 |
Description  |
|
|
TECHNICAL FIELD
This invention relates to diphosphonate-derivatized macromolecules.
Specifically, it relates to diphosphonate-derivatized macromolecules which
may be radiolabeled with technetium-99 m.
BACKGROUND OF THE INVENTION
The detection and medical/diagnostic assessment of soft-tissue tumors
currently requires a battery of relatively sophisticated diagnostic tests.
Generally a physician will utilize every appropriate diagnostic test
available when cancer is suspected. These tests utilize imaging equipment
for a visual, internal examination and laboratory tests on potential tumor
cells and secretions to determine the tumor burden. If a tumor is detected
and appears to be malignant, a biopsy is performed to arrive at a
diagnosis. Only the biopsy is taken as unequivocal evidence of malignancy.
The tests involving imaging equipment can be divided into two basic types:
those involving an external energy source, such as X-rays or sound waves,
and those involving an internal energy source, such as radioisotopes.
X-ray studies are the most useful tools in staging breast cancer. The
method is also responsible for detecting the vast majority of lung cancer
cases. Once a suspected site has been identified, precise radiographs can
offer valuable information to the physician on the exact location and
extent of tumors of the breast or lung. Unfortunately, by the time the
tumor is large enough to be detected by X-rays (1-2 cubic centimeters),
the patient's prognosis may be relatively poor. In addition to the
relatively low sensitivity of X-rays for soft-tissue tumors, serious
concerns continue to be raised about the risks associated with this
method's level of radiation exposure.
For breast cancer, the approximate location and size of the tumor can be
obtained by ultrasound techniques. Ultrasound provides an image of the
tumor from the pattern of echoes arising from high frequency sound waves
impinging on the breast. Since ultrasonic examination of large sections of
the body would be difficult to interpret and therefore of little value,
this procedure is usually employed for breast examinations after a
palpatable lump has been detected.
Gallium (Ga-67) citrate is the only radiodiagnostic agent indicated for
determining the presence and extent of certain soft-tissue tumors. Gallium
has been shown to be of diagnostic utility in tumors of the lung and
liver. In this procedure gallium is dosed intravenously; the gallium is
then scanned by a gamma camera seeking an enhanced uptake of gallium in
tumor tissue.
Gallium scanning suffers from several important drawbacks. The agent is
neither tumor nor disease specific. Gallium will not only concentrate in
many types of tumors (both benign and malignant) to some extent, but it
also will seek out any localized infection. Because of these
characteristics, the interpretation of scans obtained with gallium citrate
is extremely difficult. The scans usually exhibit low contrast and diffuse
areas of radioisotope concentration.
It has been discovered that proteins labeled with a radioisotope are useful
as radiotracers or radioscanners in humans. Examples include radiolabeled
exogenous or autologous plasma protein for diagnostics of, e.g., pulmonary
embolism; human serum albumin for blood pool imaging; radiolabeled
tumor-specific antibodies for soft tumor imaging; radiolabeled enzyme
proteins and hormone proteins for diagnosing metabolic and
endocrinological disorders. The most widely used radionuclides are
iodine-123, iodine-125, iodine-131, indium-111, gallium-67 and
technetium-99 m. The iodine isotopes, being a halogen, can irreversibly be
incorporated in protein molecules by relatively simple substitution
chemistry. The iodine isotopes are less attractive for other reasons,
especially the beta radiation emitted by these isotopes and the long half
life (8 days) of iodine-131.
Technetium-99 m is generally recognized to be the most desirable
radioisotope for radioscanning and radiotracing. Attempts to label
proteins with technetium involve either chelating of the technetium ion by
chelating groups inherently present in the protein molecule or
derivatizing the protein molecules with a chelating group prior to
labeling with the technetium. A chelate formed by technetium with
chelating groups inherently present in the protein is by its nature not
stable enough to prevent exchange of technetium with other protein
ligands. Technetium-labeled proteins of this type, therefore, often lack
the required biospecificity.
Chelator-derivatized proteins generally involve amino acetic acid compounds
as chelators (e.g. ethylenediaminetetraacetic acid (EDTA) or DTPA).
Proteins of this kind have been found to form strong chelates with
indium-111.
