SYNTHESE IN SITU DE COMPOSES RADIO-PHARMACEUTIQUES

IN SITU SYNTHESIS OF RADIOPHARMACEUTICALS

Abrégé anglais

2101642 9214492 PCTABS00015
The present invention relates to a process of making
radiopharmaceuticals in situ, i.e. wherein a radionuclide and an acyclic
ligand react with constituents of the complex forming reaction
solution to produce an administratable radiopharmaceutical agent. By
forming radiopharmarceuticals according to the present invention,
it is possible to obtain radiopharmaceuticals previously
unattainable because of problems associated with the ligand synthesis or
with the complex forming reaction.

Revendications

WO 92/14492 PCT/US92/00630
What is claimed is:
1. A method of making a radiopharmaceutical agent
comprising:
reacting a non-radioactive acyclic ligand, that is
capable of forming a cyclic ligand upon simple chemical
conversion; with a radionuclide;
wherein said acyclic ligand binds to said
radionuclide and then converts to said cyclic ligand, in
situ, to form said radiopharmaceutical agent.
2. A method of making a radiopharmaceutical agent
according to claim 1, wherein said acyclic ligand
converts to said cyclic ligand by reaction with itself,
reaction with components in the reaction solution, or by
simple rearrangement of the manner in which it binds to
the radionuclide.
3. A method of making a radiopharmaceutical agent
according to claim 1, wherein said radionuclide is a
metal radionuclide, selected from the group consisting of
Tc, Re, Co, Cu, Ni, Ru, Cr, W, Rh, Zn, In, Ga, Mo, Mn,
Pt, Pd, Os, Ir and Sm.
4. A method of making a radiopharmaceutical agent
according to claim 3, wherein said metal radionuclide is
Tc-99m, Re-186, or Re-188.

WO 92/14492 PCT/US92/00630
16
5. A method of making a radiopharmaceutical agent
according to claim 1, wherein said acyclic ligand has the
general formula:
<IMG>
wherein
W, X, Y, and Z are the same or different and are
chosen from the group consisting of S, O, PR, NR, or AsR;
wherein R is hydrogen or any straight or branched chain
radical of up to 12 carbon atoms, preferable 1-8 carbon
atoms, where one or two of the carbon atoms may be
substituted with an O, and the carbon radicals contain
hydrogens, or optionally, substituent groups such as =O,
F, Cl, Br, I, OR', CO2R', SO3R'; wherein R' is hydrogen or
any straight or branched chain radical of 1-4 carbon
atoms in which one of the carbon atoms may be substituted
by an O or S;
if W, X, Y, or Z is NR, then R may be NR or OR;
A, B, and C are the same or different straight chain
carbon radicals of 2-5 carbon atoms in which 1-4 of the
carbon atoms have been substituted in some or all
locations with R or with one of said substituent groups;
and
D contains 1-8 carbon atoms, including a 2-6 carbon
atom chain terminating in a group which allows it to form
a bond to W following reaction with a radionuclide, and
further optionally containing at least one of said
substituent groups.

WO 92/14492 PCT/US92/00630
17
6. A method of making a radiopharmaceutical agent
according to claim 1, wherein said method follows the
general reaction:
<IMG>
wherein M represents a metal radionuclide with or without
one or more additional ligands attached to the metal; and
wherein
W, X, Y, and Z are the same or different and are
chosen from the group consisting of S, O, PR, NR, or AsR;
wherein R is hydrogen or any straight or branched chain
radical of up to 12 carbon atoms, preferable 1-8 carbon
atoms, where one or two of the carbon atoms may be
substituted with an O, and the carbon radicals contain
hydrogens, or optionally, substituent groups such as =O,
F, Cl, Br, I, OR', CO2R', SO3R'; wherein R' is hydrogen or
any straight or branched chain radical of 1-4 carbon
atoms in which one of the carbon atoms may be substituted
by an O or S;
if W, X, Y, or Z is NR, then R may be NR or OR;
A, B, and C are the same or different straight chain
carbon radicals of 2-5 carbon atoms in which 1-4 of the
carbon atoms have been substituted in some or all
locations with R or with one of said substituent groups;
and
D contains 1-4 carbon atoms, including a 2-6 carbon
atom chain terminating in a group which allows it to form
a bond to W following reaction with a radionuclide and
further optionally containing at least one of said
substituent groups.

