Note: Descriptions are shown in the official language in which they were submitted.
60~;
This invention relates to the preparation of radio-
pharmaceuticals, and in particular to the preparation of
technetium-99m (9g~Tc) - labelled radiopharmaceuticals.
Radiopharmaceuticals are diagnostic or therapeutic
agents by virtue of the physical properties o~ their
constituent radionuclides. Thus, their utility is not
based on any pharmacologic action. Most clinically used
drugs of this class are diagnostic agents incorporating a
gamma-emitting nuclide which, because of its physical or
metabolic properties, localizes in a specific organ after
intravenous injection. Images reflecting organ structure
or function are then obtained by means of a scintillation
camera that detects the distribution of ionizing
radiation emitted by the radioactive drug. The principal
isotope currently used in clinical diagnostic nuclear
medicine is reactor-produced metastable technetium-99m.
Many methods have been described for the reduction
of pertechnetate (99~TcV~04-) in the preparation of 998Tc-
radiopharmaceuticals. Reducing agents which have been
used include stannous ion, electrolysis, ferrous ion,
ferrous ascorbate, formamidine sulphinic acid and sodium
borohydride (Dsutsch et al, 1983). These labelIing
procedures generally lead to the redu~tion of teshnetium
to the Tc(IV) or TctV) oxidation state. In many cases
the compound prepared contains the TcO moiety (Deutsch
1979). Because of problems experlenced with these
reducing agents, the use of a substitution route for the
production of 99~Tc-radiopharmaceuticals has been
advocated (Deutsch & Barnett, 1980). The agents normally
used for substitution reactions are TcOX52- and TcX6~- (X =
:
Cl,Br) in which technetium is in the TcV and TcrV valency
states respectively.
The present~inventors have investigated tho
preparation of 99~Tc-radiopharmaceuticals containing the
,, : . .
.
,
,
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--2--
TcN moiety, and have discovered that the TcN moiety is
extremely stable to hydrolysis and that the nitrido group
remains firmly attached to the Tc atom throughout a
number of substitution reactions.
According to the present invention, there is
provided a novel group of compounds containing the TcN
moiety, as well as methods for the preparation thereof
and methods for the preparation of 99nTc-radio- -
pharmaceuticals utilising these compounds.
According to a first aspect of the present
invention, there are provided compounds of the formula I:
R+[99mTcNX4]- I
wherein R' is a cation, preferably a soluble cation such
as sodium or another alkali metal, or ammonium, and X
represents a halogen group, particularly a chloro or
bromo group.
The compounds of this aspect of the invenkion are
characterised by the presence of the nitridotetrahalo-
technetium-99m anion in which Tc is in the TcV~ valency
state, and which has been found to ha~e particular
utility in the preparation of radiopharmaceuticals
containing the TcN moiety.
In another aspect of this invention, there is
provided a process for the preparation of compounds of
the formula I as~described above, which comprises
reaction of a compound containing the 99rTc-pertechnetate
anion (R[99nT¢o~], wherein R represents a cation such as
alkali metal, or ammonium), with an azide ion, such as
sodium azide, in the presence of a hydrohalic acid, such
as hydrochloric or hydrobromic acid.
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In another aspect, there is provided a method of
producing a 99nTcN -labelled product, which comprises
reacting a compound of the formula I with a ligand.
Suitable ligands include, for example, methylene
diphosphonate (MDP), thiourea (TU), thiomalate (TMA),
dimercaptosuccinate (DMSA), gluconate (GLUC), N-(2,6-
diisopropylphenylcarbamoylmethyl)iminodiacetate (PIPIDA),
N-(2,6-dimethylphenylcarbamoylmethyl)iminodiacetate
(~IDA) ethane-1-hydroxy-1, 1-diphosphonate (EHDP),
diethylenetriaminepentaacetate (DTPA), and cysteine
(CYS). Other ligands which may be used in accordance
with the present invention include thiouracil, diethyl-
dithiocarbamate, mercaptopyridine, mercaptopyrimidine,
thiooxine, acetylacetone, pyridoxal, oxine, tropolone and
tetracycline. Monoclonal antibodies which may also be
labelled in accordance with the present invention, and
which have been shown to retain their specificity
following labelling. This aspect of the invention is
described in greater detail hereinafter.
In yet another aspect, there is provided a 99nTcN -
labelled product which comprises the reaction product of
a compound of formula I with a ligand.
The desirability of using à subst~itution route for
the preparation of 99~Tc-radiopharmaceuticals has long
been recognised. However the method has suffered because
of the difficulty in obtaining 99~Tc in a suitable
chemical form at the Tc concentrations used for radio-
pharmaceutical production. The nitrido labelling
technique descrlbed here is a comparatively simple method
for the preparation of a wide range of radio-
pharmaceuticals based on the TcN moiety. While Tc is
initially present a= Tc ~VI) in TcNC1~-, it has been
found that reduction usually takes place to the Tc(V)
state if the ligand has the ability to act as a reducing
agent. Ligand substitution then takes place around the
" ' .
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` . ' ` ' , ' .', , ~ : . ,
_4~ 3~i~3~j
TcN2' core. In all cases studied to date, the presence
of the nitrido group has been Pound to alter the
biological behaviour of the 99~Tc-labelled ligand.
