U.S. patent number 3,784,453 [Application Number 05/098,574] was granted by the patent office on 1974-01-08 for process and apparatus for making radioactive labeled protein material.
Invention is credited to Howard J. Dworkin, Robert F. Gutkowski.
United States Patent |
3,784,453 |
Dworkin , et al. |
January 8, 1974 |
PROCESS AND APPARATUS FOR MAKING RADIOACTIVE LABELED PROTEIN
MATERIAL
Abstract
A closed sterile electrolytic system for tagging human serum
albumin and other compounds and/or substances including proteins
with a radioactive isotope, such as the 99m isotope of technetium,
producing a radioactive tracer material which is suitable for
biological studies and medical uses.
Inventors: |
Dworkin; Howard J. (Birmingham,
MI), Gutkowski; Robert F. (Sterling Heights, MI) |
Family
ID: |
22269923 |
Appl.
No.: |
05/098,574 |
Filed: |
December 16, 1970 |
Current U.S.
Class: |
424/1.37; 74/86;
204/271; 600/1; 424/1.69; 204/288.1 |
Current CPC
Class: |
A61K
51/1282 (20130101); A61K 2123/00 (20130101); Y10T
74/18544 (20150115) |
Current International
Class: |
A61K
51/12 (20060101); B01k 001/00 () |
Field of
Search: |
;136/90
;204/195R,271,286,297R,297M,131,137 ;128/1.1,1.2 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Primary Examiner: Tung; T.
Attorney, Agent or Firm: Harness, Dickey & Pierce
Claims
What is claimed is:
1. In a sealed reaction system for electrolytically tagging
compounds with radioisotopes, the combination comprising a
container defining a sterile reaction chamber and formed with an
aperture having a closure disposed in sealing relationship therein
and pierceable by a hypodermic syringe for gaining access to the
interior of said chamber, a solution in said reaction chamber
consisting essentially of a sterile non-pyrogenic aqueous mixture
at a controlled pH containing a radioactive material and a protein
compound, a pair of electrodes projecting into said chamber and
adapted to become at least partially immersed in said solution
contained therein, at least one of said electrodes comprised of a
metal selected from the group consisting of iron, zirconium and
alloys thereof, and means for electrically connecting said
electrodes to an electric power source.
2. The reaction system as defined in claim 1, in which said
electrodes are disposed in the upper portion of said chamber when
said container is in its normal upright position and in which
position said electrodes are disposed above the level of said
solution disposed within and partially filling said chamber.
3. The reaction system as defined in claim 1, in which said
container is comprised of glass and is formed with a neck in the
upper portion thereof defining said aperture in which said
pierceable closure is disposed in sealing relationship, said pair
of electrodes extending downwardly in spaced piercing relationship
through said closure to a position spaced upwardly from the bottom
wall of said container.
4. The reaction system as defined in claim 1, further including
coacting means on said vial for facilitating connection of said
electrodes to the proper polarity of electric current.
5. A process for preparing a sterile, non-pyrogenic aqueous mixture
containing a compound tagged with a radioactive isotope which
comprises the steps of preparing a closed sterile non-pyrogenic
reaction chamber having a pair of electrodes therein of which one
is comprised of a metal selected from the group consisting of
zirconium, iron and alloys thereof, introducing a sterile
non-pyrogenic aqueous mixture into said reaction chamber which
contains a radioactive material and a protein compound at a
controlled pH, immersing said electrodes in said aqueous mixture
and passing an electric current between said electrodes to effect
ionization and solution of a portion of said one electrode and an
absorption of at least a portion of said radioactive isotope on
said compound, continuing the passage of electric current until at
least a portion of said compound has become electrolytically tagged
with said radioactive material providing therewith an aqueous
sterile non-pyrogenic mixture containing an electrolytically tagged
compound suitable for use in biological studies.
6. The process as defined in claim 5, in which the pH of said
aqueous mixture is controlled within a range of about 1 to about
1.3 during the passage of electric current.
7. The process as defined in claim 5, in which said radioactive
material comprises the pertechnetate ion technetium-99m.
8. The process as defined in claim 5, in which said compound
comprises a solution of human serum albumin.
