolecithin, sphinomyelin,
20 phosphatidyl serine, phosphatidyl inositol, phosphotidyl
choline and phosphatidic acid.
This invention also teaches a second method
of making a synthetic whole hlood using the ingredients
described in this document as comprising Group B. This
25 method is also based on a process of coacervation. The
preferred procedure comprises the following steps:
5 to 15% weight to Yolume of gelatin powder is thoroughly
mixed with 5 to 15% weight to ~olume of acacia which has
been dispersed in sterile water. The solution is then
3~ stored undisturbed at 10 degrees C. for 24 hours, after
which, it is removed from refrigeration. Ordinarily,
thi~ step will produce the re~uired two phase coacervate
system. If the said system does not result lN hydro-
chloric acid may be added dropwise to the solution until
35 separation of the two phases occurs, indicating forma-
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tion of the coacer~ate system. Said system then is
adjusted to a pH in the range of 6.8 to 7.4 by adding
sodium hydroxide. If the two phase coacervate system has
formed during the 24 hour period of refrigerated storage,
5 then, as indicated above hydroc-hloric acid is not added;
however, if the pH of the said system is not in the range
of 6.8 to 7.4 it is made so by adding sodium hydroxide.
Following the step in which the pH is adjusted, the co-
acervate system is made isotonic with whole mammalian
lO blood by adding sterile water and/or sodium chloride.
The system may then be emulsified to produce droplets
ranging in size from 2 to 9 microns. ~s such, it is use-
ful as a whole blood substitute. Alternatively, the
phases are separated; the coacervate phase can be used
15 as a substitute for hematocrit. If not infused, the
compositions m~y be stored at 4 to lO degrees C.
In the preferred method, the emulsifying step
occurs after the following sequence of steps: To the
two phase coacervate system described above, such amount
20 of cholesterol is added as will result in a 1% weight to
volume concentration of cholesterol in said system. Next,
calcium chloride powder is added to a concentration of
from 3 to 5 mg.% and potassium chloride to a concentra-
tion of l to 3 rny.%. The preparation is then titrated to
25 a pH within the range of 6.8 to 7.4 by adding the ap-
propriate a~ount o~ sodium hydroxide; the pH of normal
whole human blood is preferred. Sterile water and sodium
chloride isadded as required, to render the coacervate
system isotonic with whole human blood. The system is
30 then mixed vigorously for one hour. Following this step,
the preparation is stored for 24 to 148 hours at lO
degrees C. The prep~ration is then removed from re-
frigerated storage ~nd at ambient temperature, emulsi-
fied by any of the recognized methods to produce globules
35ranging in size fro~ 2 to 9 microns. This step completes
:
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- 16 -
the preparation o~ synthetic whole blood using the com-
ponents of the previously described Group B. The com-
positions can now be infused at ambient temperature,
preferably at normal body temperature, into the recipient
or stored at 4 to 10 degrees C.
If a substitute for hematocrit is to be manu-
factured, the two phases of the coacervate system are
separated prior to emulsification, for instance, by
means of a separatory funnel. The coacervate phase of
the system is useful as a hematocrit substitute. It can
be infused at ambient temperature, preferably at
normal body temperature, into a recipient or stored at
from 4 to lO degrees C.
Five to ten percent weight to volume of stroma
free hemoglobin can be added to either the separated
coacervate phase or, prior to emulsification, to the
two phase coacerva~e system. Such addition will enhance
the intrinsic oxygen transport capability of these com-
positions.
Specific treatment regimens can be instrumented
through the claimed synthetic whole blood; i.e. hyper-
alimentation, intravenous drug therapy, etc. The in-
gredients necessary to any given treatment regimen are
added to the coacervate system prior to emulsification.
Any number of solvents may be used for Group C
compositions. T~ie solvents may be non-polar solvents or
semi-non-polar solvents. The following contains solvents
useful for this purpose: butanol, ethyl acetate, dibutanol
ketone , glycerol mono-acetate, glycerol di-acetate, gly-
cerol tri-acetate, glycerol dioleate, di-chloromethylene.
In this invention, the preferred solvent is di-chloromethy-
lene.
The manufacutre of the claimed compositions
of Group C must be carried out under ~septic conditions.