Nitrido labelling has been found to be particularly
suitable for the labelling of "soft" ligands
The present invention has particular application in
the coupling of 99~Tc to monoclonal antibodies (MAb) and
the use of the resulting complexes, for example, in the
specific detection of tumors in vivo, (see, for example,
Rhodes, et al, 1982). At present a number of
difficulties exist in the diagnostic radiolocalization of
tumors, one of which is the choice of radionuclide. Many
studies have used 131I, however this radionuclide has
serious drawbacks including a poor quality image,
significant radiation exposure due to its beta emissions,
and short biological half-life.
Technetium -99m (~Tc) has an isotope for radio-
localization offers several advantages: it has a
reasonably short hal~-life; it is cheap, easy to produce,
and is readily available. The isotope has an optimal
gamma energy (140keV) for detection with currently
available gamma scintigraphic instrumentation and
produces very little radiation exposure to patients
undergoing scanning procedures. However little use has
been made of 99~Tc for labelling antibodies, presumably
because most labelling methods used to date lead to loss
of antibody activity, due to side reactions taking place
and to transchelation reactions occurring in vl~o.
In accordance~with the present invention, compounds
of the~formula I described above have been found to
produce stable 99~Tc-labelled MAb by a substitution
reaction in the same manner as other ligands described
herein. Either whole monoclonal antibodies, or antibody
fragments (such as~Fab fragments) which react with the
,, ~
.
.
~36~3~
corresponding antigens may be labelled in accordance with
this invention. Antibody specificity is maintained in
the labelling process and the labelled product i.s table.
In addition, tests have shown tha~ when the labelled Mab
is used in tumor detection, tumors may be visualised in
as soon as two hours, and furthermore small tumors (about
0.4 cm) located near large vascular organs can be
visualised.
Preferably, in labelling monoclonal antibody in
accordance with this invention, the antibody is at least
partially reduced for example by reaction with a reducing
agent such as dithiothreitol, in order to convert
disulfide linkages into sulfhydryl residues. Such a
partial reduction procedure enables utilisation of the
preference of the TcN moiety for sulfur atoms, and thus
enables the production of more stable complexes in which
the Mab ligand is bound through sulfur atoms to the TcN
moiety. It is also noted that these sulfhydryl groups
would be removed from the sites responsible for antibody
specificity, hence formation of the complexes is less
likely to cause loss of specificity.
.
In drawings illustrating the invention:
Figure 1 is an ultraviolet absorption spectrum
showing the formation of TcNC14, complexes;
'~
Figure 2 is an ultraviolet absorption spectrum
showing the formation of TcN-~DP complexes;
Figure 3 is an ultraviolet absorption spectrum
showing the formation of PcN-DTPA compIexes: and
Figure 4 shows the clearance rates of TcN-GLUC, TcN-
HEDP, TcN-HIDA and TcN-PIPIDA complexes in bio-
distribution studies.
. .
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The following Examples illustrate the preparation of
a [99nTcNX4]- compound as well as the biological behaviour
of TcN radiopharmaceuticals containing various ligands.
EXAMPLE 1
A. Preparation of sodium
tetrachloronitridotechnetate Na[99DTcNC1~] and
99nTcN-radiopharmaceuticals.
Unless otherwise stated all solvents and chemicals
were of analytical grade. L(+)-Cysteine for biochemistry
was obtained from E.Merck, Darmstadt, Methylenediphos-
phonic acid (MDP) from Sigma Chemical Co., St. Louis and
diethylenetriaminepentaacetic acid (DTPA) from Koch-Light
Laboratories, Colnbrook. Sodium-2-gluconate was obtained
from Fluka A.G. EHDP was prepared using the method
described by Castronovo (1974).
HIDA and PIPIDA hydrochlorides were prepared by the
reaction of the N-chloroacetanilides with iminodiacetic
acid using a variation of the method of Callery et al
(1976). A mixture of N-chloroacetanilide (0.05 mole),
iminodiacetic acid (0.05 mole) and lOg anhydrous sodium
carbonate was refluxed in 30mL of 75%`aqueous ethanol for
6 hours. On cooling the solution was acidified with
concentrated hydrochloric acid and the pH adjusted to
1.5. The precipitate was filtered and recrystallised
from 50% aqueous ethanol. Use of sodium carbonate
resulted in improved yields (>60%). 99Tc in the form of
ammonium pertechnetate in 0.lM ammonium hydroxide
solution was obtained from Amersham International.
(i) A solution of 99~Tc-pertechnetate t50MCi~18
GBq for animal studies) was taken to dryness using
a rotary evaporator. Sodium azide t~20mg) was
added to the dry residue, followed by 10 ml of
,~
.
`' ' ' ' '
~3~
concentrated hydrochloric acid (sg 1.18). The
solution was refluxed for 5 minutes to complete
the reduction and destroy excess azide before
being taken to dryness in the rotary evaporator.
1 ml of ligand solution (PIPIDA 20mg/ml, pH 7, all
others 5 mg/ml, pH 71 was added followed by 2 ml
saline. If necessary, the pH was adjusted to 6-7
by the addition of 0.lM sodium hydroxide. After
filtration through a 0.22 ~m membrane filter, the
radiopharmaceutical was ready for use.
(ii) An alternative labelling procedure is to
perform the ligand replacement in a non-aqueous
solvent, such as in acetonitrile or ethanol
solution. By way of example, 10 ml of
acetonitrile is added to the dry residue after the --
azide reduction followed by 100 ~1 of the ligand
solution. ~fter heating on a water bath for 10
minutes, the acetonitrile is removed in a rotary
evaporator and the dried extract dissolved in 1 ml
o~ the ligand solution and 2 ml saline as before.