Polyphosphonates, in particular diphosphonates, are generally recognized to
be highly desirable ligands for chelating technetium. Prior to this
invention proteins derivatized with diphosphonates have not been
available. It is therefore an object of this invention to provide
diphosphonate-derivatized proteins suitable for chelating technetium-99 m.
It is a further object of this invention to provide a method of labeling
the diphosphonate-derivatized proteins utilizing technetium-99 m without
denaturization or loss of biological activity of the protein.
It is still a further object of this invention to provide a
protein-diphosphonate-technetium chelate, and to provide a method for
scintigraphic imaging such as soft tumor imaging, in humans utilizing such
chelates.
BACKGROUND ART
The use of radiolabeled proteins for soft tumor imaging is well known.
Early attempts generally involved derivatization of proteins with
iodine-123, iodine-125 or iodine-131. It is well-recognized, however, that
of the conveniently available radionuclides, technetium has by far the
best nuclear properties for diagnostic imaging (Eckelman et al., Int. J.
Appl. Radiation and Isotopes, 28 (1977), pp. 67-82; Eckelman et al.,
Cancer Research 40 (1980), pp. 3036-3042).
Attempts have been made to label proteins with technetium. Several patents
deal with "ligand exchange" and "direct labeling" techniques (U.S. Pat.
No. 4,305,922, issued to Rhodes, Dec. 15, 1981; U.S. Pat. No. 4,311,688,
issued to Burchiel et al., Jan. 19, 1982; U.S. Pat. No. 4,323,546, issued
to Crockford et al., Apr. 6, 1982). Both methods are based on the inherent
chelating properties of proteins. These complexes can be expected to be
unstable in the presence of other proteins, and are therefore not suitable
for soft tumor imaging.
Another approach has been the derivatization of proteins using bifunctional
analogs of EDTA (Leung et al., Int. J. Appl. Radiation and Isotopes, 29
(1978), pp. 687-692; Sundberg et al., J. Med. Chem, 17 (1974), pp.
1364-1367). The chelating agents are linked to the protein by a diazo
phenyl group. The compounds have been shown to chelate indium-111. This
may be due to the fact that EDTA and similar chelators probably do not
form very strong complexes with technetium (Deutsch et al., J. Nucl. Med.,
21 (1980), pp. 859-866).
U.S. Pat. No. 4,287,362, issued Sep. 1, 1981 to Yokoyama et al., discloses
a bifunctional chelating agent specifically developed for labeling
proteins with technetium. An albumin labeling efficiency of nearly 100% is
reported and the compound provides "a much higher blood level for a longer
period of time than conventional technetium-99 m labeled human serum
albumin".
SUMMARY OF THE INVENTION
This invention comprises a diphosphonate-derivatized macromolecules of the
formula
##STR1##
wherein
P is a macromolecule, selected from the group consisting of proteins,
polypeptides, polysaccharides, poly(acrylate), poly(acrylamide),
poly(methacrylate), poly(ethacrylate), poly(hydroxyalkylmethacrylate),
poly(vinyl alcohol), poly(maleic anhydride), poly(maleate) poly(amide),
poly(ethylene amine), poly(ethylene glycol), poly(propylene glycol),
poly(vinyl acetate) and poly(vinyl benzyl chloride), and further contains
one or more reactive end groups selected from the group consisting of
--NH.sub.2, --COOH, --SH, CHO, --S--S, --OH, phenol, guanidino, imidazole
or indole and mixtures thereof,
L is a linking moiety selected from the group consisting of
##STR2##
Q is a spacing group selected from the group consisting of substituted
aryl, unsubstituted aryl or C.sub.1 to C.sub.12 alkyl, where n is 0 or 1;
X is selected from the group consisting of H, OH, NH.sub.2, substituted or
unsubstituted amino, halogen or C.sub.1 -C.sub.4 alkyl; and the
pharmaceutically-acceptable salts of these derivatized macromolecules.
These derivatized macromolecules are suitable for use as anticalcification
agents which inhibits biological mineralization of bioprosthetic devices
and soft contact lenses, and also as radiographic imaging agents useful
for tumor imaging, radioassays, immunoassays, receptor binding assays or
any other scintigraphic procedure where radiolabeled macromolecules would
be used. The derivatized macromolecules are suitable for chelating heavy
metal ions, in particular, technetium-99 m. Examples of derivatized
proteins are derivatized blood proteins, derivatized enzymes, derivatized
proteinaceous hormones, derivatized antibodies and antibody fragments,
connective tissue and cytoskeletal proteins such as collagen, and myosin.