Except for those steps involving refrigeration, all other
procedures are performed at ambient temperatures. If it
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is desired, the compositions may be stored at 4 to lO
degrees C. ~AIhen infused, the comPositions Preferably
should be at a temperature that approximates normal body
temperature.
The following is a description of the preferred
method of preparing the claimed compositions of Group
C.
Disperse from 5 to 25% weight to volume of
powdered albumin in distilled water containing 0.9%
weight to volume of sodium chloride, l to 5~ weight to
volume urea and 0.1 to 10% weight to volume of lecithin.
To this ~olution, add di-chloromethylene dropwise until
globules of the coacervate phase appear. At this point,
the solution is stored, undisturbed at 4 degrees C. for
twelve hours. At the end of this period of storage, a
two phase coacervate system will have formed. The next
step consists of adding such amount of distilled water
at ambient temperature as will result in 5 to 25%
weight to volume of albumin. Such quantity of sodium
chloride is then added to the coacervate system as will
render the said system isotonic with whole mammalian
blood. At this point, there are two manufacturing op-
tions; the coacervate system can be separated into its
two component phases, or alternatively, the system can
be emulsified to produce droplets ranging in size from 2
to 9 microns. If the phases are separated, the coacer-
vate phase can be used as a hematocrit substitute. If
the system is emulsified, the composition can be used as
a whole mammalian blood substituteO Storage should be
at from ~ to lO degrees C.
In the ~referred method of manufacture, emul-
sification of the two phase coacervate system is delayed
until the procedures described immediately below are
.
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- 18 -
completed.
Add to said coacervate system such amount of
cholesterol as will result in a 0.1 to 2~ weight to
volume concentration of cholesterol in said coacervate
5 systeln. Next, add calcium chloride to a concentration
of 1 to Smg% and potassium chloride to a concentration
of 1 to 3mg~. The preparation is then titrated using
sodium bicarbonate until a pH in the range of 7.3 to
7.45 is reached. On completion of the titration step,
1~ add such quantity of distilled water as will render the
two phase coaceryate system isotonic with whole mam-
malian blood~ The preparation is now mixed vigorously
for one hour, and then stored at 10 degrees C. from
24 to 148 hours. Longer periods of storage yield greater
15 volumes of the coacervate phase. At the end o the
storage period, the preparation is then allowed to reach
room temperature. Next, the product is emulsified to
produce droplets ranging in size ~rom 2 to ~ microns. The
composition that results from these procedures con-
20 stitutes the claimed synthetic whole blood of Group C.Storage, i~ necessary, should be at 4 to 10 degrees C.
If it is desired, the two phases of the co-
acervate system described immediately above may be sep-
arated prior to the e~ulsi~ying step. If the phases are
25 separated, the separated coacervate phase is useul as
a hematocrit substitute. Five to ten per cent weight
to volume of stro~a free he~oglobin can be added as an
option, to the separated coacervate phasel or to the co-
acervate system prior to its entulsification. In either
30 preparation, the add;tion of stroma free hemoglobin will
enhance the intrinsic oxygen transport capacity o the
claimed compositions of matter.
The procedures described above indicate that
a phospholipid is a necessary component in Group C .
., .,. :
- 19~
Lecithin is the preferred phospholipid in preparing the
disclosed compositions of matter. However, any of the
following or mixtures thereof can be used in place of
lecithin: isolecithin, sphingomyelin, phosphatidyl
serine, phosphatidyl inositol, phosphatidyl choline and
phosphatidic acid.
Cholesterol is the preferred sterol in the
method(s) of manufacture of the claimed compositions of
matter. However, any of the following sterols can be
used in place of the preferred cholesterol: ergosterol,
7-dehydrocholesterol,C~sitosterol, ~ sitosterol,
v sitosterol, campesterol or mixtures thereof.
SPECIFIC EXAMPLES
Examples of the methods by means of which the
claimed com~ositions of matter may be manufactured
follow. Examples 1-7 exemplify Group A, Examples 8-15
exemplify Group B, and Examples 16-21 exemplify Group C.
Exam le 1
Twenty ive grams of albumin are added to 500
mls. of distilled water containing 0.9~ weight to volume
of sodium chloride, 3% weight to volume urea and 5Q0
mls. of a 2~ solution of lecithin. The solution is then
thoroughly mixed and stored at 4 degrees C. for 12
hours. The resulting coacervate system is then removed
: :
.