B. Animal;Distribution Studies
0.05-0.1 ml of the preparation (1-2mCij was
injected into the tail vein of Swiss mice (20-30g) and
the activity injected measurèd in an ionisation chamber.
Afker injection, mice were allowed free access to food
and water. At each time interval skudied, three mice
were sacrificed~by cervical dislocation and dissected.
:
Organs were weighed and their activities measured in the
ionisation~chamber. ~The original injected activity was
30 correc~ed~Por~the~activity found in the tail. Blood ;~
activity was calculated on~the assumption thak the
overall blood volùme represents 7% of kotal body welght.
, .
~ .; ' . ' . ~ ~ '
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-8-
C. Measurements of 99~Tc Labellina
2~L aliquots of each preparation were
chromatographed on Whatman No. 1 paper in three solvent
systems: saline, 70% methanol and methyl ethyl ketone.
After development all papers were dried and scanned in a
radiochromatogram scanner (Packard Model 7220/21). Where
appropriate, peaks were cut from the strips and counted
for gg~rc activity in a gamma counter. All preparations
contained less than 5% free pertechnetate.
D. W Spectral Studies
Preparations were made using 300~g 99~Tc added as
carrier for W spectral studies. UV spectra were
recorded on a Beckman Acta CII spectrophotometer.
Pertechnetate was reduced using the following
reduction systems:
(i) concentrated hydrochloric acid,
(ii) concentrated hydrochloric acid/potassium iodide,
(iii) concentrated hydrochloric acid/sodium azide,
(iv) concentrated hydrochloric acid/stannous chloride,
(v) concentrated hydrochloric acid/hypophosphorous
acid, ~ ~
(vi) concentrated hydrochloric acid/hydroxylamine
hydrochloride.
The spectra of all samples was measured in
hydrochloric acid solution before and after heating on a
hot plate.
W spectra~of the technetium complexes studied were
obtained after taking the above solutions to dryness in a
rotary evaporator and~dissolving the dry residue in 1 ml
ligand soIution and~2 ml water.; The pH of the solution was
adjusted to 7 using either O.lM or lM sodium hydroxide.
:
- : ' ' :
:
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;0~
E. RESULTS
The concentration of Tc in the solution used for uv
absorption measurements was determined by beta counting.
lOmL scintillation fluid (Aquassure-NEN) were added to
O.lmL aliquots of the solution for counting in a liquid
scintillation spectrometer. Counting efficiency was
determined by counting aliquots of a pertechnetate
solution standardised by uv spectrophotometry. Quenching
of the solution was checked by external standardisation.
An internal standard was added when a quench correction
was necessary.
(i) W Spectral Studies
(a) Hydrochloric Acid Studies
Addition of potassium iodide, hydroxyl-
amine hydrochloride, or hypophosphorous acid to
pertechnetate in hydrochloric acid followed by
heating all result in the formation of Tc(IV).
This is shown in Figure 1 by the characteristic
W absorption spec~rum of TcC162- which has an-
absorption maximum at 340nm and a minor peak at
305nm. Pertechnetate allowed to stand in
concentrated hydrochloric ac~d in the cold,
results in the production of TcOCl52- in which
Tc is in the Tc(v) oxidation state. The W
spectrum however indicates that the TcOC15Z-
produced also containa~Tc(~IV), the proportion
of which is increased by heating. Heating of
pertechnetate in the presence of azide results
in the formation of TCNCl4- characterised by an
absorption maximum at 395nm ( = 500m2 mol~l) and
~ free of contaminating Tc(IV).
::
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.K~
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--10--
shown in Figure 2. When MDP was added to
TCNC14- at pH 5.5, a pink colored compled
(A~x-515nm) was produced. On heating the pink
color disappeared producing a yellow complex
with an adsorption maximum at 335nm. The
spectrum of this complex was identical to that
obtained by adding MDP to TcNC14- and adjusting
the pH to 10 (A~=335nm, ~=21m2 mol~l).
(c) DTPA Complexes
When prepared at room temperature, TcN-
DTPA showed an absorption maximum at 505nm
(~=150 m2mol~l) (Figure 3). On heating the peak
disappeared producing a complex with no
signi~icant absorption in the range 300-800nm.
TcIV-DTPA produced by adding DTPA to TcC162-
showed no significant absorption maximum in the
uv-vis region.
(d) Cysteine Complexes
The~spectrum of TcN-CYS showed no
significant absorption in the uv-visible region
that could be attributed to the complex.
.
(ii) Stability of dried T~oNCl~~ Preparation
Samples of the~99nTcNCl4- preparation were taken
to;dryness ln a rotary evaporator and stored for 24
hours~(a) in air at room temperature, (b) at 80 C,
and (c~)~at room ~emperature and 100% humidity. They
were then used to prepare 99~rcN-DTPA by addition of
ligand as bafore. No significant difference in
~ chromatographic bahaviour as measured by high
; ~ perform~ance~liquid ohromatography (HPLC) was
observed in any sample indicating that th~ dry
99~TcNCl4- reagent was stable for up to 24 hours.
:
: ~: . . .:
.
~S~fi~
(iii) Animal ~istribution Studies
(a) Results of biodistribution studies of
99'TcN-MDP, 99~TcN-DTPA and 99~TcN-CYS are given in
Tables 1-3. 99~TcN-MDP was characterised by
high blood pool activity and showed
insignificant bone uptake. 9gnTcN-DTPA showed
similar renal excre~ion behaviour to 99~Tc-
DTPA(Sn) and was probably excreted by a similar
mechanism. Blood pool activity however was
higher than that observed for 99~Tc-DTPA (Sn).