WO 92/14492 PCT/US92/00630
18
7. A method of making a radiopharmaceutical agent
comprising:
providing an acyclic ligand containing at least
three and at most five amino acid groups, and having the
general formula:
<IMG>
wherein R1 to R8 are the same or different and are
selected from the group consisting of hydrogen or
substituents of any natural or synthetic amino acid; and
X is OH or <IMG> , wherein R9 and R10 have
the same meanings as R1 to R8 above;
reacting said acyclic ligand with a radionuclide;
wherein said acyclic ligand coordinates with said
radionuclide, and then undergoes internal reaction to
generate a second ligand which stays wholly or partially
coordinated to said radionuclide.
8. A method of making a radiopharmaceutical agent
according to claim 7, wherein said acyclic ligand
generates said second ligand by reaction with itself,
reaction with components in the reaction solution, or by
simple rearrangement of the manner in which it binds to
the radionuclide.

WO 92/14492 PCT/US92/00630
19
9. A method of making a radiopharmaceutical agent
according to claim 7, wherein said radionuclide is a
metal radionuclide, selected from the group consisting of
TC, Re, co, cu, Ni, Ru, Cr, W, Rh, Zn, In, Ga, Mo, Mn,
Pt, Pd, Os, Ir and Sm.
10. A method of making a radiopharmaceutical agsnt
according to claim 9, wherein said metal radionuclide is
Tc-99m, Re-186, or Re-188.
11. A kit for forming a radiopharmaceutical agent
comprising:
a ligand having the general formula:
<IMG>
wherein
W, X, Y, and Z are the same or different and are
chosen from the group consisting of S, O, PR, NR, or AsR;
wherein R is hydrogen or any straight or branched chain
radical of up to 12 carbon atoms, preferable 1-8 carbon
atoms, where one or two of the carbon atoms may be
substituted with an O, and the carbon radicals contain
hydrogens, or optionally, substituent groups such as =O,
F, Cl, Br, I, OR', CO2R', SO3R'; wherein R' is hydrogen or
any straight or branched chain radical of 1-4 carbon
atoms in which one of the carbon atoms may be substituted
by an O or S;
if W, X, Y, or Z is NR, then R may be NR or OR;
A, B, and C are the same or different straight chain
carbon radicals of 2-5 carbon atoms in which 1-4 of the
carbon atoms have been substituted in some or all
locations with R or with one of said substituent groups;

WO 92/14492 PCT/US92/00630
and
D contains 1-8 carbon atoms, including a 2-6 carbon
atom chain terminating in a group which allows it to form
a bond to W following reaction with a radionuclide and
further optionally containing at least one of said
substituent groups;
and further comprising
a reducing agent.
12. A kit according to claim 11, wherein said reducing
agent is selected from the group consisting of
dithionite, formamidine sulphinic acid, diaminomethane
disulphinate or suitable metallic reducing agents such as
Sn(II), Fe(II), Cu(I), Ti(III) or Sb(III).
13. A kit according to claim 11, further including a
pharmaceutically acceptable carrier.
14. A kit according to claim 13, wherein said carrier is
a sterile physiological saline solution.
15. A kit according to claim 11, further including
stabilizers and fillers.
16. A kit according to claim 15, wherein said
stabilizers are ascorbic acid, gentisic acid or salts of
these acids.
17. A kit according to claim 15, wherein said fillers
are glucose, lactose, mannitol, inositol, and the like.
18. A kit according to claim 11, further including a
transfer ligand.