9. The process as defined in claim 5, in which said compound
comprises particles of aggregated human serum albumin.
10. Th process as defined in claim 5, including the further step of
introducing a sterile non-pyrogenic aqueous alkaline solution into
said reaction vial at the completion of said electrolytic tagging
to adjust the pH to a level of about 7.4.
11. The process as defined in claim 5, including the further step
of extracting the resultant sterile non-pyrogenic aqueous mixture
containing the electrolytically tagged compound from said reaction
chamber with a sterile hypodermic syringe.
12. The process as defined in claim 5, including the further step
of extracting the resultant sterile non-pyrogenic aqueous mixture
containing the electrolytically tagged compound from said reaction
chamber and passing it through a bacterial filter prior to use.
13. The reaction system as defined in claim 1 wherein said
radioactive material comprises the pertechnetate ion
technetium-99m.
14. The reaction system as defined in claim 1, in which said
protein compound comprises a solution of human serum albumin.
Description
BACKGROUND OF THE INVENTION
Radioactive isotopes and particularly proteins tagged or labeled
with such radioactive isotopes have been found extremely useful in
biological studies and medical diagnoses. In particular, human
serum albumin tagged with technetium-99m has been found as
possessing extreme utility for placental localization, heart blood
pool scanning and cisternography. Unfortunately a more widespread
use of this and similar radioactive diagnostic materials has been
restricted because of the difficulty of preparing such materials
and the necessity of using such materials immediately after
preparation due to their short effective life and before tests can
be completed on the pyrogenicity and sterility thereof. Still
further problems and complications are encountered in the
preparation of radioactive labeled microaggregated and
macroaggregated human serum albumin because of the difficulty in
controlling the aggregation procedure so as to attain the desired
particle size consistent with the intended diagnostic use of the
material. The aggregation procedure, in accordance with prior art
techniques, is relatively tedious and time consuming and frequently
results in improper particle sizes, requiring the entire batch to
be discarded and a repetition of the entire preparation procedure.
In an effort to overcome the foregoing problems, highly trained and
experienced personnel have been used for preparing such radioactive
labeled protein materials utilizing relatively sophisticated
laboratory facilities. The costs and difficulties associated with
such prior practices has inhibited a more widespread adoption and
use of such materials.
The apparatus and process comprising the present invention
overcomes the foregoing problems in providing a simple and
economical system for preparing radioactive labeled protein
materials in a sterile, non-pyrogenic condition, rendering the
material ideally suited for a variety of biological studies. The
system can be furnished in a convenient kit form, facilitating the
preparation of the material by other than highly trained personnel
and without resort to sophisticated laboratory facilities.
SUMMARY OF THE INVENTION
The advantages and benefits of the present invention are achieved
by providing a closed electrolytic system including a sealed
sterilized reaction vial incorporating a pair of electrodes which
extend inwardly and are adapted to be immersed in an aqueous
solution containing a dissolved radioactive ion and a compound,
such as a protein or suspended aggregated protein particles. The
passage of electric current through the conductive solution between
the electrodes effects a complexing and absorption of the
radioactive ions on the protein constituent. In accordance with the
practice of the present invention, the several solutions employed
to form the electrolytic reaction mixture, namely: the aqueous
solution containing the radioactive material; the aqueous solution
containing the protein; the aqueous diluent, if required, to dilute
the solution to a preselected final volume; and the acidic and
basic solutions employed for pH adjustment can all be preliminarily
sterilized and subjected to sterility and pyrogenicity testing,
assuring that the final product will not produce any adverse
reactions upon injection into the blood stream. Tests conducted on
radioactive technetium-tagged human serum albumin prepared in
accordance with the closed sterile electrolytic system comprising
the present invention have evidenced a superior performance of the
tagged albumin in comparison to technetium-tagged albumin prepared
by chemical techniques and open electrolytic techniques.
In its apparatus aspects, the present invention provides a novel
sealed reaction vial within which the electrolytic tagging of a
compound with a radioactive material is effected, utilizing in one
of its embodiments, an electrode assembly adapted to be removably
mounted on a conventional standard sealed vial converting it into a
reaction vial suitable for use in accordance with the practice of
the present invention. In a preferred form of the electrode
assembly, the cap portion thereof is formed with suitable coacting
means thereon for cooperating with corresponding means on an
electrical socket for assuring proper electrical connection between
the two terminals and the polarity of the electrical power source.
