Note: Descriptions are shown in the official language in which they were submitted.
.~24~
~^ .
. ~
- 1 - . .
GELATIN BASED SYNTHETIC WHOLE BLOOI)
- AND A PROCESS FOR PREPARING THE SAME
The present invention relates to a synthetic
whole blood useful as a replacement for whole mammalian
blood and a process for preparing the same. It has now
been unexpectedly discovered that a synthetic blood may
5 be made by incorporating either microencapsulated hemo- -
globin or synthetic liposomes containing stroma free r
hemoglobin, into the coacervate phase of an appropriate
coacervate system or into an appropriate coacervate
system. It has also now been unexpectedly discovered
10 that as long as lecithin is present, a synthetic whole
blood may be made using only one gelatin or only one
modified fluid gelatin, instead of two.
It is now recognized that the physical chemical
structure of whole human blood has been successfully
15 duplicated in a composition of matter known as Synthetic
Whole Blood, as disclosed in applicants' United States
Patent 4,343,797. It is now also recognized that
Synthetic Whole Blood is a distinct entity, fundamentally
different from the preparations referred to in the
20 scientific literature as "blood substitutes".
An appropriate two-phase aqueous liquid system
~i.e. coacervate system) is fundamental to preparation r
of synthetic whole blood and its companion product,
synthetic hematocrit. U. S. Patent 4,343,797 contains
25 the comment, "In the practice of this invention the
underlying principle is that any molecule or combination
of molecules capable of forming a non-toxic, two-phase,
- aqueous liquid system can be .... used to prepare the
.
~., . . . ~,.
,
. .
:. .. . ..
: ~ ... ,. : .
- :
.~: ... . .
-2~
requisite coacervate system." Further study of the dis- F
covery of this principle by the inventors makes it~
possible to specify this principle in ~reater detall.
The present disclosure further exemplifies this principle.
One category of coacervate systems useful to
prepare synthetic whole blood contains among its prin- ~.
cipal components, (1~ a suitable protein, i.e. albumin, ~:
geIatin, modified fluid gelatin, etc.; (2) a coacervating ~;
surface active molecule such as lecithin; each of these
10 components possessing opposing surface charges; and
~3) hemoglobin in the form of synthetic liposomes con-
taining stroma free hemoglobin.or stroma free hemoglobin
per se, or microencapsulated hemoglobin~ .
The fundamental components of another category s
15 of coacervate systems useful to prepare synthetic whole ~.
blood contains (l) two similar'or two different protein
molecules, i.e..gelatin, modified fluid gelatin, etc.,
each with a surface charge that opposes the surface charge
of the other; and ~2) stroma free hemoglobin, microen- :
20 capsulated hemoglobin or synthetic liposomes containing
stroma free hemoglobin.
Appropriate physiologically useful additives
can be readily introduced into the compositions derived
from either class of the coacervate.systems described -
25 above.
A number of considerations warrant the develop-
ment of an alternative version of the Synthetic Whole
Blood preparation,. as disclosed in U.S. Patent 4,343,797.
Principal among these is the probability that a small but ~!
25 medically si~nificant number of persons may be sensitive '.'
to one or more of the ingredients.of the composition re
ferred to above.
Aside from the United States Patent 4,343,797,
the prior art that has been diligently searched fails to
30 reveal a~y reference to a preparation which can serve
as a whole blood replacement. The literature, however,
does contain more than 1500 citations to enti~ies de-
scribed as "blood substitutes". These citations refer to
. . ~ .
'
.
~?~
--3- -
studies of such substances as perfluorocarbons, albumin,
hydroethyl starch, modified gelatin, etc. (References: `~Chemical Abstracts; 1970-1982; Index Medicus; 1970-
1982).
With the single exception of the references to
the gelatin preparations used as "blood substitutes",
none of the prior art in the clinical literature appears
to-have any relevance to the presently claimed invention.
No citation was identified which hints at, suggests or
10 implies that a synthetic whole blood can be based on
gelatin.
To summarize findings from the clinical prior art,
from a physiological point of view, regarding available
gelatin "blood substitutes", the molecular structure of
15 gelatin is such that in clinical use, it can only serve as
- a plasma extender, (expand blood volume). It cannot
transport any o the physiological gases. (Reference:
Merck Index 1979). Unexpectedly, however, through their
research applicants have discovered that gelatin and/or
20 modified gelatin based coacervate systems can trans-
port essential amino acids, transport physiolog-
ically important gases and restore or maintain the
necessary osmotic pressure. There are however addi-
tional striking differences. Table I infra of thls
25 application lists 13 clinically important variables
which distinguish the claimed compositions from the
known gelatin "blood substitutes", and which show the
similarities between the claimed compositions and ~hole
human blood.