WO 92/14492 PCT/US92/00630
21
19. A kit according to claim 18, wherein said transfer
ligand is selected from the group consisting of
dicarboxylic acids, polycarboxylic acids or hydroxy
carboxylic acids, such as oxalic acid, malonic acid,
succinic acid, maleic acid, orthophthalic acid, malic
acid, lactic acid, tartaric acid, citric acid, ascorbic
acid, salicylic acid, glucoheptonic acid or derivatives
of these acids; phosphorus compounds such as
pyrophosphates; or enolates.
20. A kit according to claim 11, further including
instructions for use with a prescription for reacting the
components of said kit with a radionuclide solution.
21. A kit according to claim 11, wherein said ligand and
said reducing agent are combined, and are provided in a
lyophilized condition.
22. A method of making a radiopharmaceutical agent from
a kit, said method comprising:
providing a kit including:
an acyclic ligand having the general formula:
<IMG>
wherein
W, X, Y, and Z are the same or different and are
chosen from the group consisting of S, O, PR, NR, or AsR;
wherein R is hydrogen or any straight or branched chain
radical of up to 12 carbon atoms, preferable 1-8 carbon
atoms, where one or two of the carbon atoms may be
substituted with an O, and the carbon radicals contain
hydrogens, or optionally, substituent groups such as =O,

WO 92/14492 PCT/US92/00630
22
F, Cl, Br, I, OR', CO2R', SO3R'; wherein R' is hydrogen or
any straight or branched chain radical of 1-4 carbon
atoms in which one of the carbon atoms may be substituted
by an O or S;
if W, X, Y, or Z is NR, then R may be NR or OR;
A, B, and C are the same or different straight chain
carbon radicals of 2-5 carbon atoms in which 1-4 of the
carbon atoms have been substituted in some or all
locations with R or with one of said substituent groups;
and
D contains 1-8 carbon atoms, including a 2-6 carbon
atom chain terminating in a group which allows it to form
a bond to W following reaction with a radionuclide and
further optionally containing at least one of said
substituent groups; and further including
a reducing agent; and
combining said kit with a radionuclide solution;
wherein said acyclic ligand binds to said radionuclide
and then converts to a cyclic ligand, in situ, to form
said radiopharmaceutical agent.
23. A method of imaging using a radiopharmaceutical
agent comprising:
forming said radiopharmaceutical agent by
reacting a non-radioactive acyclic ligand, that is
capable of forming a cyclic ligand upon simple chemical
conversion; with a radionuclide;
wherein said acyclic ligand binds to said
radionuclide and then converts to said cyclic ligand, in
situ, to form said radiopharmaceutical agent;
administering said radiopharmaceutical agent to a
living being; and
scanning said living being with detection means to
detect said administered radiopharmaceutical agent.

Description

W092J1~92 -1- 2 1 ~ 1 5 ~ 2 PCT/US92~K~
IN SITU SYN~HESIS OF RADIOPHARMACEUTICALS
8ackg~ound of the Invention
The present invention relates to novel in situ
synthesis methods of forming radiopharmaceuticals.
.
The use of radiopharmaceuticals for diagnostic and
therapeutic purposes is well known in the area of
biological and medical research. In particular,
radiopharmaceuticals are used as radiographic imaging
agents for visualizing skeletal structures, organs, or
tissues. Such imaging is accomplished by preparation of
radioactive agents, which when introduced into the body of
a patient, are localized in the specific structure which is
to be studied. The localized agents may then be traced,
plotted or scintiphotographed by radiation detectors, such
as, traversing scanners or scintillation cameras. The
distribution and relative intensity of the detected
radioactive agents indicates the position of the structure
in which the agent is localized, and also shows the
presence of aberrations in structure or function,
pathological conditions or the like. In a similar manner,
radiopharmaceuticals may be used as therapeutic agents, for
providing radiation to a particular pathological condition
which is to be treated. Such treatment may be accomplished
by preparation of radioactive therapeutic agents which
again are designed to localize in a particular struçture,
organ or tissue. When such an agent is localized,
radiation may be delivered directly to the pathological
condition requiring radiation treatment.
In general, both diagnostic and therapeutic
radiopharmaceuticals are comprised of a radionuclide-
labelled compound. In the case of metal-based
radionuclides the metal can exist in it's free state, as an
ion, or in the form of a metal complex with a ligand or
. .
:,"
.~
:
... .. . .
. .
.:~ -.
..... .
.. .
: ,
-