In its narrower aspect, this invention is directed to derivatized
tumor-specific antibodies or antibody fragments chelated to technetium-99
m. This invention further provides a method of labeling the derivatized
protein with technetium by in situ reduction of a pertechnetate solution
without destroying the diphosphonate-protein bond or denaturing the
protein leading to significant loss of biological activity.
Technetium-protein chelates according to this invention are stable in the
presence of plasma proteins; antibody technetium chelates retain their
ability to bind their antigens; and the derivatized protein does not have
an excessive affinity to bone tissue.
This invention further provides a method for scintigraphic imaging using
diphosphonate-derivatized molecules chelated with technetium-99 m.
DETAILED DESCRIPTION OF THE INVENTION
This invention comprises a diphosphonate-derivatized macromolecule of the
general formula:
##STR3##
wherein
P is a macromolecule selected from the group consisting of proteins,
polypeptides, polysaccharides, poly(acrylate), poly(acrylamide),
poly(methacrylate), poly(ethacrylate), poly(hydroxyalkylmethacrylate),
poly(vinyl alcohol), poly(maleic anhydride), poly(maleate), poly(amide),
poly(ethylene amine), poly(ethylene glycol), poly(propylene glycol),
poly(vinyl acetate) and poly(vinyl benzyl chloride) preferably proteins,
and further contains one or more reactive end groups selected from the
group consisting of --NH.sub.2, --COOH, --SH, CHO, --S--S, --OH, phenol,
guanidino, imidazole or indole, and mixtures thereof;
L is a linking moiety selected from the group consisting of
##STR4##
preferably --N.dbd.N--,
##STR5##
Q is a spacing group selected from the group consisting of substitued aryl,
unsubstituted aryl or C.sub.1 to C.sub.12 alkyl, preferably an
unsubstituted aryl, where n is 0 or 1; and X is selected from the group
consisting of H, OH, NH.sub.2, substituted or unsubstituted amino, halogen
or C.sub.1 -C.sub.4 alkyl, preferably H, OH, or NH.sub.2 ; and the
pharmaceutically-acceptable salts of these derivatized macromolecules.
By "pharmaceutically-acceptable salts" as used herein is meant salts of the
diphosphonate-derivatized compounds which have the same general
pharmacological properties as the acid form from which they are derived,
and which are acceptable from a toxicity viewpoint.
Pharmaceutically-acceptable salts include alkali metal (sodium and
potassium), alkaline earth metal (calcium and magnesium), non-toxic heavy
metal (stannous and indium), and ammonium and low molecular weight
substituted ammonium (mono-,di-and triethanolamine) salts. Preferred
compounds are the sodium, potassium, and ammonium salts.
The geminal diphosphonate moieties of the present invention are linked to
the macromolecule via the reactive groups which are part of the
macromolecule. The reactive groups on macromolecules needed to link the
diphosphonate to the macromolecule are carboxyl (COOH), thiol (SH),
amino(NH.sub.2) phenol, aldehyde (CHO), alcohol (CH.sub.2 OH), guanidino,
imidazole, indole and disulfide (--S--S) groups. The number of
diphosphonates which link to the macromolecule depends upon the number of
these reactive groups on the macromolecule. For scintigraphic imaging it
is preferable to link at least one diphosphonate per macromolecule, and it
is more preferable to link as many diphosphonates on the macromolecule as
there are reactive groups, without causing significant loss of biological
activity. For inhibition of biological mineralization, depending upon
properties of the macromolecule, the optimal degree of derivatization will
range from 1% to 90%. Those macromolecules which must retain their
intrinsic biological activity will have a lower degree of derivatization
(1% to 50%) due to the need to retain their optimal functional biological
activity.