In the prior art, is a reference to gelatin
based coacervates, Veis, A. and Aranyi, C., Phase Sep- `
aration in Polyelectrolyte Systems, I; Complex Co-
acervates of Gelatin, Journal of Physical Chemistry,
Volume 64, pages 1203-1205. Examination of this prior
35 art indicates it to be a theoretical study of gelatin
based coacervate systems. It addresses only the condi-
tions under which gelatins of differing isoelectric
points will form coacervates. There is no suggestion
'' ~ .
~22~5
--4--
nor inference in this prior art that the described F
coacervate systems have any possible biological or
non-biological use. Therefore, the person ordinarily
skilled in the art cannot conclude from a study of this
5 prior art that a synthetic whole blood can be based
upon it. Given that gelatin solutions are known to be
among the available "blood substitutes", it is more
probable that this cited prior art would suggest ~-
another method of preparing the alr~ady known gelatin
lO based plasma extender.
The presently disclosed invention rests upon
the applicants' recognition of the biological utility
of a coacervate system.
However, this recognition is not of itself suf-
15 ficient to prepare an optimal synthetic whole blood,
ready to be administered to mammals, particular-ly '~umans.
Specific chemical entities, some of which do not of
themselves suggest that they are useful in the prepara-
tion of a synthetic whole blood, must be added to the
20 coacervate system employed in this invention. It is
the applicants' position that these additives alter
the chemical character and the physiological utility of
the coacervate system, resulting in not another version
of a gelatin based "blood substitute" but rather in a
25 synthetic whole blood, which can be used as a replace-
ment for whole mammalian blood.
As it now appears frequently in the literature,
the term synthetic liposomes generically covers both
stroma free hemoglobin and other stroma free hemo- '~
30 globin preparations as well as synthetic erythrocytes
or lipid encapsulated hemoglobin. Reference:
Miller, I. and Djordjevich, L.; U.S Patent 4,133,874
~1979). With regard to the Miller and Djordjevich
reference, the possibility is mentioned that the syn-
35 thetic erythrocytes they have invented can be suspended
in isotonic saline or Krebs-Ringer solution or in syn-
thetic plasma materials and used for blood transfusion
purposes. S~nce the vehicle~ given abo.e contain large
,
, ' ' . - :
::,
,:,,
:, '
12Z~J. 4~;
-- 5 --
quantities of bulk water, there is a strong likelihood
that oxygen uptake in such compositions is limited.
This stands in direct contrast with the oxygen uptake
capability of the presently disclosed invention in which ~i
microencapsulated hemoglobin or liposomes containing
stroma free hemoglobin is incorporated in the claimed
coacervate system or the coacervate phase of such a ;
system. Both the coacervate system and the coacervate
phase of the system have significant oxygen pick up.
The addition of stroma free hemoglobin in the form given
immediately above serves significantly to enhance the
oxygen uptake of these claimed compositions.
This invention provides an acceptable
substitute for whole mammalian blood, and for preparing
an acceptable substitute for whole mammalian blood.
Therefore, the present invention provides a
process for preparing a synthetic whole blood substitute,
- comprising the steps of: (a~ combining water and
lecithin and a gelatin component selected from gelatin
or modified fluid gelatin to form an aqueous solution;
(b) admixing a sufficient quantity of an electrolyte to
achieve an isotonicity equal to that of physiological
saline solution; (c) storing the solution at a temperature
of from 15 to 50C, for at least 12 hours until said
solution separates into two layers, consisting of a
- lower layer being a substantially non-polar coacervate
phase, and an upper layer being a substantially polar
equilibrium water phase; (d) separating said lower phase
from said upper phase; and (e) adjusting the p~ of said
coacervate phase to a range of from 7.2 to 7.6.
Further, the invention provides a synthetic ~-
whole blood substitute prepared according to said
process.
'
' . '
; ':
,. .~.
,. . ~ "
- 5A - F
Table I
Properties* '
.
1. Oxygen Transport
2. Carbon Dioxide Transfer ~ l
5 3. Oxygen can be held in reserve and released in L
accordance with physiological tension .