W092/1~92 2 1 0 ~ PCT/US92/~K~
group of ligands. Examples of metal radionuclides that
form complexes are Tc-99m and Re-186. The former is used
in diagnostic work and the latter is used for therapy. The
resultant radiopharmaceuticals further include appropriate
carriers and auxiliary agents, such as delivery vehicles
suitable for injection, aspiration or ingestion by the
patient, as well as physiological buffers and salts, and
the like.
Prior art methods of forming radiopharmaceuticals
generally require initial synthesis of the ligand, followed
by a separate synthesis of the radionuclide complex (i.e.
a labelling procedure). In particular,
radiopharmaceuticals of the prior art are formed by first
synthesizing the desired structure specific ligand,
according to known methods for such a ligand. The prepared
ligand, generally in a lyophilized kit also containing one
or more excipients is then reacted with a radionuclide-
containing solution under radionuclide complex-forming
reaction conditions. For example, when it is de~ired to
form a technetium-99m radiopharmaceutical, the prepared
ligand may be reacted along with a reducing agent with a
pertechnetate solution under technetium-99m complex-forming
reaction conditions. The complexes are then administered
to the patient via injection, inhalation or ingestion.
The radionuclide-containing solutions can be obtained
from a generator, as in the case of Tc-99m, or can be
supplied in saline or water by a manufacturer, as with Re-
186. With Tc-99m the radionuclide solution is eluted from
a Mo-99/Tc-99m generator system. The complex-forming
reaction is carried out at complex-forming temperatures
(e.g. 20C to 100 C) for a few minutes to several hours
when forming technetium complexes. A large excess (e.g.
greater than one hundred fold excess to metal radionuclide)
.;~ .
,r
'~'
~.......... . . - . .
.~, . : .. , . .. . . . ~ :
r , ', :,
',' , .:''~ . ',
;~ ' .:. : : : I
. ' ' : : , .' , .' ., : ~:'' :
.' ' .

wos2Jl~92 2 ~ 9 PCT/US92t~K~
of the prepared ligand is used and a sufficient amount of
reducing agent is present, if needed, to insure reduction
of the radionuclide to facilitate complexation by the
ligand.
Radiopharmaceutical agents are then prepared by
combining the radionuclide complex, in an amount sufficient
for the desired diagnostic or therapeutic purpose, with a
pharmaceutically acceptable radiological vehicle. The
radiological vehicle should be acceptable for injection,
aspiration or ingestion. Examples of such vehicles are
human serum albumin; aqueous buffer solutions, e.g.
tris(hydromethyl)aminomethane (and its salts), phosphate,
citrate, bicarbonate, etc.; sterile water; physiological
saline; and balanced ionic solutions containing chloride
and or dicarbonate salts or normal plasma cations such as
Ca'2, Na+, K+, and Mg+2.
.
~; Also, agents known as "stabilizers" can be included.
These hold the radionuclide in a stable form until it can
be reacted with the ligand. These stabilizers can include
agents known as "transfer ligands" which are particularly
useful in holding the metal stable in a reduced oxidation
state until the ligand can capture it. Examples of
~ transfer ligands include salts of glucoheptonic acid,
i~ tartaric acid, and citric acid, or other suitable ligands
~ as will be discussed in more detail below.
:..
As the above would indicate, in a standard metal
radionuclide radiopharmaceutical the ligand must be
completely presynthesized and reacted with the metal
radionuclide to give a complex in which the ligand is
essentially unchanged after complex formation with the
exception of removal of hydrogen ions or protecting groups.
Removal of these groups facilitates coordination of the
:;.
. .
. ......................................................................... .
:, .
.'.'. .' :: : ~ : ' ,
- -: ,. .
.