Suitable derivatizable macromolecules for use in this invention include
proteins, such as antibodies, antibody fragments, human serum albumin,
enzymes, proteinaceous hormones; water-soluble and water-insoluble
polysaccharides, such as cellulose, starch, dextran and agar; acrylic
homo- and copolymers, such a poly(arylate), poly(acrylamide),
poly(methacrylate), poly(ethacrylate), poly(hydroxyalkylmethacrylate),
poly(vinyl alcohol), poly(maleic anhydride) and poly(maleate);
poly(amides); poly(ethylene imine); poly(ethylene glycol) and
poly(propylene glycol); poly(vinyl acetate); and poly(vinyl benzyl
chloride).
Other macromolecules suitable for use in the present invention are
disclosed in Jakoby and Wichek (eds.), Methods in Enzymology, Vol. 34, pp.
53-76 (1974) and Mosbach (ed.) Methods in Enzymology, Vol. 44 pp. 11-148
(1976), both of which are incorporated by reference.
For scintigraphic imagings virtually any protein is suitable for use in
this invention. However, certain proteins are particularly well-suited for
specific utilities. For example, radiolabeled proteins for diagnosis of,
e.g., pulmonary embolism; human serum albumin for blood pool imaging;
radiolabeled enzyme protein and hormone proteins for diagnosing metabolic
and endocrinological disorders; and radiolabeled antibodies or antibody
fragments for soft tumor imaging.
The labeled antibodies and antibody fragments useful in the present
invention are specific to a variety of tumor-associated antigens or
markers. These markers are substances which accumulate in, on, or around
tumor cells. They may be intracellular, cell surface or cytoplasmic
markers. Tumor-specific markers and methods of raising antibodies to these
markers are well known in the art. Such tumor specific markers are
disclosed in Herberman, "Immunodiagnosis of Cancer", in Fleisher (ed.)
"The Clinical Biochemistry of Cancer," p. 347 (Am. Assn. Clin. Chem. 1979)
and in U.S. Pat. No. 4,331,647 to Goldenberg, issued May 25, 1982, and
U.S. Pat. No. 4,361,544 to Goldenberg issued Nov. 30, 1982, all of which
are incorporated by reference. Methods of raising antibodies in vitro are
disclosed in Nezlin, "Biochemistry of Antibodies" pp. 255-286 (1970),
incorporated by reference.
The diphosphonate moieties utilized in the present invention, absent a
linking moeity (L), cannot form covalent bonds with the above-mentioned
macromolecules since diphosphonates have no chemical affinity towards the
protein. L is an atom, group of atoms or a chemical bond which attaches
the geminal diphosphonate moiety separated via a spacer group, if
appropriate, to the macromolecule. L is composed of a chemically-reactive
moiety which can couple to a macromolecule by reaction with the specific
reactive end group in the macromolecule.
The linking moiety, therefore, is the result of a reactive species on the
diphosphonate forming a covalent bond with macromolecules. The
determination of the appropriate linking moiety is dependent upon the
availability of reactive groups on the macromolecule, i.e., carboxyl,
sulfhydryl, disulfide, hydroxy, guandino, imidazole, indole, sulfhydryl,
amino, phenol, aldehyde or alcohol groups. Another consideration, in the
case of proteins, is whether or not modification of one or more of these
reactive groups has a significant effect upon the biological activity of
the protein. Significant loss occurs when the derivatized antibody will no
longer localize sufficiently well on the target tissue.
Also within the scope of this invention is a procedure whereby the
macromolecule is first pre-activated toward reacting with the
diphosphonate. This is accomplished by derivatizing the macromolecule with
a reagent bearing a substituent that would further react with the
diphosphonate described in this invention.
The optimum linking moiety to be utilized, therefore, depends upon the
reactive end group on the macromolecule to which the diphosphonate moiety
is to be attached.