It is further contemplated that in addition to the electrolytic
sealed and sterilized reaction vial, one or more additional
sterilized and sealed vials containing controlled amounts of
sterilized solutions required during the course of the preparation
of the radioactive tagged protein material can be provided, forming
a kit which substantially facilitates the practice of the process
aspects of this invention.
Still further advantages and benefits of the present invention will
become apparent upon a reading of the description of the preferred
embodiments taken in conjunction with the accompanying drawings and
specific examples provided.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a side elevation view of a kit comprising an electrolytic
sealed reaction vial constructed in accordance with one embodiment
of the present invention in addition to supplemental sealed vials
containing sterilized quantities of supplemental solutions employed
in the electrolytic tagging process;
FIG. 2 is a perspective view of an electrode assembly in accordance
with an alternative embodiment of the present invention suitable
for mounting on a standard sealed vial for forming a reaction vial
suitable for use in the practice of the present invention;
FIG. 3 is a vertical cross sectional view of a standard sealed vial
having installed thereon an electrode assembly of the type shown in
FIG. 2;
FIG. 4 is a perspective view of the reaction vial shown in FIG. 3
disposed in inverted installed relationship within a socket of an
electrical power source; and
FIG. 5 is a fragmentary vertical sectional view of the electrical
disposition of the contacts and electrodes of the socket and
reaction vial, respectively, when disposed in the arrangement
illustrated in FIG. 4 .
DESCRIPTION OF THE PREFERRED EMBODIMENTS
Among the several radioactive isotopes that can be satisfactorily
used in accordance with the system comprising the present
invention, technetium-99m has been found extremely useful and has
received widespread acceptance as a radioisotope for use in various
biological studies and medical diagnoses. The tagging of this
radioisotope on proteins and specifically human serum albumin (HSA)
in accordance with the present invention has met with particular
success, producing radioactive labeled HSA in extremely high yields
and purity. While the system comprising the present invention and
the preferred embodiments thereof will be described in terms of the
use of technetium-99m and HSA as the radioactive isotope and tagged
protein carrier medium, respectively, it will be understood that
other satisfactory radioactive isotopes, as well as other compounds
including other protein materials such as fibrinogen and globulins,
can be used which are compatible with and produce satisfactory
yields in accordance with the practice of the present
invention.
The use of technetium-99m is particularly desirable and preferred
over many other radioactive isotopes in view of the relatively
short half life of the material, which is about six hours, thereby
resulting in reduced exposure of the internal organs to radiation.
The relatively short half life of this material also necessitates
local preparation of the radioactive isotope since it is ordinarily
uneconomical and impractical to effect a preparation and shipment
by the manufacturer to a remotely located attending physician.
Technetium-99m is obtained as the daughter product of molybdenum
Mo.sup.99 and is separated in the form of the pertechnetate ion
(TcO.sub.4 .sup.-). The Mo.sup.99 ion is absorbed on an alumina
column and the pertechnetate ion is separated from Mo.sup.99 by
elution with a dilute solution of hydrochloric acid or saline. Such
absorption columns containing Mo.sup.99 can be eluted or milked
daily to yield the desired quantities of the radioactive isotope
since the maximum growth of radioactivity of technetium-99m occurs
in about 23 hours. When employing saline for eluting the column,
the eluate comprises a dilute aqueous solution containing aodium
pertechnetate technetium-99m which, for the purposes of the present
invention, may range in radioactivity from a fraction of a
microcurie up to whole curie quantities and generally about 100
millicuries.
A process for preparing a sterile non-pyrogenic radioactive eluate
containing technetium-99m is described in detail in U.S. Pat. No.
3,440,423, the subject matter of which is incorporated herein by
reference as being typical of one of the various techniques well
known in the art for preparing such radioactive aqueous solutions.