99~Tc-cysteine showed rapid clearance with high
renal localization. Urinary excretion rates
for the three 99~TcN-complexes are given in
Table 4. Excretion rates of 99~TcN-MDP and
99~TcN-DTPA are significantly less than that of
the respective 99nTc(Sn) - complexes.
(b) Results of biodistribution studies of
99Yrc-GLUC, 99nTcN-HEDP, 99nTcN-HIDA and 9gnTcN--
PIDIDA~are given in Tables 5-9. All
preparations showed high blood pool activity
with the 99nTcN-GLUC and 99nTcN-HEDP preparations
cl aring slightly faster than 99nTcN-PIPIDA and
9g~TcN-HIDA. (Figure 4~. Overall, 99~TcN-GLUC~
99~TcN-HIDA and 99nTcN-HEDP showed comparable ;
patterns of biological behaviour. 99~TcN-PIPIDA
differed from the other agents in that it gave
signlf~icantly higher actlvity in the
intestines. With all preparations the
clearance into the intestines~took place
essentially in the first 30;minutes. The
higher~ clearance of 99~TCN-PIPIDA could be due
to gg~TcN-PIPIDA undergoing a slower rate of
;exchange~w1th serum proteins than 99~TCN-HIDA.
:
, . .
. .. ' ' . :
, : -
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-12-
(c) Results of biodistribution studies of
99~TcN-DMSA are given in Table 10.
(iv) Stability studies
Stability studies of the dry 99~TcNC14-
preparations have indicated that the agent is
sufficiently stable to be prepared in a central
laboratory or manufacturing site and distributed to
users for use in the preparation of 9g~TcN-labelled
radiopharmaceuticals. Preparation of the 99~TcN-
radiopharmaceuticals in most cases takes place by
simply dissolving the dry residue in a solution of
the chelate.
99~rcNC14- may also be used to label ligands that
are insoluble in water or which may be unstable in
aqueous solutions. It is possible to extract the
9snTcN- activity from the dry salt residue into
organic solvents such as acetonitrile. Labelling
may be performed in the organic solvent~which may
then be removed by evaporation. Labelling with-
9g~TcNCl4~ takes place via a substitution mechanism
and is expected~to be less susceptible to the
hydrolytic type reactions which occur with many
other~labelling proaedures. In addition, the
presence of the nitrido group modifies~the chemistry
of~the Tc atom in that~reactions with "soft" ligands
are more favoured than when other reducing agents
are~used~
EXAMPLE 2
A. ~Preparation~of 99'T~ ~belIed MAb
(i) Tumor Cell Lines
:
Two~tumor cell~lines were used: one, the E3
clonal variant of the thymoma ITT(1)75NS (Hogarth,
,
..
, . , . ~ :
'
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et al, 1982), which was obtained by three successive
rounds of cytofluorugraphic sorting of IGG(1)75NS
cells stained with monoclonal Ly-2 antibodles and
selected for the most fluorescent 1% of cells. The
murine cell line E(3) was maintained in vitro in DME
supplemented with 10% heat inactivated newborn calf
serum (Flow Laboratories, Sydney, Australia), 2mM
glutamine (Commonwealth Serum Laboratories, CSL,
Melbourne, Australia), 100 I.U./ml penicillin (CSL)
and 100mg/ml streptomycin (Glaxo, Melb., Aust.). E3
cells were washed twice in DMI (without additives)
and twice in DME containing 0.5% BSA and used in the
in vitro binding assays. The E3 cell line was
maintained in vivo by the passaging of cells from
ascites fluid produced in (B6 x BALB/c)Fl mice or
from solid tumors which grow after the subcutaneous
injection of 106 or 107 cells. The second cell line
used was a human colonic carcinoma, COLO 205 (Semple
et al, lg78). It was maintained in culture with
TPMI containing the same additives. Adherent COLO
205 cells were harvested with 0.125% trypsin (CSL)
washed with RPMI and injected subcutaneously into-
nude mice, where tumors appeared after the injection
of 2 x 106 - 1 x I07 cells.
:,
(ii) Monoclonal Antibodies ~MAb)
Two monoclonal antibodies were used: (i) anti-
Ly-2.1~(IgG2a), an antibody raised against the
murine~alloantigen Ly-2.1 (Hogarth et al, 1982) and
(ii) 250-30.6 (IgG2b), an antibody to human colonic
secretory epi~helium (Thompson, et al, 1983). ~he
monoclonal antibodies were isolated from ascitic
fluid by preaipitation with 40% saturated ammonium
sulphate, followed by dissolution in 0.01M Tris
buffer pH 8.0 and extensive dialysis against the
same buffer. The crude antibody preparations were
further purified by affinity chromatography using
:
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-14-
protein-A Sepharose (Pharmacia) and then purity
determined by gel electrophoresis and antibody
activity assayed by a rosetting test (Parish, et al,
1~78).
(iii) 99DTc-Labellinq of Antibodies
99~TcNC1~- was prepared as described above.
For labelling the MAb was first reduced with
dithiothreitol (D~T) by adding 20~1 of DTT in PBS
(115mg/ml) to 200~g of MAb (lmg/ml in PBS~ and
standing the mixture at room temperature for 30
minutes after which the reduced MAb was separated
from DTT by gel chromatography using O.lM sodium
acetate pH 4.0 as eluant on an 8cm x lcm column of
Biogel P-6 (Biorad Laboratories, Richmond, USA).