wo 92/l~g2 2 1 ~ 1 6 ~ 2 PCT/US92/~630
ligand to the metal radionuclide.
In situ reaction of ligands facilitated by metals or
metal complexes is know in the prior art. There are
; numerous examples of this type in inorganic literature
references. Some of the most well known work describes the
following reaction type:
~V/s+~ N?~S~
In this work, the ligand cyclization reaction was
facilitated by the presence of the nickel, which held the
ends of the acyclic ligand in place and allowed the
formation, in good yield, of the product ligand through
closure of the ring. The metal provided a "template" for
the ring closure of the reaction and from this effect the
term "template syntheses" has been coined for this process.
i This has also been found in syntheses involving cyclic
tetrapeptides as described in a number of references. In
these cases the ligand was the desired product with the
metal being removed at the end by standard means.
In some cases the ultimate ligand-metal complex is the
target. Such is the case in the work of Sargeson and
coworkers in which the template reaction yields a metal
complex that has unique physicochemical properties because
the metal is "locked in" and is not easily removed from the
ligand. An example of such a reaction is shown below:
. ~
: ~ .
.,
. .. ...
- . . .:
: ' , .

W092~l~92 2 ~ PCT/US92/~K~
N ~ ~ N
\ ) + 2 ~ ~ "N
~h~ro N N NH2CH2CH2NH2
An example also exists in the field of
radiopharmaceutical chemistry in which a metal complex of
a ligand is formed in situ by reaction of the ligand and a
constituent of the reaction solution with the metal
radionuclide. It should be noted that in this example, the
constituent of the solution does not end up being bound
either directly to the metal radionuclide nor to an atom of
the ligand that does bond to the radionuclide.
07Lr ~, l
Obje~~s of the Invention
It is one ob~ect of the pre~ent invention to provide
a process of making radiopharmaceuticals in situ, i.e.
wherein a radionuclide and an acyclic ligand complex react
with constituents of a complex forming reaction solution to
produce an administratable radiopharmaceutical agent.
.. . .. ~.. .. . . . ...
- -' -: . ' , .
' ~' ~ ' '~' ,' ,
.
,
., - , ~ . .
. . . .

W092/1~92 2 1 0 1 ~ ~ ~ PCT/US92/~K~
It is a further object of the present invention to
provide a process of making radiopharmaceuticals previously
unattainable because of problems associated with the ligand
synthesis or with the complex forming reaction.
Detailed Descri~tion of the Inve~ion
The present invention relates to a novel process of
forming radiopharmaceuticals, in situ. In particular,
radiopharmaceuticals may be produced in situ by reacting a
non-radioactive acyclic ligand with a radionuclide, wherein
the acyclic ligand is capable of forming a cyclic ligand
upon simple chemical conversion. The acyclic ligand first
binds to the radionuclide, and then undergoes conversion to
the cyclic ligand by reaction with itself, reaction with
components in the reaction solution, or by simple
rearrangement of the manner in which it bonds to the
radionuolide, to form the final radiopharmaceutical.
Any suitable radionuclide may be used, including a
metal radionuclide, selected from the group consisting of
Tc, Re, Co, Cu, Ni, Ru, Cr, W, Rh, Zn, In, Ga, Mo, Mn, Pt,
Pd, Os, Ir and Sm. Preferably the radionuclide is Tc-99m,
Re-186 or Re-188.
Acyclic ligands which may be used in the process
according the present invention include any known ligands
which are suitable for the formation of radionuclide
complexes. For example, ligands such as, acyclic tetra-
and penta- peptides and, also, tri- and di- peptides that
have auxiliary groups attached so that they can undergo
ring closure reactions may be used. In particular, the
acyclic ligand may be any ligand having the general
formula:
.. . . , , , ~ .:
.
-
- , ~
.: . .