In accordance with the specific arrangement described in the
aforementioned United States patent, a closed and sealed sterile
column partly filled with granular alumina having adsorbed thereon
radioactive (Mo.sup.99) ammonium molybdate is periodically eluted
with a sterile, non-pyrogenic isotonic saline to extract the
radioactive technetium-99m. The eluate is collected in a sterile
collecting container, providing a sterile, non-pyrogenic aqueous
solution containing sodium pertechnetate technetium-99m suitable
for introduction into the sterile electrolytic reaction vial of the
present invention.
The protein constituent to be tagged, such as HSA, is commercially
available conventionally in the form of a 25 percent aqueous
solution which is sterile and non-pyrogenic. It is also
contemplated, in accordance with the practice of the present
invention, that HSA in a coagulated or aggregated form can also be
electrolytically tagged with the radioactive isotope to provide a
particulate radioactive medium for scanning selected organs, such
as the lung and liver. In accordance with the system comprising the
present invention, particulated HSA, such as macroaggregated HSA,
of a particle size generally ranging from about 10 - 50 microns and
microaggregated HSA of a particle size generally ranging from about
10 to about 1,000 millimicrons can be prepared in accordance with
well known techniques by heat and pH adjustment, followed by
classification to proper particle size. The aggregated product is
subjected to sterility and pyrogenic testing preliminary to tagging
with the radioisotope. In this regard, in techniques formerly
employed for tagging such aggregated HSA, it was conventional
practice to first tag HSA in solution form, whereafter careful
aggregation was performed on the tagged solution HSA, frequently
resulting in the formation of tagged particles of improper or
undesired size. Such subsequent aggregation was performed in an
open system exposing the tagged material to possible contamination,
rendering it pyrogenic and non-sterile. In accordance with the
practice of the present invention, aggregation of the HSA prior to
tagging permits its testing for sterility and pyrogenicity,
whereafter only the acceptable aggregated HSA is tagged with the
radioisotope material. This provides for a substantial
simplification and economy in the preparation of radioactive tagged
aggregated HSA.
In addition to a source of a sterile, non-pyrogenic solution
containing the radioactive isotope and the solution or suspension
of HSA, the system comprising the present invention further
requires non-pyrogenic sterile quantities of an aqueous diluent for
providing a pre-selected volume of the reaction solution, an
aqueous acid solution for reducing the pH of the reaction solution
to within a desired range during the electrolytic tagging and an
aqueous alkaline solution for adjustment of the pH to a level
approaching that of the blood system prior to injection. The
aqueous diluent preferably comprises an isotonic saline solution
containing sodium chloride at a concentration of about 0.9 percent.
Alternatively, distilled water or other salt-containing aqueous
solutions which are compatible with the chemistry of body fluids
can be employed. The aqueous diluent is only used to increase the
volume of the eluate containing the radioisotope to provide a
satisfactory volume of liquid to facilitate subsequent electrolytic
tagging in a manner hereinafter to be described. Since the
radioactive material is usually eluted from the radioactive column
using saline, subsequent dilution of the eluate containing sodium
pertechnetate technetium-99m with saline is particularly
desirable.
The aqueous acidic solution used for reducing the pH of the
reaction solution prior to electrolytic tagging may comprise any
suitable acid or mixture of acids which is compatible with the
radioactive material and protein substance and also does not
produce any adverse effects upon subsequent injection of the
radioactive material into the blood stream. While acids such as
nitric acid and sulfuric acid forming ions, which are compatible
with the system and body fluids, can be satisfactorily employed,
hydrochloric acid is preferred in view of the presence of the
chloride anion in the other solutions and in the blood stream. For
this purpose, sterile non-pyrogenic aqueous solutions containing
hydrochloric acid at a concentration sufficient to form about a 1
normal solution are particularly satisfactory for adjusting the pH
of the reaction solution. The acidic solution is employed in an
amount sufficient to reduce the pH of the reaction solution to
within a range of about 0.8 to about 1.5, and preferably within a
range of from about 1.0 to about 1.3, at which particularly high
efficiency and yields of tagged HSA are obtained.