~Z~ 4
- 20 -
from refrigeration. When the solution reaches ambient
temperature, l gram o~ cholesterol, 0.2 gram of calcium
chloride and Q.4 gram of potassium chloride are added.
The pH of the solution is adjusted to 7.35 by adding
5 sodium bicarbonate. If the coacervate system is not
isotonic with whole hu~an bloQd, it is made so by the ad-
dition of the required amounts of sodium chloride and
distilled water. The solution is then mixed vigorously
for one hour ollowing which it is stored at a tempera-
lO ture of lO degrees C. for 148 hours. At the end of this
period, the solut~on will have separated into two dis-
tinct layers. The bottom la~er comprises the coacervate
phase of the two phase coacervate system; the upper layer
comprises the equilibrium water phase of the system.
The two phases of the system can be separated
or emulsified, for the purposes and in the manner describ-
ed previously. Infusion of the synthetic whole blood
into the circulation of the recipient may take place at
am~ient temperatures and preferably should take place
20 at normal body temperature. The storage of synthetic
whole should be at from 4 to 10 degrees C. The same
parameters govern the use and storage of the claimed
hematocrit substitute.
Example 2
Two hundred mls.of a 2% lecithin solution are
added to 200 mls.of a 4% solution of albumin. Nine
grams of sodium chloride, lO gr~ms of urea and l gram
of cholesterol are added to the above m~xture. The
remainder of the procedure follows Example 1.
30Example 3
200 mls.of 4% isolecithin solution are added to
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- 21 -
200 mls.of 5% albumin solution containing 0.9~ weight
to volume sodium chloride and 1% weight to volume urea
in a 2 liter flat bottom flask. The rest of the pro-
cedure follows E~ample l.
Example 4
500 ~ls~of 2 1/2% lecithin solution are added to
500 mls. of 5% human albumin stock solution in a 2 liter
flat bottom flask. To this mixture, ~ grams of sodium
chloride, 9 grams of urea and a.l gram of ergosterol
lO are added. The rest of the procedure follows Example 1.
Example 5
500 mls. of a 2 l/~ solution of isolecithin
are added to 500 mls. of a 5% stock solution. To this
mixture, 9 grams of sodium chloride, 9 grams of urea and
15 0.1 gram of ergosterol are added. The rest of the pro-
cedure follows Example l.
Example 6
This procedure follows Example 1 except that
50 grams of stroma free hemoglobin are added to the co-
20 acervate phase and thoroughly swirled in a flat bottomflask to achieve a uniform dispersion of the added hemo
globin.
Example 7_
The procedure follows Example l except that
25 after the manufacture of the two phase coacer~ate system
is completed, the coacervate phase is separated fro~ its
e~uilibrium water phase by means of a separatory funnel.
.~
..
~L~2~
- 22 -
The separated coace~ate phase will function as hema-
tocrit when introduced intravenously.
Example 8
Five percent weight to volume of gelatin pow-
5 der (IEP:8.2) is mixed thoroughly with ten percent weightto volume of aca~ia which has been dispersed in sterile
water. The solution is re~rigerated for 12 hours at lO
degrees C. The solution is then removea from refrigera-
tion. If the the two phases of the coacervate system
lO have not separated, lN hydrochloric acid is added drop-
wise until the two phases have separated. The solution
is adjusted to a pH in the range of 6.8-7.4 by the ad-
dition of sodium hydroxide. If the system is not iso-
tonic with whole human blood, sodium chloride and/or
15 sterile water is added to reach isotonicity. The prep-
aration is then emulsified to produce globules 2 *o 9
microns in size.
Example 9
The procedure follows Example 8 except that
20 after the two phase coacervate system is prepared, the
coacervate phase is separ~ted ~rom the equilibrium water
phase by means o a separatory funnel. The coacervate
phase can be infused intra~enously and will function as
hematocrit, or it ~ay be stored at 4 to lO degrees C
25 until needed.