WO 92tl~92 ~ 1 U 1 ~ 4 ~ PCT/US92/~K~
w~x~ 9
D Z ~ c r
wherein
W, X, Y, and Z are the same or different and are
chosen from the group consisting of S, O, PR, NR, or AsR;
wherein R is hydrogen or any straight or branched chain
radical of up to 12 carbon atoms, preferable 1-8 carbon
atoms, where one or two of the carbon atoms may be
substituted with an 0, and the carbon radicals contain
hydrogens, or optionally, substituent groups such as -O, F,
Cl, Br, I, OR', CO2R', SO3R'; wherein R' is hydrogen or any
straight or branched chain radical of 1-4 carbon atoms in
which one of the carbon atoms may be substituted by an O or
S;
if W, X, Y, or Z i6 NR, then R may al80 be NR or OR;
A, B, and C are the same or different straight chain
carbon r~dicals of 2-5 carbon atoms in which 1-4 of the
carbon atoms have been substituted in some or all locations
with R or with a sùbstituent group such as -O, F, Cl, 8r,
I, OR', CO2R', SO3R'; wherein R' is hydrogen or any straight
or branched chain radical of 1-4 carbon atoms in which one
of the carbon atoms ~ay be substituted by an O or S; and
D contains 1-8 carbon atoms, including a 2-6 carbon
atom chain terminating in a group which allows it to form
a bond to W following reaction with a radionuclide, and
further optionally contains substituent groups such as ~0,
F, Cl, Br, I, OR', CO2R', S03R'; wherein R' is hydrogen or
any straight or branched chain radical of 1-4 carbon atoms
in which one of the carbon atoms may be substituted by an
O or S.
In one preferred embodiment of the present invention,
.~ ,. .:.
~ . , . , , . : .. .
-, , : ' ' ' - ~ :' ' ' ': . . .-
..
: , . . -
- : ~ .,: ,,
:~ ., . ' .

w092/~92 PCT/US92/~K~
the acyclic ligand includes at least three and at most five
amino acid groups and has the formula:
R~
// R5
R 3_c c\
H N--I--R 6
=C H N~ ~o
\ ~NH2 1
R 2 I R ,C~ \X
wherein R~ to Rt are the same or different and are
selected from the group consisting of hydrogen or ~
substituents of any natural or synthetic amino acid; .
X is.OH or ~7c_coo~ wherein Rg and Rlo have
o
the same meanings as Rl to R8 above.
The radiopharmaceutical forming reaction will take
place in situ over a period of time by reaction of the
radionuclide and ligand complex with constituents already
present from the complex formation. In particular,
constituents such as water, Hl, OH, chloride, or ethanol
may be available to yield the radiopharmaceutical agent in
final administratable form. In addition, the complex may
simply react with itself to yield the radiopharmaceutical
agent. In particular, the reaction according to the
present invention may follow the general reaction:
.
..
, ` ' ' " ~ ~., ' ' ,
'

W092/14492 21 0 1 ~ ~ 2 PCT/US92/~K~
w--~--X--5 W5 ~ X w~ ~ x
o z c_Y ,~ ~Y ~
Z Y
D ~C J
( I l ) '
wherein M represents a metal radionuclide optionally having
one or more additional ligands attached to the metal, and
wherein A, B, C, D, W, X, Y, and Z have the same
definitions as given above in formula (I).
According to this general reaction, it is possible to
react a ligand with a radionuclide to form a radionuclide
complex, which changes over a period of time to a ;~
radiopharmaceutical agent having desirable biodistribution
properties, by further reaction of constituents pr-sent at
the time the ligand reacts with the radionuclide.
Therefore, it becomes unnecessary to carry out all of the
separate reactions which are required in the prior art.
Rather, the entire process of forming the
radiopharmaceutical agent may be performed in a single
r-action stage, by providing the constituents necessary for
such formation.
The process according to the present invention further
provides a mean6 for forming radiopharmaceuticals which
were previously unobtainable because of problems associated
with synthesis of the ligand, or with formation of the
radionuclide complex. In particular, such previously
unobtainable radiopharmaceuticals may now be formed because
the ligand does not have to be presynthesized nor does the
metal have to be complexed with the final for~ of the
.
. .,, . ~ , . . - . .
. - :: - .
,: . - . ,
.