4. Hemoglobin can be added or dispersed within the
preparation without loss of stability
5. Transfers gasses other:than 2 and CO2 ~.
10 6. Possesses both polar and non-polar properties
7. Dissolves and transports non-polar drug entities
. without loss of dosage-form stability
8. Transports enzyme systems without loss of sta~ility
9. Effect on hematocrit percent after transfusion
15 10. Essential amino acids can be transported in stable
form and desired quantity
11. Oxygen uptake ability reduced at low 2 partial
pressures
12. Transports physiologically useful lipid soluble
.. 20 entities as a stable-solution
13. Universal donor characteristics
.
~;
:
~. ~
; .,
- : '' ' .
-
:
,
-.
. : ,, , , :.
::
:,: - ~ '
.: :
-6~ S
.~
U ~~ ~0
~ ~ U n~ 0 '`
h O
C~ O Q,Q)
~¦ 0 r~lu~O R ~ ~
~1 ~ ~ U.,,.U~ ~,.,'Z'~
'~0 ~ U
r--l
E a) a) o a) o ~: ~, o o ~ o o o
q o Z ~ Z Z
H
~ O
U~ ~O ~ O
. Q)
h ~ I` ~X) ~ o
O
h
,
. - '' ~
~" ' . '`
'~ '
-7- 12~4~5
~i Z ~ Z Z Z; Z Zi Z ~ Z
- ,, o
z ~ z ~ æ z z z ~: ~ ~ z ~
: 0 ~ 0
Z 71 Z 3 Z ~Z; Z Z 1:~ ~ a Q~ Z ~
U ~ ~ :
H ,
U
' : '
,-.
, ' ` :-
. ' ~ '
OQ ~
~ . `
. ~
, . r ,
' :' ` , ' . :,
.r
'~ . ' '. , :'
,~ ,
:-~
- 8 - ~22
Some embodiments of the synthetic whole blood ~
may also include 2-15% weight to volume of a hemoglobin ~`2
selected from stroma free hemoglobin, microencapsulated
stroma free hemoglobin, or synthetic liposomes contain-
ing stroma free hemoglobin.
This invention comprises a composition of-
matter useful as a substitute for whole natural blood.
The claimed invention is comprised of a two-phase
aqueous liquid system substantially identical to the
physicochemical system of whole natural blood. A
substantially non-polar coacervate phase insoluble in
and in equilibrium with an associated substantially polar
equilibrium water phase are characteristic of both
naturally occurring whole blood and the claimed r
invention. This invention also comprises a method
of making a whole blood substitute, which yields the
two-phase system referred to above. The system is
composed of an internal suspension phase, herein referred
to as the coacervate phase, and an external suspension
phase which is the associated equilibrium water phase.
When the claimed composition is introduced intravenously,
it will disperse in the blood plasma o~ the recipient, c
and thereby contribute to the two-phase physicochemical
system of the naturally occurring whole blood. The r
physiochemical characteristics of this invention render
it sensitive to and reactive to the oxygen tension of
the recipient's blood. Further, it can readily enter
and pass through the major blood vessels, capillaries
and the microc.irculation.
The claimed synthetic whole blood can transport
and transfer oxygen and carbon dioxide much as naturally
occurring erythrocytes do, without adversely affecting
the percent of the recipient's hematocrit. In addition,
it can carry nutrients, physiological entities,
therapeutic drugs and enzyme systems.
.
9 ~2~
~ pon transfusion this invention can establish,
re-establish and/or maintain normal osmotic pressures.
The transport characteristics of this composition of , L'
matter enable it to serve as a safe and reliable ve-
5 hicle. When it is desirable to introduce enzyme systems
into the body, such systems as noted above can be
added to this invention and infused through conven-
tional intravenous methods. Enzyme systems introduced
through these compositions of matter will perform
10 their normal physiologicaI functions.
The guidelines which determine the quan-
tities of the claimed synthetic whole blood which may
be safely infused are substantially identical to those
which govern the use of whole blood.
By reason of its mode of manufacture and r
its physicochemical structure, the claimed whole blood
~,
substitute posseses a number of advantages over whole
blood. Thus, prior to infusion this invention can be
modified to meet many of the specific requirements of
20 given treatment procedures, such as hyperalimentation,
intravenous drug therapy, open heart surgery, etc. By
way of example, additional quantities of stroma free
hemoglobin or synthetic liposomes containing stroma
free hemoglobin or microencapsulated hemoglobin can
25 be incorporated in a given embodiment of this
invention so as to enable more oxygen to be carried
~or longer periods of time as would be desirable
in treatment of certain blood diseases or in instances
of prolonged surgery. ~lectrolytes can be added to L
30 the claimed substance for use in the treatment of cases
of severe burns or shock resulting from the loss of
blood. In embodiments containing added electrolytes,
adjustments to isotonicity are made following such ad-
ditions. When nutrients must be quickly introduced
35 and/or when the circulatory system is the preferred
route for nutrition, essential amino acids and other
nutritional agents can be added prior to transfusion.