The aqueous alkaline solution similarly may contain any suitable
alkaline substance which is compatible with the radioactive labeled
protein and with the chemistry of the body fluids upon injection of
the tagged serum into the blood stream. Of the various alkaline
substances that are compatible, such as sodium hydroxide, sodium
bicarbonate is particularly satisfactory in that upon
neutralization of the acid present in the reaction mixture, carbon
dioxide is evolved which passes from the solution, minimizing a
further build up in dissolved ions. The alkaline solution is
employed in amounts so as to raise the pH of the resultant
radioactive tagged HSA solution to about 7.4, corresponding to the
pH of the blood stream. While it is possible to inject the
radioactive labeled protein solution in an acidic condition without
neutralization in view of the small amounts required, the preferred
practice is to employ the alkaline constituent for raising the pH
to a level corresponding to that present in the blood stream.
Referring now in detail to the drawing, and with particular
reference to FIG. 1, a series of sealed vials are shown which are
adapted to be assembled in the form of a kit containing some of the
solutions required for preparing the tagged radioactive protein
material. The principal component of the kit comprises the sealed
reaction vial 6 which defines the closed system within which an
electrolytic tagging of the protein constituent is performed. The
reaction vial is preferably comprised of glass and is formed with a
neck portion defining an aperture or opening adapted to be sealed
by a pierceable autoclavable closure, such as a rubber stopper 8.
The rubber stopper 8 is retained in appropriate position in the
neck of the reaction vial by means of an aluminum metal collar 10
crimped around the lip portion of the neck and in overlying
relationship relative to the stopper. A pair of elongated sharpened
electrodes 12, 14 extend downwardly through the rubber stopper in
spaced substantially parallel relationship to a position spaced
from the bottom wall of the reaction vial. The portions of the
electrodes 12, 14 projecting outwardly of the stopper are adapted
to be connected to a suitable source of electric current during the
electrolytic tagging operation in a manner subsequently to be
described.
The particular mechanism for effecting the electrolytic tagging of
a compound, such as HSA, with a radioisotope, such as
technetium-99m, is not entirely understood at the present time. It
is believed, however, that during the passage of current from the
electrode 12, which is comprised of a metal or conductive material
coated with a metal selected from the group consisting of
zirconium, iron and alloys thereof, to the electrode 14, which may
be comprised of the same material as the electrode 12 or any
relatively inert, non-reactive metal, such as platinum, nickel,
stainless steel and the like, a solution of a portion of the
electrode 12 occurs such that the zirconium and/or iron ions effect
a complexing of the radioactive isotope in a manner to enhance
absorption thereof by the protein constituent. In the specific
embodiment shown in FIG. 1, the electrodes 12 and 14 extend
downwardly into the reaction chamber defined by the interior of the
vial 6 a distance of about midway such that the several solutions
can be added to the reaction vial without coming into contact or
effecting an immersion of the electrodes therein. The vial,
accordingly, can simply be inverted at the time the current is to
be applied, whereupon substantially complete immersion of the
electrodes in the solution occurs. Alternative satisfactory vial
constructions also can be employed including those in which the
electrodes extend through the walls of the vial, such as by
embedment directly in the glass.
In addition to the reaction vial 6, suitable sterile supply vials
16 and 18, as shown in FIG. 1, can be provided which are of a
construction similar to that of the reaction vial. The supply vials
16, 18 may conveniently contain adequate quantities of sterile,
non-pyrogenic aqueous solutions of the supplemental reagents
required for the preparation of the radioactive tagged protein
constituent such as the aqueous acid solution, the aqueous alkaline
solution, the aqueous diluent, the human serum albumin aqueous
mixture or suspension, and the like.
Still further flexibility and versatility is provided by the
electrode assembly as illustrated in FIGS. 2-4 of the drawing,
enabling a conversion of standard medicinal vials into sealed
electrolytic reaction vials suitable for practice of the present
invention. As shown in FIGS. 2 and 3, an electrode assembly 20
comprises a cap portion 22 having electrodes 12' and 14' affixed
thereto and projecting therefrom in substantially parallel spaced
relationship. The electrodes 12', 14' are formed with sharpened
ends, indicated at 24, to facilitate a piercing of a pierceable
closure member, such as the rubber stopper 26 shown in FIG. 3, upon
installation of the electrode assembly on a standard vial 28. The
outward projecting ends of the electrodes 12', 14' terminate in
suitable button-shaped contacts 30, 32, which are adapted to be
disposed in electrical contact with a suitable source of electric
current.
