Note: Descriptions are shown in the official language in which they were submitted.
~66~89
The present invention relates to a solid composition,
its method of manufacture and its use for treatment of hemo-
philiacs.
Time ia of considerable importance in the treatment
of he~ophiliacs both to the person administering emergency
injections and to the persons receiving such injections because
loss of blood by the hemophiliac and/or injury to the joints
is aggravated during the time required for solubilizing the
nor~ally solid composition to permit injection. The problem
therefore to which the present application is directed is to
decrease the solubilization time of the solid composition to
permit prompt injection into an injured person.
It has been the practice to freeze dry blood plasma
fractions in form of a solid composition to remove water there-
fr~m and, just prior to use, to dissolve the solid composition
in a liquid aqueous medium to form a solution which is then
injected into the patient. The blood plasma fractions are ob-
tained by removing water from blood plasma (blood from which
white and red cells and blood platelets have been removed and
which contains about 90 percent water and 10 percent solids).
The solids remaining after removal of the water from the blood
plasma are divided into blood plasma fractions, those most useful
for the purpose of this invention being the anti-hemophilic
factor (AHF, Factor VIII) fraction (which may contain fibrinogen)
and, if desired, a separate fibrinogen factor.
-2-
1066189
Solubilizing agents have been used to solubilize
blood plasma fractions but the time it takes to dissolve such
fractions leaves much to be desired. For example, U.S. Patent
2,826,533 discloses the addition of dextrose to a fibrinogen,
blood plasma, fraction and it is known that the time of
solubilization of the fibrinogen fraction can be reduced by
adding dextrose thereto. It is also known to add dextrose to
A~IF as, for example, described in the "Journal of Thrombosis
Research", Volume 1, pages 191-200, 1972, published by Pergamon
Press, Inc. which reported addition of dextrose to AHF in
order to facilitate the chromatography of AHF. The article
concluded that the yield of bovine Factor VIII from chromato-
graphy on anion exchange media can be greatly improved by the
inclusion of a low-molecular weight carbohydrate, such as
dextrose, in the solvents. In addition to the foregoing, Cutter
Laboratories, Inc. has added sufficient dextrose to its com-
mercial AHF preparation so that when the preparation is recon-
stituted according to the instructions, the resulting AHF
solution contains about one gram of dextrose per 100 milliliters
of solution.
Finally, U.S. Patent 3,057,781 discloses stabilizing
plasma with invert sugar and levulinic acid.
While dextrose has been used with blood plas~la
fractions for various purposes, as is seen from the fore~oing,
it was not appreciated before the present invention that by
critically controlling the amount of a water soluble carbohydrate
(which is free of le w linic acid) in the solid composition,
it is possible to solubilize the solid composition with an
1066~L89
aqueous medium within about 90 seconds.
More specifically, this is accomplished in accordance
with the invention by providing a solid composition comprising
AHF, characterized by including an amount of at least one
water soluble carbohydrate sufficient to solubilize the compo-
sition in an aqueous medium within about 90 seconds, the ~IF
being present in an amount sufficient to form a therapeutically
effective AHF concentration upon solubilization of the
composition.
We also provide in accordance with the invention
a method of preparing an A~F-containing composition adapted
to be rapidly solubilized upon reconstitution with a
reconstituting liquid to produce a solution containing a
therapeutically effective amount of AHF, characterized by adding
to said composition a water soluble carbohydrate in an amount
sufficient to solubilize the composition in the reconstituting
liquid within about 90 seconds.
Objects and advantages of the invention will become
apparent from the following detailed disclosure taken in
conjunction with the accompanying drawing, in which:
Fig. 1 graphically depicts the unexpected dramatic
improvement in the rate of solubilization of solid composi-
tions containing AHF which is obtained when the composition
is solubilized in the presence of from 0 to 5 grams of dextrose
per 100 ml. of reconstituted solution. The various curves
are obtained using different reconstitution temperatures and
volumes.
~66~89
The problem of reducing dissolution time of solid
compositions of the type above disclosed is solved in accordance
with the invention by the inclusion in the solid composition
of a critical amount of water soluble carbohydrate. When one
desires to use the composition of the invention, one need only
add water and because of the presence of the carbohydrate in
the solid composition, the composition dissolves in a very
short period of time, for example, within 90 seconds, and can
then be injected into the patient.