W092~1~92 PCT/US92/~K~
2 1 ~ o
ligand, both of which may be chemically and
radiopharmaceutically unfavorable processes.
A radiopharmaceutical agent according to the present
invention is generally used in the form of a composition
which is suitable for diagnostic or therapeutic functions.
In addition to the radioactive agent, such a
radiopharmaceutical composition will usually comprise a
liguid, pharmaceutically acceptable carrier material,
preferably a physiological saline solution. A
radiodiagnostic examination can be performed with such a
composition by administering the composition to the patient
and then recording the radioactive radiation emitted from
the patient by means of, for example, a gamma camera.
The present invention further relates to a method of
preparing a radiopharmaceutical agent according to the
present invention by reacting a radionuclide in the form of
a radionuclide solution in the presence of a reducing agent
and optionally a Ruitable chelator with an appropriate
compound. The reducing agent serves to reduce the metal
radionuclide in the solution which is obtained from a
generator or supplied from a manufacturer. Suitable
reducing agents are, for example, dithionite, fo mamidine
sulphinic acid, diaminomethane disulphinate or suitable
metallic reducing agents, for example, reducing metals such
as tin metal, or reducing ions such as Sn(II), Fe(II),
Cu(I), Ti(III) or Sb(III); wherein Sn(II) has proved to be
particularly suitable.
For the above-mentioned complex-forming reaction, the
radionuclide solution is reacted with a ligand having the
general formula (I) above directly, or in a two step
reaction in which the metal is first bound to a transfer
ligand and then displaced by the ligand of choice.
?

Wo92/1~92 2 1 0 1 6 ~ 2 PCT/USg~/~K~
Examples of suitable transfer ligands for the radionuclide
are dicarboxylic acids, polycarboxylic acids or hydroxy
carboxylic acids, such as oxalic acid, malonic acid,
succinic acid, maleic acid, orthophthalic acid, malic acid,
lactic acid, tartaric acid, citric acid, ascorbic acid,
salicylic acid or derivatives of these acids; phosphorus
compounds such as pyrophosphates; or enolates. Citric
acid, tartaric acid, ascorbic acid, glucoheptonic acid or
a derivative thereof are particularly suitable transfer
ligands when the radionuclide is technetium-9sm.
The present invention also relates to a kit,
comprising:
(1) A ligand according to the general formula (I); the
ligand preferably being in a dry condition, and also
preferably having an inert, pharmaceutically acceptable ^~
carrier and/or auxiliary substances added thereto; and
(2) a reducing agent and optionally a chelator;
wherein ingredients (1) and (2) may optionally be combined;-
and
further wherein instructions for use with a prescription
for carrying out the above-described method by reacting
ingredients (1) and (2) with a radionuclide solution may be
optionally included.
Examples of suitable reducing agents and chelators for
the above kit have been listed above. The radionuclide
solution can be obtained simply by the user himself from a
generator which is available to him, or as the solution is
supplied from a manufacturer. As noted above the
ingredients (1) and (2) may be combined, provided they are
compatible. Such a monocomponent kit, in which the
combined ingredients are preferably lyophilized, is
excellently suitable to be reacted by the user with the
radionuclide solution in a simple manner.