The fractions (lml) containing the protein peak
were added to the dried 99~rcNC1l salt residue and
the mixture brought to pH 3.0 with 0.2M
hydrochloric acid. After 2 minutes at room
temperature, 2 drops of O.lM sodium phosphate was
added and the pH adjusted to 7 by the careful
addition;of sodium hydroxide (lM or O.lM). - ~
Purification of the labelled MAb was then achieved
by gel chromatography with a Sephadex G-25
disposable column (PD-10, Pharmacia) equilibrated
in PBS, 500~1 aliquots were~collected and the
radiolabelled protein~peak identified by gamma
counting.
:
B. RESULTS ~ ~
(i) In vitro stability and specificity of 99nTcN-MAb
Three different specific.ity assays were
performed. The first compared the activity of 99~Tc
labelled~Ly-2.1 MAb to thymocytes from the mouse
strains RF/~ (Ly-2.1 positive and C57~L/6 (Ly-2.1
negative)). A ten fold difference was observed in
the binding of the labelled MAb to the specific
'~
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~ ?
3L~ 3~
-15-
cells (RF/J) when compared to the negative cell
controls tC57BL/6). The procedure used to couple
99~TcNC14- to MAb produced a stable complex which was
shown to be s~able and bind specifically antigen
positive target cells ten times greater than antigen
negative control cells.
In the second specificity assay two different
MAb, one directed against colonic secretory
epithelium (250-30.6) and the second the anti-Ly-2.1
MAb, were labelled with 99~TC under identical
coupling conditions and the two complexes were
tested for their ability to bind to the murine T
cell thymoma E3, which is posi-tive for the Ly-2.1
antigen but does not react with the anti-colon
antibody. The anti-Ly-2.1 MAb bound ten times more
efficiently than the 99~rcN-anti-colon complex. The
stability of the 99'TcN-MAb was demonstrated here,
where only the antibody reactive complex (99~TcN-
anti-Ly-2.1) showed increased binding on E3 target
cells. The non-reactive complex (99~TcN-anti-colon)
exhibited a significantly reduced uptake of
radioactivity on the same target cells.
The stabiIity of the TcN-MAb complexes was
examined in a third assay. Aliquots of labelled MAb
were stored at 4C overnight and~the stability of
the label was determined by binding the stored
material to thymocytes. The labelled MAb were shown
to be s~able by their retention of binding ability.
RF/J thymocytes (Ly-2.1+) bound better than 10 fold
more labelled Ly-2.1 MAb than C57BL/6 thymocytes
(Ly-2.1~). In three diPPerent specificity assays
the in vitro stabiIity of the 99~TcN-MAb complexes
was established and were shown to be chemically
stable, even when allowed to stand at 4C overnight,
blnding only to antibody reactive target cells.
: :; :
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:
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(ii) Localization
The in vivo localization and biodistribution of
TcN-MAb complexes was performed in separate studies.
In the first, the mice were dissected normal organs,
tumors and an aliquot of whole blood were counted in
a gamma counter. The solid tissues were then
weighed and the results were used to calculate the
localization ratio derived as follows: tissue
(cpm/g) / blood (cpm/g). Two groups of 16 (C57BL/6
x BALBjc)F1 mice bearing the E3 tumor (0.23 - l.llg)
were injected i.v. via the tail vein, with one of
two MAb (anti-Ly-2.1 or anti-colon carcinoma)
labelled with 99~TcNCl4- under identical conditions.
(Each mouse received 115~Ci Tc and lO~g MAb). In
the data in Table 11, 4 mice from each group were
sacrificed at different time intervals after
injection (20, 30.5, 35 hrs) and the distribution of
the specific MAb (anti-Ly-2.1) was calculated for
the individual tissues and compared to the observed
distribution of the non-reactive MAb (anti-colon).
After 20 hrs the tumor localization was observed to
be 3 times greater for the specific MAb than that
observed for the non-specific MAb. This ratio
increased to approximately 3.8 at 30.5 hrs, and 7.3
2~ at 35 hrs after injection. It is important to point
out that the E3 tumor was observed to have the
highest localization ratio (i.e. tissue (cpm/g) /
blood (cpm/g)) in the group injected with the
specific MAb (anti-Ly-2.1) with the liver, spleen
and kidney localization ratio being below or similar
to the blood ratio. However with the non-specific
antibody (250-30.6) the liver, spleen and kidney
were observed to be higher than the blood ratio,
with the liver biodistribution ratio being 5 times
greater than the blood after 30.5 hrs.
In the second study, a specific MAb (anti-Ly- -
2.1) was compared in two dif~erent tumors. Nude
~ ~ .
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38~3
-17-
mice bearing colo 205 xenografts were used as the
non reactive tumor and the E3 as the positive tumor.
The data was obtained 20 hrs after the injection of
the anti-Ly-2.1 labelled MAb (Table 12). The E3
thymoma (Ly-2.1') was observed to take up 3 times
more radioactivity than the colo 205 xenografts (Ly-
2.1-H). The two biodistribution studies illustrate
a significant increased incorporation of
radioactivity in the MAb reactive tumor when
compared to the levels of radioactivity in the blood
and that incorporated in the other normal tissues.