As will be further noted in FIGS. 2 and 3, the cap 22 is formed
with an aperture 34 centrally between the contacts 30, 32, thereby
providing access to the upper surface of the rubber stopper 26 such
as by a hypodermic needle 35, by which the several solutions are
introduced into the interior of the reaction vial and are
subsequently extracted therefrom. The cap portion 22 is also
preferably formed with a suitable coacting means, such as a
protuberance 36, which is adapted to coact with a corresponding or
complementary shaped slot 38 of an electrical socket 40, as shown
in FIG. 4, providing proper orientation of the contacts 30, 32
relative to a power source contained therein. As best seen in FIG.
5, the inverted vial incorporating the electrode assembly thereon,
when disposed in the electric socket 40, is positioned by means of
the coaction of the protuberance 36 and slot 38 so that the
contacts 30, 32 are disposed in electrical connection with contacts
42, 44, assuring that the proper polarity current is supplied to
each electrode.
In accordance with the foregoing arrangement, the electrode
assembly 20 can be sterilized, such as by autoclaving or radiation,
and installed on a standard medicinal vial 28, either prior to or
after the interior thereof has been filled with a desired amount of
the requisite solutions. Thereafter, the vial is inverted in a
manner as shown in FIGS. 4 and 5 and placed in the electric socket
40, such that electric current passes between the electrodes 12',
14' thereof for a period of time sufficient to effect a tagging of
the protein constituent with the radioactive material. The
resultant mixture thereafter can be extracted such as by means of
the hypodermic needle shown in FIG. 3 with the assurance that the
contents thereof are in a sterile non-pyrogenic condition. In
accordance with the foregoing technique, appropriate quantities of
radioactive tagged protein materials can be prepared in the
sterilized sealed system to supply the normal daily requirements of
a hospital or institution. Typically, reaction vials, such as the
vial 6 shown in FIG. 1, having a volume of 20 milliliters are
satisfactory for preparing approximately 7 milliliters of a
sterile, non-pyrogenic tagged protein suitable for medical
diagnoses and biological studies.
In order to further illustrate the sealed system and electrolytic
process for preparing a radioactive tagged protein material, the
following expample is provided. It will be understood that the
example is provided for illustrative purposes and is not intended
to be limiting of the scope of the invention as herein described
and as set forth in the subjoined claims.
EXAMPLE
Typically, a normal day's supply comprising about 7 milliliters of
a technetium-99m tagged HSA is prepared employing a reaction vial,
such as the reaction vial 6, having a volume of 20 milliliters. The
reaction vial is equipped with a zirconium anode electrode
corresponding to the electrode 12 and a stainless steel or
zirconium cathode electrode corresponding to the electrode 14 of
the reaction vial shown in FIG. 1. The entire reaction vial,
including the electrode assembly, is preliminarily sterilized, such
as by autoclaving or radiation, providing a sterile sealed system.
The interior of the vial is filled with air or other
oxygen-containing gas since it has been observed that the presence
of oxygen appears to enhance the efficiency of the electrolytic
tagging process.
Approximately 5 milliliters of an isotonic saline eluate containing
sodium pertechnetate technetium-99m having a radioactivity of about
100 millicuries is added to the interior of the reaction vial by
means of a hypodermic needle and syringe. In the event 5
milliliters of eluate are not available, the deficiency in volume
can be made up by a corresponding addition of a sterile,
non-pyrogenic isotonic saline solution. The adjustment of the pH of
the radioactive eluate in the reaction vial to within a range of
about 1 to about 1.3 is conveniently achieved by the addition of
about 0.7 milliliters of 1 normal hydrochloric acid solution
utilizing a hypodermic syringe, thereby maintaining the sterility
and non-pyrogenicity of the reaction solution. The resultant
aqueous mixture in the reaction vial is agitated for a period of
time to assure a homogeneous solution.