For example, utilizing proteins for illustration, when the reactive group
is phenolic group on tyrosine
##STR6##
then an appropriate linking moiety is a diazo group (--N.dbd.N--). This
linkage can readily be formed as follows:
##STR7##
When the reactive end group contains a primary amine, such as the amino
terminus or the epsilon amino group of lysine, an appropriate linking
group would be, for example, an amide
##STR8##
This linkage can readily be formed as follows:
##STR9##
Other appropriate linking moieties where the reactive side chain contains
a primary amine include a thiourea
##STR10##
generated from a phenylisothiocyanate diphosphonate,
##STR11##
which reacts with a protein to form:
##STR12##
a sulfonamide linkage
##STR13##
occurs by the reaction of an aryl sulfonyl halide containing
diphosphonate,
##STR14##
which reacts with the protein to form:
##STR15##
an N-carboxyanhydride-containing geminal diphosphonate,
##STR16##
reacts with an amine containing protein to form an amide linkage
##STR17##
an imidate-containing geminal diphosphonate,
##STR18##
reacts with a protein to form an amidine
##STR19##
a quinone-diphosphonate can form a disubstituted hydroquinone link as
follows:
##STR20##
Schiff base chemistry via an aldehyde-diphosphonate,
##STR21##
reacts with a protein to form an imine (--N.dbd.CH--) link. Reduction of
the imine linkage yields:
##STR22##
which contains an alkylamine linkage.
When the reactive side chain of the protein contains a carboxylic acid, as
in aspartic or glutamic acid residues or "C" terminus, the appropriate
diphosphonate contains an amino group which reacts with the protein to
form an amide. This linkage can be formed by preactivating the protein
carboxy group with a water-soluble carbodiimide, then coupling the
reactive intermediate with an omega-aminoalkyldiphosphonate, such as:
##STR23##
forming an amide
##STR24##
linkage when reacted with a protein:
##STR25##
When the reactive group on the protein is a thiol (SH), as in cystene, then
an appropriate diphosphonate contains an alkyl halide, for example,
##STR26##
wherein X=Br or I.
This diphosphonate reacts with the thiol on the protein to form a thioether
linkage (--CH.sub.2 --S--CH.sub.2 --):
##STR27##
iodoacetyl diphosphonates such as
##STR28##
reacts with the thiol group on the protein to form a thioether linkage
(--CH.sub.2 --S--CH.sub.2 --):
##STR29##
maleimide diphosphonates, such as
##STR30##
react with sulfhydryl-containing proteins to form a thioether linkage:
##STR31##
A disulfide linking group can be formed by reacting a 2-pyridine disulfide
agarose gel with a sulfhydryl-containing diphosphonate to form a disulfide
link (--S--S--) as follows:
##STR32##
A carbamate linking group can be formed by a polysaccharide, such as
reacting Sepherose.RTM. or Ficol.RTM. (Pharmacia Corporation), with
cyanogen bromide to form a reactive imidocarbonate intermediate. A
terminal amino-containing diphosphonate will react with the imidocarbonate
intermediate to form a substituted carbamate product as follows:
##STR33##
The spacer, Q, if needed, creates a space between the protein and the
diphosphonate to permit the diphosphonate to be more accessible for
radiolabeling. Suitable spacing groups are disclosed in Methods of
Enzymology, Vol. 34 pp. 26-27, incorporated herein by reference. The
spacer can be aryl or C.sub.1 to C.sub.12 alkyl. The aryl can be
substituted or unsubstitued with one or more substituents. Preferred is an
unsubstituted aryl.
The diphosphonate moieties utilized in the present invention (hereinafter
diphosphonates) have the formula:
##STR34##
where X is H, OH, NH.sub.2, substituted amino, halogen or C.sub.1 -C.sub.4
alkyl, preferred is H, OH or substituted or unsubstituted amino.
These diphosphonates are useful as anticalcification agents for
bioprosthetic devices and soft contact lenses. Bioprosthetic devices are
known to undergo biological mineralization, generally this mineral
component is composed of calcium phosphate. When this calcium builds up,
the function of the device is impaired. The diphosphonate-derivatized
macromolecules of the present invention when linked to such devices
inhibit mineralization.
Diphosphonates can be attached to proteins, such as collagen, in heart
valve and vascular graft implants. For example a water soluble
carbodiimide is added to collagen for activation, this activated
intermediate,
##STR35##
is then reacted with collagen to form:
##STR36##
a diphosphonate-derivatized insoluble protein which will resist biological
mineralization.