:
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~36~
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TABLE 1 - Biological Distribution of 9~TcN-MDP in
mice~
% Injected Dose/Organ
5Time after Injection 30 min 60 min 120 min
-
Heart 0.6(0) 0.4(0) 0.3(1)
Lung 2.9(5) 3.0(14) 1.9(15)
Liver 10.4(4) 9.4(8) 6.2(13)
Spleen 0.4(1) 0.3(1) 0.4(1)
Stomach 1.1(1) 0.8(2) 1.2(11)
Kidneys 6.9(27) 3.3(5) 2.6(7)
Intestines 4.3(4) 4.1(6) 6.1(13)
Femurs 0.3(0) 0.3(0) 0.3(0)
Blood 26.5(46) 18.8(18) 13.5(40)
% Injected ~ose/Gram Organ
Heart 4.2(2) 2.4(4) 2.0(4)
Liver 5.7(3) 4.7(5) 3.8(1)
Kidneys 13.0(54)+ 5.6(11) 5.8(18)
Femurs 1.7(2) 1.4(2) 1.5(1)
Blood 13~0(22) 8.1(3) ~. 6-7(19)+
Standard deviation of last significant~digit(s) in
brackets. : ~
* n = 3 unless otherwise stated ~.
+ 2 mice
:
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--19--
TABLE 2 - Biological Distribution of 99~TcN-DTPA in
mice~
% Injected Dose/Organ
Time after Injection 30 min 60 min 120 min
-
Heart 0.1(0) 0.1(0) 0.1(1)
Lung 0.7(1) 0.7(2) 0.4(1)
Liver 1.7(2) 1.5(3) 1.1(2)
Spleen 0.1(0) 0~1(0) 0.1(0)
Stomach 1.1(2) 1.3(4) 0-9(0)
Kidneys 1.5(1) 1.3(2) 0.8(0)
Intestines 2.1(4) 2.0(3) 2.1(2)
Femurs 0.1(0) 0.1(0) 0.1(0)
Blood 6.2(8) 4.0(5) 2.4(1)
% Injected Dose/Gram Organ
Heart 0.6(0) 0.5(1) 0.4(1)
Liver 0.8(1) 0.7(1) 0.6(1)
Kidneys 2.5(1) 2.2(4) 1.5(2)
Femurs 0.5(1) 0.4(I) 0.4(1)
Blood 2.8(5) 1.8(2) . 1.2(0)
Standard deviation of last significant digit(s) in
brackets.
' ~
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fi~3~3~
-20-
TABLE 3 - Biological Distribution of 99~TcN-CYSTEINE in
mice~
% Injected Dose/Organ
Time after Injection 30 min 60 min 120 min
Heart 0.1(0) 0.1(0) 0.1(0)
Lung 0.8(2) 0.5(1) o.~tl)
Liver 2.7(5) 1.6(1) 1.3(1)
Spleen 0.1(0) 0.1(0) 0.1(0)
Stomach 0.4(1) 0.3(3) 0.3(1)
Kidneys 4.4(5) 3.4(5) 2.8(2)
Intestines 5.3(8) 3.4(1) 4.0(3)
Femurs 0.2(0) 0.1(1) 0.1(0)
Blood 4.9(7) 2.8(4) 1.8(1)
-
% Injected Dose/Gram organ
.
Heart 0.7(1) 0.5(2) 0.4(2)
Liver 1.3(2) 0.8(2) 0.6(1)
Kidneys 7.4(11j 6.2(9) 4.8(5)
Femurs ;0.8(1) 0.6(3) 0.2(1).
Blood 2.1(2) 1.3(1) ~- 0-8(1)
: '
Standard deviatlon of last significant digit(s) in
brackets.
* n - 3
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-21-
TABLE 4 - Urinary Clearance of g9~TcN-MDP, 99~TcN-DTPA
and 99~TcN-CYS~
~ Retained Activity
99~TcN-MDP 99~TCN-DTPA 99~TCN-CYS
30 min 71.9(43) 24.8(42) 35.8(43)
60 min 51.4(14) 21.4(31) 18.7(28)
120 min 44.7(57) 14.6(6) 15.7(15)
Standard deviation of last significant digit(s) in
brackets.
* n = 3
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-22-
TABLE 5 - Biological Distribution of 9g~TcN-GLUC in mice.
% Injected Dose/0rgan
Time after Injection 30 min 60 min 120 min
Heart 0.7(0.1) 0.7(0.1)0.4(0.1)
Lung 2.2(0.6) 3.0(0.5)1.4(0.2)
Liver 6.4(1.6) 6.2(1.7)7.1(2.1)
Spleen 0.3(0.1) 0.3(0.1)0.2(0.1)
Stomach 1.4(0.4) 1.5(0.4)1.6(0.7)
Kidneys 4.7(0.6) 3.9(0.3)3.4(0-1)
Intestines 5.4(0.7) 4.1(0-8)7.0(1.4)
Femurs 0.3(0.0) 0.3(0.0)0.2(0.1)
Blood 25.7(2.3) 21.7(3-6)12.7(1.9)
% Injected Dose/Gram Organ
Heart 4.5(0.7)~ 4.6(1.0) 2.6(0.6)
Liver 3.3(0.8) 3.1(0.9) 3.3(0.8)
Kidneys 8.4(1.0) 6.6(0.7) 5.6(0.6)
Femurs 1.7(0.1) 1.3(0.1) 0.8(0.2)
Blood 12.0(1.0) 9.5(1-8)5.2(0.3)
,
Standard deviation in brackets.
n = 3.