Example lO
Fort~ mls. of a 5% weight to volume gelatin
solution (IEP: 8.2~ is thoroughly mixed with 12~ weight
to volume of acacia dispersed in sterile water. Sterile
'' "' ',
fl
- 23
water is added to this solution until a ~-olume of 150
mls. is reached. The solution is then stored in a re-
frigerator at lO degrees C for twelve hours. At the end
of this period, the two phase coacervate system should
5 be formed. If this has not occurred, lN hydrochloric
acid is added dropwise until the two phases of the system
separate. Next, the pH o the system is adjusted by ad-
dition of sodium ~ydroxide to a point in the range of 6.8
to 7.4. The coacer~ate system is rendered isotonic with
10 human blood by adding sterile water and/or sodium
chloride as may be requ-red. Following this step, such
amount of cholesterol is added as will result in a 1%
weight to ~olume concentration of cholesterol in the
solution; calcium chloride powder is added to a concen-
15 tration of 5 mg~. This step is followed by the additionof potassium chloride to a concentration of 3 mg~. The
preparation is titrated to a pH in the range of 6.8 to
7.4 by the aadition of sodium hydro~ide. If necessary,
add sodium chloride and/or sterile water as required to
20 make the coacervate system isotonic with whole human
blood. Next, mix the preparation vigorously for one
hour, then refrigerate at 10 degrees C for 148 hours.
In this examp].e, the end point purpose is preparation of
synthetic whole blood. Accordingly, the two phase
25 coacervate system is re~oYed ~rom refrigeration and em-
ulsified at ambient temperature by means of a colloid
mill to produce globules which can range in si~e from
2 to 9 microns. The emulsified composition can be in-
fused at this point or stored at ~rom 4 to lO degrees C
30 until needed.
Example ll
5~ weight to volume of gelatin powder (IEP: 8.2)
is mixea thoroughly with 5% weight to volume of acacia
'- .:': .
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~L224~
- 24 -
which has been dispersed in sterile water The solution
is refrigerated for 24 hours at 10 degrees C. If a two
phase coacervate system is not produced by the close of
the period of refrigeration, lN hydrochloric acid is
5 added dropwise at ambient temperature until the two
phase coacervate system is produced. The remainder of
the procedure ~ollows Example 10.
Example 12
5% weight to volume of gelatin powder (IEP:8.2
10 is mi~ed thoroughly with 10% weight to volume o~ acacia
which has been dispersed in sterile water. The solution
is refrigerated for 24 hour~ at 10 degrees C. If a two
phase coacervate system is not proauced by the end of
the refrigeration step, add lN hydrochloric acid at am-
15 bient temper~ture d~opwise until the two phase coacervatesystem is produce~. Ad~ust the pH of the system to a
point within the range of 6.8 to 7.4. Add such am~unt
of sodium chloride or sterile water as required to make
it isotonic with human blood. Emulsify the two phase
20 coacervate system to produce droplets ranging from 2 to
9 microns size.
Example 13
5% weig~t to ~olume of gelatin powder (IEP: 8.2)
is mixed with 10~ weight to ~olume of acacia dispersed
25 in sterile water. The solution is stored for 24 hours
at 10 degrees C. The rem~inder of the procedure follows
Example 10.
Example 14
5% weight to volume of gelatin powaer (IEP:8.2)
30 is mixed with 5~ weight to yolume of acacia which has
~ ~ .
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. -
~4144
- 25 -
been dispersed in sterile water. The remainder of the
procedure follo~s Example 10 except that 5% weight to
volume of stro~a free hemo~lobin is added prior to em-
ulsification.
Example 15
The procedures of Example 10 are followed in
their entirety except that prior to emulsification, the
two phases of the prepared coacer~ate system are sep-
arated by means of a separatory funnel. 5~ weight to
10 volume of stroma free hemoglob~n is added to the coacer-
vate phase of the two phase system. The coacervate
phase can then ~e used as a substitute fox naturall~
occurring h~matocrit.