', ' ,, ~ .
: ~ ,
10- ~22~
A significant advantage of this invention is F
that because this invention possess universal donor t
characteristics, no blood typing is necessary prior
to administration of this composition.
Other important advantages of this invention
may be enumerated as follows: the components of the ~-
claimed composition are abundant, readily available
and relatively inexpensive. Additives can be quickly
introduced to previously prepared, stored embodiments.
lO The invention can be used without the need for highly
specialized equipment or technology. The constituents
of the claimed composition of matter and the method of
preparing it eliminates the problems associated with
the storage of whole blood
In order to explain the invention more fully
the following is a general description of the prefer~red
method used to practice this invention. Specific
examples of the practice of this invention are also pro-
vided in the following section of this disclosure.
The formulation that follows specifies sub-
stantially equal proportions of two gelatins, two
modified fluid gelatins, or one gelatin and one modified
~L.
fluid gelatin, with different isoelectric points. How-
ever, in the practice of this invention unequal pro-
25 portions of the two gelatins, two modified fluid gela-
tins, or one gelatin and one modified fluid gelatin with
different isoelectric points may also be used to pre-
pare the claimed composition of matter. In the process
of manufacture, the component ingredients should be i~
30 prepared and combined under aseptic conditions.
Mix equal proportions of a 1 to 10~ weight -'
to volume solution of gelatin with an isoelectric
point of 2 to ~ with a 1 to 10~ weight to volume solu-
tion of gelatin, an isoelectric point of ~.0 to 10Ø
35 In this step, modified fluid gelatins may be used in
place of gelatin provided the requirement of differing
isoelectric points is observed. The resultant mixture
of the two gelatin solutions will be approximately 0.5
- .
' ~ ';' ' :
~ . `' ' ,,;
: ' :
- -
to 5~ weight to volume of each of said gelatins.
The mixture is then left undisturbed at 37 C for r^
24 hours. At the end of this period, the mixture .
will have separated into two layers, the lower ona of
5 which comprises the coacervate phase. The upper layer
cornprises the equilibrium water phase and may be dis-
carded. The pH of the coacervate phase is adjusted
to 7.4 by the addition, preferably dropwise, of any
nontoxic al~aline substance, preferably sodium hy-
10 droxide or sodium bicarbonate. The resulting compo-
sition can be used as a synthetic whole blood. In
the preferred procedure, 2 to 15% weight to volume of
stroma free hemoglobin, or that amount of synthetic lip-
osomes containing stroma free hemoglobin or micro-
15 encapsulated hemoglobin as will result in a 2 to 15%weight to volume of stroma free hemoglobin in the
` finished product, is added to augment the oxygen
transport capability of the composition. If
desired, l to 10% weight to volume of a nontoxic
20 ionic, or non-ionic surfactant and/or a nontoxic or-
ganic solvent may be added to the preparation, to en-
hance the oxygen transport capability of the composition.
A suitable protein such as albumin may also be added.
In such instance it is added in the amount of l to 5%
25 weight volume.
The non-ionic surfactants that may be used,
include any of the nontoxic pluronics or any of the
substances know~ as spans.
The ionic surfactants that may be used in~ h
30 clude any of the phospholipids such as lecithin, ceph-
alin, isolecithin, sphingomyelin, phosphatidyl serine,
phosphatidic acid, phosphatidyl inositol and phos- s
phatidyl choline. Other compounds known to those
skilled in the art may also be used. Lecithin is the
35 preferred phospholipid in this invention and is added in
the amount of l to 10% weight to volume.
Following the addition of any of the above,
or~any combination of the above, the preparation is
....
:
,. -~ .
.
.. ... . ..
. . . .
12 ~ 2~L~4~
subjected to vigorous shaXing for 3 minutes to achieve
uniform dissolution and dispersion of the additive(s).
It is highly desired that for the best method that when
the ingredients referred to above are added, the amounts
5 of each should be sufficient to reach the saturation
point and beyond within the coacervate phase.
If thé intended use of the composition in~
volves an open circuit, prior to infusion, oxygen should ~-
be bubbled through the preparation until the desired
10 oxygen concentration is reached. If the synthetic
whole blood composition is to be used in a closed
system, the desired level of oxygen tension is main-
tained by bubbling oxygen through the system by the usual
means.