~' j
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,
-23-
TABLE 6 - Biological Distribution of 9~TcN-HEDP in mice.
-
% Injected Dose/Organ
Time after Injection 30 min 60 min 120 min
Heart 0,5(0,0) 0.3(0.0) 0.3(0.0)
Lung 2.5(0.6) 1.8(0.3) 1.5(0.4)
Liver 12.1(0.3) 9.0(1.0) 7.7(0.5)
Spleen 0.6(0.1) 0.3(0.0) 0.3(0.1)
Stomach 0.6(0.1) 0.7(0.1) 0.6(0.1)
Kidneys 4.1(0.2) 3.2(0.2) 3.0(0.2)
Intestines6.9(0.5) 7.0(0.2) 6.8(0.6)
Femurs 0.5(0.0) 0.5(0.1) 0.4(0.1)
Blood 28.0(5.0)$ 16.2(2.0) 13.2(1.0)~
% Injected Dose/Gram Organ
-
Heart 4,3(0,5) 2.4(0.3) 2.3(0.2)
Liver 8.1(0.8) 5.5(0.7) 4.8(0.4)
Kidneys 9.1(0.4) 6.5(0.4) 6.1(0.3)
Femurs 3.4(0.5) 3.1(0.8) 2.7(0.6)
Blood 16.2(1.0)$ 7.3(1.1) 7.7(0.5)~
Standard deviation in brackets.
n = 3 unless otherwise stated.
~ n = 2
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-24-
TABLE 7 - Biological Distribution of 99~TcN-HIDA in mice.
% Injected Dose/Organ
Time after Injection30 min 60 min 120 min
Heart 0.4(0.1) 0.4(0.0) 0.2(0.0)
Lung 2.3(0.3) 2.0(0.3) 1.2(0.3)
Liver 7.2(1.4) 6.7(1.2) 3.6(0.2)
Spleen 0.4(0.1) 0.3(0.0) 0.1(0.0]
Stomach 1.1(0.2) 2.0(0.4) 1.0(0.0)
Kidneys 3.5(0.2) 3.4(0.2) 2.1(0.0)
Intestines6.8(1.0)9.2(0.4) 6.9(0.2)
Femurs 0.3(0.1) 0.4(0.0) 0.2(0.0)
Blood 25.8(1.6)20.6(1.3) 16.3(2.2)
~ Injected Dose/Gram organ
Heart 2.9(0.5) 2.7(0.4) 1.6(0.1)
Liver 4.2(0.8) 3.9(0.6) 2.1(0.1)
Kidneys 6.9(0.5) 6.7(0.4) 4.2(0.1)
Femurs 2.0(0.4) 2.3(0.1) 1.0(0.1)
Blood 13.6(0.6)10.8(0.6) 8.7(1.1)
-
Standard deviation in brackets.
n = 3.
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-25-
TABLE 8 - Biological Distribution of 99~TcN-PIPIDA in
mice.
-
% Injected Dose/Organ
Time after Injection 30 min60 min~ 120 min
Heart 0.4(0.0) 0.3(0.1)0.4(0.1)
Lung 2.3(0.3) 1.8(0.6)1.8(0.2)
Liver 14.6(1.0) 16.2(6.1)10.1(1.0)
Spleen 0.2(0.1) Q.3(0.0)0.2(0.0)
Stomach 0.7(0.1) 1.0(0.0)0.8(0.1)
Kidneys 4.6(0.5) 7.6(5.6)3.3(0.1)
Intestines12.8(1.5) 12.0(0.8)14.9(2.1)
Femurs 0,3(0.0) 0.2(0.0)0.2(0.0)
Blood 26.8(3.0) 23.2(0.4)20.0(3.7)
~ Injected Dose/Gram Organ
Heart 3.4(0.2)2.1(0.8) 2.8(0.5)
Liver 8.8(0.6)8.9(3.1) 5.8(0.8)
Kidneys 9.4(0.6)14.1(7.2j 6.3(0.2)
Femurs 2.0(0.3)1.1(0.1) 1.0(0.2)
Blood 14.8(1.~9)11.5(0.7) ~ 10.2(1.5)
Standard deviation in brackets.
n = 3.
n = 2
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-26-
TABLE 9 - Urinary Clearance of 99~TcN-GLUC, 99~TcN-HEDP,
99~TcN-HIDA and 99~TcN-PIPIDA
:
~ Retained Activity
99~TcN-GLUC 99~TcN-HEDP 99~TcN-HIDA 99~TcN-PIPIDA
30 min 72.6(4.2) 68.8(9.5~ 74.9(5.8) 79.5(1.2)
60 min 59.3(6.4) 52.2(1.2) 66.3(0.9) 73.2(2.7)
120 min 45.6(5.6) 47.2(4.5) 54.7(1.1) 62.0(2.5)
Standard deviation in brackets.
n = 3.
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.