Example 16
Twenty five grams of albumin are added to 500
mls. of distilled water containing 0.9~ weight to volume
of sodium chloride, 3~ weight to Yolume of urea and 500
mls. of a 2 1/2 solution of lecithin. Di-chlorom~thylene
is added dropwise to the solution until globules of coacer-
20 vate appear. The solution is then stored at 4 degrees
C. for twelve hours. At the end of this period, the
solution is remo~ed from storage and permitted to reach
room temperature,~after which l gram of cholesterol,
0.2 gram of calcium chloride and 0.4 gram of potassium
25 chloride are added. The pH of the solution is adjusted
to 7.35 by adding the necessary amount of sodium bi-
carbonate. The~ coacervate system is then made isotonic
with whole hu~an blood ~y addition of the required~amounts
o~ sodium chloride and distilled wate~. Following this
step, the solution is mixed vigorously~for one-hour. It
is then stored at a te~perature of 10 degrees C. for 72
'
: ~ ,. ' - ~' ,, : :
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- 26 -
hours. At the end of this period of storage, the solu-
tion will have separated into two distinct layers. The
lower layer comprises the coacervate phase of the two
phase coacervate system; the upper layer comprises the
5 equilibrium bulk water phase of the said system~
l'he t~o phases of the coacervate system can be
separated or emulsified for the purposes and in the man-
ner described prev~ously in this disclosure. Storage
of the products de~ived from this method should be at
10 4 to 10 degrees C.
Example 17
Two hundred mls. of a 2 1/2~ lecithin solution
are added to 200 mls. of a 4% solution of albumin. Nine
grams of sodium chloride and ten grams of urea are then
15 added to the above solution. Glycerol diproprionate is
then added dropwise until globules of coacervate be~in
to appear. The remainder of the procedure follows Ex-
ample 16.
Example 18
Two hundred mls of a 4% isolecithin solution
are added to 200 mls. of a 5% albumin solution containing
0.9 weight to Yolume of sodium chloride and 1% weight to
volume of urea. ~lycerol mono acetate is added dropwise
to the above solution until droplets of coacerva`te begin
25 to appear. The remainder of the procedure follow~ Ex~
ample 16.
Example 19
Five hundxed mls.o~ 2 12/% lecithin solution
ar~ added to 500 mls,of a 5% stock solution of human
30 al~umin. To this solution, 9 yrams of sodium chloride,
~22419L~
- 27 -
9 grams of urea and 0.1 gram of ergosterol are added.
Butanol is then added dropwise until globules of co-
acervate begin to appear. The remainder of the procedure
follows Example 16.
Example_20
Five hundPed mls.of 2 1j2% solution of iso-
lecithin are added to 500 mls of a 5~ stock solution of
human albumin. To t~is ~ixture, add 9 grams of sodium
chloride, 9 gra~s of urea and 0.1 gram of ergosterol.
10 Ethyl ether` is then added dropwise until globules of
coacervate appear. The'solution is then stored at 4
degrees C. for 12 hours. After this period of storage,
add such amount of dis~tilled water as will result in a
5~ weight to volume of al~)umin. Next, add such amount
15 of sodium chloride as will render the solution isotonic
with whole human blood. Follow this step by emulsifying
the solution to produce droplets which range in size
from 2 to 9 microns.
,
Example 21
This procedure follows'Example 16 with the
following exceptions: (a) glycerol dioleate is added
dropwise to the solution containing albumin, distilled
water, sodium chloride, urea and lecithin, in place of
di-chloromethylene and (b) prior to the emulsiflcation
25 step, 50 grams of stroma free hemoglobin are added and
' thoroughly swirled to achie~e a uniform dispersion through-
out the coacer~ate system.
TESTS
The foLlowing are'tests of in vivo administra-
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- 28 -
tion of the claimed synthetic whole blood and the claimed
substitute for hematocrit, prepared in accordance with the
detailed description.
TEST 1
From each of three co~mon white laboratory
rats, 4 cc of blood was remo~ed followed immediately by
infusion of 4 ce of synthetic whole blood prepared ac-
cording to Example l. This procedure was repeated after
an interval of five minutes. In effect, approximately
10 40% of the animal's total blood volume had been removed
and replaced. One rat of this series was saerificed two
hours after the experiment was completed. The lungs,
heart and other tissues revealed no gross significant
pathological changes. The remaining animals were sacrificed
15 sixty hours after the second infusion of the substitute
whole blood. Examination of the heart, lungs and other
tissues showed no pathological changes nor any signs as-
sociated with hypoxia, pulmonary edema or adverse im-
munological reaction. Blood studies of all animals in
20 this series indieated normal oxygen and carbon dioxide
tensions, and normal pH values. Neither the erythrocytes
nor the clotting mechanisms appeared to be adversely
affected.