Covalent attachment of diphosphonates to extended wear soft contact lenses
inhibit their calcification. These soft lenses are often composed of
poly(acrylamides), polyols or polycarboxylates. The lens polymer can be
reacted with a water soluble carbodimide, for example:
polymer --COOH+R--N.dbd.C.dbd.N--R'
to form a reactive intermediate:
##STR37##
This intermediate can then be reacted with a diphosphonate to form a
diphosphonate-derivatized polymer:
##STR38##
SCINTIGRAPHIC IMAGING AGENT
The diphosphonate-dervatized macromolecules are useful as scintigraphic
imaging agents when chelated with technetium-99 m. Methods of labeling the
phosphonate moiety with Tc-99 m are disclosed in Castronovo et al., "The
Phosphonate Moiety: Labeling with 99 m-Tc(Sn) After Synthetic Attachement
to Diverse Biological Compounds", Radiopharm. [International Symposium],
Chapter 7, pp. 63-70 (1975), incorporated herein by reference.
Generally, the diphosphonate-derivatized macromolecule is treated with a
solution containing stannous ion. To this mixture is added a solution of
Tc-99 m as pertechnetate. The Tc-99 m is reduced by the stannous ion
forming a coordinate covalent linkage with the diphosphonate.
As used herein the term "pertechnetate reducing agent" includes compounds,
complexes or the like, comprising a reducing ion capable of reducing
heptavalent technetium (TcO.sub.4.sup.-) to trivalent, tetravalent and/or
pentavalent technetium. Free metals, such as tin, are also known for use
as pertechnetate reducing agents, although undissolved metal must be
removed from the scanning solution prior to injection into the patient.
Suitable pertechnetate reducing agents include sodium hydrosulfite, as
well as metalic salts of sulfuric acid and hydrochloric acid, such as
stannous chloride.
The compositions herein optionally, and preferably, contain a stabilizing
amount of a stabilizer material to prevent or inhibit the oxidation of the
pertechnetate reducing agent (e.g., oxidation of Sn.sup.+2 to Sn.sup.+4)
during storage and/or to inhibit or diminish the reoxidation of reduced
technetium-99 m and/or to diminish the formation of technetium-labeled
impurities which may form during use of the compositions.
The stabilizers used herein are characterized by their toxicological
acceptability under the conditions of use, their ability to stabilize the
product for a reasonable period of storage and/or under usage conditions,
and by their substantial non-interference with the delivery of the
technetium radionuclide to, for example, soft tumors.
Stabilizers which meet the foregoing requirements and which are suitable
for intravenous injection include gentisic acid and its water-soluble
salts and esters, ascorbic acid and its water-soluble salts and esters,
and erythorbic acid and its water-soluble salts and esters. Gentisic acid,
ascorbic acid and erythorbic acid are all known, commercially-available
materials. The sodium salts of these acids are also all
commercially-available, quite water-soluble, and preferred for use herein.
As is known in the literature, stabilizer materials such as ascorbic acid
can chelate or complex with technetium and cause it to be deposited in
uncalcified soft tissue. Since the practitioner of the present invention
will wish to avoid all unnecessary deposition in soft tissue, it will be
appreciated that the amount of stabilizer materials optionally used in the
present compositions should not be so great as to overshadow the tumor
directing effect of the derivatized macromolecule, thereby interfering
with the imaging.
The scintigraphic imaging agents of the present invention are intended for
systemic or oral administration into humans or lower animals. Accordingly,
appropriate manufacturing and operating conditions are employed to provide
suitably sterile compositions.
For gastrointestinal imaging, oral administration would be the appropriate
method of administration. For soft tissue tumor imaging, the appropriate
route of administration would be intravascular or intralymphatic. For
blood pool imaging, intravenous administration would be the appropriate
method.
The compositions of the present invention can be prepared by simply dry
mixing the technetium reducing agent and the derivatized macromolecule.
The optional stabilizer can also be dry blended into such mixtures, as can
additional, non-interferring agents, such as sodium chloride.
In an alternate mode, the compositions herein can be provided in
lyophilized form. Such compositions are prepared by co-dissolving the
diphosphonate-derivatized macromolecule and the technetium reducing agent
in an aqueous solution, together with any desired optional stabilizers,
and lypholizing the composition using standard equipment. Preferably,
sterile, deoxygenated water is used in processing and the product is
stored under nitrogen. Although somewhat more complicated to manufacture
than the dry mixed product, the lypholized product offers the advantage
that water-insoluble particulate matter which might be present in the raw
materials can be removed by filtration prior to the freeze drying step.