W092/l~92 2 1 0 1 ~ 4 2 PCTtUS92/~K~
The ingredient (1) of the above kits may be delivered
as a solution, for example, in the form of a physiological
saline solution, or in some buffer solution, but is
preferably present in a dry condition, for example in a
lyophilized condition. When used as a component for an
injection liquid, it should be sterile, and, if the
ingredient (1) is present in a dry condition, the user
should use a sterile physiological saline solution as a
solvent. If desired, ingredient (1) may be stabilized in
a usual manner with suitable stabilizers such as ascorbic
acid, gentisic acid or salts of these acids, or it may be
provided with other auxiliary means such as f illers, e.g.
glucose, lactose, mannitol, inositol, and the like.
The following example set forth below describes one
embodiment according to the present invention.
EXAMPLE 1
Synthesis of a radiopharmaceutical aaent by direct
labellina
In a reaction vial are added 1 mg tetra-L-alanine, 0.2
ml phosphate buffer 0.5M pH 12, 100 ~m SnCl2.H2O dissolved
in 25 ~l HCl 0.05N, and 2 ml generator eluate containing
370 MBq 99mTc as perterhnetate. After inculbation for 10
minutes at roon temperature the reaction mixture is
analyzed by RP-HPLC on a 250 mm X 4.6 mm (I.D.) Hypersil
ODS column eluted at a rate of 1 ml/min in an isocratic way
with a mixture of ethanol - 0.025M phosphate buffer pH 5.85
(30:70). Radioactivity in the effluent is monitored with
a 2 inch NaI (tl) scintillation detector coupled to a
single channel analyzer and integrator.
The HPLC-chromatogram shows the presence of mainly one
radioactive species (peak A) and a small amount of another
compound (peak B). Electrophoresis experiments with
isolated peak A and peak B show that peak A has a free
,
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W092/1~92 2 1 0 1 6 ~ 2 PCT/US92/~K~
corboxyl group (migration distance is larger at h~gher pH)
whereas the migration distance of peak B doew not increase
with increasing pH, indicating the absence of a free
carboxyl group. Coinjection of peak B with 99Tc-cyclo-
tetra-L-alanine (structure confirmed by FAB-mass
spectrometry and single crystal X-ray analysis) shows that
peak B is 99mTc-cyclotetra-alanine as indicated in Scheme
I. Peak A is assumed to be non-cyclisized 99mTc-tetra-L-
alanine as represented in Scheme I.
Incubation of isolated peak A at pH 5.85 (phosphate
buffer) results in the gradual conversion to peak B,
whereas peak B remains stable in the same conditions.
L-~lr~-al~nin~ + Sn'~ ~ TcO,-
¦ ~H 12
t5~0 ~\ C~O ,,
C H ~ 11 H ~1~ ~ C N ~
C N ~ ~C O 011 ~ ' .C .
O CH~
Scheme I. Preparation of TcO-~ and TcO-cycl-A4
- tetra-alanine
EXAMPLE 2
Svnthesis of a ~a~iQ~h~r~aceutical agent by exchange
labellin~
In a reaction vial are added 1 mg tetra-L-alanine, 4.5
mg sodium tartrate dissolved om 0.3 ml water, 0.2 ml
phosphate buffer 0.5M pH 4, 100 ~m SnCl2.H20 dissolved in 25
~1 HCl 0.05N, and 2 ml generator eluate containing 370 MBq
99mTc as pertechnetate. The solution is heated for 10
minutes on a boiling water-bath. Analysis of the cooled
reaction mixture by HPLC indicated the presence of a
species corresponding to peak B of example 1 (cyclic 99mTc-
~ .. . . . .

W092/1~92 PCT/US92/~K~
2 ~ 14
A4), a small amount of a species corresponding to peak A ofexample 1 and a small amount of 99mTc-tartrate. The
relative amount of peak B decreases if labelling is
performed at higher pH values.
A, ~ Sn2~ ~ ~or~roto I TcO4-
1~
CU, ~COO~ Cl~
o ~ :.
The forgoing has been a discussion of the preferred
embodiments of the present invention, but is not intended ~.;
to limit the invention in any way. Rather, many
modifications, variations, and changes in detail may be
made within the scope of the present invention.
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