To the resultant solution, 0.1 milliliters of a 25 percent HSA
solution is added and the contents of the reaction vial are again
agitated to form a homogeneous solution. The zirconium anode
electrode and a stainless steel or zirconium cathode electrode,
after inversion of the vial, are thereafter connected to a direct
current power source at an amperage of 100 milliamps and at a
voltage of approximately 3 to 5 volts for a period of time
sufficient to apply 4.2 coulombs of electricity therethrough. At
the completion of the electrolytic tagging, the current source is
disconnected and the solution is gently agitated for an additional
period of time at room temperature which has been found desirable
to permit a continuance of the tagging reaction. Tests have shown
that under the foregoing conditions, about an 80 percent yield of
tagged HSA is provided based on the amount of technetium-99m
present at the conclusion of the electric current passage, while
further tagging occurs during the subsequent agitation period,
providing tagging yields of up to 95 percent and above.
At the conclusion of the subsequent agitation dwell period, 1
milliliter of an 8.4 percent sterile non-pyrogenic sodium
bicarbonate solution is injected into the reaction vial to effect
an adjustment of the pH of the solution to about 7.5. The
technetium-99m tagged HSA is now ready for use by injection into
the blood streams of humans and animals. The reaction vial serves
as a sealed and sterile storage container for the tagged HSA, from
which appropriate quantities can be extracted by a hypodermic
syringe retained in the sealed integrity of the vial.
In a similar manner, particulate HSA, such as macroaggregated and
microaggregated HSA, and albumin spheres can be tagged. It is
usually preferred when tagging aggregated protein particles to
separate the supernatant aqueous solution at the completion of the
reaction, such as by centrifugation, and replace the supernatant
reaction liquid removed with isotonic saline for injection
producing a product with 90 percent or more of the radioactivity
bound.
It is also contemplated in accordance with the practice of the
present invention that non-aggregated tagged proteins prior to
injection can be passed through a bacterial filter for further
assuring the absence of any bacterial contamination in the
material.
Sterility and pyrogenicity tests conducted on technetium-99m tagged
HSA produced in accordance with the technique as described in the
foregoing example hav shown no bacterial or pyrogenic
contamination.
Analyses of the resultant product by instant thin layer
chromatography and trichloroacetic acid precipitation revealed a
tagging efficiency of the HSA with the technetium-99m in accordance
with the foregoing example of greater than about 90 percent.
Tests have also been conducted to evaluate the performance of
technetium-99m tagged HSA prepared in accordance with the
electrolytic technique described in the foregoing example relative
to radioactive iodine-tagged HSA and HSA tagged with technetium-99m
in accordance with prior art chemical techniques. Radioiodinated
HSA, which has been in use for over 20 years as a biological tracer
for studying the blood volume and vascular spaces, was employed as
the standard for comparing the chemically and electrolytically
tagged technetium-99m HSA. The HSA tagged with technetium-99m by
chemical techniques was prepared in accordance with well known
practice, including a series of chemical steps consisting of
acidification and reduction of technetium-99m and, finally, a
purification thereof through an ion exchange resin.
The three radioactive tracer materials were injected intravenously
into a number of white rats. The performance of the chemical and
electrolytically prepared radioactive HSA materials were evaluated
by measuring the rate of their disappearance from the blood stream
in comparison to that of the radioiodinated HSA. The results of
such comparative evaluations, based on a group of 16 rats, are set
forth in the following table:
Blood Disappearance of 99mTc Albumin As A Percentage of
Radioiodinated Albumin
Elapsed Time (minutes): 10 20 30 60 90 Electro- lytically Prepared
96% 95% 90% 85% 84% 99mTc HSA Chemically Prepared 94% 90% 83% 73%
68% 99mTc HSA
it is apparent from the data as set forth in the preceding table
that the HSA tagged with 99mTc by the electrolytic technique,
comprising the present invention, is substantially superior to that
of the chemically prepared HSA. The chemically prepared material is
more susceptible to extraction from the blood stream via the
kidney, suggesting that the electrolytically tagged HSA behaves
more like natural human serum albumin and approaches the
performance of the radioiodinated HSA.
While it will be apparent that the invention herein disclosed is
well calculated to achieve the benefits and advantages as
hereinabove set forth, it will be appreciated that the invention is
susceptible to modification, variation and change without departing
from the spirit thereof.
* * * * *