The amount of carbohydrate to be added is, as noted,
critical in the sense that mere addition of carbohydrate does
not in itself result in significant solubilization time
improvements. A threshold concentration of carbohydrate must
be reached before useful improvements in solubilization time
are achieved, after which addition of more carbohydrate again
exerts no significant effect on solubilization time. This
threshold concentration will vary within the ranges disclosed
depending upon factors which are believed to include the
amount and identity of protein and salts in the AHF preparations
as well as the identity of the carbohydrate selected. The
exact optimum quantity of carbohydrate will thus vary with the
carbohydrate chosen, the method of AHF preparation and even
with separate runs using thh same preparatory method. Thus
the appropriate quantity should be determined by elementary
and conventional solubilization time assays for each lot of AHF.
The amount of carbohydrate should be sufficient to
bring the solid AHF preparation into solution within about 90
seconds, and preferably 65 seconds, the AHF being present in
the solid composition in an amount sufficient to form a thera-
peutically effective AHF concentration upon solubilization of
--5--
'1066~39
the composition. A therapeutically effective concentrationof AHF in such solutions illustratively ranges about from 3
to 100 International Units of AHF per ml. with a preferred
range of about from 3 to 40 International Units per ml. and
more commonly about 24 to about 28 units per ml. The amount
of carbohydrate present in the solid AHF composition typically
provides, upon reconstitution with water, or other suitable
reconstituting liquid, a therapeutically effectlve solution
of AHF containing at least about 2 weight units of carbohydrate
(computed in Grams) for every 100 volume units cf solution
(computed as milliliters). The a unt of carbohydrate will
generally vary about from 2 to 10 grams, preferably about 2 to
5 grams of carbohydrate per 100 ml. of solution, with about 3
grams appearing to be optimum.
To provide the desired concentration of carbohydrate
in solution, the carbohydrate is illustratively present in
the solid composition in an amount of about 1.6 to 7.5 times
the a~ount of total protein in the solid AHF composition.
Preferably, the amount of carbohydrate is about 2.0 to 5.0
times th~ weight of total protein. The preferred embodiment
is about 2.0 times the weight of total protein. This solid
composition can contain anywhere from about 2 to 200 Inter-
national Units of AHF/gm protein, and still produce as a
pr~ctical matter a solution of A~F upon reconstitution which
has a therapeutically significant effect.
The water soluble carbohydrates useful in the
invention include any which are capable of hydrating the AHF-
containing composition. This includes without limitation the
monosaccharides such as the commonly available hexoses, inclu-
ding dextrose (glucose), mannose, galactose and fructose;
~066~9
the disaccharides such as maltose, lactose and sucrose; the
trisaccharides, such as raffinose; and the short chain dextrins,
e g. dextrins having a chain length of less than about four
monosaccharide units. Mixtures of suitable carbohydrates may
also be employed. The preferred carbohydrates are dextrose,
su~rose, c~mltose and lactose, with dextrose being an especially
preferred material! The carbohydrate must be biologically
acceptable when the AHF is marketed for human administration.
The carbohydrate can be admixed with the AHF-containing composi-
tion at any point during or prior to preparation of the
lyophilized composition.
The AHF compositions, whose rate of solubility is
enhanced by the addition of carbohydrate in accordance with the
present invention, may be prepared by numerous procedures known
to those skilled in the art. In the preferred embodiment, the
solid mixture comprising AHF and fibrinogen is obtained by
starting with plasma frozen at about minus 25 degrees C. which
is then thawed to 4 or 5 degrees C, to produce a cryoprecipitate
which is collected by centrifugation.
The cryoprecipitate is suspended in heparinized,
citrated saline to which is added 3.5% by weight, of polyethylene
glycol. The resulting mixture is centrifuged and the resulting
fibrinogen precipitate is discarded and the supernatant retained.