In another mode, the compositions herein can be provided as aqueous
solutions in pharmaceutically-acceptable liquid carriers. These carriers
can be, for example, saline solution or sterile, pyrogen-free water.
Preferably, the water is deoxygenated and the composition is stored under
nitrogen, thereby minimizing undesirable oxidation of the pertechnetate
reducing agent on storage. Since the reducing agent is more prone to
oxidize in solution than in the dry powder and freeze-dried composition
forms, it is preferred that aqueous compositions contain a stabilizer.
The compositions of the present invention are prepared such that the weight
ratio of the derivatized macromolecule: technetium reducing agent is from
about 2:1 to about 100,000:1, preferably from about 2:1 to about 10,000:1.
Stabilized compositions are generally formulated such that the weight ratio
of derivatized macromolecule: stabilizer is from about 1:1 to about
10,000:1, preferably from about 1:1 to about 1,000:1.
Preferred stabilized compositions in unit dosage form contain from about
0.05 mg. to about 3 mg. of the stannous reducing agent; from about 0.25
mg. to about 1.0 mg. of the gentisate or ascorbate stabilizer; and from
about 0.01 to about 50 mg. of the diphosphonate-derivatized macromolecule.
Compositions of the foregoing type are characterized by a
physiologically-acceptable in-use solution pH in the range from about 3.5
to about 8.5, and, preferably, fall within a pH range of about 4.5 to
about 7.4.
In the case of proteins, a liquid pharmaceutical composition suitable for
scintigraphic imaging would be composed of from about 0.01% to about 20%
of the diphosphonate-derivatized protein with the remainder being
pertechnetate reducing agent, stabilizer, and a
pharmaceutically-acceptable carrier. In the case of other macromolecules,
from about 1% to about 20% of the total composition would be comprised of
the diphosphonate-dervatized macromolecule.
In the case of proteins, a lyophilized pharmaceutical composition would be
composed of from about 1% to about 50% of the diphosphonate-derivatized
protein. For other macromolecules, from about 5% to about 99% of the
composition would be comprised of the diphosphonate-derivatized
macromolecule.
In use, the compositions are mixed with a pertechnetate-99 m isotonic
solution from a commercial technetium source to yield a Tc-99 m labeled
diphosphonate-derivatized macromolecule suitable for systemic or oral
administration. The stability of such scanning agents is ample under
ordinary hospital conditions. Administration is preferably done within
about eight hours after addition of the pertechnetate solution. For
intravenous administration, the concentration of reagents and technetium
radionuclide is sufficient such that about 1 ml. of the solution is
administered to an adult of about 50-100 kg. body weight. One ml. of
solution is preferably injected intravenously over a period of about 30
seconds. For oral administration, the concentration of reagents and
technetium radionuclide is sufficient such that from about 10 ml to about
150 ml of the solution or suspension is administered to an adult of about
50-100 kg body weight. Follow up scans would consist of the same levels of
administration.
The actual structure of the reaction product formed by the 99 m
Tc/diphosphonate-derivatized macromolecule reducing agent mixture and
introduced into the body is not known with certainty.
These diphosphonate-derivatized macromolecules are useful for any
bio-analytical technique where radiolabeled proteins would be used. Such
applications include tumor imaging, radioimmunoassays, myrocardiol
infarction assays, thrombosis imaging, receptor binding assays and blood
pool imaging, and gastrointestinal imaging.
Generally, a safe and effective amount of the radiolabeled macromolecule is
administerd systemically or orally for the scintigraphic imaging
procedures of the present invention.
By "safe and effective amount" as used herein is meant an amount of the
composition high enough to provide a clinically useful scintigraphic
image, but low enough to avoid serious side effects (at a reasonable
benefit/risk ratio), within the scope of sound medical judgment. The safe
and effective amount of the composition will vary with the particular
scintigraph technique and particular clinical condition being evaluated,
the age and physical condition of the patient being treated, the severity
of the condition, the duration of treatment, the nature of concurrent
therapy and the specific diphosphonate-derivatized macromolecule employed.
Systemic administration would be appropriate for tumor imaging, myrocardial
infarction assays, thrombosis imaging, receptor binding assays, and blood
pool imaging.
The following nonlimiting examples illustrate the compounds, chelates,
compositions, methods and uses of the present invention | | |