Another embodiment of this invention also r
useful as a whole blood substitute is also claimed
which makes use of both layers. The preferred
manufacturing procedure is as follows: The claimed
two phase liquid aqueous~system is prepared in the man-
20 ner described previously. After the 24 hour period ofstorage at 37 C the two layers are separated by
means of a separatory funnel or other suitable means
but, the equilibrium water layer is retained in sterile
condition for use in a subsequent manufacturing step.
25 Following the separation procedure, the pH of the co~
acervate layer is adjusted to 7.25 to 7.4 by the drop-
wise addition of sodium hydroxide or sodium bicar-
bonate. When this step is completed, 2 to 15% weight
to volume of stroma free hemoglobin, or that amount
30 of synthet-ic liposomes containing stroma free hemo-
globin or microencapsulated hemoglobin is added so
that the stro~a free hemoglobin in the finished
product ranges from 2 to 15% weight to volume, is
added and the preparation vigourously mixed. The
35 preparation is then emulsified by adding the
previously separated equilibrium water layer and
using colloid mill or other suitable means to produce
the required emulsion. The particles of the emulsion
.
:'.: ;., - :
,
-: :
: ,
- 13 - ~ 5
can range in size from 0.5 to 9 microns in size. In
the preferred procedure, the addition of the equilib-
rium water layer and the emulsifying step follow the
addition and mixing of 2 to 15% weight to volume of
5 stroma free hemoglobin, or synthetic liposomes con-
taining stroma free hemoglobin or microencapsulated ~-
hemoglobin. Also, l to lO~ of a suitable ionic
surfactant, preferably lecithin, and l to 5~ weight
to volume of a suitable protein, preferably albumin,
lO may be added.
When preparation of the composition is com-
pleted, it may be infused to transport physiological
gases, restore or maintain osmotic pressure, trans-
port polar and non-polar drugs, carry enzyme systems,
15 nutriments, etc. Alternatively, it can be stored at
from 4 to 10 C until needed. If the composition
is to be infused into a human following refrigerated
storage it should be warmed to body temperature
(37 C).
It may be stored at conventional room tem-
peratures, if the preparation can be maintained in
completely sterile condition.
While the above description contains many
specifics these should not be construed as limitaions
25 on the scope of the invention but rather as exemplifica- _
tions of preferred embodiments. Accordingly, the scope
of this invention should not be determined by the de-
scribed embodiments but by the appended claims and
their legal equivalents. ~-
Specific Examples
Examples of how the claimed compositions of
matter may be prepared follow.
Sterile conditions are observed during all
phases of manufacture.
3~ Example l
Take 4 grams of gelatin, isoelectric point of
9 and add distilled water until a solution of lO0
mls is reached. ~ext, take 4 grams of gelatin, iso-
.'
.. ..
- 14 ~
electric point of 4, and add distilled water until a
solution of 100 mls is reached. Mix the two solutions
thoroughly and incubate, undisturbed at 37 C for ,
24 hours. Separate the resulting two layers and
5 discard the upper equilibrium water layer. Adjust the
pH of the lower (coacervate) layer to 7.~ through the
dropwise addition of sodium hydroxide, and add 10% weight
to volume of stroma free hemoglobin. Disperse the
additive by vigorous shaking for 4 minutes. If the
10 preparation is to be infused shortly after manufacture,
bubble oxygen through the composition until desired
oxygen level is reached.
Example 2
The procedure follows that of Example 1 ex-
15 cept that 5~ weight to volume of lecithin is also ad-
ded to the coacervate layer, and dispersed by shaking
the mixture.
Example 3
The procedures follows that o~ Example 1 ex-
20 cept that 2~ weight to volume of albumin is also added
to the coacervate layer and dispersed by vigorously
shaking the mixture.
Example 4 k.
The procedure follows that of Example 1 ex- -
25 cept that 5% weight to volume of stroma free hemoglobin,
5% weight to volume of lecithin, and 1~ weight to vol-
ume of albumin are added to the coacervate layer and
dispersed by means of vigorous shaking the com-
position.
Example 5
.,
Mix equal proportions of 8% weight to volume
of gelatin with an isoelectric point of 5, and a gela-
tin with an isoelectric point, of 9.5 Let the mix-
ture stand undisturbed for 24 hours at 37 C.
35 At the end of this period, separate the two layers that
will have formed and discard the upper layer. Adjust
the pH of the lower layer to 7.4 by the dropwise ad-
dition of sodium hydroxide. To this, add 5~ weight to
:.