TEST 2
In a second experiment, one rat recei~ed a
single injeetion o 6 cc. o~ a second preparation of the
substitute whole blood of Example l immediately follow-
ing removal of 6 cc. of its blood. This animal expired
approximately 70 minutes after infusion of this sample of
30 substitute blood. Tissue studies indicated signs of in-
travascular disseminated coagulation. Examination and
analysis of this sample of the synthetic whole blood
,
.
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- 29 -
yielded evidence of contamination and improper prepara-
tion.
TEST 3
Four cc. o~ a fresh prepalatiGn of the sub-
5 stitute whole bloQdi of Example lQ was administered in-
travenously to each o~ two rats in a third series with-
out withdrawal o~ ~lood from either animal. One rat
was sacrificed afte~ 48 hours; seventy two hours a~ter
infusion with su~stitute whole ~lood the second animal
10 was sacrificed. Inspection of the tissues and red blood
cells showea no pathological change or evidence of ab-
normal response. Clott;n~ mechanisms appeared`to be un-
affected.
TEST 4
.
A fourth series of tests was performed using
-two Nembutal anesthetized dogs. Approximately 10% of
the first animal's blood was withdrawn from the femoral
artery, and replaced immediately with an equal quantity
of substitute whole blood o~ Example 1. Approximately
20 40% of the blood volume was withdrawn from the second
dog and replaced with an equal quantity of the substitute
whole blood of Example 3. Samples of the circulatin~
blood were withdrawn from each animal from the site of
infusion at three minute inter~als for fifteen minutes
25 and at one hal~ hour intervals for two hours there-
after. Oxygen tension measurements were determined by
the IL Blood Gas Analyzer. Test xesults indicated an
increase in PaO2 levels oyer base line measurements.
Carbon dio~ide lavels remained within normal limits.
Mean arterial blood pressure rose to 150/88 ~rom
,~ ~ .. .
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_ 30 _ ~22~4~
135/80 after infusion in the first animal. The mean
arterial blood pressure in the second animal rose from
130/75 to 155/90, following infusion with substitute
whole blood. After 24 hours, the mean arterial blood
5 pressure stabilizéd at 130/70 in the first dog, mean
arterial blood pressure in the second dog stabilized at
145/75 twenty four hours after infusion. Following in-
fusion with synthetic wKole blood, mean heart rate in
the first animal rose to 120 beats per minute from a ~
lO base line meaSUEement of 105. Mean heart rate following
infusion of the second dog rose to 155 beats per
minute from a base line reading of 110. After 24 hours
the mean heart rate was measured at 98. The mean heart
of the second animal stabilized after 24 hours at 99
15 beats per minute. Both animals were sacrificed 96
hours a,ter infusion with substitute whole blood. Tis-
sue studies revealed no significant evidence of path-
ological change or abnormal immunological reaction in the
first animal. The second dog however exhibited equi-
20 vocal signs of intravascular disseminated coagulation insegments of the venous svstem. A third dog was exposed
to the same withdrawal and replacement procedures as
the second animal of this series. However, the infused
substitute blood in this experiment contained 10 mls. of
25 heparin. Sacrifice of this animal 96 hours after in~
fusion and study o~ the organs, tissues, and red blood
cells revealed no abnormal changes or signs of immunological-
ly undesirable response.
.
TEST 5
In a fifth test utilizing one Nembutal an-
esthetized dog, the infused substitute whole blood of
Example 1 was prepared to include stroma free hemoglobin.
Approximately 40% of the blood volume of this animal was
", ;
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- 31 -
withdrawn and replaced with the preparation described
above. Mean blood pressure after infusion rose to 150/90
from a baseline measurement of 135/80; after 24 hours,
the mean blood pressure stabilized at 140/85. Following
5 in~usion with substitute whole blood mean heart rate
rose from 110 to 145 beats per minute. After 24 hours,
the mean heart rate was measured at 105. PaO2 levels
remained increased oYer base line measurements for ap-
proximately 95 minutes. Upon restoration of base line
10 PaO2 levels, intermittent administration o~ oxygen from
an external source resulted in elevated oxygen tensions
in this animal that persisted for approximately 6 1/2
hours. This result suggests that the claimed composition
remains in the circulation, retaining its ability to
15 transport oxygen efficiently for an appreciable period of
time.