To the supernatant is added about 7.5 weight units of polyethylene
glycol (expressed as grams) per 100 volume units of supernatant
(expressed as milliliters). The resulting suspension is mixed
for about 15 minutes at room temperature and is then centrifuged
and the resulting precipitate collected. This precipitate or
10661~9
solid mixture comprises AHF and fibrinogen and can be used as
such or can be further purified by glycine fractionation. In
any event, the water soluble carbohydrate can be added to
either of such mixtures. Preferably, the solid mixture of
AHF and fibrinogen is dissolved in an aqueous medium, for
example, a dextrose citrated saline aqueous solution containing
about 0.72% sodium chloride, 0.02M sodium citrate and an
appropriate amount of the water soluble carbohydrate to produce
the desired effect of an enhanced rate of solubilization. It
is neither necessary nor desirable at this stage to add water
to the point where the solution contains about 2 weight units
of dextrose per 100 volume units of solution since such a
dilute solution may unnecessarily extend the time required for
lyophilization.
The dissolved solid mixture containing the carbohydrate
is further clarified by passing it through a coarse filter
which removes some of the fibrinogen and other insoluble pro-
teins. Thereafter, the sample is further diluted with citrated
saline, as desired, to a potency of about 3 to 75 International
Units/ml. or left as a concentrate which normally contains from
250 to 1000 International Units/ml. The dissolved product is
then sterile filtered through a "Millipore" membrane filter
having an average pore size of about 0.3 microns. The filtered
solution is filled under aseptic conditions into 10 ml. to 30 ml.
capacity vials, as desired, rapidly frozen and freeze-dried.
To administer the AHF preparation to a patient, the
normal procedure is to reconstitute the lyophilized product to
a solution containing about 3 to about 100 International Units
~rale r)l~rK
10661139
of AHF per m~., and more commonly about 24 to about 28 Units
per ml., about 2 to about 10 grams of dextrose per 100 ml.,
about 1.4 to about 1.6 grams of protein per 100 ml., about
0.6 to about 0.~ grams of fibrinogen per 100 ml., and about
0.7 gra~ to about 6 grams of salts such as NaCl, sodium
citrate, glycine and unidentified residual solids per 100 ml.
Iypically a 10 ml. vial of reconstituted ~F solution will
contain about 270 International Units of AHF, about 0.3 gram
of dextrose, about 0.15 grams of protein including about 0.07
gram of fibrinogen, about 0.51 gram of residuals and sufficient
water to 10 ml. volume.
The lyophilized product is readily soluble in
sterile water at room temperature and after the addition of
the water is almost immediately ready for administration to
he~ophillc patients as a result of the dextrose levels in the
solution.
In order to show the dramatic unexpected results
obtained by the present invention the following tests were
conducted using 10 ml. and 30 ml. capacity vials containing
the lyophilized product of the invention, the 10 ml. and 30
ml. vials being respectively filled with 10 ml. and 30 ml.
of water at room temperature and 37C. The 30 ml. vials
contained;approximately three times as much product as the
10 ml. vials. The time required for complete dissolution of
such sample was recorded. The rcsults are presented below
in Table 1 and in Fig. 1.
~066189
Table 1
grams dextrose/ Fig. 1 Curve: A B C D
100 ml. of recon- Reconstituting
stituted composi- Volume: 10 ml. 10 ml. 30 ml 30 ml.
tion Diluent Temp.: 37C Room Temp 37C Room Temp.
1176 secs. 195 secs. 100 secs. 330secs
3 35 " 85 " 50 " 65 "
48 " 55 " 52 " 75 "
None (control)185 " 210 " 105 " 210 "
The control in the above table was identical to the other
samples in all respects except that the control contained no
dextrose.
The results of Table 1 are plotted in Fig. 1. The
plotted data clearly shows the marked improvement in rate of
solubility once a dextrose concentration exceeding 2 grams per
100 mls. of reconstituted composition is obtained. Similar
desirable results are obtained with the other carbohydrates
discussed hereinabove.
In the preceding exa~ple, polyethylene glycol was used
to fractionate the blood plas'-~. However, other compounds can
be used such as ethylene oxide-propylene glycol conden~ation
products, and other procedures for fractionation can be employed
to produce a product which is rapidly soluble according to the
teachings herein.
-10 -