~2~
- 15 -
v~lume of stroma free hemoglobin. Disperse the stroma
free hemoglobin by vigorous shaking o the composition.
Example 6
The procedure follows that of Example 5 ex-
5 cept that 5% weight to volume of lecithin is added to
the coacervate layer and dispersed by vigorous shaking
of the composition.
Example 7
The procedures follows that of Example 6 ex-
10 cept that 1~ weight to volume of albumin is added to
the coacervate layer and dispersed by vigorous shaXing
of the composition.
Example 8
Thoroughly mix equal proportions of 8%
15 weight to volume of modified liquid gelatin with an iso- r
electric point of 5, and a modified liquid gelatin with
an isoelectric point of 9. Permit the mixture to stand
undisturbed at 37 C for 24 hours. At the end
of this period, separate the two layers that will have
20 formed and discard the upper layer. Adjust the pH of
the lower layer to 7.4 by the dropwise addition of sod-
ium hydroxide. Add 10% weight to volume of stroma free
hemoglobin and disperse same by vigorous shaXing for 4
minutes.
Example 9
The procedure follows that of Example 8 ex-
cept that 5% weight to volume of lecithin is added to
the coacervate layer and dispersed through vigorous
shaking of the composition.
Example 10
The procedure follows that of Example 8 ex-
cept that 1~ weight to volume of albumin is added to the s-
coacervate layer and dispersed through vigorous shaking
of the mixture.
Example 11
Thè procedure follows that of Example 8 ex-
cept that 5% weight to volume of lecithin and 1% weight
to volume of albamin are added to the coacervate layer
.. . .
... .
- ': :
'
.. , . :
.: . -':
.
~22~
- 16 -
and dispersed through vigorous shaking of the composi-
tion.
~xample 12
'
This is the procedure employing encapsulated i~
5 stroma free hemoglobin. It occurs after the coacervate ~i
system has been formed, the phases are separated, and L
2 to 5% stroma free hemoglobin has been added to the
lower coacervate layer. This procedure thus may be
applied to the resultant product of any of Examples
10 4, 5, 6, 7, 8, 9, 10 and 11. The lower coacervate
layer containing the stroma free hemoglobin is combined
with the equilibrium liquid water layer and emulsified
so that the final emulsion contains particles
(droplets) which can r~nge from 0.5 to 9 microns in ?.
- 15 size~ Next, l to 5% formaldehyde solution is added r
dropwise to the emulsified preparation until the
desired degree of shell structuring of the droplets
is achieved. The degree of structuring can range
from semi-solid or gel-like to rigid, and is achieved
20 either through the amount of formaldehyde added or
through the length of the period of storage. After
the desired degree of structuring is achieved, the
preparation is stored anywhere between 5 to 40 hours
at 20 to 40C. On removal from storage, the
25 preparation will have separated into two layers, the
bottom one of which contains microencapsulated
globules substantially spherical in shape, contain-
ing stroma free hemoglobin. The upper layer consists
of equilibrium liquid water. The two layers are
30 separated by means of a separatory funnel or other
acceptable means. The microencapsulated spheres are ~'
washed with the equilibrium liquid water, until sub-
stantially all traces of formaldehyde are completely
removed. The microencapsulated spheres containing
35 stroma free hemoglobin can then be dispersed in
physiological saline solution, in the coacervate
phase of any of the herein described coacervate systems,
or added to the coacervate phase of the two phase
- 17 - ~
coacervate system. After this step, the cornposition
is then emulsified. The resultant emulsion is pre-
pared so that the droplets can range in size from -
0.5 to 9 microns. When the microencapsulated spheres
5 containing stroma free hemoglobin are incorporated
into the two phase coacervate system as described ~;
above, the result of the proceduxe is microen-
capsulated globules containing stroma free hemoglobin
incorporated in droplets of the coacervate phase which in
lO turn is suspended in the equilibrium liquid water phase.
In practice, where optimal sustained oxygen
uptake and release is desired, minimal structuring
of the microencapsulated spheres is preferred. De-
pending upon the physiological effect to be achieved,
15 differing proportions of microencapsulated spheres
of differing degrees of shell hardness can be com-
bined. This will result in special release effects
which can be used when introducing drugs, nutrients,
enzyme systems. In other words, the composition
20 can be so prepared as to give the desired specific rate
of release of any of the components contained within
the microencapsulated spheres. The procedure to in-
corporate drugs, nutrients, enzyme systems, et cetera,
into synthetic blood containing microencapsulated
25 stroma free hemoglobin is the same as the procedure
herein described to incorporate drugs, nutrients,
enzyme systems, et cetera, into synthetic blood con-
taining microencapsulated hemoglobin.