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-27-
TABLE 10 - Biological Distribution o~ 99~TcN-DMSA
% Injected Dose/Organ
Time after Injection30 min 60 min 120 min
Heart 0.3(0,0) 0.3(0.0) 0.2(0.0)
Lung 1.6(0.1) 1.1(0.2) 1.1(0.2)
Liver 4.3(0.1) 3.9(0-3) 3.4(0.2)
Spleen 0.4(0.1) 0.4(0.1) 0.3(0.0)
Stomach 0.6(0.1) 0.7(0.2) 0.5(0.1)
Kidneys 10.3(0.5)12.3(0.8) 17.1(0.7)
Intestines5.3(0.7)5.1(0.4) 4.8(0.5)
Femurs 0.5(0.1) 0.5(0.0) 0.4(0.1)
Blood 18.0(1.7)13.5(0.9) 9.5(0.5)
Urine 39.9(11.4)57.8(4.4) 65.3(4.3)
% Injected Dose/Gram Organ
Heart 1.8(0.1) 1.8(0.2) 1.2(0.0)
Liver 2.0(0.2) 2.1(0.3) 1.7(0.2)
Kidneys 16.4(0.7)22.2(1.4) 29.0(2.9)
Femurs 2.0(0.5) 2.4(0.2) 1.9(0.4)
Blood 7.1(0.8) 6.4(0.3) 4.1(0.2)
.
5 Standard deviation in brackets.
n = 3.
f
.. :.
.
~L2~36~
-28-
TABLE 11 - Biodistribution Ratio
Tissue (cpm/g): Blood (cpm/g)
Specific MAb (anti-Ly-2.1)/Non-specific Mab (anti-colon)
Time after Injection
Organ 20 hrs 30.5 hrs 35 hrs
a2.1 30.6 a2.1 30.6 a2.1 30.6
Blood 1.00 1.00 1.00 1.00 1.00 1.00
Tumor 1.23 0.40 2.60 0.68 3.48 0.48
(0.23-l.llg)
Stomach 0.08 0.12 0.12 0.17 0.07 0.12
Spleen 0.59 1.12 0.56 1.83 0.71 2.09
Kidney 0.77 1.48 0.94 2.14 0.92 2.50
Heart 0.33 0.39 0.19 0.17 0.33 0.19
Liver 0.84 0.27 1.02 3.26 1.20 5.19
Lung 0.38 0.22 0.86 0.66 0.36 0.60
Intestine 0.09 0.16 0.15 0.20 0.09 0.19
Tail 1.45 0.90 0.78 1.92 1.14 1.66
,
T~BLE 12 - Biodistribution Ratio
20 Tissue (cpm/g): Blood (cpmjg)~
,
ITT(l)E3 COLO 205 E3/COLO 205
Blood 1.0 1.0 1.0
Tumor 1.23 0.30 ~ 4.10
(.39 - l.llg) ~ (0.5 - 1.5g)
Stomach 0.08 0.2 0.04
Spleen 0.59 0.60 0.98
Kidney 0.77 0.76 ~ 1.01
Heart 0.33 ~ 0.39 0.85
Liver 0.84 0.56 1.50
Lung 0.38 0.42 0.90
Intestlne~ o.09 ~ 0.15 0.60
:
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' ~ ,
,
:-- - ' ~ ;
2g~ 3~'i
REFEREN~ S
~E .
1. Castronovo, F.P., J.Nucl.Med. 15, 127 (1974).
2. Callery, P.S., Faith, W.C., Loberg, M.D., Fields,
A.T., Harvey, E.B. and Cooper, M.D. J.Med.Chem, 19, 962
(1976).
3. Deutsch, E., Libson, K., Jurisson, S. et al:
Technetium chemistry and technetium radiopharmaceuticals.
Prog.Inorq.Chem 30:75-139, 183.
4. Deutsch, E., Barnett, B.L.: Synthetic and
structural aspects of technetium chemistry as related to
nuclear medicine, in: Inorganic Chemistry in Biology and
Medicine (ed Martel A.E.), ACS Symp.Series No. 150,
Washington, Amer.Chem.Soc. 1980, pp.103-ll9.
5. Hogarth, P.M., Henning, M.M. and McKenzie, I.F.C.
The alloantigenic phenotype o~ radiation induced thymomas
in the mouse. J.N.C.I. lg82; 69:619-626.
6. Hogarth, P.M., Edwards, J., McKenzie, I.F.C.,
Goding, J.W. and Liew, F.Y. Monoclonal antibodies to
murine Ly-2.1 surface antigen. Immunoloay 1982; 46:135-
144.
~'
7. Parish~ C.R. and McKenzie, I.F.C. A sensitive
rosetting method for detecting subpopulations of
Lymphocytes which react with alloantisera.
J.ImmunollMethods 1978, 20:173-183.
8. Rhodes,~B.A. and Burchiel, S.W. Radiolabelling of
Antibodies with Technetium oggm. Radioimmunoimaging and
Radioimmunotherapy. Editors Burchiel, S.W. and Rhodes,
B.A. Elsevier Publishing Co. 1983, p. 207.
.
,
.. : ~ . . . . .
,
.
-30-
9. Semple, T.U., Quinn, L.A., Woods, L.F. and Moore,
G.E. Tumor and Lymphoid cell line$ from a patient with
carcinoma of the colon for a cytoxicity model. Cancer
Res. 1978; 38:1345-1355.
10. Thompson, C.H., Jones, S.L., Pihl, E. and McKenzie,
I.F.C. Monoclonal antibodies to human colon and
colorectal carcinoma. Br.J.Cancer 1983; 47:595-605.
11. Tubis, M., Krishnamurthy, G.T., Endow, J.S., Blahd,
W.H. tl975) - 9g~Tc-Penicillamine complexes:In:Subramanian
G., Rhodes, B.A., Cooper, J.F., Sodd, V.J. (eds) "Radio-
pharmaceuticalsi'. The Society of Nuclear Medicine Inc.,
New York, pp. 55-62.
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