TEST 6
A sixth test involved the withdrawal of 6cc
of blood from each of two common white laboratory rats
20 and the immediate replacement with an equal amount of
synthetic whole blood made in the method of Example 10.
Blood studies indicated elevated oxygen tensions, normal
carbon dioxide leYels and normal pH values. Both ani-
mals were sacrificed 72 hours after infusion of the syn-
25 thetic whole blood. Inspection of lungs, heart andother tissues reyealed no si~nificant sign of patho-
logical chanye or adYerse immunological response.
TEST 7
The seyenth test involved the withdrawal of
30 4 cc of blood from each o~ two common white laboratory
rats, ~ollowed by the immediate replacement of 4 cc of
:, :, . ..
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;
1~ 4~
- 32 -
the composition made in accordance with the method of
Example 15. (Substitute for hematocrit)~ Blood studies
indicated elevated oxygen tensions, normal carbon dioxide
levels and normal p~ yalues. Both animals were sacri-
ficed 48 hours after infusion of the hemabocrit sub-
stitute. Inspection of the lungs, heart and other tissues
disclosed no significant evidence of pathological change
or adverse immunological reaction.
TEST 8
From each of four common white laboratory
rats, 4 cc of blood was removed followed immediately by
infusion of 4 cc of synthetic whole blood prepared ac-
cording to Example 16. This procedure was repeated af-
15 ter an interval of five minutes. In effect~ this processresulted in the remo~al of approximately 40~ of the
animals total blood volume and the replacement with the
synthetic whole blood. One rat of this series was
sacrificed two hours after the experiment was completed.
20 The lungs, heart and other organs revealed no gro~s sig-
nificant path~logical changes. One of the remaini~g
animals was sacrificed 4~ hours afte~ the second infusion
of the substitute whole blood. The remaining two
animals were sacrificed 60 hours after the second in-
25 fusion. Examination of the heart, lungs and other organsshowed no pathological change nor any signs associated
with hypoxia, pulmonary edema or adverse immunological
reaction. Blood st~dies of all animals indicated nor-
mal oxygen and carbon dioxide tensions and normal pH
30 values. Neither erythrocytes nor clotting mechanisms
appeared to be adversely affected.
TEST 9
In the ne~t test, tWG rats received a sin~le in-
,., '' ' ''
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. . :
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- 33 -
jection of- 6 cc of synkhetic whole blood orepared ac-
cording to the method of Example 17, after 6 cc of blood
had been withdrawn. Both animals were sacrificed 48
hours after the infusion of synthetic whole blood. In-
5 spection of the heart, lunys and other organs revealedno signigicant pathological change, nor any signs as-
sociated of hypoxia, pulmonary edema or adverse im-
munological reaction. Studies of the blood of these
animals indicated normal oxygen and carbon dioxide
10 tensions and normal pH Yalues. Erythrocytes appeared
normal. Clotting mechanisms did not appear to be ad-
versely affected.
TEST 10
Three cc of a fresh preparation of the syn-
15 thetic whole blood prepared according to the method of
Example 18 was infused to each of two animals in a third
series, without withdrawal of blood from either animal.
One rat was sacrificed 36 hours after the in~usion; the
second rat was sacrificed 48 hours after the infusion
20 f synthetic whole blood. Examination of the vital
or~ans and red cells showed no patholoqical chanqe
or evidence of abnormal response. Clotting mechanisms
appeared to be unaffected.
TE~T 11
.
From each of three white laboratory rats, 4
cc of blood was withdrawn followed immediately by an
infusion of 4 cc of hematocrit substitute prepared ac-
cording to the method of Example 16. One rat was
sacrificed 36 hours after the infusion; the second rat
30 was sacrificed ~8 hours after infusion of hematocrit
substitute. Neither animal on examination of vital organs
showed any signs of pathological change. Blood studies
`~.-
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.
- 34 - ~ ~ ~4~
showed normal oxygen and carbon dioxide tensions and
normal pH values. Red cells did not appear tc be af-
~ected. Clotted mechanisms appeared to be normal.
: ~ ~
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