Example 13
Take 4 grams of gelatin, isoelectric point of
9 and add distilled water until a solution of lO0 mls -
i.5 reached. Next, take 4 grams of gelatin, isoelectric r
point of 4, and add distilled water until a solution of
100 mls is reached. Mix the two solutions thoroughly
3S and incubate, undisturbed at 37 C for 24 hours.
Separate the resulting two layers. Adjust the pH of
the lower tcoacervate) layer to 7.4 through the drop-
wise addition of sodium hydroxide, add 10%-weight to
.
,
.
.
': ' .
.
~2;2~
- 18 -
volume of stroma free hemoglobin. Disperse the ad-
ditive by vigorous shaking. ,`
The preparation is then emulsified by adding the
previously separated equilibrium water layer and using
5 a colloid mill to produce the emulsion. The desired
emulsion particle size can range from 0.54 to 9 microns
9 microns in size.
Example 14
The procedure follows that of Example 12 ex-
10 cept that 5% weight to volume of lecithin is also addedto the coacervate layer.
Example 15
The procedure follows that of Example 12 ex-
cept that 2% weight to volume of albumin is also added
15 to the coacervate layer.
Example 16
- The procedure follows that of Example 12 ex-
cept that 5% weight to volume of stroma free hemoglobin,
5~ weight to volume lecithin and 1% weight to volume
20 albumin are added to the coacervate layer.
Example 17
Mix equal proportions of 8% weight of volume
of modified gelatin, isoelectric point of 5 and with a
second modified gelatin, isoelectric point of 9.5, in-
25 cubate 24 hours at 37 C. At the end of thisperiod, separate the two layers that will have formed.
Adjust the pH of the lower coacervate layer to 7.4 by
the dropwise addition of sodium hydroxide. Add 5% weight
to the volume of stroma free hemoglobin. Disperse the
30 stroma free hemoglobin by vigorous shaking.
The previously separated equilibrium water
layer is then added to the preparation and a colloid
mill is used to produce the emulsion. The desired emul-
sion particle si~e can range from 0.5 to 9 microns.
ExampIe 18
The procedure follows that of Example 16 ex-
cept that 5~ weight to volume of lecithin is added to
the coacervate layer.
,
'' :.~ '
~ 19~ 5
Example 19
The procedure follows that of Example 16 ex-
cept that 1% weight to volume of albumin is also added
to the coacervate layer.
Example 20
The procedure follows that of Example 16 ex-
cept that 5% weight to volume of stroma free hemoglobin,
5% weight to volume of stroma free hemoglobin, 5%
weight to volume lecithin and 1% weight to volume al-0 bumin are added to the coacervate layer.
Example 21
The procedure follows that of Example 16 ex-
cept that (a) 8% weight/volume of modified fluid gela-
tin, isoelectric point of 5 is mixed with an equal amount
5 of gelatin isoelectric point of 9 and (b) instead of
stroma free hemoglobin being dispersed, synthetic lip-
osomes containing stroma free hemoglobin are dispersed
by vigorous mixing into the formed coacervate system.
Example 22
Mix equal proportions of 8~ weight to volume
of gelatin with an isoelectric point of 5, and a gelatin
with an isoelectric point, of 9.5. Let the mixture stand
undisturbed for 24 hours at 37 C. At the end of
this period, separate the two layers that will have formed
25 and discard the upper layer. Adjust the pH of the lower
layer to 7.4 by the dropwise addition of sodium hy-
droxide. To this, add 5~ weight to volume of synthetic
liposomes containing stroma free hemoglobin. Disperse
the synthetic liposomes containing stroma free hemo- ~.0 ylobin by vigorously mixing the composition.
Example 23
Thoroughly mix equal proportions of 8% weight
to volume of modified liquid gelatin with an isoelectric
point of 5, and a modified liquid gelatin with an
35 isoelectric point of 9. Permit the mixture to stand
undisturbed at 37 C for 24 hours. At the end
of this period, separate the two layers that will
have formed and discard the upper layer. Adjust the
.. . .
.
- 20 ~ ~22~
pH of the lower layer to 7.4 by the dropwise addition
of sodium hydroxide. Add 10% weight to volume of
synthetic liposomes containing stroma free hemoglobin
and disperse same by vigorous shaXing for 4 minutes.
Example 24
Mix 5 to 10~ weight/volumé of gelatin iso-
electric point 7 to 10 wi-th 1/2 to 10% weight to Yolume
of lecithin. Adjust the electrolyte concentration to ~.
give an isotonicity equal to that of physiological
10 saline solution. Incubate at 37 C for 24 hours, at
the end of which 2 layers will have separated, one of
whïch is the equilibrium water phase and the other is
the coacervate phase. Separate the resulting two layers
and discard the upper equilibrium water layer. Adjust
15 the pH of the lower (coacervate) layer to 7.4 through
the dropwise addition of sodium hydroxide. Add lQ% weight
to volume of stroma free hemoglobin. Disperse the
additive by vigorous shaking for 4 minutes. I the
preparation is to be infused shortly after manufacture,
20 bubble oxygen through the composition until desired
oxygen level is reached.
Example 25
Mix 1/2 to 10~ weight to volume of gelatin or
modified fluid gelatin isoelectric point of 5 to 10 with
25 1/2 to 10% weight to volume of lecithin~ Add by mixing
in such amounts of a salt of sodium, potassium, calcium -
and magnesium as will result in the electrolyte balance
and isotonicity of physiological saline solution. In-
cubate at 37 to 50 C for 24 to 36 hours. At the end ~;
30 of the period of incubation, the mixture will have
separated into two layers, the bottom one of which is
known as the coacervate phase. The upper layer is known r
as the equilibrium water phase. At this point the two
phases may be separated by means of a separatory funnel.
35 Stroma free hemoglobin or liposomes containing stroma
free hemoglobin or microencapsulated hemoglobin is added
to the coacervate phase in an amount that will result in
a finished product that contains 2 to 15% weight to volume
,
~,
' . ,,
- 21 - ~2~4~
of stroma free hemoglobin. The preparation descri~ed -
immediately above can be used for transfusion or stored
at from 4 to 10C. Alternatively, the coacervate
phase which contains the electrolytes and stroma frèe
5 hemoglobin or liposomes containing stroma free hemoglobin
or microencapsulated stroma free hemoglobin in the
quantities gi~en above can be combined with the
equilibrium water phase and emulsified. The emulsion
can be used for transfusion or stored at 4 to 10C.
10Example 2~
The amounts used were as follows: 5% weight
to volume gelatin isoelectric point of 7 and 7% weight
to volume of lecithin. All other ingredients were in
the amounts given above, for Example 24, and the pro- `
15 cedure followed the description given above. The two
phases were emulsified as described above. Stroma free
hemoglobin was used, in the amount of 5% weight to volume.
Example 27
This procedure is the same as Example 24 ex-
20 cept that the phases were separated and no emulsifica-
tion was used, i.e. the coacervate phase plus the de-
scribed additives constituted the composition.
Example 28 ~-~
This procedure is the same as Example 24 ex-
25 cept that modified fluid gelatin was used.
Example 29
The procedure is the same as Example 24 ex-
cept that modified fluid gelatin was used.
.
.,`:i,~.:
,
',':
:-- ., :
-:, . :
:: , ....
. . : ,-
- 22 - ~2Z~s ~-
Experiment
The following in vitro experiment was conducted
to test the oxygen carrying capacity of the claimed
composition. Control substances were comprised of (A)
5 saline solution, and (B) saline solution and 4% weight
to volume stroma free hemoglobin. The three ~ompo-
sitions according to this invention were comprised of
(C) gelatin based coacervate composition of this in-
vention, (D) gelatin based coacervate composition of
10 this invention plus 4% weight to volume o~ stroma
free hemoglobin, and (E) emulsified gelatin based co-
acervate composition of this invention plus 4% weight
to volume of stroma free hemoglobin.
Oxygen was bubbled through each for 20 minutes
15 at 3iC. The results obtained were as follows: '
Oxygen uptake
Substance (Volume to volume)
(A) Saline solution 0~
(B) Saline solution and 4% hemoglobin 1%
20 tC) Claimed composition
- (Gelatin P.I.3; Gelatin P.I.9)* 10%
(D) Claimed composition and 4% stroma free
hemoglobin (Gelatin P.I.3; Gelatin P.I.9)* 22%
(E) Emulsified claimed composition containing
4% hemoglobin 15%
(F) Claimed composition: Coacervate Phase
(Lecithin and Gelatin, P.I.9) 12%
(G) Claimed Composition: Coacervate Phase
(Lecithin, Gelatin P.I.9; 4% stroma free
hemoglobin) 19%
(H) Emulsified claimed composition containing
(Lecithin, Gelatin P.I.9 and 4% stroma free
hemoglobin? 14%
*P.I. is an abbreviation for isoelect~ic point.
